Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 62/383,330, filed September 2, 2016, which is hereby incorporated by reference in its entirety.

FIELD

[0002] Provided herein are cyclic peptide analogs, pharmaceutical compositions comprising such compounds, and methods of treating cancer with such compounds.

BACKGROUND

[0003] Cancer is a serious and debilitating disease brought on by abnormal and unchecked cell division in a patient. Current treatment strategies include chemotherapy, radiation therapy, and surger -. These treatment options may be singular treatments or combined for a more effective regimen. Unfortunately, many patients do not respond well to current chemo therapeutic regimens or develop resistance after prolonged treatment. In addition, for many chemotherapeuiics, there is a maximal lifetime level of drug that a patient may be administered. In this case, new drugs must be tried. Thus, there is a need for development of new and varied chemotherapeutic compounds to assist in the treatment of cancer.

[0004] An important aspect of cancer, as opposed to infection caused by an exogenous pathogen for example, is that the disease is caused by cells already existing in the patient. These cells are similar in many ways to healthy tissue and reside among healthy ceils in the patient. Thus, chemotherapeutic compounds, even if directly administered to a tumor, am the risk of entering and affecting healthy tissue in addition to cancerous tissue. This non-specific delivery can cause systemic and serious side effects in a patient including nausea, weakness, bleeding problems, infection, and hair loss. To avoid these systemic effects,

chemotherapeutic compounds may be conjugated to a targeting molecule that assists with the specific and direct delivery of a chemotherapeutic compound to cancerous tissue only, preventing delivery to healthy tissue. These drugs may be associated with fewer and less severe side effects than traditional therapy, and so there is a need to develop chemotherapeutics that are effective in isolation, but are also suitable for conjugation to a targeting molecule.

[0005] Various types of agents have been described for use in treatment of cancer. Many of these compounds pose challenges. For instance, many compounds described for use in treatment of cancer have problems associated with toxicity. Some compounds present challenges related to their chemical synthesis. There are also challenges associated with finding appropriate permutations of therapeutic agents for combination therapy.

Furtiiermore, only a minority of agents identified for use in treatment of cancer are suitable for conjugation to a targeting moiety. Accordingly, there remains a need for new compounds and conjugates for use in treatment of cancer.

SUMMARY

[0006] In one aspect, provided is a compound of Formula (I):

or a salt thereof, whe ein

R ¹ , R ² , R . R ^8a , R ^8b , R ⁹ , R ¹⁰ , R . R". R ^!3 , and R ¹⁴ are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

R ⁴ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl; R ^J is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, halo, or perhaloalkyl;

R° is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, halo, or perhaloalkyl;

R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl:

R ¹⁵ is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted alkyl;

n is 0, 1, or 2;

X ¹ is -N(R ^d )- or -()-:

X ² is O or S;

Y ¹ and Y ² are each independently -N(R ^d )-, -0-, or -S-;

Y ' is ~N(R ^d )~, -0-, -S-, or substituted or unsubstituted heterocycloalkyl;

Y ⁴ is -OR ^a , -NR ^b R ^c , or -SR ³ ;

each R ^a , R ^b , R ^c , and R ^d is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

W is (( \ \ ·)=..-/-(( ! ! ·),,:

Z is substituted or unsubstituted alkyl, substituted or unsubstituted substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or (Q-¾CH ₂ 0) _q ;

m, and p are each independently an integer from 0-12, inclusive; and

q is an integer from 1-12, inclusive; provided that i) when Y ⁴ is -OH, W is not ··{( ^' ] 1 · } ·· or -(CH ₂ CH ₂ 0)3(CH2)2-; and li) when V ¹ is -M I R or -N(CH ₃ )R ^C , W is not -(Π !.· ) -. -(CH ₂ ) ₆ -, or -( Η ·((Ί 1.( 1 .()) :(( ! ! .) _: - [0007] In some embodiments of Formula (I) or any variation thereof, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ ,

R ^8a , R ^8b , R ⁹ , R ¹⁰ , R ⁿ , R ¹² , R ¹³ and R ⁱ⁴ are each independently H or C C ₆ alkyl. In some embodiments, R ² is H or C _\ ~C alkyl optionally substituted with NH ₂ . In some embodiments, R ¹ , R ⁵ , R ⁶ , R ^8a , and R ^!4 are each methyl; R ² is methyl or -((Ή..) , \ Η..: R ³ , R ^8b , R ⁹ , R ⁿ , R ^!2 , and R ^iJ are each H; R ⁴ is iso-butyl ; and R ^lC' is sec-butyl ,

[0008] In some embodiments of Formula (I) or any variation thereof, R ¹⁵ is phenyl optionally substituted with halo, hydroxy, C i -Ce alkoxy, Ci-Ce alkyl, or Ci-Cr, perhaloalkyl; and n is 1.

[0009] In some embodiments of Formula (I) or any variation thereof, X ¹ is ~0~ or - N(R ^d )~, and R ^d is H or C C ₆ alkyl.

[0010] In some embodiments of Formula (I) or any variation thereof, R ⁷ is H, C _\ ~C alkyl, C ;-( ^' ,, alkenyl, or ( ^" ;-(. ^' ,·. alkynyl.

[0011] In some embodiments of Formula (1) or any variation thereof, Y ¹ is -0-. In some embodiments, Y ² is -0-. In some embodiments, X ² is -0-. In some embodiments,Y ^l , Y ² , and X are each -0-, and Y ^J is -N(R ^d )-. In some embodiments,Y ⁴ is -OR ^a or -NR ^b R ^c . In some embodiments, each R ^a , R ^b , R ^c , and R ^d is independently H or -CH ₃ .

[0012] In some embodiments of Formula (I) or any variation thereof, m and p are each 0. In some embodiments, Z is C3-C12 alkyl. In some embodiments, Z is selected from the group consisting of substituted or unsubstituted C3-C ₁₂ cycloalkyi, substituted or unsubstituted C3- C ₁₂ cycloalkenyl, substituted or unsubstituted C5-C ₁₂ aryl, substituted or unsubstituted C5-C12 heteroaryl. In some embodiments, Z is C3-C ₁₂ alkyl or (CH ₂ CH ₂ 0) _q , wherein q is an integer from 1-8, inclusive. In some embodiments, q is 1 or 2.

[0013] Provided in some embodiments are compounds selected from, the group consisting of compounds of Table 1 , or a salt thereof.

[0014] Provided in some aspects is a conjugate containing a compound of Formula (I) or any variation thereof bonded to a ligand, wherein the ligand is a polypeptide, a nucleic acid. or a targeting moiety. In some embodiments, the ligand is an antibody. In some embodiments, the compound is bonded to the ligand via a linker.

[0015] Provided in some aspects is a conjugate containg a compound of Formula (I) or any variation thereof bonded to a linker. In some embodiments, the linker is a cleavabie linker. In some embodiments,the linker is a non-cleavable linker.

[0016] Provided in some aspects is a conjugate of Formula (III):

(III)

or a salt thereof, wherein

R ¹ , R ² , R ³ , R ⁸³ , R ^8b , R ⁹ , R ^" . R ^u , R ¹² , R ¹³ , and R ^! are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl:

R ⁴ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryi, or substituted or unsubstituted heterocyclyl:

R ⁵ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryi, substituted or unsubstituted heterocyclyl, halo, or perhaloalkyl; R ⁶ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted and, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, halo, or perhaloalkyl;

R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryi, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl;

R° is substituted or unsubstituted aryi, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted alkyl;

n is 0, 1, or 2;

X ^: is -N(R ^d )~ or -0-;

X ² is O or S;

Y ¹ and Y ² are each independently -N(R ^d )-, -0-, or -S-;

Y ³ is -N(R ^d )-, -0-, -S-, or substituted or unsubstituted heterocycloalkyl;

Y ^4a is -0-, -NR ^b ~, or -S~;

each R ^b and R ^d is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

W is (CH ₂ ) _m -Z-(CH ₂ )p;

Z is substituted or unsubstituted alkyl, substituted or unsubstituted substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryi, substituted or unsubstituted heteroaryl, or (CH ₂ CH ₂ 0) _q ;

m, and p are each independently an integer from 0-12, inclusive;

q is an integer from 1-12, inclusive;

a, b, c, and d are each independently 0, 1, or 2; each L; is independently

,wherein a 1 is 0, 1 , or 2, and each R ^x is un ubstituted or substituted alkyl;

each L ₂ is independently or , wherein b 1 is an integer from 0 to 12, inclusive;

acid, cl is an integer from 0-12, inclusive, and c2 is an integer from 0- 10, mclusive;

from 0-12, inclusive, and d2 is an integer from 0-30, inclusive; and Fn is selected from the group consisting of



(IV)

or a salt thereof, wherein

R ^! , R ² , R ³ , R ^Sa , R ^8b , R ⁹ , R ^{! 0} , R . R ^!2 , R ⁱ³ , and R ¹⁴ are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenvi;

R ⁴ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenvi, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyi;

R ³ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyi, halo, or perhaloalkyl;

R ⁶ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenvi, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyi, halo, or perhaloalkyl;

R ' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cvcloalkenvl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryi, or substituted or unsubstituted heterocyclyl;

R ⁱ³ is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryi, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted alkyl:

n is 0, 1 , or 2;

X ^! is -N(R ^d )~ or -0-;

X ² is O or S;

Y ¹ and Y ² are each independently -N(R ^d )-, -0-, or -S-;

Y ³ is -N(R ^d )-, -0-, ~S~, or substituted or unsubstituted heterocycloalkyl;

Y ^4a is -0-, -NR ^b -, or -S-;

each R ^b and R ^d is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenvl.. substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cvcloalkenvl;

W is (CH ₂ ) _m -Z-(CH ₂ )p;

Z is substituted or unsubstituted alkyl, substituted or unsubstituted substituted or unsubstituted alkenvl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryi, or (CH ₂ CH ₂ Q) _q ;

m, and p are each independently a integer from 0-12, inclusive;

q is an integer from 1 -12, inclusive;

a, b, c, and d are each independently 0, 1, or 2;

in al is 0, 1, or 2, and each R ^A is unsubstituted or substituted alkyl; each L ₂ is independently , wherein b 1 is an integer from 0 to 12, inclusive:

each L ₃ is independently

_; wherein each AA is an amino acid, cl is an integer from 0- 12, inclusive, and c2 is an integer from 0-10, inclusive;

, or , wherein dl is an integer from 0-12, inclusive, and d2 is an integer from 0-30, inclusive;

mFn is selected from the group consisting of t is an integer from 1-12, inclusive; and

Ab is an antibody.

[0018] In some embodiments of Formulae (III) or (IV), i) when Y ^4a is -0-, W is not - (CH ₂ ) ₂ - or -(CH ₂ CH ₂ 0)3(CH ₂ )2-; and si) when Y ^4a is -NH- or -N(CH ₃ )-, W is not -(CH ₂ ) ₂ -, -

(CH ₂ ) ₆ ~, or -CH ₂ (CH ₂ CH ₂ 0) ₃ (CH ₂ )3-.

[0019] In some embodiments of Formulae (III) or (IV) or any variation thereof, R ¹ , R ^J , R ⁴ , R ⁵ , R ⁶ , R ^8a , R ^8b , R ⁹ , R ⁱ⁰ , R ^{! 1} , R ⁱ² , R ^!3 and R ¹⁴ are each independently H or Ci-C ₆ alkyl. In some embodiments, R ² is H or Ci-Ce alkyl optionally substituted with NH ₂ . In some embodiments, R ¹ , R ³ , R ⁶ , R ^8a , and R ¹⁴ are each methyl; R ² is methyl or -(CH ₂ ) ₄ NH ₂ ; R ^J , R ^8b , R , R ¹¹ , R ^l2 , and R ^l3 are each H; R ⁴ is iso-butyl; and R ^lC" is sec-butyl.

[0020] In some embodiments of Formulae (III) or (IV) or any variation thereof, R ¹⁵ is phenyl optionally substituted with halo, hydroxy, Ci-Ce alkoxy, Ci-Q alkyl, or C Ce perhaloalkyl; and n is 1.

[0021] In some embodiments of Formulae (III) or (IV) or any variation thereof, X ¹ is ~Q~ or -N(R ^d )-, and R ^d is H or Ci -C ₆ alkyl.

[0022] In some embodiments of Formulae (III) or (IV) or any variation thereof, R' is H, Ci-Ce alkyl, C G alkenyl, or C Ce alkynyl.

[0023] In some embodiments of Formulae (III) or (IV) or any variation thereof, Y ¹ is -0-. In some embodiments, Y ² is ~0~. In some embodiments, X ² is -0-. In some embodiments, Y ¹ , Y ¹ , and X ² are each -0-, and Y ^J is -N(R ^d )-. In some embodiments,Y ^4a is -O- or -NR ^b -. In some embodiments, each R ^b and R ^d is independently H or -CI¾.

[0024] In some embodiments of Formulae (III) or (IV) or any variation thereof, m and p are each 0. In some embodiments, Z is C3-C12 alkyl. In some embodiments, Z is selected from the group consisting of substituted or unsubstituted C3-Q 2 cycloalkyl, substituted or unsubstituted C ₃ -Ci ₂ cycloalkenyl, substituted or unsubstituted C5-C12 aryl, substituted or unsubstituted C5-C12 heteroaiyl. In some embodiments, Z is C3-C12 alkyl or (CH ₂ CH ₂ 0) _q , wherein q is an integer from 1-8, inclusive. In some embodiments, q is 1 or 2. 5] In some embodiments of Formulae (III) or (IV) or any variation thereof L] is

e mbodiments, Li . In some embodiments, al is

0 or 1. In some embodiments, each R ^x is C ₁ -C4 aikyl. In some embodiments, each R ^x is C] C4 alkyl, substituted with halo. In some embodiments, each R ^x is trichloroethyl.

In some embodiments of Formulae (III) or (IV) or any variation thereof, L ₂ is

H

V7 , ^r

b ^l . In some embodiments, b l is an integer from 1-8, inclusive.

] In some embodiments of Formulae (III) or (IV) or any variation thereof, L ₃ is . In some embodiments, L ₃ is

In some embodiments, L ₃ is

In some embodiments, cl is 2. In some embodiments, c2 is 3. In some embodiments, (AA) _ci is -Cit-Val- or -Ala-Val-.

^■ 8] In some embodiments of Formulae (III) or (IV) or any variation thereof, L ₄ is . In some embodiments, dl is any integer from 2-7, inclusive. [0029] In some embodiments of Formulae (III) or (IV) or any variation thereof a, b, c, and d are each independently 0 or 1.

[0031] Provided in some embodiments are conjugates selected from the group consisting of conjugates of Table 2, or a salt therof.

[0032] In some embodiments, the conjugate is selected from the group consisting of conjugates of Table 3, or a salt thereof. In some embodiments, b is an integer from 1 -12, inclusive, and Ab is an antibody.

[0033] In some aspects, provided are pharmaceutical compositions containing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a

pharmaceutically acceptable carrier. Provided in other embodiments are pharmaceutical compositions containing a conjugate of Formulae (III) or (IV), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition contains one or more compounds of Table 1 , or a

pharmaceutically acceptable salt thereof. In other embodiments, the pharmaceutical composition contains one or more conjugates of Table 2 or Table 3, or a pharmaceutically acceptable salt thereof.

[0034] Provided in some aspects are methods of treating cancer in an individual in need thereof, the method including administering to the individual a therapeutically effective amount of a compound of Formula (I), or a pharmaceuticaily acceptable salt thereof. In other aspects, provided are methods of treating cancer in an individual in need thereof, the method including administering to the individual a therapeutically effective amount of a conjugate of Formulae (III) or (IV), or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes administering to the individual a therapeutically effect amount of one or more compounds of Table 1, or a pharmaceutically acceptable salt thereof. In other embodiments, the method includes administering to the individual a therapeutically effect amount of one or more conjugates of Table 2 or 3, or a pharmaceutically acceptable salt thereof,

[0035] In some aspects, provided are kits containing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and instructions for use in treatment of cancer in an individual in need thereof. Provided in other embodiments are kits containing a conj ugate of Formulae (III) or (IV), or a pharmaceuticaily acceptable salt thereof, and instructions for use in treatment of cancer in an individual in need thereof. In some embodiments, the kits contain one or more compounds of Table 1, or a pharmaceutically acceptable salt thereof. In other embodiments, the kits contain one or more conjugates of Tables 2 or 3, or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0036] Figure 1 is a ^! H NMR spectrum of Compound 2 in DMSO-d6.

[0037] Figure 2 is a ^! H NMR spectrum of Compound 4 in DMSO-d6. [0038] Figure 3 is a T I NMR spectrum of Conjugate L4 in DMSO-d6.

[0039] Figure 4 is a ^! H NMR spectrum of Conjugate L5 in DMSO-d6.

[0040] Figure 5 is a ^! H NMR spectrum of Conjugate L6 in DMSO-d6.

[0041 ] Figure 6 is a Ti NMR spectrum of Conjugate L7 in DMSO-d6.

[0042] Figure 7 is a Ή NMR spectrum of Compound 14 in DMSO-d6.

[0043] Figure 8 is a Ή NMR spectrum of Compound 16 in DMSO-d6.

DETAILED DESCRIPTION

Definitions

[0044] As used herein, reference to ^" 'about ^" ' a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0045] Unless clearly indicated otherwise, ' ^" an individual" as used herein intends a mammal, including but not limited to a human, bovine, primate, equine, canine, feline, porcine, and ovine animals. Thus, the compositions and methods provided herein have use in both human medicine and in the veterinary context, including use in agricultural animals and domestic pets. The individual may be a human who has been diagnosed with or is suspected of having a condition described herein, such as cancer. The individual may be a human who exhibits one or more symptoms associated with a condition described herein, such as cancer. The individual may be a human who has a mutated or abnormal gene associated with a condition described herein, such as cancer. The individual may be a human who is genetically or otherwise predisposed to or at risk of developing a condition described herein, such as cancer.

[0046] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For puiposes of the compositions and methods provided herein, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from the condition, diminishing the extent of the condition, stabilizing the condition (e.g., preventing or delaying the worsening of the condition), preventing or delaying the spread (e.g., metastasis) of the condition, delaying or slowing the progression of the condition, ameliorating a disease state, providing a remission (whether partial or total) of a disease, decreasing the dose of one or more other medications required to treat the condition, enhancing the effect of another medication used to treat the condition, increasing the quality of life of an individual having the condition, and/or prolonging survival. A method of treating cancer encompasses a reduction of the pathological consequence of cancer. Tire methods described herein contemplate an y one or more of these aspects of treatment.

[0047] As used herein, an "at risk" individual is an individual who is at ri sk of developing a disease or condition described herein, such as cancer. An individual "at risk" may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein . "At risk" denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of a disease or condition described herein, such as cancer. An individual having one or more of these risk factors has a higher probability of developing the disease or condition than an individual without these risk factor(s).

[0048] As used herein, by ' ^" combination therapy" is meant a therapy that includes two or more different compounds. Thus, in one aspect, a combination therapy comprising a compound detailed herein and another compound is provided. In some variations, the combination therapy optionally includes one or more pharmaceutically acceptable carriers or excipients, non-pharmaceutically active compounds, and/or inert substances. In various embodiments, treatment with a combination therapy may result in an additive or even synergistic (e.g., greater than additive) result compared to administration of a single compound provided herein alone. In some embodiments, a lower amount of each compound is used as part of a combination therapy compared to the amount generally used for individual therapy. Preferably, the same or greater therapeutic benefit is achieved using a combination therapy than by using any of the individual compounds alone. In some embodiments, the same or greater therapeutic benefit is achieved using a smaller amount (e.g., a lower dose or a less frequent dosing schedule) of a compound in a combination therapy than the amount generally used for individual compound or therapy. Preferably, the use of a small amount of compound results in a reduction in the number, severity, frequency, and/or duration of one or more side-effects associated with the compound.

[0049] As used herein, the term "effective amount" intends such amount of a compound provided herein which in combination with its parameters of efficacy and toxicity, should be effective in a given therapeutic form. As is understood in the art, an effective amount may be in one or more doses, i.e. , a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. Suitable doses of any of the co-administered compounds may optionally be lowered due to the combined action (e.g. , additive or synergistic effects) of the compounds. In various embodiments, an effective amount of the composition or therapy may (i) reduce the number of cancer cells; (ii) reduce tumor size; (in) inhibit, retard, slow to some extent, and preferably stop cancer cell infiltration into peripheral organs: (iv) inhibit (e.g., slow to some extent and preferably stop) tumor metastasis: (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of a tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. In various embodiments, the amount is sufficient to ameliorate, palliate, lessen, and/or delay one or more of symptoms of a disease or condition described herein, such as cancer.

[0050] As is understood in the art, an "effective amount" may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a compound, or pharmaceutically acceptable salt thereof, may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved.

[0051] A "therapeutically effective amount" refers to an amount of a compound or salt thereof sufficient to produce a desired therapeutic outcome (e.g., reducing the severity or duration of, stabilizing the severity of, or eliminating one or more symptoms of a disease or condition described herein, such as cancer). For therapeutic use, beneficial or desired results include, e.g., decreasing one or more symptoms resulting from the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease or condition, increasing the quality of life of those suffering from the disease or condition, decreasing the dose of other medications required to treat the disease or condition, enhancing effect of another medication, delaying the progression of the disease or condition, and/or prolonging survival of patients.

[0052] It is understood that an effective amount of a compound or pharmaceutically acceptable salt thereof, including a prophylactically effective amount, may be given to an individual in the adjuvant setting, which refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) has been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgical resection), radiotherapy, and chemotherapy. However, because of their history of cancer, these individuals are considered at risk of developing cancer. Treatment or administration in the "adjuvant setting" refers to a subsequent mode of treatment.

[0053] As used herein, by "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a material that is not biologically or otherwise undesirable, e.g. , the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.

Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

[0054] "Pharmaceutically acceptable salts" are those salts which retain at least some of the biological activity of the free (non-salt) compound and which can be administered as drugs or pharmaceuticals to an individual. Such salts, for example, include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid and the like; (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine,

triethanolamine and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. Pharmaceutically acceptable salts can be prepared in situ in the manufacturing process, or by separately reacting a purified compound provided herein in its free acid or base form with a suitable organic or inorganic base or acid, respectively, and isolating the salt thus formed during subsequent purification.

[0055] The term "excipient" as used herein means an inert or inactive substance that may be used in the production of a drug or pharmaceutical, such as a tablet containing a compound provided herein as an active ingredient. Various substances may be embraced by the term excipient, including witiiout limitation any substance used as a binder, disintegrant, coating, compression/encapsulation aid, cream or lotion, lubricant, solutions for parenteral administration, materials for chewable tablets, sweetener or flavoring, suspending/gelling agent, or wet granulation agent. Binders include, e.g., carbomers, povidone, xanthan gum, etc.; coatings include, e.g., cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, enteric coatings, etc.; compression/encapsulation aids include, e.g., calcium carbonate, dextrose, fructose dc (dc ^: = "directly compressible"), honey dc, lactose (anhydrate or monohydrate; optionally in combination with aspartame, cellulose, or microcrystalline cellulose), starch dc, sucrose, etc.; disintegrants include, e.g., croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; creams or lotions include, e.g., maltodextrin, carrageenans, etc.; lubricants include, e.g., magnesium stearate, stearic acid, sodium stearyl fumarate, etc.; materials for chewable tablets include, e.g., dextrose, fructose dc, lactose (monohydrate, optionally in combination with aspartame or cellulose), etc.;

suspending/gelling agents include, e.g., carrageenan, sodium starch glycolate, xanthan gum, etc.; sweeteners include, e.g., aspartame, dextrose, fructose dc, sorbitol, sucrose dc, etc.; and wet granulation agents include, e.g., calcium carbonate, maltodextrin, microcrystalline cellulose, etc.

[0056] "Alkyl" refers to and includes saturated linear or branched univalent hydrocarbon structures and combinations thereof. Particular alkyl groups are those having 1 to 20 carbon atoms (a "C1-C20 alkyl"). More particular alkyl groups are those having 1 to 8 carbon atoms (a "d-Cg alkyl") or 1 to 6 carbon atoms (a "Cr , alkyl"). When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, "butyl" is meant to include »-butyl, sec-butyl, wo-butyl, and rert-butyl; "propyl" includes w-propyl and so-propyl. This term, is exemplified by groups such as methyl, f-butyl, w-heptyl, octyS, and the like.

[0057] "Cycloalkyl" refers to and includes cyclic univalent hydrocarbon structures. Cycloalkyl can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantyi. A cycloalkyl comprising more than one ring may be fused, spiro or bridged, or combinations thereof. A preferred cycloalkyl is a saturated cyclic hydrocarbon having from 3 to 13 annular carbon atoms. A more preferred cycloalkyl is a saturated cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a "CrCg cycloalkyl"). Examples of cycloalkyl groups include adamantyi, deeahydronaphthalenyi, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0058] "Alkenyl" refers to an unsaturated hydrocarbon group having at least one site of olefmic unsaturation (i.e., having at least one moiety of the formula C=C) and preferably having from 2 to 10 carbon atoms and more preferably 2 to 8 carbon atoms. Examples of alkenyl include but are not limited to -CH ₂ -CH=CH-CH ₃ and -CH=CH-CH=CH ₂ .

[0059] "Cyclolkenyl" refers to an unsaturated hydrocarbon group within a cycloalkyl having at least one site of olefmic unsaturation (i.e. , having at least one moiety of the formula C=C). Cycloalkenyl can consi st of one ring, such as cyclohexyl, or multiple rings, such as norboraenyl . A more preferred cycloalkenyl is an unsaturated cyclic hydrocarbon having from 3 to 8 annular carbon atoms (a "C3-C8 cycloalkenyl"). Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyciopentenyl, cyciohexenyl, and the like.

[0060] "Alkynyl" refers to an unsaturated hydrocarbon group having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C) and preferably having from 2 to 0 carbon atoms and more preferably 2 to 8 carbon atoms and the like.

[0061] The term "alkoxy" refers to an -O-alkyl group, where the O is the point of attachment to the rest of the molecule, and aikyl is as defined above.

[0062] "Heterocycle", "heterocyclic", or "heterocyclyl" refers to a saturated or an unsaturated non-aromatic group having a single ring or multiple condensed rings, and having from 1 to 10 annular carbon atoms and from 1 to 4 annular heteroatoms, such as nitrogen, sulfur or oxygen, and the like. A heterocycle comprising more than one ring may be fused, spiro or bridged, or any combination thereof. In fused ring systems, one or more of the rings can be aryl or heteroaryi. A heterocycle having more than one ring where at least one ring is aromatic may be connected to the parent structure at either a non-aromatic ring position or at an aromatic ring position , in one variation, a heterocycle having more than one ring where at least one ring is aromatic is connected to the parent structure at a non-aromatic ring position.

[0063] "Aryl" or "Ar" refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic. In one variation, the aryl group contains from 6 to 14 annular carbon atoms. An aryl group having more than one ring where at least one ring is non-aromatic may be connected to the parent structure at either an aromatic ring position or at a non-aromatic ring position. In one variation, an aryl group having more than one ring where at least one ring is non-aromatic is connected to the parent structure at an aromatic ring position.

[0064] "Heteroaryi" or "HetAr" refers to an unsaturated aromatic carbocyclic group having from 1 to 10 annular carbon atoms and at least one annular heteroatom, including but not limited to heteroatoms such as nitrogen, oxygen and sulfur. A heteroaryi group may have a single ring (e.g., pyridyl, furyl) or multiple condensed rings (e.g., indolizmyl, benzothienyl) which condensed rings may or may not be aromatic. A heteroaryi group having more than one ring where at least one ring is non-aromatic may be connected to the parent structure at either an aromatic ring position or at a non-aromatic ring position . In one variation, a heteroaryi group having more than one ring where at least one ring is non-aromatic is connected to the parent structure at an aromatic ring position.

[0065] The term "halogen" represents chlorine, fluorine, bromine, or iodine. The term "halo" represents chloro, fluoro, bromo, or iodo.

[0066] The term "substituted" means that the specified group or moiety bears one or more substituents including, but not limited to, substituents such as alkoxy, acyl, acyloxy, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyioxy, cycioaikyl, cycloalkenyl, aryl, heteroaryi, aryloxy, cyano, azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, cycioaikyl, cycloalkenyl, alkyl, alkenyl, alkynyl, heterocyclyl, aralkyl, aminosulfonyl, sulfonylamino, sulfonyl, oxo, carbonylalkyienealkoxy and the like. The term "unsubstituted" means that the specified group bears no substituents. The term "optionally substituted" means that the specified group is unsubstituted or substituted by one or more substituents. Where the term "substituted" is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system.

[0067] A composition of "substantially pure" compound means that the composition contains no more than 15% or preferably no more than 10% or more preferably no more than 5% or even more preferably no more than 3% and most preferably no more than 1% impurity, which impurity may be the compound in a different stereochemical form. For instance, a composition of substantially pure (S) compound means that the composition contains no more than 15% or no more than 10% or no more than 5% or no more than 3% or no more than 1% of the (R) form of the compound.

[0068] Any formula given herein is intended to represent compounds having structures depicted by the structural formula as well as certain variations or fonns. In particular, compounds of any formula given herein may have asymmetric centers and therefore exist in different enantiomeric forms. Ail optical isomers and stereoisomers of the compounds of the general formula, and mixtures thereof in any ratio, are considered within the scope of the formula. Thus, any formula given herein is intended to represent a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomenc forms, and mixtures thereof in any ratio. Furthermore, certain structures may exist as geometric isomers (i.e., cis and trans isomers), as tautomers, or as atropisomers. Additionally, any formula given herein is intended to refer also to any one of hydrates, solvates, and amorphous and polymorphic forms of such compounds, and mixtures thereof, even if such forms are not listed explicitly. In some embodiments, the solvent is water and the solvates are hydrates.

[0069] Any formula given herein is also intended to represent unlabeled forms as well as isotopicaily labeled forms of the compounds. Isotopicaiiy labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as ^" Ή, ¾, "C, ^l3 C, ' ⁴ C, ^!5 N, ^!8 0, ¹⁷ 0, ^{3 i} P, ³² P, ³⁵ S, ^!8 F, ³⁶ C1, and ⁿ % respectively. Substitution with heavier isotopes such as deuterium (i.e., ² H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. Isotopically labeled compounds described herein and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopicaily labeled reagent.

[0070] When referring to any formula given herein, the selection of a particular moiety from a list of possible species for a specified variable is not intended to define the same choice of the species for the variable appearing elsewhere. In other words, where a variable appears more than once, the choice of the species from a specified list is independent of the choice of the species for the same variable elsewhere in the formula, unless stated otherwise.

[0071] According to the foregoing interpretive considerations on assignments and nomenclature, it is understood that explicit reference herein to a set implies, where chemically meaningful and unless indicated otherwise, independent reference to

embodiments of such set, and reference to each and every one of the possible embodiments of subsets of the set referred to explicitly.

Compounds

[0072] Compounds and salts thereof (such as pharmaceutically acceptable salts) are detailed herein, including in the Brief Summary and in the appended claims. Also provided are the use of all of the compounds described herein, including any and all stereoisomers, including geometric isomers (cis/trans), E/Z isomers, enantiomers, diastereomers, and m ixtures thereof in any ratio including racemic mixtures, salts and solvates of the compounds described herein, as well as methods of making such compounds. Any compound described herein may also be referred to as a drug.

[0073] In one aspect, provided are compounds of Formula (I):

(I)

or a salt thereof, wherein

R ¹ , R '. Ill R ⁸³ , R ^8B , R ⁹ , R ^" . R ^U , R ¹² , R ¹³ , and R ^! are each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

R ⁴ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted axyl, substituted or unsubstituted heteroarvi, or substituted or unsubstituted heterocvclvl;

R ⁵ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocvclvl, halo, or perhaloalkyl;

R ⁶ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyciyi, halo, or perhaloalkyl;

R' is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocvclvl; R ° is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted alkyl;

n is 0, 1, or 2;

X ¹ is -\( R ^d )- or ·{)· :

X ² is O or S;

Y ¹ and Y ² are each independently -N(R ^d )-, -0-, or -S-;

Y ³ is -N(R ^d )-, -0-, -S-, or substituted or unsubstituted heterocycloalkyl;

Y is -OR ⁸ , -NR ^b R ^c , or -SR ³ ;

each R ^a , R ^b , R ^c , and R ^d is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

W is (CH ₂ ) _ffi ~Z-(CH ₂ )p:

Z is substituted or unsubstituted alkyl, substituted or unsubstituted substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or (C ^' i \ ·(. ^' ': Ι.·ί.)) ₍ ,:

m, and p are each independently an integer from 0-12, inclusive; and

q is an integer from 1-12, inclusive;

provided that i) when Y ⁴ is -OH, W is not -{CH ₂ } ₂ ~ or -(( Ή >Ci Ι.Ό) :(( ^' H <).--: and ii) when Y ⁴ is ~ HR° or -N(CH ₃ )R ^C , W is not -id ! > >-. ~! ( H · ),-.-. or -CH ₂ (CH ₂ CH ₂ 0)3(CH ₂ )3-.

[0074] In any variation of Formula (I) described herein, X ¹ may be -0-. In other embodiments of Formula (I), X ¹ is -N(R ^d )-, where R ^d is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl, in some of these embodiments, X ¹ is -N(R ^d )- where R" is Ci -Ce unsubstituted alkyl, such as methyl or ethyl. In some of these embodiments, X ¹ is -N(R ^d )- where R ^a is H or Ci-Ce alkyl. In some of these embodiments, X ¹ is -NH-.

[0075] In some embodiments of Formula (I), X" is O. In other embodiments of Formula [0076] In some embodiments of Formula (I), Y ^J , Y', and Y' are each independently - N(R ^d )-, -0-, or -S-.

[0077] In some embodiments of Formula (I), Y ¹ is -()-. In other embodiments of Formula (I), Y ^! is -S-. In yet other embodiments of Formula (I), Y ^! is -N(R ^a )-, where R ^d is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycioaikenyl. In some of these embodiments, Y ¹ is -N(R ^d )-, where R ^d is H or Cj-Ce alkyl, such as methyl or ethyl. In some embodiments, Y ¹ is -NH-. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in the (S) stereochemical configuration.

[0078] In some embodiments, Y ² is -0-, In some embodiments, Y ² is -S-. In some embodiments of Formula (I), Y ² is -N(R ^d )- where R ^d is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycioaikenyl In some of these embodiments, Y ² is -N(R ^d )-, where R is H or Cj-Ce alky], such as methyl or ethyl . In some embodiments, Y ² is -NH-. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ² may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ² may be in the (S) stereochemical configuration. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in the (S) stereochemical configuration.

[0079] In some embodiments of Formula (I), Y ³ is -N(R ^d )- where R° is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycioaikenyl . In some of these embodiments, Y ³ is -N(R°)-, where R ^d is H or Ci -Ce alkyl, such as methyl or ethyl. In some embodiments, Y ³ is -NH-. In some embodiments, Y ' is -0-. In some embodiments, Y ⁵ is -S-. In some embodiments, Y ^J is substituted or unsubstituted heterocycloalkyl. In some embodiments, Y ^J is substituted or unsubstituted pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, 1 ,3-dioxolanyl, tetrahydrothiophenyl, oxathiolanyi, sulfolanyl, piperidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, thianyl, dithianyl, trithianyl, morpholinyl, thiomorpholinyl. In some embodiments, Y ^J is piperazinyl

[0080] In some embodiments of Formula (I), Y ⁴ is -OR ^a - where R ^a is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl. In some of these embodiments, Y* is -OR ³ -, where R ^a is H or Ci-Ce alkyl, such as methyl or ethyl. In some of these embodiments, Y ⁺ is -NHR ^C , where R ^c is substituted or unsubstituted Ci-Ce alkyl. In some embodiments, Y ⁴ is -OH. In some embodiments of Formula (I), Y ⁴ is - NR ^b R ^c , where R ^b , and R ^c is each independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl. In some of these embodiments, Y ⁴ is - N ^R ⁰ , where R , and R ^c is each independently H or C ₅ ~C _& alkyl, such as methyl or ethyl. In some of these embodiments, Y ⁴ is -NHR ^C , where R° is substituted or unsubstituted Cj -Ce alkyl. In some embodiments, Y ⁴ is -NH ₂ .In some embodiments of Formula (I), Y ⁴ is -SR ^a - where R ^a is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl. In some of these embodiments, Y ⁴ is -SR ^a -, where R ^a is H or Ci-Ce alkyl, such as methyl or ethyl. In some of these embodiments, Y ⁴ is -NHR ^C , where R ^c is substituted or unsubstituted Ci -Ce alkyl. In some embodiments, Y ⁴ is -NHR ^C , where R ^c is Ci-Ce alkyl substituted with oxo. In some embodiments, Y ⁴ is -SH.

[0081] In some embodiments of Formula (I), each R ^a , R ^b , R ^c , and R ^d is independently H, or substituted or unsubstituted alkyl. In some embodiments, each R ^a , R°, R ^c , and R ^d is independently H or -CFi ₃ , In some embodiments, R ^a and R ^b are independently H or -C i -Ce alkyl. In some embodiments, R ^b and R ^c are independently H or -Cj-Ce alkyl. In some embodiments, R ^b and R ^c are each -Ci -Ce alkyl. In some embodiments, one of R ^b and R ^c is H or -Ci-Cg alkyl, and the other is -Ci-Cg alkyl substituted with oxo. In some embodiments, one of R ^b or R ^c is -Ci-C ₆ alkyl and the other is acetyl.

[0082] In some embodiments of Formula (I), W is (CH ₂ ) _m -Z-(CH ₂ )p _, where Z is selected from the group consisting of substituted or unsubstituted C3-C ₁₂ cycloalkyl, substituted or unsubstituted C3-C ₁₂ cycloalkenyl, substituted or unsubstituted C5-C ₁₂ aryl, and substituted or unsubstituted C5-C ₁₂ heteroaryl. In some embodiments, Z is C3-C ₁₂ alkyl. In some embodiments, Z is substituted or unsubstituted aryl. In some embodiments, Z is phenyl. In some embodiments, Z is substituted or unsubstituted heteroaryl. In some embodiments, Z is substituted or unsubstituted cycloalkyl . In some embodiments, Z is cyclohexyl. In some embodiments, Z is substituted or unsubstituted heterocycloalkyl. In other embodiments, Z is (CH ₂ CH ₂ 0)q. In some embodiments of Formula (I), q is an integer from 1-12, inclusi ve. In some embodiments, q is an integer from. 4-8, inclusive. In other embodiments, q is 1 or 2. In some embodiments of Formula (I), m and p are each independently an integer from 0- 12, inclusive. In some embodiments, m is an integer from 0-8, inclusive. In some embodiments, m is 0. In some embodiments, m is I . In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments of Formula (I), p is an integer from 0-8, inclusive. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, m and p are each 0.

[0083] In some embodiments, W is -C ₂ -C ₁₂ alkyl-, wherein -C ₂ -C ₁₂ alkyl- is optionally substituted. In some embodiments, W is -C ₂ -C ₁₂ alkyl-. In some embodiments, W is - (CH ₂ ) ₂ ~. In some embodiments, W is -(CH ₂ )3~. In some embodiments, W is (Ci ! · ) :-. In some embodiments, W is -(CH ₂ )5~. In some embodiments, W is -(Π¾)6-. In some embodiments, W is -(CHj)?-. In some embodiments, W is --C3-C12 alkyl-, wherein -C3-C12 alkyl- is optionally substituted. In some embodiments, W is -C3-C1 ₂ alkyl-.

[0084] In some embodiments of Formula (I), R ¹ , R ² , R ³ , R ⁴ , R ^8a , R ^8b , R ⁹ , R ¹⁰ , R ¹ R ¹² , R ^*3 , and R ^l4 are each independently H or substituted or unsubstituted alkyl, such as unsubstituted Ci-Cc, alkyl. In some embodiments, R ^! is H. In other embodiments, R ¹ is Ce alkyl, such as methyl. In some embodiments, R ² is H. In some embodiments, R ² is substituted or unsubstituted alkyl . In some embodiments, R ² is Ci-Ce alkyl, such as methyl. In some embodiments, R ² is Ci -Ce alkyl optionally substituted with NH ₂ . In yet other embodiments, R ² is which is optionally substituted. In some embodiments, R ³ is H. In other embodiments, R ³ is Ci-Ce alkyl, such as methyl. In some embodiments, R ⁴ is H. In other embodiments, R ⁴ is Cj -Ce alkyl, such as methyl. In particular embodiments, R ^* is isobutyl. In some embodiments, R ^' and R ^{' J} are both H. In other embodiments, R ' and R ^{' '} are each independently Ci-Ce alkyl, such as methyl In some embodiments, R ^8a and R ^So are both methyl . In yet other embodiments, one of R ^8a and R ^8b is H and the other is C _\ ~Ce alkyl, such as methyl. In yet other embodiments, one of R ^8a and R ^8b is H and the oilier is - (CHiWNHi, which is optionally substituted. In some embodiments, R ⁹ is H. In other embodiments, R is Ci~C _& alkyl, such as methyl. In some embodiments, R'" is H. In other embodiments, R ⁱ⁰ is C ~C alkyl, such as methyl. In particular embodiments, R ^l0 is sec- butyl . In some embodiments, R ⁹ is H. In other embodiments, R ⁹ is C Ce alkyl, such as methyl. In some embodiments, R ¹² and R ^!3 are both H. In other embodiments, R ⁱ² and R ^1J are each independently C i -Ce alkyl, such as methyl. In yet other embodiments, one of R' ² and R ^l3 is H and the other is Ci-Ce alkyl, such as methyl. In some embodiments, R ^l4 is H. In other embodiments, R ^!4 is Ci -Ce alkyl, such as methyl.

[0085] In some embodiments, R\ R ³ , R ⁴ , R ⁵ , R ⁶ , R ^8a , R ^8b , R ⁹ , R ¹⁰ , R ^{1 1} , R ⁱ² , R ¹³ and R ¹⁴ are each independently H or Cj -Ce alkyl. In some embodiments, R ¹ , R ⁵ , R ⁶ , R ^8a , and R ¹⁴ are each methyl.

[0086] In some embodiments of Formula (I), R ¹ is methyl, R ² is methyl, R ³ is H, and R ⁴ is isobutyl. In some of these embodiments, X ^! is -0-. In some of these embodiments, R ¹⁵ is substituted or unsubstituted aryl and n is 1. In particular embodiments, R ¹⁵ is substituted or unsubstituted phenyl, and n is 1 . The substituted phenyl may be a phenyl substituted at the para position. The substituted phenyl may be a phenyl substituted at the meta position. The substituted phenyl may be phenyl substituted with one or more fluoro, chloro,

trifluoromethyl, or hydroxyl groups. In particular embodiments, the substituted phenyl is 4- fluorophenyl, 4-chlorophenyl, 4-(trifluoromethyl)phenyl, or 4-hydroxyphenyl. In some embodiments, the substituted phenyl is 4-cyanophenyl. In any of the foregoing embodiments of Formula (I), n may be 0. In any of the foregoing embodiments of Formula (I), n may be 1 . In any of the foregoing embodiments of Formula (I), n may be 2.

[0087] In some embodiments of Formula (I), R ⁵ is H or Ci-Ce alkyl, such as methyl. In other embodiments of Formula (I), R^ is halo. In some such embodiments, R ⁵ is fluoro. In other such embodiments, R ⁵ is chloro. In yet other embodiments of Formula (I), R ⁵ is perhaloalkyi. In some such embodiments, R ³ is trifluoromethyl. [0088] In some embodiments of Formula (I), R ⁶ is H or Ci-Ce alkyl. In some such embodiments, R" is H, In other such embodiments, R ⁶ is methyl. In other embodiments of Formula (I) embodiments, R ⁶ is halo. In some such embodiments, R ⁶ is fluoro. In some such embodiments, R ⁶ is chloro. In yet other embodiments of Formula (I), R ⁶ is substituted or unsubstituted aryl. In particular embodiments, R ^b is substituted phenyl. In other particular embodiments, R ⁶ is unsubstituted phenyl.

[0089] In some embodiments of Formula (I), R ⁷ is H, Ci-Ce alkyl, Ci-Ce alkenyl, or Ci- C alkynyl. In some embodiments of Formula (1), R' is H or Ci-Ce alkyl. In some such embodiments, R' is methyl. In other such embodiments, R is H. In particular embodiments, R ⁷ is

When R ⁷ is sec-butyl, it may be (S)-sec-butyl or (R)-sec-butyl. In some embodiments, R is substituted or unsubstituted aryl, such as phenyl,

[0090] In some embodiments of Formula (I), R ⁵ , R ⁶ , and R ⁷ are all substituted or unsubstituted alkyl. In particular embodiments of Formula (I), R ³ and R ^D are both methyl, and R' is I r particular embodiments of Formula (I), R ³ and R ^D are both methyl, and R' is In other particular embodiments of Formula (I), R ³ is methyl, and R ⁶ and R' are both hydrogen. In yet other particular embodiments of Formula (I), R ⁵ and R ⁶ are both methyl, and R ⁷ is hydrogen.

[0091] In some embodiments of Formula (I), R ^l3 is substituted or unsubstituted aryl, substituted or unsubstituted heteroasyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclyl. In any variation of Formula (I) described herein, R ^l3 may be substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclyl. In some embodiments of Formula (I), R ^1'3 is substituted or unsubstituted aryl, such as phenyl. In some embodiments, R ³ is phenyl optionally substituted with halo, hydroxy, C _\ ~C alkoxy, Ci -Ce alkyl, or Ci-Ce perhaloalkyl. In some embodiments, R ^l5 is phenyl substituted with halo, such as fluoro or chloro. In other embodiments, R ^1'1 is phenyl substituted with hydroxyl. In other embodiments, R ⁱ⁵ is phenyl substituted with a!koxy. In other embodiments, R ¹⁵ is phenyl substituted with cyano. In other embodiments, R ^l5 is phenyl substituted with perhalomethyl. In some embodiments, R ¹³ is phenyl which is substituted at the para position. In some embodiments, R ^l5 is 4-chlorophenyl. In other embodiments, R ^1"1 is 4-cyanophenyl. In yet other embodiments, R° is unsubstituted phenyl. In any of the foregoing embodiments, n may be 1. In any of the foregoing embodiments, n may be 2. In any of the foregoing embodiments, n may be 0.

[0092] In some embodiments, the carbon bearing R ² may be in the (S) stereochemical configuration. In some embodiments, the carbon bearing R ² may be in the (R)

stereochemical configuration. In some embodiments, the carbon bearing R ⁴ may be in the (S) stereochemical configuration. In some embodiments, the carbon bearing R ⁴ may be in the (R) stereochemical configuration. In some embodiments, the carbon bearing R" may be in the (S) stereochemical configuration. In some embodiments, the carbon bearing R ⁶ may be in the (R) stereochemical configuration. In some embodiments, the carbon bearing R may be in the (S) stereochemical configuration. In some embodiments, the carbon bearing R' may be in the (R) stereochemical configuration. In some embodiments, the carbon bearing R ^8a and R ^8b may be in the (S) stereochemical configuration. In some embodiments, the carbon bearing R ^8a and R ⁸ may be in the (R) stereochemical configuration. In some embodiments, the carbon bearing R ¹⁰ may be in the (S) stereochemical configuration. In some

embodiments, the carbon bearing R ⁱ⁰ may be in the (R) stereochemical configuration. In some embodiments, the carbon bearing R ^l2 and R ^lj may be in the (S) stereochemical configuration. In some embodiments, the carbon bearing R ¹ and R" may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in the (S) stereochemical configuration.

[0093] In some variations of any of the embodiments of Formula (I) provided herein, Y ¹ , Y ² , and Y ^J are each independently -N(R ^a )-, -0-, or -S-. In some variations of any of the embodiments of Formula (I) provided herein, Y ¹ is -O- and Y ^" is -0-. In some variations, Y ¹ is -S- and Y ² is -0-. In some variations, Y ¹ is -O- and Y ² is -S-, In some variations, Y ¹ is -S- and Y ² is -S-. In some variations, Y ¹ and Y ² are each -N(R ^d )-, where each R ^d is independently H, or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl. In some variations, Y ¹ is -O- and Y ² is -N(R ^d )~, where each R ^d is independently H, or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl. In some variations, Y ¹ is -S- and Y is -N(R ^d )-, where each R ^d is independently H, or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl. In some variations, Y ¹ is— N(R°)-, where each R ^d is

independently H, or substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl, and Y" is -0-. In any of the foregoing embodiments of Formula (I), the compound may contain one of the following features: (i) Y ³ is -N(R ^d )- and Y ⁴ is - NR ^b R ^c ; (li) Y ³ is -N(R ^d )- and Y ⁴ is -OR ³ ; (ui) Y ³ is -N(R ^d )~ and Y ⁴ is -SR ^a ; (iv) Y ³ is -O- and Y ¹ is -NR ^b R ^c ; (v) Y ³ is ·<)· and Y ⁴ is OR '; (vi) Y ³ is ·()· and Y ⁴ is -SR ^a ; (vh) Y ³ is -S- and Y ⁴ is -NR ^b R ^c ; (viii) Y ³ is -S- and Y ⁴ is -OR ³ ; (ix) Y ³ is -S- and Y ⁴ is -SR ^a ; (x) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ⁴ is -OR ³ ; (xi) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ⁴ is -NR ^b R ^L ; or (xii) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ⁴ is -SR ^a . In any of the foregoing embodiments of Formula (I), the compound may further contain one of the following features: (i) X ¹ is -O- and X ² is O; (ii) X ¹ is -O- and X ² is S; (iii) X ^! is-N(R ^d )- and X ² is O; or (iv) X ¹ is-N(R ^d )~ and X ² is S. In any of the foregoing embodiments of Formula (I), W may be (CHiJm-Z-CCH j)^ where Z is selected from the group consisting of substituted or unsubstituted C3-C ₁₂ cycloalkyl, substituted or unsubstituted C3-C1 ₂ cycloalkenyl, substituted or unsubstituted C5-C ₁₂ aryl, and substituted or unsubstituted C5-C12 heteroaryl. In some embodiments, Z is C3-C12 alkyl. In some embodiments, Z is substituted or unsubstituted aryl. In some embodiments, Z is phenyl. In some embodiments, Z is substituted or unsubstituted heteroaryl. In some embodiments, Z is substituted or unsubstituted cycloalkyl. In some embodiments, Z is cyclohexyl. In some embodiments, Z is substituted or unsubstituted heterocycloalkyl. In other embodiments, Z is (€Ή ₂ €Ή ₂ 0) _¾ . In some embodiments of Formula (I), q is an integer from 1-12, inclusive. In some embodiments, q is an integer from 4-8, inclusive. In other embodiments, q is 1 or 2. In some embodiments of Formula (I), rn and p are each independently an integer from 0-12, inclusive. In some embodiments, m is an integer from 0-8, inclusive. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments of Formula (I), p is an integer from 0-8, inclusive. In some embodiments, p is 2. In some embodiments, p is 5. In some embodiments, p is 7. In some embodiments, m and p are each 0. In any of the foregoing embodiments of Formula (I), W is -C2-C12 alkyi-, wherein -C2- Ci2 aikyl- is optionally substituted. In some embodiments, W is -C2-C12 alkyl-. In some embodiments, W is -(C3¾)2-. In some embodiments, W is -(0¾)4-. In some embodiments, W is -(CH ₂ )5-. In some embodiments, W is -(CH ₂ )6-. In some embodiments, W is -(C b)?-.

[0094] In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (I), the carbon bearing Y ¹ may be in die (S) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (S) stereochemical configuration.

[0095] In some embodiments, Y . and Y ² are each -0-, X ² is O, and Y ³ is -N(R ^d )~,

[0096] In another aspect, the compound of Formula (I) is a compound of Formula (la):

(la)

or a salt thereof, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ^Sa , R ^b , R ⁹ , R ^!0 , R . R '. R ¹³ , R ¹⁴ , R ¹⁵ , n, X ¹ , X ² , W, Y ¹ , Y , Y ³ and Y ⁴ are as defined for Formula (I) and any variation or embodiment thereof, provided thai i) when Y ⁴ is -OH, W is not -(CH ₂ ) ₂ - or -(CI¾CI¾0)3(CH ₂ ) ₂ -; and ii) when V ¹ is -M IR or -N(CH ₃ )R ^C , W is not -(( ! ! _. .) -. -(CH ₂ ) ₆ -, or -( Η ·((Ί 1.( 1. ⁽ )) :(( ! ! .) _: - [0097] In another aspect, the compound of Fonnula (I) is a compound of Formula (lb):

or a salt thereof, wherein

II ^s , R ⁶ , I ⁷ , R ⁸³ , R ^8b , R ⁹ , X ² , W, Y ¹ , Y ² , Y ³ , and Y ⁴ are defined as for Formula (I) or any variation or embodiment thereof;

A is halo, perhalomethyl, cyano, nitro, amino, hydroxy, or alkoxy; and

k is 0, 1 , 2, 3, 4, or 5,

provided that i) when Y ⁴ is -OH, W is not -(CH ₂ ) ₂ - or -(CH ₂ CH ₂ 0) ₃ (CH ₂ )r; and ii) when V ¹ is -M IR or -N(CH ₃ )R ^C , W is not -(( ! !..) -. -(CH ₂ ) ₆ -, or -Π I ·(( ^" ! 1.Ό 1 ₂ ϋ) :(ίΉ ~ [0098] In some embodiments of Fonnula (lb), R ^8a is H and R ⁸ ° is methyl. In some embodiments, R ^8a and R ^5b are both H. In some embodiments, R ^8a is H and R ⁸ ° is ethyl. In some embodiments, R ⁸ and R ^8b are both methyl. In any of these embodiments, R ⁹ may be H. In any of these embodiments, R ⁹ may be methyl.

[0099] In some of the aforementioned embodiments of Formula (lb), k is 1. In some such embodiments, A is halo. In some such embodiments, A is fluoro. In some such

embodiments, A is chloro. In some such embodiments, A is trifiuoromethyl . In some such embodiments, A is hydroxy. In some embodiments, A is cyano. In particular embodiments where k is 1, A is fluoro, chloro, trifiuoromethyl, or hydroxy and is attached at the para position on the phenyl ring. In some embodiments where k is 1, A is cyano and is attached at the para position on the phenyl ring. In some embodiments where k is 1, A is cyano and is attached at the meta position on the phenyl ring. In other particular embodiments where k is 1, A is fluoro, cliloro, trifluoromethvl, or hydroxy and is attached at the meta position on the phenyl ring. In any of the foregoing embodiments of Formula (lb), the compound may contain one of the following features: i) Y ¹ and Y ² are each -0-; ii) Y ¹ is -0-, and Y ² is -S-; iii) Y ¹ is -0-, and Y ² is -N(R ^d )~; (iv) Y ¹ is -0-, and Y ² is -NH-; and (v) Y ¹ is -0-, and Y ² is - N(( I i :)- In any of the foregoing embodiments of Formula (lb), the compound may contain one of the following features: i) Y ¹ and Y ² are each -S-; ii) Y ^! is -S-, and Y ² is -0-; iii) Y ^! is - S-, and Y ² is -N(R ^d )-; (iv) Y ¹ is -S-, and Y ² is -NH-; and (v) Y ^! is -S-, and Y ² is -N(CH ₃ )-. In any of the foregoing embodiments of Formula (lb), the compound may contain one of the following features: i) Y ^! and Y ² are each -N(R ^d )-; ii) Y ¹ is ~N(R ^d )-, and Y ² is ~S~; iii) Y ¹ is - N(R ^d )-, and Y ² is -0-; (iv) Y ¹ is -N(R ^d )-, and Y ² is -NH-; (v) Y ¹ is -N(R ^d )-, and Y ² is - N( ( ! ! : )·. (vi) Y ^! is · Ν( ( ! ! : )·. and Y ² is -N(CH ₃ )-; (vn) Y ¹ is -NH-, and Y ² is -N(CH ₃ )-; (viii) Y ^l is ~N(CH ₃ )~, and Y ² is -NH-; and Y ¹ is -NH-, and Y ² is -NH-. In any of the foregoing embodiments of Formula (lb), the compound may contain one of the following features: (i) Y ³ is -N(R ^d )- and Y ⁴ is -NR ^b R°; (ii) Y ³ is -N(R ^d )- and Y ⁴ is -0R ^a ; (iii) Y ³ is -N(R ^d )- and Y ⁴ is -SR ³ ; (iv) Y ³ is -O- and Y ⁴ is -NR ^b R ^c ; (v) Y ³ is ··()·· and Y ⁴ is -OR ³ : (vi) Y ³ is ·()· and Y ⁴ is -SR ³ ; (vii) Y ³ is -S- and Y ⁴ is -NR ^b R ^c ; (viii) Y ³ is -S- and Y ⁴ is -OR ³ ; (ix) Y ³ is -S- and Y ⁴ is -SR ³ ; (x) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ^* is -OR ³ ; (xi) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ⁴ is -NR ^b R ^c ; or (xii) Y ³ is substituted or unsubstituted heterocycloalkyl and Y* is -SR ³ . In any of the foregoing embodiments of Formula (lb), the compound may further contain one of the following features: (i) X ¹ is -O- and X ² is (); i ii ) X ¹ is ·<)· and X ² is S; (iii) X ¹ is-N(R ^d )- and X ² is (); or (rv) X ^! is-N(R ^d )- and X is S. In any of the foregoing embodiments of Formula (lb), W may be (CH ₂ ) _m -Z-(CH ₂ ) _p, where Z is selected from, the group consisting of substituted or unsubstituted C3-C ₁₂

cycloalkyl, substituted or unsubstituted C ₃ -Ci ₂ cycloalkenyl, substituted or unsubstituted C5- C ₁₂ aryl, and substituted or unsubstituted Cj-Cn heteroaryi. In some embodiments, Z is C ₃ - C ₁₂ aiky l. In some embodiments, Z is substituted or unsubstituted aryl. In some

embodiments, Z is phenyl. In some embodiments, Z is substituted or unsubstituted heteroaryi. In some embodiments, Z is substituted or unsubstituted cycloalkyl. In some embodiments, Z is cyclohexyl. In some embodiments, Z is substituted or unsubstituted heterocycloalkyl. In other embodiments, Z is (CI 1 _. Ό l _. O) . In some embodiments of Formula (lb), q is an integer from 1-12, inclusive. In some embodiments, q is an integer from 4-8, inclusive. In other embodiments, q is 1 or 2. In some embodiments of Formula (lb), m and p are each independently an integer from 0-12, inclusive. In some embodiments, m is an integer from 0- 8, inclusive. In some embodiments, m is 2. In some embodiments, rn is 5. In some embodiments, m is 7. In some embodiments of Formula (lb), p is an integer from 0-8, inclusive. In some embodiments, p is 2. In some embodiments, p is 5. In some embodiments, p is 7. In some embodiments, m and p are each 0. In any of the foregoing embodiments of Formula (lb), W is -C2-C12 alkyl-, wherein -C2-C12 alkyl- is optionally substituted. In some embodiments, W is -C2-C12 alkyl-. In some embodiments, W is -(CH2)2-. In some embodiments, W is (CI !.·) :- In some embodiments, W is ί( ^" ί I ·).<-· In some embodiments, W is -(0-¾)6-. In some embodiments, W is -(CHa)?-. In any of the foregoing embodiments of Formula (lb), Y ¹ may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ¹ may be in the (S) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (S) stereochemical configuration.

[0100] In some embodiments of Formula (lb), k is 0. In some of these embodiments of Formula (lb), the compound may contain one of the following features: i) Y ¹ and Y ² are each -0-; 11) Y ^] is -0-, and Y ² is -S-; in) Y ^] is -0-, and Y ² is -N(R ^d )~: (iv) Y ^] is -0-, and Y ² is - NH-; and (v) Y ¹ is -0-, and Y ² is -N(CH ₃ )~. In some of these embodiments of Formula (lb), the compound may contain one of the following features: i) Y ¹ and Y ² are each -S-; ii) Y ¹ is - S-, and Y ² is -0-; hi) Y ¹ is -S-, and Y ² is -N(R ^d )-; (iv) Y ¹ is -S-, and Y ² is -NH-; and (v) Y ¹ is -S-, and Y ² is ~N(CH ₃ )~. In some of these embodiments of Formula (lb), the compound may contain one of the following features: i) Y ¹ and Y ² are each -N(R ^a )-; ii) Y ¹ is -N(R ^d )-, and Y ² is -S-; lii) Y ¹ is -N(R ^d )-, and Y ² is -0-; (iv) Y ¹ is -N(R ^d )-, and Y ² is -M l -: (v) Y ¹ is -N(R ^d )-, and Y ² is -N(CH ₃ )-; (vi) Y ¹ is -N(CH ₃ )-, and Y ² is -N(CH ₃ )-; (vii) Y ¹ is -M l-, and Y ² is - N(CH ₃ )-; (viii) Y ¹ is -N(CH ₃ )-, and Y ² is -NH-; and Y ¹ is - H-, and Y ² is -NH-. In any of the foregoing embodiments of Formula (lb), the compound may contain one of the follow ing features: (1) Y ³ is -N(R ^d )- and Y ⁴ is -NR ^b R ^c : (ii) Y ³ is -N(R ^d )- and Y ⁴ is -OR ^a ; (iii) Y ³ is - N(R ^d )- and Y ⁴ is -SR ^a ; (iv) Y ³ is -O- and Y ⁴ is -NR R ^c : (v) Y ³ is -O- and Y ⁴ is -OR ³ ; (vi) Y ³ is -O- and Y ⁴ is -SR ^a ; (vii) Y ³ is -S- and Y ⁴ is -NR ^b R ^c ; (viii) Y ³ is -S- and Y ⁴ is -OR ^a ; (ix) Y ³ is -S- and Y ⁴ is -SR ^a ; (x) Y' is substituted or unsubstituted heterocycloalkyl and Y ⁴ is - OR ³ ; (xi) Y ³ is substituted or unsubstituted heterocycloalkyl and Y* is -NR°R ^e ; or (xii) Y ⁵ is substituted or unsubstituted heterocycloalkyl and Y* is -SR ^a . in any of the foregoing embodiments of Formula (lb), the compound may further contain one of the following features: (i) X ¹ is ·()· and X ² is O: (ii) X ¹ is -O- and X ² is S: (iii) X ¹ is-N(R ^d )- and X ² is O; or (iv) X ¹ is-N(R ^d )~ and X ² is S. In any of the foregoing embodiments of Formula (lb), W may be (CH ₂ ) _m ~Z-(CH ₂ )n where Z is selected from the group consisting of substituted or unsubstituted C3-C ₁₂ cycloalkyl, substituted or unsubstituted C3-C ₁₂ cycloalkenyl, substituted or unsubstituted C5-C ₁ 2 aryl, and substituted or unsubstituted C5-C12 heteroaryl. In some embodiments, Z is C3-C12 alkyl. In some embodiments, Z is substituted or unsubstituted aryl. In some embodiments, Z is phenyl. In some embodiments, Z is substituted or unsubstituted heteroaryl. In some embodiments, Z is substituted or unsubstituted cycloalkyl. In some embodiments, Z is cyclohexyl. In some embodiments, Z is substituted or unsubstituted heterocycloalkyl. In other embodiments, Z is (CTfeCFkO),. In some embodiments of Formula (lb), q is an integer from 1 -12, inclusive. In some embodiments, q is an integer from 4-8, inclusive. In other embodiments, q is 1 or 2. In some embodiments of Formula (lb), m and p are each independently an integer from 0-12, inclusive. In some embodiments, m is an integer from 0-8, inclusive. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments of Formula (lb), p is an integer from 0-8, inclusive. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, m and p are each 0. In any of the foregoing embodiments of Formula (lb), W is -C ₂ -C ₁₂ alkyl-, wherein -C ₂ -C ₁₂ alkyl- is optionally substituted. In some embodiments, W is -C2-C12 alkyl-. In some embodiments, W is -(012)2-. In some embodiments, W is (CI I ·) :-. In some embodiments, W is - C k , In some embodiments, W is -(CI-^Js-- In some embodiments, W is -(CH ₂ )6-. In some embodiments, W is -(CH ₂ )7-. In some embodiments, W is --C3-C] -, alkyl-, wherein -C3-C] -, alkyl- is optionally substituted. In some embodiments, W is -C3-C12 alkyl-. In any of the foregoing embodiments of Formula (lb), Y may be in the (R) stereochemical configuration. In any of the foregoing

embodiments of Formula (lb), Y ' may be in the (S) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (S) stereochemical configuration.

[0101] In some embodiments of Formula (lb), R ⁵ is H or Ci-Ce alkyl, such as methyl. In other embodiments of Formula (lb), R ³ is halo. In some such embodiments, R ³ is fluoro. In other such embodiments, R ⁵ is chloro. In yet other embodiments of Formula (lb), R ⁵ is perhaloalkyl. In some such embodiments, R ³ is trifluoromethyl.

[0102] In some embodiments of Formula (lb), R ⁶ is H or Ci -Ce alkyl. In some such embodiments, R ⁶ is H. In other such embodiments, R ^B is methyl . In other embodiments of Formula (lb) embodiments, R ⁶ is halo. In some such embodiments, R ⁶ is fluoro. In some such embodiments, R ^D is chloro. In yet other embodiments of Formula (lb), R ^U is substituted or unsubstituted aryl. In particular embodiments, R ⁶ is substituted phenyl. In other particular embodiments, R" is unsubstituted phenyl.

[0103] In some embodiments of Formula (lb), R' is H or Ci -Ce alkyl. In some such embodiments, R is methyl. In other such embodiments, R' is H. In particular embodiments, R ⁷ is

When R ^' ' is sec-butyl, it may be (S)-sec-but l or (R)-sec-butyl. In some embodiments, R' is substituted or unsubstituted aryl, such as phenyl.

[0104] In some embodiments of Formula (lb), R ³ , R ⁶ , and R' are all substituted or unsubstituted alkyl. In particular embodiments of Formula (lb), ^ and R ⁶ are both methyl, and ' is ¹ r particular embodiments of Formula (lb), R ⁵ and R ⁶ are both methyl, and R' is In other particular embodiments of Formula (lb), R ⁵ is methyl, and R ⁶ and R ^' ' are both hydrogen. In yet other particular embodiments of Formula (lb), R ³ and R ⁶ are both methyl, and R' is hydrogen. [01Θ5] In some variations of Formula (lb), R ^s? " and R ⁸ ° are independently selected from H and methyl, R ⁹ is H, R ⁵ and R" are both methyl. In some variations, Y ¹ is -O- and Y ² is -0-. In any of the foregoing embodiments, the compound may contain one of the following features: (i) Y ³ is -N(R ^d )- and Y ⁴ is -NR ^b R ^c ; (n) Y ³ is -N(R ^d )- and Y ⁴ is -OR ^a ; (iii) Y ³ is - N(R ^d )- and Y ⁴ is -SR ^a ; (iv) Y ³ is -O- and Y ⁴ is -NR ^b R ; (v) Y ³ is -O- and Y ⁴ is ---OR ³ ; (vi) Y ³ is -O- and Y ⁴ is -SR ^a ; (vii) Y ³ is -S- and Y ⁴ is -NR R ^c ; (viii) Y ³ is -S- and Y ⁴ is -OR ^a ; (ix) Y ³ is -S- and Y ⁴ is -SR ^a ; (x) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ⁴ is - OR ^d ; (xi) Y ^J is substituted or unsubstituted heterocycloalkyl and Y ⁴ is -NR ^b R ^c ; or (xii) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ⁴ is -SR ³ . In any of the foregoing embodiments of Formula (I), the compound may further contain one of the following features: (i) X ¹ is -O- and X ² is O; (li) X ¹ is -O- and X ² is S; (iii) X ¹ is-N(R ^d )- and X ² is O; or (iv) X ¹ is-N(R ^d )- and X ² is S. In any of the foregoing embodiments of Formula (lb), W may be (CH ₂ ) _m ~Z-(CH ₂ )p _, where Z is selected from the group consisting of substituted or unsubstituted C3-C ₁₂ cycloalkyl, substituted or unsubstituted C3-C12 cycloalkenyl, substituted or unsubstituted C5-C ₁₂ aryl, and substituted or unsubstituted C5-C ₁₂ heteroaryl. In some embodiments, Z is C3-C ₁₂ aikyl. In some embodiments, Z is substituted or unsubstituted aryl. In some embodiments, Z is phenyl. In some embodiments, Z is substituted or unsubstituted heteroaryl . In some embodiments, Z is substituted or unsubstituted cycloalkyl. In some embodiments, Z is cyclohexyl. In some embodiments, Z is substituted or unsubstituted heterocycloalkyl. In other embodiments, Z is (CH2CH ₂ 0) _q . In some embodiments of Formula (lb), q is an integer from 1 -12, inclusive. In some embodiments, q is an integer from 4-8, inclusive. In other embodiments, q is 1 or 2. In some embodiments of Formula (lb), m and p are each independently an integer from 0-12, inclusive. In some embodiments, m is an integer from 0-8, inclusive. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments of Formula (lb), p is an integer from 0-8, inclusive. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, m and p are each 0. In any of the foregoing embodiments of Formula (lb), W is -C2-C12 alkyl-, wherein -C2-C12 alkyl- is optionally substituted. In some embodiments, W is -C ₂ -C ₁₂ alkyl-. In some embodiments, W is (( ^" I I ·) -. In some embodiments, W is -(CH ₂ )3 ~. In some embodiments, W is -(€112)4- · Tn some embodiments, W is -(CH ₂ )5-- In some embodiments, W is -(Cl-t - In some embodiments, W is -(CHi)?-. In some embodiments, W is -C3-C ₁₂ alkyl-, wherein -C3-C ₁₂ alkyl- is optionally substituted. In some embodiments, W is -C ₃ -Ci ₂ alkyl-. In any of the foregoing embodiments of Formula (lb), Y ^! may be in the (R) stereochemical configuration . In any of the foregoing

embodiments of Formula (lb), Y ¹ may be in the (S) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (S) stereochemical configuration. In any of the foregoing variations, the compound of Formula (lb) is further defined by having k = I . In any of the foregoing variations, the compound of Formula (lb) is further defined by having k = 0.

[0106] In another aspect, the compound of Formula (I) is a compound of Formula (Ic):

(Ic)

or a salt thereof, wherein R ⁵ , R ⁶ , R ⁷ , R ^a , R ^8b , R ⁹ , X ² , W, Y ¹ , Y ² , Y ³ , Y ⁴ , A, and k are defined as for Formula (lb) or any variation or embodiment thereof, provided that i) when Y ⁴ is -OH, W is not -(( ^' ! \; ) ·~ or -(( ^' I I (Ί I■( ⁾ } :(( f ! ·) --: and li) when Y ⁴ is -NHR ^C or -N(CH ₃ )R ^C , W is not ^• (C! ! 5;··. ··({ ! ! .),,·. or - i.-iCI Ι.·ί f I -.0) :( ! I · )

[0107] In some embodiments of Formula (lc), R ^8a is H and R ⁸ is methyl. In some embodiments, R ^' "' and R ^8b are both H. In some embodiments, R ^8a is H and R ^8b is ethyl. In some embodiments, R* ³ and R ^{' '} are both methyl. In any of these embodiments, R ^' may be H. In any of these embodiments, R ⁹ may be methyl.

[0108] In some of the aforementioned embodiments of Formula (Ic), k is 1. In some such embodiments, A is halo. In some such embodiments, A is fluoro. In some such

embodiments, A is chloro. In some such embodiments, A is trifluoromethyl. In some such embodiments, A is hydroxy. In some embodiments, A is cyano. In particular embodiments where k is 1, A is fluoro, chloro, trifluoromethyl, or hydroxy and is attached at the para position on the phenyl ring. In some embodiments where k is 1, A is cyano and is attached at the para position on the phenyl ring. In some embodiments where k is 1, A is cyano and is attached at the meta position on the phenyl ring. In other particular embodiments where rn is 1, A is fluoro, chloro, trifluoromethyl, or hydroxy and is attached at the meta position on the phenyl ring. In any of the foregoing embodiments of Formula (Ic), the compound may contain one of the followmg features: i) Y ¹ and Y ² are each -0-; li) Y ¹ is -0-, and Y ² is -S-; in) Y ¹ is -0-, and Y ² is -N(R ^d )-; (iv) Y ^! is -0-, and Y ² is -NH-; and (v) Y ¹ is -0-, and Y ² is - N(CH ₃ )~. In any of the foregoing embodiments of Formula (Ic), the compound may contain one of the following features: i) Y ¹ and Y ² are each -S-; ii) Y ^l is ~S~, and Y ² is -0-; iii) Y ^l is - S-, and Y ² is -N(R ^d )-; (iv) Y ¹ is -S-, and Y ² is -NH-; and (v) Y ¹ is -S-, and Y ² is ~N(CH ₃ )~. In any of the foregoing embodiments of Formula (Ic), the compound may contain one of the following features: i) Y ¹ and Y ² are each -N(R ^d )-; ii) Y ¹ is -N(R ^d )~, and Y ² is -S-; iii) Y ¹ is - N(R ^d )-, and Y ² is -0-; (iv) Y ¹ is -N(R ^d )-, and Y ² is -NH-; (v) Y ¹ is -N(R ^d )-, and Y ² is - ΝίΠ ! <}-·: (vi) Y ^! is -N(CH ₃ )-, and Y ² is -N(CH ₃ )-; (vn) Y ¹ is -NH-. and Y ² is -N(CH ₃ )-; (vni) Y ^! is -N(CH ₃ )-, and Y ² is -NH-; and Y ¹ is -NH-, and Y ² is -NH-. In any of the foregoing embodiments, the compound may contain one of the following features: (i) Y ³ is -N(R ^d )~ and Y ⁴ is -NR ^b R ^c ; (ii) Y ³ is -N(R ^d )- and Y ⁴ is -OR ³ ; (iii) Y ³ is -N(R ^d )~ and Y ⁴ is -SR ^a ; (iv) Y ³ is - {)· and Y ⁴ is -NR ^b R ^c ; i s ) Y ³ is 0· and Y ⁴ is OR ¹ : (vi) Y ³ is ··()·· and Y ⁴ is -SR ³ ; (vn) Y ³ is - S- and Y ⁴ is -NR ^b R ^c ; (vni) Y ³ is -S - and Y ⁴ is -OR ³ ; (ix) Y ³ is -S- and Y ⁴ is -SR ³ ; (x) Y ³ is substituted or unsubstituted heterocycloalkyl and Y ⁴ is -OR ³ ; (xi) Y ^J is substituted or unsubstituted heterocycloalkyl and Y ^* is -NR ^b R ^c ; or (xii) Y ^J is substituted or unsubstituted heterocy cloalkyl and Y ⁴ is -SR ³ . In any of the foregoing embodiments of Formula (I), the compound may further contain one of the following features: (i) X ¹ is -O- and X ² is O; (ii) X ¹ is -O- and X ² is S; (iii) X ¹ is-N(R ^d )~ and X ² is O; or (iv) X ¹ is-N(R ^d )- and X ² is S. In any of the foregoing embodiments of Formula (Ic), W may be (CH ₂ ) _m -Z-(CH ₂ ) _p . where Z is selected from the group consisting of substituted or unsubstituted C3-C ₁₂ cycloalkyL substituted or unsubstituted C3-C12 cycloalkenyl, substituted or unsubstituted C5-C12 aryl, and substituted or unsubstituted C5-C ₁₂ heteroaryl. In some embodiments, Z is C3-C12 alkyl. In some embodiments, Z is substituted or unsubstituted aryl. In some embodiments, Z is phenyl. In some embodiments, Z is substituted or unsubstituted heteroaryl . In some embodiments, Z is substituted or unsubstituted cycloalkyL In some embodiments, Z is cyclohexyl. In some embodiments, Z is substituted or unsubstituted heterocycloalkyl. In other embodiments, Z is (CFfcCFtC q. In some embodiments of Formula (Ic), q is an integer from 1-12, inclusi ve. In some embodiments, q is an integer from 4-8, inclusive. In other embodiments, q is 1 or 2. In some embodiments of Fonnula (Ic), m and p are each independently an integer from 0-12, inclusive. In some embodiments, m is an integer from 0-8, inclusive. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, rn is 8. In some embodiments of Formula (Ic), p is an integer from 0-8, inclusive. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, m and p are each 0. In any of the foregoing embodiments of Formula (Ic), W is -C2-C12 alkyl-, wherein -C ₂ - C12 alkyl- is optionally substituted. In some embodiments, W is -C2-C12 alkyl-. In some embodiments, W is -(CH ₂ ) ₂ -. In some embodiments, W is -(CH ₂ )3-. In some embodiments, W is -(CH ₂ )4-. In some embodiments, W is -(CH ₂ ).r. In some embodiments, W is -(CHjJe-. In some embodiments, W is -(Ct^) _? -. In some embodiments, W is -C3-C12 alkyl-, wherein - C3-C12 alkyl- is optionally substituted. In some embodiments, W is -C3-C ₁₂ alkyl-. In any of the foregoing embodiments of Formula (Ic), Y ¹ may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (Ic), Y ^! may be in the (S) stereochemical configuration. In any of the foregoing embodiments of Formula (lb), Y ² may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Fonnula (Ic), Y' may be in the (S) stereochemical configuration. [01Θ9] In some embodiments of Formula (Ic), k is 0. In some of these embodiments of Formula (Ic), the compound may contain one of the following features: i) Y ¹ and Y ² are each -0-; ii) Y ¹ is -0-, and Y ² is -S-; iii) Y ¹ is -0-, and Y ² is -N(R ^d )-; (iv) Y ¹ is -0-, and Y ² is - NH-; and (v) Y ¹ is -0-, and Y ² is -N(CH ₃ )-. In some of these embodiments of Formula (Ic), the compound may contain one of the following features: i) Y ^! and Y ² are each -S-; ii) Y ¹ is - S-, and V is -0-; iii) Y ^! is -S-, and V is -N(R ^d )-; (iv) Y ^] is -S-, and Y ² is -NH-; and (v) Y ^! is -S-, and Y ² is -N(CH ₃ )-, In some of these embodiments of Formula (Ic), the compound may contain one of the following features: i) Y ¹ and Y ² are each -N(R ^U )-; ii) Y ¹ is -N(R ^d )-, and Y ² is -S-; in) Y ¹ is -N(R ^d )-, and Y ² is ··()··: (iv) Y ¹ is -N(R ^d )-, and Y ² is - M i -: (v) Y ^! is -N(R ^d )-, and Y ² is -N(CH ₃ )-; (vi) Y ^: is ~N(CH ₃ )~, and Y ² is -N(CH ₃ )~; (vii) Y ¹ is - H-, and Y ² is - \(( 1 1.)-: (viii) Y ¹ is -N(CH ₃ )-, and Y ² is -NH-; and Y ^! is -NH-, and V ' is -M i-. In any of the foregoing embodiments, the compound may contain one of the following features: (i) Y ^J is -N(R ^d )~ and Y ⁴ is -NR R ^c : (ii) Y ³ is ~N(R ^d )~ and Y ⁴ is -GR ^a ; (iii) Y ³ is -N(R ^d )- and Y ⁴ is - SR ^a ; (iv) Y ³ is -O- and Y ⁴ is -NR ^b R ^c ; (v) Y ³ is -O- and Y ⁴ is -OR ³ ; (vi) Y ³ is -O- and Y ⁴ is - Sir: (vii) Y ³ is -S- and Y ⁴ is -NR ^b R ^c ; (viii) Y ³ is -S- and Y ⁴ is -OR ^a ; (ix) Y ³ is ~S- and Y ⁴ is -SR ^a ; (x) Y ³ is substituted or unsubstituted heterocycioalkyl and Y ⁴ is -OR ³ ; (xi) Y ³ is substituted or unsubstituted heterocycioalkyl and Y* is -NR ^b R ^c ; or (xii) Y ^J is substituted or unsubstituted heterocycioalkyl and Y ^÷ is -SR ^a , In any of the foregoing embodiments of Formula (Ic), the compound may further contain one of the following features: (i) X ¹ is -O- and X ² is O; (ii) X ¹ is -O- and X ² is S; (iii) X ¹ is~N(R ^d )~ and X ² is O; or (iv) X ^: is-N(R ^d )- and X is S, In any of the foregoing embodiments of Formula (Ic), W may be (CH ₂ ) _m -Z-(CH2) _p, where Z is selected from the group consisting of substituted or unsubstituted C3-C ₁₂ cycloalkyl, substituted or unsubstituted C3-C ₁₂ cycioaikenyl, substituted or unsubstituted C5- Ci ₂ aryl, and substituted or unsubstituted C5-C ₁₂ heteroaiyl. In some embodiments, Z is C ₃ ~ C 1 ₂ alkyl. In some embodiments, Z is substituted or unsubstituted aryl. In some

embodiments, Z is phenyl. In some embodiments, Z is substituted or unsubstituted heteroaiyl. In some embodiments, Z is substituted or unsubstituted cycloalkyl. In some embodiments, Z is cyclohexyl. In some embodiments, Z is substituted or unsubstituted heterocycioalkyl. In other embodiments, Z is (CFkCFkO^. In some embodiments of Formula (Ic), q is an integer from 1-12, inclusive. In some embodiments, q is an integer from 4-8, inclusive. In other embodiments, q is 1 or 2. In some embodiments of Formula (Ic), m and p are each independently an integer from 0-12, inclusive. In some embodiments, m is an integer from 0- 8, inclusive. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, rn is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments of Formula (Ic), p is an integer from 0-8, inclusive. In some embodiments, p is 0. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, m and p are eacli 0. In any of the foregoing embodiments of Fonnula (Ic), W is -C ₂ -C ₁₂ alkyl-, wherein -C ₂ -C ₁₂ alkyl- is optionally substituted. In some embodiments, W is -C2-C12 alkyl-. In some embodiments, W is -(G¾) ₂ -. In some embodiments, W is -(CH ₂ )3-. In some embodiments, W is --(CH?)/}-. In some embodiments, W is -(CH ₂ )5~. In some embodiments, W is (CI ! _. >)(,-. In some embodiments, W is (( ^' ! ! >)-- In some embodiments, W is -C ₃ -C ₁₂ alkyl-, wherein -C ₃ -C ₁₂ alkyl- is optionally substituted. In some embodiments, W is -C3-C12 alkyl-. In any of the foregoing embodiments of Formula (Ic), Y ¹ may be in the (R) stereochemical configuration. In any of the foregoing

embodiments of Formula (Ic), Y ¹ may be in the (S) stereochemical configuration. In any of the foregoing embodiments of Formula (Ic), Y ² may be in the (R) stereochemical configuration. In any of the foregoing embodiments of Formula (Ic), Y ² may be in the (S) stereochemical configurati o .

[0110] In some embodiments of Formula (Ic), R ^s is H or Cj -Ce alkyl, such as methyl. In other embodiments of Formula (Ic), R ¹ is halo. In some such embodiments, R^ is fluoro. In other such embodiments, R ⁵ is chloro. In yet other embodiments of Formula (Ic), R ^J is perhaloalkyl. In some such embodiments, R ³ is trif!uoromethyl.

[0111] In some embodiments of Formula (Ic), R ⁶ is H or C Cc, alkyl. In some such embodiments, R ⁶ is H. In other such embodiments, R ⁶ is methyl. In other embodiments of Formula (Ic) embodiments, R ⁶ is halo. In some such embodiments, R ⁶ is fluoro. In some such embodiments, R ⁶ is chloro. In yet other embodiments of Formula (Ic), R ⁶ is substituted or unsubstituted aryl. In particular embodiments, R ⁶ is substituted phenyl. In other particular embodiments, R ⁶ is unsubstituted phenyl. [0112] In some embodiments of Formula (Ic), R ' is H or Cj-Ce alkyl. In some such embodiments, R' is methyl. In other such embodiments, R ' is H. In particular embodiments, R ⁷ is

Wlien R' is sec-butyl, it may be (S)-sec-butyl or (R)-sec-butyl. In some embodiments, R ^'' is substituted or unsubstituted ajyl, such as phenyl

[0113] In some embodiments of Formula (Ic), R ^s , R ^u , and R are all substituted or unsubstituted alkyl. In particular embodiments of Formula (Ic), R ¹ and R ⁶ are both methyl, and R ' is I r particular embodiments of Formula (Ic), R ⁵ and R ⁶ are both methyl, and R' is In other particular embodiments of Formula (Ic), R ⁵ is methyl, and R ^D and R are both hydrogen. In yet other particular embodiments of Formula (Ic), R ⁵ and R° are both methyl, and is hydrogen.

[0114] In some variations of Formula (Ic), R ^8a and R ^8b are independently selected from H and methyl, R ⁹ is H, and R ^s and R ^b are both methyl. In some embodiments, R ^8a and R ^8b are both methyl. In any of the foregoing variations, the compound of Formula (Ic) is further defined by having k = 1. In any of the foregoing variations, the compound of Formula (Ic) is further defined by having k = 0.

[0115] In some variations of Formula (I), the compound is of Formula (Id):

(Id)

or a salt thereof, wherein

Y ⁴ is -OR". -NR ^b R ^c , or -SR ^a :

each R ^a , R ^b , R ^c , and R ^d is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

W is (CH ₂ ) _m -Z-(CH ₂ ) _p ;

Z is substituted or unsubstituted alkyl, substituted or unsubstituted substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or (CH ₂ CH ₂ 0) _q ;

m, and p are each independently an integer from 0-12, inclusive; and

q is an integer from 1-12, inclusive;

A is H, halo, perhalomethyi, cyano, nitro, amino, hydroxy, or alkoxy: and k is 0, 1 , 2, 3, 4, or 5;

provided that i) when Y ⁴ is -OH, W is not -(CH ₂ )2- or -(CH ₂ CH ₂ 0)3(CH ₂ )2-; and ii) when Y ⁴ is -NHR° or -N(CH ₃ )R ^C , W is not -(CH ₂ ) ₂ -, -(CH ₂ ) ₆ -, or -CH ₂ (CH ₂ CH ₂ 0) ₃ (CH ₂ ) ₃ -.

[0116] In some embodiments of Formula (Id), R ^d is H, Z is substituted or unsubstituted alkyl, and Y ⁴ is OH. In some embodiments of Formula (Id), R ^d is H, Z is substituted or unsubstituted aryl, and Y ⁴ is OH. In some embodiments of Formula (Id), R ^d is H, Z is substituted or unsubstituted cycloalkyl, and Y ^'* is OH. In any of the foregoing embodiments, A is H, halo, perhalomethyi, cyano, nitro, amino, hydroxy, or alkoxy, and k is an integer from 1 to 3. In some embodiments, A is H, halo, or cyano. In some embodiments, A is connected at the para position of the phenyl ring, and k is 1. In some embodiments, k is 0.

[0117] In some variations of Formula (I), the compound is of Formula (le):

(Ie)

or a salt thereof, wherein

Y ⁴ is -OR ^a , -NR R ^c , or -SR ³ ;

each R ^a , R ^b , R ^c , and R ^d is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynvl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl:

W is (( \ \ ·)=..-/-( ( ! ! · ),,:

Z is substituted or unsubstituted alkyl, substituted or unsubstituted substituted or unsubstituted alkenyl, substituted or unsubstituted alkynvl, substituted or unsubstituted cycloalkyL substituted or unsubstituted cycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or (CH ₂ CH ₂ 0) _q ;

m, and p are each independently an integer from 0-12, inclusive: and

q is an integer from 1-12, inclusive;

A is H, halo, perhalomethyl, cyano, nitro, amino, hydroxy, or alkoxy; and k is 0, 1, 2, 3, 4, or 5;

provided that i) when Y ⁴ is -OH, W is not -(CH ₂ ) ₂ - or -(CH ₂ CH ₂ 0) ₃ (CIi ₂ ) ₂ -; and n) when Y ⁴ is -NHR ^C or -N(CH ₃ )R ^C , W is not ~(CH ₂ } ₂ -, -(( ί I · ),·.-. or -(Ή..(( Ή..( Ί Ι.·0) :((Ί ! .) =- [011.8] In some embodiments of Formula (Ie), R ^d is H, Z is substituted or unsubstituted alkyl, and Y ⁴ is OH. In some embodiments of Formula (Ie), R" is H, Z is substituted or unsubstituted aryl, and Y ⁴ is OH. In some embodiments of Formula (Ie), ¹¹ is H, Z is substituted or unsubstituted cycloalkyl, and Y* is OH. In any of the foregoing embodiments, A is H, halo, perhalomethyl, cyano, nitro, amnio, hydroxy, or aikoxy, and k is an integer from 1 to 3. In some embodiments, A is H, halo, or cyano. In some embodiments, A is connected at the para position of the phenyl ring, and k is 1. In some embodiments, k is 0. In some embodiments, A is 4-chlorophenyl. In some embodiments, A is 4-cyanopheny3. In some embodiments, A is H.

[0119] In some embodiments, provided herein are compounds and salts thereof described in Table 1, and uses thereof.

52

53

55







60

[0120] In some variations, any of the compounds described herein, such as a compound of Formula (I), (la), (lb), (Ic), (Id), or (Ie), or any variation thereof, or a compound of Table 1 , may be deuterated (e.g., a hydrogen atom is replaced by a deuterium atom). In some of these variations, the compound is deuterated at a single site. In other variations, the compound is deuterated at multiple sites. Deuterated compounds can be prepared from deuterated starting materials in a manner similar to the preparation of the corresponding non- deuterated compounds. Hydrogen atoms may also be replaced with deuterium atoms using other methods known in the art.

[0121] Representative examples of compounds detailed herein, including intermediates and final compounds, are depicted in the tables and elsewhere herein. It is understood that in one aspect, any of the compounds may be used in the methods detailed herein, including, where applicable, intermediate compounds that may be isolated and administered to an individual.

[0122] The compounds depicted herein may be present as salts even if salts are not depicted, and it is understood that the compositions and methods pro vided herein embrace all salts and solvates of the compounds depicted here, as well as the non-salt and non-solvate form of the compound, as is well understood by the skilled artisan. In some embodiments, the salts of the compounds provided herein are pharmaceutically acceptable salts.

[0123] In one variation, the compounds herein are synthetic compounds prepared for administration to an individual. In another variation, compositions are provided containing a compound in substantially pure form. In another variation, provided are pharmaceutical compositions comprising a compound detailed herein and a pharmaceutically acceptable carrier. In another variation, methods of administering a compound are provided. The purified forms, pharmaceutical compositions and methods of administering the compounds are suitable for any compound or form thereof detailed herein.

[0124] Any variation or embodiment of R ^! , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ^8a , R* R ⁹ , R ⁱ⁰ , R ^u , R'" ^' , R ^lj , R", K ^li , n, X X", W, Y Y", Y ³ and Y ^" provided herein can be combined with every other variation or embodiment of R ¹ , R ² , R ³ , R ¹ . R ^" . R ^' \ R ⁷ , R ^Sa , R ^8b , IV. R ^]0 , R . \i R ¹³ , R , R ¹⁵ , n, X ¹ , X ² , W, Y ¹ , Y ² , Y ³ and Y\ as if each combination had been individually and specifically described.

Conjugates

[0125] The compounds described herein may be conjugated to one or more linkers and or ligands. A ligand may be directly bound to a compound of Formula (I), (la), (lb), (Ic), (Id), or (le) or any variation thereof, including any compound listed in Table 1. Alternatively, a ligand may be bound to a compound of Formula (I), (la), (lb), (Ic), (Id), or (Ie) or any variation thereof, including any compound listed in Table 1, via one or more linkers. In some variations, the ligand is a polypeptide. In some variations, the ligand is a nucleic acid. In some variations, the ligand is a targeting moiety. In some variations, the ligand is an antibody. In some variations, the antibody binds to a receptor. In some variations, the antibody binds to a receptor on the surface of a cell. [0126] Accordingly, in one aspect, provided herein are conjugates of the compounds described herein. In some variations, provided is a conjugate containing a compound of Formula (I), (la), (lb), (Ic), (Id), or (le), or any variation thereof, or any compound listed in Table 1, bonded to a ligand. In some variations, the compound of Formula (I), (la), (lb), (Ic), (Id), or (le), or any variation thereof, or any compound listed in Table 1, is directly bonded to the ligand (i.e., no linker is present). In other variations, the compound of Formula (I), (la), (lb), (Ic), (Id), or (le), or any variation thereof, or any compound listed in Table 1 , is bonded to the ligand via a linker. Any suitable ligand and/or linker can be used in the foregoing compositions, including, but not limited to, any of the linkers or ligands described herein.

[0127] In any of the conjugates provided herein, the ratio of compound to ligand may be 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, or 12: 1. In any of the conjugates provided herein, the ratio of compound to ligand may be 1 : 1 or 1 :2. In any of the conjugates provided herein, the ratio of linker to ligand may be 1 : 1 , 2: 1, 3: 1, 4: 1, 5: 1 , 6: 1 , 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, or 12: 1.

[0128] Also provided herein are conjugates containing a compound described herein bonded to a linker. The ratio of compound to linker may be 1 : 1 , 2: 1, 3: 1, 1 :2, or 1 :3. In some embodiments, the linker is additionally bonded to a ligand. In other embodiments, the linker is not bonded to a ligand. In some such embodiments, the linker contains one or more functional groups suitable for bonding to a ligand.

[0129] In some variations, provided is a conjugate containing a compound of Formula (I), (la), (lb), (Ic), (Id), or (le), or any variation thereof, or any compound listed in Table 1, bonded to a linker. Any suitable linker can be used in the foregoing compositions, including, but not limited to, any of the linkers described herein. The linker may be suitable for attachment to a ligand.

[0130] One or more linkers and/or ligands may be bonded to a compound described herein via a functional group including, but not limited to -NHR, -NHNH ₂ , -ONH ₂ , -N ₃ , - OH, -SH, or -CO ₂ H located at any chemically feasible position on the compound. In some embodiments, the conjugation site on the compound is an -OH or -NH ₂ group. In particular embodiments, the conjugation site on the compound is an -OH or -NH ₂ group located at the position corresponding to Y ⁴ . In other embodiments, the conjugation site on the compound is a phenyl ring substituted with -NHR, -NH ₂ , or OH. In particular embodiments, the functional group for conjugation is at the para position on the phenyl ring. In yet other embodiments, an amino acid unit of the compound has been replaced with a lysine unit as a site for conjugation.

[0131] Provided herein are compounds of Formula (I), (la), (lb), (Ic), (Id), or (le), or any variation thereof, or any compound listed in Table 1 , or a salt thereof, wherein the compound is substituted at any chemically feasible position with a moiety suitable for attachment to a linker and/or ligand. In some variations, provided are compounds of Formula (I), (la), (lb), (Ic), (Id), or (le), or any variation thereof, or any compound listed in Table 1, or a salt thereof, wherein the compound is substituted at any chemically feasible position with a functional group which is -NHR, -NHNH ₂ , -QNH ₂ , -N ₃ , -OH, -SH, or -C0 ₂ H. In any of the foregoing embodiments, the functional group suitable for attachment to a Sinker and/or ligand is bonded to a protecting group, which may be removed prior to reaction with a linker and/or ligand.

[0132] In some aspects, provided are conjugates of Formula (II):

and salts thereof, wherein R ^! , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ^a , R ⁸ , R ⁹ , R ¹⁰ , R ^{1 1} , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , n, W, X ¹ , X ² , Y . Y ^' . Y ^" . and Y ' are as defined for Formula (I) or any variation thereof; r is 0, 1, or 2;

s is 0, 1, or 2; and

t is an integer from 1-12, inclusive.

[0133] In other aspects, provided are conjugates of Formula (Ila):

and salts thereof, wherein R ^! , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ^8a , R ^8b , R ⁹ , R ⁱ⁰ , R ^u , R ^{1 2} , R ¹ , R ¹⁴ , R ¹⁵ , n, W, X ¹ , X ² , Y ¹ , Y ² , Y ³ , and Y ⁴ are as defined for Formula (Ta) or any variation thereof; r is 0, 1, or 2;

s is 0, 1, or 2: and

t is an integer from 1-12, inclusive.

|0134] In other aspects, provided are conjugates of Formula (lib):

(lib)

and salts thereof wherein R ⁵ , R ⁶ , R ⁷ , R ^Sa , R ^8b , R ⁹ , W, X ² , Y ¹ , Y ² , Y ³ , Y ⁴ , A and k are as defined for Formula (lb) or any variation thereof;

r is 0, 1, or 2;

s is 0, 1, or 2: and

t is an integer from 1-12, inclusive. In other as ects, provided are conjugates of Formula (lie)

Linker, H— Ligand _s

(lie)

and salts thereof, wherein R ⁵ , R ⁶ , R ⁷ , R ^Sa , R ^8b , R ⁹ , W, X ² , Y\ Y ² , Y ³ , Y ⁴ , A and k are as defined for Formula (Ic) or any variation thereof;

r is 0, 1, or 2;

s is 0, 1, or 2: and

t is an integer from 1-12, inclusive.

[0136] In other aspects, provided are conjugates of Formula (lid):

Ligandg

(nd)

and salts tliereof, wherein R ^d , W, Y ⁴ , A and k are as defined for Formula (Id) or any variation thereof:

r is 0, 1. or 2; s is 0, 1, or 2; and

t is an integer from 1-12, inclusive.

[0137] In other aspects, provided are conjugates of Formula (lie):

and salts thereof, wherein R ^a , W, Y ⁴ , A and k are as defined for Formula (Id) or any variation thereof;

q is 0, 1 , or 2;

p is 0, 1 , or 2; and

t is an integer from 1-12, inclusive.

[0138] In some embodiments of any of Formulae (II), (Ila), (lib), (lie), (lid), and (He), one or both of the following conditions apply: i) when Y ⁴ is -OH, W is not ~(CH ₂ ) ₂ - or - <α ΐ.(Ί Ι .( ⁾ > :((Ή .) -: and si) when Y ⁴ is -NHR ^C or -N(CH ₃ )R ^C , W is not -(CH ₂ ) ₂ -, -(CH ₂ ) ₆ -, or -CH ₂ (CH ₂ CH ₂ 0) ₃ (CH ₂ ) ₃ -.

[0139] In any variations of conjugates having Formulae (II), (Ila), (lib), (lie), (lid), and (He), chemically appropriate valences and chemical bonding are present in the compound at the site of conjugation to the linker and/or ligand. For example, an atom on the compound is replaced by the linker or ligand such that a chemically appropriate number of bonds is maintained for all atoms. In particular examples, an -OH moiety on the compound is replaced with an -O-linker-ligand moiety in the conjugate, or an -NH ₂ - moiety on the compound is replaced with an -NH-liiiker-ligand moiety in the conjugate. [0140] In some variations of any of the conjugates of Formulae (II), (Ila), (lib), (He), (lid), and (He), one or more linkers and/or ligands may be bonded to a compound described herein via a functional group including, but not limited to -OH, -NHR, -NH H ₂ , -ONH ₂ , - N ₃ , , -SH, or -CO ₂ H located at any chemically feasible position on the compound. In some embodiments, the conjugation site on the compound is an -OH or -NH ₂ group. In particular embodiments, the conjugation site on the compound is an ~OH or -NH ₂ group located at the position corresponding to -Y ⁴ . In other embodiments, the conjugation site on the compound is a phenyl ring substituted with -NHR, -NH ₂ , or OH. In particular embodiments, the substitution is at the para position on the phenyl ring. In yet other embodiments, an ammo acid unit of the compound has been replaced with a lysine unit as a site for conjugation.

[0141] In some variations of any of the conjugates of Formula (II), (Ila), (lib), (lie), (lid), or (lie), one or more linkers and/or ligands may be bonded to a compound described herein via a hydroxyl group (-OH). In some variations, the linkers can be carbamate linkers, silyl linkers, pyrophosphate linkers, /?-aminobenzyl (PAB) linkers, denditric-type linkers or any combinations of such linkers tliereof. In some embodiments, the imkers and/or ligands may be conjugated to a compound described herein via a primary or secondary hydroxyl group. In particular embodiments, the conjugation site on the compound is a primary or secondary hydroxy! group located at the position corresponding to -Y ⁴ . In some embodiments, the conjugation site on the compound is a primary or secondary hydroxy! group located at the position corresponding to R . In some embodiments, the conjugation site on the compound is a primary or secondary hydroxyl group located at the position corresponding to R ^8a or R ⁸ °. In some embodiments, the conjugation site on the compound is a primary or secondary hydroxyl group located at the position corresponding to R ⁱ⁵ . In other embodiments, the conjugation site on the compound is a primary or secondary hydroxyl group located at the position corresponding to R ^* . In some embodiments, the conjugation site on the compound is a phenyl ring substituted with -OH. In particular embodiments, the substitution is at the para position on the phenyl ring.

Ligands [0142] The term "ligand" as used herein refers to any molecule or moiety connected to the compounds described herein. "Ligand" may refer to one or more ligands attached to a compound. Likewise, multiple compounds may be attached to a single ligand.

[0143] Ligands may serve a number of purposes including facilitating uptake of the conjugate into a target cell or tissue and directing the conjugate to a particular cell or tissue (also referred to as conjugate targeting). A ligand may also serve to enhance the efficacy of the compounds by, for example, inhibiting or interacting with cellular factors that may inhibit or reduce the efficacy of the compounds described herein. The compound as described herein may be conjugated to one or more ligands. The term "ligand" as used herein refers to one or more ligands.

[0144] In some embodiments, a ligand is an antibody, or fragment thereof. In other embodiments, the ligand is a peptide or protein. In yet other embodiments, the ligand is another moiety useful for directing the compounds described herein to a target cell or tissue.

[0145] Antibody ligands include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, multivalent antibodies, humanized antibodies, and antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragment(s). In one embodiment the term "antibody" encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that in combination form a specific binding site for the target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgGl, IgG2, IgG3, or IgG4 antibody.

[0146] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies. A monoclonal antibody composition typically displays a single binding specificity and affinity for a particular epitope. In contrast, polyclonal antibody compositions typically include a multitude of antibodies that may be directed against different epitopes of the same target molecule. A polyclonal antibody composition may contain a plurality of monoclonal antibodies with different epitope specificities, affinities, or avidities within a single antigen that contains multiple antigenic epitopes. The term "monoclonal" indicates the character of the antibody as being obtained from, a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with any of the compositions, uses, or methods described herein may be made by the hybridoma metliod first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant D A methods (see, e.g., U.S. Pat. No. 4,816,567), The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991 ) Nature 352: 624-628 and Marks et ai. (1991) J. Mol. Biol. 222: 581-597, for example.

[0147] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. These antibodies retain the activity of a non-human antibody while being less immunogenic in humans. Humanized antibodies are chimeric antibodies which contain at least part of its sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all the non-human CDR regions, while the remaining parts of the antibody may be replaced by the corresponding human counterparts. In some embodiments, the humanized antibody retains at least one complete non-human variable domain. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), Typically that of a human immunoglobulin. See e.g., Cabilly, U.S. Pat. No. 4,816,567; Queen et al, (1989) Proc, Nat'l Acad, Sci . USA 86: 10029-10033; and Antibody Engineering: A Practical Approach (Oxford University Press 1996).

[0148] The term, "recombinant antibody", as used herein, includes all human and non- human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences.

[0149] Exemplary peptide or protein ligands include receptor ligands for targeting delivery of the conjugate to a particular cell. Receptor ligands may engage with their target receptor and provide specific targeting to a tissue or cell type that expresses that receptor. This receptor engagement may also facilitate cellular uptake of the conjugate. Exemplary peptides may also include targeting peptides to facilitate passage across the cell membrane or intracellular targeting including, but not limited to, targeting to organelles such as the nucleus, Golgi apparatus, lysosome, and mitochondria.

[0150] In some embodiments, the ligand is a antibody that is specific for a cancer cell antigen . A '"cancer cell antigen" is a peptide or molecular moiety expressed by a cancer cell that is recognized by an antibody. Antibodies specific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.

[0151] In additional embodiments, the peptide ligand is an interferon, a lymphokine, or a hormone.

[0152] Additional exemplary ligands include nucleic acids (e.g. DNA, RNA, PNA, etc.) or other molecules known in the art that would be useful for conjugation to the presently described compounds.

Linkers

[0153] In one embodiment, the compounds described herein comprise one or more linker or linking group. The term "linker", "linker molecule", "linking group", or "linker moiety" as used herein refers to a chemical moiety comprising a covalent bond and/or a chain of atoms that covalently attaches a ligand to a drug moiety or other molecule. For example, exemplar}' linkers, including their structure and synthesis, are described in WO 2004/010957, U.S. Pat. Publ. No. 2006/0074008, U.S. Pat. Publ. No. 2005/0238649, and U.S. Pat. Publ. No.

2006/0024317, U.S. Pat. Publ. Nos. 2003/0083263, 2005/0238649 and 2005/0009751 , WO 2015/095755, W.C. Widdison et a! ,, Bioconjugate Chem., 2015, 26, 2261 ; D. Shabat et al„ New J. Chem., 2012, 36, 386; F.M.H. de Groot et al, J. Org. Chem. , 2001, 66, 8815; J.M. DeSimone et aL Mai Chem. Comm., 2014, 5, 1355; and R.M. Garbaccio et al., J. Amer. Chem. Soc, 2016, 138(4), 1430, each of which is incorporated herein by reference in its entirety and for all purposes.

[0154] The linker may be cleavable or non-cleavable. Cleavable linkers are typically cleavable under intracellular conditions, such that the linker itself is degraded and releases the compound from the gand. Non-cleavable linkers do not degrade intracellularly and in this embodiment, the compound is released from the ligand via degradation of the ligand,

[0155] A conjugate as described herein may or may not comprise a linker molecule or moiety. A person of skill in the art could determine the appropriate type of linker based on the type of treatment or tissue being targeted by the conjugate. Exemplar} ⁷ linking moieties include hydrazones, peptides, chelating agents, maleimides, disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as known to one of skill in the art. Linker moieties may comprise a divalent radical such as an alkyldiyl, an arylene, a heteroarylene, moieties such as: --(CR.2)nO(CR2)n--, repeating units of alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkyiamino (e.g. polyethyleneamino, Jeffamine ^lM ); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.

[0156] Cleavable linkers may be cleaved or degraded by a cleaving agent (e.g. a protease or reducing agent) present in the intracellular environment. In some embodiments, the linker is a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long. Protein and peptide cleaving agents can include cathepsins B and D and plasmin, all of which are known to cleave dspeptides resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123). For example, a peptidyl linker that is cleavable by the thiol - dependent protease cathepsm-B, which is highly expressed in cancerous tissue, can be used (e.g., a Phe-Leu containing linker). Other examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val- Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat, No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker).

[0157] A cleavable linker may be fully cleaved by a cleaving agent to release the compound to which it is bound. Alternatively, a cleavable linker may be partially cleaved, such that a portion of the linker remains bound to the compound. For example, if a linker is bound to a compound via a group -C(0)N(CH ₃ )CH ₂ CH ₂ N(CH ₃ )-, the linker may be partially cleaved such that group -C(Q)N(CH3)CH2CH2NHC3¾ remains bound to the compound. [0158] In other embodiments, the clea able linker is pH-sensitive. Typically, the pH- sensitive linker is unstable and degrades under acidic conditions. For example, an acid-labile Hnker that is cleavable in the lysosome (e.g., a hydrazone, semicarbazone,

thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U .S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123; Neville et al. (1989) Biol. Chem. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome.

[0159] ^' Typically, the linker is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment," in the context of a linker, means that no more than about 20%, 15%, 10%, 5%, or 1% of the linkers, in a sample of compound-ligand conjugate compound, are cleaved when the antibody-drug conjugate compound is in an extracellular environment. Whether a linker is not substantially sensitive to the extracellular environment can be determined using methods known to those of skill in the relevant art.

[0160] In some embodiments, the linker contains a spacer unit. In some embodiments, the spacer unit contains a para-aminobenzoate (PAB) moiety. In some embodiments, the linker contains a stretcher unit. In some embodiments, the stretcher unit contains a maleimide moiety.

[0161] In some embodiments, the linking groups will be bifunctional, meaning they comprise two reactive sites, wherein a first reactive site may be bound to the compounds described herein and the second reactive site may be bound to the ligand. The linker may be hetero-bifunctionai, indicating that the binding moieties on either end of the linker moiety are different.

[0162] In some embodiments, conjugates of the ligand and compound are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), imiiiothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as giutaraidehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazoniiim derivatives (such as bis-(p-diazoniumbenzoyI)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro- 2,4-dinitrobenzene) . For example, a ricin immunotoxin can be prepared as described in Vitetta et al (1987) Science, 238: 1098. Carbon-14-labeled l -isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplaiy chelating agent for conjugation of radionucieotide to the compound or gand (WO94/11026).

[0163] The compounds described herein can be linked to ligands via an acid labile linker as previously described (Blattler et al, 24 Biochemistry, 1517-1524 (1985), U.S. Pat. Nos. 4,542,225, 4,569,789 and 4,764,368).

[0164] The compounds described herein can be linked to ligands via a photolabile linker as previously described (Senter et al, 42 Photochemistry and Photobiology, 231-237 (1985), U.S. Pat. No, 4,625,014).

[0165] The compounds described herein can be linked to a ligand to produce a conjugate that can be cleaved by intracellular esterases to liberate free drug. (For examples see: U.S. Pat. No, 5,208,020 and Aboud-Pirak et al, 38 Biochem. Pharmacol, 641-648 (1989), Laguzza et al, 32 J. Med. Chem., 549-555 (1989)).

[0166] The compounds described herein can also be linked to ligands via peptide spacer linkers. It has been previously shown that short peptide spacers between drags and macromolecular carriers are stable in serum but are readily hydrolyzed by intracellular lysosomal peptidases (Trouet et al, 79 Proc. Nat'l. Acad. Sci. 626-629 (1982)).

[0167] Functional groups on the compound that can serve as a handle for attachment of a linker or ligand using the coupling methods described herein include, without limitation, - OH, -NHR, -NHNH ₂ , -ONH ₂ , -N ₃ , -SH, -C0 ₂ H, and other functional groups. In any of the variations of Formulae (I), (la), (lb), (Ic), (Id), and (le) described herein, the compound maybe modified to contain one or more functional groups that can serve as a handle for attachment of a linker or ligand at any chemically feasible position. In some embodiments, any of the variations of Formulae (I), (la), (lb), (Ic), (Id), and (le) may contain one or more moieties selected from the group consisting of -OH, -NHR, -NHNH ₂ , -ONH ₂ , ~N ₃ , -SH, - CO jH at any chemically feasible position. In some embodiments, a handle for attachment of a linker or ligand can be located on an amino acid side chain. In other embodiments, a handle for attachment of a linker or ligand can be located at position Y ⁴ . In other embodiments, a handle for attachment of a linker or ligand can be located at positions R ² , R ^8a , R ^8b , R' ³ , or R\

[0168] In some variations the linker is of formula (L-Fn), where the linker is bonded to a compound provided herein via the bond shown as ^ :

(L-Fn)

wherein

a, b, c, and d are independently 0, 1, or 2;

, wherein al is 0, 1 , or 2, and each R ^x is unsubstituted substituted alkyl each Lj is b l is an integer from 0 to 12, inclusive;

each L ₃ is independently wherein each AA is an amino acid cl is an integer from 0-12, e;

from 0-12, inclusive, and d2 is an integer from 0-30, inclusive; and

Fn is selected from the group consisting of

bodiments of Formula (L-Fn), Li , wherein al is 0, 1, or 2. In some embodiments of Formula (L-Fn), L ₃

is independently or _ wherein each AA is an amino acid and cl is an integer from 0- d1

12, inclusive. In some embodiments of Formula (L-Fn), L ₄ is independently O , or . wherein d 1 is an integer from

0-12, inclusive. In some embodiments of Formula (L-Fn), Fn is selected from the group

consisting of H.

and e3 are each independently an integer from 0-12, inclusive, and R ^e is H or aikyi.

[0170] In some embodiments of Formula (L-Fn), al is 0. In some embodiments, al is 1. In some embodiments of Formula (L-Fn) bl is 1 to 8. In some embodiments, bl is 1. In some embodiments, bl is 2. In some embodiments, bl is 3. In some embodiments, bl is 4, In some ,(AA) _c1 - -

embodiments, b 1 is 5. In some embodiments, L ₃ is O In

(AA) _c1 - some embodiments, a and b are both 0 and L ₃ is O In some embodiments, b is 0 and L3 is In some embodiments, Li is

, wherein c2 is 3. In some embodiments, AA may be a natural amino acid. In some embodiments, AA may be an unnatural amino acid. In some embodiments (AA) _cl may comprise natural amino acids, unnatural amino acids, or both natural and unnatural amino acids. In some embodiments (AA)ci is -Cit-Val-. In some embodiments, (AA) _{c i} is -Ala-Val-. In some embodiments, cl is 0-8. In some embodiments, ci is 0. In some embodiments, cl is 1. In some embodiments, cl is embodiments, cl is 3. In some embodiments, cl is 4. In some embodiments, L ₄ is . In some embodiments, L ₄ is In some embodiments, L ₄ is

. In other embodiments,

is In some embodiments, dl is 1-8. In some embodiments, dl is 1. In some embodiments, dl is 2. In some embodiments, dl is 3. In some embodiments, dl is 4. In some embodiments, dl is 5 , In some embodiment, d2 is 1-30, In some embodiments, d2 is 12-28. In some embodiments, d2 is 24, In some embodiments, Fn is unsubstitiited or substituted alkyl. In some embodiments, Fn is alkvl substituted with halo. In ents, Fn is ' . In some ents, Fn is ^¾ or

[ _n some embodiments. Fn is

In other embodiments, Fn is

In some embodiments of Formula (La-Fn), al is 0. In some embodiments, al is 1 . In some embodiments of Formula La, b l is 1 to 8. In some embodiments, b l is 1. In some embodiments, b l is 2. In some embodiments, b l is 3. In some embodiments, b l is 4. In some

embodiments, b 1 is 5. In some embodiments, L ₃

some embodiments, L ₃ is

wherein c2 is 0-10. In some embodiments, c2 is 3. In some embodiments, AA may be a natural amino acid. In some embodiments, AA may be an unnatural amino acid. In some embodiments (AA) _ci may comprise natural amino acids, unnatural amino acids, or botli natural and unnatural amino acids. In some embodiments (AA) _ci is -Cit-Val-. In some embodiments, (AA) _C ; is -Ala-Val-. In some embodiments, cl is 0-8. In some embodiments, cl is 0. In some embodiments, el is 1. In some embodiments, cl is 2, In some embodiments, cl is 3. In some em ome

embodiments, L ₄ is , In some embodiments, L ₄ is . In some embodiments, L ₄ is . In other embodiments, L4 is

. In some embodiments, dl is 1-8. In some embodiments, dl is 1. In some embodiments, dl is 2. In some embodiments, dl is 3. In some embodiments, dl is 4. In some embodiments, dl is 5. In some embodiment, d2 is 1 -30, In some embodiments, d2 is 12-28. In some embodiments, d2 is 24. In some embodiments of Formula (La-Fn), Fn is unsubstitiited or substituted alkyl. in some embodiments, Fn is alkyl substituted with halo. In some embodiments, Fn is trichloroethyl (Tee). In some

I— ₃

embodiments of Formula La-Fn Fn is ^{¾ "} . In some embodiments Fn is embodiments, Fn is * ^■ or . In some

embodiments, Fn is , in some embodiments. Fn is In some

embodiments. Fn is other

embodiments, Fn is or

In some variations, the linker is of Formula (Lb-Fn):

[0174] In some embodiments of Formula (Lb-Fn), al is 0. In some embodiments, al is 1. In some embodiments of Formula (Lb-Fn), b l is 1 to 8, In some embodiments, b l is 1 . In some embodiments, b l is 2. In some embodiments, b l is 3 , In some embodiments, b l is 4. In

some embodiments, b l is 5. In some embodiments, In some embodiments, L ₃ is

wherein c2 is 0-10. In some embodiments, c2 is 3. In some embodiments, AA may be a natural amino acid. In some embodiments, AA may be an unnatural amino acid. In some embodiments (AA) _ci may comprise natural ammo acids, unnatural amino acids, or both natural and unnatural amino acids. In some embodiments (AA) _ci is -Cit-Val-. In some embodiments, (AA) _c! is -Ala-Val-. In some embodiments, cl is 0-8. In some embodiments, cl is 0. In some embodiments, cl is 1. In some embodiments, cl is 2. In some embodiments, d1

cl is 3. In some embodiments, cl is 4. In some embodiments, L ₄ is O some

O \ ^H

d

embodiments, L ₄ is O In some embodiments. L ₄ is O In some

In other embodiments, L ₄ is

. In some embodiments, dl is 1-8. In some embodiments, dl is 1. In some embodiments, dl is 2. In some embodiments, dl is 3. In some embodiments, dl is 4. In some embodiments, dl is 5. In some embodiment, d2 is 1-30. In some embodiments, d2 is 12-28. In some embodiments, d2 is 24. In some embodiments of Formula (Lb-Fn), Fn is unsubstituted or substituted alkyl. In some embodiments, Fn is alkvl substituted with halo. In some embodiments, Fn is trichloroethyi (Tee). In some

1— ¾

embodiments of Formula (Lb-Fn), Fn is ^¾ " . In some embodiments, Fn is

[0175] In some variations, the linker is of Formula (Lc-Fn): In some embodiments of Formula (Lc-Fn), al is 0. In some embodiments, al is 1. In some embodiments of Fonnula (Lc-Fn), b l is 1 to 8. In some embodiments, b l is 1. In some embodiments, b l is 2. In some embodiments, b l is 3 , In some embodiments, b i is 4. In

some embodiments, b 1 is 5. In some embodiments, L3

In some embodiments, L3 is

wherein c2 is 0-10. In some embodiments, c2 is 3. In some embodiments, AA may be a natural ammo acid. In some embodiments, AA may be an unnatural amino acid. In some embodiments (AA) _c i may comprise natural amino acids, unnatural amino acids, or both natural and unnatural amino acids. In some embodiments (AA) _c i is -Cit-Val-. In some embodiments, (AA) _C ; is -Ala-Val-. In some embodiments, cl is 0-8. In some embodiments, c l is 0. In some embodiments, c l is 1. In some embodiments, cl is 2. In some embodiments, c l is 3. In some embodiments, c l is 4. In some embodiments, L ₄ is . In some

embodiments. L4 is In some , In other embodiments, L4 is

In some embodiments, dl is 1-8. In some embodiments, dl is 1. In some embodiments, dl is 2. In some embodiments, dl is 3. In some embodiments, dl is 4. In some embodiments, dl is 5 , In some embodiment, d2 is 1-30, In some embodiments, d2 is 12-28. In some embodiments, d2 is 24, In some embodiments of Fonnula (Lc-Fn), Fn is unsubstituted or substituted alkyl. In some embodiments, Fn is alkyl substituted with halo. In some embodiments, Fn is trichloroethyi (Tee). In some embodiments of Fonnula (Lc-Fn), Fn is * ^J . In some embodiments, Fn is

[0177] In some variations, the linker is of Formula (Ld-Fn):

In some embodiments of Formula (Ld-Fn), al is 0. In some embodiments, al is 1. In some embodiments of Formula (Ld-Fn), bl is 1 to 8. In some embodiments, b l is 1. In some

ome

wherein c2 is 0-10. In some embodiments, c2 is 3. In some embodiments, AA may be a natural amino acid. In some embodiments, AA may be an unnatural amino acid. In some embodiments (AA) _c! may comprise natural amino acids, unnatural ammo acids, or both natural and unnatural amino acids. In some embodiments (AA) _cl is -Cit-Val-. In some embodiments, (AA) _C ; is -Ala-Val-. In some embodiments, cl is 0-8. In some embodiments, c l is 0. In some embodiments, e l is 1. In some embodiments, c l is 2, In some embodiments, c l is 3. In some em some

embodiments, L ₄ is , In some embodiments, L ₄ is . In some embodiments, L ₄ is . In other embodiments, L4 is

. In some embodiments, dl is 1-8. In some embodiments, dl is 1. In some embodiments, d l is 2. In some embodiments, dl is 3. In some embodiments, dl is 4. In some embodiments, dl is 5. In some embodiment, d2 is 1 -30, In some embodiments, d2 is 12-28. In some embodiments, d2 is 24. In some embodiments of Formula (Ld-Fn), Fn is unsubstituted or substituted alkyl. In some embodiments, Fn is alkyl substituted with halo. In some embodiments, Fn is trichloroethyl (Tee). In some

— ₃

n is *· . In some embodiments, Fn is mbodiments, Fn is * ^■ or . In some

e mbodiments, Fn is , in some embodiments. Fn is In some

91 some embodiments, the linker is

[0179] Exemplary conjugates having linkers of formula (L-Fn) include the following conjugates of Formula (III):

(III)

and salts thereof,

whe rein R,R _{- 3} 1¾ _; R,I¾. _; L-, ^" R _{- ;} . R R . R R . R R _? n ₅ W , X ₃

X ² , Y ^! , Y ² , andY ³ are as defined for Formula (I) or any variation thereof;

Y ^4a -0-, -NR ^b -, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl; and

Li, L ₂ , i, L4, a, b, c, d, and Fn are as defined for Formula (L-Fn) or any variation thereof,

[0180] In some aspects, provided are conjugates of Formula (IITa):

(Ilia)

and salts thereof

wherein R ¹ , R'. HZ. R ⁴ , R ^' . R ⁶ , R ⁷ , R ^8A , R. ^XI' . R '. R ^' '. R ^{; ;} . R ^' . \i . R R ^]5 , n, W, X ^: . X ^" \ Y ¹ , Y ² , and Y ^J are as defined for Formula (la) or any variation thereof;

Y ^a is -0-, -NR ^B -, or -S-, wherein R ^B is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynvl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl : and

Li, 1,2, L3, L4, a, b, c, d, and Fn are as defined for Formula (L-Fn) or any variation thereof.

[0181] In some aspects, provided are conjugates of Formula (Illb):

(Illh)

and salts thereof,

wherein R ³ , R ⁶ , R ', R ^8a , R ^sb , R ⁹ , W, X ² , Y ¹ , Y ² , Y ^J , A and k are as defined for Formula (lb) or any variation thereof;

Y ^4a is -0-, -NR ^b -, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl; and

Li, L ₂ , L3 , L4, a, b, c, d, and Fn are as defined for Formula (L-Fn) or any variation thereof

[0182] In some aspects, provided are conjugates of Formula (IIIc):

(IIIc)

and salts thereof,

wherein R ⁵ , R ⁶ , R ⁷ , R ^8a , R ^8b , R ⁹ , W, X ² , Y ^! , Y ² , Y ³ , A and k are as defined for Formula (lc) or any variation thereof;

Y ^4a is -0-, -NR -, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl; and

Li, L2, L3 , L4, a, b, c, d, and Fn are as defined for Formula (L-Fn) or any variation thereof,

[0183] In some aspects, provided are conjugates of Formula (IITd):

(Tiid)

and salts thereof,

wherein R ^d , W, A and k are as defined for Formula (Id) or any variation thereof: Y ^4a is -0-, ~NR°~, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cvcloalkyl, or substituted or unsubstituted cvcioaikenyl; and

Li, L ₂ , 1-.3 , L4, a, b, c, d, and Fn are as defined for Formula (L-Fn) or any variation thereof.

[0184] In some aspects, provided are conjugates of Formula (Me):

(Hie)

and salts thereof,

wherein R ^d , W, A and k are as defined for Formula (Id) or any variation thereof;

Y ^4a is -0-, -NR ^b -, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyi, substituted or unsubstituted alkynyl, substituted or unsubstituted cycioaikyl, or substituted or unsubstituted cycloalkenyl; and

Li, L ₂ , 1-3 , L ₄ , a, b, c, d, and Fn are as defined for Formula (L-Fn) or any variation thereof.

[0185] In some embodiments of any of Formulae (III), (Ilia), (Mb), (IIIc), (Hid), and (Me), one or both of the following conditions apply: i) when Y ⁴ is -0-, W is not -ί( ^" ί \ ·) >- or -(CH ₂ CH ₂ 0)3(CH2)2-; and ii) when Y ^4a is -NH or -N(CH ₃ ), W is not -!( ! ! ·) -. -(CH ₂ ) ₆ -, or - CH ₂ (CH ₂ CH ₂ 0) ₃ (CH ₂ ) ₃ -.

[0186] Exemplary conjugates are shown in Table 2. Also provided herein are salts of any of the conj ugates shown in Table 2. In some embodiments, a conj ugate shown in Table 2, or a salt thereof, is a precursor compound in which the linker portion may be further modified, e.g., to insert a functional group suitable for bonding to a ligand.

Table 2

98

100

101

103

109

[0187] In some variations, a conjugate of Table 2 is further bonded to a ligand, such as an antibody. In some variations, the ligand is bonded via an azide moiety on the linker. In some variations, the ligand is bonded via an alkyne moiety on the linker. In some variations, the ligand is bonded via a maleimide moiety on the linker. In some variations, the ligand is bonded via an amine moiety on the linker. In some variations, the ligand is bonded via a hydroxyl moiety on the linker. In some variations, the ligand is bonded via a suifonyl moiety on the linker. In other variations, the ligand is bonded via a thiol moiety on the linker.

[0188] In some aspects, provided are conjugates of Formula (IV):

(IV)

and salts thereof,

wherem R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ^8a , R ^8b , R ⁹ , R ¹⁰ , R ^{1 1} , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , n, W, X ^! , X ² , Y ¹ , Y ² , and Y ^J are as defined for Formula (1) or any variation thereof;

Y ^4a is -0-, -NR. ⁰ -, or ~S~, wherein R is H, substituted or unsubstituted alkyl, substituted or unsubstituted aikenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

Li, ^' La, L ₃ , L ₄ , a, b, c, and d are as defined for Formula (L-Fn) or any variation thereof; mFn is a modified form of functional group Fn as defined in Formula (L-Fn) with a valence suitable for bonding to an antibody;

t is an integer from 1-12, inclusive; and

Ab is an antibody.

[0189] In some aspects, provided are conjugates of Formula (IV a):

(IVa)

and salts thereof,

wherein R ^! , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ^8a , R ^8b , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , n, W, X ^! , X ^" , Y', Y ² , and Y ³ are as defined for Formula (la) or any variation thereof;

Y ^4a is -0-, -NR ^D -, or -S-, wherein R is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

Li, L ₂ , 1-.3 , L4, a, b, c, and d are as defined for Formula (L-Fn) or any variation thereof; mFn is a modified form of functional group Fn as defined in Formula (L-Fn) with a valence suitable for bonding to an antibody;

t is an integer from 1-12, inclusive; and

Ab is an antibody.

[0190] In some aspects, provided are conjugates of Formula (IVb):

(TVb)

and salts thereof,

wherein R ⁵ , R ⁶ , R ⁷ , R ^a , R ^sb , R ⁹ , W, X ² , Y\ Y ² , Y ³ , A and k are as defined for Formula (lb) or any variation thereof;

Y ^4a is -0-, -NR ^b -, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyi, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

Li, L ₂ , 1-3 , L ₄ , a, b, c, and d are as defined for Formula (L-Fn) or any variation thereof; mFn is a modified form of functional group Fn as defined in Formula (L-Fn) with a valence suitable for bonding to an antibody;

t is an integer from 1-12, inclusive; and

Ab is an antibody.

[0191] In some aspects, provided are conjugates of Formula (IVc):

(IVc)

and salts thereof, wherein ⁵ , R ⁶ , R', R ^8a , R ^8b , R ^y , W, X ² , Y ¹ , Y ² , Y ^J , A and k are as defined for Formula (lc) or any variation thereof;

Y ^4a is -0-, -NR. ⁰ -, or ~S~, wherein R is H, substituted or unsubstituted alkyl,

substituted or unsubstituted aikenyl, substituted or unsubstituted aikynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

Li, ^' La, L ₃ , L ₄ , a, b, c, and d are as defined for Formula (L-Fn) or any variation thereof; mFn is a modified form of functional group Fn as defined in Formula (L-Fn) with a valence suitable for bonding to an antibody;

t is an integer from 1-12, inclusive; and

Ab is an antibody.

[0192] In some aspects, provided are conjugates of Formula (IV d):

and salts thereof,

wherein R ^d , W, A and k are as defined for Formula (Id) or any variation thereof;

Y ^4a is -0-, -NR ^b -, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyi, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted cycloalkenyl;

L] , L ₂ , L3, L ₄ , a, b, c, and d are as defined for Formula (L-Fn) or any variation thereof: mFn is a modified form of functional group Fn as defined in Formula (L-Fn) with a valence suitable for bonding to an antibody;

t is an integer from 1-12, inclusive; and

Ab is an antibody.

[0193] In some aspects, provided are conjugates of Formula (IVe):

(IVe)

and salts thereof,

wherein R ^d , W, A and k are as defined for Formula (Id) or any variation thereof:

Y ^a is -0-, -NR ^b -, or -S-, wherein R ^b is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynvl, substituted or unsubstituted cycloalkyl, or substituted or unsubsti tuted cycloalkenyl;

Li, L ₂ , L ₃ , L ₄ , a, fa, c, and d are as defined for Formula (L-Fn) or any variation thereof; mFn is a modified form of functional group Fn as defined in Formula (L-Fn) with a valence suitable for bonding to an antibody;

t is an integer from 1-12, inclusive; and

Ab is an antibody.

[0194] In some embodiments of any of Formulae (I V), (IV a), (IVb), (IVc), (IV d), and (IVe), one or both of the following conditions apply: i) when V ^*a is -0-, W is not -(CH ₂ ) ₂ ~ or -(Π ΚΉ.-θΜα ΐ .)··-: and n) when Y ^a is -NH or -N(CH ₃ ), W is not -(CH ₂ ) ₂ -, -(CH ₂ ) ₆ - ₅ or - CH ₂ (CH ₂ CH ₂ 0)3(CH ₂ )3-.

[0195] In some embodiments, t is an integer from 1-8, inclusive. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In other embodiments, t is 6. In some embodiments, mFn is

117

wherein wherein el, e2, and e3 are each independently an integer from 0-12, inclusive, and R ^c is H or alkyl.

[0196] Conjugates containing an antibody can be prepared by the following general scheme.

wherein Compound denotes any compound provided herein, including any compound of Formula (I), (la), (lb), (Ic), (Id), or (le) or any variation thereof, or any compound listed in Table 1 , or a salt thereof. The partial modification of the antibody can be earned out by any method known in the art. In some embodiments, the partial modification is partial or full reduction of disulfide bonds in the antibody, for example, by any method known in the art. In other embodiments, the partial modification is attachment of one or more suitable chemical moieties by conjugation via a chemically feasible functional group. In some embodiments, the chemically feasible functional group is bonded to the the sidecham of an ammo acid within the antibody. In some embodiments, the chemically feasible functional group is a maleimide. In some embodiments, the partial modification is the installation of one or more chemical moieties containing an alkynyl or azide group, for example, by any method known in the art. In some embodiments, the linker bonds to a cysteine side chain of the antibody. In some embodiments, the linker bonds to a lysine side chain of the antibody.

[0197] Exemplaiy conjugates containing an antibody are shown in Table 3. In any of the conjugates of Table 3, t is an integer from 1-12, inclusive, and Ab is an antibody. In some variations, the antibody binds to a receptor. In some variations, the antibody binds to a receptor on the surface of a cell. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5 in some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9, In some embodiments, t is 10. In some embodiments, t is 1 1. In some embodiments, t is 12. In some variations, the antibody in any of the conjugates of Table 3 is replaced with any other suitable ligand. Also pro vided are salts of any of the conjugates shown in Table 3 or any variation thereof.

Compositions

[0198] Tlie present disclosure also provides a composition, e.g., a pharmaceutical composition, containing one or more of the compounds or conjugates described herein. The composition may be a pharmaceutical composition. The composition may be an intermediate for use in preparation of a pharmaceutical compositions. Compositions may contain one or more compounds or conjugates described herein. In some aspects, a composition may contain a synthetic intermediate that may be used in the preparation of a compound or conjugate described herein. The compositions described herein may contain any other suitable active or inactive agents.

[0199] Any of tlie compositions described herein may be sterile or contain components that are sterile. Sterilization can be achieved by methods known in the art. Any of the compositions described herein may contain one or more compounds or conjugates that are substantially pure.

[0200] In any composition containing a conjugate, wherein the conjugate comprises a ligand, the average ratio of drag to iigand in the composition is from 1 to 12, inclusi ve, where the ratio may be an integral or non-integral value. In some such compositions, the conjugate comprises an antibody, and the average drag to antibody ratio (DAR.) is from 1 to 12, inclusive, where the ratio may be an integral or non-integral value.

Pharmaceutical Formulations

[0201] The present disclosure also provides a composition, e.g., a pharmaceutical composition, containing one or more of the compounds described herein, formulated together with a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a conjugate as described herein combined with at least one other active agent.

[0202] Pharmaceutically acceptable carriers may include any and all carriers, excipients, stabilizers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the earner is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the conjugate described herein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0203] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at standard dosages and concentrations to be administered, and include buffers such as phosphate, citrate, and other organic acids: antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride: hexamethonium chloride:

benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDT ^' A; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN ^1M or polyethylene glycol (PEG).

[0204] The pharmaceutical compositions of the invention may include one or more pharmaceutically acceptable salts. A pharmaceutically acceptable salt retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N.N'dibenzylemylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0205] A pharmaceutical composition of the invention also may include a

pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisuifite, sodium sulfite and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxvtoluene (BHT), lecithin, propyl galiate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Dosages and Dosage Forms

[0206] For the prevention or treatment of disease, the appropriate dosage of conjugates and compounds described herein will depend on the type of disease to be treated, the severity and course of the disease, whether the compound or conjugate is administered for preventive or therapeutic purposes, mode of delivery, previous therapy, and the subject's clinical history. The compounds and conjugates described herein are suitably administered to a subject at one time or over a series of treatments. Depending on the type and severity of the disease, a typical daily dosage might range from about 0.0001 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.

[0207] For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1- 10 mg kg. Treatment regimens may comprise administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every 3 months or once every three to 6 months. In other embodiments, sustained release formulations are administered, which would result in less frequent administration compared to non-sustained release formulations.

[0208] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect, without being toxic to the subject. Generally, this amount will range from about 0.01 percent to about ninety -nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

Administration [0209] A composition described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for the compounds and compositions described herein include oral, sublingual, buccal, intranasal, topical, rectal, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase

"parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

Methods of Treatment

[0210] In one embodiment, the disease or condition to be treated or prevented is cancer. The term "cancer" refers to pre-cancerous conditions, non-malignant, low-grade, high-grade, and malignant cancer. Cancer of any tissue type is contemplated for treatment or prevention by the compounds disclosed herein. Exemplary types of cancer include carcinoma, lymphoma, blastema, sarcoma, leukemia, and lymphoid malignancies. More specifically, in certain embodiments the cancer is squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

[0211] Provided herein is a method of treating cancer in an individual in need thereof by administering to the individual a therapeutically effective amount of a compound, conjugate, or composition described herein. Also provided herein is the use of a compound, conjugate, or composition described herein in the manufacture of a medicament for treatment of cancer in an individual in need thereof. Also prov ided herein is the use of a compound, conjugate, or composition described herein for treatment of cancer in an individual in need thereof. Also provided herein is a compound, conjugate, or composition described herein for use in treatment of cancer in an individual in need thereof.

[0212] In one aspect, provided herein are kits containing a compound, conjugate, or composition described herein and instructions for use. The kits may contain instructions for use in the treatment of cancer in an individual in need tliereof. A kit may additionally contain any materials or equipment that may be used in the administration of the compound, conjugate, or composition, such as vials, syringes, or IV bags. A kit may also contain sterile packaging.

General Synthetic Methods

[0213] Compounds of Formula (I) will now be described by reference to illustrative synthetic schemes for their general preparation below and the specific examples that follow. Artisans will recognize that, to obtain the various compounds herein, starting materials may be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product.

Alternatively, it may be necessary or desirable to employ, in the place of the ultimately- desired substituent, a suitable group that may be carried through the reaction scheme and replaced as appropriate with the desired substituent. In addition, one of skill in the art will recognize that protecting groups may be used to protect certain functional groups (amino, carboxy, or side chain groups) from reaction conditions, and that such groups are removed under standard conditions when appropriate. Unless otherwise specified, the variables are as defined above in reference to Formula (!)

[0214] Where it is desired to obtain a particular enantiomer of a compound, this may be accomplished from a corresponding mixture of enantiomers using any suitable conventional procedure for separating or resolving enantiomers. Thus, for example, diastereomeric derivatives may be produced by reaction of a mixture of enantiomers, e.g. a racemate, and an appropriate chiral compound. The diastereorners may then be separated by any convenient means, for example by crystallization and the desired enantiomer recovered. In another resolution process, a racemate may be separated using chirai High Performance Liquid Chromatography. Alternatively, if desired a particular enantiomer may be obtained by using an appropriate chirai intermediate in one of the processes described.

[0215] Chromatography, recrystallization and other conventional separation procedures may also be used with intermediates or final products where it is desired to obtain a particular isomer of a compound or to otherwise purify a product of a reaction.

[0216] General methods of preparing compounds described herein are depicted in exemplified methods below. Variable groups in the schemes provided herein are defined as for Formula (I), (la), (lb), (Ic), (Id), (le), (II), (Ila), (lib), (lie), (lid), (He), (III), (Ilia), (Illb), (IIIc), (Hid), (Hie), (IV), (IVa), (IVb), (IVc), (IVd), (IVe), or any variation thereof. Other compounds described herein may be prepared by similar methods. For example, Scheme lb is an exemplified synthesis of the method detailed in Scheme l a, but other compounds described herein may be prepared by similar methods.

[0217] Peptide coupling, esterification, and deprotection reactions referred to herein can be carried out using methods known in the art.

Scheme la.

[0218] Protected di-peptide E-l is subjected to a peptide coupling reaction to form protected tri-peptide E-2. Protecting group PG _a is removed, followed by a further peptide coupling reaction to afford compound E-3. Protecting group PG is then removed, followed by a further peptide coupling reaction to afford compound E-4.

E-6a

[0219] Compound E-4 is reacted with compound E-5a under suitable conditions to afford compound E-6a.

Scheme 3a.

E-9b

[0220] Selective deprotection of compound E-6a to remove protecting group PG3 affords compound E-7b. This compound is subject to peptide coupling or esterification to afford compound E-8b, Removal of protecting groups PGi and PG ₄ , cycSization via a peptide coupling reaction, and removal of protecting group PG ₂ afford compounds of Formula E-9b.

Exemplified syntheses according to Schemes la through 5a are described in Schemes lb through 3b. 02211 Scheme lb.

[0222] In an exemplified synthesis, the hydrochloride salt of protected di-peptide G-l is subjected to a peptide coupling reaction to a Boc-protected phenylalanine or substituted phenylalanine to afford tri-peptide G-2. Hie Boc group is removed, followed by a peptide coupling reaction to Boc-protected alanine to afford compound G-3. The Boc-group is removed, and the compound is further reacted with D-2-hydroxyisocaproic acid (D-HICA) via a peptide coupling reaction to afford compound G-4.

Scheme 2b.

G-6a

[0223] Compound G-4 is reacted with compound G-5a under suitable esterification conditions to afford compound G-6a.

G-Sb

[0224] Compound G-6a is selectively deprotected to remove PG3, resulting in compound G~7b. G-7b is subject to peptide coupling or estenfication to afford compound G-8b.

Removal of protecting groups PG j and PG ₄ , cyclization via a peptide coupling reaction, and removal of protecting group PG? affords compound G-9a.

[0225] In another exemplified synthesis shown in Scheme 4, compound G-10 is reacted with chloro p-nitrophenylformate to form compound G-l I, which is coupled with H ₂ N- (CH2) _m -Z-(CH ₂ )p-Y ⁴ to form compound G-12.

Scheme 4

HN

\

G-12 (CH ₂ ) _m -Z-(CH ₂ ) _p -Y«

[0226] Another exemplified synthesis is shown in Scheme 5. Compound G13 is coupled to G14 to form G15, which is selectively deprotected to remove PG ₃ . H e resulting compound G16 is coupled with compound G17 to form G18, which is selectively deprotected to remove PG ₂ , resulting in compound G-19. G-19 is then coupled with G-20 to form G21, which undergoes selective deprotection of PG ₄ to form G-22, Deprotection of PG ₅ affords G- 23, which undergoes a cyclization via a petide coupling reaction to form G-24. G24 is deprotected to remove PG> to afford G-25.

Scheme 5

[0227] Another exemplified synthesis is shown in Scheme 6. Compound H-l is reacted with n-BuLi followed by (+)-B-methoxydiisopinocamphenyl borane, boron ti fluoride etherate, and H-2 to form compound H-3. Reaction with methanesulfonyl chloride in DMAP and trimethylamine and methylene chloride affords compound H-4. Compound H-4 is treated with sodium azide to produce compound H-5. Compound H-5 is treated with

Dicarbonylacetylacetonato rhodium and Bipliephos, followed by NaBH ₄ at -40 °C to produce compound H-6, which is further reduced to compound H-7 using NaBH ₄ . Compound H-7 is reduced to compound H~8 using palladium on carbon in methanol. Compound H-8 is coupled with compound H-9 using DIPEA and HATU in methylene chloride to form compound H-10. Compound H-11 is formed by reacting compound H-10 with DMP in methylene chloride. Compound H-11 is reacted with compound H-12 to form compound H- 13. Compound H-13 is treated with ^" FFA in methylene chloride to form, compound H-14, which is coupled with compound H-15 to form compound H-16. Compound H-16 is deprotected using NHiOAc and zinc followed by diethylamine and MeCN to form compound H-17, which undergoes a cyclization via coupling to form H-18.

Scheme 6,

[0228] Certain intermediates useful in the preparation of the compounds and conjugates described herein may be synthesized according to any of the following schemes or variations thereof.

Scheme 7,

Dess-Martin fj f ^TBS NaBH„, -40 °C ^{0H 0TBS}

Rfe0 ₂ e,„

CtW¾, 23 ^« C J" T ^K? *~ ""^^^ γ -

MeOH

/-7a

1. AcOH, Ac ₂ 0, DMSO "^S'^O OTBS

2. LiOH ^¾ Y' - _{R i} ~ 5-Hydroxy-26-membered lactone

l-Sa

[0229] Compound I-la is reacted with aldehyde I~2a in the presence of Β¾ΒΟ ^' ϊΐ in methylene chloride at -78 °C to form compound i-3a. Reaction with trimethylaluminum and MeNHOMe ^' HCl in methylene chloride followed by treatment with TBSCi and imidazole in DMF affords compound I-4a. Compound I-4a is treated with diisobutylaluminum hydride in methylene chloride at -78 °C to afford aldehyde I-5a. Compound I-5a is reacted with compound i-5z in the presence of boron trifluoride diethyl etherate in methylene chloride at - 78 °C to afford compound I-6a. Compound I-6a can be subjected to a Dess-Martin reaction in methylene chloride at 23 °C to afford ketone I- 7a. Further reaction with sodium borohydride in methanol at -40 °C affords compound I-8a. Compound I-8a is treated with acetic acid, acetic anhydride, and DMSO, followed by treatment with lithium hydroxide to afford compound i-9a. Compound I-9a can be further reacted as described herein to afford a 5-hydroxy-26-membered lactone compound provided herein.

i!!-Wa

[0231] Compound Πί-la is reacted with aldehyde III-lx in the presence of Β¾ΒΟΤί to form compound III-2a. Reaction with trimethylaluminum and MeNHOMe ^' HCl in methylene chloride followed by treatment with TBSCl and imidazole in DMF affords compound III-3a. Compound III-3a is treated with diisobutylaluminum hydride in methylene chloride at -78 °C to afford aldehyde III-4a. Compound III-4a is reacted with compound III-lz in the presence of boron trifluoride diethyl etherate in methylene chloride at -78 °C to afford compound III- 5a. Compound III-5a can be subjected to a Dess-Martin reaction in methylene chloride at 23 °C to afford ketone lll-6a. Further reaction with (R)-CBS and sodium borohydride in THF at -40 °C affords compound III-7a. Compound III-7a is treated with acetic acid, acetic anhydride, and DMSO to afford compound III-8a. Treatment with HF ^' pyridine in pyridine and THF at 40 °C affords compound III-9a. Compound III-9a is reacted with DPPA and DBU in toluene at 40 "C to afford compound 111-lOa. Further reaction with triphenylphosphine in water and THF at 40 °C results in compound Hl-l la. Treatment with Boc anhydride, tri methyl amine, and DMAP in methylene chloride affords compound III-12a. Compound III-12a can be further reacted as described herein to afford a 5-hydroxy-26- membered lactam compound provided herein.

[0232] Compounds containing linkers as described herein can be obtain from appropriate precursor compounds and may be prepared using methods known in the art. In some variations, linker-containing compounds of Formula (Lbl) may be prepared in accordance with the general procedure of Scheme 9.

Scheme 9,

[0233] Compounds of Formula (Ib2) may be prepared in accordance with Schemes 10- 14, below.

Scheme 1.0

[0234] Compound E-10 may be reacted with bis (2,2,2- trichloroethy phosphorochloridate under suitable conditions to afford a compound of E-1 1.

Scheme 11.

Compound E-11 may be converted to compound E-12.

Scheme 12.

[0236] Compound E-12 may be coupled with compound E-13 to form compound E-14.

Scheme 13

Compound E-14 may be deprotecied to form compound E-15.

[0238] Compound E-15 may be coupled with compound E-16 to from compounds of Formula (Ib:2). Compounds of Formula (Ib2) may be coupled to a ligand. [0239] As shown in Scheme 15, the compound may be reacted with a linker (L-Fn) at any position on the compound suitable for reaction with a Sinker to produce the compound-linker conjugate compound-L-Fn using suitable methods known in the art. In some embodiments, linker is attached via the Y ⁴ group of the compound. In Scheme 13, Compound-L-Fn may comprise any compound or conjugate described herein.

Scheme 15

L-Fn

Compound »~ Compound— L— Fn

[0240] A compound of Formula (I), wherein Y ⁴ is OH, may be reacted using suitable methods to produce a conjugate containing the compound of Formula (I) bonded to a azide- containing linker, such as compounds of Formula (Tb2).

[0241] Azide-containing compound-L-Fn conjugates provided herein can be conjugated to an antibody comprising an alkyne functional group (wherein U represents any suitable portion of a linker moiety) using methods known in the art, such as using click chemistry, as shown in Scheme 16.

[0242] Similarly, antibodies containing an azide functional group may be conjugated with alkyne-containing compound-L-Fn conjugates using click chemistry methods.

[0243] Compounds of Formula (I), may also be reacted using suitable methods to produce a conjugate containing the compound of Formula (I) bonded to a maleimide- containing linker. In some embodiments, the linker is attached via the Y ⁴ group of the compound, wherein Y ^' is -OH. More generally, any of the compounds described herein can be coupled to a maleimide-containing linker at a suitable position, as shown in Scheme 17:

Compound

[0244] Compounds or conjugates comprising maleimide groups can be conjugated to an antibody using methods known in the art, such as those described in Greg T. Hermanson, Bioconjugate Techniques, Academic Press 19, page 60. An exemplary method is provided in Scheme 18. The antibody is first subjected to reducing conditions for partial or full reduction of disulfide bonds. The modified antibody is then conjugated to the compound-L-Fn via maleimide-sulfhydryl conjugation techniques, upon which the maleimide of the compound-L- Fn is directly conjugated to the sulfur atom of a cysteine side chain in the antibody.

Scheme 18.

[0245] Bis-sulfone containing compounds can also be coupled to thiol-modified antibodies (bridged disulfide antibodies) via alkene group (ref: . Badescu, G. et al,

Bioconjugate Chem. 2014, 25, 1 124-1 136), An exemplary method is provided in Scheme 17. After subjecting the antibody to reducing conditions for partial or full reduction of disulfide bonds, the modified antibody is conjugated to the compound-L-Fn via two sulfide bonds, one via each of two geometrically close cysteine side chains within the antibody.

[0246] Alternatively, conjugation of compound-L-Fn to the antibody can be performed using a combination of the methods discussed above, such as shown in Schemes 20-2.3.

Comp

Clic

In Scheme 20, the maleimide moiety of each compound F-1 may be bound to the sulfur atom of a cysteine residue on the antibody. The modified antibody F-2 can be coupled to an azide -containing compound-L-Fn via click chemistry.

Scheme 21 1. r

red

Compound

Click C

In Scheme 21, the maleimide moiety of each compound F-3 may be bound to the sulfur atom of a cysteine residue on the antibody. Hie modified antibody F-4 can be coupled to an azide -containing compoiind-L-Fn via click chemistry.

In Scheme 22, compound F-5 is first conjugated to the antibody to fonn bridged disulfide antibody (F-6) via two sulfide bonds. The bridged disulfide antibody F-6 is then coupled to an azide-containing compound-L-Fn via click chemistry.

Scheme 23

In Scheme 23, compound F-7 is first conjugated to the antibody to form bridged disulfide antibody (F-8) via two sulfide bonds. The bridged disulfide antibody F-8 is then coupled to an alkyne-containing compound-L-Fn via click chemistry.

Scheme 23a:

[0250] In Scheme 23a, the maleimide moiety of each compound F-7a may be bound to the sulfur atom of a cysteine residue on the antibody. The modified antibody F-8a can be coupled to an cyclooctene-containing compound-L-Fn via click chemistry.

EXAMPLES

[0251] The following examples are offered to illustrate but not to limit the compositions, uses, and methods provided herein. The compounds are prepared using the general methods described above.

[0252] The following chemical abbreviations are used throughout the Examples: CDI (Ι, -carbonyldiimidazole), DCM (dichloromethane), DMAP (4-dimethylaminopyridine), DMF (dimethylformamide), DMP (Dess-Martin periodinane), EDC (l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide), ESI (electrospray ionization), Fmoc (Fluorenylmethyloxycarbonyl), HATU ( l-[Bis(dimethylamino)methylene]-lH-l,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate), HRMS (High Resolution Mass Spectrometry), Hunig's base (DIPEA, or NN-Diisopropylethylamine), LRMS (Low

Resolution Mass Spectrometry), MeCN (acetonitrile), MeOH (methanol), RP (Reversed Phase), rt (room temperature), THF (tetrahydrofuran), and TLC (thin layer chromatography).

Example 1

[0253] To a solution of kulo-2 (48 mg, 58 μηιοΐ) in DCM (5 ml.,) was added DMAP (6 mg, 50 umol), chloro p-nitrophenylformate (160 mg, 793 μτηοΐ) and diisopropylethylamine (0.5 mL). The reaction mixture was stirred for 4 h at rt, then diluted with ethyl acetate and washed with 10% citric acid solution. The aqueous lay er was extracted with ethyl acetate and the combined organic layer was washed with sodium bicarbonate solution and brine, then dried over NaS0 ₄ and concentrated in vacuo. The crude material was subjected to column chromatography to yield Intermediate 1.

Example 2

Synthesis of Compound 1

Compound 1

[0255] To the solution of (2R,5S,8R,14S, 17R,20S,21S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl)- 14-((R)-sec-butyl)-2-isobutyl-5 ,7, 10, 17,21 ,25 -hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-22 -yl (4-nitrophenyl) carbonate (2.018 μηιοΐ, 2 mg) in DCM (4.66 mmol, 0.3 ml) was added 3-aminopropan-l-ol (2.018 μηιοΐ, 1.4 μΐ,) and Hiinig's base (0.020 mmol, 3.5 μΕ). The mixture was stirred overnight and the volatile was removed. The residue was purified with RP-flash to afford

(2R,5 S,8R, 14S, 17R.20S.21 S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)- 14-((R)-sec-butyl)-2- isobutyl-5,7,10, 17,21,25-hexametl^

pentaazacyclohexacos-24-en-22-yl (3-hydroxypropyl)carbamate (0.7 mg, 37.4%). Observed HRMS (ES ) m/z: 927.5473 | M ! ! ! · .

Example 3

[0256] To the solution of (2R,5S,8R,14S ₅ 17R,20S ₅ 21S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21 ,25-hexamethy3-3,6,9, 12,15, 18,26-heptaoxo- l , 19-dioxa-4,7,10,13, 16-pentaazacyc]ohexacos-24-en-22-yl (4-nitrophenyl) carbonate (2.018 μηιοΐ, 2 mg) in DCM (7.77 mmol, 0.5 mL) was added 4-aminobutan-l-ol (0.168 mmol, 15 mg) and Hiinig 's base (0.286 mmol, 50 iiL). The mixture was stirred overnight and the volatile was removed. The residue was purified with Rp-fiash to afford

(2R,5S,8R,14S,17R,20S,21 S,22S,E)-8-benzy3-20-((E)-but-2-en-2-yl)- 14-((

isobutyl-5,7,10, 17,21,25-hexamethyl-3A

pentaazacyclohexacos-24-en-22-yi (4-hydroxybutyi)carbamate (1.7 mg, 90%). Observed HRMS (ESI) m/z: 941.562 | M H I . The ^l H NMR spectrum of Compound 2 is shown in Figure 1.

Example 4

Compound 3

[025η To the solution of (2R,5S,8R,14S, 17R,20S ₅ 21S,22S,E)-8-benzyl-20-((E)-but-2-en- 2-yl) - 14-((R)-sec-butyl)-2-isobutyl-5 ,7, 10, 17,21 ,25 -hexamethyl-3 ,6,9, 12,15, 18,26-heptaoxo- l , 19-dioxa-4,7,10,13, 16-pentaazacyc]ohexacos-24-en-22-yl (4-nitrophenyl) carbonate (5.04 μηιοΐ, 5 mg) in DCM ( 15.54 mmol, 1 mL) was added 5-aminopentan-l -ol (0.048 mmol, 5 mg) and Hiinig 's base (0.252 mmol, 0.044 mL). The mixture was stirred ovrernight, and the volatile was removed. The residue was purified with rp-fiash to afford

(2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-e n-2-yl)-14-((R)-sec-butyl)-2- isobutyl-5,7,10, 17,21,25-hexamethy«,6,9, pentaazacyclohexacos-24-en-22-yl (5-hydroxypentyl)carbamate (4.19 μιηοΐ, 4 mg, 83%). Observed HRMS (ESI) m/z: 955.5842 [M+H]+.

Example 5

[0258] To the solution of (2R,5S,8R,14S, 17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21,25-hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-22-yl (4 -nitrophenyl) carbonate (2.018 μιηοΐ, 2 mg) in DCM (7.77 rnmol , 0,5 ml.) was added 6-aminohexan-l-ol (0.017 mmoL 2 mg) and Hiinig's base (0.286 mrnol, 50 μΕ). Hie mixture was stirred overnight and the volatile was removed. The residue was purified with RP-flash to afford

(2R,5 S,8R, 14 S, 17R,20S,21 S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)- 14-((R)-sec-but -1)~2- isobutyl-5,7,10, 17,21,25 iexamethyi-3,6,9, 12, 15,18,26-heptaoxo-l, 19-dioxa-4,7, 10,13,16- pentaazacyclohexacos-24-en-22-yl (6-hydroxyhexyi)carbamate (2 mg, 102%). Observed HRMS (ESI) m/z: 969.599 [M+H]+. The 'H NMR spectrum of Compound 4 is shown in Figure 2.

Example 6

Synthesis of Compound 5

[0259] To the solution of (2R,5S,8R, 14S, 17R,20S,21 S,22S,E)-8-benzyl-20-((E)-but-2-en-

1 , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22 -yl (4-nitrophenyl) carbonate (1.01 μηιοΐ, 1 mg) in DCM (1.554 mmol, 0.1 mL) was added 7-aminoheptati- l-ol (9.91 μιηοΐ, 1.3 mg)and Hunig's base (0.0103 mmol, 1.8 μΕ). The mixture was stirred overnight and the volatile was removed. The residue was purified with RP-flash to afford

(2R,5 S,8R, 14S, 17R,2QS,21 S ₅ 22S,E)-8-benzyl-20-((E)-but-2-en-2-y 1)- 14-((R)-sec-butyl)-2- isobutyl-5,7, i0,r7,21,25 iexamethyi-3,6,9, 12, 15, 18,26-heptaoxo-i, 19"dioxa-4

pentaazacyclohexacos-24-en-22-yl (7-hydroxyheptyl)carbamate (0.6 mg, 60.5%). Observed HRMS (ESI) m/z: 983.6125 [M+H]+.

Example 7

[026Θ] To the solution of (2R,5S,8R, 14S, 17R ₅ 20S,21 S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21,25-hexamethyl-3,6,9, 12, 15, 18,26-heptaoxo- 1 , 19~dioxa-4,7, 10, 13, 16-pentaazaey clone acos-24-en-22-yi (4-nitrophenyl) carbonate (1.009 μτηοΐ, 1 rng) in DCM (3.11 mmol, 0.2 mL) was added Hiinig's base (0.0103 mmol, 1.8 μΕ) and 8-aminooctan- 1 -ol (0.0103 mmol, 1.5 mg). The mixture was stirred overnight, and the volatile was removed. The residue was purified with RP-flash to afford

(2R,5 S,8R, 14S, 17R,2QS,21 S ₅ 22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)- 14-((R)-sec-butyl)-2- isobutyl-5,7,10,r7,21,25 iexamethyl-3,6,9, 12, 15,18,26-heptaoxo-l, 19"dioxa-4,7, 10,i pentaazacyclohexacos-24-en-22-yl (8-hydroxyoctyl)carbamate (0.3 mg, 29.8%). Observed

HRMS (ESI) m/z: 997.6288 [M+H]+.

Example 8

[0261] To a solution of (2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benz5d-20-((E)-but-2-en- 2-yl)- 14-((R)-sec-butyl)-2-isobuty] -5 ,7, 10, 17,21 ,25 -hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-22 -yl (4-nitrophenyl) carbonate (0.002 g, 2.018 μηιοϊ: Intermediate 1 from example 1) in DCM (4.66 mmol, 0.3mL) was added Nl,N6-dimethylhexane-l ,6~diamine (1.455 mg, 0.010 mmol) and DIPEA (2.608 mg, 3.524 μΐ,, 0.020 mmol) and stirred for 4 hours. The solvent was removed, and the residue was purified with reverse phase flash (MeCN + 0.1 % formic acid and H ₂ 0 + 0.1% formic acid) to afford (2R,5 S,8R, 14S, 17R,20S,21 S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)-14-((R)-sec- buty3)~2-isobuty3~5,7, 10, 17,2 l,25~hexamethyl-3,6,9, 12, 15, 18,26-heptaoxo- 1, 19-dioxa- 4,7, 10,13,16-pentaazacyclohexacos-24-en-22-yl methyl(6-(methylamino)hexyl)carbamate (0.970 mg, 48.2% 0.974 μηιοΐ). Observed HRMS (ESI) m/z: 996.642 | \Μ ί | · . Example 9

[0262] To a solution of (2R,5S,8R, 14S, 17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-bu )-2-isobu -5,7J

1 , 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-22 -yl (4-nitrophenyl) carbonate (0.005 g, 5.04 μιηοΐ; Intermediate 1 from Example 1) in DCM (7.77 mmol, 0.5 mL) was added 2- (piperazin-l-yl)ethan-l-ol (6.567 mg, 6.190 μΕ, 0,050 mmol) and D1PEA (6,52 mg, 8.81 μΕ, 0.050 mmol) and stirred for 4 hours. The solvent was removed, and the residue was purified with reverse phase flash (MeCN + 0.1 % formic acid j H ₂ 0 + 0.1% formic acid) to afford (2R,5 S,8R, 14S, 17R,20S,21 S,22S,E)-8 )enzyl^

isobutyl-5,7, 10, 17,21 ,25-hexa,methyl-3,6,9, 12, 15, 18,26-heptaoxo-l , 19-dioxa-4,7, 10, 3,16- pentaazacyclohexacos-24-en-22-yl-4-(2-hydroxyethyl)piperazin e-l-carboxylate (4 mg, 4.07μηιοΙ, 81%). Observed HRMS (ESI) m/z: 982.5882 [M+H]*.

Example 10

Synthesis of Compound 10

[0263] To a solution of (2R,5S,8R, 14S, 1.7R,20S,21S ₅ 22S,E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21 ,25-hexamethy3-3,6,9, 12,15, 18,26-heptaoxo- l ,19-dioxa-4,7,10,13, 16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (5.0 mg, 5.044 μηιοΐ, 1.0 eq; Intermediate 1 from Example 1) in DCM (0.1 mL, 5.04 μιηοΐ) was added 4-(2-aminoethyl)phenol (3.460 mg, 0.025 mmoi, 5.0 eq) and DIPEA (4.41 μΕ, 0.025 mmol, 5.0 eq) and stirred overnight. The reaction was monitored by reverse phase HPLC. Tire reaction mixture was concentrated on the rotavap and the crude residue obtained was purified by reverse phase flash chromatography (acetonitrile with 0.1% formic acid/ I LO with 0.1% formic acid) to afford (2R,5S,8R, 14S,17R,20S,21S,22S _s E)-8-benzyl-20-((E)-but- 2-en~2-ylH4-((R)~see-butyl)~2-isobu^

heptaoxo- 1 , 19-dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (4- hydroxyphenethyl)carbamate (2.140 mg, 2.16 μτηοΐ, 42.9 %). Observed HRMS (ESI) m/z: 989.5665 [M+H] ⁺ .

Example I I

Synthesis of Compound 1 1

10264] To a solution of (2R,5S,8R, 14S, 17R,20S _s 21S ₅ 22S,E)-8-benzyl-20-((E)-but-2-en-

2-y3)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21 ,25-hexamethy3-3,6,9, 12,15, 18,26-heptaoxo- l ,19-dioxa-4,7,10,13, 16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (5.0 mg, 5.044 μηιοΐ, 1.0 eq; Intermediate I from Example 1) in DCM (0.1 mL, 5.04 μιηοΐ) was added 2-(2~aminoethoxy)ethan-l~o3 (5.3 mg, 0.050 mmol, 10.0 eq) and DIPEA (8.81 μΐ,, 0.050 mmol, 10.0 eq) and stirred for 2 h. The reaction was monitored by reverse phase HPLC. The reaction mixture was concentrated on the rotavap and the cmde residue obtained was purified by reverse phase flash chromatography (acetonitrile with 0.1% formic acid/ H ₂ 0 with 0.1 % fo!-mic acid) to afford (2R,5S,8R,14S,17R,20S,21 S,22S,E)-8-beaizyl-20-((E)-but- 2-en-2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21 ,25-hexamethyl-3,6,9, 2, 15,18,26- hepiaoxo- 1 , 19-dioxa-4,7, 10,13,16-pentaazacyclohexacos-24-en-22-yl (2 -(2- hydroxyethoxy)ethyl)carbamate (3.25 mg, 3.40 μιηοΐ, 67.3 %). Observed HRMS (ESI) m/z: 957.554 | Μ · Ι 1 Γ .

Example 12

[0265] To a solution of (2R,5S,8R, 14S,17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5 ₅ 7 ₅ 10, 17,2 l,25-hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyi) carbonate (5.0 mg, 5.044 μηιοΐ, 1.0 eq; Intermediate 1 from Example 1) in DCM (0.1 mL, 5.04 μηιοΐ) was added (4-(aminomethyl)phenyl)methanol (3.46 mg, 0.025 mrnol , 5.0 eq) and DIPEA (8.81 μΕ, 0.050 mrnol, 10.0 eq) and stirred overnight. The reaction was monitored by reverse phase HPLC. The reaction mixture was concentrated on the rotavap and the crude residue obtained was purified by reverse phase flash chromatography (acetonitrile with 0.1% formic acid/ H ₂ 0 with 0.1 % formic acid) to afford (2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benzyl-20-((E)-but- 2-en-2-yl)-14-((R)-sec-butyl)-2-isobutyl-5 ₅ 7 0,17,21,25-hexamethyl-3 ₅ 6,9, 12 _s 15,18 ₅ 26- heptaoxo- 1 , 19-dioxa~4,7, 10, 13,16-pentaazacyclohexacos~24-en~22-yl ( - (hydroxymethy])benzyl)carbamate (2.75 mg, 2.78 μηιοΐ, 55.1 %). Observed HRMS (ESI) m/z: 989.565 | M 1 11 . Example 13

Synthesis of Compound 13

[0266] To a solution of (2R,5S,8R, 14S, 17R,20S,21S,22S,E)-8-benzyi-20-((E)-but-2-en- 2-yl)-1 -((R)-sec-butyl)-2-isobutyU

1 , 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (0.007 g, 7.06 μιηοΐ; Intermediate 1 from Example 1) in DCM (10.88 mmol, 0.7 niL) was added 12- aminododecan-l -ol (0.014 g, 0.070 mmol) and DIPEA (9.13 mg, 12 μΕ, 0.071 mmol) and stirred for 5 hours. The solvent was removed and the residue was purified with reverse phase flash (MeCN + 0.1% formic acid j H ₂ 0 + 0.1% formic acid) to afford

(2R,5S,8R,i4S,i7R20S,21S,22S,E)~8^

isobuty]-5,7,10/! 7,21,254 examethyl-3,6,9, 12, 15, 18,26-heptaoxo-l, 19

pentaazacyclohexacos-24-en-22-yl (12-hydroxydodecyl)carbamate (0.004 g, 53.8 %, 7.06 μιηοΐ). Observed HRMS (ESI) m/z: 1053.6864 | M I I I .

Example 14

[026η Step 1 : To the solution of (2E.5S,6S.7S,8E)-7-((tert-butyldimethylsilyl)oxy)- 2,6,8-trimetliyl-5-((methylthio)methoxy)deca-2,8-dienoic acid (100 mg, 0.239 mmol), 2,2,2- trichloroethyl N-((R)-3-(4-chlorophenyl)-2-((S)-2-((R)-2-hydroxy-4-methylpe ntanan ido)-N- methylpropanamido)propanoyl)-N-methylglycyl-L-alloisoleucina te (0.205 g, 0.287 mmol), DMAP (0.029 g, 0.239 mmol) in DCM ( 1.32 g, 1 mL, 15.542 mmol) was added EDC (0.1 15 g, 0.599 mmol) at 0 C. The mixture was wanned up to rt and stirred overnight. After the completion of the reaction, the solution was diluted with citric acid solution and EtOAc. The organic phase was washed with NaHCCh aq and brine, dried overNa ₂ S04 and concentrated in vacuo. The residue was purified with flash to afford 2,2,2-trichloroethyl (2S,8R,l lS, 14R 2-((R)-see-butylH

2,6,8-trimeAyl-5-((memylthio)me1hoxy)deca-2,8-dienoyl)oxy)-8 -(4-chlorobenz 'l)-6,9, 1 1 , 16- tetramethyl-4,7,10,13-tetfaoxo-3,6,9,12-tetraazaheptadecanoa te (0.192 g, 71.91%).

[0268] Step 2: To a solution of 2,2,2-trichloroethyl (2S,8RJ lS,14R)-2-((R)-sec-butyl)- 14-(((2E,5S,6S S,8E)-7-((tert4>utyldm^

((methylmio)memox} deca-2,8-dienoyl)ox} -8-(4-chlorobenzyl)-6,9,n,16-tetramethyl- 4,7, 10, 13-tetraoxo-3,6,9, 12-tetraazaheptadecanoate (0.192 g, 0.172 mmol) in 0.05M solution (3.46mL) of 1 : 1 :6 (HF pyridine, pyridine, THF) was stirred at 60 °C for 12h. After the completion of the reaction through TLC, the mixture was diluted with EtOAc and NaHCOj aq. The organic phase was washed with brine, dried over Na?S0 ₄ and concentrated in vacuo. The residue was purified with flash to afford 2,2,2-trichloroethyl (2S,8R,1 lS, 14R)-2-((R)- sec-butyl)-8-(4-chlorobeiizylH

((meihylthio)methoxy)deca-2,8-dienoyl)oxy)-6,9, 11,16-tetramethyl-4,7, 10, 13-tetraoxo- 3,6,9,12-tetraazaheptadecanoate (148 mg, 85.86%).

Step 3: To the solution of (((9H-fluoren-9-yl)methoxy)carbonyl)-D-alanine (0.763 g, 2/453 mmol), 2,2,2-trichloroethyl (2S,8R,1 l S,14R)-2-((R)-sec-butyl)-8-(4-chlorobeaizyl)- 14-(((2E,5S,6R,7S,8E)-7-hydroxy-2,6,8-trimethyl-5-((meth^

dienoyl)oxy)-6,9, 11, 16-tetramethy 1-4,7, 10, 13-tetraoxo-3,6,9, 12-tetraazaheptadecanoate (817 mg, 0.817 mmol) and DMAP (0.099 g, 0.817 mmol) m DCM (4.224 g, 3.2 ml... 49.736 mmol) was added EDC (0.470 g, 2.453 mmol) at 0 ^° C. After stirring at 0 ^" C for 3 h, the mixture was warmed to rt and stirred overnight, diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCC and brine, dried over Na ₂ S0 ₄ and concentrated in vacuo. The residue was purified with flash to afford 2,2,2-trichloroethyl (2S..8RJ i S.141?. )- 14-(((2E,5S,6S,7S,8E)-7-(((((9H-fluoren-9^^

trimethyl-5-((methylthio)methoxy)deca-2,8-dienoyl)oxy)-2- ((R)-sec-butyl)-8-(4- chlorobenzy l)-6,9, 11,16-tetramethy 1-4,7, 10, 13-tetraoxo-3,6,9, 12-tetraazaheptadecanoate ( 1 g, 94.62%) with some Fmoc ala impurity.

[0270] Step 4: To the solution of 2,2,2-trichloroethyl (2S,8R,1 1 S, 14R)-14- (((2E,5S,6S,7S,8E)-7-(((((9H-fiuoren-9-yl)m^^

5-((memylthio)methoxy)deca-2,8-dienoyl)oxy)-2-((R)-sec-butyl )-8-(4-chlorobenzyl)-

6,9, 1 l,16~tetramethyl-4,7, 10, 13-tetraoxo-3,6,9, 12-tetraazaheptadecanoate (1 g, 0.773 mmol) in THF (17,6 g, 20 ml, 244,081 mmol) and ammonium acetate (0.308 g, 4 mL, 4 mmol) was added zinc (5 g, 76.475 mmol) at rt. The mixture was stirred for 6 h. The mixture was added citric acid solution and filtered over celite (EtOAc). The aq phase was extracted with EtOAc and the organic phase was washed with brine, dried over Na?S04 and concentrated in vacuo to use in next step.

[0271 ] Step 5: To the crude mixture N-((R)-2-((S)-2-((R)-2-(((2E,5S,6S,7S,8E)-7- (((((9H-fluoren-9-}d)melhoxy)carbonyl)-D-alanyl)oxy)-2,6,8-t rimethyl-5- ((methylthio)methox} deca-2,8-dienoj4)ox} -4-metliylpentanamido)-N- me1hylpropaiiarnido)-3-(4-chlorophenyl)propanoyl)-N-metliylg lycyl-L-alloisoleucine (0.893 g, 0.77 mmol) in MeCN (15.72 g, 20 ml, 382,919 mmol) was added diethylamine (3.5 g, 5 ml, 47.854 mmol) at rt. The mixture was stirred for 2 h. The volatile was removed, and the residue was purified with RP-flash to afford -((R.)-2~((S)-2-((R)~2-(((2E,5S,6S,7S,8E)-7- ((D-alanyl)oxy)-2,6,8-trimethyl-5-((methyltiiio)methoxy)deca -2,8-dienoyl)oxy)-4- methylpentanamido)-N-methylpropanamido

L-alloisoleucine (540 mg, 74.72%).

Compound 14

10272] Step 6: To the solution of N-((R)-2-((S)-2-((R)-2-(((2E ₅ 5S,6S,7S,8E)-7-((D- alanyl)oxy)-2,6,8 ri.methyl-5-((methylthio)methoxy)deca~2,8~dien

methylpentanamido)-N-methylpropanamido

L-alloisoleucine (0.54 g, 0.575 mmol) in DCM (1.188 kg, 900 mL, 13.988 mol) and DMF (94.399 g, 100 mL, 1.291 mol) was added HOAt (0.783 g, 5.753 mmol) and EDC (1.102 g, 5.753 mmol) at 0 C. The mixture was warmed up to rt slowly and stirred overnight. The solvent was removed and diluted with EtOAc and citric acid solution . The organic phase was washed with NaHCCh aq and brine, dried over NaiSC , concentrated in vacuo. The residue was purified with flash to afford (3R,6S,12R,15S,18R,24S,25S,26S,E)-26-((E)-but-2-en-2- yl)-6-((R)-sec-buty1)-12-(4-chto

((methylthio)methoxy)- 1 , 19-dioxa-4,7, 10,13,16-pentaazacy clohexacos-21 -ene- 2,5,8,11,14,17,20-heptaone (365 mg, 68.92%). Observed HRMS (ESI) m/z: 920.4605

[M+H . The ^! H NMR spectnim of Chloro-Kulo2-OMTM in DMSO-d6 is shown in Figure 7,

Compound IS

[0273] Step 7: To the solution of (3R,6S, 12R,15S,18R,24S,25S,26S,E)-26-((E)-but-2-en- 2-yl)-6-((R)-sec-butyl)-12-(4^^

((methyltiiio)methoxy)- 1 , 19-dioxa-4,7, 10, 13,16-pentaazacyclohexacos-21 -ene- 2,5, 8,11, 14, 17,20-heptaone (0,368 g, 0.4 mmol) in THF (8.447 g, 9.6 mL, 117.159 mmol) and water (2.4 g, 2.4 rnl, 133.222 mmol) was added 2,6-lutidine (0.857 g, 0.931 mL, 8 mmol) and silver nitrate (2.717 g, 16 mmol). The mixture was heated at 65 °Covernight. The mixture was cooled to rt and diluted with EtOAc and IN HCi, then filtered over celite. The a ueous phase was extracted with EtOAc and the organic phase was washed with NaHC0 ₃ aq and brine, dried over NaS04 and concentrated and in vacuo. The residue was purified with rp-fiash to afford (3R,6S,12R,15S,18R,24S,25S,26S,E)-26-((E)-but-2-en-2-yl)-6-( (R)-sec- hutyl)~12-(4-chlorobenzyl)~24-hy

dioxa-4,7, 10, 13, 16-pentaazac}'clohexacos-21-ene-2,5,8, l 1, 14,17,20-heptaone (257 mg, 74.67%).

Example 15

Synthesis of Intermediate 2

[0274] To a solution of (3R,6S,12R, 15S, 1 &R,24S,25S,26S )-26-((E)-bvLt-2-en-2-y\)-6- ((fl)-sec-butyl)- 12-(4-chlorobenzyl)-24-hydroxy- 18-isobut 1-3 , 10, 13,15,21 ,25 -hexamethyl- l, 19-dioxa-4,7,10,13, 16-pentaazacyclohexacos-21-ene-2,5,8,l l,14, 17,20-heptaone (0.13 g, 0, 151 renvoi ) m DCM (1 mi .. 15.54 mmo!) was added DMAP (0.018 g, 0.151 mmol), DIPEA (0.976 g, 1.319 mL, 7.553 mmol) and 4-nitrophenyl carbonochloridate (0.3()5g, 1.511mmol). The reaction was stirred for 6 h and diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCCb aq and brine, dried over a ₂ S0 ₄ , concentrated in vacuo. The residue was purified with flash to afford (2R,5S,8R,14S, 17R,20S,21 S,22S,E)-20-((E)- but-2-en-2-yl)-14-((R)-sec-butyl)-8-(4-cMoro^

3,6,9,12,15,18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13,16-pentaazacyclohexacos-24-en-22-yl (4- nitrophenyl) carbonate (0.11 g, 71%, 0.107 mmol).

Example 16

[0275] To the solution of (2R,5S,8R,14S,17R,20S.21S,22S,E)-20-((E)-but-2-en-2-yl)-14-

((R)-see-bu^)-8~(4-chlorobenzyl)-2 sobutyl-5,7, 10, 17,2 l,25-hexamethyl-3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4, 7, 10,33,16-pentaazacycl ohexacos-24-en-22-yl (4-nitrophenyl) carbonate (59 mg, 0.060 mmol) in DCM (6.600 g, 5 niL, 77.713 mmol) was added 5- aminopentan-l-ol (50 mg, 0.484 mmol) and Hunig'sbase (0.148 g, 0.2 mL, 1.145 mmol) at rt and the mixture was stirred overnight. The volatile was removed and the residue was purified with RP-flash to afford (2R,5S,8R,14S, 17R,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-buty l)-8-(4-chlorobenzyl)-2-isobutyl-5 ,7, 10, 17,21 ,25 -hexameth yl-3,6,9,12,15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13 , 16-pentaazacy clohexacos-24-en-22-yl (5 - hydroxypen1yl)earbamate (52 mg, 91.45%). Observed HRMS (ESI) m/z: 989.5447 [M+H] ⁺ . The Ti MR spectrum of Compound 16 in DMSO-d is shown in Figure 8.

Example 1 7

[0276] To a solution of (2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benzyi-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 1 , 17,21 ,25-hexamethyl-3,6,9, 2,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13, 16-pentaazacy clohexacos-24-en-22-yl (4-nitrophenyl) carbonate (3.0 mg, 2.93 umol, 1.0 eq: Intermediate 2) in DCM (0.1 mL, 5.04 μηιοΐ) was added 2-(2- aminoethoxy)ethan-l-ol (3.08 mg, 0.029 mmol, 10.0 eq) and DIPEA (5.11 μΤ, 0.029 mmol, 10.0 eq) and stirred for 4 h. Tire reaction was monitored by reverse phase HPLC. The reaction mixture was concentrated on the rotavap and the crude residue obtained was purified by reverse phase flash chromatography (acetonitrile with 0.1% formic acid/ H ₂ 0 with 0.1% formic acid) to afford (2R,5S,8R, 14S, 17R,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-

heptaoxo- 1 , 19-dioxa-4,7, 10, 13 , 16-pentaazacyclohexacos-24 -en-22-yl (2-(2- hydroxyethoxy)ethyl)carbamate (1.72 mg, 1.735 μιηοΐ, 59.3 %). Observed HRMS (ESI) m/z: 991.518 j M H j .

Example 18

Synthesis of Compound 18

[0277] To a solution of (2R,5S,8R, 14S, 17R,20S,21S,22S,E)-8-benzyi-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21 , 25-hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-22-yl (4-ni trophenyl) carbonate (3.0 mg, 2.93 μηιοΐ, 1.0 eq) in DCM (0.1 mL, 5.04 μπιοΐ; Intermediate 2) was added 4-(2- aminoethyl)phenol (4.01 mg, 0.029 mmol, 10.0 eq) and DIPEA (5.11 μΕ, 0.029 mmol, 10.0 eq) and stirred overnight. The reaction was monitored by reverse phase HPLC. The reaction mixture was concentrated on the rotavap and the crude residue obtained was purified by reverse phase flash chromatography (acetomtrile with 0.1% formic acid/ HiO with 0.1% formic acid) to afford (2R,5S,8R,14S,17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-( (R)- sec-bu1yl)-8-(4^hlorobenzyl)-2-isobutyl-5 0 7,21,25-hexametliyl ,6.9 2 5 ₅ 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13 , 16-pentaazacyclohexacos-24-en-22-yl (4- hydroxyphenethyl)carbamate (1.64 mg, 1.602 μιηοΐ, 54.8 %). Observed HRMS (ESI) m/z:

Example 19

Synthesis of Compound 19

[0278] To a solution of ((2R,5S,8R,14S, 17R,20S,21S,22S ₅ E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-butyl)-8-(4-chlorobeaizyl)-2-isobutyl-5,7, 10, 17,2 l,25-hexaniethyl~3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa~4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (0.0035 g, 3.41 μτηοΐ) in DCM (4.66 mmol, 0.3 mL) was added N 1,N6- dimethylhexane-l,6-diamine (4.923 mg, 0.034 mmol) and DIPEA (4.410 mg, 5.960 μΕ, 0.034 mmol) and stirred for 3 hours. The solvent was removed and the residue was purified with C18 reverse chromatography (MeC + 0.1% formic acid j H ₂ 0 + 0.1 % formic acid) to afford (2R,5S,8R, 14S, 17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec-butyl) -8-(4- chlorobenzy3)-2~isobuty3 -5,7, 10, 17,21 ,25 -hexamethyl-3 ,6,9, 12,15, 18,26-heptaoxo- 1,19- dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl methyl (6- (methylamino)hexyl)carbamate (0.002 g, 57%, 3.41 μιηοΐ). Observed FIRMS (ESI) m/z: 1030.6007 [M+H] ⁺ .

Example 20

Synthesis of Compound 20

[0279] To a solution of ((2R,5S,8R,14S,17R,20S,21S,22S^ 20

(ii?}-,?ec-butyi 8~(4^

beptaoxo- 1 , 19-dioxa~4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (0.003 g, 2.93 μιηοΐ: Intermediate 2) in DCM (4.66 mmol, 0.3 mL) was added Nl,N 12-dimethyldodecane-l,12-diamine (6.681 nig, 0.029 mmol) and D1PEA (3.780 mg, 5.108 μΕ, 0.029 mmol) and stirred for 3 hours. The solvent was removed and the residue was purified with C18 reverse chromatography (MeCN + 0.1 % formic acid j H ₂ 0 + 0.1% formic acid) to afford (2R,5S.8R, 14S,17R,20S,21S.22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec- but> )-8-(4-chlorobenz d)-2-isobut> -5,7, 10, 17,21,25-hexamethyl-3,6,9,12,15,18,26- beptaoxo- 1 , 19-dioxa~4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl methyl (12- (methylarnino)dodecyl)carbarnate (0.685 mg, 21%, 2.93 μτηοΐ). Observed HRMS (ESI) m/z: 1114.7044 [M+H] ⁺ .

Example 21

Synthesis of Compound 21

[0280] Step 1 : To the solution of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2- methylpropanoic acid (0.148 g, 0.455 mmol), 2,2,2-trichloroethyl (2S,8R, 1 S,14R)~2-((R)~ sec-butyl)-8~(4-chlorobenzyl) 4~((^

((meihylthio)methoxy)deca-2,8-dienoyl)oxy)-6,9, 11,16-tetramethyl-4,7, 10, 13-tetraoxo- 3,6,9, 12-tetraazaheptadecanoate (151.7 mg, 0.151 mmol; Intermediate from Example 14) and DMAP (0.018 g, 0.151 mmol) in DCM (4.224 g, 3.2 mL, 49.736 mmol) was added EDC (0.087 g, 0.455 mmol) at 0 °C. After stirring at 0 °C for 3 h, the mixture was warmed to rt and stirred overnight, diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCOs and brine, dried overNa ₂ S0 ₄ and concentrated in vacuo. The residue was purified with flash to afford 2,2,2-trichloroethyl (2S,8R,11 S,14R)-14- (((2E,5S,6S,7S,8E)-7-((2-((((9H 1uoren-9-yl)methoxy)carbonyl)amino)-2

methylpropai oyl)oxy)-2,6,84rimethyi-5-((methylthio)methoxy)deca-2,8~

((R)-sec-but> )-8-(4-cUorobenzyl)-6,9, l l, 16-tetrainethyl-4,7, 10, 13-tetraoxo-3,6,9,12^ tetraazaheptadecanoate (0.196 g, 99.05%) with some Fmoc-Isobutyric acid impurity.

[0281 ] Step 2: To a solution of 2,2,2-trichloroethyl (2S,8RJ 1 S, 14R)-14~

(((2E,5S,6S,7S,8E)-7-((2-((((9H 1uoren-9-yl)methoxy)carbonyl)amino)-2

methylpropai oyl)oxy)-2,6,84rimethyi-5-((methylthio)methoxy)deca-2,8~di

((R)-sec-but> )-8-(4-cUorobenzyl)-6,9, l l, 16-tetrainethyl-4,7, 10, 13-tetraoxo-3,6,9,12- tetraazaheptadecanoate (0.196 g, 0.150 mmol) in 0.025 M of THF (6 mL), and 0.1 13 M of 1 M NH ₄ OAc (1.3 mL) was added zinc (2.753 g, 42.120 mmol) and stirred for 4 h. The reaction was complete through TLC was filtered through celite concentrated. The resulting residue was dissolved in MeCN (2 mL) and diethyl amine was added (2 mL). After 3h the reaction was concentrated in vacuo. The residue was purified with reverse phase flash to afford N-((R)-2-((S)-2-((R)-2-(((2E,5S,6S ₅ 7S,^

2,6,8-trime1hyl-5-((methylthio)m^

methylpropanamido)-3-(4-chlorophenyl)propanoyl)-N-methylg lycyl-L-alloisoleucine (0.1 124 g, 78%, 0.118 mmol).

[0282] Step 3: To the solution ofN-((R)-2-((S)-2-((R)-2-(((2E,5S,6S,7S,8E)-7-((2-amino- 2-methylpropanoyl)oxy)-2,6,8-trimethyl-5-((methyltiiio)metho xy)deca-2,8-dienoy

methylpentanamido)-N-methyipropanam

L-alloisoleucine (0, 1 12 g, 0, 1 17 mmol) in 0.000996M of 1 : 10 (DMF 18 ml, : DCM 100 ml,) was added HOAt (0.160 g, 1.179 mmol) and EDC (0.226 g, 1.179 mmol). Tire solvent was removed and diluted with EtOAc and citric acid solution. The organic phase was washed with aqueous NaHCO _. 3 and brine, dried over NaS0 ₄ , concentrated in vacuo. The residue was purified with flash to afford (6SJ 2RJ5S, 18R,24S,25S,26S,E)-26 (E)-but-2-en-2-yl)-6-((R)- sec-buty^-12-(4-chlorobenz>'])-18-isobutyl-3,3, 10, 13, 15,21,25-heptan etliyl-24- ((methylthio)methoxy)- 1 , 19-dioxa-4,7, 10,13,16-pentaazacyclohexacos-21 -ene- 2,5,8,11,14,17,20-heptaone (0.036 g, 32.74%).

Compound 21

[0283] Step 4: To the solution of (6S, 12R,15S,18R,24S,25S,26S,E)-26-((E)-but-2-en-2- yl)-6-((R)-sec-butyl)-12-(4^oro

((methylthio)methoxy)- 1 , 19-dioxa-4,7, 10,13,16-pentaazacyclohexacos-21 -ene- 2,5,8,11,14, 17,20-heptaone (0.036 g, 0.038 mmol) in THF (0.816 g, 0.927 mL, 11.317 mmol) and water (0.231 g, 0.231 mL, 12.862 mmol) was added 2,6-lutidine (0.082 g, 0.089 mL, 0.772 mmol) and silver nitrate (0.262 g, 1.544 mmol). The mixture was heated at 65 °C for 3-4 hours. The mixture was cooled to rt and diluted with EtOAc and 1 N HQ, then filtered over ceiite. The aq phase was extracted with EtOAc and the organic phase was washed with aqueous aHCOa and brine, dried over NaS0 ₄ and concentrated and in vacuo. The residue was purified with reverse phase flash to afford (6S, 12R,15S,18R,24S,25S,26S,E)-26-((E)- but-2-en-2-yl)-6-((R)-sec-butyl)-12-(4-chlorobenzyl)-24-hydr oxy-18-isobutyl- 3 ,3 , 10, 13 , 1 ,21 ,25 -heptamethyl- 1 , 19-dioxa-4,7, 10, 13,16-pentaazacyclohexacos-21 -ene- 2,5,8,1 1 ,14, 17,20-heptaone (0.022 g, 65.1%, 0.025 mmol). Observed HRMS (ESI) m/z: 874.4734 [M+H1 ⁺ .

Example 22,

Intermediate 3 [0284] To a solution of (6S,12R, 15S.18R,24S,25S,26S.E)-26-((E)-but-2-en-2-yl)-6-((R)- sec-butyl)-12-(4-chlorobenzyl)-24-hyd^

l , 19-dioxa-4,7,10,13, 16-pentaazacyc]ohexacos-21-ene-2,5,8,l 1 ,14, 17,20-heptaone (0.007 g, 8.00 μτηοΐ) in DCM (400 μΕ, 6.22 mmol) was added N,N-dimethylpyridin-4-amine (0.977 mg, 8.004 μηιοΐ), DIPEA (0.051 g, 0.069 mL, 0.400 mmol), and 4-nitrophenyl

carbonochloridate (0.007 g, 8.00 μιηοΐ). The reaction was stirred for 6 h and diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCQi aq and brine, dried overNa ₂ S04, concentrated in vacuo. The residue was purified with flash to afford (2R,5S,8R,14S,20S,21S _s 22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec-butyl)-8-(4- ehlorobenzyl)-2-isobutyl- ,7, 10,17,17,21 ,25 -heptamethyl-3 ,6,9,12,15,18 ,26-heptaoxo- 1,19- dioxa-4,7,10, 13, 16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (0.0048 g, 57.7%, 4.62 μηιοΐ).

Example 23

Synthesis of Compound 22

[0285] To a solution of (2R,5S,8R, 14S,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-bur>'l)-8-(4-chlorobeiizyl)-2-isobutyl-5,7, 10, 17, 17,2 l,25-heptamethyl-3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (0.0048 g, 4.62 μηιοΐ) in DCM (0.7 mL, 10.88 mmol) was added 5-aminopentan-l- oi (0.00476 g, 0.046 mmol) and DIPEA (5,97 mg, 8.06 μΕ, 0.046 mmol) and stirred for 4 hours. The solvent was removed and the residue was purified with CI 8 reverse phase chromatography chromatography (MeCN + 0.1% formic acid j H ₂ 0 + 0.1% formic acid) to afford (2R,5S,8R, 14S ₅ 20S,21S _s 22S ₅ E)-20-((E)-but-2-en-2-yl)-14-((R)-sec-bu1yl)-8-(4- chlorobenzyl)-2^

dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (5-hydroxypentyl)carbamate (0.002 g, 43.2%, 4.62 μηιοΐ). Observed HRMS (EST) m/z. 1003,5538 [M+H] ⁺ .

Example 24

Synthesis of Compound 23

[0286] To a solution of (2R,5S,8R, 14S,20S,21 S,22S ₅ E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-butyl)-8-(4-chlorobenzyl)-2-isobutyl-5 ₅ 7 ₅ 10, 17, 17,21 ,25-heptamethyl-3,6 ₅ 9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10,13,16-pentaazacyclohexacos-24-en-22-}d (4-nitrophenyl) carbonate (0.0035 g, 3.37 μιηοί) in DCM (3.11 mmol, 0.2 niL) was added N1,N6- dimethylhexane~l ,6~diamme (4.856 mg, 6.070 μΕ, 0.033 mmol) and DIPEA (4.351 mg, 5.880 μΕ, 0.033 mmol) and stirred for 3 hours. The solvent was removed and the residue was purified with C 18 reverse phase chromatography (MeCN + 0.1% formic acid j H ₂ 0 + 0.1% formic acid) to afford (2R,5S,8R,14S,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec- butyl)-8-(4-chlorobenzyl)-2-isobut>l-5,7, 10,17,17,21,25-heptamethyl-3,6,9, 12, 15,18,26- heptaoxo-l,1 -dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en~22-yl methyl(6- (methylamino)hexyl)carbamate (1.1 mg, 31%, 3.37 μιηοί). Observed HRMS (ESI) m/z: 1044.6156 I NS H | ^' .

Example 25

Synthesis of Compound 24

10287] To a solution of (2R,5S,8R, 14S,20S ₅ 21S ₅ 22S,E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-butyl)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17, 17,21 ,25-heptamethyl-3,6,9, 12, 15, 18,26- heptaoxo-1 ,19-dioxa-4,7, 10, 3, 16-pentaazacyclohexacos-24-en-22-yl methyl(6- (methylamino)hexyl)carbamate (0.002 g, 1.914 μιηοΐ) in DCM (4.66 mmol, 300 μΕ) was added 2,5-dioxopyrrolidin-l-yl acetate (3.007 mg, 0.019 mmol) and DIPEA (2.474 mg, 3.343 μΕ, 0.019 mmol) and stirred for 5 hours. The solvent was removed and the residue was purified with C18 reverse phase chromatography (MeC + 0.1 % formic acid j HiO + 0.1% formic acid) to afford (2R,5S,8R,14S,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-

heptaoxo-1 ,19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl methyl(6-(N- methylacetamido)hexyl)carbamate (1.1 mg, 53%, 1.012 μηιοΐ). Observed HRMS (ESI) m/z: 1086.6287 | Μ · Η | .

Example 26

Synthesis of Compound 25

10288] To a solution of (2/^55' _s 8R 45,205,215,225^)-20-((-¾ but-2-en-2-yl)-14-((R)- iec-butyl)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17, 17,21 ,25-heptamethyl-3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4, 7, 10, 13, 16-pentaazacycl ohexacos-24-en-22-yl (4-nitrophenyl) carbonate (0.005 g, 4.81 μηιοΐ) in DCM (700 μΕ) was added 3,3'-((oxybis(ethane-2, l- diyl))bis(oxy))bis(propan- l-amine) (0.010 g, 0.010 mL, 0.048 mmol) and DIPEA (6.216 mg, 8.400 μΕ, 0.048 mmol) and stirred for 3h. ^' The solvent was removed and the residue was purified on reverse phase flash to afford (2R,5S,8R, 14S,20S,21 S,22S,E)-20-((E)-but-2-en-2- yl)-14-((R)-sec-butyl)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17, 17,21,25-heptamethyl- 3,6,9, 12, 15, 18,26-heptaoxo- 1 , 19-dioxa~4,7, 10, 13, 16-pentaazacye3ohexaeos-24~en-22~yl (3- (2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate (0.00097 g, 18%, 0.865 μ mol). Observed HRMS (ESI) m/z: 1 120.6394 | M I I I .

Example 27

Synthesis of Compound 26

[0289] To a solution of (2R,5S,8R, 14S,20S ₅ 21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)- sec¾ityl)-8-(4^1orobenzyl)-2-isoto^

heptaoxo- 1 , 1 -dioxa-4,7, 10,13,16-pentaazacyclohexacos-24-en-22-yl (4-nitrophenyl) carbonate (0.005 g, 4.81 μηιοΐ) in DCM (0.263 g, 200 .uL, 3 , 108 mmol) was added 2- (piperazin-l-yl)ethan-l-ol (6.261 mg, 5 ,901 μΕ, 0,048 mmol) and DIPEA (6.216 mg, 8.400 pL, 0.048 mmol) and stirred for 3h. The solvent was removed and the residue was purified with reverse phase flash to afford (2R,5S,8R,14S,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-but>'l)-8-(4-chlorobenzyl)-2-isobutyl-5 ,7, 10, 17,17,21 ,25 -heptamethyl- 3,6,9,12,15, 18,26-heptaoxo-l, 19-dioxa-4,7,10,13, 16-pentaazacyclohexacos-24-en-22-yl 4-(2- hydroxyethyl)piperazine-l-carboxylate (0.002 g, 40.3%, 4.81 μηιοΐ). Observed HRMS (ESI)

Example 28

Synthesis of Compound 27

((S)-2-((R)-2-hydroxy-4-methylpen

methylglycyl-L-alloisoleucinate (0.456 g, 0.647 mmol) and (2E,5S,6S,7S,8E)~7~((tert- buiyldimethylsilyl)oxy)-2,6,8 rimethyl-5-((mediyldii acid (0.225 g, 0.539 mmol) in DCM (1 raL) was added DMAP (0.065 g, 0.539 mmol) and EDC (0.258 g, 1 .349 mmol) at 0 °C. The reaction was stirred for 36 h and diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCOs aq and brine, and dried over Na ₂ S0 ₄ , concentrated in vacuo. The residue was purified with flash to afford 2,2,2-trichloroethyl (2S,8R,l l S,14R)-2-((R)-sec-butyl)-14-(((2E,5S,6S,7S,8E)-7-((tert-buty ldime ls

2,6,8-trime1hyl-5-((methylthio)memoxy)deca-2,8-dienoy])ox -8-(4-cyanobenzyl)-6,9,l l, 16- tetramethyl-4,7,10, 13-tetraoxo-3,6,9,12-tetraazaheptadecanoate (0.5658 g, 95 %, 0.513 mmol).

[0291 ] Step 2: To a solution of 2,2,2-trichloroethyl (2S,8RJ l S, ! 4R)-2-((R)-sec-butyl)- 14-(((2E,5S,6S,7S,8E)-7-((tert-butyldimed^

((memylmio)memox} deca-2,8-dienoyl)ox} -8-(4-cyanobenzyl)-6,9,l l, 16-tetramethyl- 4,7, 10, 13-tetraoxo-3,6,9, 12-tetraazaheptadecanoate (0.565 g, 0.512 mmol) was dissolved in THF (6.77 fflL), pyridine (1.615 raL) and HF pyridine (1 .615 mL). The reaction was stirred overnight at 60 °C. After the completion of the reaction through TLC, the mixture was diluted with EtOAc and NaHCOs aq. The organic phase was washed with brine, dried over Na ₂ S0 and concentrated in vacuo. The residue was purified with flash to afford 2,2,2- trichloroethyl (2S,8R,l lS,14R)-2-((R)-sec-bulyl)-8-(4-cyanobenzyl)-14-(((2E,5S,6R,7 S,8E)- 7 iydroxy"2,6,8-triniethyl-5-((methylthio)niethoxy )deca-2,8-dienoyl)oxy )-6,9, 11,16- tetramethyl-4,7,10,13-tetraoxo-3,6,9,12-tetraazaheptadecaiio ate (0.4 g, 0.404 mmol, 79%).

[0292] Step 3: To a solution of 2,2,2-trichloroethyl (2S,8RJ l S,14R)-2-((R)-sec-butyl)-8- (4-cyanobenzyl)-14-(((2E,5S ₅ 6R,7S,8E)-7-hydroxy-2,6.8-liimelhyl-5-

((methyithio)methoxy)deca-2, 8-dienoyl)oxy)-6,9, 11,16-tetramethy 1-4,7, 10,13 -tetraoxo- 3,6,9,12-tetraazaheptadecanoate (0.2 g, 0.202 mmol) in DCM (0.7mL) was added 2-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropanoic acid (0.197 g, 0.606 mmol), DMAP (0.024 g, 0.202 mmol) and EDC (0.116 g, 0.606 mmol) at 0C. The solution was stirred overnight, diluted with EtOAc and citric acid solution. The organic phase was washed with NaHC(>3 and brine, dried and concentrated in vacuo. The residue was punfied with flash to afford 2,2,2-trichloroethyl (2S,8R,1 lS, 14R)-14-(((2E,5S,6S,7S,8E)-7- ((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amm^

5-((methylthio)methoxy)deca-2,8-dienoyl)ox>')-2-((R)-sec- butyl)-8-(4-cyanobenz> )- 6,9, 1 l,16-te1xamethy]-4,7,10,13-tetraoxo-3,6,9,12-tetraazaheptade canoate (0.2495 g, 94%, 0.190 mmol).

[0293] Step 4: To a stirring solution of 2,2,2-trichloroethyl (2S,8R, 11S, 14R)-14-

(((2E,5S,6S,7S,8E)-7-((2-((((9H-fluoren-9-yl)methoxy)carb onyl)amino)-2- methylpropanoyl)oxy)-2,6,8-trimethyl-5-((m

((R)-sec-buty1)-8-(4~cyanobenzyl)-6,9, l, 16~tetraniethyl~4,7,l^

tetraazaheptadecanoate (0.2459 g, 0.190 mmol) in THF (7.58 mL, 0.025 M) and 1 M NH4OAC (1.7 mL, 0.1 13 M) was added zrac (3.471 g, 53.092 mmol) and stirred for 3 h, filtered through a pad of celite using MeCN and concentrated in vacuo. The resulting residue was dissolved in MeCN (3 mL) and diethylamine (3 mL) was added. After 1 h, the reaction was concentrated in vacuo. The residue was purified with reverse phase to afford N-((R)-2- ((S)-2-((R)-2-(((2E,5S,6S,7S,8E)-7-((2-am ^

((methylmio)methoxy)deca-2,8-dienoyl)oxy)-4-methylpentananii do)-N- me1hy]propanamido)-3-(4-cyanophenyl)propanoyl)-N-methylglycy l-L-alloisoleucine with a quantitative yield.

[0294] Step 5: To a solution of N-((R)-2-((S)-2-((R)-2-(((2E,5S,6S,7S

2-niethylpropanoy3)oxy)-2,6,8 rimethyl~5-((methy3thio)m.ethoxy)deca

me1hy]pentanamido)-N-methylpropanam^

L-aUoisoleucine (0.195 g, 0.206 mmol) in 0.000996 M solution of 1 : 10 (DMF 20 mL:DCM 180 mL) was added l-hydroxy-7-azabenzotriazole (0.281 g, 2.067 mmol) and EDC (0.396 g, 2.067 mmol) and stirred for 24 h, the solvent was removed and diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCOs aq and brine, dried over Na ₂ S0 ₄ , concentrated in vacuo. The residue was purified with reverse phase flash to afford (4- (((2R,5S,8R,14S,20S,21S,22S,E)-20~(^

5,7, 10,17,17,21,25 ieptamethyl-22~((m

dioxa-4,7,10, 13, 16-pentaazac}'clohexacos-24-en-8-yl)methyl)benzonitrile (0.1081 g, 56.5%, 0.117 mmol).

Compound 27

[0295] Step 6: To a stirring solution of 4-(((2R,5S,8R, 14S,20S,21S,22S,E)-20-((E)-but-2- en-2-yl)- 14-((R)-sec-butyl)-2-isobutyl-5 ,7, 10, 17, 17,21 ,25-heptamethyl-22- ((methylthio)methoxy)-3,6,9, 12, 15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13,16- pentaazacyclohexacos-24-en-8-yl)methyl)benzonitrile (0.108 g, 0.116 mmol) in THF (2.426 g, 2.757 mL, 33.649 mmol) and water (0,690 g, 0,690 mL, 38,323 mmol) was added 2,6- lutidine (0.250 g, 0.272 mL, 2.336 mmol) and silver nitrate (0.793 g, 4.673 mmol). The reaction was then heated to 60 °C for 1.5-2 hours. After the completion of the reaction, The mixture was cooled to rt and diluted with EtOAc and 1 N HQ, then filtered over celite. The aqueous phase was extracted with EtOAc and the organic phase was washed with aqueous NaHCC and brine, dried over NaS0 ₄ and concentrated and in vacuo. The residue was purified with rp-flash to afford 4-(((2R,5S,8R,14S,20S,21S,22S,E)-20-((E)-but-2-en-2-yI)-14- ((R)-sec-butyl)-22-hydroxy-2-isobutyl-5 ,7, 10,17,17,21 ,25 -heptamethyl-3 ,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-8-yl)methyl)benzonitrile (0.03 g, 29.7%, 0.035 mmol).

Example 29

Intermediate 4

[0296] To a stirring solution of 4-(((2R,5S,8R,14S,20S,21S,22S,E)-20-((E)-but-2-€n-2- yl)- 14-((R)-sec-but l)-22 -hydroxy -2-isobutyl-5,7, 10, 17, 17,21 ,25-heptamethyl- 3,6,9, 12, 15,18,26-heptaoxo-l, 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-8- yl) _m ethyl)benzonitrile (0.005 g, 6.820 μηιοί) in DCM (300 u! .) was added DMAP (0.833 mg, 6.820 μηιοϊ), DIPEA (0.044 g, 0.059 mL, 0.341 mmol) and 4-nitrophenyl

carbonochlondate (0.013 g, 0.068 mrnoi) and stirred for 6 h. After the completion of the reaction, the solution was diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCOs (aq) and brine, dried ove NaiSC , concentrated in vacuo. The residue was purified with reverse phase flash to afford (2R,5S,8R, 14S,20S,21 S,22S,E)-2Q-((E)-hut-2- en-2-}4)-14-((R)-sec-butyl)-8-(4-cyanobenzyl)-2-isobutyl-5,7 ,10, 17, 17,21,25-heptamethyl- 3,6,9,12, 15,18,26-heptaoxo- 1 , 1 -dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (4- nitrophenyl) carbonate (0.0025 g, 35.6%, 6.82 μπιοΐ).

Example 30

Synthesis of Compound 28

[0297] To a stirring solution of (2R,5S,8R 4S,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)- 14-((R)-sec-butyl)-8-(4 -cyanobenzyl)-2-i sobuty 1-5 ,7, 10,17, 17,21,25 -heptamethyl- 3,6,9,12,15,18,26-heptaoxo- 1 , 19-dioxa-4,7,l 0,13, 16-pentaazacyclohexacos-24-en-22-yl (4- nitrophenyl) carbonate (0.0012 g, 1.165 μηιοΐ) in DCM (200 μΕ) was added Nl , N6- dimethylhexane-l,6-diamine (1.680 mg, 0.011 mmol) and DIPEA (1.505 mg, 2.034 μΕ, 0.01 1 rnmol). The reaction was stirred for 2 h, and the solvent was removed. The residue was purified with reverse phase flash to afford ((2R,5S,8 , 14S,20S,21S,22S,E)-20-((E)-but-2-en- 2-yl)- 14-((R)-sec-butyl)-8-(4-cyanobenzyl)-2-isobutyl-5,7, 10, 1 7, 17,21 ,25-heptamethyl- 3,6,9, 12, 15, 18,26-heptaoxo-l , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl methyl(6-(methylamino)hexyl)carbamate (0.001 1 g, 91 %, 1.062 μηιοΐ). Observed HRMS (ESI) m/z: 1035.6547 [M+Hf ,

Example 31

Synthesis of Compound 29

|0298] To a stirring solution of (2R,5S,8R, 14S,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)- 14-((R)-sec-butyl)-8-(4 -cyanobenzyl)-2-i sobuty 1-5 ,7, 10, 17, 17,21,25 -heptamethyl- 3,6,9, 12, 15, 18,26-heptaoxo-1 , 19-dioxa-4,7, l 0, 13, 16-pentaazacyclohexacos-24-en-22-y] (4- nitrophenyl) carbonate (0.002 g, 2.426 μιηοΐ) in DCM ( (0.263 g, 200 μΕ, 3.108 rnmol) was added 5-aminopentan-l-ol (2.503 mg, 0.024 mmol) and D1PEA (3.136 mg, 4.238 μΕ, 0.024 mmol)and stirred for 2 h. The solvent was removed and the residue was purified with reverse phase to afford (2R,5S,8R, 14S,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec-buty1)-8- (4-cyanobenzyl)-2-isobutyl-5,7, 10, 17, 17,21 ,25-heptamethyl-3,6 ₅ 9, 12, 15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-y 1 (5 -hydroxypentyl)carbamate (0.00107 g, 44 ,3%, 2.427 μηιοΐ). Observed HRMS (ESI) m/z: 994.58 [M+H] ⁺ .

Example 32,

Synthesis of Compound

10299] Step 1 : To a reaction chamber equipped with stir bar (2E,5S,6S,7S,%E)-7-((tert-

g, 0.127 mmol) and 2,2,2-trichloroethyl methyl -D-leucinate (0.42 g, 0.153 mmol) were charged and dissolved in methylene chloride (1.27 mL, 0.1 M) followed by N, N- diisopropylethylamine (DIPEA, 0.044 mL, 0.254 mmol) addition. The reaction was cooled to 0 °C using an ice bath followed by 1 -Hydroxy- 7-azabenzotriazole (HO At, 0.022 g, 0.140 mmol) and 1 -Ethyl-3-(3KlimeihyIaminopropyI)carbodiimide (EDC, 0.027 g, 0.140 mmol) addition. The reaction was stir at 0 °C for 10 min before allowed warmed up to room- temperature for additional 2 hours and was monitored via LC/MS. Upon completion, the reaction was diluted with methylene chloride and washed with 1 Ν Hydrochloric acid solution, saturated NaHCOj solution, and brine. Combined organic layers were dried over NaiSQ/}, filtered through a filter paper, and the filtrate was concentrated in vacuo. The crude material was purified via silica gel chromatography using hexanes and ethyl acetate as eluents to yield 2,2,2-trichloroethyl N-((2E,5i.S',6 _t S ^' ,75',8£)-7-((ieri-butyldimethylsilyl)oxy)- 2,6,8-trimemyl-5-((memylthio)memox}^deca-2,8-dienoyl)-N-meth yl-Z ⁾ -leucinate as white solid (0.036 g, 0.-53 mmol, 42%).

[0300] Step 2: To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl N-

((2E,55,6S,75 8£)~7~((fer^

((methylthio)methoxy)deca-2,8-dienoyl)-N-methy]- _J D-leucinate (0.036 g, 0,053 mmol) and zinc dust (6-9 mesh, 0.976 g, 14.93 mmol) were charged. The material was suspended in tetrahydrofuran (THF, 1.06 mL, 0.25 M) followed by 1.06 mL of 1 M NH ₄ OAc solution addition. The reaction was allowed to stir at room-temperature for 8h and monitored via LC/MS. Upon completion, the reaction was diluted with ethyl acetate and 1 N Hydrochloric acid solution and the reaction was filtered through a pad of ceiite. Filtrate was washed with 1 N Hydrochloric acid solution, saturated NaHCOs solution, and brine. Combined organic layers were dried over Na2S0 ₄ , filtered and concentrated in vacuo. Crude material was purified via reverse-phase C-18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1 % formic acid) as eluents to yield N-((2E,5S,6SJS,%E)-l-((tert- butyldimethylsilyl)oxy)-2,6,8-trimethyl-5-((memyl^

methyl-Z eucine as white solid (0.011 g, 0.020 mmol, 37.9%)

[0301] Step 3: To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl N-((ii)-2- ((5)-2-amino-N-memylpropanamido)-3-(4-chlorophenyl)propaiioy l)-N-methylglycyl-Z- alloisoleucinate (0.036 g, 0.061 mmol) d N-{{2E,5S S,lS$E)-l-{{tert- butyldimethylsilyl)oxy)-2,6,8-trimethyl-5-((mem^

methyl-ZMeucine (0.011 g, 0.020 mmol) were charged and dissolved in 0.202 mL methylene chloride. The reaction was cooled to 0 °C using an ice bath followed by N, N- diisopropylethylamine (DIPEA, 0.014 mL, 0.081 mmol) addition then l -Hydroxy-7- azabenzotriazole (HOAt, 0.0094 g, 0.061 rnmol) and l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC, 0.012 g, 0.061 mmol) addition. The reaction was stir at 0 °C for 4 h before warmed up to room-temperature for additional 8h stirring and monitored via LC/MS. Upon completion, the reaction was diluted with methylene chloride and washed with 1 Ν Hydrochloric acid solution, saturated NaHCOj solution, and brine. Combined organic layers were dried over Na ₂ S0 ₄ , filtered, and concentrated in vacuo. The crude material was purified via silica gel chromatography using hexanes and ethyl acetate as eluents to yield 2,2,2-trichloroethyl N {R)-2 {S)-2^{R -2 {2E,5S,6S S,%E)-l {te ^ butyldimeihy4silyl)oxy)-A¾6,8-tetr^

methylpentanamido)-N-metiiylpropanamido)-3-(4-cM

Z-alloisoleucinate (0.015 g, 0.013 mmol, yield: 66%) as white solid.

[0302] Step 4: To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl N-((R)-2- ((¾-2-((R)-2-((2£,5S,6S S,8£)-7-^

((methylthio)me1hoxy)deca-2,8-dienamido)-4-m

3"(4-chlorophenyl)propaiioyl)-iV-methylglycyl-Z.-alloisoleuc inate (0.014 g, 0.012 mmol) was charged and dissolved in a solution of 500 μΐ, of THP/HF-pyridine/pyridine mixture (ratio: 4 to 1 to 1). The reaction chamber was sealed with Teflon and allowed to stir at 65 °C for 8 h and monitored via thin-layer-chromatography. Upon completion, the reaction was diluted with ethyl acetate followed by slow addition of saturated NaHCCh solution until gas evolution completed. The reaction was washed with saturated NaHCCh solution and brine. Combined organic layers were washed with IN Hydrochloric acid solution then brine.

Combined organic layers were dried over Na2S0 , filtered and concentrated in vacuo. Tire crude material was purified via silica gel chromatography using hexanes and ethyl acetate as eluents to yield 2,2,2-trichloroethyl N-((R)-3-(4-cnlorophenyl)-2-((5)-2-((/?)-2- ((2ii,55,6i?,75,8£)-7 iydroxy-N,2,6,8 etramethyl-5-((methy

dienamido)-4-methylpenlanamido)-A ^" -methylpropaiianiido) propanoyl)-N-methylglycyl-i- alioisoleucinate as white solid (0.005 g, 4.94 μτηοΐ, yield: 39.7%).

5] Step 5: To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl N-((i?)-3- (4^Ataro henyl)-2K^ ((methylthio)me1hox} deca-2,8-dienamido)-4-methylpentanamido)-N- methylpropanamido)propaiioyl)-N-methylglycyl-Z-alloisoleucin ate (0.01 g, 0.01 mmol) and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-methylpropan oic acid (0.0064 g, 0,02 mmol) were charged and dissolved in 0.099 mL methylene chloride followed by 4- dimethylaminopyridine (DMAP, 0.00133 g, 0.011 mmol). The reaction was cooled to 0 °C followed by i-Ethyi.-3-(3-dimethy1aminopropy])carbodiimide (EDC, 0.016 g, 0.081 mmol) addition. The reaction was kept at 0 °C and monitored via Thin-Layer-Chromatography for 4 h before it reached completion. Upon completion, the reaction was diluted with ethyl acetate and washed with 1 Ν Hydrochloric acid solution, saturated NaHCC solution and brine. Combined organic layers were dried over Na ₂ S04, filtered, and concentrated in vacuo. Grade material was purified via silica gel chromatography using hexanes and ethyl acetate as eluents to yield 2,2,2-trichloroethyl N-{{R)-2-{{S)-2-{(%S,9S, 10S, 16i^ii)-8-((£)-but-2-en-2- yl)- 1 -(9H-fluoren-9-yl)- 16-isobutyl-5 ,5,9,13,15 -pentamethyl- 10-((methylthio)methoxy)- 3,6, 14-trioxo-2,7-dioxa-4,15-diaza]ieptadec-12-en-17-amido)-N-me thylpropanamido)-3-(4- chkirophenyl)propanoyl)-A-meihylglycyl-i-alloisoleucinaie as white solid (0.002 g, 1.592 μιηοΐ, yield: 16.1%).

[0304] Step 6: To a reaction chamber equipped with stir bar, 2,2,2-trichloroethyl N-((R)- 2-((5)-2-((85 ^, ,9S, 1 OS, 16ii^ ^" )-8-((£)-but-2-en-2-yl)-l -(9H-fluoren-9-yl)- 16-isobutyl- 5,5,9, 13, 15 -pentamethyl - 10-((methylthio)methoxy)-3,6, 14-trioxo-2,7-dioxa-4, 15- diazaheptadec-12-en-17-amido)-N-methylpropan

methylglycyl- -alloisoleucinate (0.002 g, 1.592 μηιοΐ) and zinc dust (6-9 mesh, 0.031 g, 0.478 mmol) were charged and suspended in 400 μΙ_, THF and 400 μΐ, of 1 M NH ₄ OAc. The reaction was allowed to stir at room-temperature for 3 h and monitored via LC/MS. Upon completion, the reaction diluted wdth ethyl acetate and 1 N Hydrochloric acid then filtered through a pad of celite. The filtrate was washed with 1 N Hydrochloric acid, saturated NaHCCb solution, and brine. Combined organic layers were dried over a ₂ S04, filtered, and the filtrate was concentrated in vacuo. The crude material was purified via reverse-phase C- 18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluent to yield N-({R)-2-((S)-2-{(ZS,9S, IQS, 16/^.5)-8-((£)-but-2-en-2-yl)-l-(9H-fluoien-9-yl)- 16-isobutyl -5 ,5 ,9, 13, 15 -pentamethyl- 0-((methylthio)methoxy)-3 ,6, 14-tri oxo~2,7~di oxa-4, 15 - diazaheptadec- 12-en-17-amido)-N-m

methylglycyl- -alloisoleucine (0.0008 g, 0.673 μηιοΐ, yield: 42.3%).

[0305] Step 7: To a reaction chamber e uipped with stir bar N-((R)-2-((S)-2-((R)-2- ((2.¾55,65 75 8.¾ 7-((2-(((^

methylpropanoyl)oxy)-2,6,8-trimethyl-5-((methylthio)met oxy)deca-2,8-dienam

methylpentanamido)-N-methylpropanamido)-3-(4^

-alloisoleucine (0.00019 g, 1.592 μηιοΐ) was charged and dissolved in 0.159 mL of a diethylamine/acetonitrile solution mixture (ratio: 1 to 9). The reaction was allowed to stir at room-temperature for 30 niin and monitored via LC/MS. Upon completion the reaction was concentrated using a Argon stream then purified via reverse-phase C-18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluent to yield N-({R)~2- ((S)-2-((i?)-2-((2E,5S,6S 7S 8£ 7-((2-am^

((methylthio)methoxy)deca-2,8~dienam

3-(4-ch]orophenyl)propanoyl)-N-methylglycy]-L-alloisoleucine as an amorphous solid. The material was free-based by dissolving sample in saturated NaHCOj solution and extracted with a solution of methylene chloride/methanol mixture (ratio: 9 to 1). Combined organic layers were dried over Na ₂ S0 ₄ , filtered, and the filtrate was concentrated in vacuo. The crude material was purified via reverse-phase C-18 column using water (with 0.1 % formic acid) and acetonitrile (with 0.1% formic acid) as eluenis to yield .ν-((Λ)-2-((5)-2-((Λ)-2- ((2E,5S,6S,75',8E)-7-((2-amino-2-memylpropanoy

((methylthio)methoxy)deca-2,8~diena.m^

3-(4-chlorophemd)propanoyl)-N-methylglycyl-i-alloisoleucine as free base as an amorphous solid (0.00084 g, 0.8 μηιοϊ, yield: 50.5%).

[0306] Step 8: To a reaction chamber equipped with stir bar N-(( i)-2-((,S)-2-(( i)-2- ((2E,5i.S',65,75',8^-7-((2-amino-2-methylpropaiioyl)oxy)-N,2 ,6,8-tetramethyl-5- ((methylmio)memox} deca-2,8-dienamido)-4-methylpentanamido)-N-memylpropanamido) - 3-(4-chlorophenyl)propanoyl)-N-methylglycyl-Z-alloisoleucine (0.002 g, 2.18 μιηοί) was charged and dissolved in 2.18 mL methylene chloride and cooled to 0 °C using an ice bath. 1- Hydroxy-7-azabenzotriazole (HO At, 0.0034 g, 0.022 mmol) was added to the reaction mixture followed by I -Ethyl-3-{3~dimethy]arainoprop>i)carbodiimide (EDC, 0,0042 g, 0.022 mmol) addition. The reaction was allowed to stir at 0 °C for 4h before warm up to room- temperature for additional 12h stirring and was monitored via LC/MS. Upon completion, the reaction was diluted with ethyl acetate and washed with 1 M citric acid solution, saturated NaHCOs, and brine. The crude material was purified via reverse-phase C- 18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluent to yield (6S, 12R, 155, 18i ,24S,25S,26S,£)-26-((£)-but-2-en-2-yl)-6-((R)-5ec-butyl)- 12-(4- chl orobenzy])- 18-i sohuty 1-3 ,3 , 10, 13, 15, 1 ,21 ,25-octamethy] -24-((methylthio)methoxy)- 1 - oxa-4,7, 10, 13, 16, 19-hexaazacyclohexacos-21-ene-2,5,8, l 1, 14, 17,20-heptaone as an amorphous solid (0.0008 g, 0.844 μ,ηιοΐ, yield: 38,8%),

[0307] Step 9: To a reaction chamber equipped with stir bar

(65 ^* , 12R, 155, 18R,245',255 ^, ,265 ^, ,£)-26-((£)-but-2-en-2-y l)-6-((R)-rec-butyl)- 12-(4- chlorobenzyl)- 18-isobuty 1-3 ,3 , 10, 13 , 15 , 19,21 ,25 -octamethyl~24-((metliyithio)metlioxy)- 1 - oxa-4,7, 10, 13, 16, 19-hexaazacyclohexacos-21 -ene-2,5,8, l l, 14,r7,2 -heptaone (0.0008 g, 0.844 μιηοΐ) was charged and dissolved in 0.350 mL of water/THF mixture (ratio: 3 to 2 ) followed by Silver nitrate (AgN0 ₃ , 0.0057 g, 0.034 mmol), 2,6-lutidine ( 1.97 μΕ, 0.017 mmol). The reaction was sealed with Teflon and heated to 65 °C for 2h and monitored via LC/MS. Upon completion, the reaction was diluted with ethyl acetate and 1 N hydrochloric acid solution and filtered through a pad of celite. The filtrate was washed with 1 N

Hydrochloric acid solution, saturated NaHCQi, and brine. Combined organic layers were dried over NajSO,., filtered, and the filtrate was concentrated in vacuo. The crude material was purified via reverse phase C-18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluents to yield (6S 2R, l 5SA ,2AS,25S,26S,E)-26- ((£)-but-2-en-2-yl)-6-((jR)-.yec-butyl)- 12-(4-chlorobenzyl)-24-hydroxy- 18-isobutyl- 3 ,3 , 10, 13 , 15 , 19,21 ,25 -octamethy 1 - 1 -oxa-4,7, 10, 13, 16, 19-hexaazacyciohexacos-21 -ene- 2,5,8, 1 1 , 14, 17,20-heptaone as a white solid (0.0002 g, 0.237 μιηοΐ, yield: 28.0 %). Observed HRMS (ESI) m/z: 887.5075 j M 1 ! | .

Example 33

Synthesis of Compound 31

[0308] Step 1 : To a reaction chamber equipped with stir bar (2E,5S,6S,7S,8E)-7-((tert- butyldimetnylsilyl)oxy)-2,6,8-trim acid (0.055 g, 0.132 mmoi) and 2,2,2 -trichloroethyl D-leucinate (0.104 g, 0.396 mmoi) were charged and dissolved in methylene chloride (1.32 mL, 0.1 M) followed by N, N-diisopropylethylamine (DIPEA, 0.092 mL, 0.528 mmoi) addition. The reaction was cooled to 0°C using an ice bath followed by ( 1 - [Bis(dimethylamino)methylene j - 1H- 1 ,2, 3 -tnazolo [4,5 -b jpy ridmium 3 -oxid hexafluorophosphate) (HATU, 0.151 g, 0.396 mmoi) addition. The reaction was stir at 0 °C for 10 min before warmed up to room-temperature for additional 2 h stirring and was monitored via LC/MS. Upon completion, the reaction was diluted with methylene chloride and washed with 1 M citric acid solution, saturated NaHCOs solution, and brine. Combined organic layers were dried over Na. ₂ S0 ₄ , filtered, and the filtrate was concentrated in vacuo. The crude material was purified via silica gel chromatography using hexanes and ethyl acetate as eiuents to yield 2,2,2-trichioroethyl ((2E,5S,6S,7S,%E)-7-((tert- butyldimethylsilyl)oxy)-2,6,8-trimethyl-5-(^

leucinate as white solid (0.069 g, 0.105 mmoi, 79%).

[0309] Step 2: To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl

((2£,5S,6S,75,8i¾-7-((^bulyldime lsilyl)oxy)-2,6,8

((methylthio)methoxy)deca-2,8-dienoyl)-D-leucinate (0.08 g, 0.121 mmoi) and zinc dust (6-9 mesh, 2.215 g, 33.90 mmoi) were charged. The materials were suspended in tetrahydrofuran (THF, 1.20 mL, 0.2 M) followed by 1.20 mL of 1 M NH ₄ OAc solution addition. The reaction was allowed to stir at room-temperature for 2 h and monitored via LC/MS. Upon completion, the reaction was diluted with ethy l acetate and I M citric acid solution then filtered through a pad of ceiite. Filtrate was washed with 1 M citric acid solution, saturated NaHCO solution, and brine. Combined organic layers were dried over NaaSC j, filtered and the filtrate was concentrated in vacuo. Crude material was purified via reverse-phase C-18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluents to yield ((2E,5S,6S,7S 8£)-7-((ie^butyldm^

((methylthio)methoxy)deca-2,8-dienoyl)-D-]eucine as white solid (0.046 g, 0.087 mmol, 71 .8%).

[0310] Step 3 : To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl N-((R)~2- ((<S)-2-amino-N-methylpropanamid^

alioisoleucinate (0.078 g, 0.130 mmol) and ((2E,5S,6S,7S,SE)-7-((terl- butyidimethyisilyl)oxy)-2,6,8-tnniethy

(0.046 g, 0.087 mmol) were charged and dissolved in 0.347 niL methylene chloride. The reaction was cooled to 0 °C using an ice bath followed by N, N-diisopropylethylamine (DIPEA, 0.061 mL, 0.347 mmol) and (l -fBis(dime lainino)methylene]-l H-l ,2,3- tfiazolo 4,5-b]pyridinkim 3-oxid hexafluofophosphate) (HATU, 0.099 g, 0.260 mmol) addition. The reaction was stir at 0 °C for 10 min before warmed up to room-temperature for additional 4h stirring and was monitored via LC/MS. Upon completion, the reaction was diluted with methylene chloride and washed with 1 M citric acid solution, saturated NaHCOj solution, and brine. Combined organic layers were dried over Na ₂ S0 ₄ , filtered, and the filtrate was concentrated in vacuo. Tire crude material was purified via silica gel

chromatography using hexanes and ethyl acetate as eluents to yield 2,2,2-trichloroethyl N- ((/^2-((Λ>2-((^)-2-((2£ 5 ^

((methylthio)methoxy)deca-2,8~dienam^

3-(4-chlorophemd)propanoyl)-N-methylglycyl-L -alioisoleucinate as white solid (0.09 g, 0.081 mmol, yield: 93%).

[0311] Step 4: To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl J\ ^T -((R)~2- (( >2-(( )-2-((2£ 5Λ 6^

((methylthio)methoxy)deca-2,8-dienamido)-4-methylpentanamido )-N^

3-(4-chlorophenyl)propanoyl)-N-methylglycyl-Z-alloisoleuc inate (0.09 g, 0.08 mmol) was charged and dissolved in a solution of 810 μί, of THF/HF -pyridine/pyridine mixture (ratio: 4 to 1 to 1). The reaction chamber was sealed with Teflon and allowed to stir at 65 °C for 8h and monitored via thin-lay er-chromatography. Upon completion, the reaction was diluted with ethyl acetate followed by slow addition of saturated NaHCC^ solution until gas evolution was completed. The reaction was washed with saturated NaHCOs solution and brine. Combined organic layers were dried over NajSO,., filtered and the filtrate was concentrated in vacuo. The crude material was purified via silica gel chromatography using hexanes and ethyl acetate as eluents to yield 2,2,2-trichloroethyl N-(( ?)-3-(4-chlorophenyl)- 2-((S)-2~((/?)~2~((2E,5S,6i?,7^

2,8-dienamido)-4-methylpentanamido)-N-m

alloisoleucinate as white solid (0.027 g, 0.027 mmol, yield: 33.4%).

[0312] Step 5 : To a reaction chamber equipped with stir bar 2,2,2-trichloroethyl N-((R)-3- (4-chlorophenyl)-2-((S)-2-^

((methyitliio)memoxy)deca-2,8-dienamido)-4-methylpentanainid o)-N- methylpropanamido)propanoyl)-N-methylglycyl-i-alloisoleucina te (0.027 g, 0.027 mmol) and 2-((((9H-fluoren-9-yl)metlioxy)carbonyl)amino)-2-methylpropa noic acid (0.026 g, 0.081 mmol) were charged and dissolved in 0, 135 mL methylene chloride followed by 4- dimethylaminopyridine (DMAP, 0.00331 g, 0.027 mmol). The reaction was cooled to 0 °C followed by l~Ethyl-3-(3"dimethyiaminopropyl)carbodiimide (EDC, 0.016 g, 0.081 mmol) addition. The reaction was kept at 0 °C and monitored via Thin-Layer-Chromatography for 6h before it reached completion. Upon completion, the reaction was diluted with ethyl acetate and washed with 1 M citric acid solution and brine. Combined organic layers were dried over Na^SCu, filtered, and concentrated in vacuo. Crude material was purified via silica gel chromatography using hexanes and ethyl acetate as eluents to yield 2,2,2-trichloroethyl N- ((/?)-2-((S)-2-((K)-2-(^

2-methyipropanoyi)Qxy)-2,6,8-trimethy

methylpentanamido)-N-memylpropanamido)-3-(4-ch^

Z,-alloisoleucinate (0.02 g, 0.027 μιηοΐ, yield: 56.6%) as white solid.

[0313] Step 6: To a reaction chamber equipped with stir bar, 2,2,2-trichloroethyl N~((R)- 2-((S)-2-(iR)-2-(i2E,5SfiS

me1hy]propanoyl)oxy)-2,6,8-trimethyl-5-((methyl1hio)metii oxy)deca-2,8-dienamido)-4- memylpentanamido)-N-methylpropanamido)-3-(4-chlorophenyl)pro panoyl)-N-methylglycyl- -alloisoleucinate (0.014 g, 0.0107 mmol) and zinc dust (6-9 mesh, 0.196 g, 3.00 mmol) were charged and suspended in 880 μΤ THF and 880 μΤ of 1 M NR-jOAc. The reaction was allowed to stir at room-temperature for 3h and monitored via LC/MS. Upon completion, the reaction was diiuted with ethyl acetate and 1 M citric acid then filtered through a pad of ceiite. The filtrate was washed with 1 M citric acid and brine. Combined organic layers were dried over a ₂ S04, filtered, and the filtrate was concentrated in vacuo. The crude material was purified via reverse-phase C-18 column using water (with 0.1 % formic acid) and acetonitrile (with 0.1 % formic acid) as eluents to yield N-((R)~2,-((S)~2~((R)~2-

((2£,55,65,75,8 ¾-7-((2-((((9H-fluoren-9-yl)methoxy)<ar ony

memylpropanoyl)oxy)-2,6,8-trimemyl-5-((methy

methylpentanamido)-N-methylpropanami^^

L-alloiSoleucine as white solid (0.01 g, 8.52 μτηοΐ, yield: 79%).

[0314] Step 7: To a reaction chamber equipped with stir bar N-((i?)-2-((5)-2-((i?)-2- U2A;..\V.6.S.7.S.8^

memylpropanoyl)oxy)-2,6,8-trimemyl-5-((methy

methylpentanamido)-N-methylpropanam ^

L-alloisoleucine (0.0072 g, 6.13 μιηοΐ) was charged and dissolved in 0.123 mL of a diethyiamme/CH ₃ CN solution mixture (ratio: 1 to 9). The reaction was allowed to stir at room-temperature for 30 min and monitored via LC/MS. Upon completion the reaction was concentrated using a Argon stream then purified via reverse-phase C-18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluents to yield N-((R)-2- ((,5)~2-((R)-2-((2ii,5^

((methylthio)meftoxy)deca-2,8-dienamido

3-(4-chlorophenyl)propanoyl)-N-meth}'lglycyl-L-alloisoleucin e as amorphous solid. The material was free-based by dissolving sample in saturated NaHCC solution and extracted with a solution of methylene chloride/methanol mixture (ratio: 9 to 1). Combined organic layers were dried over NajSO,], filtered, and the filtrate wsa concentrated in vacuo. The crude material was purified via reverse-phase C-18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluent to yield free base as amorphous solid (0.00584 g, 6.13 μηιοΐ, yield: 68.5%).

[0315] Step 8: To a reaction chamber equipped with stir bar N-((R)-2-((S)-2-((R)-2-

((2£,55',65y7S,8fi)-7-((2-amino-2-mem^

((methylthio)methoxy)deca-2,8-die^

3-(4-ch]orophenyl)propanoyl)-N-methylglycy]-L-alloisoleucine (0,00584 g, 6. 14 μπιοΐ) was charged and dissolved in 6.14 mL methylene chloride and cooled to 0 °C using an ice bath. 1 - Hydroxy-7-azabenzotriazole (HOAt, 9.46 mg, 0.061 mraol) was added to the reaction mixture followed by l-Ethyl"3-(3-dimeihylani!nopropyl)carbodiiinide (EDC, 0.012 g, 0.061 mmol) addition. The reaction was allowed to stir at 0 °C for 6h before warm up to room- temperature for additional 12h stirring and was monitored via LC/MS. Upon completion, the reaction was diluted with ethyl acetate and washed with 1 M citric acid solution, saturated NaHCCh, and brine. The crude material was purified via reverse-phase C- 18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluents to yield (65, 12R, 15S, 18R,24S ₅ 25S ₅ 26S^-26-((i5)-but-2-en-2-yl)-6-((R)-iec-butyl)- 12-(4- chlorobenzyl)- 18-isobutyl-3,3, 10, 13, 15,21 ,25-heptamethyl-24-((methylthio)methoxy )- 1 -oxa- 4,7, 10, 13, 16, 19-hexaazacyclohexacos-21-ene-2,5, 8, 1 1, 14, 17,20-heptaone as an amorphous solid (0,0038 g, 6, 14 μιηοΐ, yield: 66.3%).

[0316] Step 9: To a reaction chamber equipped with stir bar

(65; 12R, 155, 18Λ,245,255,265^£)-26-((£)-5υΐ-2-€η-2^1)-6-((/?)-.?� �^Ηΐνί)- 12-(4- chlorobenzyl)-I 8-isobutyl-3,3, 10,13 ,15,21,25-heptamethyl-24-((rnethylthio)methoxy)-l-oxa- 4,7, 10,13 ,16,19-hexaazacyclohexacos-21-ene-2,5.8,l 1 , 14, 17,20-heptaone (0.0038 g, 4.07 μηιοΐ) was charged and dissolved in 0.122 mL of water/THF mixture (ratio: 1 to 4 } followed by Silver nitrate (AgN0 ₃ , 0.028 g, 0.163 mmol), 2,6-lutidine (9.48 μΐ., 0.081 mmol). The reaction was sealed with Teflon and heated to 65 °C for 2 h and monitored via LC/MS. Upon completion, the reaction was diluted with 0.05 N hydrochloric acid solution and filtered through a pad of celite. The filtrate was washed with 1 M citric acid solution, saturated NaHCOn, and brine. Combined organic layers were dried over NaiSO,], filtered, the filtrate was concentrated in vacuo. The erode material was purified via reverse phase C-18 column using water (with 0.1% formic acid) and acetonitrile (with 0.1% formic acid) as eluents to yield (65; i2/<155 i8 ?,245 255 265 ^^

chlorobenzyl)~24"hydroxy" 18-isobutyi-3 ,3, 10,13,15,21 ,25 -heptame thy 1- 1 -oxa-

4, 7, 10,13, 16,19-hexaazacyclohexacos-21~ene-2,5, 8,11, 14, 17,20-heptaone as a white solid

(0.002 g, 4.07 Limol, yield: 56.3%). Observed HRMS (ESI) m/z: 873.4887 [M+Hf .

Example 34

Synthesis of Compound

[0317] Step 1 : To a stirring -78 °C solution of t-BuOK (3.4 g, 30.3 mmol) in THF (9.5 mL) was added cis-2-butene (3.39 g, 37.1783 mmol) followed by the addition of n-BuLi (15.8 mL, 1.915 M in hexanes). The resultant yellow suspension was stirred at -78 °C for 30 mins, subsequently at -45 °C for 60 min and cooled to -78°C again. (+)-B- methoxydiisopinocamphenyl borane (11.52 g, 36.41 mmol) in THF (21.1615mL) was added slowly. After the addition was completed, the mixture was stirred at -78 °C for 1 hour and boron tifluoride etherate (10.67 mL, 48 %) was added dropwise. Immediately afterwards, a solution of isobutyraldehyde (7.178 niL, 74 mmol) was added dropwise. The mixture was kept at -78°C for 3 h. The reaction was quenched with saturated NaOAc and 30% H ₂ O ₂ . The resulting solution was stin-ed at -78° C for 30 mins and warmed to room temperature over 12 h. The aqueous layer was extracted with Et ₂ 0, and the combined organic layers were dried over MgS0 ₄ , and concentrated under reduced pressure yielding a colorless liquid. The liquid was purified by flash chromatography (EtOAc/n-hexane) and the colorless alcohol, A2 was obtained as a colorless liquid. (2.5g, 58%, 17.58mmol). (Ref. Organic Letters, 10(15); 3223- 3226; 2008)

[0318] Step 2: To a stirring solution of (35 ^, ,45',55)-3,5"dimetliylhept-l-en~4-ol (0.9 g, 6.33 mmo3) in DCM (31.6 m L. 6.33 mmol, 0.2 M) was added triethylamine (0.768 g, 1.058 mL. 7.592 mmol) followed by methanesulfonyl chloride (0,869 g, 0,591 mL, 7.592 mmol) at 0 °C, The reaction was allowed to stir at 0 °C for 30 mm before warming to RT. After stirring for 1 h, the solvent was removed and the residue was purified with flash to afford (3S,4S,5S}-3,5- dimethylhept-l-en-4-yl methanesulfonate (0.5032 g, 36.1 %, 2.284 mmol).

[0319] Step 3 : To a stirring solution of (35',45',5iS)-3,5-dim.ethylhept-l-en-4-yl methanesulfonate ( 1.424 g, 6.46 mmol) in DMF (6,31 mL, 6,46 mmol) was added sodium azide (1.260 g, 19.389 mmol) and heated at 65 °C overnight. After stirring overnight, the reaction was diluted with saturated sodium chloride and ethyl acetate. The organic phase was washed with brine, dried over Na ₂ S0 ₄ and concentrated in vacuo. The residue was purified with flash to afford (3S,4 ?,55)-4-azido-3,5-dimethyIhept-l-ene (0.451 g, 42 %, 2.7 mmol).

[0320] Step 4: To a stirring solution of Dicarbonyiacetylacetonato rhodium (I) (2.071 mg, 7.998 ii rno! ) in THF (3.058 g, 3.476 mL, 0.597 mmol) was added Biphephos (14 mg, 0,24 μτηοί) and (3S,4R,5S)-4-azido-3,5-dimethylhept-l-ene (0.1 g, 0.597 rnmol) in THF (5 rnL). The reaction was vacumn flushed with carbon/monoxide/hydrogen gas and stirred for 48 hours. After 48 hours, workup/purifi cation was not necessary and carried forward to aBH ₄ reaction. (Ref: Adv. Synth. Catal. 2005, 347, 1488-1494)

[0321] Step 5 : To a stirring solution of 5-azido-4,6-dimethyloctanal (0.714 g, 3.619 mmol) in Me OH (2.3.87 mL, 590 mmol) was added NaBH ₄ (0.684 g, 18.095 rnmol) at -40 °C and stirred for 2 h. The solvent was removed and the residue was purified with flash to afford (4S,5R,6S)-5-azido-4,6-dimethyioctan-l-ol (0.42 g, 58.2 %, 2.107 mmol).

[0322] Step 6: To a stirring solution of palladium on Carbon (0.027 g, 0.251 mmol) in MeOH (0.5 mL, 12.36 mmol) was added (4S,5R,6S)-5-azido-4,6-dimethyloctan-l-ol in

MeOH (0.5 mL, 12,36 mmol). The reaction was stirred for 30 min and filtered through a pad of celite using methanol to afford (4S,5R,6S)-5-amino-4,6-dimethyloctan-l-ol (0.0487 g, 88%, 0.281 mmol)

[0323] Step 7: To a stirring solution of (45',5 ?,65 ^' )-5-amino-4,6-dimethyloctan~l -ol (0.0487 g, 0.281 mmol) in DCM (0.7 mL, 10.88 mmol) was added (((9H-fluoren-9- yl)methoxy)carbonyl)-Z ⁾ -alanine (0.262 g, 0.843 mmol), DIPEA (0.145 g, 0.196 rnL, 1.124mmoi) and HATU (0.128 g, 0.337 mmol). The reaction was stirred for 1 h, the solution was diluted with citric acid solution and EtOAc, The organic phase was washed with aqueous NaHCOn and brine, dried over NaiSO and concentrated in vacuo. The residue was purified with flash to afford (9H-fluoren-9-yl)methyl ((R)-l-(((35,4R,55)-8-hydiOxy-3 ₅ 5- dimethyloctan-4-yl)aniino)~l -oxopropan~2-yl)carbamate (0.0432 g, 33 %, 0.093 mmol).

[0324] Step 8: To a stirring solution of (9H-fluoren-9-yl)methyl (( ?)- 1 -(((3S,4R,55)-8- hydroxy-3,5-dimethylocten-4-y])ainino)-l-oxopropan-2-yl)carb amate (0,0434 g, 0.093 mmol) in DCM ( 1.098 g, 0.832 rnL, 12.93 rnmol) was added DMP (0.051 g, 0.120 mmol) at 0 °C. The reaction was stirred for (h), the solution was diluted with pH 7.4 buffer, sodium thiosulfate, and Et ₂ 0. The organic phase was washed with saturated sodium bicarbonate solution, brine, and Na ₂ S0 ₄ and concentrated in vacuo. The residue was purified with flash to afford ( 9H -fluoren-9-yl)methyl ((R)-l -(((3S,4R,5S)-3 ,5 -dimethyl-8-oxooctan-4-yl)amino)- 1 - oxopropan-2-yl)carbamate (0.043 g, 100 %, 0.093 mmol).

[0325] Step 9: To a stirring solution of (9H-fluoren-9-yl)methyl (l-iCCB.S' l^^^-S^S- dimethyl-8-oxooctan-4-yl)amino)- l-oxopropan-2-yl)carbamate (0.043 g, 0.093 mmol) in THF (3 mL, 36.6 mmol) was added ferr-butyl 2-(triphenyl- ⁵ -phosphanylidene)propanoate (0.108 g, 0.278 mmol) and stirred for 3 hours, the solvent was removed and the residue was purified with flash to afford ferf-butyl (65,7/?,8S,£)-7-((/?)-2-((((9H-fluoren-9

yl)methoxy)carbonyl)amino)propanainido)-2,6,8-tjimethylde c-2-enoate (0.0415 g, 78 %, 0.072 mmol).

[0326] Step 10: To a stirring solution of fcrr-butyl (6S,7i?,8,S,ii)-7-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)-2,6,8-tjimethyldec-2- enoate (0.04 g, 0.069 mmol) in DCM (1 mL, 15.54 mmol) was added TFA (1 mL, 12.98 mmol) and stirred for 30 min. The solvent was removed and the residue was purified with flash to afford (6S,7R,8S,E)-7- ((R)-(2-((((9il-fluoren-9-yl)m

enoic acid in quantitative yield.

[0327] Step 11 : To a stirring solution of (6„S 7/?,8,S ^* ,£)-7-((i?)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)-2,6,8-1rirnethyldec-2 -enoic acid (0.045 g, 0.086 mmol) in DCM (1 mL, 15.54 mmol) was added 2,2,2-trichloroethyl N-(N-(((R)-2-hydroxy-4- methylpentanoyl)-D-alanyl)-N-meth^

(0.070 g, 0.103 mmol), DMAP (0.010 g, 0.086 mmol) and EDC (0.049 g, 0.259 mmoi) and stirred for 18 h. The reaction was diluted with citric acid solution and EtOAc. The organic phase was washed with aqueoud aHCOa and brine, dried over Na ₂ S0 ₄ and concentrated in vacuo. The residue was purified with flash to afford 2,2,2-trichloroethyl (2S,W.,\ li?,14i?)-14- (((6 ,7i?^-7-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan arnido)-2,6,8-

tetraoxo-3,6,9,12-tetraazahe tadecanoate (0.0623 g, 61 %, 0.053 mmol).

[0328] Step 12: To a solution of 2,2,2-trichloroethyl (2S,8R,11R, 14R)-14-(((6S,7R,E)-7- (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-2 ,6,8-trimethyldec-2- enoyl)oxy)-8-benzyl-2-((R)-sec-butyl)-6,9, 11,16-tetramethyl-4,7, 10, 13-tetraoxo-3,6,9, 12- tetraazaheptadecanoate (0.062 g, 0.052 mmol) In THF (1.854 g, 2.107 mL, 0.052 mmol) was added 1M ILOAc (0.113M, 470uL) and zinc (0.964 g, 14.749 mmol) and stirred for 4 h. The reaction was then filtered through a pad of celite using acetonitrile and concentrated in vacuo. The residue was dissolved in MeCN (0.5 mL) and diethylamine (0.5 mL), and stirred for 1 h, and the solvent was removed, the residue was purified with C18 reverse

chromatography (MeCN + 0.1% formic acid j H ₂ 0 + 0.1 % formic acid to afford N-(N- (((2R)-2-(((6S,7R,E)-7-(2-aminopropanamido)-2,6,8-trimethyld ec-2-enoyl)oxy)-4- methylpentanoyl)-D-alanyl)-N-methyl-D-phenylalaxiyl)-N-methy lgiy

(0.065 g, 149 %, 0.078 mmoi).

[0329] Step 13: To a solution of N-(N-(((2R)-2-(((6S,7R,E)-7-(2-am^

2,6,8-trimethyldec-2-enoyi)oxy)-4-methy

N-methylglycyl-L-alloisoleucine (0.065 g, 0.078 mmoi) in DCM/DMF was added 1- hydroxy-7-azabenzotriazole (0.106 g, 0.783 mmoi), and EDC (0.150 g, 0.783 mmoi), and stirred for 18 h. The solvent was removed and the diluted with EtOAc and citric acid solution. The organic phase was washed with NaHCOj, brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified with flash to afford

(2fl,5S,8fl,14S,17i?,20iR,21S,j¾-^

5 ,7, 10,17,21 ,25 -hexamethy 1 - 1 -oxa-4,7, 10,13,16,19-hexaazacyclohexacos-24-ene-

3,6,9,12,15,18,26-heptaone (0.0166 g, 26.2 %, 0.021 mmoi).

Example 35

Synthesis of Compound 33

[0330] Preparation of Compound 33 is very similar to the synthesis of Compound 32 with the repiacemeni of one amino acid from Fmoc-D-Ala to Fmoc-Isobutyric Acid. The

pentapeptide has also been replaced with a halogenated pentapeptide (X=C1). The last step in the synthesis is described below.

[0331] To a solution of N-((2R)-2-((2R)-2-((2R)-2-(((6S,7R,E)-7-(2-amino-2- methylpropanan ido)-2,6,8-trimethyldec-2-enoy])oxy)-4-me1hy]pentaiiamido

me1hylpropanamido)-3-(4-chlorophenyl)pro^ (0.063 g, 0.072 mmol) in DCM DMF was added l-hydroxy-7-azabenzotriazole (0.098 g, 0.718 mmol) and EDC (0.138 g, 0.718 mmol) and stirred for 18 h. The solvent was removed and the diluted with EtOAc and citric acid solution. The organic phase was washed with

NaHCC , brine, dried over sodiurn sulfate and conceniraied in vacuo. The residue was purified with flash to afford (2R,5S,8R 4S,20S,21S,E)-20-((E)-but-2-en-2-yl)-14-((R)~sec- buty3)-8-(4-chlorobenzy3)-2-isobutyl-5 ,7, 10, 17, 17,21 ,25-heptamethyl- 1 -oxa-4,7, 10,13,16, 19- hexaazacyclohexacos-24-ene-3,6,9, 12, 15,18,26-heptaone (0.008 g, 13 %, 9.33 μηιοΐ).

Example 36

[0332] To the solution of (2R,5S,8R,14S, 17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en- 2-yl)- 14-((R)-sec-butyl)-2-isobutyl-5 ,7, 10, 17,21 ,25 -hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa~4,7, 1 , 13,16-pentaazacyclohexacos-24-en -22-yl (7-hydroxyheptyl)carbamate (0.053 mmol, 52 mg) in pyridine (12.36 mmol, 1 mL) was added Bis(2,2,2-trichloroethyl) phosphorochloridate (0.212 mmol, 0.080 g). The mixture was stirred for 4 h and purfied with RP-flash to afford (2¾5S,8R,14S,17R,20S,21 S,22S,E)-8-beaizyl-20-((E)-but-2-eai-2-yl)-14- ((R)-sec-butyl)-2-isobutyl-5,7,10,17,21 ,25-hexamethyl-3,6,9,12, 15, 18,26-heptaoxo-l,19- dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (7-((bis(2,2,2- trichloroemoxy)phosphoryl)oxy)heptyl)carbamate (37 mg, 52.8%). Observed LRMS (ESI) m/z: 1323.3 [M+H]+.

Example 37

[0333] To the solution of (2R,5S,8R,14S, 17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en- 2-yl)- 14-((R)-sec-butyl)-2-isobuty] -5 ,7, 10, 17,21 ,25 -hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- 1, 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (7-((bis(2,2,2- trichloroethoxy)phosphoryl)oxy)heptyl)carbamate (0.028 mmol, 37 mg) in THF (24.41 mmol, 2 mL) and ammonium acetate (0.400 mmol, 0.4 niL) was added zinc (2.79 mmol, 0.182 g). The mixture was stirred overnight and filtered over celite. The volatile was remvoed and the residue was purified with RP-flash to afford (2R,5S.8R,14S,17R,20S,21S,22S,E)-8- benzyl-20-((E)-but-2-en-2-yl) 4-(^

3,6,9, 12, 15, 18,26-heptaoxo- 1, 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (7- (phosphonooxy)heptyl)carbamate (24 mg, 81%). Observed LRMS (ESI) m/z: 1063.5

[M+H]+.

Example 38

Synthesis of Conjugate L3

[0334] To the solution of (9H-fluoren-9-yl)methyl (2-(phosphonooxy)ethyl)carbamate (0.056 mmol, 0.021 g) in DMF (6.46 mmol, 0.5 mL) was added CDI (0.1 13 mmol, 0.018 g) and triethylamine (0.056 mmol, 7.87 μΕ). The mixture was stirred for 4 h and MeOH was added, then the volatile was removed. The residue was added DMF (6.46 mmol, 0.5 mL), zinc chloride (0.169 mmol, 0.023 g) and (2R,5S.8R, 14S, 17R,20S.21 S,22S,E)-8-benzyl-20- ((E)-but-2-en-2-yI)-14K(R)^

3,6,9, 12, 15, 18,26-heptaoxo- 1 , 19-dioxa~4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (7-

(phosphonooxy)heptyl)carbamate (5.64 μιτιοί, 6 mg) and stirred at 37 degree overnight. The mixture was purfied with RP -flash to afford (9H-fluoren-9-yl)methyl (2-((((((7-

(((((2R,5S,8R, 14S,17R,20S,21S,22S,E)-8-benzyl-20 (E)-but-2-en-2-yl) 4 (R)-sec-butyl)-

2-isobutyl-5 ,7, 0, 17,21,25 -hexamethyl-3 ,6,9, 12, 15, 18,26-heptaoxo- 1 , 19-dioxa~4,7, 10, 13, 16- pentaazacyclohexacos-24-en-22- yl)oxy)carbonyl)amino)heptyi}oxy)(hyciro

)carbamate (7.95 mg, 100%) with some impurity. Observed LRMS (ESI) m/z: 1408.5

I .M - l l ] .

Example 39

Synthesis of Conjugate L4

[0335] The solution of (9H-fluoren-9-y])methyl (2-((((((7-

(((((2R,5S,8R, 14S ₅ 17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)-14- ((R)-sec-butyl)-

2-isobu1^ -5,7,10,17,21,25-hexame l-3,6,9,12,15,18,26-heptaoxo ,19^

pentaazacyclohexacos-24-en-22- yl)oxy)carbonyl)arnino)heptyl)oxy)(hydroxy)phosphoryl)oxy)(h yd

)carbamate (1.420 μηιοΐ, 2 mg) in DMF ( 12.91 mmol, 1 mL) and diethyiamine (0.957 mmol, 0.1 mL) was stirred overnight and the volatile was removed. The residue was purifeid with RP-flash to afford (2R,5S,8R,14S,17R,20S,21 S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)-14- ((R)-sec-butyl)-2-isobutyl-5^

dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (7-(((((2- aminoethoxy)(hydroxy)phosphory^l)oxy)(hydroxy)phosphory4)oxy )heptyi)carbamate ( 1.2 mg, 71 ,2%), Observed HRMS (ESI) tn/z: 1186.5698 [M+H]+. The 'H NMR spectrum of Conjugate L4 is shown in Figure 3.

Example 40

Synthesis of Conjugate L5

[0336] To the solution of (9H-fluoren-9-yl)methyl (5-(phosphonooxy)pentyl)carbamate (0,019 mmol. 7.63 mg) in DMF (6.46 mmol . 0.5 m l .) was added CD! (0.038 mmol, 6.10 mg) and triethylamine (0.019 mmol, 2.62 μΕ). The mixture was stirred for 4 h and MeOH was added, then the volatile was removed. The residue was added DMF (6.46 mmol, 0.5 mL). zinc chloride (0.188 mmol, 0,026 g) and (2R,5S,8R, 14S, 17R,20S,21 S,22S,E)~8-benzyl-20- ((E)-but-2-en-2-yl)-14-((R)-sec-buty1)-2-isobut 1-5,7, 10, 17,21,25-hexamethyl- 3,6,9, 12, 15, 18,26-heptaoxo-l , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (7- (phosphonooxy)heptyl)carbamate (1.881 μιηοΐ, 2 mg) and stirred at 37 degree overnight. The mixture was purfied with R -flash to afford an intermediate, which was treated with MeCN (9,57 mmol, 0.5 ml) and diethylamine (0.957 mmol, 0.1 mL) for overnight and the volatile was removed and the residue was purified with RP-fiash to afford

(2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en-2 -yl)-14-((R)-sec-butyl)-2 isobutyl-5,7, 10, ! 7,21 ,25~hexame^

pentaazacyclohexacos-24-en-22-yl (7-((((((5- aminopentyl)oxy)(hydroxy)phosphoiyl)oxy)(hydroxy)phosphoiy ( 1.5 mg, 64 ,9%), Observed LRMS (ESI) m/z: 1228 ,5 [M+H]+. The 1H NMR spec! rum of Conjugate L5 is shown in Figure 4,

Example 41

Synthesis of Conjugate L6

[033η To the solution of (2R,5S,8R,14S ₅ 17R,20S ₅ 21S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl) - 14-((R)-sec-butyl)-2-isobutyl-5 ,7, 10, 17,21 ,25 -hexamethyl-3 ,6,9, 12,15, 18,26-heptaoxo- l, 19-dioxa-4,7,10, 13, 16-pentaazacyclohexacos-24-en-22-yl (7-(((((2- aminoe1hoxy)(hydroxy)phosphory])oxy)(hydroxy)phosphoryl)oxy) heptyl)carban a^ (0.674 μηιοΐ, 0.8 mg) and undefined (1.463 μτηοΐ, 1 rng) in Hunig's base (0.172 mmol, 30 μΙ_) was added Hunig's base (0.172 mmol, 30 μΕ), The mixture was stirred overnight and purified with RP-flash to afford (2R,5S,8R,14S,17R,20S,21 S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)- 14-((R)-see >utyl)-2-isobutyW

dioxa-4,7, 10,13,16-pentaazacyclohexacos-24-en-22-yl (7-(((((2-((((4-((R)-2-((R)-2-(6- azidohexanamido)-3 -methylbutanamido)-5 - ureidopentanamido)benzyl)oxy)carbonyl)amino)etiioxy)(hydroxy )phosphor>'l)oxy)(hydroxy) phosphoryl)oxy)heptyl)carbarnate (0.8 rng, 68.5%). Observed LRMS (ESI) m/z: 1730.9 | M · i 1 | . The ^! H NMR spectrum of Conjugate L6 is shown in Figure 5.

Example 42

Synthesis of Conjugate L7

[0338] To the solution of (2R,5S,8R,14S, 17R,20S,21S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-bu1yl)-2-isobutyl-5,7, l 0, 17,2 l,25-hexamethyl-3, 6,9, 12,15, 18,26-heptaoxo- 1, 19-dioxa-4,7,10, 13, 16-pentaazacyclohexacos-24-en-22-yl (7-((((((5- aminopentyl)oxy)(hydfoxy)phosphor l)oxy)(hydroxy)phosphor l)oxy)

(0.651 μηιοΐ, 0.8 mg) and undefined (1.463 μιηοΐ, 1 mg) in Hunig 's base (0. 72 mmoi, 30 μΐ,) was added Hiinig ^' s base (0.172 mmoi, 30 μΕ), The mixture was stirred overnight and purified with RP-flash to afford (2R,5S,8R,14S,17R,20S,2iS,22S,E)-8-benzyl-20-((E)-but-2- en-2-yl)~ 14-((R)-sec-butyl)-2-isobutyl-5 ,7, 10, 17,21 ,25-hexamethyl-3,6,9, 12, 15,18,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (7-((((((5-((((4-((R)-2- ((R)-2-(6-azidohexanamido)-3-methylbutanamido)-5- ureidopentanamido)benzyl)oxy)carbonyl)amino)pent5d)oxy)(hydr ox} phosphotyl)oxy)(hydro xy)phosphor}'l}oxy}heptyl)carbamate (0.8 mg, 69.3%). Observed LRMS (ESI) m/z: 1772.9 [M+H]+. The 'IT NMR. spectrum of Conjugate L7 is shown in Figure 6.

Example 43

Synthesis of Conjugate L8

[0339] To the solution of (2R,5S,8R,14S ₅ 17R,20S ₅ 21S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl) - 14-((R)-sec-butyl)-2-isobutyl-5 ,7, 10, 17,21 ,25 -hexamethyl-3 ,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13,16-pentaazacyciohexacos-24-en-22-yl (5-hydroxypentyl)carbamate (0.838 μηιοΐ, 0.8 mg) in pyridine (0.618 mmoL 50 μΕ) was added Bis(2,2,2-trichloroethyl) phosphorochloridate (3.35 μιηοΐ, 1.270 mg). The mixture was stirred for 4 h and purfied with RP-flash to afford (2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benzyl-20-((E)-but-2-en-2 -yl)-14- ((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21 ,25~faexam.ethyl-3,6,9, 12, 15 , 18,26-heptaoxo- 1 ,19- dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (5-((bis(2,2,2- tiichloroetlioxy)phosphoiyl)oxy)pentyl)carbamate. Observed LRMS (ESI) m/z: 1295.1

[M+H] ⁺

Example 44

Synthesis of Conjugate L9

[0340] To the solution of (2R,5S,8R,14S ₅ 17R,20S,21S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-bu1yl)-2-isobutyl-5,7, l 0, 17,2 l,25-hexarnethyl-3, 6,9, 12,15, 18,26-heptaoxo- l, 19-dioxa-4,7,10, 13, 16-pentaazac}'clohexacos-24-en-22-yl (5-((bis(2,2,2- trichloroethoxy)phosphor l)oxy)pentyl)carbamate (1.541 μηιοΐ, 2 mg) in THF (6.10 mmol, 0.5 ml) and ammonium acetate (0.500 mmol, 0.5 mL) was added zinc (0.765 mmol, 50 mg). The mixture was stirred overnight and filtered over celite. The volatile was removed and the residue was purified with RP-flash to afford (2R,5S,8R, 14S,17R,20S,21S,22S,E)-8-benzyl- 20-((E)-but-2-en-2-yl)-14-((R)-se^

3,6,9,i2,15,18,26-heptaoxo-l, 19-dioxa-4,7,i0,13,16~pentaaza,cyc3ohexacos-24-en-22-yl (5- (phosphonooxy)pentyl)carbamate (1 mg, 62.7%). Observed LRMS (ESI) m/z: 1035.5 | .M 1 11 - .

Example 45

Synthesis of Conjugate L10

(9,66 μηιοΐ, 3.92 mg) in DMF (6.46 mmol, 0.5 mL) was added CDI (0.019 mmol, 3.13 mg) and triethylamine (9.66 μηιοΐ, 1.346 μΕ). Hie mixture was stirred for 4 h and MeOH was added, then the volatile was removed. The residue was added DMF (6,46 mmol, 0.5 mL), (2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benzy!^

isobuty1-5,7,10, 17,21,25-hexamethy^^

pentaazacyclohexacos-24-en-22-yl (5-(phosphonooxy)pent}'i)carhamate (1.932 μιηοΐ, 2 mg) and zinc chloride (0.073 mmol, 10 mg) and stirred at 37 degree overnight. The mixture was purfied with RP-flash to afford (9H-fluoren-9-yl)methyl (5-((((((5-

(((((2R,5S.8R, 14S,17R,20S ₅ 21S.22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)-14-((R)-sec -butyl)-

2Hsobiityi-5,7, 10, 17,21,25 iexamethyl-3,6,9, 12,15,18,26-heptaoxo-l,19-dioxa-4,7, 10 pentaazacyclohexacos-24-en-22- yl)oxy)carboriyl)an ino)pentyl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)o xy)pent yl)earbamate (0.5 mg, 18.19%). Observed LRMS (ESI) m/z: 1422.7 ! M i ! | .

Example 46

Synthesis of Conjugate LI 1

10342] To the solution of (2R,5S,8R,14S ₅ 17R,20S ₅ 21S,22S ₅ E)-8-benzyl-20-((E)-but-2-en- 2-yl)-14-((R)-sec-butyl)-2-isobutyl-5,7, 10, 17,21 ,25-hexamethyl-3,6,9, 12,15, 18,26-heptaoxo- l , 19-dioxa-4,7,10, 13, 16-pentaazacyclohexacos-24-en-22-yl (5-((((((5-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)pentyl)oxy)(hydroxy)phosphor l)oxy)(hydroxy)phosph^ pentyl)-14~azaneearboxylate (0.843 μηιοΐ, 1.2 nig) in MeCN (3.83 mmol, 0.2 mL) was added diethyl amine (0.191 mmol, 20 μ,Ε). The mixture was stirred overnight and the volatile was removed.The residue was purified with RP-flash to afford

(2R,5 S,8R, 14S, 17R,20S,21 S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)- 14-((R)-sec-butyl)-2- isobutyl-5,7, 10, 17,2 l,25-hexamethyi-3,6,9, 12, 15, 18,26-heptaoxo-l, 19-dioxa-4,7, 10,13,16- pentaazacyclohexacos-24-en-22-yl (5-((((((5- aminopentyl)oxy)(hydroxy)phosphoiyl)oxy)(hydroxy)phosphoiyl) oxy)pentyl)-14- azanecarboxylate (0.7 mg, 69.1%). Observed HRMS (ESI) m/z: 1200.5949 [M+H]+.

Example 47

Synthesis of Conjugate L17

Example 48

Synthesis of Conjugate L I 8

[0344] Conjugate L 18 was prepared using a similar procedure as descnbed in Example 37. Example 49

|034S] To the solution of (9H-fluoren-9-yl)methyl ((R)- 1 -(((S)- 1 -((4- (hydroxymemyl)phenyl)amino)-l-oxo-5-ureidop^

yl)carbamate (24 mg, 0,039 mmol) in N,N-dimethylformamide (0.888 mg, 0.2 mL, 0.012 mmol) was added 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (14 mg, 0.046 mmol) and IH-tetrazole (3.278 mg, 0.104 mL, 0.046 mmol). The reaction mixture was stirred for 1 h and added (2R,5S,8R,14S,17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-( (R)-sec- butyl)-8-(4-chlorobenzy])-2-isobut l-5,7, 10, 17,21 ,2S~hexamethyl-3,6,9,12,15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13 , 16-pentaazacy clohexacos-24-en-22-yl (5 - (phosphonooxy)pentyl)carbaniate (13 mg, 0.012 mmol) and 5-(ethyithio)-lH-tetrazo3e (11.7 mg, 0.089 mmol). To the solution of crude mixture of (9H-fluoren-9-yl)methyl ((2R)-1- (((2S)-l-((4-(((((((5-(((((2R,5S,8R,14S ₅ 17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-but 4)-8^4"Chiorobenzyi)-2 sobuty4-5,7,10,17,21,25-hexainemy

heptaoxo- 1 , 19-dioxa-4,7, 10, 13,16-pentaazacy ciohexacos~24-en~22- yl)oxy)carbonyl)amino)pentyi)oxy)(hydroxy

yl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3-methy l-l -oxobutan-2-yl)carb (0.020 g, 0.012 mmol) is added diethylamine (0.07 g, 0.1 mL, 0.957 mmol) and stirred for 3 h. The mixture was purified by RP flash chromatography to

at&rd (2R,5S,8R, 14S, 17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yi)-14-((R)-sec¾ityl) -8 chlorobenzyr}-2-isobutyi-5,7, 10,17,21,25-hexamethyi-3,6,9,12,15,18,26-heptaoxo-l, 19- dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24~en -22-yl (5 -((((((4-((S)-2-((R)-2-amino-3 - methylbutanamido)-5- ureidopentanamido)benzy3)oxy)(hydroxy)phosphoiyl)oxy)(hydrox y)phosphoiyl)oxy)pe arbamate (1 mg, 5.515%).Observed LRMS (EST) m/z; 1510.4 [M+H] ⁺ .

Example 50

Synthesis of Conjugate L20

[0346] To the solution of (2R,5S,8R,14S,17R ₅ 20S,21S,22S ₅ E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-but>'l)-8-(4-chlorobenzyl)-2-isobutyl-5 ,7,10, 17,21,25-hexamethyl-3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10,13,16-pentaazacy clohexacos-24-en-22-yl (5 -((((((4-((S)-2-((R)- 2-amino-3 -methylbutanamido)-5 - ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)(hydrox y)phosphoryl)oxy)pent>'l)c arbamate ( 1 mg, 0.661 μηιοΐ), HOAt (2 mg, 0.014 mmol) and (E)-cyclooct-4-en-l -yl (2,5- dioxopyrrolidin-l-yl) carbonate (3 mg, 0.011 mmol) in DMF (0.094 g, 0.1 ml, 1.291 mmol) was added hunig'sbase (0.014 g, 20 μΕ, 0.114 mmol). The reaction was stirred overnight and purified on RP flash chromatography to

afford (2R,5S,8R, 14S, 17R,20S _s 21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec-butyl)-8 -(4- ehlorobenzyl)-2-isobutyl-5 ,7, 10, 17,21 ,25-hexamethyl-3,6,9, 12,15,18,26-heptaoxo- 1 , 19- dioxa-4,7,10, 13, 16-pentaazacyclohexacos-24-en-22-y3 (5-((((((4-((2S)-2-((2R)-2-(((((E)- cyclooct-4-en-l-yl)oxy)carbonyl)amino)-3-m

ureidopentaiiamido)benzyl)oxy)(hydroxy)phosphory4)oxy)(hy droxy)phosphory4)oxy) arbamate (0.9 mg, 81 ,76%) as white solid. Observed HRMS (ESI) m/z: 1662.7589 [M+H] ⁺ . Example 51

Synthesis of Conjugate 21

[0347] Conjugate L21 was prepared using a similar procedure as described in Examples 38 and 39.

Example 52

Synthesis of Conjugate L22

[0348] To a solution of (2R,5S,8R, 14S,17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-butyl)-8-(4-chlorobeaizyl)-2-isobutyl-5,7, 10, 17,2 l,25-hexamethyl-3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10,13,16-pentaazaey clohexacos-24-en-22-yl (5-((((((5- aminopentyl)oxy)(hydfoxy)phosphoryl)oxy) (hydroxy) phosphoryl)oxy) pentyl)carbamate (0.011 g, 8.908 μιηοΐ) in DMF (0.472 g, 0.5 rnL, 6.457 mmol) was added (9H-fluoren-9- yl)niethyl ((S)-3 -methyl- 1 -(((S) - 1 -((4-((((4- ni1xophenoxy)carbony])oxy)me1hyl)phenyl)amino)-l-oxo-5-ureid opentan-2-yl)amino)-l- oxobutan-2-yl)carbamate (0.014 g , 0.018 mmol) and DIPEA (0.011 g, 0.015 mL, 0.089 mmol). The resulting residue was dissolved in MeCN and diethylamine and stirred at RT. After 3 h the reaction was concentrated in vacuo. The residue was purified with flash chromatography to afford (2R,5S,8R,14S,17R,20S,21 S ₅ 22S,E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-but>'l)-8-(4-chlorobenzyl)-2-isobutyl-5 ,7,10, 17,21,25-hexameihyl-3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 1 ,13,16-pentaazacy clohexacos-24-en-22-yl (5 -((((((5 -((((4-((S)-2- ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)ainino)-3-methyl butananiido)-5- ureidopentanamido)benzyl)oxy)carbonyl)amino)pentyl)oxy)(hydr oxy)phosphoryl)oxy)(hydro xy)phosphoryl)ox>')pentyl)carbamate (0.004 g, 23.53 %, 9.13 μη ο3).

Example 53

Synthesis of Conjugate L23

[0349] To the solution of (2R,5S,8R, 14S ₅ 17S,20S,21 S ₅ 22S,E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-butyl)-8-(4-cUorobenzyl)-2-isobutyl-5,7, 10, 17,2 l,25-hexamethyl-3,6,9, 12, 15, 18,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (5 -((((((5 -((((4-((R)-2- ((R)-2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl)oxy)carbonyl)amino)pent}d)oxy)(hydr oxy)phospho

xy)phosphoryl)oxy)pentyl)carbamate ( 1 mg, 0.6 μηιοΐ) was added HO At (2 mg, 0.014 mmol), (E)-cyclooct-4-en-l-yl (2,5-dioxopyrroiidin-l-yi) carbonate (3 mg, 0.011 mmol) in DMF (0.094 g, 0.1 ml,, 1.291 mmol) and humg's base (0,014 g, 20 fiL, 0.1 14 mmol). The reaction was stirred overnight and purified on RP flash chromatography to afford (2R,5 S,8R, 14S, 17S,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)- 14-((R)-sec-butyl)-8-(4- chlorobenzy])-2-isobutyl-5,7, 10, 17,21 ,25-hexamethyl-3,6,9, 12, 15, 18,26-heptaoxo-l, 19- dioxa-4,7.10, 13,16-pentaazacyclohexacos-24-en-22-yl (5-((((((5-((((4-((2R)-2-((2R)-2- (((((E)-cyclooct-4-en-l-yl)oxy)carbonyl)amino)-3-methylbutan amido)-5- ureidopentaiianiido)benz> )oxy)carbonyl)aniino)pentyl)oxy)(hydroxy)phosphoryl)oxy)(hyd ro xy)phosphoiy])oxy)pentyl)carbamate (1 mg, 0.5 μηιοΐ). Observed HRMS (ESI) m/z:

896.4223 [M+H1 ²⁺ .

Example 54

Synthesis of Conjugate L24

[0350] To a solution of (2R,5S,8R,14S,17R,20S,21S,22S,E)-8-benzyi-20-((E)-but-2-en- 2-yl)- 14-((R)-sec-butyl)-2-isobutyl-5 ,7 , 10, 17,21 ,25-hexamethyl-3,6,9, 2, 5, 18,26-heptaoxo- l, 19-dioxa-4,7,10, 13, 16-pentaazacyclohexacos-24-en-22-yl (5-((((((5

aminopentyl)oxy)(hydroxy)phosphoryl)ox}^(hydroxy (3.0 mg, 2.499 μηιοΐ, 1.0 eq; Conjugate Ll l from Example 46) in DMSO (200 μΕ) was added 2,5-dioxopyrrolidin-l-yl 1 -oxo-l-(4-(3-tosyl-2-(tosylmethyl)propanoyl)phenyl)-5,8,l 1 ,14- tetraoxa-2-azaheptadecan-17-oate (2.11 mg, 2.499 499 μηιοΐ, 1.0 eq) and N-ethyl-N- isopropylpropan-2-amine (80 μΕ, 0.459 mmol, 184.0 eq) and stirred at room temp for 2 h. The reaction was monitored by reverse phase HPLC. The reaction mixture was lyophilized and the crude residue obtained was washed with hexanes followed by diethyl ether leaving behind the unreacted bis-sulfone and impurities (bis-sulfone acid) in the solvent washes. The residue obtained was then taken in the ethyl acetate to which hexanes was added resulting in the precipitation of (2R,5S,8R, 14S, 17R,20S,21 S,22S,E)-8-benzyl-20-((E)-but-2-en-2-yl)-14-

dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (5 -(((((( 1 , 17-dioxo- 1 ~(4-(3 -tosyl-2- (tosylmethyl)propanoyl)pheny])-5,8, 1 1,14-tetraoxa-2, 18-diazatricosan-23- yl)oxy)(hydroxy)phosphoiyl)oxy)(hydroxy)phosphoiyl)oxy)penty l)carbaniaie ( 2.68 mg, 1.38 μιηοΐ, 55.6%) as a major compound. Observed HRMS (ESI) m/z: 965.42 | M 1 11 ^' .

Example 55

Synthesis of Conjugate L25

[0351] To a solution of (2R,5S,8R, 14S, 17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-butyl)-8-(4-chlorobeaizyl)-2-isobutyl-5,7, 10, 17,2 l,25-hexamethyl-3,6,9, 12, 15, 18,26- heptaoxo-l, 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (5-((((((5- aminopentyl)oxy)(hydroxy)phosphoryl)oxy)(hy (3.0 mg, 2.46 μΐΐΐοΐ, 1.0 eq) in DMF (200 μΕ) was added 2,5-dioxopyrrolidin-l-yl l-oxo-l-(4-(3- tosyl-2-(tosylmethyl)propanoyl)phenyl)-5,8S01 l, 14-tetraoxa-2-azaheptadecan-17-oate (2.46 mg, 2.92 μτηοΐ, 1.2 eq) and N-ethyl-N-isopropylpropan-2-amine (80 μΕ, 0.459 mmol, 184.0 eq) and stirred at room temp for 2 h. The reaction was monitored by reverse phase HPLC. The reaction mixture was lyophilized and the crude residue obtained was washed with hexanes followed by diethyl ether leaving behind the unreacted bis-sulfone in the solvent washes. The residue obtained was then taken in the ethyl acetate to which hexanes was added resulting in the precipitation of (2R,5S,8R, 14S, 17R,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)- 14-((R)-sec-bu1yl)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17 ₅ 21,25-hexametliyl- 3,6,9, 12, 15, 18,26-heptaoxo-l , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (5- (((((( l, 17-dioxo-l-(4-(3-tosyl-2-(tosylme

diazatricosan-23- yl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)pen )carbainate ( 2.2 mg, 1.12 μτηοΐ, 41.0%) as a major compound. Observed HRMS (ESI) m/z: 982.39 [M+H] ^{2 "} .

Example 56

Synthesis of Conjugate L26

[0352] To a solution of (2R,5S,8R, 14S,17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-butyl)-8-(4-chlofobenzy 8,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yi (5-((((((4-((R)-2-((R)- 2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)(hydrox y)phosphoryl)oxy)pent>'l)c arbamate (2.39 mg, 1.582 μηιοΐ, 1.0 eq) in DMF (400 μΕ) was added 2,5-dioxopyrrolidin-l- yl l-oxo-l-(4-(3-tosyl-2-(tosylmethyl)propanoyl)phenyl)-5,8S011 ,14-tetraoxa-2- azaheptadecan- 17-oate (6.68 mg, 7.91 μηιοΐ, 5.0 eq), 1 -Hydroxy-7-azabenzoiriazoIe (0,21 mg, 1.582 μιτίοΐ, 1.0 eq) and N-ethyl-N-isopropylpropan-2-amine (60 μ\ _^ , 0.344 mmol, 218.0 eq) and stirred overnight at room temp. The reaction was monitored by reverse phase HPLC. The reaction mixture was lyophilized and the crude residue obtained was washed with hexanes followed by diethyl ether leaving behind the unreacted bis-sulfone in the solvent washes. Hie residue obtained was then purified by reverse phase flash chromatography (acetonitrile with 0.1% fornuc acid/ H ₂ 0 with 0.1% formic acid) to

afford (2R,5 S,8R, 14S, 17R,20S,21 S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec-but 'i)-8-(4- chlorobenzyl)-2-isobutyl-5,7, 10, 17,21 ,25-hexamethyl-3,6,9, 12, 15, 18,26-heptaoxo- 1, 19- dioxa-4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (5-((hydroxy((hydroxy((4- (( 19R,22R)~ 19-isopropyl- 1 , 17,20-trioxo- 1 -(4-(3 -tosyl-2-(tosylmethyl)propanoyl)phenyl)~22- (3 -ureidopropyl)-5 ,8,11,14-tetraoxa-2, 18,21 -triazatri cosan-23 - amido)benzyl)oxy)phosphor d)oxy)pliosphor\'l)oxy)pentyl)carbamate ( 2.0 mg, 0.89 μτηοΐ, 56.4%) as a major compound. Observed HRMS (ESI) m/z: 1120.45 [Μ+Ή] ^{2 "} .

Example 57

Synthesis of Conjugate L27

10353] To a solution of (2R,5S,8R, 14S,17R,20S _s 21S ₅ 22S,E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-bu^)-8~(4-chlorobenzyl)-2 sobutyl-5,7, 10, 17,2 l,25-hexamethyl-3,6,9, 12, 15, 18,26- heptaoxo-l ,19-dioxa-4,7, 10,13,16-pentaazacyc]ohexacos-24-en-22-yl (5-((((((5-((((4-((R)-2- ((R)-2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl)oxy)carbonyl)amn^^

xy)phosphoryl)oxy)pentyl)carbamate (4.0 mg, 2.439 μηιοΐ, 1.0 eq) in DMSO (200 μΕ) was added 2,5-dioxopyrrolidin- 1 -yl 1 -oxo- 1 -(4-(3-tosyl-2-(tosylmethyl)propanoyl)phenyl)- 5,8S011,14-tetraoxa-2-azaheptadecan-17-oate (4.12 mg, 4,88 μηιοΐ, 2.0 eq), l -Hydroxy~7- azabenzotriazole (0.66 mg, 4.88 μπιοΐ, 2.0 eq) and N-eAyl-N-isopropylpropan-2-araine (100 μΕ, 0.574 mmol, 235.0 eq) and stirred at room temp for 3 h. The reaction was monitored by reverse phase HPLC. The reaction mixture was lyophilized and the crude residue obtained was washed with hexanes followed by diethyl ether leaving behind the unreacted bis-sulfone in the solvent washes. The residue obtained was then purified by normal phase preparative thin-layer chromatography (dichloromethane with 0.1% formic acid/ methanol with 0.1% formic acid , 4: 1) to afford (2R,5S,8R,14S,17R,20S,21S _s 22S _s E)-20-((E)-but-2-en-2-yl)-14- ((R)-sec-bu1y4)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17,2 l,25-hexamethyl-3,6,9, 12, 15,18,26- heptaoxo-l ,19-dioxa-4,7, 10, 13,16-pentaazacyc]ohexacos-24-en-22-yl (5- ((hydroxy ((hydroxy ((5 -((((4-(( 19R.22R) - 19-isopropyl- 1 , 17,20-trioxo - 1 - (4-(3 -tosyl-2- (tosy{methyl)propanoyl)phenyl)-22-(3-ureidopropyi)-5,8, 11 , 14-tetraoxa-2, 18,21- triazatricosan-23- amido)benzyl)oxy)carbonyl)amino)pentyl)oxy

mate (1.79 mg, 0.755 μηιοΐ, 31.0%) as a major compound. Observed HRMS (ESI) m/z: 1185.00 I M ϊ ί I

Example 58

Synthesis of Conjugate L28

[0354] To the solution of (2R,5S,8R, 14S,17R,20S,21 S,22S.E)-20-((E)-but-2-en-2-yl)-

14-((R)-sec-bu1yl)-8-(4-chlorobenzyl)-2-isobutyl-5,7,10,1 7,21,25-hexametliyl-

3,6,9, 12, 15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10,13, 16-pentaazacyclohexacos-24-en-22-yl methyl(6-(methylamino)hexyl)carbamate (0.006 g, 6.306 μηιοΐ) in DCM (300 μΕ) and DIPEA (8.150 mg, 0.011 mL, 0.063 mmol) was added 1 -hydroxy -7-azabenzotriazole

(0.858 mg, 6.306 μηιοΐ) and Fmoc-Val-Cit-PABC-PNP (15 mg, 19 μιηοΐ). After stirring overnight and the starting material was consumed and then added 40% EtiNH/MeCN solution and the solution w as stirred and purified with flash to afford 8 mg of the

(2R,5S,8R,14S,17R,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-( (R)-sec-biityl)-8-(4- ehlorobenzyl)-2-isobutyl-5 ,7, 10, 17,21 ,25-hexamethyl-3,6,9, 12,15,18,26-heptaoxo- 1 , 19- dioxa-4,7,10, 13, 16-pentaazacyclohexacos-24-en-22-yl (4-((S)-2-(2-((S)-3-methyl-l- oxobutan-2-yl)hydrazinyl)-5-ureidopentanamido)benzyl) hexane-1,6- diylbis(methylcarbamate).

Example 59

Synthesis of Conjugate L29

[0355] To the solution of (2R,5S,8R,14S,17R,20S _s 21S,22S,E)-20-((E)-but-2-en-2-yl)-14-

((R)-sec-bu^)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17,2 L25-hexametliyl-3,6,9, 12, 15, 8,26- heptaoxo- 1 , 19-dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (4-((S)-2-(2-((S)-3- methyl-l-oxobutan-2-yl)hydrazinyl)-5-ureidopentanamido)benzy i) hexane-1,6- diylbis(methylcarbamate) (6 mg, 4.177 μηιοΐ) and 76-( l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yi)-3 -OXO-7, 10, 13, 16-tetraoxa~4~azanonadecan- 19-amido)-75 -oxo- 2,5,8,1 l,14, 17,20,23,26,29,32,35,38,41,44,47,5(},53,56,59,62,65,68,71-te tracosaoxa-74- azanonaheptacontan-79-oic acid (10 mg, 6.188 μηιοΐ) in DCM was added Hunig's base and HATU. The mixture was stirred overnight and purified with RP-flash chroniatography to afford (2R,5S,8R, 14S, 17R,2QS,21S,22S,E)-20~^

cWorobenzyl)-2-isobutyl-5,7, 10, 17,21 ,25-hexamethyl-3,6,9, 12,15,18,26-heptaoxo- 1,19- dioxa-4,7,10,13,16-pentaazacyclohexacos-24-en-22-yl (4-((76R,81 S,84S)-76-( l-(2,5-dioxo- 2,5-dihydro- IH-pyrrol- 1 -yl)-3 -oxo-7, 10,13,16-tetraoxa-4-azanonadecan- 19-amido)-81 - isopropyl-75,79,80-trioxo-84-(3-ureidopropyl)-

2,5,8,11,14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tet racosaoxa- 74,82,83-triazapentaoctacontan-85-amido)benzyl) he ane-l,6-diylbis(methylcarbamate) (5 mg, 39.45%). Observed HRMS (ESI) ni/z: 1 16,8314 ! \ί · Η | ' .

Example 60

Synthesis of Conjugate L30

f0356] To the solution of (2R,5S,8R,14S,20S.21S,22S ₅ E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-butyl)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17 , 17,2 l,25-heptamethyl-3,6,9, 12, 15, 18,26- heptaoxo~l ,19-dioxa-4,7, 10, 13, 16~penta.azacyc3ohexacos-24-en-22~yl methyl(6- (raethylamino)hexyl)carbaraate (0.01 g, 9.571 mol) in DCM (700 μΕ) was added DIPEA (0.012 g, 0.016 mL, 0.095 mmol), and l-hydroxy-7-azabenzotriazole (1.302 mg, 9.571 μηιοΐ) and stirred overnight at RT . The reaction was monitored by LCMS. The resulting crude material was carried forward to Fmoc deprotection using a 40% solution of EtjNH in MeCN. The material was concentrated and loaded onto C 18 column for reverse phase purification using (MeCN + 0.1% FA) & (H ₂ 0 +0.1 % FA) to afford4-((S)-2-((S)-2-amino-3- methylbutana.mido)-5-ureidopentanamido)henzyl ((2R,5S,8R,14S,20S,2 S,22S,E)-20-((E)- but-2-en-2-}d) 4 (R)-sec-butyl)-8-(4-chkirobenzyl)-2 sobutyl-5,7, 10J 7,17,21,25- heptame thyi-3 ,6,9, 12,15, 18,26-heptaoxo- 1 , 19-dioxa-4,7, 10, 13 , 16-pentaazacyclohexacos-24- en-22-yl) hexane-l,6-diylbis(methylcarbamate) (9.9 rag, 9.6 μηιοΐ).

Example 61

S nthesis of Conjugate L3

[0357] To the solution of (2R,5S,8R,14S,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)- sec-butyI)-8 4^ orobeiizyl)-2-isobutyi-5,7,10,17,17,21,25-heptame i-3,6,9,12,15,l 8,26- heptaoxo- 1 , 19-dioxa~4,7, 10, 13,16-pentaazacyclohexacos-24-en-22-yl (4 -((S)~2-(2-((S)-3 - methyl- l-oxobutan-2-yl)hydrazinyl)-5-ureidopentanamido)benzyi) hexane-1,6- diylbis(methylcarbamate) (9 mg, 6.205 μηιοΐ) and 76-(l-(2,5-dioxo-2,5 iihydro-lH~pyrrol-l- yi)-3 -OXO-7, 10, 13, 16-tetraoxa-4-azanonadecan- 19-amido)-75 -oxo- 2,5,8,1 l ,14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71 -tetracosaoxa-74- azanonaheptacontan-79-oic acid (18 mg, 0.011 mmol) in DCM was added hunigs base and HATU at 0 ^" C. The mixture was warmed up and stirred o vernight, then purified with RP-flash chromatography to afford (2R,5S,8R,14S,20S,21 S,22S,E)~20-((E)-but-2-en-2-yl)~14-((R)~ sec-butyl)-8-(4-chlorobenzyl)-2-isobutyl-5,7, 10, 17, 17,2 l,25-heptamethyl-3,6,9, 12, 15, 18,26- heptaoxo-l,19-dioxa-4,7, 10,13,16-pentaazacyclohexacos-24-en-22-yl (4-((76R,81 S,84S)-76- ( 1 -(2,5 -dioxo-2,5 -dihydro- lH-pyrrol- 1 -yl)-3 -oxo-7, 10, 13, 16-tetraoxa-4-azanonadecan- 19- amido)-81 -isopropyl-75,79,80-trioxo-84-(3-ureidopropy])- 2,5,8,11.14.17,20,23,26,29,32.35.38,41,44,47,50,53.56,59,62, 65,68,71-tettacosaoxa- 74,82,83-triazapentaoctacontan-85-amido)benzyl) hexane-l,6-diylbis(methylcarbamate) (7 mg, 37.01%). Observed HRMS (ESI) m/z: 1523 ,8465 [M+H] ²⁺ .

Example 62

Synthesis of Compound 7

[0358] This compound is prepared using a similar procedure as that described in Example 4.

Example 63

Synthesis of Conjugates L12 and L15

[0359] These compounds are prepared using similar procedures as those described in Examples 8-1 1 and 13 for the synthesis of Conjugate L6.

Example 64

Synthesis of Conjugates L13 and L16

[0360] Conjugates L13 and L16 are prepared from Conjugates L12 and L15, respectively, via click chemistry.

Example 65

Synthesis of Conjugate L14

[0361] This compound is prepared using a similar procedure as that described in Example 1 1 .

Example 66

Synthesis of Intermediate 5 (Linker- 1):

[0362] Step 1 : To the solution of 5-(tert-butyl) l-(2,5-dioxopyrrolidin-l -yl) (tert- butoxycarbonyl)glutamate ( 14 mg, 0.034 mmol) and

2,5,8,11,14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71- tetracosaoxatriheptacontan-73-amine (22 mg, 0.020 mmol) in DCM (1.32 g, 1 mL, 15.542 mmol) was added Hunig'sbase (0.074 g, 100 μL·, 0.572 mmol). Tlie mixture was stirred for 2 h and purified with RP -flash to afford tert-butyl 76-((tert-butoxycarbonyl)amino)-75-oxo- 2,5,8,l l,14, 17,20,23,26,29,32,35,38,41 ,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-74- azanonaheptacontan-79-oate (42 mg, 87,45%).

[0363] Step 2: To the solution of tert-butyl 76-((tert-butoxycarbonyl)amino)-75-oxo- 2,5,8, 1 1, 14^7,20,23,26,29,32.35.38,41 ,44,47,50,53.56,59,62,65,68 l-tettacosaoxa-74- azanonaheptacontan-79-oate (0.035 g, 0.026 mmol) in DCM (0.66 g, 0.5 mL, 7.771 m mol ) was added TFA (0.74 g, 0.5 ml, 6.489 mmol). The reaction mixture was stirred for 1 h and the volatile was removed. The residue was purified with RP -flash to afford 76-amino-75- oxo-2,5,8, 1 l, 14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tet racosaoxa-74- azanonaheptacontan-79-oic acid (32 mg, 101.1%).

[0364] Step 3 : To the solution of 2,5-dioxopyrrolidin-l-yl l-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l -yl)-3-oxo-7, 10, 13, 16-tetraoxa-4-azanonadecan-19-oate (0.013 g, 0.026

mmol) and 76-amino-75-oxo-

2,5,8, 1 l, 14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tBt racosaoxa-74- azanonaheptacontan-79-oic acid (32 mg, 0.026 mmol) in DMF (0.472 g, 0.5 mL, 6.457 mmol ) was added HOAt (3.577 mg, 0.026 mmol). The reaction mixture was stirred for 2 h and purified with RP -flash to afford 76-(l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3-oxo- 7,10, 13,16-tetraoxa-4-azanonadecan- 19-amido)-75 -oxo-

2,5,8,1 l,14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tet racosaoxa-74- azanonaheptacontan-79-oic acid (21 mg, 49.44%).

Intermediate 5 (Linker 1 )

[0365] Step 4: To the solution of 76-(l-(2,5-dioxo-2,5-diliydro-lH-pyrrol-l-yl)-3-oxo- 7, 10, 13,16-tetraoxa-4-azanonadecan - 1 -amido)-75 -oxo-

2,5,8,1 l,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,7 1-tetracosaoxa-74- azanonaheptacontan-79-oic acid (12 mg, 7.426 μπιοΐ) and 3-(4-(6-methyl-l,2,4,5-tetrazin-3- yl)phenoxy)propan-l -amine (5 mg, 0.020 mmol) in DCM (0.66 g, 0.5 mL, 7.771 mmol) and hunig'sbase (0.074 g, 0.1 ml, 0.572 mmol) was added HATU (10 rng, 0.026 mmol). The mixture was stirred overnight and diluted with RP-fiash to afford 2-(l-(2,5- dioxo-2,5 -dihydro- 1 H-pyrrol - 1 -yl)-3 -oxo-7, 10, 13 , 16-tetraoxa-4-azanonadecan- 19-amido)- N5-(3-(4-(6-methyl-l,2,4,5-tetrazin-3-yl)phenox\')propyl)-Nl - (2,5,8,1 1, 14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71- tetracosaoxatriheptacontan-73-yl)pentanediamide (Linker- 1, 4 mg, 29.22%).

Example 67

Synthesis of ntermediate 6 (Ab-Linker-1)

Intermediate 6 (Ab-Linker-1)

[0366] To the solution of Ab where Ab is a Trastuzumab Biosimilar (TBS, 0.991 mg, 0.099 mL, 0.006 μιηοΐ) in PBS was diluted with PBS (0.120 .ug, 0.1 mi .. 0.006 μηιοί) and added TCEP (0.033 mg, 0.026 rriL, 0.134 μηιοΐ) . The mixture was left at RT with mild agitation for 12 h and then added 2-(l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3-oxo- 7,10, 13, 16-tetraoxa-4-azanonadecaii-19-ainido)-N5-(3-(4-(6-methyl-l, 2,4,5-tetrazin-3- y Ijphenoxy )propy] ) -N i -

(2,5,8,11, 14, 17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71- tetracosaoxatriheptacontan-73-yl)pentanediamide (0.098 mg, 0.010 mL. 0.053 μτηοΐ) in DMSO. The mixture was further agitated overnight and then the solution was ran through a spin column (2 mL Zaba desalting spin column) to remove excess of reagents.

Example 68

Synthesis of Conjugate C20

[0367] To the solution of Intermediate-6 (Ab-Linker-1 , 0.5 mg, 50 μΐ) in PBS (50 uL) was added (2R,5S,8R, 14S ₅ 17R,20S ₅ 21S,22S,E)-20-((E)-but-2-en-2-yl)-14-((R)-sec-butyl)-8 - (4~ehlorobenzyl)-2-isobutyl~5,7, 10,17,21 ,25-hexamethy3-3,6,9,12, 15, 18,26-heptaoxo-l, 19- dioxa-4,7, 10, 13, 16-pentaazacyclohexacos-24-en-22-yl (5-((((((4-((2S)-2-((2R)-2-(((((E)- cyclooct-4-en-l -yl)oxy)carbonyl)amino)-3-methylbutanamido)-5- ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)(hydrox y)phosphoryl)oxy)pentyl)c arbamate (conjugate L20, 3.3 μηιοί, 1 μΐ,) and shaken for 15 rain and the solution was ran through spin column to remove the excess of reagent to afford Conjugate C20.

Example 69

Synthesis of Conjugate C23 [0368] To the solution of Intermediate-6 (Ab-Linker-1, 0.5 rng, 50 μΕ) diluted in PBS (50 uL) was added (2R,5S,8R,14S,17S,20S,21S,22S,E)-20-((E)-but-2-en-2-yl)-14-( (R)-sec- butyl)-8-(4~chlorobenzyl)-2-^

heptaoxo-l ,19-dioxa-4,7, 10,13,16-pentaazacyclohexacos-24-en-22-yl (5-((((((5-((((4-((2R)-

2-((2R)-2-(((((E)-cyciooct-4-en-l-yi)oxy)carbony^^

ureidopentanainido)benz 4)oxy)carbonyl)amiiio)pentyl)oxy)(hydroxy)phosphoryl)ox xy)phosphoryl)oxy)pentyl)carbamate (conjugate L23, 3.3 umol, 1 ,u.L) and shaken for 15 min and the solution was run through spin column to remove the excess of reagent to afford Conjugate C23.

Example 70

nthesis of Conjugates C24, C25, and C26

[0369] The antibody-drug conjugates C24-26, wherein Ab is Trastuzumab Biosimilar

(TBS) antibody, were synthesized using protocols similar to methods described previously (Ref: Bryant, P. et al, Mol. Pharmaceuticals, 2015, 12, 1872-1879). Briefly, the purified antibody, Trastuzumab Biosimilar (TBS, 1-2 mg) was buffer exchanged with PBS (lx solution, pH 7.5) and EDTA (20 mM) mix and diluted to a final concentration of 2.5 mg/mL. A stock solution of TCEP (1 mM) was freshly prepared in PBS, and 6 molar equiv. (relative to the antibody concentration) was added to the antibody and left the mixture on a heat block. After 1 h, the partially reduced antibody was removed from the heat block and cooled to room, temperature. A stock solution of Conjugate L24, L25, and L26 (5 mM in DMSO) was freshly prepared, and 6-8 molar equiv was added to the antibody. The reaction was incubated at RT for 15-20 h under mild agitation . Then the reaction mixture was passed through a spin column (Zeba, 2 niL) to remove small MW reagents and buffer exchanged by ultrafiltration (10 kDa MWCO) into PBS to afford the ADC, C24-C27 in PBS. The resulting ADC had an aaverage DAR around 4 by HIC. The aggregation was measured by Size Exclusion Chromatography (SEC) and it was less than 1%.

Example 71

Synthesis of Conjugates C29 and C31 [0370] The antibody-drug conjugates C29 and C31 is synthesized from L29 and L31 respectively, using protocols similar to methods described previously (Ref: Doronina,S.O. et a!, Bioconjugate Chem. 2Θ08, 19 (10), 1960-1963).

Example B 1

Inhibitory Response of Test Compounds Against BT-474 and HCC1954 Cells

[0371] BT-474 human mammary gland ductal carcinoma cells were seeded in a clear polystyrene 96-well microculture plate (Corning® Costar® 96-well flat bottom, plate, Cat.# 3997) in a total volume of 90 uL/well. After 24 hours of incubation in a humidified incubator at 37 °C with 5% C0 ₂ and 95% air, 10 \xL of 10X, serially diluted test agents in growth medium were added to each well (10 pt dose response curve, highest concentration 10 M of test agent). After 72 hours of culture in a humidified incubator at 37 °C, in an atmosphere of 5% CO _? , and 95% air, the plated cells and Cell Titer-Glo® (Promega G7571) reagents were brought to room temperature to equilibrate for 30 minutes. 100 uL of Cell Titer-Glo® reagent was added to each well . The plate was shaken for two minutes and then left to equilibrate for ten minutes. The media/Cell Titer-Glo® reagent was transferred to a white polystyrene 96-well microculture plate (Corning® Costar® ⁾ 96-well flat bottom plate, Cat.# 3917) before reading luminescence on a Tecan GENios microplate reader.

[0372] Percent inhibition of cell growth was calculated relative to untreated control wells. The iCso value for the test agents was determined using Prism 6.05 by curve-fitting of the data using the following four parameter-logistic equation:

^' Tap— E itsm

I Bottom where Top is the maxim al % of control absorbance, Bottom is the minimal % of control absorbance at the highest agent concentration, Y is the % of control absorbance, X is the agent concentration, ICso is the concentration of agent that inhibits cell growth by 50% compared to the control cells, and n s the slope of the curve. [0373] Inhibitory response of test compounds against HCC1954 cells was determined using a method analogous to that used for BT-474 cells. ICso values for various test agents in BT-474 and HCC1954 cells are shown in Table 4,

Table 4

Example B2

Inhibitor}' Response of Test Conjugates Against BT-474 and HCC1954 Cells

[0374] The inhibitory responses of test conj ugates against BT-474 and HCC1954 cells are determined using similar methods as described in Example Bl . Serially diluted test conjugates are incubated with each type of cell in a 96-well plate for 72 hours to generate a dose response curve, as described in Example Bl . The percent inhibition of ceil growth is calculated relative to untreated control wells, and the IC50 values for the test agents are determined.

[0375] While the foregoing written description of the compounds, uses, and methods described herein enables one of ordinary skill to make and use the compounds, uses, and methods described herein, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The compounds, uses, and methods provided herein should therefore not be limited by the above-described embodiments, methods, or examples, but rather encompasses all embodiments and methods within the scope and spirit of the compounds, uses, and methods provided herein.

[0376] All references disclosed herein are incorporated by reference in their entirety.