FFOOLLAATTEE--CCOO JJLliGGAATTEEDP MMOOLLEECCUULLEESS FFOORR DDEELLIIVVEERRYY OOFF TTOOXXIICC SSMMAALLLL MMOOLLEECCUULLEE IINNHHIIBBIITTOORRSS TTOO CCAANNCCEERR CCEELLLLSS AANNDD MMEETTHHOODDSS OOFF UUSSEE

This application claims the benefit of priority to U.S. Provisional Patent Application serial number 62/075,615, filed November 5, 2014, the contents of which are hereby incorporated by reference.

Background

Thapsigargin. (TG) is a naturally-occurring compound that is -highly specific for inhibiting the sarco/endoplasmic reticulum and endoplasmic reticulum Ca" ^; -ATPase pump (SERCA). The inventors recently identified TG as a modulator of Notch (Roti G., et a!., "Complementary genomic screens identify SERCA as a therapeutic target in NOTCH 1 mutated cancer." Cancer Ceil, 2013, 23(3):3 0-405.). The inhibition of SERCA lead to Notch inactivation, which inhibits T-cell acute lymphoblastic ieukemia (T-ALL) growth both in vitro and in vivo. However, inhibition of the SERCA pump induced by TG leads to an initial depletion of the endoplasmic reticulum (ER) Ca ^{"; '} pool, which ultimately results in an increase in intracellular calcium. Therefore, prolonged exposure to TG could induce apoptosis in a variet of rapidly proliferating cell types in vitro, Thapsigargin is able to kill proliferatively quiescent GO ceils, but is non-specific for any ceil types. Therefore, it is difficult to administer and deliver TG systemically due to significant nonspecific host toxicity (e.g., cardiac toxicity). Therefore, there remains a need to develop methods for selectivity delivering to rapidly proliferating cells a composition having the toxicity and activity of thapsigargin. nmaiary liie. HYfiitti.oa

in certain embodiments, the invention provides a compound, comprising a pharmacophore having the structure of formula (I):

and a celi-targetmg Iigand moiety, wherein the pharmacophore and the cell -targeting iigand moiety are covalently linked.

In certain embodiments, the ceil -targeting Iigand moiety binds to a receptor expressed on the surface of a cell In certain embodiments, die receptor is a folic acid receptor or a CD i 9 receptor.

la certain embodiments, the ceil-targetiag iigand moiety is covalently linked to the pharmacophore through a linking moiety. In eertam embodiments, the linking moiety comprises one or more bonds that are cleavable under physiologic conditions. The one or more bonds cleavable under physiologic conditions can include moieties such as amide, carbonate, carbamate, ether, ester, disulfide, sulfonate ester, sulfonamide, acetal, and/or ketal.

In certain embodiments, the cell-targeting iigand moiety comprises a residue of folic acid or a residue of an antibody.

hi certain embodiments, the cell-targeting iigand moiety is cleaved from th pharmacophore after the compound is delivered to a cell.

In certain embodiments, the compound has the structure of formula (II):

or a pharmaceutically acceptable salt thereof;

wherein R\ R _} R\ R ⁴ , R ³ , and R°, independently for each occurrence, comprises a cell-targeting iigand moiety, or is H, (CO)hydrocarbyl, COOH, hydrocarbyl,

(COj(NH)hydtOcai ^' byl, or (COXMiydrocarbyl; and

wherein at least one of R', R ^'! , R ³ , R ⁴ , R ^' \ and R* comprises a. cell-targeting iigand mo sets' .

in certain embodiments, the R ^! , R", R', R ⁴ , R ^" , or R ⁶ thai comprises a cell-targeting

Iigand moiety further comprises a linking moiety.

In certain embodiments, R\ R^, R\ R ⁴ , R ³ „ and R° ₅ independently for each occurrence, comprises a cell-targeting iigand moiety, or is H or (COihydrocarbyL

in certain embodiments, R ⁴ comprises a residue of folic acid. In certain embodiments, the compound has the structure of formula (III),

or a pharmaceutically acceptable salt thereof.

The invention also provides pharmaceutical compositions comprising a compound of any one of formulae (1), (H), or (HI), and a pharmaceutically acceptabl e exespient.

The invention also pro vides methods of treating cancer, comprising administering to a patieoi in need tiiereof a therapeutically effective amount of a compound of any one of formula (I), (11), or (111 ).

in certain embodiments, the cancer comprises cancer cells over-expressing a folic acid receptor. n certain embodiments,, the cancer is characterized by aberrant acti vity of the NOTCH! gene. In. certain embodiments, the cancer is ovarian cancer, non-small cell lung cancer, breast cancer, multiple myeloma, chronic lymphocy tic leukemia (CLL), acute lymphoblastic leukemia (ALL), B-ee!l lymphoma, meduliohlastoma, colorectal cancer, or Melanoma.

In certain embodiments, the methods of treating cancer further comprise administration of an additional chemotherapeutic agent.

In certain embodiments, the subject is a mammal, for example a human.

The invention further provides methods of inhibiting activation of NOTCH l, comprismg contacting NOTCH with an. amount of a compound of any one formulae (IX (11), or (ill) effective to inhibit NOTCHI .

Brief Description of the Drawings

FIG. 1 shows the design principle of iolate-hased drug delivery.

FIG. 2 consists of panels A-C and contains a series of images demonstrating that thapsigargin-folic acid conjugate Folate-Thap (FT) recapitulates the effect of fhapstgargin at higher concentration. FIG. 2, panel A is a bar graph showing that cells treated with the indicated dose of compounds for 24h demonstrate accumulation of FL-Notchi with reduced TM -NOTCH 1 . FIG 2, panel B is a graph showing that Foiate-Thap causes a loss of Notch in a manner similar to ihapsi argin as measured by flow cytometry. FIG. 2, panel C shows that Foiate-Thap causes arrest of cell proliferation.

FIG. 3 consists of panels A-D and shows results of experiments that determined small molecule delivery by folate, FIG. 3, panel A is an LCMS trace used to identify the existence ofThap-OH in eel! !ysate. FIG, 3, panel B depicts the structures of folate- TAMRA and folate-FiTC. FIG. 3, panel C is a graph showing folate-FiTC uptake dose- dependency, FIG. 3, panel D demonstrates that free folic acid competes with folate-FlTC, FIG. 4 shows CCLE data for the expression of FOLRl in various cell lines,

FIG . 5 is a bar graph demonstrating that uptake of the folate-eorijugated molecule is dependent on expression of the folate receptor.

FiG. 6 contains a series of graphs measuring apoptosis in a pane! of cell lines. FiG. 7 consists of panels a-3 and pictures folate receptor expression in T-ALL. Panel a tabulates expression of PR! or FR2 in 1.7 T-ALL eel! lines and in 3 primary human T-ALL samples. Data were collected using quantitative RT-PC and analyzed using the ΔΔΟΤ method. Panel b shows FR2 level in T-ALL cell lines. Protein expression is detected using an anti~FR2 antibody. Antibody specificity was confirmed including the positive control RPMl 8402-pLX FR.2 engineered to stably express high copies of FOLR2, Panel c depicts the structure of FL-TAMRA and FL-FITC. Panel d is a bar graph showing folate up-take in T-ALL cells measured using a TAMRA probe conjugated to folic acid, T-ALL cells have been treated for hours with the indicated concentrations of FL-TAMRA. Error bars denote the mean SD of 3 replicates. Statistical significance among group for treated vs. vehicle treated (DMSO) {*/* <0.05; **P <( ⁾ .ø! ; ***½0.001 ) was determined by one- way ANOVA using Bonferroni's correction for multiple comparison testing. Statistical analysis were calculated using Prism 5 Software (version 6.05). Panel e is a bar graph showing folate up-take in T-ALL cells and in peripheral blood mononuclear cells (PBMC) pre-treated with 50 ng niL phorboi 12-myristate 13-acetaie (PMA) and 1 itg oil ionoraycio for 6 hours. Treatment with indicated concentrations of FL-FITC or folic acid for 6 hours. Errors bars denote the mean of ^' FITC intensity ± SD of 2 replicates. Statistical significance comparing equiraolar doses of FL-FITC in PBMC vs. DND41 T- ALL cells (***/>< ⁽⁾ .001 , ** ^' **F:<0,OO01 ) was determined by one-way ANOVA using Bonferronfs correction for multiple comparison testing. FIG, 8 consists of panels a-f and is a series of images and graphs showing that FL- FITC uptake is Folate-receptor dependent in T-ALL by endocytosis. Panel a is a bar graph depicting FiTC fluorescence fold increase upon treatment with indicated concentrations of FL-FITC in T-ALL cells overexpressing FR isoforms. Fluorescence signal is depicted as mean fluorescence intensity relative to untreated control. Errors bars denote the mean ± SD of 2 replicates. Panel b is a bar graph depicting FiTC fluorescence fold increase upon treatment with indicated concentrations of FL-FITC in T-ALL ceils (ALL/SIL) cultured in the presence (red) or absence (black) of folic acid. Fluorescence is expressed as relative activity compared to the untreated control. Errors bars denote the mean of FITC

fluorescence intensity ± SD of 3 replicates. Statistical significance for all sample pairs in the experiment (*Ρ<0.05 _; **/*<0.( ⁾ ! ) was determined by one-way ANOVA using

Bonferroni's correction for multiple comparison testing. Pane! e is a bar graph depicting FITC fluorescence fold increase in T- ALL cells (ALL/SIL) cultured in the absence of folic acid and 10 μΜ FL-FITC or 1 uM FL-FITC and 10 uM folic acid. Fluorescence is expressed as relative activity compared to an untreated control Error bars denote the mean of fluorescence FITC intensity * SD of 2 biological replicates. Statistical significance among group (**P<O.01 ) was determined- by non-parametric t-test (Mann- Whitney). Panel d contains flow cytometry-' graphs showing FL-FITC uptake in T-ALL cells in RP!Vl I 8402 cells and RPM1 8402 overexpressing FR isoforms as measured by flo cytometry. Cells were treated with 10 μΜ FL-FITC and subsequently subjected to an acidic wash with PBS 50 mM Glycine pH 4 (blue) or no wash (red) to eliminate cell surface-bound fluorescence. Untreated cells (dotted line) were used as a control for auto fluorescence. Panel e shows the fluorescence intensity increase in T-ALL cells cultured at 37 ^a C or 4 °C upon folate- F1TC treatment as measured by flow cytometry. Experiments were performed in RPMI 8402 overexpressing FR2 isoforms. Panel d shows FiTC fluorescence in four T-ALL cell lines treated with 10 μΜ FL-FITC and prefreated with vehicle (black) or 10 μΜ filipin (red). Errors bars denote the mean ± SD of 4 ceil lines. Statistical significance for

difference in treated vs. control samples (*/ ^> <0.05) was detennined by non-parametric t-test (Matm- Whitney).

FIG. 9 consists of panels a-g and depicts that Thap-OH demonstrates anti-NOTCHi and anti-leukemia properties in T-ALL in viiro. Panel a shows the effect of Thap-OH on SERCA binding. Lysates from T-ALL cells (ALL/SIL) were co- treated with the indicated concentrations of biotinylated thapsigargin or Thap-OH for f> hours and subjected to streptavidin pulldown For 24 hours. The imrmmoblot was stained with SERCA2 and

SERCA3 antibodies. Panel b shows the effect of 24 hours of Thap-OH treatment on

NOTCH 1 cell surface staining as assessed by flow cytometry. Pane! c shows the effect of Thap-OH treatment for 24 hours on NOTCH] (N! } processing and activation in T-ALL cell lines all with HD mutations (DND41 and ALL/SIL (L1594PAPEST) _? PF382

(U 575PAPEST) and RPMl 8402 (in l584PVEL PPE). The blot was stained with an antibody against the C-terrainus of NOTCH 1 that recognizes both the furin-processed

NOTCH ί transmembrane suhunit (TM) and the unprocessed NOTCH 1 precursor (FL). The immunoblot was also stained with anti-ICNl antibody (Val 1744) and GAPDH as a loading control. Panel d shows the effect of Thap-OH treatment on eel! viability after 72 hours of treatment in NOIVHI mutated T-ALL cells (ALL/SIL, DN.D41, PF382, RPMI 8402) or WT (Loucy, MOLT16, SUPTl.1 ). Statistical significance for mutated vs WT C*P<0.05, **P<0.0 i) was determined by one-wa ANOVA with Bonferroni's correction for multiple comparison testing. Panel e shows the effect of Thap-OH treatment (24 hours) on processing of NOTCH 1 mutant (ALL/SIL) or WT (Loucy, MOLT 16) NOTCH 1. NOTCH! (N \ ) was detected with an antibody against the C-terminus of NOTCH! that recognizes the furin-processed NOTCH i transmembrane suhunit (TM) and t!ie unprocessed NOTCH ! precursor (FL). GAPDH was used as loading control. Panel £ shows the effect of Thap-OH treatment (6 and 12 hours) on NOTCH! ceil surface staining as assessed b flow

cytometry. Pane! g shows the effect of Thap-OH treatment (24 hours.) on FR2 in T-ALL cells. Iramunob ^' lot was stained with an antibody against FR2. Vincuiin was used as a loading control.

FIG. 10 consists of panels a-f and shows that JQ-FT demonstrates anti-leukemia properties in T-ALL in vitro. Panel a shows the effect of JQ-FT treatment on ceil growth. Errors bars denote mean £ SD of 4 replicates. Panel b shows the effect of JQ-FT treatment (24 hours) on NOTCH! (N l) processing and activation in T-ALL cell lines all with HD mutations. The blot was stained with an antibody against the C-terminus of NOTCH! that recognizes both the furin-processed NOTCH ! transmembrane subunit (TM) and the unprocessed NOTCH i precursor (FL). The immunoblot was also stained with anti-IC l antibody. GAPDH was used as a loading control. Panel c shows the mean expression of NOTCH 1 target genes in T-ALL cells (ALL/SIL, DND 1) treated for 24 hours with the indicated concentrations of thapsigargin, JQ-FT, Thap-OH, folic acid or the OS! compound E was determined by qRT-PCR. Error bars indicate the mean ± SD of 4 replicates. Data were analyzed using the ΔΔΟΤ method and plotted as a percentage relati ve to the control gene RPLJ3A. Statistical significance among groups for treated vs. vehicle (DMSO) samples (****P≤to.0OQl) was determined by one-way ANOVA with Bonferroni's correction for multiple comparison testing. Panel d shows the effect of JQ-FT treatment (24 hours) on NOTCHI processing in PDX cells in vitro. The blot was stained with an antibody against the C-terminus of NOTCHI (Ni) that recognizes both the furirt-proeessed NOTCH I transmembrane subunit (T ). GAPDH was used as a. loading control. Panel e shows inimunoiluorescence analysis of JQ-FT treatment (24 hours) on NOTCHI activation in permeabilized PDX cells in vitro. Ceils were probed with an anfi- OTCH! antibody (green) and nuclei were eountersfained with D PI. Panel f shows expression of indicated NOTCH! target genes in T- ALL PDX cells treated with JQ-FT for 24 hours was

determined by qRT-PCR. Error bars indicate the mean SD of 4 replicates. Data were analyzed using the A.4CT method and plotted as a percentage relati ve to the control gene RPIJ3A. Statistical significance (***/¾) .001 , ****F<0.0001 ) for treated vs. vehicle (DMSO) was determined by one-way ANOVA with Bonferroni's correction for multiple comparison testing.

FIG. 11 consists of panels a-i and shows that JQ-FT demonstrates activity in T-ALL mouse model. Panel a shows the effect of JQ-FT treatment on ceil growth (72 hours) in murine NOTCHI L160 I APBST expressing leukemia lymphobiasts. Viability data is represented as percentage relative to vehicle treatment and errors bars denote mean ± SD of 3 replicates. Statistical significance of treated vs. vehicle (DMSO) samples (** */>< ^{ ],ø! ***/*≤0.00l) was determined by one-wa ANOVA with Bonferroni's correction for multiple comparison testing. Panel b shows the effect of JQ-FT on 1CN1 levels in murine NOTCH I LI 6 IP AP.ES expressing leukemia lymphoblast cells. The immimoblot contains cell lysates statned with antl-iC 1 antibod (Vail 744) after treatment with the 1.0 μΜ of JQ-FT for 24 hows in vitro. GAPDH was used as a loading control IC l loss was quantified and bar graph (panel c) corresponds to the results of the quantification of three independent experiments. Statistical significance of treated vs. vehicle (DMSO) samples { .05) was determined by Student's t-test Expression of indicated NOTCH I target genes Nasi (panel d) andlMxI (pane! e) in murine NOTCHI LI 60 IP APEST expressing leukemia lymphobiasts treated with 10 μΜ JQ-FT for 24 hours was determined by qRT- PCR, Error bars indicate the mean ± SD of 3 replicates. Data were analyzed using the ΔΔ€Τ method and plotted as a percentage relative to the control gene Gapdh. Statistical significance for treated vs. vehicle (DMSO) (*P≤Q.QS) was determined by Student's t-test. Panel f shows a histological analysis of the spleen and die liver in a NOTCH 1 L .160 IP ΔΡΕ8Τ murine model treated with JQ-FT 60 rag/Kg or vehicle for fi ve days. The spleen and the liver of ail mice were examined; representative results for one control animal and one JQ-FT-treated animal are shown. Formalin-fixed, paraffin-embedded tissue sections were stained using the hematoxylin and eosin stain (H & E) method. Growth suppression of !ymphobiasts (dark purple) was observed in JQ-FT treated animals. Panel g shows the effect of JQ-FT on T-ALL growth in a NOTCH! LI601 P ΔΡΕ5Τ murine model. Anti- leukemic activity of JQ-FT was assessed by measuring spleen weight upon 5 days of JQ-FT treatment (60 nig/kg IP) or vehicle (65% D5W+30% PEG- 00+5% Tween-80only). The chart shows fold change mmor burden for each animal (each dot) and the horizontal bar represents the mean of the four animals per group. Statistical significance for treated vs. vehicle (*P≤Q.Q5) was determined by non-parametric t-test (Maim- Whitney). Panel h shows the antileukemic activity of JQ-FT on hone marrow NOTCH I Li 60 IP ΔΡ.Ε8Τ GFP positive leukemia cells upon 5 days of JQ-FT treatment (60mg/kg TP) or vehicle (65% D5W+3( ⁾ % PECM0O+5% Twccn-SGoniy). Error bars indicate mean ± SD of 4 replicates (of the 4 animals of each group). Statistical significance for treated vs. vehicle (*P<0.O5) was determined by non-parametric t-test (Mann-Whitney). Panel J shows the effect of JQ-FT on Notch activation in a NOTCH 1 L 160 IP ΔΡΕ8Τ murine model. The immunoblot contains splenic cell lysates stained with anti-ICN I antibody (Vail 744) after treatment with the 60 oig/ g of JQ-FT for 5 days. GAPDH was used as a loading control. ICN1 loss was quantified and bar graph (J) corresponds to the results of the quantification. Statistical significance for treated vs. vehicle (*/> 0.05) was determined by Student's t-test,

Detailed Description of the Invention

Thapsigargin is a highly specific inhibitor of sarco/endoplasmic reticulum and endoplasmic reticulum Ca~ ^! -ATPase pump (SERCA) and the Notch signaling pathway. However, thapsigargin also exhibits .non-specific toxicity in various cell types. The present invention is based, at least in part, on the discovery that chemical modification of thapsigargin yields compounds that selectively target certain cells, and in those cells can successfully impair the activation of the Notch signaling pathway, and also minimize the systemic toxicity observed for ihapsigargirt. This discovery can be of use in the treatment of diseases that rely on the Notch signaling pathway, such as certain cancers.

/. Definitions

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "art element" means one element or more than one element.

The term "aeyp is art-recognized and refers to a group represented by the general formula hydrocarbylC(0)-, preferably aSkylCiO)-.

The term "acyiamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula

hyclrocarbyiC(0)NH-

The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbyiC(0)0-, preferably aikyiC(0)0-.

The term "alkoxy" refers to an alkyl group, preferably a lower alky! group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term "aikoxyalkyl" refers to an alky! group substituted with an alkoxy group and may be represented by the general formula a!kyl-O-alkyl.

^' The term: "alkertyP, as used herein, refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyls" and "substituted aikenyls", the latter of which refers to alkeny! moieties having substituents replacing a hydrogen on one or more carbons of the alkenyi group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds.

Moreover, such substituents include all those contemplated for a!kyi groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyi groups by one or more alkyl, carhoeyely!, aryl, heterocyclyl, or heteroaryl groups is contemplated.

An "alkyl' ^' group or "alkarte" is a straight chained or branched non-aromatic hydrocarbon which is completely saturated. Typically, a straight chained or branched alky l group has from 1 to about 20 carbon atoms, preferabiy from 1 to about 10 unless oihenvtsc defined. Examples of straight chained and branched alkyl groups include methyl, ethyl, n~ propyi iso-propyl, n-buiyL sec-butyl tert-butyl, pentyl, hexyl pentyl and octyl. A C Q. straight chained or branched alkyl group is also referred to as a "lower alkyl" group. Moreover, the term "alkyi" (or "lower aikyi") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyis" and "substituted alkyis", the latter of which refers to alky moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, if not otherwise specified, can include, for example, a halogen, a hydroxy!, a carbonyl (such as a carboxyl, an alkoxyearbonyl, a formyl, or an acyi), a thiocarbonyi (such as a thioester, a mioaeetate, or a thioformate), an alkoxyl a phosphoryl, a phosphate, a phosphonate, a phosphmate, an amino, an amido, an amidine, an i ine, a eyano, a nitfo, an azido, sulfhydryl, an alkyhhio, a sulfate, a sulfonate, a su!farooyl, a sulfonamide, a sulfonyl, a hetcrocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety, it wi!i be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted aikyi may include substituted and unsubstituted forms of amino, azido, tmtno, amido, phosphoryl (including phosphonatc and phosphinate), sulfonyi (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyi groups, as well as ethers, aikylthios, carbonyls (including ketones, aldehydes, carboxylases, and esters), -(¾, -CM and the like. Exemplar substituted alkyis are described below. Cycioalky!s can be further substituted with alkyis, alkenyls, alkoxys, aikylthios, aminoal ^' fcyls, carbonyl-su.bstituted alkyis, -CF.?, -C , and the like.

The term "CW when used in conjunction with a chemical moiety, such as, acyi, acyloxy, alkyi, aikenyl, alkynyl, or alkox is meant to include groups that contain from x to y carbons in the chain. For example, the term "Cx-yalk r ^* refers to substituted or

unsttbstituted saturated, hydrocarbon groups, including straight-chain alkyi and branched- chain alkyi groups that contain from x to y carbons in the chain, including haloalkvi groups such as trifluoromethyl and 2,2,2-trifluoroethyL etc. Co aikyi indicates a hydrogen where the group is in a terminal position, a bond if internal. The terms and "C yalkynyl" refer to substituted or unsttbstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyis described above, but that contain at least one double or triple bond respectively.

^" The term "alkylamino", as used herein, refers to an amino group substituted with at least one alkyl group.

The term "aIkyithio'\ as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula aikylS-, The term "alkynyP, as used herein, refers to an aliphatic group containing at least- one triple bond and is intended to include both "uns bstituted alkynyis" and "substituted alkynyis", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are included or not included in one or more triple bonds.

Moreover, such, substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive, for example, substitution of alkynyl groups by one or more alkyl, carbocyclyL aryf heterocyciyi, or heteroaryl groups is contemplated.

The term "amide", as used herein, refers to a group

wherein each R ^!w independently represent a hydrogen or hydrocarbyl group, or two R ^* ' are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

^" The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g. , a moiety that can be represented by

_R 10 _R 10

I— N — N*-R ¹⁰

¹ \ ⁵ \

0 or f¾ ⁰

wherein each R ^H> independently represents a hydrogen or a hydrocarbyl group, or two R ^w are taken together with the atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. In certain embodiments, amine encompasses cyclic amines, including bieyclic amines. In certain embodiments, amine includes DABCO ( 1 ,4-diazabicyclo 2.2.2 Joctane).

The term "aminoa!kyr, a used herein, refers to an alkyl group substituted with an amino group.

The term "aralkyP\ as used herein, refers to an alkyl group substituted -with an ary.i group.

The term "aryi" as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7- membered ring, more preferably a 6-membered ring. The term "aryl" also includes poiycyelic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cyeloalkyls, cycloalkenyls, cycioalkynyis, aryls, heteroaryls, and/or heterocyelyls. Aryl groups include benzene, naphthalene, phenanthrenc, phenol, aniline, and the like.

The term "carbamate" is art-recognized and refers to a group

wherein R ^y and R ^l!> independently represent hydrogen or a hydrocarbyl group, such as an alkyl group, o ⁹ and ^{l u} taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.

The terms "earboeycle", and "carbocyclic" as used herein, refers to a saturated or unsaturated ring in which each atom of the ring is carbon. The term carbocycle includes both aromatic earfooeye!es and non-aroniatie earfooeyc!es. Non-aromatic carbocycles include both, cycloalkane rings, in which all carbon atoms are saturated, and cycloalkenc rings, which contain at least one double bond. ''Carbocycle" includes 5-7 mem ^' bered monocyclic and 8- 12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused, to a saturated or unsaturated ring, e.g., eyclohexane, cyciopentane, or cyclohexeue. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carhoeyc ic. Exemplary ^$ carbocycles" include cyciopentane, eyclohexane, bicyclo[2.2. ! jheptane, 1 ,5- cyclooctadiene, 1 ,2,3,4-tetrahydroeaphthalene, bicyelo 4,2.0joct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include deealin, naphthalene. 1 ,2,3,4- teiTahydronaphthakne, bicyc!o(4.2,0joctane, 4,5,6,7-tetraliydro-lH-indene and bieycloj4.1.0 jhept-3-ene. "Carbocycles" may be substituted at any one or more positions capable of bearing a hydrogen atom.

A "cycloalkyl" group is a cyclic hydrocarbon which is completely saturated. yeloaiky]" includes monocyclic and bicyclic rings. Typically, a monocyclic cyeloalky! group has from 3 to about 10 carbon atoms, more typically 3 to S carbon atoms unles otherwise defined. The second ring of a bicyeiic cyeloalkyl may be selected from: saturated, unsaturated aad aromatic rmgs. Cyeloalkyl includes bicyeiic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused cyeloalkyl" refers to a bicyeiic cyeloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyeiic cyeloalkyl may be selected from saturated, unsaturated and aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

The term "carbocyclylalkyl", as used herein, refers to an alkyl group substituted with a carbocycle group.

The term "carbonate" is art-recognized and refers to a group -GCG R wherein

R ^us represents a hydrocarbyl group.

The tenn "carboxy", as used herein, refers to a group represented by die formula

-CCbH.

The term "ester", as used herein, refers to a group -€(0)OR ^ift wherein R ^l!> represents a hydrocarbyl group.

The term "ether ^1* , as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substttuent of a hydrocarbyl group may be hydrocarbyl-O. Ethers may be either symmetrical or unsymmefrical.

Examples of ethers include, but are not limited to, heterocycle-O-heterocyc!e and aryl-O heterocycle. Ethers include "a!koxyalkyl" groups, which may be represented by the general formul a a!kyl-O-alky 1.

The terms "halo" and "halogen" as used herein means halogen and includes chloro, f!uoro. hromo, and iodo.

The terms "hetaralkyl" and "heteroaralkyr, as used herein, refers to an alky! group substituted with a hetaryl group.

The tenn "heteroalkyl", as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatoro, wherein no two heieroatoms are adjacent.

The terms "heteroaryP and "hetaryl" include substituted or unsubstit ted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6- membered rings, whose ring structures include at least one heteroatom, preferably one to lour heteroatoms, more prefer bly one or two heieroatoms. The terms "heteroary and "hetaryl" also include polycyc!ic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rmgs can be cycloalkyls, cycloalkenyls, cyeioalkynyls _f aryls, heteroaryls, and/or feeteroeyclyls. Heteroaryl groups include, for example, pyrrole, fts an, thiophene, imidazole, oxazole, tSiiazole, pyrazoSe, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

The tenn "heteroatom" as used herein means an atom of any element other than carbon or hydrogen.. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

The terms "heterocyciyi" "heteroeyele", and "heterocyclic" refer to substituted or unsubstitute . non-aromatic ring structures, preferably 3- to iO-membered rings, more preferably 3- to 7-me.rabered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyP and. "heterocyclic" also include polvcyciic ring systems having tvvo or more cyclic rings in which tvvo or more carbon arc common to two adjoining rings wherein at least one of the rings is ^' heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, lieteroaryls, and/or heteroeyelyls, Heterocyciyi groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term "heterocyclylalkyl", as used herein, refers to art alky! group substituted with a heterocycie group.

Tire tenn "hy drocarbyl", as used herein, refers to a group that is bonded through a carbon atom that does not have a™0 or ~S substituent, and typicall has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyS, and trifluoromethyi are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a ^::: 0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. ^' Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carboeycSe, heterocyciyi, alky i, alkenyl, alkynyl, and combinations thereof.

Tire tenn "hydroxyalkyl", as used herein, refers to an alkyl group substituted with a hydroxy group.

^" The tenn "lower" when used in conjunction with a chemical moiety, such as, acyl, aeyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer non-hydrogen atoms in the substituent, preferably six or fewer. A "lower alkyl", for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. Jn certain embodiments, acyl, aeyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyi, iower alkenvi iower alkynyl, or iower alkoxy, whether they appear alone or in combination with other substiiiients, such as in the recitations hydfoxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in die alkyi substituent).

The terms "potycyciyl" "polycycie", and "po!ycycHc" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, ary!s, heteroaryls, and/or heterocyclyis) in which two or more atoms are common to two adjoining rings, e.g., the rings are "fused rings". Each of the rings of the polycycie can be substi tuted or unsubstituted. in certain embodiments, each ring of the polycycie contains from 3 to it ⁾ atoms in the ring, preferably from 5 to 7.

The term: "siiyP refers to a silicon moiety with three hydrocarbyl moieties attached thereto.

The term "substituted" refers to moieties having substituents- replacing & hydrogen on one or more carbons of the backbone, it will be understood that "substitution" or

"substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, eyclization, elimination, etc. As used herein, the terra

""substituted" is contemplated to include all perraissibie substitue ts of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocycHc and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The perraissibie substituents can be one or more and the same or di fferent for appropriate organic compounds. For purposes of this in vention, the

heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbony! (such as a carboxyi, an a!koxycarbonyl, a forniyt, or an acyl), a thioearbonyi (such as a thioester, a thioacetate, or a thio formate), an alkox i, a phosphor L a phosphate, a phosphonate, a p ^' hosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryi, an alk lthio, a sulfate, a sulfonate, a sulfarnoyi, a sulfonamide, a sulfonyi, a heteroeyclyJ, an aralkyl, or an aromatic or heteroaromatic moiety, it will be understood by those skilled in the art that substiiiients can themselves be substituted, if appropriate. Unless specifically stated as "unsubstita.ed," references to chemical moieties herein are understood to include substituted variants. For example, reference to an "ary!" group or moiety implicitly includes both substituted and urvsu bsti tuted variants.

The term "sulfate" is art-recognized and refers to the group -OSQjH, or a pharmaceutically acceptable salt thereof.

The term "sulfonamide" is art-recognized and refers to the group represented by the general formulae

wherein R ^} and R' ^w indepeiidently represents hydrogen or ^' hydrocarbyl, such as aikyl, or R ^¾ and R ^>v taken together with the intervening atomis) complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term "sulfoxide' is art-recognized and refers to the group -S(0)-R ^!t \ wherein

R ^so represents a hydrocarbyl . In certain embodiments, the sulfoxide may be a stereogenie center. In certain such embodiments, the compounds may be enriched for one isomer of the sulfoxide.

The term "sulfonate" is art-recognized and refers to the group SO^H, or a pharmaceutically acceptable salt thereof. A sulfonate ester refers to a group -S(0)rOR ^{l> , wherein R ⁱ represents a hydrocarbyl

The term "sulfo«c ^,s is art-recognized and refers to the group -S{Oh-R ^,if , wherein R ^l ° represents a hydrocarbyl.

The ten "tliioalkyl' ^* , as used herein, refers to an alkyi group substituted with a thiol group.

^" The term "thioester", as used herein, refers to a group -C(0)SR ¹⁰ or -SC(O)R ⁱ⁰ wherein R ^Ui represents a hydrocarbyl.

The term "thioether", as used herein, is equivalent to an ether, wherein me oxygen is replaced with a sulfur.

The term "disulfide" refers to a group -S-S-R ⁵ ", wherein represents a hydrocarbyl. The term "ure " is art-recognized and may be represented by the genera! formula

wherein R ⁹ and R ^!u independently represent hydrogen or a hydroearbyl, such as al.kyl, or either occurrence of R ^v taken together with ⁱ and the intervening atom(s) complete a heterocycle ^' having from 4 to 8 atoms in the ring structure.

"Protecting group" refers to a group of atoms that, when a ttached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective- Groups in Organic Chemistry, 3 ^rd Ed , i 99 , John Wiley &- Sons, NY and Harrison et al., Compendium ofSjmthetic Organic Methods, Vols. 1-8, 1971 -1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, forni !, acetyl, trifluoroacetyl, benzyl, benzy!oxyearbonyl ("CBZ"), tert-butoxycarborryl ("Boc"), trimethylsiiy! ("TMS"), 2-trimethyisilyI-ethanesulfonyi C'TJES"), trityi and substituted trityi groups, aliyioxycarbonyL 9-fluorenyiraethyloxyca.ri>onyi ("FMOC"), uttro-veratryioxyearbonyi ("NVOC") and the like. Representative hydroxyJproteeting groups include, but are not limited to, those where the hydroxy! group is either acyiated (esterified) or alkylated such as benzyl and trityi ethers, as well as aikyl ethers, tetrahydropyranyl ethers, triaikySsilyl ethers (e.g., T S or TIPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.

The term '"prodrug" is intended to encompass compounds which, under physiologic conditions, are converted into the therapeutically active agents of the present invention (e.g., a compound of formula !). A common method for making a prodrug is to include one or more selected moieties which are hydrolyzed under physiologic conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxyi ic acids) are preferred prodrugs of the present invention. In certain

embodiments, some or all of the compounds of formul Ϊ in a formulation represented above can be replaced with the corresponding suitable prodrug, e.g., wherein a hydroxy 1 in the parent, compound is presented as an ester or a carbonate or carboxyiic acid present in the parent compound is presented as an ester. Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally-occurring forms of antibodies (e.g., IgG, IgA, IgM, igE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

The term "antibody" as used herein also includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen- inding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term: "antigen-binding portion" of an antibody include (i) a Fab fragment a monovalent fragme t consisting of the VL, VH, CL and CHI domains; (ti) a f (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region: (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward el al. , (1989) Nature 341 :544-546), which consists of a VH domain; and fvi) an isolated complementarity determining region CCDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain ii» which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g.. Bird et al. ( 1988) Science 242:423-426; and Huston ei al (\ 9HH) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn el al. 1998, Nature Biotechnology 16: 778), Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, hi order to generate expression vectors encodin complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab , Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodtes are also

encompassed, Diabodics are bivalent, btspeeific antibodies in which VH and VL domains arc expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domams of another chain and creating two antigen binding si tes (see e.g., Holliger, P., el al ( l 93) Froc. NatL Acad. Set USA 90:6444-6448; Poljak, R, J., et al. ( 1 94) Structure 2: 1121- 1 123).

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g,, humanized, chimeric, etc.). Antibodies may also be .folly human. The terms "monoclonal antibodies" and "monoclonal antibody composition", as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of imniunoreaeting with a particular epitope of an antigen, whereas the term "polyclonal antibodies" and "polyclonal antibody composition'" refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it imtmtnoreacts.

The terms "cancer" or "tumor" or "hypciproliferatiye disorder" refer to the presence of cells possessing characteristics typical of cancer-causing cells, such, as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a solid tumor, but such cells may exist alone within an animal, or may be a non-tirniorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobitlinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mo. chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheiiosareoma, lymphangiosarcoma, lymphangioendothe osarcoina, synovioma, mesothelioma, Ewing's tumor. leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squaraous cell carcinoma,, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, eystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal ceil carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain rumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, meduiloblastoma,

craniopharyngioma, ependymoma, pineaioma, hemangioblastoroa, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (rm/eloblastic, promyelocyte, myeloraonoeytic, monocytic and erythroieukeraia); chronic leukemia

(chrome myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's maerogSobulinemia, and heavy chain disease In certain embodiments, the cancer is characterized by aberrant activity of the NOTCH I gene or the Notch signaling pathway. In certain embodiments, the cancer is ovarian cancer, non-small eel! lung cancer, breast cancer, multiple myeloma, chronic lymphocytic leukemia (CL ^' L), acute lymphoblastic leukemia (ALL), B-cell lymphoma, meduiloblastoma, colorectal cancer, or melanoma.

The term "gene expression data" or "gene expression level" as used herein refers to information regarding the relative or absolute level of expression of a gene or set of genes in a cell or group of cells. The level of expression of a gene may be determined based on the level ofR A, such as rriRNA, encoded by the gene. Alternatively, the level of expression may be determined based on the level of a polypeptide or fragment thereof encoded by the gene. Gene expression data may be acquired for an individual ceil, or for a group of cells such as a tumor or biopsy sample. Gene expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a microarray or chip reading device. Such gene expression data can be manipulated to generate gene expression signatures.

As used herein, the term "inhibit" includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. For example, cancer is "inhibited" if at least one symptom of the cancer, such as hyperproliferao ^' ve growth, is alleviated. terminated, slowed, or prevented. As used herein, cancer is also ^' inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented

The term "modulate includes up-reguiation and down-regulation, e.g., enhancing or inhibiting a response.

The tenn "subject" refers to an healthy animal, mamma! or human, or any animal, mammal or human afflicted with a condition of interest (e.g., cancer). The term "subject" is interchangeable with "patient," in other embodiments, the subject has ovarian cancer, non-small eel! lung cancer, breast cancer, multiple myeloma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or B-ce!l lymphoma.

^' The invention provides compounds that comprise a pharmacophore derived from thapsigargin and a itgaud for a. cell surface receptor that can selectively target cancer cells and induce apoptosis via inhibition of the Notch signaling pathway (FIG. 1).

Ttiapsigargirt

In certain embodiments, the invention provides a compound comprising a pharmacophore of .formula (I) and a cell-targeting ligand moiety, wherein the

pharmacophore and the cell-targeting ligand moiety are covaJentiy linked, and where formula (I.) is represented by:

In certain embodiments, the pharmacophore derived ftom thapsigargin has the structure of formula (l.) _t wherein the wa vy bond represents sites where the pharmacophore may be derivatized by the cell-targeting ligand moiety. The pharmacophore may relate to the parent structure of thapsigargin via truncation at, for example, any hydrolyzabie bond present in the parent structure,, such as an ester bond.

Formula (i) depicts one stereochemical isomer of the pharmacophore included in the compounds of the invention. However, the pharmacophore derived from thapsigargin further encompasses enantiomers, diastereomers, and epiraers of the pharmacophore depicted in formula (I).

The pharmacophore of formula (I) may be covaiently linked to one, two, or more cell-targeting ligand moieties. In certain embodiments, one cell-targeting ligand moiety is covaiently linked to the pharmacophore of formula (!) at two different positions on the pharmacophore, as allowable by valence and molecular geometry.

In certain embodiments, the compounds of the invention provide pro-drug forms of thapsigargin and thapsigargin derivatives.

in certain embodiments, the cell-targeting iigaod. moiety binds to a receptor expressed on the surface of a cell. In certain embodiments, the receptor expressed on the surface of the cell is particular to a certain eel! type, or is over-expressed in a certain eel! type. A cell type associated with a disease or disorder, for example, a cancer, can express a receptor at a higher level than other cell types. Accordingly, the ligand-receptor recognition interaction can enable cell-selective dr g delivery.

Exemplary receptors thai can hind to the ligand moiety include folate receptors and C 19 receptors. Folate receptors include four different isoforms of the receptor. In certain embodiments, tire compounds of the invention target FRl (α)-2(β). Folate receptors may be over-expressed in certain cancers, such as in ovarian cancer cells. For example, data supports that the folate receptors are expressed in the majority of non-mucinous epithelial ovarian tumors at levels that are 10- to 100-fold higher than the normal expression of the folate receptor in the kidney, Sung, and breast epithelial cells (FIG. 4). Therefore, the compounds of the invention are advantageous due to their specificity toward cancer cells in the presence of normal ceils, minimizing systemic toxicity .

In certain embodiments, the receptor is a folic acid receptor or a CD 1 receptor. In certain embodiments, the cell-targeting ligand moiety is covaiently linked to the pharmacophore through a linking moiety, in certain embodiments, the linking moiety comprises one or more bonds that are cleavable under physiologic conditions. The one or more bonds cleavable under physiologic conditions can include moieties such as amide, carbonate, carbamate, ether, ester, disulfide, sulfonate ester, sulfonamide, acetal, ketal, or other acid- or base-labile bonds, in certain embodiments, two substitittable positions of the pharmacophore, for example adjacent suhstitutable positions, are each covaiently bound to a linking moiety, which in turn covaiently link ^' s the pharmacophore to a cell-targeting iigand moiety.

In certain embodiments, the cell-targeting ligaed moiety comprises a residue of folic acid or a residue of an antibody.

In certain embodiments, the pharmacophore derived from tliapsigargin can be chemically modified by a residue of biotin. In certain embodiments, the pharmacophore is covaiently bound to a residue of biotin. In certain such embodiments, the compound can be referred to herein as "hiotinylated thap". In certain embodiments, the biotiriylatecl map is useful in chemical sequencing.

In certain embodiments, the residue of folic acid is folic acid, substituted by a linking moiety at any sufastitutabk position (e.g., -Nj¾, -Mi, COOH), valence permitting. In certain embodiments, the residue of folic acid is folic acid, substituted by a linking moiety at either of the earboxy!ie acid moieities. In certain embodiments, the residue of folic acid is folic acid, substituted by a linking moiet at the terminal carboxylie acid moiety.

in certain embodiments, the antibody is an anti-imrmmoglobuHn antibody, or any other antibody as described herein.

In certain embodiments, the cell-targeting ligaed moiety is cleaved from the pharmacophore after the compound is delivered to a cell. For example, the conjugate can target a cell -surface receptor, be taken into the ceil via endoeytosis, and then the cell- targeting ligaed moiety can be cleaved from the conjugate, releasing the pharmacophore. In certain embodiments, the pharmacophore, once released from the conjugate, is an acti ve drag residue, In certain embodiments, the cleavage is a. result of the pH of the cytoplasmic matrix.

In certain embodiments, the cell-targeting ligaed moiety is cleaved from the pharmacophore via cleavage of one or more bonds in the linking moiety. For example, under physiological conditions, bonds such as disulfide bonds, amide linkages, and ester linkages may cleave or hydrolyze, thus separating the pharmacophore from the cell- targeting ligaed moiety. In certaiii embodiments, the compound of the invention has the s tructure of formula

or a pharmaceutically acceptable salt thereof;

wherein R\ R ^~ , R\ R , R ^" \ and R", independently for each occurrence, comprises a cell-targeting itgand. moiety, or is H, (CO)Jiydroearbyi, COOH, hydrocarbyl,

(COXNH)hydrocarbyl, or (CO)0-hydrocarbyl; and

wherein at least one of R ^! , R ^' \ R:\ R , R\ and * comprises a cell-targeting ligartd moiety.

Formula (H) depicts one stereochemical isomer of the pharmacophore included in the compounds of the invention. However, the pharmacophore derived from rhapsigargin further encompasses en antiorriers, diastereomers, and epimers of the pharmacophore depicted in formula (I I ).

In certain embodiments, the R ^! , R\ R;\ R ^"! , R\ or R ^h that comprises a cell-targeting iigand moiety further comprises linking moiet .

In certain embodiments, R\ R\ R ^J , R\ R ⁵ , and R ^' \ independently for each

occurrence, comprises a cell-targeting iigand moiety, or is H or (CQ)hydroearbyl

In certain embodiments, (CO)hydrocarbyl includes or (CO)(Cr C3«)alkenyl.

in certain embodiments, one or more of R\ R R ^"1 , R\ R or R ¹ ' comprises a residue of folic acid, in certain embodiments, one of R ¹ , R ^" , R\ R \ R ^" , or R ^h comprises a residue of folic acid. In certain embodiments, R ⁴ comprises a residue of folic acid, in certain embodiments, * comprises a residue of folic acid and a linking moiety.

Exemplary compounds provided by the invention include compounds of formulae (ΪΪΪ), (IV.), (V), (VI), (VH), (VIII), or (IX); wherein formula (ill) is represented by:

(ΊΥ>:

if) wherein formula (V) is represented by:

wherein formula (V wherein formula (VHI) is represented by:

wherein formula (IX) is represented by:

///. Pharmaceutical Compositions of (he Ccwjugftie

In certain embodiments, the invention also provides pharmaceutical compositions- comprising a compound of die invention and a pharmaceutically acceptable excipient.

The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to art animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions sueh as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters, in a preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-frce, or substantially pyrogen-free. The excipients cart be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.

In certain embodiments, the composition is a fbrai suitable for injection, systemic administration, or topical administration. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule. lyophile for reeonstitution, powder, solution,, syrup, suppository, injection or the like. The compos! HOB can also be present in a transdermal delivery system, e.g., a skirt patch.

The composition can also be present in a solution or suspension suitable for topical administration. The topically applicable form of the composition can a transdermal patch, ointment, cream, gel, suspension, liquid, elixir, or eye drop.

A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextratvs, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or exeipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery- ^* system or a sclf-microemulsifymg drug delivery system, The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metaboli able carriers that are relatively simple to make and administer.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animal without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmace tically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, exeipient, solvent or encapsulating material. Each carrier must be

"acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: ( ! ) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxyracthyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) exeipients, such as coco butter and suppository waxes: (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil com oil and soybean oil; (10) glycols, such as propylene glycol; ( 11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; {.16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a. subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules

(including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectal iy or vaginally (for example, as a pessary, cream or foam); parenteral! γ (including intramuscularly, intravenously, subcutaneousiy or intrathecally as, for example, a sterile solution or suspension); nasally; mtraperitoiieally; subcutaneousiy; transdermaliy (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound ma also be formulated for inhalation, in certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. os. 6,1 10,973, 5,763,493, 5,731,000,

5,541 ,231 , 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferabl from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid earners, or both, and thee, if necessary, shaping the product

Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a fla vored basis, usually sucrose and acacia or tragacaaih), lyophiie, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in-water or water-m-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalciam phosphate, and or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, raannitoS, and/or silicic acid; (2) binders, such as, for example, earhoxynieihyleellulose, alginates, gelatin, polyvinyl pyrroHdone, sucrose and/or acacia; (3) huraectants such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium siearate, solid polyethylene glycols, sodium iauryl sulfate, and mixtures thereof; ( 10) complexing agents, such as, modified and unmodified cyckxiextrins; and ( 11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipteats as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, dismtegrant (for example, sodium starch gl colate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound -moistened with an inert li uid diluent,

The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and grannies, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredien therein using, for example, hydroxyprop lmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the

gastrointestinal tract, optionally, in a delayed manner. Examples of embedding

compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage farms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reeonstttution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsi tiers, such as ethyl alcohol, isopropyi alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, ! ,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbifan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, .flavoring, coloring, perfuming and preservative agents. Suspensions, n addition to the active compounds,, may contain suspending agents as, for example, ethoxylated isostearyi alcohols, polyoxyethylene sorbitol and sorbitan esters, micfocrystalline cellulose, aluminum metahydr oxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.

Formulations of the pharmaceutical compositions for recta!, vaginal, or urethral administration may be presented as a. suppository, which may be prepared by mixing one or more active compounds with one or more suitable uonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as arc known in the art to be appropriate.

Alternatively or additionally, compositions can be .formulated for delivery via a catheter, stent, wire, or other intraluminal device. Deli very via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

Dosage forms for the topical administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, cream and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentomtes, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

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 cat} 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.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of

microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobiitanol, phenol sorbic acid, and the like. U may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceiiticai form ma ^¬ be brought about by the inclusion of agents that delay absorption such as aluminum tnonostearate and gelatin.

/ ί ^' . Methods of Using the Conjugates of (he [mention.

In the last decade, NOTCH! has been identified as one of the most frequently mutated genes across all cancers. In hematologic malignancies, activating NOTCH I mutations are observed in chronic lymphocytic leukemia (CLL), mantle ceil lymphoma, and at an exceptionally high rate in T-ALL, where NOTCH! mutations represent the most common actionable genetic abnormality. Targeted NOTCH I therapies, such as garoma- secretase inhibitors and receptor-blocking antibodies have entered early-stage clinical trials. However, these modalities have the liability of inhibiting normal NOTCH 1 arid NOTCH2. In addition to the known potential for gut toxicity, there is also a significant concern for secondary malignancies as Notch receptors are established context-specific tumor

suppressor genes. Thus, the development of tumor-directed inhibitors with selective activity against mutated proteins is highly desirable.

Although inhibition of SERCA proteins to selectively target mutated NOTCH! with free thapsigargin is promising, thapsigargin is poorly tolerated. The present invention provides improved methods for inhibition of SERCA proteins using a tbapsigargin-folate conjugate.

The present invention provides methods of small-molecule tola te-roedia ted delivery of thapsigargin to enable selective and target-specific drug delivery to T-ALL cells, in certain embodiments, the inhibitor, thapsigargin, is connected to folic acid with a cleavable bond and is transferred into the ceil after binding to FR on the cell surface. The expression of FR in T-ALL enabled the selective recognition of the designed molecule, JQ-FT b cancer ceils. The cieavabie bond feature of the molecule facilitated direct delivery of the inhibitory motif to the target (SERCA) and subsequently blocked mutant NOTCH 1 maturation. This strategy avoided complicated manufacturing processes, such as drug- antibody conjugation,, but still allowed selective delivery to the cancer ceil indeed, the MTD of JQ-FT in mice was 150 fold above thapsigargin, supporting the more selective uptake of the derivaiized product Importantly, in our in vivo experiments, mice were not restricted to a low folate chow to demonstrate FR-mediated antitumor effect i» vivo. Such a strategy had been reported in previously published preclinical studies testing folate-drug conjugates. JQ-FT treatment with a low folate diet is expected to have even greater efficacy in vivo.

The present invention enhances die therapeutic window of thapsigargin as a

NOTCH 1 inhibitor providing dual selectivity: leukemia over normal cell and NOTCH! mutated over WT receptors. Given the important role of mutations in NOTCH! in many cancers, JQ-FT offers a potential strategy in treating other tumors with NOTCH I mutations, such CLL and non-small cell lung cancer, Furthermore, our report demonstrated both in viiro and in vivo that the folic acid-assisted, pathway-specific drug delivery strategy could be an efficient method to solve a common drug delivery problem in the present era of targeted cancer therapy.

Accordingly, in certain embodiments, the invention provides methods of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the invention that is described herein.

In certain embodiments, the cancer cells targeted b the conjugates of the invention over-express a receptor type, in certain embodiments, the cancer cells express a receptor type at a higher level than non-cancer cell types. In certain embodiments, the cancer cells over-express a folic acid receptor.

In certain embodiments, the cancer is characterized by aberrant activity of the

NOTCH ! gene or the Notch signaling pathway.

In certain embodiments, the cancer is ovarian cancer, non-small ceil lung cancer, breast cancer, multiple myeloma, chronic lymphocytic leukemia (CLL), acute

lymphoblastic leukemia (ALL), B-eell lymphoma, roedullobiasioma, colorectal cancer, or melanoma. For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level wili depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular eompound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular

eompound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and l ike factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition req uired. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By ""therapeutically effective amount" is meant the concentration of a compound tha t is sufficient to elicit tire desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be deli ered by multiple

administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isseibaeher el aL_ ( 1996) Harrison's Principles of Internal Medicine 13 ed,, 1814- 1882, herein incorporated by reference).

in general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effec Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

The patient receiving this treatment is any animal m need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

The methods of the invention further comprise administering to the patient a therapeutically effective amount of an additional chemotherapeutie agent.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, ^" 72 hours, or a week of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

The invention further provides methods of inhibiting activation of NOTCH I , comprising contacting NOTCH I with a compound of the invention described herein in an amount of effective to inhibit NOTCML

^' The invention further provides methods of inhibiting the Notch signaling pathway, comprising contacting a c ell with a compound of the invention described herein in an amount of effective to inhibit the Notch, signaling pathway.

In certain embodiments, the inhibition of NOTCH! activation is measured b determining the level of A, such as niRNA, of NOTCH I target genes such as RES !., c- MYC, or DTX1. Alternatively, inhibition may be determined based on the level of a polypeptide or fragment thereof expressed b the gene. In certain embodiments, inhibition of NOTCH 1 will be also measured using a complex Notch off signature originally

- 3? - developed by the inventors n a Notch GE-HTS screen, in certain embodiments, NOTCH ! inhibition is measured by flow cytometry. In certain embodiments, NOTCHi inhibition is measured in heterologous sy stem in which different Notch isoforms can be co-expressed with a. specific promoter expressing luciferase.

In certain embodiments, one or more cells are contacted with biotinylated thap under conditions sufficient to inhibit NOTCHi. An antibody targeting biotin can be used to pull down the proteins bound to the thapsigargin derivative (i.e., the '"thap" moiety of biotinylated thap). Such proteins can be identified via mass spectrometry, in certain embodiments, methods that identif proteins having an affinity for the thapsigargin derivative are used in the identification of SERCA isoforms that are preferentially inhibited in cancer types such as T- ALL. in certain embodiments, methods that identify proteins having an affinity for the thapsigargin derivative are used in the identification of protein targets for thapsigargin.

Example I. Synthesis of Thapsig rgin-Folic acid Coiyugaies md Fluorescent Derivatives. Materials and Methods

Reactions were run as described in the individual procedures using standard double manifold and syringe techniques; glassware was dried by baking in an oven at 130 X tor 12h prior to use. Solvents for reactions were purchased anhydrous from Sigma-Aldrich and used as received. HPLC grade solvents were used for aqueous work ups and

chromatography. Reagents were used as received, Reaciioos were monitored by thin-layer chromatography using HMD silica gel 60 F254 (250-mieron) glass-backed plates

(visualized by UV fluorescence quenching and staining with KMnO.*) and by LC-MS using a Waters Aquiiy BEH CI 8 2 x. 50 mm. 1,7 μ ^η ι particle column (50 °C) elutmg at 1 mL/ratn with HiO acetonitriie (0.2% v/v added formic acid; 95:5 (0rain)-->5:95 (3.60rain)] using alternatirig positive/negative electrospray ionization (125-1000 amit) and UV detection (210-350 nm). Flash column chromatography was carried out using Merck grade 9385 silica gel 60 A pore size (230-400 mesh). Melting points were obtained using a capillary melting point apparatus and are uncorrected. ^! H NMR spectra were recorded at 400 MHz on a Broker spectrometer and are reported in ppm using the residual sol vent signal

(dimewylsuifoxide-dj, - 2.50 ppm; chloroform-d™ 7.27 ppm; methaaol-cU ^::: 3.31 ppm; dichloromethane-d? ^::: 5.32 ppm) as an internal standard. Data are reported as: |(δ shift). |(s ~ ^: singlet, d - doublet, dd, doublet of doublets, ddd ~ doublet of a dd, t ~ triplet, quin™ quintet, sept - septet, br - broad, ap - apparent} _; , (J - coupling constant m Hz) and (integration) ^' }} . Proton-decoupled 13C N R spectra were recorded at 5 MHz on a Bruker spectrometer and are reported in ppm using the residual solvent signal (ehiorofonn- d - 77.0 ppm; dimeihyisulfoxide-dt - 39.51. m; metijanol-d* - 49.15 ppm) as an internal standard, infrared spectra were recorded using an ATR-FTIR instrument. High resolution mass spectra were acquired by flow injection on a q ^' T ^' GF Premiere Mass Spectrometer operating in ES÷ ionization with resolution -- I 5,000.

The folate-ihapsigargin derivative of the invention was designed based on the following principles: the dual function molecule should actively bind to folate receptor (FR): the connection between folic acid and thapsigargin should be stable in serum but cleaved in the intracellular compartment; and the resulting thapsigargin derivative should potently inhibit SE ^' RCA activity. As shown below, the butyl ester bond at C8 was readily and selectively cleaved from the isolated natural product under basic conditions to produce the secondary alcohol, 8- -debutanoylthapsigargin (Thap-OH). The carboxviate of folic acid was then conjugated to the C8-a!eohol of Thap-OH to generate the ioiate-eoryugate JQ-FT.

Scheme 1. Synthesis of JQ-FT

To a solution of thapsigargin (200 rag, 0.31 raraoi) in methanol (4 raL), triethylamine (0,5 niL) was added at 23 °C. The resulting clear solution was stirred at 23 °C for 6 h. The sol ent was removed in vacuo. The crude reaction was purified directly using column chromatography (MeOH-CHaCt?, 0 to 15% gradient), and produced Thap-OH as white foam ( 1 0 mg, 94% yield). MS: mfz (MH ) ^: : 581.3.

To a solution of Thap-OH (17 rag) in DMSO ( 1 .6 raL) was added folic acid (27 mg, 0,06 mrnol), .«V, iV-dicyclohexyicarbodnmide (DCC, 9-7 mg, 0.077 mrnol), and 4- dimethyiamitK>pyridine (DMAP, 3.8 mg, 0.031 mrnol). The -reaction was stirred at 23 °C for 16 h. The reaction mixture was further diluted with methanol, and was directly purified by HPLC to afford JQ-FT as yellow powder (13 mg, 45% yield). ^X H NMR (500 MHz, DMSO-i/«) 5 ppm 0.79 - 0.89 (m, 5 11) 0.98 - l .02 (m, 1 H) 1.1 1 - 1.17 (m, 4 H) 1.19 - 1 .33 (m, 1 H) 1.44 - 1.55 (m, 3 H) 1.64 - 1.75 (m, 5 H) 1 .78 - 1.82 (m, 8 H) 1.85 - 1 .90 (m, 5 H) 1.95 - 2.02 (m, 2 H) 2.18 - 2.35 (m, 6 H) 3.07 (d, J- 1 1.60 Hz, 1 M) 4.26 - 4.32 Cm, 1 H) 4.35 (br. s., I E) 4.45 - 4.52 (ra, 3 H) 5.24 - 5.27 ( n, 1 H) .139 (t, J~ 3.5 Hz, 1 H) 52 (br. s„ 1 H) 5.59 (br. s., I H) 6.03 - 6. 9 (m, 5 H) 6.55 - 6.71 (ra, 3 H) 7.61 (d, J- 8.85 Hz, 3 H) S.25 id. ,/ = 7.63 Hz, 1 H) 8.60 - 8.71 (m, 2 H). MS: m/z (MH)*: 1004.4.

Scheme 2. Synthesis of fluorescence folic acid derivatives.

SH. (Boc)?0 (2.92 g, 13.36 mnioi) was dissolved in THF (20 mL). 3- ainiiiopropanoi ( 1 ,01 raL, 13.31 minol) was added and the reaction was stirred at room temperature for 1 h. The reaction was concentrated in vacuo to yield the desired product, AC V- 1 -076, as a clear oil

SI-2. Folic acid (502.5 mg, .1.1.4 mmoi) and DMAP (692,5 mg, 5.67 mrnol) were suspended in DMSO (5 raL). A-'.iV -Diisopropylcarbodiiminide (DIC, 350.8 uL, 2.27 mrnol) was added to the reaction Followed by a solution of SI-1 (204.9 mg, 1.17 ramol) in DMSO (2 mL). The reaction was stirred at room temperature and an additional 2 mL of DMSO were added to help soSubilize the reacfanfs. The reaction was stirred at room temperature overnight Water was added to the solution and a yellow-orange solid precipitated out. The reaction was filtered and the filtrate was collected and lyophilized. The residue was re- dissolved in methanol and purified via HPLC to give the desired product, Si-2, as a yellow- orange solid. MS: m/z 599.5 (M+l)\

SI-3. Sl-2 was suspended in 4M HC! in dioxane (6 mL) and stirred at room temperature for 3.5 h. The reaction was concentrated in vacuo, redissolved in methanol and purified via HPLC to give the desired product SI-3. MS: m/z 499,4 (Μ-ί- 1) ^" \

L-FfTC. Free amine, SI-3 (37.5 mg, 0.08 mmol) and D1PEA ( 133.4 uL, 0.77 mmol) were suspended in THF (5 mL). MeCN (i nil.) was added to the reaction to help the solubility of the reaction. FiTC (26.7mg, 0,07 mmol) was then added and the reaction was stirred at room temperature for 3 h. The reaction was purified directly via HPLC to afford the desired product, FT-FfTC as a yellow solid. ¾ NMR. (500 MHz, DMSC s) d ppm 1.18 - 1.30 (m 2 H) 1 .56 - 1.64 (m, 1 H) 1.73 - 1.82 (ax 2 H) 1.85 - 2. 13 (ra, 5 B) 2.31 - 2.45 (m, 4 H) 3.15 - 3.21 (m, 6 H) 4.03 - 4.16 (m, 3 11) 4.32 (s, 1 H) 4.49 (s, 2 H) 6.50 - 6.72 (m, 1 7 H) 6.98 (d, ,/ - 7.63 Hz, I H) 7.09 (d, ·./ = 8.24 Hz, I H) 7.17 (d, J - 8.54 Hz, 1 1:1) 7.57 - 7.69 (m, 4 H) 7.70 - 7.77 (m, 1 H) 8.08 - 8.25 (m, 3 H) 8.63 - 8.69 (m _; 1 H) 9.07 (s, 1 H) 9.97 is, 1 H). MS: m/z 887 (ΜΉ ) ^* .

FL-TAMRA. Free amine, Sl-3 ( 13.8 mg, 0,028 mmol) and DMAP (35.5 mg, 0,289 mmol) were dissolved in DMSO (2 ml.). A solution of 5,6-TAMRA succitumidyl ester ( 10.7 mg, 0,020 ramol) in DMSO ( 1 mL) was added and the reaction was stirred at room temperature overnight. The reaction was purified via HPLC to afford the desired product, FL-TAMRA as a dark purple solid. Ή NMR (500 MHz, DMSO ) d ppm 1.13 - 1 .20 (m, I H) 1.23 (s, 1 H) 1 .77 - 1 .84 (m, 1 H) 1.85 - 1.93 (m, 2 H) 1.93 - 2.01 £ra, 1 H) 2.04 - 2.18 (m. 1 H) 2,36 (s _f 1 H) 2.42 (t, J - 7.32 Hz, 2 H) 3.26 (s, 17 H) 4.10 (t, ,/ - 5.95 Hz, 3 H) 4.29 - 4,42 (in, ] H) 4.48 (s, 2 H) 5.73 - 5.77 (m, i H) 5.93 - 5.97 (in, 1 H) 6,52 - 6.58 (m, 1 H) 6.60 - 6.68 (in, 2 H) 6.95 (s, 2 H) 7,00 - 7. i 1 (m, 6 H) 7.54 - 7.69 (m, 4 H) 7.85 - 7,96 (m, 1 H) 8.1 .1 - 8.15 (m, 1 H) 8.17 (d, J= 7.93 Hz, 1 H) 8.20 - 8.25 (m, 1 H) 8.29 (dd, ,/ = 7.93, 1.83 Hz, 2 H) 8.62 - 8.72 (m, 2 H) 8.75 - 8.82 (ra, 1 H) 8.92 (s, 1 H) 9.06 (s, 1 H) 9.97 (s, 1 H). M$: m/z t L8 (M-HV\ cheme 3. Synthesis of Thap-Biotit*

To a solution of Thap-OH (17 rag) in D SO (1.6 raL) was added Biottn-PEG2-

COOH acid (27 mg, 0.06.ramol), DCC (9.7 rag, 0.077 mmol), and DMA? (3.8 mg, 0.03! mmol). The reaction was stirred at 23 °C for 16 h. The reaction mixture was further diluted with methanoi, and was directly purified by HPLC to afford Thap-Biotin as coioricss oil (13 mg, 42% yjeld). MS: m/z (M÷l) .1 144.6.

Example 2: Expression of Folate Receptor 2 in T~ALL

To establish the expression of folate receptor a!leies is human T-ALL, we analyzed the m ^' RNA transcript levels of FRl and FR2 in 17 T-ALL ceil hoes and in 3 primary leukemia samples b RT-qPCR. We observed that FR2 is abundantly expressed in all leukemia samples while FRl expression is measurable in only 2/20 cases tested (FIG. 7, panel a). To confirm stable expression of surface polypeptides, we de veloped methods for FRl and FR2 flow cytometry. Because FR-isoforms are polypeptides of 220-237 amino acids that share 68-79% sequence identity' we first evaluated the specificity of FR antibodies against FRl and FR2 using a stably transduced NOTC /-mutated T-ALL cell line (RPMI 8402) overexpressing FR.1 or FR2 and established die lack of antibody cross- reactivity by flow cytometry. Western blotting (WB) of lysates from 9 T-ALL eel! lines with the isoform-specific F.R2 antibody confirmed strong expression of FR2 across ail of the samples (FIG. 7, panel b). We did not observe a significant difference in FR2 levels among NOTCH di med (ALL/S1L DHD4L HPB-ALL KOPT KL PF382, RPMI 8402) versus WT (Loucy, MOLT 16, SUPTl 1 ) T-ALL cell lines. These results establish strong expression of FR2, supporting further a rationale for fo!ate-mediated delivery in T-ALL.

To asses functional engagement of the folate receptor on T-ALL ceils, we generated fluorescence-tagged folic acid probes, FL-TAMRA and FL-FITC as too! compounds (FIG. 7, panel c). With FL-TAMRA treatment, all tested T-ALL cell lines showed a concentration-dependent increase in fluorescence signal by flow cytometry, notably independent of NOTCH! mutational status (FIG. 7, panel d). T-ALL lines demonstrated stronger FL-FiTC labeling compared to peripheral blood mononuclear ceils (PBMC), providing support for leukemia-specific targeting (FIG. 7, panel e). Takert together, these observations indicate that functional FR2 expression is increased in T-ALL compared to normal cells, further supporting a rationale for Mate-mediated, deli ery in leukemia.

Example 3. Foiaie-Conjugates Enter T-ALL Ceils hy .FR Binding and. Active Transport

To explore the mechanism of binding and delivery of foiate-conjitgates in T-ALL, we performed competition and temperature sensitivity studies of the FL-FiTC probe. The T-ALL cell line RPMI 8402 was engineered to verexpress either FR ! or FR2 and was then, treated with increasing concentrations of FL-FITC. We observed a concentratioo -dependent increase in the FiTC signal with overexpression of either FRI or FR2 (FIG. 8, panel a). Notably, T-ALL cells overexpressmg FR2 demonstrated greater uptake of the folate- conjugate than those overexpressing FR I , We next addressed whether folic acid could compete with the FL-FITC probe for uptake. T-ALL cells were grown in medium: depleted of folic acid for 48 hours, treated with FL-FITC (t or 10 μΜ), and assessed by flow cytometry, T-ALL cells grown in folaie-depleted medium exhibited increased fluorescence compared to control cells incubated in standard folate-replete culture conditions (FIG. 8, panel b), or foiate-depleted conditions supplemented with free folate (10 Μ; FIG. 8, panel c). Acidic washing of FL-FITC incubated cells did not eliminate fluorescence intensity, supporting internalization of the folate-eonjugate fluorescence probe, as opposed to nonspecific binding to the cell surface (FIG. 8, panel d).

FL-FiTC uptake also showed an energy-dependency. T-ALL ceils with FR2 overexpression cultured at 4°C were unable to take up the FL-FITC. This observation supports an acti ve, endocy tic mechanism of uptake, as low temperature blocks endocytosis at 4°C due to altered membrane fluidity (FIG. 8, panel eh in keeping with prior reported Mate-conjugate studies. In order to determine if the endocytic proces is caveolae- tnediated, we tested FT-F1TC uptake in T-ALL cells that were pretreated with fiitpiu, a transient inhibitor of caveoiin-mediated endocytosis. The ffiipin pretreated T-ALL ceils produced significantly reduced FITC fluorescence signal (mean percentage reduction 38.9 1- 2.5) confirming that the folate conjugate ^' uptake into the ceil is mediated by caveolar transport (FIG. 8, panel f). Together, these results demonstrate that felate-conjitgared probes are internalized into T-ALL ceils by FR-depeudent, eaveoiae-mediated endocy tosis. Example 4. Thap-OH Inhibits NOTCH ! Signaling in T-ALL

To test the hypothesis that Thap-OH targets SERCA in T-ALL cells, we first performed a competitive pull-down assay in which protein iy safes were treated with a novel biotiiiviated derivative of thapsigargm (Thap-Biotm), alone or in the presence of increasing concentrations of free, competitive Thap-OH. Binding to Thap-Biotm in a complex mixture was confirmed by immunoblot, as free Thap-OH competed off biotiny!ated thapsigargm from SERCA2 and SERCA3 (Figure 9 _« panel a).

We previously described that the SERCA inhibitors thapsigargm and cye!opiazonie acid impair NOTCH 1 maturation leadin to an acovaituhttion of full-length, unprocessed polypeptides in the endoplasmic reticulum Goigi subcellular compartmen An immediate consequence of SERCA inhibition is a. decrement in NOTCH] protein display on the surface of T-ALL cells. The treatment of T-ALL ceils with Thap-OH resulted in a concentration-dependent reduction in NOTCH! expression on the ceil surface by flow cytometry, as was observed with control thapsigargm treatment. Thap-OH was again found to be less potent than the natural product, as predicted from prior studies (Figure 9, parte! b). To further support the hypothesis that Thap-OH impairs mutant. NOTCH 1 maturation, we evaluated levels of full -length, firansmembrane and activated NOTCH (1CN 1) by WB. Lysates from T-ALL cell lines treated with 1 μΜ Thap-OH were immunoblotted with an antibody specific for the cytoplasmic portion of NOTCH! that recognizes both unprocessed NOTCH 1 (FL-N! ) (-270 kDa) and the furin-processed transmembrane sitbunit (TM- 1) ( ^• -1 10 kDa). Consistent with the Row cytometry data, Thap-OH reduced the levels of the Turin-processed transmembrane NOTCH 1 suhunit, but not the unprocessed foO-iength NOTCH! precursor, in multiple T-ALL ceil lines (Figure , panel c). Moreover, Thap-OH decreased iCNl levels in T-ALL ceils, suggesting that the cleavage product retains the potent anti-NOTCHl properties observed, with SERC inhibition.

As expected, treatment with Thap-OH was associated with a decrease in T-ALL cell viability, as measured by dose-ranging ATP content assays (Figure 9, pane! d).

Furthermore, the selectivity for mutant compared to wild-type NOTCH] was retained with Thap-OH; mutant T-ALL cells were more sensitive to the effects of Thap-OH than NOTCH! WT cells (Figure 9, panel d), and there was no effect on NOTCH 1 maturation in the WT cell lines at the concentrations tested (Figure 9, panels e and f). Thus, cell lines carrying NOTCH!, alleles with HD domain mutations were more sensitive to Thap-OH than cells with WT NOTCH! alleles. Thapsigargin is a known inducer of the unfolded protein response (IJP ). As such, its derivatives may (rigger a cellular response that affects the stable expression or

trafficking of folate receptors. To exclude that FR2 is a target of UPR, we treated T-ALL cell lines with increasing concentrations of Thap-OH for 24 hours. No change in FR2 expression was observed in WB {Figure 9, panel g), indicating that FR2 is not affected by Thap-OH at concentrations targeting NOTCHi .

Together these data demonstrate that Thap-OH preferentially inhibits mutant NOTCH I receptors while sparing WT NOTCH i and FR2 expression, supporting Thap-OH as a suitable and targeted payload for Mate-conjugation. Example 5. Mechanism of Drug Delivery for T apsigargin-Folic acid Conjugates.

Both FoSate-Thap (JQ-FT) and Thap-OH were tested in T-ALL ceil lines with 24 h treatment. Similarly to thapsigargin, JQ-FT caused loss of ICNJ, loss of frans-membranc NOTCH, and accumulation of full length NOTCH Lysates were prepared after treatment with indicated doses ofthapsigargin, JQ-FT or Thap-OH for 24 hr. The blot shown was stained with an antibody against the C- enninus of NOTCHi that recognizes both the furin- processecl NOTCH t transmembrane subunit (TM) and the unprocessed NOTCH ! precursor (FL) (FIG 2 A). GAPDH was used as a loading control. Western blots were stained with antibodies specific for γ-secretase-c!eaved NOTCHI (Va!l?44 ₅ Cell Signaling, Beverly, MA, USA), or the C-terminus of NOTCHI (SC-6014 (C-20) Santa Cruz Biotechnology, Santa Cruz, CA, USA), Control stains were carried oitt with antibodies specific for GAPDH (137179, Santa Cruz Biotechnology). Blots were developed with anii-rabbi.-HRP

(NA.9340V, Amershara, Pittsburgh, PA, USA) or anti-mouse-HRP (NA9340V or

NA9310V, Amersham), Staining was quantified using ImageQuant TI V 7.0 (GE Health Care, Piscataway, NJ, USA).

The Thap-OH, on the other hand, showed much lower activity. As a further test of the idea that JQ-FT acts by preventing NOTCHi activation in a manner similar to

thapsigargin, the NOTCH.1 -dependent T- ALL cell lines RPMI.-84Q2 were transduced with empty MigRl vector or with ICNl, which lies downstream of the γ-secretase cleavage step in Notch activation. Viral supernatant production and retroviral infections were performed as described for MigRI retroviral vectors (Aster, J.C., et ai. "Essential roles for artkyriii repeat and transactivatton domains in induction of T-cell leukemia by NOTCHi Mol, Ceil. Biol. 2000; 20:7505-7515). Transduction efficiency for MigRl was monitored by assessing GFP expression with a FACScan flow cytometer (BD, Franklin Lakes, NJ, USA) (Aster et a!., Mol. Cell. Bio!. 2000; 20:7505-7515). After viral infection, GFP-positive cells were sorted by flow cytometry with a FACSAria 11 (BD, Franklin Lakes, NJ, USA). The loss of viability caused by JQ-PT was on target for NOTCH ί since overexpressing an exogenous ICNI partially rescue the observed phenotype. Therefore, JQ-FT can

recapitulate the Thapsigargin effect with the delivery mechanism of folate,

^' The NOTCH loss was measured via flow cytometry. Cell surface NOTCHl was evaluated by staining non-permeahilized ceils with monoclonal anti-human NOTCHl antibody (R&D FAB5317P, Minneapolis, MR USA), Both JQ-FT and Thap-OH show NOTCH loss, but the JQ-FT was more similar to thapsigargin at 20 «M. Thap-OH had a reduced effect (Skytte DM et al, Bioorganie Med. Chemistry, 2010, 5634) compared to Thapsigargin itself. The recapitulation of the thapsigargin effect by JQ-FT is hypothesized to be due to the delivery strategy introduced by folate. The treatment ofT-ALL cell lines with Folate- Thap (FiG, 2, panel C) produced an antiproliferation effect. Here cell, growth was assessed using the .Proraega Cell-Liter Glo ATF-based assay (Promega, Madison, W ^' L, USA). Luminescence was measured using a Fluostar Omega instrument. (BMG-Iabtech, Ortenberg, Germany ).

Treatment of a panel of cell lines with Foiate-Thap produced an apoptotic effect, as ttteasured by Caspase 2 (FIG. 6). Cells were grown in 384-welS plates and treated for 72 hours with the indicated dose of Folate-Thap, Apoptosis was measured using a

luminescence assay developed by Promega.

^' The effects of JQ-FT in a panel of T- ALL cell lines that contain acti vating mutations in the HD domain ofN ^' OTCHl and/or protein stabilizing deletions within the PEST degradation domain. In all T-ALL cell lines studied, JQ-FT impaired cell growth, leading to a G l cell cycle arrest and rapid induction of apoptosis (10 μΜ; FIG. 10, panel a). As expected based on our FL-FITC uptake studies, a greater effect on cell viability was observed in eel! Sines overexpressing F.RJ or FR2.

As observed with thapsigargin and Thap-OH, treatment of T-ALL cell lines with JQ-FT led to accumulation of full length NOTCHl (FIG. 10, panel b). A decrement of transmembrane NOTCHl (TM-N!) was confirmed by WB and flow cytometry analysis (FIG. 10, panel, b ). Loss of ICN t (FIG , 10, panel b) caused the suppression of NOTCH i target genes as measured by RT-PCR (FIG. 50, panel c). In order to establish if the effects of JQ-FT on cell viability were due to impaired NOTCHl activation, the NOTCH I - dependent T-ALL cell line RP I 8402 was transduced with MigRJ -iC l, to rescue effects on full-length NOTCH! processing, versus an empty MigRl vector control. Exogenous expression of ICNl attenuated the growth inhibitory effects of JQ-FT, in keeping with the function of ICNl downstream of ER processing and surface γ-secretase cleavage in Notch pathway activation.

To assess the translatioaai significance of these findings, we studied patient-deri ved xenografts (PDX) from T-ALL patients at the Dana-P arber Cancer institute. JQ-FT treatment of VOT '/H-mutated PDX cells in vitro resulted in loss of transmembrane NOTCH I, leading to the depletion of detectable 1C J (FIG. 10, panels d, e). in contrast no effect was observed in PDX T-ALL ceils possessing WT NOTCH! (FIG, i 0, panel d). Consistent with these results, no transcriptional changes were observed in NOTCH ! target genes in WT PDX samples while expression of canonical NOTCH ! target genes, DTX d MY were decreased in the NOTCH J mutant samples (FIG. 10, panel t). These results provide strong support for the mechanistic thesis that mutated NOTCH ! receptors are more sensitive to JQ-FT treatment in human T-ALL, prompting proof-of-eoncept studies in T- ALL models in vivo.

Example 6. Fluorescence Analysis of Folate-Thap Delivery.

in order to valida te the delivery of the Thap-OH, LCMS analysis of the cell lysate and the media was used to detect the presence of both Folate-Thap (FT) and Thap-OH. FT was not detected in either cell culture media (24 h treatment) or cell lysate. Thap-OH was detected in cell lysate, however, only after ceils were treated with FT. This data supported that FT was taken into the eel! at the folate receptor (FR) and then the folate receptor is cleaved within the cell, yielding Thap-OH. To directly visualize the folate delivery of a small molecule, ^" folic acid derivatives with appended fluorophores were designed. Both Folate-FITC, suitable for flow cytometry studies, and Foiate-TMARA, suitable for hig - throughput fluorescence plate readers, were e valuated in vitro to determine the delivery mechanism. ^' The update of Folate-FITC was observed in T-ALL treament of Folate-FITC with flow cytometry. Further, when free folic acid was added to T-ALL culture, the folic acid competed away the folate derivatives, This data further confirms that the folate derivatives hind to the FR. in the same manner as free folic acid.

Folate-FITC was further used to demonstrate that the uptake of folate-conjugaied molecules is dependent on the expression of the folate receptor (FIG. 5). T- ALL cell lines were transduced with viru coniaing particles expressing a PLX empty vector or PLX- FOLR1 or PLX-FOLR2. Transduction efficiency was confirmed by RT-qPCR measuring FOLRl or 2 transcripts. Cells were subsequently treated with the indicated doses of Folate- FITC, and fluorescence intensity was quantified by f ow-cytometry. Results were expressed as fold increase relative to untreated controls.

Example 7. JQ-FT Attenuates NOTC 1 -Driven T-ALL In Vivo.

in vitro studies provide valuable mechanistic insights but may not recapitulate tumor micro-environment and metabolic conditions in vivo. Indeed, a significant limitation of research on folate-conjugate drugs is the inconsistency between free folate

concentrations (and other middle metabolites) in culture conditions in vitro versus in vivo. High levels of free folic acid in the sera may block the binding and uptake of JQ-FT in F 2-positive T-ALL compromising the a ti-NOTCHl leukemia effect observed in vitro. To explore die therapeutic efficacy of JQ-FT in vivo, we studied effects o.n a syngeneic T* ALL mouse model carrying a NOTCH! LI 601 P APEST, a common mutation observed in the human disease. First, leukemia cells obtained from this model were treated with JQ-FT for 24 hours in vitro. Consistent with the results observed in cell lines and in PDX cells, ex vivo treatment with JQ-FT diminished the proliferation (FIG. 11 , panel a), ICNI expression (FIG. 1 1 , panels b and c) and transcription of canonical NOTCH I targets lies J, and Dtxl (FIG. 1 1 , panels d and e).

Though the JQ-FT conjugate has not, a yet, been optimized for pharmacologic properties, we are eager to explore the utility of the compound as an in vivo chemical probe, while assessing putative tolerability of folate-conjtigated thapsigargins. We first established the maximal tolerated dose (MTD) of JQ-FT as 60 mg/kg/day in mice, as administered by daily intraperitoneal injection. Notably, this tolerated concentration is 150-fold improved over our prior established MTD of unconjugated thapsigargin. We therefore initiated treatment studies of JQ-FT (60 mg/kg IP daily) in mice with established T-ALL, Following five days of treatment, an evident decrease in tumor growth was observed, confirmed pathologically by a decrease of leukemic infil tration in spleen and liver (FIG . ί I , panel f) and clinically by a reduction in spleen weight (FIG. 1 1, panel g). Bone marrow infiltration b T-ALL, the primary site of human disease, was markedly inhibited by JQ-FT, as confirmed b flow cytometric analysi iramunohistochemistry for GFP+ leukemia cells (FIG. 1 1 , panel h). Pharmacodynamic modulation of the Notch pathway was importantly validated by measuremetu of reduced 1C 1 expression in T-ALL ceils from JQ-FT treated animals, as compared to vehicle-treated controls (FIG. 1 1, panels i and j).

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, hi case of conflict, the present application, including any definitions herein, wii! control. Equivalents

While specific embodiments of the subject invention .have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.