DUAN, Jian-Xin (628 Marcie Circle, So. San Francisco, CA, 94080, US)
JIAO, Hailong (861 Comet Drive, Foster City, CA, 94404, US)
CAI, Xiaohong (1453 El Camino Real, Apartment 202Burlingame, CA, 94010, US)
DUAN, Jian-Xin (628 Marcie Circle, So. San Francisco, CA, 94080, US)
JIAO, Hailong (861 Comet Drive, Foster City, CA, 94404, US)
CLAIMS
1. A compound having a structure of the formula:
2. The compound of claim 1 having a structure of the formula:
3. The compound of claim 2 wherein each R 12 is bromo.
4. A pharmaceutically acceptable formulation comprising the compound of claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.
5. A method of treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of claim 1.
6. The method of claim 5 wherein the compound has a structure of the formula:
7. The method of claim 5, further comprising administering an anti cancer agent.
8. A method of synthesizing a compound having a structure of formula:
9. The method of claim 8 wherein Ri 2 is chloro.
10. The method of claim 8 wherein Ri 2 is bromo.
11. The method of claim 6 wherein the pyridine analog is a 4-N,N- dialkylaminopyridine.
12. A method of synthesizing a compound having a structure of formula:
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TITLE OF THE INVENTION PYROPHOSPHORAMIDE ALKYLATORS
CROSS-REFERENCES TO RELATED APPLICATIONS This application claims priority to of U.S. Patent Application No. 60/874,869, filed 13 December 2006, is incorporated herein by reference.
BACKGROUND OF THE INVENTION Field of the Invention
The present invention provides compositions and methods for treating cancer and other hyperproliferative disease conditions with pyrophosphoramide alkylators alone and in combination with other anti-cancer agents and therapies, and so relates to the fields of medicine, pharmacology, chemistry, and biology.
Description of Related Art
Phosphoramidate alkylators used in treating cancer encompass a group of compounds that have in common the ability to alkylate biologically vital macromolecules such as DNA (Hardman et al., The Pharmacological Basis of Therapeutics, 2001 , 1389-1399, McGraw-Hill, New York, USA). However, certain prodrugs currently used in cancer therapy that release phosphoramidate alkylators in vivo, such as the drugs ifosfamide and cyclophosfamide, are problematic due to side effects arising in part from toxic metabolites such as acrolein. A phosphoramidate alkylator prodrug, giufosfamide, that does not produce acrolein, has not yet been approved for treating cancer (see, US Pat. No. 5,622,936 incorporated herein by reference).
There is a need for anticancer pyrophosphoramide alkylators particularly those with reduced toxicity and/or greater efficacy compared to cyclophosfamide, giufosfamide, and ifosfamide. The present invention meets this unmet need by providing novel pyrophosphoramide alkylators and methods of therapy employing them.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides pyrophosphoramide alkylators that are anhydrides of phosphoramidate alkylators containing 1 -3 phosphoramidate alkylator moieties. In one embodiment, the present invention provides pyrophosphoramide alkylators containing two phosphoramidate alkylator moieties. In another embodiment, each of the phosphoramidate alkylator moieties is selected from the group consisting of an ifosfamide alkylator, a cyclophosfamide alkylator, an N,N,N',N'-tetrakis-2-haloalkyl phosphoramidate alkylator, and analogs of the foregoing. In another embodiment, the pyrophosphoramide alkylator is a bis-ifsofamide alkylator.
In another embodiment, the present invention provides phosphoramidate alkylator anhydrides containing two phosphoramidate alkylator moieties joined by a phosphate or a phosphoramidate moiety.
In one aspect, the present invention provides a pyrophosphoramide alkylator having a structure of formula:
R 3 and R 4 , R 4 and R 5 , R 5 and R 6 , R 6 and R 7 , R 7 and R 8 , and R 8 and R 1 form a heterocyclyl moiety; each R 9 - Rn is independently selected from the group consisting of O, S, and NR 15 ; each R 12 is independently selected from the group consisting of halo and OSO 2 R 14 ; each R^ is independently selected from the group consisting of hydrogen and C 1 -C 6 alkyl; each R 15 is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and each R 14 is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; provided that, at least two of R 1 - R 4 and at least two of R 5 - R 8 have the structure of formula:
In one embodiment, each Rg- R 11 is O. In another embodiment, the present invention provides a pyrophosphoramide alkylator having structure of formula:
H R 8
O O
R 2
-\
R 4 H "R 6 or a pharmaceutically acceptable salt thereof; wherein R 2 , R 4 , Re, and R 8 are defined as above. In another embodiment, the present invention provides a pyrophosphoramide alkylator having structure of formula:
In another aspect, the present invention provides methods of synthesizing novel pyrophosphoramide alkylators and known pyrophosphoramide compounds, comprising reacting a phosphoramidate alkylator or a phosphoramidate under Mitsunobu conditions. In another aspect, the present invention provides methods
of synthesizing novel pyrophosphoramide alkylators and known pyrophosphoramide compounds, comprising reacting a phosphoramidate alkylator or a phosphoramidate with a dialkyl or dicycloalkyl carbodiimide. In another aspect the the present invention provides methods of synthesizing novel pyrophosphoramide alkylators and known pyrophosphoramide compounds, comprising reacting a compound having a structure of formula:
In another aspect, the present invention provides a pharmaceutically acceptable formulation comprising a pyrophosphoramide alkylator and pharmaceutically acceptable diluents or excipients.
In another aspect, the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a pyrophosphoramide alkylator to a patient in need of such treatment.
These and other aspects and embodiments are described in the accompanying figure and detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 : Tumor growth delay in H460 tumor xenografts in mice, upon administration of compound 1 alone and in combination with cisplatin (CDDP)
DETAILED DESCRIPTION OF THE INVENTION This detailed description of the different aspects and embodiments of the present invention is organized as follows: Section I provides useful definitions; Section Il describes pyrophosphoramide alkylators of the present invention (part
A) and methods of synthesizing novel and known pyrophosphoramide compounds (part B); Section III describes various methods of treating cancer and other diseases of cellular hyperproliferation employing the pyrophosphoramide alkylators of the present invention; and Section IV provides illustrative examples for synthesizing pyrophosphoramide alkylators of the present invention and demonstrating in vitro and in vivo efficacy of a pyrophosphoramide alkylator of the present invention. This detailed description is organized into sections only for the convenience of the reader, and disclosure found in any section is applicable to disclosure elsewhere in the specification.
I. Definitions
The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art. refers to a position on a moiety wherein another moiety or atom is covalently bonded.
"C 1 -C 6 alkoxy" refers to a Ci-Cε alkyl covalently bonded to an oxygen atom. In other words, a C-1-C 6 alkoxy group has the general structure -0-(Cr Ce)alkyl. C I -C θ alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.
"CrCβ alkoxycarbonyl" refers to a CrC 6 alkoxy group covalently bonded to a carbonyl. In other words, a CrC 6 alkoxycarbonyl group has the general structure -C(=O)-O-(C r C 6 )alkyl.
"C 1 -Ce alkyl" refers to a substituted or unsusbstituted straight or branched chain alkyl groups having 1-6 carbon atoms. C 1 -C- 6 alkyl groups include, for
example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2- pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl. A C r C 6 alkyl substituent may be covalently bonded to an atom within a molecule of interest via any chemically suitable portion of the CrC 6 alkyl group.
"CrCβ alkylamino," refers to a CrCβ alkyl covalently bonded to -NH- . In other words, a CrC 6 alkylamino group has the general structure -NH-(Cr C 6 )alkyl. Similarly a di(C r C 6 )alkylamino group has the general structure -N-[(C r C 6 )alkyl] 2 . CrC 6 alkylamino groups include, for example, methylamino, ethylamino, propylamine and butylamino.
"CrCe alkylene" refers to a linear saturated divalent substituted or unsubstituted hydrocarbon radical or a branched saturated divalent hydrocarbon radical having 1 - 6 carbon atoms. An alkylene can be further substituted with Cr Ce alkyl and aryl groups. Alkylene groups include, for example, methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene.
"Cycloalkyl" refers to a substitutued or unsubstitutued monovalent cyclic hydrocarbon group of three to seven ring carbons. The cycloalkyl group can have one or more double bonds and can be further substituted with C 1 -C 6 alkyl and aryl groups.. More specifically, the term cycloalkyl includes, for example, cyclopropyl, cyclohexyl, cyclohexenyl, phenylcyclohexyl, 4-carboxycyclohexyl, 2- carboxamidocyclohexenyl, 2-dimethylaminocarbonyl-cyclohexyl, and the like.
"Aryl" refers to a substituted or unsusbstituted moiety that includes one or more monocyclic or fused ring aromatic systems. Such moieties include any moiety that has one or more monocyclic or bicyclic fused ring aromatic systems, including but not limited to phenyl and naphthyl.
"Halogen or halo" refers to fluorine, chlorine, bromine, and iodine.
"Heteroaryl" refers to a substituted or unsusbstituted monocyclic aromatic system having 5 or 6 ring atoms, or a fused ring bicyclic aromatic system having 8 - 20 atoms, in which the ring atoms are C, O, S, SO, SO 2 , or N and at least one of the ring atoms is a heteroatom, i.e., O, S, SO, SO 2 , or N. Heteroaryl groups include, for example, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothio- furanyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl,
benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl and xanthenyl. Unless indicated otherwise, the arrangement of the hetero atoms within the ring may be any arrangement allowed by the bonding characteristics of the constituent ring atoms.
"Heterocyclyl" refers to a substituted or unsubstituted monocyclic or fused ring multicyclic cycloalkyl group at least a portion of which is not aromatic and in which one or more of the carbon atoms in the ring system is replaced by a heteroatom selected from the group consisting of O, S, SO, SO 2 , P, or N. Examples of heterocyclyl groups include but are not limited to imidazolinyl, morpholinyl, piperidinyl, piperidin-2-only, piperazinyl, pyrrol id inyl, pyrrolidine-2- onyl, tetrahydrofuranyl, and tetrahydroimidazo [4,5-c] pyridinyl.
"C 1 -C 6 heteroalkylene" refers to a CrC 6 alkylene as defined above wherein 1 - 3 carbon atoms in the hydrocarbon radical or a branched saturated divalent hydrocarbon radical is replaced with a heteroatom. Ci-C 6 heteroalkylene groups include, for example, -CH 2 CH 2 -O-CH 2 CH 2 - and -CH 2 CH 2 -S-CH 2 CH 2 -.
"C 1 -C 6 heteroalkyl" refers to a C 1 -C 6 alkyl as defined above wherein 1 - 3 carbon atoms in the hydrocarbon radical or a branched saturated divalent hydrocarbon radical is replaced with a heteroatom. C 1 -C 6 heteroalkyl groups include, for example, -CH 2 CH 2 -O-CH 2 CH 3 and -CH 2 CH 2 -S-CH 2 CH 3 .
"Phosphoramidate alkylators" refer to phosphordiamidic acid compounds (or mustards) having up to four substituted or unsubstituted -CH 2 -CH 2 -X moieties, wherein X is selected from the group consisting of halo and an 0-SO 2 - Y moiety wherein Y is CrC 6 alkyl, CrC 6 heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. Phosphoramidate alkylators include, for example, ifosfamide alkylator, dibromoifosfamide alkylator, cyclophosfamide alkylator, and N, N, N', N' -tetrakis-2-haloalkyl phosphoramidate alkylators. Examples of other phosphoramidate alkylators are provided in PCT Patent App. Pub. No. WO 07/002931 and US Pat. No. 6,197,760 each of which is incorporated herein by reference.
"Pyrophosphoramide alkylators" refer to linear or cyclic anhydrides of phosphoramidate alkylators containing two or more phosphoramidate alkylator moieties.
"Substituent" refers to an atom or group, including, for example, amino, C-i-Cε alkylamino or di(C-ι-C 6 )alkylamino, C-i-Cβ alkoxy, C-1-C 6 alkylthio, aryl, - COOH, -CONH 2 , cyano, ethenyl, ethynyl, halo, heteroaryl, hydroxy, mono- or di(Ci-C 6 )alkylcarboxamido, mono or d^CrC^alkylsulfonamido, nitro, -OSO 2 -(Cr C 6 )alkyl, and -SO 2 NH 2 .
"Administering" or "administration of" a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration to a patient by a medical professional or may be self- administration, and/or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.
"About" refers to ± 20% of a quantity and includes, but is not limited to, ± 15%, ± 10%, and ± 5%.
"Cancer" refers to leukemias, lymphomas, and malignant tumors of potentially unlimited growth that can expand locally by invasion and systemically by metastasis. Examples of cancers include, but are not limited to cancer of the adrenal gland, bone, brain, breast, bronchi, colon and/or rectum, gallbladder,
head and neck, kidneys, larynx, liver, lung, neural tissue, pancreas, prostate, parathyroid, skin, stomach, and thyroid. Other examples of cancers include, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and in situ carcinoma, Ewing's sarcoma, epidermoid carcinomas, giant cell tumor, glioblastoma multiforma, hairy-cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemias, lymphomas, malignant carcinoid, malignant melanomas, malignant hypercalcemia, marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteo sarcoma, osteogenic and other sarcoma, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small-cell lung tumor, squamous cell carcinoma of both ulcerating and papillary type, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, topical skin lesion, veticulum cell sarcoma, and Wilm's tumor.
"Hyperproliferative disease" refers to a disease characterized by cellular hyperproliferation (e.g., an abnormally increased rate or amount of cellular proliferation). Examples of hyperproliferative diseases other than cancer include, but are not limited to, allergic angiitis and granulomatosis (Churg-Strauss disease), asbestosis, asthma, atrophic gastritis, benign prostatic hyperplasia, bullous pemphigoid, coeliac disease, chronic bronchitis and chronic obstructive airway disease, chronic sinusitis, Crohn's disease, demyelinating neuropathies, dermatomyositis, eczema including atopic dermatitis, eustachean tube diseases, giant cell arteritis, graft rejection, hypersensitivity pneumonitis, hypersensitivity vasculitis (Henoch-Schonlein purpura), irritant dermatitis, inflammatory hemolytic anemia, inflammatory neutropenia, inflammatory bowel disease, Kawasaki's disease, multiple sclerosis, myocarditis, myositis, nasal polyps, nasolacrimal duct diseases, neoplastic vasculitis, pancreatitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, periodontal disease, polycystic kidney disease, polyarteritis nodosa, polyangitis overlap syndrome, primary sclerosing cholangitis, rheumatoid arthritis, serum sickness, surgical adhesions, stenosis or
restenosis, scleritis, scleroderma, strictures of bile ducts, strictures (of duodenum, small bowel, and colon), silicosis and other forms of pneumoconiosis, type I diabetes, ulcerative colitis, ulcerative proctitis, vasculitis associated with connective tissue disorders, vasculitis associated with congenital deficiencies of the complement system, vasculitis of the central nervous system, and Wegener's granulomatosis.
"Pharmaceutically acceptable carrier, excipient, or diluent" refers to a carrier, excipient, or diluent that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier, excipient, or diluent that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable carrier, excipient, or diluent" includes one and more than one such carrier, excipient, or diluent.
"Prodrug" refers to a compound that, after administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug, relative to the drug, is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered. A prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.
"Reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) refers to decreasing the severity or frequency of the symptom(s), or elimination of the symptom(s).
"Therapeutically effective amount" of a drug refers to an amount of a drug that, when administered to a subject with cancer or another hyperproliferative disease, will have the intended therapeutic effect, e.g., alleviation, amelioration,
palliation or elimination of one or more manifestations of cancer or another hyperproliferative disease in the subject. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
"Treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer or another hyperproliferative disease; diminishment of extent of disease; delay or slowing of disease progression; amelioration, palliation, or stabilization of the disease state, or other beneficial results described more particularly below.
II.A. Compounds
Phosphoramidate alkylators have been used in treatment of cancer. Because of the reactivity and instability of the phosphoramidate alkylators, these alkylators are administered as prodrugs that are more stable than their corresponding alkylators. Upon administration, the phosphoramidate alkylator prodrugs are metabolized, releasing the alkylator, which kills cancer cells. For example, cyclophosfamide, a prodrug of cyclophosfamide alkylator, and ifosfamide, a prodrug of ifosfamide alkylator, are used in cancer treatment. However, the cyclophosfamide and ifosfamide prodrugs are problematic, because when metabolized, they produce the toxic byproduct acrolein, which can cause unwanted side effects. Glufosfamide, a prodrug of ifosfamide alkylator that does not produce acrolein, has not yet been approved for treating cancer.
The present invention arises in part out of the discovery that anhydrides of phosphoramidate alkaylators such as pyrophosphoramides are excellent prodrugs. In the phosphoramidate alkylator, an ionizable hydroxyl group, "P-OH", is converted into a "P-O-P" type, charge neutral, oxygen atom. The pyrophosphoramide alkylator is more stable to hydrolysis, aminolysis, and/or thiolysis, compared to the corresponding phosphoramidate alkylator. Upon
administering a pyrophosphoramide alkylator to a patient, the "P-O-P" linker is hydrolyzed and the phosphoramidate alkylator is released and can kill cancer cells.
In one aspect, the present invention provides anhydrides of phosphoramidate alkylators containing two or more phosphoramidate alkylator moieties. In one embodiment, the present invention provides pyrophosphoramide alkylators containing two phosphoramidate alkylator moieties. In another embodiment, the present invention provides pyrophosphoramide alkylators wherein each of the phosphoramidate alkylator moieties is the same. In another embodiment, the present invention provides pyrophosphoramide alkylators wherein each of the alkylator moieties is different. In another embodiment, each of the alkylator moieties is an ifosfamide alkylator, a dibromoifosfamide alkylator, a cyclophosfamide alkylator, an N,N,N',N'-tetrakis-2-haloalkyl phosphoramidate alkylator, or an analog of one of the foregoing. In another embodiment, the pyrophosphoramide alkylator is a bis-ifsofamide alkylator.
In one aspect, the present invention provides a pyrophosphoramide alkylator having structure of formula:
wherein the wavy line indicates the attachment to N; or together one or more pairs of substituents selected from the group consisting of Ri and R 2 , R 2 and R 3 , R 3 and R 4 , R 4 and R 5 , R 5 and R 6 , Re and R 7 , R 7 and R 8 , and R 8 and R 1 form a heterocyclyl moiety; each R 9 - Rn is independently selected from the group consisting of O, S, and NR 15 ; each R-1 2 is independently selected from the group consisting of halo and OSO 2 R 14 ; each R 13 is independently selected from the group consisting of hydrogen and C 1 -C 6 alkyl; each R 15 is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and each R 14 is independently selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; provided that, at least two of R 1 - R 4 and at least two of R 5 - R 8 are the moiety:
In one embodiment, each Rg- R 1 I is O. In another embodiment, the present invention provides a pyrophosphoramide alkylator having structure of formula:
In another embodiment, the present invention provides a pyrophosphoramide alkylator having structure of formula:
In another embodiment, the present invention provides a pyrophosphoramide alkylator having structure of formula:
by mechanism, a pyrophosphoramide alkylator containing a 4- hydroxyphenylsulfonyloxy moiety as a leaving group can exist in the extracellular region of a tumor in its charge neutral form. The charge neutral form can permeate through the cell membrane more easily than the ionized anionic form and thus, pyrophosphoramide alkylators containing one or more 4-hydroxyphenylsulfonyloxy moieties can selectively enter into and kill tumor cells compared to normal cells.
Examples of pyrophosphoramide alkylators of the present invention include:
Compound 1 Compound 2
Compound 3 Compound 4
In one embodiment, the present invention provides pharmaceutically acceptable formulations comprising the pyrophosphoramide alkylators of the present invention. In one embodiment, the pharmaceutically acceptable formulations further comprise a suitable carrier, excipient, or diluent.
H-B. Synthetic Methods
The pyrophosphoramide compounds of the present invention can be synthesized by reacting a phosphoramidate alkylator with a trisubstitutued phosphine and a dialkylazdicarboxylate under Mitsunobu reaction conditions. In one aspect, the present invention provides methods of synthesizing novel pyrophosphoramide alkylators and known pyrophosphoramide compounds comprising, reacting a phosphoramidate alkylator or a phosphoramidate; a
trisubstituted phosphine; and a dialkylazodicarboxylate. In one embodiment, the method comprises reacting a phosphoramidate alkylator having a structure of formula:
In another aspect, pyrophosphoramide alkylators are synthesized by reacting phosphoramidate alkylators, a dicycloalkyl or dialkylcarbodiimide, and optionally, amines such as a trialkylamine and/or a 4-dialkylaminopyridine. The synthesis of Compound 1 according to this method is described in Section IV.1. A.
In another aspect, the pyrophosphoramide alkylators are synthesized by reacting a compound having a structure of formula:
method of the present invention is schematically shown below and can be used for synthesizing other pyrophosphoramide alkylators of the present invention:
Yto ta I - 38%
Suitable bases useful in the method include, but are not limited to trialkyl amines and pyridines. Other pyrophosphoramide alkylator of the present invention can be synthesized following the method described above by appropriate substitution of starting material. In one embodiment, the present invention provides a method of synthesizing a pyrophosphoramide alkylator of the present invention having a structure of formula:
In one embodiment, R-12 is chloro. In another embodiment, R 12 is bromo. In another embodiment, the amine is triethyl amine. In one embodiment, the pyridine analog is a 4-N,N-dialkylaminopyridine. The synthesis of Compound 2 according to this method is described in Section IV.1. B.
Compounds 1-4 can be synthesized by a Mitsunobu reaction employing triphenyl phosphine and diisopropyl or diethyl azodicarboxylate. Pyrophosphoramide alkylators of the present invention can also be synthesized following synthetic methods reported, for example, in PCT Patent App. Pub. No. WO 07/002931 ; US Pat. No. 4,518,413; and GB 985,996 (each of which is incorporated herein by reference), modified in accordance with the teachings herein and appropriate substitution of starting material.
III. Treatment Methods
Compound 1 , a pyrophosphoramide alkylator of the present invention, was effective in treating cancer as demonstrated by treating various xenograft solid tumors in mice, when administered alone and in combination with a variety of
other anticancer agents. Suitable anticancer agents, useful in combination with Compound 1 included, CDDP and gemcitabine. Various modes of administration and dosing schedules were found to be effective in the treatment of cancer. These and other monotherapies and combination therapies for administering Compound 1 are described in Examples 5.A - C below.
Thus, in another aspect, the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a pyrophosphoramide alkylator of the present invention to a patient in need of such treatment. In one embodiment, the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a pyrophosphoramide alkylator of the present invention having structure of formula:
R 3 " RR 44 RR 55 ' R 6 or a pharmaceutically acceptable salt thereof; wherein R 1 - R 8 are defined as above, to a patient in need of such treatment. In another embodiment, the pyrophosphoramide alkylator administered has the structure of formula:
In one embodiment, the pyrophosphoramide alkylators of the present invention are administered in the form of pharmaceutically acceptable formulations. In another embodiment, the pharmaceutical formulations are
administered parenteraliy or orally (p.o.). In another embodiment, the pharmaceutical formulations are administered by i.v. or i.p. injection or by infusion.
The pyrophosphoramide alkylators of the present invention can be administered in accordance with any of a variety of dosing schedules including but not limited to daily or once every other day or once a week to the patient. Multiple daily administrations of a pyrophosphoramide alkylator of the present invention can also be employed in the methods of the invention. Depending on the dose selected by the practitioner and the convenience of the patient, the entire daily dose may be administered once daily or the daily dose may be administered in multiple smaller doses throughout the course of a day. The pyrophosphoramide alkylators of the present invention need not, however, be administered daily; for example a daily dose used for some patients or indications may be, in other patients or for other indications, given every other day, or less frequently.
In one embodiment, the daily dose is repeatedly administered over a period of time. In this embodiment, the administration of the therapeutically effective daily dose is continued for multiple days, typically for at least three consecutive days, or for at least a week, or for several weeks, or for several months, or for several years, or until cancer (or another hyperproliferative disease) or one or more of its symptoms disappears or substantially abates, or up to the rest of the patient's life. As is well understood in the field of medicine, treatment can be suspended temporarily if toxicity is observed or for the convenience of the patient without departing from the scope of the invention.
In various embodiments, the pyrophosphoramide alkylators of the present invention are administered qd, bid, tid, qid, qod, q2d, twice weekly, q7d, or qweek, and treatment is continued for a period ranging from three days to the longer periods enumerated above.
The methods of the present invention can be used for treatment of any cancer. In one embodiment, the compound of the present invention is administered for the treatment of cancer in combination with other anti cancer
agents or other anti cancer therapies. Suitable anticancer therapies useful in accordance with the present methods include, but are not limited to, radiation therapy and surgery.
In another embodiment, the present invention provides a method of treating hyperproliferative diseases characterized by cellular hyperproliferation (e.g., an abnormally increased rate or amount of cellular proliferation) other than cancer, by administering a therapeutically effective amount of a pyrophosphoramide alkylator to a patient in need of such treatment.
The invention, having been described in summary and in detail, is illustrated but not limited by the Examples below, which describe the synthesis of novel pyrophosphoramide alkylators of the present invention according to the methods of the present invention, and demonstrate the anti cancer efficacy of pyrophosphoramide alkylators.
IV. EXAMPLES
Example 1
This example describes the synthesis of a novel pyrophosphoramide alkylator of the present invention in accordance with the methods of the present invention described in Section II. B above.
A. Synthesis of Compound 1
This example describes the synthesis of compound 1 , a novel pyrophosphoramide alkylator of the present invention, from dibromoifosfamide alkylator (Br-IPM) and DCC. A mixture of Br-IPM (1.24 g, 4 mmol) and dicyclohexylcarbodiimide (DCC, 1.24 g, 6 mmol) in THF (20 ml_) was stirred at room temperature (rt). After 2 h, thin layer chromatography (15:1 (v/v) ethyl acetate/methanol) of the reaction mixture showed that the reaction was completed. Volatiles were removed from the reaction mixture in vacuo and the residue was separated by flash column chromatography on silica gel (15:1 (v/v) ethyl acetate/methanol) to yield pure compound 1 (0.92 g) as a white solid. Other pyrophosphoramide alkylators of the present invention can be synthesized employing the method described for the synthesis of compound 1.
B. Synthesis of Compound 2
This example describes the synthesis of a novel pyrophosphoramide alkylator, Compound 2, of the present invention, via the intermediacy of a compound having a structure of formula:
To a suspension of 2-chloroethylamine hydrochloride (20.3 g, 2.0 eq.) in DCM (260 ml_) was added phosphorus oxychloride (8.0 ml, 1.0 eq.) at -2O 0 C, followed by the dropwise addition of TEA (48.8 ml, 4.0 eq.) in DCM (100 ml_) over a period of 2.5 h. After the reaction mixture was allowed to come to rt from -2O 0 C and stirred for 3 h, the reaction mixture was filtered. The residue was washed with AcOEt (200 ml_). The combined filtrate was concentrated under vacuum to yield a residue that was dissolved in THF (80 ml_). Water (0.7 ml_, 0.45 eq.) was added dropwise to the THF solution followed by the addition of
TEA (6 ml_, 0.5 eq.) and DMAP (10mg) at O 0 C. the reaction mixture was allowed to come to rt from O 0 C to rt, stirred for a period of 2 h, and filtered. The volatiles were removed by vacuum and the residue was purified by flash chromatography with methanol in AcOEt 0-10% to yield Compound 2 (7.Og, 38% in two steps).
Compound 1 can be synthesized employing this method and substituting 2-chloroethylamine hydrochloride with 2-bromoethylamine hydrobromide.
Example 2 Demonstration of Antiproliferative Activity
This example demonstrates the antiproliferative activity of a pyrophosphoramide alkylator, Compound 1 , of the present invention against the H460 non small cell lung cancer cell line. H460 cells (ATCC HTB-177 (NCI- H460), 20,000 cells/well/500 μl_) were seeded in a 24 well plate in RPMI medium (Invitrogen Corp.). After 24 h, these plates were divided into 2 groups: a "control group" (no test compound) and a "treatment group" (in which the cells were incubated with the test compound, compound 1 , at various concentrations for 2 h). After the 2 h incubation, the cells in the treatment group were rinsed to remove compound 1 , further incubated for 3 days, and then stained with Alamar blue for 2 h. In the control group, at day 3, the cells were stained with Alamar blue for 2 h.
The fluorescence intensity of the treatment group at various concentrations of compound 1 was measured (fluorescence plate reader, λ ex = 550 nm and λ em = 590 nm) and plotted against the corresponding compound 1 concentration. The IC 50 of compound 1 was calculated from the plot as the concentration of compound 1 at which there was a 50% reduction in cell proliferation compared to the control group. See Biosource International Inc., Tech Application Notes, Use of Alamar Blue in the Measurement of Cell Viability and Toxicity, Determining IC 50 . The antiproliferative activity of compound 1 was determined in HT29, PC3, and MiaPaCa-2 cell lines in the same way as described above for the H460 cell line. Additionally, cells in the "treatment group" can be incubated with the test compound, compound 1 , at various concentrations
for 3 days and the antiproliferation activity of the test compound determined as described above for the 2h treatment group. The results from these tests are tabulated below (Table 1 , average value of IC 50 presented, if multiple tests were done with one cell line). This method can be employed to measure the antiproliferative activities of other compounds of the present invention.
Example 3
Demonstration of Cytotoxicity
This example demonstrates the cytotoxic property of compound 1 by determining clonogenic cell survival. Exponentially growing human colon cancer HT29 cells (obtained from the ATCC) were seeded into 60 mm notched glass plates at a density of between 2.5 and 5 x10 5 cells per plate and grown in RPMI medium supplemented with 10 % fetal bovine serum for 2 days prior to initiating drug treatment. On the day of the test, solutions of Compound 1 of known concentration were prepared in complete medium, 2 mL of the desired solution added to each plate, and the cells were incubated under an atmosphere of 95% air and 5% carbon dioxide for 2 h at 37°C. Then, the cells were rinsed to remove compound 1. The glass plates were washed with phosphate buffered saline and a solution of trypsin-EDTA and then trypsinized at 37 0 C for 5 min. Detached cells were neutralized with medium and serum, collected by centrifugation at 100xg for 5 min, resuspended at approximately 1x10 6 cells/mL, and diluted 10 fold to yield stock concentrations of treated cells for plating. The concentration of each stock was determined by counting with a Coulter Z2 particle counter. Known numbers of cells were plated, and the plates were placed in an incubator for between 7 and 10 days. Colonies were fixed and stained with a solution of 0.25% crystal violet in 95% ethanol. Colonies of greater than 50 cells were counted. The surviving fraction of treated cells was measured by comparison with colony numbers obtained from untreated cells, and the concentration of compound 1 at which 10% cells survived (or 90% cell death), or IC 90 determined. See Table 1 below. This method can be employed to measure the cytotoxicities of other compounds of the present invention.
Table 1
Clonogenic survival of 10% cells (90% cell death)
Example 4
Demonstration of in vivo Pharmacokinetic Parameters This example demonstrates various pharmacokinetic (Pk) parameters of compound 1 and the release of Br-IPM by in vivo metabolism. Male CD-1 mice (purchased from Charles River, Cambridge, MA), 7 - 8 weeks of age, were acclimatized for 3 days and handled under pathogen-free conditions. Compound 1 was formulated in PEG 400-saline for i.p. injection, and in 2% DMSO - 98% of 0.5% aqueous carboxymethylcellulose for p.o. administration. After compound 1 was administered (50 mg/kg), blood was collected by cardiac puncture at 2, 5, 15, 30, and 60 min post-administration, and stored in fluoride/oxalate containing tubes, covered by aluminum foil, in an ice bath. The blood samples were centrifuged under refrigeration, the plasma extracted and mixed with 15 μL of 98% formic acid, and stored at -7O 0 C before analysis by mass spectrometry. The Pk parameters measured are tabulated below (Table 2) and demonstrate that compound 1 releases the phosphoramidate alkylator Br-IPM in vivo and that compound 1 was bioavailable upon oral administration.
Table 2
Example 5 Demonstration of in vivo Efficacy
This example in parts A - C demonstrates the efficacy of Compound 1 for the treatment of cancer.
A. This part demonstrates the anti cancer activity of Compound 1 when administered, alone and in combination with cisplatin (CDDP), to mice bearing H460 non small cell lung tumor xenografts. Compound 1 (50 mg/kg), was formulated in vehicle (10% PEG 400/saline) and administered twice weekly (i.p., days 2 and 8, 1 cycle) alone or in combination with CDDP (i.v., 6 mg/kg, 2 h after Compound 1 administration). The tumor growth delay is tabulated below (Table 3). The results demonstrate that, a pyrophosphoramide alkylator Compound 1 , is effectively delays tumor progression in mice, alone or in combination with the platinum alkylator cisplatin.
Human equivalent doses can be estimated from doses administered to mice by dividing the mouse dose by -12. Using this conversion factor, compound 1 can be administered to humans for the treatment of cancer, such as, non small cell lung cancer, at a therapeutically effective amount of about (50/12 = ) 4 mg/kg administered once weekly, or at a therapeutically effective daily dose of about (4/7) mg/kg or about 0.6 mg/kg, alone or in combination with cisplatin. Thus, in one embodiment of the present invention, a compound of the present invention can be administered in an amount in a range of about 0.01 mg/kg - about 60 mg/kg and about 0.06 mg/kg - about 6 mg/kg for treating cancer in humans.
Table 3
B. This part demonstrates the efficacy of Compound 1 to treat cancer when administered orally. H460 xenograft tumor bearing mice were treated with Compound 1 orally at 200 mg/kg once or at 50 mg/kg, everyday for 5 days. The monotherapy, i.e., a single agent administration, of Compound 1 at 50 mg/kg daily dosing demonstrated similar tumor growth inhibition as CDDP alone. The monotherapy of Compound 1 at 200 mg/kg single dose demonstrated a higher tumor growth inhibition than Compound 1 dosed once daily. The combination therapy administering Compound 1 at 200 mg/kg single dose and at 50 mg/kg once daily dosing resulted in a similar tumor growth inhibition. The TGDs, as compared to vehicle treated tumors, to reach 500 mm 3 , were 6 and 7 days respectively, when Compound 1 was administered alone at 200 mg/kg and 50 mg/kg; they were 12 and13 days respectively when Compound 1 was administered at the same amount and in combination with CDDP. Tumor growth was inhibited 56% and 64% when Compound 1 was administered alone at 200 mg/kg and 50 mg/kg, and 84% and 85% when Compound 1 administered at the same amount was co-administered with CDDP. Animals in the drug treated groups lost weight during the treatment period, but recovered after dosing stopped, demonstrating that the administration of Compound 1 , in accordance with the present methods is effective and safe for the treatment of cancer.
C. This part demonstrated the efficacy of Compound 1 when administered orally, to treat xenografted H460 lung tumor in nude mice, by administering Compound 1 as a monotherapy, and in combination with gemcitabine. Compound 1 was formulated (2.5, 5, and 20 mg/ml_) in a vehicle (2% DMSO, 0.5% TweenδO, and 0.5% carboxymethyl cellulose (CMC) in water). Gemcitabine (6 mg/mL) was formulated in saline. The drug formulations were prepared weekly, stored at 4 0 C, and diluted prior to administration.
H460 cells were prepared in 50% matrigel and 50% PRIM medium and were implanted (1 x 10 6 . 0.2 mL/mouse) subcutaneously on the right flank of the mice. Tumor lumps were measurable on the seventh day post-implantation and were about 100 mm 3 on the eleventh day. Mice with similar tumor size were randomized for treatment and treated as follows: Compound 1 was administered orally (p.o.), once every day for 5 days/week for 2 weeks (qd x 5 x 2 weeks) at amounts of 25 and 50 mg/kg, and once every week for 2 weeks at amounts of 200 mg/kg, alone, and in combination with gemcitabile (i.p., 60 mg/kg, q3dx4); in all 6 groups of 10 mice each were thus treated. A group of 10 tumor bearing mice were used as no-treatment control. The results of tumor growth inhibition upon administration of Compound 1 alone and in combination are tabulated below.
Table 4
* The T/C ratio was calculated as T/C = (Tn-Ti)/(Cn-Ci) where Tn is tumor volume in treatment group at Day 31 , and Ti is the initial tumor volume in the treatment group, Cn is tumor volume in the vehicle control group at Day 31 , and Ci is the initial tumor volume in the vehicle control group. The tumor growth inhibition (TGI) was calculated as 1-T/C.
The drug treated groups showed weight loss during the treatment period, but recovered after drug administration was stopped. Thus, the administration of Compound 1 according to the present methods was effective and safe to treat the tumors. When Compound 1 was administered to mice bearing HT29 xenograft tumor, alone and in combination with CPT-11 , the results indicated that Compound 1 was more effective in treating mice bearing H460 xenograft tumor than mice bearing HT29 tumor.
As described in part A above, human equivalent doses can be estimated from doses administered to mice by dividing the mouse dose by -12. Using this conversion factor, Compound 1 can be administered to humans for the treatment of cancer at a daily dose of (about 25/12 mg/kg to about 200/12 mg/kg =) about 2 mg/kg to about 17 mg/kg alone and in combination with another anticancer agent
and/or anticancer therapy. Thus, in one embodiment of the present invention, a compound of the present invention can be administered in a daily dose in the range of about 0.02 mg/kg - about 1700 mg/kg and about 0.2 mg/kg - about 170 mg/kg for treating cancer in humans.
Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention, as set forth in the claims that follow. All publications and patent documents (patents, published patent applications, and unpublished patent applications) cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of publication of the same. The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims.