Inhibitors of creatine transport and uses thereof

Abstract

This invention relates to compounds that inhibit creatine transport and/or creatine kinase, pharmaceutical compositions including such compounds, and methods of utilizing such compounds and compositions for the treatment of cancer.

Claims

1. A compound having the structure: ##STR00608## wherein Q1 is ##STR00609## m is 1 or 2; R7 is hydrogen; one R8 combines with R12 with the atoms to which they are attached to form an optionally substituted C3-C4 heterocycle; a second R8, if present, is hydrogen, deuterium, halo, NH2, optionally substituted C1-C3 alkyl, or combines with an R9 and with the atoms to which they are attached to form an optionally substituted C3-C6 cycloalkyl ring; or combines with R10 or R11 and with the atoms to which they are attached to form an optionally substituted C3-C4 cycloalkyl ring; each R9 is independently hydrogen, deuterium, halo, NH2, optionally substituted C1-C3 alkyl or combines with the second R8, if present, and with the atoms to which they are attached to form an optionally substituted C3-C6 cycloalkyl ring; or combines with R10 or R11 and with the atoms to which they are attached to form an optionally substituted C3-C4 cycloalkyl ring; R10 and R11 are independently hydrogen, deuterium, optionally substituted C1-C4 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl or R10 and R11 combine with the atoms to which they are attached to form an optionally substituted C3-C6 cycloalkyl ring; or R10 or R11 combine with R8 or R9 with the atoms to which they are attached to form an optionally substituted C3-C4 cycloalkyl ring; or R10 or R11 combine with R12 with the atoms to which they are attached to form an optionally substituted C3-C4 heterocycle; R12 is hydrogen, optionally substituted C1-C6 alkyl, or R12 combines with R8 or R9 with the atoms to which they are attached to form an optionally substituted C3-C4 heterocycle, or R12 combines with R10 or R11 with the atoms to which they are attached to form an optionally substituted C3-C4 heterocycle; wherein if m is 1 and R10 is methyl then at least one of R9, and R11 is not hydrogen; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein R.sup.10 is optionally substituted C.sub.1-C.sub.4 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl.

3. The compound of claim 2, wherein R.sup.10 is methyl, ethyl, n-propyl, iso-propyl, CD.sub.3, CF.sub.3, CH.sub.2F, CHF.sub.2, CHCH.sub.2, or CCH.

4. The compound of claim 2, wherein R.sup.11 is hydrogen or methyl.

5. The compound of claim 2, wherein m is 2 and the second R.sup.8 is hydrogen.

6. The compound of claim 2, wherein R.sup.9 is hydrogen, NH.sub.2, or methyl.

7. The compound of claim 1, wherein said optionally substituted C3-C4 heterocycle is an optionally substituted C4 heterocycle.

8. A compound having the structure of Formula VIII: ##STR00610## wherein b is 0 or 1 and c is 0; Q1 is ##STR00611## R9 is hydrogen, deuterium, halo, NH2, optionally substituted C1-C3 alkyl; and R10 and R11 are, independently, hydrogen, deuterium, optionally substituted C1-C4 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, or a pharmaceutically acceptable salt thereof.

9. The compound of claim 1, wherein R10 is hydrogen or optionally substituted C1-C4 alkyl.

10. The compound of claim 9, wherein R10 is hydrogen or methyl.

11. The compound of claim 1, wherein R11 is hydrogen.

12. The compound of claim 1, wherein R9 is hydrogen, halo, hydroxyl, NH2, optionally substituted C1-C3 alkyl.

13. The compound of claim 12, wherein R9 is hydrogen, fluoro, hydroxyl, NH2, or methyl.

14. A compound having the structure: ##STR00612## or a pharmaceutically acceptable salt thereof.

15. A compound having the structure: ##STR00613## or a pharmaceutically acceptable salt thereof.

16. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-E are a set of diagrams and photographs showing that miR-483-5p, miR-551a and CKB are clinically relevant and can be therapeutically inhibited. a, miR-483-5p and miR-551a levels in 37 primary tumor samples and 30 liver metastases samples were quantified by quantitative real-time PCR. b, CKB expression levels in 37 primary tumor samples and 30 liver metastases samples were measured by quantitative real-time PCR. c, Liver metastasis in mice injected with LvM3b cells and treated with a single dose of AAV doubly expressing miR-483-5p and miR-551a one day after injection cells. d, Bioluminescent measurements of liver metastasis in mice injected with 510.sup.5 LvM3b cells and treated with cyclocreatine daily for two weeks. Mice were euthanized and livers excised for ex vivo imaging at the end of the treatment. e, Bioluminescent measurements of liver metastasis in mice injected with 510.sup.5 LvM3b cells and treated with the creatine transporter inhibitor beta-guanidinopropionic acid ((-GPA) daily for two weeks. Error bars, s.e.m; all P values are based on one-sided Student's t-tests. *P<0.05; **P<0.001; ***P<0.0001.

(2) FIG. 2 is a diagram and a photograph showing that -GPA treatment suppressed colorectal cancer metastasis. Bioluminescent measurements of liver metastasis in mice injected with 510.sup.5 LvM3b cells and treated with -GPA daily for three weeks. Mice were euthanized at three weeks and liver extracted for bioluminescent imaging and gross histology. Error bars represent the s.e.m; all P values are based on one-sided Student's t-tests. *P<0.05.

(3) FIGS. 3A-C are a set of diagrams and photographs showing that creatine transporter, SLC6a8 is required for colorectal and pancreatic cancer metastasis. a) Liver metastasis by highly aggressive LvM3b cells expressing short hairpins targeting the creatine transporter channel, SLC6a8. Liver metastasis were monitored by bioluminescent imaging and mice were euthanized three weeks after inoculation of cancer cells. Livers were extracted for gross histology. b) Liver metastasis in mice injected with 510.sup.5 SW480 cells transduced with a shRNA targeting SLC6a8. Metastatic progression was monitored by bioluminescent imaging. Mice were euthanized 28 days after injection and livers excised for bioluminescent imaging and gross histology. c) Liver metastasis in mice injected with 510.sup.5 PANC1 pancreatic cancer cells transduced with a shRNA targeting SLC6a8. Metastatic progression was monitored by bioluminescent imaging and mice were euthanized as described above. Error bars represent the s.e.m; all P values are based on one-sided Student's t-tests. *P<0.05; **P<0.001; ***P<0.0001.

(4) FIG. 4 is a diagram showing that SLC6a8 is up-regulated in liver metastases compared to primary tumors. Expression of SLC6a8 in 36 primary tumors and 30 liver metastases were quantified by quantitative real-time PCR. Error bars represent the s.e.m; all P values are based on one-sided Student's t-tests. *P<0.05.

(5) FIG. 5 is a diagram and a photograph showing that -GPA treatment suppresses survival of disseminated PANC1 pancreatic cancer cells in the liver in vivo. Bioluminescence imaging of immunodeficient mice injected with 510.sup.5 PANC1 cells with and without 10 mM -GPA-pre-treatment for 48 hr. Mice were imaged on day 1 after injection and signal was normalized to day zero. P values are based on one-sided Student's t-tests. *P<0.05.

(6) FIG. 6 is a diagram showing that -GPA enhances the cytotoxicity of Gemcitabine on PANC1 pancreatic cancer cells. Cell viability of PANC1 pancreatic cancer cells after treatment with escalating doses of Gemcitabine alone or escalating doses of Gemcitabine in combination with 10 mM -GPA. Cell viability was assayed using the WST-1 reagent. Error bars represent standard error of the mean.

(7) FIG. 7 is a diagram showing that -GPA enhances the cytotoxicity of 5-fluorouracil on LS-LvM3b colorectal cancer cells. Cell viability of Ls-LvM3b cells after treatment with escalating doses of 5-Fluorouracil alone or escalating doses of 5-Fluorouracil in combination with 10 mM -GPA. Cell viability was assayed using the WST-1 reagent. Error bars represent standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

(8) The present invention features methods for preventing or reducing aberrant proliferation, differentiation, or survival of cells. For example, compounds of the invention may be useful in reducing the risk of, or preventing, tumors from increasing in size or from reaching a metastatic state. The subject compounds may be administered to halt the progression or advancement of cancer. In addition, the instant invention includes use of the subject compounds to reduce the risk of, or prevent, a recurrence of cancer.

(9) Compounds

(10) The invention features compounds useful in the treatment of cancer. Exemplary compounds described herein include compounds having a structure according to Formulae I-Ill as described herein:

(11) ##STR00466##

(12) wherein X.sup.1 is absent, NH, or CH.sub.2;

(13) R.sup.1 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl;

(14) R.sup.2, R.sup.3, and R.sup.4 are independently hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl; and

(15) R.sup.5 and R.sup.6 are hydrogen or NH.sub.2;

(16) wherein if R.sup.5 and R.sup.6 are both hydrogen or R.sup.5 is NH.sub.2 and R.sup.6 is hydrogen then R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl,

(17) or a pharmaceutically acceptable salt thereof;

(18) ##STR00467##

(19) wherein Q.sup.1 is optionally substituted amidino or optionally substituted 2-pyridyl;

(20) X.sup.2 is S or NR.sup.12;

(21) m is 0 or 1;

(22) R.sup.7 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl;

(23) R.sup.5 and R.sup.9 are independently hydrogen, deuterium, halo, hydroxyl, NH.sub.2, optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.5 or R.sup.9 can combine with R.sup.10 or R.sup.11 to form an optionally substituted C.sub.3-C.sub.6 cycloalkyl ring or with R.sup.12 to form an optionally substituted C.sub.3-C.sub.6 heterocycle;

(24) R.sup.1 and R.sup.11 are independently hydrogen, deuterium, optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.10 or R.sup.11 can combine with R.sup.5 or R.sup.9 to form an optionally substituted C.sub.3-C.sub.6 cycloalkyl ring;

(25) R.sup.12 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.12 can combine with R.sup.5 or R.sup.9 to form an optionally substituted C.sub.3-C.sub.6 heterocycle, and

(26) wherein if R.sup.9 is halo then R.sup.5 is halo or optionally substituted C.sub.1-C.sub.6 alkyl,

(27) or a pharmaceutically acceptable salt thereof;

(28) ##STR00468##

(29) wherein Q.sup.1 is optionally substituted amidino or optionally substituted 2-pyridyl; m is 1 or 2;

(30) R.sup.7 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl;

(31) R.sup.8 and R.sup.9 are independently hydrogen, deuterium, halo, hydroxyl, NH.sub.2, optionally substituted C.sub.1-C.sub.3 alkyl, or R.sup.8 and R.sup.9 combine with the atoms to which they are attached to form an optionally substituted

(32) C.sub.3-C.sub.6 cycloalkyl ring; or R.sup.8 or R.sup.9 combine with R.sup.13 or R.sup.11 with the atoms to which they are attached to form an optionally substituted C.sub.3-C.sub.4 cycloalkyl ring; or R.sup.8 or R.sup.9 combine with R.sup.12 with the atoms to which they are attached to form an optionally substituted C.sub.3-C.sub.5 heterocycle;

(33) R.sup.13 and R.sup.11 are independently hydrogen, deuterium, optionally substituted C.sub.1-C.sub.4 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl or R.sup.13 and R.sup.11 combine with the atoms to which they are attached to form an optionally substituted C.sub.3-C.sub.6 cycloalkyl ring; or R.sup.13 or R.sup.11 combine with R.sup.8 or R.sup.9 with the atoms to which they are attached to form an optionally substituted C.sub.3-C.sub.4 cycloalkyl ring; or R.sup.13 or R.sup.11 combine with R.sup.12 with the atoms to which they are attached to form an optionally substituted C.sub.3-C.sub.4 heterocycle;

(34) R.sup.12 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.12 combines with R.sup.8 or R.sup.9 with the atoms to which they are attached to form an optionally substituted C.sub.3-C.sub.5 heterocycle, or R.sup.12 combines with R.sup.10 or R.sup.11 with the atoms to which they are attached to form an optionally substituted C.sub.3-C.sub.4 heterocycle

(35) wherein if m is 1 and R.sup.8 is hydrogen, halo, hydroxyl, or methyl then at least one of R.sup.9, R.sup.10, and R.sup.11 is not hydrogen;

(36) wherein if m is 1 and R.sup.13 is methyl then at least one of R.sup.8, R.sup.9, and R.sup.11 is not hydrogen;

(37) wherein if m is 1 and R.sup.8 is NH.sub.2 and R.sup.13 is hydrogen, methyl, or CH.sub.2CH.sub.2OH then at least one of R.sup.9 or R.sup.11 is not hydrogen;

(38) wherein if m is 1, R.sup.8 is halo, and R.sup.13 is optionally substituted C.sub.1-C.sub.4 alkyl then at least one of R.sup.9 and R.sup.13 is not hydrogen;

(39) or a pharmaceutically acceptable salt thereof; and
A-BFormula III

(40) wherein A is a inhibitor of creatine transport and/or creatine kinase comprising an amidino group; B has the structure:

(41) ##STR00469##

(42) wherein n is 0 or 1;

(43) Q.sup.2 is hydroxyl, optionally substituted amino, or SO.sub.2OH; and

(44) R.sup.13 and R.sup.14 are independently hydrogen, CO.sub.2H, or combine to form CO;

(45) wherein B is conjugated to A at one of the amidino nitrogens,

(46) or a pharmaceutically acceptable salt thereof.

(47) Other embodiments (e.g., Compounds 1-326 of Tables 1-11) as well as exemplary methods for the synthesis of these compounds are described herein.

(48) Utility and Administration

(49) The compounds described herein (e.g., a compound according to Formulae I-IX or any of Compounds 1-448 of Tables 1-11) are useful in the methods of the invention and, while not bound by theory, are believed to exert their desirable effects through their ability to inhibit creatine transport and/or creatine kinase. The compounds described herein (e.g., a compound according to Formulae I-IX or any of Compounds 1-448 of Tables 1-11) can also be used for the treatment of certain conditions such as cancer.

(50) Creatine helps supply energy to all cells in the body by increasing formation of ATP. It is taken up by tissues with high energy demands through an active transport system. The conversion of ADP to ATP by phosphate transfer from phosphocreatine is catylzed by creatine kinase. Some of the functions associated with the phosphocreatine system include efficient regeneration of energy in the form of ATP in cells with fluctuating and high energy demand, energy transport to different parts of the cell, phosphoryl transport activity, ion transport regulation, and involvement in signal transduction pathways.

(51) Creatine kinase has been shown to have elevated levels in certain tumor types. These tumor types may utilize the increased expression of creatine kinase to prevent apoptosis under hypoxic or hypoglycemic conditions. Malignant cancers with poor prognosis have also been shown to overexpress creatine kinases, which may be related to high energy turnover and failure to eliminate cancer cells by apoptosis. Inhibtion of the active transport of creatine into cancer cells may reverse these trends and result in inhibition of the cancer and/or metastasis.

(52) Treatment Methods

(53) As disclosed herein, inhibition of creatine transport and/or creatine kinase suppresses metastasis. The phosphocreatine system promotes metastasis by enhancing the survival of disseminated cancer cells in the liver by acting as an energetic store for ATP generation to endure hepatic hypoxia. Inhibition of creatine transport into cancer cells limits the amount of phosphocreatine available to use in the production of ATP. Inhibition of creatine kinase inhibits the production of ATP through conversion of phosphocreatine to creatine.

(54) Typical vascularized tumors that can be treated with the method include solid tumors, particularly carcinomas, which require a vascular component for the provision of oxygen and nutrients. Exemplary solid tumors include, but are not limited to, carcinomas of the lung, breast, bone, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, glioblastomas, neuroblastomas, Kaposi's sarcoma, and sarcomas.

(55) Treating cancer can result in a reduction in size or volume of a tumor. For example, after treatment, tumor size is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to its size prior to treatment. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor or by any reproducible means of measurement.

(56) Treating cancer may further result in a decrease in number of tumors. For example, after treatment, tumor number is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to number prior to treatment. Number of tumors may be measured by any reproducible means of measurement. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification (e.g., 2, 3, 4, 5, 10, or 50).

(57) Treating cancer can result in a decrease in number of metastatic nodules in other tissues or organs distant from the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to number prior to treatment. The number of metastatic noduless may be measured by any reproducible means of measurement. The number of metastatic nodules may be measured by counting metastatic nodules visible to the naked eye or at a specified magnification (e.g., 2, 10, or 50).

(58) Treating cancer can result in an increase in average survival time of a population of subjects treated according to the present invention in comparison to a population of untreated subjects. For example, the average survival time is increased by more than 30 days (more than 60 days, 90 days, or 120 days). An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with the compound of the invention. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with the compound of the invention.

(59) Treating cancer can also result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. For example, the mortality rate is decreased by more than 2% (e.g., more than 5%, 10%, or 25%). A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with the compound of the invention. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with the compound of the invention.

(60) Compositions

(61) Within the scope of this invention is a composition that contains a suitable carrier and one or more of the therapeutic agents described above. The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier, a dietary composition that contains a dietarily acceptable suitable carrier, or a cosmetic composition that contains a cosmetically acceptable carrier. The term pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A pharmaceutically acceptable carrier, after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be acceptable also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active compound. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

(62) As used herein, the term pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, or allergic response, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds, are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977), incorporated herein by reference. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting a free base or free acid function with a suitable reagent, as described generally below. For example, a free base function can be reacted with a suitable acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may, include metal salts such as alkali metal salts, e.g. sodium or potassium salts; and alkaline earth metal salts, e.g. calcium or magnesium salts. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts, include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hem isulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

(63) As described above, the pharmaceutical compositions of the present invention additionally include a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; natural and synthetic phospholipids, such as soybean and egg yolk phosphatides, lecithin, hydrogenated soy lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, dioleoyl lecithin, hydroxylated lecithin, lysophosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine, diastearoyl phosphatidylethanolamine (DSPE) and its pegylated esters, such as DSPE-PEG750 and, DSPE-PEG2000, phosphatidic acid, phosphatidyl glycerol and phosphatidyl serine. Commercial grades of lecithin which are preferred include those which are available under the trade name Phosal or Phospholipon and include Phosal 53 MCT, Phosal 50 PG, Phosal 75 SA, Phospholipon 90H, Phospholipon 90G and Phospholipon 90 NG; soy-phosphatidylcholine (SoyPC) and DSPE-PEG2000 are particularly preferred; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

(64) The above-described composition, in any of the forms described above, can be used for treating melanoma, or any other disease or condition described herein. An effective amount refers to the amount of an active compound/agent that is required to confer a therapeutic effect on a treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment. A pharmaceutical composition of this invention can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term parenteral as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

(65) A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, polyoxyethylated versions thereof. These oil solutions or suspensions also can contain a long chain alcohol diluent or dispersant such as, but not limited to, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants, such as, but not limited to, Tweens or Spans or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms also can be used for the purpose of formulation.

(66) A composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. In the case of tablets, commonly used carriers include, but are not limited to, lactose and corn starch. Lubricating agents, such as, but not limited to, magnesium stearate, also are typically added. For oral administration in a capsule form, useful diluents include, but are not limited to, lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.

(67) Pharmaceutical compositions for topical administration according to the described invention can be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils. Alternatively, topical formulations can be in the form of patches or dressings impregnated with active ingredient(s), which can optionally include one or more excipients or diluents. In some preferred embodiments, the topical formulations include a material that would enhance absorption or penetration of the active agent(s) through the skin or other affected areas.

(68) A topical composition contains a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. A cosmetically acceptable or dermatologically-acceptable composition or component refers a composition or component that is suitable for use in contact with human skin without undue toxicity, incompatibility, instability, or allergic response. The carrier enables an active agent and optional component to be delivered to the skin at an appropriate concentration(s). The carrier thus can act as a diluent, dispersant, solvent, or the like to ensure that the active materials are applied to and distributed evenly over the selected target at an appropriate concentration. The carrier can be solid, semi-solid, or liquid. The carrier can be in the form of a lotion, a cream, or a gel, in particular one that has a sufficient thickness or yield point to prevent the active materials from sedimenting. The carrier can be inert or possess dermatological benefits. It also should be physically and chemically compatible with the active components described herein, and should not unduly impair stability, efficacy, or other use benefits associated with the composition.

(69) Combination Therapies

(70) In some embodiments, the pharmaceutical composition may further include an additional compound having antiproliferative activity. The additional compound having antiproliferative activity can be selected from a group of antiproliferative agents including those shown in Table 12.

(71) It will also be appreciated that the compounds and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects).

(72) By antiproliferative agent is meant any antiproliferative agent, including those antiproliferative agents listed in Table 12, any of which can be used in combination with a creatine transport and/or creatine kinase inhibitor to treat the medical conditions recited herein. Antiproliferative agents also include organo-platine derivatives, naphtoquinone and benzoquinone derivatives, chrysophanic acid and anthroquinone derivatives thereof.

(73) TABLE-US-00012 TABLE 12 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazine ifosfamide altretamine hexamethylmelamine estramustine phosphate thiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomide cyclophosphamide Semustine Platinum agents spiroplatin lobaplatin (Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464 (Hoffmann-La Roche) iproplatin SM-11355 (Sumitomo) ZD-0473 (AnorMED) AP-5280 (Access) oxaliplatin cisplatin carboplatin Antimetabolites azacytidine trimetrexate Floxuridine deoxycoformycin 2-chlorodeoxyadenosine pentostatin 6-mercaptopurine hydroxyurea 6-thioguanine decitabine (SuperGen) cytarabine clofarabine (Bioenvision) 2-fluorodeoxy cytidine irofulven (MGI Pharma) methotrexate DMDC (Hoffmann-La Roche) tomudex ethynylcytidine (Taiho) fludarabine gemcitabine raltitrexed capecitabine Topoisomerase amsacrine exatecan mesylate (Daiichi) inhibitors epirubicin quinamed (ChemGenex) etoposide gimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan (Beaufour-Ipsen) 7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho) dexrazoxanet (TopoTarget) elsamitrucin (Spectrum) pixantrone (Novuspharma) J-107088 (Merck & Co) rebeccamycin analogue (Exelixis) BNP-1350 (BioNumerik) BBR-3576 (Novuspharma) CKD-602 (Chong Kun Dang) rubitecan (SuperGen) KW-2170 (Kyowa Hakko) irinotecan (CPT-11) hydroxycamptothecin (SN-38) topotecan Antitumor antibiotics valrubicin azonafide therarubicin anthrapyrazole idarubicin oxantrazole rubidazone losoxantrone plicamycin MEN-10755 (Menarini) porfiromycin GPX-100 (Gem Pharmaceuticals) mitoxantrone (novantrone) Epirubicin amonafide mitoxantrone doxorubicin Antimitotic colchicine E7010 (Abbott) agents vinblastine PG-TXL (Cell Therapeutics) vindesine IDN 5109 (Bayer) dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin (Fujisawa) A 204197 (Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D 24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258 (Aventis) combretastatin A4 (BMS) epothilone B (Novartis) isohomohalichondrin-B (PharmaMar) T 900607 (Tularik) ZD 6126 (AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi) cryptophycin 52 (Eli Lilly) IDN-5109 (Indena) vinflunine (Fabre) AVLB (Prescient NeuroPharma) auristatin PE (Teikoku Hormone) azaepothilone B (BMS) BMS 247550 (BMS) BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4 prodrug (OXiGENE) BMS 188797 (BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075 (GlaxoSmithKline) docetaxel Vinorelbine vincristine Trichostatin A paclitaxel Aromatase inhibitors aminoglutethimide YM-511 (Yamanouchi) atamestane (BioMedicines) formestane letrozole exemestane anastrazole Thymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias) synthase inhibitors ZD-9331 (BTG) CoFactor (BioKeys) DNA antagonists trabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (Baxter International) mafosfamide (Baxter International) albumin + 32P (Isotope Solutions) apaziquone (Spectrum thymectacin (NewBiotics) Pharmaceuticals) O6 benzyl guanine (Paligent) Farnesyltransferase arglabin (NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitors lonafarnib (Schering-Plough) perillyl alcohol (DOR BioPharma) BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma) zosuquidar trihydrochloride (Eli Lilly) tariquidar (Xenova) biricodar dicitrate (Vertex) MS-209 (Schering AG) Histone tacedinaline (Pfizer) pivaloyloxymethyl butyrate (Titan) acetyltransferase SAHA (Aton Pharma) depsipeptide (Fujisawa) inhibitors MS-275 (Schering AG) Metalloproteinase Neovastat (Aeterna Laboratories) CMT-3 (CollaGenex) inhibitors marimastat (British Biotech) BMS-275291 (Celltech) Ribonucleoside gallium maltolate (Titan) tezacitabine (Aventis) reductase inhibitors triapine (Vion) didox (Molecules for Health) TNF alpha virulizin (Lorus Therapeutics) revimid (Celgene) agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan (Abbott) YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca) Retinoic acid fenretinide (Johnson & Johnson) alitretinoin (Ligand) receptor agonists LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy (Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK (Progenies) Technology) adenocarcinoma vaccine (Biomira) ISF-154 (Tragen) CTP-37 (AVI BioPharma) cancer vaccine (Intercell) IRX-2 (Immuno-Rx) norelin (Biostar) PEP-005 (Peplin Biotech) BLP-25 (Biomira) synchrovax vaccines (CTL Immuno) MGV (Progenies) melanoma vaccine (CTL Immuno) -alethine (Dovetail) p21 RAS vaccine (GemVax) CLL therapy (Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCam Biotherapeutics) abatacept (BMS) MPDL3280A (Genentech) pembrolizumab MEDI4736 Hormonal and estrogens dexamethasone antihormonal agents conjugated estrogens prednisone ethinyl estradiol methylprednisolone chlortrianisen prednisolone idenestrol aminoglutethimide hydroxyprogesterone caproate leuprolide medroxyprogesterone octreotide testosterone mitotane testosterone propionate; P-04 (Novogen) fluoxymesterone 2-methoxyestradiol (EntreMed) methyltestosterone arzoxifene (Eli Lilly) diethylstilbestrol tamoxifen megestrol toremofine bicalutamide goserelin flutamide Leuporelin nilutamide bicalutamide Photodynamic talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda) agents Theralux (Theratechnologies) lutetium texaphyrin (Pharmacyclics) motexafin gadolinium hypericin (Pharmacyclics) Kinase Inhibitors imatinib (Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia) kahalide F (PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib (Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518 (Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia) Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190 (AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774 (Tarceva) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) Emodin Genistein Radicinol Radicinol Vemurafenib (B-Raf enzyme Met-MAb (Roche) inhibitor, Daiichi Sankyo) SR-27897 (CCK A inhibitor, Sanofi-Synthelabo) ceflatonin (apoptosis promotor, ChemGenex) tocladesine (cyclic AMP agonist, Ribapharm) BCX-1777 (PNP inhibitor, BioCryst) alvocidib (CDK inhibitor, Aventis) ranpirnase (ribonuclease stimulant, Alfacell) CV-247 (COX-2 inhibitor, Ivy Medical) galarubicin (RNA synthesis inhibitor, Dong-A) P54 (COX-2 inhibitor, Phytopharm) tirapazamine (reducing agent, SRI CapCell (CYP450 stimulant, Bavarian Nordic) International) GCS-100 (gal3 antagonist, GlycoGenesys) N-acetylcysteine (reducing agent, Zambon) G17DT immunogen (gastrin inhibitor, Aphton) R-flurbiprofen (NF-kappaB inhibitor, Encore) efaproxiral (oxygenator, Allos Therapeutics) 3CPA (NF-kappaB inhibitor, Active Biotech) PI-88 (heparanase inhibitor, Progen) seocalcitol (vitamin D receptor agonist, Leo) tesmilifene (histamine antagonist, YM 131-I-TM-601 (DNA antagonist, BioSciences) TransMolecular) histamine (histamine H2 receptor agonist, Maxim) eflornithine (ODC inhibitor, ILEX Oncology) tiazofurin (IMPDH inhibitor, Ribapharm) minodronic acid (osteoclast inhibitor, cilengitide (integrin antagonist, Merck KGaA) Yamanouchi) SR-31747 (IL-1 antagonist, Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai) CCI-779 (mTOR kinase inhibitor, Wyeth) aplidine (PPT inhibitor, PharmaMar) exisulind (PDE V inhibitor, Cell Pathways) gemtuzumab (CD33 antibody, Wyeth Ayerst) CP-461 (PDE V inhibitor, Cell Pathways) PG2 (hematopoiesis enhancer, AG-2037 (GART inhibitor, Pfizer) Pharmagenesis) WX-UK1 (plasminogen activator inhibitor, Wilex) Immunol (triclosan oral rinse, Endo) PBI-1402 (PMN stimulant, ProMetic LifeSciences) triacetyluridine (uridine prodrug, Wellstat) bortezomib (proteasome inhibitor, Millennium) SN-4071 (sarcoma agent, Signature SRL-172 (T cell stimulant, SR Pharma) BioScience) TLK-286 (glutathione S transferase inhibitor, TransMID-107 (immunotoxin, KS Biomedix) Telik) PCK-3145 (apoptosis promotor, Procyon) PT-100 (growth factor agonist, Point doranidazole (apoptosis promotor, Pola) Therapeutics) cafestol Chrysophanic acid kahweol Cesium oxides caffeic acid BRAF inhibitors, Tyrphostin AG PDL1 inhibitors PD-1 inhibitors MEK inhibitors CTLA-4 inhibitors bevacizumab sorafenib angiogenesis inhibitors BRAF inhibitors rituximab (CD20 antibody, Genentech urocidin (apoptosis promotor, Bioniche) carmustine Ro-31-7453 (apoptosis promotor, La Roche) Mitoxantrone brostallicin (apoptosis promotor, Pharmacia) Bleomycin -lapachone Absinthin gelonin dabrafenib CRS-207 midostaurin (PKC inhibitor, Novartis) CHS-828 (cytotoxic agent, Leo) bryostatin-1 (PKC stimulant, GPC Biotech) trans-retinoic acid (differentiator, NIH) CDA-II (apoptosis promoter, Everlife) MX6 (apoptosis promoter, MAXIA) SDX-101 (apoptosis promoter, Salmedix) apomine (apoptosis promoter, ILEX Oncology)

(74) The invention features the following numbered embodiments:

(75) 1. A compound having the structure:

(76) ##STR00470##

(77) wherein X.sup.1 is absent, NH, or CH.sub.2;

(78) R.sup.1 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl;

(79) R.sup.2, R.sup.3, and R.sup.4 are independently hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl; and

(80) R.sup.5 and R.sup.6 are hydrogen or NH.sub.2;

(81) wherein if R.sup.5 and R.sup.6 are both hydrogen or R.sup.5 is NH.sub.2 and R.sup.6 is hydrogen then R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl,

(82) or a pharmaceutically acceptable salt thereof.

(83) 2. The compound of embodiment 1, wherein R.sup.1 is hydrogen.

(84) 3. The compound of embodiments 1 or 2, wherein R.sup.3 and R.sup.4 are hydrogen.

(85) 4. The compound of any one of embodiments 1-3, wherein R.sup.2 is hydrogen or optionally substituted C.sub.1-C.sub.6 alkyl, wherein said optionally substituted C.sub.1-C.sub.6 alkyl is methyl, ethyl, isopropyl, propyl, isobutyl, or optionally substituted C.sub.1-C.sub.6 haloalkyl.

(86) 5. The compound of embodiment 4, wherein said optionally substituted C.sub.1-C.sub.6 haloalkyl is trifluoromethyl.

(87) 6. The compound of any one of embodiments 1-5, wherein R.sup.5 and R.sup.6 are both hydrogen and R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl.

(88) 7. The compound of embodiment 6, wherein said optionally substituted C.sub.1-C.sub.6 alkyl is methyl, ethyl, isopropyl, or isobutyl.

(89) 8. The compound of any one of embodiments 1-5, wherein R.sup.5 and R.sup.6 are both NH.sub.2.

(90) 9. The compound of embodiment 8, wherein R.sup.2 is hydrogen.

(91) 10. The compound of embodiment 8, wherein R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl.

(92) 11. The compound of embodiment 10, wherein said optionally substituted C.sub.1-C.sub.6 alkyl is methyl or isopropyl.

(93) 12. The compound of any one of embodiments 1-5, wherein R.sup.5 is NH.sub.2, R.sup.6 is hydrogen, and R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl.

(94) 13. The compound of embodiment 12, wherein said optionally substituted C.sub.1-C.sub.6 alkyl is methyl or isopropyl.

(95) 14. The compound of any one of embodiments 1-5, wherein R.sup.5 is hydrogen and R.sup.6 is NH.sub.2.

(96) 15. The compound of embodiment 14, wherein R.sup.2 is hydrogen.

(97) 16. The compound of embodiment 14, wherein R.sup.2 is optionally substituted C.sub.1-C.sub.6 alkyl.

(98) 17. The compound of embodiment 16, wherein said optionally substituted C.sub.1-C.sub.6 alkyl is methyl or isopropyl.

(99) 18. The compound of any one of embodiments 1-17, wherein X.sup.1 is absent.

(100) 19. The compound of any one of embodiments 1-17, wherein X.sup.1 is CH.sub.2.

(101) 20. The compound of any one of embodiments 1-17, wherein X.sup.1 is NH.sub.2.

(102) 21. The compound of embodiment 1, wherein said compound is any one of the compounds of Tables 1-3.

(103) 22. A compound having the structure:

(104) ##STR00471##

(105) wherein Q.sup.1 is optionally substituted amidino or optionally substituted 2-pyridyl;

(106) X.sup.2 is S or NR.sup.12;

(107) m is 0 or 1;

(108) R.sup.7 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl;

(109) R.sup.8 and R.sup.9 are independently hydrogen, deuterium, halo, hydroxyl, N H.sub.2, optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.8 or R.sup.9 can combine with R.sup.10 or R.sup.11 to form an optionally substituted C.sub.3-C.sub.6 cycloalkyl ring or with R.sup.12 to form an optionally substituted C.sub.3-C.sub.6 heterocycle;

(110) R.sup.10 and R.sup.11 are independently hydrogen, deuterium, optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.10 or R.sup.11 can combine with R.sup.8 or R.sup.9 to form an optionally substituted C.sub.3-C.sub.6 cycloalkyl ring;

(111) R.sup.12 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, or R.sup.12 can combine with R.sup.8 or R.sup.9 to form an optionally substituted C.sub.3-C.sub.6 heterocycle,

(112) wherein if Q.sup.1 is optionally substituted 2-pyridyl then R.sup.12 is hydrogen, and wherein if R.sup.9 is halo then R.sup.8 is halo or optionally substituted C.sub.1-C.sub.6 alkyl,

(113) or a pharmaceutically acceptable salt thereof.

(114) 23. The compound of embodiment 22, wherein R.sup.7 is hydrogen.

(115) 24. The compound of embodiments 22 or 23, wherein m is 1.

(116) 25. The compound of any one of embodiments 22-24, wherein R.sup.9 is hydrogen, deuterium, or halo.

(117) 26. The compound of embodiment 25, wherein said halo is fluoro.

(118) 27. The compound of any one of embodiments 22-26, wherein R.sup.11 is hydrogen or deuterium.

(119) 28. The compound of any one of embodiments 22-27, wherein R.sup.8 and R.sup.10 combine to form an optionally substituted C.sub.3-C.sub.6 cycloalkyl ring.

(120) 29. The compound of embodiment 28, wherein said optionally substituted C.sub.3-C.sub.6 cycloalkyl ring is cyclopropyl or cyclobutyl.

(121) 30. The compound of any one of embodiments 22-27, wherein both R.sup.10 and R.sup.11 are deuterium.

(122) 31. The compound of embodiment 30, wherein R.sup.8 and R.sup.9 are both deuterium.

(123) 32. The compound of any one of embodiments 22-27, wherein both R.sup.8 and R.sup.9 are halo.

(124) 33. The compound of embodiment 32, wherein said halo is fluoro.

(125) 34. The compound of any one of embodiments 22-27, wherein R.sup.10 is optionally substituted C.sub.1-C.sub.6 alkyl.

(126) 35. The compound of embodiment 34, wherein said optionally substituted C.sub.1-C.sub.6 alkyl is optionally substituted C.sub.1-C.sub.6 haloalkyl.

(127) 36. The compound of embodiment 35, wherein said optionally substituted C.sub.1-C.sub.6 haloalkyl is trifluoromethyl.

(128) 37. The compound of embodiment 36, wherein R.sup.8 is NH.sub.2.

(129) 38. The compound of any one of embodiments 22-27, wherein R.sup.8 is NH.sub.2.

(130) 39. The compound of embodiment 38, wherein R.sup.10 is optionally substituted C.sub.1-C.sub.6 alkyl.

(131) 40. The compound of embodiment 39, wherein said optionally substituted C.sub.1-C.sub.6 alkyl is methyl.

(132) 41. The compound of any one of embodiments 22-40, wherein Q.sup.1 is optionally substituted amidino.

(133) 42. The compound of embodiment 41, wherein said optionally substituted amidino has the structure:

(134) ##STR00472##

(135) 43. The compound of embodiments 41 or 42, wherein X.sup.2 is NR.sup.12.

(136) 44. The compound of embodiment 43, wherein R.sup.8 and R.sup.12 combine to form an optionally substituted C.sub.3-C.sub.6 heterocycle.

(137) 45. The compound of embodiment 44, wherein said optionally substituted C.sub.3-C.sub.6 heterocycle is azetidine.

(138) 46. The compound of embodiment 43, wherein R.sup.12 is hydrogen.

(139) 47. The compound of embodiments 41 or 42, wherein X.sup.2 is S.

(140) 48. The compound of any one of embodiments 22-40, wherein Q.sup.1 is optionally substituted 2-pyridyl.

(141) 49. The compound of embodiment 48, wherein said optionally substituted 2-pyridyl has the structure:

(142) ##STR00473##

(143) 50. The compound of embodiments 48 or 49, wherein X.sup.2 is NR.sup.12 and R.sup.12 is hydrogen.

(144) 51. The compound of embodiment 22, wherein said compound is any one of the compounds of Tables 4-6.

(145) 52. A compound having the structure:
A-BFormula III

(146) wherein A is a inhibitor of creatine transport comprising an amidino group;

(147) B has the structure:

(148) ##STR00474##

(149) wherein n is 0 or 1;

(150) Q.sup.2 is hydroxyl, optionally substituted amino, or SO.sub.2OH; and

(151) R.sup.13 and R.sup.14 are independently hydrogen, CO.sub.2H, or combine to form CO;

(152) wherein B is conjugated to A at one of the amidino nitrogens,

(153) or a pharmaceutically acceptable salt thereof.

(154) 53. The compound of embodiment 52, wherein R.sup.14 is hydrogen.

(155) 54. The compound of embodiment 53, wherein R.sup.13 is CO.sub.2H.

(156) 55. The compound of embodiment 53, wherein R.sup.13 is hydrogen.

(157) 56. The compound of embodiments 52, wherein R.sup.13 and R.sup.14 combine to form CO.

(158) 57. The compound of any one of embodiments 52-56, wherein n is 0.

(159) 58. The compound of any one of embodiments 52-56, wherein n is 1.

(160) 59. The compound of any one of embodiments 52-58, wherein Q.sup.2 is optionally substituted amino.

(161) 60. The compound of embodiment 59, wherein said optionally substituted amino is NH.sub.2 or

(162) ##STR00475##

(163) 61. The compound of any one of embodiments 52-58, wherein Q.sup.2 is hydroxyl.

(164) 62. The compound of any one of embodiments 52-58, wherein Q.sup.2 is SO.sub.2OH.

(165) 63. The compound any one of embodiments 52-62, wherein said inhibitor of creatine transport has the structure of a compound of any one of embodiments 1-51 or any one of the compounds of Table 7 or Table 8.

(166) 64. The compound of embodiment 52, wherein said compound is any one of the compounds of Table 9 or Table 10.

(167) 65. A method for treating cancer, comprising administering to a subject in need thereof, a compound of any one of embodiments 1-64 in an amount sufficient to treat said cancer.

(168) 66. A method of slowing the spread of a migrating cancer, comprising administering to a subject in need thereof, a compound of any one of embodiments 1-64 in an amount sufficient to slow the spread of said migrating cancer.

(169) 67. The method of embodiment 66, wherein said method comprises the suppression of metastatic colonization of said migrating cancer in the liver.

(170) 68. The method of embodiment 67, wherein said migrating cancer is metastatic cancer.

(171) 69. The method of embodiment 68, wherein the metastatic cancer comprises cells exhibiting migration and/or invasion of migrating cells.

(172) 70. The method of embodiments 68 or 69, wherein said metastatic cancer comprises cells exhibiting endothelial recruitment and/or angiogenesis.

(173) 71. The method of any one of embodiments 67-70, wherein said migrating cancer spreads via seeding the surface of the peritoneal, pleural, pericardial, or subarachnoid spaces.

(174) 72. The method of any one of embodiments 67-70, wherein said migrating cancer spreads via the lymphatic system.

(175) 73. The method of any one of embodiments 67-70, wherein said migrating cancer spreads hematogenously.

(176) 74. The method of any one of embodiments 67-70, wherein said migrating cancer is a cell migration cancer.

(177) 75. The method of embodiment 74, wherein said cell migration cancer is a non-metastatic cell migration cancer.

(178) 76. The method of embodiment 75, where said cell migration cancer is ovarian cancer, mesothelioma, or primary lung cancer.

(179) 77. A method for inhibiting proliferation or growth of cancer stem cells or cancer initiating cells, comprising contacting the cell with a compound of any one of embodiments 1-64 in an amount sufficient to inhibit proliferation or growth of said cell.

(180) 78. A method of reducing the rate of tumor seeding of a cancer comprising administering to a subject in need thereof a compound of any one of embodiments 1-64 in an amount sufficient to reduce tumor seeding.

(181) 79. A method of reducing or treating metastatic nodule-forming of cancer comprising administering to a subject in need thereof a compound of any one of embodiments 1-64 in an amount sufficient to treat said metastatic nodule-forming of cancer.

(182) 80. The method of any one of embodiments 65-79, wherein said cancer is breast cancer, colon cancer, renal cell cancer, non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, or melanoma.

(183) 81. The method of any one of embodiments 65-79, wherein said cancer is gastrointestinal cancer.

(184) 82. The method of embodiment 81, wherein said gastrointestinal cancer is esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, gallbladder cancer, colorectal cancer, anal cancer, mucosa-associated lymphoid tissue cancer, gastrointestinal stromal tumors, a cancer of the biliary tree, or a gastrointestinal carcioid tumor.

(185) 83. The method of any one of embodiments 65-82, wherein said cancer is a drug resistant cancer.

(186) 84. The method of any one of embodiments 65-83 further comprising administering an additional antiproliferative agent.

(187) 85. The method of embodiment 84, wherein said additional antiproliferative agent is capecitabine, gemcitabine, fluorouracil, FOLFOX (5-FU, leucovorin, and Eloxatin), FOLFIRI (5-FU, leucovorin, and Camptosar), EOX (Epirubicin, Oxaliplatinum, and Xeloda), Taxotere, Erbitux, Zaltrap, Vectibix, Ramucirumab, Tivozanib, Stivarga, CRS-207, or a PD-1 or PDL-1 antibody.

(188) 86. A method of treating metastatic cancer in a subject in need thereof comprising:

(189) (a) providing a subject identified to have, or to be at risk of having, metastatic cancer on the basis of the expression level of miR-483-5p and/or miR-551a is below a predetermined reference value or the expression level of CKB and/or SLC6a8 is above a predetermined reference value; and

(190) (b) administering to said subject an effective amount of a compound of any one of embodiments 1-64.

(191) 87. A method for treating metastatic cancer in a subject in need thereof, comprising contacting creatine transport channel SLC6a8 with a compound of any one of embodiments 1-64 in an amount effective to suppress metastatic colonization of said cancer.

EXAMPLES

(192) Materials and Methods

(193) Cell Culture

(194) Indicated cell-lines were purchased from ATCC and cultured in DMEM media supplemented with 10% FBS, sodium pyruvate, L-glutamine and penicillin-streptomycin antibiotics. For drug pre-treatment, cells were treated with indicated amounts of drug for 24-48 hrs.

(195) Animal Studies

(196) All animal work was conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at The Rockefeller University. 5-6 weeks old age-matched male NOD-SCID mice were used for intrahepatic colonization and liver metastasis assays.

(197) Proliferation Assay in Hypoxic Conditions

(198) 100K cells were seeded in triplicates in 6 well plates and cells were counted 5 days after seeding. Cells were cultured in cell culture chamber containing 1% oxygen.

(199) Primary Tumor Growth

(200) 110.sup.6 cells were suspended in 100 l of 1:1 PBS:Matrigel mixture and injected into the subcutaneous flanks of mice. Tumor growth was measured using digital calipers starting 7 days after injection when palpable tumors can be measured accurately. Volume of the tumors were calculated using the formula, Volume=(width).sup.2(length)/2. When treated with drugs, mice were injected with indicated amounts of drug in 300 l PBS daily until the mice were euthanized.

(201) Metastasis Assay

(202) 510.sup.6 highly metastatic cancer cells were injected into the portal circulation of immunodeficient mice. One day after inoculation of cancer cells, mice were injected indicated amounts of drug in 300 L PBS. Treatment was continued daily and metastatic progression was monitored by bioluminescent imaging until the mice were euthanized at which point livers were excised for bioluminescent imaging and gross histology. Where indicated, cancer cells were pre-treated with indicated amounts of compound for 48 hrs before injection into immunodeficient mice.

(203) In Vivo Creatine Transporter Inhibition Assay

(204) Soluble compounds were formulated in saline solution (0.9% NaCl). Some compounds were first dissolved 1 N hydrochloric acid (1.0 equivalent) to make the HCI salt followed by addition of PBS to adjust to the final volume. Less soluble compounds were first dissolved 1 or 2 N hydrochloric acid (1.0 equivalent) to make the HCI salt followed by addition of DMSO and water to adjust to the final volume resulting in a 1:1 DMSO to aqueous ratio.

(205) Studies were performed on 6-7 week old C57B16 male mice, receiving a regular diet (Purina 5001, Research Diet) and on a regular sleep rhythm (12h night/day schedule). Experiments were performed 6h after exposing to daylight. Mice were weighed and randomly divided into groups of 3 mice per group and injected i.p. with 100-200 L of dosing solution to deliver 250 mg/kg (50 mg/mL or 381 mM) -GPA equivalent (i.e. 1.91 mmol/kg) along with a vehicle control. Creatine-(methyl-d.sub.3) monohydrate (i.e. creatine-d.sub.3, Cambridge Isotope Laboratories, Catalog DLM-1302) was dissolved in 100 L 0.9% NaCl (0.2 mg/mL) and injected i.p. 7 minutes after drug injection. Volumes were adjusted based on the weight of the mice to reach a final dose of 1 mg/kg. After one hour, mice were euthanized, hearts were perfused with PBS, removed, snap-frozen in liquid nitrogen, and stored at 80 C. until further processing.

(206) Mouse hearts were thawed and weighed into 1.5 mL Eppendorf conical tubes. Typical heart weights range from 800-1200 mg. Between six to twelve 1 mm zirconia/silica beads (BioSpec Products, Inc., Bartlesville, Okla.) were added to the tubes with sufficient volume of 70% 2-propanol in water to afford a 4-fold dilution. The samples were then placed in a MiniBeadBeater (BioSpec Products, Inc.) for 2 minutes to disrupt the tissue and homogenize the sample.

(207) Aliqouts (20 L) of the homogenized hearts were transferred to a 96-well microtiter plate. Samples were extracted by the addition of acetonitrile (1.0 mL) containing 0.25 g/mL of creatine-d.sub.5 (CDN Isotopes, Pointe-Claire, Quebec) as internal standard. Samples were mixed on a rotary shaker for 10 minutes then placed in a centrifuge to spin for 10 minutes at 3000 rpm at 4 C. Supernatant (900 L) was transferred to 96-well deep well plate for analysis.

(208) Calibration standards, blanks, and quality control samples are prepared from control mouse hearts homogenized as noted above. Aliquots of homogenate were then spiked with known quantities of creatine-d.sub.3 (CDN Isotopes, Pointe-Claire, Quebec) or solvent, and processed along with the samples as noted above.

(209) Analysis was conducted by LC-MS/MS using an Acquity UPLC (Waters Corp., Milford, Mass.)/Triple Quad 5500 (AB Sciex, Framingham, Mass.) system. Five microliters of sample are injected onto a HILIC column, 2.150 mm, 3 m (Fortis Technologies, Cheshire, England) at a flow rate of 0.4 mL/min. A binary gradient of acetonitrile and 10 mM ammonium acetate was used to elute analytes from the column. The mass spectrometer was operated in positive ion electrospray in Multiple Reaction Monitoring mode for the following mass transitions:

(210) Creatine-d.sub.5: m/z 137.1/95.0

(211) Creatine-d.sub.3: m/z 135.1/93.0

(212) Data were collected and processed using Analyst 1.6.2 (AB Sciex, Framingham, Mass.). A linear calibration of the creatine-d.sub.3/creatine-d.sub.5 peak area ratio ranged from 0.05 to 10 g/mL. Data was report as g of creatine-d.sub.3 per gram of heart. Mean values and standard deviations were calculated from three heart samples and percent creatine-d.sub.3 transport inhibition was reported relative to vehicle control.

(213) TABLE-US-00013 TABLE 13 Percent Inhibition of Creatine-d.sub.3 Transport in Heart Tissue. Compound % Inhibition of Creatine-d.sub.3 Transport 219 79.0 (+/1.2) 220 1.4 (+/8.2) 258 71.8 (+/5.9) 261 24.7 (+/12.6) 358 11.1 (+/13.6) 376 39.1 (+/25.2) 125 4.5 (+/10.6) 28 (-GPA) 73.4 (+/6.7)

(214) In Vivo Selection

(215) 110.sup.6 LS174T cells expressing a luciferase reporter were suspended in a volume of 20 l 1:1 PBS/Matrigel mixture and injected intra-hepatically into the livers of NOD-SCID mice. Metastatic nodules were allowed to develop over a period of 3-4 weeks and monitored by bioluminescence imaging. Nodules formed were excised and dissociated by collagenase and hyaluronidase digestion into single cell suspension. The cells were allowed to expand in in vitro before re-injection into mice. After three re-iterations of in vivo selection, highly metastatic LvM3a and LvM3b derivative cell-lines were established.

(216) Lenti-miR Library Screening

(217) Cells were transduced with a lentivirus Lenti-miR library of 611 miRNAs (System Biosciences) at a low multiplicity of infection (MOI) such that each cell over-expressed a single miRNA. The transduced population was then injected intra-hepatically into NOD-SCID mice for in vivo selection of miRNAs that when over-expressed, either promoted or suppressed metastatic liver colonization. Genomic DNA PCR amplication and recovery of lenti-viral miRNA inserts were performed on cells prior to injection and from liver nodules according to manufacturer's protocol. miRNA array profiling allowed for miRNA insert quantification prior to and after in vivo selection.

(218) Organotypic Slice Culture System

(219) Cells to be injected were labeled with cell-tracker red or green (Invitrogen) and inoculated into livers of NOD-SCID mice through intrasplenic injection. The livers were then extracted and cut into 150 um slices using a Mcllwain tissue chopper (Ted Pella) and plated onto organotypic tissue culture inserts (Millipore) and cultured in William's E Medium supplemented with Hepatocyte Maintenance Supplement Pack (Invitrogen). After indicated time periods, the liver slices were fixed in paraformaldehyde and imaged using multi-photon microscopy.

(220) In Vivo Caspase Activation Assay

(221) To measure caspase activity in vivo, VivoGlo Caspase 3/7 Substrate (Z-DEVD-Aminoluciferin Sodium Salt, Promega) was used. The luciferin is inactive until the DEVD peptide is cleaved from by activated caspase-3 in apoptotic cells. DEVD-luciferin was injected into mice bearing colorectal cancer cells expressing luciferase. Upon activation by apoptotic cells, bioluminescence imaging can be performed to measure caspase activity in vivo. Five hours after in vivo caspase activity measurement, mice are injected with regular luciferin for normalization purposes.

(222) Adeno-Associated Viral Therapy

(223) miR-483-5p and miR-551a were cloned as a polycistron consisting of both miRNA precursor with flanking genomic sequences in tandem into the BgIII and NotI site of scAAV.GFP (Plasmid 21893, Addgene). Listed below are genomic sequences encoding for miR-483-5p and miR-551a (SEQ ID NOs: 5 and 6), corresponding precursor sequences (underlined, SEQ ID NOs: 3 and 4), and corresponding mature microRNA sequences (underlined and in bold, SEQ ID NOs: 1 and 2). Adeno-associated viruses were packaged, purified and titered using the AAV-DJ Helper Free expression system from Cell Biolabs.

(224) TABLE-US-00014 miR-551a: GGAGAACCTTCAGCTTCATGTGACCCAGAGACTCCTGTATGCCTGGCTCT GGGAGTACAGAAGGGCCTAGAGCTGACCCCTGCCCTCCGAAGCCCCTGGG GCACTAGATGGATGTGTGCCAGAGGGTAGTAGAGGCCTGGGGGTAGAGCC CAGCACCCCCTTCGCGTAGAGACCTGGGGGACCAGCCAGCCCAGCAACCC CCTCGCGGCCGACGCCTGAGGCTGTTCCTGGCTGCTCCGGTGGCTGCCAG AGGGGACTGCCGGGTGACCCTGGAAATCCAGAGTGGGTGGGGCCAGTCTG ACCGTTTCTAGGCGACCCACTCTTGGTTTCCAGGGTTGCCCTGGAAACCA CAGATGGGGAGGGGTTGATGGCACCCAGCCTCCCCCAAGCCTGGGAAGGG ACCCCGGATCCCCAGAGCCTTTCCCTGCCTATGGAGCGTTTCTCTTGGAG AACAGGGGGGCCTCTCAGCCCCTCAATGCAAGTTGCTGAG miR-483-5p: CCTGCCCCATTTGGGGGTAGGAAGTGGCACTGCAGGGCCTGGTGCCAGCC AGTCCTTGCCCAGGGAGAAGCTTCCCTGCACCAGGCTTTCCTGAGAGGAG GGGAGGGCCAAGCCCCCACTTGGGGGACCCCCGTGATGGGGCTCCTGCTC CCTCCTCCGGCTGATGGCACCTGCCCTTTGGCACCCCAAGGTGGAGCCCC CAGCGACCTTCCCCTTCCAGCTGAGCATTGCTGTGGGGGAGAGGGGGAAG ACGGGAGGAAAGAAGGGAGTGGTTCCATCACGCCTCCTCACTCCTCTCCT CCCGTCTTCTCCTCTCCTGCCCTTGTCTCCCTGTCTCAGCAGCTCCAGGG GTGGTGTGGGCCCCTCCAGCCTCCTAGGTGGTGCCAGGCCAGAGTCCAAG CTCAGGGACAGCAGTCCCTCCTGTGGGGGCCCCTGAACTGGGCTCACATC CCACACATTTTCCAAACCACTCCCATTGTGAGCCTTTGGTCCTGGTGGTG TCCCTCTGGTTGTGGGACCAAGAGCTTGTGCCCATTTTTCATCTGAGGAA GGAGGCAGC ListedbelowarethecorrespondingRNAsequences forSEQIDNOs:1-4(SEQIDNOs:7-10) (SEQIDNO:7) GACCCACUCUUGGUUUCCA (SEQIDNO:8) GGGGACUGCCGGGUGACCCUGGAAAUCCAGAGUGGGUGGGGCCAGUCUGA CCGUUUCUAGGCGACCCACUCUUGGUUUCCAGGGUUGCCCUGGAAA (SEQIDNO:9) GAAGACGGGAGGAAAGAAGGGAG (SEQIDNO:10) GAGGGGGAAGACGGGAGGAAAGAAGGGAGUGGUUCCAUCACGCCUCCUCA CUCCUCUCCUCCCGUCUUCUCCUCUC

(225) CKB, SLC6a8 Knockdown

(226) pLKO vectors expressing shRNA hairpins targeting CKB and SLC6a8 were ordered from Sigma-Aldrich. Two independent hairpins that gave the best knockdown of transcript levels were used for all experiments. These hairpin DNA and RNA sequences are listed below in Table 14:

(227) TABLE-US-00015 TABLE14 SelectedhairpinDNAandRNAsequences SEQID SEQID Name DNASequences NO RNASequences NO CKB CCGGCCCAGATTGAAACT 11 CCGGCCCAGAUUGAAACUC 15 CTCTTCACTCGAGTGAA UCUUCACUCGAGUGAAGAG GAGAGTTTCAATCTGGG AGUUUCAAUCUGGGUUUUU TTTTT CKB CCGGCCGCGGTATCTGG 12 CCGGCCGCGGUAUCUGGC 16 CACAATGACTCGAGTCAT ACAAUGACUCGAGUCAUUG TGTGCCAGATACCGCGG UGCCAGAUACCGCGGUUUU TTTTTTG UUG shSLC CCGGGCTGGTCTACAAC 19 CCGGGCUGGUCUACAACAA 20 6a8#2 AACACCTACTCGAGTAGG CACCUACUCGAGUAGGUGU TGTTGTTGTAGACCAGCT UGUUGUAGACCAGCUUUUU TTTTG G shSLC CCGGCTTATTCCCTACGT 13 CCGGCUUAUUCCCUACGUC 17 6a8#4 CCTGATCCTCGAGGATCA CUGAUCCUCGAGGAUCAGG GGACGTAGGGAATAAGTT ACGUAGGGAAUAAGUUUUU TTTG G shSLC CCGGATTACCTGGTCAAG 14 CCGGAUUACCUGGUCAAGU 18 6a8#5 TCCTTTACTCGAGTAAAG CCUUUACUCGAGUAAAGGA GACTTGACCAGGTAATTT CUUGACCAGGUAAUUUUUU TTTG G

(228) The following primers were used for quantitative qRT-PCR of SLC6a8: Forward Primer: 5-GGC AGC TAC AAC CGC TTC AAC A-3 and Reverse Primer: 5-CAG GAT GGA GAA GAC CAC GAA G-3 (SEQ ID No. 21 and 22, respectively).

(229) Cyclocreatine and Beta-Guanidiopropionic Acid Treatment

(230) Mice were treated with 10 mg of cyclocreatine or saline vehicle, administered through intra-peritoneal injection. The treatment regime started one day after inoculation of tumor cells and continued until the mice were euthanized. Beta-guanidipropionic acid was administered at a dose of 200 L of 0.5M solution through intra-peritoneal injection. Treatment regime were as that for cyclocreatine treatment.

Example 1. Synthesis of Creatine Transport Inhibitors and/or Creatine Kinase Inhibitors of the Invention

(231) Compounds of the invention may be synthesized using methods known in the art, for example using methods described in U.S. Pat. Nos. 5,321,030, 5,324,731, 5,955,617, 5,994,577, or 5,998,457 or methods described in Metabolism 1980, 29 (7), 686, J. Med. Chem. 2001, 44, 1231, J. Biol. Chem. 1972, 247, 4382, J. Chem. Inf. Model. 2008, 48 (3), 556, or J. Med. Chem. 2001, 44, 1217. Alternatively, the compounds of the invention may be synthesized using the methods described below.

(232) Abbreviations

(233) ACN acetonitrile -GPA 3-guanidinopropionic acid i.e. -guanidinopropionic acid BINAP racemic 2,2-bis(diphenylphosphino)-1,1-binaphthyl BLQ below level of quantification Boc tert-butyloxycarbonyl Br.sub.2 bromine BrCN cyanogen bromide C. degrees Celcius ca. circa or approximately CAN ceric ammonium nitrate Cbz carbobenzyloxy CH.sub.2Cl.sub.2 dichloromethane Cul copper (I) iodide Cs.sub.2CO.sub.3 cesium carbonate D.sub.2O deuterium oxide DCC dicyclohexylcarbodiimide DCI dicyclohexylcarbodiimide DCM dichloromethane or methylenechloride DIPEA diisopropylethylamine DMAP 4-dimethylaminopyridine or N,N-dimethylaminopyridine DME 1,2-dim ethoxyethane DMF N,N-dimethylformamide DMSO dimethylsulfoxide eq. equivalents ES(pos)MS electrospray positive mode mass spectrometry EtOAC ethyl acetate EtOH ethanol Et.sub.2O diethyl ether Fmoc fluorenylmethyloxycarbonyl chloride g gram(s) HPLC high performance liquid chromatography h hour H.sub.2 hydrogen gas K.sub.2CO.sub.3 potassium carbonate K.sub.3PO.sub.4 potassium phosphate tribasic KOH potassium hydroxide LC/MS liquid chromatography mass spectrometry LC/MS/MS liquid chromatography tandem mass spectrometry LiOH lithium hydroxide M molar Mel methyl iodide MeOH methanol mg milligram(s) MgSO.sub.4 magnesium sulfate min. minute(s) mL milliliters(s) mm millimeter(s) mmol millimole(s) MTS 2-methyl-2-thiopseudourea sulfate m/z mass to charge ratio N normal Na.sub.2S.sub.2O.sub.3 sodium thiosulfate Na.sub.2SO.sub.4 sodium sulfate NaH sodium hydride NaHCO.sub.3 sodium bicarbonate Nal sodium Iodide NalO.sub.4 sodium periodate NaOCH.sub.3 sodium methoxide NaOH sodium hydroxide NaNO.sub.2 sodium nitrite NBS N-bromosuccinimide NMR nuclear magnetic resonance PhthNK potassium phthalimide Pd/C palladium on carbon Pd(OAc).sub.2 palladium (II) acetate PdCl.sub.2 palladium (II) chloride psi pounds per square inch PyBOP benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate RuCl.sub.3 ruthenium trichloride hydrate SO.sub.2Cl.sub.2 sulfuryl chloride SOCl.sub.2 thionyl chloride TCl 1,1-thiocarbonyldiimidazole TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TPP triphenylphosphine TSA p-toluenesulfonic acid General method to make cyclocreatine analogs of the invention

(234) ##STR00476##

(235) Cyclocreatine analogs may be made from amino carboxylic acids or esters and reaction with aziridines in inert solvents (e.g. diethyl ether, THF, DME, ethanol, DMF, THF, etc.). These diamine intermediates are then reacted with cyanogen bromide in inert solvent (e.g. diethyl ether, THF, DMF, etc.) to form iminoimidazolidine desired products.

(236) General Method to Make 1-carboxymethyl-2-iminohexahydropyrimidine Analogs of the Invention

(237) ##STR00477##

(238) Iminohexahydropyrimidine analogs may be made from -halo carboxylic acids or esters and reaction with 1,3-diaminopropanes. These diamine intermediates may be reacted with cyanogen bromide in inert solvent (e.g. diethyl ether, THF, DMF, etc.) to form iminohexahydropyrimidine desired products.

(239) General Method to Make Dihydrazinylimidazolidine and Dihydrazinylhexahydropyrimidine Analogs of the Invention

(240) ##STR00478##

(241) Dihydrazinyl analogs may be made from -halo carboxylic acids or esters and reaction with protected hydrazinyl alkyl amino derivatives. Protected hydrazinyl alkyl amino derivatives may be made by reaction of trifluoroacetohydrazide with aziridines or 1-azido-3-halopropane followed by subsequent azide reduction under standard conditions (e.g. triphenylphosphine-acetone-water). These diamine intermediates may be reacted with 1,1-thiocarbonyldiimidazole (TCl) in polar solvent (e.g. DMF, dioxane, etc.) to form imidazolidinethiones or tetrahydro-2-pyrimidinethiones. Activation with alkyl halide (e.g. methyl iodide) and subsequent reaction with hydrazine cleaves the trifluoroacetamide protecting group and would afford the desired products.

(242) General Method to Make 2-hydrazinylimidazolidine and 2-hydrazinylhexahydropyrimidine Analogs of the Invention

(243) ##STR00479##

(244) The diaminocarboxylic acid or ester intermediates generated above may be reacted with 1,1-thiocarbonyldiimidazole (TCl) in polar solvent (e.g. DMF, dioxane, etc.) to form imidazolidinethiones or tetrahydro-2-pyrimidinethiones. Activation with alkyl halide (e.g. methyl iodide) and subsequent reaction with hydrazine would afford the desired products.

(245) General Method to Make Aminoiminoimidazolidine and Aminoiminohexahydropyrimidine Analogs of the Invention

(246) ##STR00480##

(247) Aminoiminoimidazolidine and aminoiminohexahydropyrimidine analogs may be made from -halo carboxylic acids or esters and reaction with azirindes or azetidines respectively. These -carboxy aziridines or azetidines are opened with cyanogen bromide to form cyanamide alkyl bromide intermediates (see J. Org. Chem. 1949, 14, 605 and J. Am. Chem. Soc. 2013, 135(41), 15306). Reaction with hydrazine would afford the desired cyclic products.

(248) General Method to Make imino-1,2,4-triazinanes Analogs of the Invention

(249) ##STR00481##

(250) Imino-1,2,4-triazinanes analogs may be made from -halo carboxylic acids or esters and reaction with protected hydrazinyl alkyl azido derivatives. PMB-protected hydrazinyl alkyl azide derivatives may be prepared as described in Org. Biomol. Chem. 2012, 10(30), 5811. The azide is reduced under standard conditions (e.g. triphenylphosphine-acetone-water) and the resulting diamine is treated with cyanogen bromide to form the cyclic core. Deprotection of the PMB hydrazine protecting group under standard conditions (e.g. CAN, strong acid, etc.) would afford the desired product.

(251) Synthesis of Compound 225

(252) ##STR00482##

(253) Alanine (2.0 mmol) is dissolved in diethyl ether (10 mL) and aziridine (2.0 mmol) is added. The mixture is heated to reflux for 2 h and cooled to room temperature. Cyanogen bromide (2.3 mmol) is added and the reaction is stirred at room temperature overnight. The precipitate is filtered and washed with diethyl ether to afford 2-(2-iminoimidazolidin-1-yl)propanoic acid (225).

(254) Compounds 226-228 may be synthesized using similar methods as used to make compound 225 by replacing alanine with -aminobutanoic acid, valine, or isoleucine.

(255) Synthesis of Compound 237

(256) ##STR00483##

(257) 2-Chloropropionic acid (2.0 mmol) is dissolved in diethyl ether and 1,3-diaminopropane (2.0 mmol) is added. The reaction is stirred overnight at room temperature and the precipitate is filtered and washed with diethyl ether to afford the HCl salt of 2-[(3-aminopropyl)amino]propanoic acid. The salt is dissolved in water and sodium carbonate (2.5 mmol) is added followed by cyanogen bromide (2.3 mmol). The reaction is stirred at room temperature overnight. The reaction is quenched with trifluoroacetic acid and the mixture is concentrated under reduced pressure. The residue is purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid) and the product is collected and lyophilized to afford the trifluoroacetate salt of 2-(2-imino-1,3-diazinan-1-yl)propanoic acid (237).

(258) Compounds 238-240 may be synthesized using similar methods as used to make compound 237 by replacing 2-chloropropanoic acid with 2-chlorobutanoic acid, 2-chloro-3-methylbutanoic acid, or 2-chloro-3-methylpentanoic acid.

(259) Synthesis of Compound 229

(260) ##STR00484##

Step 1:2-{[2-(trifluoroacetohydrazido)ethyl]amino}acetic acid (INT-1)

(261) ##STR00485##

(262) Trifluoroacetohydrazide (2.0 mmol) and aziridine (2.0 mmol) are dissolved in diethyl ether (5 mL) and stirred at room temperature overnight. 2-Chloroacetic acid (2.0 mmol) is added and the mixture stirred 3 h at room temperature. The precipitate is filtered and washed with diethyl ether to afford INT-1.

Step 2:2-[2-sulfanylidene-3-(trifluoroacetamido)imidazolidin-1-yl]acetic acid (INT-2)

(263) ##STR00486##

(264) Diamine INT-1 (1.5 mmol) is dissolved in DMF (5 mL) and reacted with 1,1-thiocarbonyldiimidazole (1.5 mmol) at room temperature for 3 h. The reaction is poured into 0.1 N aqueous HCl, the aqueous layer is extracted with ethyl acetate, the organic layer is dried over sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (10-90 methanol-dichloromethane) to afford INT-2.

Step 3:2-[3-amino-2-hydrazinylideneimidazolidin-1-yl]acetic acid (229)

(265) INT-2 (1.0 mmol) is dissolved in ethanol (5 mL) and methyl iodide is added (1.0 mmol). The mixture is stirred for 1 h at room temperature. Upon completion, hydrazine (5 mmol) is added and the mixture is heated to reflux for 8 h. The mixture is concentrated to remove excess hydrazine, the residue is purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid), and the product is collected and lyophilized to afford the trifluoroacetate salt of 2-[3-amino-2-hydrazinylideneimidazolidin-1-yl]acetic acid (229).

(266) Compounds 230-228 and 241-243 may be synthesized using similar methods as used to make compound 229 by replacing chloroacetic acid with 2-chloroacetic acid, 2-chloropropanoic acid, or 2-chloro-3-methylbutanoic acid. To synthesize compounds 241-243, aziridine may be replaced with 1,3-dibromopropane.

(267) Synthesis of Compound 232

(268) ##STR00487##

(269) 2-Chloropropionic acid (2.0 mmol) is dissolved in diethyl ether and aziridine (2.0 mmol) is added. The reaction is stirred overnight at room temperature and the precipitate is filtered and washed with diethyl ether to afford the HCl salt of 2-(aziridin-1-yl)propanoic acid. The salt is dissolved in water, sodium carbonate (2.5 mmol) is added followed by cyanogen bromide (2.3 mmol), and the reaction is stirred at room temperature overnight. The reaction is quenched with trifluoroacetic acid and the mixture is concentrated under reduced pressure. The residue is purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid) and the product is collected and lyophilized to afford the trifluoroacetate salt of 2-(3-amino-2-iminoimidazolidin-1-yl)propanoic acid (232).

(270) Compounds 233 and 244-246 may be synthesized using similar methods to make compound 232 by replacing 2-chloropropanoic acid with 2-chloroacetic acid, or 2-chloro-3-methylbutanoic acid. To synthesize compounds 244-246, aziridine may be replaced with azetidine.

(271) Synthesis of Compound 234

(272) ##STR00488##

Step 1:2-(2-sulfanylideneimidazolidin-1-yl)acetic acid (INT-3)

(273) ##STR00489##

(274) Glycine (2 mmol) is dissolved in diethyl ether (10 mL) and aziridine (2 mmol) is added. The mixture is heat is to reflux for 2 h and cooled to room temperature. The mixture is concentrated under reduced pressure, dissolved in DMF (5 mL), and reacted with 1,1-thiocarbonyldiimidazole (1.5 mmol) at room temperature for 3 h. The reaction is poured into 0.1 N aqueous HCl, the aqueous layer is extracted with ethyl acetate, the organic layer is dried over sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (10-90 methanol-dichloromethane) to afford INT-3.

Step 2:2-[2-hydrazinylideneimidazolidin-1-yl]acetic acid (234)

(275) INT-3 (1.0 mmol) is dissolved in ethanol (5 mL) and methyl iodide is added (1.0 mmol). The mixture is stirred for 1 h at room temperature. Upon completion, hydrazine (5 mmol) is added and the mixture is heated to reflux for 8 h. The mixture is concentrated to room excess hydrazine, the residue is purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid), and the product is collected and lyophilized to afford the trifluoroacetate salt of 2-[2-hydrazinylideneimidazolidin-1-yl]acetic acid (234).

(276) Compounds 235-336 may be synthesized using similar methods to make compound 234 by replacing glycine with alanine or valine. Compounds 247-349 may be synthesized using similar methods to make the diamine intermediate for compound 237 and then following the protocol to make 234.

(277) Synthesis of Compound 250

(278) ##STR00490##

Step 1:1-(2-azidoethyl)-1-[(4-methoxyphenyl)methyl]hydrazine (INT-4)

(279) ##STR00491##

(280) Synthesis of PMB-protected hydrazinyl ethyl azide is prepared as described in Org. Biomol. Chem. 2012, 10(30), 5811.

Step 2:2-[2-(2-aminoethyl)-2-[(4-methoxyphenyl)methyl]hydrazine-1-yl]acetic acid (INT-5)

(281) ##STR00492##

(282) 2-Chloropropionic acid (2.0 mmol) is dissolved in diethyl ether (5 mL) and INT-4 (2.0 mmol) is added. The reaction is stirred overnight at room temperature and the precipitate is filtered and washed with diethyl ether to afford the HCl salt. The salt is dissolved in water-acetone (1:10, 5 mL) and triphenylphosphine (TPP) is added. The mixture is heated to 50 C. for 14 hours.

Step 3:2-(3-imino-1,2,4-triazinan-2-yl)acetic acid (250)

(283) INT-5 (1.0 mmol) is dissolved in water, sodium carbonate (2.5 mmol) is added followed by cyanogen bromide (1.3 mmol), and the reaction is stirred at room temperature overnight. The reaction is quenched with trifluoroacetic acid and the mixture is concentrated under reduced pressure. The residue is purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid) and the product is collected and lyophilized to afford the trifluoroacetate salt of 2-(3-imino-1,2,4-triazinan-2-yl)acetic acid (250).

(284) Compounds 251-252 may be synthesized using similar methods to make compound 250 by replacing 2-chloroacetic acid with 2-chloropropanoic acid or 2-chloro-3-methylbutanoic acid.

(285) General Method to Make Guanidine Containing Compounds of the Invention

(286) ##STR00493##

(287) Guanidines are made from amines using standard guanylating agents (e.g. 2-methyl-2-thiopseudourea sulfate [MTS], cyanamide, N,N-di-Boc-1H-pyrazole-1-carboxamide, etc.). A preferred method using MTS is described in J. Med. Chem. 2001, 44, 1217, J. Chem. Soc. C 1971, 238 and Tetrahedron Lett. 1996, 37, 2483. Briefly, amines are dissolved or suspended in basic aqueous alcoholic solvent and reacted with 2-methyl-2-thiopseudourea sulfate (MTS) for 24-72 h or longer and precipitated products are isolated by filtration. If necessary, ester hydrolysis is done by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueous alcoholic solvent or THF, to afford the desired product.

(288) ##STR00494##

(289) In some case, guanidines are made from amines under anhydrous conditions using pyrazole-activated guanylating agents (e.g. 1H-pyrazole-1-carboxamidine hydrochloride, 3,5-dimethyl-1-pyrazolylformaminidium nitrate, N-Boc-1H-pyrazole-1-carboxamidine, N,N-di-Boc-1H-pyrazole-1-carboxamidine, N-(benzyloxycarbonyl)-1H-pyrazole-1-carboxamidine, and N,N-bis(benzyloxycarbonyl)-1H-pyrazole-1-carboxamidine). Methods for using pyrazole-activated guanylating agents are reviewed in Eur. J. Org. Chem. 2002, 3909. Briefly, amines are used in excess or with a base (e.g. triethylamine, diisopropylethylamine, etc.) and are dissolved in solvent (e.g. DMF, acetonitrile, THF, methanol, dichloromethane, etc.). The reaction is stirred for 4-72 h and at room temperature but in some cases heating is required. When protected pyrazole-activated guanylating agents are used (i.e. W=Boc or Cbz) products can be purified by normal phase silica gel column chromatography. If necessary, ester hydrolysis is done by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueous alcoholic solvent or THF, to provide the carboxylic acid. Finally, standard deprotection conditions are used to remove the guanidine protecting groups (i.e. TFA removal of Boc or hydrogenation of Cbz) to afford the desired product.

(290) ##STR00495##

(291) In other cases, amino alcohols are used with the method above. After guanylation, the alcohol is oxidized to the carboxylic acid using sodium metaperiodate and ruthenium (III) chloride (catalytic) in acetonitrile, ethyl acetate and water (Org. Lett. 2008, 10, 5155). Finally, standard deprotection conditions are used to remove the guanidine protecting groups (i.e. TFA removal of Boc or hydrogenation of Cbz) to afford the desired product.

(292) General Method to Make 2-aminopyridine Containing Compounds of the Invention

(293) ##STR00496##

(294) Aminopyridines may be made utilizing cross-coupling methods as described in J. Am. Chem. Soc. 2008, 130(20), 6586 and J. Org. Chem. 1996, 61(21), 7240. Briefly, amines, 2-halopyridines, and sufficient base (e.g. cesium carbonate, potassium tert-butoxide, etc.) are dissolved or suspended in polar solvent (e.g. DMF, dioxane), catalytic palladium (e.g. PdCl.sub.2 or Pd(OAc).sub.2) and phosphine ligand (e.g. BINAP) are then be added and the reaction is heated to greater than 80 C. for 4-6 h. If necessary, ester hydrolysis by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueous alcoholic solvent or THF, affords the desired product.

(295) ##STR00497##

(296) Alternatively, Buchwald amide-cross-coupling methods are used to generate desired compounds as described in see J. Am. Chem. Soc. 2002, 124, 11684 and J. Am. Chem. Soc. 2001, 123, 7727. Briefly, amines are protected as amides or carbamates (e.g. trifluoroacetamide, Boc, Cbz, etc.) and reacted with 2-bromopyridine in polar solvent (e.g. dioxane, DMF) with base (e.g. potassium phosphate tribasic, cesium carbonate, potassium tert-butoxide, etc.), racemic trans-1,2-diaminocyclohexane ligand, and catalytic copper (I) iodide. The solution is degassed for 5 minutes by bubbling nitrogen gas directly into the solution and the mixture is heated at greater than 95 C. for 6-12 hours. Amine protecting groups are removed using standard conditions and, if necessary, ester hydrolysis by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueous alcoholic solvent or THF, affords the desired product.

(297) General Method to Make Pseudothiourea Containing Compounds of the Invention

(298) ##STR00498##

(299) Pseudothioureas may be made by reaction of a thiourea with alkylhalides as described in J. Med. Chem. 2001, 44, 1217. Briefly, 3-chloropropionic acids or esters may be reacted with thiourea in polar solvent (e.g. acetone) followed by refluxing the mixture for 48 h. 3-Chloropropionic acids or esters may be made by reaction of 3-hydroxypropionic acids with thionyl chloride as described in International Patent Publication No. WO9933785.

(300) Alternatively, pseudothioureas may be made from amino compounds via alkyl bromides using methods as described in Tetrahedron: Asymmetry 1998, 9(10), 1641 and International Patent Publication No. WO2002009705. Briefly, 3-aminocarboxylic acids or esters may be converted to 3-bromocarboxylic acids or esters via activation with sodium nitrite in the presence of hydrobromic acid. The resulting bromo compound may be reacted with a thiourea in a suitable solvent (e.g. toluene or acetone) at greater than 60 C. for 4-24 h. If necessary, ester hydrolysis by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueous alcoholic solvent, would afford the desired product.

(301) General Method to Make 2-aminopseudothiourea Containing Compounds of the Invention

(302) ##STR00499##

(303) 2-Aminopseudothioureas may be made from aziridines by methods similar to those described in Chem. Comm. 2000, 7, 619 and Org. Biomol. Chem. 2005, 3(18), 3357. Briefly, N-Cbz-2-carboxyaziridines may be reacted with thiourea and borontrifluoride-etherate in inert solvent (e.g. chloroform, dichloromethane, etc). Subsequent hydrogenation in the presence of palladium on carbon, and if necessary, ester hydrolysis by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueous alcoholic solvent, would afford the desired product. 2-Carboxyaziridines may be made by methods as described in Synlett 2001, 5, 679 and Tetrahedron Lett. 2006, 47(13), 2065 and may be converted to Cbz-protected aziridines by standard methods.

(304) Many starting materials for compounds of the invention are commercially available or methods for synthesis are known in the literature. Table 15 lists starting materials for synthesis of compounds of the invention and provides literature references for uncommon reagents.

(305) TABLE-US-00016 TABLE 15 Starting materials for the synthesis of selected compounds # Structure Starting Material CAS Number Reference SM01 00 (3R)-3-aminobutyric acid 3775-73-3 Org. Process Res. Dev. 2011, 15, 1130 SM02 01 (3S)-3-aminobutyric acid 3775-72-2 Org. Process Res. Dev. 2011, 15, 1130 SM03 02 -alanine-2,2,3,3-D.sub.4 116173-67-2 J. Labelled Compd. Ra. 1988, 25(2), 217 SM04 03 -alanine-3,3-D.sub.2 116173-66-1 J. Labelled Compd. Ra. 1988, 25(2), 217 SM05 04 2,2-difluoro-3-amino- propanoic acid 428452-49-7 Tetrahedron Lett. 2003, 44(11), 2375; PCT Int. Appl., 2007062308 SM06 05 3-azetidinecarboxylic acid 36476-78-5 Commercially available SM07 06 3-amino-4,4,4- trifluorobutanoic acids 584-20-3 Commercially available SM08 07 tert-butyl (1R,2S)-2- aminocyclopropane-1- carboxylate 150626-49-6 JP 05155827 A 19930622 SM09 08 tert-butyl (1S,2R)-2- aminocyclopropane-1- carboxylate 150737-97-6 JP 05155827 A 19930622; International Patent Publication No. WO 2008123207 SM10 09 ethyl (1R,2R)-2-{[(tert- butoxy)carbonyl]amino} cyclopropane-1- carboxylate 613261-17-9 J. Org. Chem. 2003, 65(20), 7884; Org. Lett. 2013, 75(4), 772; SM11 0 ethyl (1R,2R)-2- {[(benzyloxy)carbonyl] amino}cyclopropane- 1-carboxylate 613261-16-8 J. Org. Chem. 2003, 68(20), 7884; Org. Lett. 2013, 15(4), 772; in addition protocols as described in Tetrahedron 2012, 68(47), 9566 can also be used to generate useful starting materials SM12 ethyl (1S,2S)-2- [(methoxycarbonyl) amino]cyclopropane-1- carboxylate 1356459-72-7 J. Org. Chem. 2003, 66(20), 7884; Org. Lett. 2013, 15(4), 772; in addition protocols as described in Tetrahedron 2012, 66(47), 9566 can also be used to generate useful starting materials SM13 (1R,2S)-2- aminocyclobutane-1- carboxylic acid 221158-95-8 J. Org. Chem. 2009, 74(8), 3217; J. Org. Chem. 2005, 70(20), 7963; Tetrahedron: Asymmetry 2000, 11(17), 3569 SM14 (1S,2R)-2- aminocyclobutane-1- carboxylic acid 648433-09-4 J. Org. Chem. 2009, 74(8), 3217; J. Org. Chem. 2005, 70(20), 7963 SM15 (1R,2R)-2- aminocyclobutane-1- carboxylic acid 951173-26-5 J. Org. Chem. 2009, 74(8), 3217 SM16 (1S,2S)-2- aminocyclobutane-1- carboxylic acid 951173-27-6 J. Org. Chem. 2009, 74(8), 3217 SM17 2-methyl-3- azetidinecarboxylic acid 1638771-37-5 Chiral trans isomers: Synthesis 2005, 20, 3508; Chiral cis isomers: J. Org. Chem. 2012, 77(17), 7212 SM18 2-hydroxy-3- azetidinecarboxylic acid 70807-37-3 Commercially available; International Patent Publication No. WO2011043817 SM19 2-amino-3- azetidinecarboxylic acid 138650-25-6 Commercially available SM20 3-fluoro-3- azetidinecarboxylic acid 1363380-85-1 International Patent Publication No. WO2013019561; J. Org. Chem. 2009, 74(5), 2250; or made by deprotection of 1-[(tert-butoxy)carbonyl]-3- fluoroazetidine-3-carboxylic acid [1126650-67-6] SM21 0 2-methyl-3- azetidinecarboxylic acid 1213240-07-3 Commercially available; or made by deprotection of 1-[(tert- butoxy)carbonyl]-3-methylazetidine-3- carboxylic acid [887591-62-0] SM22 (3S)-3-amino-4,4,4- trifluorobutanoic acids 151871-99-7 Chem. Comm. 2012, 45(34), 4124 SM23 (3R)-3-amino-4,4,4- trifluorobutanoic acids 151911-19-2 Chem. Comm. 2012, 45(34), 4124 SM24 (2S,3S)-2,3-diamino- 4,4,4-trifluorobutanoic acid 1632315-15-1 J. Fluorine Chem. 2015, 171, 67 SM25 ethyl (2S,3R)-2,3- diamino-4,4,4- trifluorobutanoate 1219366-64-9 International Patent Publication No. WO2010031750 SM26 (2R,3R)-2,3-diamino- 4,4,4-trifluorobutanoic acid NA Made by a similar protocol as [1632315-15-1] above but using (R)-N- [(1E)-ethylidene]-2-methylpropane-2- sulfinamide [1219607-85-8] SM27 ethyl (2R,3S)-2,3- diamino-4,4,4- trifluorobutanoate NA Made by a similar protocol as [1219366-64-9] above but using (R)-N- [(1E)-ethylidene]-2-methylpropane-2- sulfinamide [1219607-85-8] SM28 (3R)-3-amino(4,4,4- .sup.2H.sub.3)butanoic acid NA Made according to procedures described for the synthesis of chiral 3- aminobutyric acid in Helv. Chim. Acta 1988, 71, 1824 and Tetrahedron: Asymmetry 1991, 3, 183, but replacing crotonic acid with 2-butenoic-4,4,4-d.sub.3 acid [1375453-29-4] made according to J. Magn. Reson. 2011, 270(1), 107 SM29 (3S)-3-amino(4,4,4- .sup.2H.sub.3)butanoic acid NA As above SM30 (S)-3-amino-4,4- difluoro-butanoic acid 111218-68-9 Tetrahedron Asymmetry 1994, 5(6), 1119 SM31 0 (R)-3-amino-4,4- difluoro-butanoic acid 109537-89-5 Tetrahedron Asymmetry 1994, 5(6), 1119 SM32 3-amino-4- fluorobutanoic acid 77162-47-1 Syn. Comm. 1985, 15(5), 377 SM33 (3S)-3-aminopent-4- enoic acid 1389348-84-8 Made by hydrogenation of ethyl (3S)-3- aminopent-4-ynoate [149251-15-0] at 1 atm using Lindlar's catalyst and then ester hydrolysis SM34 (3R)-3-aminopent-4- enoic acid 1388637-32-8 Made by hydrogenation of ethyl (3R)-3- aminopent-4-ynoate [188853-28-3] at 1 atm using Lindlar's catalyst and then ester hydrolysis SM35 ethyl (3S)-3- aminopent-4-ynoate 149251-15-0 Bioorg. Med. Chem. Lett. 1997, 7(13), 1699; U.S. Pat. No. 5,536,869; the ester can be hydrolyzed before or after the guanylation step SM36 ethyl (3R)-3- aminopent-4-ynoate 188853-28-3 Bioorg. Med. Chem. 1999, 7(10), 2221; the ester can be hydrolyzed before or after the guanylation step SM37 3-amino-pentanoic acid 18664-78-3 Commercially available SM38 (R)-3-amino-pentanoic acid 131347-76-7 Commercially available SM39 (S)-3-aminopentanoic acid 14389-77-6 Commercially available SM40 (3R)-3-aminohexanoic acid 775551-50-3 Synthesis 2008, 7, 1153 & Chem. Commun. 2007, 8, 849 SM41 0 (3S)-3-aminohexanoic acid 91298-66-7 ChemBioChem 2009, 10(9), 1558 & Synlett 1994, 10, 795 SM42 3-amino-4- methylpentanoic acid 5699-54-7 Commercially available SM43 (S)-3-amino-4- methylpentanoic acid 40469-85-0 Commercially available SM44 (R)-3-amino-4- methylpentanoic acid 75992-50-6 Commercially available SM45 3-amino-2,2- dimethylpropan-1-ol 26734-09-8 Commercially available; requires oxidation of the alcohol after guanylation SM46 [1- (aminomethyl) cyclopropyl]methanol 45434-02-4 Commercially available; requires oxidation of the alcohol after guanylation SM47 ethyl 1- (aminomethyl) cyclobutane-1- carboxylate 911060-83-8 Commercially available; the ester can be hydrolyzed before or after the guanylation step SM48 3-amino-3- methylbutanoic acid 625-05-8 Commercially available SM49 2-(1- aminocyclopropyl) acetic acid 133616-20-3 Synlett 1991, 2, 87 SM50 2-(1-{[(tert- butoxy)carbonyl]amino} cyclobutyl)acetic acid 249762-02-5 Commercially available; requires hydrolysis of the Boc protecting group prior to guanylation SM51 0 (2R,3R)-3-amino-2- methylbutanoic acid 139344-67-5 U.S. Pat. Appl. Publ., 20110218342; Tetrahedron, 2007, 63(26), 5820 SM52 (2S,3R)-3-amino-2- methylbutanoic acid 863115-43-9 Made by an analogous protocol described in Heterocycles 1999, 50(2), 677 for [39801-26-8] but using (R)-()- N-methoxy-2-pyrrolidine carboxamide SM53 (2R,3S)-3-amino-2- methylbutanoic acid 39801-26-8 Heterocycles 1999, 50(2), 677; J. Org. Chem. 1993, 55(8), 2282 SM54 (2S,3S)-3-amino-2- methylbutanoic acid 139344-68-6 J. Am. Chem. Soc. 2005, 127(32), 11252; J. Org. Chem. 1993, 58(8), 2282 SM55 pyrrolidine-3- carboxylic acid 59378-87-9 Commercially available SM56 (3S)-pyrrolidine-3- carboxylic acid 72580-53-1 Commercially available SM57 (3R)-pyrrolidine-3- carboxylic acid 72580-54-2 Commercially available SM58 2-[(2S)-1-[(tert- butoxy)carbonyl] azetidin-2-yl]acetic acid 1289384-58-2 Made from (S)-(tert- butoxycarbonyl)azetidine-2-carboxylic acid according to the protocol described in International Patent Publication No. WO2011111875; requires hydrolysis of the Boc protecting group prior to guanylation SM59 2-[(2R)-1-[(tert- butoxy)carbonyl] azetidin-2-yl]acetic acid 1369534-61-1 Made from (R)-(tert- butoxycarbonyl)azetidine-2-carboxylic acid according to the protocol described in International Patent Publication No. WO2011111875; requires hydrolysis of the Boc protecting group prior to guanylation SM60 cis-3- aminocyclobutane-1- carboxylic acid 74316-27-1 Commercially available SM61 0 trans-3- aminocyclobutane-1- carboxylic acid 74307-75-8 Commercially available SM62 3-{[(tert- butoxy)carbonyl]amino}- 1- hydroxycyclobutane-1- carboxylic acid 1067239-17-1 International Patent Publication No. WO2011044538 & WO2008124821; requires hydrolysis of the Boc protecting group prior to guanylation SM63 ethyl 3-amino-1-{[(tert- butoxy)carbonyl]amino} cyclobutane-1- carboxylate NA ethyl 1-{[(tert-butoxy)carbonyl]amino}- 3-hydroxycyclobutane-1-carboxylate [413597-67-8] (U.S. Pat. Appl. Publ., 20060292073) is converted to the amine by activation of the hydroxyl group (e.g. tosyl chloride and pyridine), displacement with lithium azide, and reduction to the amine (i.e. catalytic hydrogenation of Pd/C or with triphenylphosphine) SM64 2-(azetidin-3-yl)acetic acid 183062-92-2 Commercially available; or made by deprotection of 2-{1-[(tert- butoxy)carbonyl]azetidin-3-yl}acetic acid [183062-96-6] prior to guanylation SM65 L-(2S)-2,3- diaminopropionic acid 4033-39-0 Commercially available SM66 -chloroalanine 51887-88-8 (D) 13215-35-5 (D/L) Org. Biomol. Chem. 2005, 3(18), 3357 SM67 (2S,3R)-2-amino-3- chlorobutanoic acid 64233-79-0 Make desired stereoisomers from L- threonine and L-allo-threonine using a similar procedure described in: International Patent Publication No. WO9933785 SM68 3-chloropropionic- 2,2,3,3-d.sub.4 acid 1219802-17-1 Commercially available SM69 ethyl 3-bromo-2,2- difluoropropionate 111773-24-1 Commercially available SM70 ethyl 3-chloro-4,4,4- trifluorobutyrate 1309602-63-8 Commercially available SM71 0 (2S,3R)-2,3- diaminobutanoic acid 25023-80-7 Org. Biomol. Chem. 2003, 1(21), 3708; Synlett 1996, 7, 621; Tetrahedron 2001, 57(39), 8267 SM72 (2S,3S)-2,3- diaminobutanoic acid 80999-51-5 Org. Biomol. Chem. 2003, 1(21), 3708; Synlett 1996, 7, 621; Tetrahedron 2001, 57(39), 8267 SM73 2,3-diaminopropionic acid 54897-59-5 Commercially available
Synthesis of Compound 219

(306) ##STR00573##

(307) (R)-Aminobutyric acid SM01 (750 mg, 7.27 mmol) and 2-methyl-2-thiopseudourea sulfate (MTS, 1.21 g, 4.36 mmol) were suspended in methanol (4 mL). After addition of 3N sodium hydroxide in water (2.62 mL, 1.09 eq.), the clear solution was stirred for 3 days at room temperature. Subsequently, the white precipitate was filtered off and washed with water/methanol (15 mL, ). The white powder was air dried for 1 hour and then put under high vacuum for 2 days to yield the (3R)-3-carbamimidamidobutanoic acid (219) as a white solid (879 mg, 83%); .sup.1H-NMR (300 MHz, D.sub.2O): 3.81 (m, 1H), 2.31 (m, 2H), 1.15 (d, 3H); ES(pos)MS m/z 146.1 (M+H.sup.+).

(308) Synthesis of Compound 220

(309) ##STR00574##

(310) (S)-Aminobutyric acid SM02 (750 mg, 7.27 mmol) was converted into the corresponding (3S)-3-carbamimidamidobutanoic acid (220) according to the procedure for compound 219. The desired product was obtained as a white powder after high vacuum drying for 3 days (756 mg, 72%); .sup.1H-NMR (300 MHz, D.sub.2O): 3.84 (m, 1H), 2.35 (m, 2H), 1.18 (d, 3H). ES(pos) MS m/z 145.9 (M+H.sup.+).

(311) Synthesis of Compound 221

(312) ##STR00575##

(313) A suspension of (2S,3R)-2,3-diaminobutanoic acid SM71 (25 mmol) and 2-methyl-2-thiopseudourea sulfate (MTS, 25 mmol) in methanol (25 mL) is stirred for 5 days at room temperature under a nitrogen atmosphere. The reaction is then cooled to 0 C., filtered through a medium porosity glass frit, and the collected solid is washed with water and dried to provide compound 221 as a mixture of regioisomers. The material is purified by preparative HPLC chromatography and the product is collected and lyophilized to afford compound 221.

(314) Synthesis of Compound 223

(315) ##STR00576##

(316) 2-chloro-pyridine (25 mmol), palladium (II) acetate (2.5 mmol), racemic 2,2-bis(diphenylphosphino)-1,1-binaphthyl (2.5 mmol) and cesium carbonate (65 mmol) are dissolved in toluene (75 mL) in a previously degassed sealed vessel. The mixture is flushed with nitrogen gas. Methyl-(R)-3-aminobutyrate SM01 (20 mmol) is added to the solution under nitrogen and the sealed mixture is heated overnight at 100 C. The reaction is cooled to room temperature, diluted with diethyl ether and washed with pH 7 buffer and water. The organic layer is concentrated and purified by silica gel column chromatography (10:90 methanol-dichloromethane) to afford (3R)-3-[(pyridin-2-yl)amino]butanoic acid (223).

(317) Synthesis of Compound 257

(318) ##STR00577##

Step 1: (1H-1,2,3-benzotriazol-1-ylmethyl)dibenzylamine (INT-6)

(319) ##STR00578##

(320) Hydroxymethylbenztriazol (10.4 g, 69.7 mmol) and dibenzylamine (13.4 mL, 69.7 mmol) were converted to INT-6 (95% yield) according to the protocol in US2009/054414.

Step 2:ethyl 3-(dibenzylamino)-2,2-difluoropropanoate (INT-7)

(321) ##STR00579##

(322) Ethyl bromodifluoro acetate (4.8 g, 23.65 mmol) and INT-6 (7.78 g, 23.7 mmol) were coupled according to the protocol in US2009/054414 to afford INT-7 (53% yield).

Step 3:ethyl 3-amino-2,2-difluoropropanoate TFA salt (INT-8)

(323) ##STR00580##

(324) The INT-7 (1.52 g, 4.59 mmol) was dissolved in ethanol (25 mL). Trifluoroacetic acid (0.372 mL, 4.85 mL) and a catalytic amount of palladium hydroxide on carbon was added and the reaction was subjected to hydrogen under atmospheric pressure for 16 h. Subsequently, the catalyst was removed by filteration through celite and washed with ethanol. The resulting filtrate was evaporated under reduced pressure and the residue was treated with toluene (50 mL) and concentrated, this was repeated twice to remove residual solvent and excess water. The residue was dried under high vacuum overnight to afford the INT-8 as a yellowish oil (used as crude in the next step).

Step 4:ethyl 3-{[(14-{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}-2,2-difluoropropanoate (INT-9)

(325) ##STR00581##

(326) The crude TFA salt of INT-8 was dissolved in dry THF (10 mL) and then treated with triethylamine (1.36 mL, 9.73 mmol) and N,N-di-Boc-1H-pyrazole-1-carboxamidine (1.57 g, 5.05 mmol). After stirring overnight at room temperature, the reaction mixture was poured into ethyl acetate (200 mL) and then washed with water (2100 mL) adjusting the pH of the aqueous layer to pH 1-2 using 1N HCl. The combined aqueous washes were then re-extracted with ethyl acetate (100 mL). The second organic phase was in turn washed with acidified water (100 mL) using 1N HCl to adjust the to pH 1-2. The combined organic phases were dried over magnesium sulfate, filtered, evaporated, and purified by silica gel column chromatography to afford INT-9 as a viscous oil that solidified upon standing (1.25 g, 70% for two steps); .sup.1H-NMR (300 MHz, DMSO-d.sub.6): 11.42 (s, 1H). 8.60 (t, 1H), 4.26 (q, 2H), 4.05 (td, 2H), 1.49 (s, 9H), 1.39 (s, 9H), 1.26 (t, 3H).

Step 5:Compound 257

(327) INT-9 (0.5 g) is dissolved in THF (5 mL) and 1N LiOH is added (2.5 mL). Once ester hydrolysis is complete the mixture is concentrated under vacuum and the residue is dissolved in 1:4 trifluoroacetic acid-dichloromethane (5 mL). The mixture is stirred at room temperature overnight to remove the Boc-protecting groups. The mixture is concentrated under vacuum, the residue is purified by preparative HPLC chromatography, and the product is collected and lyophilized to afford compound 257. Alternatively, the ester in INT-8 is hydrolyzed using lithium hydroxide to make SM05 and the resulting amino acid is guanylated using 1H-pyrazole-1-carboxamidine hydrochloride and diisopropylethylamine in DMF as shown below for compound 261.

(328) Synthesis of Compound 258

(329) ##STR00582##

(330) 3-Azetidinecarboxylic acid SM06 (2.0 g, 19.8 mmol) was suspended in water (2 mL) and then treated with 10 N sodium hydroxide solution in water (1.96 mL, 19.6 mmol). The thick yellowish solution was then treated with solid cyanamide (1.01 g, 24 mmol) and the mixture turned solid instantly. More water (5 mL) was added to ensure adequate stirring and the slurry was then stirred at room temperature for 3 days. The white solid was subsequently filtered off, washed with cold water (10 mL, 0 C.) and then air dried for 2 hours. High vacuum drying for 2 days affords compound 258 as a white solid (1.48 g, 52%); .sup.1H-NMR (300 MHz, D.sub.2O): 4.19 (t, 2H), 4.07 (dd, 2H), 3.34 (m, 1H); ES(pos) MS m/z 143.9 (M+H.sup.+).

(331) Synthesis of Compound 261

(332) ##STR00583##

(333) 3-Amino-4,4,4-trifluorobutyric acid SM07 (500 mg, 3.18 mmol) and diisopropylethylamine (1.16 mL, 6.68 mmol) were dissolved in dry DMF (3 mL). After addition of 1H-pyrazole-1-carboxamidine hydrochloride (593 mg, 3.50 mmol), the reaction mixture was stirred for 20 days at room temperature. The precipitate was filtered off, washed with methanol/water (2/1, 15 mL) and air-dried. High vacuum drying overnight affords the desired compound 261 as a white powder (135 mg, 21%); .sup.1H-NMR (300 MHz, D.sub.2O): 4.09 (m, 1H), 2.47 (dd, 1H), 2.22 (dd, 1H); ES(pos) MS m/z 200.07 (M+H.sup.+).

(334) Synthesis of Compound 275

(335) ##STR00584##

(336) Thiourea (100 mmol) and -chloro-D-alanine hydrochloride SM66 (100 mmol) in acetone (22 mL) is stirred at reflux for 48 h. Acetone (ca. 150 mL) is added, and the mixture is stirred vigorously to promote solidification. The solid is broken up, stirred until fine, filtered under nitrogen and washed with acetone. The crude solid is dissolved in warm 2-propanol (120 mL) and diluted with diethyl ether until cloudy (80 mL) and crystallization to afford (2R)-2-amino-3-(carbamimidoylsulfanyl)propanoic acid (275).

(337) Synthesis of Compound 278

(338) ##STR00585##

(339) To a flask is added cis-2-am inocyclobutane-1-carboxylic acid (18.8 mmol) and 24.5 mL of 5 N hydrobromic acid. The reaction is cooled in an ice bath to 0-5 C., followed by drop-wise addition of sodium nitrite (30.1 mmol) in 7.5 mL of water over five hours. The temperature is maintained below 5 C. during the addition. After the addition is complete, the reaction is stirred for 12 hours at room temperature. The reaction is diluted with diethyl ether (15 mL), the aqueous layer is removed and the organic phase is washed with 1 N hydrochloric acid (15 mL). The combined aqueous layers are washed with ethyl acetate (10 mL) and the combined organic extracts are dried of magnesium sulfate, filtered and concentrated under reduced pressure. The solid is recrystallized from ethyl acetate (10 mL) and hexanes (10 mL) to obtain trans-2-bromocyclobutane-1-carboxylic acid.

(340) To a suspension of thiourea (15 mmol) in toluene (50 mL) in an oil bath at 60 C. is added trans-2-bromocyclobutane-1-carboxylic acid (2.5 mmol). The reaction is stirred at 60 C. for 5 h. The toluene is then removed under reduced pressure and the resulting residue is diluted with water (25 mL) and 1 N hydrochloric acid (30 mL), to achieve pH of 1-1.5. The mixture is stirred at room temperature for 1-2 hours and then extracted with ethyl acetate (350 mL). The combined organic layers are dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting solid is dissolved in water (3.0 mL) and filtered through a 0.2 m nylon filter. The filtrate is purified by preparative HPLC (e.g. Waters PrepPak cartridge Delta-Pak C18 compression column, 15 m 25100 mm, 95:5 water-acetonitrile at 12.0 m L/m in). The product is collected and lyophilized to afford the product cis-2-(carbamimidoylsulfanyl)cyclopropane-1-carboxylic acid (278).

(341) Synthesis of Compound 286

(342) ##STR00586##

Step 1:1-Benzyl 2-methyl 3-(trifluoromethyl)aziridine-1,2-dicarboxylate (INT-10)

(343) ##STR00587##

(344) INT-10 may be made by methods as described in Synlett 2001, 5, 679 and Tetrahedron Lett. 2006, 47(13), 2065 and converted to a Cbz-protected aziridine by standard methods (see Org. Biomol. Chem. 2005, 3(18), 3357).

Step 2:methyl 2-{[(benzyloxy)carbonyl]amino}-3-(carbamimidoylsulfanyl)-4,4,4-trifluorobutanoate (INT-11)

(345) ##STR00588##

(346) INT-10 (1.0 mmol) is dissolved in dichloromethane (10 mL) and the mixture is cooled to 0 C. Borontrifluoride-etherate (1.0 mmol, 1 M dichloromethane) is added drop-wise and the reaction is warmed to room temperature. The reaction is poured into 0.1 N aqueous HCl, the aqueous layer is extracted with ethyl acetate, the organic layer is dried over sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (10-90 methanol-dichloromethane) to afford INT-11.

Step 3:2-amino-3-(carbamimidoylsulfanyl)-4,4,4-trifluorobutanoic acid (286)

(347) INT-11 (0.5 mmol) is dissolved methanol, 10% palladium on carbon (Pd/C 10 mol %) is added and the flask is vacuum purged with hydrogen gas 5 times. The mixture is stirred vigorously at room temperature under a hydrogen atmosphere for 16 h. Nitrogen gas is used to purge the flask and the mixture is filtered through Celite to remove the Pd/C. The mixture is concentrated under reduced pressure, and the residue is treated with 1N sodium hydroxide in methanol. Once ester hydrolysis is complete, the mixture is concentrated again under reduced pressure, and the resulting residue is dissolved in water (3.0 mL) and filtered through a 0.2 m nylon filter. The filtrate is purified by preparative HPLC (e.g. Waters PrepPak cartridge Delta-Pak C18 compression column, 15 m 25100 mm, 95:5 water-acetonitrile at 12.0 mL/min). The product is collected and lyophilized to afford the product 2-amino-3-(carbamimidoylsulfanyl)-4,4,4-trifluorobutanoic acid (286).

(348) Synthesis of Compound 358

(349) ##STR00589##

(350) Am inopentanoic acid SM37 (500 mg, 4.27 mmol) was converted into the corresponding guanidino derivative according to the procedure for compound (219). The desired compound 358 was obtained as a white powder after high vacuum drying (291 mg, 43%); .sup.1H-NMR (300 MHz, D.sub.2O): 3.63 (m, 1H), 2.39 (dd, 1H), 2.29 (dd, 1H), 1.52 (m, 2H), 0.85 (t, 3H); ES(pos) MS m/z 160.11 (M+H.sup.+).

(351) Synthesis of Compound 376

(352) ##STR00590##

(353) Pyrrolidine-3-carboxylic acid hydrochloride (500 mg, 3.30 mmol) was converted into the desired derivative (376) according to a modified procedure described for compound 258 where the amount of base was increased to neutralize the HCl-salt of the starting material. The final material was isolated as a white powder (198 mg, 38%); .sup.1H-NMR (300 MHz, D.sub.2O): 3.4 (m, 4H), 2.99 (m, 1H), 2.17 (m, 1H), 2.03 (m, 1H); ES(pos) MS m/z 158.09 (M+H.sup.+).

(354) Synthesis of Compound 366

(355) ##STR00591##

Step 1:tert-butyl N-[(1Z)-{[(tert-butoxy)carbonyl]imino}[(3-hydroxy-2,2-dimethylpropyl)amino]methyl]carbamate (INT-12)

(356) ##STR00592##

(357) 3-Amino-2,2-dimethylpropanol SM45 (464 mg, 4.5 mmol) was dissolved in dry THF (7 mL) and then treated with N,N-di-Boc-1H-pyrazole-1-carboxamidine (1.24 g, 4.0 mmol). After stirring for 72 hours at room temperature, the reaction mixture was poured into ethyl acetate (200 mL) and then washed with water (2100 mL) adjusting the pH of the aqueous layer to pH 1-2 using 1N HCl. The combined aqueous washes were then re-extracted with ethyl acetate (100 mL). The second organic phase was in turn washed with acidified water (100 mL) using 1N HCl to adjust the to pH 1-2. The combined organic phases were dried over magnesium sulfate, filtered, evaporated, and purified by silica gel column chromatography to afford INT-12 as a viscous oil that solidified upon standing (1.32 g, 96%); .sup.1H-NMR (300 MHz, DMSO-d.sub.6): 11.48 (s, 1H). 8.55 (t, 1H), 4.93 (t, 1H), 3.18 (d, 2H), 3.13 (d, 2H), 1.48 (s, 9H), 1.39 (s, 9H), 0.82 (s, 6H).

Step 2: 3-{[(1Z)-{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}-2,2-dimethylpropanoic acid (INT-13)

(358) ##STR00593##

(359) INT-12 (1.30 g, 3.76 mmol) was dissolved in acetonitrile (25 mL), carbon tetrachloride (25 mL), and water (40 mL). Sodium periodate (4.83 g, 22.6 mmol) and ruthenium trichloride (50 mg, catalytic) were added and the mixture was stirred for 3 h at room temperature or until TLC showed starting material had been consumed. The resulting biphasic mixture was poured into ethyl acetate (200 mL) and then washed with water (2100 mL) adjusting the pH of the aqueous layer to pH 1-2 using 1N HCl. The combined aqueous washes were then re-extracted with ethyl acetate (100 mL). The second organic phase was in turn washed with acidified water (100 m L) using 1N HCl to adjust the to pH 1-2. The combined organic phases were dried over magnesium sulfate, filtered, evaporated, and purified by silica gel column chromatography to afford INT-13 as a solid foam (1.05 g, 78%); .sup.1H-NMR (300 MHz, DMSO-d.sub.6): 11.47 (s, 1H). 8.53 (t, 1H), 3.42 (d, 2H), 1.47 (s, 9H), 1.39 (s, 9H), 1.13 (s, 6H).

Step 3:Compound 366

(360) INT-14 is dissolved in 1:4 trifluoroacetic acid-dichloromethane (5 mL). The mixture is stirred at room temperature overnight to remove the Boc-protecting groups. The mixture is concentrated under vacuum and the residue is purified by preparative HPLC chromatography to afford compound 366.

(361) In addition to the compounds described above, similar protocols are used to make numerous analogs shown in Table 16 from starting materials listed in Table 15.

(362) TABLE-US-00017 TABLE 16 Starting materials for the synthesis of selected compounds Compound # Table # Starting Material Protocol 253 4 SM08 or SM09 See 261 254 4 SM10-SM12 See 261 255 4 SM03 See 219 256 4 SM04 See 219 257 4 SM05 See 257 258 4 SM06 See 258 259 4 SM13 or SM14 See 219 260 4 SM15 or SM16 See 219 261 4 SM07 See 261 262 4 SM24-SM27 See 261 327 4 SM08 See 261 328 4 SM09 See 261 329 4 SM10 or SM11 See 261 330 4 SM12 See 261 331 4 SM13 See 219 332 4 SM14 See 219 333 4 SM15 See 219 334 4 SM16 See 219 335 4 SM17 See 258 336 4 SM17 See 258 337 4 SM17 See 258 338 4 SM17 See 258 339 4 SM18 See 258 340 4 SM19 See 258 341 4 SM20 See 258 342 4 SM21 See 258 343 4 SM22 See 261 344 4 SM23 See 261 345 4 SM24 See 261 346 4 SM25 See 261 347 4 SM26 See 261 348 4 SM27 See 261 349 4 SM28 See 219 350 4 SM29 See 219 351 4 SM30 See 261 352 4 SM31 See 261 353 4 SM32 See 219 354 4 SM33 See 219 355 4 SM34 See 219 356 4 SM35 See 219 357 4 SM36 See 219 358 4 SM37 See 358 359 4 SM38 See 219 360 4 SM39 See 219 361 4 SM40 See 219 362 4 SM41 See 219 363 4 SM42 See 261 364 4 SM43 See 261 365 4 SM44 See 261 366 4 SM45 See 366 367 4 SM46 See 366 368 4 SM47 See 261 369 4 SM48 See 261 370 4 SM49 See 261 371 4 SM50 See 261 372 4 SM51 See 219 373 4 SM52 See 219 374 4 SM53 See 219 375 4 SM54 See 219 376 4 SM55 See 376 377 4 SM56 See 376 378 4 SM57 See 376 379 4 SM59 See 258 380 4 SM58 See 258 381 4 SM60 See 219 382 4 SM61 See 219 383 4 SM62 See 219 384 4 SM63 See 219 385 4 SM64 See 258 263 5 SM65 See 223 264 5 SM72 See 223 265 5 SM71 See 223 266 5 SM08 or SM09 See 223 267 5 SM10-SM12 See 223 268 5 SM03 See 223 269 5 SM04 See 223 270 5 SM05 See 223 271 5 SM13 or SM14 See 223 272 5 SM15 or SM16 See 223 273 5 SM07 See 223 274 5 SM24-SM27 See 223 386 5 SM73 See 223 387 5 SM71 See 223 388 5 SM72 See 223 389 5 SM08 See 223 390 5 SM09 See 223 391 5 SM10 or SM11 See 223 392 5 SM12 See 223 393 5 SM13 See 223 394 5 SM14 See 223 395 5 SM15 See 223 396 5 SM16 See 223 397 5 SM17 See 223 398 5 SM17 See 223 399 5 SM17 See 223 400 5 SM17 See 223 401 5 SM18 See 223 402 5 SM19 See 223 403 5 SM20 See 223 404 5 SM21 See 223 405 5 SM22 See 223 406 5 SM23 See 223 407 5 SM24 See 223 408 5 SM25 See 223 409 5 SM26 See 223 410 5 SM27 See 223 411 5 SM28 See 223 412 5 SM29 See 223 413 5 SM30 See 223 414 5 SM31 See 223 415 5 SM32 See 223 416 5 SM33 See 223 417 5 SM34 See 223 418 5 SM35 See 223 419 5 SM36 See 223 420 5 SM38 See 223 421 5 SM39 See 223 422 5 SM45 See 223 423 5 SM46 See 223 424 5 SM47 See 223 425 5 SM48 See 223 426 5 SM49 See 223 427 5 SM50 See 223 428 5 SM51 See 223 429 5 SM52 See 223 430 5 SM53 See 223 431 5 SM54 See 223 432 5 SM58 See 223 433 5 SM59 See 223 434 5 SM60 See 223 435 5 SM61 See 223 436 5 SM62 See 223 275 6 SM66 See 275 276 6 SM67 See 275 277 6 SM67 See 275 278 6 SM08 or SM09 See 278 279 6 SM10-SM12 See 278 280 6 SM03 See 278 281 6 SM68 See 275 282 6 SM69 See 275 283 6 SM13 or SM14 See 275 284 6 SM15 or SM16 See 278 285 6 SM07 See 278 286 6 SM24-SM27 See 286
Generic Scheme to Make Sulfanyl N-amidinoprodrugs

(363) ##STR00594##

(364) Sulfanyl N-amidino prodrugs may be synthesized in several steps from homodisulfides via N-alkylthiol-phthalimide. Disulfides are commercially available or easily prepared from sulfhydryls using standard conditions (e.g. iodine-water U.S. Pat. No. 6,025,488, sodium iodide-peroxide Synthesis, 2007, 3286-3289, etc.). In step 1, disulfides are reacted with bromine or sulfuryl chloride to generate sulfenyl bromides or sulfenyl chlorides in situ at 15 C. to 0 C. in inert solvent (e.g. dichloromethane, 1,2-dichloroethane, chloroform, etc.; see J. Org. Chem. 1971, 36, 3828; and J. Org. Chem. 1986, 51 (26), 5333). The resulting mixture is then agitated for 5 to 30 minutes and subsequently transferred drop-wise to a suspension of potassium phthalimide in a suitable solvent such as 1,2-dichloroethane at 15 C. to 0 C. (see J. Med. Chem. 2009, 52 (14), 4142 and Bioorg. Med. Chem. Lett. 2007, 17, 6629). In step 2, N-alkylthiol-phthalimides are reacted with amidino or guanidine compounds to afford the desired products (see J. Med. Chem. 2009, 52 (14), 4142 and International Patent Publication No. WO2010100337).

(365) Synthesis of Compound 287

(366) ##STR00595##

Step 1: (2S)-2-amino-3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)sulfanyl]propanoic acid (INT-15)

(367) ##STR00596##

(368) L-cystine (5 mmol), phthalimide (5 mmmol), pyridine (10 mmol) are dissolved in acetonitrile (10 mL). Bromine (5 mmol) is added drop-wise to the solution at 0 C. and the mixture is warmed to room temperature and stirred overnight. The reaction is concentrated under reduced pressure and the residue is then purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid). The product is collected and lyophilized to afford the trifluoroacetate salt of INT-15.

Step 2: (2S)-2-amino-3-{[{amino[(2-carboxyethyl)amino]methylidene}amino]sulfanyl}propanoic acid (287)

(369) -Guanidinopropionic acid (1.00 mmol), INT-15 (1.15 mmol) and potassium carbonate (1.15 mmol) are dissolved in anhydrous acetonitrile (10 ml) and stirred overnight. The reaction is concentrated under reduced pressure and the residue is then purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid). The product is collected and lyophilized to afford the trifluoroacetate salt of 287.

(370) In addition to the synthesis of compound 287 described above, additional prodrugs may be made by combining different guanidine compounds (e.g. -guanidinopropionic acid, -guanidinobutanoic acid, 2-(2-iminoimidazolidin-1-yl)acetic acid, L-homocystine, etc.) with different activated thiols (e.g. derived from L-cystine, N,N-diacetyl-L-cystine, 2-(carboxymethyldisulfanyl)acetic acid, etc.) as listed in Table 17.

(371) TABLE-US-00018 TABLE 17 Starting materials for the synthesis of selected compounds Com- Disulfide Starting pound # Guanidine Compound Material [CAS] 288 -guanidinopropionic acid N,N-diacetyl-L-cystine [5545-17-5] 289 -guanidinopropionic acid 2-(carboxymethyldisulfanyl)acetic acid [505-73-7] 290 -guanidinopropionic acid 2,2-Dithiobis(ethylammonium) sulphate [16214-16-7] 291 -guanidinopropionic acid L-homocystine [626-72-2] 292 -guanidinopropionic acid 2,2-dithiodiethanesulfonic acid [45127-11-5] 293 -guanidinobutanoic acid L-cystine [56-89-3] 294 -guanidinobutanoic acid N,N-diacetyl-L-cystine [5545-17-5] 295 -guanidinobutanoic acid 2-(carboxymethyldisulfanyl)acetic acid [505-73-7] 296 -guanidinobutanoic acid 2,2-dithiobis(ethylammonium) sulphate [16214-16-7] 297 -guanidinobutanoic acid L-homocystine [626-72-2] 298 -guanidinobutanoic acid 2,2-dithiodiethanesulfonic acid [45127-11-5] 299 2-(2-iminoimidazolidin-1- L-cystine [56-89-3] yl)acetic acid 300 2-(2-iminoimidazolidin-1- N,N-diacetyl-L-cystine yl)acetic acid [5545-17-5] 301 2-(2-iminoimidazolidin-1- 2-(carboxymethyldisulfanyl)acetic yl)acetic acid acid [505-73-7] 302 2-(2-iminoimidazolidin-1- 2,2-dithiobis(ethylammonium) yl)acetic acid sulphate [16214-16-7] 303 2-(2-iminoimidazolidin-1- L-homocystine [626-72-2] yl)acetic acid 304 2-(2-iminoimidazolidin-1- 2,2-dithiodiethanesulfonic yl)acetic acid acid [45127-11-5] 305 2-(2-imino-1,3-diazinan-1- L-cystine [56-89-3] yl)acetic acid 306 2-(2-imino-1,3-diazinan-1- N,N-diacetyl-L-cystine yl)acetic acid [5545-17-5] 307 2-(2-imino-1,3-diazinan-1- 2-(carboxymethyldisulfanyl)acetic yl)acetic acid acid [505-73-7] 308 2-(2-imino-1,3-diazinan-1- 2,2-dithiobis(ethylammonium) yl)acetic acid sulphate [16214-16-7] 309 2-(2-imino-1,3-diazinan-1- L-homocystine [626-72-2] yl)acetic acid 310 2-(2-imino-1,3-diazinan-1- 2,2-dithiodiethanesulfonic yl)acetic acid acid [45127-11-5] 311 2-(1-methylguanidino)ace- L-cystine [56-89-3] tic acid 312 2-(1-methylguanidino)ace- N,N-diacetyl-L-cystine tic acid [5545-17-5] 313 2-(1-methylguanidino)ace- 2-(carboxymethyldisulfanyl)acetic tic acid acid [505-73-7] 314 2-(1-methylguanidino)ace- 2,2-dithiobis(ethylammonium) tic acid sulphate [16214-16-7] 315 2-(1-methylguanidino)ace- L-homocystine [626-72-2] tic acid 316 2-(1-methylguanidino)ace- 2,2-dithiodiethanesulfonic tic acid acid [45127-11-5] 317 2-(1,3-dimethylguani- L-cystine [56-89-3] dino)acetic acid 318 2-(1,3-dimethylguani- N,N-diacetyl-L-cystine dino)acetic acid [5545-17-5] 319 2-(1,3-dimethylguani- 2-(carboxymethyldisulfanyl)acetic dino)acetic acid acid [505-73-7] 320 2-(1,3-dimethylguani- 2,2-dithiobis(ethylammonium) dino)acetic acid sulphate [16214-16-7] 321 2-(1,3-dimethylguani- L-homocystine [626-72-2] dino)acetic acid 322 2-(1,3-dimethylguani- 2,2-dithiodiethanesulfonic acid dino)acetic acid [45127-11-5]
Synthesis of Compound 323

(372) ##STR00597##

Step 1:N-benzyl-2-oxoazetidine-1-carbothioamide

(373) ##STR00598##

(374) Azetidinone (5 mmol) is dissolved in tetrahydrofuran and the mixture is cooled in an ice bath. Sodium hydride (60% mineral oil dispersion, 6 mmol) is added followed by benzyl thioisocyanate (5 mmol) and the mixture is stirred to room temperature over 2 h. The reaction is poured into water, the aqueous layer is extracted with ethyl acetate, the organic layer is dried over sodium sulfate, concentrated under reduced pressure and purified by silica gel column chromatography (50-50 ethyl acetate-hexanes) to afford N-benzyl-2-oxoazetidine-1-carbothioamide.

Step 2:N,N-dibenzyl-2-oxoazetidine-1-carboximidamide

(375) ##STR00599##

(376) To a stirring solution of N-benzyl-2-oxoazetidine-1-carbothioamide (2.5 mmol) in methanol (5 mL) is added methyl iodide (7.0 mmol). The solution is stirred for 2 h, and solvent is removed under reduced pressure. The residue is partitioned between ethyl acetate (15 mL) and saturated aqueous bicarbonate solution (15 mL), the mixture is shaken and the organic layer is dried over sodium sulfate, concentrated under reduced pressure and purified by silica gel column chromatography (50-50 ethyl acetate-hexanes) to afford the intermediate pseudothiourea.

(377) To a stirring solution of this pseudothiourea (1 mmol) in methanol (3 mL) is added benzylamine (1 mmol). The resulting solution is heated to reflux for 12 h in a sealed tube, cooled to ambient temperature, the mixture is concentrated under reduced pressure and purified by silica gel column chromatography (50-50 ethyl acetate-hexanes) to afford N,N-dibenzyl-2-oxoazetidine-1-carboximidamide.

Step 3:2-oxoazetidine-1-carboximidamide

(378) N,N-Dibenzyl-2-oxoazetidine-1-carboximidamide (0.5 mmol) is dissolved methanol, 10% palladium on carbon (Pd/C 10 mol %) is added and the flask is vacuum purged with hydrogen gas 5 times. The mixture is stirred vigorously at room temperature under a hydrogen atmosphere for 16 h. Nitrogen gas is used to purge the flask and the mixture is filtered through Celite to remove the Pd/C. 1N Hydrochloric acid in methanol is added to precipitate the HCl salt of compound 323.

(379) Synthesis of Compound 324

(380) ##STR00600##

(381) 2-chloro-pyridine (25 mmol), palladium (II) acetate (2.5 mmol), racemic 2,2-bis(diphenylphosphino)-1,1-binaphthyl (2.5 mmol) and cesium carbonate (65 mmol) is dissolved in toluene (75 mL) in a previously degassed sealed vessel. The mixture is flushed with nitrogen gas. Azetidinone (20 mmol) is added to the solution under nitrogen and the sealed mixture is heated overnight at 100 C. The reaction is cooled to room temperature, diluted with diethyl ether, and washed with saturated bicarbonate solution and water. The organic layer is concentrated and purified by silica gel column chromatography (2:98 methanol-dichloromethane) to afford 1-(pyridin-2-yl)azetidin-2-one.

(382) Synthesis of Compound 325

(383) ##STR00601##

Step 1:3-{[{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}propanoic acid (INT-16)

(384) ##STR00602##

(385) -alanine (1.0 mmol) and N,N-di-(Boc)-1H-pyrazole-1-carboxamide (1.0 mmol) are suspended in pyridine (2.0 mL) and stirred at 25 C. for 2 days. The homogenous reaction is treated with 1N NaOH and extracted into ethyl acetate. The aqueous layer is acidified (pH 3) with 1N HCl and then extracted into ethyl acetate. The combined organic layers are washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide 3-{[{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}propanoic acid (INT-16).

Step 2: (2R,3R,4R,5R)-4-[(3-{[{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino}) methyl]amino}propanoyl)oxy]-5-(5-fluoro-2-oxo-4-{[(pentyloxy)carbonyl]amino}-1,2-Dihydropyrimidin-1-yl)-2-methyloxolan-3-yl-3-{[{(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}propanoate (INT-17)

(386) ##STR00603##

(387) INT-17 (0.7 mmol) and benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (PyBOP) (0.6 mmol) are added to a solution of capecitabine (0.30 mmol) in dichloromethane (2.0 mL). The resulting mixture is stirred at 25 C. overnight. The dichloromethane is removed under reduced pressure and the residue is taken up in ethyl acetate. The organic layer is washed with water, brine, dried over magnesium sulfate filtered, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (2:98 methanol-dichloromethane) to afford the Boc-protected coupled compound (INT-17).

Step 3: (2R,3R,4R,5R)-4-[(3-carbamimidamidopropanoyl)oxy]-5-(5-fluoro-2-oxo-4-{[(pentyloxy)carbonyl]amino}-1,2-dihydropyrimidin-1-yl)-2-methyloxolan-3-yl 3-carbamimidamidopropanoate (325)

(388) Boc-Deprotection is accomplished by standard condition using neat trifluoroacetic acid, mixtures of trifluoroacetic acid (TFA) with methylene chloride, or hydrochloride acid (HCl) in dioxane. The material from the previous step INT-17 (0.20 mmol) is treated with 4N HCl/dioxane (2.0 mmol). The mixture is stirred at 25 C. overnight, concentrated under reduced pressure, and then subjected to reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid) to afford the desired product 325.

(389) Synthesis of Compound 326

(390) ##STR00604##

Step 1:1-(benzyloxy)-3-[(3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoyl)oxy]propan-2-yl-3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoate (INT-18)

(391) ##STR00605##

(392) INT-17 (2.2 mmol) and benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (PyBOP) (2.1 mmol) are added to a solution of 3-(benzyloxy)propane-1,2-diol (1.0 mmol) in dichloromethane (10 mL). The resulting mixture is stirred at 25 C. overnight. The dichloromethane is removed under reduced pressure and the residue is taken up in ethyl acetate. The organic layer is washed with water, brine, dried over magnesium sulfate filtered, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (50:50 ethyl acetate-hexane) to afford the desired compound INT-18.

Step 2:1-[(3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoyl)oxy]-3-[(chlorocarbonyl)oxy]propan-2-yl-3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoate (INT-19)

(393) ##STR00606##

(394) INT-18 (0.5 mmol) is dissolved methanol, 10% palladium on carbon (10% Pd/C, 0.05 mmol) is added, and the flask is vacuum purged with hydrogen gas 5 times. The mixture is stirred vigorously at room temperature under a hydrogen atmosphere for 16 h. Nitrogen gas is used to purge the flask and the mixture is filtered through Celite to remove the Pd/C and the solvent is concentrated under reduced pressure. The residue is used as is in the next reaction.

(395) A mixture of triphosgene (0.25 mmol), sodium carbonate (0.5 mmol), and dimethylformamide (0.018 mmol) as a catalyst, in toluene (2 mL) is cooled to 0 C. and stirred at this temperature for 30 min. A solution of the residue from the previous reaction in toluene (2 mL) is added slowly over a period 30 min. The reaction mixture is stirred at 0 C. for 8 h. The solid sodium carbonate is removed by filtration and the solvent is concentrated under reduced pressure. The resulting residue INT-19 is used as-is in the next reaction.

Step 3:1-[({1-[(4R,6R,6aS)-2,2,6-trimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]-5-fluoro-2-oxo-1,2,3,4-tetrahydropyrimidin-4-yl}carbamoyl)oxy]-3-[(3-{[bis({[tert-butoxy)carbonyl]amino})methylidene]amino}propanoyl)oxy]propan-2-yl-3-{[bis({[tert-butoxy)carbonyl]amino})methylidene]amino}propanoate (INT-20)

(396) ##STR00607##

(397) 5-Fluoro-2,3-o-isopropylidenecytidine (5-FIPC) is made according to protocols described in International Patent Publication Nos. WO2008144980 and WO2008131062. 5-FIPC (0.25 mmol) and pyridine (0.5 mmol) are dissolved in dichloromethane (5 mL) and INT-12 is added as a solution in dichloromethane (5 mL). The mixture is stirred at room temperature for 3 h. The reaction is poured into ethyl acetate (50 mL) and washed with water, brine, dried over magnesium sulfate filtered, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (50:50 ethyl acetate-hexane) to afford the desired compound INT-20.

Step 4:1-[(3-carbamimidamidopropanoyl)oxy]-3-[({1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl}carbamoyl)oxy]propan-2-yl 3-carbamimidamidopropanoate (326)

(398) INT-20 (0.20 mmol) is treated with 4N HCl/dioxane (2.0 mmol). The mixture is stirred at room temperature overnight and then water is added and the mixture is stirred an additional 8 h. The reaction is concentrated under reduced pressure and the residue is then purified by reverse phase chromatography (acetonitrile/water with 0.05% trifluoroacetic acid) and the product is collected and lyophilized to afford the trifluoroacetate salt of compound 326.

Example 2. Determining Compound Activity

(399) The invention provides compounds that are useful for treating cancer and/or for inhibiting cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization.

(400) To determine the activity of a compound, one can contact the compound with a system containing test cells expressing a reporter gene encoded by a nucleic acid operatively liked to a promoter of a marker gene selected from the above-mentioned metastasis promoters or suppressors. The system can be an in vitro cell line model or an in vivo animal model. The cells can naturally express the gene, or can be modified to express a recombinant nucleic acid. The recombinant nucleic acid can contain a nucleic acid coding a reporter polypeptide to a heterologous promoter. One then measures the expression level of the miRNA, polypeptide, or reporter polypeptide.

(401) For the polypeptide, the expression level can be determined at either the mRNA level or at the protein level. Methods of measuring mRNA levels in a cell, a tissue sample, or a body fluid are well known in the art. To measure mRNA levels, cells can be lysed and the levels of mRNA in the lysates or in RNA purified or semi-purified from the lysates can be determined by, e.g., hybridization assays (using detectably labeled gene-specific DNA or RNA probes) and quantitative or semi-quantitative RT-PCR (using appropriate gene-specific primers). Alternatively, quantitative or semi-quantitative in situ hybridization assays can be carried out using tissue sections or unlysed cell suspensions, and detectably (e.g., fluorescent or enzyme) labeled DNA or RNA probes. Additional mRNA-quantifying methods include RNA protection assay (RPA) and SAGE. Methods of measuring protein levels in a cell or a tissue sample are also known in the art.

(402) To determine the effectiveness of a compound to treat cancer and/or inhibiting cancer cell survival, hypoxic survival, metastatic survival, or metastatic colonization, one can compare the level obtained in the manner described above with a control level (e.g., one obtained in the absence of the candidate compound). The compound is identified as being effective if (i) a metastasis suppressor's level is higher than a control or reference value or (ii) a metastasis promoter's level is lower than the control or reference value. One can further verify the efficacy of a compound thus-identified using the in vitro cell culture model or an in vivo animal model as disclosed in the examples below.

(403) The activity of compounds may also be determined by methods known in the art to determine CKB or creatine transport inhibition. For example, methods to determine inhibition of CKB are described in McLaughlin et al. J. Biol. Chem. 1972, 247:4382-4388, incorporated herein by reference. Methods to determine inhibition of creatine transport are described in Fitch et al. Metabolism, 1980, 29:686-690, Dodd et al. J. Biol. Chem. 2005, 280:32649-32654, and Dodd et al. J. Biol. Chem. 2007, 282:15528-15533, each of which is incorporated herein by reference.

Example 3. In Vivo Selection

(404) As a first step to identify molecular regulators of liver colonization by colon cancer, an in vivo selection was performed on the LS-174T human colon cancer line for enhanced liver colonization through iterative intra-hepatic injection of cancer cells into immunodeficient mice followed by surgical resection of the liver colonies and dissociation of cells. More specifically, liver colonization by 510.sup.5 LS-Parental, LvM3a and LvM3b cells was examined after direct intrahepatic injection by bioluminescence. Mice were imaged at day 21 after injection and livers extracted for ex vivo imaging and gross morphological examination. Photon flux ratios for the groups were obtained and compared. It was found that third-generation liver colonizers LS-LvM3a and LS-LvM3b displayed dramatically enhanced (>50 fold) capacity for liver colonization upon intra-hepatic injection relative to their parental line. Importantly, these derivatives also displayed significantly enhanced (>150 fold) liver metastatic capacity upon portal circulation injection in metastatic colonization assaysrevealing liver colonization capacity to be a key step in colon cancer metastatic progression. For these bioluminescence assays, all P values for the groups' respective photon flux ratios were based on one-sided Student's t-tests and found to be less than 0.05, 0.001, or 0.0001.

(405) In order to systematically identify microRNA regulators of metastatic progression, a library of lentiviral particles, each encoding one of 611 human microRNAs, was transduced into the LS-LvM3b colonizer population, the LS-174T parental line, as well as the SW620 colon cancer population. These cancer populations, containing cancer cells expressing each of 661 miRNAs, were then intra-hepatically injected into mice in order to allow for the selection of cells capable of colonizing the liver. Genomic PCR amplification of miRNA sequences, reverse-transcription, and miRNA profiling of miRNA inserts allowed for the quantification of miRNA insert representation. It was identified that several miRNAs displayed drop-out in the context of liver colonization in both colon cancer cell lines, consistent with the over-expression of these miRNAs suppressing liver colonization by colon cancer cells.

Example 4. Determination of Effect of Endogenous Levels of miRNAs

(406) In this example, assays were carried out to examine whether endogenous levels of any of these miRNAs exhibit silencing in highly metastatic derivatives relative to isogenic poorly metastatic cells. Indeed, miR-483-5p and miR-551a were found to be silenced in highly metastatic LS-LVM3a and LS-LVM3b liver colonizers relative to their parental line and the metastatic SW620 derivative relative to its isogenic parental line. Consistent with a suppressive role for these miRNAs in liver colonization, over-expression of miR-483-5p or miR-551a robustly suppressed metastatic colonization by the LS-LvM3b cells, while inhibition of endogenous miR-483-5p or miR-551a in poorly metastatic parental lines LS-174T and SW480 significantly enhanced liver metastatic colonization.

Example 5. Investigation of the Mechanism of Action of miRNAs

(407) In this example, assays were carried out to investigate the mechanism(s) by which these miRNAs exert their anti-metastatic effects. The effects of these miRNAs on metastatic progression were not secondary to modulation of proliferative capacity since miR-551a inhibition did not effect in vitro proliferation, while miR-483-5p inhibition increased proliferation. Additionally, over-expression of these miRNAs did not alter the invasive capacity or apoptotic rates of cancer cells. In order to determine the mechanism(s) by which these miRNAs impact metastasis, assays were performed to identify the time-point during the metastatic process when cells over-expressing these miRNAs display a defect in progression. Surprisingly, it was noted that as early as 24 hours after injection of cells into the portal circulation for hepatic metastatic colonization assays, cells over-expressing these miRNAs were out-competed in their representation relative to cells expressing a control hairpin.

Example 6. Organotypic Slice Culture System

(408) To elucidate the mechanism(s) by which these miRNAs suppress liver metastatic colonization, an in vitro liver organotypic slice culture system was developed. This system allowed one to study early events during liver metastasis after single-cell dissemination of colon cancer cells in the liver microenvironment. Consistent with prior studies on a significant selection on cell survival during metastatic colonization, there was a large drop-off in the numbers of cells within the liver microenvironment as a function of time. Highly metastatic LvM3b colonizer cells were significantly better at persisting in the liver microenvironment than their poorly metastatic parental lineconsistent with a positive role for intrahepatic persistence in metastatic progression.

(409) Next, assays were carried out to investigate whether the effects of this miRNA regulatory network on cancer cell persistence are caused by diminished cancer cell survival during metastatic progression. To quantify cell death in vivo, a bioluminescence-based luciferin reporter of caspace-3/7 activity was utilized.

(410) More specifically, SW480 cells whose endogenous miR-483-5p or miR-551a were inhibited and subsequently introduced into the liver of immunodeficient mice by intrasplenic injection. Then, relative in vivo caspase activity in these cells was monitored using a caspase-3 activated DEVD-luciferin. It was found that miRNA inhibition significantly reduced in vivo caspase activity in colon cancer cells during the early phase of hepatic colonization, revealing cancer survival to be the phenotype suppressed by these miRNAs.

(411) These in vivo findings were corroborated by an organotypic slice culture system. Briefly, survival of the SW480 cells in organotypic cultures (n=8) whose endogenous miR-483-5p or miR-551a were inhibited by pre-treatment with LNAs. 510.sup.5 cells were labeled with cell-tracker green (LS-Parental) or cell-tracker red (LvM3b) and introduced into the liver through intrasplenic injection. Immediately after injection, the liver was excised and 150-um slice cultures were made using a tissue chopper. Survival of the cells in organotypic cultures was monitored for up to 4 days with a multi-photon microscope. Dye-swap experiments were performed to exclude effects of dye bias. Representative images at day 0 and day 3 were shown. It was found that over-expression of both microRNAs in LS-LvM3b cells suppressed colon cancer persistence while inhibition of endogenous levels of both microRNAs enhanced persistence of poorly metastatic SW480 cells. The above findings reveal miR-483-5p and miR-551a to suppress liver metastatic colonization and metastatic cell survival in the livera phenotype exhibited by highly metastatic colon cancer cells.

Example 7. Investigation of Downstream Effectors

(412) In this example, assays were carried out to identify the downstream effectors of these miRNAs. Through transcriptomic profiling, transcripts that were down-regulated by over-expression of each microRNA and which contained 3-UTR or coding-sequence (CDS) elements complementary to the miRNAs were identified. Interestingly, Creatine Kinase Brain-type (CKB) was identified as a putative target of both miRNAs, suggesting that these miRNAs, which exhibit common in vivo and organotypic phenotypes might mediate their effects through a common target gene. Indeed, quantitative PCR validation revealed suppression of CKB transcript levels upon over-expression of the microRNAs. It was found that expression levels of CKB in highly metastatic LvM3b cells were suppressed by over-expressing miR-483-5p and miR-551a. Additionally, endogenous miR-483 and miR-551a were found to suppress endogenous CKB protein levels. For example, it was found that expression of CKB was up-regulated in poorly metastatic SW480 cells whose endogenous miR-483-5p and miR-551 aa were inhibited with LNAs. Mutagenesis and luciferase-based reporter assays revealed miR-483-5p and miR-551a to directly target the 3UTR or CDS of CKB. To that end, luciferase reporter assays of CKB coding sequence and 3-UTR were carried out. It was found that miR-483-5p and miR-551a targeted complementary regions in the 3-UTR and coding sequence of CKB respectively. The assays were performed with the complementary regions mutated as well and they were performed at least 3 times.

Example 8. Investigation of the Role of CKB in Liver Metastasis

(413) In this example, assays were carried out to examine if CKB is sufficient and necessary for liver metastatic colonization by colon cancer.

(414) Briefly, liver metastasis was examined in mice injected intrasplenically with 510.sup.5 poorly metastatic SW480 cells and CKB over-expressing cells. The mice were euthanized at 28 days after injection and livers excised for bioluminescent imaging. Similarly, liver metastasis was also examined in mice injected intrasplenically with 510.sup.5 highly aggressive LvM3b expressing a control hairpin or a hairpin targeting CKB. These mice were euthanized 21 days after injection as described above.

(415) It was found that over-expression of CKB in poorly metastatic SW480 cells was sufficient to promote liver metastasis by more than 3-folds, while CKB knockdown in metastatic LS-LvM3b cells and SW480 cells, through independent hairpin knockdown in each line robustly suppressed liver metastasis by more than 5 folds. Consistent with the effects of the miRNAs, CKB over-expression was sufficient to significantly enhance the ability of colon cancer cells to persist in the liver micro-environment and enhanced their representation in the liver, while CKB knockdown significantly reduced intra-hepatic persistence. To that end, study was carried out to examine survival of control SW480 and CKB over-expressing SW480 cells in organotypic liver slices (n=8), and organotypic slice cultures of LvM3b cells expressing a control hairpin or hairpin targeting CKB (n=8). Images taken at day 0 and day 2 showed that CKB over-expression was sufficient to significantly enhance the ability of cancer cells. In these assays, P values were found to be less than 0.001 or 0.0001 based on one-sided Student's t-tests.

(416) To investigate whether CKB acts directly downstream of miR-483-5p and miR-551a, the coding-sequence of CKB was over-expressed in cells over-expressing miR-483-5p or miR-551a. Briefly, assays were performed to examine metastatic progression in mice injected with 510.sup.5 LvM3b cells over-expressing miR-483-5p and miR-551a, with and without CKB over-expression. Liver metastases were monitored by bioluminescent imaging and mice euthanized 35 days after injection. It was found that over-expression of CKB was sufficient to rescue the suppressed liver metastatic phenotypes of cells over-expressing miR-483-5p and miR-551a. Conversely, knockdown of CKB in cells displaying endogenous miR-483-5p or miR-551a inhibition prevented the enhanced metastasis effect seen with miR-483-5p or miR-551a inhibition. To that end, assays were performed to examine liver metastasis in mice injected with 510.sup.5 SW480 cells whose endogenous miR-483-5p and miR-551a were inhibited by LNA, with and without CKB knockdown. The mice were euthanized after 28 days and liver excised for ex vivo bioluminescence imaging. The results of the above mutational, gain- and loss-of-function experiments, and epistasis analyses reveal CKB to be a direct target of miR-483-5p and miR-551a and to act as a downstream effector of these miRNAs in the regulation of colon cancer metastatic progression. In these assays, P values were found to be less than 0.05 or 0.001 based on one-sided Student's t-tests.

(417) To further confirm the roles of CKB, relative in vivo caspase activities were examined in control SW480 and CKB over-expressing cells in livers of mice. The activities were measured by bioluminescence using a caspase-3 activated DEVD-luciferin and normalized to bioluminescence signal from regular luciferin (n=3). Similar relative in vivo caspase-3 activity were also examined in SW480 cells expressing a control hairpin or hairpin targeting CKB and introduced into the livers of mice through intrasplenic injection. Caspase activities were measured on day 1, day 4 and day 7 after injection. Consistent with the above findings, CKB over-expression significantly reduced, while CKB knockdown significantly enhanced, in vivo caspase-3/7 activity in colon cancer cells during the initial phase of hepatic colonization. In these assays, P values were found to be less than 0.05 or 0.001 based on one-sided Student's t-tests. These findings reveal CKB to be a promoter of colon cancer survival during hepatic metastatic colonization.

Example 9. CKB Knockdown Experiments

(418) CKB is known to regulate the reservoir of rapidly mobilized high-energy phosphates in tissues such as the brain and kidneys by catalyzing the transfer of a high-energy phosphate group from phosphocreatine to ADP, yielding ATP and creatine. It was hypothesized that CKB generation of ATP from phosphocreatine might provide colon cancer cells with an energetic advantage during hepatic colonization. To determine if ATP, the end-product of CKB catalysis, could rescue metastasis suppression seen upon CKB knockdown, CKB knockdown cells were loaded with ATP prior to injection of cells in experimental metastasis assays. Briefly, liver metastasis was examined in mice injected with 510.sup.5 LvM3b with or without CKB knockdown and pre-treated with 100 uM ATP or vehicle. Metastatic burden was monitored by bioluminescent imaging and mice euthanized 21 days after injection. It was found that ATP loading of cells was sufficient to significantly enhance the suppressed metastasis phenotype in cells depleted of CKB by more than 10 folds. The rescue by ATP was specific since ATP loading did not enhance the metastatic activity of cells expressing a short-hairpin control.

(419) Similar studies were done to determine whether creatine and phosphocreatine could rescue the phenotype of seen upon CKB knock-down. More specifically, assays were performed to examine liver metastasis in mice injected with 510.sup.5 LvM3b cells pre-treated with 10 uM creatine, in the background of CKB knockdown. The mice were then euthanized as described above and liver extracted for ex vivo bioluminescent imaging at day 21 after injection. Also, colorectal cancer metastasis was examined in mice injected with 510.sup.5 LvM3b cells with CKB knockdown and pre-treated with 10 uM creatine-phosphate. Liver metastasis was monitored by bioluminescent imaging and mice were euthanized as described above. It was found that both creatine and creatine-phosphate rescued metastasis suppression.

(420) In order to investigate whether colon cancer metastasis could be inhibited by blocking the transport of creatine into colon cancer cells, the creatine transporter channel SLC6a8 was inhibited in LvM3b cells by expressing short hairpin targeting SLC6a8. Then liver metastasis by LvM3b cells was examined in the same manner described above. It was found that knock-down of the creatine transporter channel SLC6a8 inhibited colon cancer metastasis. These findings reveal that colon cancer cells are dependent on CKB generated ATP for their survival during hepatic colonization.

Example 10. Analysis of miRNA and CKB Expression Levels in Primary Colon Cancers and Liver Metastases

(421) In order to determine if this cooperative miRNA regulatory network controlling colon cancer metastatic progression has human pathologic relevance, the expression levels of miR-483-5p and miR-551a were analyzed in a set of 67 primary colon cancers as well as liver metastases obtained from patients at MSKCC. More specifically, miR-483-5p and miR-551a levels in 37 primary tumor samples and 30 liver metastases samples were quantified by quantitative real-time PCR. Consistent with a metastasis-suppressive role for these miRNAs during metastatic progression, miR-483 and miR-551a both displayed significantly reduced expression levels in human liver metastases relative to primary colon cancers (FIG. 1a; p<0.05 for miR-483-5p and p<0.05 for miR-551a; N=67).

(422) CKB expression levels were also examined in the 37 primary tumor samples and 30 liver metastases samples by quantitative real-time PCR. Importantly, CKB expression was found to be significantly elevated in liver metastases relative to primary colon cancers (p<0.05) and its expression was significantly anti-correlated with the miRNAsconsistent with its direct targeting by these miRNAs in human colon cancer (FIG. 1b). These findings are consistent with previous clinical histologic analyses revealing elevated levels of CKB protein in advanced stage cancer.

Example 11. Investigation of miRNA Regulatory Network as a Therapeutic Target

(423) In this example, assays were carried out to investigate the therapeutic potential of targeting this miRNA regulatory network. To this end, mice were injected with a high number (500k) of highly metastatic LvM3a cells and 24 hours later injected mice with a single intra-venous dose of adenoviral-associated virus (AAV) expressing miR-483-5p and miR-551a off a single transcript. It was found that a single therapeutic dose of adeno-associated virus (AAV) delivering both miRNAs dramatically and significantly reduced metastatic colonization by more than 5 fold (FIG. 1c).

(424) Finally, assays were carried out to determine the impact of small-molecule inhibition of CKB and restriction of creatine availability on colon cancer metastasis. Cyclocreatine, which resembles phosphocreatine, is a transition-state analog for creatine kinases. To examine the effect of cyclocreatine, bioluminescent measurements of liver metastasis were carried out in mice injected with 510.sup.5 LvM3b cells and treated with cyclocreatine daily for two weeks. The mice were then euthanized and livers excised for ex vivo imaging at the end of the treatment. It was found that, despite being a poor inhibitor of CKB (5000 uM ki), treatment of mice with cyclocreatine significantly reduced metastatic colonization and proved superior to the current standard-of-care FOLFOX chemotherapy (FIG. 1d).

(425) Similar assays were carried out using a creatine transporter inhibitor beta-guanidinopropionic acid (-GPA). Bioluminescent measurements were used to examine liver metastasis in mice injected with 510.sup.5 LvM3b cells and treated with -GPA daily for two weeks. It was found that treatment of mice with this competitive inhibitor of the creatine transporter channel also significantly reduced metastatic colonization (FIG. 1e).

(426) Using a systematic approach, two miRNAs were identified to act as suppressors of liver metastatic colonization by colon cancer cells. It was found find that these miRNAs convergently target CKBa key gene that endows cells encountering hepatic hypoxia with the ability to generate ATP from phosphocreatine reserves. The successful targeting of this pathway using 4 independent therapeutics that were more effective than the current clinical standard-of-care, and which displayed no apparent toxicity suggest promise for therapeutic targeting of this pathway in human colon cancer. The above-described combined in vivo selection/gene screening approach, which is designated as MUlti-Gene Screening of Human genes through intra-Organ Tandem Selection (MUGSHOTS) has efficiently identified robust and pathologically validated regulators of liver colonization and metastasis by colon cancer and has the potential to discover coding and non-coding regulators of metastatic colonization of any organ by any cancer type.

Example 12. Investigation of Creatine Transport as a Therapeutic Target

(427) In this example, assays were carried out to confirm the therapeutic potential of targeting the creatine transporter channel SLC6a8 by administering the small molecule -GPA, which is an inhibitor of SLC6a8. As mentioned above, administration of -GPA to mice injected with LvM3b colon cancer cells resulted in inhibition of colon cancer metastasis to the liver after two weeks of treatment (FIG. 1e). To confirm this therapeutic effect, mice injected with LvM3b colon cancer cells we treated with either 6-GPA or control vehicle (PBS) via intra-peritoneal injection daily for three weeks (FIG. 2). The mice were euthanized at three weeks and liver extracted for bioluminescent imaging and gross histology.

(428) It was found that daily treatment with -GPA led to a significant reduction in colon cancer metastasis to the liver, as assessed by in vivo bioluminescent imaging of in vivo mice, bioluminescent imaging of extracted liver, and by gross anatomical examination of extracted livers from treated mice (FIG. 2). More specifically, the average photon flux ratios as measure by the bioluminescence imaging for the control group (without treatment of -GPA) and the treated groups were about 800 and 100, respectively. P values were found to be less than 0.05 based on one-sided Student's t-tests.

Example 13. Knockdown of SLC6a8

(429) In this example assays were carried out to evaluate the therapeutic benefit of targeting the creatine transporter channel SLC6a8 with shRNA knockdown targeting SLC6a8.

(430) Briefly, mice were injected with LvM3b colon cancer cells expressing either of two independent short hairpin RNAs (shSLC6a8 #4 or shSLC6a8 #5) targeting the creatine transporter channel SLC6a8 or with control RNA (empty pLKO vector, ordered from Sigma Aldrich) (FIG. 3a). Again, liver metastasis was monitored by bioluminescent imaging and mice were euthanized three weeks after inoculation of cancer cells. Livers were extracted for gross histology. It was found that knockdown of SLC6a8 with two independent shRNAs resulted in inhibition of colon cancer metastasis (FIG. 3a).

(431) To further confirm the therapeutic benefit of knockdown of SLC6a8, another independent colon cancer cell line (SW480 colon cancer cell line) expressing a short hairpin RNA targeting SLC6a8 (shSLC6a8 #2) was injected into mice (FIG. 3b). It was found that SLC6a8 knockdown significantly inhibited metastasis of SW480 colon cancer cells (FIG. 3b).

(432) Lastly, the therapeutic benefit of targeting SLC6a8 was investigated in pancreatic cancer cells. To accomplish this, PANC1 pancreatic cancer cells expressing either an shRNA targeting SLC6a8 (shSLC6a8 #5) or a control RNA (empty pLKO vector) were injected into mice. Metastatic progression was monitored by bioluminescent imaging and mice were euthanized in the same manner described above. It was found that, at 28 days, there was a significant reduction in pancreatic cancer metastasis in the cells treated with shRNA targeting SLC6a8, revealing that SLC6a8 is a therapeutic target for pancreatic cancer.

Example 14. Correlation of Creatine Transport and Metastatic Progression

(433) In this example, it was investigated whether expression of the creatine transporter SLC6a8 in human colon cancer tumors correlated with metastatic progression.

(434) To accomplish this, quantitative real-time PCR was used to quantify the expression of SLC6a8 in 36 primary colon cancer tumors and 30 metastatic colon cancer tumors (FIG. 4). Indeed, expression of SLC6a8 was significantly higher in metastatic tumors (about 1.3) as compared with primary tumors (about 0.5), further confirming the central role of SLC6a8 in metastasis (FIG. 4). P values were found to be less than 0.05 based on one-sided Student's t-tests.

Example 15. Effect of -GPA on Pancreatic Cancer

(435) As mentioned above, it was demonstrated that inhibition of the creatine transporter SLC6a8 with shRNA mediated knock-down resulted in suppression of metastasis of both colon cancer as well as pancreatic cancer. It was also demonstrated that inhibition of SLC6a8 with the small molecule inhibitor -GPA resulted in therapeutic benefit for colon cancer metastasis in vivo. To evaluate if -GPA treatment results in therapeutic benefit in pancreatic cancer, the ability of -GPA treatment to inhibit the survival of human pancreatic cancer cells was assessed in vivo in mice.

(436) Briefly, PANC1 pancreatic cancer cells were incubated for 48 hours with and without the presence of 10 mM of -GPA, then injected into immunodeficient mice (510.sup.5 PANC1 cells each mouse; 4 mice each in the treated and untreated cohort). The mice were imaged with bioluminescence imaging on day 1 after injection and signal was normalized to day zero. Therapeutic benefit was observed as early as one day after the injections, with a significant reduction in the tumor burden of pancreatic cancer cells in vivo as assessed by bioluminescence imaging (FIG. 4) demonstrating therapeutic benefit of -GPA treatment for pancreatic cancer. More specifically, the average photon flux ratios as measure by the bioluminescence imaging for the control group (without treatment of -GPA) and the treated groups were about 2.7 and 1.6, respectively. P values were found to be less than 0.05 based on one-sided Student's t-tests.

Example 16. Combination of -GPA and Fluorouracil or Gemcitabine

(437) The above examples demonstrated that -GPA treatment alone resulted in therapeutic benefit for colon cancer and pancreatic cancer. In this example it was investigated whether -GPA treatment could enhance the therapeutic activity of the chemotherapy agents 5-Fluorouracil and Gemcitabine. To accomplish this, cell viability was performed assays to compare the cytotoxic activity of 5-Fluorouracil or Gemcitabine alone compared with combined therapy with -GPA.

(438) Briefly, 10 000 PANC1 cells were seeded in triplicate in 96-well plates and treated with various concentrations of Gemcitabine (1 nm, 10 nm, 100 nm, 1000 nm, 10000 nm, 100000 nm, and 1000000 nm) with or without 10 mM of -GPA for 48 hours. Cell viability was then assayed using the WST-1 reagent (Roche Applied Science), with absorbance at 440 nm an indicator of the number of viable cells. As shown in FIG. 6, it was found that the addition of a therapeutic concentration of -GPA enhanced the cytotoxic activity of Gemcitabine on PANC1 pancreatic cancer cells as assessed by a cell viability assay using the WST-1 reagent.

(439) Likewise, the addition of a therapeutic concentration of -GPA enhanced the cytotoxic activity of 5-Fluorouracil on Ls-LvM3b colon cancer cells. To that end, 10,000 Ls-LvM3b cells were seeded in triplicate in 96-well plates and treated with various concentrations of 5-Fluorouracil with or without 10 mM of -GPA for 48 hours. Cell viability was assayed in the same manner described above with absorbance at 440 nm an indicator of the number of viable cells. As shown in FIG. 7, these results demonstrate that -GPA enhance the therapeutic activity of commonly utilized chemotherapeutic agents for the treatment of colorectal and pancreatic cancer.

(440) The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated herein in their entireties.

Other Embodiments

(441) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

(442) All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.