CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US2016/044767, filed Jul. 29, 2016, that claims the benefit of U.S. Provisional Application No. 62/199,566, filed Jul. 31, 2015, which is incorporated herein by reference in its entirety.

BACKGROUND

The prodrug approach is a well-established strategy to improve physicochemical, biopharmaceutic and pharmacokinetic properties of potential drug molecules. Approximately 5-7% of drugs approved worldwide are prodrugs with annual sales in 2013 of $11.2 billion. Most prodrugs are simple chemical derivatives of the original molecule. Ester prodrugs, the most common prodrugs, constitute 49% of all marketed prodrugs. Reasons for the popularity of ester prodrugs include their generally straight forward synthesis, their improved lipophilicity and membrane permeability, and the ubiquitousness of estereases. An example of an approach to make an ester prodrug is capping the acidic moiety (ies) with lipophilic alkyl or alkyloxymethyl esters (i.e., pivaloyloxymethyl (POM) or propyloxy-carbonvyloxymethyl (POC); e.g., Enalapril, Adefovir). Another approach is to cap the acidic moiety(ies) with amino acids to make amides that are recognizable by amidases/peptidases in plasma for hydrolysis or to make them substrates for transporters, such as Peptide transporter 1 (PEPT1) (e.g., Pomaglumetad methionil, Valacyclovir).

Glutamine antagonists, such as 6-Diazo-5-oxo-L-norleucine (DON), and aza-serine, have been shown to exhibit broad anti-viral (Antiviral Res. 1997; 33(3):165-75; Antiviral Res. 1994; 25(3-4):269-79), anti-infective (J. Bacteriol. 1965; 89:1348-53), anti-cancer (see, e.g., Yoshioka et al., 1992; Tokushima J. Exp. Med. 39(1-2):69-76), anti-inflammatory, and immunosuppressive activities (Kulcsar et al., 2014; 111:16053-58; Maciolek et al., 2014; Curr Opin Immunol. 27:60-74; Carr et al., 2010; J Immunol. 185:1037-1044; Colombo et al., 2010; Proc Natl Acad Sci USA. 107:18868-73), as well as inhibition of convulsions (Proc R Soc Lond B Biol Sci. 1984 Apr. 24; 221(1223):145-68), multiple sclerosis (Tohoku, J. Exp. Med. 2009; 217(2):87-92), epilepsy, and viral encephalitis (J. Neurovirol. 2015 April; 21(2):159-73. doi: 10.1007/s13365-015-0314-6), in many published preclinical and several clinical studies. However, the occurrence of severe toxicity (e.g., dose limiting GI toxicities, such as oral mucosistis, gastric bleeding, nausea and vomiting, abdominal pain, leukopenia, thrombocytopenia, and the like) when administering such glutamine antagonists at therapeutic dose levels has hampered their clinical development.

Prior attempts to mitigate the severe toxicity associated with glutamine antagonists, such as DON, have been unsuccessful. For example, dividing daily dosing and administering of DON every four to six hours apparently doubled DON's toxicity potential (MgGill, et al., 1957). In another example, development of a treatment involving DON dosed with glutaminase to decrease plasma glutamine so that the DON dose could be reduced was halted after publication of a clinical trial.

SUMMARY

The presently disclosed subject matter provides prodrugs of glutamine antagonists, and pharmaceutically acceptable salts and esters thereof. In some aspects, the presently disclosed subject matter provides a prodrug of a glutamine antagonist, or a pharmaceutically acceptable salt, the prodrug having a structure of formula (I):

[Image Omitted]

wherein: X is selected from the group consisting of a bond, —O—, and —(CH₂)_n—, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; R₁is selected from the group consisting of H and a first prodrug-forming moiety capable of forming a salt or an ester; and R₂is H or a second prodrug-forming moiety capable of forming an amide linkage, a carbamate linkage, a phosphoramidate linkage or a phosphorodiamidate linkage with the nitrogen adjacent to R₂; R₂′ is selected from the group consisting of H, C₁-C₆alkyl, substituted C₁-C₆alkyl, or R₂and R₂′ together form a ring structure comprising —C(═O)-G-C(═O)—, wherein G is selected from the group consisting of C₁-C₈alkylene, C₁-C₈heteroalkylene, C₅-C₈cycloalkylene, C₆-C₁₂arylene, C₅-C₁₄heteroarylene, bivalent C₄-C₁₀heterocycle, each of which can be optionally substituted; or R₁and R₂′ together form a 4- to 6-membered heterocylic ring comprising the oxygen atom adjacent to R₁and the nitrogen atom adjacent to R₂′; provided that the compound has at least one prodrug-forming moiety selected from the group consisting of the first and the second prodrug-forming moieties.

In some aspects, the presently disclosed subject matter provides a prodrug of a glutamine antagonist, or a pharmaceutically acceptable salt, the prodrug having a structure of formula (IIA) or formula (IIB):

[Image Omitted]

wherein:

R1 is selected from the group consisting of H and C1-6 alkyl;

R11 is selected from the group consisting of H, methyl, isopropyl, sec-butvl, benzyl, p-hydroxybenzyl —CH2CH(CH3)₂, —CH2OH, —CH(OH)CH3, —CH2-3-indoyl, —CH2COOH, —CH2CH2COOH, —CH2C(O)NH2, —CH2CH2C(O)NH2, —CH2SH, —CH2CH2SCH3, —(CH2)₄NH2, —(CH2)₃NHC(═NH)NH2, and —CH2-3-imidazoyl;

R12 is selected from the group consisting of H, C1-4 alkyl, and —C(═O)R₁₃; and

R13 is C1-4 alkyl.

In some aspects, the presently disclosed subject matter provides a prodrug of a glutamine antagonist, or a pharmaceutically acceptable salt, the prodrug having a structure of formula (III):

[Image Omitted]

wherein:

R₁is selected from the group consisting of H and C_1-6alkyl;

R₃and R₄are independently selected from the group consisting of H, C₁-C₆alkyl, substituted C₁-C₆alkyl, aryl, and substituted aryl; and

R₁₀is C_1-6alkyl.

In other aspects, the presently disclosed subject matter provides a pharmaceutical composition comprising a compound of any one of formula (I), formula (IIA), formula (IIB), or formula (III), and a pharmaceutically acceptable carrier, diluent, or excipient.

In certain aspects, the presently disclosed subject matter provides a method for treating a disease or a condition, the method comprising administering to a subject in need of treatment thereof, a compound of any one of formula (I), formula (IIA), formula (IIB), or formula (III), or a pharmaceutical composition thereof, in an amount effective for treating the disease or condition. In still other aspects, the presently disclosed subject matter provides the use of a compound of any one of formula (I), formula (IIA), formula (IIB), or formula (III), or a pharmaceutical composition thereof, for treating a disease or condition. In some embodiments, the disease or condition is selected from the group consisting of an infection, cancer, an autoimmune disease, an inflammatory disease, and a neurodegenerative or neurological disease.

In yet another aspect, the presently disclosed subject matter provides a compound of any one of formula (I), formula (IIA), formula (IIB), or formula (III), or a pharmaceutically composition thereof, for use as a medicament.

In yet another aspect, the presently disclosed subject matter provides a compound of any one of formula (I), formula (IIA), formula (IIB), or formula (II), or a pharmaceutically composition thereof, for use in the treatment of a disease or condition, preferably the disease or condition is selected from the group consisting of an infection, cancer, an autoimmune disease, an inflammatory disease, and a neurodegenerative or neurological disease.

In yet another aspect, the presently disclosed subject matter provides a compound of any one of formula (I), formula (IIA), formula (IIB), or formula (III), or a pharmaceutically composition thereof, for use in the treatment of the excess and/or aberrant glutamine activity.

Applicant has found that compounds of the disclosure having formula (I), formula (IIA), formula (IIB), and formula (III) are stable in plasma, liver microsomes, liver tissue, and gastrointestinal tissue, yet these compounds are cleaved in tumor cells to liberate DON in tumor tissue. The unexpected tumor-targeted properties of compounds having formula (I), formula (IIA), formula (IIB), and formula (III) result in a surprising improvement in therapeutic index for treating cancer with DON and provide the maximum therapeutic benefit to a subject in need of such treatment.

Applicant has also found unexpectedly that compounds of the disclosure having formula (I), formula (IIA), formula (IIB), and formula (III) exhibit unexpected enhanced CSF to plasma partitioning after administration, making them uniquely useful for the treatment of CNS cancers such as glioblastoma, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor and metastatic cancer that has spread to the central nervous system (CNS).

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1 is an illustration showing exemplary amino (R) and carboxy (R′) modifications made to DON in an attempt to synthesize various slow release DON prodrugs.

FIG. 2A and FIG. 2B are illustrations demonstrating the chemical stability challenges encountered when attempting to synthesize DON prodrugs, including stability issues presented by DON esters (FIG. 2A), and DON compounds possessing a free terminal carboxylate (FIG. 2B).

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D are bar graphs demonstrating the failed chemistry and poor pharmacokinetics of certain attempts to design DON prodrugs. FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D show that most DON prodrugs having a free carboxylate group exhibited negligible exposure compared to DON (except for 26, which showed some release; FIG. 3A) when administered orally in mice, including 29 (FIG. 3B), 23 (FIG. 3C) and 30 (FIG. 3D), which showed little DON release in mouse plasma upon oral administration. Note that release of DON prodrug LTP073 was below the limit of quantitation in all mouse plasma samples at 30 minutes and 5 hours after dosing (not shown).

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D are line graphs showing percentages of DON released in plasma after 0.5 h and 5 h following administration of exemplary DON-releasing as compared to DON. FIG. 4A and FIG. 4B show the results of a pharmacokinetic study in mice showing plasma DON concentrations following administration of DON (1.6 mg/kg p.o.) and DON prodrugs dosed at 1.6 mg/kg equivalent of DON, such as 22 (FIG. 4A; 2.5 mg/kg p.o.) and 25 (FIG. 4B; 2.9 mg/kg p.o.). Compound 22 showed lesser release at 0.5 h (65% of DON), however, the levels were similar at 5 h (almost 100%) to that of DON. FIG. 4C and FIG. 4D show the results of a pharmacokinetic study in mice showing plasma DON concentrations following administration of DON (1.6 mg/kg i.p.) and DON prodrugs 17 (FIG. 4C: 3.5 mg/kg p.o.) and 6 (FIG. 4D: 3.7 mg/kg p.o.).

FIG. 5 is a bar graph showing the stability of DON prodrug 25 in phosphate-buffered saline (PBS) and simulated gastric fluid (SGF) after 30 min and 60 min.

FIG. 6 is a bar graph showing the results of an in vitro metabolic stability screen of DON prodrug 25 in mouse, monkey, pig, and human plasma after 30 min and 60 min. 25 was found to be unstable in plasma in all tested species as both the ethyl ester and leucine ester were hydrolysed by plasma esterases.

FIG. 7 is an illustration showing the generic structure of certain cell targeting DON prodrugs that comprise an isopropyl ester carboxy modification.

FIG. 8 is a bar graph showing the results of an in vitro metabolic stability screen of cell targeted phosphamide DON prodrug 4 in mouse, monkey, pig, and human plasma after 30 min and 60 min. Compound 4 was found to be unstable in mouse plasma, but stable in the plasma of all other tested species.

FIG. 9 is a bar graph showing the results of an in vitro metabolic stability screen of DON prodrug 7 in mouse, monkey, pig, and human plasma after 30 min and 60 min. Compound 7 was found to be unstable in mouse plasma, but stable in plasma of all other tested species.

FIG. 10 is a bar graph showing the results of an in vitro metabolic stability screen of DON prodrug 9 in dog, mouse, monkey, pig, and human plasma after 30 min and 60 min. Compound 9 was found to be unstable in plasma off all tested species. Metabolic identification suggested hydrolysis of leucine, but stable isopropyl ester for specific release inside lymphoid cells.

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E and FIG. 11F are bar graphs demonstrating the results of an ex vivo study comparing the accumulation of DON and DON prodrugs in plasma, blood cells, red blood cells (RBC), and peripheral blood mononuclear cells (PBMC) after 30 minutes of incubation of 20 μM of DON (FIG. 11A) and DON prodrugs 4 (FIG. 11B), 7 (FIG. 11C), 9 (FIG. 11D), 13 (FIG. 11E) and 14 (FIG. 1 IF) in whole human blood;

FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D are bar graphs demonstrating the results of ex vive studies comparing the accumulation of DON and DON prodrugs in Cynomolgous monkey plasma, blood cells, RBC and PBMC after 30 minutes of incubation of 20 μM DON (FIG. 12A) and DON prodrugs, including 20 μM 4 (FIG. 12B), 7 (FIG. 12C) and 9 (FIG. 12C), in monkey whole blood samples.

FIG. 13A, FIG. 13B, and FIG. 13C are bar graphs demonstrating the results of ex vivo studies comparing the accumulation of DON and DON prodrugs in pig plasma, blood cells, RBC and PBMC after 30 minutes of incubation of 20 μM DON (FIG. 13A) and DON prodrugs, including 20 μM 7 (FIG. 13B) and 9 (FIG. 13C), in pig whole blood samples.

FIG. 14 is a line graph showing that daily high dose treatment leads to weight loss in DON treated mice. No weight loss was observed in 25 treated mice.

FIG. 15 is a tableshowing that daily high dose treatment leads to pan-cytopeniain DON treated mice. Mice treated with 25 exhibited some leukopenia.

FIG. 16 is a table showing that renal and liver function in mice treated with either high dose DON or 25 was unaffected.

FIG. 17A, and FIG. 17B are line graphs showing that 25 and DON treatment cure lymphoma; DON mice die from treatment with DON while 25 is well tolerated.

FIG. 18A and FIG. 18B are bar graphs and FIG. 18C is an illustration showing that compound 25 (FIG. 18A) reduces C_max; (FIG. 18B) enhances tumor to gut ratio; and (FIG. 18C) causes less GI toxicity in mice vs DON at equimolar doses in mice. Ova vaccinia virus infection was given followed by daily treatment with vehicle (Veh), DON (0.8 mg/kg), and 25 (0.8 mg/kg equivalent) for 5 days. At 1 hr after last dose, animals were sacrificed, small intestines removed, perfused with DPBS pH 7.4, and rolled longitudinally using the “swiss role” technique, and fixative for paraffin wax embedding. Upper panels show hematoxylin and eosin (H&E) stained sections of jejunum-ileum at 4× magnification; lower panel shows same sections at 10× magnification. DON caused significant inflammation, consisting of multifocal crypt distortion (left circle on the bottom left picture) and dilatation with loss of columnar epithelial morphology (rightcircle circle on the bottom left picture). DON prodrug looked more similar to vehicle-treated mice, with slightly hypercellular crypts, but no distortion (left circle on the bottom right picture) and normal columnar morphology (right circle on the bottom right picture).

FIG. 19A and FIG. 19B are tables showing that no bone marrow suppression was observed (FIG. 19A) following 25 at two 14-day effective dosing schemes; (FIG. 19B) employing long term 25 dosing schemes (48 days).

FIG. 20 is an illustration showing exemplary structures of DON and DON-based prodrugs.

FIG. 21A is a bar graph and FIG. 21B is a line graph showing that DON (1) inhibits glutamine metabolism and glioblastoma multiform (GBM) tumor growth in vivo. FIG. 21A shows compound DON (1) (0.8 mg/kg, i.p.) inhibited glutamine metabolism as evidenced by increased endogenous glutamine concentrations in flank GBM tumors 2 hours post-administration relative to vehicle-treated controls; *p<0.05. FIG. 21B shows in efficacy studies, compared to Day 0 baseline, vehicle-treated mice exhibited significant growth of flank GBM tumors during the course of the experiment. By contrast, systemic administration of 1 (0.8 mg/kg, i.p, q.d. days 1-6) caused a dramatic reduction in tumor size; ***p<0.001, ****p<0.0001.

FIG. 22A and FIG. 22B are line graphs and FIG. 22C is a table showing in vivo brain and plasma pharmacokinetics of compound DON (1) following oral administration of DON (1) and 14b in mice. 1 and 14b were dosed in mice at 0.8 mg/kg equivalent, via oral gavage and plasma and brain concentrations of compound 1 were evaluated via LC/MS. Oral administration of compound 1 and 14b exhibited similar plasma and brain pharmacokinetic profiles due to complete and rapid metabolism of 14b to 1 in mouse plasma.

FIG. 23A is a bar graph, FIG. 23B is a bar graph, and FIG. 23C is a table showing in vivo pharmacokinetics of DON following intravenous (i.v.) administration of DON (1) and 14 in monkey plasma and cerebrospinal fluid (CSF). 1 and 14b were dosed in two pigtail macaques at 1.6 mg/kg equivalent of 1 via i.v. administration and plasma (0.25-6 h) and CSF (30 min) concentrations of DON were evaluated via LC/MS. Relative to 1, 14b delivered substantially lower DON plasma concentration. The reverse was observed in CSF, where 14b delivered significantly higher DON CSF concentrations, achieving a 10-fold enhanced CSF to plasma ratio at 30 minute post dose.

FIG. 24 is a bar graph showing species specific plasma stability of 14b; 14b is stable in plasma of human, pig, dog and monkeys, but rapidly metabolized in mice;

FIG. 25 is an illustration showing exemplary structures of DON and DON-based prodrugs 25, 9, 38 and 60; different N-amino acid promoeities (e.g., leucine, tryptophan) provide differential plasma and microsomal stability.

FIG. 26A, FIG. 26B, FIG. 26C, and FIG. 26D are bar graphs showing in vitro plasma stability of DON prodrugs 9, 25, 38 and 60. Metabolism occurs via removal of the N-protecting group; both ethyl and isopropyl esters are stable in plasma of pigs and humans.

FIG. 27A, FIG. 27B, FIG. 27C, and FIG. 27D are bar graphs showing in vitro liver microsomal stability of DON prodrugs 9, 25, 38 and 60; all prodrugs showed moderate-high stability in human and pig microsomes.

FIG. 28A, FIG. 28B, FIG. 28C. FIG. 28D, FIG. 28E, FIG. 28F, FIG. 28G, FIG. 28H. FIG. 28I, and FIG. 28J are bar graphs showing ex-vivo studies in whole human and pig blood of 9, 25, 38 and 60; DON prodrugs selectively deliver DON to PBMCs in both humans and pigs vs plasma; compared to DON, the PBMC/plasma ratio was enhanced 10-100+ fold;

FIG. 29A FIG. 29B, FIG. 29C, FIG. 29D and FIG. 29E are line graphs showing pig in vivo studies with DON prodrugs of 9, 25, 38 and 60; DON prodrugs selectively deliver DON to PBMCs vs plasma; compared to DON, the PBMC/plasma ratio was enhanced 6- to 10-fold.

FIG. 30A, FIG. 30B, and FIG. 30C are bar graphs showing plasma stability of compound Methyl-POM 14b and its derivatives.

FIG. 31 is an illustration showing exemplary structures of N-acylalkyloxy DON-based prodrug analogs for intracellular targeting and brain penetration; the addition of steric bulk to the “bridge” might result in a slower hydrolysis.

FIG. 32A, FIG. 32B, and FIG. 32C are line graphs showing pig i.v. studies with DON prodrugs; in vivo in pigs DON prodrugs selectively deliver DON to PBMCs vs DON.

FIG. 33 is a bar graph and two tables showing PBMC to plasma AUC(_0-t) ratio comparison DON prodrugs achieve >6 fold better ratio (based on AUC_0-t) vs DON.

FIG. 34A is a line graph and FIG. 34B is a bar graph showing monkey IV studies with DON prodrug 14b shows lower plasma but higher CSF concentrations achieving ˜6-7 fold>CSF/plasma ratio vs DON.

FIG. 35 is a bar graph showing CSF to plasma ratio at 1 h after i.v. infusion with compounds 1, 9, 14b, 25 and 60.

FIG. 36 is an illustration showing clinical findings for DON (1) and 38 over 5 days observation period DON-treated pigs exhibited more adverse clinical signs versus 38; DON (1.6 mg/kg) and 38 (1.5 mg/kg) doses were chosen to provide equivalent PBMC exposure; drugs were dosed i.v, for 2 h/day for 5 days (n=2 pigs); close clinical monitoring was performed to evaluate drug toxicity (e.g. lethargy, anorexia, diarrhea, GI-bleeding, etc); after 5 days of dosing, pigs were euthanized and tissues were harvested for PK and histopathology.

FIG. 37A, FIG. 37B, FIG. 37C, and FIG. 37D are illustrations showing that macroscopically, DON-treated pigs exhibited more severe stomach toxicity versus 38; histopathologic toxicity scoring for gastro-intestinal and other tissues is pending; and

FIG. 38 is a bar graph showing that at equivalent PBMC exposures, 38 administration delivers less DON to GI tissues versus DON administration; DON administration appeared to cause more adverse clinical effects versus 38.

FIG. 39 is a line graph illustrating that administration of DON markedly inhibits lymphoma growth in a EL4 mouse lymphoma model.

FIG. 40 is a bar graph illustrating that administration of DON has a modest effect on inhibiting melanoma growth, which is a not a bone marrow derived tumor.

FIG. 41A is a Kaplan-Meier graph and FIGS. 41B-E are line graphs showing that compound 25 (5 day dosing starting on day 7) is superior to CB-839 (a glutamase inhibitor) (30 day dosing starting day 1) in CT26 tumor model. These unexpected results suggest that 25 can be used as a single agent to treat colon cancer.

FIG. 42 is a line graph illustrating that compound 25 (4 days starting on day 6) is superior to CB-839 (continuous twice daily dosing starting on day 1 prior to engraftment) in a CT26 tumor model. FIG. 42 shows mice received daily compound 25 (1.9 mg/kg) on days 6-9 vs BID CB-839 on days 1-15. These results again demonstrate the compound 25 is superior to CB-839 and unexpectedly that compound 25 can be used as a single agent to treat cancers such as colon cancer.

FIG. 43 is a line graph illustrating that compound 25 (daily days 7-22) is superior to CB-839 (continuous twice daily dosing days 1-29) in a 4T1 breast cancer model. Mice received daily compound 25 (1.0 mg·kg/d) for days 7-22 as compared to BID CB-839 for days 1-29. These results again demonstrate the compound 25 is superior to glutamase inhibitor CB-839 and unexpectedly that compound 25 can be used as a single agent to treat cancers such as breast cancer.

FIGS. 44A-D and F are line graphs and FIG. 44E is a Kaplan-Meier graph illustrating that compound 25 dosing of 1 mg/kg following by 0.3 mg/kg leads to a complete and durable response in the MC38 tumor model. These results again demonstrate the compound 25 unexpectedly that compound 25 can be used as a single agent to treat cancers such as colon cancer

FIGS. 45A, B, and D are line graphs and FIG. 45C is a Kaplan-Meier graph illustrating that compound 25 gives a robust response and improved overall survival as compared to vehicle-treated animals in the CT26 colon cancer model.

FIGS. 46A, B, and D are line graphs and FIG. 46C is a bar graph illustrating that compound 25 provides a robust response and improved overall survival the 4T1 breast cancer model.

FIGS. 47A-47G are line graphs illustrating that mice treated with compound 25 when administered as a single agent prevents colon cancer cell proliferation upon rechallenge, demonstrating that compound 25 unexpectedly immunostimulates the mouse.

FIGS. 48A and 48C are line graphs and FIGS. 48B and 48D are Kaplan-Meier graphs showing that compound 25 inhibits colon cell proliferation and extends the survival of mice in the MC38 model.

FIG. 49A is an illustration of mice and FIG. 49B is a line graph illustrating that compound inhibits tumor growth. FIG. 49A shows mice after 30 days inoculation (8 days drug free). FIG. 49B shows tumor area vs. days post injection with 4T1 tumor cells. Mice were treated with compound 25 1 mg/kg every day (from d5-d22) or vehicle. 4T1 tumor cells (0.1 million) were injected into the mammary fat pads of 8-week-old female BALB/c mice. Mice received vehicle (PBS) or 1 mg/kg compound 25 daily from day 5 to day 22. Photos were taken on day 30 after tumor inoculation (FIG. 57A). Tumor volume was measured 2-3 times weekly until WT mice were sacrificed (when size reached to 20 mm length or necrosis occurred). Day 0: 100K 4T1 cells s.c. in 4th mammary pad. Day 5-22: Daily compound 25. The results show that compound 25 inhibited the growth of mammary carcinoma tumor cells.

FIG. 50A is an illustration of mice and FIG. 50B is a bar graph illustrating that glutamine analogue (6-diazo-5-oxo-L-norleucine (L-DON) derivative compound 25 referred to in FIGS. 50A and 50B as “JHU”) inhibits tumor growth. 4T1-Luc tumor cells (0.1 million) were injected into the mammary fat pads of 8-week-old female BALB/c mice. Mice received vehicle (PBS) or 1 mg/kg compound 25 daily from day 7. Anti-PD1 (5 mg/Kg) was administered on day 5, 8, and 12. Mice carrying 4T1-luc tumors are injected with Luciferin to measure luminescence from the tumor. IVIS imaging were taken on day 13.

FIG. 51A is a line graph and FIG. 51B is a bar graph illustrating that compound 25 inhibits tumor growth. FIG. 51B shows tumor weight (mg) on harvest day 21, 4T1-Luc tumor cells (0.1 million) were injected into the mammary fat pads of 8-week-old female BALB/c mice. Mice received vehicle (PBS) or 1 mg/kg compound 25 daily from day 7 to day 16. Anti-PD1 (5 mg/Kg) was administered on day 5, 8, 12 and 17. Tumor volume was measured 2-3 times weekly until tumors were evaluated for tumor infiltrating cells on day 21. Tumor weights were measured on day 21. On day 21, the PD1 group tumor size looks like it was reduced, however, the result was not due to a tumor size reduction, but rather because one of the large tumor mice died.

FIG. 52 is a line graph showing that compound 25 is efficacious in the MC38 tumor model. These data show unexpectedly that compound 25 is highly effective at killing colon cancer cells as a single agent.

FIG. 53 is a line graph showing compound 25 and DON are efficacious in the EL4 mouse lymphoma model. These data show unexpectedly that compound 25 is highly effective at killing lymphoma cancer cells as a single agent.

FIG. 54 is a Kaplan-Meier graph showing that DON treated mice die of toxicity, and compound 25 treated mice show no signs of toxicity in the EL4 mouse lymphoma model. These results show unexpectedly that compound 25 is highly effective at killing cancer cells but with significantly improved tolerability compared to DON.

FIG. 55 a line graph showing compound 25 administered as a single agent is more efficacious than oxaliplatin in the MC38 tumor model. These results show unexpectedly that compound 25 (referred to as “JHU”) is highly effective at killing colon cancer cells as a single agent.

FIGS. 56A-C are bar graphs showing compound 60 preferentially delivers DON to P493 tumor cells versus plasma.

FIG. 57 is a line graph showing subcutaneous administration of compound 60 unexpectedly promoted tumor regression in the MC38 tumor model as a single agent.

FIG. 58A is a line graph demonstrating different DON plasma profiles in Monkey for DON and compound 14b.

FIG. 58B is a bar graph showing that compound 14b exhibited enhanced CSF:plasma ratio of DON in Monkey.

FIG. 59A is a line graph demonstrating different DON plasma profiles in swine for DON, compound 14b and compound 47.

FIG. 59B is a bar graph showing that compounds 14b and 47 exhibited enhanced CSF delivery of DON at 60 min post-administration in swine

FIG. 59C is a bar graph showing that compounds 14b and 47 exhibited enhanced CSF:plasma ratio of DON at 60 min post-administration in swine.

The patent or application file contains at least one figure executed in color. Copies of this patent or patent application publication with color figures will be provided by the Office upon request and payment of the necessary fee.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

1. Prodrugs of Glutamine Antagonists

DON is an antibiotic that was isolated from Streptomyces in 1953. DON is a diazo analog of L-glutamine, which interferes in various reactions in the synthesis of nucleic acids and proteins in which L-glutamine donates nitrogen, blocks various glutamine recognizing enzymes, such as glutaminase, modulates brain glutamate levels, and is involved in energy metabolism, amongst others.

One strategy to improve the therapeutic index of DON for various treatment regimes, including GBM therapy, would be to increase its brain exposure while limiting its systemic exposure and thus toxicity (Upadhyay, 2014). The prodrug approach is a well-established strategy to alter the pharmacokinetic and tissue distribution of drug molecules, however synthetically this approach is challenging with DON. Given that DON's labile diazo group is critical to its glutamine antagonizing activity, the addition of promoeities must be performed under mild conditions to preserve the diazo ketone group.

The presently disclosed subject matter provides novel compositions of matter wherein pro-moieties have been added to glutamine antagonists, such as 6-diazo-5-oxo-norleucine (DON), and aza-serine.

The presently disclosed prodrugs of glutamine antagonists were prepared by masking the amine and/or the carboxylate functionalities to alter the pharmacokinetics of DON providing slower release kinetics and cellular targeting to enhance tolerability. Further, the presently disclosed prodrugs, in some embodiments, selectively target the active glutamine antagonists to specific cells or provide a slower release of DON and thus decrease the toxicity of the drug molecule.

The presently disclosed subject matter demonstrates that masking both the α-amino group and the carboxy-functionality to be derivatized enhances prodrug stability and oral bioavailability. The presently disclosed prodrugs also exhibit a stability that is comparable to free DON.

Structures of representative DON prodrugs are provided in Table 1.

TABLE 1

Structures of Representative DON Prodrugs

IOCB No./

Compound No.
Structure
MW

Compound 1 (DON)

[Image Omitted]

171.15

Compound 3

[Image Omitted]

213.24

Compound 4 (or JAM317)

[Image Omitted]

445.41

Compound 6

[Image Omitted]

391.38

Compound 7

[Image Omitted]

564.53

Compound 9

[Image Omitted]

326.39

Compound 11

[Image Omitted]

439.55

Compound 13

[Image Omitted]

369.18

Compound 14a

[Image Omitted]

385.41

Compound 14b (or 5c)

[Image Omitted]

Compound 15

[Image Omitted]

371.39

Compound 17

[Image Omitted]

375.33

Compound 20

[Image Omitted]

199.21

Compound 22

[Image Omitted]

270.28

Compound 23

[Image Omitted]

343.42

Compound 25

[Image Omitted]

312.36

Compound 26

[Image Omitted]

385.50

Compound 28

[Image Omitted]

425.52

Compound 29

[Image Omitted]

329.31

Compound 30

[Image Omitted]

343.33

Compound 31

[Image Omitted]

357.37

Compound 32

[Image Omitted]

371.39

Compound 34

[Image Omitted]

385.42

Compound 35

[Image Omitted]

327.25

Compound 36

[Image Omitted]

355.30

Compound 38

[Image Omitted]

399.45

Compound 40

[Image Omitted]

413.47

Compound 42

[Image Omitted]

371.39

Compound 44

[Image Omitted]

2.44

Compound 47

[Image Omitted]

447.49

Compound 47a

[Image Omitted]

Compound 47b

[Image Omitted]

Compound 49

[Image Omitted]

357.36

Compound 51

[Image Omitted]

618.69

Compound 52

[Image Omitted]

660.73

Compound 56

[Image Omitted]

469.54

Compound 57

[Image Omitted]

511.58

Compound 59

[Image Omitted]

511.48

Compound 60

[Image Omitted]

464.19

A

[Image Omitted]

618.54

B

[Image Omitted]

602.54

C

[Image Omitted]

530.47

D

[Image Omitted]

334.38

E

[Image Omitted]

484.51

F

[Image Omitted]

525.51

G

[Image Omitted]

509.51

LTP 073

[Image Omitted]

255.23

JAM0351

[Image Omitted]

693.66

JAM0359

[Image Omitted]

679.63

Those skilled in the art will appreciate that the representative structures of the DON prodrugs shown in Table 1 combined with the guidance disclosed herein will enable those skilled in the art to synthesize prodrugs of other glutamine analogs, such as L-DONV, aza-serine, as are exemplified in the generic structures of formula (I). In other words, it should be understood that the prodrugs of other glutamine antagonists, such as L-DONV, aza-serine, can be synthesized with the same substituents R₁, R₂and R₂′ as the DON prodrugs shown in Table 1.

Accordingly, in one aspect the presently disclosed subject matter provides a prodrug of a glutamine antagonist, or a pharmaceutically acceptable salt or ester thereof, the prodrug having a structure of formula (I):

[Image Omitted]

wherein: X is selected from the group consisting of a bond, —O—, and —(CH₂)_n—, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; R₁is selected from the group consisting of H and a first prodrug-forming moiety capable of forming a salt or an ester; and R₂is H or a second prodrug-forming moiety capable of forming an amide linkage, a carbamate linkage, a phosphoramidate linkage or a phosphorodiamidate linkage with the nitrogen adjacent to R₂; R₂′ is selected from the group consisting of H, C₁-C₆alkyl, substituted C₁-C₆alkyl, or R₂and R₂′ together form a ring structure comprising —C(═O)-G-C(═O)—, wherein G is selected from the group consisting of C₁-C₈alkylene. C₁-C₈heteroalkylene, C₅-C₈cycloalkylene, C₆-C₁₂arylene, C₅-C₁₄heteroarylene, bivalent C₄-C₁₀heterocycle, each of which can be optionally substituted; or R₁and R₂′ together form a 4- to 6-membered heterocylic ring comprising the oxygen atom adjacent to R₁and the nitrogen atom adjacent to R₂′; provided that the compound has at least one prodrug-forming moiety selected from the group consisting of the first and the second prodrug-forming moieties.

As used herein, the term “amide linkage” comprises a structure represented by the formula:

[Image Omitted]

wherein R_vis selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, and substituted heteroaryl. 10089 As used herein, the term “carbamate linkage” comprises a structure represented by the formula:

[Image Omitted]

wherein R_wis selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, and substituted heteroaryl.

As used herein, the term “phosphoramidate linkage” comprises a structure represented by the formula:

[Image Omitted]

wherein R_xand R_x′ are each independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkylamine, substituted alkylamine, heteroaryl, and substituted heteroaryl.

As used herein, the term “phosphorodiamidate linkage” comprises a structure represented by the formula:

[Image Omitted]

wherein R_yand R_zare each independently selected from the group consisting of H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, —(CR₃R₄)_mZ, —CR₃R₄)_m-Q-Z, aryl, substituted aryl, alkylamine, substituted alkylamine, heteroaryl, substituted heteroaryl, and

[Image Omitted]

In some embodiments. X is —CH₂—, and n is 1.

In other embodiments, X is —O—. In some embodiments, the prodrug compound has both the first prodrug-forming moiety and the second prodrug-forming moiety. In some embodiments, the glutamine analog is a glutamine antagonist, i.e., the prodrug is a prodrug of a glutamine analog that antagonizes a glutamine pathway. Exemplary glutamine antagonists include, without limitation, 6-diazo-5-oxo-norleucine (DON), and aza-serine, and 5-diazo-4-oxo-L-norvaline (L-DONV).

In some embodiments, the presently disclosed subject matter provides a prodrug of DON. In some embodiments, the prodrug of DON has a structure of formula (I). In some embodiments, the presently disclosed subject matter provides a prodrug of L-DONV. In some embodiments, the prodrug of L-DONV has a structure of formula (I). In some embodiments, the presently disclosed subject matter provides a prodrug of azaserine. In some embodiments, the prodrug of azaserine has a structure of formula (I).

In some embodiments, R₁of formula (I) comprises a residue PRO₁of the prodrug-forming moiety, which, together with a basic moiety and the terminal hydroxyl group forms a salt.

In some embodiments, R₁of formula (I) comprises a residue PRO₁of the prodrug-forming moiety, which, together with an alkyl group and the oxygen of an adjoining hydroxyl group forms an ester.

In some embodiments, R₁of formula (I) comprises a residue PRO₁of the prodrug-forming moiety, which, together with an alkyl group and the nitrogen adjoining the R₂′ group, forms an azlactone or an oxazolidone.

In some embodiments. R₁of formula (I) is selected from the group consisting of H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkenyl, substituted cycloalkenyl, tri(hydrocarbyl)ammonium, and tetra(hydrocarbyl)ammonium. Preferred alkyl group, cycloalkyl group, alkenyl group, alkynyl group, and cycloalkenyl group substituents include alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.

In some embodiments, R₁of formula (I) is not H. In some embodiments, R₁of formula (I) is not H when R₂and R₂′ are H. In some embodiments, R₂and R₂′ of formula (I) are each H w % when and R₁is not H.

In some embodiments, R₁of formula (I) is selected from the group consisting of a C_1-6straight-chain alkyl, a substituted C_1-6straight-chain alkyl, a C_1-6branched alkyl, a substituted C_1-6branched alkyl, tri(C₁-C₈-alkyl)ammonium, tetra(C₁-C₈-alkyl)ammonium, triphenylammonium, tri(hydroxy-C₁-C₈-alkyl)ammonium, and tetra(hydroxy-C₁-C₈-alkyl)ammonium.

In some embodiments, R₁of formula (I) is selected from the group consisting of methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl, trimethylammonium, triethylammonium, tri(hydroxyethyl)ammonium, tripropylammonium, and tri(hydroxypropyl)ammonium. In some embodiments, R₁of formula (I) is methyl. In some embodiments, R₁of formula (I) is ethyl. In some embodiments, R₁of formula (I) is isopropyl.

In some embodiments, R₂of formula (I) comprises a residue PRO₂of the second prodrug-forming moiety, which, together with a carbonyl, oxy carbonyl, or phosphonyl group and the nitrogen of the adjoining NH, forms an amide, a carbamate, phosphoramidate, or phosphorodiamidate linkage.

In some embodiments, R₂of formula (I) comprises a moiety selected from the group consisting of an amino acid, an N-substituted amino acid, a peptide, a substituted peptide, a monocyclic ring, a substituted monocyclic ring, a bicyclic ring, a substituted bicyclic ring, a purine nucleoside, a substituted purine nucleoside, a pyrimidine nucleoside, and a substituted pyrimidine nucleoside.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein:

X is selected from the group consisting of a bond, —O—, and —(CH₂)_n—, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8;

R₁is selected from the group consisting of H, C_1-6alkyl, and substituted C_1-6alkyl;

R₂is an amino acid, an N-substituted amino acid, or —C(═O)—O—(CR₃R₄)_m—O—C(═O)—R₁₀;

R₂′ is selected from the group consisting of H, C₁-C₆alkyl, and substituted C₁-C₆alkyl:

each R₃and R₄are independently H. C₁-C₆alkyl, substituted C₁-C₆alkyl, aryl, substituted aryl, —(CR₃R₄)_m—NR₅R₆, or

[Image Omitted]

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8;

R₅and R % are independently H or alkyl; and

R₁₀is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein X is —CH₂—.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, having formula (I), wherein X is —O—.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein R₁is selected from the group consisting of methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl, trimethylammonium, triethylammonium, tri(hydroxyethyl)ammonium, tripropylammonium, and tri(hydroxypropyl)ammonium.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein:

R₂is selected from the group consisting of —C(═O)—Y—(CR₃R₄)_m—NR₅R₆, and —C(═O)—O—(CR₃R₄)_m—O—C(═O)—R₁₀;

wherein:

Y is —O— or a bond;

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; and

each R₃and R₄is independently H, C₁-C₆alkyl, substituted C₁-C₆alkyl, aryl, or substituted aryl:

R₁₀is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein Y is a bond; m is 1; R₅and R₆are each H.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein R₂is an amino acid.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein the amino acid is tryptophan.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein R₂is a N-acyl amino acid.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, wherein the amino acid is tryptophan.

In another embodiment, the disclosure provides a compound having formula (IIA):

[Image Omitted]

or a pharmaceutically acceptable salt thereof, wherein:

R₁is selected from the group consisting of H and C_1-6alkyl;

R₁₁is selected from the group consisting of H, methyl, isopropyl, sec-butyl, benzyl, p-hydroxybenzyl —CH₂CH(CH₃)₂. —CH₂OH, —CH(OH)CH₃, —CH₂-3-indoyl, —CH₂COOH, —CH₂CH₂COOH, —CH₂C(O)NH₂, —CH₂CH₂C(O)NH₂, —CH₂SH, —CH₂CH₂SCH₃, —(CH₂)₄NH₂, —(CH₂)₃NHC(═NH)NH₂, and —CH₂-3-imidazoyl;

R₁₂is selected from the group consisting of H, C_1-4alkyl, and —C(═O)R₁₃; and

R₁₃is C_1-4alkyl.

In another embodiment, the disclosure provides a compound having formula (IIB):

[Image Omitted]

or a pharmaceutically acceptable salt thereof, wherein:

R₁is selected from the group consisting of H and C_1-6alkyl;

R₁₁is selected from the group consisting of H, methyl, isopropyl, sec-butyl, benzyl, p-hydroxybenzyl —CH₂CH(CH₃)₂, CH₂OH, —CH(OH)CH₃, —CH₂-3-indoyl, —CH₂COOH, —CH₂CH₂COOH, —CH₂C(O)NH₂, —CH₂CH₂C(O)NH₂, —CH₂SH, —CH₂CH₂SCH₃, —(CH₂)₄NH₂, —(CH₂)NHC(═NH)NH₂, and —CH₂-3-imidazoyl;

R₁₂is selected from the group consisting of H, C_1-4alkyl, and —C(═O)R₁₃; and

R₁₃is C_1-4alkyl.

In another embodiment, the disclosure provides a compound having formula (IIA) or formula (IIB), or a pharmaceutically acceptable salt thereof, wherein R₁is selected from the group consisting of methyl, ethyl, and isopropyl.

In another embodiment, the disclosure provides a compound having formula (IIA) or formula (IIB), or a pharmaceutically acceptable salt thereof, wherein R₁₁is selected from the group consisting of methyl, sec-butyl, benzyl, —CH₂CH(CH₃)₂, —CH(OH)CH₃and —CH₂-3-indoyl. In another embodiment. R₁₁is —CH₂-3-indoyl, i.e.,

[Image Omitted]

In another embodiment, the disclosure provides a compound having formula (IIA) or formula (IIB), or a pharmaceutically acceptable salt thereof, wherein R₁₂is selected from the group consisting of H and —C(═O)R₁₃. In another embodiment, R₁₂is —C(═O)R₁₃and R¹³is methyl.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[Image Omitted]

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, which is:

[Image Omitted]

In another embodiment, the disclosure provides a compound having formula (III):

[Image Omitted]

or a pharmaceutically acceptable salt thereof, wherein:

R₁is selected from the group consisting of H and C_1-6alkyl;

R₃and R₁are independently selected from the group consisting of H, C₁-C₆alkyl, substituted C₁-C₆alkyl, aryl, and substituted aryl; and

R₁₀is C_1-6alkyl.

In another embodiment, the disclosure provides a compound having formula (III), or a pharmaceutically acceptable salt thereof, wherein R₁is selected from the group consisting of methyl, ethyl, and isopropyl.

In another embodiment, the disclosure provides a compound having formula (III), or a pharmaceutically acceptable salt thereof, wherein R₃is selected from the group consisting of methyl and phenyl; and R₄is H.

In another embodiment, the disclosure provides a compound having formula (III), or a pharmaceutically acceptable salt thereof, wherein R₃is H and R is selected from the group consisting of methyl and phenyl.

In another embodiment, the disclosure provides a compound having formula (III), or a pharmaceutically acceptable salt thereof, wherein R₁₀is selected from the group consisting of isopropyl and tert-butyl.

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[Image Omitted]

In another embodiment, the disclosure provides a compound having formula (I), or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[Image Omitted]

In another embodiment, the disclosure provides a compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

[Image Omitted]

In another embodiment, the disclosure provides a pharmaceutical composition comprising the compound of any one of formula (I), formula (hIA), formula (IIB), or formula (III), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

In another embodiment, the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject in need thereof a compound of any one of formula (I), formula (IIA), formula (IIB), or formula (III), or a pharmaceutically acceptable salt thereof. In another embodiment, compound of any one of formula (I), formula (IIA), formula (IIB), or formula (III), or a pharmaceutically acceptable salt thereof, is administered subcutaneously to the subject. In another embodiment, the compound of formula (I), formula (IIA), formula (IIB), or formula (III) is administered in combination with one or more additional anticancer agents. The term “anticancer agent” refers to radiotherapy, e.g., gamma-radiation, neutron beam radiotherapy, and/or any chemotherapeutic drug. In another embodiment, the compound of formula (I), formula (IIA), formula (IIB), or formula (III) is administered as the only anticancer agent to the subject, i.e., the compound of formula (I), formula (IIA), formula (IIB), or formula (III) is administered to the subject as a single anticancer agent. In another embodiment, R₁is C_1-6alkyl. In another embodiment, the cancer is selected from the group consisting of non-small cell lung cancer, melanoma, squamous cell carcinoma, kidney cancer, liver cancer, pancreatic cancer, colon cancer, triple negative breast cancer, and glioma. In another embodiment, the cancer is glioblastoma.

As used herein, the term “amino acid” includes moieties having a carboxylic acid group and an amino group. The term amino acid thus includes both natural amino acids (including proteinogenic amino acids) and non-natural amino acids. The term “natural amino acid” also includes other amino acids that can be incorporated into proteins during translation (including pyrrolysine and selenocysteine). Additionally, the term “natural amino acid” also includes other amino acids, which are formed during intermediary metabolism. e.g., omithine generated from arginine in the urea cycle. The natural amino acids are summarized in Table 2:

TABLE 2

Natural Amino Acids (Used For Protein Biosynthesis)

Amino acid
3 letter code
1-letter code

Alanine
ALA
A

Cysteine
CYS
C

Aspartic Acid
ASP
D

Glutamic Acid
GLU
E

Phenylalanine
PHE
F

Glycine
GLY
G

Histidine
HIS
H

Isoleucine
ILE
I

Lysine
LYS
K

Leucine
LEU
L

Methionine
MET
M

Asparagine
ASN
N

Proline
PRO
P

Glutamine
GLN
Q

Arginine
ARG
R

Serine
SER
S

Threonine
THR
T

Valine
VAL
V

Tryptophan
TRP
W

Tyrosine
TYR
Y

The natural or non-natural amino acid may be optionally substituted. In one embodiment, the amino acid is selected from proteinogenic amino acids. Proteinogenic amino acids include glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, serine, threonine, glutamine, asparagine, arginine, lysine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine and histidine. The term amino acid includes alpha amino acids and beta amino acids, such as, but not limited to, beta alanine and 2-methyl beta alanine. The term amino acid also includes certain lactam analogues of natural amino acids, such as, but not limited to, pyroglutamine. The term amino acid also includes amino acids homologues including homocitrulline, homoarginine, homoserine, homotyrosine, homoproline and homophenylalanine.

The terminal portion of the amino acid residue or peptide may be in the form of the free acid i.e., terminating in a —COOH group or may be in a masked (protected) form, such as in the form of a carboxylate ester or carboxamide. In certain embodiments, the amino acid or peptide residue terminates with an amino group. In an embodiment, the residue terminates with a carboxylic acid group —COOH or an amino group —NH₂. In another embodiment, the residue terminates with a carboxamide group. In yet another embodiment, the residue terminates with a carboxylate ester.

As disclosed hereinabove, the term “amino acid” includes compounds having a —COOH group and an —NH₂group. A substituted amino acid includes an amino acid which has an amino group which is mono- or di-substituted. In particular embodiments, the amino group may be mono-substituted. (A proteinogenic amino acid may be substituted at another site from its amino group to form an amino acid which is a substituted proteinogenic amino acid). The term substituted amino acid thus includes N-substituted metabolites of the natural amino acids including, but not limited to, N-acetyl cysteine, N-acetyl serine, and N-acetyl threonine.

For example, the term “N-substituted amino acid” includes N-alkyl amino acids (e.g., C_1-6N-alkyl amino acids, such as sarcosine, N-methyl-alanine, N-methyl-glutamic acid and N-tert-butylglycine), which can include C_1-6N-substituted alkyl amino acids (e.g., N-(carboxy alkyl) amino acids (e.g., N-(carboxymethyl)amino acids) and N-methylcycloalkyl amino acids (e.g., N-methylcyclopropyl amino acids)); N,N-di-alkyl amino acids (e.g., N,N-di-C_1-6alkyl amino acids (e.g., N,N-dimethyl amino acid)); N,N,N-tri-alkyl amino acids (e.g., N,N,N-tri-C_1-6alkyl amino acids (e.g., N,N,N-trimethyl amino acid)); N-acyl amino acids (e.g., C_1-6N-acyl amino acid); N-aryl amino acids (e.g., N-phenyl amino acids, such as N-phenylglycine); N-amidinyl amino acids (e.g., an N-amidine amino acid, i.e., an amino acid in which an amine group is replaced by a guanidino group).

The term “amino acid” also includes amino acid alkyl esters (e.g., amino acid C_1-6alkyl esters); and amino acid aryl esters (e.g., amino acid phenyl esters).

For amino acids having a hydroxy group present on the side chain, the term “amino acid” also includes O-alkyl amino acids (e.g., C_1-6O-alkyl amino acid ethers); O-aryl amino acids (e.g., O-phenyl amino acid ethers); O-acyl amino acid esters; and O-carbamoyl amino acids.

For amino acids having a thiol group present on the side chain, the term “amino acid” also includes S-alkyl amino acids (e.g., C_1-6S-alkyl amino acids, such as S-methyl methionine, which can include C_1-6S-substituted alkyl amino acids and S-methylcycloalkyl amino acids (e.g., S-methylcyclopropropyl amino acids)); S-acyl amino acids (e.g., a C_1-6S-acyl amino acid); S-aryl amino acid (e.g., a S-phenyl amino acid); a sulfoxide analogue of a sulfur-containing amino acid (e.g., methionine sulfoxide) or a sulfoxide analogue of an S-alkyl amino acid (e.g., S-methyl cystein sulfoxide) or an S-aryl amino acid.

In other words, the presently disclosed subject matter also envisages derivatives of natural amino acids, such as those mentioned above which have been functionalized by simple synthetic transformations known in the art (e.g., as described in “Protective Groups in Organic Synthesis” by T W Greene and P G M Wuts, John Wiley & Sons Inc. (1999)), and references therein.

Examples of non-proteinogenic amino acids include, but are not limited to: citrulline, hydroxyproline, 4-hydroxyproline, Pf-hydroxyvaline, omithine, O-amino alanine, albizziin, 4-amino-phenylalanine, biphenylalanine, 4-nitro-phenylalanine, 4-fluoro-phenylalanine, 2,3,4,5,6-pentafluoro-phenylalanine, norleucine, cyclohexylalanine, α-aminoisobutyric acid, α-aminobutyric acid, α-aminoisobutyric acid, 2-aminoisobutyric acid, 2-aminoindane-2-carboxylic acid, selenomethionine, lanthionine, dehydroalanine, γ-amino butyric acid, naphthylalanine, aminohexanoic acid, pipecolic acid, 2,3-diaminoproprionic acid, tetrahydroisoquinoline-3-carboxylic acid, tert-leucine, tert-butylalanine, cyclopropylglycine, cyclohexylglycine, 4-aminopiperidine-4-carboxylic acid diethylglycine, dipropylglycine and derivatives thereof wherein the amine nitrogen has been mono- or di-alkylated.

The term “peptide” refers to an amino acid chain consisting of 2 to 9 amino acids, unless otherwise specified. In preferred embodiments, the peptide used in the present invention is 2 or 3 amino acids in length. In one embodiment, a peptide can be a branched peptide. In this embodiment, at least one amino acid side chain in the peptide is bound to another amino acid (either through one of the termini or the side chain).

The term “N-substituted peptide” refers to an amino acid chain consisting of 2 to 9 amino acids in which one or more NH groups are substituted. e.g., by a substituent described elsewhere herein in relation to substituted amino groups. Optionally, the N-substituted peptide has its N-terminal amino group substituted and, in one embodiment, the amide linkages are unsubstituted.

In one embodiment, an amino acid side chain is bound to another amino acid. In a further embodiment, side chain is bound to the amino acid via the amino acid's N-terminus, C-terminus, or side chain.

Examples of natural amino acid sidechains include hydrogen (glycine), methyl (alanine), isopropyl (valine), sec-butyl (isoleucine), —CH₂CH(CH₃)₂(leucine), benzyl (phenvlalanine), p-hydroxybenzyl (tyrosine), —CH₂OH (serine), —CH(OH)CH₃(threonine), —CH₂-3-indoyl (tryptophan), —CH₂COOH (aspartic acid), —CH₂CH₂COOH (glutamic acid), —CH₂C(O)NH₂(asparagine), —CH₂CH₂C(O)NH₂(glutamine), —CH₂SH, (cysteine), —CH₂CH₂SCH₃(methionine), —(CH₂)₄NH₂(lysine), —(CH₂)₃NHC(═NH)NH₂(arginine) and —CH₂-3-imidazoyl (histidine).

Exemplary monocyclic rings and bicyclic rings include, without limitation, benzene, pyrimidines, and purines, and more generally aryl and heteroaryl rings. Exemplary heteroaryls include, but are not limited to, pyridvl, pyrimidinyl, pyrazinyl, furanyl, thienyl, pyrazolyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, pyrrolyl, imidazolyl, indolyl, indolinolyl, and imidazopyridazinyl. Aryls include phenyl (C₆), benzyl, naphthyl (C₁₀), and biphenyl (C₁₂). Exemplary pyrimidines include, without limitation, cytosine, thymine, and uracil. Exemplary purines include, without limitation, purine, adenine, N-substituted adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, and isoguanine. Exemplary purine nucleosides include, without limitation, adenine and guanine.

In some embodiments, R₄of formula (I) is selected from the group consisting of H, alkyl, —C(═O)—Ar, —C(═O)—Y—(CR₃R₄)_m—Ar, —C(═O)—Y—(CR₃R₄)_m—NR₅R₆, —P(═O)(OR₇)_n(NHR₉)_o, —C(═O)—Y—(CR₃R₄)_m—Ar—O—C(═O)—R₈, —C(═O)—Y—(CR₃R₄)_m—Ar—O—R₈, —C(═O)—O—(CR₃R₄)_m—O—C(O)—R₁₀, —C(═O)—O—R₉, —C(═O)—Y—(CR₃R₄)_m—Ar—O—C(═O)—Ar, and —C(═O)—Y—(CR₃R₄)_m—Ar—NR₅R₆; wherein: Y is —O— or a bond; m is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; each n and o is an integer from 0 to 2 provided that the sum of n and o is 2, R₃and R₄is independently H, C₁-C₆alkyl or substituted C₁-C₆alkyl, aryl or substituted aryl, —(CR₃R₄)_m—NR₅R₆, or

[Image Omitted]

each R₅and R₆is independently H, alkyl, —C(═O)—(CR₃R₄)_mH, —C(═O)—(NR₅R₆), or —C(═O)—(CR₃R₄)_m—NR₅R₆; each R₇is independently selected from the group consisting of H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, —(CR₃R₄)_m—Z, —(CR₃R₄)_m-Q-Z, wherein Q is a monosaccharide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and wherein Z is

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or wherein R₇together with the oxygen atom to which it is attached forms a purine or pyrimidine nucleoside; each R₉is independently selected from the group consisting of H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, —(CR₃R₄)_m—Z, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and

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wherein R₁and X are as defined above, provided that R₁is not H; each R₈is independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl, substituted aryl, heteroaryl, substituted heteroaryl: each R₁₀is independently alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, monosaccharide, acylated monosaccharide, aryl, substituted aryl, heteroaryl, substituted heteroaryl; and Ar is aryl, substituted aryl, heteroaryl, or substituted heteroaryl. It should be appreciated that in addition to substitutions on the amino group of Z, one or more substitutions R₃, R₄, R₅, and/or R₆can be made to the 5 or 6 membered rings of Z.

In particular embodiments Ar is

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In other particular embodiments, Ar is and

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In yet other particular embodiments. Ar is benzyl.

In particular embodiments, the prodrug compound of formula (I) is

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In some embodiments, R₂of formula (I) is —C(═O)—Y—(CR₃R₄)_m—NR₅R₆; wherein: (i) Y is a bond; m is 1, R₅and R₆are each H; (ii) Y is a bond; m is 1; R₅is H; R₆is —C(═O)—(CR₃R₄)_mH.