This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/555,726, filed on September 8, 2017. The foregoing application is incorporated by reference herein.

This invention was made with government support under Grant No.

CA109542 awarded by National Institutes of Health - National Cancer Institute. The government has certain rights in the invention. FIELD OF THE INVENTION

This invention relates to compounds and methods for the inhibition of indoleamine 2,3 -di oxygenase.

BACKGROUND OF THE INVENTION

Immune escape by tumors is a fundamental aspect of disease progression resulting from immunoediting of tumors as they interact with the host immune system (Schreiber et al. (2011) Science 331 : 1565-1570; Dunn et al. (2004) Immunity 21 : 137- 48). Key to this process from a therapeutic standpoint is that tumors are selected to engender a tolerogenic microenvironment that actively suppresses the ability of the immune system to mount an effective response (Zou, W. (2005) Nat. Rev. Cancer 5:263-74). Ongoing progress in understanding the cellular and molecular

mechanisms that govern the pathological state of tumor immune tolerance has revealed several protein targets which provide the potential for therapeutic intervention (Muller et al. (2006) Nat. Rev. Cancer 6:613-625). One central player is the immunomodulatory enzyme indoleamine 2,3 -di oxygenase- 1, IDOl, formerly known as IDO before the discovery of a second isoform (Metz et al. (2007) Cancer Res., 67:7082-7; Ball et al. (2009) Intl. J. Biochem. Cell Biol., 41 :467-471). IDOl can contribute to immune escape when expressed directly in tumor cells or when expressed in immunosuppressive antigen presenting cells such as tolerogenic dendritic cells or tumor associated macrophages (Muller et al. (2005) Cancer Res., 65:8065-8; Munn et al. (2007) J. Clin. Invest, 117: 1147-54). For both cases, experimental results suggest that IDOl inhibition may restore an effective antitumor immune response and thus provide a method to treat malignant diseases in

combination with chemotherapeutic agents and/or immunotherapy-based strategies

(Muller et al. (2005) Nat. Med., 11 :312-9). In fact, there are currently several drugs in clinical trials testing IDOl inhibition as a strategy for the treatment of cancer. Clearly, there is an interest and a need for further development of potent IDOl inhibitors to adequately address this therapeutic opportunity (Dolusic, E.F. (2013) Exp. Opin. Thera. Patents 23 : 1367-1381; Rohrig et al. (2015) J. Med. Chem.,

58:9421-9437).

SUMMARY OF THE INVENTION

In accordance with one aspect of the instant invention, prodrugs are provided. In a particular embodiment, the prodrugs are prodrugs of an indoleamine

2,3-dioxygenase (IDO) inhibitor, particularly an IDOl inhibitor. In a particular embodiment, the IDOl inhibitor is an O-alkylhydroxylamine. In a particular embodiment, the prodrug is a carbamate prodrug of the O-alkylhydroxylamine.

Compositions comprising at least one prodrug of the instant invention and at least one pharmaceutically acceptable carrier are also provided.

In accordance with one aspect of the instant invention, methods for inhibiting the activity of indoleamine 2,3-dioxygenase (e.g., IDOl or ID02) or tryptophan 2,3- dioxygenase (e.g., TD02) are provided. Method for inhibiting, treating, and/or preventing a disease or disorder associated with IDO activity in a subject are provided. Method for inhibiting, treating, and/or preventing immunosuppression in a subject are also provided. Methods for inhibiting, treating, and/or preventing cancer in a subject are also provided. Methods for inhibiting, treating, and/or preventing an ocular disease in a subject are also provided. DETAILED DESCRIPTION OF THE INVENTION

IDOl is an extrahepatic, tryptophan (Trp) metabolizing enzyme, which catalyzes the initial and rate-limiting step along the kynurenine pathway (Sono et al. (1996) Chem. Rev., 96:2841-2888; Botting, N.P. (1995) Chem. Soc. Rev., 24:401-12; Sono et al. (1980) Biochem. Rev., 50: 173-81). The oxidative metabolism of Trp by IDOl involves the coordination of molecular oxygen to a ferrous heme iron and its subsequent addition across the C-2/C-3 bond of the indole ring. Two alkylperoxy transition or intermediate states resulting from dioxygen insertion to C-2 or C-3 carbon of the indole ring have been proposed (Sono et al. (1996) Chem. Rev., 96:2841-2888; Chung et al. (2008) J. Amer. Chem. Soc, 130: 12299-12309; Lewis- Ballester et al. (2009) Proc. Natl. Acad. Sci., 106: 17371-17376; Capece et al. (2012) J. Phys. Chem. B, 1 16: 1401-1413). A new family of IDOl inhibitors, O- alkylhydroxylamine IDOl inhibitors, which mimic the alkylperoxy species have been generated (see, e.g., WO 2009/073620 and Malachowski et al. (2016) Eur. J. Med., 108:564-576).

In accordance with the instant invention, new prodrugs are provided. In a particular embodiment, the prodrugs are IDO inhibitors. In a particular embodiment, the prodrugs are of an O-alkylhydroxylamine IDO inhibitor. In a particular embodiment, the prodrugs are carbamate prodrugs of an O-alkylhydroxylamine IDO inhibitor. In a particular embodiment, the prodrug of the instant invention comprises

the n place of a hydrogen of the amine group of an O-alkylhydroxylamine. The instant invention also encompasses

pharmaceutically acceptable salts of the prodrugs. The instant invention also encompasses derivatives of the prodrugs. For example, the -CH2- in the above moiety may comprise one or more substituents (e.g., see substituents below for term "alkyl") or replaced with an alkyl (e.g., a lower alkyl such as a C1-C3 alkyl). As another example, the benzene ring may comprise one or more substituents (e.g., X and Y as described hereinbelow).

The prodrugs of the instant invention have significant IDO inhibitory activity only upon entry into a cell. Indeed, the prodrugs of the instant invention have little to no IDO inhibitory activity in a cell-free IDO enzyme assay. However, upon entry into a cell, the protective moiety added to the O-alkylhydroxylamine IDO inhibitor is removed by inherent cell processes. Thus, the prodrugs of the instant invention allow for better delivery and distribution of the O-alkylhydroxylamine IDO inhibitors. Examples of O-alkylhydroxylamine IDO inhibitors are provided in WO 2009/073620 and Malachowski et al. (Eur. J. Med. (2016) 108:564-576) (both incorporated herein by reference; particularly for the O-alkylhydroxylamine compounds disclosed therein). In a particular embodiment, the O- alkylhydroxylamine IDO inhibitor has the general formula Ar-X-ONH ₂ , wherein Ar comprises one or more aryl groups and X is an alkyl, cycloalkyl, or alkenyl.

Examples of O-alkylhydroxylamine IDO inhibitors include, without limitation: 0-(a-cyclopropyl)benzylhydroxylamine, 0-(3 -phenyl -2-propenyl)-l - hydroxyl amine, 0-(l,2,3,4-tetrahydro-l-naphthalene)-hydroxylamine, 0-(2,3- dihydro-lH-inden-2-yl)-hydroxylamine, O-phenethylhydroxylamine, 0-(2,3-dihydro- lH-inden- 1 -yl)hydroxylamine, 0-(3 -phenylpropyl)-hydroxylamine, O- (benzyl)hydroxylamine, 0-(3-nitrobenzyl)hydroxylamine, 0-(4- carboxybenzyl)hydroxylamine, 0-(4-cyanobenzyl)hydroxylamine, 0-(2- nitrobenzyl)hydroxylamine, 0-(4-nitrobenzyl)hydroxylamine, 0-(2- carboxybenzyl)hydroxylamine, 0-(3-carboxybenzyl)hydroxylamine, 0-(4- iodobenzyl)hydroxylamine, 0-(3-chlorobenzyl)hydroxylamine, 0-(3- bromobenzyl)hydroxylamine, 0-(3-iodobenzyl)hydroxylamine, 0-(3- trifluoromethylbenzyl)hydroxylamine, 0-(2-iodobenzyl)hydroxylamine, 0-(2- chlorobenzyl)hydroxylamine, 0-(4-fluorobenzyl)hydroxylamine, 0-(3 - fluorobenzyl)hydroxylamine, 0-(4-chlorobenzyl)hydroxylamine, 0-(2- fluorobenzyl)hydroxylamine, 0-(4-bromobenzyl)hydroxylamine, 0-(2- bromobenzyl)hydroxylamine, 0-(2-trifluoromethylbenzyl)hydroxylamine, 0-[(3- methylphenyl)-methyl]-hydroxylamine, 0-(4-nitrobenzyl)hydroxylamine, 0-(2- methoxybenzyl)hydroxylamine, 0-(4-trifluoromethylbenzyl)hydroxylamine, 0-(3 - methoxybenzyl)hydroxylamine, 0-(4-phenylbenzyl)hydroxylamine, 0-(4- isopropylbenzyl)hydroxylamine, 0-(2-hydroxybenzyl)hydroxylamine, 0-(4- methoxybenzyl)hydroxylamine, 0-(4-hydroxylbenzyl)hydroxylamine, 0-(2,3- dichlorobenzyl)hydroxylamine, 0-(4-chloro-3-trifluoromethyl)benzyl)hydroxylamine, 0-(2-fluoro-5-trifluoromethylbenzyl)hydroxylamine, 0-(2,4- dichlorobenzyl)hydroxylamine, 0-(2-chloro,4-iodobenzyl)hydroxylamine, 0-(2- chloro,4-fluorobenzyl)hydroxylamine, 0-(2-chloro,6-fluorobenzyl)hydroxylamine, O- (3,5-dichlorobenzyl)hydroxylamine, 0-(2-fluoro, 4- trifluoromethylbenzyl)hydroxylamine, 0-(2,4-difluorobenzyl)hydroxylamine, 0-(3,5- difluorobenzyl)hydroxylamine, 0-(2,5-dimethoxybenzyl)hydroxylamine, 0-(3,4- dichlorobenzyl)hydroxylamine, 0-(2-trifluoromethyl,4-fluorobenzyl)hydroxylamine, 0-(3,5-diflourobenzyl)hydroxylamine, 0-(3,5-dibromobenzyl)hydroxylamine, O- (3,5-bis(triflouromethyl-benzyl)hydroxylamine, and 0-(2-hydroxy-3- methoxybenzyl)hydroxylamine hydrochloride. In a particular embodiment, the O- alkylhydroxylamine IDO inhibitor is selected from the group consisting of O- (benzyl)hydroxylamine, 0-(4-iodobenzyl)hydroxylamine, 0-(3- chlorobenzyl)hydroxylamine, 0-(3-bromobenzyl)hydroxylamine, 0-(3- iodobenzyl)hydroxylamine, 0-(3-trifluoromethylbenzyl)hydroxylamine, 0-(2- iodobenzyl)hydroxylamine, 0-(2-chlorobenzyl)hydroxylamine, 0-(4- fluorobenzyl)hydroxylamine, 0-(3-fluorobenzyl)hydroxylamine, 0-(4- chlorobenzyl)hydroxylamine, 0-(2-fluorobenzyl)hydroxylamine, 0-(4- bromobenzyl)hydroxylamine, 0-(2-bromobenzyl)hydroxylamine, 0-(2- trifluoromethylbenzyl)hydroxylamine, 0-[(3-methylphenyl)-methyl]-hydroxylamine, 0-(4-nitrobenzyl)hydroxylamine, 0-(4-cyanobenzyl)hydroxylamine, 0-(2- methoxybenzyl)hydroxylamine, 0-(4-trifluoromethylbenzyl)hydroxylamine, 0-(3 - methoxybenzyl)hydroxylamine, 0-(4-phenylbenzyl)hydroxylamine, 0-(4- isopropylbenzyl)hydroxylamine, 0-(2-hydroxybenzyl)hydroxylamine, 0-(4- methoxybenzyl)hydroxylamine, 0-(4-hydroxylbenzyl)hydroxylamine, 0-(2,3- dichlorobenzyl)hydroxylamine, 0-(4-chloro-3-trifluoromethyl)benzyl)hydroxylamine, 0-(2-fluoro-5-trifluoromethylbenzyl)hydroxylamine, 0-(2,4- dichlorobenzyl)hydroxylamine, 0-(2-chloro,4-iodobenzyl)hydroxylamine, 0-(2- chloro,4-fluorobenzyl)hydroxylamine, 0-(3-chloro,5-fluorobenzyl)hydroxylamine, O- (2-chloro,6-fluorobenzyl)hydroxylamine, 0-(3-chloro,5-fluorobenzyl)hydroxylamine, 0-(3,5-dichlorobenzyl)hydroxylamine, 0-(2-fluoro, 4- trifluoromethylbenzyl)hydroxylamine, 0-(2,4-difluorobenzyl)hydroxylamine, 0-(3,5- difluorobenzyl)hydroxylamine, 0-(2,5-dimethoxybenzyl)hydroxylamine, 0-(3,4- dichlorobenzyl)hydroxylamine, 0-(2-trifluoromethyl,4-fluorobenzyl)hydroxylamine, 0-(3,5-diflourobenzyl)hydroxylamine, 0-(3,5-dibromobenzyl)hydroxylamine, O- (3,5-bis(triflouromethyl-benzyl)hydroxylamine, and 0-(2-hydroxy-3- methoxybenzyl)hydroxylamine hydrochloride. In a particular embodiment, the O- alkylhydroxylamine IDO inhibitor is 0-(4-iodobenzyl)hydroxylamine or 0-(3- chlorobenzyl)hydroxylamine. In a particular embodiment, the O-alkylhydroxylamine IDO inhibitor is 0-(3-chlorobenzyl)hydroxylamine. In a articular embodiment, the O-alkylhydroxylamine IDO inhibitor has the H ₂ formula: , wherein n is from 1 to 10 (particularly from 1 to 6 or from 1 to 3), and wherein X and Y are independently selected from the group consisting of hydrogen, halogen, trihalomethyl, nitro, carboxy, cyano, hydroxyl, haloformyl, aldehyde, amine, imine, nitrate, sulfhydryl, sulfide, sulfonyl, methoxy, alkyl, and aryl. In a particular embodiment, X and Y are independently selected from the group consisting of hydrogen, halogen and trihalomethyl. In a particular embodiment, at least one of X and Y is hydrogen. In a particular embodiment, neither X nor Y is hydrogen.

Examples of O-alkylhydroxylamine IDO inhibitors also include, without limitation, 0-(pyridin-2-ylmethyl)hydroxylamine, 0-(pyridin-3- ylmethyl)hydroxylamine, 0-(pyridin-4-ylmethyl)hydroxylamine, O- (benzo[d][l,3]dioxol-5-ylmethyl)hydroxylamine, 0-((5-chlorobenzo[b]thiophen-3- yl)methyl)hydroxylamine, 0-(naphthalen-2-ylmethyl)hydroxylamine, 0-(quinolin-6- ylmethyl)hydroxylamine, 0-((2,3 -dihydrobenzo[b] [ 1 ,4]dioxin-6- yl)methyl)hydroxylamine, 0-(chroman-2-ylmethyl)hydroxylamine, O- (benzo[d]thiazol-2-ylmethyl)hydroxylamine, 0-((4-methyl-2-phenylpyrimidin-5- yl)methyl)hydroxylamine, 0-(benzofuran-2-ylmethyl)hydroxylamine, O- (perfluorobenzyl)hydroxylamine, 0-(3-nitrophenethyl)hydroxylamine, O- (benzylhydryl)hydroxylamine, 0-(cyclohexyl(phenyl)methyl)hydroxylamine, 0-(l,2- diphenyl ethyl )hydroxylamine, 0-(2-morpholino-l -phenyl ethyl)hydroxylamine, O- ((3',4-dichlorobiphenyl-2-yl)methyl)hydroxylamine, 0-((3'4,4'-trichlorobiphenyl-2- yl)methyl)hydroxylamine, 0-((4-chloro-4'-(trifluoromethyl)biphenyl-2- yl)methyl)hydroxylamine, 0-(5-chloro-2-(pyrimidin-5-yl)benzyl)hydroxylamine, O- (5-chloro-2-(thiophen-2-yl)benzyl)hydroxylamine, 0-(5-chloro-2-(thiophen-3- yl)benzyl)hydroxylamine, 0-((4'-chlorobiphenyl-2-yl)methyl)hydroxylamine, 0-((4'- chlorobiphenyl-3-yl)methyl)hydroxylamine, 0-((4'-methylbiphenyl-3- yl)methyl)hydroxylamine, 0-((4'-methoxybiphenyl-3-yl)m ethyl )hydroxylamine, O- (3-(pyridine-4-yl)benzyl)hydroxylamine, 0-(2-(pyridine-4-yl-benzyl)hydroxylamine, 0-(naphthalen-l-ylmethyl)hydroxylamine, 0-((4,4'-dichlorobiphenyl-2- yl)methyl)hydroxylamine, 0-((4',5-dichlorobiphenyl-3-yl)methyl)hydroxylamine, O- ((4-dichlorobiphenyl-2-yl)methyl)hydroxylamine, 0-((4-chloro-4'-methoxybiphenyl- 2-yl)methyl)hydroxylamine, 0-((2',4-dichlorobiphenyl-2-yl)methyl)hydroxylamine, 0-(5-chloro-2-(lH-indol-5-yl)benzyl)hydroxylamine, 0-(biphenyl-3- ylmethyl)hydroxylamine, 0-(biphenyl-2-ylmethyl)hydroxylamine, 0-(3-tert- butyldimethylsilyloxy)-l-phenylpropyl)hydroxylamine, 0-(l,2,3,4- tetrahydronaphthalen-l-yl)hydroxylamine, 0-(2-cyclohexyl-l- phenylethyl)hydroxylamine, 0-(2-phenoxy-l -phenlethyl)hydroxylamine, 0-(2- (benzyloxy)-l -phenyl ethyl )hydroxylamine, 0-(l,3-diphenylpropyl)hydroxylamine,

0- (3-cyclohexyl-l-phenylpropyl)hydroxylamine, methyl(aminooxymethyl)benzoate, 2-(aminooxymethyl)-N-phenylaniline, 2-(aminooxymethyl)-N-benzylaniline, 3- (aminooxymethyl)-N-benzylaniline, 4-(aminooxy)-N-methyl-4-phenylbutanamide, 4- (aminooxy)-N-cyclohexyl-4-phenylbutanamide, methyl 4-(aminooxy)-4- phenylbutanoate, 2-(aminooxy)-2-phenylethanamine, 3-(aminooxy)-3-phenylpropan-

1 - amine, 2-(aminooxy)-N-methyl-2-phenylacetamide, tert-butyl 2-(aminooxy)-2- phenylethyl(methyl)carbamate, 2-(aminooxy)-N-methyl-2-phenylethanamine dihydrochloride, 2'-(aminooxymethyl)-4'-chloro-N,N-dimethylbiphenyl-4-amine, and methyl 2'-(aminooxymethyl)-4'-chlorobiphenyl-4-carboxylate.

As explained hereinabove, prodrugs of any of the O-alkylhydroxylamine listed hereinabove are encompassed by the instant invention. In a particular embodiment, the prodrugs are carbamate prodrugs of the O-alkylhydroxylamine. In a particular embodiment, the prodrug of the instant invention comprises the moiety

in place of a hydrogen of the amine group of the O- alk lhydroxylamine. In a particular embodiment, the prodrug has the formula:

, wherein n is from 1 to 10 (particularly from 1 to 6 or from 1 to 3), and wherein X and Y are independently selected from the group consisting of hydrogen, halogen, trihalomethyl, nitro, carboxy, cyano, hydroxyl, haloformyl, aldehyde, amine, imine, nitrate, sulfhydryl, sulfide, sulfonyl, methoxy, alkyl, and aryl. In a particular embodiment, X and Y are independently selected from the group consisting of hydrogen, halogen and trihalomethyl. In a particular embodiment, at least one of X and Y is hydrogen. In a particular embodiment, neither X nor Y is hydrogen. The instant invention also encompasses pharmaceutically acceptable salts of the prodrugs. The instant invention also encompasses derivatives of the prodrugs. For example, the -CH ₂ - in the above moiety can be substituted (e.g., see substituents below for term "alkyl") or replaced with an alkyl (e.g., a C1-C3 alkyl). As another example, the benzene ring may comprise one or more substituents (e.g., X and Y as described above).

The instant invention also encompasses compositions comprising at least one prodrug of the instant invention and at least one carrier. The prodrug may be a pharmaceutically acceptable salt thereof. The composition may further comprise at least one other therapeutic compound for the inhibition, treatment, and/or prevention of a disease or disorder (see, e.g., hereinbelow). Alternatively, at least one other therapeutic compound may be contained within a separate composition(s) with at least one pharmaceutically acceptable carrier. The present invention also encompasses kits comprising a first composition comprising at least one prodrug and a second composition comprising at least one other therapeutic compound for the inhibition, treatment, and/or prevention of the disease or disorder. The first and second compositions may further comprise at least one pharmaceutically acceptable carrier.

According to another aspect of the instant, methods for using the prodrugs of the instant invention are provided. Methods for decreasing or inhibiting the activity of indoleamine 2,3 -di oxygenase (e.g., IDOl or ID02) or tryptophan 2,3 -di oxygenase (e.g., TD02) are provided. In a particular embodiment, the method comprises contacting an indoleamine 2,3-dioxygenase or tryptophan 2,3-dioxygenase, particularly within a cell, with a prodrug (or an activated prodsrug) of the instant invention (optionally contained in a composition with a carrier). The cell may be within a subject, a tissue, or in a cell culture (e.g., in vitro). In a particular

embodiment, the methods decrease or inhibit the activity of IDOL

Methods of treating, inhibiting, and/or preventing indoleamine 2,3- dioxygenase (IDO) mediated immunosuppression or any disease or disorder associated with IDO activity in a subject in need thereof are also provided. In a particular embodiment, the method comprises administering a prodrug of the instant invention (optionally contained in a composition with a carrier (e.g., a

pharmaceutically acceptable carrier)) to the subject.

In a particular embodiment, disease or disorder associated with IDO activity is an ocular disease (see, e.g., WO 2016/100851, incorporated by reference herein). In a particular embodiment, the method comprises administering a prodrug of the instant invention (optionally contained in a composition with a carrier (e.g., a

pharmaceutically acceptable carrier)) to the subject, particularly the eye. In a particular embodiment, the ocular disease is characterized by abnormal/aberrant vascularization. In a particular embodiment the ocular disease is characterized by intraocular neovascularization. The intraocular neovascularization may be, without limitation, neovascularization of the optic disc, iris, retina, choroid, cornea, and/or vitreous humour. Examples of ocular diseases include, without limitation, glaucoma, pannus, pterygium, macular edema, macular degeneration (e.g., age-related macular degeneration), retinopathy (e.g., diabetic retinopathy, vascular retinopathy, retinopathy of prematurity), diabetic retinal ischemia, diabetic macular edema, retinal degeneration, retrolental fibroplasias, retinoblastoma, corneal graft

neovascularization, central retinal vein occlusion, pathological myopia, ocular tumors, uveitis, inflammatory diseases of the eye, and proliferative vitreoretinopathy. In a particular embodiment, the ocular disease is selected from the group consisting of retinopathy (e.g., retinopathy of prematurity, diabetic retinopathy (e.g., proliferative diabetic retinopathy)) and macular degeneration (e.g., wet macular degeneration).

In a particular embodiment, the immunosuppression is associated with cancer. The immunosuppression may be tumor-specific. In a particular embodiment, the cancer that may be treated using the compositions and methods of the instant invention include, but are not limited to, prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, stomach cancer (gastric cancer), endometrial cancer, brain cancer, glioblastoma, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head and neck cancer, throat cancer, skin cancer, melanoma, basal carcinoma, mesothelioma, lymphoma, leukemia, esophageal cancer, breast cancer,

rhabdomyosarcoma, sarcoma, lung cancer, small-cell lung carcinoma, non-small-cell carcinoma, adrenal cancer, thyroid cancer, renal cancer, bone cancer, and

choriocarcinoma. In a particular embodiment, the cancer forms a tumor. In a particular embodiment, the prodrugs of the instant invention may be administered with an anti-cancer treatment to increase the effectiveness of the anti-cancer treatment. The prodrug and anti-cancer treatment may be administered sequentially (e.g., in a single composition or in separate compositions) and/or simultaneously. The anti-cancer treatment may be chemotherapy (e.g., chemotherapeutic agent) and/or radiation therapy and/or immunotherapy. In a particular embodiment, the

immunotherapy may be an antibody directed against PD-1 (e.g. pembrolizumab or nivolumab).

In a particular embodiment, the immunosuppression is associated with an infectious disease. For example, the infectious disease may be a viral infection such as an infection by the hepatitis C virus (HCV), human papilloma virus (UPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), poliovirus, varicella zoster virus, coxsackie virus, or human immunodeficiency virus (HIV). In a particular

embodiment, the immunosuppression associated with HIV-I infection. When the infectious disease is a viral infection, the prodrugs of the instant invention may be administered with an anti-viral agent (e.g., nucleoside and nucleotide reverse transcriptase inhibitors ( RTIs), non-nucleoside reverse transcriptase inhibitors (N RTIs), protease inhibitors, etc.). The prodrug and anti-viral agent may be administered sequentially (e.g., in a single composition or in separate compositions) and/or simultaneously. In a particular embodiment, the infectious disease is tuberculosis or Leishmaniasis.

The pharmaceutical preparation comprising the agents of the invention may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of the agents in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agents to be administered, its use in the pharmaceutical preparation is contemplated.

The dose and dosage regimen of the agents according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition and severity thereof for which the agent is being administered. The physician may also consider the route of administration of the agent, the pharmaceutical carrier with which the agents may be combined, and the agents' biological activity.

Selection of a suitable pharmaceutical preparation depends upon the method of administration chosen. For example, the agents of the invention may be administered by direct injection into any cancerous tissue or into the surrounding area. In this instance, a pharmaceutical preparation comprises the agents dispersed in a medium that is compatible with the cancerous tissue.

Agents may also be administered parenterally such as by injection (e.g., intravenous injection) into the blood stream, or by subcutaneous, intramuscular or intraperitoneal injection. Pharmaceutical preparations for parenteral injection are known in the art. If parenteral injection is selected as a method for administering the compounds, steps must be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect. The lipophilicity of the agents, or the pharmaceutical preparation in which they are delivered, may be increased so that the molecules can better arrive at their target locations.

Pharmaceutical compositions containing agents of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. In preparing the antibody in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. For parenterals, the carrier will usually comprise sterile water, though other ingredients, for example, to aid solubility or for preservative purposes, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient.

Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unit for the administration of the agents of the invention may be determined by evaluating the toxicity of the agents in animal models. Various concentrations of the agents of the instant invention may be administered to mice with transplanted tumors, and the minimal and maximal dosages may be determined based on the results of significant reduction of tumor size and side effects as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the agents in combination with other standard anti-cancer drugs. The dosage units of the agents may be determined individually or in combination with each anti-cancer treatment according to greater shrinkage and/or reduced growth rate of tumors.

The compositions comprising the agents of the instant invention may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient.

Definitions

The following definitions are provided to facilitate an understanding of the present invention:

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

A "therapeutically effective amount" of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, or lessen the symptoms of a particular disorder or disease. The treatment of a disorder herein may refer to curing, relieving, and/or preventing the disorder, the symptom of it, or the predisposition towards it.

"Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A "carrier" refers to, for example, a diluent, adjuvant, excipient, auxilliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described, for example, in "Remington's Pharmaceutical Sciences" by E.W. Martin.

As used herein, the term "prevent" refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition.

The term "treat" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

As used herein, the terms "host," "subject," and "patient" refer to any animal, including mammals such as humans.

The term "alkyl," as employed herein, includes straight (linear) and branched hydrocarbons containing 1 to 20 carbons, particularly 1 to 10 carbons, 1 to 6 carbons, or 1 to 3 carbons. The hydrocarbon chain of the alkyl groups may be interrupted with one or more oxygen, nitrogen, or sulfur atoms (e.g., 1 to 3 or more heteroatoms). The hydrocarbon may be unsaturated (contain one or more double or triple bonds). The alkyl group may optionally be substituted (e.g., with halo, alkyl, haloalkyl, alkoxyl, alkylthio, hydroxy, methoxy, carboxyl, oxo, epoxy, alkyloxycarbonyl,

alkylcarbonyloxy, amino, carbamoyl, urea, alkylurea, aryl, ether, ester, thioester, nitrile, nitro, amide, carbonyl, carboxylate, sulfonate, and thiol).

The term "cycloalkyl ." as employed herein, includes cyclic hydrocarbons containing 1 to about 20 carbons. The cycloalkyl may be monocyclic, bicyclic, tricyclic, or more. The cycloalkyl may be saturated or unsaturated, but not aromatic. The hydrocarbon chain of the alkyl groups may be interrupted with one or more oxygen, nitrogen, or sulfur atoms (e.g., 1 to 3 or more heteroatoms). The cycloalkyl group may optionally be substituted (e.g., with halo, alkyl, haloalkyl, alkoxyl, alkylthio, hydroxy, methoxy, carboxyl, oxo, epoxy, alkyloxycarbonyl,

alkylcarbonyloxy, amino, carbamoyl, urea, alkylurea, aryl, ether, ester, thioester, nitrile, nitro, amide, carbonyl, carboxylate, sulfonate, and thiol).

The term "aryl," as employed herein, refers to monocyclic and bicyclic aromatic groups containing about 6 to 10 carbons in the ring portion. The bicyclic aromatic group may only contain one aromatic ring. Aryl groups may be optionally substituted through available carbon atoms. The aromatic groups may be a heteroaryl (e.g., a ring system that includes at least one sulfur, oxygen, or nitrogen heteroatom ring members).

As used herein, a "prodrug" refers to an agent that is converted into the parent drug, particularly in vivo. Prodrugs are substantially, if not completely, in an inactive form that is converted to an active form (i.e., drug) - such as within the body or cells, typically by the action of, for example, endogenous enzymes or other chemicals and/or conditions.

The following example is provided to illustrate various embodiments of the present invention. It is not intended to limit the invention in any way.

EXAMPLE

All reactants and reagents were commercially available and were used without further purification unless otherwise indicated. Anhydrous THF was freshly distilled from sodium and benzophenone. All reactions were carried out under an inert atmosphere of argon or nitrogen unless otherwise indicated. Concentrated refers to the removal of solvent with a rotary evaporator at normal water aspirator pressure followed by further evacuation with a direct-drive rotary vane vacuum pump. Thin layer chromatography was performed using silica gel 60 A precoated glass or aluminum backed plates (0.25 mm thickness) with fluorescent indicator, which were cut. Developed TLC plates were visualized with UV light (254 nm), iodine, p- anisaldehyde or ninhydrin. Flash column chromatography was conducted with the indicated solvent system using normal phase silica gel 60 A, 230-400 mesh. Yields refer to chromatographically and spectroscopically pure (>95%) compounds except as otherwise indicated. Melting points were determined using an open capillary and are uncorrected. ¾ NMR spectra were recorded at 400 MHz and ¹³ C MR spectra were recorded at 100 MHz. Chemical shifts are reported in δ values (ppm) relative to an internal reference (0.05% v/v) of tetramethylsilane (TMS, 0.0) for ¾ NMR and the

CD ₃ OD solvent peak (49.0) for ¹³ C NMR. Peak splitting patterns in the ¾ NMR are reported as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The attached proton test (APT) experiment was conducted for ¹³ C NMR analysis; methylene and quaternary carbons were identified as negative peaks, while methyl and methine had positive peaks. HPLC was conducted on an Agilent 1 100 with an

Ascentis® Express C-18 column (100 x 4.6 mm, 2.7 μπι) and a mobile phase of 80:20 MeCN:H ₂ 0. GC analyses were performed on the free hydroxylamine with an EI-MS detector fitted with a 30 m x 0.25 mm column filled with cross-linked 5% PH ME siloxane (0.25 μπι film thickness); gas pressure 7.63 psi He. Analysis of samples involved heating from 70 to 250°C (10°C/minute) and finally holding at 250°C for 7 minutes.

Methods for synthesizing O-alkylhydroxylamines are known in the art (see, e.g., WO 2009/073620 and Malachowski et al. (2016) Eur. J. Med., 108:564-576). Briefly, 0-(3-chlorobenzyl)hydroxylamine hydrochloride was synthesized as follows. To a solution of 3-chlorobenzyl alcohol (1 mmol) in freshly distilled THF (5 ml) was added triphenylphosphine (1.1 mmol) and N-hydroxylphthalimide (1.1 mmol). After the solution was cooled to 0°C diisopropylazodicarboxylate (1.1 mmol) was added dropwise. The solution was allowed to warm to room temperature over 3 hours. Reaction progress was monitored by TLC (1 : 1 heptanes: ethyl acetate). Hydrazine monohydrate (1.1 mmol) was then added and the solution was allowed to stir for 30 minutes. The resulting reaction mixture was filtered to remove the white precipitate. The filtrate was concentrated and subjected to flash chromatography (1 : 1

heptanes/ethyl acetate). The resulting product was dissolved in ether and treated with HC1 (2.0 M solution in ether) to afford the HC1 salt of the O-alkylhydroxylamine. Contaminating diisopropyl hydrazinodicarboxylate could be washed away from the

HC1 salt with dichloromethane. The synthesis process yielded 0-(3- chlorobenzyl)hydroxylamine hydrochloride as white crystals in 50% yield. The compounds was found to be >95% pure. Mp =144-148°C. ¾ NMR (400 MHz, CD3OD) δ 7.49 (s, 1H, ArH), 7.45-7.35 (m, 1H, ArH), 5.04 (s, 2H, ArCH ₂ ). ¹³ C NMR (100 MHz, CD3OD) δ 139.49, 135.59, 131.42, 130.55, 130.12, 128.56, 77.02. GC t _R = 6.251 minutes. The free hydroxylamine compound can be generated by treating the hydrochloride salts with saturated NaHCCb and extracting with EtOAc.

0-(3-Chlorobenzyl)hydroxylamine (free base, 3.4 mmol) and

diisopropylethylamine (3.06 mmol) were dissolved in DCM (8 mL) in a 25 mL pear- shaped flask at 0°C in an ice bath. Benzyl chloroformate (3.06 mmol, diluted with DCM 1 mL) was added in slowly to the above solution over 10 minutes at 0°C. The reaction was monitored by GC/MS & TLC. Most of the hydroxylamine was gone after the completion of addition of 0.9 eq of DIEA and chloroform. Mostly mono acylated product was observed. The reaction mixture was cooled in an ice bath, and saturated NaHCCb was added to quench the reaction. It was partitioned with more DCM (30 mL). The organic layer was washed with water, brine and dried with anhydrous MgS0 ₄ . The organic filtrate was concentrated to obtain the crude. The crude was dissolved in DCM and dry loaded on silica gel (1.6 gram). It was then purified on 50 gram silica gel column using eluting solvent DCM/heptanes (80:20).

The desired product was eluted in fraction 9-20. Appropriate fractions were combined and concentrated under reduced pressure. It was dried under high vacuum for 5 hours to give 97-14 as a white solid, 678 mg (68% yield). ¾ MR (400 MHz, DMSO-d ⁶ ) δ 10.54 (bs, 1H, H), 7.45-7.31 (m, 9H, ArH), 5.10 (s, 2H, ArCH ₂ ), 4.79 (s, 2H, ArCH ₂ ). ¹³ C NMR (100 MHz, DMSO-d ⁶ ) δ 157.28, 139.13, 136.85, 133.39,

130.62, 128.89, 128.77, 128.51, 128.49, 128.35, 127.62, 76.81, 66.41. GC t _R = 16.965 min.

0-(3-chlorobenzyl)hydroxylamine hydrochloride and its carbamate prodrug were evaluated for inhibitory activity against human IDOl expressed endogenously in HeLa cells seeded in a 96-well plate at a density of 10,000 cells per well in 100 μΐ _^ of

DMEM, 10% FBS, and 1% Penicillin-Streptomycin. IDOl expression was induced by the addition of 100 μΐ _^ of media containing 100 ng/ml ΠΤΝΓγ. For evaluation of inhibitory activity against ID02 and TD02, these proteins were expressed exogenously in T-Rex™ 293 cells. T-Rex™ 293 cells containing a tet-regulated mouse ID02 cDNA or human TD02 cDNA were seeded in a 96-well plate at a density of 10,000 cells per well in 100 of DMEM, 10% FBS, and 1% Penicillin- Streptomycin and protein expression was induced by the addition of 100 μΐ. of media containing 20 ng/mL doxycycline. In all instances, induction was allowed to proceed for 24 hours after which the media was discarded, the wells rinsed once, and serial dilutions of compound in 200 μΙ_, of DMEM +10% FBS with the final concentration of tryptophan adjusted to 100 μΜ. Following incubation at 37 °C for an additional 20 hours (or 72 hours in the case of ID02), the assay was stopped by the addition of 50 μΐ, of 50% (w/v) TCA to each well, and the cells were fixed by incubating for 1 hour at 4 °C.

Compound IC50 values for IDOl, ID02, and TD02 were assessed from single point dilution series with the most potent compounds subsequently retested two or more times and the results reported as averages. Following the TCA fixation step, the supernatants were transferred to a round-bottomed 96-well plate and incubated at 65 °C for 15 minutes. The plates were then centrifuged at 1250 x g for 10 minutes, and 100 μΐ _^ of clarified supernatant was transferred to a new flat-bottomed 96-well plate and mixed at equal volume with 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow reaction was measured at 490 nm using a Synergy™ HT microtiter plate reader (Bio-Tek, Winooski, VT). Graphs of inhibition curves with IC50 values were generated using Prism v.5.0 (GraphPad Software, Inc.).

The IDOl, TDO, and ID02 IC50 values in the cell based assays for 0-(3- chlorobenzyl)hydroxylamine hydrochloride and its carbamate prodrug are provided in Table 1. Notably, the carbamate prodrug had an IC50 of greater than ImM in an isolated IDOl enzyme assay whereas 0-(3-chlorobenzyl)hydroxylamine

hydrochloride had an IC50 of 0.30 μΜ in the isolated IDOl enzyme assay.

Table 1 : IDOl, TDO, and ID02 IC50 values in the cell based assays

Several publications and patent documents are cited in the foregoing specification in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these citations is incorporated by reference herein. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.