NZ719822B2 - Process for the synthesis of an indoleamine 2,3-dioxygenase inhibitor - Google Patents

Abstract

The present application is directed to processes and intermediates for making 4-({2-[(aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide, which is an inhibitor of indoleamine 2,3-dioxygenase, useful in the treatment of cancer and other disorders. .

Description

PROCESS FOR THE SYNTHESIS OF AN INDOLEAMINE 2,3-DIOXYGENASE INHIBITOR This application claims the benefit of priority of US. Prov. Appl. No. 61/901,689, filed November 8, 2013, which is incorporated herein by nce in its entirety.

FIELD OF THE INVENTION The present application relates to processes and intermediates for making 4-( {2- [(aminosulfonyl)amino] ethyl} amino)-N—(3 -bromofluorophenyl)-N’-hydroxy- 1 ,2,5 — oxadiazolecarboximidamide, which is an inhibitor of amine 2,3-dioxygenase useful in the treatment of cancer and other disorders.

BACKGROUND OF THE INVENTION Tryptophan (Trp) is an essential amino acid required for the biosynthesis of proteins, niacin and the neurotransmitter 5-hydroxytryptamine (serotonin). The enzyme indoleamine 2,3—dioxygenase (also known as lNDO or IDO) catalyzes the first and rate limiting step in the degradation of L-tryptophan to N-formyl-kynurenine. In human cells, a depletion of Trp resulting from IDO ty is a prominent gamma interferon (IFN-y) —inducible antimicrobial effector ism. IFN-y stimulation s activation of IDO, which leads to a depletion of Trp, thereby arresting the growth of Trp-dependent intracellular pathogens such as Toxoplasma gondii and Chlamydia trachomatis. IDO activity also has an antiproliferative effect on many tumor cells, and IDO induction has been observed in vivo during rejection of allogeneic tumors, ting a possible role for this enzyme in the tumor rejection process (Daubener, et al., 1999, Adv. Exp. Med. Biol., 467: 517—24; , et al., 1991, FASEB J., 5: 2).

It has been observed that HeLa cells co—cultured with peripheral blood lymphocytes (PBLs) acquire an immuno-inhibitory phenotype through up-regulation of IDO activity. A reduction in PBL proliferation upon treatment with eukin-2 (1L2) was believed to result from IDO released by the tumor cells in response to IFNG secretion by the PBLs. This effect was reversed by treatment with l-methyl-tryptophan (lMT), a specific IDO inhibitor. It was proposed that IDO activity in tumor cells may serve to impair antitumor responses (Logan, et al., 2002, Immunology, 105: ).

Recently, an immunoregulatory role of Trp depletion has received much attention.

Several lines of evidence suggest that IDO is involved in induction of immune tolerance.

Studies of mammalian pregnancy, tumor resistance, chronic infections and autoimmune diseases have shown that cells expressing IDO can suppress T—cell responses and promote tolerance. rated Trp catabolism has been observed in diseases and disorders associated with cellular immune activation, such as infection, malignancy, mune diseases and AIDS, as well as during pregnancy. For example, increased levels of IFNs and elevated levels of urinary Trp metabolites have been observed in autoimmune diseases; it has been postulated that systemic or local depletion of Trp occurring in autoimmune diseases may relate to the ration and wasting symptoms of these es. In t of this hypothesis, high levels of IDO were observed in cells isolated from the synovia of arthritic joints. IFNs are also elevated in human immunodeficiency virus (HIV) patients and sing IFN levels are associated with a worsening prognosis. Thus, it was proposed that IDO is induced chronically by HIV infection, and is further increased by opportunistic infections, and that the chronic loss of Trp initiates mechanisms responsible for cachexia, dementia and diarrhea and possibly immunosuppression of AIDS patients , et al, 1991, Adv. Exp. Med. Biol, 294: ). To this end, it has recently been shown that IDO inhibition can e the levels of virus—specific T cells and, concomitantly, reduce the number of y-infected macrophages in a mouse model of HIV (Portula et al, 2005, Blood, 106: 2382—90).

IDO is believed to play a role in the immunosuppressive processes that prevent fetal rejection in utero. More than 40 years ago, it was observed that, during pregnancy, the genetically disparate mammalian conceptus survives in spite of what would be predicted by tissue transplantation immunology ar, 1953, Symp. Soc. Exp. Biol. 7: 320—38).

Anatomic separation of mother and fetus and antigenic immaturity of the fetus cannot fully explain fetal allograft survival. Recent attention has focused on immunologic tolerance of the mother. Because IDO is expressed by human syncytiotrophoblast cells and systemic tryptophan concentration falls during normal pregnancy, it was hypothesized that IDO expression at the maternal-fetal interface is necessary to prevent immunologic rejection of the fetal allografts. To test this hypothesis, pregnant mice (carrying syngeneic or allogeneic fetuses) were exposed to 1MT, and a rapid, T cell—induced rejection of all allogeneic concepti was observed. Thus, by catabolizing phan, the mammalian conceptus appears to suppresses T—cell activity and defends itself t rejection, and blocking tryptophan catabolism during murine pregnancy allows maternal T cells to provoke fetal allograft rejection (Munn, et al, 1998, Science, 281: 1191—3).

Further evidence for a tumoral immune resistance mechanism based on tryptophan degradation by IDO comes from the observation that most human tumors constitutively express IDO, and that expression of IDO by immunogenic mouse tumor cells prevents their rejection by preimmunized mice. This effect is accompanied by a lack of accumulation of specific T cells at the tumor site and can be partly reverted by systemic treatment of mice with an inhibitor of IDO, in the absence of noticeable toxicity. Thus, it was suggested that the efficacy of eutic vaccination of cancer ts might be improved by concomitant administration of an IDO inhibitor (Uyttenhove et al., 2003, Nature Med., 9: 1269—74). It has also been shown that the IDO inhibitor, l-MT, can synergize with chemotherapeutic agents to reduce tumor growth in mice, suggesting that IDO tion may also e the anti-tumor activity of conventional cytotoxic therapies (Muller et al., 2005, Nature Med., 11: 312—9).

One mechanism contributing to immunologic unresponsiveness toward tumors may be presentation of tumor antigens by tolerogenic host APCs. A subset of human IDO- expressing n-presenting cells (APCs) that coexpressed CD123 (IL3RA) and CCR6 and inhibited T-cell proliferation have also been described. Both mature and immature CD123- positive dendritic cells suppressed T—cell ty, and this IDO ssive activity was blocked by lMT (Munn, et al., 2002, Science, 297: 1867—70). It has also been demonstrated that mouse tumor-draining lymph nodes (TDLNs) contain a subset of plasmacytoid dendritic cells (pDCs) that constitutively express immunosuppressive levels of IDO. Despite comprising only 0.5% of lymph node cells, in vitro, these pDCs potently ssed T cell responses to antigens presented by the pDCs lves and also, in a dominant fashion, suppressed T cell responses to third—party ns presented by pressive APCs.

Within the population of pDCs, the majority of the functional IDO-mediated suppressor activity segregated with a novel subset of pDCs coexpressing the B-lineage marker CD19.

Thus, it was hypothesized that IDO-mediated suppression by pDCs in TDLNs creates a local microenvironment that is ly suppressive of host antitumor T cell responses (Munn, et al., 2004, J. Clin. Invest., 114(2): 280—90).

IDO degrades the indole moiety of tryptophan, serotonin and melatonin, and initiates the production of neuroactive and immunoregulatory lites, collectively known as kynurenines. By locally depleting tryptophan and sing proapoptotic kynurenines, IDO expressed by dendritic cells (DCs) can greatly affect T—cell proliferation and survival. IDO induction in DCs could be a common mechanism of deletional tolerance driven by regulatory T cells. Because such tolerogenic responses can be expected to operate in a variety of physiopathological conditions, tryptophan metabolism and kynurenine production might represent a crucial ace between the immune and nervous systems (Grohmann, et al., 2003, Trends Immunol., 24: 242—8). In states of persistent immune activation, availability of free serum Trp is diminished and, as a consequence of reduced serotonin production, serotonergic functions may also be affected (Wirleitner, et al., 2003, Curr. Med. Chem, 10: 8 1—9 1).

Interestingly, administration of eron—0t has been observed to induce neuropsychiatric side effects, such as depressive symptoms and changes in cognitive function. Direct influence on serotonergic neurotransmission may contribute to these side effects. In addition, because IDO activation leads to reduced levels of tryptophan, the precursor of serotonin (5 -HT), IDO may play a role in these sychiatric side effects by ng central 5-HT synthesis. Furthermore, kynurenine metabolites such as 3-hydroxy- kynurenine (3 -OH-KYN) and quinolinic acid (QUIN) have toxic effects on brain function. 3— OH—KYN is able to produce ive stress by increasing the production of reactive oxygen species (ROS), and QUIN may produce overstimulation of ampal N—methyl—D— aspartate (NMDA) receptors, which leads to apoptosis and hippocampal atrophy. Both ROS overproduction and hippocampal atrophy caused by NMDA overstimulation have been associated with depression (Wichers and Maes, 2004, J. Psychiatry Neurosci., 29: 11—17).

Thus, IDO activity may play a role in depression.

Small molecule inhibitors of IDO are being developed to treat or prevent IDO-related diseases such as those bed above. For example, oxadiazole and other heterocyclic IDO inhibitors are reported in US 2006/0258719 and US 2007/0185165. PCT Publication WO 99/29310 reports methods for altering T cell-mediated immunity comprising ng local extracellular concentrations of tryptophan and phan metabolites, using an inhibitor of IDO such as 1-methyl-DL-tryptophan, p-(3-benzofuranyl)-DL- alanine, p-[3— benzo(b)thienyl] —DL-alanine, and 6-nitro-L-tryptophan) (Munn, 1999). Reported in WC 03/087347, also published as European Patent 1501918, are methods of making antigen- presenting cells for enhancing or reducing T cell tolerance (Munn, 2003). Compounds having indoleamine-2,3-dioxygenase (IDO) inhibitory activity are further ed in WO 2004/094409; and US. Patent Application Publication No. 2004/0234623 is ed to methods of treating a t with a cancer or an infection by the administration of an inhibitor of amine-2,3-dioxygenase in combination with other therapeutic modalities.

In light of the mental data ting a role for IDO in immunosuppression, tumor resistance and/or rejection, chronic infections, HIV-infection, AIDS (including its manifestations such as cachexia, dementia and diarrhea), autoimmune diseases or disorders (such as rheumatoid arthritis), and immunologic tolerance and prevention of fetal rejection in utero, therapeutic agents aimed at suppression of tryptophan degradation by inhibiting IDO activity are desirable. Inhibitors of IDO can be used to activate T cells and therefore enhance T cell activation when the T cells are suppressed by pregnancy, malignancy or a virus such as HIV. Inhibition of IDO may also be an ant treatment strategy for ts with neurological or neuropsychiatric diseases or disorders such as depression.

Due to the usefulness of IDO tors, there is a need for development of new processes for making IDO inhibitors. This application is directed towards this need and others.

SUMMARY OF THE INVENTIO N The compound 4-( {2-[(aminosulfonyl)amino]ethyl} amino)-N—(3-bromo fluorophenyl)-N'-hydroxy- l ,2,5-oxadiazolecarboximidamide having Formula I: , F Q‘s/’0 N NI <1 H2N’ ‘N/\/ %N Br H H I is an inhibitor of the enzyme indoleamine 2,3-dioxygenase (also known as IDO). The compound of Formula I, as well as its preparation and use, has been described in U.S. Patent No. 8,088,803, which is incorporated herein by reference in its entirety. The intermediates and processes provided herein help satisfy the g need for the development of IDO inhibitors for the treatment of serious diseases.

The present ation provides, inter alia, intermediates and processes for preparing a compound of Formula I: Q‘s/’0 N NI <1 H2N’ ‘N/\/ %N Br H H Accordingly, the present ation es a s comprising reacting a compound of Formula F5: with an aldehyde of Formula F6: Pg1 HNACHO to afford a compound of Formula F7: H I >20 PngN/\/N7/_\(LN N\ ,N wherein Pg1 is defined infra.

The present application further provides a process comprising reacting a compound of Formula F15: with a nd of Formula F5 to afford a compound of Formula F16: 310%? H "I *0 R30 'l" \NN N / \ R1 R1 N N wherein R1 and R3 are d infra.

The present application further provides a process comprising reacting a compound of a F17: i 0‘3]? OMe R40 N’ ‘N H H/Y F17 with a compound of Formula F5 to afford a compound of Formula F18: i099 H "I a: R40 N’ ‘N/\/N N H H / \ N\ ,N wherein R4 is defined infra.

DETAILED DESCRIPTION While certain of the processes steps are rated in the Schemes shown below, it is intended that the individual process steps may be claimed individually or in any combination (e.g., in Scheme 1, steps E, F, G, H, and I may be d individually or in combination). It is not intended that the processes be limited to an overall process having each and every step in the Schemes below.

Accordingly, general scheme for the ation of the compound of Formula I is described in Scheme 1.

Scheme 1 PngNACHO o o N ’ N ’ deprotecti ng I >=O F6 H I >=O t / \ / \ N\ ,N E N\ ,N F O 0 Br Br F5 F7 F F NHPg2 ’0 O‘V’O 0:5 2 ’/ ‘NH 0 H I’ ‘NHP N’ depro ec mgt t' /\/N |N>:O ¥ K/H | >20 agent H2N N N —. / \ . organic base N\ ,N / \ N H o G Br ‘0/ F F9 Accordingly, the present application provides a s comprising reacting a compound of Formula F5: IfiN IN>ZO with an aldehyde of Formula F6: PyHN/\CH0 F6 wherein Pg1 is an amino ting group, to afford a compound of Formula F7: H I >20 PdHN/\/ V71)\NN N\,N Amino protecting groups Pg1 may be used to prevent unwanted reactions of an amino group while performing a d ormation. Amino protecting groups allow easy covalent attachment to a en atom as well as selective cleavage from the nitrogen atom.

Suitable "amino protecting groups", such as alkoxycarbonyl (such as ethoxycarbonyl, tert— butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), and the like), acyl (such as acetyl (Ac), benzoyl (B2), and the like), sulfonyl (such as methanesulfonyl, trifluoromethanesulfonyl, and the like), arylalkyl (such as benzyl, 4- methoxybenzyl, diphenylmethyl, nylmethyl (trityl), and the like), alkenylalkyl (such as allyl, prenyl, and the like), diarylmethyleneyl (such as (C6H5)2C=N, and the like), and silyl (such as tert-butyldimethylsilyl, triisopropylsilyl, and the like), are known to one skilled in the art. The chemistry of amino protecting groups can be found in Wuts and Greene, Greene is Protective Groups in Organic Synthesis, 4th Ed., pp 696—926, John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its entirety.

In some embodiments, Pg1 is ethoxycarbonyl, tert—butoxycarbonyl, oxycarbonyl, or 9-fluorenylmethyloxycarbonyl.

In some embodiments, Pg1 is C1_6 alkoxycarbonyl.

In some embodiments, Pg1 is tert—butoxycarbonyl.

Appropriate solvents for Step E include, but are not d to, methanol or ydrofuran (THF), acetonitrile and the like. Halogenated hydrocarbon solvents (i.e., halogenated alkanes, such as romethane, form, dichloroethane or tetrachloroethane) can also be used.

In some embodiments, said reacting is med in a solvent component comprising tetrahydrofuran. As used herein, a solvent ent may refer to one solvent or a mixture of solvents. In some embodiments, the solvent component is an organic solvent. In some embodiments, said reacting is performed in a solvent component comprising a halogenated hydrocarbon solvent. In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.

In some embodiments, said reacting is performed in a solvent component comprising acetonitrile.

In some embodiments, said reacting is performed in a solvent component comprising dichloromethane and acetonitrile.

In some embodiments, said reacting is performed in the presence of a reducing agent.

The reducing agent can be any compound capable of reducing an organic compound to a lower oxidation state. Reduction usually involves addition of hydrogen atoms or l of oxygen atoms from a group. For example, des such as F6 can be reduced in the presence of an amine of Formula F5 (Step E, Scheme 1) by the on of hydrogen, either in the form of hydrogen gas (H2) or using a hydride reagent (such as NaB(OAc)3H, NaBH4, LiAlH4, and the like); using triphenylphosphine; or using a combination of sodium iodide, chlorotrimethylsilane, and methanol. In some embodiments, this step can be med under acidic conditions in the presence of an acid (such as trifluoroacetic acid). In some embodiments, this step can be performed at a temperature from about -15 °C to about 30 °C, e.g., from about —15 °C to about 0 °C, from about —5 °C to about 5 °C, from about —5 °C to about 0 °C, or from about 0 °C to about 45 °C.

In some embodiments, said reducing agent is a borohydride reducing agent (e. g., c)3H, NaBH4, or other boron ning hydride reducing agent).

In some ments, said borohydride reducing agent is sodium triacetoxyborohydride.

In some embodiments, said reacting is performed in the presence of trifluoroacetic acid.

In some embodiments, the process further comprises deprotecting said compound of Formula F7 to afford a compound of Formula F8: H2N"NV—(LN / \ N\ ,N Amino deprotecting agents useful for this Step F are known to those skilled in the art, such as those in Wuts and Greene (supra). In particular, the amino protecting groups described above can be conveniently removed using many available amino deprotecting agents that are specific to the various groups mentioned above Without affecting other desired portions of the nd. The tert—butoxycarbonyl group can be removed (e. g., hydrolyzed) from the nitrogen atom, for example, by treatment with an acid (such as hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, and the like); a combination of reagents (e. g., mixture of acetyl chloride and methanol) known to generate an acid; or a Lewis acid (e. g., BF3-Et20). The benzyloxycarbonyl group can be d (e. g., hydrogenolyzed) from the nitrogen atom, for example, by treatment with hydrogen and a catalyst (such as palladium on carbon).

In some ments, the amino ecting agent is trifluoroacetic acid. In some embodiments, the amino deprotecting agent contains trifluoroacetic acid and >0.5% by volume of water, e. g., >1 .0% by volume of water, >1.5% by volume of water, >2.0% by volume of water, from about 2% to about 10% by volume of water, from about 10% to about % by volume of water, or from about 20% to about 50% by volume of water. In some embodiments, the amino deprotecting agent can be a mixture of trifluoroacetic acid and water in a tric ratio of about 98:2. In some embodiments, the amino deprotecting agent can be hydrochloric acid, optionally in a solvent (e. g., water, THF, dioxane, ethyl acetate, etc.).

In some embodiments, the t component is ethyl acetate. In some embodiments, the amino deprotecting agent can be hloric acid optionally in a solvent such as an alcohol (such as isopropanol, methanol or ethanol). Halogenated hydrocarbon solvents (e. g., dichloromethane, chloroform, dichloroethane or hloroethane) can also be used. In some embodiments, the molar ratio of hydrochloric acid and the compound of Formula F7 is about 6.0, about 5.0, about 4.0, about 3.0 about 2.0, about 1.0, or about 1.1. In some embodiments, Step F can be performed at a temperature from about —10 0C to about 60 0C, e. g., from about — °C to about 0 0C, from about 0 °C to about 25 °C, from about 25 °C to about 45 0C, or from about 45 0C to about 60 °C.

In some embodiments, said deprotecting comprises reacting the compound of Formula F7 with hydrochloric acid.

In some embodiments, said deprotecting comprises reacting the compound of Formula F7 with hydrochloric acid in a solvent ent comprising isopropanol.

In some embodiments, said deprotecting comprises reacting the nd of Formula F7 with hydrochloric acid in a solvent component comprising a halogenated hydrocarbon solvent.

In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.

In some embodiments, the invention r comprises ng said compound of Formula F8, with PgZ-NH-SOZ-X, in the presence of an organic base to afford a compound of Formula F9: NHPgZ O=S\ ,, O NH N’ O K/NH I N>:O / \ N\O,N wherein: sz is an amino protecting group; and X is halo.

In some embodiments, PgZ—NH—SOZCl can be prepared and ately used in the reaction with the nd of Formula F8. The protecting group sz could be selected from any of the protecting groups known in the art for protecting amines or sulfonamides (such as those described above for Pgl). In some embodiments, sz can be an alkoxycarbonyl group (such as tert—butoxycarbonyl).

Appropriate solvents include, but are not limited to, halogenated hydrocarbon solvents such as dichloromethane and the like. The organic base can be any base that serves to neutralize the HCl generated during the reaction of the compound of Formula F8 and the protected amino-sulfonyl chloride. The organic base can e acyclic ry amines such as _6)alkylamine (e. g., triethylamine, ropylethylamine (DIPEA) and the like), cyclic tertiary amines (e. g., N—methyl piperidine, l,4-diazabicyclo[2.2.2]octane (DABCO) and the like). In some embodiments, the organic base can be triethylamine. In some embodiments, this step can be performed at a temperature from about —15 °C to about 60 °C, e.g., from about —15 °C to about 0 °C, from about 0 °C to about 25 °C, from about 25 °C to about 45 °C, or from about 45 °C to about 60 °C.

In such embodiments, the PgZ—NH-SOZCl can be obtained by the reaction of an alcohol (such as, ethanol, tert—butyl alcohol and the like) with chlorosulfonyl isocyanate (ClS(O)2NCO).

In some embodiments, sz is ethoxycarbonyl, tert—butoxycarbonyl, benzyloxycarbonyl, or 9-fluorenylmethyloxycarbonyl.

In some ments, sz is C1_6 alkoxycarbonyl.

In some embodiments, sz is tert—butoxycarbonyl.

In some embodiments, said reacting is performed in a solvent component comprising a halogenated hydrocarbon solvent.

In some embodiments, said nated hydrocarbon solvent is dichloromethane.

In some embodiments, said organic base comprises a tri(C1_6)alkylamine.

In some embodiments, said organic base is triethylamine.

In some embodiments, X is chloro.

In some embodiments, the invention further comprises ecting said nd of Formula F9 to afford a compound of Formula F10: 0%? N H I *0 H2N’ \N/\/ #N H / \ N\ ,N In some embodiments, suitable deprotecting agents may include those described above for deprotecting the compound of Formula F7.

In some embodiments, said deprotecting ses reacting a compound of Formula F9 with hydrochloric acid. In some embodiments, said deprotecting comprises reacting a nd of Formula F9 with hydrochloric acid in a solvent component comprising an alcohol. In some embodiments, said alcohol is ethanol. In some embodiments, said deprotecting comprises reacting a compound of Formula F9 with hydrochloric acid in a solvent component comprising ethyl acetate.

In some ments, the invention further ses reacting said compound of Formula F10 with a base to afford a nd of Formula I: <29) n 1131 H H N\ ,N A base can be used for the conversion (e. g., hydrolysis) of the oxadiazolone ring in F10 to reveal the amidoxime in the compound of Formula I, optionally in a solvent (Step I, Scheme 1). The protection of the amidoxime as the oxadiazolone can be useful to prevent adverse reactions of the hydroxyl group or that of the amidoxime as a whole. The base can be either an nic base such as alkali metal hydroxide (e. g., NaOH, LiOH, KOH, 2, etc.); or an organic base such as an acyclic amine (e.g., triethylamine, diisopropylethylamine (DIPEA), etc.) or a cyclic amine (e. g., pyrrolidine, piperidine, etc).

The base can be made available in the form of a resin (such as Amberlite® and the like). In some further embodiments, the base can be provided in the form of a solution in water (e. g., about 0.5 N solution, about 1 N solution, about 1.5 N solution, about 2.5 N solution, from about 3 N to about 5 N solution, from about 5 N to about 10 N solution). In some embodiments, the base is an alkali metal hydroxide (such as, sodium hydroxide). In some embodiments, the base can be 2 N NaOH solution in water. In some embodiments, the solvent can be l or tetrahydrofuran (THF). In some embodiments, the solvent can be a mixture of ethanol and water. In some embodiments, the ng of the compound of Formula F10 with a base to afford the compound of Formula I can be performed at a temperature from about —10 0C to about 60 0C, e. g., from about —10 0C to about 20 0C, from about 0 °C to about 30 0C, from about 0 0C to about 10 0C, or from about 0 0C to about 5 °C.

In some embodiments, said base ses an alkali metal hydroxide.

In some embodiments, said alkali metal hydroxide is sodium hydroxide.

In some embodiments, said reacting is performed in a solvent component comprising tetrahydrofuran, water and ethanol.

In some embodiments, the compound of Formula F5, can be obtained in a ce of steps shown in Scheme 2. The preparation of the intermediate, 4-amino-N‘-hydroxy-1,2,5- zole-3—carboximidamide F2, has been bed in J. cycl. Chem. (1965), 2, 253, which is incorporated herein by reference in its entirety, and its conversion to the chloro oxime F3 has been described in Synth. Commun. (1988), 18, 1427, which is incorporated herein by reference in its entirety. In some embodiments, the chloro oxime of Formula F3 can be d to 3—bromo—4—fluoroaniline, optionally in a solvent (such as water), followed by addition of sodium bicarbonate, to provide an amidoxime of Formula F4. The amidooxime functionality of the compound of F4 can then be converted to an oxadiazolone or Formula F5 using N,N—carbonyldiimidazole (DCI) in a solvent (such as ethyl acetate, dioxane, THF and the like), at elevated atures such as about 50 OC, about 60 °C, about 70 °C, about 80 OC, about 90 °C, or about 100 °C.

SchemeZ N,OH ,OH 1)NaN02/HC| (aq.) NaN02 | H2N HZN NCACN —’ \ NH2 —> / / \ 2)H2NOH-HC| N\O,N HCI/AcOH (aq_) N‘o’N F1 A F2 F3 OH F H2N Br N, )L No | (\N N/\§ | >20 N B ,J \Q H2N N F / \ N N H / \ —> N\ ,N N\ ,N o o NaHCO3 (aq.) D Br C F4 F5 Altemately, the compound of Formula F10 can be obtained through a ce of steps depicted in Scheme 3.

Scheme3 | O HZN N>= / \ N\ ,N lot? *5 N . F. reductlveamlnatlon 310°? n >=o R30 '7" HO R30 "1’ WIN N / \ R1 R1 M R1 R1 N‘O/N F15 Br amino deprotecting o N’0 agent O\‘" H I >20 —>HZN’ ‘N/\/ N H / \ N N\ ,N In some embodiments, the present application provides a process, comprising reacting a compound of Formula F15: floss) R30 If ‘NACHO R1 R1 with a compound of Formula F5: l o / \ N\ ,N F5 F to afford a compound of a F16: figs? H "I >=0 R30 '1’ ‘1}1/\/ 7/_\2\N R1 R1 N N each R1 is independently an amino protecting group; and R3 is C1_6 alkyl or benzyl.

In some embodiments, R1 is C2_4 alkenyl-C1_3 alkyl or phenyl-C1_3 alkyl, wherein said phenyl—C1_3 alkyl is optionally substituted by 1, 2, or 3 independently ed C1_4 alkoxy groups.

In some embodiments, R1 is C2_4 alkenyl-C1_3 alkyl or phenyl-C1_3 alkyl, wherein said phenyl-C1_3 alkyl is optionally substituted by 1, 2, or 3 methoxy groups.

In some embodiments, R1 is allyl.

In some embodiments, R1 is 4—methoxybenzyl.

In some embodiments, R3 is C1_6 alkyl.

In some ments, R3 is tert—butyl.

In some embodiments, R3 is C1_4 alkyl.

In some embodiments, R3 is butyl.

Preferably, the reacting is performed in the presence of a reducing agent. The reducing agent can be any compound capable of ng an organic compound to a lower oxidation state. In some embodiments, the reducing agent can be hydrogen gas in the ce of a catalyst or a hydride reagent (such as NaB(OAc)3H, NaBH4, LiAlH4 and the like); using triphenylphosphine; or using a combination of sodium iodide, chlorotrimethylsilane, and methanol. In some embodiments, this step can be med in the presence of an acid such as trifluoroacetic acid. Suitable solvents for this step include isopropyl alcohol, THF, dioxane, or the like. In some embodiments, this step can be performed at a ature from about —15 0C to about 30 0C, e. g., from about —15 0C to about 0 0C, from about —5 0C to about 5 0C, from about —5 0C to about 0 0C, from about 0 to 5 0C, or from about 0 0C to about 45 °C.

In some embodiments, said reacting is performed in a solvent component comprising tetrahydrofuran.

In some embodiments, said reacting is performed in the presence of a reducing agent.

In some embodiments, said reducing agent is a dride reducing agent.

In some ments, said borohydride reducing agent is sodium triacetoxyborohydride.

In some embodiments, said reacting is performed in the presence of trifluoroacetic acid.

In some embodiments, the invention further comprises deprotecting said compound of Formula F16 to afford a nd of a F10: 0%? N H Iro HZN/ \H/V N F Treatment of a compound F16 to replace RlN with NHz can be accomplished by methods for the deprotection of particular amine protecting groups known to one skilled in the art, such as those in Wuts and Greene, Greene is tive Groups in Organic Synthesis, 4th Ed., pp 696—926, John Wiley & Sons: New York, 2006. In some embodiments, when R1 is allyl, the deprotecting agent can be a palladium catalyst (e. g., Pd(Ph3P)4, Pd/C, or Pd(dba)DPPB). In some embodiments, when the R1 is 4-methoxybenzyl, the deprotecting agent can include an organic acid (such as trifluoroacetic acid or methanesulfonic acid, and the like); an inorganic acid (such as hydrochloric acid); hydrogen and palladium; or sodium in liquid ammonia. The deprotecting can be performed at a temperature from about 30 0C to about 90 OC, e.g., from about 50 0C to about 100 0C, or from about 60 0C to about 80 °C.

WO 70007 In some embodiments, said deprotecting comprises reacting a compound of a F16 with trifluoroacetic acid.

In some embodiments, said deprotecting comprises reacting a compound of Formula F16 with is hydrochloric acid.

Compound F15 can be made by a three step s (Steps J, K and L) from chlorosulfonylisocyanate, as shown in Scheme 4.

Scheme4 3 o o R OH JL Ox‘s’l amino ting R30 /\ o HZNCHZCOZRZ COZRZ agent \\ ,0x _ ry’ ‘rTJ /S\ , H H O=C=N CI J K O O O of? R30 N N COZR —> R30 u 1?] CH0 "21 "21 L R1 R1 F14 F15 Accordingly, the present application further provides a process wherein said compound of Formula F 15 is obtained by a process comprising ng a compound of Formula F14: with a reducing agent to afford said compound of Formula F15; wherein R2 is C1_4 alkyl; and R3 is defined supra.

In some embodiments, R2 is methyl.

In some embodiments, R2 is ethyl.

In some embodiments, the reducing can be carried out with diisobutylaluminum hydride (DIBAL—H). Suitable solvents include halogenated hydrocarbon solvents such as romethane, chloroform, dichloroethane, tetrachloroethane, and the like. In some embodiments, the reduction can be performed at about room temperature e.g., from about —80 0C to about 30 0C, from about —78 0C to about 0 0C, from about 0 0C to about 30 0C, or from about 25 0C to about 30 °C.

In some embodiments, said treating is performed in a halogenated hydrocarbon solvent.

In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.

In some ments, said reducing agent is diisobutylaluminum hydride.

In some ments, said compound of Formula F14 is ed by a process comprising protecting a compound of Formula F13: RsoJLl}! \\’l/S\ nil/\COZR2 H H with one or more independently selected amino protecting agents to afford a compound of Formula F 14.

Protecting group R1 on F 14 can be selected from the various amino protecting groups known in the art (supra). In some embodiments, the amino protecting agent is allyl bromide or 4-methoxybenzyl chloride.

In some embodiments, said one or more amino protecting agents is selected from allyl bromide and 4-methoxybenzyl chloride.

In some embodiments, said ting is performed in the ce of a base.

In some embodiments, said base is ium carbonate.

In some embodiments, said protecting is performed in a solvent component comprising acetonitrile.

In some embodiments, the preparation of the compound of F 13 can be obtained by treating chlorosulfonylisocyanate with an alcohol R3OH (where R3 is defined above) and a glycine ester HZNCHZCOZRZ, wherein R2 is C1_4 alkyl. In some embodiments, this Step I is carried out in the presence of an organic acid (such as acetic acid, benzoic acid, trifluoroacetic acid). Suitable solvents for this step e dichloromethane, chloroform, dichloroethane, tetrachloroethane, and the like.

In some ments, the present ation provides a compound of Formula F 13: wherein: R2 is C1_4 alkyl; and R3 is C1_6 alkyl or benzyl.

In some embodiments, R2 is methyl.

In some embodiments, R2 is ethyl.

In some embodiments, R3 is C1_6 alkyl.

In some embodiments, R3 is tert—butyl.

In some embodiments, the compound of Formula F13 is ethyl((N—(tert— butoxycarbonyl)sulfamoyl)amino)acetate: >LOJLN,S\\0 0‘ UL\ / o/\ H O In some embodiments, the invention further provides a compound of Formula F14: o 0 JL *é' R30 If ‘ryAcozRZ R1 R1 wherein: each R1 is independently an amino protecting group; R2 is C1_4 alkyl; and R3 is C1_6 alkyl or .

In some embodiments, R1 is C2_4 alkenyl-C1_3 alkyl or -C1_3 alkyl, wherein said phenyl—C1_3 alkyl is optionally substituted by l, 2, or 3 independently selected C1_4 alkoxy .

In some embodiments, R1 is allyl.

In some embodiments, R1 is oxybenzyl.

In some embodiments, R2 is methyl.

In some embodiments, R2 is ethyl.

In some embodiments, R3 is C1_6 alkyl.

In some embodiments, R3 is tert—butyl.

In some embodiments, R3 is C1_4 alkyl.

In some embodiments, R3 is butyl.

In some embodiments, R3 is C1_4 alkyl.

In some embodiments, R3 is butyl.

In some embodiments, the nd of Formula F 14 is ethyl—2—(allyl(N—allyl—N—(tert— butoxycarbonyl)sulfamoyl)amino) acetate: O H O >L A O"S’N\)J\o/\O N’ \‘ In some embodiments, the compound of Formula F 14 is ethyl(4-methoxybenzyl(N— 4-methoxybenzyl-N-(tert—butoxycarbonyl) oyl)amino)acetate: HSCOQO O XOANI‘S’Nflo"‘bo Hsco’ : In some embodiments, the present ation provides a compound of Formula F 15: R3OJLIF’S‘NA\u/ CHO R1 n1 wherein: R3 is C1_6 alkyl or ; and each R1 is independently an amino protecting group.

In some embodiments, R1 is C2_4 alkenyl-C1_3 alkyl or phenyl-C1_3 alkyl, wherein said phenyl—C1_3 alkyl is optionally substituted by 1, 2, or 3 independently selected C1_4 alkoxy groups.

In some embodiments, R1 is allyl.

In some embodiments, R1 is 4-methoxybenzyl.

In some embodiments, R3 is C1_6 alkyl.

In some embodiments, R3 is tert—butyl.

In some embodiments, the compound of Formula F15 is tert—butyl allyl{[allyl(2- oxoethyl)amino] sulfonyl}carbamate: >LOJLN,S\\\\ ,N H In some embodiments, the compound of Formula F15 is tert—butyl(4- methoxybenzyl) { [(4-methoxybenzyl)(2-oxoethyl)amino]sulfonyl} ate: H3COQ >L AOL 0" "J1 H o N’s" H300 In some ments, the invention provides a compound of Formula F16: JL ,S\N/\/NV_2\N\W H N’O>=| 0 R30 N I1 I1 R R N/ \N n R3 is C1_6 alkyl or benzyl and each R1 is independently an amino protecting group.

In some embodiments, R1 is C2_4 alkenyl-C1_3 alkyl or phenyl-C1_3 alkyl, wherein said phenyl—C1_3 alkyl is optionally substituted by l, 2, or 3 independently selected C1_4 alkoxy groups.

In some embodiments, R1 is allyl.

In some embodiments, R1 is 4—methoxybenzyl.

In some embodiments, R3 is 01.6 alkyl.

In some embodiments, R3 is tert—butyl.

In some embodiments, R3 is C1_4 alkyl.

In some ments, R3 is butyl.

In some embodiments, the compound of Formula F16 is utyl allyl(N—allyl—N—(Z— (4-(4-(3 -bromofluorophenyl)-5 -oxo-4,5-dihydro- l ,2,4-oxadiazol-3 -yl)- l ,2,5-oxadiazol-3 - ylamino)ethyl)sulfamoyl)carbamate: >L/KQ9 H l *0 0 Ni \N/\/ 7/_\(‘\N M N\ ,N In some ments, the compound of Formula F16 is tert—butyl (4— methoxybenzyl)-(N—(4-methoxybenzyl)-N—(2-(4-(4-(3-bromofluorophenyl)—5—oxo—4,5— dihydro- l ,2,4-oxadiazol-3 -yl)- l ,2,5-oxadiazol-3 -ylamino)ethyl)sulfamoyl)carbamate: ii% n "FD O N/ \N/\/ N | | /\ 82 82 N N Scheme 5 delineates an alternative route for the preparation of the compound of Formula F 10.

Schemes _220 io‘é’ F5 F R40 N, \N/\/ N OMe / \ R40 H H N’ \N/Y reductiveamination N ,N H H ——————————————> ‘o Q Br F17 F18 amino deprotecting NIO a ent ONIO H 9 | >20 , N ——————————————> HZN ‘N/A\V/ N H / \ R N\ ,N The present application also provides a process comprising reacting a nd of Formula F17: \\ lo R4OJLN/S\ H H wherein R4 is C1_6 alkyl, C1_6 haloalkyl, , or 9H—fluorenylmethyl with a compound of Formula F5: H2N7_(H\N>:/ o N\ ,N to afford a compound of a F18: H / \ N\ ,N In some embodiments, R4 is tert—butyl.

In some embodiments, R4 is benzyl.

In some embodiments, R4 is ethyl.

In some ments, R4 is C1_3 haloalkyl.

In some embodiments, R4 is 2,2,2-trichloroethyl.

In some embodiments, R4 is 9H—fluoren—9—ylmethyl.

In this Step Q, compounds F18 can be prepared, in some embodiments, by reacting F17 with the amine compound of Formula F5 in the presence of a reducing agent.

In some embodiments, said reacting is carried out in the presence of a reducing agent.

The reducing agent can be any compound capable of ng an organic compound to a lower oxidization state, for example by use of an organosilane such as tri(C1_3 alkyl)silane (e. g., triethylsilane); elemental hydrogen or using a hydride reagent (such as NaB(OAc)3H, NaBH4, LiAlH4 and the like); using triphenylphosphine; or using a combination of sodium iodide, trimethylsilane, and methanol. In some embodiments, this step can be performed in the presence of an acid such as trifluoroacetic acid. Suitable solvents include, but are not limited halogenated hydrocarbon solvents (e.g., dichloromethane, chloroform, roethane or tetrachloroethane). In some embodiments, the halogenated hydrocarbon solvent is l,2—dichloroethane.

In some embodiments, said reducing agent is an organosilane.

In some embodiments, said reducing agent is tri(C1_3 alkyl)silane.

In some embodiments, said reducing agent is triethylsilane.

In some embodiments, said reacting is carried out in the presence of an organic acid.

In some embodiments, said organic acid is trifluoroacetic acid.

In some embodiments, said organic acid is methanesulfonic acid.

In some ments, said ng is performed in a solvent component comprising a halogenated hydrocarbon solvent.

In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.

In some embodiments, said halogenated hydrocarbon solvent is 1,2-dichloroethane.

In some embodiments, the process further comprises deprotecting said compound of a F18 to afford a compound of Formula F10: 0%? N N I #0 H2N’ ‘N/\/ 7—(LN H / \ N\ ,N In some embodiments, methods for the deprotection of particular amine protecting groups (such as carbamates) are known to one d in the art, such as those in Wuts and Greene, Greene is Protective Groups in Organic Synthesis, 4th Ed., pp 696—926, John Wiley & Sons: New York, 2006. For e, the tert—butoxycarbonyl group (e. g., when R4 is tert— butyl) can be removed (e. g., hydrolyzed) from the nitrogen atom, for example, by treatment with an acid (such as hydrochloric acid, roacetic acid, toluenesulfonic acid, and the like); a combination of ts (e. g., mixture of acetyl chloride and methanol) known to generate an acid; or a Lewis acid (e. g., BF3-Et20). The benzyloxycarbonyl group (e. g., when R4 is benzyl) can be removed (e.g., hydrogenolyzed) from the nitrogen atom, for example, by treatment with hydrogen and a catalyst (such as ium on ). The methoxycarbonyl and ethoxycarbonyl groups (i.e., when R4 is methyl or ethyl) can be removed by treatment with an inorganic base (such as KOH or ; a combination of reagents (e. g., mixture of acetyl chloride, sodium iodide and acetonitrile); or by treatment with an acid (e.g., HBr, AcOH). The 2,2,2-trichloroethoxycarbonyl group can be removed, for example by treatment with a catalyst (e. g., Zn/AcOH or Cd/AcOH). Suitable solvents for this step include, but are not d to, methanol or tetrahydrofuran (THF), acetonitrile and the like. In some 2014/064531 embodiments, the treating is performed at a temperature from about 30 0C to about 90 OC, e.g., from about 50 °C to about 100 °C, or from about 60 °C to about 80 °C.

In some embodiments, said deprotecting comprises reacting the nd of Formula F18 with zinc in the ce of acetic acid.

In some embodiments, said deprotecting is performed in a solvent component comprising tetrahydrofuran.

In some embodiments, the process further comprises reacting said compound of Formula F10 with a base to afford a compound of Formula I: ,OH F Q‘sr'o H NI a HZN/ \N/\/ N Br H H N\ [N I.

In some embodiments, said base comprises an alkali metal ide.

In some embodiments, said alkali metal hydroxide is sodium hydroxide.

In some embodiments, said reacting is performed in a solvent component comprising tetrahydrofuran, water and ethanol.

In some embodiments, the s further comprises converting said compound of Formula F18 to a compound of Formula I: ,OH F 0.00 H2N’S‘H/\/Nj/—\2\HH "I a wherein said ting comprises combining the compound of Formula F18 with a base to form a first mixture. In some embodiments, the base is N,N—bis(2-aminoethyl)ethane-1,2- diamine.

In some embodiments, the converting further comprises adding an acid to the first mixture. In some embodiments, said acid is an aqueous strong acid. In some embodiments, said aqueous strong acid is aqueous hloric acid.

In some embodiments, said converting is performed in a solvent component comprising tetrahydrofuran and ethyl acetate.

The present application also es a process comprising: i) reacting a compound of Formula F19: with a compound of Formula F5: EON/ \ N\ ,N in the presence of triethylsilane and methanesulfonic acid to afford a compound of Formula F20: O io‘B’p c o M""#QEO/\ 0 N\,N F20 Br ii) converting said compound of Formula F20 to a compound of Formula I: ,OH F H2N’S‘MNNWMH loBr 1 n said converting comprises combining said compound of Formula F20 with N,N— bis(2—aminoethyl)ethane-1,2-diamine. In some embodiments, said converting further comprises adding aqueous hydrochloric acid after said combining.

Compound F17 can be made by a one step process (Step P) from sulfonylisocyanate, as shown in Scheme 6.

Scheme6 R4OH O\\S’/O (MeOhiggeHZNI-lz OMe ’ \ —> 4 i058]? — — In some ments, the preparation of the compound of Formula F17 can be ed by treating chlorosulfonylisocyanate with 2,2-dimethoxyethanamine and alcohol R4OH (R4 is defined as above), optionally in a solvent (e.g., a halogenated hydrocarbon solvent such as dichloromethane, chloroform, dichloroethane, tetrachloroethane). In some embodiments, this step carried out in the presence of a base. The base can be either an organic base such as an acyclic amine (e. g., triethylamine, diisopropylethylamine (DIPEA), etc.) or a cyclic amine (e. g., pyrrolidine, piperidine, etc.); or an inorganic base such as alkali (e.g., NaOH, LiOH, KOH, Mg(OH)2, etc.). In some embodiments, the reaction is carried out in a solvent, for example, a halogenated hydrocarbon solvent such as dichloromethane, form, roethane, or tetrachloroethane.

In some embodiments, the present application further provides a nd of Formula F17: JL o’l 8 OMe R40 N’ ‘N H H/Y wherein R4 is C1_6 alkyl, C1_6 haloalkyl, or benzyl.

In some embodiments, R4 is tert—butyl.

In some embodiments, R4 is benzyl.

In some embodiments, R4 is ethyl.

In some embodiments, R4 is C1_3 haloalkyl.

In some embodiments, R4 is 2,2,2-trichloroethyl.

In some embodiments, R4 is 9H—fluoren—9—ylmethyl.

In some embodiments, the nd of Formula F17 is tert—butyl N—(2,2— oxyethyl)sulfamoylcarbamate.

In some embodiments, the compound of Formula F17 is benzyl N—(2,2— dimethoxyethyl)sulfamoylcarbamate.

In some embodiments, the compound of Formula F17 is ethyl — dimethoxyethyl)sulfamoylcarbamate.

In some embodiments, the compound of Formula F17 is 2,2,2—trichloroethyl N—(2,2— dimethoxyethyl)sulfamoylcarbamate.

In some ments, the compound of Formula F17 is (9H—fluorenyl)methyl N— (2,2-dimethoxyethyl)sulfamoylcarbamate.

In some embodiments, the present application further provides a nd of a F18: )qu H "(OH N’ N whrein R4 is C1_6 alkyl, C1_6 haloalkyl, or benzyl.

In some embodiments, R4 is tert—butyl.

In some embodiments, R4 is benzyl.

In some embodiments, R4 is ethyl.

In some embodiments, R4 is C1_3 haloalkyl.

In some embodiments, R4 is 2,2,2-trichloroethyl.

In some embodiments, R4 is ren—9—ylmethyl.

In some embodiments, the compound of Formula F18 is benzyl ({[2—({[4—(3 —bromo—4— fluorophenyl)oxo-4,5 -dihydro- l ,2,4-oxadiazol-3 -yl]- 1,2,5 -oxadiazol-3 - yl} amino)ethyl]amino } sulfonyl)carbamate: O O N JL 0% H I *0 O N/ \N/\/ N H H / \ N\ /N In some embodiments, the compound of Formula F18 is ethyl ({[2—({[4—(3-bromo-4— henyl)oxo-4,5-dihydro- l ,2,4-oxadiazol-3 -yl]- 1,2,5-oxadiazol yl} amino)ethyl]amino } sulfonyl)carbamate: JLO‘"?o N’ N’S‘NNNV—(LNH I >=0 H H N/ \N In some embodiments, the compound of a F18 is 2,2,2—trichloroethyl ({[2- ({[4—(3 —bromofluorophenyl)oxo-4,5-dihydro-l,2,4-oxadiazolyl]—1,2,5-oxadiazol yl} amino)ethyl]amino } sulfonyl)carbamate: In some embodiments, the compound of Formula F18 is (9H—fluoren—9—yl)methyl N— (2-((4-(4-(3 -bromofluorophenyl)—5 -oxo-4,5 -dihydro- l ,2,4-oxadiazol-3 -yl)- 1,2,5 - oxadiazolyl)amino)ethyl)sulfamoylcarbamate: O o JLoéséo H | /E0 C O WNW" H o N\O,N As used herein, the term "alkyl," when used alone or together with onal moiety terms, refers to a straight-chained or branched, saturated hydrocarbon group having from 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. Example alkyl groups include methyl, ethyl, yl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and the like.

As used herein, yl" refers to an alkyl group haVing one or more double carbon— carbon bonds. In some embodiments, said alkyl group has from 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Example alkenyl groups include ethenyl (Vinyl), yl, and the like.

As used herein, "alkenylalkyl" refers to a group of formula —alkyl-alkenyl. In some embodiments, the alkenylalkyl group is allyl.

As used herein, the term "haloalkyl," when used alone or together with an additional moiety, refers to an alkyl group tuted by one or more halogen atoms independently selected from F, Cl, Br, and I. Example haloalkyl groups include CF3, CHFZ, CH2CF3, and the like.

As used herein, the term "alkoxy" refers to an —O-alkyl group. In some embodiments, the alkyl group has 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms.

Example alkoxy groups e methoxy, ethoxy, propoxy (e.g., oxy and isopropoxy), t-butoxy, and the like.

As used herein, "trialkylamine" refers to a nitrogen atom substituted by three independently selected alkyl groups. In some embodiments, each alkyl group has from 2 to 6 WO 70007 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Example trialkylamine groups include hylamine, triethylamine, and the like.

As used herein, the term "alkoxycarbonyl" refers to a group of formula —C(O)—O- alkyl. In some embodiments, the alkyl group has from 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Example alkoxycarbonyl groups include ethoxycarbonyl, tert— butoxycarbonyl (Boc), and the like.

Halogenated hydrocarbon solvents refer to halogenated alkanes, such as dichloromethane, chloroform, dichloroethane or tetrachloroethane, wherein the alkane can be ed or ht-chained having 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms with one or more halo atoms. In some embodiments, the halogenated hydrocarbon solvent is a chlorinated alkane of l to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.

At s places in the present specification, substituents of compounds of the invention may be disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the s of such groups and .

It is intended that the compounds of the invention are stable. As used herein "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single ment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

The compounds of the invention are further intended to include all possible geometric isomers. Cis and trans geometric isomers of the compounds are described and may be isolated as a mixture of s or as separated isomeric forms.

Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton.

Compounds of the invention can also include all isotopes of atoms occurring in the ediates or final compounds. es include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

In some embodiments, the compounds of the invention, and salts thereof, are ntially isolated. By "substantially isolated" is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected.

Partial separation can include, for example, a composition enriched in the compound of the invention. Substantial separation can include compositions ning at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the ion, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

The present ation also includes salts of the compounds described herein. As used herein, "salts" refers to derivatives of the disclosed compounds n the parent compound is modified by converting an ng acid or base moiety to its salt form.

Examples of salts include, but are not limited to, mineral acid (such as HCl, HBr, H2SO4) or organic acid (such as acetic acid, benzoic acid, trifluoroacetic acid) salts of basic residues such as amines; alkali (such as Li, Na, K, Mg, Ca) or c (such as trialkylammonium) salts of acidic residues such as carboxylic acids; and the like. The salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile (ACN) are preferred.

The present application also includes pharmaceutically able salts of the compounds described herein. The "pharmaceutically acceptable salts" include a subset of the "salts" described above which are, conventional non—toxic salts of the parent compound formed, for example, from non-toxic inorganic or c acids. Lists of suitable salts are found in Remington ’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal ofPharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by nce in its entirety. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, als, itions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, ic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e. g., 1H or 13C), infrared spectroscopy, spectrophotometry (e. g., ible), or mass spectrometry; or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography. The compounds obtained by the ons can be purified by any suitable method known in the art. For example, chromatography (medium pressure) on a suitable adsorbent (e. g., silica gel, alumina and the like), HPLC, or preparative thin layer chromatography; distillation; sublimation, trituration, or recrystallization. The purity of the compounds, in general, are determined by al methods such as measuring the melting point (in case of a solid), obtaining a NMR um, or performing a HPLC separation. If the melting point decreases, if unwanted signals in the NMR spectrum are decreased, or if extraneous peaks in an HPLC trace are removed, the compound can be said to have been purified. In some embodiments, the compounds are substantially purified.

Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be y determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Greene is Protective Groups in Organic Synthesis, 4th Ed., John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials ants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent’s freezing ature to the solvent’s g temperature. A given on can be carried out in one solvent or a mixture of more than one solvent. ing on the reaction step, le solvent(s) for that particular reaction step can be selected. Appropriate ts include water, alkanes (such as pentanes, hexanes, heptanes, exane, etc., or a mixture thereof), aromatic solvents (such as benzene, toluene, xylene, etc.), alcohols (such as methanol, ethanol, panol, etc.), ethers (such as dialkylethers, methyl tert-butyl ether (MTBE), ydrofuran (THF), dioxane, etc.), esters (such as ethyl acetate, butyl acetate, etc.), halogenated hydrocarbon solvents (such as romethane (DCM), chloroform, dichloroethane, hloroethane), dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone, acetonitrile (ACN), hexamethylphosphoramide (HMPA) and N—methyl pyrrolidone (NMP). Such solvents can be used in either their wet or anhydrous forms.

Resolution of racemic mixtures of compounds can be carried out by any of numerous s known in the art. An example method includes fractional recrystallization using a "chiral resolving acid" which is an optically , salt-forming organic acid. Suitable resolving agents for onal recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids.

Resolution of racemic mixtures can also be carried out by n on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.

Methods of Use The compound of Formula I can inhibit activity of the enzyme indoleamine-2,3- dioxygenase (IDO). For example, the compound of a I can be used to inhibit activity of IDO in cell or in an individual in need of modulation of the enzyme by administering an inhibiting amount of the compound of Formula I.

The compounds of Formula I can be used in methods of inhibiting the degradation of phan in a system containing cells expressing IDO such as a tissue, living organism, or cell culture. In some ments, the present ation provides methods of altering (e. g., increasing) extracellular tryptophan levels in a mammal by administering an effective amount of the compound of Formula 1. Methods of measuring tryptophan levels and tryptophan degradation are routine in the art.

The compounds of Formula I can be used in methods of inhibiting immunosuppression such as IDO—mediated immunosuppression in a patient by administering to the patient an effective amount of the compound of Formula I. IDO-mediated immunosuppression has been associated with, for example, cancers, tumor growth, metastasis, viral infection, viral replication, etc.

The nds of Formula I can also be used in methods of treating diseases associated with ty or expression, including abnormal ty and/or overexpression, of IDO in an individual (e. g., patient) by administering to the individual in need of such treatment a eutically effective amount or dose of the compound of a I or a pharmaceutical composition thereof. Example diseases can include any disease, er or condition that is directly or indirectly linked to expression or ty of the IDO enzyme, such as over expression or abnormal activity. An IDO—associated disease can also include any disease, disorder or condition that can be prevented, ameliorated, or cured by modulating enzyme activity. Examples of IDO-associated diseases include cancer, viral infection such as HIV infection, HCV infection, depression, neurodegenerative disorders such as Alzheimer’s disease and Huntington’s disease, trauma, age-related cataracts, organ transplantation (e. g., organ transplant rejection), and autoimmune diseases including asthma, toid arthritis, multiple sclerosis, allergic inflammation, inflammatory bowel disease, psoriasis and ic lupus erythematosusor. Example cancers treatable by the s herein include cancer of the colon, pancreas, breast, prostate, lung, brain, ovary, , testes, renal, head and neck, lymphoma, leukemia, melanoma, and the like. The compound of Formula I can also be useful in the treatment of obesity and ischemia.

As used , the term "cell" is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

As used herein, the term "contacting" refers to the bringing together of ted moieties in an in vitro system or an in vivo system. For example, "contacting" the IDO enzyme with the compound of Formula I includes the stration of the compound of a I to an individual or patient, such as a human, having IDO, as well as, for example, introducing the compound of Formula I into a sample containing a ar or purified preparation containing the IDO enzyme.

As used herein, the term "individual" or "patient," used interchangeably, refers to any animal, including s, preferably mice, rats, other rodents, s, dogs, cats, swine, , sheep, horses, or primates, and most preferably humans.

As used herein, the phrase "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal se in a , system, animal, individual or human that is being sought by a researcher, narian, medical doctor or other clinician.

As used herein the term "treating" or "treatment" refers to l) preventing the disease; for example, preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; 2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), or 3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the e, condition or disorder (i.e., reversing the pathology and/or symptomatology).

Combination Therapy One or more additional ceutical agents or treatment methods such as, for example, anti-viral agents, chemotherapeutics or other anti-cancer agents, immune enhancers, immunosuppressants, radiation, anti-tumor and anti-viral vaccines, cytokine therapy (e.g., 1L2, GM—CSF, eta), and/or tyrosine kinase inhibitors can be used in ation the compound of Formula I for ent of IDO—associated diseases, disorders or conditions.

The agents can be combined with the compound of Formula I in a single dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.

Suitable antiviral agents contemplated for use in combination the compound of Formula I can comprise side and nucleotide reverse transcriptase inhibitors (NRTIs), cleoside reverse riptase tors (NNRTIs), protease inhibitors and other antiviral drugs.

Example suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir dipivoxil OM)—PMEA]; lobucavir (BMS-180194); BCH- 10652; emitricitabine [(-)-FTC]; beta-L- FD4 (also called beta-L-D4C and named beta-L-2', 3'-dicleoxyfluoro-cytidene); DAPD, ((— )-beta-D-2,6,-diamino-purine dioxolane); and sine (FddA). Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine (BHAP, 2); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442 (1 -(ethoxy-methyl)—5-(1-methylethyl)—6-(phenylmethyl)- (2,4(1H,3H)-pyrimidinedione); and (+)-calanolide A 75451) and B. Typical suitable protease inhibitors include avir (Ro 31-8959); ritonavir (ABT-53 8); indinavir (MK- 639); nelfnavir (AG-1343); amprenavir (141W94); lasinavir (EMS-234475); 0; EMS-2322623; 8; and AG-l 549. Other antiviral agents include hydroxyurea, ribavirin, IL—2, IL-12, uside and Yissum Project No.1 1607.

Suitable chemotherapeutic or other anti-cancer agents include, for example, alkylating agents (including, without limitation, nitrogen mustards, ethylenimine tives, alkyl sulfonates, nitrosoureas and triazenes) such as uracil mustard, chlormethine, cyclophosphamide (CytoxanTM), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

In the treatment of melanoma, suitable agents for use in combination with the compound of Formula I include: dacarbazine (DTIC), optionally, along with other chemotherapy drugs such as tine (BCNU) and cisplatin; the "Dartmouth regimen," which consists of DTIC, BCNU, cisplatin and tamoxifen; a combination of tin, vinblastine, and DTIC; or temozolomide. nds according to the invention may also be combined with therapy drugs, including cytokines such as interferon alpha, interleukin 2, and tumor necrosis factor (TNF) in the ent of ma.

The compound of Formula I may also be used in ation with vaccine y in the treatment of melanoma. Antimelanoma vaccines are, in some ways, similar to the anti- virus vaccines which are used to prevent diseases caused by viruses such as polio, measles, and mumps. Weakened melanoma cells or parts of melanoma cells called antigens may be injected into a patient to stimulate the body's immune system to destroy melanoma cells.

Melanomas that are confined to the arms or legs may also be treated with a combination of agents ing the compound of Formula I, using a hyperthermic isolated limb ion technique. This treatment protocol temporarily tes the circulation of the ed limb from the rest of the body and injects high doses of chemotherapy into the artery feeding the limb, thus providing high doses to the area of the tumor without exposing internal organs to these doses that might otherwise cause severe side s. Usually the fluid is warmed to 102° to 104° F. Melphalan is the drug most often used in this chemotherapy procedure. This can be given with another agent called tumor necrosis factor (TNF) (see section on cytokines).

Suitable chemotherapeutic or other anti-cancer agents include, for example, antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors) such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, guanine, fludarabine phosphate, pentostatine, and gemcitabine.

Suitable chemotherapeutic or other anti-cancer agents further include, for example, certain natural products and their derivatives (for example, vinca alkaloids, antitumor otics, enzymes, lymphokines and epipodophyllotoxins) such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara— C, paclitaxel (TAXOLTM), mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, interferons (especially IFN—a), etoposide, and teniposide.

Other cytotoxic agents include navelbene, CPT-l 1, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

Also suitable are cytotoxic agents such as epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; um coordination complexes such as cis-platin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and opoietic growth factors.

Other anti-cancer s) include antibody therapeutics such as trastuzumab (Herceptin), antibodies to costimulatory molecules such as CTLA-4, 4-1BB and PD-l, or antibodies to cytokines (IL—10, TGF—B, etc.).

Other anti-cancer agents also include those that block immune cell migration such as antagonists to chemokine receptors, including CCR2 and CCR4.

Other anti-cancer agents also include those that augment the immune system such as adjuvants or adoptive T cell transfer.

Anti—cancer vaccines include dendritic cells, synthetic peptides, DNA es and recombinant s. s for the safe and effective stration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is bed in the "Physicians' Desk Reference" (PDR, e.g., 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is orated herein by reference as if set forth in its entirety.

Pharmaceutical Formulations and Dosage Forms When employed as pharmaceuticals, the compound of a I can be administered in the form of ceutical compositions which is a combination of the compound of Formula I and a pharmaceutically acceptable carrier. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical ding ophthalmic and to mucous membranes including intranasal, l and rectal delivery), pulmonary (e. g., by inhalation or insufflation of powders or ls, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can e topical administration (eye , subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, rterial, subcutaneous, eritoneal, or intramuscular injection or infusion; or intracranial, e. g., intrathecal or intraventricular, stration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a uous perfusion pump. ceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. ceutical compositions ning the compound of a I can be prepared in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a e, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, es, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, ls (as a solid or in a liquid medium), ointments containing, for example, up to 10 % by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active nd is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, se, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: ating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening ; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, WO 70007 ned or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosage ning from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic , in association with a suitable pharmaceutical excipient.

The active compound can be effective over a wide dosage range and is generally stered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be d, the chosen route of stration, the actual compound stered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture of the compound of Formula I. When referring to these pre-formulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be y subdivided into equally ive unit dosage forms such as tablets, pills and es. This solid pre— formulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present application.

The tablets or pills containing the compound of Formula I can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist egration in the h and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the t application can be incorporated for administration orally or by injection e aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as seed oil, sesame oil, t oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic ts, or mixtures thereof, and powders.

The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the itions are administered by the oral or nasal respiratory route for local or systemic . Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure ing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an riate .

The amount of compound or composition administered to a patient will vary depending upon what is being stered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the ms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the ing clinician depending upon factors such as the ty of the disease, the age, weight and general condition of the t, and the like.

The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lized, the lyophilized preparation being combined with a e aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of n of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of the the compound of a I can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compound of Formula I in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compound of Formula I can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some l dose ranges are from about 1 ug/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, ation of the excipient, and its route of stration. Effective doses can be extrapolated from dose—response curves derived from in vitro or animal model test systems.

The compound of a I can also be formulated in combination with one or more additional active ingredients which can include any pharmaceutical agent such as iral agents, vaccines, antibodies, immune ers, immune suppressants, anti-inflammatory agents and the like.

The present application also includes pharmaceutical kits useful, for example, in the treatment or prevention of IDO-associated diseases or disorders, obesity, diabetes and other diseases referred to herein which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of the compound of Formula 1.

Such kits can further include, if d, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically able carriers, additional containers, etc., as will be readily nt to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

EXAMPLES The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will y recognize a variety of non- critical parameters which can be changed or modified to yield essentially the same results. e 1. Synthesis of 4-({2-[(Aminosulfonyl)amino]ethyl}amino)—N—(3-br0mo fluor0phenyl)-N’-hydroxy—1,2,5—oxadiazolecarb0ximidamide N,OH N,OH 1.NaN02/aq.HC| l HZN NaN02 NCACN NHZ 2.H NOH-HCI / \ H2N>\8‘\CI 2 aq.HC|/AcOH / \ N N N N \O/ \O/ 1 2 3 OH JL ,0 N’ N H2NUBr H2N>__8\NH‘ [aN AilN H2N>_8\N‘ >=o —F, N N/ \N N/ \N H20, NaHCO3 ‘0’ EtOAc ‘0’ Br Br 4 5 F F /\ H leo>=0 A3740 BocHN CHO N N\ N N BocHN/\/ HCI /\/NH —> / \ —> N\ N / \ TFA/STAB-H,THF IPA 0 N\ ,N Br Br 0 7 F 0 o 0 o 0 38: N/ 7&0 is: "0 BOCHN H2N N/ \fo \HXNQNH \ CI/S‘NHBoc Hfl/NH \ HCI N —> —> TEA,CH20I2 N/ \N BrEtOH/EtOAc N/ \N Br \0/ \O/ F F 9 10 \\ , OH H2N’SC NaOH H N: HxN NH ZO , \ Nxo/N Br 11 F Malononitrile [Aldrich, product # M1407] (320.5 g, 5 mol) was added to water (7 L), preheated to 45 OC and stirred at 45 0C for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (3 80 g, 5.5 mol, 1.1 equiv.) was added. When the temperature reached 10 0C, 6 N hloric acid (55 mL) was added. A mild exothermic reaction ensued with the temperature reaching 16 0C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 h at 16-18 0C. The reaction mixture was cooled to 13 OC and 50% aqueous hydroxylamine hydrochloride (990 g, 15 mol, 3.0 equiv.) was added all at once. The temperature rose to 26 0C. When the exothermic reaction subsided the cold bath was removed and stirring was continued for 1 h at 26 - 27 0C, then it was slowly brought to reflux. Reflux was maintained for 2 h and then the reaction mixture was d to gradually cool overnight. The reaction mixture was stirred in an ice bath and 6 N hydrochloric acid 800 mL) was added in portions over 40 min to adjust to pH 7.0. ng was continued in the ice bath at 5 °C. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 °C) to give the desired product (644 g, 90%) as an off—white solid. 13C NMR (75 MHz, CD3OD): 8 156.0, 145.9, 141.3; C3H5N502 (MW 143.10), LCMS (E1) m/e 144.0 (M + H).

Step B: 4-Amin0-N-hydr0xy-I,2,5-0xadiazoZecarb0ximidoyl chloride (3) 4-Amino-N’-hydroxy-1,2,5-oxadiazolecarboximidamide (422 g, 2.95 mol) was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 N hydrochloric acid (1.475 L, 3.0 equiv.) and the suspension was d at 42 — 45 °C until a clear solution was achieved.

Sodium chloride (518 g, 3.0 equiv.) was added and this solution was stirred in an ice/water/methanol bath. A solution of sodium nitrite (199.5 g, 0.98 equiv.) in water (700 mL) was added over 3.5 h while maintaining the temperature below 0 0C. After te addition, ng was continued in the ice bath for 1.5 h and then the reaction mixture was allowed to warm to 15 OC. The precipitate was collected by filtration, washed well with water, taken in ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g) and stirred at room temperature for 1 h. This suspension was filtered through sodium sulfate (200 g) and the te was concentrated on a rotary evaporator. The residue was dissolved in methyl tert—butyl ether (5.5 L), treated with the activated charcoal (40 g), d at room temperature for 40 min and filtered through . The solvent was d in a rotary evaporator and the resulting product was dried in a vacuum oven (45 0C) to give the desired product (256 g, 53.4%) as an ite solid. 130 NMR (100 MHz, CD3OD) 5 155.8, 143.4, 129.7; C3H3C1N4OZ (MW 162.53), LCMS (E1) m/e 163/165 (M+ + H).

Step C: 4-Amin0-N-(3-br0m0flu0r0phenyl)-N’-hydr0xy-1,2,5-0xadiazoZe carboximidamide (4) ,H F HN2% N Br NHNH/\ 4-Amino—N—hydroxy—1,2,5—oxadiazole—3 -carboximidoyl chloride (33.8 g, 208 mmol) was mixed with water (300 mL). At 60 °C, 3-bromofluoroaniline (Sigma-Aldrich) (43.6 g, 229 mmol, 1.1 equiv.) was added to the sion with stirring for 10 min. A solution of sodium bicarbonate (26.3 g, 313 mmol, 1.5 ) in water (300 mL) was added over 15 min with stirring at 60 0C. After stirring 20 min, LCMS indicated reaction completion. The reaction mixture was then cooled to room temperature and extracted with ethyl acetate (2 x 300 mL). The combined ethyl acetate solution was dried over anhydrous sodium sulfate and concentrated to give the d product (65 g, 99%) as an off—white solid, which was used in the subsequent reaction without r purification. C9H7BrFN502 (MW 316.09), LCMS (E1) m/e 8 (M+ + H).

Step D.‘ 3-(4-Amin0-I,2,5-0xadiazol—3-yl)(3-br0m0flu0r0phenyl)-1,2,4-0xadiazol— (4H)-0ne (5) ' N>:O A mixture of 4-amino-N—(3-bromofluorophenyl)—N'—hydroxy-1,2,5-oxadiazole—3- imidamide (65.7 g, 208 mmol), N,N—carbonyldiimidazole (Sigma-Aldrich) (50.6 g, 312 mmol, 1.5 equiv.), and ethyl acetate (750 mL) was heated to 60 0C and stirred for 20 min.

LCMS indicated reaction completed. The reaction was cooled to room temperature, washed with 1 N hydrochloric acid (2 x 750 mL), dried over sodium sulfate, and concentrated. The crude product was triturated with a mixture of dichloromethane, ethyl acetate, and diethyl ether to give the desired product (60.2 g, 85%) as an off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 8.05 (m, 1H), 7.69 (m, 1H), 7.57 (t, 1H, J: 8.7 Hz), 6.58 (s, 2H); C10H5BrFN503 (MW 342.08), LCMS (E1) m/e 342/344 (M+ + H).

Step E: tert—Bulyl [2-({4-[2-(3-br0m0flu0r0phenyl)0x0-2,5-dihydr0-I,2,4-0xadiazol—3- yl]-I,2, iazoZyl}amin0)ethyl]carbamate (7) H ' V—O BocHN/\/ 7/—\(LN N\ ,N To a solution of trifluoroacetic acid (20.0 mL) and tetrahydrofuran (10.0 mL) was added sodium triacetoxyborohydride (10.59 g, 49.97 mmol, 10.0 equiv.) in portions with stirring under nitrogen. This mixture was stirred for 10 min at room ature and then cooled to — 5 0C. A solution of 3—(4—amino-1,2,5—oxadiazol—3—yl)—4—(3—bromo—4— fluorophenyl)-1,2,4-oxadiazol-5(4H)-one (1.71 g, 5.0 mmol) and tert—butyl (2— oxoethyl)carbamate (Sigma-Aldrich) (1.99 g, 12.5 mmol, 2.5 equiv.) in THF (15.0 mL) was added drop—wise over 30 min with stirring while maintaining the temperature below 0 0C.

The reaction was stirred at — 5 to 0 0C and additional portions of tert—butyl (2- oxoethyl)carbamate (0.20 g, 1.2 mmol, 0.24 equiv.) in THF (1.0 mL) were added drop—wise at 20 min, 40 min at 4 h intervals. HPLC ted reaction completed after 5.25 h. The reaction mixture was poured into the ice-cold sodium onate (500 mL) and this solution was stirred at room temperature overnight. The precipitate was collected by filtration and washed with brine. The resulting residue was mixed with heptane (40 mL) and l ether (40 mL) and stirred at room temperature for 5 h. The precipitate was ted by filtration, washed with diethyl ether and dried in a vacuum oven to give the d product (1953 mg, 80.5%) as an off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 8.08 (m, 1H), 7.71 (m, 1H), 7.59 (t, 1H, J: 8.7 Hz), 6.94 (m, 1H), 6.52 (m, 1H), 3.32 (m, 2H), 3.15 (m, 2H), 1.36 (s, 9H); C17H18BrFN605 (MW 485.26); LCMS (E1) m/e 507/509 (M+ + Na).

Step F: 3-{4-[(2-Aminoethyl)amin0]-1,2,5-0xadiazol—3-yl}(3-br0m0flu0r0phenyl)- 1,2, 4-0xadiazol—5(4H)-0ne hydrochloride (8) H | >20 HCI' H2N/\/ 7%" N\ ,N Method A (preparedfrom tert—Bulyl [2-({4—[2-(3-br0m0flu0r0phenyl)0x0-2,5-dihydr0- 1,2, 4-0xadiazol—3-yU—I, 2, 5-0xadiazol—3-yl}amin0)ethyUcarbamate; Step E).‘ To a 500—mL flask was d tert—butyl [2—({4—[2—(3 —bromo—4—fluorophenyl)—5—oxo— 2,5-dihydro-1,2,4-oxadiazolyl]—1,2,5-oxadiazolyl}amino)ethyl]carbamate (20 g, 41.2 mmol) and isopropanol (255 mL). The slurry was stirred at room temperature. Hydrogen chloride gas (7.55g, 207 mmol, 5.0 equiv.) was added to the slurry with a subsurface glass tube over 16 min. Ethyl acetate (111 mL) was then added to the batch and the reaction was heated to 43 0C and stirred for 7.5 h. The batch was cooled to 19 °C and ethyl e (44 mL) was added. The slurry was filtered and the resulting residue was washed with ethyl acetate (2 x 55 mL). The isolated solid was dried under reduced pressure at 45 °C for 15 h to afford the desired product (16.61 g ,95.5% yield) as an off—white to white solid. 1H NMR (300 MHz, DMSO-dg) 5 8.11 (bs, 3H), 7.78 (m, 1H), 7.73 (m, 1H), 7.59 (t, 1H, J= 8.7 Hz), 6.74 (t, 1H, J = 6.1 Hz), 3.50 (m, 2H), 3.02 (m, 2H); C12H11BrClFN6O3, (MW 421.61; C12H10BrFN6O3 for free base, MW ), LCMS (E1) m/e 385/3 87 (M+ + H).

Method B (prepared directlyfrom min0-I,2,5-0xadiazol—3-yD(3-br0m0-4— fluorophenyD-1,2,4-0xadiazol—5(4H)-0ne; Step D): Sodium triacetoxyborohydride (2.33 g, 11.0 mmol, 11.0 equiv.) was mixed with trifluoroacetic acid (12.0 mL, 155.8 mmol, 155.8 equiv.). The resulting solution was mixed at room temperature for 30 min. A solution of 3-(4-amino-1,2,5-oxadiazolyl)(3-bromo- 4-fluorophenyl)-1,2,4-oxadiazol-5(4H)-one (5, 0.342 g, 1.0 mmol) and tert—butyl (2— oxoethyl)carbamate (Sigma-Aldrich) (1.04 g, 6.51 mmol, 6.5 equiv.) in dichloromethane (10.0 mL) and itrile (6.0 mL) was stirred under N2. This solution was cooled to —5 0C and the solution of sodium triacetoxyborohydride and trifluoroacetic acid was added drop— wise over 5 min. The reaction was stirred at room temperature for 4 h. HPLC and LC—MS (M+ — Boc + H: 385/3 87, bromide pattern) indicated a ratio of the desired product and the starting al was 4 to 1. The e was concentrated and diluted with dichloromethane (10 mL). The solution was cooled to 0 0C and 4 N sodium hydroxide was slowly added while ining the temperature at 0 — 5 0C to adjust the pH to 8 — 9. The aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined dichloromethane solution was washed with sodium bicarbonate and brine, dried over sodium sulfate and concentrated. The crude residue was then dissolved in dichloromethane (6.0 mL) and the ing on was cooled to 0 0C. 4 N hydrochloric acid in dioxane (3.0 mL) was added drop—wise at 0 — 5 0C.

The mixture was stirred at room temperature for 20 min. The precipitate was collected by filtration, washed with diethyl ether, and dried in vacuum to afford the desired product (289 mg, 54%) as an off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 8.11 (bs, 3H), 7.78 (m, 1H), 7.73 (m, 1H), 7.59 (t, 1H, J= 8.7 Hz), 6.74 (t, 1H, J= 6.1 Hz), 3.50 (m, 2H), 3.02 (m, 2H); C12H11BrClFN6O3, (MW 421.61; C12H10BrFN603 for free base, MW 385.15), LCMS (E1) m/e 385/387 (M + H).

Step G: tert—Bulyl ({[2-({4-[4-(3-br0m0flu0r0phenyl)0x0-4,5-dihydr0-I,2,4-0xadiazol— 3-yl]-I,2,5-0xadiazol—3-yl}amin0)ethyUamino}sulf0nyl)carbamate (9) 091,0 H NI O‘N,s\N/\/NV_8\N>20 7L H H N/ \N A 20-L glass reactor was charged with 3-{4-[(2-aminoethyl)amino]—1,2,5-oxadiazol- 4-(3 -bromofluorophenyl)-1,2,4-oxadiazol-5(4H)-one hydrochloride (1200 g, 2.846 mol) and dichloromethane (6.5 L) at room temperature. Triethylamine (950 g, 9.39 mol, 3.3 equiv.) was added to the batch over 7 minutes. The batch was then cooled to — 14.6 0C.

A 5-L round bottom flask was charged with tert—butanol (253 g, 3.41 mol, 1.2 equiv.) and dichloromethane (2.6 L). The solution was cooled to 0.9 0C. To this solution was added chlorosulfonyl isocyanate (463 g, 3.27 mol, 1.15 equiv.) over 43 minutes while ining the batch temperature below 10 0C. The resulting tert—butyl (chlorosulfonyl)carbamate solution was held at 3 - 5 0C for 1 h.

The on of tert—butyl (chlorosulfonyl)carbamate was added to the reactor over 73 min while maintaining the batch ature below 0 0C. The batch was then warmed to 10 0C over 1 h and was then stirred at 10 - 14 0C for 1 h. Water (4.8 L) was added to the batch and the quenched reaction mixture was stirred at room temperature for 14.5 h. The batch was allowed to settle and phases ted. The romethane solution was isolated kept in the reactor and was charged with acetic acid (513 g) over 25 min to precipitate the product. The resulting slurry was stirred at 20 0C for 2.5 h. The product was isolated by filtration and washed with dichloromethane (1.8 L). The product was dried under reduced re (—30 ian) at 45 0C for 16 h to afford the desired product (1342 g, 83.5% yield) as a white solid. 1H NMR (400 MHz, DMSO-dg): 8 10.90 (s, 1 H), 8.08 (dd, J: 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J: 8.6 Hz, 1 H), 6.58 (t, J: 5.7 Hz, 1 H), 3.38 (dd, J: 12.7, 6.2 Hz, 2 H), 3.10 2014/064531 (dd, .1: 12.1, 5.9 Hz, 2 H), 1.41 (s, 9 H). C17H19BrFN7O7S (MW 564.34), LCMS (E1) m/e 5859/5879 (M+ + Na).

Step H: N-[2-({4—[4-(3-Br0m0quor0phenyZ)0x0-4,5-dihydr0-I,2,4—0xadiazol—3-yl]- 1,2, 5-0xadiazol—3-yl}amin0)ethyUsulfamide (10) NI %o To a 20-L flask containing tert—butyl ({[2-( {4—[4-(3-bromo-4—fluorophenyl)—5—oxo— 4,5-dihydro-1,2,4-oxadiazolyl]—1,2,5-oxadiazol yl}amino)ethyl]amino}sulfonyl)carbamate (1200 g, 2.126 mol) was added l (12 L) at 20 0C. The resulting mixture was stirred at room temperature and charged with hydrogen chloride gas (472 g, 12.9 mol, 6.07 equiv.). The batch was heated to 50 0C and the ature was ined for 3 h until reaction completion. Solvent was removed by vacuum lation at 33 - 39 0C and 6 Kg of distillate was collected. Ethyl e (6.8 L, 6.1 Kg) was added to the batch and distilled to collect 5.1 Kg of distillate. Ethyl acetate (7.2 L, 6.48 Kg) was added to the batch and distilled to collect 5.1 Kg of distillate. Ethyl acetate (2.4 L, 2.14 Kg) was added to the batch to adjust the solvents ratio. 1H NMR indicated the mole ratio of ethyl acetate to ethanol was 1.0: 0.1. The solution was heated to 65 0C. n—Heptane (4.1 kg) was added to the solution at 60 — 65 °C over 43 min. The resulting slurry was stirred at 65 °C for 1 h. The slurry was cooled to 20 0C over 2.5 h and stirred at that temperature for 15 h. The product was collected by filtration and washed with n-heptane (2.42 L). The product was dried under reduced pressure at 45 °C for 65 h to afford the desired product (906 g, 91.8% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-dg) 8 8.08 (dd, J = 6.2) 7.72 (m, 1 H), 7.59 (t, J: 8.7 Hz, 1 H), 6.67 (t, J: 5.9 Hz, 1H), 6.55 (s, 2H) 6.52 (t, J: 6.0 Hz, 1 H), 3.38 (dd, J: 12.7, 6.3 Hz, 2 H), 3.11 (dd, J: 12.3, 6.3 Hz, 2H); C12H11BrFN705S (MW 464.23), LCMS (E1) m/e 485.8/487.8 (M+ — C5H802 + Na).

Step I: 4-({2-[(Amin0sulf0nyl)amin0]ethyl}amin0)-N-(3-br0m0flu0r0phenyl)-N’-hydr0xy- 1,2, 5-0xadiazolecarb0ximidamide (I I) Q19 n 1131 H H N\ ,N To a 20-L glass reactor was added N—[2—({4—[4—(3—bromo—4—fluorophenyl)—5—oxo—4,5— dihydro—l,2,4—oxadiazol—3—yl]—l,2,5—oxadiazol—3—yl}amino)ethyl]sulfamide (799.4 g, 1.72 mol) and THF (3.2 L). The resulting solution was stirred at 20 0C for 7 min. and then charged with water (1.6 L). The batch was cooled to 2 °C and was charged with 30 wt % sodium hydroxide solution (475 mL, 666.4 g, 4.99 mol, 2.9 equiv.) over 8 minutes. The batch was warmed to 20 0C and the temperature was maintained for 16 h. The batch was then charged with methyl tert—butyl ether (8.0 L) over 23 minutes. Water (2.7 L) was added and the batch was cooled to about 0 0C. The batch was then charged with 85 wt % phosphoric acid (3 70.7 g, 3.22 mol, 1.9 equiv.) over 9 minutes. The batch was warmed to 20 0C and stirred for 1 h.

The batch was allowed to settle and phases were separated. The organic layer was retained in the reactor and charged with water (2.9 L) and 85 wt% phosphoric acid (3 70.7g, 3.22 mol) and stirred at 20 °C for 1 h. The batch was allowed to settle and phases were separated. The organic layer was retained in the r and charged with water (3.2 L) and stirred at 20 0C for 1 h. The batch was allowed to settle and phases were separated. The c solution was retained in the reactor and distilled under d pressure at 20 °C to remove 3.4 Kg of distillate. l (4.8 L) was charged to the batch and the batch was distilled to a volume of 3.2 L. This lation process was repeated one more time. Ethanol (0.6 L) was added to the batch to adjust the batch volume to 4 L. The batch was stirred at 20 0C for 16 h and then d with water (6.39 L). The resultant slurry was stirred at 20 0C for 5 h. The product was collected by filtration and was washed twice with a mixture of l (529 mL) and water (1059 mL). The product was dried under reduced pressure at 45 0C for 65 h to afford the desired product (719.6 g, 95.4%) as a white solid. 1H NMR (400 MHz, DMSO—dg): 8 11.51 (s, 1 H), 8.90 (s, 1 H), 7.17 (t, J= 8.8 Hz, 1 H), 7.11 (dd, J= 6.1, 2.7 Hz, 1 H), 6.76 (m, 1 H), 6.71 (t, J= 6.0 Hz, 1 H), 6.59 (s, 2 H), 6.23 (t, J= 6.1 Hz, 1 H), 3.35 (dd, J= 10.9, 7.0 Hz, 2 H), 3.10 (dd, J: 12.1, 6.2 Hz, 2 H); C11H13BrFN7O4S (MW 438.23), LCMS (E1) m/e 437.9/439.9 (M+ + H).

Example 2. Alternate preparation of N-[2-({4-[4—(3-Bromoflu0r0phenyl)—5—0x0-4,5- dihydro-l,2,4—0xadiaz01—3-yl]-1,2,5-oxadiazol—3-yl}amin0)ethyl]sulfamide tert-BuOH O o O O allyl bromide o\ I \\,,O HZNCHZCOOEt %\ )L )‘s’: OEt —>OMor PMBCI o=c=N’S‘CI O N N N33?N/\co Et2 TEA, CHZCIZ H H ch03 | 12, CSI 13 14a, R= allyl 14b, R = PMB N\ 750 HZN N NI \N 800 (a B o /O r \ S‘N4 O O , O o 1}] H N\ V DIBAL/DCM JL \\ 40 5 F R I /\/N N 3 /\ —> R o N’ ‘N CH0 / \ | I TFA/STAB-H/THF N N R R \ , Br 15a, R = allyl 16a, R = allyl 15b, R = PMB 16b, R = PMB ’0 yo 0\S/O HZN:\S\’N H N’s\ TFA H’\, NaOH 2 R = PMB QEtOH/HZO ——8‘NH NQ‘B Step I: Ethyl {[(tert—butoxycarbonyl)-amin0]sulf0nyl}aminoacetate (13) o 0 A70JL i4 N’ ‘NAcozEt H H A solution of chlorosulfonylisocyanate (Sigma-Aldrich) (5.0 mL, 57.4 mmol) in dichloromethane (100 mL) was cooled to 0 0C. utyl l (4.26 g, 57.4 mmol, 1.0 equiv.) was added dichloromethane (100 mL) via an addition funnel. This solution was stirred at 0 0C for 30 min. Glycine ethyl ester hydrochloride (8.82 g, 63.2 mmol, 1.1 equiv.) was added followed by drop—wise addition of triethylamine (20.0 mL, 144 mmol, 2.5 equiv.) at 0 0C. This reaction mixture was stirred at room temperature for 4 h. The reaction was diluted with dichloromethane (100 mL) and washed with 0.1 N hydrochloric acid and brine.

The organic layer was dried over sodium e and trated to give the desired product (13.2 g, 81.4 %) as a crude off—white solid, which was used in the subsequent reaction without further purification. 1H NMR (300 MHZ, DMSO-dg) 5 10.88 (s, 1H), 8.07 (t, 1H, J = 6.1 Hz), 4.08 (q, 2H, J= 7.1 Hz), 3.78 (d, 2H, J= 6.1 Hz), 1.40 (s, 9H), 1.18 (t, 3H, J= 7.1 Hz).

Step 2a. Ethyl (allyl{[allyl(tert—butoxycarbonyl)amino]sulf0nyl}amin0)acetate (14a) 2014/064531 Ethyl ({[(tert—butoxycarbonyl)amino] sulfonyl}aminoacetate (1.0 g, 3.54 mmol) was mixed with potassium ate (2.45 g, 17.7 mmol, 5.0 equiv.) and acetonitrile (23.0 mL) under N2 at room temperature. Allyl bromide (1.84 mL, 21.2 mmol, 6.0 equiv.) was added drop—wise. This reaction mixture was heated to 70 0C and stirred at that temperature for 14 h.

HPLC and LCMS indicated reaction completion. The reaction was filtered and the filtrate was concentrated. The residue was dissolved in dichloromethane and washed with sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and concentrated to give the desired product (1.11 g, 87 %) as crude off—white solid, which was used in the subsequent reaction without further purification. 1H NMR (300 MHz, DMSO-dg) 5.75 (m, 2H), 5.20 (m, 4H), 4.12 (m, 6H), 3.89 (m, 2H), 1.43 (s, 9H), 1.18 (t, 3H, J = 8.7 Hz).

Step 2b. Ethyl [{[(tert—but0xycarb0nyl)(4-meth0xybenzyl)amin0]sulf0nyl}(4- ybenzybamino]acetate (14b) >LOjiNO‘SN\)J\OEt\S\\O Ethyl ({[(tert—butoxycarbonyl) amino]sulfonyl}amino)acetate (1.00 g, 4.0 mmol) was mixed with methylformamide (DMF, 6.0 mL) and stirred at room ature. Sodium iodide (0.01 g, 0.1 mmol, 0.025 equiv.), potassium carbonate (2.40 g, 20 mmol, 5.0 equiv.) and para—methoxybenzyl chloride (2.64 mL, 19.5 mmol, 4.875 equiv.) were added to the mixture. This reaction was warmed to 80 0C and d at 80 0C for 2 h. LCMS indicated reaction completion. The reaction was cooled to room temperature and d through Celite. The Celite bed was washed with dichloromethane and the combined organic filtrates were concentrated. The concentrated residue was ved in dichloromethane (20 mL) and washed with sodium bicarbonate (5 x 12 mL) and brine. The organic layer was dried over sodium sulfate and concentrated. The residue was purified on silica gel (0 — 40% ethyl e/hexane gradient elution) to give the desired product (1.39 g, 80%) as an ite solid. 1H NMR (300 MHz, DMSO-dg) 5 7.22 (m, 2H), 7.14 (m, 2H), 6.88 (m, 4H), 4.64 (s, 2H), 4.33 (s, 2H), 4.03 (q, 2H, J= 7.1 Hz), 3.92 (s, 2H), 3.72 (s, 3H), 3.71 (s, 3H), 1.39 (s, 9H), 1.14 (t, 3H, J = 7.1Hz).

Step 3a. tert—Bulyl allyl{[allyl(2-0xoethyl)amino]sulf0nyl}carbamate (15a) 0 Ow? A~OAN:S‘NACHO A solution of ethyl (allyl{[allyl(tert—butoxycarbonyl)amino]sulfonyl}amino)acetate (1.11 g, 3.05 mmol) in dichloromethane (15 mL) at -78 0C under N2 was treated with 1.0 M diisobutylaluminun hydride in dichloromethane (3.66 mL, 3.66 mmol, 1.2 equiv.). The reaction mixture was stirred at -78 0C for 1 h and then quenched with methanol (1.5 mL) and treated with a ted solution of sodium potassium tartrate (65 mL). This solution was stirred at room temperature overnight. The aqueous layer was then extracted with dichloromethane (3 x 20 mL). The combined dichloromethane solution was washed with brine, dried over sodium sulfate, filtered, and concentrated to give the desired product (0.62 g, 64 %) as a crude thick colorless oil, which was used in uent reaction without further purification. 1H NMR (300 MHz, DMSO-dg) 5 9.45 (s, 1H), 5.76 (m, 2H), 5.18 (m, 4H), 4.15 (m, 4H), 3.72 (m, 2H), 1.43 (s, 9H).

Step 3]). tert—Bulyl (4—meth0xybenzyl){[(4-meth0xybenzyl)(2- yl)amin0]sulf0nyl}carbamate (15b) A solution of ethyl [{[(tert—butoxycarbonyl)(4-methoxybenzyl)amino]sulfonyl}(4- methoxybenzyl)amino]acetate (5.30 g, 10 mmol) in dichloromethane (20.0 mL) at - 78 0C under N2 was treated with 1.0 M diisobutylaluminum hydride in dichloromethane (12.2 mL, 12.2 mmol, 1.22 equiv.). The reaction mixture was d at -78 0C for 3 h. The on was then quenched with methanol (3 mL) and treated with dichloromethane (100 mL) and a saturated solution of sodium potassium tartrate (150 mL). This solution was stirred at room temperature overnight. The aqueous layer was then extracted with dichloromethane (3 x 20 mL). The combined dichloromethane solution was washed with brine, dried over sodium sulfate and concentrated. The residue was then purified on silica gel (0 — 30% ethyl acetate/ hexane gradient elution) to give the desired t (3.45 g, 71%) as an off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 9.24 (s, 1H), 7.23 (m, 4H), 6.88 (m, 4H), 4.68 (s, 2H), 4.31 (s, 2H), 4.07 (s, 2H), 3.72 (s, 3H), 3.71 (s, 3H), 1.40 (s, 9H).

Step 4a. ulyl allyl(N-allyl-N-(2-(4-(4-(3-br0m0flu0r0phenyl)0x0-4,5-dihydr0- 1,2, 4-0xadiazol—3-yD-I,2, 5-0xadiazol—3-ylamin0)ethstulfamowarbamate (16a) Ni N / 5/ Br To a 50—mL flask was added sodium triacetoxyborohydride (1.06 g, 5.0 mmol, 1.0 equiv.), trifluoroacetic acid (TFA, 2.0 mL, 26 mmol) and tetrahydrofuran (THF, 1.0 mL) at ambient temperature. This mixture was cooled to — 5 0C under N2 and stirred at 0 — 5 0C for min. To this solution was added 3-(4-amino-1,2,5-oxadiazolyl)—4-(3 -bromo fluorophenyl)—1,2,4—oxadiazol—5(4H)-one (0.171 g, 5.0 mmol; Step D) and tert—butyl allyl{[allyl(2-oxoethyl)amino]sulfonyl}carbamate (0.398 g, 2.5 mmol, 0.5 equiv.) in THF (1.5 mL) drop—wise at 0 — 5 0C over 5 min. The resulting reaction mixture was stirred under N2 at 0 - 5 0C. At 20 min, 40 min, and 2.5 h time points, a solution of tert—butyl allyl{[allyl(2-oxoethyl)amino]sulfonyl}carbamate (0.040 g, 0.125 mmol, 0.25 equiv.) in THF (0.20 mL) was added ise at 0 — 5 0C. At 2.5 h, a solution of sodium triacetoxyborohydride (0.211 g, 1.0 mmol, 0.2 equiv.) in trifluoroacetic acid (TFA, 1.5 mL, 9.5 mmol) was added at 0 — 5 0C. The reaction was warmed to room temperature and d overnight. The on mixture was then poured into an ice-cold saturated solution of sodium ate (50 mL) and extracted with dichloromethane (3 x 20 mL). The combined dichloromethane extracts were washed with brine, dried over sodium e, and concentrated. The residue was then purified on silica gel (0 — 75% ethyl acetate/hexane gradient elution) to give the desired product (0.239 g, 74.2%) as an off—white solid. 1H NMR (300 MHz, g) 5 8.07 (m, 1H), 7.71 (m, 1H), 7.58 (t, 1H, J = 8.7 Hz), 6.62 (m, 1H), .77 (m, 2H), 5.19 (m, 4H), 4.17 (m, 2H), 3.89 (m, 2H), 3.44 (m, 2H), 3.38 (m, 2H), 1.42 (s, 9H); C23H27BrFN7O7S (MW 644.47), LCMS (E1) m/e 6 (M+ — Boc + H).

Step 4]). tert-Butyl N-(2-(4-(4-(3-br0m0flu0r0phenyl)0x0-4,5-dihydr0-I,2,4-0xadiazol— 3-yZ)-I 5-0xadiazol—3-ylamin0)ethyZ)-N-(4—meth0xybenzyl)Sulfamoyl(4- , 2, methoxybenzybcarbamate (1619) MeO OMe To a 50—mL flask was added sodium triacetoxyborohydride (0.50 g, 2.37 mmol, 4.74 equiv.), trifluoroacetic acid (TFA, 1.0 mL, 13 mmol) and tetrahydrofuran at ambient temperature.

This e was cooled to 0 —5 0C under N2 and stirred at 0 - 5 0C for 10 min. To this solution was added tert—butyl (4-methoxybenzyl){[(4—methoxybenzyl)(2— oxoethyl)amino]sulfonyl}carbamate (0.40 g, 0.84 mmol, 1.68 equiv) and 3-(4-amino-l,2,5- oxadiazol—3—yl)—4—(3—bromo—4—fluorophenyl)—1,2,4—oxadiazol-5(4H)—one (0.17 g, 0.50 mmol; Step D) in ydrofuran (THF, 1.50 mL) at 0 -5 0C. The reaction was stirred at 0 — 5 0C for 45 min and a solution of tert—butyl (4-methoxybenzyl){[(4-methoxybenzyl)(2- oxoethyl)amino]sulfonyl}carbamate (0.12 g, 0.20 mmol, 0.4 equiv.) in THF (0.50 mL) was then added at 0 - 5 0C. After stirring at 0 - 5 0C for l h, the reaction was lly warmed to room ature with stirring. At 2.5 h and 4.5 h time points, trifluoroacetic acid (0.25 mL) was added. At 5 h, a solution of tert—butyl (4—methoxybenzyl) {[(4—methoxybenzyl)(2- oxoethyl)amino]sulfonyl}carbamate (0.060 g, 0.1 mmol, 0.2 equiv.) in THF (0.20 mL) was added. At 6.5 h, a solution of sodium triacetoxyborohydride (0.060 g, 0.24 mmol, 0.48 equiv.) in trifluoroacetic acid (0.25 mL) was added. HPLC indicated approximately 4% of the 3 -(4-amino- l ,2,5-oxadiazolyl)(3 -bromofluorophenyl)— l ,2,4-oxadiazol-5(4H)-one starting material (from Step D) still remaining. The reaction mixture was stirred at room temperature ght. HPLC indicated reaction completion. The reaction mixture was poured into an ice—cold saturated solution of sodium carbonate (50 mL) and the mixture was extracted with dichloromethane (3 x 20 mL). The combined dichloromethane extracts were washed with brine, dried over sodium sulfate, and concentrated. The e was then purified on silica gel (0 — 30% ethyl acetate/hexane gradient elution) to give the desired product (0.33 g, 82.5%) as an ite solid. 1H NMR (300 MHz, DMSO-dg) 5 8.06 (m, 1H), 7.69 (m, 1H), 7.57 (t, 1H, J= 8.7 Hz), 7.22 (m, 4H), 6.87 (m, 4H), 6.48 (m, 1H), 4.72 (s, 2H), 4.36 (s, 2H), 3.70 (S, 6H), 3.39 (m, 2H), 3.31 (m, 2H), 1.37 (s, 9H); C33H35BrFN709S (MW 804.64), LCMS (E1) m/e 826/828 (M+ — Boc + Na).

Step 5: N-[2-({4—[4-(3-Br0m0flu0r0phenyl)0x0-4,5-dihydr0-I,2,4-0xadiazol—3-yl]- 1,2, 5-0xadiazol—3-yl}amin0)ethyUsulfamide (10) HZN’ ‘N/\/ #N H / \ N\ ,N To a 25-mL flask was added tert—butyl {[[2—({4—[4—(3—bromo—4—fluorophenyl)—5—oxo— 4,5-dihydro-1,2,4-oxadiazol-3 -yl]-1,2,5-oxadiazol-3 -yl} amino)ethyl](4- methoxybenzyl)amino]sulfonyl}(4-methoxybenzyl)carbamate (40.2 mg, 0.050 mmol) in trifluoroacetic acid (TFA, 0.50 mL, 6.5 mmol) at ambient temperature. This reaction mixture was heated to 70 0C under N2 and stirred for 1 h. HPLC indicated reaction completed. The on mixture was cooled to room ature and the TFA was evaporated. The residual TFA was removed by treatment with dichloromethane (3 x 10 mL) followed by evaporation in vacuum. The residue was then triturated with dichloromethane and methanol to give the desired t (20 mg, 87%) as a crude off—white solid. 1H NMR (400 MHz, DMSO-dg) 8 8.08 (dd, J: 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J: 8.7 Hz, 1 H), 6.67 (t, J: 5.9 Hz, 1H), 6.55 (s, 2H) 6.52 (t, J: 6.0 Hz, 1 H), 3.38 (dd, J: 12.7, 6.3 Hz, 2 H), 3.11 (dd, J: 12.3, 6.3 Hz, 2H); C12H11BrFN705S (MW 464.23), LCMS (E1) m/e 489.8 (M+ + Na).

Example 3. Alternate preparation of N-[2-({4-[4-(3-Brom0flu0r0phenyl)0X0-4,5- dihydro-l,2,4—0xadiazol—3-yl]-1,2,5-0xadiazol—3-yl)amino)ethyl]sulfamide HZN l N>=O NI ‘ ROH ON QBF o 0 O\\//O (MeO)2CHCH2NH2 i [as/:0 5 F O:C:N—S\ —> RD N —’_ Cl TEA, CH2C|2 H N/\(H Et381H/TFA/solvent 12, CSI 5‘: R = t'BU' 17a-d b, R = 8n- C, R = Et- d R = CCI CH - ,O ,O H N >20 H N O 0V0 ' l 0V0 ' l >20 JL \ / N Zn/HOAc x / N N —> ,S\ /\/ N , \ /\/ R0 N N / \ H2N N / \ H H R=CI3CCH2- N\ ,N H N, ,N 0 Br 0 Br 18a-d F 10 F H N 0",0 H 1 NaOH :8\ /\/ NH EtOH/HZO N~OzN Br Step Ia. tert—Bulyl N-(Z,2-dimethoxyethstulfamoylcarbamate (17a) jiOQSI? >r \NH/Y O N’ A solution of chlorosulfonylisocyanate (11.32 g, 80 mmol) in romethane (120 mL) was cooled to 0 0C. Tert—butyl alcohol (7.65 mL, 80.0 mmol, 1.0 equiv.) was added via addition funnel. The mixture was stirred at 0 0C for 1.5 h. To this mixture, a solution of aminoacetaldehyde yl acetal (8.76 mL, 80.0 mmol, 1.0 equiv.) and triethylamine (TEA, 33.4 mL, 240 mmol, 3.0 equiv.) in methylene chloride (DCM 120.0 mL) was added drop—wise via addition funnel. The reaction was warmed to room temperature and stirred overnight. The reaction was treated with 0.1 N hydrochloric acid and the organic layer was washed with brine, dried over sodium sulfate and trated to give the desired t (15.6 g, 68.5 %) as a crude off—white solid, which was used for the subsequent reaction without further purification: 1H NMR (300 MHz, DMSO-dg) 5 10.84 (s, 1H), 7.62 (t, 1H, J = 6.0 Hz), 4.38 (t, 1H, J: 5.5 Hz), 3.24 (s, 6H), 2.96 (dd, 2H, J: 5.8 Hz), , 9H).

Step II). Benzyl N-(2,2-dimethoxyethyl)sulfamoylcarbamate (17b) A solution of chlorosulfonylisocyanate (16.26 g, 114.9 mmol) in dichloromethane (100 mL) was cooled to 0 0C. Benzyl alcohol g, 115.0 mmol, 1.0 equiv.) was added via an addition funnel. The mixture was stirred at 0 0C for 0.5 h. To this mixture was added a mixture of aminoacetaldehyde dimethyl acetal (13.25 g,126.0 mmol, 1.1 equiv.) and triethylamine (TEA, 17.4 g, 172 mmol, 1.5 ) dropwise via an addition funnel at below 0C. The reaction was warmed to room temperature and stirred overnight. The reaction mixture was treated with 0.5 N hydrochloric acid (100 mL) and the collected organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuum to give the d product (23.5 g, 64.3 %) as a crude off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 11.29 (s, 1H), 7.90 (t, 1H, J= 6.0 Hz), 7.37 (m, 5H), 5.12 (s, 2H), 4.35 (t, 1H, J= 5.5 Hz), 3.21 (s, 6H), 2.97 (dd, 2H, J= 5.8 Hz).

Step 16. Ethyl N-(Z,2-dimethoxyethstulfamoylcarbamate (1 7c) 0 O"? AOJLNE‘N/fi/OMeH H A solution of chlorosulfonylisocyanate (11.32 g, 80 mmol) in romethane (120 mL) was cooled to 0 0C. Ethanol (4.67 mL, 80.0 mmol, 1.0 equiv.) was added via addition funnel. The mixture was stirred at 0 0C for 1.5 h. To this mixture was added a solution of aminoacetaldehyde yl acetal (8.76 mL,80.0 mmol, 1.0 equiv.), triethylamine (TEA, 33.4 mL, 240 mmol, 3.0 equiv.) in dichloromethane (DCM, 120.0 mL) drop-wise via addition funnel 0 0C. The reaction was warmed to room temperature and stirred ght. The reaction was treated with 0.1 N hydrochloric acid and the collected organic phase was washed with brine, dried over sodium sulfate and trated in vacuum to give the desired product (11.2 g, 55 %) as a crude off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 11.13 (s, 1H), 7.81 (t, 1H, J: 6.0 Hz), 4.37 (t, 1H, J= 5.5 Hz), 4.09 (q, 2H, J= 7.1 Hz), 3.23 (s, 6H), 2.97 (dd, 2H, J= 5.8 Hz), 1.19 (t, 3H, J: 7.1 Hz).

Step Id. 2,2,2-Trichloroethyl N-(2,2-dimeth0xyethyl)sulfamoylcarbamate (17d) /\ )L :8\ OMG CI3C O " N/Y A solution of chlorosulfonylisocyanate (6.96 mL, 80 mmol) in dichloromethane (120 mL) was cooled to 0 0C. 2,2,2—trichloroethanol (7.67 mL, 80.0 mmol, 1.0 equiv.) was added via addition funnel at 0 0C. This e was stirred at 0 0C for 1.5 h. To this mixture was then added a solution of aminoacetaldehyde dimethyl acetal (8.76 mL,80.0 mmol, 1.0 equiv.) and triethylamine (TEA, 33.4 mL, 240 mmol, 3.0 equiv.) in dichloromethane (DCM, 120.0 mL) added drop—wise via addition funnel at 0 0C. The reaction was warmed to room temperature and stirred at room temperature overnight. The reaction was treated with 0.1 N hydrochloric acid and the collected organic phase was washed with brine, dried over NaZSO4, and concentrated to give the desired product (28.01 g, 97 %) as a crude off—white solid, which was used in the subsequent reaction without further purification. 1H NMR (300 MHz, DMSO-dg) 5 11.79 (s, 1H), 8.08 (t, 1H, J= 5.9 Hz), 4.90 (s, 2H), 4.37 (t, 1H, J= 5.5 Hz), 3.23 (s, 6H), 3.00 (dd, 2H, J= 5.7 Hz).

Step 2a. Tert—bulyl ({[2-({[4-(3-br0m0-4—flu0r0phenyl)0x0-4,5-dihydr0-1,2,4-0xadiazol— I,2,5-0xadiazol—3-yl}amin0)ethyUamino}sulf0nyl)carbamate (18a) \ I? H NIO):| 0 A mixture of 3-(4-amino-1,2,5-oxadiazol-3 -yl)(3 -bromofluorophenyl)-1,2,4- oxadiazol-5(4H)—one (103 mg, 0.302 mmol, 1.5 equiv.; Step D) and tert—butyl N—(2,2— dimethoxyethyl)sulfamoylcarbamate (57.2 mg, 0.201 mmol) in dichloromethane (1.0 mL) was stirred under N2 at room temperature. To this e was added trifluoroacetic acid ( 0.50 mL, 6.5 mmol) and triethylsilane (80.2 uL, 0.502 mmol, 2.5 equiv.) drop— wise. This reaction mixture was stirred at room temperature for 2 h. HPLC indicated approximately 30% conversion. The reaction mixture was cooled to 0 0C and quenched with saturated sodium bicarbonate to pH ~ 8. The mixture was extracted in ethyl e (3 x 10 mL). The combined organic ts were washed with brine, dried over sodium sulfate and trated. The residue was purified by ative TLC (50% ethyl acetate/hexane) to give the desired product (27.5 mg, 29.5%) as an off—white solid. 1H NMR (DMSO—d6, 400 MHz): 8 10.90 (s, 1H), 8.08 (dd, J= 6.2, 2.5Hz, 1H), 7.72 (m, 1H), 7.59 (t, J= 8.6Hz, 1H), 6.58 (t, J= 5.7Hz, 1H), 3.38 (dd, J= 12.7, 6.2Hz, 2H), 3.10 (dd, J: 12.1, 5.9Hz, 2H), 1.41 (s, 9H). C17H19BrFN7O7S (MW 564.34), LCMS (E1) m/e 485.8/487.8 (M+ - CngOZ + Na).

Step 2b. Benzyl ({[2-({[4-(3-br0m0flu0r0phenyl)0x0-4,5-dihydr0-I,2,4-0xadiazol—3-yl]- 1,2,5-0xadiazol—3-yl}amin0)ethyl]amino}sulf0nyl)carbamate (181)) 000 N’0 OXN \\'I H >20 I \N/\/ N H H N| N\ /N A mixture of mino-l,2,5-oxadiazolyl)—4-(3-bromofluorophenyl)—1,2,4- oxadiazol-5(4H)—one (68 mg, 0.20 mmol; from Step D) and benzyl {[(2,2- dimethoxyethyl)amino]sulfonyl}carbamate (191 mg, 0.60 mmol, 3.0 ) in 1,2— dichloroethane (3.0 mL) was cooled to 0 0C. To this mixture was added trifluoroacetic acid (1.0 mL, 13.0 mmol) and triethylsilane (105 uL, 0.66 mmol, 3.3 equiv.) drop—wise. This reaction mixture was stirred at 0 0C for 2 h. HPLC indicated reaction completion. The on mixture was cooled to 0 0C and quenched with saturated sodium bicarbonate to pH ~ 8. and the quenched reaction mixture was ted with EtOAc (3 x 10 mL). The combined organic extracts were washed with brine, dried over sodium e and concentrated. The residue was then stirred in a mixture of heptane and diethyl ether overnight. The solids were collected by filtration, washed with heptane and dried in vacuum to give the desired product (125 mg, 99%) as a crude off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 11.31 (s, 1H), 8.05 (m, 1H), 7.87 (m, 1H), 7.68 (m, 1H), 7.56 (m, 1H), 7.32 (m, 5H), 6.54 (m, 1H), 5.07 (s, 2H), 3.29 (m, 2H), 3.09 (m, 2H); C20H17BrFN7O7S (MW 598.36), LCMS m/e 598/600 (M+ + Step 26. Ethyl ({[2-({4-[4-(3-br0m0-4—flu0r0phenyl)0x0-4,5-dihydr0-I,2,4-0xadiazol—3- yl]-I, 2, 5-0xadiazol—3-yl}amin0)ethylamino}sulf0nyl)carbamate (186) A mixture of 3-(4-amino-1,2,5-oxadiazol-3 -yl)(3 -bromofluorophenyl)-1,2,4- oxadiazol-5(4H)—one (68 mg, 0.20 mmol; from Step D) and ethyl {[(2,2- dimethoxyethyl)amino]sulfonyl}carbamate (154 mg, 0.600 mmol, 3.0 equiv.) in 1,2— dichloroethane (2.50 mL, 31.7 mmol) was stirred at 0 0C. To this mixture was added trifluoroacetic acid (1.00 mL, 13.0 mmol) and triethylsilane (105 uL, 0.66 mmol, 3.3 equiv.) = drop-wise. The reaction e was stirred at 0 0C for 3 h. HPLC indicated 97.5% conversion to the desired product. The on mixture was cooled to 0 0C and quenched with saturated sodium bicarbonate to pH ~ 8. The mixture was extracted in ethyl acetate (3 x 10 mL). The combined organic ts were washed with brine, dried over sodium sulfate and concentrated. The residue was stirred in a mixture of heptane and diethyl ether overnight.

The solids were collected by filtration, washed with heptane to give the desired t (95 mg, 88%) as a crude off—white solid. 1H NMR (300 MHz, DMSO-dg) 5 11.18 (s, 1H), 8.08 (m, 1H), 7.70 (m, 2H), 7.59 (t, 1H, J= 8.7 Hz), 6.56 (s, 1H), 4.04 (d, 2H, J= 7.2 Hz), 3.35 (m, 2H), 3.11 (m, 2H), 1.15 (t, 3H, J: 7.2 Hz); C15H15BrFN7O7S (MW 536.29), LCMS (E1) m/e 536/538 (M+ + H).

Step 2d. 2, 2, 2-Trichloroethyl ({[2-({4-[4-(3-br0m0flu0r0phenyl)0x0-4,5-dihydr0-I,2,4- oxadiazol-S-yl]-I, 2, 5-0xadiazol—3-yl}amin0)ethyl]amino}sulf0nyl)carbamate (18d) i009\ H N" #0 CI3C/\O /S\ A suspension of mino-1,2,5—oxadiazol-3—yl)—4—(3—bromo—4—fluorophenyl)—1,2,4— oxadiazol—5(4H)—one (5, 0.680 g, 1.99 mmol) and 2,2,2-trichloroethyl {[(2,2- dimethoxyethyl)amino]sulfonyl}carbamate (17d, 2.22 g, 6.17 mmol, 3.1 ) in dichloromethane (DCM, 6.0 mL) was stirred at room temperature. To this e was added triethylsilane (1.27 mL, 7.95 mmol, 4.0 equiv.) and a solution of trifluoroacetic acid (TFA, 3.0 mL, 39.0 mmol) in dichloromethane (DCM, 2.0 mL) while maintaining the reaction temperature below 30 0C. The reaction mixture became homogenous after 5 min with agitation at room temperature and was stirred at room temperature for 1 h. HPLC indicated reaction completion. The reaction was filtered and the precipitate was suspended in a mixture of dichloromethane and heptane (ratio of dichloromethane to heptane was 1 to 9 by volume). The suspension was stirred at room temperature overnight. The precipitate was collected by filtration and washed with 10% dichloromethane in heptane and dried in vacuum to give the d product (1.15 g, 90.4%) as an off—white solid, which was used in the subsequent reaction without further purification. 1H NMR (300 MHZ, DMSO-dg) 5 11.85 (s, 1H), 8.07 (m, 2H), 7.70 (m, 1H), 7.57 (t, 1H, J= 8.7 Hz), 6.56 (m, 1H), 4.88 (m, 2H), 3.37 (m, 2H), 3.16 (m, 2H); BrCl3FN7O7S (MW 639.62), LCMS (E1) m/e 638/640/642 (M+ + H).

Step 3. N-[2-({4-[4-(3-Br0m0flu0r0phenyl)0x0-4,5-dihydr0-I,2,4-0xadiazol—3-yl]- 1,2, iazol—3-yl}amin0)ethyUsulfamide (10) F A on of 2,2,2-trichloroethyl ({[2-( {4-[4-(3 -bromofiuorophenyl)—5—oxo—4,5— dihydro— 1 ,2,4—oxadiazol—3—yl]— 1 ,2,5—oxadiazol—3 — yl}amino)ethyl]amino}sulfonyl)carbamate (320 mg, 0.50 mmol; from Step Q, Method D) in tetrahydrofuran (THF, 4.0 mL) was d at room temperature. Acetic acid (0.30 mL, 5.3 mmol) and zinc flakes (160 mg, 2.5 mmol, 5.0 equiv.) were sequentially added. This reaction e was stirred at room temperature for 3 h. HPLC ted reaction completion. The on mixture was filtered through Celite and the Celite was washed with THF. The combined filtrate was concentrated in vacuum and the resulting residue was ved in ethyl acetate (20 mL). The ethyl acetate solution was washed with saturated sodium carbonate and brine, dried over sodium sulfate and concentrated. The crude material was crystallized from ethyl acetate and diethyl ether to give the desired product (147 mg, 63%) as an off—white solid.

Example 4. Alternate Preparation of 4-({2-[(Aminosulfonyl)amino]ethyl}amino)—N-(3- bromoflu0r0phenyl)-N'-hydroxy-1,2,5—oxadiazolecarboximidamide 2014/064531 0.0 O. OJOL‘N35sioCI HZN/YOMe Oésoo OH H OMe Cl/ \ —> N=C=O —> O 12,csr N' MN O HN2 ,0 I \ NI %0 O o /u\O\\S4O \0’ O JLQ‘s’p N OMe . O "I ‘N/\/H / \ O N/ \N/Y H 5 N\ (N H O o 0 OMe 13 Br 17 Et3SiH/MeSO3H DCM, RT, 4 - 48 h Oé 40 H ' HN’S\N/\/N NH N(CH20H2NH2)3(TAEA) 2 H /\ —> N N THF, RT, 20 - 24 h 11 Br Into an oven dried 2 L 4-neck round bottom flask was charged 9-fluorenylmethanol (50.0 g, 255 mmol) and anhydrous DCM (3 82 mL) at room ature. The resulting slurry was cooled in an ice—bath to about 0 — 5 °C. A solution of chlorosulfonyl isocyanate (CSI, 23.0 mL, 264 mmol) in anhydrous DCM (127 mL) was added dropwise to the slurry h an addition funnel over 22 minutes, maintaining the reaction mixture temperature at < 5°C. The resulting e was stirred at 0 - 5 0C for 1.75 h, producing a thick white slurry. A solution of aminoacetaldehyde dimethyl acetal (27.9 mL, 255 mmol) in anhydrous DCM (3 82 mL) and 4—methylmorpholine (84.0 mL, 764 mmol) were added to the mixture at about 0 - 5 0C over 71 minutes. The ing reaction e was then stirred in the ice bath for 1.5 hours. When HPLC showed the the reaction was complete, the reaction mixture was acidified by the dropwise addition of a 1.0 M phosphoric acid (H3PO4, aq., 640 mL) over 22 minutes to pH 1 - 2. Water (300 mL), EtOAc (2150 mL) and heptane (250 mL) were then added and the resulting mixture was stirred for 10 minutes. The two phases were separated and the organic phase was washed sequentially with water (500 mL), heptane (300 mL) and water (2 x 500 mL) and dried over MgSO4. The filtrate was concentrated under vacuum to dryness. The resulting solids were redissolved in EtOAc (600 mL) at 65 °C and the warm solution was filtered into a clean 3 L round bottom flask. The filtrate was cooled to room ature and stirred for 2.5 h before heptane (1200 mL) was then added via an addition funnel over 80 min. After ng overnight at room temperature, the mixture was then cooled in an ice bath for 1 h. The resulting solids were collected by ion, washed with 25% EtOAc/heptane (250 mL), and dried overnight at about 40 — 45°C under vacuum to afford 9H—fluorenylmethyl {[(2,2-dimethoxyethyl)amino]sulfonyl}carbamate (91.3 g, 88% yield) as a white powder. 1H NMR (300 MHz, DMSO—d6) 5 11.43 (s, 1H), 7.98 — 7.85 (m, 3H), 7.76 (d, J: 7.5 Hz, 2H), 7.43 (t, J: 7.2 Hz, 2H), 7.33 (td, J: 7.4, 1.1 Hz, 2H), 4.44 — 4.33 (m, 3H), 4.33 — 4.22 (m, 1H), 3.23 (s, 6H), 2.99 (t, J: 5.8 Hz, 2H) ppm.

Step 2. 9H—FZu0renylmethyl ({[2-({4—[4-(3-br0m0fluor0phenyl)0x0-4,5-dihydr0- 1,2, 4-0xadiazol—3-yU—I,2, 5-0xadiazol—3-yl}amin0)ethyUamino}sulf0nyl)carbamate O io"s"o O . O W \NN"#N/ \ o N\ ,N To a stirred suspension of 3-(4-amino-1,2,5-oxadiazolyl)(3 fluorophenyl)-1,2,4-oxadiazol-5(4H)-one (10.00 g, 29.23 mmol) in DCM (160 mL) was added methanesulfonic acid (MeSO3H, 8.46 g, 88.04 mmol) and triethylsilane (Et3SiH, 8.37 g, 71.96 mmol) at ambient temperature over 10 minutes to give a slurry. Solid ren—9— ylmethyl{[(2,2-dimethoxyethyl)amino]sulfonyl}carbamate (12.25 g, 30.14 mmol) was added portionwise (1 g/3 - 4 min; over 1 h) while ining the internal temperature at less than about 20 0C using a water bath. After the addition, the resulting mixture was stirred at about 13 to 22 0C for 3 days. Additional triethylsilane (Et3SiH, 0.1755 g, 1.51 mmol) and 9H- fluorenylmethyl{[(2,2-dimethoxyethyl)amino]sulfonyl}carbamate (0.3082 g, 0.76 mmol) were added and the resulting mixture was stirred at ambient temperature for an additional 23 h. Isopropyl alcohol (IPA, 15 mL) was added and the resulting mixture was stirred at ambient temperature for 1 h. Heptane (100 mL) was added and the mixture was stirred at ambient temperature for an additional 2 h. The solids were collected by filtration, washed with IPA/heptane (1/5; 2 x 30 mL) and heptane (2 x 30 mL), and dried under vacuum to afford 9H-fluorenylmethyl ({[2-( {4- [4-(3 -bromofluorophenyl)—5 —oxo—4,5—dihydro— 1,2,4-oxadiazol-3 -yl]-1,2,5-oxadiazolyl}amino)ethyl]amino}sulfonyl)carbamate as a white solid (18.30 g, 91.1% yield). 1H NMR (500 MHz, DMSO-d6) 5 11.44 (s, 1H), 8.07 (dd, J: 6.2, 2.5 Hz, 1H), 7.90 (t, J: 5.6 Hz, 1H), 7.88 (d, J: 7.6 Hz, 2H), 7.72 (d, J: 7.0 Hz, 2H), 7.71 (ddd, J: 8.9, 4.3, 2.6 Hz, 1H), 7.57 (dd, J: 8.7, 8.7 Hz, 1H), 7.40 (t, .1: 7.4 Hz, 2H), 7.31 (td, J: 7.4, 1.0 Hz, 2H), 6.55 (t, J: 6.0 Hz, 1H), 4.35 (d, J: 7.3 Hz, 2H), 4.25 (t, .1: 7.2 Hz, 1H), 3.39 (q, .1: 6.4 Hz, 2H), 3.15 (q, .1: 6.3 Hz, 2H); 130 NMR (126 MHz, DMSO— 616)?) 159.03 (d, J: 248.7 Hz), 156.61 (s), 155.22 (s), 151.55 (s), 148.67 (s), 143.29 (s), 140.68 (s), 133.82 (s), 133.39 (s), 130.05 (d, J: 8.5 Hz), 128.54 (d, J: 3.2 Hz), 127.73 (s), 127.07 (s), 125.24 (s), 120.11 (s), 117.42 (d, J: 24.0 Hz), 108.19 (d, J: 22.5 Hz), 66.70 (s), 46.17 (s), 43.34 (s), 40.79 (s) ppm; "P NMR (376 MHz, 6) 5 —103.99 — —107.39 (m) Step 3. 4-({2-[(Amin0sulf0nyl)amin0]ethyl}amin0)-N-(3-br0m0flu0r0phenyl)-N’-hydr0xy- 1,2, 5-0xadiazolecarb0ximidamide Into a 1 L 4-neck round bottom flask was charged 9H—fluorenylmethyl({[2-({4-[4- (3—bromo—4—fluorophenyl)—5—oxo-4,5—dihydro—1,2,4—oxadiazol—3—yl]—1,2,5—oxadiazol—3— yl}amino)ethyl]amino}sulfonyl)carbamate (25.0 g, 36.4 mmol) and anhydrous THF (250 mL) at ambient temperature to produce a homogeneous solution. The solution was then cooled to 0 — 5 °C in an ice bath before N,N-bis(2—aminoethyl)ethane—1,2-diamine (114 mL, 728 mmol) was added dropwise over 35 minutes via an addition funnel. The addition funnel was rinsed with anhydrous THF (50 mL) and the rinse was added to the reaction mixture. The cold bath was d and the reaction was lly warmed to ambient ature and stirred at ambient temperature for 2.5 h. EtOAc (400 mL) was added and the ing mixture was transferred to a 2 L 4-neck round bottom flask and cooled to about 0 — 5 0C in an ice bath. A solution of 2.0 M aqueous HCl (400 mL, 800.0 mmol) was added dropwise via an on funnel, while maintaining the internal temperature at below 10 °C. The two phases were separated and the aqueous phase was extracted with EtOAc (200 mL). The organic fractions were combined and cooled to about 6—7 °C. A solution of 2.0 M aqueous HCl (200.0 mL, 400.0 mmol) was added dropwise to the cold organic fraction, maintaining the internal temperature at below 10°C. The two phases were separated and the organic phase was washed with water (2 x 400 mL), dried over MgSO4, and concentrated under reduced pressure to a light yellow syrup. The syrup was dissolved in EtOAc (60.0 mL) to yield a homogeneous solution. To the solution was added a solution ofDCM (250.0 mL) and tert—butyl methyl ether (TBME, 100.0 mL) dropwise. The resulting slurry was stirred overnight at room temperature, then cooled in an ice bath for 1 h. The solids were collected by filtration, washed with an ice cold 250 mL solution of DCM (150 mL) and TBME (100 mL), and dried under vacuum to give 14.4 g of the crude desired product as white solids.

The crude product was dissolved in EtOAc (140.0 mL) at 60 °C and the warm solution was filtered. The e was cooled to room temperature before heptane (100.0 mL) was added dropwise over 55 min. The resulting e was then stirred overnight at room temperature. The solids were collected by filtration, washed with a 2:1 mixture of e and EtOAc (75 mL), and dried under vacuum at 40 — 50 0C to constant weight to afford 4-( {2-[(aminosulfonyl)amino]ethyl} amino)—N—(3 -bromofluorophenyl)-N'—hydroxy— 1,2,5—oxadiazolecarboximidamide (12.9 g, 81% yield) as a white solid.

Example A: Human indoleamine 2,3-dioxygenasae (IDO) enzyme assay Human indoleamine 2,3-dioxygenasae (IDO) with an N-terminal His tag was expressed in E. coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N’ -formylkynurenine. The assays were performed at room temperature as described in the literature using 95 nM IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 5 HM methylene blue and 0.2 mg/mL catalase in 50 mM ium phosphate buffer (pH 6.5). The initial reaction rates were recorded by continuously following the absorbance increase at 321 nm due to the ion ofN’—formlylkynurenine (See: Sono, M., et al., 1980, J. Biol. Chem. 255, 345). The nd of Formula I was tested in the assay of Example A and found to have an ICso of < 200 nM.

Example B: Determination of inhibitor ty in HeLa cell-based indoleamine 2,3- dioxygenase (IDO)/Kynurenine assay HeLa cells (#CCL-2) were obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA) and ely maintained in minimum essential medium (eagle) with 2 mM L—glutamine and Earle's BSS ed to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 10 % fetal bovine serum (all from Invitrogen). Cells were kept at 37 0C in a humidified incubator supplied with 5 % C02. The assay was performed as follows: HeLa cells were seeded in a 96 well culture plate at a density of 5 x 103 per well and grown overnight. On the next day, IFN- y (50 ng/mL final concentration) and serial dilutions of compounds (in total volume of 200 uL culture medium) were added into cells. After 48 hours of incubation, 140 uL of the supernatant per well was transferred to a new 96 well plate. 10 uL of 6.1 N trichloroacetic acid 9, Sigma) was mixed into each well and incubated at 50 °C for 30 min to hydrolyze N—formylkynurenine produced by indoleamine 2,3-dioxygenase to kynurenine. The reaction mixture was then centrifuged for 10 min at 2500 rpm to remove sediments. 100 uL of the supernatant per well was transferred to another 96 well plate and mixed with 100 pl of 2% (w/v) p-dimethylaminobenzaldehyde 7-7, Sigma-Aldrich) in acetic acid. The yellow color derived from Kynurenine was measured at 480 nm using a SPECTRAmax 250 microplate reader (Molecular Devices). L—kynurenine 5, Sigma) was used as rd.

The standards (240, 120, 60, 30, 15, 7.5, 3.75, 1.87 HM) were prepared in 100 uL e media and mixed with equal volume of 2 % (w/v) p-dimethylaminobenzaldehyde. The percent inhibition at individual concentrations was determined and the average values of duplicates were obtained. The data was analyzed by using nonlinear sion to generate IC50 values (Prism Graphpad). See: Takikawa O, et al., 1988, J. Biol. Chem, 263(4): 2041—8.

Example C: Determination of effect of IDO inhibitors on T cell proliferation that is suppressed by IDO-expressing dendritic cells Monocytes were collected from human peripheral mononuclear cells by leukophoresis. Monocytes were then seeded at a density of 1 x 106 well in a 96 well plate, using RPMI 1640 medium supplemented with 10 % fetal bovine serum and 2 mM L- glutamine (all from Invitrogen). Adherent cells were ed on the plate after ght culture at 37 °C. Adherent monocytes were then stimulated for 5—7 days with 100 ng/ml GM— CSF (# 300—03, PeproTech) and 250 ng/ml IL—4 (#200—04, PeproTech), followed by activation with 5 ug/mL LPS from Salmonella lyphimurium (#437650, Sigma) and 50 ng/mL IFN—y (# 285-IF, R&D Systems) for additional 2 days to induce dendritic cell maturation.

After tic cell activation, the medium was ed with completed RPMI 1640 supplemented with 100—200 U/mL IL—2 (#CYT—209, ProSpec—Tany TechnoGene) and 100 ng/mL anti—CD3 antibody (#555336, PharMingen), T cells (2—3 X 105 cells/well), and serial dilutions of IDO compounds. After incubation for 2 more days, T cell proliferation was measured by BrdU oration assay, using a colorimetric Cell Proliferation ELISA kit per manufacturer's instruction (#1647229, Roche Molecular Biochemicals). Cells were continuously cultured for 16—1 8 hrs in presence of 10 uM BrdU labeling solution. Then, the ng medium was removed, and 200 uL FixDenat per well was added to the cells and incubated for 30 minutes at room temperature. The FixDenat solution was removed and 100 uL/well anti-BrdU-POD antibody conjugate working solution was added. The reaction was carried out for 90 minutes at room temperature. The antibody conjugate was then removed, and cells were rinsed three times with 200 uL/well washing solution. Finally, 100 uL/well of substrate solution was added and the results were obtained using a microplate reader ra Max PLUS, Molecular Devices) during color development. Multiple readings at s time points were obtained to ensure the data was within the linear range. The data was routinely obtained from replicated experiments, and appropriate controls were included. See: Terness P, et a]. 2002, J. Exp. Med, 196(4): 447—57; and ku, P, et a]. 2000, J. Immunol., : 3596—9.

Example D: In vivo testing of IDO inhibitors for antitumor activity In vivo anti—tumor efficacy can be tested using modified tumor aft/xenograft protocols. For instance, it has been described in the literature that IDO inhibition can ize with cytotoxic chemotherapy in immune—competent mice (Muller, A.J., et a]. 2005, Nat. Med. —319). This synergy was shown to be dependent on T—cells by comparison of the synergistic effects of an investigational IDO inhibitor in murine tumor xenograft models (e. g. B16 and related variants, CT-26, LLC) grown in immune competent syngenic mice to that observed in syngenic mice treated with neutralizing anti-CD4 antibodies, or the same tumors grown in —compromised mice (e. g. nu/nu).

The concept of differential anti-tumor effects in -competent versus immune— compromised mice may also permit g of investigational IDO tors as single agents.

For instance, LLC tumors grow well in their ic host strain, C5 7Bl/6. However, if these mice are treated with the IDO inhibitor 1-MT (versus placebo) the formation of tumors is markedly delayed, implying that IDO inhibition was growth inhibitory (Friberg, M., et a]. 2002, Int. J. Cancer 101 : 15 1—155). Following this logic, one can examine the efficacy of IDO inhibition in the LLC xenograft tumor model grown in C5 7B1/6 immune competent mice and compare that to the effects of IDO tors on LLC tumor growth in nude or SCID mice (or C57Bl/6 mice treated with dies that neutralize T-cell activity). As the effects of relieving the tumor-mediated immune suppressive activity of IDO will likely differ depending on the immunogenic potential of different tumor models, genetic modifications can be made to the tumor cells to increase their immunogenic potential. For instance, expression of GM—CSF in B16.F10 cells increases their genic potential (Dranoff, G., et a1. 1993, Proc. Natl. Acad. Sci, USA, 90:3539—3543). As such, in some tumor models (e.g.

B16.F10) one can generate [poly]clones that express immune stimulatory proteins such as GM-CSF and test the growth tory effects of IDO inhibitors against tumors established from these tumor cells in both immune—competent and —compromised mice.

A third avenue for assessing the efficacy of IDO inhibitors in viva employs ‘pre— zation’ murine tumor allograft/xenograft . In these models, immune— competent mice are sensitized to a specific tumor antigen or antigens to mimic a therapeutic anti-tumor vaccination. This primes the mice for an umor response mediated by the immune system when mice are subsequently challenged with murine tumor cell lines (possessing similar tumor antigens to those used for immunization) in xenograft experiments.

Expression of IDO has been shown to blunt the anti-tumor response and allow xenografts to grow more rapidly. Importantly, the growth of tumors in this model is inhibited by the IDO inhibitor 1-MT (Uyttenhove, C., et a]. 2003, Nat. Med. 9:1269—1274). This model is particularly attractive as IDO activity is permissive for P815 tumor growth and specific inhibition of IDO should therefore growth inhibitory.

Lastly, therapeutic immunization may be used to evaluate the impact of IDO inhibitors in vivo. For example, it has been demonstrated using B16-BL6 cells that one can challenge Blk/6 mice with an intravenous injection of tumor cells followed by treatment with a well characterized immunogenic peptide (e. g. TRP—2) expressed by the tumor cells (Ji, et al., 2005, J. l, 175: 1456—63). Importantly, immune system modifiers, such as anti- CTL-4 antibody, can improve responses to such therapeutic zations. The impact of IDO inhibitors may be evaluated in a r manner — tumor peptide immunization with or without IDO inhibitor. cy is assess by animal survival (time to morbidity) or by the measurement of tumor metastases to the lungs and/or other organs at def1nedtimepoints.

In any/all of the above mentioned models, it may also be possible to directly and/or indirectly measure the number and/or activity of tumor ve immune cells. Methods for measuring the number and/or activity of tumor reactive immune cells are well established and can be med using techniques familiar to those schooled in the art (Current Protocols in Immunology, Vol. 4, Coligan, J. E., et al.; Immunotherapy of Cancer, Human Press, 2006, Disis, ML. and nces therein). Conceptually, a ion in the immune suppressive effects of IDO may result in increased s or vity of tumor specific immune cells.

Further, IDO inhibition may further se the number or reactivity of tumor reactive immune cells when combined with other therapeutics, for example herapeutics and/or immune modulators (e. g. anti-CTLA4 antibody).

All allograft/xenograft experiments can be performed using standard tumor techniques (reviewed by Corbett, et al., In Cancer Drug Discovery and Development: Anticancer Drug Development Guide: nical Screening, Clinical Trials, and Approval, 2nd Ed. Teicher, BA. and Andrews, P.A., Gumana Press Inc.: Totowa, NJ, 2004). The cloning and introduction of genes (e. g. IDO, GM—CSF) into tumor cell lines, can be performed using techniques familiar to those schooled in the art (reviewed in Sambrook, J. and Russel, D., Molecular Cloning: A laboratory Manual (3rd edition), Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001).

Example E: In vivo testing of IDO inhibitors in human immunodeficiency virus-1 (HIV- 1) encephalitis model 1. Cell isolation and viral infection Monocytes and PBL can be obtained by countercurrent fugal elutriation of leukopheresis packs from HIV-l, 2 and hepatitis B seronegative donors. Monocytes are ated in suspension e using Teflon flasks in Dulbecco’s ed Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10 % heat-inactivated pooled human serum, 1 % glutamine, 50 ug/mL gentamicin, 10 ug/mL ciprofloxacin (Sigma), and 1000 U/mL highly purified recombinant human macrophage colony ating factor. After seven days in culture, MDM are infected with DA at multiplicity of infection of 0.01. 2. Hu-PBL-NOD/SCID HIVE mice Four—wk old male NOD/C.B—l7 SCID mice can be purchased (Jackson Laboratory).

Animals are maintained in sterile microisolator cages under pathogen-free conditions. All s are injected intraperitoneally with rat anti-CD122 (0.25 mg/mouse) three days before PBL transplantation and twice with rabbit asialo-GMl antibodies (0.2 mg/mouse) (Wako) one day before and three days after PBL injection (20 x 106 cells/mouse). HIV—lADA—infected MDM (3 x 105 cells in 10 uL) are injected intracranially (i.c.) eight days following PBL reconstitution generating hu-PBL-NOD/SCID HIVE mice. ately following i.c. injection of HIV-1 infected MDM the hu—PBL—NOD/SCID HIVE mice are subcutaneously 2014/064531 (s.c) implanted with control (vehicle) or compound pellets (14 or 28 day slow release, Innovative Research). Initial experiments are designed to confirm the ion of virus— ic CTL in the hu PBL-NOD/SCID HIVE animals treated with IDO compounds. This is confirmed by er staining and neuropathologic analyses of MDM elimination from the brain tissue. Then, the experiment is designed to analyze human lymphocyte reconstitution, l immune responses, and neuropathological alterations. In these experiments, animals are bled on day 7 and sacrificed at 14 and 21 days after i.c. ion of human MDM. Blood collected in ontaining tubes is used for flow cytometry and plasma is used for detection of HIV-1 p24 using ELISA (Beckman CoulterTM). speciflc antibodies are detected by Western blot tests according to the manufacturer ctions (Cambridge Biotech HIV-l n blot kit, Calypte Biomedical). Similar amount of specific antibodies are detected in control and compound-treated animals. A total of three ndent experiments can be performed using three different human leukocyte donors. 3. FACScan ofperipheral blood and Spleen in ha PBL-NOD/SCID HIVE mice Two—color FACS analysis can be performed on peripheral blood at wk 1-3 and splenocytes at wk 2 and 3 after i.c. injection of human MDM. Cells are incubated with fluorochrome—conjugated monoclonal Abs (mAbs) to human CD4, CD8, CD56, CD3, IFN—y (eBioscience) for 30 min at 4 0C. To evaluate the cellular immune response, IFN—y intracellular staining is performed in combination with anti-human CD8 and FITC- conjugated anti—mouse CD45 to exclude murine cells. To determine the Ag—specific CTL, allophycocyanin-conjugated tetramer staining for HIV-lgag (p17 (aa77-85) SLYNTVATL, SL-9) and HIV-lp01 [(aa476-485) ILKEPVHGV, IL-9] is performed on phytohemaglutinin/interleukin-2 (PHA/IL-2)- stimulated splenocytes. Cells are stained following the recommendation of the NIH/National ute of Allergy and Infections Disease, National Tetramer Core Facilities. Data were analyzed with a FACS CaliburTM using CellQuest software (Becton Dickinson Immunocytometry System). 4. Histopathology and image analyses Brain tissue is collected at days 14 and 21 after i.c. injection of MDM, fixed in 4 % phosphate-buffered paraformaldehyde and embedded in paraffin or frozen at —80 0C for later use. Coronal sections from the embedded blocks are cut in order to identify the injection site.

For each mouse, 30—100 (5—um-thick) serial ns are cut from the human MDM injection site and 3—7 slides (10 sections apart) are analyzed. Brain sections are deparaff1nized with xylene and hydrated in gradient ls. histochemical staining follows a basic indirect protocol, using antigen retrieval by heating to 95 0C in 0.01 mol/L citrate buffer for min for antigen retrieval. To identify human cells in mouse brains, mAb to vimentin (1 :50, clone 3B4, Dako Corporation), which identifies all human leukocytes is used. Human MDM and CD8+ lymphocytes are detected with CD68 (1:50 dilution, clone KP 1) and CD8 (1:50 on, clone 144B) antibodies, respectively. Virus—infected cells are labeled with mAb to HIV-1 p24 (1:10, clone Kal-l, all from Dako). Reactive murine microglial cells are detected with Iba-1 antibody (1:500, Wako). Expression of human IDO ) is visualized with Abs obtained from the Department of Cell Pharmacology, Central Research Institute, Graduate School of Medicine, Hokkaido University, Sapporo, Japan. Primary antibodies are ed with the appropriate biotinylated secondary antibodies and visualized with avidin- biotin complexes (Vectastain Elite ABC kit, Vector Laboratories) and horseradish dase (HRP) coupled dextran polymer (EnVision, Dako Corporation). Immunostained sections are counterstained with s hematoxylin. Sections from which primary antibody is deleted or irrelevant IgG isotype is incorporated served as controls. Two independent observers in a blinded fashion count the numbers of CD8+ lymphocytes, CD68+ MDM and HIV-1 p24+ cells in each section from each mouse. Light microscopic examination is performed with a Nikon Eclipse 800 microscope (Nikon Instruments Inc). Semi—quantitative is for Iba1 (percentage of area occupied by immunostaining) is carried out by computer—assisted image analysis (Image—Pro®Plus, Media Cybernetics) as previously bed.

. Statistical analysis Data can be analyzed using Prism (Graph Pad) with Student t-test for comparisons and ANOVA. P—values < 0.05 were considered icant. 6. Reference Poluektova LY, Munn DH, Persidsky Y, and Gendelman HE (2002). Generation of cytotoxic T cells against infected human brain macrophages in a murine model of HIV- 1 encephalitis. J. Immunol. 168(8):3941—9. s modifications of the invention, in addition to those described herein, will be nt to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patent, patent applications, and publications, cited in the present ation is incorporated herein by reference in their entirety.

What is claimed is: 1. A process, comprising reacting a compound of a F17: wherein R4 is C1-6 alkyl, C1-6 haloalkyl, benzyl, or 9H-fluorenylmethyl with a nd of Formula F5: to afford a compound of Formula F18: 2. The process of claim 1, wherein R4 is tert-butyl. 3. The process of claim 1, wherein R4 is benzyl. 4. The process of claim 1, wherein R4 is ethyl.

. The process of claim 1, wherein R4 is 2,2,2-trichloroethyl. 6. The process of any one of claims 2-5, wherein said reacting is carried out in the presence of a ng agent. 7. The process of claim 6, wherein said reducing agent is triethylsilane. 8. The process of claim 6 or 7, wherein said reacting is carried out in the presence of an organic acid. 9. The process of claim 8, wherein said organic acid is trifluoroacetic acid.

. The process of any one of claims 2-9, further sing deprotecting said compound of Formula F18 to afford a compound of Formula F10: 11. The process of claim 10, wherein said deprotecting comprises reacting the compound of Formula F18 with zinc in the presence of acetic acid. 12. The process of claim 10 or 11, r comprising ng said compound of Formula F10 with a base to afford a compound of Formula I: 13. The process of claim 12, wherein said base is sodium hydroxide. 14. The process of claim 1, wherein R4 is 9H-fluorenylmethyl.

. The process of claim 14, wherein said ng is d out in the presence of a reducing agent. 16. The process of claim 15, wherein said reducing agent is triethylsilane. 17. The process of claim 15 or 16, wherein said reacting is carried out in the presence of an organic acid. 18. The process of claim 17, wherein said organic acid is methanesulfonic acid. 19. The process of any one of claims 14-18, further comprising converting said nd of Formula F18 to a compound of a I: wherein said converting comprises combining the nd of Formula F18 with a base to form a first mixture.

. The process of claim 19, wherein said base is N,N-bis(2-aminoethyl)ethane-1,2- diamine. 21. The s of any one of claims 19 to 20, wherein said converting further comprises adding aqueous hydrochloric acid to said first mixture. 22. A compound of Formula F17: wherein R4 is C1-6 haloalkyl, benzyl, or 9H-fluorenylmethyl. 23. A compound, which is tert-butyl N-(2,2-dimethoxyethyl)sulfamoylcarbamate. 24. The compound of claim 22, which is benzyl N-(2,2- dimethoxyethyl)sulfamoylcarbamate.

. A compound, which is ethyl N-(2,2-dimethoxyethyl)sulfamoylcarbamate. 26. The compound of claim 22, which is 2,2,2-trichloroethyl N-(2,2- dimethoxyethyl)sulfamoylcarbamate. 27. The compound of claim 22, which is (9H-fluorenyl)methyl N-(2,2- dimethoxyethyl)sulfamoylcarbamate. 28. A process, comprising: i) reacting a nd of a F19: O O O S OMe O N N H H with a compound of Formula F5: in the presence of triethylsilane and methanesulfonic acid to afford a compound of Formula F20: ii) converting said compound of Formula F20 to a compound of Formula I: wherein said converting comprises combining said compound of Formula F20 with N,N-bis(2-aminoethyl)ethane-1,2-diamine. 29. A compound of Formula F18: wherein R4 is benzyl, ethyl, C1-3 haloalkyl, 2,2,2-trichloroethyl or 9H-fluorenylmethyl.

. The compound of claim 29, wherein R4 is benzyl. 31. The compound of claim 29, wherein R4 is ethyl. 32. The nd of claim 29, wherein R4 is C1-3 kyl. 33. The compound of claim 29, wherein R4 is 2,2,2-trichloroethyl. 34. The compound of claim 29, wherein R4 is 9H-fluorenylmethyl.

Claims (34)

What is claimed is:

1. A process, comprising reacting a compound of a F17: wherein R4 is C1-6 alkyl, C1-6 haloalkyl, benzyl, or 9H-fluorenylmethyl with a nd of Formula F5: to afford a compound of Formula F18:

2. The process of claim 1, wherein R4 is tert-butyl.

3. The process of claim 1, wherein R4 is benzyl.

4. The process of claim 1, wherein R4 is ethyl.

5. The process of claim 1, wherein R4 is 2,2,2-trichloroethyl.

6. The process of any one of claims 2-5, wherein said reacting is carried out in the presence of a ng agent.

7. The process of claim 6, wherein said reducing agent is triethylsilane.

8. The process of claim 6 or 7, wherein said reacting is carried out in the presence of an organic acid.

9. The process of claim 8, wherein said organic acid is trifluoroacetic acid.

10. The process of any one of claims 2-9, further sing deprotecting said compound of Formula F18 to afford a compound of Formula F10:

11. The process of claim 10, wherein said deprotecting comprises reacting the compound of Formula F18 with zinc in the presence of acetic acid.

12. The process of claim 10 or 11, r comprising ng said compound of Formula F10 with a base to afford a compound of Formula I:

13. The process of claim 12, wherein said base is sodium hydroxide.

14. The process of claim 1, wherein R4 is 9H-fluorenylmethyl.

15. The process of claim 14, wherein said ng is d out in the presence of a reducing agent.

16. The process of claim 15, wherein said reducing agent is triethylsilane.

17. The process of claim 15 or 16, wherein said reacting is carried out in the presence of an organic acid.

18. The process of claim 17, wherein said organic acid is methanesulfonic acid.

19. The process of any one of claims 14-18, further comprising converting said nd of Formula F18 to a compound of a I: wherein said converting comprises combining the nd of Formula F18 with a base to form a first mixture.

20. The process of claim 19, wherein said base is N,N-bis(2-aminoethyl)ethane-1,2- diamine.

21. The s of any one of claims 19 to 20, wherein said converting further comprises adding aqueous hydrochloric acid to said first mixture.

22. A compound of Formula F17: wherein R4 is C1-6 haloalkyl, benzyl, or 9H-fluorenylmethyl.

23. A compound, which is tert-butyl N-(2,2-dimethoxyethyl)sulfamoylcarbamate.

24. The compound of claim 22, which is benzyl N-(2,2- dimethoxyethyl)sulfamoylcarbamate.

25. A compound, which is ethyl N-(2,2-dimethoxyethyl)sulfamoylcarbamate.

26. The compound of claim 22, which is 2,2,2-trichloroethyl N-(2,2- dimethoxyethyl)sulfamoylcarbamate.

27. The compound of claim 22, which is (9H-fluorenyl)methyl N-(2,2- dimethoxyethyl)sulfamoylcarbamate.

28. A process, comprising: i) reacting a nd of a F19: O O O S OMe O N N H H with a compound of Formula F5: in the presence of triethylsilane and methanesulfonic acid to afford a compound of Formula F20: ii) converting said compound of Formula F20 to a compound of Formula I: wherein said converting comprises combining said compound of Formula F20 with N,N-bis(2-aminoethyl)ethane-1,2-diamine.

29. A compound of Formula F18: wherein R4 is benzyl, ethyl, C1-3 haloalkyl, 2,2,2-trichloroethyl or 9H-fluorenylmethyl.

30. The compound of claim 29, wherein R4 is benzyl.

31. The compound of claim 29, wherein R4 is ethyl.

32. The nd of claim 29, wherein R4 is C1-3 kyl.

33. The compound of claim 29, wherein R4 is 2,2,2-trichloroethyl.

34. The compound of claim 29, wherein R4 is 9H-fluorenylmethyl.