indoleamine 2,3-dioxigenase inhibitor compounds and their synthesis processes

专利摘要:
INDOLEAMINE 2,3-DIOXYGENASE INHIBITORS AND THEIR SYNTHESIS PROCESS. The present application is directed to processes and intermediates to prepare 4 - ({2 - [(aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'-hydroxy-1,2,5 -oxadiazole-3-carboximidamide, which is an inhibitor of indolamine 2,3-dioxigenase, useful in the treatment of cancer and other disorders.
公开号:BR112016009786B1
申请号:R112016009786-6
申请日:2014-11-07
公开日:2021-01-05
发明作者:Ming Tao；William Frietze；David J. Meloni；Lingkai Weng；Jiacheng Zhou；Yongchun Pan
申请人:Incyte Holdings Corporation；
IPC主号:

专利说明:

[001] This application claims the priority benefit of Provisional Application US 61 / 901,689, filed on November 8, 2013, which is incorporated herein by reference in its entirety. FIELD OF INVENTION
[002] The present application concerns the processes and intermediates for preparing 4 - ({2 - [(aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'-hydroxy-1 , 2,5-oxadiazole-3-carboxyimidamide, which is an indolamine 2,3-dioxigenase inhibitor useful in the treatment of cancer and other disorders. BACKGROUND OF THE INVENTION
[003] Tryptophan (Trp) is an essential amino acid necessary for the biosynthesis of proteins, niacin and the neurotransmitter 5-hydroxytryptamine (serotonin). The enzyme indolamine-2,3-dioxigenase (also known as INDO or IDO) catalyzes the first step and rate of limitation in the degradation of L-tryptophan to N-formyl-quinurenine. In human cells, a Trp depletion resulting from IDO activity is a prominent antimicrobial effector mechanism inducible by gamma interferon (IFN-Y). Stimulation of IFN-y induces activation of IDO, which leads to Trp depletion, thereby interrupting 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, indicating a possible role for this enzyme in the tumor rejection process (Daubener, et al., 1999, Adv. Exp. Med. Biol., 467: 517-24; Taylor, et al., 1991, FASEB J., 5: 2516-22).
[004] It has been observed that HeLa cells co-cultured with peripheral blood lymphocytes (PBL), acquire an immune-inhibitory phenotype through positive regulation of IDO activity. A reduction in PBL proliferation by treatment with interleukin-2 (IL2) appears to result from the IDO released by tumor cells in response to IFNG secretion by PBLs. This effect was reversed by treatment with 1-methyl-tryptophan (1MT), a specific IDO inhibitor. It has been proposed that IDO activity in tumor cells can serve to impair antitumor responses (Logan, et al., 2002, Immunology, 105: 478-87).
[005] Recently, an immunoregulatory role of Trp depletion has received much attention. Several lines of evidence suggest that IDO is involved in inducing immune tolerance. Studies of mammalian pregnancies, tumor resistance, chronic infections, and autoimmune diseases have shown that cells that express IDO can suppress T cell responses and promote tolerance. Accelerated Trp catabolism has been observed in diseases and disorders associated with cellular immune activation, such as infection, malignancy, autoimmune diseases and AIDS, as well as during pregnancy. For example, increased levels of IFN and high levels of urinary Trp metabolites have been observed in autoimmune diseases; it has been postulated that systemic or local Trp depletion that occurs in autoimmune diseases may be related to the degeneration and wasting symptoms of these diseases. In support of this hypothesis, high levels of IDO have been observed in cells isolated from the synovium of arthritic joints. IFNs are also high in patients with human immunodeficiency virus (HIV) and increased IFN levels are associated with a worsening prognosis. Thus, it has been proposed that IDO is chronically induced by the HIV virus, and is further increased by opportunistic infections, and that chronic Trp loss initiates mechanisms responsible for cachexia, dementia and diarrhea and, possibly, the immunosuppression of AIDS patients ( Brown, et al., 1991, Adv. Exp. Med. Biol., 294: 425-35). To this end, it has recently been shown that inhibition of IDO can increase levels of virus-specific T cells and, concomitantly, reduce the number of virally infected macrophages in a mouse model of HIV (Portula et al., 2005, Blood, 106: 2382-90).
[006] IDO is believed to play a role in immunosuppressive processes that prevent fetal rejection in the uterus. More than 40 years ago, it was observed that, during pregnancy, the concept of genetically disparate mammals survives despite what would be predicted by tissue transplantation immunology (Medawar, 1953, Symp. Soc. Exp. Biol. 7: 320-38). The anatomical separation of the mother and fetus and the antigenic immaturity of the fetus cannot fully explain the survival of the fetal graft. Recent attention has focused on the mother's immune tolerance. Because IDO is expressed by human syncytiotrophoblast cells and the systemic tryptophan concentration drops during normal pregnancy, it was hypothesized that the IDO expression at the maternal-fetal interface is necessary to prevent the immune rejection of fetal allografts. To test this hypothesis, pregnant mice (carrying singene or allogeneic fetuses) were exposed to 1MT, and a rapid induced T cell rejection of all allogeneic concepts was observed. Thus, by catabolizing tryptophan, the mammalian concept appears to suppress T cell activity and defend itself against rejection, and blocking tryptophan catabolism during murine pregnancy allows maternal T cells to cause rejection of fetal allograft (Munn, et al., 1998, Science, 281: 1191-3).
[007] Additional evidence for a tumor immune resistance mechanism based on degradation of tryptophan by IDO comes from the observation that most human tumors constitutively express IDO, and that expression of IDO by immunogenic mouse tumor cells prevents its rejection by previously immunized mice. This effect is accompanied by a lack of accumulation of specific T cells at the tumor site and can be partially reversed by systemic treatment of mice with an IDO inhibitor, in the absence of evident toxicity. Thus, it has been suggested that the efficacy of therapeutic vaccination of cancer patients could 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, 1-MT, can synergize with chemotherapeutic agents to reduce tumor growth in mice, suggesting that inhibition of IDO may also increase the antitumor activity of conventional cytotoxic therapies (Muller et al ., 2005, Nature Med., 11: 312-9).
[008] A mechanism that contributes to the lack of immunological responsiveness to tumors may be the presentation of tumor antigens by tolerogenic host APCs. A subset of human cells that express antigen presenting IDO (APCs), which co-expressed CD123 (IL3RA) and CCR6 and inhibited T cell proliferation have also been described. Both mature and immature CD123 positive dendritic cells suppressed T cell activity, and this IDO suppressive activity was blocked by 1MT (Munn, et al., 2002, Science, 297: 1867-70). It has also been shown that tumor drainage lymph nodes in mice (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 suppressed T cell responses to the antigens presented by the pDCs themselves and also, in a dominant way, suppressed T cell responses to third antigen. - Genes presented by non-suppressive APC. Within the population of pDCs, most of the suppressor activity mediated by functional IDO segregated with a new subset of pDCs that co-express the CD19 lineage B marker. Thus, if the hypothesis that IDO-mediated suppression by pDCs in TDLNs create a local microenvironment that is potentially suppressive of the responses of host antitumoral T cells (Munn, et al., 2004, J. Clin. Invest., 114 (2): 280-90).
[009] IDO degrades the indole fraction of tryptophan, serotonin and melatonin, and starts the production of neuroactive and immunoregulatory metabolites, collectively known as quinurenines. By locally depleting tryptophan and increasing pro-apoptotic quinurenines, IDO expressed by dendritic cells (DCs) can greatly affect T cell proliferation and survival. The induction of IDO in DCs can be a common mechanism of tolerance to deletion directed by regulatory T cells. Since such tolerogenic responses can be expected to operate in a wide variety of pathophysiological conditions, tryptophan metabolism and quinurenine production can represent a crucial interface between the immune and nervous systems (Grohmann, et al., 2003, Trends Immunol., 24: 242-8). In states of persistent immune activation, availability of Trp-free serum is decreased and, as a consequence of reduced serotonin production, serotonergic functions can also be affected (Wirelitner, et al., 2003, Curr. Med. Chem. ., 10: 1581-91).
[0010] Interestingly, the administration of interferon-a was observed to induce neuropsychiatric side effects such as depressive symptoms and changes in cognitive function. The direct influence on serotonin neurotransmission may contribute to these side effects. In addition, because IDO activation leads to reduced levels of tryptophan, the precursor to serotonin (5-HT), IDO may play a role in these neuropsychiatric side effects, reducing central 5-HT synthesis. In addition, quinurenine metabolites, such as 3-hydroxy-quinurenine (3-OH-KYN) and quinolinic acid (QUIN) have toxic effects on brain functions. 3-OH- KYN is capable of producing oxidative stress, increasing the production of reactive oxygen species (ROS), and QUIN can produce excessive stimulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus, which leads to apoptosis and atrophy of the hippocampus. Both overproduction of ROS and atrophy of the hippocampus caused by NMDA hyperstimulation have been associated with depression (Wichers and Maes, 2004, J. Psychiatry Neurosci., 29: 11-17). Thus, IDO activity can play a role in depression.
[0011] Small molecule IDO inhibitors are being developed to treat or prevent IDO-related diseases such as those described 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 which comprises altering local extracellular concentrations of tryptophan and tryptophan metabolites, using an IDO inhibitor 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 WO 03/087347, also published as European patent 1501918, are methods of preparing antigen presenting cells to increase or reduce T cell tolerance (Munn, 2003). Compounds that have indolamine-2,3-dioxigenase (IDO) inhibitory activity are further reported in WO 2004/094409; and US Patent Application Publication 2004/0234623 refers to methods of treating a subject with cancer or infection by administering an inhibitor of indamine-2,3-dioxigenase in combination with other therapeutic modalities.
[0012] In light of experimental data indicating 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 immunological tolerance and prevention of fetal rejection in the womb, therapeutic agents aimed at suppressing the degradation of tryptophan by inhibiting IDO activity are desirable. IDO inhibitors can be used to activate T cells and therefore increase T cell activation when T cells are suppressed by pregnancy, malignancy or a virus such as HIV. Inhibition of IDO can also be an important treatment strategy for patients with neurological diseases or neuropsychiatric disorders such as depression.
[0013] Due to the usefulness of IDO inhibitors, there is a need for the development of new processes to prepare IDO inhibitors. This application is addressed to that need and others. SUMMARY OF THE INVENTION
[0014] The compound 4 - ({2 - [(aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'-hydroxy-1,2,5-oxadiazole-3-carboxyidamide with formula I:
it is an inhibitor of the enzyme indolamine-2,3-dioxigenase (also known as IDO). The Formula I compound, as well as its preparation and use, has been described in US Patent 8,088,803, which is incorporated herein by reference in its entirety. The intermediates and processes provided here help to satisfy the continuing need for the development of IDO inhibitors for the treatment of serious diseases.
[0015] The present application provides, inter alia, intermediates and processes for the preparation of a compound of Formula I:

[0016] Accordingly, the present application provides a process comprising the reaction of a compound of Formula F5:
with a Formula F6 aldehyde:
to generate a compound of Formula F7:
where Pg1 is defined below.
[0017] The present application additionally provides a process comprising the reaction of a compound of Formula F15:
with a Formula F5 compound to provide a Formula F16 compound:
where R1 and R3 are defined below.
[0018] The present application additionally provides a process comprising the reaction of a compound of Formula F17:
with a Formula F5 compound to provide a Formula F18 compound:
where R4 is defined below. DETAILED DESCRIPTION
[0019] Although some of the process steps are illustrated in the Schemes below, it is intended that the individual process steps can be claimed individually or in any combination (for example, in Scheme I, steps E, F, G, H, and I can be claimed individually or in combination). The processes are not intended to be limited to a global process with each step in the diagrams below.
[0020] Thus, the general scheme for the preparation of the compound of Formula I is described in Scheme 1. Scheme 1

[0021] Consequently, the process comprising the reaction of a compound of Formula F5:
with a Formula F6 aldehyde:
where Pg1 is an amino protecting group, to generate a compound of Formula F7:

[0022] Pg1 amino protecting groups can be used to prevent unwanted reactions from an amino group during the execution of a desired transformation. Amino-protecting groups allow easy covalent bonding of a nitrogen atom, as well as selective cleavage of the nitrogen atom. Suitable “amine protecting groups”, such as alkoxycarbonyl (such as ethoxycarbonyl, tert butoxycarbonyl (Boc), benzylxicarbonyl (Cbz), 9-fluorenylmethylxicarbonyl (Fmoc), and the like), acyl (such as acetyl (Ac), benzoyl (Bz), and the like), sulfonyl (such as methanesulfonyl, trifluoromethanesulfonyl, and the like), arylalkyl (such as benzyl, 4-methoxybenzyl, diphenylmethyl, triphenylmethyl (trityl), and the like), alkenylalkyl (such as allyl, prenyl, and the like), diarylmethylenyl (such as (C6H5) 2C = N, and the like), and silyl (such as tert-butyldimethylsilyl, tri-isopropylsilyl, and the like), are known to a person skilled in the art. The chemistry of amino protection groups can be found in Wuts and Greene, Greene's Protective Groups in Organic Synthesis, 4th Ed., Pp 696-926, John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its wholeness.
[0023] In some modalities, Pg1 is ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl or 9-fluorenylmethyloxycarbonyl.
[0024] In some modalities, Pg1 is C 1-6 alkoxycarbonyl.
[0025] In some modalities, Pg1 is tert-butoxycarbonyl.
[0026] Solvents suitable for Step E include, but are not limited to, methanol or tetrahydrofuran (THF), acetonitrile and the like. Halogenated hydrocarbon solvents (ie, halogenated alkanes, such as dichloromethane, chloroform, dichloroethane or tetrachloroethane) can also be used.
[0027] In some embodiments, said reaction is carried out in a component solvent comprising tetrahydrofuran. As used herein, a solvent component can refer to a solvent or a mixture of solvents. In some embodiments, the solvent component is an organic solvent. In some embodiments, the said reaction is carried out in a solvent component comprising a halogenated hydrocarbon solvent. In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.
[0028] In some embodiments, said reaction is carried out in a component solvent comprising acetonitrile.
[0029] In some embodiments, said reaction is carried out in a solvent component comprising dichloromethane and acetonitrile.
[0030] In some modalities, said reaction is carried out in the presence of a reducing agent.
[0031] The reducing agent can be any compound capable of reducing an organic compound to a lower oxidation state. The reduction generally involves adding hydrogen atoms or removing oxygen atoms from a group. For example, aldehydes such as F6 can be reduced in the presence of an amine of Formula F5 (Step E, Scheme 1) by adding hydrogen, either in the form of hydrogen gas (H2) or using a hydride reagent (such as NaB (OAc) 3H, NaBH 4, LiAlH4, and the like); using triphenylphosphine; or using a combination of sodium iodide, chlorotrimethylsilane, and methanol. In some embodiments, this step can be performed under acidic conditions, in the presence of an acid (such as trifluoroacetic acid). In some embodiments, this step can be performed at a temperature of about -15 ° C to about 30 ° C, for example, from about -15 ° C to about 0 ° C, from about -5 ° C at about 5 ° C, from about -5 ° C to about 0 ° C, or between about 0 ° C to about 45 ° C.
[0032] In some embodiments, said reducing agent is a borohydride reducing agent (for example, NaB (OAc) 3H, NaBH4, or other boron containing hydride reducing agent).
[0033] In some embodiments, said borohydride reducing agent is sodium triacetoxyborohydride.
[0034] In some modalities, said reaction is carried out in the presence of trifluoroacetic acid.
[0035] In some embodiments, the process further comprises deprotecting said Formula F7 compound to provide a Formula F8 compound:

[0036] Amino deprotecting agents useful for this step F are known to those skilled in the art, such as those in Wuts and Greene (above). In particular, the above described amino protecting groups can be conveniently removed using various available amino deprotecting agents that are specific to the various groups mentioned above without affecting other desired portions of the compound. The tert-butoxycarbonyl group can be removed (for example, hydrolyzate) 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 (for example, mixture of acetyl chloride and methanol) known to generate an acid; or a Lewis acid (for example, BF3 ^ Et2O). The benzyloxycarbonyl group can be removed (for example, hydrogenolysis) from the nitrogen atom, for example, by treatment with hydrogen and a catalyst (such as palladium on carbon).
[0037] In some embodiments, the amino deprotecting agent is trifluoroacetic acid. In some embodiments, the amino deprotecting agent contains trifluoroacetic acid and> 0.5% by volume of water, for example,> 1.0% by volume of water,> 1.5% by volume of water,> 2, 0% by volume of water, between about 2% to about 10% by volume of water, between about 10% to about 20% by volume of water, or from about 20% to about 50% in volume of water. In some embodiments, the amine deprotecting agent can be a mixture of trifluoroacetic acid and water in a volume ratio of about 98: 2. In some embodiments, the amino deprotecting agent can be hydrochloric acid, optionally in a solvent (for example, water, THF, dioxane, ethyl acetate, etc.). In some embodiments, the solvent component is ethyl acetate. In some embodiments, the amino deprotecting agent may be hydrochloric acid, optionally, in a solvent such as an alcohol (such as isopropanol, methanol or ethanol). Halogenated hydrocarbon solvents (eg, dichloromethane, chloroform, dichloroethane or tetrachloroethane) 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 of about -10 ° C to about 60 ° C, for example, from about -10 ° C to about 0 ° C, from about 0 ° C at about 25 ° C, from about 25 ° C to about 45 ° C, or between about 45 ° C to about 60 ° C.
[0038] In some modalities, said deprotection comprises the reaction of the compound of Formula F7 with hydrochloric acid.
[0039] In some embodiments, said deprotection comprises the reaction of the compound of Formula F7 with hydrochloric acid in a solvent component comprising isopropanol.
[0040] In some embodiments, said deprotection comprises the reaction of the compound of Formula F7 with hydrochloric acid in a solvent component comprising a halogenated hydrocarbon solvent.
[0041] In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.
[0042] In some embodiments, the invention further comprises the reaction of said compound of Formula F8, with Pg2-NH-SO2-X, in the presence of an organic base to generate a compound of Formula F9:
on what:
[0043] Pg2 is an amino protecting group; and
[0044] X is halo.
[0045] In some modalities, Pg2-NH-SO2Cl can be prepared and immediately used in the reaction with the compound of Formula F8. The protecting group Pg2 can be selected from any of the protecting groups known in the art for the protection of amines or sulfonamides (such as those described above for Pg1). In some embodiments, Pg2 may be an alkoxycarbonyl group (such as tert-butoxycarbonyl).
[0046] Suitable 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 include tertiary acyclic amines such as tri (C1-6) alkylamine (for example, triethylamine, diisopropylethylamine (DIPEA) and the like), cyclic tertiary amines (for example, N-methylpiperidine, 1,4-diazabicyclo [ 2.2] octane (DABCO) and the like). In some modalities, the organic base may be triethylamine. In some embodiments, this step can be carried out at a temperature of about - 15 ° C to about 60 ° C, for example, 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 between about 45 ° C to about 60 ° C.
[0047] In such modalities, Pg2-NH-SO2Cl can be obtained by reacting an alcohol (such as ethanol, tert-butyl alcohol and the like) with chlorosulfonyl isocyanate (ClS (O) 2NCO).
[0048] In some modalities, Pg2 is ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl or 9-fluorenylmethyloxycarbonyl.
[0049] In some modalities, Pg2 is C1-6 alkoxycarbonyl.
[0050] In some modalities, Pg2 is tert-butoxycarbonyl.
[0051] In some embodiments, said reaction is carried out in a solvent component comprising a halogenated hydrocarbon solvent.
[0052] In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.
[0053] In some embodiments, said organic base comprises tri (C1-6) alkylamine.
[0054] In some embodiments, said organic base is triethylamine.
[0055] In some modalities, X is chlorine.
[0056] In some embodiments, the invention further comprises deprotecting said Formula F9 compound to provide a Formula F10 compound:

[0057] In some embodiments, suitable deprotecting agents may include those described above for the deprotection of the Formula F7 compound.
[0058] In some embodiments, said deprotection comprises preparing to react a compound of Formula F9 with hydrochloric acid. In some embodiments, said deprotection comprises reacting a compound of Formula F9 with hydrochloric acid in a solvent component comprising an alcohol. In some modalities, the alcohol referred to is ethanol. In some embodiments, said deprotection comprises reacting a compound of Formula F9 with hydrochloric acid in a solvent component which comprises ethyl acetate.
[0059] In some embodiments, the invention further comprises reacting said compound of Formula F10 with a base to give a compound of Formula I:

[0060] A base can be used for the conversion (for example, hydrolysis) of the oxadiazolone ring to F10 to reveal the starch in the Formula I compound, optionally in a solvent (Step I, Scheme 1). The protection of amidoxime such as oxadiazolone can be useful to avoid adverse reactions of the hydroxyl group or that of amidoxima as a whole. The base can be an inorganic base such as alkali metal hydroxide (for example, NaOH, LiOH, KOH, Mg (OH) 2, etc.); or an organic base such as an acyclic amine (for example, triethylamine, diisopropylethylamine (DIPEA), etc.) or a cyclic amine (for example, pyrrolidine, piperidine, etc.). The base can be made available in the form of a resin (eg Amberlite® and the like). In some other embodiments, the base may be provided as a solution in water (for example, about 0.5 N solution, about 1 N solution, about 1.5 N solution, about 2.5 N solution, about 3 N to about 5 N solution, 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 may be a solution of 2 N NaOH in water. In some embodiments, the solvent may be ethanol or tetrahydrofuran (THF). In some embodiments, the solvent may be a mixture of ethanol and water. In some embodiments, the reaction of the Formula F10 compound with a base to generate the Formula I compound can be carried out at a temperature of about -10 ° C to about 60 ° C, for example, about -10 ° C to about 20 ° C, about 0 ° C to about 30 ° C, about 0 ° C to about 10 ° C, or between about 0 ° C to about 5 ° C.
[0061] In some embodiments, said base comprises an alkali metal hydroxide.
[0062] In some embodiments, said alkali metal hydroxide is sodium hydroxide.
[0063] In some embodiments, said reaction is carried out in a solvent component that comprises tetrahydrofuran, water and ethanol.
[0064] In some embodiments, the compound of Formula F5 can be obtained in a sequence of steps represented in Scheme 2. The preparation of the intermediate, 4-amino-N'-hydroxy-1,2,5-oxadiazole-3 - carboximidamide, has been described in J. Heterocycl. Chem. (1965), 2, 253, which is incorporated herein by reference in its entirety, and its conversion to chlorine oxime F3 has been described in Synth. Commun. (1988), 18, 1427, which is incorporated herein by reference in its entirety. In some embodiments, the chlorine oxime of Formula F3 can be coupled to 3-bromo-4-fluoroaniline, optionally in a solvent (such as water), followed by the addition of sodium bicarbonate, to provide an amidoxime of Formula F4. The starch-oxime functionality of the F4 compound 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 temperatures elevated such as about 50 ° C, about 60 ° C, about 70 ° C, about 80 ° C, about 90 ° C, or about 100 ° C. Layout 2

[0065] Alternatively, the compound of Formula F10 can be obtained through a sequence of steps described in scheme 3. Scheme 3

[0066] In some embodiments, the present application provides a process, which comprises the reaction of a compound of Formula F15:
with a compound of Formula F5:
to generate a compound of Formula F16:
on what:
[0067] each R1 is, independently, a protecting group of amine; and
[0068] R3 is C1-6 alkyl or benzyl.
[0069] In some embodiments, R1 is C2-4alkenyl-C1-3alkyl or phenyl-C1-3alkyl, wherein said phenyl-C1-3alkyl is optionally substituted by 1, 2, or 3 independently selected C1-4 alkoxy groups.
[0070] 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.
[0071] In some modalities, R1 is ally.
[0072] In some embodiments, R1 is 4-methoxybenzyl.
[0073] In some embodiments, R3 is C1-6 alkyl.
[0074] In some modalities, R3 is tert-butyl.
[0075] In some embodiments, R3 is C1-4 alkyl.
[0076] In some modalities, R3 is butyl.
[0077] Preferably, the reaction is carried out 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. In some embodiments, the reducing agent may be hydrogen gas in the presence 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 carried out 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 temperature of about -15 ° C to about 30 ° C, for example, from about -15 ° C to about 0 ° C, from about -5 ° C at about 5 ° C, from about -5 ° C to about 0 ° C, about 0 to 5 ° C, or between about 0 ° C to about 45 ° C.
[0078] In some embodiments, said reaction is carried out in a component solvent comprising tetrahydrofuran.
[0079] In some modalities, said reaction is carried out in the presence of a reducing agent.
[0080] In some embodiments, said reducing agent is a borohydride reducing agent.
[0081] In some embodiments, said borohydride reducing agent is sodium triacetoxyborohydride.
[0082] In some modalities, said reaction is carried out in the presence of trifluoroacetic acid.
[0083] In some embodiments, the invention further comprises deprotection of said compound of Formula F16 to generate a compound of Formula F10:

[0084] The treatment of an F16 compound to replace R1N with NH2 can be carried out by methods for the deprotection of specific amino protecting groups known to those skilled in the art, such as those in Wuts and Greene, Greene's Protective 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 may be a palladium catalyst (eg, Pd (Ph3P) 4, Pd / C, or Pd (dba) DPPB). In some embodiments, when R1 is 4-methoxybenzyl, the deprotecting agent may 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 protection can be carried out at a temperature of about 30 ° C to about 90 ° C, for example, from about 50 ° C to about 100 ° C, or between about 60 ° C to about 80 ° C.
[0085] In some embodiments, said deprotection comprises preparing to react a compound of Formula F16 with trifluoroacetic acid.
[0086] In some embodiments, said deprotection comprising reacting a compound of Formula F16 is with hydrochloric acid.
[0087] Compound F15 can be prepared by a three-step process (Steps J, K and L) from chlorosulfonylisocyanate, as shown in Scheme 4. Scheme 4

[0088] Therefore, the present application additionally provides a process in which said compound of Formula F15 is obtained by a process which comprises the treatment of a compound of Formula F14:
with a reducing agent to generate said compound of Formula F15; wherein R2 is C1-4alkyl; and R3 is defined above.
[0089] In some modalities, R2 is methyl.
[0090] In some modalities, R2 is ethyl.
[0091] In some modalities, the reduction can be carried out with diisobutylaluminum hydride (DIBAL-H). Suitable solvents include halogenated hydrocarbon solvents, such as dichloromethane, chloroform, dichloroethane, tetrachloroethane, and the like. In some embodiments, the reduction may be carried out at about room temperature, for example, from about -80 ° C to about 30 ° C, from about -78 ° C to about 0 ° C, from about from 0 ° C to about 30 ° C, or from about 25 ° C to about 30 ° C.
[0092] In some embodiments, said treatment is carried out in a halogenated hydrocarbon solvent.
[0093] In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.
[0094] In some embodiments, said reducing agent is diisobutylaluminum hydride.
[0095] In some embodiments, said compound of Formula F14 is obtained by a process that comprises the protection of a compound of Formula F13:
with one or more amino-protecting agents independently selected to generate a compound of Formula F14.
[0096] The protection group R1 in F14 can be selected from among the various amino protection groups known in the art (above). In some embodiments, the amino-protecting agent is allyl bromide or 4-methoxybenzyl chloride.
[0097] In some embodiments, said one or more amino protection agents are selected from allyl bromide and 4-methoxybenzyl chloride.
[0098] In some modalities, said protection is performed in the presence of a base.
[0099] In some embodiments, said base is potassium carbonate.
[00100] In some modalities, said protection is performed in a solvent component comprising acetonitrile.
[00101] In some embodiments, the preparation of the compound of F13 can be obtained by treating chlorosulfonyl isocyanate with an alcohol R3OH (where R3 is defined above) and a glycine ester H2NCH2CO2R2, where R2 is C1-4 alkyl. In some embodiments, this step J is carried out in the presence of an organic acid (such as acetic acid, benzoic acid, trifluoroacetic acid). Suitable solvents for this step include dichloromethane, chloroform, dichloroethane, tetrachloroethane, and the like.
[00102] In some embodiments, the present application provides a compound of Formula F13:
on what:
[00103] R2 is C1-4 alkyl; and
[00104] R3 is C1-6 alkyl or benzyl.
[00105] In some modalities, R2 is methyl.
[00106] In some modalities, R2 is ethyl.
[00107] In some embodiments, R3 is C1-6 alkyl.
[00108] In some modalities, R3 is tert-butyl.
[00109] In some embodiments, the compound of Formula F13 is ethyl-2- ((N- (tert-butoxycarbonyl) sulfamoyl) amino) acetate:

[00110] In some embodiments, the invention further provides a compound of Formula F14:
on what:
[00111] each R1 independently represents an amino protection group;
[00112] R2 is C1-4 alkyl; and
[00113] R3 is C1-6 alkyl or benzyl.
[00114] In some embodiments, R1 is C2-4alkenyl-C1-3alkyl or phenyl-C1-3alkyl, wherein said phenyl-C1-3alkyl is optionally substituted by 1, 2, or 3 independently selected C1-4 alkoxy groups.
[00115] In some modalities, R1 is ally.
[00116] In some embodiments, R1 is 4-methoxybenzyl.
[00117] In some modalities, R2 is methyl.
[00118] In some modalities, R2 is ethyl.
[00119] In some embodiments, R3 is C1-6 alkyl.
[00120] In some modalities, R3 is tert-butyl.
[00121] In some embodiments, R3 is C1-4 alkyl.
[00122] In some embodiments, R3 is butyl.
[00123] In some embodiments, R3 is C1-4 alkyl.
[00124] In some embodiments, R3 is butyl.
[00125] In some embodiments, the compound of Formula F14 is ethyl-2- (allyl (N-allyl-N- (tert-butoxycarbonyl) sulfamoyl) amino) acetate:

[00126] In some embodiments, the compound of Formula F14 is ethyl-2- (4-methoxybenzyl (N-4-methoxybenzyl-N- (tert-butoxycarbonyl) sulfamoyl) amino) acetate:

[00127] In some embodiments, the present application provides a compound of Formula F15:
on what:
[00128] noA no. R3 is C1-6 alkyl or benzyl; and
[00129] each R1 is, independently, a protecting group of
[00130] In some embodiments, R1 is C2-4alkenyl-C1-3alkyl or phenyl-C1-3alkyl, wherein said phenyl-C1-3alkyl is optionally substituted by 1, 2, or 3 independently selected C1-4 alkoxy groups.
[00131] In some modalities, R1 is ally.
[00132] In some embodiments, R1 is 4-methoxybenzyl.
[00133] In some embodiments, R3 is C1-6 alkyl.
[00134] In some embodiments, R3 is tert-butyl.
[00135] In some embodiments, the compound of Formula F15 is tert-butyl ally {[ally (2-oxoethyl) amino] sulfonyl} carbamate:

[00136] In some embodiments, the compound of Formula F15 is tert-butyl (4-methoxybenzyl) {[((4-methoxybenzyl) (2-oxoethyl) amino] sulfonyl} carbamate:

[00137] In some embodiments, the invention provides a compound of Formula F16:

[00138] where R3 is C1-6alkyl or benzyl and each R1 is, independently, an amino protecting group.
[00139] In some embodiments, R1 is C2-4alkenyl-C1-3alkyl or phenyl-C1-3alkyl, wherein said phenyl-C1-3alkyl is optionally substituted by 1, 2, or 3 independently selected C1-4 alkoxy groups.
[00140] In some modalities, R1 is ally.
[00141] In some embodiments, R1 is 4-methoxybenzyl.
[00142] In some embodiments, R3 is C1-6 alkyl.
[00143] In some embodiments, R3 is tert-butyl.
[00144] In some embodiments, R3 is C1-4 alkyl.
[00145] In some modalities, R3 is butyl.
[00146] In some embodiments, the compound of Formula F16 is tert-butyl ally (N-allyl-N- (2- (4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4,5 -dihydro- 1,2,4-oxadiazol-3-yl) -1,2,5-oxadiazol-3-ylamino) ethyl) sulfamoyl) carbamate:

[00147] In some embodiments, the compound of Formula F16 is (4-methoxybenzyl) - (N- (4-methoxybenzyl) -N- (2- (4- (4- (3-bromo-4-fluorophenyl) - 5 -oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl) -1,2,5-oxadiazol-3-ylamino) ethyl) sulfamoyl) tert-butyl carbamate:

[00148] Scheme 5 outlines an alternative route for the preparation of the compound of Formula F10. Layout 5

[00149] The present application also provides a process comprising the reaction of a compound of Formula F17:
wherein R4 is C1-6 alkyl, C1-6 haloalkyl, benzyl, or 9 H-fluorene-9-ylmethyl with a compound of Formula F5:
to generate a compound of Formula F18:

[00150] In some embodiments, R4 is tert-butyl.
[00151] In some modalities, R4 is benzyl.
[00152] In some modalities, R4 is ethyl.
[00153] In some embodiments, R4 is C1-3 haloalkyl.
[00154] In some embodiments, R4 is 2,2,2-trichloroethyl.
[00155] In some embodiments, R4 is 9H-fluoren-9-ylmethyl.
[00156] In this step Q, compounds F18 can be prepared, in some modalities, by means of reaction F17 with the amine compound of Formula F5 in the presence of a reducing agent.
[00157] In some modalities, said reaction is carried out in the presence of a reducing agent.
[00158] The reducing agent can be any compound capable of reducing an organic compound to a lower oxidation state, for example, by using an organosilane, such as tri (C1-3 alkyl) silane (for example, 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, chlorotrimethylsilane, 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 to, halogenated hydrocarbon solvents (eg, dichloromethane, chloroform, dichloroethane or tetrachloroethane). In some embodiments, the halogenated hydrocarbon solvent is 1,2-dichloroethane.
[00159] In some modalities, said reducing agent is an organosilane.
[00160] In some embodiments, said reducing agent is tri (C1-3 alkyl) silane.
[00161] In some embodiments, said reducing agent is triethylsilane.
[00162] In some modalities, said reaction is carried out in the presence of an organic acid.
[00163] In some embodiments, said organic acid is trifluoroacetic acid.
[00164] In some embodiments, said organic acid is methanesulfonic acid.
[00165] In some embodiments, said reaction is carried out in a solvent component comprising a halogenated hydrocarbon solvent.
[00166] In some embodiments, said halogenated hydrocarbon solvent is dichloromethane.
[00167] In some embodiments, said halogenated hydrocarbon solvent is 1,2-dichloroethane.
[00168] In some embodiments, the process further comprises deprotecting said Formula F18 compound to generate a Formula F10 compound:

[00169] In some embodiments, methods for deprotecting certain amine protecting groups (such as carbamates) are known to a person skilled in the art, such as those in Wuts and Greene, Greene's Protective Groups in Organic Synthesis, 4th Ed., pp 696-926, John Wiley & Sons: New York, 2006. For example, the tert-butoxycarbonyl group (for example, when R4 is tert-butyl) can be removed (for example, hydrolyzed) from the nitrogen atom. nio, for example, by treatment with an acid (such as hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, and the like); a combination of reagents (for example, mixture of acetyl chloride and methanol) known to generate an acid; or a Lewis acid (for example, BF3 ^ Et2O). The benzyloxycarbonyl group (for example, when R4 is benzyl) can be removed (for example, hydrogenolysis) from the nitrogen atom, for example, by treatment with hydrogen and a catalyst (such as palladium on carbon ). The methoxycarbonyl and ethoxycarbonyl groups (that is, when R4 is methyl or ethyl) can be removed by treatment with an inorganic base (such as KOH or K 2CO3); a combination of reagents (for example, a mixture of acetyl chloride, sodium iodide and acetonitrile); or by treatment with an acid (for example, HBr, AcOH). The 2,2,2-trichloroethoxycarbonyl group can be removed, for example by treatment with a catalyst (for example, Zn / AcOH or Cd / AcOH). Suitable solvents for this step include, but are not limited to, methanol or tetrahydrofuran (THF), acetonitrile and the like. In some embodiments, the treatment is carried out at a temperature of about 30 ° C to about 90 ° C, for example, from about 50 ° C to about 100 ° C, or between about 60 ° C to about 80 ° C.
[00170] In some modalities, said deprotection comprises the reaction of the compound of Formula F18 with zinc in the presence of acetic acid.
[00171] In some embodiments, said deprotection is carried out in a solvent component comprising tetrahydrofuran.
[00172] In some embodiments, the process further comprises the reaction of said compound of Formula F10 with a base to generate a compound of Formula I:

[00173] In some embodiments, said base comprises an alkali metal hydroxide.
[00174] In some embodiments, said alkali metal hydroxide is sodium hydroxide.
[00175] In some embodiments, said reaction is carried out in a solvent component that comprises tetrahydrofuran, water and ethanol.
[00176] In some embodiments, the process further comprises converting said compound of Formula F18 to a compound of Formula I:
wherein said conversion 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.
[00177] In some embodiments, the conversion further includes the addition of an acid to the first mixture. In some embodiments, said acid is a strong aqueous acid. In some embodiments, said aqueous strong acid is aqueous hydrochloric acid.
[00178] In some embodiments, said conversion is carried out from a solvent component comprising tetrahydrofuran and ethyl acetate.
[00179] The present application also provides a process comprising: i) reacting a compound of Formula F19:
with a compound of Formula F5:
in the presence of triethylsilane and methanesulfonic acid to generate a compound of Formula F20:
and ii) converting said compound of Formula F20 to a compound of Formula I:

[00180] wherein said conversion comprises combining said compound with Formula F20 N, N-bis (2-aminoethyl) ethane-1,2-diamine. In some embodiments, said conversion further comprises the addition of aqueous hydrochloric acid after said combination.
[00181] Compound F17 can be prepared by a one-step process (step P) from chlorosulfonylisocyanate, as shown in Scheme 6. Scheme 6

[00182] In some embodiments, the preparation of the compound of Formula F17 can be obtained by treating chlorosulfonylisocyanate with 2,2-dimethoxyethanamine and alcohol R4OH (R4 is defined as above), optionally in a solvent (for example, a hydrocarbon solvent) halogenated grandson, such as dichloromethane, chloroform, dichloroethane, tetrachloroethane). In some modalities, this step is performed in the presence of a base. The base can be an organic base such as an acyclic amine (for example, triethylamine, diisopropylethylamine (DIPEA), etc.) or a cyclic amine (for example, pyrrolidine, piperidine, etc.); or an inorganic base, such as alkaline salts (for example, 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, chloroform, dichloroethane, or tetrachloroethane.
[00183] In some embodiments, the present application additionally provides a compound of Formula F17:

[00184] where R4 is C1-6alkyl, C1-6haloalkyl or benzyl.
[00185] In some embodiments, R4 is tert-butyl.
[00186] In some modalities, R4 is benzyl.
[00187] In some modalities, R4 is ethyl.
[00188] In some embodiments, R4 is C1-3 haloalkyl.
[00189] In some embodiments, R4 is 2,2,2-trichloroethyl.
[00190] In some embodiments, R4 is 9H-fluoren-9-ylmethyl.
[00191] In some embodiments, the compound of Formula F17 is tert-butyl-N- (2,2-dimethoxyethyl) sulfamoylcarbamate.
[00192] In some embodiments, the compound of Formula F17 is benzyl-N- (2,2-dimethoxyethyl) sulfamoylcarbamate.
[00193] In some embodiments, the compound of Formula F17 is ethyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate.
[00194] In some embodiments, the compound of Formula F17 is 2,2,2-trichloroethyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate.
[00195] In some embodiments, the compound of Formula F17 is (9H-fluoren-9-yl) methyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate.
[00196] In some embodiments, the present application additionally provides a compound of Formula F18:

[00197] where R4 is C1-6alkyl, C1-6haloalkyl, or benzyl.
[00198] In some modalities, R4 is tert-butyl.
[00199] In some modalities, R4 is benzyl.
[00200] In some modalities, R4 is ethyl.
[00201] In some modalities, R4 is C1-3 haloalkyl.
[00202] In some embodiments, R4 is 2,2,2-trichloroethyl.
[00203] In some embodiments, R4 is 9H-fluoren-9-ylmethyl.
[00204] In some embodiments, the compound of Formula F18 is ({[2 - ({[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) benzyl carbamate:

[00205] In some embodiments, the compound of Formula F18 is ({[2 - ({[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) ethyl carbamate:

[00206] In some embodiments, the compound of Formula F18 is ({[2 - ({[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) 2,2,2-trichloroethyl carbamate:

[00207] In some embodiments, the compound of Formula F18 is N- (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) sulfamoylcarbamate of (9H-fluoren-9-yl) methyl:

[00208] As used herein, the term "alkyl", when used alone or in conjunction with the terms additional part, refers to a saturated straight or branched chain hydrocarbon group, containing 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and the like.
[00209] As used herein, "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds. In some embodiments, said alkyl group has 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples of alkenyl groups include ethylene (vinyl), propenyl, and the like.
[00210] As used herein, "alkenylalkyl" refers to a group of Formula alkyl-alkenyl. In some embodiments, the allyl group is alkenylalkyl.
[00211] As used herein, the term "haloalkyl", when used alone or in conjunction with an additional portion, refers to an alkyl group substituted by one or more halogen atoms independently selected from F, Cl , Br, and I. Exemplary haloalkyl groups include CF3, CHF2, CH2CF3, and the like.
[00212] 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. Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.
[00213] As used herein, "trialkylamine" means a nitrogen atom substituted with three independently selected groups of alkyl. In some embodiments, each alkyl group has from 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples of trialkylamine groups include trimethylamine, triethylamine, and the like.
[00214] As used herein, the term "alkoxycarbonyl" refers to a group of the general formula -C (O) -O-alkyl. In some modalities, the alkyl group has 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples of alkoxycarbonyl groups include ethoxycarbonyl, tert-butoxycarbonyl (Boc), and the like.
[00215] Halogenated hydrocarbon solvents, refer to halogenated alkanes, such as dichloromethane, chloroform, dichloroethane or tetrachloroethane, in which the alkane can be branched or straight-chain having 1 to 12 carbon atoms, 1 to 6 atoms of carbon, or 1 to 4 carbon atoms with one or more halo atoms. In some embodiments, the halogenated hydrocarbon solvent is a chlorinated alkane, from 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
[00216] In several places in the present specification, substitutes for the compounds of the invention can be developed in groups or at intervals. Specifically, it is intended that the invention includes all and each individual subcombination of the members of such groups and ranges.
[00217] The compounds of the invention are intended to be 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 effective therapeutic agent.
[00218] It is further preferred that certain features of the invention, which are, for clarity, described in the context of separate modalities, can also be provided in combination in a single modality. On the other hand, several features of the invention that are, for brevity, described in the context of a single mode, can also be provided separately or in any suitable subcombination.
[00219] The compounds of the invention are also intended to include all possible geometric isomers. Geometric cis and trans isomers of the compounds are described and can be isolated as a mixture of isomers or as separate isomeric forms.
[00220] The compounds of the invention also include tahomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond, along with the concomitant migration of a proton.
[00221] The compounds of the invention can also include all isotopes of atoms that occur in intermediates or final compounds. Isotopes include atoms that have the same atomic number, but different mass numbers. For example, hydrogen isotopes include tritium and deuterium.
[00222] In some embodiments, the compounds of the invention, and their salts, are substantially 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 containing 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 invention, or a salt thereof. Methods for isolating compounds and their salts are routine in the art.
[00223] The present application also includes salts of the compounds described herein. As used herein, "salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base portion 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 organic salts (such as trialkylammonium) of acid residues such as carboxylic acids; and the like. The salts of the present patent application can be synthesized from the parent compound that contains a basic or acidic unit by conventional chemical methods. Generally, these salts can be prepared to react to the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile (ACN) are preferred.
[00224] The present application also includes pharmaceutically acceptable salts of the compounds described herein. "Pharmaceutically acceptable salts" include a subset of "salts" described above which are conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Suitable salt lists are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety. The phrase “pharmaceutically acceptable” is used here to refer to those compounds, material forms, compositions, and / or dosage that are, within the scope of medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.
[00225] 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 (for example, 1H or 13C), infrared spectroscopy, spectrophotometry (for example, UV-visible), or mass spectrometry ; or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography. The compounds obtained by the reactions can be purified by any suitable method known in the art. For example, chromatography (medium pressure) on a suitable adsorbent (for example, silica gel, aluminum and the like), HPLC or preparative thin layer chromatography; distillation; sublimation, grinding, or recrystallization. The purity of the compounds, in general, is determined by means of physical methods, such as measuring the melting point (in the case of a solid), obtaining an NMR spectrum, or performing a separation by HPLC. If the melting point decreases, if the 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.
[00226] Preparation of compounds may involve the protection and protection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protection groups can be readily determined by a person skilled in the art. Protection group chemistry can be found, for example, in Wuts and Greene, Greene's Protective Groups in Organic Synthesis, 4th Ed., John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its wholeness.
[00227] The reactions of the processes described here can be carried out in suitable solvents that can be easily selected by a specialist in the technique of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reagents), intermediates or products at the temperatures at which the reactions are carried out, that is, temperatures that can vary from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in a solvent or in a mixture of more than one solvent. Depending on the reaction step, suitable solvents for that particular reaction step can be selected. Suitable solvents include water, alkanes (such as pentanes, hexanes, heptanes, cyclohexane, etc., or a mixture thereof), aromatic solvents (such as benzene, toluene, xylene, etc.), alcohols (such as methanol, ethanol , isopropanol, etc.), ethers (such as dialkyl ethers, methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), dioxane, etc.), esters (such as ethyl acetate, butyl acetate, etc. ), halogenated hydrocarbon solvents (such as dichloromethane (DCM), chloroform, dichloroethane, tetrachloroethane), dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone, acetonitrile (ACN), hexamethylphosphorone (HMPA) and N-Pyrrolidone and N-metholidone (NMP). Such solvents can be used in both their wet or anhydrous forms.
[00228] Resolution of racemic mixtures of compounds can be accomplished by any of the numerous methods known in the art. An exemplary method includes fractional recrystallization using a “chiral resolving acid”, which is an optically active organic acid, which forms a salt. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as forms D and L of tartaric acid, diacetyl tartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the different optically active camphor sulfonic acids. Resolution of racemic mixtures can also be carried out by eluting on a column loaded with an optically active resolving agent (eg, dinitrobenzoylphenylglycine). The composition of the suitable eluting solvent can be determined by one skilled in the art. Usage Methods
[00229] The compound of formula I can inhibit the activity of the enzyme indolamine-2,3-dioxigenase (IDO). For example, the compound of Formula I can be used to inhibit the activity of IDO in the cell or in an individual in need of modulation of the enzyme by administering an inhibitory amount of the compound of Formula I.
[00230] The compounds of Formula I can be used in methods of inhibiting the degradation of tryptophan in a system containing cells that express IDO such as a tissue, living organism, or cell culture. In some embodiments, the present application provides methods of altering (for example, increasing levels of tryptophan) extracellular in a mammal by administering an effective amount of the compound of Formula I. Methods of measuring levels of tryptophan and degradation of the tryptophan are routine in the art.
[00231] The Formula I compounds 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 Formula I compound. IDO-mediated immunosuppression has been associated with, for example, cancers, tumor growth, metastasis, viral infection, viral replication, etc.
[00232] The compounds of Formula I can also be used in methods of treating diseases associated with IDO activity or expression, including abnormal activity and / or overexpression in an individual (e.g., patient), by administration to the individual in need of such treatment of a therapeutically effective amount or dose of the compound of Formula I or a pharmaceutical composition thereof. Examples of diseases can include any disease, disorder or condition that is directly or indirectly linked to the expression or activity of the IDO enzyme, such as overexpression or abnormal activity. An illness associated with IDO can also include any disease, disorder or condition that can be prevented, ameliorated, or cured by modulating the activity of the enzyme. Examples of diseases associated with IDO include cancer, viral infection, such as HIV infection, HCV infection, depression, neurodegenerative disorders such as Alzheimer's and Huntington's disease, trauma, age-related cataracts, transplantation of organs (for example, organ transplant rejection), and autoimmune diseases, including asthma, rheumatoid arthritis, multiple sclerosis, allergic inflammation, inflammatory bowel disease, psoriasis and erythematous systemic lupus. Examples of cancers treatable by the methods here include cancer of the colon, pancreas, breast, prostate, lung, brain, ovary, cervix, testis, kidney, head and neck, lymphoma, leukemia, melanoma , and the like. The Formula I compound can also be useful in the treatment of obesity and ischemia.
[00233] As used herein, the term "cell" is intended to refer to a cell that is in vitro, ex vivo or in vivo. In some modalities, an ex vivo cell may be part of a tissue sample excited from an organism such as a mammal. In some embodiments, a cell in vitro may be a cell in a cell culture. In some embodiments, an in vivo cell is a living cell in an organism, such as a mammal.
[00234] As used herein, the term “contact” refers to the junction of fractions indicated in an in vitro system or an in vivo system. For example, "contacting" the IDO enzyme with the Formula I compound includes administering the Formula I compound to an individual or patient, such as a human, having IDO, as well as, for example, introducing the compound of Formula I in a sample containing a cell preparation or purified containing the enzyme IDO.
[00235] As used herein, the terms "individual" or "patient", used interchangeably, refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep , horses, or primates, and most preferably humans.
[00236] As used herein, the phrase "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that causes the biological or medicinal response of the tissue, system, animal, individual or human being that is being sought by an investigator, veterinarian, doctor or other clinician.
[00237] As used herein, the term "treat" or "treatment" refers to 1) disease prevention; for example, prevention of a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet suffer or present the pathology or symptoms of the disease; 2) inhibition of the disease; for example, inhibition of a disease, condition or disorder in an individual who suffers or exhibits the pathology or symptomatology of the disease, condition or disorder (i.e., interrupting the further development of the pathology and / or symptomatology), or 3) improvement of the disease; for example, improvement of a disease, condition or disorder in an individual who suffers or presents the pathology or symptomatology of the disease, condition or disorder (that is, reversing the pathology and / or symptomatology). Combination Therapy
[00238] One or more pharmaceutical agents or additional treatment methods, such as, for example, antiviral agents, chemotherapeutic agents or other anticancer agents, immune system boosters, immunosuppressants, radiation, anti-tumor and antiviral vaccines, cytokines (eg, IL2, GM-CSF, etc.), and / or tyrosine kinase inhibitors can be used in combination with the compound of Formula I for the treatment of diseases, disorders or conditions associated with IDO. 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.
[00239] Suitable antiviral agents contemplated for use in combination with the compound of Formula I may comprise nucleosides and nucleotides reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNR-TIs), protease inhibitors and other drugs antivirals.
[00240] Examples of suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir dipivoxil [bis (POM) -PMEA]; lobucavir (BMS-180194); BCH-10652; emitricitabine [(-) - FTC]; beta-L-FD4 (also called beta-L-D4C and called beta-L-2 ', 3'-dichleoxy-5-fluoro-cytidene); DAPD, ((-) - beta-D-2,6, -diamino-purine dioxolane); and lodenosine (FddA). Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine (BHAP, U-90152); 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 (NSC- 675451 ) and B. Typical suitable protease inhibitors include sa-quinavir (Ro 31-8959); ritonavir (ABT-538); indinavir (MK-639); nelfnaver (AG-1343); amprenavir (141W94); lasinavir (BMS-234475); DMP-450; BMS-2322623; ABT-378; and AG-1 549. Other antiviral agents include hydroxyurea, ribavirin, IL-2, IL-12, pentafuside and Project No. 11607 .
[00241] Suitable chemotherapeutics or other anticancer agents include, for example, alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes), such as uracil mustard, chlormetin, cyclophosphamide ( CytoxanTM), ifosfamide, melphalan, chlorambucil, polyboromann, triethylene-melamine, triethylene thiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.
[00242] In the treatment of melanoma, agents suitable for use in combination with the compound of Formula I include: da-carbazine (DTIC), optionally, together with other chemotherapy drugs such as carmustine (BCNU) and cisplatin; the “Dartmouth regime,” which consists of DTIC, BCNU, cisplatin and tamoxifen; a combination of cisplatin, vinblastine, and DTIC; or temozolomide. Compounds according to the invention can also be combined with immunotherapy drugs, including cytokines, such as alpha interferon, interleukin 2, and tumor necrosis factor (TNF) in the treatment of melanoma.
[00243] The compound of Formula I can also be used in combination with vaccine therapy for the treatment of melanoma. Antimelanoma vaccines are, in some respects, similar to the antiviral vaccines that are used to prevent diseases caused by viruses, such as polio, measles, and mumps. Weakened melanoma cells or parts of melanoma cells called antigens can be injected into a patient to stimulate the body's immune system to destroy melanoma cells.
[00244] Melanomas that are confined to the arms or legs can also be treated with a combination of agents, including the compound of Formula I, using a hyperthermic isolated limb perfusion technique. This treatment protocol temporarily separates the circulation of the involved limb from the rest of the body and injects high doses of chemotherapy into the artery that feeds the limb, thus providing high doses to the tumor area, without exposing the internal organs to these doses that would otherwise they could cause serious side effects. Normally, the fluid is heated to 102 ° to 104 ° F. Melfalan is the most used drug in this and chemotherapy procedure. This can be administered with another agent called tumor necrosis factor (TNF) (see section on cytokines).
[00245] Suitable chemotherapy or other anticancer 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, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.
[00246] Suitable chemotherapeutics or other anti-cancer agents include, for example, certain natural products and their derivatives (e.g. vinca alkaloids, anti-tumor antibiotics, enzymes, lymphokines and epipodophyllotoxins), such as vinblastine, vincristine, vinidine, bleomycin , dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ARA-C, paclitaxel (TAXOLTM), mitramycin, deoxychoformicin, mitomycin-C, L-asparaginase, interferons (especially IFN-a), etoposide, and teniposide.
[00247] Other cytotoxic agents include navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine.
[00248] Also suitable are cytotoxic agents, such as epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes, such as cisplatin and carboplatin; modifiers of the biological response; growth inhibitors; anti-hormonal therapeutic agents; leucovorin; tegafur; and hematopoietic growth factors.
[00249] Other anticancer agents include therapeutic antibodies such as trastuzumab (Herceptin), antibodies to costimulatory molecules, such as CTLA-4, 4-1BB and DP-1, or antibodies to cytokines (IL-10 , TGF-β, etc.).
[00250] Other anticancer agents also include those that block the migration of immune cells, such as chemokine receptor antagonists, including CCR2 and CCR4.
[00251] Other anti-cancer agents also include those that boost the immune system, such as adjuvants or adoptive T-cell transfer.
[00252] Cancer vaccines include dendritic cells, synthetic peptides, DNA vaccines and recombinant viruses.
[00253] Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, administration is described in the standard literature. For example, the administration of many of the chemotherapy agents is described in "Physicians' Desk Reference" (PDR, for example, 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein. by reference as shown in its entirety. Pharmaceutical Formulations and Dosage Forms
[00254] When used as pharmaceutical products, the compound of Formula I can be administered in the form of pharmaceutical 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 on whether the treatment is desired local or systemic and on the area to be treated. Administration can be topical (including ophthalmic and mucous membranes, including intranasal, vaginal and rectal), pulmonary (for example, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular release may include topical (eye drops), subconjunctival administration, periocular or intravitreal injection or introduction of a balloon catheter or ophthalmic insertions surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intra-peritoneal, or intramuscular or infusion; or intracranial, for example, intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or it can be, for example, by a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners and the like may be necessary or desirable.
[00255] Pharmaceutical compositions containing the compound of Formula I can be prepared in combination with one or more pharmaceutically acceptable carriers. In the manufacture of the compositions of the invention, the active ingredient is usually 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 vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (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.
[00256] When preparing a formulation, the active compound can be ground to generate the appropriate particle size before combining with the other ingredients. If the active compound is substantially insoluble, it can be ground to a particle size of less than 200 mesh. If the active compound is substantially soluble in water, the particle size can be adjusted by grinding to provide a substantially uniform distribution in the formulation, for example, about 40 mesh.
[00257] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydro-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide a quick, sustained or delayed release of the active ingredient after administration to the patient using procedures known in the art.
[00258] The compositions can be formulated in a unit dosage form, each dosage containing 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 amount of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
[00259] The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will generally be determined by a doctor, depending on the relevant circumstances, including the condition to be treated, the route of administration chosen, the actual compound administered, the age , weight, and individual patient's response, severity of the patient's symptoms, and the like.
[00260] To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of the compound of Formula I. When referring to these compositions of preformulation as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing, for example, from 0.1 to about 500 mg of the active ingredient of the present patent application.
[00261] Tablets or pills containing the compound of Formula I can be coated or otherwise manipulated to provide a dosage form providing the advantage of prolonged action. For example, the tablet or pill may comprise an internal dosage and an external dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and allow the internal 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 materials such as shellac, cetyl alcohol, and cellulose acetate.
[00262] The liquid forms in which the compounds and compositions of the present patent application can be incorporated for oral or injection administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical carriers.
[00263] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable solvents, aqueous or organic, or mixtures thereof, and powders. Liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered via the oral or nasal airway for local or systemic effect. Compositions can be nebulized by using inert gases. The nebulized solutions can be breathed directly from the nebulizer device or the nebulizer device can be connected to a face mask, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices that release the formulation in an appropriate manner.
[00264] The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of administration, such as prophylaxis or therapy, the condition of the patient, the mode of administration, and the like. In therapeutic applications, the compositions can be administered to a patient who is already suffering from a disease in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. Effective doses will depend on the condition being treated for the disease, as well as on the judgment of the attending physician depending on factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.
[00265] 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 they can be sterilized by filtration. The aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with an aqueous sterile vehicle prior to administration. The pH of the compound preparations will typically be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that the use of certain of the previous excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.
[00266] The therapeutic dosage of the compound of formula I may vary according to, for example, the particular use for which the treatment is made, the mode of administration of the compound, the health and condition of the patient, and the judgment medical prescription. The proportion or concentration of the Formula I compound in a pharmaceutical composition can vary depending on a number of factors, including dosage, chemical characteristics (for example, hydrophobicity) and route of administration. For example, the compound of Formula I can be provided in an aqueous solution of physiological buffer containing about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dose ranges are from about 1 μg / kg to about 1 g / kg of body weight per day. In some modalities, the dosage range is from about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage is probably dependent on such variables as the type and extent of disease or disorder progression, the general health status of the particular patient, the relative biological efficacy of the selected compound, the formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived in vitro or animal model test systems.
[00267] The compound of Formula I can also be formulated in combination with one or more additional active ingredients, which may include any pharmaceutical agent, such as antiviral agents, vaccines, antibodies, immune system boosters, immunosuppressants, agents anti-inflammatory and the like.
[00268] The present application also includes pharmaceutical kits useful, for example, in the treatment or prevention of diseases or disorders associated with IDO, obesity, diabetes and other diseases referred to herein that include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of the compound of Formula I. Such kits may further include, if desired, one or more of the various components of the conventional pharmaceutical kit, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc. ., as will be readily apparent to those skilled in the art. Instructions, as package inserts or as labels, indicating the 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
[00269] The invention will be described in more detail by means of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any way. Those skilled in the art will easily recognize a variety of non-critical parameters that can be altered or modified to produce essentially the same results.
[00270] Example 1. Synthesis of 4 - ({2- [(Aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'- hydroxy-1,2,5-oxadiazole- 3-carboxyimidamide

[00271] Step A: 4-Amino-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide (2)

[00272] Malononitrile [Aldrich, product # M1407] (320.5 g, 5 mol) was added to water (7 L), preheated to 45 ° C and stirred at 45 ° C for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol, 1.1 equiv.) Was added. When the temperature reached 10 ° C, 6 N hydrochloric acid (55 mL) was added. A moderately exothermic reaction followed with a temperature reaching 16 ° C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 h at 16-18 ° C. The reaction mixture was cooled to 13 ° C and 50% aqueous hydroxylamine hydrochloride (990 g, 15 mol, 3.0 equiv.) Was added all at once. The temperature rose to 26 ° C. When the exothermic reaction lowered the cold bath was removed and stirring was continued for 1 h at 26-27 ° C, then it was slowly brought to reflux. The reflux was maintained for 2 h and then the reaction mixture was allowed to cool gradually over night. 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. Stirring was continued in the 5 ° C ice bath. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 ° C) to generate the desired product (644 g, 90%) as an off-white solid. 13C NMR (75 MHz, CD3OD): δ 156.0, 145.9, 141.3; C3H5N5O2 (MW 143.10), LCMS (EI) m / e 144.0 (M + + H).
[00273] Step B: 4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboxyidoyl chloride (3)

[00274] 4-Amino-N'-hydroxy-1,2,5-oxadiazol-3-carboximidamide (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 stirred at 42-45 ° C until a clear solution was obtained. 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 ° C. After the addition was complete, stirring was continued in the ice bath for 1.5 h and then the reaction mixture was allowed to warm to 15 ° C. 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 filtrate was concentrated on a rotary evaporator. The residue was dissolved in methyl tert-butyl ether (5.5 L), treated with activated charcoal (40 g), stirred at room temperature for 40 min and filtered through Celite. The solvent was removed on a rotary evaporator and the resulting product was dried in a vacuum oven (45oC) to generate the desired product (256 g, 53.4%) as an off-white solid. 13C NMR (100 MHz, CD3OD) δ 155.8, 143.4, 129.7; C3H3ClN4O2 (MW 162.53), LCMS (EI) m / e 163/165 (M + + H).
[00275] Step C: 4-Amino-N- (3-bromo-4-fluorophenyl) -N'-hydroxy-1,2,5-oxadiazole-3-carboxyidamide (4)

[00276] 4-Amino-N-hydroxy-1,2,5-oxadiazole-3-carboxyidoyl chloride (33.8 g, 208 mmol) was mixed with water (300 ml). At 60 ° C, 3-bromo-4-fluoroaniline (Sigma-Aldrich) (43.6 g, 229 mmol, 1.1 equiv.) Was added to the suspension with stirring for 10 min. A solution of sodium bicarbonate (26.3 g, 313 mmol, 1.5 equiv.) In water (300 mL) was added over 15 min with stirring at 60 ° C. After stirring for 20 min, LCMS indicated completion of the reaction. 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 generate the desired product (65 g, 99%) as an off-white solid, which was used in the subsequent reaction without further purification. C9H7BrFN5O2 (MW 316.09), LCMS (EI) m / e 316/318 (M + + H).
[00277] Step D: 3- (4-Amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 (4H) -one (5)

[00278] A mixture of 4-amino-N- (3-bromo-4-fluorophenyl) -N '- hydroxy-1,2,5-oxadiazole-3-carboximidamide (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 ° C and stirred for 20 min. LCMS indicated that the reaction has 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 generate the desired product (60.2 g, 85%) as a whitish solid. 1H NMR (300 MHz, DMSO-d6) δ 8.05 (m, 1H), 7.69 (m, 1H), 7.57 (t, 1H, J = 8.7 Hz), 6.58 (s , 2H); C10H5BrFN5O3 (MW 342.08), LCMS (EI) m / e 342/344 (M + + H).
[00279] Step E: [2 - ({4- [2- (3-bromo-4-fluorophenyl) -5-oxo-2,5-dihydro- 1,2,4-oxadiazol-3-yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] tert-butyl carbamate (7)

[00280] 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 temperature and then cooled to -5 ° C. A solution of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 (4 H) -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 under a drip for 30 min with stirring, keeping the temperature below 0 ° C. The reaction was stirred at -5 to 0 ° C 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 in drops in 20 min, 40 min with 4h intervals. HPLC indicated that the reaction was complete after 5.25 h. The reaction mixture was poured into ice-cooled sodium bicarbonate (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 diethyl ether (40 ml) and stirred at room temperature for 5 h. The precipitate was collected by filtration, washed with diethyl ether and dried in a vacuum oven to generate the desired product (1953 mg, 80.5%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 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); C1yH18BrFN6O5 (MW 485.26); LCMS (EI) m / e 507/509 (M + + Na).
[00281] Step F: 3- {4 - [(2-Aminoethyl) amino] hydrochloride -1,2,5- oxadiazol-3-yl} -4- (3-bromo-4-fluorophenyl) -1,2 , 4-oxadiazole-5 (4H) -one (8)

[00282] Method A (prepared from [2 - ({4- [2- (3-bromo-4-fluorophenyl) -5-oxo-2,5-dihydro-1,2,4-oxadiazole- 3-yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] tert-butyl carbamate; Step E):
[00283] A 500 mL flask was charged [2 - ({4- [2- (3-bromo-4-fluorophenyl) -5-oxo-2,5-dihydro-1,2,4-oxadiazole -3-yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] carbamate of tert-butyl (20 g, 41.2 mmol) and isopropanol (255 ml). The slurry was stirred at room temperature. Hydrogen chloride gas (7.55 g, 207 mmol, 5.0 equiv.) Was added to the paste with a glass tube below the surface over 16 min. Ethyl acetate (111 mL) was then added to the batch and the reaction was heated to 43 oC and stirred for 7.5 h. The batch was cooled to 19 o C and ethyl acetate (44 mL) was added. The paste 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 generate the desired product (16.61 g, 95.5% yield) as a white-off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 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 385.15), LCMS (EI) m / e 385/387 (M + + H).
[00284] Method B (prepared directly from 3- (4-Amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole -5 (4H) - one; Step D):
[00285] Sodium triacetoxyborohydride (2.33 g, 11.0 mmol, 11.0 equiv.) Was mixed with trifluoroacetic acid (12.0 mL, 155.8 mmol, 155.8 eq.). The resulting solution was mixed at room temperature for 30 min. A solution of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (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 acetonitrile (6.0 ml) was stirred under N2. This solution was cooled to -5 ° C and the sodium triacetoxyborohydride and trifluoroacetic acid solution was added under a drip over 5 min. The reaction was stirred at room temperature for 4 h. HPLC and LC-MS (M + - Boc + H: 385/387, bromide standard) indicated a ratio between the desired product and the starting material was 4 to 1. The mixture was concentrated and diluted with dichloromethane (10 ml). The solution was cooled to 0 ° C and 4 N sodium hydroxide was added slowly, maintaining the temperature at 0-5 ° C 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 resulting solution was cooled to 0 ° C. 4 N hydrochloric acid in dioxane (3.0 mL) was added under a drip at 0-5 ° C. The mixture was stirred at room temperature for 20 min. The precipitate was collected by filtration, washed with diethyl ether, and dried in vacuo to generate the desired product (289 mg, 54%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 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 385.15), LCMS (EI) m / e 385/387 (M + + H).
[00286] Step G: ({[2 - ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole-3- yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] amino} sulfonyl) tert-Butyl carbamate (9)

[00287] A 20-L glass reactor was charged with 3- {4 - [(2-aminoethyl) amino] -1,2,5-oxadiazol-3-yl} -4- (3-bromo- 4-fluorophenyl) - 1,2,4-oxadiazole-5 (4H) -one (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 - 14.6oC.
[00288] A 5 L round bottom flask was loaded with tert-butanol (253 g, 3.41 mol, 1.2 equiv.) And dichloromethane (2.6 L). The solution was cooled to 0.9oC. To this solution, chlorosulfonyl isocyanate (463 g, 3.27 mol, 1.15 equiv.) Was added for 43 minutes while maintaining the batch temperature below 10oC. The resulting tert-butyl (chlorosulfonyl) carbamate solution was kept at 3 - 5oC for 1 h.
[00289] The tert-butyl (chlorosulfonyl) carbamate solution was added to the reactor over 73 min while maintaining the batch temperature below 0oC. The batch was then heated to 10 ° C over 1 h and was then stirred at 10 - 14 ° C 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 the phases separated. The dichloromethane solution was isolated kept in the reactor and charged with acetic acid (513 g) for 25 minutes to precipitate the product. The resulting slurry was stirred at 20 ° C for 2.5 h. The product was isolated by filtration and washed with dichloromethane (1.8 L). The product was dried under reduced pressure (-30 inHg) at 45 ° C for 16 h to generate the desired product (1342 g, 83.5% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 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 (dd, J = 12.1, 5.9 Hz, 2 H), 1.41 (s, 9 H). C17H19BrFN7O7S (MW 564.34), LCMS (EI) m / e 585.9 / 587.9 (M + + Na).
[00290] Step H: N- [2 - ({4- [4- (3-Bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole-3- yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] sulfamide (10)

[00291] To a 20-L flask containing ({[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) tert-butyl carbamate (1200 g, 2.126 mol) ethanol (12 L) at 20oC was added. 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 50oC and the temperature was maintained for 3 h until completion of the reaction. The solvent was removed by vacuum distillation at 33-39oC and 6 kg of distillate was collected. Ethyl acetate (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 solvent ratio. 1H NMR indicated that the molar ratio of ethyl acetate to ethanol was 1.0: 0.1. The solution was heated to 65oC. n-Heptane (4.1 kg) was added to the solution at 60-65oC over 43 min. The resulting slurry was stirred at 65 ° C for 1 h. The paste was cooled to 20oC 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 generate the desired product (906 g, 91.8% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 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); C12H11BrFN7O5S (MW 464.23), LCMS (EI) m / e 485.8 / 487.8 (M + - C5H8O2 + Na).
[00292] Step I: 4 - ({2 - [(Aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'-hydroxy-1,2,5-oxadiazole-3- carboxyimidamide (11)

[00293] To a 20-L glass reactor was added N- [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] sulfamide (799.4 g, 1.72 mol) and THF (3.2 L). The resulting solution was stirred at 20 ° C for 7 min. and then loaded with water (1.6 L). The batch was cooled to 2oC and loaded with 30% by weight sodium hydroxide solution (475 mL, 666.4 g, 4.99 mol, 2.9 equiv.) Over 8 minutes. The batch was heated to 20oC 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 ° C. The batch was then loaded with 85% by weight of phosphoric acid (370.7 g, 3.22 mol, 1.9 equiv.) Over 9 minutes. The batch was heated to 20oC and stirred for 1 h. The batch was allowed to settle and the phases were separated. The organic layer was retained in the reactor and charged with water (2.9 L) and 85% by weight of phosphoric acid (370.7g, 3.22 mol) and stirred at 20 oC for 1 h. The batch was allowed to settle and the phases were separated. The organic layer was retained in the reactor and charged with water (3.2 L) and stirred at 20oC for 1 h. The batch was allowed to settle and the phases were separated. The organic solution was retained in the reactor and distilled under reduced pressure at 20oC to remove 3.4 kg of distillate. Ethanol (4.8 L) was loaded into the batch and the mixture was distilled to a volume of 3.2 L. This distillation process was repeated once more. Ethanol (0.6 L) was added to the batch to adjust the batch volume to 4 L. The batch was stirred at 20oC for 16 h and then loaded with water (6.39 L). The resulting slurry was stirred at 20 ° C for 5 h. The product was collected by filtration and was washed twice with a mixture of ethanol (529 ml) and water (1059 ml). The product was dried under reduced pressure at 45 ° C for 65 h to generate the desired product (719.6 g, 95.4%) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 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 (EI) m / e 437.9 / 439.9 (M + + H).
[00294] Example 2. Alternative preparation of N- [2 - ({4- [4- (3- Bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole -3-yl] -1,2,5- oxadiazol-3-yl} amino) ethyl] sulfamide

[00295] Step 1: Ethyl {[(tert-butoxycarbonyl) -amino] sulfonyl} aminoacetate (13)

[00296] A solution of chlorosulfonylisocyanate (Sigma-Aldrich) (5.0 ml, 57.4 mmol) in dichloromethane (100 ml) was cooled to 0 ° C. Terc-butyl alcohol (4.26 g, 57.4 mmol, 1.0 equiv.) Was added dichloromethane (100 mL) via an addition funnel. This solution was stirred at 0 ° C for 30 min. Glycine ethyl ester hydrochloride (8.82 g, 63.2 mmol, 1.1 equiv.) Was added followed by triethylamine drip addition (20.0 mL, 144 mmol, 2.5 equiv.) At 0 ° C . 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 sulfate and concentrated to generate the desired product (13.2 g, 81.4%) as an off-white solid crude product, which was used in the subsequent reaction without further purification. 1H NMR (300 MHz, DMSO-d6) δ 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).
[00297] Step 2a. (ally {[ally (tert-butoxycarbonyl) amino] sulfonyl} amino) Ethyl acetate (14a)

[00298] ({[(tert-butoxycarbonyl) amino] sulfonyl} Ethyl aminoacetate (1.0 g, 3.54 mmol) was mixed with potassium carbonate (2.45 g, 17.7 mmol, 5.0 equiv .) and acetonitrile (23.0 mL) under N 2 at room temperature Allyl bromide (1.84 mL, 21.2 mmol, 6.0 equiv.) was added under a drip This reaction mixture was heated at 70 ° C and stirred at that temperature for 14 h. HPLC and LCMS indicated the completion of the reaction. The reaction was filtered and the filtrate was concentrated. The residue was dissolved in dichloromethane and washed with sodium bicarbonate and brine. was dried over sodium sulfate and concentrated to generate the desired product (1.11 g, 87%) as an off-white solid, which was used in the subsequent reaction without further purification. 1H NMR (300 MHz, DMSO- d6) δ 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).
[00299] Step 2b. [{[(tert-butoxycarbonyl) (4-methoxybenzyl) amino] sulfonyl} (4-methoxybenzyl) amino] Ethyl acetate (14b)

[00300] ({[(tert-butoxycarbonyl) amino] sulfonyl} amino) Ethyl acetate (1.00 g, 4.0 mmol) was mixed with N, N-dimethylformamide (DMF, 6.0 mL) and stirred at room temperature. 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 heated to 80 ° C and stirred at 80 ° C for 2 h. LCMS indicated the completion of the reaction. The reaction was cooled to room temperature and filtered through Celite. The Celite bed was washed with dichloromethane and the combined organic filtrates were concentrated. The concentrated residue was dissolved 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 acetate / hexane elution gradient) to generate the desired product (1.39 g, 80%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 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 = 7.1 Hz).
[00301] Step 3a. tert-butyl ally {[ally (2-oxoethyl) amino] sulfonyl} carbamate (15a)

[00302] A solution of (ally {[ally (tert-butoxycarbonyl) amino] sulfonyl} amino) ethyl acetate (1.11 g, 3.05 mmol) in dichloromethane (15 mL) at -78 ° C under N2 was treated with 1.0 M diisobutylaluminum hydride in dichloromethane (3.66 mL, 3.66 mmol, 1.2 equiv.). The reaction mixture was stirred at -78 ° C for 1 h and then quenched with methanol (1.5 ml) and treated with a saturated 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 generate the desired product (0.62 g, 64%) as a colorless thick crude oil, which was used in the reaction subsequent without further purification. 1H NMR (300 MHz, DMSO-d6) δ 9.45 (s, 1H), 5.76 (m, 2H), 5.18 (m, 4H), 4.15 (m, 4H), 3.72 (m, 2H), 1.43 (s, 9H).
[00303] Step 3b. (4-methoxybenzyl) {[(4-methoxybenzyl) (2-oxoethyl) amino] sulfonyl} tert-butyl carbamate (15b)

[00304] A solution of [{[(tert-butoxycarbonyl) (4-methoxybenzyl) amino] sulfonyl} (4-methoxybenzyl) amino] ethyl acetate (5.30 g, 10 mmol) in dichloromethane (20.0 ml) at - 78 ° C under N2 atmosphere, it was treated with 1.0 M of diisobutylaluminum hydride in dichloromethane (12.2 ml, 12.2 mmol, 1.22 equiv.). The reaction mixture was stirred at -78 ° C for 3 h. The reaction 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 elution gradient) to generate the desired product (3.45 g, 71%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 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).
[00305] Step 4a. tert-butyl ally (N-allyl-N- (2- (4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole- 3-yl) -1,2,5-oxadiazol-3-ylamino) ethyl) sulfamoyl) carbamate (16a)

[00306] 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. hydrofuran (THF, 1.0 mL) at room temperature. This mixture was cooled to -5 ° C under N2 and stirred at 0 -5 ° C for 10 min. To this solution was added 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 (4 H) -one (0.171 g, 5.0 mmol; Step D) and tert-butyl ally {[ally (2-oxoethyl) amino] sulfonyl} carbamate (0.398 g, 2.5 mmol, 0.5 equiv.) in THF ( 1.5 mL) under a drip at 0-5 ° C for 5 min. The resulting reaction mixture was stirred under N 2 at 0-5 ° C. At the time points of 20 min, 40 min, and 2.5 h, a solution of tert-butyl ally {[ally (2-oxoethyl) amino] sulfonyl} carbamate (0.040 g, 0.125 mmol, 0.25 equiv.) in THF (0.20 mL) was added under a drip at 0-5 ° C. In 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 ° C. The reaction was warmed to room temperature and stirred overnight. The reaction mixture was then poured into a saturated solution cooled with sodium carbonate ice (50 ml) and extracted with dichloromethane (3 x 20 ml). The combined dichloromethane extracts were washed with brine, dried over sodium sulfate, and concentrated. The residue was then purified on silica gel (0-75% ethyl acetate / hexane elution gradient) to generate the desired product (0.239 g, 74.2%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 8.07 (m, 1H), 7.71 (m, 1H), 7.58 (t, 1H, J = 8.7 Hz), 6.62 (m , 1H), 5.77 (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 (EI) m / e 544/546 (M + - Boc + H).
[00307] Step 4b. N- (2- (4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl) -1,2, Tert-butyl 5-oxadiazol-3-ylamino) ethyl) -N- (4-methoxybenzyl) sulfamoyl (4-methoxybenzyl) carbamate (16b)

[00308] To a 50 ml flask were added sodium triacetoxyborohydride (0.50 g, 2.37 mmol, 4.74 equiv.), Trifluoroacetic acid (TFA, 1.0 mL, 13 mmol) and tetrahydrofuran. hydrofuran at room temperature. This mixture was cooled to 0 -5 ° C under N2 atmosphere and stirred at 0 - 5 ° C for 10 min. To this solution were 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-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 (4 H) -one (0.17 g , 0.50 mmol; Step D) in tetrahydrofuran (THF, 1.50 mL) at 0 -5 ° C. The reaction was stirred at 0-5 ° C 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 ° C. After stirring at 0-5 ° C for 1 h, the reaction mixture was gradually warmed up to room temperature with stirring. At 2.5 h and 4.5 h time points, trifluoroacetic acid (0.25 ml) was added. In 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. In 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 that about 4% of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 ( 4 H) -one as starting material (from Step D) still remained. The reaction mixture was stirred at room temperature overnight. HPLC indicated completion of the reaction. The reaction mixture was poured into a saturated solution cooled with sodium carbonate ice (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 residue was then purified on silica gel (0-30% ethyl acetate / hexane elution gradient) to generate the desired product (0.33 g, 82.5%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 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); CssHssBrFNyOgS (MW 804.64), LCMS (EI) m / e 826/828 (M + - Boc + Na).
[00309] Step 5: N- [2 - ({4- [4- (3-Bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole-3- yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] sulfamide (10)

[00310] To a 25 ml vial was added {[[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) tert-butyl carbamate (40.2 mg, 0.050 mmol) in trifluoroacetic acid (TFA, 0.50 mL, 6.5 mmol) at room temperature. This reaction mixture was heated to 70 ° C under N2 and stirred for 1 h. HPLC indicated that the reaction was completed. The reaction mixture was cooled to room temperature and the TFA was evaporated. The residual TFA was removed by treatment with dichloromethane (3 x 10 ml) followed by evaporation in vacuo. The residue was then triturated with dichloromethane and methanol to generate the desired product (20 mg, 87%) as an off-white solid product. 1H NMR (400 MHz, DMSO-d6) δ 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); C12H11BrFN7O5S (MW 464.23), LCMS (EI) m / e 487.8 / 489.8 (M + + Na).
[00311] Example 3. Alternative preparation of N- [2 - ({4- [4- (3- Bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole -3-yl] -1,2,5- oxadiazol-3-yl) amino) ethyl] sulfamide

[00312] Step 1a. Tert-butyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate (17a)

[00313] A solution of chlorosulfonylisocyanate (11.32 g, 80 mmol) in dichloromethane (120 mL) was cooled to 0 ° C. T erc-butyl alcohol (7.65 mL, 80.0 mmol, 1.0 equiv.) Was added via an addition funnel. The mixture was stirred at 0 ° C for 1.5 h. To this mixture, a solution of dimethyl acetal aminoacetaldehyde (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 under a drip through an addition funnel. The reaction was warmed up 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 sulphate and concentrated to generate the desired product (15.6 g, 68.5%) as a solid crude product off-white, which was used for the subsequent reaction without further purification: 1H NMR (300 MHz, DMSO-d6) δ 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), 1.41 (s, 9H).
[00314] Step 1b. Benzyl-N- (2,2-dimethoxyethyl) sulfamoylcarbamate (17b)

[00315] A solution of chlorosulfonylisocyanate (16.26 g, 114.9 mmol) in dichloromethane (100 mL) was cooled to 0 ° C. Benzyl alcohol (12.44g, 115.0 mmol, 1.0 equiv.) Was added via an addition funnel. The mixture was stirred at 0 ° C for 0.5 h. To this mixture was added a mixture of dimethyl acetal aminoacetaldehyde (13.25 g, 126.0 mmol, 1.1 equiv.) And triethylamine (TEA, 17.4 g, 172 mmol, 1.5 equiv.) Under drip through addition funnel below 15 ° C. 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 vacuo to generate the desired product (23.5 g, 64 , 3%) as an off-white solid crude product. 1H NMR (300 MHz, DMSO-d6) δ 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).
[00316] Step 1c. Ethyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate (17c)

[00317] A solution of chlorosulfonylisocyanate (11.32 g, 80 mmol) in dichloromethane (120 mL) was cooled to 0 ° C. Ethanol (4.67 mL, 80.0 mmol, 1.0 equiv.) Was added via an addition funnel. The mixture was stirred at 0 ° C for 1.5 h. To this mixture was added a solution of dimethyl acetal aminoacetaldehyde (8.76 mL, 80.0 mmol, 1.0 equiv.), Triethylamine (TEA, 33.4 mL, 240 mmol, 3.0 equiv.) In dichloro- methane (DCM, 120.0 mL) under drip via addition funnel at 0 ° C. The reaction was warmed to room temperature and stirred overnight. The reaction was treated with 0.1 N hydrochloric acid and the collected organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to generate the desired product (11.2 g, 55%) as a crude product whitish. 1H NMR (300 MHz, DMSO-d6) δ 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).
[00318] Step 1d. 2,2,2-trichloroethyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate (17d)

[00319] A solution of chlorosulfonylisocyanate (6.96 mL, 80 mmol) in dichloromethane (120 mL) was cooled to 0 ° C. 2,2,2-trichloroethanol (7.67 ml, 80.0 mmol, 1.0 equiv.) Was added via addition funnel at 0 ° C. This mixture was stirred at 0 ° C for 1.5 h. To this mixture was then added a solution of dimethyl acetal aminoacetaldehyde (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 under a drip through an addition funnel at 0 ° C. 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 Na 2 SO 4, and concentrated to generate the desired product (28.01 g, 97%) as an off-white solid product. , which was used in the subsequent reaction without further purification. 1H NMR (300 MHz, DMSO-d6) δ 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).
[00320] Step 2a. ({[2 - ({[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) Terc-butyl carbamate (18a)

[00321] A mixture of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-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 mixture, trifluoroacetic acid (0.50 mL, 6.5 mmol) and triethylsilane (80.2 mL, 0.502 mmol, 2.5 equiv.) Were added under a drip. This reaction mixture was stirred at room temperature for 2 h. HPLC indicated a conversion of approximately 30%. The reaction mixture was cooled to 0 ° C and neutralized with saturated sodium bicarbonate to pH ~ 8. The mixture was extracted in ethyl acetate (3 x 10 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and concentrated. The residue was purified by preparative TLC (50% ethyl acetate / hexane) to generate the desired product (27.5 mg, 29.5%) as an off-white solid. 1H NMR (DMSO-d6, 400 MHz): δ 10.90 (s, 1H), 8.08 (dd, J = 6.2, 2.5Hz, 1H), 7.72 (m, 1H), 7 , 59 (t, J = 8.6 Hz, 1H), 6.58 (t, J = 5.7 Hz, 1H), 3.38 (dd, J = 12.7, 6.2 Hz, 2H), 3, 10 (dd, J = 12.1, 5.9 Hz, 2H), 1.41 (s, 9H). C17H19BrFN7O7S (MW 564.34), LCMS (EI) m / e 485.8 / 487.8 (M + - C5H8O2 + Na).
[00322] Step 2b. ({[2 - ({[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) benzyl carbamate (18b)

[00323] A mixture of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-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 equiv.) in 1, 2-dichloroethane (3.0 mL) was cooled to 0 ° C. To this mixture, trifluoroacetic acid (1.0 mL, 13.0 mmol) etriethylsilane (105 μL, 0.66 mmol, 3.3 equiv.) Was added under a drip. This reaction mixture was stirred at 0 ° C for 2 h. HPLC indicated completion of the reaction. The reaction mixture was cooled to 0 ° C and neutralized with saturated sodium bicarbonate to pH ~ 8 and the neutralized reaction mixture was extracted with EtOAc (3 x 10 mL). The combined organic extracts were washed with brine, dried over sodium sulfate 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 vacuo to generate the desired product (125 mg, 99%) as an off-white solid product. 1H NMR (300 MHz, DMSO-d6) δ 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 + + H).
[00324] Step 2c. ({[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) ethylamino} sulfonyl) carbamate from Ethyl (18c)

[00325] A mixture of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 (4 H ) -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 ° C. To this mixture was added trifluoroacetic acid (1.00 mL, 13.0 mmol) and triethylsilane (105 μL, 0.66 mmol, 3.3 equiv.) = Under a drip. The reaction mixture was stirred at 0 ° C for 3 h. HPLC indicated a 97.5% conversion to the desired product. The reaction mixture was cooled to 0 ° C and neutralized with saturated sodium bicarbonate to pH ~ 8. The mixture was extracted in ethyl acetate (3 x 10 ml). The combined organic extracts 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 generate the desired product (95 mg, 88%) as a whitish solid crude product. 1H NMR (300 MHz, DMSO-d6) δ 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 (EI) m / e 536/538 (M + + H).
[00326] Step 2d. ({[2 - ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl] -1,2 2,2,2-Trichlorethyl (18d), 5-oxadiazol-3-yl} amino) ethyl] amino} sulfonyl) carbamate (18d)

[00327] A suspension of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 (4 H ) 2,2,2-trichloroethyl {5 (0.6-g, 1.99 mmol, 0.699 g, 1.99 mmol) and 2,2,2-trichloroethyl {[(2,2-dimethoxyethyl) amino] sulfonyl} carbamate (17d, 2.22 g, 6.17 mmol, 3.1 equiv.) In dichloromethane (DCM, 6.0 mL) was stirred at room temperature. To this mixture 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), keeping the reaction temperature below 30 ° C. The reaction mixture became homogeneous after 5 min with stirring at room temperature and was stirred at room temperature for 1 h. HPLC indicated completion of the reaction. 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 vacuo to generate the desired 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-d6) δ 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); C15H12BrCl3FN7O7S (MW 639.62), LCMS (EI) m / e 638/640/642 (M + + H).
[00328] Step 3. N- [2 - ({4- [4- (3-Bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole-3- yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] sulfamide (10)

[00329] A solution of ({[2 - ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo- 4,5-dihydro-1,2,4-oxadiazole-3- yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] amino} sulfonyl) 2,2,2-trichloroethyl carbamate (320 mg, 0.50 mmol; from Step Q, Method D) in tetra -hydrofuran (THF, 4.0 mL) was stirred at room temperature. Acetic acid (0.30 mL, 5.3 mmol) and zinc flakes (160 mg, 2.5 mmol, 5.0 equiv.) Were added sequentially. This reaction mixture was stirred at room temperature for 3 h. HPLC indicated completion of the reaction. The reaction mixture was filtered through Celite and Celite was washed with THF. The combined filtrate was concentrated in vacuo and the resulting residue was dissolved 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 generate the desired product (147 mg, 63%) as an off-white solid.
[00330] Example 4. Alternative preparation of 4 - ({2- [(Aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'- hydroxy-1,2,5-oxadiazole -3-carboxyimidamide

[00331] Step 1. (9H-fluoren-9-yl) methyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate

[00332] In a 2 L round-bottom flask with 4 necks dried in the oven, 9-fluorenylmethanol (50.0 g, 255 mmol) and anhydrous DCM (382 mL) were loaded at room temperature. The resulting slurry was cooled in an ice bath to about 0 - 5 ° C. A solution of chlorosulfonylisocyanate (CSI, 23.0 mL, 264 mmol) in anhydrous DCM (127 mL) was added dropwise to the suspension through an addition funnel for 22 minutes, maintaining the temperature of the reaction mixture at <5 ° C . The resulting mixture was stirred at 0 - 5 ° C for 1.75 h, producing a thick white paste. A solution of dimethyl acetal aminoacetaldehyde (27.9 ml, 255 mmol) in anhydrous DCM (382 ml) and 4-methylmorpholine (84.0 ml, 764 mmol) was added to the mixture at about 0 - 5 ° C at the 71 minutes. The resulting reaction mixture was then stirred in the ice bath for 1.5 hours. When the HPLC showed that 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 a pH of 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 in vacuo to dryness. The resulting solids were again dissolved in EtOAc (600 mL) at 65 ° C and the hot solution was filtered in a clean 3 L round bottom flask. The filtrate was cooled to room temperature and stirred for 2.5 h before of heptane (1200 ml) is then added through an addition funnel over 80 min. After stirring overnight at room temperature, the mixture was then cooled in an ice bath for 1 h. The resulting solids were collected by filtration, washed with 25% EtOAc / heptane (250 mL), and dried overnight at about 40 - 45 ° C under vacuum to generate {[(2,2-dimethoxyethyl) ) amino] sulfonyl} 9H-fluoren-9-ylmethyl carbamate (91.3 g, 88% yield) as a white powder. 1H NMR (300 MHz, DMSO- d6) δ 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.
[00333] Step 2. ({[2 - ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo-4,5-dihydro-1,2,4-oxadiazole-3- yl] -1,2,5-oxadiazol-3-yl} amino) ethyl] amino} sulfonyl) 9H-Fluoren-9-ylmethyl carbamate

[00334] To a stirred suspension of 3- (4-amino-1,2,5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2,4-oxadiazole-5 ( 4H) -one (10.00 g, 29.23 mmol) in DCM (160 mL) methanesulfonic acid (Me-SO3H, 8.46 g, 88.04 mmol) and triethylsilane (Et3SiH, 8.37 g, 71.96 mmol) at room temperature over 10 minutes to generate a paste. 9H-fluoren-9-ylmethyl {[(2,2-dimethoxyethyl) amino] sulfonyl} carbamate solid (12.25 g, 30.14 mmol) was added in portions (1 g / 3 - 4 minutes; over 1 h) while maintaining the internal temperature below 20 ° C using a water bath. After the addition, the resulting mixture was stirred at about 13 to 22 ° C for 3 days. Additional triethylsilane (Et3SiH, 0.1755 g, 1.51 mmol) and 9H-fluoren-9-ylmethyl {[(2,2-dimethoxyethyl) amino] sulfonyl} carbamate (0.3082 g, 0.76 mmol) were added and the resulting mixture was stirred at room temperature for an additional 23 h. Isopropyl alcohol (IPA, 15 ml) was added and the resulting mixture was stirred at room temperature for 1 h. Heptane (100 mL) was added and the mixture was stirred at room 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 generate ({[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) 9H-fluoren-9-ylmethyl carbamate as a white solid (18.30 g, 91.1% yield). 1H NMR (500 MHz, DMSO-d6) δ 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, J = 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, J = 7.2 Hz, 1H), 3.39 (q, J = 6.4 Hz, 2H), 3.15 (q, J = 6.3 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ 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; 19F NMR (376 MHz, DMSO-d6) δ -103.99 - -107.39 (m) ppm.
[00335] Step 3. 4 - ({2 - [(Aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'-hydroxy-1,2,5-oxadiazole-3- carboxyimidamide

[00336] 9H-fluoren-9-ylmethyl ({[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 room 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 under a drip for 35 minutes through an addition funnel. The addition funnel was washed with anhydrous THF (50 ml) and the rinse was added to the reaction mixture. The cold bath was removed and the reaction mixture was gradually warmed up to room temperature and stirred at room temperature for 2.5 h. EtOAc (400 mL) was added and the resulting mixture was transferred to a 4-neck 2 L round-bottom flask and cooled to about 0 - 5 ° C in an ice bath. A 2.0 M aqueous HCl solution (400 mL, 800.0 mmol) was added under a drip through an addition funnel, while maintaining the internal temperature 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 2.0 M aqueous HCl solution (200.0 mL, 400.0 mmol) was added dripping to the cold organic fraction, keeping the internal temperature 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 generate a homogeneous solution. To the solution, a solution of DCM (250.0 mL) and tert-butyl methyl ether (TBME, 100.0 mL) was added under a drip. 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 a cold 250 ml solution of DCM ice (150 ml) and TBME (100 ml), and dried under vacuum to generate 14.4 g of the desired crude product as white solids .
[00337] The crude product was dissolved in EtOAc (140.0 ml) at 60 ° C and the hot solution was filtered. The filtrate was cooled to room temperature before heptane (100.0 mL) was added under a drip for 55 min. The resulting mixture was then stirred overnight at room temperature. The solids were collected by filtration, washed with a 2: 1 mixture of heptane and EtOAc (75 mL), and dried under vacuum at 40 - 50 oC to constant weight, to generate 4- ({2 - [(aminosulfonyl) amino] ethyl} amino) -N- (3-bromo-4-fluorophenyl) -N'- hydroxy-1,2,5-oxadiazole-3-carboximidamide (12.9 g, 81% yield) as a white solid .
[00338] Example A: Human 2,3-dioxigenasae (IDO) indolamine enzyme assay
[00339] Human 2,3-dioxygenasae (IDO) indolamine with an N-terminal His tag was expressed in E. coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the tryptophan indole core pyrrole ring to generate N'-formylquinurenine. The tests were carried out at room temperature, as described in the literature using 95 nM acid and 2 mM D-Trp, in the presence of 20 mM ascorbate, 5 μM methylene blue and 0.2 mg / mL catalase in 50 mM of potassium phosphate buffer (pH 6.5). Initial reaction rates were recorded continuously following the increase in absorbance at 321 nm due to the formation of N'-formylquinurenine (See: Sono, M., et al., 1980, J. Biol. Chem. 255, 1339-1345) . The Formula I compound was tested in the Example A test and verified to have an IC 50 of <200 nm.
[00340] Example B: Determination of the inhibitory activity in assay based on HeLa cells of indolamine 2,3-dioxigenase (IDO) assay / quinurenine
[00341] HeLa cells (# CCL-2) were obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA) and routinely maintained in minimal essential medium (Eagle) with 2 mM L-glutamine and BSS Earle's adjusted 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). The cells were maintained at 37oC in a humidified incubator supplied with 5% CO 2. The assay was performed as follows: HeLa cells were seeded in a 96-well culture dish at a density of 5 x 10 3 per well and grown overnight. The next day, IFN-Y (50 ng / mL final concentration) and serial dilutions of the compounds (in a total volume of 200 μL of culture medium) were added to cells. After 48 hours of incubation, 140 μL of the supernatant per well was transferred to a new 96-well plate. 10 μL of 6.1 N trichloroacetic acid (# T0699, Sigma) was mixed in each well and incubated at 50 ° C for 30 minutes to hydrolyze N-formylquinurenine produced by indolamine 2,3-dioxigenase to quinurenine. The reaction mixture was then centrifuged for 10 min at 2500 rpm to remove sediment. 100 μL of the supernatant per well was transferred to another 96-well plate and mixed with 100 μL of 2% (w / v) p-dimethylaminobenzaldehyde (# 15647-7, Sigma-Aldrich) in acetic acid. The yellow color derived from quinurenine was measured at 480 nm using a SPECTRAmax 250 microplate reader (Molecular Devices). L-quinurenine (# K8625, Sigma) was used as a standard. The standards (240, 120, 60, 30, 15, 7.5, 3.75, 1.87 μM) were prepared in 100 μL of culture medium and mixed with an equal volume of 2% (w / v) p- dimethylaminobenzaldehyde. The percentage of inhibition at individual concentrations was determined and mean duplicate values were obtained. The data were analyzed using non-linear regression to generate IC50 values (Prism GraphPad). See: Takikawa O, et al., 1988, J. Biol. Chem., 263 (4): 2041-8.
[00342] Example C: Determination of the effect of IDO inhibitors on T cell proliferation which is suppressed by dendritic cells expressing IDO
[00343] Monocytes were collected from human peripheral mononuclear cells by leukophoresis. The monocytes were then seeded at a density of 1 x 106 cells / 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 kept on the plate after culture overnight at 37 ° C. Adherent monocytes were then stimulated for 5-7 days with 100 ng / ml of GM-CSF (# 300-03, PeproTech) and 250 ng / ml of IL-4 (# 200-04, PeproTech), followed by activation with 5 μg / mL of LPS from Salmonella typhimurium (# 437650, Sigma) and 50 ng / mL of IFN-Y (# 285-SE, R & D Systems) for another 2 days to induce maturation of dendritic cells.
[00344] After activation of dendritic cells, the medium was replaced with complete RPMI 1640 supplemented with 100-200 U / ml IL-2 (# CYT-209, ProSpec-Tany TechnoGene) and 100 ng / ml antibody - anti-CD3 well (# 555336, PharMingen), T cells (2-3 x 105 cells / well), and serial dilutions of IDO compounds. After incubation for an additional 2 days, T cell proliferation was measured by a BrdU incorporation test using an ELISA colorimetric cell proliferation kit following the manufacturer's instructions (# 1647229, Roche Molecular Biochemicals). The cells were cultured continuously for 16-18 hours in the presence of a 10 μM labeling solution of BrdU. Then, the labeling medium was removed, and 200 μL per well of FixDenat was added to the cells and incubated for 30 minutes at room temperature. The FixDe- nat solution was removed and 100 μL / well of anti-BrdU-POD conjugated antibody working solution was added. The reaction was carried out for 90 minutes at room temperature. The antibody conjugate was then removed, and the cells were washed three times with 200 μL / well washing solution. Finally, 100 μL / well of substrate solution was added and the results were obtained using a microplate reader (Spectra Max Plus, Molecular Devices) during color development. Several readings were obtained at different points in time to ensure that the data were within the linear range. Data were routinely obtained from replicated experiments, and appropriate controls were included. See: Terness P, et al. 2002, J. Exp. Med., 196 (4): 447-57; and Hwu, P, et al. 2000, J. Immunol., 164 (7): 3596-9.
[00345] Example D: IN VIVO test of IDO inhibitors for antitumor activity
[00346] Antitumor efficacy in vivo can be tested using modified tumor allograft / xenograft protocols. For example, it has been described in the literature that inhibition of IDO may be synergistic with cytotoxic chemotherapy in immunocompetent mice (Muller, A.J., et al. 2005, Nat. Med. 11: 312-319). This synergy has been shown to be T cell dependent by comparing the synergistic effects of an investigational IDO inhibitor in murine tumor xenograft models (eg, B16 and related variants, TC-26, LLC) cultured in imenous single mice - nocompetent to that observed in syngeneic mice treated with neutralizing anti-CD4 antibodies, or the same tumors grown in immunocompromised mice (for example, nu / nu).
[00347] The concept of differential antitumor effects in immunocompetent versus immunocompromised mice may also allow testing of investigational IDO inhibitors as individual agents. For example, LLC tumors grow well in their syngeneic host strain, C57BL / 6. However, if these mice are treated with the IDO 1-MT inhibitor (versus placebo), tumor formation is significantly delayed, which implies that IDO inhibition was growth inhibitory (Friberg, M., et al. 2002 , Int. J. Cancer 101: 151-155). Following this logic, one can examine the efficacy of IDO inhibition in the LLC xenograft tumor model grown in immunocompetent C57Bl / 6 mice and compare the effects of IDO inhibitors on LLC tumor growth in naked or SCID mice (or C57B1 mice) / 6 treated with antibodies that neutralize T cell activity). As the tumor IDO-mediated immune suppressive effects of relief 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 example, the expression of GM-CSF in B16.F10 cells increases their immunogenic potential (Dranoff, G., et al. 1993, Proc. Natl. Acad. Sci., USA, 90: 3539-3543). As such, in some tumor models (eg B16.F10), a [oly] clone can be generated that express immune stimulating proteins, such as GM-CSF, and test the growth inhibitory effects of IDO inhibitors. against tumors established from these tumor cells in both immunocompetent and immunocompromised mice.
[00348] A third way to evaluate the efficacy of IDO inhibitors in vivo employs murine tumor allograft / xenograft 'preimmunization' models. In these models, immunocompetent mice are sensitized to a tumor-specific antigen or antigens to mimic an anti-tumor therapeutic vaccination. This prepares the mice for an immune-mediated antitumor response when the mice were subsequently challenged with murine tumor cell lines (having tumor antigens similar to those used for immunization) in xenograft experiments. IDO expression has been shown to decrease the antitumor response and allow xenografts to grow faster. Importantly, tumor growth in this model is inhibited by the IDO 1-MT inhibitor (Uyttenhove, C., et al. 2003, Nat. Med. 9: 1269-1274). This model is particularly attractive since the IDO activity is permissive for the growth of the P815 tumor and specific inhibition of the IDO must therefore be growth inhibitory.
[00349] Finally, therapeutic immunization can be used to assess the impact of IDO inhibitors in vivo. For example, it has been demonstrated using B16-BL6 cells that can challenge Blk / 6 mice with an intravenous injection of tumor cells, followed by treatment with a well-characterized immunogenic peptide (eg TRP-2) expressed by tumor cells (Ji, et al., 2005, J. Immunol, 175: 1456-63). Importantly, modifiers of the immune system, such as the anti-CTL-4 antibody, can improve responses to such therapeutic vaccinations. The impact of IDO inhibitors can be assessed in a similar way to tumor peptide immunization with or without IDO inhibitor. Efficacy is assessed by the animals' survival (time to morbidity) or by measuring tumor metastases to the lungs and / or other organs at defined time intervals.
[00350] In any / all of the models mentioned above, it may also be possible to directly and / or indirectly measure the number and / or activity of the tumor reactive immune cells. Methods for measuring the number and / or activity of tumor reactive immune cells are well established and can be performed using techniques familiar to those skilled in the art (Current Protocols in Immunology, Vol. 4, Coligan, JE, et al. ; Immunotherapy of Cancer, Human Press, 2006, Disis, ML and references therein). Con- ceptively, a reduction in the immunosuppressive effects of IDO may result in increased numbers or reactivity of tumor-specific immune cells. In addition, the inhibition of IDO may further increase the number or reactivity of immune cells of reactive tumor when combined with other therapeutic agents, for example, chemotherapeutic agents and / or immunomodulators (for example, anti-CTLA4 antibodies).
[00351] 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: Preclinical Screening, Clinical Trials, and Ap- proval, 2nd Ed. Teicher, BA and Andrews, PA, Gumana Press Inc .: Towa, 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 skilled 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).
[00352] Example E: IN VIVO test of IDO inhibitors in human immunodeficiency virus encephalitis model-1 (HIV-1) 1. Cell isolation and viral infection
[00353] Monocytes and PBL can be obtained by counter-current centrifugation elutriation of leukopheresis packages from seronegative donors for HIV-1, 2 and hepatitis B. Monocytes are cultured in suspension culture, using vials of Teflon in Eagle-modified Dulbecco medium (DMEM, Sigma-Aldrich) supplemented with 10% pooled human heat inactivated serum, 1% glutamine, 50 μg / gentamicin mL, 10 μg / mL ciprofloxacin (Sigma ), and 1000 U / ml of highly purified human macrophage colony stimulating factor. After seven days in culture, MDM are infected with HIV-1ADA at a multiplicity of infection of 0.01. 2. Hu-PBL-NOD / SCID HIVE mice
[00354] Four-week-old NOD / C.B-17 SCID mice can be purchased (Jackson Laboratory). The animals are kept in sterile micro-insulated cages under conditions free of pathogens. All animals are injected intraperitoneally with rat anti-CD122 (0.25 mg / mouse) three days before PBL transplantation and twice with rabbit asialo-GM1 antibodies (0.2 mg / mouse) ( Wako) one day before and three days after PBL injection (20 x 106 cells / mouse). MDM cells infected with HIV-1ADA (3 x 105 cells in 10 μL) are injected intracranially (i.c.) eight days after PBL reconstitution, generating hu-PBL-NOD / SCID HIVE mice. Immediately after i.c. injection of HIV-1 infected MDM the hu-PBL-NOD / SCID HIVE mice are implanted subcutaneously (s.c) with control (vehicle) or compound pellets (14 or 28 days of slow release, Innovative Research). The initial experiments were designed to confirm the induction of virus-specific CTL in hu-PBL-NOD / SCID HIVE animals treated with IDO compounds. This is confirmed by tetramer staining and neuropathological analysis of MDM elimination from brain tissue. Then, the assay is designed to analyze human lymphocyte replenishment, human immune responses, and neuropathological changes. In these experiments, the animals were bled on the 7th and sacrificed on the 14th and 21st days after i.c. of human MDM. The blood collected in tubes containing EDTA is used for flow cytometry and the plasma is used for the detection of HIV-1 p24 using ELISA (Beckman Coulter ™). HIV-1 specific antibodies are detected by Western blot assays according to the manufacturer's instructions (Cambridge Biotech HIV-1 Western blot kit, Calypte Biomedical). Similar amounts of virus-specific antibodies are detected in the control and compound-treated animals. A total of three independent experiments can be performed using three different donors of human leukocytes. 3. FACScan for peripheral blood and spleen in hu PBL-NOD / SCID HIVE
[00355] Two-color FACS analysis can be performed on peripheral blood in 1-3 weeks and splenocytes at week 2 and 3 after i.c. injection. of human MDM. The cells are incubated with fluorochrome-conjugated monoclonal abs (mAb) for human CD4, CD8, CD56, CD3, IFN-Y (eBioscience) for 30 min at 4 ° C. To assess the cellular immune response, intracellular staining of IFN-γ is performed in combination with human anti-CD8 and mouse anti-CD45 conjugated to FITC to exclude murine cells. To determine Ag-specific CTL, tetramer staining conjugated to allophicocyanin for HIV-1gag (p17 (aa77-85) SLYNTVATL, SL-9) and HIV-1pol [(aa476-485) ILKEPVHGV, IL-9] is performed on splenocytes stimulated with phytohemagglutinin / interleukin-2 (PHA / IL-2). The cells are stained according to the recommendation of the NIH / National Institute of Allergy and Infections Disease, National Tetrader Core Facilities. The data were analyzed with a FACS CalibratorTM using CellQuest software (Becton Dickinson Immunocytometry System). 4. Histopathological and image analysis
[00356] Brain tissue is collected on days 14 and 21 after i.c. injection MDM, fixed in 4% paraformaldehyde buffered with phosphate and embedded in paraffin or frozen at -80oC for later use. Coronal sections of embedded blocks are cut to identify the injection site. For each mouse, 30-100 serial sections (5- μm thick) are cut from the human MDM injection site and 3-7 slides (10 sections apart) are analyzed. Sections of the brain are dewaxed with xylene and hydrated in alcohol gradients. Immunohistochemical staining follows a basic indirect protocol, using antigen recovery by heating to 95 ° C in 0.01 mol / L of citrate buffer for 30 min for antigen recovery. To identify human cells in the rat brain, the vimentin mAb (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, KP 1 clone) and CD8 (1:50 dilution, 144B clone) antibodies, respectively. Virus infected cells are labeled with mAb for HIV-1 p24 (1:10, Kal-1 clone, all from Dako). Reactive murine microglial cells are detected with an Iba-1 antibody (1: 500, Wako). Human IDO expression (huIDO) is visualized with Abs obtained from the Department of Cellular Pharmacology, Central Research Institute, Hokkaido University School of Medicine, Sapporo, Japan. Primary antibodies are detected with secondary antibodies appropriate biotinylates and visualized with avidin-biotin complexes (Vectastain Elite ABC kit, Vector Laboratories) and dextran polymer coupled to horseradish peroxidase (HRP) (EnVision, DAKO Corporation). The immunostained sections are contrasted with Mayer's hematoxylin. The sections from which the primary antibody is eliminated or irrelevant IgG isotype is incorporated serving as controls. Two independent observers blindly count the numbers of CD8 +, MDM CD68 + lymphocytes and HIV-1 p24 + cells in each section of each mouse. Examination under the light microscope is performed with a Nikon Eclipse 800 microscope (Nikon Instruments Inc). Semi-quantitative analysis for Iba1 (percentage of the area occupied by immunostaining) is performed by computer-aided image analysis (Image-Pro®Plus, Media Cybernetics) as previously described. 5. Statistical analysis
[00357] Data can be analyzed using Prism (Graph Pad) with Student t- test for comparisons and ANOVA. P-values <0.05 were considered significant. 6. Reference
[00358] Poluektova LY, Munn DH, Persidsky Y, and Gendelman HE (2002). Generation of cytotoxic T cells against virus-infected human brain macrophages in a murine model of HIV-1 encephalitis. J. Immunol. 168 (8): 3941-9.
[00359] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the previous description. Such modifications are also intended to be within the scope of the appended claims. Each reference, including all patents, patent applications, and publications, cited in this patent application is incorporated herein by reference in its entirety.

权利要求:
Claims (21)
[0001]
1. Process, characterized by the fact that it comprises reacting a compound of Formula F17:
[0002]
2. Process according to claim 1, characterized by the fact that R4 is tert-butyl.
[0003]
3. Process according to claim 1, characterized by the fact that R4 is benzyl.
[0004]
4. Process according to claim 1, characterized by the fact that R4 is ethyl.
[0005]
5. Process according to claim 1, characterized by the fact that R4 is 2,2,2-trichloroethyl.
[0006]
6. Process according to any one of claims 1 to 5, characterized by the fact that said reaction is carried out in the presence of triethylsilane and trifluoroacetic acid.
[0007]
Process according to any one of claims 1 to 6, characterized by the fact that it further comprises the deprotection of said compound of Formula F18 to generate a compound of Formula F10:
[0008]
8. Process according to claim 7, characterized in that it further comprises reacting said compound of Formula F10 with a base to generate a compound of Formula I:
[0009]
9. Process according to claim 1, characterized by the fact that R4 is 9H-fluoren-9-ylmethyl.
[0010]
10. Process according to claim 9, characterized by the fact that said reaction is carried out in the presence of triethylsilane and methanesulfonic acid.
[0011]
Process according to claim 9 or 10, characterized by the fact that it further comprises converting said compound of Formula F18 to a compound of Formula I:
[0012]
12. Compound, characterized by the fact that it presents Formula F17:
[0013]
13. Compound according to claim 12, characterized by the fact that it is selected from: N- (2,2-dimethoxyethyl) benzyl sulfamoylcarbamate, 2,2,2- N- (2,2-dimethoxyethyl) sulfamoylcarbamate trichlorethyl; and (9H-fluoren-9-yl) methyl N- (2,2-dimethoxyethyl) sulfamoylcarbamate.
[0014]
14. Compound, characterized by the fact that it is N- (2,2-dimethoxyethyl) tert-butyl sulfamoylcarbamate or N- (2,2-dimethoxyethyl) ethyl sulfamoylcarbamate.
[0015]
15. Process, characterized by the fact that it comprises: i) reacting a compound of Formula F19:
[0016]
16. Compound, characterized by the fact that it presents Formula F18:
[0017]
17. A compound according to claim 16, characterized by the fact that R4 is benzyl.
[0018]
18. A compound according to claim 16, characterized by the fact that R4 is ethyl.
[0019]
19. A compound according to claim 16, characterized by the fact that R4 is C1-3 haloalkyl.
[0020]
20. Compound according to claim 16, characterized by the fact that R4 is 2,2,2-trichlorethyl.
[0021]
21. A compound according to claim 16, characterized by the fact that R4 is 9H-fluoren-9-ylmethyl.

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法律状态:
2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-01-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-02-18| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-11-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-01-05| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/11/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361901689P| true| 2013-11-08|2013-11-08|

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US61/901,689|2013-11-08|

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PCT/US2014/064531|WO2015070007A1|2013-11-08|2014-11-07|Process for the synthesis of an indoleamine 2,3-dioxygenase inhibitor|

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