Bile acid-GCPII inhibitor conjugates for treating inflammatory diseases

Abstract

A GCPII inhibitor comprising 2-(phosphonomethyl)pentanedioic acid (2-PMPA) conjugated to a bile acid, and its use for treating diseases or conditions associated with elevated GCPII levels, such as inflammatory bowel disease.

Description

[Background technology]

[0001] The folate hydrolase (FOLH1) gene encodes glutamate carboxypeptidase II (GCPII), an enzyme highly overexpressed in human inflammatory diseases, including inflammatory bowel disease (IBD). It has previously been shown that GCPII enzyme function is elevated by approximately 300% to approximately 3000% in human IBD, and that inhibition of GCPII activity using small molecule drugs in IBD mouse models results in significant disease improvement (Rais et al., 2016).

[0002] IBD is an idiopathic, chronic, and often disabling intestinal inflammatory disorder with two subtypes, Crohn's disease (CD) and ulcerative colitis (UC), each accounting for approximately 50% of IBD patients (Xavier and Podolsky, 2007; Srober et al., 2007; Sartor, 2006). IBD is a widespread gastrointestinal (GI) disorder with a prevalence of approximately 0.2% in the Western population. In the United States alone, 1.4 million patients have been diagnosed with IBD, resulting in significant suffering and medical costs.

[0003] IBD is a complex, multifactorial disease involving both genetic and environmental factors, the interplay of which leads to IBD (Xavier and Podolsky; Strober et al., 2007; Sartor, 2006; Kaser et al., 2010). Unfortunately, the pathogenesis of this mucosal dysregulation in UC and CD remains unclear (Kaser et al., 2010). Despite the increasing number of treatment options available for the management of IBD, approximately one-third of IBD patients do not respond to any therapy, and there is no cure for IBD (Hamilton et al., 2012). Anti-tumor necrosis factor (TNF)-based therapies, such as infliximab (IFX), adalimumab, and certolizumab pegol, are currently the most effective treatments for severe UC and CD (Hanauer et al., 2002; Kozuch and Hanauer, 2008; Colombel et al., 2007; Schreiber et al., 2007). However, one-third of CD patients do not respond to anti-TNF therapy, and another third lose response within 6 months of treatment (Regueiro et al., 2007; Lawrance, 2014). These non-responders exhibit more aggressive mucosal immune responses and require additional treatment (Schmidt et al., 2007). Patients with extensive disease or those at risk for short bowel syndrome due to previous resections are typically poor surgical candidates. Currently, the only approved drug for patients who have failed anti-TNF agents is natalizumab. However, natalizumab has been associated with some cases of progressive and often fatal multifocal leukoencephalopathy (PML) (Van et al., 2005). These drawbacks and complications associated with current treatment regimens highlight the importance of exploring and identifying new, more effective treatments for IBD patients. Summary of the Invention

[0004] The presently disclosed subject matter provides conjugates of 2-(phosphonomethyl)pentanedioic acid (2-PMPA) or a derivative thereof with a bile acid or a derivative thereof.

[0005] In some embodiments, the conjugate comprises a compound of formula (I): [ka] (In the formula: R1 and R2 are each independently H or -OH; and R3 is OH and R4 is selected from the group consisting of -NH-X1, -COO-X1, -C(=O)-NH-CH2-C(=O)-O-X1, and -C(=O)-NH-CH2-CH2-S(=O)2-O-X1, where X1 is -(C=O)-(CH2) m -P(=O)(OH)-X2, -(C=O)-(CH2) m -CH(COOH)-CH2-P(=O)(OH)-X2, -CH2-O-(C=O)-(CH2) m -P(=O)(OH)-X2, -CH2-OC(=O)-(CH2) m -CH(COOH)-CH2-P(=O)(OH)-X2, -CH2-OC(=O)-(CH2) m -CH(COOH)-NH-(C=O)-NH-CH(COOH)-CH2-CH(CH3)2, -CH2-OC(=O)-Ar-CH2-CH(COOH)-(CH2) m -C(=O)-NH-OH, -CH2-OC(=O)-(CH2) m -X3, -CH2-OC(=O)-Ar-CH2-X3, and a protecting group, where X2 is -OH, -CH2-CH(COOH)-(CH2) p -C(=O)-OH, and a protecting group, Ar is arylene, X3 is 2-oxotetrahydro-2H-thiopyran-3-yl, and each m and p is independently selected from the group consisting of 1, 2, 3, and 4; or R3 is -OC(=O)-O-CH2-OC(=O)-(CH2) n -CH(COOH)-CH2-P(=O)(OH)2, and -OC(=O)-CH2-CH2-P(=O)(OH)-CH2-CH(COOH)-(CH2) n-C(=O)-OH, where each n is independently an integer selected from the group consisting of 1, 2, 3, and 4, and R4 is selected from the group consisting of -NH2, -COOH, -C(=O)-NH-CH2-C(=O)-OH, and -C(=O)-NH-CH2-CH2-S(=O)2-OH. and pharmaceutically acceptable salts thereof.

[0006] In some embodiments of the compounds of formula (I) above, (a) R1 and R2 are both H; (b) R1 is H and R2 is OH; (c) R1 is OH and R2 is H; or (d) R1 and R2 are both OH.

[0007] In some embodiments, R3 is OH; and R4 is selected from the group consisting of -NH-X1, -COO-X1, -C(=O)-NH-CH2-C(=O)-O-X1, and -C(=O)-NH-CH2-CH2-S(=O)2-O-X1, where X1 is -(C=O)-CH2-CH2-P(=O)(OH)-X2, -(C=O)-CH2-CH2-CH(COOH)-CH2-P(=O)(OH)-X2, -CH2-O-(C=O)-CH2-CH2-P(=O)(OH)-X2, -CH2-OC(=O)-CH2-CH2-CH(COOH)-CH2-P(=O)(OH)-X2, -CH2- and X is selected from the group consisting of: OC(=O)-CH-CH-CH(COOH)-NH-(C=O)-NH-CH(COOH)-CH-CH(CH), -CH-OC(=O)-Ar-CH-CH(COOH)-CHCH-C(=O)-NH-OH, -CH-OC(=O)-CH-CH-X, -CH-OC(=O)-Ar-CH-X, and a protecting group, wherein X is selected from the group consisting of -OH, -CH-CH(COOH)-CH-CH-CH(=O)-OH, and a protecting group, Ar is phenyl, and X is 2-oxotetrahydro-2H-thiopyran-3-yl.

[0008] In such embodiments, the compound of formula (I) is: [ka] [ka] [ka] is selected from the group consisting of:

[0009] In some embodiments, R3 is selected from the group consisting of -OC(=O)-O-CH2-OC(=O)-CH2-CH2-CH(COOH)-CH2-P(=O)(OH)2, and -OC(=O)-CH2-CH2-P(=O)(OH)-CH2-CH(COOH)-CH2-CH2-C(=O)-OH; and R4 is selected from the group consisting of -NH2, -COOH, -C(=O)-NH-CH2-C(=O)-OH, and -C(=O)-NH-CH2-CH2-S(=O)2-OH.

[0010] In such embodiments, the compound of formula (I) is: [ka] is selected from the group consisting of:

[0011] In another aspect, the presently disclosed subject matter provides a method for treating a disease or condition associated with elevated GCPII activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate of formula (I) or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the subject of the present disclosure is a conjugate of general formula (I) for use in medicine, preferably for use in the treatment of diseases or conditions associated with increased GCPII activity.

[0013] In certain embodiments, the disease or condition associated with increased GCPII activity comprises inflammatory bowel disease. In more particular embodiments, the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD) and ulcerative colitis (UC). In certain embodiments, the method for treating the disease or condition associated with increased GCPII activity comprises inhibiting GCPII activity in the subject.

[0014] In other aspects, the presently disclosed subject matter provides pharmaceutical compositions comprising at least one compound of general formula (I), alone or in combination with one or more additional therapeutic agents, in admixture with a pharmaceutically acceptable excipient.

[0015] While certain aspects of the presently disclosed subject matter that are addressed in whole or in part by the presently disclosed subject matter have been described above, other aspects will become apparent as the description proceeds when taken in conjunction with the accompanying examples and drawings that are best described herein below. [Brief explanation of the drawings]

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Having thus generally described the subject matter of the present disclosure, reference is now made to the accompanying drawings, which are not necessarily drawn to scale. [Figure 1]These figures show that select bile acid 2-PMPA conjugates have measurable anti-colitis effects in the dextran sodium sulfate (DSS) colitis model of inflammatory bowel disease (IBD) (Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H). Colitis was induced using either 2.5% DSS (Figures 1A, 1B, and 1C) or 4.0% DSS (Figures 1D, 1E, 1F, 1G, and 1H), resulting in characteristic diarrhea, rectal bleeding, and weight loss. These parameters were assessed daily, scored, and summed to calculate the disease activity index (DAI). Mice received oral treatment with the test substance or vehicle once daily, starting on study day 0 and continuing through study day 7. Test substances LTP592 (Fig. 1B, 1D), IS-101-020 (Fig. 1G), and IS-101-077 (Fig. 1H) demonstrated anti-IBD activity as evidenced by a significant reduction in DAI in drug-treated mice on days 6 and 7 of the study. Test substances LTP582 (Fig. 1A), LTP588 (Fig. 1C), LTP1054 (Fig. 1E), and IS-101-010 (Fig. 1F) were inactive. All data are expressed as mean ± SEM. Two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001. [Figure 2]Figures 2A and 2B show that conjugates of deoxycholic acid with urea and hydroxmate-based GCPII inhibitors are protective in the dextran sodium sulfate (DSS) colitis model of inflammatory bowel disease (IBD). Colitis was induced using 4.0% DSS, resulting in characteristic diarrhea, rectal bleeding, and weight loss. These parameters were assessed daily, scored, and summed to calculate the disease activity index (DAI). Mice received oral treatment with test substance or vehicle once daily, starting on study day 0 and continuing through study day 6. The test substances IS-102-138 (Figure 2A) and TT-220420 (Figure 2B) demonstrated anti-IBD activity, as evidenced by a significant decrease in the DAI from study day 4 to study day 6 in drug-treated mice. All data are expressed as mean ± SEM. 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001. [Figure 3] Figures 3A, 3B, 3C, and 3D show IC50 values ​​for selected bile acid GCPII inhibitor conjugates. IC50 values ​​were determined for selected inhibitors using a previously described radioactive substrate-based assay with recombinant human GCPII (Rojas et al., 2002). All were found to inhibit GCPII at concentrations ranging from low nanomolar to low micromolar. Furthermore, (S)-LTP592 (Figure 3A) was found to be the active enantiomer of (R / S)-LTP592, exhibiting 150-fold greater potency than (R)-LTP592 (Figure 3B). [Figure 4]Orally administered (S)-LTP592 dose-dependently protects against dextran sulfate sodium (DSS)-induced colitis (Figures 4A, 4B, 4C, 4D, and 4F). Six-week-old male C57Bl / 6NHsd mice were challenged with 4.0% DSS in drinking water from study days 0 to 5, followed by exposure to fresh water to induce severe acute colitis. Oral (S)-LTP592 was administered once daily starting on study day 0 at doses of 1 mg / kg, 10 mg / kg, and 100 mg / kg 2-PMPA molar equivalents. (Fig. 4A) 100 mg / kg-equivalent (S)-LTP592 significantly reduced colitis severity as early as study day 3, with a dose-dependent protective trend emerging by study day 6 (n = 15 / group; ***p < 0.001, *p < 0.05 2-way ANOVA). (Fig. 4B) Correspondingly, colonic tissues collected 4 h post-dose on study day 6 showed greater than 75% inhibition of GCPII enzyme activity at effective doses of 10 mg / kg-equivalent and 100 mg / kg-equivalent (S)-LTP592, whereas GCPII was only inhibited by 40% at the ineffective dose of 1 mg / kg-equivalent (n = 8 / group; **p < 0.01 2-tailed t-test). (Fig. 4C) Surprisingly, treatment with 100 mg / kg equivalent (S)-JHU3540 also normalized colon length on study day 6 (n = 15 / group, ***p < 0.001, two-tailed t-test). (Fig. 4D, 4E, and 4F) The efficacy of 100 mg / kg equivalent (S)-LTP592 in the DSS-colitis model was driven by improvements in fecal scores (Fig. 4C) and bleeding scores (Fig. 4D) (n = 15 / group; ***p < 0.001, *p < 0.05, two-way ANOVA). [Figure 5]Orally administered (S)-LTP592 is anti-inflammatory in the DSS-colitis model (Figures 5A, 5B, 5C, 5D, 5E, 5F, and 5G). Six-week-old male C57Bl / 6NHsd mice were challenged with 4.0% DSS in drinking water from test days 0 to 6, followed by exposure to fresh water. Oral (S)-LTP592 was administered once daily starting on test day 0 at a dose of 100 mg / kg 2-PMPA molar equivalent. (Figure 5A, Figure 5B, and Figure 5C) A 100 mg / kg equivalent dose of (S)-LTP592 significantly improved colon histology on study day 6, reducing the number and depth of ulcers and the extent of inflammatory infiltrates (n = 10 / group; ***p < 0.001, two-tailed t-test), as shown in the blinded pathologist's score (Figure 5A) and representative images (Figure 5B-Figure 5C). (Figure 5D, Figure 5E, Figure 5F, and Figure 5G) A subset of mice (n = 8 / group) was euthanized on study day 5, prior to fulminant colitis. Their colons were harvested and processed for flow cytometry. As expected, 4.0% DSS caused marked colonic inflammation, including increases in all myeloid populations examined, including total CD11b+ leukocytes (Figure 5D) and neutrophils (Figure 5E), circulating monocytes (Figure 5F), and migrating monocytes (Figure 5G). All DSS-induced changes in myeloid inflammatory cell populations were attenuated by (S)-IBD3540 treatment (n=8-9 / group, **p<0.01, *p<0.05, two-tailed t-test). [Figure 6] Figure 1 shows the in vivo pharmacokinetics of LTP-592 in dogs.The concentration-time profiles and pharmacokinetic parameters of LTP-592 following intravenous (IV) and oral administration of LTP-592 in dogs are shown. [Figure 7] Figures 7A, 7B, and 7C show the in vivo pharmacokinetics of LTP-592 in mice. The concentration-time profiles of LTP-592 after intravenous (IV) and oral administration (Figure 7A), plasma (Figure 7B), and colon (Figure 7C) pharmacokinetic parameters are shown. Detailed Description of the Invention

[0017] The presently disclosed subject matter is described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter described herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it should be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0018] I. Bile Acid-GCPII Inhibitor Conjugates for Treating Inflammatory Diseases, Including Inflammatory Bowel Disease (IBD) Immune-mediated gastrointestinal disorders encompass a wide range of debilitating gastrointestinal diseases of various etiologies. Inflammatory bowel disease (IBD), one such immune-mediated gastrointestinal disorder, is a collective term used to describe two gastrointestinal disorders of unknown etiology: Crohn's disease (CD) and ulcerative colitis (UC). IBD, which occurs worldwide and reportedly affects as many as 2 million people, has a variable course and prognosis. IBD typically develops in young adulthood, with the most common symptoms being diarrhea, abdominal pain, and fever. Diarrhea ranges from mild to severe, and in UC, bleeding is often present. Anemia and weight loss are additional common signs of IBD. 10%–15% of all IBD patients require surgery within 10 years. Furthermore, IBD patients are at increased risk for developing intestinal cancer. An increased incidence of psychological issues, such as anxiety and depression, may not be surprising for this often debilitating disease that strikes people in the prime of life.

[0019] A. GCPII inhibitors containing 2-(phosphonomethyl)pentanedioic acid (2-PMPA) conjugated to bile acids In some embodiments, the subject matter of the present disclosure provides a GCPII inhibitor with anti-IBD efficacy.The GCPII inhibitor of the present disclosure is formed by conjugating 2-(phosphonomethyl)pentanedioic acid (2-PMPA), a potent GCPII inhibitor, with bile acid.The chemical formula of 2-PMPA is shown immediately below: [ka]

[0020] Endogenously abundant bile acids were selected as conjugates because they have also been reported to have direct immunomodulatory effects in various inflammatory models, such as protection in IBD models (Sipka and Bruckner, 2014; Calmus and Poupon, 2014; Ho and Steinman, 2016). Laukens et al., 2014. The presently disclosed subject matter further provides the use of such novel GCPII inhibitor / bile acid conjugates in the treatment of inflammatory bowel disease and other inflammatory diseases.

[0021] Bile acids have the following general chemical structure: [ka] During the ceremony: R'1 and R'2 are each independently H or -OH; R'3 is -OH; R'4 is selected from the group consisting of -OH, -NHCH2COOH, and -NHCH2CH2SO3H; and salts thereof.

[0022] Representative bile acids include, but are not limited to, cholic acid, glycocholic acid, deoxycholic acid, lithocholic acid, glycodeoxycholic acid, chenodeoxycholic acid (also called chenocholic acid), glycochenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, and derivatives thereof, the structures of which are shown in Table 1 immediately below.

[0023] [Table 1-1] [Table 1-2] [Table 1-3]

[0024] Thus, in some embodiments, the presently disclosed subject matter provides conjugates of 2-(phosphonomethyl)pentanedioic acid (2-PMPA) or a derivative thereof with a bile acid or a derivative thereof.

[0025] In some embodiments, the conjugate comprises a compound of formula (I): [ka] (In the formula: R1 and R2 are each independently H or -OH; and R3 is OH and R4 is selected from the group consisting of -NH-X1, -COO-X1, -C(=O)-NH-CH2-C(=O)-O-X1, and -C(=O)-NH-CH2-CH2-S(=O)2-O-X1, where X1 is -(C=O)-(CH2) m -P(=O)(OH)-X2, -(C=O)-(CH2) m -CH(COOH)-CH2-P(=O)(OH)-X2, -CH2-O-(C=O)-(CH2) m -P(=O)(OH)-X2, -CH2-OC(=O)-(CH2) m-CH(COOH)-CH2-P(=O)(OH)-X2, -CH2-OC(=O)-(CH2) m -CH(COOH)-NH-(C=O)-NH-CH(COOH)-CH2-CH(CH3)2, -CH2-OC(=O)-Ar-CH2-CH(COOH)-(CH2) m -C(=O)-NH-OH, -CH2-OC(=O)-(CH2) m -X3, -CH2-OC(=O)-Ar-CH2-X3, and a protecting group, where X2 is -OH, -CH2-CH(COOH)-(CH2) p -C(=O)-OH, and a protecting group, Ar is arylene, X3 is 2-oxotetrahydro-2H-thiopyran-3-yl, and each m and p is independently selected from the group consisting of 1, 2, 3, and 4; or R3 is -OC(=O)-O-CH2-OC(=O)-(CH2) n -CH(COOH)-CH2-P(=O)(OH)2, and -OC(=O)-CH2-CH2-P(=O)(OH)-CH2-CH(COOH)-(CH2) n -C(=O)-OH, where each n is independently an integer selected from the group consisting of 1, 2, 3, and 4, and R4 is selected from the group consisting of -NH2, -COOH, -C(=O)-NH-CH2-C(=O)-OH, and -C(=O)-NH-CH2-CH2-S(=O)2-OH. and pharmaceutically acceptable salts thereof.

[0026] In some embodiments, R1 and R2 are both H. In some embodiments, R1 is H and R2 is OH. In some embodiments, R1 is OH and R2 is H. In some embodiments, R1 and R2 are both OH.

[0027] In some embodiments, R3 is OH; and R4 is selected from the group consisting of -NH-X1, -COO-X1, -C(=O)-NH-CH2-C(=O)-O-X1, and -C(=O)-NH-CH2-CH2-S(=O)2-O-X1, where X1 is -(C=O)-CH2-CH2-P(=O)(OH)-X2, -(C=O)-CH2-CH2-CH(COOH)-CH2-P(=O)(OH)-X2, -CH2-O-(C=O)-CH2-CH2-P(=O)(OH)-X2, -CH2-OC(=O) is selected from the group consisting of -CH2-CH2-CH(COOH)-CH2-P(=O)(OH)-X2, -CH2-OC(=O)-CH2-CH2-CH(COOH)-NH-(C=O)-NH-CH(COOH)-CH2-CH(CH3), -CH2-OC(=O)-Ar-CH2-CH(COOH)-CH2CH2-C(=O)-NH-OH, -CH2-OC(=O)-CH2-CH2-X3, -CH2-OC(=O)-Ar-CH2-X3, and a protecting group; wherein X2 is selected from the group consisting of -OH, -CH2-CH(COOH)-CH2-CH2-CH(=O)-OH, and a protecting group, Ar is phenyl, and X3 is 2-oxotetrahydro-2H-thiopyran-3-yl.

[0028] In some embodiments, R1 is OH, R2 is H, R3 is OH, and R4 is COO-X1, where X1 is -CH2-OC(=O)-(CH2) m -CH(COOH)-CH2-P(=O)(OH)-X2.

[0029] In some embodiments, R1 is H and R2 is OH, or R1 is OH, R2 is OH, R3 is OH, and R4 is -NH-X1, where X1 is -(C=O)-CH2-CH2-CH(COOH)-CH2-P(=O)(OH)-X2.

[0030] In some embodiments, the compound of formula (I) is: [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0031] In some embodiments, R3 is selected from the group consisting of -OC(=O)-O-CH2-OC(=O)-CH2-CH2-CH(COOH)-CH2-P(=O)(OH)2, and -OC(=O)-CH2-CH2-P(=O)(OH)-CH2-CH(COOH)-CH2-CH2-C(=O)-OH; and R4 is selected from the group consisting of -NH2, -COOH, -C(=O)-NH-CH2-C(=O)-OH, and -C(=O)-NH-CH2-CH2-S(=O)2-OH.

[0032] In some such embodiments, the compound of Formula (I) is: [ka] is selected from the group consisting of:

[0033] Representative compounds of formula (I) are provided in Table 2 immediately below.

[0034] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0035] B. Methods of Treating Diseases or Conditions Associated with Elevated GCPII Activity

[0036] In some embodiments, the presently disclosed subject matter provides methods for treating a disease or condition associated with elevated GCPII activity. In certain embodiments, the disease or condition is inflammatory bowel disease (IBD).

[0037] Glutamate carboxypeptidase II (GCPII) is a metallopeptidase that catalyzes the hydrolysis of N-acetylaspartic acid-glutamic acid (NAAG) into N-acetylaspartic acid (NAA) and glutamic acid, and sequentially cleaves the terminal glutamic acid moiety from folate polyglutamic acid (Ristau et al., 2013; Mesters et al., 2006; Slusher et al., 2013). In certain embodiments, the compound of formula (I) of the present disclosure is a GCPII inhibitor. As used herein, a GCPII inhibitor is a molecule that reduces or inhibits the activity of GCPII. Modulation of GCPII activity can be detected by assaying the endogenous N-acetylated alpha-linked acidic dipeptidase (AALADase) activity of GCPII (Tang et al., 2003; Robinson et al., 1987; Lupoid et al., 2002; US Patent Application Publication No. 20110064657). Inhibition curves can be determined using semi-logarithmic plots, and IC 50 The value is determined at the concentration at which enzyme activity is inhibited by 50%.

[0038] GCPII inhibitors can interact directly with GCPII (e.g., through interaction with the GCPII binding site) or with another molecule, resulting in decreased activity of GCPII. The GCPII binding site contains a binuclear zinc ion and two substrate-binding pockets: the S1 (non-pharmacophore) pocket and the S1' (pharmacophore) pocket. The active site also contains a chloride ion in the S1 pocket. Near the S1 pocket, there is a funnel-shaped tunnel approximately 20 Å deep and 8–9 Å wide. Similarly, there is a narrow cavity near the S1' pocket.

[0039] GCPII activity is significantly increased in the diseased intestinal mucosa of the subject with IBD.As used herein, the term " increased GCPII activity (increased GCPII activity) " refers to the increase in GCPII activity in the subject with IBD compared with the GCPII activity in the subject without IBD, for example, about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increase.

[0040] In some embodiments, the presently disclosed subject matter provides a method for inhibiting GCPII activity. In some embodiments, the presently disclosed subject matter provides a method for inhibiting GCPII activity in a subject suffering from IBD. As used herein, the term "inhibit" refers to reducing or attenuating GCPII activity in a subject in need thereof. The term "inhibit" can also refer to reducing, suppressing, attenuating, attenuating, stopping, or stabilizing the onset or progression of a disease or condition, such as IBD. Inhibition can occur, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100%, compared to an untreated control subject or a subject without a disease or disorder.

[0041] Generally, using the methods of the present disclosure to treat IBD in a subject results in a reduction in the severity of IBD. As used herein, the terms "treat," "treating," "treatment," and the like refer to the alleviation or improvement of a disease or condition, such as IBD, and / or its associated symptoms. It should be understood that treating a disease or condition, such as IBD, does not require the complete elimination of the associated disorder, condition, or symptom, although this is not excluded. The term "reduce" refers to inhibiting, suppressing, attenuating, decreasing, stopping, or stabilizing at least one symptom of IBD (e.g., rectal prolapse, enteritis, colonic enlargement, stool inconsistency, etc.).

[0042] IBD is broadly divided into Crohn's disease and ulcerative colitis. Therefore, as used herein, the term "subject with inflammatory bowel disease" is synonymous with the term "subject diagnosed with inflammatory bowel disease" and refers to a patient with Crohn's disease or ulcerative colitis. Crohn's disease (regional enteritis) is a chronic inflammatory disease that can affect any part of the gastrointestinal tract. It generally affects the distal part of the small intestine (ileum) and the cecum. In other cases, the disease is limited to the small intestine, colon, or anorectal region. Crohn's disease sometimes affects the duodenum and stomach, and rarely the esophagus and oral cavity.

[0043] The varied clinical manifestations of Crohn's disease are, in part, a result of the disease's various anatomical locations. The most frequent symptoms of CD are abdominal pain, diarrhea, and recurrent fever. CD is commonly associated with intestinal obstruction or fistulas. Fistulas are abnormal passages between diseased intestinal loops, for example. Crohn's disease also includes complications such as inflammation of the eyes, joints, and skin; liver disease; kidney stones; or amyloidosis. Furthermore, CD is associated with an increased risk of intestinal cancer.

[0044] Several features are characteristic of the pathology of Crohn's disease. The inflammation associated with CD, known as transmural inflammation, affects all layers of the bowel wall. For example, thickening and edema typically appear throughout the entire bowel wall, and with long-standing disease, fibrosis is also seen. The inflammation characteristic of CD is also discontinuous, in that segments of inflamed tissue, known as "skip lesions," are separated by apparently normal bowel.

[0045] Additionally, linear ulcerations, edema, and inflammation of the intervening tissues contribute to the "cobblestone" appearance of the intestinal mucosa, characteristic of CD. A hallmark of Crohn's disease is the presence of discrete aggregates of inflammatory cells known as granulomas, typically found in the submucosa. While some cases of Crohn's disease exhibit typical isolated granulomas, others exhibit nonspecific transmural inflammation. Consequently, although the presence of isolated granulomas is suggestive of CD, the absence of granulomas is also consistent with the disease. Therefore, transmural or discrete inflammation, rather than the presence of granulomas, is a preferred diagnostic indicator of Crohn's disease (Rubin and Farber, 1994).

[0046] Ulcerative colitis (UC) is a disease of the large intestine characterized by cramping abdominal pain, rectal bleeding, and chronic diarrhea accompanied by loose discharge of blood, pus, and mucus. The symptoms of UC are diverse. While a pattern of exacerbations and remissions typifies the clinical course in most UC patients (70%), some UC patients experience continuous symptoms without remission. Local and systemic complications of UC include arthritis, eye inflammation such as uveitis, skin ulcers, and liver disease. Furthermore, UC, especially long-standing and extensive disease, is associated with an increased risk of colon cancer.

[0047] Several pathological features distinguish UC from other inflammatory bowel diseases. UC is typically a diffuse disease that extends over variable distances from the most distal to the proximal rectum. The term left-sided colitis describes inflammation involving the distal portion of the colon and extending to the splenic flexure. Sparing of the rectum or involvement of only the right (proximal) portion of the colon is rare in UC. The inflammatory process in UC is limited to the colon and does not involve, for example, the small intestine, stomach, or esophagus. Furthermore, UC is generally distinguished by superficial inflammation of the mucosa that protects the deeper layers of the intestinal wall. Crypt abscesses, in which degenerated intestinal crypts are filled with neutrophils, are also typical of UC (Rubin and Farber, 1994).

[0048] While Crohn's disease is a patchy disease that frequently spares the rectum, ulcerative colitis is characterized by a continuous inflammation of the colon, usually more severe distally than proximally. Ulcerative colitis inflammation is superficial, in that it is usually limited to the mucosal layer and is characterized by acute inflammatory infiltrates with neutrophils and crypt abscesses. In contrast, Crohn's disease involves the entire thickness of the bowel wall, and granulomas are often, but not always, present. Disease terminating at or in the colon distal to the ileocecal valve suggests ulcerative colitis, whereas involvement of the terminal ileum, a cobblestone appearance, and discrete ulcers or fistulas suggest Crohn's disease.

[0049] The "subject" treated by the disclosed method in many embodiments thereof is preferably a human subject, although it will be understood that the methods described herein are effective with respect to all vertebrate species intended to be encompassed by the term "subject." Thus, a "subject" can include a human subject for medical purposes, such as treatment of an existing condition or disease or prophylactic treatment to prevent the onset of a condition or disease, or an animal subject for medical, veterinary, or developmental purposes. Suitable animal subjects include, but are not limited to, primates (e.g., humans, monkeys, apes, etc.); bovines (e.g., cattle, bulls, etc.); ovines (e.g., sheep, etc.); caprines (e.g., goats, etc.); porcines (e.g., pigs, boars, etc.); equines (e.g., horses, donkeys, zebras, etc.); felines (e.g., wild cats, domestic cats, etc.); canines (e.g., roosters, etc.); lagomorphs (e.g., rabbits, hares, etc.); and rodents (e.g., mice, rats, etc.). The animal may be a transgenic animal. In some embodiments, the subject is a human, including, but not limited to, a fetus, a newborn, an infant, a juvenile, and an adult subject. Furthermore, a "subject" can include a patient suffering from or suspected of suffering from a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein. The term "subject" also refers to an organism, tissue, cell, or collection of cells from a subject.

[0050] Generally, the "effective amount" of an active agent or drug delivery device refers to the amount necessary to induce a desired biological response. As will be understood by those skilled in the art, the effective amount of an agent or device can vary depending on factors such as the desired biological endpoint, the agent being delivered, the composition of the pharmaceutical composition, the target tissue, etc.

[0051] In some embodiments, the presently disclosed subject matter provides pharmaceutical compositions comprising at least one compound of general formula (I), alone or in combination with one or more additional therapeutic agents, in admixture with a pharmaceutically acceptable excipient.

[0052] The one or more additional therapeutic agents may include other agents useful in treating inflammatory bowel disease, including, but not limited to, anti-inflammatory drugs, including, but not limited to, corticosteroids and aminosalicylates, such as mesalamine (Asacol HD, Delzicol, etc.), balsalazide (Colazar), olsalazine (Dipentam), etc.; immune system suppressants, including, but not limited to, azathioprine (Azasan, Imuran), mercaptopurine (Prinetol, Prixan), cyclosporine (Gengraf, Neoral, Sandimmune), and methotrexate (Trexall); infliximab (Remicade), adalimumab (Humira), golimumab ( These include tumor necrosis factor (TNF)-alpha inhibitors, including but not limited to Simponi, or other biologics, including but not limited to natalizumab (Tysabri), vedolizumab (Entyvio), and ustekinumab (Stelara); antibiotics, including but not limited to ciprofloxacin (Cipro) and metronidazole (Flagyl); anti-diarrheal medications, such as loperamide (Imodium AD); fiber supplements, such as psyllium powder (Metamucil) or methylcellulose (Citrucel); pain relievers, such as acetaminophen; and supplements, including but not limited to iron supplements, calcium supplements, and vitamin D supplements.

[0053] The term "combination" is used in its broadest sense and means that a subject is administered at least two agents, more specifically, a compound of Formula (I) and at least one beta-lactam antibiotic, and optionally one or more antibacterial agents. More specifically, the term "in combination" refers to the simultaneous administration of two (or more) active agents, for example, for the treatment of a single disease state. As used herein, the active agents may be administered in a single combined dosage form, simultaneously in separate dosage forms, or in separate dosage forms that are administered alternately or sequentially on the same or different days. In one embodiment of the presently disclosed subject matter, the active agents are administered in a single combined dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., where it is desirable to vary the amount of one but not the other). The single dosage form may contain additional active agents for the treatment of a disease state.

[0054] Additionally, the compounds of Formula (I) described herein can be administered alone or in combination with one or more antibacterial agents, alone or in combination with adjuvants that enhance the stability of the compounds of Formula (I), in certain embodiments facilitate administration of pharmaceutical compositions containing them, provide increased dissolution or dispersion, enhance inhibitory activity, provide adjunctive therapy with other active ingredients, etc. Advantageously, such combination therapies utilize lower doses of conventional therapeutic agents, thereby avoiding toxic and adverse side effects that can occur when these agents are used as monotherapies.

[0055] The timing of administering the compound of formula (I) and at least one additional therapeutic agent can be varied, as long as the beneficial effect of the combination of these agents is achieved.Therefore, the term "in combination" refers to administering the compound of formula (I) and at least one additional therapeutic agent simultaneously, sequentially, or any combination thereof.Therefore, the subject who is administered the combination of the compound of formula (I) and at least one additional therapeutic agent can receive the compound of formula (I) and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially (in any order), on the same day or on different days), as long as the combined effect of both agents is achieved in the subject.

[0056] When administered sequentially, the agents can be administered within 1 minute, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, or more of each other. In other embodiments, the sequentially administered agents can be administered within 1 day, 5 days, 10 days, 15 days, 20 days, or more of each other. When the compound of Formula (I) and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each containing either the compound of Formula (I) or the at least one additional therapeutic agent, or they can be administered to the subject as a single pharmaceutical composition containing both agents.

[0057] When administered in combination, the effective concentration of each agent to elicit a particular biological response may be lower than the effective concentration of each agent when administered alone, thereby allowing for a reduction in the dosage of one or more agents relative to the dosage required when administered as a single agent. The effects of multiple agents may be, but need not be, additive or synergistic. Agents may be administered multiple times.

[0058] In some embodiments, two or more agents may have a synergistic effect when administered in combination. As used herein, the terms "synergistic," "synergistic," "synergistically," and derivatives thereof, such as in "synergistic effect" or "synergistic combination" or "synergistic composition," refer to a situation in which the biological activity of the combination of a compound of Formula (I) and at least one additional therapeutic agent is greater than the sum of the biological activities of each agent when administered individually.

[0059] Synergy can be expressed as a "synergy index (SI)," which can generally be determined by the method described by F.C. Kull et al., Applied Microbiology 9, 538 (1961), from a ratio determined by the following formula: Q a / Q A +Q b / Q B = Synergy Index (SI) During the ceremony: Q A is the concentration of component A acting alone that produced the endpoint associated with component A; Q a is the concentration of component A in the mixture that produced the endpoint; Q B is the concentration of component B acting alone that produced the endpoint associated with component B; and Q b is the concentration of component B in the mixture that produced the endpoint.

[0060] In general, Q a / Q A and Q b / Q BIf the sum is greater than 1, antagonism is indicated. If the sum is equal to 1, additivity is indicated. If the sum is less than 1, synergy is indicated. The lower the SI, the greater the synergistic effect exhibited by that particular mixture. Thus, a "synergistic combination" has greater activity than would be expected based on the activity observed when the individual components are used alone. Furthermore, a "synergistically effective amount" of a component refers to the amount of that component needed to elicit a synergistic effect, for example, with another therapeutic agent present in the composition.

[0061] C. Pharmaceutical Compositions and Administration In another aspect, the present disclosure provides pharmaceutical compositions comprising a compound of general formula (I), alone or in combination with one or more additional therapeutic agents, in admixture with a pharmaceutically acceptable excipient. Those skilled in the art will recognize that pharmaceutical compositions include pharmaceutically acceptable salts of the above compounds. Pharmaceutically acceptable salts are generally well known to those skilled in the art and include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. When a compound of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent, or by ion exchange, thereby replacing one basic counterion (base) in the ionic complex with another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts.

[0062] When a compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent, or by ion exchange, thereby replacing one acidic counterion (acid) in the ionic complex with another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids, such as arginine salts, and salts of organic acids, such as glucuronic acid and galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 1977, 66, pp. 1-19). Certain compounds of the present disclosure contain both basic and acidic functional groups that allow the compounds to be converted into either base or acid addition salts.

[0063] Thus, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydrazide, hydroxybenzoate ... Examples of suitable pharmaceutically acceptable salts include hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts can be found, for example, in Remington: The Science and Practice of Pharmacy (20th Edition), Lippincott, Williams & Wilkins (2000).

[0064] For therapeutic and / or diagnostic applications, the compounds of the present disclosure can be formulated for a variety of modes of administration, including systemic administration and topical or localized administration. Techniques and formulations can generally be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0065] Depending on the specific condition to be treated, such agents can be formulated into liquid or solid dosage forms and administered systemically or locally. Agents can be delivered, for example, in time-release or sustained-release forms, as known to those skilled in the art. Formulation and administration techniques can be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes include oral, buccal, inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; intramuscular, subcutaneous, intramedullary injection, and parenteral administration, such as intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal or intraocular injection, or other modes of administration.

[0066] For injection, the agent of the present disclosure can be formulated and diluted in a physiologically compatible buffer solution, such as aqueous solution, for example, Hank's solution, Ringer's solution, or physiological saline buffer.For such transmucosal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art.

[0067] The use of pharmaceutically acceptable inert carriers to formulate the compounds disclosed herein into dosages suitable for systemic administration for the implementation of the present disclosure is within the scope of the present disclosure. By appropriate selection of the carrier and appropriate manufacturing method, the compositions of the present disclosure, particularly those formulated as solutions, can be administered parenterally, such as by intravenous injection. These compounds can be easily formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the subject (e.g., patient) to be treated.

[0068] For nasal or inhalation delivery, the agents of the present disclosure may also be formulated by methods known to those skilled in the art and may include, but are not limited to, solubilizing, diluent, or dispersing agents such as saline; preservatives such as benzyl alcohol; absorption enhancers; and fluorocarbons.

[0069] Pharmaceutical compositions suitable for use in the present disclosure include compositions containing the active ingredient in an effective amount to achieve its intended purpose. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. In general, compounds according to the present disclosure are effective over a wide dosage range. For example, in treating adult humans, dosages of 0.01-1000 mg, 0.5-100 mg, 1-50 mg / day, and 5-40 mg / day are exemplary dosages that may be used. A non-limiting dosage is 10-30 mg / day. The exact dosage will depend on the route of administration, the dosage form of the compound, the subject being treated, the subject's body weight, the bioavailability of the compound, the absorption, distribution, metabolism, and excretion (ADME) toxicity of the compound, and the preference and experience of the attending physician.

[0070] In addition to the active ingredient, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, including excipients and auxiliaries that facilitate the processing of the active compound into pharmaceutically usable preparations. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0071] Oral pharmaceutical preparations can be obtained by combining the active compound with a solid excipient, optionally grinding the resulting mixture, optionally adding suitable auxiliaries, and then processing the granular mixture to obtain tablets or dragee cores.Suitable excipients are, in particular, sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose (CMC), and / or fillers such as polyvinylpyrrolidone (PVP: povidone).If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, alginic acid or its salts (such as sodium alginate) can be added.

[0072] Sugar-coated tablet core is provided with suitable coating.For this purpose, can use concentrated sugar solution, lacquer solution and suitable organic solvent or solvent mixture, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG) and / or titanium dioxide.For identification or to characterize different combinations of dosage of active compound, dyes or pigments can be added to tablet or sugar-coated tablet coating.

[0073] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol (PEG). Additionally, stabilizers may be added.

[0074] D. Definition

[0075] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

[0076] While the following terms relating to compounds of formula (I) are believed to be well understood by those of skill in the art, the following definitions are provided to facilitate discussion of the presently disclosed subject matter. These definitions are intended to supplement and explain, but not preclude, definitions that would be apparent to one of skill in the art upon review of this disclosure.

[0077] As used herein, the terms substituted (whether preceded by the term "optionally" or not) and substituent refer to the ability to change one functional group on a molecule to another, provided that the valences of all atoms are maintained, as understood by one of ordinary skill in the art. When more than one position in any given structure can be substituted with more than one substituent selected from a particular group, the substituents can be the same or different at all positions. Substituents can also be further substituted (e.g., an aryl group substituent can have another substituent outside it, e.g., another aryl group that is further substituted at one or more positions).

[0078] Where substituents or linking groups are designated by conventional chemical formulas written from left to right, they equally encompass the chemically identical substituents that result from writing the structure from right to left, e.g., -CHO- is equivalent to -OCH-, -C(=O)O- is equivalent to -OC(=O)-, and -OC(=O)NR- is equivalent to -NRC(=O)O-.

[0079] When the term "independently selected" is used, the referenced substituents (e.g., R groups such as groups R1, R2, or variables such as "m", "n", etc.) can be the same or different. For example, R1 and R2 can both be substituted alkyl, or R1 can be hydrogen and R2 can be substituted alkyl.

[0080] As used herein with respect to a group of substituents, the terms "a," "an," or "a(n)" mean at least one. For example, if a compound is substituted with "an" alkyl or aryl, then the compound is optionally substituted with at least one alkyl and / or at least one aryl. Furthermore, if a moiety is substituted with an R substituent, the group may be referred to as "R-substituted." If a moiety is R-substituted, then the moiety is substituted with at least one R substituent, and each R substituent is optionally different.

[0081] A named "R" or group generally has the structure recognized in the art as corresponding to the named group, unless otherwise specified herein. For purposes of illustration, certain representative "R" groups, as described above, are defined below.

[0082] The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art.Therefore, when a group can be substituted with one or more of a large number of substituents, such substitutions are selected to comply with the principles of chemical bonding and to give compounds that are not inherently unstable and / or that are known to those skilled in the art to be likely to be unstable under ambient conditions, such as aqueous, neutral, and some known physiological conditions.

[0083] symbol: [ka] indicates the point of attachment of a moiety to the rest of the molecule.

[0084] When a named atom of an aromatic ring or heteroaromatic ring is defined to be "absent," the named atom is replaced with a direct bond.

[0085] The term "hydroxyl" refers to an --OH group.

[0086] The term "aryl," unless otherwise specified, means an aromatic hydrocarbon substituent which may be a single ring or multiple rings (e.g., 1 to 3 rings) fused or covalently linked together. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, and the like. The term "arylene" refers to the divalent forms of aryl and heteroaryl, respectively.

[0087] Throughout this specification and the claims, a given chemical formula or name is intended to encompass all tautomers, homologs, optical isomers and stereoisomers, as well as racemic mixtures, where such isomers and mixtures exist.

[0088] Certain compounds of the present disclosure may have asymmetric carbon atoms (optical or chiral centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms that may be defined in terms of absolute stereochemistry as (R)- or (S)-, or for amino acids as D- or L-, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those known in the art to be too unstable to synthesize and / or isolate. The present disclosure is intended to include compounds in racemic, scalaminic, and optically pure form. Optically active (R)- and (S)-, or D- and L-isomers, may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques. When a compound described herein contains an olefinic bond or other center of geometric asymmetry, unless otherwise specified, the compound is intended to include both E and Z geometric isomers.

[0089] Unless otherwise stated, structures depicted herein are meant to include all stereochemical forms of the structure, i.e., both R and S configurations of each asymmetric center. Accordingly, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[0090] It will be apparent to one of ordinary skill in the art that certain compounds of the present disclosure may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the present disclosure. As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another.

[0091] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds in which a hydrogen is replaced by deuterium or tritium, or a carbon is replaced by an isotopically enriched atom. 13 C- or 14 Compounds having this structure substituted with C-enriched carbons are within the scope of this disclosure.

[0092] The term "protecting group" refers to a chemical moiety that blocks some or all reactive moieties in a compound, preventing them from participating in a chemical reaction until the protecting group is removed, such as those listed and described in T.W. Greene, P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons (1999). When different protecting groups are used, it may be advantageous for each (different) protecting group to be removable by a different means. Protecting groups that are cleaved under completely different reaction conditions allow for the operative removal of such protecting groups. For example, protecting groups can be removed by acid, base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal, and tert-butyldimethylsilyl are acid-labile and can be used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with the hydrogenolysis-removable Cbz group and the base-labile Fmoc group. Carboxylic acid and hydroxy reactive moieties can be blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl, in the presence of acid labile groups such as tert-butyl carbamate or amines blocked with acid and base stable but hydrolytically removable carbamates.

[0093] Carboxylic acid and hydroxy reactive moieties can also be blocked with hydrolytically removable protecting groups such as benzyl groups, and amine groups capable of hydrogen bonding with acids can be blocked with base-labile groups such as Fmoc. Carboxylic acid reactive moieties can be blocked with oxidatively removable protecting groups such as 2,4-dimethoxybenzyl, and coexisting amino groups can be blocked with fluorine-labile silyl carbamates.

[0094] Allyl blocking groups are useful in the presence of acid- and base-protecting groups because the former are stable and can be subsequently removed by metal or π-acid catalysts. For example, allyl-blocked carboxylic acids can be deprotected by palladium(O)-catalyzed reactions in the presence of acid-labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate can be attached. As long as the residue is attached to the resin, its functional group is blocked and cannot react. Once released from the resin, the functional group becomes reactive.

[0095] Typical blocking / protecting groups include, but are not limited to, the following moieties: [ka]

[0096] Following long-standing patent law practice, when used in this application, including the claims, the terms "a," "an," and "the" refer to "one or more." Thus, for example, a reference to "a subject" includes a plurality of subjects unless the context clearly indicates the contrary (e.g., a plurality of subjects).

[0097] Throughout this specification and the claims, the terms "comprise," "comprises," and "comprising" are used in a non-exclusive sense unless the context requires otherwise. Similarly, the term "include" and its grammatical variations are intended to be open-ended, such that the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0098] For purposes of this specification and the appended claims, unless otherwise indicated, all numerical values ​​expressing quantities, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, quantities, properties, and other numerical values ​​used in the specification and claims are to be understood as being modified in all instances by the term "about," even if the term "about" does not explicitly appear in conjunction with that value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims need not be exact but may be approximate and / or increased or decreased as desired, reflecting tolerances, conversion factors, rounding off, measurement error, and the like, as well as other factors known to those of ordinary skill in the art, depending upon the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term "about" when referring to a value can mean encompassing variations in some embodiments of ±100%, in some embodiments of ±50%, in some embodiments of ±20%, in some embodiments of ±10%, in some embodiments of ±5%, in some embodiments of ±1%, in some embodiments of ±0.5%, and in some embodiments of ±0.1% from the stated amount, as such variations are appropriate for practicing the disclosed methods or employing the disclosed compositions.

[0099] Furthermore, when the term "about," when used in connection with one or more numerical values ​​or numerical ranges, should be understood to refer to all such numerical values, inclusive of every numerical value within that range, modifying that range by extending the boundaries above and below the numerical values ​​set forth. The recitation of numerical ranges by endpoints also includes all numbers subsumed within that range, for example, whole integers, including fractions thereof (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. [Example]

[0100] The following examples are included to provide guidance for those skilled in the art to practice representative embodiments of the presently disclosed subject matter. In light of this disclosure and the general state of the art, those skilled in the art will recognize that the following examples are intended to be illustrative only, and that numerous changes, modifications, and variations can be adopted without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples provided below are for illustrative purposes only and are not to be construed as limiting the scope of making the compounds of the present disclosure by other methods.

[0101] Example 1: Bile acid-GCPII inhibitor conjugate <1.1. Experimental Section> [ka]

[0102] Allyl-3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propanoate (LTP487) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with published data (WO2016 / 22827 A1, 2016).

[0103] 5-Allyl-1-benzyl-2-methylenepentanedioate (LTP491) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with published data (Mol. Pharm. 2017, 14, 3248).

[0104] 5-Allyl-1-benzyl-2-((diethoxyphosphoryl)methyl)pentanedioate (LTP544) [ka] The starting material LTP491 (2.54 g, 9.26 mmol, 1 equiv.) was dissolved in anhydrous DMF (40 mL). A solution of freshly ground K2CO3 (1.92 g, 13.9 mmol, 1.5 equiv.) and dibenzyl phosphite (2.55 g, 9.72 mmol, 1.05 equiv.) in anhydrous DMF (5 mL) was added, and the resulting mixture was heated to 90 °C under inert conditions for 2 h. DMF was evaporated, EtOAc (100 mL) was added, and the organic phase was washed with distilled HO (2 × 50 mL) and brine (50 mL), dried over MgSO4, and the solvent was evaporated. The crude product was purified by LC (EtOAc / cyclohexane, 1:1; Rf = 0.33; 1% KMnO4 detection) and compound LTP544 was isolated as a colorless oil in 86% yield (4.30 g). 1 H NMR (400 MHz, CDCl): δ H 1.80-2.01 (m, 3H), 2.20-2.40 (m, 3H), 2.84 (dddd, J= 19.7, 8.5, 6.9, 5.0 Hz, 1H), 4.52 (dt, J = 5.7, 1.4 Hz, 2H), 4.88-5.04 (m, 6H), 5.21 (dq, J = 10.4, 1.3 Hz, 1H), 5.27 (dq, J = 17.2, 1.5 Hz, 1H), 5.86 (ddt, J = 17.2, 10.4, 5.7 Hz, 1H), 7.23-7.38 (m, 15H). 13 C NMR (101 MHz, CDCl): δ C 28.25 (d, J C,P = 142.2 Hz), 28.46 (d, J C,P = 13.3 Hz), 31.30, 39.25 (d, J C,P = 3.7 Hz), 65.24, 66.79, 67.40 (d, J C,P = 6.4 Hz), 67.42 (d, J C,P= 6.3 Hz), 118.34, 128.09-128.66 (15C), 132.11, 135.60, 136.18 (d, J C,P = 1.8 Hz), 136.24 (d, J C,P = 1.4 Hz), 172.03, 173.66 (d, J C,P = 7.9 Hz). 31 P NMR (162 MHz, CDCl): δ P 31.94. ESI MS: 559.2 ([M + Na] + ). HR ESI MS: C 30 H 33 Calculated for O7PNa: 559.18561; observed: 559.18558.

[0105] 5-(benzyloxy)-4-((bis(benzyloxy)phosphoryl)methyl)-5-oxopentanoic acid (LTP560) [ka] The starting material LTP544 (3.60 g, 6.71 mmol, 1 equiv.) was dissolved in anhydrous THF (40 mL). Phenylsilane (1.45 g, 1.65 mL, 13.4 mmol, 2 equiv.) was added, followed by Pd(PPh3)4 (155 mg, 0.134 mmol, 2 mol%), and the reaction mixture was stirred under inert conditions at room temperature for 19 h. The THF was evaporated, and the residue was purified by LC (CHCl3 / MeOH, 20:1). Compound LTP560 was isolated as a light brown oil (2.85 g) in 86% yield. 1 H NMR (400 MHz, CDCl): δ H 1.82-1.99 (m, 3H), 2.20-2.37 (m, 3H), 2.84 (dp, J = 13.5, 6.8 Hz, 1H), 4.87-5.03 (m, 6H), 7.23-7.36 (m, 15H). 13 C NMR (101 MHz, CDCl): δC 28.12 (d, J C,P = 142.4 Hz), 28.25 (d, J C,P = 13.0 Hz), 31.13, 39.16 (d, J C,P = 3.6 Hz), 66.89, 67.68 (d, J C,P = 2.0 Hz), 67.74 (d, J C,P = 1.8 Hz), 128.20-128.73 (15C), 135.61, 136.07 (d, J C,P = 1.6 Hz), 136.13 (d, J C,P = 1.2 Hz), 173.69 (d, J C,P = 8.5 Hz), 176.61. 31 P NMR (162 MHz, CDCl): δ P 32.21. ESI MS: 519.2 ([M + Na] + ). HR ESI MS: C 27 H 29 Calculated for O7PNa: 519.15424; observed: 519.15431.

[0106] 1-Benzyl 5-(chloromethyl) 2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP576) [ka] Compound LTP560 (1.96 g, 3.95 mmol, 1 equiv.) was dissolved in DCM (30 mL; pa) and diluted with distilled water (30 mL) followed by NaHCO3 (1.26 g, 15.0 mmol, 2.8 equiv.) and BuN + HSO4 -(134 mg, 0.395 mmol, 0.1 equiv) was added. The reaction mixture was vigorously stirred at room temperature for 5 min, and finally, chloromethyl chlorosulfate (782 mg, 479 μL, 4.74 mmol, 1.2 equiv) in DCM (5 mL) was added over 1 min. The resulting mixture was stirred at room temperature for 20 h. Further DCM (20 mL) was added and the phases were separated. The aqueous phase was extracted with DCM (50 mL), and the combined organic phases were washed with saturated NaCl (50 mL), dried over MgSO4, and the DCM was evaporated. The crude product was purified by LC (EtOAc / cyclohexane, 1:1) to give the desired product (1.83 g, 85% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl): δ H 1.77-2.02 (m, 3H), 2.21-2.38 (m, 3H), 2.82 (dtt, J = 13.8, 8.2, 5.6 Hz, 1H), 4.88-5.04 (m, 6H), 5.61 (d, J = 0.8 Hz, 2H), 7.22-7.38 (m, 15H). 13 C NMR (101 MHz, CDCl): δ C 27.87 (d, J C,P = 12.5 Hz), 28.28 (d, J C,P = 142.4 Hz), 31.15, 39.10 (d, J C,P = 3.7 Hz), 66.97, 67.55 (2C, d, J C,P = 6.4 Hz), 68.73, 128.21-128.76 (15C), 135.58, 136.19 (d, J C,P = 2.2 Hz), 136.25 (d, J C,P = 1.9 Hz), 170.60, 173.53 (d, J C,P = 8.9 Hz). 31 P NMR (162 MHz, CDCl): δ P 31.95. ESI MS: 567.1 ([M + Na] + ). HR ESI MS: C 28 H 30 Calculated for O7ClPNa: 567.13099; observed: 567.13081.

[0107] 1-Benzyl 5-((((4R)-4-((3R,10S,13R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methyl) 2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP578) [ka] Compound LTP576 (500 mg, 0.917 mmol, 1 equiv.), lithocholic acid (690 mg, 1.83 mmol, 2 equiv.), and CsCO (330 mg, 1.01 mmol, 1.1 equiv.) were dissolved / suspended in anhydrous DMF (30 mL). The resulting mixture was heated (blocked) to 65 °C for 1 h under an inert atmosphere. DMF was evaporated, and the residue was dissolved in EtOAc (100 mL). It was washed with distilled HO (50 mL), saturated NaHCO (50 mL), distilled HO (50 mL), and saturated NaCl (50 mL), dried over MgSO, and the solvent was evaporated. The crude product was purified by LC (EtOAc / cyclohexane, 2:1) to give compound LTP578 as a colorless amorphous foam (592 mg, 73% yield). 1 H NMR (400 MHz, CDCl): δ H 0.63 (s, 3H), 0.89 (d, J = 6.4 Hz, 3H), 0.91 (s, 3H), 0.94-1.18 (m, 6H), 1.18-1.45 (m, 15H), 1.44-1.69 (m, 2H), 1.70-1.87 (m, 5H), 1.87-1.98 (m, 3H), 2.17-2.44 (m, 4H), 2.75-2.87 (m, 1H), 3.61 (tt, J = 10.9, 4.6 Hz, 1H), 4.85-5.04 (m, 6H), 5.64-5.70 (m, 2H), 7.21-7.39 (m, 15H). 13 C NMR (101 MHz, CDCl3): δ C 12.13, 18.33, 20.89, 23.47, 24.27, 26.50, 27.28, 28.00 (d, J). C,P = 13.0 Hz), 28.24 (d, J). C,P = 142.3 Hz), 28.26, 29.78, 30.63 (d, J). C,P = 3.4 Hz), 30.96, 31.06, 34.65, 35.34, 35.44, 35.91, 36.52, 39.13 (d, J). C,P = 3.7 Hz), 40.23, 40.49, 42.16, 42.81, 55.95, 56.55, 66.88, 67.46 (d, J). C,P = 1.1 Hz), 67.52 (d, J). C,P = 1.1 Hz), 71.85, 79.23, 128.14–128.69 (15C), 135.56, 136.15 (d, J). C,P = 2.0 Hz), 136.21 (d, J). C,P = 1.6 Hz), 171.23, 172.95, 173.53 (d, J). C,P = 8.3 Hz). 31 P NMR (162 MHz, CDCl3): δ P 31.82. ESI MS: 907.4 ([M + Na] + )。 。 HR ESI MS: C 52 H 70 O 10 Please note: 885.47011; Code: 885.47038.

[0108] 1-Benzyl 5-((((4R)-4-((3R,10S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methyl) 2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP589) [ka] Compound LTP576 (500 mg, 0.917 mmol, 1 equiv.), deoxycholic acid (720 mg, 1.83 mmol, 2 equiv.), and CsCO (330 mg, 1.01 mmol, 1.1 equiv.) were dissolved / suspended in anhydrous DMF (40 mL). The resulting mixture was heated (blocked) to 60 °C for 1 h under an inert atmosphere. DMF was evaporated, and the residue was dissolved in EtOAc (150 mL). It was washed with saturated NaHCO (100 mL), distilled water (100 mL), and saturated NaCl (100 mL). The aqueous phase was re-extracted with EtOAc (100 mL), combined, and dried over MgSO. EtOAc was evaporated. The crude product was purified by LC (gradient: EtOAc / cyclohexane, 2:1 to EtOAc) to give compound LTP589 as a colorless amorphous foam (579 mg, 70% yield). 1 H NMR (400 MHz, CDCl): δ H 0.66 (s, 3H), 0.90 (s, 3H), 0.95 (d, J = 6.2 Hz, 3H), 0.98-1.18 (m, 2H), 1.19-1.46 (m, 11H), 1.46-2.00 (m, 14H), 2.18-2.45 (m, 5H), 2.81 (dddd, J= 13.1, 11.3, 10.0, 6.0 Hz, 1H), 3.59 (tt, J = 11.0, 4.6 Hz, 1H), 3.95 (t, J = 3.0 Hz, 1H), 4.86-5.03 (m, 6H), 5.64-5.72 (m, 2H), 7.19-7.38 (m, 15H). 13 C NMR (101 MHz, CDCl): δ C12.84, 17.37, 23.26, 23.74, 26.23, 27.22, 27.54, 28.02 (d, J C,P = 13.0 Hz), 28.25 (d, J C,P (d, J C,P = 3.7 Hz), 42.17, 46.59, 47.26, 48.33, 66.91, 67.48 (d, J C,P = 2.0 Hz), 67.55 (d, J C,P = 1.9 Hz), 71.85, 73.14, 79.26, 135.57, 136.17 (d, J C,P = 1.9 Hz), 136.23 (d, J C,P = 1.5 Hz), 128.16-128.72 (15C), 171.27, 172.94, 173.58 (d, J C,P = 8.4 Hz). 31 P NMR (162 MHz, CDCl): δ P 32.04. ESI MS: 923.4 ([M + Na] + ). HR ESI MS: C 52 H 69 O 11 Calculated for PNa: 923.44697; Observed: 923.44706.

[0109] 1-Benzyl 5-((((4R)-4-((3R,7R,10S,12S,13R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methyl) 2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP584) [ka] Compound LTP576 (500 mg, 0.917 mmol, 1 equiv.), cholic acid (750 mg, 1.83 mmol, 2 equiv.), and CsCO (330 mg, 1.01 mmol, 1.1 equiv.) were dissolved / suspended in anhydrous DMF (40 mL). The resulting mixture was heated (blocked) to 65 °C for 1 h under an inert atmosphere. DMF was evaporated, and the residue was suspended in a mixture of EtOAc (150 mL) and MeOH (20 mL), stirred for 10 min, filtered, and the solvent was evaporated. The crude product was purified by LC (EtOAc / MeOH, 10:1) to give compound LTP584 as a colorless amorphous foam (479 mg, 57% yield). 1 H NMR (400 MHz, CDCl): δ H 0.67 (s, 3H), 0.88 (s, 3H), 0.96 (d, J = 6.0 Hz, 3H), 0.92-1.02 (m, 1H), 1.11 (qd, J = 12.0, 5.8 Hz, 1H), 1.19-2.08 (m, 22H), 2.12-2.48 (m, 6H), 2.74-2.86 (m, 1H), 3.39-3.47 (m, 1H), 3.83 (q, J = 3.1 Hz, 1H), 3.91-4.02 (m, 1H), 4.85-5.05 (m, 6H), 5.65-5.71 (m, 2H), 7.19-7.38 (m, 15H). 13 C NMR (101 MHz, CDCl): δ C 12.62, 17.41, 22.61, 23.32, 26.59, 27.58, 28.07 (d, J C,P = 13.1 Hz), 28.26 (d, J C,P = 141.7 Hz), 28.38, 29.82, 30.59, 31.02, 31.11, 34.77, 34.85, 35.27, 35.37, 39.18 (d, J C,P = 3.9 Hz), 39.65, 39.74, 41.58, 41.89, 46.58, 47.05, 66.94, 67.52 (d, J C,P= 2.8 Hz), 67.58 (d, J C,P = 2.6 Hz), 68.52, 72.03, 73.07, 79.27, 128.18-128.74 (15C), 135.58, 136.18 (d, J C,P = 1.8 Hz), 136.24 (d, J C,P = 1.6 Hz), 171.31, 173.01, 173.60 (d, J C,P = 8.4 Hz). 31 P NMR (162 MHz, CDCl): δ P 32.04. ESI MS: 939.4 ([M + Na] + ). HR ESI MS: C 52 H 69 O 12 Calculated for PNa: 939.44189; Observed: 939.44177.

[0110] 1-Benzyl 5-((((4R)-4-((3R,7S,10S,13R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methyl) 2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP1049) [ka] Compound LTP576 (500 mg, 0.917 mmol, 1 equiv.), ursodeoxycholic acid (720 mg, 1.83 mmol, 2 equiv.), and CsCO (330 mg, 1.01 mmol, 1.1 equiv.) were dissolved / suspended in anhydrous DMF (30 mL). The resulting mixture was heated (blocked) to 65 °C for 1 h under an inert atmosphere. DMF was evaporated, and the residue was dissolved in EtOAc (150 mL). It was washed with saturated NaHCO (100 mL), distilled HO (100 mL), and saturated NaCl (100 mL). The aqueous phase was re-extracted with EtOAc (100 mL). The organic phases were combined and dried over MgSO. EtOAc was evaporated. The crude product was purified by LC (EtOAc / cyclohexane, 2:1) to give compound LTP1049 as a colorless amorphous foam (605 mg, 73% yield). 1 H NMR (400 MHz, CDCl): δ H 0.69 (s, 3H), 0.94 (d, J = 6.3 Hz, 3H), 0.97 (s, 3H), 0.98-1.23 (m, 4H), 1.22-1.74 (m, 14H), 1.76-2.05 (m, 8H), 2.19-2.50 (m, 6H), 2.84 (dddd, J= 13.4, 11.2, 8.2, 5.6 Hz, 1H), 3.55-3.67 (m, 2H), 4.89-5.06 (m, 6H), 5.70 (qd, J = 5.6, 2.6 Hz, 2H), 7.24-7.42 (m, 15H). 13 C NMR (101 MHz, CDCl): δ C 12.25, 18.47, 21.28, 23.50, 27.00, 28.02 (d, J C,P = 12.9 Hz), 28.26 (d, J C,P = 142.3 Hz), 28.73, 30.44, 30.72, 31.03, 31.08 (2C), 34.18, 35.05, 35.28, 37.04, 37.41, 39.16 (d, J C,P= 3.1 Hz), 39.27, 40.23, 42.56, 43.85, 54.94, 55.84, 66.91, 67.49 (d, J C,P = 1.7 Hz), 67.55 (d, J C,P = 1.6 Hz), 71.39, 71.50, 79.25, 128.16-128.72 (15C), 135.57, 136.16 (d, J C,P = 2.0 Hz), 136.22 (d, J C,P = 1.6 Hz), 171.26, 172.91, 173.56 (d, J C,P = 8.4 Hz). 31 P NMR (162 MHz, CDCl): δ P 32.03. ESI MS: 923.4 ([M + Na] + ). HR ESI MS: C 52 H 69 O 11 Calculated for PNa: 923.44697; Observed: 923.44773.

[0111] 5-((((4R)-4-((3R,10S,13R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)methoxy)-5-oxo-2-(phosphonomethyl)pentanoic acid (1, LTP582) [ka] Compound LTP578 (380 mg, 0.429 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL), 10% Pd / C (137 mg, 0.129 mmol, 0.3 equiv.) was added, and the reaction mixture was saturated with H and stirred overnight (20 h) under a H atmosphere (balloon). The Pd / C was removed by filtration and washed with additional THF (2 × 10 mL). The solvent was evaporated, and the residue was dissolved in MeCN / HO 2:1 (60 mL), cooled to −78 °C, and lyophilized overnight. The desired product 1 was obtained as a colorless foam (230 mg, 87% yield). 1 H NMR (400 MHz, d6-DMSO): δ H 0.60 (s, 3H), 0.86 (d, J = 6.2 Hz, 3H), 0.87 (s, 3H), 0.82-0.93 (m, 1H), 0.96-1.41 (m, 18H), 1.44-1.99 (m, 8H), 2.05-2.10 (m, 1H), 2.17-2.44 (m, 4H), 2.51-2.63 (m, 2H), 3.36 (tt, J= 10.4, 4.5 Hz, 1H), 5.63-5.69 (m, 2H), 4.17-6.97 (bs, 4H). 13 C NMR (101 MHz, d6-DMSO): δ C 11.87, 18.06, 20.43, 23.31, 23.87, 26.92, 26.94 (d, J C,P = 154.3 Hz), 27.17 (d, J C,P = 9.5 Hz), 27.71, 28.93, 30.25, 30.30, 30.40, 30.44, 30.89, 34.23, 34.72, 35.18, 35.39, 36.31, 39.02 (d, J C,P = 2.9 Hz), 39.99, 41.56, 42.30, 55.46, 56.07, 69.89, 78.94, 171.37, 172.25, 175.54 (d, J C,P = 10.0 Hz). 31P NMR (162 MHz, d6-DMSO): δ P 25.84. ESI MS: 613.4 ([M-H] + ). HR ESI MS: C 31 H 50 O 10 Calculated value for P: 613.31471; Observed value: 613.31474.

[0112] 5-((((4R)-4-((3R,10S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)methoxy)-5-oxo-2-(phosphonomethyl)pentanoic acid (2, LTP592) [ka] Compound LTP589 (437 mg, 0.485 mmol, 1 equiv.) was dissolved in anhydrous THF (12 mL), 10% Pd / C (155 mg, 0.145 mmol, 0.3 equiv.) was added, and the reaction mixture was saturated with H and stirred overnight (16 h) under a H atmosphere (balloon). The Pd / C was removed by filtration and washed with additional THF (2 × 10 mL). The solvent was evaporated, and the residue was dissolved in MeCN / HO 2:1 (60 mL), cooled to −78 °C, and lyophilized overnight. The desired product 2 was obtained as a colorless foam (270 mg, 88% yield). 1 H NMR (400 MHz, CD3OD): δ H0.71 (s, 3H), 0.93 (s, 3H), 1.00 (d, J= 6.4, 3H), 1.07-1.23 (m, 2H), 1.24-1.55 (m, 12H), 1.56-1.64 (m, 2H), 1.74-2.10 (m, 11H), 2.11-2.23 (m, 1H), 2.30 (ddd, J = 15.7, 8.7, 7.1, 1H), 2.37-2.53 (m, 3H), 2.76 (ddt, J = 13.5, 11.3, 6.7, 1H), 3.47-3.57 (m, 1H), 3.95 (t, J = 2.7, 1H), 5.72 (d, J = 5.7, 1H), 5.74 (d, J = 5.7, 1H). 13 C NMR (101 MHz, CD3OD): δ C 13.22, 17.50, 23.72, 24.85, 27.44, 28.61, 28.88 (d, J C,P = 10.4 Hz), 29.13 (d, J C,P = 149.7 Hz), 29.49, 30.90, 31.04, 31.76, 31.85, 32.12, 34.79, 35.29, 36.43, 36.57, 37.17, 37.41, 40.62 (d, J C,P = 3.1 Hz), 43.60, 47.55, 48.07, 49.26, 72.53, 74.01, 80.41, 172.96, 174.26, 177.70 (d, J C,P = 10.0 Hz). 31 P NMR (162 MHz, CD3OD): δ P 28.43. ESI MS: 629.4 ([M - H] + ). HR ESI MS: C 31 H 50 O 11 Pについての calculated value: 629.30962; measured value: 629.30990.

[0113] 5-Oxo-2-(phosphonomethyl)-5-((((4R)-4-((3R,7R,10S,12S,13R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methoxy)pentanoic acid (3, LTP588) [ka] Compound LTP586 (444 mg, 0.484 mmol, 1 equiv.) was dissolved in anhydrous THF (12 mL), 10% Pd / C (155 mg, 0.145 mmol, 0.3 equiv.) was added, and the reaction mixture was saturated with H and stirred overnight (20 h) under a H atmosphere (balloon). The Pd / C was removed by filtration and washed with additional THF (2 × 10 mL). The solvent was evaporated, and the residue was dissolved in MeCN / HO 2:1 (60 mL), cooled to −78 °C, and lyophilized overnight. The desired product 3 was obtained as a colorless foam (305 mg, 97% yield). 1 H NMR (400 MHz, CD3OD): δ H 0.71 (s, 3H), 0.92 (s, 3H), 1.00 (d, J = 6.4 Hz, 3H), 0.96-1.04 (m, 1H), 1.05-1.20 (m, 1H), 1.22-1.69 (m, 12H), 1.70-2.08 (m, 9H), 2.08-2.54 (m, 7H), 2.67-2.82 (m, 1H), 3.32-3.42 (m, 1H), 3.80 (q, J = 3.1 Hz, 1H), 3.91-3.98 (m, 1H), 5.70-5.76 (m, 2H). 13 C NMR (101 MHz, d6-DMSO): δ C 12.34, 16.87, 22.66, 22.83, 26.23, 27.26 (d, J C,P = 5.5 Hz), 27.29, 27.84 (d, J C,P(d, J C,P (d, J C,P = 9.9 Hz). 31 P NMR (162 MHz, CD3OD): δ P 25.64. ESI MS: 669.4 ([M + Na] + ).

[0114] 5-((((4R)-4-((3R,7S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methoxy)-5-oxo-2-(phosphonomethyl)pentanoic acid (4, LTP1054) [ka] Compound LTP1048 (960 mg, 1.07 mmol, 1 equiv.) was dissolved in anhydrous THF (25 mL), 10% Pd / C (113 mg, 0.107 mmol, 0.1 equiv.) was added, and the reaction mixture was saturated with H and stirred overnight (24 h) under a H atmosphere (balloon). The Pd / C was removed by filtration and washed with additional THF (2 × 20 mL). The solvent was evaporated, and the residue was dissolved in MeCN / HO 2:1 (120 mL), cooled to −78 °C, and lyophilized overnight. The desired product 4 was obtained as a colorless foam (618 mg, 92% yield). 1 H NMR (400 MHz, CD3OD): δ H0.71 (s, 3H), 0.95 (d, J = 6.9 Hz, 3H), 0.97 (s, 3H), 0.98-1.68 (m, 19H), 1.75-2.11 (m, 7H), 2.11-2.54 (m, 5H), 2.69-2.83 (m, 1H), 3.40-3.55 (m, 2H), 5.73 (q, J= 5.7 Hz, 2H). 13 C NMR (101 MHz, CD3OD): δ C 12.68, 18.87, 22.39, 23.95, 27.94, 28.90 (d, J C,P = 10.5 Hz), 29.63, 30.18 (d, J C,P = 139.8 Hz), 31.02, 31.80, 31.94, 32.12, 35.17, 36.09, 36.55, 37.98, 38.59, 40.62 (d, J C,P = 3.0 Hz), 40.70, 41.54, 44.02, 44.45, 44.79, 56.48, 57.47, 71.95, 72.12, 80.41, 172.93, 174.19, 177.65 (d, J C,P = 10.1 Hz). 31 P NMR (162 MHz, CD3OD): δ P 28.43. ESI MS: 629.4 ([M - H] + ). HR ESI MS: C 31 H 50 O 11 P:についての Calculated value: 629.30962; Viewed value: 629.30990.

[0115]

change

[0116] 2,5-Dioxopyrrolidin-1-yl (4R)-4-((3R,10S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (LTP765) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with published data (Bioorg. Med. Chem. Lett. 2004, 14, 773).

[0117] ((4R)-4-((3R,10S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)glycine (LTP773) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with published data (Lett. Drug Des. Discov. 2012, 9, 573).

[0118] 1-Benzyl 5-(((((4R)-4-((3R,10S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)glycyl)oxy)methyl) 2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP781) [ka] Compound LTP773 (300 mg, 0.667 mmol, 1.5 equiv.), compound LTP576 (242 mg, 0.445 mmol, 1 equiv.), and CsCO (159 mg, 0.489 mmol, 1.1 equiv.) were dissolved in anhydrous DMF (12 mL), and the resulting mixture was heated to 60 °C for 1.5 h. The inorganic precipitate was filtered off and washed with EtOAc (20 mL). The organic solvent was evaporated. The crude product was purified by LC (EtOAc / MeOH, 20:1) to give compound LTP781 as a colorless amorphous solid in 35% yield (150 mg). 1 H NMR (400 MHz, CDCl): δ H 0.68 (s, 3H), 0.91 (s, 3H), 0.99 (d, J = 5.9 Hz, 3H), 0.94-1.18 (m, 2H), 1.21-1.34 (m, 2H), 1.34-2.04 (m, 20H), 2.17 (ddd, J = 14.9, 9.1, 6.3 Hz, 1H), 2.23-2.42 (m, 4H), 2.72-2.88 (m, 4H), 3.62 (tt, J = 10.5, 4.6 Hz, 1H), 3.97 (d, J = 3.2 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 4.96 (dt, J = 20.3, 12.0 Hz, 6H), 5.74 (s, 2H), 6.39 (t, J = 5.5 Hz, 1H), 7.22-7.40 (m, 15H). 13 C NMR (101 MHz, CDCl): δ C12.86, 17.51, 23.27, 23.77, 26.26, 27.24, 27.59, 28.13 (d, J = 13.4 Hz), 28.19 (d, J = 142.4 Hz), 28.76, 30.54, 31.11, 31.51, 33.07, 33.76, 34.23, 35.29, 35.34, 36.12, 36.49, 39.06 (d, J = 3.5 Hz), 41.14, 42.18, 46.61, 47.19, 48.36, 66.99, 67.65 (d, J = 3.5 Hz), 67.71 (d, J = 3.3 Hz), 71.92, 73.25, 79.47, 128.17-128.76 (15C), 135.52, 136.04 (d, J= 1.1 Hz), 136.10 (d, J = 1.2 Hz), 169.22, 171.07, 173.55 (d, J = 7.0 Hz), 174.20. 31 P NMR (162 MHz, CDCl): δ P 32.03. ESI MS: 980.6 ([M + Na] + ). HR ESI MS: C 54 H 72 O 12 Calculated for NPNa: 980.46843; Observed: 980.46883.

[0119] 5-(((((4R)-4-((3R,10S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)glycyl)oxy)methoxy)-5-oxo-2-(phosphonomethyl)pentanoic acid (5, LTP791) [ka] Compound LTP781 (128 mg, 0.134 mmol, 1 equiv.) was dissolved in anhydrous THF (4 mL), 10% Pd / C (43 mg, 0.040 mmol, 0.3 equiv.) was added, and the mixture was saturated with H and stirred overnight at room temperature under a H atmosphere (balloon). The precipitate was filtered off and washed with additional THF (2 × 10 mL). The organic solvent was evaporated, and the product was dissolved in a MeCN / HO mixture (1:1, 50 mL) and lyophilized for 3 days. The desired product 5 was obtained as a colorless foam (73 mg) in 79% yield. 1 H NMR (400 MHz, CD3OD): δ H 0.71 (s, 3H), 0.93 (s, 3H), 1.03 (d, J = 6.4 Hz, 3H), 1.22-1.69 (m, 2H), 1.22-1.69 (m, 15H), 1.69-2.24 (m, 11H), 2.26-2.38 (m, 1H), 2.38-2.56 (m, 2H), 2.75 (dq, J= 13.4, 7.0 Hz, 1H), 3.53 (tt, J = 11.0, 4.4 Hz, 1H), 3.92-4.01 (m, 3H), 5.77 (s, 2H). 13 C NMR (101 MHz, CD3OD): δ C 13.24, 17.67, 23.74, 24.86, 27.43, 28.38, 28.60, 28.81 (d, J = 10.4 Hz), 29.85, 30.30 (d, J = 141.9 Hz), 30.90, 31.00, 32.07, 33.01, 33.64, 34.75, 35.26, 36.41, 36.71, 37.14, 37.39, 40.62 (d, J = 3.2 Hz), 41.78, 43.56, 47.52, 48.05, 72.49, 74.01, 80.56, 170.07, 172.83, 177.35, 177.75 (d, J= 10.0 Hz). 31 P NMR (162 MHz, CD3OD): δ P 28.29. MALDI MS: 710.4 ([M + Na] + ). HR MALDI MS: C 33 H 53 O 12 Calculated for NPa: 686.3311; Observed: 686.3298.

[0120] [ka]

[0121] Benzyl (4R)-4-((3R,10S,13R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate (LTP761) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with published data (Org. Biomol. Chem. 2014, 12, 9592).

[0122] Benzyl (4R)-4-((3R,10S,12S,13R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate (LTP800) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with published data (Org. Biomol. Chem. 2014, 12, 9592).

[0123] Benzyl (4R)-4-((3R,7S,10S,13R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate (IS-101-001) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with published data (Org. Biomol. Chem. 2014, 12, 9592).

[0124] Benzyl (4R)-4-((3R,10S,13R)-3-(((chloromethoxy)carbonyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate (LTP772) [ka] The starting material LTP761 (400 mg, 0.857 mmol, 1 equiv.) was suspended in anhydrous EtO (10 mL), and the mixture was cooled to 0 °C and stirred under an inert atmosphere. Triethylamine (130 mg, 179 μL, 1.29 mmol, 1.5 equiv.) was added dropwise, followed by a solution of chloromethyl chlorocarbonate (155 mg, 107 μL, 1.20 mmol, 1.4 equiv.) in anhydrous EtO (4 mL). The resulting mixture was slowly heated to room temperature and stirred overnight (24 h) under inert atmosphere. EtOAc (70 mL) was added, and the organic phase was washed with distilled H2O (70 mL). The inorganic phase was then extracted with additional EtOAc (70 mL), and the combined organic phase was finally washed with saturated NaCl (50 mL), dried over MgSO4, and the solvent was evaporated. The crude product LTP772 was obtained as a colorless solid in quantitative yield (479 mg) and was used in the next step without further purification. 1 H NMR (400 MHz, CDCl): δ H0.62 (s, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.93 (s, 3H), 0.97-1.30 (m, 9H), 1.31-1.49 (m, 8H), 1.50-1.70 (m, 2H), 1.74-2.01 (m, 7H), 2.27 (ddd, J = 15.6, 9.2, 6.7 Hz, 1H), 2.40 (ddd, J = 15.0, 9.9, 4.9 Hz, 1H), 4.59-4.74 (m, 1H), 5.11 (d, J = 2.6 Hz, 2H), 5.72 (s, 2H), 7.29-7.46 (m, 5H). 13 C NMR (101 MHz, CDCl): δ C 12.15, 18.37, 20.94, 23.35, 24.29, 26.39, 26.49, 27.08, 28.28, 31.08, 31.39, 32.05, 34.65, 34.96, 35.43, 35.88, 40.21, 40.52, 41.98, 42.84, 56.06, 56.56, 66.21, 72.18, 80.15, 128.29, 128.35 (2C), 128.66 (2C), 136.25, 152.89, 174.21. ESI MS: 581.4 ([M + Na] + ). HR ESI MS: C 33 H 47 Calculated for O5ClNa: 581.30042; Observed: 581.30102.

[0125] Benzyl (4R)-4-((3R,10S,12S,13R)-3-(((chloromethoxy)carbonyl)oxy)-12-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (LTP801) [ka] The starting material LTP800 (200 mg, 0.414 mmol, 1 equiv.) was suspended in anhydrous EtO (6 mL), and the mixture was cooled to 0 °C and stirred under an inert atmosphere. Triethylamine (50 mg, 69 μL, 0.497 mmol, 1.2 equiv.) was added dropwise, followed by a solution of chloromethyl chlorocarbonate (59 mg, 41 μL, 0.456 mmol, 1.1 equiv.) in anhydrous EtO (3 mL). The resulting mixture was slowly heated to room temperature and stirred overnight (24 h) under inert atmosphere. EtOAc (50 mL) was added, and the organic phase was washed with distilled H2O (50 mL). The inorganic phase was then extracted with additional EtOAc (50 mL), and the combined organic phase was finally washed with saturated NaCl (50 mL), dried over MgSO4, and the solvent was evaporated. The crude product was purified by LC (cyclohexane / EtOAc, 6:1) to give the desired product LTP801 as a colorless solid (220 mg) in 93% yield. 1 H NMR (400 MHz, CDCl): δ H 0.65 (s, 3H), 0.93 (s, 3H), 0.96 (d, J = 6.1 Hz, 3H), 1.00-1.17 (m, 2H), 1.18-1.32 (m, 2H), 1.32-1.71 (m, 14H), 1.74-1.90 (m, 5H), 1.90-2.01 (m, 1H), 2.29 (ddd, J = 15.5, 8.9, 6.8 Hz, 1H), 2.42 (ddd, J = 14.4, 9.6, 4.8 Hz, 1H), 3.94-3.99 (m, 1H), 4.66 (dq, J = 11.3, 5.7, 4.5 Hz, 1H), 5.11 (d, J = 3.1 Hz, 2H), 5.72 (s, 2H), 7.28-7.41 (m, 5H). 13 C NMR (101 MHz, CDCl): δ C12.84, 17.42, 23.12, 23.70, 26.09, 26.37, 27.00, 27.52, 28.75, 30.96, 31.41, 31.98, 33.70, 34.18, 34.80, 35.12, 36.06, 41.94, 46.60, 47.47, 48.28, 66.22, 72.17, 73.12, 80.01, 128.29, 128.35 (2C), 128.65 (2C), 136.21, 152.90, 174.12. ESI MS: 597.4 ([M + Na] + ). HR ESI MS: C 33 H 47 Calculated for O6ClNa: 597.29534; Observed: 597.29559.

[0126] Benzyl (4R)-4-((3R,7S,10S,13R)-3-(((chloromethoxy)carbonyl)oxy)-7-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate (IS-101-002) [ka] Compound IS-101-001 (0.2 g, 0.41 mmol, 1 equiv.) was dissolved in diethyl ether (6 mL). The reaction mixture was cooled to 0 °C, and EtN (69 μL, 0.50 mmol, 1.2 equiv.) and chloromethyl chloroformate (41 μL, 0.45 mmol, 1.1 equiv.) were added. The reaction mixture was stirred at room temperature for 16 h. EtOAc (30 mL) was added, and the mixture was washed with brine (20 mL) and dried over NaSO. The volatiles were evaporated under reduced pressure, and the mixture was purified by column chromatography (cyclohexane / EtOAc, 4:1) to give the desired product IS-101-002 (0.13 g, 54% yield) as a colorless solid. 1 H NMR (400 MHz, CD3OD): δ H0.65 (s, 3H), 0.92 (d, J = 6.2 Hz, 3H), 0.96 (s, 3H), 1.01-3.48 (m, 27H), 3.51-3.62 (m, 1H), 4.58-4.67 (m, 1H), 5.06-5.16 (m, 2H), 5.72 (s, 2H), 7.29-7.40 (m, 5H). ESI MS: 597.3 ([M + Na] + ). HR ESI MS: C 33 H 47 Calculated for O6ClNa: 597.29534; Observed: 597.29542.

[0127] 1-Benzyl 5-((((((3R,10S,13R)-17-((R)-5-(benzyloxy)-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)oxy)-methyl)-2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP797) [ka] Compound LTP560 (133 mg, 0.268 mmol, 1.5 equiv.) was dissolved in anhydrous DMF (8 mL) and CsCO (64 mg, 0.197 mmol, 1.1 equiv.) was added. Finally, compound LTP772 (100 mg, 0.179 mmol, 1 equiv.) was added, and the reaction mixture was heated to 60 °C under inert conditions for 1 h. DMF was evaporated, EtOAc (70 mL) was added, and the organic phase was washed with saturated NaHCO (30 mL), distilled HO (30 mL), and saturated NaCl (30 mL) and dried over MgSO. The solvent was evaporated, and the crude product was purified by LC (cyclohexane / EtOAc, 1:1) to give the desired product LTP797 (164 mg, 90% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl): δ H0.61 (s, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.92 (s, 3H), 0.95-1.16 (m, 6H), 1.18-1.68 (m, 13H), 1.73-1.99 (m, 10H), 2.20-2.46 (m, 5H), 2.82 (dddd, J= 16.2, 13.4, 8.1, 5.8 Hz, 1H), 4.62 (ddt, J = 16.0, 11.2, 5.8 Hz, 1H), 5.11 (d, J = 2.5 Hz, 2H), 4.85-5.03 (m, 6H), 5.65-5.71 (m, 2H), 7.22-7.42 (m, 20H)。 13 C NMR (101 MHz, CDCl3): δ C 12.16, 18.39, 20.94, 23.38, 24.31, 26.41, 26.53, 27.10, 27.98 (d, J = 12.9 Hz), 28.29, 28.31 (d, J = 142.2 Hz), 29.85, 31.10, 31.43, 32.09, 34.66, 35.00, 35.45, 35.89, 39.19 (d, J = 3.6 Hz), 40.23, 40.52, 41.99, 42.85, 56.07, 56.57, 66.23, 66.95, 67.54 (2C, d, J = 6.4 Hz), 79.60, 81.82, 128.21-128.76 (20C), 135.63, 136.23 (d, J = 1.9 Hz), 136.27, 136.29 (d, J = 2.1 Hz), 153.48, 171.08, 173.60 (d, J = 8.4 Hz), 174.24。 31 P NMR (162 MHz, CDCl3): δ P 29.36。 ESI MS: 1014.5 ([M + Na] + )。 HR ESI MS: C 60 H 75 O 12Calculated for PNa: 1041.48884; Observed: 1041.48927.

[0128] 1-Benzyl 5-((((((3R,10S,12S,13R)-17-((R)-5-(benzyloxy)-5-oxopentan-2-yl)-12-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)oxy)methyl)-2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (LTP806) [ka] Compound LTP560 (259 mg, 0.522 mmol, 1.5 equiv.) was dissolved in anhydrous DMF (16 mL) and CsCO (125 mg, 0.383 mmol, 1.1 equiv.) was added. Finally, compound LTP801 (200 mg, 0.348 mmol, 1 equiv.) was added, and the reaction mixture was heated to 60 °C under inert conditions for 1 h. DMF was evaporated, EtOAc (70 mL) was added, and the organic phase was washed with saturated NaHCO (50 mL), distilled HO (50 mL), and saturated NaCl (50 mL) and dried over MgSO. The solvent was evaporated, and the crude product was purified by LC (cyclohexane / EtOAc, 2:1) to give the desired product LTP806 (320 mg, 89% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl): δ H0.64 (s, 3H), 0.91 (s, 3H), 0.95 (d, J = 5.9 Hz, 3H), 0.99-1.14 (m, 3H), 1.18-1.20 (m, 1H), 1.30-1.72 (m, 12H), 1.71-2.00 (m, 10H), 2.22-2.36 (m, 4H), 2.42 (ddd, J = 14.4, 9.7, 4.6 Hz, 1H), 2.81 (tdd, J = 13.3, 8.1, 5.8 Hz, 1H), 3.89-3.99 (m, 1H), 4.61 (tt, J = 11.3, 4.4 Hz, 1H), 4.86-5.04 (m, 6H), 5.11 (d, J = 3.1 Hz, 2H), 5.64-5.74 (m, 2H), 7.07-7.43 (m, 20H)。 13 C NMR (101 MHz, CDCl3): δ C 12.85, 17.42, 23.13, 23.72, 26.11, 26.37, 27.02, 27.53, 28.02 (d, J = 13.0 Hz), 28.28 (d, J = 142.4 Hz), 28.77, 30.99, 31.09, 31.43, 32.00, 33.68, 34.19, 34.84, 35.14, 36.07, 39.17 (d, J = 3.6 Hz), 41.94, 46.61, 47.46, 48.28, 66.23, 66.94, 67.54 (2C, d, J = 6.4 Hz), 73.09, 79.48, 81.82, 128.20-128.75 (20C), 135.62, 136.21 (d, J = 1.9 Hz), 136.24, 136.27 (d, J = 1.2 Hz), 153.46, 171.05, 173.58 (d, J = 8.3 Hz), 174.13. 31 P NMR (162 MHz, CDCl3): δ P 32.05。 ESI MS: 1057.5 ([M + Na] + )。 HR ESI MS: C60 H 75 O 13 Calculated for PNa: 1057.48375; Observed: 1057.48410.

[0129] 1-Benzyl 5-(2-(((3R,7S,10S,13R,17R)-17-((R)-5-(benzyloxy)-5-oxopentan-2-yl)-7-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-oxoethyl) 2-((bis(benzyloxy)phosphoryl)methyl)pentanedioate (IS-101-009) [ka] Compound IS-101-002 (0.11 g, 0.19 mmol, 1 equiv.), LTP592 (0.11 g, 0.23 mmol, 1.2 equiv.), and CsCO (68 mg, 0.21 mmol, 1.1 equiv.) were suspended in anhydrous DMF (10 mL). The resulting mixture was stirred at 60 °C under an inert atmosphere for 1.5 h. DMF was evaporated, and the residue was dissolved in EtOAc (50 mL), washed with brine (10 mL), and dried over NaSO. The volatiles were evaporated, and the mixture was purified by column chromatography (DCM / MeOH, 40:1) to give the desired compound IS-101-009 as a colorless solid (0.18 g, 84% yield). 1 H NMR (400 MHz, CDCl): δ H 0.65 (s, 3H), 0.92 (d, J = 6.2 Hz, 3H), 0.95 (s, 3H), 1.00-2.02 (m, 27H), 2.20-2.46 (m, 5H), 2.75-2.87 (m, 1H), 3.54 (t, J= 5.9 Hz, 1H), 4.57 (dt, J = 11.2, 6.0 Hz, 1H), 4.86-5.03 (m, 6H), 5.11 (d, J= 3.1 Hz, 2H), 5.68 (d, J = 1.5 Hz, 2H), 7.22-7.40 (m, 20H). 31P NMR (162 MHz, CDCl): δ P 32.01. ESI MS: 1057.6 ([M + Na] + ). HR ESI MS: C 60 H 76 O 13 Calculated value for P: 1035.50181; observed value: 1035.50160.

[0130] 5-((((((3R,10S,13R)-17-((R)-4-carboxybutan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)oxy)methoxy)-5-oxo-2-(phosphonomethyl)pentanoic acid (6, LTP798) [ka] Compound LTP797 (156 mg, 0.153 mmol, 1 equiv.) was dissolved in anhydrous THF (7 mL), 10% Pd / C (65 mg, 0.061 mmol, 0.4 equiv.) was added, and the mixture was saturated with H and stirred under a H atmosphere (balloon) at room temperature overnight. The precipitate was filtered off and washed with additional THF (2 × 15 mL). The organic solvent was evaporated, and the product was dissolved in a MeCN / HO mixture (1:1, 50 mL) and lyophilized for 2 days. The desired product 6 was obtained as a colorless foam (89 mg) in 88% yield. 1 H NMR (400 MHz, CD3OD): δ H 0.70 (s, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.97 (s, 3H), 1.00-1.21 (m, 5H), 1.21-1.54 (m, 13H), 1.57-1.68 (m, 2H), 1.72-2.12 (m, 8H), 2.12-2.27 (m, 2H), 2.27-2.38 (m, 1H), 2.39-2.56 (m, 2H), 2.68-2.84 (m, 1H), 4.59 (tt, J= 10.8, 4.6 Hz, 1H), 5.72 (s, 2H). 13 C NMR (101 MHz, CD3OD): δ C 12.59, 18.85, 21.95, 23.82, 25.26, 27.52, 28.12, 28.81 (d, J = 10.6 Hz), 29.20, 30.08 (d, J = 140.1 Hz), 31.93, 32.06, 32.26, 33.19, 35.34, 35.62, 35.91, 36.63, 37.13, 40.54 (d, J = 3.3 Hz), 41.42, 41.78, 43.20, 43.88, 57.37, 57.77, 80.31, 83.04, 154.77, 172.77, 177.60 (d, J = 10.0 Hz), 178.03. 31 P NMR (162 MHz, CD3OD): δ P 28.88. MALDI MS: 657.3 ([M - H] + ). HR MALDI MS: C 32 H 50 O 12 Calculated value for P: 657.3045; observed value: 657.3028.

[0131] 5-((((((3R,10S,12S,13R)-17-((R)-4-carboxybutan-2-yl)-12-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene)-3-yl)oxy)carbonyl)oxy)methoxy)-5-oxo-2-(phosphonomethyl)pentanoic acid (7, LTP808) [ka] Compound LTP806 (296 mg, 0.286 mmol, 1 equiv.) was dissolved in anhydrous THF (12 mL), 10% Pd / C (122 mg, 0.114 mmol, 0.4 equiv.) was added, and the mixture was saturated with H and stirred overnight at room temperature under a H atmosphere (balloon). The precipitate was filtered off and washed with additional THF (2 × 20 mL). The organic solvent was evaporated, and the product was dissolved in a MeCN / HO mixture (1:1, 50 mL) and lyophilized for 2 days. The desired product 7 was obtained as a colorless foam (170 mg) in 88% yield. 1 H NMR (400 MHz, CD3OD): δ H 0.72 (s, 3H), 0.96 (s, 3H), 1.01 (d, J = 6.4 Hz, 3H), 1.04-1.24 (m, 2H), 1.26-1.39 (m, 3H), 1.41-1.68 (m, 10H), 1.73-2.10 (m, 11H), 2.10-2.27 (m, 2H), 2.35 (ddt, J= 15.0, 9.7, 5.0 Hz, 1H), 2.42-2.55 (m, 2H), 2.70-2.83 (m, 1H), 3.92-4.00 (m, 1H), 4.59 (td, J = 11.1, 5.5 Hz, 1H), 5.72 (s, 2H). 13 C NMR (101 MHz, CD3OD): δ C 13.33, 17.66, 23.58, 24.78, 27.15 (d, J = 4.3 Hz), 28.02, 28.54, 28.65, 28.76, 29.60, 30.09 (d, J = 138.3 Hz), 31.04, 32.02, 32.05, 32.95, 34.51, 35.07, 35.27, 35.65, 36.48, 37.14, 40.50 (d, J = 3.1 Hz), 42.99, 47.39, 47.84, 73.72, 80.69, 83.14, 154.69, 173.29, 178.09 (d, J = 9.2 Hz), 178.50. 31 P NMR (162 MHz, CD3OD): δP 28.27. MALDI MS: 673.3 ([M - H] + ). HR MALDI MS: C 32 H 50 O 13 Calculated value for P: 673.2995; observed value: 673.3013.

[0132] 5-(2-(((3R,7S,10S,13R,17R)-17-((R)-4-carboxybutan-2-yl)-7-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-oxoethoxy)-5-oxo-2-(phosphonomethyl)pentanoic acid (8, IS-101-010) [ka] Compound IS-101-009 (3.70 g, 3.57 mmol) was dissolved in anhydrous THF (50 mL) and 10% palladium on carbon (1.52 g, 1.43 mmol, 0.4 equivalents) was added. The reaction flask was evacuated, filled with hydrogen, and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product 8 (2.2 g, 91% yield) as a colorless solid. 1 H NMR (400 MHz, CD3OD): δ H 0.72 (s, 3H), 0.92-1.02 (m, 6H), 1.04-1.62 (m, 16H), 1.67-2.55 (m, 16H), 2.75 (d, J = 7.0 Hz, 1H), 3.47 (d, J = 2.2 Hz, 1H), 4.50-4.62 (m, 1H), 5.72 (s, 2H). 31 P NMR (162 MHz, CD3OD): δ P 28.85. ESI MS: 673.3 ([M-H] + ). HR ESI MS: C32 H 50 O 13 Calculated value for P: 673.29945; observed value: 673.29917.

[0133] [ka]

[0134] (3R,7S,10S,13R,17R)-17-((R)-4-aminobutan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7-diol (TT220219) [ka] The compound was prepared according to published procedures (J. Med. Chem. 2004, 47, 4559-4569). 1 H NMR (400 MHz, CD3OD): δ H 0.73 (s, 3H), 0.98 (d, J = 6.4 Hz, 6H), 1.02-1.70 (m, 19H), 1.77-1.95 (m, 4H), 2.05 (dt, J = 12.5, 3.2 Hz, 1H), 2.57-2.81 (m, 2H), 3.43-3.55 (m, 2H). 13 C NMR (101 MHz, CD3OD): δ C 12.6, 19.4, 22.4, 24.0, 27.9, 29.8, 31.0, 35.2, 35.2, 36.1, 38.0, 38.6, 39.4, 39.7, 40.7, 41.6, 44.0, 44.5, 44.8, 56.8, 57.5, 71.9, 72.1. ESI MS: 364.3 ([M + H] + ). HR ESI MS: C 23 H 42 Calculated value for O2N: 364.32101; Observed value: 364.32088.

[0135] Benzyl 2-((bis(benzyloxy)phosphoryl)methyl)-5-(((3R)-3-((3R,7S,10S,13R,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)butyl)amino)-5-oxopentanoate (IS-101-019) [ka] Compound LTP560 (1.50 g, 3.02 mmol, 1 equiv.) was dissolved in DMF (50 mL) and HATU (1.15 g, 3.02 mmol, 1 equiv.) was added, followed by DIEA (1.58 mL, 9.07 mmol, 3 equiv.). The reaction mixture was stirred for 5 minutes, and compound TT220219 (1.10 g, 3.02 mmol, 1 equiv.) dissolved in DMF (5 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. The volatiles were evaporated, EtOAc (100 mL) was added, and the organic layer was washed with 10% aqueous KHSO (30 mL), saturated NaHCO (30 mL), and brine (30 mL). The organic portion was dried over NaSO, and the volatiles were evaporated under reduced pressure. The mixture was purified by column chromatography (DCM / MeOH, 20:1) to give the desired product IS-101-019 (2.02 g, 80% yield) as a colorless solid. 1 H NMR (400 MHz, CDCl): δ H 0.66 (s, 3H), 0.94 (d, J = 5.2 Hz, 6H), 0.98-2.11 (m, 29H), 2.32 (td, J = 16.8, 8.5 Hz, 1H), 2.73-2.86 (m, 1H), 3.02-3.13 (m, 1H), 3.15-3.29 (m, 1H), 3.52-3.78 (m, 2H), 4.79-5.09 (m, 6H), 5.41 (d, J = 5.9 Hz, 1H), 7.20-7.40 (m, 15H). 31 P NMR (162 MHz, CDCl): δ P 32.39. ESI MS: 864.5 ([M + Na] + ). HR ESI MS: C 50 H 68 Calculated for O8NNaP: 864.45748; observed: 864.45679.

[0136] 5-(((3R)-3-((3R,7S,10S,13R,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)butyl)amino)-5-oxo-2-(phosphonomethyl)pentanoic acid (9, IS-101-020) [ka] Compound IS-101-019 (2.02 g, 2.39 mmol) was dissolved in anhydrous THF (60 mL) and 10% palladium on carbon (200 mg) was added. The reaction flask was evacuated, filled with hydrogen, and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product 9 (1.25 g, 91% yield) as a colorless solid. 1 H NMR (400 MHz, CD3OD): δ H 0.72 (s, 3H), 0.91-1.01 (m, 6H), 1.02-1.73 (m, 22H), 1.74-2.33 (m, 9H), 2.66-2.79 (m, 1H), 3.06-3.18 (m, 1H), 3.18-3.29 (m, 1H), 3.41-3.55 (m, 2H). 31 P NMR (162 MHz, CD3OD): δ P 28.82. ESI MS: 570.3 ([M-H] + ). HR ESI MS: C 29 H 49 Calculated for O8NP: 570.32013; Observed: 570.31981.

[0137] [ka]

[0138] Benzyl 2-((bis(benzyloxy)phosphoryl)methyl)-5-(((3R)-3-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)butyl)amino)-5-oxopentanoate (IS-101-076) [ka] Compound LTP560 (1.70 g, 3.44 mmol, 1 equiv.) was dissolved in DMF (50 mL), and HATU (1.30 g, 3.44 mmol, 1 equiv.) was added, followed by DIEA (1.79 mL, 10.3 mmol, 3 equiv.). The reaction mixture was stirred for 5 minutes, and compound IS-100-076 (1.25 g, 3.44 mmol, 1 equiv.) dissolved in DMF (5 mL) was added. The reaction mixture was stirred at room temperature for 16 hours. The volatiles were evaporated, EtOAc (100 mL) was added, and the organic layer was washed with 10% aqueous KHSO (30 mL), saturated NaHCO (30 mL), and brine (30 mL). The organic portion was dried over NaSO, and the volatiles were evaporated under reduced pressure. The mixture was purified by column chromatography (DCM / MeOH, 20:1) to give the desired product IS-101-076 (2.05 g, 70% yield) as a colorless solid. 1 H NMR (400 MHz, CDCl): δ H 0.66 (s, 3H), 0.90 (s, 3H), 0.93-2.13 (m, 32H), 2.24-2.39 (m, 1H), 2.74-2.85 (m, 1H), 3.04-3.29 (m, 2H), 3.53-3.65 (m, 1H), 3.95 (s, 1H), 4.85-5.07 (m, 6H), 5.65 (bs, 1H), 7.23-7.38 (s, 15H). 31P NMR (162 MHz, CDCl): δ P 32.14, 32.17. ESI MS: 864.6 ([M + Na] + ). HR ESI MS: C 50 H 69 Calculated for O8NP: 842.47553; Observed: 842.47532.

[0139] 5-(((3R)-3-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)butyl)amino)-5-oxo-2-(phosphonomethyl)pentanoic acid (10, IS-101-077) [ka] Compound IS-101-076 (2.00 g, 2.37 mmol, 1 equiv.) was dissolved in anhydrous THF (50 mL) and 10% palladium on carbon (100 mg) was added. The reaction flask was evacuated, filled with hydrogen, and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product 10 (1.31 g, 96% yield) as a colorless solid. 1 H NMR (400 MHz, d6-DMSO): δ H 0.59 (s, 3H), 0.84 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.96-2.12 (m, 31H), 2.93 (d, J= 7.6 Hz, 1H), 3.02-3.19 (m, 1H), 3.30-3.42 (m, 1H), 3.79 (d, J = 2.7 Hz, 1H), 7.72 (s, 1H). 31 P NMR (162 MHz, d6-DMSO): δ P 26.61. ESI MS: 570.3 ([M-H] + ). HR ESI MS: C 29 H 49 Calculated for O8NP: 570.32013; Observed: 570.31989.

[0140] [ka]

[0141] (5-(benzyloxy)-2-((benzyloxy)carbonyl)-5-oxopentyl)phosphinic acid (IS-100-018) [ka] Compounds were prepared according to published procedures. 1 H and 13 The C NMR spectrum was consistent with previously published data (Tet. Asym. 2002, 13, 1609-1614).

[0142] Dibenzyl 2-[(benzyloxyphosphinyl)methyl]pentanedioate (IS-100-020) [ka] Compound IS-100-018 (3.15 g, 8.07 mmol, 1 equiv.) was dissolved in anhydrous THF (50 mL) and benzyl alcohol (1.1 mL, 10.5 mmol, 1.3 equiv.), followed by the addition of DCC (1.75 g, 8.47 mmol, 1.05 equiv.) and DMAP (0.09 g, 0.8 mmol, 0.1 equiv.). The reaction mixture was stirred at room temperature for 16 h. After completion, the DCC was filtered off, and the volatiles were evaporated under reduced pressure. The residue was dissolved in EtOAc (60 mL), and the organic layer was extracted with 1 M aqueous HCl (30 mL) and brine (30 mL). The organic portion was dried over Na2SO4, and the volatiles were evaporated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH, 50:1) to give the desired product IS-100-020 (3.68 g, 95% yield) as a colorless oil. 1 H and 13The 1C NMR spectrum was consistent with previously published data (J. Med. Chem. 2001, 44, 4170-4175).

[0143] Dibenzyl 2-(((benzyloxy)(3-(tert-butoxy)-3-oxopropyl)phosphoryl)methyl)pentanedioate (IS-100-021) [ka] Compound IS-100-020 (4.00 g, 8.32 mmol, 1 equiv.) was dissolved in anhydrous DCM (70 mL), EtN (5.22 mL, 37.5 mmol, 4.5 equiv.) was added, and the reaction mixture was cooled to 0 °C. Chlorotrimethylsilane (4.22 mL, 33.3 mmol, 4 equiv.) was added slowly, followed by 1 h of tert-butyl acrylate (3.62 mL, 24.9 mmol, 3 equiv.). The reaction mixture was allowed to warm slowly to room temperature and stirred for 5 days. Further DCM (100 mL) was added, and the organic layer was extracted with 1 M aqueous HCl (50 mL) and brine (50 mL). The organic portion was dried over NaSO, and the volatiles were evaporated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH, 40:1) to give the desired product IS-100-021 (2.38 g, 47% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl): δ H 1.42 (s, 9H), 1.75-1.86 (m, 1H), 1.90-2.05 (m, 4H), 2.20-2.54 (m, 5H), 2.81-2.93 (m, 1H), 4.70 (s, 1H), 4.91-5.09 (m, 5H), 7.27-7.40 (m, 15H). 31 P NMR (162 MHz, CDCl): δ P 57.42, 57.02. ESI MS: 631.3 ([M + Na] + ). HR ESI MS: C 34 H41 Calculated for O8PNa: 631.24313; observed: 631.24337.

[0144] 3-((benzyloxy)(5-(benzyloxy)-2-((benzyloxy)carbonyl)-5-oxopentyl)phosphoryl)propanoic acid (IS-100-025) [ka] Compound IS-100-021 (0.50 g, 0.82 mmol, 1 equiv.) was dissolved in DCM (1 mL) and trifluoroacetic acid (5 mL, 65.3 mmol), and the reaction mixture was stirred at room temperature for 2 h. The volatiles were evaporated under reduced pressure, and the residue was redissolved in DCM (20 mL) and evaporated three times to remove residual TFA. Crude compound IS-100-025 (0.44 g, 97% yield) was used in the next step without further purification. 1 H NMR (400 MHz, CDCl): δ H 1.80-2.13 (m, 5H), 2.22-2.40 (m, 3H), 2.42-2.66 (m, 2H), 2.79-2.92 (m, 1H), 4.91-5.10 (m, 6H), 7.23-7.37 (m, 15H). 31 P NMR (162 MHz, CDCl): δ P 59.69, 59.46. ESI MS: 551.2 ([M-H] + ). HR ESI MS: C 30 H 32 Calculated value for O8P: 551.18403; observed value: 551.18332.

[0145] 2-(((2-carboxyethyl)(hydroxy)phosphoryl)methyl)pentanedioic acid (11, IS-100-026) [ka] Compound IS-100-025 (0.44 g, 0.79 mmol, 1 equiv.) was dissolved in anhydrous THF (20 mL) and 10% palladium on carbon (60 mg) was added. The reaction flask was evacuated, filled with hydrogen (1 atm), and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product 11 (0.21 g, 90% yield) as a colorless oil. 1 H and 13 The 1C NMR spectrum was consistent with previously published data (J. Med. Chem. 1996, 39, 619-622).

[0146] [ka]

[0147] Dibenzyl 2-(((benzyloxy)(3-(chloromethoxy)-3-oxopropyl)phosphoryl)methyl)pentanedioate (IS-100-055) [ka] Compound IS-100-025 (0.68 g, 1.23 mmol, 1 equiv.) was dissolved in DCM (10 mL) and diluted with distilled water (10 mL), NaHCO3 (0.52 g, 6.2 mmol, 5 equiv.) and BuN + HSO4 - (42 mg, 0.12 mmol, 0.1 equiv) was added. The reaction mixture was vigorously stirred at room temperature for 5 min, and chloromethyl chlorosulfate (0.15 mL, 1.47 mmol, 1.2 equiv) in DCM (5 mL) was added over 1 min. The resulting mixture was stirred at room temperature for 20 h. Further DCM (20 mL) was added, and the phases were separated. The aqueous phase was extracted with DCM (50 mL), and the combined organic fractions were washed with saturated NaCl (50 mL), dried over Na2SO4, and all volatiles were evaporated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH, 30:1) to give the desired product IS-100-055 (0.43 g, 58% yield) as a colorless oil. 1H NMR (400 MHz, CDCl): δ H 1.74-1.86 (m, 1H). 1.91-2.08 (m, 4H), 2.20-2.39 (m, 3H), 2.46-2.71 (m, 2H), 2.80-2.93 (m, 1H), 4.68 (s, 1H), 4.88-5.11 (m, 5H), 5.63 (d, J = 1.2 Hz, 2H), 7.28-7.39 (m, 15H). 31 P NMR (162 MHz, CDCl): δ P 56.23, 55.81. ESI MS: 623.2 ([M + Na] + ). HR ESI MS: C 31 H 34 Calculated for O8ClPNa: 623.15720; observed: 623.15742.

[0148] Dibenzyl 2-(((benzyloxy)(3-((((4R)-4-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methoxy)-3-oxopropyl)phosphoryl)methyl)pentanedioate (IS-100-056) [ka] Compound IS-100-055 (0.12 g, 0.20 mmol, 1 equiv.), deoxycholic acid (0.16 g, 0.40 mmol, 2 equiv.), and CsCO (77 mg, 0.22 mmol, 1.1 equiv.) were suspended in anhydrous DMF (10 mL). The resulting mixture was stirred at room temperature under an inert atmosphere for 16 h. DMF was evaporated, and the residue was dissolved in DCM (50 mL), washed with 1 M aqueous HCl (10 mL) and brine (10 mL), dried over NaSO, and the volatiles were evaporated. The crude product was purified by column chromatography (DCM / MeOH, 20:1) to give the desired compound IS-100-056 as a colorless foam (0.13 g, 68% yield). 1 H NMR (400 MHz, CDCl): δ H 0.65 (s, 3H), 0.88 (s, 3H), 0.94 (d, J = 6.0 Hz, 3H), 0.97-2.66 (m, 37H), 3.54-3.62 (m, 1H), 3.94 (t, J= 3.1 Hz, 1H), 4.88-5.08 (m, 6H), 5.66-5.71 (m, 2H), 7.26-7.36 (m, 15H). 31 P NMR (162 MHz, CDCl): δ P 56.47, 56.43, 56.06, 56.02. ESI MS: 979.5 ([M + Na] + ). HR ESI MS: C 55 H 74 O 12 Calculated value for P: 957.49124; Observed value: 957.49036.

[0149] 2-(((3-((((4R)-4-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methoxy)-3-oxopropyl)(hydroxy)phosphoryl)methyl)pentanedioic acid (12, IS-100-058) [ka] Compound IS-100-056 (0.12 g, 0.012 mmol, 1 equiv.) was dissolved in anhydrous THF (15 mL) and 10% palladium on carbon (30 mg) was added. The reaction flask was evacuated, filled with hydrogen, and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product 12 (66 mg, 77% yield) as a colorless solid. 1 H NMR (400 MHz, CD3OD): δ H 0.71 (s, 3H), 0.93 (s, 3H), 1.01 (s, 5H), 1.23-1.68 (m, 17H), 1.72-2.17 (m, 12H), 2.16-2.53 (m, 6H), 2.54-2.70 (m, 2H), 2.74-2.68 (m, 1H), 3.53 (s, 1H), 3.87-4.00 (m, 2H), 4.36 (t, J = 7.1 Hz, 1H), 5.71-5.80 (m, 2H). 31 P NMR (162 MHz, CD3OD): δ P 53.75, 53.08. ESI MS: 685.3 ([M-H] + ). HR ESI MS: C 34 H 54 O 12 Calculated value for P: 685.33584; Observed value: 685.33521.

[0150] [ka]

[0151] (4R)-4-((3R,10S,12S,13R,17R)-3-((3-((benzyloxy)(5-(benzyloxy)-2-((benzyloxy)carbonyl)-5-oxopentyl)phosphoryl))propanoyl)oxy)-12-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid (IS-100-030) [ka] Compound IS-100-025 (0.35 g, 0.63 mmol, 1 equiv.) was dissolved in acetonitrile (10 mL) and benzyl deoxycholate (0.32 g, 0.66 mmol, 1.05 equiv.), DCC (0.14 g, 0.66 mmol, 1.05 equiv.), and DMAP (7.7 mg, 0.06 mmol, 0.1 equiv.) were added. The reaction mixture was stirred at room temperature for 16 h. After completion, the DCU was filtered off and the volatiles were evaporated under reduced pressure. The residue was purified by column chromatography (DCM / MeOH, 20:1) to give the partially debenzylated product IS-100-030 (0.20 g, 31% yield) as a colorless solid. 1 H NMR (400 MHz, CDCl): δ H , 0.63 (s, 3H), 0.87 (s, 3H), 0.90-2.11 (m, 36H), 2.16-2.63 (m, 6H), 2.81-2.97 (m, 1H), 3.92-4.00 (m, 1H), 4.25-4.39 (m, 1H), 5.03-5.20 (m, 6H),7.27-7.38 (m, 15H). 31 P NMR (162 MHz, CDCl): δ P 57.22, 57.13, 56.87. ESI MS: 949.5 ([M + Na] + ). HR ESI MS: C 54 H 71 O 11 Calculated for NaP: 949.46262; Observed: 949.46295.

[0152] 2-(((3-(((3R,10S,12S,13R,17R)-17-((R)-4-carboxybutan-2-yl)-12-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta)[a]phenanthren-3-yl)oxy)-3-oxopropyl)(hydroxy)phosphoryl)methyl)pentanedioic acid (IS-100-032) [ka] Compound IS-100-030 (0.18 g, 0.19 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) and 10% palladium on carbon (30 mg) was added. The reaction flask was evacuated, filled with hydrogen (1 atm), and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product (0.13 g, 89% yield) as a white solid. 1 H NMR (400 MHz, CD3OD): δ H 0.72 (s, 3H), 0.94 (s, 3H), 1.01 (d, J = 6.4 Hz, 6H), 1.24-2.45 (m, 32H), 2.49-2.64 (m, 2H), 2.74-2.86 (m, 1H), 3.98 (s, 1H), 4.26-4.39 (m, 1H). 31 P NMR (162 MHz, CD3OD): δ P 58.29, 58.27, 57.97, 57.92. ESI MS: 655.4 ([M - H] + ). HR ESI MS: C 33 H 52 O 11 Calculated value for P: 655.32527; Observed value: 655.32451.

[0153] [ka]

[0154] (2R,4aS,6S,7R)-7-((S)-4-amino-2-methylbutyl)-4a,7-dimethyltetradecahydrophenanthrene-2,6-diol (IS-100-076) [ka] Compounds were prepared according to published procedures. 1 H and 13 The 1C NMR spectrum was consistent with published data (J. Med. Pharm. Chem. 1962, 5, 281-296).

[0155] Dibenzyl 2-(((benzyloxy)(3-(((3R)-3-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)butyl)amino)-3-oxopropyl)phosphoryl)methyl)pentanedioate (IS-100-080) [ka] Compound IS-100-026 (0.1 g, 0.26 mmol, 1 equiv.) was dissolved in DCM (3 mL), and HATU (0.1 g, 0.26 mmol, 1 equiv.) was added, followed by DIEA (0.14 mL, 0.78 mmol, 3 equiv.). The reaction mixture was stirred for 5 min, and compound IS-100-076 (0.1 g, 0.26 mmol, 1 equiv.) dissolved in DCM (1 mL) was added. The reaction mixture was stirred at room temperature for 16 h. Further DCM (20 mL) was added, and the organic layer was extracted with 1 M HCl (10 mL) and brine (10 mL). The organic portion was dried over Na2SO4, and the volatiles were evaporated under reduced pressure. The mixture was purified by column chromatography (DCM / MeOH, 20:1) to give the desired product IS-100-080 (0.11 g, 47% yield) as a colorless solid. 1 H NMR (400 MHz, CDCl): δ H0.65 (s, 3H), 0.73-2.18 (m, 34H), 2.18-2.52 (m, 5H), 2.74-2.90 (m, 1H), 3.05-3.34 (m, 2H), 3.48-4.14 (m, 5H), 4.85-5.13 (m, 6H), 6.38 (s, 1H), 7.18-7.44 (m, 15H). 31 P NMR (162 MHz, CDCl): δ P 60.14, 59.91. ESI MS: 920.5 ([M + Na] + ). HR ESI MS: C 53 H 72 Calculated for O9PNa: 920.48369; observed: 920.48320.

[0156] 2-(((3-(((3R)-3-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)butyl)amino)-3-oxopropyl)(hydroxy)phosphoryl)methyl)pentanedioic acid (14, IS-100-085) [ka] Compound IS-100-080 (0.1 g, 0.11 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) and 10% palladium on carbon (30 mg) was added. The reaction flask was evacuated, filled with hydrogen (1 atm), and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product 14 (42 mg, 59% yield) as a white solid. 1 H NMR (400 MHz, CD3OD): δ H0.72 (s, 3H), 0.83-2.09 (m, 36H), 2.12-2.63 (m, 6H), 2.80 (t, J = 8.7 Hz, 1H), 3.08-3.28 (m, 2H), 3.46-3.58 (m, 1H), 3.68-3.78 (m, 1H), 3.96 (t, J = 2.9 Hz, 1H). 31 P NMR (162 MHz, CD3OD): δ P 53.71. ESI MS: 626.3 ([M-H] + ). HR ESI MS: C 32 H 53 Calculated for O9NP: 626.34634; Observed: 626.34570.

[0157] [ka]

[0158] 1-Benzyl 5-(tert-butyl)(((S)-1-(benzyloxy)-4-methyl-1-oxopentan-2-yl)carbamoyl)-L-glutamate (AN-1-96) [ka] A solution of L-glutamic acid α-benzyl ester γ-tert-butyl ester (1.70 g, 5.80 mmol, 3 equiv.) and DIEA (10.0 mL, 58.0 mmol, 30 equiv.) in anhydrous DCM (80 mL) was added dropwise (over 2 h) to a solution of triphosgene (0.57 g, 1.93 mmol, 1 equiv.) in anhydrous DCM (100 mL) under a N atmosphere at −78 °C. After stirring for an additional 30 min at −78 °C, the cooling bath was removed, and a solution of L-leucine benzyl ester (1.28 g, 5.80 mmol, 3 equiv.) and DIEA (1.04 mL, 5.80 mmol, 30 equiv.) in anhydrous DCM (20 mL) was added to the mixture at room temperature. The resulting mixture was stirred overnight (16 h), then concentrated under reduced pressure, quenched with 1 M HCl, and extracted with ethyl acetate (200 mL). The organic portion was dried over NaSO and the volatiles were evaporated under reduced pressure. The mixture was purified by column chromatography (cyclohexane / EtOAc, 3:1) to give the desired product AN-1-96 (2.50 g, 80% yield) as a colorless solid. 1 H NMR (400 MHz, CDCl): δ H 0.89 (dd, J = 6.4, 4.3 Hz, 6H), 1.41 (s, 9H), 1.44-1.71 (m, 3H), 1.85-1.98 (m, 1H), 2.03-2.15 (m, 1H), 2.19-2.38 (m, 2H), 4.47-4.57 (m, 2H), 5.04 (d, J = 8.5 Hz, 1H), 5.09-5.20 (m, 4H), 5.27 (d, J = 8.1 Hz, 1H), 7.27-7.41 (m, 10H). 13 C NMR (101 MHz, CDCl): δ C22.1, 22.9, 24.9, 28.1, 28.2, 31.6, 42.2, 51.9, 52.8, 65.5, 67.1, 67.3, 80.8, 127.1, 127.8, 128.3, 128.3, 128.4, 128.5, 128.6, 128.7, 128.7, 135.4, 135.6, 156.8, 172.5, 173.0, 174.0. ESI MS: 541.3 ([M-H] + ). HR ESI MS: C 30 H 41 Calculated for O7N2: 541.29083; observed: 541.28990.

[0159] (S)-5-(benzyloxy)-4-(3-((S)-1-(benzyloxy)-4-methyl-1-oxopentan-2-yl)ureido)-5-oxopentanoic acid (IS-102-130) [ka] Compound AN-1-96 (2.50 g, 0.82 mmol, 1 equiv.) was dissolved in DCM (1 mL), trifluoroacetic acid (5 mL) was added, and the reaction mixture was stirred at room temperature for 2 h. The volatiles were evaporated under reduced pressure, and the residue was redissolved in DCM (20 mL) and evaporated to remove residual TFA (3 times). Crude compound IS-102-130 (2.15 g, 97% yield) was used in the next step without further purification. 1 H NMR (400 MHz, CDCl): δ H 0.78-0.99 (m, 6H), 1.45-1.79 (m, 3H), 1.93-2.13 (m, 1H), 2.18-2.81 (m, 3H), 4.45-4.67 (m, 2H), 5.09-5.29 (m, 4H), 7.28-7.47 (m, 10H). ESI MS: 483.2 ([M-H] + ). HR ESI MS: C 26 H 31Calculated for O7N2: 483.21367; observed: 483.21332.

[0160] 1-Benzyl 5-(chloromethyl)(((S)-1-(benzyloxy)-4-methyl-1-oxopentan-2-yl)carbamoyl)-L-glutamate (IS-102-131) [ka] Compound IS-102-130 (2.40 g, 4.95 mmol, 1 equiv.) was dissolved in DCM (75 mL) and diluted with distilled water (75 mL), NaHCO3 (2.08 g, 24.7 mmol, 5 equiv.) and BuN + HSO4 - (0.17 g, 0.49 mmol, 0.1 equiv) was added. The reaction mixture was vigorously stirred at room temperature for 5 min, and chloromethyl chlorosulfate (0.75 mL, 7.40 mmol, 1.5 equiv) in DCM (10 mL) was added over 1 min. The resulting mixture was stirred at room temperature for 20 h. Further DCM (50 mL) was added, and the phases were separated. The aqueous phase was extracted with DCM (3 × 50 mL), and the combined organic fractions were washed with saturated NaCl (50 mL), dried over NaSO, and all volatiles were evaporated under reduced pressure. The crude product was purified by column chromatography (cyclohexane / EtOAc, 3:1) to give the desired product IS-102-131 (1.92 g, 76% yield) as a colorless solid. 1 H NMR (400 MHz, CDCl): δ H 0.82-0.98 (m, 6H), 1.43-1.75 (m, 3H), 1.91-2.07 (m, 1H), 2.14-2.29 (m, 1H), 2.35-2.54 (m, 2H), 4.47-4.62 (m, 2H), 5.02-5.26 (m, 6H), 5.64 (d, J = 1.5 Hz, 2H), 7.29-7.45 (m, 10H). 13 C NMR (101 MHz, CDCl): δ C22.1, 22.9, 24.9, 27.9, 30.2, 42.1, 51.9, 52.5, 67.2, 67.6, 68.9, 128.3, 128.4, 128.4, 128.5, 128.7, 128.7, 128.8, 135.3, 135.6, 156.8, 171.2, 172.7, 174.1. ESI MS: 533.2 ([M-H] + ). HR ESI MS: C 27 H 34 Calculated for O7ClN2: 533.20491; Observed: 533.20478.

[0161] 1-Benzyl 5-((((4R)-4-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl))pentanoyl)oxy)methyl)(((S)-1-(benzyloxy)-4-methyl-1-oxopentan-2-yl)carbamoyl)-L-glutamate (IS-102-133) [ka] Compound IS-102-131 (1.90 g, 3.55 mmol, 1 equiv.), deoxycholic acid (3.49 g, 8.88 mmol, 2.5 equiv.), tetrabutylammonium iodide (35 mg, 0.1 mmol), and CsCO (1.16 g, 3.55 mmol, 1 equiv.) were suspended in anhydrous DMF (10 mL). The resulting mixture was stirred at 50 °C under an inert atmosphere for 2 h. DMF was evaporated, and the residue was dissolved in EtOAc (100 mL). It was washed with 1 M HCl (40 mL), saturated NaHCO (40 mL), and brine (40 mL), dried over NaSO, and the volatiles were evaporated. The crude product was purified by column chromatography (EtOAc / cyclohexane, 2:1) to give the desired compound IS-102-133 as a colorless solid (1.20 g, 38% yield). 1 H NMR (400 MHz, CDCl): δH 0.67 (s, 3H), 0.88-0.93 (m, 9H), 0.96 (d, J = 6.2 Hz, 3H), 1.01-1.98 (m, 26H), 2.14-2.52 (m, 5H), 3.61 (td, J = 10.9, 5.3 Hz, 1H), 3.96 (t, J = 3.0 Hz, 1H), 4.45-4.60 (m, 2H), 5.00-5.24 (m, 6H), 5.63-5.78 (m, 2H), 7.29-7.43 (m, 10H). 13 C NMR (101 MHz, CDCl): δ C 12.9, 17.5, 22.1, 23.0, 23.3, 23.8, 24.9, 26.3, 27.2, 27.6, 27.8, 28.8, 30.3, 30.6, 31.0, 33.8, 34.3, 35.2, 35.3, 36.2, 36.6, 42.1, 42.2, 46.7, 47.3, 48.4, 51.9, 52.6, 67.1, 67.5, 72.0, 73.3, 79.8, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8, 135.3, 135.6, 156.9, 161.0, 161.8, 171.8, 172.6, 173.3, 174.0. ESI MS: 889.5 ([M - H] + ). HR ESI MS: C 51 H 73 O 11 Calculated for N2: 889.52089; Observed: 889.52073.

[0162] (2S,6S,16R)-6-Carboxy-16-((3R,10S,12S,13R,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-isobutyl-4,9,13-trioxo-10,12-dioxa-3,5-diazaheptadecanoic acid (15, IS-102-138) [ka] Compound IS-102-133 (0.89 g, 0.1 mmol, 1 equiv.) was dissolved in anhydrous THF (20 mL) and 10% palladium on carbon (50 mg) was added. The reaction flask was evacuated, filled with hydrogen (1 atm), and stirred at room temperature for 16 hours. The palladium on carbon was removed by filtration through Celite™, and the volatiles were evaporated under reduced pressure to give the desired product 15 (0.62 g, 87% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d): δ H 0.59 (s, 3H), 0.78-0.94 (m, 12H), 0.96-1.87 (m, 31H), 1.90-2.02 (m, 1H), 2.19-2.45 (m, 4H), 3.78 (s, 1H), 4.03-4.15 (m, 2H), 4.20 (bs, 1H), 4.45 (bs, 1H), 5.66 (q, J = 5.9 Hz, 2H), 6.27 (dd, J= 16.8, 8.4 Hz, 2H). 13 C NMR (101 MHz, DMSO-d): δ C 12.4, 16.8, 21.7, 22.8, 23.1, 23.5, 24.3, 26.1, 27.0, 27.2, 27.2, 28.6, 29.7, 30.2, 30.3, 30.4, 32.9, 33.8, 34.9, 35.2, 35.6, 36.3, 41.1, 41.6, 46.0, 46.1, 47.5, 50.9, 51.5, 69.9, 71.0, 79.0, 157.2, 171.2, 172.3, 173.9, 175.0. ESI MS: 731.4 ([M + Na] + ). HR ESI MS: C 37 H 60 O 11 Calculated for N2Na: 731.40893; Observed: 731.40915.

[0163] [ka]

[0164] tert-Butyl 4-((5-(3-(allyloxy)-3-oxopropyl)-2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)methyl)benzoate (TT-110320) [ka] A mixture of starting material LTP487 (6.28 g, 24.5 mmol, 1 equiv.), benzyltriethylammonium chloride (5.58 g, 24.5 mmol, 1 equiv.), and KCO (3.39 g, 24.5 mmol, 1 equiv.) in anhydrous MeCN (100 mL) was heated to 70 °C under inert atmosphere for 20 min. Next, a solution of tert-butyl 4-(bromomethyl)benzoate (8.00 g, 29.5 mmol, 1.2 equiv.) in anhydrous MeCN (100 mL) was added, and the mixture was stirred at 70 °C overnight (20 h). The solvent was evaporated, EtOAc (200 mL) was added, and the organic phase was washed with aqueous KHSO (200 mL), dried over MgSO, and concentrated. The residue was purified on a silica gel column in 20–25% acetone / hexane, followed by PR flash chromatography on a 415 g C18 Aq column with a 50–100% MeCN / HO gradient (20 min, 140 mL / min). The product, TT-110320, was isolated as a colorless solid in 71% yield (7.75 g). 1 H NMR (400 MHz, CDCl): δ H 0.74 (s, 3H), 1.53 (s, 9H), 1.58 (s, 3H), 2.37-2.44 (m, 2H), 2.44-2.51 (m, 2H), 3.34 (s, 2H), 4.55 (dm, J = 5.7 Hz, 2H), 5.21 (dm, J = 10.4 Hz, 1H), 5.29 (dm, J = 17.1 Hz, 1H), 5.81-5.93 (m, 1H), 7.20 (d, J = 8.0 Hz, 2H), 7.85 (d, J= 8.0 Hz, 2H). 13 C NMR (101 MHz, CDCl): δ C 28.22, 29.27, 29.33, 29.92, 35.29, 43.18, 56.03, 65.74, 81.31, 106.16, 118.87, 129.94, 130.43, 131.65, 131.86, 139.55, 165.29, 168.21, 170.87. ESI MS: 469.2 ([M + Na] + ). HR ESI MS: C 24 H 30 Calculated for O8Na: 469.18329; observed: 469.18320.

[0165] 5-Allyl 1-benzyl 2-(4-(tert-butoxycarbonyl)benzyl)pentanedioate (TT-250320) [ka] A solution of TT-110320 (7.75 g, 17.4 mmol, 1 equiv.) in anhydrous BnOH (45 ml) was heated to 140 °C under inert conditions for 24 h. The mixture was concentrated at 105-110 °C, and the residue was purified using RP flash chromatography on a 415 g C18 Aq column with a 50-100% MeCN / HO gradient (20 min, 150 ml / min). The product, TT-250320, was isolated as a syrup in 50% yield (3.96 g). 1 H NMR (400 MHz, CDCl): δ H1.53 (s, 9H), 1.85-2.03 (m, 2H), 2.27-2.43 (m, 2H), 2.75-2.87 (m, 2H), 2.95-3.03 (m, 1H), 4.55 (dt, J = 5.8, 1.4 Hz, 2H), 5.00 (d, J = 12.2 Hz, 1H), 5.06 (d, J = 12.2 Hz, 1H), 5.22 (dq, J = 10.4, 1.3 Hz, 1H), 5.29 (dq, J = 17.2, 1.5 Hz, 1H), 5.88 (ddt, J = 17.2, 10.4, 5.8Hz, 1H), 7.13-7.19 (m, 4H), 7.28-7.32 (m, 3H), 7.85 (dm, J = 8.4 Hz, 2H). 13 C NMR (101 MHz, CDCl): δ C 27.12, 28.31, 31.81, 38.46, 46.55, 65.29, 66.46, 80.92, 118.40, 128.32 (2C), 128.58, 128.83, 129.72, 130.43, 132.16, 135.68, 143.55, 165.72, 172. 39, 174.40. ESI MS: 475.2 ([M + Na] + ). HR ESI MS: C 27 H 32 Calculated for O6Na: 475.20911; Observed: 475.20880.

[0166] 5-(Benzyloxy)-4-(4-(tert-butoxycarbonyl)benzyl)-5-oxopentanoic acid (TT-270320) [ka] A solution of TT-250320 (3.93 g, 8.69 mmol, 1 equiv.) and dimedone (1.46 g, 10.42 mmol, 1.2 equiv.) in anhydrous THF (120 ml) was treated with a solution of Pd(PPh3)4 (400 mg, 0.35 mmol, 0.04 equiv.) in THF (40 ml). The mixture was stirred at room temperature for 90 min. The solvent was evaporated, and the residue was purified using RP flash chromatography on a 415 g C18 Aq column with a 25–100% MeCN / HO + 0.1% HCOOH gradient (30 min, 150 ml / min). The product, TT-270320, was isolated as a colorless oil in 83% yield (2.98 g). 1 H NMR (400 MHz, CDCl): δ H 1.60 (s, 9H), 1.83-2.03 (m, 2H), 2.28-2.46 (m, 2H), 2.76-2.88 (m, 2H), 2.95-3.04 (m, 1H), 5.00 (d, J = 12.2 Hz, 1H), 5.07 (d, J = 12.2 Hz, 1H), 7.13-7.20 (m, 4H), 7.28-7.32 (m, 3H), 7.85 (dm, J = 8.2 Hz, 2H). 13 C NMR (101 MHz, CDCl): δ C 26.81, 28.34, 31.61, 38.48, 46.48, 66.58, 81.02, 128.40 (2C), 128.63, 128.86, 129.78, 130.49, 135.63, 143.46, 165.79, 174.37, 178.54. ESI MS: 435 ([M + Na] + ). HR ESI MS: C 27 H 32 Calculated for O6Na: 435.17781; Observed: 435.17790.

[0167] tert-Butyl 4-(5-((benzyloxy)amino)-2-((benzyloxy)carbonyl)-5-oxopentyl)benzoate (TT-300320) [ka] A solution of TT-270320 (2.94 g, 7.13 mmol, 1 equiv.) in anhydrous DMF (15 mL) was cooled to 0 °C and then treated with DIEA (5.0 mL, 28.5 mmol, 4 equiv.), followed by a solution of HATU (2.98 g, 7.84 mmol, 1.1 equiv.) in DMF (10 mL). The resulting mixture was stirred at 0 °C for 10 min and then treated with a solution of O-benzylhydroxylamine hydrochloride (1.25 g, 7.84 mmol, 1.1 equiv.) and DIEA (1.4 mL, 7.84 mmol, 1.1 equiv.) in DMF (10 mL). The reaction mixture was stirred at room temperature for 3 h, then concentrated and purified using RP flash chromatography on a 415 g C18 Alkyl column with a 30–100% MeCN / HO gradient (30 min, 150 mL / min). The product TT-300320 was obtained as a colorless solid in 97% yield (3.58 g). 1 H NMR (400 MHz, CDCl): δ H 1.59 (s, 9H), 1.82-2.47 (m, 4H), 2.69-2.85 (m, 2H), 2.96 (dd, J = 13.3, 8.6 Hz, 1H), 4.62-4.91 (m, 2H), 4.93-5.05 (m, 2H), 7.10-7.19 (m, 4H), 7.25-7.30 (m, 3H), 7.35 (bs, 5H), 7.84 (dm, J = 8.2 Hz, 2H). 13 C NMR (101 MHz, CDCl): δ C27.77, 28.32, 30.60, 38.44, 46.57, 66.43, 78.24, 80.96, 128.44, 128.53, 128.58, 128.63, 128.74, 128.81, 129.30, 129.73, 130.43, 135.36, 135.71, 143.48, 165.75, 169.74, 174.43. ESI MS: 540.2 ([M + Na] + ). HR ESI MS: C 31 H 35 Calculated value for O6NNa: 540.23566; observed value: 540.23572.

[0168] 4-(5-((benzyloxy)amino)-2-((benzyloxy)carbonyl)-5-oxopentyl)benzoic acid (TT-010420) [ka] A solution of TT-300320 (3.52 g, 6.80 mmol, 1 equiv.) and iPrSiH (1.4 ml, 6.80 mmol, 1 equiv.) in anhydrous CHCl (40 ml) was cooled to 0 °C and treated with TFA (20 ml). The resulting mixture was stirred at room temperature for 4 h, and the volatiles were evaporated. After the addition of a small amount of MeCN and sonication, a syrupy residue crystallized. The resulting suspension was diluted with EtO and hexane. The crystals were filtered off and washed with EtO-hexane. The product TT-010420 was obtained as a colorless solid in 92% yield (2.90 g). 1 H NMR (400 MHz, CDCl): δ H 1.84-2.46 (m, 4H), 2.71-2.88 (m, 2H), 3.00 (dd, J = 13.3, 8.6 Hz, 1H), 4.65-4.92 (m, 2H), 4.93-5.09 (m, 2H), 7.14-7.22 (m, 4H), 7.26-7.31 (m, 3H), 7.36 (bs, 5H), 7.95 (dm, J = 8.2 Hz, 2H). 13 C NMR (101 MHz, CDCl): δ C 27.91, 30.66, 38.55, 46.49, 66.57, 78.33, 128.40-128.87 (m, 6C), 129.13, 129.34, 130.51, 135.30, 135.65, 144.89, 169.93, 171.38, 174.45. ESI MS: 484.2 ([M + Na] + ). HR ESI MS: C 27 H 27 Calculated value for O6NNa: 484.17306; observed value: 484.17268.

[0169] Chloromethyl (R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate (TT-170420) [ka] Deoxycholic acid (5.50 g, 14.0 mmol, 1 equiv.) and NaHCO3 (5.88 g, 70.0 mmol, 5 equiv.) were suspended in water (100 mL), followed by the addition of Bu4NHSO4 (475 mg, 1.4 mmol, 0.1 equiv.) and CHCl2 (100 mL). The resulting mixture was stirred and cooled to 0 °C. A solution of chloromethyl chlorosulfonate (2.55 mL, 25.2 mmol, 1.8 equiv.) in CHCl2 (10 mL) was added slowly, and the mixture was stirred at room temperature overnight (20 h). The organic portion was separated, and the aqueous phase was extracted with CHCl3 (2 × 100 mL). The combined organic portions were dried (MgSO4) and concentrated. The syrupy residue was chromatographed on a silica gel column in EtOAc. Compound TT-170420 was obtained as an amorphous foam in 89% yield (5.48 g). 1 H NMR (400 MHz, CDCl): δ H0.67 (s, 3H), 0.90 (s, 3H), 0.97 (d, J = 4.9 Hz, 3H), 0.99-1.89 (m, 22H), 2.25-2.48 (m, 4H), 3.56-3.67 (m, 1H), 3.95-3.99 (m, 1H), 5.66-5.72 (m, 2H). 13 C NMR (101 MHz, CDCl): δ C 12.85, 17.37, 23.25, 23.78, 26.24, 27.24, 27.59, 28.80, 30.48, 30.61, 31.16, 33.75, 34.24, 35.13, 35.32, 36.13, 36.42, 42.17, 46.62, 47.33, 48.36, 68.73, 71.99, 73.31, 172.30. ESI MS: 463.3 ([M + Na] + ). HR ESI MS: C 25 H 41 Calculated for O4ClNa: 463.25856; Observed: 463.25840.

[0170] (((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methyl 4-(5-((benzyloxy)amino)-2-((benzyloxy)carbonyl)-5-oxopentyl)benzoate (TT-200420) [ka] A mixture of hydroxamate TT-010420 (2.86 g, 6.20 mmol, 1 equiv.) and CsCO (2.02 g, 6.20 mmol, 1 equiv.) in anhydrous DMF (12 mL) was stirred and sonicated (10 min). Tetraethylammonium iodide (2.55 g, 9.92 mmol, 1.6 equiv.) was added, followed by the starting material TT-170420 (4.37 g, 9.92 mmol, 1.6 equiv.). The resulting mixture was stirred at room temperature for 4 h and filtered through Celite. The concentrate was purified using RP flash chromatography on a 415 g C18 Aq column with a 60–100% MeCN / HO gradient (25 min, 150 mL / min). Compound TT-200420 was obtained as a colorless syrup in 84% yield (4.50 g). 1 H NMR (400 MHz, CDCl): δ H 0.63 (s, 3H), 0.88 (s, 3H), 0.94 (d, J = 6.0 Hz, 3H), 0.96-1.96 (m, 27H), 2.24-2.48 (m, 3H), 2.69-2.86 (m, 2H), 2.96 (dd, J = 13.5, 8.8 Hz, 1H), 3.57 (tt, J = 10.8, 4.6 Hz, 1H), 3.90-3.95 (m, 1H), 4.63-4.89 (m, 2H), 4.94-5.04 (m, 2H), 5.94-5.99 (m, 2H), 7.13-7.19 (m, 4H), 7.24-7.29 (m, 3H), 7.35 (bs, 5H), 7.90 (d, J = 8.2 Hz, 2H). 13 C NMR (101 MHz, CDCl): δ C12.79, 17.36, 23.22, 23.71, 26.21, 27.20, 27.51, 27.81, 28.73, 30.45, 30.62 (2C), 31.14, 33.68, 34.18, 35.14, 35.30, 36.07, 36.41, 38.46, 42.13, 46.49, 46.55, 47.29, 48.27, 66.50, 71.85, 73.16, 78.18, 79.59, 128.36-128.81 (m, 6C), 129.13, 129.30, 130.32, 135.44, 135.62, 145.00, 165.16, 169.66, 173.04, 174.36. ESI MS: 888.5 ([M + Na] + ). HR ESI MS: C 52 H 67 O 10 Calculated for NNa: 888.46572; Observed: 888.46597.

[0171] 2-(4-(((((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoyl)oxy)methoxy)carbonyl)benzyl)-5-(hydroxyamino)-5-oxopentanoic acid (16, TT-220420) [ka] To a solution of starting material TT-200420 (4.42 g, 5.10 mmol, 1 equiv.) in MeOH (250 ml), Pd / C (5%, 150 mg) was added, and the mixture was stirred at room temperature under an atmosphere of H for 24 h. The catalyst was removed by filtration, and the filtrate was concentrated. The syrupy residue was chromatographed using RP flash chromatography on a 415 g C18 Aq column with a 20–100% MeCN / HO + 0.1% HCOOH gradient (30 min, 150 ml / min). The final product 16 was isolated as a colorless lyophilizate in 91% yield (3.18 g). 1 H NMR (400 MHz, CD3OD): δ H 0.62 (d, J = 2.0 Hz, 3H), 0.92 (s, 3H), 0.97 (d, J = 6.2 Hz, 3H), 1.07-1.64 (m, 16H), 1.71-1.95 (m, 10 H), 2.08-2.25 (m, 2H), 2.29-2.38 (m, 1H), 2.40-2.49 (m, 1H), 2.67-2.75 (m, 1H), 2.88 (dd, J = 13.7, 6.1 Hz, 1H), 3.01 (dd, J = 13.7, 8.6 Hz, 1H), 3.52 (tt, J = 11.2, 4.6 Hz, 1H), 3.89-3.93 (m, 1H), 5.96 (d, J = 12.5 Hz, 1H), 5.97 (d, J = 12.5 Hz, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.95 (dm, J = 8.3 Hz, 2H). 13 C NMR (101 MHz, CD3OD): δ C 13.24, 17.51, 23.77, 24.84, 27.45, 28.41, 28.63, 28.95, 29.87, 31.05, 31.40, 31.90, 31.96, 34.79, 35.29, 36.44, 36.52, 37.18, 37.40, 39.11, 43.60, 47.54, 47.76, 47.77, 48.13, 72.54, 73.99, 80.76, 128.52, 130.44, 131.02, 147.26, 166.34, 171.95, 174.30, 177.97. ESI MS: 708.4 ([M + Na] + ). HR ESI MS: C 38 H 55 O 10 Calculated value of NNaについての: 708.37182; Viewed value: 708.37158.

[0172] Chiral separation of compound LTP576 and synthesis of enantiomers 2a and 2b [ka]

[0173] Example 2: Dextran Sodium Sulfate (DSS)-Induced Colitis 2.1 Experimental Method Male C57Bl / 6NHsd mice, 6–8 weeks old, were obtained from Harlan Laboratories / Envigo (Indianapolis, IN). Animals were housed in the Johns Hopkins Animal Facility under a controlled temperature (25°C) and photoperiod (12-hour light / 12-hour dark cycle) with free access to standard chow and water (or drinking water containing DSS as specified). All animal studies were approved by the Animal Care and Use Committee of Johns Hopkins University, Baltimore, MD, and complied with all applicable institutional and government guidelines for the humane treatment of laboratory animals. Mice were housed 4–5 per cage and allowed to acclimate for at least 5 days before inclusion in experiments. Mice (n = 5–30 / group) were randomized by weight into equivalent experimental groups: (a) DSS containing vehicle or (b) DSS containing the test substance. Acute colitis was induced by administration of 2.5%–4.0% (w / v) DSS (MW 40,000–50,000; Affymetrix Santa Clara, CA) in drinking water for 5 days, followed by exposure to fresh water for 2 days. Daily oral (PO) administration of vehicle or test substance began on study day 0 and continued until sacrifice on study day 7. The disease activity index (DAI) was calculated daily using the following validated scoring system: (a) weight loss (0 = none; 1 = 1%–5% loss; 2 = 5%–10% loss; 3 = 10%–15% loss; 4 = >15% loss); (b) stool consistency (0 = normal; 1 = slightly soft; 2 = soft but still formed; 3 = soft, unformed; 4 = watery diarrhea); and (c) bleeding as assessed by the ColoScreen occult blood test (Helena Laboratories Product #5076) (0 = negative; 1 = blood test +; 2 = blood test ++; 3 = visible blood stains in the stool; 4 = rectal bleeding). All data are expressed as mean + / - standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 8.0. DAIs were compared using two-way analysis of variance (2-way ANOVA). A P value of less than 0.05 was considered statistically significant.

[0174] <2.2. Results> Test substances evaluated in the DSS colitis model included LTP582 (#1), LTP592 (#2), LTP588 (#3), LTP1054 (#4), IS-101-010 (#8), IS-101-020 (#9), and IS-101-077 (#10). The concentrations evaluated were based on the known efficacy of the parent compound 2-PMPA in this model system and the solubility of the test substances; the final concentrations tested ranged from 10 mg / kg to 100 mg / kg of 2-PMPA molar equivalent doses. The test substances LTP592 (#2; Figure 1B, Figure 1D), IS-101-020 (#9; Figure 1G), and IS-101-077 (#10; Figure 1H) demonstrated selective anti-IBD effects in DSS colitis, demonstrating significant reductions in DAI on days 6 and 7 of the study. Test substances LTP582 (#1; Fig. 1A), LTP588 (#3; Fig. 1C), LTP1054 (#4; Fig. 1E), and IS-101-010 (#8; Fig. 1F) were inactive in this model.

[0175] 2.3. GCPII enzyme activity in mouse colon tissue On day 6 of the DSS-induced colitis study, 4 hours after the final administration of vehicle or compound, mice were euthanized and 5 mm long, matched proximal colon segments were harvested and snap-frozen. Glutamate carboxypeptidase activity was measured using an optimized radioenzymatic assay as previously described (Rojas et al., 2002; Robinson et al., 1987).

[0176] 2.4. Isolation of colonic lamina propria mononuclear cells (LPMCs) and flow cytometry On day 5 of the DSS study, a subset of mice (n = 8–9 / group) was euthanized and their colons processed for flow cytometry. Specifically, the distal 3 cm of the colon was isolated, the mesenteric fat was removed, and the intestinal contents were removed. The colon was then cut longitudinally, washed with PBS, and thoroughly minced. The minced colon tissue was transferred to 5 ml of HBSS containing 10 mM HEPES containing 5% FBS and 0.2 mg / ml collagenase D (Sigma). The tissue was digested at 37°C for 20 min with constant agitation (250 rpm) and vigorously vortexed for 20 s. The resulting cell suspension containing LPMCs was filtered through a 70 μm nylon cell strainer and washed with 10 ml of PBS. The LPMC cell suspension was pelleted by centrifugation at 500g for 10 minutes at 4°C, then resuspended in 8 ml of 44% Percoll (GE Healthcare, #57-0891-01) and layered on 5 ml of 67% Percoll. The gradient was centrifuged at 500g for 20 minutes at 4°C, and the cells at the interface were collected and washed with 10 ml of PBS. The cells were transferred to a 1.5 ml low-bind tube and stained for flow cytometry analysis. Immune cells, including total leukocytes (CD11b+), neutrophils (CD11b+Ly6G+), monocytes (CD45+CD11b+Ly6ChiMHCII-CX3CR1lo), and transitional monocytes (CD11b+Ly6ChiMHCII+), were identified using previously reported markers (Nedelcovych et al., 2019; Baldwin et al., 2015).

[0177] Example 3: Pharmacokinetic evaluation 3.1. Pharmacokinetic evaluation in beagle dogs The pharmacokinetic study in dogs was conducted in accordance with the guidelines recommended in the Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee at Charles River Labs (Wilmington, MA). LTP-592 was administered orally at three dose levels: 1.67, 5.00, and 16.67 mg / kg. Additionally, LTP-592 (5 mg / kg) was also administered via the intravenous (IV) route for bioavailability measurements. Blood samples (approximately 1 mL) were collected from the jugular vein via direct venipuncture at specific time points (0.083–24 h) post-dose and placed into sodium heparin tubes. Blood samples were centrifuged at 2000 g for 15 min at 4°C. Plasma samples were collected in tubes and stored at -80°C until bioanalysis.

[0178] 3.2. Pharmacokinetic evaluation in mice All pharmacokinetic studies in mice were conducted in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee. Male CD-1 mice weighing 25–30 g were obtained from Harlan and housed under a 12-h light / dark cycle with free access to food and water. In vivo pharmacokinetic evaluation of LTP-592 was performed after oral and intravenous (IV) administration at three dose levels: 10, 30, and 100 mg / kg. Mice were sacrificed at specific time points (0.083–24 h) after drug administration. For plasma and colon collection, animals were euthanized with CO2, and blood samples were collected by cardiac puncture into heparinized microtubes. Colon samples were dissected and immediately flash-frozen (-80°C). Blood samples were centrifuged at 2000 g for 15 minutes, and plasma was removed and stored at -80°C until LC-MS / MS analysis.

[0179] <3.3.Results> The concentration-time profiles of LTP-592 at various doses in plasma are shown in Figures 6A and 6B, respectively. After intravenous (IV) administration, LTP-592 increased 5 minutes after administration (T max) with a peak plasma concentration (C max ) and total exposure (AUC 0-t ) was 9.9 h*nmol / mL. All dogs showed low levels of intact LTP-592 after oral (PO) dosing, with absolute oral bioavailability below 1% (≦1%) (FIG. 6).

[0180] The concentration-time profile of intact LTP-592 and its respective pharmacokinetic parameters in mouse plasma and colon are shown in Figures 7A, 7B, and 7C. In mice, after intravenous (IV) administration, LTP-592 increased 5 minutes after administration (T max ) with a peak plasma concentration (C max ) and total exposure (AUC 0-t ) was 0.53 h*nmol / mL. At all dose levels except 100 mg / kg, intact LTP-592 levels in plasma were below the quantification limit of 30 nM (Figure 7A). In contrast, high LTP-592 exposure (20.5-80.8 h*nmol / mL; Figures 7B and 7C) was observed in the colon after oral administration, suggesting limited gastrointestinal delivery. Thus, in both mice and dogs, the oral bioavailability of LTP-592 was low, confirming limited intestinal delivery of LTP-592 after oral administration, which is expected for clinical applications.

[0181] Example 4: Off-target safety screen of LTP-592 Eurofins SafetyScreen44™ was performed to assess off-target interactions of LTP-592 (10 μM). LTP-592 did not exhibit significant interactions with any of the targets in (Table 1).

[0182] [Table 3] References

[0183] All publications, patent applications, patents, and other references mentioned in this specification are indicative of the level of ordinary skill in the art to which the subject matter of this disclosure pertains. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. Although numerous patent applications, patents, and other references are mentioned herein, it should be understood that such references do not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0184] Rais, R.; Jiang, W.; Zhai, H.; Wozniak, KM; Stathis, M.; Hollinger, KR; Thomas, AG; Rojas, C.; Vornov, JJ; Marohn, M.; (12). Sipka, S.; Bruckner, G., The immunomodulatory role of bile acids. Int Arch Allergy Immunol 2014, 165 (1), pp. 1-8. Calmus, Y.; Poupon, R., Shaping macrophages function and innate immunity by bile acids: mechanisms and implication in cholestatic liver diseases. Clin Res Hepatol Gastroenterol2014, 38 (5), pp. 550-6. Columbus, JF; Sandborn , WJ ; Rutgeerts , P. ; Enns , R. ; Hanauer , SB ; Panaccione, R.; Schreiber , S. ; Byczkowski , D. ; Li, J.; Kent , JD ; Pollack, PF, Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial. Gastroenterology2007,132:52–65. Hamilton , MJ ; Snapper , SB ; Blumberg, RS, Update on biologic pathways in inflammatory bowel disease and their therapeutic relevance. J. Gastroenterol. 2012, 47: 1-8 Hanauer , SB ; Feagan , BG ; Lichtenstein , GR ; Mayer , LF ; Schreiber , S. ; Columbus, JF; Rachmilewitz , D. ; Wolf, DC; Olson , A. ; Bao, W.; Rutgeerts, P., Maintenance infliximab for Crohn's disease: the ACCENT I randomized trial. Lancet 2002, 359:1541–1549. Ho, PP; Steinman, L., Obeticholic acid, a synthetic bile acid agonist of the farnesoid X receptor, attenuates experimental autoimmune encephalomyelitis. Proc Natl Acad Sci USA 2016, 113(6), 1600-5. Kaser, A.; Zeissig , S. ; Blumberg, RS, Inflammatory bowel disease. Annu. Rev. Fr. Immunol. 2010, 28:573-6 Kozuch , PL and Hanauer , SB , Treatment of inflammatory bowel disease: A review of medical therapy . World J. Gastroenterol. 2008, 14:354-3 Laukens , D. ; Devisscher , L. ; Van den Bossche, L.; Hindryckx , P. ; Vandenbroucke, RE; Vandewynckel, YP; Cuvelier , C. ; Brinkman , BM ; Libert , C. ; Vandenabeele, P.; De Vos, M., Tauroursodeoxycholic acid inhibits experimental colitis by preventing early intestinal epithelial cell death. Lab Invest 2014, 94(12), 1419–30. Lawrance, IC What is left when anti-tumour necrosis factor therapy fails in inflammatory bowel diseases? World J. Gastroenterol. 2014, 20: 1248-125. Regueiro , M. ; Siemanowski , B. ; Kip, KE; Plevy, S., Infliximab dose intensification in Crohn's disease. Inflamm. Bowel Dis. 2007, 13: 1093–1099. Sartor, R.B., Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat. Clin. Pract. Gastroenterol. Hepatol. 2006, 3: 390 - 407. Schmidt, C; Giese, T.; Hermann, E.; Zeuzem, S.; Meuer, S.C.; Stallmach, A., Predictive value of mucosal TNF-alpha transcripts in steroid-refractory Crohn’s disease patients receiving intensive immunosuppressive therapy. Inflamm. Bowel Dis. 2007, 13: 65 - 70. Schreiber, S.; Khaliq-Kareemi, M.; Lawrance, I.C.; Thomsen, O.O.; Hanauer, S.B.; McColm, J.; Bloomfield, R.; Sandborn, W.J., Maintenance therapy with certolizumab pegol for Crohn’s disease. N. Engl. J. Med. 2007, 357: 239 - 250. Strober, W.; Fuss, I.; and Mannon, P., The fundamental basis of inflammatory bowel disease. J. Clin. Invest. 2007, 117: 514 - 521. Xavier, R.J. and Podolsky, D.K., Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007, 448: 427 - 434. Van, A.G.; Van, R.M.; Sciot, R.; Dubois, B.; Vermeire, S.; Noman, M.; Verbeeck, J.; Geboes, K.; Robberecht, W.; Rutgeerts, P., Progressive multifocal leukoencephalopathy after natalizumab therapy for Crohn’s disease. N. Engl. J. Med. 2005, 353: 362-368. Rojas, C.; Frazier, S. T.; Flanary, J.; Slusher, B. S., Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay. Anal Biochem 2002, 310 (1), 50-54. Robinson, M. B.; Blakely, R. D.; Couto, R.; Coyle, J. T., Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain. J Biol Chem 1987, 262 (30), 14498-14506. Kim, BH; Zhu, X.; Lovell, LE; Manning, AA; Kelschenbach, J.; Hadas, E.; Chao, W.; Prchalova, E.; Dash, RP; Wu, Y.; Alt, J.; Thomas, AG; Rais, R.; Kamiya, A.; Volsky, DJ; Slusher, BS, Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 2019. Baldwin, KT; Carbajal, KS; Segal, BM; Giger, RJ, Neuroinflammation triggered by beta-glucan / dectin-1 signaling enables CNS axon regeneration. Proceedings of the National Academy of Sciences of the United States of America 2015, 112 (8), pp. 2581-6.

[0185] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will understand that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. Compounds of formula (I): 【Chemistry 1】 (In the formula: R １ and R ２ are each independently H or —OH; and R ３ is OH, and R ４ Ha-NH-X １ , -COO-X １ , -C(=O)-NH-CH ２ -C(=O)-O-X １ and —C(═O)—NH—CH ２ -CH ２ -S(=O) ２ -O-X １ wherein X is selected from the group consisting of １ is -(C=O)-(CH ２ ) ｍ -P(=O)(OH)-X ２ , -(C=O)-(CH ２ ) ｍ -CH(COOH)-CH ２ -P(=O)(OH)-X ２ , -CH ２ -O-(C=O)-(CH ２ ) ｍ -P(=O)(OH)-X ２ , -CH ２ -OC(=O)-(CH ２ ) ｍ -CH(COOH)-CH ２ -P(=O)(OH)-X ２ , -CH ２ -OC(=O)-(CH ２ ) ｍ -CH(COOH)-NH-(C=O)-NH-CH(COOH)-CH ２ -CH(CH ３ ) ２ , -CH ２ -OC(=O)-Ar-CH ２ -CH(COOH)-(CH ２ ) ｍ -C(=O)-NH-OH, -CH ２ -OC(=O)-(CH ２ ) ｍ -X ３ , and -CH ２ -OC(=O)-Ar-CH ２ -X 3 , where X ２ is -OH and -CH ２ -CH(COOH)-(CH ２ ) ｐ -C(=O)-OH, Ar is arylene, and X ３ is 2-oxotetrahydro-2H-thiopyran-3-yl, and each m and p is independently selected from the group consisting of 1, 2, 3, and 4; or R ３ is -OC(=O)-O-CH ２ -OC(=O)-(CH ２ ) ｎ -CH(COOH)-CH ２ -P(=O)(OH) ２ and —O—C(═O)—CH ２ -CH ２ -P(=O)(OH)-CH ２ -CH(COOH)-(CH ２ ) ｎ —C(═O)—OH, where each n is independently an integer selected from the group consisting of 1, 2, 3, and 4; R ４ Ha-NH ２ , -COOH, -C(=O)-NH-CH ２ —C(═O)—OH, and —C(═O)—NH—CH ２ -CH ２ -S(=O) ２ —OH) and pharmaceutically acceptable salts thereof.

2. (a) R １ and R ２ are both H; (b) R １ is H and R ２ is OH; (c) R １ is OH, and R ２ is H; or (d) R １ and R ２ and are both OH.

3. R ３ is OH; and R ４ Ha-NH-X １ , -COO-X １ , -C(=O)-NH-CH ２ -C(=O)-O-X １ and —C(═O)—NH—CH ２ -CH ２ -S(=O) ２ -O-X １ wherein X is selected from the group consisting of １ is -(C=O)-CH ２ -CH ２ -P(=O)(OH)-X ２ , -(C=O)-CH ２ -CH ２ -CH(COOH)-CH ２ -P(=O)(OH)-X ２ , -CH ２ -O-(C=O)-CH ２ -CH ２ -P(=O)(OH)-X ２ , -CH ２ -OC(=O)-CH ２ -CH ２ -CH(COOH)-CH ２ -P(=O)(OH)-X ２ , -CH ２ -OC(=O)-CH ２ -CH ２ -CH(COOH)-NH-(C=O)-NH-CH(COOH)-CH ２ -CH(CH ３ ) ２ , -CH ２ -OC(=O)-Ar-CH ２ -CH(COOH)-CH ２ CH ２ -C(=O)-NH-OH, -CH ２ -OC(=O)-CH ２ -CH ２ -X ３ , and -CH ２ -OC(=O)-Ar-CH ２ -X 3 ; Here, X ２ is -OH and -CH ２ -CH(COOH)-CH ２ -CH ２ Ar is selected from the group consisting of —CH(═O)—OH, and X ３ The compound of claim 1, wherein is 2-oxotetrahydro-2H-thiopyran-3-yl.

4. The compound of formula (I) is as follows: 【Chemistry 2-1】 【Chemistry 2-2】 [Chemistry 2-3] 4. The compound of claim 3 selected from the group consisting of:

5. R ３ is -OC(=O)-O-CH ２ -OC(=O)-CH ２ -CH ２ -CH(COOH)-CH ２ -P(=O)(OH) ２ and —O—C(═O)—CH ２ -CH ２ -P(=O)(OH)-CH ２ -CH(COOH)-CH ２ -CH ２ -C(=O)-OH; R ４ Ha-NH ２ , -COOH, -C(=O)-NH-CH ２ —C(═O)—OH, and —C(═O)—NH—CH ２ -CH ２ -S(=O) ２ 2. The compound of claim 1, wherein the compound is selected from the group consisting of: —OH.

6. The compound of formula (I) is as follows: 【Transformation 3】 6. The compound of claim 5 selected from the group consisting of:

7. A compound according to any one of claims 1 to 6 for treating a disease or condition associated with elevated GCPII activity in a subject in need thereof.

8. The compound of claim 7, wherein the disease or condition associated with increased GCPII activity comprises inflammatory bowel disease.

9. 9. The compound of claim 8, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease (CD) and ulcerative colitis (UC).

10. A compound described in any one of claims 7 to 9, which inhibits GCPII activity in the subject.

11. A pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 1 to 6 in admixture with a pharmaceutically acceptable excipient.

12. 12. The pharmaceutical composition of claim 11, further comprising one or more additional therapeutic agents.

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