Improving the Pharmacokinetic and CYP Inhibition Profiles of

Improving the Pharmacokinetic and CYP Inhibition Profiles of...

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Improving the Pharmacokinetic and CYP Inhibition Proﬁles of Azaxanthene-Based Glucocorticoid Receptor ModulatorsIdentiﬁcation of (S)‑5-(2-(9-Fluoro-2-(4-(2hydroxypropan-2-yl)phenyl)‑5H‑chromeno[2,3‑b]pyridin-5-yl)-2methylpropanamido)‑N‑(tetrahydro‑2H‑pyran-4-yl)-1,3,4-thiadiazole2-carboxamide (BMS-341) Michael G. Yang,* T. G. Murali Dhar,* Zili Xiao, Hai-Yun Xiao, James J.-W. Duan, Bin Jiang, Michael A. Galella, Mark Cunningham, Jinhong Wang, Sium Habte, David Shuster, Kim W. McIntyre, Julie Carman, Deborah A. Holloway, John E. Somerville, Steven G. Nadler, Luisa Salter-Cid, Joel C. Barrish, and David S. Weinstein Research and Development, Bristol-Myers Squibb Company, Princeton, New Jersey 08543-4000, United States S Supporting Information *

ABSTRACT: An empirical approach to improve the microsomal stability and CYP inhibition proﬁle of lead compounds 1a and 1b led to the identiﬁcation of 5 (BMS-341) as a dissociated glucocorticoid receptor modulator. Compound 5 showed signiﬁcant improvements in pharmacokinetic properties and, unlike compounds 1a−b, displayed a linear, dose-dependent pharmacokinetic proﬁle in rats. When tested in a chronic model of adjuvant-induced arthritis in rat, the ED50 of 5 (0.9 mg/kg) was superior to that of both 1a and 1b (8 and 17 mg/kg, respectively).

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induction via GR requires a ∼6-fold higher concentration of dex than gene repression.11 These studies suggested the possibility of identifying GR agonists that could achieve a dissociated pharmacological proﬁle retaining the beneﬁcial antiinﬂammatory eﬀects while minimizing the side eﬀect proﬁle due to TA.12,13 We recently described the discovery of azaxanthene-based azole amides (1a) as partially dissociated GR modulators (Figure 1).14 In our study, cellular assays of transrepression (AP-1) and transactivation (NP-1) were used to evaluate GR ligand-dependent activities. The azole amide 1a displayed excellent transrepression activity (AP-1 EC50 18 nM) while partial agonism of TA (NP-1 EC50 99 nM, Ymax 57% relative to the eﬃcacy of dex as 100%) was maintained. Both 1a and 1b suﬀered from the risk of clinical drug−drug interactions (DDI) and nonlinear pharmacokinetics, with greater than dose proportional increases in exposure. Observed in preclinical studies, this latter risk brings with it the risk of overexposing patients to drug with only a small escalation in dose. Both issues are believed to have a common mechanistic under-

INTRODUCTION Glucocorticoids, such as prednisolone (pred) and dexamethasone (dex), have been used for the treatment of autoimmune and inﬂammatory diseases for over 60 years.1 Conformational changes induced on binding of glucocorticoids to the glucocorticoid receptor (GR) result in its translocation to the nucleus, where it exerts its transcriptional activity either by transactivation (TA) or transrepression (TR) pathways. In the TR pathway, the direct physical contact of the GR with transcription factors NFκB and AP-1 leads to the repression of major downstream proinﬂammatory factors, including proinﬂammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) and chemokines (e.g., CCL2, CCL19), leading to the observed antiinﬂammatory eﬀects with glucocorticoids.2−5 However, longterm use of glucocorticoids results in a number of undesirable side eﬀects, including diabetes/hyperglycemia,6 and osteoporosis, which are attributed to the TA pathway.7,8 As full agonists of GR, pred and dex activate both TR and TA pathways. Studies using transgenic GR dimerization-deﬁcient mice have shown that the transactivation activities (DNA binding) of GR could be separated from the transrepressive eﬀects (non-DNA binding) of GR.9,10 In addition, it has been shown that gene © XXXX American Chemical Society

Received: February 18, 2015

A

DOI: 10.1021/acs.jmedchem.5b00257 J. Med. Chem. XXXX, XXX, XXX−XXX

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thiadiazole ring in the form of carboxamide may signiﬁcantly improve the CYP inhibition proﬁle for this series of compounds, as outlined in 2−5 (Figure 1). In this paper, we describe the syntheses, structure−activity relationships (SAR), in vitro proﬁles, and in vivo ﬁndings associated with 2−5. In the current study, assays used to evaluate biological activities of GR ligands, including nuclear receptor binding, transrepression in an A549 cell line (AP-1 assay), and transactivation in a HeLa cell line (NP-1 agonist assay), have been previously reported.14,16,17 The ideal activity proﬁle of a dissociated GR agonist would be a compound that is a partial agonist in the in vitro functional assays for transrepression and one that shows little to no activity in the transactivation assay.16,18 However, compounds with a low Ymax in the transrepression AP-1 assay also show reduced potency in the human whole blood (hWB) assay. On the other hand, if the eﬃcacy (Ymax value) is too high in the AP-1 transrepression assay (90−100%), the compound typically brings in potent transactivation activity (NP-1 assay).19,20 To achieve the goal of ﬁnding a balanced and dissociated GR modulator, we screened and progressed compounds with a good partial agonist transrepression activity, i.e. EC50 40 μM), after incubating compounds 4 and 5 for over 30 min. The half-life of 5 upon incubation with human liver microsomes was similar to that of 4. However, the rat and dog liver microsomes half-lives of 5 were about 5 and 8 times better than those of 4 (Table 2). In light of their promising in vitro pharmacologic proﬁle, the morpholine amide 4 and the tertiary alcohol 5 were selected for evaluation in in vivo PK and eﬃcacy models. The pharmacokinetic properties of the morpholine amide 4 and the tertiary alcohol 5 are summarized in Table 3. A low clearance (4.8 mL/min/kg) was recorded for 4 after a single 0.5 mg/kg intravenous dose and a bioavailability of 50% was determined after a single 10 mg/kg oral dose of a solution of 4 to rats. Notably, and consistent with the improved CYP450 inhibition proﬁle, a linear dose-dependent PK proﬁle (both in terms of Cmax and AUC) was observed in rats when 4 was dosed at 30 mg/kg orally (Table 3). The pharmacokinetics of 4 was also evaluated in dogs. Given the poor microsomal stability of 4 in the dog (Table 2), it was not surprising that low exposures were recorded in the oral leg of the study, and the related iv study was not pursued (Table 3). For compound 5, low clearance (2.7 mL/min/kg) and moderate volume of distribution (1.0 L/kg) were determined after a single 1 mg/kg intravenous dose administered to rats. At oral doses of 1 and 10 mg/kg, 5 was rapidly absorbed and displayed linear dosedependent increase in AUC with an oral bioavailability of 44− 51% (Table 3). The pharmacokinetic study of 5 in dogs revealed low clearance (1.7 mL/min/kg) after a single 1 mg/kg intravenous dose, and high exposures with 38% oral bioavailability were seen after a single 5 mg/kg oral dose. The eﬃcacy of 4 and 5 in vivo was evaluated in a chronic model of adjuvant-induced arthritis (Figure 2). Male Lewis rats were ﬁrst inoculated with Mycobacterium butyricum in incomplete Freund’s adjuvant, and then both compounds were dosed preventatively from day 0 to day 21 (q.d. dosing). Prednisolone (5 mg/kg) served as the positive control in the study. A dose-dependent reduction in the onset and progression of arthritis was observed for both compounds. The ED50 values of 4 and 5 were found to be 4.6 and 0.9 mg/ kg, respectively. The potency for 5 compares well to that which had previously been measured for pred (ED50 1.0 mg/kg). The greater level of eﬃcacy achieved with 5 relative to the standard dose of pred is notable. Table 4 provides a head-to-head comparison of compound 5 with earlier lead compounds 1a and 1b.14 As illustrated in Table 4, the human whole blood potency, CYP inhibition proﬁle, and pharmacokinetic properties of compound 5 are signiﬁcantly superior to those of compounds 1a and 1b. More importantly, the improved in vitro potency and PK properties of 5 translated to a lower ED50 value in the rat AA (adjuvantinduced arthritis) model. To understand the potential in vivo beneﬁts of an improved side eﬀect proﬁle relative to steroids in humans, both 4 and 5 were evaluated in cellular assays of gene products associated with targets of glucocorticoid-mediated side eﬀects, including bone-speciﬁc alkaline phosphatase (ALP) and glutamine synthetase (GS). ALP is a marker of osteoblast diﬀerentiation22 and glutamine synthetase is a marker of muscle wasting.23 In the SAOS-2 osteosarcoma cell line, pred was found to induce

potent in the human whole blood assay (hWB EC50 509 vs 1541 nM). The metabolic stability proﬁles of 1c and 1d were improved relative to that of the unsubstituted thiadiazole 1a (Table 1). Next, we examined a series of compounds (2a−d) with a 2-substituted carboxamide oﬀ the 5-amino-1,3,4thiadiazole ring. Noticeably, the metabolic stability proﬁle of these compounds was further improved compared to that of the methyl-substituted thiadiazoles 1c,d. Once again, the C-2 ﬂuorinated compounds 2c and 2d exhibited a better human whole blood activity than the des-ﬂuoro analogs (Table 1, 2a vs 2c and 2b vs 2d). Changing the morpholine benzamide to a pyrrolidine resulted in compounds 3a−e. In general, the pyrrolidine benzamides 3a−e had moderate stability in the liver microsome assay and exhibited weak potency in the human whole blood assay; for example, a 4-fold decrease in human whole blood activity was observed for 3b vs 2c (hWB EC50: 2c, 517 nM vs 3b, 2025 nM; Table 1). However, other structural changes around 2 proved fruitful. For example, installation of a ﬂuorine at the C-9 position of the azaxanthene ring of 2b led to the ﬂuorinated analog 4, which was potent in the transrepression assay (AP-1 EC50 24 nM, Ymax 86%) while a partial dissociated proﬁle (NP-1 EC50 64 nM, Ymax 64%) was retained. In addition, the additional ﬂuorine group at the C-9 position of 4 improved the human whole blood activity vs 2b, while the in vitro metabolic stability proﬁle was maintained. The human whole blood proﬁles of the ﬂuoro analogs 2d and 4 are similar, although the in vitro metabolic stability proﬁle is marginally better for 4 (Table 1). Further structural changes led to the discovery of the tertiary alcohol 5. Speciﬁcally, compound 5 was potent in the transrepression assay (AP-1 EC50 18 nM, Ymax 84%), while a partial dissociated proﬁle (NP-1 EC50 102 nM, Ymax 53%) was maintained. In addition, the tertiary alcohol 5 showed excellent human whole blood potency (EC50 268 nM) and a promising in vitro metabolic stability proﬁle (Table 1). On the basis of the in vitro data outlined in Table 1, compounds 4 and 5 were clearly the front runners, since they had potent human whole blood activity, weaker NP-1 activity, and favorable metabolic stability. Therefore, these compounds were proﬁled further for NHR selectivity, CYP inhibition, and half-life (T1/2) determination in liver microsomes (Table 2). Both 4 and 5 showed potent GR binding aﬃnity and good Table 2. General Proﬁles of 1a, 4, and 5a 1a GR binding Ki, nM PR binding Ki, nM AR binding Ki, nM MR agonist EC50, nMb AP-1 repression EC50, nM (% dex) NP-1 agonism EC50, nM (% dex) hWB LPS/TNF EC50, nM (% dex) human/rat/dog LM T1/2, min CYP IC50, μM 3A4/2C8 2C9/2C19

4

5

1.6 >1800 >5000 >5000 18 (79)

2.6 >4000 − >5000 24 (86)

1.0 >2000 >5000 >5000 18 (85)

99 (57) 379 (91)

64 (64) 505 (91)

102 (53) 268 (84)

4.6/5.6/5.3

43/57/6

59/196/48

0.4/0.9 2.2/0.5

40/− 3.9/0.5

27/3.2 6.5/7.9

a

Values are means of at least two experiments. bIn A549 cell line, EC50 (nM)/(%maximal eﬃcacy) determination (aldosterone as positive control). C

DOI: 10.1021/acs.jmedchem.5b00257 J. Med. Chem. XXXX, XXX, XXX−XXX

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Table 3. Rat and Dog Pharmacokinetic Properties of 4 and 5 compd/species/route/dose 4/rat/iv/0.5 mg/kg 4/rat/po/10 mg/kg 4/rat/po/30 mg/kg 4/dog/po/5 mg/kg 5/rat/iv/1 mg/kg 5/rat/po/1 mg/kg 5/rat/po/10 mg/kg 5/dog/iv/1 mg/kg 5/dog/po/5 mg/kg

Cmax (nM)

Tmax (h)

8980 23454 206

1.0 2.0 0.8

625 5345

2.0 1.0

2883

2.0

AUC0−24 (nM h)

T1/2 (h)

Cl (mL/min/kg)

Vss (L/kg)

2758 27608 100736 370 9623 4885 42071 13643 25566

0.9

4.8

0.3

F (%) 50 45

4.7

2.7

1.0 51 44

7.9

1.7

1.1 38

ALP (68% above background) after a 3-day incubation (Table 5). However, 4 and 5 were signiﬁcantly less eﬃcacious than Table 5. Alkaline Phosphatase and Glutamine Synthetase Induction by Pred, 4, and 5 hGS induction compd

ALP inductiona % max ± SD (n)

EC50 ± SD, nM (n)

% pred ± SD

pred, 1f 4 5

68 ± 16 (31) 20 ± 8 (2) 21 ± 21 (10)

19 ± 11 (25) 15 ± 3.6 (3) 13 ± 4.8 (7)

100 ± 17 73 ± 17 78 ± 15

a

Induction measured after 3 days of induction at 600 nM.

pred, and the rank order for eﬃcacy was consistent with that observed in the reporter assays. Similarly, in the MG-63 osteosarcoma cell line, less eﬃcacy than pred for induction of GS was observed for 4 and 5 (73% and 78% of pred), with a similar level of potency. Although a direct relationship between glucocorticoid-mediated induction of gene expression and side eﬀects in bone and muscle has not been established, the lower level of transactivation mediated by 4 and 5 suggests that they will have a diﬀerentiated proﬁle compared to prednisolone.

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CHEMISTRY The synthetic approach for the preparation of the key intermediate 16 is outlined in Scheme 1. 2-Chloronicotinic acid was condensed with 2-ﬂuorophenol (7) using sodium phenoxide to give the corresponding phenoxypyridine acid 8 in 65% yield. Intramolecular Friedel−Crafts cyclization of phenoxypyridine acid 8 gave excellent yields of the azaxanthenone 9. Sodium borohydride reduction of 9 provided the bis benzyl alcohol 10, which was subjected to a modiﬁed Mukaiyama aldol reaction with the silylketene acetal 11 to provide the ester 12 in high yields. Oxidation of 12 with mCPBA gave the N-oxide 13, which on reaction with POCl3 provided the methyl ester 14. Saponiﬁcation of 14 provided the acid 15, which was subjected to chiral supercritical ﬂuid chromatography (SFC) to provide the homochiral acid 16. The (S) absolute stereoconﬁguration of 16 was subsequently conﬁrmed by an X-ray crystal structure of 5 (vide infra).24 The synthetic procedure for the preparation of tertiary alcohol 5 is described in Scheme 2. Suzuki−Miyaura coupling of chloride 16 with aryl boronic acid 17 gave the corresponding ketone, which on treatment with methylmagnesium bromide in THF provided the tertiary alcohol 18 in 98% yield (two steps). The HOBt-promoted coupling reaction between the acid 18 and the amine 19 yielded the ethyl ester 20 in 39% yield. It is worth mentioning that the coupling reaction was very sluggish at room temperature and heating was required to achieve the

Figure 2. Compounds 4 (top), 5 (bottom), and pred in the rat adjuvant-induced model of arthritis.

Table 4. Comparison of Azole Amide 5 to the Previous Leads 1a and 1ba

GR binding Ki, nM AP-1 EC50, nM (% dex) NP-1 EC50, nM (% dex) hWB EC50, nM (% dex) CYP IC50, μM 3A4/2C8 2C9/2C19 rat/iv/1 mg/kg: Cl (mL/min/kg)/T1/2 (h) rat/po/10 mg/kg: AUC 0−24 (nM h)/F (%) rat AA ED50, mg/kg a

1a

1b

5

1.6 18 (79) 99 (57) 379 (91)

1.9 33 (70) 242 (32) 830 (83)

1.0 17.9 (85) 102.2 (53) 268 (84)

0.4/0.9 2.2/0.5 34.0/0.4

0.6/0.5 4.2/1.3 49.5/0.6

27/3.2 6.5/7.9 2.7/4.7

904/17

673/20

42071/44

8

17

0.9

Values are means of at least two experiments. D

DOI: 10.1021/acs.jmedchem.5b00257 J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 1a

properties and revealed a linear dose-dependent PK proﬁle in rats (Table 3). Furthermore, the ED50 of 5 in the rat adjuvantinduced arthritis study was 0.9, which is superior to that of both 1a and 1b. The utility of partial agonists of GR such as 5 in treating human disease while improving on the safety proﬁle of traditional glucocorticoids needs to be further evaluated in a clinical setting.

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EXPERIMENTAL SECTION

Chemistry. All commercially available chemicals and solvents were used without further puriﬁcation. Reactions are performed under an atmosphere of nitrogen. All new compounds gave satisfactory 1H NMR, LC/MS and/or HRMS, and mass spectrometry results. 1H NMR spectra were obtained on a Bruker 400 MHz or a JEOL 500 MHz NMR spectrometer using the residual signal of deuterated NMR solvent as internal reference. Electrospray ionization (ESI) mass spectra were obtained on a Water Micromass ESI-MS single quadrupole mass spectrometer. High-resolution mass spectral analysis was performed on an LTQ-FT mass spectrometer interfaced to a Waters Acquity ultraperformance liquid chromatography. The purity of tested compounds determined by analytical HPLC was >95%, except as noted. Analytical HPLC was performed on a Shimadzu instrument with a YMC Combiscreen ODS-A 4.6 mm × 50 mm column using a gradient elution of 0−100% B/A over 4 min with 1 min hold (solvent A, 90% water/10% MeOH/0.2% H3PO4; solvent B, 90% MeOH/10% water/0.2% H3PO4), a ﬂow rate of 4 mL/min, and 220 or 254 nm as the detection wavelength, except as noted. Synthesis of (S)-2-Methyl-N-(5-methyl-1,3,4-thiadiazol-2-yl)2-(2-(4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanamide (1c, Scheme 3). Step 1: Synthesis of

Reagents and conditions: (a) step 1, NaOMe; step 2, 168 °C, 3 h, 65%. (b) Step 1, polyphosphoric acid; step 2, 160 °C, 8 h, 97%. (c) NaBH4, MeOH, 96%. (d) TiCl4, CH2Cl2, 0 °C, 79%. (e) mCPBA, CH2Cl2, 25 °C, 93%. (f) POCl3, 100 °C → 25 °C, 42%. (g) 4 N aq KOH, THF, MeOH, 65 °C, 90%. (h) Chiralcel OJ-H (5 μm), CO2/ [IPA:ACN 1:1 w 0.1% TFA] (90:10), 35 °C, 100 bar, peak 1.

a

Scheme 2a

Scheme 3

Reagents and conditions: (a) Pd(PPh3)4, DMF, K3PO4 (2 M), 90 °C. (b) MeMgBr, THF, −78 °C → 25 °C, 98% for two steps. (c) EDC, HOBt, DIEA, DMF, 40 °C → 60 °C, 39%. (d) LiOH, THF/H2O, 25 °C. (e) Tetrahydro-2H-pyran-4-amine, BOP, DIEA, 25 °C, 96%. a

(S)-2-(2-Chloro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-N-(5methyl-1,3,4-thiadiazol-2-yl)propanamide (22). The synthesis of (S)-2-(2-chloro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic acid (21) was described in ref 14. To a solution of HATU (11.02 g, 29.0 mmol) in MeCN (300 mL) was added 21 (8 g, 26.3 mmol) and Hunig’s base (13.80 mL, 79 mmol) at room temperature. The mixture was stirred for 30 min. 5Methyl-1,3,4-thiadiazol-2-amine (3.34 g, 29.0 mmol) was added and the mixture was heated to 70 °C for 4.5 h. Additional HATU (1.1 g) and 5-methyl-1,3,4-thiadiazol-2-amine (1.6 g) were added. The mixture was heated at 60 °C for additional 17 h. After cooling to room temperature, the crude material was diluted with EtOAc (100 mL) and acidiﬁed with 1 M HCl (126 mL). The resulting suspension was ﬁltered. The white solid (2.9 g) was conﬁrmed as pure product. The ﬁltrate was separated into two layers. The organic layer was washed with water (100 mL), pH 7 buﬀer solution (2 × 100 mL), sat. NaHCO3 (50 mL), and brine (50 mL), respectively. It was then dried over MgSO4, ﬁltered, and concentrated to give a dark brown solid. The solid was triturated with MeOH and ﬁltered. The solid was washed with a small amount of MeOH to give 22 as a white solid (8.4 g, 83% yield). 1H NMR (400 MHz, CDCl3): δ ppm 10.47 (br s, 1H), 7.66 (d,

desired transformation. Saponiﬁcation of the ethyl ester 20 with LiOH gave the corresponding acid, which on coupling with tetrahydro-2H-pyran-4-amine provided the ﬁnal compound 5 in 96% yield (two steps).

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CONCLUSIONS Eﬀorts aimed at improving metabolic liabilities and CYP inhibition proﬁles of the lead chemotype 1a,b led to compounds of the structural class 2−5 with signiﬁcantly improved pharmacokinetic proﬁles, while the desired dissociated proﬁle was retained. In general, adding an amide motif at the 2-position oﬀ the 5-amino-1,3,4-thiadiazole ring was found to be beneﬁcial for improving microsomal stability and CYP inhibition proﬁles. Our early lead compounds 1a and 1b exhibited greater than dose-proportional increases in exposure after oral dosing. However, we demonstrated in this study that both 4 and 5 showed an improvement in pharmacokinetic E

DOI: 10.1021/acs.jmedchem.5b00257 J. Med. Chem. XXXX, XXX, XXX−XXX

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J = 7.8 Hz, 1H), 7.35−7.28 (m, 2H), 7.24 (m, 1H), 7.11−7.05 (m, 2H), 4.73 (s, 1H), 2.76 (s, 3H), 1.21 (br s, 3H), 1.21 (br s, 3H). ESIMS: m/z 401.08 ([M + H+]). HPLC: tR = 3.88 min. Step 2: Synthesis of (S)-4-(5-(2-Methyl-1-(5-methyl-1,3,4-thiadiazol-2-ylamino)-1-oxopropan-2-yl)-5H-chromeno[2,3-b]pyridin-2yl)benzoic Acid (23). A DMF (2 mL) solution of 22 (51.5 mg, 0.128 mmol), 4-boronobenzoic acid (37.5 mg, 0.226 mmol), Pd(Ph3P)4 (21.5 mg, 0.019 mmol), and 2 M K3PO4 (320 μL, 0.640 mmol) was degassed by N2 for 3 min. The sealed tube was then heated at 100 °C for 16 h. The crude product was puriﬁed by preparative HPLC (gradient 70−100% solvent B in 30 min) to give 23 (30.7 mg, 40% yield, presumed TFA salt). 1H NMR (400 MHz, CDCl3): δ ppm 8.11−8.05 (m, 2H), 8.05−7.99 (m, 2H), 7.63−7.57 (m, 1H), 7.55− 7.50 (m, 1H), 7.40−7.31 (m, 2H), 7.22−7.10 (m, 2H), 4.51 (s, 1H), 2.79 (s, 3H), 1.29 (m, 6H). ESI-MS: m/z 487.14 ([M + H+]). HPLC: tR = 4.08 min. Step 3: Synthesis of (S)-2-Methyl-N-(5-methyl-1,3,4-thiadiazol-2yl)-2-(2-(4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanamide (1c). A MeCN (1 mL) solution of 23 (15.2 mg, 0.025 mmol, presumed TFA salt), HATU (19.6 mg, 0.052 mmol), and morpholine (10 μL, 0.115 mmol) was stirred at rt for 16 h. The crude material was puriﬁed by preparative HPLC (gradient 60−90% solvent B in 30 min) to give 1c (5.5 mg, 32% yield). 1H NMR (400 MHz, CDCl3): δ ppm 8.08 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 7.8 Hz, 1H), 7.54 (dd, J = 9.4, 7.9 Hz, 3H), 7.38−7.29 (m, 2H), 7.16−7.07 (m, 2H), 4.54 (s, 1H), 3.93−3.42 (m, 8H), 2.76 (s, 3H), 1.27 (s, 3H), 1.23 (s, 3H). ESI-MS: m/z 556.19 ([M + H+]). HPLC: tR = 3.88 min. Synthesis of (S)-2-(2-(3-Fluoro-4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-N-(5-methyl-1,3,4-thiadiazol-2-yl)propanamide (1d, Scheme 4). Step 1:

(br s, 2H), 1.13 (s, 3H), 1.08 (s, 3H). ESI-MS: m/z 477.17 ([M + H+]). HPLC: tR = 3.91 min. Step 3: Synthesis of (S)-2-(2-(3-Fluoro-4-(morpholine-4carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-N-(5methyl-1,3,4-thiadiazol-2-yl)propanamide (1d). A MeCN (1 mL) solution of 26 (19.6 mg, 0.033 mmol, presumed TFA salt), 5-methyl1,3,4-thiadiazol-2-amine (18.3 mg, 0.159 mmol), HATU (37.5 mg, 0.099 mmol), and Hunig’s base (30 μL, 0.172 mmol) was stirred at 60 °C for 16 h. The crude material was puriﬁed by preparative HPLC (gradient 70−95% solvent B in 25 min) to give 1d (18 mg, 76% yield). 1 H NMR (400 MHz, CDCl3): δ ppm 7.89−7.81 (m, 2H), 7.76 (d, J = 7.8 Hz, 1H), 7.54−7.47 (m, 2H), 7.34 (d, J = 3.8 Hz, 2H), 7.29 (br s, 1H), 7.09 (dt, J = 7.7, 4.1 Hz, 1H), 4.76 (s, 1H), 3.88−3.77 (m, 4H), 3.67 (br s, 2H), 3.38 (br s, 2H), 2.77 (s, 3H), 1.25 (br s, 3H), 1.24 (br s, 3H). ESI-MS: m/z 574.18 ([M + H+]). HPLC: tR = 3.98 min. Synthesis of (S)-N-Methyl-5-(2-methyl-2-(2-(4-(morpholine4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanamido)-1,3,4-thiadiazole-2-carboxamide (2a, Scheme 5). Step 1: Synthesis of (S)-2-Methyl-2-(2-(4-(morpholine-4-

Scheme 5

Scheme 4

carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanoic Acid (28). A mixture of 21 (150 mg, 0.494 mmol), 4-(morpholine-4carbonyl)phenylboronic acid (27) (232 mg, 0.988 mmol), 2 M aqueous solution of potassium phosphate (1.728 mL, 3.46 mmol), and DMF (5 mL) was bubbled with nitrogen for 5 min before tetrakis(triphenylphosphine)palladium(0) (57.1 mg, 0.049 mmol) was added. The mixture was bubbled with nitrogen for an additional 5 min. The reaction mixture was stirred at 90 °C under nitrogen for 3 h and then before being concentrated under reduced pressure. The solid residue was mixed with 1:1 ethyl acetate/heptane mixture (6 mL). The mixture was extracted with water (3 × 2 mL). The combined aqueous solutions were decolorized with active charcoal and neutralized to pH 7 with 6 N aqueous HCl (0.8 mL) and 10% aqueous citric acid solution. The solid was ﬁltered, washed with water (3 × 1 mL), and dried to give 28 (200 mg, 0.436 mmol, 88% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ ppm 0.99 (s, 3 H), 1.06 (s, 3 H), 3.28−3.85 (m, 8 H), 4.42 (s, 1 H), 7.05−7.11 (m, 1 H), 7.21−7.29 (m, 3 H), 7.44−7.50 (m, 3 H), 7.67 (d, J = 7.81 Hz, 1 H), 8.03 (d, J = 8.56 Hz, 2 H). ESI-MS: m/z 459.18 ([M + H+]). HPLC: tR = 3.10 min. Step 2: Synthesis of 5-Amino-N-methyl-1,3,4-thiadiazole-2carboxamide (29). To a suspesion of ethyl 5-amino-1,3,4thiadiazole-2-carboxylate (19) (106 mg, 0.612 mmol) in methanol (1.5 mL) was added methylamine solution (0.5 mL) (33 wt %) in ethanol. The mixture was stirred at rt for 6 h, heated to 70 °C, and then cooled. The mixture was concentrated. Lyophilization gave 29 (99 mg, 0.626 mmol, 100% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6): δ ppm 8.69 (q, J = 4.8 Hz, 1H), 7.70 (s, 2H), 2.73 (d, J = 4.78 Hz, 3H). ESI-MS: m/z 159.03 ([M + H+]). HPLC: tR = 0.44 min.

Synthesis of 3-Fluoro-4-(morpholine-4-carbonyl)phenylboronic Acid (25). A DMF (1.5 mL) solution of 4-borono-2-ﬂuorobenzoic acid (24) (134.3 mg, 0.730 mmol), morpholine (128 μL, 1.463 mmol), HOBT (143.5 mg, 0.937 mmol), and EDC (157.4 mg, 0.821 mmol) was stirred at room temperature for 30 min. The crude product was puriﬁed by preparative HPLC (gradient 30−60% solvent B in 30 min) to give 25 (142 mg, 77% yield). 1H NMR (400 MHz, CDCl3): δ ppm 8.78 (br s, 2H), 8.03−7.69 (m, 1H), 7.65−7.29 (m, 2H), 3.95− 3.30 (m, 8H). ESI-MS: m/z 254.09 ([M + H+]). HPLC: tR = 2.06 min. Step 2: Synthesis of (S)-2-(2-(3-Fluoro-4-(morpholine-4carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic Acid (26). A DMF (1.5 mL) solution of 21 (36.1 mg, 0.119 mmol), 25 (55.5 mg, 0.219 mmol), Pd(Ph3P)4 (17.6 mg, 0.015 mmol), and 2 M K3PO4 (300 μL, 0.600 mmol) was degassed by N2 for 3 min. The sealed tube was then heated at 100 °C for 16 h. The crude material was puriﬁed by preparative HPLC (gradient 70−100% solvent B in 30 min) to give 26 (32.5 mg, 46%, presumed TFA salt). 1H NMR (400 MHz, CDCl3): δ ppm 9.66 (br s, 1H), 7.89−7.82 (m, 2H), 7.78 (d, J = 7.8 Hz, 1H), 7.58−7.50 (m, 2H), 7.40−7.28 (m, 3H), 7.20− 7.14 (m, 1H), 4.50 (s, 1H), 3.89−3.84 (m, 4H), 3.71 (br s, 2H), 3.42 F

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Step 3: Synthesis of (S)-N-Methyl-5-(2-methyl-2-(2-(4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanamido)-1,3,4-thiadiazole-2-carboxamide (2a). A mixture of 28 (20 mg, 0.044 mmol), PyBOP (35 mg, 0.067 mmol), DIPEA (0.03 mL, 0.172 mmol), and anhydrous DMF (0.3 mL) was stirred at rt under nitrogen for 15 min before 29 (15 mg, 0.095 mmol) was added. The mixture was then sitrred at 100 °C for 1 h. Puriﬁcation using reverse-phase HPLC (YMC S5 20 × 100 mm, 10 min run; solvent A, 10% MeOH:90% H2O:0.1% TFA; solvent B, 90% MeOH, 10% H2O, 0.1% TFA) gave 2a (16 mg, 0.022 mmol, 51.5% yield) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ ppm 8.13 (d, J = 8.3 Hz, 2H), 7.75−7.66 (m, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.37 (t, J = 8.1 Hz, 1H), 7.30−7.22 (m, 2H), 7.18−7.09 (m, 1H), 4.63 (s, 1H), 3.83−3.47 (m, 8H), 2.97 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H). ESI-MS: m/z 599.20 ([M + H+]). HPLC: tR = 3.15 min. Synthesis of (S)-N-Cyclopropyl-5-(2-methyl-2-(2-(4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanamido)-1,3,4-thiadiazole-2-carboxamide (2b, Scheme 6). Step 1: Synthesis of 5-Amino-N-cyclopropyl-1,3,4-thiadiazole-2-

Scheme 7

(s, 1H), δ 9.12 (s, 1H), 7.89−8.05 (m, 3H), 7.64−7.66 (d, J = 7.9 Hz, 1H), 7.52−7.57 (t, J = 7.76 Hz, 1H), 7.36−7.39 (m, 1H), 7.30−7.32 (d, J = 7.96 Hz, 1H), 7.15−7.16 (m, 2H), 4.81(s, 1H), 3.66 (s, 4H), 3.55−3.56 (t, J = 4.0 Hz, 2H), 3.27−3.30 (m, 2H), 2.81−2.82 (m, 3H), 2.46 (m, 2H), 1.0−1.1 (m, 6H). ESI-MS: m/z 617.19 ([M + H+]). HPLC: tR = 15.14 min (SunFire C18 3.5u column 4.6 × 150 mm; solvent A, 5% MeCN:95% H2O:0.05% TFA; solvent B, 95% MeCN, 5% H2O, 0.05% TFA; gradient, 10−100% of solvent B in solvent A over 30 min; ﬂow rate, 1 mL/min; monitoring at 220 nm). Synthesis of (S)-N-Cyclopropyl-5-(2-(2-(3-ﬂuoro-4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (2d, Scheme 8). A procedure similar to that described in step 3 of

Scheme 6

Scheme 8

carboxamide (30). To a suspesion of 19 (100 mg, 0.577 mmol) in methanol (1.5 mL) was added cyclopropylamine (0.3 mL, 4.28 mmol). The mixture was stirred at rt for 6 h, heated to 70 °C, and concentrated to 0.5 mL in total volume. After water (0.5 mL) was added, the mixture was heated to 70 °C (clear solutioin) and cooled. The solid was ﬁltered, washed with cold methanol/water (1/1) mixture, and dried to give 30 (66 mg, 0.358 mmol, 62.0% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ ppm 8.90 (d, J = 4.5 Hz, 1H), 7.80 (s, 2H), 2.96−2.81 (m, 1H), 0.81−0.61 (m, 4H). ESIMS: m/z 185.04 ([M + H+]). HPLC: tR = 0.82 min. Step 2: Synthesis of (S)-N-Cyclopropyl-5-(2-methyl-2-(2-(4(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanamido)-1,3,4-thiadiazole-2-carboxamide (2b). To a stirred mixture of 28 (600 mg, 1.309 mmol), 30 (723 mg, 3.93 mmol), HOBT (200 mg, 1.309 mmol), anhydrous MeCN (15 mL), and DIPEA (0.594 mL, 3.40 mmol) was added WSCDI (753 mg, 3.93 mmol) at rt under nitrogen. The mixture was stirred at rt under nitrogen for 10 min and at 80 °C for 3 h. The reaction mixture was concentrated in vacuo and mixed with water (10 mL) and ethyl acetate (10 mL). The aqueous layer was separated and extracted with ethyl acetate (2 × 2 mL). The combined organic solutions were dried over Na2SO4 and concetrated in vacuo. The residue was dissolved in a mixture of ethyl acetate, dichloromethane, and methanol (the solid formed was ﬁltered oﬀ). Flash chromatography puriﬁcation using ISCO (12 g silica gel column, 20−100% ethyl acetate in hexanes) aﬀorded 2b (480 mg) as a foam solid. 1H NMR (400 MHz, CDCl3): δ ppm 8.13 (d, J = 8.3 Hz, 2H), 7.76−7.66 (m, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.40−7.34 (m, 1H), 7.30−7.22 (m, 2H), 7.16−7.08 (m, 1H), 4.63 (s, 1H), 3.85−3.44 (m, 8H), 2.94−2.85 (m, 1H), 1.20 (s, 3H), 1.16 (s, 3H), 0.90−0.81 (m, 2H), 0.75−0.68 (m, 2H). ESI-MS: m/z 625.22 ([M + H+]). HPLC: tR = 3.28 min. Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(morpholine-4carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-N-methyl-1,3,4-thiadiazole-2-carboxamide (2c, Scheme 7). A procedure similar to that shown in step 3 of Scheme 4 was used to prepare 2c. 1H NMR (400 MHz, DMSO-d6): δ ppm 12.92

Scheme 4 was used to prepare 2d. 1H NMR (400 MHz, DMSO-d6): δ ppm 12.96 (s., 1H), δ 9.27 (s, 1H), 7.91−8.05 (m, 3H), 7.64−7.66 (d, J = 7.9 Hz, 1H), 7.53−7.57 (t, J = 7.2 Hz, 1H), 7.31−7.53 (m, 2H), 7.15−7.16 (m, 2H), 7.15−7.16 (m, 2H), 4.81(s, 1H), 3.74−3.76 (m, 6H), 3.55 (s, 2H), 3.28 (s, 2H), 2.88−2.91 (m, 1H), 1.04−1.07 (m, 5H), 0.70−0.72 (m, 3H). ESI-MS: m/z 643.21 ([M + H+]). HPLC: tR = 10.28 min (SunFire C18 3.5u column 4.6 × 150 mm; solvent A, 5% MeCN:95% H2O:0.05% TFA; solvent B, 95% MeCN, 5% H2O, 0.05% TFA; gradient, 10−100% of solvent B in solvent A over 15 min; ﬂow rate, 1 mL/min; monitoring at 220 nm). Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (3a, Scheme 9). Step 1: Synthesis of (S)-2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)phenyl)5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic Acid (32). A mixture of 21 (800 mg, 2.63 mmol), 3-ﬂuoro-4-(pyrrolidine-1carbonyl)phenylboronic acid (31) (1.7 g, 7.17 mmol), 2 M aqueous solution of potassium phosphate (9 mL, 18.00 mmol), and DMF (24 mL) was bubbled with nitrogen for 5 min before tetrakis(triphenylphosphine)palladium(0) (300 mg, 0.260 mmol) was added. The mixture was bubbled with nitrogen for an additional 5 min. The reaction mixture was stirred at 90 °C under nitrogen for 3 h and then concentrated under reduced pressure. The residue was mixed with 1:1 ethyl acetate/heptane mixture (30 mL). The mixture was extracted with water (50 mL, 3 × 15 mL). The combined aqueous solutions were decolorized with active charcoal and neutralized to pH 6−7 with 6 N aqueous HCl (4 mL) and then 10% aqueous citric acid solution. Ethyl acetate (15 mL) was added and the mixture was stirred for 1 h. The solid was ﬁltered, washed with water (3 × 2 mL) and ethyl acetate (2 × 1 mL), and dried to give 32 (1.07 g, 2.324 mmol, 88% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ ppm 11.42 (br G

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do)-N-methyl-1,3,4-thiadiazole-2-carboxamide (3b, Scheme 10). A procedure similar to that described in step 3 of Scheme 9

Scheme 9

Scheme 10

was used to prepare 3b. 1H NMR (400 MHz, CD3OD): δ ppm 8.01− 7.89 (m, 2H), 7.80−7.66 (m, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.44−7.33 (m, 1H), 7.32−7.22 (m, 2H), 7.19−7.08 (m, 1H), 4.64 (s, 1H), 3.63 (t, J = 6.8 Hz, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.97 (s, 3H), 2.08−1.89 (m, 4H), 1.20 (s, 3H), 1.16 (s, 3H). ESI-MS: m/z 601.20 ([M + H+]). HPLC: tR = 3.41 min. Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-N,N-dimethyl-1,3,4-thiadiazole-2-carboxamide (3c, Scheme 11). A procedure similar to that described in step 3 of

s, 1H), 7.89−7.80 (m, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.57−7.46 (m, 2H), 7.36−7.28 (m, 3H), 7.18−7.09 (m, 1H), 4.49 (s, 1H), 3.70 (t, J = 6.9 Hz, 2H), 3.37 (t, J = 6.7 Hz, 2H), 2.06−1.86 (m, 4H), 1.11 (s, 3H), 1.02 (s, 3H). ESI-MS: m/z 461.18 ([M + H+]). HPLC: tR = 3.41 min. Step 2: Synthesis of (S)-Ethyl 5-(2-(2-(3-Fluoro-4-(pyrrolidine-1carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxylate (33). To a stirred mixture of 32 (400 mg, 0.869 mmol), 19 (400 mg, 2.310 mmol), HOBT (140 mg, 0.914 mmol), and anhydrous MeCN (10 mL) was added 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (340 mg, 1.774 mmol) at rt under nitrogen, followed by DIPEA (0.2 mL, 1.145 mmol). The mixture was stirred at rt for 1 h and at 80 °C for 4.5 h and then concentrated. The residue was mixed with water (15 mL). The mixture was extracted with ethyl acetate (15 mL, 3 × 10 mL). The combined organic solutions were dried over Na2SO4 and concentrated. Flash chromatography (12 g silica gel column, 20−100% ethyl acetate in hexanes) gave 33 (411 mg, 0.668 mmol, 77% yield) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ ppm 8.05 (br s, 1H), 7.91−7.83 (m, 2H), 7.59−7.51 (m, 2H), 7.34 (d, J = 3.3 Hz, 2H), 7.24 (d, J = 7.6 Hz, 1H), 7.12−7.00 (m, 1H), 4.88 (br s, 1H), 4.58 (q, J = 7.1 Hz, 2H), 3.70 (t, J = 6.8 Hz, 2H), 3.38 (t, J = 6.3 Hz, 2H), 2.05−1.91 (m, 4H), 1.50 (t, J = 7.2 Hz, 3H), 1.37 (br s, 3H), 1.27−1.22 (m, 3H). ESI-MS: m/z 616.20 ([M + H+]). HPLC: tR = 3.70 min. Step 3: Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (3a). To a stirred solution of 33 (183 mg, 0.297 mmol) in MeOH (1.5 mL) was added 1 M aqueous NaOH (1.040 mL, 1.040 mmol). The reaction mixture was stirred at rt for 1.5 h and then concentrated under reduced pressure to remove methanol. Lyophilization gave a yellow solid (207 mg; 0.297/ 0.207 = 1.4 mmol/g) which was then used as such without further puriﬁcation. To a dry ﬂask were added ammonium chloride (18 mg, 0.337 mmol), the sodium salt of (S)-5-(2-(2-(3-ﬂuoro-4-(pyrrolidine1-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxylic acid (0.029 mmol, generated from the above procedure), DIPEA (50 μL, 0.286 mmol), and anhydrous DMF (0.3 mL) at rt under nitrogen. The mixture was stirred at rt for 2 min before PyBOP (20 mg, 0.038 mmol) was added. The mixture was stirred at rt for 1 h. Puriﬁcation using reverse-phase HPLC (YMC S5 20 × 100 mm, 10 min run; solvent A, 10% MeOH:90% H2O:0.1% TFA; solvent B, 90% MeOH, 10% H2O, 0.1% TFA) gave 3a (16 mg, 0.023 mmol, 79% yield) as a white solid. 1H NMR (400 MHz, CD3OD): δ ppm 7.98−7.90 (m, 2H), 7.77−7.69 (m, 2H), 7.52 (t, J = 7.4 Hz, 1H), 7.41−7.34 (m, 1H), 7.31−7.22 (m, 2H), 7.17−7.10 (m, 1H), 4.63 (s, 1H), 3.63 (t, J = 6.9 Hz, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.07−1.90 (m, 4H), 1.20 (s, 3H), 1.16 (s, 3H). ESI-MS: m/z 587.18 ([M + H+]). HPLC: tR = 3.34 min. Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanami-

Scheme 11

Scheme 9 was used to prepare 3c. 1H NMR (400 MHz, CD3OD): δ ppm 7.99−7.90 (m, 2H), 7.78−7.70 (m, 2H), 7.53 (t, J = 7.4 Hz, 1H), 7.42−7.35 (m, 1H), 7.32−7.22 (m, 2H), 7.17−7.10 (m, 1H), 4.64 (s, 1H), 3.63 (t, J = 6.9 Hz, 2H), 3.55 (s, 3H), 3.39 (t, J = 6.7 Hz, 2H), 3.18 (s, 3H), 2.08−1.90 (m, 4H), 1.20 (s, 3H), 1.16 (s, 3H). ESI-MS: m/z 615.21 ([M + H+]). HPLC: tR = 3.44 min. Synthesis of (S)-N-Ethyl-5-(2-(2-(3-ﬂuoro-4-(pyrrolidine-1carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (3d, Scheme 12). A procedure similar to that described in step 3 of Scheme 9 was

Scheme 12

used to prepare 3d. 1H NMR (400 MHz, CD3OD): δ ppm 7.98−7.90 (m, 2H), 7.77−7.69 (m, 2H), 7.53 (t, J = 7.4 Hz, 1H), 7.42−7.34 (m, 1H), 7.31−7.23 (m, 2H), 7.16−7.10 (m, 1H), 4.64 (s, 1H), 3.63 (t, J = 7.1 Hz, 2H), 3.46 (q, J = 7.2 Hz, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.08− 1.90 (m, 4H), 1.26 (t, J = 7.2 Hz, 3H), 1.20 (s, 3H), 1.16 (s, 3H). ESIMS: m/z 615.21 ([M + H+]). HPLC: tR = 3.49 min. Synthesis of (S)-N-Cyclopropyl-5-(2-(2-(3-ﬂuoro-4-(pyrrolidine-1-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2methylpropanamido)-1,3,4-thiadiazole-2-carboxamidexamide (3e, Scheme 13). A procedure similar to that described in step 3 of Scheme 9 was used to prepare 3e. 1H NMR (400 MHz, DMSO-d6): δ ppm 7.98−7.89 (m, 2H), 7.76−7.68 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.41−7.34 (m, 1H), 7.31−7.21 (m, 2H), 7.16−7.09 (m, 1H), 4.63 (s, 1H), 3.66−3.59 (m, 2H), 3.43−3.36 (m, 2H), 2.94−2.85 (m, 1H), 2.07−1.90 (m, 4H), 1.19 (s, 3H), 1.16 (s, 3H), 0.89−0.80 (m, 2H), H

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(400 MHz, CD3OD): δ ppm 8.33 (1 H, d, J = 8.06 Hz), 7.09−7.56 (5 H, m). ESI-MS: m/z 234.05 ([M + H+]). HPLC: tR = 2.18 min (4 min gradient, YMC S5 ODS 4.6 × 50 mm, 4 mL/min, 0% B to 100% B, B = 90% MeOH/10% H2O, 0.2% H3PO4, 4 mL/min). Step 2: Synthesis of 9-Fluoro-5H-chromeno[2,3-b]pyridin-5-one (9). Compound 8 and polyphosphoric acid were combined in a 500 mL round-bottom ﬂask, which was stirred slowly on a rotary evaporator (open to the atmosphere) until a homogeneous paste was formed, and then heated at 160 °C for 8 h. Ice (∼500 g) was added portionwise with agitation to the reaction mixture to give a tancolored suspension. In a 1 L beaker, the suspension (still containing ice) was stirred while 10 N NaOH was added until the mixture reached about pH 10. The mixture was ﬁltered through a mediumporosity fritted disc ﬁlter, washed with water three times, and dried overnight on the pump to give 9 (8.95 g, 97% yield) as a gray solid. 1H NMR (400 MHz, methanol-d4): δ ppm 8.86−8.66 (m, 2H), 8.08 (dt, J = 8.1, 1.4 Hz, 1H), 7.75 (ddd, J = 10.7, 8.0, 1.5 Hz, 1H), 7.64 (dd, J = 7.8, 4.7 Hz, 1H), 7.49 (td, J = 8.1, 4.5 Hz, 1H). ESI-MS: m/z 216.04 ([M + H+]). HPLC: tR = 2.16 min (4 min gradient, YMC S5 ODS 4.6 × 50 mm, 4 mL/min, 0% B to 100% B, B = 90% MeOH/10% H2O, 0.2%H3PO4, 4 mL/min). Step 3: Synthesis of 9-ﬂuoro-5H-chromeno[2,3-b]pyridin-5-ol (10). NaBH4 (4.92 g, 130 mmol) was added to a slurry of 9 (4.0 g, 18.59 mmol) in MeOH (90 mL) and CH2Cl2 (40 mL) which was stirring at 0 °C. The reaction stirred for 2 h at 0 °C. The reaction mixture was concentrated in vacuo to remove all the methanol. The reaction was partitioned between brine and chloroform and the aq layer was backwashed with chloroform twice more until there was no product left. All the organic layers were combined, dried over sodium sulfate, ﬁltered, and concentrated in vacuo to aﬀord 10 (3.87 g, 17.82 mmol, 96% yield) as a light yellow solid. 1H NMR (400 MHz, methanol-d4): δ ppm 8.32 (dd, J = 4.8, 2.0 Hz, 1H), 8.16 (ddd, J = 7.5, 1.9, 0.7 Hz, 1H), 7.48−7.42 (m, 1H), 7.38 (dd, J = 7.5, 4.8 Hz, 1H), 7.30−7.20 (m, 2H), 5.90 (s, 1H). ESI-MS: m/z 218.05 ([M + H+]). HPLC: tR = 2.08 min. Step 4: Synthesis of Methyl 2-(9-Fluoro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoate (12). To a suspension of the crude alcohol 10 (5.0 g, 23.02 mmol) in dichloromethane (125 mL) at 0 °C was added TiCl4 (23.02 mL, 23.02 mmol) dropwise. The resulting tancolored suspension stirred for 10 min at 0 °C before (1-methoxy-2methylprop-1-enyloxy)trimethylsilane (11) (10.76 mL, 52.9 mmol) was added to the mixture dropwise. The resulting dark, homogeneous solution was allowed to stir for 3 h at 0 °C and then was quenched with the addition of saturated aqueous sodium bicarbonate, yielding gas evolution and formation of a precipitate. The mixture was ﬁltered over Celite, and the resulting organic layer of the ﬁltrate was separated, dried over sodium sulfate, and concentrated to give 12 (5.50 g, 18.25 mmol, 79% yield). 1H NMR (400 MHz, chloroform-d): δ ppm 8.27 (1 H, dd, J = 4.78, 1.76 Hz), 7.56 (1 H, dd, J = 7.55, 1.76 Hz), 7.00−7.15 (3 H, m), 6.90−6.95 (1 H, m), 4.41 (1 H, s), 3.66 (3 H, s), 1.03 (3 H, s), 1.00 (3 H, s). ESI-MS: m/z 302.17 ([M + H+]). HPLC: tR = 3.03 min. Step 5: Synthesis of 9-Fluoro-5-(1-methoxy-2-methyl-1-oxopropan-2-yl)-5H-chromeno[2,3-b]pyridine 1-Oxide (13). A solution of 12 (4.0 g, 13.28 mmol) in CH2Cl2 (100 mL) stirring at room temperature was treated with mCPBA (containing 30% m-CBA) (9.37 g, 54.3 mmol). The reaction (light yellow solution) stirred overnight for 14 h. The reaction was washed sequentially with 10% aqueous sodium sulﬁte and 1 N aqueous sodium hydroxide and then dried over sodium sulfate, ﬁltered, and concentrated to give 13 (4.37 g, 12.39 mmol, 93% yield). The crude product was taken directly to the next step. ESI-MS: m/z 318.19 ([M + H+]). HPLC: tR = 2.09 min. Step 6: Synthesis of Methyl 2-(2-Chloro-9-ﬂuoro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoate (14). A solution of 13 (4.1 g, 12.92 mmol) in POCl3 (50 mL, 536 mmol) was heated at 100 °C for 1.5 h. The reaction was cooled down to rt and stirred for 16 h. The solvent was carefully removed by distilling oﬀ the excess solvent. The excess solvent was quenched by pouring onto ice and cautiously neutralized with base. The concentrated reaction was poured slowly in small increments onto ice. With stirring, the icy slurry was neutralized

Scheme 13

0.75−0.67 (m, 2H). ESI-MS: m/z 627.21 ([M + H+]). HPLC: tR = 3.52 min. Synthesis of (S)-N-Cyclopropyl-5-(2-(9-ﬂuoro-2-(4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (4, Scheme 14). Step 1: Synthesis of 2-(2-Fluorophenoxy)nicotinic

Scheme 14

Acid (8). Sodium methoxide in MeOH (65.3 mL, 286 mmol) was added to 2-ﬂuorophenol (7) (80 g, 714 mmol), followed by 2chloronicotinic acid (22.5 g, 143 mmol). The reaction mixture was concentrated in vacuo and then dried on the pump for 2 h. The ﬂask containing the dried material was then heated in an oil bath at 168 °C for 3 h. The reaction mixture was cooled to rt and sat under nitrogen gas over one weekend. Water and diethyl ether were added to the solid, which partitioned, upon agitation, between the two layers. The purple aqueous layer was acidiﬁed with glacial acetic acid (to pH ∼5) and chilled, giving a precipitate. The solid (colorless needles) was ﬁltered, washed with water, then dried on the pump to give 8 (18 g). The aqueous layer was rendered acidic (to pH ∼ 3) with the dropwise addition of concentrated HCl. The cloudy mixture was washed with ethyl acetate (150 mL). The organic layer was washed with brine, dried (sodium sulfate), and concentrated to give a second crop of 8 (3.75 g). The total amount of 8 was 21.75 g, 65.3% yield. 1H NMR I

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mixture was stirred at rt for 10 min and at 80 °C for 6 h. The reaction mixture was cooled, concentrated, and mixed with water (30 mL) and ethyl acetate (50 mL). The organic solution was separated, dried over Na2SO4, and concentrated. The residue was dissolved in ethyl acetate and acetone, mixed with Celite (16 g) and DMF (1.6 mL), and concentrated to give an almost dry solid. Flash chromatography puriﬁcation of the solid (80 g silica gel column, 50 → 100% ethyl acetate in hexanes) aﬀorded 36 (3.15 g, 4.99 mmol, 83% yield) as a yellowish foam solid. 1H NMR (400 MHz, CD3OD): δ ppm 8.12 (d, J = 8.03 Hz, 2 H), 7.86 (br s, 1 H), 7.59 (d, J = 7.78 Hz, 1 H), 7.53 (d, J = 8.03 Hz, 2 H), 7.14 (ddd, J = 9.85, 7.84, 1.88 Hz, 1 H), 6.95−7.04 (m, 2 H), 4.77 (br s, 1 H), 4.56 (q, J = 7.28 Hz, 2 H), 3.42−3.92 (m, 8 H), 1.49 (t, J = 7.15 Hz, 3 H), 1.32 (s, 3 H), 1.25 (s, 3 H). ESI-MS: m/ z 632.19 ([M + H+]). HPLC: tR = 3.48 min. Step 11: Synthesis of (S)-N-Cyclopropyl-5-(2-(9-ﬂuoro-2-(4(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)2-methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (4). To a solid−liquid mixture of 36 (2.1 g, 3.32 mmol) and anhydrous MeOH (10 mL) was added cyclopropylamine (6 mL, 87 mmol). The solution was stirred at 40 °C under nitrogen for 18 h. The mixture was concentrated. The residue was partitioned between water (10 mL) and ethyl acetate (4 mL). The aqueous layer was separated and extracted with dichloromethane (2 × 3 mL). The combined organic solutions were dried over Na2SO4. Flash chromatography puriﬁcation (40 g silica gel column, 50 → 100% ethyl acetate in hexanes) aﬀorded a glassy solid. The solid was dissolved in ethyl acetate (15 mL) and precipitated out with hexanes (30 mL). The solid was ﬁltered, washed with a mixture of ethyl acetate and hexanes (1:2, 2 × 10 mL), and dried to give 4 (1.2 g, 1.848 mmol, 56% yield) as a white solid. 1H NMR (500 MHz, DMSO-d6): δ ppm 12.92 (s, 1 H), 9.25 (d, J = 4.67 Hz, 1 H), 8.18 (d, J = 8.25 Hz, 2 H), 7.92 (d, J = 7.97 Hz, 1 H), 7.69 (d, J = 7.97 Hz, 1 H), 7.54 (d, J = 8.25 Hz, 2 H), 7.32−7.38 (m, 1 H), 7.15 (td, J = 8.04, 5.09 Hz, 1 H), 6.96 (d, J = 7.70 Hz, 1 H), 4.86 (s, 1 H), 3.34−3.74 (m, 8 H), 2.86−2.94 (m, 1 H), 1.08 (br s, 3 H), 1.07 (br s, 3 H), 0.68−0.73 (m, 4 H). ESI-MS: m/z 643.21 ([M + H+]). HPLC: tR = 3.26 min. Synthesis of (S)-5-(2-(9-Fluoro-2-(4-(2-hydroxypropan-2-yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-N-(tetrahydro-2H-pyran-4-yl)-1,3,4-thiadiazole-2-carboxamide (5, Scheme 15). Step 1: Synthesis of (S)-2-(9-Fluoro-2-(4-(2-

by adding solid potassium carbonate in small portions. Each addition of the salt generated gas that needed to subside before more salt was introduced into the ﬂask. Once the reaction was not acidic anymore, it was diluted with methylene chloride. The layers were separated, and the aq layer was backwashed with fresh DCM twice. All the DCM layers were combined and dried over sodium sulfate, ﬁltered, and concentrated in vacuo to aﬀord the crude material as a caramel-colored gum. The reaction mixture was placed on a silica gel column. The product was eluted using 10% ethyl acetate in hexane, and the collected organic solution was concentrated in vacuo to aﬀord 14 as a white solid (1.8 g, 5.36 mmol, 41.5% yield). 1H NMR (400 MHz, methanol-d4): δ ppm 7.77 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.27−7.14 (m, 2H), 7.12−7.03 (m, 1H), 4.49 (s, 1H), 3.69 (s, 3H), 1.04 (d, J = 4.0 Hz, 6H). ESI-MS: m/z 336.07 ([M + H+]). HPLC: tR = 3.35 min. Step 7: Synthesis of 2-(2-Chloro-9-ﬂuoro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic Acid (15). To a pale yellow solution of 14 (2.33 g, 6.94 mmol) in THF (13 mL)/MeOH (22 mL) was added 4 N aqueous potassium hydroxide (17.35 mL, 69.4 mmol). The mixture was stirred at 65 °C for 10 h and then at rt for another 6 h. The reaction mixture was rendered neutral with the dropwise addition of concentrated HCl and then concentrated to half its volume in vacuo. The remaining solution was then partitioned between ethyl acetate and 1 N HCl. The organic layer was dried over sodium sulfate and concentrated to give 15 (2.0 g, 6.22 mmol, 90% yield) as a tan solid. 1H NMR (400 MHz, CD3OD): δ ppm 7.82 (d, J = 8.06 Hz, 1 H), 7.29 (d, J = 7.81 Hz, 1 H), 7.25−2.11 (m, 3 H), 4.51 (s, 1 H), 1.01 (s, 3 H), 0.96 (s, 3 H). ESI-MS: m/z 322.06 ([M + H+]). HPLC: tR = 3.11 min. Step 8: Synthesis of (S)-2-(2-Chloro-9-ﬂuoro-5H-chromeno[2,3b]pyridin-5-yl)-2-methylpropanoic Acid (16). Chiral resolution [preparative column, Chiralcel OJ-H (3 × 25 cm, 5 μm); BPR pressure, 100 bar; temperature, 35 °C; ﬂow rate, 70 mL/min; mobile phase, CO2/(IPA:ACN 1:1, 0.1% TFA) (90/10); detector wavelength, 212 nm] gave 16 (peak 1). 1H NMR (400 MHz, CD3OD): δ ppm 7.82 (d, J = 8.06 Hz, 1 H), 7.29 (d, J = 7.81 Hz, 1 H), 7.25−2.11 (m, 3 H), 4.51 (s, 1 H), 1.01 (s, 3 H), 0.96 (s, 3 H). ESI-MS: m/z 322.06 ([M + H+]). HPLC: tR = 3.11 min. The (S) absolute stereoconﬁguration of 16 was subsequently conﬁrmed by an X-ray crystal structure of 5 (vide infra). Step 9: Synthesis of (S)-2-(9-Fluoro-2-(4-(morpholine-4carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic Acid (35). A mixture of 16 (2.85 g, 5.98 mmol), morpholino(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanone (34) (3.55 g, 11.19 mmol), potassium phosphate (21.76 mL, 43.5 mmol), and DMF (60 mL) was bubbled with nitrogen for 5 min before Pd(Ph3P)4 (0.718 g, 0.622 mmol) was added. The mixture was bubbled with nitrogen for an additional 5 min and then stirred at 100 °C under nitrogen for 3 h. The mixture was concentrated under reduced pressure. The solid residue was mixed with water (40 mL) and diethyl ether (100 mL). The organic mixture was extracted with water (3 × 15 mL). The combined aqueous solutions were ﬁltered through a pad of Celite, washed with diethyl ether (30 mL), and acidiﬁed to pH 5−6 with concd aq HCl. Ethyl acetate (17 mL) was added. The mixture was stirred at rt for 20 min before heptanes (34 mL) were added. The mixture was stirred at rt for 30 min. The solid was ﬁltered, washed with water and a mixture of ethyl acetate and heptanes (1:2), and dried to give 35 (2.67 g, 5.60 mmol, 90% yield) as a brownish solid. 1H NMR (400 MHz, CD3OD): δ ppm 8.17 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.22−7.13 (m, 3H), 4.57 (s, 1H), 3.83−3.46 (m, 8H), 1.04 (s, 3H), 1.00 (s, 3H). ESI-MS: m/z 477.17 ([M + H+]). HPLC: tR = 3.07 min. Step 10: Synthesis of (S)-Ethyl 5-(2-(9-Fluoro-2-(4-(morpholine-4carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxylate (36). To a stirred mixture of 35 (2.85 g, 5.98 mmol), 19 (2.072 g, 11.96 mmol), HOBT (0.916 g, 5.98 mmol), and anhydrous MeCN (15 mL) was added WSCDI (2.293 g, 11.96 mmol) at rt under nitrogen. The mixture was stirred at rt for 5 min before DIPEA (1.358 mL, 7.78 mmol) was added. The

Scheme 15

hydroxypropan-2-yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2methylpropanoic Acid (18). The mixture of 16 (250 mg, 0.777 mmol), 4-acetylphenylboronic acid (17) (255 mg, 1.55 mmol), and potassium phosphate (2.72 mL, 5.44 mmol) in DMF (6 mL) was ﬂushed with nitrogen for 5 min. Then tetrakis(triphenylphosphine)palladium(0) (90 mg, 0.078 mmol) was added and the mixture was ﬂushed with nitrogen for another 5 min. The mixture was stirred at 90 °C for 8 h. After cooling, the mixture was ﬁltered and the ﬁltrate was added to water (15 mL) and washed with diethyl ether (2 × 20 mL). The aqueous layer was acidiﬁed with 4 N HCl to pH 5−6 and extracted with AcOEt (35 mL), which was washed with saturated J

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NH4Cl (2 × 20 mL), dried over Na2SO4, and concentrated under vacuo to give the corresponding ketone {ESI-MS: m/z 406.14 ([M + H]+)}, which was used in the next reaction without further puriﬁcation. The above intermediate was taken up in THF (5 mL), methylmagnesium bromide (2.59 mL, 7.77 mmol) was added dropwise at −78 °C, and the mixture stirred at rt for 1 h. The mixture was quenched with saturated NH4Cl (3 mL), diluted with NH4Cl (20 mL), and mixed with AcOEt (60 mL). The pH was adjusted with 4 N HCl to 5−6. The entire mixture was ﬁltered through a pad of Celite and the organic phase was separated and washed with saturated NH4Cl (2 × 30 mL), dried over Na2SO4, and concentrated under vacuo to give the crude compound 18, which was used in the next reaction without further puriﬁcation. The crude yield for the two steps was 98% yield (320 mg). 1H NMR (400 MHz, CD3OD): δ ppm 8.08−7.96 (m, 2H), 7.89 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.68−7.59 (m, 2H), 7.28−7.12 (m, 3H), 4.56 (s, 1H), 1.59 (s, 6H), 1.04 (d, J = 7.5 Hz, 6H). ESI-MS: m/z 422.17 ([M + H+]). HPLC: tR = 3.51 min. Step 2: Synthesis of (S)-Ethyl 5-(2-(9-Fluoro-2-(4-(2-hydroxypropan-2-yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxylate (20). A mixture of 18 (160 mg, 0.380 mmol), 19 (131 mg, 0.759 mmol), EDC (146 mg, 0.759 mmol), HOBt (116 mg, 0.759 mmol), and DIEA (0.332 mL, 1.898 mmol) in DMF (10 mL) was stirred at 40 °C for 16 h and then at 60 °C for 3 h. The solvent was removed and the residue was taken up in AcOEt (50 mL), washed with saturated NH4Cl (2 × 20 mL) and NaHCO3 (2 × 20 mL), dried (Na2SO4), and concentrated under vacuo to give the crude product, which was puriﬁed with ISCO ﬂash column chromatography (12 g, eluting with AcOEt/hexane = 0−50% gradient, 15 min). The yield of 20 was 85 mg (38.8% yield). 1H NMR (400 MHz, CD3OD): δ ppm 8.05 (d, J = 8.5 Hz, 2H), 7.88−7.81 (m, 1H), 7.62−7.56 (m, 3H), 7.16−7.08 (m, 1H), 6.97 (d, J = 3.5 Hz, 2H), 4.73 (s, 1H), 4.55 (d, J = 7.3 Hz, 2H), 1.65−1.61 (m, 6H), 1.51− 1.46 (m, 3H), 1.31 (s, 3H), 1.24 (d, J = 3.0 Hz, 3H). ESI-MS: m/z 577.18 ([M + H+]). HPLC: tR = 4.22 min. Step 3: Synthesis of (S)-5-(2-(9-Fluoro-2-(4-(2-hydroxypropan-2yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-N-(tetrahydro-2H-pyran-4-yl)-1,3,4-thiadiazole-2-carboxamide (5). To a mixture of 20 (7.0 g, 12.14 mmol) in THF (70 mL) was added water (5 mL) followed by lithium hydroxide hydrate (2 g, 47.7 mmol). The mixture was stirred at rt for 7 h. The mixture was concentrated under vacuo to dryness. Ethanol (50 mL) was added and the solvent was removed under vacuo (repeated twice) to give the lithium salt, which was used in the next step {ESI-MS: m/z 549.15 ([M + H+])}. To a solution of (S)-5-(2-(9-ﬂuoro-2-(4-(2-hydroxypropan-2-yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanamido)-1,3,4-thiadiazole-2-carboxylic acid (5 g, 9.11 mmol) lithium salt in DMF (60 mL) was added tetrahydro-2H-pyran-4-amine (1.383 g, 13.67 mmol), followed by BOP (8.06 g, 18.23 mmol) and DIEA (3.98 mL, 22.79 mmol), and the mixture was cooled with an ice water bath. After the addition, the ice bath was removed and the mixture was stirred at rt for 6 h. The mixture was diluted with AcOEt (300 mL); washed with saturated NH4Cl (200 mL, 2 × 120 mL), saturated NaHCO3 (3 × 120 mL), and brine (120 mL); dried over MgSO4; and concentrated under vacuo to 80 mL of volume. The solid was formed when standing at rt after 5 h. The solid was collected by ﬁltration, washed with EtOAc (50 mL), and dried under vacuo at 60 °C for 16 h to get the crystals (4.2 g). The ﬁrst 200 mL of NH4Cl wash was extracted with AcOEt (120 mL), which was washed with saturated NH4Cl (3 × 50 mL), saturated NaHCO3 (3 × 50 mL), and brine (50 mL), dried over MgSO4, and concentrated under vacuo to 15 mL of volume. The solid was formed when standing at rt for 5 h. The solid was collected by ﬁltration, washed with EtOAc (50 mL), and dried under vacuo to get the second crop of product (0.6 g). An extra 0.7 g product was recovered from the mother liquor. The total yield of 5 was 5.5 g (96% yield). 1H NMR (400 MHz, CD3OD): δ ppm 8.05− 7.95 (m, 2H), 7.79−7.52 (m, 4H), 7.27−6.96 (m, 3H), 4.65 (s, 1H), 4.21−4.06 (m, 1H), 4.04−3.92 (m, 2H), 3.53 (td, J = 11.8, 2.1 Hz, 2H), 1.91 (dd, J = 12.4, 2.3 Hz, 2H), 1.74 (qd, J = 12.0, 4.5 Hz, 2H),

1.60−1.54 (m, 6H), 1.18 (d, J = 1.5 Hz, 6H). ESI-MS: m/z 632.23 ([M + H+]). HPLC: tR = 4.03 min. Nuclear Receptor Binding Assays. The GR ligand binding assay was conducted in ﬂuorescence polarization format, which measures the competition between a test compound and a ﬂuorescently labeled ligand (GS-red) for binding to the full length or ligand binding domain of GRα (Invitrogen, catalog no. P2893). Compounds were tested in concentrations ranging from 5 μM to 85 pM. IC50 values were determined by ﬁtting the ﬂuorescence polarization signal data using a four-parameter logistic equation. The Ki values were determined by application of the Cheng−Prusoﬀ equation to the IC50 values, where Ki = IC50/(1 + ligand concentration/Kd) (Cheng, Y.; Prusoﬀ, W. H. Biochem. Pharmacol. 1973, 22, 3099−3108). The Kd used for GR was 0.3 nM, as supplied by the assay manufacturer (Invitrogen, catalog no. P2893). Data shown represent the mean values of two or more experiments. PR ligand binding assay was also conducted in ﬂuorescence polarization format, which measures the competition between a test compound and a ﬂuorescently labeled ligand for binding to the full length or ligand binding domain of the nuclear hormone receptor. Transrepression Assays. AP-1 activity is measured using an AP-1 response element (ﬁve copies) cloned into a luciferase reporter vector. This reporter is stably transfected into the human A549 lung epithelial cell line. AP-1 activity is induced by addition of phorbol myristate acetate (PMA) (15 ng/mL), and inhibition of induction by compounds is quantitated by measuring decreased luciferase activity. NFκB is measured using a truncated, NFκB-dependent E-selectin promoter (−383 bp from transcriptional start) cloned into a luciferase reporter vector. This reporter is stably transfected into the human A549 lung epithelial cell line. NFκB activity is induced using IL-1β (0.5 ng/mL), and inhibition of induction by compounds is quantitated by measuring decreased luciferase activity. Transactivation Assays: NP-1 Reporter Assay. The direct transcriptional activity is measured using a chimera between the GAL4 DNA-binding domain and the human GR ligand-binding domain (GR-LBD) cloned into a GAL4 luciferase reporter system. This reporter system is stably transfected into a NP-1 HeLa cell line (Webster, N. J. G.; Green, S.; Jin, J. R.; Chambon, P. Cell 1988, 54 (2), 199−207). The hormone-binding domains of the glucocorticoid receptor contain an inducible transcription activation function. Response to ligand/compound induced binding is quantitated by measuring luciferase activity. Direct activation of the GR-LBD by compounds (agonist) can be measured as increased luciferase activity after a 20 h incubation at 37 °C/5% CO2. Human Whole Blood Assays for TNFα and IL-1β. Human whole blood (two donors per experiment) was drawn into syringes containing ACD-A and kept at rt until initiation of the experiment (about 1 h). The blood was plated into 96-well ﬂat-bottom, polystyrene, tissue-culture-treated plates (BD Falcon) at 180 μL/ well. An amount of 10 μL of 20× test compound dilutions was added per well and mixed, and the plates were incubated overnight (about 20 h) at 37 °C with 5% CO2. The next day, 10 μL of 20× LPS (SigmaAldrich Escherichia coli 055/B5 LPS; for TNFα induction, the ﬁnal concentration of LPS was 100 ng/mL) or 20× human recombinant IL1β (R & D Systems; for IL-8 induction, the ﬁnal concentration of IL1β was 10 ng/mL) was added per well. The plates were incubated for an additional 5 h and centrifuged at low speed, and the plasmas were harvested for immediate analysis by ELISA or were frozen at −20 °C. TNFα and IL-8 induction was quantitated by ELISA (R & D Systems). Adjuvant-Induced Arthritis. Male Lewis rats (Harlan; ∼200 g) were injected intradermally at the shaved based of the tail on day 0 with 100 μL of 10 mg/mL freshly ground Mycobacterium butyricum in incomplete Freund’s adjuvant and then randomized into dose groups (n = 8/group). Test compounds were orally (po) dosed in solution (EtOH/TPGS/PEG300, 5:5:90) once daily. Baseline paw measurements were taken between days 7 and 10, and measurements were taken three times per week after disease developed (between days 11 and 14). All studies involving animals were reviewed and approved by the BMS Institutional Animal Care and Use Committee. K

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Journal of Medicinal Chemistry

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Glutamine Synthetase (GS) Induction Assay. An enzymatic assay was used to measure the induction of GS activity in the human osteosarcoma cell line MG-63. The cell line was maintained in log phase in growth medium [Dulbecco’s modiﬁed Eagle’s medium (DMEM, Invitrogen) with 10% charcoal−dextran-treated fetal calf serum (FCS, Hyclone), penicillin/streptomycin (10 000 U/mL and 10 mg/mL, respectively), and 2 mM L-glutamine]. Brieﬂy, on day 1 MG63 cells were plated in assay medium (growth medium but without phenol red and L-glutamine) at a density of 50 000 cells/well (200 μL/ well) in 96-well ﬂat-bottom tissue-culture-treated plates and incubated overnight at 37 °C with 5% CO2. On day 2, the assay medium was removed from the wells and replaced with 100 μL/well induction medium (DMEM without L-glutamine, 1% FCS, and penicillin/ streptomycin) and test compounds followed by incubation overnight at 37 °C with 5%CO2. On day 3, the induction medium was removed, and the wells were washed three times with 200 μL/well PBS. After the last wash, PBS was removed, and the cells were lysed at room temperature with 30 μL of 50 mM imidazole (pH 6.8)/0.1% Trixon100 per well. Then 30 μL of GS assay mix (imidazole, 50 mM; arsenic acid, 25 mM; ADP, 0.16 mM; L-glutamine, 50 mM; MnCl2, 2 mM; hydroxylamine, 25 mM) was added per well, and the plates were incubated at 37 °C, 5% CO2 for 90 min. At the end of the incubation period, 120 μL of ferric chloride stop solution (ferric chloride, 2.42%; TCA, 1.45%; HCl, 1.82%) was added per well, and the absorption was read at 540 nm. Alkaline Phosphatase Induction Assay. An enzymatic assay was used to measure the induction of alkaline phosphatase activity in the human osteosarcoma cell line SAOS-2. The cell line was maintained in log phase in growth medium [McCoy’s 5A (Invitrogen) with 15% fetal bovine serum (FBS, Summit) and penicillin/streptomycin (10 000 U/ mL and 10 mg/mL, respectively)]. Brieﬂy, on day 1, SAOS-2 cells were plated in assay medium (growth medium except using 15% charcoal−dextran-treated FCS) at a density of 3000 cells/well (100 μL/well) in 96-well ﬂat-bottom tissue-culture-treated plates and incubated overnight at 37 °C with 5% CO2. On day 2, the assay medium was removed from the wells and replaced with 200 μL/well induction medium (assay medium except using 1.5% charcoal−dextran treated FCS) and test compounds followed by incubation for 3 days at 37 °C with 5% CO2. After the third day of compound treatment, the induction medium was removed, and the wells were washed three times with 200 μL/well cold PBS. After the last wash, PBS was removed, and the cells were lysed at room temperature with 100 μL per well of 12.5 mM NaHCO3, 12.5 mM Tris, 0.01% sodium azide, and 0.05% Triton X-100. Then 25 μL of cell lysate was added to 100 μL of alkaline phosphatase assay mix (p-NPP diluted in 1-KPL DEA substrate buﬀer to 3 mg/mL), and the plates were incubated in the dark at room temperature for 15 min. At the end of the incubation period, 100 μL per well of 1 N HCL stop solution was added per well, and the absorbance was read at 405 nm.

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ACKNOWLEDGMENTS The authors are grateful to the following colleagues for their support of the project and their help in the preparation of this paper: Mary Ellen Cvijic, Raymond Scaringe, Ding Ren Shen, Meliisa Yarde, Dauh-Rurng Wu, Yi Tao, Ling Gao, and Peng Li.

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ABBREVIATIONS USED AA, adjuvant-induced arthritis; ALP, alkaline phosphatase; AP1, activator protein 1; AR, androgen receptor; DDI, drug−drug interactions; dex, dexamethasone; ER, estrogen receptor; GR, glucocorticoid receptor; GS, glutamine synthetase; HATU, (2(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophosphate); HOBT, hydroxybenzotriazole; iv, intravenous; mCPBA, m-chloroperoxybenzoic acid; MR, mineralocorticoid receptor; NFκB, nuclear factor kappa B; NP-1, neuropilin-1; PK, pharmacokinetic; PD, pharmacodynamic; PAMPA, parallel artiﬁcial membrane permeability assay; PR, progesterone receptor; pred, prednisolone; rt, room temperature; SAR, structure−activity relationships; SFC, supercritical ﬂuid chromatography; TA, transactivation; TNFα, tumor necrosis factor α; WSCDI, 1-(3-(dimethylamino)propyl)-3ethylcarbodiimide hydrochloride

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REFERENCES

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S Supporting Information *

The X-ray crystal structure of 5 is also available. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00257.

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Corresponding Authors

*M.G.Y.: phone, 609-252-3234; e-mail, michael.yang@bms. com. *T.G.M.D.: phone, 609-252-4158; e-mail, murali.dhar@bms. com. Notes

The authors declare no competing ﬁnancial interest. L

DOI: 10.1021/acs.jmedchem.5b00257 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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DNA binding by the glucocorticoid receptor. EMBO J. 2001, 20, 7168−7173. (11) He, Y. Z.; Yi, W.; Suino-Powell, K.; Zhou, W. E.; Tolbert, W. D.; Tang, X.; Yang, J.; Yang, H.; Shi, J.; Hou, Li.; Jiang, H.; Melcher, K.; Xu, H. E. Structures and mechanism for the design of highly potent glucocorticoids. Cell Res. 2014, 24, 713−726. (12) For recent reviews of glucocorticoid receptor modulators, see the following: (a) Berlin, M. Recent advances in the development of novel glucocorticoid receptor modulators. Expert Opin. Ther. Pat. 2010, 20, 855−873. (b) Hudson, A. R.; Roach, S. L.; Higuchi, R. I. Recent developments in the discovery of selective glucocorticoid receptor modulators (SGRMs). Curr. Top. Med. Chem. 2008, 8, 750− 765. (13) Riether, D.; Harcken, C.; Razavi, H.; Kuzmich, D.; Gilmore, T.; Bentzien, J.; Pack, E. J., Jr.; Souza, D.; Nelson, R. M.; Kukulka, A.; Fadra, T. N.; Zuvela-Jelaska, L.; Pelletier, J.; Dinallo, R.; Panzenbeck, M.; Torcellini, C.; Nabozny, G. H.; Thomson, D. S. Nonsteroidal dissociated glucocorticoid agonists containing azaindoles as steroid Aring mimetics. J. Med. Chem. 2010, 53, 6681−6698. (14) Weinstein, D. S.; Gong, H.; Doweyko, A. M.; Cunningham, M.; Habte, S.; Wang, J. H.; Holloway, D. A.; Burke, C.; Guarino, V.; Carman, J.; Somerville, J. E.; Shuster, D.; Salter-Cid, L.; Dodd, J. H.; Nadler, S. G.; Barrish, J. C. Azaxanthene based selective glucocorticoid receptor modulators: Design, synthesis, and pharmacological evaluation of (S)-4-(5-(1-((1,3,4-thiadiazol-2-yl)amino)-2-methyl-1-oxopropan-2-yl)-5H-chromeno[2,3-b]pyridin-2-yl)-2-fluoro-N,N-dimethylbenzamide (BMS-776532) and its methylene homologue (BMS791826). J. Med. Chem. 2011, 54, 7318−7333. (15) Metabolic stability is deﬁned as the percentage of parent compound remaining over 10 min of incubation time in the presence of human, rat, and mouse liver microsomes, respectively. The initial compound concentration was 0.5 μM; protein concentration was 1 mg/mL. (16) Yang, B. V.; Weinstein, D. S.; Doweyko, L. M.; Gong, H.; Vaccaro, W.; Huynh, T.; Xiao, H.; Doweyko, A. M.; McKay, L.; Holloway, D. A.; Somerville, J. E.; Habte, S.; Cunningham, M.; McMahon, M.; Townsend, R.; Shuster, D.; Dodd, J. H.; Nadler, S. G.; Barrish, J. C. Dimethyl-diphenyl-propanamide derivatives as nonsteroidal dissociated glucocorticoid receptor agonists. J. Med. Chem. 2010, 53, 8241−8251. (17) Cellular assays: AP-1 activity is measured using an AP-1 response element (containing ﬁve copies) cloned into a luciferase reporter vector. This reporter is stably transfected into the human A549 lung epithelial cell line. AP-1 activity is induced by PMA (15 ng/ mL), and inhibition of the induction by compounds is quantitated by measuring decreased luciferase activity. NP-1 GRE activation activity is measured using a GR ligand binding domain (GR-LBD) chimera cloned into a GAL4 luciferase reporter system. This reporter system is stably transfected into a HeLa cell line (NP-1). Response to ligandinduced binding is quantitated by measuring luciferase activity. Direct activation of the GR-LBD by compounds (agonist) can be measured as increased luciferase activity. (18) Gong, H.; Yang, M.; Xiao, Z.; Doweyko, A. M.; Cunningham, M.; Wang, J.; Habte, S.; Holloway, D.; Burke, C.; Shuster, D.; Gao, L.; Carman, J.; Somerville, J. E.; Nadler, S. G.; Salter-Cid, L.; Barrish, J. C.; Weinstein, D. S. Discovery of acylurea isosteres of 2-acylaminothiadiazole in the azaxanthene series of glucocorticoid receptor agonists. Bioorg. Med. Chem. Lett. 2014, 24, 3268−3273. (19) Sheppeck, J. E., II; Gilmore, J. L.; Xiao, H. Y.; Dhar, T. G. M.; Nirschl, D.; Doweyko, A. M.; Sack, J. S.; Corbert, M. J.; Malley, M. F.; Gougoutas, J. Z.; Mckay, L.; Cunningham, M. D.; Habte, S. F.; Dodd, J. H.; Nadler, S. G.; Somerville, J. E.; Barrish, J. C. Discovery of potent and selective non-steroidal indazolyl amide glucocorticoid receptor agonists. Bioorg. Med. Chem. Lett. 2013, 23, 5442−5447. (20) Gilmore, J. L.; Sheppeck, J. E., II; Wang, J.; Murali Dhar, T. G.; Cavallaro, C.; Doweyko, A. M.; Mckay, L.; Cunningham, M. D.; Habte, S. F.; Dodd, J. H.; Nadler, S. G.; Somerville, J. E.; Barrish, J. C. Synthesis and structure−activity relationships of novel indazolyl

glucocorticoid receptor partial agonists. Bioorg. Med. Chem. Lett. 2013, 23, 5448−5451. (21) Schäcke, H.; Döcke, W.-D.; Asadullah, K. Mechanisms involved in the side effects of glucocorticoids. Pharmacol. Ther. 2002, 23−43. (22) Moutsatsou, P.; Kassi, E.; Papavassiliou, A. G. Glucocorticoid receptor signaling in bone cells. Trends Mol. Med. 2001, 18, 348−359. (23) Kanda, F.; Okuda, S.; Matsushita, T.; Takatani, K.; Kimura, K. I.; Chihara, K. Steroid myopathy: Pathogenesis and effects of growth hormone and insulin-like growth factor-I administration. Horm. Res. 2001, 56 (Suppl 1), 24−28. (24) Crystals of the title compound grew as colorless blades via slow evaporation of an ethyl acetate solution. The diﬀraction data were collected at 243 K using a Bruker-AXS KappaCCD diﬀractometer with an APEX2 detector. The crystal structure was solved with the SHELXTL software package. and the atomic coordinates were reﬁned using an in-house-developed software package. All hydrogen atoms were calculated from an idealized geometry with standard bond lengths and angles and reﬁned using a riding model. The absolute conﬁguration was established through analysis of the anomalous dispersion signal using the Flack method; the Flack parameter (x) was found to be 0.08(1), illustrating that the model represents the correct enantiomer. The diﬀraction data was deposited into the Cambridge Crystallographic Data Centre (CCDC) and assigned a deposition number, CCDC 1062143.

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DOI: 10.1021/acs.jmedchem.5b00257 J. Med. Chem. XXXX, XXX, XXX−XXX

Improving the Pharmacokinetic and CYP Inhibition Profiles of

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