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Discovery of novel pyrazole-based selective aldosterone synthase (CYP11B2) inhibitors: a new template to coordinate the heme-iron motif of CYP11B2 Ryo Sakakibara, Wataru Sasaki, Yuichi Onda, Minami Yamaguchi, Hideki Ushirogochi, Yuki Hiraga, Kanako Sato, Masashi Nishio, Yasuhiro Egi, Kei Takedomi, Hidetoshi Shimizu, Tomoko Ohbora, and Fumihiko Akahoshi J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Journal of Medicinal Chemistry

Discovery of novel pyrazole-based selective aldosterone synthase (CYP11B2) inhibitors: a new template to coordinate the heme-iron motif of CYP11B2 Ryo Sakakibara*, Wataru Sasaki, Yuichi Onda, Minami Yamaguchi, Hideki Ushirogochi, Yuki Hiraga, Kanako Sato, Masashi Nishio, Yasuhiro Egi, Kei Takedomi, Hidetoshi Shimizu, Tomoko Ohbora, Fumihiko Akahoshi Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi, Toda, Saitama, 335-8505, Japan

ABSTRACT

It is necessary for aldosterone synthase (CYP11B2) inhibitors to have both high potency and high selectivity over 11β-hydroxylase (CYP11B1), a critical enzyme for cortisol synthesis. Previous studies have reported a number of CYP11B2 inhibitors, most of which have an imidazole or pyridine ring to coordinate the heme-iron motif of CYP11B2; however, highly selective inhibitors of human CYP11B2 are still needed. To expand the selectivity in humans, we

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explored alternative templates and found that pyrazoles were suitable templates for CYP11B2 inhibitors. Investigation of pyrazoles, especially N-alkyl pyrazoles, as a new template to coordinate the heme-iron motif led to a potent and highly selective CYP11B2 inhibitor 28 with an aldosterone-lowering effect at 1 mg/kg dosing in cynomolgus monkeys.

Introduction Aldosterone is an endogenous ligand of mineralocorticoid receptors and is a key component of the rennin-angiotensin-aldosterone system. Aldosterone is primarily produced in the zona glomerulosa of the adrenal cortex and plays a vital role in the regulation of fluid and electrolyte homeostasis. Recent studies suggest that aldosterone is not only an exacerbation factor of hypertension but also a risk factor for cardiovascular disorders.1 Aldosterone synthase (CYP11B2) is known to mediate the last three steps of aldosterone synthesis from 11deoxycorticosterone to aldosterone, and plasma aldosterone levels are regulated by this enzymatic activity at the adrenal cortex.2 Therefore, inhibition of CYP11B2 may be an attractive approach to the treatment of aldosterone related diseases by lowering plasma aldosterone levels. However, human CYP11B2 is 93% homologous to the 11β-hydroxylase (CYP11B1), an enzyme essential for cortisol synthesis, and this high homological identity has made it challenging to discover selective inhibitors of CYP11B2 over CYP11B1.3 CYP11B2 contains a heme-iron motif at its active site.4 All nonsteroidal CYP11B2 inhibitors have an sp2-hybridized nitrogen atom to coordinate the active site heme-iron motif.5 The crystal structure of CYP11B2 in complex with FAD286 (1, Figure 1) (PDB ID: 4FDH), the Renantiomer of the aromatase (CYP19A1) inhibitor Fadrozole, revealed the interaction between the imidazole nitrogen atom of FAD286 (1) and the heme-iron motif.4,6 LCI-699 (2, Figure 1),


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the first CYP11B2 inhibitor investigated in humans, also has an imidazole structure. Administration of LCI-699 (2) to healthy humans and patients with primary aldosteronism, essential hypertension, and resistant hypertension significantly reduced the level of aldosterone in both plasma and urine.7 However, the selectivity over CYP11B1 was not enough to avoid impairment of cortisol biosynthesis. Since LCI-699 (2) entered clinical studies, additional studies have reported a variety of CYP11B2 inhibitors to improve selectivity over CYP11B1. As shown in Figure 1, most of the reported inhibitors have one of two templates to coordinate the hemeiron motif: imidazole-based inhibitors8 like FAD286 (1) and LCI-699 (2), or pyridine-based inhibitors9 including tetrahydroisoquinoline-based inhibitors derived from substituted pyridine structures. Among a number of reported pyridine-based inhibitors, compound 3 reported by Novartis displayed high potency and selectivity over CYP11B1 and is in phase 1 clinical trials. Hoffman-la-Roche also investigated pyridine-based inhibitors, represented by compound 4, which have potent CYP11B2 inhibitory activity but poor selectivity over CYP11B1. Based on a homology model of CYP11B2 and CYP11B1, optimization of the 5,6,7,8-tetrahydroisoquinoline ring led to the discovery of a potent CYP11B2 inhibitor 5, which has high selectivity over CYP11B1 in cynomolgus monkeys. A further optimized tetrahydroisoquinoline-based inhibitor is in phase 1 clinical trials.10 The pyridine-based inhibitors 3 and 5 are potent and considerably selective over CYP11B1 in cynomolgus monkeys, but less selective in humans. To expand the selectivity in humans, we explored alternative templates other than imidazoles and pyridines and found that pyrazole was a suitable template for CYP11B2 inhibitors.


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Figure 1. Structures of representative aldosterone synthase inhibitors 1−5.

Because pyrazole is a N-containing heterocycle having different physicochemical properties compared with imidazole and pyridine, including weak basicity and hydrogen-bond acceptor ability,11 and has been applied to potent scaffolds in medicinal chemistry,12 we investigated it as a template to coordinate the heme-iron motif of CYP11B2.13 In a study of CYP2E1 metabolism, Jones et al. experimentally determined the inhibition constant of N-containing heterocycles for CYP2E1, and revealed that 1H-pyrazole derivatives bind tightly to the heme-iron motif.14 Meanwhile, N-alkyl pyrazoles have a substituent ortho to the nitrogen that could coordinate the heme-iron motif (Figure 2), but few studies have been reported on CYP11B2 inhibitors having an ortho substituent.15 Previous studies evaluating CYP-targeted inhibitors have reported that introduction of a substituent ortho to the nitrogen effectively improves selectivity over human drug-metabolizing CYP enzymes and other metalloenzymes.16 We found that pyrazoles, especially N-alkyl pyrazoles, were effective new templates to coordinate the heme-iron motif, were suitable for CYP11B2 inhibition, and discovered potent and highly selective CYP11B2 inhibitors.


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Figure 2. Illustration of the N-alkyl pyrazoles coordinating to the heme-iron motif.

Chemistry The synthesis of a series of N-alkyl pyrazole compounds 6−13 is shown in Scheme 1. Commercially available N-methylpyrazole-4-boronic acid pinacol ester (33) was reacted with 4bromobenzonitrile under Suzuki-Miyaura coupling conditions using a microwave reactor to produce compound 6. Similarly, Suzuki-Miyaura coupling of 4-cyanophenylboronic acid with various 1,5-disubstituted 4-bromopyrazoles 34a−34g produced compounds 7−10, 11a, 12 and 13, respectively. Reductive amination of aldehyde 11a with dimethylamine produced compound 11. Tri substituted benzene compounds 14−19 and imidazole compound 20 were also prepared under Suzuki-Miyaura conditions (Scheme 2).


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Scheme 1. Synthesis of aldosterone synthase inhibitors 6−13.

Reagents and conditions: (a) 4-bromobenzonitrile, Pd(PPh3)4, NaHCO3, DME, H2O, reflux 5 h, 78%; (b) 4-cyanophenylboronic acid, Pd(OAc)2, 2-dicyclohexylphosphino-2',6'dimethoxybiphenyl, K2CO3, MeCN, H2O, MW, 100 °C, 30 min or 4-cyanophenylboronic acid, PdCl2(PPh3)2, Na2CO3, 1,4-dioxane, H2O, MW, 150 °C, 10 min, 57%−75%; (c) dimethylamine, NaBH(OAc)3, 1,2-dichloroethane, rt, 18 h, 67%. Scheme 2. Synthesis of aldosterone synthase inhibitors 14−20.

Reagents and conditions: (a) PdCl2(PPh3)2, Na2CO3, 1,4-dioxane, H2O, MW, 100 °C or 150 °C, 10 min, 52%−95%; (b) (4-cyano-2-methyl-phenyl)boronic acid, PdCl2(PPh3)2, Na2CO3, 1,4dioxane, H2O, MW, 150 °C, 10 min, 73%.


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A series of C-5 substituted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole compounds was synthesized using the withasomnine alkaloid methodology (Scheme 3).17 Sydnone (38) was synthesized in three steps from enantiomerically-pure protected hydroxyproline 37.17 Deprotection of the N-Boc group of hydroxyproline and N-nitrosation, followed by cyclodehydration with trifluoroacetic anhydride, produced compound 38. Product 39 was synthesized by regioselective cycloaddition of trimethylsilyl-substituted alkynylboronate with sydnone (38).17 Suzuki-Miyaura coupling of compound 39 with 4-bromo-3-methyl-benzonitrile, followed by removal of the trimethylsilyl group, produced target compound 23. After removal of the benzyl group from 39, Suzuki-Miyaura coupling of compound 40 with an aryl bromide, followed by desilylation of 41, produced compound 21. Compound 21 was treated with methyl iodide to produce compound 22. O-alkylation of alcohol 21 with ethyl chloroacetate, followed by hydrolysis of ester, produced compound 24, and condensation of 24 and ammonia produced compound 25. Nucleophilic aromatic substitution of either 2-chloro-pyrazine or 2-chloropyrimidines with compound 21 produced target compounds 26−30. A racemic alcohol was synthesized by oxidation of alcohol 21 with (R)-configuration, followed by reduction of the resulting ketone 42. Alcohol 31 with (S)-configuration was separated using chiral chromatography of the racemic alcohol. Compound 32 was synthesized by nucleophilic aromatic substitution of 2-chloro-5-fluoro-pyrimidine with alcohol 31.


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Scheme 3. Synthesis of aldosterone synthase inhibitors 21−32.

Reagents and conditions: (a) TFA, CHCl3, rt, 16 h; (b) NaNO2, AcOH, rt, 15 h; (c) TFAA, THF, rt, 15 h, 55% in 3 steps; (d) trimethyl-[2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)ethynyl]silane, 1,2-dichlorobenzene, 185 °C, 21 h, 53%; (e) 4-bromo-3-methyl-benzonitrile, PdCl2(PPh3)2, Na2CO3, 1,4-dioxane, H2O, MW, 150 °C, 15 min; (f) TBAF in THF, THF, rt to 70 °C, 3 days, 71% in 2 steps; (g) H2, Pd(OH)2/C, EtOH, rt, 16 h, 76%; (h) 4-bromo-3-methylbenzonitrile, PdCl2(PPh3)2, Na2CO3, DME, H2O, 80 °C, 15 h, 56%; (i) TBAF in THF, THF, 80 °C, 17 h, 76%; (j) MeI, NaH, DMF, rt, 4 h, 45%; (k) ethyl chloro acetate, NaH, THF, 0 °C to rt, 2 h, 50%; (l) NaOH aq., THF, EtOH, rt, 1.5 h, 100%; (m) NH3 aq., WSC, Nhydroxylsuccinimide, chloroform, rt, 40 min, 70%; (n) ArCl, NaH, DMF, and/or THF, rt, 1–16 h, 29%−91%; (o) 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one, CHCl3, rt, 4 h, 73%; (p) NaBH4, MeOH, rt, 4 h, then Chiralpak ID column (n-hexane/propan-2-ol/THF/diethyl amine, 70/15/15/0.1), 45%; (q) 2-chloro-5-fluoro-pyrimidine, NaH, THF, rt, 2.5 h,100%.

Results and Discussion To begin our investigation of new CYP11B2 inhibitors featuring a pyrazole template to coordinate the heme-iron motif, we replaced the pyridine moiety of compound 4 with various Nalkyl pyrazoles while keeping the benzonitrile ring structure common to imidazole-based


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CYP11B2 inhibitors, such as FAD286 and LCI-699. In vitro hCYP11B2 and hCYP11B1 inhibitory activity and selectivity factor (the ratio between the IC50 values against CYP11B2 and CYP11B1) are shown in Table 1. Unfortunately, 1-methylpyrazole (6) had no CYP11B2 inhibitory activity. However, compound 7, obtained by introduction of a methyl group at the C-5 position of the pyrazole moiety of compound 6, showed a potency of CYP11B2 IC50 = 290 nM. Improvements in potency were observed with the ethyl (8) group and trifluoromethyl (9) group at the C-5 position, while introduction of hydrophilic substituents, such as the hydroxymethyl (10) group and dimethylaminomethyl (11) group, resulted in the loss of CYP11B2 inhibitory activity. Moreover, further enlargement of the methyl group at the N-1 position of the pyrazole ring to an ethyl group produced compound 12 (CYP11B2 IC50 = 72 nM), which demonstrated improved potency. These increases in CYP11B2 inhibitory activities following the introduction of alkyl groups at the N-1 and C-5 positions were due to hydrophobic interactions between the alkyl groups and the active site cavity of CYP11B2, and previous reports that state that the active site cavity of CYP11B2 is lined with nonpolar and aromatic residues favorable for binding steroid substrates support this observation.4 However, compounds 7−10 and 12 showed no CYP11B1 inhibitory activities. We anticipated that incorporation of a cyclic ring structure at the 1,5-position of the pyrazole ring would introduce favorable conformational constraint for interaction with the CYP11B2 cavity. In fact, the cyclized compound, 5,6-dihydro-4Hpyrrolo[1,2-b]pyrazole (13), showed dramatically improved potency (CYP11B2 IC50 = 13 nM) and high selectivity over CYP11B1 (Selectivity Factor = 440).


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Table 1. Structure-Activity Relationship (SAR) Data for Pyrazole Derivatives 6–13.

Microsomal metabolic R compd R stability (h)/(mky) (%)a Me H >1000 NT NT 6 Me Me 290 >10000 >35 62/42 7 Me Et 150 >10000 >67 73/21 8 Me CF3 280 >10000 >36 NT/0.9 9 Me CH2OH >1000 >10000 104/90 10 Me CH2NMe2 >1000 NT 90/54 11 Et Me 72 >10000 >140 43/10 12 -CH2CH2CH213 5700 440 12/3 13 LCI-699 (2) 1.2 22 18 NT a The percentage of the remaining substrate after 20 min incubation with NADPH is calculated from its concentration after 20 min divided by that at incubation time zero. h = human, mky = cynomolgus monkey. 1

2

hCYP11B2 IC50 (nM)

hCYP11B1 IC50 (nM)

Selectivity Factor

The substituent effects of the benzene ring are listed in Table 2. Introducing a halogen group, such as fluoro (14) or chloro (15), to the benzene ring at the meta position with respect to the nitrile group enhanced the CYP11B2 inhibitory activities. Furthermore, introduction of a methyl group at the meta position with respect to the nitrile group led to the most potent compound 16 (CYP11B2 IC50 = 3.7 nM), whereas a methoxy (17) group decreased the CYP11B2 inhibitory activities. Introduction of a methyl (18) or fluoro (19) group at the ortho position with respect to the nitrile group led to decreased CYP11B2 inhibitory activities in comparison with substituents at the meta position.


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Table 2. Structure-Activity Relationship (SAR) Data for Pyrazole Derivatives 13−19 and Imidazole Derivative 20.

Microsomal R metabolic stability compd R (h)/(mky) (%)a H H 13 5700 440 12/3 13 F H 9.2 1200 130 9/0.4 14 Cl H 5.4 480 89 10/0.3 15 Me H 3.7 350 95 22/10 16 OMe H 34 2200 65 4/0.4 17 H Me 6.7 590 88 0.4/0.3 18 H F 26 2100 81 1/0.4 19 2.5 58 23 75/75 20 a The percentage of the remaining substrate after 20 min incubation with NADPH is calculated from its concentration after 20 min divided by that at incubation time zero. h = human, mky = cynomolgus monkey. 3

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hCYP11B2 IC50 (nM)

hCYP11B1 IC50 (nM)

Selectivity Factor

To understand the structure-activity relationships (SAR) of these inhibitors, compound 16 was docked into human CYP11B2 using GOLD18 (Figure 3). The docking study revealed that the nitrogen of the 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole moiety was directly coordinated to the heme iron and that the benzonitrile ring engaged in aromatic interaction with the Trp116 side chain, occupying a similar position to that of FAD286. The methyl group at the meta position with respect to the nitrile group contributed to the stabilization of the twisted form between the pyrazole ring and the benzene ring, and interacted with the side chains of Phe231 and Thr318.These interactions would explain the reason for which compound 16 was 3.5 times as potent as compound 13. The hydrophobic interaction of the dihydropyrrole moiety with the Ile488 and Thr318 side chains may also contribute to potency improvement.


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Figure 3. Overlay of the compound 16 docking model (in green) and the crystal structure of CYP11B2 (in yellow) in complex with FAD286 (in orange) (PDB ID: 4FDH).

Replacement of the pyrazole moiety with an imidazole moiety (20) led to an increase in CYP11B2 inhibitory activity but decreased selectivity over CYP11B1 (Selectivity Factor = 23, Table 2). Compound 16 has a fused structure similar to that of imidazole-based inhibitor LCI699, with a 6,7-dihydro-5H-pyrrolo[1,2-c]imidazole moiety, and pyridine-based inhibitor 5, with a 5,6,7,8-tetrahydroisoquinoline moiety. Compared with these inhibitors, pyrazole-based inhibitor 16 had high selectivity over CYP11B1. From the above, it is presumed that the electronic character and steric effect of 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole could attenuate the magnitude of the interaction between pyrazole and the heme-iron motif. It is thought that CYP11B2 inhibitory activity is improved through the increased magnitude of the CYP11B2specific hydrophobic interactions, which may also lead to improved selectivity over CYP11B1. And our results may be consistent with studies of fugal CYP51 inhibitors suggesting that the magnitude of the Fe-N interaction could be tuned by modulating the heterocycles’ electronic character and that weaker coordination may help minimizing off-target inhibitory activity,19 and


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with another study suggesting that introducing a substituent ortho to the nitrogen to coordinate the heme-iron motif would weaken the magnitude of the Fe-N interaction.16 We found that compound 16 had inhibitory activity toward CYP11B2, with high potency and selectivity over CYP11B1; however, the metabolic stability of a series of N-alkyl pyrazole derivatives 13−19 was unsatisfactory for oral dosing. To improve metabolic stability, we attempted to introduce a hydrophilic substituent at the C-5 position of the 5,6-dihydro-4Hpyrrolo[1,2-b]pyrazole ring based on the results of the docking study (Figure 3). The introduction of a hydroxyl group with (R)-configuration at the C-5 position of 5,6-dihydro-4Hpyrrolo[1,2-b]pyrazole produced 21, with significantly improved metabolic stability, but slightly reduced inhibitory activity (CYP11B2 IC50 = 12 nM). Moreover, compound 21 displayed increased selectivity over CYP11B1 (Selectivity Factor = 160) compared with unsubstituted compound 16.


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Table 3. Structure-Activity Relationship (SAR) Data for Pyrazole Derivatives 16, 21−32.

compd

R5

R6

hCYP11B2 IC50 (nM)

16 21 22 23 24 25

H OH OMe OCH2Ph OCH2COOH OCH2CONH2

H H H H H H

3.7 12 3.2 2.3 >1000 14

350 1900

(CYP11B2) Inhibitors - ACS Publications - American Chemical Society

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