Accepted Manuscript 6,6-Fused heterocyclic ureas as highly potent TRPV1 antagonists Wei Sun, Hyo-Shin Kim, Sunho Lee, Aeran Jung, Sung-Eun Kim, Jihyae Ann, Suyoung Yoon, Sun Choi, Jin Hee Lee, Peter M. Blumberg, Robert FrankFoltyn, Gregor Bahrenberg, Klaus Schiene, Hannelore Stockhausen, Thomas Christoph, Sven Frormann, Jeewoo Lee PII: DOI: Reference:

S0960-894X(14)01394-8 http://dx.doi.org/10.1016/j.bmcl.2014.12.086 BMCL 22331

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

3 December 2014 23 December 2014 25 December 2014

Please cite this article as: Sun, W., Kim, H-S., Lee, S., Jung, A., Kim, S-E., Ann, J., Yoon, S., Choi, S., Lee, J.H., Blumberg, P.M., Frank-Foltyn, R., Bahrenberg, G., Schiene, K., Stockhausen, H., Christoph, T., Frormann, S., Lee, J., 6,6-Fused heterocyclic ureas as highly potent TRPV1 antagonists, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.086

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Graphical Abstract

6,6-Fused Heterocyclic Ureas as Highly Potent TRPV1 Leave this area blank for abstract info. Antagonists Wei Sun, Hyo-Shin Kim, Sunho Lee, Aeran Jung, Sung-Eun Kim, Jihyae Ann, Suyoung Yoon, Sun Choi, Jin Hee Lee, Peter M. Blumberg, Robert Frank-Foltyn, Gregor Bahrenberg, Klaus Schiene, Hannelore Stockhausen, Thomas Christoph, Sven Frormann, Jeewoo Lee*

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com

6,6-Fused Heterocyclic Ureas as Highly Potent TRPV1 Antagonists Wei Sun a,b, Hyo-Shin Kim b, Sunho Lee b, Aeran Jung b, Sung-Eun Kim b, Jihyae Ann b, Suyoung Yoon b, Sun Choi c, Jin Hee Lee c, Peter M. Blumberg d, Robert Frank-Foltyn e, Gregor Bahrenberg e, Klaus Schiene e , Hannelore Stockhausen e, Thomas Christoph e, Sven Frormann e, Jeewoo Lee b,* a

Shenyang Pharmaceutical University, Shenyang 110016, China Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea c National Leading Research Laboratory of Molecular Modeling & Drug Design, College of Pharmacy, Graduate School of Pharmaceutical Science and Global Top 5 Research Program, Ewha Womans University, Seoul 120-750, Korea d Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA e Grunenthal Innovation, Grunenthal GmbH, D-52078 Aachen, Germany b

ARTICLE INFO

ABSTRACT

Article history: Received Revised Accepted Available online

A series of N-[{2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)-pyridin-3-yl}methyl] N’-(6,6-fused heterocyclic) ureas have been investigated as hTRPV1 antagonists. Among them, compound 15 showed highly potent TRPV1 antagonism to capsaicin, with Ki(ant) = 0.2 nM, as well as antagonism to other activators, and it was efficacious in a pain model. A docking study of 15 with our hTRPV1 homology model indicates that there is crucial hydrogen bonding between the ring nitrogen and the receptor, contributing to its potency.

Keywords: Vanilloid Receptor 1 TRPV1 Antagonist Analgesic Molecular Modeling

2009 Elsevier Ltd. All rights reserved.

The transient receptor potential V1 (TRPV1) receptor1 is a molecular integrator of nociceptive stimuli, including protons, heat, inflammatory endogenous mediators and natural vanilloids such as capsaicin and resiniferatoxin. Since the receptor activation leads to an increase in intracellular Ca2+ that results in excitation of primary sensory neurons and ultimately the central perception of pain, TRPV1 antagonists have been actively developed as novel analgesic and antiinflammatory agents, particularly for chronic pain and inflammatory hyperalgesia.2 Although hyperthermia associated with TRPV1 antagonism is in some instances problematic, TRPV1 is still one of most attractive drug targets for the treatment of neuropathic pain. The clinical development of TRPV1 antagonists in the pharmaceutical industry has been extensively reviewed.3 Previously, the Abbott group reported a series of ureas with a variety of 6,6-fused nitrogen heterocycles as TRPV1 antagonists, among which the 5-isoquinoline-containing compound 1 exhibited good oral bioavailability and in vivo activity in animal models (Figure 1).4 Similarly, the Johnson & Johnson group had described N-isoquinoline-5-yl-N’-aralkyl ureas and amide antagonists, among which compound 2 exhibited excellent potency in receptor binding and antagonism

(Figure 1).5 The pharmacophoric analysis of these leads indicates that the 5-isoquinolinyl urea as the A/B-region and 4-substituted benzyl group as the C-region represent principal pharmacophores, respectively. As part of our continuing program for the discovery of novel TRPV1 antagonists for the treatment of neuropathic pain, we have investigated a set of novel antagonists with a N-6,6fused heteroaryl urea moiety as the A/B-region and a library of substituted heteroaryls as the C-region, which was designed based on the our pharmacophore model with the above leads.

Figure 1. Discovery of potent TRPV1 antagonists

The extensive and systematic structure-activity relationship investigation of the series indicated that the 5-isoquinolinyl urea moiety as the A/B-region and a series of {2-substituted-6(trifluoromethyl)-pyridin-3-yl}methyl moieties as the C-region6 contributed high potency in antagonism. Among them, we identified compound 15 to be highly potent for antagonism of capsaicin, with an IC50 = 0.2 nM, as well as a potent antagonist for stimulation by heat or N-arachidonoyl dopamine (NADA), and it is thus about 90 fold more potent than the parent compound 1 under the same conditions. In this paper, we report the structure activity relationships of the A-region with 6,6-fused heterocycles in the series while the B/C-region was fixed as the N-[{2-(4-methylpiperidin-1-yl)6-(trifluoromethyl)-pyridin-3-yl}methyl]urea group. Further biological characterization and molecular modeling of compound 15 will also be described. The target compounds (3-21) were synthesized in general by the carbamylation of the aryl amine A-region with phenyl or trichloromethyl chloroformates, followed by coupling with the pyridylmethyl amine of the C-region, as represented in Scheme 1.

Scheme 1. Reagents and conditions: (a) Method A: PhOCOCl, pyridine, CH3CN-THF or Method B: CCl3COCl, TEA, CH2Cl2; (b) Method A: R-NH2, TEA, DMSO or Method B: R-NH2, DBU, CH3CN

The C-region amine, 2-(4-methylpiperidin-1-yl)-6(trifluoromethyl)-pyridin-3-methylamine, was synthesized by the nucleophilic addition of 4-methylpiperidine to 2-chloro-6trifluoromethyl-3-pyridinecarbonitrile which was prepared in three steps starting from ethyl vinyl ether as shown in Scheme 2.

aminoquinazoline for 11 and 12 were synthesized from the anilines, 1,3-diaminobenzene and 1,4-diaminobenzene, respectively, by Chilin’s method8 (Scheme 4). 8-Amino isoquinoline for 14 was prepared by catalytic hydrogenation from commercially available 5-bromo-8-nitro isoquinoline (Scheme 5).4 4-Amino quinoline for 17 was prepared from commercially available 4-nitroquinoline N-oxide using Fe/AcOH reduction (Scheme 6). 4-Amino isoquinoline for 18 was prepared from commercially available 4-bromoisoquinoline in two steps (Scheme 7). 5-Amino quinoxaline for 20 was prepared from 3nitro-1,2-diaminobenzene according to the literature procedure (Scheme 8). 10

Scheme 3. Reagents and conditions: (a) NaBH4, BF3 etherate, 1,2dimethoxyethane, >99%; (b) oxalyl chloride, DMSO, NEt3, CH2Cl2, 50%; (c) NH2NH2·H2O, CH2Cl2, EtOH, 51%; (d) H2, 10% Pd/C, MeOH, 95%

Scheme 4. Reagents and conditions: (a) ClCO2Et, NEt3, THF, 90-97%; (b) (1) HMTA, TFA and (2) KOH, K3Fe(CN)6, EtOH, H2O, 32-47%; (c) CCl3COCl, NEt3, CH2Cl2, 75-78%; (d) R-NH2, DBU, MeCN, 32-39%

Scheme 5. Reagents and conditions: (a) H2, 10% Pd/C, MeOH, >99%

Scheme 6. Reagents and conditions: (a) Fe, AcOH, >99%

Scheme 2. Reagents and conditions: (a) (CF3CO)2O, pyridine, CHCl3, 50%; (b) NCCH2CONH2, K2CO3, toluene, 35%; (c) POCl3, 90%; (d) 4methylpiperidine, K2CO3, 18-C-6, CH3CN, 90%; (e) 2M BH3-SMe2 in THF, 70%

The A-region 6,6-fused aryl amines were either obtained from commercial sources or prepared by the modification of known methods as illustrated in Schemes 3-8. The A-region amines for the syntheses of 3-9, 13, 15, 16, 19, and 21 were commercially available. 6-Amino phthalazine for 10 was prepared from 4-nitro phthalic acid in four steps according to the literature procedure (Scheme 3).7 7-Aminoquinazoline and 6-

Scheme 7. Reagents and conditions: (a) benzophenoneimine, Pd(OAc)2, NEt3, Cs2CO3, DPPF, BINAP, toluene, 58%; (b) 2M HCl, THF, EtOH, 97%

Scheme 8. Reagents and conditions: (a) glyoxal, EtOH, 90%; (b) Fe, AcOH, 76%

Table 1. In vitro hTRPV1 antagonism to capsaicin

The synthesized compounds were evaluated for their ability to inhibit the effect of the given concentration of capsaicin (100 nM) on hTRPV1 as determined using a fluorescence imaging plate reader (FLIPR) and inhibitory values were expressed as (f) Ki(ant).6a The results are presented in Table 1, together with the potencies of the parent antagonists 1 and 2. The A-region of the synthesized compounds has one of the two geometries, –fused (compounds 3-12) or –fused (compounds 13-21), depending on the relative position of the Aregion to the B-region urea. Among the –fused analogues, compounds 3-9 include 7 different mono-aza heterocycles and their antagonism appears to be sensitive to the position of the nitrogen. Generally, whereas the distal aza-compounds 4-6 showed good antagonism with Ki(ant) values ranging from 7-9 nM, the proximal aza-compounds 3 and 9 were weak or inactive although compounds 7 and 8 were exceptional. The diaza analogues phthalazine 10 and quinazoline 11 showed similar potency to the distal aza surrogates, whereas quinazoline 12 was found to be an agonist.

R

R

(f) Ki(ant) (nM) 13.8

2

-Fused

The SAR analysis indicates that a hydrogen bonding interaction between the ring nitrogen of the A-region and the receptor is critical for antagonism, as indicated by the positional sensitivity of the ring nitrogen for activity, and further indicates that the ring nitrogen needs to be present at the distal area of the ring. Additional hydrogen bonding at the proximal area may also be important because compounds 8 and 18, containing a proximal nitrogen at the same position, also showed good potency.

The analgesic activity of compound 15 was examined in the formalin animal model. When administered orally in mice, compound 15 was found to be efficacious at 0.3 and 10 mg/kg, respectively, where it showed 18% and 73% maximal possible effect (% MPE). The detailed in vivo evaluation will be presented elsewhere.

18.3

1

Among the –fused A-region analogues, compounds 13-19 also represent the 7 different mono-aza analogues as above. As found for the –fused derivatives, the distal aza-analogues 14-16 also exhibited excellent antagonism in the subnanomolar range. In particular, isoquinolin-5-yl urea 15 displayed highly potent antagonism with Ki = 0.2 nM, which was about 90 fold more potent than compound 1 under the same conditions. The structural comparision of the C-regions between compounds 1 and 15 indicated that the 4-methylpiperidinyl moiety in compound 15 contributes high potency in antagonism probably due to enhanced specific binding interactions with the receptor. On the other hand, the proximal aza-analogues 17-19 showed quite different functional profiles. Whereas compound 18 showed potent antagonism, compounds 17 and 19 exhibited agonism and loss of activity, respectively. The diaza analogues quinoxaline 20 and naphthyridine 21 displayed reduced potency.

Since compound 15 was identified as the most potent antagonist to capsaicin in this series, its antagonism to other activators including pH, N-acetyl dopamine (NADA), and heat was also evaluated as shown in Table 2. The potency of compound 15 for antagonism to capsaicin was ca. 70-90 fold higher than reference compounds 1 and 2 under the same conditions. In addition, compound 15 also antagonized Nacetyldopamine (NADA) and heat with high potency. However, it appeared not to antagonize pH activation under our system.

(f) Ki(ant) (nM)

-Fused

3

NE

13

91.6

4

7.1

14

0.4

5

8.4

15

0.2

6

7.2

16

0.7

7

WE

17

AG

8

9.5

18

1.8

9

WE

19

NE

10

8.9

20

48.1

11

7.8

21

NE

12

AG

AG: agonism, WE: weakly effective (7: 40% inhibition at 1 uM, 9: 10% inhibition at 1 uM), NE: not effective a

Table 2. in vitro antagonism of compound 15 for various activators of hTRPV1 CAP (f) Ki (nM)

pH IC50 (nM)

NADA (f) Ki (nM)

HEAT(45oC) IC50 (nM)

0.2

NE

0.02

0.55

(A)

(B)

(C)

LP 0.14

Leu547 Thr550

0.12 0.09 0.07 0.05 0.03 0.01 -0.02 -0.04

Leu515

-0.06 -0.08 -0.11

Tyr511

-0.13 -0.15 -0.17 -0.19

Ser512

Figure 2. Flexible docking result of 15 in the hTRPV1 model (A) Binding mode of 15. The key interacting residues are marked and displayed as a capped-stick with carbon atoms in white. The helices are colored in gray and the helices of the neighboring monomer are displayed as a line ribbon. The ligand is depicted as a ball-and-stick with carbon atoms in magenta and its van der Waals surface is presented with lipophilic potential property (LP) which ranges from brown (highest lipophilic area) to blue (highest hydrophilic area). Hydrogen bonds are drawn in black dashed lines and non-polar hydrogens are undisplayed for clarity. (B) Surface of hTRPV1 and 15. The Fast Connolly surface of hTRPV1 was generated by MOLCAD and colored by the lipophilic potential. For clarity, the surface of hTRPV1 is Z-clipped and that of the ligand is in its carbon color. (C) Lipophilic potential property of 15.

In order to investigate the key interactions of compound 15 with the receptor, we carried out the docking study of compound 15 with the human TRPV1 homology model, which was built on our rat TRPV1 homology model as reported recently11 and modified to include the sequence differences between rat and human TRPV1 in the vicinity of the binding site. As shown in Figure 2, compound 15 fitted well to the binding site of hTRPV1 which has a deep bottom hole, surrounded by Tyr511 and Ser512, and an upper hydrophobic region containing Leu547. The isoquinoline group of 15 in the A-region occupied the deep bottom hole and formed a hydrogen bonding with Ser512. In addition, its urea group as the B-region interacted with Tyr511 by hydrogen bonding. The C-region of 15 oriented toward the upper region of the binding site and the hydrophobic CF3 group fitted very well, making the hydrophobic interaction with Leu547. This docking result identified binding interactions between the three regions of 15 and hTRPV1 and the high potency of 15 can be understood in terms of criticial hydrogen bonding between the isoquinoline nitrogen and Ser512.

In summary, we have investigated a series of N-[{2-(4methylpiperidin-1-yl)-6-(trifluoromethyl)-pyridin-3-yl}methyl]N’-(6,6-fused heterocylic) ureas as hTRPV1 antagonists. Among them, compound 15 showed highly potent TRPV1 antagonism to capsaicin, which was 70-90 fold more potent than lead compounds 1 and 2; it further antagonized against stimulation by heat and was efficacious in the formalin pain model. Molecular modeling analysis with our hTRPV1 homology model provides the binding mode of compound 15 with the receptor in which hydrogen bonding between the A-ring nitrogen and Ser512 is critical for high potency.

Acknowledgments This research was supported by Grants from Grunenthal, the Korea Health Technology R&D Project (HI13D2358), National Research Foundation of Korea (R11-2007-107-02001-0), the National Leading Research Lab program (2011-0028885), and in part by the Intramural Research Program of NIH, Center for Cancer Research, NCI (Project Z1A BC 005270).

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