ACE inhibitors for treating metabolic syndrome

Bioorganic & Medicinal Chemistry Letters 27 (2017) 2313–2318

Contents lists available at ScienceDirect

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

Approaches towards the development of chimeric DPP4/ACE inhibitors for treating metabolic syndrome Jitendra A. Sattigeri ⇑, Sachin Sethi, Joseph A. Davis, Shahadat Ahmed, Geeta V. Rayasam, Balasaheb G. Jadhav, Satya M. Chilla, Dhrubajyoti Datta, A. Gadhave, Vamshi K. Tulasi, Tarun Jain, Sreedhara Voleti, Biju Benjamin, Sunitha Udupa, Garima Jain, Yogender Singh, Kona Srinivas, Vinay S. Bansal, Abhijit Ray, Pradip K. Bhatnagar, Ian A. Cliffe New Drug Discovery Research, R&D III, Ranbaxy Laboratories Limited, Plot 20, Sector 18, Udyog Vihar, Gurgaon, Haryana 122015, India

a r t i c l e

i n f o

Article history: Received 23 January 2017 Revised 24 March 2017 Accepted 12 April 2017 Available online 13 April 2017 Keyword: DPP4-ACE dual inhibitor

a b s t r a c t Designing drug candidates exhibiting polypharmacology is one of the strategies adopted by medicinal chemists to address multifactorial diseases. Metabolic disease is one such multifactorial disorder characterized by hyperglycaemia, hypertension and dyslipidaemia among others. In this paper we report a new class of molecular framework combining the pharmacophoric features of DPP4 inhibitors with those of ACE inhibitors to afford potent dual inhibitors of DPP4 and ACE. Ó 2017 Elsevier Ltd. All rights reserved.

Diabetes is a growing epidemic in both the developing and developed world. It has been estimated that there are
420 million diabetics globally with a projected global prevalence of 640 million in 2040.1 More than 70% of people suffering from Type 2 diabetes are hypertensive (blood pressure  140/90 mmHg).2 Additionally, studies have identified the risk of developing type 2 diabetes is much higher in people with hypertension than in normotensives.3 An ideal treatment for this subset of diabetics would not only address their hyperglycemia but also the hypertensive component of the disease. DPP4 (dipeptidyl peptidase-4) inhibitors and ACE (angiotensin converting enzyme) inhibitors have been the mainstay for treating hyperglycaemia and hypertension in these patients. However, patient compliance becomes an issue when multiple drugs need to be taken on a long-term basis. In addition, there is the risk of enhanced drug-drug interactions when multiple drugs are prescribed. As a consequence, availability of a single therapeutic agent which would simultaneously ameliorate the pharmacological processes contributing to both hypertension and diabetes could be a significant step forward in the treatment of metabolic syndrome. Here in, we report a designed multiple ligand approach to identify dual inhibitors which incorporate all the structural elements required for inhibition of DPP4 and ACE. One of the strategies for designing ligands capable of binding to two phylogenetically diverse biological targets is to merge the structural elements of two pharmacophores.4 Herein, the identifi⇑ Corresponding author. E-mail address: [email protected] (J.A. Sattigeri). http://dx.doi.org/10.1016/j.bmcl.2017.04.036 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

Fig. 1. Key structural features for DPP4 and ACE inhibition: (a) Enalapril binding in ACE (PDB ID – 1UZE5) (b) Sitagliptin binding in DPP4 (PDB ID – 1X706).

2314

J.A. Sattigeri et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2313–2318

cation of molecular points on both ligands which would allow for merging of the two structures is the most critical step. Indeed, a detailed analysis of the pharmacophoric features, the size of the enzyme active sites and knowledge of the tolerability of the respective lead compounds to structural variations is an important exercise which aids in the design of dual merged ligands. In our work we critically evaluated ligand-DPP4 and ligand-ACE co-crystal structures5,6 (Fig. 1) to design dual inhibitors. Thus, ACE inhibitors (Fig. 1a) need a carboxy group to bind to the S20 site, a zinc chelating group (COOH or SH), and a hydrophobic group that fits into the S1 pocket. ACE crystal structure further suggests that the S20 pocket is big enough to accommodate large groups. From the structures of fosinoprilat (2) and zofenopril (3) (Chart 1),7 it appears that position 4 of the proline ring in enalaprilat (1) is tolerant to further substitution without loss of activity and that these groups are well tolerated in S20 pocket. DPP4 inhibitors, on the other hand, require a hydrophobic moiety (e.g. trifluorophenyl) or an electrophilic group (e.g. CN) to interact with a serine at the S1 site and a basic amine to form a salt bridge with the glutamic acid diad at the S2 site. A similar analysis of crystal structure of the DPP4 enzyme (Fig. 1b) suggests that the S2 pocket can

accommodate large groups as in sitagliptin (4) and similar compound (5), (Chart 1).8 Thus, based on the structural aspects of the ligands and enzymes, it appears that enalaprilat and sitagliptin can be merged to form a dual inhibitor by removing the relatively unessential triazolopiperidine of 4 and forming an amide bond with an amino group placed at the fourth position of the proline residue of 1. Thus, the proposed dual inhibitor ligands 6a–d incorporate all the essential structural elements required for DPP4 and ACE inhibition (Scheme 1). Molecular docking simulations14 were carried out for ligands 6a–d in the ACE crystal structure (1O86)5 and DPP4 crystal structure (2G5P).6 Comparison of the binding mode of 6a–d (Fig. 2) with enalaprilat (Fig. 1a) in ACE shows that the phenethyl group (6a–d, enalaprilat) occupies the S1 pocket and the adjacent carboxylic acid group (6a–d, enalaprilat) lies in the metal binding region. The R groups of 6a–d and the methyl group of enalaprilat fit into the S10 pocket and the pyrrolidine group with the carboxylic acid (6a–d, enalaprilat) positions itself into the S20 pocket. Comparison of the binding mode of 6a–d (Fig. 3) with sitagliptin (Fig. 1b) in DPP4 shows that the 2,4,6-triflurophenyl group and the basic amine moiety (6a–d, sitagliptin) binds in the S1 pocket. The

Chart 1. Representative DPP4 and ACE inhibitors.

Scheme 1. DPP4-ACE dual inhibitors from the merger of enalaprilat 1 and sitagliptin 4.

J.A. Sattigeri et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2313–2318

2315

Fig. 2. The complex of ACE with 6a–d. ACE protein is shown as a white color Ca trace. The active site channel is generated using MOLCAD and is shown as a white transparent surface. The zinc atom is shown as a white ball in the Metal Binding Region. 6a (yellow), 6b (orange), 6c (green) and 6d (cyan) are shown as ball and stick model.

Fig. 3. The complex of DPP4 with four unique conformations of 6a–d. DPP4 protein is shown as a white Ca trace. The active site channel is generated using MOLCAD and is shown as a white transparent surface. 6a (yellow), 6b (orange), 6c (green) and 6d (cyan) are shown as ball and stick model. The S1 pocket has a well-defined alignment of poses but different poses are generated in the S2 pocket.

S1 pocket is closed and offers tight binding to the docked poses of the ligands, all of which maintain the same alignments and amino acid interactions, as have been reported for other known DPP4 inhibitors.9 Fig. 1b shows that the S2 pocket is wide enough to accommodate bulkier substitutions. Molecular docking supports this hypothesis and it was observed that the ACE pharmacophore of 6a–d binds into the S2 pocket of DPP4 (Fig. 3). The ligands 6a–d were synthesized by standard synthetic and peptide coupling methods (Scheme 2). Amine 710was coupled with b-amino acid 810using EDCI to afford 9 from which the Cbz group was removed to yield the key intermediate 10. The proposed

DPP4-ACE dual inhibitors 6a–d were synthesized from 10 in a simple three-step protocol by coupling the intermediate 10 with 13a–d under EDCI coupling conditions followed by sequential deprotection of the Boc and ester groups of 11a–d. The homophenylalanine derivatives 13a–d were prepared according to a literature procedure.11 Merged ligands 6a–d have high MW (>500) and PSAs (162 Å2) (Table 1). These compounds exhibit gradual increase in c Log Ps with increasing alkyl chain length from CH3 to n-Bu (4.23–5.78 respectively). Also, the number of rotatable bonds increases from 14 to 17 on going from 6a to 6d respectively.

2316

J.A. Sattigeri et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2313–2318

Scheme 2. Synthesis of 6a–d.15 (a)EDCI, HOBt, TEA, DMF (dry), 72%; (b) Pd/C, MeOH, ammonium formate, 62%; (c) EDCI, HOBt, TEA, 13a–d, DMF (dry), 41–63% (d) TFA, DCM (dry), 59–75%; (e) LiOH, THF, water, 63–97%.

Table 1 Calculated molecular properties of 1, 4 and designed merged ligands 6a–d. Compound

Mol Wt

c Log P

PSA Å2

Rot Bond

1 4 6a 6b 6c 6d

348.39 407.3 578.58 592.61 606.63 620.66

3.63 2.06 4.23 4.74 5.25 5.76

107 77 162 162 162 162

8 5 14 15 16 17

Next, compounds 6a–d were evaluated in vitro for their ability to inhibit12,13 ACE and DPP4. For this purpose, plasma of Wistar rat, ob/ob mouse and human was used as enzyme source for ACE and DPP4. Table 2 shows the inhibitory activities for compounds 6a–d and the reference standards 1 and 4. As expected compound 1 inhibited ACE potently (IC50 < 11 nM) and did not inhibit DPP4 even at concentration of 100 lM. Likewise, 4 showed potent inhibition of DPP4 (IC50 < 50 nM) across species but showed only weak inhibition potential for ACE enzyme. To our delight, compounds 6a–d, incorporating the features of both ACE and DPP4 inhibitors,

2317

J.A. Sattigeri et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2313–2318 Table 2 Inhibition of DPP4 and ACE by 1, 4 and 6a–d. F F

NH2 F

O

O

N H

OH O

N

R O

H N OH

DPP4 Inhibition* (IC50 nM)

Compound

R

1 4 6a 6b 6c 6d

– – Me Et n-Pr n-Bu

ACE Inhibition* (IC50 nM)

Rat (Wistar)

Mouse (ob/ob)

Human

Rat (Wistar)

Mouse (ob/ob)

Human

N.D. 33 155 (0.61) 319 (5.6) 1230 (18.4) 4170 (40.9)

N.D. 46 217 (0.85) 488 (8.6) 2470 (36.9) 7170 (70.3)

>100 lM 20 255 57 67 102

9.6 N.D. 334 (2.1) 24 (2.8) 893 (17.5) 2800 (10.0)

11.5 N.D. 58 (0.37) 100 (11.6) 2023 (39.7) 2210 (7.9)

2.5 11 lM 158 8.6 51 279

Data represents average values of at least two independent experiments done in triplicates. Values in parenthesis indicate fold shift in IC50s on using rat/mouse enzymes over that observed with human enzymes. N.D. – Not determined. * Plasma from Wistar rat, ob/ob mouse and human was used as source of DPP4 and ACE enzymes.

exhibited dual inhibition of human ACE and DPP4. Compounds 6b (ACE IC50 = 8.6 nM; DPP4 IC50 = 57 nM) and 6c (ACE IC50 = 51 nM; DPP4 IC50 = 67 nM) were found to be the most potent dual inhibitors of human ACE and DPP4. Compounds 6a and 6d also displayed dual inhibition of ACE and DPP4 but at relatively higher concentrations (IC50 > 100 nM). Compound 6a moderately inhibited human ACE. The inter-species shifts in ACE IC50 values for compound 6a were not significant (within the range of experimental error). Compound 6b exhibited a very potent inhibition of human ACE (IC50 = 8.9 nM). However, 6b showed a considerable shift (12-fold) in mouse ACE inhibition (IC50 = 100 nM) and up to 3-fold shift in rat ACE inhibition (IC50 = 24 nM). Compounds 6c and 6d also exhibited a considerable loss of inhibitory potential against rat ACE (seventeen- and ten-fold, respectively) and mouse ACE (forty- and eight-fold, respectively). While compound 6a exhibited similar inhibitory potencies against DPP4 across all three species, a progressive loss in rat and mouse DPP4 inhibitory potency was observed for compounds 6b–d with the greatest loss being seen for mouse DPP4 (
2-fold more than that for rat DPP4). The plasma protein binding

Table 3 Plasma protein binding data (%) for compounds 6a–d, 1 and 4. Plasma

6a

6b

6c

6d

1

4

Human Mouse Rat

29.3 49 42

36.4 87.1 59.7

40.3 98.5 43.6

67.3 98.6 41.7

65.9 57.6 54.4

47.0 29.3 41.3

(PPB) for 6a–d was found to increase in human and mouse plasma with increasing length of R (Table 3) which can be correlated with increasing c log P (Table 1); the inherent PPB in mouse plasma being higher than in human plasma. Thus, the upward shift in IC50 of compound 6b–d in mouse ACE and DPP4 (plasma being used as enzyme source) can be explained to a certain extent based on increased PPB in mouse plasma. However, the shift in IC50s in rat ACE and DPP4 (plasma being used as enzyme source) cannot be explained based on PPB. These unusual shifts were not noted for the reference compounds 1 and 4. Compounds 6a–d were profiled for their inhibitory activity against dipeptidyl peptidase-IV Activity and/or Structure Homologues (DASH) [e.g. DPP2, DPP8 and DPP9] and non-DASH [eg. post-proline cleaving enzyme (PPCE), neutral endopeptidase (NEP), aminopeptidase P (APP), aminopeptidase N (APN)] enzymes. Compounds 6b–d exhibited over one thousand-fold selectivity for DPP4 over DPP2, DPP8 and DPP9 enzymes (Table 4), while Compound 6a was approximately one hundred-fold selective. Compounds 6b–d exhibited high selectivity (>1000 fold) for nonDASH enzymes PPCE, NEP, APP and APN. While 6a showed >1000 fold selectivity for NEP, APP and APN, it was only
100 fold selective for PPCE. Compounds 6a–d showed that they had no liability for inhibiting common cytochrome P-450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) up to a concentration of 10 lM. Furthermore, the compounds were found to be stable to human, mouse, rat and dog liver microsomes. These compounds had very low Caco-2 permeabilities, which is to be expected from the higher values of calculated molecular properties.

Table 4 Selectivity against DASH and non-DASH enzymes. Compound

4 6a 6b 6c 6d €

IC50 (lM) DPP8€

DPP9€

DPP2

PPCE

NEP

APP

APN

>100 27 (105) 69 (1210) 57 (850) >100 (>1000)

>100 44 (172) >100 (>1700) >100 (>1500) >100 (>1000)

>100 >100 >100 >100 >100

>100 31.6 >100 >100 >100

>100 >100 >100 >100 >100

>100 >100 >100 >100 >100

>100 >100 >100 >100 >100

DASH: DPP4 structure and activity homologues; Numbers in parenthesis represent the fold selectivity over human DPP4.

2318

J.A. Sattigeri et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2313–2318

Table 5 Plasma exposures of compounds in Wistar rats following oral dosing (30 mpk).

6a 6b 6c 6d

Tmax (h)

Cmax (lg/mL)

AUC0-24h (lg.h/mL)

AUC0-1 (lg.h/mL)

1.50 ± 0.87 1.67 ± 0.58 3.3 ± 1.2 2.0 ± 00

0.26 ± 0.09 0.55 ± 0.25 2.5 ± 0.51 1.33 ± 0.29

0.61 ± 0.27 2.36 ± 0.8 21.49 ± 4.11 13.94 ± 1.81

1.17§ 2.86 ± 0.67 23. 49 ± 4.75 16.77 ± 1.31

Values are presented as mean ± SD, n = 3. Mean values of two rats are reported because the terminal phase of one rat was not well defined.

§

Despite these compounds showing very low Caco2 permeability, the plasma exposure profiles of compounds 6a–d were studied at 30 mpk in Wistar rats following oral dosing (Table 5). Compound 6a exhibited a very low Cmax. and AUC (0.26 lg/mL and 0.61 lg.h/mL, respectively). Compound 6b exhibited a slightly improved plasma exposure (Cmax = 0.55 lg/mL; AUC = 2.36 lg.h/mL), while compound 6c exhibited good plasma exposure (Cmax = 2.51 lg/mL; AUC = 23.49 lg.h/mL). Compound 6d also exhibited a good Cmax and AUC values (Cmax = 1.33 lg/mL; AUC = 13.94 lg.h/mL); however these concentrations were lower than those for 6c. To conclude, rational de novo design and synthesis has been used to identify compounds having a potent and balanced in vitro potency against both human DPP4 and ACE. The compounds have a favourable in vitro DMPK profile. While compound 6b exhibited moderate oral plasma exposure, compounds 6c and 6d exhibited high plasma oral exposure in rats. We have thus demonstrated that a merged multiple ligand approach is a feasible strategy for simultaneously inhibiting DPP4 and ACE enzymes. Acknowledgements Authors would like to thank Dr. Tridib Chaira for PK studies, Dr. Vanya Shah, for generating the plasma protein binding data, Mr. M. Bandgar for providing a few intermediates. We wish thank Mr. Abhishek Gupta, Mr. Varun Jain and Mr. Sreekumar V.B for recording the MS and HPLCs.

6.

7.

8.

9. 10.

11.

12.

References 1. Reusch JE, Manson JE. Management of type 2 diabetes in 2017: getting to goal. JAMA. 2017;317:1015–1016. 2. (a) National High Blood Pressure Education program working group report on hypertension in diabetes . Hypertension. 1994;23:145–158; (b) Sowers JR, Epstein M, Frohlich ED. Diabetes, hypertension, and cardiovascular disease: an update. Hypertension. 2001;37:1053–1059. 3. Gress TW, Nieto FJ, Shahar E, Wofford MR, Brancati FL. Hypertension and antihypertensive therapy as risk factors for type 2 diabetes mellitus. N Engl J Med. 2000;342:905–912. 4. (a) Morphy R, Rankovic Z. Designed multiple ligands. an emerging drug discovery paradigm. J Med Chem. 2005;48:6523–6543; (b) Peters JU. Polypharmacology – Foe or friend. J Med Chem. 2013;56:8955–8971. 5. (a) Natesh R, Schwager SLU, Sturrock ED, Acharya KR. Crystal structure of the human angiotensin-converting enzyme-lisinopril complex. Nature. 2003;421:551–554; (b) Natesh R, Schwager SLU, Evans HR, Sturrock ED, Acharya KR. Structural

13.

14.

15.

details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme. Biochemistry. 2004;43:8718–8724. (a) Pei Z, Li X, Longenecker K, et al. Discovery, structure-activity relationship and pharmacological evaluation of (5-substituted-pyrrolidinyl-2-carbonyl)-2cyanopyrrolidines as potent dipeptidyl peptidase IV inhibitors. J Med Chem. 2006;49:3520–3535; (b) Kim D, Wang L, Beconi M, et al. (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6dihydro[1,2,4]triazolo[4,3-a]pyrazin- 7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan2-amine: A potent, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J Med Chem. 2005;48:141–151. Krapcho J, Turk C, Cushman DW, et al. Angiotensin-converting enzyme inhibitors. Mercaptan, carboxyalkyl dipeptide, and phosphinic acid inhibitors incorporating 4-substituted prolines. J Med Chem. 1988;31:1148–1160. (a) Biftu T, Feng D, Qian X, et al. (3R)-4-[(3R)-3-Amino-4-(2,4,5-trifluorophenyl) butanoyl]-3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one, a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Bioorg Med Chem Lett. 2007;17:49–52; (b) Kim D, Kowalchick JE, Brockunier LI, et al. Discovery of potent and selective dipeptidyl peptidase IV inhibitors derived from b-aminoamides bearing substituted triazolopiperazines. J Med Chem. 2008;51:589–602. Kuhn B, Hennig M, Mattei P. Molecular recognition of ligands in dipeptidyl peptidase IV. Curr Top Med Chem. 2007;7:609–619. (a) Yelin EA, Onoprienko VV, Kudelina IA, Miroshnikov AI. The synthesis of isomeric 4-prolinylamines and 4,40 -diprolinylamines. Russ J Bioorg Chem. 2000;26:774–783(b) Dreher SD, Ikemoto N, Njolito E, Rivera NR, Tellers DM. Process to chiral beta- amino acid derivatives. WO 2004/085661. (a) Bhagwat SS, Fink CA, Gude C, et al. 4-Substituted proline derivatives that inhibit angiotensin converting enzyme and neutral endopeptidase 24.11. Bioorg Med Chem Lett. 1994;4:2673–2676; (b) Iwasaki G, Kimura R, Numao N, Kondo K. A practical and diastereoselective synthesis of angiotensin converting enzyme inhibitors. Chem Pharm Bull. 1989;37:280–283. (a) Schwager SL, Carmona AK, Sturrock ED. A high-throughput fluorimetric assay for angiotensin I-converting enzyme. Nat Protocols. 2006;4:1961–1964; (b) Leiting B, Pryor KD, Wu JK, et al. Catalytic properties and inhibition of proline-specific depeptidyl peptidase II, IV and VII. Biochem J. 2003;371:525–532; (c) Lankas GR, Leiting B, Roy RS, et al. Dipeptidyl peptidase IV inhibition for the treatment of type 2 diabetes: Potential importance of selectivity over dipeptidyl peptidases 8 and 9. Diabetes. 2005;54:2988–2994; (d) Brandt I, Joossens J, Chen X, et al. Inhibition of dipeptidyl-peptidase IV catalyzed peptide truncation by vildagliptin ((2S)-{[3-hydroxyadamantan-1-yl) amino]acetyl}-pyrrolidine-2-carbonitrile). Biochem Pharmacol. 2005;70:134–143. Villhauer EB, Brinkman JA, Naderi GB, et al. 1-[[(3-Hydroxy-1-adamantyl) amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J Med Chem. 2003;46:2774–2789. All the molecular modeling calculations (ligand preparation, protein preparation and molecular docking) were performed using modules available within the SYBYL7.3 suite of programs. SYBYL, Tripos Inc., www.tripos.com. All new compounds were characterized by 1H NMR and MS. The purity of compounds was further confirmed by HPLC before submission for biological evaluation.