Discovery of furan and dihydrofuran-fused tricyclic benzo[d]imidazole derivatives as potent and orally efficacious microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors: Part-1

Bioorganic & Medicinal Chemistry Letters 27 (2017) 5131–5138

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Discovery of furan and dihydrofuran-fused tricyclic benzo[d]imidazole derivatives as potent and orally efficacious microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors: Part-1 Nagarajan Muthukaman a, Macchindra Tambe a, Sanjay Deshmukh a, Dnyandeo Pisal a, Shital Tondlekar a, Mahamadhanif Shaikh a, Neelam Sarode a, Vidya G. Kattige b, Monali Pisat b, Pooja Sawant b, Srinivasa Honnegowda b, Vikas Karande b, Abhay Kulkarni b, Dayanidhi Behera c, Satyawan B. Jadhav c, Ramchandra R. Sangana c, Girish S. Gudi c, Neelima Khairatkar-Joshi b, Laxmikant A. Gharat a,⇑ a

Chemical Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra 400709, India Biological Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra 400709, India c Drug Metabolism and Pharmacokinetics, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra 400709, India b

a r t i c l e

i n f o

Article history: Received 14 August 2017 Revised 18 October 2017 Accepted 25 October 2017

Keywords: Benzofuran Prostanoid Inflammation Scaffold hopping Hyperalgesia PGE2 Osteoarthritis

a b s t r a c t This letter describes the synthesis and biological evaluation of furan and dihydrofuran-fused tricyclic benzo[d]imidazole derivatives as novel mPGES-1 inhibitors, capable of inhibiting an increased PGE2 production in the disease state. Structure-activity optimization afforded many potent mPGES-1 inhibitors having <50 nM potencies in the A549 cellular assay and adequate metabolic stability in liver microsomes. Lead compounds 8l and 8m demonstrated reasonable in vitro pharmacology and pharmacokinetic properties over other compounds. In particular, 8m revealed satisfactory oral pharmacokinetics and bioavailability in multiple species like rat, guinea pig, dog and cynomolgus monkey. In addition, the representative compound 8m showed in vivo efficacy by inhibiting LPS-induced thermal hyperalgesia with an ED50 of 14.3 mg/kg in guinea pig. Ó 2017 Elsevier Ltd. All rights reserved.

Prostaglandin E2 (PGE2) is a lipid mediator, produced in abundance by most mammalian tissues and regulates multiple biological processes under pathophysiological conditions.1 In addition to being a key mediator of pathological inflammation, PGE2 also plays an important role in cellular physiological events such as neuronal functions via prostanoid E receptors (EPRs), female reproduction, vascular hypertension, kidney function and gastric mucosal protection. PGE2 has also been shown to support tumor growth by inducing angiogenesis, modulating tumor-cell apoptosis, suppressing immune surveillance and to induce colon carcinogenesis in the presence of bile acid.2 Increased levels of PGE2 in the synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) was observed from published studies.3 The role of prostaglandins (PGs) in inflammatory pain is well established. Specifically PGE2 is one of the most endogenous substances known and mediates a number of inflammatory processes including redness, swelling

⇑ Corresponding author. E-mail address: [email protected] (L.A. Gharat). https://doi.org/10.1016/j.bmcl.2017.10.062 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

and pain. Binding of PGs to prostanoid receptors (EP1, EP2, EP3 and EP4) sensitizes pain specific neurons to stimulate pain in central nociceptive systems and mPGES-1 expression was strongly upregulated in the brain and spinal cord during inflammation.4a–d Therefore, mPGES-1 enzyme which is functionally coupled to COX-2 results in an increased formation of PGE2, which has major role in nociception and analgesic activity.4e Taking into consideration the multiple roles of PGE2, targeting the PGE2 synthesis pathway is of importance to several inflammationdriven diseases such as arthritis, fever, cardiovascular dysfunction, periodontitis, pain, inflammatory bowel and bone disorders.5 The biosynthesis of PGE2 proceeds from arachidonic acid (AA), which is metabolized into the unstable endoperoxide PGH2 by COX-1 or COX-2. PGH2 is further isomerized into PGE2 by terminal PGE synthases (PGES), such as membrane PGES (mPGES-1 and mPGES-2) and cytosolic PGES (cPGES).6 mPGES-1 is a member of the Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism (MAPEG) superfamily.6 Also, mPGES-1

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Fig. 1. Structure of known mPGES-1 inhibitors.

requires glutathione as an essential cofactor for its activity and is predominantly up-regulated and functionally coupled to COX-2.7 mPGES-1 expression is very low in most normal tissues. However, constitutive and copious expression is detected in a limited number of organs including the lungs, kidneys and reproductive organs. mPGES-1 is an inducible isoform, which plays a predominant role under inflammatory conditions8 and its expression can be specifically induced by lipopolysaccharide (LPS), IL-1b and TNF-a in various cell types with or without induction of COX-2.9 Genetic deletion studies have highlighted the importance of COX and mPGES-1 in different mouse models of arthritis, reducing incidence, severity and pain in knock-out mice under the relevant experimental settings.10a,10b Because of its key role in PGE2 synthesis, mPGES-1 has attracted attention as a potential drug target in the areas of inflammatory diseases.10 The primary limitations related to the use of commercially available anti-inflammatory drugs (NSAIDs and Coxibs), which decreases PGE2 levels by blocking COX enzymes, are having cardiovascular, gastrointestinal, and renal side effects.9b Therefore, there is a strong need to develop safer alternatives for long-term therapies use. In this aspect, mPGES-1 inhibitors could epitomize a valuable pharmacological approach without affecting the formation of PGH2 pharmacologically produced by the COXs. The other terminal enzyme mPGES-2 is not essential for PGE2 synthesis in in vivo as reported in mPGES2-deficient mouse study.11 Numerous mPGES-1 inhibitor chemotypes have been identified in the course of research towards this target12 and few are exemplified in Fig. 1. Among these, MF-63 (1) was the first mPGES-1 inhibitor to be identified as an in vivo active (oral) with superior enzyme and moderate cellular potency (hIC50: 1 nM and A549 cell IC50: 420 nM).3a,11b,13 MF-63 also exhibited similar potency in guinea pig (IC50: 0.9 nM) and was not active in both mouse and rat enzyme due to significant differences in the protein sequence of the substrate binding site.14 Furthermore, MF-63 selectively suppressed the biosynthesis of PGE2 without affecting other prostaglandins in the in vivo models (LPS-induced pyresis, hyperalgesia, and iodoacetate-induced OA pain model)13a tested in guinea pig. Next, PF-4693627 (2) provided acceptable enzyme and human whole blood potency (IC50 values 3 nM and 109 nM)15 and prolonged mPGES-1 inhibition using PF-4693627 (2) did not affect the renal function as disclosed recently.16 Other inhibitors such as aminobenzimidazole-5-carboxamide (3, hIC50: 3 nM),17,18 pyridine-3-carboxamide (4, hIC50: 0.9 nM),19 aminobenzimidazole-5-

carboxamide (5, hIC50: 8 nM)20 and quinazolinone (6, hIC50: 5 nM)21 also demonstrated sufficient mPGES-1 potency. Further, Eli Lilly (LY3023703)22 and our group (GRC27864)23 has moved mPGES-1 inhibitors to human clinical development for inflammatory diseases. In a prior study inspired from benzimidazole-5-carboxamide 3, we disclosed the structure-activity relationship (SAR) of aminobenzimidazole-5-carboxamide 5 as a potent mPGES-1 inhibitor with oral in vivo efficacy in a pain model of hyperalgesia.20 Also, no further updates were available to compound 3 other than published patents.17,18 Consequently, as part of our on-going drug discovery program, we designed novel scaffold from the known structure 3 by applying ring closure scaffold hopping strategy24 and this resulted rigid tricyclic benzimidazole core 7 and 8 which was expected to retain similar mPGES-1 potency as exemplified in Fig. 2.25 In this manuscript, we describe the synthetic chemistry approach towards optimization of tricyclic 1H-benzofuro[4,5-d] imidazole series 7 and 8 and its pharmacological profile. The syntheses of furan-fused benzo[d]imidazole derivative 7 and dihydrofuran-fused benzo[d]imidazole derivative 8 are depicted in Schemes 1 and 2.25 Commercially available 4-aminosalicylic acid 9 was acetylated with Ac2O, followed by nitration in TFA using sodium nitrite (NaNO2) to afford nitrated compound 10.26 Esterification of compound 10 with conc. H2SO4 in methanol at 90 °C gave N-deacetylated ester product 11 in 75% yield.26 Next, the furan-fused intermediate 13 was obtained by alkylation of alcohol 11 using propargyl bromide, followed by intramolecular cyclization27 in N,N-dimethylaniline at high-temperature. Ironaq. HCl mediated nitro-reduction of compound 13 provided diamine 14 in 55% yield. Then, the aryl isocyanate 15 prepared from the corresponding anilines,28 reacted with 1,2-diamino compound 14 in the presence of N,N0 -diisopropylcarbodiimide (DIC) furnished tricyclic compound29 16 and further ester hydrolysis provided key acid intermediate 17 for further analoging. The carboxylic acid 17 was coupled with both aliphatic and aromatic amines using either acid chloride or TBTU coupling method to provide amide derivatives 7a–l (Table 1) in moderate yield as shown in Scheme 1. The furan-fused tricyclic benzo[d]imidazole analogs 7a–l were screened for mPGES-1 potency in order to understand scaffold viability. The inhibitory activity of aliphatic amide derivatives, such as isopentyl, cyclohexylmethyl and cyclohexyl derived amides 7a–e gave reasonable mPGES-1 potency with IC50 values in the range of 11–82 nM (Table 1).30 Despite having adequate potency, 7a–e revealed poor metabolic stability in human and guinea pig liver

N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 5131–5138

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Fig. 2. Structure 7 and 8: R1 = alkyl, cycloalkyl, aryl and heteroaryl; X = Cl, F; Y = C or N.

Scheme 1. Reagents and conditions: (a) Ac2O, EtOH, reflux; (b) NaNO2, TFA, 5–0 °C, 2 h, 70% for two steps; (c) MeOH, conc.H2SO4, reflux; 80%; (d) propargyl bromide, K2CO3, DMF, 80 °C, 8 h, 55%; (e) N,N-dimethylaniline, CsF, 230 °C, 24 h, 35%; (f) Fe, aq.HCl, 0 °C to rt, 1 h, 55%; (g) thiophosgene, DIPEA, DCM, 0 °C to rt, 4–6 h; 60–80%; (h) N,N0 diisopropylcarbodiimide, Ar-NCS, CH3CN, rt, 24 h, 60–75%; (i) 10% NaOH, MeOH, 60 °C, 8–10 h, 60–70%; (j) SOCl2, DCM, reflux, 2 h, then DIPEA, DCM, aryl amine/alkyl amine, 4–6 h, 30–55%; or TBTU, HOBT, DIPEA, 2 h, then aryl amine/alkyl amine, rt, 8–10 h, 40–60%.

Scheme 2. Reagents and conditions: (a) methallyl chloride, K2CO3, DMF, 80 °C, 9 h, 55%; (b) AlCl3, DCM, 70 °C to rt, 18 h, 73%; (c) Fe, aq.HCl, 0 °C to rt, 2 h, 45%; (d) thiophosgene, DIPEA, DCM, 0 °C to rt, 4–6 h; 60–80%; (e) N,N0 -diisopropylcarbodiimide, Ar-NCS, CH3CN, rt, 24 h, 40–60%; (f) 10% NaOH, MeOH, 60 °C, 8–10 h, 70–75%; (g) SOCl2, DCM, reflux, 2 h, then DIPEA, DCM, aryl amine/alkyl amine, 4–6 h, 30–55%; or TBTU, HOBT, DIPEA, 2 h, then aryl amine/alkyl amine, rt, 10–14 h, 30–60%.

microsomes (<50% remaining after 60 min incubation).30 Next, the introduction of 4-fluorobenzyl amine derived amides 7g and 7f (IC50s: 58 nM and 157 nM) demonstrated 4–12-fold reduced enzyme potency in comparison to 7b–d. In order to improve meta-

bolic stability and retain superior potency, substituted anilines were introduced. Compounds bearing para- and ortho-fluorophenyl, para- and meta-trifluoromethylphenyl derived amides 7h–l demonstrated single digit mPGES-1 enzyme potency with

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Table 1 SAR of furan-fused benzo[d]imidazole.

Compd

R1

X

Y

mPGES-1 IC50 (nM)a,b

7a 7b 7c d 7d 7e 7f 7g 7h 7i 7j 7k d 7l

Isopentyl Cyclohexylmethyl Cyclohexylmethyl Cyclohexyl Cyclohexyl 4F-Benzyl 4F-Benzyl 4F-Phenyl 2F-Phenyl 4CF3-Phenyl 4CF3-Phenyl 3CF3-Phenyl

Cl Cl F Cl Cl Cl Cl F Cl Cl Cl Cl

N N C N C N C C C N C C

82 13 11 12 26 157 58 22 10 6.5 9.4 4.6

Met. stability (% remaining)c HLM

GPLM

– 55 19 19 – – – 66 75 – 95 79

– 0.4 0.9 29 – – – 78 85 45 60

a MF-63 (1) was used as a positive control in this experiment. See Refs. 3a, 10 and 14 for the literature reported potency. b IC50 values are derived from graphs plotted with data from a minimum of two experiment in duplicates. c Percentage of test compound remaining after 60 min incubation with liver microsomes (human and guinea pig) at 37 °C. MS experiment was conducted in triplicates (see Supporting info (SI) for details. d A549 cell potency of 7d (cell IC50: 39 nM) and 7l (cell IC50: 18 nM).

IC50s < 10 nM except 7h which gave slightly lower enzyme potency (IC50: 22 nM). These compounds revealed favorable metabolic stability in human and guinea pig liver microsomes. Couple of compounds from this series, 7d and 7l were further evaluated for PGE2 release cell potency (A549)30 and the cell IC50s were exemplified as 39 nM and 18 nM, respectively (Foot note in Table 1). The metabolically stable and potent compound 7l was further assessed in the LPS-stimulated human whole blood (HWB) assay30 and cytochrome P450 (CYP) assay. Thus, 7l displayed poor human whole blood potency (HWB IC50: 1890 nM) and no CYP isoforms liability against CYP3A4, CYP2C9 and CYP2C19 (<50% inhibition at 10 mM concentration) tested (Table 3). Few aniline derived amides 7j–l exhibited poor intrinsic solubility probably attributed due to its near planar tricyclic scaffold.31 Consequently, in order to improve compound solubility while maintaining the overall molecular size and similar shape of furan-fused benzo[d]imidazole series 7, we sought to reduce the overall aromatic ring count by increasing the sp3 carbon count in our new design through the saturation of furan ring of benzimidazole system.32 This modification led to dihydrofuran-fused benzimidazole 8, which expected to offer favorable enzyme, cell, human whole blood potency and improved solubility. The dihydrofuran-fused benzimidazole analogs 8a–w (Table 2) were prepared starting from advanced intermediate 11 as shown in Scheme 2. The intermediate 11 was alkylated using methallyl chloride in the presence of K2CO3 in DMF at 80 °C to afford compound 18 in 55% yield. Claisen rearrangement of 18 in N,N-dimethylaniline and subsequent treatment with formic acid at 100 °C gave dihydro benzofuran derivative 19,33 followed by nitro reduction in Fe/aq.HCl to afford diamine compound 20. Isothiocyanate 15,28 prepared from the corresponding aryl aniline using thiophosgene and diisopropylethyl amine, reacted with diamine 20 in the presence of N,N0 -

diisopropylcarbodiimide to afford tricyclic dihydrobenzofuro aminoimidazole derivative 21 in 40–60% yield (two steps).29 The ester derivative 21 was hydrolyzed to carboxylic acid derivative 22 in the presence of 10% NaOH solution, followed by coupling with various alkyl and aryl amines (Table 2) using either acid chloride method or TBTU mediated coupling to afford amide derivatives 8a–w in 30–60% yield. The biological activity of dihydrofuran-fused benzimidazole analogs 8a–w were evaluated for mPGES-1 potency and metabolic stability. Since, alicyclic amides 7a–e were metabolically less stable (Table 1), we introduced few more alicyclic amides, including fluorinated in the dihydrofuran series 8 in order to afford metabolically stable analogs with reduced aromatic ring counts.32 Among the synthesized alicyclic amide derivatives 8a–e, potent analogs 8a–b and 8e were metabolically less stable (<50%) in both human and guinea pig liver microsomes (HLM and GPLM).30 But, cyclopropylmethyl derived amide 8c exhibited good metabolic stability (>70%) even though it has 11-fold lower potency compared to the potent mPGES-1 inhibitor 8a (Table 2). Next, fluorinated amide derivatives 8f–h were introduced in order to improve metabolic stability. Among them, 8h gave adequate metabolic stability and satisfactory mPGES-1 enzyme and cell potency. Further, introduction of simple aniline derived amide 8i provided single digit enzyme and cell potency. However, it showed poor stability in HLM and adequate stability in GPLM. Next, 4-fluorophenyl derived amides 8j, 8k and 4-trifluoromethylphenyl derived amides 8l–n unveiled single digit enzyme potency and acceptable A549 cell potency along with more than adequate metabolic stability (Table 2). In addition, para- and meta-substituted cyclopropylphenyl amides 8o and 8p33 also provided adequate metabolic stability along with satisfactory enzyme and cell potency (IC50s: <20 nM). The introduction of di-substituted aryl amides such as, 2-fluoro-4-trifluoromethylphenyl derived amide 8q and 4-fluoro-3-trifluoromethylphenyl derived amide 8r furnished significant mPGES-1 potency (IC50s: 3.7 and 4.7 nM) and acceptable metabolic stability in liver microsomes. Among the substituted pyridine derived amides 8s–w tested, 8s (IC50: 6.7 nM) and 8w (IC50: 13 nM) displayed poor metabolic stability even though they possessed satisfactory mPGES-1 potency. In contrast, analogs 8t–v provided reasonable enzyme and cell potency along with adequate metabolic stability in comparison with other analogs (Table 2). Having identified several mPGES-1 inhibitors with satisfactory enzyme, A549 cell potency and adequate metabolic stability, compounds such as 8h, 8l–n and 8t were further evaluated for PGE2 release human whole blood (HWB) potency and most relevant human cytochrome P450 (CYP) inhibition study. Among these, only compounds 8h, 8l–n were demonstrated good HWB potency (Table 3). Further, compounds 8n and 8u having pyridine ring in the scaffold strongly inhibited CYP isoforms 3A4 and 2C9, whereas 8h, 8p and 8t revealed higher CYP2C9 and CYP2C19 liability at 10 mM concentration which is not desirable. Other potent mPGES-1 inhibitors 8l and 8m strongly inhibited only CYP2C9 isoform. Potent compounds 8l–n were further evaluated for hERG activity and displayed moderate activity in the hERG channel35 (patch clamp assay, 38–56% inhibition @ 10 mM test concentration), suggesting lower likelihood of QTc prolongation effect36 in humans as disclosed in Table 3. Based on the preliminary data disclosed in Tables 2 and 3, compounds 8l and 8m were chosen for further profiling due to its favorable enzyme, cell and HWB potency, acceptable CYP inhibition and metabolic stability, and moderate hERG liability compared to other potent mPGES-1 inhibitors. Therefore, the identified preclinical leads (8l and 8m) were further evaluated in guinea pig (in vivo efficacy model species) and showed single digit enzyme potency (GPIC50s: 2.6 and 6.3 nM) and adequate whole blood potency (guinea pig whole blood IC50 of 8m: 222 nM). In addition,

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Compd

R1

X

Y

mPGES-1 IC50 (nM)a,b

A549, 2% FBS PGE2 IC50 (nM)c

8a 8b 8c 8d 8e 8f 8g 8h 8i 8j 8k 8l 8m 8n e 8o e 8p 8q 8r 8s 8t 8u 8v 8w

Cyclohexylmethyl Cyclopentylmethyl Cyclopropylmethyl Cyclopropylmethyl Cyclohexyl 4,4-Difluorocyclohexyl CF3CH2– CF3CF2CH2– Phenyl 4F-phenyl 4F-phenyl 4CF3-Phenyl 4CF3-Phenyl 4CF3-Phenyl 4Cy-Phenyl 3Cy-Phenyl 2F, 4CF3-Phenyl 4F, 3CF3-phenyl 6CH3O-3-Pyridyl 6CHF2O-3-Pyridyl 6CF3-3-Pyridyl 5Cl-2-Pyridyl 2,6-Dimethyl-3-pyridyl

Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl F Cl F Cl F F Cl Cl Cl F Cl F Cl

N C N C N C C C C C C C C N C C N N C C C C C

2.2 2.3 25 12 6.3 47 23 6.2 4.1 3.5 2.8 3.7 3.9 5.8 4.2 7.7 3.7 4.7 6.7 20 17 21 13

7.8 – – 47 12 – – 12 4.1 11 20 4.4 10 7.2 4.3 19 12 – – 33 20 29 –

Metabolic stability (% remaining)d HLM

GPLM

37 1 89 38 3 – 36 61 37 59 73 97 87 89 86 89 100 98 6 71 75 72 29

0.4 6 71 41 1 – 29 53 65 100 100 65 78 100 58 73 85 73 44 52 34 89 9

a

MF-63 (1) was used as a positive control in this experiment. See Refs. 3a, 10 and 14 for the literature reported potency. IC50 values are derived from graphs plotted with data from a minimum of two experiment in duplicates. c IC50 values represent the concentration to inhibit 50% of PGE2 relative to vehicle control and derived from graphs plotted with data from a minimum of two experiments in duplicates. d Percentage of test compound remaining after 60 min incubation with liver microsomes (human and guinea Pig). At 37 °C. MS experiment was conducted in triplicates (see Supporting info (SI) for details. HLM: human liver microsomes; GPLM: guinea pig liver microsomes. e See Ref. 34 for details. b

Table 3 HWB, CYP and hERG data of selected mPGES-1 inhibitors. Compd

HWB IC50 (nM)a,b

7l 8h e 8l e 8m 8n 8p 8t 8u

1890 302 234 275 164 – 1335 –

CYP% inhibition @ 10 mM concentrationc 3A4

2C9

2C19

15 0.3 16 42 87 39 34 64

40 81 82 72 57 69 77 75

40 88 41 51 33 85 55 39

hERG% inhi. @10 mMd

– – 51 56 38 – – –

a MF-63 (1) was used as a positive control in this experiment. See Refs. 3a, 10 and 14 for the literature reported potency. b Lipopolysaccharide (LPS) stimulated human whole blood (HWB) cell assay. IC50 values represent the concentration to inhibit 50% of PGE2 relative to vehicle control and minimum of two experiments in duplicates. c Cytochrome P450 (CYP)% inhibition as compared to control (no inhibitor) and conducted in triplicates (see SI for details). d For hERG assay, see Ref. 35. e For CYP1A2 and CYP2D6 data, see Table 4.

leads 8l and 8m were found to be >1000-fold selective for mPGES1 over COX-1, COX-2, mPGES2 and cPGES enzymes. As established earlier for other known mPGES-1 inhibitors,13–21 our leads 8l and 8m were not active against rat and mouse enzymes as illustrated in Table 4. Further, both leads demonstrated more than adequate metabolic stability, favorable CYP inhibition except CYP2C9 liability, high plasma protein binding (PPB) and low PAMPA permeability.30 In general, most of the approved COX-2 inhibitors (Coxibs) were having adequate CNS penetration which contributed to its analgesic activity.37 The lead compound 8m tested for CNS penetration revealed sufficient brain penetration and the brain to plasma (B/P) ratio 0.22 was obtained. Hence, CNS penetration of 8m may positively contribute to its analgesic activity in the efficacy model. Leads 8l and 8m were further evaluated against wide range of receptors, enzymes assays and ion channel binding (CEREP safety screen panel) to understand off-target liability.38 The CEREP safety data of 8l and 8m revealed moderate binding against Na+, Cl channels, 5-LO (50–65% inhibition @ 10 mM concentration) and strong binding activity against benzodiazepines (BZD) as disclosed in Table 4. The BZD potency of 8l and 8m had only 30-fold margin over their mPGES-1 enzyme potency, which probably may

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Table 4 Summary of in vitro data of pre-clinical lead 8l and 8m.

Parametersa

8l

8m

In vitro pharmacology: mPGES-1 hIC50 A549 cell IC50 hWBA Guinea pig IC50 Guinea pig WBA Rat mPGES-1 inhibition Mouse mPGES-1 inhibition COX-1 inhibition COX-2 inhibition mPGES-2 inhibition cPGES inhibition

3.7 nM 4.4 nM 234 nM 2.6 nM NT 4.5% @10 mM 10.4% @ 10 mM 11.8% @ 10 mM 0% @ 10 mM 16.2% @ 10 mM 1.4% @ 10 mM

3.9 nM 10 nM 275 nM 6.3 nM 222 nM 14.7% @10 mM 8.2% @ 10 mM 44.2% @ 10 mM 3.2% @ 10 mM 11.8% @ 10 mM 0.01% @ 10 mM

97/65/88/96/90/85 17/5/16/82/41 >99.5/99.1/>99.5 NT NT 51%@ 10 mM Na+ channel: 57%@ 10 mM (IC50: 1.4 mM) BZD (peripheral): 80%@10 mM (IC50: 120 nM) 5-LO: 63%@ 10 mM (IC50: 2.6 mM) EP3(h) agonist: 51%@10 mM FP (h) agonist: 59%@10 mM

87/78/84/92/92/84 23/18/42/72/51 99.2/>99.5/>99.5 0.22 0.13 (low) 56%@ 10 mM Na+ channel: 67%@10 mM (IC50: 2.4 mM) Cl- channel: 62%@10 mM (IC50: 6.0 mM) BZD (peripheral): 86%@10 mM (IC50: 110 nM) 5-LO: 58%@10 mM (IC50: 6.1 mM) A3(h) agonist: 76%@10 mM FP (h) agonist: 61%@10 mM

20 mg/mL 0.3 mcg/mL 0.6 mcg/mL Nil 0.1 mcg/mL

25 mg/mL 0.9 mcg/mL 0.6 mcg/mL Nil 0.2 mcg/mL

ADME properties Metabolic stability (% remaining), H/GPig/R/M/D/Mk CYP (% inhibition @10 mM), 1A2/2D6/3A4/2C9/2C19 PPB (% bound), H/R/G.Pig Rat CNS b/p ratio PAMPA (Pe x 106)cm/s In vitro safety properties: hERG inhibition (Patch clamp) b POR (CEREP panel data; Potential off-target liability)

c

Solubility data Solubility in DMSO Solubility at FaSSIF (pH 6.0) Solubility at FeSSIF (pH 5.2) Solubility in acetate buffer (pH 4.5) 0.1 N HCl

NT: Not tested. a See supporting information for experimental procedure. b POR data presented here for 8l and 8m, which showed >50% inhibition at 10 mM test concentration against receptors, ion channels and enzyme targets. See Ref. 37. c Fasted state simulated intestinal fluid (FaSSIF) and Fed state simulated intestinal fluid (FeSSIF).

induce some toxicity during compound development. As intrinsic solubility is an important parameter for drug development, current leads 8l and 8m were further subjected to solubility study and revealed moderate to low solubility in the aqueous media as shown in Table 4. Although both 8l and 8m provided comparable in vitro pharmacology and pharmacokinetics profile (DMPK), we chose compound 8m for further profiling due to slightly lesser lipophilicity and molecular weight. After accomplishing favorable in vitro properties, advanced lead 8m was further assessed for oral pharmacokinetics (PK) in rat, guinea pig, dog and cynomolgus monkey, and the results were summarized in Table 5. Lead 8m was found to have low intravenous half-life (T1/2 = 1.1 h) in rat (1 mg/kg dose), as a result of high clearance (Cl = 37 mL/min/kg), low volume of distribution (Vz = 3.6 L/ kg), respectively. Besides, 8m demonstrated 100% oral bioavailability (%F), acceptable Cmax and mean AUC along with adequate CNS brain penetration (Tables 4 and 5) in 10 mg/kg oral dose in rat. In addition, dose ranging study of 8m (30 and 100 mg/kg) in rat

using methyl cellulose (MC) suspension revealed dose proportional increase in Cmax and mean AUC as shown in Table 5. Similarly, a dose range oral PK studies of 8m (10 and 30 mg/kg) in guinea pig provided gradual increase in Cmax and mean AUC with saturation at 100 mg/kg. The dog PK study39 of 8m (10 mg/kg) displayed increase in Cmax, mean AUC and very slow absorption (Tmax = 12 h) in comparison to rodent data. The intravenous and oral PK study of 8m in cynomolgus monkey39 (1 mg/kg i.v, and 10 mg/kg oral) revealed lower clearance (Cl = 8.03 mL/min/kg), volume of distribution (Vz = 1.7 L/kg), lower half-life (T1/2 = 2.5 h), and 78% bioavailability in comparison to rat data and comparable Cmax and mean AUC was observed with respect to dog PK data. Thus, compound 8m demonstrated satisfactory PK data across species. Having established favorable pharmacokinetics in rat, guinea pig, dog and monkey, the advanced lead 8m was further evaluated for in vivo efficacy in acute pain model.30 The lead compound 8m was studied at 3, 10 and 30 mg/kg in LPS-induced hyperalgesia pain model in guinea pig and the results are summarized in

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N. Muthukaman et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 5131–5138 Table 5 Oral pharmacokinetic (PK) data of 8m in rat, guinea pig, dog and cynomolgus monkey. Parametersa

Rat (n = 3)b,c

Dose (mg/kg) Cmax (ng/mL) AUC0-24 (ng.h/mL) Tmax (h) %F T1/2 (h) CL (mL/min/kg) Vz (L/kg)

10 546 ± 88 6505 ± 103 2 100 1.12 ± 0.17 37 ± 2 3.59 ± 0.34

Guinea Pig (n = 3)d 30 2531 ± 652 32370 ± 13656 2 100 – – –

100 5546 ± 980 107911 ± 24201 8 – – – –

10 426 ± 71 5078 ± 983 4 ND ND ND ND

30 742 ± 104 11628 ± 2176 4 – – – –

100 736 ± 130 11675 ± 2719 2 – – – –

Dog (n = 3)e

Cyno monkey (n = 3)f

10 1093 ± 105 21998 ± 3045 12 ND ND ND ND

10 908 ± 127 16696 ± 9907 6 78 2.49 ± 0.63 8.03 ± 1.66 1.67 ± 0.112

-: not applicable; ND: not determined. a Cmax, AUC0-24, Tmax, %F, T1/2, CL and Vz were determined in male Sprague-Dawley rats. Rat and cynomolgus monkey i.v dose is 1 mg/kg. b Vehicle for rat Oral dosing- 0.5% methylcellulose (MC) suspension; Vehicle for rat i.v- 20% NMP + 20% Ethanol + 60% PEG 200). The data represented is mean ± SD (n = 3). c Rat CNS b/p ratio = 0.22 (brain concentration: 58.9 ng/mL/plasma concentration: 268.9 ng/mL). d Vehicle for guinea pig oral dosing- 0.5% methylcellulose suspension. The data represented is mean ± SD (n = 3). e Vehicle for beagle dog oral dosing-2.5 lL/mL Tween 80 + 0.5 (w/v) methyl cellulose (MC) suspension. See Ref. 39 f Vehicle for cynomolgus monkey oral dosing-2.5 mL/mL Tween 80 + 0.5 (w/v) methyl cellulose (MC) suspension. Vehicle for cynomolgus monkey i.v dosing-10% NMP + 10% ethanol + 10% PEG + 70% premix solvent (v/v mixture of 3:2 PEG200: Milli-Q water). See Ref. 39.

Fig. 3. Analgesic effects of 8m in the guinea pig hyperalgesia pain model. Data are expressed as percentage of hyperalgesia, with the naive group (injected intraplantarly with saline) as 0% and the vehicle-treated LPS-injected group as 100%. Results are shown as Mean ± S.E.M. (n = 5–8/group).

Table 6 Plasma concentration of 8ma at PD time point. Dose (mg/kg, po, od)

% Hyperalgesia inhibition

Plasma concentration@ PD time point (ng/mL)b

Hyperalgesia ED50 (mg/kg)

3 10 30

0 42 61

43 116 192

14.3

a

Guinea pig whole blood IC50: 222 nM. Concentrations are means of n = 5–8 animals per dose group. The data represented is from a single experiment. Study protocol is provided in the SI.

and centrally mediated PGE2 synthesis13,40 and showed efficient analgesic activity in the LPS-induced hyperalgesia pain model when administered orally. In conclusion, we have demonstrated the optimization of furan and dihydrofuran-fused tricyclic benzo[d]imidazole analogs as mPGES-1 inhibitor with favorable enzyme and cell potency. The most potent analogs 8l and 8m displayed satisfactory in vitro potency in human, guinea pig enzymes and selectivity over COX1, COX-2, mPGES-2 and cPGES enzymes. Compounds 8l and 8m were metabolically stable in across species tested (human, guinea pig, mouse, rat, dog and monkey liver microsomes) and had liability only for CYP2C9, CYP2C19, and hERG channel. No significant off-target liability for compounds 8l and 8m were observed in CEREP panel assay except BZD. The advanced lead 8m demonstrated adequate CNS penetration and satisfactory oral PK in rat, guinea pig, dog and cynomolgus monkey along with high oral bioavailability in rat and monkey. Further, lead 8m exhibited in vivo efficacy in hyperalgesia pain model with ED50 of 14.3 mg/ kg. Since, current series suffer from CYP and hERG channel liabilities, further SAR optimization is necessary to overcome these liabilities without altering the core scaffold. Consequently, SAR optimization of dihydrofuran-fused tricyclic benzo[d]imidazole mPGES-1 inhibitors free from CYP and hERG liabilities will be reported in the forthcoming publication. Acknowledgements We thank Drs Sravan Mandadi and Sanjib Das for the review of this manuscript and valuable inputs. We also thank scientists from the analytical support group for their help in compound characterization.

b

A. Supplementary data Fig. 3 and Table 6. Injection of LPS into the plantar region of guinea pig right hind paw caused a significant increase in thermal hyperalgesic response compared to saline injected animals. The lead compound 8m was significantly inhibited hyperalgesic response in a dose-dependent manner with maximum inhibition of 61% at 30 mg/kg in comparison with NSAID standard, Naproxen (Fig. 3). The calculated ED50 was found to be 14.3 mg/kg. As depicted in Table 6, the plasma concentration was measured at 8.5 h (pharmacodynamics time point) post compound treatment 8m (30 mg/kg) and was found to be 192 ng/ml (equivalent to 371 nM). This is roughly 2-fold over guinea pig whole blood potency (GPWB IC50: 222 nM). Overall, lead compound 8m suppresses both peripheral

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