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. 2011 Jul 29;2(10):752–757. doi: 10.1021/ml2001399

4-Methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide: A Potent and Selective Agonist of S1P1

Lewis D Pennington †,*, Kelvin K C Sham †, Alexander J Pickrell †, Paul E Harrington †, Michael J Frohn †, Brian A Lanman †, Anthony B Reed †, Michael D Croghan †, Matthew R Lee ‡, Han Xu §, Michele McElvain §, Yang Xu ⊥, Xuxia Zhang ∥, Michael Fiorino ∥, Michelle Horner #, Henry G Morrison +, Heather A Arnett ∥, Christopher Fotsch †, Min Wong ∥, Victor J Cee †
PMCID: PMC4018088  PMID: 24900263

Abstract

graphic file with name ml-2011-001399_0006.jpg

The sphingosine-1-phosphate-1 receptor (S1P1) and its endogenous ligand sphingosine-1-phosphate (S1P) cooperatively regulate lymphocyte trafficking from the lymphatic system. Herein, we disclose 4-methoxy-N-[2-(trifluoromethyl)biphenyl-4-ylcarbamoyl]nicotinamide (8), an uncommon example of a synthetic S1P1 agonist lacking a polar headgroup, which is shown to effect dramatic reduction of circulating lymphocytes (POC = −78%) in rat 24 h after a single oral dose (1 mg/kg). The excellent potency that 8 exhibits toward S1P1 (EC50 = 0.035 μM, 96% efficacy) and the >100-fold selectivity that it displays against receptor subtypes S1P2–5 suggest that it may serve as a valuable tool to understand the clinical relevance of selective S1P1 agonism.

Keywords: Sphingosine-1-phosphate-1 receptor agonist, peripheral lymphocyte count, immunosuppression, multiple sclerosis

Sphingosine-1-phosphate (1, S1P) and its affiliated G-protein-coupled receptors S1P1–5 are involved in modulating myriad physiological processes including cardiovascular function, pulmonary endothelium integrity, and cell motility (Figure 1).1,2 S1P (1) is derived biosynthetically from sphingosine (2) via phosphorylation mediated by either sphingosine kinase 1 or sphingosine kinase 2 (SphK1 or SphK2).3 Several years ago, S1P (1) and S1P1 were shown to regulate lymphocyte trafficking from the lymphatic system.4,5 At high concentrations of 1 or upon exposure to synthetic agonists of S1P1, binding to S1P1 located on the cell surface of lymphocytes in lymphatic tissues induces receptor internalization (RI) and prevents lymphocyte egress to the periphery and the central nervous system (CNS).6,7 Synthetic agonists of S1P1 are under intense investigation as potential therapies for medical conditions that may benefit from immunosuppression, such as multiple sclerosis (MS).8,9

Figure 1.

Figure 1

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Endogenous and synthetic agonists of S1P1.

Fingolimod (3, FTY720)10,11 bears a dihydroxyamino polar headgroup akin to that of 2 and is likewise phosphorylated by SphK2 to give its bioactive congener 4 (FTY720-P),12,13 the first non-natural agonist of S1P1 to be reported (Figure 1). Notably, 4 displays agonist activity toward several S1P receptor subtypes,14 and until recently, its activation of S1P3 was believed to be exclusively responsible for its bradycardia side effects in humans and preclinical animal models.15,16 A short time ago, our laboratory disclosed 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]pyridin-2-yl)benzyl)azetidine-3-carboxylic acid (5, AMG369), a potent and selective dual agonist of S1P1/S1P5 bearing a polar headgroup that does not require bioactivation.17,18 Much more rare are S1P1 agonists that do not contain a polar headgroup,8,9 such as 5-(4-phenyl-5-(trifluoromethyl)thiophen-2-yl)-3-(3-(trifluoromethyl)phenyl)-1,2,4-oxadiazole (6, SEW2871),19,20 a compound that is exquisitely selective for S1P1 versus the other S1P receptor subtypes.21 The agonism of S1P1 by 4, 5, and 6 has been shown to effect reduction of circulating lymphocytes in vivo,11,17,22 and 3 (FTY720) was recently approved by the U.S. Food and Drug Administration for the treatment of relapsing forms of MS.23,24 As part of an effort to discover structurally novel agonists of S1P1, we set out to search for atypical chemotypes like 6 that lack a polar headgroup.25,26

During a high-throughput screening (HTS) campaign, we identified N-(3-chloro-4-(piperidin-1-yl)phenylcarbamothioyl)-2-methoxybenzamide (7, Scheme 1).27 The auspicious potency of 7 toward S1P1 (EC50 = 0.43 μM, 87% efficacy),28 coupled with its excellent selectivity against S1P3 (EC50 > 25 μM)29 and reasonable molecular and physicochemical properties (MW = 404, PSA = 54 Å2, cLogP = 4.5, and cLogD7.4 = 4.7),30 prompted us to investigate analogues that might exhibit improved physicochemical and biological profiles. The highlights of this lead optimization effort and its fruition in the discovery of a potent, selective, and orally bioavailable agonist of S1P1 lacking a polar headgroup, 4-methoxy-N-(2-(trifluoromethyl)biphenyl-4-ylcarbamoyl)nicotinamide (8) are the subject of this report (Scheme 1).

Scheme 1. Evolution of HTS Hit 7 to Carbamoylnicotinamide 8.

Scheme 1

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Carbamoylnicotinamide 8 was prepared as detailed in Scheme 2. Commercially available 2-(trifluoromethyl)biphenyl-4-amine (9)31 was treated with phosgene at elevated temperature to afford isocyanate 10,32 which was used immediately in the next step of the synthesis without purification. Deprotonation of known 4-methoxynicotinamide (11)33 with NaH followed by addition of 10 gave carbamoylnicotinamide 8 in 35% overall yield for the two-step sequence.34 This expedient route provided a multigram quantity of 8 that was sufficiently pure (>99%, HPLC) for an in vivo toxicology study (vide infra).

Scheme 2. Synthesis of Carbamoylnicotinamide 8.

Scheme 2

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Table 1 summarizes the lead optimization endeavor through which carbamothioylbenzamide 7 evolved to carbamoylnicotinamide 8.35 Although initially wary of the potential chemical lability of the carbamothioylbenzamide moiety of 7,36 we speculated that its intramolecular hydrogen-bonding network37 might help impart hydrolytic stability and conformational rigidity to it and its carbamoylbenzamide analogues.38−40 However, the potential toxicity of the carbamothioylbenzamide moiety or its possible metabolites41 prompted us to exchange the sulfur atom with oxygen to give carbamoylbenzamide 12. Whereas 12 exhibits reasonable potency toward S1P1 (EC50 = 0.23 μM, 120% efficacy), its cell permeability is diminished (Papp < 1.0 × 10–6 cm/s). Postulating that the chloro and piperidinyl substituents of 12 may bind to the same region of S1P1 as do the trifluoromethyl and phenyl groups on the thiophene ring of 6,25 we replaced the Cl atom in 12 with a CF3 group to afford 13. Although the improved potency of 13 was encouraging (EC50 = 0.013 μM, 120% efficacy), we desired to improve its stability against rat liver microsomes (CLint = 26 μL/min/mg). Suspecting that the piperidine ring of 13 may be the primary site of its metabolism,42 we replaced this ring with a phenyl group to provide 14. Gratifyingly, carbamoylbenzamide 14 displays enhanced microsomal stability (CLint = 15 μL/min/mg) and exhibits good potency toward S1P1 (EC50 = 0.068 μM, 110% efficacy). In an attempt improve the potency of 14, we replaced the CF3 substituent in 14 with an isopropyl group to give 15, which does indeed exhibit potent agonism of S1P1 (EC50 = 0.0054 μM, 120% efficacy). We also sought to improve the solubility and cell permeability of 14 by inserting a polar N atom into the scaffold, exemplified by 2-pyridinyl derivative 16, which shows the desired improvements (Sol. = 8.5–28 μg/mL; Papp = 7.4 × 10–6 cm/s), in addition to excellent potency on S1P1 (EC50 = 0.013 μM, 110% efficacy). Interestingly, 2-thiazolyl congener 17 displays greater cell permeability as compared to 16 (Papp = 12 × 10–6 cm/s) but has lower solubility and microsomal stability (Sol. = < 1.0–12 μg/mL; CLint = 33 μL/min/mg). To examine the effect of replacing the left-hand heterocyclic and (hetero)aromatic rings present in 7 and 12–17 with acyclic groups, isopropyl analogue 18 was prepared. Analogue 18 exhibits both excellent potency toward S1P1 (EC50 = 0.014 μM, 110% efficacy) and microsomal stability (CLint < 14 μL/min/mg). The 2,2,2-trifluoroethoxy compound 19 also shows potent activity toward S1P1 (EC50 = 0.0068 μM, 83% efficacy) but has lower cell permeability and microsomal stability (Papp < 1.0 × 10–6 cm/s; CLint = 25 μL/min/mg). Finally, inspired by 2-pyridinyl analogue 16, we desired to probe the effect of placing a polar N atom into the right-hand ring of carbamoylbenzamide 14. Capitalizing on the ready availability of 4-methoxynicotinamide (11),33 carbamoylnicotinamide 8 was prepared (Scheme 2). Indeed, 8 displays good potency toward S1P1 (EC50 = 0.035 μM, 96% efficacy), excellent stability against both human and rat liver microsomes (CLint < 14 μL/min/mg), reasonable cell permeability (Papp = 2.3 × 10–6 cm/s), and moderate solubility (Sol. = 6.6–17 μg/mL).

Table 1. S1P1 Structure–Activity Relationship, Physicochemical and Microsomal Stability Profiles, and PLC Activitya.

graphic file with name ml-2011-001399_0004.jpg

  X Y R1 R2 hS1P1, (μM)b EC50 (% eff.) sol. (μg/mL)c HCl, PBS, SIF Papp (ER)d (× 10–6 cm/s) CLint, RLMe (μL/min/mg) PLC, POCf (Cplasma, ng/mL)
7 S CH Cl N-piperidinyl 0.43 (87) <1.0, < 1.0, 27 1.8 (1.0) 29 NDg
12 O CH Cl N-piperidinyl 0.23 (120) 3.6, < 1.0, 8.5 <1.0 (NR)h 36 NDg
13 O CH CF3 N-piperidinyl 0.013 (120) 3.0, < 1.0, 4.1 <1.0 (NR)h 26 NDg
14 O CH CF3 phenyl 0.068 (110) <1.0, < 1.0, 23 <1.0 (NR)h 15 –20 (4.9)i,j
15 O CH i-Pr phenyl 0.0054 (120) <1.0, < 1.0, 24 1.2 (1.0) <14 +3.0 (7.0)i
16 O CH CF3 2-pyridinyl 0.013 (110) 28, 19, 8.5 7.4 (1.1) 15 –14 (13)i
17 O CH CF3 2-thiazolyl 0.038 (130) 1.1, < 1.0, 12 12 (1.4) 33 –18 (1.0)i
18 O CH CF3 isopropyl 0.014 (110) <1.0, < 1.0, 6.9 2.1 (1.0) <14 –16 (BQL)i,k
19 O CH CF3 CF3CH2O 0.0068 (83) <1.0, < 1.0, 2.2 <1.0 (NR)h 25 +4.0 (1.0)i
8 O N CF3 phenyl 0.035 (96) 17, 6.6, 16 2.3 (1.0) <14 –67 (64)l
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a

See the Supporting Information for experimental details.

b

Data represent an average of at least two determinations; see ref (28).

c

Solubility in 0.010 M aqueous hydrogen chloride (HCl), phosphate-buffered saline (PBS) at pH 7.4, or simulated intestinal fluid (SIF).

d

Apparent permeability (Papp) through porcine proximal tubule cells (LLC-PK1 cell line) and efflux ratio (ER).

e

Estimated intrinsic clearance (CLint) determined by incubation of test compound with rat liver microsomes (RLM) for 30 min and measurement of % turnover.

f

Percent-of-control (POC) reduction vs vehicle in PLC 24 h after a single oral dose (1 mg/kg; vehicle = 20% captisol, 1% HPMC, and 1% pluronic F68, pH 2.1 with MSA) administered to female Lewis rats (N = 5/group) and total compound concentration in plasma (Cplasma) at the 24 h time point.

g

ND, not determined.

h

NR, not reportable.

i

The measured POC reduction in PLC is not statistically significant (P > 0.05 vs vehicle by the ANOVA/Dunnett's multiple comparison test).

j

See ref (43).

k

BQL, below the quantifiable limit.

l

The measured POC reduction in PLC is statistically significant (P < 0.05 vs vehicle by the ANOVA/Dunnett's multiple comparison test).

We desired next to examine the effect of the S1P1 agonists in Table 1 on circulating lymphocyte levels in vivo. Female Lewis rats were administered a single oral dose of each compound (1 mg/kg); after 24 h, lymphocyte counts in blood and compound concentrations in plasma were measured. The 24 h time point was selected for our studies, as it was found to mitigate the effects of dosing on lymphocyte counts.17 Notably, carbamoylnicotinamide 8 reaches >5-fold higher concentration in plasma than the other compounds tested in Table 1 (64 ng/mL) and is also the only one that effects statistically significant (P < 0.05 vs vehicle by the ANOVA/Dunnett's multiple comparison test) reduction in circulating lymphocytes (POC = −67%). Given their comparable in vitro profiles, it is unclear why compounds 14–16 and 18 do not achieve higher plasma levels and realize statistically significant reduction of peripheral lymphocyte counts (PLCs).43

Because of its robust activity in our preliminary in vivo study, carbamoylnicotinamide 8 was selected for further examination. As mentioned previously, compound 8 exhibits potent activation of S1P1 internalization (EC50 = 0.035 μM, 96% efficacy; Table 1);28 gratifyingly, it does not display appreciable agonism of receptor subtypes S1P2–4 (EC50 > 25 μM)44 and shows only weak activation of S1P5 (EC50 = 4.3 μM, 58% efficacy; Table 2).44 As the agonism of both S1P1 and S1P5 in oligodendrocytes by 4 has been suggested to contribute to its efficacy in the treatment of MS,45 S1P1-selective agonists such as 8 may serve as valuable tool compounds to examine the clinical relevance of S1P5 agonism.

Table 2. S1P Receptor Subtype Selectivity of 8a.

EC50 (μM)
hS1P2 hS1P3 hS1P4 hS1P5 (% eff.)
>25 >25 >50 4.3 (58)
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a

See the Supporting Information for experimental details (data represent an average of at least two determinations; see ref (44)).

A pharmacokinetic profile of carbamoylnicotinamide 8 was undertaken in preparation for more detailed studies in vivo (Table 3). In male Sprague–Dawley rats (N = 3) given a single intravenous dose (1 mg/kg), 8 displays low clearance (CL = 0.25 L/h/kg), moderate volume of distribution (Vdss = 6.3 L/kg), and a long half-life (T1/2 = 19 h). Although the plasma protein binding of 8 was found to be moderately high (PPB = 95%), its excellent bioavailability (% F = 140) and dose-proportional plasma exposure over a wide range of doses (AUC0→48 h, 1 mg/kg iv =3400 ng h/mL; AUC0→48 h, 3 mg/kg po =13000 ng h/mL; AUC0→48 h, 100 mg/kg po = 350000 ng h/mL) enabled a subsequent in vivo toxicology study (vide infra).

Table 3. Pharmacokinetic Profile of Carbamoylnicotinamide 8a.

  1 mg/kg iv
      
PPBb (%) rat CLc (L/h/kg) Vdssc (L/kg) T1/2c (h) AUC0→48 hc (ng h/mL) AUC0→48 hd (ng h/mL) 3 mg/kg po Fe (%) AUC0→48 hf (ng h/mL) 100 mg/kg po
95 0.25 6.3 19 3400 13000 140 350000
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a

In vivo experiments were conducted using male Sprague–Dawley rats (N = 3/group).

b

Percent rat plasma protein binding (PPB) of 8 measured in vitro following separation by ultracentrifugation.

c

Clearance (CL), volume of distribution (Vdss), half-life (T1/2), and area under the plasma concentration–time curve (AUC0→48 h) determined following a single intravenous dose (1 mg/kg; vehicle = DMSO).

d

AUC0→48 h determined following a single oral dose (3 mg/kg; vehicle = 20% HPBCD, 1% HPMC, and 1% pluronic F68, pH 2.1, with MSA).

e

Percent bioavailability (F) calculated using AUC0→∞ values determined from the 1 (iv) and 3 mg/kg (po) doses.

f

AUC0→48 h determined following a single oral dose (100 mg/kg; vehicle = 20% HPBCD, 1% HPMC, and 1% pluronic F68, pH 2.1, with MSA).

We next sought to evaluate the pharmacodynamic effects of 8 in more detail (Figure 2). Following a single oral dose in female Lewis rats (0.30, 1.0, or 3.0 mg/kg), 8 exhibits dose-proportional plasma exposure (17, 63, and 160 ng/mL, respectively) and concomitant dose-dependent reduction in circulating lymphocytes at the 24 h time point. Statistically significant lymphocyte depletion is realized for the 1.0 and 3.0 mg/kg doses (78 and 81% reduction in PLCs; P < 0.001 and P < 0.01 vs vehicle by the ANOVA/Dunnett's multiple comparison test, respectively). This dose-dependent decrease in circulating lymphocytes reaching a plateau at 81% maximal reduction is consistent with the S1P1 agonist activity of 8.19,22

Figure 2.

Figure 2

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Female Lewis rats (vehicle, 0.30, and 1.0 mg/kg dose groups: N = 5/group; 3.0 mg/kg dose group: N = 3; vehicle = 20% HPBCD, 1% HPMC, and 1% pluronic F68, pH 2.1, with MSA) administered a single oral dose of carbamoylnicotinamide 8 (0.30, 1.0, or 3.0 mg/kg) showed dose-proportional plasma exposure (black circles represent average plasma concentration ± SE) and dose-dependent reduction in circulating lymphocytes (gray bars represent average blood lymphocyte counts ± SE) at the 24 h time point (statistical significance: ***P < 0.001 and **P < 0.01 vs vehicle by the ANOVA/Dunnett's multiple comparison test, respectively); see the Supporting Information for experimental details.

The potential safety of carbamoylnicotinamide 8 was initially examined in vitro. Encouragingly, 8 displays no appreciable activity against either hERG (IC50 > 10 μM) or hCYPs 3A4 and 2D6 (IC50 > 30 μM) and exhibits negligible induction of hPXR (POC = +3.7%, 10 μM). An in vivo toxicology study of 8 was subsequently conducted. In male Sprague–Dawley rats (N = 3/group) dosed orally at 50 or 200 mg/kg (qd) for 4 days, no mortality, adverse clinical signs, or changes in body weight were observed. However, increases in total bilirubin (both doses) and cholesterol (high dose only) suggest altered bile uptake, processing, or excretion.

In conclusion, carbamoylnicotinamide 8 is a rare example of an S1P1 agonist lacking a polar headgroup. Despite its modest solubility and cell permeability, 8 is orally bioavailable and induces dose-dependent reduction in lymphocyte count in rat 24 h after a single oral dose. The excellent potency that 8 exhibits toward S1P1 and the remarkable selectivity that it displays against receptor subtypes S1P2–5 suggest that it may serve as a valuable tool to examine the proposed clinical relevance of S1P5 agonism for treating MS and the cardiovascular safety issues associated with S1P3 agonism.

Acknowledgments

We thank Laura Scott (HTS and Molecular Pharmacology, Amgen) for executing the HTS campaign that identified carbamothioylbenzamide 7, Kevin Turney (Analytical Research and Development, Amgen) and Christopher Wilde (Molecular Structure, Amgen) for confirmation of the structure assignment of carbamoylnicotinamide 8 using HRMS and NMR methods, respectively, and Ronya Shatila and Elizabeth Tominey (Pharmacokinetics and Drug Metabolism, Amgen) for in vivo pharmacokinetic studies. All live animal studies were conducted in an AAALAC-accredited facility and husbandry procedures met all of the recommendations of the Guide for the Care and Use of Laboratory Animals. All work using research animals was conducted under Institutional Animal Care and Use Committee (IACUC) approved protocols.

Glossary

Abbreviations

EC50

molar concentration of compound that produces half maximal response

RI

receptor internalization

PSA

polar surface area

cLogP

calculated log P (logarithm of the octanol/water partition coefficient)

cLogD7.4

calculated log D (logarithm of the octanol/water distribution coefficient at pH 7.4)

Papp

apparent permeability

CLint

intrinsic clearance

PLC

peripheral lymphocyte count

HPMC

hydroxypropyl methylcellulose

MSA

methanesulfonic acid

Cplasma

total compound concentration in plasma

HPBCD

hyroxypropyl β-cyclodextrin

CL

clearance

Vdss

volume of distribution at steady state

T1/2

half-life of compound in plasma

AUC

area under the plasma concentration–time curve

F

bioavailability

hERG

human ether-a-go-go related gene product (Kv11.1 potassium ion channel)

hCYP 3A4

human cytochrome P450 subtype 3A4

hCYP 2D6

human cytochrome P450 subtype 2D6

IC50

molar concentration of compound that produces half maximal inhibition

hPXR

human pregnane X receptor

qd

quaque die (once per day dosing)

Supporting Information Available

Experimental procedures and characterization data for compounds 8 and 12–19, as well as experimental details and statistical analysis for key biological assays. This material is available free of charge via the Internet at http://pubs.acs.org.

Supplementary Material

ml2001399_si_001.pdf (144.1KB, pdf)

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