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. 2014 Sep 8;5(10):1082–1087. doi: 10.1021/ml5003426

Discovery of a Potent and Selective DGAT1 Inhibitor with a Piperidinyl-oxy-cyclohexanecarboxylic Acid Moiety

Shuwen He 1,*, Qingmei Hong 1, Zhong Lai 1, David X Yang 1, Pauline C Ting 1, Jeffrey T Kuethe 1, Timothy A Cernak 1, Kevin D Dykstra 1, Donald M Sperbeck 1, Zhicai Wu 1, Yang Yu 1, Ginger X Yang 1, Tianying Jian 1, Jian Liu 1, Deodial Guiadeen 1, Arto D Krikorian 1, Lisa M Sonatore 1, Judyann Wiltsie 1, Jinqi Liu 1, Judith N Gorski 1, Christine C Chung 1, Jack T Gibson 1, JeanMarie Lisnock 1, Jianying Xiao 1, Michael Wolff 1, Sharon X Tong 1, Maria Madeira 1, Bindhu V Karanam 1, Dong-Ming Shen 1, James M Balkovec 1, Shirly Pinto 1, Ravi P Nargund 1, Robert J DeVita 1
PMCID: PMC4207266  PMID: 25349648

Abstract

graphic file with name ml-2014-003426_0011.jpg

We report the discovery of a novel series of DGAT1 inhibitors in the benzimidazole class with a piperdinyl-oxy-cyclohexanecarboxylic acid moiety. This novel series possesses significantly improved selectivity against the A2A receptor, no ACAT1 off-target activity at 10 μM, and higher aqueous solubility and free fraction in plasma as compared to the previously reported pyridyl-oxy-cyclohexanecarboxylic acid series. In particular, 5B was shown to possess an excellent selectivity profile by screening it against a panel of more than 100 biological targets. Compound 5B significantly reduces lipid excursion in LTT in mouse and rat, demonstrates DGAT1 mediated reduction of food intake and body weight in mice, is negative in a 3-strain Ames test, and appears to distribute preferentially in the liver and the intestine in mice. We believe this lead series possesses significant potential to identify optimized compounds for clinical development.

Keywords: DGAT1, inhibitor, benzimidazole, ACAT1, A2A receptor, cyclohexanecarboxylic acid, lipid tolerance test, epimerization, metabolite, Ames test, skin

DGAT1 inhibitors have emerged as potential therapeutic agents against diabetes and obesity.1 DGAT (acyl CoA:diacylglycerol acyltransferase) catalyzes the final and committed step in the synthesis of triglyceride: the formation of triacylglycerol from diacylglycerol and acyl-CoA. DGAT1 is one of the isoforms and shares only limited homology with DGAT2 in the terms of amino acid sequence.2−4 DGAT1 has more sequence homology to acyl CoA:cholesterol acyltransferase (ACAT1 and ACAT2), which play a crucial role in cholesterol homeostasis.5 DGAT1 has attracted much attention since the disclosure of the phenotype of DGAT1 knockout mice, which were shown to be viable and resistant to diet-induced obesity.6 DGAT1 knockout mice were also reported to have enhanced insulin sensitivity compared to wild-type mice.7 The data, taken together, has prompted significant research effort in identifying small molecule DGAT1 inhibitors as a potential treatment for obesity and diabetes (Figure 1)8−16 In a phase II clinical trial, 3-week dosing of LCQ-908 (pradigastat) at 20 mg daily resulted in a 40% reduction in fasting triglyceride levels in patients with familial chylomicronemia syndrome, thus demonstrating the clinical proof of concept that inhibition of DGAT1 in humans leads to reductions of plasma triglycerides.17 Recent reports on the clinical results for AZD-7687 demonstrated the ability of DGAT1 inhibitors to attenuate postprandial triacylglyceride excursion. However, the gastrointestinal side effects could hinder further development of DGAT1 inhibitors as a novel treatment for diabetes and obesity.18,19

Figure 1.

Figure 1

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Structures of selected DGAT1 inhibitors.

Recently, we disclosed a series of novel DGAT1 inhibitors in the benzimidazole class bearing a pyridyl-oxy-cyclohexanecarboxylic acid moiety.20 A representative of this series, 1A, is a potent DGAT1 inhibitor with excellent selectivity against ACAT1 (Figure 2). Furthermore, 1A significantly reduces triglyceride excursion in lipid tolerance (LTT) in both mice and dogs. However, 1A undergoes cis/trans isomerization in vivo, which could complicate the further development.

Figure 2.

Figure 2

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Representative DGAT1 inhibitors of the pyridyl-oxy-cyclohexanecarboxylic acid series.

During the profiling of 1A as the lead compound, other potential issues for this structural series were uncovered. First, 1A has low aqueous solubility. In the high throughput solubility test, all the salt forms prepared, free form, formate, and ammonium salt, have solubility less than 10 μM in PBS buffers at pH 2 and 7, respectively. Second, 1A appears to be tightly bound to plasma proteins. The unbound free fraction of 1A in human plasma is only 0.36%. In mouse plasma, the unbound free fraction is slightly higher (1.7%). Finally, this series of compounds were shown to have an off-target interaction with the A2A receptor.21 Compound 2A, an earlier lead compound with a chloro substitution on the benzimidazole ring, was screened against a panel of known biological targets (Figure 2). In this test, 2A had an IC50 of 247 nM in a radioligand based competition binding assay with recombinant human A2A receptor. The activity was later confirmed in a similar in-house human A2A receptor binding assay. In this assay, 1A had IC50 of 334 nM, which is only ∼160-fold selectivity against DGAT1. Consistent with this result, in an A2A cAMP functional assay, compound 1A displayed antagonist activity (IC50 = 269 nM with 101% inhibition at 30 μM). Given the cis/trans isomerization issue and the potential issues discussed above, further profiling of 1A was discontinued.

While profiling 1A and related compounds, we continued to explore other structural variations to present both the benzimidazole and the carboxylic acid pharmacophores in the molecule. We became interested in a new design, which incorporates a piperidinyl linker instead of a pyridyl linker (e.g., structure 3A in Figure 3). We believed the piperidiny linker would impart more flexibility to the structure, as well as a more basic piperidine nitrogen, which could improve aqueous solubility with molecules of essentially the same molecular weight. As an advantage to alternative designs, the symmetrical nature of this piperidinyl linker would not impose additional stereochemical complications to the synthesis. Furthermore, given that many known A2A receptor modulators have multiple aromatic rings in their structures, we expected that the saturation of the pyridyl ring system could decrease the number of aromatic rings and potentially reduce the affinity with the A2A receptor.22

Figure 3.

Figure 3

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Design of the piperidinyl-oxy-cyclohexanecarboxylic acid series.

In addition, the compounds with a piperidinyl linker series (e.g., 3A) might have a lower risk to cause adverse effects in the skin. DGAT1 is known to be expressed in the skin of mice and human.23 DGAT1 KO mice were shown to display deficiency in skin and fur, including sebaceous gland atrophy and hair loss. These findings raised concerns that pharmacological inhibition of DGAT1 in skin could lead to undesirable adverse effects in humans. The calculated logD for 3A is about one unit lower than that of 1A (0.35 vs 1.36, Figure 3). Given that the skin tissue is largely lipophilic, we believed that an inhibitor with lower logD is expected to distribute less in the skin and therefore has a lower tendency to elicit undesirable effect.24

The synthesis for this series of compounds focused on the preparation of the piperidine substructures 8A and its trans isomer 8B (Scheme 1). The initial synthetic route involved saturating a pyridyl ring to furnish the required piperidinyl intermediates. The Mitsunobu reaction of commercially available ethyl 4-hydroxycyclohexanecarboxylate 6 (a cis and trans mixture) and 4-hydroxypyridine followed by SFC separation gave the cis and trans isomers (7A and 7B). Saturation of the pyridyl ring to a piperidinyl ring turned out to be challenging, which we rationalized to be due to the electron donating effect of the alkoxy substituent on 4-position of the pyridyl ring. Eventually, we were able to hydrogenate 7A to piperidine 8A with frustratingly long reaction time at 45 psi in a Parr shaker at room temperature. The trans isomer 7B behaved similarly in the hydrogenation step to give 8B. Coupling of 8A with 2-(6-fluoropyridin-3-yl)-5-fluoro-1H-benzimidazole 9 gave ethyl ester 10A. The coupling reaction condition was identified from a comprehensive screening of a variety of solvents and bases. Eventually, we identified that the coupling worked best with a weak inorganic base such as NaHCO3 in polar aprotic solvents (such as DMSO and NMP).25 The coupling chemistry thus developed proved to be pivotal for the rapid progression of this structural series. Under the same conditions, 8B was coupled with 6-fluoronicotinaldehyde to give aldehyde intermediate 11B. Oxidative condensation of 11B with 4-fluorobenzene-1,2-diamine afforded ester 10B. Finally, hydrolysis of ethyl esters 10A and 10B provided compounds 3A and 3B, respectively.

Scheme 1. Synthesis of 3A and 3B.

Scheme 1

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Reagents and conditions: (a) 1. PPh3, 4-hydroxypyridine, DIAD, THF, 55 °C, 2 days; 2. SFC (ChiralPak AD-H), 7A (14% for two steps), 7B (23% for two steps); (b) 6-fluoronicotinaldehyde, potassium peroxymonosulfate, DMF–water, 69%; (c) PtO2, TsOH, H2 (45 psi), EtOH, RT, 5 days; (d) NaHCO3, DMSO, 110 °C, 43% for two steps; (e) LiOH, THF–water, 83%; (f) PtO2, TsOH, H2 (45 psi), EtOH, 5 days; (g) NaHCO3, DMSO, 110 °C, 27% for two steps; (h) potassium peroxymonosulfate, 4-fluorobenzene-1,2-diamine, DMF–water, 18%; (i) LiOH, THF–water, 70%.

The difficulty in preparing the piperidine pieces (8A and 8B) by the route shown above significantly hampered the synthesis and profiling of this series of compounds. To overcome this synthetic problem, we developed a new route starting with benzyl 4-oxopiperidine-1-carboxylate (Scheme 2). Reductive etherification of this starting material with ethyl 4-hydroxycyclohexanecarboxylate (cis and trans) gave a mixture of 12A and 12B.26 Removal of Cbz-protecting group using standard conditions furnished a mixture of 8A and 8B. Following the chemistry described above, 5A and 5B were prepared. During the reductive etherification reaction, trans/cis configuration in ethyl 4-hydroxycyclohexanecarboxylate was transferred to the product with complete retention. Therefore, scale-up of this series of compounds could begin with pure cis- or trans-ethyl 4-hydroxycyclohexanecarboxylate to provide the desired intermediate in cis or trans form, respectively.27

Scheme 2. Reductive Etherification Route.

Scheme 2

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Reagents and conditions: (a) 1. ethyl 4-hydroxycyclohexanecarboxylate (cis/trans mixture), triethylamine, TMSCl, 0 °C; 2. benzyl 4-oxopiperidine-1-carboxylate, Et3SiH, TMSOTf, −78 to 0 °C, 91%; (b) 1. H2, Pd/C; 2. NaHCO3, NMP, 110 °C; 3. SFC (ChiralPak AD-H), 13A (37% for three steps), 13B (33% for three steps); (d) LiOH, THF–water, RT, 73%; (e) LiOH, THF–water, RT, 81%.

Applying the chemistry described above, we prepared the compounds with several different substitutions on the benzimidazole ring in both the cis and trans series. The compounds and their profiles are listed in Table 1. Most of the compounds in piperidinyl series maintain potent inhibition on both human and mouse DGAT1 except for 3B, the least potent analogue in this series.28 The compounds are slightly less potent on mouse DGAT1 than on human DGAT1. The potency loss appears to be more severe for the compounds in the trans series. In contrast, in the pyridyl linked series reported earlier (e.g., 1A), there is less differentiation of potencies (human vs mouse and cis vs trans).16 However, the piperidinyl series shows much improved off-target selectivity. All the compounds in the piperdinyl series are now highly selective against ACAT1.29 In contrast, in the pyridyl linker series, a small substituent on benzimidazole (e.g., F or H) is required to offer good ACAT selectivity, while maintaining potent DGAT1 inhibition. Furthermore, all the piperidinyl linked compounds possess improved selectivity against A2A as compared to 1A and 2A of the pyridyl linker series. In particular, 3A, 4B, 5A, and 5B achieve >1000-fold selectivity (the ratios of hDGAT1 IC50 and hA2AKi). Finally, the piperidinyl series displays improved aqueous solubility and increased free fractions in plasma. For example, the TFA salt of 5B has solubility of 130 and 151 μM in PBS buffer at pH 2 and 7, respectively. The solubility of the free form is 167 μM and 153 μM at pH 2 and 7, respectively. The free fraction of 5B is 9.0% and 7.8% in human and mouse plasma, respectively.30

Table 1. Profiles of Compounds 3–5.

graphic file with name ml-2014-003426_0010.jpg

compd cis/trans X human DGAT1 IC50 (nM) mouse DGAT1 IC50 (nM) human ACAT1 IC50 (% inh. at 10 μM) hA2A binding Ki (μM) mouse LTT (10 mpk) triglyceride reduction @18 h hERG binding (Ki, μM)
3A cis F 5.8 26 >10 μM (−2%) >10 55% 11
3B trans F 15 110 >10 μM (5%) 9.7 ND >60
4A cis Cl 2.2 11 >10 μM (13%) ND 52% 15
4B trans Cl 2.9 29 >10 μM (4%) 3.5 75% 4.0
5A cis CF3 2.5 8.0 >10 μM (22%) 8.0 75% 7.0
5B trans CF3 3.9 23 >10 μM (23%) 4.6 89% 10
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The compounds were subsequently tested in rodent lipid tolerance test (LTT), an in vivo lipid excursion assay, to evaluate their ability to inhibit plasma triglyceride accumulation. In mouse LTT screening at 10 mpk oral dose, 3A and 4A show modest efficacy reducing lipid excursion (∼50% at 20 h time point).31 Because of its poor potency on mouse DGAT1, 3B was excluded from mouse LTT screening. However, 5A and 5B with a CF3 substituent have the best efficacy. Similarly, 4B with a chloro substituent on benzimidazole ring gave excellent efficacy in mouse LTT screening. However, it suffers from slightly increased off-target activity on hERG ion channel in a competitive binding assay with radiolabeled MK-499 (Ki = 4.0 μM).32 Because of its excellent efficacy observed in mouse LTT screening, 5B was also tested in rat LTT model (Figure 4).33 At 1 mpk and 3 mpk, 5B reduces lipid excursion in rat by ∼80%. The corresponding plasma concentrations of 5B were 20 and 58 nM.34 In this assay, reduction of lipid excursion by the positive control, Cpd A (a known DGAT1 inhibitor from the literature), at 10 mpk was normalized to 100%.35

Figure 4.

Figure 4

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Compound 5B reduces lipid excursion in rat LTT.

To establish the target engagement on DGAT1, 5B was evaluated in wild type (WT) vs DGAT1 knockout (KO) mice for the effect on body weight and food intake (Figure 5).36 In a six day study with daily dosing at 10 mpk, 5B reduced cumulative body weight gain and food intake significantly in WT mice as compared to vehicle treated groups starting from day 3, while the body weight and food intake of 5B treated KO mice were indistinguishable from the vehicle treated group. This result strongly suggests that the reduction of body weight and food intake was due to the inhibition of DGAT1.

Figure 5.

Figure 5

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Effect on body weight and food intake of 5B in DGAT1 KO vs WT mice.

During the profiling of 5B, there was some concern around the possible mutagenicity due to the presence of a benzimidazole moiety in the structure.37 Initial in silico prediction by MultiCASE and DEREK indicated that the probability of mutagenicity was low.38 Indeed, 5B was tested and shown to be negative in a 3-strain microbial mutagenesis assay. Over a concentration range of 30 to 5000 μg/plate, 5B did not produce any 2-fold or greater increases in revertants relative to the solvent control in any of the three strains tested (TA1535, TA98, and TA100), either with or without metabolic activation. This result offered us more confidence on the overall potential for this structure class.

With the increasing interest in 5B as a potential development candidate, 5B was screened against a panel of more than 100 biological targets (receptors and enzymes). In this set of assays, 5B showed an excellent selectivity profile with IC50 >10 μM against all targets except A2A.

As 5B was extensively profiled as a potential development candidate, 1A in the pyridyl linked series was shown to isomerize to its trans isomer in vivo. Immediately, 5B was evaluated for this possibility. In a hepatocyte incubation study, 5B does isomerize but at different levels across the species (Table 2). In dog and monkey, the isomerization was minimal, while in mouse and human there was more isomerization. The isomerization was most pronounced in rat hepatocytes. As a comparison, the isomerization from 5A (the cis isomer) to trans-5B occurs more extensively in rat and human.

Table 2. Conversion of 5B to 5A and Vice Versa in Hepatocyte Incubationa.

conversion of 5B (parent) to 5A (metabolite)
species 5A peak area as % of 5B
mouse 3.3%
rat 34.6%
dog 0%
monkey 0%
human 5.7%
conversion of 5A (parent) to 5B (metabolite)
species 5B peak area as % of 5A
rat 67.0%
human 26.1%
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a

Compound was added with final concentration of 10 μM into hepatocytes diluted to 1 × 106 cell/mL in a buffer. The incubation was performed at 37 °C for 120 min. The plates were centrifuged, and the supernatant was analyzed by LC–MS/MS.

In addition to the in vitro hepatocyte incubation study, 5B was also tested for its in vivo pharmacokinetics in rat and dog (Table 3). Consistent with the in vitro data, about half of trans-5B was converted to the cis isomer 5A in rat in vivo as indicated by the AUCs in plasma. Less isomerization occurred in dog (∼5%).

Table 3. Pharmacokinetic Data for 5B in Rat and Doga,b.

PK parameters rat dog
F (%) 8 126
Cl (mL min–1 kg–1) 5.82 88.9
Vdss (L kg–1) 0.19 4.54
t1/2 (h) 0.91 1.16
oral dose (mg kg–1) 2 4
AUC (μM·h) 5B 1.00 1.94
AUC (μM·h) metabolite 5A 0.97 0.11
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a

Compound 5B was dosed in Sprague–Dawley rats as a solution in EtOH/PEG400/water (10:50:40) at 1 mg/kg, iv, and 2 mg/kg, po.

b

Compound 5B was dosed in beagle dogs as a solution in 30% captisol (pH 8) at 1 mg/kg, iv, and in 0.5 methylcellulose at 4 mg/kg, po.

Finally, 5B was dosed orally in mice to determine the distribution in various tissues of interest (Table 4). Considering the isomerization of 5B to 5A, the drug levels of 5A were also measured. Compound 5B showed much higher drug levels in liver and small intestine compared with the concentrations in plasma at both 3 and 24 h time points. The level of 5B in skin is much lower compared to liver and intestine, but still high compared with the IC50 on DGAT1 (17- and 7-fold over mouse DGAT1 IC50 at 3 and 24 h, respectively). The tissue distribution of 5A (the metabolite of 5B from isomerization) tracks that of 5B albeit at lower concentrations. On the basis of this study, 5B demonstrated favorable distributions in the target organs (liver and small intestine). However, 5B is present in the skin at a significant concentration. Furthermore, the isomerization of 5B to an active metabolite (5A) could complicate the development process.39 Therefore, although there were many desirable attributes associated with 5B, the profiling of 5B as a potential development candidate was discontinued.

Table 4. Mouse Tissue Distribution after Oral Dosing of 5Ba.

  concentration of 5B (μM)
 concentration of 5A (μM)
tissue 3 h 24 h 3 h 24 h
plasma 0.324 <0.01 0.054 <0.01
liver 21.6 4.02 6.91 0.672
intestine (small) 36.6 0.711 5.026 0.076
fat (adipose) 0.237 <0.01 0.021 <0.010
skin 0.404 0.167 0.046 0.018
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a

Compound 5B was dosed in C57BL/6 mice as a solution in EtOH/PEG400/water (10:50:40) at 10 mg/kg, po.

In summary, we have described the discovery of a novel series of DGAT1 inhibitors in the benzimidazole class with a piperdinyl-oxy-cyclohexanecarboxylic acid moiety. This series of compounds abolish the ACAT1 off-target activity associated with the earlier pyridyl-oxy-cyclohexanecarboxylic acid series. Meanwhile, the liability of A2A binding affinity is significantly reduced by the introduction of the piperidinyl linker. The series shows improved aqueous solubility and higher free fraction in plasma. The most interesting compound in this series to date, 5B, was shown to possess an excellent selectivity profile by screening it against a panel of more than 100 biological targets. In addition to its favorable in vitro profile, 5B significantly reduced lipid excursion in LTT assays in mouse and rat, respectively. In a WT vs DGAT1 KO mouse study, 5B demonstrated DGAT1 mediated reduction in food intake and body weight. Upon oral dosing in mice, 5B seemed to distribute in liver and small intestine preferentially. Furthermore, the risk of mutagenicity of 5B was low based on a 3-strain Ames test. All the exciting data collected for 5B helped us to make a critical decision to deprioritize the previous pyridyl linked series and instead focus on this new series. Additional efforts focusing on addressing cis/trans isomerization issue and further reducing the distribution of the DGAT1 inhibitor in skin while maintaining other desirable attributes will be disclosed in the future.

Acknowledgments

We thank Mr. Thomas J. Novak and Dr. Li-Kang Zhang at Merck Research Laboratories for measuring the high resolution mass.

Supporting Information Available

Syntheses and characterization data for the new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.

Supplementary Material

ml5003426_si_001.pdf (283.6KB, pdf)

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