Design and Synthesis of Cannabinoid Receptor 1 Antagonists for Peripheral Selectivity

Abstract

Antagonists of cannabinoid receptor 1 (CB1) have potential for the treatment of several diseases such as obesity, liver disease and diabetes. Recently, development of several CB1 antagonists was halted due to adverse central nervous system (CNS) related side effects observed with rimonabant, the first clinically approved CB1 inverse agonist. However, recent studies indicate that regulation of peripherally expressed CB1 with CNS-sparing compounds is a viable strategy to treat several important disorders. Our efforts aimed at rationally designing peripherally restricted CB1 antagonists have resulted in compounds that have limited blood-brain barrier (BBB) permeability and CNS exposure in preclinical in vitro and in vivo models. Typically, compounds with high topological polar surface areas (TPSAs) do not cross the BBB passively. Compounds with TPSAs higher than rimonabant (rimonabant TPSA = 50) and excellent functional activity with limited CNS penetration were identified. These compounds will serve as templates for further optimization.

Introduction

The endocannabinoid system (ECS) consists of receptors, transporters, endocannabinoids, and the enzymes involved in synthesis and degradation of endocannabinoids.¹ There have been two cannabinoid receptors (CBRs) identified to date, CB1 and CB2. CB1 and CB2 are both G protein-coupled receptors (GPCRs) and their primary function is to activate inhibitory G proteins (Gi/o).^1–2 The ECS is responsible for many important physiological processes and regulation of these processes holds promise for the treatment of several diseases. ECS components are under evaluation for the treatment of obesity, liver disease, diabetes, pain and inflammation.²

The CB1 receptor is expressed throughout the body, however, it is found at much greater concentrations in the central nervous system (CNS). There has been great interest in the use of CB1 antagonists for the treatment of metabolic disorders, such as obesity and diabetes. Rimonabant (SR141716A, 1, Figure 1), a potent and selective CB1 inverse agonist/antagonist, was clinically approved to treat obesity in Europe. Unfortunately, 1 produced serious CNS-related side effects such as anxiety, depression and suicidal ideation in patients, leading to its withdrawal from European markets and denial of approval in the United States.³ Upon discovery of rimonabant’s side effects several other CB1 antagonists, such as taranabant, otenabant (2), and ibipinabant, were pulled from development due to regulatory concerns.⁴

Figure 1.

CB1 antagonists

A strategy to take advantage of the therapeutic potential of CB1 antagonism and avoid the CNS-related adverse effects is to generate CB1 antagonists that do not cross the blood-brain barrier (BBB). This strategy is being pursued by several groups and a small set of CB1 antagonists that do not cross the BBB have been reported (3–6, Figure 2).⁵ However, none of these peripherally restricted CB1 antagonists have been fully characterized or their efficacy demonstrated clinically.

Figure 2.

Reported CB1 antagonists that are selective for the periphery

Our group has pursued a two-pronged strategy to develop peripherally restricted CB1 antagonists. The first strategy involved development of CB1 antagonists that have a permanent charge. Charged compounds do not normally cross the BBB unless they are acted upon by a transporter.⁶ Results for this strategy have been previously reported.⁷ The second strategy was to target compounds with high topological polar surface areas (TPSAs). It has been shown that compounds with higher TPSAs have lower permeability into the CNS.⁸ Higher TPSAs can be achieved by adding polar groups, such as sulfonamide or sulfamide, or by replacing existing functional groups with more polar functional groups. This strategy led to the identification of compounds 7 and 8 that we have previously reported. While these compounds were promising, their selectivity for CB1 over CB2 was only modest. Here we describe our ongoing efforts towards designing peripherally restricted CB1 antagonists with improved properties. This study evolved to include a more empirical approach that was based more on SAR than on computational parameters. We have been able to design, synthesize, and characterize highly selective CB1 antagonists that appear to be peripherally restricted.

Results

Ligand design and pharmacological characterization

Compounds were synthesized, purified, characterized and tested as has been described under the “Experimental Section”. All compounds were tested in vitro as antagonists using a calcium mobilization assay as has been previously described.⁷ The ability of each compound to antagonize functional activation of CB1 was quantitatively measured and expressed as its apparent dissociation affinity constant (Ke). Compounds that were found to be potent (Ke ~100 nM or less) using the functional assay were subsequently characterized using radioligand displacement of either [³H]SR141716A or [³H]CP55940 at CB1 and CB2. Equilibrium dissociation constant (Ki) values were determined at each receptor.

During our studies of charged compounds, carboxylic acids were examined due to the fact that they are negatively charged at the physiological pH. Around the same time, carboxylic acid 9 (Figure 3) was reported by another group to be a CB1 antagonist.^5d This finding led to the synthesis and evaluation of carboxylic acid 10 (Table 1). Compound 10 was only moderately active (Ke = 1170 nM). However, examination of the structure of 2 revealed a primary amide at the 4-position of the piperdine ring (Figure 1). This amide was in a similar position as the carboxylic acid functionality of compound 10, leading to the decision to convert carboxylic acid 10 to amide 11 (Figure 3). Amide 11 lacked the charged nature of a carboxylic acid but it did have hydrogen bonding ability that could lower its permeability into the CNS. Compound 11 was found to be a potent CB1 antagonist having a Ke of 0.44 nM and was also highly selective for CB1 over CB2 (CB2:CB1 of 1600). Interestingly, the 4-phenylpiperidine-4-carboxamide group was also reported on a closely related pyrrole scaffold.^5c Compound 11 was advanced into a Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-mdr1) model of BBB penetration, which is widely used to predict in vivo permeability of compounds.⁹ The potency, selectivity, and relatively low permeability of compound 11 across the MDCK-mdr1 cells (apical (A) to basal (B), 8%) made it an interesting starting point for further modifications towards designing potent and selective CB1 antagonists that do not cross the BBB.

Figure 3.

Design of compound 11

Table 1.

Pharmacological assessment of compound 11 with rimonabant 1 and otenabant 2.

Compd	TPSA	Ke (nM) CB1	Ki (nM) CB1 [3H]SR141716 (1)	Ki (nM) CB1 [3H]CP55940	Ki (nM) CB2 [3H]CP55940	CB2:CB1 ratio	MDCK-mdr1a
1	50	1.1		6.2	313	50.6	15%
2		8.7					90%
10	75	1170
11	81	0.4	0.4	3.4	5504	1600	8%

^aCompound’s permeability was measured from apical to basal sides of the membrane.

Compound 11 served as a starting point for several modifications to the amine portion of the pyrazole C-3 carboxamide (Figure 4, Table 2). One of the first modifications of compound 11 studied was the replacement of the phenyl group. Compound 12 was targeted because it represented a hybrid of compounds 11 and 2. Compound 12 also closely resembles the Sanofi-aventis compound 5. However, compound 12’s potency (Ke CB1 = 91 nM) and selectivity (ratio CB2:CB1 of 28.3) did not warrant further investigation of this compound. Next, the reversal of the primary amide of compound 11 became of interest. To realize this, both compounds 13 and 14 needed to be synthesized as precursors of reverse amide compound 15. However, both compound 13 and 14 were interesting in their own right. Compound 13 added the additional functionality of a carbamate; this was a functionality that had not been pursued in our laboratory, and it also had good potency (Ke CB1 = 20.2 nM) and selectivity (ratio CB2:CB1 of ~50). Compound 14 was of interest because it replaced the primary amide of 11 with a primary amine. This maintained the possibility of hydrogen bonding, and increased the basicity of the molecule. This amine group proved to be detrimental to potency (Ke CB1 = 485 nM). However, the amine group in compound 14 also allowed for the introduction of different functionalities. The amine group of compound 14 was used to make the reverse amide compound 15 which was only weakly active (Ke CB1 = 201 nM). Sulfonamide 16, which was also made from amine 14, had higher TPSA compared to compound 11and was potent but only moderately selective for CB1 over CB2 (Ke CB1 = 3.5 nM, ratio of CB2:CB1 of 5.64). Finally, amine 14 also allowed for the synthesis of urea 17a. Urea 17a proved to be a potent CB1 antagonist (Ke CB1 = 2.4 nM) and had good selectivity against CB2 (ratio CB2:CB1 of ~425). Compound 17a was advanced into the in vitro model of BBB permeability (MDCK-mdr1, apical to basal) and was predicted not to cross the BBB (Table 2, <1% transported). These results spawned the synthesis of a small library of ureas 17b–17k, which had potencies (Ke) ranging from 0.5 nM to >10,000 nM against CB1. Several of these compounds were very selective with five of the ten compounds being over 100-fold selective for CB1 over CB2 (Table 2).

Figure 4.

Ligand design around compound 11

Table 2.

Pharmacological assessment of analogues of compound 11

Compd	TPSA	Ke (nM) CB1	Ki (nM) CB1 [3H]CP55940	Ki (nM) CB2 [3H]CP55940	CB2:CB1 ratio	MDCK-mdr1a
12	93	91	78.4	2217	28.3
13	76	20.2	42.3	2110	49.9	<1%
14	64	485	104	1127	10.8
15	67	201	62.6	214	3.4
16	93	3.5	7.3	41	5.6	3%
17a	79	2.4	47.1	20,000	424.6	<1%
17b	79	2.1	43	17126	398	<1%
17c	103	>10,000
17d	79	89	614	20,000	33
17e	82	264	1489	16289	11
17f	79	0.5	38.8	2414	62	<1%
17g	79	0.7	13.5	4914	364
17h	79	10.8	15	182	12
17i	79	0.4	7.6	293	39
17j	79	12	792	20,000	25
17k	79	0.4	15.5	2760	178	<1%

^aCompound’s permeability was measured from apical to basal sides of the membrane.

The positive results for compound 17a also led to the exploration of 4- and 3-aminopiperdine and cyclohexyl amides as different spacers between the pyrazole amide and their polar functionality (Figure 5, Table 3). Structurual series 18 was chosen because it offered similar spacing to compound 17a and presented the opportunity for rapid derivatization off the piperidine nitrogen. Due to the positive results observed for compounds 18a–l (7 out of the 12 compounds had Ke <100 nM against CB1, and 4 analogues having CB2:CB1 ratios greater than 100), other amino-piperidine linkers were explored. The 1,3-disubstituted aminopiperdine series 19 and 20 explored the importance of the effect of an alternative juxtaposition of substituents and the introduction of stereochemistry versus the 4-aminopiperdine linker of structurual series 18. Both enantiomers were explored to examine the effect of chirality on potency and selectivity. Positive results were obtained with compounds 19a–j, with three of the ten analogues having Ke values below 100 nM at CB1, and two analogues were over 100-fold selective for CB1 versus CB2. Compounds 20a–j were also of interest with three of the ten analogues having Ke (CB1) less than 30 nM; in addition three of the ten analogues were over 100-fold selective for CB1 vs CB2. Finally, since sulfonamide 7 and sulfamide 8 have been found to be potentially useful in the development of periphery restricted CB1 antagonists,⁷ ureas of structure 21 were targeted. In general these compounds were only weakly active and compounds 21a–e were not pursued further.

Figure 5.

Exploration of different spacers

Table 3.

Pharmacological assessment of different spacers and functional groups

Compd	TPSA	Ke CB1 (nM)	Ki (nM) CB1 [3H]CP55940	Ki (nM) CB2 [3H]CP55940	CB2:CB1 ratio	MDCK-mdr1a
18a	76	4.7	2.9	2510	877.6	<1%
18b	59	5115
18c	67	195	65.3	2236	34.2
18d	93	269
18e	119	592
18f	79	78	14.7	3349	227.8	<1%
18g	93	4097
18h	79	20.5	165	5693	35
18i	79	16.7	86	9791	114	14%
18j	79	66.5	184	8459	46
18k	79	78.1	187	8721	47
18l	76	3	9.2	2997	326	<1%
19a	76	842
19b	58	1879
19c	67	86	39.1	4350	111.3	14%
19d	93	62	77.9	10893	140	<1%
19e	79	280
19f	79	265
19g	79	70	271	7795	29
19h	79	230
19i	79	206
19j	76	75	65.5	5420	83	<1%
20a	76	52	22.3	6720	302	<1%
20b	58	1174
20c	67	530
20d	93	7	11.84	1248	105.5	<1%
20e	79	171
20f	79	31.3	97	4135	43
20g	79	27	134	3090	23
20h	79	118	125	3031	24
20i	79	268
20j	76	10.4	17.5	2507	143	<1%
21a	88	135
21b	88	351
21c	88	486
21d	88	274
21e	88	150

^aCompound’s permeability was measured from apical to basal sides of the membrane.

Synthesis

Compound 10 (Scheme 1) was prepared by first making the acid chloride of the readily available acid 22.¹⁰ Acid 22 was treated with oxalyl chloride and a catalytic amount of dimethylformamide (DMF) in dichloromethane to form the desired acid chloride. This acid chloride was then treated with amino acid 23 in the presence of triethylamine to yield compound 10 in 67% yield. Carboxylic acid 10 was converted to amide 11 by the use of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), triethylamine, and ammonium chloride in 46% yield.

Scheme 1.

Synthesis of compound 11

Reagents and conditions: (a) 1) oxalyl chloride, DMF (cat.), dichloromethane, 2) 23, triethylamine, dichloromethane; (b) (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), triethylamine, ammonium chloride, THF.

Amino amide 12 was made by reacting acid 22 with commercially available piperidine 24 under BOP coupling conditions, and this reaction produced compound 12 in 46% yield (Scheme 2). The protected amine 13 was made by the coupling of acid 22 and readily available amine 25.¹¹ The Boc group of compound 13 was removed using 30% trifluoroacetic acid (TFA) in dichloromethane to yield amine 14 in 87% yield. Amine 14 was used as a common intermediate for compounds 15, 16, and 17a–k. Acetyl amide 15 was made by reacting amine 14 with acetic anhydride and pyridine in 71% yield. Sulfonamide 16 was formed by the reaction of 14 with methanesulfonyl chloride and triethylamine in 65% yield. Urea 17a was synthesized in 45% yield by reacting amine 14, tert-butyl isocyanate, and triethylamine at 40°C in THF. Synthesis of ureas 17b–k was accomplished by reacting amine 14, the appropriate isocyanate, and triethylamine in tetrahydrofuran (THF) at room temperature. These reactions proceeded in yields that ranged from 58%–74%.

Scheme 2.

Synthesis of analogues of compound 11

Reagents and conditions: (a) amine, BOP, triethylamine, THF; (b) 30% TFA in dichloromethane; (c) acetic anhydride, pyridine; (d) methanesulfonyl chloride, triethylamine, dichloromethane; (e) isocyanate, triethylamine, THF, rt or 40 °C.

Compounds 18a, 19a, and 20a were synthesized by the coupling of the appropriate commercially available amine to acid 22 in the presence of BOP and triethylamine in yields ranging from 88%–96% (Scheme 3). Treatment of compounds 18a, 19a, and 20a with trifluoroacetic acid in dichloromethane produced the amines 18b, 19b, and 20b. The amines 18b, 19b, and 20b were reacted with acetic anhydride and pyridine to produce amides 18c, 19c, and 20c respectively in yields ranging from 81%–87%. Sulfonamides 18d, 19d, and 20d were made by reacting amines 18b, 19b, and 20b with methanesulfonyl chloride and triethylamine in THF. Ureas 18f,h–l, 19e–j, and 20e–j were made by reacting the appropriate amine with the appropriate isocyanate in dichloromethane or THF. The N-tert-butylpiperidine carboxamide 18f was converted to the unsubstituted piperidine carboxamide 18g in 81% yield by stirring 18f in 50% TFA in dichloromethane.¹² The sulfamide 18e was synthesized from the reaction of 18b with excess sulfamide in dioxane at 90° C in 67% yield (Scheme 4).⁷ Ureas 21a–e were made as a mixture of cis/trans isomers from the previously described amine 26 in yields ranging from 71%-98%.⁷

Scheme 3.

Synthesis of compounds with different spacers and functional groups.

Reagents and conditions: (a) amine, BOP, triethylamine, THF; (b) 30% TFA in dichloromethane; (c) acetic anhydride, pyridine; (d) methanesulfonyl chloride, triethylamine, THF; (e) isocyanate, triethylamine, THF, rt; (f) ethyl chloroformate, triethylamine, THF; (g) 50% TFA in dichloromethane.

Scheme 4.

Synthesis of compounds with different spacers and functional groups continued.

Reagents and conditions: (a) sulfamide, dioxane, 90° C; (b) isocyanate, triethylamine, THF, rt.

In vitro metabolic stability and in vivo evaluation of brain penetration

A small set of compounds that were potent, selective and were predicted not to penetrate the CNS as determined using the MDCK-mdr1 assay were tested for in vitro metabolic stability (Table 4). Stability was measured in human plasma and human hepatic S9 fractions to gauge any metabolic liabilities that might be present with these compounds. All compounds tested had good stability in plasma. Stabilities of compounds in S9 fractions were more variable than stabilities in plasma. However, all compounds except 17b displayed metabolic stabilities similar to or greater than compound 1.

Table 4.

Pharmacological assessment of select compounds in for in vitro metabolic stability.

Compd	TPSA	Ke CB1 (nM)	CB2:CB1 [3H]CP55940	MDCK-mdr1 A to B	In vitro metabolic stability
					S9 (% remaining 120 min)	Plasma (% remaining 60 min)
1	50	1.1	50.6	15%	47	>90
7	101	113	16	<1%	37	>90
8	127	106	17	<1%	62	>90
13	76	20.2	49.9	<1%	88	>90
17a	79	2.4	424.6	<1%	88	>90
17b	79	2.09	398	<1%	18	82
18a	76	4.7	877.6	<1%	>90	>90
18f	79	78	227.8	<1%	67	71

Compounds were prioritized and progressed into in vivo experiments in mice for analysis of brain penetration (Table 5). Compound 13 was not progressed due to its relatively low selectivity compared with other compounds found in Table 4, and compound 17b was not progressed due to its relatively low stability in S9 fractions. Ureas 17a and 18f along with carbamate 18a were chosen and evaluated in vivo. Urea 17a was bioavailable with either oral or intra-peritoneal (ip) dosing. Brain levels of 17a were below the lower limit of quantitation when dosed orally and its brain to plasma ratio was 0.03 with ip dosing at one hour. Carbamate 18a was also bioavailable with either oral or ip dosing. When dosed by ip injection, carbamate 18a had a brain to plasma ratio of 0.02 at one hour. Urea 18f was also bioavailable with oral or intra-peritoneal dosing. However, brain to plasma ratios for urea 18f were 0.16 with oral dosing at one hour and 0.38 with intraperitoneal dosing at one hour. Since unperfused brains were examined and because the volume of blood in the unperfused brain is ~ 2–4%¹³, these promising results indicated that 17aand 18a had little to no permeability into the brain as expected while 18f was not selective for the periphery.

Table 5.

In vivo evaluation of select compounds.

Comd	R	Route of administration	Sacrifice Time (min)	Plasma Conc. (ng/mL)	Brain Conc. (ng/mL)	Brain:Plasma
17a		Oral	30	3.72	NA	NA
		Oral	60	10.3	LOQa	NA
		ip	60	386	12.4	0.0320
18a		Oral	30	LOQa	NAb	NAb
		Oral	60	13.2	LOQa	NAb
		ip	60	197	4.20	0.0214
18f		Oral	30	5.08	NA	NA
		Oral	60	28.0	4.46	0.160
		ip	60	67.4	25.5	0.379

^aLOQ: Below limit of quantitation,

^bNA: not applicable.

Discussion

In this publication we report our ongoing efforts to produce peripherally selective CB1 antagonists that may be useful in treating a wide range of clinical indications. We have now identified several highly selective CB1 antagonists that are metabolically stable with limited oral bioavailability. The addition of polarity to 3-carboxamide position has been found to be advantageous. Sulfonamide 7 came out of our efforts to synthesize CB1 antagonists that had high TPSAs.⁷ Those efforts focused on compounds that contained sulfonamide and sulfamide functionality because of the relatively large TPSA for these functional groups. Continued efforts along those lines have yielded more positive results, including two new sulfonamides, 19d, and 20d, that are more selective than 7. Sulfonamide 7 demonstrated modest selectivity for CB1 versus CB2 (16 fold), but both 19d and 20d demonstrated over 100-fold selectivity. This improvement in selectivity came with an increase in potency as well; sulfonamide 20d is 16-fold more potent than 7.

Sulfonamides and sulfamides were not the only polar functionalities to be utilized. Amides, ureas, and carbamates were also synthesized to increase the polarity. Examples of all three have been found to be potent and selective. However, it should be noted that polar groups caused a loss of activity unless they were accompanied by additional lipophilicity. This is best demonstrated by comparing compounds 18g to 18h. The unsubstituted piperidine carboxamide 18g contained no additional lipophilicity and had poor activity at CB1 (18g, Ke CB1 = 4097 nM). With the addition of an ethyl group, such as found in compound 18h, the activity was significantly increased (18h, Ke CB1 = 20.5 nM). The enhancing effects of lipophilicity on potency at CB1 could be also seen in compounds 13–17k.

The shape of the functional group seemed to have impact on potency. The linear (4-aminopiperdine) linker seemed to be favored over the bent (3-aminopiperidine) linker for potency. Structure 18 was found to be the most potent analogue in 6 out of the 10 examples where structures 18–20 possesed the same substituent. However, it should be noted that those 6 analogues were all ureas or carbamates. Of the analogues that favored the bent (3-aminopiperidine) linker only one contained a urea (20g). Amine (19b and 20b), amide (19c), and sulfonamide (20d) substituents favored the 3-aminopiperdine linker. Of the two bent (19, (3S)-3-aminopiperdine; 20, (3R)-3-aminopiperdine) linkers, the R enantiomer (20) was the most favored for activity at CB1. Analogues of structure 20 were found to be more potent than their corresponding analogues of structure 19 seven out of ten times. The 4-amino-4-phenylpiperidine linker present in compounds 13–17k was by far the most potent linker tested. However, it was difficult to determine if the improved potency observed with the 4-amino-4-phenylpiperidine linker was due to shape or the greater lipophilicity present with this linker.

While maintaining a desirable profile in the MDCK-mdr1 assay, which is predictive of brain penetration, gains were made in selectivity over our previously reported sulfonamide 7. Fifteen compounds with over 100-fold selectivity (CB1:CB2 Ki vs CP55940) have been identified. These compounds, at least in part, were designed to increase the TPSA over currently known CB1 antagonists in hopes of limiting exposure to the CNS. They were found to have limited permeability (<1%) in the MDCK-mdr1 permeability assay, which serves as in vitro measure of CNS permeability Based on data from the MDCK-mdr1 permeability assay, a set of compounds were chosen for both plasma and metabolic stability studies in human plasma and hepatic S9 fractions. These studies demonstrated that most compounds tested were at least as stable as 1 with low loss of the parent molecule even after two hours of incubation. Further evidence that some of these compounds do not penetrate the CNS was seen in an in vivo pharmacokinetics (PK) assay on compounds 17a, 18a and 18f. Of these, 17a and 18a had little to no CNS penetration as demonstrated by a very low brain:plasma ratio. Further, both compounds demonstrated limited but clearly detectable oral absorption. Future studies will be aimed at improving the oral bioavailability of this class of compounds as well as exploring other scaffolds. Efficacy studies in disease models where these compounds may be useful are being planned as are more detailed PK studies to establish compound half-lives and dosing regimens.

In conclusion, a series of highly potent and selective CB1 antagonists were synthesized and evaluated leading to the identification of two compounds with limited brain penetration. These compounds will serve as templates for further refinement to enhance their oral bioavailability and to examine the role of peripheral CB1 receptors in various diseases such as obesity, liver fibrosis and diabetes.

Experimental Section

Compound synthesis and characterization

Chemistry

Reactions were conducted under N₂ atmospheres using oven-dried glassware. All solvents and chemicals used were reagent grade. Anhydrous tetrahydrofuran, dichloromethane, and N,N-dimethylformamide (DMF) were purchased from Aldrich and used as such. Unless otherwise mentioned, all reagents and chemicals were purchased from commercial vendors and used as received. Flash column chromatography was carried out on a Teledyne ISCO CombiFlash Companion system using RediSep Rf prepacked columns. Purity and characterization of compounds were established by a combination of HPLC, TLC, and NMR analytical techniques described below. ¹H and ¹³CNMR spectra were recorded on a Bruker Avance DPX-300 (300 MHz) spectrometer and were determined in CHCl₃-d or MeOH-d4 with tetramethylsilane (TMS) (0.00 ppm) or solvent peaks as the internal reference unless otherwise noted. Chemical shifts are reported in ppm relative to the solvent signal, and coupling constant (J) values are reported in hertz (Hz). Thin-layer chromatography (TLC) was performed on EMD precoated silica gel 60 F254 plates, and spots were visualized with UV light or I₂ detection. Low-resolution mass spectra were obtained using a Waters Alliance HT/Micromass ZQ system (ESI). All test compounds were greater than 95% pure as determined by HPLC on an Agilent 1100 system using an Agilent Zorbax SB-Phenyl, 2.1×150 mm, 5 μm column with gradient elution using the mobile phases (A) H₂O containing 0.05% CF₃COOH and (B) Methanol. A flow rate of 1.0 mL/min was used.

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidine-4-carboxylic acid (10)

A 2 M solution of oxalyl chloride in dichloromethane (3 eq., 0.19 mL, 0.377 mmol) was added to 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (22) (1 eq., 48 mg, 0.126 mmol) in dichloromethane (5 mL). Next, 2 drops of anhydrous N,N-dimethylformamide was added and the reaction was stirred for 2 h. The reaction was concentrated in vacuo. The reaction mixture was dissolved in dichloromethane (5 mL). Triethylamine (3 eq., 0.05 mL, 0.377 mmol) and 4-carboxy-4-phenylpiperidin-1-ium chloride (23) (1.5 eq., 45.7 mg, 0.189 mmol) was added and the reaction was stirred for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–10% methanol/dichloromethane with 1% acetic acid to yield pure desired product (10) (48 mg, 67%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.87 – 2.09 (m, 2 H), 2.15 (s, 3 H), 2.61 (t, J=16.18 Hz, 2 H), 3.21 (t, J=12.03 Hz, 1 H), 3.47 (t, J=11.94 Hz, 1 H), 4.26 (d, J=13.61 Hz, 1 H), 4.57 (d, J=13.56 Hz, 1 H), 7.05 (d, J=8.34 Hz, 2 H), 7.12 – 7.45 (m, 10 H), [M + H]⁺ 568.4.

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidine-4-carboxamide (11)

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidine-4-carboxylic acid (22) (1 eq., 12.7 mg, 0.024 mmol), ammonium chloride (10 eq., 12.7 mg, 0.24 mmol), benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP)(1 eq., 10.5 mg, 0.024 mmol), and triethylamine (10.1 eq., 0.03 mL, 0.024 mmol) was stirred in tetrahydrofuran (5 mL) for 3 days. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (11) (6 mg, 44%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.92 – 2.31 (m, 5 H), 2.46 (d, J=13.94 Hz, 2 H), 3.65 (t, J=10.36 Hz, 1 H), 3.75 – 3.90 (m, 1 H), 4.02 (d, J=13.38 Hz, 1 H), 4.23 (d, J=13.00 Hz, 1 H), 5.24 (br. s., 2 H), 7.07 (d, J=8.38 Hz, 2 H), 7.12 – 7.20 (m, 1 H), 7.20 – 7.49 (m, 9 H), [M + H]⁺ 567.5.

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-(ethylamino)piperidine-4-carboxamide (12)

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (22) (1 eq., 20 mg, 0.052 mmol), triethylamine (3 eq., 0.02 mL, 0.157 mmol), 4-(ethylamino)-4-piperidinecarboxamide (1 eq., 9 mg, 0.052 mmol), and benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) (1eq., 23 mg, 0.052 mmol) was stirred in tetrahydrofuran (5 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% CMA 80(chloroform, methanol, ammonium hydroide 80:18:2)/ethyl acetate and precipitated from ethyl acetate with hexane to yield pure desired product (12) (13 mg, 46%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.10 (t, J=6.97 Hz, 3 H), 1.61 – 1.80 (m, 2 H), 2.08 – 2.26 (m, 5 H), 2.45 – 2.63 (m, 2 H), 3.68 (td, J=8.85, 4.43 Hz, 2 H), 3.96 – 4.19 (m, 2 H), 5.40 (br. s., 1 H), 7.07 (d, J=8.29 Hz, 2 H), 7.12 – 7.19 (m, 1 H), 7.20 – 7.36 (m, 3 H), 7.44 (d, J=1.98 Hz, 1 H), [M + H]⁺ 534.5.

tert-Butyl-N-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)carbamate (13)

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (22) (1 eq., 201 mg, 0.53 mmol), triethylamine (3 eq., 0.22 mL, 0.157 mmol), tert-butyl N-(4-phenylpiperidin-4-yl)carbamate (25) (1 eq., 146 mg, 0.53 mmol), and Benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) (1eq., 233 mg, 0.53 mmol) was stirred in tetrahydrofuran (10 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (13) (295 mg, 87%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.37 (br. s., 9 H), 2.11 (dd, J=12.67, 4.00 Hz, 2 H), 2.21 (s, 3 H), 2.24 – 2.34 (m, 1 H), 2.34 – 2.56 (m, 1 H), 3.26 (t, J=12.01 Hz, 1 H), 3.56 (t, J=12.39 Hz, 1 H), 4.32 (d, J=13.75 Hz, 1 H), 4.64 (d, J=13.56 Hz, 1 H), 4.96 (br. s., 1 H), 7.08 (d, J=8.38 Hz, 2 H), 7.13 – 7.49 (m, 10 H), [M + H]⁺ 639.7.

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-amine (14)

tert-Butyl N-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)carbamate (13) (1 eq., 243 mg, 0.380 mmol) was stirred in dichloromethane (7 mL) and trifluoroacetic acid (3 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–50% CMA 80/ethyl acetate to yield pure desired product (14) (178 mg, 87%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.64 – 1.95 (m, 2 H), 2.10 – 2.29 (m, 5 H), 3.50 – 3.66 (m, 1 H), 3.70 – 3.88 (m, 1 H), 4.03 – 4.19 (m, 1 H), 4.42 (d, J=13.28 Hz, 1 H), 7.04 – 7.10 (m, 2 H), 7.13 – 7.20 (m, 1 H), 7.20 – 7.40 (m, 6 H), 7.40 – 7.50 (m, 3 H), [M + H]⁺ 539.4.

N-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)acetamide (15)

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-amine (14) (1 eq., 35.3 mg, 0.066 mmol) was stirred in a mixture of acetic anhydride (2 mL) and pyridine (2 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (15) (27 mg, 71%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 2.01 (s, 3 H), 2.05 – 2.19 (m, 2 H), 2.21 (s, 3 H), 2.34 (d, J=14.60 Hz, 1 H), 2.66 (d, J=13.85 Hz, 1 H), 3.15 – 3.34 (m, 1 H), 3.52 (t, J=11.68 Hz, 1 H), 4.26 (d, J=13.75 Hz, 1 H), 4.54 (d, J=13.75 Hz, 1 H), 6.10 (s, 1 H), 7.03 – 7.11 (m, 2 H), 7.14 – 7.19 (m, 1 H), 7.19 – 7.41 (m, 8 H), 7.44 (d, J=2.17 Hz, 1 H), [M + H]⁺ 581.0.

N-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)methanesulfonamide (16)

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-amine (14) (1 eq., 36.5 mg, 0.068 mmol), methanesulfonyl chloride (2 eq., 0.01 mL, 0.135 mmol), and triethylamine (3 eq., 0.03 mL, 0.203 mmol) was stirred in tetrahydrofuran for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (16) (27 mg, 65%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 2.18 (d, J=4.52 Hz, 6 H), 2.21 – 2.37 (m, 2 H),2.39 – 2.61 (m, 2 H), 3.65 (t, J=10.69 Hz, 1 H), 3.86 (t, J=10.93 Hz, 1 H), 4.07 – 4.20 (m, 2 H), 4.29 (d, J=13.75 Hz, 1 H), 5.30 (s, 1 H), 7.03 – 7.11 (m, 2 H), 7.15 – 7.21 (m, 1 H), 7.21 – 7.38 (m, 4 H), 7.38 – 7.47 (m, 2 H), 7.47 – 7.55 (m, 2 H), [M + H]⁺ 617.3.

3-tert-Butyl-1-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)urea (17a)

1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-amine (14) (1 eq., 39.3 mg, 0.073 mmol), tert-butyl isocyanate (1.5 eq., 0.013 mL, 0.109 mmol), and triethylamine (3.0 eq., 0.03 mL, 0.218 mmol) was stirred in dichloromethane (5 mL) for 16 h. Next, tetrahydrofuran (5 mL) and an additional 0.02 mL of tert-butyl isocyanate were added and the reaction was stirred for 16 h. Finally, the reaction was heated to 40° C for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (17a) (21 mg, 45%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.15 (s, 9 H), 1.93 – 2.17 (m, 4 H), 2.20 (s, 3 H), 2.42 (br. s., 1 H), 3.13 – 3.35 (m, 1 H), 3.58 (br. s., 1 H), 4.25 (br. s., 1 H), 4.44 (s, 1 H), 4.52 – 4.69 (m, 1 H), 5.13 (s, 1 H), 7.03 – 7.10 (m, 2 H), 7.13 – 7.37 (m, 7 H), 7.38 – 7.46 (m, 3 H), [M + H]⁺ 638.7.

1-Benzyl-3-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)urea (17b)

Amine 14 (1 eq., 26.1 mg, 0.048 mmol), benzyl isocyanate (1.5 eq., 9.7 mg, 0.073 mmol), and triethylamine (3.0 eq., 0.02 mL, 0.145 mmol) was stirred in THF (2 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield 17b (24 mg, 74%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.79 – 2.13 (m, 3 H), 2.17 (s, 3 H), 2.53 (br. s., 1 H), 3.13 (br. s., 1 H), 3.48 (br. s., 1 H), 4.21 (d, J=5.75 Hz, 3 H), 4.49 (d, J=13.47 Hz, 1 H), 5.24 (br. s., 1 H), 5.53 (s, 1 H), 6.94 – 7.49 (m, 17 H), [M + H]⁺ 672.4.

3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-(4-cyanophenyl)urea (17c)

Following a procedure similar to the preparation of 17b, 17c was obtained from 14 and the appropriate isocyanate in 74% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.98 (br. s., 2 H), 2.11 – 2.41 (m, 4 H), 2.87 (d, J=13.47 Hz, 1 H), 3.40 (br. s., 1 H), 3.64 (br. s., 1 H), 3.97 – 4.24 (m, 1 H), 4.58 (d, J=13.19 Hz, 1 H), 6.79 (s, 1 H), 6.90 – 7.52 (m, 16 H), 8.56 (br. s., 1 H), [M + H]⁺ 683.8.

3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-(4-fluorophenyl)urea (17d)

Following a procedure similar to the preparation of 17b, 17d was obtained from 14 and the appropriate isocyanate in 67% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.95 (br. s., 2 H), 2.08 – 2.34 (m, 4 H), 2.83 (d, J=13.37 Hz, 1 H), 3.23 – 3.41 (m, 1 H), 3.58 (t, J=12.29 Hz, 1 H), 4.21 (d, J=13.56 Hz, 1 H), 4.58 (d, J=13.28 Hz, 1 H), 6.31 (br. s., 1 H), 6.82 (t, J=8.62 Hz, 2 H), 6.93 – 7.48 (m, 14 H), 7.78 (br. s., 1 H), [M + H]⁺ 676.3.

3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-[4-(dimethylamino)phenyl]urea (17e)

Following a procedure similar to the preparation of 17b, 17e was obtained from 14 and the appropriate isocyanate in 58% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.86 – 2.15 (m, 3 H), 2.21 (s, 3 H), 2.62 (d, J=13.47 Hz, 1 H), 2.90 (s, 6 H), 3.11 (br. s., 1 H), 3.47 (br. s., 1 H), 4.29 (d, J=13.37 Hz, 1 H), 4.59 (d, J=13.37 Hz, 1 H), 5.40 (br. s., 1 H), 6.50 (br. s., 1 H), 6.66 (d, J=8.67 Hz, 2 H), 6.94 – 7.49 (m, 14 H), [M + H]⁺ 701.6.

1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-hexylurea (17f)

Following a procedure similar to the preparation of 17b, 17f was obtained from 14 and the appropriate isocyanate in 72% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.84 (t, J=6.78 Hz, 3 H), 1.04 – 1.38 (m, 8 H), 1.95 – 2.17 (m, 3 H), 2.17 – 2.25 (m, 3 H), 2.51 (br. s., 1 H), 2.90 – 3.11 (m, 2 H), 3.24 (br. s., 1 H), 3.55 (br. s., 1 H), 4.28 (d, J=13.56 Hz, 1 H), 4.48 – 4.65 (m, 2 H), 5.18 – 5.38 (m, 1 H), 6.96 – 7.51 (m, 12 H), [M − H]⁻ 666.8.

1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-(propan-2-yl)urea (17g)

Following a procedure similar to the preparation of 17b, 17g was obtained from 14 and the appropriate isocyanate in 73% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.82 – 1.03 (m, 6 H) 1.94 – 2.24 (m, 6 H) 2.46 (d, J=13.47 Hz, 1 H) 3.26 (t, J=11.26 Hz, 1 H) 3.56 (t, J=12.10 Hz, 1 H) 3.68 – 3.85 (m, 1 H) 4.16 – 4.37 (m, 2 H) 4.59 (d, J=13.47 Hz, 1 H) 5.05 (s, 1 H) 6.94 – 7.49 (m, 12 H), [M + H]⁺ 624.7.

1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-ethylurea (17h)

Following a procedure similar to the preparation of 17b, 17h was obtained from 14 and the appropriate isocyanate in 73% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.78 – 1.04 (m, 3 H), 1.92 – 2.25 (m, 6 H), 2.51 (d, J=13.56 Hz, 1 H), 2.98 – 3.15 (m, 2 H), 3.24 (br. s., 1 H), 3.55 (br. s., 1 H), 4.29 (br. s., 1 H), 4.44 – 4.68 (m, 2 H), 5.23 (s, 1 H), 6.90 – 7.49 (m, 12 H), [M + H]⁺ 610.1.

1-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-3-propylurea (17i)

Following a procedure similar to the preparation of 17b, 17i was obtained from 14 and the appropriate isocyanate in 71% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.74 (t, J=7.39 Hz, 3 H), 1.21 – 1.40 (m, 2 H), 2.05 – 2.29 (m, 6 H), 2.35 (br. s., 1 H), 3.02 (q, J=6.72 Hz, 2 H), 3.28 (br. s., 1 H), 3.57 (br. s., 1 H), 4.07 (t, J=5.27 Hz, 1 H), 4.34 (d, J=13.66 Hz, 1 H), 4.63 (d, J=14.32 Hz, 1 H), 4.74 (s, 1 H), 7.00 – 7.54 (m, 12 H), [M + H]⁺ 624.8.

3-(1-{[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)-1-cyclohexylurea (17j)

Following a procedure similar to the preparation of 17b, 17j was obtained from 14 and the appropriate isocyanate in 69% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.78 – 0.98 (m, 2 H), 1.06 (d, J=9.89 Hz, 1 H), 1.16 – 1.35 (m, 2 H), 1.50 (d, J=8.76 Hz, 3 H), 1.73 (d, J=10.83 Hz, 2 H), 2.00 – 2.27 (m, 6 H), 2.43 (d, J=13.56 Hz, 1 H), 3.26 (br. s., 1 H), 3.37 – 3.66 (m, 2 H), 4.15 – 4.40 (m, 2 H), 4.62 (br. s., 1 H), 4.99 (s, 1 H), 6.90 – 7.55 (m, 12 H), [M + H]⁺ 664.9.

3-Butyl-1-(1-{[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl]carbonyl}-4-phenylpiperidin-4-yl)urea (17k)

Following a procedure similar to the preparation of 17b, 17k was obtained from 14 and the appropriate isocyanate in 71% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.76 – 0.86 (m, 3 H), 1.08 – 1.21 (m, 2 H), 1.28 (dq, J=14.40, 7.10 Hz, 2 H), 1.94 – 2.26 (m, 6 H), 2.50 (d, J=13.47 Hz, 1 H), 3.04 (q, J=6.56 Hz, 2 H), 3.15 – 3.32 (m, 1 H), 3.55 (t, J=12.15 Hz, 1 H), 4.27 (d, J=13.56 Hz, 1 H), 4.48 – 4.74 (m, 2 H), 5.33 (s, 1 H), 6.98 – 7.49 (m, 12 H), [M + H]⁺ 638.6.

tert-Butyl-4-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (18a)

Benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) (1eq, 490 mg, 1.11 mmol) was added to a solution of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (22) (1eq., 422 mg, 1.11 mmol), tert-butyl 4-amino-1-piperidinecarboxylate (1eq., 222 mg, 1.11 mmol), and triethylamine (3 eq., 0.46 mL, 3.32 mmol) in tetrahydrofuran (5 mL). The reaction mixture was stirred for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (18a) (548 mg, 88%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.33 – 1.51 (m, 9 H), 1.93 – 2.10 (m, 2 H), 2.37 (s, 3 H), 2.91 (t, J=11.82 Hz, 2 H), 3.89 – 4.23 (m, 2 H), 6.84 (d, J=8.19 Hz, 1 H), 7.00 – 7.12 (m, 2 H), 7.19 – 7.36 (m, 4 H), 7.43 (d, J=1.32 Hz, 1 H), [M + H]⁺ 563.6.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide (18b)

tert-Butyl 4-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (18a) (1 eq., 531 mg, 0.941 mmol) was stirred in dichloromethane (4 mL) and trifluoroacetic acid (1 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% CMA 80/ethyl acetate to yield pure desired product (18b) (415 mg, 95%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.44 (qd, J=11.81, 3.86 Hz, 2 H), 1.92 – 2.08 (m, 2 H), 2.37 (s, 3 H), 2.64 – 2.85 (m, 2 H), 2.98 – 3.21 (m, 2 H), 3.92 – 4.19 (m, 1 H), 6.85 (d, J=8.29 Hz, 1 H), 7.06 (d, J=8.38 Hz, 2 H), 7.28 (s, 4 H), 7.43 (s, 1 H), [M + H]⁺ 463.5.

N-(1-Acetylpiperidin-4-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (18c)

5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide (18b) (1 eq., 34 mg, 0.073 mmol) was stirred in pyridine (1 mL) and acetic anhydride (1 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (18c) (30 mg, 81%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.28 – 1.49 (m, 2 H), 1.87 – 2.13 (m, 5 H), 2.30 (s, 3 H), 2.62 – 2.82 (m, 1 H), 3.05 – 3.24 (m, 1 H), 3.75 (d, J=13.56 Hz, 1 H), 3.98 – 4.25 (m, 1 H), 4.49 (d, J=13.37 Hz, 1 H), 6.80 (d, J=8.01 Hz, 1 H), 6.99 (d, J=8.48 Hz, 2 H), 7.13 – 7.29 (m, 4 H), 7.36 (d, J=1.51 Hz, 1 H), [M + H]⁺ 505.5.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(1-methanesulfonylpiperidin-4-yl)-4-methyl-1H-pyrazole-3-carboxamide (18d)

Methanesulfonyl chloride (2 eq., 0.01 mL, 0.15 mmol) was added to 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide (18b) (1 eq., 35 mg, 0.076 mmol) and triethyamine (3 eq., 0.03 mL, 0.227 mmol) in tetrahydrofuran (2 mL). The reaction was stirred for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (18d) (36 mg, 87%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.53 – 1.78 (m, 2 H), 2.06 – 2.22 (m, 2 H), 2.37 (s, 3 H), 2.67 – 3.00 (m, 5 H), 3.82 (d, J=12.24 Hz, 2 H), 4.01 – 4.17 (m, 1 H), 6.88 (d, J=8.01 Hz, 1 H), 7.07 (s, 2 H), 7.19 – 7.36 (m, 4 H), 7.43 (d, J=1.70 Hz, 1 H), [M + H]⁺ 543.6.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1-sulfamoylpiperidin-4-yl)-1H-pyrazole-3-carboxamide (18e)

5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide (18b) (1 eq., 38 mg, 0.082 mmol) and sulfamide (5 eq., 39 mg, 0.41 mmol) was heated to 90° C in dioxane (2 mL) for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield desired product (18e) (30 mg, 67%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.60 – 1.78 (m, 2 H), 2.07 – 2.21 (m, 2 H), 2.37 (s, 3 H), 2.86 (t, J=10.83 Hz, 2 H), 3.74 (d, J=12.24 Hz, 2 H), 4.00 – 4.16 (m, 1 H), 4.37 (s, 2 H), 6.87 (d, J=7.91 Hz, 1 H), 7.06 (d, J=8.38 Hz, 2 H), 7.20 – 7.37 (m, 4 H), 7.43 (d, J=1.32 Hz, 1 H), [M + H]⁺ 542.7.

1-N-tert-Butyl-4-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,4-diamido (18f)

5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide (18b) (1 eq., 38 mg, 0.082 mmol), tert-butyl isocyanate (1.5 eq., 0.014 mL, 0.123 mmol), and triethylamine (3eq., 0.034 mL, 0.246 mmol) were stirred in dichloromethane for 16h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield pure desired product (18f) (42 mg, 91%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.27 (d, J=7.16 Hz, 9 H), 1.40 – 1.73 (m, 2 H), 2.05 (s, 2 H), 2.37 (s, 2 H), 2.92 (t, J=11.44 Hz, 2 H), 3.87 (d, J=13.37 Hz, 2 H), 4.00 – 4.18 (m, 1 H), 4.33 (s, 1 H), 6.84 (d, J=7.91 Hz, 1 H), 7.05 (d, J=8.48 Hz, 2 H), 7.20 – 7.35 (m, 4 H), 7.42 (s, 1 H), [M + H]⁺ 562.4.

4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,4-diamido (18g)

1-N-tert-butyl-4-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,4-diamido (18f) (1eq., 33 mg, 0.059 mmol) was stirred in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) overnight. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% CMA 80/ethyl acetate to yield pure desired product (18g) (24 mg, 81%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.41 – 1.60 (m, 2 H), 1.99 – 2.12 (m, 2 H), 2.37 (s, 3 H), 3.01 (t, J=11.77 Hz, 2 H), 3.94 (d, J=13.09 Hz, 2 H), 4.03 – 4.24 (m, 1 H), 4.65 (br. s., 2 H), 6.90 (d, J=7.91 Hz, 1 H), 7.06 (d, J=8.38 Hz, 2 H), 7.22 – 7.37 (m, 4 H), 7.43 (s, 1 H), [M + H]⁺ 506.4.

4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-ethylpiperidine-1,4-diamido (18h)

Following a procedure similar to the preparation of 17b, 18h was obtained from 18b and the appropriate isocyanate in 97% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.15 (t, J=7.21 Hz, 3 H), 1.35 – 1.60 (m, 2 H), 1.93 – 2.14 (m, 2 H), 2.38 (s, 3 H), 2.97 (t, J=11.54 Hz, 2 H), 3.16 – 3.38 (m, 2 H), 3.94 (d, J=13.47 Hz, 2 H), 4.04 – 4.25 (m, 1 H), 4.46 (br. s., 1 H), 6.87 (d, J=8.01 Hz, 1 H), 7.08 (s, 2 H), 7.23 – 7.37 (m, 4 H), 7.44 (d, J=1.22 Hz, 1 H), [M + H]⁺ 534.4.

4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-(propan-2-yl)piperidine-1,4-diamido (18i)

Following a procedure similar to the preparation of 17b, 18i was obtained from 18b and the appropriate isocyanate in 99% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.16 (d, J=6.50 Hz, 6 H), 1.49 (dd, J=11.68, 3.11 Hz, 2 H), 1.98 – 2.11 (m, 2 H), 2.38 (s, 3 H), 2.95 (t, J=11.68 Hz, 2 H), 3.86 – 4.02 (m, 3 H), 4.11 (dd, J=13.70, 6.92 Hz, 1 H), 4.27 (d, J=7.16 Hz, 1 H), 6.86 (d, J=7.91 Hz, 1 H), 7.07 (d, J=8.38 Hz, 2 H), 7.25 – 7.36 (m, 4 H), 7.44 (s, 1 H), [M + H]⁺ 548.5.

4-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-propylpiperidine-1,4-diamido (18j)

Following a procedure similar to the preparation of 17b, 18j was obtained from 18b and the appropriate isocyanate in 95% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.86 – 0.98 (m, 3 H), 1.43 – 1.60 (m, 4 H), 1.94 – 2.13 (m, 2 H), 2.38 (s, 3 H), 2.97 (t, J=11.77 Hz, 2 H), 3.20 (q, J=6.69 Hz, 2 H), 3.94 (d, J=13.37 Hz, 2 H), 4.09 (d, J=6.78 Hz, 1 H), 4.51 (br. s., 1 H), 6.87 (d, J=8.01 Hz, 1 H), 7.07 (d, J=8.29 Hz, 2 H), 7.23 – 7.37 (m, 4 H), 7.44 (s, 1 H), [M + H]⁺ 548.6.

1-N-Butyl-4-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,4-diamido (18k)

Following a procedure similar to the preparation of 17b, 18k was obtained from 18b and the appropriate isocyanate in 76% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.94 (t, J=7.16 Hz, 3 H), 1.28 – 1.41 (m, 2 H), 1.42 – 1.58 (m, 4 H), 1.95 – 2.12 (m, 2 H), 2.38 (s, 3 H), 2.97 (t, J=12.24 Hz, 2 H), 3.16 – 3.33 (m, 2 H), 3.94 (d, J=13.37 Hz, 2 H), 4.03 – 4.23 (m, 1 H), 4.47 (br. s., 1 H), 6.86 (d, J=7.91 Hz, 1 H), 7.07 (d, J=8.29 Hz, 2 H), 7.23 – 7.36 (m, 4 H), 7.44 (s, 1 H), [M + H]⁺ 562.4.

Ethyl-4-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (18l)

Following a procedure similar to the preparation of 20j, 18l was obtained from 18b and ethyl chloroformate in 76% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.27 (d, J=14.22 Hz, 3 H), 1.47 (dd, J=11.63, 3.44 Hz, 2 H), 1.93 – 2.14 (m, 2 H), 2.38 (s, 3 H), 2.98 (br. s., 2 H), 4.14 (q, J=6.97 Hz, 4 H), 6.86 (d, J=8.10 Hz, 1 H), 7.06 (s, 2 H), 7.22 – 7.38 (m, 4 H), 7.44 (d, J=1.51 Hz, 1 H), [M + H]⁺ 535.3.

tert-Butyl-(3S)-3-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (19a)

Following a procedure similar to the preparation of 18a, 19a was obtained from 22 and an appropriate amine in 96% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.31 (s, 9 H), 1.49 (dd, J=12.62, 5.84 Hz, 1 H), 1.65 (d, J=5.09 Hz, 2 H), 1.82 (d, J=9.04 Hz, 1 H), 2.29 (s, 3 H), 3.32 (br. s., 3 H), 3.48 – 3.73 (m, 1 H), 3.93 – 4.14 (m, 1 H), 6.99 (d, J=8.38 Hz, 3 H), 7.12 – 7.27 (m, 4 H), 7.33 (s, 1 H), [M + H]⁺ 563.4.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-[(3S)-piperidin-3-yl]-1H-pyrazole-3-carboxamide (19b)

Following a procedure similar to the preparation of 18b, 19b was obtained from 19a in >99%. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.42 – 1.95 (m, 4 H), 2.26 (s, 3 H), 2.86 (dd, J=12.15, 8.76 Hz, 2 H), 3.09 (d, J=12.81 Hz, 1 H), 3.40 (dd, J=12.20, 2.97 Hz, 1 H), 4.13 – 4.32 (m, 1 H), 6.99 (s, 2 H), 7.09 – 7.28 (m, 5 H), 7.33 (s, 1 H), [M + H]⁺ 463.7.

N-[(3S)-1-Acetylpiperidin-3-yl]-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (19c)

Following a procedure similar to the preparation of 18c, 19c was obtained from 19b in 87% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.62 (s, 4 H), 2.01 – 2.08 (m, 3 H), 2.30 (s, 3 H), 3.10 – 3.29 (m, 2 H), 3.81 (d, J=13.19 Hz, 2 H), 4.00 (d, J=6.59 Hz, 1 H), 6.86 (d, J=7.06 Hz, 1 H), 6.99 (d, J=8.38 Hz, 2 H), 7.15 – 7.28 (m, 4 H), 7.36 (s, 1 H), [M + H]⁺ 505.6.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-[(3S)-1-methanesulfonylpiperidin-3-yl]-4-methyl-1H-pyrazole-3-carboxamide (19d)

Following a procedure similar to the preparation of 18d, 19d was obtained from 19b in 73% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.58 – 1.73 (m, 2 H), 1.83 (d, J=10.46 Hz, 2 H), 2.29 (s, 3 H), 2.74 (s, 3 H), 2.99 – 3.25 (m, 3 H), 3.45 (dd, J=11.73, 3.16 Hz, 1 H), 4.24 (br. s., 1 H), 6.84 – 7.11 (m, 3 H), 7.14 – 7.28 (m, 4 H), 7.35 (s, 1 H), [M + H]⁺ 543.5.

(3S)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-ethylpiperidine-1,3-diamido (19e)

Following a procedure similar to the preparation of 17b, 19e was obtained from 19b and the appropriate isocyanate in 80% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.04 (t, J=7.39 Hz, 3 H), 1.55 (d, J=7.44 Hz, 3 H), 1.91 (br. s., 1 H), 2.29 (s, 3 H), 2.95 – 3.30 (m, 4 H), 3.60 (br. s., 1 H), 3.97 (br. s., 1 H), 4.63 (br. s., 1 H), 6.90 (d, J=6.97 Hz, 1 H), 6.99 (d, J=8.38 Hz, 2 H), 7.15 – 7.29 (m, 4 H), 7.35 (s, 1 H), [M − H]⁻ 534.4.

(3S)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-(propan-2-yl)piperidine-1,3-diamido (19f)

Following a procedure similar to the preparation of 17b, 19f was obtained from 19b and the appropriate isocyanate in 78% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.01 (d, J=6.50 Hz, 3 H), 1.07 (d, J=6.50 Hz, 3 H), 1.41 – 1.66 (m, 3 H), 1.88 (d, J=6.40 Hz, 1 H), 2.30 (s, 3 H), 3.10 – 3.27 (m, 2 H), 3.40 – 3.61 (m, 2 H), 3.87 (d, J=6.59 Hz, 1 H), 3.93 – 4.04 (m, 1 H), 4.43 (d, J=7.06 Hz, 1 H), 6.81 – 7.05 (m, 3 H), 7.10 – 7.28 (m, 4 H), 7.35 (s, 1 H), [M − H]⁻ 548.7.

(3S)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-propylpiperidine-1,3-diamido (19g)

Following a procedure similar to the preparation of 17b, 19g was obtained from 19b and the appropriate isocyanate in 78% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.71 – 0.87 (m, 3 H), 1.35 – 1.47 (m, 2 H), 1.48 – 1.63 (m, 3 H), 1.90 (br. s., 1 H), 2.29 (s, 3 H), 2.95 – 3.27 (m, 4 H), 3.58 (br. s., 2 H), 3.98 (d, J=6.69 Hz, 1 H), 4.68 (br. s., 1 H), 6.86 – 7.05 (m, 3 H), 7.12 – 7.29 (m, 4 H), 7.35 (s, 1 H), [M + H]⁺ 548.6.

(3S)-1-N-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (19h)

Following a procedure similar to the preparation of 17b, 19h was obtained from 19b and the appropriate isocyanate in 85% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.74 – 0.94 (m, 3 H), 1.09 – 1.32 (m, 2 H), 1.33 – 1.46 (m, 2 H), 1.46 – 1.63 (m, 3 H), 1.90 (br. s., 1 H), 2.29 (s, 3 H), 3.05 – 3.21 (m, 4 H), 3.58 (t, J=14.32 Hz, 2 H), 3.84 – 4.06 (m, 1 H), 4.65 (br. s., 1 H), 6.90 (d, J=6.88 Hz, 1 H), 6.99 (d, J=8.38 Hz, 2 H), 7.16 – 7.29 (m, 4 H), 7.35 (s, 1 H), [M − H]⁻ 562.5.

(3S)-1-N-tert-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (19i)

Following a procedure similar to the preparation of 17b, 19i was obtained from 19b and the appropriate isocyanate in 77% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.07 – 1.29 (m, 9 H), 1.42 – 1.60 (m, 2 H), 1.66 (s, 1 H), 1.80 – 1.95 (m, 1 H), 2.30 (s, 3 H), 3.10 – 3.31 (m, 2 H), 3.46 (d, J=2.45 Hz, 2 H), 3.99 (br. s., 1 H), 4.51 (s, 1 H), 6.99 (d, J=8.29 Hz, 3 H), 7.15 – 7.31 (m, 4 H), 7.34 (s, 1 H), [M + H]⁺ 562.4.

Ethyl-(3S)-3-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (19j)

Following a procedure similar to the preparation of 20j, 19j was obtained from 19b and ethyl chloroformate in 67% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.01 – 1.20 (m, 3 H), 1.40 – 1.75 (m, 3 H), 1.91 (br. s., 1 H), 2.30 (s, 3 H), 3.16 (br. s., 2 H), 3.47 – 3.64 (m, 1 H), 3.81 (d, J=10.93 Hz, 1 H), 3.93 – 4.17 (m, 3 H), 6.88 (d, J=8.01 Hz, 1 H), 6.99 (d, J=8.38 Hz, 2 H), 7.15 – 7.28 (m, 4 H), 7.35 (s, 1 H), [M + H]⁺ 535.5.

tert-Butyl-(3R)-3-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (20a)

Following a procedure similar to the preparation of 18a, 20a was obtained from 22 and an appropriate amine in 89% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.28 – 1.42 (m, 9 H), 1.47 – 1.61 (m, 1 H), 1.70 (d, J=5.18 Hz, 2 H), 1.87 (d, J=8.95 Hz, 1 H), 2.35 (s, 3 H), 3.38 (br. s., 3 H), 3.60 (br. s., 1 H), 3.95 – 4.21 (m, 1 H), 7.04 (d, J=8.38 Hz, 3 H), 7.19 – 7.33 (m, 4 H), 7.39 (s, 1 H), [M + H]⁺ 563.3.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-[(3R)-piperidin-3-yl]-1H-pyrazole-3-carboxamide (20b)

Following a procedure similar to the preparation of 18b, 20b was obtained from 20b in >99%. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.54 – 2.01 (m, 4 H), 2.33 (s, 3 H), 2.80 – 3.00 (m, 2 H), 3.17 (d, J=12.62 Hz, 1 H), 3.38 – 3.57 (m, 1 H), 4.22 – 4.41 (m, 1 H), 6.50 (br. s., 2 H), 7.06 (d, J=8.38 Hz, 2 H), 7.18 – 7.35 (m, 5 H), 7.41 (s, 1 H), [M + H]⁺ 463.7.

N-[(3R)-1-Acetylpiperidin-3-yl]-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (20c)

Following a procedure similar to the preparation of 18c, 20c was obtained from 20b in 87% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.72 (br. s., 4 H), 2.11 – 2.17 (m, 3 H), 2.38 (s, 3 H), 3.18 – 3.37 (m, 2 H), 3.89 (d, J=13.19 Hz, 2 H), 4.09 (d, J=6.59 Hz, 1 H), 6.95 (d, J=7.06 Hz, 1 H), 7.08 (d, J=8.29 Hz, 2 H), 7.20 – 7.40 (m, 4 H), 7.44 (s, 1 H), [M + H]⁺ 505.5.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-[(3R)-1-methanesulfonylpiperidin-3-yl]-4-methyl-1H-pyrazole-3-carboxamide (20d)

Following a procedure similar to the preparation of 18d, 20d was obtained from 20b in 78% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.68 – 1.82 (m, 2 H), 1.91 (d, J=10.36 Hz, 2 H), 2.38 (s, 3 H), 2.83 (s, 3 H), 3.11 – 3.34 (m, 3 H), 3.53 (dd, J=11.73, 3.06 Hz, 1 H), 4.33 (br. s., 1 H), 7.00 – 7.19 (m, 3 H), 7.23 – 7.38 (m, 4 H), 7.44 (s, 1 H), [M − H]⁻ 541.6.

(3R)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-ethylpiperidine-1,3-diamido (20e)

Following a procedure similar to the preparation of 17b, 20e was obtained from 20b and the appropriate isocyanate in 76% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.06 – 1.21 (m, 3 H), 1.49 – 1.74 (m, 3 H), 1.99 (br. s., 1 H), 2.38 (s, 3 H), 3.04 – 3.34 (m, 4 H), 3.57 – 3.79 (m, 2 H), 4.07 (d, J=6.50 Hz, 1 H), 4.72 (br. s., 1 H), 6.98 (d, J=6.97 Hz, 1 H), 7.07 (d, J=8.38 Hz, 2 H), 7.23 – 7.36 (m, 4 H), 7.44 (s, 1 H), [M + H]⁺ 534.5.

(3R)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-(propan-2-yl)piperidine-1,3-diamido (20f)

Following a procedure similar to the preparation of 17b, 20f was obtained from 20b and the appropriate isocyanate in 69% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.09 (d, J=6.50 Hz, 3 H), 1.17 (s, 3 H), 1.51 – 1.78 (m, 3 H), 1.98 (br. s., 1 H), 2.38 (s, 3 H), 3.18 – 3.41 (m, 2 H), 3.63 (dd, J=13.47, 2.73 Hz, 2 H), 3.95 (d, J=6.59 Hz, 1 H), 4.07 (d, J=6.78 Hz, 1 H), 4.51 (d, J=7.16 Hz, 1 H), 6.90 – 7.14 (m, 3 H), 7.21 – 7.37 (m, 4 H), 7.43 (s, 1 H), [M + H]⁺ 548.6.

(3R)-3-C-5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-1-N-propylpiperidine-1,3-diamido (20g)

Following a procedure similar to the preparation of 17b, 20g was obtained from 20b and the appropriate isocyanate in 87% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.81 (t, J=7.44 Hz, 3 H) 1.36 – 1.47 (m, 2 H), 1.48 – 1.65 (m, 3 H), 1.82 – 1.96 (m, 1 H), 2.29 (s, 3 H), 2.98 – 3.24 (m, 4 H), 3.46 – 3.68 (m, 2 H), 3.86 – 4.09 (m, 1 H), 4.68 (t, J=4.99 Hz, 1 H), 6.91 (d, J=6.97 Hz, 1 H), 6.99 (d, J=8.38 Hz, 2 H), 7.15 – 7.28 (m, 4 H), 7.35 (s, 1 H), [M + H]⁺ 548.8.

(3R)-1-N-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (20h)

Following a procedure similar to the preparation of 17b, 20h was obtained from 20b and the appropriate isocyanate in 72% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.82 (t, J=7.16 Hz, 3 H) 1.24 (dd, J=14.93, 7.21 Hz, 2 H), 1.32 – 1.45 (m, 2 H), 1.47 – 1.64 (m, 3 H), 1.90 (br. s., 1 H), 2.29 (s, 3 H), 2.95 – 3.26 (m, 4 H), 3.42 – 3.70 (m, 2 H), 3.98 (d, J=6.69 Hz, 1 H), 4.65 (br. s., 1 H), 6.90 (d, J=6.88 Hz, 1 H), 6.98 (d, J=8.38 Hz, 2 H), 7.14 – 7.28 (m, 4 H), 7.35 (s, 1 H), [M + H]⁺ 562.3.

(3R)-1-N-tert-Butyl-3-C-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-piperidine-1,3-diamido (20i)

Following a procedure similar to the preparation of 17b, 20i was obtained from 20b and the appropriate isocyanate in 70% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.22 – 1.38 (m, 9 H), 1.61 (d, J=8.01 Hz, 2 H), 1.71 – 1.79 (m, 1 H), 1.89 – 2.04 (m, 1 H), 2.38 (s, 3 H), 3.23 – 3.37 (m, 2 H), 3.54 (d, J=2.73 Hz, 2 H), 4.07 (d, J=6.78 Hz, 1 H), 4.59 (s, 1 H), 6.94 – 7.13 (m, 3 H), 7.20 – 7.39 (m, 4 H), 7.43 (s, 1 H), [M + H]⁺ 562.5.

Ethyl-(3R)-3-[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-amido]piperidine-1-carboxylate (20j)

Amine 20b (1.0 eq., 19.2 mg, 0.041 mmol), ethyl chloroformate (1.5 eq., 6.7 mg, 0.062 mmol), and triethyamine (3.0 eq., 0.02 mL, 0.124 mmol) were stirred in THF (2 mL) at room temp. for 16 h. The reaction was concentrated in vacuo. The crude reaction material was then purified by silica gel column chromatography using 0–100% ethyl acetate/hexane to yield 20j (15 mg, 68%). ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.08 – 1.22 (m, 3 H), 1.42 – 1.74 (m, 3 H), 1.91 (br. s., 1 H), 2.30 (s, 3 H), 3.17 (br. s., 2 H), 3.56 (d, J=13.19 Hz, 1 H), 3.82 (d, J=10.74 Hz, 1 H), 3.94 – 4.15 (m, 3 H), 6.88 (d, J=7.91 Hz, 1 H), 6.94 – 7.04 (m, 2 H), 7.15 – 7.28 (m, 4 H), 7.36 (s, 1 H), [M + H]⁺ 535.4.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-[(4-{[(ethylcarbamoyl)amino]methyl}cyclohexyl)methyl]-4-methyl-1H-pyrazole-3-carboxamide (21a)

Following a procedure similar to the preparation of 17b, 21a was obtained from 26 and the appropriate isocyanate in 91% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.01 – 1.12 (m, 3 H), 1.21 – 1.63 (m, 8 H), 1.72 (br. s., 2 H), 2.29 (s, 3 H), 2.94 (br. s., 1 H), 3.04 (br. s., 1 H), 3.09 – 3.23 (m, 2 H), 3.30 (t, J=6.73 Hz, 1 H), 4.48 (br. s., 2 H), 6.85 – 7.06 (m, 3 H), 7.15 – 7.27 (m, 4 H), 7.36 (s, 1 H), [M + H]⁺ 576.6.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-{[4-({[(propan-2-yl)carbamoyl]amino}methyl)cyclohexyl]methyl}-1H-pyrazole-3-carboxamide (21b)

Following a procedure similar to the preparation of 17b, 21b was obtained from 26 and the appropriate isocyanate in 84% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.07 (d, J=6.40 Hz, 6 H), 1.25 – 1.61 (m, 8 H), 1.64 – 1.78 (m, 2 H), 2.30 (s, 3 H), 2.93 (br. s., 1 H), 3.04 (br. s., 1 H), 3.16 – 3.24 (m, 1 H), 3.25 – 3.36 (m, 1 H), 3.77 (br. s., 1 H), 4.21 (br. s., 1 H), 4.38 (br. s., 1 H), 6.81 – 7.06 (m, 3 H), 7.12 – 7.28 (m, 4 H), 7.36 (s, 1 H), [M + H]⁺ 590.5.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-[(4-{[(propylcarbamoyl)amino]methyl}cyclohexyl)methyl]-1H-pyrazole-3-carboxamide (21c)

Following a procedure similar to the preparation of 17b, 21c was obtained from 26 and the appropriate isocyanate in 84% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.78 – 0.89 (m, 3 H), 1.25 – 1.53 (m, 10 H), 1.71 (br. s., 2 H), 2.29 (s, 3 H), 2.94 (br. s., 1 H), 3.05 (br. s., 3 H), 3.19 (s, 1 H), 3.29 (t, J=6.73 Hz, 1 H), 4.49 (br. s., 2 H), 6.82 – 7.05 (m, 3 H), 7.12 – 7.28 (m, 4 H), 7.35 (s, 1 H), [M + H]⁺ 590.4.

N-[(4-{[(Butylcarbamoyl)amino]methyl}cyclohexyl)methyl]-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (21d)

Following a procedure similar to the preparation of 17b, 21d was obtained from 26 and the appropriate isocyanate in 98% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 0.69 – 0.91 (m, 3 H), 1.08 – 1.62 (m, 12 H), 1.64 – 1.84 (m, 2 H), 2.29 (s, 3 H), 2.94 (s, 1 H), 2.99 – 3.13 (m, 3 H), 3.19 (s, 1 H), 3.29 (t, J=6.73 Hz, 1 H), 4.47 (d, J=5.56 Hz, 2 H), 6.81 – 7.06 (m, 2 H), 7.13 – 7.29 (m, 4 H), 7.36 (s, 1 H), [M + H]⁺ 604.6.

N-[(4-{[(tert-Butylcarbamoyl)amino]methyl}cyclohexyl)methyl]-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (21e)

Following a procedure similar to the preparation of 17b, 21e was obtained from 26 and the appropriate isocyanate in 71% yield. ¹H NMR (300 MHz, CHLOROFORM-d) d ppm 1.34 (d, J=2.07 Hz, 9 H), 1.38 – 1.66 (m, 8 H), 1.82 (br. s., 2 H), 2.38 (s, 3 H), 2.98 (br. s., 1 H), 3.09 (br. s., 1 H), 3.28 (s, 1 H), 3.38 (t, J=6.78 Hz, 1 H), 4.25 (br. s., 2 H), 6.93 – 7.12 (m, 3 H), 7.23 – 7.37 (m, 4 H), 7.44 (s, 1 H), [M + H]⁺ 604.6.

Calcium mobilization and radioligand displacement assays

Each compound was pharmacologically characterized using a functional fluorescent CB1 activated Gαq₁₆-coupled intracellular calcium mobilization assay in CHO-K1 cells as has been described in our previous publications and apparent affinity (Ke) values were determined.⁷ Briefly, CHO-K1 cells were engineered to co-express human CB1 and Gq_α16. Activation of CB1 by an agonist then leads to generation of inositol phospahatase 3 (IP₃) and activation of IP₃ receptors, which leads to mobilization of intracellular calcium. Calcium flux was monitored in a 96-well format using the fluorescent dye Calcein-4 AM in an automated platereader (Flexstation, Molecular Devices). The antagonism of a test compound was measured by its ability to shift the concentration response curve of the synthetic CB1 agonist CP55940 rightwards using the equation:

$$\text{Ke}=[\text{Ligand}]/[\text{DR}-1]$$

where DR is the EC₅₀ ratio of CP55940 in the presence or absence of a test agent.¹⁴

Further characterization of select compounds was performed using radioligand displacement of [³H]SR141716 and equilibrium dissociation constant (Ki) values were determined as has been described previously.^{7, 15} Selectivity of these compounds at CB1 versus CB2 was also determined by obtaining Ki values at either receptor using displacement of [³H]CP55940 in membranes of CHO-K1cells over-expressing either receptor. Data reported are average values from 3–6 measurements.

MDCK-mdr1 permeability assays

MDCK-mdr1 cells obtained from the Netherlands Cancer Institute were grown on Transwell type filters (Corning) for 4 days to confluence in DMEM/F12 media containing 10% fetal bovine serum and antibiotics as has been described previously.⁷ Compounds were added to the apical side at a concentration of 3.16 μM in a transport buffer comprising of 1X Hank’s balanced salt solution, 25 mM D-glucose and buffered with HEPES to pH 7.4. Samples were incubated for 1 hr at 37°C and carefully collected from both the apical and basal side of the filters. Compounds selected for MDCK-mdr1 cell assays were infused on an Applied Biosystems API-4000 mass spectrometer to optimize for analysis using multiple reaction monitoring (MRM). Flow injection analysis was also conducted to optimize for mass spectrometer parameters. Samples from the apical and basolateral side of the MDCK cell assay were dried under nitrogen on a Turbovap LV. The chromatography was conducted with an Agilent 1100 binary pump with a flow rate of 0.5 mL/min. Mobile phase solvents were A, 0.1% formic acid in water, and B, 0.1% formic acid in methanol. The initial solvent conditions were 10% B for 1 minute, then a gradient was used by increasing to 95% B over 5 minutes, then returning to initial conditions. Data reported are average values from 2–3 measurements.

In vitro stability testing

In vitro testing for metabolic stability was conducted in pooled samples of mixed gender human plasma from BioChemed Services, Winchester, VA and human mixed gender pooled hepatic S9 fraction supplied by Xenotech, LLC, Lenexa, KS. Identity of the donors was unknown.

For the hepatic S9 metabolism studies, all samples were tested at 10 μM final concentration in a 1 ml volume containing 1 mg/ml S9. Samples were incubated in a buffer containing 50 mM potassium phosphate, pH 7.4 with 3 mM MgCl2 and a NADPH regeneration system comprising of NADP (1 mM), glucose-6-phosphate (5 mM) and glucose-6-phosphate dehydrogenase (1 unit/ml). Triplicate samples were incubated for 0, 15, 30, 60 and 120 min. Reactions were terminated by addition of 3 volumes of acetonitrile and processed as described for the MDCK-mdr1 assays but standard curves were prepared in blank matrix for each compound for quantitative assessment.

The plasma stability studies were conducted at 37°C in a volume of 1 ml plasma per sample. All compounds were tested at 10 μM final concentration at 0, 30 and 60 min after a 5 min pre-incubation. Reactions were terminated by addition of acetonitrile and analyzed as described above.

Evaluation of compounds in vivo

Male Sprague Dawley rats aged 7–8 weeks at time of dosing were acquired from Charles River Laboratories and were dosed by two routes: ip or oral. Oral doses were formulated in corn oil and ip doses were formulated in 1:1:18 ethanol:cremophor:saline, both at 10 mg/kg. Plasma and brain were taken from all rats at 1 hour post-dose. At 30 minutes post-dose, tail vein blood was collected only from rats dosed orally.

Samples were prepared and analyzed as follows: Plasma (50 μL) was mixed with 10 uL of internal standard, reserpine (1 μg/mL), 10 μL of acetonitrile, and 300 μL of acetonitrile, vortexed, and centrifuged at 9000g for 5 minutes. Supernatant, (100 μL) was mixed with 900 μL of 50:50 methanol:water in autosampler vials. For 30 minute plasma samples, the supernatant was injected without dilution. The left lobe of the brain was homogenized with 50:50 ethanol:water (3:1, v/v) using a Potter Elvehjem type homogenizer. Homogenate (50 μL) was mixed with 10 μL of internal standard, reserpine (1 μg/mL), 10 μL of acetonitrile, and 300 μL of acetonitrile, vortexed and centrifuged at 9000 g for 5 minutes. Supernatant was transferred to inserts and injected without dilution. Standards were prepared as above for each compound in blank plasma, blank liver homogenate, and blank brain homogenate. Standards used were within 15% of nominal, except for 20% at LOQ. Compounds for LC-MS/MS analyses were supplied at 1 mg/mL in methanol. The stock solutions were further diluted to ~100 ng/mL. The 100 ng/mL solutions were used to optimize the mass spectrometer for MRM transitions and mass spectrometer parameters. Infusion and flow injection optimization were also performed.

ABBREVIATIONS USED

CB1

Cannabinoid Receptor 1

CB2

Cannabinoid Receptor 2

CNS

Central Nervous System

BBB

Blood-Brain Barrier

TPSA

Topological Polar Surface Area

ECS

Endocannabinoid System

CBR

Cannabinoid Receptors

Ke

apparent affinity constant

MDCK-mdr1

Madin-Darby canine kidney cells transfected with the human MDR1 gene

A

Apical

B

basal

BOP

Benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate

CHO-K1

Chinese Hamster Ovary Cells

IP₃

Inositol Phospahatase 3

MRM

Multiple Reaction Monitoring

LOQ

Below Limit of Quantitation

NA

not applicable