Indazole Partial Agonists Targeting Peripheral Cannabinoid Receptors

George Amato; Lucas Laudermilk; Vineetha Vasukuttan; Elaine A Gay; Ann M Decker; Rodney Snyder; Yun Lan Yue; Scott Runyon; Rangan Maitra

doi:10.1016/j.bmc.2025.118327

. Author manuscript; available in PMC: 2026 Jul 21.

Published in final edited form as: Bioorg Med Chem. 2025 Jul 21;129:118327. doi: 10.1016/j.bmc.2025.118327

Indazole Partial Agonists Targeting Peripheral Cannabinoid Receptors

George Amato ¹, Lucas Laudermilk ¹, Vineetha Vasukuttan ¹, Elaine A Gay ¹, Ann M Decker ¹, Rodney Snyder ¹, Yun Lan Yue ¹, Scott Runyon ¹, Rangan Maitra ^1,^*

PMCID: PMC12439760 NIHMSID: NIHMS2100732 PMID: 40716175

Abstract

Considerable efforts have been made to produce full agonists of cannabinoid receptors (CBRs), but there has been limited reports on partial agonists and the associated structure activity relationship (SAR) studies. Partial agonists of peripheral CBRs may have unique pharmacological profiles that provide prolonged efficacy through delayed or limited development of tolerance, absent or limited psychiatric effects, and good therapeutic index. Some potential therapeutic applications include inflammatory diseases, gastrointestinal (GI) disorders and pain. In this report, we show the SAR developed in the conversion of indazole full agonists to peripheral partial agonists. Compound 45 is a partial agonist of CBRs with 50% Effective Concentration (EC₅₀) of ~150 nM at human (h)CB1 and 35 nM at hCB2 along with % Maximum Efficacy (E_max) of ~32 at hCB1 and ~17 at hCB2 respectively. This compound demonstrated peripheral selectivity and oral absorption in mouse pharmacokinetic (PK) studies with a half-life of ~7.3 hours in plasma and <10% brain penetrance.

Keywords: CB1, cannabinoid, peripheral, CB2, partial, agonist

Graphical Abstract

graphic file with name nihms-2100732-f0072.jpg

1. Introduction

Selective modulation of the peripheral G protein coupled cannabinoid receptors (CBRs) has potential therapeutic applications in treating inflammation, GI disorders, pain and other important medical conditions.^1–8 (−)-Trans-Δ⁹-tetrahydrocannabinol (THC), a constituent of marijuana believed to be a main contributor to its therapeutic effects, is a partial agonist of both CB1 and CB2.⁹ The centrally mediated psychoactive effects of THC, however, limit its potential therapeutic uses. Partial agonists of peripheral CBRs are expected to have unique pharmacological profiles that favor development of efficacious compounds with a good therapeutic index.^{10, 11} A peripheral CB2 selective partial agonist was recently shown to have a protective effect in a mouse model of nephrotoxicity and to have a safe in vivo profile.¹² While considerable efforts have been made to produce selective antagonists or full agonists of CBRs, there are limited reports on partial agonists. The lack of needed SAR to control partial versus full agonism makes this a challenging issue.

Our efforts in this field began with a project to make peripheral analogs of BAY 59–3074 (1, Figure 1), a centrally penetrating partial agonist of CBRs.¹³ While some success was achieved, the resulting SAR did not provide a clear path to compounds with desired profile.¹⁴ We noted a previous patent application describing indazole compounds as full CB1 agonists with polar functionality that should favor peripheral selectivity (2, Figure 1)¹⁵. Other reports on indazoles as potent centrally acting cannabinoid receptor full agonists have also appeared.^{16, 17} Recently, the discovery of biased indazole peripheral full agonists was published.¹⁸ We sought to discover novel indazole compounds (2, Figure 1) that would favor peripheral selectivity and possess partial agonism (target efficacy of 20–50%) of both CB1 and CB2 receptors. We have previously reported our early efforts directed at related pyrazole partial agonists, which resulted in lead compound 3.¹⁹ Pyrazole 3 is a partial agonist of hCB1 and has peripheral selectivity in mice, but CBR potency and PK need further improvements.

2. Results and Discussion

2.1. Compound Design and Synthesis

Reported crystal structures of hCB1 and hCB2 combined with docking studies of THC at the orthosteric binding site, show the potential polar and nonpolar receptor binding regions of importance.^20–23 We envisioned that indazoles 2 may be binding to hCB1 with the nonpolar R₁ group in a lipophilic channel and the amide linker directing R₂ towards a mixed polarity channel that leads to the extracellular space. The location of R₂ in the mixed polarity channel is consistent with SAR generated by Pfizer, showing that polar functionality is allowed at this position.¹⁵ Furthermore, the binding location of the amide linker and R₂ is hypothesized to be near the aromatic toggle residues that are important for efficacy (PHE200 and TRP356 in the CB1 binding site). Modification of R₂ should therefore allow for the tuning of both efficacy and polarity to achieve our goal of discovering peripherally selective partial agonists. While centrally acting indazole CB1 agonists are known, there are no reports of indazoles as peripherally selective CB1 partial agonists.

Indazole 4 (Table 1) was reported¹⁶ in the literature as a potent full agonist of hCB1 and hCB2, and our results confirm this.¹⁹ Compound 4 is an intriguing starting point because of its drug like features: an indazole core, three hydrogen bond donors, a cLogP of 2.9, a TPSA of 88, and a MW of 368. Analyses of clinical compounds indicate that a TPSA of 80–140 Å, the presence of 1–3 hydrogen bond donors, a cLogP of <5, and a MW of 450–600 Da will generally improve the likelihood of identifying a compound with good oral bioavailability and minimal CNS penetration.^24–26 Furthermore, the indazole core is considered to be a privileged core that is used in drug development and is amendable to varied synthetic functionalization. In this report we show how changes to R₂ led to the conversion of a full agonist to a partial agonist. We also report on the effects of changes to R₁ on potency, efficacy, and metabolic stability.

Table 1. Right Side Amide Groups.


#	-R	hCB1 Ca EC₅₀ (nM)	hCB1 Ca E_max^a (%)	hCB2 Ca EC₅₀ (nM)	hCB2 Ca E_max^a (%)
4		3.4 ± 0.6	120 ± 3	17 ± 2	77 ±2
5		5545 ± 435	123 ± 7	516 ± 19	54 ± 1
6		12 ± 2	120 ± 1	30 ± 1	73 ± 3
7		178 ± 55	104 ± 3	42 ± 9	69 ± 10
8		337 ± 33	115 ± 2	59 ± 7	66 ± 8
9		1244 ± 17	89 ± 2	63 ± 6	71 ± 7
10		>10000		477 ± 142	61 ± 6
11		182 ± 6	111 ± 2	21 ± 3	84 ± 5
12		360 ± 88	112 ± 2	360 ± 48	70 ± 2
13		282 ± 73	103 ± 2	74 ± 19	77 ± 3
14		20 ± 9	92 ± 3	16 ± 1	82 ± 6
15		>10,000		>10,000
16		168 ± 13	89 ± 3	190 ± 88	46 ± 6
17		32 ± 15	96 ± 1	101 ± 34	68 ± 9

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E_max was measured as a percentage of the maximum CP55,940 signal in CHO cells expressing hCB1 or hCB2 receptor and G_αq16 using a calcium mobilization assay.

Our synthesis of indazole 4 was previously reported.¹⁹ Indazoles 7–9 (in Table 1), indazoles 18, 20, 21 and 28 (in Table 2) and 37 (in Table 3) were synthesized as shown in Scheme 1. Commercially available 1H-indazole-3-carboxylic acid methyl ester was N-alkylated (reaction a), followed by hydrolysis of the ester (reaction b) to provide the intermediate carboxylic acid 60. The carboxylic acid 60 was coupled to an amine (reaction c) to provide the amides 61 (7–9, 18, 20, 21, 28, 37).

Table 2. Right Side Aryl Amide Groups.


#	R	hCB1 Ca EC₅₀ (nM)	hCB1 Ca E_max^a (%)	hCB2 Ca EC₅₀ (nM)	hCB2 Ca E_max^a (%)
18		181 ± 20	91 ± 4	168 ± 24	53 + 5
19		423 ± 190	105 ± 1	52 ± 22	79 ± 3
20		38 ± 14	104 ± 6	172 ± 12	32 ± 2
21		>10,000		1383 ± 149	13 ± 1
22		26 ± 2	85 ± 6	50 + 5	48 + 4
23		433 ± 84	23 ± 2	660 ± 135	25 ± 2
24		25 ± 5	24 ± 1	27 ± 7	41 ± 2
25		>10,000	>10,000	68 ± 1	67 ± 1
26		590 ± 152	40 ± 9	160 ± 34	34 ± 2
27		2741 ± 939	25 ± 5	96 ± 6	44 ± 5

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E_max was measured as a percentage of the maximum CP55,940 signal in CHO cells expressing the hCB1 or hCB2 receptor and G_αq16 using a calcium mobilization assay.

Table 3. Right Side Non-Aryl Amide Groups.


#	R₂	hCB1 Ca EC₅₀ (nM)	hCB1 Ca E_max^a (%)	hCB2 Ca EC₅₀ (nM)	hCB2 Ca E_max^a (%)
28		17 ± 1	111 ± 2	28 ± 8	78 ± 6
29		950 ± 108	79 ± 5	178 ± 45	29 ± 10
30		671 ± 161	91 ± 4	>10000	>10000
31		139 ± 11	75 ± 5	29 ± 4	37 ± 3
32		168 ± 23	109 ± 3	150 ± 6	41 ± 3
33		239 ± 76	110 ± 4	170 ± 24	36 ± 5
34		369 ± 57	44 ± 4	28 ± 13	13 ± 2
35		40 ± 5	109 ± 6	29 ± 4	62 ± 2
36		2371 ± 452	70 ± 1	132 ± 49	59 ± 2
37		23 ± 2	111 ± 3	14 ± 3	95 ± 3
38		541 ± 52	105 ± 1	59 ± 1	74 ± 6
39		>10000	>10000	207 ± 66	66 ± 5
40		36 ±14	115 ± 6	22 ± 1	61 ± 6
41		803 ± 73	117 ± 3	33 ± 1	93 ± 7
42		39 ± 1	142 ± 11	41 ± 14	102 ± 3

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E_max was measured as a percentage of the maximum CP55,940 signal in CHO cells expressing the hCB1 or hCB2 receptor and G_αq16 using a calcium mobilization assay.

Scheme 1. — Reagents and conditions: (a) 4-F-BnBr, K₂CO₃, MeCN, 60 °C; (b) 2 N NaOH, dioxane, 50 °C; (c) RNH₂, HBTU, NEt₃, MeCN, 50 °C.

Carboxylic acid product 5 (in Table 1) and the amide products 6 and 10–16 (in Table 1) were prepared as shown in Scheme 2. The intermediate carboxylic esters 62 were prepared from 60 via a standard amide coupling reaction with an amine as shown in Scheme 1 (reaction c). Hydrolysis of esters 62 (reaction a) provided the carboxylic acids 63, including the product 5. Coupling of the carboxylic acids 63 to an amine (reactions b or c) provided the amides 16 and 64 (6, 10–15).

The product 17 was prepared as shown in Scheme 3. The carboxylic acid 60 (from Scheme 1), was coupled to an amine (reaction a) to provide the amide 65. The alcohol of amide 65 was oxidized to the aldehyde (reaction b1) and then used in a reductive amination (reaction b2) to provide 17.

The product 19 was prepared as shown in Scheme 4. The amide 66 was prepared from 60 via a standard amide coupling reaction with an amine as shown in Scheme 1 (reaction c). The ester was hydrolyzed to the carboxylic acid (reaction a) which was then converted to the carboxamide 19 via an amide coupling reaction (reaction b).

The phenyl carboxamides 68 (22–24, 27 in Table 2; 43, 45–55, 58, 59 in Table 4) were prepared as shown in Scheme 5. The intermediate phenyl bromides 67 were prepared from commercially available 1H-indazole-3-carboxylic acid methyl ester, starting with N-alkylation of the indazole (reaction a), followed by ester hydrolysis (reaction b) and then a standard amide coupling (reaction c). For analogs 22–24, 27, 43, 45–52, 58 and 59 the aryl bromide was converted to an aryl nitrile using a palladium catalyzed reaction (reaction d1). The aryl nitrile was then hydrolyzed to an aryl carboxamide 68 using aqueous base and hydrogen peroxide (reaction d2). For analogs 53–55 the aryl bromides 67 were directly converted to the carboxamides via lithiation with n-BuLi followed by reaction with trimethylsilyl-isocyanate (TMS-NCO, reaction e).

Table 4. Left-Side Modifications with Optimized Right Side Amide Group.


#	R	hCB1 Ca EC₅₀ (nM)	hCB1 Ca E_max^a (%)	hCB2 Ca EC₅₀ (nM)	hCB2 Ca E_max^a (%)	Ki hCB1 hCB2 (nM) ^b	HLM Half-life (HL, min) Clearance (Cl, μL/min/mg)
24		25 ± 5	24 ± 1	27 ± 7	41 ± 2	4 10	HL: 123 Cl: 5
43		>10000		>10000
44		234 ± 154	15 ± 3	>10000		98 290	HL: >200 Cl: <5
45		151 ± 32	32 ± 2	35 ± 6	17 ± 4	18 27	HL: >200 Cl: <5
46		>10000		>10000
47		243 ± 37	77 ± 1	>10000
48		893 ± 192	67 ± 4	613 ± 351	40 ± 9	130 110	HL: 22 Cl: 58
49		383 ± 163	51 ± 1	189 ± 10	29 ± 11
50		15 ± 3	86 ± 5	51 ± 4	38 ± 5	2.5 3.0	HL: >200 Cl: <5
51		141 ± 14	92 ± 1	63 ± 10	57 ± 3	150 56	HL: 188 Cl: 3.3
52		202 ± 16	30 ± 6	175 ± 32	43 ± 5	250 250	HL: 32 Cl: 19
53		463 ± 187	47 ± 3	3416 ± 151	33 ± 7	54 75	HL: 160 Cl: 7.9
54		84 ± 6	33 ± 7	84 ± 7	35 ± 5	68 130	HL: 61 Cl: 10
55		156 ± 35	73 ± 4	341 ± 145	40 ± 1	210 300	HL: 102 Cl: 6
56		731 ± 161	24 ± 9	760 ± 175	10 ± 3	460 780	HL: 180 Cl: 7
57		742 ± 50	94 ± 2	>10000		250 130
58		>10000		>10000
59		>10000		2557 ± 492	11 ± 7

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E_max was measured as a percentage of the maximum CP55,940 signal in CHO cells expressing the hCB1 or hCB2 receptor and G_αq16 using a calcium mobilization assay.

Radioligand displacement was measured using [³H]CP55940 in CHO cell membrane preparations overexpressing hCB1 or hCB2 receptors.

Scheme 5. — Reagents and conditions: (a) RBr, K₂CO₃, MeCN, 60 °C; (b) 2 N NaOH, dioxane, 50 °C; (c) R’NH₂, HBTU, NEt₃, MeCN, 50 °C; (d) (1) Zn(CN)₂, Pd(PPh₃)₄, DMF, 70 °C; (2) 6 N NaOH, 50% H₂O₂, EtOH, rt; (e) n-BuLi, THF, -78 °C then TMS-NCO, rt.

The acetanilide 25 (in Table 2), the urea 26 (in Table 2) and the piperidines 71 (in Table 3) were prepared as shown in Scheme 6. The aniline 69, prepared from 60 via an amide coupling reaction as shown in Scheme 3 (reaction a), served as the intermediate to acetanilide 25 and urea 26. Treatment of aniline 69 with acetyl chloride (reaction a) or TMS-NCO (reaction b) provided acetanilide 25 or urea 26 respectively. The Boc protected piperidine intermediates 70, prepared from 60 via a standard amide coupling reaction as shown in Scheme 1 (reaction c), were used to synthesize the piperidine derivatives 71. Hydrolysis of the Boc group with aqueous acid (reaction c) was followed by reaction with TMS-NCO (reaction d) to provide ureas 71a (29–31, 36, 38, 39), or alkylation with chloroacetamide (reaction e) to provide carboxamides 71b (32–35, 40–42).

Phenyl carboxamides 44, 56 and 57 (in Table 4) were prepared using an alternate procedure (Scheme 7) to allow for indazoles with a nitrile on the left side. This procedure began with an amide coupling (reaction a) to provide the aryl bromide 72. Palladium catalyzed conversion of the bromide to a nitrile (reaction b), followed by hydrolysis of the nitrile using aqueous base and hydrogen peroxide (reaction c), provided the carboxamide 73. Finally, the indazole nitrogen was alkylated with an alkyl halide, using potassium carbonate as the base (reaction d) to provide the targets 44, 56 and 57.

2.2. Pharmacological Evaluation

All target compounds were evaluated in fluorescent calcium mobilization assays with CHO cells expressing the human (h)CB1 or hCB2 receptor and G_αq16 as has been described in our previous publications (Tables 1–3).^{27, 28} Both the potency (EC₅₀) and efficacy (E_max), which is indicative of partial agonism compared to the full-agonist CP55940 were determined. Select compounds were also evaluated in radioligand binding assays for hCB1 and hCB2 to determine their binding affinities at each receptor as described previously.^{27, 28}

We began this quest by evaluating the effects of small changes to the right side of the known indazole 4, a potent full agonist of both hCB1 and hCB2 (Table 1), looking for ways to reduce the full agonism to partial agonism without decreasing potency. We previously showed that the S isomer is about 100 times more potent than the R isomer and so our efforts focused on the S isomer.¹⁹ Replacement of the carboxamide with a carboxylic acid (5) results in a large loss of potency. Methylation of the carboxamide (6) has little effect on either potency or efficacy. Increasing the size of the isopropyl group to phenyl (7) or benzyl (8) results in somewhat lower potency and little effect on efficacy. Replacement of the valinamide of 4 with 1-aminocyclopentane-1-carboxamide (9) resulted in a small reduction of hCB1 efficacy (~10%), accompanied by a large loss of potency. Interestingly, the α,α-dimethyl substituted analog 10 was inactive at hCB1. The N-methyl carboxamide of 10 (compound 11) is significantly more potent, and various aryl amides were tried, resulting in compounds such as 12 that have improved potency, but high efficacy. Compound 13, an analog of 11 with an added polar acetamide, resulted in little to no change in potency or efficacy. However, cyclized versions of 13, the isomeric prolinamides 14 (S isomer) and 15 (R isomer) resulted in isomers with very different activities. While the R isomer is inactive, the S isomer is highly potent against both hCB1 and hCB2 (~20 nM at each receptor). Although the efficacy of 14 is too high, the excellent physical properties (cLogP = 2.2, TPSA = 110, HBD = 3, HBA = 8, MW = 451.5, rotatable bonds < 10) prompted us to further characterize it, to support investigation of additional prolinamide analogs. Compound 14 is very potent in the hCB1 and hCB2 binding assays (Ki = 4 & 6 nM, respectively) and is stable in the human liver microsome (HLM) stability assay, having a high half-life and low clearance (HL = 610 min; Cl = 2.0 μL/min/mg). These excellent results prompted us to explore additional compounds with the prolinamide group. Tying up the α,α-dimethyl group into a cyclopropyl group (16), provided partial agonism at hCB2, but not at hCB1 and potency was ~8-fold less at both receptors. Replacement of the carbonyl with a methylene (17) resulted in similar potency and efficacy. In summary, these early results showed that modifications of the right side could convert a full agonist to a partial agonist, but maintaining good potency was challenging. We felt this warranted a broader exploration of the right side with both aryl and nonaryl groups as described below.

It was envisioned that the accessibility of a wide range of substituted benzyl groups could provide a way to further probe the effects of right-side modifications on both potency and efficacy. Benzyl amides such as 18, were found to be full agonists of hCB1 and partial agonists of hCB2 (Table 2). We looked at adding a polar carboxamide to pick up an additional binding interaction and decrease the potential for CNS penetration. Of the ortho, meta and para carboxamides (19, 20 & 21), the meta carboxamide 20 was found to have the most desirable combination of activities. It was clearly the most potent agonist of hCB1 (EC₅₀ = 38 nM, E_max = 100%) and it is a partial agonist of hCB2 with good potency (EC₅₀ = 170 nM, E_max = 32%). We then proceeded to add alpha methyl groups with the hope of improving activity at hCB1. As observed with the starting valinamide 4, stereochemistry significantly affects potency and efficacy. Looking at the monomethylated isomers, the S isomer (22) has better potency on both hCB1 and hCB2, while the R isomer (23) has the better efficacy. We were excited to find that the α,α-dimethyl compound (24) has the better potency of the S isomer and the better efficacy of the R isomer, thus providing a potent partial agonist of both hCB1 and hCB2. Additionally, the physical properties of 24 (cLogP = 4.0, TPSA = 90, HBD = 3, HBA = 6, MW = 430.5, rotatable bonds < 10) favor peripheral restriction. Further characterization of 24 revealed it to be potent binders of both hCB1 and hCB2 (Ki = 4 & 10 nM, respectively) and to have good HLM stability (HL = 123 min; Cl = 5 μL/min/mg). Replacing the carboxamide of 24 with a urea (26) also produced a partial agonist, but hCB1 potency was reduced. Tying the α,α-dimethyl of 24 into a cyclopropyl (27) also produced a partial agonist, but again, hCB1 potency was negatively affected. In summary, we have successfully used right side modifications to convert a full agonist to a partial agonist of both hCB1 and hCB2. Simultaneously, these compounds have good HLM stability and physical properties that favor peripheral selectivity.

In addition to using aryl groups for right side modifications, we also explored nonaryl groups to find compounds with the desired profile. The initial goal was to employ functionalized piperidines to access the polar binding region of the receptor. The cyclohexylmethylene analog 28 is a potent full agonist of both hCB1 and hCB2, providing a good starting point to incorporate substituted piperidines. We started with 3-substitute piperidines, which based on the aryl analogs, were predicted to be most likely to access the polar binding site. While the unfunctionalized 3-substituted piperidines (R & S isomers) are inactive (data not shown), functionalization of the piperidine nitrogen with a carboxamide provides the isomeric compounds 29 and 30, both of which full agonists of hCB1. The S isomer (29) is a partial agonist, while the R isomer (30) is inactive at hCB2. Analogous to the aryl work above, the α,α-dimethyl group was installed to provide 31 (racemic), which once again resulted in improved potency and efficacy, but the efficacy at hCB1 continued to be high. Installation of a methylene spacer between the piperidine nitrogen and the carboxamide was investigated. The unsubstituted isomers 32 and 33 have similar activity. They are both full agonists of hCB1 and partial agonists of hCB2. Incorporation of the α,α-dimethyl group (34), again resulted in improved efficacy, but similar potency. The corresponding 4-substituted piperidines 35 and 36 were also tested, but the right balance of potency and efficacy was not found. We were interested in connecting the piperidine directly to the amide linker of the indazole. The simple cyclohexyl amide 37 (Table 3) is a potent full agonist of both hCB1 and hCB2, providing a good starting point. Using a 3-substituted piperidine, a carboxamide was connected to the piperidine nitrogen either directly (38 & 39) or via a methylene spacer (40 & 41). As observed previously, stereochemistry of alpha to the amide plays a role in the potency, efficacy, and selectivity. The most interesting compound was the S isomer with a methylene spacer, compound 40, a full agonist of hCB1 (EC₅₀ = 36 nM, E_max = 115%) and a partial agonist of hCB2 (EC₅₀ = 22 nM, E_max = 61%). With the hope of reducing the hCB1 efficacy, the alpha methylated analog 42 was prepared as a racemic mixture which could not be separated. While this compound is a potent agonist of both hCB1 and hCB2, the efficacy is high at both receptors. To probe the potential of these nonaryl analogs, the best compounds were further profiled. Compounds 31 and 34 were assessed for hCB1 and hCB2 binding, and HLM stability, which we used as deciding points on whether or not to make and test the individual isomers. In each case, the binding data indicated high affinity at each receptor (31: hCB1/hCB2 Ki = 49/27 nM; 34: hCB1/hCB2 Ki = 48/21 nM), but the compounds were not stable in the human liver microsomal (HLM) stability assay as indicated by short half-life (HL) (31: HL = 26 minutes; 34: HL <20 minutes). In summary, we were able to show how modification of the right side with nonaryl groups also leads to compounds that are partial agonists of both hCB1 and hCB2 but they lack metabolic stability.

Partial agonist 24 has good HLM stability but has a short plasma half-life in mice (see PK studies below in Table 6). Metabolite identification revealed that oxidation of the N-benzyl group was occurring in mice. We sought to develop SAR for the left side to help us circumvent this problem. Using the highly favored right-side aryl carboxamide group, which was optimized for good potency and the desired efficacy, we investigated a variety of left-side groups (>20), a subset of which is shown in Table 4. We found that the lipophilic pocket with which this group interacts is particularly sensitive to the size, shape, and polarity of the group, favoring nonpolar cyclic groups. Changes to this region affect both potency and efficacy. The size/shape sensitivity is illustrated by the loss of both hCB1 and hCB2 activity upon switching from a 4-fluoro benzyl (24) to a 4-chloro benzyl (43) or 4-fluoro phenethyl (46). The more polar 4-cyano benzyl analog (44), however, does still have functional hCB1 activity and binds to both hCB1 and hCB2, albeit with reduced affinities. This compound has improved HLM stability compared to 24, but it lacks functional hCB2 activity. Like the 4-fluorobenzyl analog 24, the 2,4-difluorobenzyl analog 45 is a partial agonist of both hCB1 and hCB2 but is about 10-fold less potent. It has good CB receptor binding and improved HLM stability. A variety of cycloalkyl analogs were prepared as exemplified by compounds 48–51. In general, cycloalkyl analogs are less potent than benzyl analogs and have lower HLM stability. A notable exception is the difluorocyclohexyl analog 50, which is among the most potent binders to both hCB1 and hCB2 (2.5 & 3.0 nM, respectively). This compound has excellent HLM stability and is a good partial agonist of hCB2, but it is closer to a full agonist of hCB1. The activity of the tetrahydropyran 51 was also of interest due to the high polarity of this group. Like compound 50, this compound has good HLM stability and is a partial agonist of hCB2 but is closer to a full agonist of hCB1 and has lower hCB1 potency and binding, a general issue with compounds bearing a polar left side group. A set of acyclic left side groups was also investigated. The early n-pentyl analog 52 is a partial agonist of both hCB1 and hCB2, but the receptor binding affinity and HLM stability are not ideal. A variety of compounds with fluoro, cyano or ether substitution were evaluated. Of the fluorinated compounds, the 5-fluoropentyl analog 54 proved the most interesting, with good hCB1 and hCB2 potency and the desired efficacy in the functional assay. The hCB binding and HLM stability of 54 were also improved compared to 52. The more polar cyano and ether analogs (56–59) were weakly active or inactive in the hCB1 functional assay. In summary, this exploration of left side groups has shown that potency, efficacy, and HLM stability can be affected by the nature of these groups. Of particular interest, the 2,4-difluorobenzyl analog 45 is a partial agonist of both hCB1 and hCB2 with improved HLM stability and only moderately less potent than 24. The difluorocyclohexyl group of 50 contributes to excellent hCB potency and HLM stability, but high hCB1 efficacy needs to be addressed by re-optimization of the right-side group and/or modifications to the indazole core.

Table 6. Mouse PK Data.

#	Structure	Dose^a mg/kg	Max. Conc.^b ng/mL	Brain/Plasma^c	Plasma Half-life (HL, h) Clearance (Cl, μL/min/mg)
24		IP 2.5	Plasma: 72 at 0.25 h Liver: 650 at 0.25 h Brain: 1.4 at 0.25 h	0.02	HL: 1.1 Cl: >10,000
45		PO 3	Plasma: 160 at 0.5 h Liver: 920 at 0.5 h Brain: 11 at 0.5 h	0.07	HL: 7.3 Cl: 5,200

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^a.

Formulated in 1%NMP/0.3% Tween80/CMC

^b.

Multiple time points: 0.25, 0.5, 1, 2, 4, & 8 hours for IP; 0.5, 1, 2, 4, 8, & 24 hours for po.

^c.

The maximum plasma and brain concentrations were used.

2.3. Cytochrome P450 (CYP) Induction and Off Target Activity

Compounds 24 and 45 were investigated for the potential to significantly induce CYP enzymes, which might pose a liability in chronic use and polypharmacy situations. Isoform-specific CYP induction assays using real-time RT-PCR in HepaRG cells were performed to assess potential for induction of CYP3A4, CYP1A2 and CYP2B6. These three isoforms are important contributors to human CYP-mediated drug metabolism. As shown in Table 5, compound 24 does not have an effect on these CYPs. Compound 45 has little effect on CYP2B6 and CYP1A2 but does show potential to induce CYP3A4. Compounds 50, 51 and 54 were also tested (data not shown) and found to have a profile similar to that of 45.

Table 5. CYP Induction Potential of 24 and 45.

#	CYP1A2 Induction^* (% of Omeprazole)	CYP2B6 Induction^* (% of Phenobarbital)	CYP3A4 Induction^* (% of Rifampicin)
24	<1	<1	<1
45	20	6	66

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Data are expressed as % of positive control compound Omeprazole, Phenobarbital or Rifampicin.

2.4. Pharmacokinetic Studies

While 24 has good HLM stability, in mouse PK it was found to have a short plasma half-life (Table 6). The short half-life was investigated by LC/MS/MS analysis after incubation of 24 with mouse liver microsomes, revealing oxidative cleavage of the 4-fluorobenzyl group. An exploration of alternative left side groups led us to 45. Although 45 is somewhat less potent than 24, it has better HLM stability and a longer plasma half-life in mice. Compound 45 is orally bioavailable and like 24, it has little CNS penetration. Administration of the difluorocyclohexyl analog 50, the tetrahydropyran analog 51 or the 5-fluoropentyl analog 54 to mice at 3 mg/kg po, resulted in low plasma levels (< 30 ng/mL; data not shown).

3. Summary and Conclusions

Peripherally restricted CB1 partial agonists could become important tools in treating various inflammatory diseases, GI disorders and pain. Compound 1 is a centrally acting partial agonist that was shown by Bayer to effectively treat neuropathic and inflammatory pain in animal models.²⁹ Potential advantages in safety were highlighted for this partial agonist versus full agonists. We believe that targeting only the peripheral cannabinoid receptors with a partial agonist will result in compounds with a safer profile. Partial agonists, however, are typically discovered serendipitously and hence SAR to control efficacy is lacking.

In this report, we highlight how a CB receptor full agonist was converted to a peripherally selective partial agonist. Specific SAR for both potency and efficacy is noted that applies to this class of cannabinoid agonists. The indazole right side amide and the attached group was found to be a key component for achieving partial agonism of both hCB1 and hCB2 and attaining physical properties favoring a peripherally selective compound. Docking studies with the hCB1 crystal structure (not shown) indicate that this right-side group is near the aromatic toggle residues (PHE200 and TRP356) that are important for efficacy and is directed towards a mixed polarity channel that leads to the extracellular space. Compound 24, with its α,α-dimethylbenzylamide and a meta carboxamide was a key discovery. In sharp contrast to the valinamide group of the CB receptor potent full agonist 4, the α,α-dimethylbenzyl group favors partial agonism of both hCB1 and hCB2. A combination of the α,α-dimethyl group with a piperidine, compound 34, also resulted in partial agonism of both hCB1 and hCB2. We found that while the α,α-dimethylbenzyl group favors partial agonism via direct interaction at the “toggle switch” area, other parts of the molecule also affect efficacy. For example, while the 3-substituted piperidine 43 is a partial agonist of hCB1, the regioisomeric 4-substituted piperidine 35 is a full agonist. We hypothesize that binding in other regions can affect how the molecule interacts at the “toggle switch” area, believed to be the source of controlling efficacy. Left side groups were also found to affect efficacy. Benzyl groups and unbranched chains favor lower efficacy while saturated rings favor higher efficacy, especially at hCB1.

Compound 24 has physical properties that favor peripheral selectivity and mouse PK show that this compound is peripherally selective. While 24 has good HLM stability, the plasma half-live in mice is short due to rapid oxidative debenzylation of the indazole. To circumvent this issue, alternative left side groups were explored. We found that adding an ortho fluoro to the benzyl group, compound 45, improved metabolic stability while maintaining hCB receptor efficacy and losing only a small amount of hCB receptor potency. Encouragingly, this compound is orally absorbed and has a good half-life in mice (~7 hr) and is peripherally selective (Brain:Plasma Cmax ratio ~0.7). We plan to further develop the SAR for controlling efficacy and to identify compounds with better overall properties and peripheral restriction, by investigating modifications of the indazole core and new right-side groups.

4. Experimental

Chemistry General.

Purity and characterization of compounds were established by a combination of LC/MS, NMR, HPLC and TLC analytical techniques, as described below. ¹H NMR spectra were recorded on a Bruker Avance DPX-300 (300 MHz) or a JEOP 400YH (400 MHz) spectrometer and were determined in chloroform-d (7.26 ppm) with tetramethylsilane (TMS, 0.00 ppm) as the internal reference, unless otherwise stated. ¹³C NMR spectra were recorded on a JEOP 400YH (100 MHz) spectrometer and were determined in chloroform-d (77.2 ppm) using the solvent as the internal reference. Chemical shifts are reported in ppm and coupling constant (J) values are reported in hertz (Hz). TLC was performed on precoated silica gel 60 F254 plates, and spots were visualized with UV light or I₂ detection. LC/MS was performed with an Agilent InfinityLab MSD single quadrupole mass spectrometer equipped with an API-ES and an Agilent Infinity II 1260 HPLC equipped with an Agilent Infinity 1260 variable wavelength detector and a Phenomenex Synergi 2.5 μm Hydro-RP 100A C18 30x2 mm column. LC/MS, HPLC Method: starting with a flow rate of 0.6 mL/min for 0.4 minutes at 20% solvent B followed by a 1.3 minute gradient of 20–95% solvent B at 0.6 mL/min followed by 2 minutes at 95% solvent B with a flow rate of 0.6 mL/min for 0.3 minutes and then a gradual ramp up of the flow rate to 1.2 mL/min at the end (solvent A, water with 0.1% formic acid; solvent B, acetonitrile with 0.1% formic acid and 5% water; absorbance monitored at 254 and 280 nm). LC/MS, MS Method: using atmospheric pressure ionization-electrospray, positive and negative ions were monitored in the range of 70–700. HPLC method: a Waters 2695 Separation Module equipped with a Waters 2996 Photodiode Array Detector and a Phenomenex Synergi 4 μm Hydro-RP 80A C18 250x4.6 mm column using a flow rate of 1 mL/min starting with 1 minute at 5% solvent B, followed by a 15 minute gradient of 5–95% solvent B, followed by 9 minutes at 95% solvent B (solvent A, water with 0.1% TFA; solvent B, acetonitrile with 0.1% TFA and 5% water; absorbance monitored at 220 and 280 nm). All compounds tested in biological assays have a purity of >95% as determined by HPLC. Optical rotations were measured in triplicate with a Rudolph Autopol IV using a 50 mm cell filled with a 10 mg/mL solution of the compound.

General Procedure A: N-Alkylation of Indazoles (Scheme Reactions 1a, 5a & 7d).

A mixture of an indazole (2 mmol), an alkyl bromide (2.2 mmol, 1.1 equiv; an alkyl chloride was used to make final products 53 & 57), K₂CO₃ (830 mg, 3 equiv) and MeCN (6 mL) was stirred at rt for 15 min and then at 60 °C for 15 h (80 °C was used to make final products 46, 49–55, 57 & 59; a sealed reaction vessel was used to make final product 53). Ethyl acetate (20 mL) was added, followed by water (4 mL) and brine (8 mL). After 10 min, the aqueous layer was removed. Celite (2.5 g) and toluene (2 mL) were added to the organic layer and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient provided the purified N-alkylated indazole.

General Procedure B: Carboxylic Acids from Hydrolysis of Carboxy Methyl Esters (Scheme Reactions 1b, 2a, 4a & 5b).

To a solution of a N-alkyl indazole 3-carboxy methyl ester (1 mmol) in dioxane (2 mL; scheme reaction 1b & 5b) or dioxane/EtOH (1/1, 2 mL; scheme reactions 2a & 4a) was added 2 N NaOH (1 mL, 2 equiv). The resulting mixture was stirred at rt (scheme reactions 2a & 4a) or heated at 50 °C (scheme reaction 1b) for 15 h. The mixture was acidified with 6 N HCl (0.37 mL, 2.1 equiv). Ethyl acetate (10 mL) was added, followed by brine (2 mL). The aqueous layer was saturated with NaCl, and the layers were separated. The aqueous layer was extracted with EtOAc (1x2 mL). The combined organic layers was dried (Na₂SO₄, 20 min) and filtered. Toluene (1 mL) was added, and the solvent evaporated to provide the N-alkyl indazole 3-carboxylic acids.

General Procedure C: Amides from Carboxylic Acids (Scheme Reactions 1c, 2c, 3a, 5c & 7a).

To a heterogeneous mixture of a carboxylic acid (0.2 mmol), HBTU (84 mg, 1.1 equiv) and MeCN (1 mL; scheme reactions 1c, 2c & 3a) or DMF (1 mL; scheme reaction 6a) was added NEt₃ (0.061 mL, 2.2 equiv). The mixture was stirred at rt for 30 min. An amine (0.22 mmol, 1.1 equiv) was added and after 30 min, the mixture was stirred at rt (scheme reactions 3a & 6a) or heated at 50 °C (scheme reactions 1c & 2c) for 15 h. Water (0.8 mL) was added and after 5 min, EtOAc (4 mL) was added, followed by brine (0.8 mL). After 10 min, the aqueous layer was removed, and the organic layer was washed with brine (0.8 mL; scheme reaction 6a only) and then 0.5 M NaHCO₃ solution (0.8 mL; all reactions). Celite (600 mg) was added to the organic layer and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient provided the purified amide.

General Procedure D: Aryl Nitriles from Aryl Bromides (Scheme Reactions 5d1 & 7b).

Using low light and nitrogen, Pd(PPh₃)₄ (70 mg, 0.2 equiv) was added to a solution of an aryl bromide (0.3 mmol) and Zn(CN)₂ (55 mg, 1.5 equiv) in DMF (1 mL). The mixture was stirred at rt for 10 min and then heated at 70–75 °C for 20 h. Ethyl acetate (4 mL) was added, followed by water (1 mL) and then saturated NaHCO₃ solution (2 mL). After 10 min, the aqueous layer was removed, and the organic layer washed with brine (2x1 mL). Celite (800 mg) was added to the organic layer and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient provided the purified nitrile .

General Procedure E: Aryl Carboxamides from Aryl Nitriles (Scheme Reactions 5d2 & 7c).

To a mixture of an aryl nitrile (0.2 mmol) in EtOH (0.8 mL) was added 50% H₂O₂ (0.2 mL) followed by 6 N NaOH (0.2 mL). The mixture was vigorously stirred at rt for 20 h. Ethyl acetate (3 mL) was added, followed by brine (0.6 mL). After 10 min, the aqueous layer was removed, celite (500 mg) was added to the organic layer and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 4% MeOH provided the purified aryl carboxamide.

General Procedure F: Aryl Carboxamides from Aryl Bromides (Scheme Reaction 5e).

To a solution of an aryl bromide (0.2 mmol) in THF (1 mL) at -78 °C (IPA/dry ice bath) was added drop wise 2.5 M n-BuLi in hexanes (0.16 mL, 2 equiv). After 5 min, TMS-NCO ( 0.041 mL, 1.5 equiv) was added all at once. After 1 h, 2 N HCl (0.24 mL) was added drop wise and then the bath was removed. The mixture was stirred at rt for 30 min. Ethyl acetate (4 mL) was added, followed by brine (0.6 mL). After 10 min, the aqueous layer was removed, celite (600 mg) was added to the organic layer and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 4% MeOH provided the purified aryl carboxamide.

General Procedure G: Deprotection of Boc Piperidines (Scheme Reaction 6c).

To a mixture of a Boc protected piperidine (0.2 mmol) and ethanol (1 mL) was added 6 N HCl (0.5 mL). The mixture was stirred at rt for 10 min and then at 55 °C for 15 h. Chloroform (3 mL) was added, followed by brine (0.5 mL) and then slow addition of 6 N NaOH (0.55 mL). After 10 min, the organic and aqueous layers were separated. The aqueous layer was saturated with NaCl and extracted with CHCl₃ (2x1 mL). The combined organic layers was dried (Na₂SO₄ for 20 min) and filtered. Toluene (1 mL) was added and the solvent evaporated, providing the deprotected piperidine.

General Procedure H: Piperidine Ureas from Reaction with TMS-NCO (Scheme Reaction 6d).

To a solution of a piperidine (0.15 mmol) in DCE (1 mL) was added TMS-NCO (0.025 mL, 1.2 equiv). The mixture was stirred at rt for 15 h. Water (0.1 mL) and EtOAc (1 mL) were added. After 15 min, celite (600 mg) was added and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 6% MeOH provided the purified piperidine urea.

General Procedure I: Alkylation of Piperidines with Chloroacetamide (Scheme Reaction 6e).

A mixture of a piperidine (0.2 mmol), chloroacetamide (23 mg, 1.2 equiv), K₂CO₃ (83 mg, 3 equiv) and MeCN (1 mL) was stirred at rt for 15 min and then heated at 50 °C for 15 h. EtOAc (3 mL) was added, followed by water (0.8 mL) and then brine (0.8 mL). After 10 min, the aqueous layer was removed, celite (600 mg) was added and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 8% MeOH provided the purified carboxamide.

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-3-methylbutanoic Acid (5).

The title compound was prepared by the general procedure B to provide 55 mg (100%) of a white crystalline solid, mp 148–149 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.27–7.41 (m, 3H), 7.18 (dd, J = 8.4, 5.4 Hz, 2H), 6.93–7.07 (m, 2H), 5.59 (s, 2H), 4.76 (dd, J = 8.6, 5.2 Hz, 1H), 2.32–2.48 (m, 1H), 0.94–1.22 (m, 6H). LC/MS (m/z) 370.4 (M+1), 368.2 (M-1), >95% at 2.57 min. HPLC >99% at 15.42 min.

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-N,3-dimethylbutanamide (6).

To a solution of 5 (45 mg, 0.12 mmol), HBTU (50 mg, 1.1 equiv) and THF (1 mL) was added NEt₃ (0.020 mL, 1.2 equiv). The mixture was stirred at rt for 30 min and then cooled in an ice bath. Methylamine (2 M/MeOH, 0.18 mL, 3 equiv) was added and after 30 min, the mixture was stirred at rt for 15 h. Water (0.4 mL) was added, followed by EtOAc (4 mL) and then saturated NaHCO₃ solution (0.8 mL). After 10 min, the aqueous layer was removed, celite (600 mg) was added and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 2% MeOH provided 30 mg (65%) of a white crystalline solid, mp 163–164 °C. R_f = 0.39 (2% MeOH/40% EtOAc/30% CH₂Cl₂/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.33 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.27–7.42 (m, 3H), 7.15–25 (m, 2H), 6.93–7.07 (m, 2H), 6.30–6.43 (m, 1H), 5.58 (s, 2H), 4.46 (dd, J = 8.8, 7.4 Hz, 1H), 2.85 (d, J = 4.0 Hz, 3H), 2.21–2.48 (m, 1H), 1.05 (d, J = 8.8 Hz, 6H). LC/MS (m/z) 383.4 (M+1), 381.2 (M-1), >95% at 2.53 min. HPLC 97% at 15.09 min.

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-2-phenylacetamide (7).

The title compound was prepared by the general procedure C to provide 25 mg (42%) of a white crystalline solid, mp 162–164 °C. R_f = 0.38 (2% MeOH/50% EtOAc/30% CH₂Cl₂/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.30 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 6.8 Hz, 1H), 7.56 (d, J = 6.8 Hz, 2H), 7.29–7.48 (m, 6H), 7.14–7.24 (m, 2H), 6.89–7.07 (m, 2H), 6.09 (br s, 1H), 5.79 (d, J = 6.8 Hz, 1H), 5.67 (br s, 1H), 5.58 (s, 2H). LC/MS (m/z) 403.4 (M+1), 401.2 (M-1), >95% at 2.54 min. HPLC >99% at 15.17 min.

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-3-phenylpropanamide (8).

The title compound was prepared by the general procedure C to provide 18 mg (29%) of a white amorphous solid, mp 146–147 °C. R_f = 0.32 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.24–7.42 (m, 8H), 7.18 (dd, J = 8.4, 5.4 Hz, 2H), 6.89–7.08 (m, 2H), 6.01 (br s, 1H), 5.55 (s, 2H), 5.41 (br s, 1H), 4.85–5.01 (m, 1H), 3.14–3.40 (m, 2H). LC/MS (m/z) 417.4 (M+1), 415.2 (M-1), >98% at 2.61 min. HPLC >98% at 15.20 min.

N-(1-Carbamoylcyclopentyl)-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (9).

The title compound was prepared by the general procedure C to provide 38 mg (67%) of a white crystalline solid, mp 135–136 °C. R_f = 0.18 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.36 (d, J = 8.1 Hz, 1H), 7.27–7.47 (m, 4H), 7.09–7.22 (m, 3H), 6.91–7.07 (m, 2H), 5.58 (s, 2H), 5.37 (br s, 1H), 2.36–2.60 (m, 2H), 2.09–2.30 (m, 2H), 1.77–1.92 (m, 4H). LC/MS (m/z) 381.4 (M+1), 379.2 (M-1), >98% at 2.52 min. HPLC >99% at 14.38 min.

2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-2-methylpropanamide (10).

The title compound was prepared by the general procedure C to provide 22 mg (69%) of a white crystalline solid, mp 191–193 °C. R_f = 0.23 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃, CD₃OD) δ 8.32 (d, J = 8.1 Hz, 1H), 7.27–7.44 (m, 3H), 7.20 (dd, J = 8.0, 5.4 Hz, 2H), 6.95–7.09 (m, 2H), 5.59 (s, 2H), 1.72 (m, 6H). LC/MS (m/z) 355.4 (M+1), 353.2 (M-1), >98% at 2.45 min. HPLC >98% at 13.64 min.

2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-N,2-dimethylpropanamide (11).

To a solution of 63 (X, Y = Me, 43 mg, 0.12 mmol), HBTU (50 mg, 1.1 equiv) and THF (1 mL) was added NEt₃ (0.020 mL, 1.2 equiv). The mixture was stirred at rt for 30 min and then cooled in an ice bath. Methylamine (2 M/MeOH, 0.18 mL, 3 equiv) was added and after 30 min, the mixture was stirred at rt for 15 h. Water (0.4 mL) was added, followed by EtOAc (4 mL) and then saturated NaHCO₃ solution (0.8 mL). After 10 min, the aqueous layer was removed, celite (600 mg) was added and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 2% MeOH provided 12 mg (27%) of a white amorphous solid, mp 178–179 °C. R_f = 0.28 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.36 (d, J = 8.1 Hz, 1H), 7.27–7.46 (m, 4H), 7.18 (dd, J = 8.4, 5.4 Hz, 2H), 6.96–7.07 (m, 2H), 6.92 (br s, 1H), 5.58 (s, 2H), 2.86 (d, J = 4.9 Hz, 3H), 1.72 (s, 6H). LC/MS (m/z) 369.4 (M+1), >98% at 2.49 min. HPLC >98% at 14.16 min.

3-[2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-2-methylpropanamido]benzamide (12).

The title compound was prepared by the general procedure C to provide 51 mg (90%) of a white crystalline solid, mp 170–172 °C. R_f = 0.37 (5% MeOH/75% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃, CD₃OD) δ 8.33 (d, J = 8.1 Hz, 1H), 8.03 (s, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.31–7.45 (m, 4H), 7.13–7.26 (m, 2H), 6.94–7.08 (m, 2H), 5.60 (s, 2H), 1.79 (s, 6H). LC/MS (m/z) 474.0 (M+1), 472.2 (M-1), >97% at 2.64 min. HPLC >99% at 14.21 min.

N-(Carbamoylmethyl)-2-({1-[(4-fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-N,2-dimethylpropanamide (13).

The title compound was prepared by the general procedure C to provide 17 mg (33%) of a white crystalline solid, mp 192–194 °C. R_f = 0.17 (5% MeOH/75% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.25 (d, J = 8.1 Hz, 1H), 7.74 (br s, 1H), 7.28–7.47 (m, 4H), 7.12–7.24 (m, 2H), 6.98–7.05 (m, 2H), 5.60 (s, 2H), 5.38 (br s, 1H), 4.15 (s, 2H), 3.21 (s, 3H), 1.70 (s, 6H). LC/MS (m/z) 448.0 (M+1+Na), 424.2 (M-1), >98% at 2.56 min. HPLC >99% at 13.27 min.

N-{1-[(2S)-2-Carbamoylpyrrolidin-1-yl]-2-methyl-1-oxopropan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (14).

The title compound was prepared by the general procedure C to provide 37 mg (68%) of a white crystalline solid, mp 183–184 °C. R_f = 0.12 (5% MeOH/75% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.29 (d, J = 8.1 Hz, 1H), 7.72 (br s, 1H), 7.28–7.46 (m, 4H), 7.13–7.26 (m, 2H), 6.96–7.09 (m, 2H), 5.60 (s, 2H), 5.35 (br s, 1H), 4.69 (dd, J = 8.0, 6.3 Hz, 1H), 3.72–3.92 (m, 1H), 3.31–3.51 (m, 1H), 2.12–2.31 (m, 1H), 1.92–2.05 (m, 1H), 1.80–1.91 (m, 2H), 1.75 (s, 3H), 1.62 (s, 3H). LC/MS (m/z) 474.0 (M+1+Na), 450.2 (M-1), >97% at 2.58 min. HPLC >99% at 13.72 min. [α]²³_D 41.4 (c 1.0, CDCl₃).

N-{1-[(2R)-2-Carbamoylpyrrolidin-1-yl]-2-methyl-1-oxopropan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (15).

The title compound was prepared by the general procedure C to provide 45 mg (83%) of a white crystalline solid, mp 188–190 °C. R_f = 0.12 (5% MeOH/75% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.29 (d, J = 8.1 Hz, 1H), 7.71 (br s, 1H), 7.28–7.47 (m, 4H), 7.13–7.26 (m, 2H), 6.96–7.10 (m, 2H), 5.60 (s, 2H), 5.31 (br s, 1H), 4.69 (dd, J = 8.2, 6.1 Hz, 1H), 3.75–3.92 (m, 1H), 3.29–3.50 (m, 1H), 2.14–2.34 (m, 1H), 1.93–2.01 (m, 1H), 1.81–1.91 (m, 2H), 1.75 (s, 3H), 1.62 (s, 3H). LC/MS (m/z) 474.0 (M+1+Na), 450.2 (M-1), >97% at 2.58 min. HPLC >99% at 13.71 min. [α]²³_D -39.8 (c 1.0, CDCl₃).

N-{1-[(2S)-2-Carbamoylpyrrolidine-1-carbonyl]cyclopropyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (16).

The title compound was prepared by the general procedure C to provide 45 mg (100%) of a white crystalline solid, mp 154–156 °C. R_f = 0.18 (7% MeOH/73% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, J = 8.1 Hz, 1H), 7.30–7.51 (m, 4H), 7.13–7.22 (m, 2H), 6.97–7.08 (m, 2H), 5.58 (s, 1H), 4.65 (t, J = 7.1 Hz, 1H), 3.87 (br s, 1H), 3.49–3.65 (m, 1H), 2.19–2.32 (m, 1H), 2.06–2.17 (m, 1H), 1.82–2.00 (m, 3H), 1.27–1.40 (m, 2H), 1.02 (m, 1H). LC/MS (m/z) 450.2 (M+1+22), 448.0 (M-1+46), >98% at 3.78 min. HPLC >99% at 13.34 min. [α]²³_D 75.1 (c 1.0, CDCl₃).

N-{1-[(2S)-2-Carbamoylpyrrolidin-1-yl]-2-methylpropan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (17).

To a solution of 65 (34 mg, 0.10 mmol) in CH₂Cl₂ (1 mL) was added Des Martin periodane (85 mg, 2 equiv). The mixture was stirred at rt for 2 h and then 0.5 M NaHCO₃ (0.6 mL) was added, followed by Na₂S₂O₃ (32 mg, 2 equiv). The mixture was stirred at rt for 30 min, and then the aqueous layer was removed and the organic layer dried with Na₂SO₄ (20 min). The mixture was filtered, L-prolinamide (46 mg, 4 equiv) and toluene (0.5 mL) were added, and the solvent evaporated. To a solution of the resulting residue in DCE (1 mL) was added Na(OAc)₃BH (128 mg, 6 equiv). After 30 min, AcOH (0.034 mL, 6 equiv) was added and stirring continued at rt for 15 h. EtOAc (4 mL) was added, followed by brine (0.3 mL), water (0.3 mL) and then 6 N NaOH (0.12 mL, 7 equiv). After 5 min, 0.8 M NaHCO₃ (0.3 mL) was added and stirring continued for 30 min. The aqueous layer was removed, celite (600 mg) was added, and the solvent was evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 4% MeOH provided 22 mg (50%) of a white amorphous hygroscopic solid. R_f = 0.11 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.38 (d, J = 7.9 Hz, 1H), 7.29–7.46 (m, 3H), 7.10–7.20 (m, 3H), 6.95–7.07 (m, 2H), 5.55 (s, 2H), 5.44 (br s, 1H), 3.21–3.39 (m, 2H), 2.88–3.11 (m, 2H), 2.47–2.59 (m, 1H), 2.05–2.16 (m, 1H), 1.70–1.82 (m, 2H), 1.52 (s, 3H), 1.55 (s, 3H). LC/MS (m/z) 438.2 (M+1), >98% at 2.62 min. HPLC 99% at 12.84 min. [α]²³_D -15.7 (c 1.0, CDCl₃).

N,1-Bis[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (18).

The title compound was prepared by the general procedure C to provide 35 mg (62%) of a white crystalline solid, mp 119–120 °C. R_f = 0.51 (30% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.42 (d, J = 8.1 Hz, 1 H), 7.28–7.48 (m, 6H), 7.10–7.23 (m, 2H), 6.92–7.10 (m, 4H), 5.55 (s, 2H), 4.66 (d, J = 6.0 Hz, 2H). LC/MS (m/z) 378.4 (M+1), >95% at 2.72 min. HPLC >99% at 17.26 min.

N-[(2-Carbamoylphenyl)methyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (19).

The methyl ester 66 (170 mg, 0.41 mmol) was hydrolyzed using general procedure B to provide 150 mg of a colorless residue (40% of desired intermediate carboxylic acid by LC/MS). To a solution of the residue in dioxane (1.2 mL) was added HBTU (160 mg, 1.1 equiv) followed by NEt₃ (0.061 mL, 1.2 equiv). After 30 min, ammonium carbonate (54 mg, 3 equiv) was added, followed by NEt₃ (0.12 mL, 2.4 equiv). The mixture was stirred at rt for 20 h. Water (0.4 mL) was added, followed by EtOAc (4 mL) and then brine (0.8 mL). After 5 min, the aqueous layer was removed and the organic layer washed with 0.8 M NaHCO₃ (0.6 mL). Celite (1 g) was added to the organic layer and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 2% MeOH provided 21 mg (13%) of a white amorphous solid, mp 169–170 °C. R_f = 0.31 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl_3, CD₃OD) δ 8.35 (d, J = 8.1 Hz, 1H), 8.14 (t, J = 6.0 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.40–7.50 (m, 1H), 7.22–7.38 (m, 4H), 7.11–7.21 (m, 2H), 6.92–7.05 (m, 2H), 6.85 (br s, 1H), 5.84 (br s, 1H), 5.55 (s, 2H), 4.77 (d, J = 6.4 Hz, 2H). LC/MS (m/z) 403.0 (M+1), 401.0 (M-1), >97% at 2.67 min. HPLC 98% at 14.72 min.

N-[(3-Carbamoylphenyl)methyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (20).

The title compound was prepared by the general procedure C to provide 54 mg (90%) of a white crystalline solid, mp 138–140 °C. R_f = 0.28 (4% MeOH/60% EtOAc/hexanes; blue with UV). ¹H NMR (300 MHz, CDCl_3, CD₃OD) δ 8.37 (d, J = 8.1 Hz, 1H), 7.86 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.52–7.61 (m, 1H), 7.32–7.50 (m, 4H), 7.18 (dd, J = 8.4, 5.4 Hz, 2H), 6.91–7.09 (m, 2H), 5.57 (s, 2H), 4.63–4.79 (m, 2H). LC/MS (m/z) 403.4 (M+1), >97% at 2.48 min. HPLC >99% at 14.17 min.

N-[(4-Carbamoylphenyl)methyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (21).

The title compound was prepared by the general procedure C to provide 37 mg (61%) of a white crystalline solid, mp 139–141 °C. R_f = 0.23 (4% MeOH/60% EtOAc/hexanes; blue with UV). ¹H NMR (300 MHz, CDCl_3, CD₃OD) δ 8.34 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.29–7.54 (m, 5H), 7.14–7.25 (m, 2H), 6.93–7.08 (m, 2H), 5.59 (s, 2H), 4.72 (s, 2H). LC/MS (m/z) 403.4 (M+1), >97% at 2.46 min. HPLC >99% at 14.00 min.

N-[(1S)-1-(3-Carbamoylphenyl)ethyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (22).

The title compound was prepared by the general procedure E to provide 13 mg (82%) of a white amorphous solid, mp 102–103 °C. R_f = 0.22 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J = 8.1 Hz, 1H), 7.93 (s, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.28–7.49 (m, 5H), 7.10–7.20 (m, 2H), 6.92–7.07 (m, 2H), 5.56 (s, 2H), 5.27–5.48 (m, 1H), 1.65 (d, J = 7.0 Hz, 3H). LC/MS (m/z) 417.4 (M+1), >97% at 2.50 min. HPLC 99% at 14.60 min.

N-[(1R)-1-(3-Carbamoylphenyl)ethyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (23).

The title compound was prepared by the general procedure E to provide 25 mg (95%) of a white amorphous solid, mp 100–101 °C. R_f = 0.22 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J = 8.1 Hz, 1H), 7.93 (s, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.28–7.48 (m, 5H), 7.11–7.21 (m, 2H), 6.94–7.07 (m, 2H), 5.57 (s, 2H), 5.27–5.48 (m, 1H), 1.65 (d, J = 7.0 Hz, 3H). LC/MS (m/z) 417.4 (M+1), >97% at 2.50 min. HPLC >98% at 14.60 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (24)

The title compound was prepared by the general procedure E to provide 87 mg (84%) of a white crystalline solid, mp 158–159 °C. R_f = 0.27 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.28 (d, J = 8.1 Hz, 1H), 8.00 (s, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.59 (d, J = 7.4 Hz, 1H), 7.46 (s, 1H), 7.28–7.41 (m, 3H), 7.13–7.25 (m, 3H), 6.95–7.08 (m, 2H), 5.58 (s, 2H), 1.85 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 169.8, 162.6 (d, J = 246 Hz), 161.7, 147.9, 141.0, 138.2, 133.7, 131.8, 129.0, 128.8, 128.7, 127.2, 125.4, 124.4, 123.2, 122.9, 115.9 (d, J = 21 Hz), 109.4, 55.6, 52.9, 29.8. LC/MS (m/z) 431.0 (M+1), 475.2 (M-1+HCO₂H) >98% at 2.70 min. HPLC >99% at 15.07 min.

N-[2-(3-Acetamidophenyl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (25).

To a solution of the aniline 69 (21 mg, 0.052 mmol) in DCE (0.5 mL) was added acetyl chloride (0.005 mL, 1.3 equiv) followed by pyridine (0.007 mL, 1.5 equiv). The mixture was stirred at rt for 15 h. EtOAc (3 mL) was added, followed by water (0.2 mL) and then brine (0.4 mL). After 10 min, the aqueous layer was removed, celite (500 mg) was added to the organic layer, and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 2% MeOH provided 21 mg (91%) of a white crystalline solid, mp 157–158 °C. R_f = 0.38 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.31 (d, J = 7.7 Hz, 1H), 7.57 (br s, 1H), 7.28–7.50 (m, 6H), 7.11–7.24 (m, 4H), 6.95–7.09 (m, 2H), 5.59 (s, 2H), 2.05 (s, 3H), 1.81 (s, 6H). LC/MS (m/z) 445.0 (M+1), 489.2 (M-1+HCO₂H), >98% at 2.75 min. HPLC >99% at 15.58 min.

N-{2-[3-(Carbamoylamino)phenyl]propan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (26).

To a solution of the aniline 69 (80 mg, 0.20 mmol) in DCE (1 mL) was added TMS-NCO (0.042 mL, 1.5 equiv). The mixture was stirred at rt for 1 h and then at 60 °C for 20 h. Water (0.1 mL), MeOH (0.1 mL) and EtOAc (1 mL) were added. After 15 min, celite (600 mg) was added and the solvent evaporated. Flash chromatography using silica gel with an EtOAc/hexanes gradient containing up to 2% MeOH provided 56 mg (63%) of a white crystalline solid, mp 143–144 °C. R_f = 0.38 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃, CD₃OD) δ 8.24 (d, J = 8.1 Hz, 1H), 7.54 (s, 1H), 7.31–7.43 (m, 3H), 7.14–7.27 (m, 5H), 6.97–7.10 (m, 2H), 5.60 (s, 2H), 1.80 (s, 6H). LC/MS (m/z) 446.0 (M+1), 490.2 (M-1+HCO₂H), >98% at 2.70 min. HPLC >99% at 14.99 min.

N-[1-(3-Carbamoylphenyl)cyclopropyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (27).

The title compound was prepared by the general procedure E to provide 56 mg (69%) of a white crystalline solid, mp 122–124 °C. R_f = 0.32 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.36 (d, J = 8.1 Hz, 1H), 7.83 (s, 1H), 7.73 (s, 1H), 7.53–7.64 (m, 2H), 7.27–7.41 (m, 4H), 7.13–7.21 (m, 2H), 6.95–7.06 (m, 2H), 5.56 (s, 2H), 1.36–1.54 (m, 4H). LC/MS (m/z) 429.2 (M+1), 473.2 (M-1+46), >98% at 2.91 min. HPLC 99% at 14.76 min.

N-(Cyclohexylmethyl)-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (28).

The title compound was prepared by the general procedure C to provide 44 mg (80%) of a white crystalline solid, mp 106–107 °C. R_f = 0.55 (30% EtOAc/hexanes; blue with UV). ¹H NMR (300 MHz, CDCl₃) δ 8.41 (d, J = 8.1 Hz, 1H), 7.27–7.44 (m, 3H), 7.16 (dd, J = 8.5, 5.3 Hz, 2H), 7.09 (br s, 1H), 6.93–7.05 (m, 2H), 5.57 (s, 2H), 3.34 (t, J = 6.6 Hz, 2H), 1.60–1.93 (m, 6H), 1.12–1.37 (m, 3H), 0.91–1.11 (m, 2H). LC/MS (m/z) 366.4 (M+1), >97% at 2.86 min. HPLC >99% at 18.92 min.

N-{[(3S)-1-Carbamoylpiperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (29).

The title compound was prepared by the general procedure H to provide 43 mg (70%) of a white amorphous solid, mp 85–87 °C. R_f = 0.14 (5%MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 88.37 (d, J = 8.1 Hz, 1H), 7.26–7.44 (m, 3H), 7.18 (dd, J = 8.3, 5.5 Hz, 2H), 6.93–7.06 (m, 2H), 5.57 (s, 2H), 4.58 (br s, 2H), 3.70–3.84 (m, 1H), 3.54–3.67 (m, 1H), 3.40–3.50 (m, 2H), 2.90–3.18 (m, 2H), 1.84–2.00 (m, 2H), 1.47–1.76 (m, 2H), 1.33–1.45 (m, 1H). LC/MS (m/z) 410.4 (M+1), >98% at 2.41 min. HPLC >99% at 13.90 min. [α]²³_D 2.6 (c 1.0, CDCl₃).

N-{[(3R)-1-Carbamoylpiperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (30).

The title compound was prepared by the general procedure H to provide 54 mg (88%) of a white amorphous solid, mp 75–77 °C. R_f = 0.14 (5%MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.37 (d, J = 8.1 Hz, 1H), 7.29–7.44 (m, 3H), 7.18 (dd, J = 8.3, 5.5 Hz, 2H), 6.93–7.08 (m, 2H), 5.57 (s, 2H), 4.57 (br s, 2H), 3.70–3.84 (m, 1H), 3.56–3.68 (m, 1H), 3.38–3.49 (m, 2H), 2.89–3.19 (m, 2H), 1.85–1.99 (m, 2H), 1.47–1.77 (m, 2H), 1.33–1.45 (m, 1H). LC/MS (m/z) 410.4 (M+1), >98% at 2.41 min. HPLC >99% at 13.89 min. [α]²³_D -2.3 (c 1.0, CDCl₃).

N-[2-(1-Carbamoylpiperidin-3-yl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (31).

The title compound was prepared by the general procedure H to provide 51 mg (78%) of a white crystalline solid, mp 96–98 °C. R_f = 0.19 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.33 (d, J = 8.1 Hz, 1H), 7.28–7.43 (m, 3H), 7.11–7.22 (m, 2H), 6.96–7.08 (m, 2H), 6.89 (s, 1H), 5.57 (s, 2H), 4.61 (br s, 2H), 4.05–4.19 (m, 1H), 3.89–4.03 (m, 1H), 2.47–2.79 (m, 2H), 1.91–2.03 (m, 1H), 1.55–1.81 (m, 3H), 1.51 (s, 3H), 1.42 (s, 3H), 1.24–1.38 (m, 1H). LC/MS (m/z) 438.4 (M+1), >97% at 2.68 min. HPLC >99% at 15.37 min.

N-{[(3S)-1-(Carbamoylmethyl)piperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (32).

The title compound was prepared by the general procedure I to provide 39 mg (61%) of a white crystalline solid, mp 158–160 °C. R_f = 0.11 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.38 (d, J = 8.1 Hz, 1H), 7.27–7.46 (m, 3H), 7.08–7.22 (m, 4H), 6.91–7.07 (m, 2H), 5.57 (s, 2H), 5.46 (br s, 1H), 3.44–3.65 (m, 1H), 3.27–3.44 (m, 1H), 2.89–3.11 (m, 2H), 2.93–2.88 (m, 2H), 2.09–2.37 (m, 2H), 1.95–2.07 (m, 1H), 1.69–1.84 (m, 2H), 1.50–1.66 (m, 1H), 1.10–1.26 (m, 1H). LC/MS (m/z) 421.4 (M+1), >98% at 2.14 min. HPLC >99% at 12.03 min. [α]²³_D 12.5 (c 1.0, CDCl₃).

N-{[(3R)-1-(Carbamoylmethyl)piperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (33).

The title compound was prepared by the general procedure I to provide 37 mg (58%) of a white crystalline solid, mp 161–163 °C. R_f = 0.11 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.38 (d, J = 8.1 Hz, 1H), 7.27–7.45 (m, 3H), 7.07–7.21 (m, 4H), 6.93–7.07 (m, 2H), 5.57 (s, 2H), 5.39 (br s, 1H), 3.45–3.66 (m, 1H), 3.29–3.44 (m, 1H), 2.90–3.09 (m, 2H), 2.64–2.89 (m, 2H), 2.10–2.39 (m, 2H), 1.95–2.08 (m, 1H), 1.52–1.85 (m, 3H), 1.06–1.31 (m, 1H). LC/MS (m/z) 424.4 (M+1), >98% at 2.14 min. HPLC >99% at 12.02 min. [α]²³_D -10.8 (c 1.0, CDCl₃).

N-{2-[1-(Carbamoylmethyl)piperidin-3-yl]propan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (34).

The title compound was prepared by the general procedure I to provide 54 mg (80%) of a white amorphous solid, mp 171–172 °C. R_f = 0.19 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.36 (d, J = 8.1 Hz, 1H), 7.28–7.43 (m, 2H), 7.09–7.23 (m, 3H), 6.88–7.07 (m, 3H), 5.56 (s, 2H), 5.32 (br s, 1H), 2.93–3.07 (m, 2H), 2.80–2.91 (m, 1H), 2.58–2.72 (m, 1H), 1.96–2.19 (m, 2H), 1.58–1.96 (m, 4H), 1.44 (s, 3H), 1.48 (s, 3H), 1.04–1.21 (m, 1H). LC/MS (m/z) 452.6 (M+1), >97% at 2.44 min. HPLC >99% at 12.94 min.

N-{2-[1-(Carbamoylmethyl)piperidin-4-yl]propan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (35).

The title compound was prepared by the general procedure I to provide 56 mg (83%) of a white crystalline solid, mp 101–103 °C. R_f = 0.15 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.38 (d, J = 8.1 Hz, 1H), 7.28–7.43 (m, 3H), 7.09–7.20 (m, 3H), 6.94–7.06 (m, 2H), 6.85 (s, 1H), 5.56 (s, 2H), 5.52 (br s, 1H), 2.87–3.07 (m, 4H), 2.14–2.36 (m, 2H), 1.72–1.84 (m, 3H), 1.37–1.54 (m, 8H). LC/MS (m/z) 452.6 (M+1), >97% at 2.42 min. HPLC >99% at 12.77 min.

N-[2-(1-Carbamoylpiperidin-4-yl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (36).

The title compound was prepared by the general procedure H to provide 60 mg (92%) of a white amorphous solid, mp 69–71 °C. R_f = 0.15 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.37 (d, J = 7.9 Hz, 1H), 7.28–7.42 (m, 3H), 7.10–7.21 (m, 2H), 6.94–7.06 (m, 2H), 6.86 (s, 1H), 5.57 (s, 2H), 4.52 (br s, 2H), 3.95–4.07 (m, 2H), 2.75–2.91 (m, 2H), 2.45–2.61 (m, 1H), 1.73–1.84 (m, 3H), 1.45 (s, 6H), 1.26–1.41 (m, 1H). LC/MS (m/z) 438.4 (M+1), >97% at 2.66 min. HPLC >99% at 15.00 min.

N-Cyclohexyl-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (37).

The title compound was prepared by the general procedure C to provide 44 mg (84%) of a white amorphous solid, mp 137–138 °C. R_f = 0.52 (30% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.41 (d, J = 8.1 Hz, 1H), 7.23–7.42 (m, 3H), 7.16 (dd, J = 8.4, 5.4 Hz, 2H), 6.95–7.06 (m, 2H), 6.91 (d, J = 7.9 Hz, 1H), 5.56 (s, 2H), 3.92–4.13 (m, 1H), 2.00–2.14 (m, 2H), 1.72–1.90 (m, 2H), 1.60–1.72 (m, 1H), 1.15–1.54 (m, 5H). LC/MS (m/z) 352.4 (M+1), >97% at 2.79 min. HPLC >99% at 18.19 min.

N-[(3S)-1-Carbamoylpiperidin-3-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (38).

The title compound was prepared by the general procedure H to provide 52 mg (88%) of a white crystalline solid, mp 199–201 °C. R_f = 0.14 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J = 7.9 Hz, 1H), 7.28–7.47 (m, 3H), 7.13–7.23 (m, 2H), 6.94–7.11 (m, 3H), 5.58 (s, 2H), 4.63 (s, 2H), 4.03–4.23 (m, 1H), 3.72–3.84 (m, 1H), 3.59–3.72 (m, 1H), 3.21–3.40 (m, 2H), 2.03–2.15 (m, 1H), 1.67–1.90 (m, 3H). LC/MS (m/z) 396.4 (M+1), 394.2 (M-1), >97% at 2.40 min. HPLC >97% at 13.82 min. [α]²³_D 4.3 (c 1.0, CDCl₃).

N-[(3R)-1-Carbamoylpiperidin-3-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (39).

The title compound was prepared by the general procedure H to provide 41 mg (69%) of a white crystalline solid, mp 198–199 °C. R_f = 0.14 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.35 (d, J = 8.1 Hz, 1H), 7.27–7.46 (m, 3H), 7.12–7.22 (m, 2H), 6.93–7.12 (m, 3H), 5.57 (s, 2H), 4.74 (s, 2H), 4.03–4.23 (m, 1H), 3.71–3.86 (m, 1H), 3.57–3.71 (m, 1H), 3.20–3.38 (m, 2H), 1.99–2.14 (m, 1H), 1.59–1.87 (m, 3H). LC/MS (m/z) 396.4 (M+1), 394.2 (M-1), >97% at 2.40 min. HPLC >97% at 13.83 min. [α]²³_D -4.2 (c 1.0, CDCl₃).

N-[(3S)-1-(Carbamoylmethyl)piperidin-3-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (40).

The title compound was prepared by the general procedure I to provide 58 mg (95%) of a white crystalline solid, mp 167–168 °C. R_f = 0.17 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.38 (d, J = 7.9 Hz, 1H), 7.28–7.47 (m, 3H), 7.08–7.22 (m, 3H), 6.93–7.07 (m, 3H), 5.57 (s, 3H), 4.20–4.40 (m, 1H), 2.93–3.12 (m, 3H), 2.59–2.74 (m, 1H), 2.31–2.46 (m, 2H), 1.86–2.01 (m, 1H), 1.65–1.86 (m, 2H), 1.42–1.61 (m, 1H). LC/MS (m/z) 410.4 (M+1), >97% at 2.13 min. HPLC 98% at 11.99 min. [α]²³_D 7.8 (c 1.0, CDCl₃).

N-[(3R)-1-(Carbamoylmethyl)piperidin-3-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (41).

The title compound was prepared by the general procedure I to provide 52 mg (85%) of a white crystalline solid, mp 164–165 °C. R_f = 0.17 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.38 (d, J = 8.1 Hz, 1H), 7.28–7.48 (m, 3H), 7.08–7.21 (m, 3H), 6.92–7.07 (m, 3H), 5.63 (br. s., 1H), 5.57 (s, 2H), 4.21–4.41 (m, 1H), 2.90–3.13 (m, 3H), 2.59–2.75 (m, 1H), 2.31–2.46 (m, 2H), 1.63–2.01 (m, 3H), 1.44–1.61 (m, 1H). LC/MS (m/z) 410.4 (M+1), >97% at 2.13 min. HPLC 98% at 12.01 min. [α]²³_D -13.4 (c 1.0, CDCl₃).

N-[1-(Carbamoylmethyl)-3-methylpiperidin-3-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (42).

The title compound was prepared by the general procedure I to provide 65 mg (100%) of a white amorphous hygroscopic solid. R_f = 0.33 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃, CD₃OD) δ 8.38 (d, J = 7.9 Hz, 1H), 7.29–7.48 (m, 4H), 7.08–7.19 (m, 2H), 6.95–7.08 (m, 3H), 5.63 (br s, 1H), 5.55 (s, 2H), 3.05 (br s, 2H), 2.89–3.01 (m, 1H), 2.80 (br s, 1H), 2.40–2.54 (m, 1H), 2.15–2.38 (m, 2H), 1.79 (br s, 2H), 1.64 (br s, 1H), 1.59 (s, 3H), 1.21–1.42 (m, 1H). LC/MS (m/z) 424.6 (M+1), >97% at 2.40 min. HPLC >98% at 12.53 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[(4-chlorophenyl)methyl]-1H-indazole-3-carboxamide (43).

The title compound was prepared by the general procedure E to provide 19 mg (73%) of a white crystalline solid, mp 213–214 °C. R_f = 0.37 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃, CD₃OD) δ 8.25 (d, J = 8.1 Hz, 1H), 7.94 (s, 1H), 7.66 (d, J = 7.7 Hz, 2H), 7.19–7.46 (m, 7H), 7.14 (d, J = 8.3 Hz, 2H), 5.60 (s, 2H), 1.86 (s, 6H). LC/MS (m/z) 447.0 (M+1), 491.0 (M-1+46), >98% at 2.80 min. HPLC >99% at 15.96 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[(4-cyanophenyl)methyl]-1H-indazole-3-carboxamide (44).

The title compound was prepared by the general procedure A to provide 34 mg (84%) of a white amorphous solid, mp 197–199 °C. R_f = 0.27 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, J = 8.1 Hz, 1H), 8.01 (s, 1H), 7.54–7.73 (m, 4H), 7.33–7.46 (m, 3H), 7.20–7.32 (m, 4H), 5.68 (s, 2H), 1.86 (s, 6H). LC/MS (m/z) 438.0 (M+1), 482.2 (M-1+46), >97% at 2.72 min. HPLC 99% at 14.28 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[(2,4-difluorophenyl)methyl]-1H-indazole-3-carboxamide (45).

The title compound was prepared by the general procedure E to provide 104 mg (77%) of a white crystalline solid, mp 156–158 °C. R_f = 0.26 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.31 (d, J = 8.3 Hz, 1H), 8.02 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.37–7.48 (m, 4H), 7.21–7.27 (m, 1H), 7.02–7.11 (m, 1H), 6.76–6.94 (m, 2H), 6.20 (br s, 1H), 5.72 (br s, 1H), 5.64 (s, 3H), 1.88 (s, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 169.8, 162.8 (d, J = 250 Hz), 161.6, 160.4 (d, J = 250 Hz), 147.9, 141.0, 138.5, 133.7, 130.5, 128.8, 128.7, 127.4, 125.4, 124.4, 123.2, 123.1, 123.0, 119.2 (d, J = 20 Hz), 112.0 (d, J = 22 Hz), 109.2, 104.2 (t, J = 25 Hz), 55.6, 46.3, 29.8. LC/MS (m/z) 449.0 (M+1), 493.0 (M-1+HCO₂H), >98% at 2.73 min. HPLC >99% at 15.27 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[2-(4-fluorophenyl)ethyl]-1H-indazole-3-carboxamide (46).

The title compound was prepared by the general procedure E to provide 59 mg (89%) of a white amorphous solid, mp 142–143 °C. R_f = 0.35 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.25 (d, J = 7.9 Hz, 1H), 7.99 (s, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.29–7.46 (m, 3H), 7.13–7.22 (m, 2H), 7.00–7.11 (m, 2H), 6.88–6.99 (m, 2H), 4.59 (t, J = 7.2 Hz, 2H), 3.22 (t, J = 7.2 Hz, 2H), 1.86 (s, 6H). LC/MS (m/z) 445.2 (M+1), 489.2 (M-1+46), >98% at 2.92 min. HPLC >99% at 15.55 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[1-(4-fluorophenyl)ethyl]-1H-indazole-3-carboxamide (47).

The title compound was prepared by the general procedure E to provide 32 mg (83%) of a white crystalline solid, mp 197–198 °C. R_f = 0.40 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.27 (d, J = 8.1 Hz, 1H), 8.02 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.48 (s, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.17–7.31 (m, 5H), 7.01 (t, J = 8.6 Hz, 2H), 6.46 (br s, 1H), 5.85 (q, J = 6.8 Hz, 2H), 2.50 (br s, 1H), 2.06 (d, J = 7.0 Hz, 3H), 1.80–1.95 (m, 6H). LC/MS (m/z) 445.0 (M+1), 489.2 (M-1+HCO₂H), >98% at 2.75 min. HPLC >99% at 15.70 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(cyclopentylmethyl)-1H-indazole-3-carboxamide (48).

The title compound was prepared by the general procedure E to provide 77 mg (73%) of a white crystalline solid, mp 193–194 °C. R_f = 0.40 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.27 (d, J = 7.9 Hz, 1H), 7.98 (s, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 7.3 Hz, 1H), 7.32–7.47 (m, 4H), 7.16–7.24 (m, 1H), 4.32 (d, J = 7.3 Hz, 2H), 2.49–2.71 (m, 1H), 1.86 (s, 6H), 1.54–1.79 (m, 6H), 1.36 (br s, 2H). LC/MS (m/z) 445.0 (M+1), >98% at 3.03 min. HPLC 99% at 16.40 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(2-cyclopentylethyl)-1H-indazole-3-carboxamide (49).

The title compound was prepared by the general procedure E to provide 93 mg (86%) of a white amorphous solid, mp 152–153 °C. R_f = 0.45 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.27 (d, J = 8.1 Hz, 1H), 7.98 (br s, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.59 (d, J = 7.3 Hz, 1H), 7.32–7.47 (m, 4H), 7.20 (t, J = 6.3 Hz, 1H), 4.40 (t, J = 7.3 Hz, 2H), 1.93–2.08 (m, 3H), 1.86 (br s, 8H), 1.47–1.70 (m, 4H), 1.14–1.29 (m, 2H). LC/MS (m/z) 419.0 (M+1), >98% at 3.11 min. HPLC >99% at 17.57 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[(4,4-difluorocyclohexyl)methyl]-1H-indazole-3-carboxamide (50).

The title compound was prepared by the general procedure F to provide 47 mg (43%) of a white crystalline solid, mp 114–116 °C. R_f = 0.19 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.26 (d, J = 8.1 Hz, 1H), 8.00 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.32–7.47 (m, 4H), 7.16–7.25 (m, 1H), 4.28 (d, J = 7.4 Hz, 2H), 2.06–2.26 (m, 3H), 1.86 (s, 6H), 1.57–1.78 (m, 4H), 1.21–1.55 (m, 4H). LC/MS (m/z) 455.0 (M+1), 499.2 (M-1+HCO₂H), >98% at 2.73 min. HPLC >99% at 15.34 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(oxan-4-ylmethyl)-1H-indazole-3-carboxamide (51).

The title compound was prepared by the general procedure E to provide 31 mg (62%) of a white crystalline solid, mp 149–150 °C. R_f = 0.14 (2% MeOH/70% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.27 (d, J = 8.1 Hz, 1H), 8.00 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.33–7.49 (m, 4H), 7.15–7.24 (m, 1H), 4.28 (d, J = 7.2 Hz, 2H), 3.91–4.05 (m, 2H), 3.25–3.47 (m, 2H), 2.25–2.41 (m, 1H), 1.87 (s, 6H), 1.42–1.59 (m, 4H). LC/MS (m/z) 421.0 (M+1), 465.2 (M-1+HCO₂H), >98% at 2.56 min. HPLC >99% at 13.38 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-pentyl-1H-indazole-3-carboxamide (52).

The title compound was prepared by the general procedure F to provide 34 mg (48%) of a white amorphous solid, mp 110–111 °C. R_f = 0.28 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.26 (d, J = 8.3 Hz, 1H), 7.98 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.33–7.48 (m, 4H), 7.16–7.24 (m, 1H), 4.39 (t, J = 7.2 Hz, 2H), 1.92–2.04 (m, 2H), 1.86 (s, 6H), 1.28–1.45 (m, 4H), 0.92 (t, J = 6.7 Hz, 3H). LC/MS (m/z) 393.0 (M+1), >98% at 2.78 min. HPLC 99% at 16.21 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(4,4-difluoropentyl)-1H-indazole-3-carboxamide (53).

The title compound was prepared by the general procedure E to provide 86 mg (77%) of a white amorphous solid, mp 132–133 °C. R_f = 0.36 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.28 (d, J = 8.3 Hz, 1H), 7.99 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.33–7.46 (m, 4H), 7.18–7.25 (m, 1H), 4.46 (t, J = 7.0 Hz, 2H), 2.21 (quin, J = 7.3 Hz, 2H), 1.82–1.94 (m, 8H), 1.61 (t, J = 16.6 Hz, 3H). LC/MS (m/z) 429.0 (M+1), 473.0 (M-1+46), >98% at 2.92 min. HPLC 96% at 14.76 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(5-fluoropentyl)-1H-indazole-3-carboxamide (54).

The title compound was prepared by the general procedure F to provide 34 mg (30%) of a white amorphous solid, mp 100–102 °C. R_f = 0.18 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.27 (d, J = 8.1 Hz, 1H), 7.98 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.33–7.47 (m, 4H), 7.15–7.24 (m, 1H), 4.53 (t, J = 5.8 Hz, 1H), 4.34–4.47 (m, 3H), 1.96–2.12 (m, 2H), 1.87 (s, 6H), 1.25–1.80 (m, 6H). LC/MS (m/z) 411.0 (M+1), 455.2 (M-1+HCO₂H), >97% at 2.68 min. HPLC 99% at 14.66 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(5,5,5-trifluoropentyl)-1H-indazole-3-carboxamide (55).

The title compound was prepared by the general procedure F to provide 14 mg (12%) of a white amorphous solid, mp 131–133 °C. R_f = 0.21 (2% MeOH/60% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃, CD₃OD) δ 8.23 (d, J = 8.1 Hz, 1H), 7.94 (s, 1H), 7.66 (d, J = 7.7 Hz, 2H), 7.36–7.46 (m, 3H), 7.19–7.27 (m, 1H), 4.45 (t, J = 6.9 Hz, 2H), 2.01–2.25 (m, 4H), 1.86 (s, 6H), 1.57–1.70 (m, 2H). LC/MS (m/z) 447.0 (M+1), 491.0 (M-1+HCO₂H), >98% at 2.72 min. HPLC 99% at 14.93 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(4-cyanobutyl)-1H-indazole-3-carboxamide (56).

The title compound was prepared by the general procedure A to provide 37 mg (100%) of a white amorphous solid, mp 155–156 °C. R_f = 0.14 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.29 (d, J = 8.3 Hz, 1H), 7.99 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.34–7.47 (m, 4H), 7.17–7.24 (m, 1H), 4.46 (t, J = 6.6 Hz, 2H), 2.39 (t, J = 6.9 Hz, 2H), 2.11–2.27 (m, 2H), 1.87 (s, 6H), 1.65–1.74 (m, 2H). LC/MS (m/z) 404.0 (M+1), 448.2 (M-1+46), >98% at 2.64 min. HPLC 99% at 13.20 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(4-cyano-4,4-dimethylbutyl)-1H-indazole-3-carboxamide (57).

The title compound was prepared by the general procedure A to provide 31 mg (77%) of a white amorphous solid, mp 163–164 °C. R_f = 0.29 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.29 (d, J = 8.3 Hz, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.34–7.49 (m, 4H), 7.17–7.25 (m, 1H), 4.44 (t, J = 6.8 Hz, 2H), 2.13–2.32 (m, 2H), 1.87 (s, 6H), 1.50–1.63 (m, 2H), 1.35 (s, 6H). LC/MS (m/z) 432.0 (M+1), 476.0 (M-1+46), >98% at 2.89 min. HPLC >99% at 14.38 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(2-ethoxyethyl)-1H-indazole-3-carboxamide (58).

The title compound was prepared by the general procedure E to provide 110 mg (100%) of a white amorphous solid, mp 124–125 °C. R_f = 0.32 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.26 (d, J = 8.1 Hz, 1H), 7.98 (s, 1H), 7.67 (d, J = 7.3 Hz, 1H), 7.60 (d, J = 7.3 Hz, 1H), 7.33–7.54 (m, 4H), 7.14–7.24 (m, 1H), 4.58 (t, J = 5.6 Hz, 2H), 3.90 (t, J = 5.6 Hz, 2H), 3.45 (q, J = 6.8 Hz, 2H), 1.86 (s, 6H), 1.12 (t, J = 7.0 Hz, 3H). LC/MS (m/z) 395.0 (M+1), 439.0 (M-1+46), >98% at 2.82 min. HPLC 96% at 13.60 min.

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-(3-methoxypropyl)-1H-indazole-3-carboxamide (59).

The title compound was prepared by the general procedure E to provide 76 mg (96%) of a white amorphous solid, mp 129–131 °C. R_f = 0.27 (5% MeOH/65% EtOAc/hexanes; UV active). ¹H NMR (300 MHz, CDCl₃) δ 8.26 (d, J = 7.9 Hz, 1H), 7.99 (br s, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.60 (d, J = 7.3 Hz, 1H), 7.32–7.49 (m, 4H), 7.20 (t, J = 7.3 Hz, 1H), 4.52 (t, J = 6.6 Hz, 2H), 3.32 (br s, 5H), 2.16–2.29 (m, 1H), 1.86 (s, 6H). LC/MS (m/z) 395.0 (M+1), >98% at 2.80 min. HPLC 99% at 13.56 min.

Testing for Pan-assay Interference (PAIN)

The compounds synthesized are considered low risk for PAIN as they are analogs of previously well-characterized indazole compounds that are specific for cannabinoid receptors. Additionally, compounds were manually inspected to identify structural similarities related to known PAIN compounds^30–32.

Calcium mobilization and radioligand displacement assays

Each compound was biologically characterized using a functional fluorescent hCB1 of hCB2 activated Gαq₁₆-coupled intracellular calcium mobilization assay in CHO-K1 cells, as has been described in our previous publications and EC₅₀ and E_max values were determined.^{33, 34} Briefly, CHO-K1 cells were engineered to co-express hCB1 or hCB2 and G_αq16. Activation of hCB1 or hCB2 by an agonist then leads to generation of inositol phosphatase 3 (IP₃) and activation of IP₃ receptors, which leads to mobilization of intracellular calcium that is detected using a fluorescent dye. Calcium flux was monitored in a 96-well format using the Calcium 5 assay kit in an automated plate reader (FLIPR Tetra, Molecular Devices) using manufacturer’s instructions.

Further characterization of select compounds was performed using radioligand displacement of [³H]CP55940 and equilibrium dissociation constant (Ki) values were determined as described previously.^{33, 34} Selectivity of these compounds at hCB1 versus hCB2 was also determined by obtaining Ki values at either receptor in membranes of CHO cells over-expressing either receptor. Data reported are average values and standard error from 4–6 measurements.

Human Microsomal Stability Studies

Human microsomal stability assays were performed as described previously.³⁵ Briefly, test compounds were incubated at a 1 μM final concentration with 0.5 mg/ml pooled human liver microsomes from 200 unidentified donors (Xenotech, LLC, Lenexa, KS) in a 100 mM phosphate buffer (pH 7.4) containing 3 mM MgCl₂, 1 mM nicotinamide adenine dinucleotide phosphate (NADPH), 5 mM uridine diphosphate glucuronic acid (UDPGA), and 50 μg/ml alamethicin. Triplicate samples were incubated for up to 120 min. Samples were removed at regular intervals. Reactions were terminated by addition of 3 volumes of MeOH and processed for LC-MS by centrifugation. Standard curves were prepared in blank matrix for each compound for quantitative assessment. Intrinsic clearance rate was calculated for each compound using the formula: Clint (μl/min/mg) = 0.693/(t_1/2 X microsomal protein concentration). Data reported are average values from 3 measurements.

Cytochrome P450 induction assay in HepaRG cells

HepaRG cells (Invitrogen) were plated in 96-well rat tail I collagen coated plates in HepaRG culture medium per the manufacturer’s recommendations. Six hours after plating, the culture medium was removed and replaced with HepaRG serum-free induction medium per the manufacturer’s recommendations. The cells were incubated for 2 days at 37°C, 5% CO₂. On the third day, HepaRG cells were treated with test compounds and controls for 24 hrs. Test compound dilutions (10 μM final) were prepared in HepaRG serum-free induction medium with a final concentration of 1% DMSO. The positive controls were CYP450 isoform-specific inducers (50 μM omeprazole – CYP1A2, 1 mM phenobarbital – CYP2B6, 10 μM rifampicin – CYP3A4). For the gene expression assays, mRNA was harvested from the HepaRG cells using commercially available kits (Invitrogen^™ TaqMan^™ Fast Advanced Cells-to-CT^™ Kit or Qiagen^™ RNeasy Mini Kit), reverse transcribed using reverse transcriptase and amplified by PCR using established and validated Taqman assays for the specific CYP450 isoforms (Table 7). Relative fold gene expression was calculated using the ΔΔCt method and GAPDH as the housekeeping gene.³⁶ Percent of positive control was calculated based on the fold induction of the CYP450 isoform-specific positive control.

Table 7. Primers used for amplification.

Probe Name	Vendor	Assay ID
GAPDH-VIC	Fisher Scientific/SSI	Hs02758991_g1
CYP1A2-FAM	Fisher Scientific/SSI	Hs00167927_m1
CYP2B6-FAM	Fisher Scientific/SSI	Hs04183483_g1
CYP3A4-FAM	Fisher Scientific/SSI	Hs00604506_m1

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Pharmacokinetic Testing

All animal studies were approved by institutional animal use committee (IACUC) and followed ethical guidelines mandated by the sponsor (National Institutes of Health, USA). Male or female C57BL/6 mice were procured from Jackson Laboratories at 9–10 weeks of age and allowed to acclimate to the facility. Animals were dosed with compounds in a vehicle comprised of 1% NMP and 0.3% Tween 80 in 0.5% sodium carboxymethylcellulose (medium viscosity; deionized water). Animals were humanely euthanized at multiple time-points (0.5, 1, 2, 4, 8 & 24 h for po; 0.25, 0.5, 1, 2, 4, & 8 h for ip) and samples were removed. Pharmacokinetic analyses were performed as has been described in our previous publications using Phoenix WinNonlin (Certara).³⁴ All LC/MS/MS analysis for pharmacokinetic studies was performed on an API 4000 Triple Quadrupole Mass Spectrometer with Turboion Spray source with a Phenomenex Luna C18 column. 24: 431.026 → 269.8 (DP=76; CE=17; CXP=6); 45: 449.104 → 288.129 (DP=81; CE=13; CXP=26).

Plasma, brain and liver samples were prepared for LC/MS/MS analysis as follows. Plasma: 40 μL of plasma, 10 μL of acetonitrile, and 150 μL of 100 ng/mL Reserpine in acetonitrile with 0.1% formic acid were vortexed and centrifuged at 4000 RPM for 10 minutes. Fifty μL of the supernatant was diluted with 50 μL of water prior to LC/MS/MS analysis. Brain and Liver: Tissues were homogenized with 50:50 EtOH:H2O (1:5, v/v) using a Geno Grinder bead mill. Forty μL of the homogenate, 10 μL of acetonitrile, and 150 μL of 100 ng/mL Reserpine in acetonitrile with 0.1% formic acid were vortexed and centrifuged at 4000 RPM for 10 minutes. Fifty μL of the supernatant was diluted with 50 μL of water prior to LC/MS/MS analysis.

Supplementary Material

NIHMS2100732-supplement-1.pdf^{(1.1MB, pdf)}

File 1. Calculated Properties of Compounds

File 1. HPLC and NMR data for lead compounds

NIHMS2100732-supplement-2.docx^{(19.3KB, docx)}

Highlights.

Important structural features that control CBR partial versus full agonism of indazoles were discovered
Indazole partial agonists of both CB1 and CB2
Compound 45 is a partial agonist of both CB1 and CB2 with good HLM stability
In mice, compound 45 is orally absorbed and peripherally selective with <10% brain penetrance

Acknowledgements

We express our gratitude to the NIDA drug supply program for providing radiolabeled probes and control compounds and to Dr. Brian Thomas for supplying the CB1 cells. This research was funded by research grants AA022235 and DK100414 to RM from NIH. We thank Ms. Taylor Rosa for assistance.

Abbreviations Used:

CB1: cannabinoid receptor 1
CB2: cannabinoid receptor 2
CBRs: cannabinoid receptors
CHO: Chinese hamster ovary cells
EtOAc: ethyl acetate
EtOH: ethanol
HBTU: N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
HLM: human liver microsomes
IP₃: inositol phosphatase 3
NASH: nonalcoholic steatohepatitis
THC: (−)-trans-Δ⁹-tetrahydrocannabinol
TPSA: topological polar surface area

Footnotes

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Declaration of Interest Statement

The authors do not have any known conflicts of interest.

CRediT author statement

George Amato (conceptualization, investigation, data curation, formal analysis, writing), Lucas Laudermilk (investigation, data curation, writing), Vineetha Vasukuttan (investigation, data curation, formal analysis), Elaine A. Gay (investigation, data curation, formal analysis), Ann M. Decker (investigation, data curation, formal analysis), Rodney Snyder (investigation, data curation, formal analysis), Yun Lan Yue (investigation, data curation), Scott Runyon (supervision, funding acquisition) and Rangan Maitra (conceptualization, funding acquisition, supervision, data curation, formal analysis, writing)

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9.Pertwee RG; Howlett AC; Abood ME; Alexander SP; Di Marzo V; Elphick MR; Greasley PJ; Hansen HS; Kunos G; Mackie K; Mechoulam R; Ross RA, International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB(1) and CB(2). Pharmacol Rev 2010, 62 (4), 588–631. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Pertwee RG, Emerging strategies for exploiting cannabinoid receptor agonists as medicines. British journal of pharmacology 2009, 156 (3), 397–411. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Järbe TUC; LeMay BJ; Halikhedkar A; Wood J; Vadivel SK; Zvonok A; Makriyannis A, Differentiation between low- and high-efficacy CB1 receptor agonists using a drug discrimination protocol for rats. Psychopharmacology 2014, 231 (3), 489–500. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Mukhopadhyay P; Baggelaar M; Erdelyi K; Cao Z; Cinar R; Fezza F; Ignatowska‐ Janlowska B; Wilkerson J; van Gils N; Hansen T; Ruben M; Soethoudt M; Heitman L; Kunos G; Maccarrone M; Lichtman A; Pacher P; Van der Stelt M, The novel, orally available and peripherally restricted selective cannabinoid CB(2) receptor agonist LEI‐101 prevents cisplatin‐induced nephrotoxicity. British journal of pharmacology 2016, 173 (3), 446–458. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.De Vry J; Denzer D; Reissmueller E; Eijckenboom M; Heil M; Meier H; Mauler F, 3-[2-Cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59–3074): A Novel Cannabinoid CB1/CB2 Receptor Partial Agonist with Antihyperalgesic and Antiallodynic Effects. Journal of Pharmacology and Experimental Therapeutics 2004, 310 (2), 620–632. [DOI] [PubMed] [Google Scholar]
14.Amato G; Vasukuttan V; Harris D; Laudermilk L; Lucitti J; Runyon S; Maitra R, Structure-Activity Relationship Development Efforts towards Peripherally Selective Analogs of the Cannabinoid Receptor Partial Agonist BAY 59–3074. Molecules 2022, 27 (17). [DOI] [PMC free article] [PubMed] [Google Scholar]
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16.Banister SD; Moir M; Stuart J; Kevin RC; Wood KE; Longworth M; Wilkinson SM; Beinat C; Buchanan AS; Glass M; Connor M; McGregor IS; Kassiou M, Pharmacology of Indole and Indazole Synthetic Cannabinoid Designer Drugs AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, and 5F-ADBICA. ACS Chemical Neuroscience 2015, 6 (9), 1546–1559. [DOI] [PubMed] [Google Scholar]
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20.Shao Z; Yin J; Chapman K; Grzemska M; Clark L; Wang J; Rosenbaum DM, High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 2016, 540, 602. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Hua T; Vemuri K; Pu M; Qu L; Han GW; Wu Y; Zhao S; Shui W; Li S; Korde A; Laprairie RB; Stahl EL; Ho JH; Zvonok N; Zhou H; Kufareva I; Wu B; Zhao Q; Hanson MA; Bohn LM; Makriyannis A; Stevens RC; Liu ZJ, Crystal Structure of the Human Cannabinoid Receptor CB1. Cell 2016, 167 (3), 750–762.e14. [DOI] [PMC free article] [PubMed] [Google Scholar]
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24.Lipinski CA; Lombardo F; Dominy BW; Feeney PJ, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews 1997, 23 (1), 3–25. [DOI] [PubMed] [Google Scholar]
25.Ghose AK; Herbertz T; Hudkins RL; Dorsey BD; Mallamo JP, Knowledge-Based, Central Nervous System (CNS) Lead Selection and Lead Optimization for CNS Drug Discovery. ACS chemical neuroscience 2012, 3 (1), 50–68. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Kerns EH; Di L, Drug-like properties : concepts, structure design, and methods : from ADME to toxicity optimization. Elsevier Academic Press: Amsterdam ; Boston, 2008; p xix, 526 p. [Google Scholar]
27.Fulp A; Zhang Y; Bortoff K; Seltzman H; Snyder R; Wiethe R; Amato G; Maitra R, Pyrazole antagonists of the CB1 receptor with reduced brain penetration. Bioorg Med Chem 2016, 24 (5), 1063–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Amato G; Wiethe R; Manke A; Vasukuttan V; Snyder R; Runyon S; Maitra R, Functionalized 6-(piperidin-1-yl)-8,9-diphenyl purines as inverse agonists of the CB1 receptor - SAR efforts towards selectivity and peripheralization. Bioorg Med Chem 2019, 27 (16), 3632–3649. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.De Vry J; Denzer D; Reissmueller E; Eijckenboom M; Heil M; Meier H; Mauler F, 3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59–3074): a novel cannabinoid Cb1/Cb2 receptor partial agonist with antihyperalgesic and antiallodynic effects. J Pharmacol Exp Ther 2004, 310 (2), 620–32. [DOI] [PubMed] [Google Scholar]
30.Baell JB; Holloway GA, New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 2010, 53 (7), 2719–40. [DOI] [PubMed] [Google Scholar]
31.Baell JB; Nissink JWM, Seven Year Itch: Pan-Assay Interference Compounds (PAINS) in 2017-Utility and Limitations. ACS Chem Biol 2018, 13 (1), 36–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Dahlin JL; Walters MA, How to Triage PAINS-Full Research. Assay Drug Dev Technol 2016, 14 (3), 168–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
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34.Fulp A; Bortoff K; Zhang Y; Seltzman H; Mathews J; Snyder R; Fennell T; Maitra R, Diphenyl purine derivatives as peripherally selective cannabinoid receptor 1 antagonists. J Med Chem 2012, 55 (22), 10022–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Fulp A; Bortoff K; Seltzman H; Zhang YA; Mathews J; Snyder R; Fennell T; Maitra R, Design and Synthesis of Cannabinoid Receptor 1 Antagonists for Peripheral Selectivity. J Med Chem 2012, 55 (6), 2820–2834. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Livak KJ; Schmittgen TD, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25 (4), 402–8. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS2100732-supplement-1.pdf^{(1.1MB, pdf)}

File 1. Calculated Properties of Compounds

File 1. HPLC and NMR data for lead compounds

NIHMS2100732-supplement-2.docx^{(19.3KB, docx)}

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[R9] 9.Pertwee RG; Howlett AC; Abood ME; Alexander SP; Di Marzo V; Elphick MR; Greasley PJ; Hansen HS; Kunos G; Mackie K; Mechoulam R; Ross RA, International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB(1) and CB(2). Pharmacol Rev 2010, 62 (4), 588–631. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Pertwee RG, Emerging strategies for exploiting cannabinoid receptor agonists as medicines. British journal of pharmacology 2009, 156 (3), 397–411. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Järbe TUC; LeMay BJ; Halikhedkar A; Wood J; Vadivel SK; Zvonok A; Makriyannis A, Differentiation between low- and high-efficacy CB1 receptor agonists using a drug discrimination protocol for rats. Psychopharmacology 2014, 231 (3), 489–500. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Mukhopadhyay P; Baggelaar M; Erdelyi K; Cao Z; Cinar R; Fezza F; Ignatowska‐ Janlowska B; Wilkerson J; van Gils N; Hansen T; Ruben M; Soethoudt M; Heitman L; Kunos G; Maccarrone M; Lichtman A; Pacher P; Van der Stelt M, The novel, orally available and peripherally restricted selective cannabinoid CB(2) receptor agonist LEI‐101 prevents cisplatin‐induced nephrotoxicity. British journal of pharmacology 2016, 173 (3), 446–458. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.De Vry J; Denzer D; Reissmueller E; Eijckenboom M; Heil M; Meier H; Mauler F, 3-[2-Cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59–3074): A Novel Cannabinoid CB1/CB2 Receptor Partial Agonist with Antihyperalgesic and Antiallodynic Effects. Journal of Pharmacology and Experimental Therapeutics 2004, 310 (2), 620–632. [DOI] [PubMed] [Google Scholar]

[R14] 14.Amato G; Vasukuttan V; Harris D; Laudermilk L; Lucitti J; Runyon S; Maitra R, Structure-Activity Relationship Development Efforts towards Peripherally Selective Analogs of the Cannabinoid Receptor Partial Agonist BAY 59–3074. Molecules 2022, 27 (17). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Ingrid Price Buchler MJH, Hegde Sridhar Gajanan, Hockerman Susan Landis, Jones Darin Eugene, Kortum Steven Wade, Rico Joseph Jerase, TenBrink Ruth Elizabeth, Wu Kun Ken Indazole Derivatives. 2011. [Google Scholar]

[R16] 16.Banister SD; Moir M; Stuart J; Kevin RC; Wood KE; Longworth M; Wilkinson SM; Beinat C; Buchanan AS; Glass M; Connor M; McGregor IS; Kassiou M, Pharmacology of Indole and Indazole Synthetic Cannabinoid Designer Drugs AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, and 5F-ADBICA. ACS Chemical Neuroscience 2015, 6 (9), 1546–1559. [DOI] [PubMed] [Google Scholar]

[R17] 17.Banister SD; Longworth M; Kevin R; Sachdev S; Santiago M; Stuart J; Mack JBC; Glass M; McGregor IS; Connor M; Kassiou M, Pharmacology of Valinate and tert-Leucinate Synthetic Cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and Their Analogues. ACS Chemical Neuroscience 2016, 7 (9), 1241–1254. [DOI] [PubMed] [Google Scholar]

[R18] 18.Rangari VA; O’Brien ES; Powers AS; Slivicki RA; Bertels Z; Appourchaux K; Aydin D; Ramos-Gonzalez N; Mwirigi J; Lin L; Mangutov E; Sobecks BL; Awad-Agbaria Y; Uphade MB; Aguilar J; Peddada TN; Shiimura Y; Huang X-P; Folarin-Hines J; Payne M; Kalathil A; Varga BR; Kobilka BK; Pradhan AA; Cameron MD; Kumar KK; Dror RO; Gereau RW; Majumdar S, A cryptic pocket in CB1 drives peripheral and functional selectivity. Nature 2025, 640 (8057), 265–273. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Amato G; Runyon S; Vasukuttan V; Decker AM; Gay EA; Laudermilk L; Maitra R, Discovery of 1,3-disubstituted pyrazole peripheral cannabinoid receptor partial agonists. Bioorg Med Chem Lett 2023, 93, 129430. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Shao Z; Yin J; Chapman K; Grzemska M; Clark L; Wang J; Rosenbaum DM, High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 2016, 540, 602. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Hua T; Vemuri K; Pu M; Qu L; Han GW; Wu Y; Zhao S; Shui W; Li S; Korde A; Laprairie RB; Stahl EL; Ho JH; Zvonok N; Zhou H; Kufareva I; Wu B; Zhao Q; Hanson MA; Bohn LM; Makriyannis A; Stevens RC; Liu ZJ, Crystal Structure of the Human Cannabinoid Receptor CB1. Cell 2016, 167 (3), 750–762.e14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Amato GS; Manke A; Harris DL; Wiethe RW; Vasukuttan V; Snyder RW; Lefever TW; Cortes R; Zhang Y; Wang S; Runyon SP; Maitra R, Blocking Alcoholic Steatosis in Mice with a Peripherally Restricted Purine Antagonist of the Type 1 Cannabinoid Receptor. Journal of Medicinal Chemistry 2018, 61 (10), 4370–4385. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Li X; Hua T; Vemuri K; Ho J-H; Wu Y; Wu L; Popov P; Benchama O; Zvonok N; Locke K. a.; Qu L; Han GW; Iyer MR; Cinar R; Coffey NJ; Wang J; Wu M; Katritch V; Zhao S; Kunos G; Bohn LM; Makriyannis A; Stevens RC; Liu Z-J, Crystal Structure of the Human Cannabinoid Receptor CB2. Cell 2019, 176 (3), 459–467.e13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Lipinski CA; Lombardo F; Dominy BW; Feeney PJ, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews 1997, 23 (1), 3–25. [DOI] [PubMed] [Google Scholar]

[R25] 25.Ghose AK; Herbertz T; Hudkins RL; Dorsey BD; Mallamo JP, Knowledge-Based, Central Nervous System (CNS) Lead Selection and Lead Optimization for CNS Drug Discovery. ACS chemical neuroscience 2012, 3 (1), 50–68. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Kerns EH; Di L, Drug-like properties : concepts, structure design, and methods : from ADME to toxicity optimization. Elsevier Academic Press: Amsterdam ; Boston, 2008; p xix, 526 p. [Google Scholar]

[R27] 27.Fulp A; Zhang Y; Bortoff K; Seltzman H; Snyder R; Wiethe R; Amato G; Maitra R, Pyrazole antagonists of the CB1 receptor with reduced brain penetration. Bioorg Med Chem 2016, 24 (5), 1063–70. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Amato G; Wiethe R; Manke A; Vasukuttan V; Snyder R; Runyon S; Maitra R, Functionalized 6-(piperidin-1-yl)-8,9-diphenyl purines as inverse agonists of the CB1 receptor - SAR efforts towards selectivity and peripheralization. Bioorg Med Chem 2019, 27 (16), 3632–3649. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] 29.De Vry J; Denzer D; Reissmueller E; Eijckenboom M; Heil M; Meier H; Mauler F, 3-[2-cyano-3-(trifluoromethyl)phenoxy]phenyl-4,4,4-trifluoro-1-butanesulfonate (BAY 59–3074): a novel cannabinoid Cb1/Cb2 receptor partial agonist with antihyperalgesic and antiallodynic effects. J Pharmacol Exp Ther 2004, 310 (2), 620–32. [DOI] [PubMed] [Google Scholar]

[R30] 30.Baell JB; Holloway GA, New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem 2010, 53 (7), 2719–40. [DOI] [PubMed] [Google Scholar]

[R31] 31.Baell JB; Nissink JWM, Seven Year Itch: Pan-Assay Interference Compounds (PAINS) in 2017-Utility and Limitations. ACS Chem Biol 2018, 13 (1), 36–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] 32.Dahlin JL; Walters MA, How to Triage PAINS-Full Research. Assay Drug Dev Technol 2016, 14 (3), 168–74. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R33] 33.Fulp A; Bortoff K; Seltzman H; Zhang Y; Mathews J; Snyder R; Fennell T; Maitra R, Design and synthesis of cannabinoid receptor 1 antagonists for peripheral selectivity. J Med Chem 2012, 55 (6), 2820–34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] 34.Fulp A; Bortoff K; Zhang Y; Seltzman H; Mathews J; Snyder R; Fennell T; Maitra R, Diphenyl purine derivatives as peripherally selective cannabinoid receptor 1 antagonists. J Med Chem 2012, 55 (22), 10022–32. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R35] 35.Fulp A; Bortoff K; Seltzman H; Zhang YA; Mathews J; Snyder R; Fennell T; Maitra R, Design and Synthesis of Cannabinoid Receptor 1 Antagonists for Peripheral Selectivity. J Med Chem 2012, 55 (6), 2820–2834. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Livak KJ; Schmittgen TD, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25 (4), 402–8. [DOI] [PubMed] [Google Scholar]

PERMALINK

Indazole Partial Agonists Targeting Peripheral Cannabinoid Receptors

George Amato

Lucas Laudermilk

Vineetha Vasukuttan

Elaine A Gay

Ann M Decker

Rodney Snyder

Yun Lan Yue

Scott Runyon

Rangan Maitra

Abstract

Graphical Abstract

1. Introduction

Figure 1.

2. Results and Discussion

2.1. Compound Design and Synthesis

Table 1. Right Side Amide Groups.

Table 2. Right Side Aryl Amide Groups.

Table 3. Right Side Non-Aryl Amide Groups.

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

Table 4. Left-Side Modifications with Optimized Right Side Amide Group.

Scheme 5.

Scheme 6.

Scheme 7.

2.2. Pharmacological Evaluation

Table 6. Mouse PK Data.

2.3. Cytochrome P450 (CYP) Induction and Off Target Activity

Table 5. CYP Induction Potential of 24 and 45.

2.4. Pharmacokinetic Studies

3. Summary and Conclusions

4. Experimental

Chemistry General.

General Procedure A: N-Alkylation of Indazoles (Scheme Reactions 1a, 5a & 7d).

General Procedure B: Carboxylic Acids from Hydrolysis of Carboxy Methyl Esters (Scheme Reactions 1b, 2a, 4a & 5b).

General Procedure C: Amides from Carboxylic Acids (Scheme Reactions 1c, 2c, 3a, 5c & 7a).

General Procedure D: Aryl Nitriles from Aryl Bromides (Scheme Reactions 5d1 & 7b).

General Procedure E: Aryl Carboxamides from Aryl Nitriles (Scheme Reactions 5d2 & 7c).

General Procedure F: Aryl Carboxamides from Aryl Bromides (Scheme Reaction 5e).

General Procedure G: Deprotection of Boc Piperidines (Scheme Reaction 6c).

General Procedure H: Piperidine Ureas from Reaction with TMS-NCO (Scheme Reaction 6d).

General Procedure I: Alkylation of Piperidines with Chloroacetamide (Scheme Reaction 6e).

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-3-methylbutanoic Acid (5).

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-N,3-dimethylbutanamide (6).

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-2-phenylacetamide (7).

(2S)-2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-3-phenylpropanamide (8).

N-(1-Carbamoylcyclopentyl)-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (9).

2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-2-methylpropanamide (10).

2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-N,2-dimethylpropanamide (11).

3-[2-({1-[(4-Fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-2-methylpropanamido]benzamide (12).

N-(Carbamoylmethyl)-2-({1-[(4-fluorophenyl)methyl]-1H-indazol-3-yl}formamido)-N,2-dimethylpropanamide (13).

N-{1-[(2S)-2-Carbamoylpyrrolidin-1-yl]-2-methyl-1-oxopropan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (14).

N-{1-[(2R)-2-Carbamoylpyrrolidin-1-yl]-2-methyl-1-oxopropan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (15).

N-{1-[(2S)-2-Carbamoylpyrrolidine-1-carbonyl]cyclopropyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (16).

N-{1-[(2S)-2-Carbamoylpyrrolidin-1-yl]-2-methylpropan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (17).

N,1-Bis[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (18).

N-[(2-Carbamoylphenyl)methyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (19).

N-[(3-Carbamoylphenyl)methyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (20).

N-[(4-Carbamoylphenyl)methyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (21).

N-[(1S)-1-(3-Carbamoylphenyl)ethyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (22).

N-[(1R)-1-(3-Carbamoylphenyl)ethyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (23).

N-[2-(3-Carbamoylphenyl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (24)

N-[2-(3-Acetamidophenyl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (25).

N-{2-[3-(Carbamoylamino)phenyl]propan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (26).

N-[1-(3-Carbamoylphenyl)cyclopropyl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (27).

N-(Cyclohexylmethyl)-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (28).

N-{[(3S)-1-Carbamoylpiperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (29).

N-{[(3R)-1-Carbamoylpiperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (30).

N-[2-(1-Carbamoylpiperidin-3-yl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (31).

N-{[(3S)-1-(Carbamoylmethyl)piperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (32).

N-{[(3R)-1-(Carbamoylmethyl)piperidin-3-yl]methyl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (33).

N-{2-[1-(Carbamoylmethyl)piperidin-3-yl]propan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (34).

N-{2-[1-(Carbamoylmethyl)piperidin-4-yl]propan-2-yl}-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (35).

N-[2-(1-Carbamoylpiperidin-4-yl)propan-2-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (36).

N-Cyclohexyl-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (37).

N-[(3S)-1-Carbamoylpiperidin-3-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (38).

N-[(3R)-1-Carbamoylpiperidin-3-yl]-1-[(4-fluorophenyl)methyl]-1H-indazole-3-carboxamide (39).