The Design and Synthesis of Potent and Selective Inhibitors of Trypanosoma brucei Glycogen Synthase Kinase 3 for the Treatment of Human African Trypanosomiasis

Abstract

Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3β enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture.

Introduction

Human African trypanosomiasis (HAT) or African sleeping sickness is a serious life threatening disease.¹ Around 60 million people in 36 African countries are currently in constant threat of infection. Although the reported number of cases has dropped over recent years, the actual number of unreported cases is estimated to be around 70000–80000.² HAT is caused by infection with Trypanosoma brucei, a vector-borne parasite, which is transmitted by the bite of tsetse flies. The symptoms of the disease occur in two main stages. In the first stage, known as the hemolymphatic phase, the parasites multiply in blood, subcutaneous tissues, and lymph, causing headaches, fever, itching, joint pains, and swelling of lymph nodes. In the second stage, or neurological phase, the trypanosomes cross the blood–brain barrier and invade the central nervous system. This phase entails confusion, change of behavior, reduced coordination, sensory disturbances, disturbance of sleep cycle, and finally death. Most available drugs for HAT display severe toxic side effects, require long periods of administration, and/or are expensive due to the logistics to reach rural African areas.³ Further, resistance to all in use drugs has been observed in the laboratory and/or in the field,⁴ resulting in an urgent requirement for better, safer, and inexpensive therapeutic alternatives to the current treatments.

Genetic knockdown studies have identified several proteins that are essential for the survival of the parasite, including members of the protein kinase (PK) family.⁵⁻⁸ In Trypanosoma brucei PKs are essential in many fundamental cellular processes, e.g., proliferation, differentiation, and cell cycle control, and can therefore be considered as potential drug targets for the treatment of HAT.^7,9−12

In the T. brucei genome there are two kinases that are highly homologous to human glycogen synthase kinase 3 (HsGSK3): TbGSK3 short and TbGSK3 long.¹³ RNA interference (RNAi) knockdown of TbGSK3 has shown that TbGSK3 short is critical for cell growth, with a role in the control of mitosis and/or cytokinesis.^7,13

The ability to selectively inhibit TbGSK3 over the off-target HsGSK3 is highly desirable because mouse knockout studies revealed that the disruption of the murine GSK3β gene causes embryonic lethality; consequently, nonselective inhibitors are not applicable for use in infants and women of child bearing age.^14,15

From a homology perspective, TbGSK3 is not only very closely related to HsGSK3β but also to other human PKs such as cyclin dependent kinase 1 (HsCDK1) and cyclin dependent kinase 2 (HsCDK2).¹⁶HsCDK2 and HsCDK1 are essential for G1/G2 progression and S/M-phase entry of the cell cycle. Off-target inhibition of these human kinases will therefore result in cell cycle arrest and reduction of cellular proliferation and as such potentially lead to severe side effects.

Over the past decade, various groups and pharmaceutical companies have identified multiple series of HsGSK3β inhibitors.^16,17 Recently, Astex Therapeutics and researchers at the University of Osaka have developed a series of aminopyrazoles that are potent inhibitors of HsGSK3β.¹⁸⁻²⁰ Co-crystal structures of this series with HsGSK3β are not available to date; however, complex structures with the closely related HsCDK2 have been determined.¹⁹ In all structures, the pyrazole scaffold forms two hydrogen bonds to the hinge region of HsCDK2 (Figure 1). Further, the NH group of the 3-position amide forms an additional hydrogen-bond interaction to the backbone of Leu83. A water-mediated hydrogen bond from the amide carbonyl oxygen atom to the backbone NH of Asp145 is also observed. The R¹ residues (Figure 2) access the gatekeeper region between the gatekeeper residue Phe80 and the catalytic Asp145 (Figure 1). The R² substituents occupy the hydrophobic pocket II, formed by the backbone of the linker region, Leu83, Phe82, and side chains of Ile10, Asp86, and Leu134. Finally, an intramolecular hydrogen bond between the R¹-NH and R²-carbonyl group is present. The similarity of HsCDK2, HsGSK3β, and TbGSK3 indicates that aminopyrazoles will also bind into the ATP-binding site of the latter enzyme.^13,16

Figure 1.

Co-crystal structure (2vu3) of AT7519¹⁹ (carbon atoms in gray) bound to CDK2. The binding pocket of CDK2 is shown in light-blue surface representation. Key: red sphere, water molecule; black dashed lines, protein–ligand and water–ligand hydrogen bonds; yellow stick, hydrogen atom..

Figure 2.

Generic binding mode of the R¹ and R² substituted aminopyrazole scaffold (carbon atoms in gray).

Herein, we describe the design, synthesis, and biological evaluation of aminopyrazole inhibitors which bind to TbGSK3 short. The inhibitors were also tested against the closely related off-targets HsGSK3β and HsCDK2 and evaluated against a panel of mammalian protein kinases. The most potent compound has nanomolar affinity for TbGSK3 short, is selective over HsGSK3β and HsCDK2, and clean in the kinase panel. By using computer-aided molecular modeling, we were able to rationalize the observed selectivity profile. Enzyme affinity correlated with inhibition of T. b. brucei proliferation, albeit a 100-fold offset in potency, was found. In light of these results, we discuss the value of TbGSK3 short as a drug target for HAT.

Results

Starting Point

The aminopyrazole derivatives developed by Astex Therapeutics and Yumiko Uno et al. for inhibition of HsCDK2 and HsGSK3β enzymes were chosen as a starting point for the investigation of TbGSK3 short inhibitors.¹⁸⁻²⁰ Aminopyrazoles analogues were generated by substituting at either R¹ or R² position (Figure 2) using two synthetic routes (Scheme 1A,B).¹⁹

Scheme 1. Synthetic Routes for 50 R¹ and R² Substituted Aminopyrazoles (4a–z and 9a–x).

Reagents and conditions used in routes A and B: (a) trans-4-methoxycyclohexylamine, EDC, HOBt, DMF, rt; (b) 10% Pd/C, H₂, DMF, rt; (c) R¹COOH, EDC, HOBt, DIPEA, rt; (d) SOCl₂, MeOH, 0°C, rt; (e) 10% Pd/C, H₂, EtOH, rt; (f) 2,6-dimethoxybenzoylchloride, Et₃N, dioxane, rt; (g) NaOH, dioxane, H₂O, rt; (h) R₂NH₂, polystyrene-bound carbodiimide, HOBt, acetonitrile, MW, 100 °C.

Differences in the ATP Binding Pockets of TbGSK3, HsGSK3β, and HsCDK2

A structural model of TbGSK3 was built to assess the differences in the binding sites of TbGSK3 short, HsGSK3β, and HsCDK2 and to guide ligand design. An overlay of 42 HsGSK3 crystal structures showed that there is low flexibility in the ATP binding site. Only the regions including Phe67 and Arg141 showed some mobility. Phe67 either points toward or away from the hinge region. Arg141 also spans a number of distinct conformations, including examples where it occupies space in the binding site (1J1B, 1J1C, 1O9U, 2O5K) and therefore could influence docking results. However, to allow for ligands of a significant size, we have used examples with Arg141 pointing out of the binding site. Therefore, two homology models for TbGSK3 were generated representing both states of Phe67. As we were mainly interested in aminopyrazoles with less extended R¹ groups, the crystal structure with Phe67 pointing toward the hinge (with structure 1r0e as a representative) was more suited as model system. The selection of 1r0e instead of other members of this group (with Phe67 pointing toward the hinge) was arbitrary. For this analysis, all residues that are located within 6 Å of the ligand bound to the template structure (1r0e) were considered.

The binding pockets of TbGSK3 and HsGSK3β differ by nine amino acid residues (Table 1, Figure 3A). Of the amino acid side chains that point toward the ligand, the most significant differences are the replacement of Tyr134 in HsGSK3β with Phe103, Leu132 with Met101, Gln72 with Leu36, and Tyr140 with His109. The binding pockets of TbGSK3 and HsCDK2 are more diverse. Here, in total 16 out of 26 amino acids were found to be different (Table 1, Figure 3B), the most important of these differences being the replacement of Lys20 in CDK2 with Leu36, Phe80 with Met101, His84 with Pro105, Lys89 with Arg110, and Ala144 with Cys170. Interestingly, most of the amino acid differences occurred in the hydrophobic pocket II and the gatekeeper region. Therefore, we decided to direct the optimization of the lead scaffold toward suitable interactions with amino acids which are located in these subpockets of the ATP binding site.

Table 1. Differences in the Binding Pockets of TbGSK3, HsGSK3β, and HsCDK2^a.

TbGSK3	HsGSK3β	HsCDK2
V25	V61	K9
A26	I62	I10
G27	G63	G11
Q28	N64	E12
G29	G65	G13
T30	S66	T14
F31	F67	Y15
V34	V70	V18
L36	Q72	K20
A47	A83	A31
K49	K85	K33
E61	E97	I52
M65	M101	L55
V77	V110	V64
M101	L132	F80
E102	D133	E81
F103	Y134	F82
104	V135	L83
P105	P136	H84
E106	E137	Q85
T107	T138	D86
H109	Y140	K88
R110	R141	K89
K154	K183	K129
H156	Q185	Q131
N157	N186	N132
L159	L188	L134
C170	C199	A144
D171	D200	D145

^aAmino acids of hGSK3β or HsCDK2 which differ in TbGSK3 short are shown in boldface.

Figure 3.

Superposition of the binding sites of the homology model of TbGSK3 short (blue carbon atoms) with (A) the HsGSK3β crystal structure (PDB code 1r0e) and (B) the HsCDK2 crystal structure (PDB code 2vu3). The solvent accessible surface of TbGSK3 short is shown in light blue. Only residues that differ between the binding pockets are shown. For orientation, the ligands bound to crystal structures are also displayed. Amino acid residue labels are for TbGSK3.

Chemistry

The synthesis of R¹ and R² substituted aminopyrazole derivatives started from 4-nitro-pyrazole-3-carboxylic acid 1 and is described by two different routes (Scheme 1) based on previous work from Wyatt et al.¹⁹ In route A, 1 was coupled with trans-4-methoxycyclohexylamine using EDC as the activating agent. Reduction of the subsequent intermediate 2 by hydrogenation in the presence of palladium on carbon generated amino pyrazole 3. The conversion to compounds 4a–z was accomplished by coupling of 3 with a suitable selection of carboxylic acids. In route B, after esterification of the carboxyl group of 1, the nitro group of intermediate 5 was reduced to afford amine 6. Treatment of 6 with 2,6-dimethoxybenzoyl chloride under standard conditions, followed by base hydrolysis of the ester, provided acid 8. In the final step, 8 was coupled with appropriate amines in a microwave reaction using polystyrene-bound carbodiimide to yield final compounds 9a–x.

Activity and Selectivity of R¹ Substituted Compounds

Twenty-six R¹ substituted aminopyrazole analogues (Table 2) were made according to the synthetic route shown in Scheme 1A. A range of R¹ groups varying in size and polarity was chosen to probe whether the differences in the gate keeper region between TbGSK3, HsGSK3, and HsCDK2 could be exploited to derive selective and potent TbGSK3 inhibitors (Figure 3).

Table 2. Kinase Inhibitory Activity and Antiproliferative Efficacy of R¹ Substituted Aminopyrazoles.

	IC50 (μM)	IC50 (μM)	IC50 (μM)			EC50 (μM)
compd	TbGSK3a	HsGSK3βa	ratio HsGSK3β/TbGSK3	HsCDK2a	ratio HsCDK2/TbGSK3	T. b. bruceib	MRC5c
4a	0.50	0.008	0.16	1.0	2	16	>50
4b	0.69	0.008	0.0012	0.38	0.55	32	>50
4c	0.39	0.14	0.35	0.82	2.1	23	>50
4d	0.50	0.21	0.42	0.85	1.7	22	40
4e	0.23	0.2	0.87	0.29	1.3	7.3	3.1
4f	0.024	<0.005	>0.21	0.038	<1.6	1.3	0.8
4g	0.020	<0.005	0.25	0.014	<0.7	0.4	1.0
4h	0.018	<0.013	<0.72	0.1	5.6	2.3	3.3
4i	0.053	0.02	0.38	0.07	1.3	2.8	3.4
4j	0.004	<0.013	<3.3	0.1	25	0.9	1.5
4k	0.011	0.02	1.8	>10	>910	4.4	35
4l	0.066	0.03	0.45	>10	>150	5.8	16
4m	0.002	<0.005	<2.5	0.19	95	0.5	31
4n	0.003	0.09	30	3.1	1000	5.9	>50
4o	0.053	<0.005	<0.09	0.22	4.2	3.8	11
4p	0.024	<0.005	<0.21	0.083	3.5	2.6	0.6
4q	0.057	<0.005	<0.08	0.63	11	9.6	13
4r	0.016	<0.005	<0.31	0.27	17	1.1	2.5
4s	0.019	<0.005	<0.26	0.13	6.8	2.7	>50
4t	0.070	<0.005	<0.071	1.0	14	2.0	20
4u	0.013	<0.005	<0.38	nd	nd	1.2	0.8
4v	0.063	<0.005	<0.080	0.042	0.67	1.9	1.0
4w	0.094	<0.005	<0.053	0.15	1.6	2.6	5.9
4x	0.10	0.042	0.42	2.2	22	2.9	34
4y	0.007	<0.005	<0.72	0.01	1.4	0.3	0.1
4z	0.92	0.005	0.0058	22	24	>50	28

^aData represents the average of two or more experiments.

^bConcentration required to inhibit the growth of T. b. brucei in culture by 50% over 72 h.

^cConcentration required to inhibit the growth of MRC5 cells in culture by 50% over 72 h.

Enzyme Activity

All compounds showed good potency against TbGSK3 (<1 μM). An unsubstituted phenyl ring (4f) provided on average a 20-fold improvement of inhibition potency relative to saturated six-membered ring systems (4a and 4b) and benzyl groups (4c, 4d, and 4e). In general, a variety of different aryl and heteroaryl rings (4g–4y compared to 4a and 4b) in the R¹ position led to significantly improved potency against TbGSK3. Additionally, a wide variety of substituents were tolerated on the phenyl ring. In general, ortho-substituted phenyl rings gave the best improvement in activity compared to the unsubstituted phenyl group (4j, 4k, 4m, and 4n). The methoxyphenyl moieties in 4j, 4m, and 4n, which had TbGSK3 IC₅₀ values of 4, 2, and 3 nM, respectively, were the most favorable substituents. These derivatives were approximately 10-fold more potent than the unsubstituted phenyl compound 4f. Only the 2,4,6-trimethoxyl derivative 4n showed >10-fold selectivity over HsGSK3. Interestingly, this was also the most selective compound for HsCDK2 (>1000-fold).

To rationalize the observed selectivity, all analogues were docked into the binding sites of TbGSK3, HsGSK3β, and HsCDK2 and their poses were visually analyzed. For most compounds, a binding mode similar to that observed for AT7519¹⁹ in HsCDK2 (Figure 1) was predicted in TbGSK3, HsGSK3β, and HsCDK2. One important difference between HsCDK2, TbGSK3, and HsGSK3β is the gatekeeper residue (Table 1, Figure 3A,B). While HsGSK3 and TbGSK3 enzymes have Leu or Met, respectively, in this position, in HsCDK2 Phe is present. As a consequence, the gatekeeper region of HsCDK2 (located between Phe80 and Asp145) is more restricted compared to the other two enzymes. This resulted in a higher energy, out of plane conformation of the amide group of 4f when binding into this pocket (Figure 4), while a low energy conformation was found when binding into T. brucei and human GSK3 (not shown). Further, without induced fit adaptations, the bulky R¹-substituents such as the 2,6-dimethoxybenzamide group of 4m, the 2,4,6-trimethoxybenzamide groups 4n, and the phenylaminobenzamide groups of 4k and 4l can only be accommodated by the gatekeeper region of TbGSK3 and HsGSK3β but not the narrower HsCDK2 gatekeeper region. These observations might explain the reduced binding affinity of 4f for HsCDK2 compared to HsGSK3. Of note, this explanation is further supported by the report by Wyatt et al.,¹⁹ which found that the aryl groups which are located in the same position need to twist in order to provide potent CDK2 activity.

Figure 4.

Predicted binding mode of 4f in HsCDK2. Putative hydrogen bonds are shown as black dotted lines. Docking results suggested that the phenyl ring of compound 4f needs to be significantly twisted out of plane by approximately 60° compared to the amide in order to fit into the gatekeeper region..

Antiparasitic Activity

All R¹ substituted compounds were tested for their ability to inhibit the proliferation of bloodstream form (BSF) T. b. brucei in culture. As an initial indication of potential toxicity, compounds 4a–4z were additionally tested against proliferating human fetal lung fibroblast cells (MRC5 cell line). Four compounds (4g, 4j, 4m, and 4y) had EC₅₀ values <1 μM and a further 11 compounds had EC₅₀ values <3 μM against BSF T. b. brucei (Table 2). The EC₅₀ values correlated well with enzyme activity (R² = 0.73, Figure 5). However, a 100-fold drop from enzyme to cellular activity was observed. Selectivity over the MRC5 cells was achieved with compounds 4m (60-fold), 4s (>19-fold), 4x (12-fold), and 4n (>9-fold), however, the majority of compounds showed a poor selectivity over MRC5 cells.

Figure 5.

Correlation between the inhibition of recombinant TbGSK3 and bloodstream form T. b. brucei proliferation by R¹ substituted aminopyrazole derivatives (4a–z).

Activity and Selectivity of R² Substituted Compounds

As 4m was the most potent inhibitor of TbGSK3 and proliferation of T. b. brucei cells, we retained the 2,6-dimethoxybenzamide group at position R¹ for optimization of the R² substituent. R² substituted aminopyrazole analogues (9a–9x) were made according to the synthetic route shown in Scheme 1B to explore the structural requirements for improvement of antiparasitic activity and selectivity over the closely related human kinases.

Enzyme Activity

The majority of variations led to potent TbGSK3 inhibitors, indicating that chemical diversity at this position was well tolerated (Table 3). One of the SAR trends observed was that six-membered saturated rings (9c) and seven-membered saturated rings (9d) were favored over their three- and four-membered equivalents (9a and 9b). Further, it was noted that the replacement of the cyclohexane of 9c with a phenyl ring or 4-pyridine, to give 9k or 9l, gave a 6-fold decrease in potency against TbGSK3. The 2-pyridine analogue (9m), on the other hand, was much less active (50-fold) against TbGSK3. Homologation of aromatic (9g) and saturated six-membered (9j) rings by one carbon atom produced inhibitors with 1 nM activity for TbGSK3. For aliphatic side chain derivatives 9r–9w, the pentanyl and 1-isopropoxypropanyl analogues had IC₅₀ values of 1 nM. The impact of replacing the amide group (3-position) with carboxylic acid and ester groups was investigated with compounds 8 and 7. Compound 8 containing a carboxylate group in the 4-position showed a dramatic loss in activity (IC₅₀ >50 μM). The ester group of compound 7 on the other hand was better tolerated (IC₅₀ 0.5 μM). Interestingly, compared with 4a–4z, a majority of R² substituted analogues (9a–9x) showed selectivity over HsCDK2 and HsGSK3β. These are the most selective TbGSK3 inhibitors described to date.

Table 3. Kinase Inhibitory Activity and Antiproliferative Efficacy of R² Substituted Aminopyrazoles.

	IC50 (μM)	IC50 (μM)	IC50 (μM)			EC50 (μM)
compd	TbGSK3a	HsGSK3βa	ratio HsGSK3β/TbGSK3	HsCDK2a	ratio HsCDK2/TbGSK3	T. b. bruceib	MRC5c
9a	0.012	0.22	18	>10	>830	19	>50
9b	0.008	0.08	10	2.4	300	12	>50
9c	0.001	0.05	50	1.2	1200	4.1	35
9d	0.001	0.02	20	2.0	2000	4.5	42
9e	0.018	nd	nd	>10	560	16	>50
9f	0.081	0.45	5.6	>10	120	50	>50
9g	0.001	0.33	330	>10	10000	5.9	50
9h	0.015	0.32	21	>10	670	20	>50
9i	0.14	0.87	6.2	>10	71	>50	>50
9j	0.001	nd	nd	nd	nd	7.7	>50
9k	0.006	0.07	12	4.3	720	11.5	>50
9l	0.004	0.12	30	1.3	330	8.2	>50
9m	0.32	0.94	2.9	>10	31	>50	>50
9n	0.002	0.07	35	1.6	800	6.4	34
9o	0.001	nd	nd	>4.8	4800	6.7	45
9p	0.006	0.14	23	4.7	780	12	>50
9q	0.008	0.08	10	>10	1300	8.9	38
9r	0.034	0.3	8.8	>10	290	43	>50
9s	0.001	0.1	100	4.8	4800	7.3	>50
9t	0.001	nd	nd	>10	10000	6.6	>50
9u	0.33	0.47	1.4	>10	30	>50	>50
9v	0.32	0.66	2.1	>10	31	>50	>50
9w	0.054	0.63	12	>10	190	>50	>50
9x	0.002	<0.005	<2.5	0.19	95	0.5	31
8	>50	>10		>10		>50	>50
7	0.52	4.5	8.7	>10	19	>50	>50

^aData represents the average of two or more experiments.

^bConcentration required to inhibit the growth of T. b. brucei in culture by 50% over 72 h.

^cConcentration required to inhibit the growth of MRC5 cells in culture by 50% over 72 h.

The predicted binding mode of 9g in TbGSK3 offers an explanation for the observed selectivity (Figure 6). In the highest scoring docking pose, the core scaffold adopts a similar binding mode as observed for AT7519 in HsCDK2 (Figure 1).¹⁹ In addition, the docking results suggested that the hydrophobic pocket II of TbGSK3 was occupied by the N-benzylamide group of 9g in such a way that its phenyl moiety formed T-shaped edge-to-face interactions with the side chain of Phe103 and hydrophobic interactions with Leu36 and Ala26. In hGSK3β, Phe103 is replaced with a Tyr (Table 1, Figure 3A), resulting in steric clash and electrostatic repulsion toward the benzyl moiety of 9g. Further, Leu36 is substituted with Gln72 in hGSK3β and Lys20 in HsCDK2, diminishing hydrophobic interactions between the benzyl moiety of 9g and these residues. Overall, these changes together with differences in the gatekeeper region of HsCDK2 (see above) are likely to be responsible for the high selectivity of 9g for TbGSK3 over hGSK3 and HsCDK2. Similar observations regarding the R² group placement and selectivity were also made for compound 9h supporting this model.

Figure 6.

Proposed binding mode of 9g in the homology model of TbGSK3 (blue carbon atoms) overlaid on the HsGSK3β crystal structure (pink carbon atoms). Both ligand and protein are represented as sticks and color coded by atom types. Ligand carbon atoms are shown in gray, protein carbon atoms of TbGSK3 are shown in blue, and HsGSK3β carbon atoms in salmon. Amino acid residue labels are for TbGSK3. Hydrogen bonds and hydrophobic interactions are shown as black dotted lines, with interaction distances in angstroms. TbGSK3 amino acids which are involved in hydrophobic interactions with the benzyl group are marked in bold. The gold sphere represents the center of the phenyl ring..

Antiparasitic Activity

The R² substituted compounds were tested against BSF T. b. brucei and MRC5 cells. As for the R¹-substituted analogues, a good correlation between the EC₅₀ and IC₅₀ values and a 100-fold drop in activity between the biochemical and cell assay was observed (Figure 7). Compound 9c had an EC₅₀ for T. b. brucei of 4 μM (Table 3). Limited selectivity (>7-fold) over MRC5 cells was achieved with compounds 9c, 9d, 9g, 9s, and 9t. It was found that the compounds showed selective inhibition of TbGSK3 over HsGSK3 (>20-fold) and HsCDK2 (>1200-fold).

Figure 7.

Correlation between the inhibition of recombinant TbGSK3 and bloodstream from T. b. brucei proliferation using R² substituted aminopyrazole derivatives (9a–x).

Human Kinase Selectivity Profile

PK inhibitors frequently inhibit multiple kinases, often leading to off-target toxic effects. To assess the selectivity of the aminopyrazole inhibitors, remaining activity at 10 μM concentration was measured for compounds 4f, 4m, and 4y against a panel of 80 human PKs and for compound 9g against 124 human PKs. Compounds 4m and 9g were found to be highly specific (Table 4). Compound 4m inhibited only two PKs, namely GSK3β and CDK2, at more than 80%. 9g showed activity against three PKs: GSK3β, MAPKAP-K2, and MINK1 at more than 80%. Compound 4f was found to inhibit seven PKs and compound 4y 15 PKs by greater than 80% at 10 μM.

Table 4. Kinase Profiling against a Panel of Mammalian Kinases^a.

PKs	4f	4m	4y	9g
MKK1	54	83	17	76
ERK2	18	51	2	92
JNK1	31	77	8	71
JNK2	51	82	15	63
ERK8	14	27	7	47
MAPKAP-K2	92	33	90	13
GSK3b	33	0	0	10
CDK2	1	9	1	38
MELK	32	100	13	96
DYRK1A	6	84	1	79
DYRK2	3	63	1	28
DYRK3	24	100	2	80
PIM1	46	100	13	92
PIM3	19	94	1	96
HIPK2	14	100	4	93
IGF-1R	96	100	15	79
MINK1	nd	nd	nd	18

^aNumbers represent average percentage of activity compared to the control at 10 μM. In this table, only kinases with activity values <20% are shown (for full table see Supporting Information). PK activity values <20% are marked in bold.

Discussion

In this work, we exploited the knowledge of the previously described aminopyrazoles inhibitors of HsCDK2 and HsGSK3β²⁰ to identify selective inhibitors of the TbGSK3 short isoform. This kinase has been shown using genetic manipulation studies to be essential for the survival of the T. b. brucei parasite.¹³ However, we wanted to confirm if antiparasitic activity could be gained using selective, small molecule inhibitors of TbGSK3. The ability to selectively inhibit TbGSK3 over HsGSK3 and HsCDK2 is essential to avoid potential side effects. Therefore, more than 50 aminopyrazole derivatives were synthesized and screened against TbGSK3, HsGSK3β, HsCDK2, and proliferating T. b. brucei and human cells in culture.

The results (Table 2 and Table 3) showed that almost all compounds were highly potent TbGSK3 inhibitors. The activity could be rationalized using the homology models and subsequent molecular docking studies (Figure 4 and Figure 6). The aminopyrazole derivatives make three H-bond interactions with the kinase hinge region, driving much of the potency of the compounds against the three kinases studied. Selectivity could be derived from substitution at both R¹ and R² positions.

From a homology perspective, HsCDK2 is the most closely related kinase to HsGSK3β.¹⁶ Although, the enzymes only share approximately 33% amino acid identity, their ATP binding pockets are highly conserved,¹⁶ resulting in the majority of known HsCDK2 inhibitors also potently inhibiting HsGSK3β. The results demonstrated that the required profile could be achieved, with several compounds with high affinity (<18 nM) for TbGSK3 showing high selectivity (>500-fold) over HsCDK2. Docking studies provided a number of important insights into the binding modes and the selectivity profile of aminopyrazole derivatives. First, the docking results suggested that if the phenyl ring of compound 4f is planar with the amide group at R¹ position it cannot bind into the truncated gate keeper region of HsCDK2, defined by Phe80 in HsCDK2, compared to Met101 in TbGSK3. To fit into this region of HsCDK2, the phenyl ring needs to significantly twist out of plane of the amide, with a torsion angle of approximately 60°, resulting in reduced binding affinity (Figure 4). To stabilize this twist, di-ortho-substituents on the R¹ phenyl group are required to cause a steric/electronic clash with the carbonyl of the amide bond. However, this region of the pocket in HsCDK2 is narrow (in a plane perpendicular to the hinge backbone and the pyrazole core), only allowing small ortho-substituents (such as in compound 9g) on the phenyl group. In contrast, the wider gatekeeper regions of hGSK3 and TbGSK3 can tolerate large substituents such as the ortho-dimethoxy groups of compound 4m or 4n and the ortho-phenylaminobenzamide groups of 4k or 4l. Second, the highest increase in selectivity (>10000-fold) over HsCDK2 was achieved by accessing the hydrophobic pocket II. Exploitation of hydrophobic interactions in these two pockets not only reliably increased ligand-binding affinity but also impacted on the selectivity profile of these compounds. On the basis of the biological results of compound 9g and structural modeling studies, we have shown that selectivity over HsGSK3 can be achieved by exploiting the Phe103Tyr, Leu36Gln, and Ala26Ile active site differences in the hydrophobic pocket II of TbGSK3 enzyme (Figure 6). Taken together, the region between the gate keeper residue and the catalytic aspartate of the DFG loop, together with the hydrophobic pocket II, are the key areas to exploit to achieve high selectivity over HsCDK2.

The TbGSK3 short enzyme IC₅₀ values correlated well with the T. b. brucei antiproliferative EC₅₀ activities of the described substituted aminopyrazole inhibitors (Figures 5 and 7), indicating that the compounds act on target. However, a 100-fold drop in cell activity was observed, compared to that in the TbGSK3 assay (1 μM). The calculated physical properties (MW < 473; log P −0.4–3.6; PSA < 130 Å) of the series of compounds suggest this loss of activity was not driven by lack of cellular penetration. In addition, the compound series was observed to be highly chemically stable under the range of synthetic conditions used during the chemistry campaign, suggesting that chemical degradation was not responsible for the loss of activity in the proliferation assay. Although metabolism by the parasite cannot be ruled out, the high degree of correlation between enzyme inhibition and antiparasitic activity suggests this is not the case, as it would be not expected that all compounds be metabolized to a constant extent. Therefore, the drop of activity was probably due to the high ATP concentration (millimolar range) in the cell compared to the kinase assay conditions.²¹ Furthermore, chemical proteomic profiling conducted in parasite cell extracts confirmed that compound 4m binds the endogenous TbGSK3 short with nanomolar affinity and very few other kinase targets with much lower affinity in the micromolar range.²²

Conclusion

In this study, we have developed a series of substituted aminopyrazole amides as TbGSK3 short inhibitors starting from a compound series initially designed by Astex Therapeutics to inhibit HsCDK2 and HsGSK3β. SAR investigation and optimization successfully provided 18 low nanomolar (IC₅₀ <10 nM) inhibitors of TbGSK3 with high selectivity (>10000-fold) over HsCDK2. With compound 9g, we have shown that good (330-fold) selectivity over HsGSK3 can be achieved by targeting the hydrophobic pocket II. Compound 9g is the most selective TbGSK3 inhibitor described to date.^13,23−25 In addition, 9g proved to be highly selective against a panel of 124 human PKs, showing >90% inhibition at 10 μM against only one PK, HsGSK3β. Molecular modeling has also shown that despite overall conservation in sequence and conformation between the three PKs (HsGSK3β, HsCDK2, and TbGSK3), the binding pockets have distinct features that determine their specificity for particular compounds. Further, we have shown that enzymatic inhibition correlates well with cell efficacy over a wide range of concentrations and a representative member of this series binds the endogenous TbGSK3 with nanomolar potency, indicating that compounds definitely act on target.²² However, a general 100-fold drop in activity between target and cellular activities resulted at best in compounds with low micromolar antiparasitic activity. Taken together, this data suggests that specific ATP competitive hinge binders of TbGSK3 short require low picomolar potency to obtain nanomolar antiproliferative activity against T. brucei. This leads us to the conclusion that alternative strategies are required. First, non-ATP competitive approaches to inhibition of TbGSK3, through irreversible hinge binders or allosteric inhibitors, could be pursued. However, these approaches have potential downsides, through the introduction of a reactive functionality or an increased chance of resistance causing mutations, respectively. Second, a polypharmacology approach through the inhibition of a number of essential T. brucei kinases in addition to TbGSK3 could be investigated, although obtaining selectivity over human kinases would be more problematical. However, the aminopyrazole compounds (4a–4z and 9a–9x) reported here represent an excellent start for chemistry optimization of selective TbGSK3 short inhibitors and an outstanding probe for studying the physiological functions of TbGSK3 short in T. brucei parasites.

Experimental Section

Molecular Modeling

Homology Modeling

Sequence alignments between T. brucei and HsGSK3β were generated using ClustalW.²⁶ Subsequently, Modeler 9.2²⁷ was used to build homology models of TbGSK3 short, whereas the HsGSK3β crystal structure (PDB code 1r0e) served as template. Modeler was run with default settings, and only the highest-scoring structure was used for further analysis and modeling.

Ligand Docking

FlexX 2.0.1 (BioSolveIT GmbH) was used to dock ligands flexible into protein binding sites.²⁸ The active sites were defined as the areas within 7 Å of the co-crystallized ligands of HsCDK2 (PDB code 2vu3)¹⁹ and HsGSK3β (PDB code 1r0e)²⁹ or the equivalent residues in the homology model of TbGSK3. In all three structures, protonation states of amino acids and the orientations of the protons of hydroxyl and amine groups of active-site residues were manually assigned using the FlexX GUI. A highly conserved water molecule (H₂O 82 in 1r0e or H₂O 2134 in 2vu3) was kept in all three protein structures used for docking. Docking was carried out using default settings, and only the highest scoring binding modes were visually analyzed.

All figures of protein binding sites were prepared using PyMol.³⁰

Potency Screen Assays

For compound potency determinations, a radiometric 96-well Flashplate assay (PerkinElmer) was adopted. Compounds were solubilized in DMSO at a top concentration of 3 mM and serially diluted to achieve 10-point titration of final assay concentrations from 30 μM to 0.3 nM with a final DMSO concentration of 1% (v/v). The reaction mixtures contained 1 μM biotinylated GSP2 substrate, 1 μM ATP, 3.7 KBq/well [γ-33P]-ATP and 2.5 nM TbGSK3 in the TbGSK3 kinase assay buffer. GSK3 inhibitors were screened for selectivity assessment also against HsGSK3β. For HsGSK3 assay, the reaction mixes contained 1 μM biotinylated GSP2 substrate, 2 μM ATP, 7.4 KBq/well [γ-33P]-ATP and 15 nM HsGSK3β in the TbGSK3 kinase assay buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 5 mM DTT, 0.02% CHAPS, 2 U/mL heparin). For HsCDK2/cyclin A assay, the reaction mixtures contained 1 mM CDK5 biotinylated peptide substrate (Biotin-C₆-PKTPKKAKKL), 1 μM ATP, 7.4 KBq/well [γ-³³P]-ATP and 2 nM HsCDK2/cyclin A in the kinase assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 2 mM DTT, 100 mM NaCl, 0.2 mM EGTA, 0.02% (v/v) Brij35).

Statistical Evaluation of Assay Reproducibility

The statistical significance of the compound potency (IC₅₀) was based on the performance of standard molecules which have been tested to a high replication. In the case of TbGSK3 short assay, the standard compound GW8510 was tested 93 times across 9 independent runs. The average pIC₅₀ value was 8.26 with a SD (standard deviation) of 0.23. The minimum significant ratio (MSR) of 0.4 was evaluated considering the following formula:

where SD is the standard deviation and N the number of replicate values routinely used for the assay (2 in our case).³¹ This implies that a difference of >0.4 in pIC₅₀ can be considered statistically significant for this assay.

In the case of HsGSK3 assay, the standard compound GW8510 was tested 47 times across 5 independent runs, with an average pIC₅₀ value of 8.10 and a SD of 0.21. This implies that a difference of >0.3 in pIC₅₀ can be considered statistically significant for this assay.

For the HsCDK2 assay, the analysis was performed using two different standards (GW8510 and staurosporine) tested respectively five times in a single run and 19 times in 2 independent runs. This implies that a difference of >0.3 in pIC₅₀ can be considered statistically significant for this assay.

Mammalian Kinase Profiling

Selected compounds were screened against a panel of mammalian kinases routinely run by the Division of Signal Transduction Therapy (DSTT) at the University of Dundee in duplicate at 10 μM.³² Enzymes included in the panel and assay conditions are reported in the literature. All biochemical assays are run below the K_m^app for the ATP for each enzyme, allowing comparison of inhibition across the panel.

Trypanosome and MRC5 Proliferation Assay

Measurement of inhibition of the proliferation of MRC5 (human lung fibroblast) cells and T. b. brucei bloodstream stage cells was performed using a modification of the cell viability assay previously described.³³ Compounds (50 μM to 0.5 nM) were incubated with 2 × 10³ cells/well in 0.2 mL of the appropriate culture medium (MEM with 10% fetal bovine serum for MRC5 cells) in clear 96-well plates. Plates were incubated at 37 °C in the presence of 5% CO₂ for 69 h. Resazurin was then added to a final concentration of 50 μM, and plates were incubated as above for a further 4 h before being read on a BioTek flx800 fluorescent plate reader.

Chemistry

General Experimental Details

¹H and ¹³C NMR spectra were recorded on either a Bruker Avance DPX 300 or 500 MHz spectrometer. Chemical shifts (δ) are expressed in parts per million (ppm) and coupling constants (J) are in hertz (Hz). Signal splitting patterns are described as singlet (s), broad singlet (br s), doublet (d), triplet (t), quartet (q), quintuplet (quin), sextuplet (sex), septet (sept), multiplet (m), or combinations thereof. LCMS (liquid chromatography mass spectrometry) analyses were performed with either an Agilent HPLC 1100 series connected to a Bruker Daltonics MicrOTOF or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LCMS, and both instruments were connected to an Agilent diode array detector. LCMS chromatographic separations were conducted with a Phenomenex Gemini C18 column, 50 mm × 3.0 mm, 5 μm particle size; mobile phase/acetonitrile +0.1% HCOOH 80:20 to 5:95 over 3.5 min, and then held for 1.5 min; flow rate 0.5 mL min^–1. High resolution electrospray measurements (HRMS) were performed with a Bruker Daltonics MicrOTOF mass spectrometer. Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 plates using UV light and/or KMnO₄ for visualization. Column chromatography was performed using RediSep 4 or 12 g silica prepacked columns. When applicable, all glassware was oven-dried overnight and all reactions were carried out under dry and inert conditions (Argon atmosphere).

All in this work synthesized compounds had a measured purity of greater than 95% (measured on analytical HPLC-MS system). M⁺ data are given below to substantiate the purity and integrity of the compounds. ¹H NMR, ¹³C NMR, and HRMS experiments were also used to confirm compound identity and purity.

N-((1r,4r)-4-Methoxycyclohexyl)-4-nitro-1H-pyrazole-3-carboxamide (2)

A mixture of 4-nitro-3-pyrazolecarboxylic acid (1) (2.33 g, 14.8 mmol), trans-4-methoxy-cyclohexylamine (2.39 g, 18.5 mmol), EDC (3.55 g, 18.5 mmol), and HOBt (2.50 g, 18.5 mmol) in DMF (75 mL) was stirred at ambient temperature for 16 h. The mixture was reduced in vacuo and partitioned between saturated aqueous sodium bicarbonate and EtOAc. The organic layer was washed (water, brine), dried (MgSO₄), and reduced in vacuo to give a yellow oil, which was purified by column chromatography, eluting 0–100% EtOAc in petroleum ether to give 2. Yield: 3.12 g (solid), 62%. ¹H NMR (DMSO-d₆ DMSO-d₆) δ (ppm) 14.02 (s, 1H), 8.73 (s, 1H), 8.57 (d, J = 7.81 Hz, 1H), 3.74 (m, 1H), 3.24 (s, 3H), 3.11 (m, 1H), 1.95 (dd, J = 55.8, 10.9 Hz, 4H), 1.27 (m, 4H). ¹³C NMR (DMSO-d₆) δ (ppm) 159.04, 141.49, 132.17, 131.44, 77.40, 55.01, 47.58, 29.67, 29.30. LRMS (ES⁺): m/z 269 [M + H]⁺.

4-Amino-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (3)

A solution of 2 (1.13 g, 4.2 mmol) in DMF (100 mL) was treated with 10% palladium on carbon then shaken under hydrogen at room temperature and atmospheric pressure for 5 h. The reaction mixture was diluted with EtOAc, filtered through Celite, washing with further EtOAc, and the filtrate reduced in vacuo to give crude 3 as brown oil. Yield: 982 mg, 98%. ¹H NMR (CD₃OD) δ (ppm) 7.23 (s, 1H), 3.84 (m, 1H), 3.37 (s, 3H), 3.23 (m, 1H), 2.07 (dd, J = 45.8, 11.2 Hz, 4H), 1.38 (m, 4H). ¹³C NMR (MeOD-d₄) δ (ppm) 165.58, 134.15, 133.13, 118.22, 79.72, 56.15, 48.58, 31.37. LRMS (ES⁺): m/z 239 [M + H]⁺.

General Method for Variation of Substituent R¹: Example 4-Benzamido-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4f)

A mixture of benzoic acid (0.051 g, 0.42 mmol), 3 (0.1 g, 0.42 mmol), EDC (0.096 g, 0.5 mmol), and HOBt (0.068 g, 0.5 mmol) in DMF (10 mL) was stirred at ambient temperature for 16 h. The mixture was reduced in vacuo and partitioned between saturated aqueous sodium bicarbonate and EtOAc. The organic layer was washed (water, brine), dried (MgSO₄), and reduced in vacuo to give a creamy solid 4f, which was purified by column chromatography. Evaporation of the appropriate fraction yielded the desired compound as an amorphous solid. Yield: 39 mg, 27%. ¹H NMR (CDCl₃) δ (ppm) 10.65 (s, 1H), 8.52 (s, 1H), 8.01 (d, J = 7.2 Hz, 2H), 7.57 (t, J = 7.2 Hz, 1H), 7.51 (m, 2H), 6.86 (d, J = 8.3 Hz, 1H), 4.01 (m, 1H), 3.39 (s, 3H), 3.22 (m, 1H), 2.16 (m, 4H), 1.42 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 164.36, 163.21, 133.54, 133.28, 132.03, 128.83, 127.24, 123.74, 120.82, 78.13, 55.91, 47.48, 30.69, 30.11. LRMS (ES⁺): m/z 343 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₃N₄O₃ [M + H]⁺ 343.1765, found 343.1751.

4-(4,4-Difluorocyclohexanecarboxamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4a)

Yield: 90 mg (solid) 56%. ¹H NMR (CDCl₃) δ (ppm) 9.86 (s, 1H), 8.35 (s, 1H), 6.83 (d, J = 8.3 Hz, 1H), 3.95 (m, 1H), 3.39 (s, 3H), 3.21 (m, 1H), 2.44 (m, 1H), 2.22 (m, 2H), 2.11 (m, 6H), 1.94 (m, 2H), 1.81 (m, 2H), 1.40 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 171.61, 163.21, 133.28, 123.22, 122.55 (t, J = 239.4 Hz), 120.71, 78.07, 55.91, 47.50, 42.74, 32.85 (t, J = 23.5 Hz), 30.35, 25.73. LRMS (ES⁺): m/z 385 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₇F₂N₄O₃ [M + H]⁺ 385.2046, found 385.2036.

N-(4-Methoxycyclohexyl)-4-(tetrahydro-2H-pyran-4-carboxamido)-1H-pyrazole-3-carboxamide (4b)

Yield: 94 mg (solid), 64%. ¹H NMR (CDCl₃) δ (ppm) 9.86 (s, 1H), 8.34 (s, 1H), 6.81 (d, J = 8.1 Hz, 1H), 4.07 (m, 2H), 3.95 (m, 1H), 3.47 (m, 2H), 3.39 (s, 3H), 3.20 (m, 1H), 2.59 (m, 1H), 2.13 (d, J = 11.0 Hz, 4H), 1.91 (m, 4H), 1.40 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 171.97, 163.30, 133.22, 123.23, 120.71, 78.12, 67.26, 55.92, 47.50, 42.20, 30.64, 30.12, 29.10. LRMS (ES⁺): m/z 351 [M + H]⁺. HRMS (ES⁺): calcd for C₁₇H₂₇N₄O₄ [M + H]⁺ 351.2027, found 351.2011.

4-(2-(2-Fluorophenyl)acetamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4c)

Yield: 56 mg (solid), 71%. ¹H NMR (CD₃OD) δ (ppm) 8.20 (s, 1H), 7.38 (t, 7.7 Hz, 1H), 7.34–7.26 (m, 1H), 7.18–7.05 (m, 2H), 3.83 (m, 1H), 3.80 (s, 2H), 3.33 (s, 3H), 3.16 (m, 1H), 2.06 (br d, J = 11.5 Hz, 2H), 1.97 (br d, J = 12.0 Hz, 2H), 1.34 (m, 4H). ¹³C NMR (CD₃OD) δ (ppm) 169.57, 164.14 (d, J = 159.5 Hz), 134.23, 132.98, 130.64 (d, J = 10.9 Hz), 125.75, 123.77, 123.20 (d, J = 18.2 Hz), 121.96, 116.48 (d, J = 21.7 Hz), 79.69, 56.14, 48.68, 37.61, 31.41, 31.27. LRMS (ES⁺): m/z 375 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₄FN₄O₃ [M + H]⁺ 375.1827, found 375.1817.

N-(4-Methoxycyclohexyl)-4-(2-(2-methoxyphenyl)acetamido)-1H-pyrazole-3-carboxamide (4d)

Yield: 43 mg (solid), 53%. ¹H NMR (DMSO-d₆) δ (ppm) 13.13 (s, 1H), 9.76 (s, 1H), 8.15 (s, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.31–7.23 (m, 2H), 7.02 (d, J = 8.0 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 3.82 (s, 3H), 3.79–3.70 (m, 1H), 3.24 (s, 3H), 3.07 (m, 1H), 2.01 (d, J = 11.4 Hz, 2H), 1.79 (d, J = 11.4 Hz, 2H), 1.45 (m, 2H), 1.21 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 167.30, 162.43, 157.02, 132.38, 130.90, 128.59, 123.21, 122.30, 120.44, 119.70, 110.88, 77.70, 55.41, 55.04, 46.70, 38.17, 30.26, 29.73. LRMS(ES⁺): m/z 387 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₇N₄O₄ [M + H]⁺ 387.2027, found 387.2028.

4-(2-(2,6-Dichlorophenyl)acetamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4e)

Yield: 63 mg (solid), 81%. ¹H NMR (DMSO-d₆) δ (ppm) 13.18 (br s, 1H), 9.86 (s, 1H), 8.14 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 8.2 Hz, 2H), 7.36 (t, J = 8.2 Hz, 1H), 4.10 (s, 2H), 3.76 (m, 1H), 3.24 (s, 3H), 3.08 (s, 1H), 2.01 (m, 2H), 1.81 (m, 2H), 1.45 (m, 2H), 1.19 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 170.16, 162.31, 135.49, 131.62, 129.68, 128.30, 128.10, 122.23, 122.09, 77.68, 55.04, 46.77, 38.23, 30.25, 29.74. LRMS (ES⁺): m/z 425 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₃Cl₂N₄O₃ [M + H]⁺ 425.1142, found 425.1147.

4-(2-Fluorobenzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4g)

Yield: 97 mg (solid), 64%. ¹H NMR (CDCl₃) δ (ppm) 10.89 (d, J = 12.2 Hz, 1H), 8.45 (s, 1H), 8.07 (m, 1H), 7.44 (m, 1H), 7.22 (m, 1H), 7.13 (m, 1H), 6.73 (d, J = 8.3 Hz, 1H), 3.96 (m, 1H), 3.29 (s, 3H), 3.11 (m, 1H), 2.05 (m, 4H), 1.31 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 162.80, 160.84 (d, J = 246.9 Hz), 160.53, 133.81 (d, J = 10.9 Hz), 131.77, 124.79, 123.19, 121.42, 120.65 (d, J = 10.7 Hz), 116.46 (d, J = 21.8 Hz), 78.19, 55.88, 47.27, 30.75, 30.14. LRMS (ES⁺): m/z 361 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₂FN₄O₃ [M + H]⁺ 361.1670, found 361.1645.

N-(4-Methoxycyclohexyl)-4-(2-(trifluoromethyl)benzamido)-1H-pyrazole-3-carboxamide (4h)

Yield: 67 mg (solid), 78%. ¹H NMR (DMSO-d₆) δ (ppm) 13.40 (s, 1H), 10.22 (s, 1H), 8.32 (s, 1H), 8.29 (d, J = 8.6 Hz, 1H), 7.90–7.88 (m, 1H), 7.84–7.81 (m, 1H), 7.78–7.75 (m, 2H), 3.72 (m, 1H), 3.23 (s, 3H), 3.06 (m, 1H), 2.00 (br d, J = 12.5 Hz, 2H), 1.76 (br d, J = 12.5 Hz, 2H), 1.45 (m, 2H), 1.14 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 163.34, 162.73, 135.06, 133.01, 132.78, 130.73, 128.39, 126.63 (d, J = 5.2 Hz) 126.04 (d, J = 29.1 Hz), 123.57 (d, J = 276.3 Hz), 122.06, 120.30, 77.66, 55.06, 46.97, 30.28, 29.67. LRMS (ES⁺): m/z 411 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₂F₃N₄O₃ [M + H]⁺ 411.1639, found 411.1621.

4-(2-Ethylbenzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4i)

Yield: 63 mg (solid), 81%. ¹H NMR (CDCl₃) δ (ppm) 10.04 (s, 1H), 8.39 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.30 (d, 7.7 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 3.92 (m, 1H), 3.36 (s, 3H), 3.16 (m, 1H), 2.90 (q, J = 7.6 Hz, 2H), 2.08 (br d, J = 10.0 Hz, 4H), 1.35 (m, 4H), 1.26 (t, J = 7.6 Hz, 3H). ¹³C NMR (CDCl₃) δ (ppm) 167.60, 163.29, 142.86, 134.89, 133.19, 130.65, 129.69, 127.15, 126.11, 123.25, 120.94, 78.18, 55.86, 74.45, 30.58, 30.12, 26.44, 15.84. LRMS (ES⁺): m/z 371 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₇N₄O₃ [M + H]⁺ 371.2078, found 371.2078.

4-(2-Methoxybenzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4j)

Yield: 35 mg (solid), 45%. ¹H NMR: (DMSO-d₆) δ (ppm) 13.25 (s, 1H), 11.77 (s, 1H), 8.44 (s, 1H), 8.15 (s, 1H), 8.04 (s, 1H), 7.63 (s, 1H), 7.29 (s, 1H), 7.18 (s, 1H), 4.15 (s, 3H), 3.88 (m, 1H), 3.30 (s, 3H), 3.16 (m, 1H), 2.08 (m, 2H), 1.91 (m, 2H), 1.55 (m, 2H), 1.29 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.43, 160.94, 157.47, 133.49, 131.33, 122.29, 120.83, 120.59, 120.28, 112.32, 77.73, 56.05, 55.05, 46.72, 30.28, 29.83. LRMS (ES⁺): m/z 373 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₅N₄O₄ [M + H]⁺ 373.1870, found 373.1873.

N-(4-Methoxycyclohexyl)-4-(2-(phenylamino)benzamido)-1H-pyrazole-3-carboxamide (4k)

Yield: 41 mg (solid), 45%. ¹H NMR (DMSO-d₆) δ (ppm) 13.32 (br s, 1H), 10.93 (s, 1H), 9.53 (s, 1H), 8.31 (s, 1H), 8.28 (d, J = 8.5 Hz, 1H), 7.68 (dd, J = 8.0, 1.4 Hz, 1H), 7.43 (m, 1H), 7.33–7.28 (m, 3H), 7.16–7.14 (m, 2H), 7.01–6.96 (m, 2H), 3.82 (m, 1H), 3.35 (s, 1H), 3.25 (s, 3H), 3.09 (m, 1H), 2.03 (br d, J = 12.0 Hz, 2H), 1.81 (br d, J = 12.0 Hz, 2H), 1.48 (m, 2H), 1.20 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 164.84, 162.97, 144.57, 141.62, 132.81, 129.33, 128.04, 122.32, 122.00, 120.01, 119.63, 119.26, 118.30, 116.49, 77.74, 55.06, 46.95, 30.33, 29.73. LRMS (ES⁺): m/z 434 [M + H]⁺. HRMS (ES⁺): calcd for C₂₄H₂₈N₅O₃ [M + H]⁺ 434.2187, found 434.2165.

4-(2-((2,3-Dimethylphenyl)amino)benzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4l)

Yield: 53 mg (solid), 55%. ¹H NMR (DMSO-d₆) δ (ppm) 13.35 (br s, 1H), 10.89 (s, 1H), 9.53 (s, 1H), 8.33 (s, 1H), 7.63 (dd, J = 8.1, 1.4 Hz, 1H), 7.34 (m, 1H), 7.11–7.09 (m, 2H), 6.99–6.97 (m, 1H), 6.87 (m, 1H), 6.82 (dd, J = 8.4, 1.0 Hz, 1H), 3.84 (m, 1H), 3.35 (s, 1H), 3.25 (s, 3H), 3.09 (m, 1H), 2.29 (s, 3H), 2.14 (s, 3H), 2.03 (br d, J = 12.0 Hz, 2H), 1.82 (br d, J = 12.0 Hz, 2H), 1.49 (m, 2H), 1.21 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 165.36, 163.08, 147.05, 138.87, 137.74, 132.95, 132.72, 130.25, 127.52, 125.96, 125.74, 122.41, 120.79, 119.98, 117.44, 115.36, 114.63, 77.74, 55.06, 46.95, 30.33, 29.75, 20.25, 13.64. LRMS (ES⁺): m/z 462 [M + H]⁺. HRMS (ES⁺): calcd for C₂₆H₃₂N₅O₃ [M + H]⁺ 462.2500, found 462.2503.

4-(2,6-Dimethoxybenzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4m)

Yield: 73 mg, 43% (solid). ¹H NMR (DMSO-d₆) δ (ppm) 13.28 (s, 1H), 9.75 (s, 1H), 8.29 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 8.5 Hz, 1H), 6.75 (d, J = 8.5 Hz, 2H), 3.76 (s, 6H), 3.70 (m, 1H), 3.22 (s, 3H), 3.06 (m, 1H), 2.00 (m, 2H), 1.76 (m, 2H), 1.45 (m, 2H), 1.13 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.79, 161.21, 156.88, 132.21, 131.08, 122.52, 119.81, 115.03, 104.28, 77.66, 55.81, 55.08, 46.93, 30.29, 29.66. LRMS (ES⁺): m/z 403 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₇N₄O₅ [M + H]⁺ 403.1976, found 403.1960.

N-(4-Methoxycyclohexyl)-4-(2,4,6-trimethoxybenzamido)-1H-pyrazole-3-carboxamide (4n)

Yield: 76 mg (solid), 84%. ¹H NMR (CD₃OD) δ (ppm) 8.36 (s, 1H), 6.17 (s, 2H), 3.84 (s, 3H), 3.82 (s, 6H), 3.35 (s, 3H), 3.22 (m, 1H), 2.08 (m, 4H), 1.36 (m, 4H). ¹³C NMR (CD₃OD) δ (ppm) 164.90, 164.72, 164.35, 160.74, 134.07, 124.36, 122.58, 108.39, 92.09, 79.74, 57.05, 56.89, 56.62, 48.71, 31.61, 31.40. LRMS (ES⁺): m/z 433 [M + H]⁺. HRMS (ES⁺): calcd for C₂₁H₂₉N₄O₆ [M + H]⁺ 433.2082, found 433.2065.

4-(2,4-Difluorobenzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4o)

Yield: 114 mg (solid), 72%. ¹H NMR (DMSO-d₆) δ (ppm) 13.24 (s, 1H), 10.89 (d, J = 10.2 Hz, 1H), 8.33 (s, 1H), 8.09 (q, J = 8.7 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.39 (m, 1H), 7.24 (m, 1H), 3.82 (m, 1H), 3.25 (s, 3H), 3.10 (m, 1H), 2.02 (d, J = 10.3 Hz, 2H), 1.84 (d, J = 11.6 Hz, 2H), 1.47 (q, J = 12.0 Hz, 2H), 1.22 (q, J = 12.5 Hz, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 164.3 (q, J = 250.6, 10.9 Hz), 162.71, 160.3 (q, J = 250.8, 14.5 Hz), 158.03, 133.26, 133.18, 132.82, 122.12, 120.50, 117.39, 117.31, 112.59, 112.42, 104.96, 104.75, 104.52, 79.12, 78.84, 78.58, 77.71, 55.07, 46.89, 30.09, 29.76;. LRMS (ES⁺): m/z 379 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₁F₂N₄O₃ [M + H]⁺ 379.1576, found 379.1567.

4-(3,5-Difluorobenzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4p)

Yield: 157 mg (solid), 99%. ¹H NMR (DMSO-d₆) δ (ppm) 13.39 (s, 1H), 10.77 (s, 1H), 8.34 (d, J = 8.5 Hz, 1H), 8.30 (s, 1H), 7.59 (m, 1H), 7.52 (m, 2H), 3.84 (m, 1H), 3.24 (s, 3H), 3.09 (m, 1H), 2.02 (d, J = 10.7 Hz, 2H), 1.81 (d, J = 10.7 Hz, 2H), 1.49 (q, J = 11.8 Hz, 2H), 1.20 (q, J = 11.8 Hz, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.8, 162.5(q, J = 250.7, 14.4 Hz), 160.3, 137.16 127.75, 126.59, 124.12, 122.13, 118.94, 110.2 (q, J = 22.0, 14.5 Hz), 107.5 (t, J = 27.0 Hz), 77.71, 55.06, 46.99, 30.29, 29.76. LRMS (ES⁺): m/z 379 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₁F₂N₄O₃ [M + H]⁺ 379.1576, found 379.1568.

4-(3,5-Dichlorobenzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4q)

Yield: 147 mg (solid), 85%. ¹H NMR (CDCl₃) δ (ppm) 10.34 (s, 1H), 8.23 (s, 1H), 7.60 (d, J = 1.9 Hz, 2H), 7.31 (t, J = 1.8 Hz, 1H), 7.05 (s, 1H), 6.58 (d, 1H), 3.77 (m, 1H), 3.15 (s, 3H), 2.98 (m, 1H), 1.91 (m, 4H), 1.18 (m, 4H). ¹³C NMR (DMSO-d₆) δ (ppm) 163.12, 161.74, 136.46, 135.73, 131.89, 128.34, 126.03, 125.79, 121.09, 78.10, 55.91, 47.48, 30.69, 30.08. LRMS (ES⁺): m/z 411 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₁Cl₂N₄O₃ [M + H]⁺ 411.0985, found 411.0966.

4-(4-(Difluoromethoxy)benzamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4r)

Yield: 96 mg (solid), 56%. ¹H NMR (CDCl₃) δ (ppm) 10.65 (s, 1H), 8.48 (s, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.3 Hz, 1H), 6.62 (t, J = 73.2 Hz, 1H), 4.00 (m, 1H), 3.39 (s, 3H), 3.22 (m, 1H), 2.15 (m, 4H), 1.42 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 163.28, 163.20, 154.0 (t, J = 2.9 Hz), 133.56, 130.28, 129.24, 123.64, 120.74, 119.22, 118.94, 115.47, 112.01, 78.11, 55.94, 47.53, 30.67, 30.12. LRMS (ES⁺): m/z 409 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₃F₂N₄O₄ [M + H]⁺ 409.1682, found 409.1649.

N-(4-Methoxycyclohexyl)-4-(4-(pyrrolidin-1-yl)benzamido)-1H-pyrazole-3-carboxamide (4s)

Yield: 73 mg (solid), 42%. ¹H NMR (CDCl₃) δ (ppm) 10.44 (s, 1H), 8.49 (s, 1H), 7.89 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.3 Hz, 1H), 6.62 (d, J = 8.8 Hz, 2H), 4.01 (m, 1H), 3.39 (m, 7H), 3.20 (m, 1H), 2.15 (m, 4H), 2.06 (m, 4H), 1.41 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 164.74, 163.48, 150.26, 133.26, 128.99, 124.26, 120.52, 119.42, 111.20, 78.20, 55.92, 47.66, 47.39, 30.73, 30.18, 25.46. LRMS (ES⁺): m/z 412 [M + H]⁺. HRMS (ES⁺): calcd for C₂₂H₃₀N₅O₃ [M + H]⁺ 412.2343, found 412.2338.

N-(4-Methoxycyclohexyl)-4-(4-(4-methylpiperazin-1-yl)benzamido)-1H-pyrazole-3-carboxamide (4t)

Yield: 37 mg (solid), 20%. ¹H NMR (CDCl₃) δ (ppm) 10.48 (s, 1H), 8.47 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 6.83 (m, 1H), 4.00 (m, 1H), 3.39 (s, 3H), 3.37 (t, J = 5 Hz, 4H), 3.20 (m, 1H), 2.60 (t, J = 5 Hz, 4H), 2.38 (s, 3H), 2.14 (m, 4H), 1.41 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 164.17, 163.44, 153.54, 133.33, 128.81, 124.05, 122.92, 120.61, 114.27, 78.17, 55.92, 54.79, 47.62, 47.41, 46.11, 30.72, 30.17. LRMS (ES⁺): m/z 441 [M + H]⁺. HRMS (ES⁺): calcd for C₂₃H₃₃N₆O₃ [M + H]⁺ 441.2609, found 441.2597.

4-(Benzofuran-2-carboxamido)-N-(4-methoxycyclohexyl)-1H-pyrazole-3-carboxamide (4u)

Yield: 134 mg (solid), 83%. ¹H NMR (CDCl₃) δ (ppm) 10.76 (s, 1H), 8.53 (s, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 0.9 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 4.07 (m, 1H), 3.40 (s, 3H), 3.22 (m, 1H), 2.16 (m, 4H), 1.43 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 163.03, 156.21, 155.22, 148.18, 133.76, 129.23, 127.56, 127.24, 123.77, 122.59, 121.20, 112.41, 111.23, 78.15, 55.91, 47.41, 30.74, 30.14. LRMS (ES⁺): m/z 383 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₃N₄O₄ [M + H]⁺ 383.1714, found 383.1703.

N-(3-((4-Methoxycyclohexyl)carbamoyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-2-carboxamide (4v)

Yield: 115 mg (solid), 72%. ¹H NMR (DMSO-d₆) δ (ppm) 13.30 (s, 1H), 11.01 (s, 1H), 8.83 (d, J = 7.0 Hz, 1H), 8.37 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.09 (t, J = 6.9 Hz, 1H), 3.85 (m, 1H), 3.26 (s, 3H), 3.11 (m, 1H), 2.04 (d, J = 10.3 Hz, 2H), 1.84 (d, J = 10.7 Hz, 2H), 1.49 (m, 2H), 1.23 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.70, 158.15, 146.85, 141.15, 132.80, 128.99, 124.60, 122.00, 120.08, 119.14, 114.53, 97.79, 77.74, 55.06, 46.95, 30.33, 29.79. LRMS (ES⁺): m/z 383 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₃N₆O₃ [M + H]⁺ 383.1826, found 383.1812.

N-(3-((4-Methoxycyclohexyl)carbamoyl)-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine-3-carboxamide (4w)

Yield: 11 mg (solid), 7%. ¹H NMR (MeOD-d₄) δ (ppm) 9.57 (d, J = 6.9 Hz, 1H), 8.31 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 9.0 Hz, 1H), 7.57 (td, J = 6.9, 1.2 Hz, 1H), 7.19 (t, J = 6.9, 1.2 Hz, 1H), 3.94 (m, 1H), 3.39 (s, 3H), 3.27 (m, 1H), 2.12 (m, 4H), 1.50 (m, 2H), 1.39 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.89, 156.52, 147.37, 136.35, 127.61, 127.41, 122.12, 119.89, 117.57, 117.46, 114.43, 77.71, 55.07, 46.90, 30.30, 29.76. LRMS (ES⁺): m/z 383 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₃N₆O₃ [M + H]⁺ 383.1826, found 383.1811.

N-(3-((4-Methoxycyclohexyl)carbamoyl)-1H-pyrazol-4-yl)-2-methylimidazo[1,2-a]pyridine-3-carboxamide (4x)

Yield: 47 mg (solid), 28%. ¹H NMR (CDCl₃) δ (ppm) 10.88 (s, 1H), 10.24 (s, 1H), 9.58 (d, J = 6.9 Hz, 1H), 8.51 (s, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 6.99 (t, J = 6.9 Hz, 1H), 6.80 (d, J = 7.9 Hz, 1H), 4.01 (m, 1H), 3.39 (s, 3H), 3.21 (m, 1H), 3.02 (s, 3H), 2.14 (m, 4H), 1.41 (m, 4H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.73, 157.27, 146.18, 145.49, 127.61, 127.42, 122.35, 120.04, 116.31, 114.46, 113.62, 77.74, 55.06, 46.77, 30.24, 29.81, 16.52. LRMS (ES⁺): m/z 397 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₅N₆O₃ [M + H]⁺ 397.1983, found 397.1972.

N-(4-Methoxycyclohexyl)-4-(5-phenylfuran-2-carboxamido)-1H-pyrazole-3-carboxamide (4y)

Yield: 107 mg (solid), 62%. ¹H NMR (CDCl₃): δ (ppm) 10.61 (s, 1H), 8.49 (s, 1H), 7.84 (m, 2H), 7.48 (t, J = 7.5 Hz, 2H), 7.38 (t, J = 7.4 Hz, 1H), 7.31 (t, J = 3.6 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 3.6 Hz, 1H), 4.07 (m, 1H), 3.40 (s, 3H), 3.22 (m, 1H), 2.16 (m, 4H), 1.42 (m, 4H). ¹³C NMR (CDCl₃) δ (ppm) 163.03, 156.39, 155.72, 146.33, 133.74, 129.49, 128.94, 128.80, 124.72, 122.85, 120.91, 117.13, 107.30, 78.18, 55.88, 47.25, 30.78, 30.14. LRMS (ES⁺): m/z 409 [M + H]⁺. HRMS (ES⁺): calcd for C₂₂H₂₅N₄O₄ [M + H]⁺ 409.1870, found 409.1832.

N-(3-((4-Methoxycyclohexyl)carbamoyl)-1H-pyrazol-4-yl)-5-phenyloxazole-4-carboxamide (4z)

Yield: 71 mg (solid), 41%. ¹H NMR (DMSO-d₆) δ (ppm) 13.30 (s, 1H), 11.20 (s, 1H), 8.68 (s, 1H), 8.38 (s, 1H), 8.30 (m, 2H), 8.14 (d, J = 8.4 Hz, 1H), 7.58–7.51 (m, 3H), 3.83 (m, 1H), 3.26 (s, 3H), 3.11 (m, 1H), 2.04 (d, J = 10.5 Hz, 2H), 1.84 (d, J = 11.0 Hz, 2H), 1.49 (q, J = 12.5 Hz, 2H), 1.23 (q, J = 11.5 Hz, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.64, 157.34, 151.99, 150.43, 132.90, 130.23, 128.50, 128.07, 127.92, 126.62, 121.89, 120.02, 77.74, 55.06, 47.00, 30.33, 29.76. LRMS (ES⁺): m/z 410 [M + H]⁺. HRMS (ES⁺): calcd for C₂₁H₂₄N₅O₄ [M + H]⁺ 410.1823, found 410.1807.

Methyl 4-Nitro-1H-pyrazole-3-carboxylate (5)

A 100 mL three-necked round-bottomed flask equipped with a magnetic stirring bar and fitted with a dropping funnel was charged with 4-nitro-1H-pyrazole-3-carboxylic acid (4.0 g, 25.5 mmol) and methanol (40 mL). The flask was cooled to 0 °C, and thionyl chloride (2.1 mL, 28.9 mmol) was added to the vigorously stirred solution over a period of 10 min. The mixture was stirred for an additional 12 h at room temperature, after which time TLC indicated complete consumption of the starting acid. The reaction mixture was concentrated under reduced pressure at 40 °C and the residue treated with toluene and reconcentrated (3 × 20 mL) under reduced pressure at 40 °C to give methyl ester 5 as an off-white solid. Yield: 4.42 g, 99%. ¹H NMR (DMSO-d₆) δ (ppm) 14.39 (br s, 1H), 9.98 (s, 1H), 3.90 (s, 3H). ¹³C NMR (DMSO-d₆) δ (ppm) 161.15, 138.13, 133.20, 130.90, 52.84. LRMS (ES⁺): m/z 172 [M + H]⁺.

Methyl-4-amino-1H-pyrazole-3-carboxylate (6)

A 100 mL round-bottomed flask equipped with digital thermometer and stirrer was charged with 10% palladium on carbon (0.621 g) under argon. In a separate vessel, a slurry of methyl ester 5 (4.42 g, 25.8 mmol) in ethanol (45 mL) was warmed to 35 °C to effect dissolution and the solution added to the catalyst under argon. Following a nitrogen–hydrogen purge sequence, an atmosphere of hydrogen was introduced and the reaction mixture maintained at 30 °C until the reaction completion (6 h) was noted by ¹H NMR analysis. Following a purge cycle, the reaction mixture under argon was filtered and the liquors concentrated under reduced pressure to give amine 6 as a solid. Yield: 3.57 g, 98%. ¹H NMR (DMSO-d₆) δ (ppm) 12.83 (br s, 1H), 7.10 (s, 1H), 4.83 (br s, 2H), 3.78 (s, 3H). ¹³C NMR (DMSO-d₆) δ (ppm) 160.39, 136.94, 128.43, 115.59, 50.88. LRMS (ES⁺): m/z 142 [M + H]⁺.

Methyl-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxylate (7)

A solution of amine 6 (3.57 g, 25.3 mmol) in 1,4-dioxane (50 mL) under argon was treated with triethylamine (4.3 mL, 31 mmol) followed by 2,6-dimethoxybenzoyl chloride (6.13 g, 30.6 mmol) such that the internal temperature was maintained in the range 20–25 °C. The reaction mixture was stirred at 25 °C until the reaction was complete (12 h) by TLC analysis. The reaction mixture was filtered, the filter-cake washed with 1,4-dioxane, and the combined filtrates progressed to next stage without further isolation.

To obtain analytical data for compound 7 and also to determine the yield of this reaction, a 2 g sample was taken out of the homogeneous filtrate solution (total weight of this solution is 91g). The 2 g sample was then concentrated under reduced pressure until dryness. The crude product (∼192 mg) was purified by column chromatography (DCM/MeOH). Evaporation of the appropriate fractions yielded finally the desired compound 7 as an amorphous solid (161 mg). Therefore, in the whole filtrate contained 7.33 g of compound 7. A 5 mg sample was used for to obtain analytical data; the rest was redissolved for use in the next reaction. Yield: 7.33 g, 95%. ¹H NMR (DMSO-d₆) 13.68 (br s, 1H), 9.16 (s, 1H), 8.31 (s, 1H), 7.41 (t, J = 8.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H), 3.83 (s, 3H), 3.77 (s, 6H). ¹³C NMR (DMSO-d₆) δ (ppm) 163.86, 161.55, 157.07, 131.27, 129.97, 123.61, 120.41, 114.66, 104.35, 55.84, 51.63. LRMS (ES⁺): m/z 306 [M + H]⁺. HRMS (ES⁺): calcd for C₁₄H₁₆N₃O₅ [M + H]⁺ 306.1084, found 306.1081.

4-(2,6-Dimethoxybenzamido)-1H-pyrazole-3-carboxylic Acid (8)

To a solution of sodium hydroxide (3.32 g, 83 mmol) in water (20 mL) was charged a solution of ester 7 in one portion (7.33 g, 24.0 mmol; the solution of crude 7 from the previous reaction, plus 156 mg of redissolved pure 7). The reaction mixture was stirred at 25 °C until completion as determined by TLC analysis. The reaction mixture was concentrated under reduced pressure at 45 °C, the oily residue diluted with water and acidified to pH 1 with concentrated hydrochloric acid, such that the temperature was maintained below 30 °C. The resulting precipitate was collected by filtration, washed with water, pulled dry on the filter, and subsequently washed with heptanes. The filter cake was charged to a 200 mL rotary evaporator flask and drying completed azeotropically with toluene. Yield: 6.22 g, 89%. ¹H NMR (DMSO-d₆) 13.44 (br s, 2H), 9.17 (br s, 1H), 8.29 (s, 1H), 7.40 (t, J = 8.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H), 3.77 (s, 6H). LRMS (ES⁺): m/z 292 [M + H]⁺. HRMS (ES⁺): calcd for C₁₃H₁₄N₃O₅ [M + H]⁺ 292.0928, found 292.0920.

General Method for Variation of Substituent R²: Example N-Cyclohexyl-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxamide (9c)

A mixture of carboxylic acid (50 mg, 0.17 mmol, 1.2 equiv), amine (14 mg, 0.14 mmol, 1.0 equiv), hydroxybenzotriazole (19 mg, 0.14 mmol, 1.0 equiv), polymer supported-carbodiimide (105 mg, 0.14 mmol, 1.0 equiv), and acetonitrile was heated by microwave irradiation for 10 min at 100 °C. The final product (9c) was isolated from the reaction mixture by filtering through a short column of Si-carbonate under gravity, which scavenged the excess carboxylic acid and hydroxybenzotriazole. No further purification was required. Removal of the solvent under reduced pressure yielded the required compounds as amorphous solids. Yield: 49 mg (solid), 67%. ¹H NMR (CD₃OD) δ (ppm) 8.33 (s, 1H), 7.42 (t, J = 8.5 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 3.86 (s, 6H), 3.82 (m, 1H), 1.88 (m, 4H), 1.68 (d, J = 12.8 Hz, 1H), 1.40 (m, 4H), 1.27 (m, 1H). ¹³C NMR (CD₃OD) δ (ppm) 165.02, 164.71, 159.24, 134.17, 132.97, 123.89, 121.82, 115.64, 105.24, 56.50, 49.36, 33.77, 26.58, 26.19. LRMS (ES⁺): m/z 373 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₅N₄O₄ [M + H]⁺ 373.1870, found 373.1850.

N-Cyclopropyl-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxamide (9a)

Yield: 42 mg (solid), 64%. ¹H NMR (CD₃OD) δ (ppm) 8.32 (s, 1H), 7.42 (t, J = 8.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 2H), 3.86 (s, 6H), 2.79 (m, 1H), 0.80 (m, 2H), 0.65 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 161.32, 161.20, 131.10, 122.44, 122.30, 115.01, 104.28, 55.82, 22.13, and 22.02 (d, rotamers), 5.55. LRMS (ES⁺): m/z 331 [M + H]⁺. HRMS (ES⁺): calcd for C₁₆H₁₉N₄O₄ [M + H]⁺ 331.1401, found 331.1385.

N-Cyclobutyl-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxamide (9b)

Yield: 56 mg (solid), 94%. ¹H NMR (DMSO-d₆) δ (ppm) 13.28 (s, 1H), 9.71 (s, 1H), 8.60 (d, J = 8.1 Hz, 1H), 8.30 (s, 1H), 7.40 (t, J = 8.4 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H), 4.37 (sex, J = 8.3, 1H), 3.76 (s, 6H), 2.13 (m, 4H), 1.63 (m, 2H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.53, 161.23, 156.93, 132.14, 131.07, 122.61, 119.84, 115.07, 104.32, 55.82, 54.86, 43.39, 29.81, 14.62. LRMS (ES⁺): m/z 345 [M + H]⁺. HRMS (ES⁺): calcd for C₁₇H₂₁N₄O₄ [M + H]⁺ 345.1557, found 345.1548.

N-Cycloheptyl-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxamide (9d)

Yield: 60 mg (solid), 90%. ¹H NMR (CDCl₃) δ (ppm) 12.19 (br s, 1H), 9.94 (s, 1H), 8.41 (s, 1H), 7.29 (t, J = 8.5 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.5 Hz, 2H), 4.05 (m, 1H), 3.80 (s, 6H), 2.00–1.94 (m, 2H), 1.66–1.45 (m, 10H). ¹³C NMR (CDCl₃) δ (ppm) 161.75, 160.98, 155.95, 131.18, 129.57, 121.16, 119.75, 112.84, 102.24, 54.19, 48.30, 33.28, 26.12, 22.39. LRMS (ES⁺): m/z 387 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₇N₄O₄ [M + H]⁺ 387.2027, found 387.2043.

N-(Bicyclo[2.2.1]heptan-2-yl)-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxamide (9e)

Yield: 49 mg (solid), 52%. ¹H NMR (DMSO-d₆) δ (ppm) 11.80 (br s, 1H), 9.72 (s, 1H), 8.28 (s, 1H), 8.00 (br d, J = 6.9 Hz, 1H), 7.40 (t, J = 8.4 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 3.76 (s, 6H), 3.66 (m, 1H), 2.21 (br s, 1H), 2.15 (br s, 1H), 1.63–1.37 (m, 5H), 1.16–0.99 (m, 3H). ¹³C NMR (CD₃OD) δ (ppm) 165.11, 164.82, 159.28, 133.97, 133.00, 124.01, 122.33, 115.79, 105.34, 56.59, 53.91, 43.80, 40.38, 37.06, 36.28, 29.28, 27.54. LRMS (ES⁺): m/z 385 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₅N₄O₄ [M + H]⁺ 385.1870, found 385.1857.

4-(2,6-Dimethoxybenzamido)-N-morpholino-1H-pyrazole-3-carboxamide (9f)

Yield: 34 mg (solid), 53%. ¹H NMR (DMSO-d₆) δ (ppm) 13.30 (s, 1H), 9.66 (s, 1H), 9.55 (s, 1H), 8.31 (s, 1H), 7.39 (t, J = 8.4 Hz, 1H), 6.75 (d, J = 8.6 Hz, 2H), 3.76 (s, 6H), 3.62 (m, 4H), 2.84 (m, 4H). ¹³C NMR (DMSO-d₆) δ (ppm) 161.26, 161.18, 156.93, 131.41, 131.10, 122.90, 119.80, 115.01, 104.32, 65.98, 55.82, 54.31. LRMS (ES⁺): m/z 376 [M + H]⁺. HRMS (ES⁺): calcd for C₁₇H₂₂N₅O₅ [M + H]⁺ 376.1615, found 376.1620.

4-(2,6-Dimethoxybenzamido)-N-(3-(dimethylamino)propyl)-1H-pyrazole-3-carboxamide (9g)

Yield: 41 mg (solid), 63%. ¹H NMR (DMSO-d₆) δ (ppm) 13.15 (brs, 1H), 9.70 (s, 1H), 8.99 (t, J = 6.3 Hz, 1H), 8.32 (s, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.31–7.29 (m, 4H), 7.22 (m, 1H), 6.74 (d, J = 8.4 Hz, 2H), 4.41 (d, J = 6.4 Hz, 2H), 3.75 (s, 6H). ¹³C NMR (DMSO-d₆) δ (ppm) 163.46, 161.29, 156.93, 139.45, 131.95, 131.10, 128.21, 127.26, 126.71, 122.58, 120.18, 115.01, 104.32, 55.82, 41.66. LRMS (ES⁺): m/z 381 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₁N₄O₄ [M + H]⁺ 381.1557, found 381.1543.

4-(2,6-Dimethoxybenzamido)-N-(pyridin-2-ylmethyl)-1H-pyrazole-3-carboxamide (9h)

Yield: 28 mg (solid), 42%. ¹H NMR (DMSO-d₆) δ (ppm) 13.33 (br s, 1H), 9.66 (s, 1H), 8.96 (t, J = 5.9, 1H), 8.50 (m, 1H), 8.34 (s, 1H), 7.74 (td, J = 7.7, 1.9 Hz, 1H), 7.39 (t, J = 8.4 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.25 (m, 1H), 6.73 (d, J = 8.4 Hz, 2H), 4.53 (d, J = 5.6 Hz, 2H), 3.75 (s, 6H). ¹³C NMR (DMSO-d₆) δ (ppm) 163.65, 161.32, 158.14, 156.93, 148.75, 136.68, 131.89, 131.10, 122.61, 122.06, 120.84, 120.18, 114.98, 104.32, 55.82, 43.54. LRMS (ES⁺): m/z 382 [M + H]⁺.

4-(2,6-Dimethoxybenzamido)-N-((3-methylpyridin-2-yl)methyl)-1H-pyrazole-3-carboxamide (9i)

Yield: 30 mg (solid), 44%. ¹H NMR (DMSO-d₆) δ (ppm) 13.30 (br s, 1H), 9.68 (s, 1H), 8.72 (t, J = 5.0 Hz, 1H), 8.39 (d, J = 5.0 Hz, 1H), 8.34 (s, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 8.4 Hz, 1H), 7.23 (dd, J = 7.6, 5.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 4.54 (d, J = 5.0 Hz, 2H), 3.76 (s, 6H), 2.29 (s, 3H). ¹³C NMR (DMSO-d₆) δ (ppm) 163.28, 161.29, 156.96, 153.98, 145.81, 137.64, 132.10, 131.13, 130.64, 122.47, 122.24, 120.04, 114.98, 104.32, 55.84, 40.96, 17, 13. LRMS (ES⁺): m/z 396 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₂N₅O₄ [M + H]⁺ 396.1666, found 396.1656.

N-(Cyclohexylmethyl)-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxamide (9j)

Yield: 56 mg (solid), 84%. ¹H NMR (CD₃OD) δ (ppm) 8.35 (s. 1H), 7.37 (t, J = 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 2H), 3.82 (s, 6H), 3.17 (d, J = 7.0 Hz, 2H), 1.77–1.64 (m, 5H), 1.55 (m, 1H), 1.28–1.13 (m, 3H), 1.00–0.89 (m, 2H). ¹³C NMR (CD₃OD) δ (ppm) 165.64, 165.00, 159.22, 134.15, 132.99, 123.89, 122.10, 115.69, 105.31, 56.58, 46.09, 39.42, 32.00, 27.57, 27.03. LRMS (ES⁺): m/z 386 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₇N₄O₄ [M + H]⁺ 387.2027, found 387.2008.

4-(2,6-Dimethoxybenzamido)-N-phenyl-1H-pyrazole-3-carboxamide (9k)

Yield: 48 mg (solid), 76%. ¹H NMR (DMSO-d₆) δ (ppm) 13.51 (s, 1H), 10.31 (s, 1H), 9.65 (s, 1H), 8.40 (s, 1H), 7.78 (d, J = 7.7 Hz, 2H), 7.41 (t, J = 8.5 Hz, 1H), 7.31 (t, J = 7.8 Hz, 2H), 7.09 (t, J = 7.3 Hz, 1H), 6.76 (d, J = 8.5 Hz, 2H), 3.77 (s, 6H). ¹³C NMR (DMSO-d₆) δ (ppm) 162.29, 161.40, 157.02, 138.10, 132.32, 131.19, 128.49, 123.81, 123.02, 120.69, 120.37, 114.98, 104.40, 55.88. LRMS (ES⁺): m/z 367 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₁₉N₄O₄ [M + H]⁺ 367.1401, found 367.1402.

4-(2,6-Dimethoxybenzamido)-N-(pyridin-4-yl)-1H-pyrazole-3-carboxamide (9l)

Yield: 10 mg (solid), 16%. ¹H NMR (DMSO-d₆) δ (ppm) 13.63 (s, 1H), 10.73 (s, 1H), 9.55 (s, 1H), 8.43 (d, J = 6.4 Hz, 3H), 7.83 (d, J = 5.5 Hz, 2H), 7.42 (t, J = 8.3 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 3.78 (s, 6H). LRMS (ES⁺): m/z 368 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₁₈N₅O₄ [M + H]⁺ 368.1353, found 368.1347.

4-(2,6-Dimethoxybenzamido)-N-(pyridin-2-yl)-1H-pyrazole-3-carboxamide (9m)

Yield: 10 mg (solid), 16%. ¹H NMR (DMSO-d₆) δ (ppm) 13.61 (s, 1H), 9.63 (s, 1H), 9.49 (s, 1H), 8.42 (s, 1H), 8.38 (br d, J = 4.6 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.83 (m, 1H), 7.42 (t, J = 8.4 Hz, 1H), 7.18 (m, 1H), 6.77 (d, J = 8.5 Hz, 2H), 3.78 (s, 6H). ¹³C NMR (DMSO-d₆) δ (ppm) 161.87, 161.55, 157.01, 150.45, 148.29, 138.41, 131.62, 131.30, 123.04, 120.90, 120.15, 114.66, 113.86, 104.35, 55.84. LRMS (ES⁺): m/z 368 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₁₈N₅O₄ [M + H]⁺ 368.1353, found 368.1335.

4-(2,6-Dimethoxybenzamido)-N-((1r,4r)-4-hydroxycyclohexyl)-1H-pyrazole-3-carboxamide (9n)

Yield: 51 mg (solid), 77%. ¹H NMR (DMSO-d₆) δ (ppm) 13.21 (br s, 1H), 9.75 (s, 1H), 8.28 (s, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 8.4 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 4.53 (br d, J = 3.8 Hz, 1H), 3.76 (s, 6H), 3.66 (m, 1H), 3.40–3.33 (m, 1H), 1.82 (m, 2H), 1.73 (m, 2H), 1.44 (m, 2H), 1.19 (m, 2H). ¹³C NMR (DMSO-d₆) 162.67, 161.25, 156.91, 131.07, 122.56, 115.10, 104.32, 68.11, 55.82, 47.00, 34.19, 30.00. LRMS (ES⁺): m/z 389 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₅N₄O₅ [M + H]⁺ 389.1819, found 389.1800.

cis-/trans-(4-(2,6-Dimethoxybenzamido)-N-(4-methylcyclohexyl)-1H-pyrazole-3-carboxamide) (9o)

Yield: 49 mg (solid), 74%. ¹H NMR (CD₃OD) δ (ppm) 8.36 (s, 0.5H), 8.33 (s, 0.5H), 7.39 (t, J = 8.5 Hz, 1H), 6.73 (d, J = 8.5 Hz, 2H), 4.05 (m, 0.5H), 3.84 (s, 6H), 3.76 (m, 0.5H), 1.96–1.32 (m, 8H), 1.06 (m, 1H), 0.98 (d, J = 6.5 Hz, 1.5 H), 0.92 (d, J = 6.6 Hz, 1.5H). ¹³C NMR (CD₃OD) δ (ppm) 165.06, 165.02, 164.76, 164.64, 159.25, 134.07, 134.01, 132.97, 123.99, 123.91, 122.30, 122.12, 115.70, 115.66, 105.39, 105.29, 56.54, 49.52, 46.86, 35.15, 33.67, 33.12, 31.53, 31.01, 30.09, 22.63, 21.25. LRMS (ES⁺): m/z 387 [M + H]⁺. HRMS (ES⁺): calcd for C₂₀H₂₇N₄O₄ [M + H]⁺ 387.2027, found 387.2007.

tert-Butyl 4-(4-(2,6-Dimethoxybenzamido)-1H-pyrazole-3-carboxamido)piperidine-1-carboxylate (9p)

Yield: 50 mg (solid), 41%. ¹H NMR (CD₃OD) δ (ppm) 8.35 (s, 1H), 7.37 (t, J = 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 2H), 4.08–3.99 (m, 3H), 3.82 (s, 6H), 2.90 (m, 2H), 1.89 (m, 2H), 1.51 (m, 2H), 1.46 (s, 9H). ¹³C NMR (CD₃OD) δ (ppm) 165.00, 164.89, 159.22, 156.39, 134.00, 132.99, 124.03, 122.16, 115.72, 105.35, 81.17, 56.61, 47.67, 44.39, and 43.61 (br d, rotamers), 32.69, 28.79. LRMS (ES⁺): m/z 474 [M + H]⁺. HRMS (ES⁺): calcd for C₂₃H₃₂N₅O₆ [M + H]⁺ 474.2347, found 474.2324.

N-(1-Benzylpyrrolidin-3-yl)-4-(2,6-dimethoxybenzamido)-1H-pyrazole-3-carboxamide (9q)

Yield: 69 mg (solid), 89%. ¹H NMR (CD₃OD) δ (ppm) 8.32 (s, 1H), 7.36 (t, J = 8.4 Hz, 1H), 7.32–7.22 (m, 5H), 6.68 (d, J = 8.4 Hz, 2H), 4.52 (m, 1H), 3.78 (s, 6H), 3.58 (d, J = 2.5 Hz, 2H), 2.76 (m, 2H), 2.58 (dd, J = 10.0, 4.3, 1H), 2.43 (q, J = 8.1 Hz, 1H), 2.27 (m, 1H), 1.75 (m, 1H). ¹³C NMR (CD₃OD) δ (ppm) 165.06, 165.00, 159.22, 139.26, 133.92, 132.99, 130.33, 129.50, 128.49, 124.01, 122.16, 115.67, 105.32, 61.26, 61.17, 56.58, 53.83, 49.19, 32.54. LRMS (ES⁺): m/z 450 [M + H]⁺. HRMS (ES⁺): calcd for C₂₄H₂₈N₅O₄ [M + H]⁺ 450.2136, found 450.2113.

4-(2,6-Dimethoxybenzamido)-N-(2-methoxyethyl)-1H-pyrazole-3-carboxamide (9r)

Yield: 39 mg (solid), 65%. ¹H NMR (CD₃OD) δ (ppm) 8.35 (s. 1H), 7.38 (t, J = 8.4 Hz, 1H), 6.72 (d, 8.4 J = 8.4 Hz, 2H), 3.83 (s, 6H), 3.53 (s, 4H), 3.37 (s, 3H). ¹³C NMR (CD₃OD) δ (ppm) 165.76, 165.05, 159.26, 134.10, 132.99, 115.71, 105.32, 72.07, 59.03, 56.56, 39.52. LRMS (ES⁺): m/z 349 [M + H]⁺. HRMS (ES⁺): calcd for C₁₆H₂₁N₄O₅ [M + H]⁺ 349.1506, found 349.1504.

4-(2,6-Dimethoxybenzamido)-N-pentyl-1H-pyrazole-3-carboxamide (9s)

Yield: 52 mg (solid), 84%. ¹H NMR (DMSO-d₆) δ (ppm) 13.23 (br s, 1H), 9.74 (s, 1H), 8.38 (br s, 1H), 8.29 (s, 1H), 7.39 (t, J = 8.5 Hz, 1H), 6.75 (d, J = 8.5 Hz, 2H), 3.76 (s, 6H), 3.19 (m, 2H), 1.49 (quint, J = 7.2 Hz, 2H), 1.31–1.22 (m, 4H), 0.85 (t, J = 7.0 Hz, 3H). ¹³C NMR (DMSO-d₆) δ (ppm) 163.39, 161.20, 156.93, 132.25, 131.07, 122.35, 119.79, 115.05, 104.32, 55.82, 37.95, 28.77, 28.57, 21.78, 13.87. LRMS (ES⁺): m/z 361 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₅N₄O₄ [M + H]⁺ 361.1870, found 361.1868.

4-(2,6-Dimethoxybenzamido)-N-(3-isopropoxypropyl)-1H-pyrazole-3-carboxamide (9t)

Yield: 64 mg (solid), 95%. ¹H NMR (CD₃OD) δ (ppm) 8.35 (s, 1H), 7.38 (t, J = 8.5 Hz, 1H), 6.71 (d, J = 8.5 Hz, 2H), 3.83 (s, 6H), 3.58 (sept, J = 6.1 Hz, 1H), 3.53 (t, J = 6.1 Hz, 2H), 3.45 (t, J = 6.6 Hz, 2H), 1.83 (quin, J = 6.4 Hz, 2H), 1.15 (d, J = 6.1 Hz, 6H). ¹³C NMR (CD₃OD) δ (ppm) 165.58, 165.00, 159.22, 134.17, 132.99, 123.82, 121.99, 115.69, 105.32, 73.08, 67.50, 56.61, 38.06, 30.72, 22.49. LRMS (ES⁺): m/z 391 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₇N₄O₅ [M + H]⁺ 391.1976, found 391.1961.

4-(2,6-Dimethoxybenzamido)-N-(3-(dimethylamino)propyl)-1H-pyrazole-3-carboxamide (9u)

Yield: 49 mg (solid), 76%. ¹H NMR (CD₃OD) δ (ppm) 8.23 (s, 1H), 7.26 (t, J = 8.4 Hz, 1H), 6.59 (d, J = 8.4 Hz, 2H), 3.71 (s, 6H), 3.26 (t, J = 7.5 Hz, 2H), 2.26 (t, J = 7.5 Hz, 2H), 2.11 (s, 6H), 1.65 (q, J = 7.5 Hz, 2H). ¹³C NMR (CD₃OD) δ (ppm) 165.70, 164.94, 159.22, 134.15, 132.99, 123.92, 122.05, 115.75, 105.35, 58.25, 56.61, 45.52, 38.23, 28.32. LRMS (ES⁺): m/z 376 [M + H]⁺. HRMS (ES⁺): calcd for C₁₈H₂₆N₅O₄ [M + H]⁺ 376.1979, found 376.1965.

4-(2,6-Dimethoxybenzamido)-N-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrazole-3-carboxamide (9v)

Yield: 57 mg (solid), 86%. ¹H NMR (CD₃OD) δ (ppm) 8.34 (s, 1H), 7.38 (t, J = 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 2H), 3.83 (s, 6H), 3.51 (t, J = 6.9 Hz, 2H), 2.69 (t, J = 6.9 Hz, 2H), 2.59 (m, 4H), 1.80 (m, 4H). ¹³C NMR (CD₃OD) δ (ppm) 165.69, 164.97, 159.22, 134.09, 132.99, 123.89, 122.01, 115.72, 105.32, 56.58, 56.32, 55.08, 38.64, 24.31. LRMS (ES⁺): m/z 388 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₆N₅O₄ [M + H]⁺ 388.1979, found 388.1965.

4-(2,6-Dimethoxybenzamido)-N-(2-morpholinoethyl)-1H-pyrazole-3-carboxamide (9w)

Yield: 37 mg (solid), 53%. ¹H NMR (CD₃OD) δ (ppm) 8.34 (s, 1H), 7.40 (t, J = 8.4 Hz, 1H), 6.73 (d, J = 8.4 Hz, 2H), 3.84 (s, 6H), 3.70 (t, J = 4.5 Hz, 4H), 3.5 (t, J = 6.6, 2H), 2.57 (t, J = 6.6 Hz, 2H), 2.52 (br t, J = 4.5 Hz, 4 Hz). ¹³C NMR (CD₃OD) δ (ppm) 165.67, 165.00, 159.23, 134.06, 132.96, 123.86, 122.02, 115.72, 105.32, 67.80, 58.60, 56.55, 54.67, 36.50. LRMS (ES⁺): m/z 404 [M + H]⁺. HRMS (ES⁺): calcd for C₁₉H₂₆N₅O₅ [M + H]⁺ 404.1928, found 404.1929.

Glossary

Abbreviations Used

GSK

glycogen synthase kinase

HAT

human African trypanosomiasis

CDK

cyclin dependent kinase

T. brucei

Trypanosoma brucei

T. b. brucei

Trypanosoma brucei brucei

SAR

structure–activity relationships

PK

protein kinase

Supporting Information Available

Kinase profiling of 4f, 4m, 4y, and 9g against a panel of mammalian kinases; calculated physicochemical properties of all synthesized aminopyrazole derivatives (PDF and PDB). This material is available free of charge via the Internet at http://pubs.acs.org.

Author Present Address

^§ Johannes Gutenberg-Universität Mainz Institut für Pharmazie und Biochemie Staudinger Weg 5 D-55128 Mainz, Germany

The authors declare no competing financial interest.