Discovery of a Novel Class of Potent and Selective Tetrahydroindazole-based Sigma-1 Receptor Ligands

Abstract

The sigma-1 and sigma-2 receptors have been shown to play important roles in CNS diseases, cancer, and other disorders. These findings suggest that targeting these proteins with small-molecule modulators may be of important therapeutic value. Here we report the development of a new class of tetrahydroindazoles that are highly potent and selective ligands for sigma-1. Molecular modeling was used to rationalize the observed structure-activity relationships and identify key interactions responsible for increased potency of the optimized compounds. Assays for solubility and microsomal stability showed this series possesses favorable characteristics and is amenable to further therapeutic development. The compounds described herein will be useful in the development of new chemical probes for sigma-1 and to aid in future work therapeutically targeting this protein.

Graphical Abstract

1. INTRODUCTION

The sigma receptors were first defined as an opioid receptor subtype.¹ However, work over the last several decades has shown that the sigma receptors are non-opioid proteins that have since been divided into two subtypes, sigma-1 and sigma-2. Sigma-1 was first cloned in 1997² and is a widely-expressed 25 kD protein with no other mammalian protein homologs.³ The most closely related protein is yeast C8-C7 sterol isomerase;² however, replacement of sterol isomerase by sigma-1 fails to reproduce isomerase function in yeast. Adding to the questions about its function, sigma-1 knockout mice display a mild depressive-like behavior⁴ but otherwise grow normally with no overt phenotype.⁵

Recent work to study the role of sigma-1 has shown that it regulates the activity of a wide range of proteins⁶ by acting as a chaperone localized in the endoplasmic reticulum (ER) membrane.⁷ It has been shown that sigma-1 regulates a variety of protein functions by direct binding, including those of opioid receptors,⁷ TRPV1,⁸ dopamine receptors,⁹ potassium channels,¹⁰ kinases,¹¹ and others^12–13 to modulate their functions. These results help explain the observed effects of sigma-1 ligands on opioid analgesia in vivo,^14–15 as well as in models of pain¹⁶ and alcohol abuse.¹⁷ Besides CNS disorders, sigma-1 has also been shown to be involved in mediating intracellular calcium levels in breast and colon cancer cells through its association to potassium channels.¹⁰ In the context of cancer, it has been shown that sigma-1 ligands reduce the proliferation of breast cancer cell lines by inducing ER stress.¹⁸ More recently, it was shown that sigma-1 is involved in the regulation and degradation of PD-L1 by autophagy, suggesting a potential link between this target and immunotherapy.¹⁹

Because of the important roles of the sigma-1 receptor in CNS diseases and cancer, there have been a number of efforts aimed at developing potent and selective ligands to modulate its function. Among the ligands that have been developed are series of alkoxyisoxazoles²⁰ and alkoxypyrazoles²¹ that displayed potent and selective binding to sigma-1 and showed significant analgesic effects in mouse models of pain.^20–21 Several other series of sigma-1 ligands based on aminotriazole and pyrimidine scaffolds have also been reported with efficacy in in vivo models of pain.^22–23 There have been other scaffolds developed as potent and selective sigma-1 ligands for CNS and cancer indications as well (Figure 1).^{19, 24–29}

Figure 1.

Examples of known sigma-1 ligands.

During the course of our studies on the biological activity of a class of tetrahydroindazoles,³⁰ we noted that one compound possessed moderate inhibition of the sigma-1 receptor. As this protein has been shown to be involved in multiple important diseases, we initiated a thorough assessment of the structure-activity relationships (SAR) around this class of compounds. Molecular modeling was then carried out to rationalize our observed SAR and build a platform to support further medicinal chemistry. These efforts led to the identification of a new ligand that potently inhibits the sigma-1 receptor with high selectivity against sigma-2. These results suggest that this series of sigma-1 ligands may serve as an attractive starting point for further lead optimization to develop potential therapeutics for neurological diseases, cancer, and other conditions, as well as provide useful new tool compounds for studying the roles of these two proteins.

2. RESULTS

Biological Screening.

Compounds were screened in a radioligand binding assay against sigma-1 and sigma-2 by the University of North Carolina Psychoactive Drug Screening Program (PDSP).³¹ Binding was assessed by displacement of [³H]- Pentazocine for sigma-1 and [³H]-DTG for sigma-2. New molecules were first screened at a single concentration of 10 μM (pK_i = 5) and those that showed > 50% inhibition were subsequently tested across a dose range to determine pK_i.

Structure-Activity Relationships.

The initial hit ligand (7a) inhibited sigma-1 and sigma-2 receptors with pK_i of 5.8 and 5.7 M, respectively (Table 1). Initially, the possibility of diversifying the linker between the tetrahydroindazole and the fluorobenzyl group was explored. On replacing the nitrogen on the C-5 position of the tetrahydroindazole with an oxygen or a sulfur (compounds 9 and 11, respectively), activity against both sigma receptors was completely lost. We then sought to determine if other hydrogen-bond donors could also retain sigma-1 activity. Substituting the amine with an amide (13) or a sulfonamide (14) similarly led to a complete loss of activity, thus highlighting the importance of a basic nitrogen for the activity of this class of compounds. While retaining the amine at the C-5 position of the tetrahydroindazole, substituents on the R₂-benzyl group were studied. The trifluorophenethyl analog (7b) had similar activity towards sigma-1 as the parent (7a), but was a more potent ligand for sigma-2. Replacement of the trifluoromethyl group with a methylendioxy (7c) saw a significant improvement in potency for sigma-1. Several analogs were synthesized with piperidine amides in the R₁-position as well. Notably, the hydroxyphenethyl derivative (7d) displayed potent binding to sigma-2 and high selectivity over sigma-1, suggesting the hydroxyl moiety was interacting with key differences between the two proteins. Besides alkyl chains on R₃, a number of phenyl substituents were also studied. The electron-withdrawing fluorobenzyl derivative (7aa) showed potent inhibition of sigma-1 and no activity against sigma-2. A variety of other R₂ analogs also showed excellent binding to sigma-1, including those with electron-withdrawing groups (7ac and 7ae) and both hydrogen-bond acceptor and donor substituents (7ad and 7af). When R₃ was a benzyl group and fluorobenzyl amine was at R₂ (7ah), the compound was found to have similar potency as the phenyl derivative (7aa). Finally, it was also found that nitrogen-containing groups such as pyridine were well-tolerated R₂ substituents (7ag).

Table 1.

Sigma Binding Affinity of Dimethyl and Piperidine Amides

Cpd	R1	R2	pKi ± SEM (M)
			R3	Sigma-1	Sigma-2
7a			n-Pr	5.8 ± 0.05	5.7 ± 0.06
9			n-Pr	<5	<5
11			n-Pr	<5	<5
13			n-Pr	<5	<5
14			n-Pr	<5	<5
7b			n-Pr	5.6 ± 0.08	6.2 ± 0.1
7c			n-Pr	6.3 ± 0.09	6.4 ± 0.1
7d			n-Pr	<5	6.6 ± 0.1
7aa			Ph	6.8 ± 0.05	<5
7ab			Ph	6.2 ± 0.08	6.3 ± 0.1
7ac			Ph	6.5 ± 0.05	<5
7ad			Ph	6.3 ± 0.05	6.5 ± 0.1
7ae			Ph	6.9 ± 0.06	7.0 ± 0.1
7af			Ph	6.7 ± 0.05	<5
7ag			Bn	6.6 ± 0.09	<5
7ah			Bn	6.6 ± 0.09	<5

Building upon on the initial SAR, effects on changing the regiochemistry of the tetrahydroindazole N-1 substituent were next studied. Comparing the activity between the two regioisomers, it was noted that the N-2 regioisomer was generally more potent against both sigma receptors compared to the N-1 regioisomer. N-2 derivative 7ba was over 5-fold and 60-fold more potent against sigma-1 and sigma-2 receptors, respectively, compared to N-1 regioisomer 7aa. Comparing compound 7bb with 7ab, it was found that the N-2 isomer was significantly more potent against both receptors. The methylenedioxy analog (7bd) also possessed much higher affinity for both sigma-1 and sigma-2 compared with its N-1 derivative (7ad). When the fluorophenyl R₂ substituent was tested, the resulting compounds possessed selectivity towards sigma-1, including compound 7bf which is the most potent and selective sigma-1 ligand found in this series with pK_i of 7.8 (K_i = 17 nM), along with pKi <5 (K_i >10 μM) against sigma-2 (Table 2).

Table 2.

Sigma Binding Affinity of N-1 and N-2 aryl Tetrahydroindazole Regioisomers

Cpd	R2	R3	pKi ± SEM (M)
			Sigma-1	Sigma-2
7ba		Ph	7.5 ± 0.05	6.8 ± 0.09
7bb		Ph	7.4 ± 0.05	6.9 ± 0.09
7bc		Ph	6.7 ± 0.05	<5
7bd		Ph	7.2 ± 0.07	7.5 ± 0.1
7be		p-F(Ph)	7.0 ± 0.09	6.6 ± 0.1
7bf		p-F(Ph)	7.8 ± 0.09	<5
7bg		4-CONH2-Ph	6.2 ± 0.08	<5
7bh		4-OH-Ph	6.5 ± 0.08	6.6 ± 0.1

Molecular Modeling of Sigma-1 Ligands.

To better understand the binding mode of this series of sigma-1 ligands and rationalize the SAR, molecular modeling of the initial hit compound (7a) and the most potent compound (7bf) was carried out (Figure 2).

Figure 2.

Docked poses of compound 7a (Left) and 7bf (Right) in the sigma-1 crystal structure. Both compounds show potential hydrogen bonds between the critical C-5 amine and Tyr120 and Glu172. Compound 7bf also shows additional interactions with Thr181 and Tyr103, as well as more complete occupancy of the hydrophobic pocket.

Glide-XP docking of both ligands was carried out using the published sigma-1 crystal structure³² and the default docking parameters. Three docked poses for each ligand were generated. Analysis of the lowest-energy binding poses of the most potent compound (7bf) showed potential hydrogen bonding with Glu172, Thr181 and Tyr120, a π-π interaction with Tyr103, and more completely filled the hydrophobic pocket. While analyzing the binding pose of 7a, we found that it lacked the potential hydrogen bond with Thr181 and had no π-π interaction with Tyr103. These residues were shown to form critical interactions with the reported co-crystal structure ligand as well.³² To calculate the estimated protein-ligand binding energy, the force-field based Molecular Mechanical/Generalize Born Surface Area (MM-GBSA)^33–34 was used. As per the requirements of MMGBSA computations, three low energetic conformers for each ligand were generated using the Glide-XP module. The average MM-GBSA ΔG was found to be −9.98 and −21.64 kcal/mol for 7a and 7bf, respectively.

Solubility and micorosomal stability.

To evaluate the pharmaceutical properties and potential for future development, a selection of 6 of the most potent and/or selective ligands were chosen for microsomal stability and solubility analyses. The thermodynamic solubility of compounds in pH=7.4 PBS buffer were evaluated in a high-throughput manner on a Synergy HTX plate reader. With the exception of compound (7be), all compounds had solubilities >100 μM, and two of the compound had solubilities >1 mM in PBS buffer (Table 3). Notably, the two least soluble compounds (7be and 7bf) both possessed 2-p-F-Ph-tetrahydroindazoles. The same set of compounds tested in the solubility assay were screened for their in vitro stability towards mouse liver microsomes to evaluate their potential in vivo metabolic stability. Each compound was measured after a 60-minute incubation at 37 °C. Two of the compounds were nearly-completely consumed by liver microsomes after 60 minutes (7ab and 7ba), while the others retained significant amounts of parent compound after treatment with liver microsomes. (Table 3).

Table 3.

In vitro metabolism and solubility

Cpd	Microsomal Stabilitya	Solubility (μM)b
7a	21 ± 0.2	1783
7ab	0.3 ± 0.1	1567
7ba	7 ± 0.9	508
7bd	24 ± 1.4	655
7be	21 ± 1.0	ND
7bf	25 ± 0.2	110

^aMouse liver microsomes, % remaining after 60-minute incubation at 37 °C (reactions were run in duplicate).

^bSolubility measured in PBS buffer at pH=7.4.

Chemistry.

The synthetic route to access compounds 7a – 7d is shown in Scheme 1. Commercially available 1,4-dioxaspiro[4.5]decan-8-one (1) was acylated with diethyloxalate in the presence of LDA at −78 °C to afford 2 in good yield. Cyclization with propyl hydrazine afforded tetrahydroindazole 3 in 82% yield. Hydrolysis of the ester afforded acid 4 which was coupled to either dimethylamine or piperidine to afford the amides (5a and 5b), respectively. Removal of the ketal protecting group followed by reductive amination gave the final compounds (Scheme 1).

Scheme 1.

Synthesis of N-propyl tertiary amide analogs^a

^aReagents and conditions: (a) diethyloxalate, LDA, THF, −78 °C to rt, 16 h, 68%; (b) propylhydrazine, K₂CO₃, EtOH, rt, 3 h, 82%; (c) NaOH, EtOH, rt, 4 h, 84%; (d) R¹NH, EDC, HOBt, TEA, DCM, rt, 8 h, 44% for 5a and 67% for 5b; (e) 3N HCl, THF, 50 °C, 2 h, 73% for 6a and 85% for 6b; (f) R₂NH₂, AcOH, 1,2-DCE, 30 min, then NaBH(OAc)₃, rt, overnight 32–95%.

For tetrahydroindazoles with alternative C-5 linkers, the route shown in Scheme 2 was used. Sodium borohydride reduction of ketone 6a afforded secondary alcohol 8, which was O-alkylated to give compound 9. For thioether linkers, alcohol 8 was converted to a tosyl group (10) which was displaced with a thiol to afford sulfide 11. The tosylate group in compound 10 was also displaced with sodium azide and subsequently reduced to the primary amine (12). Treatment of the amine with either an acid or sulfonyl chloride afforded the amide (13) and sulfonamide (14), respectively.

Scheme 2.

Synthesis of indazole analogs with alternative C-5 linkers^a

^aReagents and conditions: (a) NaBH₄, MeOH, rt, 2 h, 79%; (b) TsCl, DMAP, TEA, DCM, 0 °C to rt, 8 h, 84%; (c) (4-fluorophenyl)methanethiol, NaOMe, EtOH, 60 °C, 6 h, 22%; (d) NaN₃, DMF, 70 °C, 8 h, then H₂, Pd/C, EtOH, 14 psi, 3 h, 56%; (e) 4-fluorobenzoic acid, EDCI, DMAP, DCM, 8 h, 48%; (f) 4-fluorobenzene-1-sulfonyl chloride, TEA, DCM, 0 °C to rt, 4 h, 55%; (g) 1-(bromomethyl)-4-fluorobenzene, NaH, DMF, 60 °C, 8 h, 49%.

The synthesis of N-1 and N-2 aryl tetrahydroindazole regioisomers is shown in Scheme 3. Cyclization of 2 with hydrazine afforded pyrazole 3a in quantitative yield. Ester hydrolysis provided acid 4a which was coupled with piperidine to provide the amide (5c). Next, a variety of N-substituents were installed using a Chan-Lam coupling which afforded stereoisomers 6c and 6d in a 1:1 ratio. Each individual regioisomer was separated at this stage using reverse-phase preparative HPLC. Finally, removal of the ketal protecting group followed by reductive amination gave the final compounds (7aa – 7ah and 7ba −7bh, Scheme 3).

Scheme 3.

Synthesis of N-1 and N-2 aryl indazole regioisomers^a

^aReagents and conditions: (a) NH₂NH₂, AcOH, EtOH,70 °C, 3 h, > 99%; (b) NaOH, EtOH, rt, 3 h, 73%; (c) piperidine, EDCI, HOBt, TEA, DMF, 8 h, 53%; (d) arylboronic acid, Cu(OAc)₂, Py., TEA, DMF, 48 h, 36–38%; (e) 3N HCl, THF, 50 °C, 52–73%; (f) R₂NH₂, AcOH, 1,2-DCE, NaBH(OAc)₃, 23–69%

3. CONCLUSION

Based on our initial identification of tetrahydroindazole 7a with moderate activity against sigma-1 and sigma-2 receptors, an SAR study was carried out to improve the potency and selectivity of this series as novel sigma-1 ligands. To improve the potency and selectively towards sigma-1 for this scaffold, we substituted different aryl groups on the N-1 and N-2 of the tetrahydroindazole and identified compound 7bf with pK_i of 7.8 M against sigma-1 and over 500-fold selectivity against sigma-2. We found that a wide range of substituents off of the saturated ring were tolerated, including electron-withdrawing and -donating aromatic rings and basic amines. Our SAR also indicated that a basic amine was a required linker for the R₂ substituents, as ether, thioether, sulfonamide, and amides were completely inactive. It was also found that in general, N-2 substituted tetrahydroindazoles possessed greater potency for sigma-1 than did the N-1 variations.

Furthermore, these compounds were shown to have good to excellent thermodynamic solubility with two of the tested compounds having solubility of >1 mM in PBS buffer. Several of these tetrahydroindazoles also showed moderate mouse liver microsomal stability after 60 minutes. Tetrahydroindazole substituents were found to significantly affect microsomal stability, suggesting this scaffold can be further developed into compounds with even better microsomal stability. Finally, we also carried out molecular modeling of this scaffold for the sigma-1 receptor to help rationalize the observed SAR. Our modeling highlighted several important interactions present in the optimized sigma-1 ligand (7bf) and binding energy calculations supported its observed increased binding. Notably, our molecular modeling showed both compounds 7a and 7bf formed hydrogen bond interactions with Glu172 and Tyr120, thus providing rationale for our observation that the basic amine linker is required.

Sigma-1 and sigma-2 receptors have been implicated in a wide variety of diseases, including cancer and CNS disorders. Because of the potential for developing new therapeutics targeting sigma-1, there has been substantial effort to develop potent ligands for this protein. Here we report a novel and highly drug-like scaffold that can be modified to produce compounds that are potent and selective ligands for the sigma-1 receptor. These molecules can be used as selective chemical probes to help characterize the biological effects of this protein. In addition, the presented compounds are excellent starting points for future medicinal chemistry optimization to develop them into compounds with in vivo efficacy for CNS disorders or cancer as they possess excellent solubility and moderate microsomal stability. Further studies to profile their effects on sigma-1-related phenotypes and disease are currently underway.

4. EXPERIMENTAL SECTION

Solubility

A selection of 6 of the most potent and/or selective ligands were chosen for microsomal stability and solubility analysis. From stock solutions in DMSO, concentrations of 500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.9, 0.9 μM solutions in acetonitrile was prepared in a 96-well plate while keeping the DMSO concentration constant at 0.5 %. Individual standard solubility curves for each compound were developed from the UV-absorption measured on a Synergy HTX plate reader. 1 mg of each compound in 1 mL of PBS (pH = 7.4) was stirred at 500 rpm for 16 hours at room temperature. After this time, the solution was allowed to equilibrate for 30 minutes. The solution was then filtered through 0.2 μm filter and the UV-absorption of the filtrate was measured. The solubility was extrapolated from the standard solubility curve previously generated for each compound.

Mouse Liver Microsomal (MLM) Stability

In a 96-well plate, 100 μL of Mouse Liver microsomes (0.4 mg/mL, Corning, cat# 452702) with co-factor (NADPH) in Phosphate Buffer (0.5 M) was added to 10 μM of the compounds. Vera-pamil-HCl was run as the control. At time = 0 min (T₀), the reactions were immediately quenched and mixed with 2x the volume of ice-cold acetonitrile. The other reactions were incubated at 37 °C with shaking for 60 min. At time 60 min (T₆₀), the reactions were stopped by mixing with 2x volume of ice-cold acetonitrile. The samples were centrifuged to remove the precipitated protein and the supernatants were analyzed by Acquity-H UPLC/MS in SRM mode to quantitate the remaining parent compound at both T₀ and T₆₀. The percent of the parent compound remaining was calculated from the formula:

$$\%\text{ parent compound remaining}=(\text{concentration at }60\text{ min}/\text{concentration at }0\text{ min})*100$$

All the reactions were run in duplicate and the results are the mean of the runs.

Protein Preparation and Docking Protocols

Schmidt et al.³² has solved the co-crystal structures of sigma-1 with two different small molecule ligands, having the accession codes 5HK1 and 5HK2 in the protein database. For our in-silico studies, we used 5HK1 as it has better resolution than the other structure. Analysis of this crystal structure revealed that it has missing or uncertain (high B values) atoms on the side chains of the active site residues and some of the residues show restricted torsions. The structure was then subjected to Prime validations implemented in the Schrodinger suite in order to correct the irrelevant side chains, add missing atoms, eliminate partial occupant rotamers, fix the undesired orientation of Asn, Gln and His residues, and finally to replace the “b” values by the optimized potential for liquid simulations (OPLS3) charges. Next, the protein-preparation module was applied in order to verify the suitability of the protein for docking simulations. The docking engine is built upon a grid-based technique and hence a 12 ų grid was generated to include the reported critical residues Tyr103, Leu105, Glu172, Trp164, Phe107, Met93 and Leu95. To carry out the docking simulations of our compounds, we used the Glide-XP module³⁵ implemented in the Schrodinger platform. For the Glide docking, the receptor van der Waals scaling was set at 0.80 and the ligand van der Waals was scaled at 0.5.

Ligand Preparation

In order to perform the docking simulations of the compounds, we used the Lig-Prep module of the Schrodinger suite. Compounds 7a and 7bf were subjected to protonation and deprotonation at physiological pH 7.4 ± 1, which generated low energetics 3D structures suitable for Glide-docking. The Glide-XP docking followed by MMGBSA computations were carried out for these two ligands in order to compute and compare the docked poses and binding energy.

MMGBSA

To calculate the estimated binding energy, various computational tools have been developed, each with trade-offs between computational costs and accuracy.^36–37 These methods include the fast but inaccurate scoring functions based on coarse physical approximations³⁶, to the computationally most-intense free energy perturbation techniques.^{33, 38} To develop a reasonable approach that provides good accuracy while also restraining computational costs, a force-field based method has been introduced called Molecular Mechanical/Generalize Born Surface Area (MM-GBSA).^33–34 This method computes the free energy of binding from the difference between the free energies of protein, ligand, and the complex in solution. The MM-GBSA method is based on the molecular mechanics (MM) energy (i.e. a combination of the bond, angle, and torsion energy terms along with the Coulomb and van der Waals energy), polar and non-polar solvation terms, and an entropy term. This method can be described as follows:

$$\Delta \text{G}=\Delta \text{E}(\text{MM})+\Delta \text{G}(\text{sol},\text{p})+\Delta \text{G}(\text{sol},\text{np})-\text{T}\Delta \text{S}$$

where E = combination of all MM energies, G(sol, p) = polar contribution to the solvation free energy, G(sol, np) = non-polar contribution to the solvation free energy computed through the solvent accessible surface area (SASA), and S = entropic contribution.

Chemistry

Unless otherwise noted, all materials for the synthetic chemistry portion were obtained from commercial suppliers (Combi-Blocks, CombiPhos, Fisher Scientific, Sigma-Aldrich, or VWR) and used without further purification. The ¹H-and ¹³C-NMR spectra were recorded on a Bruker AVANCE 500 MHz spectrometer using CDCl₃ or CD₃OD as the solvent. Chemical shifts are expressed in ppm (δ scale) and referenced to residual protonated solvent. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s (broad singlet). Thin layer chromatography (TLC) was performed on glass backed Merck silica gel 60 F₂₅₄ plates, column chromatography was performed using KP-SIL silica gel (Biotage, USA), and flash column chromatography was performed on Biotage prepacked columns using the automated flash chromatography system Biotage Isolera One. The purities of all the final compounds were of > 95% as determined by UPLC analysis unless otherwise indicated. High Resolution Mass analysis was performed on an Agilent 6210A LC-TOF.

Preparative HPLC Purification

All compounds that are designated as being purified by preparative HPLC were purified according to the same method, as described below. Purification was carried out using a Gilson GX-271 preparative scale reverse phase HPLC system with Gilson model 159 UV-VIS detector and Phenonemex Kinetex 5 μm, C18, 100 Å, 50 mm × 30 mm column. Compounds were eluted using a gradient elution of A:B (90:10 to 0:100) over 5 min at a flow rate of 50.0 mL/min, where solvent A was H₂O (with 0.1% formic acid) and solvent B was CH₃CN (with 0.1% formic acid). The product fractions were combined and dried using a Genevac EZ-2 Centrifugal Evaporator.

General procedure A: synthesis of amides

To a solution carboxylic acid (1.0 equiv) in DCM (0.1 M) was added EDC (1.1 equiv) and HOBT (1.0 equiv). The reaction mixture was stirred at RT for 30 min and then amine (1.0 equiv) and TEA (1.1 equiv) were added. The mixture was then stirred at RT for 8 h, diluted with DCM and washed with H₂O and brine. The organic extract was dried over with anhydrous Na₂SO₄, filtered and concentrated in vacuo to give a residue which was purified by a short silica gel plug (10% MeOH in DCM) to give the amide.

General procedure B: removal of ketal protecting group

Ketals (1.0 equiv) were dissolved in THF (0.8 M) then 3N HCl (5.0 equiv) was added to the mixture. The resulting solution was heated at 50 °C for 2 h. The solvents were evaporated and EtOAc was added. The organic layer was washed with saturated NaHCO₃ and H₂O. The combined organic layer was dried over anhydrous Na₂SO₄ and evaporated in vacuo to give the crude product which was purified by a short silica gel plug using 0–10% MeOH in DCM to give the product.

General procedure C: reductive amination

A solution of the ketone (1 equiv) and acetic acid (1.5 equiv) in DCE (1.5 mL) was stirred at room temperature. The amine (2 equiv.) was added, followed by 2 equiv. NaBH(OAc)₃. The mixture was stirred at room temperature overnight, quenched with saturate NaHCO₃ aqueous solution (3 mL), and extracted with DCM (3 mL x 3). The organic layers were combined, dried over Na₂SO₄, concentrated and purified by preparative HPLC, as described in the general methods.

General procedure D: Chan-Lam coupling

A solution of tetrahydroindazole (1 equiv.), phenylboronic acid (2 equiv.), copper acetate (1 equiv.), pyridine (2 equiv.) and TEA (2 equiv.) in dry DMF was stirred at room temperature for 48 h. The mixture was poured into water and extracted with ethyl acetate. The organic layers were combined, washed with water and brine sequentially, dried over Na₂SO₄, concentrated and purified by preparative HPLC, as described in the general methods.

Ethyl 2-(8-hydroxy-1,4-dioxaspiro[4.5]dec-7-en-7-yl)-2-oxoacetate (2).

To a solution of 1,4-dioxaspiro[4.5]decan-8-one 1 (7.81 g, 50 mmol) in anhydrous THF (150 mL) cooled to −78 °C under a N₂ atmosphere was added LDA (27.5 mL, 55 mmol). After stirring for 15 minutes, diethyl oxalate (7.47 mL, 55 mmol) was added in portions over 10 min. The reaction was gradually warmed to RT and stirred for 16h. The reaction mixture was quenched with 1N HCl and the resulting mixture was extracted with EtOAc, washed with brine and dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography (0–100% EtOAc in Hexanes) to yield the ethyl 2-(8-hydroxy-1,4-dioxaspiro[4.5]dec-7-en-7-yl)-2-oxoacetate 2 (8.7 g, 68%) as a thick yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 15.35 (s, 1H), 4.33 (q, J = 7.22 Hz, 2H), 3.97 – 4.02 (m, 4 H), 2.74 (s, 2 H), 2.68 (t, J = 6.87 Hz, 2H), 1.91 (t, J = 6.87 Hz, 2H), 1.37 (t, J = 7.17 Hz, 3H). LCMS (ESI) m/z: [M+H]⁺ 257.16.

Ethyl 1’-propyl-1’,4’,6’,7’-tetrahydrospiro[[1,3]dioxolane-2,5’-indazole]-3’-carboxylate (3).

Propylhydrazine·2HCl (2 g, 13.6 mmol) was added to a solution of ethyl 2-oxo-2-(8-oxo-1,4-dioxaspiro[4.5]decan-7-yl)acetate 2 (3.49 g, 13.6 mmol) and K₂CO₃ (3.76 g, 27.2 mmol) in EtOH (84 mL). The reaction mixture was stirred at RT for 3 h and then concentrated in vacuo. The residue was re-dissolved in EtOAc and H₂O and the layers separated. The aqueous layer was extracted with EtOAc and the combined organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue obtained was purified by silica gel chromatography (0–60% EtOAc in Hexanes) to give ethyl 1’-propyl-1’,4’,6’,7’-tetrahydrospiro[[1,3]dioxolane-2,5’-indazole]-3’-carboxylate 3 (3.3 g, 82%) as a thick oil. ¹H NMR (500 MHz, CDCl₃) δ 4.37 (q, J = 7.22 Hz, 2H), 3.97 – 4.08 (m, 6H), 2.98 (s, 2H), 2.78 (t, J = 6.56 Hz, 2H), 1.98 (t, J = 6.56 Hz, 2H), 1.86 (m, 2H), 1.34 – 1.40 (m, 3H), 0.92 (t, J = 7.48 Hz, 3H). LCMS (ESI) m/z: [M + H]⁺ 295.41.

Ethyl 1,4,6,7-tetrahydrospiro[indazole-5,2’-[1,3]dioxolane]-3-carboxylate (3a).

A solution of 2 (7.03 g, 27.4 mmol), hydrazine monohydrate (2.7 mL, 64%, 34.9 mmol), and acetic acid (16 mL, 279 mmol) in ethanol (80 mL) was heated to 65 °C for 90 min. After cooling down to room temperature, solvent was evaporated, residues dissolved in water (200 mL), neutralized with NaHCO₃, and extracted with ethyl acetate (150 mL X 4). The organic layers were combined, dried over Na₂SO₄, concentrated in vacuo to provide the product as light yellow oil (6.92 g, 100%), which was used in the next step without further purification.

1’-Propyl-1’,4’,6’,7’-tetrahydrospiro[[1,3]dioxolane-2,5’-indazole]-3’-carboxylic acid (4).

To a mixture of ethyl 1’-propyl-1’,4’,6’,7’-tetrahydrospiro[[1,3]dioxolane-2,5’-indazole]-3’-carboxylate 3 (3.3 g, 11.21 mmol) in EtOH (50 mL) was added a 2M solution of NaOH (50 mL) and the reaction mixture stirred at RT for 4 h. The EtOH was evaporated and the aqueous mixture was washed with diethyl ether. The pH of the aqueous layer was adjusted to 5–6. The aqueous layer was then extracted with EtOAc (×3). The combined organic extract was dried using anhydrous Na₂SO4, filtered and concentrated under reduced pressure to yield 1’-propyl-1’,4’,6’,7’-tetrahydrospiro[[1,3]dioxolane-2,5’-indazole]-3’-carboxylic acid 4 (2.5 g, 84%) as an off-white solid. ¹H NMR (500 MHz, CD₃OD) δ 3.99 – 4.06 (m, 6H), 2.90 (s, 2H), 2.81 (t, J = 6.56 Hz, 2H), 1.94 – 2.00 (m, 2H), 1.79 – 1.90 (m, 2H), 0.92 (t, J = 7.32 Hz, 3H). LCMS (ESI) m/z: [M + H]⁺ 267.37.

1,4,6,7-Tetrahydrospiro[indazole-5,2’-[1,3]dioxolane]-3-carboxylic acid (4a).

A solution of 3a (6.92 g, 27.4 mmol) in ethanol (80 mL) was stirred at room temperature. NaOH (10%, 80 mL) aqueous solution was added, and the mixture was stirred at room temperature overnight. Ethanol was carefully evaporated; the resulting aqueous solution was acidified with concentrated HCl to pH~4. The white solid was collected by filtration, dried in vacuo to afford the product 4a (4.48 g, 73%). ¹H NMR (500 MHz, CD₃OD) δ 4.04 − 3.97 (m, 2H), 3.96 (s, 2H), 3.35 (s, 1H), 2.97 (s, 1H), 2.83 − 2.77 (m, 1H), 2.57 − 2.50 (m, 2H), 1.94 (t, J = 6.7 Hz, 1H), 1.87 − 1.82 (m, 1H). ¹³C NMR (126 MHz, CD₃OD) δ 170.4, 116.5, 110.1, 108.5, 65.5, 34.4, 33.4, 32.5, 30.9, 30.8. LCMS (ESI) m/z: [M + H]⁺ 255.21.

N,N-dimethyl-1’-propyl-1’,4’,6’,7’-tetrahydrospiro[[1,3]dioxolane-2,5’-indazole]-3’-carboxamide (5a).

Prepared according to General procedure A using carboxylic acid 4 (0.3 g, 1.13 mmol) to afford N,N-dimethyl-1’-propyl-1’,4’,6’,7’-tetrahydrospiro[[1,3]dioxolane-2,5’-indazole]-3’-carboxamide 5a (145 mg, 44%) as a pale yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 3.97 – 4.07 (m, 4H), 3.92 (t, J = 7.32 Hz, 2H), 3.32 (br. s, 3H), 3.05 (br. s, 3H), 2.93 (s, 2H), 2.77 (t, J = 6.56 Hz, 2H), 1.99 (t, J = 6.56 Hz, 2H), 1.83 (m, J = 7.32 Hz, 2H), 0.93 (t, J = 7.32 Hz, 3H). LCMS (ESI) m/z: [M + H]⁺ 294.30.

Piperidin-1-yl(1-propyl-1,4,6,7-tetrahydrospiro[indazole-5,2’-[1,3]dioxolan]-3-yl)methanone (5b).

Prepared according to General procedure A using carboxylic acid 4 (200 mg, 0.751 mmol) and piperidine (77mg, 0.901 mmol) to afford 5b (168 mg, 67%) as a yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 4.01 (dddd, J = 13.9, 8.3, 6.6, 4.0 Hz, 4H), 3.92 (t, J = 7.2 Hz, 2H), 3.82 (s, 2H), 3.67 (s, 2H), 2.90 (s, 2H), 2.76 (t, J = 6.6 Hz, 2H), 1.99 (t, J = 6.6 Hz, 2H), 1.83 (h, J = 7.4 Hz, 2H), 1.71 − 1.51 (m, 6H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 163.5, 142.5, 137.3, 116.7, 108.8, 64.8, 51.0, 48.3, 43.4, 32.2, 31.4, 26.9, 25.0, 23.6, 19.7, 11.4.

Piperidin-1-yl(1,4,6,7-tetrahydrospiro[indazole-5,2’-[1,3]dioxolan]-3-yl)methanone (5c).

A solution of 4a (3.4 g, 15.2 mmol), EDCI (3.3 g, 17.2 mmol), HOBt (2.3 g, 15.0 mmol) in dry DMF (25 mL) was stirred at room temperature for 30 min, then a solution of piperidine (3 mL, 30.4 mmol) and trimethylamine (4 mL, 28.7 mmol) in DMF (25 mL) was added. The mixture was stirred at room temperature overnight. The mixture was poured into water (150 mL) and extracted with ethyl acetate (150 mL × 6). The organic layers were combined, washed with water (150 mL) and brine (150 mL) sequentially, dried over Na₂SO₄, concentrated and further purified by flash silica gel column, with EA/hex, (gradient up to 99:1), to provide the product as pale yellow solid (2.1 g, 48%).

N,N-dimethyl-5-oxo-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (6a).

Prepared according to General procedure B using 5a (320 mg, 1.1 mmol) to afford N,N-dimethyl-5-oxo-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide 6a (230 mg, 85%) as yellow waxy solid. ¹H NMR (500 MHz, CDCl₃) δ 3.90 (td, J = 7.0, 1.1 Hz, 2H), 3.77 − 3.63 (m, 4H), 2.70 − 2.65 (m, 3H), 2.53 − 2.38 (m, 3H), 1.80 (q, J = 7.3 Hz, 2H), 1.55 (d, J = 7.8 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 207.5, 163.9, 142.6, 138.0, 116.5, 77.2, 77.2, 77.0, 76.7, 60.8, 55.4, 50.7, 25.8, 24.8, 23.5, 21.1, 11.2. LCMS (ESI) m/z: [M + H]⁺ 250.24.

3-(piperidine-1-carbonyl)-1-propyl-1,4,6,7-tetrahydro-5H-indazol-5-one (6b).

Prepared according to General Procedure B using 5b (240 mg 0.702 mmol) to afford 6b (177 mg, 85%) as a yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 4.06 − 3.94 (m, 3H), 3.90 (t, J = 7.3 Hz, 1H), 3.82 (s, 2H), 3.65 (s, 2H), 2.89 (s, 1H), 2.75 (t, J = 6.6 Hz, 1H), 2.68 (t, J = 7.0 Hz, 1H), 1.98 (t, J = 6.6 Hz, 1H), 1.82 (hept, J = 7.6 Hz, 2H), 1.71 − 1.49 (m, 6H), 0.91 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 208.6, 163.5, 142.4, 137.1, 116.5, 108.7, 64.6, 50.9, 32.0, 31.3, 24.8, 23.5, 23.4, 19.6, 11.2.

5-((4-Fluorobenzyl)amino)-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (7a).

Prepared according to General procedure C using 6a (150 mg, 0.6 mmol) to afford 7a (114 mg, 53%) as light pink solid (FA salt). ¹H NMR (500 MHz, CD₃OD) δ 7.42 – 7.51 (m, 2H), 7.08 – 7.17 (m, 2H), 4.05 (s, 2H), 4.00 (t, J = 7.02 Hz, 2H), 3.26 (s, 3H), 3.10 – 3.24 (m, 2H), 3.08 (s, 3H), 2.82 – 2.92 (m, 1H), 2.64 – 2.74 (m, 1H), 2.52 (dd, J = 15.56, 9.46 Hz, 1H), 2.22 – 2.33 (m, 1H), 1.95 (s, 3H), 1.80 – 1.87 (m, 3H), 0.88 – 0.93 (m, 3H); ¹³C NMR (126 MHz, CD₃OD) δ 167.0, 163.4, 143.3, 140.0, 132.6, 132.5, 116.9, 116.7, 116.1, 55.1, 51.9, 50.3, 39.6, 36.1, 28.0, 27.1, 24.5, 23.0, 20.7, 11.5. HRMS m/z calcd for C₂₀H₂₇FN₄O [M + H]⁺ 359.4689; found: 359.4670.

N,N-Dimethyl-1-propyl-5-((4-(trifluoromethyl)phenethyl)amino)-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (7b).

Prepared according to General procedure C using 6a (30 mg, 0.120 mmol), to afford 7b (19 mg, 37 %) as white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.88 (s, 2H, FA), 8.36 (s, 1H), 7.46 (d, J = 7.7 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 3.82 (t, J = 7.5 Hz, 2H), 3.23 (d, J = 15.7 Hz, 6H), 3.12 − 2.99 (m, 3H), 2.95 (s, 3H), 2.81 − 2.66 (m, 2H), 2.57 (q, J = 11.1, 8.8 Hz, 1H), 2.38 − 2.29 (m, 1H), 1.93 (h, J = 8.2, 6.5 Hz, 1H), 1.72 (h, J = 7.6 Hz, 2H), 0.81 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.5, 164.7, 142.0, 141.2, 137.4, 129.6, 129.4, 129.3, 125.8, 125.8, 125.8, 125.8, 114.9, 54.4, 51.0, 46.3, 39.2, 36.0, 32.6, 26.0, 25.0, 23.5, 19.8, 11.2. HRMS m/z calcd for C₂₂H₂₉F₃N₄O [M + H]⁺: 423.2366; found: 423.2371.

5-((2-(Benzo[d][1,3]dioxol-5-yl)ethyl)amino)-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (7c).

Prepared according to General procedure C using 6a (30 mg, 0.120 mmol), to afford 7c (17 mg, 36%) as white solid (FA salt). ¹H NMR (500 MHz, CDCl₃) δ 9.01 (s, 2H, FA), 8.41 (s, 1H), 6.68 − 6.62 (m, 3H), 3.89 (t, J = 7.3 Hz, 2H), 3.41 − 3.26 (m, 4H), 3.26 − 3.17 (m, 1H), 3.12 (tt, J = 10.7, 5.7 Hz, 1H), 3.03 (s, 3H), 2.93 (ddd, J = 17.0, 10.9, 5.5 Hz, 2H), 2.86 − 2.72 (m, 2H), 2.63 (ddd, J = 16.5, 10.5, 5.5 Hz, 1H), 2.45 − 2.35 (m, 1H), 2.00 (q, J = 7.8, 7.4 Hz, 1H), 1.79 (h, J = 7.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 167.4, 164.7, 148.0, 146.7, 142.0, 137.5, 130.6, 121.9, 114.9, 109.2, 108.6, 101.1, 54.3, 51.0, 46.9, 39.2, 36.0, 32.4, 25.9, 24.7, 23.5, 19.8, 11.3. HRMS m/z calcd for C₂₂H₃₀N₄O₃ [M + H]⁺: 399.2391; found: 399.2401.

(5-((4-Hydroxyphenethyl)amino)-1-propyl-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7d)

Prepared according to General procedure C 7d as white solid (FA salt). ¹H NMR (500 MHz, CDCl₃) δ 8.44 (s, 1H, FA), 7.09 − 7.00 (m, 2H), 6.81 − 6.71 (m, 2H), 3.93 (dd, J = 7.8, 6.4 Hz, 2H), 3.88 − 3.74 (m, 2H), 3.74 − 3.46 (m, 2H), 3.40 − 3.28 (m, 1H), 3.26 − 3.05 (m, 3H), 3.01 − 2.75 (m, 3H), 2.66 (tt, J = 15.1, 7.9 Hz, 2H), 2.42 − 2.28 (m, 1H), 2.02 − 1.91 (m, 1H), 1.81 (q, J = 7.3 Hz, 2H), 1.75 − 1.48 (m, 6H), 0.91 (t, J = 7.4 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 169.2, 165.1, 157.5, 143.4, 139.1, 131.2, 128.9, 117.2, 115.5, 55.3, 55.0, 52.4, 51.5, 49.9, 48.2, 45.0, 33.3, 28.2, 27.4, 27.2, 26.1, 25.8, 24.8, 21.0, 12.5.

5-Hydroxy-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (8)

To a solution of 6 (100 mg, 0.4 mmol) in MeOH (3 mL) was added NaBH₄ (45.5 mg, 1.2 mmol) and stirred for 2 h. The reaction was quenched with NaBHCO₃, and extracted with EtOAc (3x). The organic layers were combined, washed with brine and dried over Na₂SO₄, concentrated and purified by by flash silica gel column, with EA/hex, (gradient up to 99:1), to provide 8 (80 mg, 79%) as yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 4.10 (tt, J = 6.6, 4.2 Hz, 1H), 3.89 (t, J = 7.2 Hz, 2H), 3.25 (s, 3H), 3.01 (d, J = 15.1 Hz, 4H), 2.76 − 2.62 (m, 2H), 2.55 (dt, J = 15.9, 6.7 Hz, 1H), 1.96 − 1.88 (m, 2H), 1.79 (p, J = 7.3 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.4, 142.5, 137.8, 115.8, 66.5, 50.8, 39.1, 35.7, 30.6, 29.9, 23.5, 18.5, 11.2. LCMS (ESI) m/z: [M + H]⁺ 252.42.

5-((4-Fluorobenzyl)oxy)-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (9)

To a solution of 5-hydroxy-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide 8 (50 mg, 0.199 mmol) in DMF was added NaH (60% in mineral oil, 11.94 mg, 0.298 mmol) and 1-(bromomethyl)-4-fluorobenzene (0.037 mL, 0.298 mmol). The reaction mixture was heated at 80 °C overnight. The reaction was cooled to RT, quenched with saturated NH₄Cl, and extracted with EtOAc (3x). The organic layers were combined, washed with brine and dried over Na₂SO₄, concentrated and purified by preparative HPLC as described in the general methods to afford 9 (35 mg, 49%) as a light yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 7.32 − 7.27 (m, 2H), 7.04 − 6.96 (m, 2H), 4.61 (d, J = 11.9 Hz, 1H), 4.51 (d, J = 11.9 Hz, 1H), 3.92 (dd, J = 7.8, 6.5 Hz, 2H), 3.90 − 3.78 (m, 1H), 3.31 (s, 3H), 3.16 − 3.01 (m, 4H), 2.87 − 2.70 (m, 2H), 2.58 (dt, J = 15.8, 6.8 Hz, 1H), 2.08 − 1.94 (m, 2H), 1.82 (q, J = 7.3 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.2, 162.3 (d, J = 254.9), 142.6, 137.9, 134.6 (d, J = 2.9), 129.4 (d, J = 8.1), 116.0, 115.3 (d, J = 19.3), 73.5, 69.6, 50.9, 39.1, 35.8, 27.9, 27.3, 23.6, 18.9, 11.3. HRMS m/z calcd for C₂₀H₂₆FN₃O₂ [M + H]⁺: 360.2082; found: 360.2085.

3-(Dimethylcarbamoyl)-1-propyl-4,5,6,7-tetrahydro-1H-indazol-5-yl 4-methylbenzenesulfonate (10).

A dry flask containing 4-methylbenzene-1-sulfonyl chloride (91 mg, 0.48 mmol), DMAP (38.9 mg, 0.32 mmol), TEA (0.133 ml, 0.96 mmol) in DCM (1.5 ml) was cooled to 0 °C and then 8 (80 mg, 0.32 mmol) in DCM (1 ml) was added dropwise. The reaction was allowed to warm to RT and then stirred for 8 h. The reaction was diluted with DCM and washed with water and brine. The organic layer was dried, concentrated and purified via biotage to afford 10 (109 mg, 84%) as an off white fluffy solid. ¹H NMR (500 MHz, CDCl₃) δ 7.81 − 7.75 (m, 2H), 7.33 (d, J = 8.1 Hz, 2H), 4.91 − 4.86 (m, 1H), 3.92 (td, J = 7.0, 1.3 Hz, 2H), 3.30 (s, 3H), 3.03 (s, 3H), 2.93 (dd, J = 17.0, 4.7 Hz, 1H), 2.85 (d, J = 4.9 Hz, 1H), 2.83 − 2.73 (m, 1H), 2.68 − 2.59 (m, 1H), 2.44 (s, 3H), 2.32 − 2.22 (m, 1H), 2.05 − 1.95 (m, 1H), 1.81 (h, J = 7.3 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 164.7, 144.8, 142.6, 137.1, 134.1, 130.0, 127.8, 114.4, 77.7, 50.9, 4.1, 35.8, 28.2, 27.9, 23.5, 21.8, 17.7, 11.3. LCMS (ESI) m/z: [M + H]⁺ 406.46.

5-((4-Fluorobenzyl)thio)-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (11)

A solution of NaOEt (70.5 mg, 1.04 mmol) in EtOH (3 mL) was heated at 60 °C until a clear solution was formed. (4-Fluorophenyl)methanethiol (0.064 mL, 0.52 mmol) was then added and was stirred for 10 mins before addition of 3-(dimethylcarbamoyl)-1-propyl-4,5,6,7-tetrahydro-1H-indazol-5-yl 4-methylbenzenesulfonate 10 (140 mg, 0.35 mmol) in EtOH (3 mL). The stirring was continued at 60 °C for 6 h. The reaction was cooled to RT, quenched with saturated NH₄Cl, and extracted with EtOAc (3x). The organic layers were combined, washed with brine and dried over Na₂SO₄, concentrated and purified by preparative HPLC according to the general method to afford 11 (18 mg, 22%) as a clear oil. ¹H NMR (500 MHz, CDCl₃) δ 7.39 − 7.28 (m, 2H), 7.13 − 6.75 (m, 2H), 3.91 (t, J = 7.2 Hz, 2H), 3.78 (s, 2H), 3.30 (s, 3H), 3.18 (dd, J = 16.2, 5.1 Hz, 1H), 3.07 (s, 3H), 2.93 (tdd, J = 8.0, 6.5, 2.8 Hz, 1H), 2.71 (dq, J = 16.4, 5.0, 4.1 Hz, 2H), 2.60 − 2.43 (m, 1H), 2.10 (dq, J = 10.8, 2.7 Hz, 1H), 1.82 (dq, J = 14.6, 7.4 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.2, 161.9 (d, J = 247.0), 142.3, 137.7, 134.3 (d, J = 3.2), 130.5 (d, J = 8.7), 116.9, 115.5 (d, J = 21.7), 50.9, 39.3, 39.1, 35.8, 34.3, 28.8, 28.5, 23.6, 20.5, 11.3. HRMS m/z calcd for C₂₀H₂₆FN₃OS [M + H]⁺: 373.2035; found: 373.2039.

5-Amino-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (12)

A solution of 10 (140 mg, 0.35 mmol) and sodium azide (112 mg, 1.72 mmol) in DMF (15 ml) was heated at 70 °C overnight. The reaction mixture was then diluted with EtOAc and washed with brine. The organic layer was dried with sodium sulfate, concentrated and purified via biotage to afford 5-azido-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide as a white solid (80 mg, 84%) which was used immediately for the next step. ¹H NMR (500 MHz, CDCl₃) δ 3.97 − 3.85 (m, 3H), 3.32 (s, 3H), 3.15 − 3.09 (m, 1H), 3.07 (s, 3H), 2.84 − 2.70 (m, 2H), 2.67 − 2.58 (m, 1H), 2.09 − 1.93 (m, 2H), 1.83 (h, J = 7.3 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 164.7, 142.5, 137.1, 115.1, 56.7, 50.8, 38.9, 35.7, 27.3, 27.1, 23.4, 18.7, 11.1. LCMS (ESI) m/z: [M + H]⁺ 277.31.

To a solution of 5-azido-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (90 mg, 0.326 mmol) in EtOH (6 ml) was added Pd/C (30 mg, 0.326 mmol). A balloon filled with hydrogen was attached to the reaction flask via a needle and the reaction was stirred for 3 h at RT. The reaction mixture was filtered through a pad of Celite, washed with EtOH, concentrated and purified by silica gel column to afford 12 (55 mg, 56%) as a clear oil. ¹H NMR (500 MHz, CDCl₃) δ 3.91 (t, J = 7.2 Hz, 2H), 3.56 (p, J = 7.3 Hz, 1H), 3.29 (s, 3H), 3.18 (dd, J = 15.9, 5.0 Hz, 1H), 3.03 (s, 3H), 2.92 − 2.75 (m, 2H), 2.67 (pd, J = 12.2, 9.5, 4.1 Hz, 1H), 2.38 − 2.25 (m, 1H), 2.16 − 2.03 (m, 1H), 1.81 (h, J = 7.4 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.0, 141.8, 137.5, 114.7, 51.1, 47.6, 39.3, 36.1, 26.7, 26.6, 23.5, 19.2, 11.3. LCMS (ESI) m/z: [M + H]⁺ 251.34.

5-(4-Fluorobenzamido)-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (13).

Prepared according to General procedure A using 12 (25 mg, 0.1 mmol) to afford 13 (18 mg, 48%) as white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.06 (td, J = 7.9, 1.8 Hz, 1H), 7.44 (tdd, J = 7.4, 5.2, 1.9 Hz, 1H), 7.24 (d, J = 7.5 Hz, 1H), 7.08 (dd, J = 12.1, 8.3 Hz, 1H), 6.72 (dd, J = 12.8, 7.5 Hz, 1H), 4.45 (ddt, J = 9.8, 4.8, 2.4 Hz, 1H), 3.96 (t, J = 7.1 Hz, 2H), 3.31 (s, 3H), 3.15 (dd, J = 16.0, 5.2 Hz, 1H), 3.06 (s, 3H), 2.79 − 2.60 (m, 3H), 2.22 (dq, J = 10.0, 3.0 Hz, 1H), 2.05 − 1.92 (m, 1H), 1.83 (q, J = 7.3 Hz, 2H), 1.74 (q, J = 6.9, 6.5 Hz, 1H), 0.91 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.0, 163.0, 160.6 (d, J = 246.7), 142.8, 137.7, 133.3 (d, J = 9.2), 132.0, 124.9 (d, J = 3.3), 121.4, 121.3, 116.1 (d, J = 25.0), 115.9, 50.9, 46.1, 39.1, 35.8, 29.8, 28.2, 27.9, 23.6, 19.4, 11.2. HRMS m/z calcd for C₂₀H₂₅FN₄O₂ [M + H]⁺ 395.1854; found: 395.1860.

5-((4-Fluorophenyl)sulfonamido)-N,N-dimethyl-1-propyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (14).

Triethylamine (0.056 mL, 0.399 mmol) was added to a solution of 12 (20 mg, 0.080 mmol) in DCM (1 mL). The reaction mixture was cooled to 0 °C and then 4-fluorobenzene-1-sulfonyl chloride (15.55 mg, 0.080 mmol) was added in portions. The reaction was warmed to RT and stirred until completion. The reaction mixture was washed with 1M HCl and water, dried, concentrated and purified by preparative HPLC according to the general method to afford 14 (18 mg, 55%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.08 − 7.91 (m, 2H), 7.26 (t, J = 8.6 Hz, 2H), 4.00 (t, J = 7.1 Hz, 2H), 3.71 − 3.59 (m, 1H), 3.38 (s, 3H), 3.13 (s, 3H), 2.92 (dd, J = 16.2, 5.1 Hz, 1H), 2.81 (dt, J = 16.5, 6.3 Hz, 1H), 2.69 (td, J = 15.7, 15.2, 6.8 Hz, 2H), 2.15 − 2.05 (m, 2H), 1.89 (p, J = 7.3 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 164.8, 164.2 (d, J = 253.1), 142.5, 137.6, 136.9 (d, J = 4.2), 129.8 (d, J = 9.4), 116.4 (d, J = 22.1), 115.2, 50.9, 49.3, 39.1, 35.9, 29.0, 28.7, 23.5, 18.7, 11.3. HRMS m/z calcd for C₁₉H₂₅FN₄O₃S [M + H]⁺ 409.1704; found: 409.1706.

(1-Phenyl-1,4,6,7-tetrahydrospiro[indazole-5,2’-[1,3]dioxolan]-3-yl)(piperidin-1-yl)methanone (6ca).

Prepared according to General procedure D from 5c to afford 6ca as pale yellow oil (38%). ¹H NMR (500 MHz, CDCl₃) δ 7.29 (d, J = 7.6 Hz, 2H), 7.24 (t, J = 7.8 Hz, 2H), 7.13 (t, J = 7.4 Hz, 1H), 3.93 − 3.76 (m, 4H), 3.68 (t, J = 5.3 Hz, 2H), 3.50 (t, J = 5.1 Hz, 2H), 2.78 (s, 2H), 2.73 (t, J = 6.5 Hz, 2H), 1.78 (t, J = 6.5 Hz, 2H), 1.53−1.31 (m, 6H).

(2-Phenyl-2,4,6,7-tetrahydrospiro[indazole-5,2’-[1,3]dioxolan]-3-yl)(piperidin-1-yl)methanone (6da).

Prepared according to General procedure D from 5c to afford 6da as pale yellow oil (37%). ¹H NMR (500 MHz, CDCl₃) δ 7.33 (dd, J = 7.9, 1.6 Hz, 2H), 7.24 (t, J = 7.9 Hz, 2H), 7.13 (dd, J = 13.9, 6.4 Hz, 1H), 3.96 − 3.78 (m, 4H), 3.58 (s, 1H), 3.29 (s, 1H), 2.99 − 2.68 (m, 5H), 2.52 (d, J = 16.1 Hz, 1H), 1.87 (t, J = 6.5 Hz, 2H), 1.40 (s, 1H), 1.32 (q, J = 5.7 Hz, 2H), 1.25 (s, 1H), 1.10 (s, 1H), 0.65 (s, 1H).

4-(3-(Piperidine-1-carbonyl)-6,7-dihydrospiro[indazole-5,2’-[1,3]dioxolan]-2(4H)-yl)benzamide (6db).

Prepared according to General procedure D from 5c to afford 6db as pale yellow oil (17%). ¹H NMR (500 MHz, CDCl₃) δ 7.92 − 7.74 (m, 2H), 7.63 − 7.43 (m, 2H), 4.00 (s, 4H), 3.78 − 3.40 (m, 2H), 3.21 − 2.75 (m, 5H), 2.64 (d, J = 16.2 Hz, 1H), 2.00 (t, J = 6.8 Hz, 2H), 1.74 − 1.39 (m, 4H), 1.32 (d, J = 12.3 Hz, 1H), 0.97 (d, J = 22.0 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 168.8, 163.9, 161.2, 149.7, 142.6, 133.6, 131.5, 128.6, 121.9, 117.2, 108.3, 64.7, 47.5, 42.9, 31.7, 31.1, 26.2, 25.3, 24.2, 21.2. LCMS (ESI) m/z: [M + H]⁺ 411.29.

(2-(4-Hydroxyphenyl)-2,4,6,7-tetrahydrospiro[indazole-5,2’-[1,3]dioxolan]-3-yl)(piperidin-1-yl)methanone (6dc).

Prepared according to General procedure D from 5c to afford 6dc as pale yellow oil (20%). ¹H NMR (500 MHz, CDCl₃) δ 7.31 − 7.12 (m, 2H), 6.93 − 6.74 (m, 2H), 4.03 (s, 4H), 3.65 (dd, J = 12.1, 7.2 Hz, 1H), 3.42 (t, J = 10.4 Hz, 1H), 3.15 − 2.94 (m, 2H), 2.94 − 2.81 (m, 3H), 2.65 (d, J = 15.9 Hz, 1H), 2.02 (q, J = 6.3 Hz, 2H), 1.64 − 1.44 (m, 3H), 1.44 − 1.21 (m, 2H), 0.89 (d, J = 16.3 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 161.5, 157.0, 148.2, 133.7, 131.6, 124.6, 115.7, 115.6, 108.3, 64.6, 47.6, 42.8, 31.5, 30.8, 25.8, 25.1, 23.9, 20.8. LCMS (ESI) m/z: [M + H]⁺ 384.41.

(5-((4-Fluorobenzyl)amino)-1-phenyl-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7aa).

Prepared according to General procedure C to afford 7aa as a colorless oil (84%). ¹H NMR (500 MHz, CDCl₃) δ 8.09 (s, 1H, FA), 7.36 − 7.20 (m, 6H), 7.20 − 7.11 (m, 1H), 6.80 (t, J = 8.4 Hz, 2H), 3.96 (d, J = 12.9 Hz, 1H), 3.85 (d, J = 12.9 Hz, 1H), 3.70 (t, J = 5.3 Hz, 2H), 3.47 (t, J = 5.3 Hz, 2H), 3.15 (q, J = 8.3, 5.2 Hz, 2H), 2.80 − 2.66 (m, 1H), 2.57 (dq, J = 9.7, 5.4, 4.4 Hz, 2H), 2.00 (d, J = 12.5 Hz, 1H), 1.64 (dd, J = 11.9, 6.9 Hz, 1H), 1.55−1.32 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 166.8, 163.0 (d, J = 245.2), 162.8, 143.8, 139.1, 137.9, 132.1 (d, J = 7.7), 129.3, 127.7, 127.6 (d, J = 3.1), 123.2, 116.3, 115.9 (d, J = 21.1), 52.9, 48.3, 47.8, 43.5, 26.7, 26.0, 25.8, 24.6, 24.4, 21.5. HRMS m/z calcd for C₂₆H₂₉FN₄O [M + H]⁺ 432.2398; found: 432.2404.

(5-(Benzylamino)-1-phenyl-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7ab).

Prepared according to General procedure C to afford 7ab as a colorless oil (61%). ¹H NMR (500 MHz, CDCl₃) δ 8.12 (s, 1H, FA), 7.35−7.19 (m, 6H), 7.19−7.09 (m, 3H), 7.08 (d, J = 7.1 Hz, 1H), 3.93 (d, J = 13.0 Hz, 1H), 3.83 (d, J = 13.0 Hz, 1H), 3.68 (t, J = 5.3 Hz, 2H), 3.47 (t, J = 5.4 Hz, 2H), 3.20−3.07 (m, 2H), 2.79−2.66 (m, 1H), 2.57 (qd, J = 10.8, 4.7 Hz, 2H), 2.12 − 1.99 (m, 1H), 1.78 − 1.63 (m, 1H), 1.57 − 1.35 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 167.1, 162.8, 143.9, 139.2, 137.9, 132.1, 129.9, 129.3, 128.9, 128.9, 127.6, 123.2, 116.4, 52.5, 48.4, 48.3, 43.4, 26.8, 26.1, 25.8, 24.7, 24.5, 21.6. HRMS m/z calcd for C₂₆H₃₀N₄O [M + H]⁺ 415.2492; found: 415.2488.

(1-Phenyl-5-((4-(trifluoromethyl)phenethyl)amino)-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7ac).

Prepared according to General procedure C to afford 7ac as a colorless oil (72%). ¹H NMR (500 MHz, CDCl₃) δ 8.21 (s, 1H, FA), 7.35 (d, J = 7.9 Hz, 2H), 7.30 − 7.21 (m, 4H), 7.21 − 7.12 (m, 3H), 3.70 (t, J = 5.3 Hz, 2H), 3.46 (q, J = 4.4 Hz, 2H), 3.34 (q, J = 9.3, 7.8 Hz, 1H), 3.15 (td, J = 12.6, 10.4, 5.5 Hz, 2H), 3.01 (dtd, J = 50.4, 13.0, 12.0, 5.3 Hz, 3H), 2.82 − 2.61 (m, 3H), 2.21 (d, J = 12.3 Hz, 1H), 1.91 − 1.76 (m, 1H), 1.47 (dq, J = 25.6, 5.4, 5.0 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 167.1, 162.8, 143.8, 140.9, 139.0, 137.9, 129.3, 129.1, 127.7, 125.7, 125.7, 125.7, 125.7, 123.2, 116.2, 54.0, 48.3, 46.3, 43.5, 32.4, 26.7, 25.9, 25.8, 24.6, 24.6, 21.5. HRMS m/z calcd for C₂₈H₃₁F₃N₄O [M + H]⁺ 497.1523; found: 497.2526.

(5-((2-(Benzo[d][1,3]dioxol-5-yl)ethyl)amino)-1-phenyl-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7ad).

Prepared according to General procedure C to afford 7ad as a colorless oil (82%). ¹H NMR (500 MHz, CDCl₃) δ 8.23 (s, 1H, FA), 7.29 − 7.20 (m, 4H), 7.20−7.13 (m, 1H), 6.61 − 6.35 (m, 3H), 5.73 (s, 2H), 3.69 (t, J = 5.3 Hz, 2H), 3.47 (p, J = 6.5, 5.2 Hz, 2H), 3.35 − 3.21 (m, 1H), 3.13 (dd, J = 15.6, 5.1 Hz, 1H), 3.06 (td, J = 11.0, 5.9 Hz, 1H), 2.96 (td, J = 11.1, 5.3 Hz, 1H), 2.87 − 2.61 (m, 5H), 2.27 − 2.15 (m, 1H), 1.91 − 1.73 (m, 1H), 1.46 (dq, J = 27.9, 6.0, 5.3 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 167.3, 162.8, 147.9, 146.5, 143.9, 139.1, 137.9, 130.5, 129.3, 127.6, 123.2, 121.8, 121.6, 116.3, 109.1, 108.5, 108.4, 100.9, 53.9, 48.3, 46.8, 43.4, 32.5, 26.7, 26.2, 25.8, 24.6, 24.6, 21.6. HRMS m/z calcd for C₂₈H₃₂N₄O₃ [M + H]⁺ 473.2547; found: 473.2553.

(5-((4-Fluorophenethyl)amino)-1-phenyl-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7ae).

Prepared according to General procedure C to afford 7ae as a colorless oil (78%). ¹H NMR (500 MHz, CDCl₃) δ 8.24 (s, 1H, FA), 7.33 − 7.21 (m, 4H), 7.21 − 7.14 (m, 1H), 6.98 (dd, J = 8.4, 5.2 Hz, 2H), 6.77 (t, J = 8.4 Hz, 2H), 3.70 (t, J = 5.2 Hz, 2H), 3.46 (q, J = 4.6 Hz, 2H), 3.30 (q, J = 7.2, 6.4 Hz, 1H), 3.18 − 3.05 (m, 2H), 3.05 − 2.94 (m, 1H), 2.86 (t, J = 12.2 Hz, 2H), 2.78 − 2.58 (m, 3H), 2.20 (d, J = 11.3 Hz, 1H), 1.81 (t, J = 9.5 Hz, 1H), 1.47 (dq, J = 24.9, 5.7, 5.2 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ δ 167.35, 162.79, 161.86 (d, J = 245.31 Hz), 143.9, 139.1, 137.9, 132.5 (d, J = 3.2 Hz), 130.3 (d, J = 7.9 Hz), 129.3, 127.7, 123.2, 116.3, 115.6 (d, J = 23.0 Hz), 53.9, 48.3, 46.7, 43.4, 31.9, 26.7, 26.1, 25.8, 24.6, 21.5. HRMS m/z calcd for C₂₇H₃₁FN₄O [M + H]⁺ 447.2555; found: 447.2563.

(5-((3-Hydroxyphenethyl)amino)-1-phenyl-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7af).

Prepared according to General procedure C to afford 7af as a colorless oil (55%). ¹H NMR (500 MHz, CDCl₃) δ 8.32 (s, 1H, FA), 7.47 − 7.28 (m, 5H), 6.97 (t, J = 7.8 Hz, 1H), 6.75 (s, 1H), 6.69 − 6.62 (m, 1H), 6.57 (d, J = 7.4 Hz, 1H), 3.81 (q, J = 4.8 Hz, 2H), 3.66 − 3.51 (m, 2H), 3.51 − 3.40 (m, 1H), 3.32 − 3.13 (m, 3H), 2.89 (dq, J = 35.3, 9.6, 8.4 Hz, 3H), 2.74 (t, J = 4.8 Hz, 2H), 2.33 (d, J = 12.1 Hz, 1H), 2.02 − 1.81 (m, 1H), 1.60 (dq, J = 27.6, 6.9, 6.2 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 167.8, 162.8, 157.8, 143.6, 139.0, 138.1, 137.8, 129.8, 129.3, 127.7, 123.2, 119.6, 116.1, 115.9, 114.5, 54.1, 48.4, 46.5, 43.6, 32.4, 26.7, 25.7, 25.5, 24.5, 24.4, 21.5. HRMS m/z calcd for C₂₇H₃₂N₄O₂ [M + H]⁺ 445.2598; found: 445.2586.

(1-Benzyl-5-((2-(pyridin-4-yl)ethyl)amino)-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7ag).

Prepared according to General procedure C to afford 7ag as a colorless oil (53%). ¹H NMR (500 MHz, CDCl₃) δ 8.60 − 8.47 (m, 2H), 8.24 (s, 3H), 7.33 − 7.27 (m, 3H), 7.23 − 7.19 (m, 2H), 7.15 − 7.06 (m, 2H), 3.85 (qt, J = 12.6, 5.0 Hz, 2H), 3.64 (pt, J = 12.6, 5.2 Hz, 2H), 3.40 − 3.17 (m, 4H), 3.06 (dddd, J = 20.6, 13.2, 10.3, 5.3 Hz, 2H), 2.83 (dd, J = 15.4, 9.6 Hz, 1H), 2.69 (ddd, J = 16.6, 6.0, 3.2 Hz, 1H), 2.48 (ddd, J = 16.7, 10.4, 5.9 Hz, 1H), 2.29 (d, J = 13.4 Hz, 1H), 2.03 − 1.88 (m, 1H), 1.74 − 1.54 (m, 7H). ¹³C NMR (126 MHz, CDCl₃) δ 165.7, 163.0, 148.9, 147.0, 142.2, 137.7, 135.7, 128.9, 128.1, 127.1, 124.4, 115.1, 54.3, 53.6, 48.4, 45.5, 43.6, 31.9, 26.7, 25.8, 25.6, 24.7, 24.6, 19.5. HRMS m/z calcd for C₂₇H₃₃N₅O [M + H]⁺ 444.2785; found: 444.2763.

(1-Benzyl-5-((4-fluorobenzyl)amino)-4,5,6,7-tetrahydro-1H-indazol-3-yl)(piperidin-1-yl)methanone (7ah).

Prepared according to General procedure C to afford 7ah as a colorless oil (62%). ¹H NMR (500 MHz, CDCl₃) δ 8.24 (s, 1H), 7.49 − 7.38 (m, 2H), 7.35 − 7.27 (m, 4H), 7.17 − 7.08 (m, 2H), 7.04 − 6.92 (m, 2H), 4.08 (d, J = 13.0 Hz, 1H), 3.93 (d, J = 13.0 Hz, 1H), 3.85 (q, J = 6.3 Hz, 2H), 3.65 (dq, J = 13.5, 8.0, 6.7 Hz, 2H), 3.28 (dd, J = 15.3, 5.1 Hz, 1H), 3.20 − 3.08 (m, 1H), 2.78 (dd, J = 15.3, 9.7 Hz, 1H), 2.57 (ddd, J = 16.5, 5.9, 2.8 Hz, 1H), 2.37 (ddd, J = 16.5, 10.6, 5.8 Hz, 1H), 2.13 − 1.99 (m, 1H), 1.82 − 1.49 (m, 8H). ¹³C NMR (126 MHz, CDCl₃) δ 166.56, 164.0, 163.3, 142.5, 137.9, 136.1, 132.2, 132.1, 129.0, 128.4, 128.2, 127.3, 116.0, 115.8, 115.6, 53.7, 53.2, 48.4, 48.1, 43.6, 26.9, 26.0, 25.9, 24.8, 24.7, 19.8. HRMS m/z calcd for C₂₇H₃₁FN₄O [M + H]⁺ 447.2555; found: 447.2566.

(5-((4-Fluorobenzyl)amino)-2-phenyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)(piperidin-1-yl)methanone (7ba).

Prepared according to General procedure C to afford 7ba as a colorless oil (59%). ¹H NMR (500 MHz, CDCl₃) δ 8.23 (s, 2H, FA), 7.42 (dt, J = 21.7, 7.7 Hz, 8H), 7.35 − 7.27 (m, 1H), 4.21 − 3.90 (m, 2H), 3.86 − 3.63 (m, 1H), 3.48 − 3.10 (m, 3H), 3.10 − 2.79 (m, 5H), 2.79 − 2.59 (m, 2H), 2.22 (d, J = 13.1 Hz, 1H), 1.90 (d, J = 15.1 Hz, 1H), 1.55 (s, 1H), 1.44 (p, J = 5.8 Hz, 2H), 1.35 (s, 1H), 1.21 (d, J = 24.9 Hz, 1H), 0.63 (s, 1H). ¹³C NMR (126 MHz, CDCl₃)δ 166.4, 162.1, 161.2, 148.3, 139.5, 133.6, 131.9, 131.9, 129.3, 127.7, 127.4, 122.8, 116.2, 116.0, 114.1, 53.3, 47.9, 47.4, 42.8, 26.8, 25.5, 25.1, 24.1, 23.5, 21.5. HRMS m/z calcd for C₂₆H₂₉FN₄O [M + H]⁺ 433.2398; found: 433.2404.

(5-(Benzylamino)-2-phenyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)(piperidin-1-yl)methanone (7bb).

Prepared according to General procedure C to afford 7bb as a colorless oil (40%). ¹H NMR (500 MHz, CDCl₃ δ 8.32 (s, 1H, FA), 7.62 − 7.18 (m, 10H), 4.24 − 4.07 (m, 1H), 3.77 (tt, J = 18.0, 8.4 Hz, 1H), 3.48 − 3.30 (m, 2H), 3.28 − 3.07 (m, 1H), 3.07 − 2.82 (m, 4H), 2.82 − 2.56 (m, 2H), 2.31 (d, J = 16.8 Hz, 1H), 2.09 − 1.82 (m, 1H), 1.56 (s, 1H), 1.47 (p, J = 6.2 Hz, 2H), 1.36 (s, 1H), 1.32 − 1.16 (m, 1H), 0.67 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 166.9, 161.2, 148.6, 139.2, 129.4, 129.2, 129.0, 128.9, 128.0, 122.8, 114.5, 52.9, 47.5, 42.9, 26.7, 25.5, 24.9, 24.2, 23.9, 21.2. HRMS m/z calcd for C₂₆H₃₀N₄O [M + H]⁺ 415.2492; found: 415.2497.

(2-Phenyl-5-((4-(trifluoromethyl)phenethyl)amino)-4,5,6,7-tetrahydro-2H-indazol-3-yl)(piperidin-1-yl)methanone (7bc).

Prepared according to General procedure C to afford 7bc as a colorless oil (63%). ¹H NMR (500 MHz, CDCl₃) δ 8.39 (s, 1H, FA), 7.54 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 7.39 (t, J = 7.8 Hz, 2H), 7.36 − 7.27 (m, 3H), 3.88 − 3.66 (m, 1H), 3.47 − 3.23 (m, 3H), 3.23 − 3.05 (m, 4H), 3.04 − 2.58 (m, 5H), 2.52 − 2.30 (m, 1H), 2.12 − 1.89 (m, 1H), 1.52 (d, J = 13.3 Hz, 1H), 1.41 (qd, J = 8.0, 5.2 Hz, 2H), 1.32 (s, 1H), 1.21 (d, J = 15.7 Hz, 1H), 0.59 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) 167.3, 161.1, 148.4, 141.1, 139.5, 133.6, 129.3, 129.0, 127.7, 125.7, 125.1, 122.9, 122.7, 114.6, 114.2, 54.4, 47.4, 46.5, 42.8, 32.8, 27.3, 25.5, 25.0, 24.0, 23.9, 21.6, 21.5. HRMS m/z calcd for C₂₈H₃₁F₃N₄O [M + H]⁺ 497.2523; found: 497.2526.

(5-((2-(Benzo[d][1,3]dioxol-5-yl)ethyl)amino)-2-phenyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)(piperidin-1-yl)methanone (7bd).

Prepared according to General procedure C to afford 7bd as a colorless oil (74%). ¹H NMR (500 MHz, CDCl₃) δ 8.40 (s, 1H, FA), 7.45 (d, J = 7.8 Hz, 2H), 7.39 (dd, J = 8.7, 7.0 Hz, 2H), 7.34 − 7.27 (m, 1H), 6.77 − 6.56 (m, 3H), 5.91 (d, J = 4.6 Hz, 2H), 3.79 (dd, J = 14.0, 6.6 Hz, 1H), 3.31 (q, J = 14.1, 10.9 Hz, 2H), 3.24 − 3.01 (m, 3H), 3.01 − 2.82 (m, 5H), 2.82 − 2.62 (m, 2H), 2.41 (d, J = 14.0 Hz, 1H), 2.12 − 1.86 (m, 1H), 1.53 (s, 1H), 1.47 − 1.38 (m, 2H), 1.33 (s, 1H), 1.27 − 1.12 (m, 1H), 0.61 (d, J = 13.0 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) 167.3, 161.1, 148.5, 147.9, 146.6, 139.6, 133.7, 130.6, 129.3, 127.6, 122.7, 121.7, 121.6,114.2, 109.0, 108.5, 108.4, 101.0, 100.9, 54.3, 47.4, 47.0, 42.8, 39.3, 35.2, 32.6, 27.2, 25.5, 25.0, 24.1, 23.8, 21.6. HRMS m/z calcd for C₂₈H₃₂N₄O₃ [M + H]⁺ 473.2547; found: 473.2552.

(2-(4-Fluorophenyl)-5-((2-(pyridin-4-yl)ethyl)amino)-4,5,6,7-tetrahydro-2H-indazol-3-yl)(piperidin-1-yl)methanone (7be).

Prepared according to General procedure C to afford 7be as a colorless oil (54%) ¹H NMR (500 MHz, CDCl₃) δ 8.55 (s, 2H, FA), 8.18 (s, 1H), 7.57 − 7.32 (m, 4H), 7.08 (t, J = 8.3 Hz, 2H), 3.78 (d, J = 26.7 Hz, 1H), 3.55 − 3.10 (m, 7H), 3.10 − 2.61 (m, 6H), 2.52 (s, 1H), 2.21 − 1.88 (m, 1H), 1.64 − 1.03 (m, 6H), 0.56 (d, J = 26.5 Hz, 1H). HRMS m/z calcd for C₂₆H₃₀FN₅O [M + H]⁺ 448.2507; found: 448.2511.

(5-((4-Fluorobenzyl)amino)-2-(4-fluorophenyl)-4,5,6,7-tetrahydro-2H-indazol-3-yl)(piperidin-1-yl)methanone (7bf).

Prepared according to General procedure C to afford 7bf as a colorless oil (61%) ¹H NMR (500 MHz, CDCl₃) δ 8.20 (s, 1H), 7.64 − 7.51 (m, 2H), 7.49 − 7.34 (m, 2H), 7.16 − 7.07 (m, 2H), 7.01 (q, J = 9.7, 8.3 Hz, 2H), 4.31 − 4.05 (m, 2H), 3.88 − 3.59 (m, 1H), 3.42 − 3.11 (m, 2H), 3.09 − 2.73 (m, 4H), 2.73 − 2.58 (m, 1H), 2.37 (s, 1H), 2.16 − 1.87 (m, 1H), 1.47 (p, J = 6.4, 6.0 Hz, 3H), 1.27 (d, J = 57.1 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 165.6, 162.8, 162.2, 161.1, 160.9, 148.5, 135.9, 133.8, 132.3, 132.2, 127.2, 124.7, 116.4, 116.3, 116.1, 114.3, 52.9, 47.9, 47.6, 43.0, 26.5, 25.9, 25.3, 24.2, 23.6, 21.7. HRMS m/z calcd for C₂₆H₂₈F₂N₄O [M + H]⁺ 451.2304; found: 451.2038.

4-(5-(Phenethylamino)-3-(piperidine-1-carbonyl)-4,5,6,7-tetrahydro-2H-indazol-2-yl)benzamide (7bg).

Prepared according to General procedure C to afford 7bg as a colorless oil (63%). ¹H NMR (500 MHz, CDCl₃) δ 8.35 (s, 1H, FA), 8.06 − 7.55 (m, 2H), 7.41 (dd, J = 40.7, 8.2 Hz, 2H), 7.33 − 7.09 (m, 4H), 6.70 (s, 1H), 3.68 (ddd, J = 35.5, 14.8, 8.2 Hz, 1H), 3.58 − 3.34 (m, 2H), 3.34 − 3.06 (m, 5H), 3.06 − 2.86 (m, 3H), 2.86 − 2.66 (m, 2H), 2.40 (d, J = 47.7 Hz, 1H), 2.14 (d, J = 16.0 Hz, 1H), 1.63 – 1.14 (m, 5H), 0.84 (d, J = 14.5 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 168.8, 166.9, 160.9, 148.9, 142.2, 136.7, 136.5, 133.7, 131.8, 128.9, 128.7, 128.6 127.1, 121.8, 121.6, 114.6, 54.2, 47.4, 46.8, 42.9, 32.6, 26.4, 26.0, 25.1, 24.0, 23.5, 21.3, 20.7. HRMS m/z calcd for C₂₈H₃₃N₅O₂ [M + H]⁺ 472.2707; found: 472.2716.

(2-(4-Hydroxyphenyl)-5-(phenethylamino)-4,5,6,7-tetrahydro-2H-indazol-3-yl)(piperidin-1-yl)methanone (7bh).

Prepared according to General procedure C to afford 7bh as a colorless oil (76%). ¹H NMR (500 MHz, CDCl₃) δ 8.36 (s, 1H, FA), 7.31 (dd, J = 8.5, 6.4 Hz, 2H), 7.28 − 7.15 (m, 5H), 6.85 (d, J = 8.5 Hz, 2H), 3.71 (h, J = 8.2, 6.0 Hz, 1H), 3.41 − 3.31 (m, 2H), 3.21 (qd, J = 13.0, 11.7, 6.7 Hz, 2H), 3.00 (tt, J = 45.0, 20.9 Hz, 6H), 2.82 − 2.54 (m, 2H), 2.35 (d, J = 12.5 Hz, 1H), 2.09 − 1.87 (m, 1H), 1.61 − 1.41 (m, 3H), 1.41 − 1.16 (m, 2H), 0.76 (s, 1H). ¹³C NMR (126MHz, CDCl₃)δ 166.8, 161.2, 157.2, 147.6, 136.4, 133.7, 131.4, 128.8, 128.6, 127.1, 124.5, 1 15.9, 113.3, 54.2, 53.9, 47.6, 46.4, 42.9, 32.5, 26.2, 25.5, 24.9, 23.9, 23.5, 21.2, 20.9. HRMS m/z calcd for C₂₇H₃₂N₄O₂ [M + H]⁺ 445.2598; found: 445.2606.

ABBREVIATIONS

CNS

central nervous system

DIPEA

N,N-diisopropylethylamine

DMF

dimethylformamide

LDA

Lithium diisopropylamide

EDC

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

DMAP

4-Dimethylaminopyridine

FA

formic acid

HPLC

high-performance liquid chromatography

TFA

trifluoroacetic acid

THF

tetrahydrofuran

AcOH

acetic acid

Ki

inhibitory constant

QSAR

Quantitative Structure Activity Relationship

PBS

Phosphate buffered saline

MLM

Mouse Liver Microsomal