Asymmetric Synthesis of the TRPV1 Modulator AMG8562 via Lipase-Catalyzed Kinetic Resolution

Abstract

The transient receptor potential vanilloid-1 (TRPV1) channel is a key mediator of pain perception and responds to various stimuli such as heat, low pH, and inflammation. Among TRPV1 antagonists, (R,E)-N-(2-hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)­phenyl)-acrylamide (AMG8562) shows promise as a nonopioid analgesic, attenuating pain behaviors in rodent models without eliciting hyperthermic side effects commonly associated with other TRPV1 antagonists. Despite its potential, there has been limited research regarding the synthesis of AMG8562. Here, we present an asymmetric synthesis of AMG8562 featuring a lipase-catalyzed kinetic resolution of racemic 4-nitroindan-2-ol. This approach enables scalable access to enantiomerically pure material and provides a platform for the synthesis of structurally diverse AMG8562 analogues with potential for improved pharmacological properties.

Introduction

The transient receptor potential vanilloid-1 (TRPV1) channel is a polymodal cation channel involved in nociception. It is primarily expressed in the central and peripheral nervous system, and it is activated by a variety of painful stimuli, including heat (>43 °C), protons (pH < 5.3), and tissue damage, as well as by modulators, such as capsaicin, resiniferatoxin, substance P (SP), nerve growth factor (NGF), and prostaglandins. Genetic and pharmacological investigations have elucidated that TRPV1 agonists induce pain responses, , whereas mice with TRPV1 gene deletion exhibit reduced pain behaviors. − Furthermore, TRPV1 antagonists have been demonstrated to attenuate pain behaviors in rodent models of pain. , Based on this evidence, pharmacological targeting of TRPV1 has attracted particular interest, with TRPV1 antagonists becoming candidates for nonopioid pain management.

The pharmacophore of capsaicin-like TRPV1 ligands is well-defined, characterized by an aromatic “head” region, a linker “neck”, and a hydrophobic “tail” region (Figure A). However, shortly after the onset of clinical trials for such compounds, it was discovered that modulation of TRPV1 was accompanied by significant hyperthermia in both preclinical − and clinical trials. − This side-effect has been linked to TRPV1 modulation via ligand-induced conformational changes in the S4–S5 linker region, inducing hyperthermia by disrupting the tonic activation of TRPV1 by protons in the trunk. This disruption affects thermoregulatory reflexes, including thermogenesis and cutaneous vasoconstriction, leading to altered core body temperature. , Attempts to mitigate this effect have included structural modifications such as minimizing central nervous system penetration. However, these attempts have failed to eliminate hyperthermia. For example, peripherally restricted TRPV1 antagonists with low brain-to-plasma ratios still induce hyperthermia in preclinical models. Thus, when Lehto et al. reported that (R,E)-N-(2-hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)­phenyl)-acrylamide (AMG8562) (1, Figure A), an N-aryl trans-cinnamide, significantly reduced pain behaviors in rodent models while not inducing hyperthermia, AMG8562 (1) garnered a significant amount of attention. It was notable that while AMG8562 (1) acted as a TRPV1 antagonist while bypassing the transient hyperthermic effect in rats, its (S)-enantiomer AMG8563 (2) showed similar antagonist capabilities but elicited a maximal increase in temperature of 1.2 °C at 40 min postadministration. This phenomenon of the two enantiomers of an TRPV1 antagonist eliciting divergent physiological responses underscores the critical role of stereochemistry in their pharmacological activity.

1.

(A) Structure of AMG8562 (1) and its (S)-enantiomer AMG8563 (2); (B) retrosynthesis of AMG8562 (1).

We were intrigued by the potential of AMG8562 (1) to provide insights on how to eliminate the on-target hyperthermic effect observed with other TRPV1 antagonists. While AMG8562 (1) is expected to have a hyperthermic effect in humans, studying its lack of such an effect in rodent models is crucial for guiding TRPV1 antagonist design to achieve similar outcomes in humans. Herein, we describe an asymmetric synthesis of AMG8562 (1) that leverages a lipase-catalyzed kinetic resolution of the head fragment, enabling access to AMG8562 (1) for further study. We found that while the head fragment follows Kazlauskas’ rule for the acylation of secondary racemic alcohols, the differences in the substituents next to the hydroxyl group are more subtle than what would be expected for the observed enantioselectivity; the enantiomeric excess (ee) achieved is significantly high compared to previously reported substrates.

Results and Discussion

The chiral (R)-4-aminoindan-2-ol ((R)-4, Figure B), the key fragment for the asymmetric synthesis of AMG8562 (1), is a reported structural motif in TRPV1 antagonists. It has previously been separated from its racemate using chiral high-performance liquid chromatography (HPLC). But separating desired chiral building blocks from racemic mixtures by chiral HPLC may present a challenge in the development of AMG8562 derivatives, primarily the limited scalability of chiral HPLC separation technique. Therefore, there is a need for alternative methods for the preparation of chiral secondary alcohols in high enantiomeric excess (ee). Toward this goal, we explored enzymatic chiral resolution. Figure B illustrates our approach to a convergent synthesis of AMG8562 (1) which could be realized by coupling α,β-unsaturated carboxylic acid 3 with (R)-4-aminoindan-2-ol ((R)-4). Carboxylic acid 3 is derived from the commercially available 2-fluoro-4-(trifluoromethyl)­benzoic acid (5), while (R)-4-aminoindan-2-ol ((R)-4) is obtained from lipase-catalyzed kinetic resolution of racemic 4-nitroindan-2-ol (rac-6) followed by NO₂ reduction.

To this end, we prepared racemic 4-nitroindan-2-ol (rac-6) for kinetic resolution and subsequent NO₂ reduction (Scheme ). Starting with commercially available 4-nitroindan-1-one (7), NaBH₄ reduction to the corresponding nitroindanol in 97% yield followed by elimination of benzylic alcohol in the presence of a catalytic amount of PTSA yielded nitroindene 8 (94%). Epoxidation by m-CPBA to give epoxide 9 (91%) followed by regioselective epoxide opening using NaBH₃CN and ZnI₂ furnished racemic 4-nitroindan-2-ol (rac-6) in 81% yield.

1. Preparation of Racemic 4-Nitroindan-2-ol (rac-6)­ .

^a Reagents and conditions: (a) NaBH₄, THF/MeOH (10:1), 25 °C, 0.5 h, 97%; (b) PTSA, benzene, reflux, 15 h, 94%; (c) m-CPBA, CH₂Cl₂, 0 °C, 16 h, 91%; (d) NaBH₃CN, ZnI₂, 1,2-dichloroethane, reflux, 1 h, 81%.

For the chiral resolution of secondary alcohols, methods such as chiral HPLC, enzymatic resolution, and diastereomeric salt formation are conventionally used in general. The chiral resolution method using HPLC requires a relatively expensive instrumental setting, and it exhibits limited scalability. The diastereomeric salt formation method requires stoichiometric or even excess amounts of a chiral resolving agent, increasing cost, especially for large-scale applications. Often, multiple recrystallizations are needed to achieve high enantiomeric purity, leading to significant material loss and time consumption. Therefore, we opted for the enzymatic kinetic resolution method to prepare (R)-4-nitroindan-2-ol ((R)-6) from rac-6.

A kinetic resolution of rac-6 utilizing lipase from Pseudomonas fluorescens and vinyl acetate showed initial promise, , as early tests showed approximately 50% of the substrate was acetylated within the first 24 h (Table ). Conversion of rac-6 to the corresponding acetate 10 plateaued thereafter, with minimal or little additional conversion observed after 48 h. After isolating acetate 10 from remaining 4-nitroindan-2-ol, we conducted a Mosher ester analysis to determine the absolute configuration of C2-OH in the unreacted 4-nitroindan-2-ol. The Mosher ester analysis of the remaining unreacted substrate revealed the remaining substrate was the (S)-enantiomer of rac-6 with >99% ee (see the Supporting Information for details). This indicates that the lipase-catalyzed kinetic resolution of rac-6 follows Kazlauskas’ rule.

1. Preparation of Enantiopure (R)-6 by Enzymatic Kinetic Resolution of rac-6 Using Amano AK Lipase from Pseudomonas fluorescens .

entry	time (h)	product	ee of (R)-10	yield of (R)-10	product	ee of (S)-6	yield of (S)-6	E	conversion
1	3 h	(R)-enriched 10	99%	41%	(S)-enriched 6	74%	56%	445	0.43
2	6 h	(R)-enriched 10	98%	46%	(S)-enriched 6	99%	48%	525	0.50
3	24 h	(R)-enriched 10	86%	49%	(S)-enriched 6	>99%	46%	69	0.54
4	48 h	(R)-enriched 10	82%	52%	(S)-enriched 6	>99%	45%	52	0.55
5	5 h	(R)-enriched 10	>99%	38%	(S)-enriched 6	57%	61%	355	0.37

^aReagents and conditions: (a) Amano AK lipase from Pseudomonas fluorescens, vinyl acetate, toluene, 40 °C, 3–48 h; (b) K₂CO₃, MeOH, 40 °C, 1 h, 63%; (c) H₂, Pd/C (10 wt %), MeOH, 25 °C, 12 h, 86% for (R)-4, 76% for (S)-4.

^bAll reactions were performed with a 0.16 M solution of rac-6 in toluene at 40 °C, using 2 eq. of vinyl acetate and 10 wt % of Amano lipase.

^cThe ee was determined by a chiral HPLC separation of the corresponding alcohol obtained upon hydrolysis of (R)-10.

^dIsolated yield.

^eThe ee was determined by a chiral HPLC separation of (S)-6 from kinetic resolution.

^fCalculated according to Chen et al. using the equation: E = ln­[(1 – c)­(1 – ee_s)]/ln­[(1 – c)­(1 + ee_s)], where c (conversion) was determined from the enantiomeric excess of the recovered (S)-6 (ee_s) and the product (R)-10 (ee_p), using the formula c = ee_s/(ee_s + ee_p).

^gThe reaction was performed on a 50 mg scale.

^hThe reaction was performed on an 862 mg scale.

Various reaction times for lipase-catalyzed kinetic resolution were attempted to maximize the yield and optical purity of acetate 10 (Table ). Optical purity was measured by separation on a Chiralpak IA column, monitoring at 254 nm (see the Supporting Information for details). For each condition, the acetate product 10 and unreacted nitroindanol 6 were first separated by SiO₂-column chromatography; chiral HPLC analysis was performed after hydrolysis of acetate 10 to the corresponding secondary alcohol, as well as on the remaining nitroindanol substrate. Gratifyingly, a kinetic resolution for 3 h under the conditions in Table (entry 1), followed by hydrolysis with K₂CO₃, gave (R)-6 in high enantiomeric excess (99% ee). Under similar conditions, a gram-scale kinetic resolution of rac-6 afforded the desired (R)-10 in 38% yield with >99% ee (entry 5). Reduction with H₂ and Pd/C (10 wt %) of (R)-6 furnished (R)-4-aminoindan-2-ol ((R)-4) in 86% yield. The corresponding (S)-4-aminoindan-2-ol ((S)-4), the head fragment of AMG8563 (2, enantiomer of AMG8562), was also obtained by purification of enantiopure unreacted substrate (S)-4 afforded by the kinetic resolution, followed by reduction of the nitro group (Table ).

With aminoindanol (R)-4 in hand, we turned our attention to synthesizing α,β-unsaturated acid 3 for amide formation with (R)-4 (Scheme ). Starting with commercially available 2-fluoro-4-(trifluoromethyl) benzoic acid (5) in MeCN, heating with piperidine in a pressurized reaction tube yielded the substitution product 11 in 76% yield. Initially, conversion of carboxylic acid 11 to aldehyde 13 was thought to be possible through LiAlH₄ reduction of a Weinreb amide intermediate, as reported by Nahm et al. However, the addition of CDI to 11, followed by N,O-dimethylhydroxylamine hydrochloride and Et₃N, showed no formation of the corresponding Weinreb amide in THF or CH₂Cl₂. We decided to shift our efforts on a reduction to alcohol 12, followed by mild oxidation to aldehyde 13. Initial attempts in directly reducing carboxylic acid 11 to benzyl alcohol 12 with LiAlH₄ resulted in poor yields (<25%) due to the formation of unidentifiable side products; to explore milder reducing conditions, we first converted carboxylic acid 11 to a mixed anhydride using ethyl chloroformate, followed by NaBH₄ reduction. Gratifyingly, this method yielded alcohol 12 in 69% yield, which was oxidized by MnO₂ to furnish the corresponding aldehyde 13 in 82% yield (91% BRSM).

2. Preparation of Carboxylic Acid 3 and Completion of the Synthesis of AMG8562 (1)­ .

^a Reagents and conditions: (a) piperidine, MeCN, 200 °C, 6 h, 76%; (b) ClCO₂Et, Et₃N, THF, −10 °C, 0.5 h; NaBH₄, THF, MeOH, 0 °C, 1.5 h, 69%; (c) MnO₂, CH₂Cl₂, 0 to 25 °C, 16 h, 82% (91% BRSM); (d) methyl (triphenylphosphoranylidene)­acetate, toluene, 100 °C, 16 h, 81% (E/Z = 9.8:1); (e) 1 N LiOH, THF/MeOH (2:1), 25 °C, 1 h, 93%; (f) (R)-4, DMTMM, MeOH, 25 °C, 24 h, 87%.

To install the desired α,β-unsaturated (E)-alkene, we treated the aldehyde with methyl (triphenylphosphoranylidene) acetate (Scheme ). Successful production of α,β-unsaturated methyl ester 14 (81%) was observed, with a E/Z ratio of 9.8:1, which were separated by SiO₂ column chromatography. Finally, hydrolysis of 14 furnished the desired α,β-unsaturated carboxylic acid 3 in 93% yield. To achieve the formation of the desired amide 1, DCC and DMAP were added to a solution of 3 and (R)-4 in CH₂Cl₂ (see the Supporting Information for details). Unexpectedly, this resulted in esterification between 3 and (R)-4. The same ester product was also observed utilizing DCC without DMAP. To circumvent this side reaction, we first protected the alcohol moiety of aminoindanol (R)-4 as the corresponding TBS ether, followed by DCC/DMAP to afford the corresponding TBS-protected amide. This was followed by desilylation to complete the asymmetric synthesis of AMG8562 (1). Fortunately, it was later discovered that using the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) in MeOH proved to be more efficient, directly producing AMG8562 (1, 87%, 99% ee) without the need for a protecting group. The enantiomeric AMG8563 (2, 99% ee) was prepared in a similar manner.

Conclusions

In summary, the ability of AMG8562 (1) to act as a TRPV1 antagonist in rodent models without causing hyperthermia makes it a valuable tool for studying the role of TRPV1 in pain signaling and body temperature regulation. AMG8562 (1) is also a lead compound for developing nonaddictive analgesics. We highlight the asymmetric synthesis of AMG8562 (1), leveraging a lipase-catalyzed chiral resolution of the head fragment, leading to a synthetic route for its preparation on a large-scale. Specifically, this study underscores the application of lipase from Pseudomonas fluorescens with AMG8562’s head fragment, demonstrating the enzyme’s versatility in handling substrates with subtle substituent differences. We expect that this work will facilitate the in-depth study of AMG8562 (1) by providing a more straightforward and efficient synthesis, thereby making it more accessible for researchers investigating TRPV1-related mechanisms and therapeutic applications. Our synthesis will also allow a generation of a diverse set of AMG8562 (1) for structure–activity relationship (SAR) and preclinical studies. Future work should focus on further understanding the stereochemical influences on the pharmacological activity of TRPV1 antagonists.

Experimental Section

General Methods

All reactions were conducted in oven-dried glassware under nitrogen. Unless otherwise stated, all reagents were purchased from commercial suppliers (Sigma-Aldrich, VWR, TCI, or Ambeed) and used without further purification. All solvents were American Chemical Society (ACS) grade or better and used without further purification. Analytical thin layer chromatography (TLC) was performed with glass-backed silica gel (60 Å) plates with fluorescent indication (Whatman). Visualization was accomplished by UV irradiation at 254 nm and/or by staining with p-anisaldehyde solution or potassium permanganate solution followed by heating. Flash column chromatography was performed by using silica gel (particle size 230–400 mesh, 60 Å) purchased from Silicycle. All ¹H NMR and ¹³C NMR spectra were recorded with a Bruker 500 (500 MHz) spectrometer. All NMR δ values are given in parts per million (ppm) and are referenced to the residual isotopomer solvent signals (CDCl₃: δ 7.26 ppm, CD₃OD: δ 3.31 ppm, (CD₃)₂SO: δ 2.50 ppm) for ¹H NMR spectra, or the solvent signals (CDCl₃: δ 77.16 ppm, CD₃OD: δ 49.00 ppm, (CD₃)₂SO: δ 39.52 ppm) for ¹³C NMR spectra. Coupling constants (J) are given in Hertz (Hz) and multiplicities are indicated using the conventional abbreviation (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet or overlap of nonequivalent resonances, br = broad). Electrospray ionization (ESI) mass spectrometry (MS) was recorded with an Agilent 6224 series (LC/MS–TOF) spectrometer to obtain the molecular masses of the compounds. Optical rotation values were measured with a Rudolph Research Analytical (A21102 API/1W) polarimeter. Chiral HPLC analyses were performed on a Daicel Chiralpak IA column (Daicel Chiral Technologies) under the specified conditions.

4-Nitro-2,3-dihydro-1H-inden-1-ol (S1)

To a solution of 4-nitroindan-1-one (7) (950 mg, 5.36 mmol) in THF (9.5 mL, 0.56 M) was slowly added NaBH₄ (80.0 mg, 2.11 mmol) and MeOH (0.95 mL). After being stirred at 25 °C for 0.5 h, the reaction mixture was concentrated in vacuo, diluted with H₂O, and extracted three times with 50% EtOAc in hexanes. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 1:1) to afford S1 (934 mg, 97%) as a clear oil: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 7.4 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 5.33 (t, J = 6.5 Hz, 1H), 3.57 (ddd, J = 18.3, 8.8, 4.5 Hz, 1H), 3.34–3.24 (m, 1H), 2.60 (dddd, J = 13.2, 8.4, 7.1, 4.5 Hz, 1H), 2.03 (dddd, J = 12.9, 8.8, 6.9, 5.7 Hz, 1H). HRMS (ESI-TOF) m/z: [M + NH₄]⁺ Calcd for C₉H₁₃N₂O₃ 197.0921; Found 197.0920.

7-Nitro-1H-indene (8)

To a solution of S1 (934 mg, 5.21 mmol) in benzene (21 mL, 0.25 M) was added PTSA (90.0 mg, 0.521 mmol). After stirring at reflux for 15 h, the reaction mixture was washed with saturated aqueous NaHCO₃. The aqueous layer was extracted with 10% EtOAc in hexanes. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 20:1) to afford 8 (790 mg, 94%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.06 (d, J = 8.1 Hz, 1H), 7.69 (dd, J = 7.4, 0.9 Hz, 1H), 7.46 (t, J = 7.8 Hz, 1H), 6.94 (dt, J = 5.5, 1.8 Hz, 1H), 6.76 (dt, J = 5.6, 1.9 Hz, 1H), 3.94 (t, J = 1.9 Hz, 2H).

5-Nitro-1a,6a-dihydro-6H-indeno­[1,2-b]­oxirene (rac-9)

To a solution of 8 (790 mg, 4.90 mmol) in CH₂Cl₂ (25 mL, 0.20 M) was added m-CBPA (purity ≤ 77%, 2.09 g, 9.33 mmol) slowly under stirring at 0 °C. The reaction mixture was stirred at 0 °C for 16 h, diluted with 20% aqueous Na₂CO₃ solution, and extracted with CH₂Cl₂. The organic portions were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 9:1) to afford rac-9 (790 mg, 91%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.13 (dd, J = 8.3, 1.0 Hz, 1H), 7.80 (dd, J = 7.3, 1.0 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 4.37 (dd, J = 2.8, 1.4 Hz, 1H), 4.24 (t, J = 2.8 Hz, 1H), 3.76 (dd, J = 19.8, 1.3 Hz, 1H), 3.42 (dd, J = 19.8, 2.9 Hz, 1H).

4-Nitro-2,3-dihydro-1H-inden-2-ol (rac-6)

To a solution of rac-9 (790 mg, 4.46 mmol) in 1,2-dichloroethane (11.6 mL, 0.38 M) was slowly added zinc iodide (2.10 g, 6.58 mmol) and sodium cyanoborohydride (2.12 g, 33.74 mmol) under stirring at 25 °C. The reaction mixture was refluxed for 1 h, then cooled to 0 °C, and diluted with 20% aqueous Na₂CO₃ solution to quench the excess sodium cyanoborohydride. After stirring for 0.5 h, the solution was extracted with CH₂Cl₂, and the organic part was dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 4:1) to afford rac-6 (650 mg, 81%) as a yellow-white solid: ¹H NMR (500 MHz, CDCl₃) δ 8.03 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 4.81–4.78 (m, 1H), 3.64 (dd, J = 18.5, 5.7 Hz, 1H), 3.48 (dd, J = 18.4, 2.4 Hz, 1H), 3.28 (dd, J = 16.8, 5.9 Hz, 1H), 3.03 (dd, J = 16.8, 2.4 Hz, 1H); HRMS (ESI-TOF) m/z: [M + NH₄]⁺ Calcd for C₉H₁₃N₂O₃ 197.0921; Found 197.0924.

(R)-4-Nitro-2,3-dihydro-1H-inden-2-yl Acetate ((R)-10)

To a solution of rac- 6 (50 mg, 0.279 mmol) in toluene (1.7 mL, 0.16 M) was slowly added vinyl acetate (0.0514 mL, 0.558 mmol) and Amano Lipase from Pseudomonas fluorescens (5 mg, ∼ 20,000 U/g). The reaction mixture was stirred at 40 °C for 3 h and then concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 6:1) to afford acetate (R)-10 (25.3 mg, 41%, 99% ee) as a white solid and the remaining (S)-enriched alcohol 6 (28.0 mg, 56%, 74% ee) as a yellow-white solid; [Large-Scale Kinetic Resolution] To a solution of rac- 6 (862 mg, 4.81 mmol) in toluene (30 mL, 0.16 M) was slowly added vinyl acetate (0.89 mL, 9.62 mmol) and Amano Lipase from Pseudomonas fluorescens (86.2 mg, ∼ 20,000 U/g). The reaction mixture was stirred at 40 °C for 5 h and then concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 6:1) to afford acetate (R)-10 (398.6 mg, 38%, >99% ee) as a white solid and the remaining (S)-enriched alcohol 6 (527.4 mg, 61%, 57% ee) as a yellow-white solid.

For acetate (R)-10: ¹H NMR (500 MHz, CDCl₃) δ 8.05 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 7.4 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 5.61–5.57 (m, 1H), 3.73 (dd, J = 19.1, 6.3 Hz, 1H), 3.58 (dd, J = 19.0, 2.4 Hz, 1H), 3.40 (dd, J = 17.5, 6.4 Hz, 1H), 3.11 (dd, J = 17.5, 2.3 Hz, 1H), 2.02 (s, 3H); HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₁₁H₁₁NO₄Na 244.0580; Found 244.0584.

For (S)-enriched alcohol 6: ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 7.0 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 4.82–4.79 (m, 1H), 3.65 (dd, J = 18.5, 5.7 Hz, 1H), 3.49 (dd, J = 18.5, 2.4 Hz, 1H), 3.29 (dd, J = 16.8, 5.9 Hz, 1H), 3.04 (dd, J = 16.8, 2.4 Hz, 1H), 1.68 (s, 1H).

(R)-4-Nitro-2,3-dihydro-1H-inden-2-ol ((R)-6)

To a solution of (R)-10 (300 mg, 1.37 mmol) in MeOH (50 mL, 0.03 M) was slowly added K₂CO₃ (1.127 g, 8.16 mmol). The reaction mixture was stirred at 40 °C for 1 h, dried in vacuo, diluted with H₂O, and extracted with CH₂Cl₂. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 4:1) to afford (R)-6 (154 mg, 63%, 99% ee) as a yellow-white solid: $[\alpha {]}_{\mathrm{D}}^{25}$ −133.1 (c 0.1, MeOH); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 7.4 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 4.82–4.79 (m, 1H), 3.65 (dd, J = 18.6, 5.7 Hz, 1H), 3.49 (dd, J = 18.6, 2.3 Hz, 1H), 3.29 (dd, J = 16.8, 5.9 Hz, 1H), 3.04 (dd, J = 16.9, 2.4 Hz, 1H), 1.71 (s, 1H).

(R)-4-Amino-2,3-dihydro-1H-inden-2-ol ((R)-4)

To a solution of (R)-6 (154 mg, 0.859 mmol) in MeOH (3.7 mL, 0.23 M) was slowly added Pd/C (10 wt %, 37 mg) at 25 °C. The reaction mixture was evacuated then backfilled with hydrogen gas three times and attached to a hydrogen source. The reaction mixture was then stirred at 25 °C for 12 h. The catalyst was filtered over a pad of Celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 2:3) to afford (R)-4 (110 mg, 86%, 99% ee) as a white crystalline solid: ¹H NMR (500 MHz, CDCl₃) δ 7.02 (t, J = 7.7 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 6.54 (d, J = 7.8 Hz, 1H), 4.73 (br s, 1H), 3.58 (br s, 2H), 3.21 (dd, J = 16.5, 6.0 Hz, 1H), 3.05 (dd, J = 15.9, 6.1 Hz, 1H), 2.90 (dd, J = 16.5, 3.3 Hz, 1H), 2.73 (dd, J = 15.9, 3.1 Hz, 1H), 1.66 (br s, 1H); HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₉H₁₂NO 150.0913; Found 150.0912.

(S)-4-Amino-2,3-dihydro-1H-inden-2-ol ((S)-4)

To a solution of (S)-6 (250 mg, 1.40 mmol; $[\alpha {]}_{\mathrm{D}}^{25}$ +134.9, c 0.1, MeOH) in MeOH (6 mL, 0.23 M) was slowly added Pd/C (10 wt %, 60 mg) at 25 °C. The reaction mixture was evacuated then backfilled with hydrogen gas three times and attached to a hydrogen source. The reaction mixture was then stirred for 12 h at 25 °C. The catalyst was filtered over a pad of Celite and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 2:3) to afford (S)-4 (158 mg, 76%, 99% ee) as a white crystalline solid: ¹H NMR (500 MHz, CDCl₃) δ 7.02 (t, J = 7.6 Hz, 1H), 6.70 (d, J = 7.4 Hz, 1H), 6.54 (d, J = 7.9 Hz, 1H), 4.75–4.71 (m, 1H), 3.58 (br s, 2H), 3.21 (dd, J = 16.4, 6.1 Hz, 1H), 3.05 (dd, J = 15.8, 6.1 Hz, 1H), 2.90 (dd, J = 16.4, 3.2 Hz, 1H), 2.73 (dd, J = 15.9, 3.1 Hz, 1H), 1.69 (br s, 1H).

2-(Piperidin-1-yl)-4-(trifluoromethyl)­benzoic Acid (11)

In a heavy wall pressure vessel, a solution of 2-fluoro-4-(trifluoromethyl)­benzoic acid (5) (940 mg, 4.52 mmol) in MeCN (7 mL, 0.65 M) was added piperidine (1.42 g 16.7 mmol). The reaction mixture was then placed in a 200 °C sand bath and stirred for 6 h. The reaction mixture was then cooled to 25 °C and dried in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 1:1) to afford 2-(piperidin-1-yl)-4-(trifluoromethyl)­benzoic acid (11, 940 mg, 76%) as a white crystalline solid: ¹H NMR (500 MHz, DMSO) δ 8.11 (d, J = 8.1 Hz, 1H), 7.99 (s, 1H), 7.68 (dd, J = 8.2, 1.7 Hz, 1H), 3.13 (t, J = 5.4 Hz, 4H), 1.75–1.70 (m, 4H), 1.63–1.59 (m, 2H); ¹³C NMR (125 MHz, DMSO) δ 166.25, 151.09, 132.94 (q, ² J _CF = 32.0 Hz, C₄), 131.77, 128.77, 123.46 (q, ¹ J _CF = 271.8 Hz, CF₃), 122.36, 119.43, 53.21, 25.49, 22.33; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₃H₁₅F₃NO₂ 274.1049; Found 274.1055.

2-(Piperidin-1-yl)-4-(trifluoromethyl)­phenyl)­methanol (12)

To a cooled solution of 11 (920 mg, 3.367 mmol) in anhydrous THF (40 mL, 0.08 M) at –10 °C was added Et₃N (1.022 g, 10.10 mmol) followed by ethyl chloroformate (731 mg, 6.734 mmol). The mixture was stirred at –10 °C for 0.5 h, then filtered through a cotton plug. The filter cake was washed with anhydrous THF (40 mL). The crude solution containing the mixed anhydride was used in immediately in the following step without further concentration or purification. To a cooled (0 °C) solution of the crude mixed anhydride in THF was added NaBH₄ (1.010 g, 26.70 mmol), followed by dropwise addition of MeOH (5.0 mL). After stirring at 0 °C for 1.5 h, the reaction mixture was quenched with a saturated solution of NH₄Cl (30.0 mL), and the aqueous part was extracted three times with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 6:1) to afford 12 (605 mg, 69%) as a clear oil: ¹H NMR (500 MHz, CDCl₃) δ 7.41 (s, 1H), 7.35 (d, J = 7.4 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 5.37 (br s, 1H), 4.84 (s, 2H), 2.94 (t, J = 5.2 Hz, 4H), 1.80–1.76 (m, 4H), 1.64–1.60 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 153.24, 139.47, 130.42 (q, ² J _CF = 32.2 Hz, C₄), 128.74, 124.15 (q, ¹ J _CF = 272.2 Hz, CF₃), 121.14 (q, ³ J _CF = 4.0 Hz, C₅), 117.53 (q, ³ J _CF = 3.7 Hz, C₃), 63.91, 53.87, 26.59, 23.90; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₃H₁₇F₃NO 260.1257; Found 260.1264.

2-(Piperidin-1-yl)-4-(trifluoromethyl)­benzaldehyde (13)

To a cooled solution of 12 (600 mg, 2.31 mmol) in anhydrous CH₂Cl₂ (45 mL, 0.05 M) at 0 °C was slowly added activated MnO₂ (2.4 g, 27.8 mmol). The mixture was allowed to warm up to 25 °C and stirred at 25 °C for 16 h. The solids were removed by vacuum filtration, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 7:1) to afford aldehyde 13 (490 mg, 82%, 91% BRSM) as a clear yellow oil: ¹H NMR (500 MHz, CDCl₃) δ 10.29 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.38 (s, 1H), 7.33 (d, J = 8.1 Hz, 1H), 3.14 (t, J = 4.5 Hz, 4H), 1.85–1.80 (m, 4H), 1.67–1.62 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 190.67, 156.78, 135.89 (q, ² J _CF = 32.2 Hz, C₄), 130.78, 130.01, 123.73 (q, ¹ J _CF = 273.2 Hz, CF₃), 118.34 (q, ³ J _CF = 3.8 Hz, C₅), 116.05 (q, ³ J _CF = 3.7 Hz, C₃), 55.56, 26.15, 23.96; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₃H₁₅F₃NO 258.1100; Found 258.1106.

Methyl (E)-3-(2-(Piperidin-1-yl)-4-(trifluoromethyl)­phenyl)­acrylate (14)

In a heavy wall cylindrical pressure vessel, a solution of 13 (490 mg, 1.90 mmol) in toluene (40 mL, 0.05 M) was added methyl (triphenylphosphoranylidene)­acetate (830 mg 2.48 mmol). The reaction mixture was then placed in a 100 °C sand bath and stirred for 16 h. The reaction mixture was quenched with H₂O, and the aqueous part was extracted three times with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 50:1) to afford 14 (484 mg, 81%) as a clear yellow oil, along with the (Z)-isomer (14’, 49.5 mg, 8.3%): Compound 14: ¹H NMR (500 MHz, CDCl₃) δ 8.10 (d, J = 16.1 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.35 (s, 1H), 7.31 (d, J = 8.2 Hz, 1H), 6.46 (d, J = 16.1 Hz, 1H), 3.83 (s, 3H), 3.01 (t, J = 5.3 Hz, 4H), 1.88–1.83 (m, 4H), 1.66–1.60 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 167.51, 153.96, 141.48, 132.36 (q, ² J _CF = 32.2 Hz, C₄), 131.93, 128.36, 124.10 (q, ¹ J _CF = 272.9 Hz, CF₃), 119.13, 118.86 (q, ³ J _CF = 3.8 Hz, C₅), 115.82 (q, ³ J _CF = 3.7 Hz, C₃), 54.18, 51.82, 26.26, 24.13; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₆H₁₉F₃NO₂ 314.1362; Found 314.1371; Compound 14’: ¹H NMR (500 MHz, CDCl₃) 7.57 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 6.9 Hz, 1H), 7.20 (s, 1H), 7.14 (d, J = 12.3 Hz, 1H), 6.02 (d, J = 12.3 Hz, 1H), 3.70 (s, 3H), 2.95 (t, J = 5.3 Hz, 4H), 1.72–1.68 (m, 4H), 1.62–1.55 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 166.76, 153.11, 141.86, 132.74, 131.53 (q, ² J _CF = 32.0 Hz, C₄), 131.36, 124.27 (q, ¹ J _CF = 270.4 Hz, CF₃), 119.28, 118.04, 114.83, 53.82, 51.50, 26.46, 24.20; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₆H₁₉F₃NO₂ 314.1362; Found 314.1370.

(E)-3-(2-(Piperidin-1-yl)-4-(trifluoromethyl)­phenyl)­acrylic Acid (3)

To a solution of 14 (484 mg, 1.54 mmol) in THF (40 mL, 0.04 M) was added MeOH (20 mL) and aqueous 1 N LiOH (30 mL). The reaction mixture was then stirred at 25 °C for 1 h before it was neutralized with 1 N HCl (20 mL). The resulting mixture was diluted with EtOAc and extracted three times with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 1:1) to afford 3 (431 mg, 93%) as a yellow solid: ¹H NMR (500 MHz, CDCl₃) δ 8.20 (d, J = 16.1 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.36 (s, 1H), 7.35 (d, J = 6.9 Hz, 1H), 6.47 (d, J = 16.1 Hz, 1H), 3.05 (t, J = 5.2 Hz, 4H), 1.89 (p, J = 5.6 Hz, 4H), 1.67–1.63 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 172.95, 154.23, 143.76, 132.81 (q, ² J _CF = 32.2 Hz, C₄), 131.52, 128.67, 124.08 (q, ¹ J _CF = 272.2 Hz, CF₃), 118.92 (q, ³ J _CF = 3.7 Hz, C₅), 118.66, 115.93 (q, ³ J _CF = 3.7 Hz, C₃), 54.34, 26.30, 24.15; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₅H₁₇F₃NO₂ 300.1206; Found 300.1211.

(R,E)-N-(2-Hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)­phenyl)­acrylamide (1, AMG8562)

To a solution of 3 (120 mg, 0.402 mmol) and (R)-4 (50.0 mg, 0.335 mmol) in MeOH (6.5 mL) was added DMTMM (139 mg, 0.503 mmol). The reaction mixture was then stirred at 25 °C for 24 h. The reaction solvent was removed in vacuo and the residue was diluted with a saturated solution of NaHCO₃ (10 mL). The aqueous portion was extracted three times with EtOAc. The combined organic layers were washed five times with a saturated solution of Na₂CO₃, dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 2:1) to afford AMG8562 (1, 125 mg, 87%, 99% ee) as a yellow solid: $[\alpha {]}_{\mathrm{D}}^{25}$ −34.5 (c 0.15, MeOH); ¹H NMR (500 MHz, CD₃OD) δ 8.02 (d, J = 15.8 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 6.96 (d, J = 15.8 Hz, 1H), 4.63–4.59 (m, 1H), 3.16 (dd, J = 16.4, 6.1 Hz, 2H), 2.95 (t, J = 5.3 Hz, 4H), 2.88 (ddd, J = 16.6, 7.3, 3.5 Hz, 2H), 1.80–1.76 (m, 4H), 1.62–1.57 (m, 2H); ¹³C NMR (125 MHz, CD₃OD) δ 166.42, 154.32, 143.86, 138.69, 135.47, 135.27, 133.94, δ 132.89 (q, ² J _CF = 31.7 Hz, C₄), 129.39, 128.15, 125.43 (q, ¹ J _CF = 270.1 Hz, CF₃), 124.08, 122.98, 122.32, 120.39, 116.79, 73.20, 55.30, 43.39, 41.24, 27.10, 24.88; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₄H₂₆F₃N₂O₂ 431.1941; Found 431.1949.

(S,E)-N-(2-Hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)­phenyl)­acrylamide (2, AMG8563)

To a solution of 3 (145 mg, 0.484 mmol) and (S)-4 (60.0 mg, 0.402 mmol) in MeOH (6.0 mL) was added DMTMM (189 mg, 0.683 mmol). The reaction mixture was then stirred at 25 °C for 24 h. The reaction solvent was removed in vacuo and the residue was diluted with a saturated solution of NaHCO₃ (10 mL). The aqueous portion was extracted three times with EtOAc, then the combined organic layers were washed five times with a saturated solution of Na₂CO₃, dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 2:1) to afford AMG8563 (2, 163 mg, 94%, 99% ee) as a yellow solid: $[\alpha {]}_{\mathrm{D}}^{25}$ +37.1 (c 0.15, MeOH); ¹H NMR (500 MHz, CD₃OD) δ 8.02 (d, J = 15.8 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.30 (s, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.14 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.94 (d, J = 15.8 Hz, 1H), 4.63–4.59 (m, 1H), 3.16 (ddd, J = 16.5, 6.2, 3.4 Hz, 2H), 2.97–2.84 (m, 6H), 1.80–1.75 (m, 4H), 1.62–1.57 (m, 2H); ¹³C NMR (125 MHz, CD₃OD) δ 166.52, 154.72, 143.94, 138.89, 135.61, 135.32, 134.07, 132.95 (q, ² J _CF = 31.9 Hz, C₄), 129.38, 128.21, 125.51 (q, ¹ J _CF = 270.0 Hz, CF₃), 123.95, 123.06, 122.41, 120.28, 116.81, 73.26, 55.30, 43.44, 41.29, 27.21, 25.00; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₄H₂₆F₃N₂O₂ 431.1941; Found 431.1947.

Determination of Enantiomeric Excess of 1 and 2 by Chiral HPLC

Enantiomeric excess (ee) was determined by chiral HPLC analysis on a Daicel Chiralpak IA column (250 × 4.6 mm ID, particle size 3 μm, Daicel Chiral Technologies) using a Shimadzu LC-20 series HPLC system equipped with a UV/vis detector set at 254 nm. The mobile phase consisted of hexanes/EtOAc (65:35 v/v) prepared using HPLC-grade solvents obtained from Sigma-Aldrich. Flow rate was maintained at 1.5 mL/min. Samples were prepared by dissolving approximately 0.3 to 0.4 mg of the compound in 10 mL of hexanes/EtOAc (65:35 v/v), filtering through a 0.22 μm PTFE syringe filter, and injecting 40 μL into the column. The column temperature was maintained at 25 °C. The retention times were 10.3 min for AMG8562 (1) and 17.2 min for AMG8563 (2). Peak areas were integrated for each sample. All data were processed using Shimadzu LabSolutions software.

The data underlying this study are available in the published article and its online Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c08033.

• Preparation of AMG8562 (1) by DCC/DMAP coupling, Mosher ester analysis of (S)-6, lipase-catalyzed kinetic resolution of rac-6, and copy of ¹H NMR spectra, ¹³C NMR spectra, and chiral HPLC chromatograms (PDF)

The authors declare no competing financial interest.