Concise Syntheses of Microsomal Metabolites of a Potent OXE (Oxoeicosanoid) Receptor Antagonist

Abstract

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most potent eosinophil chemoattractant among lipid mediators, and its actions are mediated by the selective oxoeicosanoid (OXE) receptor. Our group previously developed a highly potent indole-based OXE antagonist, S-C025, with an IC₅₀ value of 120 pM. S-C025 was converted to a number of metabolites in the presence of monkey liver microsomes. Complete chemical syntheses of authentic standards enabled us to identify that the four major metabolites were derived by the oxidation at its benzylic and N-methyl carbon atoms. Herein we report concise syntheses of the four major metabolites of S-C025.

Introduction

Arachidonic acid (AA) is metabolized into a variety of oxidized products that play important roles in human diseases.¹⁾ One of these products is 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) formed by the 5-lipoxygenase (5-LO) pathway²⁾ (Fig. 1). 5-Oxo-ETE is the most potent chemoattractant for human eosinophils among lipid mediators³⁾ and stimulates a variety of responses in these cells, such as actin polymerization, calcium mobilization, increased surface expression of CD11b, degranulation, superoxide production, and transendothelial migration.^4–6) These actions are all mediated by the selective G_i-protein coupled oxoeicosanoid (OXE) receptor that is highly expressed in eosinophils, neutrophils, and basophils.^7,8) Therefore, 5-oxo-ETE is likely to be an important mediator in eosinophilic disorders (such as asthma, allergic rhinitis, and eosinophilic esophagitis) and preventing its effects with a selective antagonist could be a useful therapeutic approach. We designed and synthesized a series of indole-based OXE receptor antagonists as structural mimics of 5-oxo-ETE.^9–13) We used monkeys as an animal model to investigate in vivo metabolism, pharmacokinetics, and efficacy of these antagonists because rodents lack an ortholog of the OXE receptor.^14–16) These studies resulted in the identification of S-C025 (Fig. 1) as a novel OXE receptor antagonist with in vitro potency in the picomolar range and good plasma lifetime in cynomolgus monkeys.^17,18)

Fig. 1. S-C025: Structure and Monkey Liver Microsomes Metabolism Sites

To better understand the metabolic stability of S-C025, we recently investigated its metabolism by monkey liver microsomes.¹⁹⁾ Four major metabolites were isolated from microsomal incubations. We speculated that these metabolites could have resulted from oxidation of benzylic and N-methyl carbon atoms of the antagonist.¹⁹⁾ Unambiguous identification of these metabolites required the complete syntheses of authentic standards. Herein we report concise syntheses of the four major metabolites of S-C025 (vide infra).

Results and Discussion

During the aforementioned investigations, we detected two metabolites that were rapidly produced by the incubation of S-C025 with monkey liver microsomes in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Based on their UV spectra and chromatographic behaviors, we hypothesized that these compounds were formed by hydroxylation of the N-methyl group (1, Fig. 2) and the methylene carbon atom adjacent to the phenyl group (3). Time course data suggested that compound 1 subsequently lost its hydroxymethyl unit to give compound 2, and that compound 3 was further oxidized to the corresponding ketone (4). Therefore, we embarked on the syntheses of these four compounds to confirm our hypothesis, especially to conclusively demonstrate that liver microsomes hydroxylate the N-methyl group of S-C025 (Fig. 2). It is worthy of mention that although hydroxylation of indole N-methyl group in zafirlukast (brand name—Accolate) is reported on the basis of LC/MS data,²⁰⁾ its verification by the synthetic standard is lacking. Ismail et al. have reported indole N-methyl-hydroxylation as a metabolic pathway in the case of alosetron (brand name—Lotronex), which is also without a synthetic standard.²¹⁾

Fig. 2. Metabolism of S-C025 by Monkey Liver Microsomes

We chose N-methyl hydroxylated 1 for our first synthetic target. Hsu et al. reported the reduction of 1-indolyl carbamates to hemiaminals using LiAlH₄ with a good generality.²²⁾ As such, we synthesized model carbamate 10 from 5-chloro-1H-indole-2-carbaldehyde (5) in three steps and subjected it to their method to make hemiaminal 12 (Chart 1). However, in our case, LiAlH₄ reduction of 10 gave free (NH)-indole 8 as the major product. The desired compound (12) was not observed in this reduction reaction. Since our result can be rationalized by pK_a values of indole NH and ethanol (21.0 and 29.8 in dimethyl sulfoxide (DMSO), respectively),^23,24) we did not investigate the reduction approach further.

Chart 1. Attempted Synthesis of Model Hemiaminal 12 by the Reduction of 10

Next, we evaluated the addition of free (NH)-indole 8 to formaldehyde in the presence of tetrabutylammonium fluoride (TBAF).²⁵⁾ To our delight, this method yielded desired hemiaminal 12 although corresponding polymeric compounds (13) also formed as shown in Chart 2. It was hardly practical to isolate 12 from some of the polymeric compounds for its full characterization, thus was not performed.

Chart 2. Hydroxymethylation of a Model Free (NH)-Indole (8) with Formaldehyde

Encouraged by the above result, we decided to prepare fully functionalized intermediate 15 to move forward with our synthetic study as illustrated in Chart 3. Enantiomerically pure fragment 14²⁶⁾ was successfully coupled at the 3-position of 8 using Me₂AlCl to give 15 in 91% yield. To our delight, hydroxymethylation of 15 with formaldehyde proceeded cleanly in contrast to the model study (vide supra).²⁵⁾ Hemiaminal 16 was found to be stable for standard purification by silica gel chromatography and isolated in 94% yield.

Chart 3. Synthesis of Fully Functionalized Precursor 16

The final step toward the synthesis of 1 was to hydrolyze the methyl ester of 16. We subjected 16 to a general basic hydrolysis condition. However, the rapid deformylation took place in the presence of LiOH to provide 15 in >90% yield. As such, we next evaluated an acid-catalyzed hydrolysis reaction on 16, but the rapid deformylation resulted again. Since 16 was prepared in the presence of rather basic TBAF and it is stable to silica gel chromatography, the rapid deformylations under both basic and acid conditions were somewhat surprising to us at first glance. Nonetheless, we speculated that the ketone conjugated at the 3-position of its indole ring might have assisted the cleavage of the hemiaminal group under both basic and acidic conditions, as illustrated in Charts 4 and 5, respectively.

Chart 4. Base-Catalyzed Deformylation

Chart 5. Acid-Catalyzed Deformylation

The aforementioned hydrolysis studies indicated that it would be prudent to keep a reaction condition neutral to avoid the decomposition of the hemiaminal unit, which led us consider an enzymatic ester hydrolysis approach. Porcine liver esterase (PLE) is considered to lower the kinetic barrier to an ester hydrolysis through stabilization of a tetrahedral transition state by cooperative noncovalent interactions with specific site residues.^27,28) As such, we thought that we could mechanistically distinguish the ketone-conjugated hemiaminal and the methylester of 16 by PLE, and proceeded to test it in phosphate buffer (pH, 7.0). To our delight, it successfully hydrolyzed the methyl ester in 16 and afforded desired acid 1 in 17% yield (Chart 6). This is the first example of PLE-mediated ester hydrolysis in the presence of a highly labile hemiaminal functional group, although the PLE-mediated ester hydrolysis is known.^29–31)

Chart 6. Synthesis of 1 Using Porcine Liver Esterase (PLE)

Next, we evaluated acetone and acetonitrile as a co-solvent to increase homogeneity of the reaction medium so as to improve the yield (Entries 1–3, Table 1). While acetone adversely affected the yield, acetonitrile was clearly beneficial, affording the product in 26% yield. For all three cases, the hydrolysis reaction proceeded cleanly but did not go to completion. For example, we recovered 16 in 45% yield along with deformylated acid 2 and ester 15 that accounted for about 30% of the starting material used. As 2 and 15 were not in the crude reaction mixture right after the PLE hydrolysis reaction, 1 and 16 subsequently underwent deformylation possibly catalyzed by the carboxylic acid of 1 (vide infra). This highly labile nature of the hemiaminal unit of 1 could be attributed to its ketone directly conjugated to the nitrogen atom. Therefore, we tested the PLE hydrolysis on readily available N-hydroxymethyl indoles 17, 19, and 21, in which a carbonyl group is attached at the 2-position of the indole ring, and obtained acids 18, 20 and 22 in 61, 91, and 97% yields, respectively (Entries 4–6). These compounds were stable unlike 1. These results showed that a carbonyl group at the 3-position of an indole ring makes its N-hydroxymethyl unit extremely labile due to the direct conjugation. Indole rings that bear a carbonyl group at its 3-position and an alkyl group at its nitrogen atom are a structural motif that is often found in medicinally relevant molecules.^32–42) As such, our studies presented above are expected to be of general interests to scientists working in chemical synthesis, medicinal chemistry and related fields.

Table 1. Investigation of PLE Mediated Hydrolysis of Methyl Esters^a)

a) Reaction conditions: PLE, 0.05 M phosphate buffer, pH = 7.0. b) Isolated yield.

We treated 15 with excess LiOH (14 equivalents (equiv.)) to hydrolyze its methyl ester and obtained corresponding acid 2 in 93% yield (Chart 7). Notably, purified 1 converted into 2 on standing, while the corresponding methyl ester (16) was stable under the identical condition.⁴³⁾ These results indicates a possibility that 2 may have spontaneously formed from metabolite 1 non-enzymatically. Indole-N-demethylation is reported as a metabolic pathway for the antiviral drug umifenovir (brand name—Arbidol).⁴⁴⁾ Ondansetron (brand name—Zofran) is an indole-N-methyl-based drug used to treat nausea and vomiting caused by chemotherapy.⁴⁵⁾ The N-desmethylated metabolites of ondansetron have been reported.^45,46) However, the indole-N-CH₂–OH (hemiaminal) metabolites of umifenovir and ondansetron have not been reported. Our studies presented herein point out possibilities that the indole-N-CH₂–OH derivatives of umifenovir and ondansetron might have formed in their metabolic pathways but degraded into the reported N-desmethylated compounds due to a highly labile nature of the indole-3-carbonylated-N-hemiaminals as we demonstrated by the chemical synthesis. Overall, the identification of compound 1 as a metabolite of S-C025 using a synthetic standard contributed to a better understanding of the metabolism of an indole-N-methyl chemotype to the corresponding indole-N-desmethylated compound.

Chart 7. Synthesis of 2

Benzylic hydroxylation metabolite 3 was synthesized from the alcohol 23¹⁵⁾ in six steps as illustrated in Chart 8. The benzylic hydroxyl group was introduced by a Grignard reaction between the aldehyde 24 and phenyl magnesium bromide, which provided racemic alcohol 25. It was protected as a tert-butyldimethylsilyl (TBDMS) ether (26), and then coupled with optically pure fragment 14 using Me₂AlCl to give fully functionalized 27 as an inseparable mixture of diastereomers. Subsequently, the TBDMS group of 27 was removed with HF·Pyridine and the methyl ester 28 was hydrolyzed by LiOH·H₂O to provide compound 3 (1 : 1 mixture of diastereomers). The chiral column (amylose-2 column) fully resolved those diastereomers, and thus we could confirm that the microsomal benzylic hydroxylation of S-C025 was non-stereoselective.¹⁹⁾

Chart 8. Synthesis of 3

We tried to oxidize the benzylic alcohol 3 into metabolite 4 by either pyridinium chlorochromate (PCC) or MnO₂. However, these reactions gave complex mixtures of compounds that were inseparable by the TLC. Therefore, we did not attempt to analyze the mixtures and decided to prepare 4 with a different synthetic approach. We oxidized the corresponding benzylic alcohol (25) to the ketone 29 before the fragment coupling (Chart 9). The Me₂AlCl-catalyzed acylation of 29 did not work with optically pure acid chloride 14 but proceeded with 3-methylglutaric anhydride (30), affording racemic 4.

Chart 9. Synthesis of Racemic 4

Compounds 1–4 were identical to the four S-C025 metabolites shown in Table 2 on the bases of their HPLC retention times, UV spectra, and mass spectra, as reported separately.¹⁹⁾ We reported the HPLC conditions and HPLC charts of synthetic 1, 2, 3, and racemic 4 in Ref. 19.

Table 2. Properties of Synthetic and Microsomal S-C025 Metabolites (Data from Ref. 19)^a)

Compound	Retention time (min)b)		[M − H]−
	Synth	Micr	Synth	Micr	Theoretical
1	24.65	24.62	468.1942	NDc)	468.1947
2	27.45	27.41	438.1836	438.1837	438.1841
3d)	18.85	18.88	468.1943	468.1945	468.1947
rac-4	23.51	23.58	466.1786	466.1787	466.1791

a) See Ref. 19 for experimental details. b) RP-HPLC with a water/acetonitrile gradient. c) Compound 1 was too labile to permit mass spectral analysis of the microsomal product. d) Using the chiral HPLC co-chromatography (amylose-2 column) of the microsomal metabolite (3) and the synthetic 3, we found out that the microsomal metabolite was a mixture of diastereomers due to the non-stereoselective benzylic hydroxylation. See Ref. 19 for a detailed discussion.

Conclusion

In conclusion, the concise chemical syntheses of the four major microsomal metabolites of S-C025 were developed. These synthetic samples made it possible to unambiguously identify the structures of the four metabolites.¹⁹⁾ Although the synthesis of the hemiaminal 1 proved to be quite challenging due to its inherent lability, its synthesis was successfully accomplished by the PLE-mediated selective hydrolysis of the methyl ester. Since indole rings that bear a carbonyl group at its 3-position and an alkyl group at its nitrogen atom are a structural motif that is often found in medicinally relevant molecules, our synthetic studies presented above are expected to be of general interests to scientists working in chemical synthesis, medicinal chemistry and related fields. Furthermore, structural elucidation of the S-C025 metabolites provided useful information for the future design of metabolically resistant OXE receptor antagonists.

Experimental

Chemistry

All reactions were carried out under an argon atmosphere using oven-dried glassware and anhydrous solvents unless otherwise mentioned. All reagents were purchased from commercial sources and were used as received. Reaction progress was monitored by using a Merck TLC Silica gel 60F₂₅₄ plate. ¹H-NMR and ¹³C-NMR spectra were recorded at ambient temperature on a BRUKER AMX 400 MHz or BRUKER 500 MHz spectrometer using tetramethysilane as an internal standard. High-resolution mass spectra (HR-MS) were recorded on an AccuTOF mass spectrometer or at USF Mass Spec and Peptide Core Facility in Department of Chemistry at University of South Florida. Pig Liver Esterase was purchased from Millipore Sigma U.S.A. (Esterase from porcine liver, product number–E3019).

(E)-5-Chloro-2-(6-phenylhex-1-en-1-yl)-1H-indole (7)

To a stirred suspension of triphenyl(5-phenylpentyl)phosphonium bromide (6) (7.9 g, 16.141 mmol) in tetrahydrofuran (THF) (50 mL) was added LiHMDS (1.0 M solution in THF, 16.0 mL) at −78 °C. The mixture was warmed to room temperature (r.t.) and stirred for 30 min. 5-Chloro-1H-indole-2-carbaldehyde (5) (1.125 g, 6.264 mmol) in THF (15 mL) was then added dropwise at −78 °C. The reaction mixture was allowed to warm to r.t. and stirred for 2 h. Aqueous saturated NH₄Cl solution (25 mL) was added to quench the reaction, and the aqueous phase was extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, and the solvents were evaporated under reduced pressure. The resulting crude material was purified by silica gel chromatography (10% EtOAc/hex) to afford 7 as a yellow solid, 1.785 g, 92%. ¹H-NMR (400 MHz, CDCl₃) δ: 8.05 (s, 1H), 7.48 (s, 1H), 7.30–7.22 (m, 2H), 7.20–7.17 (m, 4H), 7.08–7.06 (m, 1H), 6.38–6.31 (m, 2H), 6.04 (dt, J = 16.0 Hz, 6.9 Hz, 1H), 2.64 (t, J = 7.6 Hz, 2H), 2.29–2.20 (m, 2H), 1.72–1.65 (m, 2H), 1.56–1.48 (m, 2H); ¹³C-NMR (100 MHz, CDCl₃) δ: 142.5, 137.8, 134.7, 130.9, 130.1, 128.4 (2C), 128.3 (2C), 125.7, 125.5, 122.3, 120.6, 119.7, 111.3, 100.9, 35.8, 32.8, 31.0, 28.8; HR-MS (electrospray ionization (ESI)) m/z Calcd for [C₂₀H₂₀ClN + H]⁺: 310.1362. Found 310.1393.

5-Chloro-2-(6-phenylhexyl)-1H-indole (8)

To a stirred solution of the olefin 7 (800 mg, 2.582 mmol) in EtOH (10 mL) was added palladium on activated charcoal (10% Pd basis, 80 mg). H₂ was purged into the reaction mixture through a balloon, and the reaction mixture was stirred at r.t. for 12 h. The reaction mixture was filtered through a pad of Celite. The Celite pad was washed with CH₂Cl₂, the filtrates were combined and concentrated to dryness. The resulting crude product was purified by silica gel chromatography (10% EtOAc/hexane) to afford 8 as a yellow solid, 756 mg, 94%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.84 (s, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.29–7.25 (m, 3H), 7.19–7.15 (m, 4H), 6.16 (s, 1H), 2.71 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 7.6 Hz, 2H), 1.74–1.54 (m, 4H), 1.44–1.32 (m, 4H); ¹³C-NMR (100 MHz, CDCl₃) δ: 142.7, 141.5, 134.2, 130.0, 128.4 (2C), 128.3 (2C), 125.7, 125.2, 121.1, 119.2, 111.2, 99.3, 35.9, 31.3, 29.1, 29.03, 28.97, 28.2; HR-MS (ESI) m/z Calcd for [C₂₀H₂₂ClN + H]⁺: 312.1519. Found 312.1557.

Methyl (S)-5-(5-Chloro-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoate (15)

To a stirred solution of 8 (100 mg, 0.321 mmol) in CH₂Cl₂ (4 mL) was added Me₂AlCl (0.52 mL, 1.0 M solution in hexanes) at 0 °C. After stirring the reaction mixture at r.t. for 15 min, the acyl chloride 14 (0.453 mmol) in CH₂Cl₂ (2 mL) was added at 0 °C. The reaction mixture was allowed to warm to r.t. and stirred for 12 h. Aqueous saturated NH₄Cl solution (5 mL) was added to quench the reaction, the two layers separated, the aqueous phase extracted EtOAc (3 × 5 mL), and the combined organic extracts were washed with brine, dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (15% EtOAc/hex) to afford 15 as a yellow viscous oil, 133 mg, 91%. ¹H-NMR (400 MHz, CDCl₃) δ: 8.34 (s, 1H), 7.93 (d, J = 1.2 Hz, 1H), 7.29–7.24 (m, 3H), 7.19–7.15 (m, 4H), 3.68 (s, 3H), 3.13 (t, J = 7.8 Hz, 2H), 3.01 (dd, J = 16.2 Hz, 6.4 Hz, 1H), 2.88 (dd, J = 16.3 Hz, 6.9 Hz, 1H), 2.79–2.70 (m, 1H), 2.60 (t, J = 7.7 Hz, 2H), 2.53 (dd, J = 14.4 Hz, 5.9 Hz, 1H), 2.32 (dd, J = 15.2 Hz, 7.6 Hz, 1H), 1.77–1.61 (m, 4H), 1.48–1.36 (m, 4H), 1.10 (d, J = 6.6 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 195.4, 173.2, 149.4, 142.6, 132.9, 128.4 (2C), 128.3 (2C), 127.8, 127.6, 125.7, 122.6, 120.6, 113.6, 111.9, 51.5, 49.1, 41.1, 35.9, 31.3, 29.4, 29.0, 28.9, 28.8, 26.5, 20.4; HR-MS (ESI) m/z Calcd for [C₂₇H₃₂ClNO₃ + H]⁺: 454.2149. Found 454.2130.

Methyl (S)-5-(5-Chloro-1-(hydroxymethyl)-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoate (16)

The method for the synthesis of 16 was adapted from a published procedure.²⁵⁾ To a stirred suspension of 15 (100 mg, 0.220 mmol) and formaldehyde (37 wt% aqueous solution, 0.8 mL) in THF/H₂O:1/1 (0.6 mL) was added TBAF (1.0 M solution in THF, 0.05 mL) at r.t., and the reaction mixture was stirred for 48 h and then extracted with a solution of EtOAc/hex:1 : 1 (3 × 3 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated. The resulting crude mixture was purified using silica gel column chromatography (25% EtOAc/hex) to afford 16 as a pale-yellow viscous oil, 100 mg, 94%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.82 (d, J = 1.3 Hz, 1H), 7.36–7.14 (m, 7H), 5.58–5.57 (m, 2H), 3.65 (s, 3H), 3.09 (t, J = 7.7 Hz, 2H), 2.88 (dd, J = 16.1 Hz, 6.3 Hz, 1H), 2.78–2.64 (m, 2H), 2.58 (t, J = 7.6, 2H), 2.44 (dd, J = 15.0 Hz, 5.8 Hz, 1H), 2.25 (dd, J = 15.0 Hz, 7.5 Hz, 1H), 1.64–1.55 (m, 4H), 1.47–1.31 (m, 4H), 1.03 (d, J = 6.5 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 195.5, 173.2, 149.8, 142.7, 134.1, 128.4 (2C), 128.3, 128.3 (2C), 127.3, 125.6, 122.8, 120.7, 114.4, 110.7, 66.2, 51.5, 49.4, 41.0, 35.9, 31.4, 29.9, 29.7, 29.0, 26.4, 26.0, 20.3; HR-MS (ESI) m/z Calcd for [C₂₈H₃₄ClNO₄+H]⁺:484.2255. Found 484.2295.

(S)-5-(5-Chloro-1-(hydroxymethyl)-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic Acid (1)

To a stirred suspension of 16 (20 mg, 0.041 mmol) in phosphate buffer, pH 7.0 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 3.1 mL) was added acetonitrile (0.2 mL). Esterase from porcine liver (lyophilized powder, ≥15 units/mg solid, 6 mg) was dissolved in phosphate buffer, pH 7.00 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 0.5 mL) and added to the reaction mixture. The pH of the reaction mixture was monitored by using a pH spear, and maintained at 7.0 by manually adding 0.01 M aqueous NaOH solution using a syringe. The mixture was stirred at r.t. for 48 h. Water (4 mL) was added, and the reaction mixture was acidified to pH 6.0 by adding 1M aqueous HCl. The mixture was filtered through celite. The filtrate (aqueous layer) was extracted with EtOAc (3 × 4 mL), the combined organic extracts were washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified using flash column (10% MeOH/CH₂Cl₂), then further purified by preparative TLC (50% EtOAc/Hex) to yield 1 as a yellow viscous oil, 5 mg, 26%. ¹H-NMR (400 MHz, CD₂Cl₂) δ: 7.95 (d, J = 1.9 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.25–7.11 (m, 6H), 5.64 (s, 2H), 3.17 (t, J = 8.0 Hz, 2H), 2.99 (m, 2H), 2.67 (m, 1H), 2.61 (t, J = 7.8 Hz, 2H), 2.52 (dd, J = 15.2 Hz, 5.6 Hz, 1H), 2.33 (dd, J = 15.1 Hz, 7.3 Hz, 1H), 1.69–1.36 (m, 8H), 1.12 (d, J = 6.7 Hz, 3H). The title compound decomposed during the ¹³C-NMR experiment in CD₂Cl₂ due to its highly labile nature as discussed in the text. HR-MS (ESI) m/z Calcd for [C₂₇H₃₂ClNO₄ − H]⁻: 468.1947. Found 468.1942.¹⁹⁾

Methyl 1-(Hydroxymethyl)-5-methoxy-1H-indole-2-carboxylate (17)

To a stirred solution of methyl 5-methoxy-1H-indole-2-carboxylate (51.3 mg, 0.250 mmol), K₂CO₃ (34.6 mg, 0.250 mmol), paraformaldehyde (75 mg, 2.5 mmol) in CHCl₃ (2 mL), was added tetrabutylammonium bromide (TBAB) (4 mg, 0.013 mmol). The reaction mixture was stirred at r.t. for 3 h. Added water (3 mL) to quench the reaction. Extracted with EtOAc (3 × 3 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated. The resulting crude mixture was purified using silica gel column chromatography (12% EtOAc/hex) to afford 17 as a white solid, 41 mg, 70%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.39 (d, J = 9.9 Hz, 1H), 7.23 (s, 1H), 7.05 (m, J = 7.6, 2H), 5.74 (d, J = 8.3 Hz, 2H), 4.53 (t, J = 8.3 Hz, 1H), 3.94 (s, 3H), 3.84 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 163.7, 155.1, 134.4, 127.3, 126.4, 117.6, 111.6, 111.2, 102.7, 68.1, 55.8, 52.3. HR-MS (ESI): the title compound underwent dehydration; m/z Calcd for [C₁₂H₁₃NO₃]⁺: 218.0817. Found: 218.0810.

1-(Hydroxymethyl)-5-methoxy-1H-indole-2-carboxylic Acid (18)

To a stirred suspension of 17 (4.7 mg, 0.020 mmol) in phosphate buffer, pH 7.0 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 3.1 mL) was added acetone (0.2 mL). Esterase from porcine liver (lyophilized powder, ≥15 units/mg solid, 3 mg) was dissolved in phosphate buffer, pH 7.00 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 0.5 mL) and added to the reaction mixture. The pH of the reaction mixture was monitored by using a pH spear, and maintained at 7.0 by manually adding 0.01 M aqueous NaOH solution using a syringe. The mixture was stirred at r.t. for 48 h. Water (4 mL) was added, and the reaction mixture was acidified to pH 6.0 by adding 1M aqueous HCl. The mixture was filtered through celite. The filtrate (aqueous layer) was extracted with EtOAc (3 × 4 mL), the combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated, and purified using silica gel column chromatography (10% MeOH/CH₂Cl₂+0.5% AcOH) to afford 18 as a white powder, 2.7 mg, 61%. ¹H-NMR (500 MHz, CD₃OD) δ: 7.39 (d, J = 9.0 Hz, 1H), 7.06 (d, J = 2.1 Hz, 1H), 6.98 (s, 1H), 6.89 (dd, J = 9.0, 2.4 Hz, 1H), 5.77 (s, 2H), 3.81 (s, 3H); ¹³C-NMR (125 MHz, CD₃OD) δ: 180.1, 155.8, 134.3, 128.4, 115.4, 111.7, 109.0, 103.5, 68.5, 56.1 (two aromatic carbon peaks overlap to give one signal). HR-MS (ESI): the title compound underwent dehydration; m/z Calcd for [C₁₁H₁₀NO₃]⁺: 204.0661. Found: 204.0650.

Methyl 5-Chloro-1-(hydroxymethyl)-1H-indole-2-carboxylate (19)

To a stirred solution of methyl 5-chloro-1H-indole-2-carboxylate (148 mg, 0.706 mmol), K₂CO₃ (97.6 mg, 0.706 mmol), paraformaldehyde (211.9 mg, 7.060 mmol) in CHCl₃ (2 mL), was added TBAB (11.4 mg, 0.035 mmol). The reaction mixture was stirred r.t. for 3 h. Added water (3 mL) to quench the reaction. Extracted with EtOAc (3 × 3 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated. The resulting crude mixture was purified using silica gel column chromatography (12% EtOAc/hex) to afford 19 as a white solid, 84.8 mg, 50%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.66 (d, J = 1.9 Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.34 (dd, J = 8.9, 2.0 Hz, 1H), 7.26 (s, 1H), 5.77 (d, J = 8.4 Hz, 2H), 4.49 (t, J = 8.4 Hz, 1H), 3.97 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 163.5, 137.2, 128.3, 127.0, 127.0, 126.5, 122.1, 111.5, 111.5, 68.2, 52.6. HR-MS (ESI): the title compound underwent dehydration; m/z Calcd for [C₁₁H₉ClNO₂]⁺: 222.0322. Found: 222.0306.

5-Chloro-1-(hydroxymethyl)-1H-indole-2-carboxylic Acid (20)

To a stirred suspension of 19 (4.8 mg, 0.020 mmol) in phosphate buffer, pH 7.0 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 3.1 mL) was added acetone (0.2 mL). Esterase from porcine liver (lyophilized powder, ≥15 units/mg solid, 3 mg) was dissolved in phosphate buffer, pH 7.00 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 0.5 mL) and added to the reaction mixture. The pH of the reaction mixture was monitored by using a pH spear, and maintained at 7.0 by manually adding 0.01 M aqueous NaOH solution using a syringe. The mixture was stirred at r.t. for 24 h. Water (4 mL) was added, and the reaction mixture was acidified to pH 6.0 by adding 1M aqueous HCl. The mixture was filtered through celite. The filtrate (aqueous layer) was extracted with EtOAc (3 × 4 mL), the combined organic extracts were washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified using silica gel column chromatography (10% MeOH/CH₂Cl₂+0.5% AcOH) to afford 20 as a white powder, 4.1 mg, 91%. ¹H-NMR (500 MHz, CD₃OD) δ: 7.55 (d, J = 1.7 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.19 (dd, J = 8.8, 1.9 Hz, 1H), 6.99 (s, 1H), 5.81 (s, 2H); ¹³C-NMR (125 MHz, CD₃OD) δ: 180.0, 169.5, 137.3, 129.1, 126.7, 124.6, 121.7, 112.4, 108.4, 68.5. HR-MS (ESI): the title compound underwent dehydration; m/z Calcd for [C₁₀H₇ClNO₂]⁺: 208.0165. Found: 208.0148.

Methyl 1-(Hydroxymethyl)-1H-indole-2-carboxylate (21)

To a stirred solution of methyl 1H-indole-2-carboxylate (43.8 mg, 0.250 mmol), K₂CO₃ (34.6 mg, 0.250 mmol), paraformaldehyde (75 mg, 2.5 mmol) in CHCl₃ (2 mL) was added TBAB (4 mg, 0.013 mmol). The reaction mixture was stirred r.t. for 3 h. Added water (3 mL) to quench the reaction. Extracted with EtOAc (3 × 3 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated. The resulting crude mixture was purified using silica gel column chromatography (12% EtOAc/hex) to afford 21 as a white solid, 40 mg, 78%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.69 (d, J = 7.9 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.34 (s, 1H), 7.19 (t, J = 7.4 Hz, 1H), 5.80 (d, J = 8.3 Hz, 2H), 4.54 (t, J = 8.3 Hz, 1H), 3.96 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 163.8, 138.9, 127.2, 126.2, 126.1, 123.0, 121.4, 112.4, 110.3, 69.0, 52.4. HR-MS (ESI): The title compound underwent dehydration; m/z Calcd for [C₁₁H₁₀NO₂]⁺: 188.0712. Found: 188.0705.

1-(Hydroxymethyl)-1H-indole-2-carboxylic Acid (22)

To a stirred suspension of 21 (4.1 mg, 0.020 mmol) in in phosphate buffer, pH 7.0 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 3.1 mL) was added acetone (0.2 mL). Esterase from porcine liver (lyophilized powder, ≥15 units/mg solid, 3 mg) was dissolved in phosphate buffer, pH 7.00 (potassium phosphate monobasic, sodium hydroxide buffer, 0.05 M aqueous, 0.5 mL) and added to the reaction mixture. The pH of the reaction mixture was monitored by using a pH spear, and maintained at 7.0 by manually adding 0.01 M aqueous NaOH solution using a syringe. The mixture was stirred at r.t. for 12 h. Water (4 mL) was added, and the reaction mixture was acidified to pH 6.0 by adding 1M aqueous HCl. The mixture was filtered through celite. The filtrate (aqueous layer) was extracted with EtOAc (3 × 4 mL), the combined organic extracts were washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified using silica gel column chromatography (10% MeOH/CH₂Cl₂+0.5% AcOH) to afford 22 as a white powder, 3.7 mg, 97%. ¹H-NMR (400 MHz, CD₃OD) δ: 7.59 (d, J = 7.9 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.13 (s, 1H), 7.08 (t, J = 7.5 Hz, 1H), 5.88 (s, 2H); ¹³C-NMR (100 MHz, CD₃OD) δ: 163.2, 140.5, 129.5, 127.8, 126.3, 123.4, 122.0, 112.9, 111.6, 67.7. HR-MS (ESI): The title compound underwent dehydration; m/z Calcd for [C₁₀H₈NO₂]⁺: 174.0555. Found: 174.0547.

(S)-5-(5-Chloro-2-(6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic Acid (2)

To a stirred suspension of 15 (10 mg, 0.022 mmol) in THF/H₂O:4/1 (2 mL) was added LiOH.H₂O (13 mg, 0.310 mmol) and MeOH (2 drops) at r.t., and the reaction mixture was stirred for 16 h. The reaction mixture was acidified to pH 1–2 by adding 1M aqueous HCl solution. The aqueous layer was extracted with EtOAc (3 × 2 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (5% MeOH/CH₂Cl₂) to yield the desired product 2 as a yellow viscous oil, 9 mg, 93%. ¹H-NMR (400 MHz, CDCl₃) δ: 8.54 (s, 1H), 7.93 (d, J = 1.3 Hz, 1H), 7.28–7.24 (m, 3H), 7.19–7.14 (m, 4H), 3.11 (t, J = 7.8 Hz, 2H), 3.04–2.91 (m, 2H), 2.76–2.68 (m, 1H), 2.61–2.53 (m, 3H), 2.37 (dd, J = 15 Hz, 6.9 Hz, 1H), 1.76–1.58 (m, 4H), 1.47–1.35 (m, 4H), 1.15 (d, J = 6.7 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 195.8, 176.5, 149.7, 142.6, 132.9, 128.4 (2C), 128.3 (2C), 128.0, 127.6, 125.7, 122.7, 120.6, 113.6, 111.9, 48.8, 35.8, 31.2, 29.7, 29.4, 29.0, 28.9, 28.7, 26.5, 20.5; HR-MS (ESI) m/z Calcd for [C₂₆H₃₀ClNO₃ − H]⁻: 438.1841 found 438.1836.¹⁹⁾

6-(5-Chloro-1-methyl-1H-indol-2-yl)hexanal (24)

To a stirred solution of the alcohol 23 (100 mg, 0.376 mmol) in CH₂Cl₂ (20 mL) was added PCC (71 mg, 0.329 mmol) at 0 °C. The reaction mixture was allowed to warm to r.t. and stirred for 18 h. Al₂O₃ (165 mg) was added, and the mixture was stirred for additional 20 min. The mixture was filtered through Celite, the residue washed with CH₂Cl₂, the combined filtrate concentrated, and the crude material was purified using silica gel column chromatography (2% EtOAc/hex) to yield the desired product (24) as a yellow viscous oil, 56 mg, 56%. ¹H-NMR (400 MHz, CDCl₃) δ: 9.77 (s, 1H), 7.47 (s, 1H), 7.15 (d, J = 12.9 Hz, 1H), 7.08 (d, J = 12.9 Hz, 1H), 6.17 (s, 1H), 3.63 (s, 3H), 2.72 (t, J = 11.3 Hz, 2H), 2.46 (t, J = 10.7 Hz, 2H), 1.78–1.66 (m, 4H), 1.50–1.43 (m, 2H); ¹³C-NMR (100 MHz, CDCl₃) δ: 202.5, 142.4, 135.8, 128.8, 124.9, 120.7, 119.1, 109.7, 98.5, 43.8, 29.6, 28.9, 28.2, 26.6, 21.8; HR-MS (ESI) m/z Calcd for [C₁₅H₁₈ClNO + H]⁺: 264.1155. Found 264.1156.

6-(5-Chloro-1-methyl-1H-indol-2-yl)-1-phenylhexan-1-ol (25)

To a stirred solution of PhMgBr (0.3 mL, 3.0 M solution in Et₂O) in THF (1 mL) was added the aldehyde 24 (100 mg, 0.379 mmol) in THF (1 mL) at 0 °C. The reaction mixture was allowed to warm to r.t. and stirred for 2 h. Aqueous saturated NH₄Cl solution (2 mL) was added, the aqueous layer extracted with EtOAc (3 × 3 mL), the combined organic extracts washed with brine and dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (30% EtOAc/heptane) to afford the alcohol 25 as a light-yellow viscous oil, 100 mg, 77%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.46 (s, 1H), 7.37–7.25 (m, 5H), 7.15–7.06 (m, 2H), 6.15 (s, 1H), 4.68–4.65 (m, 1H), 3.61 (s, 3H), 2.68 (t, J = 11.2 Hz, 2H), 1.84–1.67 (m, 4H), 1.51–1.31 (m, 4H); ¹³C-NMR (100 MHz, CDCl₃) δ: 144.8, 142.7, 135.8, 128.9, 128.5 (2C), 127.6, 125.9 (2C), 124.9, 120.6, 119.1, 109.6, 98.4, 74.6, 38.9, 29.6, 29.2, 28.3, 26.8, 25.6; HR-MS (ESI) m/z Calcd for [C₂₁H₂₄ClNO + H]⁺: 342.1624. Found 342.1624.

2-(6-((tert-Butyldimethylsilyl)oxy)-6-phenylhexyl)-5-chloro-1-methyl-1H-indole (26)

To a stirred solution of the alcohol 25 (400 mg, 1.170 mmol) in CH₂Cl₂ (15 mL) was added imidazole (300 mg, 4.407 mmol) and tert-Butyldimethylsilyl chloride (TBDMSCl) (330 mg, 2.189 mmol) at r.t. The reaction mixture was stirred at r.t. for 14 h. H₂O (20 mL) was added, the two layers separated, and the aqueous phase was extracted with CH₂Cl₂ (3 × 5 mL). The combined organic extracts were dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (10% EtOAc/hex) to furnish the product (26) as a yellow viscous oil, 347 mg, 87%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.45 (S, 1H), 7.30–7.20 (m, 5H), 7.13–7.05 (m, 2H), 6.14 (s, 1H), 4.63–4.60 (m, 1H), 3.58 (s, 3H), 2.65 (t, J = 11.1 Hz, 2H), 1.75–1.52 (m, 4H), 1.45–1.31 (m, 4H), 0.87 (s, 9H), 0.00 (s, 3H), −0.16 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 145.8, 142.8, 135.8, 128.9, 128.0 (2C), 126.8, 125.9 (2C), 124.9, 120.6, 119.1, 109.6, 98.4, 75.0, 40.9, 29.6, 29.3, 28.4, 26.8, 25.9 (3C), 25.4, 18.3, −4.6, −4.9; HR-MS (ESI) m/z Calcd for [C₂₇H₃₈ClNOSi + H]⁺: 456.2489. Found 456.2486.

Methyl (3S)-5-(2-(6-((tert-Butyldimethylsilyl)oxy)-6-phenylhexyl)-5-chloro-1-methyl-1H-indol-3-yl)-3-methyl-5-oxopentanoate (27)

To a stirred solution of 26 (124 mg, 0.272 mmol) and the acyl chloride 14 (84 mg, 0.470 mmol) in CH₂Cl₂ (12 mL) was added Me₂AlCl (0.3 mL, 1.0 M solution in hexanes, 0.3 mmol) dropwise at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and then at r.t. for 10 h. Aqueous saturated NH₄Cl solution (10 mL) was added, the aqueous layer extracted with EtOAc (3 × 10 mL), the combined organic extracts washed with brine and dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (15% EtOAc/hex) to yield the desired product (27) as a yellow viscous oil, 78 mg, 48%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.92–7.81 (m, 1H), 7.26–7.20 (m, 7H), 4.61 (t, J = 5.3 Hz, 1H), 3.68–3.66 (m, 6H), 3.14–2.73 (m, 5H), 2.50–2.31 (m, 2H), 1.70–1.00 (m, 11H), 0.86 (s, 9H), −0.01 (s, 3H), −0.17 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 195.0, 173.3, 150.8, 145.9, 135.2, 128.1 (2C), 127.9, 127.0, 126.9, 126.0 (2C), 122.3, 120.4, 113.3, 110.7, 75.2, 51.6, 49.5, 41.2, 41.0, 29.8, 29.7, 29.2, 26.6, 26.4, 26.0 (3C), 25.6, 20.5, 18.4, −4.5, −4.8; HR-MS (ESI) m/z Calcd for [C₃₄H₄₈ClNO₄Si + H]⁺: 598.3119. Found 598.3126.

Methyl (3S)-5-(5-Chloro-2-(6-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5-oxopentanoate (28)

To a stirring solution of 27 (55 mg, 0.092 mmol) in CH₃CN (5 mL) was added hydrogen fluoride pyridine (0.3 mL, pyridine approx. 30%, hydrogen fluoride approx. 70%) at 0 °C. The reaction mixture was allowed to warm to r.t., and stirred for 5 h. Aqueous saturated NaHCO₃ solution (8 mL) was added, the aqueous layer was extracted with EtOAc (3 × 5 mL), the combined organic extracts were washed with brine, dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (40% EtOAc/hex) to yield the product (28), pale-yellow viscous oil, 26 mg, 58%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.91 (d, J = 1.6 Hz, 1H), 7.38–7.14 (m, 7H), 4.72–4.69 (m, 1H), 3.73–3.59 (m, 6H), 3.24–3.01 (m, 2H), 2.96–2.88 (m, 1H), 2.83–2.68 (m, 1H), 2.58–2.49 (m, 1H), 2.37–2.31 (m, 1H), 2.22–2.20 (m, 1H), 1.89–1.25 (m, 8H), 1.12 (d, J = 6.7 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 195.0, 173.2, 150.6, 144.9, 135.1, 128.4 (2C), 127.9, 127.5, 126.9, 125.8 (2C), 122.2, 120.4, 113.3, 110.6, 74.5, 51.5, 49.4, 41.1, 38.9, 29.6, 29.4, 28.8, 26.5, 26.1, 25.4, 20.4. HR-MS (ESI) m/z Calcd for [C₂₈H₃₄ClNO₄ + H]⁺: 484.2254. Found 484.2251.

(3S)-5-(5-Chloro-2-(6-hydroxy-6-phenylhexyl)-1-methyl-1H-indol-3-yl)-3-methyl-5-oxopentanoic Acid (3)

To a stirred suspension of 28 (15 mg, 0.031 mmol) in THF/H₂O: 4/1 (4 mL) was added LiOH.H₂O (25 mg, 0.596 mmol) and MeOH (2 drops). The reaction mixture was stirred at r.t. for 48 h followed by acidification to pH 6–7 by adding aqueous HCl. The aqueous layer was extracted with EtOAc (3 × 4 mL), the combined organic extracts washed with brine, dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (5% MeOH/CH₂Cl₂) to afford the desired product as a pair of diastereomers (3), yellow viscous oil, 9 mg, 62%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.94–7.93 (m, 1H), 7.37–7.22 (m, 7H), 4.75–4.71 (m, 1H), 3.71 (s, 3H), 3.25–2.95 (m, 4H), 2.80–2.68 (m, 1H), 2.57–2.51 (m, 1H), 2.41–2.36 (m, 1H), 2.13–1.26 (m, 8H), 1.17 (d, J = 6.7 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 195.6, 175.5, 151.0, 144.7, 135.2, 128.5 (2C), 128.1, 127.5, 127.0, 125.9 (2C), 122.4, 120.5, 113.1, 110.7, 74.5, 49.0, 41.1, 29.7, 29.3, 29.1, 28.7, 26.7, 26.1, 25.1, 20.7; HR-MS (ESI) m/z Calcd for [C₂₇H₃₂ClNO₄ − H]⁻: 468.1947. Found 468.1943.¹⁹⁾

6-(5-Chloro-1-methyl-1H-indol-2-yl)-1-phenylhexan-1-one (29)

To a stirred solution of the alcohol 25 (25 mg, 0.073 mmol) in CH₂Cl₂ (1 mL) was added MnO₂ (127 mg, 1.460 mmol) at once, and the reaction mixture was stirred at r.t. for 28 h. The mixture was filtered through Celite, the residue washed with CH₂Cl₂, and the combined filtrate was concentrated and purified using silica gel column chromatography (12% EtOAc/hex) to afford the desired product (29) as a light-yellow solid, 19 mg, 77%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.96–7.94 (m, 2H), 7.65–7.44 (m, 4H), 7.16–7.07 (m, 2H), 6.18 (s, 1H), 3.64 (s, 3H), 2.99 (t, J = 10.2 Hz, 2H), 2.73 (t, J = 10.5 Hz, 2H), 1.86–1.73 (m, 4H), 1.59–1.50 (m, 2H); ¹³C-NMR (100 MHz, CDCl₃) δ: 200.3, 142.6, 137.1, 135.8, 133.0, 128.8, 128.6 (2C), 128.0 (2C), 124.9, 120.7, 119.1, 109.7, 98.5, 38.4, 29.6, 29.0, 28.3, 26.7, 24.0; HR-MS (ESI) m/z Calcd for [C₂₁H₂₂ClNO + H]⁺: 340.1468. Found 340.1468.

5-(5-Chloro-1-methyl-2-(6-oxo-6-phenylhexyl)-1H-indol-3-yl)-3-methyl-5-oxopentanoic Acid (rac-4)

To a stirred solution of 29 (40 mg, 0.118 mmol) in CH₂Cl₂ (4 mL) was added Me₂AlCl (0.4 mL, 1.0 M solution in hexanes) at 0 °C. After stirring the reaction mixture at r.t. for 20 min, CH₃NO₂ (1 mL) was added followed by 3-methylglutaric anhydride (30) (38 mg, 0.297 mmol). The reaction mixture was stirred at r.t. for 16 h. Aqueous saturated NH₄Cl solution (5 mL) was added and the reaction mixture was acidified to pH 3–4 by adding aqueous HCl solution. The aqueous layer was extracted with EtOAc (3 × 5 mL), the combined organic extracts washed with brine (10 mL) and dried over Na₂SO₄, concentrated, and purified using silica gel column chromatography (1% MeOH/CH₂Cl₂) to furnish rac-4, 23 mg, 42%. ¹H-NMR (400 MHz, CDCl₃) δ: 7.98–7.96 (m, 2H), 7.58–7.45 (m, 4H), 7.25–7.22 (m, 2H), 3.72 (s, 3H), 3.25–3.16 (m, 2H), 3.01–2.95 (m, 4H), 2.76–2.68 (m, 1H), 2.52 (dd, J = 14.8 Hz, 5.7 Hz, 1H), 2.39 (dd, J = 14.8 Hz, 6.7 Hz, 1H), 1.85–1.53 (m, 6H), 1.16 (d, J = 6.6 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 200.1, 196.5, 175.7, 150.9, 137.6, 136.9, 133.1, 128.6 (2C), 128.1 (2C), 127.0, 122.5, 120.5, 116.7, 113.1, 110.7, 48.9, 40.9, 38.3, 29.7, 29.3, 28.9, 26.7, 26.2, 23.9, 20.7; HR-MS (ESI) m/z Calcd for [C₂₇H₃₀ClNO₄ − H]⁻: 466.1791. Found 466.1786.¹⁹⁾

Acknowledgments

This work was supported by the American Asthma Foundation (J.R., Grant 12-0049), and the National Heart, Lung, and Blood Institute (J.R., Grant R01HL081873), the Canadian Institutes of Health Research (W.S.P., Grants MOP-6254 and PP2-133388), and by AmorChem (Montreal, QC, Canada). The Meakins-Christie Laboratories-MUHC-RI are supported in part by a Centre grant from Le Fond de la Recherche en Santé du Québec as well as by the J. T. Costello Memorial Research Fund. J.R. also wishes to acknowledge the National Science Foundation for the AMX-360 (Grant CHE-90-13145) and Bruker 400 MHz (Grant CHE-03-42251) NMR instruments. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.

Conflict of Interest

J.R. and W.S.P. have applied for a patent covering S-C025.

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