Deuteration of Indole Compounds: Synthesis of Deuterated Auxins, Indole-3-acetic Acid-d5 and Indole-3-butyric Acid-d5

Abstract

In this study, we describe a practical and facile synthesis of deuterium-labeled indoles via acid-catalyzed hydrogen–deuterium exchange. 3-Substituted indoles were efficiently deuterated through treatment with 20 wt % D₂SO₄ in CD₃OD at 60–90 °C. A deuterium incorporation reaction of 3-unsubstituted indoles was accomplished through treatment with CD₃CO₂D at 150 °C. The in situ preparation of a 20 wt % D₂SO₄/CH₃OD/D₂O solution enabled a large-scale and low-cost synthesis of auxins, indole-3-acetic acid-d5 and indole-3-butyric acid-d5.

Introduction

Deuterium compounds are widely used as an efficient tool to understand the biosynthesis of natural products, reaction mechanism, pharmacokinetics, and metabolism of bioactive compounds, by taking advantage of their features in NMR and mass spectroscopies. Besides their use in chemical kinetics, deuterated drugs have recently attracted much attention because of the deuterium pharmacokinetic isotope effect, which lowers the rate of metabolism and the distribution of metabolites.¹ Indeed, deutetrabenazine² has recently been approved by the United States Food and Drug Administration, and several deuterated compounds are currently being tested in clinical trials. An indole skeleton is one of the most common structures observed in biologically active molecules. Thus, deuterated indole compounds are in demand not only by organic chemists but also by bioorganic chemists. Particularly, polydeuterium-labeled indoles are useful for tracing the biotransformation of bioactive indoles. To access a deuterium-labeled indole, a hydrogen–deuterium (H–D) exchange reaction is considered more effective than the multistep synthesis from originally deuterium-labeled small synthons.³ Although several examples of selective deuterium incorporation at the reactive positions of indoles have been reported,⁴ there are limited examples of the H–D exchange reaction of all hydrogen attached to indoles.^5,6 Oba’s group⁴ and Kańska’s group⁶ have reported the H–D exchange reaction of l-tryptophan to prepare pentadeuterium-labeled l-tryptophan with high isotopic purity under acidic conditions. These methods require expensive deuterium reagents, such as CF₃CO₂D, or repeated operations, resulting in reduced optical purity. However, 3-unsubstituted indoles cannot be exposed to acidic conditions because of their high reactivity at the 3-position.⁷ The polydeuteration of indoles, including 3-unsubstituted indoles, has been achieved using a transition metal catalyst.⁸ Although such catalytic conditions are effective for small-scale preparations, the required pyrophoric and flammable reagents and/or high-pressure conditions might not be suitable for large-scale synthesis.

In the course of our study on the biological role and biosynthesis of auxins such as indole acetic acid (IAA, 1) and indole butyric acid (IBA, 2) in plants,⁹ we required deuterium-labeled IAA (3) and IBA (4). Thus, we attempted to prepare a labeled compound via Fischer indole synthesis as a key step from commercially available nitrobenzene-d5 (Scheme 1).¹⁰ Although we successfully prepared the desired deuterium-labeled IBA (4), the deuteration degree was dramatically reduced under the Fischer indole synthesis condition, 1% H₂SO₄ in the CH₃OH solution.¹¹ To our surprise, all deuteriums attached to the indole at C4–C7 positions were partially replaced by protons. The H–D replacement at the C2 position, usually reactive under acidic conditions, also occurred and demonstrated a slight D incorporation (C2:13%, C4:95%, C5:49%, C6:91%, and C7:76% D incorporation). These results indicate that the deuteriums of IBA methyl ester 5 and CH₃OH proton were exchanged under acidic conditions. This inspired a new idea for preparing deuterium-labeled indoles: indole compounds would incorporate deuterium from CD₃OD in the presence of an appropriate acid catalyst. This method could be applicable to biologically important indole compounds, such as IAA (1), tryptophan (6), and other substituted indoles. In this manuscript, we describe a practical and facile method to prepare deuterium-labeled indoles via acid-catalyzed H–D exchange reactions.

Scheme 1. Synthesis of IBA-d5 via Fischer Indole Synthesis.

Results and Discussion

On the basis of the aforementioned idea, we first attempted the deuteration of IBA (2). Thus, 2 was dissolved in a variety of H₂SO₄/CD₃OD solutions, and the reaction was monitored by ¹H NMR spectra. The treatment of 2 with 2.4 wt % H₂SO₄ in CD₃OD promoted the incorporation of deuteriums at the C2 and C4–C7 positions, even at room temperature, albeit with low D content. Upon continuous stirring at 60 °C for 65 h, IBA CD₃ ester 7 with good D incorporation (77% average D incorporation at C2 and C4–C7) was obtained (Table 1, entry 1).¹² The use of 9.7 wt % H₂SO₄ accelerated D incorporation. After reaction at 60 °C for 110 h, the average D incorporation reached a high of 91% (entry 2). This D content of the resulting IBA indicates that the H–D exchange reaction was completed because the deuterium content in the whole reaction system was calculated to be 91%. Further increasing the H₂SO₄ proportion to 20 wt % accelerated the reaction rate, resulting in a complete reaction at 45 h in 98% yield, without generating any other side products (82% average D incorporation, entry 3). Since the D content is reflected in the H⁺/D⁺ ratio of the reaction mixture, increasing the proportion of H₂SO₄ accelerated the reaction rate but decreased D incorporation. The reaction progress under a light shielding condition indicates that the reaction proceeded via a cationic pathway rather than a radical pathway (entries 3 and 4). Using 20 wt % D₂SO₄ instead of H₂SO₄ as the acid accelerated the reaction rate and improved D incorporation (97% average D incorporation, entry 5). Increasing the IBA concentration from 0.1 to 0.3 M slightly decreased the yield and D incorporation (93% average D incorporation, entry 6).

Table 1. Optimization of Reaction Conditions.

entry	H2SO4 (%)	time (h)	D content (%)a	yield (%)b
1	2.4	65	77c	n.d.d
2	9.7	110	91c	n.d.d
3	20	45	82e	98
4f	20	45	81c	n.d.d
5	20g	18	97e	99
6h	20g	18	93e	93

^aIndicated as the average D incorporation at C2 and C4–C7.

^bIsolated yield.

^cD content was calculated based on the ¹H NMR spectra of the reaction mixture.

^dNot determined.

^eD content was calculated based on the ¹H NMR spectra of the isolated product.

^fWith light shielding.

^gD₂SO₄ was used instead of H₂SO₄.

^hThe reaction was performed in a 0.3 M solution.

Once we achieved optimized reaction conditions, the substrate scope in the H–D exchange reaction was investigated, and the results are summarized in Table 2. 3-Substituted indole compounds, such as IAA (1), l-tryptophan (6), N,O-dimethyl-IBA (12), indomethacin (14), and yohimbine hydrochloride (16), were effectively deuterated in good yields with high D incorporation (>91% yield, >95% average D incorporation at C2, C4–7, entries 1–3 and 5–7). The H–D exchange reaction of l-tryptophan (6, >99% ee) was relatively slower than those of other indoles, presumably due to the presence of the amino group (entry 2). The optical purity was determined by chiral high-performance liquid chromatography (HPLC) analysis and was almost maintained (98% ee). When 6 was treated at 90 °C under the same acidic conditions, the reaction time was shortened to 20 h, and the corresponding deuterated tryptophan CD₃ ester (9) was obtained in 99% yield without reducing the optical purity (>99% ee, entry 3). As predicted, introduction of an electron-withdrawing group reduced the reactivity; however, the reaction proceeded well and provided deuterated indole 11 with good D incorporation except for the C7 position (entry 4). 3-Substituted N-methylindole (12) was smoothly deuterated with high D incorporation (entry 5). An acyl group on indole nitrogen was removed under this reaction condition and then the H–D exchange reaction proceeded smoothly because of increasing the electron density (entry 6). In this reaction, C2 methyl protons were replaced with deuterium.¹³ As a result, it was found that various 3-substituted indoles can be effectively deuterated with a high degree of deuterium. Meanwhile, the H–D exchange reaction of carbazole (18) was remarkably slow and required more than 120 h at 90 °C to complete the reaction. Repeating the H–D exchange reaction resulted in increased D incorporation, but the yield decreased (entries 8, 9). Treatment of harmine hydrochloride (20), a naturally occurring β-carboline alkaloid, under this H–D exchange reaction provided deuterated harmine 21 in good yield (entry 10). However, only C6 and C8 protons were selectively exchanged to deuterium due to low electron density at other positions. Contrary to the results of 3-substituted indoles, C3-unsubstituted indoles, such as 2-methylindole (22) and 5-methylindole (23), resulted in complex mixtures under these conditions (entries 10 and 11). High nucleophilicity at the C3 position could cause side reactions.

Table 2. H–D Exchange Reaction of Indoles and Carbazole.

^aD content was calculated based on the ¹H NMR spectra of the isolated product.

^bIsolated yield.

^cIsolated yield of CD₃ ester.

^d98% ee.

^eThe reaction was conducted in a sealed tube.

^f>99% ee.

^gReactions were performed in a 0.05 M solution.

^hD content was calculated based on ¹H NMR spectroscopy after N-methylation.

ⁱAfter isolating deuterated carbazole (70% average D incorporation), the H–D exchange reaction of this product was performed again.

Therefore, for the H–D exchange reaction of 3-unsubstituted indoles, we considered using weaker Brønsted acid, such as acetic acid and substituted acetic acid, which could act as both an acid catalyst and a source of deuterium (Table 3). After screening several reaction conditions, we found that treating 3-unsubstituted indoles with CD₃CO₂D at 150 °C effectively promotes the incorporation of deuteriums at C2–C7 positions. Using more-acidic α-halogenated acetic acids, such as ClCH₂CO₂D, CCl₃CO₂D, and CF₃CO₂D, resulted in the production of indole dimers and oligomers.⁷ The H–D exchange reactions of 3-unsubstituted indoles with CD₃CO₂D are summarized in Table 3. The condition was found to be applicable to the indole itself and the substituted indoles bearing an alkyl group at each position. After reacting for 110 h, the corresponding deuterated indoles were obtained with moderate to good D incorporation and yield. In all cases, the C3 position of the indole was highly reactive, and the H–D exchange occurred even when dissolved in CD₃CO₂D and CDCl₃ and also during purification by silica gel column chromatography. Therefore, the C3 deuterium was partially protonated during the isolation process. Introducing an electron-donating group increased D incorporation, but the yield decreased because of undesired oligomerisation.⁷ However, the substituent at around the C2 and C3 positions suppressed that side reaction due to their steric hindrance. In the reaction of 2-methylindole, methyl protons were replaced with deuterium (entry 3),¹³ whereas the alkyls on the benzene ring were intact (entries 4–6).

Table 3. H–D Exchange Reaction of 3-Unsubstituted Indoles^a.

^aReactions were performed in CD₃CO₂D (0.1 M) in a sealed tube at 150 °C for 110 h.

^bD content was calculated based on the ¹H NMR spectra of the isolated product.

^cIsolated yield.

^dD content was calculated based on ¹H NMR spectroscopy after N-methylation.

^eIndicated as average D incorporation.

As described above, we developed a facile protocol to introduce deuteriums into the indole moiety. However, the method required a somewhat large amount of expensive deuterium reagents and is not necessarily efficient for large-scale synthesis. Thus, we proceeded to study the use of an inexpensive deuterium oxide as the deuterium source. After examining several reaction conditions, we found that the treatment of IBA (2) with 20 wt % D₂SO₄ in CD₃OD/D₂O (7/3, 0.2 M) at 95 °C for 5 h followed by saponification with aqueous LiOH effectively provided deuterated IBA (4, 97% average D incorporation at C2, C4–C7) in 99% yield (Table 4, entry 1). Next, we turned our attention to the use of HC(OCH₃)₃ as a source of CH₃OD and the in situ preparation of this solvent system (entry 2). Indeed, a D₂SO₄/CH₃OD/D₂O mixture was prepared by treating HC(OCH₃)₃ with D₂O in the presence of D₂SO₄, followed by removing HCO₂CH₃ via common distillation in an Ar atmosphere. In this media, the reaction of 2 proceeded as smoothly as under original conditions and successfully provided deuterated IBA (4) in a high yield with a high D content (95% yield, 97% average D incorporation).¹⁴ The effective deuteration of IAA (1) with this procedure was also attained (entry 3). The reaction conditions provide economical ways to access deuterium-labeled 3-substituted indoles in quantities greater than 1 g.¹⁵

Table 4. Synthesis of Polydeuterated IAA and IBA in an Economical Way^a.

entry	substrate	solvent	time (h)	D content (%)b	yield (%)c
1	2	70% CD3OD/D2O	5	97	99
2d	2	CH(OCH3)3/D2Oe	5	97	95
3d	3	CH(OCH3)3/D2Oe	14	97	94

^aReactions were performed in 20 wt % D₂SO₄ in CD₃OD/D₂O (7/3, 0.2 M). After the reaction was completed, the mixture was poured into an aqueous LiOH solution to hydrolyze.

^bIndicated as the average D content at C2 and C4–C7, which were calculated based on the ¹H NMR spectra of the isolated product.

^cIsolated yield.

^dReactions were performed on a 1 gram scale of the substrate.

^eIn situ preparation of 20 wt % D₂SO₄ in CD₃OD/D₂O (7/3).

Conclusions

In summary, we developed a practical and facile protocol for preparing polydeuterium-labeled indoles through the H–D exchange reaction. These procedures are operationally and economically feasible and can be performed not only by organic chemists but also by researchers in other fields. We believe that the results are valuable to encourage researchers to further investigate chemical biology and drug development. In our laboratories, we are currently investigating the metabolism of indole plant hormones using deuterium-labeled indoles.

Experimental Section

General Information

All reagents and solvents were purchased from Tokyo Chemical Industry Co., Ltd.; FUJIFILM Wako Pure Chemical Corporation; Kanto Chemical Co., Ltd.; Sigma-Aldrich Co., LLC; and ISOTEC and used without further purification. Unless otherwise noted, all reactions were conducted without any inert gas. Chromatography was carried out with Wakogel C-200 silica gel (FUJIFILM Wako Pure Chemical Corporation, granule, 0.075–0.150 mm). NMR spectra were recorded at 600, 500, and 400 MHz for ¹H and 150, 125, and 100 MHz for ¹³C on JEOL JNM-ECA600, -ECA500, and -ECZ400R spectrometers, respectively. Chemical shifts were reported in parts per million (ppm, δ) relative to the residual solvent peaks of CD₃OD (3.31 ppm for ¹H NMR, 49.0 ppm for ¹³C NMR) and (CD₃)₂CO (2.05 ppm for ¹H NMR, 206.26 ppm for ¹³C NMR), and coupling constants (J values) were given in Hertz. High-performance liquid chromatography was carried out with a PU–2089 plus HPLC pump (JASCO), an LC–NetII/ADC (JASCO), and a UV–2075 plus UV/vis detector (JASCO). High-resolution mass spectra (HRMS) were measured on a JEOL Accu TOF T-100 equipped with an electrospray ionization (ESI) unit.

Preparation of Deuterated 3-Substituted Indole CD₃ Ester (Tables 1 and 2)

A solution of 3-substituted indoles with 20 wt % D₂SO₄ in CD₃OD (0.1 M) was heated at 60 °C in an NMR tube or 90 °C in a sealed tube. The reaction was monitored by ¹H NMR spectroscopy. After the reaction was completed, the reaction mixture was slowly poured into a saturated aqueous NaHCO₃ solution (10 mL). The resulting mixture was extracted with Et₂O (3 × 10 mL). The combined organic extracts were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give deuterated 3-substituted indole CD₃ ester.

Deuterated IBA CD₃ Ester (7)

Treatment of IBA (2) (10.7 mg, 0.053 mmol) with 20 wt % D₂SO₄ in CD₃OD at 60 °C afforded deuterated IBA CD₃ ester 7 (11.8 mg, 99% yield, 97% average D incorporation at C2 and C4∼C7). ¹H NMR (500 MHz, CD₃OD) δ 7.51 (s, 0.04H), 7.31 (s, 0.03H), 7.07 (s, 0.02H), 7.00–6.97 (0.05H), 2.77 (t, J = 7.5 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H), 1.99 (tt, J = 7.5, 7.5 Hz, 2H), ¹³C NMR (125 MHz, CD₃OD) δ 176.1, 138.1, 128.7, 122.8 (t, J = 27.4 Hz,), 121.7 (t, J = 23.8 Hz), 118.9 (t, J = 23.3 Hz, 2C), 115.3, 111.8 (t, J = 23.3 Hz), 51.1 (m), 34.4, 26.8, 25.5. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₃H₈D₈NO₂]⁺ 226.1683, found 226.1684.

Deuterated IAA CD₃ Ester (8)

Treatment of IAA (1) (8.8 mg, 0.050 mmol) with 20 wt % D₂SO₄ in CD₃OD at 60 °C afforded deuterated IAA CD₃ ester (9.9 mg, 99% yield). ¹H NMR (500 MHz, CD₃OD) δ 7.51 (s, 0.07H), 7.34 (s, 0.04H), 7.15 (s, 0.03H), 7.10 (s, 0.03H), 7.01 (s, 0.03H), 3.76 (s, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 174.9, 137.9, 128.5, 124.4 (t, J = 26.8 Hz), 122.0 (t, J = 23.8 Hz), 119.4 (t, J = 23.3 Hz), 118.9 (t, J = 23.8 Hz), 111.9 (t, J = 24.4 Hz), 108.3, 51.7 (m), 31.9. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₁H₄D₈NO₂]⁺ 198.1370, found 198.1369.

Deuterated l-Trp-CD₃ Ester (9)

Treatment of l-tryptophan (6) (11.2 mg, 0.055 mmol) with 20 wt % D₂SO₄ in CD₃OD at 90 °C in a sealed tube afforded deuterated l-Trp CD₃ ester (9) (12.5 mg, 99% yield, >99% ee). ¹H NMR (500 MHz, CD₃OD) δ 7.52 (s, 0.10H), 7.35 (s, 0.04H), 7.11–7.09 (0.07H), 7.02 (m, 0.03H), 3.88 (t, J = 6.3 Hz, 1H), 3.24 (dd, J = 14.3, 5.3 Hz, 1H), 3.16 (dd, J = 14.3, 7.0 Hz, 1H). ¹³C NMR (125 MHz, CD₃OD): δ 176.2, 138.1, 128.6, 124.5 (t, J = 27.4 Hz), 122.0 (t, J = 23.9 Hz), 119.4 (t, J = 24.4 Hz), 118.7 (t, J = 23.9 Hz), 112.0 (t, J = 23.8 Hz), 110.1, 55.8, 51.8 (m), 31.0. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₂H₇D₈N₂O₂]⁺ 227.1636, found 227.1637. Chiral HPLC analysis: CHIRAL-PAK IB N-5, i-PrOH (0.1% diethylamine): hexane (0.1% diethylamine) = 3: 7, UV 282 nm, flow rate 1.0 mL/min, 25 °C, l-isomer t_R = 10.4 min, D isomer t_R = 9.5 min.

Preparation of 3-(5-Chloroindol-3-yl)-propanoic Acid Methyl Ester (10)

According to Hong and Wang’s report, 3-(5-chloroindol-3-yl)-propanoic acid was prepared.¹⁶ To a solution of crude 3-(5-chloroindol-3-yl)-propanoic acid (710 mg, 3.2 mmol) in methanol (9.6 mL) was added H₂SO₄ (0.5 mL) at 0 °C under an Ar atmosphere. The reaction mixture was stirred at room temperature for 6 h and then 1N NaOH was added to neutralize. The mixture was extracted with Et₂O (3 × 25 mL). The combined organic extracts were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/AcOEt = 5:1) to give 3-(5-chloroindol-3-yl)-propanoic acid methyl ester (10) (731 mg, 96%) as a red brown solid. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (brs, 1H), 7.56 (d, J = 2.3 HZ, 1H), 7.26 (d, J = 8.5 Hz, 1H), 7.14 (dd, J = 8.5, 2.3 Hz, 1H), 7.03 (m, 1H), 3.68 (s, 3H), 3.06 (m, 2H), 2.70 (t, J = 7.6 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 173.6, 134.6, 128.3, 125.1, 122.9, 122.3, 118.2, 114.7, 112.1, 51.6, 34.6, 20.4. IR (neat, ATR) 3370, 1716, 1459, 1441, 1205, 1158, 1096, 1053, 895, 799 cm^–1. HRMS (ESI-TOF) m/z [M – H]⁻ calcd for [C₁₂H₁₁ClNO₂]⁻ 236.0478, found 236.0481.

Deuterated 3-(5-Chloroindol-3-yl)-propanoic Acid CD₃ Ester (11)

Treatment of 3-(5-chloroindol-3-yl)-propanoic acid methyl ester (10) (23.8 mg, 0.10 mmol) with 20 wt % D₂SO₄ in CD₃OD at 90 °C in a sealed tube afforded deuterated 3-(5-chloroindol-3-yl)-propanoic acid CD₃ ester (11) (22.2 mg, 92% yield, 88% average D incorporation at C2, C4 and C6∼C7). ¹H NMR (400 MHz, CD₃OD) δ 7.48 (s, 0.02H), 7.28 (s, 0.42H), 7.07 (s, 0.02H), 7.04 (m, 0.02H), 3.01 (t, J = 7.5 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 175.6, 136.4, 129.5, 125.2, 124.5 (t, J = 27.3 Hz), 122.1 (td, J = 25.0, 10.2 Hz), 118.3 (t, J = 25.0 Hz), 114.6, 113.0 (t, J = 24.9 Hz), 51.3 (sept, J = 22.5 Hz), 35.9, 21.5. HRMS (ESI-TOF) m/z [M – H]⁻ calcd for [C₁₂H₄D₇ClNO₂]⁻ 243.0918, found 243.0915.

Deuterated N-methyl-IBA CD₃ Ester (13)

Treatment of N-methyl-IBA methyl ester (12) (13.9 mg, 0.06 mmol) with 20 wt % D₂SO₄ in CD₃OD at 60 °C afforded deuterated N-methyl-IBA CD₃ ester (13) (14.3 mg, 99% yield). ¹H NMR (600 MHz, CD₃OD) δ 7.51 (s, 0.05H), 7.28 (s, 0.08H), 7.13 (s, 0.08H), 7.01 (s, 0.08H), 6.90 (d, J = 2.1 Hz, 0.07H), 3.72 (d, J = 2.1 Hz, 3H), 2.75 (t, J = 7.5 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.97 (tt, J = 7.5, 7.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.1, 138.6, 129.2, 127.3 (t, J = 27.2 Hz), 121.8 (t, J = 23.0 Hz), 119.2 (t, J = 23.0 Hz), 119.0 (t, J = 24.4 Hz), 114.8, 109.7 (t, J = 23.0 Hz), 51.2 (m), 34.4, 32.5, 26.9, 25.3. HRMS (ESI-TOF) m/z [M + Na]⁺ calcd for [C₁₄H₉D₈NNaO₂]⁺ 262.1666, found 262.1665.

Deuterated 2-(5-Methoxy-2-methylindole-3-acetic Acid) CD₃ Ester (15)

Treatment of indomethacin (14) (17.9 mg, 0.05 mmol) with 20 wt % D₂SO₄ in CD₃OD at room temperature afforded deuterated 2-(5-methoxy-2-methyl-indole-3-acetic acid) CD₃ ester (15) (10.8 mg, 91% yield). ¹H NMR (600 MHz, CD₃OD) δ 7.11 (s, 0.03H), 6.92 (s, 0.03H), 6.67 (s, 0.03H), 3.79 (s, 3H), 3.65 (s, 2H), 2.31 (m, 0.1H). ¹³C NMR (100 MHz, CD₃OD) δ 174.9, 155.0, 135.0, 132.1, 130.1, 111.6 (t, J = 24.5 Hz), 110.9 (t, J = 24.5 Hz), 104.4, 100.9 (t, J = 24.0 Hz), 56.3, 51.6 (quint, J = 22.3 Hz), 30.9. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₃H₇D₉NO₃]⁺ 243.1700, found 243.1697.

Deuterated Yohimbine (17)

Treatment of yohimbine hydrochloride (16) (39.1 mg, 0.1 mmol) with 20 wt % D₂SO₄ in CD₃OD at room temperature afforded deuterated yohimbine (17) (32.8 mg, 91% yield). ¹H NMR (600 MHz, CD₃OD) δ 7.37 (s, 0.03H), 7.29 (s, 0.03H), 7.03 (s, 0.03H), 6.96 (s, 0.03H), 4.23 (m, 1H), 3.39 (dd, J = 12.0, 3.0 Hz, 1H), 3.10 (dd, J = 11.6, 5.7 Hz, 1H), 2.98 (m, 1H), 2.90 (dd, J = 11.1, 2.4 Hz, 1H), 2.72 (dd, J = 15.3, 4.2 Hz, 1H), 2.62 (ddd, J = 11.6, 4.8, 4.2 Hz, 1H), 2.46 (ddd, J = 12.4, 3.0, 3.0 Hz, 1H), 2.34 (dd, J = 11.4, 3.0 Hz, 1H), 2.23 (dd, J = 11.1, 10.8 Hz, 1H), 1.99 (m, 1H), 1.91 (m, 1H), 1.64 (m, 1H), 1.56–1.46 (2H), 1.38 (m, 1H), 1.17 (ddd, J = 12.4, 12.0, 12.0 Hz, 1H). ¹³C NMR (100 MHz, CD₃OD) δ 175.3, 138.0, 135.5, 128.2, 121.4 (t, J = 24.9 Hz), 119.2 (t, J = 23.1 Hz), 118.2 (t, J = 25.0 Hz), 111.7 (t, J =24.5 Hz), 107.7, 68.6, 62.2, 61.9, 54.2, 53.8, 51.4 (quint, J = 22.3 Hz), 41.1, 37.4, 34.6, 33.4, 24.4, 22.4. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₂₁H₂₀D₇N₂O₃]⁺ 362.2461, found 362.2475.

Deuterated Carbazole (19)

A solution of carbazole (18) (15.7 mg, 0.094 mmol) with 20 wt % D₂SO₄ in CD₃OD (1.9 mL, 0.05 M) was heated at 90 °C for 120 h in a sealed tube. The reaction mixture was slowly poured into a saturated aqueous NaHCO₃ solution (30 mL). The resulting suspension was filtered and washed with H₂O to collect the precipitate, which was subsequently dried under reduced pressure. The obtained deuterated carbazole (14.4 mg, 90% yield, 70% average D incorporation at C1–C8) was treated with 20 wt % D₂SO₄ in CD₃OD (1.9 mL, 0.05 M) at 90 °C for 48 h in a sealed tube. Deuterated carbazole (19, 10.0 mg, 61% yield, 98% average D incorporation at C1–C8) was isolated through the same procedure as described above. ¹H NMR (400 MHz, acetone-d6) δ 10.3 (brs), 8.11 (s), 7.51(s), 7.38 (s), 7.17 (s). ¹³C NMR (150 MHz, acetone-d6) δ 141.1, 126.0 (t, J = 24.5 Hz), 124.0, 120.6 (t, J = 24.5 Hz), 119.2 (t, J = 24.4 Hz), 111.4 (t, J = 25.9 Hz). HRMS (ESI-TOF) m/z [M – H]⁻ calcd for [C₁₂D₈N]⁻ 174.1159, found 174.1152.

N-Methylation of Deuterated Carbazole

To a solution of deuterated carbazole (19) (5.1 mg, 0.03 mmol) in THF (300 μL) was added NaH (60% oil dispersion, 1.5 mg, 0.06 mmol) at 0 °C under an Ar atmosphere. After being stirred at 0 °C for 20 min, methyl iodide (4 μL, 0.06 mmol) was added and stirred at rt for 1 h. The reaction was quenched with a saturated aqueous NH₄Cl solution (5 mL) and extracted with AcOEt (5 mL × 3). The combined organic extracts were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give crude N-methylcarbazole (30). Deuterium content was calculated based on the ¹H NMR spectra of this crude mixture. ¹H NMR (400 MHz, acetone-d6) δ 8.14 (s, 0.04H) 7.54 (s, 0.04H), 7.46 (s, 0.03H), 7.20 (s, 0.06H), 3.91 (s, 3H). HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₃H₄D₈N]⁺ 190.1472, found 190.1473.

Deuterated Harmine (21)

Treatment of harmine hydrochloride (20) (24.9 mg, 0.10 mmol) with 20 wt % D₂SO₄ in CD₃OD at 90 °C afforded deuterated harmine (21) (20.3 mg, 94% yield). ¹H NMR (600 MHz, CD₃OD) δ 8.14 (d, J = 5.7 Hz, 1H), 7.97 (s, 1H), 7.78 (d, J = 5.7 Hz, 1H), 7.03 (s, 0.02H), 6.85 (d, J = 8.4 Hz, 0.02H), 3.90 (s, 3H), 2.76 (s, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 162.5, 144.1, 142.0, 137.9, 136.3, 130.2, 123.4, 116.3, 113.3, 110.7 (t, J = 25.0 Hz), 95.2 (t, J = 24.5 Hz), 56.0, 19.5. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₃H₁₁D₂N₂O]⁺ 215.1153, found 215.1146.

General Procedure of the H–D Exchange Reaction of 3-Unsubstituted Indole (Table 3)

A solution of 3-unsubstituted indoles in CD₃CO₂D (0.1 M) was heated at 150 °C for 110 h in a sealed tube. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/AcOEt = 7/1) to give deuterated indoles.

Deuterated Indole (24)

A solution of indole (29.3 mg, 0.25 mmol) in CD₃CO₂D (2.5 mL, 0.1 M) was heated at 150 °C to afford deuterated indole (24) (22.8 mg, 77% yield). ¹H NMR (600 MHz, CD₃OD) δ 7.53 (m, 0.86H), 7.36 (m, 0.68H), 7.20 (s, 0.02H), 7.08 (m, 0.72H), 6.99 (m, 0.57H), 6.42 (s, 0.39H). ¹³C NMR (100 MHz, CD₃OD) δ 137.6, 129.7–129.2, 125.4–125.0, 122.2–121.8, 121.3–120.8, 120.0–119.4, 112.3–111.7, 102.2–101.7. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₈H₁D₅N]⁺ 121.0814, found 121.0813.

N-Methylation of Deuterated Indole

To a solution of deuterated indole (24) (15.1 mg, 0.13 mmol) in THF (1.3 mL) was added NaH (60% oil dispersion, 8 mg, 0.20 mmol) at 0 °C under an Ar atmosphere. After being stirred at 0 °C for 20 min, methyl iodide (17 μL, 0.27 mmol) was added and stirred at rt for 1 h. The reaction was quenched with a saturated aqueous NH₄Cl solution (5 mL) and extracted with AcOEt (5 mL × 3). The combined organic extracts were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give crude N-methylindole (31). Deuterium content was calculated based on the ¹H NMR spectra of this crude mixture. ¹H NMR (600 MHz, acetone-d6) δ 7.57–7.53 (0.86H), 7.39–7.35 (0.68H), 7.21–7.19 (0.02H), 7.18–7.12 (0.58H), 7.06–7.00 (0.72H), 6.41 (s, 0.57H), 3.82 (s, 3H).

Deuterated 1-Ethylindole (25)

A solution of 1-ethylindole (20.5 mg, 0.14 mmol) in CD₃CO₂D (1.4 mL, 0.1 M) was heated at 150 °C to afford deuterated 1-ethylindole (25) (16.6 mg, 81% yield). ¹H NMR (400 MHz, acetone-d6) δ 7.56–7.53 (0.74H), 7.44–7.40 (0.53H), 7.28 (d, J = 3.2 Hz, 0.12H), 7.16–7.11 (0.49H), 7.03–6.98 (0.26 H), 6.43–6.41 (0.81 H), 4.24 (q, J = 7.4 Hz, 2H), 1.41 (t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 135.6, 128.5, 126.7 (t, J = 27.8 Hz), 121.2 (t, J = 11.1 Hz), 120.9, 119.1, 109.1, 100.8, 40.9, 15.4. HRMS (ESI-TOF) m/z [M – H]⁻ calcd for [C₁₀H₆D₄N]⁻ 148.1064, found 148.1069.

Deuterated 2-Methylindole (26)

A solution of 2-methylindole (22) (14.7 mg, 0.11 mmol) in CD₃CO₂D (1.1 mL, 0.1 M) was heated at 150 °C to afford deuterated 2-methylindole (26) (13.8 mg, 92% yield). ¹H NMR (500 MHz, CD₃OD) δ 7.39–7.35 (0.34H), 7.23 (s, 0.30H), 6.99–6.96 (0.04H), 6.93–6.89 (0.10H), 6.09 (s, 1H), 2.38–2.35 (m, 0.14H). ¹³C NMR (125 MHz, CD₃OD) δ 137.9, 136.4, 130.5, 121.1–118.8 (3C), 110.9 (t, J = 24.4 Hz), 100.1 (m), 12.7 (m). HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₉H₁D₇N₁]⁺ 137.1096, found 137.1094.

Deuterated 4-Methylindole (27)

A solution of 4-methylindole (12.4 mg, 0.10 mmol) in CD₃CO₂D (1.0 mL, 0.1 M) was heated at 150 °C to afford deuterated 4-methylindole (27) (13.0 mg, 96% yield). ¹H NMR (600 MHz, CD₃OD) δ 7.21–7.16 (0.11H), 6.97 (s, 0.67H), 6.80–6.76 (0.04H), 6.45 (s, 0.53H), 2.50 (s, 3H).¹³C NMR (125 MHz, CD₃OD) δ 137.2, 130.3, 129.1, 124.8–124.1, 122.4–121.5, 119.7 (t, J = 20.3 Hz), 109.5 (t, J = 19.2 Hz), 100.6–100.1, 18.9. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₉H₃D₅N₁]⁺ 135.0971, found 135.0970.

Deuterated 5-Methylindole (28)

A solution of 5-methylindole (23) (13.1 mg, 0.10 mmol) in CD₃CO₂D (1.0 mL, 0.1 M) was heated at 150 °C to afford deuterated 5-methylindole (28) (7.7 mg, 57% yield). ¹H NMR (600 MHz, CD₃OD) δ 7.31 (s, 0.07H), 7.24 (s, 0.33H), 7.16–7.14 (0.02H), 6.93–6.89 (0.03H), 6.32 (s, 0.28H), 2.39 (m, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 135.9, 129.6, 128.7, 125.6–124.8, 123.8–123.1, 120.4 (t, J = 22.7 Hz), 111.4 (t, J = 23.8 Hz), 101.3 (t, J = 26.3 Hz), 21.5. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₉H₃D₅N]⁺ 135.0971, found 135.0965.

Deuterated 7-Ethylindole (29)

A solution of 7-ethylindole (13.7 mg, 0.10 mmol) in CD₃CO₂D (1.0 mL, 0.1 M) was heated at 150 °C to afford deuterated 7-ethylindole (29) (11.5 mg, 78% yield). ¹H NMR (600 MHz, CD₃OD) δ7.38–7.33 (0.16H), 7.19 (s, 0.03H), 6.94–6.88 (0.27H), 6.41 (s, 0.44H), 2.89 (q, J = 7.6 Hz, 2H), 1.33 (t, J = 7.6 Hz, 3H). ¹³C NMR (125 MHz, CD₃OD): δ 136.2, 129.3 (t, J = 9.6 Hz), 127.9, 125.0 (t, J = 9.6 Hz), 120.5 (t, J = 19.1 Hz), 120.0, 118.8 (t, J = 15.6 Hz), 102.5–102.3, 25.2, 14.7. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₀H₅D₅N]⁺ 149.1127, found 149.1123.

General Procedure of the Economically Efficient H–D Exchange Reaction of 3-Substituted Indole (Table 4)

To a mixture of HC(OCH₃)₃ (51 mL, 0.47 mol) and D₂O (24.5 mL, 1.35 mol) was added D₂SO₄ (6.5 mL, 0.12 mol) dropwise at 0 °C under an Ar atmosphere. After being stirred at rt for 20 min, generated HCO₂CH₃ (28.6 mL, 0.47 mol) was removed by general distillation under an Ar atmosphere to prepare 20 wt % D₂SO₄ in CH₃OD/D₂O (7/3) solution. A solution of IAA (1) or IBA (2) (1.0 g) in 20 wt % D₂SO₄ in a CH₃OD/D₂O (7/3) solution (0.2 M) was heated at 95 °C for 5–14 h. The mixture was cool to ambient temperature and poured into a 6 wt % LiOH aqueous solution (100 mL). After being stirred at rt for 1 h, 6 N HCl was added to the mixture to be an acidic solution (pH = 2). The resulting mixture was extracted with Et₂O (100 mL × 3). The combined organic extracts were washed with brine (100 mL), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/AcOEt = 1/1) to provide deuterated IAA (3) or IBA (4) (97% average D incorporation at C2 and C4–C7).

Deuterated IBA (4)

A solution of IBA (2) (1.0 g, 4.92 mmol) in 20 wt % D₂SO₄ in a CH₃OD/D₂O (7/3) solution (24.6 mL, 0.2 M) was heated at 95 °C for 5 h to provide deuterated IBA (4) (967 mg, 95% yield, 97% average D incorporation at C2 and C4–C7) as colorless crystals. ¹H NMR (600 MHz, CD₃OD) δ 7.53 (s, 0.05H), 7.32 (s, 0.03H), 7.07 (s, 0.03H), 7.01 (s, 0.03H), 6.98 (s, 0.03H), 2.79 (t, J = 7.8 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.99 (tt, J = 7.8, 7.5 Hz, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 177.8, 138.1, 128.7, 122.7 (t, J = 27.3 Hz), 121.7 (t, J = 23.0 Hz), 119.0 (t, J = 23.0 Hz), 118.9 (t, J = 23.0 Hz), 115.4, 111.8 (t, J = 24.5 Hz), 34.5, 26.9, 25.5. HRMS (ESI-TOF) m/z [M+H]⁺ calcd for [C₁₂H₉D₅NO₂]⁺ 209.1338, found 209.1332.

Deuterated IAA (3)

A solution of IAA (1) (1.0 g, 5.7 mmol) in 20 wt % D₂SO₄ in a CH₃OD/D₂O (7/3) solution (28.6 mL, 0.2 M) was heated at 95 °C for 14 h to provide deuterated IAA (3) (959 mg, 94% yield, 95% average D incorporation at C2 and C4–C7) as brown solid. ¹H NMR (600 MHz, CD₃OD) δ 7.54 (s, 0.03H), 7.33 (s, 0.03H), 7.15 (s, 0.03H), 7.09 (s, 0.02H), 7.01 (0.03H), 3.73 (s, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.5, 137.9, 128.6, 124.4 (t, J = 27.3 Hz), 121.9 (t, J = 23.0 Hz), 119.3 (t, J = 24.5 Hz), 119.0 (t, J = 24.5 Hz), 111.8 (t, J = 23.0 Hz), 108.7, 31.9. HRMS (ESI-TOF) m/z [M + H]⁺ calcd for [C₁₀H₅D₅NO₂]⁺ 181.1025, found 181.1030.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c02940.

• Details of the results and ¹H and ¹³C NMR spectra and MS spectra for all deuterated compounds (PDF)

The authors declare no competing financial interest.