Synthesis and Structure Revision of Dimeric Tadalafil Analogue Adulterants in Dietary Supplements

Abstract

A number of phosphodiesterase 5 (PDE5) inhibitors approved by authorities have been used successfully in the treatment of erectile dysfunction. These medicines must be prescribed carefully due to their adverse effects, but they and their analogues are being illegally added to dietary supplements. These illegal dietary supplements pose a significant risk to public health. Several dimeric tadalafil analogues have been synthesized for use as reference standards in the inspection of functional foods that are mainly advertised as sexual enhancement products. During the course of this synthesis, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) was proven to be the reagent of choice for amide coupling to produce these dimeric tadalafil analogues. Moreover, the trans-isomer structures tentatively assigned for the isolated dimeric tadalafil analogues (bisprehomotadalafil and bisprecyclopentyltadalafil) found in dietary supplements are now revised to cis-isomer structures.

Tadalafil (1, Cialis^®), a phosphodiesterase 5 (PDE5) inhibitor, has been widely used for the treatment of erectile dysfunction (ED). In spite of its excellent efficacy and safety profile, the administration of tadalafil (1) to patients who are concurrently using any form of organic nitrates or guanylate cyclase stimulators is contraindicated due to its hypotensive effects.^1–3) Tadalafil (1) should also not be prescribed in patients with Stevens–Johnson syndrome or exfoliative dermatitis because hypersensitivity reactions to this ED drug have been reported in such patients.⁴⁾ However, tadalafil (1) and its analogues have been detected in a number of dietary supplements over the last decade. These illegal products are mainly advertised as sexual enhancement products and sold without any declarations on the label of the pharmacological and toxicological effects arising from the PDE5 inhibitory activity. Most of the tadalafil analogues detected in illegal dietary supplements were generated by removal of an N-methyl group or its replacement by another group such as amino, ethyl, butyl, octyl or cyclopentyl.^5–14) Epimeric tadalafil analogues^5,15) and precursor-related analogues of tadalafil (1) have also been identified.^16–18) These analogues were presumably designed for use in adulteration to avoid identification by authorities. Recently, we found a new type of tadalafil analogues in dietary supplements, exemplified by 2a,b and 3, which bear two (pre)tadalafil moieties (Fig. 1). We have named these trans-bisprehomotadalafil, trans-bisprecyclopentyltadalafil and bisprenortadalafil, respectively.^5,19,20) The relative stereochemistry of these dimeric analogues was tentatively assigned based on no observation of nuclear Overhauser effect (NOE) between the protons at stereogenic centers in the molecules and by comparison of their spectral data with those of monomeric tadalafil derivatives. Their structures, thus, need to be confirmed clearly by synthesizing the molecules or X-ray crystallography.

Fig. 1. Structures of Tadalafil (1) and Dimeric Tadalafil Analogues (2a,b and 3)

To reduce human exposure to these illegal products will require a method for the rapid screening of dietary supplements. Reference standards are needed to establish and operate screening systems for detecting these dimeric tadalafil analogues. As a part of our ongoing efforts to protect public health from illegal drug analogues, we report herein the synthesis and structural revision of several dimeric tadalafil analogues.

Results and Discussion

Since dimeric tadalafil analogues bearing two (pre)tadalafil moieties were detected together with their monomeric analogues, we surmised that these dimeric tadalafil analogues were generated from either stereoisomer of chloroacetamide 4 as by-products during the synthesis of their monomeric analogues and were not properly removed during purification (Fig. 2). Presumably, dialkylation of amines 5a–c with chloroacetamide 4 (with or without subsequent intramolecular amidation) might produce this type of dimeric tadalafil analogue. We initially attempted to improve the formation of trans-bisprehomotadalafil (trans-2a) from the trans-isomer of chloroacetamide 4 by modification of the reaction conditions for tadalafil (1).^21,22)

Fig. 2. Proposed Mechanism for Generation of Tadalafil (1), trans-Tadalafil (trans-1) and Dimeric Tadalafil Analogues (2a,b, trans-2a,b, 3 and trans-3)

Contrary to our expectations, only trace amounts of trans-bisprehomotadalafil (trans-2a) were obtained under a variety of conditions, and the HPLC retention time for this product was different from that of the previously isolated compound. Thus, considering that the isolated bisprehomotadalafil (2a) might be a cis-isomer, we devised a new synthetic route that produces both the cis- and trans-isomers of the dimeric tadalafil analogues without forming the monomeric analogues (Chart 1). We postulated that the coupling of diacid 8a with an excess of aminoester 9 and trans-9 would afford dimeric tadalafil analogues 2a and trans-2a, respectively. Aminoesters 9 and trans-9 were prepared according to literature procedures.^21,22) Dialkylation of amines 5a,b with bromoacetate 6 produced diester 7a,b, from which the benzyl groups were removed by hydrogenolysis to produce diacids 8a,b in high yields.²³⁾ With these diacids 8a,b in hands, we moved next to couple them with amines 9 and trans-9. Initial attempts using N,N′-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) [with hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt) or 4-dimethylaminopyridine (DMAP)] and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) produced only trace amounts of the dimeric analogues 2a and trans-2a, with nearly quantitative recovery of the starting materials 9 and trans-9 under various conditions. We also tried to couple amines 9 and trans-9 to an acid chloride generated from diacid 8a by using thionyl chloride or oxalyl chloride, but we could not observe the formation of 2a and trans-2a.

Chart 1. Synthesis of Bisprehomotadalafils (2a and trans-2a) and Bisprecyclopentyltadalafil (2b)

We ascribed these poor results to steric hindrance in amines 9 and trans-9 due to the bulky substituents in their piperidine rings. We investigated several other amide coupling reagents (Table 1). Significant amounts of dimeric tadalafil analogues 2a and trans-2a were obtained when bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP)²⁴⁾ and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) were used to activate diacid 8a (Table 1, Entries 1–5).²⁵⁾ Further investigation revealed that among the reagents examined, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) was the most efficient in producing these dimeric tadalafil analogues 2a and trans-2a in moderate to good yields (Table 1, Entries 6–10).²⁶⁾ Contrary to the other coupling reagents examined, amine 9 was almost completely consumed by activation of diacid 8a with HATU. The spectral data for the synthesized bisprehomotadalafil (2a), a cis-isomer, was in good agreement with those of the isolated compound that was originally reported as a trans-isomer.¹⁹⁾ Bisprecyclopentyltadalafil (2b), a cis-isomer, was also synthesized in moderate yield using HATU and proven to be identical to the isolated compound that had tentatively been assigned as the trans-isomer (Table 1, Entry 11).⁵⁾ These results were further confirmed by performing extensive NOE experiments. In contrast to 2a (isolated and synthesized) in CDCl₃, NOE between the protons at stereogenic centers in 2a were clearly observed when acetone-d₆ was used as an NMR solvent (Fig. S36). We also observed NOE between the protons at C-6 and C-12a when we carried out NOE experiments with a large amount of 2b (synthesized) in acetone-d₆. (Fig. S37).

Table 1. Coupling Reactions of Diacids 8a,b with Aminoester 9 or trans-9

Entry	Amine	Diacid	Reaction conditionsa,b)	Product	Yield (%)c)
1	trans-9	8a	PyBroP, i-Pr2EtN, CH2Cl2, reflux, 48 h	trans-2a	5
2	trans-9	8a	PyBroP, i-Pr2EtN, (CH2)2Cl2, reflux, 48 h	trans-2a	5
3	9	8a	PyBroP, i-Pr2EtN, CH2Cl2, reflux, 48 h	2a	54d)
4	9	8a	PyBroP, i-Pr2EtN, (CH2)2Cl2, reflux, 48 h	2a	8
5	9	8a	DMT-MM, NMM, CH2Cl2, rt, 48 h	2a	47e)
6	trans-9	8a	HATU, i-Pr2EtN, CH2Cl2, reflux, 62 h	trans-2a	10
7	trans-9	8a	HATU, i-Pr2EtN, (CH2)2Cl2, reflux, 62 h	trans-2a	32
8	9	8a	HATU, i-Pr2EtN, CH2Cl2, reflux, 48 h	2a	12
9	9	8a	HATU, i-Pr2EtN, (CH2)2Cl2, reflux, 48 h	2a	11
10	9	8a	HATU, i-Pr2EtN, CH2Cl2, rt, 48 h	2a	68
11	9	8b	HATU, i-Pr2EtN, CH2Cl2, rt, 48 h	2b	60

a) The reactions were conducted with diacids 8a,b (1.0 molar equiv.), amine 9 and trans-9 (2.0 molar equiv.), coupling reagents (3.0 molar equiv.) and bases (6.0 molar equiv.). b) PyBroP, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DMT-MM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; NMM, N-methylmorpholine. c) Isolated. d) Based on recovered starting material (70% conversion). e) Based on recovered starting material (50% conversion).

We next sought to synthesize bisprenortadalafil (3). Coupling of aminoester 9 and the readily available diacid 10 using HATU gave diamide 11 in 54% yield²⁷⁾ (Chart 2). Removal of the benzyloxycarbonyl (Cbz) group in 11 with catalytic hydrogenolysis under basic conditions concomitant with intramolecular amidation of the resulting amine succeeded to give bisprenortadalafil (3) in 75% yield. The spectral data for the synthesized bisprenortadalafil (3) were in good accordance with those for the isolated compound.²⁰⁾

Chart 2. Synthesis of Bisprenortadalafil (3)

Conclusion

We have accomplished the synthesis of dimeric tadalafil analogues 2a,b and 3, and found that HATU is an effective coupling reagent for diamide formation from the sterically hindered amine 9 during the course of the synthesis. The structures of the isolated dimeric tadalafil analogues 2a,b, which had tentatively been assigned as trans-isomers, were revised to cis-isomers. We believe that these synthesized dimeric tadalafil analogues will be highly useful as reference standards to aid in inspections of illegally adulterated dietary supplements that contain this type of tadalafil analogue.

Experimental

General Information

All chemicals, solvents and reagents were obtained from commercial suppliers and used without further purification. All reactions were conducted under a nitrogen or argon atmosphere in oven-dried glassware with magnetic stirring. All the reactions were monitored by TLC using Merck pre-coated TLC plates (silica gel 60GF254, 0.25 mm), and flash column chromatographic separations were carried out using silica gel (Merck Kieselgel 60, 230–400 mesh) packed in glass columns. ¹H- and ¹³C-NMR spectra were recorded on Bruker Avance II 600 MHz and Bruker DPX 400 MHz spectrometers. The mass spectra were obtained using JEOL JMS-700 and Waters Xevo G2 QTOF mass spectrometers. The IR spectra were recorded on a Frontier FT-IR spectrophotometer. Optical rotation data were collected by using a Jasco P-2000 digital polarimeter.

Benzyl 2,2′-(Ethylazanediyl)diacetate (7a)

To a stirred suspension of bromoacetate 6 (11.2 mL, 48.7 mmol) and KHCO₃ (5.5 g, 54.9 mmol) in N,N-dimethylformamide (DMF) (100 mL) was slowly added ethylamine 5a (70% aqueous solution, 2.0 mL, 22.1 mmol) over 5 min at 0°C. After 30 min at 0°C, the mixture was stirred overnight at room temperature and concentrated in vacuo. Saturated aqueous NaHCO₃ (50 mL) was added to the residue. The resulting mixture was extracted with CH₂Cl₂ (3×50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous MgSO₄, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (hexane–EtOAc, 9 : 1) to give aminoester 7a (8.0 g, 95%) as a colorless oil: ¹H-NMR (400 MHz, CDCl₃) δ: 7.41–7.28 (m, 10H), 5.14 (s, 4H), 3.61 (s, 4H), 2.79 (q, J=7.2 Hz, 2H), 1.07 (t, J=7.2 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 171.1, 135.7, 128.5, 128.3, 66.2, 54.6, 48.3, 13.0; IR (KBr, cm⁻¹) 3424, 3100, 3012, 2969, 1694, 1472, 1404, 1363; high resolution (HR)-MS-electron ionization (EI): m/z Calcd for C₂₀H₂₃NO₄ (M⁺) 341.1627. Found 341.1626.

2,2′-(Ethylazanediyl)diacetic Acid (8a)

A mixture of aminoester 7a (2.50 g, 7.3 mmol) and 5% Pd/C (350 mg) in MeOH (50 mL) was stirred under a hydrogen atmosphere for 12 h at room temperature. The mixture was filtered through a pad of Celite, and washed with aqueous MeOH (50%, 50 mL). The filtrate was concentrated in vacuo. Traces of water in the resulting residue were removed by co-evaporation with isopropyl alcohol to yield diacid 8a (1.13 g, 95%) as an off-white solid: ¹H-NMR (400 MHz, D₂O) δ: 3.99 (s, 4H), 3.38 (q, J=7.2 Hz, 2H)), 1.31 (t, J=7.2 Hz, 3H); ¹³C-NMR (100 MHz, D₂O) δ: 169.9, 55.6, 52.2, 9.0; IR (KBr, cm⁻¹) 2970, 1746, 1455, 1168; HR-MS-EI: m/z Calcd for C₆H₁₁NO₄ (M⁺) 161.0688. Found 161.0689. The diacid 8a was pure enough to provide spectral data, and was subjected to the next step without further purification.

Benzyl 2,2′-(Cyclopentylazanediyl)diacetate (7b)

Aminoester 7b was synthesized according to the procedure described for 7a, and was obtained as a pale yellow oil (yield: 94%): ¹H-NMR (600 MHz, CDCl₃) δ: 7.41–7.28 (m, 10H), 5.14 (s, 4H), 3.67 (s, 4H), 3.31–3.28 (m, 1H), 1.85–1.74 (m, 2H), 1.75–1.61 (m, 2H), 1.57–1.46 (m, 2H), 1.45–1.34 (m, 2H); ¹³C-NMR (150 MHz, CDCl₃) δ: 171.5, 135.7, 128.5, 128.2, 66.2, 63.8, 54.0, 31.1, 23.5; IR (KBr, cm⁻¹) 2956, 1747, 1455, 1152, 1001; HR-MS-EI: m/z Calcd for C₂₃H₂₇NO₄ (M⁺) 381.1940. Found 381.1939.

2,2′-(Cyclopentylazanediyl)diacetic Acid (8b)

Diacid 8b was synthesized according to the procedure described for 8a, and was obtained as a white solid (yield: 87%): ¹H-NMR (400 MHz, D₂O) δ: 3.99 (s, 4H), 3.97–3.88 (m, 1H), 2.16–2.03 (m, 2H), 1.84–1.64 (m, 6H); ¹³C-NMR (100 MHz, D₂O) δ: 170.2, 68.4, 55.6, 27.5 23.7; IR (KBr, cm⁻¹) 3425, 3031, 2975, 2956, 1737, 1633, 1413, 1333, 1233; HR-MS-EI: m/z Calcd for C₉H₁₅NO₄ (M⁺) 201.1001. Found 201.1001.

(1R,1′R,3R,3′R)-Dimethyl-2,2′-(2,2′-(ethylazanediyl)bis(acetyl))bis(1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate) (2a)

A mixture of diacid 8a (0.4 g, 2.5 mmol), diisopropylethylamine (2.7 mL, 15.5 mmol) and HATU (3.0 g, 7.9 mmol) in anhydrous CH₂Cl₂ (50 mL) was stirred at room temperature for 30 min. Amine 9 (1.8 g, 5.2 mmol) was added and the reaction mixture was stirred for 48 h at room temperature. The mixture was poured into ice-cold water (20 mL) and diluted with CH₂Cl₂ (100 mL). The organic layer was separated, washed with brine, dried over anhydrous MgSO₄, and concentrated at reduced pressure. The residue was semi-purified by flash column chromatography on silica gel (hexane–EtOAc, 1 : 1). The crude solid was recrystallized from isopropylalcohol to afford bisprehomotadalafil (2a, 1.4 g, 68%) as an off-white solid: [α]_D²⁵ −160.2 (c=0.1, CHCl₃); ¹H-NMR (600 MHz, CDCl₃) δ: 7.73 (s, 2H), 7.54 (d, J=7.2 Hz, 2H), 7.25 (d, J=7.8 Hz, 2H), 7.18 (t, J=7.3 Hz, 2H), 7.13 (t, J=7.4 Hz, 2H), 6.88 (s, 2H), 6.82 (s, 2H), 6.60 (d, J=7.9 Hz, 2H), 6.55 (d, J=7.5 Hz, 2H), 5.86 (s, 4H), 5.62 (s, 2H), 3.80 (d, J=14.5 Hz, 2H), 3.64 (d, J=15.7 Hz, 2H), 3.35 (d, J=14.1 Hz, 2H), 3.17 (s, 6H), 3.08 (dd, J=15.4, 6.7 Hz, 2H), 2.79 (br s, 1H), 2.69–2.63 (m, 1H), 1.07 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 171.0, 170.5, 147.5, 147.1, 136.3, 133.5, 130.1, 126.4, 122.9, 122.4, 119.7, 118.6, 111.0, 110.1, 107.8, 107.5, 101.0, 57.5, 52.1, 52.0, 51.4, 49.3, 21.5, 11.9; IR (KBr, cm⁻¹) 3403, 2951, 2899, 1738, 1645, 1488, 1440, 1237, 1038; HR-MS-electrospray ionization (ESI) (negative): m/z Calcd for C₄₆H₄₂N₅O₁₀ [M−H]⁻ 824.2932. Found 824.2944.

(1R,1′R,3S,3′S)-Dimethyl-2,2′-(2,2′-(ethylazanediyl)bis(acetyl))bis(1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate) (trans-2a)

A mixture of diacid 8a (115 mg, 0.71 mmol), diisopropylethylamine (0.68 mL, 3.87 mmol) and HATU (814 mg, 2.14 mmol) in anhydrous dichloroethane (10 mL) was stirred at room temperature for 30 min. Amine trans-9 (500 mg, 1.42 mmol) was added and the reaction mixture was refluxed for 62 h. The mixture was poured into ice-cold water (5 mL) and diluted with CH₂Cl₂ (50 mL). The organic layer was separated, washed with brine, dried over anhydrous MgSO₄, and concentrated at reduced pressure. The residue was semi-purified by flash column chromatography on silica gel (hexane–EtOAc, 1 : 1). The crude solid was recrystallized from isopropylalcohol to give trans-bisprehomotadalafil (trans-2a, 190 mg, 32%) as an off-white solid: [α]_D²⁵ +62.6 (c=0.05, CHCl₃); ¹H-NMR (600 MHz, CDCl₃) δ: 8.25 (s, 2H), 7.50 (d, J=7.0 Hz, 2H), 7.13 (d, J=24.8 Hz, 6H), 6.81 (s, 2H), 6.71 (d, J=12.7 Hz, 4H), 6.37 (s, 2H), 5.91 (s, 4H), 4.95 (s, 2H), [3.60 (s), 3.48 (br d, J=11.9 Hz), 3.19 (br d, J=12.6 Hz) and 3.11 (br d, J=14.8 Hz), 14H], 2.67 (s, 2H), 0.84 (s, 3H); ¹³C-NMR (150 MHz, CDCl₃) δ: 173.0, 171.9, 148.2, 147.3, 136.4, 135.2, 133.5, 126.4, 122.1, 120.1, 119.7, 118.3, 111.4, 108.3, 107.3, 106.8, 101.2, 56.7, 56.0, 53.9, 52.3, 49.1, 22.5, 11.5; IR (KBr, cm⁻¹) 3399, 2953, 2904, 1737, 1654, 1488, 1441, 1239, 1038; HR-MS-ESI (negative): m/z Calcd for C₄₆H₄₂N₅O₁₀ [M−H]⁻824.2932. Found 824.2936.

(1R,1′R,3R,3′R)-Dimethyl-2,2′-(2,2′-(cyclopentylazanediyl)bis(acetyl))bis(1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate) (2b)

Bisprecyclopentyltadalafil (2b) was synthesized according to the procedure described for 2a, and was obtained as a white solid (yield: 60%): [α]_D²⁵ +155.0 (c=0.1, CHCl₃); ¹H-NMR (600 MHz, (CD₃)₂CO) δ: 9.89 (s, 2H), 7.56 (d, J=7.7 Hz, 2H), 7.34 (d, J=8.0 Hz, 2H), 7.12 (t, J=7.4 Hz, 2H), 7.06 (t, J=7.4 Hz, 2H), 6.90 (s, 2H), 6.85 (s, 2H), 6.68 (d, J=8.0 Hz, 2H), 6.53 (d, J=7.9 Hz, 2H), 6.16 (d, J=6.5 Hz, 2H), 5.94 (d, J=4.2 Hz, 4H), 3.97 (d, J=14.7 Hz, 2H), 3.61 (d, J=15.8 Hz, 2H), 3.52 (d, J=14.7 Hz, 2H), 3.41–3.30 (m, 1H), 3.16–3.11 (m, 8H), 1.79 (s, 2H), 1.64–1.36 (m, 6H); ¹³C-NMR (150 MHz, (CD₃)₂CO) δ: 172.3, 171.6, 148.2, 147.8, 137.8, 135.5, 131.5, 127.6, 123.6, 122.6, 119.8, 119.0, 112.0, 110.6, 108.3, 108.1, 101.9, 64.8, 57.1, 52.6, 52.1, 51.8, 30.5, 27.3, 24.5, 24.3, 22.1; IR (KBr, cm⁻¹): 3396, 2951, 1740, 1650, 1487, 1439, 1236, 1039; HR-MS-ESI (negative): m/z Calcd for C₄₉H₄₆N₅O₁₀ [M−H]⁻ 864.3245. Found 864.3265.

Dimethyl-2,2′-(2,2′(((benzyloxy)carbonyl)azanediyl)bis(acetyl))(1R,1′R,3R,3′R)-bis(1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate) (11)

Carbamate 11 was synthesized according to the procedure described for 2a and was obtained as an off-white solid (yield: 54%): [α]_D²⁵ −128.5 (c=0.2, CHCl₃); ¹H-NMR (characterization of the distinguishable two rotamers was performed; 1 : 1.2 rotamer ratio; asterisks denote peaks of the minor rotamer detected separately from those of the major rotamer, 600 MHz, CDCl₃) δ: [8.07* (s), 7.83 (s), 2H)], 7.53 (dd, J=12.4, 7.8 Hz, 2H), 7.32–7.27 (m, 2H), 7.18 (d, J=5.0 Hz, 4H), 7.15 (s, 5H), 6.86 (d, J=18.3 Hz, 2H), 6.78 (s, 2H), 6.62–6.53 (m, 4H), 5.89–5.79 (m, 4H), 5.07 (d, J=12.3 Hz, 1H), 4.99 (d, J=12.3 Hz, 1H), 4.80 (d, J=6.5 Hz, 1H), 4.70–4.57 (m, 2H), 4.53 (d, J=16.9 Hz, 1H), 4.34 (m, 2H), [3.50 (d, J=15.8 Hz) and 3.37* (d, J=14.8 Hz), 2H], [3.14 (s), 3.00* (s), 6H], 2.81–2.75 (m, 2H); ¹³C-NMR (100 MHz, CDCl₃) δ: 170.3, 170.1, 168.9, 168.6, 156.5, 147.4, 147.3, 147.1, 147.0, 136.4, 136.3, 135.8, 133.3, 133.1, 129.6, 128.3, 128.0, 127.8, 126.3, 123.0, 122.5, 122.3, 119.7, 119.5, 118.7, 118.6, 111.0, 110.9, 110.1, 110.0, 107.9, 107.5, 107.5, 107.3, 101.01, 100.97, 68.1, 67.6, 52.2, 52.1, 52.0, 51.8, 50.1, 49.5, 23.9, 21.3, 21.1; IR (KBr, cm⁻¹): 3394, 3316, 2949, 1742, 1700, 1658, 1487, 1439, 1237; HR-MS-ESI (negative): m/z Calcd for C₅₂H₄₄N₅O₁₂ [M−H]⁻ 930.2986. Found 930.3016.

Methyl(1R,3R)-1-(benzo[d][1,3]dioxol-5-yl)-2-(2-((6R,12aR)-6-(benzo[d][1,3]dioxol-5-yl)-1,4-dioxo-3,4,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-2(1H)-yl)acetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (3)

A mixture of dimer 11 (0.60 g, 0.64 mmol), 5% Pd/C (0.24 g) and five drops of Et₃N was stirred under a hydrogen atmosphere at room temperature for 3 d. The mixture was filtered through a pad of Celite, and the filtrate was concentrated at reduced pressure. The crude solid was recrystallized from methanol to yield bisprenortadalafil (3, 0.37 g, 75%) as an off-white solid: [α]_D²⁵ −61.0 (c=0.1, CHCl₃); ¹H-NMR (600 MHz, (CD₃)₂CO) δ: 10.21 (s, 1H), 9.95 (s, 1H), 7.61 (d, J=7.6 Hz, 1H), 7.58 (d, J=7.8 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.33 (d, J=7.9 Hz, 1H), 7.13 (t, J=7.4 Hz, 1H), 7.11–7.03 (m, 3H), 6.93 (s, 1H), 6.90 (s, 1H), 6.90–6.87 (m, 1H), 6.82 (s, 1H), 6.70 (d, J=8.1 Hz, 1H), 6.68 (d, J=8.1 Hz, 1H), 6.56 (d, J=7.6 Hz, 1H), 6.33 (s, 1H), 5.94 (s, 2H), 5.89 (dd, J=9.5, 0.9 Hz, 2H), 5.23 (d, J=6.8 Hz, 1H), 4.88 (d, J=16.2 Hz, 1H), 4.59–4.52 (m, 2H), 4.40 (d, J=16.9 Hz, 1H), 3.98 (d, J=16.9 Hz, 1H), 3.68–3.60 (m, 2H), 3.18–3.11 (m, 5H); ¹³C-NMR (150 MHz, (CD₃)₂CO) δ: 171.6, 168.7, 168.5, 168.1, 148.6, 148.4, 148.1, 147.7, 138.0, 137.9, 137.7, 135.1, 135.0, 131.2, 127.6, 127.4, 123.8, 122.8, 122.6, 121.0, 120.2, 120.0, 119.2, 119.1, 112.2, 112.1, 110.7, 108.7, 108.3, 108.2, 108.1, 106.6, 102.1, 102.0, 56.7, 56.6, 52.9, 52.5, 52.3, 52.1, 48.7, 24.0, 22.2; IR (KBr, cm⁻¹) 3402, 2897, 1740, 1664, 1487, 1440, 1306, 1204; HR-MS-ESI (negative): m/z Calcd for C₄₃H₃₄N₅O₉ [M−H]⁻ 764.2357. Found 764.2375.

Acknowledgments

This research was supported by a Research Grant (15182MFDS523) from the Ministry of Food and Drug Safety (MFDS) in Korea and a 2015 Research Grant (D1000298-01-01) from Kangwon National University. We thank the Central Laboratory of Kangwon National University for providing us with technical assistance on the spectroscopic experiments.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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