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Journal of Food and Analysis, Vol. 15, No. 3, 2007, Pages 220-227 藥物食品分析 第十五卷 第三期

Isolation and Identification of a Analogue in a Dietary Supplement

Kuo-Chih Lai1, Yi-Chu Liu1, Mu-Chang Tseng1, Yun-Lian Lin2 and Jer-Huei Lin1*

1. Bureau of Food and Drug Analysis, Department of Health, Executive Yuan, 161-2, Kunyang St., Nangang, Taipei 115, Taiwan, R.O.C. 2. Natiearch Institute of Chinese Medicine, 155-1, Sec. 2, Linong St., Beitou, Taipei 112, Taiwan, R.O.C.

(Received: August 31, 2006; Accepted: March 13, 2007)

ABSTRACT

A vardenafil analogue was found to be added illegally into a dietary supplement marketed for erectile dysfunction. Its structure was determined as 2-(2-ethoxyphenyl)-5-methyl-7-propyl-imidazo[5,1-f][1,2,4]triazin-4(3H)-one. The sample was extracted with ethanol and isolated by column chromatography. The structure was identified with a series of 1-D and 2-D NMR techniques and LC/MS/MS. Having compared with structure of vardenafil, the data showed that the ethylpiperazinyl and sulfonyl of vardenafil were removed from the phenyl group. Since its structure was similar to that of vardenafil, side effects of vardenafil might associate with this analogue. This vardenafil analogue has been included in the inspection list of illegal adulterants in Taiwan.

Key words: vardenafil analogue, LC/MS/MS, NMR

INTRODUCTION using a Waters 2690 Alliance LC module, equipped with 996 photodiode array detector and Micromass Quattro and vardenafil, two PDE (phosphodies- Ultima tandem mass. The NMR spectra were recorded 1 terase) inhibitors, are used popularly as orally effective on a Bruker AMX-400 spectrometer (400 MHZ for H, (1,2) 13 in the treatment of male erectile dysfunction , 100 MHZ for C) with dimethylsulfoxide-d6 as solvent. despite their association with several serious side All chemicals were of analytical grades. effects(3). A substantial identification system for silde- nafil in health foods was reported using three different II. Extraction and Isolation analytical methods, i.e. TLC, HPLC/MS and HPLC/PDA in Japan(4). Four analogues, homosildenafil, acetildena- Test samples were obtained from local markets and fil, hydroxyhomosildenafil and piperidenafil were found from many functional foods marketed for penis erectile dysfunction in Korea(5), Netherlands(6) and USA(7). O N OO HN In our laboratory we have found adulterants such N (8) (9) S as sildenafil , homosildenafil, , hydroxy- N N (10) R N homosildenafil , , vardenafil and piperide- O nafil in many dietary supplements (Figure 1). Another Sildenafil R = H unknown compound A related to vardenafil was found in Homosildenafil R = CH3 Hydroxyhomosildenafil R = CH2OH a dietary supplement marketed for enhancing male sex ability. Although its molecular weight and UV spectra were different from those of sildenafil, vardenafil and the above sildenafil analogues, the data of NMR indicated a vardenafil analogue was inferred.

Acetildenafil Tadalafil MATERIALS AND METHODS O OO HN N S N I. Equipments N N R O

The melting point was determined on a Fisher-Johns Vardenafil R = NCH2CH3 melting point apparatus. The LC/MS/MS was performed Piperidenafil R = CH2

* Author for correspondence. Tel: +886-2-26531239; Figure 1. Structure of sildenafil, vardenafil, tadalafil, vardenafil Fax: +886-2-26531244; E-mail: [email protected] analogue and sildenafil analogues. 221

Journal of Food and Drug Analysis, Vol. 15, No. 3, 2007 consumer service centers of the local health bureaus. Six rate was 0.3 mL/min, the injection volume was 10 µL, capsules of sample (3.1 g) were extracted with ethanol, and the running time was 40 min. The eluate was moni- and the leftover was removed by filtration. The filtrate tored by a photo-diode array detector and scan range was was dried under reduced pressure using a rotary evapo- 200~350 nm. rator at 40°C. The residue was subjected to a silica gel The analytical condition of tandem mass was as column chromatography using n-hexane with increasing follows: positive ion eletrospray (ES+) modes, daughter amount of ethyl acetate as solvent. The target fractions ion: 313.2, capillary voltage: 3.0 kV, cone voltage: 60 V, were collected and recrystallized with methanol to yield collision energy: 20 eV, source temperature: 120°C, and the white crystal compound A (47.0 mg). desolvation temperature: 300°C.

III. NMR Correlation Data of Compound A RESULT AND DISCUSSION The purified compound A was identified with a series of 1-D and 2-D NMR spectroscopic techniques, A silica gel column chromatography using n-hexane including 1H, 13C, DEPT, COSY, HMQC and HMBC. containing increasing amounts of ethyl acetate as The data are shown in Table 1. solvent was applied to the separation and purification of compound A from one dietary supplement which claimed IV. Analysis Condition of LC/MS/MS to enhance male sex ability. The isolated compound A was obtained as white crystal from methanol. The melt- HPLC of LC/MS/MS was carried out on a column ing point was between 144 and 145°C. 1 13 of Cosmosil 5C18-AR (4.6 × 150 mm, 5 µm) with metha- Table 1 shows the H-NMR, C-NMR, DEPT, nol/0.25% formic acid (70:30) as mobile phase. The flow 1H-1H COSY and HMBC spectral data of compound A.

Figure 2. 1H spectrum of vardenafil analogue A. 222

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Table 1. NMR correlation of vardenafil analogue A 13 1 No. C (δC) H (δH) DEPT COSY HMBC 2 147.5 - 0 - H-6’ 3 - 11.51 (1H, s) - - - 4 155.1 - 0 - - 4a 113.6 - 0 - H-11 5 137.5 - 0 - H-11 7 144.3 - 0 - H-8/H-9

8 27.1 2.81 (2H, t, J = 7.6 HZ) 2 H-9 H-9/H-10 9 20.3 1.71 (2H, m) 2 H-8/H-10 H-8/H-10

10 13.7 0.91 (3H, t, J = 7.4 HZ) 3 H-9 H-8/H-9 11 14.2 2.49 (3H, s) 3 - - 1’ 120.1 - 0 - H-6’/H-5’/H-3’ 2’ 156.8 - 0 - H-6’/H-4’/H-3’/H-7’

3’ 112.7 7.14 (1H, d, J = 8.4 HZ) 1 H-4’ H-5’/H-4’

4’ 132.4 7.50 (1H, dd, J = 7.6, 8.4 HZ) 1 H-3’/H-5’ H-6’/H-5’/H-3’

5’ 120.3 7.04 (1H, dd, J = 7.2, 6.8 HZ) 1 H-6’/H-4’ H-6’/H-4’/H-3’

6’ 130.3 7.52 (1H, d, J = 7.6 HZ) 1 H-5’ H-5’/H-4’

7’ 64.0 4.09 (2H, q, J = 7.0 HZ) 2 H-8’ H-8’

8’ 14.5 1.30 (3H, t, J = 7.0 HZ) 3 H-7’ H-7’ δ 13 1 ppm in DMSO-d6 , J in HZ, 100 MHZ for C, 400 MHZ for H DEPT is the number of attached protons.

Figure 3. DEPT spectrum of vardenafil analogue A. 223

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Figure 4. 1H-1H COSY spectrum of vardenafil analogue A. H-11 H-10

LIN-M312 H-H COSY H-8’ H-7’ H-8

ppm H-9

ppm

5 0.5

1.0 10

1.5 123456789101112 ppm 2.0

2.5 ppm

H-5’ 3.0 7.1 H-3’ 7.2 3.5

7.3 4.0

7.4 4.5 4.5 3.54.0 2.53.0 1.52.0 1.0 0.5 ppm H-4’ 7.5 H-6’ 7.6 7.17.27.37.47.57.6 ppm

Figure 5. Partial 1H-1H COSY spectrum of vardenafil analogue A. 224

Journal of Food and Drug Analysis, Vol. 15, No. 3, 2007 H-11 H-10 H-8’ H-4’ H-5’ H-6’ H-3’ H-9 H-7’ H-8

LIN-M312 HMQC ppm

C-10 10 C-11 C-8’ C-9 20

C-8 30

40

50

60 C-7’ 70

80

90

100

C-3’ 110 C-4a C-1’ 120 C-5’ C-6’ 130 C-4’ C-5 140 C-7 C-2 150 C-4 C-2’ 6.06.57.07.58.0 5.5 4.55.0 3.03.54.0 2.5 1.52.0 0.51.0 ppm

Figure 6. HMQC spectrum of vardenafil analogue A.

H-11 H-8’ H-10 H-4’ H-3’ H-7’ H-8 LIN-M312 HMBC H-9 H-6’ H-5’ H-3 ppm C-10 C-11 C-8’ C-9 20 C-8 30

40

50

60 C-7’ 70

80

90

100

C-3’ 110 C-4a C-1’ C-5’ 120

C-6’ 130 C-4’ C-5 140 C-7 C-2 150 C-4 C-2’ 123456799101112 ppm

Figure 7. HMBC spectrum of vardenafil analogue A. 225

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The spectra were similar to that of vardenafil, except the peaks at δH 2.81, δH 1.71 and δH 4.09 were assigned as the ethylpiperazinyl and sulfonyl group were removed from methylene for H2-8, H2-9 and H2-7’, respectively. phenyl moiety, was named as vardenafil analogue A. The 13C-NMR and DEPT (Figure 3) spectra indi- The 1H-NMR, DEPT, 1H-1H COSY, HMQC and HMBC cated three primary carbons, three secondary carbons, spectra of vardenafil analogue A are shown in Figures 2, four tertiary carbons and seven quaternary carbons. One 3, 4, 5, 6 and 7, respectively. The spectroscopic number- carbon peak at δC 155.1 belonged to lactam. Three methy- ing used is given in Figure 8. All signals were assigned lene signals were shown at δC 27.1, δC 20.3 and δC 64.0 unequivocally according to the various NMR spectro- for C-8, C-9 and C-7’. Four methine peaks at δC 130.3, δC scopic data. 120.3, δC 132.4 and δC 112.7 were assigned as the aromatic The 1H-NMR (Figure 2) spectrum showed charac- carbon for C-6’, C-5’, C-4’ and C-3’, respectively. 1 1 1 1 teristics of an amide at δH 11.51 (1H, s), four aromatic In the H- H COSY (Figure 4) and Partial H- H protons at δH 7.52 (1H, d, J = 7.6 HZ), 7.04 (1H, d, d, J = 6.8, 7.2 HZ), 7.50 (1H, d, d, J = 7.6, 8.4 HZ) and 7.14 (1H, Vardenafil d, J = 8.4 HZ), respectively. Two triplet peaks at δH 0.91 Vardenafil-0429 324 (5.505) 11: Diode Array 100 221.4 5.99e4 and δH 1.30 were assigned as the methyl group for H3-10 and H3-8’. One singlet peak at δH 2.49 was assigned as Vardenafil the methyl group for H3-11. A methine signal of phenyl % 206.4 at δH 7.04 in this vardenafil analogue A was significantly different from vardenafil, which lacked this signal. Three

0 nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

O 11 W HM1-1a 1963 (32.828) 2: Diode Array 220.4 100 2.31e6 3 54a 248.4 HN 4 N 6 6' Vardenafil analogue A 1' 1 N % 5' 2 N 4b 7 9 2' 8 4' 7' O 8' 10 0 nm 3' 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Figure 8. Structure of vardenafil analogue A. Figure 9. UV spectra of vardenafil and vardenafil analogue A.

M nlfd3 1342 Vardenafil, ES+,D489, 80/35/3, 100/300C, 489 > 151 > 312 Library 100 151.0 312.0 255

Vardenafil

299.0

% 489.0

284.0 376.0 169.0 113.0 283.0 377.0 99.0 114.0149.0 329.0 339.0 256.0 0 m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500

W HM1-1-ES+D313 1954 (32.611) Cn (Top,4, Ht); Sm (Mn, 2x0.75); Sb (1,40.00 ); Cm (1876:1997-(1654:1811+2026:2216)) 100 151.1 1.52e6

Vardenafil analogue A %

123.1 169.1 94.1 110.0 82.2 135.1 313.2 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

Figure 10. The fragmentation of vardenafil and vardenafil analogue A. 226

Journal of Food and Drug Analysis, Vol. 15, No. 3, 2007

COSY (Figure 5) spectra, the correlation of H-6’/H-5’, between H-6’ (δH 7.52) and C-2 (δC 147.5) suggested that H-5’/H-6’, H-4’, H-4’/H-5’, H-3’ and H-3’/H-4’ exhibited the triazin ring linked to the benzene ring. the ethylpiperazinyl and sulfonyl group of vardenafil The UV spectrum of vardenafil analogue A was were removed from phenyl moiety. shown as λmax at 248.4, 220.4 nm (Figure 9). Both the In the HMQC (Figure 6) and HMBC (Figure 7) molecular weight and absorption of UV spectrum were spectra, the correlation of H-7’/C-2’ exhibited the attach- different from those of vardenafil. ment of the ethoxy group (δH 4.09, 1.30) to phenolic The [M+H] of vardenafil analogue A founded at carbon (δC 156.8). The correlation of H-8 and H-9/C-7 m/z 313.2, corresponding to the molecular formula showed the linkage of the propyl group (δH 2.81, 1.71, C17H20O2N4, 176 a.m.u. less than vardenafil ([M+H]: m/z 0.91) to pyrazolic carbon (δC 144.3). The correlation of 489.0) referred to ethylpiperazinyl and sulfonyl group. H-11/C-4a exhibited the attachment of a methyl group (δH The fragmentation of vardenafil analogue A is shown in 2.49) to the pyrimidine ring (δC 113.6). The correlation Figure 10. Possible fragmentation pathways of vardena-

H

O O O

N NH N NH NH NH N N N O O O

N N N m/z 313 m/z 313 m/z 313

O O O

N NH N NH NH NH N N N O OH O N N N m/z 313 m/z 313 m/z 313

O O O

N NH N NH NH NH N NH N O OH O

N N N m/z 313 m/z 285 m/z 313

O O NH NH N NH N NH N NH NH O O OH N m/z 313 N m/z 285 N m/z 193

O O

N NH N NH NHNH NH NH N O OH O NH N N m/z 313 m/z 285 m/z 193

NH NH NH N OH O

N N m/z 123 N m/z 151 m/z 94

Figure 11. The possible fragmentation pathways of vardenafil analogue A at the LC/MS/MS. 227

Journal of Food and Drug Analysis, Vol. 15, No. 3, 2007 fil analogue A at the LC/MS/MS electrospray positive 5. Shin, M. H., Hong, M. K., Kim, W. S., Lee, Y. J. and (ES+) are shown in Figure 11. Jeoung, Y. C. 2003. Identification of a new analogue of Based on the mass and NMR spectroscopic data, the sildenafil added illegally to a functional food marketed structure of compound A was an analogue of vardenafil for penile erectile dysfunction. Food Addit. Contam. 20: and was determined as 2-(2-ethoxyphenyl)-5-methyl- 793-796. 7-propyl-imidazo[5,1-f ][1,2,4]triazin-4(3H)-one. 6. Blok-Tip, L., Zomer, B., Bakker, F., Hartog, K. D., Hamzink, M., ten Hove, J., Vredenbregt, M. and de Kaste, D. 2004. Structure elucidation of sildenafil REFERENCES analogues in herbal products. Food Addit. Contam. 21: 737-748. 1. Terrett, N. K., Bell, A. K., Brown, D. and Ellis, P. 1996. 7. Reepmeyer, J. C. and Woodruff, J. T. 2006. Use of Sildenafil (Viagra) a potent and selective inhibitor of liquid chromatography–mass spectrometry and a hydro- type-5 cyclic GMP phosphodiesterase with utility for lytic technique for the detection and structure elucida- the treatment of male erectile dysfunction. Bioorg. Med. tion of a novel synthetic vardenafil designer drug added Chem. Lett. 6: 1819-1824. illegally to a “natural” herbal dietary supplement. J. 2. Boolell, M., Allen, M. J., Ballard, S. A., Gepi-Attee, Chromatogr. A 1125: 67-75. S., Muirhead, G. J., Naylor, A. M., Osterloh, I. H. and 8. Tseng, M. C. and Lin, J. H. 2002. Determination of Gingell, C. 1996. Sildenafil: an orally active type-5 sildenafil citrate adulterated in a dietary supplement cyclic GMP specific phosphodiesterase inhibitor capsule by LC/MS/MS. J. Food Drug Anal. 10: 112- for treatment of penile erectile dysfunction. Int. J. 119. Impotence Res. 8: 47-52. 9. Lai, K. C., Liu, Y. C., Tseng, M. C. and Lin, J. H. 2006. 3. Beavo, J. A. 1995. Cyclic nucleotide phosphodiesteras- Isolation and identification of a sildenafil analogue es functional implication of multiple isoforms. Physiol. illegally added in dietary supplements. J. Food Drug Rev. 75: 725-748. Anal.14: 19-23. 4. Takako, M., Sutemi, S., Kiyoko, K., Fusako, I., 10. Lin, M. C., Liu, Y. C. and Lin, J. H. 2006. Identification Jyunichi, N., Hisashi, K. and Ichiro, Y. 2001. Identifi- of a sildenafil analogue adulterated in two herbal food cation system for sildenafil in health foods. Yakugaku supplements. J. Food Drug Anal.14: 260-264. Zasshi 121: 765-769.