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Journal of Food and Analysis, Vol. 15, No. 2, 2007, Pages 133-138 藥物食品分析 第十五卷 第二期

Isolation and Identification of a Analogue in a Functional Food Marketed for Penile

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, Kuen Yang St., Nankang, Taipei 115, Taiwan, R.O.C. 2. National Research Institute of Chinese Medicine, 155-1, Sec. 2, Li Nung St., Peitou, Taipei 112, Taiwan, R.O.C.

(Received: May 5, 2006; Accepted: December 11, 2006)

ABSTRACT

A vardenafil analogue was found to be added illegally to a dietary supplement marketed for erectile dysfunction. The structure was determined as 2-[2-ethoxy-5-(piperidine-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f ]-[1,2,4]triazin-4- one and named as piperidenafil. The sample was extracted with ethanol and isolated by column chromatography. The structure was identified by a series of 1-D and 2-D NMR techniques, mass and LC/MS/MS. As compared with the structure of vardenafil, it is shown that the ethylpiperazine was switched to piperidine. Since its structure was similar to that of vardenafil, side effects of vardenafil might be associated with this analogue. Basic on the law, foods using any illegal additive such as synthetic chemical compounds shall not be sold, manufactured, imported, processed, used, prepared, stored or transported. Therefore, this vardena- fil analogue has been included in the inspection list of illegal health-related substances in Taiwan.

Key words: vardenafil analogue, piperidenafil, isolated, identified, NMR, LC/MS/MS

INTRODUCTION , and vardenafil in many dietary supplements. A substantial identification system for Recently, and vardenafil (Figure 1) are sildenafil in health foods was reported using three differ- used popularly as an orally drug in the treatment of male ent analytical methods, i.e. TLC, HPLC/MS and HPLC/ erectile dysfunction(1,2), in spite of its association with PDA in Japan(6). Three sildenefil analogues, namely several serious side effects(3). homosildenafil, and hydroxyhomosildenafil, In our laboratory we have found adulterants such were found from many functional foods marketed for as sildenafil(4), homosildenafil, acetildenafil(5), hydroxy- penis erectile dysfunction in Korea(7) and Netherlands(8). Another unknown suspicious compound related to vardenafil was found in a dietary supplement marketed O for enhancing the male sex ability. Although its molecu- lar weight and UV spectra were different from those of N O O HN sildenafil, vardenafil and sildenafil analogues, the data of N NMR inferred a vardenafil analogue. S This analogue was found from an herbal dietary N N supplement in USA in 2006 and was named as piperide- (9) (10) N nafil or piperidino vardenafil . O Sildenafil MATERIALS AND METHODS O I. Equipments OO HN N S N The melting point was determined on a Fisher-Johns N N melting point apparatus. The LC/MS/MS was performed N using a Waters 2690 Alliance LC module, equipped with O 996 photodiode array detector and Micromass Quattro Vardenafil Ultima tandem mass. The NMR spectra were recorded 1 Figure 1. Structures of sildenafil and vardenafil. on a Bruker AMX-400 spectrometer (400 MHZ for H, 100 MH for 13C) with dimethylsulfoxide-d as solvent. * Author for correspondence. Tel: +886-2-26531239; Z 6 Fax: +886-2-26531244; E-mail: [email protected] The infrared (IR) spectrum was recorded in the range 134

Journal of Food and Drug Analysis, Vol. 15, No. 2, 2007 of 400~4000 cm-1 on a Jasco FT-IR-480 plus spectrom- IV. Analysis Condition of LC/MS/MS eter using KBr pellets. The mass was acquired on a THERMO Polaris Q with a DIP module. All chemicals The HPLC of LC/MS/MS was performed out on a used were of analytical grade. Cosmosil 5 C18-AR column (4.6 × 150 mm, 5 μm) using acetonitrile/methanol/1% acetic acid (17:25:58) as the II. Extraction and Isolation mobile phase. The flow rate was 0.5 mL/min, the injec- tion volume was 10 μL and the running time was 35 min. Test samples were randomly obtained from local The eluate was monitored by a photo-diode array detec- markets and the consumer service centers of the local tor and the scan range was 200 to 350 nm. health bureaus by the health officers. Eight capsules of The analytical condition of tandem mass was as sample (4.2 g) were extracted with ethanol, and the left- follows: positive ion electrospray (ES+) modes; daugh- over was removed by filtration. The filtrate was dried ters ion: 460; capillary voltage: 3.0 kV; cone voltage: under reduced pressure using a rotary evaporator at 40°C. 80 V; collision energy: 25 eV; source temperature: 100°C; The residue was subjected to silica gel column chroma- desolvation temperature: 300°C. tography using chloroform containing methanol gradient. The fractions were collected and re-crystallized with methanol to yield the solid compound A (85 mg). RESULTS AND DISCUSSION

III. NMR Correlation Data of Compound A Silica gel column chromatography using chloroform containing methanol gradient was employed to the sepa- The isolated compound A was identified by a series ration and purification of compound A from a dietary of 1-D and 2-D NMR spectroscopic methods, including supplement enhancing the male sex ability. The isolated 1H, 13C, DEPT, COSY, HMQC and HMBC. The data are compound A was obtained as colorless crystal from showed in Table 1. methanol. The melting point was between 185 and 186°C. The UV spectrum of compound A by LC/MS/MS

Table 1. NMR correlation of vardenafil analogue (A)

13 a 1 b No. C (δC) H (δH) DEPT COSY HMBC 1 144.4 – 0 - H-11/H-12 3 137.6 – 0 - H-10 4 155.0 – 0 - - 5 – 11.67 (1H, s) - - - 6 146.1 – 0 - H-15 9 113.7 – 0 - H-10 10 14.2 2.48 (3H, s) 3 - -

11 27.1 2.81 (2H, t, J = 7.6 HZ) 2 H-12 H-12/H-13 12 20.2 1.71 (2H, m) 2 H-11/H-13 H-11/H-13

13 13.7 0.91 (3H, t, J = 7.4 HZ) 3 H-12 H-11/H-12 14 127.0 – 0 - H-15/H-18 15 129.9 7.83 (1H, s) 1 - H-17 16 120.7 – 0 - H-18

17 131.9 7.85 (1H, d, J = 7.6 HZ) 1 H-18 H-15

18 113.2 7.36 (1H, d, J = 8.0 HZ) 1 H-17 - 19 160.1 – 0 - H-15/H-17/H-18/H-20

20 64.9 4.18 (2H, q, J = 6.8 HZ) 2 H-21 H-21

21 14.2 1.32 (3H, t, J = 6.8 HZ) 3 H-20 H-20 25 22.9 1.36 (2H, m) 2 H-24, H-26 H-23, H-27/ H-24, H-26 23,27 46.5 2.90 (4H, br. m) 2,2 H-24, H-26 H-27, H-23 24,26 24.6 1.53 (4H, br. m) 2,2 H-23, H-27 H-23, H-27 a 13 1 δppm in DMSO-d6 , J in HZ, 100 MHZ for C, 400 MHZ for H. bDEPT is the number of attached protons. 135

Journal of Food and Drug Analysis, Vol. 15, No. 2, 2007

-1 showed as λmax at 213 nm (Figure 2). Both the molecular characteristics of an amine at 3317 cm , an aromatic ring weight and absorption spectrum of UV/VIS were differ- at 1593 and 1470 cm-1, an αβ-unsaturated lactam at 1695 ent from those of vardenafil. cm-1 and an ether group at 1250 and 1031 cm-1, all the The mass of compound A founded at m/z 459.11, same as those of vardenafil. 1 13 1 1 corresponding to the molecular formula C22H29O4N5S, The H-NMR, C-NMR, DEPT, H- H COSY and + 29 a.m.u. less than vardenafil referred to CH3CH2 . The HMBC spectral data of compound A are shown in Table fragmentation of compound A was shown in Figure 3. 1, which were similar to that of vardenafil, except the The proposed fragmentation pathways of compound A ethylpiperazine was switched to piperidine. The 1H-1H at the LC/MS/MS electrospray positive (ES+) were illus- COSY, HMQC and HMBC spectra of vardenafil analogue trated in Figure 4. (A) were shown in Figures 5, 6 and 7, respectively. The The IR spectrum showed absorption bands with the spectroscopic numbering used is given in Figure 8. All

9428455 2: Diode Array HH-7-1-LCa 262 (4,477) 7.77e5 213.4 100

Vardenafil

% Vardenafil analogue (A)

0 nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 Figure 2. UV spectra of vardenafil and vardenafil analogue (A).

2003.10.14 Vadenafil-2 204 (5.159) Cm (188:227-144:181) 1: Daughters of 489ES + 151.2 2.27e7 100

Vardenafil

312.2

%

299.2 489.2 376.1 113.3 169.2 284.1 329.2 0

HH-7-1-LCa 245 (4.129) Cm (224:307-98:181) 1: Daughters of 460ES + 151.2 4.92e7 100

Vardenafil analogue (A) 169.2 312.3 % 284.2

256.2 460.4 329.3 0 m/z 100 150 200 250 300 350 400 450 500 Figure 3. The LC/MS/MS fragmentations of vardenafil and vardenafil analogue (A). 136

Journal of Food and Drug Analysis, Vol. 15, No. 2, 2007

signals were assigned unequivocally according to vari- δH 7.83 (1H, s), 7.85 (1H, d, J = 7.6 HZ), 7.36 (1H, d, J = ous NMR spectroscopic data. 8.0 HZ). Two multiple peaks at δH 2.90 and δH 1.53 were 1 The H-NMR spectrum showed characteristics of assigned as the piperidine methylene for H4-23,27 and an amide at δH 11.67 (1H, s), three aromatic protons at H4-24,26, respectively. One peaks at δH 1.36 (2H, m),

H

O O N N O S N S S O O O

O O O

N NH HN NH HN NH N N N O O O N m/z 460 N m/z 460 N m/z 460

O OH N N S S O O O O O

N NH N NH HN NH N N N O O O N N N m/z 460 m/z 311 m/z 460

OH OH N N S S O O

O OH

N NH N NH HN NH HN NH N N N N O O O O

N N N N

m/z 460 m/z 432 m/z 193 m/z 193

HO N S O OH HO HN NH N NH N NH N N N O O O N N N m/z 432 m/z 283 m/z 151 Figure 4. The possible fragmentation pathways of compound A at the LC/MS/MS. 137

Journal of Food and Drug Analysis, Vol. 15, No. 2, 2007 further proved with 2D NMR data, was assigned as the vardenafil (Figure 1). piperidine methylene for H2-25. In the HMQC (Figure 6) and HMBC (Figure 7) The 13C-NMR and DEPT spectra indicated three spectra, the correlation of H-24,26/C-25 indicated that primary carbons, which was one less than vardenafil, two methylenyl groups (δH 1.53) attached to a methyle- eight secondary carbons, three tertiary carbons and nyl carbon (C-25). The correlation of H-20/C-19 exhib- eight quaternary carbons, the same as that of vardenafil. ited the attachment of the ethoxy group (δH 4.18, 1.32) One carbon peak at δC 155.0 belonged to lactam. One to the phenolic carbon (δC 160.1). The correlation of methylene signal at δC 22.9 was obviously different from H-11, H-12/C-1 showed the linkage of the propyl group (δH 2.81, 1.71, 0.91) to pyrazolic carbon (δC 144.4). The correlation of H-10/C-9 exhibited the attachment of

COSY spectrum of Sample II in DMSO, MW 459, Av400 BBO-Z, NRICM a methyl group (δH 2.48) to the pyrimidine ring (δC 113.7). The correlation between H-15(δH 7.83) and C-6 (δC 146.1) suggested that the triazin ring is linked to the ppm phenolic ring. Based on the mass, infrared spectrum and NMR H-13 1 H-21,H-25 spectroscopic data, the structure of compound A was H-24,H-26 H-12 2 determined as 2-[2-ethoxy-5-(piperidine-1-sulfonyl)- H-10 H-23,H-27 3 phenyl]-5-methyl-7-propyl-3H -imidazo[5,1- f ] - [1,2,4]triazin-4-one. Compound A was a vardenafil 4 H-20 analogue (Figure 8), in which a piperidine replaces 5 ethylpiperazine in vardenafil. This analogue was found 6 in an herbal dietary supplement in USA in 2006 and was 7 named as piperidenafil or piperidino vardenafil. In this H-18 H-17 H-15 8

9 BC spectrum of Sample II in DMSO, MW 459, Av400 BBO-Z, NRICM H-10

10 H-15 H-13 H-18 H-24,H-26 H-23,H-27 H-20 H-17 H-21 H-25 H-11

11 H-12

12 ppm C-13 12 11 10 9 8 7 6 5 4 3 2 1 C-10 C-21 ppm C-12 C-25 20 C-24,26 Figure 5. 1H-1H COSY spectrum of vardenafil analogue (A). C-11 40 C-23,27

60 C-20 QC spectrum of Sample II in DMSO, MW 459, Av400 BBO-Z, NRICM H-23,H-27 H-24,H-26 H-21 H-13 H-10

H-15 80 H-20 H-18 H-11 H-17 H-25 H-12

ppm NRICM 100 C-18 C-13 C-9 C-21 C-10 C-16 120 C-12 20 C-14 C-25 C-15 C-24,26 C-17 C-11 30 C-3 140 C-1 C-6 40 C-4 C-23,27 C-19 160 50

60 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm C-20 70 Figure 7. HMBC spectrum of vardenafil analogue (A). 80

NRICM 90

100 O 10 110 C-18 C-19 5 9 3 120 C-16 OO NH 4 C-14 C-15 130 N C-17 27 15 C-3 S 14 N 140 6 C-1 N N 8 C-6 150 22 1 C-4 23 11 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 25 19 O 20 Figure 6. HMQC spectrum of vardenafil analogue (A). Figure 8. Structure of vardenafil analogue (A). 138

Journal of Food and Drug Analysis, Vol. 15, No. 2, 2007 report, the isolation procedures along with a series of 1-D 6. Takako, M., Sutemi, S., Kiyoko, K., Fusako, I., and 2-D NMR spectral data, the fragmentation of mass, Jyunichi, N., Hisashi, K. and Ichiro, Y. 2001. Identifi- the infrared spectrum, the absorption of UV spectra and cation system for sildenafil in health foods. Yakugaku the analytical condition of LC/MS/MS were established Zasshi 121: 765-769. and elucidated. 7. Shin, M. H., Hong, M. K., Kim, W. S., Lee, Y. J. and Jeoung, Y. C. 2003. Identification of a new analogue of sildenafil added illegally to a functional food marketed REFERENCES for penile erectile dysfunction. Food Addit. Contam. 20: 793-796. 1. Terrett, N. K., Bell, A. K., Brown, D. and Ellis, P. 1996. 8. Blok-Tip, L., Zomer, B., Bakker, F., Hartog, K. D., Sildenafil (Viagra) a potent and selective inhibitor of Hamzink, M., ten Hove, J., Vredenbregt, M. and de type-5 cyclic GMP phosphodiesterase with utility for Kaste, D. 2004. Structure elucidation of sildenafil the treatment of male erectile dysfunction. Bioorg. Med. analogues in herbal products. Food Addit. Contam. Chem. Lett. 6: 1819-1824. 21:737-748. 2. Boolell, M., Allen, M. J., Ballard, S. A., Gepi-Attee, 9. Reepmeyer, J. C. and Woodruff, J. T. 2006. Use of S., Muirhead, G. J., Naylor, A. M., Osterloh, I. H. and liquid chromatography–mass spectrometry and a hydro- Gingell, C. 1996. Sildenafil: an orally active type-5 lytic technique for the detection and structure elucida- cyclic GMP specific phosphodiesterase inhibitor tion of a novel synthetic vardenafil designer drug added for treatment of penile erectile dysfunction. Int. J. illegally to a “natural” herbal dietary supplement. J. Impotence Res. 8: 47-52. Chromatogr. A 1125: 67-75. 3. Beavo, J. A. 1995. Cyclic nucleotide phosphodiesteras- 10. Gratz, S. R., Gamble, B. M. and Flurer, R. A. 2006. es functional implication of multiple isoforms. Physiol. Accurate mass measurement using Fourier transform Rev. 75: 725-748. ion cyclotron resonance mass spectrometry for structure 4. Tseng, M. C. and Lin, J. H. 2002. Determination of elucidation of designer drug analogs of tadalafil, sildenafil citrate adulterated in a dietary supplement vardenafil and sildenafil in herbal and pharmaceutical capsule by LC/MS/MS. J. Food Drug Anal. 10: 112- matrices. Rapid Commun. Mass Spectrom. 20: 2317- 119. 2327. 5. Lai, K. C., Liu, Y. C., Tseng, M. C. and Lin, J. H. 2006. Isolation and identification of a sildenafil analogue illegally added in dietary supplements. J. Food Drug Anal. 14: 19-23.