**2.2 Extraction and isolation**

The water contents of the alüminyum can (250 ml) were extracted with the mixture of dichloromethane-methanol (95:5) and evaporated, residue was directly carried out to a open column with silica gel 60 (0.04-0.063 mm) and eluted with dichloromethane-isopropanol (97:3). Fractions were collected and analyzed by TLC. All of the fractions having the target compound were collected and the solvent was evaporated and crystallization of the residue from ethanol gave 0.021g of white powder compound was obtained, m.p: 189-190oC, Anal. Calcd. for C24H34N6O4S. 0.5 HOH: C 56.34, H 6.90, N 16.42, S 6.27. Found: C 56.47, H 6.95, N 16.21, S 6.28.

## **2.3 Structure identification**

#### **2.3.1 NMR correlation data of dimethylhomosildenafil**

Dimethylhomosildenafil was dissolved in CDCl3 and subjected to 1D and 2D NMR spectroscopic analysis (1H, 13C, DEPT, homo-COSY, HSQC and HMBC). The data are shown in Table 2.

#### **2.3.2 Analysis condition of HPLC/MS**

LC-MS coupled with positive and negative (ESI+) Electro Spray method was used to determine its molecular weight. The HPLC of LC/MS was carried out on a column XTerra® MS C-18 (4.6 X250 mm,5 µm) with Acetonitrile: Methanol:0.05 M Ammonium acetate in water (55:20:25) as mobile phase. The flow rate was 0.9 mL/min, the injection volume was 5 µL and the appropriate running time (at least 15 min). The eluate was monitored by a photo-diode array detector at 254 nm. The analytical condition of mass was as follows: capillary voltage :3.41 kV, cone voltage : 26 V, source temperature : 100 oC : desolvation temperature : 350oC. The HPLC chromatogram of dimethylhomosildenafil is given in Fig 2 with 5.02 min. rt values. This method was carried out all the given HPLC-MS analysis in this text.

**Table 3** shows the 1H-NMR, 13C-NMR, DEPT, COSY, HSQC and HMBC spectral data of isolated compound **1**, which were similar to that of dimethylsildenafil. The spectroscopic numbering used is given in Table 3. The difference of this compound, than dimethylsildenafil is related with ether protons connected to the C-19. Here is one more metyhlene group as propoxy. The 3,5-dimethyl protons of piperazine were observed at δH 1.05 (d, 6H) as expected, in the HMBC spectrum the correlation of H-28,29/H-24,26, DEPT and HSQC results indicated that dimethyl group attached to C24-26. Since the 2,6 diequatorial methyl groups would be lower energetic form, so the configuration is established as a cis diequatorial methyl configuration as shown in Table 1 as it is in the

VARIAN MERCURY 400 MHz FT spectrometer, with CDCl3 as solvent. Chemical shifts (δ) are in ppm relative to TMS. The LC/MS were taken on a Waters Micromass ZQ connected with Waters Alliance HPLC, using ESI(+) method, with C-18 column. Elemental analyses were performed by Leco CHNS-932. The infrared spectrum was recorded in the 600-3600

The water contents of the alüminyum can (250 ml) were extracted with the mixture of dichloromethane-methanol (95:5) and evaporated, residue was directly carried out to a open column with silica gel 60 (0.04-0.063 mm) and eluted with dichloromethane-isopropanol (97:3). Fractions were collected and analyzed by TLC. All of the fractions having the target compound were collected and the solvent was evaporated and crystallization of the residue from ethanol gave 0.021g of white powder compound was obtained, m.p: 189-190oC, Anal. Calcd. for C24H34N6O4S. 0.5 HOH: C 56.34, H 6.90, N 16.42, S 6.27. Found: C 56.47, H 6.95, N

Dimethylhomosildenafil was dissolved in CDCl3 and subjected to 1D and 2D NMR spectroscopic analysis (1H, 13C, DEPT, homo-COSY, HSQC and HMBC). The data are shown

LC-MS coupled with positive and negative (ESI+) Electro Spray method was used to determine its molecular weight. The HPLC of LC/MS was carried out on a column XTerra® MS C-18 (4.6 X250 mm,5 µm) with Acetonitrile: Methanol:0.05 M Ammonium acetate in water (55:20:25) as mobile phase. The flow rate was 0.9 mL/min, the injection volume was 5 µL and the appropriate running time (at least 15 min). The eluate was monitored by a photo-diode array detector at 254 nm. The analytical condition of mass was as follows: capillary voltage :3.41 kV, cone voltage : 26 V, source temperature : 100 oC : desolvation temperature : 350oC. The HPLC chromatogram of dimethylhomosildenafil is given in Fig 2 with 5.02 min. rt values. This method was carried out all the given HPLC-

**Table 3** shows the 1H-NMR, 13C-NMR, DEPT, COSY, HSQC and HMBC spectral data of isolated compound **1**, which were similar to that of dimethylsildenafil. The spectroscopic numbering used is given in Table 3. The difference of this compound, than dimethylsildenafil is related with ether protons connected to the C-19. Here is one more metyhlene group as propoxy. The 3,5-dimethyl protons of piperazine were observed at δH 1.05 (d, 6H) as expected, in the HMBC spectrum the correlation of H-28,29/H-24,26, DEPT and HSQC results indicated that dimethyl group attached to C24-26. Since the 2,6 diequatorial methyl groups would be lower energetic form, so the configuration is established as a cis diequatorial methyl configuration as shown in Table 1 as it is in the

cm-1 range using a Jasco FT-IR-420 spectrometer and KBr pellets.

**2.3.1 NMR correlation data of dimethylhomosildenafil** 

**2.2 Extraction and isolation** 

**2.3 Structure identification** 

MS analysis in this text.

**2.3.2 Analysis condition of HPLC/MS** 

16.21, S 6.28.

in Table 2.

methisosildenafil (Reepmeyer & Avignon, 2009). The IR spectrum of dimethylhomosildenafil is given in Fig 11. This compound also must be put on the inspection list for illegal health-related substances because of the unknown safety and toxicity profile.

Fig. 11. IR spectrum of Dimethylhomosildenafil.

Chromatographic Separation and Identification of Sildenafil

**3. Conclusion** 

the real drug.

**5. References** 

15:467-480.

**4. Acknowledgment** 

spectrometers and elemental analyzer used in this work.

and Yohimbine Analogues Illegally Added in Herbal Supplements 65

Oral PDE5 inhibitors are the treatment of choice for MED**.** The physiological mechanism of erection involves release of nitric oxide (NO) in the corpus cavernosum as a result of sexual stimulation. NO then activates the enzyme guanylate cyclase, which results in increased levels of cyclic guanosine monophosphate (cGMP), leading to smooth muscle relaxation in blood vessels supplying the corpus cavernosum and allowing inflow of blood. Nafil derivatives have no direct relaxant effect on isolated human corpus cavernosum, but enhance the effect of NO by inhibiting PDE5, which is responsible for degredation of cGMP in the corpus cavernosum. When sexual stimulation causes local release of NO, inhibition of PDE5 by nafil analogues causes increased levels of cGMP in the corpus cavernosum, resulting in smooth muscle relaxation and inflow of blood to the corpus cavernosum. This mode of action means that PDE5 inhibitors are ineffective without sexual stimulation. The PDE-5 inhibitors have helped many men with MED, and the FDA indicate that Sildenafil citrate, Vardenafil HCl and Tadalafil are safe and well-tolerated when taken as directed by men who have gotten approval from their doctors. These drugs must not been used without medical examination or prescription. PDE-5 inhibitors also increase the risk of a variety of cardiovascular diseases, including heart attack, myocardial infarction, and sudden death. The medication may interact with other drugs which should be mortal, e.g. synergic effect with alpha-blockers. Other side effects associated with PDE-5 drugs, such as priapism, severe hypotension, increased intraocular pressure and sudden hearing loss and blidness. The PDE-5 inhibitors must not buy over the internet or other non-standart source, otherwise, the men run several risk, such as it should be counterfeit product that does not have the legal structural compound which has been untested for safety or no same purity as

We thank Prof. Dr. Erden Banoğlu (Gazi University, Faculty of Pharmacy, Ankara) for providing sample of Homothiomethisosildenafil which was also isolated from the herbal dietary supplement by him. Central Instrumental Analysis Lab. of Pharmacy, Faculty of Ankara University provided support for acquisition of the IR, NMR, HPLC-MS

Albersen, M.; Shindel, A. W.; Mwamukonda, K. B. & Lue, T. F. (2010). The future is today:

Bell, A. S. & Palmer, M. J. (2011). Novel phosphodiesterase type 5 modulators: a patent survey

Chen, J.; Chiou, W. F.; Chen, C. C. & Chen, C. F. (2000). Effect of the plant-extract osthole on the relaxation of rabbit corpus cavernosum tissue *in vitro*. *J. Urol*. 163:1975-80. Chiou, W. F.; Huang, Y. L.; Chen, C. F. & Chen, C. C. (2001). Vasorelaxing Effect of Coumarins from *Cnidium monnieri* on Rabbit Corpus Cavernosum. *Planta Med*. 67:282-284.

(2008-2010), *Expert Opinion on Therapeutic Patents* 21:1631-1641.

emerging drugs for the treatment of erectile dysfunction, *Expert Opin. Emerging Drugs*


δ ppm in CDCl3, *J* in Hz a) Number in DEPT is the number of attached protons.

Table 3. NMR data of Dimethylhomosildenafil.
