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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Pyrazolopyrimidines in ‘all-natural’ products for erectile dysfunction treatment: the unreliable quality of dietary supplements a
a
b
Nicholas Schramek , Uwe Wollein & Wolfgang Eisenreich a
Bavarian Health and Food Safety Authority, Oberschleißheim, Germany
b
Lehrstuhl für Biochemie, Technische Universität München, Garching, Germany Published online: 17 Dec 2014.
Click for updates To cite this article: Nicholas Schramek, Uwe Wollein & Wolfgang Eisenreich (2014): Pyrazolopyrimidines in ‘all-natural’ products for erectile dysfunction treatment: the unreliable quality of dietary supplements, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2014.992980 To link to this article: http://dx.doi.org/10.1080/19440049.2014.992980
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Food Additives & Contaminants: Part A, 2014 http://dx.doi.org/10.1080/19440049.2014.992980
Pyrazolopyrimidines in ‘all-natural’ products for erectile dysfunction treatment: the unreliable quality of dietary supplements Nicholas Schrameka*, Uwe Wolleina and Wolfgang Eisenreichb a
Bavarian Health and Food Safety Authority, Oberschleißheim, Germany; bLehrstuhl für Biochemie, Technische Universität München, Garching, Germany
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(Received 8 September 2014; accepted 24 November 2014) A herbal food supplement advertised as a potency pill was screened for the presence of PDE5 inhibitors. The resulting signals were characterised by UV, LC-MS in ESI-negative mode, and NMR spectroscopy using 1D and 2D experiments. Several substances were identified, bearing the basic chemical structure of sildenafil, but were not supposed to exhibit PDE5 inhibition. These compounds may be process-related impurities or by-products of different reaction steps in the synthesis of PDE5 analogues. As they were found to be present in different capsules at different concentrations, this is an example of the unreliable quality of dietary supplements. Keywords: dietary supplements; sildenafil analogue; impurities; LC-MS; NMR; reaction pathway
Introduction Medicinal products are highly regulated to ensure high quality and safety. Of particular importance is the identification and quantification of pharmaceutical impurities in medicinal products, active ingredients and pharmaceutical excipients, as they may influence the efficacy and safety of pharmaceutical products even in small amounts (Roy 2002). The European pharmacopoeia limits impurities for pharmaceutical use in the general text 5.10 ‘Control of impurities in substances for pharmaceutical use’, which is equal to the ICH guideline ‘Impurities in New Drug Substances’ ICH Q3A (R2) from 2006 (ICH Q3A (R2) Impurities in New Drug Substances 2006). Specific thresholds for reporting, identification and quantification have been established in the general monograph ‘Substances for Pharmaceutical Use’ (Ph.Eur. 8.0/2034). All these regulatory documents are useful tools in the control of impurities in products having an authorisation to enter the legal supply chain. But there is a black market where all these regulations are futile. On the one hand, there are counterfeit and imitation medicinal products bearing a high risk to human health since their actual content is unknown (Ham 2003; Deisingh 2005). On the other hand, a huge and still growing market exists for dietary supplements intended to maintain and promote health (Crowley & FitzGerald 2006; Marik & Flemmer 2012). Contamination and inaccurate labelling of dietary supplements is known to be a problem and could be a threat to human health (Slifman et al. 1998; Maughan 2005). This is especially true for lifestyle preparations such as slimming agents or sexual enhancers. Their usual pure natural ingredients are often expanded by one or several non*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
declared chemical compounds (Blok-Tip et al. 2004; Yuen et al. 2007; Venhuis et al. 2008; Tang et al. 2011; Wollein et al. 2011; Venhuis & De Kaste 2012; Khazan et al. 2013; Patel et al. 2014). These kinds of reports all cover active pharmaceutical ingredients (API) or analogues added to herbal supplements, not monitored by authorities. Moreover, there seems to be high variability within single packages of dietary supplements (Venhuis et al. 2014). In this study, we identified most of the variable ‘impurities’ bearing substructures of PDE5 inhibitors, present in identical samples of a herbal dietary supplement. Five of them were published for the first time. Furthermore, we showed that the content of identical products can vary greatly in the composition of illegally added substances. Material and methods Reference standards of sildenafil citrate were obtained from Pfizer (Berlin, Germany). All LC solvents were obtained in LC-MS grade (Chromasolv®) from Fluka (Buchs, Switzerland). Ammonium formate, methylene chloride and sodium hydroxide were purchased from Merck (Darmstadt, Germany). All the samples analysed in this study carried the identical trade name, batch number and expiration date. They were advertised as pure herbal. Irrespective of that, the samples originated from different sources. Sample A was taken by the local competent authority from an erotic store. Sample B was a counter sample received from the stakeholder during official investigations. Sample C was part of the products confiscated in the course of law
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N. Schramek et al.
enforcement actions. Sample D was a composite sample prepared by mixing 10 different capsules from sample C. The content of one capsule (samples A–C) was mixed with 10 ml of methylene chloride and 2 ml of 2 M sodium hydroxide solution. The organic layer was collected using a separation funnel. The aqueous layer was extracted again with 5 ml of methylene chloride. The combined organic solvents were dried under a stream of nitrogen. The residue was reconstituted in 1 ml of acetonitrile. This solution was suitably diluted with the same solvent for LC-DAD analysis and for LC-MS analysis. All samples and solutions were filtered before use through a 0.45 µm Spartan filter from Whatman (Dassel, Germany). For the composite sample D the content of 10 capsules was mixed with 100 ml of methylene chloride and 20 ml of 2 M sodium hydroxide solution. The organic layer was collected using a separation funnel. The aqueous layer was extracted again with 50 ml of methylene chloride. The combined organic solvents were dried under a stream of nitrogen. The residue was reconstituted in 10 ml of acetonitrile. LC-DAD analysis was performed on an Elite La Chrom system, equipped with a L-2455 diode array detector (VWR, Darmstadt, Germany) operated in a detection range from 200 to 350 nm. For chromatographic separation, a Zorbax-SB C18 column (250 mm × 4.6 mm × 5.0 µm; Agilent Deutschland GmbH, Böblingen, Germany) was used. The elution conditions were as follows: gradient elution with solvent A – 10 mM ammonium formate, and solvent B – acetonitrile. Chromatography was started with 20% of solvent B which was raised to 80% within 40 min. Solvent B was subsequently decreased within 5 min to 20% again to allow reconditioning the analytical column at 20% of solvent B for an additional of 5 min. The column temperature was kept at 40°C, the flow rate was set to 1.0 ml min–1 with an injection volume being 10 µl of each sample. Isolation of the compounds was carried out using the chromatographic system described above, but with a semiprep Zorbax-SB C18 column (250 mm × 9 mm × 5.0 µm; Agilent Deutschland GmbH), a flow rate of 5.0 ml min–1 and an injection volume of 400 µl. The eluate was collected after detection of peaks in the chromatographic run. The solvent was removed under reduced pressure and the residue was subjected to NMR analysis. LC-MS studies were carried out by using a Shimadzu prominence LC-20 (Shimadzu, Kyoto, Japan) system connected to an AB Sciex 5500 Qtrap™ triple quadrupole linear ion-trap mass spectrometer (AB Sciex, Foster City, CA, USA) operated in electrospray-negative mode by Analyst software. Separation conditions were comparable with the parameters mentioned under LC-DAD, but using a Luna C18 column (150 mm × 2.0 mm × 3 µm; Phenomenex, Torrance, CA, USA) at 28°C and a flow rate of 0.2 ml min–1. The MS parameters were: entrance potential: −10 V; declustering potential: −10 V; collision energy: −10 V; collision cell exit potential: −11 V; curtain
gas: 30 psi; ion spray voltage: −4.5 kV; source temperature: 600°C; ion source gas 1: 40 psi; and ion source gas 2: 60 psi. The MS operated in an enhanced MS scan (EMS) mode, which uses Q3 in a scan range between 100 and 500 Da with a scan rate of 10 000 Da s–1. Fragmentation patterns were obtained by operating the MS in enhanced product ion (EPI) scan mode with a collision energy of 45 V with an additional spread of 15 V. Fragmentation was carried out on [M – 1] – parent ion obtained from prior performed EMS. Each of the purified compounds was dissolved in 0.6 ml of CD2Cl2. NMR experiments with detection of 1H (1D 1HNMR, 2D H,H-COSY, H-NOESY, HSQC and HMBC) were performed with a Bruker AVANCE-I 500 MHz spectrometer, equipped with an inverse 5 mm 1H/13C probe head. NMR experiments with detection of 13C (1D 13C NMR with 1H decoupling, DEPT-135, DEPT-90) were carried out with a Bruker AVANCE-III spectrometer, equipped with a 5 mm CP-QNP X-detect cryoprobehead at 125.6 MHz 13C-NMR frequency. Data were processed with MNova software (Mestrelab Research SL, Santiago de Compostela, Spain). Chemical shifts are reported in ppm.
Results LC-DAD Using HPLC-DAD, a herbal dietary supplement sold in an erotic shop and taken as an official sample by the competent authority was screened for illegally added PDE5 inhibitors. The chromatogram showed several signals with retention times from 10 to nearly 27 min (Figure 1A). The signals at 11.7 min (compound 2) and 12.2 min (compound 3) were identified previously as isopiperazinonafil and piperazinonafil, respectively (Wollein et al. 2011). Additionally, two other samples of the same dietary supplement (identical trade name, batch and expiration date) were tested. One was the counter sample from the stakeholder (sample B; Figure 1B), the other sample was obtained from the product confiscated by the police (sample C; Figure 1C). Finally, a composite sample (mixture of 10 different capsules of the confiscated product; sample C) was tested (sample D; Figure 1D). All samples showed very similar chromatograms with respect to the retention times of the different components, but were very different regarding the signal intensities. To elucidate the identity of the signals, the samples were subjected to LC-MS analysis. Moreover, using semipreparative HPLC, the compounds characterised by peaks at 10.1, 11.1, 13.1, 15.5, 16.8–17.0, 17.6, 20.8, 24.2 and 26.7 min (Figure 1A/D) were isolated and subjected to NMR analysis. For the UV spectra of all of the signals obtained from the LC-DAD runs of the different sample, see the Supplementary data online.
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Food Additives & Contaminants: Part A
3
Figure 1. LC-DAD run (wavelength 254 nm) of different capsules of the same product. (A) Sample A: official sample from an erotic store, taken by local competent authority; (B) sample B: counter sample submitted by the stakeholder; (C) sample C: confiscated product during law enforcement action; and (D) sample D: mixed sample of 10 capsules of the confiscated product (sample C).
Table 1. Identified compounds and corresponding retention times. Compounds 2, 3, 4, 4a, 7, 8, 10 and 13 were described earlier (Instrumental Analysis Data of Illegal Compounds in Food 2010; Wollein et al. 2011; Venhuis & De Kaste 2012; Kim et al. 2013). The identity of compound 4a could not be verified. Compound number 1 2 3 4 4a 5 6 7 8 9 10 11 12 13
Name 5-(5-(1,2-Dihydroxyethyl)-2-ethoxyphenyl)-1-methyl-3-propyl1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (dihydroxydenafil) Isopiperazinonafil Piperazinonafil Oxoacetildenafil Dioxoacetildenafil 5-(2-Ethoxy-5-(2-ethoxy-1-hydroxyethyl)phenyl)-1-methyl-3propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (hydroxyethoxydenafil) 5-(2-Ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7Hpyrazolo[4,3-d]pyrimidin-7-one (Z)-dichlorodenafil Hydroxyacetildenafil 4-Ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1Hpyrazolo[4,3-d]pyrimidin-5-yl)benzaldehyde (aldenafil) Gendenafil 5-(2-Ethoxy-5-(2-thioxobutyl)phenyl)-1-methyl-3-propyl-1,6dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (thioxobutyldenafil) 5-(5-Chloro-2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7Hpyrazolo[4,3-d]pyrimidin-7-one (chlorofil) (E)-dichlorodenafil
LC-MS Chromatographic conditions lead to comparable retention times than pointed out in Figure 1A and Table 1. Operating Q3 in ESI-negative mode combined with an enhanced scan experiment at a 10 000 Da s–1 scanning rate, for each signal its accurate [M – 1] – could be observed. Compounds 2, 3
Retention time (min) (LC-DAD)
[M – 1] – (LC-MS)
10.1
371.1
11.7 12.2 13.1 13.4 15.5
481.2 481.2 479.2 493.2 399.2
20.8
311.2
26.7 11.1 16.8
405.1 481.0 339.2
17.0 17.6
353.0 397.1
24.2
345.0
27.1
405.1
and 8, appearing at 11.7, 12.2 and 11.1 min respectively have already been reported (Instrumental Analysis Data of Illegal Compounds in Food 2010; Wollein et al. 2011). Compound 1 at 10.1 min could be assigned to [M – 1] – of m/z 371 (Figure 2C). A sildenafil substructure could be postulated by showing a neutral loss of ethene from the
N. Schramek et al.
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4
Figure 2. Mass pattern of (A) compound 6, (B) compound 12, (C) compound 1, (D) compound 5, (E) compounds 9 and 10 (gendenafil) (Lin et al. 2008), (F) compounds 7 and 13 ((Z)-/(E)-dichlorodenafil) (Kim et al. 2013), (G) compound 4 (oxoacetildenafil) (Instrumental Analysis Data of Illegal Compounds in Food 2010), (H) compound 4a (dioxoacetildenafil) (Venhuis & De Kaste 2012), and (I) compound 11.
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Food Additives & Contaminants: Part A ethoxy-moiety (m/z 343), and an additional loss of the substituent in position 3 of the phenylring to give the ethoxyphenyl-pyrazolopyrimidine basic ion with an m/z of 311. Between 13.1 and 13.4 min, two nearly unseparated signals occur, which were labelled as compound 4 and 4a. Compound 4, giving also a great intensity in LC-DAD runs at 13.1 min, was identified to be oxoacetildenafil (Instrumental Analysis Data of Illegal Compounds in Food 2010) as its [M – 1]– was only two units lower than piperazinonafil (Figure 2G). Due to the low intensity of the signal of compound 4a, it was not possible to isolate the substance. Based on LC-DAD data and considering a 14 Da higher mass value in comparison with compound 4 (Figure 2G), compound 4a (Figure 2H) could be assigned to dioxoacetildenafil (Venhuis & De Kaste 2012), although no analytical data for this compound could be found in the literature. Compound 5 at 15.5 min could be assigned to [M – 1] – of m/z 399 (Figure 2D). Its fragmentation pattern, again, shows the characteristic daughter ions for sildenafil substructures (m/z 371, 311). Compound 6 at 20.8 min corresponds to 312 Da (Figure 2A), which is fully in line with the pyrazolopyrimidine basic structure of sildenafil. Compound 7 at 26.7 min shows characteristic [M – 1] – isotope mass pattern together with [(M – 1) + 2] – and [(M – 1) + 4] –, which derives from the assemblage of two chloro substituents (Figure 2F). These signals are identical to the mass data published for (Z)-dichlorodenafil (Kim et al. 2013). Interestingly, compound 13 at 27.1 min is represented by the same mass pattern, which could be assigned to the corresponding (E)-isomer. The parent ion of compound 12 shows similar isotope mass pattern to compounds 7 and 13 but with respect to the isotope ratios of the [(M – 1) + 2] – and [(M – 1) + 4] – and is suggested to have a single chloro moiety in position 3 of the phenyl substituent, which is represented by its m/z of 345 and 347 (Figure 2B). Compounds 9 and 10, which elute very closely to each other at 16.8–17.0 min, are together represented in Figure 2E. An m/z ratio of 353 together with its typical formal loss of ethene (m/z 325) could be attributed to the [M – 1] – of compound 10, which is congruent to published data of gendenafil (Lin et al. 2008). Compound 9 is suggested to be the analogue formyl derivative of compound 10, showing an [M – 1] – of m/z 339 with, again, a neutral loss of ethene forming a m/z 311 daughter ion. At 17.6 min compound 11 elutes with a corresponding [M – 1] – of m/z 397 (Figure 2I), which shows in the enlarged display of its parent ion the typical isotope pattern of sulphur ([(M – 1) + 2] – and in very low intensity due to the low amount of the analyte its [(M – 1) + 4] – signal. NMR 1
H- and 13C-NMR data of the investigated compounds are summarised in Table 2; the 1H- and 13C-NMR spectra are shown in Figures 3 and 4. The 2D-NMR data (H,H-COSY
5
and HMBC) of compounds 1, 5 and 9–12 are summarised in Table 3. The signals in the aromatic region between 8.5 and 7 ppm of the 1H-NMR spectra of all isolated compounds resemble the coupling pattern of the 1,2,4-substituted phenyl ring of sildenafil. The signals at about 4.3 ppm (H-20) and 1.7 ppm (H-21) could be assigned to the O-ethyl moiety and the signals at about 2.9 ppm (H-11), 1.8 ppm (H-12) and 1.0 ppm (H-13) represent the 1-propyl group. HMBC correlations from C-19 to H-20 and C-1 to H-11 and H-12 confirmed the positions of the ethoxy and the propyl moiety, respectively. All observed compounds therefore carry the basic chemical structure of sildenafil. The 1H-NMR spectrum of compound 6 shows four aromatic proton signals and H,H-COSY data shows correlation between these four signals. This indicates an orthosubstitution pattern and is consistent with a 1,2-substituted phenyl ring. The 13C-NMR spectrum shows 17 different carbon signals. LC-MS experiments revealed an m/z = 311. Compound 6 could therefore be assigned to 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7Hpyrazolo[4,3-d]pyrimidin-7-one (C17H21N4O2), the basic pyrazolopyrimidine structure of sildenafil (Ellis & Terrett 1994). Compared with published NMR data, compounds 4, 7, 8 and 10 could be assigned to oxoacetildenafil (oxohongdenafil) (Instrumental Analysis Data of Illegal Compounds in Food 2010), dichlorodenafil ((Z)-isomer) (Kim et al. 2013), hydroxyacetildenafil (hydroxyhongdenafil) (Hou et al. 2006), and gendenafil (Lin et al. 2008), respectively. Compounds 7 and 13 could not be isolated separately (Figure 1D). Consequently the 1H-NMR spectrum of compound 7 (Figure 3E) shows additional signals with very low intensities. While the signal at 6.79 ppm represents the vinyl proton of the (Z)-isomer (main component) the small signal at 6.57 ppm could be assigned to the (E)-isomer of dichlorodenafil (Kim et al. 2013). On the basis of the integrals of both vinyl protons a ratio of 96% (Z)-isomer and 4% (E)-isomer could be estimated. The 1H- and 13C-NMR spectra of compound 1 are shown in Figures 3F and 4F. Compared with compound 6 two additional carbon atoms appear. Using HSQC correlations (data not shown) the proton signal (dd, 1H) at 4.89 ppm could be assigned to the carbon atom at 73.9 ppm (C-22). The two proton signals at 3.66 and 3.77 ppm (each ddd, 1H) could be assigned to one carbon atom at 67.1 ppm (C-31), which is a typical situation for diastereotopic protons in a chiral molecule. Together with H, H-COSY correlations between this three proton signals (Table 3) an ethyl moiety with substituents at both carbon atoms could be expected. HMBC correlations from C-15, C-16 and C-17 to H-22, and additionally from C-16 to H31, confirmed the position of the ethyl moiety. Compound 1 has a mass 60 Da higher compared with compound 6 (Table 1), which is indicative for a C2H4O2 substituent.
7.51
7.05
16 17
18
3.66 3.78 3.80
4.30 1.61 4.89
13
C
67.1
156.2 65.5 14.5 73.9
113.1
134.2 130.1
148.1 138.4 124.4 38.0 27.6 22.3 13.8 119.8 128.5
146.3 153.6
δ
2
Notes: 1(Z)-isomer, compound 7. (E)-isomer, compound 13.
31
19 20 21 22 24 25 26 27 28 29 30
8.44
4.24 2.93 1.85 1.05
11.08
δ 1H
Compound 1
3.90
3.39 2.93 3.43 1.11 3.41
3.29
4.37 1.61
7.14
8.11
9.04
4.27 2.96 1.86 1.03
10.81
δ 1H C
13
62.8
45.8 49.8 41.0 11.9
160.0 65.9 14.4 194.1 57.2 165.7
112.8
129.4 132.3
147.4 138.3 124.5 38.0 27.7 22.3 13.8 120.3 131.7
146.4 153.5
δ
Oxoacetildenafil compound 4
3.62
3.47 1.22
4.29 1.57 4.47
7.08
7.44
8.38
4.21 2.90 1.84 1.01
11.07
δ 1H C
13
67.8
64.1 15.7
156.9 66.1 15.1 82.4
113.8
133.1 131.0
148.7 139.0 125.0 38.6 28.1 22.9 14.4 120.6 130.0
146.8 154.2
δ
Compound 5
4.28 1.58
7.08
7.13 7.46
8.45
4.21 2.88 1.84 1.01
11.08
δ 1H 13
C
157.1 65.8 15.0
113.5
122.0 132.8
148.9 139.1 124.9 38.3 28.1 22.6 14.2 120.8 131.3
146.8 154.2
δ
Compound 6
6.79a 6.57b
4.31 1.60
7.64a 7.72b 7.07a 7.10b
8.62a 8.72b
4.21 2.91 1.86 1.01
10.93
δ 1H C
13
116.3
3.94
3.20 2.92 3.02 3.85
2.92 3.20
4.30 1.55
7.09
113.7 157.7 66.2 14.9 129.8
8.03
8.86
4.18 2.88 1.82 1.00
11.00
δ 1H C
13
63.3
53.0 50.6 59.9 56.6
160.5 66.3 14.8 194.3 50.6 53.0
113.1
129.6 132.5
148.0 138.8 124.9 38.5 28.1 22.8 14.3 121.1 131.9
146.7 154.1
δ
Hydroxyacetildenafil compound 8
135.0 130.9
148.0 138.9 124.9 38.6 28.2 22.7 14.2 120.9 129.7
146.7 154.1
δ
(Z)1-/(E)2dichlorodenafil compounds 7/13
4.35 1.62 10.01
7.21
8.00
8.91
4.22 2.91 1.85 1.02
10.83
δ 1H 13
C
161.3 66.6 14.9 190.9
113.9
131.0 133.1
147.7 138.9 125.0 38.6 28.2 22.8 14.3 121.4 134.4
146.9 154.0
δ
Compound 9
NMR data of identified compounds 1 and 4–12. Positions 1–31 indicate either a hydrogen or a carbon signal.
1 4 5 6 8 9 10 11 12 13 14 15
Position
δppm in CD2Cl2
Table 2.
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2.62
4.36 1.60
7.13
8.07
8.99
4.22 2.92 1.85 1.02
10.83
δ 1H C
13
26.8
160.3 66.4 14.9 196.7
113.3
131.5 132.7
148.0 138.9 125.0 38.6 28.2 22.8 14.3 120.7 132.4
146.9 154.0
δ
Gendenafil compound 10
3.65 1.27
4.37 1.61 4.74
7.14
8.08
9.00
4.22 2.92 1.85 1.27
10.81
δ 1H
13
C
195.4
67.7 15.5
160.5 66.4 14.9 74.2
113.4
129.3 132.4
147.9 138.8 125.0 38.6 28.2 22.9 14.3 120.8 132.0
147.0 154.0
δ
Compound 11
1
H
4.27 1.58
7.02
7.42
8.44
4.21 2.89 1.83 1.01
11.01
δ
13
C
155.7 66.9 15.0
115.1
127.2 132.3
147.6 138.8 125.0 38.9 28.5 23.3 14.7 122.1 130.7
147.0 154.0
δ
Compound 12
6 N. Schramek et al.
H-12
H-11/H-13
H-12
12
13
H-21 H-20 H-31
20 21 22
H-15/H-18 H-17
H-17
H-22
H-29 H-22
H-15/H-18 H-17
H-17
H-12
H-11/H-13
H-22
H-15/H-17/ H-18/H-20 H-21 H-21 H-20 H-20 H-15/H-29/ H-31 H-22/H-30
H-17/H-22 H-18/H-22/ H-31 H-15/H-22
H-18
H-11/H-12
H-11/H-13
H-12
H,H-COSY
H-15/H-18 H-17
H-17
H-12
H-11/H-13
H-12
H,H-COSY
H-15 H-15/H-17 H-15/H-17/ H-18/H-20 H-21 H-20 H-15/H-17/ H-31
H-15/H-17/ H-18 H-17/H-18 H-18/H-31
H-11/H-12
H-11/H-13
H-10/H-11/ H-12 H-15/H-18 H-11 H-10 H-12/H-13
HMBC
Compound 10 (gendenafil)
H-15H-17/ H-18/H-20 H-21 H-21 H-20 H-20 H-15/H-17
H-15/H-22
H-17/H-22 H-18/H-22b
H-15/H-18
H-11/H-12
H-11/H-13
H-15/H-18 H-11 H-10 H-12/H-13
H-11/H-12
HMBC
Compound 9
Note: Due to the very low amount isolated, only a very weak HMBC spectrum could be acquired. b 2 ( JCH = 25 Hz).
30 31
H-22
H-18 H-17
17 18 19
29
H-17/H-22 H-18/H-22/ H-31 H-15/H-22
H-17
15 16
H-12
H-11/H-13
H-12
H-15/H-18 H-11 H-10 H-12/H-13
H-11/H-12
HMBC
Compound 5 H,H-COSY
H-15/H-17/ H-18/H-20 H-21 H-21 H-20 H-20 H-15/H-17/ H-31 H-31 H-30
H-18
H-11/H-12
14
a
H-15/H-18 H-11 H-10 H-12/H-13
6 8 9 11
H-11/H-13
H-11/H-12
HMBC
1
Position H,H-COSY
Compound 1
H-29
H-30
H-21 H-20
H-15/H-18 H-17
H-17
H-12
H-11/H-13
H-12
H-22
H-22/ H-30
H-21
H-21 H-20
H-15/H-18 H-17
H-17
H-10 H-12/ H-12 H-13 H-11/ H-11/H-13 H-13 H-11/ H-12 H-12
H-15/H-17/ H-18/H-20 H-21 H-20
H-15
H-15/H-17/ H-18 H-17/H-18 H-15/H-18
H-11/H-12
H-11/H-13
H-15/H-18 H-11 H-10 H-12/H-13
H-11/H-12
HMBC
Compound 12
H,H-COSY HMBCa H,H-COSY
Compound 11
Table 3. 2D-NMR data of compounds 1, 5 and 9–12. Positions 1–31 indicate either a hydrogen or a carbon signal. δppm in CD2Cl2 .
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Figure 3. 1H-NMR spectra of (A) compound 6, (B) compound 12, (C) compounds 9 and 10, (D) compound 11, (E) compound 7 ((Z)-dichlorodenafil (Kim et al. 2013)) and compound 13 ((E)-dichlorodenafil (Kim et al. 2013)), (F) compound 1, (G) compound 5, (H) compound 4 (oxoacetildenafil; Instrumental Analysis Data of Illegal Compounds in Food 2010), and (I) compound 8 (hydroxyacetildenafil (Hou et al. 2006)). *Formate.
Compound 1 could therefore be assigned to 5-(5-(1,2dihydroxyethyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1,6dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one and was named dihydroxydenafil. The 1H- and 13C-NMR spectra of compound 5 are shown in Figures 3G and 4G. Compared with compound 1, two additional carbon signals at 64.1 ppm (C-29) and 15.7 ppm (C-30), and two additional proton signals at 3.47 ppm (m, 2H, H-29) and 1.22 ppm (t, 3H, H-30) appear. LC-MS data revealed a mass 28 Da higher compared with compound 1. Together with H,H-COSY correlations between H-29 and H-30 these data are indicative of a second ethyl moiety. HMBC correlations from C-22 to H-29 confirmed the position of this ethyl moiety. Compound 5 could therefore be assigned to 5-(2-ethoxy-5-(2-ethoxy-1-hydroxyethyl)phenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one and was named hydroxyethoxydenafil. The 1H-NMR spectrum of compound 10 (gendenafil) is shown in Figure 3C. The smaller signals originate from compound 9 since compounds 9 and 10 could not be fully separated. The corresponding 13C-NMR and HMBC spectra are shown in Figures 4C and 5. Compared with the known structure of compound 10, the signal of the methyl
group at 2.62 ppm (H-31) is missing in compound 9. Instead a proton signal at 10.01 ppm (H-22) appears. The 13C-NMR signal of the carbonyl group (C-22) is shifted to a higher field. Compound 9 shows HMBC correlations between C-15/17 and H-22 (Figure 5, green signals). Moreover, a correlation between C-16 and H-22 is shown. Together with LC-MS data this is consistent with an aldehyde moiety at C-16. Compound 9 could therefore be assigned to 4-ethoxy-3-(1-methyl-7-oxo-3propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl) benzaldehyde and was named aldenafil. The 1H-NMR spectrum of compound 12 (Figure 3B) was found to be nearly similar to that of compound 6, except the signal at 7.13 ppm (H-16) is missing (Figure 3B). LC-MS data revealed an m/z of 345 for compound 12. Together with the isotopic pattern, chlorine as a substituent seems plausible. Consequently the 13C-NMR signal of C-16 is shifted towards a lower field (Figure 4B). Compound 12 could therefore be assigned to 5-(5-chloro-2-ethoxyphenyl)-1methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin7-one and was named chlorofil. The 1H-NMR spectrum of compound 11 is shown in Figure 3D. Compared with compound 6, an additional
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Figure 4. 13C-NMR spectra of (A) compound 6, (B) compound 12, (C) compounds 9 and 10, (D) compound 11, (E) compound 7 ((Z)-dichlorodenafil (Kim et al. 2013)) and compound 13 ((E)-dichlorodenafil (Kim et al. 2013)), (F) compound 1, (G) compound 5, (H) compound 4 (oxoacetildenafil; Instrumental Analysis Data of Illegal Compounds in Food 2010), and (I) compound 8 (hydroxyacetildenafil (Hou et al. 2006)). *Formate.
singlet at 4.74 ppm (3H), a quartet at 3.65 ppm (2H) and a triplet at 1.27 ppm (3H) appears. A 13C-NMR signal at about 195 ppm (Figure 4D) together with H,H-COSY (Figure 6) and HMBC correlations (Table 3) indicates a butyl moiety bearing a carbonyl group. LC-MS experiments revealed an m/z of 397 and an [(M – 1) + 4] – signal [Figure 2I], indicative for the presence of a 34S isotope. This is well in line with a 2-thioxobutyl moiety. Compound 11 could therefore be assigned to 5-(2-ethoxy-5-(2-thioxobutyl)phenyl)-1methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin7-one and was named thioxobutyldenafil. The chemical structure of compound 14 could not be safely determined. Based on NMR spectroscopic data this compound seems to bear the well-known sildenafil/pyrazolopyrimidine base structure. A neutral loss of ethene (−28 Da) from the ethoxy moiety may underline this
assumption. Unfortunately the substituent in position 5 of the 2-ethoxyphenyl moiety remains unclear.
Discussion In the present study, 14 different chemical compounds, bearing the same basic pyrazolopyrimidine structure like sildenafil (Figure 7), were isolated from different samples of the same dietary supplement, sold as a pure herbal product. Seven of them have already been published (piperazinonafil, isopiperazinonafil, oxoacetildenafil, (Z)-/(E)-dichlorodenafil, gendenafil and hydroxyhomoacetildenafil) (Lin et al. 2008; Instrumental Analysis Data of Illegal Compounds in Food 2010; Wollein et al. 2011; Kim et al. 2013). Compound 6 could be assigned to 5-(2-ethoxyphenyl)-
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Figure 5.
(colour online) HMBC spectrum of compound 9 (green) and compound 10 (red, gendenafil; Lin et al. 2008).
Figure 6.
H,H-COSY spectrum of compound 11.
1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, the commercially available pyrazolopyrimidine basic structure of sildenafil. The chemical structures of another five analogues were elucidated using LC-MS, UV and NMR spectroscopic data (Table 1). Although some of the compounds like piperazinonafil, isopiperazionafil and the acetildenafil derivatives found in the present study may exhibit PDE5 inhibition, most of them were not expected to. More likely, they are by-products of the synthesis of a sildenafil analogue. For the synthesis of
sildenafil and its analogues many different routes are described (Kim et al. 2001, 2013; Ley et al. 2002; Yu et al. 2003; Li et al. 2004; Xia et al. 2005; Abdel-Jalil, Khanfar, Abu-Safieh et al. 2005; Abdel-Jalil, Khanfar, Al-Gharabli et al. 2005; Medrasi et al. 2013). Based on published procedures a hypothetical reaction scheme towards pyrazolopyrimidines bearing an acetyl bridge, explaining the existence of the compounds found in the present study and earlier by others, is shown in Figure 8. The Friedel–Crafts-type acylation of commercially available 6 with chloroacetyl chloride
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Figure 7. Chemical structures of sildenafil analogues found in the present study. Compound 2, isopiperazinonafil (Wollein et al. 2011); compound 3, piperazinonafil (Wollein et al. 2011); compound 4, oxoacetildenafil (Instrumental Analysis Data of Illegal Compounds in Food 2010); compound 4a, dioxoacetildenafil (Venhuis & De Kaste 2012); compounds 7/13, (Z)-/(E)-dichlorodenafil (Kim et al. 2013); compound 8, hydroxyacetildenafil (Hou et al. 2006); and compound 10, gendenafil (Lin et al. 2008).
leads to compound 6b, known as chlorodenafil which was found earlier in dietary supplements (Instrumental Analysis Data of Illegal Compounds in Food 2010). Reaction of 6b with pyrimidine or pyrimidinone derivatives gives sildenafil analogues of the acetildenafil type. The existence of 10 could be explained by assuming a synthesis of chloroacetyl chloride using chloroacetic acid and phosphorus pentachloride or phosphoryl chlorid. Chloroacetic acid is known to contain considerable amounts of acetic acid which gives acetyl chloride with chlorinating reagents. The chlorination of 6b with remnants of the chlorinating reagent explains the existence of 7 (Kagan et al. 1983; Kim et al. 2013), hydrolysis could give 6a. Formation of 6a could also be explained via direct α-hydroxylation of 10 (Jones 1991; Chan Lee et al. 1997), although this seems not very likely. Since most synthesis routes require the use of strong bases, formation of 5 and 6d seems plausible if ethanol is used as solvent. Depending on the starting material and the target molecule, oxidation and/or reduction reactions are also very common in the synthesis of sildenafil analogues, which could explain the formation of compounds 1 and 5. Since isopiperazinonafil (compound 2) bears a very untypically structure, a completely different synthesis route
is required. Based on a patented procedure towards the formation of phenylacetic acid derivatives, a hypothetical reaction scheme towards pyrazolopyrimidines bearing a 2-oxoethyl bridge is shown in Figure 9 (Stelzer 1995). Reaction of 6 with sulfonyloxyacetic acid derivatives leads to 6f, which could be converted to 6h using pyrimidinone derivatives. A final reduction step could explain the formation of 2. As described above, the use of strong bases and ethanol as solvent explains the formation of 6g. The following conversion of the carbonyl group into a thiocarbonyl group using, for example, phosphorous pentasulfide gives compound 11 (Kim et al. 2002).
Conclusions Recently, Venhuis et al. (2014) demonstrated that there can be fundamental differences in the composition of dosage units within one package of an adulterated dietary supplement. The present example shows the difficulties a laboratory could have in the determination of the ‘real’ composition of an adulterated dietary supplement. Although exact determination as well as the quantification of all of the (illegally added) substances in an adulterated
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Figure 8. Hypothetical synthesis route towards pyrazolopyrimidinones bearing an acetyl instead of a sulfonyl bridge (e.g. compound 4). Reaction intermediates 6b, 6c, 7, 13 and 10 were found earlier and are known as chlorodenafil, hydroxychlorodenafil, (Z)-/(E)dichlorodenafil and gendenafil, respectively (Lin et al. 2008; Instrumental Analysis Data of Illegal Compounds in Food 2010; Kim et al. 2013). Reaction intermediates 6a and 6d have not been found in the present study. The dashed reaction arrow indicates a possible (e.g. (Jones 1991; Chan Lee et al. 1997)) but not mandatory direct α-hydroxylation. R1 , ethoxy group; R2 , pyrazolopyrimidine moiety; R3 , alkyl group; R4 , hydroxyethyl moiety.
Figure 9. Hypothetical synthesis routes towards compounds 2 and 11 based on a patented method for the preparation of phenylacetic acid derivatives (Stelzer 1995). Compounds 6f, 6g and 6h are hypothetical intermediates and have not been found in the present study. R1 , ethoxy group; R2 , pyrazolopyrimidine moiety; R3 , alkyl group.
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Food Additives & Contaminants: Part A dietary supplement may not be necessary in every case to prohibit the placing on the market, a different composition of samples can lead to substantial enforcement difficulties. This is particularly true if, like in the present study, the composition of official samples taken from the police or from competent authorities differ significantly from the composition of other samples (with respect to illegally added substances). Moreover, this example shows the difficulties for all parties – competent authorities as well as distributors – to fulfil their legal obligations. According to article 17 of the regulation (EC) No. 178/2002 of the European Parliament and of the Council, food business operators should ensure their products satisfy the requirements of food law. Competent authorities should monitor and verify that the relevant requirements of food law are fulfilled by food business operators. Even if both of them perform random checks on a product, they cannot be sure of its quality. Many dietary supplements therefore are and remain a high risk for human health unless they are properly regulated. Since they bear a greater resemblance to pharmaceuticals than to usual ‘foods’, the regulatory requirements should be further adapted. With a pre-market approval, an obligation to define product specifications, and to monitor each batch (using average samples) for quality and compliance with these specifications, some of the difficulties with dietary supplements could possibly be solved. Acknowledgement The authors thank Christine Schwarz and Rosalia Spirkl for expert technical assistance.
Supplemental data Supplemental data for this article can be accessed here: http://dx. doi.org/10.1080/19440049.2014.992980.
References Abdel-Jalil RJ, Khanfar M, Abu-Safieh K, Al-Gharabli S, ElAbadelah M, Voelter W. 2005. An efficient one-pot synthesis of pyrazolopyrimidines, intermediates for potential phosphodiesterase inhibitors. Monatsh Chem. 136:619–624. Abdel-Jalil RJ, Khanfar M, Al-Gharabli S, El-Abadelah MM, Eichele K, Anwar MU, Voelter W. 2005. High throughput synthesis of pyrazolopyrimidines via copper-catalysed cyclization and x-ray study. Heterocycles. 65:1821–1827. Blok-Tip L, Zomer B, Bakker F, Hartog KD, Hamzink M, Ten Hove J, Vredenbregt M, De Kaste D. 2004. Structure elucidation of sildenafil analogues in herbal products. Food Addit Contam. 21:737–748. Chan Lee J, Park C, Choi Y. 1997. A New Procedure for αHydroxylation of Ketones Using Thallium(III) pNitrobenzenesulfonate (TNS). Synth Commun. 27:4079–4084. Crowley R, FitzGerald LH. 2006. The impact of cGMP compliance on consumer confidence in dietary supplement products. Toxicology. 221:9–16. Deisingh AK. 2005. Pharmaceutical counterfeiting. Analyst. 130:271–279.
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Ellis P, Terrett NK, inventors. 1994. Pyrazolopyrimidinones for the treatment of impotence WO 1994028902 A1. Ham M. 2003. Health RISKS OF COUNTERFEIT PHARMACEUTICALS. Drug Saf. 26:991–997. Hou P, Zou P, Low M-Y, Chan E, Koh H-L. 2006. Structural identification of a new acetildenafil analogue from pre-mixed bulk powder intended as a dietary supplement. Food Addit Contam. 23:870–875. ICH Q3A (R2) Impurities in New Drug Substances O. 2006. Instrumental Analysis Data of Illegal Compounds in Food [Internet]. 2010. Korean National Institute for Food and Drug Safety Evaluation. [cited 2013 Nov 13]. Available from: http://nifds.go.kr Jones AB, editor. 1991. Chapter 2.3: oxidation adjacent to C=X bonds by hydroxylation methods. Oxford: Pergamon Press. Kagan J, Arora SK, Bryzgis M, Dhawan SN, Reid K, Singh SP, Tow L. 1983. Reaction of phosphorus pentachloride with 2acetylthiophene and acetophenone. J Org Chem. 48:703–706. Khazan M, Hedayati M, Askari S, Azizi F. 2013. Adulteration of products sold as Chinese Herbal medicines for weight loss with thyroid hormones and PCP. J Herbal Med. 3:39–43. Kim D-K, Lee JY, Lee N, Ryu DH, Kim J-S, Lee S, Choi J-Y, Ryu J-H, Kim N-H, Im G-J, et al. 2001. Synthesis and phosphodiesterase inhibitory activity of new sildenafil analogues containing a carboxylic acid group in the 5′-sulfonamide moiety of a phenyl ring. Bioorg Med Chem. 9:3013–3021. Kim J-H, Kim Y, Choi KI, Kim DH, Nam G, Seo JH, inventors. 2002. Preparation of novel pyrazolopyrimidinethiones as phosphodiesterase V inhibitors for treating erectile dysfunction WO2002102802A1. Kim JY, Hwang IG, Oh JH, Kang IH, Kwon SW, Kim D. 2013. Efficient synthesis of dichlorodenafil, an unapproved sildenafil analogue appearing in non-prescription supplements. Chem Pharm Bull (Tokyo). 61:572–575. Ley SV, Baxendale IR, Brusotti G, Caldarelli M, Massi A, Nesi M. 2002. Solid-supported reagents for multi-step organic synthesis: preparation and application. Il Farmaco. 57:321–330. Li J-J, Johnson DS, Sliskovic DR, Roth BD, editors. 2004. Chapter 13, PDE 5 inhibitors for erectile dysfunction: sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis). In: Contemporary drug synthesis. Hoboken, NJ: John Wiley & Sons, Inc.; p. 189–199. Lin M-C, Liu Y-C, Lin Y-L, Lin J-H. 2008. Isolation and identification of a novel sildenafil analogue adulterated in dietary supplements. J Food Drug Anal. 16:15–20. Marik PE, Flemmer M. 2012. Do dietary supplements have beneficial health effects in industrialized nations: what is the evidence? J Parenter Enteral Nutr. 36:159–168. Maughan RJ. 2005. Contamination of dietary supplements and positive drug tests in sport. J Sports Sci. 23:883–889. Medrasi HY, Al-Sheikh MA, Salaheldin AM. 2013. Enaminonitriles in heterocyclic synthesis: a route to 1,3diaryl-4-aminopyrazole derivatives. Molecules. 18:535–544. Patel DN, Li L, Kee C-L., Ge X, Low M-Y., Koh H-L. 2014. Screening of synthetic PDE-5 inhibitors and their analogues as adulterants: analytical techniques and challenges. J Pharm Biomed Anal. 87:176–190. Roy J. 2002. Pharmaceutical impurities–a mini-review. AAPS PharmSciTech. 3:1–8. Slifman NR, Obermeyer WR, Aloi BK, Musser SM, Correll WA, Cichowicz SM, Betz JM, Love LA. 1998. Contamination of botanical dietary supplements by digitalis lanata. N Engl J Med. 339:806–811. Stelzer U, inventor. 1995. Preparation of phenylacetic acids EP665212A1.
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Tang MHY, Chen SPL, Ng SW, Chan AYW, Mak TWL. 2011. Case series on a diversity of illicit weight-reducing agents: from the well known to the unexpected. Br J Clin Pharmacol. 71:250–253. Venhuis BJ, De Kaste D. 2012. Towards a decade of detecting new analogues of sildenafil, tadalafil and vardenafil in food supplements: a history, analytical aspects and health risks. J Pharm Biomed Anal. 69:196–208. Venhuis BJ, Zomer G, De Kaste D. 2008. Structure elucidation of a novel synthetic thiono analogue of sildenafil detected in an alleged herbal aphrodisiac. J Pharm Biomed Anal. 46:814–817. Venhuis BJ, Zwaagstra ME, Keizers PH, De Kaste D. 2014. Dose-to-dose variations with single packages of counterfeit medicines and adulterated dietary supplements as a potential source of false negatives and inaccurate health risk assessments. J Pharm Biomed Anal. 89:158–165.
Wollein U, Eisenreich W, Schramek N. 2011. Identification of novel sildenafil-analogues in an adulterated herbal food supplement. J Pharm Biomed Anal. 56:705–712. Xia GX, Li JF, Lai SA, Peng AM, Zhang SJ, Wei XH, Chen XJ, Shen JS, Ji RY. 2005. Design and synthesis of pyrido[1,2-e] purin-4(3H)-one derivatives as potential PDE 5 inhibitors. Chin Chem Lett. 16:1283–1286. Yu G, Mason H, Wu X, Wang J, Chong S, Beyer B, Henwood A, Pongrac R, Seliger L, He B, et al. 2003. Substituted pyrazolopyridopyridazines as orally bioavailable potent and selective PDE5 inhibitors: potential agents for treatment of erectile dysfunction. J Med Chem. 46:457–460. Yuen YP, Lai CK, Poon WT, Ng SW, Chan AY, Mak TW. 2007. Adulteration of over-the-counter slimming products with pharmaceutical analogue–an emerging threat. Hong Kong Med J. 13:216–220.
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Pyrazolopyrimidines in ‘all-natural’ products for erectile dysfunction treatment: the unreliable quality of dietary supplements a
a
b
Nicholas Schramek , Uwe Wollein & Wolfgang Eisenreich a
Bavarian Health and Food Safety Authority, Oberschleißheim, Germany
b
Lehrstuhl für Biochemie, Technische Universität München, Garching, Germany Published online: 17 Dec 2014.
Click for updates To cite this article: Nicholas Schramek, Uwe Wollein & Wolfgang Eisenreich (2014): Pyrazolopyrimidines in ‘all-natural’ products for erectile dysfunction treatment: the unreliable quality of dietary supplements, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2014.992980 To link to this article: http://dx.doi.org/10.1080/19440049.2014.992980
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Food Additives & Contaminants: Part A, 2014 http://dx.doi.org/10.1080/19440049.2014.992980
Pyrazolopyrimidines in ‘all-natural’ products for erectile dysfunction treatment: the unreliable quality of dietary supplements Nicholas Schrameka*, Uwe Wolleina and Wolfgang Eisenreichb a
Bavarian Health and Food Safety Authority, Oberschleißheim, Germany; bLehrstuhl für Biochemie, Technische Universität München, Garching, Germany
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(Received 8 September 2014; accepted 24 November 2014) A herbal food supplement advertised as a potency pill was screened for the presence of PDE5 inhibitors. The resulting signals were characterised by UV, LC-MS in ESI-negative mode, and NMR spectroscopy using 1D and 2D experiments. Several substances were identified, bearing the basic chemical structure of sildenafil, but were not supposed to exhibit PDE5 inhibition. These compounds may be process-related impurities or by-products of different reaction steps in the synthesis of PDE5 analogues. As they were found to be present in different capsules at different concentrations, this is an example of the unreliable quality of dietary supplements. Keywords: dietary supplements; sildenafil analogue; impurities; LC-MS; NMR; reaction pathway
Introduction Medicinal products are highly regulated to ensure high quality and safety. Of particular importance is the identification and quantification of pharmaceutical impurities in medicinal products, active ingredients and pharmaceutical excipients, as they may influence the efficacy and safety of pharmaceutical products even in small amounts (Roy 2002). The European pharmacopoeia limits impurities for pharmaceutical use in the general text 5.10 ‘Control of impurities in substances for pharmaceutical use’, which is equal to the ICH guideline ‘Impurities in New Drug Substances’ ICH Q3A (R2) from 2006 (ICH Q3A (R2) Impurities in New Drug Substances 2006). Specific thresholds for reporting, identification and quantification have been established in the general monograph ‘Substances for Pharmaceutical Use’ (Ph.Eur. 8.0/2034). All these regulatory documents are useful tools in the control of impurities in products having an authorisation to enter the legal supply chain. But there is a black market where all these regulations are futile. On the one hand, there are counterfeit and imitation medicinal products bearing a high risk to human health since their actual content is unknown (Ham 2003; Deisingh 2005). On the other hand, a huge and still growing market exists for dietary supplements intended to maintain and promote health (Crowley & FitzGerald 2006; Marik & Flemmer 2012). Contamination and inaccurate labelling of dietary supplements is known to be a problem and could be a threat to human health (Slifman et al. 1998; Maughan 2005). This is especially true for lifestyle preparations such as slimming agents or sexual enhancers. Their usual pure natural ingredients are often expanded by one or several non*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
declared chemical compounds (Blok-Tip et al. 2004; Yuen et al. 2007; Venhuis et al. 2008; Tang et al. 2011; Wollein et al. 2011; Venhuis & De Kaste 2012; Khazan et al. 2013; Patel et al. 2014). These kinds of reports all cover active pharmaceutical ingredients (API) or analogues added to herbal supplements, not monitored by authorities. Moreover, there seems to be high variability within single packages of dietary supplements (Venhuis et al. 2014). In this study, we identified most of the variable ‘impurities’ bearing substructures of PDE5 inhibitors, present in identical samples of a herbal dietary supplement. Five of them were published for the first time. Furthermore, we showed that the content of identical products can vary greatly in the composition of illegally added substances. Material and methods Reference standards of sildenafil citrate were obtained from Pfizer (Berlin, Germany). All LC solvents were obtained in LC-MS grade (Chromasolv®) from Fluka (Buchs, Switzerland). Ammonium formate, methylene chloride and sodium hydroxide were purchased from Merck (Darmstadt, Germany). All the samples analysed in this study carried the identical trade name, batch number and expiration date. They were advertised as pure herbal. Irrespective of that, the samples originated from different sources. Sample A was taken by the local competent authority from an erotic store. Sample B was a counter sample received from the stakeholder during official investigations. Sample C was part of the products confiscated in the course of law
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enforcement actions. Sample D was a composite sample prepared by mixing 10 different capsules from sample C. The content of one capsule (samples A–C) was mixed with 10 ml of methylene chloride and 2 ml of 2 M sodium hydroxide solution. The organic layer was collected using a separation funnel. The aqueous layer was extracted again with 5 ml of methylene chloride. The combined organic solvents were dried under a stream of nitrogen. The residue was reconstituted in 1 ml of acetonitrile. This solution was suitably diluted with the same solvent for LC-DAD analysis and for LC-MS analysis. All samples and solutions were filtered before use through a 0.45 µm Spartan filter from Whatman (Dassel, Germany). For the composite sample D the content of 10 capsules was mixed with 100 ml of methylene chloride and 20 ml of 2 M sodium hydroxide solution. The organic layer was collected using a separation funnel. The aqueous layer was extracted again with 50 ml of methylene chloride. The combined organic solvents were dried under a stream of nitrogen. The residue was reconstituted in 10 ml of acetonitrile. LC-DAD analysis was performed on an Elite La Chrom system, equipped with a L-2455 diode array detector (VWR, Darmstadt, Germany) operated in a detection range from 200 to 350 nm. For chromatographic separation, a Zorbax-SB C18 column (250 mm × 4.6 mm × 5.0 µm; Agilent Deutschland GmbH, Böblingen, Germany) was used. The elution conditions were as follows: gradient elution with solvent A – 10 mM ammonium formate, and solvent B – acetonitrile. Chromatography was started with 20% of solvent B which was raised to 80% within 40 min. Solvent B was subsequently decreased within 5 min to 20% again to allow reconditioning the analytical column at 20% of solvent B for an additional of 5 min. The column temperature was kept at 40°C, the flow rate was set to 1.0 ml min–1 with an injection volume being 10 µl of each sample. Isolation of the compounds was carried out using the chromatographic system described above, but with a semiprep Zorbax-SB C18 column (250 mm × 9 mm × 5.0 µm; Agilent Deutschland GmbH), a flow rate of 5.0 ml min–1 and an injection volume of 400 µl. The eluate was collected after detection of peaks in the chromatographic run. The solvent was removed under reduced pressure and the residue was subjected to NMR analysis. LC-MS studies were carried out by using a Shimadzu prominence LC-20 (Shimadzu, Kyoto, Japan) system connected to an AB Sciex 5500 Qtrap™ triple quadrupole linear ion-trap mass spectrometer (AB Sciex, Foster City, CA, USA) operated in electrospray-negative mode by Analyst software. Separation conditions were comparable with the parameters mentioned under LC-DAD, but using a Luna C18 column (150 mm × 2.0 mm × 3 µm; Phenomenex, Torrance, CA, USA) at 28°C and a flow rate of 0.2 ml min–1. The MS parameters were: entrance potential: −10 V; declustering potential: −10 V; collision energy: −10 V; collision cell exit potential: −11 V; curtain
gas: 30 psi; ion spray voltage: −4.5 kV; source temperature: 600°C; ion source gas 1: 40 psi; and ion source gas 2: 60 psi. The MS operated in an enhanced MS scan (EMS) mode, which uses Q3 in a scan range between 100 and 500 Da with a scan rate of 10 000 Da s–1. Fragmentation patterns were obtained by operating the MS in enhanced product ion (EPI) scan mode with a collision energy of 45 V with an additional spread of 15 V. Fragmentation was carried out on [M – 1] – parent ion obtained from prior performed EMS. Each of the purified compounds was dissolved in 0.6 ml of CD2Cl2. NMR experiments with detection of 1H (1D 1HNMR, 2D H,H-COSY, H-NOESY, HSQC and HMBC) were performed with a Bruker AVANCE-I 500 MHz spectrometer, equipped with an inverse 5 mm 1H/13C probe head. NMR experiments with detection of 13C (1D 13C NMR with 1H decoupling, DEPT-135, DEPT-90) were carried out with a Bruker AVANCE-III spectrometer, equipped with a 5 mm CP-QNP X-detect cryoprobehead at 125.6 MHz 13C-NMR frequency. Data were processed with MNova software (Mestrelab Research SL, Santiago de Compostela, Spain). Chemical shifts are reported in ppm.
Results LC-DAD Using HPLC-DAD, a herbal dietary supplement sold in an erotic shop and taken as an official sample by the competent authority was screened for illegally added PDE5 inhibitors. The chromatogram showed several signals with retention times from 10 to nearly 27 min (Figure 1A). The signals at 11.7 min (compound 2) and 12.2 min (compound 3) were identified previously as isopiperazinonafil and piperazinonafil, respectively (Wollein et al. 2011). Additionally, two other samples of the same dietary supplement (identical trade name, batch and expiration date) were tested. One was the counter sample from the stakeholder (sample B; Figure 1B), the other sample was obtained from the product confiscated by the police (sample C; Figure 1C). Finally, a composite sample (mixture of 10 different capsules of the confiscated product; sample C) was tested (sample D; Figure 1D). All samples showed very similar chromatograms with respect to the retention times of the different components, but were very different regarding the signal intensities. To elucidate the identity of the signals, the samples were subjected to LC-MS analysis. Moreover, using semipreparative HPLC, the compounds characterised by peaks at 10.1, 11.1, 13.1, 15.5, 16.8–17.0, 17.6, 20.8, 24.2 and 26.7 min (Figure 1A/D) were isolated and subjected to NMR analysis. For the UV spectra of all of the signals obtained from the LC-DAD runs of the different sample, see the Supplementary data online.
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Figure 1. LC-DAD run (wavelength 254 nm) of different capsules of the same product. (A) Sample A: official sample from an erotic store, taken by local competent authority; (B) sample B: counter sample submitted by the stakeholder; (C) sample C: confiscated product during law enforcement action; and (D) sample D: mixed sample of 10 capsules of the confiscated product (sample C).
Table 1. Identified compounds and corresponding retention times. Compounds 2, 3, 4, 4a, 7, 8, 10 and 13 were described earlier (Instrumental Analysis Data of Illegal Compounds in Food 2010; Wollein et al. 2011; Venhuis & De Kaste 2012; Kim et al. 2013). The identity of compound 4a could not be verified. Compound number 1 2 3 4 4a 5 6 7 8 9 10 11 12 13
Name 5-(5-(1,2-Dihydroxyethyl)-2-ethoxyphenyl)-1-methyl-3-propyl1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (dihydroxydenafil) Isopiperazinonafil Piperazinonafil Oxoacetildenafil Dioxoacetildenafil 5-(2-Ethoxy-5-(2-ethoxy-1-hydroxyethyl)phenyl)-1-methyl-3propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (hydroxyethoxydenafil) 5-(2-Ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7Hpyrazolo[4,3-d]pyrimidin-7-one (Z)-dichlorodenafil Hydroxyacetildenafil 4-Ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1Hpyrazolo[4,3-d]pyrimidin-5-yl)benzaldehyde (aldenafil) Gendenafil 5-(2-Ethoxy-5-(2-thioxobutyl)phenyl)-1-methyl-3-propyl-1,6dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (thioxobutyldenafil) 5-(5-Chloro-2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7Hpyrazolo[4,3-d]pyrimidin-7-one (chlorofil) (E)-dichlorodenafil
LC-MS Chromatographic conditions lead to comparable retention times than pointed out in Figure 1A and Table 1. Operating Q3 in ESI-negative mode combined with an enhanced scan experiment at a 10 000 Da s–1 scanning rate, for each signal its accurate [M – 1] – could be observed. Compounds 2, 3
Retention time (min) (LC-DAD)
[M – 1] – (LC-MS)
10.1
371.1
11.7 12.2 13.1 13.4 15.5
481.2 481.2 479.2 493.2 399.2
20.8
311.2
26.7 11.1 16.8
405.1 481.0 339.2
17.0 17.6
353.0 397.1
24.2
345.0
27.1
405.1
and 8, appearing at 11.7, 12.2 and 11.1 min respectively have already been reported (Instrumental Analysis Data of Illegal Compounds in Food 2010; Wollein et al. 2011). Compound 1 at 10.1 min could be assigned to [M – 1] – of m/z 371 (Figure 2C). A sildenafil substructure could be postulated by showing a neutral loss of ethene from the
N. Schramek et al.
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Figure 2. Mass pattern of (A) compound 6, (B) compound 12, (C) compound 1, (D) compound 5, (E) compounds 9 and 10 (gendenafil) (Lin et al. 2008), (F) compounds 7 and 13 ((Z)-/(E)-dichlorodenafil) (Kim et al. 2013), (G) compound 4 (oxoacetildenafil) (Instrumental Analysis Data of Illegal Compounds in Food 2010), (H) compound 4a (dioxoacetildenafil) (Venhuis & De Kaste 2012), and (I) compound 11.
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Food Additives & Contaminants: Part A ethoxy-moiety (m/z 343), and an additional loss of the substituent in position 3 of the phenylring to give the ethoxyphenyl-pyrazolopyrimidine basic ion with an m/z of 311. Between 13.1 and 13.4 min, two nearly unseparated signals occur, which were labelled as compound 4 and 4a. Compound 4, giving also a great intensity in LC-DAD runs at 13.1 min, was identified to be oxoacetildenafil (Instrumental Analysis Data of Illegal Compounds in Food 2010) as its [M – 1]– was only two units lower than piperazinonafil (Figure 2G). Due to the low intensity of the signal of compound 4a, it was not possible to isolate the substance. Based on LC-DAD data and considering a 14 Da higher mass value in comparison with compound 4 (Figure 2G), compound 4a (Figure 2H) could be assigned to dioxoacetildenafil (Venhuis & De Kaste 2012), although no analytical data for this compound could be found in the literature. Compound 5 at 15.5 min could be assigned to [M – 1] – of m/z 399 (Figure 2D). Its fragmentation pattern, again, shows the characteristic daughter ions for sildenafil substructures (m/z 371, 311). Compound 6 at 20.8 min corresponds to 312 Da (Figure 2A), which is fully in line with the pyrazolopyrimidine basic structure of sildenafil. Compound 7 at 26.7 min shows characteristic [M – 1] – isotope mass pattern together with [(M – 1) + 2] – and [(M – 1) + 4] –, which derives from the assemblage of two chloro substituents (Figure 2F). These signals are identical to the mass data published for (Z)-dichlorodenafil (Kim et al. 2013). Interestingly, compound 13 at 27.1 min is represented by the same mass pattern, which could be assigned to the corresponding (E)-isomer. The parent ion of compound 12 shows similar isotope mass pattern to compounds 7 and 13 but with respect to the isotope ratios of the [(M – 1) + 2] – and [(M – 1) + 4] – and is suggested to have a single chloro moiety in position 3 of the phenyl substituent, which is represented by its m/z of 345 and 347 (Figure 2B). Compounds 9 and 10, which elute very closely to each other at 16.8–17.0 min, are together represented in Figure 2E. An m/z ratio of 353 together with its typical formal loss of ethene (m/z 325) could be attributed to the [M – 1] – of compound 10, which is congruent to published data of gendenafil (Lin et al. 2008). Compound 9 is suggested to be the analogue formyl derivative of compound 10, showing an [M – 1] – of m/z 339 with, again, a neutral loss of ethene forming a m/z 311 daughter ion. At 17.6 min compound 11 elutes with a corresponding [M – 1] – of m/z 397 (Figure 2I), which shows in the enlarged display of its parent ion the typical isotope pattern of sulphur ([(M – 1) + 2] – and in very low intensity due to the low amount of the analyte its [(M – 1) + 4] – signal. NMR 1
H- and 13C-NMR data of the investigated compounds are summarised in Table 2; the 1H- and 13C-NMR spectra are shown in Figures 3 and 4. The 2D-NMR data (H,H-COSY
5
and HMBC) of compounds 1, 5 and 9–12 are summarised in Table 3. The signals in the aromatic region between 8.5 and 7 ppm of the 1H-NMR spectra of all isolated compounds resemble the coupling pattern of the 1,2,4-substituted phenyl ring of sildenafil. The signals at about 4.3 ppm (H-20) and 1.7 ppm (H-21) could be assigned to the O-ethyl moiety and the signals at about 2.9 ppm (H-11), 1.8 ppm (H-12) and 1.0 ppm (H-13) represent the 1-propyl group. HMBC correlations from C-19 to H-20 and C-1 to H-11 and H-12 confirmed the positions of the ethoxy and the propyl moiety, respectively. All observed compounds therefore carry the basic chemical structure of sildenafil. The 1H-NMR spectrum of compound 6 shows four aromatic proton signals and H,H-COSY data shows correlation between these four signals. This indicates an orthosubstitution pattern and is consistent with a 1,2-substituted phenyl ring. The 13C-NMR spectrum shows 17 different carbon signals. LC-MS experiments revealed an m/z = 311. Compound 6 could therefore be assigned to 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1,6-dihydro-7Hpyrazolo[4,3-d]pyrimidin-7-one (C17H21N4O2), the basic pyrazolopyrimidine structure of sildenafil (Ellis & Terrett 1994). Compared with published NMR data, compounds 4, 7, 8 and 10 could be assigned to oxoacetildenafil (oxohongdenafil) (Instrumental Analysis Data of Illegal Compounds in Food 2010), dichlorodenafil ((Z)-isomer) (Kim et al. 2013), hydroxyacetildenafil (hydroxyhongdenafil) (Hou et al. 2006), and gendenafil (Lin et al. 2008), respectively. Compounds 7 and 13 could not be isolated separately (Figure 1D). Consequently the 1H-NMR spectrum of compound 7 (Figure 3E) shows additional signals with very low intensities. While the signal at 6.79 ppm represents the vinyl proton of the (Z)-isomer (main component) the small signal at 6.57 ppm could be assigned to the (E)-isomer of dichlorodenafil (Kim et al. 2013). On the basis of the integrals of both vinyl protons a ratio of 96% (Z)-isomer and 4% (E)-isomer could be estimated. The 1H- and 13C-NMR spectra of compound 1 are shown in Figures 3F and 4F. Compared with compound 6 two additional carbon atoms appear. Using HSQC correlations (data not shown) the proton signal (dd, 1H) at 4.89 ppm could be assigned to the carbon atom at 73.9 ppm (C-22). The two proton signals at 3.66 and 3.77 ppm (each ddd, 1H) could be assigned to one carbon atom at 67.1 ppm (C-31), which is a typical situation for diastereotopic protons in a chiral molecule. Together with H, H-COSY correlations between this three proton signals (Table 3) an ethyl moiety with substituents at both carbon atoms could be expected. HMBC correlations from C-15, C-16 and C-17 to H-22, and additionally from C-16 to H31, confirmed the position of the ethyl moiety. Compound 1 has a mass 60 Da higher compared with compound 6 (Table 1), which is indicative for a C2H4O2 substituent.
7.51
7.05
16 17
18
3.66 3.78 3.80
4.30 1.61 4.89
13
C
67.1
156.2 65.5 14.5 73.9
113.1
134.2 130.1
148.1 138.4 124.4 38.0 27.6 22.3 13.8 119.8 128.5
146.3 153.6
δ
2
Notes: 1(Z)-isomer, compound 7. (E)-isomer, compound 13.
31
19 20 21 22 24 25 26 27 28 29 30
8.44
4.24 2.93 1.85 1.05
11.08
δ 1H
Compound 1
3.90
3.39 2.93 3.43 1.11 3.41
3.29
4.37 1.61
7.14
8.11
9.04
4.27 2.96 1.86 1.03
10.81
δ 1H C
13
62.8
45.8 49.8 41.0 11.9
160.0 65.9 14.4 194.1 57.2 165.7
112.8
129.4 132.3
147.4 138.3 124.5 38.0 27.7 22.3 13.8 120.3 131.7
146.4 153.5
δ
Oxoacetildenafil compound 4
3.62
3.47 1.22
4.29 1.57 4.47
7.08
7.44
8.38
4.21 2.90 1.84 1.01
11.07
δ 1H C
13
67.8
64.1 15.7
156.9 66.1 15.1 82.4
113.8
133.1 131.0
148.7 139.0 125.0 38.6 28.1 22.9 14.4 120.6 130.0
146.8 154.2
δ
Compound 5
4.28 1.58
7.08
7.13 7.46
8.45
4.21 2.88 1.84 1.01
11.08
δ 1H 13
C
157.1 65.8 15.0
113.5
122.0 132.8
148.9 139.1 124.9 38.3 28.1 22.6 14.2 120.8 131.3
146.8 154.2
δ
Compound 6
6.79a 6.57b
4.31 1.60
7.64a 7.72b 7.07a 7.10b
8.62a 8.72b
4.21 2.91 1.86 1.01
10.93
δ 1H C
13
116.3
3.94
3.20 2.92 3.02 3.85
2.92 3.20
4.30 1.55
7.09
113.7 157.7 66.2 14.9 129.8
8.03
8.86
4.18 2.88 1.82 1.00
11.00
δ 1H C
13
63.3
53.0 50.6 59.9 56.6
160.5 66.3 14.8 194.3 50.6 53.0
113.1
129.6 132.5
148.0 138.8 124.9 38.5 28.1 22.8 14.3 121.1 131.9
146.7 154.1
δ
Hydroxyacetildenafil compound 8
135.0 130.9
148.0 138.9 124.9 38.6 28.2 22.7 14.2 120.9 129.7
146.7 154.1
δ
(Z)1-/(E)2dichlorodenafil compounds 7/13
4.35 1.62 10.01
7.21
8.00
8.91
4.22 2.91 1.85 1.02
10.83
δ 1H 13
C
161.3 66.6 14.9 190.9
113.9
131.0 133.1
147.7 138.9 125.0 38.6 28.2 22.8 14.3 121.4 134.4
146.9 154.0
δ
Compound 9
NMR data of identified compounds 1 and 4–12. Positions 1–31 indicate either a hydrogen or a carbon signal.
1 4 5 6 8 9 10 11 12 13 14 15
Position
δppm in CD2Cl2
Table 2.
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2.62
4.36 1.60
7.13
8.07
8.99
4.22 2.92 1.85 1.02
10.83
δ 1H C
13
26.8
160.3 66.4 14.9 196.7
113.3
131.5 132.7
148.0 138.9 125.0 38.6 28.2 22.8 14.3 120.7 132.4
146.9 154.0
δ
Gendenafil compound 10
3.65 1.27
4.37 1.61 4.74
7.14
8.08
9.00
4.22 2.92 1.85 1.27
10.81
δ 1H
13
C
195.4
67.7 15.5
160.5 66.4 14.9 74.2
113.4
129.3 132.4
147.9 138.8 125.0 38.6 28.2 22.9 14.3 120.8 132.0
147.0 154.0
δ
Compound 11
1
H
4.27 1.58
7.02
7.42
8.44
4.21 2.89 1.83 1.01
11.01
δ
13
C
155.7 66.9 15.0
115.1
127.2 132.3
147.6 138.8 125.0 38.9 28.5 23.3 14.7 122.1 130.7
147.0 154.0
δ
Compound 12
6 N. Schramek et al.
H-12
H-11/H-13
H-12
12
13
H-21 H-20 H-31
20 21 22
H-15/H-18 H-17
H-17
H-22
H-29 H-22
H-15/H-18 H-17
H-17
H-12
H-11/H-13
H-22
H-15/H-17/ H-18/H-20 H-21 H-21 H-20 H-20 H-15/H-29/ H-31 H-22/H-30
H-17/H-22 H-18/H-22/ H-31 H-15/H-22
H-18
H-11/H-12
H-11/H-13
H-12
H,H-COSY
H-15/H-18 H-17
H-17
H-12
H-11/H-13
H-12
H,H-COSY
H-15 H-15/H-17 H-15/H-17/ H-18/H-20 H-21 H-20 H-15/H-17/ H-31
H-15/H-17/ H-18 H-17/H-18 H-18/H-31
H-11/H-12
H-11/H-13
H-10/H-11/ H-12 H-15/H-18 H-11 H-10 H-12/H-13
HMBC
Compound 10 (gendenafil)
H-15H-17/ H-18/H-20 H-21 H-21 H-20 H-20 H-15/H-17
H-15/H-22
H-17/H-22 H-18/H-22b
H-15/H-18
H-11/H-12
H-11/H-13
H-15/H-18 H-11 H-10 H-12/H-13
H-11/H-12
HMBC
Compound 9
Note: Due to the very low amount isolated, only a very weak HMBC spectrum could be acquired. b 2 ( JCH = 25 Hz).
30 31
H-22
H-18 H-17
17 18 19
29
H-17/H-22 H-18/H-22/ H-31 H-15/H-22
H-17
15 16
H-12
H-11/H-13
H-12
H-15/H-18 H-11 H-10 H-12/H-13
H-11/H-12
HMBC
Compound 5 H,H-COSY
H-15/H-17/ H-18/H-20 H-21 H-21 H-20 H-20 H-15/H-17/ H-31 H-31 H-30
H-18
H-11/H-12
14
a
H-15/H-18 H-11 H-10 H-12/H-13
6 8 9 11
H-11/H-13
H-11/H-12
HMBC
1
Position H,H-COSY
Compound 1
H-29
H-30
H-21 H-20
H-15/H-18 H-17
H-17
H-12
H-11/H-13
H-12
H-22
H-22/ H-30
H-21
H-21 H-20
H-15/H-18 H-17
H-17
H-10 H-12/ H-12 H-13 H-11/ H-11/H-13 H-13 H-11/ H-12 H-12
H-15/H-17/ H-18/H-20 H-21 H-20
H-15
H-15/H-17/ H-18 H-17/H-18 H-15/H-18
H-11/H-12
H-11/H-13
H-15/H-18 H-11 H-10 H-12/H-13
H-11/H-12
HMBC
Compound 12
H,H-COSY HMBCa H,H-COSY
Compound 11
Table 3. 2D-NMR data of compounds 1, 5 and 9–12. Positions 1–31 indicate either a hydrogen or a carbon signal. δppm in CD2Cl2 .
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N. Schramek et al.
Figure 3. 1H-NMR spectra of (A) compound 6, (B) compound 12, (C) compounds 9 and 10, (D) compound 11, (E) compound 7 ((Z)-dichlorodenafil (Kim et al. 2013)) and compound 13 ((E)-dichlorodenafil (Kim et al. 2013)), (F) compound 1, (G) compound 5, (H) compound 4 (oxoacetildenafil; Instrumental Analysis Data of Illegal Compounds in Food 2010), and (I) compound 8 (hydroxyacetildenafil (Hou et al. 2006)). *Formate.
Compound 1 could therefore be assigned to 5-(5-(1,2dihydroxyethyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1,6dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one and was named dihydroxydenafil. The 1H- and 13C-NMR spectra of compound 5 are shown in Figures 3G and 4G. Compared with compound 1, two additional carbon signals at 64.1 ppm (C-29) and 15.7 ppm (C-30), and two additional proton signals at 3.47 ppm (m, 2H, H-29) and 1.22 ppm (t, 3H, H-30) appear. LC-MS data revealed a mass 28 Da higher compared with compound 1. Together with H,H-COSY correlations between H-29 and H-30 these data are indicative of a second ethyl moiety. HMBC correlations from C-22 to H-29 confirmed the position of this ethyl moiety. Compound 5 could therefore be assigned to 5-(2-ethoxy-5-(2-ethoxy-1-hydroxyethyl)phenyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one and was named hydroxyethoxydenafil. The 1H-NMR spectrum of compound 10 (gendenafil) is shown in Figure 3C. The smaller signals originate from compound 9 since compounds 9 and 10 could not be fully separated. The corresponding 13C-NMR and HMBC spectra are shown in Figures 4C and 5. Compared with the known structure of compound 10, the signal of the methyl
group at 2.62 ppm (H-31) is missing in compound 9. Instead a proton signal at 10.01 ppm (H-22) appears. The 13C-NMR signal of the carbonyl group (C-22) is shifted to a higher field. Compound 9 shows HMBC correlations between C-15/17 and H-22 (Figure 5, green signals). Moreover, a correlation between C-16 and H-22 is shown. Together with LC-MS data this is consistent with an aldehyde moiety at C-16. Compound 9 could therefore be assigned to 4-ethoxy-3-(1-methyl-7-oxo-3propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl) benzaldehyde and was named aldenafil. The 1H-NMR spectrum of compound 12 (Figure 3B) was found to be nearly similar to that of compound 6, except the signal at 7.13 ppm (H-16) is missing (Figure 3B). LC-MS data revealed an m/z of 345 for compound 12. Together with the isotopic pattern, chlorine as a substituent seems plausible. Consequently the 13C-NMR signal of C-16 is shifted towards a lower field (Figure 4B). Compound 12 could therefore be assigned to 5-(5-chloro-2-ethoxyphenyl)-1methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin7-one and was named chlorofil. The 1H-NMR spectrum of compound 11 is shown in Figure 3D. Compared with compound 6, an additional
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Figure 4. 13C-NMR spectra of (A) compound 6, (B) compound 12, (C) compounds 9 and 10, (D) compound 11, (E) compound 7 ((Z)-dichlorodenafil (Kim et al. 2013)) and compound 13 ((E)-dichlorodenafil (Kim et al. 2013)), (F) compound 1, (G) compound 5, (H) compound 4 (oxoacetildenafil; Instrumental Analysis Data of Illegal Compounds in Food 2010), and (I) compound 8 (hydroxyacetildenafil (Hou et al. 2006)). *Formate.
singlet at 4.74 ppm (3H), a quartet at 3.65 ppm (2H) and a triplet at 1.27 ppm (3H) appears. A 13C-NMR signal at about 195 ppm (Figure 4D) together with H,H-COSY (Figure 6) and HMBC correlations (Table 3) indicates a butyl moiety bearing a carbonyl group. LC-MS experiments revealed an m/z of 397 and an [(M – 1) + 4] – signal [Figure 2I], indicative for the presence of a 34S isotope. This is well in line with a 2-thioxobutyl moiety. Compound 11 could therefore be assigned to 5-(2-ethoxy-5-(2-thioxobutyl)phenyl)-1methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin7-one and was named thioxobutyldenafil. The chemical structure of compound 14 could not be safely determined. Based on NMR spectroscopic data this compound seems to bear the well-known sildenafil/pyrazolopyrimidine base structure. A neutral loss of ethene (−28 Da) from the ethoxy moiety may underline this
assumption. Unfortunately the substituent in position 5 of the 2-ethoxyphenyl moiety remains unclear.
Discussion In the present study, 14 different chemical compounds, bearing the same basic pyrazolopyrimidine structure like sildenafil (Figure 7), were isolated from different samples of the same dietary supplement, sold as a pure herbal product. Seven of them have already been published (piperazinonafil, isopiperazinonafil, oxoacetildenafil, (Z)-/(E)-dichlorodenafil, gendenafil and hydroxyhomoacetildenafil) (Lin et al. 2008; Instrumental Analysis Data of Illegal Compounds in Food 2010; Wollein et al. 2011; Kim et al. 2013). Compound 6 could be assigned to 5-(2-ethoxyphenyl)-
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Figure 5.
(colour online) HMBC spectrum of compound 9 (green) and compound 10 (red, gendenafil; Lin et al. 2008).
Figure 6.
H,H-COSY spectrum of compound 11.
1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one, the commercially available pyrazolopyrimidine basic structure of sildenafil. The chemical structures of another five analogues were elucidated using LC-MS, UV and NMR spectroscopic data (Table 1). Although some of the compounds like piperazinonafil, isopiperazionafil and the acetildenafil derivatives found in the present study may exhibit PDE5 inhibition, most of them were not expected to. More likely, they are by-products of the synthesis of a sildenafil analogue. For the synthesis of
sildenafil and its analogues many different routes are described (Kim et al. 2001, 2013; Ley et al. 2002; Yu et al. 2003; Li et al. 2004; Xia et al. 2005; Abdel-Jalil, Khanfar, Abu-Safieh et al. 2005; Abdel-Jalil, Khanfar, Al-Gharabli et al. 2005; Medrasi et al. 2013). Based on published procedures a hypothetical reaction scheme towards pyrazolopyrimidines bearing an acetyl bridge, explaining the existence of the compounds found in the present study and earlier by others, is shown in Figure 8. The Friedel–Crafts-type acylation of commercially available 6 with chloroacetyl chloride
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Food Additives & Contaminants: Part A
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Figure 7. Chemical structures of sildenafil analogues found in the present study. Compound 2, isopiperazinonafil (Wollein et al. 2011); compound 3, piperazinonafil (Wollein et al. 2011); compound 4, oxoacetildenafil (Instrumental Analysis Data of Illegal Compounds in Food 2010); compound 4a, dioxoacetildenafil (Venhuis & De Kaste 2012); compounds 7/13, (Z)-/(E)-dichlorodenafil (Kim et al. 2013); compound 8, hydroxyacetildenafil (Hou et al. 2006); and compound 10, gendenafil (Lin et al. 2008).
leads to compound 6b, known as chlorodenafil which was found earlier in dietary supplements (Instrumental Analysis Data of Illegal Compounds in Food 2010). Reaction of 6b with pyrimidine or pyrimidinone derivatives gives sildenafil analogues of the acetildenafil type. The existence of 10 could be explained by assuming a synthesis of chloroacetyl chloride using chloroacetic acid and phosphorus pentachloride or phosphoryl chlorid. Chloroacetic acid is known to contain considerable amounts of acetic acid which gives acetyl chloride with chlorinating reagents. The chlorination of 6b with remnants of the chlorinating reagent explains the existence of 7 (Kagan et al. 1983; Kim et al. 2013), hydrolysis could give 6a. Formation of 6a could also be explained via direct α-hydroxylation of 10 (Jones 1991; Chan Lee et al. 1997), although this seems not very likely. Since most synthesis routes require the use of strong bases, formation of 5 and 6d seems plausible if ethanol is used as solvent. Depending on the starting material and the target molecule, oxidation and/or reduction reactions are also very common in the synthesis of sildenafil analogues, which could explain the formation of compounds 1 and 5. Since isopiperazinonafil (compound 2) bears a very untypically structure, a completely different synthesis route
is required. Based on a patented procedure towards the formation of phenylacetic acid derivatives, a hypothetical reaction scheme towards pyrazolopyrimidines bearing a 2-oxoethyl bridge is shown in Figure 9 (Stelzer 1995). Reaction of 6 with sulfonyloxyacetic acid derivatives leads to 6f, which could be converted to 6h using pyrimidinone derivatives. A final reduction step could explain the formation of 2. As described above, the use of strong bases and ethanol as solvent explains the formation of 6g. The following conversion of the carbonyl group into a thiocarbonyl group using, for example, phosphorous pentasulfide gives compound 11 (Kim et al. 2002).
Conclusions Recently, Venhuis et al. (2014) demonstrated that there can be fundamental differences in the composition of dosage units within one package of an adulterated dietary supplement. The present example shows the difficulties a laboratory could have in the determination of the ‘real’ composition of an adulterated dietary supplement. Although exact determination as well as the quantification of all of the (illegally added) substances in an adulterated
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Figure 8. Hypothetical synthesis route towards pyrazolopyrimidinones bearing an acetyl instead of a sulfonyl bridge (e.g. compound 4). Reaction intermediates 6b, 6c, 7, 13 and 10 were found earlier and are known as chlorodenafil, hydroxychlorodenafil, (Z)-/(E)dichlorodenafil and gendenafil, respectively (Lin et al. 2008; Instrumental Analysis Data of Illegal Compounds in Food 2010; Kim et al. 2013). Reaction intermediates 6a and 6d have not been found in the present study. The dashed reaction arrow indicates a possible (e.g. (Jones 1991; Chan Lee et al. 1997)) but not mandatory direct α-hydroxylation. R1 , ethoxy group; R2 , pyrazolopyrimidine moiety; R3 , alkyl group; R4 , hydroxyethyl moiety.
Figure 9. Hypothetical synthesis routes towards compounds 2 and 11 based on a patented method for the preparation of phenylacetic acid derivatives (Stelzer 1995). Compounds 6f, 6g and 6h are hypothetical intermediates and have not been found in the present study. R1 , ethoxy group; R2 , pyrazolopyrimidine moiety; R3 , alkyl group.
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Food Additives & Contaminants: Part A dietary supplement may not be necessary in every case to prohibit the placing on the market, a different composition of samples can lead to substantial enforcement difficulties. This is particularly true if, like in the present study, the composition of official samples taken from the police or from competent authorities differ significantly from the composition of other samples (with respect to illegally added substances). Moreover, this example shows the difficulties for all parties – competent authorities as well as distributors – to fulfil their legal obligations. According to article 17 of the regulation (EC) No. 178/2002 of the European Parliament and of the Council, food business operators should ensure their products satisfy the requirements of food law. Competent authorities should monitor and verify that the relevant requirements of food law are fulfilled by food business operators. Even if both of them perform random checks on a product, they cannot be sure of its quality. Many dietary supplements therefore are and remain a high risk for human health unless they are properly regulated. Since they bear a greater resemblance to pharmaceuticals than to usual ‘foods’, the regulatory requirements should be further adapted. With a pre-market approval, an obligation to define product specifications, and to monitor each batch (using average samples) for quality and compliance with these specifications, some of the difficulties with dietary supplements could possibly be solved. Acknowledgement The authors thank Christine Schwarz and Rosalia Spirkl for expert technical assistance.
Supplemental data Supplemental data for this article can be accessed here: http://dx. doi.org/10.1080/19440049.2014.992980.
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