Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement

Accepted Manuscript Title: Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement Authors: Takahiro Doi, Kazunaga Takahashi, Midori Yamazaki, Akiko Asada, Akihiro Takeda, Kyohei Kiyota, Takaomi Tagami, Yoshiyuki Sawabe, Tetsuo Yamano PII: DOI: Reference:

S0731-7085(18)31276-7 https://doi.org/10.1016/j.jpba.2018.08.031 PBA 12162

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

1-6-2018 14-8-2018 16-8-2018

Please cite this article as: Doi T, Takahashi K, Yamazaki M, Asada A, Takeda A, Kiyota K, Tagami T, Sawabe Y, Yamano T, Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.08.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characterization of a new illicit phosphodiesterase-type-5 inhibitor identified in the softgel shell of a dietary supplement

aDepartment

of Hygienic Chemistry, Osaka Institute of Public Health, 1-3-69

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Nakamichi, Higashinari-ku, Osaka 537-0025, Japan

Prefectural Institute of Public Health, 666-2 Nitona-cho, Chuo-ku, Chiba,

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bChiba

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Chiba 260-8715, Japan *

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Graphical anstract

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E-mail: [email protected]

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Kyohei Kiyota,a Takaomi Tagami,a Yoshiyuki Sawabe,a Tetsuo Yamanoa

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Takahiro Doi,a, * Kazunaga Takahashi,b Midori Yamazaki,b Akiko Asada,a Akihiro Takeda, a

1N HCl These compounds are mainly detected from the softgel shell.

Softgel Shell

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Incubation in boiled water to dissolve.

Softgel Content

Extraction of acetildenafil-like analogs as hydrochloride salts.

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1N HCl

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Cutting

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Washed with diethyl ether

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Highlights

A novel sildenafil analog is found from a dietary supplement together with noracetildenafil, and the structure

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was investigated by UV spectroscopy and high-resolution MS analysis. We

synthesized

1-methyl-5-{5-[2-(4-methylpiperazin-1-yl)acetyl]-2-propoxyphenyl}-3-propyl-1,6-

dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one as the reference standard. The illicit sildenafil analogs were mainly detected from the shell of the capsule sample.

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Abstract 2

A new sildenafil analog has been identified in the softgel shell of a dietary supplement. The compound was investigated by UV spectroscopy and high-resolution MS analysis, leading to the proposed structure 1-methyl-5-{5-[2-(4-methylpiperazin-1-yl)acetyl]-2-propoxyphenyl}-3-propyl1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one. A synthetic reference compound with the

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proposed structure was prepared, and the two sets of analytical data were compared, confirming the

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structure of the new compound. The compound was named propoxyphenyl noracetildenafil from its

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structure and similarity with the known compound.

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Keywords propoxyphenyl noracetildenafil; phosphodiesterase-type-5 inhibitor; softgel shell;

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dietary supplement

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1. Introduction

Analogs of approved erectile dysfunction (ED) drugs are often synthesized for use in illicit dietary

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supplements to escape detection by analytical laboratories. Accordingly, several analogous compounds of approved phosphodiesterase-type-5 (PDE-5) inhibitors such as sildenafil (Viagra) have been detected as illicit adulterants in dietary supplements [1]. A common class of PDE-5inhibitor analogs is the acetildenafil-like analogs, which were first reported in 2004 and are structurally similar to sildenafil but with a sulfonyl group replaced by an acetyl group [2]. Since the 3

first report of the detection of acetildenafil in herbal products, nine more analogs have been detected [1]. Furthermore, the detection of propoxyphenyl aildenafil and propoxyphenyl thioaildenafil, both of which are sildenafil analogs, in powdered health supplements was reported in 2012 [3], and nine more analogous compounds have been identified in dietary supplements since then [1]. On the other

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hand, it has been confirmed that adulterants can be compounded into the outer skin of capsules

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instead of in the contents of the capsule themselves [4].

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Unlike the approved drugs, these illicit adulterants are often not sufficiently assessed for

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efficacy and safety, and their ingestion can have unexpected adverse effects. Several cases of

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harmful effects caused by counterfeit drug products have been confirmed[5], including a fatality

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due to a dietary supplement containing PDE-5 analogs [6].

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Identification of PDE-5 inhibitor analogs in dietary supplements is usually performed via high-

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resolution(HR)MS, and NMR analysis after purification [1]. However, HRMS can only be used to

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obtain estimated or proposed structures. Furthermore, the compound under investigation is required in milligram quantities for NMR study, and structure elucidation is difficult in cases where the

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target compound lacks hydrogen and/or carbon atoms. In this study, a novel acetildenafil analog, which was detected in the softgel shells of a dietary

supplement, has been detected. HRMS and UV spectroscopy were used to obtain a proposed structure of the compound. However, for the reasons outlined above, its structure was confirmed by synthesizing the proposed compound and comparing the analytical data for the two. The new analog 4

has been named propoxyphenyl noracetildenafil (Fig. 1a).

2. Experimental 2.1. Samples and reagents

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The dietary supplement samples were soft black capsules containing a black viscous liquid, which

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were purchased over the Internet. An aluminum bag containing two softgels was enclosed in a paper box, and the supplement was sold as food containing extract of cordyceps fruit body. Anhydrous

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potassium carbonate and potassium tert-butoxidce was purchased from Tokyo Chemical Industry

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Co., Ltd. (Tokyo, Japan). 1-Methylpiperazine, 2-n-propoxybenzoic acid, and 4-amino-2-methyl-5-

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propylpyrazole-3-carboxamide hydrochloride were purchased from Combi-Blocks Inc. (San Diego,

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CA, USA). Deuterated chloroform (CDCl3) containing 0.05% (v/v) tetramethylsilane (TMS) was

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purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). All other reagents

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used in this study were purchased from Wako Pure Chemical Industries (Osaka, Japan).

2.2. Instrumentation

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2.2.1. HPLC-PDA analysis HPLC analysis was performed on a 2695 Alliance HPLC system (Waters, Milford, MA, USA) or a Prominence HPLC system (Shimadzu, Kyoto, Japan) equipped with a diode array detector (DAD). HPLC conditions were described elsewhere [7]. A Cosmosil 3C18-EB column (250 mm × 4.6 mm i.d., 5 µm particle size) (Nacalai Tesque, Kyoto, Japan) was used. The mobile phase was composed 5

of solvent A (0.1% formic acid aqueous solution) and solvent B (0.1% formic acid acetonitrile solution). The gradient elution program started with solution A at 70% followed by a linear decrease to 30% over 50 min. The flow rate was 1.0 mL/min, and the injection volume was 50 L. The

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column temperature was 40 °C.

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2.2.2. LC-HRMS analysis

LC-HRMS experiments were performed on a 1290VL LC system coupled with a 6530B

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accurate-mass quadrupole time-of-flight MS instrument (Agilent Technologies, Santa Clara, CA,

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USA). LC conditions were as described previously [8]. An ACQUITY UPLC HSS T3 column

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(100 mm × 2.1 mm i. d., 1.8 µm particle size) (Waters, Milford, MA, USA) was used. The mobile

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phase was composed of solvent A (0.1% formic acid aqueous solution) and solvent B (0.1% formic

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acid acetonitrile solution) at a concentration of 30% B. The instrumentation parameters for MS

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analysis were as described in a previous report [9]. The flow rate of the mobile phase was

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0.3 mL/min, and the injection volume was 1 L. The column temperature was 40 °C. Data analysis including assignment of the molecular formulae was performed using Agilent Mass Hunter Workstation software (Qualitative Analysis version B.05.00) (Agilent Technologies).

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Stock standard solutions were prepared at approximately 100 μg/mL in methanol. When

necessary, the sample solutions and the stock standard solutions were diluted by the mobile phase for the LC-HRMS analysis.

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2.3. NMR NMR spectra were recorded using an ECZS-400 spectrometer (JEOL Resonance, Tokyo, Japan) with CDCl3 containing 0.05% (v/v) TMS as the solvent. The chemical shifts (δ) are reported in ppm relative to TMS (1H: δ = 0 ppm, 13C: δ = 0 ppm) or the solvent (13C: δ = 77.0 ppm) as an internal

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standard. The spectra were assigned using 1H-NMR, 13C-NMR, 1H–13C heteronuclear multiple

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quantum coherence, 1H–13C heteronuclear multiple-bond correlation, and 1H–1H correlation

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spectroscopy.

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2.4. Preparation of sample solutions

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For the initial screening of the capsule sample, a sample solution was prepared by dissolving an

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entire capsule. To a new 15 mL polypropylene tube containing a single capsule, 3 mL of distilled

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water was added, and the tube was incubated in boiled water for 5 min. Then, 7 mL of methanol

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was added, and the solution was filtered through 0.45 μm polyethersulfone filters (Dainippon Seiki,

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Kyoto, Japan) to make the stock sample solutions for initial screening. Sample solutions of the softgel shell and the capsule contents separately were prepared as follows:

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A softgel shell was cut with surgical scissors on a glass dish, and the contents were washed out thoroughly using diethyl ether. After drying, the softgel shell was transferred to a new 15 mL tube, and 3 mL of 1 M HCl was added. The tube was incubated in boiled water for 5 min to allow the softgel to dissolve. The solution was made up to exactly 5 mL with 1 M HCl and filtered through 0.45 μm polyethersulfone filters to make the stock sample solutions of the softgel shell. The diethyl 7

ether solution in the glass dish was transferred to a new glass tube and extracted twice with 3 mL of 1 M HCl. The aqueous layers were combined and made up to 10 mL with 1 M HCl, affording the stock sample solution of the capsule content. These stock solutions were diluted with 50% methanol or 25% acetonitrile in H2O when necessary. The remaining ether layer was made up to 10 mL with

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more ether (organic layer, capsule contents).

2.5 Chemical synthesis

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The chemical syntheses were performed following the method summarized in Fig. 1b [10, 11].

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Amidation of compound 1 with 4-amino-2-methyl-5-propylpyrazole-3-carboxamide hydrochloride

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yielded compound 3. Intramolecular cyclization of 3 gave compound 4. Subsequent Friedel-Crafts

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acylation with 2-bromoacetyl bromide gave compound 5. Compound 5 was reacted with 1-

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methylpiperazine to yield the target compound (6). Detail of the synthetic procedures are shown in

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Supplementary 1.

3. Results and Discussion

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3.1. Analysis of the dietary supplement The results of the initial HPLC-PDA analysis are shown in Supplementary 2. Two major peaks are observed in the UV chromatogram of the sample solution derived from whole softgel, and their UV spectra indicate that they are acetildenafil analogs (Supplementary 2a, and 2b). The peak at 6.67 min (Compound A) was ascribed to noracetildenafil by direct comparison of its HPLC-PDA 8

and LC-HRMS data with those of standard reference solutions (Supplementary 2, Fig. 2a, and data not shown). In the mass spectrum of the unknown peak (Compound B), [M + H] + is observed at m/z 467.2776, which were equivalent to C24H35N6O3+ (calculated exact mass 467.2765), suggesting that the molecular formulae of the unknown compound to be C25H34N6O3 (Fig. 2b). There are two

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known acetildenafil-like analogs previously detected in dietary supplements with the same

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molecular formulae, that is, acetildenafil and dimethylacetildenafil (Fig. 1a) [2, 12]. However, the difference in retention times reveals that Compound 1 is neither acetildenafil nor

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dimethylacetildenafil (Fig. 3). In the MS/MS spectrum of Compound B, there are ions at m/z

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70.0656, 97.0764, and 113.1073, which are similar to those of noracetildenafil (m/z 70.0658,

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97.0766, and 113.1076 (Fig 2c)). These are reported to be the diagnostic ions for the

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methylpiperazine moiety of noracetildenafil [10], which suggests that the unknown compound has a

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similar methylpiperazine moiety in its substructure (Supplementary 3). Focusing on the aromatic

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portions of these compounds, similar ions are observed at m/z 297.1358 and 297.1340 in the

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MS/MS spectra of Compound B and noracetildenafil, respectively. Those ions are diagnostic ions for the acetildenafil family, and the reported structure of the ion is shown in Fig. 4. Comparing the

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structure of noracetildenafil with that of the ion, the ethyl group of the ethoxy phenyl moiety is likely to be lost during fragmentation. Taken together, these factors indicate that the unknown compound is propoxyphenyl noracetildenafil, in which the ethoxy group of noracetildenafil is replaced with a propoxyphenyl group (Supplementary 3).

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3.2. Synthesis of propoxyphenyl noracetildenafil Most PDE-5-inhibitor analogs are identified using LC-HRMS and/or NMR analysis of the compounds after isolation and purification. However, in this study, the amounts of the detected compounds are very limited, so it was unlikely that enough of the purified compound would be

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available to confirm the structure by NMR or X-ray diffraction. The reference standard of

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propoxyphenyl noracetildenafil was not commercially available as well, and consequently, we decided to synthesize propoxyphenyl noracetildenafil.

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The synthetic route to the target compounds is shown in Fig. 1. Except for the self-

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cyclization reaction and the purification step, most of the reactions were performed as previously

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described [10]. We reasoned that the crude reaction mixture following the synthesis of 6 would

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potentially contain, as well as the desired product, the starting materials Compound 5, 1-

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methylpiperazine, and potassium carbonate. We assumed from its structure that Compound 5 would

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not form an organic salt but that the introduced methylpiperazine moiety would allow the target

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Compound 6 to form an organic salt upon the addition of an acid such as HCl. Furthermore, 1methylpiperazine is known to be soluble in water even as the free base. Thus, the target compound

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is easily extracted to an organic phase upon basic/neutral aqueous workup, with the 1methylpiperazine and potassium carbonate remaining in the aqueous phase.

3.3. Extraction of softgel shell and capsule contents In another screening of the same sample performed at the Chiba Prefectural Institute of Public 10

Health, it was indicated that the detected PDE-5-inhibitor analogs were contained in the softgel shells rather than in their contents. However, this screening analysis was performed without the chemical properties of the detected compounds being known, making it very difficult to determine whether the detected compounds were really derived from the softgel shells. In fact, both

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noracetildenafil and an unknown acetildenafil analog were observed in both the shells and contents

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of the capsules. A screening analysis at the Osaka Institute of Public Health was performed by

dissolving the whole capsule at once, so we have attempted in the current study to reveal whether

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the detected compounds are derived from the softgel shell or the contents.

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In compound preparation method in Section 3.2, the free base of propoxyphenyl

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noracetildenafil is distributed to the organic phase and its hydrochloride salt is distributed to the

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aqueous phase. To separate the capsule content from the shell thoroughly, the capsule contents were

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washed out with diethyl ether, in which the softgel shell does not dissolve. The dried shell was then

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added to 1 M HCl headed in boiled water to allow the softgel shell and any acetildenafil-like

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compounds therein to dissolve. The diethyl ether suspension of the capsule contents was separated with 1 M HCl, most likely distributing the acetildenafil-like analogs in the capsule contents to the

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aqueous phase.

The analytical data for the propoxyphenyl noracetildenafil standard are shown in Fig. 4.

The retention time and MS(/MS) spectra of the peaks in the chromatogram of the sample solution are in accordance with those of the propoxyphenyl noracetildenafil solution, indicating that the dietary supplement is adulterated with propoxyphenyl noracetildenafil. HPLC-PDA analysis of 11

reference standards and sample solutions was performed, and the peak areas of the UV chromatograms were compared to estimate the approximate amounts of the detected compounds. The amounts of noracetildenafil and propoxyphenyl noracetildenafil detected in the softgel shell constitute more than 98% of the total contents (data not shown), which indicates that the

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manufacturer of the dietary supplement intentionally loaded these adulterants into the softgel shells

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to avoid detection in analytical laboratories. For the organic layer of the capsule contents, no peaks for acetildenafil analogs were found in the UV chromatogram (data not shown).

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In this study, we have reported the characterization of a novel PDE-5-inhibitor analog,

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propoxyphenyl noracetildenafil, which we found blended into the capsule material of a dietary

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supplement together with noracetildenafil. The PDE-5 inhibitor tadalafil has been previously

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detected in hard capsule shells [4, 13]. In hard capsules, powdery or solid contents are enclosed, so

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it is relatively easy to separate the shells and their contents. However, in the present case, both the

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shell and the viscous liquid contents of the soft capsule are colored black, making it very difficult to

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distinguish the contents from the shells visually.

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4. Conclusion

The soft shell of a dietary supplement was found to contain a novel sildenafil analog, propoxyphenyl noracetildenafil, in which the ethoxy group of noracetildenafil is replaced with a propoxy group. This compound was characterized using HPLC-PDA and LC-HRMS, and its structure was confirmed using a reference standard that we synthesized. 12

To the best of our knowledge, this is the first report to describe the detection of an ED drug other than tadalafil in the capsule casing of a dietary supplement. However, it is worthy of mention that tadalafil was previously only found in hard capsule cases. Our findings are not only useful for preparing reference standards for illicit ED drug analogs but also provide an initial warning

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regarding a novel risk. Furthermore, the data provided, the synthetic method for the reference

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standard, and the sample preparation method for the softgel shells may be of benefit to the detection

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of other novel PDE-5 inhibitors in analytical laboratories.

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References

[1] C.L. Kee, X. Ge, V. Gilard, M. Malet-Martino, M.Y. Low, A review of synthetic phosphodiesterase type 5 inhibitors (PDE-5i) found as adulterants in dietary supplements, J. Pharm. Biomed. Anal. 147 (2018)

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250-277.

[2] L. Blok-Tip, B. Zomer, F. Bakker, K.D. Hartog, M. Hamzink, J. Ten Hove, M. Vredenbregt, D. De Kaste,

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Structure elucidation of sildenafil analogues in herbal products, Food. Addit. Contam. 21 (2004) 737-748. [3] C.L. Kee, X. Ge, H.L. Koh, M.Y. Low, Isolation and characterization of propoxyphenyl linked sildenafil and thiosildenafil analogues in health supplements, J. Pharm. Biomed. Anal. 70 (2012) 265-272.

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[4] B.J. Venhuis, J. Tan, M.J. Vredenbregt, X. Ge, M.Y. Low, D. de Kaste, Capsule shells adulterated with tadalafil, Forensic Sci. Int. 214 (2012) e20-22. [5] W.T. Poon, Y.H. Lam, C.K. Lai, A.Y. Chan, T.W. Mak, Analogues of erectile dysfunction drugs: an underrecognised threat, Hong Kong Med. J. 13 (2007) 359-363.

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[6] E.L. Bakota, A.T. Kelly, J.P. Walterscheid, D.R. Phatak, A Case Report of Fatal Desmethyl Carbodenafil Toxicity, J. Anal. Toxicol. 41 (2017) 250-255. [7] T. Tagami, A. Aoyama, A. Takeda, A. Asada, T. Doi, K. Kajimura, Y. Sawabe, Simultaneous identification of 18 illegal adulterants in dietary supplements by using high-performance liquid chromatography-mass spectrometry, Shokuhin Eiseigaku Zasshi 55 (2014) 34-40. [8] T. Doi, T. Tagami, A. Takeda, A. Asada, Y. Sawabe, Evaluation of carboxamide-type synthetic cannabinoids as CB 1/CB 2 receptor agonists: difference between the enantiomers, Forensic Toxicol. 36

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(2018) 51-60. [9] T. Doi, A. Asada, A. Takeda, T. Tagami, M. Katagi, S. Matsuta, H. Kamata, M. Kawaguchi, Y. Satsuki, Y. Sawabe, Identification and characterization of α-PVT, α-PBT, and their bromothienyl analogs found in illicit drug products, Forensic Toxicol. 34 (2016) 76-93. [10] C. Mustazza, A. Borioni, A.L. Rodomonte, M. Bartolomei, E. Antoniella, P. Di Martino, L. Valvo, I. Sestili, E. Costantini, M.C. Gaudiano, Characterization of Sildenafil analogs by MS/MS and NMR: a guidance for detection and structure elucidation of phosphodiesterase-5 inhibitors, J. Pharm. Biomed. Anal. 96 (2014) 170-186.

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[11] A.S. Bell, D. Brown, N.K. Terrett, Pyrazolopyrimidone antianginal agents, PfizerResearch and Development Company, EP0526004 A1, 1992.

[12] A.H. GÖKER, M. Coşkun, M. Alp, Isolation and identification of a new acetildenafil analogue used to adulterate a dietary supplement: dimethylacetildenafil, Turkish J. Chem. 34 (2010) 157-164.

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[13] A. Lanzarotta, J.B. Crowe, M. Witkowski, B.M. Gamble, A multidisciplinary approach for the analysis of an adulterated dietary supplement where the active pharmaceutical ingredient was embedded in the

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capsule shell, J. Pharm. Biomed. Anal. 67-68 (2012) 22-27.

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Figure Legends

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Fig. 1 Structures of acetildenafil, noracetildenafil, dimethylacetildenafil, and Compound B

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(propoxyphenyl noracetildenafil) (a), and synthetic procedure for the preparation of the

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propoxyphenyl noracetildenafil standard (b).

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Fig. 2 LC-HRMS analysis of the sample solution derived from whole softgel. Extracted ion chromatograms (EICs) at m/z 453.2609, and 467.2765 (Δ = ±0.5 mDa) (a), MS1

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spectra (b), and MS2 spectra (c) of each peak obtained by LC-HRMS. Fig. 3 Extracted ion chromatograms at m/z 467.2765 (Δ = ±0.5 mDa) for acetildenafil (upper panel) and dimethylacetildenafil (lower panel) obtained by LC-HRMS. Fig. 4 Extracted ion chromatograms at m/z 467.2765 (Δ = ±0.5 mDa), MS1 spectra, and MS2 spectra of propoxyphenyl noracetildenafil obtained by LC-HRMS. 14

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(a)

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(a) EIC at m/z 453.2609 (Δ = ±0.5 mDa)

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x10 5 4 3 2 1 0

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2.4 2.8 3.2 3.6 4 4.4 4.8 Retention time (min) EIC at m/z 467.2765 (Δ = ±0.5 mDa)

x104 3

Compound B

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(b)

2.4 2.8 3.2 3.6 4 4.4 4.8 Retention time (min)

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0.4 0.8 1.2 1.6 2

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1

Compound A [Noracetildenafil] (2.071 min)

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x102 1 0.8 0.6 0.4 0.2 0

453.2605 [M+H]+

2+

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[M+2H] 227.1332

150

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3.289

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0

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0.4 0.8 1.2 1.6

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Compound A 2.071 (Noracetildenafil)

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m/z

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x101 6

467.2776 + [M+H]

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2+

2 0

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400

M ED PT CC E A

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450

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[M+2H] 234.1428

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Compound B (3.289 min)

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m/z

(c) 453.2609

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400

450 m/z

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MS2 Spectrum (Compound B) 467.2745

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297.1358 325.1299

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ED PT CC E A

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297.1340 325.1289 396.2042

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x10 70.0656 1 97.0764 0.8 113.1073 0.6 0.4 166.0954 0.2 0 100 150 200

MS2 Spectrum (Noracetildenafil)

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2 x10 70.0658 97.0766 1 0.8 113.1076 0.6 0.4 166.0938 0.2 0 100 150 200

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450 m/z

EIC at m/z 467.2765 (Δ = ±0.5 mDa)

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x10 8

2.448

6 4 2 2

2.4 2.8 3.2 3.6 4 4.4 4.8 Retention time (min)

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0.4 0.8 1.2 1.6

EIC at m/z 467.2765 (Δ = ±0.5 mDa) 2.226

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5

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2.4 2.8 3.2 3.6 4 4.4 4.8 Retention time (min)

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0.4 0.8 1.2 1.6

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x10 1 0.8 0.6 0.4 0.2 0

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0

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EIC at m/z 467.2765(Δ = ±0.5 mDa) 3.245

2.4 2.8 3.2 3.6

4 4.4 4.8 Retention time (min)

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2

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0.4 0.8 1.2 1.6

MS1 spectrum (Propoxyphenyl noracetildenafil)

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467.2762 + [M+H]

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[M+2H] 234.1415

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150

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x10 6 5 4 3 2 1 0

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450

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550 m/z

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2 x101 97.0763 MS spectrum (Propoxyphenyl noracetildenafil) 1 70.0655 467.2754 113.1072 0.8 0.6 297.1338 325.1285 0.4 166.0974 0.2 0 100 150 200 250 300 350 400 450 m/z