Bioactivation of 2-(Alkylthio)-1,3,4-Thiadiazoles and 2-(Alkylthio)-1,3

Nov 13, 2012 - When 5-substituted 2-(methylthio)-1,3,4-thiadiazoles and ... by thermoinactivation (0–45%), suggesting that the sulfoxidation step is...
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Bioactivation of 2‑(Alkylthio)-1,3,4-Thiadiazoles and 2‑(Alkylthio)-1, 3-Benzothiazoles Yanou Yang,* Feng Qiu, Jonathan L. Josephs, W. Griffith Humphreys, and Yue-Zhong Shu Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States ABSTRACT: Certain functional groups/structural motifs are known to generate chemically reactive metabolites that can covalently modify essential cellular macromolecules and, therefore, have the potential to disrupt biological function and elicit idiosyncratic adverse drug reactions. In this report, we describe the bioactivation of 5-substituted 2-(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles, which can be added to the growing list of structural alerts. When 5-substituted 2-(methylthio)-1,3,4-thiadiazoles and 2-(methylthio)-1,3-benzothiazole were incubated with pooled human liver microsomes in the presence of NADPH and GSH, unusual GSH adducts were formed. Characterization of these GSH adducts by highresolution mass spectrometry indicated the replacement of the methylthio- group by GSH, and NMR experiments ascertained the proposed structures. On the basis of the metabolic profile change in incubation samples with/without GSH, we proposed that the GSH adduct formation involved two steps: (1) enzymatic oxidation of the alkylthio- group to form sulfoxide and sulfone and (2) nucleophilic displacement of the formed sulfoxide and sulfone by GSH. The proposed mechanism was confirmed by the formation of the same GSH adduct from the incubation of synthetically prepared sulfoxide and sulfone compounds in buffer. We found the sulfur oxidation step was significantly inhibited (80−100%) by preincubation with 1-aminobenzotriazole but was much less affected by thermoinactivation (0−45%), suggesting that the sulfoxidation step is primarily catalyzed by cytochrome P450s and not by flavin monooxygenases. We also investigated the presence of this bioactivation pathway in more than a dozen compounds containing 2-(alkylthio)-1,3,4-thiadiazole and 2-(alkylthio)-1,3-benzothiazoles. The common GSH adduct formation pathway demonstrated by current studies raises a new structural alert and potential liability in drug safety when 2-alkylthio derivatives of 1,3-benzothiazoles and 1,3,4-thiadiazoles are incorporated in drug design.



INTRODUCTION Covalent modification of cellular proteins by chemically reactive compounds/metabolites has the potential to disrupt biological function and elicit serious adverse drug reactions.1−3 Although there is no consistent link between chemically reactive metabolites and toxicity, removing the chemical alerts as early as possible in the drug discovery process can eliminate perceived reactive metabolite-mediated chemical liabilities and the need for extensive safety evaluation beyond standard practices.4,5 Therefore, it is essential to conduct a thorough examination of chemical reactivity of new chemical entities from a safety standpoint. Thanks to this early avoidance strategy, a growing list of functional groups/ structural motifs have been reported to possess intrinsic chemical reactivity or generate chemically reactive metabolites.6−10 Five-membered aromatic heterocycles, including benzothiazoles and thiadiazoles, are of great medicinal chemistry interest due to their occurrence in pharmacologically active molecules.11 Compounds containing benzothiazoles and thiadiazoles are reported to have a wide range of pharmacological activities such as antitumor, antimicrobial, antidiabetic, anticonvulsant, and antiinflammatory activities.12−17 The diverse biological utilities have motivated new efforts in searching for novel analogues of benzothiazole and thiadiazole compounds with improved biological and other druglike properties. © XXXX American Chemical Society

In this paper, we present a previously unreported bioactivation pathway associated with alkylthio- derivatives of 1,3,4thiadiazole1 and 1,3-benzothiazole. This bioactivation pathway was first observed for a 4,5-substituted 2-(methylthio)-1,3,4thiadiazole compound among related molecules on which we performed biotransformation studies. When this compound was incubated with pooled human liver microsomes (HLM) in the presence of NADPH and GSH, an unusual GSH adduct was formed. Structural identification of the GSH adduct by ultra high-pressure liquid chromatography/high-resolution accurate mass spectrometry (UHPLC/HRMS) indicated the displacement of the alkylthio- group by GSH. Upon further investigations, the GSH displacement reaction was also observed with 5-substituted 2-(methylthio)-1,3,4-thiadiazole and 2-(methylthio)benzothiazole. NMR data unambiguously confirmed the nucleophilic displacement by GSH. The GSH adduct formation could be attributed to the enzymatic oxidation of the alkylthio group by cytochrome P450s and/or flavin monooxygenases (FMOs) to form the sulfoxide and sulfone, which are excellent leaving groups and are readily displaced upon nucleophilic attack by GSH. We also investigated the presence of this bioactivation Received: September 19, 2012

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equal volume of acetonitrile followed by centrifugation. The supernatant was transferred for UHPLC-UV/MS analysis. To determine the participation of cytochrome P450 enzymes in the oxidation of the alkylthio- group, HLMs (1 mg/mL) were preincubated with 1 mM 1-ABT for 20 min in the presence of 2 mM NADPH in 100 mM phosphate buffer, and then, compounds 1−3 were added to the incubation mixture at a final concentration of 30 μM and incubated for 45 min. The formation of the corresponding sulfoxides and sulfones was compared with the formation in the control incubation samples omitting 1-ABT during a preincubation step. To investigate the involvement of FMOs in the sulfoxidation step, HLMs were immersed in 50 °C water bath for 90 s and then incubated individually with compounds 1−3 in 100 mM phosphate buffer in the presence of 2 mM NADPH. Benzydamine was included in the incubation as a positive control. The formation of the corresponding sulfoxides and sulfones was compared with the formation in the control incubation samples containing HLMs without heat treatment. UHPLC-UV/HRMS Methods. All incubation samples were analyzed using an Accela UHPLC system coupled to a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Chromatographic separations were performed with an Acquity UPLC High Strength Silica T3 1.8 μm (2.1 mm × 100 mm) column at a flow rate of 0.6 mL/min. The column was eluted with water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). Because of the wide range of retention properties of the compounds, three solvent gradient programs were employed as follows: (1) 0−0.5 min, 98% A; 0.5−8 min, 98% A to 5% A; 8−10.5 min, 5% A; and 10.5−12 min, 98% A; (2) 0−0.5 min, 98% A; 0.5−8 min, 98% A to 30% A; 8−10.5 min, 30% A; and 10.5−12 min, 98% A; (3) 0−0.5 min, 98% A; 0.5−8 min, 98% A to 50% A; 8−10.5 min, 50% A; and 10.5−12 min, 98% A. Gradient 1 was used for sample analysis of compounds 1, 1A, 1C, and 3A while gradient 2 was used for 2A, 2B, 2C, 2D, 3, 3B, 3C, and 3D, and gradient 3 was used for 1B, 2, and 2E. The column and samples were maintained at temperatures of 55 and 5 °C, respectively. The UV data were collected for a scan range of 200− 600 nm using an in-line Accela PDA detector (Thermo Fisher Scientific,

pathway in more than a dozen compounds containing 2-(alkylthio)1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles. The facile GSH adduct formation demonstrated by current studies raises a new structural alert and potential liability when 2-alkylthio derivatives of 1,3-benzothiazoles and 1,3,4-thiadiazoles are incorporated in drug design.



EXPERIMENTAL PROCEDURES

Materials. HLMs were obtained from BD Gentest (Woburn, MA). NADPH, reduced glutathione, 1-aminobenzotriazole (1-ABT), and benzydamine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Compounds used in this paper were available from the following commercial sources: compounds 1, 2, 2C, 2E, 3, 3A, 3B, and 3C were from Sigma-Aldrich; compounds 1B, 1C, 2A, 2B, and 3D were from Aurora Fine Chemicals (San Diego, CA); compound 1A was from the Florida Center for Heterocyclic Compounds (Gainesville, FL); compounds 2D, 2-(methylsulfinyl)benzo[d]thiazole, and 2-(methylsulfonyl)benzo[d]thiazole were from Ryan Scientific, Inc. (Mt. Pleasant, SC). HPLC grade water and acetonitrile were purchased from EMD Chemicals (Gibbstown, NJ). Incubations. Each compound was incubated individually with three replicates at 37 °C with HLM at a concentration of 30 μM in 100 mM phosphate buffer (pH 7.4) in the presence of both NADPH (2 mM) and GSH (5 mM) and only NADPH or GSH. The protein concentration of liver microsomes used in the incubation was 1 mg/mL. Incubation samples (100 μL) were taken at 0 and 75 min and quenched with an

Figure 1. Chemical structures of test compounds 1−3.

Figure 2. LC/UV chromatograms of incubations of compound 1 (30 μM) in (A) HLM in the presence of NADPH and GSH at 0 min, (B) HLM in the presence of GSH at 75 min, (C) HLM in the presence of NADPH at 75 min, and (D) HLM in the presence of NADPH and GSH at 75 min. The MS and MS2 spectra of the GSH adduct at retention time 4.8 min are shown in E and F. B

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Figure 3. LC/UV chromatograms of incubations of compound 2 (panels A−C) and 3 (panels D−F) at 30 μM in (A and D) HLM in the presence of NADPH and GSH at 0 min, (B and E) HLM in the presence of NADPH at 75 min, and (C and F) HLM in the presence of NADPH and GSH at 75 min.

Table 1. Accurate Masses, MS Fragments, and the Proposed Chemical Compositions of GSH Adducts Detected from Incubations of Compounds 1−3 with HLM in the Presence of NADPH and GSHa test compds (PH+)

GSH adduct

RT (min)

observed accurate mass of GSH adduct (GH+)

1 (300.0624)

1G

4.9

559.1420

2 (223.0358)

2G1

3.7

498.1117

2G2

4.0

498.1117

2G3

5.5

482.1162

3G

4.1

441.0899

3 (182.0093) a

key fragments of GSH adduct

proposed composition of GSH adduct (accurate mass)

430.1000 (GH+-129) 286.0468 (PH+-CH2) 369.0684 (GH+-129) 225.0150 (PH++O−CH2) 369.0684 (GH+-129) 225.0150 (PH++O−CH2) 353.0739 (GH+-129) 209.0202 (PH+-CH2) 312.0474 (GH+-129) 167.9938 (PH+-CH2)

P-CH2S + GSH (559.1428) P-CH2S + O + GSH (498.1112) P-CH2S + O + GSH (498.1112) P-CH2S + GSH (482.1163) P-CH2S + GSH (441.0897)

Notations: GH+, [GSH adduct + H] +; PH+, [parent + H] +; and P, formula of parent compound. incubation flask. The quenched incubation solution was centrifuged, and the supernatant was concentrated to 5 mL, which was then injected onto a Waters Sunfire column (4.6 mm × 150 mm, 3.5 μm). The column was eluted with water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) at a flow rate of 1 mL/min. Employed was the following solvent gradient program: 0−5 min, 90% A; 5−15 min, 90% A to 50% A; 15−18 min, 50% A to 10% A; and 18−20, 90% A. Under the above conditions, GSH adduct 3G elutes at 10.8 min. Fractions from 10 to 11.5 min were collected in a 96-well plate at 0.25 min per well. Multiple injections (80 μL each) and chromatography were repeated under the same conditions in an automated fashion, and the eluent from the column was collected into different wells. The solvents in the collected wells corresponding to the GSH adduct were combined and evaporated to dryness under a stream of nitrogen. NMR Experiment. The purified GSH adduct from compound 3 (3G, Figure 4) was dissolved in 20 μL of CD3OD and transferred to a 1.7 mm NMR tube. NMR data were acquired on a 600 MHz Bruker Avance III NMR spectrometer (Bruker Biospin, Billerica, MA) equipped with a 1.7 mm cryoprobe. One-dimensional (1D) proton,

San Jose, CA). The HPLC eluent was directly coupled to an LTQ/ Orbitrap mass spectrometer (Thermo Fisher Scientific). Mass spectrometric analysis was conducted with an electrospray ionization source. Mass calibration was performed daily according to the manufacturer's guidelines. The capillary temperature was 275 °C, the capillary voltage was 34 V, the source voltage was 4 kV, and the tube lens voltage was 108 V. Three scan events were used as follows: (1) m/z 100−1000 full-scan MS operated at a target mass resolution of 15000 (fullwidth at half-maximum as defined at m/z 400), (2) data-dependent MS2 scan with mass of 7500 on the most intense ion from the full-scan event, and (3) data-dependent scan MS3 with a mass resolution of 7500 on the most intense ion from the MS2 scan. All data were processed in the Qual browser module of Xcalibur (ThermoFisher Scientific). GSH Adduct Isolation for NMR. Scale-up reactions were conducted to isolate adequate amounts of the GSH adduct 3G as shown in Figure 4 for NMR characterization. The GSH adduct scale-up incubation mixture contained 100 μM compound 3, 2 mM NADPH, 5 mM GSH, and 2 mg/mL microsome protein/mL in potassium phosphate buffer (100 mM, pH 7.4) with a total volume of 20 mL. The reaction was stopped at 2 h by adding an equal volume of cold ACN into the C

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two-dimensional (2D) 1H−13C hetronuclear multiple quantum coherence (HMQC), and 2D 1H−13C heteronuclear multiple bond

coherence (HMBC) were acquired to determine the structure of this GSH adduct. A 1D proton was acquired at 600.33 MHz frequency using 16 scans with 20 ppm spectral width and 2.66 s acquisition time. The spectrum was processed using 64 k zero filling. Two-dimensional HMQC data were acquired at 600.33 MHz proton and 150.96 MHz 13C frequencies using 1024 × 128 acquisition data points with 13 ppm of proton and 180 ppm of carbon-13 spectra widths. Two-dimensional HMBC data were acquired at 600.33 MHz proton and 150.97 MHz 13C frequencies using 1024 × 128 acquisition data points with 13 ppm of proton and 240 ppm of carbon-13 spectra widths. The data were processed using zero filling in the F2 dimension and linear prediction in the F1 dimension formed a 2048 × 1024 data matrix.



RESULTS AND DISCUSSION GSH Adduct Formation Detected by UHPLC/HRMS. During our screening for bioactivation potential of 4,5-substituted-1,3,4-thiadiazoles, we incubated compound 1 (structure in Figure 1) with HLM fortified with NADPH in the presence and absence of GSH. As shown in Figure 2, the UV peak at RT 4.9 min was only observed in incubation samples supplemented with NADPH plus GSH, but not in samples omitting GSH,

Figure 4. Proposed GSH adduct structures formed from test compounds 1−3. GSH adduct detected for compound 1 at RT 4.9, 1G; GSH adduct detected for compound 2 at RT 5.5, 2G3; GSH adduct detected for compound 2 at RT 3.7 and RT 4.0, 2G1 and 2G2; and GSH adduct detected for compound 3 at RT 4.1, 3G.

Figure 5. 2D HMQC data (A) and 2D HMBC data (B) of GSH adduct 3G. D

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Table 2. Summary of Oxidative Metabolites Which Showed Decreased Formation upon Addition of GSH into the Incubations

1

Calculated from the metabolite LC/UV peak area in +NADPH + GSH sample as % of +NADPH sample. 2n = 3.

GSH adducts with common chemical formula of parent + GSHS-CH2. Two additional GSH adducts, GSH adduct 2G1 and 2G2 as shown in Figure 3C, were observed in incubation samples from compound 2. Both adducts showed the same protonated molecular ion, [M + H]+ at m/z 498, an addition of 275 Da to compound 2 [M + H]+ at m/z 223. The chemical formula derived from high-resolution MS data indicated the chemical formula of GSH adducts 1 and 2 to be parent + O + GSH-S-CH2. Because the common parts of the structures among three compounds are 2-(methylthio) thiazole, displacement of the methylthio group by GSH would match the common GSH adduct formula parent + GSH-S-CH2. The proposed structures for all of the GSH adducts formed from the proposed displacement reaction for compounds 1−3 are shown in Figure 4. This postulated displacement reaction is also consistent with the common fragments [P + H]+-CH2 that are present in the MS2 spectra of all of the GSH adducts detected for compounds 1−3. GSH Adduct Structure Identification by NMR. To confirm the proposed GSH adduct structure, we isolated the GSH

which indicated that it was a GSH adduct peak. The full scan data (Figure 2E) showed a protonated molecular ion, [M + H]+ at m/ z 559, an addition of 259 Da to compound 1 [M + H]+ at m/z 300. The chemical formula derived from high-resolution MS data indicated the addition of GSH (305 Da) minus (S + CH2) (46 Da) to the parent molecule. MS2 fragmentation (Figure 2F) of m/z 559 ions revealed ions at m/z 430, corresponding to a neutral loss of 129 Da, consistent with loss of pyroglutamic acid from the glutathione moiety. This confirmed that RT 4.9 is a GSH adduct peak. The formation of GSH adduct 1G required oxidative metabolism since it was not detected when NADPH was absent from the incubation (Figure 2B). To investigate whether other methyl sulfides with similar structures also form GSH adduct, we incubated compounds 2 and 3 (structures in Figure 1) with HLM fortified with NADPH and GSH. LC/UV chromatograms from these incubation samples are displayed in Figure 3, and the observed high-resolution m/z values and their key fragments for detected GSH adducts are listed in Table 1. Both compound 2 and 3 formed NADPH-dependent E

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Figure 6. CID spectra of compound 1 and its oxidative metabolites 1M1, 1M2, and 1M3.

compound 1−3 with HLM supplemented with NADPH. Their structures were assigned based on high-resolution full scan MS data and MS2 fragmentation patterns. Metabolite assignment for compound 1 was shown as an example (Figure 6). It becomes obvious that the common biotransformation leading to the metabolites was the oxidation on the 2-methylthio moiety. The most rational oxidation position is the sulfur, resulting in sulfoxide and sulfone attached to the thiadiazole and benzothiazole ring, which can be readily displaced by GSH through nucleophilic attack due to their high intrinsic electrophilicity. Thus, when GSH is present in the incubation samples, the sulfone and sulfoxide metabolite formation were partially or completely consumed as intermediates to the GSH adduct, shunting the metabolism to GSH adduct formation. There have been previous reports on nucleophilic displacement of methylsulfone/ sulfonamide by GSH under nonenzymatic and/or enzymatic conditions.8,18−23 To examine this hypothesis, we incubated the corresponding synthetically prepared sulfoxide and sulfone of compound 3 individually with GSH in buffer. As shown in Figure 7, the

adduct (3G, Figure 4) formed from compound 3 and conducted a NMR experiment. The results are shown in Figure 5. In Figure 5A, 1D proton and 2D HMQC showed both proton and carbon13 assignments for this GSH adduct. The chemical shifts and the proton integrals fit the proposed structure. In Figure 5B, the 2D long-range HMBC correlations revealed most connections for the proposed structure. Among them, the key correlations from proton 11 to carbon 2 were observed as underlined cross-peaks and arrows in the structure, confirming the linkage of GSH motif to the core of compound 3. Mechanism of GSH Adduct Formation. During our metabolite profiling of incubation samples of compounds 1−3 with HLM supplemented with NADPH, we repeatedly noticed that the amounts of certain oxidative metabolites were dramatically reduced or completely diminished when GSH is present in the incubation samples, in comparison to those in the absence of GSH (Figures 2 and 3). The observation indicated that these oxidative metabolites may be related to the GSH adduct formation. Table 2 listed the extent of decrease of the oxidative metabolites when GSH is present in the incubations of F

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Figure 7. GSH adduct formation from synthetically prepared sulfoxide (3M1) and sulfone (3M2) of compound 3. The LC/UV chromatograms are (A) 3M1 in buffer at 0 min, (B) 3M1 in buffer in the presence of 5 mM GSH at 0 min, (C) 3M2 in buffer at 0 min, and (D) 3M2 in buffer in the presence of 5 mM GSH at 0 min.

Figure 8. Proposed mechanism for GSH adduct formation.

sulfoxide (3M1) and sulfone (3M2) corresponding to compound 3 both formed a GSH adduct at RT 4.1 min immediately after being in contact with GSH in buffer (0 min). The sulfone showed complete conversion to GSH adduct, while the sulfoxide showed 12% remaining, which indicates that sulfone is a better leaving group than the corresponding sulfoxide. The retention time and MS fragmentation pattern of this GSH adduct formed from the sulfone and sulfoxide in buffer matched those of the GSH adduct detected when

compound 3 (Figure 3F) was incubated with HLM supplemented with NADPH and GSH. On the basis of the combined evidence, we propose that the GSH adduct formation of compounds 1−3 consists of two steps (Figure 8): (1) sulfoxide and sulfone formation from the oxidation of the sulfide and (2) nucleophilic displacement of the sulfoxide and sulfone formed from step 1 by GSH. Step 1 is an enzymatic and NADPH-dependent process, while step 2 can proceed spontaneously in buffer. The magnitude/propensity of G

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Table 3. Effect of Heat Inactivation and 1-ABT Treatment on the Sulfoxide and Sulfone Formation % inhibition by 1-ABT (mean ± SDa) % inhibition by heat treatment (mean ± SDa) a

1M1

1M2

1M3

2M1

2M2

3M1

3M2

81.5 ± 7.7 2.2 ± 0.2

79.0 ± 6.5 2.2 ± 0.2

100.0 ± 0.0 0.0 ± 0.0

100.0 ± 0.0 30.3 ± 7.5

100.0 ± 0.0 45.1 ± 8.7

89.8 ± 5.3 0.0 ± 0.0

93.1 ± 3.6 22.8 ± 1.9

n = 3.

Table 4. GSH Adduct Formation of Different Thiadiazole Analogues

reactions in both steps 1 and 2 for a specific compound determines its amount of GSH adduct formation. Because cytochrome P450s and FMOs are known to catalyze sulfoxidation,24 we decided to further investigate the enzyme class involved in the step 1 for compound 1−3 by (1) mild heat treatment (50 °C for 90 s) in the presence of NADPH and (2) preincubation with 1-ABT (1 mM, 20 min of preincubation) on the formation of the sulfoxides and sulfones in HLM from compounds 1−3. As shown in Table 3, preincubation with 1-ABT inhibited 80−100% sulfoxide and sulfone formation, although FMO-mediated N-oxidation for a positive control compound, benzydamine, was inhibited by over 95% under the same conditions. These data suggested that mainly cytochrome P450s are involved in the NADPH-dependent sulfoxidation (step 1) for the test compounds used in current studies, while the contribution from FMOs appears to be minor. Structure−Bioactivation Relationship. To further extend the investigation of GSH adduct formation for 2-(alkylthio)1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles, we incubated a dozen analogues of compounds 1−3 with HLM supplemented with NADPH and GSH for 75 min and analyzed the incubation samples by UHPLC/HRMS. The amount of GSH adduct formation was expressed by percentage of peak area of the GSH adduct as total peak area from all of the peaks detected in LC/UV chromatograms (Tables 4 and 5). All tested 4,5substituted-2-(alkylthio)-1,3,4-thiadiazoles (compound 1 analogues) showed robust GSH adduct formation ranging from 20 to 40%, and 5-substituted-2-(alkylthio)-1,3,4-thiadiazoles (compound 2 analogues) had larger variation in its GSH adduct formation. While analogues with aromatic ring structure substitution at 5-positions have 30−80% GSH adduct formation, the analogues with linear structure substitutions had much less amount of GSH adduct formation (≤4%). Table 5 summarizes the GSH adduct formation of 2-(alkylthio)-benzothiazoles (compound 3 analogues). All of the analogues tested in this group showed significant amount of GSH adduct formation (16−100%). The proposed GSH adduct formation mechanism for these compounds suggests that the amount of final GSH adduct depends primarily upon the amount of sulfoxide and sulfone metabolized from the sulfide moiety. For example, while compound 3 was completely converted to sulfoxide and sulfone after 75 min of incubation with HLM in the presence of NADPH (Figure 3E), only 30% of compound 1 was metabolized (Figure 2C) with much less sulfoxide and sulfone formation. The observed GSH adduct formation from compound 1 was consequently far less than that of compound 3. In a different scenario, if fast metabolic turnover of a parent compound is due to the presence of another “metabolically softer” group that shifts the metabolism away from the sulfide moiety, a net smaller fraction of formation of sulfoxide/ sulfone intermediates as a part of total metabolism would result in less GSH displacement products. As shown with compound 2 in Figure 3, the formation of two nonsulfoxidation metabolites at RT 6.3 min and RT 6.8 min (Figure 3B), which cannot be displaced by GSH, competed with the sulfoxide/sulfone formation from compound 2 and led to less GSH adduct formation. The sulfoxide and sulfone leaving group property could come into play as well.

*

n = 3.

Table 5. GSH Adduct Formation of Different Benzothiazole Analogues

*

n = 3.

This can be illustrated by the differences in the amount of sulfoxide/ sulfone still detected in the presence of GSH (Table 2). Most of the H

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sulfoxides and sulfones were completely displaced by GSH, while some of them show slow displacement reaction, as shown by 1M1 and 1M2. In addition, the degree of electron deficiency of the heterocycles, when combined with a robust sulfoxide or sulfone leaving group, could enhance the formation of GSH adducts. For instance, in the case of 3C and 3D in Table 5, larger amounts of GSH adducts were formed when electron-withdrawing group CHF2SO2- or NO2- were present on the benzothiazole system, whereas smaller amounts of GSH adduct was formed when electron-donating group NH2- (3B) was present.



CONCLUSIONS We have reported a new bioactivation pathway common to 5-substituted 2-(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)1,3-benzothiazoles, which can be added to the growing list of structural alerts for bioactivation. High-resolution MS data suggested that the GSH adduct formed from this bioactivation pathway is a result of displacement of the alkyl sulfide moiety by GSH, and the structure was confirmed by NMR data. The proposed mechanism for this GSH adduct formation consists of two steps: (1) enzymatic oxidation of alkylthio group to form the sulfoxide and sulfone and (2) nucleophilic displacement of the sulfoxide and sulfone by GSH. Investigation of this bioactivation pathway on more than a dozen commercially available compounds containing 5-substituted 2-(alkylthio)-1,3,4-thiadiazole and 2-(alkylthio)-1,3-benzothiazoles structural motifs indicated that this pathway is widespread in these chemotypes. These findings can assist medicinal chemists in making necessary chemical modifications and applying thoughtful drug design to avoid potential liability related to 2-(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles. Should they be incorporated in drug candidate molecules, the extent of GSH adduct formation ought to be carefully examined in conjunction with broader toxicological and drug safety profiles.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ABBREVIATIONS 1-ABT, 1-aminobenzotriazole; FMO, flavin monooxygenases; UHPLC/HRMS, ultra high-pressure liquid chromatography/highresolution accurate mass spectrometry; HLM, pooled human liver microsomes; HMQC, hetronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond coherence



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dx.doi.org/10.1021/tx3003998 | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX