Forced Oxidative Degradation Pathways of the Imidazole Moiety of Daclatasvir

Journal of Pharmaceutical Sciences 108 (2019) 3312-3318

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Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Forced Oxidative Degradation Pathways of the Imidazole Moiety of Daclatasvir Yande Huang*, Bao-Ning Su, Jonathan Marshall, Scott A. Miller Chemical & Synthetic Development, Global Product Development and Supply, Bristol-Myers Squibb Company, New Brunswick, New Jersey 08901

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 April 2019 Revised 14 May 2019 Accepted 17 May 2019 Available online 27 May 2019

Daclatasvir hydrochloride (DCV) is the active pharmaceutical ingredient of Daklinza, a marketed product for the treatment of hepatitis C viral infection. The intrinsic stability of daclatasvir was evaluated via a forced degradation study. DCV was found to be stable in the solid state. In solution, its carbamate moiety is susceptible to basic hydrolysis, whereas its imidazole is liable to base-mediated autoxidation to form degradants 1 and 3, 7-8, respectively. The imidazole moiety can also be oxidized to form degradants 6-7 in the presence of hydrogen peroxide or azobisisobutyronitrile. The chloro-adduct degradant 9 was also observed in hydrogen peroxide solution. Furthermore, the imidazole moiety is sensitive to photodegradation in solution. Degradants 2-8 were observed in a solution of DCV exposed to high intensity light/UV light; the formation of degradants 2 and 5-8 was postulated through 4 degradation pathways. The degradants 3 and 4 were deemed to be secondary degradants of 7 and 5, respectively. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Keywords: forced conditions oxidation(s) photodegradation degradation products stability nuclear magnetic resonance (NMR) spectroscopy liquid chromatographyemass spectrometry (LC-MS)

Introduction Forced degradation is widely used during pharmaceutical development to study the intrinsic stability and potential degradation pathways of active pharmaceutical ingredients (APIs) and drug products (DPs) as a valuable tool to facilitate stability indicating analytical method development and to satisfy regulatory requirements. However, as the exact forced degradation conditions are not specified in regulatory guidance,1 developers need some freedom to conduct forced degradation experiments that would be appropriate for their APIs and DPs. Alsante et al.2 have established a roadmap for when and how to conduct relevant experiments, such as acid and base hydrolysis, higher temperature and humidity, oxidation, and photostability in the course of pharmaceutical development. Additionally, the regulatory guidance1 states that it

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Current address for Dr Su: WuXi AppTec Co., Ltd, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.05.022. * Correspondence to: Yande Huang (Telephone: þ1-732-227-7405). E-mail address: [email protected] (Y. Huang).

may not be necessary to examine certain degradation products if it has been demonstrated that they were not formed under accelerated or long-term storage conditions. It is generally true that forced degradation will generate more degradants than those from accelerated or long-term storage conditions. In contrast, certain degradants observed from accelerated or long-term storage conditions may not be formed during a forced degradation study because it is not easy to mimic certain microenvironments. On the other hand, the accelerated or long-term stability data may not be available at early stages of development. Therefore, it would be appropriate to conduct a thorough forced degradation investigation to generate indepth stability knowledge of APIs and DPs to aid in DP formulation design and the selection of appropriate packaging and storage conditions to minimize the potential risk of DP stability liability. Daclatasvir hydrochloride (DCV) is a direct-acting antiviral agent specifically targeting the hepatitis C virus (HCV) NS5A replication complex. It has been marketed as Daklinza, usually in combination with other antiviral agents, such as sofosbuvir,3 asunaprevir,4 and beclabuvir,5 for the treatment of HCV infections. The dimeric structure of DCV shown in Figure 1 consists of 2 identical phenylimidazole moieties, 2 amide, and 2 carbamate functional groups, which might be susceptible to degradation. During the pharmaceutical development of DCV, forced degradation experiments were conducted to aid in developing a stability indicating analytical HPLC

https://doi.org/10.1016/j.xphs.2019.05.022 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Y. Huang et al. / Journal of Pharmaceutical Sciences 108 (2019) 3312-3318

method; the results are summarized in Table 1. It was found that DCV was stable under the stress conditions of 80 C/75% relative humidity, 80 C, high intensity light/UV light (solid), and 0.1N HCl solution for 7 days. When DCV was stressed in a basic solution of 0.01N NaOH for 7 days, degradants 1, 3, 7, and 8 were observed. Under oxidative degradation conditions, degradants 6, 7, and 9 were detected in a solution of 0.3% H2O2 at ambient temperature for 7 days, whereas degradants 6, 7, and 10 were observed in a solution of azobisisobutyronitrile (AIBN) at 40 C for 2 days. When a solution of DCV at 1 mg/mL in acetonitrile (ACN)/water (1:1 v/v) was exposed to high-intensity light/UV light for 8h, degradants 2-7 were generated. To delineate the degradation pathways of DCV, the structures of degradants 1-10 were elucidated. It is worth mentioning that accurate mass liquid chromatographyemass spectrometry (LC-MS/ MS) with the aid of authentic markers was sufficient for definitive structure elucidation. In this study, degradants 1-2 and 9 were identified via accurate mass LC-MS/MS and comparison with their synthetic markers. Degradant 10 was deemed an artifact derived from the reaction of DCV with AIBN radical initiator based on its accurate mass LC-MS/MS data (vide infra). As the markers of degradants 3-8 were not available for comparison, it was essential to isolate each individual degradant from the stressed solutions for nuclear magnetic resonance (NMR) structure elucidation. Therefore, a solution of DCV at a higher concentration (15 mg/mL) was irradiated for a longer time (30 h) to enhance the formation of the degradants 3-8 significantly, as shown in Table 1 (entry 10). In this article, details of the isolation and structure elucidation of degradants 3-8 are presented. It is interesting to observe degradants 5 (benzaldehyde) and 7 (ketoimide) from the photooxidative degradation of the imidazole moiety of DCV. Such structures have not previously been reported from the in-depth studies of the photosensitized oxygenation of imidazole derivatives in the literature.6-12 Degradant 9 was formed through the reaction of DCV with its chloride counter-ion mediated by hydrogen peroxide. It was somewhat surprising to observe photodegradants 3, 7, and 8 in basic hydrolysis solution. We postulate this is via autoxidation of DCV, which is accelerated under basic conditions. The plausible forced degradation mechanisms of the imidazole moiety of DCV to form degradants 2-9 are proposed.

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method are shown in Supporting Information (SI-1). To enrich degradants 3-8 for isolation and structure elucidation, DCV (750 mg) dissolved in 50 mL of a solution of ACN/H2O (1:1 v/v) was dispensed into 5 20-mL Scintillation vials (each with 10 mL headspace), capped, and stressed in an Atlas light box (Atlas Materials Testing Technology LLC., Chicago, IL) at an energy level of 900 KJ/m2 h for 30 h. The solutions were combined for HPLC analysis and isolation. Analytical HPLC Method for Scale-Up Isolation A new analytical HPLC method, with shorter run time compared with the stability indicating method shown in Supporting Information SI-1, was developed for scale-up to separate degradants 2-8 in the photostressed solution. A Shimadzu LC-10AD separation module equipped with a Shimadzu SPD-10A UV/Vis detector (Shimadzu Corporation, Columbia, MD) and coupled with a Micromass ZQ single quadrupole mass detector (Waters, Milford, MA) and operated by Shimadzu ClassVP software was used for this separation. A SunFire C18 column (4.6 mm  50 mm, 2.1 mm particle size; Waters) was used at ambient temperature. The flow rate was set at 1.0 mL/min using mobile phases A (water/ACN/TFA, 1900:100:1 v/v/v) and B (water/ACN/TFA, 100:1900:1 v/v/v). The mobile phase gradient was set as follows: linear from 20% B to 50% B in 10 min. The column was flushed with 100% B and fully equilibrated through the Shimadzu ClassVP programming after each injection. Injection volume was 10.0 mL of diluted photolysis solution (0.03 mL diluted to 1.5 mL with water/ACN,1:1 v/v). UV detection was set at 306 nm. The MS data were acquired in positive electrospray ionization (ESI)(þ) mode with a flow rate of 0.4 mL/min split to the mass detector. The mass spectrometer settings were capillary voltage 3.3 kV, cone voltage 30 V, source temperature 125 C, desolvation temperature 275 C, and desolvation gas flow 550 L/h. Preparative HPLC Method

DCV and degradants 1-2 and 9 markers were synthesized by the Chemical & Synthetic Development Department, Bristol-Myers Squibb (New Brunswick, NJ). HPLC grade of water (HPLC plus), ACN (for HPLC, gradient grade), and methanol (MeOH, UHPLC, for mass spectrometry), as well as DMSO-d6 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Trifluoroacetic acid (TFA) was obtained from Thermo Scientific (Rockford, IL).

A Shimadzu preparative HPLC equipped with 2 LC-8A pumps, a SCL-8A System controller, a SPD-10A UV-Vis detector, a FRC-10A Fraction collector, and a SIL-10A autoinjector controlled with Shimadzu Class VP software (Shimadzu Corporation, Columbia, MD) was used for isolation of the degradants 3-8. The autoinjector was equipped with a 5.0 mL sample loop. A Sunfire Prep C18 OBD column (19  150 mm, 5 mm particle size, Waters) was employed at ambient temperature. The flow rate was set at 17 mL/min using mobile phases A (water/TFA, 2000:1 v/v) and B (ACN/TFA, 2000:1 v/v). The mobile phase gradient was set as follows: linear from 20% B to 50% B in 10 min. The column was flushed with 100% B and fully equilibrated through the Shimadzu ClassVP programming after each injection. Injection volume was 1000 mL of concentrated photolysis solution (rotary evaporated to remove most of the ACN and filtered through a 0.45 mm filter). The UV detection was set at 306 nm.

Forced Degradation Experiments

Isolation of Degradants 3-8

The forced degradation conditions for experiments listed in Table 1 and the pertinent stability indicating analytical HPLC

A typical LC-UV chromatogram of the photolysis solution using the newly developed method is depicted in Figure 2. The mass spectrometry data were used to assign peaks to degradants 2-8 based on their molecular weight. The elution order of degradants 2-8 was the same as that (Table 1) using the stability indicating HPLC method except that degradant 3 and DCV was reversed. Furthermore, degradants 4 and 5 were well resolved in this new method. When scaled-up for preparative HPLC isolation, however, degradant 3 overlapped with DCV owing to overloaded sample on the column and was not collected. A typical preparative HPLC chromatogram is shown in Supporting Information SI-2. Degradants

Experimental Materials

Figure 1. Structure of DCV.

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Table 1 Degradation Profiles of DCV Under Forced Degradation Conditions on Stability Indicating HPLC Methoda Condition/Time

1b

2

3

4&5c

6

7

8

9

10

1. Control 2. Heat (80 C/75% RH, 7d) 3. Heat (80 C, 7d) 4. UV light, solid (7d) 5. Acid (0.1 N HCl 7d) 6. Base (0.01N NaOH 7d) 7. Hydrogen Peroxide (0.3%, 7d) 8. AIBN (40 C, 2d) 9. UV light solution-1 (8h) 10. UV light solution-2 (30h)

ed e e e e 0.62 e e e e

e e e e e e e e 0.22 1.41

e e e e e 3.11 e e 0.52 0.41

e e e e e e e e 0.57 10.67

e e e e e e 0.27 4.28 1.29 4.94

e e e e e 0.19 0.12 0.32 2.18 5.62

e e e e e 0.18 e e e 0.46

e e e e e e 0.26 e e e

e e e e e e e 2.41 e

AIBN, azobisisobutyronitrile; DCV, daclatasvir hydrochloride; RT, retention time. a All values of degradants 1-10 are reported as area % by HPLC. (see Supporting Information SI-1 for detail experimental conditions). b 1 (RT ¼ 7.22 min, m/z ¼ 681); 2 (RT ¼ 9.62 min, m/z ¼ 490); DCV (RT ¼ 11.63 min, m/z ¼ 739); 3 (RT ¼ 13.67 min, m/z ¼ 519); 4 (RT ¼ 15.42 min, m/z ¼ 491); 5 (RT ¼ 15.42 min, m/z ¼ 475); 6 (RT ¼ 19.34 min, m/z ¼ 744); 7 (RT ¼ 19.92 min, m/z ¼ 772); 8 (RT ¼ 22.16, min, m/z ¼ 753); 9 (RT ¼ 22.54 min, m/z ¼ 773), 10 (RT ¼ 22.65 min, m/z ¼ 822). c 4&5 co-eluted (RT ¼ 15.42 min). d Either not detected or less than reporting limit of 0.05%.

4-8 were well resolved and collected in individual fractions 1-5. Fractions from multiple injections were pooled and freeze-dried to afford 3-20 mg of degradants 4-8 as fluffy solids. For the sake of mechanistic investigation, the stability of the isolated degradants 6 and 7 was further evaluated by diluting the NMR samples with ACN/ water (1:1 v/v). The resulting solutions were placed in a 50 C oven for 17 h and monitored by LC-MS. It was found that degradant 6 was stable (Supporting Information SIs 3 and 4), whereas degradant 7 was partially hydrolyzed to form degradant 3. (Supporting Information SIs 5 and 6). Therefore, degradant 3 was isolated from the hydrolysis of the degradant 7, as described below. The pooled fraction 4 (degradant 7) from a repeated experiment was transferred to 2 40-mL scintillation vials, capped, and placed in a 50 C oven for 24 h. Under such conditions with the presence of 0.05% TFA, degradant 7 was significantly hydrolyzed to form degradant 3. The resulting solutions were combined and freeze-dried. The lyophile was reconstituted with ACN/water (1:1 v/v) for the isolation of degradant 3 using the same preparative HPLC method. A typical HPLC chromatogram is shown in Supporting Information SI-7. Fractions from multiple injections were pooled and freeze-dried to afford 3.4 mg of degradant 3 as a yellowish solid. Accurate Mass LC-MS Method The accurate mass LC-MS analysis was performed on a Waters Acquity UPLC system coupled with a Thermo Orbitrap mass spectrometer and operated by Xcaliber software (Thermo Fisher

Scientific Inc., Waltham, MA). The isolates of degradants 3-8 were analyzed via a loop-injection with a flow rate of 1.0 mL/min using an isocratic elution of 50% mobile phase A (water/TFA, 2000:1 v/v) and 50% B (ACN/TFA, 2000:1 v/v) for 1.0 min. The MS data were acquired in ESI(þ) mode with a flow rate of 0.4 mL/min split to the mass detector. The mass spectrometer settings were spray voltage 3.5 kV, sheath gas flow 20 units, auxiliary gas flow 2 units, capillary temperature 275 C, probe heater temperature 100 C, S-lens RF level 50 V, and a resolution of 70 k.

NMR Analysis The 1-D and 2-D NMR spectra with 1H detection were acquired on a Bruker Avance II 600 NMR spectrometer using a Bruker 5-mm inverse triple resonance CryoProbe with a z-gradient (Bruker Biospin Inc., Billerica, MA). 13C NMR spectra were obtained on a Bruker Avance 500 NMR instrument using a 3-mm Broadband Observe (BBO) probe. All experiments were conducted at 25 C. The isolates (3-20 mg) of degradants 4-8 were dissolved in DMSOd6 (0.2-0.25 mL) and placed in 3-mm NMR tubes. For degradant 3, neat TFA (2 mL) was added to DMSO-d6 sample solution to sharpen and better resolve the proton peaks around the imidazole moiety. The 1H and 13C chemical shift values are reported relative to DMSO-d6 (d ¼ 2.49 and 39.5 ppm, respectively). The 15N NMR chemical shifts are externally referenced to liquid ammonia (NH3) at d 0.0.

0 0

1

2

3

4

Wavelength = 306 nm; ESI(+) Pk # 1 2 3 4 5 6 7 8

Retention Time 2.01 2.55 3.43 4.24 5.79 6.66 7.23 7.82

Name Deg-2 Deg-3 DCV Deg-4 Deg-5 Deg-6 Deg-7 Deg-8

m/z 490 519 739 491 475 744 772 753

5 Minutes

Area 39583 11437 2142896 245338 55018 139019 158204 12817

6

7

7.82

7.23

6.66

5.79

3.43

2.55

2.01

50

4.24

100

8

9

1

Area Percent 1.406 0.406 76.124 8.715 1.954 4.939 5.620 0.455

Figure 2. A typical LC-UV/MS chromatogram of a photolyzed solution of DCV for 30h acquired on the analytical method developed for scale-up isolation.

Y. Huang et al. / Journal of Pharmaceutical Sciences 108 (2019) 3312-3318

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Table 2 Accurate Mass and Key 1H,15N, and13C NMR Chemical Shifts (ppm) of Degradants 3-8 Compound

3

4

5

6

7

8

DCV

Empirical formula: Theory: [M þ H]þ Found: [M þ H]þ H-4 NH-1 NH-3 N-1 N-3 N-7 N-13 C-2 C-4 C-5

C28H31N4O6 519.2238 519.2244 e e e e e e e e 188.2 166.1

C27H31N4O5 491.2289 491.2284 e e e e e e e e 167.1 e

C27H31N4O4 475.2340 475.2331 10.05 e e e e e e e 192.7 e

C39H50N7O8 744.3715 744.3704 e

C40H50N7O9 772.3665 772.3658 e 12.22

C40H49N8O7 753.3719 753.3716 e 10.40

164.1

143.3

131.8 87.4 173.4 187.4 169.9

125.6 107.6 89.5 157.8 163.8

C40H51N8O6 739.3926 739.3928 e 15.5a 15.0a 171.0b 175.5b 131.3 85.3 149.3 126.4 114.9

11.20 155.7 132.2 88.1 172.9 166.1 e

NMR, nuclear magnetic resonance. a Interchangeable assignments. b Interchangeable assignments.

Results and Discussion Structure Elucidation of Degradants 3-8 The empirical formulae of degradants 3-8 were determined via accurate mass LC-MS analysis of the isolated samples, as shown in Table 2. The structures of degradants 3-8 depicted in Figure 3 were elucidated by interpretation of 1D 1H and 13C and 2D COSY, 1H-13C HSQC, 1H-13C heteronuclear multiple bond correlation (HMBC), and 1 H-15N HMBC NMR data (Supporting Information SIs 8-45 and Tables S1-S18). The key 1H, 13C, and 15N chemical shifts reflecting the changes of degradants 3-8 from the imidazole moiety of DCV are listed in Table 2. Degradant 3 was assigned as a pyruvic acid based on its chemical shifts of C-4 (188.2 ppm) and C-5 (166.1 ppm). The chemical shift of C-4 (167.1 ppm) of degradant 4 is consistent with a benzoic acid structure. The aldehyde moiety of degradant 5 was clearly established with the chemical shifts of H-4 (10.05 ppm) and C-4 (192.7 ppm). The imide structure of degradant 6 was assigned based on the chemical shifts of NH-3 (11.20 ppm), C-2 (172.9 ppm), C-4 (166.1 ppm), and N-3 (155.7 ppm). The ketoimide structure of degradant 7 was deduced from the characteristic chemical shifts of NH-1 (12.22 ppm), C-2 (173.4 ppm), C-4 (187.4 ppm), C-5 (169.9 ppm), and N-1 (164.1 ppm). For degradant 8, the spirane structure is supported by 1 H-13C HMBC correlations of NH-1 (10.40 ppm), H-6 (4.31 ppm), H10b (1.01 ppm), and H-12 (4.34 ppm) to C-2 (89.5 ppm). The newly formed chiral center of degradant 8 was determined with the aid of 2D Rotating-frame Overhauser Enhancement SpectroscopY NMR data (Supporting Information SI-45), where NH-1 (10.40 ppm) showed NOE correlations with H-6 (4.31 ppm). Proposed Photooxidative Degradation Mechanism of DCV In-depth studies of photosensitized oxidation of aryl and alkyl substituted imidazoles are well documented in the literature.6-12 An unstable endoperoxide structure is usually postulated as the first intermediate from the [4 þ 2] cycloaddition of the imidazole moiety with singlet oxygen, which can then undergo further rearrangement to form a variety of degradants, including the hydroperoxides isolated from the reaction of triaryl substituted imidazoles with singlet oxygen.6,7 The first observation of a 2,5-endoperoxide of 1,4dimethylimidazole was reported by Ryang et al. using low temperature NMR spectroscopy.8 The hydroperoxides could further rearrange to form a putative dioxetane, which would decompose to form diaroylarylamidines.9 By using 13C and 15N isotopic labeling of a model compound (4,5-diphenylimidazole) under photosensitized conditions, Kang et al.10 observed a 2,5-endoperoxide structure as

the first transient intermediate by NMR spectroscopy at 100 C. The 2,5-endoperoxide was then decomposed to form a 2-hydroperoxide as the second transient intermediate at 88 C, which underwent dehydration to form an imidazolone at 80 C. Kai et al.11 conducted dye-sensitized photooxidation of 2,4-disubstituted imidazoles in methanol to form isomeric imidazolinones through a sequence of 2,5-endoperoxide to hydroperoxide and dioxetane to imidazolinones. Seburg et al.12 reported a postulated 2,5-endoperoxide of the 4-chloro-imidazole moiety of losartan intermediate could decompose to release cyanogen chloride via retro [4 þ 2] cycloaddition to facilitate the degradation of losartan in an extemporaneous suspension formulation. For DCV containing 2 identical 4-aryl-2-alkylimidazole moieties, only 1 of the 2 imidazole moieties is subjected to photooxidative degradation at low degradation of DCV (<25%). The photooxidative degradation of the imidazole moiety under high intensity light/UV light cannot be exclusively attributed to singlet oxygen oxidation. Other pathways, such as the reaction of the excited triplet state of DCV with ground state of triplet oxygen, cannot be unequivocally eliminated. Nevertheless, either pathway would lead to the formation of the putative 2,5-endoperoxide as the first intermediate depicted in Figure 4. It is worth mentioning that when DCV absorbs light energy, only 1 of the 2 imidazole moieties is likely excited to the triplet state. The other imidazole would putatively remain at ground state. Otherwise, the molecule would be doubly excited to an extremely high energetic state, which is highly unlikely. If the singlet oxygen reacts with 1 of the 2 imidazole moieties at the ground state, the probability of the other imidazole moiety reacting with another singlet oxygen is less likely at low overall degradation of DCV. The unstable 2,5-endoperoxide would rearrange to 5hydroperoxide, which could either undergo pathway a of dehydration to form an imidazolone intermediate, followed by intramolecular Michael addition to yield degradant 8 or cyclize to form the putative dioxetane intermediate. The dioxetane could decompose through pathways b-d to yield degradants 2-7. In pathway b, the hydride shift from C-5 to C-4 position is postulated as the key step, although the driving force of the hydride shift is not well understood, leading to the formation of the aldehyde (degradant 5). Degradant 5 is further oxidized to the carboxylic acid (degradant 4). The formation of degradants 2 and 6 can be attributed to the 4membered dioxetane ring opening through pathway c. Degradant 2 is highly unlikely to be formed from the hydrolysis of imide degradant 6 because isolated degradant 6 in a solution of ACN/ water is stable at 50 C for 17h (Supporting Information SIs 3 and 4). In pathway d, it is postulated that the breaking of the peroxide bond of the dioxetane along with the 5-membered heterocycle ring

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Figure 3. Structures of degradants 1-10 and their formation conditions.

opening is initiated by the leaving of the acidic proton H-5, resulted in the formation of the ketoacylamidine intermediate, which hydrolyzes to yield ketoimide degradant 7. Degradant 7 is readily hydrolyzed to form degradant 3, as evidenced by the follow-up stability study of the isolated degradant 7 (Supporting Information SIs 5 and 6). Degradant 3 was detected at 8.6 area% initially in a diluted NMR sample of degradant 7 and increased substantially to 22.0% after 17 h at 50 C.

Proposed Autoxidation Mechanism of DCV Under Basic Conditions As shown in Table 1, not only the hydrolytic degradant 1 but also the oxidative degradants 3, 7, and 8 were detected under basic conditions. It is postulated that the autoxidation of DCV can form the same 5-hydroperoxide and dioxetane intermediates as those under photolysis shown in Figure 4. The basic conditions would accelerate the dehydration of the 5-hydroperoxide in pathway a to

Y. Huang et al. / Journal of Pharmaceutical Sciences 108 (2019) 3312-3318

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Figure 4. Proposed photodegradation mechanisms of DCV.

form degradant 8. They also favor the deprotonation (H-5) of the dioxetane intermediate in pathway d, leading to the formation of degradants 3 and 7. Proposed Oxidative Degradation Mechanisms of DCV Mediated by Hydrogen Peroxide and AIBN Degradants 6 and 7 were also observed under hydrogen peroxide and AIBN conditions. A free radicaleinitiated mechanism can be

postulated (Supporting Information SIs 46 and 47) to form the same dioxetane intermediate depicted in Figure 4, which would undergo degradation pathways c and d to afford degradants 6 and 7, respectively. Additionally, degradant 9 was detected under hydrogen peroxide, whereas degradant 10 appeared under AIBN conditions. A plausible formation mechanism for degradant 9 is illustrated in Figure 5. The chloride counter-ion in DCV could react with hydrogen peroxide to form hypochlorous acid, which could react with DCV to afford degradant 9. In fact, the degradant 9 marker

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Figure 5. Proposed formation mechanism of degradant 9.

was synthesized via the reaction of DCV with N-chlorosuccinimide (data not shown), which would proceed via a similar mechanism as the one proposed for the formation of degradant 9. Degradant 10 was deemed to be an artifact based on the accurate mass LC-MS/MS data (Supporting Information SIs 48-50), where the loss of a key component, C4H6N, in MS2 experiment apparently derived from the AIBN-free radical initiator, (CH3)2CCN. Therefore, the definitive structure elucidation of degradant 10 was not conducted.

Acknowledgments

Conclusion

References

In-depth intrinsic stability knowledge of DCV has been acquired through forced degradation studies. The solid state of DCV was deemed stable under thermal and photostressed conditions. DCV was also stable in solution under acidic conditions. However, in basic solution, the carbamate functional group of DCV is susceptible to base hydrolysis to form degradants 1. Additionally, autoxidation of the imidazole moiety of DCV was accelerated under basic condition to form degradants 3, 7-8. The oxidative degradants 6-7 were also formed in solution in the presence of hydrogen peroxide or AIBN. Degradant 9, a chlorinated DCV, was derived from the reaction of DCV with its chloride counter-ion mediated by hydrogen peroxide. Degradants 2-8 were observed in a solution of DCV exposed to high intensity light/UV light. Four plausible photodegradation mechanisms initiated by the oxygenation of the imidazole moiety of DCV were postulated to delineate such transformations. Degradants 3 and 4 were confirmed to be secondary degradants of 7 and 5, respectively. In contrast, degradant 2 was deemed to be concurrently formed along with 6 via degradation pathway c rather than a secondary degradant of 6. The elucidation of the structure of degradant 8 as a spirane instead of its putative imidazolone precursor was unexpected. Overall, such intrinsic stability knowledge of DCV are valuable for making scientifically informed decisions on formulation design, DP packaging, and storage conditions.

The authors would like to acknowledge the daclatasvir project team members for their collaborations, especially Dr. Li Li for conducting stability indicating method development, and Drs. James Chadwick and Mark S. Bolgar for manuscript review and discussions.

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