TOF, LC–MSn, NMR and LC–NMR in characterization of stress degradation products: Application to cilazapril

Accepted Manuscript Title: Use of LC-MS/TOF, LC-MSn , NMR and LC-NMR in characterization of stress degradation products: Application to cilazapril Author: Mallikarjun Narayanam Archana Sahu Saranjit Singh PII: DOI: Reference:

S0731-7085(15)00215-0 http://dx.doi.org/doi:10.1016/j.jpba.2015.03.038 PBA 10031

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

29-9-2014 26-3-2015 29-3-2015

Please cite this article as: M. Narayanam, A. Sahu, S. Singh, Use of LC-MS/TOF, LC-MSn , NMR and LC-NMR in characterization of stress degradation products: Application to cilazapril, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.03.038 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.

Highlights Forced degradation of cilazapril carried out according to ICH and WHO guidelines.

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Seven unknown degradation products detected by HPLC.

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Five hydrolytic products identified by using LC/TOF-MS, LC-MSn and LC-NMR.

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Two isolated oxidative degradants characterized through MS and NMR.

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Pathway and mechanism of degradation of the drug outlined.

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Use of LC-MS/TOF, LC-MSn, NMR and LC-NMR in characterization of

Mallikarjun Narayanama, Archana Sahub, Saranjit Singhb*

Present address: Biocon-BMS R&D Centre (BBRC), Biocon Park, Bangalore, India

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a

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stress degradation products: Application to cilazapril

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Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India

*Corresponding author. Tel.: +91-172-2292031; fax: +91-172-2214692 e-mail: [email protected] (Saranjit Singh)

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Abstract Forced degradation studies on cilazapril were carried out according to ICH and WHO guidelines.

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Significant degradation of the drug was observed in acid and base conditions, resulting primarily in cilazaprilat. In neutral conditions, five degradation products were formed, while under

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oxidative condition, two degradation products were generated. In total, seven degradation

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products were formed, which were separated on an Inertsil C-18 column using a stabilityindicating HPLC method. Structure elucidation of the degradation products was done LC-MS/TOF, LC-MSn, on-line H/D

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by using sophisticated and hyphenated tools like,

exchange, LC-NMR and NMR. Initially comprehensive mass fragmentation pathway of the drug

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was laid down. Critical comparison of mass fragmentation pathways of the drug and the hydrolytic degradation products allowed structure characterization of the latter. 1D and 2D

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proton LC-NMR studies further confirmed the proposed structures of hydrolytic degradation

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products. The oxidative degradation products could not be characterized using LC-MS and LC-

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NMR tools. Hence these degradation products were isolated using preparative HPLC and extensive 1D (1H,

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C, DEPT) and 2D (COSY, TOCSY, HETCOR and HMBC) NMR studies

were performed to ascertain their structures. The degradation pathways and mechanisms of degradation of the drug were outlined.

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Keywords: Cilazapril, stress studies, degradation products, LC-MS/TOF and MSn, LC-NMR, NMR 1. Introduction

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Stress testing is an integral part of the drug development process, aimed at understanding of the intrinsic stability behaviour of the drug, identification of its degradation products and

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establishing the degradation pathway. The ICH stability testing guideline Q1A(R2) [1] requires

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stress testing of drugs to be performed under hydrolysis, oxidation, thermal and photoconditions. In this study, degradation behaviour of cilazapril was investigated by using LC-MS [2-4] and

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LC-NMR [5-8] tools. To elucidate the structures of oxidative degradation products, the latter were isolated, and subjected to 1D and 2D NMR studies.

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Cilazapril (CL) is an angiotensin converting enzyme inhibitor (ACE) used in the treatment of

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hypertension and congestive heart failure. It is a prodrug that is converted to an active

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metabolite, cilazaprilat, upon hydrolysis. There is no report on the degradation behaviour of cilazapril according to ICH [1] and WHO [9] regulatory guidelines. Hence, the aim of the

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present study was to identify and characterize its degradation products, to establish its degradation pathway, and propose mechanistic explanation for the same. These studies are important with respect to building up of QbD knowledge space [10], which requires information on all possible degradation products of a drug. The knowledge space requirement is also well highlighted in ICH guideline in the statement ‘This discussion can be limited to those impurities that might reasonably be expected based on knowledge of the chemical reactions and conditions involved’ [11]. Even the WHO latest stability testing guideline [9] clearly mentions that ‘For an API, when available, it is acceptable to provide the relevant data published in the scientific

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literature to support the identified degradation products and pathways’. A similar statement exists in EMEA guideline on stability testing of existing drug substances and products [12].

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2. Experimental 2.1. Drug and reagents

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Pure CL was obtained as a gift sample from Ranbaxy Research Laboratories (S.A.S. Nagar,

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India). Analytical reagent (AR) grade sodium hydroxide (NaOH) was purchased from RFCL Ltd. (New Delhi, India), hydrochloric acid (HCl) from LOBA Chemie Pvt. Ltd. (Mumbai, India) and

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hydrogen peroxide (H2O2) from s.d. Fine-chem Ltd. (Boisar, India). Buffer salts and all other chemicals of AR grade were bought from local suppliers. HPLC grade acetonitrile (ACN) was

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procured from J.T. Baker (Phillipsburg, NJ, USA). Deuterated acetonitrile (CD3CN), methanol

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(CD3OD) and water (D2O) of 99.9% purity were obtained from Aldrich (California, Missouri,

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Wycombe, England).

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USA). Water for HPLC studies was obtained from ultra-pure water purification unit (Elga,

2.2. Apparatus and equipments

Precision water baths equipped with MV controller (Julabo, Seelbach, Germany) were used for degradation studies in the solution state. A Dri-Bath (Thermolyne, Iowa, USA) was used for solid-state thermal stress studies. Photostability studies were carried out in a chamber (KBWF 240, WTC Binder, Tuttlingen, Germany) set at 25 °C and ambient humidity. It was equipped with an illumination bank on inside top, consisting of a combination of two UV (OSRAM L18 W/73) and four white fluorescent (Philips, Trulite) lamps, as recommended by ICH guideline Q1B [13]. A lux meter (model ELM 201, Escorp, New Delhi, India) and a near UV-A radiometer

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(model 206, PRC Krochmann GmbH, Berlin, Germany) were used to measure visible illumination and near UV energy, respectively.

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pH/Ion analyzer (MA 235, Mettler Toledo, Schwerzenbach, Switzerland) was used to check and adjust pH of the buffers solutions. Other equipments used were sonicator (3210, Branson

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Ultrasonics Corporation, Danbury, Connecticut, USA), precision analytical balance (AG 135,

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Mettler Toledo, Schwerzenbach, Switzerland) and autopipettes (Eppendorf, Hamburg, Germany).

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The separation of the drug and its degradation products was achieved using a liquid chromatograph (LC), which was equipped with a photodiode array detector and was controlled

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by CBM-20A software, version 3 (LC-Solution series, Shimadzu, Kyoto, Japan). An Inertsil

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ODS-3V C-18 column (250 mm x 4.6 mm i.d., particle size 5 μ) (LCGC Chromatography

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Solution, Hyderabad, India) was employed for the analysis of the stressed samples.

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MS/TOF and LC-MS/TOF studies were carried out by using LC-micrOTOF-Q-MS (Bruker Daltonik, Bremen, Germany), in which LC part consisted of 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) comprising of an on-line degasser (G1379A), binary pump (G131A), auto injector (G1313A), column oven (G1316A) and PDA detector (G1315B). The controlling software used was Hystar (version 3.1) from Bruker. All masses were corrected by the use of 5 mM sodium formate calibrant solution. For MSn experiments, LTQ-XL-MS 2.5.0 (Thermo, CA, USA) instrument was used. The mass spectra were acquired and processed using Xcalibur software (version 2.0). The mass parameters used were: mass range, 50-1000 amu; vaporizer temperature, 200 °C; helium gas flow rate (used

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to improve the ion trapping efficiency), 0.5 ml/min; scan rate for product ions, 11000 amu/s; and sample infusion flow rate, 10 l/min.

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NMR and LC-NMR spectra of the drug and the degradation products were recorded by using JEOL ECA 500 MHz spectrometer. It was equipped with Oxford superconducting magnet and 5

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mm BBO probe along with z axis. The data were acquired and processed using Delta software

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(version 4.6).

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2.3. Stress studies

These studies were carried out under hydrolytic, oxidative, photolytic and thermal stress

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conditions according to ICH [1] and WHO [9] recommendations. Acidic and alkaline hydrolytic studies were carried out in different molar concentrations of HCl and NaOH, while neutral

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hydrolysis was performed in a mixture of water and ACN (50:50). The drug concentration was 2

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mg/ml, while the temperature of the study was 80 °C. The oxidative study was carried out in

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15% H2O2 and 10 mole% AIBN at room temperature and 40 °C, respectively. Photodegradation studies were carried out by exposing the solid drug in the form of a thin layer and also drug solutions prepared in 0.1N HCl, 0.01N NaOH and equal mixture of H2O and ACN (2 mg/ml). Dark controls were kept concurrently for comparison. Thermal stress testing was carried out by sealing the drug in a glass ampoule and heating the same in a thermostatic block at 60 °C for 21 d. The stressors, choice of their concentration and preparation of samples were based on our previous publications [14, 15].

2.4. Preparation of samples for HPLC analyses

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At suitable time intervals, samples were withdrawn and appropriately neutralized and diluted twice with the solvent before injecting into HPLC. The final drug concentration was 1 mg/ml.

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The injection volume was 10 µl in all the cases.

2.5. HPLC method optimization

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To achieve an acceptable separation of the drug and the degradation products in the stressed

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samples, the components of the mobile phase, their proportions, buffer concentration, pH, flow rate and column temperature were duly optimized. A desirable separation and resolution was

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obtained using a mobile phase composed of ACN and 10 mM phosphate buffer (pH 3.0), which

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was run in a gradient mode at a flow rate of 1 ml/min and temperature of 30 ºC (Table S1). 2.6. MS/TOF, MSn and H/D exchange studies on the drug

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In order to get a complete fragmentation pattern of the drug, MS/TOF and MSn studies were performed in an ESI positive mode. Various MS/TOF parameters were sequentially altered to

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obtain initially the molecular ion peak of the drug and then its fragments. The optimized MS/TOF parameters are listed in Table S2. The same parameters were used to obtain molecular ion and fragmentation data of all the degradation products. MSn studies on the drug and its degradation products helped to trace the origin of each individual fragment. The fragmentation of various precursor ions was achieved at different collision energies and by employing other optimized parameters (Table S2). MSn studies were carried out up to MS6. H/D exchange studies were also conducted using the MSn system, by injecting the solution containing 10 ppm drug in D2O. The purpose was to confirm the number of labile hydrogens present in various fragments.

2.7. LC-MS/TOF studies on the degradation products

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LC-MS/TOF studies were carried out on the stressed samples containing the degradation products by duly modifying the LC method (Section 2.5) by replacing phosphate buffer with ammonium formate, pH 3.0. The exact masses of molecular ion peaks and fragment peaks of

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each degradation product were recorded by using MS/TOF parameters optimized for the drug (Table S2). On-line H/D exchange studies on each degradation product were conducted on the

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MSn system by using an additional loop, where D2O was injected during elution of the peak of

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interest.

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2.8. Isolation of oxidative degradation products

Due to their low concentrations, the oxidative degradation products were isolated using

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preparative high performance liquid chromatography (Prominence, Shimadzu, Kyoto, Japan)

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equipped with a reversed-phase Pheonomenex Luna ODS column (250 mm × 21.2 mm, 10 µ).

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The mobile phase was acetonitrile (A) and water adjusted to pH 3.0 using formic acid (B). It was pumped at a flow rate of 21 ml/min in a gradient manner, according to the program given in

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Table S1. The collected fractions were evaporated to dryness under vacuum.

2.9. NMR studies on the drug and oxidative degradation products The solutions of drug and isolated degradation products were prepared in suitable solvents like CD3OD and D2O. 1H NMR,

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C NMR, DEPT, 2D COSY, TOCSY, HETCOR and HMBC

spectra were acquired using a JEOL ECA 500MHz NMR spectrometer equipped with a PFG 5mm BBO probe. 1H NMR spectra were acquired with a 10 kHz sweep width using 16K timedomain points with an acquisition time of 3.56 s. The

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C NMR spectra were recorded at 125

MHz with decoupling or off-resonance partial proton decoupling; spectral width 39 KHz, 32K

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data points with digital resolution of 1.19 Hz. 2D COSY and TOCSY spectra were acquired using 1H sweep width 5 kHz in f1 and f2 dimensions, with 2048 points in f2, 512 complex increments in f1, 2 scans per increment with 50 [ms] mixing time. The two-dimensional C-H

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NMR correlated spectrum was recorded at 125.76 MHz using the pulse sequence acquisition under the following conditions: spectral width in f1 = 5.8 KHz, f2 = 24 KHz; and 256

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experiments with 32 acquisition scans and 2048 data points in fl and 202 data points in f2. The

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signal was processed using a sine bell function. The data were referenced either to methoxy

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signal of CH3OH at  3.30 ppm or HOD signal at  4.80 ppm.

3.0. LC-NMR studies on the drug and hydrolytic degradation products

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The hydrolytic stressed samples were subjected to LC-NMR studies to obtain 1H-NMR and

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COSY data. An optimized LC-NMR method (Table S3) was developed at a flow rate of 0.5

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ml/min. In this case, a mixture of NMR grade ACN and phosphate buffer prepared in D2O (pH, 3.0) was used for the method development (Table S3). All the resolved peaks (drug and

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degradation products) were collected in the fraction loop using terminal cube and sent to inverse 3 mm flow probe equipped with 1H {13C} channels with pulsed-field gradient along the z-axis. The active sample volume of the probe was approximately 60 μl and the transfer time from the UV cell to the active volume was 45 s at a flow-rate of 0.5 ml/min. One dimensional 1H NMR spectra were recorded using the WET pulse sequences, with attenuated power at 63 and 62.5 [dB] for solvent suppression of the acetonitrile and HOD signal, which gave digital resolution of 0.76 Hz per point for the drug and its degradation products. Spectra were acquired with 15 KHz spectral width and 16K data points. Depending on the concentration of the degradation products, a total number of scans for CL-I, CL-III&IV, CL-V and CL-VI were 512, 1024, 256 and 2096 to

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get the appropriate signal to noise-ratio of 1H LC-NMR spectra. The chemical shifts were referenced to the methyl signal of the residual CH3CN at  1.93 ppm or methoxy signal of

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residual CH3OH at  3.30 ppm.

3. Results and discussion

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3.1. HPLC evaluation of the stressed samples

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For HPLC analysis, samples were made by mixing of 40% oxidative, 40% neutral and remaining ratio with other stressed samples. LC separation between the drug and its degradation products is

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shown in Fig. 1, which was recorded at a wavelength of 210 nm. Significant degradation of the

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drug was observed in acid and base conditions, resulting primarily in CL-V. This degradation product was also formed as a single product in photolytic and thermal conditions. In the case of

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neutral hydrolysis, five degradation products (CL-I, CL-III to CL-VI) were formed, while under

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oxidative conditions (30% H2O2), only two degradation products (CL-II and CL-VII) were

3.2.

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generated. No drug decomposition was observed in the presence of AIBN.

Mass studies on the drug

The fragmentation pathway of the drug was established by performing MS/TOF studies. The MS/TOF line spectrum of the drug is shown in Figure 2. MSn data are given in Table 1. Elemental composition calculator was used to obtain the most probable molecular formulae, losses, RDB and error in mmu for each fragment. The same are listed in Table 2, along with H/D exchange data. The fragmentation pathway of the drug is shown in Figure 3. As depicted, the molecular ion (m/z 418) in MS2 got fragmented into an ion of m/z 211 upon the loss of C12H17NO2. The latter reduced into products of m/z 183, 169 (loss of CH2CO), 143 (loss of

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C4H4O) and 140 (loss of C2HO2N). During MS/TOF studies, the product ion of m/z 183 was found as two fragments of the same nominal mass, but with different mass defects (Fig. 2). The one with accurate m/z of 183.1149 was formed from parent of m/z 211 upon the loss of CO,

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while the fragment of m/z 183.0719 was obtained from the same parent through the loss of ethylene. The ion of m/z 183.1149 was further reduced to a fragment of m/z 165 upon the loss of

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water, and the latter further formed an ion of m/z 137 upon elimination of ethylene (C2H4). The

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fragment of m/z 183.0719 also dissociated to the same mass ion of m/z 137, through the loss of HCOOH. Thus the ion of m/z 137 had two different possible structures (j and j’), though it had

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the same exact mass. The ion of m/z 169 lost ethylene (C2H4) to form a product of m/z 141. The smallest fragment of m/z 114 was derived from m/z 143 upon the loss of CH2NH. The sodium

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adduct of the fragment of m/z 211 was also observed with m/z of 233, with its origin from

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cilazapril sodium. The ion of m/z 189 was derived in parallel from the ion of m/z 233 through

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the loss of CO2. The structures of all the fragments were justified by the number of labile hydrogens established to be present through H/D exchange studies. Here critical consideration

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was given on the number of labile hydrogen(s) present, the migration of mobile deuterium, and the movement of protons/deuterium from neutral losses.

3.3.

NMR studies on the drug

Figures 4 and 5 show 1H and 13C NMR peak assignments and multiplicity for the drug, based on COSY, TOCSY, HETCOR and HMBC connectivities (Figs. S1-S6). The prominent signals in CD3OD included: i) one triplet and multiplet at δ 1.30 and 4.24 ppm, corresponding to methyl and methylene protons at positions H-23 and H-24, respectively, representing the ester linkage; ii) two pairs of multiplets at δ 1.36 to 1.78 ppm corresponding to non-equivalent methylene

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protons at positions H-4a and H-4b, respectively; iii) a cluster of multiplets emerging from δ 1.76 to 2.41 ppm, corresponding to protons of positions H-5a, H-8, H-9 and H-14; iv) two multiplets originating at δ 2.41 and 2.53 ppm due to non-equivalent protons of methylene for the positions

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H-5b and H-7a, respectively; v) one doublet of doublet arising in the range from δ 2.98 to 3.11 ppm due to methylene protons at positions H-6a and H-6b, respectively; vi) one multiplet at δ

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3.58 ppm for position H-7b; vii) three separate triplets at δ 3.71, 4.50 and 4.79 ppm of methine

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protons for positions H-13, H-10 and H-3, respectively and viii) one multiplet at δ 7.23 ppm

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corresponding to aromatic ring for positions H-17 to H-21. The data are compiled in Table 3. Minor changes were monitored in 1H chemical shifts of the drug in D2O as compared to CD3OD

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(Fig. 4), with a difference from ±0.04 to ±0.30 ppm. For example, the position H-13 emerged at 4.00 ppm (3.71 ppm in CD3OD) and the proton at 10th position was not observed due to merging

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with HOD signal. Chemical shifts were further verified using COSY (Fig. S2) and TOCSY (Fig.

C NMR spectrum of the drug (Fig. 5) showed a total of twenty two carbons in the structure.

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S4) connectivities. The data are included in Table 3.

The position of methylene, methyl and methine carbons were differentiated based on DEPT 135 measurement (Fig. 5). Further, HETCOR and HMBC studies were carried out to assign the connectivities between carbons and protons. Three signals arose at δ 175.7, 172.6 and 172.8 ppm for three carbonyls for positions C-2, C-11 and C-22, respectively. Another three methine signals were observed at δ 53.0, 59.4 and 60.8 ppm for positions C-3, C-10 and C-13, respectively. Six signals at δ 17.4, 26.5, 53.2, 51.9, 26.3 and 28.5 ppm corresponding to positions C-4 to C-9 for methylene carbons and two methylene groups for positions C-14 and C-15 were found at δ 34.6 and 32.5 ppm, respectively. Other methylene and methyl carbon signals appeared at δ 62.9 and

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14.5 ppm for C-23 and C-24 (ester group). Aromatic carbon signals, i.e., C-16 to C-21 appeared at δ 141.9, 129.5, 129.6, 127.3, 129.6 and 129.5 ppm, respectively (Table 3).

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3.4. Characterization of the degradation products based on LC-MS/TOF, LC-MSn, LC-NMR, 1D and 2D NMR data

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LC-MS/TOF line spectra of all the degradation products are shown in Fig. S7. The

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corresponding MSn data of degradation products are listed in Table S4. The observed mass values of the molecular ion and its fragments, along with error in mmu, and H/D exchange data

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are listed in Table 4. Overlaid LC-NMR spectra of cilazapril and its hydrolytic degradation products CL-I, CL-III to CL-VI are shown in Fig. S8. Overlaid NMR spectra of CL-II and CL-

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VII are shown in Fig. 6. LC-1H NMR data of the drug and degradation products CL-I, CL-III to

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CL-VI are compiled in Table 5. Comparative NMR data of CL-II and CL-VII are listed in Table

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degradation products.

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6. The coalesced information from all these tools was used for structural elucidation of all the

3.4.1. CL-I and CL-IV

As highlighted from the LC-MS/TOF line spectrum in Fig. S7, CL-I had an accurate mass of m/z 180.1047, while the same for CL-IV was m/z 208.1350. The mass difference of 28 Da suggested that CL-I was rather generated upon ester hydrolysis of CL-IV. The fragmentation pathways of both the degradation products are shown together in Fig. 7. As shown, CL-I and CL-IV ionized to a common fragment of m/z 134 upon the loss of HCOOH and C3H6O2, respectively. This ion lost ammonia to generate a product of m/z 117, which subsequently lost H2 to form a fragment of

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m/z 115. H/D exchange studies indicated the presence of four labile hydrogens in CL-I and three in CL-IV (Fig. S9).

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LC-NMR data of CL-I indicated the presence of an aromatic region and protons at positions H13, H-14 and H-15, while all other protons (H-3 to H-10, H-23 and H-24) disappeared as

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compared to the drug (Fig. S8). LC-NMR data of CL-IV was similar to CL-I, except for the

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presence of protons H-23 and H-24 in the former, highlighting the presence of an intact ester linkage (Figs. S10, S11). Based on the above data, CL-I was characterized as 2-amino-4-

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phenylbutanoic acid and CL-IV as ethyl 2-amino-4-phenylbutanoate [16].

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3.4.2. CL-II

The accurate mass of molecular ion of this degradation product was determined to be m/z

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242.1117 (Fig. S7). Elemental composition calculator suggested its molecular formula as

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C10H16N3O4+. H/D exchange data indicated the presence of four labile hydrogens in the structure.

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Its mass fragmentation (Fig. 8) was initiated through the loss of H2O and CO forming ions of m/z 224 and 214, respectively. The ion of m/z 224 further lost CO and C2HNO to generate products of m/z 196 and 169, respectively. The product ion of m/z 196 underwent further fragmentation to form ions of 178 (loss of H2O) and 152 (loss of CO2). The ion of m/z 214 yielded a fragment of m/z 197 through the loss of ammonia. Based on the above data, the structure of CL-II could be proposed

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8-amino-6,10-dioxooctahydro-1H-pyridazino[1,2-a][1,2]diazepine-1-carboxylic

acid. To verify the postulated structure, specific help was sought of 1D and 2D NMR data. However, CL-II was obtained in very low quantity in pure form due to secondary degradation during

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enrichment . Therefore, only 1H (Fig. 6), COSY (Fig. S12) and TOCSY (Fig. S13) data could be acquired. After 1D and 2D NMR measurements, stability of this degradants had been checked by injecting the sample into HPLC. The following salient observations were made from 1H NMR

In total, eleven protons appeared in the 1H NMR spectrum. This highlighted that CL-II did

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i)

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data on CL-II, in comparison to the drug:

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not have an ethyl 4-phenyl butanoate moiety. The disappearance of aromatic protons from positions H-16 to H-21 in NMR spectrum also confirmed the absence of this moiety. The

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absence of two protons in pyridazine ring for position 7 also supported the structure of CL-II, which was proposed by the mass data. However, H-3 was not observed during proton NMR

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studies, perhaps to suppression of the HOD region.

ii) TOCSY correlations were found between positions H-6a, 6b (δ 2.87 to 2.91 ppm) and H-5a,

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5b and H-4a, 4b; positions H-8a, 8b and H-9a, 9b and H-10, except for position H-7. It

3.4.3.

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structure.

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clearly indicated that oxidation occurred at position H-7, thus supporting the proposed

CL-III, CL-V and CL-VI

LC-MS/TOF spectra of CL-III, CL-V and CL-VI (Fig. S7) indicated their accurate masses as m/z 229.1163, 390.2024 and 418.2329, respectively. The corresponding calculated molecular formulae were C10H17N2O4, C20H28N3O5 and C22H32N3O5, respectively. All the degradation products had similar fragmentation profile among themselves and the drug. As highlighted, they fragmented to form a product ion of m/z 211, similar to the fragment formed in case of the drug. The same further fragmented through pathways a→c, d, e, g, i; c→f→j; d→j; e→h and g→k, as seen during dissociation of the drug (Fig. 3).

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LC-1H NMR studies on CL-III indicated the presence of H-3 to H-10 protons, while aromatic protons and ester linkage were absent. This indicated that ethyl 2-amino-4-phenylbutanoate moiety was removed in the case of this degradation product. The proton at position 10 was

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deshielded from  4.79 ppm to 4.86 ppm, indicating that hydrolysis took place at this proton. Based on the above data, the structure of this degradation product was assigned as 9-hydroxy-10acid.

H/D

exchange

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oxooctahydro-1H-pyridazino[1,2-a][1,2]diazepine-1-carboxylic

data

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supported the proposed structure by the presence of three labile hydrogens. 2D COSY NMR

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spectrum (Fig. S14) also substantiated the same.

CL-V was obtained in most of the stress conditions. H/D exchange studies indicated the presence

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of four labile hydrogens. LC-NMR studies highlighted the absence of H-23 and H-24 protons of ester linkage, whereas rests of the protons were similar as in the drug. Chemical shifts of all

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other protons and COSY correlations (Fig. S15) were also same as drug. Based on these data, the

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[17].

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product was safely assumed to be cilazaprilat, which is a known active metabolite of the drug

CL-VI had same mass and even same number of labile hydrogens as compared to the drug, suggesting the possibility of diastereomer. LC-NMR studies also indicated that chemical shifts of almost all protons were similar to the drug. Although there were three chiral centers at positions 3, 10 and 13, the configuration for position 13 was indicated to have changed from S to R, due to the presence of an ester group. Describing stereochemistry by using coupling constant between positions 13 and 14 from 1H LC-NMR was not possible in this case, as position 13 was a triplet and position 14 was not clearly observed due to acetonitrile suppression region. Based on the available data, a tentative structure assigned to this degradation product was (1S, 9S)-9-[(2R)-(1-

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ethoxy-1-oxo-4-phenylbutan-2-yl)amino]-10-oxo-octahydro-1H-pyridazino[1,2-a][1,2]diazepine -1-carboxylic acid.

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3.4.4. CL-VII This degradation product was formed in oxidative conditions and had an experimental accurate

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m/z of 432.2124 (Fig. S7), 14 Da mass higher than the drug. It fragmented in mass studies to

us

form product ions of m/z 414 (loss of H2O), 404 and 276 (C6H8N2O3). In HR-MS study, the fragment of m/z 404 was observed as two lines of m/z 404.2184 (loss of CO) and 404.1801 (loss

an

of C2H4). Both these fragments further lost C2H4 and CO, respectively, to generate a common ion of m/z 376. The product ion of m/z 414 reduced to form ions of m/z 386 (loss of CH2N radical),

M

340 (loss of C3H6O2), 211 (loss of C12H13NO2) and 209 (loss of C12H15NO2). The ion of m/z 340

d

lost ethylene to form a product ion of m/z 312. The fragment of m/z 211 was isomeric to the

te

same mass ion formed in the case of the drug and its subsequent fragmentation routes below m/z 211, viz., m/z 211→183, 169, 143, 140; 183→165→137; 183→137; 169→141; 143→114, were

Ac ce p

similar to the drug. The ion of m/z 209 further lost H2O and CO to form product ions of m/z 191 and 181, respectively. The fragmentation pathway of this degradation product is shown in Fig.9. 1

H (Fig. 6), 13C (Fig. 10), DEPT-135 (Fig. 10) and two dimensional COSY, TOCSY, HETCOR

and HMBC NMR studies (Figs. S16-S19) of isolated CL-VII highlighted its following characteristic features, as compared to the drug: i)

1

H NMR studies indicated the presence of twenty seven protons, with only two protons

being absent in comparison to the drug. All other chemical shifts were also almost similar

Page 18 of 41

to the drug, except for the positions 9, 10 and 13. Even the connectivities in case of the degradation product were the same as the drug, except for positions 9 and 10. ii) The absence of proton at position 10 was supported by COSY and TOCSY data.

13

C

ip t

NMR study established the position of carbon losing this proton at δ 155.58 ppm. HMBC study confirmed its position based on correlation with position H-9.

cr

iii) In both 1H and 13C NMR studies, position 13 showed deshielding behaviour (δ 5.33 ppm

us

and δ 72.37 ppm) than the drug (δ 3.71 ppm and δ 60.78 ppm).

iv) An interesting observation was made for position H-9 in 1H NMR study. A peak arose at

13

an

δ 2.66 ppm with single integral. It suggested conversion of methylene to methine. The C NMR spectrum indicated its δ as 27.72 ppm. DEPT-135 also confirmed the same as a

M

methine. In TOCSY, it showed correlations with H-7 and H-8, while in HMBC it showed

d

correlation with C-10.

Ac ce p

22.

te

v) In HMBC, correlations were found between H-13 to C-14, H-14 to C-15 and H-13 to C-

Eventually, on the basis of mass and NMR data, the structure of CL-VII was proposed as 8-(1ethoxy-1-oxo-4-phenylbutan-2-ylcarbamoyl)-9-oxooctahydropyridazino[1,2-a]pyridazine-1carboxylic acid.

3.5.

Degradation pathway and mechanism of degradation of cilazapril

The conversion of the drug to cilazaprilat (CL-V) involved typical acid-base catalysis of the ester. In the case of neutral conditions, the attack of water occurred at 10th position resulting in the formation of CL-III and CL-IV. The latter further underwent ester hydrolysis to form CL-I. Epimerization of the drug under neutral conditions resulted in the formation of CL-VI. CL-II was

Page 19 of 41

formed by oxidation at α carbon to nitrogen (7th position), which further underwent Ndealkylation (Fig. 11). The formation of CL-VII involved initial formation of hydroxyl amine, which lost water to generate an imine. The same underwent ring shortening, followed by

ip t

oxidation at the 10th position [18-20].

cr

4. Conclusion

us

The study provided useful information regarding degradation behavior of cilazapril under different stress conditions. In neutral conditions, the drug yielded five degradation products,

an

which were characterized using LC-MS/TOF, LC-MSn and LC-NMR data. Two additional

te

Acknowledgments

C,

d

DEPT-135, HETCOR and HMBC data.

13

M

oxidative degradation products were isolated and characterized using 1H, COSY, TOCSY,

The authors thank Prof. A.K. Chakraborti, Dr I.P Singh, Dr M.S. Gill, T. Handa (NIPER, SAS

Ac ce p

Nagar, India) and Dr R.P. Shah (BMS-Biocon Research Centre, Bangalore, India) for their valuable inputs.

References

[1] ICH, Stability testing of new drug substances and products, Q1A(R2), International Conference on Harmonisation, IFPMA, Geneva, 2003. [2] M. Narayanam, T. Handa, P. Sharma, S. Jhajra, P.K. Muthe, P.K. Dappili, R.P. Shah, S. Singh, Critical practical aspects in the application of liquid chromatography-mass spectrometric

Page 20 of 41

studies for the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 87 (2014) 191-217. [3] M. Narayanam, S. Singh, Characterization of stress degradation products of fosinopril by

ip t

using LC-MS/TOF, MSn and on-line H/D exchange, J. Pharm. Biomed. Anal. 92 (2014) 135143.

cr

[4] S. Singh, T. Handa, M. Narayanam, A. Sahu, M. Junwal, R.P. Shah, A critical review on the

us

use of modern sophisticated hyphenated tools in the characterization of impurities and degradation products, J. Pharm. Biomed. Anal. 69 (2012) 148-173.

electrocyclized

an

[5] F.G. Vogt, L. Wu, M.A. Olsen, W.M. Clark, A spectroscopic and computational study of an photo-degradation

product

of

6-(2-(5-chloro-2-(2,4-

M

difluorobenzyloxy)phenyl)cyclopent-1-enyl)picolinic acid, J. Mol. Struct. 984 (2010) 246–261.

d

[6] P. Novak, P. Tepes, M. Ilijas, I. Fistric, I. Bratos, A. Avdagic, Z. Hamersak, V.G. Markovic,

te

M. Dumic, LC-NMR and LC-MS identification of an impurity in a novel antifungal drug icofungipen, J. Pharm. Biomed. Anal. 50 (2009) 68–72.

Ac ce p

[7] S. Provera, L. Rovatti, L. Turco, S. Mozzo, A. Spezzaferri, S. Bacchi, A. Ribecai, S. Guelfi, A. Mingardi, C. Marchioro, D. Papini, A multi-technique approach using LC-NMR, LC-MS, semi-preparative HPLC, HR-NMR and HR-MS for the isolation and characterization of low-level unknown impurities in GW876008, a novel corticotropin-release factor 1 antagonist, J. Pharm. Biomed. Anal. 53 (2010) 517–525. [8] T. Murakami, H. Konno, N. Fukutsu, M. Onodera, T. Kawasaki, F. Kusu, Identification of a degradation product in stressed tablets of olmesartan medoxomil by the complementary use of HPLC hyphenated techniques, J. Pharm. Biomed. Anal. 47 (2008) 553–559

Page 21 of 41

[9] WHO, Stability Testing of Active Pharmaceutical Ingredients and Finished Pharmaceutical Products, World Health Organisation, Geneva, 2009. [10] S. W. Baertschi, P. J. Jansen, K. M. Alsante, Stress Testing: A Predicting Tool, in: S.W.

Degradation, Informa Healthcare, London (UK), 2011, pp 10-49.

ip t

Baertschi, K.M. Alsante, R.A. Reed (Eds.), Pharmaceutical Stress Testing: Predicting Drug

cr

[11] ICH, Impurities in new drug substances Q3A(R2), International Conference on

us

Harmonisation, IFPMA, Geneva, 2006, pp. 2.

[12] EMEA, Guideline on stability testing: Stability testing of existing active substances and

an

related finished products, CPMP, London, 2003, pp. 4/18.

[13] ICH, Photostability testing of new drug substances and products Q1B, International

M

Conference on Harmonisation, IFPMA, Geneva, 1996.

d

[14] M. Bakshi, S. Singh, Development of validated stability-indicating assay methods-critical

te

review, J. Pharm. Biomed. Anal. 28 (2002) 1011-1040. [15] S. Singh, M. Bakshi, Guidance on conduct of stress tests to determine inherent stability of

Ac ce p

drugs, Pharm. Technol. On-line. 24 (2000) 1-14. [16] M. Narayanam, A. Sahu, S. Singh, Characterization of stress degradation products of benazepril by using sophisticated hyphenated techniques, J. Chromatogr A. 1271 (2013) 124136.

[17] J. Prieto, R. Jimenez, R. Alonso, Quantitative determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine by high-performance liquid chromatography with amperometric detection, J. Chromatogr B. 714 (1998) 285-292.

Page 22 of 41

[18] S. W. Baertschi, K.M. Alsante, R.A. Reed (Eds.), Pharmaceutical Stress Testing: Predicting Drug Degradation, Informa Healthcare, London (UK), 2011. [19] S. W. Hovorka, M.J. Hageman, C. Schöneich, Oxidative degradation of a sulfonamide-

ip t

containing 5, 6-dihydro-4-hydroxy-2-pyrone in aqueous/organic cosolvent mixtures, Pharm Res. 19 (2002) 538-545.

cr

[20] L. Min, Organic chemistry of drug degradation, Royal Society of Chemistry, 29 ed.,

Ac ce p

te

d

M

an

us

Croydon, 2012.

Page 23 of 41

Precursor ion (m/z) 418 211

MS4

183 169 143 165

MS5

Product ion(s) (m/z) 211 183, 169, 165, 143, 141*, 140*, 137*, 114* 165, 137* 141* 114* 137*

cr

MSn MS2 MS3

ip t

Table 1 MSn data of cilazapril

Ac ce p

te

d

M

an

us

*Ions could not be captured for further fragmentation due to low intensity

Page 24 of 41

Observed only in MSn studies

ip t 8.5 8.5 4.5 4.5 3.5 3.5 4.5 3.5 4.5 2.5 3.5 2.5 4.5 4.5 2.5

cr

0.1 -2.4 -0.2 0.5 -0.3 2.1 -4.5 -0.4 0.2 -0.7 0.3 0.3 -

Possible parent ion

Difference from parent ion

Molecular formula for the loss

H/D exchange data

Number of labile hydrogen(s)

[M+Na]+ [M+H]+ [211+Na]+ a a a c a e a d f g

207.1262 207.1230 43.9900 27.9933 28.0363 42.0114 18.0125 68.0274 46.0007 28.0312 -

C12H17NO2 C12H17NO2 CO2 CO C2H4 C2H2O H2O C4H4O C2H4 C2HNO2 HCOOH C2H4 CH2NH

442 421 233 212 189 184 184 170 165 144 142 140 137 137 115

2 3 0 1 0 1 1 1 0 1 1 0 0 0 1

us

RDB

M an

C22H31N3NaO5+ C22H32N3O5+ C10H14N2NaO3+ C10H15N2O3+ C9H14N2ONa+ C9H15N2O2+ C8H11N2O3+ C8H13N2O2+ C9H13N2O+ C6H11N2O2+ C6H9N2O2+ C8H14NO+ C7H9N2O+ C7H9N2O+ C5H8NO2+

Error in mmu

Ac

#

440.2157 418.2312 233.0895 211.1082 189.0995 183.1149 183.0719 169.0968 165.1024 143.0808 141 140 137.0712 137.0712 114

ce pt

[M+Na]+ [M+H]+ [211+Na]+ a b c d e f g h# i# j j’ k#

Exact mass of most probable structure 440.2156 418.2336 233.0897 211.1077 189.0998 183.1128 183.0764 169.0972 165.1022 143.0815 141.0659 140.1070 137.0709 137.0709 114.0550

ed

Table 2 Interpretation of mass data of cilazapril Peak code MS/TOF Best possible data molecular formula

Page 25 of 41

ip t

DEPT

COSY

HMBC

CH CH2 CH2 CH2 CH2 CH2 CH2 CH CH CH2 CH2 CH CH CH CH CH CH2 CH3

H-4 H-3, H-5 H-4, H-6 H-5 H-8 H-7, H-9 H-8, H-10 H-9 H-14 H-13, H-15 H-14 H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 H-24 H-23

C-2, C-4, C-5 C-2, C-3, C-5, C-6 C-3, C-4, C-6 C-4, C-5, C-7 C-6, C-8 C-7, C-9 C-7, C-8, C-8, C-9, C-11, C-13 C-10, C-14, C-15, C-22 C-13, C-15, C-22 C-13, C-14, C-16 C-16 C-16 C-16 C-16 C-16 C-24 C-23

4.84 (t) 1.39-1.68 (m) 1.82-2.30 (m) 3.00-3.06 (m) 2.56-3.28 (m) 1.68-1.83 (m) 1.89-2.06 (m) * 4.00 (t) 2.32 (m) 2.76- 2.86 (m) 7.32 (m) 7.32 (m) 7.32 (m) 7.32 (m) 7.32 (m) 4.25 (q) 1.28 (t)

ce pt

4.79(t) 1.36-1.78 (m) 1.76-2.41 (m) 2.98-3.11 (m) 2.53-3.58 (m) 1.76-2.13 (m) 1.65-2.13 (m) 4.50 (t) 3.71 (t) 2.14 (m) 2.75 (m) 7.23 (m) 7.23 (m) 7.23 (m) 7.23 (m) 7.23 (m) 4.24 (m) 1.30 (t)

Ac

2 3 4a,b 5a, b 6a, b 7a, b 8a, b 9a, b 10 11 12 13 14 15a, b 16 17 18 19 20 21 22 23 24

C ( ppm) 175.7 53.0 17.4 26.5 53.2 51.9 26.3 28.5 59.4 172.6 60.8 34.6 32.5 141.9 129.5 129.6 127.3 129.6 129.5 172.8 62.9 14.5

Cilazapril (D2O)

1

ed

H ( ppm)

13

us

Cilazapril (CD3OD) 1

M an

Position

cr

Table 3 NMR chemical shift assignments of cilazapril (CL) in CD3OD and D2O solvent

H ( ppm)

COSY

TOCSY

H-4 H-3, H-5 H-4, H-6 H-5 H-8 H-7, H-9 H-8, H-10 * H-14 H-13, H-15 H-14 H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 H-24 H-23

H-4, H-5, H-6 H-3, H-5, H-6 H-3, H-4, H-6 H-3, H-4, H-5 H-8, H-9 H-7, H-9 H-7, H-8 * H-14, H-15 H-13, H-15 H-13, H-14 H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 H-24 H-23

*signals could not be observed due to merging with HOD region

Page 26 of 41

ip t

cr

Table 4 LC-MS/TOF and on-line H/D exchange data of CL-I to CL-VII along with their possible molecular formula and major fragments in ESI positive mode MS/TOF values for [M+H]+

Nitrogen rule

Probable molecular formula (exact mass; error in mmu; RDB)

m/z after H/D exchange in ESI +ve mode

Number of labile hydrogen(s)

Experimental mass of fragments in ESI +ve mode (error in mmu)

CL-I

180.1047

Odd

184

4

CL-II

242.1117

Odd

C10H14NO2+ (180.1019; 2.8; 4.5) C10H16N3O4+ (242.1135; -1.8; 4.5)

246

4

CL-III

229.1163

Even

C10H17N2O4+ (229.1183; -2.0; 3.5)

232

3

CL-IV

208.1350

Odd

211

3

CL-V

390.2024

Odd

C12H18NO2+ (208.1332; 1.8; 4.5) C20H28N3O5+ (390.2023; 0.1; 8.5)

394

4

CL-VI

418.2329

Odd

C22H32N3O5+ (418.2336; -0.7; 8.5)

421

3

CL-VII

432.2124

Odd

435

3

134.0984 (2.0), 117.0725 (2.6), 115.0579 (3.7) 224.1020 (-1.0), 214.1143 (-4.3), 196.1032 (-4.9), 178.0926 (-4.9), 169.0918 (-5.4), 152.1131 (-5.1) 211.1044 (-3.3), 183.1108 (-2.0), 183.0764 (0.0), 169.0984 (1.2), 165.1029 (0.7), 143.0807 (-0.8) 134.0976 (1.2), 117.0720 (2.1), 115.0555 (1.3) 211.1073 (-0.4), 183.1072 (-5.6), 183.0728 (-3.6), 169.0958 (-1.4), 165.1030 (0.8), 143.0789 (-2.6) 211.1105 (2.8), 183.1136 (0.8), 183.0792 (2.8), 169.0976 (0.4), 165.1009 (1.3), 143.0836 (2.1) 414.2003 (-2.0), 404.2184 (0.4), 404.1801 (-1.5), 386.1922 (8.6), 376.1837 (-3.0), 340.1627 (-2.9), 312.1319 (-2.4), 276.1556 (-3.8), 211.1052 (-2.5), 209.0902 (-1.9), 183.1135 (0.7), 183.0791 (2.7), 169.0968 (-0.4), 165.1002 (-2.0), 143.0863 (4.8)

M an

ed

ce pt

Ac

us

Degradation products

C22H30N3O6+ (432.2129; -0.5; 9.5)

Page 27 of 41

ip t

2.29 (m)

10 13 14

4.79 3.92 (t) 2.30 (m)

15 17 18

2.76,2.86 (m) 7.30 (m) 7.30 (m)

19

7.30 (m)

20

7.30 (m)

21 23 24

7.30 (m) 4.26 (q) 1.30 (t)

5 6 7

COSY

1

COSY

-

-

-

-

-

H-4 H-3, H-5 H-4, H-6 H-5

H-8

-

-

H-7, H-9 H-8, H-10 H-9 H-14 H-13, H-15 H-14

-

-

-

-

3.76 (t) *

-

5.01 (t) 1.391.71 (m) 1.782.27 (m) 3.003.07 (m) 2.573.25 (m) 1.691.77 (m) 1.782.28 (m) 4.86 (m) -

2.70

-

-

-

7.30 (m) 7.30 (m)

H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 -

-

-

2.692.80 (m) 7.30 (m) 7.30 (m)

-

-

7.30 (m)

-

-

7.30 (m)

-

-

7.30 (m) 4.23 (q) 1.28 (t)

H-4 H-3, H-5 H-4, H-6 H-5

H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 H-24 H-23

H ( ppm)

7.30 (m)

7.30 (m)

7.30 (m) -

1

H ( ppm)

COSY

-

-

-

-

-

-

H-8

-

-

H-7, H-9 H-8, H-10 H-9 -

-

-

-

-

M an

9

3 4

1

CL-IV

H ( ppm) -

COSY

ed

8

H ( ppm) 4.78 (m) 1.401.66 (m) 1.742.29 (m) 2.993.08 (m) 2.563.34 (m) 1.78 (m)

CL-III

ce pt

1

CL-I

us

Cilazapril

Ac

Position

cr

Table 5 LC-1H NMR peak assignments for the drug and its hydrolytic degradation products CL-I and CL-III to CL-VI along with multiplicity and their COSY correlation

4.03 (t) 2.22 (m)

H-14 H-13, H-15 H-14 H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 H-24 H-23

CL-V 1

H ( ppm) 4.21 1.381.67 (m) 1.71 (m) 2.983.07 (m) 2.583.32 (m) 1.682.28 (m) 1.832.32 (m) 4.06 3.60 (t) * 2.712.77 (m) 7.30 (m) 7.30 (m) 7.30 (m) 7.30 (m) 7.30 (m) -

CL-VI COSY H-4 H-3, H-5 H-4, H-6 H-5 H-8 H-7, H-9 H-8, H-10 H-9 H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 -

1

H ( ppm) 4.79 (m) 1.401.67 (m) 1.67 (m) 2.983.07 (m) 2.553.32 (m) 1.67 2.34 4.69 (m) 3.86 (t) 2.32 (m) 2.722.85 (m) 7.30 (m) 7.30 (m) 7.30 (m) 7.30 (m) 7.30 (m) 4.27 (q) 1.31 (t)

COSY H-4 H-3, H-5 H-4, H-6 H-5 H-8 H-7, H-9 H-8, H-10 H-9 H-14 H-13, H-15 H-14 H-18 H-17, H-19 H-18, H-20 H-19, H-21 H-20 H-24 H-23

*Signal not observed due to merging with CH3CN region

Page 28 of 41

Table 6 NMR chemical shift assignments of CL-II in D2O and CL-VII in CD3OD solvent CL-II

* 1.28-1.53 (m) 1.58-2.13 (m) 2.87-2.91 (m) -

* H-5

* H-5, H-6

H-4, H6 H-5

H-4, H-6

-

-

H-9

10 11 12 13 14

1.66-2.03 (m) 2.28-2.34 (m) 3.25 -

# -

H-9, H10 H-8, H10 H-8, H-9

15a, b

-

-

16 17 18

-

-

19

-

-

20

-

-

7a, b 8a, b 9a, b

5 6

H-8

H-4, H-5

DEPT

COSY

HMBC

CH CH2

H-4 H-3, H-5

C-4, C-5 C-3, C-5

25.83

CH2

H-4, H-6

#

52.92

CH2

H-5

#

51.53

CH2

5.33 (m) 2.30-2.44 (m) 2.58-2.80 (m) 7.23 (m) 7.23 (m)

155.58 164.86 72.37 31.82

21.76 27.72

M

6a, b

C ( ppm) 173.54 50.94 17.25

H-8

#

CH2

H-7, H-9

#

CH

H-8

#

CH CH2

H-14 H-13, H15 H-14

C-14, C-15, C-22 C-13, C-15, C-16, C-22 C-13, C-14, C-16, C-17, C-21 C-16, C-18, C-19 C-16, C-17, C-20

32.92

CH2

d

5a, b

142.54 129.40 129.40

CH CH

7.18 (m)

127.07

CH

7.23 (m)

129.40

CH

Ac ce p

2 3 4a,b

13

H ( ppm) 4.80 (m) 1.46-1.82 (m) 1.82-2.46 (m) 3.06-3.19 (m) 2.46-3.49 (m) 1.93-2.32 (m) 2.66 (m)

ip t

TOCSY

cr

COSY

H ( ppm)

3 4

CL-VII 1

us

1

an

Position

te

1 2

21 7.23 (m) 129.40 CH 22 168.41 23 4.21 (m) 63.20 CH2 24 1.26 (t) 14.44 CH3 *signals could not be observed due to merging with either HOD or CD3OD region # not observed clearly

H-18 H-17, H19 H-18, H20 H-19, H21 H-20 H-24 H-23

C-16, C-17, C-18, C-20 C-16, C-17, C-19, C-21 C-16, C-17, C-20 C-22, C-24 C-23

7

29 Page 29 of 41

mAU 2000 1750 1500

ip t

1250 1000 750

CL

H2O2

CL-VII

CL-I

CL-II CL-III

0

11 12 13 14 15 16 17 18

15.0

CL-I

CL-II

CL-III

Stress conditions

N

O

N

CL-IV

25.0

N

30.0

min

CL-V

CL-VI

CL-VII

A, B, N, P, T

N

O

M

Degradation products

20.0

an

10.0

Key: CL: cilazapril; A: acid; B: base; N: neutral; P: photo; O: oxidative; T: thermal

Fig. 1. Chromatogram showing separation of cilazapril (CL) and its degradation products

d

10

5.0

te

9

0.0

CL-VI

Ac ce p

7 8

CL-IV CL-V

us

250

cr

500

19 20 21 22

30 Page 30 of 41

d

te

1 j

3

2

100 b

0 183.1 m/z

150 200 250

0 300 350

400

+

ip t

440.2157 [M+Na] 456.1894 [M+K]+

[M+H]+

cr

418.2312

233.0895 [211+Na]+ 249.0630 [211+K]+

189.0995

4

169.0968 e165.1024 f

143.0808 g

5

137.0712

a

211.1082

c

d

183.0719 183.1149

2

us

an

27

M

23 24 25 26

Ac ce p

Intens. +MS, 2.5min #150 Intens. x10 4 x10 5 3

+MS, 2.1min #128

1

m/z

Fig. 2. MS/TOF mass spectrum of cilazapril

31

Page 31 of 41

N N

N N H2N

NH2

CL

O O

CL-VI

NaO

NH 2

ip t

Cilazapril sodium

-C12H 17NO 2

HO

-C12H17NO 2 N N

-C10H 13NO2

OO

OO

HO

N N

NH 2

O

HO

-C2H4

N N

N N

O

OO

HO

183.1128 (184) c -H2O

HO

183.0764 (184) d -HCOOH

165.1022 (165) f

O

169.0972 (170) e

N NH

HO

137.0709 (137) j

HO

O

141.0659 (142) h

N O

NH N HO

O ONa

233.0897 (233)

-CO 2

NH N ONa

O

143.0815 (144) g

140.1070 (140) i

189.0998 (189) b

-CH2NH

N N

O

-C2HO 2N

-C4H 4O

-C2H4

Ac ce p

O

N N H

N N

N N

-C2H 2O

te

HO

CL-V

an

-CO

OO

233.0897 (233)

M

CL-III

390.2023 (394)

211.1077 (212) a

d

229.1183 (232)

NaO

us

OH 2

OO

HO

N N

O

440.2156 (442)

Cilazapril (CL) and CL-VI

-H 2O

H2N O

418.2336 (421)

N N

OO

cr

OO

HO

NH2 HO

O

114.0550 (115) k

-C2H4 N N

O

28

137.0709 (137) j'

29 Fig. 3. Mass fragmentation pathway of cilazapril (CL) and its degradation products CL-III, CL-V and CLVI. The exact masses (m/z) are given below each structure along with H/D exchange data, which are 30 shown in brackets. The fragments in the dotted box were seen only in MSn studies. 31 32

32 Page 32 of 41

6

7

8

21

CL in CD3OD

19 18

16 17 5 N 9 15 4 3 N 11 10 14 2 NH 13 O 12 22 HO O O 1 O 23 24

24

HOD signal CH3OH signal

cr

ip t

20

13

23

6 15

an

10

us

17, 18, 19, 20, 21

6

5

CL in D2O

M

7

14 8

Ac ce p

17, 18, 19, 20, 21

te

d

24

3

23 13 66

15 15

14 5

85 8 9 9

4

33 34

Fig. 4. Overlaid 1H NMR spectra of cilazapril (CL) in CD3OD and D2O

35 36 37 33 Page 33 of 41

20 21

8

Solvent signal

18

16 17 15 4 3 N 11 10 14 2 NH 13 O 12 22 O HO O 1 O 23 24 N

9

ip t

5

17, 18, 20, 21

cr

7

6

19

19 16

24

14

d

M

17, 18, 20, 21

us

22 11

15

an

2

13 23 10 6 3 7

38 39 40

Ac ce p

te

19

13

10

3 24

67 23

14 15

Fig. 5. Overlaid 13C NMR and DEPT-135 spectra of cilazapril (CL) in CD3OD

41

34 Page 34 of 41

O 6 7 8 N 9 3 N 1110 NH2 12

OO

ip t

2 HO 1

us

6

HOD Signal

9

8

19

21 18 16 17 8 5 N 15 14 H 4 3 N 11 9 10 N 13 22 O 1 2 O O O HO O 23 24

43 44

4

CL-VII 24

d

te

Ac ce p

13

42

4

M

7

17, 18, 19, 20, 21

8

an

20

5

5

10

6

CL-II

cr

5 4

Residual ACN

Solvent signal

Residual ACN

23

14 3

7

15 915 6 6 5

14

5 8 4

4

Fig. 6. Overlaid 1H NMR spectra of oxidative degradation products, CL-II in D2O and CL-VII in CD3OD

35 Page 35 of 41

OH

O

H 3N

O

H3N 180.1019

208.1332

CL-I

CL-IV -C 3H6O 2

-HCOOH

H3N

ip t

O

134.0964

cr

-NH 3

an

-H 2

us

117.0699

45

115.0542

Ac ce p

te

d

M

46 Fig. 7. Common fragmentation pathway of CL-I and CL-IV. The exact masses are given below each structure 47

36 Page 36 of 41

O N N OO

H 2O

NH 2

242.1135

-CO

-H2O

N N

N N HO

HO

224.1030

NH 2

HO

169.0972

N N

50 51

O

O

197.0921

d 152.1182

te

49

HO

NH 2

NH 2

178.0975

N N

Fig. 8. Fragmentation pathway of CL-II. The fragment of m/z 197 was only seen in MSn studies. The exact masses are given below each structure

Ac ce p

48

-NH 3

M

-CO 2

N N O

O

an

N NH

196.1081 -H2O

O

us

-C2HNO

N N O

O

214.1186

-CO

HO

NH 3

cr

NH 2

OO

ip t

CL-II

37 Page 37 of 41

N N HO

O

N N

H N OH O

O

O HO

O

O

N OH 2 O O

432.2129 CL-VII

O

O

O

O

O

414.2023

O

O HO

N N

-C12H 15NO2

HO

386.1836

O

O

HO

340.1656

HO

O

O

-C2H4

N N HO

O

HO

O

O

183.0764

O

Ac ce p

HO

O

169.0972

-C4H4O

N NH HO

137.0709

HO

O

-CO

O

141.0659

HO

O

H N OH

HO

O

-C2HO2N

N H O 140.1070

-CH 2NH

N N O

O

143.0815

-C2H4

N N

HO

376.1867

N N H

HO

O

404.1816

N N

O 181.0972

-C2H 2O

-HCOOH

te N N

165.1022

-CO

N N

183.1128 -H 2O

O

O

-C2H 4

N N

O

209.0921

-CO

H N

312.1343

HO

211.1077

-C2H4

N N

O

N N O

HO

H N

OH

O 191.0815

an

O

N N

N

M

O

O

O

d

HO

N N

N

O

OH O

O

-H 2O

N

O

N N

H N

404.2180

276.1594

-C12H 13NO 2

-C3H6O 2

-CH 2N

N N

H N

cr

HO

N

-C2H 4

-CO

us

N N

-C6H 8N 2O3

ip t

-H2O

NH 2 HO

O

114.0550

-C2H 4 N N

O

52 53 54

137.0709

Fig. 9. Fragmentation pathway of CL-VII. Fragments in dotted box were only seen in case of MSn studies. Exact masses are given below each structure

38 Page 38 of 41

19

16 10

14

7 63

13

5 9

8

4

us

2

11

24

15

23

19 22

ip t

17, 18, 20, 21

cr

20

21 18 6 7 16 17 8 5 N 15 14 4 3 N 11 9 10 H N 13 22 O 1 2 O O O HO O 23 24

an

17, 18, 20, 21

24

13

55 56 57

Ac ce p

te

d

M

19

3

9 5

67 14 23

4 8

15

Fig. 10. Overlaid 13C NMR and DEPT-135 spectra of CL-VII in CD3OD

58 59 60

39 Page 39 of 41

N N HO

O

O

HN O O

Ester hydrolysis

H 2O

Epimerization H 2O

H +/OH- /H 2O

H2O 2

O OH

N N

O

O H 2O

HN HO

O

O

N

O

HO

HN

O

O

O

N

O O

O

HO

HO

N

NH

O

O

N

O

HO

O

O

HN O O

O

CL-V H 2O

N

H

HN

cr

N

N

H 2O 2

OH-

H 2O

OH OH

us

HO

N

ip t

Cilazapril

O

N

HO

O

N H 2N

HO O

O

O

HN

O

HO

O

O

OH

N O

N-Dealkylation

HO

61 62 63 64

Ac ce p

CL-I

O

HO

HO

O

O

O

O

CL-II

HO

O O

NH 2

N N HO

O

O

N O O

H 2O 2

N

N

N

N

O

N

N

H+

HN

te

O

O

d

HO

HO

H

O O

O

N

H2N

N

N

N

H 2O

N

O O

M

CL-IV

Ester hydrolysis

HN

O

H+

CL-III

O

H

an

N

N

OH-

N

O

HN O

HO

O

N HN

O

Cilazapril

H N

N O

O O

CL-VI

HO

O

O

O

O

O

CL-VII

Fig. 11. Degradation pathway and mechanisms of degradation of cilazapril (CL) to products (CL-I to CL-VII)

65

40 Page 40 of 41

us

cr

i

*Graphical Abstract

H 2N

M an

O HO N

CL-I

N HO

O

O

OH

CL-III

O N N HO

ed

Oxidative

N

ce pt

H 2N O O

Ac

CL-IV

O

NH2

O

CL-II

N

HO

O

O

HN O O

N

Cilazapril

H N

N N

HO

O

O

O

O O

H N

N HO

O

O

O

O O

CL-VII

CL-VII

N N

N N HO

O

O

HO

HN

O

O

HN

O HO

O O

CL-VI

CL-V

Page 41 of 41