Nagaraju Rajana1,3* ,V. Pallavi1, P. Madhavan1, H. Rama Mohan1,J. Moses Babu2,K. Basavaiah3
1Technology Development Center, Custom Pharmaceutical Services, Dr. Reddy’s Laboratories Ltd, Miyapur, Hyderabad 500049, India,
2Integrated Product Development Organization, Dr. Reddy’s Laboratories, Innovation Plaza, Bachupally, Telangana 500072, India,
3Department of Inorganic & Analytical Chemistry, Andhra University, Visakhapatnam (AP),530003 India.
*Corresponding Author E-mail: nagarajurajana@drreddys.com, nagarajrajana@gmail.com
ABSTRACT:
During reverse phase chromatography, the chiral fragment–B of Boceprevir i.e methyl (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride was separated along its cis isomer, acid impurity and ester impurity. The R and S isomers of Trans isomers were separated by chiral chromatography; the chiral S isomer was characterized by the 1D NMR, FT-IR, Mass, SOR, X ray studies and differential scanning colorimetry.
KEYWORDS: Boceprevir, Absolute configuration, 1D NMR, Chiral chromatography.
INTRODUCTION:
Boceprevir is a protease inhibitor ,it cures the hepatitis caused by hepatitis C virus genotype 1,it binds to the HCV nonstructural protein 3 active site[1],The chiral fragment of Boceprevir drug substance is plays key role for the activity on chronic hepatitis C infection[2]. It is having two isomers among these two the S isomer is active than R- isomer, FDA approved this concept on 13 May 2011[3]. A UPLC–MS/MS method for the simultaneous plasma quantification of all isomeric forms of the new anti-HCV protease inhibitors boceprevir and telaprevir[4].Development and validation of RP– HPLC method for the analysis of Boceprevir and related impurities in bulk and pharmaceutical dosage forms[5].Rapid RP-HPLC method development and validation for the quantitative estimation ribavirin in tablets[6]. Highly sensitive determination of HCV protease inhibitors boceprevir (SCH 503034) and telaprevir (VX 950) in human plasma by LC-MS/MS[7]. Development and validation of stability-indicating hptlc method for determination of boceprevir[8].As per the author knowledge no method was explained the absolute configuration of Chiral fragment of Boceprevir, but in this work the author as proposed that the absolute configuration of Chiral fragment of Boceprevi-B, this was confirmed Normal phase chromatography, ID NMR, SOR, FT-IR ,Thermal analysis PXRD and single crystal.[9]
METHODS AND MATERIALS:
The Dichloro methane, Toluene, Ethyl acetate, Diethyl amine, Di isopropyl ethyl ether reagents were used for single crystal formation from Rankem and merk make GC grade. The di-potassium hydrogen phosphate and acetonitrile used for the reverse phase from Rankem and Merck make GC grade. The Hexane and Ethanol reagents for normal phase chromatography were purchased from Rankem and Merck make GC grade. The 1200 series Agilent HPLC was used for the normal and reverse phase separations. Thermal analysis has been performed by using waters TA Q2000 series DSC instrument. Tzero lid and pan used for the analysis. The data was interpreted by using the TA Universal analysis. The analysis was performed with 4 mg sample. The heat flow was used from 30°C to 300°C with 10°C/min ramp. The specific optical rotation was performed by using the Jasco P-2000 model polarimeter, the polarizer was Glan-Tylor Prism, and the path length was 100 mm, sodium lamp with 589 nm and 5 cycles. The powder XRD was performed on Cu K-ALPHA1/50 kv/34 mA with scintillation counter and RINT2000 wide angle goniometer. The FT-IR spectrum was recorded in the solid state potassium medium in Perkin Elmer FT-IR spectrophotometer. The NMR experiments were done in 400MHz Varian Mercury Plus FT-NMR spectrometer using DMSO-d6 solvent. The ESI Mass was performed on a LC-Mass spectrometer Agilent Model No.6410 with Mass hunter software and Agilent Model No.1200 infinity HPLC system.The single crystal X-ray was performed by using Bruker Smart Apex CCD diffractometer by using the SHELXTL-PLUS software.
RESULTS AND DISCUSSION:
The Normal phase chromatography was used for total study to differentiate the S and R isomer of Trans isomers and cis isomers. The column used in this study was Chiral Pak- IC, (250 x 4.6) mm, 5 mm particle size, the isocratic mobile phase was 0.05% Di ethyl amine in (n-Hexane: Ethanol: 90:10) (%v/v).The column temperature, detection, column load ,flow were 15°C, 210nm20 mL with 5.0 mg/mL, 0.6 mL/min ,the run time was 30 min ,the diluents was used in this study was (n-Hexane: Ethanol: 70:30) (%v/v), the all 4 isomers were well resoluted [2].
Synthesis of Boceprevir Fragment-B single crystal.
methyl(1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxylate hydrochloride was synthesized by slow evaporation technique, in this procedure the pure 15 mg Boceprevir Fragment-b was taken in 25 mL volumetric flask containing 7.5 mL of Dichloro methane and added 7.5 mL of di isopropyl ether and properly mixed by vortex mixer and covered with parafilm and made it holes for slow evaporation after 2-6 hrs, needle shaped crystal were obsereved by microscopic image Figure.3. These crystals were used for chiral purity and NMR, Mass, FT-IR and Thermal analysis and it can be used for the crystallography study. The following trails were performed for synthesis of single crystal.
Solvent Boceprevir Fragment- B Results (Crystal type)
Dichloro methane 15mg/15mL Spikes type of crystal
Toluene 15mg/15mL No crystal formation
Ethyl acetate 15mg/15mL Spikes type of crystal
Diethyl amine 15mg/15mL No distinct crystal
Dichloro methane +Ethyl acetate 15mg/15mL Spikes type of crystal
Dichloro methane +DIPE 15mg/15mL Needle shape single crystal with 0.16 x 0.14 x 0.05 mm3
Figure.3 Needle shape single crystal by Microscopy.
The Differential scanning calorimetry study stated that the Trans isomer was still it was in the single crystal and powder X ray studies reveals the crystallinity of the Boceprevir fragment-B crystal. The Specific optical rotation, 1H NMR, FT-IR and Mass stated that the chemicalmoiety was not changed. The free base mass number confirms the structure. The Onset, peak temperatures were 162.48°C 163.9°C respectively [Figure.4]. The specific optical rotation was -29.0500 ° at 25°C temperature with 1.0 % w/v in chloroform [Figure.5].The crystallinity was confirmed by compact intensities and 33 peaks [Figure.7]. The FT-IR bands and 1H NMR chemical shift values confirmed the structure of compound [Figure.8]. The free base mass number 170[M+H] confirms the structure [Figure.9]. The single crystal study was performed to confirm the structure, Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radii. Dashed line indicates hydrogen bond.
Table 1. Crystal data and structure refinement for Boceprevir Fragment-B.
Identification code DCM-DIPE-1/CBIC8 (IICT file code: AT21m)
Empirical formula C9H16 NO2+. Cl-
Formula weight 205.68
Temperature 294(2) K
Wavelength 0.71073 Å
Crystal system Orthorhombic
Space group P 212121
Unit cell dimensions a = 7.2827(5) Å = 90°.
b = 8.8683(6) Å = 90°.
c = 16.3243(11) Å = 90°.
Volume 1054.31(12) Å3
Z 4
Density (calculated) 1.296 Mg/m3
Absorption coefficient 0.332 mm-1
F(000) 440
Crystal size 0.16 x 0.14 x 0.05 mm3
qrange for data collection 2.50 to 25.00°.
Index ranges -8<=h<=8, -10<=k<=10, -19<=l<=19
Reflections collected 10105
Independent reflections 1855 [R(int) = 0.0232]
Completeness to q = 25.00° 100.0 %
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 1855 / 0 / 129
Goodness-of-fit on F2 1.072
Final R indices [I>2sigma(I)] R1 = 0.0295, wR2 = 0.0819
R indices (all data) R1 = 0.0302, wR2 = 0.0825
Absolute structure parameter 0.00(7)
Largest diff. peak and hole 0.164 and -0.161 e.Å-3
Measurement Bruker Smart Apex CCD diffractometer
Software Used SHELXTL-PLUS
Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) For Boceprevir Fragment-B. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
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x y z U(eq)
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C(1) 7518(2) 7877(2) 2348(1) 34(1)
C(2) 7793(2) 6577(2) 2924(1) 38(1)
C(3) 6601(3) 6833(2) 3667(1) 46(1)
C(4) 5612(3) 8301(3) 3562(1) 52(1)
C(5) 8660(3) 6857(2) 3747(1) 44(1)
C(6) 9657(3) 8296(3) 3923(2) 59(1)
C(7) 9493(4) 5486(3) 4141(1) 71(1)
C(8) 7036(2) 7382(2) 1482(1) 39(1)
C(9) 5671(4) 8183(3) 250(1) 58(1)
N(1) 5969(2) 8799(2) 2705(1) 40(1)
O(1) 7293(3) 6150(2) 1216(1) 60(1)
O(2) 6330(2) 8524(1) 1063(1) 45(1)
Cl(1) 7398(1) 12050(1) 2954(1) 51(1)
Table 3. Bond lengths [Å] and angles [°] for Boceprevir Fragment-B.
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C(1)-C(2) 1.501(2)
C(1)-N(1) 1.510(2)
C(1)-C(8) 1.522(2)
C(1)-H(1) 0.9800
C(2)-C(5) 1.505(2)
C(2)-C(3) 1.509(3)
C(2)-H(2) 0.9800
C(3)-C(4) 1.498(3)
C(3)-C(5) 1.506(3)
C(3)-H(3) 0.9800
C(4)-N(1) 1.490(3)
C(4)-H(4A) 0.9700
C(4)-H(4B) 0.9700
C(5)-C(6) 1.496(3)
C(5)-C(7) 1.503(3)
C(6)-H(6A) 0.9600
C(6)-H(6B) 0.9600
C(6)-H(6C) 0.9600
C(7)-H(7A) 0.9600
C(7)-H(7B) 0.9600
C(7)-H(7C) 0.9600
C(8)-O(1) 1.191(2)
C(8)-O(2) 1.326(2)
C(9)-O(2) 1.443(2)
C(9)-H(9A) 0.9600
C(9)-H(9B) 0.9600
C(9)-H(9C) 0.9600
N(1)-H(1N) 0.91(3)
N(1)-H(2N) 0.87(3)
C(2)-C(1)-N(1) 105.85(13)
C(2)-C(1)-C(8) 113.02(13)
N(1)-C(1)-C(8) 110.03(14)
C(2)-C(1)-H(1) 109.3
N(1)-C(1)-H(1) 109.3
C(8)-C(1)-H(1) 109.3
C(1)-C(2)-C(5) 119.23(14)
C(1)-C(2)-C(3) 108.14(15)
C(5)-C(2)-C(3) 59.95(12)
C(1)-C(2)-H(2) 118.3
C(5)-C(2)-H(2) 118.3
C(3)-C(2)-H(2) 118.3
C(4)-C(3)-C(5) 118.43(19)
C(4)-C(3)-C(2) 108.40(15)
C(5)-C(3)-C(2) 59.91(12)
C(4)-C(3)-H(3) 118.5
C(5)-C(3)-H(3) 118.5
C(2)-C(3)-H(3) 118.5
N(1)-C(4)-C(3) 106.31(16)
N(1)-C(4)-H(4A) 110.5
C(3)-C(4)-H(4A) 110.5
N(1)-C(4)-H(4B) 110.5
C(3)-C(4)-H(4B) 110.5
H(4A)-C(4)-H(4B) 108.7
C(6)-C(5)-C(7) 114.34(17)
C(6)-C(5)-C(2) 121.07(17)
C(7)-C(5)-C(2) 114.65(16)
C(6)-C(5)-C(3) 120.83(19)
C(7)-C(5)-C(3) 115.3(2)
C(2)-C(5)-C(3) 60.14(12)
C(5)-C(6)-H(6A) 109.5
C(5)-C(6)-H(6B) 109.5
H(6A)-C(6)-H(6B) 109.5
C(5)-C(6)-H(6C) 109.5
H(6A)-C(6)-H(6C) 109.5
H(6B)-C(6)-H(6C) 109.5
C(5)-C(7)-H(7A) 109.5
C(5)-C(7)-H(7B) 109.5
H(7A)-C(7)-H(7B) 109.5
C(5)-C(7)-H(7C) 109.5
H(7A)-C(7)-H(7C) 109.5
H(7B)-C(7)-H(7C) 109.5
O(1)-C(8)-O(2) 125.03(16)
O(1)-C(8)-C(1) 124.57(16)
O(2)-C(8)-C(1) 110.38(14)
O(2)-C(9)-H(9A) 109.5
O(2)-C(9)-H(9B) 109.5
H(9A)-C(9)-H(9B) 109.5
O(2)-C(9)-H(9C) 109.5
H(9A)-C(9)-H(9C) 109.5
H(9B)-C(9)-H(9C) 109.5
C(4)-N(1)-C(1) 109.43(14)
C(4)-N(1)-H(1N) 108.4(15)
C(1)-N(1)-H(1N) 108.5(19)
C(4)-N(1)-H(2N) 108.5(17)
C(1)-N(1)-H(2N) 106.3(17)
H(1N)-N(1)-H(2N) 116(2)
C(8)-O(2)-C(9) 116.29(15)
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Table 4. Anisotropic displacement parameters (Å2x 103)forBoceprevir Fragment-B. The anisotropic displacement factor exponent takes the form: -22[ h2a*2U11 + ... + 2 h k a* b* U12 ]
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U11 U22 U33 U23 U13 U12
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C(1) 35(1) 31(1) 38(1) -2(1) 0(1) -3(1)
C(2) 47(1) 31(1) 37(1) -1(1) -3(1) 1(1)
C(3) 56(1) 45(1) 38(1) 5(1) 5(1) -8(1)
C(4) 48(1) 68(1) 41(1) -4(1) 7(1) 8(1)
C(5) 56(1) 38(1) 39(1) -2(1) -8(1) 5(1)
C(6) 63(1) 54(1) 62(1) -10(1) -18(1) -5(1)
C(7) 108(2) 51(1) 52(1) -2(1) -27(1) 22(1)
C(8) 42(1) 38(1) 37(1) 2(1) 3(1) 0(1)
C(9) 78(2) 58(1) 39(1) -2(1) -12(1) 10(1)
N(1) 45(1) 33(1) 41(1) -5(1) -4(1) 4(1)
O(1) 86(1) 49(1) 45(1) -11(1) -8(1) 22(1)
O(2) 62(1) 37(1) 36(1) 3(1) -5(1) -2(1)
Cl(1) 50(1) 39(1) 64(1) -1(1) -5(1) 2(1)
Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103) forBoceprevir Fragment-B.
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x y z U(eq)
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H(1) 8637 8491 2334 41
H(2) 7857 5558 2694 46
H(3) 5926 5974 3895 55
H(4A) 4306 8170 3653 63
H(4B) 6071 9042 3949 63
H(6A) 8986 9127 3695 89
H(6B) 10859 8256 3683 89
H(6C) 9766 8426 4505 89
H(7A) 9379 5561 4725 106
H(7B) 10768 5420 3995 106
H(7C) 8864 4599 3953 106
H(9A) 6697 8053 -113 88
H(9B) 4917 8997 57 88
H(9C) 4961 7271 264 88
H(1N) 6320(40) 9780(30) 2716(15) 69(7)
H(2N) 5010(40) 8600(30) 2413(15) 58(7)
Table 6.Torsion angles [°] for Boceprevir Fragment-B.
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N(1)-C(1)-C(2)-C(5) -72.4(2)
C(8)-C(1)-C(2)-C(5) 167.10(15)
N(1)-C(1)-C(2)-C(3) -7.27(18)
C(8)-C(1)-C(2)-C(3) -127.76(15)
C(1)-C(2)-C(3)-C(4) -1.1(2)
C(5)-C(2)-C(3)-C(4) 112.8(2)
C(1)-C(2)-C(3)-C(5) -113.83(16)
C(5)-C(3)-C(4)-N(1) 74.2(2)
C(2)-C(3)-C(4)-N(1) 9.1(2)
C(1)-C(2)-C(5)-C(6) -15.1(3)
C(3)-C(2)-C(5)-C(6) -110.1(2)
C(1)-C(2)-C(5)-C(7) -158.74(19)
C(3)-C(2)-C(5)-C(7) 106.2(2)
C(1)-C(2)-C(5)-C(3) 95.08(19)
C(4)-C(3)-C(5)-C(6) 14.7(3)
C(2)-C(3)-C(5)-C(6) 110.5(2)
C(4)-C(3)-C(5)-C(7) 159.08(16)
C(2)-C(3)-C(5)-C(7) -105.11(18)
C(4)-C(3)-C(5)-C(2) -95.81(18)
C(2)-C(1)-C(8)-O(1) -18.3(3)
N(1)-C(1)-C(8)-O(1) -136.42(19)
C(2)-C(1)-C(8)-O(2) 163.07(14)
N(1)-C(1)-C(8)-O(2) 44.99(18)
C(3)-C(4)-N(1)-C(1) -13.9(2)
C(2)-C(1)-N(1)-C(4) 13.18(19)
C(8)-C(1)-N(1)-C(4) 135.60(16)
O(1)-C(8)-O(2)-C(9) 6.3(3)
C(1)-C(8)-O(2)-C(9) -175.15(16)
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Table 7.Hydrogen bonds for Boceprevir Fragment-B [Å and °].
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D-H...A d(D-H) d(H...A) d(D...A) <(DHA)
___________________________________________________________________________________________________________
N(1)-H(1N)...Cl(1) 0.91(3) 2.20(3) 3.0924(17) 170(2)
N(1)-H(2N)...Cl(1)#1 0.87(3) 2.31(3) 3.0938(18) 151(2)
___________________________________________________________________________________________________________
Symmetry transformations used to generate equivalent atoms:
#1 -x+1,y-1/2,-z+1/2 .
STERIOCHEMISTRY OBSERVED USING PLATON (A.L. SPEK. J.APPL.CRYST. 36, 7-13, 2003)
C(1) Chiral: S
C(2) Chiral: R
C(3) Chiral: S
Figure.1. Typical Boceprevir Fragment-B Chromatogram (Chemical analysis)
Figure.2.Blank chromatogram (Chiral analysis)
Figure.3 Typical Boceprevir Fragment-B chromatogram (Chiral analysis)
Figure.4DSC thermogram
Figure.5SOR Results
Figure.6A) FT-IR Spectrum B) 1H NMR Spectrum.
Figure.7. A)Powder XRD diffractogram results of Boceprevir Fragment-B, B) Peak search tables results of Boceprevir Fragment-B.
Figure.8. Mass spectrum of Boceprevir Fragment-B
CONCLUSION:
The single crystal of methyl (1R,2S,5S)-6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxylate hydrochloride was synthesized and identified the S isomer by chiral chromatography, spectroscopy and single crystal study. The crystallography study was done for the absolute configuration of the Boceprevir fragment-B of Boceprevir drug substance; this is key starting material for Boceprevir drug substance.
CONFLICT OF INTEREST:
No conflict of interest.
Dr. Reddy’s Laboratory IPDO IPM-00528 has been allotted for the attached research article.
ACKNOWLEDGEMENTS:
Authors thanks to PRD colleagues of custom pharmaceutical services Dr. Reddy’s Laboratories Hyderabad, for providing the high pure Boceprevir fragment-B and also thanks to A R& D colleagues of custom pharmaceutical services Dr. Reddy’s Laboratories Hyderabad. Thank to Dr. B. Sridhar for executing single crystal analysis and also thank to Dr. K. Ravi Kumar Laboratory of X-ray Crystallography IICT, Hyderabad for verifying the single crystal analysis data.
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3. Development and validation of RP-HPLC method for the analysis of boceprevir and related impurities in bulk and pharmaceutical dosage forms. Shiny Ganji, Dr. D. Satyavati.
4. Rapid RP-HPLC method development and validation for the quantitative estimation ribavirin in tablets G. Raveendra Babu, A. Lakshmana Rao, J. Venkateswara Rao.
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6. Development and validation of stability-indicating HPTLC method for determination of boceprevir Mrinalini C. Damle and Sunny R. Salunke.
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Received on 15.04.2018 Modified on 27.04.2018
Accepted on 30.04.2018 ©AJRC All right reserved
AsianJ.ResearchChem.2018;11(3):671-680.
DOI: 10.5958/0974-4150.2018.00120.7