CC BY
22DOI: 10.17516/1998-2836-0199 УДК 547.455.522
Ribose Moieties Acylation and Characterization of Some Cytidine Analogs
Kazi M. Ranaa, Jannatul Ferdousa, Anowar Hosenb and Sarkar M.A. Kawsar*a
aLaboratory of Carbohydrate and Nucleoside Chemistry (LCNC) Department of Chemistry, Faculty of Science University of Chittagong Chittagong, Bangladesh bCentre for Advanced Research in Sciences
University of Dhaka Dhaka, Bangladesh
Received 03.10.2020, received in revised form 10.11.2020, accepted 03.12.2020
Abstract. Modification of naturally occurring nucleosides is an important area in the search for new agents with therapeutic potential. In this study, nucleoside molecules, that is, cytidine analogs bearing ribose moieties were successfully synthesized to obtain 5'-O-acyl cytidine (2), which in turn was converted into 2',3'-di-O-acyl cytidine (3-7) through direct acylation. Similarly, several cytidine analogs (8-15) were formed using the aforementioned technique. Physicochemical properties and spectroscopic methods were used to characterize the newly synthesized cytidine analogs. X-ray powder diffraction was employed for quantitatively identifying crystalline compounds. Hence, these synthesized derivatives can be used as potential antimicrobial agents and promising drug candidates.
Keywords: ribose, acylation, analogs, cytidine, spectroscopy.
Citation: Rana K.M., Ferdous J., Hosen A., Kawsar S.M.A. Ribose moieties acylation and characterization of some cytidine analogs, J. Sib. Fed. Univ. Chem., 2020, 13(4), 465-478. DOI: 10.17516/1998-2836-0199
© Siberian Federal University. All rights reserved
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). Corresponding author E-mail address: [email protected]
*
Ацилирование рибозных фрагментов и характеристики некоторых аналогов цитидина
К.М. Ранаа, Дж. Фердоуса, А. Хосен6, С.М.А. Кавсара
аЛаборатория химии карбогидратов и нуклеозидов (LCNC) Департамент химии, Факультет наук Университет Читтагонг Бангладеш, Читтагонг бЦентр перспективных исследований в науке
Университет Дакка Бангладеш, Дакка
Аннотация. Модификация природных нуклеозидов - важная область в поиске новых агентов с терапевтическим потенциалом. В этом исследовании нуклеозидные молекулы, являющиеся аналогами цитидина, с рибозными фрагментами были успешно использованы для синтеза 5-0-ацил цитидина (2), который в свою очередь был превращен в 2,3-ди-0-ацил цитидин (3-7) путем прямого ацилирования. Точно так же, с использованием вышеописанного метода, получено несколько аналогов цитидина (8-15). Синтезированные новые аналоги цитидина были охарактеризованы физико-химическими методами. Для количественной идентификации кристаллических соединений применили метод порошковой рентгеновской дифракции. Эти синтезированные производные могут быть использованы как потенциальные антимикробные агенты и перспективные лекарственные препараты.
Ключевые слова: рибоза, ацилирование, аналоги, цитидин, спектроскопия.
Цитирование: Рана, К.М. Ацилирование рибозных фрагментов и характеристики некоторых аналогов цитидина / К.М. Рана, Дж. Фердоус, А. Хосен, С.М.А. Кавсар // Журн. Сиб. федер. ун-та. Химия, 2020. 13(4). С. 465-478. DOI: 10.17516/1998-2836-0199
Introduction
Nucleosides and their analogues are of enormous importance. They are an established class of clinically useful medicinal agents, possessing antiviral and anticancer activity [1-3]. A number of different types of nucleosides have been synthesized from time to time which are reported to be biologically active, for example, ribavirin or virazole is an important such nucleoside. It has been reported as one of the most powerful synthetic antiviral agent's active against DNA viruses [4]. Virazole has been approved by the FDA for the treatment of viral infections [5]. The 5-azacytidine has been shown to possess promising activity against adult non-lymphocytic leukemia. It can either be synthesized chemically or produced microbiologically. It mainly affects the synthesis and function of DNA [6]. 2',3'-Dideoxycytidine also been reported to inhibit HIV and its clinical trials have been successfully carried out at NIH in AIDS patients [7]. Structural modifications of nucleosides have
Fig. 1. Structure of cytidine
given rise to widely used drugs such as zidovudine [8] and acyclovir [9], which demonstrates that this strategy offers interesting opportunities to synthesize new therapeutically useful compounds.
Cytidine (Fig. 1) is a nucleoside that consists of a sugar part, ribose, which is linked to the pyrimidine base cytosine via a P-glycosidic bond. Cytidine is a component of RNA and a precursor for uridine. When RNA-rich food is consumed, RNA is broken down into ribosyl pyrimidines (cytidine and uridine) and its basic elements are released for absorption from intestine [10]. RNA-rich foods are considered good cytidine sources. The supplementation of dietary cytidine (5')-diphosphocholine protects against the development of memory deficits [11].
Modifications in the sugar moiety of nucleosides have resulted in various effective therapeutic applications. In the last few years, many researchers have investigated the selective acylation of the hydroxyl groups of the ribose moieties of nucleosides and nucleotides by using various methods [12, 13]. Different methods for nucleoside acylation have been successfully developed and employed [14, 15]. Among those, a direct method is most encouraging for nucleoside acylation [16].
Encouraged by the literature [17, 18] and our findings [19-21], we synthesized some selectively acylated analogs of cytidine (schemes 1-4) containing various substituents in a single molecular framework and X-ray diffraction studies on them for the first time.
Experimental
Materials and methods
Thin layer chromatography (TLC) was performed on Kieselgel GF254, and visualization was achieved by spraying plates with 1% H2SO4 followed by heating the plates at 150-200 °C until coloration occurred. Melting points (m.p.) were determined using an electrothermal melting point apparatus and were uncorrected. Evaporation was performed using a Buchi rotary evaporator under diminished pressure. Analytical grade solvents were employed and purified using standard procedures. Infrared (IR) spectral analyses were conducted using a Fourier transform IR (FTIR) spectrophotometer (IR Prestige-21, Shimadzu, Japan) within 200-4000 cm-1 at the Department of Chemistry, University of Chittagong, Bangladesh. The mass spectra of the synthesized compounds were recorded through liquid chromatography electrospray ionization-tandem mass spectrometry in the positive ionization mode (LC/ESI(+)-MS/MS) by using a system comprising a JASSO LC (JASCO, Japan). A Brucker advance DPX 400 MHz with tetramethylsilane as an internal standard was used to record 'H-NMR
spectra in CDCl3 (5 in ppm) at WMSRC, JU, Bangladesh. XRD patterns were obtained using an XRD-53 analyzer, Rigaku, Japan diffractometer, with a back monochromator and Cu target and Ka (X = 1.5406 nm) in 20 = 2°-70° at CARS, Dhaka University, Bangladesh. Column chromatography was performed with silica gel G6o. CHCl3/CH3OH was employed as the solvent system for TLC analyses was in different proportions. All reagents used were commercially available (Aldrich) and used without further purification unless otherwise specified.
Synthesis
Over past several years, our laboratory has been synthesizing nucleoside derivatives containing various acyl groups to explore their antimicrobial properties [30, 31].
In dry DMF (A,A-dimethylformamide) (3 ml), a cytidine (1) (70 mg, 0.287 mmol) solution was cooled to -5 °C when decanoyl chloride (65 mg, 1.1 molar eq.) was added. The solution was stirred at this temperature for 5 to 6 h and then was allowed to stand at room temperature overnight. Reaction progress was monitored through TLC, which indicated the complete conversion of the starting material into a single product. A few pieces of ice were added to the flask to stop the reaction. Subsequently, the solvent was evaporated using a high pressure vacuum evaporator, and the resulted product was passed through silica gel column chromatography and eluted with (1:24), which provided the decanoyl derivative (2) (92 mg). The recrystallization of (CHCl3-C6Hi4) led to the formation of the title derivative (2) as needles. The compound was sufficiently pure for subsequent use without further treatment and identification.
5'-O-Decanoylcytidine (2): Yield 83.8% as crystalline solid, M.P. 85-87 °C (CHCl3-C6H14), Rf = 0.51 (CHCl3/CH3OH = 24/1, v/v). FTIR: vmax 1731, 1714 (-CO), 3550 (-NH), 3420 cm-1 (-OH). *H -NMR (400 MHz, CDCl3): 5H 9.02 (1H, s, -NH), 7.44 (1H, d, J = 7.8 Hz, H-6), 6.56 (1H, d, J = 3.0 Hz, H-1'), 6.49 (1H, s, 2'-OH), 6.01 (1H, dd, J = 2.4 and 12.3 Hz, H-5'a), 5.56 (1H, dd, J = 2.4 and 12.3 Hz, H-5'b), 5.45 (1H, s, 3'-OH), 4.85 (1H, dd, J = 2.4 and 5.6 Hz, H-4'), 4.60 (2H, s, -NH2), 4.45 (1H, d, J = 3.2 Hz, H-2'), 4.31 (1H, dd, J = 3.6 and 5.8 Hz, H-3'), 3.80 (1H, d, J = 7.2 Hz, H-5), 2.38 {2H, m, CH3(CH2)7CH2CO-}, 1.59 {2H, m, CH3(CH2)6CH2CH2CO-}, 1.32 {12H, m, CH3(CH2)6(CH2)2CO-}, 0.85 {3H, m, CH3(CH2)8CO-}. MS [M+1]+ 398.10.
Anal Calcd. for C19H31N3O6 : % C, 72.54, H, 7.81; found: % C, 72.53, H, 7.82.
General procedure for the direct 2',3'-di-O-acylation of 5-O-decanoylcytidine (2) derivatives (3-7)
In DMF (3 ml), octanoyl chloride (0.185 ml, 4 molar eq.) was added to a cooled (0 °C) and stirred solution of the decanoyl derivative (2) (110 mg, 0.276 mmol). The mixture was stirred at 0 °C for 8 h and then allowed to stand overnight at room temperature. TLC analyses showed the complete conversion of reactants into a single product. A few pieces of ice were added to the reaction flask to eliminate the excess reagent, and the reaction mixture was evaporated using the high-pressure vacuum evaporator to remove the solvent. The percolation of the resulting product achieved by passing through a silica gel column with the CHCl3-CH3OH eluant led to the formation of the octanoyl derivative (3) (163 mg) as a crystalline solid.
A similar reaction and purification procedure was employed to prepare compounds (4), (5), (6), and (7).
5'- O-Decanoyl-2',3'-di- O-octanoylcytidine (3): Yield 77.6% as a white crystalline solid, M.P. 89-91 °C (CHCl3-C6H14), Rf = 0.51 (CHCl3/CH3OH = 24/1, v/v). FTIR: vmax 1729, 1716 (-CO), 3470 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.18 (1H, s, -NH), 7.27 (1H, d, J = 7.8 Hz, H-6), 6.54 (1H, d, J = 3.2 Hz, H-1'), 5.45 (1H, m, H-2'), 4.82 (1H, dd, J = 3.5 and 5.6 Hz, H-3'), 4.67 (1H, dd, J = 2.4 and 12.2 Hz, H-5'a), 4.58 (2H, s, -NH2), 4.55 (1H, dd, J = 2.4 and 12.2 Hz, H-5'b), 4.38 (1H, dd, J = 2.4 and 5.5 Hz, H-4'), 3.70 (1H, d, J = 7.2 Hz, H-5), 2.37 {4H, m, 2xCH3(CH2)5CH2CO-}, 1.64 {4H, m, 2xCH3(CH2)4CH2CH2CO-}, 1.29 {16H, m, 2xCH3(CH2)4(CH2)2CO-}, 0.89 {6H, m, 2xCH3(CH2)6CO-}. MS [M+1]+ 650.09.
Anal Calcd. for C35H59O8N3: % C, 64.71, H, 9.09; % found: C, 64.73, H, 9.06.
5'- O-Decanoyl-2',3'-di- O-palmitoylcytidine (4): Yield 91.2% as a white crystalline solid, M.P. 78-79 °C (CHCl3-C6H14), Rf = 0.54 (CHCl3/CH3OH = 24/1, v/v). FTIR: vmax 1696 (-CO), 3500 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5h 9.10 (1H, s, -NH), 7.29 (1H, d, J = 7.7 Hz, H-6), 6.52 (1H, d, J = 3.2 Hz, H-1'), 5.48 (1H, m, H-2'), 4.86 (1H, dd, J = 3.5 and 5.6 Hz, H-3'), 4.67 (1H, dd, J = 2.4 and 12.1 Hz, H-5'a), 4.58 (2H, s, -NH2), 4.56 (1H, dd, J = 2.4 and 12.1 Hz, H-5'b), 4.41 (1H, dd, J = 2.4 and 5.5 Hz, H-4'), 3.81 (1H, m, H-5), 2.36 {4H, m, 2xCH3(CH2)13CH2CO-}, 1.27 {52H, m, 2xCH3(CH2)13CH2CO-}, 0.91 {6H, m, 2xCH3(CH2)14CO-}. MS [M+1]+ 874.30.
Anal Calcd. for C51H91O8N3: % C, 70.10, H, 10.42; found: % C, 70.13, H, 10.40.
5'-O-Decanoyl-2',3'-di-O-stearoylcytidine (5): Yield 85.3% as crystalline solid, M.P. 9597 °C (CHCl3-C6H14), Rf = 0.50 (CHCl3/CH3OH = 23/1, v/v). FTIR: vmax 1709 (-CO), 3490 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.08 (1H, s, -NH), 7.28 (1H, d, J = 7.7 Hz, H-6), 6.68 (1H, d, J = 3.2 Hz, H-1'), 6.48 (1H, m, H-2'), 6.21 (1H, dd, J = 3.5 and 5.6 Hz, H-3'), 6.07 (1H, dd, J = 2.4 and 12.1 Hz, H-5'a), 5.51 (1H, dd, J = 2.4 and 12.1 Hz, H-5'b), 5.21 (1H, dd, J = 2.4 and 5.5 Hz, H-4'), 4.88 (2H, s, -NH2), 4.11 (1H, m, H-5), 2.36 {4H, m, 2xCH3(CH2)15CH2CO-}, 1.65 {4H, m, 2xCH3(CH2)14CH2CH2CO-}, 1.27 {56H, m, 2xCH3(CH2)14CH2CH2CO-}, 0.90 {6H, m, 2XCH3(CH2)16CO-}. MS [M+1]+ 930.23.
Anal Calcd. for C55H99O8N3: % C, 71.0, H, 10.60; % found: C, 71.03, H, 10.63.
5- O-Decanoyl-2',3'-di- O-(triphenylmethyl)cytidine (6): Yield 84.3% as a white crystalline solid, M.P. 102-104 °C (CHCl3-C6H14), Rf = 0.52 (CHCl3/CH3OH = 22/1, v/v). FTIR: vmax 1686 (-CO), 3496 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.02 (1H, s, -NH), 7.58 (12H, m, 2xAr-H), 7.36 (18H, m, 2xAr-H), 7.22 (1H, d, J = 7.6 Hz, H-6), 6.61 (1H, d, J = 3.2 Hz, H-1'), 5.47 (1H, d, J = 3.2 Hz, H-2'), 4.88 (1H, dd, J = 3.6 and 5.6 Hz, H-3'), 4.68 (1H, dd, J = 2.3 and 12.1 Hz, H-5'a), 4.60 (2H, s, -NH2), 4.57 (1H, dd, J = 2.4 and 12.1 Hz, H-5'b), 4.49 (1H, dd, J = 2.4 and 5.5 Hz, H-4'), 3.92 (1H, m, H-5). MS [M+1]+ 882.12.
Anal Calcd. for C57H59O6N3: % C, 77.60, H, 6.71; % found: C, 77.62, H, 6.73.
5'-O-Decanoyl-2',3'-(4-terf-butylbenzoyl)cytidine (7): Yield 90.4% as crystalline solid, M.P. 109-111 °C (CHCl3-C6H14), Rf = 0.52 (CHCl3/CH3OH = 24/1, v/v). FTIR: vmax 1712 (-CO), 3501 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.03 (1H, s, -NH), 8.06 (4H, m, 2xAr-H), 7.51 (4H, m, 2xAr-H), 7.28 (1H, d, J = 7.7 Hz, H-6), 6.68 (1H, d, J = 3.2 Hz, H-1'), 5.57 (1H, m, H-2'), 4.96 (1H, dd, J = 3.5 and 5.6 Hz, H-3'), 4.66 (1H, dd, J = 2.4 and 12.1 Hz, H-5'a), 4.54 (2H, s, -NH2), 4.52 (1H, dd, J = 2.4 and 12.1 Hz, H-5'b), 4.25 (1H, dd, J = 2.4 and 5.5 Hz, H-4'), 4.01 (1H, m, H-5), 1.33, 1.28 {18H, 2xs, 2X(CH3)3C-}. MS [M+1]+ 718.11.
Anal Calcd. for C41H55O8N3: % C, 68.60, H, 7.60; % found: C, 68.63, H, 7.58.
In dry DMF (3 ml), a cytidine (1) (70 mg, 0.287 mmol) solution was cooled to -5 °C and treated with 1.1 molar equivalent of triphenylmethyl chloride (85 mg) with continuous stirring at the same temperature for 5 to 6 h. Stirring was continued overnight at room temperature. Reaction progress was monitored through TLC. A few pieces of ice were added to the flask to terminate the reaction. Subsequently, the solvent was evaporated using the high-pressure vacuum evaporator. The resulting syrupy mass was purified through silica gel column chromatography (with CHCl3-CH3OH, 1:24 eluant) to acquire the title compound (8, 107 mg) as a crystalline solid.
5'- O-(Triphenylmethyl)cytidine (8): Yield 81.2% as a white crystalline solid, M.P. 75-77 °C) (CHCl3-C6H14), Rf = 0.50 (CHCl3/CH3OH = 24/1, v/v). FTIR: vmax 1701 (-CO), 3547 (-NH), 3416-3468 (br) cm-1 (-OH). *H -NMR (400 MHz, CDCl3): 5H 9.01 (1H, s, -NH), 7.35 (6H, m, Ar-H), 7.30 (9H, m, Ar-H), 7.23 (1H, d, J = 7.8 Hz, H-6), 6.61 (1H, d, J = 3.0 Hz, H-1'), 6.48 (1H, s, 2'-OH), 5.30 (1H, dd, J = 2.2 and 12.2 Hz, H-5'a), 5.26 (1H, dd, J = 2.2 and 12.2 Hz, H-5'b), 5.15 (1H, s, 3'-OH), 4.85 (1H, dd, J = 2.2 and 5.6 Hz, H-4'), 4.50 (2H, s, -NH2), 4.25 (1H, d, J = 3.2 Hz, H-2'), 4.01 (1H, dd, J = 3.6 and 5.8 Hz, H-3'), 3.91 (1H, d, J = 7.2 Hz, H-5). MS [M+1]+ 486.08.
Anal Calcd. for C28H27O5N3: % C, 69.28, H, 5.56; % found: C, 69.27, H, 5.53.
General procedure for the direct 2',3'-di-O-acylation of 5'-O-(triphenylmethyl)cytidine derivatives (9-15)
The triphenylmethyl derivative (8, 60 mg, 0.124 mmol) was dissolved in dry DMF (3 ml), and the solution was cooled to 0 °C when hexanoyl chloride (0.067 ml, 4 molar eq.) was added. The mixture was stirred at 0 °C for 6 h and at room temperature overnight. The conventional work-up procedure and subsequent chromatographic purification with the CHCl3-CH3OH (1:24) eluant led to the formation of 2',3'-di- O-hexanoyl derivative (9) (157 mg).
2,3-Di-O-hexanoyl-5-O-(triphenylmethyl)cytidine (9): Yield 81.5% as crystalline solid, M.P. 100-102 °C, Rf = 0.52 (CHCl3/CH3OH = 22/1, v/v). FTIR: vmax 1710 (-CO), 3503 cm-1 (-NH). 'H-NMR (400 MHz, CDCl3): 5H 9.08 (1H, s, -NH), 7.32 (6H, m, Ar-H), 7.28 (9H, m, Ar-H), 7.21 (1H, d, J = 7.6 Hz, H-6), 6.51 (1H, d, J = 3.2 Hz, H-1'), 5.47 (1H, m, H-2'), 4.88 (1H, dd, J = 3.2 and 5.4 Hz, H-3'), 4.61 (1H, dd, J = 2.2 and 12.2 Hz, H-5'a), 4.54 (2H, s, -NH2), 4.48 (1H, dd, J = 2.2 and 12.2 Hz, H-5'b), 4.35 (1H, dd, J = 2.2 and 5.4 Hz, H-4'), 3.81 (1H, d, J = 7.1 Hz, H-5), 2.31 {4H, m, 2xCH3(CH2)3CH2CO-}, 1.62 {4H, m, 2xCH3(CH2)2CH2CH2CO-}, 1.26 {8H, m, 2xCH3(CH2)2CH2CH2CO-}, 0.88 {6H, m, 2xcH3(ch2)4co-}. MS [M+1]+ 682.05.
Anal Calcd. for C40H47O7N3: % C, 70.40, H, 6.90; % found: C, 70.42, H, 6.91.
A similar reaction and purification method were employed to synthesize compounds (10) (164 mg), (11) (152 mg), (12) (109.5 mg), (13) (100.4 mg), (14) (132 mg), and (15) (111 mg).
2', 3-Di-O-heptanoyl-5 -O-(triphenylmethyl)cytidine (10): Yield 78.9% as crystalline solid, M.P. 98-99 °C (CHCI^Hm), Rf = 0.50 (CHCl3/CH3OH = 24/1, v/v). FTIR: vm£K 1715 (-CO), 3506 cm-1 (-NH). 'H-NMR (400 MHz, CDCl3): 5H 9.03 (1H, s, -NH), 7.30 (6H, m, Ar-H), 7.28 (9H, m, Ar-H), 7.20 (1H, d, J = 7.4 Hz, H-6), 6.44 (1H, d, J = 3.2 Hz, H-1'), 5.50 (1H, d, J = 3.2 Hz, H-2'), 5.24 (1H, dd, J = 3.2 and 5.4 Hz, H-3'), 5.06 (1H, dd, J = 2.2 and 12.2 Hz, H-5'a), 4.65 (2H, s, -NH2), 4.02 (1H, dd, J = 2.1 and 12.1 Hz, H-5'b), 3.95 (1H, dd, J = 2.2 and 5.3 Hz, H-4'), 3.80 (1H, m, H-5), 2.41 {4H, m, 2xCH3(CH2)4CH2CO-}, 1.62 {4H, m, 2xCH3(CH2)3CH2CH2CO-}, 1.35 {12H, m, 2xCH3(CH2)3CH2CH2CO-}, 0.91 {6H, m, 2xCH3(CH2)5CO-}. MS [M+1]+ 710.14.
Anal Calcd. for C42H53O7N3: % C, 71.00, H, 7.10; % found: C, 71.02, H, 7.09.
2',3'-Di-O-lauroyl-5'-O-(triphenylmethyl)cytidine (11): Yield 93.2% as a white crystalline solid, M.P. 107-109 °C (CHCl3-C6H14), Rf = 0.50 (CHCl3/CH3OH = 23/1, v/v). FTIR: vmax 1735 (-CO), 3500 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.00 (1H, s, -NH), 7.36 (6H, m, Ar-H), 7.31 (9H, m, Ar-H), 7.28 (1H, d, J = 7.3 Hz, H-6), 6.47 (1H, d, J = 3.3 Hz, H-1'), 5.57 (1H, d, J = 3.2 Hz, H-2'), 5.27 (1H, dd, J = 3.2 and 5.4 Hz, H-3'), 5.11 (1H, dd, J = 2.1 and 12.1 Hz, H-5'a), 4.58 (2H, s, -NH2), 4.11 (1H, dd, J = 2.2 and 12.2 Hz, H-5'b), 3.99 (1H, dd, J = 2.1 and 5.2 Hz, H-4'), 3.85 (1H, d, J = 7.1 Hz, H-5), 2.37 {4H, m, 2xCH3(CH2)9CH2CO-}, 1.68 {4H, m, 2xCH3(CH2)8CH2CH2CO-}, 1.29 {32H, m, 2xCH3(CH2)8CH2CH2CO-}, 0.89 {6H, m, 2xCH3(CH2)10CO-}. MS [M+1]+ 850.23.
Anal Calcd. for C52H71O7N3: % C, 73.50, H, 8.36; % found: C, 73.49, H, 8.35.
2',3'-Di-O-myristoyl-5'-O-(triphenylmethyl)cytidine (12): Yield 88.75.3% as a crystalline solid, M.P. 96-98 °C (CHCl3-C6H!4), Rf = 0.51 (CHCl3/CH3OH = 24/1, v/v). FTIR: vm£K 1726 (-CO), 3470 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.09 (1H, s, -NH), 7.38 (6H, m, Ar-H), 7.33 (9H, m, Ar-H), 7.29 (1H, d, J = 7.4 Hz, H-6), 6.40 (1H, d, J = 3.2 Hz, H-1'), 5.51 (1H, d, J = 3.2 Hz, H-2'), 5.20 (1H, dd, J = 3.1 and 5.2 Hz, H-3'), 5.01 (1H, dd, J = 2.1 and 12.1 Hz, H-5'a), 4.59 (2H, s, -NH2), 4.10 (1H, dd, J = 2.1 and 12.1 Hz, H-5'b), 3.89 (1H, dd, J = 2.1 and 5.2 Hz, H-4'), 3.87 (1H, d, J = 7.1 Hz, H-5), 2.38 {4H, m, 2xCH3(CH2)„CH2CO-}, 1.68 {4H, m, 2xCH3(CH2)10CH2CH2CO-}, 1.29 {40H, m, 2xCH3(CH2)10CH2CH2CO-}, 0.95 {6H, m, 2xCH3(CH2)12CO-}. MS [M+1]+ 906.04.
Anal Calcd. for C56H79O7N3: % C, 74.25, H, 8.73; % found: C, 74.27, H, 8.74.
2',3'-Di-O-pivaloyl-5- O-(triphenylmethyl)cytidine (13): Yield 91.4% as crystalline solid, M.P. 96-97 °C (CHCl3-C6H14), Rf = 0.55 (CHCl3/CH3OH = 24/1, v/v). FTIR: vmax 1718 (-CO), 3501 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.04 (1H, s, -NH), 7.33 (6H, m, Ar-H), 7.30 (9H, m, Ar-H), 7.20 (1H, d, J = 7.5 Hz, H-6), 6.36 (1H, d, J = 3.1 Hz, H-1'), 5.50 (1H, d, J = 3.1 Hz, H-2'), 5.10 (1H, dd, J = 3.2 and 5.2 Hz, H-3'), 5.00 (1H, dd, J = 2.2 and 12.2 Hz, H-5'a), 4.89 (2H, s, -NH2), 4.11 (1H, dd, J = 2.2 and 12.1 Hz, H-5'b), 3.92 (1H, dd, J = 2.2 and 5.4 Hz, H-4'), 3.90 (1H, d, J = 7.1 Hz, H-5), 1.26 {18H, s, 2x(CH3)3CCO-}. MS [M+1]+ 654.01.
Anal Calcd. for C38H43O7N3: % C, 69.83, H, 6.58; % found: C, 69.84, H, 6.60.
2',3'-Di-O-(4-chlorobenzoyl)-5'-O-(triphenylmethyl) cytidine (14): Yield 78.3% as a white crystalline solid, M.P. 99-101 °C (CHCl3-C6H14), Rf = 0.52 (CHCl3/CH3OH = 22/1, v/v). FTIR: vmax 1711 (-CO), 3505 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.01 (1H, s, -NH), 7.98 (4H, m, Ar-H), 7.58 (4H, m, Ar-H), 7.32 (6H, m, Ar-H), 7.28 (9H, m, Ar-H), 7.21 (1H, d, J = 7.5 Hz, H-6), 6.44 (1H, d, J = 3.1 Hz, H-1'), 5.58 (1H, d, J = 3.2 Hz, H-2'), 5.12 (1H, dd, J = 3.2 and 5.2 Hz, H-3'), 5.02 (1H, dd, J = 2.2 and 12.2 Hz, H-5'a), 4.60 (2H, s, -NH2), 4.10 (1H, dd, J = 2.1 and 12.1 Hz, H-5'b), 4.00 (1H, dd, J = 2.2 and 5.4 Hz, H-4'), 3.92 (1H, d, J = 7.1 Hz, H-5). MS [M+1]+ 763.22.
Anal Calcd. for C42H33O7N3Cl2: % C, 66.14, H, 4.33; % found: C, 66.12, H, 4.32.
2',3'-Di-O-cinnamoyl-5'-O-(triphenylmethyl)cytidine (15): Yield 88.5% white crystalline solid, M.P. 102-104 °C (CHCl3-C6H14), Rf = 0.50 (CHCl3/CH3OH = 22/1, v/v). FTIR: vmax 1678 (-CO), 3492 cm-1 (-NH). 1H-NMR (400 MHz, CDCl3): 5H 9.02 (1H, s, -NH), 7.85 (4H, m, Ar-H), 7.40 (6H, m, Ar-H), 7.60, 7.57 (2x1H, 2xd, J = 16.1 Hz, 2xPhCH=CHCO-), 7.36 (6H, m, Ar-H), 7.31 (9H, m, Ar-H), 7.28 (1H, d, J = 7.4 Hz, H-6), 6.45 (1H, d, J = 3.2 Hz, H-1'), 6.48, 6.42 (2x1H, 2xd, J = 16.1 Hz, 2xPhCH=CHCO-), 5.68 (1H, d, J = 3.3 Hz, H-2'), 5.34 (1H, dd, J = 3.3 and 5.3 Hz, H-3'), 4.97 (1H, dd, J = 2.2 and 12.2 Hz, H-5'a), 4.61 (2H, s, -NH2), 4.31 (1H, dd, J = 2.2 and
12.2 Hz, H-5b), 4.15 (1H, dd, J = 2.2 and 5.4 Hz, H-4), 3.62 (1H, d, J = 7.1 Hz, H-5). MS [M+1]+ 743.30.
Anal Calcd. for C46H36O7N3: % C, 74.39, H, 4.85; % found: C, 74.41, H, 4.84.
X-ray powder diffraction
X-ray powder diffraction was performed using Rigaku Dmax2200PC diffractometer (Rigaku Corporation, Tokyo, Japan) and Cu Ka-radiation [22]. The X-ray intensity was measured in the range of 5° < 29 < 90° with a scan speed of 2°min-1. The peak position of the 002 coke peak was measured. By using Bragg's law, the interlayer d-spacing was calculated. The improved Langford method was employed to calculate the stacking disorder degree, P.
In this study, regioselective decanoylation (Fig. 2 and 3) and triphenylmethylation (Fig. 4 and 5) of cytidine (1) were performed using the direct method. The resulting decanoylation and triphenylmethylation products were converted into numerous analogs by employing various acylating agents.
Characterization and selective decanoyl of cytidine
Cytidine 1 was initially converted into the 5'-O-decanoylcytidine derivative 2 through treatment with dry pyridine, and this product after the reaction with decanoyl chloride, followed by solvent removal and silica gel column chromatographic purification, produced 5;-O-decanoyl derivative (2) with 83.8% yield as needles and m.p. of 85-87 °C). The FTIR spectrum of compound 2 showed the following absorption bands: 1731, 1714 cm-1 (due to -CO), 3420 cm-1 (due to -OH), and 3550 cm-1 (due to -NH) stretching. In its 'H-NMR spectrum, two two-proton multiplets observed at S 2.38 {CH3(CH2)7C#2CO-} and S 1.59 {CH3(CH2)6C^2CH2CO-}, a twelve-proton multiplet appearing at S 1.32 {CH3(C^2)6(CH2)2CO-}, and a three-proton multiplet seen at S 0.85 {C^C^^CO-} were caused by the presence of one decanoyl group in the molecule. The downfield shift of C-57 proton to S 6.01 (as dd, J = 2.4 and 12.3 Hz, H-5'a) and to S 5.56 (as dd, J = 2.4 and 12.3 Hz, H-5'b) from their general values [23] in the precursor compound (1) and the resonances of other protons in their anticipated positions indicated the presence of the decanoyl group at position 5;. The formation of 5'-O-decanoylcytidine (2) might be caused by the high reactivity of the sterically less hindered
Results and discussion
Chemistry
Fig. 2. Reagents and conditions: (a) dry C6H5N, -5 °C, 6 to 7 h; 2 = decanoyl derivative
Fig. 3. (b) dry pyridine, 0 °C to room temperature, DMAP, stir for 6-8 h, Rj = different acyl halides (3-7)
Fig. 4. Reagents and conditions: (c) dry C6H5N, 0-5°C, 6 h; 8 = triphenylmethyl derivative
primary hydroxyl group of the ribose moiety of cytidine (1). Mass spectrometry provided a molecular ion peak at m/z [M+1]+ 398.10, which corresponded to the aforementioned molecular formula. From the complete analysis of FTIR, 'H-NMR, and elemental data, the structure of this compound was assigned as 5'-O-decanoylcytidine (2).
Furthermore, the structure of compound (2) was confirmed through the preparation of its octanoyl derivatives (3) (77.6%) as the crystalline solid with the m.p. of 89-91 °C. In its 'H-NMR spectrum, two four-proton multiplets appearing at 5 2.37 {2 x CH3(CH2)5CH2CO-} and 1.64 {2 x CH3(CH2)4C#2CH2CO-}, sixteen-proton multiplet observed at 5 1.29 {2xCH3(C#2)4(CH2)2CO-}, and six-proton multiplet obtained at 5 0.89 {2 x C#3(CH2)6CO-} were caused by the presence of two octanoyl groups in the molecule. The downfield shift of H-2' and H-37 protons to 5 5.45 and 5 4.82 from their precursor values (2) [24] indicated the attachment of two octanoyl groups at positions 2 and 37. The structure of octanoyl derivatives (3) was confirmed as 5'-O-decanoyl-2',3'-di- O-octanoylcytidine (3)through the complete analysis of their FTIR, 1H-NMR, and elemental data.
Through the palmitoylation of compound (2) by using palmitoyl chloride as acylating agent in C5H5N at room temperature, we isolated compound 4 in good yield. The following resonance peaks ascertained the presence oftwo palmitoyl groups in the molecule: 5 2.36 {4H, m, 2 x CH3(CH2)13C#2CO-}, 5 1.27 {52H, m, 2 x C^CH^C^CO-}, and 5 0.91 {6H, m, 2 x C^CH^CO-}. The introduction of palmitoyl groups at position 2 and 37 was indicated by appearance of H-27 and H-37 resonance peaks
at 5 5.48 (as m) and 5 4.86 (as dd, J = 3.5 and 5.6 Hz), which were deshielded considerably from their precursor diol (2) peaks. The decanoyl derivative 2 was further transformed easily into the 2',3'-di-O-stearoyate 5, 2',3'-di-O-(triphenylmethyloate) 6, and 2',3'-(4-tert-butylbenzoate) 7.
Characterization and selective triphenylmethylation of cytidine
Cytidine (1) was then transformed into the 5'-O-(triphenylmethyl)cytidine derivative 8 through a treatment with a unimolecular amount of triphenylmethyl chloride in anhydrous pyridine at -5 °C. The conventional work-up procedure, followed by solvent removal and silica gel column chromatographic purification, produced high yields of the triphenylmethyl derivative (8) as the crystalline solid. In its 1H-NMR spectrum, two characteristic six-proton multiplets appearing at 5 7.35 (Ar-H) and nine-proton multiplets observed at 5 7.30 (Ar-H) were caused by three phenyl protons of the triphenylmethyl group in the molecule. The downfield shift of C-57 proton to 5 5.30 (as dd, J = 2.2 and 12.2 Hz, H-5'a) and to 5.26 (as dd, J = 2.2 and 12.2 Hz, H-5'b) from their usual values (~4.00 ppm) in the precursor compound (1) and the resonances of other protons at their anticipated positions showed the presence of the triphenylmethyl group at position 5;. This finding is in accordance with the mechanism proposed by Kawsar et al. [25] based on similar nucleoside derivatives.
The preparation and identification of hexanoyl derivative 9 further supported the structure of compound 8. The 1H-NMR spectra exhibited two four-proton multiplets at 5 2.31 {2 x CH3(CH2)3C#2CO-} and at 5 1.62 {2 x C^C^^C^C^CO-}, eight-proton multiplet at 5 1.26 {2 x CH3(C#2)2CH2CH2CO-}, and six-proton multiplet at 5 0.88 {2 x CflsCCH^CO-}, which showed the attachment of two hexanoyl groups indicating the formation of 2',3'-di-O-hexanoate 9.
Compound 8 was then converted into heptanoyl derivative 10 by using similar procedures, and a high yield of heptanoate 10 was isolated as needles, (m.p. 98-99 °C). From the complete analysis of the FTIR, 1H-NMR, and elemental data, the structure of this compound was confirmed as 2',3'-di-O-heptanoyl -5'-O-(triphenylmethyl)cytidine (10). Similarly, compound 8 was converted into numerous acylated derivatives (11-15) to obtain newer compounds for antimicrobial evaluation studies. The structures of these derivatives were ascertained through the complete interpretation of their FTIR and 1H-NMR spectra.
Fig. 5. Reagents and conditions: (d) anhydr. pyridine, 0 °C to room temperature, DMAP, stir for 6 to 7 h, R2 = several acyl halides (9-15)
Thus, cytidine (1) acylation by applying the direct method was unique, and the reaction provided a single mono-substituted product in reasonably high yields. These newly synthesized products may be used as important precursors to modify cytidine molecules at different positions. All the prepared products were employed as test compounds for evaluating their antimicrobial and anticancer activities and for computational investigations.
XRD measurements
XRD is mainly performed for quantitatively identifying crystalline compounds, whereas single crystal XRD is conducted for structure determination. If h, k, and l represent the miller indices, the rules of the determination of crystal lattice type are as follows (Table 1).
The XRD patterns of the pure compounds synthesized under the optimized conditions were obtained in the 20 range of (0°-50°). The peaks observed at 20 of 19.653 and 21.506 (h,k,l:112 & 220), 6.010 and 21.472 (h,k,l:320 & 100), 8.386 and 20.212 (h,k,l:100 & 100), and 7.427 and 12.360 (h,k,l:100 & 111) corresponded to compounds 4, 5, 13, and 15, respectively. These peaks indicated the formation of typical phases of compounds 4, 5, 13, and 15. According to the phase analysis, the compounds synthesized using this method have high purity, and no impurities were detected in the XRD pattern. Moreover, compounds 4, 5, 13, and 15 show many lines with high intensity in their XRD patterns, which indicated that all the compounds are highly crystalline (Fig. 6). By applying the rules (Table 1) of the determination of the lattice type, we assigned the lattice structures to the synthesized
Table 1. Rules of the determination of crystal lattice type
Lattice type Rules for reflection to be observed
Primitive, P None
Body centered, I hkl; h+k+l= 2n
Face centered, F hkl; h,k,l either all odd or all even
Side centered, C hkl; h+k= 2n
Rhombohedral hkl; -h+k+l= 3n or h-k+l= 3n
20 30
2-Theta (deg)
Fig. 6. XRD pattern of 2',3'-di-0-pivaloyl-5'-0-(triphenylmethyl)cytidine (13)
compounds. Compound 4 satisfied the rule, h + k + l = 2n and was assigned the body centered lattice, while compounds 5, 13, and 15 were assigned primitive, for which no rule was provided.
Conclusion
In conclusion, an efficient method was proposed for synthesizing cytidine analogs. Moreover, acylation reactions are highly promising because considerably high yields of a single mono-substituted product were isolated through all the reactions. XRD pattern showed that the compounds 4, 5, 13 and 15 exhibited many lines with high intensity and suggested these compounds are well crystalline.
Author Contributions
S.M.A.K. designed and planned the experiments; K.M.R., and A.H. performed the synthetic experiment and determined XRD. S.M.A.K. interpreted the data and wrote the paper. All authors have read and approved the final version of the manuscript.
Acknowledgment
The authors are grateful to the Ministry of Science and Technology (MOST), the Government of the People's Republic of Bangladesh for providing financial support (Ref.: 39.00.0000. 09.06.024.19/ Phy's-544-560, 2019-2020) to conduct this research.
Declaration of interest
The authors declare no conflict of interest.
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