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28Macrocycles
Макроциклы
Макрогетэроцикль]
http://mhc-isuct.ru
Paper Статья
DOI: 10.6060/mhc224868a
First Macrocyclic 1,2,3-Triazolyl Uridine Analogues
Olga V. Andreeva,® Mayya G. Belenok, Bulat F. Garifullin, Irina Yu. Strobykina, Liliya F. Saifina, Alexandra D. Voloshina, Vyacheslav E. Semenov, and Vladimir E. Kataev
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences, 420088 Kazan, Russian Federation
@Corresponding author E-mail: [email protected]
Two macrocyclic 1,2,3-triazolyl uridine analogues were obtained for the first time by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. A 29-membered macrocycle consisted of one uracil molecule, two D-ribofuranose molecules and four 1,2,3-triazole molecules. A 58-membered macrocycle consisted of two uracil molecules, four D-ribofuranose molecules and eight 1,2,3-triazole molecules. Both macrocycles demonstrated moderate cytotoxicity against eight human cancer cell lines and normal cell line WI-38.
Keywords: Nucleoside analogues, macrocycles, uridine, 1,2,3-triazole, click chemistry, CuAAC reaction.
Первые макроциклические 1,2,3-триазоловые аналоги уридина
О. В. Андреева,® М. Г. Беленок, Б. Ф. Гарифуллин, И. Ю. Стробыкина, Л. Ф. Сайфина, А. Д. Волошина, В. Э. Семенов, В. Е. Катаев
Институт органической и физической химии им. А.Е.Арбузова ФИЦ Казанский научный центр, 420088 Казань, Российская Федерация
ovandreeva [email protected]
Катализируемой медью реакцией азид -алкинового циклоприсоединения (CuAAC) впервые получены два маро-циклических 1,2,3-триазоловых аналогов уридина. 29-Членный макроцикл состоял из одной молекулы ура-цила, двух молекул D-рибофуранозы и четырех молекул 1,2,3-триазола. 58-Членный макроцикл состоял из двух молекул урацила, четырех молекул D-рибофуранозы и восьми молекул 1,2,3-триазола. Оба макроцикла продемонстрировали умеренную цитотоксичность в отношении восьми линий раковых клеток человека и линии нормальных клеток Ш-38.
Ключевые слова: Аналоги нуклеозидов, макроциклы, уридин, 1,2,3-триазол, клик химия, CuAAC реакция.
Introduction
Almost immediately after the discovery of the fundamental role of nucleic acids in the vital activity of the cell, a large-scale study of the properties of not only nucleosides as essential components of nucleic acids, but also their various derivatives, called nucleoside analogues, began. Hundreds of nucleoside analogues have been synthesized, many of which were approved for use as antiviral and anticancer drugs.[1,2] Numerous approaches to obtaining nucleoside analogues were described in the literature: the intro-
duction of various substituents into the pyrimidine (purine) moiety of a native nucleoside or into a ribofuranose residue; the replacement of a nucleic base with another hetero-cycle; the replacement of a ribofuranose residue with a cyclopentane (cyclopentene) ring or a heterocyclic ring containing sulfur or nitrogen, and so on.[3]
Cyclic derivatives of native nucleosides are very interesting representatives of nucleoside analogues. They can be classified into four families: (i) spironucleosides in which a carbocyclic/heterocyclic ring is inserted into the ribofuranose residue of the nucleoside, usually at the C4'
position (Figure 1a); (ii) bicyclic nucleoside analogues in which two atoms of the ribofuranose residue are connected by an alkyl or ether bridge (Figure 1b); (iii) cyclic nucleoside analogues in which one atom of the ribofuranose residue and one atom of the nucleic base are connected by an alkyl or ether bridge (Figure 1c); (iv) macrocyclic nucleoside analogues in which several nucleic bases are connected by any linkers (Figure 1d,e,f).
Chemists' interest in the synthesis of spirocyclic nucleoside analogues (Figure 1a) arose after the discovery in 1961 of the naturally occurring spirocyclic nucleoside analogue hydantocidine which showed potent herbicidal activity and in which the imidizolidine-2,5-dione cycle was inserted into the ribofuranose residue at the anomeric car-bon.[4] Since then, various series of spirocyclic nucleoside analogues, not only C4'-spironucleosides, but also anomeric spiro-, C2'-spiro- and C3'-spironucleosides have been synthesized and some of them have shown excellent antiviral activities.[5]
Bicyclonucleoside analogues (Figure 1b) possessing a bicyclic sugar ring instead of the natural D-ribofuranose ring have attracted the spotlight of nucleoside chemists worldwide due to their conformationally restricted structure of the ribofuranose ring[6,7] that increases binding affinity to complementary RNA or DNA which allows them to be used for the synthesis of antisense oligonucleotides.!8-10] For the past time a large series of bicyclic nucleoside analogues possessing a cycloprorane ring,[6'11'12] an oxirane ring,[6'13'14] an oxetane ring,[15] a cyclobutane ring[16] fused to the C2'-C3' bond of various 2' ,3'-dideoxynucleoside analogues, 2'-C,4'-C-bridged bicyclonucleosides,[17] nucleoside analogues containing a 2' -O,4'-C-methylene-linked bicyclic furanose moiety[18-20] has been synthesized. Several bicyclic nucleoside analogues showed anti-HIV activity [11-13,15] and good growth inhibition activity against human tumor cell lines.[9]
Cyclic nucleoside analogues (Figure 1c), in which a pyrimidine or purine nucleic base is connected to a ribofuranose residue, in addition to a glycoside bond, by a polymethylene or ether chain, mainly through the C5'
atom, have been synthesized and studied for more than 50 years.[21-23] The first cyclic nucleoside analog, 2',3' -O-isopropylidene-N3,5' -cycloadenosine, was accidentally obtained by Todd's group in 1951.[24] Of course, such compound aroused the interest of chemists with its unusual structure, and in 1969 the Ikehara's group for the first time prepared a large series of various 8-cyclopurine nucleosides.!2^ Interestingly, N3,5' -cycloxanthosine synthesized by the Holmes group in 1963[26] appeared to be a natural compound that was isolated from the sea sponge Eryus sp. from the Great Australian Bight by the Trotter group in 2005.[27] Note that many cyclic nucleoside analogues have
shown antiviral activity, as a rule, against hepatitis C virus [21-23,28-30]
In order to study supramolecular assemblies formed by amphiphilic molecular systems containing nucleic bases due to hydrogen bonds, stacking and hydrophobic interactions, a small series of macrocyclic nucleoside analogues (Figure 1d,e,f) was synthesized.[31-34] The literature provides data on the preparation of 12- and 13-membered macrocycles containing two uridine or guanidine moieties (Figure 1d),[32-34] 12-, 16-, 17- membered macrocycles containing three uridine moieties (Figure 1e),[31-33] and a 21-membered macrocycle containing four uridine moieties (Figure 1f).[33] Polymethylene chains,[31,33] urea, thiourea, carbodiimide moieties,[34] and isoxazolidine cycles[32] were used as linkers in the listed macrocyclic nucleoside analogues.
Recently, we synthesized a series of 1,2,3-triazole nucleoside analogues[35,36] and being interested in the work on the synthesis ofmacrocyclic nucleoside analogues,[31-34] we also decided to prepare several macrocycles, but to use for this aim not the ring-closing metathesis strategy,[33] but the strategy of a copper-catalyzed 1,3-dipolar cycloaddition.[37] Herein, we report on the synthesis of a 29-membered macrocycle formed from one uracil molecule, two D-ribofuranose molecules and four 1,2,3-triazole molecules as well as a 58-membered macrocycle formed from two uracil molecules, four D-ribofuranose molecules and eight 1,2,3-triazole molecules.
Q/yNuc
Hd \>h
a
NUC ""<yj......"Nuc
linker
HO
OH
HO^
O^Nuc
HO
V7
(L
Nuc iELA>Nuc
-..........-I linker L ^ N uc
ho' K AS '"'oh
/w
Nuc
HO®""
0 Nuc
X 'OH Nuc..... .0. .....U
O. Nuc
HO OH
c
Figure 1. Schematic representation of families of cyclic nucleoside analogues (Nuc stands for a nucleic base).
Experimental
Chemistry
General Methods
'H and 13C spectra were recorded on 400 MHz, 500 MHz and 600 MHz Brucker Avance. 13C NMR spectra were obtained in the above instrument operating at 100.6 MHz. Mass spectra (MALDI) were recorded in a positive ion mode on a Bruker Ul-traflex III TOF/TOF mass spectrometer for 10-3 mg/mL solutions in CHCl3. Flash chromatography was performed on silica gel 60 (40-63 ^m, Buchi, Sepacore). Thin-layer chromatography was carried out on plates with silica gel (Sorbfil, Russia). Spots of compounds were visualized by using ultraviolent fluorescence under a short wavelength (254 nm) followed by heating the plates (at ca. 150 °C) after immersion in a solution of 5% H2SO4 and 95% H2O. All reactions sensitive to air and/or moisture were carried out under argon atmosphere with anhydrous solvents. Anhydrous solvents were purified and dried (where appropriate) according to standard procedures.
Uracil and propargyl bromide were purchased from Sigma-Aldrich; D-ribose was purchased from Acros. Compounds 3, 5-9 were prepared as described earlier.[36,38] Their spectral data were in keeping with published ones.[36,38]
Synthesis
Azido 5-mesyloxy-2,3-O-isopropylidene-fi-D-ribofuranoside (10). Azide 9 (2.23 g, 10.4 mmol) was dissolved in anhydrous methylene chloride (50 mL) and triethylamine (3 mL). The reaction mixture was cooled to 0oC and methanesulfonyl chloride (1.8 g, 15.7 mmol) was added dropwise. The reaction was stirred at room temperature for 16 hours, then washed with water, saturated solution of sodium bicarbonate, water again, dried over anhydrous sodium sulfate and evaporated under vacuum to afford 10 as a yellow syrup (2.64 g, 87%). Found, %: C 36.82, H 5.21, N 14.39, S 10.89. C9H15N3O6S requires, %: C 36.86, H 5.16, N 14.33, S 10.93. m/z ESI MS: [(M+H)+] calcd for C9H16N3O6S: 294.08, found 294.90 >H NMR (CDCb) Sh ppm: 5.54 (1H, s, H1), 4.7 (1H, dd, J = 5.9, 0.9 Hz, H-3), 4.45-4.51 (2H, m, H-2, H-4), 4.234.31 (2H, m, H-5a,b), 3.06 (3H, s, Ms), 1.46 (3H, s, CH3), 1.29 (3H, s, CH3). 13C NMR (CDCb) Sc ppm: 113.50 (C6), 97.06 (C1), 85.11 (C4), 84.56 (C3), 81.34 (C2), 67.72 (C5), 37.57 (CMs), 26.36, 24.88 (C7, C8).
1,3-Bis{1-(5-mesyloxy-2,3-O-isopropylidene-fi- D-ribofurano-syl)-1H-1,2,3-triazol-4-yl]methyl}pyrimidine-2,4-dion (11). To a solution of 1,3-bis(propargyl)uracil 3 (0.3 g, 1.6 mmol) and azide 10 (0.94 g, 3.2 mmol) in a mixture of 1:1 tert-butanol/water (50 mL) was added freshly prepared solution of sodium ascorbate (0.63 g, 3.2 mmol) in 2 mL water and CuSO4*5^O (0.79 g, 3.2 mmol) in 2 mL water. The reaction mixture was stirred at 40oC for 48 h, then was concentrated under reduced pressure. The residue was taken up in methylene chloride, washed successively with water, dried over anhydrous sodium sulphate and concentrated under vacuum. The residue was purified by flash chromatography on a silica gel (eluent chloroform:methanol from 200:1 to 20:1) to afford 11 as a white foam (0.6 g, 49%). Found, %: C 43.38, H 4.99, N 14.48, S 8.25. C78H38N8O14S2 requires, %: C 43.41, H 4.94, N 14.46, S 8.28. MALDI MS: calcd for [(M+H)+]: 775.2, found 775.1. >H NMR (CDCb) Sh ppm: 7.90 (1H, s, H-9 (H-12)), 7.77 (1H, s, H-12 (H-9)), 7.43 (1H, d, J = 8.1 Hz, H-6), 6.14 (1H, d, J = 0.8 Hz, H-1' (H-1' ' )),6.09 (1H, s, H-1' ' (H-1' )),5.74 (1H, d, J = 7.8 Hz, H-5), 5.49-5.45 (2H, m, H-3' , H-3' '), 5.24-5.18 (2H, m, 2H-10), 5.05-4.95 (4H, m, 2H-7, H-2 ', H-2 ' ' ), 4.6-4.53 (2H, m, H-4' , H-4 ' ' ), 4.21-4.08 (4H, m, H-5'a,b, H-5' 'a,b), 2.91 (3H, s, Ms), 2.85 (3H, s, Ms), 1.57 (3H, s, CH3), 1.56 (3H, s, CH3), 1.38 (3H, s, CH3), 1.37 (3H, s, CH3). 13C NMR (CDCb) Sc ppm: 162.51 (C4), 151.21 (C2), 143.76, 142.63 (C8, C11), 142.49 (C6), 123.79, 123,65 (C9, C12), 114.32, 114.11 (C6', C6''), 102.03 (C5), 93.63,
93.38 (C1', C1" ), 86.03, 85.95 (C4' , C4" ), 84.36, 84.31 (C3' , C3" ), 81.48, 81.37 (C2' , C2' ' ), 67.91 (C5' , C5' ' ), 44.10, 36.08 (C7, C10), 37.58, 37.37 (2CMs), 26.82, 26.79, 25.14, 25.13 (4CH3).
1,3-Bis {1-(5-azido-5-deoxy-2,3-O-isopropylidene-ß-D-ribofu-ranosyl)-1H-1,2,3-triazol-4-yl]methyl}pyrimidine-2,4-dion (12) To a solution of 11 (1 g, 1.3 mmol) in dry DMF sodium azide (0.7 g, 10.7 mmol) was added. The mixture was heated at 90oC 20 h., then cooled, diluted with acetone, washed with water, saturated solution of NaHCO3, water again. The resulting solution was dried over anhydrous sodium sulphate and concentrated under vacuum. The residue was purified by flash chromatography on a silica gel (eluent chloroform:methanol from pure chloroform to 50:1) to afford 12 as a white foam (0.43 g, 50%). Found, %: C 46.69, H 4.86, N 29.39. C26H32N14O8 requires, %: C 46.71, H 4.82, N 29.33. MALDI MS: calcd for [(M+H)+]: 670.6, found 670.6. 'H NMR (CDCb) Sh ppm: 7.90 (1H, s, H-9 (H-12)), 7.78 (1H, s, H-12 (H-9)), 7.40 (1H, d, J = 7.8 Hz, H-6), 6.12 (1H, s, H-1' (H-1'')), 6.07 (1H, s, H-1'' (H-1')),5.71 (1H, d, J= 7.8 Hz, H-5), 5.49-5.45 (2H, m, H-3' , H-3 ' ' ), 5.23-5.16 (2H, m, 2H-10), 5.024.93 (2H, m, 2H-7), 4.87-4.82 (2H, m, H-2 ' , H-2 ' ' ), 4.46-433 (2H, m, H-4 ' , H-4 ' ' ), 3.37-3.17 (4H, m, H-5'a,b, H-5''a>), 1.55 (3H, s, CH3), 1.54 (3H, s, CH3), 1.35 (3H, s, CH3), 1.35 (3H, s, CH3). 13C NMR (CDCb) Sc ppm: 162.35 (C4), 151.12 (C2), 143.73, 142.61,
142.24 (C8, C11, C6), 124.16, 124,06 (C9, C12), 114.18, 113.96 (C6' , C6'' ), 102.11 (C5), 93.49, 93.11 (C1' , C1' ' ), 87.45, 87.29 (C4' , C4'' ), 84.35, 84.31 (C3' , C3' ' ), 82.38, 82.22 (C2' , C2'' ), 52.40 (C5' , C5'' ), 44.08, 35.83 (C7, C10), 26.77, 26.74, 25.11 (4CH3).
Synthesis of macrocyclic 1,2,3-triazolyl uridine analogues. To a solution of sodium ascorbate (0.24 g, 1.2 mmol) and CuSO4*5H2O (0.3 g, 1.2 mmol) in 100 mL mixture of 1:1 tert-butanol/water 12 (0.41 g, 0.6 mmol) in 50 mL mixture of 1:1 tert-butanol/water and 1,7-octadiyne (0.065 g, 0.6 mmol) in 50 mL mixture of 1:1 tert-butanol/water were contemporaneously dropped. The reaction mixture was stirred at 40oC for 60 h, then it was concentrated under reduced pressure. The residue was taken up in methylene chloride, washed successively with water, dried over anhydrous sodium sulphate and concentrated under vacuum. The residue was purified by flash chromatography on a silica gel (eluent chloroform:methanol from pure chloroform to 1:1)
42,43,112,113-di-O-isopropylideno-1(3,1)-uracila-3(4,1),5(1,4), 10(4,1),12(1,4)-tetratriazola-4(1,5),11(5,1)-di-ß-D-ribofuranosa-cyclotriskaidecaphan (13). A white foam (0.1 g, 21%). Found, %: C 52.78, H 5.42, N 25.28. C34H42N14O8 requires, %: C 52.71, H 5.46, N 25.31. MALDI MS: calcd for [(M+Na)+]: 797.8, found 797.9. >H NMR (CDCb) Sh ppm: 7.68 (1H, s, H-9 (H-12)), 7.59 (1H, s, H-12 (H-9)), 7.46 (1H, d, J = 7.7 Hz, H-6), 6.88 (1H, s, H-19 (H-21)), 6.56 (1H, s, H-21 (H-19)), 6.13 (1H, d, J = 1.4 Hz H-1' (H-1' ' )), 6.07 (1H, s, H-1'' (H-1' )), 5.83 (1H, d, J = 8.1 Hz, H-5), 5.51 (1H, d, J = 5.9 Hz H-3' (H-3 ' ' )), 5.37-4.92 (7H, m, H-3' ' (H-3' ), 2H-10, 2H-7, H-2 ' , H-2 ' ' ), 4.76-4.67 (2H, m, H-4 ' , H-4' ' ), 4.52-4.43 (2H, m, H-5' a, H-5 ' ' a), 4.38-4.25 (2H, m, H-5'b, H-5' ' b), 2.78-2.66 (4H, m, 2H-23, 2H-26), 1.69-1.6 (4H, m, 2H-24, 2H-25), 1.57 (3H, s, CH3), 1.55 (3H, s, CH3), 1.39 (6H, s, 2CH3). 13C NMR (CDCb) Sc ppm: 162.36 (C4), 151.34 (C2), 147.87, 147.62, 144.04, 142.93, 142.53 (C6, C8, C11, C20, C22), 123.49, 123.14, 122.47, 122.31 (C9, C12, C19, C21), 114.74, 114.31 (C13, C16),
102.25 (C5), 93.48, 93.15 (C1', C1'' ), 86.63, 85.83 (C4' , C4' ' ), 84.06, 84.03 (C3', C3' ' ), 81.98, 81.66 (C2' , C2' ' ), 51.09, 51.03 (C5' , C5' ' ), 44.22, 36.21 (C7, C10), 28.36, 28.29, 26.82, 26.76 (C23, C24, C25, C26), 25.18, 24.91 (4CH3).
42,43,112,113,172,173,242,243-tetra-O-isopropylideno-1(3,1), 14(3,1)-diuracila-3(4,1),5(1,4),10(1,4),12(1,4),16(4,1),18(1,4), 23(4,1)-octatriazola-4(1,5),11(5,1),17(1,5),24(5,1)-tetra-ß-D-ribo-furanosacyclotetracosahexaphan (14). A white foam (0.11 g, 23%). Found, %: C 52.73, H 5.41, N 25.36. C68H84N28O16 requires, %: C 52.71, H 5.46, N 25.31. MALDI MS: calcd for [(M+H)+]: 1550.61, found 1550.6. >H NMR (CDCb) Sh ppm: 7.94, 7.93, 7.79, 7.78 (4H, 4s, H-9, H-12, H-39, H-42), 5.521 (1H, d, J = 7.7 Hz, H-6 (H-36)), 5.519 (1H, d, J = 7.7 Hz, H-36 (H-6)), 6.94,
6.93, 6.81, 6.80 (4H, 4s, H-19, H-21, H-49, H-51), 6.13 (2H, d, J = 2.2 Hz, H-1' , H-1' ' ' (H-1' ', H-1' '' ' )), 6.07 (2H, s, H-1' ' , H-1' ''' (H-1' , H-1' '' )), 5.75 (1H, d, J = 8.1 Hz, H-5 (H-35)), 5.74 (1H, d, J = 8.1 Hz, H-35 (H-5)), 5.46-5.41 (4H, m, H-3' , H-3 ' ' , H-3 ' ' ', H-3' '' ' ), 5.27-5.14 (4H, m, H-2 ' , H-2 ' ' , H-2' ' ', H-2 ' ' ' '), 5.05-4.96 (8H, m, 2H-10, 2H-40, 2H-7, 2H-37), 4.72-4.64 (4H, m, H-4 ', H-4 ' ', H-4 ' ' ' , H-4 ' ' ' ' ), 4.53-4.44 (4H, m, H-5 ' a, H-5' ' a, H-5' ' ' a, H-5' '' 'a), 4.31-4.20 (4H, m, H-5'b, H-5 ' 'b, H-5' ''b, H-5' '' 'b), 2.68-2.57 (8H, m, 2H-23, 2H-26, 2H-27, 2H-30), 1.65-1.58 (8H, m, 2H-24, 2H-25, 2H-28, 2H-29), 1.54 (6H, s, 2CH3), 1.53 (6H, s, 2CH3), 1.37 (6H, s, 2CH3), 1.36 (6H, s, 2CH3). 13C NMR (CDCb) 5c ppm: 162.43 (C4, C34), 151.27 (C2, C32), 147.87, 147.81, 147.70, 147.65 (C8, C11, C38, C41), 143.79 (C6, C36), 142.87, 142.85, 142.74 (C20, C22, C50, C52), 124.43, 124.39, 124.05, 124.04 (C9, C12, C39, C42), 122.12, 122.10, 121.79, 121.75 (C19, C21, C49, C51), 114.37, 114.18 (C13, C16, C43, C46), 101.97 (C5, C35), 93.15, 93.12, 93.04, 93.02 (C1' , C1'' , C1''' , C1''' ' ), 87.11, 86.89, 86.87 (C4' , C4'' , C4''' , C4''' ' ), 84.37, 84.34, 84.27, 84.24 (C3', C3'' , C3''', C3''' ' ), 82.20, 82.07 (C2', C2'', C2''', C2''''), 51.47, 51.43 (C5', C5'', C5''', C5''''), 44.17, 44.15, 36.04 (C7, C37, C10, C40), 28.61, 28.53, 26.75, 26.74 (C23, C24, C25, C26, C27, C28, C29, C30), 25.15, 25.13, 25.10 (8CH3).
Biological Study
Cells and Materials
For experiments, we used tumor cell cultures M-HeLa clone 11 (epithelioid carcinoma of the cervix, subline HeLa., clone M-HeLa), HuTu 80, human duodenal adenocarcinoma; PC3 - prostate adenocarcinoma cell line from ATCC (American Type Cell Collection, USA; CRL 1435); MCF7 - human breast adenocarcinoma (pleural fluid); Hep G2, human liver carcinoma; PANC-1, human pancreatic carcinoma; A549, human lung carcinoma; A375, human amelanotic melanoma cell line; WI38, VA 13 subline 2RA, human embryonic lung from the collection of the Institute of Cytology, Russian Academy of Sciences (St. Petersburg).
MTT assay
The cytotoxic effect on cells was determined using the col-orimetric method of cell proliferation - the MTT test. NADP-H-dependent cellular oxidoreductase enzymes can, under certain conditions, reflect the number of viable cells. These enzymes are capable of reducing the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to insoluble blue-violet formazan, crystallizes inside the cell. The amount of form-azan formed is proportional to the number of metabolically-active cells. Cells were seeded on a 96-well Nunc plate at a concentration of 5 ■ 103 cells per well in a volume of 100 ¡L of medium and cultured in a CO2 incubator at 37 °C until a monolayer was formed. Then the nutrient medium was removed and 100 ¡L of solutions of the test drug in given dilutions were added to the wells, which were prepared directly in the nutrient medium with the addition of DMSO (5% v/v) to improve solubility. After 48 h incubation of cells with the tested compounds, the nutrient medium was removed from the p lates, and 100 ¡iL of the nutrient medium without serum containing MTT at a concentration of0.5 mg/mL was added and incubated for 4 h at 37 °C. Formazan cry stals were added 100 ¡L of DMSO to each well. Absorbance was recorded at 540 nm on an Invitrologic microplate reader (Russia). Experiments for all compounds were repeated three times.
Results and Discussion
Synthesis
The synthesis oftarget macrocycles was carried out in four stages. At the first stage (Scheme 1), in accordance with the procedure already described,[36] uracil 1 was con-
verted by a reaction with an excess of sodium hydride in dimethylformamide (DMF) into disodium salt 2, which was alkylated in situ with propargyl bromide to afford 1,3-bis(propargyl)uracil 3 in 70% yield. At the second stage (Scheme 2), using the methods previously reported,!36! firstly, commercial D-ribose 4 was converted to tetraacetyl D-ribofuranose 6, which was involved in a reaction with trimethylsilylazide (TMSN3) in the presence of tin tetrachloride to obtain azide 2,3,5-triacetyl-P-D-ribofuranose 7. Secondly, the acetyl protection of monosaccharide 7 was removed by the treatment with sodium methylate in methanol and a 2,3-0-isopropylidene protecting group was introduced by the reaction with 2,2-dimethoxypropane (DMP) in boiling acetone in the presence of ^-toluenesulfonic acid (TsOH) according to a known procedure[38] to afford compound 9. Then 5-mesyloxy-1-azido-1-deoxy-2,3-(3-isopropylidene-P-D-glucofuranose 10 was obtained by the reaction of azide 9 with mesyl chloride (MsCl) in the presence of Et3N (Scheme 2). On the third stage, a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was utilized to couple 1,3-bis(propargyl)uracil 3 with azide 10 (Scheme 3) by analogy with the procedure previously described.[36] The 1,3-disubstituted uracil derivative 11 obtained in 49% yield was treated with sodium azide in DMF at 80 oC for 12 h to give the 5 ',5 "-diazide 12 in moderate yield (50%). The 1H and 13C NMR spectra of 12 confirmed the presence of 1,2,3-triasole rings. The signals within the range 7.78 - 7.9 ppm in the 1H NMR spectrum of 12 belonged to the triazolyl protons H-9 and H-12 in accordance with the literature data.[35,36] The 13C NMR spectrum of 11 showed signals within the range 142.24 -143.73 ppm and signals within the range 124.06 - 124.16 ppm attributing to the 1.2.3-triazolyl C8, C11 carbons and C9, C12 carbons, respectively, in keeping with known da-ta.[35,36] At the fourth stage, the CuAAC reaction of 1,7-octadiine with 5 ',5 ''-bisazide 12 was carried out to obtaine target macrocycles (Scheme 4). The reagents were introduced into the reaction in equimolar quantities in the presence of two moles of CuSO4 and sodium ascorbate in a 1:1 tBuOH / H2O solution at a concentration of 10-3 mol/L. The reaction mixture was stirred for 60 hours at a temperature of 40 °C. Macrocycles 13 and 14 were isolated by preparative flash chromatography on silica gel in 21 and 23% yields respectively. The structure of the obtained macrocycles 13 and 14 was confirmed by 1H and 13C NMR spectroscopy and MALDI mass spectrometry. In the 1H NMR spectrum of macrocycle 13, there are four signals for the triazolyl protons at 6.56, 6.88, 7.59, 7.68 ppm, two sets of signals for the protons of the ribofuranose residue and the signals for the protons at the C5 and C6 atoms of the uracil moiety in the corresponding integral intensities. The protons at the C1' and C1'' atoms of the D-ribofuranose residues resonated at 6.07 and 6.13 ppm as singlets, that indicated P-orientation of the glycosidic bonds in macrocycle 13. All the above signals also presented in the 1H NMR spectrum of macrocycle 14 but with the doubled quantity. Two anomeric protons of macrocycle 14 resonated in the 1H NMR spectrum as doublets at 6.13 ppm with vitinal constants of 3.3 Hz, and two other anomeric protons of macrocycle 14 resonated as a broad singlet with an integral intensity corresponding to two protons. This indicated the P-orientation of the four glycosidic bonds. The 13C NMR spectra of both
macrocycles are also consistent with the assumed structures. In the MALDI spectrum of macrocycle 13, there is only one signal of 797 m/z which corresponds to the mass of [M+Na]+. In the MALDI spectrum of macrocycle 14, there is only one signal of 1550 m/z, which corresponds to the mass [M+H]+.
o
£
NH
N^O H
NaH DMF
P Na 2
t
^^Br fy-0
iTl,
DMF
Scheme 1. Synthesis of 1,3-dipropargyluracil.
a OH Aco-^ OMe
1. MeOH, H+ \ /
u | OH 2. AC2O / Py V-r
r\LI -^ - ' I .
AC2O / AcOH,
HO'" Y "'OH 2.AC20/ Py Y-f 5% H2SO4
OH AcO OAc AcO OAc AcO OAc
4 5 6 7
Ac0-1 „ Y3 1. MeONa HO. N3 ™PH HO> „ N3 MsCI, MsO-^ N3
2.MeQH) , VSI E'3N ,
y-f V~r acetone* Y~f CH2CI2, (I
Ac6 ¿Ac HO OH ref'UX ¿y!» O O
7 8 H3C CH3 H3c CH3
9 10
Scheme 2. Synthesis of 5-methanesulfonyloxy-1-azido-1-deoxy-2,3-O-isopropylidene-P-D-glucofuranose.
3
MsO. N<
"I „ "a Me Me
CuS04i
Na L-ascorbate tBuOH / H20
ivib ivi
A
O
N OMs
h-4 0
o
N
NaN3 DMF
Me Me
A
rLii'
N. s" N3
MsO^ 1 80°C, 24 h N3—* Ï
n4- 0
4-
12
10
y
Me Me
y
Me Me
Scheme 3. Synthesis of compound 12 possessing the uracil moiety coupled to two 1,2,3-triazol-4-yl-p-D-ribofaranosyl fragments.
H3C CH-a
Uox O _3
^ •• Ci,, «»
12
cuso4,
Na L-ascorbate tBuOH / H20
O »
o po^GH.
."Aa »JjT I
o CH.
Scheme 4. Synthesis of29- and 58-membered macrocyclic 1,2,3-triazolyl uridine analogues.
Table 1. Cytotoxicity (IC50, |JM) of macrocycles 13 and 14.
IC50,* pM
Compounds
Cancer cell lines
M-HeLa
HuTu80
PC3
MCF-7 HepG2 PANC-1
A549
А375
Normal cell line
WI-38
13
14
70.0±5.5 54.6±4.3
57.7±4.5 68.8±5.4
86.7±6.6 100±8.2
69.0±5.3 55.4±4.3
>100 >100
100±8.7 59.1±4.6
>100 >100
43.4±3.5 39.5±3.1
40.3±3.1 45.3±5.5
*IC50 represents the concentration at which a compound exerts half of its maximal inhibitory effect. The experiments were performed in triplicate. Results are expressed as the mean ± standard deviation.
Cytotoxicity
Macrocycles 13 and 14 were evaluated for in vitro cytotoxicity against eight human cancer cell lines: M-HeLa cervical epitheloid carcinoma, HuTu-80 duodenal adenocarcinoma, PC3 prostate adenocarcinoma, MCF-7 breast adenocarcinoma, Hep G2 human liver carcinoma, PANC-1 pancreatic carcinoma, A549 pulmonary adenocarcinoma, A375, human amelanotic melanoma cell line, as well as a diploid human cell strain WI-38 composed of fibroblasts. The resulting data expressed as concentrations causing the inhibition of the growth of 50% of cells in the experimental population (IC50) are presented in Table 1. As one can see, the tested compounds demonstrated moderate activity in the range of IC50 values of 39-87 pM against most of the cell lines used, including the normal cell line WI-38. The exception was HepG2 and A549 cell lines, for which both macrocycles were completely inactive.
Conclusions
Two macrocyclic 1,2,3-triazolyl uridine analogues were obtained for the first time by a copper-catalyzed az-ide-alkyne cycloaddition (CuAAC) reaction. A 29-membered macrocycle consisted of one uracil molecule, two D-ribofuranose molecules and four 1,2,3-triazole molecules. A 58-membered macrocycle consisted of two uracil molecules, four D-ribofuranose molecules and eight 1,2,3-triazole molecules. The ability of the macrocycles to form supramolecular assemblies is in progress and will be reported in due course. Both macrocycles demonstrated moderate cytotoxicity against eight human cancer cell lines and normal cell line WI-38.
Acknowledgements. The authors are grateful to the Assigned Spectral-Analytical Center of FRC Kazan Scientific Center of RAS for technical assistance during research.
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Received 19.12.2022 Accepted 14.01.2023
