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5N-Макроциклы
N-Macrocycles
Шкрогэтароцмклы
Статья
Paper
http://macroheterocycles.isuct.ru
DOI: 10.6060/mhc210945s
Synthesis of Macrocyclic Peptide-Diterpenoid Conjugates
by a Sequential Arylation/Peptide Coupling/Click Macrocyclization
Procedure
Mariia A. Gromova,a,b Yurii V. Kharitonov,b Tatyana S. Golubeva,c and Elvira E. Shultsb
'•Novosibirsk State Pedagogical University (NSPU), 630126 Novosibirsk, Russia
^Novosibirsk Institute of Organic Chemistry SB RAS (NIOCH SB RAS), 630090 Novosibirsk, Russia
cThe Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences,
630090 Novosibirsk, Russia
@Corresponding author E-mail: schultz@nioch.nsc.ru
An approach relying on a sequential selective arylation of isopimaric acid/peptide coupling/CuAAC-based macrocyclization procedure of alkynyl peptides to azidoditerpenoid, was developed for the preparation of novel triazole-linked peptide - tricyclic diterpenoid conjugates. The process has comprised the synthesis of azidoditerpenoid bearing the azido group at the side chain by the cross-coupling reaction of isopimaric acid with 1-azido-2-iodobenzene as well as the preparation of a small library of dipeptides featuring varied amino acid sequence (Gly-Gly, Gly-Val, Val-Gly and Val-Val). This approach has shown great chemical efficiency and gave macrocyclic compounds in good yields. It was shown that the yield of macrocyclization products depends on the substituent in the dipeptide fragment. The cytotoxicity of the synthesized compounds was evaluated using the conventional MTT assays. It has been found that the introduction of a dipeptide fragment into the structure of isopimaric acid and macrocyclization have a significant effect on toxicity towards human cancer cellsMCF-7 andDU-145.
Keywords: Isopimaric acid, diterpenoids, dipeptide macrocycles, cross-coupling reaction, CuAAC reaction,
Синтез макроциклических пептидо-дитерпеноидных конъюгатов последовательностью реакций арилирования, образования амидной связи и азид-алкинового циклоприсоединения
М. А. Громова,a Ю. В. Харитонов,13 Т. С. Голубева,с Э. Э. Шульць@
aФедеральное государственное бюджетное образовательное учреждение высшего образования Новосибирский государственный педагогический университет, 630126 Новосибирск, Россия
ъНовосибирский институт органической химии им. Н.Н. Ворожцова Сибирского отделения Российской академии наук, 630090 Новосибирск, Россия
сФедеральное государственное бюджетное научное учреждение Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук, 630090 Новосибирск, Россия @E-mail: schultz@nioch.nsc.ru
Описано получение макроциклических дипептидов, содержащих фрагменты 1,2,3-триазола и трициклического дитерпеноида пимаранового типа. Процесс включает синтез азидодитерпеноида по реакции кросс-сочетания изопимаровой кислоты с 1-азидо-2-иодбензолом, получение небольшой библиотеки алкинилзамещенных дипептидов с различной аминокислотной последовательностью (Gly-Gly, Gly-Val, Val-Gly и Val-Val) и внутримолекулярную реакцию азид-алкинового циклоприсоединения. Предложенный подход показал высокую химическую эффективность и привел к образованию макроциклических соединений с хорошими выходами. Показано, что выход продуктов макроциклизации зависит от строения аминокислот в дипептидном
cytotoxicity.
фрагменте. С помощью МТТ теста получены данные о цитотоксичности новых соединений. Установлено, что введение дипептидного фрагмента в структуру изопимаровой кислоты и макроциклизация оказывают значительное влияние на токсичность в отношении опухолевых клеток человекаMCF-7, DU-145.
Ключевые слова: Макроциклические пептиды, изопимаровая кислота, дитерпеноиды, реакция кросс-сочетания, CuAAC реакция, цитотоксичность.
Introduction
Peptide macrocycles are a promising class of compounds due to their capacity to target complex protein-protein interactions. With a size range between small molecules and biologics, that when optimized, have the potential to adopt some of the favourable properties from each: the high potency and selectivity, and the higher bioavail-ability and cellular permeability associated with smaller molecules.[1,2] This unique place in chemical space, makes them particularly applicable to modulating targets previously thought of as undruggable, such as protein-protein interactions.[3] The use of peptides to improve the efficacy of a known small molecule or drug, in the form of a peptide-drug conjugate is well known.[4-6] Conjugation to natural biomolecules such as fatty acids, steroids or heteroaromatic rings is a well-established technique to modulate peptide properties producing hybrids that show significant potential in addressing the limitations of peptides as therapeutic agents or tools in chemical biology.[7-9] Steroid scaffold was selected as a fragment limiting the conformational mobility of macrocyclic peptides. Important advantages of steroid compounds are the possibility to achieve conformational restriction and to couple peptides in a way to achieve loop like amphiphilic conformation for the desired activity.[1011]
As a promising scaffold for the synthesis of macro-cyclic peptides, we consider the use of natural compounds of the pimarane type diterpenoid series. Like steroids, these compounds are characterized by the presence of a rigid polycyclic skeleton with a given stereochemistry. In this work, isopimaric acid 1 is proposed as a structural basis for the synthesis of macrocyclic peptides. The performed synthetic transformations of isopimaric acid indicate the possibility of carrying out the transformation along the path of complication of the initial molecule with high stereochemical control.[12-18]
As oligopeptides, we used dipeptides containing the amino acid residues of valine and glycine. The choice of the latter is determined both for revealing the effect of steric factors on the yield of target macrocyclic products and data on the perspective biological activity of compounds containing them. Among the tricyclic diterpenoids, such peptide conjugates are represented by antimicrobial peptoids with a dehydroabietylamine fragment.[19] It was found that the combination of N-alkylated Gly-Gly-Gly and the indicated dehydroabietylamine, which does not possess antibacterial activity, leads to an increase in the target biological effect. Macrocyclic di- and tri-peptides were considered as a new structural template that can be exploited for design of proteasome inhibitors[20] and of potential chemotherapeu-tics that specifically inhibit human cathepsin D proteases.[21]
Depending on the intended function, conjugation of the small molecule motif can occur directly on an amino
acid side chain, through the amide backbone or through the cyclization linkage itself. The Cu(I)-catalysed azide-alkyne cycloaddition reaction (CuAAC) is a clear candidate for peptide stapling, given its widespread popularity as a biocompatible ligation technique. 1,2,3-Triazoles are interesting depeptidizing element as they are physiologically stable. The triazole-containing analogues were shown to be more potent tyrosinase inhibitors than the homodetic counterparts.[22] Incorporation of one to two triazole moieties into somatostatin-binding peptide macrocycles generated conformationally heterogeneous analogues.[23] Additionally, the 1,2,3-triazole linkage has scarcely been used for pep-tide-steroid conjugation and only to assemble short apolar tripeptides onto bile acid scaffolds, despite the increasing interest of these type of conjugates in diverse applications such as HIV inhibitors and immunogens for vaccine development.[11-24] A review of the usefulness of triazole ring in peptide macrocycles was published.[25] 1,2,3-Triazole found application in the structural modification of natural products.[26] We have recently shown that the CuAAC reaction of labdane type[27-31] and pimarane-type[32] diacetylenes with various diazides led to optically active 1,2,3-triazole-containing macroheterocyclic diterpenoids in good yields. Among the synthesized macrocyclic compounds, substances were found that have high cytotoxicity to human tumor cells.[30] In the current study we have designed and synthesized a small series of isopimaric acid incorporated mac-roheterocycles connected at C-16 and C-18 with an aryl, triazolyl, dipeptide and amide linker group, differing with respect to the amino acid sequence (Gly-Gly, Gly-Val, Val-Gly and Val-Val). The cytotoxicity of the synthesized compounds toward cancer cell lines in vitro was studied.
Experimental
General methods
1H and 13C NMR spectra were registered on Bruker AV-400 (1H: 400.13 MHz, 13C: 100.61 MHz), Bruker AV-300 (1H: 300.13 Mz, 13C: 75.47 MHz), Bruker AV-600 (1H: 600.30 MHz, 13C: 150.95 MHz) (Bruker BioSpin GmbH, Rheinstetten, Germany) instruments. Deuterochloroform (CDCl3) was used as a solvent, with residual CHCl3 (SH = 7.24 ppm) or CDCl3 (SC = 77.0 ppm). In the description of the 1H and 13C NMR spectra, the atoms numeration system given in macrocycle 11 was used. The IR spectra were recorded by means of the KBr pellet (or film) technique on a Bruker Vector-22 spectrometer. The UV spectra were obtained on an HP 8453 UV-Vis spectrometer (Hewlett-Packard, Waldbronn, Germany). The mass spectra were recorded on a Thermo Scientific DFS high resolution mass spectrometer (evaporator temperature 240-270 °C, EI ionization at 70 eV). Melting points were determined using termosystem Mettler Toledo FP900 (USA). The optical rotation was measured on a polarimeter PolAAr3005 in ethanol at 20-25 °C.
co2h
Reaction products were isolated by column chromatography on silica gel 60 (0.063-0.200 mm, Merck KGaA) and eluted with petroleum ether-ether (10:1; to 1:1) and chloroform-ethanol (100:1; to 25:1). The reaction progress and the purity of the obtained compounds were controlled by TLC on Sorbfil UV-254 plates (detection under UV light or by spraying with a 10 % aqueous solution of H2SO4, followed by heating to 100 °C). Isopimaric acid 1 was isolated from Pinus sibirica R. Mayr sap by the previously described method.[33] 1-Azido-2-iodobenzene 2 was synthesized by reported method.[34] In the NMR 1H and 13C spectra the assignments marked with the same symbols *, # are interchangeable.
Synthesis
(1R,4aR,4bS, 7S, 10aR)-7-((E)-2-Azidostyryl)-1,4a, 7-trimethyl-1,2,3,4,4a, 4b, 5,6,7,8,10,10a-dodecahydrophenanthrene-1-carboxylic acid (3, C26H33N3O2). A mixture of isopimaric acid 1 (0.50 g, 1.65 mmol), 1-azido-2-iodobenzene 2 (0.41 g, 1.65 mmol), Pd(OAc)2 (0.07 g, 0.33 mmol) and Ag2CO3 (0.91 g, 3.30 mmol) in t-BuOH (5 mL) was stirred at 80 °C for 12 h (control TLC). After cooling, the mixture was diluted with CHCl3 (50 mL), the organic layer was separated, washed with water (3x15 mL), dried over MgSO4, and evaporated in vacuo. The residue was purified by column chromatography (eluent petroleum ether-Et2O, from 1:10 to 1:1) to afford compound 3 (0.42 g, 60 %).
Brown oil. [a]D25 = +8.2 (c 0.27 in CHCl3). HRMS (EI) (m/z): [(M+H)+] calcd for C26H33O2: 419.2567, found 419.2558. IR (film) v cm-1: 748 m, 970 m, 1151 m, 1189
max 55 5
m, 1284 s, 1384 m, 1448 m, 1484 m, 1699 vs, 2088 s, 2121 vs, 2651 w, 2819 m, 2848 s, 2865 s, 2925 s. UV-
io
Vis (ethanol) 1max (lge) nm: 319 (3.00), 307 (3.20), 264 (3.94), 262 (3.94). 1H NMR^CDC^, 298 K) SH ppm: 0.91 (3H, s, CH3-17), 0.96 (3H, s, CH3-20), 1.11 (1H, m, H-1), 1.26 (3H, s, CH3-19), 1.29 (1H, m, H-11), 1.43 (2H, m, H-12,11), 1.54, 1.57, 1.59 (4H, m, H-2,2,6,12), 1.67 (1H, m, H-3), 1.76 (2H, m, H-9,3), 1.83 (1H, m, H-1), 1.86 (1H, m, H-5), 1.95, 2.01, 2.04 (3H, m, H-6,14,14), 5.35 (1H, d J = 3.0 Hz, H-7), 6.16 (1H, d J = 16.3 Hz, H-15), 6.53 (1H, d J = 16.3 Hz, H-16), 7.06 (1H, t J = 7.6 Hz, H-4'), 7.10 (1H, d J = 7.6 Hz, H-3'), 7.22 (1H, t J = 7.6 Hz, H-5'), 7.46 (1H, d J = 7.6 Hz, H-6'). 13C NMR (CDCl3, 298 K) SC ppm: 15.20 (C20), 16.99 (C19), 17.80 (C2), 19.90 (C11), 21.74 (C17), 25.06 (C6), 34.88 (C10), 36.21 (C13), 36.76 (C12), 36.83 (C3), 38.66 (C1), 44.84 (C5), 46.18 (C4), 46.26 (C14), 51.81 (C9), 118.25 (C6'), 119.23 (C4'), 121.11 (C7), 124.64 (C16), 126.19 (C3'), 137.83 (C5'), 129.72 (C2'), 135.26 (C8), 136.57 (C1'), 144.13 (C15), 185.36 (C18).
Synthesis of compounds 9a-d. Glycine (1.00 g, 13.3 mmol) or L-valine (1.56 g, 13.3 mmol) was dissolved in a mixed solvent of potassium hydroxide aqueous solution and 1,4-dioxane (40 mL, 1:1 V/V). (Boc)2O (3.7 mL, 16.0 mmol) was added to the reaction solution. After the reaction system was stirred at room temperature for 12 hours, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed sequentially with 10 % sodium hydrogen sulfate solution and brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give N-Boc-glycine 4a and N-Boc-L-valine 4b as white solids, which on NMR spectra were pure enough for next step. A solution of N-Boc-Gly 4a or N-Boc-L-Val 4b (1.00 mmol), hydroxybenzotriazole (1.00 mmol), N,N'-dicyclohexylcarbodiimide (1.30 mmol) in CH2Cl2 (10 mL) was stirred at room temperature for 24 h, then propargyl amine hydrochloride 5 (1.00 mmol) and Et3N (2.00 mmol) were added, then the mixture was stirred at room temperature for 1 h and heated under reflux for 2 h. The formed precipitate was filtered and the mother liquid was concentrated in vacuo. The resulting residue was purified by column chromatography
(eluting with petroleum ether-Et2O, 1:10 ^ 1:1) to give compounds 6a (yield 81 %), or 6b (yield 89 %), which after being checked with TLC and NMR spectra were used for next step directly. The 1H and 13C NMR and mass-spectra of Boc-N-propargylamino-Glycine 6a was identical to that described in [35].
A stirred solution of compound 6a or 6b (0.5 mmol) in CH2Cl2 (6 mL) was treated with trifluoroacetic acid (0.7 mL, 10 mmol) in the conditions described in [36,37]. After stirring for 2 h, the reaction mixture was diluted with anhydrous toluene and the solution was removed in vacuo. The procedure was repeated three times to completely remove trifluoroacetic acid residues. The dipeptide products 7a (yield 87 %) and 7b (yield 88 %) on the TLC and NMR data, were pure enough for next step.
A solution of N-Boc-Gly 4a or N-Boc-L-Val 4b (1.00 mmol), hydroxybenzotriazole (1.00 mmol), N,N'-dicyclohexylcarbodi-imide (1.30 mmol) in CH2Cl2 (10 mL) was stirred at room temperature for 24 h, then ammonium trifluoroacetate 7a or 7b (1.00 mmol) and Et3N (2.00 mmol) was added. The mixture was stirred at room temperature for 1 h and heated under reflux for 2 h. The formed precipitate of the side product was filtered off, and the filtrate was concentrated under vacuum. Di-peptide products (8a-d, yield 63-81 %) were checked by NMR analyses and if necessary were purified by column chromatography (elut-ing with petroleum ether-Et2O, from 1:10 to 1:1) (for 8a,c,d). 1H and 13C NMR spectra of the compounds Boc-N-propargylamino-Gly-Glycine 8a[38] and Boc-N-propargylamino-Val-Glycine 8d[35] were identical to the literature data. The crude product 8b were used in the next step without further purification.
tert-Butyl(2-(((S)-3-methyl-1-oxo-1-(prop-2-yn-1-ylamino) butan-2-yl)amino)-2-oxoethyl)carbamate (Boc-N-propargyl-amino-Gly-Valine) (8c, 0.20 g, yield 63 %).
White solid. M.p. 125.1-126.7 °C. [a]D25 = -15.2 (c = 0.33 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for C15H25N3O4: 311.1840, found 311.1838. IR (film) v cm-1: 1168 m, 122(5 m, 1247 m, 1367
max
m, 1392 m, 1446 m, 1517 s, 1648 vs, 1675 s, 1702 s, 2129 vw, 2971 m, 3297 s. 1H NMR (CDCl3, 298 K) SH ppm: 0.88 (3H, d J = 8.2 Hz, CH(CH3)2), 0.90 (3H, d J = 8.2 Hz, CH(CH3)2), 1.38 (9H, s, OC(CH3)3), 2.05 (1H, br.s, CH(CH3)2), 2.18 (1H, s, C=CH), 3.84-4.03 (4H, m, 2CH2), 4.39 (1H, t J= 8.2 Hz, CH), 5.83 (1H, br.s, NH), 7.42 (1H, br.s, NH), 7.79 (1H, br.s, NH). 13C NMR (CDCl3, 298 K) SC ppm: 18.07, 18.96 (CH(CH3)2), 28.11 (C(CH3)3), 28.71 (CH2), 31.12 (CH(CH3)2), 43.94 (CH2), 58.12 (CH), 71.22 (C=CH), 79.28 (OC(CH3)3), 79.77 (C=CH), 156.02 (C02C(C H3)3), 169.98 (CO), 171.09 (CO3.
To a stirred solution of compound 8a-d (0.5 mmol) in CH2Cl2 (6 mL) trifluoroacetic acid (10 mmol) was added. After stirring for 2 h, the reaction mixture was diluted with anhydrous toluene and the solution was removed in vacuo. This procedure was repeated three time for completely remove acid residues. The resulting crude product was purified by column chromatography (eluting with CHCl3-MeOH, 10:1 to 10:3) to give the compounds 9a-d (yield 67-92 %). 1H and 13C NMR spectra of the compound 9a (yield 76 %) are consistent with previous report.[3839] The compound 9d (yield 90 %) was used for the next step without purification.
(S)-3-Methyl-1-(((S)-3-methyl-1-oxo-1-(prop-2-yn-1-ylamino)butan-2-yl)amino)-1-oxobutan-2-aminium 2,2,2-trifluo-roacetate (9b, 0.12 g, 67 %).
White solid. M.p. 147.4 °C. [a]D25 = +30.0 (c = 0.10 in CHCl3:MeOH 1:1). ESI-HRMS (3m/z): [(M+H)+] -CF3CO2H calcd for C^O^: 253.1785, found 253.1782. IR (film) v cm-1: 659 m, 750 m, 1230
max
m, 1288 m, 1386 m, 1448 m, 1469 m, 1484 s, 1513 s, 1643 vs,
+hW
cf3c02"
h ^
+h3n cf3co2"
2088 m, 2121 s, 2871 m, 2927 m, 2962 s, 3307 m, 3370 m. 'H NMR (CDCl3-CD3OD, 298 K) SH ppm: 0.74-0.89 (12H, m, 2CH(CH3)2), 1.96 (1H, q J = 6.8 Hz, CH(CH3)2), 2.01 (1H, q J = 6.5 Hz, CH(CH3)2), 2.14 (1H, s, C=CH), 3.13 (1H, br.s, CH and CH3OH), 3.84 (1H, d J = 17.0 Hz, CH2), 3.95 (1H, d J = 17.0 Hz, CH2), 4.06 (1H, d J = 6.9 Hz, CH), 7.88 (1H, m, NH). 13C NMR (CDCl3-CD3OD, 298 K) SC ppm: 16.06, 17.93, 18.87, 19.23 (2CH(CH3)2), 28.432 (CH2), 30.70 (2CH(CH3)2), 57.74 (CH), 59.78 (CH), 70.95 (C=CH), 78.88 (C=CH), 171.18 (CO), 174.87 (CO).
2-(((S)-3-Methyl-1-oxo-1-(prop-2-yn-1-ylamino)butan-2-yl) amino)-2-oxoethan-1-aminium 2,2,2-trifluoroacetate (9c, 0.34 g, 92 %).
O White solid. M.p.
191.4-192.8 °C. [a]D25 = -20.5 (c = 0.40 in CHCl3:MeOH 1:1). ESI-HRMS (m/z): [(M+H)+] -CF3CO2H calcd for C10H17O2N3: 211.1315, found 211.1314. IR (film) v cm-1: 725 m, 1132$ m, 1203
v ' max
s, 1257 m, 1432 m, 1471 m, 1556 s, 1643 vs, 1672 s, 2439 w, 2960 m, 3087 m, 3288 s. 1H NMR (CDCl3-CD3OD, 298 K) SH ppm: 0.78 (3H, d J = 5.8 Hz, CH(CH3)2), 0.80 (3H, d J = 5.8 Hz, CH(CH3)2), 1.95 (1H, q J = 5.8 Hz, CH(CH3)2), 2.12 (1H, s, C=CH), 3.59 (2H, m, CH2), 3.81 (2H, m, CH2), 4.02 (1H, br.s, CH and CH3OH), 8.05 (1H, br. s, NH), 8.19 (1H, br.s, NH). 13C NMR (CDCl3-CD3OD, 298 K) 5 H ppm: 17.15, 18.27 (CH(CH3)2), 28.10 (CH2), 30.22 (CH(CH3)2), 39.90 (CH2), 58.71 (CH), 70.59 (C=CH), 78.56 (C=CH), 165.90 (CO), 171.226 (CO).
General procedure for the synthesis of carboxamides 10a-d. A solution of isopimaric acid derivatives 3 (0.50 g, 1.19 mmol) in anhydrous CH2Cl2 (10 mL) under a stream of argon was cooled in ice, stirred vigorously for 15 min, and treated with oxalyl chloride (0.20 ml, 2.38 mmol) in CH2Cl2 (10 mL), catalytic amount of DMF (two drops). The reaction mixture was stirred for 4 h at ambient temperature, then the solvent was removed in vacuum. The residue was treated with CH2Cl2 (10 mL). The solvent was removed again. This procedure was repeated four times. The formed acid chloride derivative of diterpenoid 3 was used for the next step. A stirred solution of acid chloride of compound 3 in anhydrous CH2Cl2 (15 mL) was treated under argon with Et3N (0.33 ml, 2.38 mmol) and then, portion wise, with 9a-d (1.31 mmol) at ambient temperature. The reaction mixture was stirred for 24 h ^H NMR control), treated with 10 ml of water and extracted with methylene chloride (4 x 10 mL). The combined extract was dried over MgSO4, filtered and evaporated. The residue was subjected to column chromatography on silica gel (eluent CHCl3-MeOH) to give compounds 10a-d.
(1R,4aR,4bS, 7S,10aR)-7-((E)-2-Azidostyryl)-1,4a, 7-tri-methyl-N-(2-oxo-2-((2-oxo-2-(prop-2-yn-1-ylamino)ethyl)amino) ethyl)-1,2,3,4,4a,4b,5,6,7,8,10,10a-dodecahydrophenanthrene-1-carboxamide (10a, 0.41 g, 86 %).
White solid. M.p. 86.1 °C. [a]D25 = +20.4 (c 0.33 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for CH^ON: 570.3313, found
33 42 3 6
570.3309. IR (film) v cm-1: 661
max
m, 750 m, 970 w, 1243 m, 1284 s, 1384 m, 1419 m, 1446 m, 1484 m, 1521 s, 1652 vs, 2086 m, 2121 s, 2846 m, 2865 m, 2923 m, 3070 w, 3301 s, 3388 m. UV-Vis (ethanol) (lge) nm: 241 (4.15), 263 (4.09). 1H NMR (CDCl3, 298 K) 5H
l
ppm: 0.88 (3H, s, CH3-17), 0.92 (3H, s, CH3-20), 1.10 (1H, m, H-1), 1.28 (4H, m, CH3-19, H-11), 1.38-1.41 (2H, m, H-12,11), 1.53 (4H, m, H-2,2,6,12), 1.73-1.78 (3H, m, H-3,9,3), 1.84 (1H, m, H-1), 1.88 (2H, m, H-5,6), 1.95 (2H, m, H-14,14), 2.19 (1H, br.s, C=CH), 3.92 (6H, m, 2H-5",8",2"), 5.28 (1H, br.s, H-7), 6.10 (1H, d J = 16.2 Hz, H-15), 6.47 (1H, d J = 16.2 Hz, H-16), 7.04 (3H, m, H-3',4', NH-1"), 7.18 (1H, t J = 7.5 Hz, H-5'), 7.42 (2H, m, H-6', NH-4"), 7.54 (1H,
br.s, NH-7'').13C NMR (CDCl3, 298 K) SC ppm: 15.16 (C20), 17.07 (C19), 17.88 (C2), 19.75 (C11), 21.66 (C17), 24.71 (C6), 28.84 (C8"), 34.92 (C10), 36.10 (C13), 36.63 (C12), 37.06 (C3), 38.54 (C1), 42.79 (C5"), 43.64 (C2"), 45.48 (C5), 46.12 (C4,14), 51.78 (C9), 71.22 (C=CH), 79.31 (C=CH), 118.16 (C6'), 119.16 (C4'), 120.99 (C7), 124.57 (C16), 126.10 (C3'), 127.75 (C5'), 129.59 (C2'), 135.26 (C8), 136.45 (C1'), 143.94 (C15), 168.75 (C6''), 170.14 (C3''), 180.12 (C18).
(1R,4aR,4bS, 7S, 10aR)-7-((E) -2-Azidostyryl)-1,4a, 7-trimethyl-N-((S)-3-methyl-1-(((S)-3-methyl-1-oxo-1-(prop-2-yn-1-ylamino)butan-2-yl)amino) -1 -oxobutan-2-yl) -1,2,3,4,4a,4b, 5,6,7,8,10,10a-dodecahydrophenanthrene-1-carbox-amide (10b, 0.41 g, 53 %).
White solid. M.p. 174.3 °C. [a]D25 = -28.8 (c 0.23 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for CH^ON: 654.4252, found
39 54 3 6
654.4263. IR (film) v cm-1: 663 w,
v ' max
684 w, 719 w, 1230 m, 1376 w, 1384 w, 1465 m, 1548 s, 1635 vs, 2119 vw, 2852 m, 2871 m, 2923 s, 2960 s, 3077 w, 3284 s, 3378 m. UV-Vis (ethanol) 1max (lge) nm: 242 (4.21), 264 (4.15). H NMR (CDCl3, 298 K) SH ppm: 0.83-0.92 (12H, m, 2CH(CH3)2), 0.88 (3H, s, CH3-17), 0.92 (3H, s, CH3-20), 1.11 (1H, m, H-1), 1.32 (3H, s, CH3-19), 1.39-1.44 (2H, m, H-11,12), 1.47-1.60 (5H, m, H-11,2,2,6,12), 1.71-1.83 (3H, m, H-3,9,3), 1.90-2.00 (4H, m, H-1,5,6,14), 2.04 (1H, m, H-14), 2.09 (2H, m, 2CH(CH3)2), 2.14 (1H, t J = 2.4 Hz, C=CH), 3.98 (1H, m J = 5.2, 2.4 Hz, H-8"), 4.03 (1H, m J = 5.2, 2.4 Hz, H-8"), 4.34 (1H, d J = 8.2 Hz, H-5'')*, 4.38 (1H, d J = 7.3 Hz, H-2'')*, 5.28 (1H, d J = 3.9 Hz, H-7), 6.10 (1H, d J = 16.3 Hz, H-15), 6.48 (1H, d J = 16.3 Hz, H-16), 6.72 (1H, br. s, NH-1"), 7.03 (1H, t, J = 8.3 Hz, H-4'), 7.04 (1H, d J = 8.3 Hz, H-3'), 7.18 (1H, t J = 8.3 Hz, H-5'), 7.43 (1H, d J = 8.3 Hz, H-6'), 7.47 (1H, m, NH-4'')*, 7.54 (1H, m, NH-7")* 13C NMR (CDCl3, 298 K) SC ppm: 15.22 (C20), 17.46 (C19), 18.00 (C2), 18.05, 18.68, 19.07, 19.28 (2CH(CH3)2), 19.78 (C11), 21.65 (C17), 24.85 (C6), 28.72 (C8''), 29.95, 30.37 (2CH(CH3)2), 34.94 (C10), 36.20 (C13), 36.65 (C12), 37.20 (C3), 38.58 (C1), 45.08 (C5), 46.12 (C4), 46.21 (C14), 51.88 (C9), 58.33 (C2'), 59.09 (C5'), 70.99 (C=CH), 79.49 (C=CH), 118.21 (C6), 119.19 (C4), 121.12 (C7), 124.61 (C16), 126.16 (C3), 127.78 (C5'), 129.68 (C2'), 135.16 (C8), 136.52 (C1'), 144.04 (C15), 171.02 (C6''), 171.99 (C3'), 178.76 (C18).
(1R,4aR,4bS, 7S, 10aR)-7-((E)-2-Azidostyryl)-1,4a, 7-trimethyl-N-(2-(((S)-3-methyl-1-oxo-1-(prop-2-yn-1-ylamino) butan-2-yl)amino)-2-oxoethyl)-1,2,3,4,4a,4b,5,6,7,8,10,10a-dodecahydrophenanthrene-1-carboxamide (10^ 0.34 g, 50 %).
White oil. [a]D25 = +14.8 (c 0.57 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for C36H48O3N6: 612.3782, found 612.3780. IR (film) v cm-1: 634 w, 659 m,
max
752 m, 970 w, 1232 m, 1286 m, 1338 w, 1386 m, 1448 m, 1484 m, 1523 s, 1650 ws, 2088 m, 2121 s, 2848 m, 2869 m, 2925 s, 2960 m, 3068 w, 3307 s. UV-Vis (ethanol) 1max (lge) nm: 242 (4.13), 263 (4.07). 1H NMR (CDCl3, 298 K) SH ppm: 0.90 (3H, s, CH3-17), 0.90 (6H, d J = 8.7 Hz, CH(CH3)2), 0.93 (3H, s, CH3-20), 1.13 (1H, m, H-1), 1.29 (3H, s, CH3-19), 1.40 (2H, m, H-11,12), 1.50-1.56 (5H, m, H-11,2,2,6,12), 1.75-1.83 (3H, m, H-3,9,3), 1.91 (2H, m, H-1,5), 1.96 (3H, m, H-6,14,14), 2.09 (1H, m J = 8.7 Hz, CH(CH3)2), 2.18 (1H, t J = 2.5 Hz, C=CH), 3.98 (4H, m, 2H-2'',8''), 4.36 (1H, t J = 8.7, Hz, H-5'), 5.29 (1H, d J = 3.9 Hz, H-7), 6.12 (1H, d J = 16.3 Hz, H-15), 6.50 (1H, d J = 16.3 Hz, H-16), 7.04 (1H, t J = 7.8 Hz, H-4'), 7.07 (1H, d J =7.8 Hz, H-3'), 7.11 (1H, t J =7.4 Hz, NH-1"), 7.19 (1H, t J = 7.8 Hz, H-5'), 7.43 (1H, d J = 7.8 Hz, H-6'), 7.48 (1H, d J = 8.7 Hz, NH-4')*, 7.52 (1H, t J = 5.1 Hz, NH-7"). 13C NMR (CDCl 298 K) SC
ppm: 15.20 (C20), 17.13 (C19), 17.91 (CH(CH3)2), 17.95 (C2), 19.04 (CH(CH3)2), 19.76 (C11), 21.67 (C17), 24.75 (C6), 28.82 (C8''), 30.85 (CH(CH3)2), 34.94 (C10), 36.15 (C13), 36.64 (C12), 37.03 (C3), 38.52 (C1), 43.(68 (C2''), 45.45 (C5), 46.12 (C14), 46.16 (C4), 51.79 (C9), 58.39 (C5'), 71.20 (C=CH), 79.31 (C=CH), 118.17 (C6'), 119.18 (C4'), 121.04 (C7), 124.58 (C16), 126.12 (C3'), 127.76 (C5'), 129.62 (C2'), 135.19 (C8), 136.47 (C1), 143.98 (C15), 169.88 (C6'), 170.97 (C3'), 179.69 (C18).
(1R,4aR,4bS,7S,10aR)-7-((E)-2-Azidostyryl)-1,4a,7-tri-methyl-N-((S)-3-methyl-1-oxo-1-((2-oxo-2-(prop-2-yn-1-ylamino) ethyl)amino)butan-2-yl)-1,2,3,4,4a, 4b,5,6,7,8,10,10a-dodecahy-drophenanthrene-1-carboxamide (10d, 0.63 g, 86 %).
White oil. [a]D25 = +6.0 (c 0.23 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for C36H48O3N6: 612.3782, found 612.3780. IR (film) v cm-1: 632 w, 659 m,
max
680 m, 752 s, 970 m, 1160 m, 1232 m, 1286 s, 1336 m, 1369 m, 1386 m, 1448 m, 1484 s, 1513 s, 1641 ws, 2088 s, 2121 ws, 2846 m, 2869 m, 2925 s, 2960 s, 3070 w, 3305 s, 3544 w. UV-Vis (ethanol) l
max
(lge) nm: 242 (4.16), 263 (4.10). 1H NMR (CDCl3, 298 K) SH ppm: 0.90 (3H, s, CH3-17), 0.93 (6H, m, CH(CH3)2), 0.94 (3H, s, CH3-20), 1.11 (1H, m, H-1), 1.30 (3H, s, CH3-19), 1.41 (1H, m, H-11), 1.51-1.58 (6H, m, H-12,11,2,2,6,12), 1.75-1.81 (3H, m, H-3,9,3), 1.92 (2H, m, H-1,5), 1.97, 2.00, 2.06 (3H, m, H-6,14,14), 2.17 (1H, t J = 2.4 Hz, C=CH), 2.26 (1H, br.s, CH(CH3)2), 3.84 (1H, dd J = 16.8, 2.4 Hz, H-5''), 3.97 (1H, dd J = 5.5, 2.4 Hz, H-8"), 3.99 (1H, dd J = 5.5, 2.4 Hz, H-8' ), 4.05 (1H, dd J = 16.8, 2.4 Hz, H-5"), 4.15 (1H, t J = 7.4 Hz, H-2''), 5.35 (1H, d J = 4.4 Hz, H-7), 6.13 (1H, d J = 16.1 Hz, H-15), 6.50 (1H, d J = 16.1 Hz, H-16), 6.51 (1H, d J = 7.4 Hz, NH-1"), 7.03 (1H, t J = 8.5 Hz, H-4'), 7.07 (1H, d J = 8.5 Hz, H-3'), 7.20 (1H, td J = 8.5, 1.2 Hz, H-5'), 7.26 (1H, d J = 5.5 Hz, NH-7''), 7.43 (2H, m, H-6', NH-4'). 13C NMR (CDCl3, 298 K) SC ppm: 15.20 (C20), 17.25 (C19), 17.90 (C2), 18.65 (CH(CH3)2), 19.22 (CH(CH3)2), 19.77 (C11), 21.68 (C17), 24.81 (C6), 28.87 (C8"), 30.25 (CH(CH3)2), 34.98 (C10), 36.13 (C13), 36.66 (C12), 37.20 (C3), 38.54 (C1), 42.95 (C5''), 45.19 (C5), 46.17 (C4), 46.27 (C14), 51.79 (C9), 59.55 (C2''), 71.22 (C=CH), 79.29 (C=CH), 118.21 (C6'), 119.21 (C4'), 120.88 (C7), 124.61 (C16), 126.14 (C3'), 127.79 (C5'), 129.64 (C2'), 135.36 (C8), 136.51 (C1'), 144.00 (C15), 168.54 (C6''), 172.19 (C3''), 179.65 (C18).
General procedure for the synthesis of macroheterocycles 11a,c,d. A solution of sodium ascorbate (0.12 g, 0.60 mmol) in H2O (1.0 mL) and a solution of CuSO4x5H2O (0.03 g, 0.12 mmol) in H2O (1.0 mL) were added by vigorously stirring to a solution of compounds 10a,c,d (0.60 mmol) in CH2Cl2 (60 mL). The heterogeneous mixture was heated at 40 °C under stirring for 80 h (TLC-and 1H NMR control). After cooling, 5 mL of water was added. The organic layer was separated, washed with H2O (3x30 mL), dried over MgSO4, and evaporated in vacuo. The residue was purified by a column chromatography (eluent CHCl3-MeOH) to afford compounds 11a,c,d.
(14Z,52S,54aS,54bR, 58R,58aR,510Z, 3E)-52,54b,58-Trimethyl-51,52,53,54,54a, 54b, 55,56,57,58,58a, 59-dodecahydro-11H-7,10,13-triaza-1(1,4)-triazola-5(2,8)-phenanthrena-2(1,2)-benzenacyclotetra-decaphan-3-ene-6,9,12-trione (11a, 0.18 g, 53 %).
White solid. M.p. 138.1 °C. [a]D25 = +60.8 (c 1.00 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for C„H ON ■ 570.3313,
33 42 3 6
found 570.3307. IR (film) v
max
cm-1: 757 s, 1238 m, 1259 m, 1365 m, 1382 m, 1413 m, 1446 m, 1465 m, 1519 s, 1664 vs, 2852 m, 2923 s, 3077 w, 3309 m, 3338 m, 3349 m, 3367 m, 3430 m. UV-Vis (ethanol) l (lge) nm: 252
(3.93). 1H NMR (CDCl3, 298 K) SH ppm: 0.81 (3H, s, CH3-511), 0.85 (3H, s, CH3-513), 1.20 (4H, s, CH3-512, H-55), 1.29 (1H, m, H-54), 1.53-1.58 (6H, m, H-57,57, 54,56,59,53), 1.70-1.74 (2H, m, H-54a,58a), 1.81-1.88 (5H, m, H-53,51,51,55,59), 1.98 (1H, m, H-56), 3.40 (1H, dd J = 16.6, 2.3 Hz, H-11)*, 3.52 (1H, dd J = 16.9, 3.0 Hz, H-8)*, 3.96 (1H, m, H-11)#, 4.27 (1H, m, NH-10), 4.49 (2H, m, 2H-14), 4.91 (1H, dd J = 16.9, 8.0 Hz, H-8)#, 5.22 (1H, d J = 4.5 Hz, H-510), 5.73 (1H, d J = 16.1 Hz, H-3), 6.17 (1H, d J = 16.1 Hz, H-4), 6.49 (1H, dm J = 8.0 Hz, NH-7), 7.32 (1H, m, H-23), 7.42 (2H, m, H-24,25), 7.57 (1H, d J = 7.6 Hz, H-26), 7.76 (1H, s, H-15), 7.92 (1H, dd J = 7.7, 4.4 Hz, NH-13). 13C NMR (CDCl3, 298 K) SC ppm: 15.25 (C513), 16.95 (C512), 18.14 (C56), 19.96 (C54), 23.21 (C511), 24.51 (C59), 33.74 (C54b), 35.89 (C14), 36.29 (C57), 37.10 (C53), 37.76 (C52), 38.90 (C55), 41.60 (C11), 42.78 (C8), 43.62 (C51), 46.45 (C58), 48.67 (C58a), 53.20 (C54a), 118.41 (C3), 119.99 (C510), 124.90 (C15), 126.02 (C23), 126.14 (C26), 127.70 (C24), 129.85 (C25), 132.95 (C22), 134.43 (C21), 136.95 (C510a), 143.93 (C14), 145.97 (C4), 168.69 (C12), 170.69 (C9), 178.82 (C6).
(14Z,52S,54aS,54bR,58R,58aR,510Z, 3E,11S)-11 -Isopropyl-52,54b, 58-trimethyl-51,52,53,54,54a,54b,55,56,57,58,58a,59-dodecahydro-11H-7,10,13-triaza-1 (1,4)-triazola-5(2,8) -phenanthrena-2(1,2) -benzena-cyclotetradecaphan-3-ene-6,9,12-trione (11c, 0.07 g, 19 %).
White solid. M.p. 170.6 °C. [a]D25 = +44.9 (c 0.27 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for C,,H„O,N,: 612.3782,
36 48 3 6
found 612.3786. IR (film) v
max
cm-1: 759 s, 970 m, 1043 m, 1195 m, 1226 m, 1263 m, 1334 w, 1369 m, 1384 m, 1465 s, 1521 s, 1658 vs, 1726 m, 2852 s, 2923 vs, 3066 w, 3324 s. UV-Vis (ethanol) l
max
(lge) nm: 245 (3.94), 254 (3.95). 1H NMR (CDCl3, 298 K) SH ppm: 0.82 (3H, s, CH3-511), 0.88 (3H, d J = 6.9 Hz, CH(CH3)2), 0.92 (3H, s, CH3-513), 0.95 (3H, d J = 6.9 Hz, CH(CH3)2), 1.24 (4H, s, CH3-512, H-55), 1.27 (1H, m, H-54), 1.53-1.60 (6H, m, H-57,57,54,56,59,53), 1.65-1.68 (2H, m, H-51,54a), 1.74-1.82 (3H, m, H-58a,53,55), 1.86 (1H, m, H-51), 1.95 (1H, m, H-59), 1.97 (1H, m, H-56), 2.21 (1H, dq J = 12.6, 6.9 Hz, CH(CH3)2), 3.81 (1H, dd J = 15.4, 4.6 Hz, H-8), 3.97 (1H, dd J = 15.4, 4.6 Hz, H-8), 4.29 (1H, m, H-11), 4.43 (1H, dd J = 15.3, 5.6 Hz, H-14), 4.79 (1H, dd J = 15.3, 5.6 Hz, H-14), 5.31 (1H, br.s, H-510), 5.88 (1H, d J = 16.3 Hz, H-3), 6.26 (1H, d J = 16.3 Hz, H-4), 6.35 (1H, t J = 5.6 Hz, NH-13), 6.52 (1H, d J = 8.2 Hz, NH-10), 6.89 (1H, t J = 4.6, NH-7), 7.33 (1H, dd J = 7.8, 1.3 Hz, H-23), 7.40 (1H, t J = 7.8 Hz, H-24), 7.44 (1H, dt, J = 7.8, 1.3 Hz, H-25), 7.65 (1H, d J = 7.8 Hz, H-26), 7.70 (1H, s, H-15). 13C NMR (CDCl3, 298 K) SC ppm: 14.94 (C513), 17.28 (C512),
17.70 (CH(CH3)2), 18.04 (C56), 19.18 (CH(CH3)2), 19.82 (C54), 24.91 (C59), 26.57 (C511), 30.54 (CH(CH3)2), 34.84 (C14), 34.95 (C57),
35.71 (C53), 36.93 (C52), 37.49 (C54b), 38.64 (C55), 42.96 (C8), 43.82 (C51), 46.05 (C58), 46.61 (C58a), 52.27 (C54a), 58.34 (C11), 118.81 (C3), 120.76 (C510), 124.11 (C15), 125.90 (C23), 126.32 (C26), 127.50 (C24), 129.81 (C25), 133.24 (C22), 134.66 (C21), 135.08 (C510a), 143.49 (C14), 145.85 (C4), 169.66 (C12), 170.45 (C9), 179.83 (C6).
(14Z, 52S, 54aS, 54bR, 58R, 58aR, 510Z, 3E, 8S)-8-Isopropyl-52,54b, 58-trimethyl-51,52,53,54,54a,54b, 55,56,57,58,58a,59-dodecahydro-11H-7,10,13-triaza-1 (1,4)-triazola-5(2,8) -phenanthrena-2(1,2)-ben-zenacyclotetradecaphan-3-ene-6,9,12-trione (11d, 0.17 g, 46 %).
White solid. M.p. 175.5 °C. [a]D25 = +49.3 (c 0.63 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for C H ON : 612.3782, found
36 48 3 6
612.3784. IR (film) v cm-1: 757 s,
max
971 w, 1045 w, 1191 m, 1236 m, 1255 m, 1367 m, 1384 m, 1467 m, 1508 s, 1660 vs, 2852 m, 2869 m, 2927 s, 2956 s, 3077 v, 3307 m, 3440 m. UV-Vis (ethanol) l
max
(lge) nm: 241 (3.89), 251 (3.90). 1H
NMR (CDCl3, 298 K) SH ppm: 0.76 (3H, s, CH3-511), 0.83 (3H, d J = 7.0 Hz, CH(CH3)2), 0.85 (3H, s, CH3-513), 0.88 (3H, d J = 6.6 Hz, CH(CH3)2), 1.20 (4H, s, CH3-512, H-55), 1.26 (1H, m, H-54), 1.51-1.65 (7H, m, H-57,57,54,56,56,59,53), 1.67-1.73 (2H, m, H-55,54a), 1.76-1.83 (3H, m, H-58a,53,51), 1.87 (1H, d J = 13.1 Hz, H-51), 1.94 (1H, m, H-59), 2.43 (1H, m, CH(CH3)2), 3.35 (1H, dd J = 16.5, 2.6 Hz, H-11), 4.19 (1H, dd J = 14.9, 4.2 Hz, H-14), 4.53 (1H, dd J = 9.2, 4.8 Hz, H-8), 4.59 (1H, dd J = 16.5, 9.0 Hz, H-11), 4.97 (1H, dd J = 14.9, 8.4 Hz, H-14), 5.14 (1H, d J = 4.4 Hz, H-510), 5.70 (1H, d J = 16.1 Hz, H-3), 6.13 (1H, d J = 16.1 Hz, H-4), 6.51 (1H, dd J = 9.0, 2.6 Hz, NH-10), 6.62 (1H, d J = 9.2 Hz, NH-7), 7.30 (1H, t J = 7.5 Hz, H-24), 7.37 (1H, d J = 7.5 Hz, H-23), 7.40 (1H, t J = 7.5 Hz, H-25), 7.53 (1H, d J = 7.5 Hz, H-26), 7.65 (1H, s, H-15), 8.18 (1H, dd J = 8.4, 4.2 Hz, NH-13). 13C NMR (CDCl3, 298 K) SC ppm: 15.24 (C513), 16.85 (C512), 17.22 (CH(CH3)2), 18.25 (C56), 19.46 (CH(CH3)2), 19.86 (C54), 22.92 (C511),
24.49 (C59), 29.05 (CH(CH3)2), 33.61 (C14), 35.10 (C57), 36.28 (C53),
36.50 (C52), 37.00 (C54b), 38.71 (C55), 41.52 (C11), 43.54 (C51), 46.81 (C58), 45.58 (C58a), 52.99 (C54a), 57.99 (C8), 118.32 (C3), 120.07 (C510), 124.31 (C15), 125.90 (C23), 126.13 (C26), 127.66 (C24), 129.62 (C25), 132.64 (C22), 134.40 (C21), 136.57 (C510a), 143.91 (C14), 145.97 (C4), 168.65 (C12), 172.07 (C9), 178.46 (C6).
(14Z,52S,54aS,54bR,58R,58aR,510Z, 3E, 8S,11S)-8,11-Diiso-propyl-52,54b, 58-trimethyl-51,52,53,54,54a,54b,55,56,57,58,58a,59-dodecahydro-1H-7,10,13-triaza-1(1,4)-triazola-5(2,8)-phenan-threna-2(1,2)-benzenacyclotetradecaphan-3-ene-6,9,12-trione (11b). A solution of compound 10b (0.60 mmol) in DMF (6 mL) was cooled in ice under argon and sequentially treated with CuI (0.02 g, 0.12 mmol) and DIPEA (0.21 mL, 1.20 mmol). The reaction mixture was stirred for 24 h (TLC-control), then diluted with CHCl3 (50 mL) and washed with 2N H2SO4 (15 mL), H2O (3 x 50 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. Column chromatography of the residue (eluent CHCl3-MeOH) afforded compound 11b (0.11 g, 28 %).
White solid. M.p. 202.2 °C. [a]D25 = +39.3 (c 0.30 in CHCl3). ESI-HRMS (m/z): [(M+H)+] calcd for C,_H,.O,N, : 654.4252, found
39 54 3 6
654.4249. IR (film) v cm-1: 759
max
m, 970 w, 1041 w, 1157 w, 1189 m, 1226 m, 1263 m, 1290 m, 1317 w, 1336 w, 1367 m, 1384 m, 1467 s, 1504 s, 1656 vs, 2852 m, 2869 m, 2927 s, 2960 s, 3317 m, 3400 m.
UV-Vis (ethanol) 1max (lge) nm: 239 (3.20), 251 (3.19). 1H NMR (CDCl3, 298 K) SH ppm: 087-0.92 (12H, m, 2CH(CH3)2), 0.90 (3H, s, CH3-511), 0.93 (3H, s, CH3-513), 1.10 (1H, m, H-55), 1.21 (3H, s, CH3-512), 1.27-1.31 (1H, m, H-54), 1.50-1.64 (7H, m, H-57,57,54,56,56,59,53), 1.69-1.78 (5H, m, H-55,54a,58a,53,51), 1.87-1.94 (2H, m, H-51,59), 2.10 (1H, m, CH(CH3)2), 2.26 (1H, m, CH(CH3)2), 4.09 (1H, t J = 8.0 Hz, H-11), 4.40 (2H, m, 2H-14), 4.79 (1H, dd, J = 15.1, 6.5 Hz, H-8), 5.33 (1H, m, H-510), 5.86 (1H, d J = 16.1 Hz, H-3), 6.00 (1H, d J =8.0 Hz, NH-10), 6.25 (1H, d J = 16.1 Hz, H-4), 6.57 (1H, d J = 6.5 Hz, NH-7), 6.71 (1H, br.s, NH-13), 7.37 (1H, t J = 7.6 Hz, H-24), 7.44 (2H, m, H-23,25), 7.63 (1H, d J = 7.6 Hz, H-26), 7.68 (1H, s, H-15). 13C NMR (CDCl3, 298 K) SC ppm: 15.19 (C513), 16.92 (C512), 17.45, 18.31 (CH(CH3)2), 18.26 (C56), 19.33, 19.39 (CH(CH3)2), 20.07 (C54), 23.82 (C511), 24.64 (C59), 30.23, 30.82 (2CH(CH3)2), 34.84 (C14), 35.07 (C57), 36.15 (C53), 36.15 (C52), 37.31 (C54b), 37.48 (C55), 44.49 (C51), 46.91 (C58), 48.16 (C58a), 53.55 (C54a), 58.26 (C8)*, 58.92 (C11)*, 118.91 (C3), 121.56 (C510), 124.21 (C15), 125.79 (C23), 126.27 (C26), 127.65 (C24), 129.84 (C25), 132.94 (C22), 134.41 (C21), 134.62 (C510a), 143.79 (C14), 145.93 (C4), 170.39 (C12), 171.74 (C9), 179.27 (C6).
Cytotoxicity studies
Human tumor cell lines, MCF-7 (breast cancer), DU145 (prostate cancer), and hTERT (telomerase reverse transcriptase,
non-cancer control) were used in the work. Cells were cultivated in RPMI-1640 medium containing bovine serum (10 %), L-gluta-mine (2 mM), gentamicin (80 ¡ig/mL), and lincomycin (30 mg/mL) at 37 °C with 5 % CO2 in an incubator for 72 h. Tested compounds were dissolved in DMSO and added at the required concentrations to cell cultures. Three wells were used for each concentration (0.1, 1, 10, and 100 ¡g/mL). Control cells were incubated without adding tested compounds. GI50 values were determined using the standard MTT assay[30,40,41] based on reduction of colorless tetrazolium
bromide [3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide, MTT reagent] by mitochondrial and cytoplasmic dehy-drogenases of metabolically active living cells to form blue forma-zan crystals. The amount of formazan in the DMSO was measured by spectrometry. A reduction of optical density in samples as compared with a control was indicative of cytotoxicity. Optical density (D) of samples was measured on a Bio-Rad 680 microplate reader (USA) at 490 nm. The mean and error of the mean were calculated for each analyte concentration. Cell survival (%) was plotted as a function of concentration of tested cytotoxic compound to determine the dose inhibiting by 50 % cell survival (GI50) and the standard error (SE) of GI50. Results were processed statistically using Microsoft Excel 2007, Statistica 6.0, and Graph-Pad Prism 5.0 software. Results were given as means ± deviations from the means. The statistical significance (p) was estimated using the Student t-criterion. Differences were considered statistically significant for p < 0.05. Experimental results are given as the means of three independent experiments.
Results and Discussion
Synthesis
In the present research to synthesize the novel diterpe-noid heteromacrocyclic derivatives a methodology has been adopted, involving the cross-coupling reaction of isopimaric acid (1) with azido-iodobenzene (2) followed by the amide coupling of 16-(2-azidophenyl)isopimaric acid (3) with ammonium 2,2,2-trifluoroacetate salt of alkynyl dipeptides and intramolecular reaction of azide-alkyne cycloaddition of the resulting conjugates. The key diterpenoid derivative 3 was synthesized by the cross-coupling reaction of isopimaric acid (1) with 1-azido-2-iodobenzene (2) in the presence of Pd(OAc)2 (1 mol%) and Ag2CO3 (1 equiv.) in the previously described conditions (Scheme 1).[18,32] The (E)-16-(2-azidophenyl) substituted derivative 3 was isolated in 60 % yield after column chromatography.
To synthesize the peptide precursors, as illustrated in Scheme 2, Boc-protected glycine 4a or L-valine 4b were coupled with propargylamine hydrochloride 5 to provide alkynyl amino acid derivatives 6a,b (81-88 %). After removal of the protecting groups by treating with DCM/ TFA, amino compounds 7a,b (yield 87-88 %) were further coupled with Boc-protected glycine 4a or L-valine 4b using HOBt and DCC to obtain Boc-protected alkynyl dipeptides 8a-d (63-81 %). Subsequently, treatment with TFA led to complete removal of the Boc group to provide ammonium 2,2,2-trifluoroacetate dipeptides 9a-d (67-92 % yield).
The synthesis of 16-(2-azidophenyl)-isopimarate amides 10a-d is outlined in Scheme 3. Treatment of isopi-maric acid derivative 3 with an excess of oxalyl chloride in the presence of a catalytic amount of dimethylformamide gave the corresponding acid chloride, which was reacted without isolation with TFA salts of alkynyldipeptides 9a-d.
Scheme 1. Synthesis of 16-(2-azidophenyl)isopimaric acid (3).
X/OH + HCI - i S # i
u u CF.COV
6a: R=H, 81%; 3 2 7a: R=H, 87%;
4a: R=H, 5 6b. R=/.pr 88o/o 7b: R=/.pr, 88°/
4b: R=/-Pr
OR OR
4a, b + 7a,b - BocHN^X^J^N^^ ^N^X,, J^N^i^
8c: R^H, R= /-Pr, 63% 8d: R^i-Pr, R=H 70%
a: HOBt, DCC, CH2CI2, rt, 24 h, then 5, Et3N, reflux, 2 h.; b: TFA, CH2CI2, rt, 2 h.
R1 o CF3C02" "1 0
8a: R=R1= H, 81% 9a. r1=r2= H, 76%
8b: R=R1= /-Pr, 70% 9b. r1=r2= /.p^ 67o/o
9c: R^H, R=/-Pr, 92% 9d: R,=/-Pr, R2=H 90%
Scheme 2. Synthesis of alkynyldipeptide 2,2,2-trifluoroacetate ammonium salts 9a-d.
The reaction was carried out at room temperature in anhydrous methylene chloride in the presence of triethylamine. Terpenoid amides 10a-d were isolated in 50-86 % yields after column chromatography.
Intramolecular macrocyclization of azidoalkynes 10a-d was performed using Cu-catalyzed cycloaddition reaction of alkyne with azide (CuAAC-reaction). We have found that compounds 10a,c,d with a dipeptide fragment Gly-Gly, Gly-Val and Val-Gly could be transformed in the corresponding macroheterocycles 11a,c,d by reflux in aq. CH2Cl2 in the presence of copper sulfate and sodium ascorbate (NaAsc). The reaction was carried out under conditions of strong dilution (0.01 M alkynazide 10a,c,d solution in CH2Cl2) (TLC-control). Macrocyclic compounds 11a,c,d were isolated by column chromatography in the yield of 19-53 % (Scheme 3). When macrocyclization of amide 10b with the Val-Val fragment was carried out under mentioned conditions, the formation of a macrocyclic product was not observed; the reaction was characterized with the formation of a complex mixture. The desired intramolecular azide-alkyne cycloaddition proceeded more smoothly when the reaction was carried out in the presence of CuI and DIPEA in DMF at ambient temperature. After stirring the reaction mixture for 24 h in argon at room temperature (TLC-control), subsequent treatment and column chromatography, compound 11b was isolated in the 28 % yield.
As it was observed, the yields of macrocyclization products depend on the nature of the dipeptide fragment. An increase in the steric volume leads to a decrease
in the yield of the macrocyclic product, and this effect is more pronounced in the presence of a more bulky substituent in the amino acid residue.
The cytotoxicity of macrocyclic compounds 11a-d, azidoalkynes 10a-d, terpenoid azide 3, and compound 1 was studied in cell cultures MCF-7 (breast cancer) and DU-145 (prostate cancer). Immortalized hTERT human fibroblasts were used as a non-cancerous control. MTT assays were performed for quantitative evaluation of in vitro cytotoxicity.[40] Doxorubicin was used as the positive control. The cytotoxicity was determined by measuring the concentration inhibiting human tumor cell viability by 50 % (GI50). The results are presented in Table 1. The SAR revealed that the substituent at C-16 position of isopimaric acid 1 has a great influence on the cytotoxicity. Compound 3 with azidophenyl substituent showed high potency against breast cancer cell line MCF-7. Modification of isopimaric acid amide with peptide substituent can improve the anticancer activity. A remarkable increase in activity and selectivity towards prostate cancer cell line DU-145 was observed for compounds 10a and 11a with a dipeptide featuring with Gly-Gly amino acid sequence. Characteristically, the amino acid sequence has a great influence on the cytotoxicity. It should be noted that macrocyclic compounds 11c,d are more cytotoxic towards MCF7 cells than their acyclic precursors 10c,d. Among all derivatives macrocyclic compound 11d shows the higher cytotoxicity towards normal cell lines (Table 1).
10a-d
b or c
19-53%
О R
cf3c02" ri
a, 50-86%
9a-d
O^^NH HN
w ъ
Ri о 10a-d
10
11
10a, 11a: H H 60% 53% b
10b, 11b: CH(CH3)2 CH(CH3)2 53% 28% c
10c, 11c: H CH(CH3)2 50% 19% b
10d, 11d: CH(CH3)2 H 86% 46% b
11a-d
a: (COCI)2, CH2CI2, 5 h, then 9a-d, Et3N, CH2CI2, 24 h;
b: c11so4, NaAsc, CH2CI2-H20, 40°C, 80 h; c: Cul, DIPEA, DMF, rt, 24 h.
Scheme 3. Synthesis of macroheterocyclic compounds 11a-d.
Table 1. Cytotoxicity of isopimaric acid (1), compound 3, alkynyl dipeptide derivatives 10a-d and macroheterocycles 11a-d.
Compound Growth inhibition of cells (GI50, ^M)*
hTERT Lung Fibroblasts MCF7 DU145
1 ND > 100 135.56 ± 25.76
3 72.77 ± 3.47 10.25 ± 2.17 > 100
10a > 100 > 100 17.94 ± 2.22
10b 68.73 ± 5.44 44.67 ± 2.84 > 100
10c 23.95 ± 0.93 53.21 ± 2.51 > 100
10d 84.56 ± 9.74 > 100 > 100
11a 61.33 ± 4.78 > 100 40.08 ± 3.74
11b 63.18 ± 1.46 42.35 ± 0.98 > 100
11c 64.77 ± 1.94 15.88 ± 2.31 > 100
11d 15.23 ± 2.44 55.33 ± 3.42 75.44 ± 4.38
Doxorubicin 3.03 ± 0.44 3.55 ± 1.02 4.88 ± 0.63
*GI50: concentration inhibiting human tumor cell viability by 50 % is observed after 72 h incubation. The experimental results are given as the average values of data obtained from three independently conducted experiments.
Conclusions
We have shown that sequential arylation/amide coupling/CuAAC-based macrocyclization approach could be considered as a powerful procedure for the conjugation of alkynyl peptides to azidoditerpenoids. This approach was used for obtaining macrocyclic compounds with dipeptide fragment containing the amino acid sequence of glycine and valine. We have also found that macrocycliza-
tion of isopimaric acid derivatives with dipeptide substituent leads to a noticeable change in cytotoxicity in relation to normal and cancer cells. The results reported herein pave the way for the further access of new targeted anticancer agents. In addition, due to their simplicity and effectiveness, it is likely that the studied transformations of tricyclic diterpenoid core would find use in the development of new compounds and could be used as scaffolds toward accessing other libraries of bioactive compounds.
Acknowledgements. The reported study was funded by RFBR, project number 19-33-60043. Authors would like to acknowledge the Multi-Access Chemical Research Center SB RAS for spectral and analytical measurements.
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Received 16.09.2021 Accepted 22.10.2021
