ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 1, pp. 83–86. © Pleiades Publishing, Ltd., 2015. Original Russian Text © S.F. Vasilevsky, D.S. Baranov, A.I. Govdi, I.V. Sorokina, T.G. Tolstikova, 2015, published in Bioorganicheskaya Khimiya, 2015, Vol. 41, No. 1, pp. 97–101.
The Synthesis and AntiInflammatory Activity of Propargylamino Derivatives of Naphthoquinonlevopimaric Acid S. F. Vasilevskya, b, 1, D. S. Baranova, A. I. Govdia, I. V. Sorokinac, and T. G. Tolstikovac aVoevodsky
Institute of Chemical Kinetics and Combustion, Siberian Branch, Russian Academy of Sciences, ul. Institutskaya 3, Novosibirsk, 630090 Russia b Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia c Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences, pr. akad. Lavrent’eva 9, Novosibirsk, 630090 Russia Received April 4, 2014; in final form, June 30, 2014
Abstract—Naphthoquinonlevopimaric acid was modified with propargylamino residues and antiinflamma tory activities of the synthesized Mannich bases was studied. Keywords: acetylenes, antiinflammatory activity, naphthoquinonlevopimaric acid, Mannich bases DOI: 10.1134/S1068162015010148 1
INTRODUCTION
NLPA 4aminobut2ynyl esters, and the evaluation of their antiinflammatory properties.
During the last years synthetic transformations of natural compounds performed with the goal of prepar ing their biologically active analogues are becoming a topical course of bioorganic chemistry. The synthesis of natural metabolite analogues, diterpene resin acids produced by coniferous trees, is of particular interest. Levopimaric acid (LPA), a major component of pine soft resin, is the most available and promising com pound among them. Its essential advantage is its high concentration in soft resin of Pinus sylvestris (27%) and its subvariety Pinus hamata (36%) [1].
RESULTS AND DISCUSSION The LPA molecule is a convenient base for modifi cations because it contains a conjugated diene frag ment, which can be used in the Diels–Alder reaction, and a carboxy group, which is easily subjected to func tionalization. Considering all these facts, for the synthesis of the target compound, prop2ynyl1carboxylate (II), we used a double modification of the LPA molecule con sisting of diene synthesis with 1,4naphtoquinone fol lowed by esterification of the carboxy group with a propargyl residue. It is noteworthy that the first stage (diene synthesis with quinone) is not only the first modification stage but also the best way of LPA isolation from a complex mixture of pine soft resin. This approach is simple and includes mixing of quinone and soft resin. An addi tional advantage of the adducts is their greater stability if compared with the initial LPA. NLPA (I) was obtained as described in [8] and the key compound prop2ynyl1 carboxylate (II), by the NLPA interaction with propargyl bromide in the pres ence of K2CO3 in a yield of 30% (Scheme 1). It is known that propargyl amines display valuable medicinal properties. Some of them are effective anti cancer agents, HIV reverse transcriptase inhibitors, and some are essential synthetic intermediates [3, 9]. Among these compounds, inhibitors of mammalian squalene epoxidase were found [10].
Various synthetic LPA derivatives display a wide spectrum of biological effects, particularly, anti inflammatory and antiulcer activities [1], and play an important role in protection from insects and patho genic microorganisms [2]. At the same time, the latest monograph on this topic [1] (chapter 3, 142 refer ences) covering the period of 1933–2010 and describ ing methods of synthesis of LPA analogues does not have any data on the preparation of LPA acetylene derivatives. Acetylene derivatives are known to be important natural biologically active compounds [3, 4]. Moreover, the introduction of alkynyl residues is widely used for modifications of natural compounds including triterpenoids [5, 6]. As a part of our scien tific program on studies of plant metabolites produced by plants and trees of the Siberian region [6, 7], we report the synthesis of new acetylene derivatives of naphthoquinonlevopimaric acid (NLPA), namely, 1 Corresponding author: phone: +7 (383) 3333347; fax: +7 (383)
3307350; email:
[email protected].
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The study of antiinflammatory effects of compounds (II) and (IIIa–d) using a histamine inflammatory model Compound
Inflammatory index, %
Edema size vs the control, %
27.3 ± 1.5d 19.8 ± 1.9a 17.4 ± 1.2b 16.0 ± 1.5b 18.6 ± 1.5a 20.1 ± 1.4a, c 15.1 ± 1.1b
100 72.5 63.7 58.6 68.1 73.6 55.3
Control (IIIa) (IIIb) (IIIc) (IIId) (II) Indomethacinum
Antiinflammatory effect vs the control, % 0 27.5 36.3 41.4 31.9 26.4 44.7
a p < 0.01, b p < 0.001, reliable relative to the control group; c p < 0.05, d p < 0.001, reliable relative to indomethacinum.
CH3
CH3 17
CH3 CH3
O
CH3
CH3 5 CH3
CH3 1,4naphtoquinone 3 2
4 1
7 6a 7a 12a 11a 12
16 15
4b 4a 12b 14a 13 14
CH3
O
H
6
H
8 11
9 10
CH3 CH3
Br
O (II)
CH3 COOH
OH
CH3
Me2CO, K2CO3
O
O
O
(I)
CH3
O
CH2R2, CuCl
O
1,4dioxane
CH3 CH3 CH3
O
R=
N
(IIIa),
N
O
O
O (IIIb),
R
N
(IIIa–d)
(IIIc),
N
(IIId)
Scheme 1. Preparation of acetylene derivatives of naphthoquinonpimaric acid (II) and Mannich bases (IIIa–d). The compounds of this type can be prepared using the Mannich reaction. In a classical version it is an interaction of three components, a terminal alkyne, formaldehyde (generated in situ from paraformalde hyde), and a secondary amine [11]. For the synthesis of compounds (IIIa–d) we used bisaminomethanes obtained separately. The reaction was performed in dioxane in the presence of CuCl at room temperature in an inert atmosphere (0.5 h) to give aminoalkylation products (IIIa–d) in yields of 88–96% (the scheme). The stereochemistry of the starting NLPA (I) was reliably confirmed [8]. Since the NLPA ester (II) and Mannich bases (IIIa–d) are synthesized at moderate temperatures (55 and 20°C respectively), we suppose that the stereochemistry of the molecule is retained.
The structures of the synthesized acetylenes and the products of their transformation were confirmed by ana lytical and spectral methods (IR, 1H and 13C NMR). Antiinflammatory activity of NLPA derivatives (IIIa–d) was studied on a standard model of hista mineinduced inflammation in the mouse paw. The results shown in the table demonstrated that all the Mannich bases synthesized (IIIa–d) manifested a reliable antiinflammatory effect (26–41%) relative to the control following intraperitoneal administration at a dose of 20 mg/kg. For all the compounds tested except acetylene (II) the edema size did not have a sig nificant difference from that following the administra
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THE SYNTHESIS AND ANTIINFLAMMATORY ACTIVITY
tion of the reference compound at a similar dose (see the Experimental section). The experimental data showed that the Mannich bases with morpholine (IIIb) and diethylamine (IIIc) residues had a marked activity, whereas compounds with pyrrolidine (IIIa) and piperidine (IIId) frag ments as well as alkyne1 (II) displayed a moderate antiinflammatory activity. EXPERIMENTAL Chemistry IR spectra were registered on a Bruker Vector 22 spectrometer in KBr tablets. NMR spectra (δ, ppm, J, Hz) were registered on a Bruker AV 400 spectrometer in CDCl3 at 400.13 (1H NMR) and 100.61 MHz (13C NMR). Mass spectra were registered on a DFS mass spectrometer (Thermo Electron Corporation) using the direct injection at a the temperature of ion ization chamber of 220–270°C in the ionization chamber and an ionization voltage of 70eV. For col umn chromatography Al2O3 (50150 μm, TU609 391675) was used; TLC was performed on Silufol 60 F254 plates (Merck). Propargyl bromide was purchased from Aldrich (United States); di(N,Ndiethylamino) and di(Npiperidino)methane were obtained as described in [12]; and di(Nmorpholino) and di(Npyr rolidino)methane, as described in [13] and [14] respec tively. (1R,4aR,4bS,6R,6aR,12aR,12bS)16Isopropyl 1,4adimethyl1,2,3,4,4a,4b,5,6a,7,12,12a,13,14,14a pentadecahydro2H(6,12betheno)benzo[b]chrysen 7,12dion1carboxylic acid (I) was obtained as described in [8]; mp 174–177°C (mpref 175–178°C). Prop2ynyl {(1R,4aR,4bS,6R,6aR,12aR,12bS) 16isopropyl1,4adimethyl1,2,3,4,4a,4b,5,6a,7,12, 12a,13,14,14apentadecahydro2H(6,12betheno) benzo[b]chrysen7,12dion}1carboxylate (II). A mix ture of naphthoquinonpimaric acid (7 g, 15.2 mmol), propargyl bromide (3.6 g, 30.4 mmol), and K2CO3 (4.8 g, 45.6 mmol) in acetone (190 mL) was refluxed for 7 h. The reaction mixture was filtered, evaporated in vacuo, and the residue was purified by column chro matography on Al2O3 eluting with methylene chloride to give 2.3 g (30%) of the target (II); mp 175–176°C (hexane). 1H NMR: 0.49 (3H, d, J 6.7, CH3), 0.53 (3H, s, CH3), 0.85 (3H, d, J 6.7, CH3), 0.94 (1H, m, CH), 1.14 (3H, s, CH3), 1.25 (2H, m, CH2), 1.35– 1.58 (6H, m, CH2), 1.65 (1H, m, CH), 1.72–1.90 (5H, m, CH2, CH), 2.48 (1H, t, J 2.4, ≡CH), 2.87 (1H, d, J 8.6, CH), 3.05 (1H, br s, CH), 3.25 (1H, dd, J1 2.9, J2 8.6 CH), 4.68 (2H, m, OCH2), 5.06 (1H, br s, =CH), 7.61 (2H, m, ArH), 7.71 (1H, m, ArH), 7.79 (1H, m, ArH). 13C NMR: 16.23, 16.82, 17.10, 19.11, 20.73, 22.07, 28.21, 33.24, 35.02, 36.48, 37.82, 38.33, 42.24, 42.45, 47.28, 49.35, 52.23, 52.26, 56.23, 59.41, 74.75, 78.10, 125.19, 125.90, 125.93, 133.26, 134.02, 136.91, 138.70, 147.60, 177.93, 198.57, 198.86. Found, %: C 79.69; H 7.62. C33H38O4. Calc., %: С RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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79.48; H 7.68. IR, ν, cm–1: 1680, 1722 (C=O); 2131, 3290 (C≡CH). The synthesis of Mannich bases. Diaminomethane (0.44 mmol) was added to a solution of propargyl naphthoquinonpimaroate (II) (200 mg, 0.4 mmol) and CuCl (10 mg) in 1,4dioxane (10 mL) in an argon atmosphere, the mixture was kept for 0.5 h, and poured in toluene (20 mL). The mixture was washed with saturated NH4Cl (2 × 20 mL) and dried with Na2SO4. The product was purified by column chroma tography on Al2O3 eluting with ethyl acetate. [4(Pyrrolidin1yl)but2ynyl]{(1R,4aR,4bS,6R, 6aR,12aR,12bS)16isopropyl1,4adimethyl1,2,3,4,4a, 4b,5,6a,7,12,12a,13,14,14apentadecahydro2H(6,12b etheno)benzo[b]chrysen7,12dion}1carboxylate (IIIa). The yield of 220 mg (95%); mp 167–168°C (hexane). 1 H NMR: 0.49 (3H, d, J 6.9, CH3), 0.53 (3H, s, CH3), 0.85 (3H, d, J 6.9, CH3), 0.94 (1H, m, CH), 1.12 (3H, s, CH3), 1.25 (2H, m, CH2), 1.36–1.57 (6H, m, CH2), 1.63 (1H, m, CH), 1.72–1.87 (9H, m, CH2, CH), 2.64 (4H, m, N(CH2)2), 2.86 (1H, d, J 8.6, CH), 3.05 (1H, br s, CH), 3.24 (1H, dd, J1 2.9, J2 8.5, CH), 3.50 (2H, t, J 1.7, CH2N), 4.71 (2H, t, J 1.8, OCH2), 5.06 (1H, br s, =CH), 7.60 (2H, m, ArH), 7.70 (1H, m, ArH), 7.78 (1H, m, ArH). 13C NMR: 16.22, 16.84, 17.13, 19.11, 20.72, 22.07, 23.95, 28.18, 33.24, 35.11, 36.47, 37.85, 38.33, 42.23, 42.44, 43.35, 47.29, 49.36, 52.24, 52.71, 52.74, 56.28, 59.41, 78.69, 82.58, 125.20, 125.91, 125.93, 133.23, 134.00, 136.91, 138.69, 147.60, 177.99, 198.43, 198.86. MS, m/z: found 581.3502 [M]+. C38H47NO4. Calc. M 581.3500. IR, ν, cm–1: 1678, 1716 (C=O). [4(Morpholin4yl)but2ynyl]{(1R,4aR,4bS,6R, 6aR,12aR,12bS)16isopropyl1,4adimethyl1,2,3,4, 4a,4b,5,6a,7,12,12a,13,14,14apentadecahydro2H (6,12betheno)benzo[b]chrysen7,12dion}1carbox ylate (IIIb). The yield 230 mg (96%); mp 170–171°C (hexane). 1H NMR: 0.48 (3H, d, J 6.9, CH3), 0.53 (3H, s, CH3), 0.85 (3H, d, J 6.9, CH3), 0.94 (1H, m, CH), 1.12 (3H, s, CH3), 1.20–1.29 (2H, m, CH2), 1.37–1.56 (6H, m, CH2), 1.60–1.65 (1H, m, CH), 1.72–1.88 (5H, m, CH2, CH), 2.60 (4H, t, J 4.6, N(CH2)2), 2.86 (1H, d, J 8.6, CH), 3.05 (1H, br s, CH), 3.24 (1H, dd, J1 2.9, J2 8.5, CH), 3.38 (2H, t, J 1.9, CH2N), 3.78 (4H, t, J 4.6, (CH2)2O), 4.71 (2H, m, OCH2), 5.05 (1H, br s, =CH), 7.60 (2H, m, ArH), 7.69 (1H, m, ArH), 7.78 (1H, m, ArH). 13C NMR: 16.21, 16.86, 17.12, 19.12, 20.72, 22.07, 28.20, 33.24, 35.13, 36.43, 37.86, 38.34, 42.23, 42.44, 47.32, 47.63, 49.40, 52.23, 52.48, 52.60, 56.28, 59.41, 67.05, 79.86, 81.43, 125.18, 125.90, 125.93, 133.24, 134.01, 136.90, 138.67, 147.64, 177.95, 198.41, 198.82. MS, m/z: found 597.3440 [M]+. C38H47NO5. Calc. M 597.3449. IR, ν, cm–1: 1680, 1715 (C=O). [4Diethylaminobut2ynyl]{(1R,4aR,4bS,6R,6aR, 12aR,12bS)16isopropyl1,4adimethyl1,2,3,4,4a, 4b,5,6a,7,12,12a,13,14,14apentadecahydro2H(6,12b etheno)benzo[b]chrysen7,12dion}1carboxylate (IIIc). The yield 220 mg (94%); mp 140–141°C (hexane). 1H NMR: 0.49 (3H, d, J 6.9, CH ), 0.53 (3H, s, CH ), 3 3 Vol. 41
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0.85 (3H, d, J 6.9, CH3), 0.94 (1H, m, CH), 1.10 (6H, m, N(CH2CH3)2), 1.13 (3H, s, CH3), 1.25 (2H, m, CH2), 1.37–1.56 (6H, m, CH2), 1.63 (1H, m, CH), 1.74–1.87 (5H, m, CH2, CH), 2.57 (4H, q, J 7.3, N(CH2CH3)2), 2.86 (1H, d, J 8.6, CH), 3.05 (1H, br s, CH), 3.24 (1H, dd, J1 2.8, J2 8.6, CH), 3.49 (2H, t, J 1.9, CH2N), 4.70 (2H, t, J 1.9, OCH2), 5.06 (1H, br s, =CH), 7.60 (2H, m, ArH), 7.69 (1H, m, ArH), 7.78 (1H, m, ArH). 13C NMR: 12.80, 16.23, 16.84, 17.14, 19.11, 20.72, 22.08, 28.18, 33.24, 35.13, 36.55, 37.87, 38.33, 41.06, 42.23, 42.45, 47.29, 47.38, 49.34, 52.24, 52.72, 56.31, 59.43, 79.13, 81.56, 125.20, 125.90, 125.92, 133.22, 134.00, 136.91, 138.70, 147.61, 177.96, 198.40, 198.84. MS, m/z: found 583.3655 [M]+. C38H49NO4. Calc. M 583.3656. IR, ν, cm–1: 1678, 1718 (C=O). [4(Piperidin1yl)but2ynyl]{(1R,4aR,4bS,6R, 6aR,12aR,12bS)16isopropyl1,4adimethyl1,2,3,4, 4a,4b,5,6a,7,12,12a,13,14,14apentadecahydro2H (6,12betheno)benzo[b]chrysen7,12dion}1carbox ylate (IIId). The yield 210 mg (88%); mp 138–139°C (hexane). 1H NMR: 0.49 (3H, d, J 6.9, CH3), 0.53 (3H, s, CH3), 0.85 (3H, d, J 6.9, CH3), 0.94 (1H, m, CH), 1.13 (3H, s, CH3), 1.25 (2H, m, CH2), 1.37– 1.56 (8H, m, CH2), 1.60–1.68 (5H, m, CH2, CH), 1.75–1.88 (5H, m, CH2, CH), 2.25 (4H, br s, N(CH2)2), 2.86 (1H, d, J 8.6, CH), 3.05 (1H, br s, CH), 3.24 (1H, dd, J1 2.9, J2 8.6, CH), 3.34 (2H, t, J 1.9, CH2N), 4.71 (2H, t, J 1.9, OCH2), 5.06 (1H, br s, =CH), 7.60 (2H, m, ArH), 7.69 (1H, m, ArH), 7.78 (1H, m, ArH). 13C NMR: 16.23, 16.85, 17.14, 19.12, 20.73, 22.08, 24.07, 26.10, 28.20, 33.25, 35.12, 36.50, 37.87, 38.34, 42.24, 42.45, 47.31, 48.06, 49.37, 52.25, 52.78, 53.49, 56.29, 59.43, 79.24, 82.34, 125.22, 125.91, 125.93, 133.23, 134.00, 136.93, 138.71, 147.62, 178.00, 198.42, 198.86. MS, m/z: found 595.3650 [M]+. C39H49NO4. Calc. M 595.3656. IR, ν, cm–1: 1678, 1724 (C=O). Biology The antiinflammatory activity of the compounds synthesized was evaluated according to the reduction of the edema in the mouse paw (56 male animals) induced by 0.01% histamine solution following sub planar administration (ICN BioPharmaceutical). The compounds under study were administered into stom ach at a dose of 20 mg/kg as a watertween suspension 1 h prior to flogogen injection. Control animals were also administered this watertween suspension. Indometacin (Fluka) at a dose of 20 mg/kg served as a reference compound. The edema size was estimated 6 h after the administration of the compounds tested on the basis of the difference between masses of healthy and inflamed paws. For each animal the inflammation index was calculated as a ratio of this difference to the mass of the healthy paw. The results were treated using a STATISTIKA 8 software package program packet. The differences were considered reliable with the prob
ability p < 0.05. The edema size was calculated for each experimental group relative to the control group, which was taken as 100%. An antiinflammatory effect was taken as the difference between the relative edema size in the control group and that in the experimental group. ACKNOWLEDGMENTS The work was supported by the Integration grant of the Siberian Branch of the Russian Academy of Sci ences no. 51 (2012–2014); the Russian Foundation for Basic Research, grant no. 130300129a (2013– 2015); the Ministry of Education and Science of the Russian Federation (2014–2016); and the Chemical Service Center of Russian Academy of Sciences. REFERENCES 1. Tolstikov, G.A., Tolstikova, T.G., Shul’ts, E.E., Tol stikov, S.E, and Khvostov, M.V., Smolyanye kisloty khvoinykh Rossii. Khimiya, farmakologiya (Resin Acids in Conifers of Russia: Chemistry and Pharmacology), Trofimov, B.A., Ed., Novosibirsk: GEO, 2011. 2. Trapp, S. and Croteau, R., Annu. Rev. Plant Physiol. Plant Mol. Biol., 2001, vol. 52, pp. 689–724. 3. Dembitsky, V.M. and Levitsky, D.O., Nat. Prod. Com mun., 2006, vol. 1, no. 5, pp. 405–429. 4. Galm, U., Hager, M.H., Van Lanen, S.G., Ju, J., Thorson, J.S., and Shen, B., Chem. Rev., 2005, vol. 105, pp. 739–758. 5. Vasilevsky, S.F., Govdi, A.I., Shults, E.E., Shakirov, M.M., Sorokina, I.V., Tolstikova, T.G., Baev, D.S., Tolstikov, G.A., and Alabugin, I.V., Bioorg. Med. Chem., 2009, vol. 17, pp. 5164–5169. 6. Vasilevsky, S.F., Govdi, A.I., Sorokina, I.V., Tolsti kova, T.G., Tolstikov, G.A., Mamatuyk, V.I., and Ala bugin, I.V., Bioorg. Med. Chem. Lett., 2011, vol. 21, pp. 62–65. 7. Vasilevskii, S.F., Govdi, A.I., Shul’ts, E.E., Shakirov, M.M., Alabugin, I.V., and Tolstikov, G.A., Dokl. Akad. Nauk, 2009, vol. 424, no. 5, pp. 631–634. 8. Vafina, G.V., Fazlyev, R.R., Galin, F.Z., and Spi rikhin, L.V., Zh. Org. Khim., 2009, vol. 45, pp. 515– 519. 9. Dembitskii, V.M., Tolstikov, G.A., and Tolstikov, A.G., Khim. Interesah Ustoich. Razvit., 2003, vol. 11, no. 2, pp. 341–348. 10. Musso, D.L., Clarke, M.J., Kelley, J.L., Boswell, G.E., and Chen, G., Org. Biomol. Chem., 2003, vol. 1, pp. 498–506. 11. Vasilevskii, S.F., Slabuka, P.A., Izyumov, E.G., Shvarts berg, M.S., and Kotlyarevskii, I.L., Izv. Akad. Nauk SSSR, Ser. Khim., 1972, no. 11, pp. 2524–2529. 12. Feldman, J.R. and Wagner, E.C., Org. Chem., 1942, vol. 7, p. 31. 13. Bailey, P.S., Nowlin, G., and Bost, W., J. Am. Chem. Soc., 1951, vol. 51, p. 4078. 14. Putochin, N.J., Chem. Ber., 1922, vol. 55, p. 2749.
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