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10-1-2009
On the Synthesis and Anticancer Testing of alpha,beta-Unsaturated Ketones as Analogs of Combretastatin-A4 Sameer Chavda Hope College
Ryan Davis Hope College
Amanda Ferguson Hope College
Camille Riddering Hope College
Kristin Dittenhafer Hope College See next page for additional authors
Follow this and additional works at: http://digitalcommons.hope.edu/faculty_publications Part of the Chemistry Commons Recommended Citation Published in: Letters in Drug Design and Discovery, Volume 6, Issue 7, October 1, 2009, pages 531-537. Copyright © 2009 Bentham Science Publishers, Sharjah, U Arab Emirates. The final published version is available at: http://www.benthamscience.com/contentsJCode-LDDD-Vol-00000006-Iss-00000007.htm
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Sameer Chavda, Ryan Davis, Amanda Ferguson, Camille Riddering, Kristin Dittenhafer, Hilary Mackay, Balaji Babu, Moses Lee, Adam Siegfried, William Pennington, Miriam Shadfan, Susan L. Mooberry, Bijay K. Mishra, and Hari N. Pati
This article is available at Digital Commons @ Hope College: http://digitalcommons.hope.edu/faculty_publications/16
Digital Commons @ Hope College Faculty Bibliography
10-1-2009
On the Synthesis and Anticancer Testing of α,βUnsaturated Ketones as Analogs of Combretastatin-A4 Sameer Chavda Hope College
Ryan Davis Hope College
Amanda Ferguson Hope College
Camille Riddering Hope College
Kristin Dittenhafer Hope College See next page for additional authors
This Article is brought to you for free and open access by Digital Commons @ Hope College. It has been accepted for inclusion in Faculty Bibliography by an authorized administrator of Digital Commons @ Hope College.
Authors
Sameer Chavda, Ryan Davis, Amanda Ferguson, Camille Riddering, Kristin Dittenhafer, Hilary Mackay, Balaji Babu, and Moses Lee
On the Synthesis and Anticancer Testing of α,β-Unsaturated Ketones as Analogs of Combretastatin-A4 Sameer Chavdaa, Ryan Davisa, Amanda Fergusona, Camille Ridderinga, Kristin Dittenhafera, Hilary Mackaya, Balaji Babua, Moses Lee,a,* Adam Siegfriedb, William Penningtonb, Miriam Shadfan,c Susan L. Mooberryc, Bijay K. Mishrad, and Hari N. Patid a
Department of Chemistry and the Division of Natural and Applied Sciences, Hope College 49423,
USA b c
Department of Chemistry, Clemson University, SC 29634, USA
Department of Pharmacology, , University of Texas Health Science Center at San Antonio, San
Antonio, TX, 78229 d
Department of Chemistry, Sambalpur University, Jyoti Vihar, Orissa-768 019, India
*Address correspondence to this author at the Division of Natural and Applied Sciences and Department of Chemistry, Hope College, Holland, MI, 49423, USA; Tel: +1 616 395 7190; Fax: +1 616 395 7923; E-mail:
[email protected] Running title: α,β-Unsaturated Ketones as Analogs of Combretastatin-A4
Abstract – Twenty-one α,β-unsaturated ketone analogs of combretastatin-A4 (CA-4) that were designed for good solubility in aqueous media were synthesized. Compounds defined as Type A were derived from phenylacetone, in which sub-class I contained ortho-, meta- or no substituents, sub-class II contained para-substituents, and sub-class III consisted of two substituents. Type B compounds were derived from cyclopropyl 2-fluorobenzyl ketone. The cis-configuration of the target compounds was ascertained through a single crystal X-ray crystallographic analysis of the fluorine-containing compound 8f. Five of the analogs, 8c, 8j and 8l of Type A and 9d and 9i of Type B, were shown to display modest cytotoxic potency (IC50 in the 3.8 – 21 µM range) against the growth of murine melanoma (B16) and leukemia (L1210) cells in culture. Compounds 8j, 8l and 9i were further tested against MDA-MB-435 human melanoma cells. The cyclopropanecontaining compound 9i was the most potent; within IC50 value of 2.4 µM. Even though no appreciable effects on interphase microtubules were observed when A-10cells were treated with 30 µM 8j or 8l, compound 9i caused extensive microtubule depolymerization at a this concentration. These results suggest that compound 9i of Type B has a similar mechanism of action as CA-4 whilst compounds 8j and 8l of Type B are likely to have a different mechanism of action. Keywords: combretastatin, cytotoxicity, tubulin, microtubule, cancer
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INTRODUCTION Vascular disrupting agents (VDAs) act as effective anti-tumor agents because they initiate rapid shutdown of tumor vasculature, leading to tumor necrosis,1 possibly mediated through the vascular endothelial cadherin signaling pathway.1b The combretastatins are one class of VDAs that rapidly shutdown tumor vasculature. Combretastatin-A4, (CA-4 or 1 as shown in Fig. (1) a natural product which consists of a core cis-stilbene moiety, was originally isolated from the African Willow tree, Combretum caffrum, and is known to inhibit tubulin polymerization via interaction with the colchicine binding site of tubulin.2 CA-4 causes the rapid destruction of aberrant tumor vasculature and these effects are probably mediated through direct effects on tumor endothelial cells. Despite the effectiveness of CA-4 as an antitumor agent, one major drawback leading to the impairment of its antitumor activity is its lack of bioavailability and poor solubility in biological media.3 As a result there is a significant interest in the design of structural analogs of 1 that exhibit more pharmacologically beneficial properties. Derivatives, such as, compounds 2 (CA4P),4 3 (an amino analog of CA-4) and 4 (an amino acid derivative of CA-4P, AVE-8062)5 depicted in Fig. (1) are examples of more water-soluble pro-drugs of CA-4. CA-4P is presently undergoing clinical trials.4 In addition, analogs of combretastatins incorporating a heterocycle capable of mimicking the rigidity of the π framework, maintaining the cis-configuration of the aromatic rings (necessary for a good fit into the colchicine binding site in tubulin), as well as increasing the polarity of the molecules so as to improve water solubility have been reported.6 A class of compounds possessing these criteria reported previously from our laboratory include the 1,2,3-triazole derivatives of combretastatin, which were found to possess levels of cytotoxicity in the micro-molar range against the growth of B16 murine melanoma cells.7 Although the potencies of the representative triazole analogs shown in Fig (2) range from poor to moderate, we believe that compounds bearing the general structure -phenyl-heterocycle-phenyl- or –phenyl-π-phenyloffer favorable solubility in aqueous media and maintain the geometry necessary to fit the colchicines binding site in tubulin, and this model warrants further investigation. With this information in hand, we have synthesized a series of α,β-unsaturated ketones 8a-m and 9a-j, which contain the cis-stilbene core present in the combretastatins [Fig (3)]. Even though such enone-containing molecules can potentially undergo Michael reactions with biological nucleophiles such as glutathione,8 they are worthy of further investigation because α,β-enones,
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such as chalcones analgues of CA-4, have been found to exhibit potent cytotoxicity against cancer cells in culture.9 In addition, to the best of our knowledge, these types of combretastatin analogs have not been investigated to date for anticancer activity. RESULTS AND DISCUSSION A series of α,β-unsaturated ketone analogs of CA-4 which conform to the criteria mentioned above were synthesized: the incorporation of the cis-stilbene core was required to maintain biological activity and the presence of the polar acetyl functionality on the stilbene core to help promote solubility in aqueous media. Twenty-one α,β-unsaturated ketones were synthesized and assessed for their ability to inhibit the growth of L1210 and B16 cells (murine leukemia and melanoma cell lines). Three compounds were also selected for testing against the growth of human melanoma MDA-MB-435 cells and depolymerization of microtubules in rat A10 aortic cells. Two categories of compounds were synthesized: Type A (derived from phenylacetone10 8e) sub-classes I (ortho-, meta- and non substituted 8a-d), II (para-substituted; 8f-8k, Table 1), III (consisting of two disubstituted analogs 8l and 8m, Table 2) and Type B (para-, di- and trisubstituted) derived from cyclopropyl 2-fluorobenzyl ketone11 (Table 3, 9a-e and 9g-j). The Type A compounds were synthesized using a known literature procedure10c by acid catalyzed condensation of the appropriate aldehyde with phenylacetone in good to excellent yield [65-75%, Fig (3), Tables 1 and 2]. Compounds 8a, 8f, 8g, 8h and 8j have been reported previously.12 The Type B compounds were synthesized by refluxing a mixture of cyclopropyl 2-fluorobenzyl ketone, sodium methoxide and the appropriate aryl aldehyde in methanol to give the corresponding α,βunsaturated ketones in good yields [60-75%, Fig (3), Table 3]. Due to commercial availability constraints, we were limited mainly to a series of mono- and disubstituted aldehydes (Tables 1, 2 and 3). The geometry of the aromatic rings about the double bond in these compounds was exclusively found to be cis as ascertained from the X-ray structure of compound 8f [Fig (4)]. Further evidence for this geometry arises from the NMR chemical shift of the benzylidene proton in these compounds, which appears as a singlet at approximately δ 7.60. In comparison, for the trans isomer, this particular proton appears to be slightly shifted downfield at approximately δ 8.00.13
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With a range of the target molecules in hand, their ability to inhibit the growth of murine B16 and L1210 melanoma cell lines was assessed. These cells were continuously exposed for three days with the growth inhibition properties of Type A and B compounds studied using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.14 The concentrations needed to inhibit growth of B16 and L1210 cells for compounds 8a-8m are shown in Tables 1, 2 and 3. The results from these studies display several interesting trends based upon the substitution pattern of the aromatic ring (derived from the benzaldehyde reactant) and the nature of the substituent. As a control, for categories A and B, phenylacetone 8e and cyclopropyl 2-fluorobenzyl ketone 9f were tested for cytotoxicity against both cell lines and, as a result, both gave IC50 values >100 µM suggesting that having two aromatic rings on these molecules is necessary for biological activity. For the Type A compounds, it appears that ortho-substitution of a Cl atom in the Ar ring moderately affects potency (compound 8b) in relation to its non-substituted counterpart 8b. Incorporation of a slightly less polar and larger Br atom in the meta position dramatically results in an increase in cytotoxicity, with IC50 values of around 4 µM, in both B16 and L1210 cell lines, for compound 8c, which is significantly more active than its unsubstituted analog 8a, which gave an IC50 of >100 µM. Meta- substitution of an electron-donating methoxy group moderately diminishes the potency (compound 8d, IC50 values 67 (B16) and 56 (L1210) µM). In comparison to ortho- and meta- substitution, a fluorine or bromine atom in the para- position of the Ar ring (8f and 8g; Table 1; Type A sub-class II) results in very little cytotoxicity. Incorporation of an electron-withdrawing nitro group into the para-position of the Ar ring (compound 8j) has the most significant positive effect on cytotoxicity (IC50 values 14.4 (B16) and 21.4 (L1210) µM) in comparison to compounds 8h, 8i, 8j and 8k presumably suggesting that the electronic influence (due to inductive and resonance effects) of the para-nitro group enhances the compound’s interactions with its cellular targets. It is interesting to note that 8k, containing a para-substituted dimethylamino moiety capable of exerting an electronic influence through electron donating resonance effects, shows low potency (>100 µM for B16 and L1210 lines). This suggests that substituent size could possibly be a factor contributing to the potency of these compounds as well as substituent polarizability in the para-position. Based on the cytotoxic behavior of the Type A sub-classes I and II compounds, we were curious to probe whether 2,6 and 3,4 disubstitution of the Ar ring could enhance cytotoxicity (8l
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and 8m, Table 2; Type A sub-class III). Interestingly, the presence of a phenoxy group in the meta-position adjacent to a fluorine atom in the para-position in 8l resulted in an increase in potency (~11 µM) in comparison to its para-fluorinated counterpart 8f (Table 1), this could be due to potential interaction of the non-polar phenoxy group phenyl ring with its biomolecular target in cells. On the other hand, 2,6-dimethoxy substitution of the Ar ring on 8m furnished poor cytotoxicity in both cell lines. Based on the observations (from the Type A sub-classes I and II) that the presence of a fluorine substituent on the aromatic ring(s) does not diminish potential bioactivity, we chose to use cyclopropyl 2-fluorobenzyl ketone as the main framework for the Type B compounds. In this study, we were curious to see whether the cyclopropyl moiety would further enhance the cytotoxicity potency of these analogs. The cytotoxicity results for the Type B compounds, 9a-j, against B16 and L1210 cell lines are given in Table 3. The simple non-substituted compound 9a showed low potency (IC50 >100 µM for both cell lines). For the other compounds, several reoccurring trends were observed in addition to an enhancement of cytotoxicity. The cytotoxicity for both cell lines, 9c > 9g > 9b, suggest that these results are related to the polarity of the halogen atom in the para-position of the Ar ring (Table 3), and it is roughly in line with 8f and 8g (Type A; sub-class II). In addition, the cyclopropyl group seems to moderately enhance the potency of 9b, 9c and 9g in comparison to 8f and 8g (Table 2). This is also true for 9d (Table 3) which contains a para-nitro group on the Ar ring, for which the cytotoxicity is slightly enhanced in comparison to 8j (IC50 for 8j 14.4 (B16) and 21.4 µM verses 4.1 (B16) and 4.5 µM for 9d). A similar scenario exists for 9i which contains a 2,6-dimethoxyphenyl ring (Table 3; IC50 values 50.0 (B16) and 6.0 µM (L1210) when compared to 8m (IC50 values >100 (B16) and 80.0 (L1210) µM). Contrary to these observations, the cyclopropyl group does not seem to enhance the potency of 9h (Table 3; IC50 values 54.7 (B16) and 43.0 (L1210) µM) compared to 8i (Table 1; IC50 values 45.0 (B16) and 46.0 (L1210) µM). Three compounds, 8j and 8l of Type A, and 9i of Type B, were selected for further biological evaluation to provide mechanistic information. These compounds were evaluated for: cytotoxicity in MDA-MB-435, human melanoma cells and microtubule disrupting effects. Compounds 8j, 8l and 9i were effective in MDA-MB-435 cells and gave IC50 values of 9.6, 23.1, and 2.4 µM, respectively. Interestingly, these widely varied structures had relatively minor differences in cytotoxic potency. Furthermore, the results from human cancer cells are within the
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same range of the results obtained from murine cells. The three compounds were further tested for microtubule disrupting effects in A-10 cells using an immunofluorescence assay.14,15 The results given in Fig (5) are striking for Type B compound 9i, which caused 50% microtubule loss at 30 µM. This result is consistent with a classic microtubule depolymerizer, 14,15 including compounds previously studied in our laboratories.14,15 Interestingly, the Type A compounds 8j and 8l did not show effects on microtubule structures at 30 µM. Even though the exact mechanism of action by these compounds is unknown, the findings suggest that the strained and rigid nature of the cyclopropane ring in 9i could help orient the biologically active portion of the molecule within the binding site for more efficient interaction with its cellular target, presumably the colchicine bidding site of tubulin. On the other hand, tubulin may not be the site of action by compounds 8j and 8l, suggesting that small changes in the molecules can have an impact on the mechanism of action. Given their cytotoxicity, compounds 8c, 9d and 9i are worthy of further biological investigations. EXPERIMENTAL Proton NMR spectra were recorded using a 400 MHz Varian AVANCE-400 FT NMR using an internal deuterium lock. Chemical shifts are quoted in parts per million downfield from tetramethylsilane. Infrared spectra were recorded on a Perkin Elmer 100 FT-IR spectrophotometer with DRS (Diffuse Reflectance Sampler). Mass spectra were recorded using an API 2000 spectrometer, ion source (ESI /APCI). Characterization data for compounds 8a, 8f, 8g, 8h and 8j was found to be consistent with that of reported previously.12 General method of preparation of type A compounds: Hydrochloric acid (35%, 0.5 mL) was added drop wise to a solution of Phenylacetone (500 mg, 1 equiv.) and the appropriate aryl aldehyde (2 equiv.) in ethanol (20 mL) at room temperature. The reaction mixture was stirred under reflux for 2-3 hrs. Completion of the reaction was determined by TLC (Hex: EtOAc: 9:1) after which ethanol was removed under vacuum. The resulting crude residue obtained was purified by column chromatography using Hexane- Ethyl acetate (9:1) as eluent.
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8a: Colorless oil; 1H NMR (400 MHz, CDCl3) δ 2.30(s, 3H), 7.01(m, 2H), 7.14-7.33(m, 8H), 7.64(s, 1H); IR (KBr): 3064, 1718, 1600, 1493, 1450, 1317, 1234, 1176, 759, 702cm-1; ESI (APCI)-MS: m/z 223(M+1). 8b: Colorless oil; 1H NMR (400 MHz, CDCl3) δ 2.39(s, 3H), 6.70(d, J=8.0Hz, 1H), 6.83(m, 1H), 7.08( m, 3H), 7.27(m, 3H), 7.33 (d, J =8.0Hz, 1H), 7.85(s, 1H); IR (KBr): 3054, 2929, 1677, 1593, 1440, 1233, 756, 700cm-1; ESI (APCI)-MS: m/z 257(M+1). 8c: Colorless oil; 1H NMR (400 MHz, CDCl3) δ 2.31(s, 3H), 6.92(m, 1H), 6.99(m, 1H), 7.147.43(m, 7H), 7.54(s, 1H); IR (KBr): 3063, 2997, 1961, 1658, 1428, 1390, 1279, 1234, 1166, 1072, 895, 700 cm-1; ESI (APCI)-MS: m/z 303 (M+2). 8d: Colorless oil; 1H NMR (400 MHz, CDCl3) δ 2.30(s, 3H), 3.44(s, 3H), 6.46(t, J=2.0Hz, 1H), 6.72(m, 2H), 7.09(m, 1H), 7.16(m, 3H), 7.31(m, 2H), 7.60(s, 1H); IR (KBr): 3061, 2995, 1715, 1657, 1596, 1485, 1433, 1391, 1290, 1159, 948, 794 cm-1; ESI (APCI)-MS: m/z 253(M+1). 8i: Light yellow solid; mp: 47-49 ºC; 1H NMR (400 MHz, CDCl3) δ 2.28(s, 3H), 3.74(s, 3H), 6.67(d, J= 8.8Hz, 2H), 6.97(d, J= 8.4Hz, 2H), 7.18(m,2H), 7.38(m,3H), 7.62(s, 1H); IR (KBr): 2927, 2835, 1650, 1599, 1509, 1355, 1308, 1255, 1169, 1032, 829, 700cm-1; ESI (APCI)-MS: m/z 253(M+1). 8j: Off white solid; mp: 109-111 ºC; 1H NMR (400 MHz, CDCl3) δ 2.31(s, 3H), 7.16(m, 4H), 7.41(m, 3H), 7.62(s, 1H), 7.99(d, J=8.8Hz, 2H); IR (KBr): 3112, 3058, 1673, 1661, 1590, 1517, 1350, 1230, 861, 703 cm-1; ESI (APCI)-MS: m/z 268(M+1). 8k: Semisolid; 1H NMR (400 MHz, CDCl3) δ 2.26(s, 3H), 2.93(s, 6H)), 6.44(d, J=8.8Hz, 2H), 6.69(d, J=8.8Hz, 2H), 7.37(m, 3H), 7.62(s, 1H), 7.73(d, J=8.4Hz, 2H); IR (KBr): 2914, 2819, 2740, 1898, 1669, 1597, 1526, 1440, 1370, 1317, 1234, 1165, 1067, 1004, 944, 816, 726, 703 cm1
; ESI (APCI)-MS: m/z 266(M+1).
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General method of preparation for type B compounds: A mixture of cyclopropyl 2fluorobenzyl ketone (500 mg, 1 equiv.), sodium methoxide (2 equiv.), and the appropriate aryl aldehyde (1.2 equiv.) in methanol (10 ml) was stirred at 60 ºC for overnight. Completion of the reaction was monitored by TLC (Hex: EtOAc : 19:1). Methanol was removed under vacuum, residue was dissolved in water and ethyl acetate mixture and neutralized by dilute HCl. Ethyl acetate layer was separated and dried over sodium sulphate. Ethyl acetate was removed under vacuum, and the crude compound obtained was purified by column chromatography using Hexane- Ethyl acetate (9:1) as eluent. In all cases, the RF values were ~ 0.5 in (95:5; hexanes-ethyl acetate). 9a: Colorless oil; 1H NMR (400 MHz, CDCl3) δ 0.87(m, 2H), 1.18(m, 2H), 2.12(m, 1H), 7.127.34(m, 8H), 7.40(m, 1H), 7.80(s, 1H); IR (KBr): 3067, 2989, 2951, 2833, 1954, 1698, 1655, 1579, 1501, 1463, 1246,1229,1124, 1001, 933, 831, 765cm-1; ESI (APCI)-MS: m/z 267(M+1). 8b: Colorless oil; 1H NMR (400 MHz, CDCl3) δ 0.87(m, 2H), 1.16(m, 2H), 2.05(m, 1H), 7.05(d, J=8.4Hz, 2H), 7.15-7.20(m, 5H), 7.38(m, 1H), 7.74(s, 1H); IR (KBr): 3080, 3003, 2950, 2836, 1956, 1699, 1657, 1510, 1461, 1248, 1224,1120, 934, 833, 773cm-1; ESI (APCI)-MS: m/z 285(M+1). 9c: Colorless oil; 1H NMR (400 MHz, CDCl3) δ 0.86(m, 2H), 1.17(m, 2H), 2.07(m, 1H), 6.96(m, 3H), 7.16(m, 4H), 7.34(m, 1H), 7.68(s, 1H); IR (KBr): 3088, 2994, 2960, 2841, 1950, 1693, 1658, 1581, 1510, 1459, 1239, 1218, 1134, 1100, 933, 835, 771cm-1; ESI (APCI)-MS: m/z 347(M+2). 9d: Yellow coloured semisolid; 1HNMR (400 MHz, CDCl3) δ 0.89(m, 2H), 1.18(m, 2H), 2.03(m,1H), 7.12(m, 3H), 7.22(d, J= 8.8Hz, 2H), 7.39(m, 1H), 7.76(s, 1H), 8.02(d, J= 9.2Hz, 2H); IR (KBr): 3055, 2997, 2960, 2841, 1918, 1809, 1657, 1600, 1480, 1377, 1250, 1229, 971, 825, 780cm-1; ESI (APCI)-MS: m/z 312(M+1). 9e: Off white solid; mp: 94-96 ºC; 1H NMR (400 MHz, CDCl3) δ 0.86(m, 2H), 1.16(m, 2H), 2.11( m, 1H), 3.57(s, 6H), 3.81(s, 3H), 6.37(s, 2H), 7.18( m, 3H), 7.35(m, 1H), 7.72(s, 1H); IR (KBr):
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3074, 2996, 2941, 2835, 1955, 1698, 1649, 1578, 1507, 1460, 1247, 1225, 1130, 1007, 935, 833, 770cm-1 ; ESI (APCI)-MS: m/z 357(M+1). 9g: Colorless oil; 1H NMR: (400 MHz, CDCl3) δ 0.87(m, 2H), 1.15(m, 2H), 2.05(m, 1H), 7.00(d, J=8.4Hz, 2H), 7.13-7.18(m, 5H), 7.38(m, 1H), 7.72(s, 1H); IR (KBr): 3073, 2991, 2953, 2828, 1954, 1701, 1646, 1577, 1501, 1463, 1225, 1127, 1009, 934, 831, 775cm-1; ESI (APCI)-MS: m/z 301(M+1). 9h: Colorless oil; 1H NMR: (400 MHz, CDCl3) δ 0.85(m, 2H), 1.13(m, 2H), 2.09(m, 1H), 2.28(s, 3H), 6.99(m, 4H), 7.12(m, 3H), 7.36(m, 1H), 7.78(s, 1H); IR (KBr): 3077, 3001, 2943, 2835, 1961, 1692, 1645, 1589, 1506, 1459, 1259, 1230, 1003, 935, 829, 776cm-1; ESI (APCI)-MS: m/z 281( M+1). 9i: Off white solid; mp: 102-103 ºC; 1H NMR (400 MHz, CDCl3): δ 0.87(m, 2H), 1.17(m, 2H), 2.23(m, 1H), 3.53(s, 6H), 6.38(d, J= 8.4Hz, 2H), 6.88(m, 2H), 7.01(m,1H), 7.14(m, 2H), 7.76(s, 1H); IR (KBr): 3061, 3006, 2952, 2836, 1923, 1810, 1667, 1596, 1472, 1384, 1255, 1225, 1113, 967, 824, 775cm-1; ESI (APCI)-MS: m/z 327(M+1). 9i: Colorless oil; 1H NMR (400 MHz, CDCl3): δ 0.84(m, 2H), 1.13(m, 2H), 2.05(m, 1H), 5.91(s, 2H), 6.40(s, 1H), 6.67(d, J=8.4Hz, 1H), 6.77(d, J=8.0Hz, 1H), 7.14-7.21(m, 3H), 7.38(m, 1H), 7.71(s, 1H); IR (KBr): 3080, 2999, 2931, 2841, 1953, 1683, 1654, 1577, 1497, 1461, 1253, 1128, 999, 931, 772cm-1; ESI (APCI)-MS: m/z 311(M+1). CONCLUSION This paper reports a systematic study on the synthesis and biological evaluation of a range of α, β-unsaturated ketones as combretastatin analogs. The results show, first, for good levels of cytotoxicity (i) a non-polar substituent in the meta-position of the Ar ring (in particular a nonpolar) halogen or (ii) an electron-withdrawing group with some degree of steric bulk in the paraposition of Ar ring is required. In addition, incorporation of a strained cyclopropyl moiety adjacent to the carbonyl functionality appeared to enhance these effects, including that of microtubule delpolymerization. We believe that the Type B compounds represent an alternate class of
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compounds to combretastatins worthy of further investigation. Likewise Type A compounds should also be further investigated since the compounds exert cytotoxicity through a mechanism different from Type B. ACKNOWLEDGEMENT The authors thank Conjura Pharmaceuticals, LLC for support. REFERENCES AND NOTES [1] (a) Hinnen, P.; Eskens, F. A. L. M. Vascular disrupting agents in clinical development. Br. J. Cancer, 2007, 96, 1159-1165; (b) Vincent, L.; Kermani, P.; Young, L. M.; Cheng, J.; Zhang, F.; Shido, K.; Lam, G.; Bompais-Vincent, H.; Zhu, Z.; Hicklin, D. J.; Bohlen, P.; Chaplin, D. J.; May, C.; Rafii, S. Hemin-activated macrophages home to the pancreas and protect from acute pancreatitis via heme oxygenase-1 induction. J. Clin. Invest., 2005, 115, 2992-3006. [2] (a) Pettit, G. R.; Bux, S.; Singh, B.; Niven, M. L.; Hamel, E.; Schimdt, J. M. Isolation, structure, and synthesis of Combretastatins A-1 and B-1, potent new inhibitors of microtubule assembly, derived from Combretum caffrum. J. Nat. Prod., 1987, 50, 119-131. (b) H.P. Hsieh J.P. Liou, Y.T. Lin, N. Mahindroo, J.Y. Chang, Y.N. Yang, S.S. Chern, U.K. Tan, C.W. Chang, T.W. Chen, C.H. Lin, Y.Y. Chang, C.C. Wang. Structure–activity and crystallographic analysis of benzophenone derivatives—the potential anticancer agents. Bioorg Med Chem Lett., 2003, 13 101-105. [3] Galbraith, S. M.; Maxwel, R. J.; Lodge, M. A.; Tozer, G. M.; Wilson, J.; Taylor, N. J.; Stirling, J. J.; Sena, L.; Padhani, A. R.; Rustin, G. S. Combretastatin A4 phosphate has tumor antivascular activity in rat and man as demonstrated by dynamic magnetic resonance imaging. J. Clin. Oncol., 2003, 15, 2831-2848. [4] (a) Hadimani, M.B.; Hua, J.; Jonklaas, K. M. D.; Kessler, R. J.; Sheng, Y.; Olivares, A.; Tanpure, R. P.; Weiser, A.; Zhang, J.; Edvardsen, K.; Kane, R. R.; Pinney, K. G. Synthesis, in vitro, and in vivo evaluation of phosphate ester derivatives of Combretastatin A-4. Bioorg. Med.
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Chem. Lett., 2003, 13, 1505-1508; (b) Bedford, S. B.; Quarterman, C. P.; Rathbone, D. L.; Slack, J. A.; Griffin, R. J.; Stevens, M. F. G. Synthesis of water-soluble prodrugs of the cytotoxic agent Combretastatin A4. Bioorg. Med. Chem. Lett., 1996, 6, 157-160. [5] Tozer, G.M.; Kanthou, C.; Parkins, C.S.; Hill, S.A. The biology of the combretastatins as tumour vascular targeting agents. Int. J. Exp. Pathol., 2002, 83, 21-38. [6] (a) Pettit, G. R.; Singh, S. B.; Boyd, M. R.; Hamel, E.; Pettit, R. K.; Schimdt, J. M.; Hogan, F. Antineoplastic agents. 291. Isolation and synthesis of Combretastatins A-4, A-5, and A-6. J. Med. Chem., 1995, 38, 1666-1672; (b) Hamel, E. Antimitotic natural products and their interactions with tubulin. Med. Res. Rev., 1996, 16, 207-231; (c) Gaukoger, K.; Hadfield, J. A.; Hepworth, L. A.; Lawrence, N. J.; McGown, A. T. Novel syntheses of cis and trans isomers of combretastatin A-4. J. Org. Chem., 2001, 66, 8135-8138. [7] Pati, H. N.; Wicks, M.; Holt. H. L.; LeBlanc, R.; Weisbruch, P.; Forrest, L.; Lee, M. A stereospecific route for the preparation of trans-Combretastatin analogs: synthesis and cytotoxicity. Letters in Drug Design and Discovery, 2004, 1, 275-278. [8] Dimmock, J. R.; Kandepu, N. M.; Hetherington, M.; Quail, J. W.; Pugazhenthi, U.; Sudom, A. M.; Chamanklah, M.; Rose, P.; Pass, E.; Allen.; Halleran, S.; Szydlowski, J.; Mutus, B.; Tanus, M.; Manavathu, E.; Myers, T. G.; De Clereq; E; Balzarini, J. Cytotoxic activities of mannich bases of chalcones and related compounds. J. Med. Chem., 1998, 41, 1014-1026. [9] (a) Dickson, J.; Flores, F.; Stewart, M.; LeBlanc, R.; Pati, H. N.; Lee, M.; Holt, H. Synthesis and cytotoxic properties of chalcones: an interactive and investigative undergraduate laboratory project at the interface of chemistry and biology. J. Chem. Ed., 2006, 83, 934-936; (b) Lawrence, N. J.; Rennison, D.; McGown, A. T.; Ducki, S.; Gul. L.; Hadfield, J. A.; Khan. N. J. Linked parallel synthesis and MTT bioassay screening of substituted chalcones. J. Comb. Chem., 2001, 3, 421-426; (c) Lawrence, N. J.; McGown, A. T.; Ducki, S.; Hadfield, J. A. The interaction of chalcones with tubulin.. Anti-Cancer Drug Design, 2000, 15, 135-141; (c) Ducki, S.; Forrest.; S.;
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Hadfield, J. A.; McGown, A. T.; Rennison, D. Potent antimitotic and cell growth inhibitory properties of substituted chalcones. Bioorg. Med. Chem. Lett., 1998, 8, 1051-1056. [10] (a) Renfrow, W.B. The use of potassium t-amyloxide for the alkylation of acetoacetic ester and its alkyl substitution products. J. Am. Chem. Soc., 1944, 66, 144-146; (b) Walker, H. G.; Hauser, C. R. The carbethoxylation of ethyl phenylacetate and of ethyl α-phenyl-n-butyrate using sodium amide. J. Am. Chem. Soc., 1946, 68, 672-673; (c) Zimmerman, H. E.; Wilson, D. W. Singlet photochemistry of vinyl cyclopropenes: regioselectivity and mechanism; mechanistic and exploratory organic photochemistry. J. Org. Chem., 1995, 60, 692-697. [11] (a) US patent: number 5436342, page 32; (b) US patent 5288726, page 30.
[12] (a) Fine, A.; Pulaski, P. Reexamination of the Claisen-Schmidt condensation of phenylacetone with aromatic aldehydes. J. Org. Chem., 1973, 38, 1747-1749; (b) Elba, M. E.; Darwish, A. I.; Mohamed, A. A.; El-Sadany, S. K. Reactions of (E)-4-(p-substituted phenyl)-3phenyl-3-buten-2-ones with hydrazine derivatives. Egypt. J. Chem., 2000, 43, 483-500; Chem. Abs., 2001, 134, 366472; (c) Abramovitch, R. A.; Obach, A. The condensation of 1-phenyl-2propanone with aromatic aldehydes. Can. J. Chem., 1959, 37, 502-504. [13] Using NMR chemical shift prediction package within ChemDraw software, the benzylidene proton for compound cis-8m appears at δ 7.60 whilst the two aromatic protons adjacent to the methoxy groups on the 2,6-dimethoxyphenyl ring
appear as a doublet at δ 6.37. NMR data
obtained for cis 8m (400 MHz, CDCl3) shows that the benzylidene proton appears at δ 7.74 whilst the doublet corresponding aromatic protons appears at δ 6.37. For trans-8m, the predicted data shows the benzylidene proton shifted downfield to δ 8.00 whilst the aromatic protons still appear δ 6.33. [14] (a) LeBlanc, R.; Dickson, J.; Brown, T.; Stewart, M.; Pati, H.; VanDerveer, D.; Arman, H.; Harris, J.; Pennington, W.; Holt, H.; Lee, M. Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combretastatins. Bioorg. Med. Chem., 2005, 13, 6025-6034; (b) Ruprich, J.; Prout, A.; Dickson, J.; Younglove, B., Nolan, L.; Baxi, K.; LeBlanc, R.; Forrest, L.; Hills, P.; Holt, H.; Mackay, H.; Brown, T.; Mooberry, S.; Lee, M. Design, Synthesis and biological testing of 13
cyclohexenone derivatives of Combretastatin-A4. Letters in Drug Design and Discovery, 2007, 4, 144-148. [15] Lee, L.; Davis, R.; Vanderham, J.; Hills, P.; Mackay, H.; Brown, T.; Mooberry, S.L.; Lee, M. 1,2,3,4-Tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4. Eur. J. Med Chem., 2008, 43, 2011-2015.
Legends for Figures Figure 1. Structures of combretastatin-A4 (1), CA-4P (2), an amino analog of CA-4 (3) and its derivative AVE-8062 (4). Figure 2. Structures of triazoles 5, 6 and 7 and their IC50 values against the growth of B16 murine melanoma cells. Figure 3. Synthesis of Type A and B compounds. Figure 4. X-ray structure of compound 8f. Figure 5. Effects of 9i on cellular microtubules in A-10 cells. A, vehicle control, and B, 60 µM compound 9i. Cells were treated for 18 h and cellular microtubules visualized by indirect immunofluorescence techniques.
14
Figure 1
H3CO H3CO
R OCH3
OCH3
1, R=OH, cis-CA-4 2, R=OPO32-, water soluble derivative CA-4P 3, R=NH2 4, R=NHCOCH(NH2)CH2OH; AVE-8062
15
Figure 2
N
N
NH 5, R1=R3=H, R2=OCH3, R4=CH3, IC50=>100 µMolar 6, R1=R3=H, R2=OCH3, R4=NH2, IC50=>100 µMolar 7, R1=R2=R3=OCH3, R4=NH2, IC50= 56 µMolar
R1 R3
R2
R4
16
Figure 3
O Ph
O
(i) Arylaldehyde CH3
CH3
(ii) HCl (35%)
Ar
Reflux in EtOH, 3 hrs 8e
O
8a-d, 8f-m O
NaOMe, Arylaldehyde MeOH, reflux 12 hrs
F
Ph
F Ar
9a-9e, 9g-j
9f
17
Figure 4
18
Figure 5
A
B
19
Tables 1 (top) and 2 (bottom). Cytotoxicity data for compounds 8a-k (Type A subclasses I and II) and compounds 8l and 8m (Type A; sub-class III). Type A: Sub-class I (o-, m- & nosubstitution) Structure
IC50 (µM) B16
L1210
Type A: Sub-class II (para-substitution) Structure
O
IC50 (µM) B16
L1210
>100
>100
70
>100
45.0
46.0
>100
49.7
14.4
21.4
>100
>100
O CH3
CH3
Ar
Ar
Ar =
Ar = >100
>100 F
8a
8f Cl
45
45 Cl
8g
8b Br
4.2
3.8 CH3
8c
8h OMe
67
56 OCH3
8d
8i
Phenylacetone
>100
>100 NO2
8e
8j NMe2
8k
Type A: Sub-class
IC50 (µM)
IC50 (µM)
III (disubstitution) Structure OPh
B16
L1210
11.3
11.8
OMe
F
8l
MeO
8m
20
B16
L1210
>100
80.0
Table 3. Cytotoxicity data for compounds 9a-j (Type B) Type B: Structure
IC50 (µM) B16
L1210
>100
>100
IC50 (µM) B16
L1210
>100
>100
51.7
61.0
54.7
43.0
50.0
6.0
65
41
O F Ar
Ar = F
9a
O
9f 60.3
58.0
F
Cl
9b
9g 37.0
51.0
Br
CH3
9c
9h 4.1
4.5
OMe
NO2
MeO
9d
9i OMe
43.0
51.0 O
OMe
O
OMe
9j
9e
21
