Bioorganic & Medicinal Chemistry Letters 25 (2015) 2078–2081
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and activities towards resistant cancer cells of sulfone and sulfoxide griseofulvin derivatives Frédéric Liéby-Muller ⇑, Quentin Heudré Le Baliner, Serge Grisoni, Emmanuel Fournier, Nicolas Guilbaud, Frédéric Marion ⇑ Institut de Recherche Pierre Fabre, CRDPF/CROE, 3 avenue Hubert Curien, BP 13562, 31035 Toulouse Cedex 1, France
a r t i c l e
i n f o
Article history: Received 12 March 2015 Revised 26 March 2015 Accepted 29 March 2015 Available online 3 April 2015 Keywords: Griseofulvin Sulfone Sulfoxide Cytotoxicity
a b s t r a c t Griseofulvin, an antifungal drug, has been shown in recent years to have anti-proliferative activities. We report here the synthesis of new analogs of griseofulvin, substituted in 20 by a sulfonyl group or in 30 by a sulfinyl or sulfonyl group. These compounds exhibit good anti-proliferative activities against SCC114 cells, an oral squamous carcinoma cell line showing pronounced centrosome amplification, and unexpected cytotoxic activities on HCC1937 cells, a triple negative breast cancer cell line resistant to microtubule inhibitors. Ó 2015 Elsevier Ltd. All rights reserved.
Griseofulvin 1 (Fig. 1) is a natural product initially isolated from Penicillium griseofulvum,1 and used to treat fungal infections in humans and animals. Griseofulvin is also of interest as anti-cancer agent, due to its low toxicity and efficacy in inhibiting proliferation of cancer cells.2 Although several studies have suggested that tubulin is the main target of griseofulvin, the exact mechanism of action remains still unclear.3 In order to increase the anti-tumor properties of griseofulvin, several 20 -oxygen and 20 -sulfur analogs of griseofulvin were synthesized by Clausen and co-workers.4 Among these compounds, GF-15 2 (Fig. 1) has been reported to have tumor growth inhibition through centrosomal clustering inhibition.5 Based on these observations, and since a part of our research programs is dedicated to the quest for anti-cancer natural products, we considered that griseofulvin could provide a good molecular basis to initiate a medicinal chemistry program. However, the research field of anti-mitotic microtubule-interfering agents is a mature area that counts several marketed drugs but is still dynamic owing to recent approvals in the last decade.6 One of the major clinical issues faced by these chemotherapeutic agents is intrinsic or acquired-drug resistance of tumor cells which has different origins, such as efflux protein expression or tubulin mutations, among others. Having these considerations in mind, we ⇑ Corresponding authors. Tel.: +33 5 34 50 69 38; fax: +33 5 34 50 30 54 (F.L-M.); tel.: +33 5 34 50 60 57 (F.M.). E-mail addresses: [email protected] (F. Liéby-Muller), [email protected] (F. Marion). http://dx.doi.org/10.1016/j.bmcl.2015.03.081 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
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O Cl
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5'
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Figure 1. Structures of griseofulvin 1 and GF-15 2.
conducted a program to identify potent anti-cancer griseofulvin derivatives that could overcome resistance. We report here our efforts in the synthesis of 20 -sulfone, 30 -sulfoxide and 30 -sulfone griseofulvin analogs, as well as their antiproliferative activities, notably against a resistant cancer cell line. 20 -Demethoxy-20 -sulfonylgriseofulvin analogs 5a–c were obtained in a three steps synthetic process, starting from commercial griseofulvin (Scheme 1). Griseofulvin 1 was reacted with lithium chloride and phosphoryl chloride in refluxing dioxane,4 affording the 20 -vinyl chloride 3 in 40% yield, after separation of its 40 -vinyl chloride isomer. The addition of thiols on 3 was performed using an excess of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in refluxing dioxane.4 20 -Sulfur compounds 4a–c were obtained with an average yield of 80% for the three compounds, then oxidized to 20 -sulfone by addition of
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O
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20 -Demethoxy-30 -sulfinyl griseofulvin analogs 10a–b and 20 demethoxy-30 -sulfonyl griseofulvin analogs 11a–b were obtained through a five steps synthetic sequence (Scheme 3). Griseofulvin 1 was reduced in the presence of Pd/C 10% and hydrogen in ethyl acetate to afford intermediate 6. Elimination of the methoxy group was performed with sulfuric acid 2 N, in refluxing ethanol to afford 7.7 Epoxidation of the resulting double bound with hydrogen peroxide led to compound 8 with an overall yield of 94% over three steps, starting from commercial griseofulvin. When 8 was treated with thiols in basic conditions, 30 -vinyl sulfur derivatives 9a–b were obtained with 15–25% yields. Although the conversion seemed to be complete (monitoring of the reaction by LCMS), these disappointing yields were obtained after purification and were maybe due to a lack of solubility or instability of the compounds over silica gel. Moreover, in our hands, the reaction was found to be poorly reproducible and adaptable to a range of thiols, and sodium hydroxide at different concentrations (1 N for 9a or 0.1 N for 9b) was eventually found to give the best results. Thereby, the use of other bases, such as sodium hydride or sodium ethoxide, did not allow us to improve the reaction yields, leading to complex mixtures. Oxidation of 30 -vinyl sulfur 9a with a stoichiometric amount of meta-chloroperbenzoic acid (m-CPBA) led to 10a with a moderate yield of 30%. 9b was oxidized with sodium periodate and led to sulfoxide 10b, as a mixture of two diastereoisomers, with moderate yields of 35%. Oxidation of 30 -vinyl sulfur 9a and 9b with an excess of m-CPBA led to the formation of sulfones 11a and 11b, respectively, with 79% and 45% yield. Compounds 5a–d, 10a–b and 11a–b8 were evaluated for their antiproliferative activities against HCC1937 cells,9 a triple negative breast cancer cell line resistant to most microtubule inhibitors.10 These derivatives were compared to reference compounds such as paclitaxel, epothilone B, vinorelbine, griseofulvin and GF-15 (Table 1). Compounds with IC50 values higher than 10 lM were regarded as inactive. 20 -Vinyl sulfones 5a–d were found to be active on the HCC1937 cell line, having IC50 values in the micromolar range, whereas references were found to be inactive. The degree of oxidation of the 20 -sulfur has a real impact on the antiproliferative activities for this particular cell line as parent compounds 4a– b were inactive. The size and aromaticity of the substituent borne
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i O
O
O
O
O
O
Cl
Cl 1
3 ii
R O OS O
O
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O
O
R
OS
iii O
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a R = Bn b R = -C 2H 4 -NMe2 c R = Ph
5 a-c
O
O
O Cl
4 a-c
Scheme 1. Reagents and conditions: (i) LiCl (3 equiv), POCl3 (5 equiv), 1,4-dioxane, 100 °C, 1 h, 40%; (ii) R-SH (1–2 equiv), DBU (2.5 equiv), 1,4-dioxane, 100 °C, 18 h; (iii) oxone (6 equiv), H2O/MeOH/THF 5:1:1, rt, 16 h, 8–26% over 2 steps.
O
O Cl
R O OS O
O i
O
O
O
O
O
O
Cl
Cl 5c R = Ph 5d R = Me
3
Scheme 2. Reagents and conditions: (i) R-SO2Na (1 equiv), DMF, rt, 16 h, 86%.
an excess of aqueous oxone. Derivatives 5a–c were obtained in yields of 11–32%. An alternative route using sulfinates as nucleophiles was found to improve the overall yield of the process. For example, starting from 20 -vinyl chloride 3, addition of sodium phenylsulfinate or methylsulfinate in DMF directly led, respectively, to compounds 5c or 5d with 85% yield each (Scheme 2).
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Cl 6
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8
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Cl
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O
iv O
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R S
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vi O
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R O S O O
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Scheme 3. Reagents and conditions: (i) Pd/C 10%, EtOAc, H2, rt, 6 h, 100%; (ii) H2SO4 2 N (10 vol), EtOH, 80 °C, 16 h, 99%; (iii) NaOH 2 N (1.4 equiv), H2O2 (1 equiv), EtOH, 0 °C, 1 h, 95%; (iv) R = Ph: PhSH (1.2 equiv), NaOH 1 N (30 vol), THF, 70 °C, 15% or R = Bn: BnSH (1.2 equiv), NaOH 0.1 N (30 vol), THF, rt, 25%; (v) R = Bn: mCPBA (1 equiv), CH2Cl2, 0 °C, 1 h, 30%; R = Ph: NaIO4 (1.5 equiv), MeOH/H2O 2:1, 50 °C, 16 h, 35% (vi) mCPBA (2.7 equiv), CH2Cl2, rt, 0.5–1 h, 45–79%.
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Table 1 IC50 values of synthesized griseofulvin analogs and references on HCC1937 and SCC114 cell lines IC50 (lM)
Compounds HCC1937 4a 4b 5a 5b 5c 5d 10a 10b 11a 11b Paclitaxel Epothilone B Vinorelbine Griseofulvin GF-15 2
version, at http://dx.doi.org/10.1016/j.bmcl.2015.03.081. These data include MOL files and InChiKeys of the most important compounds described in this article.
a
>10 >10a 1.4 5.9 1.6 >10b n.d. 0.4 0.9 0.7 >10a >10a >10a >10a >10a
SCC114 n.d. 1.45 0.4 2.8 1 1.9 0.02 0.25 n.d. 0.4 0.0003 0.00017 0.00007 6.4 0.98
n.d.: not determined. a 0% inhibition at 10 lM. b 40% inhibition at 10 lM.
by the sulfone seems also to affect the antiproliferative activity, 5a and 5c being more active than 5b and 5d.11 Substitution of position 30 is of particular relevance regarding the cytotoxic activities, sulfoxide 10b and sulfones 11a–b being the most active derivatives, with sub-micromolar IC50 values. Introduction of an electron withdrawing group in the 30 position lead to quite a reactive Michael acceptor and it is less likely with 20 substituted compounds so this reactivity difference could explain the various pharmacological activities observed between 20 and 30 series. The cytotoxic activities of these compounds were also evaluated against the oral squamous carcinoma cell line SCC114,9 known to have pronounced centrosome amplification and to be sensitive to declustering agents.3c,12 The aim of evaluating this particular cell line was to assess if newly synthesized derivatives would be endowed with similar activities as the related griseofulvin. All tested compounds were found to be more active than griseofulvin, and equipotent or more active than GF-15, 5a and 11b having IC50 values of 0.4 lM, 10b having an IC50 value of 0.25 lM and 10a being the most active compound, with an IC50 value of 20 nM (Table 1). Mechanistically, these results suggest that these compounds may inhibit cell growth by inducing, at least partially, centrosomal declustering. Such compound characterization awaits confirmation with complementary studies. In conclusion, several new 20 and 30 griseofulvin analogs were synthesized through a short synthetic process. Their anti-proliferative activities were evaluated on HCC1937 cells, a resistant cancer cell line, as well as SCC114 cells, a centrosomal clustering dependant cell line. A significant potency gain was obtained by introducing constrained sulfones on position 20 , the increase of activity being even better with the introduction of sulfoxides or sulfones on position 30 . These new 20 and 30 griseofulvin analogs have evidenced interesting cytotoxic properties not only against cells with centrosomal amplification but also unexpected cytotoxic properties against a cell line intrinsically resistant to anti-mitotic and clinically used anticancer drugs. The reactive nature of the newly synthesized compounds may be involved in the mode of action especially for the 30 derivatives. Investigations to precise the origin of the observed activity of these series is underway and will be reported in due course. Supplementary data Supplementary data (1H and 13C NMR spectra of 5a–d, 10a–b and 11a–b) associated with this article can be found, in the online
References and notes 1. (a) Oxford, A. E.; Raistrick, H.; Simonart, P. Biochem. J. 1939, 33, 240; (b) Birch, A. J.; Massy-Westropp, R. A.; Rickards, R. W.; Smith, H. J. Chem. Soc. 1958, 360; for a review on the chemistry of griseofulvin, see: (c) Petersen, A. B.; Ronnest, M. H.; Larsen, T. O.; Clausen, M. H. Chem. Rev. 2014, 114, 12088. 2. Ho, Y.-S.; Duh, J.-S.; Jeng, J.-H.; Wang, Y.-J.; Liang, Y.-C.; Lin, C.-H.; Tseng, C.-J.; Yu, C.-F.; Chen, R.-J.; Lin, J.-K. Int. J. Cancer 2001, 91, 393. 3. (a) Mir, L.; Oustrin, M.-L.; Lecointe, P.; Wright, M. FEBS Lett. 1978, 88, 259; (b) Panda, D.; Rathinasamy, K.; Santra, M. K.; Wilson, L. Prod. Natl. Acad. Sci. U.S.A. 2005, 102, 9878; (c) Rebacz, B.; Larsen, T. O.; Clausen, M. H.; Rønnest, M. H.; Löffler, H.; Ho, A. D.; Krämer, A. Cancer Res. 2007, 67, 6342; (d) Ranthinasamy, K.; Jindal, B.; Asthana, J.; Singh, P.; Balaji, P. V.; Panda, D. BMC Cancer 2010, 10, 213. 4. (a) Rønnest, M. H.; Rebacz, B.; Markworth, L.; Terp, A. H.; Larsen, T. O.; Krämer, A.; Clausen, M. H. J. Med. Chem. 2009, 52, 3342; (b) Clausen, M. H.; Krämer, A.; Larsen, T. O.; Rebacz, B. WO2009/000937. 5. Raab, M. S.; Breitkreutz, I.; Anderhub, S.; Rønnest, M. H.; Leber, B.; Larsen, T. O.; Weiz, L.; Konotop, G.; Hayden, P. J.; Podar, K.; Fruehauf, J.; Nissen, F.; Mier, W.; Haberkorn, U.; Ho, A. D.; Goldschmidt, H.; Anderson, K. C.; Clausen, M. H.; Krämer, A. Cancer Res. 2012, 72, 5374. 6. (a) Dumontet, C.; Jordan, M. A. Nat. Rev. Drug Disc. 2010, 9, 790; (b) Mukhtar, E.; Adhami, V. M.; Mukhtar, H. Mol. Cancer Ther. 2014, 13, 275. 7. Oda, T.; Sato, Y. Chem. Pharm. Bull. 1983, 31, 934. 8. 5a: light brown solid, 1H NMR (CDCl3, 400 MHz) d: 0.96 (d, 3H, J = 6.8 Hz), 2.39 (dd, 1H50 , J = 17.6, 4.8 Hz), 2.74 (m, 1H60 ), 3.07 (dd, 1H50 , J = 17.6, 14.0 Hz), 3.99 (s, 3H), 4.06 (s, 3H), 4.49 (d, 1H, J = 13.6 Hz), 4.69 (d, 1H, J = 13.6 Hz), 6.19 (s, 1H), 6.52 (s, 1H30 ), 7.37 (m, 3H), 7.49 (m, 2H); 13C NMR (125 MHz, CDCl3) d: 195.2, 189.7, 168.2, 164.5, 158.0, 151.7, 137.6, 130.9, 129.4, 129.0, 127.1, 105.2, 97.1, 90.4, 89.1, 62.9, 57.1, 56.5, 39.6, 39.1, 14.3; LCMS (ES, m/z): 477.1 [M+H]+ 5b: yellow solid, 1H NMR (CDCl3, 400 MHz) d: 0.95 (d, 3H, J = 6.8 Hz), 2.27 (s, 6H), 2.50 (dd, 1H50 , J = 17.4, 4.6 Hz), 2.66 (dt, 1H, J = 13.2, 5.2 Hz), 2.87 (m, 1H+1H60 ), 3.14 (dd, 1H50 , J = 17.4, 14.2 Hz), 3.22 (m, 1H), 3.77 (ddd, 1H, J = 14.4, 8.8, 6.0 Hz), 3.98 (s, 3H), 4.03 (s, 3H), 6.16 (s, 1H), 6.88 (s, 1H30 ); 13C NMR (100 MHz, CDCl3) d: 195.22, 189.12, 167.68, 163.80, 157.35, 152.94, 134.42, 104.60, 96.44, 89.70, 88.90, 56.36, 55.85, 54.26, 52.02, 44.41, 39.23, 38.77, 13.63; LCMS (ES, m/z): 458.1 [M+H]+. 5c: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.83 (d, 1H, J = 6.8 Hz), 2.44 (dd, 1H50 , J = 17.2, 4.8 Hz), 2.77 (m, 1H60 ), 3.11 (dd, 1H50 , J = 17.2, 14.0 Hz), 4.00 (s, 3H), 4.05 (s, 3H), 6.18 (s, 1H), 7.11 (s, 1H30 ), 7.54 (t, 2H, J = 7.6 Hz), 7.65 (t, 1H, J = 7.6 Hz), 7.83 (d, 2H, J = 7.6 Hz); 13C NMR (100 MHz, CDCl3) d: 195.6, 189.7, 168.2, 164.3, 157.7, 154.9, 139.0, 135.5, 133.8, 129.1, 128.4, 105.0, 96.9, 89.9, 89.1, 56.8, 56.2, 39.6, 39.2, 14.0; LCMS (ES, m/z): 463.0 [M+H]+. 5d: light yellow solid, 1H NMR (CDCl3, 400 MHz) d:0.98 (d, 3H, J = 6.8 Hz), 2.52 (dd, 1H50 , J = 17.2, 4.8 Hz), 2,93 (m, 1H60 ), 3.18 (dd, 1H50 , J = 17.2, 14.0 Hz), 3.22 (s, 3H), 3.98 (s, 3H), 4.03 (s, 3H), 6.16 (s, 1H), 6.98 (s, 1H30 ); 13C NMR (100 MHz, CDCl3) d: 195.7, 189.6, 168.3, 164.5, 158.0, 154.1, 136.0, 105.2, 97.2, 90.4, 89.1, 57.0, 56.5, 44.9, 39.9, 39.5, 14.3; LCMS (ES, m/z): 400.8 [M+H]+. 10a: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.97 (d, 3H, J = 6.7 Hz), 2.60 (dd, 1H50 , J = 17.4, 5.0 Hz), 2.88–2.96 (m, 1H60 ), 3.27 (dd, 1H50 , J = 17.4, 13.8 Hz), 3.90 (d, 1H, J = 13.0 Hz), 4.01 (s, 3H), 4.03 (s, 3H), 4.35 (d, 1H, J = 13.0 Hz), 6.14 (s, 1H), 6.87 (s, 1H20 ), 7.20 (d, 2H, J = 7.7 Hz), 7.26–7.34 (m, 3H); 13C NMR (100 MHz, CDCl3) d: 194.5, 190.4, 168.5, 164.9, 157.9, 146.8, 145.1, 130.9, 128.8, 128.5, 128.3, 105.1, 97.8, 92.2, 89.8, 59.4, 57.1, 56.5, 40.7, 38.3, 14.2; LCMS (ES, m/z): 461.1 [M+H]+. 10b: white solid, 1H NMR (400 MHz, CDCl3) d: 0.92 (d, 1.3H, J = 6.9 Hz), 0.95 (d, 1.6H, J = 6.9 Hz), 2.38 (dd, 0.52H50 , J = 17.1, 5.2 Hz), 2.45 (dd, 0.5H50 , J = 17.1, 5.2 Hz), 2.69–2.79 (m, 0.4H60 ), 2.93–3.03 (m, 0.6H60 ), 3.07–3.25 (m, 1H50 ), 4.00 (s, 3H), 4.05 (s, 3H), 6.17 (s, 1H), 7.33 (s, 0.4H20 ), 7.35 (s, 0.5H20 ), 7.46–7.48 (m, 3H), 7.69–7.71 (m, 1.1H), 7.79 (m, 0.9H); 13C NMR (125 MHz, CDCl3) d: 193.7, 193.1, 190.9, 190.8, 168.6, 168.4, 165.0, 164.9, 158.0, 157.9, 147.7, 147.1, 143.9, 143.5, 142.9, 142.4, 131.9, 131.7, 129.4, 129.2, 126.3, 125.7, 105.2, 105.0, 97.7, 97.5, 92.0, 91.3, 89.9, 89.7, 57.1, 56.5, 41.3, 40.3, 38.7, 37.1, 31.9, 29.0, 22.7, 14.3, 13.9; LCMS (ES, m/z): 447.0 [M+H]+. 11a: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.93 (d, 3H, J = 6.7 Hz), 2.63 (dd, 1H50 , J = 17.6, 5.3 Hz), 2.80 (m, 1H60 ), 3.21 (dd, 1H50 , J = 17.6, 13.7 Hz), 3.98 (s, 3H), 4.03 (s, 3H), 4.56 (d, 1H, J = 13.7 Hz), 4.63 (d, 1H, J = 13.7 Hz), 6.14 (s, 1H), 7.25–7.29 (m, 2H), 7.31 (s, 1H20 ), 7.33 (m, 3H); 13C NMR (100 MHz, CDCl3) d: 191.1, 188.6, 167.8, 164.6, 157.5, 152.9, 138.7, 130.4, 128.4, 128.3, 126.5, 104.3, 97.2, 90.1, 89.5, 60.3, 56.5, 55.9, 39.6, 36.7, 29.1, 13.5; LCMS (ES, m/z): 477.1 [M+H]+. 11b: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.92 (d, 3H, J = 6.7 Hz), 2.46 (dd, 1H50 , J = 17.2, 4.9 Hz), 2.86 - 2.94 (m, 1H60 ), 3.13 (dd, 1H50 , J = 17.2, 14.0 Hz), 4.00 (s, 3H), 4.06 (s, 3H), 6.18 (s, 1H), 7.52 (t, 2H, J = 7.4 Hz), 7.62 (t, 1H, J = 7.4 Hz), 7.78 (s, 1H20 ), 8.01 (d, 2H, J = 7.4 Hz); 13C NMR (100 MHz, CDCl3) d: 189.7, 189.5, 168.0, 164.8, 157.7, 151.0, 141.5, 138.7, 133.5, 128.6, 128.5, 104.6, 97.3, 90.4, 89.6, 56.7, 56.1, 40.1, 37.1, 13.7; LCMS (ES, m/z): 463.0 [M+H]+. 9. SCC-114 and HCC-1937 cells were grown in MEM (SIGMA—M7278) or RPMI (SIGMA—R0883) media supplemented with 10% Fetal Bovine Serum respectively. Antiproliferative activity was measured using the ATPlite assay (Perkin Elmer) on exponentially growing adherent cells that were seeded in 96
F. Liéby-Muller et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2078–2081 well plates with 103 cells/well for SCC-114 and 2103 cells/well for HCC-1937. Cell viability was evaluated by dosing the ATP released by viable cells after an incubation period of 72 h for SCC-114 cells or 48 h for HCC-1937 cells at 37 °C in 5% CO2 incubator. Luminescence was measured using a TopCount NXT™ luminescence counter (Perkin–Elmer). The IC50 values were determined by curve fitting analysis (non linear regression model with a sigmoidal dose response, variable hill slope coefficient), performed with the algorithm provided by the GraphPad Software.
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10. Kadra, G.; Finetti, P.; Toiron, Y.; Viens, P.; Birnbaum, D.; Borg, J.-P.; Bertucci, F.; Gonçalves, A. Breast Cancer Res. Treat. 2012, 132, 1035. 11. Although the activity of 5 d was reported to be inactive in Table 1, it was found to induce 40% inhibition at 10 lM, when no inhibition was induced with the reference compounds at the same concentration. 12. Quintyne, N. J.; Reing, J. E.; Hoffelder, D. R.; Gollin, S. M.; Saunders, W. S. Science 2005, 307, 127.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and activities towards resistant cancer cells of sulfone and sulfoxide griseofulvin derivatives Frédéric Liéby-Muller ⇑, Quentin Heudré Le Baliner, Serge Grisoni, Emmanuel Fournier, Nicolas Guilbaud, Frédéric Marion ⇑ Institut de Recherche Pierre Fabre, CRDPF/CROE, 3 avenue Hubert Curien, BP 13562, 31035 Toulouse Cedex 1, France
a r t i c l e
i n f o
Article history: Received 12 March 2015 Revised 26 March 2015 Accepted 29 March 2015 Available online 3 April 2015 Keywords: Griseofulvin Sulfone Sulfoxide Cytotoxicity
a b s t r a c t Griseofulvin, an antifungal drug, has been shown in recent years to have anti-proliferative activities. We report here the synthesis of new analogs of griseofulvin, substituted in 20 by a sulfonyl group or in 30 by a sulfinyl or sulfonyl group. These compounds exhibit good anti-proliferative activities against SCC114 cells, an oral squamous carcinoma cell line showing pronounced centrosome amplification, and unexpected cytotoxic activities on HCC1937 cells, a triple negative breast cancer cell line resistant to microtubule inhibitors. Ó 2015 Elsevier Ltd. All rights reserved.
Griseofulvin 1 (Fig. 1) is a natural product initially isolated from Penicillium griseofulvum,1 and used to treat fungal infections in humans and animals. Griseofulvin is also of interest as anti-cancer agent, due to its low toxicity and efficacy in inhibiting proliferation of cancer cells.2 Although several studies have suggested that tubulin is the main target of griseofulvin, the exact mechanism of action remains still unclear.3 In order to increase the anti-tumor properties of griseofulvin, several 20 -oxygen and 20 -sulfur analogs of griseofulvin were synthesized by Clausen and co-workers.4 Among these compounds, GF-15 2 (Fig. 1) has been reported to have tumor growth inhibition through centrosomal clustering inhibition.5 Based on these observations, and since a part of our research programs is dedicated to the quest for anti-cancer natural products, we considered that griseofulvin could provide a good molecular basis to initiate a medicinal chemistry program. However, the research field of anti-mitotic microtubule-interfering agents is a mature area that counts several marketed drugs but is still dynamic owing to recent approvals in the last decade.6 One of the major clinical issues faced by these chemotherapeutic agents is intrinsic or acquired-drug resistance of tumor cells which has different origins, such as efflux protein expression or tubulin mutations, among others. Having these considerations in mind, we ⇑ Corresponding authors. Tel.: +33 5 34 50 69 38; fax: +33 5 34 50 30 54 (F.L-M.); tel.: +33 5 34 50 60 57 (F.M.). E-mail addresses: [email protected] (F. Liéby-Muller), [email protected] (F. Marion). http://dx.doi.org/10.1016/j.bmcl.2015.03.081 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
O
OO 2'
O
O Cl
3' 4'
6'
5'
O
OO
O O
O
O
Cl Griseofulvin 1
GF-15 2
Figure 1. Structures of griseofulvin 1 and GF-15 2.
conducted a program to identify potent anti-cancer griseofulvin derivatives that could overcome resistance. We report here our efforts in the synthesis of 20 -sulfone, 30 -sulfoxide and 30 -sulfone griseofulvin analogs, as well as their antiproliferative activities, notably against a resistant cancer cell line. 20 -Demethoxy-20 -sulfonylgriseofulvin analogs 5a–c were obtained in a three steps synthetic process, starting from commercial griseofulvin (Scheme 1). Griseofulvin 1 was reacted with lithium chloride and phosphoryl chloride in refluxing dioxane,4 affording the 20 -vinyl chloride 3 in 40% yield, after separation of its 40 -vinyl chloride isomer. The addition of thiols on 3 was performed using an excess of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in refluxing dioxane.4 20 -Sulfur compounds 4a–c were obtained with an average yield of 80% for the three compounds, then oxidized to 20 -sulfone by addition of
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O
O
OO
20 -Demethoxy-30 -sulfinyl griseofulvin analogs 10a–b and 20 demethoxy-30 -sulfonyl griseofulvin analogs 11a–b were obtained through a five steps synthetic sequence (Scheme 3). Griseofulvin 1 was reduced in the presence of Pd/C 10% and hydrogen in ethyl acetate to afford intermediate 6. Elimination of the methoxy group was performed with sulfuric acid 2 N, in refluxing ethanol to afford 7.7 Epoxidation of the resulting double bound with hydrogen peroxide led to compound 8 with an overall yield of 94% over three steps, starting from commercial griseofulvin. When 8 was treated with thiols in basic conditions, 30 -vinyl sulfur derivatives 9a–b were obtained with 15–25% yields. Although the conversion seemed to be complete (monitoring of the reaction by LCMS), these disappointing yields were obtained after purification and were maybe due to a lack of solubility or instability of the compounds over silica gel. Moreover, in our hands, the reaction was found to be poorly reproducible and adaptable to a range of thiols, and sodium hydroxide at different concentrations (1 N for 9a or 0.1 N for 9b) was eventually found to give the best results. Thereby, the use of other bases, such as sodium hydride or sodium ethoxide, did not allow us to improve the reaction yields, leading to complex mixtures. Oxidation of 30 -vinyl sulfur 9a with a stoichiometric amount of meta-chloroperbenzoic acid (m-CPBA) led to 10a with a moderate yield of 30%. 9b was oxidized with sodium periodate and led to sulfoxide 10b, as a mixture of two diastereoisomers, with moderate yields of 35%. Oxidation of 30 -vinyl sulfur 9a and 9b with an excess of m-CPBA led to the formation of sulfones 11a and 11b, respectively, with 79% and 45% yield. Compounds 5a–d, 10a–b and 11a–b8 were evaluated for their antiproliferative activities against HCC1937 cells,9 a triple negative breast cancer cell line resistant to most microtubule inhibitors.10 These derivatives were compared to reference compounds such as paclitaxel, epothilone B, vinorelbine, griseofulvin and GF-15 (Table 1). Compounds with IC50 values higher than 10 lM were regarded as inactive. 20 -Vinyl sulfones 5a–d were found to be active on the HCC1937 cell line, having IC50 values in the micromolar range, whereas references were found to be inactive. The degree of oxidation of the 20 -sulfur has a real impact on the antiproliferative activities for this particular cell line as parent compounds 4a– b were inactive. The size and aromaticity of the substituent borne
O Cl
i O
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Cl 1
3 ii
R O OS O
O
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R
OS
iii O
Cl
a R = Bn b R = -C 2H 4 -NMe2 c R = Ph
5 a-c
O
O
O Cl
4 a-c
Scheme 1. Reagents and conditions: (i) LiCl (3 equiv), POCl3 (5 equiv), 1,4-dioxane, 100 °C, 1 h, 40%; (ii) R-SH (1–2 equiv), DBU (2.5 equiv), 1,4-dioxane, 100 °C, 18 h; (iii) oxone (6 equiv), H2O/MeOH/THF 5:1:1, rt, 16 h, 8–26% over 2 steps.
O
O Cl
R O OS O
O i
O
O
O
O
O
O
Cl
Cl 5c R = Ph 5d R = Me
3
Scheme 2. Reagents and conditions: (i) R-SO2Na (1 equiv), DMF, rt, 16 h, 86%.
an excess of aqueous oxone. Derivatives 5a–c were obtained in yields of 11–32%. An alternative route using sulfinates as nucleophiles was found to improve the overall yield of the process. For example, starting from 20 -vinyl chloride 3, addition of sodium phenylsulfinate or methylsulfinate in DMF directly led, respectively, to compounds 5c or 5d with 85% yield each (Scheme 2).
O
O
OO
O
OO
i O
O
O
O
ii O
O
O
Cl
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Cl 1
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Cl 6
7 iii
O
O
O
O
O
Cl
O O
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R S O
10 a-b
O
8
O v
9 a-b
O a R = Bn b R = Ph
Cl
O
Cl 9 a-b
O
O
iv O
O
O
R S
O
vi O
O
R O S O O
Cl 11 a-b
Scheme 3. Reagents and conditions: (i) Pd/C 10%, EtOAc, H2, rt, 6 h, 100%; (ii) H2SO4 2 N (10 vol), EtOH, 80 °C, 16 h, 99%; (iii) NaOH 2 N (1.4 equiv), H2O2 (1 equiv), EtOH, 0 °C, 1 h, 95%; (iv) R = Ph: PhSH (1.2 equiv), NaOH 1 N (30 vol), THF, 70 °C, 15% or R = Bn: BnSH (1.2 equiv), NaOH 0.1 N (30 vol), THF, rt, 25%; (v) R = Bn: mCPBA (1 equiv), CH2Cl2, 0 °C, 1 h, 30%; R = Ph: NaIO4 (1.5 equiv), MeOH/H2O 2:1, 50 °C, 16 h, 35% (vi) mCPBA (2.7 equiv), CH2Cl2, rt, 0.5–1 h, 45–79%.
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Table 1 IC50 values of synthesized griseofulvin analogs and references on HCC1937 and SCC114 cell lines IC50 (lM)
Compounds HCC1937 4a 4b 5a 5b 5c 5d 10a 10b 11a 11b Paclitaxel Epothilone B Vinorelbine Griseofulvin GF-15 2
version, at http://dx.doi.org/10.1016/j.bmcl.2015.03.081. These data include MOL files and InChiKeys of the most important compounds described in this article.
a
>10 >10a 1.4 5.9 1.6 >10b n.d. 0.4 0.9 0.7 >10a >10a >10a >10a >10a
SCC114 n.d. 1.45 0.4 2.8 1 1.9 0.02 0.25 n.d. 0.4 0.0003 0.00017 0.00007 6.4 0.98
n.d.: not determined. a 0% inhibition at 10 lM. b 40% inhibition at 10 lM.
by the sulfone seems also to affect the antiproliferative activity, 5a and 5c being more active than 5b and 5d.11 Substitution of position 30 is of particular relevance regarding the cytotoxic activities, sulfoxide 10b and sulfones 11a–b being the most active derivatives, with sub-micromolar IC50 values. Introduction of an electron withdrawing group in the 30 position lead to quite a reactive Michael acceptor and it is less likely with 20 substituted compounds so this reactivity difference could explain the various pharmacological activities observed between 20 and 30 series. The cytotoxic activities of these compounds were also evaluated against the oral squamous carcinoma cell line SCC114,9 known to have pronounced centrosome amplification and to be sensitive to declustering agents.3c,12 The aim of evaluating this particular cell line was to assess if newly synthesized derivatives would be endowed with similar activities as the related griseofulvin. All tested compounds were found to be more active than griseofulvin, and equipotent or more active than GF-15, 5a and 11b having IC50 values of 0.4 lM, 10b having an IC50 value of 0.25 lM and 10a being the most active compound, with an IC50 value of 20 nM (Table 1). Mechanistically, these results suggest that these compounds may inhibit cell growth by inducing, at least partially, centrosomal declustering. Such compound characterization awaits confirmation with complementary studies. In conclusion, several new 20 and 30 griseofulvin analogs were synthesized through a short synthetic process. Their anti-proliferative activities were evaluated on HCC1937 cells, a resistant cancer cell line, as well as SCC114 cells, a centrosomal clustering dependant cell line. A significant potency gain was obtained by introducing constrained sulfones on position 20 , the increase of activity being even better with the introduction of sulfoxides or sulfones on position 30 . These new 20 and 30 griseofulvin analogs have evidenced interesting cytotoxic properties not only against cells with centrosomal amplification but also unexpected cytotoxic properties against a cell line intrinsically resistant to anti-mitotic and clinically used anticancer drugs. The reactive nature of the newly synthesized compounds may be involved in the mode of action especially for the 30 derivatives. Investigations to precise the origin of the observed activity of these series is underway and will be reported in due course. Supplementary data Supplementary data (1H and 13C NMR spectra of 5a–d, 10a–b and 11a–b) associated with this article can be found, in the online
References and notes 1. (a) Oxford, A. E.; Raistrick, H.; Simonart, P. Biochem. J. 1939, 33, 240; (b) Birch, A. J.; Massy-Westropp, R. A.; Rickards, R. W.; Smith, H. J. Chem. Soc. 1958, 360; for a review on the chemistry of griseofulvin, see: (c) Petersen, A. B.; Ronnest, M. H.; Larsen, T. O.; Clausen, M. H. Chem. Rev. 2014, 114, 12088. 2. Ho, Y.-S.; Duh, J.-S.; Jeng, J.-H.; Wang, Y.-J.; Liang, Y.-C.; Lin, C.-H.; Tseng, C.-J.; Yu, C.-F.; Chen, R.-J.; Lin, J.-K. Int. J. Cancer 2001, 91, 393. 3. (a) Mir, L.; Oustrin, M.-L.; Lecointe, P.; Wright, M. FEBS Lett. 1978, 88, 259; (b) Panda, D.; Rathinasamy, K.; Santra, M. K.; Wilson, L. Prod. Natl. Acad. Sci. U.S.A. 2005, 102, 9878; (c) Rebacz, B.; Larsen, T. O.; Clausen, M. H.; Rønnest, M. H.; Löffler, H.; Ho, A. D.; Krämer, A. Cancer Res. 2007, 67, 6342; (d) Ranthinasamy, K.; Jindal, B.; Asthana, J.; Singh, P.; Balaji, P. V.; Panda, D. BMC Cancer 2010, 10, 213. 4. (a) Rønnest, M. H.; Rebacz, B.; Markworth, L.; Terp, A. H.; Larsen, T. O.; Krämer, A.; Clausen, M. H. J. Med. Chem. 2009, 52, 3342; (b) Clausen, M. H.; Krämer, A.; Larsen, T. O.; Rebacz, B. WO2009/000937. 5. Raab, M. S.; Breitkreutz, I.; Anderhub, S.; Rønnest, M. H.; Leber, B.; Larsen, T. O.; Weiz, L.; Konotop, G.; Hayden, P. J.; Podar, K.; Fruehauf, J.; Nissen, F.; Mier, W.; Haberkorn, U.; Ho, A. D.; Goldschmidt, H.; Anderson, K. C.; Clausen, M. H.; Krämer, A. Cancer Res. 2012, 72, 5374. 6. (a) Dumontet, C.; Jordan, M. A. Nat. Rev. Drug Disc. 2010, 9, 790; (b) Mukhtar, E.; Adhami, V. M.; Mukhtar, H. Mol. Cancer Ther. 2014, 13, 275. 7. Oda, T.; Sato, Y. Chem. Pharm. Bull. 1983, 31, 934. 8. 5a: light brown solid, 1H NMR (CDCl3, 400 MHz) d: 0.96 (d, 3H, J = 6.8 Hz), 2.39 (dd, 1H50 , J = 17.6, 4.8 Hz), 2.74 (m, 1H60 ), 3.07 (dd, 1H50 , J = 17.6, 14.0 Hz), 3.99 (s, 3H), 4.06 (s, 3H), 4.49 (d, 1H, J = 13.6 Hz), 4.69 (d, 1H, J = 13.6 Hz), 6.19 (s, 1H), 6.52 (s, 1H30 ), 7.37 (m, 3H), 7.49 (m, 2H); 13C NMR (125 MHz, CDCl3) d: 195.2, 189.7, 168.2, 164.5, 158.0, 151.7, 137.6, 130.9, 129.4, 129.0, 127.1, 105.2, 97.1, 90.4, 89.1, 62.9, 57.1, 56.5, 39.6, 39.1, 14.3; LCMS (ES, m/z): 477.1 [M+H]+ 5b: yellow solid, 1H NMR (CDCl3, 400 MHz) d: 0.95 (d, 3H, J = 6.8 Hz), 2.27 (s, 6H), 2.50 (dd, 1H50 , J = 17.4, 4.6 Hz), 2.66 (dt, 1H, J = 13.2, 5.2 Hz), 2.87 (m, 1H+1H60 ), 3.14 (dd, 1H50 , J = 17.4, 14.2 Hz), 3.22 (m, 1H), 3.77 (ddd, 1H, J = 14.4, 8.8, 6.0 Hz), 3.98 (s, 3H), 4.03 (s, 3H), 6.16 (s, 1H), 6.88 (s, 1H30 ); 13C NMR (100 MHz, CDCl3) d: 195.22, 189.12, 167.68, 163.80, 157.35, 152.94, 134.42, 104.60, 96.44, 89.70, 88.90, 56.36, 55.85, 54.26, 52.02, 44.41, 39.23, 38.77, 13.63; LCMS (ES, m/z): 458.1 [M+H]+. 5c: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.83 (d, 1H, J = 6.8 Hz), 2.44 (dd, 1H50 , J = 17.2, 4.8 Hz), 2.77 (m, 1H60 ), 3.11 (dd, 1H50 , J = 17.2, 14.0 Hz), 4.00 (s, 3H), 4.05 (s, 3H), 6.18 (s, 1H), 7.11 (s, 1H30 ), 7.54 (t, 2H, J = 7.6 Hz), 7.65 (t, 1H, J = 7.6 Hz), 7.83 (d, 2H, J = 7.6 Hz); 13C NMR (100 MHz, CDCl3) d: 195.6, 189.7, 168.2, 164.3, 157.7, 154.9, 139.0, 135.5, 133.8, 129.1, 128.4, 105.0, 96.9, 89.9, 89.1, 56.8, 56.2, 39.6, 39.2, 14.0; LCMS (ES, m/z): 463.0 [M+H]+. 5d: light yellow solid, 1H NMR (CDCl3, 400 MHz) d:0.98 (d, 3H, J = 6.8 Hz), 2.52 (dd, 1H50 , J = 17.2, 4.8 Hz), 2,93 (m, 1H60 ), 3.18 (dd, 1H50 , J = 17.2, 14.0 Hz), 3.22 (s, 3H), 3.98 (s, 3H), 4.03 (s, 3H), 6.16 (s, 1H), 6.98 (s, 1H30 ); 13C NMR (100 MHz, CDCl3) d: 195.7, 189.6, 168.3, 164.5, 158.0, 154.1, 136.0, 105.2, 97.2, 90.4, 89.1, 57.0, 56.5, 44.9, 39.9, 39.5, 14.3; LCMS (ES, m/z): 400.8 [M+H]+. 10a: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.97 (d, 3H, J = 6.7 Hz), 2.60 (dd, 1H50 , J = 17.4, 5.0 Hz), 2.88–2.96 (m, 1H60 ), 3.27 (dd, 1H50 , J = 17.4, 13.8 Hz), 3.90 (d, 1H, J = 13.0 Hz), 4.01 (s, 3H), 4.03 (s, 3H), 4.35 (d, 1H, J = 13.0 Hz), 6.14 (s, 1H), 6.87 (s, 1H20 ), 7.20 (d, 2H, J = 7.7 Hz), 7.26–7.34 (m, 3H); 13C NMR (100 MHz, CDCl3) d: 194.5, 190.4, 168.5, 164.9, 157.9, 146.8, 145.1, 130.9, 128.8, 128.5, 128.3, 105.1, 97.8, 92.2, 89.8, 59.4, 57.1, 56.5, 40.7, 38.3, 14.2; LCMS (ES, m/z): 461.1 [M+H]+. 10b: white solid, 1H NMR (400 MHz, CDCl3) d: 0.92 (d, 1.3H, J = 6.9 Hz), 0.95 (d, 1.6H, J = 6.9 Hz), 2.38 (dd, 0.52H50 , J = 17.1, 5.2 Hz), 2.45 (dd, 0.5H50 , J = 17.1, 5.2 Hz), 2.69–2.79 (m, 0.4H60 ), 2.93–3.03 (m, 0.6H60 ), 3.07–3.25 (m, 1H50 ), 4.00 (s, 3H), 4.05 (s, 3H), 6.17 (s, 1H), 7.33 (s, 0.4H20 ), 7.35 (s, 0.5H20 ), 7.46–7.48 (m, 3H), 7.69–7.71 (m, 1.1H), 7.79 (m, 0.9H); 13C NMR (125 MHz, CDCl3) d: 193.7, 193.1, 190.9, 190.8, 168.6, 168.4, 165.0, 164.9, 158.0, 157.9, 147.7, 147.1, 143.9, 143.5, 142.9, 142.4, 131.9, 131.7, 129.4, 129.2, 126.3, 125.7, 105.2, 105.0, 97.7, 97.5, 92.0, 91.3, 89.9, 89.7, 57.1, 56.5, 41.3, 40.3, 38.7, 37.1, 31.9, 29.0, 22.7, 14.3, 13.9; LCMS (ES, m/z): 447.0 [M+H]+. 11a: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.93 (d, 3H, J = 6.7 Hz), 2.63 (dd, 1H50 , J = 17.6, 5.3 Hz), 2.80 (m, 1H60 ), 3.21 (dd, 1H50 , J = 17.6, 13.7 Hz), 3.98 (s, 3H), 4.03 (s, 3H), 4.56 (d, 1H, J = 13.7 Hz), 4.63 (d, 1H, J = 13.7 Hz), 6.14 (s, 1H), 7.25–7.29 (m, 2H), 7.31 (s, 1H20 ), 7.33 (m, 3H); 13C NMR (100 MHz, CDCl3) d: 191.1, 188.6, 167.8, 164.6, 157.5, 152.9, 138.7, 130.4, 128.4, 128.3, 126.5, 104.3, 97.2, 90.1, 89.5, 60.3, 56.5, 55.9, 39.6, 36.7, 29.1, 13.5; LCMS (ES, m/z): 477.1 [M+H]+. 11b: white solid, 1H NMR (CDCl3, 400 MHz) d: 0.92 (d, 3H, J = 6.7 Hz), 2.46 (dd, 1H50 , J = 17.2, 4.9 Hz), 2.86 - 2.94 (m, 1H60 ), 3.13 (dd, 1H50 , J = 17.2, 14.0 Hz), 4.00 (s, 3H), 4.06 (s, 3H), 6.18 (s, 1H), 7.52 (t, 2H, J = 7.4 Hz), 7.62 (t, 1H, J = 7.4 Hz), 7.78 (s, 1H20 ), 8.01 (d, 2H, J = 7.4 Hz); 13C NMR (100 MHz, CDCl3) d: 189.7, 189.5, 168.0, 164.8, 157.7, 151.0, 141.5, 138.7, 133.5, 128.6, 128.5, 104.6, 97.3, 90.4, 89.6, 56.7, 56.1, 40.1, 37.1, 13.7; LCMS (ES, m/z): 463.0 [M+H]+. 9. SCC-114 and HCC-1937 cells were grown in MEM (SIGMA—M7278) or RPMI (SIGMA—R0883) media supplemented with 10% Fetal Bovine Serum respectively. Antiproliferative activity was measured using the ATPlite assay (Perkin Elmer) on exponentially growing adherent cells that were seeded in 96
F. Liéby-Muller et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2078–2081 well plates with 103 cells/well for SCC-114 and 2103 cells/well for HCC-1937. Cell viability was evaluated by dosing the ATP released by viable cells after an incubation period of 72 h for SCC-114 cells or 48 h for HCC-1937 cells at 37 °C in 5% CO2 incubator. Luminescence was measured using a TopCount NXT™ luminescence counter (Perkin–Elmer). The IC50 values were determined by curve fitting analysis (non linear regression model with a sigmoidal dose response, variable hill slope coefficient), performed with the algorithm provided by the GraphPad Software.
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10. Kadra, G.; Finetti, P.; Toiron, Y.; Viens, P.; Birnbaum, D.; Borg, J.-P.; Bertucci, F.; Gonçalves, A. Breast Cancer Res. Treat. 2012, 132, 1035. 11. Although the activity of 5 d was reported to be inactive in Table 1, it was found to induce 40% inhibition at 10 lM, when no inhibition was induced with the reference compounds at the same concentration. 12. Quintyne, N. J.; Reing, J. E.; Hoffelder, D. R.; Gollin, S. M.; Saunders, W. S. Science 2005, 307, 127.
