Article pubs.acs.org/jnp
cis-Restricted 3‑Aminopyrazole Analogues of Combretastatins: Synthesis from Plant Polyalkoxybenzenes and Biological Evaluation in the Cytotoxicity and Phenotypic Sea Urchin Embryo Assays Dmitry V. Tsyganov,† Leonid D. Konyushkin,† Irina B. Karmanova,† Sergei I. Firgang,† Yuri A. Strelenko,† Marina N. Semenova,‡,§ Alex S. Kiselyov,⊥ and Victor V. Semenov*,† †
N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991, Moscow, Russian Federation Institute of Developmental Biology, RAS, Vavilov Street, 26, 119334, Moscow, Russian Federation § Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus ⊥ ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, California 92121, United States ‡
S Supporting Information *
ABSTRACT: We have synthesized a series of novel cis-restricted 4,5polyalkoxydiaryl-3-aminopyrazole analogues of combretastatins via short synthetic sequences using building blocks isolated from dill and parsley seed extracts. The resulting compounds were tested in vivo in the phenotypic sea urchin embryo assay to reveal their antimitotic and antitubulin effects. The most potent aminopyrazole, 14a, altered embryonic cell division at 10 nM concentration, exhibiting microtubule-destabilizing properties. Compounds 12a and 14a displayed pronounced cytotoxicity in the NCI60 anticancer drug screen, with the ability to inhibit growth of multidrug-resistant cancer cells. microvasculature.2,8−10 Despite this promising “dual” therapeutic potential, CA4P was reported to display a number of considerable dose-limiting side effects.11,12 Several research teams have attempted to improve the safety−efficacy profile of this compound class.13,14 Combretastatin analogues exhibit at least three structural commonalities. These are the trimethoxy ring A, the ring B substituted at C3 and C4, and a cis-olefin tether of variable length between the two rings reported to be essential for structural rigidity and ultimately activity of these compounds (Figure 1). A number of combretastatin analogues featuring modified A and B rings and bridge isosteres have been synthesized and evaluated in biological assays.13−16 Among numerous reported cyclic analogues of combretastatins, fivemembered heterocycles have been described as active, nonisomerizable, and metabolically stable isosteres for the cisolefin bridge.13,14 It was reported recently that 4,5-diaryl-3-aminopyrazole derivatives of CA4 showed strong cytotoxicity against five human cancer cell lines, inhibited purified tubulin polymerization, and caused cell cycle arrest in the G2/M phase.17 However, this study did not elaborate further on (i) the relationship between the NH2 group position and the resulting antimitotic activity (e.g., potency of II vs III, Figure 1) and (ii) the optimal number and position of methoxy groups in ring A. Moreover, tetramethoxy-, methylenedioxy-, and ethylenediox-
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ntimitotic drugs have been successfully used as chemotherapeutic agents for the treatment of cancer. The majority of known antimitotic drugs selectively target microtubules, responsible for the formation of the mitotic spindle, which is essential for proper chromosomal separation during cell division. It is generally agreed that agents affecting tubulin polymerization impair microtubule dynamics and consequently arrest cells during mitosis.1−4 Natural combretastatins, namely, combretastatins A-2 and A4 (Figure 1; CA2, CA4), are antimitotics found in the bark of
Figure 1. Structures of combretastatins and their 3-aminopyrazole analogues.
Combretum caf frum Kuntze (Combretaceae).5 It is generally recognized that these agents bind to the colchicine binding site of tubulin and affect its assembly.6,7 Several phosphorylated prodrugs including CA4 disodium phosphate (CA4P, Zybrestat) and combretastatin A-1 phosphate (Oxi4503) show promise in the late stages of clinical trials. Notably, in addition to affecting tubulin dynamics, they also disrupt tumor © XXXX American Chemical Society and American Society of Pharmacognosy
Received: April 25, 2013
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dx.doi.org/10.1021/np400310m | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Scheme 1. Synthesis of Precursors for cis-Restricted 4,5-Polyalkoxydiaryl-3-aminopyrazolesa
Reagents and conditions: (a) KOH, 100 °C, 40 min; ref 18; (b) O3, CHCl3−MeOH−pyridine (80:20:3 v/v), −15 °C, 1−2 h; ref 18; (c) CO(NH2)2·H2O2, CH3OH, reflux, 1.5 h; ref 18; (d) SOCl2, MeOH, reflux 1 h; (e) NaOH, Cu, H2O, reflux, 27 h; (f) BrCH2CH2Br, K2CO3, DMF, 100−105 °C, 5 h; (g) NaBH4, MeOH, rt, 6−7 h; (h) SOCl2, C6H6, 48−50 °C, 40 min; (i) KCN, CH3CN, 50 °C, 10 h. a
Scheme 2. Synthesis of 4,5-Polyalkoxydiaryl-3-aminopyrazoles 12 and 14a
a
Reagents and conditions: (a) NaH, THF, reflux, 5−6 h; (b) N2H4·2HCl, EtOH, reflux, 12−18 h.
restricted 4,5-(polymethoxy)diaryl-3-aminopyrazole analogues of CA4 and CA2 have been accessed via short synthetic sequences from easily available dill and parsley extracts.19
yaryl substituents have not been evaluated. For example, we have shown previously that CA4, CA2, and ethylenedioxy analogues of CA2, I (Figure 1), exhibited comparable antimitotic microtubule-destabilizing activity in the sea urchin embryo model.18 Considering the literature evidence, studies summarized in this paper were aimed at (i) the development of robust synthetic protocols for the synthesis of amino pyrazole derivatives from natural building blocks and (ii) the assessment of their structure−activity relationship in vivo using the phenotypic sea urchin embryo assay. Specifically, both the number and arrangement of OCH3 substituents on the B ring and position of the NH2 group in the pyrazole ring were investigated. Toward this goal, a series of conformationally
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RESULTS AND DISCUSSION The targeted diaryl-3-aminopyrazoles I and II were conveniently assembled in 18−65% overall yields from the key intermediate polyalkoxybenzoic acids 4 and respective aromatic acetonitriles 10 (Scheme 1) following the initial base-mediated condensation to furnish α-ketonitriles 11 and 13 (Scheme 2). These were subsequently cyclized into pyrazoles 12 and 14 with N2H4·2HCl.17 Notably, similar reactions conducted with N2H4·H2O resulted only in trace amounts of 12 and 14. Hydrazides of the corresponding polymethoxybenzoic acids B
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Table 1. 1H NMR Spectroscopic Dataa of 3-Aminopyrazoles 12 and 14 position Ring A 2″ 3″ 4″ 5″ 6″ Ring B 2′ 3′ 4′ 5′ 6′ NH2b NHb
12a 6.71 3.59 3.65 3.59 6.71
12b
s s s s s
7.30 d 7.02 d 3.78 s 7.02 d 7.30 d 4.33 11.75
(8.7) (8.7) (8.7) (8.7)
12c
12d
6.73 d (1.5) 3.69 s 6.02 s
6.61 d (2.1) 3.58 s 4.20 m
6.53 d (1.5)
6.50 d (2.1)
3.67 s 6.42 s
7.28 d 7.00 d 3.79 s 7.00 d 7.28 d 4.37 13.85
7.27 d 7.00 d 3.79 s 7.00 d 7.27 d 4.30 11.80
7.07 d 6.86 d 3.70 s 6.86 d 7.07 d 4.34 11.70
(8.8) (8.8) (8.8) (8.8)
(8.7) (8.7) (8.7) (8.7)
3.39 s 6.01 s
position
12e 3.37 s 3.90 s 5.99 s 6.32 s
(8.7) (8.7) (8.7) (8.7)
7.07 d 6.84 d 3.71 s 6.84 d 7.07 d 4.45 11.70
(8.8) (8.8) (8.8) (8.8)
14a
14b
Ring A 2′ 6.58 s 3′ 3.67 s 4′ 3.68 s 5′ 3.67 s 6′ 6.58 s Ring B 2″ 7.35 d (8.6) 3″ 6.98 d (8.6) 4″ 3.77 s 5″ 6.98 d (8.6) 6″ 7.35 d (8.6) NH2 4.42 NH 11.82
14d
6.49 d (1.4) 3.74 s 5.98 s
3.5 s 6.03 s
6.38 d (1.4)
3.68 s 6.32 s
7.28 d 6.94 d 3.76 s 6.94 d 7.28 d 4.40 11.91
7.27 d 6.91 d 3.74 s 6.91 d 7.27 d 4.38 11.87
(8.8) (8.8) (8.8) (8.8)
14e 3.39 s 3.94 s 6.04 s 6.41 s
(8.7) (8.7) (8.7) (8.7)
7.33 d 6.96 d 3.75 s 6.96 d 7.33 d 4.42 11.90
(8.8) (8.8) (8.8) (8.8)
a 500.13 MHz in DMSO-d6. Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses (Hz). bNH2 and NH signals were broadened due to exchange of protons in DMSO.
Table 2. 13C NMR Spectroscopic Dataa of 3-Aminopyrazoles 12 and 14 position Ring A 1″ 2″ OCH3-2″ 3″ OCH3-3″ 4″ OCH3-4″ 5″ OCH3-5″ 6″ O(CH2)nO Ring B 1′ 2′ 3′ 4′ OCH3-4′ 5′ 6′ 3 4 5
12a
12b
12c
124.1 105.4
121.8 108.0
119.7 104.3
152.5 55.7 137.1 60.0 152.5 55.7 105.4
143.1 56.1 135.5
148.6 55.6 134.0
148.4
141.7
101.4 101.7 121.5 131.4 114.3 158.8 55.1 114.3 131.4 149.0 102.9 141.4
127.6 131.5 114.1 158.6 55.2 114.1 131.5 139.5 98.7 149.9
12d 113.0 135.0 59.2 138.7 141.0
12e 118.4 144.9 60.5 137.1 59.7 137.6
108.9 64.0
135.3 59.2 102.3 102.1
143.8 103.7 101.5
121.8 131.2 114.4 158.9 55.2 114.4 131.2 149.4 102.6 141.7
121.4 130.5 114.1 158.7 55.0 114.1 130.5 149.2 99.3 142.1
126.0 129.1 113.7 156.9 54.8 113.7 129.1 150.0 104.5 140.0
position Ring A 1′ 2′ OCH3-2′ 3′ OCH3-3′ 4′ OCH3-4′ 5′ OCH3-5′ 6′ O(CH2)nO Ring B 1″ 2″ 3″ 4″ OCH3-4″ 5″ 6″ 3 4 5
14a
14b
14d
14e
122.5 106.5
127.2 109.1
152.8 55.7 135.5 60.0 152.8 55.7 106.5
143.3 56.1 133.3
118.3 136.1 59.3 138.8
123.8 146.0 60.6 137.4 59.8 135.4
129.3 128.7 113.8 158.8 55.2 113.8 128.7 150.5 99.9 140.0
148.5
138.8
103.4 101.1
135.6 56.3 110.4 101.5
143.9 102.8 100.9
123.2 128.7 113.9 159.0 55.2 113.9 128.7 149.6 104.2 141.5
123.2 127.8 113.8 158.9 55.1 113.8 127.8 149.8 100.5 142.1
127.8 126.4 113.4 157.1 54.9 113.4 126.4 146.0 105.8 140.1
a
1
125.76 MHz in DMSO-d6. Chemical shifts (δ) are in ppm relative to TMS. Assignments are based on 1H−1H NOESY, 1H−13C HMBC, and H−13C HSQC NMR experiments.
were the main products. The 1H NMR spectra for α-ketonitriles 11 and 13 are shown in Figures S1−S9 of the Supporting Information. 1H and 13C NMR spectroscopic data for pyrazoles 12 and 14 are presented in Tables 1 and 2 and Figures S10− S40 of the Supporting Information. 13C NMR signals for 12 and 14 were assigned by 1H−1H NOESY, 1H−13C HMBC, and 1 H−13C HSQC techniques. All of the resulting pyrazole derivatives were tested in the phenotypic sea urchin embryo assay developed in our laboratory.20 The screen has been shown to reliably and reproducibly assess antiproliferative and microtubule-destabilizing activities of compounds. Moreover, these data were consistent with results obtained via conventional purified tubulin polymerization and cell-based assays.21−23 Notable
advantages of this in vivo system include the possibility to both identify cell-permeable molecules with antimitotic or nonspecific cytotoxic activity and/or to establish their mode of action.24 The assay allows for the simultaneous monitoring of (i) fertilized egg test for antimitotic activity displayed by cleavage alteration/arrest and (ii) behavioral monitoring of a free-swimming blastula treated immediately after hatching. During the course of the extensive validation of this assay system at least three phenotypic parameters were observed; namely, lack of forward movement, settlement to the bottom of the culture vessel, and rapid spinning of an embryo around the animal−vegetal axis correlate with a microtubule-destabilizing activity caused by a molecule.21−23 C
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and cancer cell assay systems the antiproliferative activity of aminopyrazoles decreased in the following order: 14a > 12a > 14b > 12b ≈ 12c > 12e. Thus, cis-restricted 4,5-polyalkoxydiaryl-3-aminopyrazole analogues of natural antimitotic combretastatins were prepared starting from the natural allylpolyalkoxybenzenes extracted from dill and parsley seeds and from synthetic derivatives of vanillin. Biological effects of the resulting pyrazoles were evaluated in vivo in the phenotypic sea urchin embryo assay to reveal their antimitotic and antitubulin activity. In these series, pyrazoles substituted with ortho-amino and polyalkoxybenzene moieties were consistently more potent than their symmetric regioisomers (compare 14a and 12a; 14b and 12b), although the exact structural rationale for this observations is not clear. Molecules 12a and 14a, with a 3,4,5-trimethoxy ring A, exhibited the highest antimitotic microtubule-destabilizing effect in the sea urchin embryo assay. These compounds also showed a considerable cytotoxicity against a panel of 60 human cancer cell lines. In addition, both 12a and 14b inhibited growth of multi-drug-resistant P-glycoprotein-overexpressing ovarian cancer cells, suggestive of their potential as anticancer agents.
In this assay system, compounds 12a and 14a substituted with a 3,4,5-trimethoxyaryl moiety exhibited the highest activity (10−100 nM, Table 3). In our hands, type-III 5-aminoTable 3. Effects of 4,5-(Polymethoxy)diaryl-3aminopyrazoles on Sea Urchin Embryos and Their Cytotoxicity against Human Cancer Cells EC, μMa
NCI60 screen
compound
cleavage alteration
cleavage arrest
embryo spinning
CA4 CA2 If 12a 12b 12c 12d 12e 14a 14b 14d 14e
0.002 0.002 0.002 0.1 1 1 4 >4 0.01 0.2 >4 >4
0.01 0.01 0.01 1 >4 >4 >4 >4 0.05 >4 >4 >4
0.05 0.05 0.05 >10 >5 >5 >4 >4 1 >10 >4 >4
mean GI50, μMb
mean GI, %c
0.0032d 2.66e NDg 0.302 75.84 18.06 32.00 NDg 3.38 0.046 89.22 4.37 45.98 NDg NDg
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a
The sea urchin embryo assay was conducted as described previously.20 Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values. bGI50: concentration required for 50% cell growth inhibition. c GI, %: inhibition of cell growth at 10 μM concentration in the singledose screen; presented for compounds with low cytotoxicity. dData from ref 25. eMean value for seven human cancer cell lines.26 fData from ref 18. gND: not determined.
EXPERIMENTAL SECTION
General Experimental Procedures. NMR spectra were collected on a Bruker DR-500 instrument [working frequencies of 500.13 MHz (1H) and 125.76 MHz (13C)]. Mass spectra were obtained on a Finnigan MAT/INCOS 50 instrument (70 eV) using direct probe injection. Elemental analysis was accomplished with the automated Perkin-Elmer 2400 CHN microanalyzer. Compound purity was determined by NMR, HPLC, and elemental analyses. Polyalkoxybenzoic styrenes 2b,d,e, aldehydes 3b,d,e, acids 4a,b,d,e, and corresponding acid methyl esters 5b,d,e were synthesized according to previously reported procedures.18 3,4-Dihydroxy-5-methoxybenzaldehyde (7). This was prepared as described earlier.27 Bromovanillin 6 (105 g, 0.454 mol) was added to a vigorously stirred suspension of NaOH (130 g, 3.25 mol) and copper powder (550 mg, 8.65 mmol in 1.5 L of H2O). The resulting mixture was refluxed for 27 h and cooled to room temperature; the pH was adjusted to 7 with 1 M H2SO4 followed by the extraction with EtOAc (7 × 100 mL). Organic extracts were combined, refluxed over activated carbon, filtered, and dried over anhydrous Na2SO4. Solvent was removed under reduced pressure, and the residue was crystallized from toluene (250 mL) to yield 7 (49 g, 64% yield) as a tan powder: mp 129−131 °C (lit.27 mp 132−133 °C); 1H NMR (DMSO-d6) δ 9.7 (1H, s, CHO), 9.50 (1H, s, ArOH), 9.44 (1H, s, ArOH), 7.04 (1H, d, J = 1.7 Hz, ArH), 7.00 (1H, d, J = 1.7 Hz, ArH), 3.84 (3H, s, OCH3); EIMS m/z 168 [M]+ (100), 167 (87), 125 (24), 97 (30), 53 (21), 51 (35), 39 (39); anal. C 57.25; H 4.91%, calcd for C8H8O4, C 57.14; H 4.80%. 3,4-Ethylendioxy-5-methoxybenzaldehyde (3c). Dry K2CO3 (16.8 g, 122 mmol) was added to a vigorously stirred solution of 7 (14 g, 83 mmol) and 1,2-dibromoethane (22.5 g, 10.3 mL, 120 mmol) in DMF (110 mL). The resulting mixture was stirred for 5 h at 100−105 °C and left overnight at room temperature. The reaction mixture was diluted with water (600 mL), followed by extraction with EtOAc. The EtOAc extract was washed with water, and solvent was evaporated in vacuo. The residue (14.2 g) was crystallized from EtOH (40%) to afford 3c (13.3 g, 68 mmol, 82% yield) as a light brown powder: mp 78−80 °C; 1H NMR (DMSO-d6) δ 9.78 (1H, s, CHO), 7.11 (1H, d, J = 1.6 Hz, ArH), 7.10 (1H, d, J = 1.6 Hz, ArH), 4.28−4.34 (4H, octet, OCH2CH2O), 3.85 (3H, s, OCH3); EIMS m/z 194 [M]+ (100), 193 (38), 138 (28), 123 (42), 95 (29), 66 (21), 51 (26), 39 (67), 38 (25); anal. C 61.78; H 5.08%, calcd for C10H10O4, C 61.85; H 5.19%. 3,4-Ethylenedioxy-5-methoxybenzoic Acid (4c). Aqueous NaOH (6 M, 18 mL) was added dropwise to a stirred solution of the urea− hydrogen peroxide complex (1:1; 75 g) and aldehyde 3c (10 g, 5.15
pyrazoles were consistently more potent than the corresponding type-II analogues (compare 14a and 12a; 14b and 12b). It is worth noting that the majority of synthesized compounds did not affect sea urchin embryo spinning. The only notable exception was 14a. This molecule caused embryo spinning at 1 μM, suggesting that it is a microtubule-destabilizing agent. At the same concentration, aminopyrazole 12a caused formation of tuberculate arrested eggs, the effect also associated with a microtubule disruption activity. Substitution of trimethoxy groups in 12a and 14a with methylenedioxy (12b and 14b) or ethylenedioxy moieties (12c) reduced activity of the resultant compounds. Apiol and dillapiol derivatives with tetraalkoxy ring A, 12d, 12e, 14d, and 14e, showed only modest potency in the phenotypic assay. The outcome of the sea urchin embryo assay was supported using a conventional cell-based screen. The cytotoxicity of molecules 12a, 12b, 12c, 12e, 14a, and 14b was assessed in the NCI60 anticancer drug screen (Table 3; Table S1, Supporting Information). The most cytotoxic aminopyrazoles, 12a and 14a, exhibited mean GI50 values of 0.302 and 0.046 μM, respectively (Table 3). The melanoma MDA-MB-435 cell line was the most sensitive toward tested molecules. Compound 12a displayed high cytotoxicity against leukemia HL-60(TB), K-562, and SR, colon cancer KM12 and SW-620, and ovarian cancer OVCAR-3 cells, with GI50 values all less than 0.1 μM. Aminopyrazole 14a significantly inhibited growth of non-smallcell lung cancer NCI-H522 and melanoma MDA-MB-435 cells with GI50 values less than 0.01 μM (Table S1, Supporting Information). Notably, 12a, 14a, and 14b were more cytotoxic against multi-drug-resistant NCI/ADR-RES ovarian cancer cells as compared to the parent OVCAR-8 line. In both sea urchin D
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(43); anal. C 62.72; H 4.68; N 7.44%, calcd for C10H9NO3, C 62.82; H 4.74; N 7.33%. 2,5-Dimethoxy-3,4-methylenedioxyphenylacetonitrile (10d): 19.5 g, 88% yield; off-white solid; mp 97−98 °C (lit.18 mp 96−98 °C); 1H NMR (CDCl3) δ 6.49 (1H, s, H-6), 5.99 (2H, s, OCH2O), 3.97 (3H, s, OCH3), 3.87 (3H, s, OCH3-7), 3.61 (2H, s, CH2); EIMS m/z 221 [M]+ (72), 206 (80), 180 (3), 148 (10), 120 (14), 108 (11), 107 (13), 105 (12), 93 (31), 92 (20), 80 (49), 77 (79), 65 (64), 39 (100); anal. C 59.61; H 4.95; N 6.47%, calcd for C11H11NO4, C 59.73; H 5.01; N 6.33%. 2,3-Dimethoxy-4,5-methylenedioxyphenylacetonitrile (10e): 14.4 g, 65% yield; beige solid; mp 60−61 °C; 1H NMR (CDCl3) δ 6.52 (1H, s, ArH-6), 5.94 (2H, s, OCH2O), 4.04 (3H, s, OCH3), 3.88 (3H, s, OCH3), 3.62 (2H, s, CH2); EIMS m/z 221 [M]+ (54), 206 (100), 191 (13), 179 (12), 105 (21), 93 (16), 92 (13), 80 (27), 77 (68), 65 (30), 53 (54); anal. C 59.58; H 4.96; N 6.48%, calcd for C11H11NO4, C 59.73; H 5.01; N 6.33%. General Procedure for the Synthesis of α-Cyanoketones (11, 13). A 60% suspension of NaH (25 mmol) in mineral oil was added to a solution of phenylacetonitrile (10.0 mmol) in anhydrous THF (20 mL) at room temperature under an argon atmosphere. The resulting mixture was stirred for 30 min, and a solution of methyl benzoate (10 mmol) in anhydrous THF (20 mL) was added dropwise during 5 min. The reaction mixture was refluxed for 5−6 h, cooled, quenched with ice water (200 mL), acidified with 3 N HCl, and extracted with CH2Cl2 (3 × 20 mL). The organic layers were combined, washed with water, 10% NaHCO3 and water, dried over anhydrous Na2SO4, and concentrated in vacuo to obtain the crude product. The resulting αcyanoketones were purified by column chromatography (SiO2, EtOAc−hexanes). 2-(4-Methoxyphenyl)-3-oxo-3-(3,4,5-trimethoxyphenyl)propanenitrile (11a): 0.5 g, 65% yield; off-white solid; mp 94−96 °C (lit.31 mp 90−92 °C); 1H NMR (CDCl3) δ 7.35 (2H, d, J = 8.7 Hz, H2′,6′), 7.18 (2H, s, H-2″,6″), 6.93 (2H, d, J = 8.7 Hz, H-3′,5′), 5.47 (1H, s, HC−CN), 3.91 (3H, s, OCH3-4′), 3.86 (6H, s, 2 × OCH33″,5″), 3.80 (3H, s, OCH3-4″) (Figure S1, Supporting Information); EIMS m/z 341 [M]+ (3), 196 (31), 195 (100), 152 (22), 147 (11), 146 (26), 137 (19), 122 (16), 109 (19), 107 (12), 92 (12), 91 (13), 81 (26), 77 (34), 76 (16), 66 (27); anal. C 66.98; H 5.67; N 3.97%, calcd for C19H19NO5, C 66.85; H 5.61; N 4.10%. 2-(4-Methoxyphenyl)-3-oxo-3-(3-methoxy-4,5methylenedioxyphenyl)propanenitrile (11b): 2.4 g, 59% yield; oil; 1H NMR (CDCl3) δ 7.33 (2H, d, J = 8.7 Hz, H-2′,6′), 7.25 (1H, s, 1H, H4″), 7.08 (1H, s, H-6″), 6.91 (2H, d, J = 8.7 Hz, H-3′,5′), 6.06 (2H, s, OCH2O), 5.43 (1H, s, HC−CN), 3.91 (3H, s, OCH3-4′), 3.79 (3H, s, OCH3-3″) (Figure S2, Supporting Information); EIMS m/z 325 [M]+ (3), 180 (6), 179 (100), 151 (42), 146 (25), 103 (13), 95 (65), 93 (18), 91 (14), 78 (26), 77 (13), 65 (18), 63 (15); anal. C 66.52; H 4.69; N 4.27%, calcd for C18H15NO5, C 66.46; H 4.65; N 4.31%. 2-(4-Methoxyphenyl)-3-oxo-3-(3-methoxy-4,5ethylenedioxyphenyl)propanenitrile (11c): 0.41 g, 65% yield, oil; 1H NMR (CDCl3) δ 7.34 (2H, d, J = 8.7 Hz, H-2′,6′), 7.15 (1H, d, J = 2.0 Hz, H-5″), 7.13 (1H, d, J = 2.0 Hz, H-7″), 6.91 (2H, d, J = 8.7 Hz, H3′,5′), 5.47 (1H, s, HC−CN), 4.37 (2H, m, OCH2), 4.27 (2H, m, OCH2), 3.89 (3H, s, OCH3-4′), 3.79 (3H, s, OCH3-3″) (Figure S3, Supporting Information); EIMS m/z 339 [M]+ (1), 194 (20), 193 (100), 165 (18), 146 (35), 137 (12), 109 (11), 107 (19), 103 (11), 91 (11), 66 (33); anal. C 67.40; H 5.09; N 4.26%, calcd for C19H17NO5, C 67.25; H 5.05; N 4.13%. 2-(4-Methoxyphenyl)-3-oxo-3-(2,5-dimethoxy-3,4methylenedioxyphenyl)propanenitrile (11d): 1.82 g, 61% yield; oil; 1 H NMR (CDCl3) δ 7.27 (2H, d, J = 8.8 Hz, H-2′,6′), 6.99 (1H, s, H6″), 6.88 (2H, d, J = 8.8 Hz, H-3′,5′), 6.06 and 6.08 (2H, 2s, OCH2O), 5.86 (1H, s, HC−CN), 4.06 (3H, s, OCH3-4′), 3.84 (3H, s, OCH32″), 3.79 (3H, s, OCH3-5″) (Figure S4, Supporting Information); EIMS m/z 355 [M]+ (1), 210 (22), 209 (100), 194 (23), 166 (12), 146 (21), 136 (8), 121 (9), 106 (13), 103 (9), 95 (15), 93 (17), 91 (11), 78 (13), 77 (13), 65 (23); anal. C 64.33; H 4.87; N 3.83%, calcd for C19H17NO6, C 64.22; H 4.82; N 3.94%.
mmol) in 200 mL of MeOH at room temperature. The resulting mixture was stirred at 65 °C for 1 h, cooled to 40 °C, treated with 7.5 g of fine urea−hydrogen peroxide complex (1:1), and heated at 65 °C for an additional 1 h. The pH was adjusted to 3 with 18% HCl, and MeOH was removed in vacuo. The residue was diluted with water (200 mL) and extracted with CHCl3. The CHCl3 extract was washed with water, refluxed over activated carbon, filtered, and dried over anhydrous MgSO4. The resulting extract was concentrated in vacuo to afford 5c (9.2 g, 4.38 mmol, 85% yield) as an off-white solid: mp 212− 213 °C; 1H NMR (DMSO-d6) δ 12.73 (1H, s, COOH), 7.11 (1H, d, J = 1.9 Hz, ArH), 7.07 (1H, d, J = 1.9 Hz, ArH), 4.25−4.30 (4H, octet, OCH2CH2O), 3.80 (3H, s, OCH3); EIMS m/z 210 [M]+ (100), 195 (26), 154 (31), 139 (35), 111 (35), 69 (30), 67 (23), 66 (44), 55 (29), 53 (27), 51 (28), 50 (32), 45 (26), 39 (61), 38 (50); anal. C 57.26; H 4.75%, calcd for C10H10O5, C 57.14; H 4.80%. 3,4-Ethylenedioxy-5-methoxybenzoic Acid Methyl Ester (5c). A mixture of 4c (1g, 4.76 mmol) and SOCl2 (0.85 g, 7.12 mmol) in 15 mL of MeOH was refluxed for 1 h and concentrated in vacuo to yield methyl ester 5c (0.96 g, 90% yield (95% purity)) as an off-white solid: mp 106−108 °C (MeOH); 1H NMR (DMSO-d6) δ 7.11 (1H, d, J = 1.9 Hz, ArH), 7.09 (1H, d, J = 1.9 Hz, ArH), 4.26−4.31 (4H, octet, OCH2CH2O), 3.82 (3H, s, COOCH3), 3.81 (3H, s, OCH3); EIMS m/ z 224 [M]+ 224; anal. C 58.89; H 5.28%, calcd for C11H12O5, C 58.93; H 5.39%. 3-Methoxy-4,5-methylenedioxybenzyl Alcohol (8b). This was synthesized according to a literature procedure:28 mp 64−66 °C (lit.28 mp 64−66 °C). 2,5-Dimethoxy-3,4-methylenedioxybenzyl Alcohol (8d). This was synthesized according to a literature procedure:29 mp 88 °C (lit.29 mp 85 °C). 2,3-Dimethoxy-4,5-methylenedioxybenzyl Alcohol (8e). A suspension of aldehyde 3e (10.0 g, 47.6 mmol) in MeOH (150.0 mL) was stirred for 10 min, and NaBH4 (2.05 g, 54.7 mmol) was added in small portions over 30 min at room temperature. After stirring for 6−7 h, the pH was adjusted to 5 with glacial CH3COOH. Solvent was removed in vacuo followed by the addition of water. The mixture was filtered, washed with H2O (2 × 100 mL), and dried to afford the title compound in 88.4% yield (8.75 g, 41.2 mmol) as colorless crystals: mp 51−53 °C; 1H NMR (DMSO-d6, 500 MHz) δ 6.60 (1H, s, H-6), 5.95 (2H, s, OCH2O), 4.98 (1H, t, J = 5.3 Hz, OH), 4.40 (2H, d, J = 5.03 Hz, CH2), 3.91 (3H, s, OCH3), 3.6 (3H, s, OCH3); EIMS m/z 212 [M]+ (87), 197 (15), 195 (21), 139 (96), 137 (50), 111 (52), 109 (33), 107 (23), 96 (23), 95 (24), 93 (34), 83 (24), 81 (29), 79 (35), 77 (29), 69 (24), 68 (50), 67 (21), 66 (28), 65 (51), 55 (51), 53 (89), 52 (21), 51 (42), 50 (28), 40 (25), 39 (100), 38 (28).; anal. C 56.71; H 5.78%, calcd for C10H12O5, C 56.60; H 5.70%. General Procedure for the Synthesis of Polyalkoxybenzyl Chlorides (9b,d,e). Neat SOCl2 (8 mL, 110 mmol) was added dropwise to a stirred solution of polyalkoxybenzyl alcohol (43.3 mmol) in dry benzene (70 mL) at room temperature. The resulting mixture was warmed to 45−50 °C, stirred for 40 min, and concentrated in vacuo. Traces of SO2Cl2 were removed by coevaporation of the residue with benzene (50 mL) in vacuo. The resulting crude benzyl chloride (94−100% yield) was used for synthesis of the corresponding nitriles without further purification. General Procedure for the Synthesis of Polyalkoxyphenylacetonitriles (10b,d,e). A solution of crude chloride (above, 0.1 mol) in dry acetonitrile (120 mL) was treated with dibenzo-18-crown-6 (0.5 g, 1.4 mmol) followed by addition of dried KCN (15 g, 0.23 mol). The reaction mixture was stirred for 10 h at 50 °C, concentrated in vacuo, and treated with water (200 mL). The residue was filtered and recrystallized from EtOH to yield the corresponding nitrile. 3,4,5Trimethoxyphenylacetonitrile (10a) was purchased from Acros. 3-Methoxy-4,5-methylenedioxyphenylacetonitrile (10b): 16.3 g, 85% yield; light brown solid; mp 88−90 °C (lit.30 mp 89−90 °C); 1H NMR (DMSO-d6, 500 MHz) δ 6.65 (1H, d, J = 1.5 Hz, H-4), 6.60 (1H, d, J = 1.5 Hz, ArH-6), 6.00 (2H, s, OCH2O), 3.90 (2H, s, CH2), 3.83 (3H, s, OCH3); EIMS m/z 191 [M]+ (100), 190 (43), 176 (11), 160 (5), 146 (23), 133 (16), 90 (51), 78 (19), 77 (17), 64 (20), 63 E
dx.doi.org/10.1021/np400310m | J. Nat. Prod. XXXX, XXX, XXX−XXX
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2-(4-Methoxyphenyl)-3-oxo-3-(2,3-dimethoxy-4,5methylenedioxyphenyl)propanenitrile (11e): 1.5 g, 65% yield; offwhite solid; mp 122−124 °C; 1H NMR (CDCl3) δ 7.27 (2H, d, J = 8.8 Hz, H-2′,6′), 6.86 (2H, d, J = 8.8 Hz, H-3′,5′), 6.75 (1H, s, H-4″), 5.98 (2H, 2s, OCH2O), 5.86 (1H, s, HC−CN), 4.03 (3H, s, OCH3-4′), 3.90 (3H, s, OCH3-2″), 3.78 (3H, s, OCH3-3″) (Figure S5, Supporting Information); EIMS m/z 355 [M]+ (4), 209 (10), 194 (17), 166 (33), 165 (22), 146 (59), 123 (18), 121 (14), 103 (27), 95 (17), 93 (16), 91 (28), 77 (29), 76 (25), 65 (86); anal. C 64.36; H 4.88; N 3.78%, calcd for C19H17NO6, C 64.22; H 4.82; N 3.94%. 3-(4-Methoxyphenyl)-3-oxo-2-(3,4,5-trimethoxyphenyl)propanenitrile (13a): 2.21 g, 65% yield; yellow oil (lit.31 mp 94−96 °C); 1H NMR (CDCl3) δ 7.95 (2H, d, J = 8.6 Hz, H-2′,6′), 6.94 (2H, d, J = 8.6 Hz, H-3′,5′), 6.61 (2H, s, H-4″,6″), 5.46 (1H, s, HC−CN), 3.87 (3H, s, OCH3-4′), 3.85 (6H, s, 2 × OCH3-3″,5″), 3.83 (3H, s, OCH3-4″) (Figure S6, Supporting Information); EIMS m/z 341 [M]+; anal. C 66.92; H 5.70; N 4.01%, calcd for C19H19NO5, C 66.85; H 5.61; N 4.10%. This intermediate was used in the next steps without further purification. 3-(4-Methoxyphenyl)-3-oxo-2-(3-methoxy-4,5methylenedioxyphenyl)propanenitrile (13b): 1.8 g, 69% yield; offwhite solid; mp 121−123 °C; 1H NMR (CDCl3) δ 7.93 (2H, d, J = 9.0 Hz, H-2″,6″), 6.93 (2H, d, J = 9.0 Hz, H-3″,5″), 6.59 (2H, s, H-4′,6′), 5.97 (2H, s, OCH2O), 5.43 (1H, s, HC−CN), 3.89 (3H, s, OCH3-4″), 3.86 (3H, s, OCH3-3′) (Figure S7, Supporting Information); EIMS m/ z 325 [M]+ (1), 136 (13), 135 (100), 107 (17), 92 (10), 77 (16), 64 (6); anal. C 66.51; H 5.68; N 4.24%, calcd for C18H15NO5, C 66.46; H 4.65; N 4.31%. 3-(4-Methoxyphenyl)-3-oxo-2-(2,5-dimethoxy-3,4methylenedioxyphenyl)propanenitrile (13d): 1.6 g, 72% yield; offwhite solid; mp 97−99 °C; 1H NMR (CDCl3) δ 7.95 (2H, d, J = 9.0 Hz, H-2″,6″), 6.92 (2H, d, J = 9.0 Hz, H-3″,5″), 6.56 (1H, s, H-6′), 5.98 and 6.00 (2H, 2s, OCH2O), 5.94 (1H, s, HC−CN), 3.99 (3H, s, OCH3-4″), 3.86 (3H, s, OCH3-2′), 3.83 (3H, s, OCH3-5′) (Figure S8, Supporting Information); EIMS m/z 355 [M]+ (16), 324 (2), 220 (6), 136 (10), 135 (100), 107 (7), 92 (12), 77 (14), 43 (36); anal. C 64.29; H 4.86; N 3.84%, calcd for C19H17NO6, C 64.22; H 4.82; N 3.94%. 3-(4-Methoxyphenyl)-3-oxo-2-(2,3-dimethoxy-4,5methylenedioxyphenyl)propanenitrile (13e): 1.9 g, 57% yield; offwhite solid; mp 118−120 °C; 1H NMR (CDCl3) δ 7.94 (2H, d, J = 9.0 Hz, H-2″,6″), 6.90 (2H, d, J = 9.0 Hz, H-3″,5″), 6.55 (1H, s, H-4′), 5.96 (1H, s, HC−CN), 5.89 and 5.93 (2H, 2s, OCH2O), 4.03 (3H, s, OCH3-4″), 3.89 (3H, s, OCH3-2′), 3.85 (3H, s, OCH3-3′) (Figure S9, Supporting Information); EIMS m/z 355 [M]+ (15), 324 (1), 220 (5), 136 (19), 135 (100), 107 (21), 104 (7), 92 (32), 77 (50), 64 (16); anal. C 64.32; H 4.88; N 3.82%, calcd for C19H17NO6, C 64.22; H 4.82; N 3.94%. General Procedure for the Synthesis of Diaryl-3-aminopyrazoles (12, 14). A suspension of α-cyanoketone (3 mmol) and hydrazine dihydrochloride (15 mmol) in EtOH (20 mL) was refluxed for 12−18 h with stirring until disappearance of the starting material (TLC). After cooling, the resulting precipitate was filtered, washed with ethanol, and dried to afford the targeted 3-aminopyrazole as a respective hydrochloride salt. Alternatively, if precipitation was not observed, the reaction solvent was removed in vacuo, and the residue was treated with saturated aqueous NaHCO3 and filtered. The resulting product was purified by SiO2 column chromatography (EtOAc−EtOH, 2:1) to afford 12a−e and 14a,b,d,e. 4-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3amine (12a): 0.18 g, 18% yield (12% for the last two steps); light yellow solid; mp 174−178 °C (lit.17 mp 169−171 °C); 1H and 13C NMR, see Tables 1 and 2 and Figures S10 and S11, Supporting Information; EIMS m/z 355 [M]+ (35), 340(11), 162 (13), 154 (10), 140 (10), 127 (11), 113 (12), 38 (34), 36 (100), 35 (20); anal. C 64.27; H 5.89; N 11.74%, calcd for C19H21N3O4, C 64.21; H 5.96; N 11.82%. 4-(4-Methoxyphenyl)-5-(3-methoxy-4,5-methylenedioxyphenyl)1H-pyrazol-3-amine (12b): 1.7 g, 40% yield (24% for the last two steps); off-white solid; mp 191−196 °C; 1H and 13C NMR, see Tables 1 and 2 and Figures S12−S15, Supporting Information; EIMS m/z 339 [M]+ (21), 179(21), 140 (10), 139 (13), 133 (10), 77 (15), 76
(17), 75 (9), 63 (15), 53 (18), 51 (11), 44 (17), 43 (16), 38 (35), 36 (100); anal. C 63.86; H 5.14; N 12.44%, calcd for C18H17N3O4, C 63.71; H 5.05; N 12.38%. 4-(4-Methoxyphenyl)-5-(3-methoxy-4,5-ethylenedioxyphenyl)1H-pyrazol-3-amine (12c): 0.77 g, 65% yield (42% for the last two steps); light brown solid; mp 216−218 °C; 1H and 13C NMR, see Tables 1 and 2 and Figures S16−S20, Supporting Information; EIMS m/z 353 [M]+ (100), 226 (17), 77 (16), 44 (30), 43 (37), 39 (16), 32 (89), 29 (57); anal. C 64.66; H 5.36; N 11.96%, calcd for C19H19N3O4, C 64.58; H 5.42; N 11.89%. 4-(4-Methoxyphenyl)-5-(2,5-dimethoxy-3,4-methylenedioxyphenyl)-1H-pyrazol-3-amine (12d): 0.07 g, 22% yield (21% for the last two steps); light brown solid; 1H and 13C NMR, see Tables 1 and 2 and Figures S21 and S22, Supporting Information; EIMS m/z 369 [M]+ (68), 238 (20), 210 (23), 198 (51), 194 (22), 184 (31), 170 (49), 169 (46), 168 (63), 156 (24), 155 (25), 154 (34), 153 (34), 152 (21), 148 (32), 147 (43), 146 (100), 142 (23), 141 (36), 140 (41), 139 (41), 135 (27), 134 (23), 128 (24), 127 (33), 126 (33), 125 (22), 116 (29), 115 (30), 83 (28), 77 (53), 76 (33), 75 (20), 69 (39), 63 (26), 45 (27), 44 (30), 43 (62), 39 (28); anal. C 61.87; H 5.24; N 11.45%, calcd for C19H19N3O5, C 61.78; H 5.18; N 11.38%. 4-(4-Methoxyphenyl)-5-(2,3-dimethoxy-4,5-methylenedioxyphenyl)-1H-pyrazol-3-amine (12e): 0.21 g, 24% yield (10% for the last two steps); light brown solid; mp 182−188 °C; 1H and 13C NMR, see Tables 1 and 2 and Figures S23− S26, Supporting Information; EIMS m/z 369 [M]+ (100), 154 (11), 146 (29), 43 (15); anal. C 61.92; H 5.29; N 11.47%, calcd for C19H19N3O5:, C 61.78; H 5.18; N 11.38%. 5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3amine (14a): 0.73 g, 68% yield (44% for the last two steps); brown solid; mp 162−165 °C (lit.17 mp 174−176 °C); 1H and 13C NMR, see Tables 1 and 2 and Figures S27−S30, Supporting Information; EIMS m/z [M]+ 355; anal. C 64.15; H 5.88; N 11.90%, calcd for C19H21N3O4, C 64.21; H 5.96; N 11.82%. 5-(4-Methoxyphenyl)-4-(3-methoxy-4,5-methylenedioxyphenyl)1H-pyrazol-3-amine (14b): 0.45 g, 55% yield (38% for the last two steps); light brown solid; mp 128−132 °C; 1H and 13C NMR, see Tables 1 and 2 and Figures S31−S34, Supporting Information; EIMS m/z 339 [M]+ (100), 223(12), 167 (12), 166 (11), 140 (16), 139 (15), 53 (18), 43 (16); anal. C 63.66; H 5.11; N,12.49%, calcd for C18H17N3O4, C 63.71; H 5.05; N 12.38%. 5-(4-Methoxyphenyl)-4-(2,5-dimethoxy-3,4-methylenedioxyphenyl)-1H-pyrazol-3-amine (14d): 0.56 g, 35% yield (25% for the last two steps); light brown solid; mp 132−134 °C; 1H and 13C NMR, see Tables 1 and 2 and Figures S35−S38, Supporting Information; EIMS m/z 369 [M]+ (35), 135 (100), 77 (19), 36 (16), 32 (36); anal. C 61.65; H 5.07; N 11.44%, calcd for C19H19N3O5, C 61.78; H 5.18; N 11.38%. 5-(4-Methoxyphenyl)-4-(2,3-dimethoxy-4,5-methylenedioxyphenyl)-1H-pyrazol-3-amine (14e): 0.17 g, 15% yield (9% for the last two steps); light brown solid; 1H and 13C NMR, see Tables 1 and 2 and Figures S39 and S40, Supporting Information; EIMS m/z [M]+ 369; anal. C 61.85; H 5.10; N 11.31%, calcd for C19H19N3O5, C 61.78; H 5.18; N 11.38%. Sea Urchin Embryo Assay (ref 20). Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean Sea on the Cyprus coast in March−May and October−December, 2012, and kept in an aerated seawater tank. Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater and fertilized by adding drops of diluted sperm. Embryos were cultured at room temperature under gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The embryos were observed with a Biolam light microscope (LOMO, St. Petersburg, Russia). For treatment with the test compounds, 5 mL aliquots of embryo suspension were transferred to six-well plates and incubated as a monolayer at a concentration up to 2000 embryos/mL. Stock solutions of compounds were prepared in DMSO at 10 mM concentration followed by a 10-fold dilution with 95% EtOH. This procedure enhanced the solubility of the test compounds in the salt-containing medium (seawater), as evidenced by F
dx.doi.org/10.1021/np400310m | J. Nat. Prod. XXXX, XXX, XXX−XXX
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microscopic examination of the samples. The maximal tolerated concentrations of DMSO and EtOH in the in vivo assay were determined to be 0.05% and 1%, respectively. Higher concentrations of either DMSO (≥0.1%) or EtOH (>1%) caused nonspecific alteration and retardation of the sea urchin embryo development independent of the treatment stage. Combretastatins A-4 and A-2 (synthesized according to ref 18) served as reference compounds. The antiproliferative activity was assessed by exposing fertilized eggs (8−20 min after fertilization, 45−55 min before the first mitotic cycle completion) to 2-fold decreasing concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5−5.5 h after fertilization. The effects were estimated quantitatively as an effective threshold concentration, resulting in cleavage alteration and embryo death before hatching or full mitotic arrest. At these concentrations all tested microtubule destabilizers caused 100% cleavage alteration and embryo death before hatching, whereas at 2-fold lower concentrations the compounds failed to produce any effect. For microtubuledestabilizing activity, the compounds were tested on free-swimming blastulae just after hatching (8−10 h after fertilization), which originated from the same embryo culture. Embryo spinning was observed after 15 min to 20 h of treatment, depending on the structure and concentration of the compound. Both spinning and lack of forward movement were interpreted to be the result of the microtubule-destabilizing activity of a molecule. Video illustrations are available at http://www.chemblock.com. Both sea urchin embryo assay and DTP NCI60 cell line activity data are available free of charge via the Internet at http://www.zelinsky.ru.
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ASSOCIATED CONTENT
S Supporting Information *
H NMR spectra of α-cyanoketones 11 and 13 (Figures S1− S9). 1H, 13C, 1H−1H NOESY, 1H−13C HMBC, and 1H−13C HSQC NMR spectra of 3-aminopyrazoles 12 and 14 (Figures S10−S40). Cell growth inhibition of compounds 12a and 14b in 60 human cancer cell lines (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org. 1
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AUTHOR INFORMATION
Corresponding Author
*Tel: +7 916 620 9584. Fax: +7 499 137 2966. E-mail: vs@ zelinsky.ru. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant from Chemical Block Ltd. We thank the National Cancer Institute (NCI) (Bethesda, MD, USA) for screening compounds 12a, 12b, 12c, 12e, 14a, and 14b by the Developmental Therapeutics Program at NCI (Anticancer Screening Program; http://dtp.cancer.gov).
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REFERENCES
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