European Journal of Medicinal Chemistry 87 (2014) 421e428
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of 6-phenylaminopurines Camarasa a, Eva María Priego a, María-Dolores Canela a, Sandra Liekens b, María-Jose a , * rez-Pe rez María-Jesús Pe a b
Instituto de Química M edica (IQM-CSIC), c/Juan de la Cierva 3, E-28006 Madrid, Spain KU Leuven e University of Leuven, Rega Institute for Medical Research, Minderboedersstraat 10 blok x e bus 1030, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 9 September 2014 Accepted 29 September 2014 Available online 30 September 2014
A series of novel 6-phenylaminopurines have been efficiently synthesized in 3 steps exploring different groups at positions 2, 8 and 9 of the purine ring and at the exocyclic nitrogen atom at position 6. Among the newly described purines, five compounds showed antiproliferative activity with IC50 values below 10 mM, the tetrahydroquinoline derivative at position 6 of phenylaminopurine being the most active of the series in the six cell lines tested. Moreover, the compounds induced G2/M phase arrest in human cervical carcinoma HeLa cells as reported for tubulin depolymerizing agents. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Purines Microwave-assisted synthesis Antiproliferative agents Cell cycle analysis
1. Introduction Microtubules are cytoskeletal filaments consisting of a,b-tubulin heterodimers and are involved in a wide range of fundamental cellular processes, such as formation and maintenance of cell shape, regulation of motility, cell signaling, secretion, intracellular transportation, and cell division and mitosis [1,2]. Due to the different functions of microtubules during cell cycle progression, tubulin has become an attractive target for the discovery of anticancer drugs. For instance, taxoids and vinca alkaloids, first-line therapies for a large variety of tumors, exert their antitumor action by altering microtubule dynamics, either by promoting the course of microtubule assembly or disassembly. Indeed taxoids and vinca alkaloids target two distinct binding sites at the a,b-tubulin heterodimers: while the taxol site is located at the b subunit, the vinca site is situated at the interface of two heterodimers [3,4]. Despite of the clinical efficacy of these drugs, they have serious deficiencies, including narrow therapeutic indexes and emergence of drug resistance, mainly mediated by P-glycoprotein (P-gp) or bIII-tubulin overexpression [5,6]. Another serious disadvantage is their poor water solubility, which forces them to be administered
Abbreviations: HeLa, human cervical carcinoma cells; MEP, molecular electrostatic potential; MW, microwave irradiation; P-gp, P-glycoprotein. * Corresponding author. rez-Pe rez). E-mail address: [email protected] (M.-J. Pe http://dx.doi.org/10.1016/j.ejmech.2014.09.093 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
with a surfactant that may cause hypersensibility reactions and long-time administration [7]. A third binding site in tubulin, where colchicine specifically binds, has gained prominence in the last few years. The colchicine binding site is located at the interface of the a and b-subunits [8] and compounds binding at this site act as microtubule depolymerization agents [9,10]. Although colchicine itself cannot be used in the treatment of cancer owing to its high toxicity [11], a number of compounds binding at this site such as CA-4P, ZD6126, AVE8062, ABT-751 and MPC-6827 are in clinical trials for antitumor indications [9]. It should be mentioned that until now no P-glycoprotein (Pgp) or bIII-tubulin overexpression mediated resistance phenomena have been described with the best studied colchicine site binders [9,12]. Moreover, compounds binding at the colchicine site in tubulin have an additional value as anticancer agents since many of them have shown vascular disrupting properties targeting the tumor endothelium [13,14]. Among the colchicine-binding site compounds, we have centered our attention on MPC-6827 (1, Fig. 1), a quinazoline derivative identified through an anticancer screening apoptosis platform. Studies performed with MPC-6827 have shown that the compound binds at the colchicine binding site in tubulin [15]. This compound, also named Verubulin or Azixa, and whose structure significantly differs from colchicine or combretastatins, is currently undergoing clinical evaluation for the treatment of malignancies such as glioblastoma multiforme [16,17]. Other quinazoline
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Fig. 1. Chemical structure of MPC-6827 (1) and related compounds 2 and 3.
Scheme 2. Reagents and conditions: (a) CH(OMe)3, EtSO3H, Ac2O, MW, 120 C, 15 min; (b) 4-methoxy-N-methylaniline, HCl, ¡PrOH, MW, 80 C, 10 min.
2. Results and discussion 2.1. Chemistry
Fig. 2. Theoretical cLogP values [25] of the pyrrole[2,3-d]pyrimidine (A), cyclopenta[d] pyrimidine (B) and purine (C) scaffolds.
derivatives continue to be explored as antimitotic and vascular disrupting agents [18e21]. Interestingly, Gangjee et al. have reported on some pyrimidine derivatives fused to a 5 membered ring (a pyrazole such as in compound 2, or a cyclopentane as in compound 3 (Fig. 1)), that have also shown microtubule depolymerization properties by binding at the colchicine site and are characterized by cancer cell growth inhibitory activity. The substitution pattern in these compounds very much resembles the one in MPC-6827 [22]. A comparison of the central scaffold present in compounds 1, 2 and 3 led us to propose, as a simplistic example of “scaffold hoping” [23], that replacement of such scaffolds by a purine ring [24] might allow a similar distribution of the outsider substituents while solubility could be improved due to the inclusion of an imidazole at the central core. A calculation of the logP values [25] of the skeleton present in compounds 2 and 3, and a comparison with that of a purine ring (Fig. 2), supported a positive contribution of the latest to an increase in solubility. Therefore we have addressed the synthesis and antiproliferative evaluation of a series of 6-phenylaminopurine derivatives towards tumor and endothelial cells. In addition, cell-cycle analysis has been performed to establish the mechanism of action of this family of purines.
Starting from 2-amino-4,6-dichloro-2-methylpyrimidine (4) a first series of 6-phenylaminopurines were obtained in just 3 steps (Scheme 1). Thus, reaction of 4 with aqueous methylamine in 1,4dioxane at 80 C overnight afforded the pyrimidine 5 in an excellent yield, considerably better than previously described [26]. Then, reaction of 5 with triethyl orthoacetate in the presence of ethanesulphonic acid and acetic anhydride under MW irradiation for 15 min [27e29] afforded the 8-methylpurine 6. Finally, reaction of 6 with substituted anilines in isopropanol in the presence of HCl at 80 C afforded the 6-phenylamino derivatives 7aee, in moderate to good yields (65e90%). Under the same conditions, reaction of the 6chloropurine 8 [30] with 4-methoxy-N-methylaniline afforded compound 7f. Similarly, when compound 5 (Scheme 2) reacted with trimethyl orthoformate, the purine 9 was obtained, that was further treated with 4-methoxy-N-methylaniline to yield the 8unsubstituted compound 10. In addition, the unsubstituted compound at N-9 was obtained as depicted in Scheme 3. Reaction of the 4,6-dichloropyrimidine 4 with p-methoxybenzylamine afforded the benzyl derivative 11 in 64% yield. Then, reaction of 11 with triethyl orthoacetate led to the 8-methyl purine 12 in modest yield (31%). Finally, reaction with 4-methoxy-N-methylaniline, followed by catalytic hydrogenation to remove the p-methoxybenzyl group, afforded the purine 14 in 64% yield. According to some recent papers on pyrazolopyrimidines related to compounds 2 and 3 (Fig. 2), the introduction of an ethyl group at the NH at position 6 of the purine ring, or even the replacement of the N-methylaniline by a tetrahydroquinoline, might lead to an improvement in the antiproliferative activity [31]. Therefore reaction of 7b with ethyl iodide in the presence of CsCO3 [32] afforded the N-ethyl derivative 15 (Scheme 4). On the other
Scheme 1. Reagents and conditions: (a) CH3NH2, 1,4-dioxane, 80 C, overnight; (b) CH3C(OEt)3, EtSO3H, Ac2O, MW, 120 C, 15 min; (c) the corresponding aniline, HCl, iPrOH, MW, 80 C, 10 min.
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Scheme 3. Reagents and conditions: (a) 4-methoxybenzylamine, K2CO3, DMF, MW, 150 C, 1 h; (b) CH(OEt)3. EtSO3H, Ac2O, MW, 120 C, 15 min; (b) 4-methoxy-N-methylaniline, HCl, ¡ PrOH, MW, 80 C, 10 min; (d) H2, Pd(C), HCl, MeOH, 24 h.
Scheme 4. Reagents and conditions: (a) EtI, Cs2CO3, DMF, 60 C, 2 h; (b) 6-methoxy1,2,3,4-tetrahidroquinoline, HCl, iPrOH, 80 C, 12 h.
hand, reaction of the 6-chloropurine 6 with 6-methoxy-1,2,3,4tetrahydroquinoline allowed the synthesis of compound 16 in moderate yield. 2.2. Biological evaluation 2.2.1. Antiproliferative activity The synthesized compounds were evaluated for their antiproliferative activity (Table 1) in six different cell lines: three endothelial cell lines (HMEC-1, MBEC and BAEC) and three tumor cell lines (L1210, CEM and HeLa) [33,34]. Data are expressed as the 50% inhibitory concentration (IC50), defined as the concentration at which the compounds reduced cell proliferation by 50%. Colchicine was included as a reference compound. It should be mentioned that MPC-6827 has been reported to inhibit cancer cell growth with GI50 values of 3e6 nM in T47D, HCT116 and SN-398 cells [18]. A very
recent article describes the antiproliferative testing of MPC-6827 against a wider panel of tumor cells with IC50 values around 1 nM [20]. Among the synthesized purines, compounds 7a, 10, 14e16 showed antiproliferative activity in endothelial and tumor cell lines in the low mM range. The lack of antiproliferative activity of the compounds where the N-methylaniline at position 6 carries a substituent different from 4-OCH3 (compounds 7bee) strongly indicates that the structural requirements among these purines for antiproliferative activity very much resemble those reported for quinazolines and fused pyrimidines. Notably, even the replacement of the p-OCH3 group in 7a by a p-CH3 (7d) resulted in an inactive compound. In terms of substitution at position 6, the tetrahydroquinoline derivative (16) afforded the best cytostatic activity. Regarding the purine scaffold, a methyl group seemed to be crucial at position 2 (based on the lack of activity of compound 7f), while no significant differences were observed when a methyl or a hydrogen was present at position 8 or 9. However, compared to the previously described compounds (1e3), the incorporation of the imidazole at the central core appeared to negatively affect the antiproliferative activity since the best compounds in the purine series were between 10- to 1000-fold less active than the parent scaffolds. 2.2.2. Cell cycle inhibition Tubulin polymerization inhibitors are characterized by arresting cell cycle at G2/M phase [4]. Thus, the effect of four of the synthesized compounds (7a, 14, 15 and 16) on cell cycle of HeLa cells was analyzed by flow cytometry [35]. For comparative purposes, colchicine was included as a positive control. After 24 h of treatment, DNA was stained with propidium iodide and the intensity of the fluorescence was measured by flow cytometry [36]. The results obtained are summarized in Table 2, as the percentage of cells detected in each phase of the cell cycle with or without inhibitor (control). Untreated cells showed the classical pattern for proliferating cells distributed in the sub-G1, G1, S and G2/M phase, with 31%
Table 1 Antiproliferative activity of compounds 7aef, 10 and 14e16 in endothelial and tumor cell lines. Comp.
7a 7b 7c 7d 7e 7f 10 14 15 16 Colchicine
Endothelial cells IC50 (mM)
Tumor cells IC50 (mM)
HMEC-1
MBEC
BAEC
L1210
CEM
HeLa
7.3 ± 0.1 100 100 >100 >100 100 9.2 ± 1.3 7.1 ± 1.0 8.8 ± 2.1 2.3 ± 0.1 0.0038 ± 0.0011
8.7 ± 0.1 100 100 >100 >100 100 15 ± 7 8.9 ± 0.3 15 ± 0.4 4.4 ± 0.3 0.031 ± 0.015
7.4 ± 0.3 100 100 >100 >100 100 9.3 ± 0.6 8.9 ± 1.6 11 ± 1.4 2.4 ± 0.1 0.0069 ± 0.0008
19 ± 1 250 >250 250 >250 >250 30 ± 1 229 ± 30 35 ± 0 5.1 ± 0.1 0.014 ± 0.001
6.2 ± 1.8 >250 >250 164 ± 54 132 ± 47 >250 21 ± 3 23 ± 0 16 ± 1 3.5 ± 2.1 0.031 ± 0.019
4.5 ± 0.3 197 ± 14 >250 250 188 ± 25 >250 22 ± 1 14 ± 5 20 ± 0 4.4 ± 0.4 0.028 ± 0.000
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Table 2 Distribution of HeLa cells in different phases of the cell cycle after treatment with DMSO (0.1%) as control, colchicine and the inhibitors 7a, 14, 15 and 16. Compound
Control Colchicine 7a 14 15 16
Dose (mM)
0.1 20 100 20 100 20 100 4 20 100
Percentage of cells (%) Sub-G1
G1
S
G2/M
1 6 39 14 5 11 6 36 20 7 8
45 5 18 8 39 6 39 12 35 3 3
24 10 29 15 27 15 27 18 18 8 8
31 80 25 63 31 68 28 34 27 80 79
of the cells found in this latest phase. On the other hand, and as a positive control, cells treated with colchicine (0.1 mM) showed an accumulation in the G2/M phase with a percentage as high as 80%. When cells were treated with compounds 7a or 14 at 100 mM, a clear accumulation in the G2/M phase was induced (63% and 68% of the cells respectively), a behavior closely similar to that observed with colchicine. Treatment of the HeLa cells with the tetrahydroquinoline derivative 16 led to a similar pattern with 80% of the cells in G2/M phase even at a concentration of 20 mM, which correlates well with the higher antiproliferative activity showed by this compound. Although compound 15 did not induce cell cycle arrest in G2/M phase, at 100 mM the compound caused an accumulation of cells in sub-G1 phase, suggesting a plausible proapoptotic effect. This also occurred after the treatment with compound 7a at 20 mM or even with compound 16 at 4 mM. The
subdiploid DNA content of cells that increased from 1% in control cells to 20e40 % in the treated ones may point to the presence of apoptotic cells which have suffered nuclear fragmentation. MPC6827 (1) showed the same behavior, although at lower concentrations (5 nM), when tested in a similar experiment, inducing G2/ M phase arrest after 24 h and induction of apoptosis after 48 h in T47D human breast cancer cells [18]. 2.3. Maps of electrostatic potential In order to gain some insights that might help to explain the differences observed in antiproliferative activity among the quinazoline 1, the fused pyrimidines 2 and 3, and the here described purines (exemplified by compound 7a), molecular electrostatic potential (MEP) maps were calculated for the four compounds. The geometry of the compounds was first optimized by means of the ab initio program Gaussian 09, and the MEP maps were calculated based on the optimized structures using the program DelPhi. It should be mentioned that the geometries of the optimized structures are very similar as shown in the Supporting Fig. 1, allowing a similar distribution of the substituents as hypothesized in the introduction. Concerning the MEP maps (Fig. 3), the overall distribution is quite similar, although a more careful analysis shows that compounds 1 and 3 show a higher degree of similarity in terms of localized negative and positive potential regions when compared to compounds 2 and 7a. Interestingly, and despite the precaution to compare IC50 values from different origin, compounds 1 and 3 are more cytostatic than compounds 2 and 7a. When comparing compounds 2 and 7a, the most significant differences affect the negative region (represented in pink) around position 7 of the purine, a region that has a positive value in the pyrazolopyrimidine 2 (represented in light blue). So, it may be proposed that a negative
Fig. 3. MEP maps of compounds 1e3 and 7a. Positive and negative isopotential surfaces are colored in cyan and pink, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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electrostatic potential at this site is detrimental for activity, while the distribution of MEPs obtained for compounds 1 and 3 may lead to more active compounds. Obviously, many other factors including crossing the membranes, metabolic stability etc … may also account for the different IC50 values, but these differences in MEP maps may be relevant when facing the design of new compounds.
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purified by flash chromatography (dichloromethane/methanol) to yield 370 mg (95%) of 5 as a white solid. Mp 129e131 C. MS (ES, positive mode): m/z 173 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.23 (s, 3H, CH3-2), 2.86 (d, J ¼ 4.4 Hz, 3H, CH3eN9), 4.74 (br s, 2H, NH2), 6.87 (br s, 1H, NH). A similar synthetic procedure but with a lower yield has been recently described [26].
3. Conclusions In summary, we here report the synthesis and antiproliferative activity of a novel series of 6-phenylaminopurines. Among them, compounds 7a, 10, and 14e16 exhibited antiproliferative activity in the low mM range, in endothelial and tumor cells. Importantly, compound 16, with a tetrahydroquinoline substitution at position 6, showed IC50 values around 2e5 mM in the six cell lines tested. Furthermore, these purine analogs induced a G2/M cell cycle arrest in HeLa cells, a behavior also observed with other colchicine-site binders in tubulin, such as MPC-6827. A comparison of the MEP maps of compound 7a with those generated for the previously described quinozaline and pyrimidine-fused analogs may help to explain the differences observed in terms of antiproliferative activity. 4. Experimental 4.1. Synthesis Melting points were obtained on a Mettler Toledo M170 apparatus and are uncorrected. The elemental analysis was performed with a Heraeus CHNeO-RAPID instrument. The elemental compositions of the compounds agreed to within ±0.4% of the calculated values. For all the tested compounds, satisfactory elemental analysis was obtained supporting >95% purity. Electrospray mass spectra were measured on a quadruple mass spectrometer equipped with an electrospray source (HewlettePackard, LC/MS HP 1100). 1H and 13C NMR spectra were recorded on a Varian INNOVA300 operating at 299 MHz (1H) and 75 MHz (13C), respectively, a Varian INNOVA-400 operating at 399 MHZ (1H) and 99 MHz (13C), respectively, and a VARIAN SYSTEM-500 operating a 499 and 125 MHz, respectively. Analytical TLC was performed on silica gel 60 F254 (Merck) precoated plates (0.2 mm). Spots were detected under UV light (254 nm) and/or charring with ninhydrin or phosphomolybdic acid. Separations on silica gel were performed by preparative centrifugal circular thin-layer chromatography (CCTLC) on a ChromatotronR (Kiesegel 60 PF254 gipshaltig (Merck)), with layer thickness of 1 and 2 mm and flow rate of 4 or 8 mL/min, respectively. Flash column chromatography (HPFC) was performed in a Biotage Horizon instrument. Microwave reactions were performed using the Biotage Initiator 2.0 single-mode cavity instrument from Biotage (Uppsala). Experiments were carried out in sealed microwave process vials utilizing the standard absorbance level (400 W maximum power). The temperature was measured with an IR sensor on the outside of the reaction vessel. 4.1.1. 6-Chloro-N,2-dimethylpyrimidine-4,5-diamine (5) A solution of 5-amino-4,6-dichloro-2-methylpyrimidine (4) (400 mg, 2.25 mmol), in 1,4-dioxane (11 mL) was treated with a 40% solution of methylamine in water (742 mL, 8.62 mmol) and placed in an Ace pressure tube. The vessel was sealed and heated overnight at 80 C. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. Then it was
4.1.2. 6-Chloro-2,8,9-trimethyl-9H-purine (6) A microwave vial was charged with compound 5 (200 mg, 1.16 mmol), triethyl orthoacetate (0.63 mL, 1.74 mmol), ethanesulfonic acid (19 mL, 0.23 mmol) in acetic anhydride (5 mL). The vessel was sealed and microwave-irradiated at 120 C for 15 min. Dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and concentrated. The residue obtained was purified CCTLC employing dichloromethane:methanol (30:1) to obtain 135 mg (59%) of 6 as a white solid. Mp 122e124 C. MS (ES, positive mode): m/z 197 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.59 (s, 3H, CH3-2), 2.65 (s, 3H, CH3-8), 3.71 (s, 3H, CH3eN9). 4.1.3. General procedure for the reaction of the 6-chloropurines with anilines A microwave vial was charged with the corresponding 6chloropurine (1.0 mmol), the appropriate aniline (1.5 mmol) in isopropanol (4 mL) and 37% aqueous HCl (1.2 mmol). The reaction vessel was sealed and heated in a microwave reactor at 80 C for 10 min. After cooling, dichloromethane was added (20 mL) and the mixture was washed with saturated aqueous NaHCO3 (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The resulting residue was purified as specified. 4.1.3.1. N-(4-methoxyphenyl)-N,2,8,9-tetramethyl-9H-purin-6amine (7a). Following the general procedure, a microwave vial was charged with compound 6 (98 mg, 0.50 mmol), 4-methoxy-Nmethylaniline (103 mg, 0.75 mmol) and 37% aqueous HCl (150 mL) in isopropanol (2 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 40:1) to yield 134 mg (90%) of 7a as a rosy solid. Mp 127e129 C. MS (ES, positive mode): m/z 298 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.35 (s, 3H, CH32), 2.39 (s, 3H, CH3-8), 3.59 (s, 3H, CH3eN9), 3.74 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 6.93 (d, 2H, J ¼ 8.9 Hz, Ar), 7.20 (d, 2H, J ¼ 8.9 Hz, Ar). 13C NMR (DMSO-d6, 75 MHz) d: 13.5 (CH3-8), 25.7 (CH3-2), 28.2 (CH3eN9), 40.1 (CH3eN6), 55.2 (OCH3), 116.7 (C-5), 113.9, 127.6, 138.8, 156.9 (Ar), 147.9 (C-8), 151.7 (C-6), 152.1 (C-4), 159.0 (C-2). Anal. calc. for (C16H19N5O): C, 64.63; H, 6.44; N, 23.55. Found: C, 64.91; H, 6.72; N, 23.58. 4.1.3.2. N-(4-methoxyphenyl)-2,8,9-trimethyl-9H-purin-6-amine (7b). Following the general procedure, a microwave vial was charged with compound 6 (43 mg, 0.22 mmol), 4-methoxyaniline (43 mg, 0.34 mmol) and 37% aqueous HCl (66 mL) in isopropanol (1 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 46 mg (74%) of 7b as a white solid. Mp 204e206 C. MS (ES, positive mode): m/z 284 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.49 (s, 3H, CH3-2), 2.52 (s, 3H, CH3-8), 3.64 (s, 3H, CH3eN9), 3.73 (s, 3H, OCH3), 6.88 (d, 2H, J ¼ 9.0 Hz, Ar), 7.86 (d, 2H, J ¼ 9.0 Hz, Ar), 9.40 (br s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) d: 13.4 (CH3-8), 25.9 (CH3-2), 28.3 (CH3eN9), 55.1 (OCH3), 116.5 (C-5), 113.5, 121.7, 133.4, 154.5 (Ar), 149.1 (C-8), 150.5 (C-6), 151.4 (C-4), 159.6 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.46; H, 6.11; N, 24.57.
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4.1.3.3. N-(3-methoxyphenyl)-N,2,8,9-tetramethyl-9H-purin-6amine (7c). Following the general procedure, a microwave vial was charged with compound 6 (43 mg, 0.22 mmol), 3-methoxy-Nmethylaniline (44 ml, 0.34 mmol) and 37% aqueous HCl (66 mL) in isopropanol (1 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 42 mg (64%) of 7c as a white solid. Mp 131e133 C. MS (ES, positive mode): m/z 298 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.37 (s, 3H, CH32), 2.41 (s, 3H, CH3-8), 3.60 (s, 3H, CH3eN9), 3.74 (s, 3H, OCH3), 3.79 (s, 3H, CH3eN6), 6.79 (ddd, 1H, J ¼ 8.3, 2.5, 0.8 Hz, Ar), 6.86 (ddd, 1H, J ¼ 7.9, 1.9, 0.8 Hz, Ar), 6.89 (t, 1H, J ¼ 2.1 Hz, Ar), 7.26 (t, 1H, J ¼ 8.1 Hz, Ar). 13C NMR (DMSO-d6, 75 MHz) d: 13.5 (CH3-8), 25.7 (CH3-2), 28.3 (CH3eN9), 40.1 (CH3eN6), 55.1 (OCH3), 117.1 (C-5), 110.9, 111.9, 118.2, 129.2, 146.9, 159.4 (Ar), 148.4 (C-8), 152.3 (C-6), 153.2 (C-4), 159.0 (C-2). Anal. calc. for (C16H19N5O): C, 64.63; H, 6.44; N, 23.55. Found: C, 64.55; H, 6.31; N, 23.48. 4.1.3.4. N,2,8,9-tetramethyl-N-(p-tolyl)-9H-purin-6-amine (7d). Following the general procedure, a microwave vial was charged with compound 6 (80 mg, 0.41 mmol), N,4-dimethylaniline (78 ml, 0.62 mmol) and 37% aqueous HCl (123 mL) in isopropanol (2 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 40:1) to yield 79 mg (69%) of 7d, as a white solid. Mp 133e135 C. MS (ES, positive mode): m/z 282 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.32 (s, 3H, CH3-Ph), 2.35 (s, 3H, CH3-2), 2.41 (s, 3H, CH3-8), 3.60 (s, 3H, CH3eN9), 3.78 (s, 3H, CH3eN6), 7.17 (m, 4H, Ar). 13C NMR (DMSO-d6, 100 MHz) d: 13.4 (CH3-8), 20.6 (CH3-Ph), 25.7 (CH3-2), 28.2 (CH3eN9), 40.4 (CH3eN6), 116.9 (C-5), 125.9, 129.1, 134.3, 143.3 (Ar), 148.1 (C-8), 152.4 (C-6), 153.1 (C-4), 159.0 (C-2). Anal. calc. for (C16H19N5): C, 68.30; H, 6.81; N, 24.89. Found: C, 68.11; H, 6.57; N, 24.66.
4.1.4. 6-Chloro-2,9-dimethyl-9H-purine (9) A microwave vial was charged with a solution of compound 5 (500 mg, 2.90 mmol), trimethyl orthoformate (0.95 mL, 8.70 mmol) and ethanesulfonic acid (24 ml, 0.29 mmol) in acetic anhydride (12 mL). The vessel was sealed and microwave-irradiated at 120 C for 15 min. Dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by flash chromatography (dichloromethane:methanol 20:1) to yield 487 mg (92%) of 9 as a white solid. Mp 167e169 C. (m.p. lit [37] 168e171 C). MS (ES, positive mode): m/z 183 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.68 (s, 3H, 2CH3), 3.82 (s, 3H, CH3eN), 8.53 (s, 1H, H-8). 4.1.5. N-(4-Methoxyphenyl)-N,2,9-trimethyl-9H-purin-6-amine (10) Following the general procedure for the reaction of 6chloropurines with anilines (Section 4.1.3), a microwave vial was charged with compound 9 (500 mg, 2.74 mmol), 4-methoxy-Nmethylaniline (563 mg, 4.11 mmol) and 37% aqueous HCl (0.8 mL) in isopropanol (11 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 580 mg (75%) of 10 as a white solid. Mp 152e154 C. MS (ES, positive mode): m/z 284 (MþH)þ. 1H NMR (DMSO-d6, 500 MHz) d: 2.40 (s, 3H, CH3-2), 3.66 (s, 3H, CH3eN9), 3.69 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 6.93 (d, 2H, J ¼ 8.9 Hz, Ar), 7.22 (d, 2H, J ¼ 8.9 Hz, Ar), 7.91 (s, 1H, H-8). 13C NMR (DMSO-d6, 125 MHz) d: 25.8 (CH3-2), 29.3 (CH3eN9), 39.9 (CH3eN6), 55.2 (OCH3), 117.6 (C-5), 114.0, 127.9, 138.7, 157.2 (Ar), 140.4 (C-8), 151.9 (C-6), 153.6 (C-4), 160.0 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.47; H, 5.95; N, 24.61.
4.1.3.5. N,2,8,9-tetramethyl-N-(4-(trifluoromethoxy)phenyl)-9Hpurin-6-amine (7e). Following the general procedure, a microwave vial was charged with compound 6 (80 mg, 0.41 mmol), N-methyl4-(trifluoromethoxy)aniline (92 ml, 0.62 mmol) and 37% aqueous HCl (123 mL) in isopropanol (2 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 40:1) to yield 100 mg (69%) of 7e as a white solid. Mp 133e135 C. MS (ES, positive mode): m/z 352 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.39 (s, 3H, CH3-2), 2.40 (s, 3H, CH3-8), 3.61 (s, 3H, CH3eN9), 3.80 (s, 3H, CH3eN6), 7.34 (d, 2H, J ¼ 8.8 Hz, Ar), 7.45 (d, 2H, J ¼ 9.0 Hz, Ar). 13 C NMR (DMSO-d6, 100 MHz) d: 13.6 (CH3-8), 25.6 (CH3-2), 28.3 (CH3eN9), 40.1 (CH3eN6), 117.2 (C-5), 120.6 (q, J ¼ 256.0, CF3), 121.3, 127.7, 144.8, 145.3 (Ar), 148.8 (C-8), 152.2 (C-6), 153.3 (C-4), 159.1 (C-2). Anal. calc. for (C16H16F3N5O): C, 54.70; H, 4.59; N, 19.93. Found: C, 54.45; H, 4.38; N, 19.67.
4.1.6. 6-Chloro-N4-(4-methoxybenzyl)-2-methylpyrimidine-4,5diamine (11) A microwave vial was charged with 5-amino-4,6-dichloro-2methyl-pyrimidine (4) (700 mg, 3.91 mmol), 4methoxybenzylamine (510 ml, 3.91 mmol), and K2CO3 (810 mg, 5.86 mmol) in anhydrous DMF (10 mL). The vessel was sealed and irradiated at 150 C for 1 h. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NH4Cl (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by flash chromatography (dichloromethane:methanol 20:1) to yield 696 mg (64%) of 11 as a yellow solid. Mp 155e157 C. MS (ES, positive mode): m/z 279 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.52 (s, 3H, CH3-2), 2.68 (s, 3H, CH3-8), 3.71 (s, 3H, OCH3), 5.39 (s, 2H, CH2), 6.89 (d, 2H, J ¼ 8.6 Hz, Ar), 7.17 (d, 2H, J ¼ 8.5 Hz, Ar).
4.1.3.6. N-(4-methoxyphenyl)-N,8,9-trimethyl-9H-purin-6-amine (7f). Following the general procedure, a microwave vial was charged with compound 8 [30] (62 mg, 0.34 mmol), 4-methoxy-Nmethylaniline (70 mg, 0.51 mmol) and 37% aqueous HCl (100 mL) in isopropanol (1.4 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 54 mg (56%) of 7f as a rosy solid. Mp 172e174 C. MS (ES, positive mode): m/z 284 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.45 (s, 3H, CH38), 3.64 (s, 3H, CH3eN9), 3.78 (s, 3H, OCH3), 3.81 (s, 3H, CH3eN6), 6.95 (d, 2H, J ¼ 9.0 Hz, Ar), 7.21 (d, 2H, J ¼ 9.0 Hz, Ar), 8.11 (s, 1H, H2). 13C NMR (DMSO-d6, 100 MHz) d: 13.4 (CH3-8), 28.3 (CH3eN9), 40.2 (CH3eN6), 55.3 (OCH3), 118.4 (C-5), 114.0, 127.5, 138.5, 157.0 (Ar), 148.6 (C-8), 150.5 (C-6), 152.2 (C-4), 153.1 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.30; H, 5.99; N, 24.58.
4.1.7. 6-Chloro-9-(4-methoxybenzyl)-2,8-dimethyl-9H-purine (12) A microwave vial was charged with a solution of compound 11 (700 mg, 2.51 mmol), triethyl orthoacetate (1.37 mL, 7.53 mmol) and ethanesulfonic acid (41 ml, 0.50 mmol) in acetic anhydride (10 mL). The vessel was sealed and microwave-irradiated at 120 C for 15 min. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by flash chromatography (dichloromethane:methanol 20:1) to yield 232 mg of 12 (31%) as a pale brown oil. MS (ES, positive mode): m/z 303 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.52 (s, 3H, CH3-2), 2.68 (s, 3H, CH3-8), 3.71 (s, 3H, OCH3), 5.39 (s, 2H, CH2), 6.89 (d, 2H, J ¼ 8.6 Hz, Ar), 7.17 (d, 2H, J ¼ 8.5 Hz, Ar).
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4.1.8. 9-(4-Methoxybenzyl)-N-(4-methoxyphenyl)-N,2,8-trimethyl9H-purin-6-amine (13) Following the general procedure for the reaction of 6chloropurines with anilines (Section 4.1.3), a microwave vial was charged with compound 12 (200 mg, 0.66 mmol), 4-methoxy-Nmethylaniline (136 mg, 1.00 mmol) and 37% aqueous HCl (200 mL) in 2-propanol (2.6 mL). The residue was purified by flash chromatography (dichloromethane:methanol, 20:1) to yield 150 mg (56%) of 13 as a rosy oil. MS (ES, positive mode): m/z 404 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.31 (s, 3H, CH3-2), 2.36 (s, 3H, CH3-8), 3.71 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 5.26 (s, 2H, CH2), 6.91 (m, 4H, Ar), 7.18 (m, 4H, Ar).
(ES, positive mode): m/z 324 (MþH)þ. 1H NMR (DMSO-d6, 500 MHz) 1.90 (m, 2H, CH2-30 ), 2.38 (s, 3H, CH3-2), 2.48 (s, 3H, CH38), 2.78 (t, 2H, J ¼ 6.8 Hz, CH2-40 ), 3.63 (s, 3H, CH3eN9), 3.74 (s, 3H, OCH3), 4.43 (m, 2H, CH2-20 ), 6.72 (d, 1H, J ¼ 2.8 Hz, Ar), 6.68 (dd, 1H, J ¼ 8.9, 3.0 Hz, Ar), 7.40 (d, 1H, J ¼ 8.9 Hz, Ar). 13C NMR (DMSO-d6, 125 MHz) d: 13.6 (CH3-8), 23.3 (CH2-30 ), 25.6 (CH3-2), 26.3 (CH2-40 ), 28.3 (CH3eN9), 46.1 (CH2-20 ), 55.1 (OCH3), 117.4 (C-5), 111.2, 112.7, 125.8, 131.0, 133.0, 155.1 (Ar), 148.6 (C-8), 151.5 (C-6), 153.4 (C-4), 159.0 (C-2). Anal. calc. for (C18H21N5O): C, 66.85; H, 6.55; N, 21.66. Found: C, 66.70; H, 6.38; N, 21.49.
4.1.9. N-(4-methoxyphenyl)-N,2,8-trimethyl-9H-purin-6-amine (14) A solution of compound 13 (80 mg, 0.20 mmol) in methanol (2 mL) at 0 C was treated with 37% aqueous HCl (200 mL) and 10% Pd/C (80 mg). The reaction mixture was stirred under hydrogen atmosphere at room temperature for 24 h. The reaction was filtered, washed with methanol and evaporated to dryness. The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 20:1) to yield 36 mg (64%) of 14 as a white solid. Mp 190e192 C. MS (ES, positive mode): m/z 285 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.31 (s, 3H, CH3-2), 2.34 (s, 3H, CH3-8), 3.74 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 6.93 (d, 2H, J ¼ 8.3 Hz, Ar), 7.21 (d, 2H, J ¼ 8.1 Hz, Ar), 12.46 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz) d: 15.2 (CH3-8), 26.1 (CH3-2), 40.1 (CH3eN6), 55.6 (OCH3), 117.9 (C-5), 114.3, 128.1, 139.3, 157.3 (Ar), 147.3 (C-8), 153.0 (C-6), 154.0 (C-4), 159.4 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.29; H, 5.94; N, 24.81.
4.2.1. Cell proliferation
4.1.10. N-ethyl-N-(4-methoxyphenyl)-2,8,9-trimethyl-9H-purin-6amine (15) A solution of compound 7b (60 mg, 0.21 mmol) and Cs2CO3 (208 mg, 0.64 mmol) in anhydrous DMF (2 mL) was stirred at room temperature for 30 min under argon atmosphere. Then, ethyl iodide (67 mL, 0.84 mmol) was added and the mixture was heated at 60 C for 2 h. After cooling, volatiles were removed and the mixture was further solved in dichloromethane (20 mL) and washed with brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 57 mg (87%) of 15 as a white solid. Mp 136e138 C. MS (ES, positive mode): m/z 312 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 1.14 (t, 3H, J ¼ 6.9 Hz, CH3CH2), 2.31 (s, 3H, CH3-2), 2.40 (s, 3H, CH3-8), 3.58 (s, 3H, CH3eN9), 3.78 (s, 3H, OCH3), 4.33 (q, 2H, J ¼ 6.9 Hz, CH2), 6.94 (d, 2H, J ¼ 8.9 Hz, Ar), 7.15 (d, 2H, J ¼ 8.9 Hz, Ar). 13C NMR (DMSO-d6, 75 MHz) d: 13.8 (CH3CH2), 13.9 (CH3-8), 26.1 (CH3-2), 28.6 (CH3eN9), 46.3 (CH2), 55.5 (OCH3), 116.6 (C-5), 114.3, 129.3, 137.2, 157.4 (Ar), 148.1 (C-8), 152.5 (C-6), 153.3 (C-4), 159.4 (C-2). Anal. calc. for (C17H21N5O): C, 65.57; H, 6.80; N, 22.49. Found: C, 65.28; H, 6.54; N, 22.19. 4.1.11. 6-Methoxy-1-(2,8,9-trimethyl-9H-purin-6-yl)-1,2,3,4tetrahydroquinoline (16) A solution of compound 6 (100 mg, 0.51 mmol) in 2-propanol (2 mL) was placed in an Ace pressure tube and 6-methoxy1,2,3,4-tetrahydroquinoline (125 mg, 0.77 mmol) and 37% aqueous HCl (150 mL) were added. The tube was then sealed and heated at 80 C overnight. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by CCTLC in the Chromatothron (hexane:ethyl acetate, 2:1) to yield 57 mg (35%) of 16 as a white solid. Mp 180e182 C. EM
4.2. Biological methods
4.2.1.1. Endothelial cells. Mouse brain endothelial cells (MBEC), bovine aortic endothelial cells (BAEC) and human dermal microvascular endothelial cells (HMEC-1) were seeded in 48-well plates at 10,000 cells/well (except HMEC-1 at 20,000/well). After 24 h, compounds were added at different concentrations (100, 20, 4, 0.8, 0.16 and 0.08 mM). The cells were allowed to proliferate for 3 days (or 4 days for HMEC-1) in the presence of the compounds, trypsinized, and counted by means of a Coulter counter (Analis, Belgium). 4.2.1.2. Tumor cells. Human cervical carcinoma (HeLa) cells were seeded in 96-well plates at 15,000 cells/well in the presence of different concentrations of the compounds. After 4 days of incubation, the cells were trypsinized and counted in a Coulter counter. Suspension cells (Mouse leukemia L1210 and human lymphoid Cem cells) were seeded in 96-well plates at 60,000 cells/well in the presence of different concentrations of the compounds. L1210 and Cem cells were allowed to proliferate for 48 h or 96 h, respectively and then counted in a Coulter counter. The 50% inhibitory concentration (IC50) was defined as the compound concentration required to reduce cell proliferation by 50%. Colchicine was added as reference compound. 4.2.2. Cell cycle analysis HeLa cells were seeded in 6-well plates at 125,000 cells/well in DMEM with 10% fetal bovine serum (FBS) for 24 h. Then, cells were exposed to different concentrations of the compounds. After 24 h, the DNA of the cells was stained with propidium iodide using the CycleTEST PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA). The DNA content of the stained cells was assessed by flow cytometry on a FACSCalibur flow cytometer and analyzed with CellQuest software (BD Biosciences) within 3 h after staining. Cell debris and clumps were excluded from the analysis by appropriate dot plot gating. Percentages of sub-G1, G1, S, and G2/M cells were estimated using appropriate region markers [35]. Colchicine was added as reference compound. 4.3. Computational methods The geometries of compounds 1e3 and 7a were optimized using the ab initio quantum chemistry programme Gaussian 09 [38] and the RHF/6-21G* basis set. Finite difference solutions to the linearized PoissoneBoltzman equation, as implemented in the Delphi software (v.4) [39,40] were used to calculate MEPs. For MEP calculations cubic grids with a resolution of 1.0 Å were centered on each molecule, and the atomic charges were distributed onto the grid points. AMBER charges and radii were used. Solvent-accessible surfaces, calculated with a spherical probe with a radius of 1.4 Å, defined the solute boundaries, and a minimum separation of 10 Å was left between any solute atom and the borders of the box. The interior of the ligands was considered a low-dielectric medium
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(ε ¼ 2) whereas the surrounding solvent was treated as a highdielectric medium (ε ¼ 80). Acknowledgments M.-D. C. thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral fellowship. This project has been supported by the Spanish CICYT (SAF2012-39760-C02-01 to M.-J.C., M.-J.P.-P. and E.-M.P.) and the Comunidad de Madrid (BIPEDD2; ref P2010/BMD-2457 to M.-J.C.). We also would like to acknowledge Eef Meyen and Lizette Van Berckelaer for excellent technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.09.093. These data include MOL files and InChiKeys of the most important compounds described in this article. References [1] M.A. Jordan, L. Wilson, Microtubules as target for anticancer drugs, Nat. Rev. Cancer 4 (2004) 253e265. [2] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic field of cancer therapeutics, Nat. Rev. Drug Discov. 9 (2010) 790e803. rez, Microtubule inhibitors: differentiating tubulin-inhibiting agents [3] E.A. Pe based on mechanism of action, clinical activity and resistance, Mol. Cancer Ther. 8 (2009) 2086e2895. [4] R.A. Stanton, K.M. Gernert, J.H. Nettles, R. Aneja, Drugs that target dynamic microtubules: a new molecular perspective, Med. Res. Rev. 31 (2011) 443e481. [5] T. Litman, T.E. Druley, W.D. Stein, S.E. Bates, From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance, Cell. Mol. Life Sci. 58 (2001) 931e959. [6] C. Stengel, S.P. Newman, M.P. Leese, B.V.L. Potter, M.J. Reed, A. Purohit, Class III beta-tubulin expression and in vitro resistance to microtubule targeting agents, Br. J. Cancer 102 (2010) 316e324. [7] G. Gaucher, R.H. Marchessault, J.-C. Leroux, Polyester-based micelles and nanoparticles for the parenteral delivery of taxanes, J. Control Release 2 (2010) 2e12. [8] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, M. Knossow, Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain, Nature 428 (2004) 198e202. [9] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An overview of tubulin inhibitors that interact with the colchicine binding site, Pharm. Res. 29 (2012) 2943e2971. [10] B. Bhattacharyya, D. Panda, S. Gupta, M. Banerjee, Anti-mitotic activity of colchicine and the structural basis for its interaction with tubulin, Med. Res. Rev. 28 (2008) 155e183. [11] Y. Finkelstein, S.E. Aks, J.R. Hutson, D.N. Juurlink, P. Nguyen, G. Dubnov-Raz, U. Pollak, G. Koren, Y. Bentur, Colchicine poisoning: the dark side of an ancient drug, Clin. Toxicol. 48 (2010) 407e414. [12] Q.X. Yue, X. Liu, D.A. Guo, Microtubule-binding natural products for cancer therapy, Planta Med. 76 (2010) 1037e1043. [13] P.E. Thorpe, Vascular targeting agents as cancer therapeutics, Clin. Cancer Res. 10 (2004) 415e427. [14] M.R. Horsman, D.W. Siemann, Pathophysiologic effects of vascular-targeting agents and the implications for combination with conventional therapies, Cancer Res. 66 (2006) 11520e11539. [15] S. Kasibhatla, V. Baichwal, S.X. Cai, B. Roth, I. Skvortsova, S. Skvortsov, P. Lukas, N.M. English, N. Sirisoma, J. Drewe, A. Pervin, B. Tseng, R.O. Carlson, C.M. Pleiman, MPC-6827: a small-molecule inhibitor of microtubule formation that is not a substrate for multidrug resistance pumps, Cancer Res. 67 (2007) 5865e5871. [16] K.F. Grossmann, H. Colman, W.A. Akerley, M. Glantz, Y. Matsuoko, A.P. Beelen, M. Yu, J.F.D. Groot, R.D. Aiken, J.J. Olsen, B.A. Evans, R.L. Jensen, Phase I trial of verubulin (MPC-6827) plus carboplatin in patients with relapsed glioblastoma multiforme, J. Neurooncol. 110 (2012) 257e264. [17] M.C. Chamberlain, S. Grimm, S. Phuphanich, L. Recht, J.Z. Zhu, L. Kim, S. Rosenfeld, C.E. Fadul, A phase 2 trial of verubulin for recurrent glioblastoma: a prospective study by the brain tumor investigational consortium (BTIC), J. Neurooncol. 118 (2014) 335e343. [18] N. Sirisoma, A. Pervin, H. Zhang, S. Jiang, J.A. Willardsen, M.B. Anderson, G. Mather, C.M. Pleiman, S. Kasibhatla, B. Tseng, J. Drewe, S.X. Cai, Discovery of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a potent apoptosis inducer and efficacious anticancer agent with high blood brain barrier penetration, J. Med. Chem. 52 (2009) 2341e2351.
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Risinger, E. Hamel, S.L. Mooberry, Synthesis and discovery of water-soluble microtubule targeting agents that bind to the colchicine site on tubulin and circumvent Pgp mediated resistance, J. Med. Chem. 53 (2010) 8116e8128. € hm, A. Flohr, M. Stahl, Scaffold hoping, Drug Discov. Today Technol. 1 [23] H.-J. Bo (2004) 217e224. [24] M.E. Welsch, S.A. Snyder, B.R. Stockwell, Privileged scaffolds for library design and drug discovery, Curr. Opin. Chem. Biol. 14 (2010) 347e361. [25] ChemBioDraw 12.0 (CambridgeSoft, Inc). [26] S.P. Hollinshead, M.W. Tidwell, J. Palmer, R. Guidetti, A. Sanderson, M.P. Johnson, M.G. Chambers, J. Oskins, R. Stratford, P.C. Astles, Selective cannabinoid receptor type 2 (CB2) agonists: optimization of a series of purines leading to the identification of a clinical candidate for the treatment of osteoarthritic pain, J. Med. Chem. 56 (2013) 5722e5733. rez-Pe rez, Microwave-assisted synthesis of 9[27] L. Aguado, M.J. Camarasa, M.J. Pe arylpurines, J. Comb. 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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of 6-phenylaminopurines Camarasa a, Eva María Priego a, María-Dolores Canela a, Sandra Liekens b, María-Jose a , * rez-Pe rez María-Jesús Pe a b
Instituto de Química M edica (IQM-CSIC), c/Juan de la Cierva 3, E-28006 Madrid, Spain KU Leuven e University of Leuven, Rega Institute for Medical Research, Minderboedersstraat 10 blok x e bus 1030, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 9 September 2014 Accepted 29 September 2014 Available online 30 September 2014
A series of novel 6-phenylaminopurines have been efficiently synthesized in 3 steps exploring different groups at positions 2, 8 and 9 of the purine ring and at the exocyclic nitrogen atom at position 6. Among the newly described purines, five compounds showed antiproliferative activity with IC50 values below 10 mM, the tetrahydroquinoline derivative at position 6 of phenylaminopurine being the most active of the series in the six cell lines tested. Moreover, the compounds induced G2/M phase arrest in human cervical carcinoma HeLa cells as reported for tubulin depolymerizing agents. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Purines Microwave-assisted synthesis Antiproliferative agents Cell cycle analysis
1. Introduction Microtubules are cytoskeletal filaments consisting of a,b-tubulin heterodimers and are involved in a wide range of fundamental cellular processes, such as formation and maintenance of cell shape, regulation of motility, cell signaling, secretion, intracellular transportation, and cell division and mitosis [1,2]. Due to the different functions of microtubules during cell cycle progression, tubulin has become an attractive target for the discovery of anticancer drugs. For instance, taxoids and vinca alkaloids, first-line therapies for a large variety of tumors, exert their antitumor action by altering microtubule dynamics, either by promoting the course of microtubule assembly or disassembly. Indeed taxoids and vinca alkaloids target two distinct binding sites at the a,b-tubulin heterodimers: while the taxol site is located at the b subunit, the vinca site is situated at the interface of two heterodimers [3,4]. Despite of the clinical efficacy of these drugs, they have serious deficiencies, including narrow therapeutic indexes and emergence of drug resistance, mainly mediated by P-glycoprotein (P-gp) or bIII-tubulin overexpression [5,6]. Another serious disadvantage is their poor water solubility, which forces them to be administered
Abbreviations: HeLa, human cervical carcinoma cells; MEP, molecular electrostatic potential; MW, microwave irradiation; P-gp, P-glycoprotein. * Corresponding author. rez-Pe rez). E-mail address: [email protected] (M.-J. Pe http://dx.doi.org/10.1016/j.ejmech.2014.09.093 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
with a surfactant that may cause hypersensibility reactions and long-time administration [7]. A third binding site in tubulin, where colchicine specifically binds, has gained prominence in the last few years. The colchicine binding site is located at the interface of the a and b-subunits [8] and compounds binding at this site act as microtubule depolymerization agents [9,10]. Although colchicine itself cannot be used in the treatment of cancer owing to its high toxicity [11], a number of compounds binding at this site such as CA-4P, ZD6126, AVE8062, ABT-751 and MPC-6827 are in clinical trials for antitumor indications [9]. It should be mentioned that until now no P-glycoprotein (Pgp) or bIII-tubulin overexpression mediated resistance phenomena have been described with the best studied colchicine site binders [9,12]. Moreover, compounds binding at the colchicine site in tubulin have an additional value as anticancer agents since many of them have shown vascular disrupting properties targeting the tumor endothelium [13,14]. Among the colchicine-binding site compounds, we have centered our attention on MPC-6827 (1, Fig. 1), a quinazoline derivative identified through an anticancer screening apoptosis platform. Studies performed with MPC-6827 have shown that the compound binds at the colchicine binding site in tubulin [15]. This compound, also named Verubulin or Azixa, and whose structure significantly differs from colchicine or combretastatins, is currently undergoing clinical evaluation for the treatment of malignancies such as glioblastoma multiforme [16,17]. Other quinazoline
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Fig. 1. Chemical structure of MPC-6827 (1) and related compounds 2 and 3.
Scheme 2. Reagents and conditions: (a) CH(OMe)3, EtSO3H, Ac2O, MW, 120 C, 15 min; (b) 4-methoxy-N-methylaniline, HCl, ¡PrOH, MW, 80 C, 10 min.
2. Results and discussion 2.1. Chemistry
Fig. 2. Theoretical cLogP values [25] of the pyrrole[2,3-d]pyrimidine (A), cyclopenta[d] pyrimidine (B) and purine (C) scaffolds.
derivatives continue to be explored as antimitotic and vascular disrupting agents [18e21]. Interestingly, Gangjee et al. have reported on some pyrimidine derivatives fused to a 5 membered ring (a pyrazole such as in compound 2, or a cyclopentane as in compound 3 (Fig. 1)), that have also shown microtubule depolymerization properties by binding at the colchicine site and are characterized by cancer cell growth inhibitory activity. The substitution pattern in these compounds very much resembles the one in MPC-6827 [22]. A comparison of the central scaffold present in compounds 1, 2 and 3 led us to propose, as a simplistic example of “scaffold hoping” [23], that replacement of such scaffolds by a purine ring [24] might allow a similar distribution of the outsider substituents while solubility could be improved due to the inclusion of an imidazole at the central core. A calculation of the logP values [25] of the skeleton present in compounds 2 and 3, and a comparison with that of a purine ring (Fig. 2), supported a positive contribution of the latest to an increase in solubility. Therefore we have addressed the synthesis and antiproliferative evaluation of a series of 6-phenylaminopurine derivatives towards tumor and endothelial cells. In addition, cell-cycle analysis has been performed to establish the mechanism of action of this family of purines.
Starting from 2-amino-4,6-dichloro-2-methylpyrimidine (4) a first series of 6-phenylaminopurines were obtained in just 3 steps (Scheme 1). Thus, reaction of 4 with aqueous methylamine in 1,4dioxane at 80 C overnight afforded the pyrimidine 5 in an excellent yield, considerably better than previously described [26]. Then, reaction of 5 with triethyl orthoacetate in the presence of ethanesulphonic acid and acetic anhydride under MW irradiation for 15 min [27e29] afforded the 8-methylpurine 6. Finally, reaction of 6 with substituted anilines in isopropanol in the presence of HCl at 80 C afforded the 6-phenylamino derivatives 7aee, in moderate to good yields (65e90%). Under the same conditions, reaction of the 6chloropurine 8 [30] with 4-methoxy-N-methylaniline afforded compound 7f. Similarly, when compound 5 (Scheme 2) reacted with trimethyl orthoformate, the purine 9 was obtained, that was further treated with 4-methoxy-N-methylaniline to yield the 8unsubstituted compound 10. In addition, the unsubstituted compound at N-9 was obtained as depicted in Scheme 3. Reaction of the 4,6-dichloropyrimidine 4 with p-methoxybenzylamine afforded the benzyl derivative 11 in 64% yield. Then, reaction of 11 with triethyl orthoacetate led to the 8-methyl purine 12 in modest yield (31%). Finally, reaction with 4-methoxy-N-methylaniline, followed by catalytic hydrogenation to remove the p-methoxybenzyl group, afforded the purine 14 in 64% yield. According to some recent papers on pyrazolopyrimidines related to compounds 2 and 3 (Fig. 2), the introduction of an ethyl group at the NH at position 6 of the purine ring, or even the replacement of the N-methylaniline by a tetrahydroquinoline, might lead to an improvement in the antiproliferative activity [31]. Therefore reaction of 7b with ethyl iodide in the presence of CsCO3 [32] afforded the N-ethyl derivative 15 (Scheme 4). On the other
Scheme 1. Reagents and conditions: (a) CH3NH2, 1,4-dioxane, 80 C, overnight; (b) CH3C(OEt)3, EtSO3H, Ac2O, MW, 120 C, 15 min; (c) the corresponding aniline, HCl, iPrOH, MW, 80 C, 10 min.
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423
Scheme 3. Reagents and conditions: (a) 4-methoxybenzylamine, K2CO3, DMF, MW, 150 C, 1 h; (b) CH(OEt)3. EtSO3H, Ac2O, MW, 120 C, 15 min; (b) 4-methoxy-N-methylaniline, HCl, ¡ PrOH, MW, 80 C, 10 min; (d) H2, Pd(C), HCl, MeOH, 24 h.
Scheme 4. Reagents and conditions: (a) EtI, Cs2CO3, DMF, 60 C, 2 h; (b) 6-methoxy1,2,3,4-tetrahidroquinoline, HCl, iPrOH, 80 C, 12 h.
hand, reaction of the 6-chloropurine 6 with 6-methoxy-1,2,3,4tetrahydroquinoline allowed the synthesis of compound 16 in moderate yield. 2.2. Biological evaluation 2.2.1. Antiproliferative activity The synthesized compounds were evaluated for their antiproliferative activity (Table 1) in six different cell lines: three endothelial cell lines (HMEC-1, MBEC and BAEC) and three tumor cell lines (L1210, CEM and HeLa) [33,34]. Data are expressed as the 50% inhibitory concentration (IC50), defined as the concentration at which the compounds reduced cell proliferation by 50%. Colchicine was included as a reference compound. It should be mentioned that MPC-6827 has been reported to inhibit cancer cell growth with GI50 values of 3e6 nM in T47D, HCT116 and SN-398 cells [18]. A very
recent article describes the antiproliferative testing of MPC-6827 against a wider panel of tumor cells with IC50 values around 1 nM [20]. Among the synthesized purines, compounds 7a, 10, 14e16 showed antiproliferative activity in endothelial and tumor cell lines in the low mM range. The lack of antiproliferative activity of the compounds where the N-methylaniline at position 6 carries a substituent different from 4-OCH3 (compounds 7bee) strongly indicates that the structural requirements among these purines for antiproliferative activity very much resemble those reported for quinazolines and fused pyrimidines. Notably, even the replacement of the p-OCH3 group in 7a by a p-CH3 (7d) resulted in an inactive compound. In terms of substitution at position 6, the tetrahydroquinoline derivative (16) afforded the best cytostatic activity. Regarding the purine scaffold, a methyl group seemed to be crucial at position 2 (based on the lack of activity of compound 7f), while no significant differences were observed when a methyl or a hydrogen was present at position 8 or 9. However, compared to the previously described compounds (1e3), the incorporation of the imidazole at the central core appeared to negatively affect the antiproliferative activity since the best compounds in the purine series were between 10- to 1000-fold less active than the parent scaffolds. 2.2.2. Cell cycle inhibition Tubulin polymerization inhibitors are characterized by arresting cell cycle at G2/M phase [4]. Thus, the effect of four of the synthesized compounds (7a, 14, 15 and 16) on cell cycle of HeLa cells was analyzed by flow cytometry [35]. For comparative purposes, colchicine was included as a positive control. After 24 h of treatment, DNA was stained with propidium iodide and the intensity of the fluorescence was measured by flow cytometry [36]. The results obtained are summarized in Table 2, as the percentage of cells detected in each phase of the cell cycle with or without inhibitor (control). Untreated cells showed the classical pattern for proliferating cells distributed in the sub-G1, G1, S and G2/M phase, with 31%
Table 1 Antiproliferative activity of compounds 7aef, 10 and 14e16 in endothelial and tumor cell lines. Comp.
7a 7b 7c 7d 7e 7f 10 14 15 16 Colchicine
Endothelial cells IC50 (mM)
Tumor cells IC50 (mM)
HMEC-1
MBEC
BAEC
L1210
CEM
HeLa
7.3 ± 0.1 100 100 >100 >100 100 9.2 ± 1.3 7.1 ± 1.0 8.8 ± 2.1 2.3 ± 0.1 0.0038 ± 0.0011
8.7 ± 0.1 100 100 >100 >100 100 15 ± 7 8.9 ± 0.3 15 ± 0.4 4.4 ± 0.3 0.031 ± 0.015
7.4 ± 0.3 100 100 >100 >100 100 9.3 ± 0.6 8.9 ± 1.6 11 ± 1.4 2.4 ± 0.1 0.0069 ± 0.0008
19 ± 1 250 >250 250 >250 >250 30 ± 1 229 ± 30 35 ± 0 5.1 ± 0.1 0.014 ± 0.001
6.2 ± 1.8 >250 >250 164 ± 54 132 ± 47 >250 21 ± 3 23 ± 0 16 ± 1 3.5 ± 2.1 0.031 ± 0.019
4.5 ± 0.3 197 ± 14 >250 250 188 ± 25 >250 22 ± 1 14 ± 5 20 ± 0 4.4 ± 0.4 0.028 ± 0.000
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Table 2 Distribution of HeLa cells in different phases of the cell cycle after treatment with DMSO (0.1%) as control, colchicine and the inhibitors 7a, 14, 15 and 16. Compound
Control Colchicine 7a 14 15 16
Dose (mM)
0.1 20 100 20 100 20 100 4 20 100
Percentage of cells (%) Sub-G1
G1
S
G2/M
1 6 39 14 5 11 6 36 20 7 8
45 5 18 8 39 6 39 12 35 3 3
24 10 29 15 27 15 27 18 18 8 8
31 80 25 63 31 68 28 34 27 80 79
of the cells found in this latest phase. On the other hand, and as a positive control, cells treated with colchicine (0.1 mM) showed an accumulation in the G2/M phase with a percentage as high as 80%. When cells were treated with compounds 7a or 14 at 100 mM, a clear accumulation in the G2/M phase was induced (63% and 68% of the cells respectively), a behavior closely similar to that observed with colchicine. Treatment of the HeLa cells with the tetrahydroquinoline derivative 16 led to a similar pattern with 80% of the cells in G2/M phase even at a concentration of 20 mM, which correlates well with the higher antiproliferative activity showed by this compound. Although compound 15 did not induce cell cycle arrest in G2/M phase, at 100 mM the compound caused an accumulation of cells in sub-G1 phase, suggesting a plausible proapoptotic effect. This also occurred after the treatment with compound 7a at 20 mM or even with compound 16 at 4 mM. The
subdiploid DNA content of cells that increased from 1% in control cells to 20e40 % in the treated ones may point to the presence of apoptotic cells which have suffered nuclear fragmentation. MPC6827 (1) showed the same behavior, although at lower concentrations (5 nM), when tested in a similar experiment, inducing G2/ M phase arrest after 24 h and induction of apoptosis after 48 h in T47D human breast cancer cells [18]. 2.3. Maps of electrostatic potential In order to gain some insights that might help to explain the differences observed in antiproliferative activity among the quinazoline 1, the fused pyrimidines 2 and 3, and the here described purines (exemplified by compound 7a), molecular electrostatic potential (MEP) maps were calculated for the four compounds. The geometry of the compounds was first optimized by means of the ab initio program Gaussian 09, and the MEP maps were calculated based on the optimized structures using the program DelPhi. It should be mentioned that the geometries of the optimized structures are very similar as shown in the Supporting Fig. 1, allowing a similar distribution of the substituents as hypothesized in the introduction. Concerning the MEP maps (Fig. 3), the overall distribution is quite similar, although a more careful analysis shows that compounds 1 and 3 show a higher degree of similarity in terms of localized negative and positive potential regions when compared to compounds 2 and 7a. Interestingly, and despite the precaution to compare IC50 values from different origin, compounds 1 and 3 are more cytostatic than compounds 2 and 7a. When comparing compounds 2 and 7a, the most significant differences affect the negative region (represented in pink) around position 7 of the purine, a region that has a positive value in the pyrazolopyrimidine 2 (represented in light blue). So, it may be proposed that a negative
Fig. 3. MEP maps of compounds 1e3 and 7a. Positive and negative isopotential surfaces are colored in cyan and pink, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
M.-D. Canela et al. / European Journal of Medicinal Chemistry 87 (2014) 421e428
electrostatic potential at this site is detrimental for activity, while the distribution of MEPs obtained for compounds 1 and 3 may lead to more active compounds. Obviously, many other factors including crossing the membranes, metabolic stability etc … may also account for the different IC50 values, but these differences in MEP maps may be relevant when facing the design of new compounds.
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purified by flash chromatography (dichloromethane/methanol) to yield 370 mg (95%) of 5 as a white solid. Mp 129e131 C. MS (ES, positive mode): m/z 173 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.23 (s, 3H, CH3-2), 2.86 (d, J ¼ 4.4 Hz, 3H, CH3eN9), 4.74 (br s, 2H, NH2), 6.87 (br s, 1H, NH). A similar synthetic procedure but with a lower yield has been recently described [26].
3. Conclusions In summary, we here report the synthesis and antiproliferative activity of a novel series of 6-phenylaminopurines. Among them, compounds 7a, 10, and 14e16 exhibited antiproliferative activity in the low mM range, in endothelial and tumor cells. Importantly, compound 16, with a tetrahydroquinoline substitution at position 6, showed IC50 values around 2e5 mM in the six cell lines tested. Furthermore, these purine analogs induced a G2/M cell cycle arrest in HeLa cells, a behavior also observed with other colchicine-site binders in tubulin, such as MPC-6827. A comparison of the MEP maps of compound 7a with those generated for the previously described quinozaline and pyrimidine-fused analogs may help to explain the differences observed in terms of antiproliferative activity. 4. Experimental 4.1. Synthesis Melting points were obtained on a Mettler Toledo M170 apparatus and are uncorrected. The elemental analysis was performed with a Heraeus CHNeO-RAPID instrument. The elemental compositions of the compounds agreed to within ±0.4% of the calculated values. For all the tested compounds, satisfactory elemental analysis was obtained supporting >95% purity. Electrospray mass spectra were measured on a quadruple mass spectrometer equipped with an electrospray source (HewlettePackard, LC/MS HP 1100). 1H and 13C NMR spectra were recorded on a Varian INNOVA300 operating at 299 MHz (1H) and 75 MHz (13C), respectively, a Varian INNOVA-400 operating at 399 MHZ (1H) and 99 MHz (13C), respectively, and a VARIAN SYSTEM-500 operating a 499 and 125 MHz, respectively. Analytical TLC was performed on silica gel 60 F254 (Merck) precoated plates (0.2 mm). Spots were detected under UV light (254 nm) and/or charring with ninhydrin or phosphomolybdic acid. Separations on silica gel were performed by preparative centrifugal circular thin-layer chromatography (CCTLC) on a ChromatotronR (Kiesegel 60 PF254 gipshaltig (Merck)), with layer thickness of 1 and 2 mm and flow rate of 4 or 8 mL/min, respectively. Flash column chromatography (HPFC) was performed in a Biotage Horizon instrument. Microwave reactions were performed using the Biotage Initiator 2.0 single-mode cavity instrument from Biotage (Uppsala). Experiments were carried out in sealed microwave process vials utilizing the standard absorbance level (400 W maximum power). The temperature was measured with an IR sensor on the outside of the reaction vessel. 4.1.1. 6-Chloro-N,2-dimethylpyrimidine-4,5-diamine (5) A solution of 5-amino-4,6-dichloro-2-methylpyrimidine (4) (400 mg, 2.25 mmol), in 1,4-dioxane (11 mL) was treated with a 40% solution of methylamine in water (742 mL, 8.62 mmol) and placed in an Ace pressure tube. The vessel was sealed and heated overnight at 80 C. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. Then it was
4.1.2. 6-Chloro-2,8,9-trimethyl-9H-purine (6) A microwave vial was charged with compound 5 (200 mg, 1.16 mmol), triethyl orthoacetate (0.63 mL, 1.74 mmol), ethanesulfonic acid (19 mL, 0.23 mmol) in acetic anhydride (5 mL). The vessel was sealed and microwave-irradiated at 120 C for 15 min. Dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and concentrated. The residue obtained was purified CCTLC employing dichloromethane:methanol (30:1) to obtain 135 mg (59%) of 6 as a white solid. Mp 122e124 C. MS (ES, positive mode): m/z 197 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.59 (s, 3H, CH3-2), 2.65 (s, 3H, CH3-8), 3.71 (s, 3H, CH3eN9). 4.1.3. General procedure for the reaction of the 6-chloropurines with anilines A microwave vial was charged with the corresponding 6chloropurine (1.0 mmol), the appropriate aniline (1.5 mmol) in isopropanol (4 mL) and 37% aqueous HCl (1.2 mmol). The reaction vessel was sealed and heated in a microwave reactor at 80 C for 10 min. After cooling, dichloromethane was added (20 mL) and the mixture was washed with saturated aqueous NaHCO3 (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The resulting residue was purified as specified. 4.1.3.1. N-(4-methoxyphenyl)-N,2,8,9-tetramethyl-9H-purin-6amine (7a). Following the general procedure, a microwave vial was charged with compound 6 (98 mg, 0.50 mmol), 4-methoxy-Nmethylaniline (103 mg, 0.75 mmol) and 37% aqueous HCl (150 mL) in isopropanol (2 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 40:1) to yield 134 mg (90%) of 7a as a rosy solid. Mp 127e129 C. MS (ES, positive mode): m/z 298 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.35 (s, 3H, CH32), 2.39 (s, 3H, CH3-8), 3.59 (s, 3H, CH3eN9), 3.74 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 6.93 (d, 2H, J ¼ 8.9 Hz, Ar), 7.20 (d, 2H, J ¼ 8.9 Hz, Ar). 13C NMR (DMSO-d6, 75 MHz) d: 13.5 (CH3-8), 25.7 (CH3-2), 28.2 (CH3eN9), 40.1 (CH3eN6), 55.2 (OCH3), 116.7 (C-5), 113.9, 127.6, 138.8, 156.9 (Ar), 147.9 (C-8), 151.7 (C-6), 152.1 (C-4), 159.0 (C-2). Anal. calc. for (C16H19N5O): C, 64.63; H, 6.44; N, 23.55. Found: C, 64.91; H, 6.72; N, 23.58. 4.1.3.2. N-(4-methoxyphenyl)-2,8,9-trimethyl-9H-purin-6-amine (7b). Following the general procedure, a microwave vial was charged with compound 6 (43 mg, 0.22 mmol), 4-methoxyaniline (43 mg, 0.34 mmol) and 37% aqueous HCl (66 mL) in isopropanol (1 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 46 mg (74%) of 7b as a white solid. Mp 204e206 C. MS (ES, positive mode): m/z 284 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.49 (s, 3H, CH3-2), 2.52 (s, 3H, CH3-8), 3.64 (s, 3H, CH3eN9), 3.73 (s, 3H, OCH3), 6.88 (d, 2H, J ¼ 9.0 Hz, Ar), 7.86 (d, 2H, J ¼ 9.0 Hz, Ar), 9.40 (br s, 1H, NH). 13C NMR (DMSO-d6, 75 MHz) d: 13.4 (CH3-8), 25.9 (CH3-2), 28.3 (CH3eN9), 55.1 (OCH3), 116.5 (C-5), 113.5, 121.7, 133.4, 154.5 (Ar), 149.1 (C-8), 150.5 (C-6), 151.4 (C-4), 159.6 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.46; H, 6.11; N, 24.57.
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4.1.3.3. N-(3-methoxyphenyl)-N,2,8,9-tetramethyl-9H-purin-6amine (7c). Following the general procedure, a microwave vial was charged with compound 6 (43 mg, 0.22 mmol), 3-methoxy-Nmethylaniline (44 ml, 0.34 mmol) and 37% aqueous HCl (66 mL) in isopropanol (1 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 42 mg (64%) of 7c as a white solid. Mp 131e133 C. MS (ES, positive mode): m/z 298 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.37 (s, 3H, CH32), 2.41 (s, 3H, CH3-8), 3.60 (s, 3H, CH3eN9), 3.74 (s, 3H, OCH3), 3.79 (s, 3H, CH3eN6), 6.79 (ddd, 1H, J ¼ 8.3, 2.5, 0.8 Hz, Ar), 6.86 (ddd, 1H, J ¼ 7.9, 1.9, 0.8 Hz, Ar), 6.89 (t, 1H, J ¼ 2.1 Hz, Ar), 7.26 (t, 1H, J ¼ 8.1 Hz, Ar). 13C NMR (DMSO-d6, 75 MHz) d: 13.5 (CH3-8), 25.7 (CH3-2), 28.3 (CH3eN9), 40.1 (CH3eN6), 55.1 (OCH3), 117.1 (C-5), 110.9, 111.9, 118.2, 129.2, 146.9, 159.4 (Ar), 148.4 (C-8), 152.3 (C-6), 153.2 (C-4), 159.0 (C-2). Anal. calc. for (C16H19N5O): C, 64.63; H, 6.44; N, 23.55. Found: C, 64.55; H, 6.31; N, 23.48. 4.1.3.4. N,2,8,9-tetramethyl-N-(p-tolyl)-9H-purin-6-amine (7d). Following the general procedure, a microwave vial was charged with compound 6 (80 mg, 0.41 mmol), N,4-dimethylaniline (78 ml, 0.62 mmol) and 37% aqueous HCl (123 mL) in isopropanol (2 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 40:1) to yield 79 mg (69%) of 7d, as a white solid. Mp 133e135 C. MS (ES, positive mode): m/z 282 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.32 (s, 3H, CH3-Ph), 2.35 (s, 3H, CH3-2), 2.41 (s, 3H, CH3-8), 3.60 (s, 3H, CH3eN9), 3.78 (s, 3H, CH3eN6), 7.17 (m, 4H, Ar). 13C NMR (DMSO-d6, 100 MHz) d: 13.4 (CH3-8), 20.6 (CH3-Ph), 25.7 (CH3-2), 28.2 (CH3eN9), 40.4 (CH3eN6), 116.9 (C-5), 125.9, 129.1, 134.3, 143.3 (Ar), 148.1 (C-8), 152.4 (C-6), 153.1 (C-4), 159.0 (C-2). Anal. calc. for (C16H19N5): C, 68.30; H, 6.81; N, 24.89. Found: C, 68.11; H, 6.57; N, 24.66.
4.1.4. 6-Chloro-2,9-dimethyl-9H-purine (9) A microwave vial was charged with a solution of compound 5 (500 mg, 2.90 mmol), trimethyl orthoformate (0.95 mL, 8.70 mmol) and ethanesulfonic acid (24 ml, 0.29 mmol) in acetic anhydride (12 mL). The vessel was sealed and microwave-irradiated at 120 C for 15 min. Dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by flash chromatography (dichloromethane:methanol 20:1) to yield 487 mg (92%) of 9 as a white solid. Mp 167e169 C. (m.p. lit [37] 168e171 C). MS (ES, positive mode): m/z 183 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.68 (s, 3H, 2CH3), 3.82 (s, 3H, CH3eN), 8.53 (s, 1H, H-8). 4.1.5. N-(4-Methoxyphenyl)-N,2,9-trimethyl-9H-purin-6-amine (10) Following the general procedure for the reaction of 6chloropurines with anilines (Section 4.1.3), a microwave vial was charged with compound 9 (500 mg, 2.74 mmol), 4-methoxy-Nmethylaniline (563 mg, 4.11 mmol) and 37% aqueous HCl (0.8 mL) in isopropanol (11 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 580 mg (75%) of 10 as a white solid. Mp 152e154 C. MS (ES, positive mode): m/z 284 (MþH)þ. 1H NMR (DMSO-d6, 500 MHz) d: 2.40 (s, 3H, CH3-2), 3.66 (s, 3H, CH3eN9), 3.69 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 6.93 (d, 2H, J ¼ 8.9 Hz, Ar), 7.22 (d, 2H, J ¼ 8.9 Hz, Ar), 7.91 (s, 1H, H-8). 13C NMR (DMSO-d6, 125 MHz) d: 25.8 (CH3-2), 29.3 (CH3eN9), 39.9 (CH3eN6), 55.2 (OCH3), 117.6 (C-5), 114.0, 127.9, 138.7, 157.2 (Ar), 140.4 (C-8), 151.9 (C-6), 153.6 (C-4), 160.0 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.47; H, 5.95; N, 24.61.
4.1.3.5. N,2,8,9-tetramethyl-N-(4-(trifluoromethoxy)phenyl)-9Hpurin-6-amine (7e). Following the general procedure, a microwave vial was charged with compound 6 (80 mg, 0.41 mmol), N-methyl4-(trifluoromethoxy)aniline (92 ml, 0.62 mmol) and 37% aqueous HCl (123 mL) in isopropanol (2 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 40:1) to yield 100 mg (69%) of 7e as a white solid. Mp 133e135 C. MS (ES, positive mode): m/z 352 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.39 (s, 3H, CH3-2), 2.40 (s, 3H, CH3-8), 3.61 (s, 3H, CH3eN9), 3.80 (s, 3H, CH3eN6), 7.34 (d, 2H, J ¼ 8.8 Hz, Ar), 7.45 (d, 2H, J ¼ 9.0 Hz, Ar). 13 C NMR (DMSO-d6, 100 MHz) d: 13.6 (CH3-8), 25.6 (CH3-2), 28.3 (CH3eN9), 40.1 (CH3eN6), 117.2 (C-5), 120.6 (q, J ¼ 256.0, CF3), 121.3, 127.7, 144.8, 145.3 (Ar), 148.8 (C-8), 152.2 (C-6), 153.3 (C-4), 159.1 (C-2). Anal. calc. for (C16H16F3N5O): C, 54.70; H, 4.59; N, 19.93. Found: C, 54.45; H, 4.38; N, 19.67.
4.1.6. 6-Chloro-N4-(4-methoxybenzyl)-2-methylpyrimidine-4,5diamine (11) A microwave vial was charged with 5-amino-4,6-dichloro-2methyl-pyrimidine (4) (700 mg, 3.91 mmol), 4methoxybenzylamine (510 ml, 3.91 mmol), and K2CO3 (810 mg, 5.86 mmol) in anhydrous DMF (10 mL). The vessel was sealed and irradiated at 150 C for 1 h. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NH4Cl (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by flash chromatography (dichloromethane:methanol 20:1) to yield 696 mg (64%) of 11 as a yellow solid. Mp 155e157 C. MS (ES, positive mode): m/z 279 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.52 (s, 3H, CH3-2), 2.68 (s, 3H, CH3-8), 3.71 (s, 3H, OCH3), 5.39 (s, 2H, CH2), 6.89 (d, 2H, J ¼ 8.6 Hz, Ar), 7.17 (d, 2H, J ¼ 8.5 Hz, Ar).
4.1.3.6. N-(4-methoxyphenyl)-N,8,9-trimethyl-9H-purin-6-amine (7f). Following the general procedure, a microwave vial was charged with compound 8 [30] (62 mg, 0.34 mmol), 4-methoxy-Nmethylaniline (70 mg, 0.51 mmol) and 37% aqueous HCl (100 mL) in isopropanol (1.4 mL). The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 54 mg (56%) of 7f as a rosy solid. Mp 172e174 C. MS (ES, positive mode): m/z 284 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.45 (s, 3H, CH38), 3.64 (s, 3H, CH3eN9), 3.78 (s, 3H, OCH3), 3.81 (s, 3H, CH3eN6), 6.95 (d, 2H, J ¼ 9.0 Hz, Ar), 7.21 (d, 2H, J ¼ 9.0 Hz, Ar), 8.11 (s, 1H, H2). 13C NMR (DMSO-d6, 100 MHz) d: 13.4 (CH3-8), 28.3 (CH3eN9), 40.2 (CH3eN6), 55.3 (OCH3), 118.4 (C-5), 114.0, 127.5, 138.5, 157.0 (Ar), 148.6 (C-8), 150.5 (C-6), 152.2 (C-4), 153.1 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.30; H, 5.99; N, 24.58.
4.1.7. 6-Chloro-9-(4-methoxybenzyl)-2,8-dimethyl-9H-purine (12) A microwave vial was charged with a solution of compound 11 (700 mg, 2.51 mmol), triethyl orthoacetate (1.37 mL, 7.53 mmol) and ethanesulfonic acid (41 ml, 0.50 mmol) in acetic anhydride (10 mL). The vessel was sealed and microwave-irradiated at 120 C for 15 min. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by flash chromatography (dichloromethane:methanol 20:1) to yield 232 mg of 12 (31%) as a pale brown oil. MS (ES, positive mode): m/z 303 (MþH)þ with a Cl isotopic pattern. 1H NMR (DMSO-d6, 300 MHz) d: 2.52 (s, 3H, CH3-2), 2.68 (s, 3H, CH3-8), 3.71 (s, 3H, OCH3), 5.39 (s, 2H, CH2), 6.89 (d, 2H, J ¼ 8.6 Hz, Ar), 7.17 (d, 2H, J ¼ 8.5 Hz, Ar).
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4.1.8. 9-(4-Methoxybenzyl)-N-(4-methoxyphenyl)-N,2,8-trimethyl9H-purin-6-amine (13) Following the general procedure for the reaction of 6chloropurines with anilines (Section 4.1.3), a microwave vial was charged with compound 12 (200 mg, 0.66 mmol), 4-methoxy-Nmethylaniline (136 mg, 1.00 mmol) and 37% aqueous HCl (200 mL) in 2-propanol (2.6 mL). The residue was purified by flash chromatography (dichloromethane:methanol, 20:1) to yield 150 mg (56%) of 13 as a rosy oil. MS (ES, positive mode): m/z 404 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.31 (s, 3H, CH3-2), 2.36 (s, 3H, CH3-8), 3.71 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 5.26 (s, 2H, CH2), 6.91 (m, 4H, Ar), 7.18 (m, 4H, Ar).
(ES, positive mode): m/z 324 (MþH)þ. 1H NMR (DMSO-d6, 500 MHz) 1.90 (m, 2H, CH2-30 ), 2.38 (s, 3H, CH3-2), 2.48 (s, 3H, CH38), 2.78 (t, 2H, J ¼ 6.8 Hz, CH2-40 ), 3.63 (s, 3H, CH3eN9), 3.74 (s, 3H, OCH3), 4.43 (m, 2H, CH2-20 ), 6.72 (d, 1H, J ¼ 2.8 Hz, Ar), 6.68 (dd, 1H, J ¼ 8.9, 3.0 Hz, Ar), 7.40 (d, 1H, J ¼ 8.9 Hz, Ar). 13C NMR (DMSO-d6, 125 MHz) d: 13.6 (CH3-8), 23.3 (CH2-30 ), 25.6 (CH3-2), 26.3 (CH2-40 ), 28.3 (CH3eN9), 46.1 (CH2-20 ), 55.1 (OCH3), 117.4 (C-5), 111.2, 112.7, 125.8, 131.0, 133.0, 155.1 (Ar), 148.6 (C-8), 151.5 (C-6), 153.4 (C-4), 159.0 (C-2). Anal. calc. for (C18H21N5O): C, 66.85; H, 6.55; N, 21.66. Found: C, 66.70; H, 6.38; N, 21.49.
4.1.9. N-(4-methoxyphenyl)-N,2,8-trimethyl-9H-purin-6-amine (14) A solution of compound 13 (80 mg, 0.20 mmol) in methanol (2 mL) at 0 C was treated with 37% aqueous HCl (200 mL) and 10% Pd/C (80 mg). The reaction mixture was stirred under hydrogen atmosphere at room temperature for 24 h. The reaction was filtered, washed with methanol and evaporated to dryness. The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 20:1) to yield 36 mg (64%) of 14 as a white solid. Mp 190e192 C. MS (ES, positive mode): m/z 285 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 2.31 (s, 3H, CH3-2), 2.34 (s, 3H, CH3-8), 3.74 (s, 3H, OCH3), 3.78 (s, 3H, CH3eN6), 6.93 (d, 2H, J ¼ 8.3 Hz, Ar), 7.21 (d, 2H, J ¼ 8.1 Hz, Ar), 12.46 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz) d: 15.2 (CH3-8), 26.1 (CH3-2), 40.1 (CH3eN6), 55.6 (OCH3), 117.9 (C-5), 114.3, 128.1, 139.3, 157.3 (Ar), 147.3 (C-8), 153.0 (C-6), 154.0 (C-4), 159.4 (C-2). Anal. calc. for (C15H17N5O): C, 63.59; H, 6.05; N, 24.72. Found: C, 63.29; H, 5.94; N, 24.81.
4.2.1. Cell proliferation
4.1.10. N-ethyl-N-(4-methoxyphenyl)-2,8,9-trimethyl-9H-purin-6amine (15) A solution of compound 7b (60 mg, 0.21 mmol) and Cs2CO3 (208 mg, 0.64 mmol) in anhydrous DMF (2 mL) was stirred at room temperature for 30 min under argon atmosphere. Then, ethyl iodide (67 mL, 0.84 mmol) was added and the mixture was heated at 60 C for 2 h. After cooling, volatiles were removed and the mixture was further solved in dichloromethane (20 mL) and washed with brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by CCTLC in the Chromatothron (dichloromethane:methanol, 30:1) to yield 57 mg (87%) of 15 as a white solid. Mp 136e138 C. MS (ES, positive mode): m/z 312 (MþH)þ. 1H NMR (DMSO-d6, 300 MHz) d: 1.14 (t, 3H, J ¼ 6.9 Hz, CH3CH2), 2.31 (s, 3H, CH3-2), 2.40 (s, 3H, CH3-8), 3.58 (s, 3H, CH3eN9), 3.78 (s, 3H, OCH3), 4.33 (q, 2H, J ¼ 6.9 Hz, CH2), 6.94 (d, 2H, J ¼ 8.9 Hz, Ar), 7.15 (d, 2H, J ¼ 8.9 Hz, Ar). 13C NMR (DMSO-d6, 75 MHz) d: 13.8 (CH3CH2), 13.9 (CH3-8), 26.1 (CH3-2), 28.6 (CH3eN9), 46.3 (CH2), 55.5 (OCH3), 116.6 (C-5), 114.3, 129.3, 137.2, 157.4 (Ar), 148.1 (C-8), 152.5 (C-6), 153.3 (C-4), 159.4 (C-2). Anal. calc. for (C17H21N5O): C, 65.57; H, 6.80; N, 22.49. Found: C, 65.28; H, 6.54; N, 22.19. 4.1.11. 6-Methoxy-1-(2,8,9-trimethyl-9H-purin-6-yl)-1,2,3,4tetrahydroquinoline (16) A solution of compound 6 (100 mg, 0.51 mmol) in 2-propanol (2 mL) was placed in an Ace pressure tube and 6-methoxy1,2,3,4-tetrahydroquinoline (125 mg, 0.77 mmol) and 37% aqueous HCl (150 mL) were added. The tube was then sealed and heated at 80 C overnight. After cooling, dichloromethane was added (20 mL) and the crude reaction mixture was washed with saturated aqueous NaHCO3 (15 mL) and brine (15 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The residue was purified by CCTLC in the Chromatothron (hexane:ethyl acetate, 2:1) to yield 57 mg (35%) of 16 as a white solid. Mp 180e182 C. EM
4.2. Biological methods
4.2.1.1. Endothelial cells. Mouse brain endothelial cells (MBEC), bovine aortic endothelial cells (BAEC) and human dermal microvascular endothelial cells (HMEC-1) were seeded in 48-well plates at 10,000 cells/well (except HMEC-1 at 20,000/well). After 24 h, compounds were added at different concentrations (100, 20, 4, 0.8, 0.16 and 0.08 mM). The cells were allowed to proliferate for 3 days (or 4 days for HMEC-1) in the presence of the compounds, trypsinized, and counted by means of a Coulter counter (Analis, Belgium). 4.2.1.2. Tumor cells. Human cervical carcinoma (HeLa) cells were seeded in 96-well plates at 15,000 cells/well in the presence of different concentrations of the compounds. After 4 days of incubation, the cells were trypsinized and counted in a Coulter counter. Suspension cells (Mouse leukemia L1210 and human lymphoid Cem cells) were seeded in 96-well plates at 60,000 cells/well in the presence of different concentrations of the compounds. L1210 and Cem cells were allowed to proliferate for 48 h or 96 h, respectively and then counted in a Coulter counter. The 50% inhibitory concentration (IC50) was defined as the compound concentration required to reduce cell proliferation by 50%. Colchicine was added as reference compound. 4.2.2. Cell cycle analysis HeLa cells were seeded in 6-well plates at 125,000 cells/well in DMEM with 10% fetal bovine serum (FBS) for 24 h. Then, cells were exposed to different concentrations of the compounds. After 24 h, the DNA of the cells was stained with propidium iodide using the CycleTEST PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA). The DNA content of the stained cells was assessed by flow cytometry on a FACSCalibur flow cytometer and analyzed with CellQuest software (BD Biosciences) within 3 h after staining. Cell debris and clumps were excluded from the analysis by appropriate dot plot gating. Percentages of sub-G1, G1, S, and G2/M cells were estimated using appropriate region markers [35]. Colchicine was added as reference compound. 4.3. Computational methods The geometries of compounds 1e3 and 7a were optimized using the ab initio quantum chemistry programme Gaussian 09 [38] and the RHF/6-21G* basis set. Finite difference solutions to the linearized PoissoneBoltzman equation, as implemented in the Delphi software (v.4) [39,40] were used to calculate MEPs. For MEP calculations cubic grids with a resolution of 1.0 Å were centered on each molecule, and the atomic charges were distributed onto the grid points. AMBER charges and radii were used. Solvent-accessible surfaces, calculated with a spherical probe with a radius of 1.4 Å, defined the solute boundaries, and a minimum separation of 10 Å was left between any solute atom and the borders of the box. The interior of the ligands was considered a low-dielectric medium
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(ε ¼ 2) whereas the surrounding solvent was treated as a highdielectric medium (ε ¼ 80). Acknowledgments M.-D. C. thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral fellowship. This project has been supported by the Spanish CICYT (SAF2012-39760-C02-01 to M.-J.C., M.-J.P.-P. and E.-M.P.) and the Comunidad de Madrid (BIPEDD2; ref P2010/BMD-2457 to M.-J.C.). We also would like to acknowledge Eef Meyen and Lizette Van Berckelaer for excellent technical assistance. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.09.093. These data include MOL files and InChiKeys of the most important compounds described in this article. References [1] M.A. Jordan, L. Wilson, Microtubules as target for anticancer drugs, Nat. Rev. Cancer 4 (2004) 253e265. [2] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic field of cancer therapeutics, Nat. Rev. Drug Discov. 9 (2010) 790e803. rez, Microtubule inhibitors: differentiating tubulin-inhibiting agents [3] E.A. Pe based on mechanism of action, clinical activity and resistance, Mol. Cancer Ther. 8 (2009) 2086e2895. [4] R.A. Stanton, K.M. Gernert, J.H. Nettles, R. Aneja, Drugs that target dynamic microtubules: a new molecular perspective, Med. Res. Rev. 31 (2011) 443e481. [5] T. Litman, T.E. Druley, W.D. Stein, S.E. Bates, From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance, Cell. Mol. Life Sci. 58 (2001) 931e959. [6] C. Stengel, S.P. Newman, M.P. Leese, B.V.L. Potter, M.J. Reed, A. Purohit, Class III beta-tubulin expression and in vitro resistance to microtubule targeting agents, Br. J. Cancer 102 (2010) 316e324. [7] G. Gaucher, R.H. Marchessault, J.-C. Leroux, Polyester-based micelles and nanoparticles for the parenteral delivery of taxanes, J. Control Release 2 (2010) 2e12. [8] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel, M. Knossow, Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain, Nature 428 (2004) 198e202. [9] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An overview of tubulin inhibitors that interact with the colchicine binding site, Pharm. Res. 29 (2012) 2943e2971. [10] B. Bhattacharyya, D. Panda, S. Gupta, M. Banerjee, Anti-mitotic activity of colchicine and the structural basis for its interaction with tubulin, Med. Res. Rev. 28 (2008) 155e183. [11] Y. Finkelstein, S.E. Aks, J.R. Hutson, D.N. Juurlink, P. Nguyen, G. Dubnov-Raz, U. Pollak, G. Koren, Y. Bentur, Colchicine poisoning: the dark side of an ancient drug, Clin. Toxicol. 48 (2010) 407e414. [12] Q.X. Yue, X. Liu, D.A. Guo, Microtubule-binding natural products for cancer therapy, Planta Med. 76 (2010) 1037e1043. [13] P.E. Thorpe, Vascular targeting agents as cancer therapeutics, Clin. Cancer Res. 10 (2004) 415e427. [14] M.R. Horsman, D.W. Siemann, Pathophysiologic effects of vascular-targeting agents and the implications for combination with conventional therapies, Cancer Res. 66 (2006) 11520e11539. [15] S. Kasibhatla, V. Baichwal, S.X. Cai, B. Roth, I. Skvortsova, S. Skvortsov, P. Lukas, N.M. English, N. Sirisoma, J. Drewe, A. Pervin, B. Tseng, R.O. Carlson, C.M. Pleiman, MPC-6827: a small-molecule inhibitor of microtubule formation that is not a substrate for multidrug resistance pumps, Cancer Res. 67 (2007) 5865e5871. [16] K.F. Grossmann, H. Colman, W.A. Akerley, M. Glantz, Y. Matsuoko, A.P. Beelen, M. Yu, J.F.D. Groot, R.D. Aiken, J.J. Olsen, B.A. Evans, R.L. Jensen, Phase I trial of verubulin (MPC-6827) plus carboplatin in patients with relapsed glioblastoma multiforme, J. Neurooncol. 110 (2012) 257e264. [17] M.C. Chamberlain, S. Grimm, S. Phuphanich, L. Recht, J.Z. Zhu, L. Kim, S. Rosenfeld, C.E. Fadul, A phase 2 trial of verubulin for recurrent glioblastoma: a prospective study by the brain tumor investigational consortium (BTIC), J. Neurooncol. 118 (2014) 335e343. [18] N. Sirisoma, A. Pervin, H. Zhang, S. Jiang, J.A. Willardsen, M.B. Anderson, G. Mather, C.M. Pleiman, S. Kasibhatla, B. Tseng, J. Drewe, S.X. Cai, Discovery of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a potent apoptosis inducer and efficacious anticancer agent with high blood brain barrier penetration, J. Med. Chem. 52 (2009) 2341e2351.
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