Article pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Pyrazolo[3,4‑d]pyrimidines Active in Vivo on the Bcr-Abl T315I Mutant Marco Radi,†,‡ Cristina Tintori,† Francesca Musumeci,§ Chiara Brullo,§ Claudio Zamperini,† Elena Dreassi,† Anna Lucia Fallacara,†,∥ Giulia Vignaroli,† Emmanuele Crespan,⊥ Samantha Zanoli,⊥ Ilaria Laurenzana,# Irene Filippi,△ Giovanni Maga,⊥ Silvia Schenone,*,§ Adriano Angelucci,○ and Maurizio Botta*,†,◆ †
Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy Dipartimento di Farmacia, Università degli Studi di Parma, Viale delle Scienze 27/A, 43124 Parma, Italy § Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV 3, 16132 Genova, Italy ∥ Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy ⊥ Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy # Laboratory of Preclinical and Translational Research, IRCCS-Referral Cancer Center of Basilicata (CROB), Rionero in Vulture (PZ), Italy △ Dipartimento di Medicina Molecolare e dello Sviluppo, Università degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy ○ Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università dell’Aquila Via Vetoio, 67100 Coppito, L’Aquila, Italy ◆ Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United States ‡
S Supporting Information *
ABSTRACT: Starting from our in-house library of pyrazolo[3,4-d]pyrimidines, a cross-docking simulation was conducted on Bcr-Abl T315I mutant. Among the selected compounds (2a−e), the 4-bromo derivative 2b showed the best activity against the Bcr-Abl T315I mutant. Deeper computational studies highlighted the importance of the bromine atom in the para position of the N1 side chain phenyl ring for the interaction with the T315I mutant. A series of 4-bromo derivatives was thus synthesized and biologically evaluated. Compound 2j showed a good balance of different ADME properties, high activity in cell-free assays, and a submicromolar potency against T315I Bcr-Abl expressing cells. In addition, it was converted into a water-soluble formulation by liposome encapsulation, preserving a good activity on leukemic T315I cells and avoiding the use of DMSO as solubilizing agent. In vivo studies on mice inoculated with 32D-T315I cells and treated with 2j showed a more than 50% reduction in tumor volumes.
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INTRODUCTION Chronic myeloid leukemia (CML) is a hematological malignancy caused by the constitutively activated kinase BcrAbl, resulting from the Philadelphia chromosome (Ph) translocation.1 Since 2001, the Bcr-Abl tyrosine kinase (TK) inhibitor imatinib (IM) has represented the first line therapy for CML, being particularly active in the chronic phase of the disease, with impressive response and survival rates.2 However, many patients eventually develop resistance that is frequently associated with mutations in the Bcr-Abl kinase domain.3 The second-generation Bcr-Abl inhibitor nilotinib4 and the dual Src/Bcr-Abl inhibitor dasatinib,5 both approved by FDA for IM-resistant CML treatment, are active against many IM© 2013 American Chemical Society
resistant mutants but proved to be ineffective against the T315I mutation of the gatekeeper residue, which accounts for 15− 20% of clinically observed mutations.6,7 Preclinical and clinical studies showed that ATP-competitive dual Src/Bcr-Abl inhibitors bosutinib (SKI-606),8 bafetinib ( I N NO - 4 0 6 , NS -1 8 7 ) , 9 3 -[ 2 -[ 2 -c y c l o p e n t y l - 6- (4 dimethylphosphorylanilino)purin-9-yl]ethyl]phenol (AP23464),10 and 6-(2,6-dichlorophenyl)-2-{[3(hydroxymethyl)phenyl]amino}-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (PD166326)11 inhibit Bcr-Abl more Received: February 14, 2013 Published: June 7, 2013 5382
dx.doi.org/10.1021/jm400233w | J. Med. Chem. 2013, 56, 5382−5394
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Figure 1. Selected second- and third-generation Bcr-Abl inhibitors.
sensitive and imatinib resistant CML patients, these last bearing the three most frequent Bcr-Abl mutations (including T315I).22 We decided to get further insights into this type of compounds with the aim of obtaining other active derivatives and of evaluating their activity in vivo. In the present work, we describe the rational development of novel pyrazolo[3,4-d]pyrimidines able to inhibit the Bcr-Abl T315I mutant both in vitro and in vivo. A combination of in silico and in vitro studies highlighted the importance of a bromine atom in the para position of the N1 side chain phenyl ring for the interaction with the hydrophobic region I of the T315I mutant. Following this computational study, we synthesized a small collection of 4-bromo derivatives, which led to the identification of compound 2j as the most promising inhibitor showing significant reduction of tumor volume in mice inoculated with 32D-T315I cells.
efficiently than IM and overcome resistance caused by most mutations, with the notable exception of T315I. Optimization of CML treatment remains an area of active research, and much effort has been devoted to the development of more effective TK inhibitors such as ponatinib (AP24534).12 This compound inhibits both native and mutant Bcr-Abl, including T315I, acting as a pan-Bcr-Abl inhibitor also targeting other TKs.13 The T315I point mutation precludes drug binding to the Abl catalytic domain by either direct steric hindrance or stabilization of its active conformation, with the DFG-loop in the “in” disposition (DGF-in).14 The development of new drugs targeting the open and active conformation of the T315I mutant is therefore a major challenge in CML therapy.15 Despite the past efforts to develop selective targeted inhibitors for the treatment of cancer, the main strategy has recently turned to find compounds acting on multiple targets in order to face the drug resistance, often connected with the activation of alternative signaling pathways.16 Furthermore, Abl shares significant sequence homology and remarkable structural resemblance with Src family members in their active state and several ATP-competitive Src inhibitors targeting the active conformation showed to be also potent Abl inhibitors.17a In this context, our research group has deeply investigated a class of pyrazolo[3,4-d]pyrimidine derivatives acting as dual inhibitors of Src and Abl kinases. These compounds showed potent antiproliferative and proapoptotic activity toward different cancer cell lines depending on the nature and position of substituents on the heterocyclic core.18−21 Recently, we identified two pyrazolo-pyrimidine based BcrAbl inhibitors, namely 1a and 1b (Figure 1), which were effective against CD34+ cells collected from both imatinib-
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RESULTS AND DISCUSSION Computational Studies on Bcr-Abl T315I Mutant. A cross-docking approach was employed in this study for the in silico screening of our in-house library of pyrazolo[3,4d]pyrimidines (consisting of about 300 structurally characterized compounds with purity >98%) to select the most promising potential binders of the Bcr-Abl T315I mutant for biological investigation. Two crystal structures were used for this purpose, the structure of the T315I mutant of Abl kinase bound by the pyrrolo-pyridine inhibitor 3 (PPY-A) (Figure 1) (1.95 Å resolution, PDB code 2Z60)23 and that of the T315I mutant Abl kinase domain in complex with another pyrrolopyridine 4 (SX7) (2.01 Å resolution, PDB code 3DK7).24 In 5383
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contrast to other inhibitors cocrystallized with the T315I Abl mutant, such as danusertib (PHA-739358),25 ponatinib (AP24534),26 5-[(5-{[4-{[4-(2-hydroxyethyl)piperazin-1-yl]methyl}-3-(trifluoromethyl)phenyl]carbamoyl}-2methylphenyl)ethynyl]-1-methyl-1H-imidazole-2-carboxamide (AP24589),27 and rebastinib (DCC2036),28 compounds 3 and 4 have a shape that more closely resembles that of our pyrazolo[3,4-d]pyrimidine compounds, including 1a,b (Figure 1). Compounds analyzed herein are already known dual Src/Abl inhibitors with activity ranging from 0.02 to 100 μM. From a structural point of view, these compounds are related to the Src inhibitors PP1 and PP2 but bear a different substitution pattern on the position 1, 3, 4, and 6 of the pyrazolo[3,4-d]pyrimidine nucleus. In agreement with our previously reported simulations,20 docking studies within the ATP binding site of T315I Abl gave two different outcomes depending on the presence or the absence of an alkylthio substituent at position 6 of the pyrazolo-pyrimidine ring. Docking calculations on the C6substituted compounds within the ATP binding site of the T315I Abl mutant did not converge to a reasonable solution, while profitable contacts could be established by the same compounds within the wild-type enzyme, in agreement with the binding mode described elsewhere.29 Unfavorable binding poses within the T315I Abl catalytic pocket were due to the steric hindrance caused by the residue Ile315, which precludes the access of the C6-substituted pyrazolo[3,4-d]pyrimidines, as shown by our earlier results.20 Instead, a well-defined binding mode was found for C6-unsubstituted compounds within the two X-ray structures of T315I Abl mutant used for calculations (Figure 2A). In detail, the C6-unsubstituted derivatives are involved in two hydrogen bonds with the Met318 of the hinge region by means of the atom N5 and the exocyclic amino group at C4. Moreover, the N1 side chain is located within a hydrophobic pocket formed by residues Lys271, Met290, Phe382, Ile313, Ile315, Val299, Val256, and Tyr253 (hydrophobic region I), while substituents at C4 interact with Leu248, Gly321, and Phe317 (hydrophobic region II). Because the amino acid in position 315 belongs to the hydrophobic region I, where the interactions between the C6-unsubstituted-pyrazolopyrimidines and the protein are exclusively hydrophobic, its mutation to isoleucine does not affect the correct placement of the C6-unsubstituted derivatives within the binding site. As a consequence, the binding mode proposed for the compounds within the T315I Abl binding pocket is the same of that previously found in the wild-type enzyme.29 Comparable results were obtained using the two proteins 2Z60 and 3DK7 in terms of both chemscore values and docking poses. Examination of the results indicated that all the inhibitors showed chemscore values ranging from 25 to 37 and from 26 to 38 in 2Z60 and 3DK7, respectively. However, slight differences between the two X-ray structures were observed in the interaction of C6unsubstituted ligands with hydrophobic region I (Figure 2B). While in 2Z60 the ligand adopted an extended conformation with the N1-2-chloro-2-phenylethyl substituent immersed in the deep pocket, in 3DK7, the N1-side chain was rotated by ∼50° with the terminal phenyl group exposed to the solvent. The different placement of the substituent at N1 in the two crystallographic structures was mainly due to the different conformation adopted by the conserved DFG motif that affects the shape of the pocket and consequently the pattern of interactions between the N1 side chain and the protein.
Figure 2. (A) Schematic representation of the binding mode of C6unsubstituted pyrazolo[3,4-d]pyrimidines within the T315I Abl mutant binding site. (B) Binding mode of compound 2b within 2Z60 (cyano) and 3DK7 (magenta) structures. Slight differences can be observed in the orientation of the ligand within the hydrophobic region I of the two structures.
According to the above-described docking results, the inhouse available C6-unsubstituted compounds 2a−e were selected for in vitro evaluation against the isolated T315I Abl together with compound 1b, used as a reference (Table 1). These compounds were selected for testing according to the predicted score values, the number of clusters obtained, the binding poses observed, and their molecular structure in order to evaluate the importance of the R2 substituent. Biological results are reported in Table 1. It is noteworthy that compound 2b, in which R2 is a bromine atom, proved to be the most active against the T315I Abl mutant (36 nM) with a comparable activity against Abl wild-type (55 nM). To rationalize these biological data, the binding free energies between Abl T315I and ligands (1b and 2a−e) were calculated by employing the molecular mechanics/generalized Born surface area (MMGBSA) scoring method on the docking poses (Table S2, Supporting Information). Two interesting aspects emerged from this analysis: (a) all compounds had a higher affinity for protein 2Z60 (compared to 3DK7) giving more stable complexes; (b) the ΔG values obtained with the use of 2Z60 structure better correlate with the experimental activities. In fact, the lowest binding energy (−53.2 KJ/mol) was found for compound 2b, the most active of the series. On the other hand, no correlation between calculated and experimental data was obtained by using the 3DK7 structure. Overall, these results highlighted that 2Z60 was the most reliable structure for in silico studies on our pyrazolo-pyrimidine inhibitors and thus we decided to use only this one in the following calculations. 5384
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Table 1. Enzymatic Activities, Cellular Activities, and ADME Properties
Ki (μM)a
compd
R1
R2
Abl (WT)
1b 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n 2o 2p 2q 2r 2s 2t 2u 2v 2w 2x 2y 2z
CH2C6H4-4F C6H4-3F CH2C6H4-3F CH2C6H4-3F CH2C6H4-3F C6H4-3Cl C6H4-3Br C6H4-3Br C6H4-3F C6H5 C6H5 CH2C6H5 CH2C6H4-3Cl CH2C6H4-2Cl CH2C6H4-4Cl CH2C6H3-2,5Cl CH2C6H3-3,4Cl CH2C6H4-4F CH2C6H3-2,5F CH2C6H3-3,4F CH2C6H4-4Br CH2C6H4-2Br CH2C6H4-3Br CH2C6H4-3OCH3 CH2C6H3-3,5CF3 C6H5 C6H5
Cl F Br F H F F H H H Br Br Br Br Br Br Br Br Br Br Br Br Br Br Br CH3 I
0.08 0.732 0.055 0.1 0.04 0.18 0.656 0.260 1.440 0.89 0.610 1.308 0.021 0.005 0.140 3.342 1.100 2.530 2.719 NA 3.000 NA 1.264 4.750 1.810 0.060 0.050
IC50 (μM)b
Abl (T315I)
c-Src
P210
parental
T315I
0.16 2.430 0.036 23.6 16.7 2.69 3.24 1.280 3.000 37.5 0.090 0.575 0.970 0.002 0.029 1.5 0.497 0.006 1.080 2.710 0.099 1.343 0.673 3.400 1.155 1.900 0.580
0.8 0.160 0.045 1.5 0.08 1.4 0.340 0.395 2.350 0.11 0.064 0.618 0.070 0.620 0.006 0.610 0.500 1.080 0.165 1.700 1.097 0.723 0.770 0.150 0.637 ND ND
2.7 >10 3.1 ND ND 7.5 >10 7.2 >10 ND 2.1 ND 4.7 0.8 ND ND ND 3.1 ND ND ND ND ND ND ND ND ND
0.5 >10 >10 ND ND >10 >10 >10 >10 ND >10 ND >10 >10 ND ND ND >10 ND ND ND ND ND ND ND ND ND
3.0 1.7 0.91 ND ND 3.8 3.3 1.7 8.3 ND 0.12 ND 4.5 0.22 ND ND ND 0.15 ND ND ND ND ND ND ND ND ND
c
−6
PAMPA Papp 10
d
cm/s (%MR)
16.6 (64) 13.9 (5.4) ND ND ND ND 6.8 (29.3) 3.20 (60) 7.5 (29.5) ND 6.64 (32.1) ND 9.1 (23.3) 8.78 (21) 4.24 (45.8) ND ND 10.7 (21.8) ND ND 3.41 (50.7) ND ND ND ND ND ND
solub H2O (μg mL−1)
met stab human (%)e
