Design, synthesis and biological evaluation of 2-amino-N-(2-aminophenyl)thiazole-5-carboxamide derivatives as novel Bcr-Abl and histone deacetylase dual inhibitors

Abstract

In recent studies, combinations of histone deacetylase (HDAC) inhibitors with kinase inhibitor showed additive and synergistic effects. Herein we present a novel design approach for cancer drug development by combination of breakpoint cluster Abl (Bcr-Abl) and HDAC inhibitory activity, two independent pharmacological activities, in one molecule. The designed compounds were synthesized and tested, showing inhibitory activity against Bcr-Abl and HDAC1. The representative dual Bcr-Abl/HDAC inhibitors, compounds 6a and 6m, showed potent antiproliferative activities against human leukemia cell line K562 and prostate cancer cell line DU145 in cellular assays. This work may lay the foundation for developing dual Bcr-Abl/HDAC inhibitors as potential anticancer therapeutics.

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Introduction

In the past few years, the development of mechanism-based targeted antitumor drugs has made significant progress. However, their clinical effectiveness is generally unsustainable in long-term treatment and relapse is almost inevitable after treatment.¹ In addition, acquired drug resistance always limits the use of these agents. Since cancer is a disease with complex signaling networks, a therapeutic agent inhibiting only one key target in a tumor may not completely shut off the core hallmark capability, allowing some cancer cells to survive with residual function.¹ To address these problems, one particularly promising approach is incorporating the elements that simultaneously tackle multiple cancer-fighting targets into one molecule to obtain new chemical entity. This kind of therapeutic regimens can have superior efficacy and fewer side effects than single-target treatments.²

Protein kinases play key roles in the processes of governing cellular proliferation, differentiation and evasion from apoptosis as well. The Bcr-Abl tyrosine fusion protein is a constitutively active kinase, which is expressed in 95% of chronic myelogenous leukemia (CML), representing an extremely attractive target for CML therapy.³ Imatinib (the first generation of Bcr-Abl inhibitor), Nilotinib and Dasatinib (the second generation of Bcr-Abl inhibitors) inhibit this aberrant Bcr-Abl chimeric protein and interrupts the subsequent signal transduction that eventually lead to deregulated proliferation (Fig. 1). However, resistance to those drugs frequently stem from amplification of Bcr-Abl gene, the point mutations and increased efflux mediated by the multidrug resistance P-glycoprotein.^4,5 Additionally, serious side effects such as blood clotting in cardiac valves, chambers and cerebral vessels also exist.⁶ As a result, there is an urgent need for new approaches to combat the inevitable drug resistance and side effects.

Fig. 1 Representative structures of Bcr/Abl inhibitors.

Histone lysine acetylation is an epigenetic marker associated with gene transcriptional activation and repression.⁷ Histone deacetylases (HDACs) are a family of enzymes that catalyze the deacetylation of lysine residues located in the NH₂ terminal tails of core histones and promote a more closed chromatin structure where transcription is repressed.^8–10 They play an important role in epigenetic regulation of gene transcription and also control other cellular functions, such as proliferation, cell death, and motility.^11–13 HDAC inhibitors have been applied to the treatment of human cancers.¹⁴ Zolinza (vorinostat, SAHA) was approved by the FDA for the treatment of cutaneous T-cell lymphoma (CTCL).¹⁵ A benzamide HDAC inhibitor, MS-275, is reported to be a selective HDAC inhibitor targeting class I HDACs, which is a moderately potent HDAC inhibitor currently in phase II^16,17 (Fig. 2). Several small molecule HDAC inhibitors have entered clinical trials for the treatment of a variety haematological and solid tumors.¹⁸

Fig. 2 Representative structures of HDAC inhibitors.

Preclinical data with numerous cancer cell lines has shown synergistic and additive effects when combining HDAC inhibitors with various antitumor agents, including Bcr-Abl inhibitors.^19–23 HDAC inhibitors vorinostat and/or pracinostat (SB939) can also induce apoptosis in Bcr/Abl-expressing cells.²⁴ This evidence suggests that simultaneous Bcr-Abl and HDAC inhibition could be a promising approach in cancer therapy. The rationale for the dual-inhibitor design originated from our deep insights into previously reported X-ray crystal structure of Dasatinib bound to Abl (Fig. 3A) and MS-275 docked to HDAC1 homolog (Fig. 3B).^25,26 The 2-aminothiazole of Dasatinib occupies the ATP-binding pocket of Abl with three hydrogen bonds. A pair of hydrogen bonds was formed in the hinge region of the ATP-binding site between the 2-amino hydrogen of Dasatinib and the carbonyl oxygen of Met318 and between the 3-nitrogen of the thiazole ring of Dasatinib and the amide nitrogen of Met318. A hydrogen bond was also formed between the hydroxyl oxygen of Thr315 and the amide nitrogen of Dasatinib. It is suggested that the 2-aminothiazole motif is critical for binding to the protein. The pyrimidine occupies a hydrophobic cleft created by Leu248 and Gly321 and the piperazine group points toward the surface-exposed portion of the hinge region. The 2-chloro-6-methyl phenyl ring of Dasatinib is orthogonal to the thiazole carboxamide group and probes into a mostly hydrophobic pocket composed of the Thr315, Met290, Val299, Ile313, and Ala380.²⁵ In the case of HDAC, the docking results on MS-275 revealed that the 2-aminophenyl ring was inserted into the particle hydrophobic pocket composed of Ile313, Ala269, Val299 and Tyr297. 2-Aminophenylbenzamide moiety forms four hydrogen bonds with His131, Asp168, Gly140 and His140, respectively. The benzamide benzene and linked carbamate of MS-275 are modeled to extend through the channel to the solvent exposed region of the enzyme. The capping pyridine group binding to the protein surface. It is well established that the pharmacophore of HDACi (Fig. 2) consists of a capping group, an appropriate linker and a zinc-binding group (ZBG). Generally, the ZBG plays a significant role in the binding efficiency between HDACi and enzyme. 2-Aminophenylbenzamide is one of the most potent zinc ion chelating group among all types of ZBGs and shows the potent selectivity to HDAC1.¹⁷ Given the known binding modes of the Abl inhibitor Dasatinib and HDAC1 inhibitor MS-275, it was envisaged that amalgamation of these pharmacophores would be a viable strategy to single molecules retaining both activities. We selected the 2-aminophenylamide motif of MS-275 for combination with the 2-(pyrimidin-4-yl)aminothiazole core structure of Dasatinib since the hydrophobic cavities of Abl and HDAC1 share considerable similarities (Fig. 3). In the meantime, the 2-(pyrimidin-4-yl)aminothiazole core structure of Dasatinib may direct into the tubular channel to make it well occupied which may increase the affinity to HDAC1. The fused molecules were expected to retain the essential interactions with both proteins to exert desired biological functions.

Fig. 3 Design of dual inhibitors of Abl/HDAC (A) X-ray crystal structures of Dasatinib bound to Abl (PDB entry: 2GQG), (B) MS-275 docked to an HDAC1 homolog (PDB entry: 1C3R). The proteins are shown as white surface or cartoon. Dasatinib and MS-275 are shown in sticks. The hydrogen bonds were denoted by black dash lines. Figures were prepared using PyMOL.

Results and discussion

Chemistry

The synthetic route of our novel inhibitors are described in Schemes 1 and 2. 4,6-Dichloropyrimidine (1) reacted with methyl 2-aminothiazole-5-carboxylate (2) to form compound 3. Compound 3 was coupled with various amines to obtain intermediates 4a–k in basic condition. Hydrolysis of 4a–k yielded carboxylic acids 5a–k, which were than reacted with benzene-1,2-diamine or 4-fluorobenzene-1,2-diamine to form compounds 6a–k, 6m and 6n.

Scheme 1 Reagent and conditions: (a) Cs₂CO₃, DMF, 25 °C; (b) amine, n-BuOH, DIPEA, 120 °C, 12 h; (c) NaOH, H₂O, MeOH; (d) benzene-1,2-diamine or 4-fluorobenzene-1,2-diamine, TBTU, DIPEA, DMF, 4 h.

Scheme 2 Reagent and conditions: (a) aniline, TFA, reflux, 6 h; (b) NaOH, H₂O, MeOH; (c) benzene-1,2-diamine, TBTU, DIPEA, DMF, r.t., 4 h.

Compound 3 reacted with aniline to obtain intermediates 4l in acidic condition, which was then converted to compound 6l by the same method described above.

Biological assays

In vitro HDAC inhibition. HDAC1 enzymatic inhibitory activities of these N-(pyrimidin-4-yl)thiazol-2-amine derivatives were evaluated using MS-275 as a positive control. The results were calculated and tabulated as IC₅₀ values in Table 1.

Table 1 IC₅₀ values of compounds 6a–n against HDAC1 and Bcr-Abl^a

Compd	R^1	R^2	IC50^b of HDAC1 (μM)	IC50^b of Bcr-Abl (nM)
a ND: not determined.b Values were the average of three experiments, SD < 10%.
6a		H	0.15	21.2
6b		H	0.87	56.5
6c		H	0.61	210
6d		H	0.17	83.6
6e		H	0.25	55.9
6f		H	0.37	42.1
6g		H	0.28	29.8
6h		H	0.24	61.4
6i		H	0.42	50.5
6j		H	0.27	36.6
6k		H	0.56	172
6l		H	0.65	98.0
6m		F	0.81	17.8
6n		F	1.20	87.1
MS-275			0.19	ND
Dasatinib			ND	4.2

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All of the hybrids maintained anti-HDAC1 activity. Interestingly, compounds 6a and 6d exhibited better inhibitory activities against HDAC1 than MS-275. According to the data in Table 1, we found that heterocycle group of R¹ might have a great impact on the effectiveness of the set of compounds against HDAC1. For example, the compounds with anilino or benzyl amino group of R¹ (6c, 6k and 6l) showed poor inhibition on HDAC1. Replacement of the pyridyl group with a phenyl group (such as 6d, 6e and 6c) resulted in the decrease of enzymatic inhibitory activity. Besides, the compound 6b (IC₅₀ = 0.87 μM) showed poorer inhibitory activity against HDAC1 than compound 6j (IC₅₀ = 0.27 μM) without phenyl substituent. These results may suggest a preference for a certain hydrophilic group as a surface recognition cap group.

In addition, the fluorine substituent on the para-position of the amide also decreased the enzyme inhibitory activity. For instance, compound 6m (IC₅₀ = 0.81 μM) showed poorer inhibitory activity against HDAC1 than compound 6a (IC₅₀ = 0.15 μM) without the fluorine substituent. Moreover, the inhibitory activities of compound 6n also confirmed this conclusion. This result demonstrated that the fluorine substituent on the para-position of the amide is helpless for the ZBG to chelate with the Zn²⁺ ion located at the bottom of the active site of HDAC1.

In order to further ascertain the selectivity of HDAC1 inhibition, three representative compounds 6a, 6j and 6m with better HDAC1 inhibition were selected and evaluated against recombinant human HDAC1, HDAC3, HDAC6 and HDAC8 enzymes, using MS-275 as the positive control compound. As shown on Table 2, it is noteworthy that this set of structures show promising selectivity for HDAC1, as they are almost no activity in the inhibition of HDAC3, HDAC6 and HDAC8.

Table 2 Inhibition of HDAC isoforms by compound 6a, 6j and 6m^a

Compd	IC50^b (μM)
	HDAC1	HDAC3	HDAC6	HDAC8
a NA: no activity observed at 50 μM concentration tested.b Values were the average of three experiments, SD < 10%.
6a	0.126	NA	28.27	NA
6j	0.26	NA	NA	47.97
6m	0.810	40.48	NA	NA
MS-275	0.141	12.06	NA	NA

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In vitro Bcr-Abl inhibition. The data in Table 1 also demonstrated that compounds 6a–6n exhibited the potential inhibitory activity against Bcr-Abl, though they inhibited constitutively active Abl kinase in the biochemical assay with lower potency than that of Dasatinib. Compound 6m is the most active with an IC₅₀ of 17.8 nM. The activities of compounds 6b, 6c, 6d, 6e, 6h, 6k, 6l and 6n against Bcr-Abl were found to be less potent than other synthesized compounds. These results illustrated that the aromatic group of R¹ declined in Bcr-Abl inhibition. Compounds 6m and 6n relatively exhibited higher inhibitory activities than compounds 6a and 6l against Bcr-Abl. These results suggested that the fluorine substituent on the para-position of the amide was favorable for Bcr-Abl inhibition.

In vitro cell growth inhibition. To test the anticancer activities of the synthesized compounds, we evaluated antiproliferative activities of compounds 6a–d, 6f, 6g, 6j and 6m against human leukemia cell line (K562), human hepatocellular carcinoma cell line (Hepg2) and prostate cancer cell line (DU145) by applying the MTT colorimetric assay. The IC₅₀ values were summarized on Table 3. According to the inhibition data, all tested compounds showed obvious anti-proliferative activities. Moreover, DU145 was the most sensitive cell line to our synthesized Abl-Bcr/HDAC1 inhibitors and compounds 6g, 6m showed the better anti-proliferative activity in DU145 cell line than Dasatinib and MS-275. In addition, compounds 6a and 6m exhibited the most potent anti-proliferative activities in K562 cell line.

Table 3 Antiproliferative activities of compounds 6a–d, 6f, 6g, 6j and 6m^a

Compd	IC50^b (μM)
	K562	Hepg2	DU145
a ND: not determined.b Values were the average of two experiments, SD < 10%.
6a	2.56	16.53	5.79
6b	ND	ND	2.56
6c	ND	ND	6.75
6d	11.30	32.67	3.58
6f	8.25	18.56	3.58
6g	22.62	ND	1.75
6j	48.23	17.44	9.60
6m	2.62	10.44	0.60
Dasatinib	7.44	15.47	2.43
MS-275	21.30	9.56	3.75

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Molecular docking studies. For further understanding of the interaction between these inhibitors and proteins (Bcr-Abl and HDAC) and guiding the structure–activity relationships, we docked the representative compound 6a, the most potent compound of this series, in the active site of Abl (PDB code: 2GQG) (Fig. 4) and HDAC homolog (PDB code: 1C3R) (Fig. 5), revealing excellent shape complementarity between ligand and the binding pocket. As shown in Fig. 4A, compound 6a has a binding mode similar to that of Dasatinib as we expected. The aminothiazole ring of 6a occupied the ATP binding pocket. The three hydrogen bonds of the amino-NH and thiazyl-N with the backbone of Met318 (hinge), of the amide-NH with the side chain of Thr315 (hinge) are reproduced. The substituted pyrimidine moiety also projects into the solvent. 2-Aminophenyl moiety is aligned with a mainly hydrophobic cavity composed of the Thr315, Met290, Val299, Ile313, and Ala380 (Fig. 4B). The fluorine substitution of 2-aminophenyl moiety could increase the van der Waals interactions with the hydrophobic cavity. This may illustrate that compounds 6m and 6n relatively exhibited higher anti-Bcr-Abl inhibition than compounds 6a and 6l. As shown in Fig. 5, the 2-aminophenylthiazole carboxamide moiety of the compound 6a adopted a deeper position in the binding pocket to enable coordination of the amino nitrogen and carbonyl oxygen with the zinc ion. The hydrogen bond of the 2-amino substituent with the imidazolyl nitrogen of His131 may contribute to the tight fit. However, the fluorine substitution of 2-aminophenyl moiety could reduce the coordination of 2-amino nitrogen with the zinc ion due to the electronegativity of fluorine. For example, compounds 6m and 6n showed poorer inhibitory activity against HDAC1 than compounds 6a and 6l. The thiazyl ring of the thiazole carboxamide moiety is stacked between Phe198 and Phe141. The substituted pyrimidine moiety of 6a projects outward into the solvent, corresponding to the fact that the SARs of the 2-aminophenylbenzamides do not strongly depend on variations in this region.

Fig. 4 The docking model of 6a bound to Abl (PDB entry 2GQG). 6a is represented by tube and coloured by atom type (C, yellow; N, blue; O, red). The hydrogen bonds were denoted by black dash lines. Figures were prepared using PyMOL. (A) The protein is shown as ribbons. The key amino acids forming the pocket are represented by tube with carbon atoms colored in light blue, oxygen atoms in red and nitrogen atoms in blue. (B) The protein is shown as surface. The green area is the hydrophobic cavity composed of the Thr315, Met290, Val299, Ile313, and Ala380.

Fig. 5 The docking model of 6a bound to HDAC1 (PDB entry 1C3R). 6a is represented by tube and coloured by atom type (C, yellow; N, blue; O, red). The hydrogen bonds were denoted by black dash lines. Figures were prepared using PyMOL. (A) The protein is shown as ribbons. The key amino acids forming the pocket are represented by tube with carbon atoms colored in light blue, oxygen atoms in red, nitrogen atoms in blue and zinc in grey. (B) The protein is shown as surface.

Conclusions

In summary, we designed and synthesized a series of novel inhibitors with combinations of MS-275 with the Bcr-Abl inhibitor Dasatinib. The chimeric HDAC/kinase inhibitory compounds of the present study are based on the Dasatinib scaffold, bearing benzamide head groups which transfer the HDAC inhibitory property and determine the selectivity profile. As expected, these compounds exhibited distinct inhibitory activity against Bcr-Abl and HDAC1 and potent antiproliferative activities in vitro, especially against DU145. Compound 6g and 6m showed the most potent inhibitory activity against DU145. Compounds 6a and 6m exhibited higher potency against K562 than MS-275 and Dasatinib. By combining two distinct pharmacophores into one molecule, we have demonstrated the example of Bcr-Abl/HDAC dual inhibitors as a promising approach to search for efficient anticancer multi-target agents.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No. 81202410), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20120096120010) and Fundamental Research Funds for the Central Universities (Grant No. JKPZ2013003).

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Footnote

† Electronic supplementary information (ESI) available: Details of experimental procedure, spectral data of all novel compounds. See DOI: 10.1039/c6ra21271a

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