Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line

doi:10.1016/j.ejmech.2016.07.034

European Journal of Medicinal Chemistry

Volume 123, 10 November 2016, Pages 1-13

https://doi.org/10.1016/j.ejmech.2016.07.034 Get rights and content

Highlights

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Some pyrazolo[3,4-d]pyrimidines were prepared as dual Src-Abl inhibitors.
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Compounds 7a, 7b revealed good activity against the T315I mutant Abl and Src.
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Compound 10b exerted equal inhibition against both Abl forms.
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10b, The rigide analog of 7b showed a decreased activity against both enzymes.
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These compounds showed promising cytotoxic activity against K-562 cell line.

Abstract

Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for further structural optimization to obtain better activity.

Graphical abstract

Introduction

Cancer is considered as a health burden worldwide due to its clinical and economic outcomes. It is the second leading cause of death after the cardiovascular diseases [1], [2]. The new cancer cases are expected to increase by as much as 15 million per year by 2020, according to the World Health Organization, unless further preventive measures are followed [3]. The Agency for Healthcare Research and Quality estimates that the direct medical costs (total of all health care expenditures) for cancer in the US in 2013 were $74.8 billion [1]. Chronic myeloid leukemia (CML) is a type of cancer that starts in certain blood-forming cells of the bone marrow. In CML, a genetic change takes place forming an abnormal gene called Bcr-Abl, which turns the cell into a CML cell. The leukemia cells grow and divide, building up in the bone marrow and spilling over into the blood. In time, the cells can also settle in other parts of the body, including the spleen. CML is a fairly slow growing leukemia, but it can also change into a fast-growing acute leukemia that is hard to treat [1]. The future of cancer treatment depends on development of targeted agents that specifically block key proteins involved in progression of specific types of cancer [4]. Therefore, the first-line treatment for CML was the selective inhibition of Bcr-Abl kinase activity by imatinib mesylate (Gleevec) I. However, resistance to Gleevec has been documented in patients through mutations both in and outside the Bcr-Abl kinase domain leading to interference with imatinib binding to Bcr-Abl [5]. As a consequence, there was a growing need for developing second-generation small molecule inhibitors able to treat Gleevec-resistant CML [6], [7], [8], [9].

Second generation Abl inhibitors were synthesized through rational drug design approaches to overcome resistance to I [10]. Recently, Novartis disclosed Nilotinib (Fig. 1, II), structurally related to I, which showed promising results in preclinical studies and was 10–25-fold more potent compared to I in cellular assays. Compound II also inhibited the growth of cell lines expressing Bcr-Abl mutants resistant to I [11]. On the other hand, unlike I and its derivatives, III (Fig. 1) interacted with the peptide substrate binding site of Abl and inhibited wild type kinase 10-fold more potently than I, and it also had activity against kinase domain mutations resistant to I [12].

Furthermore, many 4-amino-substituted pyrazolo[3,4-d]pyrimidines were synthesized and found to be active on different oncogenic tyrosine kinases and cancer cell lines depending on the nature and position of substituents on the heterocyclic core [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. Compound IV was an inhibitor of Abl (wild type) with K_i = 0.04 μM [22], while compound V had K_i = 0.025 μM on Abl T315I (mutant form) (Fig. 1) [14]. Although the most widely proposed mechanism for imatinib resistance is the presence of kinase domain mutations [5], cells from patients resistant to imatinib express an activated form of the (Src Family Kinases, SFKs) Lyn [24]. Moreover, disease progression and resistance to imatinib are correlated to over-expression and up-regulation of Hck (another SFK) and Lyn in CML blast-crisis patients [25], [26]. Therefore, dual Src and Abl inhibitors might be useful in the treatment of patients who have relapsed on imatinib. Fortunately, Abl is known to share significant sequence homology with Src and in its active conformation it bears remarkable structural resemblance with most SFKs [27]. As a result, many compounds originally developed as Src inhibitors frequently exhibited potent inhibition of Abl kinase [28], [29]. Thus, several second generation Bcr-Abl kinase inhibitors targeting both Src and Abl kinases were synthesized in order to overcome imatinib resistance, such as dasatinib VI and bosutinib VII (Fig. 2) [30], [31]. Additionally, many pyrazolo[3,4-d]pyrimidines that were designed originally as Src inhibitors, exhibited potent inhibition of Abl kinase as PP2, VIII [32] and compound IX (Fig. 2) that revealed Ki values 0.22 and 0.19 μM against Src and Abl enzymes, respectively [19].

It was found that most of the reported compounds carried substitutions at positions 1, 4 and 6 of the pyrazolo[3,4-d]pyrimidine core, therefore, this work aimed to explore the effect of the substitution of a phenyl ring at position 5 in addition to the functional moieties at positions 1 and 6 hoping to develop a better structure activity relationship. Moreover, some pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines were prepared to study the effect of this structure rigidification on the biological activity. Furthermore, compounds with promising activity on the enzyme have been screened against Leukemia K-562 cell lines. Moreover, the activity of the most active compound against both Abl and Src kinases has been validated through molecular modeling technique.

Section snippets

Chemistry

The target compounds were synthesized as outlined in Scheme 1, Scheme 2. Ethyl ethoxy methylene cyanoacetate 1 [33] was reacted with phenyl hydrazine according to the reported procedure [34] to obtain ethyl 5-amino-1-phenyl-1H-pyrazolo-4-carboxylate 2. The pyrazolo[3,4-d]pyrimidine core of 3 was formed from the reaction of 2 with phenyl isothiocyanate [35]. The reaction of 3 with methyl iodide in dry acetone in the presence of potassium carbonate afforded 4. ¹H NMR spectrum showed the presence

Conclusion

Some novel pyrazolo[3,4-d]pyrimidines and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines were synthesized through facile procedures. They revealed mild to moderate activity against both Src and Abl (wt) kinases. Compounds 7a, 7b and 10b were the most active compared to the other derivatives against both enzymes and the mutant Abl type T135I, and the leukemia K-562 cell line. However, one point of interest in the present study, is the identification of chemical scaffolds (compounds 7a, 7b and

Chemistry

Melting points were determined by open capillary tube method using Electrothermal 9100 melting point apparatus and were uncorrected. Elemental microanalyses were performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University. The infrared spectra (IR) were recorded as potassium bromide discs on Schimadzu FT-IR 8400S and Bruker FT-IR 12243136 spectrophotometers at Faculty of Pharmacy, Cairo University and Central Research Lab, Nahda University, respectively. ¹H NMR spectra

References (39)

H. Frankish
Lancet
(2003)
T. O'Hare et al.
Curr. Opin. Genet. Dev.
(2006)
C. Walz et al.
Crit. Rev. Oncol. Hematol.
(2006)
E. Weisberg et al.
Cancer Cell
(2005)
S. Schenone et al.
Eur. J. Med. Chem.
(2008)
C. Tintori et al.
Eur. J. Med. Chem.
(2009)
N.J. Donato et al.
Blood
(2003)
Y. Dai et al.
Biol. Chem.
(2004)
E. Dreassi et al.
Eur. J. Med. Chem.
(2009)
J.H. Hanke et al.
J. Biol. Chem.
(1996)

M. Chauhan et al.

Bioorg. Med. Chem.

(2013)

Cancers Facts and Figures

(2016)

D. Belpomme et al.

Int. J. Oncol.

(2007)

J.B. Gibbs

Science

(2000)

S.W. Cowan-Jacob et al.

Mini-Rev. Med. Chem.

(2004)

T. O'Hare et al.

Clin. Cancer Res.

(2005)

J. Cortes

Curr. Opin. Hematol.

(2006)

M. Azam et al.

Mol. Diagn. Ther.

(2006)

K. Gumireddy et al.

Proc. Natl. Acad. Sci. U.S.A.

(2005)

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Research paperNovel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemistry

Conclusion

Chemistry

Lancet

Curr. Opin. Genet. Dev.

Crit. Rev. Oncol. Hematol.

Cancer Cell

Eur. J. Med. Chem.

Eur. J. Med. Chem.

Blood

Biol. Chem.

Eur. J. Med. Chem.

J. Biol. Chem.

Bioorg. Med. Chem.

Cancers Facts and Figures

Int. J. Oncol.

Science

Mini-Rev. Med. Chem.

Clin. Cancer Res.

Curr. Opin. Hematol.

Mol. Diagn. Ther.

Proc. Natl. Acad. Sci. U.S.A.

Research paper
Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line