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Journal of Enzyme Inhibition and Medicinal Chemistry Volume 31, 2016 - Issue 6
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Short Communication

Synthesis of new thieno[2,3-b]pyridine derivatives as pim-1 inhibitors

Bassem H. NaguibPharmaceutical Chemistry Department, Faculty of Pharmacy, The British University in Egypt, Cairo, Egypt and ;Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
&
Hala B. El-NassanPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, EgyptCorrespondence[email protected]
Pages 1718-1725 | Received 27 Jul 2015, Accepted 14 Feb 2016, Published online: 22 Mar 2016

Abstract

Three series of 5-bromo-thieno[2,3-b]pyridines bearing amide or benzoyl groups at position 2 were prepared as pim-1 inhibitors. All the prepared compounds were tested for their pim-1 enzyme inhibitory activity. Two compounds (3c and 5b) showed moderate pim-1 inhibitory activity with IC50 of 35.7 and 12.71 μM, respectively. Three other compounds (3d, 3g and 6d) showed poor pim-1 inhibition. The most active compounds were tested for their cytotoxic activity on five cell lines [MCF7, HEPG2, HCT116, A549 and PC3]. Compound 3g was the most potent cytotoxic agent on almost all the cell lines tested.

Introduction

Pim kinases are a class of constitutively active serine/threonine kinases. The name pim came from the identification of pim-1 gene as a proviral insertion site of the Moloney murine leukemia virus-induced T-cell lymphomaCitation1. There are three known pim kinases [pim-1, -2, and -3] which are highly homologous (60–70%) in their kinase domains and have very closely related ATP binding siteCitation2,Citation3.

Pim kinases are involved in many biological processes such as cell survival, proliferation, differentiation, migration, metabolism and apoptosisCitation2–8. Besides, pim-1 was reported to act as a positive regulator of cell cycle progression at G1/S and G2/M transitionsCitation9,Citation10. Pim kinases are able to suppress apoptosis via direct phosphorylation of BCL-2-associated agonist of cell death (BAD) and thereby can act as oncogenic survival factorsCitation2,Citation4.

Overexpression of pim-1 and pim-2 were reported in hematologic cancers such as acute myeloid leukemia (AML), murine leukemia and lymphoma as well as solid cancers such as colon, bladder, stomach, pancreas and prostate cancersCitation2–7,11–13.

Therefore, targeting pim kinases may represent a promising and successful strategy to fight cancer. Indeed, many pim inhibitors especially pim-1 inhibitors had been designed and synthesized. Such inhibitors showed promising anticancer activity in vitro and in vivo, although only two of them have reached clinical trials against leukemia, lymphoma, bladder and prostate cancersCitation4,Citation11. These inhibitors belonged to several chemical classes including thiazolesCitation14, thiazolidinonesCitation15, pyridonesCitation16, pyrazinesCitation4,5,8,17,18, imidazopyridazinesCitation11,Citation19,Citation20, benzo-naphthyridinesCitation21, benzothienopyrimidinesCitation22, benzofuropyrimidinesCitation23 and benzofuran 2-carboxylic acidsCitation24.

Examining the crystal structure of pim-1 kinaseCitation25,Citation26 pointed out that the enzyme has several unique features that differentiate it from other kinases of known structures. The most important difference is the presence of a proline base at position 123 within the hinge region which prevents the formation of the second hydrogen bond between the hinge backbone and the adenine moiety of ATPCitation26. Besides, the hinge region (residues 121–126) contains Val126 insertion which enlarges the adjacent binding pocketCitation26. These differences allow for the development of selective pim inhibitors.

In 2011, Xiang et al.Citation24 reported the discovery of benzofuran-2-carboxylic acids (I, ) as potent and selective pim-1 inhibitors. The authors revealed the importance of the presence of hydrophobic groups at position 5, especially bromo group or heterocyclic groups. According to the X-ray crystallography, these groups fit into a hydrophobic pocket created by the hinge residue Pro125 and surrounded by Ala65, Arg122, Leu44 and Leu174 residues and analogs lacking such a hydrophobic group at position 5 showed limited or no pim-1 inhibitory activity. The 2-carboxylic acid group made a salt-bridge interaction with Lys67 and H-bond with the amino acids Glu89 and Asp186 via water molecule. Besides, a vacant space was present at the ribose-binding region which allowed for extra substitution at position 7 of the benzofuran core. This can accommodate a methoxy group or other larger basic groups. Although these derivatives have potent pim-1 enzyme inhibition and good ADME properties, no further anti-proliferative activity testing was done.

Figure 1. The design of the target compounds as pim-1 kinase inhibitors.

Figure 1. The design of the target compounds as pim-1 kinase inhibitors.

In the light of these findings, we thought of preparing selective pim-1 inhibitors through structure modifications of the active benzofuran-2-carboxylic acids. This was achieved via bioisosteric replacement of the benzofuran ring with thieno[2,3-b]pyridine ring. The bromine at position 5 was kept constant to ensure hydrophobic interaction within the pim-1 ATP active site. However, the acid group was replaced with amidic or benzoyl groups, both functional groups cannot form a salt bridge like the carboxylic acid group but they can form H-bonds with Lys67. Three series of compounds were synthesized by varying the substituents at position 2 [secondary aromatic amide (CONHAr), tertiary aliphatic amide (CONRR) and benzoyl groups (COAr)] to examine the effect of these modifications on the enzyme activity ().

All the prepared compounds were tested for their pim-1 enzyme inhibitory activity and the most active compounds were further tested for their anti-proliferative activity on five different cell lines. To the best of our knowledge, this is the first published work on thieno[2,3-b]pyridine derivatives as pim-1 inhibitors.

Results and discussion

Chemistry

Scheme 1 outlines the synthesis of the target thieno[2,3-b]pyridine derivatives 3a–h, 5a,b and 6a–d.

Scheme 1. Synthesis of the target compounds 3a–h, 5a,b and 6a–d.

Scheme 1. Synthesis of the target compounds 3a–h, 5a,b and 6a–d.

The synthesis of the target compounds was accomplished via reacting 5-bromo-4,6-dimethyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (1)Citation27 with 2-chloro-N-(substituted phenyl)acetamides 2a–hCitation28–30, 2-chloro-1–(4-substituted phenylpiperazin-1-yl)ethanones 4a,bCitation31 or substituted phenacyl bromides in sodium ethoxide to give 3-amino-N-(substituted phenyl)-thieno[2,3-b]pyridine-2-carboxamides 3a–h, (3-amino-thieno[2,3-b]pyridin-2-yl)(4-substituted phenylpiperazin-1-yl)methanones 5a,b and (3-amino-thieno[2,3-b]pyridin-2-yl)(substituted phenyl)methanones 6a–d, respectively.

The formation of compounds 3a–h and 5a,b was confirmed by IR spectra that indicated the disappearance of the C≡N band as well as the appearance of the NH/NH2 bands at 3493–3178 cm − 1 together with an amidic C=O band at 1635–1631 cm − 1. Whilst, IR spectra of compounds 6a–d showed the NH2 band at 3495–3234 cm − 1 and the C=O band at 1616–1595 cm − 1. The appearance of the C=O band at such low frequency might be attributed to the formation of intramolecular H-bond with the vicinal amino group as well as the conjugation with the neighboring heterocyclic ring.

1H NMR spectra of compounds 3a–h showed two exchangeable singlet signals at δ 6.99–7.26 ppm and δ 9.39–9.85 ppm corresponding to the NH2 and NH protons, respectively. While, 1H NMR spectra of compounds 5a,b and 6a–d showed an exchangeable singlet signal δ 5.91–6.71 ppm and δ 8.13–8.20 ppm corresponding to their NH2 protons, respectively.

13C NMR spectra of compounds 3a–f and 5b revealed the presence of the amidic C=O carbon signal at δ 164.2–167.3 ppm. While, 13C NMR spectrum of compound 6d showed the ketone C=O carbon signal at δ 188.1 ppm.

Kinase inhibitory activity

All the compounds were tested for their inhibitory activity against pim-1 kinase at 50 μM using Staurosporine at 1 μM as a positive control and the results are displayed in and represented graphically in .

Figure 2. Percentage inhibition of the test compounds against pim-1 kinase.

Figure 2. Percentage inhibition of the test compounds against pim-1 kinase.

Table 1. Results of pim-1 kinase inhibition achieved by the test compounds at 50 μM.

The results indicated that the most potent compounds were 5b (4-methoxyphenylpiperazine derivative) and 3c (3-chloro-4-fluorophenylamino derivative) that showed 85% and 73% inhibition, respectively. Besides, three other compounds, 3d, 3 g and 6d, showed moderate inhibition (43–34%). The rest of the compounds displayed weak inhibition of the enzyme that ranged between 8% and 27%.

Examining the structures of the compounds highlighted the following SAR as pim-1 inhibitors:

  • Regarding the phenylamino series (3a–3 h), it was found that the presence of para substituent enhanced the enzyme inhibition relative to the unsubstituted derivative 3a. Furthermore, the presence of 3,4-disubstitution (compounds 3c and 3d) greatly improved the enzyme inhibition.

  • Substitution of the phenylpiperazine ring greatly enhanced the pim-1 inhibition and afforded the most potent compound in this work.

  • Regarding the benzoyl series (6a–6d), substitution of the phenyl ring slightly increased the pim-1 inhibition.

  • The IC50 of the most active compounds (3c, 3d, 3 g, 5b and 6d) was determined. Compounds 3c and 5b displayed pim-1 IC50 values of 35.7 and 12.71 μM, respectively. The other compounds 3d, 3 g and 6d displayed high pim-1 IC50 values (>100 μM).

In vitro cytotoxic activity

The most active pim-1 inhibitors in this study, compounds 3c, 3d, 3 g, 5b and 6d were screened for their cytotoxic activity against five cell lines using MTT methodCitation32,Citation33. The cell lines examined were human breast adenocarcinoma (MCF7), human hepatocellular carcinoma (HEPG2), human colon adenocarcinoma (HCT116), human non-small lung cancer cells (A549) and human prostate cancer cells (PC3). The results in terms of IC50 in μM are displayed in and represented graphically in .

Figure 3. IC50 in μM of compounds 3c, 3d, 3 g, 5b and 6d on five cell lines.

Figure 3. IC50 in μM of compounds 3c, 3d, 3 g, 5b and 6d on five cell lines.

Table 2. Results of log p and in vitro cytotoxic screening of compounds 3c, 3d, 3 g, 5b and 6d on five cell lines.

The results indicated that all the compounds except compound 6d showed moderate to weak cytotoxic activity on all the cell lines tested with IC50 values between 32 and 302 μM. Compound 6d did not display significant anti-proliferative activity and its IC50 values were >1000 μM against all the tested cell lines.

MCF7 was the most sensitive cell line toward the effect of all the compounds followed by HEPG2, PC3, HCT116 then A549 cell line.

Compound 3 g was the most active on all the cell lines tested except HCT116 where compound 3d was the most active. The order of cytotoxic activity of the compounds on MCF7, HEPG2, A549 and PC3 cell lines was: compound 3 g, 3c, 5b then 3d.

In the light of pim-1 percentage inhibition and IC50 values, the expected order was 5b, 3c, 3d, 3 g then 6d. The variation in the results of cytotoxic screening can be accounted for by examining log p of the compounds ()Citation34. Calculated log p of the compounds showed that compound 3 g has the least log p (2.79) while compound 6d has the highest log p (5.34). The other three compounds have log p values between 4.29 and 4.92. Thus, it seemed that reducing log p of the compounds might improve its cytotoxic activity. Further optimization of the compounds is currently done to reduce their lipophilicity and improve their pim-1 inhibitory activity and cytotoxic activity.

Conclusion

Three series of 5-bromo-thieno[2,3-b]pyridines carrying 2-amide or 2-benzoyl groups were synthesized as pim-1 inhibitors. All the compounds were tested for their pim-1 enzyme inhibitory activity. Two compounds (3c and 5b) showed moderate pim-1 inhibitory activity with IC50 of 35.7 and 12.71 μM, respectively. Besides, three compounds (3d, 3 g and 6d) showed poor pim-1 inhibition with IC50 >100 μM. The most active compounds were tested for their cytotoxic activity on five cell lines [MCF7, HEPG2, HCT116, A549 and PC3]. Compound 3 g was the most potent cytotoxic agent on almost all the cell lines tested. The results of the cytotoxic screening were in good agreement with the calculated log p of the compounds. This indicated that reducing log p of the compounds might improve their cytotoxic activity. This is the first published work on the use of thieno[2,3-b]pyridine derivatives as pim-1 inhibitors. Further work is currently done to improve the enzyme inhibition and the physicochemical properties of these derivatives.

Experimental part

General

Melting points were determined using a Griffin apparatus and were uncorrected. IR spectra were recorded on Shimadzu IR 435 spectrophotometer and values were represented in cm −1. 1H NMR and 13C NMR spectra were carried out on Bruker 400 MHz and 100 MHz spectrophotometer, respectively, Faculty of Pharmacy, Cairo University, Cairo, Egypt, using TMS as an internal standard and chemical shifts were recorded in ppm on δ scale. Elemental analyses were carried out at the regional center for mycology and biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of the reactions and to check the purity of the products. All the reagents and solvents were purified and dried by standard techniques.

5-Bromo-4,6-dimethyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (1)Citation27, 2-chloro-N-(substituted phenyl)acetamides 2a–hCitation28–30 and 2-chloro-1–(4-substituted phenylpiperazin-1-yl)ethanones 4a,bCitation31 were prepared according to the published methods.

General procedure for the synthesis of 3-amino-5-bromo-N-(substituted phenyl)-4,6-dimethylthieno[2,3-b] pyridine-2-carboxamides 3a–h, (3-amino-5-bromo-4,6-dimethylthieno[2,3-b]pyridin-2-yl)(4-substituted phenylpiperazin-1-yl)methanones 5a,b and (3-amino-5-bromo-4,6-dimethylthieno[2,3-b]pyridin-2-yl) (substituted phenyl)methanones 6a–d

A mixture of 5-bromo-4,6-dimethyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (1) (0.24 g, 1 mmol) and 2-chloro-N-(substituted phenyl)acetamide 2a–h, 2-chloro-1–(4-substituted phenylpiperazin-1-yl)ethanone 4a,b or substituted phenacyl bromide (1 mmol) in sodium ethoxide solution (0.05 g Na metal in 15 mL absolute ethanol) was heated under reflux for 4 h. The solvent was concentrated under reduced pressure, and the solid formed was filtered, dried and crystallized from the suitable solvent.

3-Amino-5-bromo-4,6-dimethyl-N-phenylthieno[2,3-b] pyridine-2-carboxamide (3a)

Crystallized from ethyl acetate. Yield: 62%; m.p.: 269–270 °C; IR (cm − 1): 3325, 3207, 3184 (NH/NH2), 2953, 2926 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.71 (s, 3H, CH3), 2.90 (s, 3H, CH3), 7.04 (s, 2H, NH2, D2O exchangeable), 6.07–7.11 (t, 1H, Ar-H), 7.31–7.35 (t, 2H, Ar-H), 7.66–7.68 (d, 2H, Ar-H), 9.54 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 20.4, 27.0 (CH3), 99.4, 121.3, 121.7, 124.1, 124.8, 128.8, 139.1, 144.7, 149.0, 157.3, 157.8 (aromatic carbons), 164.4 (C=O); Anal. Calcd for C16H14BrN3OS: C, 51.07; H, 3.75; N, 11.17. Found: C, 51.24; H, 3.78; N, 11.39.

3-Amino-5-bromo-N-(4-chlorophenyl)-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (3b)

Crystallized from acetic acid. Yield: 72%; m.p.: 282–283 °C; IR (cm − 1): 3309, 3236, 3213 (NH/NH2), 2956, 2920 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.70 (s, 3H, CH3), 2.89 (s, 3H, CH3), 7.07 (s, 2H, NH2, D2O exchangeable), 7.37–7.39 (d, 2H, Ar-H, J = 8.84 Hz), 7.71–7.73 (d, 2H, Ar-H, J = 8.84 Hz), 9.63 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 20.4, 27.0 (CH3), 98.9, 121.3, 123.2, 124.7, 127.7, 128.7, 138.1, 144.7, 149.3, 157.4, 158.0 (aromatic carbons), 164.4 (C=O); Anal. Calcd for C16H13BrClN3OS: C, 46.79; H, 3.19; N, 10.23. Found: C, 46.93; H, 3.26; N, 10.45.

3-Amino-5-bromo-N-(3-chloro-4-fluorophenyl)-4,6-dimethylthieno [2,3-b]pyridine-2-carboxamide (3c)

Crystallized from ethanol. Yield: 85%; m.p.: 251–252 °C; IR (cm − 1): 3415, 3300, 3269 (NH/NH2), 2974, 2854 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.68 (s, 3H, CH3), 2.87 (s, 3H, CH3), 7.04 (br s, 2H, NH2, D2O exchangeable), 7.07–7.97 (m, 3H, Ar-H), 9.65 (br s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 20.4, 27.0 (CH3), 117.0, 119.2, 121.3, 122.0, 123.1, 124.7, 137.0, 144.7, 149.2, 152.4, 154.8, 157.4, 157.9 (aromatic carbons), 164.5 (C=O); Anal. Calcd for C16H12BrClFN3OS: C, 44.83; H, 2.82; N, 9.80. Found: C, 45.01; H, 2.85; N, 9.97.

3-Amino-5-bromo-4,6-dimethyl-N-(3,4-dimethylphenyl)thieno[2,3-b]pyridine-2-carboxamide (3d)

Crystallized from ethyl acetate. Yield: 90%; m.p.: 259–260 °C; IR (cm − 1): 3493, 3408, 3325 (NH/NH2), 2953, 2920 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.18 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.68 (s, 3H, CH3), 2.88 (s, 3H, CH3), 7.00 (s, 2H, NH2, D2O exchangeable), 7.05–7.07 (d, 1H, Ar-H, J = 8.10 Hz), 7.36–7.38 (d, 1H, Ar-H, J = 8.10 Hz), 7.44 (s, 1H, Ar-H), 9.39 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 19.2, 20.0 (CH3), 20.4, 26.9 (pyridine CH3), 99.7, 119.4, 121.2, 123.1, 124.9, 129.7, 131.9, 136.4, 136.7, 144.6, 148.7, 157.2, 157.7 (aromatic carbons), 164.2 (C=O); Anal. Calcd for C18H18BrN3OS: C, 53.47; H, 4.49; N, 10.39. Found: C, 53.71; H, 4.54; N, 10.52.

3-Amino-5-bromo-N-(4-methoxyphenyl)-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (3e)

Crystallized from toluene. Yield: 87%; m.p.: 268–269 °C; IR (cm−1): 3311, 3219, 3188 (NH/NH2), 2972, 2920 (CH-aliphatic), 1631 (C=O); 1H NMR (DMSO-d6) δ ppm 2.70 (s, 3H, CH3), 2.89 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 6.89–6.92 (d, 2H, Ar-H, J = 8.98 Hz), 6.99 (s, 2H, NH2, D2O exchangeable), 7.53–7.56 (d, 2H, Ar-H, J = 8.98 Hz), 9.42 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 20.3, 27.0 (CH3), 55.6 (CH3O), 99.6, 114.0, 121.3, 123.7, 124.9, 131.9, 144.6, 148.6, 156.1, 157.2, 157.7 (aromatic carbons), 164.2 (C=O); Anal. Calcd for C17H16BrN3O2S: C, 50.25; H, 3.97; N, 10.34. Found: C, 50.37; H, 4.01; N, 10.52.

3-Amino-5-bromo-4,6-dimethyl-N-(4-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide (3f)

Crystallized from acetic acid. Yield: 68%; m.p.: 253–254 °C; IR (cm − 1): 3294, 3213, 3178 (NH/NH2), 2947, 2916 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.27 (s, 3H, CH3), 2.69 (s, 3H, CH3), 2.88 (s, 3H, CH3), 7.00 (s, 2H, NH2, D2O exchangeable), 7.12–7.14 (d, 2H, Ar-H, J = 8.28 Hz), 7.53–7.55 (d, 2H, Ar-H, J = 8.28 Hz), 9.44 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 20.3, 20.9, 27.0 (CH3), 99.6, 121.3, 121.9, 124.8, 129.2, 133.1, 136.5, 144.6, 148.8, 157.2, 157.7 (aromatic carbons), 164.3 (C=O); Anal. Calcd for C17H16BrN3OS: C, 52.31; H, 4.13; N, 10.77. Found: C, 52.49; H, 4.21; N, 10.89.

3-Amino-5-bromo-4,6-dimethyl-N-(4-sulfamoylphenyl)thieno[2,3-b]pyridine-2-carboxamide (3g)

Crystallized from ethanol. Yield: 88%; m.p.: 246–247 °C; IR (cm − 1): 3419, 3408 (NH/NH2), 2900, 2800 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.65 (s, 3H, CH3), 2.86 (s, 3H, CH3), 7.14 (s, 2H, NH2, D2O exchangeable), 7.26 (s, 2H, NH2, D2O exchangeable), 7.76–7.78 (d, 2H, Ar-H, J = 8.22 Hz), 7.86–7.88 (d, 2H, Ar-H, J = 8.22 Hz), 9.85 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 19.7, 26.7 (CH3), 112.8, 120.2, 122.6, 126.3, 127.8, 130.4, 133.3, 142.4, 143.2, 154.4, 157.2 (aromatic carbons), 167.3 (C=O); Anal. Calcd for C16H15BrN4O3S2: C, 42.20; H, 3.32; N, 12.30. Found: C, 42.39; H, 3.30; N, 12.41.

3-Amino-5-bromo-4,6-dimethyl-N-[4-(pyrimidin-2-ylsulfamoyl)phenyl]thieno[2,3-b]pyridine-2-carboxamide (3h)

Crystallized from ethanol. Yield: 79%; m.p.: 296–297 °C; IR (cm − 1): 3388, 3300, 3246, 3215 (NH/NH2), 2900, 2920 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.70 (s, 3H, CH3), 2.89 (s, 3H, CH3), 5.40 (s, 1H, NH, D2O exchangeable), 7.07 (s, 2H, NH2, D2O exchangeable), 6.39–8.12 (m, 7H, Ar-H), 9.64 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 20.4, 27.0 (CH3), 99.2, 110.0, 112.3, 120.3, 121.3, 124.7, 127.6, 140.0, 142.2, 144.8, 149.3, 157.4, 157.6, 164.4 (aromatic carbons), 164.9 (C=O); Anal. Calcd for C20H17BrN6O3S2: C, 45.03; H, 3.21; N, 15.75. Found: C, 45.24; H, 3.19; N, 15.89.

(3-Amino-5-bromo-4,6-dimethylthieno[2,3-b]pyridin-2-yl)(4-phenylpiperazin-1-yl)methanone (5a)

Crystallized from toluene. Yield: 64%; m.p.: 169–170 °C; IR (cm − 1): 3400, 3205 (NH2), 2924, 2835 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.68 (s, 3H, CH3), 2.88 (s, 3H, CH3), 3.21 (s, 4H, piperaznyl CH2), 3.75 (s, 4H, piperaznyl CH2), 5.91 (s, 2H, NH2, D2O exchangeable), 6.79–7.26 (m, 5H, Ar-H); Anal. Calcd for C20H21BrN4OS: C, 53.94; H, 4.75; N, 12.58. Found: C, 54.12; H, 4.82; N, 12.74.

(3-Amino-5-bromo-4,6-dimethylthieno[2,3-b]pyridin-2-yl)[4–(4-methoxyphenyl)piperazin-1-yl]methanone (5b)

Crystallized from acetic acid. Yield: 79%; m.p.: 252–253 °C; IR (cm − 1): 3417, 3267 (NH2), 2953, 2821 (CH-aliphatic), 1635 (C=O); 1H NMR (DMSO-d6) δ ppm 2.64 (s, 3H, CH3), 2.84 (s, 3H, CH3), 3.06 (s, 4H, piperaznyl CH2), 3.68 (s, 3H, OCH3), 3.75 (s, 4H, piperaznyl CH2), 6.71 (s, 2H, NH2, D2O exchangeable), 6.80–6.82 (d, 2H, Ar-H, J = 8.82 Hz), 6.89–6.92 (d, 2H, Ar-H, J = 8.82 Hz); 13C NMR (DMSO-d6) δ ppm 20.2, 26.5 (CH3), 45.4, 50.5 (piperazine carbons), 55.6 (CH3O), 102.7, 114.7, 121.2, 121.7, 126.0, 138.4, 142.6, 144.1, 154.3, 157.5, 158.8 (aromatic carbons), 165.3 (C=O); Anal. Calcd for C21H23BrN4O2S: C, 53.06; H, 4.88; N, 11.79. Found: C, 53.18; H, 4.92; N, 12.01.

(3-Amino-5-bromo-4,6-dimethylthieno[2,3-b] pyridin-2-yl)(phenyl)methanone (6a)

Crystallized from ethanol. Yield: 52%; m.p.: 198–199 °C; IR (cm − 1): 3483, 3286 (NH2), 2953, 2918 (CH-aliphatic), 1610 (C=O); 1H NMR (DMSO-d6) δ ppm 2.68 (s, 3H, CH3), 2.90 (s, 3H, CH3), 7.52–7.75 (m, 5H, Ar-H), 8.13 (s, 2H, NH2, D2O exchangeable); Anal. Calcd for C16H13BrN2OS: C, 53.20; H, 3.63; N, 7.75. Found: C, 53.29; H, 3.69; N, 7.89.

(3-Amino-5-bromo-4,6-dimethylthieno[2,3-b] pyridin-2-yl)(3-bromophenyl)methanone (6b)

Crystallized from ethyl acetate. Yield: 25%; m.p.: 250–251 °C; IR (cm − 1): 3429, 3296 (NH2), 2900, 2800 (CH-aliphatic), 1616 (C=O); 1H NMR (DMSO-d6) δ ppm 2.67 (s, 3H, CH3), 2.87 (s, 3H, CH3), 7.50–7.86 (m, 4H, Ar-H), 8.20 (s, 2H, NH2, D2O exchangeable); Anal. Calcd for C16H12Br2N2OS: C, 43.66; H, 2.75; N, 6.36. Found: C, 43.93; H, 2.73; N, 6.58.

(3-Amino-5-bromo-4,6-dimethylthieno[2,3-b] pyridin-2-yl)(4-bromophenyl)methanone (6c)

Crystallized from ethyl acetate. Yield: 70%; m.p.: 239–240 °C; IR (cm − 1): 3491, 3234 (NH2), 2956, 2922 (CH-aliphatic), 1595 (C=O); 1H NMR (DMSO-d6) δ ppm 2.69 (s, 3H, CH3), 2.90 (s, 3H, CH3), 7.68–7.70 (d, 2H, Ar-H, J = 8.44 Hz), 7.74–7.76 (d, 2H, Ar-H, J = 8.44 Hz), 8.18 (s, 2H, NH2, D2O exchangeable); Anal. Calcd for C16H12Br2N2OS: C, 43.66; H, 2.75; N, 6.36. Found: C, 43.87; H, 2.81; N, 6.43.

(3-Amino-5-bromo-4,6-dimethylthieno[2,3-b] pyridin-2-yl)(4-chlorophenyl)methanone (6d)

Crystallized from ethanol. Yield: 85%; m.p.: 232–233 °C; IR (cm − 1): 3495, 3234 (NH2), 2900, 2800 (CH-aliphatic), 1595 (C=O); 1H NMR (DMSO-d6) δ ppm 2.68 (s, 3H, CH3), 2.89 (s, 3H, CH3), 7.59–7.61 (d, 2H, Ar-H, J = 8.10 Hz), 7.75–7.77 (d, 2H, Ar-H, J = 8.10 Hz), 8.17 (br s, 2H, NH2, D2O exchangeable); 13C NMR (DMSO-d6) δ ppm 20.7, 27.2 (CH3), 104.0, 121.3, 123.4, 129.1, 129.7, 136.3, 139.8, 146.1, 152.8, 159.8, 159.9 (aromatic carbons), 188.1 (C=O); Anal. Calcd for C16H12BrClN2OS: C, 48.56; H, 3.06; N, 7.08. Found: C, 48.81; H, 3.11; N, 7.13.

Kinase inhibitory activity

Materials and methods

The kinase inhibitory activity of the synthesized compounds was determined using the Kinexus compound profiling service, Canada. All the compounds were tested for their inhibitory activity against pim-1 kinase at 50 μM. The kinase used was cloned, expressed and purified using proprietary methods. Quality control testing is routinely performed to ensure compliance to acceptable standards. 33P-ATP was purchased from PerkinElmer. All other materials were of standard laboratory grade.

Kinase protein assays

A radioisotope assay format was used for profiling evaluation of protein kinase target and all assays were performed in a designated radioactive working area. Protein kinase assays were performed at ambient temperature for 20–30 min in a final volume of 25 μL according to the following assay reaction components: Component 1; 5 μl of diluted active protein kinase (∼10–50 nM final concentration in the assay). Component 2; 5 μl of stock solution of substrate. Component 3; 5 μl of kinase assay buffer. Component 4; 5 μl of the test compound, Staurosporine at 1 μM or 10% DMSO. Component 5; 5 μl of 33P-ATP (250 μM stock solution, 0.8 μCi).

The assay was initiated by the addition of 33P-ATP and the reaction mixture incubated at ambient temperature for 30 min. After the incubation period, the assay was terminated by spotting 10 μL of the reaction mixture onto a Multiscreen phosphocellulose P81 plate. The Multiscreen phosphocellulose P81 plate was washed 3 times for approximately 15 min each in a 1% phosphoric acid solution. The radioactivity on the P81 plate was counted in the presence of scintillation fluid in a Trilux scintillation counter. Blank control was set up that included all the assay components except the addition of the appropriate substrate (replaced with equal volume of assay dilution buffer). The corrected activity for protein kinase target was determined by removing the blank control value. The results were displayed in terms of percent inhibition and IC50 for the most active compounds. and show the obtained results.

In vitro cytotoxic activity

Cell culture

Cancer cells from different cancer cell lines were purchased from American type Cell Culture collection (ATCC, Manassas, VA). The cell lines used in this study were human breast adenocarcinoma (MCF7), human hepatocellular carcinoma (HEPG2), human colon adenocarcinoma (HCT116), human non-small lung cancer cells (A549) and human prostate cancer cells (PC3). The cell lines were grown on the appropriate growth medium Dulbecco's modified Eagle's medium (DMEM) or Roswell Park Memorial Institute medium (RPMI 1640) supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin and 10% of heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO2 atmosphere at 37 °C.

Cytotoxicity assay by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

Exponentially growing cells from different cancer cell lines were trypsinized, counted and seeded at the appropriate densities (2000–10 000 cells/0.33 cm2 well) into 96-well microtiter plates. The cells were incubated in a humidified atmosphere at 37 °C for 24 h. Then, the cells were exposed to different concentrations of compounds 3c, 3d, 3g, 5b and 6d (0.1, 10, 100 and 1000 μM) for 72 h. The viability of the treated cells was determined using MTT technique. The media were removed; cells were incubated with 200 μL of 5% MTT solution/well (Sigma Aldrich, St. Louis, MO) and were allowed to metabolize the dye into colored-insoluble formazan crystals for 2 h. The remaining MTT solution were discarded from the wells and the formazan crystals were dissolved in 200 μL/well acidified isopropanol for 30 min, covered with aluminum foil and with continuous shaking using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc., Kalamazoo, MI) at room temperature. The absorbance was measured at 570 nm using a Stat FaxR 4200 plate reader (Awareness Technology, Inc., Palm City, FL). The cell viability were expressed as percentage of control and the concentration that induces 50% of maximum inhibition of cell proliferation (IC50) was determined for each compound using Graph Pad Prism version 5 software (Graph Pad software Inc., San Diego, CA)Citation32,Citation33. The results are given in and represented graphically in .

Acknowledgments

The authors thank Dr. Tamer Abdelghany, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt, for carrying out the cytotoxic screening. The authors are grateful to Kinexus lab, Canada, for performing the kinase inhibitory assay.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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