Accepted Manuscript Novel thiazole-thiophene conjugates as adenosine receptor antagonists: Synthesis, biological evaluation and docking studies Dhaivat H. Pandya, Jayesh A. Sharma, Hitesh B. Jalani, Amit N. Pandya, V. Sudarsanam, Sonja Kachler, Karl Norbert Klotz, Kamala K. Vasu PII: DOI: Reference:
S0960-894X(15)00052-9 http://dx.doi.org/10.1016/j.bmcl.2015.01.040 BMCL 22380
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
28 October 2014 16 January 2015 20 January 2015
Please cite this article as: Pandya, D.H., Sharma, J.A., Jalani, H.B., Pandya, A.N., Sudarsanam, V., Kachler, S., Klotz, K.N., Vasu, K.K., Novel thiazole-thiophene conjugates as adenosine receptor antagonists: Synthesis, biological evaluation and docking studies, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/ 10.1016/j.bmcl.2015.01.040
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Novel thiazole-thiophene conjugates as adenosine receptor antagonists: Synthesis, biological evaluation and docking studies† Dhaivat H. Pandyaa¶, Jayesh A. Sharmaa, Hitesh B. Jalania, Amit N. Pandyaa, V. Sudarsanama, Sonja Kachlerb, Karl Norbert Klotzb, Kamala K. Vasua* Department of Medicinal Chemistry, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad-380 054, Gujarat, India. b
Institut für Pharmakologie und Toxikologie, Julius-Maximilians-Universität Würzburg, Germany. †Communication
Ref. No.: PERD250714
*Corresponding author. Tel.: + 91 79 27439375; Fax: + 91 79 27450449 E-mail: [email protected] ¶
Dhaivat H. Pandya is registered research scholar in Institute of Science, Nirma University.
Abstract: Here we report novel thiazole-thiophene conjugates as adenosine receptor antagonists. All the molecules were evaluated for their binding affinity for adenosine receptors. Most of the molecules were found to interact with the A1, A2A and A3 adenosine receptor subtypes with good affinity values. The most potent and selective compound 8n showed an A3 Ki value of 0.33 µM with selectivity ratios of > 90 versus the A1 and > 30 versus the A2 subtypes. For compound 8n docking studies into the binding site of the A3 adenosine receptor are provided to visualize its binding mode.
Key Words: adenosine receptors, thiazoles, thiophenes, molecular docking
1
Adenosine regulates a variety of cellular functions through interaction with the four G-protein coupled receptor (GPCR) subtypes A1, A2A, A2B and A3. The A1 and A3 adenosine receptors couple to Gi and thereby inhibit adenylyl cyclase (AC) with a consequent decrease of cellular cAMP levels. The A2A and A2B receptor couple to Gs and mediate a stimulation of AC resulting in enhanced intracellular cAMP levels.1,2 Adenosine receptor subtypes are a target of great interest because of their pathological involvement in numerous diseases. Activation of adenosine receptors is beneficial in many conditions like epilepsy, pain, cancer, etc. whereas inhibition of adenosine receptors is helpful in Parkinson’s disease, Alzheimer’s disease, asthma, diabetes and cancer.1,2,3 Development of adenosine receptor antagonists is a major focus of medicinal chemistry since several decades. Many selective antagonists have been developed so far and are helpful in pathological conditions like renal failure (A1), Parkinson’s disease (A2A), diabetes (A2B), asthma and chronic obstructive pulmonary disease COPD (A2B/A3), glaucoma and cancer (A3). The traditional xanthine derivatives like caffeine and theophylline are naturally occurring nonselective antagonists. Selective antagonists have been developed by modifying the xanthine or adenine scaffolds, or heterocyclic moieties with mono-, bi- or tricyclic ring systems.4 Several of them are in preclinical or clinical trials and the selective A2A receptor antagonist Istradefylline got approval in Japan for the treatment of Parkinson’s disease (Figure 1). The major problem associated with xanthine derivatives is related to pharmacokinetic issues. Consequently, there is a need for new non-xanthine molecules. Structures incorporating thiazole, thiophene and benzothiazinones have been developed as selective adenosine receptor antagonists.5–7
INSERT FIGURE-1 HERE As published in many reports, aminothiazoles and aminothiophenes are two important structural elements in adenosine receptor antagonists. Such 2-aminothiophene substituents are also found in modulation of adenosine receptors.8 Based on similar observations, Aurelio et al published 2aminothiophene derivatives as adenosine receptor modulators9. Aminothiazole compounds as adenosine receptor antagonists have been reported from our group to show high affinity and selectivity.5,7 So keeping in mind the importance of both moieties, i.e. thiophene and thiazole, here we have designed novel thiazole-thiophene conjugates with an amide spacer (Figure-2).
2
INSERT FIGURE-2 HERE
The synthesis of thiazole-thiophenes (8a-8r, Table-1) was carried out by a four-step reaction. The respective starting derivatives of 2-chloroacetamidothiophene and amidinothiourea were prepared
separately.
The
reaction
between
2-chloroacetamidothiophenes
(4)
and
amidinothioureas (7) was carried out to get the final thiazole-thiophene conjugate compounds (Scheme-1). For the preparation of 2-chloroacetamidothiophene, 2-aminothiophenes (3) were prepared by the Gewald reaction10 utilizing various carbonyl compounds (1), active methylene nitriles (2) and elemental sulphur in presence of morpholine as a base. This 2-aminothiophene (3) was further reacted with chloroacetyl chloride in THF with triethyl amine as a base at 0-50C to furnish 2chloroacetamidothiophene derivatives (4a-d, Scheme-1).
Our group generated many novel biologically important ligands from the intermediate amidinothiourea (7).11–13 Various isothiocyanates were reacted with different amidine derivatives in THF to give the amidinothiourea (7a-h, Scheme-1).
The amidinothiourea was reacted with 2-chloroacetamidothiophenes in DMF to give the final thiazolyl-thiophenes (8a-r, Table-1). The reaction takes place by the S-alkylation of amidinothiourea followed by the 5-exo-trig cyclization and removal of secondary amine to furnish the desired thiazolyl-thiophenes (Scheme-1).
INSERT SCHEME HERE
All the molecules were screened for their binding affinity for human adenosine receptor subtypes by radioligand binding at hA1, hA2A, hA3 as previously described by Klotz et al.14,15 Interestingly, 15 out of 18 synthesized thiazole-thiophene conjugates showed micromolar or sub micromolar affinity towards hA3 adenosine receptors. None of the compounds showed measurable interaction with the hA2B subtype (Ki > 10 µM, not shown). Compounds 8c, 8d and 8n showed the highest affinity (Ki 2.2, 0.52, 0.33 µM, respectively) and high selectivity (≥ 1490fold) towards the hA3 adenosine receptor (Table 1). Compound 8k also showed high A3 3
affinity but was not very selective (≤ tenfold) due to significant affinity for the A1 and A2A subtypes (Ki 2.3 and 3.4 µM, respectively) (Table-1). The three compounds 8c, 8d and 8n with the highest A3 affinity and selectivity were characterized by the presence of a 4-methylphenyl or 4-methoxyphenyl group at R4 and R5 position. These substituents might be responsible for good A3 affinity and selectivity. Replacement of 4-methylphenyl in R4 position for an unsubstituted phenyl resulted invariably in nonselective compounds (e.g. 8a, 8e, 8k) (Table-1). Also, a dimethylamino group in R5 position produced nonselective ligands (Table-1).
INSERT TABLE-1 HERE
To see the possible interactions of the newly synthesized ligands with the A3 adenosine receptor flexible docking studies into an A3 homology model (PDB id: 1OEA) were carried out in Surflex-Dock, SYBYL X-2.0. The three A3-selective compounds 8c, 8d and 8n, and the nonselective ligand 8k, showing the highest affinity, were investigated. All four molecules were found to interact with the receptor (Supplymentary figure S1) with the amino acids ASN250, PHE168, GLN167 and SER170 through hydrogen bonding. The common interaction site of the four active molecules with the receptor was the amide spacer present in the molecule. All of these four molecules were found to interact through hydrogen bonding to the amino acid ASN250 which is reported to be responsible for antagonist interaction.16 In the most potent molecule 8n, the carbonyl oxygen of –CONH2 present on the 3-position of the thiophene ring interacted with a hydrogen of the ASN250 amido group. The carbonyl oxygen of the amide linker present between the thiazole and thiophene has shown three interactions, namely with the second amido hydrogen of ASN250 and with the two amido hydrogens of GLN167. The amine spacer hydrogen present of the 2nd position of thiazole ring was found to interact with the hydroxyl oxygen of the SER170. Figure-3 shows the highest consensus score (CScore = 4) pose of the molecule 8n in the cavity of the ligand binding site. The study documents interaction of the new compounds with ligand binding domain of the A3 receptor and also shows the important role of the amide spacer between the two ring systems which may be responsible for good antagonist affinity. INSERT FIGURE 3 HERE
4
In conclusion, novel thiazole-thiophene conjugates were designed, synthesized, characterized and evaluated for their binding to adenosine receptors. Most of the novel compounds synthesized were found to interact with adenosine receptors showing that the chosen thiazole-thiophene conjugate structure is favorable for the interaction with adenosine recpetors. In particular, some were found to bind with high affinity and selectivity to the A3 adenosine receptor. The most potent and selective molecule 8n bound to the A3 subtype with a Ki-value of 0.33 µM. The four compounds revealing the highest A3 affinity were docked into a homology model of the A3 adenosine receptor confirming their similar interaction with the ligand binding domain.
Acknowledgement We would like to acknowledge B.V. Patel PERD Centre, for providing research facilities. We also thank Industrial Commissioner (IC) of Gujarat and Indian Council of Medical Research (ICMR) for their financial assistance. We thank Prof. C. J. Shishoo and Dr. Manish Nivsarkar, Directors, B.V. Patel PERD Centre, for their constant encouragement and support. References: 1.
Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K. N.; Linden, J. Pharmacol. Rev. 2001, 53, 527–552.
2.
Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Linden, J.; Müller, C. E. Pharmacol. Rev. 2011, 63, 1–34.
3.
Chen, J.-F.; Eltzschig, H. K.; Fredholm, B. B. Nat. Rev. Drug Discov. 2013, 12, 265–286.
4.
Müller, C. E.; Jacobson, K. A. Biochim. Biophys. Acta 2011, 1808, 1290–1308.
5.
Scheiff, A. B.; Yerande, S. G.; El-Tayeb, A.; Li, W.; Inamdar, G. S.; Vasu, K. K.; Sudarsanam, V.; Müller, C. E. Bioorg. Med. Chem. 2010, 18, 2195–2203.
6.
Gütschow, M.; Schlenk, M.; Gäb, J.; Paskaleva, M.; Alnouri, M. W.; Scolari, S.; Iqbal, J.; Müller, C. E. J. Med. Chem. 2012, 55, 3331–3341.
7.
Inamdar, G. S.; Pandya, A. N.; Thakar, H. M.; Sudarsanam, V.; Kachler, S.; Sabbadin, D.; Moro, S.; Klotz, K.-N.; Vasu, K. K. Eur. J. Med. Chem. 2013, 63, 924–934.
8.
Göblyös, A.; Ijzerman, A. P. Biochim. Biophys. Acta 2011, 1808, 1309–1318.
5
9.
Aurelio, L.; Christopoulos, A.; Flynn, B. L.; Scammells, P. J.; Sexton, P. M.; Valant, C. Bioorg. Med. Chem. Lett. 2011, 21, 3704–3707.
10.
Gewald, K.; Schinke, E.; Böttcher, H. Chem. Ber. 1966, 99, 94–100.
11.
Kaila, J. C.; Baraiya, A. B.; Vasu, K. K.; Sudarsanam, V. Tetrahedron Lett. 2008, 49, 7220–7222.
12.
Giri, R. S.; Thaker, H. M.; Giordano, T.; Chen, B.; Nuthalapaty, S.; Vasu, K. K.; Sudarsanam, V. Eur. J. Med. Chem. 2010, 45, 3558–3563.
13.
Kaila, J. C.; Baraiya, A. B.; Pandya, A. N.; Jalani, H. B.; Sudarsanam, V.; Vasu, K. K. Tetrahedron Lett. 2010, 51, 1486–1489.
14.
Klotz, K.-N.; Hessling, J.; Hegler, J.; Owman, C.; Kull, B.; Fredholm, B. B.; Lohse, M. J. Naunyn. Schmiedebergs. Arch. Pharmacol. 1997, 357, 1–9.
15.
Klotz, K.-N.; Falgner, N.; Kachler, S.; Lambertucci, C.; Vittori, S.; Volpini, R.; Cristalli, G. Eur. J. Pharmacol. 2007, 556, 14–18.
16.
Wei, J.; Li, H.; Qu, W.; Gao, Q. Neurochem. Int. 2009, 55, 637–642.
6
Figure 3: The most active molecule 8n in the active site of adenosine A3 receptor showing hydrogen interactions (PDB Id: 1OEA)
Table 1: Binding affinity (Ki) of synthesized compounds at adenosine receptors with selectivity
No 8a 8b 8c 8d 8e 8f 8g 8h 8i 8j 8k
R1
R2
Ki (µM) hA2Ab 6.45
R3
R4
R5
-(CH2)4-
-COOEt
C6H4
-N(Me)2
-(CH2)4-
-COOEt
4-MeC6H4
Me
> 30
> 30
-(CH2)4-
-COOEt
4-MeC6H4
4-MeC6H4
> 30
> 30
-(CH2)4-
-COOEt
4-OMe C6H4
4-MeC6H4
> 10
> 10
-(CH2)4-
-COOEt
C6H4
4-MeC6H4
-(CH2)4-
-COOEt
4-Me C6H4
-N(Me)2
-(CH2)4-
-COOEt
CH3OCO
-N(Me)2
> 100
> 100
-(CH2)4-
-CN
C6H4
-N(Me)2
> 30
> 30
-(CH2)4-
-CN
4-MeC6H4
-N(Me)2
> 100
> 10
-(CH2)4-
-CONH2
4-MeC6H4
-N(Me)2
-(CH2)4-
-CONH2
C6H4
4-MeC6H4
a
hA1 40.8
(36.2 – 45.9)
7.75 (6.42 – 9.35)
23.5 (18.8 – 29.3)
32.4 (21.4 – 49.3)
c
hA3 4.12
(3.91 – 10.6) (3.94 – 4.32)
28.4
8.22 (6.96 – 9.70)
2.16 (1.25 – 3.73)
0.520 (347 – 778)
4.09
(17.1 – 47.2) (2.28 – 7.33)
18.0
4.71
(13.6 – 12.8) (3.91 – 5.67)
22.3
> 30 35.6 (25.3 – 50.2)
34.0 (30.4 – 38.0)
7.13
(15.8 – 31.5) (6.40 – 7.95)
2.38
3.44
0.334
(1.68 – 3.38)
(2.10 – 5.62)
(236 – 473)
Selectivity hA1/hA3 hA2A/hA3 9.9
1.6
> 3.7
> 3.7
> 14
> 14
> 19
> 19
1.9
6.9
5.0
3.8
> 3.3
> 3.3
> 0.84
> 0.84
> 2.9
> 0.29
4.5
3.1
7.1
10
8l 8m 8n 8o 8p 8q 8r
-(CH2)4-
-CN
4-MeC6H4
4-MeC6H4
> 10
> 10
-(CH2)4-
-CN
4-OMeC6H4
4-MeC6H4
> 30
> 30
-(CH2)4-
-CONH2
4-MeC6H4
4-MeC6H4
> 30
> 10
-(CH2)4-
-CN
4-MeC6H4
Me
> 30
-COOMe
Me
-COOEt
4-OMeC6H4
-N(Me)2
-COOMe
Me
-COOEt
4-MeC6H4
Me
-COOMe
Me
-COOEt
4-MeC6H4
-N(Me)2
14.0 (12.0 – 16.5)
5.67
> 10 9.56 (6.05 – 15.1)
0.33 (239 – 456)
3.87
(5.01 – 6.41) (3.01 – 4.99)
9.66
2.73
(7.30 – 12.8) (2.11 – 3.55)
> 30
> 30
>30
19.0
26.6
3.91
(15.4 – 23.6)
(19.8 – 35.9) (2.98 – 5.11)
Data are expressed as geometric means with 95% confidence intervals in parentheses. a Displacement of specific [3H]CCPA binding at human A1 receptors expressed in CHO cells. b Displacement of specific [3H]NECA binding at human A2A receptors expressed in CHO cells. c Displacement of specific [3H]HEMADO binding at human A3 receptors expressed in CHO cells.
-
-
> 3.1
> 3.1
> 90
> 30
> 7.8
1.5
5.1
3.5
-
0.33
4.9
6.8
S0960-894X(15)00052-9 http://dx.doi.org/10.1016/j.bmcl.2015.01.040 BMCL 22380
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
28 October 2014 16 January 2015 20 January 2015
Please cite this article as: Pandya, D.H., Sharma, J.A., Jalani, H.B., Pandya, A.N., Sudarsanam, V., Kachler, S., Klotz, K.N., Vasu, K.K., Novel thiazole-thiophene conjugates as adenosine receptor antagonists: Synthesis, biological evaluation and docking studies, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/ 10.1016/j.bmcl.2015.01.040
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Novel thiazole-thiophene conjugates as adenosine receptor antagonists: Synthesis, biological evaluation and docking studies† Dhaivat H. Pandyaa¶, Jayesh A. Sharmaa, Hitesh B. Jalania, Amit N. Pandyaa, V. Sudarsanama, Sonja Kachlerb, Karl Norbert Klotzb, Kamala K. Vasua* Department of Medicinal Chemistry, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad-380 054, Gujarat, India. b
Institut für Pharmakologie und Toxikologie, Julius-Maximilians-Universität Würzburg, Germany. †Communication
Ref. No.: PERD250714
*Corresponding author. Tel.: + 91 79 27439375; Fax: + 91 79 27450449 E-mail: [email protected] ¶
Dhaivat H. Pandya is registered research scholar in Institute of Science, Nirma University.
Abstract: Here we report novel thiazole-thiophene conjugates as adenosine receptor antagonists. All the molecules were evaluated for their binding affinity for adenosine receptors. Most of the molecules were found to interact with the A1, A2A and A3 adenosine receptor subtypes with good affinity values. The most potent and selective compound 8n showed an A3 Ki value of 0.33 µM with selectivity ratios of > 90 versus the A1 and > 30 versus the A2 subtypes. For compound 8n docking studies into the binding site of the A3 adenosine receptor are provided to visualize its binding mode.
Key Words: adenosine receptors, thiazoles, thiophenes, molecular docking
1
Adenosine regulates a variety of cellular functions through interaction with the four G-protein coupled receptor (GPCR) subtypes A1, A2A, A2B and A3. The A1 and A3 adenosine receptors couple to Gi and thereby inhibit adenylyl cyclase (AC) with a consequent decrease of cellular cAMP levels. The A2A and A2B receptor couple to Gs and mediate a stimulation of AC resulting in enhanced intracellular cAMP levels.1,2 Adenosine receptor subtypes are a target of great interest because of their pathological involvement in numerous diseases. Activation of adenosine receptors is beneficial in many conditions like epilepsy, pain, cancer, etc. whereas inhibition of adenosine receptors is helpful in Parkinson’s disease, Alzheimer’s disease, asthma, diabetes and cancer.1,2,3 Development of adenosine receptor antagonists is a major focus of medicinal chemistry since several decades. Many selective antagonists have been developed so far and are helpful in pathological conditions like renal failure (A1), Parkinson’s disease (A2A), diabetes (A2B), asthma and chronic obstructive pulmonary disease COPD (A2B/A3), glaucoma and cancer (A3). The traditional xanthine derivatives like caffeine and theophylline are naturally occurring nonselective antagonists. Selective antagonists have been developed by modifying the xanthine or adenine scaffolds, or heterocyclic moieties with mono-, bi- or tricyclic ring systems.4 Several of them are in preclinical or clinical trials and the selective A2A receptor antagonist Istradefylline got approval in Japan for the treatment of Parkinson’s disease (Figure 1). The major problem associated with xanthine derivatives is related to pharmacokinetic issues. Consequently, there is a need for new non-xanthine molecules. Structures incorporating thiazole, thiophene and benzothiazinones have been developed as selective adenosine receptor antagonists.5–7
INSERT FIGURE-1 HERE As published in many reports, aminothiazoles and aminothiophenes are two important structural elements in adenosine receptor antagonists. Such 2-aminothiophene substituents are also found in modulation of adenosine receptors.8 Based on similar observations, Aurelio et al published 2aminothiophene derivatives as adenosine receptor modulators9. Aminothiazole compounds as adenosine receptor antagonists have been reported from our group to show high affinity and selectivity.5,7 So keeping in mind the importance of both moieties, i.e. thiophene and thiazole, here we have designed novel thiazole-thiophene conjugates with an amide spacer (Figure-2).
2
INSERT FIGURE-2 HERE
The synthesis of thiazole-thiophenes (8a-8r, Table-1) was carried out by a four-step reaction. The respective starting derivatives of 2-chloroacetamidothiophene and amidinothiourea were prepared
separately.
The
reaction
between
2-chloroacetamidothiophenes
(4)
and
amidinothioureas (7) was carried out to get the final thiazole-thiophene conjugate compounds (Scheme-1). For the preparation of 2-chloroacetamidothiophene, 2-aminothiophenes (3) were prepared by the Gewald reaction10 utilizing various carbonyl compounds (1), active methylene nitriles (2) and elemental sulphur in presence of morpholine as a base. This 2-aminothiophene (3) was further reacted with chloroacetyl chloride in THF with triethyl amine as a base at 0-50C to furnish 2chloroacetamidothiophene derivatives (4a-d, Scheme-1).
Our group generated many novel biologically important ligands from the intermediate amidinothiourea (7).11–13 Various isothiocyanates were reacted with different amidine derivatives in THF to give the amidinothiourea (7a-h, Scheme-1).
The amidinothiourea was reacted with 2-chloroacetamidothiophenes in DMF to give the final thiazolyl-thiophenes (8a-r, Table-1). The reaction takes place by the S-alkylation of amidinothiourea followed by the 5-exo-trig cyclization and removal of secondary amine to furnish the desired thiazolyl-thiophenes (Scheme-1).
INSERT SCHEME HERE
All the molecules were screened for their binding affinity for human adenosine receptor subtypes by radioligand binding at hA1, hA2A, hA3 as previously described by Klotz et al.14,15 Interestingly, 15 out of 18 synthesized thiazole-thiophene conjugates showed micromolar or sub micromolar affinity towards hA3 adenosine receptors. None of the compounds showed measurable interaction with the hA2B subtype (Ki > 10 µM, not shown). Compounds 8c, 8d and 8n showed the highest affinity (Ki 2.2, 0.52, 0.33 µM, respectively) and high selectivity (≥ 1490fold) towards the hA3 adenosine receptor (Table 1). Compound 8k also showed high A3 3
affinity but was not very selective (≤ tenfold) due to significant affinity for the A1 and A2A subtypes (Ki 2.3 and 3.4 µM, respectively) (Table-1). The three compounds 8c, 8d and 8n with the highest A3 affinity and selectivity were characterized by the presence of a 4-methylphenyl or 4-methoxyphenyl group at R4 and R5 position. These substituents might be responsible for good A3 affinity and selectivity. Replacement of 4-methylphenyl in R4 position for an unsubstituted phenyl resulted invariably in nonselective compounds (e.g. 8a, 8e, 8k) (Table-1). Also, a dimethylamino group in R5 position produced nonselective ligands (Table-1).
INSERT TABLE-1 HERE
To see the possible interactions of the newly synthesized ligands with the A3 adenosine receptor flexible docking studies into an A3 homology model (PDB id: 1OEA) were carried out in Surflex-Dock, SYBYL X-2.0. The three A3-selective compounds 8c, 8d and 8n, and the nonselective ligand 8k, showing the highest affinity, were investigated. All four molecules were found to interact with the receptor (Supplymentary figure S1) with the amino acids ASN250, PHE168, GLN167 and SER170 through hydrogen bonding. The common interaction site of the four active molecules with the receptor was the amide spacer present in the molecule. All of these four molecules were found to interact through hydrogen bonding to the amino acid ASN250 which is reported to be responsible for antagonist interaction.16 In the most potent molecule 8n, the carbonyl oxygen of –CONH2 present on the 3-position of the thiophene ring interacted with a hydrogen of the ASN250 amido group. The carbonyl oxygen of the amide linker present between the thiazole and thiophene has shown three interactions, namely with the second amido hydrogen of ASN250 and with the two amido hydrogens of GLN167. The amine spacer hydrogen present of the 2nd position of thiazole ring was found to interact with the hydroxyl oxygen of the SER170. Figure-3 shows the highest consensus score (CScore = 4) pose of the molecule 8n in the cavity of the ligand binding site. The study documents interaction of the new compounds with ligand binding domain of the A3 receptor and also shows the important role of the amide spacer between the two ring systems which may be responsible for good antagonist affinity. INSERT FIGURE 3 HERE
4
In conclusion, novel thiazole-thiophene conjugates were designed, synthesized, characterized and evaluated for their binding to adenosine receptors. Most of the novel compounds synthesized were found to interact with adenosine receptors showing that the chosen thiazole-thiophene conjugate structure is favorable for the interaction with adenosine recpetors. In particular, some were found to bind with high affinity and selectivity to the A3 adenosine receptor. The most potent and selective molecule 8n bound to the A3 subtype with a Ki-value of 0.33 µM. The four compounds revealing the highest A3 affinity were docked into a homology model of the A3 adenosine receptor confirming their similar interaction with the ligand binding domain.
Acknowledgement We would like to acknowledge B.V. Patel PERD Centre, for providing research facilities. We also thank Industrial Commissioner (IC) of Gujarat and Indian Council of Medical Research (ICMR) for their financial assistance. We thank Prof. C. J. Shishoo and Dr. Manish Nivsarkar, Directors, B.V. Patel PERD Centre, for their constant encouragement and support. References: 1.
Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K. N.; Linden, J. Pharmacol. Rev. 2001, 53, 527–552.
2.
Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Linden, J.; Müller, C. E. Pharmacol. Rev. 2011, 63, 1–34.
3.
Chen, J.-F.; Eltzschig, H. K.; Fredholm, B. B. Nat. Rev. Drug Discov. 2013, 12, 265–286.
4.
Müller, C. E.; Jacobson, K. A. Biochim. Biophys. Acta 2011, 1808, 1290–1308.
5.
Scheiff, A. B.; Yerande, S. G.; El-Tayeb, A.; Li, W.; Inamdar, G. S.; Vasu, K. K.; Sudarsanam, V.; Müller, C. E. Bioorg. Med. Chem. 2010, 18, 2195–2203.
6.
Gütschow, M.; Schlenk, M.; Gäb, J.; Paskaleva, M.; Alnouri, M. W.; Scolari, S.; Iqbal, J.; Müller, C. E. J. Med. Chem. 2012, 55, 3331–3341.
7.
Inamdar, G. S.; Pandya, A. N.; Thakar, H. M.; Sudarsanam, V.; Kachler, S.; Sabbadin, D.; Moro, S.; Klotz, K.-N.; Vasu, K. K. Eur. J. Med. Chem. 2013, 63, 924–934.
8.
Göblyös, A.; Ijzerman, A. P. Biochim. Biophys. Acta 2011, 1808, 1309–1318.
5
9.
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6
Figure 3: The most active molecule 8n in the active site of adenosine A3 receptor showing hydrogen interactions (PDB Id: 1OEA)
Table 1: Binding affinity (Ki) of synthesized compounds at adenosine receptors with selectivity
No 8a 8b 8c 8d 8e 8f 8g 8h 8i 8j 8k
R1
R2
Ki (µM) hA2Ab 6.45
R3
R4
R5
-(CH2)4-
-COOEt
C6H4
-N(Me)2
-(CH2)4-
-COOEt
4-MeC6H4
Me
> 30
> 30
-(CH2)4-
-COOEt
4-MeC6H4
4-MeC6H4
> 30
> 30
-(CH2)4-
-COOEt
4-OMe C6H4
4-MeC6H4
> 10
> 10
-(CH2)4-
-COOEt
C6H4
4-MeC6H4
-(CH2)4-
-COOEt
4-Me C6H4
-N(Me)2
-(CH2)4-
-COOEt
CH3OCO
-N(Me)2
> 100
> 100
-(CH2)4-
-CN
C6H4
-N(Me)2
> 30
> 30
-(CH2)4-
-CN
4-MeC6H4
-N(Me)2
> 100
> 10
-(CH2)4-
-CONH2
4-MeC6H4
-N(Me)2
-(CH2)4-
-CONH2
C6H4
4-MeC6H4
a
hA1 40.8
(36.2 – 45.9)
7.75 (6.42 – 9.35)
23.5 (18.8 – 29.3)
32.4 (21.4 – 49.3)
c
hA3 4.12
(3.91 – 10.6) (3.94 – 4.32)
28.4
8.22 (6.96 – 9.70)
2.16 (1.25 – 3.73)
0.520 (347 – 778)
4.09
(17.1 – 47.2) (2.28 – 7.33)
18.0
4.71
(13.6 – 12.8) (3.91 – 5.67)
22.3
> 30 35.6 (25.3 – 50.2)
34.0 (30.4 – 38.0)
7.13
(15.8 – 31.5) (6.40 – 7.95)
2.38
3.44
0.334
(1.68 – 3.38)
(2.10 – 5.62)
(236 – 473)
Selectivity hA1/hA3 hA2A/hA3 9.9
1.6
> 3.7
> 3.7
> 14
> 14
> 19
> 19
1.9
6.9
5.0
3.8
> 3.3
> 3.3
> 0.84
> 0.84
> 2.9
> 0.29
4.5
3.1
7.1
10
8l 8m 8n 8o 8p 8q 8r
-(CH2)4-
-CN
4-MeC6H4
4-MeC6H4
> 10
> 10
-(CH2)4-
-CN
4-OMeC6H4
4-MeC6H4
> 30
> 30
-(CH2)4-
-CONH2
4-MeC6H4
4-MeC6H4
> 30
> 10
-(CH2)4-
-CN
4-MeC6H4
Me
> 30
-COOMe
Me
-COOEt
4-OMeC6H4
-N(Me)2
-COOMe
Me
-COOEt
4-MeC6H4
Me
-COOMe
Me
-COOEt
4-MeC6H4
-N(Me)2
14.0 (12.0 – 16.5)
5.67
> 10 9.56 (6.05 – 15.1)
0.33 (239 – 456)
3.87
(5.01 – 6.41) (3.01 – 4.99)
9.66
2.73
(7.30 – 12.8) (2.11 – 3.55)
> 30
> 30
>30
19.0
26.6
3.91
(15.4 – 23.6)
(19.8 – 35.9) (2.98 – 5.11)
Data are expressed as geometric means with 95% confidence intervals in parentheses. a Displacement of specific [3H]CCPA binding at human A1 receptors expressed in CHO cells. b Displacement of specific [3H]NECA binding at human A2A receptors expressed in CHO cells. c Displacement of specific [3H]HEMADO binding at human A3 receptors expressed in CHO cells.
-
-
> 3.1
> 3.1
> 90
> 30
> 7.8
1.5
5.1
3.5
-
0.33
4.9
6.8
