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Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines
Accepted Manuscript Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines Michelle van Rensburg, Euphemia Leung, Natalie A. Haverkate, Chatchakorn Eurtivong, Lisa I. Pilkington, Jóhannes Reynisson, David Barker PII: DOI: Reference:
S0960-894X(16)31276-8 http://dx.doi.org/10.1016/j.bmcl.2016.12.009 BMCL 24498
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
9 August 2016 14 November 2016 2 December 2016
Please cite this article as: van Rensburg, M., Leung, E., Haverkate, N.A., Eurtivong, C., Pilkington, L.I., Reynisson, J., Barker, D., Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.009
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.
Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines Michelle van Rensburga, Euphemia Leungb, Natalie A. Haverkate,a Chatchakorn Eurtivonga, Lisa I. Pilkington,a Jóhannes Reynissona and David Barker*a a b
School of Chemical Sciences, University of Auckland, New Zealand
Auckland Cancer Society Research Centre and Department of Molecular Medicine and Pathology, University of Auckland, New Zealand
*To whom correspondence should be addressed: School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. E-mail: [email protected], Tel. 64-9-373-7599, Fax. 64-9-373-7422
Keywords: cancer; thieno[2,3-b]pyridine; phospholipase C; tyrosyl-DNA phosphodiesterase I; antiproliferative
Abstract 3-Amino-2-arylcarboxamide-thieno[2,3-b]pyridines are a known class of antiproliferative compounds with activity against the phospholipase C enzyme. To further investigate the structure activity relationships of these derivatives a series of analogues were prepared modifying key functional groups. It was determined that modification of the 3-amino and 2aryl carboxamide functionalities resulted in complete elimination of activity, whilst modification at C-5 allowed compounds of greater activity to be prepared.
The potent antiproliferative activity of thieno[2,3-b]pyridines has driven substantial synthetic interest in this area.1–7 Activity of these compounds has been reported to be due to their ability to inhibit phospholipase C (PLC), an enzyme that plays a key role in cell signalling pathways involved in cell proliferation and motility.8,9 More recently, reported interactions with tyrosyl-DNA phosphodiesterase I (TDP1) may also account for the antiproliferative activity exhibited by this class of compounds.10 Previously, a range of thieno[2,3-b]pyridine analogues were prepared and tested for their antiproliferative activity against the National Cancer Institute’s human tumour cell lines (NCI-60).3 These analogues consisted of two main types, those containing a fused cyclohexyl ring adjacent to the pyridine ring (derivative 1) and those lacking this moiety (derivatives 2 and 3, Figure 1). These two groups contained further variations, with the former group differing by the incorporation of a ketone or alcohol functionality at C-5, whilst the latter group included modifications to the 3-amino group.
1
R 3 5
NH 2 2
N
S
X
H N
NH 2
Y
H N N
O
S
X
O
1
2 N N
R = (=O) or OH X = various substitutions Y = H, Br or Cl
Y
X N O
S
N
3
Figure 1. First group of thieno[2,3-b]pyridine analogues prepared previously.3
Of these analogues, derivatives 1 were found to be most active, with derivatives 2 and 3 showing poor to no activity.3 After the identification of potent activity in derivatives 1, docking studies of compounds related to 1 were performed and suggested that the 3-amino and 2-aryl carboxamide moieties of the ligand are involved in hydrogen bonding within the PLC enzyme site and are therefore important for activity.1 The only investigation of the importance of the 3-amino group has been undertaken on derivatives 3. However, due to the poor activity results of the parent compounds containing the 3-amino group (derivatives 2), it could not be concluded if this group is important for activity.
Our interest was therefore to further develop an understanding of the structure activity relationship of active thieno[2,3-b]pyridines. We have previously reported thieno[2,3b]pyridine 4 which has been the subject of two studies to further determine its biological activity (Figure 2).1,11 The related, 2-chlorophenyl derivative 5, was found to have similar activity against tumour cell lines MDA-MB-435, MDA-MB-468, NCI-H522, SF-295 and K562.2 Due to promising results, 5 was selected for further testing at the NCI to establish its activity, which due to its potency led to in vivo toxicity testing. Subsequently, it has been found to be non-toxic in non-tumoured animals.
O
NH 2 H N N
S
O
X
Y
4 X = H, Y = Cl 5 X = Cl, Y = H
Figure 2. Active thieno[2,3-b]pyridines 4 and 5.
The encouraging activity seen for thieno[2,3-b]pyridine 5 prompted our interest to develop a series of derivatives to investigate the structural activity relationship, determining what moieties are essential for activity. Docking studies of thienopyridine 5 against the binding 2
site of PLC-δ1 show hydrogen bonding interactions between the 2-carboxamide and 3-amino groups with Asn312, His311, Arg549 and Glu341, as well as hydrogen bonding interactions between the ketone group at C-5 with Lys438 and Arg549 (Figure 3).
Figure 3. The docked configuration of derivative 5 to the binding site of PLC-δ1 using ChemPLP. (A) The protein surface is rendered. The phenyl group occupies a lipophilic cavity to the left hand side. Red depicts positive partial charge, blue depicts negative partial charge and grey depicts lipophilic/neutral areas. (B) Hydrogen bonds are depicted as green dotted lines. The amino acids bolded in stick display style contribute to hydrogen bonds with derivative 5.
To validate the importance of these interactions to biological activity, synthetic focus was directed to include modifications to the 3-amino, 2-aryl carboxamide, C-5 substituent and the size of the fused cycloalkyl ring. As such, a group of sixteen derivatives were targeted for synthesis (Figure 4). R
R
NH 2
N N N
H N n N
S
O
n N
Cl
5 R (=O), n = 1 6a R = H, n = 1 6b R = H, n = 2 6c R = H, n = 3 6d R = H, n = 5 7 R = OH, n = 1
R
S
8 R (=O), n = 1 9a R = H, n = 1 9b R = H, n = 2 9c R = H, n = 3 9d R = H, n = 5 10 R = OH, n = 1
R
NH 2
N N N
H N N
S
O
O Cl
N
Cl
11 R (=O) 12 R = OH
S
O Cl
13 R (=O) 14 R = OH
Figure 4. Compounds of interest to further investigate SAR of the thieno[2,3-b]pyridine family. 3
Derivatives 6a-d were first prepared by the reaction of ketone 15a-d with either sodium methoxide or sodium ethoxide with methyl- or ethyl formate to give the corresponding salts 16a-d (Scheme 1).12,13 Salts 16a-d were then reacted with cyanothioacetamide and piperidinium acetate, followed by acidification with acetic acid to give carbonitriles 17a-d.12 Carbonitriles 17a-d proceeded in a coupling reaction with 2-chloro bromoacetamide 19, itself obtained from the reaction of 2-chloroaniline 18 with bromoacetyl bromide and triethylamine to give the desired thienopyridines 6a-d.
i
ii
O Na
n O 15a-d
CN
n O 16a-d
iii
H 2N
n N H 17a-d
H N
Br O
Cl 18
S
iv Cl
19 NH 2
an=1 bn=2 cn=3 dn=5
H N n N
S
O
Cl
6a-d
Scheme 1. Reagents and conditions: (i) Methyl formate (1 equiv.), Na, MeOH; or Na (1 equiv), ethyl formate (1 equiv), EtOH (cat.), Et2O, r.t., 24 h; (ii) cyanothioacetamide (1.1 equiv.), piperidinium acetate, water, reflux, 4 h, AcOH, r.t., 12 h, 17a 34%, 17b 67%, 17c 62%, 17d 64% (yields over two steps); (iii) bromoacetyl bromide (1 equiv.), Et3N (1.1 equiv.), CH2Cl2, 0 ˚C, 1 h, 19 80%; (iv) Na2CO3 (1.06 equiv.), EtOH, 100 ˚C, 18 h, 6a 78%, 6b 91%, 6c 14%, 6d 52%.
Derivative 5 was prepared in 40% yield by using a similar approach, coupling 2-chloro bromoaniline 19 with carbonitrile 21, which is derived from diketone 20 (Scheme 2).3 Alcohol derivative 7 was successfully obtained in 89% yield from the reduction of 5 with NaBH4.
4
O
O O
O
CN
i N 21 H
20
OH
NH 2
ii
H N
S
S
N
5
O
Cl
NH 2
iii
H N S
N
O
Cl
7
Scheme 2. Reagents and conditions: (i) DMFDMA (1 equiv.), DMF, 24 h; cyanothioacetamide (1 equiv.), NaH (2 equiv.), DMF, 24 h; conc. HCl, 24 h, 21 82%; (ii) 19, Na2CO3 (1.06 equiv.), EtOH, 100 ˚C, 18 h, 5 40%; (iii) NaBH4 (1 equiv.), MeOH/THF, 2 h, 7 89%.
Derivatives 11 and 12 were prepared starting from 2,4-pentandione 22 (Scheme 3).
O O
O
O CN
i N 23 H
22 OH
NH 2
ii
S
H N N
S 11
O
Cl
NH 2 H N
iii N
S
O
Cl
12
Scheme 3. Reagents and conditions: (i) DMFDMA (1 equiv.), dioxane, 24 h; Na (2 equiv.), MeOH, cyanothioacetamide (1 equiv.), 1 h, r.t., 4 h reflux, 23 97%; (ii) 19, Na2CO3 (2.0 equiv.), EtOH, 100 ˚C, 48 h, 11 98%; (iii) NaBH4 (1 equiv.), MeOH/THF, 2 h, 12 68%.
With the successful preparation of derivatives 5, 6a-d, 7, 11 and 12, modification of the 3amino group was achieved through diazotisation using sodium nitrite to give the corresponding thienotriazinones 8, 9a-d, 10, 13 and 14 (Scheme 4).
5
R
R
NH 2 H N n
N
S
N n N
O
Cl 5 R = (=O), n = 1 6a R = H, n = 1 6b R = H, n = 2 6c R = H, n = 3 6d R = H, n = 5 7 R = OH, n = 1
R
O
O Cl
N N
i
H N S
S
8 R = (=O), n = 1 9a R = H, n = 1 9b R = H, n = 2 9c R = H, n = 3 9d R = H, n = 5 10 R = OH, n = 1
R
NH 2
N
N N
i
N N
Cl
S
O Cl
13 R = (=O) 14 R = OH
11 R = (=O) 12 R = OH
Scheme 4. Reagents and conditions: (i) NaNO2, AcOH, H2O, 0 ˚C - r.t., 2-7 d, 8 20%, 9a 56%, 9b 31%, 9c 53%, 9d 76%, 10 43%, 13 81%, 14 93%.
Due to the promising preliminary activity of derivative 7 (see Table 1), acetate derivatives were prepared to investigate further modifications to C-5 (Scheme 5).
6
O O
NH 2 HN S
N OH
24a
NH 2 H N N
S
O Cl
O
i
+
Cl
O
7
O
N N S
N
O Cl
24b
O OH
N N N N
S
O
i
N N N
O Cl
N
10
S
O Cl
25
Scheme 5. Reagents and conditions: (i) acetic anhydride (2 equiv.), DMAP, pyridine, 18-72 h, 24a 41%, 24b 56%, 25 91%.
Derivatives 5, 6a, 7, 8, 9a and 10 were accepted for testing against the National Cancer Institute’s human tumour cell lines (NCI-60), allowing preliminary analysis of some compounds at at 10 μM concentration (Table 1).14 Table 1. The NCI mean percentage (%) of growth arrest at 10 μM as well as their percentage (%) of growth arrest against select tumour cell lines, as compared to untreated cells (100% growth). Compound 5 6a 7 8 9a 10
NCI Mean 48 99 36 96 89 97
HCT116 76 107 30 96 88 102
MDA-MB-468 25 91 26 86 88 91
7
MDA-MB-231 70 100 58 94 81 86
Independently, we assessed all nineteen derivatives, 5-14, 24a, 24b and 25, against three human tumour cell lines, HCT116, MDA-MB-468 and MDA-MB-231 at 1 μM. Cell proliferation was measured using a thymidine incorporation assay (Table 2).15
Table 2. Antiproliferative activity of compounds 5-25. aValues represent relative growth (%) versus control. All compounds were tested at 1 μM against the specified cell lines. Compound 5 6a 6b 6c 6d 7 8 9a 9b 9c 9d 10 11 12 13 14 24a 24b 25
HCT116a 99 94 94 71 91 11 97 99 99 90 97 96 87 97 98 100 3 92 98
MDA-MB-468a 94 95 81 100 78 14 91 81 90 85 82 80 104 96 96 118 7 64 93
MDA-MB-231a 104 119 101 56 104 30 97 94 105 92 91 91 100 108 103 100 10 82 95
The two most active compounds, 7 and 24a, were further tested to establish their IC50 values (Table 3).
Table 3. IC50 values calculated for compounds 7 and 24a. Compound 7 24a
HCT116 616 ± 3 184 ± 7
IC50 (nM) MDA-MB-468 MDA-MB-231 546 ±22 675 ± 21 181 ± 4 201 ± 1
K562 431 ± 68 192 ± 18
It was determined that a pyridine-fused cycloalkyl ring is required for activity as derivatives 11-14 were all inactive. Of the analogues containing a fused ring without further substituents at C-5, only the eight-membered analogue, 6c, was mildly active with all others showing no activity at 1 μM. It was determined that acetate (24a) and then alcohol (7) substitution on a cyclohexane ring resulted in more active compounds, over ketone groups (5) or nonsubstitution (6a-d) (Table 2), with 24a and 7 exhibiting IC50 values in the nano-molar range (Table 3). In all cases, modification of the 3-amino and 2-aryl carboxamide moieties (8-10, 13, 14, 24b and 25) resulted in inactive compounds, with direct comparisons able to be made 8
between compound 7 (11-30% relative growth across the three tumour cell lines) and 10 (8096%), as well as between compounds 24a (3-10%), 24b (64-93%) and 25 (93-98%) which confirms that these groups are essential for bioactivity. These results are in line with molecular modelling which shows hydrogen bonding between thienopyridine ligands and amino (Glu341 and Lys438) within the PLC active site. The incorporation of hydrogen bonding groups at C-5 had mixed results with similar activities seen between derivatives 5 and 6, while derivative 7 had increased activity compared to 5. Interestingly, the incorporation of an acetate functionality in derivative 24a, which modelling suggests should have reduced hydrogen bonding, had similar activity results to that of derivative 7.
Twenty two thieno[2,3-b]pyridine derivatives were successfully prepared to allow investigations into the SAR of this class of compounds. Synthetic focus was directed to aid in the determination of the importance of the 5-keto functionality on the ring fused to the pyridine ring, as well as the importance of the 3-amino and 2-carboxamide moieties on the activity of the compound. Cytotoxicity results showed that modifications are tolerated at C-5 but not at C-2/C-3, which resulted in the loss of activity in all cases. Additionally the size or absence of the fused cycloalkyl ring effects the activity of the compounds suggesting a defined binding pocket with specific important interactions. Interestingly, acetate derivative 24a was found to be more active than derivative 5, a result which is not consistent with the predicted binding pattern from docking studies. It is possible that the addition of an acetate group in derivative 24a may increase cell permeability. This may be a beneficial discovery as concurrent studies have shown that thienopyridines have inherently poor aqueous solubility.13
Acknowledgements This work was supported by the Auckland Medical Research Fund, Auckland Cancer Society, the Auckland Cancer Society Research Centre, and the Faculty Research Development Fund from the University of Auckland.
Supplementary data Supplementary data (experimental details for the synthesis of compounds) associated with this article is available.
References (1) (2) (3) (4)
Feng, L.; Reynisdóttir, I.; Reynisson, J. Eur. J. Med. Chem. 2012, 54, 463–469. Arabshahi, H. J.; Leung, E.; Barker, D.; Reynisson, J. MedChemComm 2014, 5 (2), 186–191. Hung, J. M.; Arabshahi, H. J.; Leung, E.; Reynisson, J.; Barker, D. Eur. J. Med. Chem. 2014, 86, 420–437. Wang, J.; Luo, C.; Shan, C.; You, Q.; Lu, J.; Elf, S.; Zhou, Y.; Wen, Y.; Vinkenborg, J. L.; Fan, J.; Kang, H.; Lin, R.; Han, D.; Xie, Y.; Karpus, J.; Chen, S.; Ouyang, S.; Luan, C.; Zhang, N.; Ding, H.; Merkx, M.; Liu, H.; Chen, J.; Jiang, H.; He, C. Nat. Chem. 2015, 7 (12), 968–979. 9
(5) (6)
(7) (8) (9) (10) (11) (12)
(13) (14) (15)
Deng, X.-Q.; Wang, H.-Y.; Zhao, Y.-L.; Xiang, M.-L.; Jiang, P.-D.; Cao, Z.-X.; Zheng, Y.-Z.; Luo, S.-D.; Yu, L.-T.; Wei, Y.-Q.; Yang, S.-Y. Chem. Biol. Drug Des. 2008, 71 (6), 533–539. Zeng, X.-X.; Zheng, R.-L.; Zhou, T.; He, H.-Y.; Liu, J.-Y.; Zheng, Y.; Tong, A.-P.; Xiang, M.L.; Song, X.-R.; Yang, S.-Y.; Yu, L.-T.; Wei, Y.-Q.; Zhao, Y.-L.; Yang, L. Bioorg. Med. Chem. Lett. 2010, 20 (21), 6282–6285. Zhou, R.; Huang, W.-J.; Guo, Z.-Y.; Li, L.; Zeng, X.-R.; Deng, Y.-Q.; Hu, F.-Y.; Tong, A.-P.; Yang, L.; Yang, J.-L. Oncol. Rep. 2012, 28 (1), 225–231. Kadamur, G.; Ross, E. M. Annu. Rev. Physiol. 2013, 75, 127–154. Kölsch, V.; Charest, P. G.; Firtel, R. A. J. Cell Sci. 2008, 121 (Pt 5), 551–559. Reynisson, J.; Court, W.; O’Neill, C.; Day, J.; Patterson, L.; McDonald, E.; Workman, P.; Katan, M.; Eccles, S. A. Bioorg. Med. Chem. 2009, 17 (8), 3169–3176. Leung, E.; Hung, J. M.; Barker, D.; Reynisson, J. Med Chem Commun 2014, 5 (1), 99–106. Leung, E.; Pilkington, L. I.; van Rensburg, M.; Jeon, C. Y.; Song, M.; Arabshahi, H. J.; De Zoysa, G. H.; Sarojini, V.; Denny, W. A.; Reynisson, J.; Barker, D. Bioorg. Med. Chem. 2016, 24 (5), 1142–1154. Pilkington, L. I.; Haverkate, N. A.; van Rensburg, M.; Reynisson, J.; Leung, E.; Barker, D. Synlett 2016. DOI: 10.1055/s-0036-1588619. Shoemaker, R. H. Nat. Rev. Cancer 2006, 6 (10), 813–823. Reynisson, J.; Jaiswal, J. K.; Barker, D.; D’mello, S. A. N.; Denny, W. A.; Baguley, B. C.; Leung, E. Y. Cancer Cell Int. 2016, 16, 18.
10
Graphical Abstract
Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines Michelle van Rensburga, Euphemia Leungb, Natalie A. Haverkate,a Chatchakorn Eurtivonga, Lisa I. Pilkington,a Jóhannes Reynissona and David Barker*a a b
School of Chemical Sciences, University of Auckland, New Zealand
Auckland Cancer Society Research Centre and Department of Molecular Medicine and Pathology, University of Auckland, New Zealand
11
S0960-894X(16)31276-8 http://dx.doi.org/10.1016/j.bmcl.2016.12.009 BMCL 24498
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
9 August 2016 14 November 2016 2 December 2016
Please cite this article as: van Rensburg, M., Leung, E., Haverkate, N.A., Eurtivong, C., Pilkington, L.I., Reynisson, J., Barker, D., Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.009
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.
Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines Michelle van Rensburga, Euphemia Leungb, Natalie A. Haverkate,a Chatchakorn Eurtivonga, Lisa I. Pilkington,a Jóhannes Reynissona and David Barker*a a b
School of Chemical Sciences, University of Auckland, New Zealand
Auckland Cancer Society Research Centre and Department of Molecular Medicine and Pathology, University of Auckland, New Zealand
*To whom correspondence should be addressed: School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. E-mail: [email protected], Tel. 64-9-373-7599, Fax. 64-9-373-7422
Keywords: cancer; thieno[2,3-b]pyridine; phospholipase C; tyrosyl-DNA phosphodiesterase I; antiproliferative
Abstract 3-Amino-2-arylcarboxamide-thieno[2,3-b]pyridines are a known class of antiproliferative compounds with activity against the phospholipase C enzyme. To further investigate the structure activity relationships of these derivatives a series of analogues were prepared modifying key functional groups. It was determined that modification of the 3-amino and 2aryl carboxamide functionalities resulted in complete elimination of activity, whilst modification at C-5 allowed compounds of greater activity to be prepared.
The potent antiproliferative activity of thieno[2,3-b]pyridines has driven substantial synthetic interest in this area.1–7 Activity of these compounds has been reported to be due to their ability to inhibit phospholipase C (PLC), an enzyme that plays a key role in cell signalling pathways involved in cell proliferation and motility.8,9 More recently, reported interactions with tyrosyl-DNA phosphodiesterase I (TDP1) may also account for the antiproliferative activity exhibited by this class of compounds.10 Previously, a range of thieno[2,3-b]pyridine analogues were prepared and tested for their antiproliferative activity against the National Cancer Institute’s human tumour cell lines (NCI-60).3 These analogues consisted of two main types, those containing a fused cyclohexyl ring adjacent to the pyridine ring (derivative 1) and those lacking this moiety (derivatives 2 and 3, Figure 1). These two groups contained further variations, with the former group differing by the incorporation of a ketone or alcohol functionality at C-5, whilst the latter group included modifications to the 3-amino group.
1
R 3 5
NH 2 2
N
S
X
H N
NH 2
Y
H N N
O
S
X
O
1
2 N N
R = (=O) or OH X = various substitutions Y = H, Br or Cl
Y
X N O
S
N
3
Figure 1. First group of thieno[2,3-b]pyridine analogues prepared previously.3
Of these analogues, derivatives 1 were found to be most active, with derivatives 2 and 3 showing poor to no activity.3 After the identification of potent activity in derivatives 1, docking studies of compounds related to 1 were performed and suggested that the 3-amino and 2-aryl carboxamide moieties of the ligand are involved in hydrogen bonding within the PLC enzyme site and are therefore important for activity.1 The only investigation of the importance of the 3-amino group has been undertaken on derivatives 3. However, due to the poor activity results of the parent compounds containing the 3-amino group (derivatives 2), it could not be concluded if this group is important for activity.
Our interest was therefore to further develop an understanding of the structure activity relationship of active thieno[2,3-b]pyridines. We have previously reported thieno[2,3b]pyridine 4 which has been the subject of two studies to further determine its biological activity (Figure 2).1,11 The related, 2-chlorophenyl derivative 5, was found to have similar activity against tumour cell lines MDA-MB-435, MDA-MB-468, NCI-H522, SF-295 and K562.2 Due to promising results, 5 was selected for further testing at the NCI to establish its activity, which due to its potency led to in vivo toxicity testing. Subsequently, it has been found to be non-toxic in non-tumoured animals.
O
NH 2 H N N
S
O
X
Y
4 X = H, Y = Cl 5 X = Cl, Y = H
Figure 2. Active thieno[2,3-b]pyridines 4 and 5.
The encouraging activity seen for thieno[2,3-b]pyridine 5 prompted our interest to develop a series of derivatives to investigate the structural activity relationship, determining what moieties are essential for activity. Docking studies of thienopyridine 5 against the binding 2
site of PLC-δ1 show hydrogen bonding interactions between the 2-carboxamide and 3-amino groups with Asn312, His311, Arg549 and Glu341, as well as hydrogen bonding interactions between the ketone group at C-5 with Lys438 and Arg549 (Figure 3).
Figure 3. The docked configuration of derivative 5 to the binding site of PLC-δ1 using ChemPLP. (A) The protein surface is rendered. The phenyl group occupies a lipophilic cavity to the left hand side. Red depicts positive partial charge, blue depicts negative partial charge and grey depicts lipophilic/neutral areas. (B) Hydrogen bonds are depicted as green dotted lines. The amino acids bolded in stick display style contribute to hydrogen bonds with derivative 5.
To validate the importance of these interactions to biological activity, synthetic focus was directed to include modifications to the 3-amino, 2-aryl carboxamide, C-5 substituent and the size of the fused cycloalkyl ring. As such, a group of sixteen derivatives were targeted for synthesis (Figure 4). R
R
NH 2
N N N
H N n N
S
O
n N
Cl
5 R (=O), n = 1 6a R = H, n = 1 6b R = H, n = 2 6c R = H, n = 3 6d R = H, n = 5 7 R = OH, n = 1
R
S
8 R (=O), n = 1 9a R = H, n = 1 9b R = H, n = 2 9c R = H, n = 3 9d R = H, n = 5 10 R = OH, n = 1
R
NH 2
N N N
H N N
S
O
O Cl
N
Cl
11 R (=O) 12 R = OH
S
O Cl
13 R (=O) 14 R = OH
Figure 4. Compounds of interest to further investigate SAR of the thieno[2,3-b]pyridine family. 3
Derivatives 6a-d were first prepared by the reaction of ketone 15a-d with either sodium methoxide or sodium ethoxide with methyl- or ethyl formate to give the corresponding salts 16a-d (Scheme 1).12,13 Salts 16a-d were then reacted with cyanothioacetamide and piperidinium acetate, followed by acidification with acetic acid to give carbonitriles 17a-d.12 Carbonitriles 17a-d proceeded in a coupling reaction with 2-chloro bromoacetamide 19, itself obtained from the reaction of 2-chloroaniline 18 with bromoacetyl bromide and triethylamine to give the desired thienopyridines 6a-d.
i
ii
O Na
n O 15a-d
CN
n O 16a-d
iii
H 2N
n N H 17a-d
H N
Br O
Cl 18
S
iv Cl
19 NH 2
an=1 bn=2 cn=3 dn=5
H N n N
S
O
Cl
6a-d
Scheme 1. Reagents and conditions: (i) Methyl formate (1 equiv.), Na, MeOH; or Na (1 equiv), ethyl formate (1 equiv), EtOH (cat.), Et2O, r.t., 24 h; (ii) cyanothioacetamide (1.1 equiv.), piperidinium acetate, water, reflux, 4 h, AcOH, r.t., 12 h, 17a 34%, 17b 67%, 17c 62%, 17d 64% (yields over two steps); (iii) bromoacetyl bromide (1 equiv.), Et3N (1.1 equiv.), CH2Cl2, 0 ˚C, 1 h, 19 80%; (iv) Na2CO3 (1.06 equiv.), EtOH, 100 ˚C, 18 h, 6a 78%, 6b 91%, 6c 14%, 6d 52%.
Derivative 5 was prepared in 40% yield by using a similar approach, coupling 2-chloro bromoaniline 19 with carbonitrile 21, which is derived from diketone 20 (Scheme 2).3 Alcohol derivative 7 was successfully obtained in 89% yield from the reduction of 5 with NaBH4.
4
O
O O
O
CN
i N 21 H
20
OH
NH 2
ii
H N
S
S
N
5
O
Cl
NH 2
iii
H N S
N
O
Cl
7
Scheme 2. Reagents and conditions: (i) DMFDMA (1 equiv.), DMF, 24 h; cyanothioacetamide (1 equiv.), NaH (2 equiv.), DMF, 24 h; conc. HCl, 24 h, 21 82%; (ii) 19, Na2CO3 (1.06 equiv.), EtOH, 100 ˚C, 18 h, 5 40%; (iii) NaBH4 (1 equiv.), MeOH/THF, 2 h, 7 89%.
Derivatives 11 and 12 were prepared starting from 2,4-pentandione 22 (Scheme 3).
O O
O
O CN
i N 23 H
22 OH
NH 2
ii
S
H N N
S 11
O
Cl
NH 2 H N
iii N
S
O
Cl
12
Scheme 3. Reagents and conditions: (i) DMFDMA (1 equiv.), dioxane, 24 h; Na (2 equiv.), MeOH, cyanothioacetamide (1 equiv.), 1 h, r.t., 4 h reflux, 23 97%; (ii) 19, Na2CO3 (2.0 equiv.), EtOH, 100 ˚C, 48 h, 11 98%; (iii) NaBH4 (1 equiv.), MeOH/THF, 2 h, 12 68%.
With the successful preparation of derivatives 5, 6a-d, 7, 11 and 12, modification of the 3amino group was achieved through diazotisation using sodium nitrite to give the corresponding thienotriazinones 8, 9a-d, 10, 13 and 14 (Scheme 4).
5
R
R
NH 2 H N n
N
S
N n N
O
Cl 5 R = (=O), n = 1 6a R = H, n = 1 6b R = H, n = 2 6c R = H, n = 3 6d R = H, n = 5 7 R = OH, n = 1
R
O
O Cl
N N
i
H N S
S
8 R = (=O), n = 1 9a R = H, n = 1 9b R = H, n = 2 9c R = H, n = 3 9d R = H, n = 5 10 R = OH, n = 1
R
NH 2
N
N N
i
N N
Cl
S
O Cl
13 R = (=O) 14 R = OH
11 R = (=O) 12 R = OH
Scheme 4. Reagents and conditions: (i) NaNO2, AcOH, H2O, 0 ˚C - r.t., 2-7 d, 8 20%, 9a 56%, 9b 31%, 9c 53%, 9d 76%, 10 43%, 13 81%, 14 93%.
Due to the promising preliminary activity of derivative 7 (see Table 1), acetate derivatives were prepared to investigate further modifications to C-5 (Scheme 5).
6
O O
NH 2 HN S
N OH
24a
NH 2 H N N
S
O Cl
O
i
+
Cl
O
7
O
N N S
N
O Cl
24b
O OH
N N N N
S
O
i
N N N
O Cl
N
10
S
O Cl
25
Scheme 5. Reagents and conditions: (i) acetic anhydride (2 equiv.), DMAP, pyridine, 18-72 h, 24a 41%, 24b 56%, 25 91%.
Derivatives 5, 6a, 7, 8, 9a and 10 were accepted for testing against the National Cancer Institute’s human tumour cell lines (NCI-60), allowing preliminary analysis of some compounds at at 10 μM concentration (Table 1).14 Table 1. The NCI mean percentage (%) of growth arrest at 10 μM as well as their percentage (%) of growth arrest against select tumour cell lines, as compared to untreated cells (100% growth). Compound 5 6a 7 8 9a 10
NCI Mean 48 99 36 96 89 97
HCT116 76 107 30 96 88 102
MDA-MB-468 25 91 26 86 88 91
7
MDA-MB-231 70 100 58 94 81 86
Independently, we assessed all nineteen derivatives, 5-14, 24a, 24b and 25, against three human tumour cell lines, HCT116, MDA-MB-468 and MDA-MB-231 at 1 μM. Cell proliferation was measured using a thymidine incorporation assay (Table 2).15
Table 2. Antiproliferative activity of compounds 5-25. aValues represent relative growth (%) versus control. All compounds were tested at 1 μM against the specified cell lines. Compound 5 6a 6b 6c 6d 7 8 9a 9b 9c 9d 10 11 12 13 14 24a 24b 25
HCT116a 99 94 94 71 91 11 97 99 99 90 97 96 87 97 98 100 3 92 98
MDA-MB-468a 94 95 81 100 78 14 91 81 90 85 82 80 104 96 96 118 7 64 93
MDA-MB-231a 104 119 101 56 104 30 97 94 105 92 91 91 100 108 103 100 10 82 95
The two most active compounds, 7 and 24a, were further tested to establish their IC50 values (Table 3).
Table 3. IC50 values calculated for compounds 7 and 24a. Compound 7 24a
HCT116 616 ± 3 184 ± 7
IC50 (nM) MDA-MB-468 MDA-MB-231 546 ±22 675 ± 21 181 ± 4 201 ± 1
K562 431 ± 68 192 ± 18
It was determined that a pyridine-fused cycloalkyl ring is required for activity as derivatives 11-14 were all inactive. Of the analogues containing a fused ring without further substituents at C-5, only the eight-membered analogue, 6c, was mildly active with all others showing no activity at 1 μM. It was determined that acetate (24a) and then alcohol (7) substitution on a cyclohexane ring resulted in more active compounds, over ketone groups (5) or nonsubstitution (6a-d) (Table 2), with 24a and 7 exhibiting IC50 values in the nano-molar range (Table 3). In all cases, modification of the 3-amino and 2-aryl carboxamide moieties (8-10, 13, 14, 24b and 25) resulted in inactive compounds, with direct comparisons able to be made 8
between compound 7 (11-30% relative growth across the three tumour cell lines) and 10 (8096%), as well as between compounds 24a (3-10%), 24b (64-93%) and 25 (93-98%) which confirms that these groups are essential for bioactivity. These results are in line with molecular modelling which shows hydrogen bonding between thienopyridine ligands and amino (Glu341 and Lys438) within the PLC active site. The incorporation of hydrogen bonding groups at C-5 had mixed results with similar activities seen between derivatives 5 and 6, while derivative 7 had increased activity compared to 5. Interestingly, the incorporation of an acetate functionality in derivative 24a, which modelling suggests should have reduced hydrogen bonding, had similar activity results to that of derivative 7.
Twenty two thieno[2,3-b]pyridine derivatives were successfully prepared to allow investigations into the SAR of this class of compounds. Synthetic focus was directed to aid in the determination of the importance of the 5-keto functionality on the ring fused to the pyridine ring, as well as the importance of the 3-amino and 2-carboxamide moieties on the activity of the compound. Cytotoxicity results showed that modifications are tolerated at C-5 but not at C-2/C-3, which resulted in the loss of activity in all cases. Additionally the size or absence of the fused cycloalkyl ring effects the activity of the compounds suggesting a defined binding pocket with specific important interactions. Interestingly, acetate derivative 24a was found to be more active than derivative 5, a result which is not consistent with the predicted binding pattern from docking studies. It is possible that the addition of an acetate group in derivative 24a may increase cell permeability. This may be a beneficial discovery as concurrent studies have shown that thienopyridines have inherently poor aqueous solubility.13
Acknowledgements This work was supported by the Auckland Medical Research Fund, Auckland Cancer Society, the Auckland Cancer Society Research Centre, and the Faculty Research Development Fund from the University of Auckland.
Supplementary data Supplementary data (experimental details for the synthesis of compounds) associated with this article is available.
References (1) (2) (3) (4)
Feng, L.; Reynisdóttir, I.; Reynisson, J. Eur. J. Med. Chem. 2012, 54, 463–469. Arabshahi, H. J.; Leung, E.; Barker, D.; Reynisson, J. MedChemComm 2014, 5 (2), 186–191. Hung, J. M.; Arabshahi, H. J.; Leung, E.; Reynisson, J.; Barker, D. Eur. J. Med. Chem. 2014, 86, 420–437. Wang, J.; Luo, C.; Shan, C.; You, Q.; Lu, J.; Elf, S.; Zhou, Y.; Wen, Y.; Vinkenborg, J. L.; Fan, J.; Kang, H.; Lin, R.; Han, D.; Xie, Y.; Karpus, J.; Chen, S.; Ouyang, S.; Luan, C.; Zhang, N.; Ding, H.; Merkx, M.; Liu, H.; Chen, J.; Jiang, H.; He, C. Nat. Chem. 2015, 7 (12), 968–979. 9
(5) (6)
(7) (8) (9) (10) (11) (12)
(13) (14) (15)
Deng, X.-Q.; Wang, H.-Y.; Zhao, Y.-L.; Xiang, M.-L.; Jiang, P.-D.; Cao, Z.-X.; Zheng, Y.-Z.; Luo, S.-D.; Yu, L.-T.; Wei, Y.-Q.; Yang, S.-Y. Chem. Biol. Drug Des. 2008, 71 (6), 533–539. Zeng, X.-X.; Zheng, R.-L.; Zhou, T.; He, H.-Y.; Liu, J.-Y.; Zheng, Y.; Tong, A.-P.; Xiang, M.L.; Song, X.-R.; Yang, S.-Y.; Yu, L.-T.; Wei, Y.-Q.; Zhao, Y.-L.; Yang, L. Bioorg. Med. Chem. Lett. 2010, 20 (21), 6282–6285. Zhou, R.; Huang, W.-J.; Guo, Z.-Y.; Li, L.; Zeng, X.-R.; Deng, Y.-Q.; Hu, F.-Y.; Tong, A.-P.; Yang, L.; Yang, J.-L. Oncol. Rep. 2012, 28 (1), 225–231. Kadamur, G.; Ross, E. M. Annu. Rev. Physiol. 2013, 75, 127–154. Kölsch, V.; Charest, P. G.; Firtel, R. A. J. Cell Sci. 2008, 121 (Pt 5), 551–559. Reynisson, J.; Court, W.; O’Neill, C.; Day, J.; Patterson, L.; McDonald, E.; Workman, P.; Katan, M.; Eccles, S. A. Bioorg. Med. Chem. 2009, 17 (8), 3169–3176. Leung, E.; Hung, J. M.; Barker, D.; Reynisson, J. Med Chem Commun 2014, 5 (1), 99–106. Leung, E.; Pilkington, L. I.; van Rensburg, M.; Jeon, C. Y.; Song, M.; Arabshahi, H. J.; De Zoysa, G. H.; Sarojini, V.; Denny, W. A.; Reynisson, J.; Barker, D. Bioorg. Med. Chem. 2016, 24 (5), 1142–1154. Pilkington, L. I.; Haverkate, N. A.; van Rensburg, M.; Reynisson, J.; Leung, E.; Barker, D. Synlett 2016. DOI: 10.1055/s-0036-1588619. Shoemaker, R. H. Nat. Rev. Cancer 2006, 6 (10), 813–823. Reynisson, J.; Jaiswal, J. K.; Barker, D.; D’mello, S. A. N.; Denny, W. A.; Baguley, B. C.; Leung, E. Y. Cancer Cell Int. 2016, 16, 18.
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Graphical Abstract
Synthesis and antiproliferative activity of 2-chlorophenyl carboxamide thienopyridines Michelle van Rensburga, Euphemia Leungb, Natalie A. Haverkate,a Chatchakorn Eurtivonga, Lisa I. Pilkington,a Jóhannes Reynissona and David Barker*a a b
School of Chemical Sciences, University of Auckland, New Zealand
Auckland Cancer Society Research Centre and Department of Molecular Medicine and Pathology, University of Auckland, New Zealand
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