2-Methylacrylamide as a bioisoster of thiourea group for 1,3-dibenzylthioureido TRPV1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2018.04.044

In order to replace thiourea group with the more drug-like moiety for 1,3-dibenzylthioureas having TRPV1 antagonist activity, we introduced a set of functional groups between the two aromatic rings based on bioisosteric replacement. The synthesized bioisosteres of 1,3-dibenzylthioureas were tested for their antagonist activities on TRPV1 by ⁴⁵Ca²⁺-influx assay using neonatal rat cultured spinal sensory neurons. Among the tested 14 kinds of bioisosters, 2-methylacrylamide group was the best candidate to replace thiourea group. Compound 7c, 2-methylacrylamide analog of ATC-120, showed as potent as ATC-120 in its antagonist activity. In addition, 2-methylacrylamide analog 7e having vinyl moiety showed the most potent activity with 0.022 μM of IC₅₀ value, indicating that thiourea group of 1,3-dibenzylthioureas could be replaced to 2-methylacrylamide without loss of their potencies.

Full text of DOI:10.1016/j.bmcl.2018.04.044

Accepted Manuscript
2-Methylacrylamide as a Bioisoster of Thiourea Group for 1,3-Dibenzylthiour-
eido TRPV1 Receptor Antagonists
Seol Rin Park, Juhyun Kim, Sun Young Lee, Young-Ho Park, Hee-Doo Kim
PII:
S0960-894X(18)30356-1
https://doi.org/10.1016/j.bmcl.2018.04.044
BMCL 25791
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 February 2018
13 April 2018
16 April 2018
Please cite this article as: Park, S.R., Kim, J., Lee, S.Y., Park, Y-H., Kim, H-D., 2-Methylacrylamide as a Bioisoster
of Thiourea Group for 1,3-Dibenzylthioureido TRPV1 Receptor Antagonists, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.04.044
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2-Methylacrylamide as a Bioisoster of Thiourea Group
for 1,3-Dibenzylthioureido TRPV1 Receptor Antagonists
Seol Rin Park, a Juhyun Kim, a Sun Young Lee,a Young-Ho Park,b Hee-Doo Kim* a
a College of Pharmacy, Sookmyung Women’s University, Seoul 04310, Korea,
bAmorePacific R & D Center, Youngin-Si, Gyeonggi-do 17014, Korea
ABSTRACT
In order to replace thiourea group with the more drug-like moiety for 1,3-dibenzylthioureas having
TRPV1 antagonist activity, we introduced a set of functional groups between the two aromatic rings
based on bioisosteric replacement. The synthesized bioisosteres of 1,3-dibenzylthioureas were tested for
their antagonist activities on TRPV1 by 45Ca2+-influx assay using neonatal rat cultured spinal sensory
neurons. Among the tested 14 kinds of bioisosters, 2-methylacrylamide group was the best candidate to
replace thiourea group. Compound 7c, 2-methylacrylamide analog of ATC-120, showed as potent as
ATC-120 in its antagonist activity. In addition, 2-methylacrylamide analog 7e having vinyl moiety
showed the most potent activity with 0.022 M of IC50 value, indicating that thiourea group of 1,3-
dibenzylthioureas could be replaced to 2-methylacrylamide without loss of their potencies.
Key Words: 2-methylacrylamide, bioisoster, TRPV1, 1,3-dibenzylthioureas, antagonist, 45Ca2+-influx
assay
The transient receptor potential vanilloid-1 (TRPV1) is a ligand-gated nonselective cation channel with
high Ca2+ permeability,1 emerging as an attractive target for the treatment of chronic and inflammatory
pain.2 Capsaicin, resiniferatoxin,3 and SDZ-2494824 represent the most well-known agonists to date.
However, due to their undesirable side effects such as pungency and/or hypothermia responses,5 recent
efforts have been focused on the discovery of novel antagonists.6 We and co-workers discovered the
potent antagonists (MK-056,7a SC-0030,7b,c and ATC-1207d) by changing phenolic hydroxyl group of
SDZ-249482 to the corresponding methanesulfonylamido group. (Figure 1) Over the past few years, we
have demonstrated that a series of 1,3-dibenzylthioureas having methanesulfonylamido group were potent
TRPV1 antagonists active against multiple activators.8 In these SAR studies, we have found that thiourea
moiety of 1,3-dibenzylthioureas is very important pharmacophore for their high potencies. However, in
view of drug-like properties, there is a need to develop the more drug-like moiety than is thiourea. Thus,
we decide to investigate the new pharmacophoric alternatives to replace thiourea group of the 1,3-
dibenzylthiourea series.
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Figure 1. Structure of capsaicin and 1,3-dibenzylthioureas
A number of functional groups including urea, amide, acrylamide and glycolamide were chosen as
bioisosteres of thiourea. ATC-120 was also chosen as reference compounds in order to clarify the effect
of bioisosteric replacement. The target compounds were synthesized via the route outlined in Scheme 1-5.
4-Methanesulfonamido--methylbenzyamines 5a~c were coupled with 4-tert-butylbenzenes 6a~e having
the requisite functional groups. (S)-4-Methanesulfonamido--methylbenzylamine (5a) and (S)-3-Fluoro-
4-methanesulfonamido--methylbenzylamine (5b) were prepared according to the previously reported
methods.7d, 9 (S)-3-Vinyl-4-methanesulfonamido--methylbenzyamine (5c) was prepared via the route
outlined in Scheme 1. Treatment of 1 with iodine monochloride produced 2 regioselectively in 47% yield.
The iodide 2 was then converted the vinyl compound 3 using by Stille’s coupling, followed by
methanesulfonylation and deprotection to give the (S)-3-vinyl-4-methanesulfonamido--
methylbenzyamine (5c).
Scheme 1. Synthesis of chiral amine 5c: a) ICl, CH2Cl2, 47%; b) Bu3SnCH=CH2, LiCl, Pd(PPh3)4, DMF,
reflux, 72%; c) (CH3SO2)2O, pyridine, CH2Cl2, 47%; d) CF3CO2H, CH2Cl2, 100%.
At first, we made urea analog 7a as a thiourea bioisoster of ATC-120, as shown in Scheme 2. (S)-4-
Methanesulfonamido--methylbenzylamine 5a was treated with 4-tert-butylbenzylisocyanate 6a under
basic condition followed by deprotection to give the urea analog 7a in 17% yield.
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Scheme 2. Synthesis of urea 7a: a) TEA, CH2Cl2, then CF3CO2H, 17 %.
Next, we focused on the design and synthesis of amide analogs due to their drug-like properties. Amides,
acrylamides, thioamides, and thioacrylamides were designed and prepared via the route outlined in
Scheme 3. (S)-4-Methanesulfonamido--methylbenzylamines (5a~c) were treated with (E)-3-[4-(tert-
butyl)phenyl]acrylic acid (6b) or (E)-3-[4-(tert-butyl)phenyl]-2-methylacrylic acid (6c) with an aid of
coupling agent DEPC under basic condition in DMF to produce the corresponding (methyl)acrylamides
7b~e in 55~94% yields. Double bond reduction of (methyl)acrylamides 7b~c by hydrogenolysis gave the
(methyl)amides 7f~g in good yields. Treatment of 7b or 7f with Lawesson’s reagent gave the
corresponding thio(acryl)amide 7h or 7i respectively.
Scheme 3. Synthesis of amides and thioamides: a) DEPC, TEA, DMF, 55~94 %; b) H2, Pd/C, quant.; c)
Lawesson’s reagent, toluene, reflux, 87%; d) Lawesson’s reagent, toluene, reflux, 87 %.
We also designed the glycolamides 7j,k and its analogs 7l and 7m as a bioisoster of thiourea ATC-120.
Synthese of 7j~m are outlined in Scheme 4. (S)-4-Methanesulfonamido--methylbenzylamines (5a~b)
were treated with 2-[4-(tert-butyl)phenoxy]acetic acid (6d) with an aid of coupling agent DEPC under
basic condition in DMF to produce the corresponding glycolamides 7j,k. Treatment of 7j with
Lawesson’s reagent gave the corresponding thioglycolamide 7l in 88% yield. By reacting with cyanamide
and HgCl2, thioglycolamide 7l could be converted to the corresponding N-cyanoacetimidamide 7m in
98% yield.
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Scheme 4. Synthesis of glycolamides and its analogs: a) DEPC, TEA, DMF, 77~88 %; b) Lawesson’s
reagent, toluene, reflux, 88 %; c) HgCl2, H2NCN, TEA, DMF, 98%.
Finally, we designed propiolamide as a bioisoster of thiourea of ATC-120. (S)-4-Methanesulfonamido-
-methylbenzylamine (5a) was treated with 2-[4-(tert-butyl)phenyl] propiolic acid (6e) with an aid of
coupling agent DEPC under basic condition in DMF to produce the corresponding propiolamide 7n, as
shown in Scheme 5.
Scheme 5. Synthesis of propiolamide: a) DEPC, TEA, DMF, 61 %.
The prepared bioisosters for ATC-120 were tested for their antagonist activities on TRPV1 by 45Ca2+
-
influx assay using neonatal rat cultured spinal sensory neurons.10 The results are summarized in Table 1.
ATC-120 was used as reference compound. As is anticipated, urea analog 7a showed 13-fold decrease in
antagonist activity compared to thiourea analog ATC-120. Amide analogs 7f, methyl-branched amide 7g,
and thioamide 7h were less potent than thiourea analog ATC-120, but more active than urea analog 7a.
When an oxygen atom is introduced to -position in place of CH2 into the amide 7f, the resulting
glycolamide 7j showed drastic increase of antagonistic potency with an IC50 of 0.096 M, but still less
potent (1/2 fold) as compared to thiourea ATC-120. Both N-cyanoacetimidamide 7m and sulfur analogs
7l of glycolamide 7j exhibited 3 to 7 fold less potent antagonistic potency compared to glycolamide 7j.
Thus, we explored the modification of the aromatic ring attached to methansulfonamide group. When we
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replaced the hydrogen atom with fluoride atom at X position of 7j (Table 1), the antagonist activity
increased up to 0.071 M of IC50 value. However, we could not find out the better compounds than
glycolamide 7j from the modification study. Thus, we turned our attention to acrylamide analogs aiming
that introduction of double bond could restrict the rotation around both amide bond and tert-butylated
phenyl ring, thereby increasing the % population of bioactive conformation. trans-Acrylamide 7b showed
0.16 M of IC50, 1.5 fold more potent than saturated amide 7f, indicating that trans-conformation might
be closer to the bioactive conformation. Next, we introduced triple bond between amide and 4-tert-
butylphenyl ring, providing propiolamide 7n, proved better antagonist with IC50 value of 0.1 M.
However, there is no space to modify around triple bond on propiolamide 7n, we needed to explore the
acrylamide further. Methyl-branching at -position of acrylamide 7b, providing 7c, has an impact on the
improvement in activity with IC50 value of 0.046 M. It means that antagonistic potency increased
approximately 4-fold compared to 7b, comparable to that of thiourea analog ATC-120. Encouraged with
the result, we explored the modification of the aromatic ring attached to methansulfonamide group.
Substitution at X-position of 7c with fluorine atom, providing 7d, resulted in equipotent activity with
parent compound 7c. The best result obtained by introducing vinyl group at X-position of 7c to provide
compound 7e with IC50 value of 0.022 M, representing 2-fold increase in antagonistic potency compared
to thiourea ATC-120.
It is also notable that all thioamides including thioacrylamides and
thioglycolamides studied here showed very weak antagonistic activities.
Table 1. 45Ca2+ -Influx activity of the bioisosters of 1,3-dibenzylthioureido TRPV1 receptor antagonist
45Ca2+ influx activity (M)a
Compound
X
Y
Z
Agonist (EC50)
> 100
Antagonist(IC50)
ATC-120
H
H
H
H
H
H
H
H
S
O
O
O
S
-NHCH2-
-NHCH2-
0.05
0.68
0.24
0.27
0.30
0.096
0.68
0.21
> 100
7a
7f
-CH2CH2-
> 100
-CHMeCH2- (racemic)
-CH2CH2-
> 100
7g
7h
7j
> 100
O
S
-CH2O-
> 100
-CH2O-
> 100
7l
NCN
-CH2O-
> 100
7m
- 5 -

F
O
O
S
-CH2O-
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.071
0.16
7k
7b
7i
H
-CH=CH- (trans)
-CH=CH- (trans)
-C≡C-
H
5.0
H
O
O
O
O
0.10
7n
7c
7d
7e
H
-C(Me)=CH- (trans)
-C(Me)=CH- (trans)
-C(Me)=CH- (trans)
0.046
0.041
0.022
F
H2C=CH
aEC50 (the concentration of derivatives necessary to produce 50% of the maximal response) and IC50 values (the
concentration of derivatives necessary to reduce to 0.5 M capsaicin by 50%) were estimated with at least 3
replicates at each concentration. Each compound was tested in two independent experiments. Antagonist data were
fitted with a sigmoid function.
In summary, we have designed and synthesized a series of bioisosters of 1,3-dibenzylthiourea TRPV1
antagonist ATC-120, focusing on the replacement of thiourea functionality to improve drug-likeness.
Among the tested 14 kinds of bioisosters, 2-methylacrylamide group was the best candidate to replace
thiourea group. Compound 7c, 2-methylacrylamide analog of ATC-120, showed as potent as ATC-120 in
its antagonist activity. In addition, 2-methylacrylamide analog 7e showed the most potent activity with
0.022 M of IC50 value, indicating that the less druggable thiourea group of 1,3-dibenzylthioureas could
be replaced to the more drug-like 2-methylacrylamide group without loss of their potencies. This
bioisosteric replacement might enable us to jump into the new chemical space of TRPV1 related
antagonists.
Acknowledgements
This research was supported by the grant from AmorePacific Corporation, and by Basic Science Research
Program through the National Research Foundation of Korea (NRF) funded by Ministry of Education
(2016008310).
References
1. (a) Szallasi, A.; Cortright, D. N.; Blum, C. A.; Eid, S. R. Nat. Rev. Drug Disc. 2007, 6, 357; (b)
Caterina, M. J.; Schumacher, M. A.; Tominaga, M.; Rosen, T. A.; Levine, J. D.; Julius, D. Nature 1997,
389, 816; (c) Tominaga, M.; Tominaga, T. Pfluegers Arch. 2005, 451, 143; (d) Cortright, D. N.;
Szallasi, A. Eur. J. Biochem. 2004, 271, 1814.
2. (a) Tominaga, M.; Caterina, M. J.; Malmberg, A. B.; Rosen, T.; Gilbert, H.; Skinner, K.; Rauman, B.
E.; Basbaum, A. I.; Julius, D. Neuron 1998, 21, 531; (b) Caterina, M. J.; Julius, D. Annu. Rev. Neurosci.
- 6 -

2001, 24, 487; (c) Breitenbucher, J. P.; Chaplan, S. R.; Carruthers, N. I. Annu. Rep. Med. Chem. 2005,
40, 185; (d) Szallasi, A.; Cruz, F.; Geppetti, P. Trends Mol. Med. 2006, 12, 545; (e) Westaway, S. M. J.
Med. Chem. 2007, 50, 2589.
3. Szallasi, A.; Blumberg, P. M. Neurosci. 1989, 30, 515.
4. Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.; Campbell, E. A.; Dray, A.; James, I. F.; Masdin, K. J.;
Perkins, M. N.; Winter, J. J. Med. Chem. 1993, 36, 2381.
5. (a) Wrigglesworth, R.; Walpole, C. S. J. Drugs Future 1998, 23, 531; (b) Jancsó, N.; Jancso-Gábor,
A.; Szolcsányi, J. Br. J. Pharmacol. Chemother. 1967, 31, 138; (c) Petsche, U.; Fleischer, E.; Lembeck,
F.; Handwerker, H. O. Brain Res. 1983, 265, 233; (d) Dray, A.; Bettany, J.; Forster, P. Br. J.
Pharmacol. 1990, 101, 727; (e) Dray, A.; Bettany, J.; Reuff, A.; Walpole, C. S. J.; Wrigglesworth, R.
Eur. J. Pharmacol. 1990, 181, 289.
6. For recent reviews, see: (a) Kym, P. R.; Kort, M. E.; Hutchins, C. W. Biochem. Pharmacol. 2009, 78,
211; (b) Gunthorpe, M. J.; Chizh, B. A. Drug Discovery Today 2009, 14, 56; (c) Khairatkar, N.;
Szallasi, A. Trends Mol. Med. 2009, 15, 14; (d) Lambert, D. G. Br. J. Anaesth. 2009, 102, 153. (e)
Voight, E. A.; Kort, M. E. Expert Opin. Ther. Pat. 2010, 20, 1107; (f) Szolcsányi, J.; Sándor, Z. Trend
Phrmacol. Sci. 2012, 33, 646; (g) Szallsi, A.; Sheta, M. Expert Opin. Invest. Drug 2012, 21,1351; (h)
Lee, Y.; Hong, S.; Cui, M.; Sharma, P. K.; Lee, J.; Choi, S. Expert Opin. Ther. Pat. 2015, 25, 291.
7. (a) Park, H.-G.; Choi, J.-Y.; Choi, S.-H.; Park, M.-K.; Lee, J.; Suh, Y.-G.; Cho, H.; Oh, U.; Lee, J.;
Kang, S.-U.; Lee, J.; Kim, H.-D.; Park, Y.-H.; Jeong, Y. S.; Choi, J. K.; Jew, S.-S. Bioorg. Med. Chem.
Lett. 2004, 14, 787; (b) Suh, Y.-G.; Lee, Y.-S.; Min, K.-H.; Park, O.-H.; Seung, H.-S.; Kim, H.-D.;
Park, H.-G.; Choi, J.-Y.; Lee, J.; Kang, S.-W.; Oh, U.; Koo, J.-y.; Joo, Y.-H.; Kim, S.-Y.; Kim, J. K.;
Park, Y.-H. Bioorg. Med. Chem. Lett. 2003, 13, 4389; (c) Min, K. H.; Suh, Y.-G.; Park, M.-K.; Park,
H.-G.; Park, Y.-H.; Kim, H.-D.; Oh, U.; Blumberg, P. M.; Lee, J. [published erratum appears in Mol.
Pharmacol. 2003, 63, 958] Mol. Pharmacol. 2002, 62, 947; (d) Ryu, C. H.; Jang, M. J.; Jung, J. W.;
Park, J.-H.; Choi, H. Y.; Suh, Y.-G.; Oh, U.; Park, H.-G.; Lee, J.; Koh, H.-J.; Mo, J.-H.; Joo, Y. H.;
Park, Y.-H.; Kim, H.-D. Bioorg. Med. Chem. Lett. 2004, 14, 1751.
8. (a) Suh, Y.-G.; Lee, Y.-S.; Min, K.-H.; Park, O.-H.; Kim, J.-K.; Seung, H.-S.; Seo, S.-Y.; Lee, B.-Y.;
Nam, Y.-H.; Lee, K.-O.; Kim, H.-D.; Park, H.-G.; Lee, J.; Oh, U.; Lim, J.-O.; Kang, S.-U.; Kil, M.-J.;
Koo, J.-Y.; Shin, S. S.; Joo, Y.-H.; Kim, J. K.; Jeong, Y.-S.; Kim, S.-Y.; Park, Y.-H. J. Med. Chem
2005. 48. 5823; (b) Chang, M.; Park, S.-R.; Kim, J.; Jang, M.; Park, J. H.; Park, J. E.; Park, H.-G.; Suh,
Y.-G.; Jeong, Y. S.; Park, Y.-H.; Kim, H-D. Bioorg. Med. Chem. 2010, 18, 111; (c) Jang, M.; Ryu, C.
H.; Park, Y.-H.; Kim, H.-D. Arch. Pharm. Res. 2012, 35, 321.
- 7 -

9. (a) Lee, J.; Lee, J.; Kang, M.; Shin, M.; Kim, J.-M.; Kang, S.-U.; Lim, J.-O.; Choi, H.-K.; Suh, Y.-G.;
Park, H.-G.; Oh, U.; Kim, H.-D.; Park, Y.-H.; Ha, H.-J.; Kim, Y.-H.; Toth, A.; Wang, Y.; Tran, R.;
Pearce, L. V. J.; Lundberg, D. J.; Blumberg, P. M. J. Med. Chem. 2003, 46, 3116; (b) Suh, Y.-G.; Lee,
Y.-S.; Min, K.-H.; Park, O.-H.; Kim, J.-K.; Seung, H.-S.; Seo, S.-Y.; Lee, B.-Y.; Nam, Y.-H.; Lee, K.-
O.; Kim, H.-D.; Park, H.-G.; Lee, J.; Oh, U.; Lim, J.-O.; Kang, S.-U.; Kil, M.-J.; Koo, J.; Shin, S. S.;
Joo, Y.-H.; Kim, J. K.; Jeong, Y.-S.; Kim, S.-Y.; Park, Y.-H. J. Med. Chem. 2005, 48, 5823.
10. Wood, J. N.; Winter, J.; James, I. F.; Rang, H. P.; Yeats, J.; Bevan, S. J. Neurosci. 1988, 8, 3208.
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Highlights
- 1,3-Dibenzylthioureas having methanesulfonylamide are potent TRPV1 antagonists.
- Thiourea moiety of 1,3-dibenzylthioureas could be replaced to 2-methylacrylamide.
- This replacement enable us to design the more druggable TRPV1 related antagonists.
- 10 -