Pyrrole derivatives as potent inhibitors of lymphocyte-specific kinase: Structure, synthesis, and SAR

Article abstract of DOI:10.1016/j.bmcl.2009.11.014

We have described the synthesis, enzyme inhibitory activity, structure-activity relationships, and proposed binding mode of a novel series of pyrrole derivatives as lymphocyte-specific kinase (Lck) inhibitors. The most potent analogs exhibited good enzyme inhibitory activity (IC₅₀s <10 nM) for Lck kinase inhibition.

Full text of DOI:10.1016/j.bmcl.2009.11.014

Bioorganic & Medicinal Chemistry Letters 20 (2010) 108–111
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrrole derivatives as potent inhibitors of lymphocyte-specific kinase: Structure,
synthesis, and SAR
*
Tetsuo Takayama , Hiroki Umemiya, Hideaki Amada, Tetsuya Yabuuchi, Fumiyasu Shiozawa,
Hironori Katakai, Akiko Takaoka, Akie Yamaguchi, Mayumi Endo, Masakazu Sato
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd, 403, Yoshino-Cho 1-Chome, Kita-Ku, Saitama-Shi, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have described the synthesis, enzyme inhibitory activity, structure–activity relationships, and pro-
posed binding mode of a novel series of pyrrole derivatives as lymphocyte-specific kinase (Lck) inhibitors.
The most potent analogs exhibited good enzyme inhibitory activity (IC50s <10 nM) for Lck kinase
inhibition.
Received 9 September 2009
Revised 5 November 2009
Accepted 7 November 2009
Available online 12 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Lck inhibitor
Kinase inhibitor
Lymphocyte-specific kinase (Lck), a Src family tyrosine kinase
predominantly expressed in T lymphocytes, plays a critical role
in development and activation of T-cells, including T-cell antigen
receptor signaling.1–3 Lck activity leads to production of cytokines
ano group in compound 6 with KOH gave the acid amide analog.
Deprotection of the 4-methoxybenzyl group in this compound
was achieved using trifluoroacetic acid (TFA)-triflic acid in the
presence of dimethyl sulfide to give pyrrole derivative 1. Alterna-
tively, compound 6 was treated with polyphosphoric acid in the
presence of thioanisole to give pyrrole derivatives.
With compound 1 as the initial lead, the effect of substituents
on the benzoyl group at the 5-position of the pyrrole ring was stud-
ied, and these results are summarized in Table 1. Introduction of an
ortho substituent on the benzoyl group resulted in an increase in
potency (7–11) against Lck, whereas the ortho-methoxy substitu-
ent analog 12 showed similar potency compared to compound 1.
Introduction of meta or para substituent resulted in a slight de-
crease in potency compared to the corresponding ortho substituent
such as interleukin-2 and IFNc. Therefore, an inhibitor against Lck
could be a potential immunosuppressive agent for treatment of
inflammatory diseases such as rheumatoid arthritis, atopic derma-
titis, asthma, and organ transplant rejection.
Several groups have previously reported the synthesis and char-
acterization of Lck inhibitors.4–12
Our efforts identified compound 1 as a potent Lck inhibitor
(Fig. 1). Molecular modeling was performed to understand the
binding mode of the Lck binding site. In this study, we describe
the synthesis, enzyme inhibitory activity,13 cellular activity against
mixed lymphocyte reaction (MLR14), and structure–activity rela-
tionships (SAR) of a series of pyrrole derivatives.
The synthesis of pyrrole derivatives is outlined in Scheme 1.
Pyrrole derivatives were synthesized from commercially available
4-phenoxybenzoic acid 2. The starting material was reacted with
thionyl chloride under reflux to give the acid chloride analog. This
compound was subsequently treated with malononitrile in the
presence of diisopropylethylamine (DIPEA) to give compound 3.
Methylation of the hydroxyl group in compound 3 with dimethyl
sulfate gave compound 4. This compound was treated with 4-
methoxybenzylamine to give compound 5. The resulting interme-
diate 5 was reacted with the appropriate a-bromoacetophenone in
the presence of K2CO3 to give compound 6.15 Hydrolysis of the cy-
* Corresponding author. Tel.: +81 48 669 3064; fax: +81 48 652 7254.
E-mail address: tetsuo.takayama@po.rd.taisho.co.jp (T. Takayama).
Figure 1. Activity for initial lead 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.014

T. Takayama et al. / Bioorg. Med. Chem. Lett. 20 (2010) 108–111
109
Scheme 1. Synthesis of pyrrole derivatives. Reagents and conditions: (a) SOCl2, reflux; (b) malononitrile, DIPEA, toluene, THF, rt; (c) dimethyl sulfate, NaHCO3, dioxane, H2O,
100 °C; (d) 4-methoxybenzylamine, MeOH, rt; (e) R1COCH2Br, K2CO3, acetone, rt; (f) KOH, tBuOH, reflux; (g) dimethyl sulfide, TFA, CF3SO3H, 50 °C; (h) thioanisole, PPA, 100 °C.
Table 1
In vitro data of pyrrole derivatives with R2–R6 substitution modifications
(13–20). In particular, the para-phenoxy substituent analog 21
exhibited decreased potency. As a general trend, ortho substitution
was much more favored over meta or para substitution. Introduc-
tion of 2,3-, 2,4-, or 2,6-disubstituents showed similar potency to
the corresponding ortho substituent analogs (22, 24–27 vs 7, 8,
10). The 2,6-difluoro substituent analog 22 was identified as the
most potent inhibitor against MLR in this series. On the other hand,
both the 3,5-disubstituted analogs 28 and 29, and the 2,5-disubsti-
tuted analogs 30 and 31 showed decreased potency.
Subsequently, we focused on developing SAR with respect to
the phenoxy group. These compounds were prepared according
to Schemes 2 and 3. Compound 32 was synthesized by a method
similar to that described in Scheme 1, using commercially available
4-methoxybenzoic acid. Demethylation of the methoxy group in
compound 32 with BBr3 gave compound 33. The resulting com-
pound 33 was reacted with various halides in the presence of
K2CO3 to give compounds 34–36. Compound 37 was also prepared
by a method similar to that described in Scheme 1, using commer-
cially available 4-nitrobenzoic acid. Reduction of the nitro group
with Fe in compound 37 gave compound 38. This compound was
treated with various halides to give compounds 40, 42, 44–46.
Alternatively, compound 38 was coupled to benzoic acid in the
presence of WSC–HCl and HOBt–H2O to give compound 39. Com-
pound 41 was prepared by reductive amination of compound 38
and cyclohexanal in the presence of NaBH(OAc)3.
Table 2 outlines the SAR observed with a substituent on the
phenyl group at the 2-position of the pyrrole ring. Modification
of the substitution on the phenyl ring resulted in a dramatic
change in potency. Replacement of the phenoxy group of com-
pound 22 by a methoxy or pyrimidine-2-yloxy group resulted in
a >100-fold loss of potency (32 and 34). Introduction of the benzyl-
oxy group was tolerable (35); however, the 2,6-dichlorobenzyloxy
analog 36 displayed significantly reduced activity. Both the amino
analog 38 and the benzoylamino analog 39 were also significantly
less potent. The benzylamino analog 40 was equipotent with the
benzyloxy analog 35. However, surprisingly, introduction of a
2,6-dichlorobenzylamino group resulted in an increase in potency,
whereas the 2,6-dichlorobenzyloxy analog was significantly less
potent (42 vs 36). Replacement of the benzylamino group by a
cyclohexylamino group resulted in a ninefold loss of activity (41
Compound
R2
R3
R4
R5
R6
IC50 (nM)
Lcka MLRb
1
7
8
H
F
H
H
H
H
H
H
H
F
Cl
Br
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Cl
Br
Me
PhO
H
F
H
H
Cl
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Me
Cl
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
11
—
1.5
1.8
2.5
2.7
3.5
9.4
5.0
6.3
6.2
4.0
10
—
Cl
Br
Me
CF3
MeO
H
H
H
H
H
H
H
H
H
F
F
Cl
Me
Cl
Me
H
H
Cl
Me
69
150
180
68
—
—
—
—
420
—
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
7.0
5.9
2.8
49
—
—
180
—
1.3
1.8
1.2
2.6
2.3
1.8
38
31
62
45
110
—
F
Cl
Me
H
H
H
H
H
H
H
H
H
H
—
—
Cl
Me
H
13
—
19
—
H
H
10
—
a
Mean values from at least two independent experiments. IC50 values were
determined from full eight-point, half-log concentration–response curves.
Mean values from at least two independent experiments. IC50 values were
determined from full nine-point, half-log concentration–response curves.
b

110
T. Takayama et al. / Bioorg. Med. Chem. Lett. 20 (2010) 108–111
Scheme 2. Reagents and conditions: (a) BBr3, 1,2-dichloroethane, reflux; (b) various R7-halide, K2CO3, DMF, rt to 80 °C.
Scheme 3. Reagents and conditions: (a) Fe, NH4Cl aq, 2-PrOH, 80 °C; (b) preparation of 39: benzoic acid, WSC–HCl, HOBt–H2O, DMF, 80 °C; preparation of 40,42,44–46:
various R8-halide, Et3N, DMF, 100 °C; preparation of 41: cyclohexanal, AcOH, NaBH(OAc)3, THF, rt.
vs 40), and N-methylation of the aniline moiety of compound 42
led to a 100-fold loss of activity (43). The importance of 2,6-disub-
stitution in the phenyl ring of the benzylamino moiety was demon-
strated by the finding that the 2,5-disubstituted analog 44 was
100-fold less potent than the 2,6-disubstituted analog 42.
The most potent analog 22 was docked into the molecular mod-
el, which was developed based on the published X-ray co-crystal
structure of Lck and imatinib (PDB code: 2PL0).16,17 As shown in
Figure 2, several hydrogen bonding interactions are thought to be
involved: the 4-NH2 of the pyrrole ring is in H-bond contact with
the Met319 main-chain carbonyl, the carbonyl of the 3-carboxam-
ide is in H-bond contact with the Met319 main-chain NH, and the
NH of the 3-carboxamide is in H-bond contact with the Glu317
main-chain carbonyl. We believe that ortho substitution of the
phenyl ring at the 5-position of pyrrole enables the phenyl ring
to adopt a preferred orientation wherein the ring is skewed out
of plane with respect to the pyrrole ring. This conformation allows
the ortho substituent of the phenyl ring to fit into the hydrophobic
pocket consisting of Leu251, Gly252, and Val259. Furthermore, this
Table 2
In vitro data of pyrrole derivatives with X and Y modifications
Compound
X
Y
IC50 (nM)
Lcka MLRb
22
32
34
35
36
38
39
40
41
42
43
44
45
46
O
O
O
OCH2
OCH2
NH
NHCO
NHCH2
NHCH2
NHCH2
NMeCH2
NHCH2
NHCH2
NHCH2
Ph
Me
1.3
31
—
—
>1000
>1000
>1000
—
—
—
31
>1000
—
140
56
>100
>100
6.1
>100
>100
>100
6.4
74
0.92
92
2-Pyrimidinyl
Ph
2,6-Dichlorophenyl
H
Ph
Ph
Cyclohexyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2-Chloro-6-methylphenyl
93
2.7
3.6
a
Mean values from at least two independent experiments. IC50 values were
determined from full eight-point, half-log concentration–response curves.
Mean values from at least two independent experiments. IC50 values were
determined from full nine-point, half-log concentration–response curves.
Figure 2. Model of 22 (orange) bound to the ATP-binding site of Lck. Oxygen atoms
are shown in red and nitrogen atoms in blue. Hydrogen bonds are shown as cyan
dotted lines.
b

T. Takayama et al. / Bioorg. Med. Chem. Lett. 20 (2010) 108–111
111
Table 3
Kinase selectivity IC50 values (nM)
LcK
1.3
Src
23
Csk
4.5
ZAP-70
>10,000
MEK1
PKA
PKB
PKC
Abl
CAMK2
>10,000
CDK1
>10,000
22
>10,000
>10,000
>10,000
>10,000
>10,000
9. Sabat, M.; VanRens, J. C.; Brugel, T. A.; Maier, J.; Laufersweiler, M. J.;
Golebiowski, A.; De, B.; Easwaran, V.; Hsieh, L. C.; Rosegen, J.; Berberich, S.;
Suchanek, E.; Janusz, M. J. Bioorg. Med. Chem. Lett. 2006, 16, 4257.
10. Bamborough, P.; Angell, R. M.; Bhamra, I.; Brown, D.; Bull, J.; Christopher, J. A.;
Cooper, A. W. J.; Fazal, L. H.; Giordano, I.; Hind, L.; Patel, V. K.; Ranshaw, L. I.;
Sims, M. J.; Skone, P. A.; Smith, K. J.; Vickerstaff, E.; Washington, M. Bioorg. Med.
Chem. Lett. 2007, 17, 4363.
11. Martin, M. W.; Newcomb, J.; Nunes, J. J.; Boucher, C.; Chai, L.; Epstein, L. F.;
Faust, T.; Flores, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Kim, J. L.;
Middleton, S.; Morgenstern, K.; Oliveira-dos-Santos, A.; Patel, V. F.; Powers, D.;
Rose, P.; Tudor, Y.; Turci, S. M.; Welcher, A. A.; Zack, D.; Zhao, H.; Zhu, L.; Zhu,
X.; Ghiron, C.; Ermann, M.; Johnston, D.; Saluste, C.-G. P. J. Med. Chem. 2008, 51,
1637.
12. DiMauro, E. F.; Newcomb, J.; Nunes, J. J.; Bemis, J. E.; Boucher, C.; Chai, L.;
Chaffee, S. C.; Deak, H. L.; Epstein, L. F.; Faust, T.; Gallant, P.; Gore, A.; Gu, Y.;
Henkle, B.; Hsieh, F.; Huang, X.; Kim, J. L.; Lee, J. H.; Martin, M. W.; McGowan,
D. C.; Metz, D.; Mohn, D.; Morgenstern, K. A.; Oliveira-dos-Santos, A.; Patel, V.
F.; Powers, D.; Rose, P. E.; Schneider, S.; Tomlinson, S. A.; Tudor, Y. -Y.; Turci, S.
M.; Welcher, A. A.; Zhao, H.; Zhu, L.; Zhu, X. J. Med. Chem. 2008, 51, 1681.
13. Inhibition of Lck activity was measured using a recombinant human Lck kinase
domain, expressed as a GST fusion in insect cells. Lck (5 ng/well) was incubated
in kinase buffer (50 mM Hepes, 50 mM KCl, 25 mM MgCl2, 5 mM MnCl2,
phenyl ring is presumed to be in contact with the phenyl group in
the side chain of Phe383 from the DFG motif by a interaction.
p–p
Compound 22, the most potent analog, was tested for selectivity
against several kinases, and these results are summarized in Table
3.18 Compound 22 exhibited >17-fold selectivity versus the Src
family member Src and >3-fold versus Csk. Compound 22 was inac-
tive for other kinases, including ZAP-70, MEK-1, PKA, PKB, PKC, Abl,
CAMK2 and CDK1 (IC50 >10 lM).
In summary, we have described the synthesis, enzyme inhibi-
tory activity, SAR, and proposed Lck binding mode of a novel class
of pyrrole derivatives, as represented by 1. Optimization of 1 led to
compound 22 with excellent enzymatic activity against Lck
(IC50 = 1.3 nM).
References and notes
1. Molina, T. J.; Kishihara, K.; Siderovskid, D. P.; van Ewijk, W.; Narendran, A.;
Timms, E.; Wakeham, M. A.; Paige, C. J.; Hartmann, K.-U.; Veillette, A.;
Davidson, D.; Mak, T. W. Nature 1992, 357, 161.
2. Straus, D. B.; Weiss, A. Cell 1992, 70, 585.
3. Levin, D. S.; Anderson, S. J.; Forbushm, K. A.; Perlmutter, R. M. EMBO J. 1993, 12,
1671.
4. Goldberg, D. R.; Butz, T.; Cardozo, M. G.; Eckner, R. J.; Hammach, A.; Huang, J.;
Jakes, S.; Kapadia, S.; Kashem, M.; Lukas, S.; Morwick, T. M.; Panzenbeck, M.;
Patel, U.; Pav, S.; Peet, G. W.; Peterson, J. D.; Prokopowicz, A. S.; Snow, R. J.;
Sellati, R.; Takahashi, H.; Tan, J.; Tschantz, M. A.; Wang, X.-J.; Wang, Y.; Wolak,
J.; Xiong, P.; Moss, N. J. Med. Chem. 2003, 46, 1337.
5. Chen, P.; Doweyko, A. M.; Norris, D.; Gu, H. H.; Spergel, S. H.; Das, J.; Moquin, R.
V.; Lin, J.; Wityak, J.; Iwanowicz, E. J.; McIntyre, K. W.; Shuster, D. J.; Behnia, K.;
Chong, S.; Fex, H.; Pang, S.; Pitt, S.; Shen, D. R.; Thrall, S.; Stanley, P.; Kocy, O. R.;
Witmer, M. R.; Kanner, S. B.; Schieven, G. L.; Barrish, J. C. J. Med. Chem. 2004, 47,
4517.
100
concentration of 5
acid- -tyrosine, 4:1) were added and incubated at 25 °C for 60 min and stopped
with 100 L of 100 mM phosphoric acid. The mixture was transferred to a
l
M Na3VO4, 0.01% CHAPS, 1 mM DTT) with test compound. The final
l
M ATP, 0.1 Ci/well M poly ( -glutamic
l
c
33P-ATP, and 10
l
L
L
l
MultiScreen-HA mixed cellulose ester membrane plate and harvested by
filtration. Scintillation cocktail was added, and radioactivity was measured on a
Packard Topcount instrument.
14. To isolate na T-cells from C57BL/6 mice (Charles River Japan), spleen and
lymph node cell suspensions were passed through nylon wool columns.
Nonadherent T-cells (3 Â 105 cells/well) were cocultured with mitomycin-C
treated spleen cells from BALB/c mice (Charles River Japan) (2 Â 105 cells/well).
These cultures were incubated at 37 °C in 5% CO2 for 72 h. Cell proliferation
was assayed by pulsing the cells with 3H-thymidine for the last 4 h. 3H-
Thymidine incorporation into DNA was measured by Topcount. Compounds
were added at the start of the culture.
15. General procedure for preparation of pyrrole derivatives (1, 7–31). To
a
6. Borhani, D. W.; Calderwood, D. J.; Friedman, M. M.; Hirst, G. C.; Li, B.; Leung, A.
K. W.; McRae, B.; Ratnofsky, S.; Ritter, K.; Waegell, W. Bioorg. Med. Chem. Lett.
2004, 14, 2613.
mixture of 5 (1 mmol) and K2CO3 (3 mmol) in acetone (7.5 ml) was added
R1COCH2Br (1.2 mmol), stirred at room temperature for 15 h. H2O was added,
and the mixture was extracted with AcOEt. The organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography to give 6. Compound 6 (1 mmol) was
treated with thioanisole (10 mmol) and PPA (3 g), stirred at 100 °C for 2 h, then
cooled to room temperature. H2O was added, and the mixture was extracted
with AcOEt. The organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by column
chromatography to give 1, 7–31.
7. Martin, M. W.; Newcomb, J.; Nunes, J. J.; McGowan, D. C.; Armistead, D. M.;
Boucher, C.; Buchanan, J. L.; Buckner, W.; Chai, L.; Elbaum, D.; Epstein, L. F.;
Faust, T.; Flynn, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Lee, J. H.;
Metz, D.; Middleton, S.; Mohn, D.; Morgenstern, K.; Morrison, M. J.; Novak, P.
M.; Oliveira-dos-Santos, A.; Powers, D.; Rose, P.; Schneider, S.; Sell, S.; Tudor, Y.;
Turci, S. M.; Welcher, A. A.; White, R. D.; Zack, D.; Zhao, H.; Zhu, L.; Zhu, X.;
Ghiron, C.; Amouzegh, P.; Ermann, M.; Jenkins, J.; Johnston, D.; Napier, S.;
Power, E. J. Med. Chem. 2006, 49, 4981.
8. DiMauro, E. F.; Newcomb, J.; Nunes, J. J.; Bemis, J. E.; Boucher, C.; Buchanan, J.
L.; Buckner, W. H.; Cee, V. J.; Chai, L.; Deak, H. L.; Epstein, L. F.; Faust, T.; Gallant,
P.; Geuns-Meyer, S. D.; Gore, A.; Gu, Y.; Henkle, B.; Hodous, B. L.; Hsieh, F.;
Huang, X.; Kim, J. L.; Lee, J. H.; Martin, M. W.; Masse, C. E.; McGowan, D. C.;
Metz, D.; Mohn, D.; Morgenstern, K. A.; Oliveira-dos-Santos, A.; Patel, V. F.;
Powers, D.; Rose, P. E.; Schneider, S.; Tomlinson, S. A.; Tudor, Y. -Y.; Turci, S. M.;
Welcher, A. A.; White, R. D.; Zhao, H.; Zhu, L.; Zhu, X. J. Med. Chem. 2006, 49,
5671.
16. Jacobs, M. D.; Caron, P. R.; Hare, B. J. Proteins 2008, 70, 1451.
17. The Binding Model was Examined and Visualized Using MOETM (Molecular
Operating Environment) Version 2007.09, Chemical Computing Group:
Montreal, Canada.
18. Lck, Src, and Csk were purchased from Upstate (NY, USA). Kinase assays for
ZAP-70, MEK1, PKA, PKB, PKC, Abl, CAMK2 and CDK1 were run by Cerep (Paris,
France) using the Kinase profiler service according to the manufacturer’s
procedures.