Disubstituted pyrimidines as Lck inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.07.102

We have developed a family of 4-benzimidazolyl-N-piperazinethyl-pyrimidin-2-amines that are subnanomolar inhibitors of Lck. A subset of these Lck inhibitors, with heterocyclic substituents at the benzimidazole C5, are also low-nanomolar inhibitors of cell

Full text of DOI:10.1016/j.bmcl.2009.07.102

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5440–5443
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Disubstituted pyrimidines as Lck inhibitors
*
Julianne A. Hunt , Richard T. Beresis, Joung L. Goulet, Mark A. Holmes, Xinfang J. Hong, Ernest Kovacs,
Sander G. Mills, Rowena D. Ruzek, Frederick Wong, Jeffrey D. Hermes, Young-Whan Park, Scott P. Salowe,
Lisa M. Sonatore, Lin Wu, Andrea Woods, Dennis M. Zaller, Peter J. Sinclair
Merck Research Laboratories, Merck & Co., PO Box 2000, Rahway, NJ 07065, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a family of 4-benzimidazolyl-N-piperazinethyl-pyrimidin-2-amines that are subn-
anomolar inhibitors of Lck. A subset of these Lck inhibitors, with heterocyclic substituents at the benz-
imidazole C5, are also low-nanomolar inhibitors of cellular IL2 release.
Received 27 May 2009
Revised 20 July 2009
Accepted 22 July 2009
Available online 25 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Lck
Src
Protein tyrosine kinase
Autoimmune disease
Rheumatoid arthritis
Pyrimidine
P56Lck (Lck) is a member of the Src family of non-receptor pro-
tein tyrosine kinases. Lck is expressed almost exclusively in T-cells
and its catalytic activity is required for T-cell signal transduction.1
A potent Lck kinase inhibitor would inhibit T-cell activation, and
thus could be useful as an immunosuppressive agent for the pre-
vention of graft rejection following organ transplantation, or for
treating autoimmune diseases such as rheumatoid arthritis and
psoriasis. Therefore, Lck is an attractive target for therapeutic
intervention.2
(5), as well as of the des-methyl analog (6) underscore the impor-
tance of the benzylic methyl substitution, as well as the preferred
(S) stereochemistry.
The increased potency of the m-nitrobenzylamine analog 7 rel-
ative to 1 prompted further exploration of this position. As inhibi-
tory activity was not dependent on the presence of an aromatic
N
N
N
N
High-throughput screening for a nascent medicinal chemistry
program aimed at identifying an Lck inhibitor disclosed disubsti-
N
CH3
N
H3C
N
O
tuted pyrimidine 1 as a promising lead.3 Optimization of the
a-
N
N
H
N
N
H
N
N
H
methylbenzylamine substructure of 1 led to the piperidine naph-
thyl urea 2, which was highly potent against Lck activity in vitro,
and moderately active in our cellular assay for IL2 release. Optimi-
zation of the benzimidazole substructure of compound 1 resulted
in C5-substituted inhibitors such as pyridine 3, which was a potent
inhibitor of Lck activity in vitro and a moderate inhibitor of cellular
IL2 release. Combination of the optimized substructures of 2 and 3
proved synergistic: Compound 4 is a potent inhibitor of both Lck
activity and cellular IL2 release (Fig. 1).
Me
2, Lck IC50=1 nM
1, Lck IC50=153 nM
IL2 IC50=111 nM
IL2 IC50=ND
N
N
N
N
N
N
Our initial structure–activity investigations were directed at the
2-benzylamino group of our screening lead, compound 1. As shown
in Table 1, the diminished activities of the enantiomeric compound
N
CH3
N
H3C
N
O
N
N
H
N
N
H
N
N
H
Me
4, Lck IC50=0.1 nM
3, Lck IC50=3 nM
IL2 IC50=8 nM
IL2 IC50=98 nM
* Corresponding author. Tel.: +1 732 594 0463; fax: +1 732 594 3178.
E-mail address: julianne_hunt@merck.com (J.A. Hunt).
Figure 1. Disubstituted pyrimidines as inhibitors of Lck.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.102

J. A. Hunt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5440–5443
5441
Table 1
Table 3
2-Aminopyrimidine derivatives
Ureas, carbamates, and amides
N
N
N
N
N
R1
N
CH3
O
R2
*
*
N
N
H
N
N
H
N
XR
R1
R2
Lck IC50 (nM)
4
Compd
4
Compd
X
R
Lck IC50 (nM)
1
5
6
7
8
(S)-Me
(R)-Me
H
(S)-Me
(S)-Me
Ph
Ph
Ph
153
1770
827
47
13
14
15
16
NH
NH
NH
CH2
O
Me
Cyclohexyl
Ph
Ph
Ph
242
1260
11
800
46
m-NO2–Ph
Cyclohexyl
243
17
18
NH
NH
NH
2-Naphthyl
1-Naphthyl
1-Naphthyl
40
1
0.4
19 (rac)
20 (S,S)a
ring (note cyclohexyl-substituted compound 8), we chose to inves-
tigate heterocyclic replacements and, in particular, piperidines. All
three of the regioisomeric methylamino-N-Cbz-piperidines were
prepared in order to determine the optimum regioisomer. The
Cbz-substituted piperidine 10, with the nitrogen at the 3 position,
exhibited activity comparable to that of 6, the des-methyl analog of
1 (Table 2); this result dovetailed with the results for m-nitroben-
zylamine 7 (Table 1).
a
Proposed stereochemistry. The (S)-methyl enantiomer is more active in the a-
methylbenzylamine series, and the (S,S)-methylpiperazine is the more potent
enantiomer (see below).
more accessible des-methyl compounds 21–25.7 As shown in Table
4, piperazine 25 was tenfold more potent than the corresponding
des-methyl piperidine naphthyl urea, 22.
We turned next to the installation of an
a
-methyl group on
Introduction of the potency-enhancing methyl group into com-
pound 25 provided compounds 26a and 26b (Table 5). Comparison
of diastereomers 26a and 26b shows that the relative configuration
of 26a, with the two methine protons syn, significantly enhances
the potency of the compound as an inhibitor of both Lck activity
and cellular IL2 release. Thus, the remaining analogs shown in Ta-
ble 5 (27–35) were synthesized in the same relative configuration
as 26a. None of these analogs improved on 26a in either biochem-
ical or cellular potency. Taken together, the data in Tables 3 and 5
pointed to the 1-naphthyl urea as the optimal substituent at this
position.
Separation of the enantiomers of 26a via chiral HPLC (Chiralpak
OD) showed that the (S,S) enantiomer—compound 36 (Table 6)—
was approximately sixfold more potent than the racemate 26a. Ta-
ble 6 summarizes the biochemical and cellular activity of several
(S,S)-piperazines. Replacement of the R1 methyl in compound 6
with an ethyl (37) did not significantly change the potency, but lar-
ger alkyl groups, such as isobutyl (38) or hexyl (39) led to signifi-
cantly weaker inhibitors of IL2 release. Other changes at R1, such
as pyridyl, ethyl ester, and acetyl substituents, were also detrimen-
tal to inhibitor potency and cellular activity.
piperidine 10. As expected, addition of the
a-methyl group re-
sulted in a marked increase in activity. The more active diastereo-
mer 12a showed inhibitory activity comparable to our lead 1.
The piperidine scaffold was examined further via derivatization
of 12a. Removal of the Cbz group followed by methylation or ben-
zylation under standard conditions resulted in loss of activity.5
Recognizing the significance of the carbonyl linkage to the piperi-
dine, we next prepared a variety of urea, amide, and carbamate
analogs using standard acylation chemistry.
As shown in Table 3, the activity of phenyl urea 15 was signif-
icantly improved over that of our lead compound 1. Phenyl carba-
mate 17 was fourfold less potent than 15, while the phenyl
acetamide 16 lost much of its potency. These results indicated a
preference for a pendant urea group. Therefore, we assessed a
number of substituted aryl urea compounds. Among the analogs
prepared, the 1-naphthyl urea compound 19 proved the most ac-
tive. The individual enantiomers of 19 were separated by chiral
HPLC (Chiralcel OJ) and the more active enantiomer, 20, exhibited
subnanomolar potency. Unfortunately, although compound 20 was
an extremely potent inhibitor of Lck activity, its activity in our cel-
lular assay for IL2 release was poor (IC50 = 1758 nM).6
Although piperazine 36 is the most potent inhibitor of Lck activ-
ity in this series (IC50 = 0.06 nM), it is only a 111-nM inhibitor of
cellular IL2 release, corresponding to a nearly 2000-fold shift in po-
In an attempt to improve the activity of our Lck inhibitors in our
cellular assay, we turned to heterocyclic replacements of the piper-
idine ring. For our initial screen, we prepared the synthetically
Table 4
Des-methyl heterocycles
Table 2
Piperidine regioisomers
N
N
N
N
N
O
N
R
X
N
N
H
N
N
H
N
N
H
Y
Z
X
n
4
4
Compd
R
X
Y
Z
Lck IC50 (nM)
Compd
X
N
Lck IC50 (nM)
9
10
11
12a
12b
H
H
H
Me
Me
NCbz
CH2
CH2
CH2
CH2
CH2
NCbz
CH2
NCbz
NCbz
CH2
CH2
NCbz
CH2
CH2
>20,000
650
>20,000
144
21
22
23
24
25
CH2
CH2
O
NH
NCH3
1
2
2
2
2
>10,000
106
203
110
10
2723

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J. A. Hunt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5440–5443
Table 5
Table 8
Racemic piperazines
C5-Substituted benzimidazoles
R
N
N
N
N
N
N
R2
N
N
H
N
H
N
N
N
R1
4
6
Compd
R
Lck IC50 (nM)
IL2 IC50 (nM)
R1
R2
Lck IC50 (nM)
IL2 IC50 (nM)
4
6
Compd
N
26a
26b
27
28
29
30
31
32
33
34
35
Me
Me
Bn
SO2Me
SO2Ph
Cbz
Me
Me
Me
1-Np urea
1-Np urea
1-Np urea
1-Np urea
1-Np urea
1-Np urea
2-Np urea
SO2Me
0.25
17.4
2.6
361
3
3
98
>103
>103
>103
>104
>104
>103
>103
>104
>104
>104
N
11.8
26.5
>103
75.2
22.5
501
49
50
1.5
1
111
N
NH2
N
N
167
Bn
Me
H
Me
Me
>103
>103
O
ity against Lck, as exemplified by the 2-methyl analog 43. Substitu-
tion of the 5- and 6-positions of the benzimidazole proved more
promising. The regioisomeric methyl- (44 and 45) and amino-
(47 and 48) analogs maintained activity, with the 6-methyl and
5-amino-regioisomers each being more potent than the parent
compound.
Encouraged by the activity of compound 48, and cognizant of
the synthetic utility of the amino group, we prepared a variety of
alkylamine, amide, sulfonamide, and urea derivatives, but none
of these compounds showed cellular activity.7 However, when
we explored heterocyclic substituents at the benzimidazole C5,
we eventually discovered a handful of compounds, shown in Table
8, with promising activity in our IL2 assay.
When these benzimidazole C5 substituents were incorporated
into the piperazine naphthyl urea series, the resulting compounds
were highly potent inhibitors of both Lck activity and cellular IL2
release. Compounds 51, 52, and 4 showed much less dramatic
shifts in potency from the enzyme to the cell, as compared to the
parent piperazine 36 (Table 9). In particular, pyridine-substituted
compound 4 was a 0.12-nM inhibitor of Lck activity and an 8-nM
inhibitor of IL2 release (corresponding to a 70-fold shift). Com-
pound 4 is the most potent inhibitor of cellular IL2 release pre-
pared to date in our laboratories.
Table 6
(S,S)-Piperazines
N
N
N
N
Me
O
N
H
N
N
H
N
R
4
6
Compd
R
Lck IC50 (nM)
IL2 IC50 (nM)
36
37
38
39
40
41
42
Me
Et
i-Bu
Hexyl
0.06
0.07
0.2
111
154
538
>103
546
847
>104
1.8
0.2
4-Pyridyl
CH2CO2Et
Ac
0.45
115
tency from the enzyme to the cell. We turned our attention to the
benzimidazole portion of the inhibitor in hopes of addressing this
potency shift.
Concurrent with our studies on substitutions for the 2-benzyl-
amino group of lead compound 1, we also examined the effect of
modifications of the benzimidazole moiety, as shown in Table 7.
Substitution at C2 of the benzimidazole severely diminished activ-
Table 9
C5-Substituted benzimidazole (S,S)-piperazines
R
N
Table 7
Substituted benzimidazoles
N
N
Me
O
R5
R6
N
R2
N
H
N
N
H
N
N
N
Me
N
4
6
Compd
R
Lck IC50 (nM)
0.4
IL2 IC50 (nM)
N
N
H
N
N
51
42
16
8
4
Compd
R2
R5
R6
Lck IC50 (nM)
O
1
H
CH3
H
H
H
H
H
CH3
H
CH3
H
H
H
CH3
CH3
H
153
N
43
44
45
46
47
48
>20,000
128
52
4
0.07
0.12
N
NH2
58
2073
86
N
H
H
NH2
H
NH2
19.5

J. A. Hunt et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5440–5443
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2. (a) 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.; Poers, 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. and references therein; (b) Burchat, A.; Borhani, Q. W.; Caldewood, D. J.;
Hirst, G. C.; Li, B.; Stachlewitz, R. F. Bioorg. Med. Chem. Lett. 2006, 16, 118. and
references therein; (c) Maier, J. A.; Brugel, T. A.; Sabat, M.; Golebiowski, A.;
Laufersweiler, M. J.; VanRens, J. C.; Hopkins, C. R.; De, B.; Hsieh, L. C.; Brown, K.
K.; Easwaran, V.; Janusz, M. J. Bioorg. Med. Chem. Lett. 2006, 16, 3646. and
references therein; (d) 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.; Ching, S.; de Fex, H. F.; 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. and references therein.
3. For a recent report of disubstituted pyrimidines as Lck inhibitors, see Ref. [2a].
4. Park, Y.-W.; Cummings, R. T.; Wu, L.; Zheng, S.; Cameron, P. M.; Woods, A.;
Zaller, D. M.; Marcy, A. I.; Hermes, J. D. Anal. Biochem. 1999, 269, 94.
5. Hunt, J. A., et al. Unpublished data.
6. To evaluate inhibition of downstream signaling events, compounds were
assayed for their ability to block antigen-stimulated IL-2 release in stable
Jurkat cell transformants expressing a TCR specific for the MART-1 antigen (Cole,
D. J.; Weil, D. P.; Shilyansky, J.; Custer, M.; Kawakami, Y.; Rosenberg, S. A.;
Nishimura, M. I. Cancer Res., 1995, 55, 748).
In summary, we have developed a family of 4-benzimidazolyl-
1-piperazinethyl-substituted pyrimidin-2-amines as Lck kinase
inhibitors. We have shown that the preferred piperazine substitu-
ents are N1-naphthyl urea and N4-methyl, and we have deter-
mined the optimal relative and absolute stereochemistry of these
chiral compounds. Further, we have shown that compound 4,
which combines the optimized piperazine-ethyl moiety at the
pyrimidine C2 with the optimized benzimidazolyl substituent at
the pyrimidine C4, is a potent inhibitor of both Lck kinase activity
and cellular IL2 release.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.102.
References and notes
1. (a) Alberola-Ila, J.; Takaki, S.; Kerner, J. D.; Perlmutter, R. M. Annu. Rev. Immunol.
1997, 15, 125; (b) Bolen, J. B.; Brugge, J. S. Annu. Rev. Immunol. 1997, 15, 371; (c)
Chan, A. C.; Desai, D. M.; Weiss, A. Annu. Rev. Immunol. 1994, 12, 555.
7. See Supplementary data for synthetic schemes and additional data.