Design and synthesis of novel pyrimidine analogs as highly selective, non-covalent BTK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.11.037

BTK is a promising target for the treatment of multiple diseases such as B cell malignances, asthma, and rheumatoid arthritis. Here, we report the discovery of a series of novel pyrimidine analogs as potent, highly selective, non-covalent inhibitors of BTK. Compound 25d demonstrated higher affinity to an unactivated conformation of BTK that resulted in an excellent kinase selectivity. Compound 25d showed a good oral bioavailability in mice, and significantly inhibits the PCA reaction in mice.

Full text of DOI:10.1016/j.bmcl.2017.11.037

Accepted Manuscript
Design and synthesis of novel pyrimidine analogs as highly selective, non-co-
valent BTK inhibitors
Wataru Kawahata, Tokiko Asami, Takayuki Irie, Masaaki Sawa
PII:
S0960-894X(17)31136-8
https://doi.org/10.1016/j.bmcl.2017.11.037
BMCL 25443
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 October 2017
22 November 2017
23 November 2017
Please cite this article as: Kawahata, W., Asami, T., Irie, T., Sawa, M., Design and synthesis of novel pyrimidine
analogs as highly selective, non-covalent BTK inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi:
https://doi.org/10.1016/j.bmcl.2017.11.037
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design and synthesis of novel pyrimidine analogs as highly selective, non-
covalent BTK inhibitors
Wataru Kawahata∗, Tokiko Asami, Takayuki Irie and Masaaki Sawa
Research and Development, Carna Biosciences, Inc., 3rd Floor, BMA, 1-5-5 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
BTK is a promising target for the treatment of multiple diseases such as B cell malignances,
asthma, and rheumatoid arthritis. Here, we report the discovery of a series of novel pyrimidine
analogs as potent, highly selective, non-covalent inhibitors of BTK. Compound 25d
demonstrated higher affinity to an unactivated conformation of BTK that resulted in an excellent
kinase selectivity. Compound 25d showed a good oral bioavailability in mice, and significantly
inhibits the PCA reaction in mice.
Revised
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
Bruton’s tyrosine kinase
Kinase inhibitor
Kinase selectivity
Pyrimidine
Passive cutaneous anaphylaxis
Bruton's tyrosine kinase (BTK) is a member of the Tec family
of non-receptor tyrosine kinases, and plays a crucial role in both
B-cell receptor (BCR) and Fc receptor signaling.1 The
implication of BTK in a number of pathological processes has
been reported, and thus BTK is considered as a promising
therapeutic target for the treatment of a wide array of diseases
involving B-cell and/or macrophage activation, such as B-cell
malignancies, asthma and rheumatoid arthritis.2, 3 Ibrutinib, the
first FDA-approved covalent BTK inhibitor, has demonstrated
impressive response rates in patients with mantle cell lymphoma
and chronic lymphocytic leukemia,4 and several covalent BTK
inhibitors are currently being evaluated in clinical trials for B-cell
malignancies.5 However, it should be difficult to utilize these
covalent inhibitors for non-oncology indications because of
safety concerns associated with a potential immunogenicity of
protein-inhibitor adducts.6 Therefore, the development of a
highly selective and non-covalent BTK inhibitor for non-
oncology indications is highly demanded.
benzene ring of aminopyrimidine 1 resulted in a dramatic
improvement of BTK inhibition (2a, IC50 = 44.7 nM) while the
inhibitory activity for SYK was diminished (Fig. 1). Therefore
we chose compound 2a as the lead compound for further
optimization study.
O
O
N
H
N
H
N
N
N
N
Me
H
N
H
N
HN
HN
N
N
1
2a
BTK : 0.44 µM
SYK : >10 µM
BTK : 7.3 µM
SYK : 0.36 µM
H3 pocket
X
During the course of study on the SYK/BTK dual inhibitor
program at Carna, aminopyrimidine analog 1 has been identified
as a good starting point for developing non-covalent BTK
inhibitors because 1 has multiple sites for substitution which
allow to access to the specific binding sites in BTK.
Aminopyrimidine 1 showed moderate SYK inhibitory activity
(IC50 = 0.36 µM) but marginal inhibitory activity against BTK
(IC50 = 7.3 µM). Installation of a methyl group on the central
N
N
Z
R
HN
Ar
hinge region
solvent accesible region
Figure 1. Structures of aminopyrimidine analogs and lead optimization
strategy.
———
∗ Corresponding author. Tel.: +81 78 302 7040; fax: +81 78 302 7086; e-mail: wataru.kawahata@carnabio.com (W. Kawahata).

O
O
HN
NH2
O
F
O
F
HN
Me
Me
Br
a
b
Me
Br
HN
Cl
N
+
Br
Cl
O
a
b
B
O
O
O
7
8
9
5
3
4
O
F
O
F
O
F
c
d
O
Cl
N
B
N
O
Cl
N
O
O
OAc
OAc
OH
10
HN
HN
11
12
Me
N
Me
N
H2N
O2
N
O2N
c
d
O
g, h
e, f
N
CN
CN
N
N
N
N
H
OH
H
HN
Cl
Ar
13
14
15i
6
: Ar = 1H-indazol-6-yl
2a
2b : Ar = 1H-indazol-5-yl
2c
O
F
: Ar = 1H-indole-6-yl
2d : Ar = 3,4,5-trimethoxyphenyl
2e
H2N
Cl
H2N
Cl
H2
N
i
j, k
N
: Ar = 4-morpholinocarbonylphenyl
N
N
N
N
N
N
R
Cl
16
HN
HN
Ar
Ar
: Ar = 4-morpholinophenyl
18a
: Ar = 4-morpholinophenyl
17a
Scheme 1. Preparation of 2-amino-4-(2-methylphenyl)pyrimidine derivatives
2a 2e. Reagents and conditions: (a) 4-tert-butylbenzoyl chloride,
17e : Ar = 1H-pyrazol-4-yl
17f : Ar = 1-methyl-1H-pyrazol-4-yl
17i
18e : Ar = 1H-pyrazol-4-yl
18f : Ar = 1-methyl-1H-pyrazol-4-yl
18i
: Ar = 5-cyano-1-methyl-1H-pyrrol-3-yl
–
: Ar = 5-cyano-1-methyl-1H-pyrrol-3-yl
triethylamine, CH2Cl2, rt, 94%; (b) bis(pinacolato)diboron, KOAc,
PdCl2(PPh3)2-CH2Cl2 adduct, 1,4-dioxane, reflux, 71%; (c) 2,4-
dichloropyrimidine, Pd(PPh3)4, benzene/MeOH, reflux, 72%; (d) Ar-NH2, cat.
HCl, BuOH, 110°C, 8 - 23%.
Scheme 2. Preparation of 2,4-diamino-pyrimidine derivatives 18a, 18e, 18f
and 18i. Reagents and conditions: (a) CuI, K2CO3, DMF, 120°C, 49%; (b)
NaBH4, CH2Cl2/IPA, 0 °C, 94%; (c) acetyl chloride, pyridine, CH2Cl2, rt,
74%; (d) bis(pinacolato)diboron, KOAc, Pd(dba)2, X-Phos, 1,4-dioxane,
70°C, 88%; (e) ammonia, CDI, THF, rt, quant.; (f) propylphosphonic
anhydride, triethylamine, EtOAc, rt, 68%; (g) MeI, NaH, DMF, 0°C, 80%;
(h) H2, Pd-C, MeOH/THF, rt, quant.; (i) Ar-NH2, DME, 120°C, 10-22%; (j)
12, K2CO3, Pd(PPh3)4, DME/water, 110°C; (k) K2CO3, EtOH, 70°C, 6-34%, 2
steps.
Table 1. Effects of 2-arylamino group on BTK inhibitory potency.
O
N
H
N
N
Me
H2N
H2N
O2N
HN
O
O
a, b
c, d, b
Ar
N
H
N
N
N
IC50 (nM)a
SYK
H
Comp.
Ar
19
14g
20
14j
BTK
45
>10000
>10000
>10000
2a
O
F
O
F
H2
N
f, g
e
H2N
N
N
OAc
21
12
16
+
2b
2c
40
52
N
N
N
N
R
HN
Cl
Ar
: Ar = 4-(4-methylpiperazin-1-yl)phenyl
: Ar = 4-(morpholinomethyl)phenyl
: Ar = 4-methoxyphenyl
18b
18c
18d
18g
: Ar = 1-methyl-1H-pyrazol-3-yl
18h : Ar = 1-methyl-1H-pyrrol-3-yl
18j : Ar = 5-acetyl-1-methyl-1H-pyrrol-3-yl
>10000
>10000
6.1
9.0
2d
2e
Scheme 3. Alternative synthetic method for 2,4-diamino-pyrimidine
derivatives 18b-d, 18g, 18h and 18j. Reagents and conditions: (a) MeI,
K2CO3, DMA, 60°C, 85%; (b) H2, Pd-C, EtOH/1,4-dioxane, rt, 90-96%; (c)
Nitric acid, Acetic anhydride, -10°C, 22%; (d) MeI, NaH, DMF, 0°C, 78%;
(e) K2CO3, Pd(PPh3)4, DME/water, 110°C, 39%; (f) Ar-NH2, 2M-HCl aq, 2-
PrOH, 150°C; (g) K2CO3, EtOH, 70°C, 6-32%, 2 steps.
a
Values are the mean of two separate experiments. See References and
notes for experimental details..
indazole with
a benzene ring resulted in a significant
The syntheses of 2-amino-4-(2-methylphenyl)pyrimidine
derivatives 2a – 2e are shown in Scheme 1. Commercially
available 3-bromo-2-methylaniline 3 was coupled with 4-tert-
butylbenzoyl chloride, then the bromo group was converted to a
boronate ester by treating with bis(pinacolato)diboron. Boronate
ester 5 was then coupled with 2,4-dichloropyrimidine by Suzuki-
coupling to yield 2-chloropyrimidine 6. Substitution of Cl in 6
with appropriate aromatic amines gave the desired compounds 2a
– 2e.
improvement of inhibitory potencies for BTK without increasing
SYK potency. Namely, compound 2d and 2e exhibited a single-
digit nanomolar IC50 values for BTK, but no activity against SYK
up to 10 µM. Unfortunately, those compounds showed poor
stabilities in human and mouse microsomes presumably due to
the lability of the amide bond linking the terminal benzene, and
hydroxylation on the tert-butyl group. Young et al identified a
tetrahydrobenzothiophene as the isostere of the tert-butyl
benzene to improve ADME property.8 Recently Lou et al
reported that the replacement of the tert-butyl benzoylamide with
a 6-cyclopropyl- isoquinolone group resulted the improved
microsomal stability.9 In addition, they found that hydroxylation
of the methyl group on the central benzene ring increased the
inhibitory potency for BTK by making a hydrogen bond with
BTK. Furthermore, our docking model with BTK suggested that
At first, we examined the effects of the 2-arylamino group on
BTK inhibitory potency, because the modeling of compound 2a
with BTK suggested that 2-arylamino group was exposed to the
solvent accessible regions, which provided opportunities for
installing various substituents (Fig 1). As shown in Table 17, no
significant changes of the inhibitory potencies for BTK were
observed with bicyclic analogs 2b and 2c. Replacement of the

O2
N
H2N
23a : R = ethyl
a or b or c, d
Table 2. Inhibitory profiles of 2,4-diamino-pyrimidine derivatives 18a-j
23c
23d
23f
: R = tert.-butyl
: R = cyclopropyl
: R = cyclopropylmethyl
: R = 2,2-difluoroethyl
N
N
N
H
against BTK[A] and BTK[U].
N
R
23g
O
F
22
H2N
N
H2
N
e, f, d
N
N
N
OH
N
OH
HN
Ar
IC50 (nM)a
23e
Ar
Comp.
18a
H2
N
Cl
BTK[A]
BTK[U]
O
F
N
N
H2N
g
h, i
12.9
0.6
N
16
HN
N
N
R
N
HN
N
Ar
5.3
>10000
39
0.4
309
2.5
0.6
18b
18c
18d
18e
R
: R = ethyl
24c : R = tert.-butyl
24d : R = cyclopropyl
24e
: R = 1-(hydroxymethyl)cyclopropyl
: R = ethyl
25a
25c : R = tert.-butyl
25d : R = cyclopropyl
25e
: R = 1-(hydroxymethyl)cyclopropyl
24a
O
F
H2N
j, k
N
21
N
N
R
8.1
HN
Ar
: Ar = isopropyl
: Ar = cyclopropylmethyl
: Ar = 2,2-difluoroethyl
25b
25f
25g
3.7
0.4
18f
Scheme 4. Preparation of N-substituted pyrazole derivatives 25a-g. Reagents
and conditions: (a) alkyl halide, K2CO3, 77-98%, for 23a, 23d, 23f; (b) tert-
butyl acetate, H2SO4, 1,4-dioxane, rt, 72%, for 23c; (c) 2,2-difluoroethanol,
DIAD, Ph3P, THF, rt, 81% for 23g; (d) H2, Pd-C, EtOH, rt, 10%-quant.; (e)
methyl 2,4-dibromobutyrate, NaH, DMA, 0°C, 51%; (f) LiBH4, THF, rt,
quant.; (g) aminopyrazole, 2M-HCl aq., PrOH, 100°C, 25-44%; (h) 12,
K2CO3, Pd(PPh3)4, DME/water, 110°C; (i) K2CO3, EtOH, 70°C, 4-64%, 2
steps; (j) aminopyrazole, 2M-HCl aq, 2-PrOH, 150°C; (k) K2CO3, EtOH,
70°C, 10-39%, 2 steps.
245
62
13.9
17
18g
18h
18i
20.2
1.7
5.9
0.8
18j
We previously reported that compounds that preferentially
bind to an unactivated state of BTK would show high selectivity
to BTK.10, 11 Therefore two conformationally different BTKs, an
activated form of BTK (BTK[A])12 and an unactivated form of
BTK (BTK[U])13 were used for further evaluation of compounds
to select compounds that inhibit BTK[U] strongly. Because the
substituted benzene analogs 2d and 2e displayed potent
inhibitory activity, the SAR study for the aryl group Ar was
performed with 2,4-diamino-pyrimidine analogs, and the results
were shown in Table 2.14 Attaching a morpholine group to the
phenyl ring directly (18a) still showed potent inhibitory activity
for both BTK[A] and BTK[U]. Introduction of more basic
piperazine group (18b) in place of the morpholine increased
potency for BTK[A]. Interestingly, insertion of methylene group
between morpholine and phenyl ring (18c) resulted in a
significant loss of potency. p-Methoxyphenyl analog 18d showed
modest activity. It was noteworthy that simple pyrazole
derivative 18e exhibited comparable potency with 18b. This
replacement of the phenyl moiety with pyrazole ring provided
further opportunity to install the substituents because of its
smaller molecular weight, and thus we focused on the
optimization of the pyrazole moiety. Methylation of the pyrazole
(18f) further increased the potency for BTK[A]. Surprisingly,
switching of substitution position from 4 to 3 at the pyrazole ring
resulted in a significant loss of activities, especially for BTK[A].
The binding model suggested that the pyrazole ring was exposed
to the solvent accessible region and no interaction with BTK was
observed, therefore this decrease of potency might be due to the
changes in the distribution of electron density at the
aminopyrimidine moiety which would affect the hydrogen
bonding with the hinge region. Because this series of compounds
preferentially binds to BTK[U] by interacting with H3 pocket,
18g could retain the potency for BTK[U] with a slight decrease.
a
Values are the mean of two separate experiments. See References and notes
for experimental details.
an introduction of amino group at 4-position of the pyrimidine
ring can form a hydrogen bond with the hinge region of BTK.
Thus, we decided to introduce these structures into our
compounds for further optimization.
The syntheses of 2,4-diamino-pyrimidine derivatives 18a, 18e,
18f and 18i are shown in Scheme 2. Isoquinolone 89 was coupled
with 2-bromo-6-chlorobenzaldehyde 7 by Cu-catalyzed cross
coupling, and the resulting benzaldehyde 9 was reduced using
NaBH4 to produce benzylalcohol 10. Acetylation of the hydroxyl
group with acetyl chloride followed by conversion of the chloride
to the corresponding boronate ester afforded the common
intermediate 12. Pyrrole-2-carboxylic acid 13 was coupled with
ammonia followed by a dehydration reaction which gave
cyanopyrrole 14. Methyl group was introduced by treating with
MeI, and reduction of the nitro group afforded arylamine 15i.
Substitution of Cl in 4-amino-2,6-dichloropyrimidine 16 with
appropriate arylamines gave the corresponding 2-arylamino
pyrimidines 17a, 17e, 17f and 17i. Those compounds were then
coupled with boronate ester 12 by Suzuki-coupling, followed by
deprotection of the hydroxyl group furnished the desired products
18a, 18e, 18f and 18i. The alternative synthetic method of 2,4-
diamino-pyrimidine derivative 18b-d, 18g, 18h and 18j are
shown in Scheme 3. N-methyl pyrrole-3-amine 14g was obtained
by N-methylation with MeI, and subsequent hydrogenation of the
nitro group. N-methyl-5-acetyl-pyrrole-3-amine 14j was also
obtained in a manner similar to 14g. Compound 16 was coupled
with boronate ester 12 by Suzuki-coupling and the resulting
intermediate 21 was coupled with appropriate arylamines. Finally
the acetyl group was removed to give 18b-d, 18g, 18h and 18j.

Table 3. Inhibitory activity of N-substituted pyrazole analogs 18f and 25a-g for BTKs and physicochemical profiles.
O
F
H2N
N
N
N
OH
HN
N
N
R
IC50 (nM)a
Caco2 permeability
(Papp×106/cm·s-1)
Aqueous solubility
(µM)
Comp.
18f
R
BTK[A]
3.7
BTK[U]
0.4
6.3
5.4
18
10
0.4
0.2
NT
25a
2.6
3.7
4.0
25b
11
2.8
12
0.6
0.3
0.6
5.7
6.9
0.7
NT
9.8
NT
25c
25d
25e
3.4
0.3
6.1
3.1
6.3
2.1
25f
3.3
0.2
25g
a Values are the mean of two separate experiments. See References and notes for experimental details.
The pyrrole analog 18h showed 17-fold and 43-fold decrease of
inhibitory activity for BTK[A] and BTK[U] respectively.
Installment of electron withdrawing groups on the 5-position of
the pyrrole (18i and 18j) resulted in increase in the potencies
especially for BTK[U].
To confirm the inhibitory potency in vitro, we evaluated the
ability of compounds to inhibit autophosphorylation of BTK in
cells. As shown in Figure 3, 18f and 25d potently inhibited anti-
IgM induced phosphorylation of BTK in cells. To select a
compound for further evaluations in vivo, Caco-2 membrane
permeability and aqueous solubility (pH 7.4)18 were measured.
As shown in Table 3, methyl analog 18f and cyclopropyl analog
25d have good permeability and aqueous solubility, and thus
those two compounds were subjected to mouse PK studies. As
shown in Table 4, both compounds showed good oral
bioavailability with a similar pharmacokinetics parameters. As
cyclopropyl analog 25d exhibited superior Cmax and AUC
compared to 18f, we selected 25d for further evaluations.
Because N-methyl pyrazole analog 18f showed the most
potent inhibitory activity for BTK, further optimization of the
substituent at 1-position of the pyrazole was performed. The
syntheses of N- substituted pyrazole derivatives 25a-g are shown
in Scheme 4.15 N-substituted pyrazole amine 23a and 23c-g were
prepared from 4-nitropyrazole 22. For 23e, 4-nitropyrazole was
alkylated with methyl 2,4-dibromobutyrate in the presence of
NaH to provide the cyclopropanecarboxylate ester which was
converted to the corresponding alcohol by reducing with LiBH4,
followed by hydrogenation of the nitro group gave 23e. Further
steps to the desired products were executed in a manner similar
to the synthesis of 18a-j.
To investigate the kinase selectivity, compoud 25d was
screened over a panel of 307 kinases.19 Compound 25d
demonstrated a high selectivity profile, only inhibited 2 kinases
within the panel (BMX and TEC, 70% and 92% inhibitions at
0.3µM concentration, respectively).
All of the compounds in the series exhibited significant
inhibitory activity against BTK[U] with IC50 values below 1 nM
(Table 3). The increase of steric bulk (compounds 25c and 25e)
resulted in a decrease of inhibitory activity for BTK[A], although
no significant changes of activities for BTK[U] were observed in
these compounds. These results suggested that BTK[U] has little
sensitivity to the N-substituents of this chemical series. To better
understand the observed SAR, the binding model of the 25d-
BTK[U] complex was constructed.16 As shown in Figure 2, the
cyclopropyl group at the isoquinolone moiety occupied the large
hydrophobic pocket, called H3 pocket which is formed in
BTK[U].17 The pyrimidine N1 and 2-amino group formed
bidentate hydrogen bonds with the backbone amide of hinge
Met477. In addition, the 6-NH2 group of the pyrimidine ring was
positioned at the hydrogen bond distance with the side chain of
Thr474 and the main chain carbonyl of Glu475. The cyclopropyl
group at the pyrazole group seems to expose to the solvent
region, supporting the observed results that broader range of
substituents at this position were tolerable.
To study the ability of 25d to inhibit acute immune reaction in
vivo, 25d was subjected to the passive cutaneous anaphylaxis
(PCA) model in mice.20 Oral administration of compound 25d at
30 mg/kg significantly inhibited the PCA reaction, and its
inhibition rate was 46% as compared to vehicle control.
In conclusion, we have discovered
a novel series of
pyrimidine analogs as highly selective, non-covalent inhibitors of
BTK. Compound 25d preferentially bound to the unactivated
conformation of BTK, which resulted in the high kinase
selectivity.
Compound
25d
strongly
inhibited
autophosphorylation of BTK in cells and also demonstrated
excellent efficacy in a mouse PCA model. Unfortunately 25d was
found to inhibit hERG channel that hindered further development
of this compound. Currently, further optimization to eliminate the
hERG issue is underway.

Table 4. Pharmacokinetics parameters of 18f and 25d in ICR Mice.
t1/2
hr
Tmax
hr
Cmax
ng/mL
2952
AUC(0-t)
ng/mL*hr
3458
AUC(0-∞)
ng/mL*hr
3476
Vz
L/kg
0.93
-
CL
MRT(0-∞)
hr
F
Dosage and
route
Comp.
18f
L/hr/kg
%
IV-2 mg/kg
PO-10 mg/kg
IV-2 mg/kg
1.12
0.96
1.09
1.31
0.083
0.50
0.083
1.00
0.58
1.13
-
4541
12746
12809
-
0.63
-
1.91
73.70
-
2259
3147
3171
1.00
-
1.36
25d
PO-10 mg/kg
5941
18736
19084
2.24
120.35
5. Wu J, Liu C, Tsui ST, Liu D. Second-generation inhibitors of
Bruton tyrosine kinase. J Hematol Oncol. 2016; 9: 80-86.
6. Barf T, Kaptein AIrreversible protein kinase inhibitors: balancing
the benefits and risks. J Med Chem. 2012; 55: 6243-6262.
7. Enzymatic activity of SYK and BTK was measured using 1 µM
FITC-labeled substrate peptide (Blk/Lyntide for SYK and Srctide
for BTK, respectively) in the presence of compounds in assay
buffer consisting of 20 mM HEPES (pH7.5), 0.01% Triton X-100,
2 mM DTT, 5 mM MgCl2. For SYK or BTK assays, 25 µM ATP
or 75 µM ATP was added respectively. The reaction was
terminated by adding of EDTA. The reaction product was
quantified by using a LabChip EZ Reader II (Caliper Life
Sciences). Inhibitory activity of compounds were evaluated in a
10-dose with 3-fold serial dilution starting at 10 µM for SYK and
BTK in duplicates (N = 2), and IC50 values were calculated from
dose-response curves.
8. Young WB, Barbosa J, Blomgren P, Bremer MC, Crawford JJ,
Dambach D, Gallion S, Hymowitz SG, Kropf JE, Lee SH, Liu L,
Lubach JW, Macaluso J, Maciejewski P, Maurer B, Mitchell SA,
Ortwine DF, Di Paolo J, Reif K, Scheerens H, Schmitt A, Sowell
CG, Wang X, Wong H, Xiong JM, Xu J, Zhao Z, Currie KS.
Potent and selective Bruton's tyrosine kinase inhibitors: discovery
of GDC-0834. Bioorg Med Chem Lett. 2015; 25: 1333-1337.
9. Lou Y, Han X, Kuglstatter A, Kondru RK, Sweeney ZK, Soth M,
McIntosh J, Litman R, Suh J, Kocer B, Davis D, Park J,
Frauchiger S, Dewdney N, Zecic H, Taygerly JP, Sarma K, Hong
J, Hill RJ, Gabriel T, Goldstein DM, Owens TD. Structure-based
drug design of RN486, a potent and selective Bruton's tyrosine
kinase (BTK) inhibitor, for the treatment of rheumatoid arthritis. J
Med Chem. 2015; 58, 512-516.
Figure 2. Docking study of 25d bound to the unactivated conformation of
BTK (PDB ID: 3OCS).
10. Asami T, Kawahata W, Sawa M. TR-FRET binding assay
targeting unactivated form of Bruton's tyrosine kinase. Bioorg
Med Chem Lett. 2015; 25: 2033-2036.
11. Kawahata W, Asami T, Fujii I, Sawa M. 'Turn On/Off'
fluorescence probe for the screening of unactivated Bruton's
tyrosine kinase. Bioorg Med Chem Lett. 2015; 25: 2141-2145.
12. Biotinylated BTK having a N-terminal DYKDDDDK tag was
treated with 3 mM ATP at 4 °C overnight in buffer solution
consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10%
Glycerol, 0.05% Brij35, 1 mM DTT and 10 mM MgCl2. The
mixture was then run through the 10-DG column to give
biotinylated BTK [A].
Figure 3. Inhibition of autophosphorylation of BTK stimulated with anti-IgM
in Ramos cells. Ramos cells were treated with compounds at 30 nM for 1h
and then stimulated with anti-IgM for 10 min. The phosphorylation levels of
BTK (Tyr223) were analyzed by western blotting.
13. Biotinylated BTK having a N-terminal DYKDDDDK tag was
treated with His-lambda phosphatase (11400U) at 4 °C overnight
in buffer solution consisting of 50 mM Tris-HCl (pH 7.5), 150
mM NaCl, 10% Glycerol, 0.05% Brij35, 1 mM DTT. The mixture
was purified by a DYKDDDDK tag antibody agarose gel column,
then the buffer was exchanged using the 10-DG column to give
biotinylated BTK [U].
Acknowledgments
We thank Mr. Fumio Nakajima for the docking study. We also
gratefully acknowledge Carna’s Contract Research Department
for providing assay kits and selectivity profiling.
14. Enzymatic activity of BTK[A] and BTK[U] was measured using 1
µM FITC-labeled Srctide peptide in the presence of compounds in
assay buffer consisting of 20 mM HEPES (pH7.5), 0.01% Triton
X-100, 2 mM DTT, 5 mM MgCl2. For biotinylated BTK [A] or
biotinylated BTK [U] assays, 25 µM ATP or 50 µM ATP was
added respectively. The reaction was terminated by adding of
EDTA. The reaction product was quantified by using a LabChip
EZ Reader II (Caliper Life Sciences). Inhibitory activity of
compounds were evaluated in a 10-dose with 3-fold serial dilution
starting at 10 µM for BTK [A] and BTK [U] in duplicates (N = 2),
and IC50 values were calculated from dose-response curves.
15. Data for 25d: : 1H NMR (400 MHz, DMSO-d6) δ 8.96 (s, 1H),
7.99 (s, 1H), 7.56 - 7.52 (m, 2H), 7.42 (s, 1H), 7.39 (dd, J = 6.3,
2.9 Hz, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.27 (d, J = 1.6 Hz, 1H),
References and notes
1. Ellmeier W, Abramova A, Schebesta A. Tec family kinases:
regulation of FceRI-mediated mast-cell activation. FEBS J. 2011;
278: 1990-2000.
2. Hendriks RW, Yuvaraj S, Kil LP. Targeting Bruton’s tyrosine
kinase in B cell malignancies. Nat Rev Cancer. 2014; 14: 219-232.
3. Horwood NJ, Urbaniak AM, Danks L. Tec family kinases in
inflammation and disease. Int Rev Immunol. 2012; 31: 87-103.
4. Davids MS, Brown JR. Ibrutinib: a first in class covalent inhibitor
of Bruton’s tyrosine kinase. Future Oncol. 2014; 10: 957-967.

6.99 (dd, J = 13.2, 1.6 Hz, 1H), 6.93 – 6.69 (m, 2H), 6.61 (dd, J =
7.5, 2.1 Hz, 1H), 6.06 (s, 1H), 5.15 (s, 1H), 4.34 - 4.22 (m, 1H),
4.14 - 4.01 (m, 1H), 3.66 - 3.57 (m, 1H), 2.12 - 2.02 (m, 1H), 1.14
- 1.06 (m, 2H), 1.04 - 0.96 (m, 2H), 0.95 - 0.81 (m, 4H). MS:
524.2 [M+H]+.
16. Molecular modeling activities were performed using the suite of
programs within the Discovery Studio release 2.1 (Accelrys
Software). The compound 25d was flexibly docked into the active
site of BTK (PDB ID: 3OCS) using CDOCKER Module with a
standard parameter setup.
17. Di Paolo JA, Huang T, Balazs M, Barbosa J, Barck KH, Bravo BJ,
Carano RA, Darrow J, Davies DR, DeForge LE, Diehl L,
Ferrando R, Gallion SL, Giannetti AM, Gribling P, Hurez V,
Hymowitz SG, Jones R, Kropf JE, Lee WP, Maciejewski PM,
Mitchell SA, Rong H, Staker BL, Whitney JA, Yeh S, Young WB,
Yu C, Zhang J, Reif K, Currie KS. Specific Btk inhibition
suppresses B cell- and myeloid cell-mediated arthritis. Nat. Chem.
Biol. 2011; 7, 41-50.
18. Solubility of compounds were determined by kinetic solubility
measurement method. DMSO stock solution of test compound
was added into 100 mM phosphate buffer (pH 7.4), and the
solution was shaken for 1 hour at room temperature. Samples are
centrifuged to precipitate un-dissolved particles, and
concentrations of the supernatant was determined by LC-UV or
LC-MS/MS.
19. Kinase panel assay (QuickScout™) was conducted by Contract
Research Department of Carna Biosciences. The detailed
experimental procedure is available at
http://www.carnabio.com/output/pdf/ProfilingProfilingBook_en.p
df.
20. Mice were passively sensitized by intradermal injection of 10 µL
anti-DNP IgE in both ears. After 46 hours, compound 25d or
vehicle were administered. Two hours later, DNP-BSA/dye
mixture was administered (0.25 mL/mouse, i.v.). The mice were
then sacrificed 30 min after the injection, and both ears of each
mouse were collected, and weighed. A pair of the ears was
dissolved by 1N KOH (0.7mL) at 37°C overnight, then 9.3 mL of
an acetone/0.6N Phosphoric Acid (13:5) mixture was added to the
suspension, and the mixture was shaken. The extracts were
centrifuged (3,000rpm, 10min) to remove precipitates, then
supernatant was filtered by a 0.2µm filter. Absorbance at 620 nm
was measured to determine the amount of the dye.

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Graphical Abstract
Design and synthesis of novel pyrimidine
analogs as highly selective, non-covalent
BTK inhibitors
Leave this area blank for abstract info.
Wataru Kawahata, Tokiko Asami, Takayuki Irie and Masaaki Sawa
O
O
F
H2N
N
H
N
N
N
N
N
OH
H
N
HN
HN
N
N
N
25d
BTK selective inhibitor
1
SYK/BTK dual inhibitor
SYK IC50 : >10 µM50
SYK IC50 : 0.36 µM50
BTK IC50
BTK[A] IC50
BTK[U] IC50
:
:
2.8 nM50
0.3 nM50
:
7.3 µM50