Highly potent and selective pyrazolylpyrimidines as Syk kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.09.011

A series of pyrazolylpyrimidine scaffold based Syk inhibitors were synthesized and evaluated for their biological activities and selectivity. Lead optimization efforts provided compounds with potent Syk inhibition in both enzymatic and TNF-α release assay.

Full text of DOI:10.1016/j.bmcl.2015.09.011

Accepted Manuscript
Highly potent and selective pyrazolylpyrimidines as Syk kinase inhibitors
Jang-Sik Choi, Hae-jun Hwang, Se-Won Kim, Byung Il Lee, Jaekyoo Lee, Ho-
Juhn Song, Jong Sung Koh, Jung-Ho Kim, Phil Ho Lee
PII:
S0960-894X(15)30045-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.09.011
BMCL 23092
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 June 2015
2 September 2015
5 September 2015
Please cite this article as: Choi, J-S., Hwang, H-j., Kim, S-W., Lee, B.I., Lee, J., Song, H-J., Koh, J.S., Kim, J-H.,
Lee, P.H., Highly potent and selective pyrazolylpyrimidines as Syk kinase inhibitors, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.09.011
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Highly potent and selective pyrazolylpyrimidines as Syk kinase inhibitors
Jang-Sik Choia,b, Hae-jun Hwangb, Se-Won Kimb, Byung Il Leec, Jaekyoo Leed, Ho-Juhn Songd, Jong Sung
Kohd, Jung-Ho Kimb, *, Phil Ho Leea,*
a Department of Chemistry, Kangwon National University, Chuncheon 200-701, Republic of Korea
b Oscotec Inc., 694-1 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
c Biomolecular Function Research Branch, Division of Convergence Technology, Research Institute, National
Cancer Center, Goyang, Gyeonggi 410-769, Republic of Korea
d Genosco, 767C Concord Avenue, 2nd Floor, Cambridge, MA 02138, USA
* Corresponding authors. Tel.; +82 33 2508493; fax: +82 33 2595667
E-mail address: phlee@kangwon.ac.kr, jhkim@oscotec.com
Keywords: Spleen tyrosine kinase; Pyrazolylpyrimidine; Syk inhibitors; Rheumatoid arthritis; Structure-activity
relationship
ABSTRACT
A series of pyrazolylpyrimidine scaffold based Syk inhibitors were synthesized and evaluated for their
biological activities and selectivity. Lead optimization efforts provided compounds with potent Syk inhibition in
both enzymatic and TNF-α release assay.
Spleen tyrosine kinase (Syk) is an intracellular non-receptor protein tyrosine kinase that is involved in
immunoreceptor signaling events, mainly in B-cell receptor, Fc receptors, integrin and lectin receptors.1 Syk is
most widely expressed in hematopoietic cells like B cells, mast cells, macrophages and neutrophils but is also
expressed in other tissues.2
Upon engagement with these receptors Syk performs an important function in signal transduction which
mediates diverse cellular responses, including proliferation, differentiation and phagocytosis.3 Dysregulation
and/or misregulation of different signal transduction pathways of Syk in different cell types have been
implicated in numerous diseases and disorders e.g., allergic rhinitis, asthma, autoimmune diseases, rheumatoid
arthritis (RA), osteopenia, osteoporosis, COPD and various leukemia and lymphomas.4-8 Thus, the inhibition of
Syk activity may offer a therapeutic option for treatment of many diseases associated with Syk activity.
Over the last few years a number of ATP-competitive Syk inhibitors of varied chemotypes have been
described9 and it has been reported that pharmacological inhibition of Syk kinase activity modulates mast cell
degranulation, leukocyte immune function and suppress inflammation in vivo.10,11 Aminopyrimidine core
scaffold is well recognized in discovery of kinase inhibitor as it provides an essential hinge binding motif.
Several aminopyrimidine based Syk inhibitors are know in the literature. Few examples are Astellas compound
1
12 , Vertex’s compound 213, Rigel’s compound 3 (R-788), and Portola’s compound 414 (PRT062607). Clinical
development of R-788 for rheumatoid arthritis was halted due to limited efficacy in patients inactive to biologic

TNF inhibitors.15 Other few molecules are in early phases of clinical studies, including R-343 for asthma16 and
PRT062607 for autoimmune diseases. PRT062607 (also known as BIIB-057, 4) was the only selective Syk
inhibitor that has been evaluated in clinical studies. However, a planned phase II trial in rheumatoid arthritis
(RA) was withdrawn prior to patient enrollment. Recently, Syk inhibitors with different scaffolds were also
reported. Among them are thiazolopyrimidine and triazolopyridine derivative from Hoffmann-La Roche17,
triazine amide compounds from Novartis18, and phenyl carboxamide compounds from Merck.19 These
compounds were discontinued partly due to issues related to cellular permeability, solubility and or toxicity.
In our efforts to discover novel, potent and selective Syk inhibitors with improved pharmacological profiles,
cheminfomatic analysis and high throughput screening of in-house focused chemical library of pyrimidine
scaffold were carried out. Pyrrolylpyrimidine derivative 520 was identified as a potent Syk inhibitor and was
selected for further optimization.
Herein, we wish to report our findings on pyrrolylpyrimidine and pyrazolylpyrimidine derivatives as potent
and selective Syk inhibitors with good pharmacological profiles.
Schemes 1 & 2 outline the general synthetic procedures20, 21 for the final arylaminopyrimidine compounds.
The pyrrolylpyrimidine and pyrazolylpyrimidine derivatives 12a-y were synthesized starting from 4-chloro-2-
(methylthio)pyrimidine 6. The coupling reaction of pyrimidine 6 with pyrrole or pyrazole esters gave pyrrolyl-
and pyrazolylpyrimidine esters 7. Activation of the sulfide group of the compounds 7 via oxidation with
mCPBA to sulfones 8, followed by displacement of methanesulfonyl group with a variety of arylamines
afforded a series of 2-arylaminopyrimidine 9. Subsequent reduction of the esters 9 using lithium
aluminumhydride gave the intermediate alcohols 10, which were further oxidized with PDC to afford pyrrole-
and pyrazole aldehydes 11. Finally, the desired compounds 12a-y were successfully obtained from reductive
amination of pyrrole- and pyrazole aldehydes 11 with appropriate amines.

Scheme 1. Reagent and conditions: (a) pyrrole (pyrazole) ester, K2CO3, CH3CN, reflux, 80%; (b) mCPBA,
CH2Cl2, rt, 83%; (c) R1NH2, c-HCl, EtOH, reflux, 30-50%; (d) LiAlH4, THF, 0℃, 90%; (e) PDC, CH2Cl2, rt,
90%; (f) R2NH, NaBH(OAc)3, Et3N, CH2Cl2, rt, 70-90%
Similarly, 2-indolylamino substituted pyrazolylpyrimidine derivatives 16a-m were prepared according to the
synthetic method shown in Scheme 2. Compounds of general structure 16a-m were synthesized in three-step
procedures starting from 2,4-dichloropyrimidine 13. The compound 13 was reacted with pyrazole aldehyde22 in
acetonitrile to give pyrimidine pyrazole aldehyde derivative 14 as a mixture with 2-substituted and 2,4-
disubstituted compounds. Then, the desired 4-substituted isomer 14 was isolated as a single regioisomer by a
silica gel column chromatography and used in the following reaction. Further coupling reaction of the
compound 14 with various aniline derivatives via a Buchwald-Hartwig condition or acid catalyzed condition
afforded 2-anilino pyrimidine pyrazole aldehyde derivative 15. Finally, the desired compounds 16a-m were
prepared from the compound 15 by reductive amination with appropriate amines.
Scheme 2. Reagent and conditions: (a) pyrazole aldehyde, K2CO3, CH3CN, reflux, 40-45%; (b) R1NH2,
Pd(OAc)2, Xantphos, K2CO3, dioxane, reflux or R1NH2, c-HCl, EtOH, reflux, 40-50% ; (c) R2NH, NaBH(OAc)3,
TEA, CH2Cl2, rt, 60-80%
Compounds 12a-y and 16a-m were tested for inhibitory activity against Syk kinase and selectivity against a
small panel of other key kinases. Kinase screening activity revealed that 3,5-disubstituted aniline is optimal for
Syk inhibitory activity. Syk inhibitory activity data of 2-aniline substituted pyrimidine derivatives with a
concomitant substitution of various aminomethyl groups at pyrrole or pyrazole moiety are shown in Table 1.
Compound 5, 3,5-dimethylanilino compound with substitution on 4 position with aminopyrrole, showed potent
Syk inhibitory activity with an IC50 value of 2.3 nM. The first modification was focused on introducing different

heteroaryl group as a linker and different amine group instead of pyrrole linker to improve its selectivity and
potency. Pyrrole or pyrazole linker with 4-5 membered cyclic aminomethyl substituted compounds (5, 12a, 12c
and 12d) showed generally high inhibitory activity against Syk. However, Syk inhibitory activity was not
improved when a bigger 6-membered cyclic amine was employed (compound 12g). Interestingly, 4,4-
difluoropiperidine analogue 12h was about 133-fold less active than compound 5. The decreased activity of 4,4-
difluoropiperidine derivative 12h might be due to the lowered basicity of tertiary amine by strong electron-
withdrawing difluoro substitution. Acyclic amino groups attached compounds were also investigated. N,N-
dimethylamine derivative 12e strongly inhibited Syk similar to cyclic amine derivatives. Second modification
performed on 2-position of pyrimidine. The replacement of 3,5-dimethylaniline by trimethoxyaniline such as in
compound 12b and 12f resulted in a 7-fold and 12-fold decrease in activity against Syk, respectively.
Derivatives 12i and 12j with cyclopropyl substituent in R3 position of pyrrole ring showed similar Syk
inhibitory activity compared to corresponding methyl analogs 5 and 12a. Interestingly, replacing the hydroxyl
group in pyrrolidine or azetidine with an amino or carboxyl group (compounds 12K and 12l) showed modest
decrease in Syk inhibitory activity in comparison to compound 5.
Modifications at 4’-position of aniline portion of the compound 5 was undertaken in order to improve
physicochemical properties and activity and the data is presented in Table 2. Substitution of hydroxyethyloxy
group to 4’-position of the compound 5 leaded to the compound 12n of which kinetic solubility (25 mM DMSO
stock) was increased to 120 µM compared to 70 µM of the compound 5. Cell permeability of the compound 12n
in Caco-2 cell was also increased with Papp of 10.3 x 10-6 cm/s compared to 5 with Papp of 2.0 x 10-6 cm/s.
Inhibitory activity of the compound 12n against CYP 3A4 isozyme was improved with IC50 value of >10 µM
compared to the compound 5 with IC50 value of 5.1 µM. All these improvement are partly dues to a decrease in
lipophilicity based on the comparison of the calculated cLogP values of 12n (3.64) and 5 (4.59) using
ChemBioDraw software. Changes at 4’-position of aniline with hydroxyethyloxy, pyrrolidinylamidomethoxy,
pyrrolidinylethoxy, methoxy and methanesulfonate, 12m (IC50 5.0 nM), 12o (IC50 7.3 nM), 12s (IC50 5.7 nM),
12t (IC50 6.3 nM), respectively, had lower Syk inhibitory activities. Azetidinol substituted derivatives, 12m, 12o
were 2-3.5 fold more potent than pyrrolidinol derivatives. C4 position with heterocycle (pyrrole or pyraz
ole) on pyrimidine ring had similar activities (12r vs 12q). It was interesting to see that 3,5-dimethoxy
aniline derivatives, 12v, 12w were 7-8 fold less active than 3,5-dimethyl aniline substituted derivative,
12o, 12p. Moreover, larger group like 4’-phenoxy substituted compounds 12x (IC50 144.5 nM), 12y (IC50
377 nM) lost its inhibitory activity significantly.
Further elaboration of C2-substitution at pyrimidine scaffold by employing indoline, indole and indazole
was undertaken. Derivatives of compound 16 were similarly prepared from corresponding aminoindoline,
aminoindole and aminoindazole following Scheme 1 or 2 above. Syk inhibitory activity data of compound 16
derivatives are shown in Table 3. There was not much difference in activity by varying N-alkyl substituent at
indole ring (16b and 16d). Cyclopropyl variant at R3 position of compound 16c showed about 2.5-4 fold
increase in activity (16b and 16d). Modification on C5 position of pyrimidine with flourine resulted in dramatic
loss of inhibitory activity as seen in compound 16l compared to non-substituted derivative 16i. Further

derivatization with 4-fluoropyrimidine compound was not carried out. Surprisingly 3’-chloro substitution at
indole or indazole ring (16e-16g and 16k) showed excellent Syk inhibitory activity with IC50 values of 0.7 nM,
1.0 nM, 1.0 nM and 0.7 nM, respectively. These compounds (16f and 16g) also showed high cellular activity in
THP-1 cell with IC50’s of 38 nM and 44 nM, respectively (Table 4). However, similar electron withdrawing CN
substitution at 3’-position of indole (16m) was not highly active partly due to hydrophilic nature of CN
compared to chlorine atom.

Table 1
Syk inhibitory activity23 of compounds 5 and 12a-12l
a IC50 values are averages of two or three experiments.

Table 2
Syk inhibitory activity of compounds 12m-12y
a IC50 values are averages of two or three experiments.

Table 3
Syk inhibitory activity of compounds 16a-16m
a IC50 values are averages of two or three experiments.

To evaluate the kinase selectivities compounds 16f, 16g and 16h kinase profiling was performed against an
extended panel of tyrosine and serine/threonine kinases (Table 4). Within the protein tyrosine kinase (PTK)
group, 16f, 16g and 16h was selective (>50-fold) over representatives from the ZAP-70, JAK2, JAK3, Aurora
B, KDR and RET families. Compound 16f was further tested in a kinase selectivity panel (298 kinases at 0.1
and 0.01 µM concentration), and the kinases most potently inhibited were Syk (100 and 96%, respectively) and
MLK1 (91 and 44%, respectively).24
Table 4
In vitro selectivity of Syk inhibition23, 25 over other tyrosine kinases
IC50 (µM)
THP-1
0.077
IC50 (nM)
JAK3
Compound
SYK
2.3
ZAP-70 JAK2
Aurora B KDR
RET
NT
5
NT
115.3
111.9
162.4
138.2
349.1
NT
NT
16f
0.038
1.0
53.85
73.42
995.8
78.12
1711
NT
191.9
NT
67.8
9594
NT
16g
0.044
1.0
478.1
16h
0.056
1.7
418.2
985.2
653
NT: not tested
In order to understand the binding mode of our Syk inhibitor X-ray co-crystal structure26 of compound 16h
bound to the kinase domain of Syk was solved (Fig. 1). From the crystal structure, pyrimidine N1 nitrogen of
the compound 16h serves as a hydrogen bond acceptor for the amine nitrogen of Ala451 in the hinge region and
aniline nitrogen forms hydrogen bond with Ala451 carbonyl oxygen. Tertiary amine nitrogen of azetidinol ring
forms a hydrogen bond with Asp512 carbonyl oxygen as ammonium ion type. Hydroxyl group in azetidinol
forms another hydrogen bond with the carbonyl group of Arg498. In addition to hydrogen bondings compound
16h was confirmed to have many hydrophobic interactions with the amino acid residues around the binding site.
First, the indole ring is exposed to the existing Gly454, Pro455 and the solvent region, and a pyrimidine makes
hydrophobic interactions with each of Ala400 and Leu501. The pyrazole ring moiety and azetidinol have
interaction with Val385 and Asp512, respectively. Therefore, our Syk inhibitors are expected to show good
activity and selectivity for Syk based on these interactions with kinase domain of Syk.

Figure 1. X-ray co-crystal structure of the Syk kinase domain with compound 16h (PDB code 4RSS).
Pharmacokinetic studies were performed for the highly active and selective compounds in order to evaluate
pharmaceutical profiles. Compounds 16f, 16g and 16h have good human liver microsomal stabilities (>60 min,
respectively) and good Caco-2 cell permeability (Papp in (cm/s) 10-6 16f AB: 2.7, BA: 3.8; 16g AB: 6.2, BA:
7.3; 16h AB: 6.2, BA: 9.9). These compound showed >10 µM of IC50 values for five CYP isozymes. These
compounds demonstrated high human plasma protein binding (% free fraction, <0.4%). Compound 16f (IC50
24.2 µM) and 16h (IC50 12.5 µM) have moderate hERG inhibition27 up to 10 µM and also negative in an Ames
assay.28 In vivo pharmacokinetic studies for the most potent 3,5-dimethyl aniline compound (5) and 4-alkoxy-
substituted aniline compound (12s) showed poorly bioavailability (F = 7.6% and 7.8%, respectively) in rats.
However, C-2 indole and indazole substituted compounds (16f and 16h) had good pharmacokinetic properties
with moderate and high bioavailabilites (F = 28.4% and 61%, respectively) (Table 5). The compound 16f also
had high bioavailability of 46.4% in vivo PK studies in dogs (Table 6). Thus, compound 16f was selected as the
lead compound for further development.
Table 5
Pharmacokinetic parameters for 5, 12s, 16f and 16h in male Sprague-Dawley rats (iv 10 mg/kg; po 10 mg/kg),
where values are means of n = 3
Compound
5a
CLc (mL/min/kg)
Vssd (L/kg)
4.3
T1/2e (h)
2.6
Ff (%)
7.6
AUClast g(ng h/mL)
43
70
26
21
6240
550
12sa
5.6
1.2
7.8
16fb
3.3
3.6
28.4
61
3233
4000
16hb
3.7
3.4
a PEG400/Saline/DMSO (40:55:5)
b 20% SBECD + 0.5% acetic acid, iv 5mg/kg.
c Clearance.
d Volume of distribution at steady state
e Terminal half life.

f Bioavailability.
gArea under curve from time of dosing to last observation
Table 6
Pharmacokinetic parameters of compound 16f in doga
Compound
16f
Route
IVb
5
PO b
10
Dose (mg/kg)
CL (mL/min/kg)
Vss (L/kg)
40.4
7.1
4.6
2093
_
_
_
T1/2 (h)
_
AUClast (ng h/mL)
F (%)
1941
46.4
a Each value is an average of data from three animals.
b 20% HPBCD in saline
Evaluation of in vivo efficacy of the compound 16f was studied in a mouse Collagen Induced Arthritis (CIA)
model. The model mice were treated orally with 10 and 30 mg/kg of the compound 16f after arthritis induction
for 3 weeks. The compound 16f showed a significant reduction in the average clinical score with 30% and 50%
respectively at doses of 10 and 30 mg/kg.29 From these studies it can be safely concluded that 16f significantly
reduced the severity and development of arthritis.
A series of novel pyrazolylpyrimidine derivatives as Syk inhibitors have been reported. The strong Syk
inhibitory activity was achieved when the 2 position of the pyrimidine ring was substituted with 3,5-dimethyl
aniline and 4-alkoxy-substituted aniline (12a-y). These compounds, however, had poor pharmacokinetic
properties. To improve drug like properties, indole or indazole amine was introduced at 2 position on the
pyrimidine ring. Compounds (16a-m) gave the better activities and improved pharmacokinetic properties. On
the basis of the SAR studies of indole and indazole amine substituted derivatives we have optimized the
structures and finally identified 16f as the most potent Syk inhibitor with desirable pharmaceutical profiles.
Compound 16f had excellent Syk inhibitory activity with IC50 of 1.0 nM and an oral bioavailability of 46.4% in
dogs. Based on these results it was decided to develop compound 16f for treatment of autoimmune diseases such
as rheumatoid diseases.
Acknowledgment
This work was supported by the Korea Drug Development Fund (KDDF) grant funded by the Korea
government (MSIP, MOTIE, MW) (No. KDDF2011-06) and the National Research Foundation of Korea (NRF)
grant funded by the Korea government (MSIP) (2011-0018355).

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2011060295A1, 2011.

21. Compounds 16a-m were prepared according to procedures described in PCT Int. Appl. WO 2013109882A1,
2013.
22. Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Romagnoli, R.; Baccioli, G.; Zaid, A. N.; Pineda de las Infantas, M.
J. Synthesis 1997, 10, 1140.
23. Cell free kinase assay: Compounds of the invention were initially diluted to 10mM in 100% DMSO
(CALBIOCHEM) for storage and made into kinase buffer solution to create a compound concentration
ranging from 1uM and 10uM. Serial dilutions of compounds of the invention were dispensed into a 96-well
plate (GREINER BIOSCIENCES) at 6 micro L each. Purified full-length human SYK, ZAP70 and truncated
human RET and JAK2 (CARNA BIOSCIENCES) were diluted in kinase buffer and added to the compound
solutions and pre-incubated for 30 minutes at room temperature (1 hour for PYK2). Next, ATP (TEKNOVA)
and substrate solution (suggested manufacture substrates of PerkinElmer™, for example, Ulight™-TK
peptide for SYK, Ulight™-PolyGT for ZAP70, FAK, and PYK2 (PERKINELMER)) was added (12uL each)
to the wells containing the compound solution and enzyme. The reaction mixture was incubated for 1 hour (2
hours for PYK2). Following the incubation, the stop solution made with EDTA, water, and Lance detection
buffer (PERKINELMER) was added (12 micro L each) to stop phosphorylation. Following the addition of the
stop solution and 5 minutes of shaking, the detection solution containing the Europium-labeled antibody
(suggested manufacture substrates of PerkinElmer™, for example, PT66 for SYK, ZAP70 and PYK), water,
and Lance detection buffer was added (12mmicro L each) to the reaction mixture and incubated again for 50
minutes. Substrate phosphorylation was a function of the 665nm emission measured following the addition of
the detection solution and 50 minutes of incubation. The IC50 value of test compound was calculated at
Gradpad Prism 5 unless specified otherwise.
24. Kinases with > 70% inhibition at 0.1 µM: Abl(Q252H)(h), EGFR(T790M)(h), EGFR(T790M, L858R)(h),
Flt3(D835Y)(h), Plk3(h), Ret(V804L)(h), Ret(V804M)(h); kinases with > 90% inhibition at 0.1 µM:
MLK1(h); kinase with <25% inhibition at 0.1 µM: Fgr(h), Fyn(h), Lck(h), Lyn(h), Yes(h), Src(1-530)(h),
Src(T341M)(h), Hck(h), Blk(h); kinases with <50% inhibition at 0.01 µM: Abl(Q252H)(h),
EGFR(T790M)(h), EGFR(T790M, L858R)(h), Flt3(D835Y)(h), Plk3(h), Ret(V804L)(h), Ret(V804M)(h),
MLK1(h).
25. TNF-a release assay : Compounds of the invention were tested for their TNF-α release effect on THP-1 cells.
For SYK dependent TNF-α release assay (i.e., via IgG stimulation), THP-1 cells derived from human
monocytic cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). This cell
line was maintained with an Roswell Park Memorial Institute (RPMI) medium (GIBCO) containing 10 fetal
bovine serum (FBS; GIBCO) and 0.05 mM 2-mercaptoethanol. The THP-1 cells were seeded at 1x 105
cells/100 micro L/well into human IgG (10 micro g/well, INVITROGEN)-coated 96 well culture plate, and
serially diluted compound was then added. After an 18-hour incubation period at 37℃, supernatants were
collected for the determination of the TNF-α level by enzyme-linked immunosorbent assay (ELISA), and the
remaining cells were subjected to an MTT (yellow tetrazolium salt) assay to determine the cytotoxic effects
of compound. For SYK independent TNF-α release assay (i.e., via lipopolysaccharide (LPS)-stimulation),
THP-1 cells derived from human monocytic cells were obtained from the American Type Culture Collection

(ATCC, Manassas, VA). This cell line was maintained with an RPMI medium (GIBCOÔ) containing 10 fetal
bovine serum (FBS, GIBCO) and 0.05 mM 2-mercaptoethanol. The THP-1 cells were seeded at 1x 105
cells/100 micro L /well into 96-well culture plates, and treated with lipopolysaccharide (1 micro g/ml), and
serially diluted compound was then added. After an 18-hour incubation period at 37°C, supernatants were
collected for the determination of the TNF-α level by ELISA, and the remaining cells were subjected to an
MTT assay to determine the cytotoxic effects.
26. The structure was deposited in the Protein Data Bank ID code 4RSS. Binding mode was analyzed using
program Ligand Explorer in PDB.
27. hERG testing was performed at Millipore IonChannelProfilerTM and ChanTest® Manual Patch Clamp assay.
28. Neither toxicity nor mutagenic activity has been observed at 5000 ug/plate in 4 Samonella typhimurium
strains and 1 Escherichia coli strain (TA98, TA100, TA1535, TA1537 and WP2 uvrA ± liver S9 fraction).
29. The Collagen Induced Arthritis (CIA) mouse model was carried out at Oscotec Inc. (Republic of Korea).
Male mice of DBA1/J strain were injected intradermally with bovine type II collagen and Complete Freund’s
Adjuvant at day 0 and 21 (boost at day 21). Mice develop inflammation 3 weeks after the first collagen
injection. A chronic inflammation in the joints of the animals is achieved within 7–10 days after disease
onset. Disease progression was monitored by scoring the severity of arthritis in four paws. Animals were
euthanized on Day 42. The compound was administered orally continued once daily and twice daily through
study.

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Highly potent and selective
pyrazolylpyrimidines as Syk kinase
inhibitors
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Jang-Sik Choi, Hae-jun Hwang, Se-Won Kim, Byung il Lee, Jaekyoo Lee, Ho-Juhn Song, Jong Sung Koh,
∗
Jung-Ho Kim*, Phil Ho Lee
Indole substituted pyrazolylpyrimidine compound (16f) was identified as a potent and selective Syk inhibitor
with good pharmacological profiles.