Discovery and SAR of novel [1,6]naphthyridines as potent inhibitors of Spleen Tyrosine Kinase (SYK)

Article abstract of DOI:10.1016/S0960-894X(03)00163-X

The discovery of novel 5,7-disubstituted[1,6]naphthyridines as potent inhibitors of Spleen Tyrosine Kinase (SYK) is discussed. The SAR reveals the necessity for a 7-aryl group with preference towards para substitution and that this in combination with 5-aminoalkylamino substituents further improved the potency of the compounds. The initial SAR as well as a survey of the other positions is discussed in detail.

Full text of DOI:10.1016/S0960-894X(03)00163-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1415–1418
Discovery and SAR of Novel [1,6]Naphthyridines as Potent
Inhibitors of Spleen Tyrosine Kinase (SYK)
Charles L. Cywin,a,* Bao-Ping Zhao,b Daniel W. McNeil,a Matt Hrapchak,a
Anthony S. Prokopowicz, III,a Daniel R. Goldberg,a Tina M. Morwick,a Amy Gao,a
Scott Jakes,a Mohammed Kashem,a Ronald L. Magolda,a Richard M. Soll,b
Mark R. Player,b Mark A. Bobko,b James Rinker,b Renee L. DesJarlaisb
and Michael P. Wintersb
aBoehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06801-0368, USA
b3-Dimensional Pharmaeuticals, Inc., 665 Stockton Drive, Suite 104, Exton, PA 19341, USA
Received 2 January 2003; accepted 11 February 2003
Abstract—The discovery of novel 5,7-disubstituted[1,6]naphthyridines as potent inhibitors of Spleen Tyrosine Kinase (SYK) is
discussed. The SAR reveals the necessity for a 7-aryl group with preference towards para substitution and that this in combination
with 5-aminoalkylamino substituents further improved the potency of the compounds. The initial SAR as well as a survey of the
other positions is discussed in detail.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
the kinase activity of SYK.4 Amast cell line lacking
SYK was unable to signal without transfection of active
SYK.5 Additionally, inhibition of the antigen-stimu-
lated phosphorylation of SYK by piceatannol (a non-
selective SYK inhibitor) inhibited functional responses
in mast cells, including mediator release.6 These findings
support the hypothesis that inhibiting SYK activity
would attenuate FceRI-mediated release of pro-inflam-
matory mediators and cytokines from mast cells. As
part of our continuing efforts to identify new ther-
apeutics directed at asthma and allergic disorders we
sought to find novel inhibitors of SYK. Compound 1
and its analogue 2 were identified as a potentially inter-
esting series for further SAR work which is the subject
of this report (Fig. 1).
Asthma and allergic disorders remain areas with unmet
medical needs. Mast cells play an important part in the
pathophysiology of these disorders by releasing pro-
inflammatory mediators and cytokines. Activation of
mast cells by antigen-mediated aggregation of FceRI
(the high-affinity receptor for IgE) results in a series of
signaling events leading to the release of mediators,
including histamine, proteases, leukotrienes and cyto-
kines. These mediators play key roles in the etiology and
symptoms of asthma and allergic disorders. Acrucial
downstream event in the signaling pathway following
the activation of mast cells is activation of spleen tyr-
osine kinase (SYK).1ꢀ3 SYK acts as a central initiator of
the FceRI-mediated signaling pathways and has been
shown to be necessary for antigen-induced mediator
release.3,4 It has been shown that clustering of SYK
chimera, introduced into a transmembrane protein in a
mast cell line, was sufficient to stimulate the events
leading to mediator release normally induced by clus-
tering of FceRI and that these events were dependent on
Results and Discussion
General chemistry
The compounds were synthesized as shown in Scheme 1.
A2-methylnicotinic acid derivative ( I) is treated with
lithium diisopropylamide (LDA) in THF at about ꢀ78
to 0 ꢁC to form the dianion.7 Subsequent addition of a
nitrile (II) at ꢀ78 ꢁC, and continuing the reaction at
*Corresponding author. Tel.: +1-203-791-6401; fax: +1-203-791-
5868; e-mail: ccywin@rdg.boehringer-ingelheim.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00163-X

1416
C. L. Cywin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1415–1418
8-positions (data not shown), resulted in compounds
with generally reduced potency. While the 3-position
tolerated small substituents like bromo (13) and methyl
(data not shown), larger substituents like phenyl were
considerably less potent. Based on the initial SAR we
focused on determining the SAR of the 5-amino and
7-aryl substituents more thoroughly.
We undertook an analysis of the substituent tolerance at
the ortho, meta and para positions of the 7-phenyl group
(Table 2). It was quickly discerned that ortho substitu-
tion was not tolerated (16–18). Small meta substituents
generally had little effect on potency (19–22) while lar-
ger aryl substituents (23–24) led to less potent com-
pounds. The para position proved to be the most
fruitful avenue of exploration with a larger variety of
substituents being tolerated. The para-position appears
to have both a steric requirement and an electronic
component. Small electron withdrawing substituents
Figure 1.
ambient temperature until completion provides the
[1,6]naphthyridin-5-ol (III), which may also exist as the
tautomeric 6H-[1,6]naphthyridin-5-one (IIIa).8 The
[1,6]naphthyridin-5-ol is then treated with POCl3, with
or without base (i.e., N,N-diethylaniline) at 100–135 ꢁC,
to give the 5-chloro-[1,6]naphthyridine (IV). This is then
reacted with an excess of the desired amine (R0R00NH)
at 100–140 ꢁC, in a sealed reaction vessel, to provide the
5-amino substituted compounds (V) shown in the
tables.9 Alternatively, the 5-substituted ethers were
available by reacting IV with the desired alcohol
(R0OH), in the presence of sodium hydride in DMF at
room temperature to give ethers VI.
Table 1. Initial SAR Survey
Structure–activity relationships
Compd X Y R2 R3
R5
R7
SYK IC50 (mM)
Arapid survey of the molecule was undertaken to
determine the best path for further SAR studies (Table
1).10 Compounds 1–5 show that the [1,6]naphthyridine
core system is essential for maintaining potency. The
5-amino substituent appeared to directly correlate with
activity (compounds 1, 2, 6, 7) and appeared to be a
good site for further exploration. Most notably, the
requirement for a substituted amine and the dependence
of activity on chain length was observed. The 7-aryl
moiety also appeared essential. Replacement by t-butyl
(8) and other alkyl substituents resulted in loss of activity.
While variation of the 7-aryl group could be beneficial
(9), substitution at the 2-position, as well as the 4-, and
1
2
3
4
5
6
7
8
N C
N C
N N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NH(CH2)2NH2 4-MeOPh
1.5
0.49
>30 mg/mL
>30 mg/mL
40
17
0.14
>30 mg/mL
0.44
0.19
NHCH3
NHCH3
NHCH3
NHCH3
NH2
4-MeOPh
4-MeOPh
4-MeOPh
4-MeOPh
4-MeOPh
C C
C N
N C
N C
N C
N C
N C
N C OH
N C CF3
N C
N C
NH(CH2)3NH2 4-MeOPh
NH(CH2)3NH2 t-Bu
NH(CH2)3NH2 2-Thienyl
NH(CH2)2NH2 4-Me2NPh
NH(CH2)3NH2 4-MeOPh
NH(CH2)3NH2 4-MeOPh
9
10
11
12
13
14
>30 mg/mL
>30 mg/mL
0.12
H
H
Br NH(CH2)3NH2 4-MeOPh
Ph NH(CH2)3NH2 4-MeOPh
3.6
Scheme 1. General synthetic method.

C. L. Cywin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1415–1418
1417
Table 2. 7-aryl SAR
Table 3. 5-Position SAR
Compd
R
SYK IC50 (mM)
Compd
R
SYK IC50 (mM)
15
16
17
18
19
20
21
22
23
24
7
25
26
27
28
29
30
31
H
o-Br
o-OCH3
o-SCH3
m-OCH3
m-F
m-Cl
0.41
25
>30 mg/mL
>30 mg/mL
38
39
10
29
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
NHCH3
N(CH3)2
NH(CH2)2NH2
NH(CH2)3NH2
NH(CH2)4NH2
NH(CH2)5NH2
O(CH2)3OH
O(CH2)4NH2
NH(CH2)4OH
0.23
0.42
0.19
0.034
0.019
0.21
0.20
0.16
0.20
0.072
0.078
0.89
1.7
0.50
0.85
0.26
0.83
1.5
3.8
0.14
1.1
m-OCF3
m-(thien-3-yl)a
m-(tolu-4-yl)a
p-OCH3
N(CH3)(CH2)3NH2
NH(CH2)3NHCH3
N(CH3)(CH2)3NHCH3
NH(CH2)3N(CH3)2
N(CH3)(CH2)3N(CH3)2
NHCH2CH(OH)CH2NH2
NHCH2C(CH3)2CH2NH2
NHCH2c-Hex
3-Pyridylmethylamino
3-Pyridylmethylamino
(3-Aminomethylbenzylamino)
(2-Aminobenzylamino)
(4-Aminobenzylamino)
p-F
p-Cl
1.2
1.6
p-(thien-3-yl)a
p-(tolu-4-yl)a
p-N(CH3)2
p-N(CH2CH3)2
p-(N-morpholinyl)
0.90
0.034
0.020
0.008
0.071
0.42
0.26
0.063
0.034
0.090
8.6
0.043
0.31
5.9
32
33
34
35
36
37
p-(N(CH3)(CH2)2N(CH3)2)
p-(N(CH3)(CH2)3N(CH3)2)
1.3
0.50
0.33
0.85
13
p-Br
p-CH3
p-SCH3
p-CH(CH3)2
aPrepared via the corresponding bromides via Suzuki couplings.
like fluoro and chloro (25–26) were detrimental to activity
however small electron donating groups like methyl (35)
and methoxy (7) were beneficial. The p-dimethylamino
(29) is preferred to the isosteric p-isopropyl (37) group
indicating that the nitrogen, as well as the oxygen and
sulfur atom of 7 and 36, might be acting additionally as
hydrogen bond acceptors. Further exploration of the
p-amino substituent showed that larger substitution can
be beneficial in conjunction with an additional polar
functionality. For example, the p-morpholino compound
31 shows an IC50 less than 10 nM. There also appears to
be a size restriction as shown by 32 and 33.
The key to activity proved to be the proper positioning
of the second basic amine group. Compare compounds
53 and 55 to 57 (which can be considered ring con-
strained versions of 41). The loss of activity in 57 is
likely a result of both positioning as well as the basicity
of the distal amine.
Conclusions
It has been shown that suitable 5,7-disubstituted-
[1,6]naphthyridines are potent inhibitors of the tyrosine
kinase SYK. The SAR has shown that a 7-aryl and pre-
ferably para-substituted aryl group as well as 5-alkyldia-
mines have synergistic effects on activity. The potencies
ranged from high micromolar to low nanomolar. These
compounds are of further interest and additional SAR and
biological evaluations will be the subject of future reports.
We next explored SAR at the 5-position. Since we had
already determined that an unsubstituted amine (6) was
weakly active, the next step was to determine the effects
of substitution (Table 3). Compound 39 showed that
disubstitution on nitrogen was permitted albeit with
slight loss in activity. 5-Aminoalkylamines of 3 to 4
carbons in length were optimal. Aclear preference for
propylenediamine and butylenediamine is shown by
comparing 10, 29, 40, and 41. Examining substitution of
the diamino group showed that both amines could be
replaced individually or together by oxygen (42–44)
with approximately a log loss in potency. Mono-
methylation of the propylenediamine at either nitrogen
was tolerated (45–46). However, either di- or trimethy-
lation resulted in a ꢂ10–100-fold loss in activity (47–
49). Substitution of the central carbon of the propyle-
nediamine was also tolerated (50–51). Other groups
such as cyclohexylmethylamino (52) proved detrimental.
Acknowledgements
The authors would like to thank Joachim Heider who
originally synthesized 1 and 2.
References and Notes
1. Turner, M.; Schweighoffer, E.; Colucci, F.; Di Santo, J. P.;
Tybulewicz, V. L. Immunology Today 2000, 21, 148.
2. Siraganian, R. P.; Zhang, J.; Suzuki, K.; Sada, K. Mole-
cular Immunology 2001, 38, 1229.

1418
C. L. Cywin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1415–1418
3. Taylor, J. A.; Karas, J. L.; Ram, M. K.; Green, O. M.;
Seidel-Dugan, C. Mol. Cell Biol. 1995, 15, 4149.
4. Rivera, V. M.; Brugge, J. S. Mol. Cell. Biol. 1995, 15, 1582.
5. Zhang, J.; Berenstein, E. H.; Evans, R. L.; Siraganian, R. P.
J. Exp. Med. 1996, 184, 71.
6. Oliver, J. M.; Burg, D. L.; Wilson, B. S.; McLaughlin, J. L.;
Geahlen, R. L. J. Biol. Chem. 1994, 269, 29697.
7. For a recent report of analogous chemistry to produce iso-
quinol-1-ones see: Brun, E. M.; Gil, S.; Mestres, R.; Parra, M.
Synthesis 2000, 273.
400 MHz) d 9.03 (dd, 1H), 8.54 (d, 1H), 8.13 (s, 1H), 8.08 (d,
2H), 7.46 (dd, 1H), 6.81 (d, 2H), 3.05 (s, 6H). Amixture of the
chloride (10 mg, 0.035 mmol) and 1,3-diaminopropane (200
mL, 2.4 mmol) was stirred at 100 ꢁC for 5 h. After cooling to rt,
H2O was added and the mixture was extracted with CH2Cl2
(2ꢃ3 mL). Purification by preparative TLC afforded 29 (8.5
mg). 1H NMR (CDCl3, 400 MHz) d 8.85 (dd, 1H), 8.10 (d,
2H), 8.07 (d, 1H), 7.50 (s, 1H), 7.21 (dd, 1H), 6.81 (d, 2H),
3.87 (t, 2H), 3.02 (s, 6H), 2.97 (t, 2H), 1.90 (quint, 2H); MS
(m/e) calcd for C19H23N5 321, found 322 (M+ H).
8. Alternatively the method of Ames, D. E.; Dodds, W. D. J.
Chem. Soc., Perkin Trans. 1, 705, could be employed for the
synthesis of III 1972.
10. The kinase activity was measured using DELFIA(Dis-
sociation Enhanced Lanthanide Fluoroimmunoassay). The
kinase assay was performed in buffer (50 mM HEPES, pH 7.0,
25 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 100 mM Na3VO4,
0.2% BSA, 0.01% CHAPS). Test samples initially dissolved in
DMSO at 5 mg/mL, were pre-diluted for dose response with
the assay buffer in 96-well microtiter plates and a 25 mL ali-
quots of these diluted samples were added to a neutravidin
coated 96-well white plate (PIERCE). A25 mL volume of
diluted enzyme (0.6 ng/mL final conc.) and a 50 mL volume of
a mixture of substrates containing 200 nM ATP and 3.6 ng/mL
PGTYR-biotin (CIS Biointernational) in the assay buffer was
sequentially added and the assay plates were incubated for 30
min at RT. Following incubation, the plates were washed
three times with 300 mL wash buffer (50 mM Tris–HCl, pH
7.4, 150 mM NaCl, 0.05% Tween 20, 0.2% BSA). A 100 mL
aliquot of Eu-labeled anti-phosphotyrosine (Eu3+-PT66,
Wallac CR04-100) diluted in DELFIAAssay Buffer (1 nM
final conc.) was added to each well and incubated for 30 min
at rt. The plates were washed four times with 300 mL of wash
buffer and 100 mL of DELFIAEnhancement Solution (Wal-
lac) was added to each well. After 10 min or longer, time-
resolved fluorescence was measured on the LJL’s Analyst
(excitation at 360 nm, emission at 620 nm, EU 400 Dichroic
Mirror) after a delay time of 250 ms.
9. Atypical experimental (preparation of 29). LDA(2 M
(THF), 12.5 mL, 25 mmol) was added to 2-methylnicotinic
acid (1.37 g, 10 mmol) in THF (20 mL) at ꢀ78 ꢁC. The mix-
ture was stirred at ꢀ78 ꢁC for 30 min then slowly warmed to
0 ꢁC over 1.5 h. The mixture was cooled to ꢀ78 ꢁC and a
solution of 4-(dimethylamino)benzonitrile (2.19 g, 15 mmol) in
THF (10 mL) was added. The mixture was warmed to rt and
stirred for 16 h. H2O (20 mL) was added and the THF was
removed in vacuo. EtOAc (10 mL) was added and the solution
was allowed to stand for 2 h. The precipitate was collected by
filtration to afford the 7-(4-dimethylaminophenyl)-[1,6]naph-
1
thyridin-5-ol (1.2 g). H NMR (DMSO-d6, 400 MHz) d 11.56
(b, 1H), 8.86 (dd, 1H), 8.45 (dd, 1H), 7.72 (dd, 2H), 7.39 (dd,
1H), 6.81 (m, 3H), 2.99 (s, 6H), MS (m/e) calcd for
C16H15N3O 265, found 266 (M+H). Amixture of naphthyr-
idinol (1.2 g, 5 mmol), POCl3 (10 mL) and N,N-diethylaniline
(0.15 g, 1.0 mmol) were stirred at 110 ꢁC for 16 h then cooled
to rt. Excess POCl3 was evaporated and the residue neu-
tralized with aq Na2CO3 and extracted with CH2Cl2. The
residue from the organic layer was purified by flash chroma-
tography (hexanes/EtOAc) to afford 5-chloro-7-(4-dimethyl-
aminophenyl)-[1,6]naphthyridine (1.1 g). 1H NMR (CDCl3,