Identification of 2-amino-5-(thioaryl)thiazoles as inhibitors of nerve growth factor receptor TrkA

Article abstract of DOI:10.1016/j.bmcl.2007.11.076

2-Amino-5-(thioaryl)thiazoles are potent inhibitors of TrkA (e.g., 20h, TrkA IC₅₀ = 0.6 nM) that show anti-proliferative effect in cellular assays. A proposed inhibitor binding mode to TrkA active site is consistent with key SAR observations.

Full text of DOI:10.1016/j.bmcl.2007.11.076

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 634–639
Identification of 2-amino-5-(thioaryl)thiazoles as inhibitors
of nerve growth factor receptor TrkA
Soong-Hoon Kim,a,* John S. Tokarski,b Kenneth J. Leavitt,a Brian E. Fink,a
Mark E. Salvati,a Robert Moquin,a Mary T. Obermeier,d George L. Trainor,a
Gregory G. Vite,a Linda K. Stadnick,c Jonathan S. Lippy,c Dan You,c
Matthew V. Lorenzic and Ping Chena
aDepartment of Oncology Chemistry, Bristol-Myers Squibb Research and Development, PO Box 4000,
Princeton, NJ 08543-4000, USA
bComputer Aided Drug Design, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
cDiscovery Biology, Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
dPharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, PO Box 4000,
Princeton, NJ 08543-4000, USA
Received 3 October 2007; revised 19 November 2007; accepted 19 November 2007
Available online 28 November 2007
Abstract—2-Amino-5-(thioaryl)thiazoles are potent inhibitors of TrkA (e.g., 20h, TrkA IC50 = 0.6 nM) that show anti-proliferative
effect in cellular assays. A proposed inhibitor binding mode to TrkA active site is consistent with key SAR observations.
Ó 2007 Elsevier Ltd. All rights reserved.
TrkA (Troponyosin-receptor kinase A) is a cell surface
receptor kinase containing an extracellular, a trans-
membrane, and a cytoplasmic kinase domain. The bind-
ing of a neurotrophin triggers oligomerization of the
receptors, phosphorylation of tyrosine residues in the ki-
nase domain, and activation of intercellular signaling
pathways, including Ras/MAPK cascade, PI3K/AKT,
and IP3-dependent Ca2+ release. The interaction of
TrkA, along with related TrkB and C, with neurotro-
phins (NGF, BDNF, and NT-3) plays an important role
for homeostasis of neural network, including differenti-
ation, proliferation, and survival.1–3 Several lines of evi-
dence also suggest that the interplay of TrkA with NGF
plays a critical role in the prognosis and outcome in sev-
eral tumors.
als of Trk inhibitors may provide valuable insights into
anti-tumor efficacy and Trk inhibition.7–9 In addition,
other research groups have reported selective inhibitors
of TrkA.10,11 We report potent (TrkA IC50 < 0.001 lM)
2-amino-5-(thioaryl)thiazole inhibitors of TrkA, and
provide evidence that their anti-proliferative effect
against cell lines is due to selective inhibition of TrkA.
Screening of an in-house kinase deck with the criteria of
(1) having activity against kinases at a pre-determined
IC50 cut-off, and (2) maximizing structural diversity
led to several potent hits. Among them, 2-amino-5-
(thioaryl)thiazole 1 appeared especially promising due
to its potent TrkA inhibition (TrkA IC50 = 0.05 lM,
Fig. 1); and as a class, 2-amino-5-(aryl)thiazoles have
For instance, an over-expression of TrkA correlates with
an aggressive phenotype and poor clinical outcome in
malignant melanomas,4 ovarian carcinoma,5 and pan-
creatic cancer.6 In view of this, the on-going clinical tri-
4
Br
N
NAc
N
1
S
HN
3
N
S
2
O
Keywords: TrkA; Troponyosin-receptor kinase A; Nerve growth fact-
or; NGF; Kinase inhibitor; 2-Amino-5-(thioaryl) thiazoles.
1: TrkA IC50 = 0.05 μM
Figure 1. Structure of initial TrkA screening hit.
*
Corresponding author. Tel.: +1 609 252 4079; fax: +1 609 252
3993; e-mail: soonghoon.kim@bms.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.076

S.-H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 634–639
635
shown favorable oral pK as selective inhibitors of Ikt
(Interleukin-2-inducible T cell kinases).12,13 The next
set of TrkA inhibitors were designed toward establishing
SAR around both pyridine and thioaryl rings; their syn-
theses are shown in Schemes 1–5.
NaNO2 and HCl, which is displaced with potassium eth-
ylxanthate to afford xanthate 4. Hydrolysis to thioacid
5, followed by condensation with bromo-thiazole 912
with NaOMe in methanol, gave the aminothiazole acid
10. Both primary and secondary amides were prepared
using BOP as the coupling reagent; yields ranged from
37% to 91% for a range of amides. For additional substi-
tuted pyridines (6: R1 = H, Me, Br) and thioaryls (4:
X = H, Me), the same synthetic route was employed
(Scheme 1).
The majority of compounds in Tables 1–4 follows a gen-
eralized synthetic route shown in Scheme 1, established
previously for Ikt inhibitors.12,13 Nitro ester 2 is re-
duced, converted to its transient diazo-compound with
c
X
b
a
X
X
X
S
HS
CO2H
EtO
S
CO2Me
H2N
CO2Me
O2N
CO2Me
5
4
3
2
d
f
e
S
N
N
R1
R1
R1
R1
Br
S
S
N
N
NH2
N
N
N
N
H
N
NH2
H
H
8
9
7
6
g
R1
6
4
R1
h
X
N
X
N
1
N
N
NR2R3
HN
HN
OH
S
S
S
S
O
O
10
11 and 20
Scheme 1. For R1 = H, X = Me (compounds 11h, 11j–11t, 19a, 20a, and 20c). Reagents and conditions: (a) H2 (balloon), 10% Pd (C), EtOAc, rt,
1.5 h, 96%; (b) NaNO2, HCl, H2O; NaHCO3(s), KSCSOEt, H2O, 45 °C, 1.5 h, 30%; (c) KOH, MeOH–H2O, rt, 18 h, 78%; (d) SCNCOPh, acetone,
100%; aq NaOH, 73%; (e) ClCH2CHO, aq ethanol, 79%; (f) Br2, AcOH, 86%; (g) NaOMe, MeOH, 52 °C, 75%; (h) BOP, DIEA, DMF, rt (37–91%).
Compounds 1, 11a–b (R1 = 6-Br, X = H); 11c, 11t–w, 20d, and 20g (R1 = 6-Br, X = methyl); 11d (R1 = H, X = H); 11f (R1 = 5-Br, X = H); and 11h
(R1 = methyl, X = H) were prepared following the same general synthetic scheme.
b
N
N
a
N
NAc
O
H2N
H2N
HN
N
S
S
S
S
CO2Me
S
S
O
14
13
12
Scheme 2. Reagents: (a) KOH, MeOH/H2O, 87%; N-acetylpiperazine, BOP, DIEA, DMF, 100%. (b) Ac2O, DMAP, DCM, 25%.
a
b
N
N
N
N
N
N
HN
HN
OH
HN
S
S
S
S
NH
S
S
NH2
O
CN
O
10a (R1 = H, X = Me)
16
15
Scheme 3. Reagents and conditions: (a) DPPA, TEA, 1,4-dioxane, rt; Me3Si(CH2)2OH, 75 °C; TFA, DCM, rt, 29% for three steps. (b)
NCCH2CO2H, BOP, DIEA, DMF, rt, 13%.

636
S.-H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 634–639
Ac
R
a
b
N
N
N
OH
N
HN
OH
S
S
S
S
O
18
10b (R1 = Br, X = Me)
17
Scheme 4. Reagents and conditions: (a) CDI, THF, rt, 19 h; NaBH4, H2O, 76% for two steps. (b) MnO2, DCE, 60 °C; N-acetyl piperazine,
NaCNBH3, ACN, 2%.
R
R
a
NH
b
N
N
N
N
N
HN
OH
S
S
S
S
O
O
O
19a (R1 = H, X = Me)
19b (R1 = Br, X = Me)
10a (R1 = H, X = Me)
10b (R1 = Br, X = Me)
20b, 20e, 20f, 20h, 20i, 20j
Scheme 5. Reagents and conditions: (a) Piperazine, BOP, DIEA, DMF, 51%. (b) BOP, DIEA, DMF, rt (20b: 49%; 20e: 75%); 20h: N-Boc glycine,
BOP, DIEA, DMF, rt then TFA, DCM (41% for two steps); 20i: N-Boc-L-alanine, BOP, DIEA, DMF, rt then TFA, DCM (65% for two steps); 20j:
N-Boc-D-alanine, BOP, DIEA, DMF, rt then TFA, DCM (67% for two steps); Compound 20f was prepared from 20d (BH3-THF, 68 °C, 25%).
Table 3. SAR of acyclic amides
Table 1. SAR of terminal arylthio ring
R
N
N
4
Br
X
HN
N
O
N
S
R1
S
1
S
HN
S
R1
TrK A
IC50 (lM)14
3
N
Compound
R
2
NAc
11j
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
H
CONH2
CONHCH2CH3
0.79
Compound
X
Regioisomer
Trk A IC50 (lM)14
11k
11l
>5.0
2.0
CONHCHMe2
CONHCH2CN
1
H
H
—
1,3-Meta
1,2-Ortho
1,4-Para
1,3-Meta
0.050
1.9
1.3
11m
11n
11o
11p
11q
11r
11s
11t
11u
11v
11w
16
0.044
1.2
11a
11b
11c
CONHCH2CCH
CONHCH2CH2CN
CONHCH2CH2OH
CONHCH2CH2NHMe
CONHCH2CH2NHAc
CONHBn
0.065
0.76
0.19
Me
0.001
0.32
>5.00
0.002
0.067
0.010
0.031
0.060
Table 2. SAR of substituted pyridine ring
CON(Me)CH2CN
CONHCH2CH2CN
CON(Me) CH2CH2CN
X
N
R
NAc
HN
N
CON(Me)CH2CH2NHMe
NHCOCH2CN
S
S
O
Compound
X
R
TrK A IC50 (lM)14
Scheme 3 shows synthesis of a reverse amide of com-
pound 11m. Curtuis re-arrangement of compound 10a,
followed by TFA de-protection, gave aniline 15. Cyano-
acetic acid was coupled with compound 15 to give com-
pound 16. The synthesis of compound 18, which lacks
one amide carbonyl group from potent inhibitor 11c,
is shown in Scheme 4. The key step was the reductive
amination with N-acetyl piperazine, following MnO2
oxidation of alcohol 17.
11d
11e
11f
11g
11h
11c
13
H
H
H
H
2-Pyridyl
2-(6-Bromo) Pyridyl
2-(5-Bromo) Pyridyl
0.24
0.050
0.52
2-(6-Methyl) Pyridyl 1.1
0.080
Me 2-Pyridyl
Me 2-(6-Bromo) Pyridyl
Me
Me Acetyl
0.001
>5.0
>5.0
H
14
Synthesis of substituted piperazines (Table 5) is shown
in Scheme 5, following standard acylation chemistry
from compounds 19a and 19b.
Several compounds were designed to address specific
SAR questions. Synthesis of compounds 13 and 14,
which lack the pyridine ring, is shown in Scheme 2.
Previously described ester 1213 is hydrolyzed and cou-
pled to N-acetylpiperazine to give aminothiazole 13,
which is acylated with acetic anhydride to give com-
pound 14.
As illustrated in Table 1, the 1,3-meta substitution (1:
TrkA IC50 = 0.05 lM)14 pattern in the thiophenyl ring
is favored over others (1 vs 11a–b). In addition, a 50-fold

S.-H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 634–639
637
Table 4. SAR of substituted piperazines
ly, the effect of changes on the thioaryl and pyridine
rings appears additive. Compound 11c, which has both
an ortho-methyl group and a C-6 bromine group, shows
potent inhibition (TrkA IC50 = 0.001 lM).
4
N
R1
R
N
N
HN
N
S
S
n
As shown in Table 3, acyclic amides (11j–w, 16) are
typically not as potent inhibitors of TrkA. Neverthe-
less, a subset of compounds show potent activity
and consistent SAR, suggestive of a common pharma-
cophore. The 100-fold increase in potency from ethyl
amide 11k (IC50 > 5 lM) to isosteric nitrile 11m
(IC50 = 0.044 lM) is suggestive of a productive bind-
ing interaction of the nitrile group in the active site.
This observation is further supported by the less ac-
tive compound 11n (IC50 = 1.2 lM), which contains a
non-basic nitrile isostere. Compound 16, a reverse
amide isostere of 11m, shows nearly identical activity
(IC50 = 0.060 lM). Within a given series, an N-methyl-
ation appears to increase potency. Thus, there is a 7-
fold increase from compound 11u (IC50 = 0.067 lM)
to compound 11v (IC50 = 0.010 lM). N-methyl 11t
(IC50 = 0.002 lM) is a potent inhibitor of TrkA.
Z
Compound
R
R1
TrK A IC50 (lM)14
11h
18a
20ab
19a
20b
20c
20d
20e
20f
20g
20h
20i
H
COMe
COMe
COMe
H
0.080
0.72
1.1
H
H
H
0.16
0.21
H
CO2Me
SO2Me
COMe
COEt
H
0.91
Br
Br
Br
Br
Br
Br
Br
0.001
0.021
0.23
CH2Me
CH2CONH2
0.15
COCH2NH2
COCHMe(S)NH2
COCHMe(R)NH2
0.0006
0.001
0.001
20j
a Z = H, H for compound 18; all others are Z = Oxygen.
b n = 2 for compound 20a; all others are n = 1.
The role of substituted piperazine, a cyclic amide present
in potent inhibitor 11c, was explored and is summarized
in Table 4.
A 7-membered homopiperazine 20a
Table 5. Antiproliferative effects of Trk A inhibitors18
(IC50 = 0.72 lM) is less active than the piperazine 11h
(IC50 = 0.08 lM). A 9-fold loss in potency is observed
when the amide carbonyl group next to the phenyl
group is removed (18: IC50 = 0.72 lM), compared to
control 11h. The nature of distal N-4 carbonyl group
also appears to play an important role in overall inhibi-
Compound Trk A
V3.1
CD8-Trk SHSY5Y
IC50 (lM)
IC50 (lM) IC50 (lM) A IC50
(lM)
20j
0.001
0.001
0.01
3.61
2.28
3.32
3.0
0.23
0.25
0.37
0.39
0.46
0.55
1.15
>10
1.78
1.14
1.17
0.75
1.66
3.06
4.26
>10
11c
11v
11t
11w
20h
20i
tion. Thus, compounds with a carbonate (20b: IC50
=
0.002
0.03
0.21 lM), or a sulfonamide (20c: IC50 = 0.91 lM), have
lower potency than 11h, even if both possess a basic car-
bonyl group. A more dramatic effect is seen if the exocy-
clic N-4 carbonyl group is removed entirely; a 200-fold
in drop in potency is observed for compound 20f
(IC50 = 0.23 lM), underscoring the importance of the
carbonyl group in compound 20d to overall inhibition.
Compound 20h, the most potent compound in our stud-
ies, shows <1 nM inhibition against TrkA (IC50
0.0006 lM), superior to its steric isostere 20e (IC50
0.021 lM). Finally, a chiral center adjacent to the car-
bonyl group does not affect TrkA inhibition; the enan-
3.06
4.08
3.52
>10
0.0006
0.001
>5
11k
increase in potency is observed from X = H (1) and
X = methyl (11c). This increase in potency by ortho-sub-
stitution appears general, as demonstrated by a 4-fold
difference in potency between compound 11d (X = H,
TrkA IC50 = 0.24 lM, Table 2) and compound 11h
(X = Me, TrkA IC50 = 0.08 lM).
=
=
tiomeric pair 20i (IC50 = 0.001 lM) and 20j (IC50
0.001 lM) shows the same level of inhibition.
=
Next, the role of the pyridine ring in overall inhibition
was explored (Table 2). Compound 13 that lacks the
pyridine ring is essentially devoid of TrkA inhibition
To better understand the SAR of these inhibitors, a
binding model of 20i in a homology model of the kinase
domain of TrkA was generated. The TrkA homology
model was built using the crystal structure of Muscle-
specific kinase (MuSK)15 as a template with the struc-
ture prediction algorithm implemented in the software
Prime.16 Compound 20i was manually docked in the
ATP binding site of the model (see Fig. 2) using the
small molecule X-ray structure geometry of an analog
of 20i (data not shown), and the crystal structures of ki-
nase complexes of related chemotypes as guidance. A
pair of hydrogen bond interactions is proposed between
the nitrogen of the thiazole ring and the backbone NH
of Met592 (part of the hinge region) and between the
anilino NH and the carbonyl oxygen of Met592; similar
(IC50 > 5 lM)
compared
to
control
(11h:
IC50 = 0.08 lM). Furthermore, the lack of activity of
compound 14 (IC50 > 5 lM), with a basic carbonyl
group, suggests that the pyridine provides more than a
polar interaction through its basic nitrogen. This obser-
vation is further supported by the narrow SAR of the
pyridine ring. Thus, a 5-fold increase from 11d to 11e
(IC50 = 0.05 lM) is observed from a hydrogen to a bro-
mine at C-6 position. The less polar methyl analog 11g
shows reduced TrkA inhibition (IC50 = 1.1 lM). Fur-
thermore, the position of the bromine is critical. Com-
pound 11f (IC50 = 0.52 lM) with bromine at C-5
shows 10-fold decrease in activity relative to 11e. Final-

638
S.-H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 634–639
versus 11h. However, one cannot rule out that the se-
ven-membered ring may also direct the terminal N-4
carbonyl toward unfavorable protein contacts. A dra-
matic loss of activity (>200-fold loss) is observed when
the carbonyl group is removed (compound 20f) from
the control compound 20d. While it is not clear whether
this is the consequence of simple hybridization, the re-
moval of the carbonyl group may remove a potential
interaction with the conserved Lys544. In addition, the
terminal amino group has the opportunity to hydrogen
bond with the conserved Asp668 and/or Glu560.
As shown in Table 5, a select set of TrkA inhibitors
were evaluated in cell based proliferation assay.18
CD8-TrkA is rat kidney epithelium cells (RKE) with
constitutively activated TrkA; V3.1 is its control
TrkA-null cell line whose proliferation is not dependent
on TrkA signaling pathway. SH-SY5Y is a human neu-
roblastoma cell-line that expresses TrkA. Inactive
TrkA inhibitor 11k (IC50 > 5 lM) shows essentially
no anti-proliferative effect in both CD8-Trk A and
SH-SY5Y cells (IC50 > 10 lM). In contrast, potent
inhibitors of TrkA (20j, 11c, 11v, 11t, 11w, 20h, and
Figure 2. Binding model of 20i in the ATP binding site of a homology
model of TrkA. Carbon atoms of 20i are in green. Carbon atoms of
protein are in cyan. Hydrogen bonds are indicated by block dotted
lines. Image created with Pymol from DeLano Scientific LLC, San
Carlos, CA, USA. http://www.pymol.org.
interactions are also observed with another aminothia-
zole kinase inhibitor.17 The pyridine ring points toward
the surface-exposed portion of the hinge region. The
thioaryl group probes a mostly hydrophobic pocket
which includes the gatekeeper residue Phe589 and
Leu564 of helix-aC. The choice of using MuSK tyrosine
kinase as the template was reinforced by the high se-
quence homology with TrkA and by the observation
that the kinase was in an inactive conformation; i.e.,
the activation loop folds back toward the ATP binding
site creating a larger pocket near the gatekeeper than
is normally found with an active conformation. The
inactive conformation allows the substituted thioaryl
group to occupy the buried specificity region vacated
by the portion of the activation loop following Asp668
of the DFG motif. This model is consistent with some
key SAR observations. With respect to the terminal pyr-
idine ring, both compounds 11d (C-6 substituent is
hydrogen) and 11g (C-6 substituent is methyl) are 5-
to 10-fold less active than compound 11e (C-6 substitu-
ent is bromine). The model suggests that the hydrogen is
too small, and the methyl group not polarized to maxi-
mize polar and van der Waals interactions with the sur-
face-exposed specificity pocket which includes residues
Tyr591 and Arg599. The difference in activity of the
methyl-substituted thioaryl ring analog 11c and the
un-substituted 11e may due to the methyl filling in a
small pocket near Val573. The ortho-methyl may also
reduce the conformational space toward available space.
The size of the buried specificity pocket is limited as evi-
denced by the loss of activity of the larger ring of 20a
20i)
show
consistent
anti-proliferative
effects
(IC50 < 0.5 lM) in CD8-TrkA cells with a ꢀ10-fold
window against the vector V3.1. In the SH-SH-5Y
cells, the level of anti-proliferation is 2- to 8-fold less
than that observed for the CD8-TrkA cells. Further
evidence that proliferation is linked to TrkA inhibition
comes from the measurement of phospho-TrkA levels.
In CD8-TrkA cells, the level of phospho-TrkA and
phospho-ERK1 and
2 (a downstream kinase) is
down-regulated with increasing concentration of inhib-
itor 11t (data not shown), consistent with the mecha-
nism of inhibition, and supports the observation that
proliferation is likely due to TrkA inhibition.
Compounds evaluated in proliferation assay were also
tested against a limited set of kinases, as shown in
Table 6. All compounds show good selectivity: a 6-
to 500-fold window against CDK2, as well as against
Met, IGF1R, and VEGFR, consistent with the obser-
vation that anti-proliferative effects are driven by
TrkA inhibition.
In summary, we have identified 2-amino-5-(thioa-
of
ryl)thiazoles
as
potent
inhibitors
TrkA
(IC50 < 1 nM) and provided structural basis for the
key SAR observations. The intrinsic biochemical po-
tency against TrkA and corresponding anti-prolifera-
tive effects and biochemical markers, together with
the kinase selectivity, are suggestive that TrkA inhibi-
tion is responsible for their cellular response. The
Table 6. Kinase selectivity of TrkA inhibitors
Compound
Trk A IC50 (lM)
CDK2 IC50 (lM)
IGF1R IC50 (lM)
Met IC50 (lM)
VEGFR IC50 (lM)
11c
11w
20h
20i
0.001
0.03
0.49
0.18
0.54
0.52
0.23
>1.0
>1.0
>1
>1.0
>1.0
0.43
>1.0
0.17
>25
8%b
NT
NT
NT
0.0006
0.001
0.001
0.36
0.7
20j
b % inhibition at 2 lM.

S.-H. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 634–639
639
12. Das, J.; Liu, C.; Moquin, R.; Lin, J.; Furch, J. A.; Spergel,
S. H.; McIntyre, K. W.; Shuster, D. J.; O’Day, K. D.;
Penhallow, B.; Hung, C.-H.; Kanner, S. B.; Lin, T.-A.;
Dodd, J. H.; Barrish, J. C.; Wityak, J. Biog. Med. Chem.
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evaluation of these compounds in in vivo models will
be reported in due course.
References and notes
13. Das, J.; Furch, J. A.; Liu, C.; Moquin, R.; Lin, J.; Spergel,
S. H.; McIntyre, K. W.; Shuster, D. J.; O’Day, K. D.;
Penhallow, B.; Hung, C.-H.; Doweyko, A. M.; Kamath,
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14. Values are means of three experiments with stan-
dard deviation of less than 15%. Assay conditions:
ATP concentration at 10 lM, reaction time = 35 min,
enzyme concentration (lg/well) = 0.008, Substrate
concentration (lg/well) = 0.625 at 30 °C.
15. Till, J. H.; Becerra, M.; Watty, A.; Lu, Y.; Ma, Y.;
Neubert, T. A.; Burden, S. J.; Hubbard, S. R. Structure
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D.; Wittekind, M.; Kiefer, S. E.; Kish, K.; Lee, F. Y.
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