2-(2-Chloro-6-fluorophenyl)acetamides as potent thrombin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.09.013

2-(2-Chloro-6-fluorophenyl)acetamides having 2,2-difluoro-2-aryl/heteroaryl-ethylamine P3 and oxyguanidine P1 substituents are potent thrombin inhibitors (K_i = 0.9-33.9 nM). 2-(5-Chloro-pyridin-2-yl)-2,2-difluoroethylamine was the best P3 subst

Full text of DOI:10.1016/j.bmcl.2007.09.013

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6266–6269
2-(2-Chloro-6-fluorophenyl)acetamides as potent
thrombin inhibitors
Lily Lee, Kevin D. Kreutter, Wenxi Pan, Carl Crysler, John Spurlino, Mark R. Player,*
Bruce Tomczuk and Tianbao Lu
Drug Discovery, Johnson & Johnson, Pharmaceutical Research and Development, Spring House, PA, USA
Received 3 August 2007; revised 30 August 2007; accepted 4 September 2007
Available online 8 September 2007
Abstract—2-(2-Chloro-6-fluorophenyl)acetamides having 2,2-difluoro-2-aryl/heteroaryl-ethylamine P3 and oxyguanidine P1 substit-
uents are potent thrombin inhibitors (Ki = 0.9–33.9 nM). 2-(5-Chloro-pyridin-2-yl)-2,2-difluoroethylamine was the best P3 substitu-
ent, yielding the most potent inhibitor (Ki = 0.7 nM). Replacing the P3 heteroaryl group with a phenyl ring or replacing the difluoro
substitution with dimethyl or cyclopropyl groups in the linker reduced the affinity for thrombin significantly. The aminopyridine P1s
also provided an increase in potency.
Ó 2007 Elsevier Ltd. All rights reserved.
The discovery of small molecule inhibitors of thrombin,
a key serine protease in the coagulation cascade, contin-
ues to be an important goal for anti-thrombotic ther-
apy.1 Thrombin, a trypsin-like peptidase, mediates the
cleavage of fibrinogen to fibrin and the activation of
platelets, leading to the formation of blood clots.2 Inhi-
bition of thrombin would provide effective treatment for
venous or arterial thromboembolism, e.g., prevention of
postoperative deep venous thrombosis (DVT) and
pulmonary embolism (PE), prevention of stroke during
atrial fibrillation, and treatment of acute coronary
syndrome (ACS).3 The inhibition of thrombin has been
clinically validated by the availability of Argatroban
and the recombinant hirudins, used to treat heparin
induced thrombocytopenia.
The synthesis of 2-(2-chloro-6-fluorophenyl)acetamides
was achieved using the chemistry depicted in Scheme 1
(compounds 2–4 and 9–11).5
First, diethyl malonate was deprotonated with NaH and
reacted with 1,2,3-trifluoro-4-nitro-benzene (I). Subse-
quent decarboxylation using LiCl6 gave the ester II.
Then, the aromatic nucleophilic substitution (SNAr)
was accomplished by the displacement of the aryl
fluoride (II) with the requisite aryl/heteroaryl amine.7
Following protection of the aniline group as a trifluoro-
acetate, conversion of the nitro group to chloride IV was
accomplished by reduction to the corresponding aniline
and a Sandmeyer reaction. Deprotection of the aniline
and ester, followed by coupling to the O-guanidine
segment, completed the synthesis to render target
compounds (V).
Pyridinones have been shown to be suitable P2 scaf-
folds,4 providing the appropriately spaced hydrogen
bonding partners to interact with the backbone
Gly216, a key binding interaction in the thrombin active
site (Fig. 1). Herein, we describe a series of 2-(2-chloro-
6-fluorophenyl)acetamides capable of maintaining the
Gly216 hydrogen bonding interaction, and exploration
of the P3 group to provide potent thrombin inhibitors.
Alternatively, the acetamides were synthesized using the
chemistry shown in Scheme 2 (compounds 1, 8, and 14–
16). 1,2,3-Trifluoro-4-nitro-benzene (I) was treated with
diethyl malonate sodium salt, followed by acetic acid/
hydrochloric acid mediated decarboxylation and sapon-
ification to yield acid VI. The acid was reduced using
sodium borohydride. Then, the aniline group was intro-
duced via SNAr of t-Bu-amine and VII followed by
deprotection of the tert-butyl group. The alcohol (VIII)
was protected as the acetate and the P3-unit was intro-
duced using the requisite acid chloride, giving
compound X. The 2-Cl substituent was similarly con-
verted from its nitro precursor via reduction to the
Keywords: Thrombin inhibitor; Anti-coagulant; Trypsin; Serine
protease.
*
Corresponding author. Tel.: +1 610 458 6980; e-mail: mplayer@
prdus.jnj.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.013

L. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6266–6269
6267
P3
P2
P1
P3
P2
P1
(Z)
N
R
R
A
O
A
O
Y
Y
Z
N
H
N
H
Z
N
H
N
H
arginine
mimic
arginine
mimic
O
F
O
H
N
O
H
N
gly 216
gly 216
Figure 1. Replacement of pyridinone scaffold with aryl fluoride.
NO2
NO2
NO2
NO2
NO2
F
b
a
a
O
b
CO2H
F
OH
F
F
F
F
F
OEt
F
F
F
F
F
VI
VII
I
I
II
NO2
NO2
NO2
Cl
c, d
e
R
X
X
R
O
H2N
OAc
O
c
H2N
OH
F
X
X
N
H
N
F
OEt
X
X
OEt
VIX
F
F
F
TFA F
F
F
VIII
NH2
III X = CH, N; R = H, Cl
IV X = CH, N; R = H, Cl
O
NO2
g
f
O
Ar
Ar
N
H
OAc
OH
Ar
Cl
N
OAc
F
F
R
X
F
H
O
NH
O
F
F
F
d
XI
X
N
H
N
H
N
H
NH2
X
X
Cl
F
Cl
F
F
O
j
h, i
V X = CH, N; R = H, Cl
Ar
N
H
N
H
OH
F
F
F
F
F
F
Scheme 1. Reagents and conditions: (a) i—NaH (2 equiv), diethyl
malonate (2 equiv), THF, 0 °C–rt, 3 h; ii—LiCl, H2O, DMSO, 150 °C,
2 h, 78%; (b) ArCF2CH2NH2 (0.95 equiv), DIEA (1.2 equiv), DMSO,
81–85%; (c) i—TFAA (5 equiv), DIEA (1.2 equiv), DCM, 99%; ii—Zn
(11 equiv), AcOH (22 equiv), DCM, 99%; iii—tert-butyl nitrite
(1.5 equiv), CuCl2 (1.2 equiv), CH3CN, 48–52%; (d) i—NaOH
(2.5 equiv), MeOH; ii—H2NCH2CH2ONHC(=NBoc)NHBocÆHCl
(2 equiv), BOP (6 equiv), DIEA (6 equiv), DMF, 83%; iii—TFA,
DCM, 82–85%.
XIII
XII
Cl
O
k, l
Ar
F
N
H
OH
F
F
XIV
Scheme 2. (a) i—NaCH(CO2Et)2; ii—concd HOAc, 4 N HCl, reflux,
14 h, 58%; (b) NaBH4, BF3, 99%; (c) t-BuNH2, toluene, reflux, 16 h,
62%; (d) i—concd HCl, EtOH, reflux, 2 h; ii—NaHCO3, 93%; (e)
AcCl, THF, 0 °C, DIEA, 69%; (f) ArCF2COCl, DIEA, DCM, 92%; (g)
Pd/C, H2, EtOH, 92%; (h) i—K2CO3 (aq), MeOH, ii—HCl, 92%; (i)
i—HOAc, concd HCl, NaNO2; ii—CuCl, concd HCl, 48%; (j) BH3,
THF, reflux, 20 h, 81%; (k) SO3Æpyridine, DMSO, DIEA, 99%; (l) i—
NaClO2, H2O, DMSO, NaH2PO4; ii—HCl aq, 51%.
aniline, deprotection of the acetate group and a Sand-
meyer reaction using CuCl (compound XII). The acet-
amide was reduced using a borane reduction, giving
the desired P3 segment. Oxidation then provided the
necessary carboxylic acid (XIV), ready for attachment
to the P1 moiety.
(Fig. 2). As expected, the aniline-NH contributes to a
hydrogen bonding interaction with the thrombin back-
bone (Gly216, 3.05 A). The amide NH also makes
important hydrogen bonding interactions with the back-
Compounds 1–18 were evaluated for the inhibition
of thrombin using a standard chromogenic assay8
(Tables 1 and 2). In summary, the SAR directed us to
electron-deficient mono- and bicyclic 2,2-difluoroethyl-
amine moieties as the most effective P3s. For example,
switching from a phenyl group (1, Ki = 47.1 nM) to a
2-pyridyl group (9, Ki = 8.6 nM) increases the potency
more than fivefold. This is due to better edge to face
r–p interactions of the electron-deficient aryl groups
with the backbone p-rich Trp215 in the P3 region.9 This
effect is also seen in compounds 3 and 4. The p-Cl group
in compound 11 increases the binding affinity for throm-
bin almost fivefold compared with compound 9. Biaryl
rings with an electron withdrawing substituent are also
sufficient to achieve nM potency, such as compound 8.
9
˚
˚
˚
bone protein (Ser214, 3.01 A; Ser195 3.11 A), and the O-
guanidine moiety, an arginine mimic, effectively anchors
the ligand with multiple (4) hydrogen bonding interac-
˚
˚
˚
tions (Gly219, 2.90 A; Asp189, 2.69 A, 3.19 A; Ala190,
˚
3.25 A).
An interesting insight from the crystal structure was the
observed distance between the P2 fluorine and the back-
˚
bone nitrogen of Gly216 (F–N distance 3.17 A). This
˚
equates to a F–H distance of 2.14 A, as N–H bond
10
˚
lengths are 1.03 A. While organofluorine may only
rarely be a H-bond acceptor,11 F–H distances of
˚
62.35 A are regarded as strong evidence that such an
Several H-bond interactions were revealed in the X-ray
crystal structure of compound 1 in human thrombin
H-bond exists.11,12 It should be noted, however, that
while the CF–HN hydrogen bond is undoubtedly

6268
L. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6266–6269
Table 1. Effect of R1 on potency of 2-fluoro-4-chloro-3-phenyl
acetamide O-guanidines
Table 2. Replacement of O-guanidine P1 with aminopyridines
Compound
Ki (nM)
Cl
Cl
O
NH
NH2
O
O
R1
N
H
N
H
14
10.3
3.3
2.8
0.7
0.9
N
H
N
H
F
F
F
F
F
F
N
NH2
Compound
R1
Ki (nM)
Cl
O
1
47.1
NH
15
16
17
18
N
H
F
F
N
H
F
F
N
NH2
N
2
3
4
5
6
33.9
2.7
NH
Cl
F
F
F
O
N
H
N
H
F
F
B
N
NH2
NH
A
F
F
N
Cl
Cl
Cl
O
B
N
H
N
N
H
4.7
NH
F
A
F
F
F
F
N
NH2
N
Cl
NH
O
1281
58.0
S
O
O
N
H
N
O
N
H
F
F
F
N
NH2
N
NH
NH
Me Me
contributing to the potency of the series, it is also possi-
ble that the P2 (and P3) fluorines are providing potency
by increasing the strength of the aniline NH–CO hydro-
gen bond via increased aniline NH acidity. Indeed, the
N
F
7
8
128
3.1
Me Me
13
calculated pKa of the gem-difluoro aniline NH of 2
(ꢀ1.1) is 3 pKa units lower than the fourfold less potent
gem-dimethyl aniline NH of 7 (2.2), which is in line with
the known effect of b-fluorine on amine pKa.14
NH
F
F
9
8.6
N
NH
F
F
N
10
11
12
13
169
1.8
NH
F
F
Cl
N
NH
F
F
57.0
142
N
NH
NH
N
Figure 2. X-ray crystal structure of compound 1 in human thrombin
(PDB ID: 2R2M).

L. Lee et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6266–6269
6269
7. (a) 3-Dimensional Pharmaceuticals Inc.; Pan, W., Lu, T.,
Markotan, T., Tomczuk, B. Aminopyridyl-, Aminoguan-
idinyl- and Alkoxyguanidinyl-Substituted Phenyl Aceta-
mides as Protease Inhibitors, WO0228825; (b) 3-
Dimensional Pharmaceuticals, Inc.; Kreutter, K., Lee,
L., Lu, T., Mohan, V., Patel, S., Huang, H., Xu, G.
Substituted Phenyl Acetamides and their Use as Protease
Inhibitors, WO04091613.
Aminopyridines have been reported to be suitable argi-
nine (P1) surrogates.15 A brief survey revealed that
replacement of the O-guanidine group in compound 1
with a methyl aminopyridine P1 (compound 14) and a
dimethyl aminopyridine (compound 15) increased the
potency 4- and 14-fold, respectively. The methyl amino-
pyridine also improved the potency of compound 11,
giving compound 17 with sub-nM potency
(Ki = 0.7 nM). Interestingly, the corresponding N-oxide
compound 18 has comparable potency to 17.9
8. Dissociation constants were determined at 37 °C in a 96-
well format using a Molecular Devices plate reader.
Varied concentrations of inhibitors in 10 ll dimethylsulf-
oxide were added to wells with 280 ll of assay buffer (pH
7.5) which contained 50 mM Hepes, 0.2 M NaCl, 1%
dimethylsulfoxide, 0.05% n-octyl b-D-glucopyranoside,
and substrate, and incubated for 15 min at 37 °C. Reac-
tions were initiated by the addition of 10 ll of enzyme in
assay buffer without dimethylsulfoxide and substrate, and
the change in absorbance at 405 nm was monitored for
15 min. IC50s were obtained from the inverse slope of plots
of the ratio of initial velocity in the absence of inhibitor to
initial velocity in the presence of inhibitor as a function of
inhibitor concentration. All velocities were corrected for
background substrate conversion (no enzyme addition).
The dissociation constant (Ki) was calculated using the
equation Ki = IC50/(1 + S/Km) where S is the substrate
concentration (Cheng, Y.; Prusoff, W. H. Biochem. Phar-
macol. 1973, 22, 3099). Km for the enzyme–substrate pair
was determined from Hanes–Wolf plots using the same
final buffer conditions as Ki determinations. Respective
substrate, substrate concentration, and Km values for
human a-thrombin were: succinyl-Ala-Ala-Pro-Arg-p-
nitroanilide, 100, and 320 lM. Within-run assay coeffi-
cient of variation (CV) was <10%; between-run CV was
<20%.
In conclusion, we have developed a series of non-pep-
tidic, 2-(2-chloro-6-fluorophenyl)acetamides which have
excellent potency against human thrombin. Proper
selection of the P3 substituent was critical in attaining
sub-nM potency. The O-guanidine moiety was a suitable
arginine replacement. Replacement of the O-guanidines
with aminopyridines further improved the thrombin
potency.
References and notes
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