Phenolic P2/P3 core motif as thrombin inhibitors - Design, synthesis, and X-ray co-crystal structure

Article abstract of DOI:10.1016/j.bmcl.2005.10.082

Prototypical thrombin inhibitors were synthesized based on a trisubstituted phenol as a core motif. A naphthylsulfonamide analogue showed excellent antithrombin activity. An X-ray co-crystal structure showed the expected interactions.

Full text of DOI:10.1016/j.bmcl.2005.10.082

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1032–1036
Phenolic P2/P3 core motif as thrombin inhibitors—Design,
synthesis, and X-ray co-crystal structure
Stephen Hanessian,a,* Eric Therrien,a Willem A. L. van Otterlo,a Malken Bayrakdarian,a
Ingemar Nilsson,b,* Ola Fjellstro¨mb and Yafeng Xuec
aDepartment of Chemistry, Universite´ de Montre´al, C.P. 6128, Succ. Centre-Ville, Montre´al, PQ, Canada H3C 3J7
bAstraZeneca R&D Mo¨lndal, Medicinal Chemistry Mo¨lndal, Sweden
cAstraZeneca R&D Mo¨lndal, Structural Chemistry Laboratory Mo¨lndal, Sweden
Received 21 September 2005; revised 14 October 2005; accepted 24 October 2005
Available online 15 November 2005
Abstract—Prototypical thrombin inhibitors were synthesized based on a trisubstituted phenol as a core motif. A naphthylsulfona-
mide analogue showed excellent antithrombin activity. An X-ray co-crystal structure showed the expected interactions.
Ó 2005 Elsevier Ltd. All rights reserved.
The proteolytic activity of the enzyme thrombin on
endogenous proteins such as fibrinogen, and the
involvement of other serine proteases such as Factor
VIIa and Factor Xa in a series of complex biochemical
events leads to blood clots, with serious thrombotic
health consequences when procoagulant activities are
unnaturally high.1 Selective inhibitors of key coagula-
tion factors such as thrombin could offer a unique meth-
od for the management of cardiovascular thrombotic
states in the heart and elsewhere. The availability of
X-ray crystallographic data on thrombin with a host
of small molecules offers attractive options for the de-
sign and synthesis of structure-based potential inhibi-
tors.2 Indeed, several peptidomimetic type drug
candidates known to interact with thrombin at specific
sites are in advanced stages of clinical studies.3 More re-
cent efforts have focused on non-peptidic inhibitors
based on heteroaromatic or heterocyclic core struc-
tures.4 Such comparatively ‘‘simpler’’ molecules are
equally effective in inhibiting thrombin, since they are
deployed with requisite functionality for optimal bind-
ing in the active site.
We have reported on the design, synthesis, and X-ray
co-crystal structure of indolizidinones with thrombin.5
Low to moderate nanomolar inhibition was observed,
thus validating the initial design concept which focused
on constrained and conformationally rigidified ana-
logues of the natural substrate D-Phe-Pro-Arg. We have
also reported on related indolizidinones designed to tar-
get Factor VIIa.6 The marine natural products Dysino-
sin A7 and Oscillarin8 recently synthesized in our
laboratories exhibit inhibitory activity against thrombin
and Factor VIIa.
We now report on the synthesis and antithrombin activ-
ity of a series of achiral sulfonamides appended to a tri-
substituted phenolic core structure represented by the
generic expression 1 (Fig. 1). The requisite P1, P2, P3
interactions with their respective subsites in the enzyme
as suggested by molecular modeling augured well
for anticipating in vitro activity as well. During the
S2
P2
Me
O
P3
N
N
H
H
S3
OH
Keywords: Thrombin; Inhibitor; Trypsin; Phenol; Sulfonamide.
P1
*
Corresponding authors. Tel.: +514 343 6738; fax: +514 343 5728
(S.H.); tel.: +46 31 7761161; fax: +46 31 7763839 (I.N.); e-mail
addresses: stephen.hanessian@umontreal.ca; ingemar.nilsson@
astrazeneca.com
1
S1
Figure 1. Prototype of phenolic core with variations in the P1 and P3
subunits.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.082

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1032–1036
1033
biological evaluation of our set of compounds, a patent
appeared from the 3-D Pharmaceuticals Inc. group, in
which related compounds were reported but limited data
were provided on their antithrombotic activity.9
over trypsin (Table 1, entries 1–6). Unfortunately, no
clear-cut SAR could be observed by varying the position
of aromatic substituents (Table 1, entries 2–10). The
benzyl sulfonamide analogue displayed similar activity
as the analogous phenylsulfonamide (Table 1, compare
entries 7 and 11). Amides and ureas were also included
and proved to be detrimental to antithrombin activity
(Table 1, entries 12–14). Although the activities could
not be directly correlated with the nature or position
of aromatic substitution on the arylsulfonamide part,
we were intrigued that the 1-naphthylsulfonamide
showed excellent antithrombin activity with a 67-fold
selectivity over trypsin (Table 1, entry 1).
The phenolic core containing a diverse set of P3 append-
ages was synthesized as shown in Scheme 1, by a some-
what obvious protocol. Thus, the commercially
available 2-nitro-5-methyl phenol 2 was O-allylated
and the product subjected to an ortho-Claisen rearrange-
ment followed by O-benzylation to give 3 in excellent
overall yield. Oxidative cleavage of the double bond
and subsequent treatment of the resulting carboxylic
acid with diazomethane yielded the corresponding meth-
yl ester. Chemoselective reduction of the nitro group
with Pt/C led to the amine 4. Sulfonylation or acylation
of 4 with the desired P3 moiety afforded 5. Hydrolysis of
the ester followed by debenzylation yielded 6, which was
submitted to amide formation and subsequent deprotec-
tion to give the desired analogue 7. In order to provide
sufficient diversity in the library, different aromatic P1
substituents were also prepared using the general proto-
col (Fig. 2).
In Tables 2–4 are listed the IC50 values of another set of
substituted phenolic sulfonamides with simultaneous
variations of the aromatic substituents and the P1
subunits (series B, Table 2). Sulfonamides in the 2-ami-
no-5-aminomethyl-6-methyl pyridines were almost
equipotent with IC50Õs in the 190–590 nM range against
thrombin and with excellent selectivity. The same trend
was observed with the 2-amino-4-aminomethyl pyridine
P1 subunit (series C, Table 3), as well as with the 2-ami-
nomethyl-3-fluoro-6-methyl pyridine P1 subunit (series
D, Table 4), although the activity against thrombin
was greatly reduced compared to the 4-amidinobenzyl
series A (Table 1).
The sets of sulfonamides and amides harboring the phe-
nolic core motif exhibited excellent purities by LC/MS.
The IC50 values against thrombin and trypsin of the
phenolic sulfonamides and amides in the 4-amidinoben-
zyl P1 series (A) are listed in Table 1.
Determination of the X-ray crystal structure of com-
pound 8 in complex with thrombin revealed the same
general binding mode as observed for CVS16954c,10
(Figs. 3 and 4). The 4-amidinobenzyl group occupies
Several mono- and disubstituted sulfonamides exhibited
IC50 values in the range of 16–82 nM with selectivity
a) allyl bromide, K2CO3
a) RuCl3, NaIO4
b) CH2N2
Me
Me
b) 170 oC (neat)
c) BnBr, K2CO3
O2N
O2N
a) H2, Pt/C, THF
(92%, three steps)
OH
2
OBn
(90%, three steps)
3
a) R'SO2Cl, pyr.
OR
Me
Me
O
a) NaOH, THF/H2O
O
W
H2N
OMe
b) H2, Pd/C, MeOH
OMe
b) R'COCl, pyr.
OR
c) R'NCO, pyr.
R'
N
H
OBn
OBn
4
5
Me
Me
O
a) EDC, HOBt, i-Pr2NEt
DMF, R"NH2
O
W
W
R"
R'
N
OH
R'
N
N
H
b) H2, Pd/C, MeOH/HCl
OR HCl/MeOH
H
H
OH
OH
6
7
Scheme 1. General protocol for the synthesis of the phenolic analogues.
A
B
D
C
Me
F
R" =
NH2
Me
N
NH2
N
N
NH2
NH
Figure 2. Different P1 substituents.

1034
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1032–1036
Table 1. IC50 values for the 4-amidinobenzyl P1 subunit (series A)
Me
R5
O
R4
R3
W
N
N
H
H
OH
NH
R1
HCl
R2
NH2
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Ratioa
Thrombin
Trypsin
1
2
8
9
SO2
SO2
CH@CH–CH@CH2
Me
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.017
0.025
0.082
0.016
0.039
0.050
0.097
0.12
0.25
0.13
0.19
4.7
1.1
67
H
H
H
0.11
0.68
0.28
0.34
0.91
0.60
2.8
4.5
8.4
17
3
10
11
12
13
14
15
16
17
18
19
20
21
SO2
SO2
F
Cl
H
4
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
H
5
SO2
SO2
OMe
OMe
H
8.7
18
6
7
SO2
SO2
H
6.1
23
8
CF3
H
H
9
SO2
SO2
Me
Cl
H
1.1
4.4
7.2
17
10
11
12
13
14
H
0.94
3.1
SO2CH2
COCH2
CO
H
H
H
4.0
—
0.86
—
1.0
H
H
1.9
2.4
CONHCH2
H
H
2.7
a Ratio of (IC50 trypsin)/(IC50 thrombin).
Table 2. IC50 values for the 2-amino-5-aminomethyl-6-methyl pyridine P1 subunit (series B)
Me
R5
O
Me
R4
R3
W
N
H
N
N
H
HCl
NH2
OH
R1
R2
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Ratioa
Thrombin
Trypsin
1
2
3
4
23
24
25
26
SO2
SO2
Me
Me
H
Cl
H
F
H
H
H
H
H
H
H
H
H
0.19
0.26
0.59
0.57
>44
>44
>44
>44
222
212
75
H
SO2
SO2CH2
Me
H
H
H
78
a Ratio of (IC50 trypsin)/(IC50 thrombin).
Table 3. IC50 values for the 2-amino-4-aminomethyl pyridine P1 subunit (series C)
Me
R5
O
R4
R3
W
NH2
HCl
N
N
H
H
OH
N
R1
R2
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Ratioa
Thrombin
Trypsin
1
2
28
29
SO2
SO2
Cl
Cl
H
H
H
H
Cl
H
H
H
1.0
3.0
>44
>44
44
15
a Ratio of (IC50 trypsin)/(IC50 thrombin).

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1032–1036
1035
Table 4. IC50 values for the 2-aminomethyl-3-fluoro-4-methyl pyridine P1 subunit (series D)
Me
R5
O
F
R4
R3
W
Me
N
N
H
H
OH
N
R1
R2
Entry
Compound
W
R1
R2
R3
R4
R5
IC50 (lM)
Thrombin
Ratioa
Trypsin
1
2
3
30
31
32
SO2
SO2
H
H
H
Cl
Cl
Cl
Me
H
H
H
H
H
H
H
20
14
15
>44
>44
>44
2.2
3.2
3.0
SO2CH2
H
a Ratio of (IC50 trypsin)/(IC50 thrombin).
the S1-pocket with a salt bridge between the amidino
˚
moiety and Asp 189 (d = 2.62 and 2.97 A). Similarly,
Proximal
pocket
the H-bond from the P1–P2 amide linkage to the C@O
of Ser 214 is conserved (d = 3.07 A). The P2-group of 8
˚
aligns well with that of CVS1695 creating hydrophobic
interaction with the proximal pocket (S2-pocket). The
A-ring of the P3-naphthyl group of 8 occupies almost
the same space as the phenyl ring of CVS1695 with
interactions in the lipophilic distal (D) pocket (S3-pock-
et). The major difference is the absence of hydrogen
bonds between the 2-aminophenol group and Gly 216
Ser
214
Distal
pocket
˚
˚
(d = 4.33 A phenol-O to NH of Gly 216 and 3.90 A
NH to C@O of Gly 216). This may partially explain
the slightly lower potency of this series of compounds
compared to similar P2-motifs recently disclosed, which
in general exhibit an antiparallel b-sheet hydrogen bond
network to Gly 216.11 This difference is in line with the
stronger H-bond donating capability of the amide oxy-
gen in CVS1695 and similar P2-motifs compared to that
of the phenolic oxygen in our series.12 This also provides
an explanation why removal of the phenolic hydroxyl
group (as in the P2 phenyl analogue of the benzylsulf-
onamide 18) only reduced the potency two times (IC50
0.41 lM thrombin, 3.3 lM trypsin).
Gly
216
Asp
189
Figure 3. X-ray crystal structure of 8 bound in thrombin active site.
Essential H-bonds to Asp 189 and Ser 214, and to sidechains of the
proximal and distal pockets involved in lipophilic interactions are
shown (PDB ID code 2BDY).
The B-ring of the naphthyl group is evidently too big to
be accommodated in the D pocket and merely interacts
with bulk water. Notably, compound 8 is four times as
active as phenyl analogue 14 (IC50 0.017 and
0.097 lM). Most likely, this is due to intramolecular sta-
bilization of the torsion around the N–S–C–C bond of
the flanking B-ring. In agreement with this, improved
potency is also observed for compound 9 having an
ortho-methyl group (IC50 0.025 lM).
In conclusion, we have shown that a trisubstituted phenol
can be used as central P2 scaffold and that substituted phe-
nylsulfonamides can be most effective P3 subunits.
Depending on the nature of the P1 subunit, activity and
selectivity over trypsin can be nicely modulated. The X-
ray crystal structure of the 1-naphthylsulfonamide ana-
logue 8 corroborates the anticipated interactions in the
binding site of thrombin (Figs. 3 and 4, PDB ID code
2BDY).
Figure 4. Superposition of the X-ray crystal structures of 8 (white) and
CVS1695 (magenta) in complexes with thrombin.

1036
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1032–1036
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Clearly, the 4-amidinobenzyl P1 subunit (A) is favored
for thrombin with some selectivity against trypsin. Selec-
tivity is dramatically increased with less basic P1 sub-
units at the expense of a 3- to 20-fold loss of
antithrombin activity compared to the amidine series.
The simplicity of these structures and their relative ease
of synthesis should pave the way to a better understand-
ing of the relative roles of the P2/P3 subunits, and the
subtle influences of aromatic substitutions on the
bioactivity.13
˚
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Acknowledgments
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Pellicore, J. M.; Pal, S.; Wallace, A. A.; Clayton, F.
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K. E.; Barrow, J. C.; Cutrona, K. J.; Glass, K. L.;
Krueger, J. A.; Kuo, L. C.; Lewis, S. D.; Lucas, B. J.;
McMasters, D. R.; Morrissette, M. M.; Nantermet, P.
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H. G.; Lewis, S. D.; Lucas, B. J.; Krueger, J. A.; Miller-
Stein, C.; White, R. B.; Wong, B.; McMasters, D. R.;
Wallace, A. A.; Lynch, J. J., Jr.; Yan, Y.; Chen, Z.;
Kuo, L.; Gardell, S. J.; Shafer, J. A.; Vacca, J. P.; Lyle,
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Young, M. B.; Barrow, J. C.; Glass, K. L.; Lundell, G.
F.; Newton, C. L.; Pellicore, J. M.; Rittle, K. E.;
Selnick, H. G.; Stauffer, K. J.; Vacca, J. P.; Williams, P.
D.; Bohn, D.; Clayton, F. C.; Cook, J. J.; Krueger, J.
A.; Kuo, L. C.; Lewis, S. D.; Lucas, B. J.; McMasters,
D. R.; Miller-Stein, C.; Pietrak, B. L.; Wallace, A. A.;
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Med. Chem. 2004, 47, 2995; (e) Burgey, C. S.; Robinson,
K. A.; Lyle, T. A.; Sanderson, P. E. J.; Lewis, S. D.;
Lucas, B. J.; Krueger, J. A.; Singh, R.; Miller-Stein, C.;
White, R. B.; Wong, B.; Lyle, E. A.; Williams, P. D.;
Coburn, C. A.; Dorsey, B. D.; Barrow, J. C.; Stranieri,
M. T.; Holahan, M. A.; Sitko, G. R.; Cook, J. J.;
McMasters, D. R.; McDonough, C. M.; Sanders, W.
M.; Wallace, A. A.; Clayton, F. C.; Bohn, D.; Leonard,
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We thank the NSERC of Canada and AstraZeneca
(Mo¨lndal, Sweden) for financial assistance through the
Medicinal Chemistry Chair program. W.A.L.v.O thanks
the NRF (South Africa) for partial post-doctoral sup-
port. We also thank Sofi Nielsen at AstraZeneca for
IC50 determinations.
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