From natural products to achiral drug prototypes: Potent thrombin inhibitors based on P2/P3 dihydropyrid-2-one core motifs

Article abstract of DOI:10.1016/j.bmcl.2009.07.107

A series of dihydropyrid-2-ones was synthesized and tested for inhibitory activity against serine protease enzymes. Moderate to low nanomolar inhibitory activities were obtained against thrombin and excellent selectivity against trypsin was observed.

Full text of DOI:10.1016/j.bmcl.2009.07.107

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5429–5432
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
From natural products to achiral drug prototypes: Potent thrombin
inhibitors based on P2/P3 dihydropyrid-2-one core motifs
a
a
a
b
Stephen Hanessian a, , Eric Therrien , Jianbin Zhang , Willem van Otterlo , Yafeng Xue ,
*
David Gustafsson c, Ingemar Nilsson d, , Ola Fjellström
e
*
a Department of Chemistry, Université de Montréal, C. P. 6128, Succ. Centre-Ville, Montréal, P. Q., Canada H3C 3J7
b AstraZeneca R&D Mölndal, Cell Protein & Structural Sciences, 431 83 Mölndal, Sweden
c AstraZeneca R&D Mölndal, Scientific Advisory Group, 431 83 Mölndal, Sweden
d AstraZeneca R&D Mölndal, Medicinal Chemistry, 431 83 Mölndal, Sweden
e AstraZeneca R&D Mölndal, Lead Generation, 431 83 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dihydropyrid-2-ones was synthesized and tested for inhibitory activity against serine protease
enzymes. Moderate to low nanomolar inhibitory activities were obtained against thrombin and excellent
selectivity against trypsin was observed.
Received 8 July 2009
Revised 21 July 2009
Accepted 22 July 2009
Available online 26 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Thrombin inhibitors
Dihydropyridone
Blood coagulation is a complex multifaceted process that in-
volves cellular and proteinaceous components.1 Through a series
of ordered events, primary hemostasis caused by platelet aggrega-
tion is followed by activation of plasma coagulation factors causing
clot formation in blood vessels (secondary hemostasis). The cas-
cade of biochemical events leading to blood coagulation involves
a stepwise activation of trypsin-like proteases. The multifunctional
serine protease thrombin (Factor IIa)2 is responsible for the final
cleavage of fibrinogen to fibrin, which undergoes cross-linking to
form a strong fibrin clot. Under normal physiological conditions,
the blood coagulation system is balanced by anticoagulation on
the one hand, and fibrinolysis on the other.3 A pathogenic imbal-
ance that favors the activation of the coagulation system may lead
to thrombosis in humans.4 Inhibition of thrombin is widely viewed
as an effective therapeutic means to prevent the formation of blood
clots and related thromboembolic disorders.5
revealed the importance of a hydrophobic interaction in the S3 site.
Based on these observations, hybrid molecules were designed and
synthesized, showing superb in vitro inhibition of thrombin.10 We
also reported on the design and synthesis of P2/P3 phenolic core
motifs11 as well as P2/P3 pyrid-2-one core motifs (Fig. 1).12
Further refinement of the core motif led to the synthesis of mol-
ecules containing a dihydropyrid-2-one with impressive inhibitory
activities against thrombin (Scheme 1).13
Protection of 4-methyl piperidine 1 as the N-Boc derivative, fol-
lowed by oxidation with NaIO4 and RuO2 led to the lactam 2. For-
mation of the Li enolate and alkylation with ethyl 2-bromoacetate
gave 3 in 69% yield. Introduction of an endocyclic double bond was
achieved by formation of an enolate, addition of PhSeBr and oxida-
tive elimination. The desired product 4 was accompanied by the
a,b-unsaturated ester 5. Increasing the size of the ester (e.g., CO2t-
Bu) favors the endo:exo ratio in a 2:1 fashion albeit in lower total
In previous studies we have shown that chlorodysinosin A, a
natural product belonging to the aeruginosin family6 exhibits low
nanomolar in vitro inhibitory activity against thrombin, trypsin,
and tissue factors VIIa and Xa (Fig. 1).7
yield (64%).
Removal of the N-Boc group of 4 and treatment with t-BuONO
and pyridine in Et2O gave the N-nitroso product 6. Reduction with
Zn/AcOH followed by treatment with a diverse set of arylsulfonyl
chlorides gave the corresponding N-sulfonamides 7a–i. Hydrolysis
of the ethyl ester, coupling with the N-Cbz-4-amino-methylbenz-
amidine, followed by hydrogenolysis, afforded a set of compounds
(8a–i) that were evaluated for their enzymatic in vitro activity
against thrombin and trypsin.
An X-ray co-crystal structure of chlorodysinosin7 (and related
aeruginosins such as dysinosin A8 and oscillarin,9 with thrombin,
* Corresponding authors. Tel.: +1 514 343 6738; fax: +1 514 343 5728 (S.H.);tel.:
+46 31 7761161; fax: + 46 31 7763839 (I.N.).
E-mail addresses: stephen.hanessian@umontreal.ca (S. Hanessian), ingemar.
nilsson@astrazeneca.com (I. Nilsson).
Compounds 8a–i exhibited remarkably different levels of inhib-
itory activity against thrombin (Table 1). The weakest inhibitor
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.107

5430
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5429–5432
H
H
H
HO
HO
O
O
N
N
HN
HN
HO
H
R
N+
N
O
O
NH2
NH2
NH
NH
Ph
H2N
HN
-
O
OSO3
O
Ph
OMe
OH
R = Cl, Chlorodysinosin A
(thrombin IC50 = 5.8 nM)
R = H, Dysinosin A
Oscillarin
(thrombin IC50 = 28 nM)
(thrombin IC50 = 46 nM)
H
Me
O
O
R
N
HN
N
N
R1
R2
H
H
H
OH
NH2
NH
O
NH
Phenolic P2/P3 core
(thrombin IC50 = 16 - 4 700 nM)
NH2
O
Ph
HN
OH
Me
O
R1 = Cl, R2 = i-Pr
(thrombin IC50 = 1.6 nM)
R1 = Me, R2 = Et
(thrombin IC50 = 3.3 nM)
R1, R2 = cyclohexyl
(thrombin IC50 = 3.4 nM)
R
N
N
N
H
H
O
NH2
NH
Pyrid-2-one P2/P3 core
(thrombin IC50 = 23 - 2 471 nM)
Figure 1. Structures of natural and unnatural products and their in vitro inhibitory activity.
Me
Me
Me
CO2Et
a, b
c
HN
N
N
Boc
Boc
1
2
3
O
O
Me
CO2Et
Me
CO2Et
d, e
f, g
+
N
N
Boc
Boc
4
5
O
O
Me
Me
CO2Et
O O
S
h, i
j-l
N
CO2Et
N
ON
R
N
H
6
7a-i
O
O
Me
O
O O
S
N
R
N
N
H
H
8a-i
O
NH2
.HCl
NH
Scheme 1. Reagents and conditions: (a) Boc2O, Et3N, DCM, 35 °C, 73%; (b) RuO2ÁxH2O, NaIO4, EtOAc/H2O, 83%; (c) LiHMDS, BrCH2CO2Et, THF, À78 °C, 69%; (d) LiHMDS, PhSeBr,
À78 °C; (e) H2O2, pyridine, DCM, 0 °C, 4, 35%, 5, 41%; (f) TFA, DCM; (g) t-BuONO, pyridine, Et2O, reflux, 91%; (h) Zn, AcOH, 61%; (i) RSO2Cl, pyridine, DCM; (j) LiOH, THF/H2O;
(k) EDC, HOBt, i-Pr2NEt, N-Cbz-4-amino-methylbenzamidine, DMF; (l) H2, Pd/C, MeOH/HCl.
was the benzylsulfonamide 8a. In contrast, the corresponding phe-
nylsulfonamide 8b was five times more active (Table 1, entries 1
and 2). Single or double substitution at the ortho, meta, and para
positions were well tolerated regardless of the electronic nature
of the substituents (Table 1, entries 3, 4, 6, 8). An electron with-
drawing ortho-F substituent (8e) was less favorable (Table 1, entry
5). By far, the best combination of substituents were found to be at
the two ortho-positions, exemplified by the chloro analogue 8g,
with an IC50 of 3 nM against thrombin, and excellent selectivity
against trypsin (Table 1, entry 7). A naphthylsulfonyl group was
also well tolerated (Table 1, entry 9).
There is a remarkably improved inhibition for the dihydropyrid-
2-one series compared to the pyrid-2-one series.12 Included in Ta-
ble 1 are the IC50 values of identical compounds in both series ex-
cept for the nature of the P2/P3 heterocyclic core. Between 25- and
100-fold enhancement of inhibitory activity was observed in each

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5429–5432
5431
Table 1
Inhibitory activities against thrombin and trypsin and measured pKa of sulfonamide N for dihydropyrid-2-ones 8a–i and corresponding pyrid-2-ones (9a–d, 9h, 9i)
Me
Me
R5
O
R5
O
R4
R3
W
N
R4
R3
W
N
N
H
N
H
N
H
N
H
O
NH2
NH
O
NH2
NH
R1
R1
8a-i
9a,b,c,d,h,i
R2
R2
Entry
Compd
W
R1
R2
R3
R4
R5
Thrombina
Trypsinb
(IC50 nM)
pKa
1
2
3
4
5
6
7
8
9
8a (9a)
8b (9b)
8c (9c)
8d (9d)
8e
8f
8g
8h (9h)
8i (9i)
CH2SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
H
H
H
Me
F
H
H
OMe
H
H
H
H
H
H
H
H
H
H
Cl
H
H
Me
H
H
H
H
Me
H
Cl
H
H
H
H
H
H
H
H
241 (3960)
42 (2470)
11 (1050)
6 (578)
106
— (14,900)
766 (5,770)
860 (15,600)
374 (7130)
683
1250
286
117 (2020)
8.2 (5.8)
— (—)
— (5.9)
8.8 (—)
8.7 (—)
—
—
—
— (6.7)
Cl
Cl
10
3
Me
H
26 (1240)
OMe
CH@CH–CH@CH
44 (1180)
915 (17,100)
H
a
Human thrombin.
Bovine trypsin.
b
case with improved selectivity against trypsin compared to the
pyrid-2-one series.
In view of the otherwise virtual identity of these pairs of com-
pounds, we attribute the greatly enhanced activity in the dihydro-
pyrid-2-one series mainly to the higher pKa of the sulfonamide NH
group (see 8i vs 9i in Tables). It is possible that this results in a bet-
ter antiparallel H-bonded interaction with Gly 216 (Fig. 2).6–9,14 At
physiological pH (pH ꢀ7.4), the corresponding pyridones are
deprotonated with a delocalised negative charge at the sulfon-
amide N and may be so also in the complex with thrombin. Thus,
a direct H-bond with the Gly 216 carbonyl group is not possible.
It should be noted that the thrombin assay is run at pH 7.4.
The X-ray structure of compound 9i in complex with thrombin
has been solved at 1.61 Å resolution. As seen in Figure 3, there are
no direct H-bonds between the ligand and Gly 216 in thrombin.
The distances between the sulfonamide nitrogen and the amide
oxygen of Gly 216 and between the pyrid-2-one oxygen and amide
nitrogen of Gly 216 are 4.36 and 3.87 Å, respectively. The corre-
sponding distances in the X-ray of a dihydropyrid-2-one naphthyl
Figure 3. Co-crystal structure of 9i with thrombin. The long non hydrogen bond
distances to Gly 216 are highlighted as well as the favorable interactions between
the benzamidine and Asp 189.
Me
O
O O
S
N
N
N
H
H
O
N
H2
8i
analogue with a different P1 moiety are 3.08 and 3.01 Å, indicative
of direct H-bonding (data not shown). The pKa of 9i sulfonamide
group is 5.8 and in the crystallization conditions the pH is 7.2–
7.4, which is similar to pH 7.4 used in the thrombin activity mea-
surements. It is therefore likely that the sulfonamide group in the
pyrid-2-one 9i is negatively charged, whereas it is neutral for the
dihydropyrid-2-one analogues. These data support the hypothesis
that pKa of the sulfonamide nitrogen strongly influences the affin-
ity to thrombin.
In conclusion, we have shown that structural information de-
rived from co-crystal complexes of natural product inhibitors of
thrombin can be used for the design and synthesis of totally achiral
congeners. Dihydropyrid-2-ones with IC50 values below 10 nM
against thrombin have been achieved which is a dramatic improve-
ment compared to the closely related pyrid-2-one analogues. We
attribute this to the pKa differences of the sulfonamide nitrogen
resulting in a productive H-bonded interaction with Gly 216 for
the dihydropyrid-2-ones. Subtle differences in the nature of sub-
O
NH2
O
H
N
O
N
H
Asp 189
Gly 216
Me
O
O O
S
N
O
N
N
H
H
NH2
NH2
9i
O
X X
O
H
N
O
N
H
Asp 189
Gly 216
Figure 2. Proposed interactions of sulfonamide inhibitor 8i and 9i with Gly 216 and
Asp 189 in thrombin.

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S. Hanessian et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5429–5432
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Acknowledgments
We thank the NSERC of Canada, FQRNT (Québec) and AstraZen-
eca (Mölndal, Sweden) for financial assistance through the Medic-
inal Chemistry Chair program. The authors also acknowledge
Daniel Simard for his technical assistance.
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