Targeting thrombin and factor VIIa: Design, synthesis, and inhibitory activity of functionally relevant indolizidinones

Article abstract of DOI:10.1016/S0960-894X(02)00612-1

Guided by molecular modeling, docking experiments, and available X-ray crystal structure data on the serine protease Factor VIIa and thrombin, a series of indolizidinone derivatives was designed and synthesized having diverse functionality at the P₁, P₂, and P₃ sites.

Full text of DOI:10.1016/S0960-894X(02)00612-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2907–2911
Targeting Thrombin and Factor VIIa: Design, Synthesis, and
Inhibitory Activity of Functionally Relevant Indolizidinones
Stephen Hanessian,a,* Eric Therrien,a Kenneth Granbergb and Ingemar Nilssonb
a
´
´
´
Department of Chemistry, Universite de Montreal, PO Box 6128, Station Centre-ville, Montreal, QC, Canada H3C 3J7
¨
bAstraZeneca R&D Molndal, Medicinal Chemistry, Molndal, Sweden
¨
Received 7 May 2002; accepted 11 July 2002
Abstract—Guided by molecular modeling, docking experiments, and available X-ray crystal structure data on the serine protease
Factor VIIa and thrombin, a series of indolizidinone derivatives was designed and synthesized having diverse functionality at the
P1, P2, and P3 sites.
# 2002 Elsevier Science Ltd. All rights reserved.
Thrombin [Factor IIa (FIIa)] is the ultimate enzyme in
the blood coagulation cascade, and is capable of cleav-
ing fibrinogen to soluble fibrin, which in turn can poly-
merize to form a fibrin plug or to stabilize a clot.1
Although direct inhibition of FIIa has been well studied,
and has resulted in promising clinical drug candidates,2
other enzymes in the serine protease family have
become targets for intensive research in response to the
medical need for new anticoagulants with improved
safety profiles over existing antithrombotic therapies.3
Attention has been drawn most recently to Factor VIIa
(FVIIa)4 because of its role as initiator of the cascade in
complex with tissue factor (TF) upon blood vessel
damage. Other important targets are FXa as the ultimate
step in FIIa generation, FIXa and FXIa for their roles in
maintaining the coagulation process after intiation.4
While a major part of the synthetic effort has been
directed at inhibitors of FIIa, little is known about
molecules that may interact with other enzymes in the
cascade such as FVIIa.6 We recently reported the
design, synthesis, and X-ray co-crystal structure with
FIIa of a IC50=20 nM inhibitor, 2.8 The validity of
molecular modeling prior to performing synthesis was
evident when it was predicted that the presence of a
benzyl group as in 2 rather than a methyl (22, FIIa
IC50=270 nM, Table 1, entry 1) was beneficial for the
P3–S3 interaction. Furthermore, a tertiary hydroxyl
group at C-3, and the lactam carbonyl appeared to
provide the requisite interaction with Gly 216.
Molecular modeling9 of the indolizidinone motif in 2
with FVIIa indicated that 2 will bind in a similar con-
formation as observed in the X-ray of 2 and FIIa and
that productive interactions could be had by further
functionalization with specific groups in the P2 region
(Fig. 2, left panel). Comparison of the S2 proximal
pocket in FIIa and FVIIa indicates that whereas a small
cavity exists in the latter, the same volume is partially
occupied by Leu 99 in FIIa.8 Also, the transition
between S2 and S3 is clearer in FVIIa compared to FIIa,
with a canyon that leads to S4, having Thr 99 at the
bottom and Thr 98 and Pro 170I on the sides. As seen in
The first X-ray structure of a TF/FVIIa complex with
d-Phe-Phe-Arg-chloromethyl ketone (PPACK II),5
(Fig. 1), has been instrumental in the design and synth-
esis of prototypical inhibitors.6 Generally, such inhib-
itors incorporate a basic P1 guanidino, amidino, or
amino group which interact with Asp 189 in the S1
pocket. Irreversible inhibitors such as PPACK II, an
electrophilic substrate that mimics the oxyanion hole,
are susceptible to an attack by Ser 195.7 Potency is
gained by the incorporation of complimentary lipophilic
residues at P2 and P3 in inhibitors.
Figure 2(yellow capped sticks), the P -Phe group of
2
PPACK II partly occupies the small S2 cavity in FVIIa.
We argued that suitable substituents in the 7-position of
the indolizidinone could fill this cavity and provide
selectivity over FIIa. Modeling also indicated that a C-5
substituent should have an effect on the location of the
*Corresponding author. Tel.: +1-514-343-6738; fax: +1-514-343-
5728; e-mail: stephen.hanessian@umontreal.ca
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00612-1

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S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2907–2911
Figure 1.
Table 1. Inhibition of FIIa, FVIIa, Plasmin, and FXIa, measured as IC50 (mM) unless notedb
Entry
Compda
Factor IIa
Factor VIIa
Plasmin
—
Factor XIa
1
ref 10
ref 10
ref 10
0.27
>10
20% (Inhib. at 8 mM)
2
3
4
5
6
7
0.020
0.11
1.1
—
78% (Inhib. at 8 mM)
0.5271
35
0.17
4.6
141
11
>133
>133
>133
0.21
0.42
1.6
0.38
37
7.2109
—
—
aR=
bFormate salt.

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2907–2911
2909
Figure 2. Left—Connolly surface map of the X-ray structure of Factor VIIa with PPACK II (yellow)7 and docked inhibitor 2 (purple); right—
proposed inhibitor 4 (purple) with PPACK II (yellow).
indolizidinone in its interaction with FIIa. On the other
hand a C-(5R)-methoxy group nicely fits into the S3–S4
canyon of FVIIa without moving the indolizidinone
ring. We thus chose to synthesize a series of 3-alkyl-
stereoselectively ethylated with ethyl triflate to 8. Imi-
nium ion chemistry11 in conjunction with 2-trimethyl-
silyloxy furan8,12 afforded 9 as the major isomer (Scheme
1). Cleavage of the N-Boc group,13 ring expansion from
lactone to lactam and O-methylation gave 10. We were
now faced with the challenge of introducing C- and N-
branching corresponding to the P3 subsite with high
stereocontrol. The Li lactam enolate prepared with
t-BuLi was first alkylated with 1-bromomethyl cyclo-
hexene to afford 11 as a major isomer. Reformation of a
Li lactam enolate and treatment with trisyl azide14
afforded the anticipated product 12. Desilylation gave
X-ray quality crystalline alcohol 13, thus corroborating
(3S)-amino-(5R)-methoxy-(7R)-ethyl
indolizidinones
with variation in the 3-substituents (Fig. 1), to evaluate
their inhibitory effect on FVIIa in comparison to FIIa,
plasmin, and FXIa. Figure 2(right panel) shows the
superposition of the docked analogue 4 over the co-
crystal structure of PPACK II with FVIIa.
The readily available l-pyroglutamic acid was trans-
formed in four high yielding steps into 710 which was
Scheme 1. (a) LiHMDS, EtOTf, THF, À78 ꢀC, 92%; (b) Dibal-H, toluene, À78 ꢀC; (c) Ac2O, Et3N, DMAP, CH2Cl2, 98% (two steps); (d)
BF3.OEt2, 2-(trimethylsilyloxy)furan, CH2Cl2, À78 ꢀC, 95%; (e) H2 1 atm, 10% Pd/C, EtOAc, 99%; (f) B-bromocatecholborane, CH2Cl2; (g) tolu-
ene, reflux, 71% (two steps); (h) KH, MeI, THF, 80%; (i) t-BuLi, 1-bromomethyl-cyclohexene, THF, À78 ꢀC, 72%; (j) t-BuLi, Trisyl-N3, THF,
À78 ꢀC, 78%; (k) TBAF, THF, 80%; (l) Swern oxidation; (m) t-BuOH, NaClO2, NaH2PO4, H2O, 90% (two steps); (n) N-Cbz-protected 4-amino-
methylbenzamidine, BOP reagent, i-Pr2NEt, CH2Cl2, 50%; (o) H2 1 atm, 10% Pd/C, MeOH, HCl 37% (1 drop), 85%.

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S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2907–2911
the expected stereochemistry resulting from enolate
chemistry. Oxidation and further functional group
manipulation led to 3 as the dihydrochloride.
azide group with propane 1,3-dithiol15 followed by
protection gave the corresponding N-Boc derivative 21
in excellent overall yield from 10. Transformation to the
carboxylic acid and amide formation took place
uneventfully. However cleavage of the N-Boc group
with TFA under normal conditions (0 ꢀC or rt),
caused migration of the exo-methylene group. For-
tunately, cleavage without rearrangement took place
with 98% formic acid to give the intended analogue
5 (Scheme 4).
We deemed it necessary to also prepare the epimeric
amine 6 for comparison. In order to control the stereo-
chemistry at the tertiary carbon atom, we capitalized on
the steric bias offered by intermediate 9 (Scheme 2).
Thus, sequential alkylation and azidation afforded 15
and 16, respectively. Ring expansion to 17, oxidation
and functionalization as described in Scheme 1 afforded
the epimeric amine dihydrochloride 6.
Table 1 lists the IC50 values of inhibition for FIIa,
FVIIa, plasmin and FXIa. The 3-benzyl analogue 4
proved to be a better FVIIa inhibitor than the corre-
sponding cyclohexenylmethyl (3) or 3-methyl propenyl
(5) analogues (Table 1, entries 5, 4, and 6, respectively).
The inverted stereochemistry at C-3 results, as expected
in a loss of activity (Table 1, entries 4 and 7). It is of
interest that the introduction of the C-5 methoxy group
reduces the activity of FIIa while slightly improving the
affinity for FVIIa (Table 1, compare entry 2with entries
3 and 5). We believe this is due to a smooth fit of the C-
5 methoxy in the canyon clearly present in FVIIa in
contrast to FIIa. Eventually this preference for FVIIa
compared to FIIa may be further improved by slightly
larger hydrophobic groups. In contrast, the C-7 ethyl
substituent exhibits a negligible effect on the selectivity
for FVIIa versus FIIa (Table 1, entries 3 and 5). This
may be due to a better hydrophobic interaction of the
C-7 ethyl group in the S2 pocket of FIIa than antici-
pated, which may in part compensate for the displace-
ment of the indolizidinone in the docked model,
compared to the IC50=20 nM FIIa inhibitor 2.8 Selec-
tivity was achieved over plasmin and FXIa with this
In order to compare the effect of a tertiary hydroxyl as in
2, with the corresponding amine, we prepared analogue
4 as shown in Scheme 3, following the same protocol
discussed above for 3. The benzyl and azide groups were
introduced with high stereoselectivity affording 18,
which was converted to the corresponding acid 19 and
further functionalized to the intended 4.
The synthesis of the 3-methylpropene analogue 5 pre-
sented certain operational challenges due to the pre-
sence of the double bond, thus excluding the use of
catalytic reduction and strongly acidic conditions. The
common bicyclic lactam intermediate 10 was converted
to the 3-methylpropenyl azido analogue 20 via Li eno-
late chemistry as described above. Reduction of the
Scheme 2. (a) LiHMDS, 1-bromomethyl-cyclohexene, THF, À78 ꢀC,
74%; (b) t-BuLi, trisyl-N3, THF, À78 ꢀC, 72%; (c) B-bromoca-
techolborane, CH2Cl2; (d) toluene, reflux, 50% (two steps); (e) KH,
THF, Me2SO4 94%; (f) HF.pyr, MeCN, 75%; (g) Dess–Martin,
CH2Cl2; (h) t-BuOH, NaClO2, NaH2PO4, H2O, 70% (two steps); (i)
N-Cbz-protected 4-aminomethylbenzamidine, EDC, HOBt, i-Pr2NEt,
DMF, 55%; (j) H2, 40psi, 10% Pd/C, MeOH, HCl 37% (1 drop),
quant.
Scheme 3. (a) t-BuLi, BnBr, THF, À78 ꢀC, 78%; (b) t-BuLi, Trisyl-
N3, THF, À78 ꢀC, 60%; (c) TBAF, THF, 76%; (d) TPAP, NMO,
CH2Cl2; (e) t-BuOH, NaClO2, NaH2PO4, H2O, 60% (two steps); (f)
N-Cbz-protected 4-aminomethylbenzamidine, EDC, HOBt, i-Pr2NEt,
DMF, 65%; (g) H2, 1 atm, 10% Pd/C, MeOH, HCl 37% (1 drop),
Scheme 4. (a) t-BuLi, 3-bromo-2-methyl-propene, THF, À78 ꢀC, 83%; (b) t-BuLi, Trisyl-N3, THF, À78 ꢀC, 72%; (c) HS(CH2)3SH, Et3N, MeOH;
(d) Boc2O, Et3N, CH2Cl2, 67% (two steps); (e) TBAF, THF, 98%; (f) PDC, wet DMF; (g) N-Boc-protected 4-aminomethylbenzamidine, EDC,
HOBt, i-Pr2NEt, DMF, 61% (two steps); (h) HCO2H, quant.

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2907–2911
2911
class of compounds. Further optimization of the size
and complimentary polarity, with respect to the S2
pocket of FVIIa, and of the C-7 substituent could per-
haps lead to the anticipated selectivity and for better
inhibitory activity against FVIIa.
5. (a) Banner, D. W.; D’Arcy, A.; Chene, C.; Winkler, F. K.;
Guha, A.; Konigsberg, W. H.; Nemerson, Y.; Kirchhofer, D.
Nature 1996, 380, 41. (b) Kirchhofer, D.; Banner, D. W.
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Acknowledgements
Financial assistance through the NSERC Medicinal
Chemistry Chair program is gratefully acknowledged.
We thank Dr. Michel Simard for X-ray structure ana-
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measured by Johanna Deinum, Karolina Akesson, and
Erik Ryberg at the Department of Biochemistry and
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