Design and synthesis of macrocyclic indoles targeting blood coagulation cascade Factor XIa

Article abstract of DOI:10.1016/j.bmcl.2010.09.141

The synthesis of a series of novel macrocyclic compounds designed to target blood coagulation Factor XIa is described. The compounds were evaluated for their inhibition of a small set of serine proteases. Several compounds displayed modest activity and go

Full text of DOI:10.1016/j.bmcl.2010.09.141

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6925–6928
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of macrocyclic indoles targeting blood coagulation cascade
Factor XIa
Stephen Hanessian a, , Andreas Larsson a,c, Tomas Fex b, Wolfgang Knecht b, Niklas Blomberg b
⇑
a Department of Chemistry, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal, QC, Canada H3C 3J7
b AstraZeneca R&D Mölndal, Medicinal Chemistry, Mölndal, Sweden
c Department of Chemistry, Umeå University, 901 87 Umeå, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of novel macrocyclic compounds designed to target blood coagulation Factor XIa
is described. The compounds were evaluated for their inhibition of a small set of serine proteases. Several
compounds displayed modest activity and good selectivity for Factor XIa. Within the series, a promising
lead structure for developing novel macrocyclic inhibitors of thrombin was identified.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 August 2010
Revised 27 September 2010
Accepted 28 September 2010
Available online 8 October 2010
Keywords:
Enzyme inhibitor
Thrombin
Macrocycle
Despite a widespread drug discovery effort, thromboembolic
disease remains one of the major causes of morbidity and mortality
in the world.1 The primary strategy for its treatment and preven-
tion is largely based on anticoagulant therapy. The only oral anti-
coagulant in clinical use is Warfarin, a highly efficacious vitamin
K antagonist that regrettably suffers from response variation and
a need for clinical monitoring.2 Alternative treatments include var-
ious heparin analogs, and the direct thrombin inhibitor hirudin
which besides the disadvantages of intravenous administration
also displays problems with dosage and severe side-effects.3 While
extensive resources have been expended in the search for safer
agents primarily for entities inhibiting one of the blood Factors
IIa, VIIa and Xa,4 inhibition of the blood Factor XIa has received
much less attention.
Factor XIa is a trypsin-like serine protease involved in the
intrinsic pathway of the blood coagulation cascade.5 Its role as a
major contributor in the amplification of the thrombotic response
without affecting the initiation of clot formation has raised the
hypothesis that specific inhibitors of Factor XIa may be beneficial
for having an effective treatment of thrombosis without a signifi-
cant risk of bleeding.6
rigid structures can potentially exhibit a better pharmacokinetic
profile, such as increased metabolic stability and better membrane
permeability.7a,b
Nature has also provided the aeruginosin chlorodysinosin A (3
in Fig. 1),8 as well as macrocyclic peptides like micropeptin 103
(1)9a and the cyclotheonamides 2, a family of metabolites found
in marine sponges of the genus Theonella, which exhibit nanomolar
inhibition of Factor IIa.9b,c Known synthetic macrocyclic inhibitors
include derivatives of the cyclotheonamides10 and the highly
potent pyrazinone 4 and proline-based 5.11
Small molecule inhibitors of Factor XIa are scarcely found in the
literature, and the ones that are reported sometimes suffer from
weak potency and/or lack of selectivity vis-à-vis other serine pro-
teases.12 The known serine protease inhibitor 613 was modeled
into the active site of Factor XIa using FRED (OpenEye Scientific
Software).14
The model revealed a ligand orientation having an extensive
hydrogen bond network between the three amide motifs and the
protein backbone (Ser214, Gly216 and Gly218), with the leucine
and cyclohexyl glycine side-chains occupying S2 and S3, respec-
tively (Fig. 2). A relatively short distance between the methylene
adjacent to the sulphonamide, and the benzamidine moiety could
be observed, thereby allowing the incorporation of a bridge. In
light of this observation, we hypothesized that the introduction
of a four-carbon bridge would lead to a macrocycle that incorpo-
rates the structural features of 6, thereby creating a new class of
potentially selective Factor XIa inhibitors. To the best of our knowl-
edge, no macrocyclic inhibitors of Factor XIa are known to date. To
further enhance the selectivity and the DMPK properties it was
decided to exchange the benzamidine to a 5-chloroindole (Fig. 3),
Macrocyclic compounds are abundant in nature, many of which
have high affinity for enzyme receptors.7 Macrocyclic structures
are often superior to their linear equivalents in terms of enhanced
potency due to conformational preorganization that lowers the
entropy of the molecular recognition event. In addition, the more
⇑
Corresponding author.
E-mail address: stephen.hanessian@umontreal.ca (S. Hanessian).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.141

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S. Hanessian et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6925–6928
NH2
O
O
O
1
Micropeptin 103 ( )
O
O
O
IC50(Thrombin): 8.7 µM
N
H
N
H
O
O
NH
HN
HN
NH
NH
N
H
HO
NH
S
O
O
O
H
H
OH
N
O
N
N
N
Cl
H
O
O
O
O
N
H
7
N
6
NH
O
HN
NH2
OH
O
O
H
HO
HO
O
HN
O
O
H2N
HN
N
NH
O
Cl
N
H
O
Cl
OH
O
O
H
O
O
NH
NH
NHCHO
X
NH2
NH2
H
N
NH
8 X = SO2, R = H
9 X = CO, R = H
10 X = CO, R = Ph
N
N
N
Boc
R
11
OSO3
O
OMe
Cyclotheonamide A (2)
IC50(Thrombin): 1.5 nM
Figure 3. Retrosynthetic analysis.
HN
3
Chlorodysinosin A ( )
IC50(Thrombin): 5.7 nM
H2N
NH
O
we deemed it necessary to alter the N-Ts protection. Thus, elabora-
tion of protecting groups as described in Scheme 1 followed by
azidation using diphenyl phosphoryl azide and DBU according to
Thompson’s procedure17 afforded the azide 18 in 70% yield over
five-steps. A Staudinger reaction completed the synthesis of the
indole building block 11. The resulting free amine was highly
unstable in neat conditions and was used as a crude compound
in the following coupling reactions.
Ph
Cl
O
N
H
N
H
NH
N
N
O
N
H
H
N
O
O
O
N
HN
O
N
Pyrazinone-based DTI (4)
Ki (Thrombin): 0.09 nM
H
5
Proline-based DTI ( )
Ki (Thrombin): 1.3 nM
Cl
Cl
N-Boc-D-cyclohexylglycine (19) was converted to the dipeptide
20, the amine was sulfonylated with but-3-en-1-sulfonyl chlo-
ride18 and the ester hydrolyzed to afford 8 (Scheme 2). The initial
couplings between the indole 11 and the dipeptide 8 showed a sus-
ceptibility to epimerization of the leucine alpha carbon when using
benzotriazole-based coupling reagents (HOBt, HOAt, HATU) in
combination with carbodiimidies (DIC, EDC). This could fortu-
nately be diminished by using Goodman’s phosponium based
benzotriazinone reagent, DEPBT,19 which gave 21 as a single
diastereomer in good yield. Ring-closing metathesis was carried
out using Grubbs’ second generation catalyst and proceeded
smoothly upon heating in dimethoxyethane to give macrocycle
23 in a 9:1 E/Z isomeric ratio.
Figure 1. Natural macrocyclic, linear peptidic, and non-natural macrocyclic
thrombin inhibitors.
R
Ts
N
N
b, c
d, e
Cl
Cl
12 R = H
13 R = Ts
14
a
Figure 2. (a) Model of compound 6 in complex with Factor XIa. (b) Compound 6.
The hydrogen bond network to the Factor XIa backbone is shown (dotted arrows).
R1
N
Boc
N
i, j
since Factor XIa is known to tolerate neutral P1 motifs.12f A small
library was designed to also investigate the role and importance
of the sulphonamide and the aryl moiety protruding towards a cav-
ity formed by Tyr143, Leu146, Arg147, and Lys192. It was antici-
pated that a viable route to the desired structures could involve
an amide coupling between building blocks 8–10 and the indole
11, and subsequent macrocyclization by ring-closing metathesis.
Readily available N-tosyl 5-chloroindole15 was halogenated and
transformed to 14 via metalation, trans-metalation and allylation
of the resulting 2-iodo-5-chloroindole.16 Sequential formylation
and reduction furnished 15 in excellent yields. In order to provide
a traceless and straightforward deprotection of the final product,
Cl
Cl
OR2
R
15 R1 = Ts, R2 = H
= Ts, R2 = TBS
17 R1 = Boc, R2 = TBS
18 R = N3
f
k
1
16
11 R = NH2
R
g, h
Scheme 1. Synthesis of indole building block. Reagents and conditons: (a) NaOH,
TsCl, n-Bu4NHSO4, CH2Cl2, quant.; (b) t-BuLi, Et2O, À78 °C, then I2, 79%; (c) i-PrMgBr,
CuCN–2LiCl (cat.), THF, À30 °C, then allylBr, 90%; (d) TiCl4, Cl2CHOCH3, CH2Cl2,
À78 °C, 98%; (e) NaBH4, EtOH/CH2Cl2, quant.; (f) TBS–OTf, 2,6-lutidine, CH2Cl2, 97%;
(g) Mg, MeOH, 0 °C; (h) (Boc)2O, DMAP, CH2Cl2, 83% (two-steps); (i) TBAF, THF, 96%;
(j) DBU, DPPA, THF, 91%; (k) PPh3, H2O, THF, quant.

S. Hanessian et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6925–6928
6927
R1
O
O
O
H
N
H
N
a, b
c, d
a, b
c
Boc
OH
N
H
H2N
HCl
OMe
N
H
OH
CO2H
O
O
R1
O
26 R1 = H
27 R1 = Ph
9 R1 = H
10 R1 = Ph
19
20
O
O
O
Cl
H
e
S
N
O
O
N
H
OH
H
N
e
O
N
H
N
H
8
R1
O
N
R2
Cl
O
O
O
1
2
1 = Ph, R2 = Boc
29
R
H
N
g
28
R = H, R = Boc,
S
d
N
H
N
H
30 R1 = H, R2 = H, 31 R1 = Ph, R2 =H
O
N
R
O
O
21 R = Boc
f
O
O
22
R = H
N
N
H
g
H
O
NH
HN
O
NH
HN
O
O
R1
R1
O
O
Cl
Cl
N
H
N
H
N
HN
R2
32 R1 = H, R2 = Boc
i
O
O
O
O
NH
HN
NH
HN
S
S
36 R1 = H
37 R1 = Ph
1 = Ph, R2 = Boc
33
R
Cl
Cl
f
N
HN
34 R1 = H, R2 = H
R
35 R1 = Ph, R2 =H
23 R = Boc
24
25
h
Scheme 3. Synthesis of amide analogs. Reagents and conditons: (a) Compound 20,
DEPBT, i-Pr2NEt, THF; (b) 0.1 M LiOH aq, THF; (c) compound 11, DEPBT, i-Pr2NEt,
THF; (d) t-BuNH2, MeOH, microwave 130 °C; (e) Grubbs 2nd gen cat., DME, reflux;
(f) t-BuNH2, MeOH, microwave 130 °C; (g) H2 1 atm, 3% PtO2, MeOH, quant.
R = H
Scheme 2. Synthesis of macrocyclic sulfones. Reagents and conditons: (a) H2N–
Leu–OMe, HOAt, EDC, i-Pr2NEt, CH2Cl2, 74%; (b) 30% v/v TFA/CH2Cl2, quant.; (c) but-
3-en-1-sulfonyl chloride, Et3N, DMAP, CH2Cl2, 68%; (d) 0.1 M LiOH aq, THF, quant.;
(e) compound 11, DEPBT, i-Pr2NEt, THF, 78%; (f) t-BuNH2, MeOH, microwave 130 °C,
90%; (g) Grubbs 2nd gen cat., DME, reflux, 90%; (h) t-BuNH2, MeOH, microwave
130 °C, 69%; (i) H2 1 atm, 3% PtO2, MeOH, quant.
macrocycles 36 and 37, giving Kic values between 50 and
380
to compounds showing low or no activity against FXIa, such as the
sulphonamide 24 and the amide 36 (Kic = 10.6 and 1.7 M, respec-
lM. Activity against thrombin was slightly better and limited
l
Unexpectedly, attempted removal of the Boc-protection using
acidic conditions resulted in decomposition. Various aqueous bases
and neutral conditions known for indole carbamate deprotection
also gave unsatisfactory results. Gratifyingly, Boc-cleavage took
place using t-butyl amine in MeOH20 heated to 130 °C under
microwave conditions to give the free indole macrocycle 24. Using
palladium catalysts for reduction of the double bond resulted in
extensive dehalogenation of the indole moiety. In contrast,
catalytic PtO2 furnished the final saturated macrocycle 25
quantitatively.
tively). The selectivity against activated protein C (APC, a down
regulator of the coagulation cascade) and trypsin was also investi-
gated. Interestingly, the sulphonamide series (22, (E)-24, and 25)
showed no activity against APC. The acyclic amide 30 displayed
modest to low activity against Factor XIa, but with a four-fold
selectivity against APC. Unfortunately, trypsin activity (Kic between
20 nM and 1.2 lM) was observed for these compounds.
Although the macrocyclic compounds were designed to target
Factor XIa, promising results for thrombin inhibition were ob-
served. Compound 36 had a low micromolar activity against
thrombin with a 300-fold selectivity against APC. A docking
study23 of 36 revealed a high similarity as compared to the crystal
structure of the proline-based macrocycle 5 in thrombin prepared
by Nantermet et al.11 (Fig. 4).The excellent selectivity of 5 against
The synthesis of the two macrocyclic amide analogs was accom-
plished essentially as described for 25, utilizing the commercially
available 4-pentenoic acid 26 and (S)-2-phenyl-4-pentenoic acid
27,21 respectively as coupling partners (Scheme 3).
Coupling of 26 and 27 mediated by DEPBT, afforded 9 and 10,
respectively. Further coupling with 5-chloroindole 11, led to bis-al-
kenes 28 and 29. Finally, ring-closing metathesis (giving predomi-
nantly the E isomer, 95:5–99:1), deprotection and hydrogenation
gave macrocyclic bis-amides 36 and 37.
The linear and macrocyclic compounds 22, (E)-24, 25, 30, 31,
(E)-35, 36, and 37, were tested for their inhibitory activity on a
small panel of serine proteases including FXIa, FIXa, and FIIa
(Table 1).22
thrombin versus trypsin (Ki 1.3 nM and 21.5 lM, respectively)
was attributed in part, to the presence of heteroatoms in the tether
between the P1 motif and cyclohexyl glycine residue.11b
In conclusion, we have described the design and synthesis of a
series of macrocyclic compounds intended to display activity
against Factor XIa. This goal was only partially achieved with ana-
log 25 (Table 1). On the other hand, macrocyclic chloroindole 36
displayed a promising activity against thrombin and good selectiv-
ity against APC. The lack of selectivity versus trypsin can be ad-
dressed by an introduction of heteroatoms in the alkane bridge
as for 5. Both compounds represent good starting points for further
As anticipated, the presence of a 5-chloroindole as a P1 motif
was tolerated by FXIa, with the exception of the saturated

6928
S. Hanessian et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6925–6928
Table 1
Inhibition of FXIa, FIXa, FIIa, APC, and trypsin measured as IC50 (lM) and Kic (l
M) for compounds 22, (E)-24, 25, 30, 31, (E)-35, 36, and 37a
b
c
b
c
b
c
b
c
Entry
FXIa IC50
FXIa Kic
FIIa IC50
FIIa Kic
APC IC50
APC Kic
Trypsin human IC50
Trypsin human Kic
22
24
25
30
31
35
36
37
n.d.
n.d.
n.d.
67.9
n.d.
n.d.
n.d.
n.d.
10.6
n.d.
n.d.
10.6
n.d.
n.d.
n.d.
n.d.
1.7
n.d.
n.d.
n.d.
284.1
103.5
144.5
395.3
n.d.
n.d.
n.d.
n.d.
227.3
82.8
115.6
316.2
n.d.
1.16
2.69
2.43
2.17
0.16
0.26
10.0
2.31
0.142
0.328
0.296
0.264
0.020
0.031
1.220
0.281
767.8
175.9
103.1
145.9
135.8
n.d.
383.9
87.9
51.6
73.0
67.9
n.d.
n.d.
n.d.
n.d.
a
b
c
Compounds were tested in a concentration range from 300
l
M to 0.015 lM. IC50 and Kic values are reported as the average of three, four experiments SD.
IC50 values were determined by non-linear fit (using Sigma Plot 8) to all data from the concentration curve.
For better comparisons between the different proteases, the IC50 values were converted into Kic using the equation: Kic = IC50/(1 + [S]/Km) with the respective substrate
M).
concentration and Km values for each protease and assay. (n.d. = not determined, no inhibition detected at 300
l
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refinements towards macrocyclic compounds with higher activity
and selectivity.
14.
A
conformational ensemble generated in MOE (Molecular Operating
Environment, CCG, Montréal, Canada) using stochastic conformational
a
search (default settings) was docked in the active site of Factor XIa (PDB
code: 1ZSL) using the rigid docking program FRED (OpenEye Scientific
Software, New Mexico, USA).
Acknowledgments
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P. J. Org. Chem. 2000, 65, 4618; (b) Hanessian, S.; Giroux, S.; Larsson, A. Org. Lett.
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We thank NSERC (Canada) and AstraZeneca for financial
support and the Wenner-Gren foundation (Sweden) for financial
assistance to A.L.
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J. J. J. Org. Chem. 1993, 58, 5886.
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required. No inhibition of FIXa could be detected at 300
l
M.
23.
A
conformational ensemble generated in MOE (Molecular Operating
Environment, CCG, Montréal, Canada) using
a stochastic conformational
search (default settings except 60 kJ energy cutoff, 0.9 RMS tolerance, and
amide bond rotation allowed), was docked in the active site of thrombin (PDB
code: 1NT1) using the rigid docking program FRED (OpenEye Scientific
Software, New Mexico, USA).