Biphenylsulfonyl-thiophene-carboxamidine inhibitors of the complement component C1s

Article abstract of DOI:10.1016/j.bmcl.2008.01.064

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein target

Full text of DOI:10.1016/j.bmcl.2008.01.064

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1603–1606
Biphenylsulfonyl-thiophene-carboxamidine inhibitors
of the complement component C1s
Jeremy M. Travins, Farah Ali, Hui Huang, Shelley K. Ballentine, Ehab Khalil,
Heather R. Hufnagel, Wenxi Pan, Joan Gushue, Kristi Leonard, Roger F. Bone,
Richard M. Soll, Renee L. DesJarlais, Carl S. Crysler, Nisha Ninan, Jennifer Kirkpatrick,
Maxwell D. Cummings, Norman Huebert, Christopher J. Molloy, Michael Gaul,
Bruce E. Tomczuk and Nalin L. Subasinghe*
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, PA 19341, USA
Received 9 December 2007; revised 14 January 2008; accepted 16 January 2008
Available online 19 January 2008
Abstract—Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury,
acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein
targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including
anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement ther-
apy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of
a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical path-
way of complement is described.
Ó 2008 Elsevier Ltd. All rights reserved.
The complement cascade is a major component of the
innate immune system in mammals and other vertebrate
species.1 It plays a major role in the destruction of
invading microorganisms and the clearance of immune
complexes. However, unregulated complement activa-
tion leading to acute inflammation and tissue damage
has been implicated in the pathology of many disease
states.2 Activation of the classical pathway has been
implicated in humorally-mediated graft rejection,3 ische-
mia-reperfusion injury (IRI),4 hereditary angioedema
(HAE),5 vascular leak syndrome,6 and acute respiratory
distress syndrome (ARDS).7 C1-esterase inhibitor (C1-
Inh) has been used for many years as a replacement ther-
apy for patients with hereditary angioedema caused by a
deficiency of C1-Inh.8 Although C1 esterase inhibitor
has been the only complement inhibitor approved
for clinical use, a humanized monoclonal antibody
(eculizumab) against terminal complement protein C5
has recently been approved for the treatment of patients
with paroxysmal nocturnal hemoglobinuria (PNH).9
Small-molecule complement inhibitors have yet to enter
the marketplace. Compstatin, a synthetic 13 amino acid
cyclic peptide that blocks all three known complement
activation pathways, is currently undergoing clinical
development for the treatment of age-related macular
degeneration (ARMD).10 We have previously reported
the discovery of a novel series of arylsulfonylthioph-
ene-2-carboxamidine inhibitors11 of the complement
component C1s, a trypsin-like serine protease that is
present as a proenzyme within the first component of
complement in the classical pathway.12 We have contin-
ued to optimize the potency and physicochemical prop-
erties of these compounds in order to identify a
candidate compound suitable for dosing as an intrave-
nous agent for the treatment of acute complement med-
iated diseases such as IRI and ARDS.
O
O
S
MeS
Br
S
HN
NH2
1
Keywords: C1s; Complement; Protease; Inhibitor; Amidine; Pharma-
cokinetic; Suzuki.
Our previous work identified compound 1, which has a
Ki of 0.36 lM for C1s.11 Further substitution at the 3-
position of the phenyl ring provided compound 3 with
*
Corresponding author. Tel.: +1 610 458 6066; fax: +1 610 458
8249; e-mail: NSUBASIN@prdus.jnj.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.064

1604
J. M. Travins et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1603–1606
Table 1. SAR from Suzuki coupling library
a Ki of 0.26 lM. This observation led to more extensive
exploration of the structure–activity relationship (SAR)
at the 3-position of the phenyl ring. Molecular modeling
studies using the published C1s structure13,14 suggested
two possible binding modes for substituents originating
from the 3-position of the phenyl ring A (Fig. 1). To fur-
ther explore the SAR at this position, a small library of
biaryl compounds was synthesized via parallel synthesis
(Table 1).15 Compound 1 was treated with tert-butyl-
oxycarbonyl anhydride in THF to protect the amidine
moiety. The protected amidine 2 (Scheme 1) was cou-
pled to a series of aryl and heteroaryl boronic acids
using Suzuki coupling conditions to give compounds
3–18.16 Compound 19 was prepared by treating com-
pound 1 with 3-methyl-2-pyridylzinc bromide. Replace-
ment of bromo with phenyl to give compound 3
provides only a marginal improvement in activity. Fur-
ther substitution on compound 3 involving the introduc-
tion of small hydrophobic or hydrophilic groups at the
3-position (5, 8, 11) or the 4-position (6, 9, 12) of the dis-
tal aryl ring (ring B) did not have a significant effect on
activity. However, addition of small hydrophobic
groups at the 2-position of ring B generated compounds
with significantly improved potency. For example, as
shown in Table 1, when R1 is 2-methyl (7) or 2-chloro
(14) there is approximately 9- and 7-fold enhancement
in activity, respectively, in comparison to compound 3.
R1
R2
Z
O
O
MeS
B
S
A
S
HN
NH2
Compound
Z
R1
H
R2
Kia (nM)
3
4
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
H
260
610
490
350
30
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-OH
H
5
H
6
H
7
H
8
H
1850
250
460
150
190
120
40
9
H
10
11
12
13
14
15
16
17
18
19
H
3-OH
4-OH
H
H
2-CH2OH
2-Cl
H
H
2-CHCH2
2-CH3
2-CH3
H
140
50
6-CH2OH
6-CO2H
NH2
150
20
2-CH3
2-CH3
—
40
a Ki values were determined as described in Ref. 11. Between-run
coefficient of variation was <24%.
Molecular modeling suggests a preference for binding
conformations in which ring B is orthogonal to the
proximal aryl ring (ring A), and this applies to both pos-
sible binding modes (Fig. 1). The improved potency ob-
served with compounds 7 and 14 is likely due to both a
conformational effect and enhanced binding contacts.
Having a substituent at the ortho position of ring B (4,
7, 10, 13, and 14) can result in the biphenyl dihedral
being closer to the predicted bound conformation than
in the unsubstituted case, 3. In either of the plausible
binding modes the ortho substituent could be directed
toward the protein surface, or away from it and out to-
ward solvent (Fig. 1). Modeling studies also suggested
that a small hydrophobic residue at the 2-position can
O
S
O
O
O
O
S
R3
S
Br
S
a, b
S
S
t-Bu
HN
N
H
2
O
HN
NH2
3-19
Scheme 1. Reagents and conditions: (a) R3B(OH)2, Pd(PPh3)4, 2 M
Na2CO3, EtOH, toluene, 80 °C, 12 h or R3ZnBr, Pd(PPh3)4, THF,
80 °C, 1 h; (b) 50% TFA/DCM.
make a favorable interaction with the protein surface
and this is consistent with the improved potency ob-
served with compounds 7 and 14, compared to the
unsubstituted compound 3. However, larger hydropho-
bic residues would make this an unfavorable binding
conformation due to steric-clash with the protein sur-
face. Introducing larger hydrophobic residues (4 and
15) or hydrophilic residues (10 and 13) at this position
has little effect on activity, fully consistent with a bind-
ing mode in which these substituents can be directed to-
ward the solvent.
Compounds 3–15 did not have sufficient aqueous solu-
bility at physiological pH to be evaluated as potential
intravenous agents. To mitigate the poor aqueous solu-
bility of compound 7, the most potent in the series, a
pyridyl ring was introduced instead of the phenyl ring
to give compound 19. While compound 19 had the re-
quired solubility properties, it had poor pharmacoki-
netic properties, a poor in vitro safety profile,17 and
potent hERG K channel blocking in the patch-clamp as-
say18 (Table 2). In an alternative approach, 7 was
further substituted with hydrophilic residues at the
Figure 1. Binding models of 2 in the active site of C1s. These models
are derived from the crystal structure of an analog of 2 bound to a C1s
homolog (unpublished results), which shows that the thiophene
amidine occupies the S1 subsite. The C1s crystal structure14 is depicted
with a Connolly surface19 (cyan) and the two binding models with
green and magenta carbons, respectively. The figure was prepared with
PyMOL.20

J. M. Travins et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1603–1606
1605
Table 2. Amine substituted derivatives of compound 18: potency, solubility, and in vitro safety
Compound
R5
Z
Kia (nM) Aqueous
solubility (lM)
Activity in SEP (% inhibition at 10 lM)b Inhibition of hERG
(% inhibition at 10 lM)18
19
30
H
N
C
45
22
A = 69; B = 74; C = 95; D = 76; E = 83
A = 2; B = 1; C = 9; D = 11; E = 17
66.0
O
O
4
42
40
39
—
43
35
0.5
5.8
*
N
H
OH
O
H
N
H
N
*
31
32
33
34
C
C
C
C
22
64
12
14
A = 0; B = 15; C = 0; D = 26; E = 5
A = 7; B = 5; C = 9; D = 21; E = 11
A = 23; B = 47; C = 31; D = 51; E = 73
A = 9; B = 28; C = 40; D = 54; E = 53
OH
N
5
O
N
H
N
H
N
NH
*
1.0
N
2
O
O
O
S
O
O
0.9
*
*
N
H
O
O
S
3
18.0
N
H
NovaScreen uses a criteria of 50% inhibition or greater to qualify a compound as active. A = adrenergic, alpha 1, non-selective; B = dopamine
transporter; C = muscarinic, non-selective, peripheral; D = opiate, non-selective; E = sodium, site 2.
a Ki values were determined as described in Ref. 11. Between-run coefficient of variation was <24%.
b Compounds were tested in the NovaScreen General Side Effect Profile (SEP).17
6-position of the phenyl ring B. Since the 6-position ap-
peared to be solvent-exposed this was a reasonable strat-
egy to follow. While introduction of 6-hydroxymethyl
(16) and 6-carboxy (17) groups reduced potency 2- and
6-fold, respectively, 6-amino (18) substitution did not
change potency. The 6-amino group was further substi-
tuted with a series of hydrophilic groups (Table 2) with
the hope of improving solubility as well as the pharma-
cologic side-effect profile.
of base to give 22. The ester 22 was hydrolyzed to the
acid 27, which was deprotected with TFA to give tar-
geted compound 31. Compound 20 was treated with 4-
nitrophenyl chloroformate and pyridine to give the 4-
nitrophenyl carbamate which was reacted with 3-amino-
propionitrile to give the urea 23. Compound 23 was
treated with azidotrimethylsilane and dibutyltin oxide
to give the tetrazole 28, which was converted to the ami-
dine 32 with TFA. Compound 20 was reacted with bro-
moacetylbromide to give the amide 24, which was
treated with sodium methanesulfinate to give the sulfone
29. The sulfone 29 was deprotected with TFA to give the
amidine 33. Compound 20 was treated with 4-methane-
sulfonyl-butyrylchloride to give the sulfone 25, which
was converted to the amidine 34 with TFA.
The tert-butyloxycarbonyl-protected scaffold, 20 was
synthesized according to Scheme 1 and functionalized
according to Scheme 2 to give compounds 30–34.15
Compound 20 was treated with 5-chlorocarbonyl-penta-
noic acid methyl ester in the presence of base to give the
ester 21. The ester 21 was hydrolyzed to give the acid 26
which was deprotected with 50% TFA in DCM to give
the final amidine 30. Compound 20 was treated with
6-isocyanato-hexanoic acid ethyl ester in the presence
Incorporating either the carboxylic acid or the sulfone
moiety (30, 31, 33, and 34) provided compounds with
acceptable aqueous solubility. Compound 19 was tested
in a ‘side-effect’ profile panel of 54 target assays from
NovaScreen.17 The compound showed significant activ-
ity at 5 receptor targets (Table 2). In contrast, com-
pounds 30, 31, and 32 proved to be completely inactive
in these assays. Incorporating sulfone moieties provided
compounds that had some activity in 2 out of 54 assays.
Compounds 30–33 were completely devoid of any hERG
activity, while compound 34 had weak potency.
O
S
O
S
O
O
O
O
S
S
O
NH2
HN
R4
S
S
a, b, c,
d or e
R4
t-Bu
t-Bu
HN
HN
N
H
N
H
O
O
R4
21 -(CH2)4CO2Me
20
f
26
27
-(CH2)4CO2H
-HN(CH2)5CO2H
N
g
-HN(CH2)5CO2Et
22
Compounds 19, 31, and 34 were dosed intravenously in
rat pharmacokinetic studies and proved to have very
high clearances (2- to 4-fold rat hepatic blood flow)
and short half-lives (Table 3).
N
h
-HN(CH2)2CN
28 -HN(CH2)2
23
NH
N
i
-CH2Br
-(CH2)3S(O)2Me
-CH2S(O)2Me
29
24
25
Scheme 2. Reagents and conditions: (a) MeO2C(CH2)4COCl, DIEA;
(b) EtO2C(CH2)5NCO, TEA, DCM, 3 h; (c) i—p-nitrophenyl chloro-
formate, pyridine, DCM, 4 h; ii—NC(CH2)3NH2, DCM, 48 h; (d)
BrCH2COBr, DIEA, CHCl3; (e) CH3(O)2S(CH2)3COCl, DIEA; (f)
LiOH, 60 °C, 12 h; (g) 1 N NaOH, 2 h; (h) (CH3)3SiN3, dibutyltin
oxide, toluene, 70 °C, 16 h, 80 °C 4 h; (i) CH3SO2Na, EtOH, 16 h.
In summary, we have discovered a novel series of potent
and selective C1s inhibitors. These are among the most
potent compounds reported to date. More lipophilic
compounds in this series such as 19 exhibit significant
hERG channel binding activity while more hydrophilic

1606
J. M. Travins et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1603–1606
Table 3. Rat pharmacokinetic properties for selected compoundsa
10. Mulakala, C.; Lambris, John D.; Kaznessis, Y. Bioorg.
Med. Chem. 2007, 15, 1638.
Compound
T1/2 (h)
Vd (L/kg)
Cl (mL/min/kg)
11. Subasinghe, N. L.; Travins, J. M.; Ali, F.; Huang, H. I.;
Ballentine, S. K.; Marugan, J. J.; Khalil, E.; Hufnagel, H.
R.; Bone, R. F.; DesJarlais, R. L.; Crysler, C. S.; Ninan,
N.; Cummings, M. D.; Molloy, C. J.; Tomczuk, B. E.
Bioorg. Med. Chem. Lett. 2006, 16, 2200.
12. Cooper, N. R. Adv. Immunol. 1985, 37, 151; Arlaud, G.
J.; Gaboriaud, C.; Thielens, N. M.; Rossi, V. Biochem.
Soc. Trans. 2002, 30, 1001; Arlaud, G. J.; Gaboriaud,
C.; Thielens, N. M.; Budayova-Spano, M.; Rossi, V.;
Fontecilla-Camps, J. C. Mol. Immunol. 2002, 39, 383;
Gal, P.; Ambrus, G.; Zavodszky, P. Immunobiology
2002, 205, 383.
13. Gaboriaud, C.; Rossi, V.; Bally, I.; Arlaud, G. J.;
Fontecilla-Camps, J. C. EMBO J. 2000, 19, 1755.
14. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.;
Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E.
Nucleic Acids Res. 2000, 28, 235.
19
31
34
2.0
0.2
1.8
15
283.0
228.5
129.6
2.65
2.03
a Three animals were used for each dosing group. The rats were dosed
2 mg/kg intravenous.
compounds such as 30 are devoid of significant hERG
activity. Incorporating hydrophilic residues also im-
proved selectivity against the NovaScreen SEP panel.
However, these compounds have poor in vivo pharma-
cokinetic properties. Further structural modifications
to improve in vivo pharmacokinetic properties are in
progress.
15. For detailed experimentals please see: Subasinghe, N.;
Khalil, E.; Leonard, K.; Ali, F.; Hufnagel, H. R.;
Travins, J. M.; Ballentine, S. K.; Wilson, K. T.;
Cummings, M. D.; Pan, W.; Gushue, J.; Meegalla, S.;
Wall, M.; Chen, J.; Rudolph, M. J.; Huang, H. 3-
Dimensional Pharmaceuticals, Inc., USA. PCT Int. Appl.
WO 2003099805, 2003.
16. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
17. Compounds were tested in the General Side Effect Profile
offered by NovaScreen Biosciences, 7170 Standard Drive,
Hanover, MD 21076-1334, USA.
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