Design and synthesis of polyethylene glycol-modified biphenylsulfonyl- thiophene-carboxamidine inhibitors of the complement component C1s

Article abstract of DOI:10.1016/j.bmcl.2012.06.030

Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, an

Full text of DOI:10.1016/j.bmcl.2012.06.030

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5303–5307
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of polyethylene glycol-modified biphenylsulfonyl-thiop-
hene-carboxamidine inhibitors of the complement component C1s
⇑
Nalin L. Subasinghe , Ehab Khalil, Jeremy M. Travins, Farah Ali, Shelley K. Ballentine, Heather R. Hufnagel,
Wenxi Pan, Kristi Leonard, Roger F. Bone, Richard M. Soll, Carl S. Crysler, Nisha Ninan,
Jennifer Kirkpatrick, Michael X. Kolpak, Karen A. Diloreto, Stephen H. Eisennagel, Norman D. Huebert,
Christopher J. Molloy, Bruce E. Tomczuk, Michael D. Gaul
Janssen Pharmaceutical Research & Development, L.L.C., Welsh & McKean Roads, Spring House, PA 19477, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with
activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-
reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The
utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their
poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with
good potency and good in vivo pharmacokinetic properties.
Received 22 April 2012
Revised 5 June 2012
Accepted 11 June 2012
Available online 16 June 2012
Keywords:
Complement inhibitors
C1s inhibitors
Ó 2012 Elsevier Ltd. All rights reserved.
Pegylation
Pegylated small molecule
Polyethylene glycol-modified
Pharmacokinetics
Polyethylene glycol-modified small
molecule
Pegylated molecule pharmacokinetics
Pegylated molecule PK
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 comple-
ment activation leading to acute inflammation and tissue damage
has been implicated in the pathology of many disease states.2 Only
a handful of therapeutic agents have been approved for the treat-
ment of disease states whose etiology involves uncontrolled acti-
vation of complement. Eculizumab,3 a humanized monoclonal
antibody against the terminal complement protein C5 is currently
being used to treat paroxysmal nocturnal hemoglobinuria (PNH)
and atypical hemolytic uremic syndrome (aHUS), a rare and
chronic blood disease that disproportionately affects children.
Compstatin,4 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).
We have previously reported the discovery of a novel series of
arylsulfonylthiophene-2-carboxamidine inhibitors5 of the comple-
ment component C1s. These inhibitors have potential utility in the
treatment of diseases associated with activation of the classical
complement pathway, such as humorally mediated graft rejec-
tion,6 ischemia-reperfusion injury (IRI),7 vascular leak syndrome,8
and acute respiratory distress syndrome (ARDS).9 Compounds with
good C1s potency and selectivity were identified from this series,
but rapid clearance and poor systemic exposure prevented further
advancement. Renal clearance is a major contributor to the elimi-
nation of many organic cationic molecules. This could also be a sig-
nificant route of elimination for these basic amidines, which are
positively charged at physiological pH.10 Conjugation of organic
molecules to high molecular weight (>20 kDa) polyethylene glycol
(PEG) is known to reduce renal clearance.11 Pegylation was pur-
sued as a potential avenue to reduce clearance and improve sys-
temic exposure of these amidines.
⇑
Corresponding author. Tel.: +1 215 628 7824; fax: +1 215 540 4612.
E-mail address: NSUBASIN@its.jnj.com (N.L. Subasinghe).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.030

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N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5303–5307
Table 1
In vitro potency and in vivo pharmacokinetic parameters of pegylated compounds15
O
S
S
O
Linker
PEG
HN
S
R1
H2N
NH
Compd
Linker
R1
PEG size (kDa)
Kia (nM)
Rat PK
Cl (mL/min/kg)
Vss (L/kg)
t1/2
AUCinf
O
H
N
S
S
5
6
H
H
0.75
10
55
95
Below level of quantitation in <30 min
O
O
O
O
H
N
7.2
0.4
0.09
0.3
0.1
5.9
139
H
S
N
7
H
20
40
20
20
270
365
79
2305
O
O
NH
NH2
NH
NH2
NH
NH2
H
N
8
0.34
0.58
0.17
0.32
5.67
6.5
3110
1735
N
HN
HN
HN
5
H
O
O
H
N
9
N
5
H
O
O
O
10
60
N
10 H
11
12
13
Structure shown below
Structure shown below
Structure shown below
20
20
35
39
29
18
0.64
0.09
1.6
1570
a
Ki values were determined as described in Ref. 16. Between-run coefficient of variation was <24%.
While pegylation of peptides and proteins is a well estab-
B
4
lished strategy to improve in vivo exposure and reduce clear-
ance,12 pegylation of small molecules has been less explored.
Often, high molecular weight PEG-conjugation of small mole-
cules leads to significant reduction in potency. To overcome this,
reversible pegylation strategies have also been explored.13 Here
we describe the pegylation14 of small molecule C1s inhibitors
via stable covalent linkages. The purity of pegylated molecules
was established by HPLC, and loading was determined by 1H
NMR.14
Linkage site: Based on structure–activity relationship (SAR) stud-
ies on the small molecule 1,5 substitution on site A and site B were
considered to be least likely to interfere with receptor binding.
Compounds 4 and 5 were synthesized by treating the correspond-
ing Boc-protected amidine bromoacetamides 2 and 3 with thiol-
O
O
S
O
S
O
S
S
R1
6
S
S
A
HN
HN
NH2
NH2
1
2 R1 = 4-NHCOCH2Br
3 R1 = 6-NHCOCH2Br
Ki = 30 nM
O
4 R1 = 4
NH(CO)CH2S
Ki = 260 nM
N
PEG750
H
O
6
5 R1 = NH(CO)CH2S
N
H
PEG750
Ki = 55 nM
functionalized
750 Da
polyethylene
glycol,
mPEG750-
PEG size: While pegylation was likely to improve pharmacoki-
netic (PK) properties, it was also possible that conjugation of the
small-molecule inhibitor to a large PEG molecule would lead to
some loss of potency. We synthesized several pegylated analogs
(Table 1) with varying PEG sizes to identify the optimal balance be-
tween acceptable pharmacokinetic properties and potency. Com-
NHCO(CH2)2SH (Rapp Polymere, GMBH, Tübingen, Germany) in
the presence of triethyl amine. The Boc groups were removed by
treating with trifluroacetic acid (TFA) in dichloromethane (DCM)
and the product was purified by reverse-phase HPLC. Considering
that the loss of potency for compound 5 was less compared with
compound 4 (ꢀ2-fold vs ꢀ9-fold), site A was chosen as the site
of pegylation for all the other PEG constructs.
pounds
6 and 7 were synthesized in a similar manner to

N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5303–5307
5305
O
O
O
SiMe3
e
O
SiMe3
CO2Me
HN
HN
a,b,c
d
NO2
NH2
NO2
Br
15
Br
Br
17
16
O
O
S
S
S
O
SiMe3
O
O
S
S
HN
O
O
H2N
S
Br
BocHN
O
NH
BocHN
NH2
19
NH
B
Me3Si
NH
O
O
f
20
18
O
O
5CO2H
O
S
5CO2H
O
S
O
S
S
S
O
N
H
O
N
H
HN
HN
i
S
g,h
O
NH2
NH
HN
HN
NHBoc
Me3Si
NHBoc
22
21
O
5CO2H
NH
O
S
S
S
j
O
N
H
HN
BocN
NHBoc
S
k,l
HN
NHBoc
BocN
NHBoc
O
23
O
O
S
S
5
O
NH
PEG
NH
N
H
HN
S
H2N
HN
PEG = mPEG40kDa
NH2
NH
8
9 PEG = mPEG20kDa
Scheme 1. Reagents and conditions: (a) NaOH; (b) DPPA, DIEA, dioxane 90 °C; (c) TMSEtOH; (d) Fe, NH4Cl, EtOH, 85 °C 14 h; (e) Pd(OAc)2, TEA, pinacolborane, dioxane, 80 °C,
1 h; (f) Pd(PPh3)4, EtOH, toluene, Na2CO3, 80 °C, 16 h; (g) OCN(CH2)nCO2Et, DCM, 40 min; (h) LiOH, THF; (i) TBAF, THF, 40 °C, 4 h; (j) MeOH, AcOH, 40 °C, 2 h; (k) mPEG-NH2,
DIC, DMAP, 6 h; (l) TFA, DCM.
compound 5, using mPEG10k-NHCO(CH2)2SH and mPEG20k
-
NHCO(CH2)2SH, respectively. Compound was synthesized
8
NH2
NO2
O
O
S
O
S
O
according to Scheme 1. Ester 15 was hydrolyzed and converted
to the urethane 16 via a Curtius rearrangement. Compound 16
was reduced and converted to the pinacol boronate 18, which
was coupled with aryl bromide 195 to give the biaryl amidine 20.
Amidine 20 was treated with 6-isocyanato-hexanoic acid ethyl es-
ter, followed by hydrolysis to give compound 21. The Teoc group
was removed and the aniline was converted to the protected gua-
nidine 23. PEG amine was coupled to the activated acid and the Boc
groups were removed by treating with TFA in DCM to give the
pegylated compound 8.
S
S
a,b,c
S
O
S
HN
HO
NH2
HN
HN
10
NH
Boc
NH
Boc
25
24
O
Scheme 2. Reagents and conditions: (a) TEA, ClOC(CH2)10CO2Et; (b) Fe, EtOH,
NH4Cl, 75 °C, 1 h; (c) NaOH, MeOH.
The 750 Da pegylated compound 5 was cleared very rapidly,
reaching levels below quantitation within 30 min following infu-
sion. While increasing the PEG size to 10 kDa (6) did not provide
an improvement in pharmacokinetic properties, the 20 kDa pegy-
lated compound 7 demonstrated a favorable pharmacokinetic pro-
file with reduced clearance and an excellent t1/2. Increasing the PEG
size to 40 kDa (8) did not provide a significant enhancement in
pharmacokinetic properties over compound 7.
Linker length: There was a concomitant loss in potency with in-
crease in PEG size, as exemplified by compounds 5–7.
Steric bulk of the PEG molecule interfering with receptor bind-
ing was thought to be one reason for this loss of potency. An at-
tempt was made to install linkers of varying lengths between the
inhibitor and the PEG molecule to reduce steric crowding (Table
1, compounds 9–10). Compound 9 was synthesized according to

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N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5303–5307
H
H
N
NH
NH2
N
NH
NH2
H
N
O
S
O
O
S
S
H2N
O
S
O
S
O
S
NH
S
S
HN
O
HN
O
S
H
N
NH
NH
HN
HN
HN
HN
NH2
NH2
O
NH
NH
O
5
O
O
H2N
H2N
n
n
O
O
NH
O
O
O
O
n
O
O
NH
NH2
O
O
N
H
PEG20kDa
H
N
HN
n
O
S
O
NH2
n
S
5
HN
HN
HN
S
O
S
O
HN
NH
O
NH2
NH
S
O
HN
S
S
H2N
N
H
S
NH2
S
O
O
N
NH
H
12
11
Tetravalent (4 Arm) PEG20kDa
HN
H
N
H
NH
NH2
N
O
O
S
O
S
O
S
S
S
NH
O
O
H
N
H
S
HN
N
NH
NH
HN
O
O
HN
NH
NH2
H2N
H
N
H
N
O
NH2
H2N
O
O
HN
O
HN
NH
O
O
NH
O
N
H
O
HN
O
N
H
NH2
NH
S
S
NH2
S
O
S
S
O
PEG35kDa
S
O
N
NH
HN
N
H
H
13
Scheme 1 in a manner similar to compound 8, using a 20 kDa PEG-
amine. In order to synthesize compound 10, a 10 methylene linker
was attached to the aniline 24 according to Scheme 2, and the acid
25 was treated in a manner similar to compound 22 (Scheme 1).
Increasing the linker length from 5 (9) to 10 (10) methylenes
provided only a marginal increase in potency. It is quite possible
that these hydrophobic methylene linkers do not exist exclusively
in an extended conformation in an aqueous environment, instead
preferring a collapsed globular conformation as the linker length
is increased.17 Exploring linkers that would be in a solvated ex-
tended conformation in an aqueous environment, it became evi-
dent that the PEG-chain itself could serve this purpose.18
Multivalent PEGs: The expected route of delivery for these cova-
lently pegylated compounds is by intravenous infusion. Attaching
multiple small molecules to a single PEG was an approach that
was pursued to reduce the dosage size and volume. Although there
are several examples19 of two pharmacologically active small mol-
ecules linked by short PEG linkers demonstrating synergistic
enhancement in potency, our constructs were not designed to ex-
plore this possibility. Compounds 11 and 12 were synthesized
according to Scheme 1, using a 20 kDa bis-aminoethyl PEG and a
20 kDa tetravalent amine PEG. Bis-Boc–protected lysine succini-
mide ester was coupled to both ends of a 35 kDa bivalent amine
PEG, and the Boc groups were removed to provide a tetravalent
amine PEG, which was treated according to Scheme 1 to give com-
pound 13. Compounds 11 and 13 containing 2 and 4 active moie-
appears to be a function of the volume of distribution (Vss). Com-
pound 11 has a significantly reduced Vss compared with compound
9 (0.09 vs 0.32). For both compounds, volume of distribution is
smaller than the combined plasma and extracellular fluid vol-
umes,20 suggesting limited intracellular and tissue distribution.
With a significantly reduced Vss, compound 11 would appear to
be distributed mostly within the central compartment, which
would facilitate elimination and lead to a reduced t1/2
.
In summary, we have explored a pegylation strategy to improve
the pharmacokinetic properties of a potent C1s inhibitor. This work
has led to the identification of a compound (9) that has good po-
tency and acceptable pharmacokinetic properties for parenteral
dosing.
References and notes
1. (a) Walport, M. J. N. Engl. J. Med. 2001, 1058, 344; (b) Walport, M. J. N. Engl. J.
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W.; Gushue, J.; Leonard, K.; Bone, R. F.; Soll, R. M.; DesJarlais, R. L.; Crysler, C. S.;
Ninan, N.; Kirkpatrick, J.; Cummings, M. D.; Huebert, N.; Molloy, C. J.; Gaul, M.;
Tomczuk, B. E.; Subasinghe, N. L. Bioorg. Med. Chem. 2008, 18, 1603.
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8. Lissoni, P.; Barni, S.; Cattaneo, G.; Archili, C.; Crispino, S.; Tancini, G.; D’Angelo,
L.; Magni, S.; Fiorelli, G. Int. J. Biol. Markers 1990, 5, 195.
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10. (a) Kamenecka, T. M.; Park, Y.; Lin, L. S.; de Laszlo, S.; McCauley, E. D.; Van
Riper, G.; Egger, L.; Kidambi, U.; Mumford, R. A.; Tong, S.; Tang, W.; Colletti, A.;
Teffera, Y.; Stearns, R.; MacCoss, M.; Schmidt, J. A.; Hagmann, W. K. Bioorg. Med.
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ties
per
molecule,
respectively,
demonstrate
additive
enhancement in potency by being two and fourfold more potent
than the comparable monomeric compound (9). Bifunctionalized
analog 11 has low clearance but significantly reduced t1/2 in com-
parison with the monofunctionalized analog 9. Considering that
clearance is similar for compound 9 and 11, the difference in t1/2
11. Fishburn, C. S. J. Pharm. Sci. 2008, 97, 1520.

N. L. Subasinghe et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5303–5307
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12. Greenwald, R. B.; Choe, Y. H.; McGuire, J.; Conover, C. D. Adv. Drug Delivery Rev.
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precipitation. A portion of the material was subjected to HPLC purification (C18
column, 10–55% CH3CN in H2O (0.1% TFA) over 30 min). The two batches of
PEG (HPLC purified vs precipitation only) were identical by 1H NMR analysis
and analytical HPLC showed <0.5% difference in purity between the two. The
purity of the precipitated PEGylated compound was determined to be >99.2%
13. Yonit, M.; Keren, S.; Mati, F.; Yoram, S. J. Med. Chem. 2008, 51, 4300.
14. (a) For detailed experimentals on multiple PEG constructs, please see:
Subasinghe, N.; Khalil, E.; Ali, F.; Hufnagel, H. Rae; Ballentine, S.; Travins, J.
M.; Leonard, K. A.; Bone, R. F. US7482376 B2 A61K 20090127.
(b) Representative example of pegylation: Synthesis of compound 9.
Diisopropylcarbodiimide (0.2 M in DCM, 1.125 mL) was added dropwise to a
(HPLC, Rf = 6.18 min, C18, 5l, k 215 nm, gradiant 5–100% B in 8 min. A = 0.1%
TFA/H2O; B = 0.1% TFA/CH3CN; 1.2 ml/min). 1H NMR (CDCl3/CD3OD) (with PEG
suppression at d 3.62): d 8.24 (s, 1H), 8.03 (ddd, 1H, J = 1.2, 1.9, 7.9 Hz), 7.82 (t,
1H, J = 1.2 Hz), 7.69 (t, 1H, J = 7.7 Hz), 7.58 (d, 1H, J = 1.9 Hz) 7.49 (dt, 1H, J = 1.3,
7.9 Hz), 6.88 (d, 1H, J = 1.9 Hz), 4.22 (s, DOH), 3.81 (m, PEG satellite), 3.62 (m,
PEG methylenes), 3.45 (m, PEG satellite), 3.36 (s, 3H, PEG-OMe terminus), 3.03
(t, 1H, J = 6.7 Hz) 2.67 (s, 3H), 2.15 (t, 2H, J = 7.4 Hz), 1.97 (s, 3H), 1.55 (m, 2H),
1.37 (m, 2H), 1.22 (m, 2H). Small molecule loading on PEG was determined by
using the relative 1H NMR integrals of the PEG-OMe and compound SMe peaks.
15. PK analysis was done using HPLC with evaporative light scattering detection
(ELSD). Quinidine was used as the internal standard.
solution
of
6-(3-{3’-[5-(tert-Butoxycarbonylamino-imino-methyl)-2-
methylsulfanyl-thiophene-3-sulfonyl]-4-(N,N0-bis-tert-butoxycarbonyl)-
guanidino-6-methyl-biphenyl-2-yl}-ureido)-hexanoic acid (Compound 23,
210 mg, 0.23 mmol) and N,N-dimethylaminopyridine (32 mg, 0.26 mmol) in
DCM (23 mL) at 0 °C. The solution was stirred for 30 min at 0 °C and mPEG20K-
NH2 (3.25 g, 0.15 mmol) was added. The solution was warmed to room
temperature and was stirred for 6 h (ninhydrin negative on TLC). DCM (10 mL)
was added followed by the slow addition of Et2O to induce a slow precipitation.
The solid was collected via filtration and washed with DCM/Et2O to yield
ꢀ3.3 g of crude PEGylated compound. The solid mass was redissolved in 20%
MeOH in DCM and Et2O was added slowly to induce precipitation. The solid
was collected via filtration and the solid cake was dried under high vacuum.
Analysis of this material by HPLC showed purity ꢀ98%, and was devoid of
small-molecule starting material impurities (according to HPLC analysis of the
filtrate). The dried PEGylated compound was redissolved in DCM (8 mL) and
H2O (0.1 mL) was added followed by TFA (8 mL). After stirring for 1.5 h MeOH
was added (4 mL) followed by slow addition of Et2O to induce gradual
16. 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,
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