Discovery and structural modification of 1-phenyl-3-(1-phenylethyl)urea derivatives as inhibitors of complement

Article abstract of DOI:10.1021/ml300005w

A series of 1-phenyl-3-(1-phenylethyl)urea derivatives were identified as novel and potent complement inhibitors through structural modification of the original compound from high-throughput screening. Various analogues (7 and 13-15) were synthesized and identified as complement inhibitors, with the introduction of a five- or six-carbon chain (7c, 7d, 7k, 7l, and 7o) greatly improving their activity. Optimized compound 7l has an excellent inhibition activity with IC₅₀ values as low as 13 nM. We demonstrated that the compound 7l inhibited C9 deposition through the classical, the lectin, and the alternative pathways but had no influence on C3 and C4 depositions.

Full text of DOI:10.1021/ml300005w

Letter
pubs.acs.org/acsmedchemlett
Discovery and Structural Modification of 1-Phenyl-3-
(1-phenylethyl)urea Derivatives as Inhibitors of Complement
Mei Zhang,†,‡,⊥ Xiao-Ying Yang,§,⊥ Wei Tang,§,⊥ Tom W. L. Groeneveld,∥,⊥ Pei-Lan He,§
Feng-Hua Zhu,§ Jia Li,‡ Wei Lu,† Anna M. Blom,*,∥ Jian-Ping Zuo,*,§ and Fa-Jun Nan*,‡
†Department of Chemistry and Institute of Medicinal Chemistry, East China Normal University, 3663 North Zhong Shan Road,
Shanghai 200062, People's Republic of China
‡Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China
§Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People's Republic of China
∥Department of Laboratory Medicine, Section of Medical Protein Chemistry, Lund University, S-205 02 Malmo, Sweden
̈
S
* Supporting Information
ABSTRACT: A series of 1-phenyl-3-(1-phenylethyl)urea derivatives were identified as
novel and potent complement inhibitors through structural modification of the original
compound from high-throughput screening. Various analogues (7 and 13−15) were
synthesized and identified as complement inhibitors, with the introduction of a five- or
six-carbon chain (7c, 7d, 7k, 7l, and 7o) greatly improving their activity. Optimized
compound 7l has an excellent inhibition activity with IC50 values as low as 13 nM. We
demonstrated that the compound 7l inhibited C9 deposition through the classical, the
lectin, and the alternative pathways but had no influence on C3 and C4 depositions.
KEYWORDS: high-throughput screening, structural modification, complement inhibitors, C9
he complement system plays an important role in protect-
ing the body against the intrusion of cellular and viral
potency, and short half-life. There is an urgent need for the
development of organic small molecule complement inhibitors
for effectively treating complement-related diseases. In this paper,
we report 1-phenyl-3-(1-phenylethyl) urea derivatives as novel
and potent inhibitors of the complement system through struc-
tural modification based on screening hit 1 (Figure 1).
T
pathogens, as well as in inflammation.1 There are at least 25
complement proteins, which are a complex collection of plasma
proteins and membrane cofactors. Complement is activated by
three pathways: the classical pathway, the alternative pathway,
and the lectin pathway. Under physiological conditions, com-
plement activation is regulated by a series of membrane-bound
and soluble complement inhibitors, involved in protecting the
body against excessive complement activation. Accumulating
data suggest that inappropriate activation of complement is
involved in the pathogenesis of many inflammatory, auto-
immune, neurodegenerative, and infectious diseases.2,3 Moreover,
with the development of improved animal models and genome-
wide association studies, it has become apparent that some
common and often untreatable diseases such as age-related
macular degeneration are largely due to mutations in the com-
plement system. The successful use of Soliris (Eculizumab), a
monoclonal antibody specific for C5, and other complement
inhibitors indicates that targeting complement is a promising
strategy in many clinical situations.4,5
Figure 1. High-throughput screening hit 1.
Through high-throughput screening (HTS) in the fetal com-
plement hemolytic assay, 1-(3,4-dimethoxyphenyl)-3-(1-
phenylethyl)urea (hit 1) was discovered as a hit compound that
showed a moderate complement inhibition activity, with a half-
maximal inhibitory concentration (IC50) value of 50 μM for the
hemolytic assay. This prompted us to start a chemistry program
to further explore this class of compounds. In this communication,
In recent years, several organic small molecule inhibitors of
complement have been discovered.6−8 In contrast to protein
inhibitors, low molecular weight drugs are cheap and have oral
bioavailability and pharmacokinetics advantages. However,
most of these inhibitors have proved to be plagued by a
variety of problems, including poor selectivity, high toxicity, low
Received: January 5, 2012
Accepted: February 26, 2012
Published: February 27, 2012
© 2012 American Chemical Society
317
dx.doi.org/10.1021/ml300005w | ACS Med. Chem. Lett. 2012, 3, 317−321

ACS Medicinal Chemistry Letters
Letter
we would like to report a series of potent complement inhibitors
discovered through this effort.
Table 1. Compounds 5a−e
We initially prepared compounds 2a−d (Figure 2), with
modification of the right-hand side of compound 1, in which
compd
R1
R2
X
5a
5b
5c
5d
5e
H
H
C
C
C
C
N
H
Cl
H
OMe
H
OMe
H
H
None of these compounds showed any activity against com-
plement in hemolytic assay at a concentration of 100 μg/mL.
These results indicate that the substituent in the left-hand
phenyl group is more sensitive. To understand the contribution
of the two methoxyl groups to complement inhibition activity, a
focused library was designed by replacement of the methyl with
various substituted groups, including H, benzyl, Et, allyl, and
carboxylalkyl- and amino-substituted alkyls (Table 2).
Figure 2. Compounds 2a−d.
the S-phenylethylamino group in compound 1 was replaced
with biphenylmethylamino (2a), naphthylmethylamino (2b),
R-phenylethylamino (2c), and N-methylbenzylamino (2d) groups.
Compounds 2a−d were synthesized according to Scheme 1.
Table 2. Compounds 6a−k
a
Scheme 1. Synthesis of Compounds 2a−d
compd
R1
H
R2
IC50 (μM)
6a
Me
35.28% inhibition
at 100 μg/mL
6b
benzyl
Me
32.71% inhibition
at 100 μg/mL
6c
6d
6e
6f
Et
Me
Me
Me
H
2.86 0.38
1.75 0.21
6.20 0.31
a
Reagents and conditions: (a) DPPA, Et3N, benzene, then reflux.
allyl
(b) DCM, 45−80% (two steps).
EtO2CCH2−
Me
14.62% inhibition
at 100 μg/mL
Dimethoxybenzoic acid 3 was converted to the corresponding aryl
isocyanate 4 with diphenyl phosphorazidate (DPPA) and triethyl-
amine (Et3N) via a modified Curtius rearrangement,9−11 followed
by treatment of isocyanate 4 with the corresponding amines to
afford the desired compounds 2a−d.
Among these ureas, compounds 2a and 2b did not show any
complement inhibition at 100 μg/mL concentration, while
compounds 2c and 2d showed 16.95 and 43.8% inhibition at
100 μg/mL, respectively. These initial attempts indicated that
the phenylethyl subunit in hit 1 is essential for effective inhibi-
tion of complement activation. It is worth mentioning that com-
pound 2c, a R-enantiomer of compound 1, showed very weak
inhibition (16.95% inhibition at 100 μg/mL) as compared to
compound 1 (IC50 = 50 μM) in the hemolytic assay. These
results indicated that the S-configuration of the chiral center in
this position is very critical.
Then, we switched our focus to the left-hand side benzene
ring subunit of compound 1. Five compounds (5a−e) were
prepared (Table 1), in which the dimethoxyphenyl in com-
pound 1 was replaced with various substituted phenyls and
pyridine. The syntheses of compounds 5a−e in Table 1 were
similar to compounds 2a−d. Substituted benzoic acid was
converted to the corresponding aryl isocyanate. Treatment of
the isocyanate with 1-phenylethylamine led to the desired com-
pounds 5a−e.
a
6g
6h
6i
Me
Me
Me
Me
Me
Benzyl
Et
NA
NA
NA
NA
Allyl
6j
EtO2CCH2−
BocNHCH2CH2− NA
6k
a
Showed no inhibition at 100 μg/mL concentration.
The inhibitory activities of these compounds are summarized
in Table 2. The activity of these compounds varied greatly.
3-Position methyl substituted with Et (6c), allyl (6d), or ethyl
acetate (6e) resulted in ca. 9−28-fold improvement of activity
as compared with compound 1. Substitution with other sub-
stituents such as benzyl (6b) or H (6a) resulted in weaker
activity as compared with compound 1. 4-Position methyl sub-
stituted with benzyl, Et, allyl, or other groups all led to inactive
compounds (6f−k). 3-Position methyl replacements were
tolerated. The above results also indicate that the size of the
substituent in the 3-position has a great impact on the activity
and therefore provides a suitable point for further modification
to improve the activity.
A follow up-focused library (outlined in Scheme 2) changing
the methyl for a series of 3-alkyl groups was then synthesized.
In this library, we kept both the 4-methoxy in the left-hand side
benzene ring and the S-phenylethyl in the right-hand side.
318
dx.doi.org/10.1021/ml300005w | ACS Med. Chem. Lett. 2012, 3, 317−321

ACS Medicinal Chemistry Letters
Letter
a
Scheme 2. Synthesis of Compounds 7a−x
a
Reagents and conditions: (a)Benzyl bromide, K2CO3, acetone, 98%. (b) 30% H2O2, NaH2PO4, NaClO2, acetonitrile, H2O, 72%. (c) DPPA, Et3N,
benzene, then reflux. (d) (S)-α-Phenylethylamine, DCM, 48% (two steps). (e) Hydrogen, 5% palladium on activated carbon, DMF, 85%. (f) Alkyl
bromide, K2CO3, 18-crown-6, DMF, 80−95%.
The syntheses of compounds 7a−x in Table 3 were similar to
those of compounds 2a−d in Scheme 2. Benzylation of starting
material isovanillin 8 was achieved to benzaldehyde 9, which
was then oxidized to carboxylic acid 10 applying a known
method.12 Carboxylic acid 10 was converted to isocyanate 11.
Treatment of the isocyanate with (S)-α-phenylethylamine
yielded compound 6b. Hydrogenolytic cleavage of the benzyl
ether group of compound 6b using H2/Pd/C provided com-
pound 6a. Treatment of compound 6a with various alkyl
bromides in the presence of K2CO3 provided corresponding
compounds 7a−x.
The inhibitory activities of these compounds toward com-
plement are summarized in Table 3. Most of this series of
compounds showed potent inhibition activity, with the trends
of the length of side chain significantly affecting the activity and
heteroatom introduction in the side chain removing the activity.
Among 24 compounds tested (7a−7x), 21 of them were more
active than the lead molecule 1. In particular, compounds with
a five- or six-carbon chain (7c, 7d, 7k, 7l, and 7o) showed a
dramatic increase of up to 1000-fold improvement of activity as
compared with compound 1. Longer side chains (7g, 7h)
resulted in no inhibition because of poor solubility. The overall
structure−activity relationship (SAR) is alkane > alkyl > alkynyl >
cycloalkane > heterocycloalkane. The compound BCX1470 was
used as the positive control. It is a potent inhibitor of C1s and
factor D in vitro, and it prevents the initial phase of IC-
mediated inflammation and associated systemic complement
activation in rats.13
Figure 3. Compounds 12a−f.
or extension of the key methyl group to inhibitory activity
(Figure 4).
As a further extension of the SAR on the substituent posi-
tions on the left-hand side benzene ring, we synthesized several
compounds, 12a−f, with different substituted positions of the
methoxyl of C5 alkyloxyl group, as outlined in Figure 3.
Compounds 12c−f showed no inhibitory activity against
complement. Compounds 12a and 12b exhibited an IC50 of 3.2
and 0.27 μM in hemolytic assays, respectively, which were all weaker
than those of the closely related compound 7c (IC50 = 0.019 μM).
These results, combined with the data from the series of 6c and
7a−r, indicated that the 4-methoxyl group, as well as an appropriate
size alkyl substituent in the 3-position, is essential for effective
inhibition of complement. Hydrophobic interaction of 3- and 4-
position groups with target proteins might be responsible for an
increase in potency, which may be worth further investigation for
confirmation.
Having synthesized the potent inhibitor 7b, we then moved
our attention to the right-hand side modification for further
improvement of potency. Compounds 13a and 13b were de-
signed to examine the length between the phenylethyl and the
N atom, while 13c, 13d, 13e, and 13f were designed to
determine the contribution of the configuration and restriction
Figure 4. Compounds 13a−f.
Compounds 13a−d showed no activity. Compound 13e
exhibited an IC50 of 0.105 μM, and compound 13f was 0.42 μM.
The two compounds were all weaker than that of the closely
related compound 7b (IC50 = 0.033 μM). The results also indicate
that the size of the connection had a great impact on inhibition
activity. We also synthesized compounds 14a−g, in which the N
atom of the amide bond was alkylated with different size groups
(Table 4).
Except for compound 14a with a methyl group on the right-
hand side N atom, whose activity (IC50 = 0.019 μM) was similar
to compound 7l (IC50 = 0.013 μM), the other compounds exhibit
10−200-fold decrease in activity as compared with compound 7l.
319
dx.doi.org/10.1021/ml300005w | ACS Med. Chem. Lett. 2012, 3, 317−321

ACS Medicinal Chemistry Letters
Letter
Table 3. Compounds 7a−x
Table 4. Compounds 14a−h
compd
R1
R2
IC50 (μM)
7l
H
H
0.013 0.001
0.019 0.047
0.54 0.123
14a
14b
14c
14d
14e
14f
14g
14h
H
Me
Me
H
a
Me
Me
NA
H
CH2CH2OH
CH2CHOHCH2OH
H
0.323 0.026
0.368 0.087
1.58 0.307
0.223 0.047
7.16 0.02
H
CH2CH2CH2OH
H
H
CH2CH2CH2OH
CH2CH2CH2NH2
a
Showed no inhibition at 100 μg/mL concentration.
Table 5. Compounds 15a−l
compd
R
IC50 (μM)
7l
H
0.013 0.001
1.32 0.083
3.39 0.173
0.368 0.059
1.69 0.48
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
3-Br
4-Br
2-NO2
4-NO2
4-OH
3-OH
4-F
0.139 0.029
0.077 0.009
0.402 0.038
a
4-CF3
4-CN
4-CO2Me
4-CO2H
4-Me
NA
1.26 0.059
0.535 0.051
0.761 0.178
0.327 0.040
a
Showed no inhibition at 10 μg/mL concentration.
this position is flat, almost of these modifications resulted in similar
or weaker activities as compared with that of 7l, which has an
unsubstituted phenyl group. Compounds with electron-donating
groups exhibited better activity, while compounds with electron-
withdrawing groups resulted in ca. 30−100-fold decrease in
activity. Further studies are needed to clarify the role of SAR.
Compound 7l was selected for studies delineating the
mechanism of inhibitory action on complement. The results
indicated that compound 7l did not interfere with the C4 and
C3 activation but inhibited the membrane attack complex
formation (C9 deposition) by the three pathways. The IC50 was
approximately 3.68, 4.46, and 3.59 μg/mL for the classical,
lectin, and alternative pathways, respectively. There was almost
90% inhibition at 10 μg/mL (Figure 5).
In summary, we have discovered a novel series of complement
inhibitors based on 1-(3,4-dimethoxyphenyl)-3-(1-phenylethyl)urea.
Extensive SAR studies were carried out on both the left-hand
dimethoxyphenyl moiety and the right-hand phenylethylamine
moiety of the initial HTS lead compound 1, which resulted in the
identification of potent inhibitors with IC50 values as low as 13 nM
(7l). We also demonstrated that the compound 7l inhibited C9
a
Showed no inhibition at 100 μg/mL concentration.
This indicates that the free left-hand side NH is necessary to keep
the high potency of inhibition activity, while the right-hand side
NH tolerates small size substitutions.
We further studied the right-hand side substituent on the ben-
zene ring by keeping all of the favored substituted groups, including
3-isohexyl, 4-methyl, two free NH as well as S-configuration methy-
lphenylamine and with various substitutions on the benzyl ring.
Among the synthesized compounds 15a−l (Table 5), a compound
with a substituted hydroxyl exhibited better activity (15f), while
compounds with 3-bromine, 4-fluorine, or methyl substitutions
resulted in ca. 30−100-fold decrease in activity. Overall, the SAR in
320
dx.doi.org/10.1021/ml300005w | ACS Med. Chem. Lett. 2012, 3, 317−321

ACS Medicinal Chemistry Letters
Letter
Author Contributions
⊥The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. These authors contributed equally.
Funding
This work was supported by The National Natural Science
Foundation of China (Grants 30725049, 81021062, and
81072652) and by the grant of Shanghai Science and
Technology Committee (No. 11431921102).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
DCM, dichloromethane; DPPA, diphenylphosphoryl azide;
Et3N, triethylamine; HTS, high-throughput screening; NHS,
normal human serum
REFERENCES
■
(1) Barrington, R.; Zhang, M.; Fischer, M.; Carroll, M. C. The role of
complement in inflammation and adaptive immunity. Immunol. Rev.
2001, 180, 5−15.
(2) Kirschfink, M. Targeting complement in therapy. Immunol. Rev.
2001, 180, 177−189.
(3) Caliezi, C.; Wuillemin, W. A.; Zeerleder, S.; Redondo, M.; Eisele,
B.; Hack, C. E. C1-esterase inhibitor: An anti-inflammatory agent and
its potential use in the treatment of diseases other than hereditary
angioedema. Pharmacol. Rev. 2000, 52, 91−112.
(4) Wagner, E.; Frank, M. M. Therapeutic potential of complement
modulation. Nat. Rev. Drug Discovery 2010, 9, 43−56.
(5) Ricklin, D.; Lambris, J. D. Complement-targeted therapeutics.
Nat. Biotechnol. 2007, 25, 1265−1275.
(6) Holland, M. C. H.; Morikis, D.; Lambris, J. D. Synthetic small
molecule complement inhibitors. Curr. Opin. Invest. Drugs 2004,
5 (11), 1164−1173.
(7) Kozlov, L. V.; Burdelev, O. O.; Bureeva, S. V.; Kaplun., A. P.
Artificial inhibition of the complement system. Russ. J. Bioorg. Chem.
2007, 33 (5), 449−473.
(8) Qu, H.; Ricklin, D.; Lambris, J. D. Recent developments in low
molecular weight complement inhibitors. Mol. Immunol. 2009, 47,
185−195.
(9) Shioiri, T.; Ninomiya, K.; Yamada, S.-i. Diphenylphosphoryl
azide. New convenient reagent for a modified Curtius reaction and for
peptide synthesis. J. Am. Chem. Soc. 1972, 94, 6203−6205.
(10) Ninomiya, K.; Shioiri, T.; Yamada, S. Phosphorus in organic
synthesisVII: Diphenyl phosphorazidate (DPPA). A new conven-
ient reagent for a modified curtius reaction. Tetrahedron 1974, 30,
2151−2157.
(11) Rigby, J. H.; Balasubramanian, N. Preparation of highly
substituted 2-pyridones by reaction of vinyl isocyanates and enamines.
J. Org. Chem. 1989, 54, 224−228.
(12) Dalcanale, E.; Montanari, F. Selective oxidation of aldehydes to
carboxylic acids with sodium chlorite-hydrogen peroxide. J. Org. Chem.
1986, 51, 567−569.
(13) Szalai, A. J.; Digerness, S. B.; Agrawal, A.; Kearney, J. F.; Bucy,
R. P.; Niwas, S.; Kilpatrick, J. M.; Babu, Y. S.; Volanakis, J. E. The
Arthus reaction in rodents: Species-specific requirement of comple-
ment. J. Immunol. 2000, 164, 463−468.
Figure 5. Compound 7l inhibited C9 deposition through the classical,
the lectin, and the alternative pathways but had no influence on C3
and C4 depositions. Normal human serum (NHS) was added to IgM-
coated plates (classical pathway), mannan-coated plates (lectin
pathway), or zymosan-coated plates (alternative pathway) and then
incubated with various concentrations of compound 7l. Deposited
C3 (A), C4 (B), and C9 (C) were measured by ELISA.
deposition but had no influence on the preceding C3 and C4
deposition by the classical, the lectin, and the alternative pathway.
These compounds could serve as leads for the development of small
molecule agents targeting the complement system.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and characterization of new chemical
entities. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Fax: (+86)21-508-00-954. E-mail: anna.blom@med.lu.se
(A.M.B.), jpzuo@mail.shcnc.ac.cn (J.-P.Z.), or fjnan@mail.
shcnc.ac.cn (F.-J.N.).
321
dx.doi.org/10.1021/ml300005w | ACS Med. Chem. Lett. 2012, 3, 317−321