The design, synthesis and in vitro immunosuppressive evaluation of novel isobenzofuran derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.11.078

The synthesis and biological evaluation of a series of novel isobenzofuran-based compounds are described. The compounds were evaluated for their immunosuppressive effects of T-cell proliferation and IMPDH type II inhibitor activity in vitro, as well as their structure-activity relationships were assessed. Several compounds demonstrated highly efficacious immunosuppressive properties, especially compounds 2d, 2e, 2h and 2j, which were superior to MPA, while compounds 2k, 2m, 2n, 4c and 5d exhibited an equipotent inhibitory activity compared to MPA. Generally, it was obviously demonstrated that α,β-unsaturated amides proved more potent than the diamide and urea series. The present study provides a guide for further research on development of safe and effective immunosuppressive agents.

Full text of DOI:10.1016/j.bmcl.2011.11.078

Bioorganic & Medicinal Chemistry Letters 22 (2012) 53–56
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The design, synthesis and in vitro immunosuppressive evaluation of novel
isobenzofuran derivatives
⇑
Na Yang, Qing-He Wang, Wen-Qian Wang, Jian Wang, Feng Li, Shen-Peng Tan, Mao-Sheng Cheng
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of a series of novel isobenzofuran-based compounds are
described. The compounds were evaluated for their immunosuppressive effects of T-cell proliferation
and IMPDH type II inhibitor activity in vitro, as well as their structure–activity relationships were
assessed. Several compounds demonstrated highly efficacious immunosuppressive properties, especially
compounds 2d, 2e, 2h and 2j, which were superior to MPA, while compounds 2k, 2m, 2n, 4c and 5d
exhibited an equipotent inhibitory activity compared to MPA. Generally, it was obviously demonstrated
Received 13 September 2011
Revised 21 October 2011
Accepted 19 November 2011
Available online 28 November 2011
Keywords:
Isobenzofuran
Immunosuppressive agents
T-cell proliferation assay
MPA
that
a,b-unsaturated amides proved more potent than the diamide and urea series. The present study
provides a guide for further research on development of safe and effective immunosuppressive agents.
Ó 2011 Elsevier Ltd. All rights reserved.
IMPDH type II
Immunosuppressive agents are drugs that inhibit immune sys-
tem activity and have important clinical roles through preventing
the rejection of transplanted organs and tissues such as the bone
marrow, heart, kidney and liver. Also they treat autoimmune dis-
eases or diseases that are most likely of autoimmune origin, such
as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, sys-
temic lupus erythematosus, focal segmental glomerulosclerosis,
Crohn’s disease, pemphigus and ulcerative colitis, and other non-
autoimmune inflammatory diseases. With the application of corti-
costeroids, cyclosporine,1,2 mycophenolate mofetil,3 sirolimus,4
FK5065,6 and other immunosuppressants,7–10 the clinical success
rate of organ transplantation has greatly improved. However, these
drugs have side-effects and risks11,12 due to their poor selectivity.
Therefore, it is desirable to develop novel immunosuppressants
that are effective and safe and that exhibit reduced toxicity.13
Inosine-50-monophosphate dehydrogenase (IMPDH)14–16 is a
key enzyme in the de novo guanosine nucleotide synthesis and
has emerged as an attractive target in the quest for the discovery
of drugs in the immunosuppressive, antiviral, antibacterial and
anticancer therapeutic areas. Two isoforms of the enzyme have
been identified and designated as type I and type II, which possess
84% sequence identity. IMPDH inhibitors are used in immunosup-
pressive chemotherapy (MPA, mizoribine) and antiviral chemo-
therapy (ribavirin). In addition, tiazofurin (Tiazole) was granted
orphan drug for treatment of chronic myelogenous leukemia.
Several compounds such as amides, oxazolyl indoles, quinazoli-
nones, acridones etc., have been reported to inhibit IMPDH activity
at low micromolar concentrations.16
Mycophenolic acid (MPA; Fig. 1) acts as a non-competitive,
selective, and reversible inhibitor of IMPDH and blocks the re-
sponse of B- and T-cells to mitogens or antigens. MPA displays
antiviral and anticancer activities in some in vitro tests.17 Recently,
interest in MPA as an anticancer drug has revived due to its antian-
giogenic activity.18 MPA also induces the differentiation or apopto-
sis of several cancer cell lines.19–22 Mycophenolate mofetil (MMF;
Fig. 1), which is a prodrug of MPA, has been used in organ and stem
cell transplantation and in autoimmune diseases as a highly effec-
tive immunosuppressive agent.23,24 Unfortunately, dose-limiting
gastrointestinal (GI) toxicity is observed following the oral admin-
istration of MMF or MPA.25,26 There is a continuing effort to
identify and develop new inhibitors that differ from MMF with
improved GI tolerability.
In this report, we have outlined the synthesis and biological eval-
uation of three new classes of isobenzofuran-based compounds,
which are indicated by the compounds 2, 4 and 5 ( Fig. 2). The
compounds were evaluated for in vitro immunosuppressive effects
on T-cell proliferation and IMPDH type II inhibition. In addition,
their structure–activity relationships were assessed. The results of
the present study provide insights into the development of safe
and effective immunosuppressive agents.
The synthesis of the analogs 2, 4 and 5 are shown in Scheme 1.
The important intermediate 5-aminoisobenzofuran-1(3H)-one 1
was prepared from phthalimide according to the reported method
⇑
Corresponding author. Tel.: +86 24 23986413; fax: +86 24 23995043.
E-mail address: mscheng@syphu.edu.cn (M.-S. Cheng).
by Wang.27 The
a,b-unsaturated amides 2 were synthesized
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.078

54
N. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 53–56
Figure 1. Chemical structures of MPA and MMF.
Figure 2. Structures of a,b-unsaturated amide 2, urea 4 and diamide 5.
Scheme 1. Reagents and conditions: (a) HNO3, condH2SO4 (87%); (b) condHCl, SnCl2, 50 °C (91%); (c) Zn/NaOH, CuSO4, H2O, reflux (64%); (d) cinnamic acid, (COCl)2, CH2Cl2,
0 °C (91%); (e) ClCOOCH2CCl3, DIEA/THF, 0 °C (45–72%); (f) R2R3NH, DIEA/DMSO, 0 °C (26–85%); (g) (COCl)2, CH2Cl2, R4R5NH, 0 °C (53–87%).
following the reaction of various synthetic
ylic acids with equivalent of oxalyl chloride in dichloromethane
a
,b-unsaturated carbox-
As described in Table 1, the phenyl substituted analog 2a
showed an IC50 value of 7.24 M to T-cell proliferation. The analogs
l
(CH2Cl2) at 0 °C and subsequently reacted with the intermediate
1 in the presence of pyridine. The starting a,b-unsaturated carbox-
2l and 2m demonstrated that the bulky substituents at the para-
position of the benzene ring were favored over the unsubstituted
2a. Introducing a methoxy group to the ortho-position obtained
ylic acids were obtained via the Knoevenagel reaction28 from com-
mercially available substituted benzoic aldehyde and malonic acid.
The compound 1 reacted with 2,2,2-trichloroethylchloroformate
yielded 3. The subsequent reaction of 3 with various amines
yielded the analog 4. The acylation of commercially available
amine with ethyl oxalyl chloride yielded the corresponding oxa-
lamic acid ethyl ester, which were hydrolyzed to generate oxalam-
ic acid. The diamides 5 were prepared from oxalamic acid and 1,
which used the same procedure for the amides 2.
the analog 2j which displayed an IC50 value of 0.039
to the 0.28 M of MPA. The dimethoxyl analog 2d had an IC50 value
of 0.029 M, indicating the electron-donating substituent at the
lM compared
l
l
benzene ring was essential to retain or increase the activity. How-
ever, the thiomethoxy-substituted analog 2i showed a 60-fold low-
er activity compared to the methoxy-substituted and dimethoxyl
analogs 2d and 2j, which exhibited steric and electronic effects.
The acetoxy substituted analogs 2h, 2k and 2n possessed notice-
ably enhanced activities compared to the analog 2a. The analog
The compounds were tested for their ability to alter the prolif-
erative response of mouse splenocytes to concanavalin A, which is
a T-cell mitogen.29 The structure–activity relationships for the
immunosuppressive activities are summarized in Tables 1–3. The
2h exhibited an IC50 value of 0.03 lM and immunosuppressive
activity that was superior to MPA. The analog 2o with a saturated
double bond was prepared to determine the necessity of the
double bond in the amide. This structure led to a fivefold decrease
in potency compared to the analog 2i. The phenyl ring of 2a was
results indicate that the series of
a,b-unsaturated amides exhibit
increased potency compared to the urea and diamide series.

N. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 53–56
55
Table 1
Inhibition of T cells proliferation by the
a,b-unsaturated amide analogues
H
R1
N
O
O
O
Compd
R1
Double bond
T cells proliferationa IC50
(lM)
MPA
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
—
Phenyl
2-Furanyl
2-Pyrido
—
0.28
7.24
9.00
17.85
0.029
0.034
>100
43.09
0.03
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Unsaturated
Saturated
3,4-Dimethoxylphenyl
m-Fluoropheny
o-Bromophenyl
p-Cyanophenyl
m-Acetoxyphenyl
3,4,5-TriMethoxylphenyl
o-Methoxylphenyl
3-Methoxyl-4-acetoxyphenyl
p-Isopropylphenyl
p-Tert-Butylphenyl
1.73
0.039
0.136
1.59
0.72
0.18
3,4-DiAcetoxyphenyl
3,4,5-TriMethoxylphenyl
7.74
a
IC50 values are mean of three experiments, standard deviation below 20%.
Table 2
replaced by heterocycles to give the analogs 2b and 2c which
showed a decreased anti-proliferative activity. The analog 2g with
strong electron-withdrawing cyano-group exhibited a decreased
potency compared to the analog 2a. The analog 2e with m-fluorine
substituted showed an increased activity compared to the analog
2f with o-bromine. These results imply that the presence of a large
steric group at the 2-position decreases activities.
Inhibition of T cells proliferation by urea analogues
R3
R2
H
N
N
O
O
O
The bioactivity results of the urea series 4a–j were described in
Compd
R2
R3
H
T cells proliferationa IC50
(lM)
Table 2. Overall, this series was less potent than the
rated amide series. Compound 4a displayed an IC50 value of
7.0 M. The analog 4b (R3 = methyl) exhibited loss of activity,
a,b-unsatu-
4a
4b
4c
4d
4e
4f
4g
4h
4i
Phenyl
Phenyl
7.09
>100
0.90
2.26
2.67
4.19
2.45
1.37
12.71
58.12
Methyl
o-Methylphenyl
m-Methylphenyl
p-Methylphenyl
p-Fluoropheny
p-Chlorophenyl
p-Iodophenyl
Cyclohexyl
H
H
H
H
H
H
H
H
l
mainly because of lacking an amide hydrogen which functions as
hydrogen-bond donor. This result partially supports the essential
role of the free amide group as a hydrogen bonding donor. Substi-
tution at the meta- and para-positions, such as that in the com-
pounds 4d and 4e resulted in an overall increase in potency
relative to the compound 4a. Interestingly, the analogs 4f, 4g and
4h with halogen at the para-positions exhibited increased potency,
which reflected the effects of the substituent size. This result sug-
gests that a sterically bulky group is favorable for the activity. The
replacement of the arylamine 4a with the aliphatic amine 4j
showed significantly decreased activity. The cyclohexyl substituted
amide 4i exhibited a slight decrease in activity with an IC50 value of
12.71 lM compared to compound 4a. This result implies that the
benzene ring-conjugated system is essential to retain or increase
the activity.
A series of diamide analogs 5a–j were also investigated. As
shown in Table 3, the compound 5a displayed an IC50 value of
4j
n-Propyl
a
IC50 values are mean of three experiments, standard deviation below 20%.
Table 3
Inhibition of T cells proliferation by the diamide analogues
O
H
N
R4
N
O
O
R5
O
Compd R4
R5
T cells proliferationa IC50
(lM)
36.07 lM. The aryl–amine group of 5a was replaced by aliphatic-
amine to give 5c which showed 12-fold enhancement of the inhib-
itory activity of T-cell proliferation. The analogs 5d and 5e with
electron-withdrawing groups at the para-positions exhibited high-
er activities than the analog 5a, whereas the analog 5g with elec-
5a
5b
5c
5d
5e
5f
5g
5h
5i
Phenyl
Benzyl
Tert-Butyl
p-Fluoropheny
p-Chlorophenyl
p-Hydroxylphenyl
p-Methoxylphenyl
o-Methylphenyl
3,4,5-
H
36.07
14.29
3.40
0.57
2.24
5.03
153.33
5.51
Methyl
H
H
H
H
H
H
tron-donating groups showed
a fivefold decrease in activity
compared to the patent compound. However, the p-hydroxyl ana-
log 5f exhibited a significantly increased activity mainly because
the hydroxyl group acted as a hydrogen bonding donor. This result
partially supports the essential role of the free hydroxyl group as a
hydrogen bonding donor. Substitution at the meta-positions, such
as that in compound 5j, resulted in an overall increase in potency
Methyl
3.28
TriMethoxylbenzyl
m-Nitrophenyl
5j
H
1.82
a
IC50 values are mean of three experiments, standard deviation below 20%.

56
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The compounds were tested (concentration range: 0.1–10 mg/
mL) for their ability to inhibited IMPDH type II.30 In this assay,
MPA inhibited IMPDH type II with an IC50 of 1.5
amide-based inhibitors that were evaluated in the current study
inhibited IMPDH type II with an IC50 of less than 10 M. The com-
pounds 2d, 2e, 2h, 2m and 2n inhibited IMPDH type II with IC50
values of 2.1, 4.5, 8.8, 3.2 and 2.8 M, respectively.
lM. Some of the
l
l
In summary, we have synthesized 35 isobenzofuran-derived
analogs and evaluated their activities on T-cell proliferation and
IMPDH type II. Generally, the
a,b-unsaturated amide series were
more potent than the diamide and urea series. The structure–activ-
ity relationship of isobenzofuran-derived analogs for immunosup-
pressive activity was also evaluated. From the lymphoproliferation
assay results, the analogs 2d, 2e, 2h and 2j proved more potent
activities than MPA, whereas the analogs 2k, 2m, 2n, 4c and 5d
exhibited an equipotent inhibitory activity compared to MPA. In
addition, the ability of the compounds to inhibit IMPDH type II
activity in vitro was tested. The analogs 2d, 2e, 2m and 2n inhib-
ited IMPDH type II as effectively as MPA. Further evaluation of
the immunosuppressant efficacy using other immunological tests
is currently under way to elucidate their potential therapeutic
use and establish the relationship between efficacy and toxicity
in vivo.
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Acknowledgment
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The authors would like to thank New Drug Research& Develop-
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providing biological data.
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Supplementary data
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Supplementary data associated with this article can be found, in
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