Thalidomide as a nitric oxide synthase inhibitor and its structural development

Article abstract of DOI:10.1248/cpb.52.498

Thalidomide has been found to exhibit weak nitric oxide synthase (NOS)-inhibitory activity. Structural development studies of thalidomide showed that some N-2,6-dimethylphenylhomophthalimide analogs possess NOS-inhibiting activity.

Full text of DOI:10.1248/cpb.52.498

498
Communications to the Editor
Chem. Pharm. Bull. 52(4) 498—499 (2004)
Vol. 52, No. 4
least three times. The result showed that thalidomide at 1 mM
inhibits NOS by about 23% (Table 1). Although the NOS-in-
hibitory activity of thalidomide was weak, one or more of the
thalidomide metabolites or decomposition products might
possess more potent activity. Such considerations led us to
investigate the NOS-inhibitory activity of thalidomide-re-
lated compounds.
Thalidomide as a Nitric Oxide Synthase
Inhibitor and Its Structural Development
Rumiko SHIMAZAWA, ,1) Hiroko SANO, Aya TANATANI,
*
Hiroyuki MIYACHI, and Yuichi HASHIMOTO
Institute of Molecular & Cellular Biosciences, The University of
Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan.
Received February 5, 2004; accepted March 8, 2004
Thalidomide (1) is chemically labile and not suitable for a
lead compound. So we chose N-phenyl(homo)phthalimide or
N-phenylquinazolinedione derivatives as lead compounds,
based on our previous studies on peptidase inhibitors and
COX inhibitors derived from thalidomide.4,12—16,19,20) Substi-
tuted phthalimide and homophthalimide compounds were
prepared by routine organic synthetic methods, i.e., conden-
sation of appropriate amines with phthalic (3) or homo-
phthalic anhydride (4—11). Quinazolinedione derivatives
(12—14) were prepared by the condensation of 2,6-dialkyl-
anilines and methyl anthranilate in the presence of triphos-
gene.15) The 4-methoxy hemiacetal analog of 6 (15) was ob-
tained quantitatively by the light-dependent autoxidation of 6
in methanol.12,25) Compound 16 was prepared from the tricar-
bonyl analog, which was afforded by recrystallization of the
hemiacetal (15) in water, by reaction of the carbonyl group at
the benzyl position with (diethylamino)sulfur trifluoride.12,25)
The 2-dihydro derivative (17) was prepared by treatment of
the amide, which was prepared by the acylation of 2,6-di-
ethylaniline with 2-nitrobenzoyl chloride followed by reduc-
tion of the nitro group, with formalin and sodium hydroxide
in ethanol, followed by treatment with triphosgene.15) The 4-
dihydro analog (18) was prepared by treatment of 2-amino-
N-(2,6-diethylphenyl)benzenemethanamine, which was pre-
pared from 2-nitrobenzyl bromide and 2,6-diethylaniline,
with triphosgene.15) The structures of all the compounds were
confirmed by analysis data, and the analysis data were con-
sistent with expectation.
Phthalimide derivatives with a five-membered ring, includ-
ing compound 3, did not show apparent NOS-inhibitory ac-
tivity in the concentration range examined. The ring expan-
sion of the phthalimide moiety to a six-membered ring, i.e.,
homophthalimide derivatives (4—11), resulted in appearance
of NOS-inhibitory activity, which was more potent than that
of thalidomide (Table 1). N-Phenylhomophthalimide (4) at
1 mM inhibited NOS activity to the extent of about 42%, and
introduction of alkyl groups at the ortho-positions of the N-
phenyl substituent of 4 (compounds 5—9) enhanced the
NOS-inhibitory activity (61—79% inhibition). Although the
most effective alkyl substituent could not be deduced from
the investigated alkyl groups, the preferred site of substitu-
tion seems to be ortho. Quinazolinedione derivatives (12—
14), which are bioisosters of the corresponding homophthal-
imide derivatives (5—9) in peptidase-inhibitory activity as-
says,15) were unexpectedly inactive. Interestingly, reduction
of the 3-carbonyl group (substituent Y in Table 1) of 13, i.e.,
compound 17, resulted in appearance of NOS-inhibitory ac-
Thalidomide has been found to exhibit weak nitric oxide syn-
thase (NOS)-inhibitory activity. Structural development studies
of thalidomide showed that some N-2,6-dimethylphenylhomo-
phthalimide analogs possess NOS-inhibiting activity.
Key words thalidomide; nitric oxide synthase (NOS); inhibitor; struc-
tural development
Thalidomide (1) is a hypnotic/sedative drug that was with-
drawn from the market because of its severe teratogenic-
ity.2—4) In spite of this, research into thalidomide was not
halted, and the drug has since been established to be effective
for the treatment of various diseases, including leprosy,
myeloma and AIDS.3—5) Although thalidomide affects pro-
duction of various cytokines, the prevailing hypothesis was
that the effectiveness of thalidomide in these diseases is
elicited entirely through regulation of tumor necrosis factor
(TNF)-a production.
We have been engaged in structural development studies
of thalidomide, and have demonstrated that thalidomide is in
fact a multi-target drug. Our studies resulted in the creation
of TNF-a production regulators (including bi-directional
ones, as well as pure inhibitors and enhancers),3,4,6—8) andro-
gen antagonists,3,4,9,10) peptidase inhibitors,4,11—15) glucosi-
dase inhibitors,16,17) thymidine phosphorylase inhibitors18)
and cyclooxygenase inhibitors.19,20) Nevertheless, not all of
the beneficial pharmacological effects elicited by thalidomide
can be fully interpreted in terms of the above activities, indi-
cating the existence of other target molecules/phenomena.
We suspected that nitric oxide synthase (NOS) might be an-
other target molecule of thalidomide, because the drug is
effective against diabetes and has hypoglycemic effect,
whereas NO acts as disease-progressing factor.
The family of nitric oxide synthases (NOS), which in-
cludes neuronal NOS (nNOS), endothelial NOS (eNOS) and
inducible NOS (iNOS),21) catalyzes the conversion of L-argi-
nine to L-citrulline and nitric oxide (NO), an important cellu-
lar messenger molecule which has been implicated in both
physiological and pathophysiological functions. In this paper,
we describe evaluation of the NOS-inhibitory activity of
thalidomide and its analogs.
First we investigated NOS-inhibitory activity of thalido-
mide (1). Inhibitory activity was assayed by monitoring ni-
trite/nitrate production using Ichimori’s protocol with a little
modifications.22,23) Although the quantitative values differed
from experiment to experiment, the results were basically re-
producible, and average inhibition rate are presented in Table
1. In the assay system, the IC50 value of NG-nitro-L-arginine
methyl ester (NNA, 2), which is a well-known analog of the
endogenous NOS substrate, L-arginine, was determined to be
Fig. 1. Structures of Thalidomide (1) and NG-Nitro-L-arginine Methyl
10 mM. The assay was performed in triplicate, and repeated at Ester (NNA: 2)
To whom correspondence should be addressed. e-mail: shimazaw@nihs.go.jp
© 2004 Pharmaceutical Society of Japan

April 2004
499
Table 1. NOS-Inhibitory Activity of Compounds 1—18
Inhibition rate
Compound
X
Y
Z
R1
R2
1 mM
100 mM
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H2
O
—
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3Ј-Me
H
5Ј-Me
H
Ͻ10%
42Ϯ8%
66Ϯ18%
64Ϯ5%
(69Ϯ6%)a)
79Ϯ12%
61Ϯ17%
44Ϯ14%
31Ϯ17%
Ͻ10%
Ͻ10%
Ͻ10%
71Ϯ16%
74Ϯ5%
(50Ϯ3%)a)
Ͻ10%
Ͻ10%
63Ϯ10%
31Ϯ5%
26Ϯ9%
58Ϯ20%
25Ϯ2%
17Ϯ6%
Ͻ10%
2Ј-Me
2Ј-Et
2Ј-iPr
2Ј-Me
2Ј-Me
2Ј-Me
3Ј-Me
2Ј-Me
2Ј-Et
2Ј-iPr
2Ј-Et
2Ј-Et
2Ј-Et
2Ј-Et
6Ј-Me
6Ј-Et
6Ј-iPr
6Ј-Et
6Ј-iPr
5Ј-Me
5Ј-Me
6Ј-Me
6Ј-Et
6Ј-iPr
6Ј-Et
6Ј-Et
6Ј-Et
6Ј-Et
NH
NH
NH
C(OH)OMe
CF2
41Ϯ18%
42Ϯ12%
46Ϯ3%
NH
NH
Thalidomide (1)
NNA (2)
23Ϯ8%
87Ϯ7%
Ͻ10%
74Ϯ28%
a) Inhibitory activity of 7 and 17 was measured at 0.5 mM instead of 1 mM, because these compounds showed poor solubility.
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tivity, while reduction of another carbonyl group at position
1, i.e., compound 18, did not affect the activity. We cannot
interpret these phenomena at this stage.
Chemically, homophthalimide derivatives are autoxidized
at the benzylic methylene position in the tetrahydroisoquino-
line ring to give a hemiacetal analog (15) in methanol or a
tricarbonyl analog in water.12,24,25) Therefore, N-phenylho-
mophthalimides (4—11) might inhibit the production from
NO of nitrite/nitrate, which are the products measured di-
rectly by Ichimori’s protocols,22,23) through self-oxidization.
However, the inhibitory activity of non-oxidizable analogs,
i.e., the hemiacetal analog (15: already oxidized) and difluo-
rinated analog (16: blocked at the benzylic position) is al-
most the same as that of the corresponding homophthalimide
compound 6. This result suggests that N-phenylhomophthal-
imides (4—11) inhibit NOS directly, but do not merely in-
hibit production of nitrite/nitrate from NO.
In conclusion, we found firstly that thalidomide (1) has
NOS-inhibitory activity, even though the activity is weak,
and secondly, that some N-phenylhomophthalimide deriva-
tives possess NOS-inhibitory activity which is more potent
than that of thalidomide. Although the structure–activity re-
lationships remain to be investigated, and the structure has
not been optimized yet, the prepared compounds show mod-
erate activity and represent a new class of non-amino-acid-
based NOS inhibitors. Further structural development aiming
at improvement of the NOS-inhibiting activity and at NOS
isoform selectivity is in progress.
Acknowledgment The work described in this paper was partially sup-
ported by Grants-in-Aid for Scientific Research from The Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan.
References and Notes
1) Present address: Pharmaceuticals and Medical Devices Evaluation
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