Suppression of inducible nitric oxide synthase expression by yakuchinones and their analogues

Article abstract of DOI:10.1248/cpb.54.377

Analogues of yakuchinones were synthesized as inhibitors of nitric oxide production in lipopolysaccharideactivated macrophage cell line, RAW 264.7 cells. We prepared stronger inhibitors than the original natural molecules, yakuchinones A and B reported from Alpinia oxyphylla. From the limited structural activity relation study of analogues, we concluded that the optimal length of linker between two aryl groups and the presence of enone moiety in the linker were identified as essential for the activity. The IC₅₀ value of the most potent structure was 0.92 μM. The active analogues suppressed the expression of inducible nitric oxide synthase protein and mRNA.

Full text of DOI:10.1248/cpb.54.377

March 2006
Notes
Chem. Pharm. Bull. 54(3) 377—379 (2006)
377
Suppression of Inducible Nitric Oxide Synthase Expression by
Yakuchinones and Their Analogues
Hwa Jin LEE, Ji Sun KIM, Joung Wha YOON, Hee-Doo KIM, and Jae-Ha RYU*
College of pharmacy, Sookmyung Women’s University; 52 Hyochangwon-Gil, Yongsan-Gu, Seoul 140–742, Republic of
Korea. Received September 28, 2005; accepted November 12, 2005
Analogues of yakuchinones were synthesized as inhibitors of nitric oxide production in lipopolysaccharide-
activated macrophage cell line, RAW 264.7 cells. We prepared stronger inhibitors than the original natural mole-
cules, yakuchinones A and B reported from Alpinia oxyphylla. From the limited structural activity relation study
of analogues, we concluded that the optimal length of linker between two aryl groups and the presence of enone
moiety in the linker were identified as essential for the activity. The IC50 value of the most potent structure was
0.92 mM. The active analogues suppressed the expression of inducible nitric oxide synthase protein and mRNA.
Key words nitric oxide; yakuchinone analogues; inhibitor; iNOS; expression
Diarylheptanoid is a family of natural plant metabolites calcium-regulated constitutive isoforms, endothelial (eNOS)
whose characteristic structural feature is the presence of two and neuronal (nNOS) have important roles in regulation of
aromatic rings tethered by a linear seven-carbon chain. The blood pressure and neurotransmission,19) whereas the in-
diarylheptanoids exhibit a broad range of biological activities ducible isoform (iNOS) is calcium-independent and induced
including anti-tumor,1,2) anti-inflammatory,3,4) anti-oxidant,5,6) by LPS and various cytokines such as IFN-a, IL-1b, and
antihepatoxic,7) antifungal,8,9) and related effects. For in- TNF-a.20) Low concentrations of NO produced by iNOS
stance, curcumin, a yellow pigment from tumeric (Curcuma possess beneficial roles in antimicrobial activity of
longa, Zingiberaceae), has been shown to inhibit tumorigene- macrophages against pathogens,21) while the overproduction
sis during both initiation and promotion stages in several ani- of NO and its derivatives, such as peroxynitrite and nitrogen
mal models, possibly through the inhibition of cyclooxyge- dioxide, have been suggested to be mutagenic in vivo and to
nase and lipoxygenase and blocking the formation of arachi- provoke the pathogenesis of septic shock and various inflam-
donic acid metabolites.10—12) It was reported that the substitu- matory processes.22) Furthermore, NO and its oxidized forms
tion pattern on the aromatic moiety of curcumin has crucial have also been shown to be carcinogenic.23) Thus, inhibitors
effects on the gene expression of inducible forms of cy- of iNOS enzyme activity or its expression can be used as po-
clooxygenase (COX-2) and nitric oxide synthase (iNOS).13) tential therapeutic tools for management of NO-related disor-
Yakuchinone A and yakuchinone B (Fig. 1), diarylheptanoids ders.
from Alpinia oxyphylla (Zingiberaceae) have been reported
In the present study, we tried to obtain potent inhibitors of
to show potent anti-inflammatory and anti-tumor promo- iNOS expression by the structural modification of yakuchi-
tional activities through the inhibition of COX-2 and iNOS none that can serve as a new lead for the development of
expression.14,15) Yakuchinone B and the structural analogues anti-inflammatory drug. Our efforts toward the design of
have been extensively studied as inhibitors of acyl-CoA: cho- novel yakuchinone analogues have focused on the modifica-
lesterol O-acyltransferase that can be therapeutic agents for tion of the conjugation and the length of linker between two
hypercholesterolemia and atherosclerosis.16) Yakuchinone B aromatic rings.
has also been reported to show inhibitory activity of tyrosi-
nase. The a,b-unsaturated carbonyl conjugated moiety and Results and Discussion
aromatic ring of yakuchinone B play important roles in the
For the evaluation of inhibitory activity of yakuchinone
competitive inhibition of tyrosinase.17) The overall biological analogues against NO production, NO released from culture
activity of yakuchinone B is stronger than yakuchinone A media was quantitated after incubation with samples during
that might come from the presence of enone group in the LPS-activation of RAW 264.7 cells. When the cells were
linker. We tried to optimize the structure of yakuchinone for treated with 1 mg/ml LPS for 20 h, the NO production was
the inhibitory activity of nitric oxide production in activated markedly increased to 30—40 mM, while basal level was
macrophages.
1.0—2.5 mM. The inhibitory potencies, expressed as the IC50
The critical role of nitric oxide (NO) in various pathologi- values, of the synthetic analogues are shown in Table 1. The
cal conditions has led to the discovery of new therapeutic most essential structural requirement for the activity is the
agents. NO, a gaseous free radical, is produced through the enone moiety in the linker. The activity of compounds 1, 4
oxidation of arginine by nitric oxide synthase (NOS).18) The and 7 are more potent than the respective non-enone type
structures 2, 5 and 8, respectively. The length of linker is pro-
portional to the activity up to six-carbon, and then the activ-
ity declined with the seven-carbon diarylheptanoid, com-
pound 1. Generally there are many reports of diarylhep-
tanoids from plants with diverse biological activities. Diaryl-
hexanoid can be another target for getting valuable bio-active
Fig. 1. Structures of Yakuchinone B (1) and A (2)
structures. Compound 3 with enone moiety in the diaryl-
∗ To whom correspondence should be addressed. e-mail: ryuha@sookmyung.ac.kr
© 2006 Pharmaceutical Society of Japan

378
Vol. 54, No. 3
Chart 1. Preparation of Yakuchinone A, B, and Their Analogues
Table 1. Inhibitory Activities of Yakuchinone Analogues on NO Produc-
tion in LPS-Activated Macrophages
Compounds
X
n
Y
IC50 (mM)a)
1
2
3
4
5
6
7
8
CHꢀCH (trans)
CH2–CH2
CHꢀCH (trans)
CHꢀCH (trans)
CH2–CH2
CHꢀCH (trans)
CHꢀCH (trans)
CH2–CH2
4
4
3
2
2
0
0
0
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Methyl
Methyl
1.38ꢁ0.35
16.81ꢁ1.47
0.92ꢁ0.07
3.73ꢁ0.14
ꢂ50
4.91ꢁ0.85
9.62ꢁ1.26
ꢂ50
Fig. 2. (A) Effects of Yakuchinone Analogues on the Expression of iNOS
Protein in LPS-Activated Macrophages
RAW 264.7 cells were treated with synthetic compounds 1—6 (5 mM) for 20 h during
LPS (1 mg/ml) activation. Cell lysates were prepared and the iNOS and b-actin protein
levels were determined by Western blotting. The blot is the representative result of three
separate experiments.
a) IC50 values represents meansꢁS.D. (nꢀ3).
hexa-type linker between two aromatic rings showed the
most potent inhibition of LPS-induced NO production
(IC50ꢀ0.92 mM). Compound with diaryl-type showed more
potent activity compared with non-diaryl type (compound 6
vs. 7).
(B) Effects of Yakuchinone Analogues on the Expression of mRNA in LPS-
Activated Macrophages
RAW 264.7 cells were pre-treated with synthetic compounds 1—6 (5 mM) for 30 min,
and then treated with LPS (1 mg/ml) for 6 h. mRNA level for iNOS and b-actin were
determined by RT-PCR from total RNA extracts. The gel shown is the representative re-
sult of three separate experiments.
These results indicate some structural requirements of
yakuchinone analogues for the inhibition of LPS-induced
NO production in macrophages as follows: (1) the enone and B have been reported to be strong inhibitors of COX-2
moiety, especially the conjugated double bond located be- and iNOS expression through the suppression of NF-kB acti-
tween aromatic ring and carbonyl group is essential; (2) the vation in 12-O-tetradecanoylphorbol-13-acetate (TPA)
structure with diarylhexa type showed the most potent activ- treated mouse skin.14) Here, we report the inhibitory effects
ity; (3) the diaryl structure tends to increase activity. To elu- of synthetic yakuchinone analogues on iNOS expression in
cidate the activity mechanisms of active synthetic analogues, activated macrophages. These results imply that the yakuchi-
we examined the effects of diaryl compounds 1—6 on the none analogues may have anti-inflammatory and cancer
expression of iNOS protein and mRNA in LPS-activated chemopreventive potential through the similar mechanism of
RAW 264.7 cells. The induced iNOS was detected in West- yakuchinones reported.
ern blot analysis after 20 h incubation with 1 mg/ml of LPS.
In conclusion, we prepared analogues of yakuchinone A
Compounds 1, 3, 4 and 6 with enone moiety apparently re- and B, and evaluated the inhibitory activity of NO production
duced amounts of iNOS expression at 5 mM (Fig. 2(A)). At in LPS-activated macrophages. Diarylhexanoid with enone
RT-PCR analysis, the level of iNOS mRNA expression was moiety located in linker between two aromatic rings showed
increased dramatically by LPS-activation for 6 h. The induc- the most potent activity. These active compounds suppressed
tion of mRNA was suppressed by the treatment of com- the LPS-induced upregulation of iNOS protein and mRNA.
pounds 1, 3, 4 and 6 at 5 mM as shown in Fig. 2(B). The pre- The optimized structures can be lead compounds for the de-
treatment of test compounds for 30 min prior to LPS-stimula- velopment of anti-inflammatory and cancer chemopreventive
tion showed more distinct inhibition of mRNA expression drugs.
than simultaneous treatment. The Western blot and RT-PCR
Experimental
analysis indicated that the inhibition of NO production by
Preparation of Yakuchinone Analogues The general synthetic route to
yakuchinone analogues was correlated with the suppression
of the iNOS protein and mRNA expression. Yakuchinone A
yakuchinone A, B, and their analogues is outlined in Chart 1. The commer-
cially available cinnamaldehyde I was protected with tert-butyldimethylsilyl

March 2006
379
(TBS) chloride to give the TBS ether II. The alkylation of II with Grignard
reagent, followed by oxidation of allylic alcohol functionality with MnO2
gave the enone IV. Deprotection of compound IV with tetrabutylammonium
fluoride (TBAF) in THF gave the enones 1,24) 3,25) 4,25) 626) and 7,27) which
were further reduced to the ketones 2,28) 525) and 829) by catalytic hydrogena-
tion. Reaction progresses were checked by TLC and the structures of final
products were confirmed by the analyses of NMR spectra.
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macrophage cell line, RAW 264.7 cells, in 10% fetal bovine serum (FBS)-
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by standard method. The membrane was probed with antibody for anti-
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Taq DNA polymerase (Promega, Madison, WI, U.S.A.) and primers (sense 24) Itokawa H., Ajyma R., Ikuta A., Phytochemistry, 21, 241—243 (1982).
and antisense). The sense and antisense primers for iNOS were 5ꢅ-ATGTC- 25) 1-(4-Hydroxy-3-methoxyphenyl)-6-phenylhex-1-en-3-one (>B>3): 1H-
CGAAGCAAACATCAC-3ꢅ and 5ꢅ-TAATGTCCAGGAAGTAGGTG-3ꢅ, re-
spectively. The sense and antisense primers for b-actin were 5ꢅ-TGTGATG-
GTGGGAATGGGTCAG-3ꢅ and 5ꢅ-TTTGATGTCACGCACGATTTCC-3ꢅ,
respectively. The PCR amplification was performed under following condi-
tions; 25 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s
and extension at 72 °C for 30 s, using thermal cycler (Gene Amp PCR sys-
tem 2400: Applied Biosystems, Foster City, CA, U.S.A.). The amplified
PCR products were separated on 1% agarose gel.
NMR (300 MHz, CDCl3) d 7.36 (1H, d, Jꢀ16.1), d 7.24—7.10 (5H,
m), d 7.00 (1H, dd, Jꢀ8.1, 1.8), d 6.96 (1H, d, Jꢀ1.8), d 6.84 (1H, d,
Jꢀ8.1), d 5.82 (1H, s), d 3.86 (3H, s), d 2.64—2.56 (4H, m), d
2.00—1.90 (2H, m).
(E)-1-(4-Hydroxy-3-methoxyphenyl)-5-phenylpent-1-en-3-one
(4):
1H-NMR (300 MHz, CDCl3) d 7.48 (1H, d, Jꢀ16.1), d 7.32—7.18
(5H, m), d 7.07 (1H, dd, Jꢀ8.2, 1.9), d 7.03 (1H, d, Jꢀ1.9), d 6.92
(1H, d, Jꢀ8.2), d 6.59 (1H, d, Jꢀ16.1), d 5.90 (1H, s), d 3.93 (3H, s),
d 3.00 (4H, m).
Acknowledgements This work was supported by the Korea Research
Foundation grant funded by the Korean Government (Grant No. R06-2002-
015-01001-0) and grant of the Research Center for Women’s Diseases from
the Korea Science and Engineering Foundation (KOSEF).
1-(4-Hydroxy-3-methoxyphenyl)-5-phenylpentan-3-one (5): 1H-NMR
(300 MHz, CDCl3) d 7.20—d 7.06 (5H, m), d 6.74 (1H, d, Jꢀ7.9), d
6.59 (1H, d, Jꢀ1.9), d 6.56 (1H, dd, Jꢀ7.9, 1.9), d 5.41 (1H, s), d
3.78 (3H, s), d 2.81 (2H, t, Jꢀ7.4), d 2.74 (2H, t, Jꢀ7.1), d 2.66—
2.58 (4H, m).
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