A new lead compound for abscisic acid biosynthesis inhibitors targeting 9-cis-epoxycarotenoid dioxygenase

Article abstract of DOI:10.1016/j.bmcl.2004.04.035

9-cis-Epoxycarotenoid dioxygenase (NCED), a key enzyme in abscisic acid (ABA) biosynthesis, cleaves the olefinic double bond of 9-cis-epoxycarotenoid. Several analogues of nordihydroguaiaretic acid (NDGA) were designed and synthesized, and their efficacy

Full text of DOI:10.1016/j.bmcl.2004.04.035

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3033–3036
A new lead compound for abscisic acid biosynthesis inhibitors
targeting 9-cis-epoxycarotenoid dioxygenase
Sun-young Han,a,b Nobutaka Kitahata,a,b Tamio Saito,c Masatomo Kobayashi,d
Kazuo Shinozaki,c Shigeo Yoshidaa and Tadao Asamia,*
aRIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan
bDepartment ofBiological and Environmental Sciences, Graduate School ofScience and Engineering, Saitama University,
Shimo-Okubo 255, Saitama 338-8570, Japan
cRIKEN Tsukuba Institute, Koyadai 3-1-1, Ibaraki, Tsukuba 305-0074, Japan
dRIKEN Tsukuba Institute BioResource Center, Koyadai 3-1-1, Ibaraki, Tsukuba 305-0074, Japan
Received 17March 2004; revised 14 April 2004; accepted 15 April 2004
Abstract—9-cis-Epoxycarotenoid dioxygenase (NCED), a key enzyme in abscisic acid (ABA) biosynthesis, cleaves the olefinic
double bond of 9-cis-epoxycarotenoid. Several analogues of nordihydroguaiaretic acid (NDGA) were designed and synthesized, and
their efficacy as inhibitors of NCED was examined. One of the synthesized compounds (20) was found to be an inhibitor of this
enzyme, and inhibited ABA accumulation and stomatal closing, suggesting that 20 should be ABA biosynthesis inhibitor.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Plants can respond to environmental stresses, such as
drought, cold, and high salt, and can control aspects of
their growth and development. One important regulator
of these responses is plant hormone abscisic acid
(ABA).1–3 The endogenous concentration of ABA
increases over 10-fold within a few hours of drought
stress and decreases dramatically to normal levels
following rehydration.4 In higher plants ABA is syn-
thesized via oxidative cleavage of epoxycarotenoids,5–7
which is the key regulatory step in ABA biosynthesis in
response to environmental stresses (Fig. 1).8–10 The
Figure 1. Cleavage of epoxycarotenoids by NCED.
enzyme, 9-cis-epoxycarotenoid dioxygenase (NCED),
catalyzes the cleavage of 9-cis-epoxycarotenoids to
apocarotenoid (C25) and xanthoxin (C15)8 and is
up-regulated by water-deficit stress.11
biosynthesis pathway. Since carotenoids are the precur-
sors of ABA in plants, carotenoid biosynthesis inhibitors
should also prevent the biosynthesis of ABA.13;16;17
Fluridone and norflurazon have been used to identify
ABA functions in plants as ABA biosynthesis inhibi-
tors.12–15 Both inhibit phytoene desaturase, which con-
verts phytoene to phytofluene in the carotenoid
However, the inhibition of carotenoid biosynthesis using
fluridone and norflurazon causes lethal damage during
plant growth because carotenoids play an important role
in protecting photosynthetic organisms against photo-
oxidation damage and absorb light energy in plants.18
Therefore, the use of these phytoene desaturase inhibi-
tors in the investigation of ABA functions is limited to
narrow physiological aspects.
Keywords: Abscisic acid; Plant hormone; Inhibitor; Carotenoid; Di-
oxygenase; Biosynthesis.
* Corresponding author. Tel.: +81-48-467-9525; fax: +81-48-462-4674;
e-mail: tasami@riken.jp
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.035

3034
S. Han et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3033–3036
In view of the indispensable nature of carotenoids and
the importance of ABA functions in plants, it is
worthwhile synthesizing and evaluating specific inhibi-
tors of ABA biosynthesis that would be useful tools
for functional studies of ABA biosynthesis and the
effects of ABA in higher plants as was seen in the
case of brassinosteroid biosynthesis inhibitors.19–22 In
this context, we started finding ABA biosynthesis
inhibitors.
2. Results and discussion
In order to develop novel specific ABA biosynthesis
inhibitors, NCED is an attractive target because it is the
key regulatory enzyme in the ABA biosynthesis path-
way.23;24 Therefore, to find a lead compound for ABA
biosynthesis inhibitor, firstly we tested the inhibitory
activity of NDGA (nordihydroguaiaretic acid) against
NCED in vivo because NDGA was reported to decrease
ABA content in treated-plants.25 An enzymaic test was
performed according to the method described by Iuchi
et al.26 When NCED expressed in E. coli was incubated
with 90-cis-neoxanthin, the main products of this reac-
tion were C25-apocarotenoid and xanthoxin. To quan-
tify the molar amounts of the products, the C25-products
were analyzed by HPLC using all-trans-violaxanthin as
an internal standard. In this test we found that NDGA
inhibited about 45% of NCED activity at 100 lM. Then
we started the modification of the chemical structure of
NDGA to increase its specificity as ABA biosynthesis
inhibitor because NDGA has been reported to inhibit
lipoxygenases and several events in cells27;28 and there-
fore it is easily supposed that NDGA should not be a
specific ABA biosynthesis inhibitor. Whitman et al.
reported that the alkylation of the hydoxyl groups on
the phenyl moiety of NDGA reduced its inhibitory
activity against lipoxygenases.29 On the basis of this
observation, we anticipated that the alkylation of
phenol group could reduce the lipoxygenase inhibitory
activity of NDGA and therefore strengthen the speci-
ficity of NDGA derivatives to ABA biosynthesis
inhibition. In our preliminary test, chemicals possessing
nitrogen atom instead of carbon atom in the chain
connecting two phenyl groups showed significant
activity. Thus, in order to develop ABA biosynthesis
inhibitors targeting NCED, a number of compounds
were synthesized as summarized in Figures 2 and 3.30–33
4-{[2-(4-Hydroxyphenyl)ethylimino]methyl}benzene-1,2-
diol (1) was prepared by the condensation of 4-(2-amino-
ethyl)phenol with 3,4-dihydroxybenzaldehyde (Fig. 2).
Compounds 2 and 3 were prepared by the same method
used for compound 1. 4-{[2-(4-Hydroxyphenyl)ethyl-
amino]methyl}benzene-1,2-diol (4) was produced from
the reduction of compound 1 using sodium borohydride
(NaBH4). Compounds 5, 6, 11, and 12 were prepared
using the method described for compound 4. The
reaction of 4-[2-(3,4-dimethoxy-benzylamino)ethyl]-
phenol (5) with iodomethane in the presence of NaH
produced (3,4-dimethoxybenzyl)[2-(4-methoxy-phenyl)-
ethyl]methylamine (7). Compound 8 was prepared by
the method described for compound 7.
Figure 2. Syntheses of designed compounds and structure of NDGA:
(A) benzene, reflux; (B) NaBH4, EtOH; (C) 60% NaH, alkyl halide,
DMF.
Figure 3. Syntheses of designed compounds: (A) (1) NEt3, methyl
chloroformate, )7 ꢁC, THF, (2) NaBH4, MeOH, 1 h, 10 ꢁC, THF, (3)
1 N HCl; (B) (1) DMSO, CH2Cl2, oxaly chloride, )78 ꢁC, (2) starting
material, CH2Cl2, (3) NEt3; (C) benzene, reflux; (D) EtOH, NaBH4;
(E) 60% NaH, alkyl halide, DMF.
Compounds 17 and 18 were prepared from the reaction
of (E)-3-(3,4-dimethoxy-phenyl)propenal (14) with the
corresponding amines, followed by reduction using
NaBH4 (Fig. 3). Compounds 19 and 20 were also pre-
pared from the reaction of [3-(3,4-dimethoxyphenyl)-
allyl](4-fluorobenzyl)amine (18) with iodomethane or
methyl bromoacetate using the method described for 7.
1
The structures of the compounds were confirmed by H
NMR, 13C NMR, and elemental analysis.
In NCED test a few chemicals showed inhibitory
activity among the synthesized chemicals. In Table 1

S. Han et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3033–3036
3035
Table 1. NCED inhibitory activity of synthesized compounds
IC50 (lM)
inhibitor of stomatal closure. The co-application of
10 lM ABA and 100 lM 20 produced a response similar
to that with ABA alone (Fig. 4). In contrast, NDGA
abolished the promotion of stomatal closure when
co-applied with ABA, although it reduced stomatal
closure in 0.4 M mannitol. This observation should be
due to the side effects of NDGA.
NDGA
6
183 28
160 35
87 14
7
17
20
145 19
55
9
NCED activity was estimated in the presence of 23 lM 90-cis-neo-
xanthin at pH 7.0 at 20 ꢁC. The product, C25-apocarotenoid, was
analyzed by HPLC at 440 nm wavelength.
The ABA content of spinach leaves was determined
after incubation in 0.4 M mannitol. Spinach leaf slices
were incubated for 2 h in 10 mM HEPES (pH 6.5 with
KOH) with inhibitor at 100 lM. Subsequently, the leaf
slices were incubated for 4 h in 10 mM HEPES with
0.4 M mannitol, and with or without inhibitor. Fol-
lowing incubation, the slices were homogenized and
extracted in 80% methanol with 200 mg/L BHT. The
ABA purified by HPLC (Shiseido CAPCELL PAK C18
column, 4.6 · 250 mm) was methylated with diazome-
thane. GC–MS analysis was carried out on a JEOL
Auto-mass JMS-AM 150 mass spectrometer connected
to a Hewlett-Packard 5890-A-II gas chromatograph
with a capillary DB-1 (J&W Scientific, Folsom, CA)
column (0.25 mm · 15 m, 0.25 mm film thickness). [13C2]-
ABA was used as an internal standard.36 The GC–SIM
responses at m=z 190 (base peak of ABA) and m=z 192
(base peak of 13C2-ABA) were monitored.
chemicals possessing NCED inhibitory activity are lis-
ted. The inhibitory activity was estimated by comparing
the amount of resulted C25-apocarotenoid in chemical-
treated reaction medium and nontreated reaction med-
ium. Among the chemicals compound20 showed the
most potent activity. This indicates that NDGA, com-
pounds 6, 7, 17, and 20 are NCED inhibitors and
therefore can be ABA biosynthesis inhibitors in vivo.
Next, to test the efficacy of NDGA and synthesized
chemicals as an ABA biosynthesis inhibitor, stomatal
closure assay was examined. In guard cells, ABA regu-
lates stomatal apertures by inhibiting stomatal opening
and inducing stomatal closure in response to drought
stress.34 The ABA levels increased 10-fold more in
water-deficit stressed tissues than in nonstressed tis-
sues.35 A high concentration of mannitol is expected to
mimic the effect of water-deficit stress and increase the
ABA concentration. That is, incubation in 0.4 M man-
nitol imposes osmotic stress on guard cells. As illus-
trated in Figure 4, we treated epidermal cells from
spinach with 100 lM NCED inhibitors, NDGA or
10 lM ABA to assess the effect of this inhibitor on
stomatal closure. Treatment with 0.4 M mannitol caused
stomatal closure similar to that seen with 10 lM ABA.
Under these conditions, NDGA, compounds 7, 17, and
20 inhibited stomatal closure. Other compounds were
inactive. Compound 20 was found to be the most potent
NDGA, compounds 7, 17, and 20 at 100 lM were
examined for ABA biosynthesis inhibitory activities un-
der osmotic stress three times. As shown in experiment 1
of Table 2, the ABA content in leaf slices treated with
0.4 M mannitol increased 7.8-fold to 22.7 ng/g FW in
10 mM HEPES (pH 6.5). Treatment with compounds 7
and 17 resulted in no significant reductions in ABA
biosynthesis under osmotic stress. Nevertheless, com-
pound 20, which results from the replacement of the
N-methyl group of compound 19 by N-acetic acid methyl
ester, inhibited about 38% of ABA accumulation
(14.0 ng/g FW). NDGA was less active than compound
20. These results demonstrate that compound 20 is the
strongest ABA biosynthesis inhibitor among the tested
compounds. The test was repeated in triplicate under the
same experimental conditions and similar results were
obtained as shown in Table 2. In a previous study,25
NDGA significantly inhibited ABA accumulation under
similar conditions as used here; however, this compound
did not show clear inhibitory activity in our experiments.
Table 2. The effect of compounds on ABA accumulation in spinach
leaf slices
Experiment
1
2
3
ABA
(ng/g FW)
ABA
(ng/g FW)
ABA
(ng/g FW)
Control 2.90
0.4 M mannitol 8.4 22.717 134.9
11.3
17.8
100 lM NDGA
100 lM 7
100 lM 17
100 lM 20
20.5
22.3
26.1
14.0
165.3
174.4
128.5
123.5
.1168.7137
82.8
Figure 4. The effect of compounds on closing of stomatal aperture.
Epidermal strips were immersed in 0.4 M mannitol with or without
100 lM inhibitors or 10 lM ABA and incubated for 3 h. The data show
the mean SE of three independent experiments measuring at least 100
stomata. All solutions included DMSO at ca. 0.02%.
65.6
Spinach leaf slices were treated with each 100 lM of NDGA, com-
pound 7, 17, or 20 in 0.4 M mannitol solution (10 mM HEPES buffer).
FW ¼ fresh weight.

3036
S. Han et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3033–3036
13. Thompson, A. J.; Jackson, A. C.; Parker, R. A.; Morpeth,
D. R.; Burbidge, A.; Taylor, I. B. Plant Mol. Biol. 2000,
42, 833–845.
14. Moreno-Fonseca, L. P.; Covarrubias, A. A. Plant Mol.
Biol. 2001, 45, 501–515.
15. Ullah, H.; Chen, J.-G.; Wang, S.; Jones, A. M. Plant
Physiol. 2002, 129, 897–907.
16. Gamble, P. E.; Mullet, J. Eur. J. Biochem. 1986, 160, 117–
121.
Eventually we found that 20 should be an ABA bio-
synthesis inhibitor. On the basis of this study, it can be
estimated that the essential structures required for
NCED inhibitory activity of this chemical group should
be an amine moiety, which is substituted with phen-
ylalkyl group, phenylalkenyl group, and carbo-
alkoxyalkyl group. As there still remains a lot of room
for structural modification of this chemical group, the
further study on structure–activity relationships will
lead us to find more potent and specific NCED inhibi-
tors. The result thus obtained will play an important
role for designing new inhibitors. Recently ‘chemical
genetics’ has been used as a new tool for dissecting and
understanding biological systems in plants.37;38 Devel-
opment of chemicals that induce phenotypes of interest
is now emerging as a useful way to study biological
systems in plants and this would be a complement to
classical biochemical and genetic methods. In this con-
text ABA biosynthesis inhibitor could be a new player in
this field.
17. Yoshioka, T.; Endo, T.; Satoh, S. Plant Cell Physiol. 1998,
39, 307–312.
18. Britton, G. In Carotenoids; Britton, G., Liaaen-Jensen, S,
Pfander, H., Eds.; Birkhauser: Austin, 1998; pp 117–121.
19. Asami, T.; Yoshida, S. Trends Plant Sci. 1999, 4, 348–353.
20. Asami, T.; Min, Y. K.; Fujioka, S.; Takatsuto, S.; Nagata,
N.; Yamagishi, K.; Murofushi, N.; Yamaguchi, I.; Yosh-
ida, S. Plant Physiol. 2000, 123, 93–100.
21. Nagata, N.; Min, Y. K.; Nakano, T.; Asami, T.; Yoshida,
S. Planta 2000, 211, 781–790.
22. Nagata, N.; Asami, T.; Yoshida, S. Plant Cell Physiol.
2001, 42, 1017–1023.
23. Burbidge, A.; Grieve, T.; Jackson, A.; Thompson, A.;
Taylor, I. J. Exp. Bot. 1997, 48, 2111–2112.
24. Iuchi, S.; Kobayashi, M.; Taji, T.; Naramoto, M.; Seki,
M.; Kato, T.; Tabata, S.; Kakubari, Y.; Yamaguchi-
Shinozaki, K.; Shinozaki, K. Plant J. 2001, 27, 325–333.
25. Creelman, R. A.; Bell, E.; Mullet, J. E. Plant Physiol.
1992, 99, 1258–1260.
26. Iuchi, S.; Kobayashi, M.; Yamaguchi-Shinozaki, K.;
Shinozaki, K. Plant Physiol. 2000, 123, 553–562.
27. Kemal, C.; Louis-Flamberg, P.; Krupinski-Olsen, R.;
Shorter, A. L. Biochemistry 1987, 26, 7064–7072.
28. Merigout, P.; Kepes, F.; Perret, A.-M.; Satiat-Jeunemai-
tre, B.; Moreau, P. FEBS Lett. 2002, 518, 88–92.
29. Whitman, S.; Gezginci, M.; Timmermann, B. N.; Holman,
T. R. J. Med. Chem. 2002, 45, 2659–2661.
Acknowledgements
We thank Mrs. M. Kobayashi and A. Hanada (RIKEN,
Plant Science Center) for helpful technical assistance in
the ABA measurements. This research was supported in
part by the Bioarchitect Research Program at RIKEN,
funded by the Science and Technology Agency of Japan.
References and notes
1. Shinozaki, K.; Yamaguchi-Shinozaki, K. Molecular Res-
ponses to Cold, Drought, Heat and Salt Stress in Higher
Plants; R.G. Landes Company: Basel, 1999.
2. Zhu, J. K. Trends Plant Sci. 2001, 6, 66–71.
3. Finkelstein, R. R.; Gampala, S. S. L.; Rock, C. D. Plant
Cell 2002, S15–S45.
30. Soai, K.; Yokoyama, S.; Mochida, K. Synthesis 1987,
647–648.
31. Shishido, K.; Yamashita, A.; Hiroya, K.; Fukumoto, K.
J. Chem. Soc., Perkin Trans. 1 1990, 469–475.
32. Cushman, M.; He, H.-M.; Lin, C. M.; Hamel, E. J. Med.
Chem. 1993, 36, 2817–2821.
4. Zeevaart, J. A. D. Plant Physiol. 1980, 66, 672–678.
5. Zeevaart, J. A. D.; Creelman, R. A. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 1988, 39, 439–473.
6. Parry, A. D.; Babiano, M. J.; Horgan, R. Planta 1990,
182, 118–128.
7. Li, Y.; Walton, D. C. Plant Physiol. 1990, 92, 551–559.
8. Schwartz, S. H.; Tan, B. C.; Gage, D. A.; Zeevaart, J. A.
D.; McCarty, D. R. Science 1997, 276, 1872–1874.
9. Tan, B. C.; Schwartz, S. H.; Zeevaart, J. A. D.; McCarty,
D. R. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12235–
12240.
33. Tanaka, N.; Tamai, T.; Mukaiyama, H.; Hirabayashi, A.;
Muranaka, H.; Akahane, S.; Miyata, H.; Akahane, M.
J. Med. Chem. 2001, 44, 1436–1445.
34. Uno, Y.; Furihata, T.; Abe, H.; Yoshida, R.; Shinozaki,
K.; Yamaguchi-Shinozaki, K. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 11632–11637.
35. Creelman, R. A.; Zeevaart, J. A. D. Plant Physiol. 1985,
77, 25–28.
36. Asami, T.; Sekimata, K.; Wang, J. M.; Yoneyama, K.;
Takeuchi, Y.; Yoshida, S. J. Chem. Res. (S) 1999, 658–
659.
10. Qin, X.; Zeevaart, J. A. D. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 15354–15361.
11. Bray, E. A. Plant Cell Environ. 2002, 25, 153–161.
12. Grappin, P.; Bouinot, D.; Sotta, B.; Miginiac, E.; Jullien,
M. Planta 2000, 210, 279–285.
37. Asami, T.; Nakano, T.; Nakashita, H.; Sekimata, K.;
Shimada, Y.; Yoshida, S. J. Plant Growth Regul. 2003, 22,
336–349.
38. Blackwell, H. E.; Zhao, Y. Plant Physiol. 2003, 133, 448–
455.