Selectivity improvement of an azole inhibitor of CYP707A by replacing the monosubstituted azole with a disubstituted azole

Article abstract of DOI:10.1016/j.bmcl.2010.07.067

The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme (CYP701A), inhibits multiple P450 enzymes including ABA 8′-hydroxylase (CYP707A), a key enzyme in ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron atom via sp² nitrogen and interacting with surrounding protein residues through a lipophilic region. We hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orientation for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI because the added functional group may interact with the active site to restrict orientation of the molecule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acrylate, strongly inhibited recombinant CYP707A3, with no growth-retardant effect.

Full text of DOI:10.1016/j.bmcl.2010.07.067

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5506–5509
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selectivity improvement of an azole inhibitor of CYP707A by replacing the
monosubstituted azole with a disubstituted azole
a
a
a
b
Yasushi Todoroki a, , Kumi Naiki , Hikaru Aoyama , Minaho Shirakura , Kotomi Ueno ,
*
Masaharu Mizutani b, Nobuhiro Hirai c
a Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
b Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
c Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The plant growth-retardant uniconazole (UNI), a triazole inhibitor of gibberellin biosynthetic enzyme
(CYP701A), inhibits multiple P450 enzymes including ABA 80-hydroxylase (CYP707A), a key enzyme in
ABA catabolism. Azole P450 inhibitors bind to a P450 active site by both coordinating to the heme-iron
atom via sp2 nitrogen and interacting with surrounding protein residues through a lipophilic region. We
hypothesized that poor selectivity of UNI may result from adopting a distinct conformation and orienta-
tion for different active sites. Based on this hypothesis, we designed and synthesized novel UNI analogs
with a disubstituted azole ring (DSI). These analogs were expected to have higher selectivity than UNI
because the added functional group may interact with the active site to restrict orientation of the mole-
cule in the active site. DSI-505ME and DSI-505MZ, which have an imidazolyl group with a methyl 5-acry-
late, strongly inhibited recombinant CYP707A3, with no growth-retardant effect.
Received 21 May 2010
Revised 14 July 2010
Accepted 16 July 2010
Available online 21 July 2010
Keywords:
Plant growth regulator
P450 inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
Uniconazole is an azole-containing cytochrome P450 inhibitor
developed as a plant growth retardant in the 1980s.1,2 UNI has
since been used as a plant growth regulator in agriculture and hor-
ticulture. The main site of action of UNI is suggested to be ent-kau-
rene oxidase (CYP701A), which catalyzes the three-step oxidation
of ent-kaurene to ent-kaurenoic acid,3 a biosynthetic precursor of
the plant hormone gibberellin (GA). This has prompted researchers
to use UNI as a chemical tool inhibiting GA biosynthesis. However,
UNI also inhibits brassinosteroid biosynthesis4,5 and alters the
level of other plant hormones, such as auxins, cytokinins, ethylene,
and abscisic acid (ABA).6 Recently, Kitahata et al.7 and Saito et al.8 re-
vealed that UNI strongly inhibits ABA 80-hydroxylase (CYP707A9,10),
a key enzyme in ABA catabolism (Fig. 1).
Azole-type P450 inhibitors including UNI bind to the target
P450 active site by both coordinating to the heme-iron atom and
interacting with surrounding protein residues. Because heme
co-ordination is a common property of azole-containing inhibitors,
their affinity, and specificity for individual P450 enzymes depend
on structural properties other than the azole group. UNI may be
small and flexible enough to embed itself into various substrate-
binding pockets, where UNI may adopt a distinct conformation
and orientation for different active sites. Ketoconazole, a well-
known antifungal drug, binds to various P450 active sites in different
conformations and orientations ( Fig. 2). In homology models for
CYP707A3 and CYP701A3, the active site void spaces that are close
to the heme differ in size and shape (Fig. 3). Although these models
have low reliability because of the low sequence similarity (ꢀ25%) to
ABA catabolism
HO
O
ABA 8'-hydroxylase
(CYP707A)
OH
OH
CO2H
CO2H
O
S-(+)-ABA
Cl
8'-hydroxy-ABA
5'
4'
3'
6'
1'
3
HO
2'
1"
1
2"
2
N
N
3"
4"
5"
N
S-(+)-UNI
ent-kaurene oxidase
(CYP701A)
CO2H
ent-kaurene
ent-kaurenoic acid
GA biosynthesis
* Corresponding author. Tel./fax: +81 54 238 4871.
E-mail address: aytodor@agr.shizuoka.ac.jp (Y. Todoroki).
Figure 1. UNI is a potent inhibitor of ABA 80-hydroxylase (CYP707A) and ent-
kaurene oxidase (CYP701A).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.067

Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5506–5509
5507
Figure 2. Overlaid structures of ketoconazole-binding P450 active sites: black,
3LD6 (human CYP51); pink, 2V0M (human CYP3A4); blue, 1JIN (bacterial
CYP107A1); green, 2JJP (bacterial CYP113A1). The figures were manually drawn
using Chimera after automatic overlay.
template proteins, we speculated that UNI may inhibit various P450
enzymes in a manner similar to the case of ketoconazole. Based on
this speculation, in previous works, we generated selective inhibi-
tors of CYP707A by elongating or conformationally freezing the
UNI molecule.11,12 In this study, we focused on restricting azole ori-
entation by using a disubstituted azole ring for the monosubstituted
ring of UNI. A functional group introduced into the azole of UNI can
have a new interaction with the active site residues, causing advan-
tageous or disadvantageous reorientation in binding to the active
site. This reorientation depends on the active site profile, which
will be different for different P450 enzymes. We designed and
synthesized seven UNI analogs with a disubstituted imidazole
(DSI) (Fig. 4), which has an oxidized functional group at C200 or C500,
in hopes of electrostatic or hydrogen-bonding interactions in addi-
Figure 3. Ligand binding sites in the homology models of CYP707A3 (top) and
CYP701A3 (bottom). Molecular surfaces were calculated and displayed with PyMOL.
Cl
Cl
HO
N
OH
HO
ref. 11
iii
iv
DSI-505
DSI-502
DSI-503
+
(trace, E/Z mixture)
N
MeO2C
N
DSI-201
N
DSI-505MZ (8%)
Cl
Cl
Cl
Cl
HO
HO
O
HO
O
O
MeO2
C
HO
i
N
DSI-505
(6%, E/Z mixture)
N
N
DSI-501
+
22%
N
N
N
N
N
DSI-501
DSI-502
DSI-503
DSI-505ME (32%)
Cl
Cl
Cl
O
HO
ii
HO
DSI-504
O
MeO2C
HO2C
89%
N
N
N
N
N
N
Scheme 1. Synthesis of DSI. Reagents and conditions: (i) NaH, MeI, DMF, 0 °C, 22%;
(ii) NaH, EtI, DMF, 0 °C, 89%; (iii) NaH, (CF3CH2O)2P(O)CH2CO2Me, THF, 0–75 °C, 8%
(DSI-505MZ) and trace (DSI-505); (iv) NaH, (CF3CH2O)2P(O)CH2CO2Me, THF, 0 °C to
rt, 32% (DSI-505ME) and 6% (DSI-505).
DSI-504
DSI-505M
DSI-505
Figure 4. Chemical structures of DSI.

5508
Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5506–5509
Table 1
Inhibitory activity of DSI against recombinant CYP707A3 and rice seedling elongation
Compound
Structural properties
CYP707A3
Rice growth
IC50 M)
2-Az
3-OX
Inhibitionc (%)
KI (nM)
(l
N
N
N
N
N
UNIa
H
100
10
0.18
2.3
IMIb
H
H
100
0.6
N
d
DSI-201
OH
31
–
>100
N
HO
N
DSI-501
DSI-502
DSI-503
H
95
91
95
160
240
190
5.4
N
O
O
N
H
10
N
N
CH3
9.6
N
O
N
DSI-504
C2H5
62
–
32
N
MeO2C
MeO2C
N
N
DSI-505MZ
H
98
220
>100
N
N
DSI-505ME
DSI-505
H
H
100
120
>100
>100
HO2C
N
31
–
N
The values are averages of two independent experiments.
a
Ref. 11.
Ref. 13.
b
c
Inhibition ratio of compounds (10
Not measured.
l lM).
M) in the 80-hydroxylation of ABA (5
d
tion to steric effects. We checked the enzyme selectivity of the DSI by
examining both the inhibitory activity of DSI compounds against re-
combinant CYP707A and the plant growth-retardant effect caused
mainly by inhibition of gibberellin biosynthetic enzymes including
CYP701A and CYP88A.
DSI-201, DSI-501, and DSI-502 were prepared by a method
reported earlier.11 Oxidation of DSI-201 with MnO2 gave not the
aldehyde but the tautomeric isomer, the cyclic hemiacetal, which
was previously reported as a conformationally restricted UNI
analog.11 Other disubstituted UNI analogs were synthesized from
DSI-501 according to Scheme 1. Methylation and ethylation of
DSI-501 with methyl iodide and ethyl iodide, respectively, gave
DSI-503 and DSI-504 with 22% and 89% yield. DSI-505MZ and
DSI-505ME were prepared by a Horner–Emmons reaction of DSI-
502 with the phosphonoate. This reaction gave a mixture of E
and Z isomers of DSI-505M, DSI-505ME, and DSI-505MZ. The major
product was the E-isomer at room temperature, whereas it was
converted into the Z-isomer by rising the reaction temperature to
75 °C. The free acid DSI-505 yielded a E/Z mixture (1:2) by partial
alkaline hydrolysis during aqueous work-up of the reaction.
The inhibitory activity against ABA 80-hydroxylase was exam-
ined using recombinant Arabidopsis CYP707A3 co-expressed with
Arabidopsis P450 reductase (ATR2) in Escherichia coli.12 The activity
was evaluated based on the decrease in the enzymatic product,
phaseic acid, caused by addition of a test compound at a concentra-
tion two times higher (10
lM) than the substrate S-(+)-ABA
(5
l
M).12 For compounds showing >90% activity, the inhibition
constant (KI) was measured. The plant growth-retardant effect
was examined using rice seedlings to estimate the concentration
giving 50% inhibition (IC50). The results are summarized in Table 1.
DSI-201 was much less effective in both assays. This compound
is substituted at C200 adjacent to N300, which actually coordinates to
the heme iron. The C200 hydroxymethyl group may interfere with
N–Fe co-ordination. DSI-501, DSI-502, and DSI-503 were less

Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5506–5509
5509
We tested the effect of DSI-505ME on early growth of lettuce in
the presence and absence of ABA. DSI-505ME exhibited no signifi-
cant effect at the tested concentrations of 0.1–100
enhanced the effect of 10 M ABA upon simultaneous application
of 100 M inhibitor (Fig. 5). This suggests that DSI-505ME acts as
lM, whereas it
l
l
an inhibitor of CYP707A in vivo as well as in vitro to keep a high
concentration of ABA.
Finally, we found a more selective azole inhibitor than UNI by
replacing the monosubstituted azole with the disubstituted one.
This design strategy may be useful for increasing the selectivity
of azole CYP inhibitors.
Acknowledgments
We thank Toray Industries Inc., Tokyo, Japan, for the gift of (+)-
ABA. Part of this research was carried out using an instrument at
the Center for Instrumental Analysis of Shizuoka University. This
research was supported by a Grant-in-Aid for Scientific Research
(No. 22580118) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
Supplementary data
Figure 5. Enhanced effect of DSI-505ME on ABA activity in an early growth assay of
Supplementary data (the synthetic procedure for new com-
pounds, their 1H NMR and high-resolution MS data, and the proce-
dures for homology modeling, enzyme assay, and biological assays
are given) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2010.07.067.
lettuce.
effective than UNI by a factor of 20–50 in both assays. The
hydroxymethyl, formyl, and methoxymethyl groups at C500 may
have a similar unfavorable effect on binding to the active sites of
CYP707A and CYPs involved in plant growth. DSI-504, with the
C500 ethoxymethyl group, was less effective than DSI-501, DSI-
502, and DSI-503 in both assays. This may be because of the larger
steric effect by substituents at both C500 and C3. DSI-505MZ and
DSI-505ME exhibited CYP707A inhibitory activity equivalent to
that of DSI-501, DSI-502, and DSI-503 in spite of its larger C500 sub-
stituent, whereas it had little inhibitory activity on rice seedling
growth. This suggests that the methyl acrylate moiety at C500 is tol-
erated in the CYP707A active site, but not in those of CYPs involved
in plant growth. Considering that DSI-505MZ and DSI-505ME were
less effective CYP707A inhibitors than UNI and the inhibitory
potency was largely independent of the acrylate E/Z geometry,
the acrylate at C500 may function as a steric obstacle, and neither
an electrostatic nor a hydrogen-bonding donor or acceptor, for
the amino acid residues in the active site; its sterically unfavorable
effect may be too small for CYP707A to allow the inhibitor azole to
orient to the heme iron, but too large for CYPs involved in plant
growth to allow it. On the other hand, the free acid DSI-505 (E/Z
mixture) exhibited little inhibitory activity in either assay. Because
the P450 active site is generally hydrophobic, the carboxylate may
not be accepted. We cannot discuss the structure–activity relation-
ships in greater detail due to the lack of structural characterization
of CYP707A and the CYPs involved in plant growth.
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