Discovery of Plant Growth Stimulants by C-H Arylation of 2-Azahypoxanthine

Article abstract of DOI:10.1021/acs.orglett.8b02407

A series of new AHX derivatives were synthesized by Pd-catalyzed C-H arylation. Their rice-growth-promoting activity was evaluated in vivo. Among the synthesized compounds, C8 phenyl-substituted AHX showed remarkable growth-promoting activity on rice. The

Full text of DOI:10.1021/acs.orglett.8b02407

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Discovery of Plant Growth Stimulants by C−H Arylation of
2‑Azahypoxanthine
Hiroyuki Kitano,† Jae-Hoon Choi,‡ Ayaka Ueda,† Hideto Ito,†,§ Shinya Hagihara,†,∥
Toshiyuki Kan,⊥ Hirokazu Kawagishi,*,‡,¶ and Kenichiro Itami*,†,§
†Institute of Transformative Bio-Molecules (WPI-ITbM) and Graduate School of Science, Nagoya University, Chikusa, Nagoya
464-8601, Japan
‡Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
§JST-ERATO, Itami Molecular Nanocarbon Project, Nagoya University, Chikusa, Nagoya 464-8602, Japan
∥Center for Sustainable Resource Science, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
⊥School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 Japan
¶Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
S
* Supporting Information
ABSTRACT: A series of new AHX derivatives were synthesized by Pd-
catalyzed C−H arylation. Their rice-growth-promoting activity was evaluated
in vivo. Among the synthesized compounds, C8 phenyl-substituted AHX
showed remarkable growth-promoting activity on rice. The present study
shows the power and significant opportunity of C−H functionalization
chemistry to rapidly transform biologically active natural products into more
active compounds.
forming fungus and a plant.2 More recently, we disclosed that
airy rings are naturally occurring ring-shaped thick growth
For necrotic spots of grasses, which are seen on the lawn molecular entities of this interaction are plant-growth-
stimulating compound 2-azahypoxanthine (AHX) and plant-
growth inhibitor imidazole-4-carboxamide (ICA) identified
from one of the fairy-ring-forming fungi, Lepista sordida.3
These compounds have been named “fairy chemicals” (FCs).4
Regulation of plant growth by FCs was observed in all the
plants tested, regardless of their species. Furthermore,
treatment of FCs increased the yields of rice, wheat, and
other various types of crops in greenhouse and/or field
experiments.3,5 We also reported that AHX and AOH are
endogenously produced in plants by a new purine metabolic
pathway.3b,6 Based on the results above, we have hypothesized
that FCs are a new family of plant hormone.7
(Figure 1). Sometimes fruiting bodies of fungi are also seen in
the fairy rings. The term “fairy rings” has its origin in the myths
and superstitions associated with their occurrence in the
Middle Ages. The first scientific article about this phenomenon
was reported in 1675.1 In 1995, Couch reported that this
mysterious ring is due to the interaction between a fairy-ring-
Although AHX has an interesting plant-growth activity, the
mode of action of how AHX enhances plant growth has been
poorly understood. Even the structure−activity relationship
(SAR) of AHX derivatives has not yet been defined due to the
difficulty in chemical derivatization of AHX. AHX has a 1,2,3-
triazin-4(3H)-one structure that is rarely seen in natural
products. There are only three hydrogen atoms to be
substituted in AHX (Figure 1a). Initially, we synthesized
N,N′-dialkyl AHX by a simple alkylation reaction and
evaluated their plant-growth-promoting activity (Figure 2).
However, most N,N′-dialkyl AHX did not show noticeable
Figure 1. Fairy chemical AHX. (a) Structure and features of AHX.
(b) Plant growth caused by AHX. Picture: fairy ring on the turfgrass
field, University of Shizuoka, Japan. Reprinted with permission by Dr.
Makoto Inai. Copyright Dr. Makoto Inai.
Received: July 28, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b02407
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Table 1. Pd/Cu-Mediated C−H Phenylation of DMB-
Protected AHX
Figure 2. Brief summary of SAR of AHX derivatives.
a
entry
deviation from standard conditions
NMR yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
50, 2a/2a′ = 1.4:1
52, 2a/2a′ = 1.4:1
49, 2a/2a′ = 1.1:1
43, 2a/2a′ = 1.4:1
46, 2a/2a′ = 1.3:1
21, 2a/2a′ = 2.5:1
44, 2a/2a′ = 1.9:1
43, 2a/2a′ = 1.7:1
17, 2a/2a′ = 3.3:1
17, 2a/2a′ = 2.4:1
37, 2a/2a′ = 1.3:1
1, 2a
growth-promoting activity (see Supporting Information (SI)
for details). From these results, we decided to focus on the
functionalization of the remaining C−H bonds of the
imidazole ring. Despite several preliminary attempts at C−H
functionalizations, such as halogenation and alkylation, the
instability of the AHX skeleton and the lower reactivity at a C8
position disturbed the derivatization of AHX. Therefore, an
alternative appropriate synthetic method was required for
obtaining C8-substituted AHX derivatives. Herein, we report
the application of C−H arylation chemistry to synthesize AHX
derivatives and the evaluation of their plant-growth-promoting
activity.8 The easy access to these derivatives allowed us to
rapidly discover a novel plant-growth stimulator that shows
activity stronger than that of the original AHX (Figure 2). This
compound might be a key for identifying the target of AHX.
Although extensive screening of reaction conditions was
conducted to synthesize arylated AHX in one step by metal-
catalyzed C−H arylation,9 none of the examined conditions
was successful (see SI for details). Thus, we decided to use a
protecting group for N−H moieties to accomplish the
synthesis (Scheme 1). We chose 2,4-dimethoxybenzyl
PCy3·HBF4 (10 mol %) was added
PtBu3·HBF4 (10 mol %) was added
BINAP (5 mol %) was added
Na2CO3 instead of K3PO4
K2CO3 instead of K3PO4
Cs2CO3 instead of K3PO4
DMA instead of DMF
1,4-dioxane instead of DMF
toluene instead of DMF
iodobenzene instead of 4a
without CuI
without Pd(OAc)2
1.1 mmol scale, reaction time 15 h
11, 2a/2a′ = 1.8:1
b
71 , 2a/2a′ = 2.6:1
a
Determined by 1H NMR using CH2Br2 as an internal standard.
Isolated yield.
b
independent reactions using isomerically pure 1 and 1′
revealed that both isomers have almost the same reactivity,
giving a mixture of 2a and 2a′ (1: 56% yield (2a/2a′ = 1.4:1),
1′: 38% yield (2a/2a′ = 1.2:1); see SI for details). These
results suggest that migration of DMB group between N7 and
N9 positions occurs during the reaction.
Scheme 1. Protection of AHX with a DMB Group
Listed in Table 1 are the effects of reaction parameters in the
Pd-catalyzed C−H arylation of DMB-protected AHX. The
addition of ligands such as PCy3, PtBu3, and BINAP had no
beneficial effect on the yield of 2a/2a′ (43−52% yield; entries
2−4). The product yield depends on the base employed,
increasing in the order of K2CO3 < Cs2CO3 < Na2CO3 <
K3PO4 (entries 1, 5, 6, and 7). The polarity of solvents is also
important. Whereas DMF and DMA resulted in moderate
yields (50 and 43% yields; entries 1 and 8), the reactions in
1,4-dioxane and toluene showed significant decrease in yield
(entries 9 and 10). The use of iodobenzene instead of
bromobenzene resulted in the decrease of the yield of 2a/2a′
(37% yield; entry 11). Whereas the absence of CuI almost shut
down the reaction (entry 12), the absence of Pd(OAc)2 still
gave the target material in 11% yield (entry 13), suggesting
that copper-mediated (non-palladium-catalyzed) C−H aryla-
tion also occurs in this reaction. Notably, conducting the
reaction in larger scale increased the yield (71% yield; entry
14).
Using the optimized conditions, we synthesized a range of
arylated AHX derivatives via Pd-catalyzed arylation with
bromoarenes 4a−h (Scheme 2). The reactions with sterically
hindered bromoarenes, such as 2-bromotoluene (4b) and 1-
bromonaphthalene (4f), gave the corresponding products in
good yields (92 and 78%). The reactions with bromobenzene
(4a), 3-bromotoluene (4c), 4-bromotoluene (4d), 4-bromo-
biphenyl (4e), 1-bromo-2-fluorobenzene (4g), and 1-bromo-3-
chlorobenzene (4h) resulted in moderate yields of C−H
(DMB) as a protecting group because it can be deprotected
easily under mild oxidative conditions. First, we protected the
two nitrogen atoms of AHX by the DMB group. This
protection reaction afforded a separable mixture of re-
gioisomers 1 and 1′ in a ratio of 4.2:1. The structures of
these isomers were identified by the heteronuclear multiple
bond coherence and heteronuclear multiple quantum
correlation (see SI for details). The low yield of 1/1′ is
attributed to the lower stabilities of both protecting-group-free
AHX and DMBCl.
Next, we examined the palladium-catalyzed C−H arylation
of DMB-protected AHX (1 and 1′) with bromoarenes. Based
on the C−H arylation reactions of related imidazole substrates
reported by Miura10 and Rossi,9a we identified reaction
conditions for the coupling of DMB-protected AHX and
bromoarenes using a Pd/Cu catalytic system. For example,
treatment of DMB-protected AHX (1/1′ = 4.2:1) with
bromobenzene (2.0 equiv) in the presence of Pd(OAc)2 (5
mol %), CuI (2.0 equiv), and K3PO4 (2.5 equiv) in DMF at
140 °C for 12 h afforded the expected C−H arylation products
(2a/2a′ = 1.4:1) in 50% yield (Table 1, entry 1). The ratio of
DMB regioisomers changed during the reaction. The
B
DOI: 10.1021/acs.orglett.8b02407
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 2. Synthesis of Arylated AHX 3 through C−H
a
Arylation and Deprotection
a
Reaction conditions for C−H arylation: Pd(OAc)2 (5 mol %), ArBr
(2.0 equiv), CuI (2.0 equiv), K3PO4 (2.5 equiv), DMF, 140 °C, 12 h.
Reaction conditions for deprotection of DMB group: CAN (8.0
Figure 3. Plant-growth-regulating activities of AHX-related com-
pounds. (a) Chemical structures of the reported AHX-related
compounds. (b) Effect of Xan, HX, IMI, EtC, AHX, AOH, ICA,
and AICA on the growth of rice. Germinated seeds were treated with
0.1 mM solution of Xan, HX, IMI, EtC, AHX, AOH, ICA, and AICA
and incubated for a week in a test tube. Results are the mean
standard deviation (n = 12). Asterisk indicates a value that is
significantly different from that of the control (Student’s t-test, p <
0.05).
b
c
equiv), CH3CN, H2O, rt. Reaction time was 15 h. 1/1′ = 4.4:1.
d
Single isomer 2g was used as a starting material. CAN =
Ce(NH4)2(NO3)6. DMB = 2,4-dimethoxybenzyl.
arylation products (35−71%). These results suggest that the
influence of the steric hindrance on the aryl group could affect
the ratio of DMB regioisomers.
Next, we examined the deprotection of DMB groups from
the C−H arylated AHX derivatives 2 (Scheme 2). Treatment
of 2 with ceric ammonium nitrate led to the formation of the
corresponding deprotected compounds (3a−h) in moderate to
low yield (8−42%). In addition, we also conducted the
deprotection reaction of isomerically pure 2a and 2a′
independently. Both isomers gave the desired compound 3a
in similar yields (35 and 20%) (see SI for details).
With the newly synthesized compounds (3a−h) in hand, we
investigated the SAR of AHX analogues and derivatives in
terms of growth-promoting activity. The growth-promoting
activity was investigated using rice (Oryza sativa L. cv.
Nipponbare). The activities of reported AHX-related com-
pounds (Figure 3a) showed good agreement with the
previously reported results.11 Xanthine (Xan), hypoxanthine
(HX), imidazole (IMI), ethyl imidazole-4-carboxylate (EtC),
and 5-aminoimidazole-4-carboxamide (AICA) did not show
shoot or root-growth-regulating activity. ICA exhibited growth-
inhibitory activity in both shoot and root, and AHX and 2-aza-
8-oxohypoxanthine (AOH) showed growth-promoting activity
in roots (Figure 3b).
Figure 4. Effect of arylated AHXs 3a−h on the growth of rice.
Germinated seeds were treated with 0.1 mM solution of 3a−h and
incubated for a week in a test tube. Results are the mean standard
deviation (n = 12). Asterisk indicates a value that is significantly
different from that of the control (Student’s t-test, p < 0.05). Number
sign indicates a value that is significantly different from that of the
AHX-treated group (Student’s t-test, p < 0.05).
The plant-growth-regulating activities of the arylated AHX
derivatives 3, synthesized in this study, are summarized in
Figure 4 and Figure 5. To our delight, all the arylated AHXs
tend to have a root-growth-promoting activity stronger than
that of the original AHX. The introduction of a phenyl (3a), o-
methylphenyl (3b), 4-biphenylyl (3e), 1-naphthyl (3f), or m-
chlorophenyl (3h) moiety at the C8 position of AHX was
found to be highly effective for the rice root growth activity.
Substitutions on the phenyl group with the m-methyl (3c), p-
methyl (3d), or o-fluoro (3g) group resulted in activity weaker
than that of 3a. Although further studies toward the mode of
action of AHX is necessary, it is obvious that C−H
functionalization chemistry12 disclosed a novel chemical
space of biological importance which is only accessible by
synthetic chemistry.
Figure 5. Images of rice seedlings after the growth-promotion assay.
Germinated seeds were treated with 0.1 mM solution of compounds.
In summary, C−H arylation reaction of DMB-protected
AHX made it possible to discover novel plant-growth-
promoting compounds. Moreover, the newly synthesized
arylated AHX derivatives showed growth-promoting activity
stronger than that of the natural plant growth promoter AHX.
These series of compounds have immense potential for
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DOI: 10.1021/acs.orglett.8b02407
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Organic Letters
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applications in agriculture. Future identification of target(s) of
AHX and its arylated derivatives may provide the molecular
basis of their interesting plant-growth activity including fairy-
ring formation. Moreover, the present work emphasizes that,
with the recent advent of game-changing C−H functionaliza-
tion chemistry,12 there are significant opportunities to use
these technologies to accelerate agricultural science.
ASSOCIATED CONTENT
* Supporting Information
(4) (a) Mitchinson. Nature 2014, 505, 298. (b) Kawagishi, H. Biosci.,
Biotechnol., Biochem. 2018, 82, 752.
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(5) (a) Asai, T.; Choi, J.-H.; Ikka, T.; Fushimi, K.; Abe, N.; Tanaka,
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b02407.
Detailed experimental procedures for chemical synthesis
and growth-promotion assay, spectral data for all
1
compounds including scanned images of H, 13C NMR
(7) Santner, A.; Calderon-Villalobos, L. I.; Estelle, M. Nat. Chem.
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spectra (PDF)
Accession Codes
(8) (a) For our previous study on the use of C−H arylation
chemistry to discover stomata-controlling molecules, see: Ziadi, A.;
Uchida, N.; Kato, H.; Hisamatsu, R.; Sato, A.; Hagihara, S.; Itami, K.;
Torii, K. U. Chem. Commun. 2017, 53, 9632. (b) For related study on
the discovery of new biologically active compounds by C−H
arylation, see: Oshima, T.; Yamanaka, I.; Kumar, A.; Yamaguchi, J.;
Nishiwaki-Ohkawa, T.; Muto, K.; Kawamura, R.; Hirota, T.; Yagita,
K.; Irle, S.; Kay, S. A.; Yoshimura, T.; Itami, K. Angew. Chem., Int. Ed.
2015, 54, 7193.
CCDC 1845158−1845159 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Authors
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■
*E-mail: itami@chem.nagoya-u.ac.jp.
*E-mail: kawagishi.hirokazu@shizuoka.ac.jp.
ORCID
Hideto Ito: 0000-0002-4034-6247
Shinya Hagihara: 0000-0003-0348-7873
Toshiyuki Kan: 0000-0002-9709-6365
Hirokazu Kawagishi: 0000-0001-5782-4981
Kenichiro Itami: 0000-0001-5227-7894
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by JST-ERATO (No. JPMJER1302
to K.I.), JST-PRESTO (No. JPMJPR15Q9 for S.H.), JSPS
KAKENHI (Grant Nos. JP26810057, JP16H00907,
JP17K1955, JP18H02019 (H.I.), JP16H06192 (J.-H.C.),
JP17H06350 (S.H.), JP17H06402 (H.K.)), and DAIKO
Foundation (H.I.). We also thank Dr. K. Okamoto (Ushio
Chemix Co. Ltd.) for providing AHX. We thank Dr. Y. Segawa
(Nagoya University) and Mr. K. Kato (Nagoya University) for
assistance with X-ray crystal structure analysis. ITbM is
supported by the World Premier International Research
Center Initiative (WPI), Japan.
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DOI: 10.1021/acs.orglett.8b02407
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