Structure-activity relationships of natural quinone vegfrecine analogs with potent activity against VEGFR-1 and -2 tyrosine kinases

Article abstract of DOI:10.1038/s41429-021-00445-y

A series of analogs of vegfrecine, a natural quinone vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor, was synthesized via oxidative amination of 2,5-dihydroxybenzamide with functionalized arylamine followed by ammonolysis and

Full text of DOI:10.1038/s41429-021-00445-y

The Journal of Antibiotics
https://doi.org/10.1038/s41429-021-00445-y
SPECIAL FEATURE: ARTICLE
Structure-activity relationships of natural quinone vegfrecine
analogs with potent activity against VEGFR-1 and -2 tyrosine kinases
Hayamitsu Adachi1 Chisato Nosaka2 Sonoko Atsumi2 Koichi Nakae2 Yoji Umezawa2 Ryuichi Sawa2
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Yumiko Kubota2 Chie Nakane2 Masabumi Shibuya3 Yoshio Nishimura2
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Received: 30 March 2021 / Revised: 10 May 2021 / Accepted: 10 May 2021
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2021
Abstract
A series of analogs of vegfrecine, a natural quinone vascular endothelial growth factor receptor (VEGFR) tyrosine kinase
inhibitor, was synthesized via oxidative amination of 2,5-dihydroxybenzamide with functionalized arylamine followed by
ammonolysis and substitution of the quinone ring. The inhibitory activities of the analogs against the VEGFR-1 and -2
tyrosine kinases were assayed in vitro with the aim to identify a compound suitable to treat cancer and inflammatory
diseases. Alterations of the functionality of the phenyl group, substitution of the quinone ring, and oxidative cyclization of
the 1-carboxamide-2-aminoquinone moiety to form an isoxazole quinone ring were examined. Introduction of halo- and
alkyl-substituents at the 5′-position of the phenyl ring resulted in potent inhibition of the VEGFR-1 and -2 tyrosine kinases.
In particular, structural modification at C-5′ on the phenyl ring was shown to significantly affect the selectivity of the
inhibition between the VEGFR-1 and -2 tyrosine kinases. Compound 8, 5′-methyl-vegfrecine, showed superior selectivity
toward the VEGFR-2 tyrosine kinase over the VEGFR-1 tyrosine kinase.
Introduction
leads to weak receptor phosphorylation and no significant
mitogenic response to ECs. The VEGFR-1 signal was also
reported to stimulate angiogenesis via recruitment of endo-
thelial and monocyte progenitor cells from bone marrow into
the tumor vasculature. Furthermore, VEGFR-1 is functionally
expressed in monocytes and macrophages, which plays a
crucial role in promoting inflammation [19–23]. Blocking the
VEGFR-1 signal inhibits pathological angiogenesis involved
in cancer, atherosclerosis, arthritis, ocular neovascular disease,
and tumor metastasis [24–27].
Extensive studies of small-molecule VEGFR inhibitors
have been performed for synthetic molecules, and current
drug development in the field of VEGFR-targeted drug
heads to the multi-kinase inhibitor as shown in Sorafenib,
Sunitinib, Axitinib, and Regorafenib, possessing VEGFRs
inhibitory activity. Even for this situation, explore of natural
receptor tyrosine kinase inhibitors have been continued for
biologically validated starting point [28]. As natural pro-
ducts targeted to VEGFRs, capsicodendrin [29], acetyl-11-
keto-β-boswellic acid [30], 3′-O-acetylhamaudol [31], tet-
ramethylpyrazine [32], (1 S, 2E, 4 S, 6 R, 7E, 11E)-2,7,11-
cembratriene-4,6-diol [33], and chebulagic acid [34] were
isolated from natural sources.
Vascular endothelial growth factors (VEGFs) and their
receptors are important regulators of angiogenesis, both in
normal physiological and pathological processes [1–4].
Angiogenesis in physiological processes plays a crucial role
in embryonic development, stimulating proliferation as well
as survival and migration of endothelial cells (ECs), whereas
angiogenesis in pathological processes is associated with
inflammation [5], rheumatoid arthritis [6], ocular neovascu-
larization [7], psoriasis [8], and tumor development [9].
The VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-2
(KDR), exist almost exclusively in ECs [10–13] and are
recognized to be a promising target of cancer therapy [14–17].
VEGFR-2 is strongly autophosphorylated in ECs upon VEGF
stimulation and mediates a mitogenic response [18]. In con-
trast to VEGFR-2, activation of VEGFR-1 with VEGF only
* Hayamitsu Adachi
adachih@bikaken.or.jp
1
Institute of Microbial Chemistry (BIKAKEN), Shizuoka, Japan
2
Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan
In the course of our screening for VEGFR-1 tyrosine
kinase inhibitors from microbial metabolites focusing on
3
Jobu University, Gunma, Japan

H. Adachi et al.
cancer and inflammatory disease, vegfrecine (1), a novel
inhibitor of the VEGF receptor tyrosine kinase, was isolated
from cultured Streptomyces sp [35, 36]. Compound 1
exhibited potent in vitro inhibitory activity against VEGFRs
tyrosine kinases, but only weak inhibitory activity against
platelet-derived growth factor receptors, fibroblast growth
factor receptor, and epidermal growth factor receptor.
In the present study, we characterized the structure-activity
relationships of VEGFR-1 and -2 tyrosine kinase inhibitors
based on the structure of compound 1. In particular, 5′-
functionalization on the phenyl ring moiety of 1 affected the
selectivity of the inhibition between the VEGFR-1 and
VEGFR-2 tyrosine kinases and led to identification of a new
VEGFR-2 inhibitor with enhanced potency.
acetate and washed with water, dried over MgSO4, and
filtered. The filtrate was evaporated to give a crude oil,
which was subjected to column chromatography on silica
gel. Elution with a mixture of hexane and ethyl acetate gave
the TBS derivative.
General oxidative amination procedure for the syntheses of
1-carbamoyl hydroquinones 31–39
For the preparation of 31–39, a solution of 30, the amines
23, 14, 15, 24–29, and NaIO4 (3 equiv.) in MeOH– H2O
(2:1) was stirred overnight at room temperature. The reac-
tion mixture was diluted with ethyl acetate, washed with
water, dried over MgSO4, and filtered. The filtrate was
evaporated to give a crude oil, which was subjected to
column chromatography on silica gel. Elution with a mix-
ture of toluene and acetone gave the desired quinone.
Experimental procedures
Materials and Instrumentation
General ammonolysis procedure for the syntheses of 3 and 4
NMR spectra were recorded using a JNM-ECA600 and a
JNM-ECS-400 spectrometer (JEOL, Tokyo, Japan) with
TMS as an internal standard. HRESIMS spectra were
measured using an LTQ Orbitrap XL and Q Exactive mass
spectrometer (Thermo Fisher Scientific, San Jose, CA,
USA). Compounds 14–22 were purchased from commercial
sources. Compound 30 was prepared as described in the
literature [37]. Vegfrecine (1) was already synthesized via
31 as described in the literature [35].
To a solution of 32 or 33 in a mixture of chloroform (2 ml)
was added a 7 M ammonia in methanol solution (1 ml), and
the reaction mixture was stirred for 1 h at room temperature.
The reaction mixture was diluted with ethyl acetate, washed
with water, dried over MgSO4, and filtered. The filtrate was
evaporated to give a solid that was subjected to preparative
thin-layer chromatography on silica gel developed with a
mixture of chloroform and methanol (50:1) to give 3 or 4.
Molecular modeling
2-Amino-5-((2-methoxyphenyl)amino)-3,6-dioxocyclohexa-
1,4-diene-1-carboxamide (3)
Vascular endothelial growth factor receptor 1 (VEGFR-1:
EC 2.7.10.1, Tyrosine-protein kinase receptor FLT (Flt-1))
provided from UniProtKB was adapted to the docking
study. The human sequence was taken from the SwissProt
Data Bank (accession number P17948). First compound 2
was docked, and its position was optimized by the DIS-
COVER program (Insight II, Accelrys), using the consistent
valence force field (CVFF). Compound 1 was superimposed
onto the position of 2, and its position was optimized. The
results indicated that compound 1 fit the binding site well
with a LigScore2 of 5.44.
1
Compound 3 was prepared in 70% yield: H NMR (600
MHz, DMSO-d6) δ 10.97 (1H, s), 9.14 (1H, br s), 8.93 (1H,
br s), 8.60 (1H, s), 7.39 (1H, s), 7. 30 (1H, d, J = 7.9 Hz), 7.
26 (1H, dt, J = 0.9 and 7.8 Hz), 7.13 (1H, d, J = 7.9 Hz),
6.99 (1H, dt, J = 0.9 and 7.6 Hz), 5.47 (1H, s), and 3.80
(3H, s); 13C NMR (100 MHz, DMSO-d6) δ 176.6, 175.5,
169.4, 157.3, 152.3, 127.4, 124.3, 123.3, 120.7, 112.1,
96.5, 95.8, and 55.7; HRESIMS (positive) m/z 288.0978
(M + H)+ (calcd for C14H14N3O4 288.0979).
2-Amino-5-((2-chlorophenyl)amino)-3,6-dioxocyclohexa-
1,4-diene-1-carboxamide (4)
Chemical synthesis
General protection of 16–22 with a tert-butyldimethylsilyl
(TBS) group for the syntheses of 23–29
Compound 4 was prepared in 84% yield: 1H NMR
(400 MHz, DMSO-d6) δ 10.98 (1H, s), 9.50 (1H, br s), 9.00
(1H, br s), 8.65 (1H, s), 7.62 (1H, d, J = 8.0 Hz), 7.34–7.48
(4H, m), and 5.15 (1H, s); 13C NMR (150 MHz, DMSO-d6)
δ 176.5, 175.7, 169.4, 157.1, 150.1, 134.9, 130.2, 129.5,
128.6, 128. 3, 127.8, 96.5, and 96.1; HRESIMS (positive)
m/z 292.0500 (M + H)+ (calcd for C13H11ClN3O3
292.0497).
To a solution of commercial amine 16–22 in N, N-dime-
thylformamide was added tert-butyldimethylsilyl chloride
(1.5 equiv.) and imidazole (2 equiv.) at room temperature,
and the reaction mixture was stirred overnight at room
temperature. The reaction mixture was diluted with ethyl

Structure-activity relationships of natural quinone vegfrecine analogs with potent activity against. . .
General ammonolysis and TBS-deprotection procedure for
6.94 (1H, d, J = 8.7 Hz), and 5.43 (1H, s); 13C NMR (150
the syntheses of 5–10
MHz, DMSO-d6) δ 176.5, 176.0, 169.4, 157.0, 150.2,
148.2, 127.1, 125.6, 124.2, 122.1, 117.5, 96.6, and 96.4;
HRESIMS (positive) m/z 308.0428 (M + H)+ (calcd for
C13H11ClN3O4, 308.0433).
To a solution of diarylaminoquinone 34–39 in chloroform
(2 ml) was added a 7 M ammonia in methanol solution (1
ml), and the reaction mixture was stirred for 1 h at room
temperature. The reaction proceeded quantitatively. The
reaction mixture was diluted with ethyl acetate, washed
with water, dried over MgSO4, and filtered. The filtrate was
evaporated to give a solid that was dissolved in tetra-
hydrofuran. To a solution of the 2-amino derivative was
added acetic acid (38 equiv.) and tetrabutylammonium
fluoride (1.1 equiv.), and the mixture was stirred overnight
at room temperature. The reaction mixture was diluted with
ethyl acetate, washed with water, dried over MgSO4, and
filtered. The filtrate was evaporated to give a solid that was
suspended in methanol. The suspension was filtered to give
the alcohol.
2-Amino-5-((2-hydroxy-5-methylphenyl)amino)-3,6-
dioxocyclohexa-1,4-diene-1-carboxamide (8)
Compound 8 was prepared in 63% yield (2 steps): 1H NMR
(400 MHz, DMSO-d6) δ 10.99 (1H, s), 9.9 (1H, br s), 9.18
(1H, br s), 8.97 (1H, br s), 8.64 (1H, br d, J = 2.9 Hz), 7.43
(1H, br d, J = 2.9 Hz), 7.06 (1H, d, J = ~1 Hz), 6.92 (1H,
dd, J = 1 and 8.3 Hz), 6.84 (1H, dd, J = 8.3 Hz), 5.56
(1H, s), 2.22 (3H, s); 13C NMR (600 MHz, DMSO-d6) δ
176.6, 175.6, 169.4, 157.2, 148.2, 148.1, 128.3, 127.7,
124.2, 124.0, 116.0, 96.4, 95.9, and 20.0; HRESIMS
(positive) m/z 288.0978 (M + H)+ (calcd for C14H14N3O4
288.0979).
2-Amino-5-((3-hydroxyphenyl)amino)-3,6-dioxocyclohexa-
1,4-diene-1-carboxamide (5)
2-Amino-5-((5-(tert-butyl)-2-hydroxyphenyl)amino)-3,6-
dioxocyclohexa-1,4-diene-1-carboxamide (9)
Compound 5 was prepared in 68% yield (2 steps): 1H NMR
(400 MHz, DMSO-d6) δ 10.95 (1H, s), 9.70 (1H, s), 9.54
(1H, br s), 8.92 (1H, br s), 8.66 (1H, d, J = 3.7 Hz), 7.43
(1H, d, J = 3.7 Hz), 7.21 (1H, t, J = 8.0 Hz), 6.5 ~ 6.79
(1H, br d, J = 7.8 Hz), 6.73 (1H, t, J = 2.2 Hz), and 5.73
(1H, s); 13C NMR (150 MHz, DMSO-d6) δ 176.8, 176.0,
169.5, 158.0, 156.9, 149.1, 138.3, 130.0, 114.8, 113.2,
110.9, 96.5, and 95.7; HRESIMS (positive) m/z 274.0822
(M + H)+ (calcd for C13H12N3O4 274.0822).
1
The compound 9 was prepared in 30% yield (2 steps): H
NMR (400 MHz, DMSO-d6) δ 10.98 (1H, s), 9.87 (1H, br
s), 9.16 (1H, br s), 8.94 (1H, br s), 8.66 (1H, d, J = 3.8 Hz),
7.41 (1H, d, J = 3.8 Hz), 7.18 (1H, d, J = 2.3 Hz), 7.15
(1H, dd, J = 2.3 and 8.5 Hz), 6.87 (1H, d, J = 8.5 Hz), 5.40
(1H, s), and 1.23 (9H, s); 13C NMR (150 MHz, DMSO-d6)
δ 176.6, 175.4, 169.5, 157.3, 148.7, 148.6, 141.7, 124.4,
123.5, 121.5, 115.9, 96.4, 95.7, 33.8, and 31.2; HRESIMS
(positive) m/z 330.1450 (M + H)+ (calcd for C17H20N3O4
330.1448).
2-Amino-5-((2-(hydroxymethyl)phenyl)amino)-3,6-
dioxocyclohexa-1,4-diene-1-carboxamide (6)
2-Amino-5-((4-hydroxy-[1,1′-biphenyl]-3-yl)amino)-3,6-
Compound 6 was prepared in 77% yield (2 steps): 1H NMR
(400 MHz, DMSO-d6) δ 10.96 (1H, s), 9.62 (1H, br s), 8.91
(1H, br s), 8.62 (1H, d, J = 2.3 Hz), 7.45 (1H, d, J =
5.0 Hz), 7.40 (1H, d, J = 2.3 Hz), 7.35 (1H, dt, J = 1 and
5.0 Hz), 7.29 (1H, d, J = 4.8 Hz), 7.27 (1H, dt, J = 1 and
dioxocyclohexa-1,4-diene-1-carboxamide (10)
1
The compound 10 was prepared in 65% yield (2 steps): H
NMR (400 MHz, DMSO-d6) δ 10.97 (1H, d, J = 4.8 Hz),
10.21 (1H, s), 9.24 (1H, s), 8.93 (1H, d, J = 5.5 Hz), 8.63
(1H, d, J = 3.7 Hz), 7.56 (2H, m), 7.45 (1H, dd, J = 2.3 and
6.9 Hz), 7.35–7.45 (4H, m), 7.27 (1H, m) 7.02 (1H, d, J =
8.5 Hz), and 5.44 (1H, s); 13C NMR (150 MHz, DMSO-d6)
δ 176.6, 175.7, 169.4, 157.2, 150.5, 148.7, 139. 3, 131.6,
128.8, 126.8, 126.1, 125.9, 124.6, 123.0, 116.8, 96.4, and
96.2; HRESIMS (positive) m/z 350.1132 (M + H)+ (calcd
for C19H16N3O4 350.1135).
4.8 Hz), 5.44 (1H, s), 5.42 (1H, br s), and 4.44 (2H, s); 13
C
NMR (150 MHz, DMSO-d6) δ 176.5, 175.9, 169.4, 157.1,
149.4, 136.6, 135.3, 128.5, 127.9, 126.5, 124.5, 96.5, 95.4,
and 60.3; HRESIMS (positive) m/z 288.0973 (M + H)+
(calcd for C14H14N3O4 288.0979).
2-Amino-5-((5-chloro-2-hydroxyphenyl)amino)-3,6-
dioxocyclohexa-1,4-diene-1-carboxamide (7)
5-((2-Hydroxyphenyl)amino)-2-(methylamino)-3,6-
dioxocyclohexa-1,4-diene-1-carboxamide (11)
Compound 7 was prepared in 75% yield (2 steps): 1H NMR
(400 MHz, DMSO-d6) δ 10.98 (1H, s), 9.22 (1H, br s), 8.97
(1H, s), 8.63 (1H, d, J = 3.7 Hz), 7.44 (1H, d, J = 3.0 Hz),
7.27 (1H, d, J = 2.8 Hz), 7.17 (1H, dd, J = 2.5 and 8.7 Hz),
To a solution of 31 (16 mg, 0.0398 mmol) in chloroform
(1.4 ml) was added a methylamine (40%) methanol solution

H. Adachi et al.
(0.4 ml), the reaction mixture was stirred overnight at room
temperature. The reaction mixture was diluted with ethyl
acetate, washed with water, dried over MgSO4, and filtered.
The filtrate was evaporated to give a solid that was sub-
jected to preparative thin layer chromatography on silica gel
developed with a mixture of chloroform and methanol
(50:1) to give the TBS derivative of 2-methylamino veg-
frecine (8.2 g, 51%). To a solution of the TBS derivative in
tetrahydrofuran (1 ml) was added acetic acid (38 μl, 0.062
mmol) and a tetrabutylammonium fluoride (1.0 M) solution
in THF, and the reaction mixture was stirred for 1 h. The
reaction proceeded quantitatively. The reaction mixture was
diluted with ethyl acetate, washed with water, dried over
MgSO4, and filtered. The filtrate was evaporated to give a
solid that was suspended in methanol. The suspension was
filtered, and the solid was washed with methanol to give 11
(4.7 mg, 95%).
7.47 (1H, d, J = ~1.0 Hz), 7.01–7.15 (2H, m), 6.83 (1H, d,
J = 8.0 Hz), and 6.77 (1H, dt, J = ~1.0 Hz and 8.0 Hz); 13C
NMR (100 MHz, DMSO-d6) δ 175.2, 171.2, 169.2, 156.7,
153.9, 152.3, 145.3, 127.8, 127.3, 118.2, 115.2, 103.1, and
95.4; HRESIMS (negative) m/z 306.0279 (M-H)- (calcd for
C13H9ClN3O4 306.0276).
3-Amino-5-((2-hydroxyphenyl)amino)benzo[c]isoxazole-4,7-
dione (13)
Ammonolysis of 31 gave the 2-amino-TBS derivative
quantitatively as described in the literature [35]. The 2-
amino-TBS derivative (56 mg, 0.145 mmol) was dissolved
in a mixture of tetrahydrofuran (2 ml) and chloroform (4
ml). To this solution was added Pb(OAc)4 (257 mg, 0.580
mmol), and the reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was diluted with ethyl
acetate, washed with water, and dried over MgSO4 to give a
solid that was subjected to preparative thin-layer chroma-
tography on silica gel developed by a mixture of chloroform
and methanol (20:1) to give the benzo[c]isoxazole deriva-
tive (31 mg, 56%). To a solution of the benzo[c]isoxazole
derivative (20 mg, 0.0518 mmol) in tetrahydrofuran (1 ml)
was added acetic acid (112 μl, 1.97 mmol) and tetra-
butylammonium fluoride in THF (57 μl, 0.0569 mmol), and
the reaction mixture was stirred for 1 h at room temperature.
The reaction mixture was diluted with ethyl acetate, washed
with water, dried over MgSO4, and filtered. The filtrate was
evaporated to give a solid that was suspended in methanol.
The suspension was filtered to give the alcohol 13 (9.8
mg, 70%).
1
Compound 11 was prepared in 48% yield (2 steps): H
NMR (600 MHz, DMSO-d6, 60 °C) δ 9.95 (1H, s), 9.03
(1H, br s), 8.86 (1H, br s), 7.31 (1H, br s), 7.26 (1H, d, J =
6.0 Hz), 7.09 (1H, t, J = 6.0 Hz), 6.97 (1H, d, J = 6.0 Hz),
6.88 (1H, t, J = 6.0 Hz), 5.60 (1H, s), and 3.40 (2H, d, J =
o
6.0 Hz); 13C NMR (150 MHz, DMSO-d6, 60 C) δ179.0,
175.1, 170.5, 156.9, 150.0, 146.8, 126.5, 124.7, 123.1,
119.2, 115.9, 97.6, 96.7, and 33.6; HRESIMS (positive) m/z
310.0796 (M + Na)+ (calcd for C14H13N3O4Na 310.0798).
2-Amino-4-chloro-5-((2-hydroxyphenyl)amino)-3,6-
dioxocyclohexa-1,4-diene-1-carboxamide (12)
Ammonolysis of 31 gave the TBS derivative quantitatively
as described in the literature [35]. The TBS derivative (10
mg, 0.0258 mmol) was dissolved in a mixture of methanol
and chloroform (4:3) (1.4 ml). To this solution was added
N-chlorosuccinimide (6.4 mg, 0.0518 mmol), and the reac-
tion mixture was stirred overnight at room temperature. The
reaction mixture was diluted with ethyl acetate, washed
with water, and dried over MgSO4 to give a solid that was
subjected to preparative thin layer chromatography on silica
gel developed with a mixture of chloroform and methanol
(20:1) to give the 4-chloro derivative (6.5 mg, 65%). A
solution of the 4-chloro derivative (10 mg, 0.0236 mmol),
acetic acid (51 μl, 0.9 mmol, 38 equiv.), and tetra-
butylammonium fluoride (26 μl, 0.0259 mmol, 1.1 equiv.)
in tetrahydrofuran (1 ml) was stirred for 1 h at room tem-
perature. The reaction mixture was diluted with ethyl
acetate, washed with water, dried over MgSO4, and filtered.
The filtrate was evaporated to give a solid that was sus-
pended in methanol. The suspension was filtered to give the
alcohol 12 (5 mg, 69%).
1
Compound 13 was prepared in 37% yield (3 steps): H
NMR (400 MHz, DMSO-d6) δ 8.7-9.4 (3H, br m), 8.92
(1H, br s), 7.13 (1H, d, J = 1.6 and 6.4 Hz), 7.11 (1H, dd,
J = 1.6 and 8.0 Hz), 6.95 (1H, dd, J = 1.3 and 8.1 Hz), 6.86
(1H, dt, J = 1.3 and 7.7 Hz), and 5.49 (1H, s); 13C NMR
(150 MHz, DMSO-d6) δ 176.2, 172.1, 168.3, 156.3, 150.8,
149.5, 127.3, 124.7, 124.6, 119.4, 116.2, 101.7, and 91.2;
HRESIMS (positive) m/z 294.0489 (M + Na)+ (calcd for
C13H9N3O4Na 294.0485).
In vitro tyrosine kinase assay
The kinase domain of Flt-1 was expressed as a GST-fusion
protein by infection of Sf9 cells with engineered baculo-
viruses. GST-Flt-1 was purified from infected Sf9 cell
lysates on glutathione sepharose. The enzyme KDR was
purchased from Invitrogen Co. Ltd (Carlsbad, CA, USA).
The assays were performed in 96-well filter plates. The
assay buffers were 20 mM Tris-HCl, 3 mM MnCl2, 3 mM
MgCl2, 50 μg ml−1 poly GluTyr, 100 mM NaCl, 1 μM
Na3VO4, 1 μM DTT, and 0.5% BSA at pH 7.4 for Flt-1 and
20 mM Tris-HCl, 1 mM MnCl2, 10 mM MgCl2, 50 μg ml−1
1
Compound 12 was prepared in 43% yield (3 steps): H
NMR (400 MHz, DMSO-d6) δ 11.07 (1H, s), 9.79 (1H, br
s), 9.36 (1H, s), 9.16 (1H, br s), 8.53 (1H, d, J = 4.0 Hz),

Structure-activity relationships of natural quinone vegfrecine analogs with potent activity against. . .
Fig. 1 Chemical structures of vegfrecine (1), SU5416 (2) and vegfrecine analogs 3–13
poly GluTyr, 100 mM NaCl, 1 μM Na3VO4, 1 μM DTT, and
0.5% BSA at pH 7.4 for KDR. Each kinase (55 ng/20 μμl
buffer) and the compound dissolved in a 2% DMSO buffer
solution (10 μl) were added to a well, and the reaction was
started by addition of a 4 µM ATP/10 µCi/ml 32P-ATP
buffer solution (10 μl). After incubation for 20 min at room
temperature, the reaction was stopped by the addition of
30% TCA. Filter plates were washed three times with 15%
TCA and radioactivity was measured on a scintillation
counter (Tri-Carb 2800TR, PerkinElmer, Waltham,
MA, USA).
in the absence or presence of test samples. After 2 days
incubation, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) (10 µl of 5 mg ml−1) was added
to each well, and the plate was further incubated for 2 h.
The resulting MTT formazan was solubilized with 10%
sodium dodecyl sulfate overnight and the absorbance at
570 nm was measured using the ARBO-SX 1420 multi-
labeled counter (PerkinElmer, Waltham, MA, USA).
Results and discussion
Cell culture
Molecular modeling
Flt-1-overexpressing NIH3T3-Flt-1 cells and KDR-
overexpressing NIH3T3-KDR cells were grown in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with
10% fetal calf serum, 2 mmol glutamine and 200 µg ml−1
G418.
The structures of the vegfrecine analogs 3–13 are sum-
marized in Fig. 1. In order to gain insight into the structural
design of these analogs, a docking study of vegfrecine (1)
into the active site of the VEGFR-1 tyrosine kinase was
conducted in silico and the binding was optimized based on
the docking of semaxanib (SU5416, 2) [38] with VEGFR-1.
Compound 1 was superimposed onto 2 in silico. The amide
moiety and benzene ring of 2 were also superimposed onto
the carbamoyl and quinone ring moiety of 1, respectively.
The 1-carbamoyl and 2-amino groups, the carbonyl oxygen
on the quinone ring, and the hydroxyl group on the benzene
Cytotoxicity assay
Cell viability was measured by the MTT colorimetric
method. The cells were seeded in 96-well microplates at
densities of 3 × 103 cells in 100 µl growth medium per well

H. Adachi et al.
hydroxy, and hydroxymethyl groups (compounds 3–6,
respectively). As observed from the docking study, there
was sufficient space to incorporate some functional groups
at 5′-position on the phenyl ring and the introduction of
non-polar groups was expected to increase hydrophobicity
and improve the transport of the compound through the cell
membrane (compounds 7–10).
D1040
Oxidative amination of the amide 30 [37] with 23, 14,
15, and 24–29 gave the corresponding amino benzoqui-
nones (31 [35]–39). Treatment of the amino benzoquinones
32 and 33 with ammonia resulted in amine substitution at
the 2-position to produce 3 and 4, respectively. In a similar
fashion, ammonolysis of 34–39 followed by deprotection
yielded 5–10, respectively.
In order to increase the electron density of the quinone
ring, treatment of 31 with methylamine followed by
deprotection of the TBS group gave the corresponding 2-
NHMe analog 11. On the other hand, we introduced an
electron-withdrawing group at the 4-position of the quinone
ring to reduce its electron density. Compound 31 was
converted to the 4-chloro analog 12 using N-chlor-
osuccinimide after ammonolysis.
The effects of the orientation of the 1-carbamoyl and 2-
amino groups were also investigated. Ammonolysis of 31
gave the 1-carbamoyl-2-aminoquinone derivative that in turn,
was subjected to oxidative cyclization between the 1-
carobamoyl group and 2-amino group using lead acetate to
give the isoxazole ring-fused quinone. The TBS group of the
isoxazole quinone derivative was deprotected to provide 13.
Fig. 2 Docked structure of vegfrecine (1) with VEGFR-1. Color
version is available online from https://www.nature.com/articles/
s41429-021-00445-y.
ring of 1 provided favorable interactions with the distal
region of the backbone of the enzyme (Fig. 2). The NH of
Asp1040 seemed to be involved in interactions with the
carbonyl oxygen at C-6 and the carbonyl oxygen of the 1-
carbamoyl group. The carboxyl group of Glu878 was
directed to the hydrogens of the amino group at C-2 and the
carbamoyl amino group at C-1 of 1. The Lysine861 amide
NH existed at the near place of carbonyl oxygen at C-3. The
carbonyl oxygen of Glu910 was found in proximity to the
2′-hydroxyl group of 1. Thus, alteration of the 1-carbamoyl
and 2-amino groups on the quinone ring and the 2′-hydro-
xyl group of the phenyl ring should directly affect the
inhibitory activity. The binding sites of the inhibitor were
positioned on the surface of the protein. The benzene ring
moiety at the 5′-position faced outward and could accom-
modate a functional group that could increase the compound’s
hydrophobicity and improves its transport through the cell
membrane. This finding encouraged us to introduce a non-
polar substituent at the 5′-position of the benzene ring of 1.
Biological evaluation
Table 1 shows the biological results for vegfrecine (1),
SU5416 (2) and the analogs 3–13 including their in vitro
inhibitory activity toward the tyrosine kinases and their
cytotoxicity in cells. In place of the hydroxy group on the
phenyl ring of 1, compounds 3 and 4 having a methoxy and
chloro group on the phenyl ring, respectively, lost inhibitory
potency against the autophosphorylation of the VEGFR-1
and -2 tyrosine kinases in vitro. Additionally, the positional
change of the hydroxyl group on the phenyl ring was
evaluated. The 3′-hydroxy analog 5 and 2′-hydroxymethyl
analog 6 decreased inhibitory activities of both the VEGFR-
1 and VEGFR-2 tyrosine kinases. Therefore, the 2′-phenol
hydroxy group seems to be indispensable for inhibition as
predicted by its hydrogen-bonding interaction with the
enzyme backbone observed in the docking study.
Chemical synthesis
The syntheses of the analogs were carried out by oxidative
amination [35, 39] of 2,5-dihydroxybenzamide [40] (30) in
the presence of the amines 23, 14, 15, and 24–29
(Scheme 1). The introduction of a functional group to a
quinone ring is known for the naturally occurring quinone
nakijiquinone that is inhibitor against insulin-like growth
factor 1 receptor, tyrosine kinase receptor Tie-2, and
VEGFR-3 [41]. We first focused on modifying the func-
tionality of the phenyl ring. The hydroxy group of the
phenyl ring was changed to methoxy, chloro, meta-
The 5′-substituted analogs 7–10 were shown to potently
inhibit the VEGFR-1 and -2 tyrosine kinases. In particular,
the inhibitory activity of 8 was more potent than that of the
parent 1 against the VEGFR-2 tyrosine kinase. Variation of
the functional group at C-5′ affected the selectivity of 1
against the VRGFR-1 and VEGFR-2 tyrosine kinases.

Structure-activity relationships of natural quinone vegfrecine analogs with potent activity against. . .
O
O
OH
R4
O
H
N
R5
CONH2
NH2
H
N
R4
2'
CONH2
CONH2
1
5
b
3'
6
3
c
1'
6'
32, 33
4
4'
2
N
H
5'
R5
R4
R5
OH
O
3, 4
30
31 - 39
3
4
(R4 = 2'-OMe, R5 = H)
(R4 = 2'-Cl, R5 = H)
31
32
33
34
35
36
37
38
39
(R4 = 2'-OTBS, R5 = H)
(R4 = 2'-OMe, R5 = H)
(R4 = 2'-Cl, R5 = H)
(R4 = 3'-OTBS, R5 = H)
(R4 = 2'-CH2OTBS, R5 = H)
(R4 = 2'-OTBS, R5 = Cl)
(R4 = 2'-OTBS, R5 = CH3)
(R4 = 2'-OTBS, R5 = tert-Bu)
(R4 = 2'-OTBS, R5 = Ph)
O
H
c, d
R6
CONH2
N
34-39
NH2
R5
O
5-10
5
6
7
8
9
(R6 = 3'-OH, R5 = H)
(R6 = 2'-CH2OH, R5 = H)
(R6 = 2'-OH, R5 = Cl)
(R6 = 2'-OH, R5 = CH3)
(R6 = 2'-OH, R5 = tert-Bu)
10
(R6 = 2'-OH, R5 = Ph)
Scheme 1 Reagents and Conditions: a TBSCl, imidazole, DMF, r.t. b
amines 23, 14, 15 and 24–29, NaIO4, MeOH - H2O (2:1), r.t. c 2.3 N
NH3, CHCl3–MeOH (2:1), r.t. d TBAF, AcOH in THF, r.t. e MeNH2,
CHCl3–MeOH (3:1), r.t. f N-chlorosuccimide, MeOH–CHCl3 (4:3), r.t.
g Pb(OAc)4. CHCl3 in THF (2:1), r.t
While the selectivity of 1 toward the inhibition of
VEGFR-1 and -2 is almost equal, the selectivities of 7–10
depend on the C-5′ functionality. In particular, compound 8
exhibited superior selectivity toward the VEGFR-2 tyrosine
kinase over the VEGFR-1 tyrosine kinase. Thus, mod-
ification at C-5′ is thought to have an influence on the
selectivity of 1 against the VEGFR-1 and -2 tyrosine
kinases.
Compound 11 possessing an electron-donating NHMe
group at C-2 decreased inhibitory activity against both of
VEGFR-1 and -2 tyrosine kinases, whereas the inhibitory
activity of 12 having an electron-reducing Cl substituent was

H. Adachi et al.
Table 1 Inhibitory activities of vegfrecine (1), SU5416 (2) and
compounds 3–13
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