The first synthesis of the antiangiogenic homoisoflavanone, cremastranone

Article abstract of DOI:10.1039/c4ob01604a

An antiangiogenic homoisoflavanone, cremastranone, was synthesized for the first time. This scalable synthesis, which includes selective demethylation, could be used to develop lead molecules to treat angiogenesis-induced eye diseases. Synthetic cremastra

Full text of DOI:10.1039/c4ob01604a

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Biomolecular Chemistry
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The first synthesis of the antiangiogenic
homoisoflavanone, cremastranone†
Cite this: Org. Biomol. Chem., 2014,
12, 7673
Bit Lee,a Halesha D. Basavarajappa,b,c Rania S. Sulaiman,b,d,e Xiang Fei,a
Received 28th July 2014,
Accepted 18th August 2014
Seung-Yong Seo*‡a and Timothy W. Corson*‡b,c,d
DOI: 10.1039/c4ob01604a
www.rsc.org/obc
An antiangiogenic homoisoflavanone, cremastranone, was syn- incomplete patient response.2–5 Hence there is a critical
thesized for the first time. This scalable synthesis, which includes requirement for new therapies. Towards this goal, we are pur-
selective demethylation, could be used to develop lead molecules suing development of small molecules as drug leads for these
to treat angiogenesis-induced eye diseases. Synthetic cremastra- diseases, based on natural products.
none inhibited the proliferation, migration and tube formation
Homoisoflavanones are a small class of naturally occurring
ability of human retinal microvascular endothelial cells, important oxygen heterocycles that have been isolated from many
steps in pathological angiogenesis.
geographically diverse plant genera. These compounds are
categorized into four types: 3-benzyl-4-chromanones, 3-benzyl-
idene-4-chromanones, 3-benzyl-3-hydroxy-4-chromanones and
scillascillins.6 In general, these compounds are reported to
show anti-inflammatory, anti-oxidative, anti-bacterial, anti-
cancer, anti-histaminic, anti-viral, and anti-phosphodiesterase
activities. Cremastranone (1), 5,7-dihydroxy-3-(3-hydroxy-4-
Retinopathy of prematurity (ROP), proliferative diabetic retino-
pathy (DR) and neovascular (“wet”) age-related macular
degeneration (wet AMD) are three major ocular diseases,
affecting children, adults and elderly people.1 These diseases
are characterized by abnormal formation of blood vessels in
the eye through the process of pathological angiogenesis. The
blood vessels formed during pathological angiogenesis in the
eye are fragile, porous and not completely differentiated. As a
result haemorrhage, retinal detachment, fibrotic scarring and
rapid photoreceptor degeneration occur in the eye leading to
vision loss.
methoxybenzyl)-6-methoxychroman-4-one, is
a
3-benzyl-4-
chromanone that was isolated from the plants Muscari
armeniacum,7 Chionodoxa luciliae,8 Scilla natalensis,9 Merwilla
plumbea,10 and Cremastra appendiculata (D. Don)11 (1; Fig. 1).
The bulb of the orchid C. appendiculata is used in East Asia as
a
traditional medicine, taken internally to treat several
Nearly 2 million people are affected by wet AMD in the
United States, while about 75% of diabetic patients show clini-
cal symptoms of DR.1 Blocking pathological angiogenesis in
the eyes is the current treatment strategy for these diseases.
The current mainstay of therapy is the use of anti-VEGF bio-
logics. However, these treatments are associated with vision
damaging side effects, unfavourable cost to benefit ratio, and
cancers, and applied externally for skin lesions.
Cremastranone (1) was previously reported to possess anti-
angiogenic activity both in vitro and in vivo and was identified
as a potent inhibitor of the proliferation of human umbilical
vein endothelial cells (HUVECs).11 Also 1 inhibited vascular
tube formation and new vessel growth induced by basic fibro-
blast growth factor. Its anti-angiogenic properties were further
confirmed in vivo in the laser-induced choroidal neovasculari-
aCollege of Pharmacy and Gachon Institute of Pharmaceutical Sciences,
Gachon University, Incheon, South Korea. E-mail: syseo@gachon.ac.kr
bEugene and Marilyn Glick Eye Institute, Department of Ophthalmology,
Indiana University School of Medicine, Indianapolis, Indiana, USA.
E-mail: tcorson@iupui.edu
cDepartment of Biochemistry and Molecular Biology, Indiana University School of
Medicine, Indianapolis, Indiana, USA
dDepartment of Pharmacology and Toxicology, Indiana University School of
Medicine, Indianapolis, Indiana, USA
eDepartment of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
†Electronic supplementary information (ESI) available: Synthetic methodology,
compound characterization, and biological methods. See DOI: 10.1039/
c4ob01604a
‡Equal contributors.
Fig. 1 The structures of cremastranone (1) and its congeners.
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zation and oxygen induced retinopathy mouse models,12,13 began with an aldol condensation of 6′-hydroxy-2′,3′,4′-tri-
used for testing novel therapies for wet AMD and ROP, respecti- methoxy-acetophenone 3 with 3-benzyloxy-4-methoxybenzalde-
vely. Moreover, injection of compound 1 into the vitreous of hyde 4. Catalytic hydrogenation of the chalcone 5 and the
normal adult mice showed no short-term cytotoxic or inflam- cyclization of the dihydrochalcone 6 with formaldehyde led to
matory effects on the retina, nor did it induce apoptosis of the desired 4-chromanone 8 after byproducts (e.g. 7) were
retinal cells.13 These results suggest that proliferative ocular treated with K2CO3. Finally, in order to remove two methyl
vascular diseases such as ROP, DR, and AMD may be targeted groups selectively, the 4-chromanone 8 would be demethylated
using compound 1 or its derivatives. Other recent studies have with a variety of reagents. However, the selective removal of
suggested that 1 has potent anti-inflammatory activity in the two methyl groups under most conditions was not satisfactory.
context of UV-induced skin inflammation14 and allergy.15
Among them, the demethylation using an excess of TMSI in
So far, the syntheses of 5,7-dihydroxy-6-methoxyflavones CHCl3 gave the best conversion,20 but its product was not iden-
and 5,7-dihydroxy-6-methoxyhomoisoflavanones have been tical to cremastranone but rather its regioisomer, called
reported (Fig. 1).16,17 But in spite of cremastranone’s interest- SH-11052 (2). Its structure was elucidated by 2D-NMR spectro-
ing biological activities and the synthesis of these congeners, scopy techniques such as NOESY and HMBC.18 To overcome
to the best of our knowledge, synthesis of 1 has not yet been the unwanted result, the alternative 4-chromone 9 cyclized by
reported. To allow future profiling and development of this DMF and PCl5 was demethylated and hydrogenated.21 Simi-
homoisoflavanone and its analogs, we sought to develop a larly, the desired product was not afforded from 9. Surpris-
scalable synthesis suitable for quickly securing multi-gram ingly, treatment of 8 with BBr3 led to the other isomer
quantities. During our previous synthetic approach toward demethylated on the C4′ position (10)22 (eqn (1), Scheme 2).
cremastranone, the regioisomer (SH-11052, 2) was generated The next trial was to remove the intermediates which were
due to the challenge of selective deprotection of methyl phenyl demethylated only on the C5 position or protected with a
ethers.18 Herein, we describe a short and efficient synthesis of benzoyl group on the C3′ position (eqn (2) and (3), Scheme 2).
cremastranone (1) featuring a solution to the issue of selective Unfortunately, the free OH group at the C7 position could not
deprotection of the methyl phenyl ether. Moreover, we show be generated from any intermediates bearing 5,6,7-trimethoxy-
for the first time that synthetic cremastranone has biological 4-chromanone.
properties consistent with the natural-source compound, in a
disease relevant cell model.
Finally, our key solution for an alternative route to crema-
stranone (1) was the generation of the phenol group on the C7
Our plan for the preparation of cremastranone entailed position prior to the C6 and C4′ positions. Thus 4′-benzyloxy-
4-chromanone formation and deprotection of a methyl aryl 6′-hydroxy-2′,3′-dimethoxyacetophenone 15 was chosen as the
ether.19 This strategy would allow significant flexibility in the starting material instead of 3 (Scheme 3). As reported before,
synthesis of 1 and thereby establish a platform that leads to a 15 was prepared from 2′,4′,6′-trihydroxyacetophenone with
variety of derivatives for further structure–activity relationship several steps.23 Aldol condensation of 15 with isovanillin, fol-
studies. In addition, we envisioned that our synthesis would lowed by catalytic hydrogenation of the chalcone 16 under H2
become very useful for the syntheses of similar interesting and Pd/C afforded the dihydrochalcone 17. With the dihydro-
homoisoflavanones.
As shown in Scheme 1 and described previously,18 our directly attempted using N,N-dimethylformamide dimethyl
initial approach toward the synthesis of cremastranone (1) acetal. However, the methylation of phenol group
chalcone 17 in hand, the formation of the 4-chromone was
a
accompanied this reaction. To overcome the side reaction, the
Scheme 1 The initial synthetic approach to cremastranone (1).
Reagents and conditions: (a) KOH, MeOH, 0 °C, 54%; (b) Pd/C, HCO2Na,
HCO2H, 60 °C, 79%; (c) formalin, NaOH, 60 °C; (d) K2CO3, EtOH, 47% for
2 steps; (e) TMSI (4 eq.), CHCl3, 50 °C, 49%; (f) PCl5, BF3–OEt2, DMF,
62%; (g) HBr, AcOH then H2, Pd/C, MeOH.
Scheme 2 The study of the demethylation of 5,6,7-trimethoxy-4-
chromanone. Reagents and conditions: (a) BBr3 (or BCl3) (2.5 eq.),
CH2Cl2, −78 °C, 65%; (b) TMSI, CHCl3, reflux, 38%; (c) TMSI (4 eq.),
CHCl3, reflux, 52%.
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spectra match those of the reported natural product (ESI
Table 1†).7 To date, the configuration at C3 for natural-source
compound 1 has not been reported. Further experiments are
necessary to determine the absolute configuration of the C3
position in compound 1.
Previously, cremastranone isolated from C. appendiculata
was tested for its anti-proliferative activity against an endo-
thelial cell model, human umbilical vein endothelial cells
(HUVECs), and the 50% growth inhibitory concentration (GI50)
value was reported to be 1.5 µM.11 Hence, we tested the anti-
proliferative activity of synthetic cremastranone 1 on HUVECs
in an alamarBlue fluorescence cell proliferation assay and the
GI50 was observed to be 377 nM. The slightly higher potency of
synthetic 1 might be attributed to a difference in the prolifer-
ation assays used or compound purity. Furthermore, we tested
the anti-proliferative activity of 1 against a more ocular disease
relevant endothelial cell model, human retinal microvascular
endothelial cells (HRECs). We observed clear dose dependent
inhibition of HREC proliferation by 1 in complete medium
Scheme 3 First synthesis of ( )-cremastranone (1). Reagents and con-
ditions: (a) isovanillin, KOH, EtOH, rt, 53%; (b) H2, Pd/C, MeOH, 94%; (c)
K2CO3, benzyl bromide, acetone, reflux, 89%; (d) (CH3)2NCH(OCH3)2,
toluene, reflux, 80%; (e) H2, Pd/C, MeOH, 87%; (f) TMSI (2 eq.), CHCl3,
60 °C, 87%.
dihydrochalcone 17 was carefully treated with benzyl bromide with a GI50 of 217 nM (Fig. 2a). In addition we also tested the
to afford the bis(benzyl ether) 18 which was converted to the proliferation of HRECs activated by vascular endothelial
corresponding 4-chromone 19. To this end, catalytic hydrogen- growth factor, a potent inducer of angiogenesis, in the pres-
ation of 19 led to the desired 4-chromanone 20. Finally, methyl ence of 1 and found the GI50 to be comparable (276 nM).
groups were removed by 2 equivalents of TMSI to give crema- Previously we reported a GI50 of 43 µM for the regioisomer
stranone (1). Thus the antiangiogenic homoisoflavanone SH-11052 (2),18 which differed only in the positions of hydroxyl
cremastranone is available in 6 steps and 26.8% overall yield and methoxy groups at C6 and C7 as compared to 1. The
from the acetophenone 15. Although our synthetic cremastra- importance of positions of the groups on the ‘A’ ring suggests
none is a racemate, its chemical shifts in 1H- and 13C-NMR that a structure–activity relationship can be established.
Fig. 2 Synthetic cremastranone (1) blocks in vitro angiogenesis of human retinal microvascular endothelial cells (HRECs) in a dose-dependent
manner. (a) The effect of 1 on proliferation of HRECs was tested using an alamarBlue fluorescence assay. (b, e) In an EdU incorporation assay, 1
blocked DNA synthesis as evidenced by a decrease in incorporation of EdU (pink) into the nuclei of HRECs (nuclei, blue). (c, f) Tube formation by
HRECs on Matrigel was blocked by 1; the extent of tube formation was measured as tubule length. (d) Migration of HRECs in a scratch-wound assay
was inhibited by 1. Original magnifications: 40×. The data points in all graphs indicate mean SEM; ** represents p < 0.01; *** represents p < 0.001
(ANOVA with Dunnett’s post hoc tests). Each of the panels is representative of three independent experiments.
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We then measured the endothelial cell specificity of 1 by Graduate Scholarship from Kemin Health to HDB. This publi-
measuring its effects on the proliferation of non-endothelial cation was also supported in part by an unrestricted grant
ocular cell lines, Y79 (retinoblastoma cell line), 92-1 (uveal from Research to Prevent Blindness, Inc.
melanoma cell line) and ARPE-19 (retinal pigmented epithelial
cells). The anti-proliferative potency of 1 on these non-endo-
thelial cell lines was significantly lower than that on endo-
thelial cells with GI50 values of 9.8 µM, 47 µM, and >250 µM
on Y79, 92-1, and ARPE-19 cells, respectively, indicating that
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Acknowledgements
This work was supported by grants from the International
Retinal Research Foundation, Ralph W. and Grace
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methoxy-4-chromanone with 3,4-dihydroxybenzaldehyde,
olefin reduction, and mono-demethylation on the C5 is
identical to compound 10. For detailed scheme and experi-
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