Synthesis and biological evaluation of novel gigantol derivatives as potential agents in prevention of diabetic cataract

Article abstract of DOI:10.1371/journal.pone.0141092

As a continuation of our efforts directed towards the development of natural anti-diabetic cataract agents, gigantol was isolated from Herba dendrobii and was found to inhibit both aldose reductase (AR) and inducible nitric oxide synthase (iNOS) activity, which play a significant role in the development and progression of diabetic cataracts. To improve its bioefficacy and facilitate use as a therapeutic agent, gigantol (compound 14f) and a series of novel analogs were designed and synthesized. Analogs were formulated to have different substituents on the phenyl ring (compounds 4, 5, 8, 14a-e), substitute the phenyl ring with a larger steric hindrance ring (compounds 10, 17c) or modify the carbon chain (compounds 17a, 17b, 21, 23, 25). All of the analogs were tested for their effect on AR and iNOS activities and on D-galactose-induced apoptosis in cultured human lens epithelial cells. Compounds 5, 10, 14a, 14b, 14d, 14e, 14f, 17b, 17c, 23, and 25 inhibited AR activity, with IC50 values ranging from 5.02 to 288.8 μM. Compounds 5, 10, 14b, and 14f inhibited iNOS activity with IC50 ranging from 432.6 to 1188.7 μM. Compounds 5, 8, 10, 14b, 14f, and 17c protected the cells from D-galactose induced apoptosis with viability ranging from 55.2 to 76.26%. Of gigantol and its analogs, compound 10 showed the greatest bioefficacy and is warranted to be developed as a therapeutic agent for diabetic cataracts.

Full text of DOI:10.1371/journal.pone.0141092

RESEARCH ARTICLE
Synthesis and Biological Evaluation of Novel
Gigantol Derivatives as Potential Agents in
Prevention of Diabetic Cataract
Jie Wu1☯, Chuanjun Lu2,3☯, Xue Li1, Hua Fang1, Wencheng Wan1, Qiaohong Yang1,
Xiaosheng Sun1, Meiling Wang1, Xiaohong Hu1, C.-Y. Oliver Chen4, Xiaoyong Wei1,4
*
1 School of Basic Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006,
China, 2 College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, 310014, China,
3 Institute of Drug Synthesis and Pharmaceutical Processing, School of Pharmaceutical Sciences, Sun Yat-
sen University, Guangzhou, 510006, China, 4 Antioxidants Research Laboratory, Jean Mayer USDA Human
Nutrition Research Center on Aging, Tufts University, Boston, MA, 02111, United States of America
a11111
☯ These authors contributed equally to this work.
* jidewowxy@163.com
Abstract
OPEN ACCESS
As a continuation of our efforts directed towards the development of natural anti-diabetic
cataract agents, gigantol was isolated from Herba dendrobii and was found to inhibit both
aldose reductase (AR) and inducible nitric oxide synthase (iNOS) activity, which play a sig-
nificant role in the development and progression of diabetic cataracts. To improve its bioeffi-
cacy and facilitate use as a therapeutic agent, gigantol (compound 14f) and a series of
novel analogs were designed and synthesized. Analogs were formulated to have different
substituents on the phenyl ring (compounds 4, 5, 8, 14a-e), substitute the phenyl ring with a
larger steric hindrance ring (compounds 10, 17c) or modify the carbon chain (compounds
17a, 17b, 21, 23, 25). All of the analogs were tested for their effect on AR and iNOS activi-
ties and on D-galactose-induced apoptosis in cultured human lens epithelial cells. Com-
pounds 5, 10, 14a, 14b, 14d, 14e, 14f, 17b, 17c, 23, and 25 inhibited AR activity, with IC50
values ranging from 5.02 to 288.8 μM. Compounds 5, 10, 14b, and 14f inhibited iNOS activ-
ity with IC50 ranging from 432.6 to 1188.7 μM. Compounds 5, 8, 10, 14b, 14f, and 17c pro-
tected the cells from D-galactose induced apoptosis with viability ranging from 55.2 to
76.26%. Of gigantol and its analogs, compound 10 showed the greatest bioefficacy and is
warranted to be developed as a therapeutic agent for diabetic cataracts.
Citation: Wu J, Lu C, Li X, Fang H, Wan W, Yang Q,
et al. (2015) Synthesis and Biological Evaluation of
Novel Gigantol Derivatives as Potential Agents in
Prevention of Diabetic Cataract. PLoS ONE 10(10):
e0141092. doi:10.1371/journal.pone.0141092
Editor: Rong Wen, Univeristy of Miami, UNITED
STATES
Received: April 19, 2015
Accepted: October 5, 2015
Published: October 30, 2015
Copyright: © 2015 Wu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the National
Natural Science Foundation of China (81274157,
81102674), Guangzhou Science & Technology
Planning Project (2014J4100082), Guangdong
Natural Science Foundation (S2011010005661),
Guangdong Science & Technology Planning Project
(2011B031700076, 2009B090300335) and the U.S.
Department of Agriculture (USDA)/Agricultural
Research Service (Cooperative Agreement No. 1950-
5100-087).
Introduction
Gigantol (4-[2-(3-hydroxy-5-methoxyphenyl)ethyl]-2-methoxyphenol, PubChem CID:
10221179) is a naturally occurring 1,2-diphenylethane(bibenzyl) found in Herba dendrobii [1].
The literature has shown that gigantol has several bioactions, e.g. anti-carcinogenic [2–5], anti-
oxidant [6], anti-aging [7], anti-coagulating [8], anti-mutagenic [9], antispasmodic [10–12],
and anti-inflammatory [13]. Although the structure of gigantol is different from that of more
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
Competing Interests: The authors have declared
that no competing interests exist.
extensively studied aldose reductase (AR) inhibitors, such as carboxylic acids, spirohydantoin
derivatives, and compounds with sulfonyl groups [14–16]. Previous studies have shown that
gigantol extracted from dendrobii prevented and inhibited development of cataracts through
its inhibitory effect on the activity of AR and inducible nitric oxide synthase (iNOS) [17].
Cataracts are the leading cause of visual impairment and blindness worldwide [18]. The
development and progression of cataracts are attributed to a wide range of risk factors, e.g.
aging, genetics, radiation, medications, and diseases. Among these factors, chronic hyperglyce-
mia is understood to increase the risk of cataracts because hyperglycemic conditions increase
osmotic pressure and induce oxidative damage in lenses, partially through the activation of AR
and iNOS [19–22]. AR converts glucose to sorbitol, whose accumulation inside cells in turn
causes fluid accumulation, elevates osmotic pressure, and induces lens swelling and degeneration
of hydropic lens fibers [23–25]. All of these events enable cataract development. Furthermore,
peroxynitrites are formed from superoxides and nitric oxides when iNOS expression and activity
is up-regulated by the hyperglycemic condition involved in pathogenesis of cataracts [26].
Due to increasing number of patients with diabetes worldwide, the incidence of diabetic cat-
aracts is steadily increasing [27]. Even though cataract surgery is an effective cure, this opera-
tion may not be the best option for all patients because of surgery related health concerns,
complications, and costs [28, 29]. For this reason, it is necessary to develop pharmacological
therapies for diabetic cataract treatment and prevention. In this context, gigantol could be a
suitable drug candidate for the treatment and prevention of diabetic cataracts. However, the
limited availability of gigantol from its natural source, Herba dendrobii and other plants, may
limit its development and use in diabetic cataract prevention. Thus, to continue investigating
applicability of gigantol in diabetic cataracts, chemical synthesis of gigantol and its analogs
becomes a viable approach. In addition to serving as a therapeutic agent for diabetic cataracts,
some of these analogs could be valuable drug candidates for tumor therapy, local anesthetics,
antidepressants, or antipsychotics, and smooth muscle relaxants [30]. Because the bioactivity
and bioefficacy of these analogs have not been assessed in diabetic cataracts, the main objective
of the study was to synthesize gigantol and its analogs and then assess their effect on the devel-
opment and progression of diabetic cataracts through modulation of AR and iNOS. The gigan-
tol analogs were synthesized by using different substituents on the phenyl ring (compounds 4,
5, 8, 14a–e), substituting the phenyl ring with a larger steric hindrance ring (compounds 10,
17c), and changing the carbon chain (compounds 17a, 17b, 21, 23, 25). Their bioactions were
assessed by determining their capability to inhibit AR and iNOS activity and ameliorate D-
galactose-induced death of cultured human lens epithelial cells (HLECs).
Results and Discussion
Synthesis of gigantol and its analogs
The routes of synthesis of gigantol analogs are shown in Figs 1 and 2. Compounds 5 and 8
were synthesized in six steps according to previously reported procedures (Fig 1) [31]. Using
commercially available 3,5-dimethoxybenzaldehyde as the starting material, compound 2 was
synthesised through reduction, bromination, and reaction with triethylphosphite. Compound
2 served as the starting compound. Wittig olefination, followed by hydrogenation and demeth-
ylation, produced compounds 5 and 8. The synthesis of compounds 10, 14, and 14f was similar
to that of compound 4, except that the starting material was first protected by chloromethyl
methyl ether (MOMCl) and benzyl bromide, respectively (Fig 1). Compounds 17a–c were syn-
thesized in one pot. Amine reacted with aldehyde to produce imine, and NaBH4 was then
added to produce the target compounds (Fig 2). Intermediate compound 19 was synthesized
by aldol condensation, followed by hydrogenation and demethylation to yield compound 21
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
Fig 1. Synthesis of 4, 5, 8, 10, 14, and gigantol. Reagents and conditions: a. NaBH4, MeOH; b. PBr3, pyridine, 0°C; c. P(OEt)3, 120°C; d. different
aldehydes, CH3ONa, 0°C to room temperature (RT), 12 h; e. Pd/C, H2, RT, 12 h; f. BBr3, CH2Cl2, -20°C, 2 h; RT, 4 h; g. NaH, ethanethiol, DMF, N2, reflux; h.
MOMCl, i-Pr2NEt, CH2Cl2, 0°C, 1 h; RT, 12 h; i. diethyl naphthalen-1-ylmethylphosphonate, CH3ONa, 0°C, 1 h; rt, 12 h; j. 2 M HCl, methanol, 50°C, 1 h; k.
BnBr, 18-crown-6, K2CO3, reflux, 9 h.
doi:10.1371/journal.pone.0141092.g001
Fig 2. Synthesis of 17, 21, 23, and 25. Reagents and conditions: a. TsOH, ethanol; 0°C, NaBH4; b. K2CO3, ethanol; c. Pd/C, H2, RT, 12 h; d. BBr3, CH2Cl2,
-20°C, 2 h; RT, 4 h. e. Et3N, CH2Cl2; f. 180°C, neat, N2.
doi:10.1371/journal.pone.0141092.g002
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
(Fig 2). As shown in Fig 2, compound 23 was generated by reacting 4-methoxyaniline with
4-methylbenzene-1-sulfonyl chloride followed by the addition of BBr3. Compound 25 was pro-
duced by the reaction of 2-(4-hydroxyphenyl)acetic acid and 2-(3,4-dimethoxyphenyl)ethana-
mine with stirring at 180°C without solvent under N2. The purity of all synthesized
compounds was determined by HPLC.
Biological activities of gigantol and analogs
Evaluation of AR inhibitory properties. AR has been acknowledged as a validated dia-
betic cataract inducer [32–36]. Thus, we tested the potential of gigantol and its analogs for pre-
vention and treatment of diabetic cataract by assessing their capability to inhibit AR activity.
Table 1 shows that compounds 14a–e were more potent than synthetic gigantol (compound
14f) except compound 14c. Results showed that most synthetized compounds were capable of
inhibiting AR activity with their IC50 values ranging from 5.02 to 347.35 μM in a dose-depen-
dent manner, which were at least 5 times lower than the extracted gigantol. Among these syn-
thetic compounds, compounds 23 (5.02 μM), 14a (17.28 μM), 14e (21.83 μM), and 10
(31.86 μM) displayed potency in AR inhibition [37, 38]. Of all tested compounds, sulfonamide
compound 23 appeared to be the best inhibitor, suggesting that the N-sulfonylation link might
play a critical role in the binding of the compound to the AR catalytic site because sulfonyl
group has been reported as an important pharmacophore of AR inhibitors [39–42]. As syn-
thetic gigantol (compound 14f) exhibited intermediate potency, we found that substituting one
of the phenyl ring in gigantol with a larger steric hindrance naphthalene ring made the com-
pound 10 9-fold more potent. In order to study the role of the 4-hydroxy-3-methoxyphenyl
ring in the AR inhibition, we synthesized compounds 14a-e by keeping 4-hydroxy-3-methoxy-
phenyl ring and placing different substituents on the other phenyl ring, and results showed
that significance of the 4-hydroxy-3-methoxyphenyl ring in AR inhibition. These results sug-
gest that compounds 10, 14a, 14e, and 23 can be considered as lead compounds for further
development of new diabetic cataract drugs.
Assessment of anti-iNOS inhibitory properties. The role of iNOS in the development of
diabetic cataracts has been well documented [21]. Results showed that compounds 5, 10, 14b,
and 14f inhibited iNOS in a dose-dependent manner with IC50 values ranging from 432.6 to
1188.7 μM (Table 2). Although the IC50 of compounds 5, 10, 14b, and 14f was larger than that
of the extracted gigantol, these compounds remain good candidates for the development of dia-
betic cataract drugs because of their superior AR inhibitory effect.
Table 1. Inhibitory effect of gigantol and its analogs on AR activity1.
Compound
IC50 (μM)
Compound
IC50 (μM)
Extractive gigantol
2516.6 10.35
347.35 3.74
288.80 2.16
513.38 2.33
31.17 1.51
17.28 1.72
125.94 1.3
534.35 5.44
39.20 2.13
14e
14f
17a
17b
17c
21
21.83 5.47
176.06 3.21*
NA
4
5
8
173.73 3.38
242.67 5.67
556.34 4.37
5.02 2.57
54.44 2.39
10
14a
14b
14c
14d
23
25
1The results are expressed as mean SD (n = 3).
Abbreviation: NA, no activity
*P < 0.01, vs. Extractive gigantol.
doi:10.1371/journal.pone.0141092.t001
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
Table 2. Inhibitory effect of gigantol and its analogs on iNOS activity1.
Compound
IC50 (μM)
Compound
I C50 (μM)
Extractive gigantol
32.23 2.61
14e
14f
17a
17b
17c
21
NA
4
NA
432.6 2.37
NA
680.07 3.28
5
NA
NA
NA
NA
NA
NA
8
10
1188.7 3.31
NA
14a
14b
14c
14d
433.57 4.23
NA
23
25
NA
1The results are expressed as mean SD (n = 3). Abbreviation: NA, no activity.
doi:10.1371/journal.pone.0141092.t002
Evaluation of the effects on D-galactose-induced cell death in HLECs
Galactose toxicity causes the sequential death of different LEC populations in the lenses of
galactosemic rats, starting with those in the central and peripheral mitotic zone, followed by
the central (non-mitotic) LECs, and eventually the remaining LECs [43, 44]. In this study, the
protective effect of the synthetic compounds was tested at concentrations 0.1, 0.5, and
1 μgÁmL-1 on D-galactose-induced apoptosis of the cultured HLECs. Results showed that
extracted gigantol (1.0 μgÁmL-1, 5.28 μM)) and compounds 5 (0.5 μgÁmL-1, 1 μM), 8
(1.0 μgÁmL-1, 4.39 μM), 10 (0.5 μgÁmL-1, 0.89 μM), 14b (0.1 μgÁmL-1, 0.366 μM), 14f
(1.0 μgÁmL-1, 5.28 μM), and 17c (0.1 μgÁmL-1, 0.388 μM) protected HLECs from apoptosis. Of
all compounds tested, compound 10 showed the most efficacious protection against apoptosis
with the cell survival reaching 72.26% (P<0.05) (Fig 3). Given that apoptosis of HLEC contrib-
utes greatly to cataract formation, protecting HLECs against programmed cell death appears to
Fig 3. Gigantol analogs at 0.1, 0.5, and 1.0 μgÁmL-1 on viability of HLECs treated with 250 mmolÁL-1 D-
galactose for 72 h. Cell viability was determined by the MTT assay in the absence (Con) and presence (all
other groups) of D-galactose. Ext-G refers to gigantol extracted from dendrobii. Viability (mean SD, n = 3) is
expressed as the percentage of viable cells in the treatment to those of the Con. #P < 0.01 vs. Con, *P < 0.05
vs. D-galactose.
doi:10.1371/journal.pone.0141092.g003
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
be one of the therapeutic strategies for cataract treatment [45]. Results show that extracted
gigantol and compounds 5, 8, 10, 14b, 14f, and 17c were the most effective in the protection of
LEC against D-galactose-induced apoptosis.
Conclusions
Gigantol and its synthetic analogs were evaluated for their potential to treat diabetic cataracts.
Synthetic compounds were designed by placing different substituents on the phenyl ring (com-
pounds 4, 5, 8, 14a-e), substituting the phenyl ring with a larger steric hindrance ring (com-
pounds 10, 17c), and changing the carbon chain (compounds 17a, 17b, 21, 23, 25). We found
compounds 4, 5, 10, 14b, 14d, 14e, 14f, 17c, 23, and 25 inhibited AR activity with the IC50
value ranging from 5.02 to 347.35 μM and compounds 5, 10, 14b, and 14f inhibited iNOS
activity with the IC50 values ranging from 432.6 to 1188.7 μM. Furthermore, compounds 5, 8,
10, 14b, 14f, and 17c and the extracted gigantol protected HLECs from apoptosis. Among all
of the test compounds, only synthesized gigantol and its analogs 5, 10, and 14b were effective
to inhibited AR and iNOS activities and reduced HLEC apoptosis. Of these 3 compounds, com-
pound 10 (3-methoxy-5-(2-(naphthalen-1-yl)ethyl)phenol) showed the most pronounced pro-
tective effect against apoptosis, and further studies are needed to test its candidacy as a diabetic
cataract drug, starting with the bioavailability, efficacy, and toxicity in in vivo experiments.
Materials and Methods
Synthesis
Mass spectrometry was performed on an Agilent LC–MS 6120 system equipped with an ESI
mass spectrometer. Melting points were determined using an SRS OptiMelt Automated Melt-
ing Point System. NMR spectra were generated on a Bruker Avance III spectrometer (S1 File),
using tetramethylsilane(TMS) as an internal standard. The purity of synthesized compounds
was verified by an HPLC system equipped with an Eclipse Plus C8 column (4.6 × 150 mm,
5 μm). Compound 12 was synthesized according to the previously reported procedure [46].
General procedure 1. Phosphate (1.5 equiv.) and sodium methoxide (3 equiv.) were
mixed in DMF and stirred for 30 min at 0°C. Then, 1 equiv. aldehyde was added to DMF under
nitrogen. The resulting mixture was stirred overnight at room temperature, quenched by addi-
tion of ice-cold water, and extracted using ethyl acetate. After the removal of organic solvents,
the crude product was purified using silica gel chromatography with ethyl acetate/petroleum
ether as the eluent.
General procedure 2. To compound dissolved in methanol, 10% Pd/C was added and the
resulting mixture was stirred overnight at room temperature under hydrogen. The weight of
compound to Pd/C was 10:1. After the reaction mixture was filtrated and concentrated, the
crude product was purified either by recrystallization or silica gel chromatography to yield the
target product.
General procedure 3. BBr3 (4 equiv.) was added dropwise to a 1 equiv. solution of com-
pound in dried CH2Cl2 at -20°C under nitrogen. The resulting solution was slowly warmed to
room temperature and stirred overnight. Then, ice-cold water was slowly added and the mix-
ture filtered to yield the crude product. The final product was obtained after purification using
recrystallization or silica gel chromatography.
General procedure 4. p-Toluenesulfonic acid (0.2 equiv) was added to a solution of amine
(1.0 equiv) and aldehyde (1.0 equiv) in ethanol, and the resulting mixture was stirred at room
temperature. When the raw material faded (as monitored by TLC), NaBH4 (3.0 equiv) was
added at 0°C. The mixture was stirred for 1 h, followed by addition of water and extraction
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
using ethyl acetate. Finally, all solvents were removed under reduced pressure to obtain the
crude product, which was purified by flash chromatography on silica gel.
(E)-1,2-bis(3,5-dimethoxyphenyl)ethane (3). Compound 3 was synthesized according to
the general procedure 1. The crude product was purified by column chromatography (petro-
leum ether: ethyl acetate = 30:1) to yield 3 (0.51 g, 85%) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 7.01 (s, 2H), 6.67 (d, J = 2.2 Hz, 4H), 6.41 (t, J = 2.2 Hz, 2H), 3.83 (s, 12H). 13C NMR
(101 MHz, CDCl3) δ 161.0, 139.2, 129.2, 104.7, 100.2, 55.4. LC/MS (ESI): 254.1[M+H]+. HPLC
purity: 97.1%.
1,2-bis(3,5-dimethoxyphenyl)ethane (4). Compound 4 was synthesized according to the
general procedure 2. The crude product was purified by column chromatography (petroleum
ether: ethyl acetate = 40:1) to yield 4 (0.26 g, 87%) as a white solid. mp: 104.6–107.9°C. 1H
NMR (400 MHz, CDCl3) δ 6.36–6.35 (m, 4H), 6.33–6.31 (m, 2H), 3.77 (d, J = 1.1 Hz, 12H),
2.85 (s, 4H). 13C NMR (101 MHz, CDCl3) δ 160.8, 144.1, 106.5, 98.0, 55.3, and 38.0. LC/MS
(ESI): 303.1 [M+H]+. HPLC purity: 96.3%.
5,5'-(ethane-1, 2-diyl)dibenzene-1,3-diol5,5'-(ethane-1,2-diyl)dibenzene -1,3-diol (5).
Compound 5 was synthesized according to the general procedure 3. The crude product was
purified by recrystallization with MeOH to yield 5 (0.11 g, 68%) as a white solid. mp: 245.9–
248.1°C. 1H NMR (400 MHz, MeOD): δ 6.15 (s, 4H), 6.09 (s, 2H), 2.69(s, 4H). 13C NMR (101
MHz, MeOD): δ 159.3, 145.6, 108.0, 101.2, 39.0. LC/MS (ESI): 247.1 [M+H]+. HPLC purity:
99.4%.
5-4-(dimethylamino)phenethyl)benzene-1,3-diol (8). Compound 8 was synthesized
according to previously reported procedures [32]. The crude product was purified over silica
gel column chromatography (petroleum ether: ethyl acetate = 5:1) yielding 8 (0.09 g, 90%) as a
gray solid. mp: 152.5–154.1°C. 1H NMR (400 MHz, MeOD) δ 7.29 (d, J = 8.6 Hz, 2H), 7.22 (d,
J = 8.6 Hz, 2H), 5.99 (dd, J = 5.7, 1.6 Hz, 3H), 3.10 (s, 6H), 2.85–2.76 (m, 2H), 2.65 (t, J = 7.6
Hz, 2H). 13C NMR (101 MHz, MeOD) δ 159.4, 144.9, 143.7, 143.3, 131.5, 120.3, 108.2, 101.4,
46.4, 38.6, 37.8. LC/MS (ESI): 258.1 [M+H]+. HPLC purity: 97.4%.
3-hydroxy-5-methoxybenzaldehyde (9). Compound 9 was synthesized using previously
reported procedures [47]. The characteristic of the compound is white solid and mp: 67–68°C,
the yield was 53%. 1H NMR (400 MHz, MeOD) δ 9.83 (s, 1H), 6.93 (ddd, J = 14.6, 2.2, 1.3 Hz,
2H), 6.65 (t, J = 2.3 Hz, 1H), 3.82 (s, 3H). 13C NMR (101 MHz, MeOD) δ 194.0, 162.9, 160.6,
140.1, 110.0, 108.8, 106.7, 56.0.
3-methoxy-5-(2-(naphthalen-1-yl)ethyl)phenol (10). Compound 10 was synthesized
with compound 9 according to the general procedure 1 after protecting by MOMCl, following
by deprotection and hydrogenation to produce the title product. The crude product was puri-
fied over silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) yielding 10
(0.20 g, 72%) as a white solid. mp: 103.9–106.7°C. 1H NMR (400 MHz, CDCl3) δ 8.08 (d,
J = 8.1 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.60–7.44 (m, 2H), 7.39 (t,
J = 7.6 Hz, 1H), 7.30 (d, J = 6.9 Hz, 1H), 6.45–6.21 (m, 3H), 4.87 (br, 1H), 3.76 (s, 3H), 3.46–
3.24 (m, 2H), 3.08–2.85 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 160.9, 156.8, 144.8, 137.7,
133.9, 131.8, 128.9, 126.8, 126.0, 125.9, 125.6, 125.5, 123.6, 108.0, 106.8, 99.2, 55.3, 37.1, 34.7.
LC/MS (ESI): 279.1 [M+H]+. HRMS calcd for C19H18O2 [M+H]+: 279.1380, found: 279.1381.
HPLC purity: 95.0%.
(E)-2-(benzyloxy)-5-(4-(benzyloxy)-3-methoxystyryl)-1,3-dimethoxybenzen (13a).
Compound 13a was synthesized according to the general procedure 1. The crude product was
purified by column chromatography (petroleum ether: ethyl acetate = 20:1) to yield 13a (0.25
g, 52%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 6.9 Hz, 2H), 7.45 (d, J = 7.2
Hz, 2H), 7.34 (ddd, J = 22.4, 13.2, 7.1 Hz, 6H), 7.07 (d, J = 1.7 Hz, 1H), 7.01–6.95 (m, 1H), 6.89
(dd, J = 18.3, 8.4 Hz, 3H), 6.70 (s, 2H), 5.18 (s, 2H), 5.02 (s, 2H), 3.95 (s, 3H), 3.87 (s, 6H).
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
(E)-2-(benzyloxy)-1-(4-(benzyloxy)-3-methoxystyryl)-3-methoxybenzene (13b). Com-
pound 13b was synthes-ized according to the general procedure 1. The crude product was puri-
fied by column chromatography (petroleum ether: ethyl acetate = 15:1) to yield 13b (0.15 g,
33%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.53–7.46 (m, 2H), 7.46–7.41 (m, 2H),
7.41–7.26 (m, 7H), 7.21 (d, J = 7.9 Hz, 1H), 7.06 (q, J = 8.0 Hz, 1H), 7.02–6.95 (m, 2H), 6.86
(ddd, J = 8.8, 8.2, 6.8 Hz, 3H), 5.16 (s, 2H), 5.01 (s, 2H), 3.89 (s, 3H), 3.87 (s, 3H).
(E)-(4-(4-(benzyloxy)-3-methoxystyryl)-1,2-phenylene)bis(oxy)bis(methylene) diben-
zene (13c). Compound 13c was synthesized according to the general procedure 1. The crude
product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to
yield 13c (0.24 g, 45%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.50–7.41 (m, 6H),
7.41–7.28 (m, 9H), 7.12 (s, 1H), 7.04 (s, 2H), 6.92 (s, 2H), 6.85 (s, 3H), 5.20 (s, 2H), 5.17 (s,
4H), 3.94 (s, 3H).
(E)-1-(benzyloxy)-4-(3,5-dimethoxystyryl)-2-methoxybenzene (13d). Compound 13d
was synthesized according to general procedure 1. The crude product was purified by column
chromatography (petroleum ether: ethyl acetate = 10:1) to yield 13d (0.15 g, 40%) as a white
solid. 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 7.1 Hz, 2H), 7.37 (dd, J = 8.1, 6.6 Hz, 2H), 7.31
(d, J = 7.2 Hz, 1H), 7.08 (d, J = 1.9 Hz, 1H), 6.99 (dd, J = 17.3, 9.1 Hz, 2H), 6.92–6.85 (m, 2H),
6.65 (d, J = 2.2 Hz, 2H), 6.38 (t, J = 2.2 Hz, 1H), 5.18 (s, 2H), 3.95 (s, 3H), 3.83 (s, 6H).
(E)-1-(benzyloxy)-2-(4-(benzyloxy)-3-methoxystyryl)-4-chlorobenzene (13e). Com-
pound 13e was synthesized according to the general procedure 1. The crude product was puri-
fied by column chromatography (petroleum ether: ethyl acetate = 10:1) to yield 13e (0.21 g,
46%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 2.5 Hz, 1H), 7.44 (d, J = 7.4
Hz, 4H), 7.41–7.26 (m, 7H), 7.09 (ddd, J = 16.4, 7.9, 4.0 Hz, 3H), 7.01–6.93 (m, 1H), 6.85 (d,
J = 8.5 Hz, 2H), 5.17 (s, 2H), 5.12 (s, 2H), 3.91 (s, 3H).
4-(4-hydroxy-3-methoxyphenethyl)-2,6-dimethoxyphenol (14a). Compound 14a was
synthesized according to the general procedure 2. The crude product was purified by column
chromatography (petroleum ether: ethyl acetate = 5:1) to yield 14a (0.10 g, 66%) as a blue
solid. mp: 78.2–79.5°C. 1H NMR (400 MHz, CDCl3) δ 6.83 (d, J = 7.9 Hz, 1H), 6.67 (d, J = 7.9
Hz, 1H), 6.61 (s, 1H), 6.35 (s, 2H), 5.52 (br, 2H), 3.82 (d, J = 3.6 Hz, 9H), 2.81 (s, 4H). 13
C
NMR (101 MHz, CDCl3) δ 146.9, 146.3, 143.8, 133.7, 132.92, 132.88, 121.1, 114.3, 111.4, 105.3,
56.3, 55.9, 38.4, 37.9. LC/MS (ESI): 305.1 [M+H]+. HPLC purity: 99.7%.
4-(2-hydroxy-3-methoxyphenethyl)-2-methoxyphenol (14b). Compound 14b was syn-
thesized according to the general procedure 2. The crude product was purified by column chro-
matography (petroleum ether: ethyl acetate = 5:1) to yield 14b (0.09 g, 99%) as a brown oil. 1H
NMR (400 MHz, CDCl3) δ 6.82 (d, J = 8.0 Hz, 1H), 6.72 (ddd, J = 8.9, 8.3, 3.9 Hz, 5H), 5.77 (br,
1H), 5.53 (br, 1H), 3.86 (s, 3H), 3.82 (s, 3H), 2.96–2.79 (m, 4H). 13C NMR (101 MHz, CDCl3) δ
146.4, 146.3, 143.7, 143.6, 134.4, 127.8, 122.5, 121.1, 119.3, 114.2, 111.3, 108.6, 56.0, 55.9, 35.7,
32.3. LC/MS (ESI): 273.0 [M-H]-. HPLC purity: 95.0%.
4-(4-hydroxy-3-methoxyphenethyl)benzene-1,2-diol (14c). Compound 14c was synthe-
sized according to the general procedure 2. The crude product was purified by column chroma-
tography (petroleum ether: acetone = 5:1) to yield 14c (0.06 g, 53%) as a red oil. 1H NMR (400
MHz, CDCl3) δ 6.82 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.66 (dd, J = 8.0, 1.9 Hz, 2H),
6.62–6.57 (m, 2H), 5.49 (br, 1H), 5.35 (br, 2H), 3.83 (s, 3H), 2.78 (t, J = 3.3 Hz, 4H). 13C NMR
(101 MHz, CDCl3) δ 146.3, 143.6, 143.4, 141.6, 135.0, 133.8, 121.0, 121.0, 115.7, 115.2, 114.2,
111.2, 55.9, 37.7, 37.5. LC/MS (ESI): 259.0 [M-H]-. HPLC purity: 96.3%.
4-(3,5-dimethoxyphenethyl)-2-methoxyphenol (14d). Compound 14d was synthesized
according to the general procedure 2. The crude product was purified by column chromatogra-
phy (petroleum ether: ethyl acetate = 10:1) to yield 14d (0.10 g, 90%) as a blue oil. 1H NMR
(400 MHz, CDCl3) δ 6.83 (d, J = 8.0 Hz, 1H), 6.69 (dd, J = 8.0, 1.7 Hz, 1H), 6.63 (d, J = 1.6 Hz,
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
1H), 6.32 (dd, J = 7.4, 2.0 Hz, 3H), 5.54 (br, 1H), 3.83 (s, 3H), 3.76 (s, 6H), 2.83 (s, 4H). 13
C
NMR (101 MHz, CDCl3) δ 160.7, 146.3, 144.2, 143.8, 133.7, 121.0, 114.3, 111.2, 106.6, 98.0,
55.9, 55.3, 38.6, 37.4. LC/MS (ESI): 289.1 [M+H]+. HPLC purity: 96.9%.
4-chloro-2-(4-hydroxy-3-methoxyphenethyl)phenol (14e). Compound 14e was synthe-
sized according to the general procedure 2. The crude product was purified by column chroma-
tography (petroleum ether: ethyl acetate = 4:1) to yield 14e (0.05 g, 46%) as a colorless oil. 1H
NMR (400 MHz, CDCl3) δ 7.06 (d, J = 1.6 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 6.84 (d, J = 8.0 Hz,
1H), 6.68 (t, J = 8.3 Hz, 2H), 6.61 (s, 1H), 5.52 (br, 1H), 4.82 (br, 1H), 3.82 (s, 3H), 2.83 (s, 4H).
13C NMR (101 MHz, CDCl3) δ 152.3, 146.4, 143.9, 133.4, 130.1, 129.8, 127.0, 125.4, 121.0,
116.7, 114.4, 111.2, 55.9, 35.7, 32.5. LC/MS (ESI): 277.0 [M-H]-. HRMS calcd for C15H15O3Cl
[M-H]-: 277.0637, found: 277.0644. HPLC purity: 99.9%.
4-[2-(3-hydroxy-5-methoxyphenyl)ethyl]-2-methoxyphenol (14f, gigantol). Compound
14f was synthesized according to the general procedure 2. The crude product was purified by
column chromatography (petroleum ether: ethyl acetate = 5:1) to yield gigantol (0.50 g, 68%)
as a white solid. 1H NMR (400 MHz, CDCl3) δ 6.83 (d, J = 8.0 Hz, 1H), 6.68 (dd, J = 8.0, 1.8
Hz, 1H), 6.62 (d, J = 1.8 Hz, 1H), 6.31 (s, 1H), 6.25 (d, J = 2.0 Hz, 2H), 5.47 (s, 1H), 3.84 (s,
3H), 3.75 (s, 3H), 2.80 (dt, J = 11.8, 5.9 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 160.9, 156.6,
146.3, 144.6, 143.8, 133.6, 121.0, 114.2, 111.2, 108.1, 106.9, 99.1, 55.9, 55.3, 38.3, 37.3. HPLC
purity: 98.5%.
3-methoxy-5-((4-methoxyphenylamino)methyl)phenol (17a). Compound 17a was syn-
thesized according to the general procedure 4. The crude product was purified by column chro-
matography (petroleum ether: ethyl acetate = 3:1) to yield 17a (0.19 g, 74%) as a yellow oil. 1H
NMR (400 MHz, CDCl3) δ 6.77 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 8.9 Hz, 2H), 6.49 (s, 1H), 6.42
(s, 1H), 6.29 (s, 1H), 4.18 (s, 2H), 3.75 (s, 3H), 3.73 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
161.1, 157.3, 152.4, 142.3, 142.2, 115.0, 114.8, 107.0, 105.5, 100.3, 55.9, 55.3, 49.4. LC/MS (ESI):
260.1 [M+H]+. HRMS calcd for C15H17NO3 [M+H]+: 260.1281, found: 260.1287. HPLC purity:
95.3%.
4-((3,4-dimethoxyphenethylamino)methyl)phenol (17b). Compound 17b was synthe-
sized according to the general procedure 4. The crude product was purified by column chroma-
tography (petroleum ether: ethyl acetate = 5:1) to yield 17b (0.24 g, 82%) as a white solid. mp:
116.7–118.6°C. 1H NMR (400 MHz, CDCl3) δ 7.05 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.1 Hz, 1H),
6.76–6.72 (m, 2H), 6.62 (d, J = 8.5 Hz, 2H), 3.87 (s, 3H), 3.84 (s, 3H), 3.71 (s, 2H), 2.93 (t,
J = 7.0 Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 156.3, 149.0, 147.5,
131.9, 129.7, 129.6, 120.6, 115.9, 112.0, 111.5, 55.9, 55.8, 53.2, 50.2, 35.1. LC/MS (ESI): 288.1
[M+H]+. HPLC purity: 98.0%.
2-((benzo[d]thiazol-2-ylamino)methyl)-5-methoxyphenol (17c). Compound 17c was
synthesized according to the general procedure 4. The crude product was purified by column
chromatography (petroleum ether: ethyl acetate = 4:1) to yield 17c (0.19 g, 65%) as a white
solid. mp: 184.3–185.7°C. 1H NMR (400 MHz, MeOD) δ 7.57 (dd, J = 7.9, 0.7 Hz, 1H), 7.44 (d,
J = 7.6 Hz, 1H), 7.26 (td, J = 7.8, 1.2 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H), 7.06 (td, J = 7.8, 1.1 Hz,
1H), 6.48–6.36 (m, 2H), 4.49 (s, 2H), 3.74 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 167.8, 160.8,
156.8, 150.4, 131.5, 129.7, 126.0, 121.9, 120.8, 118.1, 117.8, 106.2, 103.2, 55.2, 44.1. LC/MS
(ESI): 287.0 [M+H]+. HRMS calcd for C15H14N2O2S [M+H]+: 287.0849, found: 287.0863.
HPLC purity: 97.6%.
(E)-3-(3,5-dimethoxyphenyl)-1-(4-fluorophenyl)prop-2-en-1-one (19). A mixture of 1-
(4-fluorophenyl)ethanone (2 mmol) and 3,5-dimethoxybenzaldehyde (2.2 mmol) in ethanol
was added to K2CO3 (20 mmol) and stirred at room temperature overnight. After thorough
extraction with EtOAc, the combined organic layer was washed with brine, dried over anhy-
drous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
compound 19 was purified using silica gel column chromatography with EtOAC/petroleum
ether (1/20) as eluent to produce 0.51 g yellow oil, yield: 89%. 1H NMR (400 MHz, CDCl3) δ
8.05 (t, J = 6.0 Hz, 2H), 7.72 (d, J = 15.7 Hz, 1H), 7.44 (d, J = 15.7 Hz, 1H), 7.18 (t, J = 7.9 Hz,
2H), 6.77 (s, 2H), 6.54 (s, 1H), 3.84 (s, 6H).
1-(3-(4-fluorophenyl)propyl)-3,5-dimethoxybenzene (20). Compound 20 was synthe-
sized according to the general procedure 2. The crude product was purified by column chroma-
tography (petroleum ether: ethyl acetate = 10:1) to yield 20 (0.23 g, 48%) as a colorless oil. 1H
NMR (400 MHz, CDCl3) δ 7.18–7.01 (m, 2H), 7.00–6.85 (m, 2H), 6.43–6.17 (m, 3H), 3.74 (s,
6H), 2.69–2.42 (m, 2H), 2.71–2.38 (m, 2H), 2.13–1.65 (m, 1H), 2.05–1.79 (m, 1H).
5-(3-(4-fluorophenyl)propyl)benzene-1,3-diol (21). Compound 21 was synthesized
according to the general procedure 3. The crude product was purified by column chromatogra-
phy (petroleum ether: ethyl acetate = 4:1) to yield 21 (0.19 g, 92%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) δ 7.06 (s, 2H), 6.92 (t, J = 8.4 Hz, 2H), 6.22 (s, 2H), 6.18 (s, 1H), 2.55–2.51
(m, 2H), 2.45–2.41 (s, 2H), 1.81 (d, J = 4.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 162.4,
160.0, 156.4, 145.7, 137.7, 137.7, 129.7, 127.7, 115.1, 114.9, 108.3, 100.4, 35.1, 34.4, 32.4. LC/MS
(ESI): 247.1 [M+H]+. HRMS calcd for C15H15O2F [M-H]-: 245.0983, found: 245.0987. HPLC
purity: 98.8%.
N-(4-hydroxyphenyl)-4-methylbenzenesulfonamide (23). Compound 23 was synthe-
sized according to the general procedure 3. The crude product was purified by column chroma-
tography (petroleum ether: ethyl acetate = 5:1) to yield 23 (0.17 g, 63%) as a yellow oil. 1H
NMR (400 MHz, MeOD) δ 7.31 (d, J = 8.2 Hz, 2H), 7.04 (d, J = 7.9 Hz, 2H), 6.61 (d, J = 8.8 Hz,
2H), 6.38 (d, J = 8.8 Hz, 2H), 3.15–3.03 (m, 3H). 13C NMR (101 MHz, CDCl3) δ 159.2, 147.3,
140.4, 133.0, 132.7, 130.9, 128.9, 119.1, 24.0. LC/MS (ESI): 262.0 [M-H]-. HPLC purity: 99.8%.
N-(3,4-dimethoxyphenethyl)-2-(4-hydroxyphenyl)acetamide (25). 2-(4-hydroxyphenyl)
acetic acid (1.0 mmol) and 2-(3,4-dimethoxyphenyl) ethanamine (1.0 mmol) were mixed and
stirred at 180°C for 4 hours under N2. Crude product 25 was purified by recrystallization using
acetonitrile to produce 0.25 g gray solid. Yield: 80%, mp: 151.7–153.2°C. 1H NMR (400 MHz,
MeOD) δ 7.02 (d, J = 8.4 Hz, 2H), 6.80 (dd, J = 15.8, 4.9 Hz, 2H), 6.71 (d, J = 8.5 Hz, 2H), 6.68–
6.62 (m, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.39 (t, J = 7.1 Hz, 2H), 3.35 (s, 2H), 2.71 (t, J = 7.0 Hz,
2H). 13C NMR (101 MHz, MeOD) δ 174.6, 157.5, 150.4, 149.1, 133.3, 131.1, 127.6, 122.3, 116.4,
113.8, 113.2, 56.6, 55.4, 43.2, 42.0, 35.9. LC/MS (ESI): 316.1 [M+H]+. HPLC purity: 98.1%.
Biological assay
AR inhibition assays. AR was purchased from Prospec-TanyTechnogene Ltd. (Israel). AR
activity was evaluated as previously described [48, 49]. Briefly, the reaction mixture was com-
posed of enzyme solution (20 mmolÁL-1, 20 μL), NADPH (0.104 mmolÁL-1, 50 μL; Italian Roth,
Italy), DL-glyceraldehyde (10 mmolÁL-1, 50 μL; Sigma, U.S.), buffer phosphate (0.1 molÁL-1),
and selected concentrations of test compounds. The reaction was initiated by the addition of
DL-glyceraldehyde as the substrate, and PBS was used as the blank control. The use of
NAPDH, in parallel to AR activity, was monitored at room temperature for 10 min at 40 s
intervals at 340 nm in an EnSpire™ Multimode Plate Reader. AR inhibition was expressed as
inhibition rate (%) = [1−(A2 − A0)/(A1 − A0)] ×100% [50], where A0 represents the decrease in
NADPH absorbance without AR, substrate, or compounds; A1 represents the decrease in
NADPH absorbance prior to the test compounds addition; A2 represents decrease in NADPH
absorbance following the test compounds addition. All assays were performed in triplicate.
iNOS inhibition assays. iNOS produces NO by catalyzing a reaction involving L-Arg and
oxygen. Its activity was assessed by monitoring NO production using a Nitric Oxide Synthase
Assay Kit (Nanjing Jiancheng Bioengineering Institute, China), which was developed based on
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Synthesis and Evaluation of Gigantol Derivatives in Prevention of DC
the method of Fröhlich et al. [51]. The magnitude of iNOS inhibition is expressed as inhibition
rate which was calculated using the following equation: (%) = [(B1 − B2)/(B1 − B0)] × 100%, B0
and B1 represented the absorbance values obtained for the blank (control solution) and stan-
dard, respectively, and B2 represented the absorbance of the test compounds. All assays were
performed in triplicate.
Cell viability assays. Human lens epithelial cells (HLECs; SRA 01/04) were obtained from
Dr. Fu Shang, USDA HNRCA at Tufts University, Boston, MA, U.S. and Zhongshan Ophthal-
mic Center, Sun Yat-sen University, Guangzhou, P. R. China [52, 53] and cultured in minimal
essential medium (MEM) supplemented with 20% fetal bovine serum (FBS) and a cocktail of
Penicillin-Streptomycin at 37°C in a humid environment containing 5% CO2 [52]. Cells were
harvested at 80% confluency by trypsinization, and then fresh culture medium was added to
generate single-cell suspensions for use in cell viability assays. HLECs were then seeded in
96-well cell culture grade microplates at a density of 1 × 105ÁmL-1. After 24 h of incubation, the
cells were treated for 72 h with 250 mmolÁL-1 D-galactose with and without test compounds
(0.1, 0.5, or 1.0 μgÁmL-1) [45].
Cell viability was assessed using an MTT assay. After the treatments, 20 μL of 5 mgÁmL-1
MTT solution was added to each well, followed by incubation for 4 h at 37°C. The resulting for-
mazan crystals were then dissolved in 150 μL DMSO and absorbance measured at 570 nm
using an EnSpire™ Multimode Plate Reader. The results are presented as percentages of cell sur-
vival as compared to the untreated control group, and all assays were performed in triplicate.
Statistical methods. Statistical analyses and data processing were performed using the
SPSS v.16.0 statistical software. Cell viability data are presented as mean SD. One-way
ANOVA was performed to assess statistical significance between the test compounds. P < 0.05
was considered statistically significant.
Supporting Information
S1 File. Contents: 1H and 13C NMR spectra of target compounds.
(DOCX)
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81274157,
81102674), Guangzhou Science & Technology Planning Project (2014J4100082), Guangdong
Natural Science Foundation (S2011010005661), Guangdong Science & Technology Planning
Project (2011B031700076, 2009B090300335) and the U.S. Department of Agriculture (USDA)/
Agricultural Research Service (Cooperative Agreement No. 1950-5100-087).
Author Contributions
Conceived and designed the experiments: XW. Performed the experiments: CL JW XL. Ana-
lyzed the data: CL JW. Contributed reagents/materials/analysis tools: HF. Wrote the paper:
XW JW CL XL HF WW QY XS MW XH CYOC.
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