Studies on the synthesis and antiproliferative activities of 13-cis-retinoyl sugar derivatives

Article abstract of DOI:10.1080/07328300600966497

New retinoyl sugar derivatives of 13-cis-retinoic acid were synthesized in three ways in this paper in order to enhance pharmacal effects, especially antiproliferative activities of 13-cis-retinoic acid. Their structures were confirmed by IR, ¹H-NMR, ¹³C-NMR, and MS spectra and their antiproliferative activities were determined in vitro using human cancer lines. Results showed that some compounds possessed potential antitumor activities. Copyright Taylor & Francis Group, LLC.

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Studies on the Synthesis and Antiproliferative
Activities of 13‐cis‐Retinoyl Sugar Derivatives
Jian‐Nan Xiang a b c , Li‐Hui Jiang a , Chao‐Yue Chen d , Zhi‐ying Fu c ,
Jun‐Fei Duan a , Xiao‐Xiao He b c & Ke‐Min Wang a b c
a College of Chemistry and Chemical Engineering, Hunan University,
Changsha, China
b Bio‐medicine Engineering Center, Hunan University, Changsha, China
c State Key Laboratory of Chem/Biosensing and Chemometrics, Hunan
University, Changsha, China
d Department of Chemical Engineering, Anhui University of Science and
Technology, Huainan, Anhui, China
Version of record first published: 02 Feb 2007.
To cite this article: Jian‐Nan Xiang , Li‐Hui Jiang , Chao‐Yue Chen , Zhi‐ying Fu , Jun‐Fei Duan , Xiao‐Xiao
He & Ke‐Min Wang (2006): Studies on the Synthesis and Antiproliferative Activities of 13‐cis‐Retinoyl Sugar
Derivatives, Journal of Carbohydrate Chemistry, 25:7, 595-614
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Journal of Carbohydrate Chemistry, 25:595–614, 2006
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300600966497
Studies on the Synthesis
and Antiproliferative
Activities of 13-cis-Retinoyl
Sugar Derivatives
Jian-Nan Xiang
College of Chemistry and Chemical Engineering, Bio-medicine Engineering Center, and
State Key Laboratory of Chem/Biosensing and Chemometrics, Hunan University,
Changsha, China
Li-Hui Jiang
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, China
Chao-Yue Chen
Department of Chemical Engineering, Anhui University of Science and Technology,
Huainan, Anhui, China
Zhi-ying Fu
State Key Laboratory of Chem/Biosensing and Chemometrics, Hunan University,
Changsha, China
Jun-Fei Duan
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, China
Received June 27, 2006; accepted August 14, 2006.
Address correspondence to Jian-Nan Xiang, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082, China. E-mail: jnxiang@ hnu.cn
595

J.-N. Xiang et al.
596
Xiao-Xiao He
Bio-Medicine Engineering Center and State Key Laboratory of Chem/Biosensing and
Chemometrics, Hunan University, Changsha, China
Ke-Min Wang
College of Chemistry and Chemical Engineering, Bio-medicine Engineering Center, and
State Key Laboratory of Chem/Biosensing and Chemometrics, Hunan University,
Changsha, China
New retinoyl sugar derivatives of 13-cis-retinoic acid were synthesized in three ways in
this paper in order to enhance pharmacal effects, especially antiproliferative activities
of 13-cis-retinoic acid. Their structures were confirmed by IR, 1H-NMR, 13C-NMR,
and MS spectra and their antiproliferative activities were determined in vitro using
human cancer lines. Results showed that some compounds possessed potential antitu-
mor activities.
Keywords 13-cis-Retinoic acid, Retinoylation, Retinoyl sugar derivatives, Synthesis,
Antiproliferative activities
INTRODUCTION
Retinoids include active metabolites of vitamin A (retinol, 1) as well as a
diverse spectrum of natural or synthetic derivatives. They play an essential
role in vertebrate growth and development, supporting cell differentiation,
embryonic development, the immune response, and reproduction.[1–3] All-
trans-retinoic acid (2, ATRA), 13-cis-retinoic acid (3, 13-cis-RA), and other
retinoids are currently used for treatment of dermatological disorders and che-
motherapeutic agent against various endothelial cancers, breast cancer, and
endometrial cancer.[4–7] The actions of retinoids are mediated through
binding and activation of the retinoic acid receptors (RARs) or retinoid X recep-
tors (RXRs), which function as ligand-dependent transcription factors.[8]
However, retinoids, including retinoic acids, have been found to be too toxic
at high dosage levels to be of practical value for cancer prevention in higher
mammals. Side effects such as teratogenicity, hepatotoxicity, and headaches
have been observed as a result when the most of these compounds were
used.[9] Researchers have been pursuing the discovery and synthesis of novel
retinoic acid analog in order to increase their therapeutic efficiency and/or
reduced toxicity (Sch. 1).[10]
It is known that carbohydrates and their derivatives have a significant bio-
logical role and occur widely in all living matter and participate in many
biological functions.[11] Nishikawa et al.[12] have prepared a series of saturated
fatty acyl derivatives of 2,3,4,6-tetra-O-acetyl-b-D-glucopyranose (4). These
agents to be tested were administrated to mice by intraperitoneal injection
and with high antitumor activity.

13-cis-Retinoyl Sugar Derivatives
597
Scheme 1: Structures of retinol, all-trans-RA, and 13-cis-RA.
In the literatures [4] and [13–14], all-trans-retinoyl glucuronide (ARAG,
5), which readily formed from ATRA (2) in several tissues, play important
roles in cellular differentiation, proliferation, and other cellular events.
Additionally, numerous reports have indicated that the biological activity of
5 is similar to that of 2, otherwise without the toxic side effects of 2,[15] and
can function as a nontoxic substitute in a variety of clinic settings.[16] On the
other hand, Winum et al.[17] have reported that three glycosyl conjugates of
4-hydroxyphenylretinamide (6) have been synthesized and tested on a broad
variety of tumor cells (Sch. 2).
These retinoids with potential antiproliferative properties have led us to
investigate the influence of a glycosyl moiety on the activity and the toxicity
of retinoids. The carbohydrate moiety can be expected to play the role of
drug carrier and improve the activity and selectivity of compounds for cancer-
ous cell lines. There have reports that O-Aryl glycosides have good biological
activities. For example, gastrodin is the bioactive component of Gastrodia
elata blume and is widely used for the treatment of rheumatism, headache,
and vertigo.[18] Therefore, sugar derivatives with structures of the glycosyl
moiety both directly and indirectly linked with 13-cis-RA were designed in
our study, and 10 sugar conjugates (8a–8d, 9, 11a, 17a, 17b, 18, and 19) of
13-cis-RA were synthesized in three ways. Their structures were characterized
and bioactivities were evaluated by MTT method.
RESULTS AND DISCUSSION
Synthesis of Compound 7a–7d
The synthesis of compounds 2,3,4,6-tetra-O-acetyl-s-D-glucopyranosyl
bromide (7a), 2,3,4-tri-O-acetyl-s-D-xylopyranosyl bromide (7b), 2,3,6,20,30,40,
60-hepta-O-acetyl-s-lactosylbromide(7c),and2,3,6,20,30,40,60-hepta-O-acetyl-s--
maltobiosyl bromide (7d) were performed as shown in Scheme 3. Bromo-deri-
vatives of glucose, xylose, lactose, and maltobiose have been prepared in two
steps as described in the literature[19] in high yield. Crystallization of the
crude products from anhydrous ether afforded pure products. The purity and
identity of these compounds were confirmed by HPLC/MS and 1H NMR.
They were used in the next synthetic sequence as soon as possible.

J.-N. Xiang et al.
598
Scheme 2: Structures of 4, 5 (ARAG), and 6.
Synthesis of Compounds 8a–8d and 9
1-O-retinoyl-2,3,4,6-tetra-O-acetyl-b-D-glucopyranose (8a), 1-O-retinoyl-
2,3,4-tri-O-acetyl-b-D-xylopyranose (8b), 1-O-retinoyl-2,3,6,20,30,40,60-hepta-O-
acetyl-b-lactose (8c), and 1-O-retinoyl-2,3,6,20,30,40,60-hepta-O-acetyl-b-maltose
(8d) were synthesized by the reaction of 7a–7d with 13-cis-RA in the presence
of potassium hydroxide and 4-dimethylaminopyridine (DMAP) as a catalyst in
53% to 65% yields. 1-O-retinoyl-b-D-glucopyranose (9) was obtained by refluxing
8a in methanol using dibutyltin oxide as a catalyst in 50% yield.[20]
Synthesis of Compound 10a and 11a
1,2:3,4-diisopropylidenegalactose (10a) was prepared[19] first by conden-
sation of acetone with galactose in the presence of anhydrous cupric sulfate
and concentrated sulfuric acid. Then 6-O-13-cis-retinoyl-1,2:3,4-diisopropene-
galactose (11a) was obtained from the reaction of 13-cis-RA with 10a when
N,N-dicyclohexylcarbodiimide (DCC) was used as dehydrator and DMAP as
catalyst in 73% yield (Sch. 4).
Synthesis of Compound 17a, 17b, 18, and 19
2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (12) was prepared
according to the similar procedure for 7a. Glycosyl bromides were reacted
Scheme 3: The synthetic route to glycosyl retinoates 8a–9.

13-cis-Retinoyl Sugar Derivatives
599
Scheme 4: The two-step synthetic route to glycosyl retinoate 11a.
respectively with the vanillin (or p-hydroxylbenzaldehyde, or o-hydroxylbenz-
aldehyde) to give glycoside 13a (13b, or 14) in good yields. The reduction of
the carbonyl group was facile with sodium borohydride to give 15a (15b, or
16). Then the target compounds 17a (17b, or 18) were synthesized by esterifica-
tion of 15a (15b, or 16) with 13-cis-RA in mild reaction conditions by use of DCC
as reagent under catalysis of DMAP. The 17a was deprotected to yield 19 by the
same method as 9.
Confirming the Configuration for 8a–8d and 9
In the IR spectra of compounds 8a–8d, there are two wide and strong
shoulder peaks of sugar ring at 1000 to 1100 cm21 and 1200 to 1300 cm21
.
Also, the strong carbonyl (C55O) absorption peak takes place at 1720 cm21 [21]
.
In the NMR spectra, identical data were in all respects to the previously made
Scheme 5: The synthetic route of glycoside derivatives of 13-cis-RA.

J.-N. Xiang et al.
600
materials. The mass spectra of (8a–8d, 9) indicate their [M þ Na]þ peaks. All of
these data identify the basic structure of the sugar ester.
Compounds 8a–8d and 9 were confirmed to be b-anomeric pyranosyl deriva-
tives by IR and 1H NMR spectra, which are presented in Table 1. For example, for
8a, there was a medium intensity absorption peak at 909 cm21 in IR spectrum,
which was the shear vibration characteristic absorption of b-anomeric pyranosyl
derivatives. At the same time, the 1H NMR spectrum notably exhibited a large
coupling constant (3J1–2 ¼ 8.4 Hz) for the C1–H signal of sugar ring in 8a,
indicating this compound was b-configuration. The literature[21] indicates that
3
the coupling constant value of C1–H and C2–H of sugar ring are J1–2 ¼ 7 to
10 Hz in b-anomer while 3J1–2 ¼ 2.5 to 3.5 Hz in a-anomer.
Confirming the Structure of Compound 11
1H NMR spectrum of compound 10a matched data reported in the litera-
ture[22] and its structure was confirmed to be a six-ring structure (10a) and
not a five-ring structure (10b) (shown as Sch. 6) because the chemical shift
value of H-1 peak of the galactose sugar ring appears in d ¼ 5.57 ppm higher
field, not in d ¼ 5.80 ppm lower field. Compound 11a should be a six-ring
structure and not a five-ring structure (11b) since esterification reaction of
10a does not involve conversion of the sugar ring configuration.
Confirming the Configurations for Compounds 17a,
17b, 18, and 19
Compounds 17a, 17b, and 18 were confirmed to be b-configuration by IR
1
and H NMR spectra data, which are presented in Table 2. Take 17a as an
example, There was a medium intensity absorption peak in 908 cm21 in IR
spectra, which was the shear vibration characteristic absorption of
b-anomeric pyranosyl derivatives. Besides, C1–H signal of sugar ring in 17a
1
in H NMR spectrum had a chemical shift value smaller than 5.32 ppm and
exhibited a large coupling constant (3J1–2 ¼ 8.0 Hz) for indicating this
compound was b-configuration.[20] The 13C NMR spectra were also in accord-
ance with their expected structures.
Optimal Reaction Conditions
The reported reaction of 13-cis-RA to react with thionyl chloride (PCl5/PCl3 or
other carbonyl chlorides) to form retinoyl chloride, which in turn is reacted with
alcohols to produce retinoates, failed in our laboratory because hydrogen chloride
in solution of retinoyl chloride isomerizes the polyene chain.[23,24] Our method
was established by use of DCC as the dehydrator and DMAP as the catalyst.
It presents the advantages of mild conditions, high yield, and ease of operation.

1
Table 1: The characteristic H NMR, IR, and MS data of compound 8a–8d and 9.
1H NMR (C1–H)
IR
d
3J1–2
(Hz)
Shoulder peak of
C1–H
MS
Compounds
(ppm)
sugar ring (cm21
)
(cm21
)
[M þ Na]þ
Configuration
8a
8b
8c
8d
9
5.77
5.75
5.74
8.4
6.8
8.0
8.4
1044–1082, 1232
1044–1081, 1229
1068, 1230
—
909
903
910
—
653
581
941
941
485
b
b
b
b
b
5.77
Overlapped
1026, 999
827

J.-N. Xiang et al.
602
Scheme 6: The possible configurations for compound 10 and 11.
Sugar esters were mostly conveniently prepared using the Koenigs-Knorr
reaction,[25] phase-transfer catalysis,[26] carbonyl halide method,[27] and enzy
me-catalyzed method.[28] Recently, phase transfer catalysis has proven to be a
very useful method on various synthetic transformations. This methodology
allows synthetic modifications under mild conditions that were heretofore
reserved for very drastic reagents and reaction media.[29] In the present
paper, four glycosyl esters (8a–8d) were synthesized by reaction of sugar
bromide (7a–7d) with 13-cis-RA in the presence of potassium hydroxide and
DMAP as phase-transfer catalyst. This method has advantages of mild
reaction conditions and easy separation of products.
Glucovanillin was extracted from green pods, which have sedative and
anticonvulsant actions, retrieval, and antioxidant. It belongs to O-glucoside,
in which the phenolic hydroxyl group is linked to the glucose.[30] Therefore,
the vanillyl alcohol, and p-hydroxybenzyl alcohol, and o-hydroxybenzyl
alcohol were used as spaces to link the glycosyl and retinoic acid, respectively.
Four glucoside derivatives (17a, 17b, 18, and 19) of 13-cis-RAwere synthesized
by a series of synthetic steps such as acetylation, glycosidation, esterification,
and deprotection. The synthetic procedure of glycosidation was modified with
DMAP as phase-transfer catalyst and simpler workup procedure than the
methods[5] reported and give higher yields.
The reaction condition of deacetylation was crucial in our synthetic route
(Sch. 3 and Sch. 5) because there were two kinds of ester functions both in
8a and 17a. The deacetylation must be carried out without breaking of
retinoyl. According to the literature[20] retinoates 8a and 17a suffered selec-
tively deacetylation by using dibutyltin oxide as catalyst in methanol and the
target compounds 9 and 19 were prepared successfully with good yield.
Biological Assays
8a was tested for antiproliferative activities toward three different cancer
cell lines: human lung cancer cell line (A549), human liver cancer cell line
(BEL7404), and human tongue cancer cell line (Tca). The antiproliferative
activities were summarized in terms of the IC50 values in Table 3. It was

1
Table 2: The characteristic H NMR, IR, and MS data of compound 17a, 17b, 18, and 19.
1H NMR (C1–H)
IR
MS
d
3J1–2
(Hz)
Shoulder peak of
C1-H
Compounds
(ppm)
sugar ring (cm21
)
(cm21
)
[M þ Na]þ
[M þ H]þ
Configuration
17a
17b
18
4.96
5.08
Overlapped
Overlapped
8.0
7.6
—
1044–1070, 1231
1045, 1230
—
908
908
—
789.5
759.4
759.3
622.0
—
b
b
b
b
737.1
737.0
600.0
19
—
1026, 1003
825

J.-N. Xiang et al.
604
Table 3: Antiproliferative activity of 8a.a
Human
cell lines
A549
BEL7404
Tca
IC50 (mM)
32.4
.100
71.1
a IC50: The concentration of drug required for 50%
inhibition.
found that 8a possessed selective antiproliferative activity toward A549.
Therefore, the material retinoic acid 3 and compounds 8b–8d, 11a, and
17a–19 were also tested for antiproliferative activity against A549. The anti-
proliferative activities are summarized in terms of their IC50 values in Table 4.
Based on the data in Table 4, the glycosyl esters 8b–8d showed lower
antiproliferative activity than 3. Antiproliferative activity of 8a and 3 were
similar. 8a was hydrolyzed to yield the 1-O-retinoyl-b-D-glucopyranose (9),
which exhibited interesting antiproliferative activities and was stronger than
3. The result was in accordance with what we expected. The compound 9
showed higher antiproliferative activity than 8a, with perhaps the former
presenting better water soluability.
It should be noted that four glucoside derivatives—17a, 17b, 18, and 19—
exhibited interesting antiproliferative activities and were stronger than 3.
There have reports that gastrodin and glucovanillin are natural compounds
with biological activities.[18,30] 15a and 15b were the derivatives of gastrodin
and glucovanillin, respectively, and were obtained after the glucose was
linked to 4-hydroxylbenzylalcohol and 2-methoxyl-4-hydroxylbenzylalcohol,
respectively (see Sch. 5). 17a and 17b were achieved by the reaction of 15a
and 15b with 13-cis-RA. On the other hand, the novel structure of 17a and
17b presented the characteristics for antiproliferative activity, and among
them the bifunctional group compounds such as 4-hydroxylbenzylalcohol
were used as space to link glycosyl and 13-cis-RA, just like the structure of 6.
Unfortunately, the unobvious difference means that antiproliferative activity
presented small differentiation between the glycosyl molecules and acetylated
glycosyl molecules with linking space, for example, 17a and 19. Perhaps both
the bioactive component group and the structure improved the bioactivity of
13-cis-RA.
Table 4: The antiproliferative activity of compound 8b–8d, 9, 11a, and 17a–19
against A549.
Compound
3
8b
8c
8d
9
11a
17a
17b
18
19
IC50 (mM)
33.3 35.8 42.0 32.8 23.6
21.4
13.0
13.6
21.7 10.2

13-cis-Retinoyl Sugar Derivatives
605
Further investigations of structure–activity relationships of these kinds
of compounds are currently underway in our group.
EXPERIMENTAL SECTION
13-cis-RA was purchased from Sigma Chemical Co. DCC and DMAP were
purchased from Alfa Aesar. The sugars and all other reagents were of the
highest commercially available quality. All retinoids were stored at 2208C
under dry nitrogen atmosphere. All operations involved in the preparation, iso-
lation, purification, and transfer of retinoids were carried out under an atmos-
phere of dry nitrogen. All operations were also performed in dim light or
photographic darkroom light with containers wrapped with aluminum foil or
black cloths. All reactions were monitored by TLC.
Melting points were measured with a XRC-1 melting point apparatus
and are uncorrected. FT infrared (IR) spectra were recorded in KBr disks
1
using FD-5DX spectrometer. H NMR and 13C NMR spectra were obtained
on a Varian INOVA-400 Spectrometer, using CDCl3 as a solvent; TMS (d
0.00 ppm) was used as an internal standard. All NMR chemical shifts are
reported as d values in parts per million (ppm) and coupling constants (J )
are given in hertz (Hz). The splitting pattern abbreviations are as follows: s,
singlet; d, doublet; br, broad peak; and m, multiplet. Mass spectra were
carried out on a ZAB-HS mass spectrometer.
A General Procedure for Compound 8a–8d
13-cis-RA (1.200 g, 4.0 mmol) and DMAP (0.489 g, 4.0 mmol) were suspended
in a mixture of 40 mL dichloromethane and 4% aqueous KOH (5 mL, about
4.0 mmol). The suspension was slowly heated to completely dissolve, the reactant
vigorously stirred, and a solution of 7 (4.0 mmol) in dichloromethane (10 mL)
added dropwise. The reaction solution was heated to reflux and stirred for 6 h
under nitrogen atmosphere. The solution was washed with 4% aqueous NaOH
(15 mL) and distilled water (20 mL ꢀ 3). The organic layer was dried over with
anhydrous Na2SO4 and filtrated, and the filtrate was distilled under reduced
pressure to remove the solvent. The residue was purified by column chromato-
graphy (silica gel 60, mesh size 200–300, ethyl acetate/petroleum ether, v/v).
1-O-retinoyl-2,3,4,6-tetra-O-acetyl-b-D-glucopyranose (8a) was synthesized via
the procedure described. The crude product was purified by column chromato-
graphy (eluent: ethyl acetate: petroleum ether ¼ 1:3, Rf ¼ 0.38) to give 8a as
1
yellow solid, the yield: 61.4%, m.p. 122–1248C. H NMR (400 MHz, CDCl3) d:
1.03 (s, 6H, 2 ꢀ CH3); 1.46–1.49 (m, 2H, H on C2); 1.59–1.65 (m, 2H, H on
C3); 1.72 (s, 3H, CH3 on C5); 1.97–2.09 (m, 20H, 4 ꢀ COCH3 and 2 ꢀ CH3 and
CH2); 3.84–3.88 (m, 1H, glycosyl-ring-H); 4.11–4.31 (m, 2H, glycosyl-ring-H);

J.-N. Xiang et al.
606
5.13–5.30 (m, 3H, glycosyl-ring-H); 5.62 (s, 1H, H14); 5.77 (d, 1H, J ¼ 8.4 Hz,
glycosyl-ring-H1); 6.16 (d, 1H, J ¼ 16.0 Hz, H8); 6.25 (d, 1H, J ¼ 11.6 Hz, H10);
6.31 (d, 1H, J ¼ 16.0 Hz, H7); 7.06 (dd, 1H, J ¼ 11.6 Hz, J ¼ 11.6 Hz, H11);
7.71 (d, 1H, J ¼ 15.2 Hz, H12). 13C NMR (400 MHz, CDCl3) d: 12.9, 19.2, 20.2,
20.6, 21.1, 21.7, 28.9, 33.1, 34.3, 61.0, 66.9, 67.8, 67.9, 71.0, 71.6, 88.1, 91.7,
114.16, 128.8, 129.2, 130.1, 130.3, 133.7, 137.3, 137.6, 141.0, 154.9, 163.7,
169.5, 170.0, 170.2,170.3. MS: m/z ¼ 653 ([M þ Na]þ), 581, 493, 372, 353, 331,
270, 211. IR (KBr), n/cm21: 3437 (broad), 2930 (CH3), 1749 (C55O), 1606–
1579, 1440–1378, 1044–1082, 1232 (shoulder peak of sugar ring), 977, 909.
1-O-retinoyl-2,3,4-tri-O-acetyl-b-D-xylopyranose (8b) was prepared, following the
same procedure, and the reaction time was prolonged to 9 h. After purification by
column chromatography (eluent:ethylacetate: petroleumether ¼ 1:3, Rf ¼ 0.52),
8b was obtained as a pale yellow solid, the yield: 65.0%, m.p. 93–948C. 1H NMR
(400 MHz, CDCl3) d: 1.03 (s, 6H, 2 ꢀ CH3); 1.46–1.49 (m, 2H, H on C2); 1.59–1.65
(m, 2H, H on C3); 1.72 (s, 3H, CH3 on C5); 2.00–2.10 (m, 17H, 3 ꢀ COCH3 and
2 ꢀ CH3 and CH2); 3.54 (dd, 1H, J ¼ 12.0 Hz, J ¼ 12.0 Hz, glycosyl-ring-H);
4.16 (dd, 1H, J ¼ 12.0 Hz, J ¼ 12.0 Hz, glycosyl-ring-H); 5.00 (tt, 1H,
J ¼ 8.4 Hz, J ¼ 8.8 Hz, glycosyl-ring-H); 5.09 (t, 1H, J ¼ 7.6 Hz, glycosyl-ring-
H); 5.24 (t, 1H, J ¼ 8.4 Hz, glycosyl-ring-H); 5.61 (s, 1H, H14); 5.75 (d, 1H,
J ¼ 6.8 Hz, glycosyl-ring-H1); 6.15 (d, 1H, J ¼ 16.4 Hz, H8); 6.24 (d, 1H,
J ¼ 11.6 Hz, H10); 6.30 (d, 1H, J ¼ 15.6 Hz, H7); 7.05 (dd, 1H, J ¼ 12.0 Hz,
J ¼ 11.2 Hz, H11); 7.73 (d, 1H, J ¼ 14.8 Hz, H12). MS: m/z ¼ 581 ([M þ Na]þ),
523, 465, 299, 281. IR (KBr), n/cm21: 3437 (broad), 2939 (CH3), 1749 (C55O),
1608–1580, 1440–1377, 1229, 1044–1081(shoulder peakofsugar ring), 975, 903.
1-O-retinoyl-2,3,6,20,30,40,60-hepta-O-acetyl-b-lactose (8c) was synthesized via
the same procedure used for 8b. The crude product was purified by column
chromatography (eluent: ethyl acetate: petroleum ether ¼ 1:1, Rf ¼ 0.56), 8c
as yellow solid, the yield: 55.6%, m.p. 130–1328C. 1H NMR (400 MHz,
CDCl3) d: 1.03 (s, 6H, 2 ꢀ CH3); 1.46–1.49 (m, 2H, H on C2); 1.59–
1.65 (m, 2H, H on C3); 1.72 (s, 3H, CH3 on C5); 1.97–2.16 (m, 29H,
7 ꢀ COCH3 and 2 ꢀ CH3 and CH2); 3.77–3.90 (m, 3H, glycosyl-ring-H);
4.06–4.92 (m, 5H, glycosyl-ring-H); 4.93–5.36 (m, 5H, glycosyl-ring-H); 5.59
(s, 1H, H14); 5.74 (d, 1H, J ¼ 8.0 Hz, glycosyl-ring-H1); 6.16 (d, 1H,
J ¼ 16.0 Hz, H8); 6.25 (d, 1H, J ¼ 11.2 Hz, H10); 6.31 (d, 1H, J ¼ 16.0 Hz,
H7); 7.05 (dd, 1H, J ¼ 11.6 Hz, J ¼ 11.2 Hz, H11); 7.71 (d, 1H, J ¼ 15.2 Hz,
H12). 13C NMR (400 MHz, CDCl3) d: 12.9, 19.2, 20.5, 20.7, 20.8, 20.9, 21.1,
21.8, 29.0, 33.1, 34.3, 39.6, 60.9, 61.9, 66.6, 69.0, 70.6, 70.7, 71.0, 72.8, 73.4,
75.8, 91.1, 101.0, 114.2, 128.8, 129.2, 130.1, 130.3, 133.7, 137.3, 137.6, 141.0,
154.8, 163.6, 169.1, 169.7, 170.1, 170.2, 170.4. MS: m/z ¼ 941 ([M þ Na]þ),
659, 641, 619, 331. IR (KBr), y/cm21: 3455 (broad), 2950 (CH3), 1753 (C55O),
1609–1583, 1443–1376, 1230, 1068 (shoulder peak of sugar ring), 977, 910.

13-cis-Retinoyl Sugar Derivatives
607
1-O-retinoyl-2,3,6,20,30,40,60-hepta-O-acetyl-b-maltose (8d) was prepared accor-
ding to the procedure described for the synthesis of 8c. After purification by
column chromatography (eluent: ethyl acetate: petroleum ether ¼ 1:1,
Rf ¼ 0.68), 8d was obtained as viscous syrup, the yield: 53.1%, 1H NMR
(400 MHz, CDCl3) d: 1.00 (s, 6H, 2 ꢀ CH3); 1.43–1.46 (m, 2H, H on C2);
1.56–1.62 (m, 2H, H on C3); 1.69 (s, 3H, CH3 on C5); 1.94–2.14 (m, 29H,
7 ꢀ COCH3 and 2 ꢀ CH3 and CH2); 3.83–4.08 (m, 4H, glycosyl-ring-H);
4.20–4.23 (m, 2H, glycosyl-ring-H); 4.42 (d, 1H, J ¼ 12.8 Hz, glycosyl-ring-
H); 4.87 (dd, 1H, J ¼ 10.4 Hz, J ¼ 4.0 Hz, glycosyl-ring-H); 4.98–5.43 (m, 4H,
glycosyl-ring-H); 5.57 (s, 1H, H14); 5.77 (d, 1H, J ¼ 8.4 Hz, glycosyl-ring-H1);
6.13 (d, 1H, J ¼ 16.0 Hz, H8); 6.23 (d, 1H, J ¼ 11.6 Hz, H10); 6.28 (d, 1H,
J ¼ 16.4 Hz, H7); 7.04 (dd, 1H, J ¼ 11.6 Hz, J ¼ 15.6 Hz, H11); 7.69 (d, 1H,
J ¼ 15.2 Hz, H12). MS: m/z ¼ 941 ([M þ Na]þ), 919 ([M þ H]þ), 619, 331.
Synthesis of the Compound 1-O-retinoyl-b-D-
glucopyranose (9)
8a (1.890 g, 3.0 mmol) was dissolved in methanol (50 mL), and then a
catalytic amount of dibutyltin oxide was added. The reaction mixture was
stirred and refluxed for 16 h under nitrogen atmosphere. The reaction
solution was filtered. The solvent was removed from the filtrate. The residue
was purified by column chromatography (eluent: methanol: chloroform ¼ 1:8)
to give the title compound 9 (0.711 g, 51.5%) as a yellow syrup. 1H NMR
(400 MHz, DMSO-d6) d: 1.01 (s, 6H, 2 ꢀ CH3); 1.44–1.45 (m, 2H, H on C2);
1.56–1.59 (m, 2H, H on C3); 1.69 (s, 3H, CH3 on C5); 1.97–1.99 (m, 6H, CH3
on C19 and CH3 on C20); 2.09–2.11 (m, 2H, H on C4); 3.67–3.69 (m, 1H,
glycosyl-ring-H); 4.52 (d, 1H, J ¼ 4.0 Hz, glycosyl-ring-H); 4.65 (d, 1H,
J ¼ 4.8 Hz, glycosyl-ring-H); 4.87 (d, 1H, J ¼ 7.2 Hz, glycosyl-ring-H); 5.09
(d, 1H, J ¼ 4.0 Hz, glycosyl-ring-H); 5.33–5.35 (m, 2H, glycosyl-ring-H); 5.70
(s, 1H, H14); 6.11 (d, 1H, J ¼ 16.0 Hz, H8); 6.22 (d, 1H, J ¼ 11.6 Hz, H10); 6.30
(d, 1H, J ¼ 16.0 Hz, H7); 7.08 (dd, 1H, J ¼ 11.6 Hz, J ¼ 12.0 Hz, H11); 7.65
(d, 1H, J ¼ 15.2 Hz, H12). 13C NMR (400 MHz, DMSO-d6) d: 12.7, 18.7, 20.6,
21.6, 28.8, 32.6, 33.9, 38.9, 40.1, 60.2, 68.0, 69.5, 73.3, 76.2, 94.4, 115.6,
128.4, 128.8, 129.5, 129.7, 130.2, 130.4, 133.3, 137.0, 137.2, 140.5, 152.9,
164.2. MS: m/z ¼ 485.0 ([M þ Na]þ). IR (KBr), n/cm21: 3427(broad), 2935,
1649(C55O), 1454, 1375, 1236, 1157, 1026 (shoulder peak of sugar ring), 999,
827, 766.
Synthesis of the Compound 6-O-13-cis-retinoyl-1,2:3,4-
diisopropylidenegalactose (11a)
13-cis-RA (1.801 g, 4.2 mmol), 1,2:3,4-diisopropylidenegalactose (1.040 g,
4.0 mmol), and a catalytic amount of DMAP were dissolved in anhydrous

J.-N. Xiang et al.
608
dichloromethane (50 mL) at 08C. A solution of DCC (0.911 g, 4.4 mmol) in
anhydrous dichloromethane (15 mL) was added dropwise to the above
mixture. The reaction solution was slowly warmed to room temperature and
stirred for 10 h. The reaction mixtures were diluted with dichloromethane
(30 mL) and cooled at 08C overnight, and the mixture was filtered to remove
the by product urine. Reaction mixture concentrated in vacuo. The residue
was purified by column chromatography (elute: ethyl acetate: petroleum
ether ¼ 1 : 12, Rf ¼ 0.45) to give title compound 1.590 g (yield 73.2%) of the
orange yellow syrup. 1H NMR (400 MHz, CDCl3) d: 1.03 (s, 6H, 2 ꢀ CH3);
1.25–1.30 (m, 2H, H on C2); 1.33 (s, 3H, CH3); 1.35 (s, 3H, CH3); 1.45–1.48
(m, 2H); 1.46 (s, 3H, CH3); 1.52 (s, 3H, CH3); 1.61 (s, 3H, CH3); 1.71 (s, 3H,
CH3 on C5); 1.99 (s, 3H, CH3); 2.00–2.05 (m, 2H); 2.07 (s, 3H, CH3); 4.06
(m, 1H); 4.21–4.36 (m, 4H, glycosyl-ring-H); 4.63 (dd, 1H, J ¼ 7.6 Hz,
J ¼ 2.8 Hz); 5.56 (d, 1H, J ¼ 5.2 Hz); 5.70 (s, 1H); 6.14 (d, 1H, H8,
J ¼ 15.6 Hz); 6.25 (d, 1H, J ¼ 5.6 Hz); 6.29 (s, 1H); 6.98 (dd, 1H, H11,
J ¼ 15.2 Hz, J ¼ 11.6 Hz); 7.76 (d, 1H, H12, J ¼ 15.2 Hz). 13C NMR
(400 MHz, CDCl3) d: 12.8, 19.2, 20.8, 21.7, 24.7, 25.3, 25.5, 26.4, 28.9, 29.0,
30.7, 30.8, 32.7, 32.8, 33.1, 34.2, 39.5, 49.4, 57.7, 119.8, 128.3, 129.4, 130.0,
130.1, 131.3, 137.4, 137.6, 139.3, 147.1, 154.2. MS: m/z ¼ 543 ([M þ H]þ).
A General Synthetic Method for Compound 13a,
13b, and 14
3-Hydroxy-4-methoxylbenzaldehyde (2.990 g, 22 mmol) was dissolved in
dichloromethane (75 mL), and then 4% KOH solution (30 mL, about
24 mmol) and DMAP (1.223 g, 10 mmol) were added. The reaction was slowly
heated to reflux with stirring. A solution of 7a (8.220 g, 20 mmol) in dichloro-
methane (40 mL) was added dropwise with magnetic stirring. The solution
was stirred for an additional 5 h. The solution was slowly cooled to 08C and
the organic layer was separated. The organic layer was washed with 4%
NaOH solution (30 mL ꢀ 2) and distilled water (40 mL ꢀ 3). Then the
organic layer was dried with MgSO4 and concentrated in vacuo. The residue
was recrystallized in anhydrous ethanol. The product 13a was obtained as
1
white solid, the yield: 68.4%, m.p. 144–1468C. H NMR (400 MHz, CDCl3) d:
2.05–2.09 (m, 12H, 4 ꢀ COCH3); 3.84–3.88 (m, 1H, H-50); 3.90 (s, 3H, OCH3);
4.20 (dd, 1H, J ¼ 12.0 Hz, J ¼ 2.4 Hz, H-60); 4.28 (dd, 1H, J ¼ 12.4 Hz,
J ¼ 5.2 Hz, H-60); 5.11 (d, 1H, J ¼ 7.2 Hz, H-10); 5.16–5.21 (m, 1H, H-40);
5.29–5.36 (m, 2H, H-20, H-30); 7.22 (d, 1H, J ¼ 8.0 Hz, Ar-H); 7.42–7.44
(m, 2H, Ar-H); 9.90 (s, 1H, CHO).
Compound 13b was synthesized with the same procedure used for
13a. 4-Hydroxybenzaldehyde was used as starting material. 13b is obtained
as white solid, the yield: 61.2% m.p. 117–1188C. 1H NMR (400 MHz, CDCl3) d:
2.06 (s, 3H, COCH3); 2.07 (s, 6H, 2 ꢀ COCH3); 2.08 (s, 3H, COCH3); 3.92–3.96

13-cis-Retinoyl Sugar Derivatives
609
(m, 1H); 4.19 (dd, 1H, J ¼ 12.4 Hz, J ¼ 2.0 Hz); 4.30 (dd, 1H, J ¼ 12.0 Hz,
J ¼ 5.2 Hz); 5.17–5.20 (m, 1H,); 5.23 (m, 1H); 5.30–5.36 (m, 2H); 7.11 (d, 2H,
J ¼ 8.0 Hz, Ar-H); 7.86 (d, 2H, J ¼ 8.0 Hz, Ar-H); 9.93 (s, 1H, CHO).
Compound 14 was prepared according to the procedure described for the
synthesis of 13a except that 2-hydroxybenzaldehyde and compound 12 were
used as starting materials to afford 14 as white solid, the yield: 64.3%, m.p.
87–908C. 1H NMR (400 MHz, CDCl3) d: 2.01 (s, 3H, COCH3); 2.05 (s, 6H,
2 ꢀ COCH3); 2.18 (s, 3H, COCH3); 4.09–4.24 (m, 3H); 5.13 (dd, 2H,
J ¼ 9.6 Hz, J ¼ 4.0 Hz); 5.47 (d, 1H, J ¼ 3.2 Hz); 5.58 (dd, 1H, J ¼ 10.4 Hz,
J ¼ 8.0 Hz); 7.11 (d, 1H, J ¼ 8.0 Hz); 7.16 (t, 1H, J ¼ 7.6 Hz); 7.55 (t, 1H,
J ¼ 8.4 Hz); 7.84 (d, 1H, J ¼ 7.6 Hz); 10.34 (s, 1H, CHO).
A General Synthetic Method for Compound 15a,
15b, and 16
Compound 15a (3.860 g, 8.0 mmol) was suspended in propan-2-ol (200 mL)
and heated to 508C with stirring. Then the solution was cooled to 268C. NaBH4
(0.150 g, 3.95 mmol) was added with magnetic stirring. The reaction mixture
was stirred for an additional hour at 258C and then poured into ice-water
(80 mL). The pH was adjusted to about 6.0 to 7.0. The mixture was extracted
with chloroform (150 mL ꢀ 3). The combined organic layers were washed
with distilled water (70 mL ꢀ 2), dried with MgSO4, and evaporated by
vacuum. The residue was crystallized in anhydrous ethanol and yielded 15a
as a white solid (16 is yellow viscous syrup), the yield: 86.1%, m.p. 191–
1
1938C. H NMR (400 MHz, CDCl3) d: 2.02–2.07 (m, 12H, 4 ꢀ COCH3); 3.72–
3.76 (m, 1H, H-50); 3.81 (s, 3H, OCH3); 4.14 (dd, 1H, J ¼ 12.0 Hz, J ¼ 2.4 Hz,
H-60); 4.26 (dd, 1H, J ¼ 12.4 Hz, J ¼ 4.8 Hz, H-60); 4.62 (s, 2H, Ar-CH2O);
4.91 (d, 1H, J ¼ 7.6 Hz, H-10); 5.12–5.16 (m, 1H, H-40); 5.25–5.27 (m, 2H,
H-20, H-30); 6.81–6.85 (m, 1H, Ar-H); 6.92 (d, 1H, J ¼ 1.6 Hz, Ar-H); 7.06–
7.09 (m, 1H, Ar-H).
Compound 15b was prepared following the same procedure used for 15a.
13b was used as starting material and obtained as white solid, the yield:
1
83.4%, m.p. 170–1718C. H NMR (400 MHz, CDCl3) d: 2.04 (s, 3H, COCH3);
2.06 (s, 3H, COCH3); 2.07 (s, 3H, COCH3); 2.09 (s, 3H, COCH3); 3.80–3.92
(m, 1H, H-50); 4.15–4.18 (m, 1H, H-60); 4.27–4.30 (m, 1H, H-60); 4.65 (s, 2H,
Ar-CH2O); 5.07 (d, 1H, J ¼ 7.6 Hz, H-10); 5.16–5.19 (m, 1H, H-40); 5.28–5.31
(m, 2H, H-20, H-30); 6.99 (d, 2H, J ¼ 8.4 Hz, Ar-H); 7.31 (d, 2H, J ¼ 8.4 Hz, Ar-H).
A General Synthetic Method for Compound 17a,
17b, and 18
13-cis-RA (1.260 g, 4.2 mmol), compound 15a (1.940 g, 4.0 mmol), and a
catalytic amount of DMAP were dissolved in anhydrous dichloromethane

J.-N. Xiang et al.
610
(50 mL) at 08C. A solution of DCC (0.911 g, 4.4 mmol) in anhydrous
dichloromethane (15 mL) was added dropwise to the above mixture. The
reaction solution was slowly warmed to rt and stirred for 10 h. The reaction
mixture was diluted with dichloromethane (30 mL) and cooled at 08C over-
night, and the mixture was filtered to remove the byproduct urine. The
solvent was evaporated off under reduced pressure. The remaining residue
was purified by column chromatography (elute: ethyl acetate: petroleum
1
ether ¼ 1:2) to give 17a as yellow solid, the yield: 67.1% m.p. 154–1558C. H
NMR (400 MHz, CDCl3) d: 1.03 (s, 6H, 2 ꢀ CH3); 1.46–1.49 (m, 2H, H on C2);
1.59–1.65 (m, 2H, H on C3); 1.72 (s, 3H, CH3 on C5); 2.00–2.08 (m, 20H,
4 ꢀ COCH3 and 2 ꢀ CH3 and CH2); 3.74–3.81 (m, 1H); 3.83 (s, 3H, OCH3);
4.15 (dd, 1H, J ¼ 12.4 Hz, J ¼ 2.4 Hz); 4.28 (dd, 1H, J ¼ 12.0 Hz, J ¼ 4.8 Hz);
4.93 (d, 1H, J ¼ 8.0 Hz, H-10); 5.09 (s, 2H, Ar-CH2O); 5.14–5.18 (m, 1H,
H-40); 5.27–5.30 (m, 2H, H-20, H-30); 5.68 (s, 1H, H14); 6.15 (d, 1H,
J ¼ 16.0 Hz, H8); 6.24–6.30 (m, 2H, H10 and H7); 6.88–6.93 (m, 2H, Ar-H);
7.01 (dd, 1H, J ¼ 15.2 Hz, J ¼ 7.2 Hz, H11); 7.09 (d, 1H, J ¼ 8.0 Hz, Ar-H);
7.79 (d, 1H, J ¼ 15.2 Hz, H12). 13C NMR (400 MHz, CDCl3) d: 12.9, 19.2, 20.6,
20.7, 20.8, 21.0, 21.7, 28.9, 33.1, 34.2, 39.5, 60.0, 65.2, 68.3, 71.1, 71.9, 72.5,
100.8, 112.6, 115.8, 120.0, 120.7, 128.7, 129.0, 130.1, 130.2, 132.6, 133.1,
137.4, 137.6, 140.1, 145.8, 150.6, 152.1, 166.0, 169.4, 170.3, 170.6. IR (KBr),
n/cm21: 3466, 2961, 1755, 1637, 1609, 1515, 1231, 1044, 1070, 1045, 908.
MS: m/z ¼ 789.5 ([M þ Na]þ).
Compound 17b was prepared following a similar procedure as described
for 17a. After purification by column chromatography, 17b was obtained as
1
yellow powder solid, the yield: 74.0%, m.p. 154–1558C. H NMR (400 MHz,
CDCl3) d: 1.03 (s, 6H, 2 ꢀ CH3); 1.46–1.49 (m, 2H, H on C2); 1.58–1.65
(m, 2H, H on C3); 1.72 (s, 3H, CH3 on C5); 2.00–2.08 (m, 20H, 4 ꢀ COCH3
and 2 ꢀ CH3 and CH2); 3.84–3.88 (m, 1H, H-50); 4.16 (dd, 1H, J ¼ 12.4 Hz,
J ¼ 2.4 Hz, H-60); 4.29 (dd, 1H, J ¼ 12.0 Hz, J ¼ 5.2 Hz, H-60); 5.08 (d, 1H,
J ¼ 7.6 Hz, H-10); 5.10 (s, 2H, Ar-CH2O); 5.17 (t, 1H, J ¼ 9.2 Hz, H-40); 5.25–
5.33 (m, 2H, H-20, H-30); 5.66 (s, 1H, H14); 6.15 (d, 1H, J ¼ 16.0 Hz, H8);
6.23–6.30 (m, 2H, H10 and H7); 6.98 (d, 2H, J ¼ 8.8 Hz, Ar-H); 6.97–7.03 (m,
1H, H11); 7.32 (d, 2H, J ¼ 8.8 Hz, Ar-H); 7.79 (d, 1H, J ¼ 15.6 Hz, H12). IR
(KBr), n/cm21: 3468, 2925, 1750, 1611, 1514, 1230, 1045, 908. MS: m/
z ¼ 759.4 ([M þ Na]þ), 737.1 ([M þ H]þ).
According to the procedure described for the synthesis of 17a. After column
chromatography, compound 18 was obtained as yellow viscous syrup, the yield:
55.4%. 1H NMR (400 MHz, CDCl3) d: 1.03 (s, 6H, 2 ꢀ CH3); 1.45–1.48 (m, 2H,
H on C2); 1.59–1.64 (m, 2H, H on C3); 1.72 (s, 3H, CH3 on C5); 2.00–2.19
(m, 20H, 4 ꢀ COCH3 and 2 ꢀ CH3 and CH2); 4.08–4.27 (m, 3H); 5.07–5.16
(m, 3H); 5.23 (d, 1H, J ¼ 13.6 Hz); 5.47 (d, 1H, J ¼ 3.2 Hz); 5.56 (dd, 1H,
J ¼ 10.4 Hz, J ¼ 8.0 Hz); 5.70 (s, 1H, H14); 6.15 (d, 1H, J ¼ 16.4 Hz, H8);
6.24–6.30 (m, 2H, H10 and H7); 7.00 (dd, 1H, J ¼ 15.2 Hz, J ¼ 11.2 Hz, H11);

13-cis-Retinoyl Sugar Derivatives
611
7.08–7.12 (m, 2H, Ar-H); 7.26 (t, 1H, J ¼ 8.4 Hz, Ar-H); 7.36 (d, 1H, J ¼ 7.2 Hz,
Ar-H); 7.81 (d, 1H, J ¼ 15.6 Hz, H12). MS: m/z ¼ 759.3 ([M þ Na]þ), 737.0
([M þ H]þ).
Synthesis of the Compound 19
Compound 19 was synthesized with the same procedure used for 9. 17a
was used as starting material. 19 was obtained as yellow viscous syrup, the
1
yield: 61.2%. H NMR (400 MHz, DMSO-d6) d: 1.02 (s, 6H, 2 ꢀ CH3); 1.44–
1.46 (m, 2H, H on C2); 1.56–1.58 (m, 2H, H on C3); 1.68 (s, 3H, CH3 on C5);
1.99–2.01 (m, 6H, CH3 on C19 and CH3 on C20); 2.05–2.07 (m, 2H, H on
C4); 3.19 (d, 1H, J ¼ 8.4 Hz, glycosyl-ring-H); 3.27–3.32 (m, 2H, glycosyl-
ring-H); 3.44–3.48 (m, 3H, glycosyl-ring-H); 3.67 (d, 1H, J ¼ 11.2 Hz,
glycosyl-ring-H); 3.77 (s, 3H, OCH3); 5.03 (s, 2H, Ar-CH2O); 5.73 (s, 1H,
H14); 6.21 (d, 1H, J ¼ 16.0 Hz, H8); 6.28–6.32 (m, 2H, H10 and H7); 6.89
(d, 1H, J ¼ 8.0 Hz, Ar-H); 7.02–7.11 (m, 3H, Ar-H and H11); 7.72 (d, 1H,
J ¼ 15.2 Hz, H12). 13C NMR (400 MHz, DMSO-d6) d: 12.6, 18.7, 20.5, 21.5,
28.8, 32.6, 33.8, 40.4, 55.7, 60.6, 64.9, 69.6, 73.2, 76.9, 77.0, 100.0, 112.8,
115.2, 115.8, 120.8, 128.2, 128.9, 129.7, 129.9, 130.2, 132.8, 136.9, 137.2,
140.1, 146.3, 148.8, 151.8, 165.3. IR (KBr), n/cm21: 3426, 2956, 1653, 1514,
1454, 1381, 1271, 1151, 1026 (shoulder peak of sugar ring), 1003, 825, 764.
MS: m/z ¼ 622.0 ([M þ Na]þ), 600.0 ([M þ H]þ).
Antiproliferative Activities Assays
The antiproliferative activities of 3, 8a–8d, 11a, and 17a–19 were
assessed by use of the MTT assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazol-ium bromide) assay is a simple nonradioactive colorimetric
assay to measure cell cytotoxicity, proliferation, or viability. MTT is a yellow,
water-soluble, tetrazolium salt. Metabolically active cells are able to convert
this dye into a water-insoluble dark blue formazan by reductive cleavage of
the tetrazolium ring. Formazan crystals then can be dissolved and quantified
by measuring the absorbance of the solution at 570 nm, and the resultant
value is related to the number of living cells.
The effect of 8a on the cells’ proliferation efficiency was determined after
24 h incubation with cells. To determine cell proliferation, the A549 cell
lines, BEL7404 cell lines, and Tca cell lines were individually plated at a
density of 1 ꢀ 104 cells/well in 96 well plates at 378C in 5% CO2 atmosphere.
After 24 h of culture, the medium in the wells was replaced with the fresh
medium containing 8a of varying concentrations respectively. The 8a concen-
tration given upon is the final concentration in the well. Every concentration
added five wells as parallel control. After 24, 10 mL of MTT dye solution
(5 mg/mL in phosphate buffer pH 7.4) was added to each well and incubated

J.-N. Xiang et al.
612
for 4 h at 378C and 5% CO2 for exponentially growing cells and 10 min for
steady-state confluent cells. The formazan crystals were solubilized with
100 mL of DMSO and the solution was vigorously mixed to dissolve the
reacted dye. The absorbance of each well was read on a microplate reader
(DYNATECH MR7000 instruments) at 570 nm. The spectrophotometer was
calibrated to zero using culture medium without cells. We selected inhibitory
effect to evaluate side effects of the silica-coated fluorescent 8a to cells prolifer-
ation. The inhibitory effect of 8a was calculated as percentage inhibition in
comparison to the value obtained in untreated well to which no 8a was
added. All the other compounds were determined by the same method except
that the cancer cell line is only A549.
CONCLUSION
Ten 13-cis-RA analog were synthesized in three ways by structural modifi-
cations with glycosyl groups in order to decrease toxicities of 13-cis-RA and
enhance its antiproliferative activities. Secondly, a new retinoylation method
using of DCC as reagent under catalysis DMAP was developed after evaluated
various reaction conditions. The O-glycosylation method was investigated
using DMAP as phase-transfer catalyst. Furthermore, deacetylation of
glycosyl retinoates was studied using catalytic amount of dibutyltin oxide as
catalyst in methanol. Finally, 8a was tested by MTT assay on growth of three
different tumor cell lines. Among the tested cell lines, 8a possessed selective
antiproliferative activity toward A549. Therefore, the antiproliferative activi-
ties of compounds 8b–8d, 11a, and 17a–19 against A549 cell line were evalu-
ated. The results show that 9, 17a, 17b, and 19 demonstrated stronger
potential antitumor activities.
ACKNOWLEDGEMENTS
The project was supported by the National Natural Science Foundation of
China No. 20472018(NSFC), and Natural Science Foundation of Hunan
Province (No. 03JJY3016). We thank the Chinese key laboratory of Chem/
Biosensing and Chemometrics for the biological evaluation of the compounds.
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