Synthesis, DNA intercalation and 3D QSAR analysis of cis-2,4,5-trisubstituted-1,3-dithiolanes as a novel class of antitumor agents

Article abstract of DOI:10.1016/j.bmc.2009.06.011

Acid-catalyzed transacetalation of dimethyl (2R,3S)-2,3-dimercapto-succinate and 1,1,3,3-tetramethoxypropane provided cis-4,5-dimethoxycarbonyl-2-(2′,2′-dimethoxyethyl)-1,3-dithiolane (2) in 77% yield. The esterification of 2 and l-amino acids provided 18

Full text of DOI:10.1016/j.bmc.2009.06.011

Bioorganic & Medicinal Chemistry 17 (2009) 6085–6095
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, DNA intercalation and 3D QSAR analysis of cis-2,4,5-trisubstituted-
1,3-dithiolanes as a novel class of antitumor agents
b
a
c
c
Fei Huang a, Ming Zhao b, , Xiaoyi Zhang , Chao Wang , Keduo Qian , Reen-Yen Kuo ,
*
Susan Morris-Natschke c, Kuo-Hsiung Lee c, , Shiqi Peng
a,b,
*
*
a College of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
b College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
c Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Acid-catalyzed transacetalation of dimethyl (2R,3S)-2,3-dimercapto-succinate and 1,1,3,3-tetramethoxy-
propane provided cis-4,5-dimethoxycarbonyl-2-(20,20-dimethoxyethyl)-1,3-dithiolane (2) in 77% yield.
Received 17 March 2009
Revised 3 June 2009
Accepted 8 June 2009
Available online 13 June 2009
The esterification of 2 and
noacyloxymethyl)-1,3-dithiolane analogs (5a–r). Five compounds (5b,c,e,k,p) exhibited remarkable anti-
tumor activity in in vivo assays. The in vivo antitumor potency of 5e,k,p at 44.64 mol/kg was similar to
that of cytarabine at 89.28 mol/kg. Several different assay systems, including UV–vis of CT DNA with or
L-amino acids provided 18 active antitumor cis-2-carbonylmethyl-4,5- di(L-ami-
l
l
Keywords:
1,3-Dithiolane
Antitumor
DNA-recognition
3D QSAR
without the representative compound 5d and CD spectra of CT DNA with or without representative com-
pounds 5b,f,i demonstrated that DNA is the target of 5a–r. A 3D QSAR model was established to elucidate
quantitative relationships between in vivo antitumor activity and analog structures. An equation with r2
equal to 0.992 was built to predict antitumor activity of unknown cis-2,4,5-trisubstituted-1,3-dithiolane
analogs.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
ety, which influenced the activity of some small molecules with
DNA recognizing capacity,22,23 and dimethylaminosulfonates of al-
Although several chemotherapeutic drugs including platinum
agents, groove binders, alkylating and intercalating agents are rou-
tinely used in the clinic for treating cancers, their use still suffers
from toxicity and intrinsic or acquired drug resistance. Thus, the
discovery of new compounds with potent antitumor activity is still
one of the most important goals in medicinal chemistry. Over the
past several decades, numerous studies have focused on the design
of DNA recognizing molecules. The interaction of a small molecule
with DNA initially causes conformational changes in the double
helix of DNA, subsequently interrupts replication, transcription,
and repair, and finally kills the fast growing cells.1–3 In the design
of small molecules with DNA recognizing capacity, various struc-
tures such as harmine and its derivatives,4–8 anthracyclines,9 chro-
mium(III) complexes,10 ellagic acids,11 neutral red,12 platinum
compounds,13,14 MLN944,15 a naphthoquinone,16 mithramycin,17
and flavonoids18 have attracted much interest. Structures contain-
ing sulfur have also been explored as antitumor molecules. Some
examples are 1,3-dithiane rings, which greatly influenced in vivo
effects of some antitumor compounds,19–21 a dithiazolidinone moi-
kane diols or alkenyl thiosulfates, which themselves showed anti-
tumor efficacy.24,25 The structures of these moieties and
compounds are shown in Figure 1. In the present study, we pre-
pared a class of novel 2,4,5-trisubstituted-1,3-dithiolanes and
found that they interact with DNA to exhibit antitumor activity.
We also analyzed 3D QSAR for the new compounds.
2. Results and discussion
2.1. Synthesis of cis-2,4,5-trisubstituted-1,3-dithiolanes
As depicted in Scheme 1, acid-catalyzed transacetalation of di-
methyl (2R,3S)-2,3-dimercaptosuccinate (1) and 1,1,3,3-tetrameth-
R'
S
S
O
S
S
S
R
N
N
CH CH CH
SNa
3
H
Alkenyl thiosulfates
Dithiazolidinone
1,3-Dithiane ring
CH CH CH
2
O
S
SNa
CH -SO -O-(CH ) -O-SO -CH
3
3
2
3
2
2 n
2
* Corresponding authors. Tel./fax: +86 10 83911528 (S.P.); tel./fax: +86 10
82802482 (M.Z.); tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.-H.L).
E-mail addresses: khlee@unc.edu (K.-H. Lee), sqpeng@email.bjmu.edu.cn (S.
Peng).
Alkyl thiosulfates
Alkyldiols dimethylsulfonate
Figure 1. Structures of active antitumor moieties and compounds containing
sulfur.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.011

6086
F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
HS
HS
CO H
2
CO Me
HS
HS
MeO C
2
HOH C
2
2
S
S
S
S
H
H
ii
OMe
OMe
OMe
OMe
iii
i
MeO C
2
HOH C
2
H
H
H
H
CO H
2
CO Me
2
2
3
1
iv
AA-OH C
2
Boc-AA-OH C
2
S
S
S
H
O
H
OMe
v
AA-OH C
2
Boc-AA-OH C
2
H
H
H
S
4a-r
H
OMe
H
5a-r
Scheme 1. Synthetic route to cis-2,4,5-trisubstituted-1,3-dithiolanes, (i) HCl in MeOH (4 N); (ii) concd HCl, CHCl3, 40 °C or BF3ꢀEt2O, CH2Cl2, 0 °C; (iii) LiAlH4/THF, 40 °C; (iv)
DMAP/DCC/CH2Cl2, room temperature; (v) HCl in EtOAc, 0 °C; In 4a and 5a AA = Ala, in 4b AA = NG,NG-(Boc)2-Arg, in 5b AA = Arg, in 4c and 5c AA = Asn, in 4d and 5d AA = Gln,
x
in 4e and 5e AA = Gly, in 4f AA = imidazole-N-Boc-His, in 5f AA = His, in 4g and 5g AA = Ile, in 4h and 5h AA = Leu, in 4i AA = N -Boc-Lys, in 5i AA = Lys, in 4j and 5j AA = Met,
in 4k and 5k AA = Phe, in 4l and 5l AA = Pro, in 4m and 5m AA = Ser, in 4n and 5n AA = Thr, in 4o and 5o AA = Val, in 4p AA = O-Boc-Tyr, in 5p AA = Tyr, in 4q and 5q
AA = Glu(OtBu), in 4r and 5r AA = Trp.
oxypropane provided cis-4,5-dimethoxycarbonyl-2-(20,20-dimeth-
oxyethyl)-1,3-dithiolane (2). The specific catalyst, solvent, and
temperature significantly affected the yield of 2. With concen-
trated HCl as the catalyst and CHCl3 as the solvent at 40 °C for
48 h, the yield of 2 was 77%. When the reaction was carried out
at 25 °C or 0 °C for 48 h, the product was obtained in low yield.
With BF3ꢀEt2O as the catalyst and CH2Cl2 as the solvent at 25 °C
or 0 °C for 10 min, the yields of 2 were 33% and 75%, respectively.
However, with diethyl ether or THF instead of CH2Cl2 for 6 h, the
reaction provided no desirable product. The data are listed in Table
1. To assign the configuration of 2, a NOE difference experiment
was performed. Positive NOE signals were observed between the
methoxy groups at the 2-, 4- and 5-positions. The reported litera-
ture values for the coupling constants of ring protons, H-2 and H-5,
and CH2 of 2-dimethoxyethyl of cis-1,3-dioxanes were 9.5, 6.3 and
7.9 Hz, respectively, while those for ring protons, H-2 and H-5, and
CH2 of 2-dimethoxyethyl of trans-1,3-dioxanes were 3.9, 7.8 and
3.0–5.3 Hz, respectively.26,27 Here we found that the coupling con-
stant of ring protons, H-2 and H-4, and CH2 of 2-dimethoxyethyl of
2 were 7.0, 8.0 and 7.0 Hz, respectively. Based on the results of NOE
difference experiment and comparison of the coupling constants of
the corresponding protons, we assigned a cis-configuration to 2.
Compounds 3, 4a–r and 5a–r, which were derived from 2, were
also assigned as having cis-configurations.
the activity of the compounds. The dose-dependent in vivo antitu-
mor effects of 5e,k,p were also evaluated.
2.2.1. In vivo antitumor activity of 5a–r
The in vivo assay was carried out according to standard proce-
dures. In the experiments, the tumor weights of S180 mice were
used to express the activities of 5a–r (44.64
0.2 ml of NS), with 0.2 mL of NS as the negative control and cyt-
arabine (89.28 mol/kg/day in 0.2 mL of NS) as the positive con-
lmol/kg/day in
l
trol. The treatment was carried out for seven consecutive days.
The tumor weights of mice receiving 5a–r are listed in Table 2,
while the inhibition percents of the tumor growth are shown
with Figure 2. The tumor weights of mice receiving 5a–r ranged
from 0.529 g to 1.493 g. Among the tested compounds, the tumor
weights of mice treated with 5b,c,e,k,p were significantly lower
than that of NS-receiving mice (1.240 g, p < 0.05–0.01). In addi-
tion, the antitumor potency of 5e,k,p at 44.64
substantially equal to that of cytarabine (0.582 g, p > 0.05) at
89.28 mol/kg/day. Thus, the data confirmed that the designed
lmol/kg/day was
l
2,4,5-trisubstituted-1,3-dithiolane analogs possess antitumor
ability.
Compound 2 was reduced with LiAlH4 in anhydrous THF to give
Table 2
cis-4,5-dihydroxymethyl-2-(20,20-dimethoxyethyl)-1,3-dithiolane
Effect of 5a–r on tumor growth of S180 mice
(3) in 80% yield. Esterification of 3 with Boc-L-amino acids, pro-
Compda
Tumor weight
moted by a catalytic amount of DMAP, gave cis-4,5-di(N-Boc-L-
NS
5a
5b
5c
5d
5e
5f
5g
1.240 0.448
1.213 0.884
0.778 0.265c
0.855 0.371b
1.061 0.400
0.723 0.203d
1.076 0.462
1.064 0.351
1.296 0.596
0.989 0.409
1.139 0.300
0.582 0.250c
0.693 0.248e
1.446 0.757
1.493 0.926
1.087 0.400
1.114 0.470
0.529 0.173e
1.051 0.608
0.954 0.339
1.206 0.414
aminoacyloxymethyl)-2-(20,20-dimethoxyethyl)-1,3-dithio- lanes
(4a–r) in yields ranging from 16% to 94%. Removal of the Boc and
dimethyl acetal protecting groups by using HCl in EtOAc gave the
target cis-2-carbonylethyl-4,5-di(
L-aminoacyloxymethyl)-1,3-dith-
iolanes (5a–r) in yields ranging from 89% to 98%.
2.2. Antitumor activity of 5a–r
5h
5i
5j
We evaluated in vivo antitumor activity of 5a–r by using S180
mice as the animal model. Tumor weight was used to correlate
Cytarabine
5k
5l
5m
5n
5o
5p
5q
5r
4
Table 1
Effect of reaction condition on the transacetalation
Catalyst
Solvent
CHCl3
Temp. (°C)
Time
Yield (%)
HCl (concentrated)
0
25
40
48 h
48 h
48 h
15
30
77
a
Tumor weight is expressed by X SD g; inhibition is expressed by X SD%; NS
mol/kg; cytarabine: dose =
l
Compared to NS p < 0.05.
Compared to NS p < 0.01.
BF3ꢀEt2O
CH2Cl2
Ether
THF
0
25
0
25
0
10 min
10 min
6 h
6 h
6 h
75
33
0
0
0
(normal saline) = vehicle; n = 10; 5a–r: dose = 44.64
89.28 mol/kg.
l
b
c
d
e
Compared to NS p < 0.01, to cytarabine p > 0.05.
Compared to NS p < 0.01, to cytarabine p > 0.05.
25
6 h
0

F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
0.2
6087
DNA & 5d
(4 to
40 μΜ)
0.1
Figure 2. Inhibition percentage of the tumor growth of 5a–r treated mice.
DNA alone
240
0.0
2.2.2. Dose-dependent in vivo antitumor action of 5e,k,p
Compounds with the highest in vivo antitumor activity, namely
5e,k,p, were also evaluated for dose-dependent action. Potency in-
creased sequentially for 5e,k,p at 0.45, 4.46, and 44.64 lmol/kg.
Therefore, compounds 5e,k,p exhibited dose-dependent anticancer
200
280
320
360
Figure 4. UV titration spectra of 10 lM solution of CT DNA in PBS buffer with
10 mM solution of 5d in PBS buffer (final concentration 4 ꢁ 10ꢂ6
,
8 ꢁ 10ꢂ6
,
1.2 ꢁ 10ꢂ5
,
1.6 ꢁ 10ꢂ5
,
2.0 ꢁ 10ꢂ5
,
2.4 ꢁ 10ꢂ5
,
2.8 ꢁ 10ꢂ5
,
3.2 ꢁ 10ꢂ5 and
4.0 ꢁ 10ꢂ5 M; pH 7.4).
action (Fig. 3).
2.3. Spectral evidence for interaction of 5a–r with DNA
from 4 to 40 lM), and the spectra were recorded. Addition of 5a–
r induced a general hyperchromic effect. As a representative exam-
ple, the UV titration spectra of CT DNA with 5d are shown in Figure
4. Clearly, addition of 5d enhanced the intensity of CT DNA. In addi-
tion, a bathochromic shift of 8 nm also occurred in the CT DNA
spectra.
DNA spectra are widely used to study the interactions of small
molecules with DNA. To reveal the interactions of 5a–r with DNA
in our studies, CT DNA was selected as the model molecule of
DNA, and interaction experiments of 5a–r toward CT DNA were
performed, with which the interactions of 5a–r toward DNA were
explored. For some important experiments, for example, UV–vis
and CD, tests of this simulation system were performed. The re-
sults demonstrated that these experiments not only provide
important spectroscopic evidence for the interaction of 5a–r to-
ward CT DNA, but also prove that this simulation system should
be generally useful to define the interaction of cis-2,4,5-trisubsti-
tuted-1,3-dithiolanes toward DNA.
2.3.2. Effect of 5a–r on the CD spectra of CT DNA
According to general understanding, the circular dichroic (CD)
spectrum of CT DNA is characterized by positive and negative
bands, of which the former is due to base stacking and the latter
is due to right-handed helicity. Interactions of small molecules
with CT DNA usually result in intensity changes of the two
bands.28,29 To observe the effects of 5a–r on the CD spectrum of
CT DNA, tests were performed over 200–750 nm. Two mixed solu-
tions (5a–r excess solution and 5a–r stoichiometric solution) of CT
DNA and 5a–r in PBS buffer (pH 7.4) were prepared. The CD spectra
of both solutions showed significant intensity decreases in the po-
sitive bands and intensity increases in the negative bands. Repre-
sentative CD spectra of CT DNA alone and CT DNA with 5b,f,i
over 200–300 nm are shown in Figure 5. It was clear that the CT
DNA spectra were greatly influenced by addition of 5b,f,i. These
changes in the spectra of CT DNA most likely are results of interac-
tions of CT DNA and 5a–r.
2.3.1. UV–vis spectra of CT DNA and 5a–r show induced
hyperchromic effect
In the UV–vis experiments, spectra of solutions of CT DNA in
PBS (pH 7.4, final concentration 10
lM) were recorded. To 2.5 mL
of this solution, sequential aliquots (10
l
L of each aliquot) of
10 mM 5a–r, which was transparent in the UV region of 200–
360 nm, in PBS (pH 7.4) were added (final concentration ranging
2.4. 3D QSAR analysis of the in vivo activity of 5a–r
To gain insight into the correlation between the antitumor
activity and the structures of 5a–r, the tumor inhibition of S180
mice receiving 5a–r were selected as the activity parameter for
3D QSAR analysis. As described below, the equation (r2 = 0.992)
that was generated by using the Cerius2 QSAR module following
standard procedures demonstrated that the tumor inhibition of
5a–r correlated quantitatively with structure. This equation should
be capable of predicting the in vivo antitumor activity of unknown
cis-2,4,5- trisubstituted-1,3-dithiolanes.
2.4.1. Alignment of 5a–r
To establish valid 3D-QSAR models, a proper alignment proce-
dure for 5a–r was determined using the target model align strategy
in the align module within Cerius.2 Based on the assumption that
each compound exerts activity at the same binding site on DNA,
the structures of 5a–r were aligned in a pharmacologically active
Figure 3. Dose-dependent action of 5e,k,p (a) inhibition is expressed by X SD%;
n = 10; (b) compared to 4.46
group p < 0.01; (d) compared to 0.45
4.46 mol/kg group p < 0.05.
lmol/kg group p < 0.01; (c) compared to 0.45
lmol/kg
l
mol/kg group p < 0.05; (e) compared to
l

6088
F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
80000
250000
200000
150000
100000
50000
60000
DNA&5i
40000
DNA&5b
DNA
DNA
DNA&5b
20000
0
0
-50000
-100000
-150000
-200000
DNA&5f
DNA&5f
-20000
DNA&5i
200 220 240 260 280 300
200 220 240 260 280 300
Wavelength[nm]
5b,f,i excesssolutions
Wavelength[nm]
5b,f,i stoichiometricsolutions
Figure 5. Effect of 5b,f,i on the CD signals of CT DNA.
orientation. To obtain a consistent alignment, the 1,3-dithiolan-1-
ylaldehyde moiety was selected as a common pharmacophore for
the template to superimpose 5a–r. The method used for perform-
ing the alignment was the maximum common subgraph (MCS).
MCS looks at molecules as points and lines, and uses techniques
from graph theory to identify patterns.30 A rigid fit of atom pairings
was performed to superimpose each structure onto the target
model 1,3-dithiolan-1-ylaldehyde. A stereoview of aligned 5a–r
used for molecular field generation is shown in Figure 6.
2.4.2.1. MFA model for tumor weights of the mice receiving
5a–r. Eq. 1 expresses the MFA model for predicted tumor
weights of the mice receiving 5a–r in terms of the descriptors pro-
ton, methyl, and hydroxyl groups (alignment shown in Fig. 7).
Figure 8 shows the straight-line correlation of the actual tumor
weights found in the S180 mouse model with the tumor weights
calculated using Eq. 1.
Activity ¼ 21:46 þ 0:91ðHþ=953Þ ꢂ 0:32ðHþ=956Þ
ꢂ 0:30ðCH3=204Þ þ 0:82ðCH3=401Þ þ 0:27ðCH3=641Þ
þ 1:54ðCH3=710Þ þ 0:16ðHOꢂ=548Þ ꢂ 0:70ðHOꢂ=577Þ
ꢂ 0:31ðHOꢂ=625Þ ꢂ 0:21ðHOꢂ=760Þ ꢂ 0:21ðHOꢂ=807Þ
2.4.2. QSAR module of Cerius2 based MFA of 5a–r
After energy-minimization using MMFF94 (Merck Molecular
Force Field), Molecular Field Analysis (MFA) was performed using
the QSAR module of Cerius.231 A five-step procedure, which in-
cluded generating conformers, minimizing energy, matching
atoms and aligning molecules, setting preferences, and analyzing
regression, was automatically performed by MFA. Molecular elec-
trostatic and steric fields were created by using proton and methyl
groups, respectively, as probes. These fields were sampled at each
point of a regularly spaced grid of 1 Å. An energy cut-off of
30.0 kcal/mol was set for both electrostatic and steric fields. To-
tally, 672 grid points were generated. Although spatial and struc-
tural descriptors such as dipole moment, polarizability, radius of
gyration, number of rotatable bonds, molecular volume, principal
moment of inertia, AlogP98, number of hydrogen bond donors
and acceptors, and molar refractivity were also considered, only
the highest variance holder proton, methyl and hydroxyl descrip-
tors were used. Regression analysis was carried out using the ge-
netic partial least squares (G/PLS) method consisting of 50,000
generations with a population size of 100. The number of compo-
nents was set to 5. Cross-validation was performed with the
leave-one-out procedure. PLS analysis was scaled, with all vari-
ables normalized to a variance of 1.0.
ꢂ 0:25ðHOꢂ=891Þ ꢂ 0:23ðHOꢂ=938Þ
ð1Þ
In Eq. 1, the data points (n), correlation coefficient (r), square
correlation coefficient (r2), cross-validated correlation coefficient
(r2cv), bootstrap correlation coefficient (rB2S) and least square error
(LSE) were 18, 0.996, 0.992, 0.077, 0.213 and 3.029, respectively.
The equation contains one term of H+/953 with a positive coeffi-
cient, which means that an electron-donating group at this posi-
tion will increase tumor inhibition, and one term of H+/956 with
a negative coefficient, which means that electron-withdrawing
groups at this position will increase tumor inhibition. Eq. 1 in-
cludes three terms of CH3/401, CH3/641 and CH3/710 with positive
coefficients, which means that a large group at these sites will in-
crease tumor inhibition, and one term of CH3/204 with a negative
coefficient, which means that a large group at this site will de-
crease tumor inhibition. Eq. 1 also contains one term of HOꢂ/548
with a positive coefficient, which means that an electron-donating
group at this position will increase tumor inhibition, and six terms
of HOꢂ/577, HOꢂ/625, HOꢂ/760, HOꢂ/807, HOꢂ/891 and HOꢂ/938
with negative coefficients, which means that electron-withdraw-
ing groups at these positions will increase tumor inhibition.
Figure 6. Alignment stereoview of 5a–r used for molecular field generation.
Figure 7. Alignment stereoview of 5a–r reflecting tumor inhibition in mice.

F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
4. Experimental
6089
4.1. General
Statistical analysis of all biological data was carried out by use
of ANOVA test with p < 0.05 as significant cut-off. Protected amino
acids with L-configuration used were purchased from Sigma Chem-
ical Co. All coupling and deprotective reactions were carried out
under anhydrous conditions. Chromatography was performed on
Qingdao Silica Gel H. The purity of the intermediates and the prod-
ucts was measured on TLC (Merck silica gel plates of type 60 F254
,
0.25 mm layer thickness) and HPLC (Waters, C18 column
4.6 ꢁ 150 mm). Melting points were determined in capillary tubes
on an electrothermal SM/XMP apparatus without correction. UV
spectra were measured on Shimadzu UV 2550. ESI-MS was deter-
mined by Micromass Quattro micro TM API, Waters Co. 1H (300
and 500 MHz) and 13C (75 and 125 MHz) NMR spectra were re-
corded on Bruker AMX-300 and AMX-500 spectrometers for
DMSO-d6 solution or CDCl3 solution with tetramethylsilane as
internal standard. Optical rotations were determined on P-1020
Jasco instrument.
Figure 8. Graph of tested tumor weight against predicted tumor weight of mice
receiving 5a–r.
Examples of these factors are shown Figure 9. Compounds 5k,p
(A), which have electron-releasing groups near H+/953 and HOꢂ/
625, electron-withdrawing groups near HOꢂ/938, large groups
near CH3/401 and CH3/641, and small groups near CH3/204 showed
increased antitumor activity, and 5d,f (B), which have electron-
withdrawing groups near H+/956 and HOꢂ/548, and small groups
near CH3/641 and CH3/401 showed significantly decreased tumor
inhibition activity.
Table 3
Chemical, physical and related data of 4a–r
3. Conclusion
Yield (%)
Mp (°C)
½
a 2D5
ꢃ
(c 1.0, CH3OH)
ESI-MS [M+H]+
4a
4b
4c
4d
4e
4f
4g
4h
4 i
4j
91
43
16
94
82
73
84
73
85
88
91
74
48
55
73
82
79
81
Oil
101–104
Oil
Oil
Oil
71–72
Oil
Oil
98–101
Oil
113–115
102–103
Oil
ꢂ23.41
ꢂ29.16
ꢂ46.56
ꢂ46.49
2.70
ꢂ21.70
ꢂ60.71
ꢂ41.76
ꢂ60.99
ꢂ17.57
ꢂ9.48
597
1167
683
711
569
929
681
681
911
717
749
649
629
657
653
981
825
827
In summary, 18 cis-2-carbonylmethyl-4,5-di-
ethyl)-1,3-dithiolanes (5a–r) were synthesized from cis-2-carbon-
ylmethyl-4,5-dihydroxymethyl-1,3-dithiolane and -amino acids
in this study. In an in vivo antitumor assay in S180 mice model,
5e,k,p (AA = Gly, -Phe or -Tyr) exhibited the highest antitumor
potency. In addition, the in vivo activity of 5e,k,p at 44.64 mol/
kg were comparable to that of cytarabine at 89.28 mol/kg. Com-
pounds whose amino acid residues contain aromatic rings had
the greatest antitumor activity. To explore the possible molecular
target of the newly synthesized analogs, UV–vis and CD spectra
were performed. Results from both studies demonstrated that
DNA was the target of these compounds. A 3D QSAR model was
also established in this study, and an equation with r2 equal
0.992 was built to predict the in vivo antitumor activity of un-
known cis-2,4,5-trisubstituted-1,3-dithiolane analogs.
L-aminoacyloxym-
L
L
L
l
l
4k
4l
ꢂ92.15
ꢂ45.23
ꢂ15.96
ꢂ33.37
ꢂ50.07
21.63
4m
4n
4o
4p
4q
4r
61–62
Oil
Oil
Oil
Oil
ꢂ20.93
Figure 9. Electrostatic and steric environments of 5k,p (A) and 5d,f (B) within the grid with 3D points of Eq. 1.

6090
F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
Table 4
4.2. Synthesis
Elementary analysis data of 4a–r
4.2.1. cis-2-(2,2-Dimethoxyethyl)-4,5-dimethoxycarbonyl-1,3-
dithiolane (2)
Formula
Calcd
H
Found
H
C
N
C
N
To a solution of 1.81 g (11.0 mmol) of 1,1,3,3-tetra-methoxy-
propane (TMOP) and 2.1 g (10.0 mmol) of dimethyl (2R,3S)-2,3-
dimercaptosuccinate in 50 mL of CHCl3, 1 mL of concentrated HCl
was added. The mixture was stirred at 40 °C for 48 h. TLC analysis
(CHCl3/MeOH/HOAc, 10/1/0.2) indicated complete disappearance
of starting materials. The mixture was neutralized with saturated
aqueous solution of NaHCO3 to pH 7 and extracted with water
three times. The organic layer was dried over anhydrous Na2SO4
overnight, and then filtered. The filtrate was evaporated under vac-
uum to give 3.70 g of yellow oil, which was purified by chromatog-
raphy on silica gel (petroleum ether–EtOAc, 3:1) to yield 2.4 g
(77%) of the title compound as colorless oil. ESI-MS (m/e): 311
[M+H]+. 1H NMR (CDCl3): d 4.63 (t, J = 7.0 Hz, 1H), 4.49 (d,
J = 8.0 Hz, 2H), 4.41 (t, J = 6.0 Hz, 1H), 3.77 (s, 6H), 3.35 (s, 6H),
2.14 (t, J = 7.0 Hz, 2H). Anal. Calcd for C11H18O6S2: C, 42.57; H,
5.85. Found: C, 42.80; H, 5.71.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
C25H44N2O10S2
C51H90N8O18S2
C27H46N4O12S2
C29H50N4O12S2
C23H40N2O10S2
C41H64N6O14S2
C31H56N2O10S2
C31H56N2O10S2
50.32
52.47
47.49
49.00
48.57
53.00
54.68
54.68
54.04
48.58
59.34
53.68
47.76
49.37
53.35
57.53
53.86
59.54
7.43
7.77
6.79
7.09
7.09
6.94
8.29
8.29
8.19
7.31
7.00
7.46
7.05
7.37
8.03
6.99
7.82
6.58
4.69
9.60
8.21
7.88
4.93
9.05
4.11
4.11
6.15
3.91
3.74
4.32
4.46
4.27
4.29
2.86
3.40
6.77
50.11
52.69
47.71
49.23
48.35
52.77
54.46
54.89
53.83
48.36
59.55
53.46
47.54
49.42
53.12
57.30
53.65
59.31
7.30
7.91
6.94
7.22
6.96
6.80
8.15
8.42
8.04
7.20
7.16
7.33
6.91
7.29
7.90
6.84
7.70
6.44
4.44
9.85
7.97
8.14
4.70
8.81
4.35
4.34
6.38
3.66
3.98
4.10
4.69
4.23
4.03
3.13
3.65
6.53
C
41H74N4O14S2
C29H52N2O10S4
C37H52N2O10S2
C29H48N2O10S2
C25H44N2O12S2
C27H48N2O12S2
C29H52N2O10S2
C47H68N2O16S2
C
37H64N2O14S2
C41H54N4O10S2
Table 5
1H NMR data of 4a–r except R
O
O
O
l
i
f
g
j
k
NH
h
S
S
e
a
c
O
O
O
O
O
O
b
d
R
R
NH
O
Ha
Hb
Hc
Hd
He
Hf
Hh
Hi
Hl
4a
4b
4c
4d
4e
4f
3.35, 6H, s
4.55, 1H, t,
J = 7.0
4.51, 1H, t,
J = 7.0
4.52, 1H, t,
J = 6.5
4.53, 1H, t,
J = 6.5
4.53, 1H, t,
J = 7.0
4.55, 1H, t,
J = 7.0
4.57, 1H, t,
J = 7.0
4.50, 1H, t,
J = 7.0
4.54, 1H, t,
J = 7.0
4.55, 1H, t,
J = 7.0
4.56, 1H, t,
J = 7.0
4.55, 1H, t,
J = 6.5
4.50, 1H, t,
J = 6.0
4.52, 1H, t,
J = 7.0
4.56, 1H, t,
J = 7.0
4.54, 1H, t,
J = 7.5
2.15, 2H, t,
J = 5.5
2.14, 2H, t,
J = 5.5
2.13, 2H, t,
J = 6.0
2.13, 2H, t,
J = 6.0
2.14, 2H, t,
J = 6.0
2.13, 2H, t,
J = 6.0
2.15, 2H, t,
J = 6.5
2.13, 2H, t,
J = 6.5
2.34, 2H, t,
J = 6.5
2.15, 2H, t,
J = 6.5
2.16, 2H, t,
J = 5.0
2.16, 2H, t,
J = 5.5
2.13, 2H, t,
J = 5.0
2.13, 2H, t,
J = 5.0
2.16, 2H, t,
J = 6.0
2.15, 2H, t,
J = 6.5
4.44, 1H, t,
J = 6.0
4.42, 1H, t,
J = 8.0
4.41, 1H, t,
J = 6.0
4.42, 1H, t,
J = 5.5
4.42, 1H, t,
J = 6.0
4.42, 1H, t,
J = 6.0
4.44, 1H, t,
J = 6.0
4.40, 1H, t,
J = 6.0
4.33, 2H, t,
J = 8.5
4.31, 2H, t,
J = 7.5
4.33, 2H, t,
J = 6.0
4.33, 2H, t,
J = 6.5
3.87, 2H, t,
J = 5.0
4.32, 2H, q,
J = 6.5
4.27, 2H, q,
J = 6.0
4.25, 2H, q,
J = 6.0
3.94, 2H, m,
J = 5.5
3.96, 2H, m,
J = 6.5
4.34, 2H, m,
J = 8.0
4.29, 2H, q,
J = 5.5
4.23, 2H, m,
J = 5.0
4.23, 2H, q,
J = 4.0
4.25, 2H, q,
J = 4.5
4.30, 2H, q,
J = 5.5
4.40, 4H, d,
J = 7.0
4.40, 4H, m
3.92, 2H, m,
J = 9.0
3.86, 2H, m,
J = 5.5
3.90, 2H, m,
J = 5.5
3.90, 2H, m,
J = 5.5
3.90, 4H, t,
J = 4.5
3.90, 2H, m,
J = 5.0
3.90, 2H, q,
J = 7.5
3.88, 2H, q,
J = 6.0
3.90, 2H, q,
J = 7.0
3.90, 2H, m,
J = 6.5
3.91, 2H, m,
J = 5.0
3.90, 2H, m,
J = 8.0
3.90, 2H, m,
J = 5.5
3.90, 2H, q,
J = 6.0
3.91, 2H, q,
J = 7.0
3.79, 2H, m
5.25, 5.13, 2H,
s
5.62, 5.55, 2H,
s
5.81, 5.63, 2H,
s
5.82, 5.65, 2H,
s
1.46 18H, s
3.35, 6H, s
3.29, 6H, s
3.29, 6H, s
3.33, 6H, s
3.35, 6H, s
3.36, 6H, s
3.33, 6H, s
3.35, 6H, s
3.34, 6H, s
3.36, 6H, s
3.35, 6H, s
3.36, 6H, s
3.36, 6H, s
3.36, 6H, s
3.35, 6H, s
3.51, 6H, s
3.31, 6H, s
1.45 18H, s
1.43 18H, s
1.43 18H, s
1.44 18H, s
1.46 18H, s
1.46 18H, s
1.43 18H, s
1.46 18H, s
1.44 18H, s
1.46 18H, s
1.46 18H, s
1.46 18H, s
1.46 18H, s
1.46 18H, s
1.42 18H, s
1.44 18H, s
1.46 18H, s
4.36, 4H, t,
J = 5.0
4.37, 4H, t,
J = 5.5
4.36, 4H, t,
J = 7.0
4.39, 4H, t,
J = 7.0
4.40, 4H, t,
J = 6.5
4.36, 4H, t,
J = 8.5
4.36, 4H, d,
J = 6.5
4.38, 4H, d,
J = 6.0
4.41, 4H, d,
J = 6.0
4.39, 4H, d,
J = 5.0
4.37, 4H, d,
J = 6.0
4.37, 4H, d,
J = 6.0
4.40, 4H, d,
J = 6.5
4.45, 4H, d,
J = 6.0
4.37, 4H, m,
J = 9.0
4.32, 4H, m,
J = 7.0
5.24, 2H, s
7.24, 7.11, 2H,
s
5.22, 5.08, 2H,
s
5.27, 5.00, 2H,
s
5.40, 5.24, 2H,
s
5.43, 5.25, 2H,
s
5.26, 5.14, 2H,
s
4g
4h
4i
4.45, 1H, m
4j
4.41, 1H, t,
J = 7.0
4.45, 1H, t,
J = 6.5
4.44, 1H, t,
J = 4.5
4.41, 1H, t,
J = 5.0
4.41, 1H, t,
J = 5.0
4.44, 1H, t,
J = 5.5
4.52, 1H, t,
J = 8.5
4.15, 1H, t,
J = 9.0
4k
4l
—
4m
4n
4o
4p
4q
4r
5.50, 5.40, 2H,
s
5.50, 5.41, 2H,
s
5.21, 5.07, 2H,
s
5.26, 5.11, 2H,
s
5.42, 5.28, 2H,
s
5.42, 5.28, 2H,
s
4.54, 1H, t,
J = 7.0
4.53, 1H, t,
J = 7.0
2.15, 2H, t,
J = 9.5
2.02, 2H, t,
J = 6.0
4.34, 2H, m
3.86, 2H, m,
J = 7.5
3.72, 2H, m,
J = 7.5
4.38, 1H, t,
J = 6.0
3.88, 2H, q,
J = 6.0

F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
6091
Table 6
NMR data of R in 4a–r
Structure of Ra
1H
13C
4a
4b
CH3
1.42 (d, J = 7.0, 6H)
18.36
i
h
g
f
d
O
H
N
e
HN
a: 1.84 (m, 4H); b: 1.28 (m, J = 7.0, 4H); c: 3.48 (m, J = 4.5,
4H); d: 9.31 (s, 2H); f: g: 9.44 (s, 2H); j: 1.53 (s, 36H)
a: 28.34; j: 28.19; b: 22.08; c: 39.11; e: 158.86; h: 155.13,
154.59; i: 79.83, 79.66
O
a
H2C
N
O
c
b
O
O
b
4c
a: 2.72 (d, J = 5.5, 4H); c: 6.58 (s, 2H), 6.14 (s, 2H)
a: 36.71; b: 175.42
H2C
a
NH2
c
O
4d
H2C
a
a: 1.25 (m, J = 3.0, 4H); b: 2.37 (t, J = 4.5, 4H); d: 6.60 (s, 2H),
6.16 (s, 2H)
a: 27.38; b: 33.84; c: 176.13
c
NH
2
b
d
a
CH2
b
d
4f
a: 2.94 (dd, J = 11.5, J = 3.5, 4H); c: 7.49 (s, 2H); d: 6.88 (s, 2H);
g: 1.46 (s, 18H)
a: 34.59; b: 126.75; c: 134.66; d: 118.83; e: 151.22; f: 80.03;
g: 27.21
O
e
N
g
N
c
O
f
H C
b
CH
3
3 d
4g
4h
a: 1.90 (m, J = 3.0, 2H); b: 0.96 (d, J = 8.0, 6H); c: 1.24 (q,
J = 9.0, 4H); d: 0.94 (t, J = 3.5, 6H)
a: 37.78; b: 15.65; c: 25.02; d: 11.57
a: 24.82; b: 41.48; c: 22.87
HC
CH2
a
c
c
CH3
a
b
a: 1.58 (m, J = 3.0, 4H); b: 1.64 (m, J = 3.0, 2H); c: 0.94 (d,
J = 7.5, 12H)
H2C
CH
CH3
a
H2C
H
c
e
h
N
f
O
4i
a: 1.84 (m, J = 7.0, 4H); b: 1.66 (m, 4H); c: 1.72 (m, 4H); d:
3.14 (m, 4H); e: 4.78 (s, 1H), 4.74 (s, 1H); h: 1.46 (s, 18H)
a: 31.02; b: 22.67; c: 29.57; d: 41.53; f: 155.62; g: 79.59; h:
21.71
g
b
d
O
a
b
c
CH3
4j
a: 1.97 (q, J = 7.5, 4H); b: 2.56 (t, J = 5.5, 4H); c: 2.11 (s, 6H)
a: 31.79; b: 30.07; c: 15.47
H2C CH2
S
c
d
a
4k
b
d
a: 2.89 (dd, J = 10.5, J = 4.5, 4H); c: 7.24 (d, J = 8.5, 4H), d: 7.16
(d, J = 4.0, 4H); e: 7.27 (t, J = 4.5, 2H);
a: 38.18; b: 135.96; c: 127.11; d: 128.64; e: 129.31
e
CH2
c
b
4l
b: 2.00 (m, J = 7.0, 4H); c: 1.89 (m, J = 5.5, 4H); d: 2.25 (m,
J = 8.0, 4H)
b: 29.90; c: 23.63; d: 42.61
a: 60.77
a
N
a
b
4m
4n
a: 4.13 (m, 4H); b: 4.55 (s, 2H)
OH
H2C
c
a
OH
a: 4.34 (m, 2H); b: 1.33 (d, J = 5.5, 6H); c: 4.59 (s, 2H)
a: 70.74; b: 24.57
HC
b
CH3
CH3
a
b
4o
4p
a: 2.14 (m, J = 6.0, 2H); b: 1.00 (d, J = 7.5, 6H), 0.90 (d, J = 7.5,
6H)
a: 31.08; b: 17.10
HC
CH3
O
c
d
a
CH2
h
a: 3.14 (dd, J = 8.0, J = 5.0, 4H); c: 7.14 (d, J = 8.0, 4H); d: 7.12
(d, J = 8.0, 4H); h: 1.57 (s, 18H)
a: 37.52; b: 133.63; c: 121.45; d: 130.27; e: 150.18; f: 151.83;
g: 83.54; h: 24.84
b
e
O
g
f
O
b
d
a
c
O
a
e
4q
a: 2.24 (m, J = 7.0, 4H); b: 2.51 (t, J = 12.5, 4H); e: 1.42 (s, 18H)
a: 27.30; b: 30.30; c: 172.48; d: 82.1; e: 29.80
H2C
O
c
e
d
f
CH2
4r
a: 3.24 (dd, J = 12.0, J = 4.0, 4H); b: 9.11 (s, 1H), 9.05 (s, 1H);
e: 7.55 (d, J = 7.0, 2H), g: 7.36 (d, J = 8.0, 2H), f and h: 7.17
(m, J = 4.5, 4H); j: 7.00 (d, 2H)
a: 30.89; c: 109.25; d: 127.26; e: 118.77; f: 119.78; g: 122.31;
h: 111.65; i: 136.23; j: 123.36
j
g
N
i
h
b
H
a
Compound 4e has no R.

6092
F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
Table 7
13C NMR data of 4a–r except R
O
O
O
l
i
f
g
j
k
NH
h
S
S
e
a
c
O
O
O
O
O
O
b
d
R
R
NH
O
Ca
Cb
Cc
Cd
Ce
Cf
Cg
Ch
Cj
Ck
Cl
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
53.47
55.47
53.21
53.89
53.43
55.46
54.24
53.56
54.06
53.48
54.73
53.70
53.41
53.69
53.41
54.71
54.01
53.51
103.42
103.28
103.23
103.17
103.45
103.37
103.45
103.48
103.43
103.39
103.42
103.46
103.44
103.37
103.44
103.44
103.41
103.31
46.12
46.37
46.53
46.29
45.76
46.33
46.27
45.94
46.33
46.11
46.23
46.33
45.35
44.75
46.26
46.18
46.39
46.57
40.94
40.93
40.75
40.44
40.42
40.72
41.02
40.35
40.29
40.92
40.87
41.00
45.35
40.81
41.00
40.86
40.93
40.82
47.15
47.22
47.29
47.48
46.18
47.84
47.31
47.12
47.24
47.15
47.25
47.46
47.22
47.27
47.29
47.23
47.10
47.41
63.91
64.01
63.66
63.59
63.67
63.49
63.83
63.77
63.54
63.92
63.64
65.80
63.15
63.99
63.78
63.77
63.92
63.76
172.81
173.03
171.93
171.94
171.88
172.14
171.84
172.51
172.24
171.80
171.35
172.32
171.02
170.50
171.82
171.38
171.61
172.06
49.30
53.56
50.40
53.34
47.39
54.01
53.44
52.92
53.38
52.89
53.56
63.90
57.11
58.42
58.59
53.35
53.57
56.12
155.18
155.88
155.90
155.58
155.66
155.47
155.65
155.56
156.11
155.48
155.17
154.36
155.73
156.11
155.73
155.19
155.82
156.08
80.02
80.03
80.07
79.44
79.89
79.97
79.90
79.90
80.02
80.07
80.05
79.95
80.08
79.90
79.90
80.16
80.15
80.11
28.33
28.34
28.23
28.41
28.22
28.33
28.32
28.32
28.33
28.31
28.28
28.39
28.40
28.31
28.31
28.29
28.30
28.46
Table 8
Table 9
Chemical, physical and related data of 5a–r
Elementary analysis data of 5a–r
Yield (%)
Mp (°C)
½
a 2D5
ꢃ
(c 1.0, H2O)
ESI-MS [M+H]+
Formula
Calcd
H
Found
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
91
95
90
93
89
97
98
90
91
97
96
97
92
93
94
94
94
95
151–153
132–133
170–172
181–183
181–182
202–204
175–176
165–166
124–126
154–155
168–169
Oil
176–178
150–151
166–167
121–122
143–145
>220
ꢂ14.63
351
521
437
465
323
483
435
435
465
471
503
403
383
411
407
535
467
581
C
N
C
H
N
9.14
11.70
6.37
6.50
15.40
6.03
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
C13H22N2O5S2
C19H36N8O5S2
C15H24N4O7S2
C17H28N4O7S2
44.55
43.83
41.27
43.95
40.98
47.29
52.51
52.51
49.11
43.38
59.74
50.72
40.83
43.89
50.22
56.16
43.77
59.98
6.33
6.97
5.54
6.08
5.63
5.43
7.89
7.89
7.81
6.42
6.02
6.51
5.80
6.38
7.44
5.66
5.62
5.55
7.99
21.52
12.84
12.06
8.69
17.41
6.45
6.45
12.06
5.95
5.57
6.96
7.32
6.82
6.89
5.24
6.00
44.34
43.05
41.51
43.72
40.77
47.50
52.30
52.28
48.90
43.14
59.95
50.50
40.60
43.66
50.00
56.38
43.54
59.74
6.20
6.81
5.70
5.93
5.50
5.59
7.74
7.72
7.66
6.28
6.17
6.37
5.94
6.21
7.28
5.80
5.50
5.41
7.75
21.77
12.60
12.31
8.92
17.66
6.69
6.69
12.32
5.71
5.84
6.70
7.10
6.60
7.14
5.49
6.26
9.91
C
11H18N2O5S2
C19H26N6O5S2
C19H34N2O5S2
C19H34N2O5S2
C19H36N4O5S2
C17H30N2O5S4
C25H30N2O5S2
C17H26N2O5 S2
3.30
14.09
10.16
3.90
ꢂ16.80
2.97
ꢂ16.33
1.50
C
13H22N2O7S2
C15H26N2O7S2
C17H30N2O5S2
C25H30N2O7S2
C17H26N2O9S2
C29H32N4O5S2
5.00
3.77
6.47
9.65
4.2.2. cis-2-(2,2-Dimethoxyethyl)-4,5-dihydroxymethyl-1,3-
dithiolane (3)
colorless oil, which was purified by chromatography on silica gel
(CHCl3–MeOH, 40:1) to yield 1.24 g (80%) of the title compound
as colorless oil. ESI-MS (m/e): 255 [M+H]+. 1H NMR (CDCl3) d 5.01
(t, J = 5.0 Hz, 2H), 4.43 (t, J = 7.0 Hz, 1H), 3.76 (d, J = 5.0 Hz, 4H),
3.73 (t, J = 5.0 Hz, 1H), 3.49 (t, J = 5.0 Hz, 2H), 3.35 (s, 6H), 2.15 (t,
J = 7.0 Hz 2H). Anal. Calcd for C9H18O4S2: C, 42.50; H, 7.13. Found:
C, 42.81; H, 7.00.
A suspension of 432 mg (12.0 mmol) of LiAlH4 in 30 mL of anhy-
drous THF was stirred vigorously at 40 °C for 1 h, after which a
solution of 1.70 g (5.0 mmol) of 2 in 20 mL of anhydrous THF
was added. The reaction was continued for 4 h, at which time
TLC analysis (petroleum ether–EtOAc, 3:1) indicated complete dis-
appearance of 2. The reaction mixture was stirred vigorously,
cooled in an ice bath, quenched by slowly adding crushed ice,
and neutralized by adding a solution of 2 mL of concentrated
hydrochloric acid in 30 mL of water. The formed suspension was
filtered, the filtrate was evaporated under vacuum, the residue
was extracted with EtOAc (30 mL ꢁ 3), and the organic layer was
washed successively with satd aq NaCl, aq NaHCO3 (5%), and satd
aq NaCl. The organic layer was dried over anhydrous Na2SO4 and
filtered, and the filtrate was evaporated under vacuum to give a
4.2.3. General procedure for preparing cis-2-(2,2-dimethoxy-
ethyl)-4,5-di(N-Boc-
L
-aminoacy-loxymethyl)-1,3-dithiolanes
(4a–r)
A solution of 254 mg (1.0 mmol) of 3, 2.2 mmol of Boc-
L-amino
acid, 453 mg (2.2 mmol) of DCC and 26.8 mg (0.22 mmol) of DMAP
in 20 mL of anhydrous CH2Cl2 was stirred at room temperature for
0.5 h. TLC analysis (petroleum ether–EtOAc, 3:1) indicated complete

F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
6093
Table 10
1H NMR data of 5a–r except R
O
h
e
f
NH2
g
S
S
d
b
O
O
a
c
R
R
O
NH2
O
Ha
Hb
Hc
Hd
He
Hg
Hh
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
9.58, 1H, s
9.59, 1H, s
9.58, 1H, s
9.60, 1H, s
9.60, 1H, s
9 58, 1H, s
9.59, 1H, s
9.59, 1H, s
9.59, 1H, s
9.58, 1H, s
9.58, 1H, s
9.58, 1H, s
9.58, 1H, s
9.58, 1H, s
9.58, 1H, s
9.58, 1H, s
9.59, 1H, s
9.58, 1H, s
3.09, 2H, dd, J = 10.5, J = 3.0
2.88, 2H, dd, J = 11.5, J = 3.0
3.10, 2H, dd, J = 10.0, J = 3.0
3.08, 2H, dd, J = 10.5, J = 3.0
3.09, 2H, dd, J = 10.5, J = 3.0
3.10, 2H, dd, J = 11.5, J = 3.0
3.12, 2H, dd, J = 10.5, J = 2.5
3.10, 2H, dd, J = 10.0, J = 3.0
3.10, 2H, m
4.24, 1H, t, J = 6.0
4.38, 1H, t, J = 5.0
4.24, 1H, t, J = 6.0
4.30, 1H, t, J = 6.5
4.24, 1H, t, J = 6.0
4.23, 1H, t, J = 6.5
4.34, 1H, t, J = 6.0
4.29, 1H, t, J = 6.5
4.22, 1H, t, J = 6.5
4.33, 1H, t, J = 6.0
4.23, 1H, t, J = 7.0
4.34, 1H, t, J = 7.5
4.23, 1H, t, J = 6.0
4.22, 1H, t, J = 6.5
4.34, 1H, t, J = 5.5
4.25, 1H, t, J = 6.5
4.34, 1H, t, J = 6.0
4.04, 1H, t, J = 10.0
3.74, 2H, m, J = 5.0
4.07, 2H, m, J = 6.0
3.74, 2H, m, J = 5.0
3.82, 2H, m, J = 5.5
3.83, 2H, m, J = 5.0
3.74, 2H, m, J = 6.0
3.92, 2H, m, J = 4.5
3.82, 2H, m, J = 5.0
3.80, 2H, , m, J = 7.0
3.90, 2H, m, J = 6.5
3.74, 2H, m, J = 6.0
3.94, 2H, m, J = 5.5
3.74, 2H, m, J = 5.0
3.74, 2H, m
4.28, 4H, d, J = 5.0
4.47, 4H, d, J = 6.0
4.28, 4H, , d, J = 5.0
4.34, 4H, d, J = 5.5
4.26, 4H, d, J = 7.0
4.27, 4H, d, J = 6.0
4.37, 4H, d, J = 7.0
4.34, 4H, d, J = 7.0
4.26, 4H, d, J = 6.5
4.38, 4H, d, J = 6.0
4.27, 4H, d, J = 5.0
4.48, 4H, d, J = 7.0
4.28, 4H, d, J = 5.0
4.28, 4H, d, J = 5.0
4.38, 4H, d, J = 6.5
4.29, 4H, d, J = 5.5
4.38, 4H, d, J = 6.5
4.25, 4H, d, J = 6.0
3.82, 2H, m, J = 7.0
4.25, 2H, m, J = 3.0
3.84, 2H, m, J = 7.0
4.21, 2H, m, J = 5.5
3.88, 4H, m, J = 5.5
3.82, 2H, m, J = 7.0
4.08, 2H, m, J = 6.5
3.9, 2H, m, J = 6.5
3.99, 2H, m, J = 5.5
4.01, 2H, m, J = 5.0
3.82, 2H, m, J = 8.0
4.31, 2H, m, J = 6.0
3.83, 2H, m, J = 7.0
3.88, 2H, m, J = 5.0
4.25, 2H, m, J = 4.5
4.43, 2H, m
8.71, 4H, s
8.72, 4H, s
8.71, 4H, s
8.73, 4H, s
8.67, 4H, s
9.00, 4H, s
8.74, 4H, s
8.73, 4H, s
8.84, 4H, s
8.71, 4H, s
8.71, 4H, s
8.70, 2H, s
8.71, 4H, s
8.71, 4H, s
8.70, 4H, s
8.73, 4H, s
8.77, 4H, s
8.70, 4H, s
3.10, 2H, m
3.19, 2H, dd, J = 11.5, J = 5.0
3.15, 2H, dd, J = 11.5, J = 5.0
3.09, 2H, dd, J = 10.5, J = 3.0
3.09, 2H, dd, J = 9.5, J = 3.5
3.09, 2H, m, J = 7.0
3.09, 2H, dd, J = 10.0, J = 6.5
3.12, 2H, dd, J = 10.5, J = 2.5
3.06, 2H, dd, J = 10.0, J = 4.5
3.91, 2H, m, J = 7.0
3.98, 2H, m, J = 5.5
3.92, 2H, m, J = 4.5
3.87, 2H, m
4.08, 2H, m, J = 6.5
3.98, 2H, m, J = 3.5
disappearance of 3. The reaction mixture was filtered, the filtrate
was washed successively with satd aq NaCl, aq NaHCO3 (5%), aq
KHSO4 (5%), and satd aq NaCl. The organic layer was dried over anhy-
drous Na2SO4 and filtered. The filtrate was evaporated under vac-
uum to give colorless oil, which was purified by silica gel
chromatography (petroleum ether–EtOAc, 5:1) to yield title
stirred for 1 h. TLC analysis (CHCl3–MeOH, 10:1) indicated com-
plete disappearance of 4a–r. The reaction mixture was evaporated
under vacuum to remove excess HCl, and the residue was tritu-
rated with anhydrous diethyl ether and then purified by silica gel
chromatography (CHCl3–MeOH, 25:1) to give cis-2-carbonylmeth-
yl-4,5-di(
ylmethyl-4,5-di(
carbonylmethyl-4,5-di(
cis-2- carbonylmethyl-4,5-di(
lane (5d), cis-2-carbonyl-methyl-4,5-di(
dithiolane (5e), cis-2-carbonylmethyl-4,5- di(N-Boc-
oxymethyl)-1,3-dithiolane (5f), cis-2-carbonylmethyl-4,5-di(
eucinyloxymethyl)-1,3-dithiolane (5g), cis-2-carbonylmethyl-4,5-
di( -leucinyl-oxymethyl)-1,3-dithiolane (5h), cis-2-carbonylmeth-
yl-4,5-di( -lysyloxymethyl)-1,3-dithiolane (5i), cis-2-carbonyl-
methyl-4,5-di( -methionyloxymethyl)-1,3-dithiolane (5j), cis-2-
carbonylmethyl-4,5-di( -phenylalaninyloxymethyl)-1,3-dithiolane
(5k), cis-2-carbonylmethyl-4,5-di( -prolinyloxymethyl)-1,3-dithio-
lane (5l), cis-2-carbonylmethyl-4,5-di( -serinyloxymethyl)-1,3-
dithiolane (5m), cis-2-carbonylmethyl-4,5-di( -threoninyloxymeth-
yl)-1,3-dithiolane (5n), cis-2-carbonylmethyl-4,5-di( -valinyloxym-
ethyl)-1,3-dithiolane (5o), cis-2-carbonylmethyl-4,5-di( -tyrosylo-
xymethyl)-1,3-dithiolane (5p), cis-2-carbonylethyl-4,5-di( -gluta-
moyloxymethyl)-1,3-dithiolane (5q), and cis-2-carbonylmethyl-4,5-
di( -tryptophanyloxymethyl)-1,3-dithiolane (5r). Their chemical,
L
-alanyloxymethyl)-1,3- dithiolane (5a), cis-2-carbon-
-arginyloxymethyl)-1,3-dithiolane (5b), cis-2-
-asparaginyloxymethyl)-1,3-dithiolane (5c),
-glutaminyloxymethyl)-1,3-dithio-
-glycinyloxymethyl)-1,3-
-histidinyl-
-isol-
compounds cis-2-(2,2-dimethoxyethyl)-4,5-di(N-Boc-
L
-alanyloxy-
L
methyl)-1,3-dithiolane (4a), cis-2-(2,2-dimethoxyethyl)-4,5-di(NG,
L
a
NG,N -triBoc-
L
-arginyloxymethyl)-1,3-dithiolane (4b), cis-2-(2,2-
-asparaginyloxymethyl)-1,3-dithio-
lane (4c), cis-2-(2,2-dimethoxyethyl)-4,5-di(N-Boc- -glutaminyl-
oxymethyl)-1,3-dithiolane (4d), cis-2-(2,2-dimethoxyethyl)-4,5-
di(N-Boc- -glycinyloxymethyl)-1,3-dithiolane (4e), cis-2-(2,2-
dimethoxyethyl)-4,5-di(N,N-di-Boc- -histidinyl-oxymethyl)-1,3-di-
thiolane (4f), cis-2-(2,2-dimethoxyethyl)-4,5-di(N-Boc- -isoleuci-
nyloxymethyl)-1,3-dithiolane (4g) cis-2-(2,2-dimethoxyethyl)-4,5-
di(N-Boc- -leucinyloxymethyl)-1,3-dithiolane (4h), cis-2-(2,2-
dimethoxyethyl)-4,5-di(N ,N -diBoc-
lane (4i), cis-2-(2,2-dimethoxyethyl)-4,5-di(N-Boc-
oxymethyl)-1,3-dithiolane (4j), cis-2-(2,2-dimethoxyethyl)-4,
5-di(N-Boc- -phenylalaninyloxymethyl)-1,3-dithiolane (4k), cis-2-
(2,2-dimethoxyethyl)-4,5-di(N-Boc- -prolinyloxymethyl)-1,3-dithi-
olane (4l), cis-2-(2,2-dimethoxyethyl)-4,5-di(N-Boc- -serinyloxym-
ethyl)-1,3-dithiolane (4m), cis-2-(2,2-dimethoxyethyl)-4,5-di(N-
Boc- -threoninyloxymethyl)-1,3-dithiolane (4n), cis-2-(2,2-dime-
thoxyethyl)-4,5-di(N-Boc- -valinyloxymethyl)-1,3-dithiolane (4o),
cis-2-(2,2-dimethoxyethyl)-4,5-di(O,N-diBoc- -tyrosyloxymethyl)-
1,3-dithiolane (4p), cis-2-(2,2-dimethoxyethyl)-4,5-di(N-Boc-
tert-butylester- -glutamoyloxymethyl)-1,3-dithiolane (4q), and
cis-2-(2,2-dimethoxyethyl)-4,5-di(N-Boc- -tryptophanyloxymeth-
L
dimethoxyethyl)-4,5-di(N-Boc-
L
L
L
L
L
L
L
L
L
L
L
L
L
x
a
L-lysyloxymethyl)-1,3-dithio-
L
L
-methionyl-
L
L
L
L
L
L
L
L
L
L
L
physical, spectral and elemental analysis data are listed in Tables
8–12.
L
c
-
L
4.3. In vivo anticancer assay
L
yl)-1,3-dithiolane (4r). Their chemical, physical, spectral and ele-
mental analysis data are listed in Tables 3–7.
Male ICR mice, purchased from Peking University Health Sci-
ence Center, were maintained at 21 °C with a natural day/night cy-
cle in a conventional animal colony. The mice were 10–12 weeks
old at the beginning of the experiments. The tumor used was
S180, which forms solid tumors when injected subcutaneously.
S180 cells for initiation of subcutaneous tumors were obtained
from the ascitic form of the tumors in mice, which were serially
4.2.4. General procedure for preparing cis-2-carbonylmethyl-
4,5-di-L-amino-acyloxymethyl)-1,3-dithiolanes (5a–r)
At 0 °C, a solution of 0.1 mmol of 4a–r in 5 mL of anhydrous
EtOAc was treated with 15 mL of EtOAc containing HCl (6 M) and

6094
F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
Table 11
NMR data of R in 5a–r
Structure of Ra
1H
13C
5a
5b
CH3
1.53 (d, J = 7.5, 6H)
20.62
g
f
NH2
e
HN
a: 3.20 (m, J = 5.5, 4H); b: 2.17 (m, J = 2.5, 4H); c: 4.02 (m, 4H); d: 8.98
(s, 2H); f: 9.44 (s, 2H); g: 8.44 (s, 4H)
a: 31.91; b: 25.08; c: 38.22; e: 158.86
a
d
NH
H2C
c
b
O
b
5c
a: 2.72 (d, J = 5.5, 4H); c: 6.56 (s, 2H), 6.15 (s, 2H)
a: 34.76; b: 169.25
H2C
a
NH2
c
O
5d
H2C
a
a: 1.55 (m, J = 3.0, 4H); b: 2.67 (t, J = 4.5, 4H); d: 6.60 (s, 2H), 6.16 (s,
2H)
a: 30.68; b: 34.61; c: 169.27
c
NH2
b
d
a
CH2
b
d
5f
a: 3.34 (d, J = 7.0, 4H); c: 9.13 (s, 2H); d: 7.59 (s, 2H); e: 14.9 (s, 1H),
14.6 (s, 1H)
a: 34.59; b: 126.95; c: 134.56; d: 118.73
a: 36.26; b: 14.33; c: 25.71; d: 12.01
N
NH
e
c
H C
b
CH
3
3 d
5g
a: 1.99 (m, 2H); b: 0.95 (d, J = 4.0, 6H); c: 1.48 (m, 4H); d: 0.91 (t,
J = 6.5, 6H)
HC
CH2
a
c
c
CH3
a
b
5h
5i
a: 1.54 (m, 4H); b: 1.88 (m, 2H); c: 0.98 (d, J = 4.0, 12H)
a: 25.44; b: 44.86; c: 22.71
H2C
CH
CH3
c
a
e
H2C
NH2
a: 2.84 (m, J = 6.0, 4H); b: 1.63 (m, 4H); c: 1.88 (m, J = 7.0, 4H); d: 3.75
(m, J = 4.0, 4H); e: 8.25 (s, 4H)
a: 34.61; b: 21.75; c: 29.63; d: 38.67
b
d
S
a
H2C
b
CH2
c
5j
a: 1.97 (q, J = 7.5, 4H); b: 2.56 (t, J = 5.5, 4H); c: 2.11 (s, 6H)
a: 34.71; b: 30.07; c: 18.15
CH3
c
d
e
a
5k
b
a: 3.19 (dd, J = 10.5, J = 4.5, 4H); c: 7.30 (d, J = 4.5, 4H), d: 7.16 (d, J = 4.0,
4H); e: 7.27 (t, J = 8.5, 2H);
a: 36.22; b: 135.24; c: 127.73; d: 129.08; e: 129.95
CH2
c
b
a
5l
b: 1.99 (m, J = 6.0, 4H); c: 1.90 (m, J = 6.0, 4H); d: 3.26 (m, J = 6.0, 4H)
a: 4.11 (d, J = 6.0, 4H); b: 4.52 (s, 2H)
b: 33.67; c: 25.24; d: 46.74
a: 64.37
d
N
a
b
5m
OH
H2C
c
a
OH
5n
5o
5p
5q
a: 4.34 (m, 2H); b: 1.33 (d, J = 5.5, 6H); c: 4.58 (s, 2H)
a: 2.18 (m, J = 6.0, 2H); b: 2.12 (d, J = 7.5, 12H)
a: 71.59; b: 20.39
HC
b
CH3
a
HC
CH3
CH3
b
a: 29.78; b: 18.97
c
d
a
CH2
b
a: 3.20 (dd, J = 8.0, J = 5.0, 4H); c: 7.14 (d, J = 8.0, 4H); d: 7.12 (d, J = 8.0,
4H); f: 10.17 (s, 2H)
a: 37.52; b: 133.63; c: 130.27; d: 121.45; e: 150.18
a: 27.80; b: 31.30; c: 177.31
e
HO
f
b
a
e
c
OH
a: 2.24 (m, J = 7.0, 4H); b: 2.51 (t, J = 9.5, 4H); e: 12.34 (s, 2H)
H2C
O
c
e
a
d
f
CH2
5r
a: 3.34 (dd, J = 12.0, J = 4.0, 4H); b: 10.94 (s, 2H); e: 7.42 (d, J = 2.5, 2H),
g: 7.26 (d, J = 10.5, 2H), f and h: 7.17 (m, J = 4.5, 4H); j: 6.82 (d, J = 7.5,
2H)
a: 26.20; c: 106.19; d: 126.65; e: 118.11; f: 119.61;
g: 122.31; h: 112.24; i: 136.41; j: 125.34
j
g
N
i
h
b
H
a
Compound 5e has no R.

F. Huang et al. / Bioorg. Med. Chem. 17 (2009) 6085–6095
6095
Table 12
Canvas Program (Model J-810, Jasco, Japan) over the range of
200–750 nm.
13C NMR data of 5a–r except R
O
h
e
f
NH2
g
S
S
d
b
Acknowledgements
O
O
a
c
R
R
This work was finished in Beijing Area Major Laboratory of Pep-
tide and Small Molecular Drugs; supported by PHR (IHLB,
KZ200810025010) and Special Project (2008ZX09401-002) of
China.
O
NH2
O
Ce
Ca
Cb
Cc
Cd
Cf
Cg
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
203.96
204.28
203.15
203.41
204.39
203.21
204.15
203.79
203.85
203.19
204.01
204.18
203.48
203.89
203.44
203.17
203.51
204.17
57.49
54.16
53.75
55.41
56.47
56.46
56.50
56.44
56.46
56.33
56.69
59.73
56.44
59.44
57.82
56.96
56.47
64.73
44.66
43.17
44.71
45.57
44.21
44.74
44.75
44.86
45.36
44.39
45.02
44.36
44.51
44.45
45.81
45.88
40.93
43.59
48.81
48.64
49.13
50.50
49.03
51.50
49.83
50.03
50.59
50.35
50.54
50.46
50.32
50.72
50.55
50.53
50.55
50.32
66.03
65.78
65.38
64.63
65.35
65.01
64.37
64.76
64.93
64.71
64.55
65.25
65.74
65.31
65.38
65.38
65.82
65.58
170.20
50.74
52.45
50.30
53.78
44.21
54.09
53.93
50.82
52.16
52.69
53.60
56.39
56.19
56.68
56.06
54.72
53.98
56.29
References and notes
172.31
171.13
173.68
167.63
168.16
168.76
169.15
169.43
168.89
168.87
169.34
168.93
166.92
168.85
169.38
172.46
169.55
1. Momekov, G.; Bakalova, A.; Karavanova, M. Curr. Med. Chem. 2005, 12, 2177.
2. Hurley, L. H. Nat. Rev. Cancer 2002, 2, 188.
3. Martinez, R.; Chacon-Garcia, L. Curr. Med. Chem. 2005, 12, 127.
4. Meester, C. Mutat. Res. 1995, 339, 139.
5. Funayama, Y.; Nishio, K.; Wakabayashi, K.; Nagao, M.; Shimoi, K.; Ohira, T.;
Hasegawa, S.; Saijo, N. Mutat. Res. Lett. 1999, 9, 3319.
6. AUTHOR: PLEASE PROVIDE MORE DETAILS.
7. Al-Allaf, T. A. K.; Rashan, L. J. Eur. J. Med. Chem. 1998, 33, 817.
8. Cao, R.; Peng, W.; Chen, H.; Ma, Y.; Liu, X.; Hou, X.; Guan, H.; Xu, A. Biochem.
Biophys. Res. Commun. 2005, 338, 1557.
9. Li, N.; Yang, X. Biochem. Biophys. Res. Commun. 2005, 331, 947.
10. Vijayalakshmi, R.; Kanthimathi, M.; Subramanian, V.; Nair1, B. U. Biochem.
Biophys. Res. Commun. 2000, 271, 731.
11. Thulstrup, P. W.; Thormann, T.; Spanget-Larsen, J.; Bisgaard, H. C. Biochem.
Biophys. Res. Commun. 1999, 265, 416.
12. Jiang, X.; Shang, L.; Wang, Z.; Dong, S. Biophys. Chem. 2005, 118, 42.
13. Desoize, B.; Madoulet, C. Crit. Rev. Oncol. Hematol. 2002, 42, 317.
14. Zorbas-Seifried, S.; Jakupec, M. A.; Kukushkin, N. V.; Groessl, M.; Hartinger, C.
G.; Semenova, O.; Zorbas, H.; Kukushkin, H. V. Y.; Keppler, B. K. Mol. Pharmacol.
2007, 71, 357.
15. Dai, J.; Punchihewa, C.; Mistry, P.; Ooi, A. T.; Yang, D. J. Biol. Chem. 2004, 279,
46096.
16. Yang, F.; Chen, Y.; Duan, W.; Zhang, C.; Zhu, H.; Ding, J. Int. J. Cancer 2006, 119,
transplanted once per week. Subcutaneous tumors were implanted
by injecting 0.2 mL of NS containing 4 ꢁ 107 viable tumor cells un-
der the skin on the right oxter. Twenty-four hours after implanta-
tion, the tumor-bearing mice were randomized into 20
experimental groups (10 per group). All mice were given a daily
1184.
17. Rodrıguez, D.; Quiros, L. M.; Salas, J. A. J. Biol. Chem. 2004, 279, 8149.
18. Chianga, L.; Ng, L. T.; Linc, I. C.; Kuo, P.; Lin, C. Cancer Lett. 2006, 237, 207.
19. Hansch, C.; Leo, A.; Schmidt, C.; Jow, P. Y. C. J. Med. Chem. 1980, 23, 1095.
20. Montgomery, J. A.; McCaleb, G. S.; Johnston, T. P.; Mayo, J. G.; Laster, W. R., Jr. J.
Med. Chem. 1977, 20, 291.
ip injection of cytarabine (positive control, 89.28
0.2 ml of NS), or NS (negative control, 0.2 ml), or 5a–r
(44.64 mol/kg/day in 0.2 ml of NS) for seven consecutive days.
lmol/kg/day in
21. Johnston, T. P.; McCaleb, G. S.; Clayton, S. D.; Frye, J. L.; Krauth, C. A.;
Montgomery, J. A. J. Med. Chem. 1977, 20, 279.
l
Twenty-four hours after the last administration, all mice were sac-
rificed by diethyl ether anesthesia, and the tumors were dissected
and weighed. The inhibitory rate of tumor growth was calculated
using the equation: Inhibition = (tumor weight from negative con-
trol mice) ꢂ (tumor weight from 5a–r or cytarabine treated mi-
ce) ꢄ (tumor weight from negative control mice).
22. Janovec, L.; Sabolova, D.; Kozurkova, M.; Paulikova, H.; Kristian, P.; Ungvarsky,
J.; Moravcikova, E.; Bajdichova, M.; Podhradsky, D.; Imrich, J. Bioconjugate
Chem. 2007, 18, 93.
23. Kawakami, M.; Koya, K.; Ukai, T.; Tatsuta, N.; Ikegawa, A.; Ogawa, K.; Shishido,
T.; Chen, L. B. J. Med. Chem. 1997, 40, 3151.
24. Sanyal, U.; Nanda, R.; Samanta, S.; Pain, A.; Dutta, S.; Verma, A. S.; Rider, B. J.;
Agrawal, K. C. Cancer Lett. 2000, 155, 89.
25. Chang, H.; Yamato, O.; Yamasaki, M.; Ko, M.; Maede, Y. Cancer Lett. 2005, 223,
47.
4.4. UV and CD spectral studies on 5a–r
26. Bi, L.; Zhao, M.; Wang, C.; Peng, S.; Winterfeldt, E. Eur. J. Org. Chem. 2000, 2669.
27. Bi, L.; Zhang, Y.; Zhao, M.; Wang, C.; Chan, P.; Tok, J. B.-H.; Peng, S. Bioorg. Med.
Chem. 2005, 13, 5640.
28. Chauhan, M.; Arjmand, F. J. Organomet. Chem. 2007, 692, 5156.
29. Kumar, R. S.; Arunachalam, S. Polyhedron 2007, 26, 3255.
30. Shaikh, A. R.; Ismael, M.; Carpio, C. A. D.; Tsuboi, H.; Koyama, M.; Endou, A.;
Kubo, M.; Broclawikc, E.; Miyamoto, A. Bioorg. Med. Chem. Lett. 2006, 16, 5917.
31. Doddareddy, M. R.; Cho, Y. S.; Koh, H. Y.; Pae, A. N. Bioorg. Med. Chem. 2004, 12,
3977.
UV–vis spectra of 5a–r (pH 3.5–4.0, aqueous solution, 0.1 mM,
25 °C) were recorded with a Shimadzu 2550UV–visible spectro-
photometer over the range of 200–800 nm. Circular dichroism
(CD) spectra of 5a–r (pH 3.5–4.0, aqueous solution, 1 mM, 25 °C)
were obtained on a JASCO J-810 spectropolarimeter with JASCO