1,2,4-Triazole d-ribose derivatives: Design, synthesis and antitumoral evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.10.028

Herein we report the design, synthesis and characterization of novel 1,2,4-triazole d-ribose derivatives, as well as their synthetic precursors. The antitumoral activity against T cell lymphoma cell line of these products was studied. Structures containin

Full text of DOI:10.1016/j.ejmech.2011.10.028

European Journal of Medicinal Chemistry 47 (2012) 104e110
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,2,4-Triazole
D
-ribose derivatives: Design, synthesis and antitumoral evaluation
,
Romina E. Avanzo a, Claudia Anesini b, Mirta L. Fascio a, María I. Errea c, Norma B. D’Accorso a
*
a CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria,
1428 Buenos Aires, Argentina
b Instituto de Química y Metabolismo del Fármaco (IQUIMEFA-UBA-CONICET), Facultad de Farmacia y Bioquímica, UBA, Junín 956 2do piso, Ciudad Autónoma de Buenos Aires,
Argentina
c Instituto Tecnológico de Buenos Aires, Madero 399, Ciudad de Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the design, synthesis and characterization of novel 1,2,4-triazole
well as their synthetic precursors.
D-ribose derivatives, as
Received 18 August 2011
Received in revised form
12 October 2011
Accepted 13 October 2011
Available online 21 October 2011
The antitumoral activity against T cell lymphoma cell line of these products was studied. Structures
containing a 1,2,4-triazolic ring linked by sulfur to the carbohydrate moiety showed a moderate anti-
proliferative activity. The presence of the second heterocyclic ring did not show significant changes in
their biological activity. Meanwhile, structures with 3-thiobenzyl-5-substituted-1,2,4-triazole ring linked
by nitrogen leads to compounds with a biphasic behavior, stimulating cell proliferation at low concen-
trations and inhibiting it at higher ones. An increment in the polarity was associated with a decrease in
the activity of the evaluated compounds.
Keywords:
1,2,4-Triazole
Isoxazoline
A preliminary antitumoral screening pointed the 1,2,4-triazolic structures linked to protected sugars as
promising leaders for further studies.
D
-ribose
Antitumoral activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
a 1,2,4-triazole ring and an isoxazoline ring as the second phar-
macophore in some of them. The antitumoral activity of the new
The triazole derivatives and their nucleoside analogs have
shown strong cytotoxicity against several human cancer cells [1].
For instance, Peng and col. reported two novel N-aryltriazoles as
potent apoptosis-related antiproliferative activity against a drug-
resistant pancreatic cancer cell line [2]. It has also been reported
that the biological activity of compounds containing a triazolic ring
in their structure is due to the high dipolar moment of this
heterocycle which allows strong hydrogen bonding interaction
with the biological target [3]. Furthermore, this heterocycle has
been reported to be stable to acidic and basic hydrolysis as well as
reductive/oxidative conditions [4].
On the otherhand, it has beenproved that the isoxazoline ring acts
as inhibitor of tumor cells and enhances the antiviral activity of iso-
carbonucleosides [5]. In addition, Kamal et al. have recently described
a new 3,5-diarylisoxazoline linked 2,3-dihydroquinazolinone as
a good candidate to further develop potential anticancer agents [6].
Considering the potential antiviral and/or antitumoral activity of
diheterocyclic compounds [7e9], we focused our attention in the
design and synthesis of potential antitumoral structures containing
triazole derivatives was performed on a T cell lymphoma cell line,
which “in vivo” malignancy in relation to the invasive and the
metastatic potential is known [10].
2. Results and discussion
2.1. Chemistry
Tosyl derivative (1) was obtained by treatment of allyl 2,3-O-
cyclopentylidene-
b-D-ribofuranoside [11] with tosyl chloride in
pyridine. The synthetic strategy of this first step was the func-
tionalization of the terminal hydroxyl group of the carbohydrate
derivate with a good living group for subsequent nucleofilic
displacements.
In the first case, compound 1 was treated with sodium 1,2,4-
triazolyl-3-thiolate to obtain allyl 2,3-O-cyclopentylidene-5-
deoxy-5-S-[3(1,2,4-triazolyl)]-
b
-D-ribofuranoside
(2).
This
compound was subjected to a 1,3-dipolar cycloaddition reaction by
treatment with 3,4,5-trimethoxy benzaldoxime in the presence of
chloramine-T using t-butanol/water as a solvent. The diheterocyclic
product (4), was obtained as a diasteroisomeric mixture in a ratio
1:1, estimated by 1H-NMR spectroscopy.
* Corresponding author. Tel./fax: þ54 11 4576 3346.
E-mail address: norma@qo.fcen.uba.ar (N.B. D’Accorso).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.028

R.E. Avanzo et al. / European Journal of Medicinal Chemistry 47 (2012) 104e110
105
On the other hand, with the aim of obtaining triazoline thiones
derivatives, compound 1 was also converted into the corresponding
azido derivate (6) by nucleofilic displacement of the tosylate group
with sodium azide. The azido derivative was transformed into the
1,2,4-triazoline-3-thione derivative (9) using the synthetic pathway
that we reported in a previous work [12].
In Scheme 1 we show, the synthetic pathway applied to obtain
the heterocyclic compounds. The physical and spectroscopical
characterization of the final products and the corresponding
precursors are shown in Section 4.
2.2. Proliferation assays
With the object to obtain a diheterocycle structure from
compound 9, the procedure described above for compound 2 was
applied, but, in this case, the reaction produced several side-
products and the expected diheterocycle could not be isolated. To
avoid this problem, previously to 1,3-dipolar cycloaddition reaction,
compound 9 was protected with a benzyl group to give the corre-
sponding derivative 11. From this compound, now the diheterocyclic
12 could be obtained satisfactorily (yield 78%).
The antiproliferative action on the lymphoma cell line of
compounds 2e5, 9e13 was analyzed. Since our aim was to perform
a preliminary antitumoral screening some epimeric mixtures were
included. The bioactivity of the analyzed compounds on cell lines
BW 5147 is summarized in Table 1, as well as their lipophilic
parameters.
From Table 1 it can be observed:
Compounds 2, 4, 9 and 12 were subjected to deprotection
reaction, by treatment with a solution of acetic acid 80% as are
described in the experimental section [11].
ꢀ Compounds 2e4 showed inhibitory activities over the range of
measured concentrations.
Scheme 1. Reagents and conditions: i) TsCl pyridine; ii) triazolyl sodium tiolate, DMF, 50 ꢁC, 18 h; iii) CH3CO2H 80%, 55 ꢁC, 24 h; iv) R1HC^NOH, chloramine-T, EtOH, H2O, 3 h; v)
CH3CO2H 80%, 40 ꢁC, 24 h; vi) NaN3, DMF, 80 ꢁC, 2 h; vii) CS2. (Ph)3P, dioxane, 24 h; viii) R1CONHNH2, dioxane, reflux, 3 h; ix) NaEtO, EtOH, reflux, 3 h; x) CH3CO2H, 80%, 40 ꢁC, 24 h;
xi) BnCl, K2CO3, acetone; xii) R1HC^NOH, chloramine-T, EtOH, H2O, 4 h; xiii) CH3CO2H, 80%, 40 ꢁC, 24 h.

106
R.E. Avanzo et al. / European Journal of Medicinal Chemistry 47 (2012) 104e110
Table 1
Effect of different compounds upon lymphoma cell line proliferation. Cells were incubated with different compounds in different concentrations during 24 h.
Comp.
Concentration (
1
m
g/mL)
logP
ClogP
10
ꢂ9 ꢄ 1
29.5
50
100
2
%
c
%
c
%
c
%
c
%
c
%
c
%
c
%
c
ꢂ7 ꢄ 0.8
2.9
ꢂ29 ꢄ 2*
147.5
ꢂ43 ꢄ 4**
295.0
1.95
0.78
ꢂ0.95
1.60
3
ꢂ2.4 ꢄ 0.2
ꢂ3 ꢄ 0.2
ꢂ14 ꢄ 1**
ꢂ38 ꢄ 3**
ꢂ2.45
0.90
ꢂ0.52
1.91
0.18
1.63
1.97
1.17
3.7
36.6
183.2
366.3
4
ꢂ5 ꢄ 0.5
1.8
ꢂ21 ꢄ 2
ꢂ24 ꢄ 1*
91.2
ꢂ45 ꢄ 4**
182.1
18.2
5
93 ꢄ 5**
51 ꢄ 3**
20.7
43 ꢄ 4*
29 ꢄ 2
ꢂ0.13
1.80
2.1
103.7
207.0
9
49 ꢄ 4*
2.2
44 ꢄ 4*
70 ꢄ 5**
111.2
123 ꢄ 10**
22.2
222.2
10
11
12
13
ꢂ1.5 ꢄ 0.1
6 ꢄ 0.5
26.1
0
4.4 ꢄ 0.3
2 ꢄ 0.2
260.8
n/d
ꢂ0.25
5.13
2.6
130.4
37 ꢄ 3*
1.85
ꢂ13 ꢄ 1
92.7
18.5
184.8
215 ꢄ 2**
60 ꢄ 6*
ꢂ18 ꢄ 1.9
ꢂ45 ꢄ 4**
5.96
1.3
13.4
66.8
134.0
%
c
143 ꢄ 15**
1.5
210 ꢄ 20**
14.6
ꢂ52 ꢄ 2.6**
73.3
ꢂ67 ꢄ 4.5**
146.0
3.91
Results were expressed as cell proliferation percentage of basal (%). Concentration expressed in
experimental.
mM (c). ClogP was calculated using ChemBio3D Ultra 11.0, logP data is
*p < 0.05 and **p < 0.01 are significant differences respect to basal accordingly to ANOVA þ Dunnett’s test.
n/d: not determined.
ꢀ Compound 5 showed an increment in cell proliferation, which
4. Experimental protocols
decreased at higher concentrations. This stimulating effect
could be related to an increment in the polarity (logP: ꢂ0.52)
respect to compound 4 (log P: 0.90) or to a conformational
change of the carbohydrate moiety.
4.1. Chemistry
4.1.1. General remarks
ꢀ Compound 10 did not show differences significantly respect to
Synthesis of compounds 1e13 was carried out using reagents as
purchased, without further purification. Solvents were reagent
grade and, in most cases, dried and distillated before use according
to standard procedures. Analytical TLC was conducted on Silica Gel
60G (Merck) on precoated plates and visualization was made by UV
light and ethanol/sulfuric acid (10:1) or cerium molybdate followed
by heating. Column-chromatographic separations were performed
on Silica Gel (240e400 mesh, Merck). Elemental analysis was
performed on an Exeter Analytical CE-440 elemental analyzer.
Optical rotations were recorded at 20 ꢁC on a Perkin Elmer 343
polarimeter, and melting points were uncorrected. 1H, 13C NMR
spectra were recorded on a Bruker AC-200 spectrometer, operating
at 200, 50 MHz respectively; or a Bruker AMX-500 spectrometer,
operating at 500, 125 MHz respectively. Assignments of the 1H and
13C NMR spectra were confirmed with the aid of two dimensional
basal percentage over all concentrations.
ꢀ Compounds 11e13 exerted a biphasic action, stimulating cell
proliferation at low concentrations and inhibiting it at higher
ones. Such behavior could be interpreted as evidence for the
existence of separate modulatory drug binding sites.
ꢀ It is worth noting that compound 9 augmented cell prolifera-
tion (stimulation) as the concentration increased. This
compound has a completely different behavior than its deriv-
ative 11, likely due to the protection of thiol group which
inhibits the tautomeric equilibrium between thiol and thione.
Compounds 2 and 4 exhibited moderate activity against the
lymphoma cell line tested. The inhibitory pattern was similar
between them and no stimulation activity was observed. The
inhibitory action may be attributed to the 1,2,4-triazol ring linked
through sulfur to C-5 of the carbohydrate moiety. Deprotection of
compound 2 (logP 1.95) reduced its antiproliferative activity (see
Table 1, compound 3) may be due to the increase of the hydro-
philicity (logP ꢂ2.45). Changes in the hydrophilicity could also
justify the different behavior observed for compounds 3 and 5, as
well as for compound 9 and 10 (See Table 1).
techniques 1H, 13C (COSY, HSQC). Chemical shifts (
d) are reported in
parts per million downfield from tetramethyl silane as internal
standard. High-resolution mass spectra HRMS were obtained by
Electrospray Ionization (ESI) and Q-TOF detection.
4.1.2. Allyl 2,3-O-cyclopentylidene-5-tosyl-
b-
D-ribofuranoside (1)
To a solution of 2.73 g of allyl 2,3-O-cyclopentylidene-
b-D
-ribo-
furanoside [11] dissolved in anhydrous pyridine (5 mL), 2.44 g of
tosyl chloride was added with continuous stirring under Ar atmo-
sphere. The mixture was kept at room temperature during one
night. The crude product was extracted with methylene chloride
and water, washed with hydrogen chloride (5%), then sodium
bicarbonate (5%) and finally with water, then was purified by
3. Conclusion
To summarize, we designed a synthetic pathway to obtain
novel triazolic derivatives with or without an isoxazoline ring
from
D
-ribose. Compounds 2e4, 11e13 presented moderate
column
compound 1 as white solid (3.25 g, 7.9 mmol, 69%); mp: 55e56 ꢁC,
a 2D0
ꢂ 44:4 (c 1.1, chloroform). Anal. Calcd for C20H26O7S: C, 58.52;
H, 6.38. Found: C, 58.37; H, 6.54. 1H NMR (500 MHz, CDCl3)
: 7.78
(d, 2H, J 8.4 Hz, aromatic protons), 7.34 (d, 2H, J 8.0 Hz, aromatic
protons), 5.77 (m, 1H, H-20), 5.21 (m, 1H, H-30b), 5.16 (m, 1H, H-30a),
5.08 (s, 1H, H-1), 4.55 (dd, 1H, J3,2 6.0 Hz, J3,4 0.7 Hz, H-3), 4.51 (d,
chromatography
(cyclohexane:acetone)
affording
inhibitory activity against BW 5147 lymphoma cell line at high
concentrations but only compounds 2e4 showed inhibitory
behavior in the range of measured concentrations. These results
suggest that this kind of compounds may be considered as
promising leader molecules for further synthetic and biological
exploration.
½
ꢃ
d

R.E. Avanzo et al. / European Journal of Medicinal Chemistry 47 (2012) 104e110
107
1H, J2,3 6.0 Hz, H-2), 4.31 (ddd,1H, J4,5b 7.7 Hz, J4,5a 6.7 Hz, J4,3 0.9 Hz,
H-4), 4.03 (dd, 1H, J5b,5a 10.0 Hz, J5b,4 7.8 Hz, H-5b), 4.03 (m, 1H, H-
10b), 3.99 (dd, 1H, J5a,5b 10.0 Hz, J5a,4 6.7 Hz, H-5a), 3.87 (m, 1H, H-
10a), 2.44 (s, 3H, CH3), 1.92e1.62 (m, 8H, cyclopentylidene protons);
was evaporated and the residue was purified by flash column
chromatography using toluene:ethyl acetate, as eluent. Compound
4 was obtained as a diastereoisomeric pair as a 1:1 unresolved
mixture of epimers (129.3 mg, 0.24 mmol, 42%). 1H NMR
13C NMR (125 MHz, CDCl3)
d
: 145.2 (aromatic carbon), 133.4 (C-20),
(500 MHz, CDCl3) d: 8.14, 8.12 (s, 1H, H-3 of triazolyl group), 6.89
132.8, 130.0 and 128.1 (aromatic carbons), 122.4 (quaternary carbon
of cyclopentylidene ring), 117.8 (C-30), 107.1 (C-1), 84.9 (C-2), 83.4
(C-4), 81.3 (C-3), 69.4 (C-5), 68.2 (C-10), 35.9, 35.8, 23.7, 23.2
(cyclopentylidene carbons), 21.8 (CH3).
(s, 2H, trimethoxyphenyl protons), 5.17, 5.16 (s, 1H, H-1), 4.92 (m,
1H, H-500 isoxazoline ring), 4.67, 4.66 (d, 1H, J3,2 5.8 Hz, H-3), 4.64,
4.61 (d, 1H, J2,3 6.0 Hz, H-2), 4.47 (m, 1H, H-4), 4.00, 3.93 (dd, 1H,
J1 b,1 a 10.8, 10.7 Hz, J1 b,5 3.5, 6.0 Hz, H-10b), 3.88, 3.87, 3.87 (s, 9H,
0
0
0
00
0
0
0
00
methoxyl protons), 3.59, 3.58 (dd, 1H, J1 a,1 b 10.8, 10.7 Hz, J1 a,5
4.1.3. Allyl 2,3-O-cyclopentylidene-5-deoxy-5-S-(1,2,4-triazol-3-
3.9, 3.0 Hz, H-10a), 3.43, 3.41 (dd, 1H, J5b,5a 13.3, 13.2 Hz, J5b,4 7.0,
00
00
00
00
yl)-
b
-
D
-ribofuranoside (2)
7.4 Hz, H-5b), 3.41, 3.39 (dd, 1H, J4 b,4 a 16.5, 16.6, J4 b,5 6.7, 6.9 Hz,
To a solution of sodium ethoxide in ethanol, prepared adding
metallic sodium (approximately 243.9 mg) to ethanol (20 mL), was
added 1,2,4-triazolyl-5-thiol (1.03 g). The reaction mixture was
heated at 50 ꢁC during 10 min and then the solvent was evaporated
at reduced pressure. The resulting solid was added at a solution of
compound 1 (1.04 g) in DMF (7 mL) and heated under argon
atmosphere at 50 ꢁC for 18 h. The solution was evaporated,
extracted with dichloromethane and a solution of sodium chloride,
dried with sodium sulfate, filtered and evaporated. The crude
product was purified by flash column chromatography using
cyclohexane:acetone, as eluent. Compound 2 was obtained as
a crystalline solid (679.4 mg, 2.00 mmol, 79%), mp: 149e151 ꢁC,
H-400b isoxazoline ring), 3.28, 3.26 (dd, 1H, J5b,5a 13.3,13.3 Hz, J5b,4
10.9, 11.3 Hz, H-5a), 3.23, 3.15 (dd, 1H, J4 a,4 b 16.6, 16.6 Hz, J4 a,5
7.6, 7.0 Hz, H-400a isoxazoline ring), 1.90e1.63 (m, 8H, cyclo-
pentylidene protons); 13C NMR (50 MHz, CDCl3)
: 156.6, 156.5 (C-
00
00
00
00
d
5 of triazolyl ring), 153.6 (C-300 isoxazoline ring), 153.4, 140.0,
124.7, 104.2 (trimethoxyphenyl carbons), 147.5, 147.3 (C-3 of tri-
azolyl ring), 122.3 (quaternary carbon of cyclopentylidene ring),
108.7, 108.7 (C-1), 85.3, 85.2, 85.0 (C-2, C-4), 83.1 (C-3), 79.6, 79.4
(C-500 isoxazoline ring) 68.7 (C-10), 61.0, 56.4 (OCH3), 37.4, 37.1 (C-
400 isoxazoline ring), 36.0, 35.8, 35.7, 35.6 (C-5 and cyclo-
pentylidene carbons), 23.7, 23.2 (cyclopentylidene carbons).
HRMS (ESI) m/z (M þ Na) calcd for C25H32N4NaO8S 571.1833, found
571.1839; m/z (M þ H) calcd for C25H33N4O8S 549.2014, found
549.2026.
½
a 2D0
H, 6.24; N, 12.38. Found: C, 53.05; H, 6.42; N, 12.23. 1H NMR
ꢃ
ꢂ 73:0 (c 3.0, acetone). Anal. Calcd for C15H21N3O4S: C, 53.08;
(500 MHz, (CD3)2CO)
d
: 8.45 (s, 1H, NH), 8.37 (s, 1H, H-3 0of triazolyl
group), 5.93 (m, 1H, H-20), 5.30 (d, 1H, J3 b,2 17.2 Hz, H-3 b), 5.15 (d,
4.1.6. ((4R,S)-3-(3,4,5-trimethoxyphenyl)-isoxazolin-5-yl)methyl 5-
deoxy-5-S-(1,2,4-triazol-3-yl)-b-D-ribofuranoside (5)
0
0
0
0
0
1H, J3 a,2 10.6 Hz, H-3 a), 5.10 (s, 1H, H-1), 4.80 (d, 1H, J3,2 5.9 Hz, H-
3), 4.64 (d, 1H, J2,3 5.9 Hz, H-2), 4.43 (t br, 1H, J 7.6 Hz, H-4), 4.25 (dd,
Compound 4 (230.0 mg, 0.42 mmol) was dissolved in 80% acetic
acid and heated to 40 ꢁC for 48 h. Then, the solution was evaporated
and the residue was purified by column chromatography using
cyclohexane:acetone as eluent, giving 5 as a white solid (114.3 mg,
0.24 mmol, 57%). Anal. Calcd for C20H26N4O8S: C, 49.78; H, 5.43; N,
11.61. Found: C, 49.39; H, 5.59; N, 11.26. 1H NMR (500 MHz, CDCl3)
1H, J1 b,1 a 13.1 Hz, J1 b,2 4.9 Hz, H-10b), 4.01 (dd, 1H, J1 a,1 b 12.8 Hz,
0
0
0
0
0
0
J1 a,2 5.6 Hz, H-10a), 3.38 (dd, 1H, J5b,5a 13.7 Hz, J5b,4 6.7 Hz, H-5b),
3.27 (dd, 1H, J5a,5b 13.9 Hz, J5a,4 9.1 Hz, H-5a), 1.84e1.62 (m, 8H,
0
0
cyclopentylidene protons); 13C NMR (50 MHz, CDCl3)
d: 157.1 (C-5
of heterocyclic ring), 146.5 (C-3 of heterocyclic ring), 133.5 (C-20),
122.2 (quaternary carbon of cyclopentylidene ring), 117.8 (C-30),
107.5 (C-1), 85.2 (C-2), 85.1 (C-4), 83.1 (C-3), 68.3 (C-10), 35.8
(cyclopentylidene carbon), 35.7 (C-5 and cyclopentylidene carbon),
23.7 and 23.2 (cyclopentylidene carbons).
d
: 8.31 (br s, 1H, H-3 of triazolyl group), 7.01 (s, 2H, trimethox-
yphenyl protons), 4.96 (s, 1H, H-1), 4.94e4.87(m, 1H, H-500 iso-
xazoline ring), 4.22e4.17 (m, 2H, H-2, H-4), 4.00, 3.98 (dd, 1H, J3,2
4.2 Hz, J3,4 0.8 Hz, H-3), 3.88, 3.87, 3.77 (s, 9H, methoxyl protons),
3.83, 3.80 (dd, 1H, J1 b,1 a 10.9, 10.6 Hz, J1 b,5 4.8, 4.6 Hz, H-10b),
0
0
0
00
4.1.4. Allyl 5-deoxy-5-S-(1,2,4-triazol-3-yl)-b-D-ribofuranoside (3)
3.62e3.57 (m, 1H, H-10a), 3.55, 3.54 (dd, 1H, J5b,5a 13.7, 13.6 Hz, J5b,4
00
00
00
00
Compound 2 (251.4 mg, 0.74 mmol) was dissolved in 80% acetic
acid and heated to 55 ꢁC during 24 h. Then, the solution was
evaporated and the residue was purified by column chromatog-
raphy using cyclohexane:acetone as eluent. Finally, compound 3
was obtained as a white solid (80.0 mg, 40%); mp: 122e123 ꢁC,
4.9, 4.7 Hz, H-5b), 3.51, 3.49 (dd, 1H, J4 b,4 a 16.9, 16.8, J4 b,5 4.4, 4.5
Hz, H-400b isoxazoline ring), 3.44e3.39 (m, 1H, H-5a), 3.35, 3.29 (dd,
1H, J4 a,4 b 16.8, 16.9 Hz, J4 a,5 7.6, 7.4 Hz, H-400a isoxazoline ring);
00
00
00
00
13C NMR (125 MHz, CDCl3)
d: 157.0, 157.0 (C-5 of heterocyclic ring),
154.4, 142.4, 140.8, 126.4, 126.3, 105.1 (trimethoxyphenyl carbons),
147.2 (C-3 of triazolyl ring), 108.6 (C-1), 83.1, 83.0, (C-4), 80.7, 80.6
(C-500 isoxazoline ring), 76.3 (C-3), 75.0, 74.9 (C-2), 69.6, 69.1 (C-10),
60.6, 56.6 (OCH3), 38.0, 37.7 (C-400 isoxazoline ring), 36.7, 36.7 (C-5).
½
a 2D0
ꢃ
ꢂ 36:6 (c 1.0, methanol). Anal. Calcd for C10H15N3O4S: C, 43.95;
H, 5.53; N, 15.37. Found: C, 44.31; H, 5.32; N, 15.23. 1H NMR
(500 MHz, (CD3)2CO) : 8.30 (s, 1H, H-3 of triazolyl group), 5.92 (m,
d
1H, H-20), 5.26 (m, 1H, H-30b), 5.12 (m, 1H, H-30a), 4.90 (br s, 1 H, H-
1), 4.23e4.16 (m, 3H, H-2, H-4, H-10b), 4.01 (dd, 1H, J2,3 4.7 Hz, J2,1
0.7 Hz, H-3), 3.94 (m, 1H, H-10a), 3.50 (dd, 1H, J5b,5a 13.5 Hz, J5b,4
5.2 Hz, H-5b), 3.34 (dd, 1H, J5a,5b 13.5 Hz, J5a,4 6.6 Hz, H-5a); 13C
4.1.7. Allyl 2,3-O-cyclopentylidene-5-deoxy-5-azide-b-D-
ribofuranoside (6)
To a solution of compound 1 (318.5 mg) in DMF (8 mL) we added
sodium azide (312.5 mg). The reaction mixture was heated under
argon atmosphere at 80 ꢁC for 3 h. Then it was filtered and washed
with ethyl ether. The product was evaporated under reduced
pressure, suspended in water and extracted with ethyl ether. The
extract was dried with sodium sulfate, filtered and concentrated.
The residue was purified by flash column chromatography using
cyclohexane:acetone, as eluent. Compound 6 was obtained as
NMR (50 MHz, CD3OD) d: 158.3 (C-5 of heterocyclic ring), 147.8 (C-3
of heterocyclic ring),135.5 (C-20), 117.3 (C-30),107.9 (C-1), 82.7 (C-4),
76.5 (C-3), 75.3 (C-2), 69.2 (C-10), 37.5 (C-5).
4.1.5. ((4R,S)-3-(3,4,5-trimethoxyphenyl)-isoxazolin-5-yl)methyl 5-
deoxy-5-S-(1,2,4-triazol-3-yl)-2,3-O-cyclopentylidene-b-D-
ribofuranoside (4)
To a solution of 3,4,5-trimethoxy benzaldoxime (194.3 mg) in
t-buthanol:water 1:1, chloramine-T (195.1 mg) was added in small
a transparent syrup (161.9 mg, 0.58 mmol, 74%); ½a D25
ꢂ 65:4 (c 1.7,
ꢃ
chloroform). Anal. Calcd for C13H19 N3O4: C, 55.50; H, 6.81. Found: C,
portions. This solution was slowly added to compound
2
55.53; H, 6.74. 1H NMR (500 MHz, CDCl3) : 5.88 (m, 1H, H-20), 5.29
d
(191.6 mg, 0.56 mmol) dissolved in t-buthanol:water 1:1 and the
(m, 1H, H-30b), 5.21 (m, 1H, H-30a), 5.15 (s, 1H, H-1), 4.60 (d, 1H, J2,3
6.1 Hz, H-2), 4.56 (dd, 1H, J3,2 6.0 Hz, J3,4 1.1 Hz, H-3), 4.31 (ddd, 1H,
reaction mixture was heated at 45 ꢁC for 2 h. Then, the solution

108
R.E. Avanzo et al. / European Journal of Medicinal Chemistry 47 (2012) 104e110
J4,5b 7.8 Hz, J4,5a 6.8 Hz, J4,3 1.1 Hz, H-4), 4.21 (m, 1H, H-10b), 3.99 (m,
1H, H-10a), 3.47 (dd, 1H, J5b,5a 12.6 Hz, J5b,4 7.9 Hz, H-5b), 3.28 (dd,
1H, J5a,5b 12.5 Hz, J5a,4 6.7 Hz, H-5a), 1.94e1.70 (m, 8H, cyclo-
reaction mixture was refluxed, with continuous stirring, during
24 h. The solution was neutralized with HCl 1 N, evaporated and
the crude residue was further purified by flash column chroma-
tography using cyclohexane:acetone as eluent, affording
compound 9 as white crystals (312.9 mg, 0.70 mmol, 81%); mp:
pentylidene protons); 13C NMR (125 MHz, CDCl3) : 133.6 (C-20),
d
122.4 (quaternary carbon of cyclopentylidene ring), 117.8 (C-30),
107.6 (C-1), 85.3 (C-2), 85.2 (C-4), 82.0 (C-3), 68.2 (C-10), 53.9 (C-5),
36.0, 35.9, 23.7 and 23.3 (cyclopentylidene carbons).
53e54 ꢁC,
C21H24ClN3O4S: C, 56.06; H, 5.38. Found: C, 56.07; H, 5.69. 1H NMR
(500 MHz, CDCl3) : 7.60 (d, 2H, J 8.8 Hz, aromatic protons), 7.53 (d,
½
a 2D5
ꢃ
ꢂ 54:0 (c 1.0, chloroform). Anal. Calcd for
d
4.1.8. Allyl 2,3-O-cyclopentylidene-5-deoxy-5-isothiocyanate-
ribofuranoside (7)
b
-
D
-
2H, J 8.7 Hz, aromatic protons), 5.83 (m, 1H, H-20), 5.27 (m, 1H, H-
30b), 5.19 (m, 1H, H-30a), 5.08 (s, 1H, H-1), 4.93 (dd, 1H, J3,2 6.0 Hz,
J3,4 0.7 Hz, H-3), 4.68 (dd, 1H, J5b,5a 14.3 Hz, J5b,4 9.3 Hz, H-5b), 4.57
(d, 1H, J2,3 6.0 Hz, H-2), 4.36 (ddd, 1H, J4,5b 9.3 Hz, J4,5a 4.6 Hz, J4,3
0.8 Hz, H-4), 4.07 (m, 1H, H-10b), 4.05 (dd, 1H, J5a,5b 14.4 Hz, J5a,4
4.5 Hz, H-5a), 3.90 (m, 1H, H-10a), 1.85e1.61 (m, 8H, cyclo-
To a solution of compound 6 (158.2 mg), carbon disulfide
(0.4 mL) in dioxane anhydrous (5 mL) was added 169.0 mg of tri-
phenylfosfine. The reaction mixture was stirred at room temper-
ature during 24 h, under Ar atmosphere. Then the solvent was
evaporated and the crude product was extracted with ethyl ether.
The extract was dried with sodium sulfate, filtered and concen-
trated, dissolved in cyclohexane and filtered off. The solvent was
evaporated. The residue was purified by flash column chroma-
tography using cyclohexane:acetone, as eluent. Compound 7 was
obtained as a transparent syrup (131.6 mg, 0.44 mmol, 79%);
pentylidene protons);13C NMR (125 MHz, CDCl3)
d: 168.4 (CS) and
150.9 (CN) (heterocyclic carbons), 137.7 (aromatic carbon), 133.4
(C-20), 130.1, 129.8 and 124.1 (aromatic carbons), 122.4 (quaternary
carbon of cyclopentylidene ring), 117.5 (C-30), 107.4 (C-1), 84.9 (C-
2), 83.7 (C-4), 81.7 (C-3), 68.4 (C-10), 46.8 (C-5), 35.9, 35.8, 23.6, 23.1
(cyclopentylidene carbons).
½
a 2D5
56.55; H, 6.44; N, 4.70. Found: C, 56.47; H, 6.51; N, 4.86. IR (KBr,
cmꢂ1): 2207 and 2089 (N]C]S). 1H NMR (500 MHz, CDCl3)
ꢃ
ꢂ 21:6 (c 1.0, chloroform). Anal. Calcd for C14H19NO4S: C,
4.1.11. Allyl 5-deoxy-5-[(5-p-chlorophenyl-3-thionyl)-1,2,4-
triazolin-4-yl]-b-D-ribofuranoside (10)
d:
5.86 (m, 1H, H-20), 5.29 (m, 1H, H-30b), 5.20 (m, 1H, H-30a), 5.15 (s,
1H, H-1), 4.60 (d, 1H, J2,3 6.0 Hz, H-2), 4.55 (dd, 1H, J3,2 6.0 Hz, J3,4
0.7 Hz, H-3), 4.37 (ddd, 1H, J4,5b 7.8 Hz, J4,5a 7.0 Hz, J4,3 0.9 Hz, H-4),
4.20 (m, 1H, H-10b), 3.99 (m, 1H, H-10a), 3.68 (dd, 1H, J5b,5a 14.3 Hz,
J5b,4 7.9 Hz, H-5b), 3.55 (dd, 1H, J5a,5b 14.3 Hz, J5a,4 7.0 Hz, H-5a),
1.92e1.65 (m, 8H, cyclopentylidene protons); 13C NMR (125 MHz,
Compound 9 (95.9 mg, 0.21 mmol) was dissolved in 80% acetic
acid and heated to 40 ꢁC for 24 h. Then, the solution was evaporated
(as described for compound 3) and the residue was purified by
column chromatography using cyclohexane:acetone as eluent,
giving 10 as a white solid (50.8 mg, 0.13 mmol, 62%); mp:
149e152 ꢁC (dec), ½a 2D0
ꢂ 15:3 (c 1.0, methanol). Anal. Calcd for
ꢃ
CDCl3)
d
:
133.4 (C-20), 122.6 (quaternary carbon of cyclo-
C16H18ClN3O4S: C, 50.06; H, 4.73; N, 10.95. Found: C, 50.00; H, 5.07;
pentylidene ring), 118.0 (C-30), 107.5 (C-1), 85.1 (C-2), 84.5 (C-4),
81.7 (C-3), 68.5 (C-10), 48.1 (C-5), 35.9, 35.8, 23.7, 23.2 (cyclo-
pentylidene carbons).
N, 10.63. 1H NMR (200 MHz, CD3OD)
d: 7.73 (d, 2H, J 8.6 Hz,
aromatic protons), 7.54 (d, 2H, J 8.6 Hz, aromatic protons), 5.73 (m,
1H, H-20), 5.14 (m, 1H, H-30b), 5.10 (m, 1H, H-30a), 4.72 (s, 1H, H-1),
4.58 (dd, 1H, J5b,5a 17.4 Hz, J5b,4 7.3 Hz, H-5b), 4.34e4,20 (m, 2H, H-4
and H-5a), 4.07 (dd, 1H, J3,2 4.6 Hz, J3,4 7.0 Hz, H-3), 3.81 (d, 1H, J2,3
4.6 Hz, H-2), 3.70 (m, 1H, H-10b), 3.58 (m, 1H, H-10a); 13C NMR
4.1.9. Allyl 5-(p-clorobenzohydrazinecarbothionyl) amino-2,3-O-
cyclopentylidene-5-deoxy-b-D-ribofuranoside (8)
A
suspension of p-chloro-benzoic hydrazide (275.3 mg,
(50 MHz, CD3OD) d: 169.7 (CS) and 153.2 (CN) (heterocyclic
1.61 mmol) in dry dioxane was heated until dissolution and
compound 7 was added (432.6 mg, 1.45 mmol). The resulting
solution was kept at room temperature for 24 h under Ar atmo-
sphere. The solvent was evaporated and the crude product was
purified by flash chromatography using cyclohexane:acetone as
eluent. The p-chloro substituted thiosemicarbazide (8) was ob-
tained as white crystals (623.6 mg, 1.33 mmol, 92%); mp:
carbons), 138.3 (aromatic carbon), 135.7 (C-20), 132.2, 130.7 and
126.9 (aromatic carbons), 117.3 (C-30), 108.3 (C-1), 80.3 (C-4), 76.3
(C-3), 74.6 (C-2), 69.6 (C-10), 49.7 (C-5).
4.1.12. Allyl 5-deoxy-5-[(5-p-chlorophenyl-3-thiobenzyl)-1,2,4-
triazol-4-yl]-2,3-O-cyclopentylidene-b-D-ribofuranoside (11)
A mixture of compound 9 (501.3 mg, 1.1 mmol), benzyl bromide
(0.2 mL) and anhydrous potassium carbonate (153.6 mg) in
acetone was stirred at room temperature for 17 h. The reaction
mixture was filtered, and the filtrate was evaporated. The residue
was purified by flash column chromatography using toluene:ethyl
acetate as eluent, affording compound 11 as a syrup (567.6 mg,
119e121 ꢁC,
C21H26ClN3O5S: C, 53.90; H, 5.60; N, 8.98. Found: C, 53.68; H, 5.64;
N, 8.95. 1H NMR (500 MHz, CDCl3)
: 9.10 (NH), 8.72 (NH), 7.82 (d,
2H, J 8.6 Hz, aromatic protons), 7.59 (NH), 7.45 (d, 2H, J 8.6 Hz,
½
a 2D5
ꢃ
ꢂ 49:2 (c 1.0, chloroform). Anal. Calcd for
d
aromatic protons), 5.62 (m, 1H, H-20), 5.14 (dd, 1H, J3 b,2 17.2 Hz,
0
0
J3 b,3 a 1.3 Hz, H-30b), 5.10 (dd, 1H, J3 a,2 10.3 Hz, J3 a,3 b 1.2 Hz, H-30a),
5.07 (s, 1H, H-1), 4.62 (d, 1H, J2,3 6.4 Hz, H-3), 4.53 (d, 1H, J3,2 6.2 Hz,
H-2), 4.37 (br signal,1H, J4,5b 5.8 Hz, J4,5a 4.6 Hz, H-4), 4.11 (br signal,
1.05 mmol, 94%); ½a D25
ꢃ
ꢂ 55:7 (c 1.0, methanol). Anal. Calcd for
0
0
0
0
0
0
C28H30ClN3O4S: C, 62.27; H, 5.60; N, 7.78. Found: C, 61.89; H, 6.00;
N, 7.93. 1H NMR (500 MHz, CDCl3)
d: 7.45e7.20 (9H, aromatic
0
1H, H-5b), 4.04 (dd, 1H, J1 b,1 a 12.8 Hz, J1 b,2 5.4 Hz, H-1 b), 3.88 (dd,
protons), 5.72 (m, 1H, H-20), 5.16 (m, 1H, H-30b), 5.12 (m, 1H, H-30a),
4.92 (s, 1H, H-1), 4.40 (d, J12.8 Hz, thiobenzyl proton), 4.37 (d, J
12,8 Hz, thiobenzyl proton), 4.30 (d, J 6.0 Hz, H-2), 4.17 (dd, 1H, J3,2
6.0 Hz, J3,4 0.9 Hz, H-3), 4.01 (ddd, 1H, J4,5b 9.2 Hz, J4,5a 5.3 Hz, J4,3
0.8 Hz, H-4), 3.92 (dd, 1H, J5b,5a 14.5 Hz, J5b,4 9.3 Hz, H-5b), 3.92 (m,
1H, H-10b), 3.76 (m, 1H, H-10a), 3.74 (dd, 1H, J5a,5b 14.5 Hz, J5a,4
5.3 Hz, H-5a), 1.74e1.44 (m, 8H, cyclopentylidene protons); 13C
0
0
0
0
1H, J1 a,1 b 12.9 Hz, J1 a,2 6.4 Hz, H-10a), 3.63 (br d, 1H, J5b,5a 14.2 Hz,
0
0
0
0
H-5a), 1.88e1.63 (m, 8H, cyclopentylidene protons); 13C NMR
(50 MHz, CDCl3) d: 182.6 (CS), 166.2 (CO), 139.2 (aromatic carbon),
133.0 (C-20), 129.4, 129.3 and 129.1 (aromatic carbons), 122.1
(quaternary carbon of cyclopentylidene ring), 118.5 (C-30), 107.3 (C-
1), 85.3 (C-2), 85.0 (C-4), 81.6 (C-3), 68.6 (C-10), 47.7 (C-5), 35.8, 35.7,
23.6, 23.1 (cyclopentylidene carbons).
NMR (50 MHz, CDCl3) d
: 154.6, 151.3 (CN), 133.4 (C-20), 136.8, 136.5,
130.2, 129.6, 129.3, 128.8, 128.0, 125.7 (aromatic carbons), 122.6
(quaternary carbon of cyclopentylidene ring), 117.7 (C-30), 107.4 (C-
1), 84.9 (C-2), 83.8 (C-4), 81.1 (C-3), 68.6 (C-10), 46.8 (C-5), 39.0
(CH2 benzyl carbon), 35.8, 35.8, 23.6, 23.2 (cyclopentylidene
carbons).
4.1.10. Allyl 5-deoxy-5-[(5-p-chlorophenyl-3-thionyl)-1,2,4-
triazolin-4-yl]-2,3-O-cyclopentylidene-b-D-ribofuranoside (9)
Thiosemicarbazide (8) (401.3 mg, 0.86 mmol) was dissolved in
ethanol and sodium metallic was added (79.8 mg, 3.47 mmol). The

R.E. Avanzo et al. / European Journal of Medicinal Chemistry 47 (2012) 104e110
109
4.1.13. ((4R,S)-3-(3,4,5-trimethoxyphenyl)-isoxazolin-5-yl)methyl
5-deoxy-5-[(5-p-chlorophenyl-3-thiobenzyl)-1,2,4-triazol-4-yl]-
has a TCRab as determined by flow cytometric analysis. Cells were
cultured at optimal concentrations of 3 ꢅ 105 cells/mL in RPMI 1640
medium (GIBCO) supplemented with 10% fetal calf serum (FCS),
2 mM glutamine and antibiotics [13]. Cells were cultured at a final
volume of 0.2 mL in 96-well flat-bottom microtiter plates (Nunc).
2,3-O-cyclopentylidene-
b-D-ribofuranoside (12)
To homogeneous solution of compound 11 (393.2 mg,
a
0.73 mmol) and 3,4,5-trimethoxy benzaldoxime (320.0 mg,
1.45 mmol) in ethanol/water, chloramine-T (665.9 mg) were slowly
added. The reaction mixture was kept at room temperature for 5 h.
Then, the solution was evaporated, the crude was dissolved in
acetone and filtered off. The residue was purified by flash column
chromatography using toluene:ethyl acetate, as eluent. Compound
12 was obtained as a diastereoisomeric pair as a 1:1 unresolved
mixture of epimers (425.3 mg, 0.57 mmol, 78%). 1H NMR (500 MHz,
4.2.2. Analyzed compounds
The compounds analyzed were 2e5, 9e13. Compound 13 was
dissolved in dimethyl sulfoxide (DMSO) and the others in different
concentrations of ethanol:water mixtures. Compound 2 was dis-
solved in ethanol 12%, compound 4 in ethanol 15%, and compounds
3, 5, 9e12 in ethanol 10%. The final ethanol concentration in wells
was 0.5% and DMSO 1%.
CDCl3) d: 7.53e7.20 (m, 9H, aromatic protons), 6.85 (s, 1 H, trime-
thoxyphenyl proton), 6.82 (s, 1H, trimethoxyphenyl proton), 4.94,
4.93 (s, 1H, H-1) 4.82e4.74 (m, 1H, H-500 isoxazoline ring), 4.45, 4.45
(d, 1H, J 12.7 Hz, thiobenzyl proton), 4.39, 4.37 (d, 1H, J 12.7 Hz,
thiobenzyl proton), 4.23e4.00 (m, 4H, H-2, H-3, H-4, H-5b), 3.89 (s,
9H, methoxy protons), 3.94, 3.85 (dd, 1H, J5b,5a 14.2, 14.7 Hz, J5b,4
4.2.3. Proliferation assays
The effects of different compounds on tumoral lymphocytes
proliferation was evaluated by the uptake of tritiated thymidine
[3H]TdR. Cells were cultured during 24 h in presence or absence
(basal) of different concentrations of compounds 2e5, 9e13 (1, 10,
4.0, 4.8 Hz, H-5a), 3.85e3.61 (m,1H, H-10b), 3.42, 3.39 (dd,1H, J1 a,1 b
0
0
11.1, 10.7 Hz, J1 a,4 4.2, 3.6 Hz, H-1 a), 3.32, 3.25 (dd, 1H, J4 b,4 a 16.5,
50 and 100 mg/mL) and then pulsed with 0.75 m
Ci/well of [3H]TdR
0
0
00
00
00
00
00
00
16.4, J4 b,5 10.8, 11.0 Hz, H-4 b isoxazoline ring), 3.17, 2.95 (dd, 1H,
(S ¼ 25 Ci/mmol) for the last 16 h previously to culture sacrifice by
freezing as previously described [14]. Blanks corresponding to
ethanol 0.5% and DMSO 1% were assayed.
Results were expressed as cpm or as cell proliferation (% of
basal): [cpm basal ꢂ cpm treated/cpm basal ꢅ 100]. Data represent
the mean ꢄ SEM of three experiments performed in triplicate. It is
important to note that the blanks did not modify cell proliferation
by their self (proliferation cpm (mean ꢄ SEM): Basal: 5487 ꢄ453,
Ethanol 0.5%: 5520 ꢄ 400; DMSO 1%: 5500 ꢄ 500).
J4 a,4 b 16.4 Hz, 16.5 Hz, J4 a,5 8.5, 7.4 Hz, H-400a isoxazoline ring),
00
00
00
00
1.69e1.49 (m, 8H, cyclopentylidene protons); 13C NMR (125 MHz,
CDCl3)
d
: 156.4, 156.1 (C-300 isoxazoline ring), 154.5, 153.6, 151.7,
151.5 (triazolyl carbons), 153.5, 140.2e124.7, 104.2 (aromatic
carbons) 122.6, 122.5 (quaternary carbon of cyclopentylidene ring),
108.5, 108.4 (C-1), 84.7, 84.7 (C-2), 84.0, 83.9 (C-4), 81.0 (C-3), 79.6,
79.4 (C-500 isoxazoline ring), 69.3, 68.2 (C-10), 61.1, 61.1, 56.5, 56.4
(OCH3), 46.4, 46.4 (C-5), 38.6, 38.4 (thiobenzyl carbon), 37.4, 36.3
(C-400 isoxazoline ring), 35.7, 35.6, 23.6, 23.1, 23.1 (cyclopentylidene
carbons). HRMS (ESI) m/z (M þ Na) calcd for C38H41ClN4NaO8S
771.2226, found 771.2235.
4.2.4. Statistical analysis
Data was analyzed using one way analysis of variance and
Dunnett’s test. Significant difference was determined when
p ꢆ 0.05.
4.1.14. ((4R,S)-3-(3,4,5-trimethoxyphenyl)-isoxazolin-5-yl)methyl
5-deoxy-5-[(5-p-chlorophenyl-3-thiobenzyl)-1,2,4-triazol-4-yl]-
b-
D
-ribofuranoside (13)
Compound 12 (138.6 mg, 0.18 mmol) was dissolved in 80% acetic
Acknowledgments
acid and heated to 40 ꢁC for 48 h. Then, the solution was evaporated
and the residue was purified by column chromatography using
cyclohexane:acetone as eluent, giving 13 as a white solid (74.6 mg,
Research was supported by Agencia Nacional de Promoción
Científica y Tecnológica, Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET) and Universidad de Buenos Aires
(UBA), Argentina. C.A. and N.B.D. are members of Research Career
from CONICET.
0.11 mmol, 59%). 1H NMR (500 MHz, CDCl3)
d: 7.76e7.13 (m, 9H,
aromatic protons), 7.02, 7.00 (s, 1 H, trimethoxyphenyl proton), 4.81
(s,1H, H-1), 4.76e4.70 (m,1H, H-500 isoxazoline ring), 4.46e4.42 (m,
thiobenzyl protons), 4.29e4.25 (m, 1H, H-5b), 4.17, 4.13 (dd, 1H,
J5b,5a 15.0, 15.1 Hz, J5b,4 7.7, 7.8 Hz, H-5a), 3.83, 3.78 (d, 1H, J3,2 3.5,
4.5 Hz, H-3), 4.00e3.96 (m, 2H, H-2, H-4), 3.88, 3.87, 3.76, 3.76 (s,
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0
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9H, methoxy protons), 3.55, 3.48 (dd, 1H, J1 b,1 a 10.9, 10.7 Hz, J1 b,2
4.8, 4.1 Hz, H-10b), 3.42, 3.35 (dd, 1H, J1 a,1 b 11.9, 10.6 Hz, J1 a,4 3.7,
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00
00
00
00
11.0 Hz, H-400b isoxazoline ring), 3.10, 3.09 (dd, 1H, J4 a,4 b 16.9, 16.8
00
00
Hz, J4 a,5 7.7, 7.9 Hz, H-400a isoxazoline ring); 13C NMR (125 MHz,
00
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CDCl3)
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carbons), 154.5, 138.4e126.3, 105.2, 105.2 (aromatic carbons), 108.8,
108.7 (C-1), 81.5, 81.3 (C-4), 80.7, 80.5 (C-500 isoxazoline ring), 75.5
(C-3), 73.6 (C-2), 69.9, 69.5 (C-10), 60.7, 56.6 (OCH3), 49.0, 48.7 (C-5),
38.8 (thiobenzyl carbon), 37.8, 37.5 (C-400 isoxazoline ring). HRMS
(ESI) m/z (M þ Na) calcd for C33H35ClN4NaO8S 705.1756, found
705.1770.
4.2. Pharmacological studies
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