Synthesis and preliminary biological evaluation of a compound library of triazolylcyclitols

Article abstract of DOI:10.1016/j.bmc.2013.04.084

A small library of compounds was prepared by a combination of toluene dioxygenase (TDO)-catalyzed enzymatic dihydroxylation and copper(I)-catalyzed Hüisgen cycloaddition. Some compounds were obtained by coupling an alkyne and a conduritol derivative, while more complex structures were obtained by a double Hüisgen reaction of a dialkyne and two molecules of the cyclitol. The compounds were fully characterized and subjected to preliminary biological screening.

Full text of DOI:10.1016/j.bmc.2013.04.084

Bioorganic & Medicinal Chemistry 21 (2013) 4225–4232
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and preliminary biological evaluation of a compound
library of triazolylcyclitols
Gonzalo Carrau a, Carine C. Drewes b, Ana Lúcia B. Shimada b, Ana Bertucci a, Sandra H. P. Farsky b,
Helio A. Stefani c, David Gonzalez a,
⇑
a Depto. de Química Orgánica, Facultad. de Química, Universidad de la República, UdelaR, Montevideo, Uruguay
b Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil
c Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of compounds was prepared by a combination of toluene dioxygenase (TDO)-catalyzed
enzymatic dihydroxylation and copper(I)-catalyzed Hüisgen cycloaddition. Some compounds were
obtained by coupling an alkyne and a conduritol derivative, while more complex structures were
obtained by a double Hüisgen reaction of a dialkyne and two molecules of the cyclitol. The compounds
were fully characterized and subjected to preliminary biological screening.
Received 15 February 2013
Revised 23 April 2013
Accepted 30 April 2013
Available online 14 May 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Hygromycin
Biocatalysis
Hüisgen cycloaddition
Antifungal
Immunosuppressant
1. Introduction
ingly investigated as drug candidates since the emergence of the
‘click chemistry’ concept.12–14 Moreover, amides and triazoles are
Hygromycin A (1) is a natural antibiotic amide compound iso-
lated in 1953 from Streptomyces hygroscopicus.1 It exhibits moder-
ate activity against gram-positive and gram-negative bacteria.1,2 It
shows excellent activity against Treponema hyodysenteriae, the
microorganism responsible for porcine dysentery.3 and also exhib-
its immunosuppressant activity since it inhibits the proliferation of
lymphocytes.4 The structure of hygromycin A consists of three
chemically well-defined substructures or residues, including cycli-
tol, cynnamoyl and furanose. This constitution makes it ideal for
sequential structure variation and combinatorial approaches. In
fact scientists from Pfizer have disclosed studies on several analogs
with modifications in the three subunits.5–7 Among the findings, it
is noteworthy that there is a structure of an analogue that lacks the
sugar subunit but shows the same potency as the natural antibiotic
(Fig. 1).8
bioisosteric and that has allowed the substitution of the former
group by a triazole ring in several bioactive molecules.15,16 This
precedent prompted us to envision a class of molecules of moder-
ate complexity (Fig. 1) that can relate to hygromycin A and could
be rapidly built by combining azidocyclitols and alkynes in a diver-
sity-oriented fashion.
2. Results and discussion
2.1. Synthesis
Our strategy involved the initial preparation of azidocyclitols 6
and 7. The chemoenzymatic routes to these azides (or their chlo-
rine analogues) have been previously described,9,17–19 and their
application to cyclitol synthesis was also demonstrated in a recent
review.20 trans-Hydroxyazide 6 was prepared by a short and effi-
cient sequence that delivered the desired compound in four steps
and an overall yield of 82%9 from homochiral bacterial metabolite
5, which was ultimately derived from bromobenzene (4) in a high-
yield biotransformation (Scheme 1).21,22 In a similar fashion, but
involving a double inversion sequence, cis-bromoazide 7 was pre-
pared in overall yield of 78%.17
Since 2008, our groups in Brazil and Uruguay have been jointly
involved in the synthesis of small libraries of natural product-like
compounds by combined enzymatic and transition metal cataly-
sis.9–11 These compounds typically contained two subunits with
one polar cyclitol derivative and an apolar aromatic or alkenyl res-
idue. In addition, triazole-containing structures have been increas-
Azides 6 and 7 are remarkable synthons that allow for further
functionalization by metal catalyzed coupling at the vinyl bromine,
⇑
Corresponding author. Tel.: +598 2924 4543; fax: +598 2929 1906.
E-mail address: davidg@fq.edu.uy (D. Gonzalez).
URL: http://www.lbb.fq.edu.uy (D. Gonzalez).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.084

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G. Carrau et al. / Bioorg. Med. Chem. 21 (2013) 4225–4232
Figure 1. Structure of hygromycin A (1), a known synthetic analogue (2) and the proposed structurally related structures (3).
dendrimers centered in an aromatic ring, and in 2011 Ehlers
et al.24 synthesized triaryl
-helix mimics by a similar strategy.
a
Previuosly, in 2010 the group of Fokin had reacted a dialkyne with
benzyl azide in the absence of a metal catalyst.25 In the light of
these precedents, we decided to explore the simultaneous reaction
of more complex secondary azides to build triazolylconduritol con-
jugates. Four dimers (10–13) (Scheme 2) were prepared by direct
double cycloaddition between 2 equiv of the azide and one equiv-
alent of the dialkyne. Two extra structures (14 and 15) were syn-
thesized by sequential cycloadditon of two different conduritols
with the dialkyne with intermediate isolation of the triazolylcon-
duritols 16–19 (Schemes 3 and 4).
Scheme 1. Strategy used for the synthesis of the epimeric azidocyclitols.
The reaction between azide 7 and alkyne 9 was used to find the
best reactions conditions for double or single cycloaddition. A ser-
ies of experiments were performed as depicted in Table 1. We
searched for conditions to maximize the yield and minimize the
reaction time with a minimum excess of any of the reactants.
The use of Cu(II) and sodium ascorbate in a t-BuOH/H2O (1:1) mix-
ture was a better option than Cu(I) in THF (entries a and b), and the
reaction was much faster at reflux conditions (90 °C) than at room
temperature (entries c and e). The use of microwave heating did
esterification of the hydroxyl group or modification of the azide by
reduction or cycloaddition. In this research, we explored their reac-
tion with dialkynes 8 and 9 in order to prepare up to 10 combined
structures by Hüisgen cycloaddition.
The simultaneous double Huisgen cycloaddition of a dialkyne
has recently been used to prepare complex structures. In 2008,
Wyszogrodzka and Haag23 described the preparation of glycerol
Br
O
∗
N
Br
O
N
CuSO4.5H2O (20mol %),
sodium ascorbate (40mol %)
N
O
O
OH
+
∗
Br
N3
t-BuOH:H2O (1:1), reflux
OH
∗
O
N
N
m-
p-
8
9
β-N3
α-N3
6
7
N
O
HO
2.05 Eq.
1 Eq
compounds 10-13
O
O
Br
Br
O
O
Br
Br
O
O
O
O
OH
OH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
OH
OH
10
(90%)
11
12
13
(91%)
(72%)
(93%)
Br
Br
N
N
N
N
N
N
N
N
N
N
HO
O
HO
O
O
O
HO
HO
O
O
Br
Br
O
O
Scheme 2. Double Hüisgen cycloaddition.

G. Carrau et al. / Bioorg. Med. Chem. 21 (2013) 4225–4232
4227
Br
O
O
Br
CuSO4.5H2O (20 mol %),
sodium ascorbate (40 mol %)
∗
N
O
O
N
N
∗
OH
N3
t-BuOH:H2O (1:1), reflux
OH
8 m or
9 p
1 Eq
6 or 7
compounds 16-19
0.7 Eq.
Br
Br
Br
Br
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
N
N
OH
OH
OH
OH
16 (60%)
10 (33%)
17 (52%)
12 (31%)
18 (58%)
11 (34%)
19 (55%)
13 (45%)
Scheme 3. Monomer synthesis.
Br
O
Br
N
Br
O
O
CuSO4.5H2O (10 mol%),
sodium ascorbate (20 mol%)
N
N
O
O
OH
N
O
N
N
Br
OH
N3
t-BuOH:H2O (1:1), reflux
OH
O
N
N
N
N
O
HO
16 or 17
7
14 or 15
1.2 Eq.
1 Eq
Br
Br
O
O
O
O
N
N
N
N
N
OH
OH
14
(86%)
15
(63%)
Br
OH
O
N
N
N
N
N
N
O
HO
O
O
Br
Scheme 4. Mixed-dimer synthesis.
Table 1
Optimal conditions for double or single cycloaddition
Entry
Catalyst (solvent)
Molar ratio
Temp (°C)
Reaction time (h:min)
Product (yield)
(azide/alkyne)
a
b
c
CuI (1 equiv), (THF)
2.05/1
2.05/1
2.05/1
2.05/1
25–66
25
25
72
19
72
1:45
19 (10%), degradation products
19 (56%), 13 (41%)
19 (47%), 13 (22%)
13 (85%)
CuSO4, sodium ascorbate (t-BuOH/H2O/1:1)
CuSO, sodium ascorbate (t-BuOH/H2O/1:1)
CuSO4, sodium ascorbate (t-BuOH/H2O/1:1)
d
MW oven: 300 at 80 °C 300 at
100 °C 450 at 110 °C
Oil bath/reflux
e
f
g
CuSO4, sodium ascorbate (t-BuOH/H2O/1:1)
CuSO4, sodium ascorbate (t-BuOH/H2O/1:1)
CuSO4, sodium ascorbate (t-BuOH/H2O/1:1)
2.05/1
0.71/1
0.71/1
1:40
0:45
72
19 (4%), 13 (93%)
19 (55%), 13 (45%)
19 (55%), 13 (45%)
Oil bath/reflux
25
not result in an improvement (entry d) compared to traditional
heating. Just a stoichiometric ratio of 2:1 was enough to obtain
double cycloaddition in 93% yield and with only 4% of the product
of mono cycloaddition. In fact the formation of the dimer was very
favorable. We were also interested in obtaining the products of sin-
gle cycloaddition (16–19), but even with a 40% excess of the dial-
kyne (entries f and g) double cycloaddition was unavoidable.
Using a larger excess of the alkyne was impractical, but fortunately
the chromatographic separation of monomers and dimers was
straightforward.

4228
G. Carrau et al. / Bioorg. Med. Chem. 21 (2013) 4225–4232
Table 2
Final deprotection results
otic hygromycin A. The strategy is simple and allows for further
diversification by choosing different cyclitols and dialkynes.
Entry
Starting material
Deprotection method
Product
Yield (%)
2.2. Biological evaluation
a
b
c
d
e
f
g
h
i
16
17
18
19
10
12
11
13
14
15
Dowex resin
Dowex resin
Dowex resin
Dowex resin
Dowex resin
p-TsOH
Dowex resin
Dowex resin
Dowex resin
Dowex resin
20
21
22
23
24
25
26
27
28
29
79
52
57
86
100
57
65
46
79
66
In order to perform a preliminary evaluation of the compound
library, we studied the compounds in three different experiments
to search for antifungal and immunosuppressant activity. The for-
mer was evaluated in a bioautographic agar overlay test that pro-
vides qualitative information about in vitro antifungal activity with
a very low detection limit.26 Immunosuppressant activity was
studied in two separate experiments: cell viability by an MTT assay
j
and
antiproliferative
activity
on
concanavalin-activated
lymphocytes.
2.3. Antifungal assay
The antifungal screening was performed on seven ATCC classi-
fied yeast and filamentous fungi strains: Candida albicans (Ca) ATCC
90022, Candida parapsilosis (Cp) ATCC 22919, Candida glabrata (Cg)
ATCC 15126, Cryptococcus neoformans (Cn) ATCC 14116, Trichophy-
ton rubrum (Tr) ATCC 28188, Trichophyton mentagrophytes (Tm)
ATCC 9533 and Microsporum gypseum (Mg) ATCC 24102. The assay
was performed using an in-house modification of the agar overlay
method described by Rahalison et al.27 In all cases negative con-
trols (DMSO 1%) and antifungal controls (amphotericin B and keto-
conazol) were including in the same plate.
Table 3 shows that five of the tested compounds exhibited
selective qualitative activity in this screening. Only one of the stud-
ied strains (C. parapsilosis ATCC 22919) was sensitive to the pres-
ence of more than one compound. Growth of this yeast strain
was inhibited by two monomeric compounds (22 and 23) and
one dimeric structure (27). Two other dimers were also active.
Trans dimer 25 inhibited the growth of the skin fungus T. rubrum
and M. gypseum, while the mixed dimer 28 was active against
the yeast strain C. glabrata and the fungus strain of T. mentagro-
phytes. Although this is a qualitative assay, it seems that cis struc-
Scheme 5. Deprotection of the alcohols.
With the monomers in hand, we were able to prepare com-
pounds with different cyclitols at the extremes. This was per-
formed by mixing the compounds 16 or 17 with azidoconduritol
7 under the optimal reaction conditions determined previously.
These reactions rendered mixed structures 14 and 15 with 86%
and 57% yields, respectively (Scheme 4).
For biological evaluation purposes, we were interested in test-
ing the free alcohols rather than the acetonides. To obtain these
compounds named 20–29 we proceeded to full deprotection of
the ten compounds. For nine derivatives, this was achieved in var-
ied yield by boiling the compounds for 1–5 h in a 10:1 methanol/
water mixture in the presence of Dowex 50WX2-100 resin in the
acidic form (Table 2). After the reaction was completed, the resin
was separated by filtration and the final compounds recovered
from the solution. The ‘para’ hexahydroxy derivatives were less
soluble in methanol than the ‘meta’ analogs. Compound 25 in par-
ticular was insoluble in all common volatile solvents and, thus, was
impossible to separate from the resin after deprotection. Alterna-
tively, this compound was obtained using p-toluenesulfonic acid
instead of the resin (entry f). Since the product precipitated out
of the solution after the completed reaction, it was recovered by fil-
tration and washed with methanol to remove traces of the acid
(Scheme 5).
tures and dimeric compounds present
a stronger potential
compared to monomeric and trans molecules.
2.4. Immunosuppressant study
Cell viability was monitored by the MTT assay.28 This assay,
which measures the viable cells based on their mitochondrial
dehydrogenase activity where MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] will be re-
duced to formazan. The tested compounds (200 nM) did not alter
mouse splenic lymphocyte viability when incubated at 37 °C for
72 h. On the other hand, treatment with 10% DMSO added as a po-
sitive control experiment and incubated under the same conditions
induced toxicity to lymphocytes (Fig. 2).
In summary, by this approach we were able obtain ten solid
compounds of moderately complex structure related to the antibi-
Table 3
Results of the antifungal screening
Strain
Monomers
21
Dimers
20
22
23
24
25
26
27
28
29
trans
trans
cis
cis
trans/trans
trans/trans
cis/cis
cis/cis
cis/trans
cis/trans
Ca
Cp
Cg
Cn
Tr
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
+
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Tm
Mg
ꢀ
+
ꢀ

G. Carrau et al. / Bioorg. Med. Chem. 21 (2013) 4225–4232
4229
120
100
80
60
40
20
0
Vehicle DMSO
20
21
22
23
24
25
26
27
28
29
Compounds (200 nM)
Figure 2. Effect of the library on mouse splenic lymphocyte viability. The results are expressed as mean SEM of cells of three animals in triplicate.
0.25
0.20
0.15
0.10
0.05
0.00
Vehicle ConA
20
21
22
23
24
25
26
27
28
29
Compound (200 nM)
Figure 3. Effects of the library on mouse splenic lymphocyte proliferation. The results are expressed as mean SEM of cells of three animals in triplicate.
2.5. Cell proliferation study
allowed combining monomeric Hüisgen products to build mixed
dimers with increased molecular complexity.
Concanavalin A induces mouse cell proliferation. Active com-
pounds will significantly decrease cell proliferation and eventually
exhibit a dose–response relationship. All compounds tested were
treated for 1 h and incubated in the presence of concanavalin A.
The results were compared with an experiment containing only
the promoter and inactive vehicle. As observed in Figure 3, for all
compounds tested cell proliferation was comparable to the control
experiment. In fact, for concentrations above 0.002 nM, prolifera-
tion was up to 30% more for the tested compounds compared to
the control. In conclusion, the library did not exhibit antiprolifera-
tive activity against mouse splenic lymphocytes in this test.
Compounds were biologically evaluated and demonstrated
promising antifungal activity but lacked the immunosuppressant
action described for hygromycin A. Our future goal is to continue
elaborating on the basis of this strategy in order to build other
triazolyl-inositol compounds and hygromycin A analogs.
4. Experimental section
4.1. General methods
All non-hydrolytic reactions were carried out under a nitrogen
atmosphere with standard techniques for the exclusion of moisture.
The commercially available reagents were purchased from
Sigma–Aldrich and used without further purification. Melting
points were determined either on a capilar DPX 300 Bruker Advance
apparatus or on a Bruker Avance DPX-400, and are uncorrected.
Infrared spectra (IR) were recorded on a Shimadzu FT-IR model
IRprestige-21. High-resolution mass spectra were performed on a
Waters Xevo G2 QToF (ESI+mode). Nuclear magnetic resonance
spectra were recorded either on a DPX 300 Bruker Advance or on
3. Conclusions
A small library of compounds inspired in the natural bioactive
compound hygromycin A was constructed following an efficient
chemoenzymatic strategy. A double Hüisgen cycloaddition allowed
for rapid complexity building and resulted in isomeric compounds
of molecular weight 624 kDa and eight stereocenters synthesized
in six steps from achiral starting materials. A related strategy

4230
G. Carrau et al. / Bioorg. Med. Chem. 21 (2013) 4225–4232
00 00
a Bruker Avance DPX-400 instrument. Chemical shifts (d) are
given in parts per million downfield from tetramethylsilane, and
coupling constants (J) are reported in Hertz. Optical rotation
values were determined on a Kruss Optronic GmbH P8000 on a
0.5 dm cell (concentration is expressed as W/V percentage).
Analytical TLC was performed on silica gel 60F-254 plates and
visualized with UV light (254 nm) and/or anisaldehyde–H2SO4–
AcOH as detecting agent. Purifications by column chromatography
were performed on Merck 60 silica gel (0.040–0.063 mm).
J = 8.4 Hz, 2H, H3 ,5 ), 6.06 (s, 1H, H3), 5.69 (d, J = 5.7 Hz, 1H, OH1),
5.55–5.49 (m, 1H, H2), 4.97 (s, 1H, H1), 4.82 (dd, J = 4.7, 1.8 Hz,
1H, H5), 4.65 (t, J = 4.7 Hz, 1H, H6), 3.15 (s, 1H, CC-H), 1.49 (s, 6H,
13
0
CH3).
C NMR (75 MHz, CDCl3) d (ppm): 146.3 (C4 ), 132.7
(C2 ,6 ), 129.5 (Cquaternary arom), 128.1 (Cquaternary arom), 124.6
(C3 ,5 ), 124.2 (C3), 122.1 (C4), 120.5 (C5 ), 110.8 (Cisopropylidene),
83.4 (Ar-CC-H), 78.3 (Ar-CC-H), 76.6 (C6), 75.9 (C5), 68.3 (C1), 60.3
00 00
00 00
0
(C2), 27.9 (CH3), 26.6 (CH3). FT-IR (mmax/cm): 3400.0–3000.0 (OH),
3298.3 (CC-H), 2985.1 (CH3), 2931.8 (CH3), 2891.1 (CH), 1654.9
(C@C), 1234.4 (C–N), 1084.0 (C–O–C–O–C), 1057.0 (C–O–C–O–C).
ESI-MS: Calcd for [C19H18BrN3O3+H]+: 416.0610; found: 416.0611
(Error: 0.2 ppm).
4.2. General procedure for double Hüisgen cycloaddition
(compounds 10–13)
To a mixture of azide 6 or 7 (0.24 mmol, 2.05 equiv) and the cor-
responding diethynylbenzene (m or p, 0.12 mol, 1 equiv) in t-BuOH
(1.5 mL), a solution of sodium ascorbate (0.048 mmol, 40 mol %)
and CuSO4 (0.024 mmol, 20 mol %) in H2O (miliQ, 1.5 mL) was
added. The resulting system was heated at reflux temperature until
total consumption of the alkyne and the single cycloaddition prod-
ucts. Then NH4Cl was added and the mixture was extracted with
five portions of AcOEt, dried over MgSO4, filtered and concentrated
under reduced pressure. Purification was accomplished by flash
chromatography with a gradient of AcOEt/hexanes mixtures.
4.6. General procedure of cycloaddition for the synthesis of
heterodimers 14 and 15
To a mixture of azide 6 or 7 (0.17 mmol, 1.2 equiv) and the cor-
responding alkyne (16–19) 0.14 mmol, 1 equiv) in t-BuOH
(1.5 mL), a solution of sodium ascorbate (0.029 mmol, 20 mol %)
and CuSO4 (0.014 mmol, 10 mol %) in H2O (miliQ, 1.5 mL) was
added. The resulting system was heated at reflux temperature until
total consumption of the alkyne. Then NH4Cl was added and the
mixture was extracted with five portions of AcOEt, dried over
MgSO4, filtered and concentrated under reduced pressure. Purifica-
tion was accomplished by flash chromatography. A gradient elu-
tion was performed, starting with a solution of AcOEt/Hexanes
4:6, and then the polarity of the solution is increased until finally
pure AcOEt was used.
4.3. (100R,200R,500S,600S)-1,3-Bis-(10-(400-bromo-500,600-isopropyliden-
edioxy-cyclohexa-300-ene-100-ol-200-yl)-10-H-[10,20,30]-triazol-40-yl)-
benzene (11)
White solid (91%). Mp = 269.3–270.1 °C (decomposition). ½a D22
ꢁ
+22.2 (c 1.70, DMSO). 1H NMR (300 MHz, CDCl3) d (ppm): 7.90 (s,
4.7. (100R,1IVR,200R,2IVS,500S,5IVS,600S,6IVS)-1-(10-(400-bromo-500,600-
isopropylidenedioxy-cyclohexa-300-ene-100-ol-200-yl)-10-H-
0
2H, H5 ), 7.42 (s, 1H, H2), 7.20 (d, J = 7.7 Hz, 2H, H4,6), 7.07 (t,
J = 7.7 Hz, 1H, H5), 6.20 (d, J = 4.7 Hz, 2H, OH1 ), 6.16 (s, 2H, H3 ),
[10,20,30]-triazol-40-yl)-3-(1000-(4IV-bromo-5IV,6IV
-
00
00
5.58 (s, 2H, H2 ), 5.17 (s, 2H, H1 ), 4.88 (d, J = 3.1 Hz, 2H, H5 ),
isopropylidenedioxy-cyclohexa-3IV-ene-1IV-ol-2IV-yl)-1000-H-
[1000,2000,3000]-triazol-4000-yl)-benzene (14)
00
00
00
4.70 (t, J = 4.3 Hz, 2H, H6 ), 1.52 (s, 12H, CH3). 13C NMR (75 MHz,
00
0
00
CDCl3) d (ppm): 146.2 (C4 ), 129.6 (C1,3), 129.3 (C5), 128.6 (C4 ),
00
0
124.5 (C4,5), 124.1 (C3 ), 121.4 (C2), 120.7 (C5 ), 110.7 (Cisopropyli-
White solid (86%). Mp = 176.1–177.8 °C (decomposition). ½a D22
ꢀ1.4 (c 1.40, DMSO). 1H NMR (300 MHz, MeOD) d (ppm): 8.44 (s,
ꢁ
00
00
00
00
dene), 77.4 (C6 ), 75.7 (C5 ), 68.1 (C1 ), 60.4 (C2 ), 27.9 (CH3), 26.7
(CH3). FT-IR ( max/cm): 3300.0–3000.0 (OH), 2982.0 (CH3), 2931.8
(CH3), 2897.1 (CH), 1647.2 (C@C), 1238.3 (C–N), 1084.0 (C–O–C–
O–C), 1057.0 (C–O–C–O–C). ESI-MS: Calcd for [C28H30Br2N6O6+H]+:
705.0672; found: 705.0667 (Error: ꢀ0.7 ppm).
0
000
m
2H, H5 ,5 ), 8.25 (s, 1H, H2), 7.84 (t, J = 7.9 Hz, 2H, H4,6), 7.53 (t,
J = 7.9 Hz, 1H, H5), 6.48 (d, J = 2.2 Hz, 1H, H3IV), 6.43 (d, J = 1.6 Hz,
00
00
1H, H3 ), 5.63 (dd, J = 4.8, 2.8 Hz, 1H, H2 ), 5.23 (d, J = 8.7 Hz, 1H,
00
H2IV), 4.96 (d, J = 6.3 Hz, 1H, H5IV), 4.86–4.82 (m, 1H, H5 ), 4.63–
00 00
4.53 (m, 2H, H1 ,6 ), 4.44 (dd, J = 8.5, 6.3 Hz, 1H, H6IV), 4.24–4.11
4.4. General procedure of cycloaddition for the synthesis of
triazoles (compounds 16–19)
(m, 1H, H1IV), 1.62 (s, 3H, CH3), 1.55 (s, 3H, CH3), 1.52 (s, 3H,
CH3), 1.51 (s, 3H, CH3). 13C NMR (75 MHz, MeOD) d (ppm): 148.3
0
000
0
000
(C4 o C4 ), 148.2 (C4 o C4 ), 132.4 (C1 o C3), 132.2 (C3IV), 131.3
00
00
To a mixture of azide 6 or 7 (1.72 mmol, 1 equiv) and the corre-
sponding diethynylbenzene (m or p, 2.41 mmol, 1.4 equiv) in t-
(C1 o C3), 130.6 (C5), 127.5 (C4 ), 126.8 (C3 ), 126.3 (C4 o C6),
0
000
126.3 (C4 o C6), 123.9 (C2), 123.1 (C4IV), 122.8 (C5 o C5 ), 122.7
0
000
BuOH (10 mL),
a
solution of sodium ascorbate (0.69 mmol,
(C5 o C5 ), 111.9 (Cisopropylidene), 111.6 (Cisopropylidene), 79.4 (C6IV),
00
00
00
40 mol %) and CuSO4 (0.35 mmol, 20 mol %) in H2O (miliQ,
10 mL) was added. The resulting system was heated at reflux tem-
perature until total consumption of the azide. Then NH4Cl was
added and the mixture was extracted with five portions of AcOEt,
dried over MgSO4, filtered and concentrated under reduced pres-
sure. Purification was accomplished by flash chromatography.
The single cycloaddition product was eluted with a solution of
AcOEt/Hexanes 3:7, and then the double cycloaddition product
was eluted after a quick gradient, with a solution of AcOEt/Hexanes
8:2.
78.6 (C5IV), 78.1 (C6 ), 77.4 (C5 ), 72.8 (C1IV), 69.7 (C1 ), 64.6 (C2IV),
00
61.1 (C2 ), 28.4 (CH3), 28.0 (CH3), 26.6 (CH3), 26.1 (CH3). FT-IR
(mmax/cm): 3600.0–3000.0 (OH), 2985.8 (CH3), 2935.7 (CH3),
1651.1 (C@C), 1222.9 (C–N), 1076.3 (C–O–C–O–C), 1053.0 (C–O–
C–O–C). ESI-MS: Calcd for [C28H30Br2N6O6+H]+: 705.0672; found:
705.0667 (Error: ꢀ0.7 ppm).
4.8. General procedure for the deprotection of acetonides and
diacetonides
To a solution of the corresponding acetonide (65 mg) in 3.3 mL
of MeOH/H2O (10:1), DOWEX-H+ 50X2-100 resin (650 mg) was
added. The resulting system was heated at reflux temperature until
total consumption of the acetonide. Then the resin was filtered and
washed sequentially with MeOH, NH4OH(ac), MeOH and AcOEt.
The solution was concentrated under reduced pressure, and the
product was purified by flash chromatography with a gradient of
AcOEt/MeOH mixtures.
4.5. (1R,2R,5S,6S)-4-Bromo-5,6-isopropylidenedioxy-2-(40-(400-
ethynylbenzene-100-yl)-10-H-[10,20,30]-triazol-10-yl)-cyclohexa-3-
ene-1-ol (19)
White solid. Mp = 188.7–189.5 °C. Yield (7p) = 55%;
R
(7p7) = 45%. ½a 2D2
ꢁ
+ 4.6 (c 1.26, AcOEt). 1H NMR (300 MHz, CDCl3)
0
00 00
d (ppm): 7.70 (s, 1H, H5 ), 7.33 (d, J = 8.3 Hz, 2H, H2 ,6 ), 7.24 (d,

G. Carrau et al. / Bioorg. Med. Chem. 21 (2013) 4225–4232
4231
4.9. (100S,200S,300R,400S)-1,3-Bis-(10-(600-bromo-cyclohexa-500-ene-
7.2) and washed (400ꢂg) in incomplete RPMI media. The cells were
resuspended in complete RPMI media containing 10% FBS (Sigma),
and the numbers and viability assessed by the trypan blue exclu-
sion method.
100,200,300-triol-400-yl)-10-H-[10,20,30]-triazol-40-yl)-benzene (24)
White solid (100%). Mp = 70.1–75.7 °C (decomposition). ½a D22
ꢀ24.7 (c 1.17, MeOH). 1H MNR (300 MHz, MeOD) d (ppm): 8.49
ꢁ
0
(s, 2H, H5 ), 8.31 (d, J = 1.5 Hz, 1H, H2), 7.86 (dd, J = 7.7, 1.7 Hz,
4.12. Cell proliferation study
00
2H, H4,6), 7.55 (t, J = 7.8 Hz, 1H, H5), 6.30 (d, J = 2.5 Hz, 2H, H5 ),
5.19 (dd, J = 8.4, 2.4 Hz, 2H, H4 ), 4.47 (d, J = 4.0 Hz, 2H, H1 ), 4.22
A suspension containing 1 ꢂ 106 cells/mL was prepared and
00
00
00
00
(dd, J = 10.3, 8.5 Hz, 2H, H3 ), 3.88 (dd, J = 10.3, 4.2 Hz, 2H, H2 ).
100 lL were added to wells on the culture plate, which was kept
13
0
C NMR (75 MHz, MeOD) d (ppm): 148.5 (C4 ), 132.4 (C1,3), 130.7
at 37 °C and 5% CO2. The treatments were added at concentrations
of 0.002–200 nM for 1 h. In addition, the cells were incubated with
00
00
0
(C5), 129.5 (C5 ), 127.6 (C6 ), 126.4 (C4,6), 123.9 (C2), 122.2 (C5 ),
00
00
00
00
74.6 (C1 ), 72.95 (C2 ), 71.3 (C3 ), 66.5 (C4 ). FT-IR (
m
max/cm):
or without concanavalin A (0.5
lg/mL). After 48 h of incubation,
3600.0–3000.0 (OH), 2920.2 (CH), 1658.8 (C@C), 1230.6 (C–N),
1095.6 (C–N). ESI-MS: Calcd for [C22H22Br2N6O6+H]+: 625.0046;
found: 625.0046 (Error: 0.0 ppm)
Full experimental data for all the compounds prepared is avail-
able in the Supplementary data.
25 l of 0.01% resazurin was added to all wells. The resazurin is a
l
blue compound, non-toxic, permeable and non-fluorescent using
reduction reactions from metabolically active cells to convert res-
azurin into a molecule called red fluorescent resofurin. The amount
of fluorescence and also the variation in coloration produced dur-
ing the assay is proportional to the number of living cells.
Twenty-four hours after the addition of resazurin, the optical den-
sity (OD) of each well was measured at 570 and 600 nm, and pro-
liferation was assessed by subtracting the values obtained from the
OD of the two readings.
4.10. Bioautography (antifungal assay)
Samples of the compounds, at a concentration of 10 mg/mL
were spotted on silica gel plates usually used for thin layer chro-
matography, including solvent (DMSO 1%) as a negative control
and antifungal controls (amphotericin B and ketoconazol). Fresh
cultures of filamentous fungi and yeasts were used to prepare sus-
pensions with concentrations of 106 cells or spores/mL. Plates in-
side Petri dishes received thin films of Sabouraud agar inoculated
with the fungal suspensions at a ratio of 50:1. After the necessary
time and the best growth conditions for each fungus, zones around
antifungal compounds that did not show growth could be seen in
the case of filamentous fungi and antibiotics. In the case of yeasts,
it is necessary to first dye with a tetrazolium salt (MTT: 3-(4,5-
dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide. Unco-
lored zones show inhibition, since the color reagent recognizes
mitochondrial activity in living cells.
Acknowledgments
The authors acknowledge ANII (FCE-2011-6669), CAPES (31.24-
10-0) and FAPESP (2012/00424-2). G.C. acknowledges ANII for a
graduate student scholarship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.084.
References and notes
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4232
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