Synthesis and antitubercular evaluation of new bis-1,2,3-triazoles derived from D-mannitol

Article abstract of DOI:10.1080/07328303.2010.511749

Five new bis-1,2,3-triazole derivatives from D-mannitol, namely 2,3,4,5-tetra-O-acetyl-1,6-dideoxy-1,6-bis-(4-substituted-1H-1,2,3-triazol-1-yl) -D-mannitol (4), have been synthesized from 2,3,4,5-tetra-O-acetyl-1,6-diazido- 1,6-dideoxy-D-mannitol (3) and

Full text of DOI:10.1080/07328303.2010.511749

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Synthesis and Antitubercular Evaluation
of New Bis-1,2,3-Triazoles Derived from
D-Mannitol
Marcelle de L. Ferreira a b , Marcus V. N. de Souza a , Solange M. S.
V. Wardell a , James L. Wardell a , Thatyana R. A. Vasconcelos b ,
Vitor F. Ferreira a b & Maria C. S. Lourenço c
a Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos-Far
Manguinhos , Fundação Oswaldo Cruz , 21041-250, Rio de Janeiro,
RJ, Brazil
b Universidade Federal Fluminense, Instituto de Química,
Departamento de Química Orgânica, Outeiro de S. João Batista s/n°
Centro , 24020-150, Niterói, RJ, Brazil
c Fundação Oswaldo Cruz, Instituto de Pesquisas Clínicas Evandro
Chagas, Departamento de Bacteriologia, Av. , Brasil, 4365,
Manguinhos, Rio de Janeiro, RJ, Brazil
Published online: 29 Sep 2010.
To cite this article: Marcelle de L. Ferreira , Marcus V. N. de Souza , Solange M. S. V. Wardell ,
James L. Wardell , Thatyana R. A. Vasconcelos , Vitor F. Ferreira & Maria C. S. Lourenço (2010)
Synthesis and Antitubercular Evaluation of New Bis-1,2,3-Triazoles Derived from D-Mannitol, Journal
of Carbohydrate Chemistry, 29:6, 265-274, DOI: 10.1080/07328303.2010.511749
To link to this article: http://dx.doi.org/10.1080/07328303.2010.511749
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Journal of Carbohydrate Chemistry, 29:265–274, 2010
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2010.511749
Synthesis and Antitubercular
Evaluation of New
Bis-1,2,3-Triazoles Derived
from D-Mannitol
Marcelle de L. Ferreira,1,2 Marcus V. N. de Souza,1
Solange M. S. V. Wardell,1 James L. Wardell,1
Thatyana R. A. Vasconcelos,2 Vitor F. Ferreira,2
and Maria C. S. Lourenc¸ o3
1Fundac¸a˜o Oswaldo Cruz, Instituto de Tecnologia em Fa´rmacos-Far Manguinhos,
Fundac¸a˜o Oswaldo Cruz, 21041-250, Rio de Janeiro, RJ, Brazil
2Universidade Federal Fluminense, Instituto de Qu´ımica, Departamento de Qu´ımica
Orgaˆnica, Outeiro de S. Joa˜o Batista s/n◦ Centro 24020-150, Nitero´i, RJ, Brazil
3Fundac¸a˜o Oswaldo Cruz, Instituto de Pesquisas Cl´ınicas Evandro Chagas,
Departamento de Bacteriologia, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ,
Brazil
Five new bis-1,2,3-triazole derivatives from D-mannitol, namely 2,3,4,5-tetra-
O-acetyl-1,6-dideoxy-1,6-bis-(4-substituted-1H-1,2,3-triazol-1-yl)-D-mannitol (4), have
been synthesized from 2,3,4,5-tetra-O-acetyl-1,6-diazido-1,6-dideoxy-D-mannitol (3)
and alkynes, employing copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC)
methodology. Evaluation of their in vitro antitubercular activity against Mycobacterium
tuberculosis H37Rv using the Alamar Blue susceptibility test indicated poor activities.
However, this study has provided information about the SAR of D-mannitol derivatives
in the search for new anti-TB drugs based on this carbohydrate.
Keywords D-mannitol; 1,2,3-Triazole; Antitubercular activity; Drugs.
INTRODUCTION
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tu-
berculosis that infects more than 2 billion people, equal to one-third of the
Received November 29, 2009; accepted July 27, 2010.
Address correspondence to Vitor F. Ferreira, Universidade Federal Fluminense, Insti-
tuto de Qu´ımica, Departamento de Qu´ımica Orgaˆnica, Outeiro de S. Joa˜o Batista s/n◦
Centro 24020-150, Nitero´i, RJ, Brazil. E-mail: cegvito@vm.uff.br
265

M. de L. Ferreira et al.
266
world’s population. According to the World Health Organization (WHO), 1.77
million people died from TB in 2007. Because of the spread of HIV infection
and the advent of resistant strains of TB bacilli, the global scene is even
more alarming. For instance, there were an estimated 511,000 new multidrug-
resistant (MDR) TB cases (strains that are resistant to first-line drugs) in
2007. The spread of MDR-TB can cost between 100 and 1,400 times the cost
of standard treatment, and its mismanagement led to the appearance of an-
other resistant strain called extensively drug-resistant TB (XDR-TB), which
occurs when there is resistance to first- and second-line drugs. The increase
of XDR-TB cases can restrict the treatment options and consequently further
threatens to make TB incurable.[1] Due to the seriousness of this situation, we
urgently need to develop new drugs to treat MDR-TB and XDR-TB. In this
context, 1,2,3-triazoles are a significant class of heterocyclic compounds, which
show a wide range of pharmacological activities, such as trypanocidal,[2,3] anti-
HIV,[4] antiplatelet,[5] anticonvulsant,[6] and antimicrobial.[7,8]
After investigating amino-alcohols and Schiff bases derived from D-
mannitol as possible antitubercular agents (Sch. 1),[9,10] we investigated sym-
metric bis-1,2,3-triazoles from D-mannitol because of the promising anti-TB
activity exhibited by this heterocyclic nucleus.[11,12] Based on our previous work
and the vital importance of lipophilicity for the biological activity of anti-TB
compounds, we proposed another important modification in the D-mannitol
core, that is, the acetylation of the hydroxyl groups. This modification will in-
crease the lipophilicity of the derivatives, which could facilitate their entry into
the intracellular environment. We now report the synthesis and evaluation
of the antitubercular activity of 2,3,4,5-tetra-O-acetyl-1,6-dideoxy-1,6-bis-(4-
substituted-1H-1,2,3-triazol-1-yl)-D-mannitol derivatives (4a–e), formed using
copper (I)-catalyzed azide-alkyne cycloaddition methodology (CuAAC), known
as “click chemistry.”
RESULTS AND DISCUSSION
Synthesis of the Key Intermediate 2,3,4,5-Tetra-O-acetyl-1,6-
diazido-1,6-dideoxy-D-mannitol (3)
The synthesis of 1,6-diazido-1,6-dideoxy-D-mannitol 2 was obtained in five
steps in 50% overall yield, as we previously described.[10] Next, the key inter-
mediate 3 was prepared by acetylation of 2, using acetic anhydride and pyri-
dine (95%)[13] in 48% overall yield, as shown in Scheme 2. Characterization of
1
3 was achieved from spectroscopic data. H and 13C NMR data were identical
to those described.[13]

Synthesis of New Bis-1,2,3-triazoles Derived from D-mannitol
267
OH OH
O
O
O
O
HO
OH
HO
OH
OTs
O
O
OH OH
TsO
D
-mannitol
R2NH
OH OH
NH2
O
O
H2N
OH OH
HO
OH
R
O
H
R
N
N
R
R
µg/mL
R= n-Bu: MIC= 50.0
R= t-Bu: MIC= Resistant
NO2
OH OH
NO2
N
N
OH OH
R
N
O2N
OH OH
R
N
R
R
µg/mL
µg/mL
µg/mL
o-NO2 : MIC= 12.5
m-NO2 : MIC= 25.0
p-NO2 : MIC= 25.0
OH OH
R= -Bu
n
: MIC= Resistant
R= t-Bu: MIC= Resistant
Scheme 1: Amino-alcohols and Schiff bases derived from D-mannitol previously synthesized
by our research group.
OH OH
OH OH
OAc OAc
HO
a
N3
N3
OH
N3
N3
[13]
[10]
OH OH
OH OH
OAc OAc
D-mannitol
2
3
Scheme 2: Reagents and conditions: (a) Ac2O, Py, rt, 20 h, 95%.

M. de L. Ferreira et al.
268
N3
N
OAc
OAc
N
R / Ar
AcO
AcO
N
OAc
OAc
a
N
6
N
4´
2
4
1
3
5
N
5´
4´
OAc
OAc
5´
Ar / R
N3
4a-e
3
Scheme 3: Reagents and conditions: (a) CH2Cl2/H2O, CuSO4.5H2O, sodium ascorbate,
different alkynes, 72%–91%.
Synthesis of 2,3,4,5-Tetra-O-acetyl-1,6-dideoxy-1,6-bis-(4-
substituted-1H-1,2,3-triazol-1-yl)-D-mannitol Derivatives
(4a–e)
The 2,3,4,5-tetra-O-acetyl-1,6-dideoxy-1,6-bis-(4-substituted-1H-1,2,3-triazol-
1-yl)-D-mannitol derivatives (4a–e) were obtained by coupling 3 with differ-
ent alkynes, employing the CuAAC methodology (Sch. 3 and Table 1). This
methodology has several advantages over older reactions, in that it is more
rapid (mainly if we compare alkynes that are poor 1,3-dipolar acceptors), is
highly efficient, can be performed in various solvents (including water), and is
a regioselective process, since copper(I) binds to terminal alkynes to produce
the 1,4-adduct exclusively.[14,15]
The selection of the substituents in the 1,2,3-triazole nucleus was chosen
with the purpose of analyzing the influence of these substituents on the biologi-
cal activity. Because of this, we synthesized 1,2,3-triazoles from alkynes having
(1) an aromatic apolar group, for example, 4a; (2) polar groups, for example, 4b
and 4d; and (3) apolar groups, for example, 4c and 4e.
All these compounds were identified by spectroscopic means. In general,
1
the H NMR spectra occur with the 1,2,3-triazole protons ( N CH C ) as
singlets in the range of 7.70 to 8.55 ppm, while the C C signals in the 13C
Table 1: Identification and yields of the 1,2,3-triazole derivatives (4a–e)
Entry
Substituents
Yield (%)
4a
4b
4c
4d
4e
Ar = phenyl
85
82
91
72
78
R = 1-hydroxy-cyclohexyl
R = 1-cyclohexen-1-yl
R = 2-hydroxy-4-methylpentan-2-yl
R = hexyl

Synthesis of New Bis-1,2,3-triazoles Derived from D-mannitol
269
Table 2: In vitro activity of compounds 2, 3, and 4a–e against M. tuberculosis H37Rv
strain (ATCC 27294, susceptible to both rifampin and isoniazid)
Entry
Substituents
MIC (µg/mL)a
clogPb
2
-------------
-------------
Ar = phenyl
>100.0c
>100.0
>100.0
>100.0
>100.0
>100.0
>100.0
3.12
–0.43
2.08
3.10
2.86
3.54
2.61
5.49
–0.71
3
4a
4b
R = 1-hydroxy-cyclohexyl
R = 1-cyclohexen-1-yl
R = 2-hydroxy-4-methylpentan-2-yl
R = hexyl
4c
4d
4e
Ethambutol
-------------
aMinimum inhibitory concentration.
bCalculated using online www.molinspiration.com site.
cThe strain is considered resistant to the tested substance.
ꢁ
NMR spectra occur between 155.6 and 146.9 ppm (C4 ) and 120.5 and 122.8
ꢁ
ppm (C5 ).
Formation of the 1,4-regioisomer in each case was confirmed by NOE NMR
analysis of the 1,2,3-triazole ( N CH C ) and NCH2 protons, which clearly
indicated their close proximity.
Antimycobacterial Activity
The antimycobacterial test of derivatives 2, 3, and 4a–e against M. tuber-
culosis ATTC 27294 was performed[16] using the micro plate Alamar Blue as-
say (MABA).[17] This methodology is nontoxic, uses a thermally stable reagent
and shows good correlation with proportional and BACTEC radiometric meth-
ods.[18] The results are shown in Table 2.
In conclusion, we synthesized five new bis-1,2,3-triazole derivatives from
D-mannitol (4a–e) prepared by CuAAC methodology (“click chemistry”). The
bis-1,2,3-triazole derivatives 2, 3, and 4a–e did not exhibit any antitubercular
activity in spite of their increased lipophilicity (see clogP values in Table 2).
Thus, lipophilicity was not so important for the modulation of the biological
activity of the D-mannitol derivatives, as we had hoped. Other molecular char-
acteristics such as the larger size and volume of these substances could be af-
fecting their interactions with the active site of their cellular target. However,
despite these results, we have obtained important information about the SAR
of D-mannitol derivatives, which will be useful in the search for new anti-TB
drugs based on this carbohydrate.

M. de L. Ferreira et al.
270
EXPERIMENTAL
General Procedures
Melting points were determined on a Buchi apparatus and are uncorrected.
Infrared spectra were recorded on a Thermo Nicolet Nexus 670 spectrometer
in KBr disks and frequencies are expressed in cm−1. Mass spectra (MS) were
carried out using a Waters model ZQ-LC/MS 2000. NMR spectra were recorded
at ambient temperature on a Bruker Avance 500 spectrometer operating at 400
MHz (1H) and 100 MHz (13C) in deuterated dimethyl sulfoxide. Chemical shifts
are reported in ppm (δ) relative to tetramethylsilane. Elemental analyses were
performed at ICSN, CNRS, Gif-sur-Yvette, France.
1,6-Diazido-1,6-dideoxy-D-mannitol (2)
Yield: 95% (white crystalline solid); mp: 93◦C (91–93◦C, ethanol)[10]
2,3,4,5-Tetra-O-acetyl-1,6-diazido-1,6-dideoxy-D-mannitol (3)
Yield: 95% (brown oil); [α]D = +32.0◦ (c = 1.00, CH2Cl2), [α]D = +33.0◦
(c = 1.00, CH2Cl2)[13]
General Procedure for the Preparation of 2,3,4,5-tetra-O-acetyl
-1,6-dideoxy-1,6-bis-(4-substituted-1H- 1,2,3-triazol-1-yl)-
D-mannitol Derivatives (4a–e)
Compound 3 (180.0 mg, 0.45 mmol) in 10 mL CH2Cl2/H2O 1:1 is reacted
with CuSO4.5H2O (5.4 mg, 0.02 mmol) and sodium ascorbate (12 mg, 0.06
mmol) and the desired alkyne (0.90 mmol). The mixture was maintained un-
der agitation at rt until the total formation of the product, as shown by thin
layer chromatography. The organic phase was extracted with dichloromethane,
dried with Na2SO4, and concentrated under reduced pressure. The residue ob-
tained was purified by column chromatography on silica gel using as eluent a
gradient mixture of hexane/ethyl acetate with increasing polarity up to 100%
ethyl acetate.
2,3,4,5-Tetra-O-acetyl-1,6-dideoxy-1,6-bis-(4-phenyl-1H-1,2,3-triazol-1-yl)-D-
mannitol (4a)
Yield: 85% (white solid); mp: 107◦C (ethanol). 1H NMR (400 MHz; DMSO-
d6); δ (ppm): 1.93 (6H, s; COCH3); 2.13 (6H, s; COCH3); 4.59 (2H, dd, J = 14.6
ꢁ
and 8.6 Hz; H1’ and H6 ); 4.77 (2H, dd, J = 14.6 and 2.8 Hz; H1 and H6); 5.29
(2H, dd, J = 8.6 and 2.8 Hz; H2 and H5); 5.37 (2H, d, J = 7.2 Hz; H3 and H4);
ꢁ
ꢁ
ꢁ
7.33 (2H, dd, 7.4 and 7.4 Hz; H9 ); 7.45 (4H, dd, 7.4 and 7.4 Hz; H8 and H11 );
7.83 (4H, d, 7.4 Hz; H7 and H10 ); 8.55 (2H,s; H5 ). 13C NMR (100 MHz, DMSO-
ꢁ
ꢁ
ꢁ
d6); δ (ppm): 20.3 (COCH3); 20.6 (COCH3); 49.7 (C1 and C6); 68.2 (C3 and C4);

Synthesis of New Bis-1,2,3-triazoles Derived from D-mannitol
271
ꢁ
ꢁ
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68.7 (C2 and C5); 122.1 (C5 ); 125.1 (C7 and C10 ); 127.9 (C9 ); 128.9 (C8 and
ꢁ
ꢁ
ꢁ
C11 ); 130.5 (C6 ); 146.4 (C4 ); 169.0 (COCH3); 169.7 (COCH3). MS/ESI: [M+23]:
627.4; [M + 1]: 605.5. Anal. Calcd for C30H32N6O8.H2O.CH2Cl2: C, 52.62; H,
5.13. Found: C, 52.20; H, 5.10.
2,3,4,5-Tetra-O-acetyl-1,6-dideoxy-1,6-bis[4-(cyclohexen-1-yl)-1H-1,2,3-triazol-
1-yl]-D-mannitol (4b)
Yield: 91% (white solid); mp: 126–127◦C (ethanol). H NMR (400 MHz;
1
ꢁ
ꢁ
DMSO-d6); δ (ppm): 1.60 (2H, m; H9 ); 1.68 (2H, m; H10 ); 1.91 (6H, s; COCH3);
ꢁ
ꢁ
2.10 (6H, s; COCH3); 2.14 (2H, m; H11 ); 2.23 (2H, m; H8 ); 4.48 (2H, dd, J =
ꢁ
14.6 and 8.4 Hz; H1’ and H6 ); 4.65 (2H, dd, J = 14.6 and 2.9 Hz; H1 and H6);
5.20 (2H, dd, J = 8.4 and 2.9 Hz; H2 and H5); 5.28 (2H, d, J = 7.2 Hz; H3
and H4); 6.39 (2H, sl; H7 ); 8.00 (2H, s; H5 ). 13C NMR (100 MHz, DMSO-d6);
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
δ (ppm): 20.3 (COCH3); 20.6 (COCH3); 21.8 (C9 ); 21.9 (C10 ); 24.6 (C11 ); 25.7
ꢁ
ꢁ
(C8 ); 49.3 (C1 and C6); 68.1 (C3 and C4); 68.7 (C2 and C5); 120.5 (C5 ); 123.5
ꢁ
ꢁ
ꢁ
(C7 ); 127.2 (C6 ); 148.0 (C4 ); 168.9 (COCH3); 169.7 (COCH3). MS/ ESI: [M+H]:
[M+23]: 635.5; [M+1]: 613.5. Anal. Calcd for C30H40N6O8: C, 58.81; H, 6.58.
Found: C, 59.14; H, 6.12.
2,3,4,5-Tetra-O-acetyl-1,6-dideoxy -1,6-bis[4-(1-hydroxy-cyclohexyl)-1H-1,2,3-
triazol-1-yl]-D-mannitol (4c)
Yield: 82% (white solid); mp: 84–86◦C (ethanol). 1H NMR (400 MHz;
ꢁ
ꢁ
ꢁ
ꢁ
DMSO-d6); δ (ppm): 1.37 (8H, m; H8 and H10 ); 1.63 (8H, m; H7 and H11 );
ꢁ
1.85 (4H, m; H9 ); 1.88 (6H, s; COCH3); 2.09 (6H, s; COCH3); 4.47 (2H, dd, J =
ꢁ
14.5 and 8.6 Hz; H1’ and H6 ); 4.65 (2H, dd, J = 14.5 and 3.0 Hz; H1 and H6);
4.85 (2H, s; OH); 5.19 (2H, dd, J = 8.6 and 3.0 Hz; H2 and H5); 5.28 (2H, d, J =
7.2 Hz; H3 and H4); 7.81 (2H,s; H5 ). 13C NMR (100 MHz, DMSO-d6); δ (ppm):
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
20.3 (COCH3); 20.6 (COCH3); 21.6 (C8 and C10 ); 25.2 (C9 ); 37.9 (C7 and C11 );
ꢁ
ꢁ
49.1 (C1 and C6); 67.9 (C6 ); 68.2 (C3 and C4); 68.7 (C2 and C5); 121.8 (C5 ); 155.6
ꢁ
(C4 ); 168.7 (COCH3); 169.7 (COCH3). MS/ESI: [M+H]: [M+23]: 671.4; [M+1]:
649.5. Anal. Calcd for C30H40N6O8.H2O: C, 54.04; H, 6.95; N, 12.61. Found: C,
53.88; H, 6.76; N, 12.33.
2,3,4,5-Tetra-O-acetyl-1,6-dideoxy-1,6-bis-[4-(2-hydroxy-4-methylpentan-2-yl)-
1H-1,2,3-triazol-1-yl]-D-mannitol (4d)
Yield: 85% (white solid); mp: 98–99◦C (ethanol). 1H NMR (400 MHz;
ꢁ
ꢁ
DMSO-d6); δ (ppm): 0.66 (6H, d, J = 6.4 Hz; H9 ); 0.80 (6H, d; J = 6.4 Hz; H11 );
ꢁ
ꢁ
1.40 (6H, s; H10 ); 1.55 (4H, m; H7 ); 1.87 (6H, s; COCH3); 2.10 (6H, s; COCH3);
ꢁ
4.48 (2H, dd, J = 14.3 and 8.5 Hz; H1’ and H6 ); 4.65 (2H, dd, J = 14.3 and 2.8
ꢁ
Hz; H1 and H6); 4.91 (2H, m, H6 ); 5.19 (2H, dd, J = 8.5 and 2.8 Hz; H2 and
13
ꢁ
H5); 5.27 (2H, d, J = 7.3 Hz; H3 and H4); 5.75 (2H, s; OH) 7.77 (2H,s; H4 ). C
ꢁ
NMR (100 MHz, DMSO-d6); δ (ppm): 20.3 (COCH3); 20.6 (COCH3); 23.7 (C9 or
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
C9 or C11 ); 24.2 (C9 or C9 or C11 ); 24.4 (C9 or C9 or C11 ); 30.0 (C10 ); 49.0 (C1

M. de L. Ferreira et al.
272
ꢁ
ꢁ
ꢁ
ꢁ
and C6); 51.4 (C7 ); 68.1 (C3 and C4); 68.8 (C2 and C5 ); 69.9 (C6 ); 121.8 (C5 );
ꢁ
155.3 (C4 ); 168.7 (COCH3); 169.7 (COCH3). MS/ESI: [M+23]: 549.2; [M+1]:
526.1. Anal. Calcd for C32H52N6O10.1.5H2O: C, 53.68; H, 7.21; N, 12.12. Found:
C, 54.03; H, 7.25; N, 12.58.
2,3,4,5-Tetra-O-acetyl-1,6-dideoxy-1,6-bis[4-(1-hexyl)-1H-1,2,3-triazol-1-yl]-D-
mannitol (4e)
Yield: 72% (white solid); mp: 92–94◦C (ethanol). 1H NMR (400 MHz;
ꢁ
ꢁ
DMSO-d6); δ (ppm): 0.84 (6H, t, J = 6.5 Hz); 1.25 (12H, m; H10/H10 ; H9/H9 ;
ꢁ
ꢁ
and H8/H8 ); 1.54 (4H, m; H7/H7 ); 1.89 (6H, s; COCH3); 2.09 (6H, s; COCH3);
ꢁ
2.57 (4H, t, J = 7.3 Hz; H6 and H6 ); 4.45 (2H, dd, J = 14.5 and 8.3 Hz; H1’ and
ꢁ
H6 ); 4.64 (2H, dd, J = 14.5 and 2.6 Hz; H1 and H6); 5.18 (2H, dd, J = 8.3 and
13
ꢁ
2.6 Hz; H2 and H5); 5.28 (2H, d, J = 7.2 Hz; H3 and H4); 7.80 (2H,s; H4 ). C
ꢁ
NMR (100 MHz, DMSO-d6); δ (ppm): 13.9 (C11 ); 20.3 (COCH3); 20.6 (COCH3);
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
22.0 (C10 ); 24.8 (C9 ); 28.0 (C8 ); 38.9 (C7 ); 31.0 (C6 ); 49.1 (C1 and C6); 68.1
ꢁ
ꢁ
(C3 and C4); 68.7 (C2 and C5); 121.5 (C5 ); 146.9 (C4 ); 168.7 (COCH3); 169.7
(COCH3). MS/ESI: [M+23]: 533.4; [M+1]: 510.2. Anal. Calcd for C30H48N6O8:
C, 58.05; H, 7.79; N, 13.54. Found: C, 57.68; H, 8.02; N, 13.97.
Antimycobacterial Activity
Briefly, 200 µL of sterile deionized water was added to all outer perimeter
wells of sterile 96 well plates (Falcon, 3072: Becton Dickinson, Lincoln Park,
NJ) to minimize evaporation of the medium in the test wells during incubation.
The 96 plates received 100 µL of the Middlebrook 7H9 broth (Difco Laborato-
ries, Detroit, MI) and a serial dilution of the compounds 2, 3, and 4a–e was
made directly on the plate. The final drug concentrations tested were 0.01 to
100.0 µL/mL. Plates were covered and sealed with parafilm and incubated at
37◦C for 5 days. After this time, 25 µL of a freshly prepared 1:1 mixture of
Alamar Blue (Accumed International, Westlake, OH) reagent and 10% Tween
80 was added to the plate and incubated for 24 h. A blue color in the well was
interpreted as no bacterial growth, and a pink color was scored as growth. The
minimal inhibition concentration was defined as the lowest drug concentra-
tion, which prevented a color change from blue to pink.
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