Synthesis of Leubethanol derivatives and evaluation against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2012.04.059

Twenty-five derivatives of the natural diterpene leubethanol, including several potential pro-drugs, with changes in the functionality of the aliphatic chain or modifications of the phenolic group, were synthesized and tested in vitro by the MABA techniqu

Full text of DOI:10.1016/j.bmc.2012.04.059

Bioorganic & Medicinal Chemistry 20 (2012) 4155–4163
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of Leubethanol derivatives and evaluation against
Mycobacterium tuberculosis
Jonathan Perez-Meseguer a, Esther del Olmo b, , Blanca Alanis-Garza a, Ricardo Escarcena b,
⇑
Elvira Garza-González a, Ricardo Salazar-Aranda a, Arturo San Feliciano b, Noemí Waksman de Torres a,
⇑
a Department of Analytical Chemistry, Faculty of Medicine, Universidad Autónoma de Nuevo Leon, PO Box 2316, Sucursal Tecnológico, Monterrey, NL 64841, Mexico
b Departamento de Química Farmacéutica, Facultad de Farmacia, CIETUS, Universidad de Salamanca, Campus Unamuno, E-37007 Salamanca, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-five derivatives of the natural diterpene leubethanol, including several potential pro-drugs, with
changes in the functionality of the aliphatic chain or modifications of the phenolic group, were synthe-
sized and tested in vitro by the MABA technique for their activity against the H37Rv strain of Mycobac-
terium tuberculosis. Several compounds showed antimycobacterial potencies similar to that of the lead
compound and two of them displayed higher selectivity indexes.
Received 27 January 2012
Revised 22 April 2012
Accepted 27 April 2012
Available online 4 May 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Leubethanol
SAR
Mycobacterium tuberculosis
MABA
Cytotoxicity
1. Introduction
Due to those facts mentioned above, new anti-TB drugs and bet-
ter therapeutic strategies against TB are urgently and permanently
Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis
(MTB), has been widespread since ancient times and remains a
worldwide major health problem. According to the latest report
of World Health Organization on tuberculosis,1 9.4 million new
cases were reported in 2009, 14 million prevalent cases, 1.3 million
deaths among HIV-negative people and 0.38 million deaths among
HIV-positive people. Most cases occurred in the Southeast Asian,
African and Western Pacific regions (35%, 30% and 20%, respec-
tively). An estimated 11–13% of incident cases were HIV-positive;
the African Region accounted for 80% of these cases approximately.
Mycobacteria are able to survive in a latent state in infected
individuals, thereby serving as a reservoir, waiting for the opportu-
nistic reactivation.2 It is estimated that there are around 2 billion
people, almost one-third of the world’s population, infected with
latent or active MTB.3 An additional contributing factor is the con-
tinued emergence of new MDR-MTB strains,4 often associated to
poor compliance to treatments and, since 2006, to the officially
recognized appearance of MTB strains with extended resistance
(XDR-MTB),5 now spread to more than 58 countries.6 Furthermore,
TB treatment protocols are prolonged up to six or more months,
complex and rather expensive, and most times end in the poor
patient compliance.
needed. The new drug candidates should shorten the standard reg-
imens, being effective enough against MDR-TB. Though several
compounds are currently in advanced phases of clinical assay, for
some 40 years no new compounds have been brought into the
market for TB treatment. However, in recent years, an emphasized
research activity for the development of new TB drugs has been
being produced. Some compounds are presently in clinical devel-
opment, while others are being investigated pre-clinically, in an at-
tempt to discover new molecules for a target-based treatment of
TB.7
Waksman and co-workers8 recently reported on the structure
assignment and antimycobacterial activity of leubethanol (1,
Fig. 1), a natural serrulatane diterpenoid isolated from the metha-
nol extract of Leucophyllum frutescens (Fam.: Scrophulariaceae).
The compound displayed a certain activity (MIC = 25.1
the H37Rv strain, sensitive to the first-line anti-TB agents, with
similar potency (MIC = 21.9 M), against the MDR-MTB strain CI-
lM) against
l
BIN/UMF15:99, a clinical isolate that had resulted resistant to all
five first-line anti-TB drugs: streptomycin, isoniazid, rifampin, eth-
ambutol, and pyrazinamide (SIREP-resistance). The present study
was intended to explore the possibility of obtaining better antimy-
cobacterial compounds through modification of the leubethanol
structure. Thus, several derivatives of leubethanol with changes
in the functionality of the side-chain along with several phenolic
ethers and esters (Fig. 1) were prepared and evaluated against MTB.
⇑
Corresponding author.
E-mail address: olmo@usal.es (E. del Olmo).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.059

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J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
20
14,15-dehydroLeub; R2= CH3
OR1
R1 = H; R2= CH3
R1
9
1
4
7
14,15-dihydroLeub ; 14,15-epoxyLeub
15-hydroxy-14,15-dihydroLeub
14-amino,15-hydroxy-14,15-dihydroLeub
2
H; CH3-; CH3(CH2)3-; CH3(CH2)5-
4-ClBn; 4-NO2Bn; 3,5-diNO2-Bn
Br(CH2)4-; Br(CH2)6-
19
CH3
10
5
15-amino,14-hydroxy-14,15-dihydroLeub
11
Leub(CH2)4-; Leub(CH2)6-
CH3CO- ; CH3(CH2)4CO-
PhCO-; 4-NO2PhCO-
18
14,15-dehydroLeub; R1 = H; R2 = CHO
14,15-dehydroLeub; R1 = H; R2 = CH2OH
14
15
R2
2-furoyl
17
leubethanol = Leub
(14,15-dehydro; R1 = H; R2= CH3
)
Figure 1. Global structure and variants of leubethanol derivatives.
and 8a,b, respectively. We finally treated leubethanol with sele-
nium dioxide to obtain the allylic alcohol 9 and the ,b-unsatu-
rated aldehyde 10.
Relating to the phenolic function, its masking through the for-
mation of ethers and esters was considered opportune taking into
account the antimycobacterial results found by Rodriguez and Ra-
mirez,10 for some natural products closely related to leubethanol.
Erogorgiane, another natural serrulatane diterpenoid without any
phenolic hydroxyl in its structure, was reported to inhibit up to
2. Chemistry
a
Aiming to investigate the influence of how the structural
changes could affect the antimycobacterial activity of leubethanol
derivatives, the two main parts of the molecule, the olefinic bond
in the side-chain and the phenolic fragment, were taken into con-
sideration. Changes at the side chain included hydrogenation,
epoxidation followed by oxirane aperture and allylic oxidation
(Scheme 1). Hydrogenation of 1 at ambient temperature and pres-
sure in the presence of Pd-C gave dihydroleubethanol 2 almost
quantitatively. The treatment of 1 with MCPBA gave the mixture
of the epimeric oxiranes 3a,b, whose reduction with lithium alumi-
num hydride led to the tertiary alcohol 4. The mixture of epoxides
3a,b was used to prepare some b-aminoalcohol derivatives, for
which, in accordance with our previous experience on lipidic ami-
noalcohols and diamines,9 good antimycobacterial activities were
expected. When the oxirane mixture 3a,b was treated with sodium
azide, the nucleophilic attack occurred at both C-14 and C-15 posi-
tions leading to the four possible hydroxyazides, which were chro-
matographed and characterized as mixtures 5a,b (14-OH,15-N3)
and 6a,b (14-N3,15-OH). These azide mixtures were reduced sepa-
rately with NaBH4/MeOH in the presence of a catalytic amount of
Pd-C, to give mixtures of the corresponding b-aminoalcohols 7a,b
96% of MTB growth at a 46.2 lM concentration, while its 7-hydro-
xyl derivative attained a 77% inhibition at a similar concentration.
These findings suggested that the 8-hydroxyl group of leubethanol
would not be an essential function for the antimycobacterial activ-
ity. Therefore, several types of ethers and esters with different
chemical-metabolic stability or with expected bioactivity signifi-
cance were considered for the blocking of the phenolic group at
C-8, also aiming to introduce some extra fragments on the leubeth-
anol structure that could promote additional interactions with the
mycobacterial target and lead to more efficacious compounds.
One group of derivatives was constituted by simple alkyl ethers
with variable chain size (methyl, n-butyl, n-hexyl), for which a con-
siderable stability was expected and by haloalkyl derivatives (4-
bromobutyl and 6-bromohexyl), which would be able to alkylate
OH
OH
OH
OH
ii
i
iii
90%
65%
99%
CH3
CH3
CH3
CH3
14
OH
O
16
leubethanol (1)
2
3
4
vi
iv
OH
OH
OH
+
CH3
CH3
CH3
R1
R2
R1
R2
R1
OH N3
N3 OH
R2
v
v
7
8
OH NH2
NH2 OH
5
6
H
OH
5, 6
10
9
O
i) H2/Pd-C, EtOH; ii) MCPBA, CHCl3; iii) LiAlH4, THF; iv) NaN3 /EtOH, 24h at 70¼C;
v) THF/Pd-C; NaBH4/MeOH; vi) SeO2/ EtOH
Scheme 1. Synthesis of leubethanol derivatives modified at the side chain.

J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
4157
irreversibly any nucleophilic function close to the target biomole-
cule in the mycobacteria. Another group of ethers were of benzylic
nature (4-chloro, 4-nitro and 3,5-dinitro), chemically and metabol-
ically less stable, whose syntheses were proposed on the basis of
an observed enhancement of the antimycobacterial activity caused
by the introduction of a benzyl group in a series of aminoalcohol
derivatives previously obtained by us.9 Furthermore, it would be
expected that the incorporation of a m-dinitrophenyl substituent
could act in two different ways. On one hand it would add to leu-
bethanol its intrinsic antimicrobial toxicity, and additionally due to
its reported character of potential antigenic response inducer, it
could also act as an immuno-killer promoting death of the MTB-in-
fected cell by attracting antibodies to it.11
Leubethanol was also treated with acetic anhydride in pyridine
to provide the acetate 11k, while other ester derivatives 11m to
11p were prepared in good yield by treatment with sodium hy-
dride followed by the appropriate acyl chloride.
3. Bioactivity
The anti-MTB activity was assessed in vitro against the H37Rv
strain (ATTC 27294),12 susceptible to all SIREP anti-TB drugs,
according to a modified Microplate Alamar Blue Assay (MABA).13
Ethambutol (EMB) was used as the reference drug and the assays
were performed by triplicate independent experiments. Cytotoxic-
ity on Vero cells14 was also measured for those compounds with an
appreciable in vitro anti-MTB effect.
The hydroxyl group was transformed into alkyl and benzyl
ethers in presence of potassium carbonate by reaction with the
appropriate iodinated/brominated alkyl or arylalkyl reagent to pro-
vide compounds 11a to 11f in medium to good yields (Scheme 2).
Interestingly the reaction of 1 with 3,5-dinitrobenzyl bromide, in
addition to the expected ether 11f, led to compound 12 in 15%
yield. The HRMS of compound 12 denoted a molecular weight of
466.3596, in agreement with the molecular formula C27H34N2O5.
Its IR spectrum showed the absorption of the free hydroxyl group
at 3400 cmꢀ1, while its 1H NMR spectrum, in addition to the aro-
matic signals associated to the m-dinitrobenzyl group, showed
only one signal in the aromatic region (d 6.54 ppm), correlating
with a methine signal at d 134.8 ppm in the 13C NMR spectrum.
The identity of compound 12 was ascertained on the light of sev-
eral 1D- and 2D- 1H/13C NMR experiments and particularly of
NOE-difference experiments. Thus, irradiation of the signal for
the benzylic protons (d 4.14 ppm) generated NOE enhancements
on the signals associated to those 20,60-aromatic protons (d
8.13 ppm) of the dinitrobenzyl group, to the methyl singlet Me-
19 (d 2.05 ppm), to the methyl doublet Me-18 (d 0.80 ppm) and
to the methine multiplet CH-11 (d 2.64 ppm). The last two effects
can only occur if the dinitrobenzyl fragment is attached to position
C-5 and would support the unexpected structure proposed for
compound 12 in Scheme 2. The treatment of 1 with sodium hy-
dride followed by reaction with 1,4-dibromobutane and 1,6-dibro-
mohexane allowed us to obtain the bromoethers 11g and 11i, in
addition to the pseudo-dimeric diethers 11h and 11j.
4. Results and discussion
Those MIC values found for the compounds represented in
Schemes 1 and 2 are shown in Tables 1 and 2, respectively. MIC
and CC50 values are expressed in
lM units, rather than in lg/mL
units, most commonly found in the literature related with this type
of evaluations, to provide more adequate comparisons of antimy-
cobacterial potencies. It is also relevant to note that MIC values
in tables refer to the total killing of the cultured mycobacteria,
whereas CC50 values refer to only 50% killing of Vero cells, thus real
SI values should actually be higher than those calculated and
shown in Tables 1 and 2.
As it can be observed in Table 1 the hydrogenation of the side-
chain double bond slightly decreased the activity and the toxicity
of compound 2, whereas the introduction of oxygen or nitrogen
containing functions in the C14–C15 fragment (compounds 3–8),
decreased the antimycobacterial activity by a factor of three times
or more. Furthermore, no improvement of cytotoxicity on Vero
cells was observed for these compounds. As can also be observed
for compounds 9 and 10, the introduction at C-16 of an allylic hy-
droxyl group or a conjugated aldehyde, failed to improve the anti-
mycobacterial/cytotoxicity properties of the natural compound
leubethanol.
Related to the results observed for leubethanol ethers 11a–11j
to and esters 11k–11p, with modifications of the phenolic hydroxyl
OR
OH
R
R
CH3-
CH3(CH2)3-
CH3(CH2)5-
11a
11b
11c
CH3CO-
CH3(CH2)4CO 11m
11k
vii) or viii)
ix) or x)
CH3
CH3
11n
11o
11p
CO-
11d
11e
Cl
CH2-
CH2-
O2N
O
CO-
O2N
O2N
CO-
leubethanol (1)
11
CH2-
11f
vii) K2CO3/ R-X; viii) NaH /Br-(CH2)4/6-Br
ix) Ac2O/Pyr; x) NaH/ R-COCl
O2N
Br(CH2)4-
11g
11h
11i
LeubO(CH2)4-
Br(CH2)6-
OH
1
LeubO(CH2)6-
11j
19
CH3
4
11
5
NO2
18
NO2
12
Scheme 2. Synthesis of ethers, esters and pseudo-dimeric leubethanol derivatives.

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J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
Table 1
Table 2
Antimycobacterial activity, cytotoxicity and selectivity indexes of leubethanol
derivatives modified at the side chain
Antimycobacterial activity, cytotoxicity and selectivity indexes of ethers, esters,
pseudo-dimeric ethers and compound 12
20
OH
OH
OH
OR
OH
1
4
1
4
7
2
19
CH3
CH3
CH3
OH
CH3
10
11
CH3
NO2
5
5
NO2
18
R1
R2
14
H
1711a-p16
12
2 - 8
9
10
O
Compd
R
MTB H37Rv
MIC ( M)
Vero cells CC50 Selectivity
Compd R1
R2
H
MTB H37Rv
MIC ( M)
Vero cells CC50 Selectivity
l
(lM)
index (SI)
l
(lM)
index (SI)
Leub
11a
11b
11c
11d
11e
11f
H
CH3–
25.1
>200
123
nd
nd
nd
nd
nd
nd
nd
nd
4.9
–
–
–
–
Leub
D14
25.1
30.3
123
192
4.9
6.3
2
H
CH3(CH2)3–
CH3(CH2)5–
4-ClBn
4-NO2Bn
3,5-diNO2Bn
Br(CH2)4–
Leub-O–
(CH2)4–
Br(CH2)6–
Leub-O–
(CH2)6–
CH3CO–
CH3(CH2)4CO– >200
>200
>200
>200
>200
>200
>200
200
O
H
3
103
661
6.4
C
C
4
H
OH
N3
OH
NH2
OH
—
200
181
90
98
98
25.8
25.8
7.5
nd
231
127
141
158
132
116
–
1.3
1.4
1.4
1.6
5.1
4.5
>67
–
–
–
5
6
7
OH
N3
OH
11g
11h
8
9
NH2
—
11i
11j
>200
190
nd
nd
–
–
10
EMB
—
—
>500
11k
11m
11n
11o
48.7
>1100
nd
>22.6
–
–
–
MIC and CC50: rounded media values of three experiments; nd = no determined.
SI: selectivity index calculated by the equation: SI = CC50 (Vero)/MIC H37Rv).
–: not calculated; EMB = ethambutol.
PhCO
4-NO2PhCO
>200
>200
nd
nd
11p
>200
nd
–
leubethanol (Table 2), practically all the derivatives resulted inac-
tive and only the acetate 11k retained approximately one half of
12
EMB
—
17.2
7.5
>770
>500
>45
>67
the antimycobacterial activity of leubethanol (MIC: 48.7
lM vs
MIC and CC50: rounded media values of three experiments; nd = no determined.
Selectivity index calculated by the equation: SI = CC50 (Vero)/MIC H37Rv).
–: Not calculated; EMB = ethambutol.
25.1 M, respectively), that could be associated either to its intrin-
l
sic antimycobacterial ability or to its potential pro-drug character
and the possible hydrolysis during the in vitro evaluation period.
Regarding its cytotoxicity, it is interesting to note that compound
Gel 60 (0.04–0.063 mm). Reactions were monitored by TLC using
Merck 60F254 silica gel plates. Compounds were detected visually
under UV irradiation (254 nm) and by spraying with sulfuric acid
and phosphomolybdic acid reagents followed by heating at
100 °C. 1H NMR and 13C NMR spectra were obtained with a Brucker
AC 200 spectrometer (200 and 50.3 MHz, respectively). Chemical
shifts were recorded in parts per million (ppm, d) and were re-
ported relative to the solvent peak or TMS. High resolution mass
spectra (HRMS) were measured with a QSTAR XL quadrupole
time-of-flight mass spectrometer, by direct injection on the sample
dissolved in MeOH and Ionization voltage of 5500 V. Infrared (IR)
spectra were measured on a Nicolet Impact 410 spectrophot-
ometer. The absorbances were measured with a Bio-Rad Bench-
mark model microplate reader. Middlebrook 7H9 broth medium
was obtained from DifcoÒ, resazurin from ProbiotekÒ, and
11k showed a fair decrease in cytotoxicity (CC50: 1100
lM) with
respect to leubethanol (CC50: 123 M), which leads to a higher
l
selectivity index (SI: 22.6 for 11k vs 4.9 for leubethanol). These re-
sults, indicate the convenience of the presence of the free phenolic
group at C-8 of the serrulatane skeleton, in contrast to the findings
of Rodriguez and Ramirez10 for the hydrocarbon erogorgiane and
its 7-hydroxyderivative. Significantly, and to confirm the anterior
statement, the dinitrobenzyl derivative 12, while maintaining the
free hydroxyl group, showed a slightly increase of the antimyco-
bacterial potency (17.2 lM for 12 vs 25.1 lM for leubethanol)
and in spite of containing two nitro groups, resulted around one or-
der of magnitude less cytotoxic than the parent compound (SI = 45
for 12 vs 4.9 for leubethanol).
In summary, the results found in this research and, in particular,
those SI calculated for compounds 11k and 12, could support the
continuation of further research focused on establishing the mech-
anism of action and the target molecule or the affected pathway in
the mycobacteria, previous to carry out an eventual structure opti-
mization of the side-chain functionality through preparation and
evaluation of a larger number of substances, oriented to define a
good candidate for in vivo assays.
0.45 l
m pore size, 13 mm diameter PTFE from Millipore MillexÒ.
Isoniazid, rifampicin, penicillin, streptomycin, and MTT were ob-
tained from SigmaÒ and fetal bovine serum from HycloneÒ.
5.1.1. Leubethanol isolation
Dry grounded root bark (2.0 kg) of Leucophyllum frutescens was
extracted with methanol and the extract (146 g) fractionated by si-
lica gel liquid chromatography using a gradient of n-hexane and
ethyl acetate (AcOEt) as eluents. Fraction hexane/AcOEt (20:1,
56 g) was fractionated in a reverse phase Lobar RP-18 column
using aqueous methanol 80%, 90% and 100%. Methanol 100% frac-
tion was chromatographed in parallel on silica flash columns and
eluted with hexane/AcOEt (95:5) to obtain 8.5 g (5.9% from MeOH
extract) of the serrulatane leubethanol.
5. Experimental section
5.1. General
All commercial chemicals and solvents used were reagent
grade. Flash column chromatography was done using Merck Silica

J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
4159
5.1.1.1. Leubethanol (1).
Yellow oil. IR (
.
m
): 3463, 2953, 2923,
was purified by silica flash chromatography using hexane/ethyl
acetate (85:15) as eluent to provide compound 4.
2866, 1618, 1576, 838 cmꢀ1
1H NMR (200 MHz, CDCl3):
d
ppm = 1.01 (d, J = 6.8 Hz, 3H), 1.23 (d, J = 7.0 Hz, 3H), 1.10–1.31
(m, 2H), 1.50–1.97 (m, 5H), 1.59 (s, 3H), 1.69 (s, 3H), 1.82–2.01
(m, 2H), 2.27 (s, 3H), 2.61 (m, 1H), 3.09 (m, 1H) 4.84 (br s, OH),
5.03 (t, J = 6.4 Hz, 1H), 6.44 (s, 1H), 6.61 (s, 1H); 13C NMR
(50.3 MHz, CDCl3) d (ppm) 17.7 (CH3), 18.8 (CH3), 19.5 (CH2),
21.3 (CH3), 21.3 (CH3), 25.8 (CH3), 26.3 (CH2), 26.7 (CH), 27.6
(CH2), 33.5 (CH2), 38.5 (CH), 42.5 (CH), 113.3 (CH), 122.6 (CH),
125.0 (CH), 126.3 (C), 131.2 (C), 135.2 (C), 141.1 (C), 153.1 (C).
HRMS (ESI) for C20H30O: calcd 286.2297; found: 286.2391.
5.1.4.1. 15-Hydroxy-14,15-dihydroleubethanol (4).
26 mg (66% after chromatography); yellow oil. IR ( ): 3378, 2953,
2930, 1579, 1459, 1258 cmꢀ1 1H NMR (200 MHz, CDCl3) d (ppm),
0.96 (d, J = 6.8 Hz, 3H), 1.10–1.30 (m, 2H), 1.18 (s, 2 ꢁ 3H), 1.20
(d, J = 6.8 Hz, 3H), 1.31–1.51 (m, 2H), 1.55–2.03 (m, 7H), 2.24 (s,
3H), 2.57 (m, 1H), 3.05 (m, 1H), 5.08 (br s, OH), 6.42 (s, 1H), 6.57
(s, 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 19.0 (CH3), 19.3
(CH2), 21.2 (2 ꢁ CH3), 22.5 (CH2), 26.6 (CH), 27.4 (CH2), 29.2
(CH3), 29.3 (CH3), 34.0 (CH2), 38.6 (CH), 42.4 (CH), 44.1 (CH2),
71.3 (C), 113.3 (CH), 122.6 (CH), 126.4 (C), 135.1 (C), 140.9 (C),
Yield:
m
.
5.1.2. Synthesis of compound 2
To a solution of leubethanol (57 mg, 0.20 mmol) in 2 mL of ab-
solute ethanol a catalytic amount of Pd/C was added and the mix-
ture maintained under hydrogen atmosphere, at room temperature
and stirred for 3 h. The crude mixture was filtered in a filter plate,
washed with ethyl acetate and the organic layer concentrated un-
der vacuum. The residue was purified by silica flash chromatogra-
phy using hexane/ethyl acetate (95:5) as eluent to give compound
2.
153.1(C). HRMS (ESI) for
304.4506.
C20H32O2: calcd 304.2402; found:
5.1.5. Synthesis of compounds 5 and 6
To a stirring solution of 3a,b (0.29 mmol) in 3 mL ethanol, NaN3
(2.9 mmol) and NH4Cl (3.5 mmol) were added and the mixture
maintained for 24 h at 70 °C. The ethanol was removed under va-
cuum, and the solid dissolved in ethyl acetate. The organic layer
was washed with water and then dried over Na2SO4. Elimination
of the organic solvent gave a crude mixture of compounds 5a,b
and 6a,b that were purified by silica flash chromatography using
hexane/ethyl acetate (9:1) as solvent.
5.1.2.1. 14,15-Dihydroleubethanol (2).
Yield: 57 mg (99%
after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm) 0.86 (d, J = 6.5 Hz, 6H), 0.97 (d, J = 6.8 Hz, 3H), 1.10–1.30
(m, 2H), 1.23 (d, J = 7.0 Hz, 3H) 1.50–2.03 (m, 8H), 2.27 (s, 3H),
2.58 (m, 1H), 3.08 (m, 1H), 4.87 (br s, OH), 6.45 (s, 1H), 6.61 (s,
1H); 13CNMR (50.3 MHz, CDCl3) d (ppm) 19.0 (CH3), 19.6 (CH2),
21.2 (CH3), 21.3 (CH3), 22.6 (CH3), 22.9 (CH3), 25.6 (CH2), 26.7
(CH), 27.4 (CH2), 28.0 (CH), 33.8 (CH2), 34.2 (CH2), 38.5 (CH), 42.5
(CH), 113.3 (CH); 122.8 (CH), 126.3 (C), 135.1 (C), 141.2 (C),
5.1.5.1.
(5a,b).
15-Azo,14-hydroxy-14,15-dihydroleubethanol
Yield: 42 mg (42% after chromatography); yellow oil.
IR (m .
): 3378, 2953, 2930, 2105, 1579, 1258, 1062, 843 cmꢀ1 1H
NMR (200 MHz, CDCl3) d (ppm) 0.95 (d, J = 6.8 Hz, 3H), 0.99 (d,
J = 6.8 Hz, 3H), 1.05–1.15 (m, 2H), 1.20 (d, J = 6.8 Hz, 2 ꢁ 3H),
1.23 (s, 2 ꢁ 3H), 1.24 (s, 2 ꢁ 3H), 1.47–1.54 (m, 4H), 1.62–1.95
(m, 12H), 2.24 (s, 2 ꢁ 3H), 2.60 (m, 2 ꢁ 1H), 3.05 (m, 2 ꢁ 1H),
3.22 (m, 2 ꢁ 1H), 4.83 (br s, 2 ꢁ OH), 6.42 (s, 2 ꢁ 1H), 6.56 (s,
2 ꢁ 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 18.9 (CH3), 19.0
(CH2), 19.3 (CH3), 19.6 (CH2), 21.2 (6 ꢁ CH3), 22.8 (2 ꢁ CH3), 26.6
(2 ꢁ CH), 27.3 (CH2), 27.4 (CH2), 29.3 (CH2), 29.6 (CH2), 29.9
(CH2), 30.9 (CH2), 38.6 (CH), 38.8 (CH), 42.3 (2 ꢁ CH), 65.4
(2 ꢁ C), 77.4 (CH), 77.8 (CH), 113.4 (2 ꢁ CH), 122.3 (CH), 122.4
(CH), 126.2 (C), 126.5 (C), 135.2 (C), 135.3 (C), 140.7 (C), 140.9
(C), 153.2 (2 ꢁ C). HRMS (ESI) for C20H31N3O2: calcd 345.2416;
found 345.4791.
153.1 (C). HRMS (ESI) for
288.6235.
C20H32O: calcd 288.2453; found:
5.1.3. Synthesis of compound 3
To a cooled solution of leubethanol (229 mg, 0.8 mmol) in 2 mL
of CHCl3, solutions of MCPBA (0.9 mmol) in 2 mL CHCl3 and NaH-
CO3 (0.9 mmol) in 1 mL CHCl3 were added under stirring on ice
bath for 15 min. The reaction was maintained with stirring at room
temperature for 12 h. The crude mixture was washed with a 40%
solution of NaHSO3 and the solvent removed under vacuum. The
resulting solid was dissolved in ethyl acetate, washed with 5%
NaHCO3, water and dried over Na2SO4. The product was purified
by silica flash chromatography using hexane/ethyl acetate (8:2)
5.1.5.2.
(6a,b).
14-Azo,15-hydroxy-14,15-dihydroleubethanol
as eluent to give compound 3 as mixture of
a
and b epoxides.
Yield: 38 mg (38%); yellow oil. 1H NMR (200 MHz,
CDCl3) d (ppm) 0.96 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.9 Hz, 3H),
1.16 (s, 3H), 1.17 (s, 3H), 1.19 (s, 2 ꢁ 3H), 1.20 (d, J = 6.8 Hz,
2 ꢁ 3H), 2.24 (s, 2 ꢁ 3H), 1.32–1.95 (m, 18H), 2.60 (m, 2 ꢁ 1H),
3.05 (m, 4 ꢁ 1H), 4.81 (br s, 2 ꢁ OH), 6.43 (s, 2 ꢁ 1H), 6.58 (s,
2 ꢁ 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 18.9 (CH3), 19.1
(CH2), 19.4 (CH3), 19.7 (CH2), 21.2 (4 ꢁ CH3), 25.3 (2 ꢁ CH3), 26.5
(2 ꢁ CH), 26.6 (2 ꢁ CH3), 27.3 (2 ꢁ CH2), 28.2 (2 ꢁ CH2), 30.9
(CH2), 31.7 (CH2), 38.6 (2 ꢁ CH), 42.2 (2 ꢁ CH), 73.6 (CH), 73.2
(CH), 73.8 (2 ꢁ C), 113.5 (2 ꢁ CH), 122.5 (CH), 122.6 (CH), 126.2
(C), 126.5 (C), 135.2 (2 ꢁ C), 140.6 (2 ꢁ C), 153.1 (2 ꢁ C). HRMS
(ESI) for C20H31N3O2: calcd 345.2416; found 345.3954.
5.1.3.1. 14,15-Epoxyleubethanol (3a,b).
Yield: 217 mg (90%
after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm), 0.98 (d, J = 6.8 Hz, 3H), 1.1–1.5 (m, 4H), 1.20 (d, J = 6.8 Hz,
3H), 1.23 (s, 3H), 1.29 (s, 3H), 1.70–1.98 (m, 5H), 2.23 (s, 3H),
2.62 (m, 1H), 3.01 (m, 1H), 3.12 (m, 1H), 5.30 (br s, OH), 6.44 (s,
1H), 6.57 (s, 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 18.8
(2 ꢁ CH3), 19.2/19.3, (CH2), 21.2 (2 ꢁ CH3), 24.9 (CH3), 26.6 (CH),
27.3 (CH2), 27.5/27.6 (CH2), 30.0/30.2 (CH2), 38.4/38.5 (CH), 42.4
(CH), 58.6/59.0 (C), 64.8/65.1 (CH), 113.4 (CH), 122.1/122.3 (CH),
126.4 (C), 135.1 (C), 140.6/140.7 (C), 153.4 (C). HRMS (ESI) for
C20H30O2: calcd 302.2246; found: 302.4126.
5.1.6. Procedure for the synthesis of compounds 7 and 8
To a stirring solution of 5a,b (0.12 mmol), NaBH4 (0.36 mmol)
and a catalytic amount of Pd/C in 1 mL of dry THF, dry methanol
(4 mL) drop by drop was added. The mixture was maintained at
room temperature for 12 h. The reaction product was filtered over
celite, washed with ethyl acetate and the organic solvents removed
under vacuum. The resulting solid was dissolved in ethyl acetate
and washed with water. Elimination of the ethyl acetate gave a
5.1.4. Synthesis of compound 4
To a stirring solution of 3 (40 mg, 0.13 mmol) in 2 mL of dry
THF, LiAlH4 (0.26 mmol) were added and maintained for 6 h at
room temperature. Then, a solution of water-saturated ether was
added and the resulting white solid filter off. The organic layer
was concentrated under vacuum and the solid dissolved with
ether, washed with 10% NaHCO3, 2 N HCl and water. The product

4160
J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
crude that was purified by silica flash chromatography using di-
chloromethane/methanol (95:5) as eluent.
2.12–2.38 (m, 2H), 2.24 (s, 3H), 2.63 (m, 1H), 3.09 (m, 1H), 4.82
(br s, OH), 6.35 (1H, t, J = 7.9 Hz) 6.45 (s, 1H), 6.57 (s, 1H), 9.34
(s, 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 9.2 (CH3), 18.8 (CH3),
19.0 (CH2), 21.2 (2 ꢁ CH3), 26.6 (CH), 27.6 (2 ꢁ CH3), 31.9 (CH2),
38.6 (CH), 42.1 (CH), 113.4 (CH), 122.1 (CH), 126.5 (C), 135.4 (C),
139.3 (C), 140.5 (C), 153.3 (C), 155.9 (CH), 195.7 (CH). HRMS
(ESI) for C20H28O2: calcd 300.4351; found 300.5863.
5.1.6.1.
(7a,b).
15-Amino,14-hydroxy-14,15-dihydroleubethanol
Yield: 26 mg (68%); yellow oil. 1H NMR (200 MHz,
CDCl3 + CD3OD)
d (ppm) 0.88 (d, J = 6.8 Hz, 3H), 0.98 (d,
J = 6.8 Hz, 3H), 1.30–1.95 (m, 18H), 1.15 (d, J = 6.8 Hz, 2 ꢁ 3H),
1.34 (s, 2 ꢁ 3H), 1.40 (s, 2 ꢁ 3H), 2.19 (s, 2 ꢁ 3H), 2.58 (m,
2 ꢁ 1H), 3.03 (m, 2 ꢁ 1H), 3.38 (m, 2 ꢁ 1H), 6.44 (s, 2 ꢁ 1H), 6.52
(s, 2 ꢁ 1H); 13C NMR (50.3 MHz, CDCl3 + CD3OD) d (ppm) 22.8
(2 ꢁ CH3), 22.9 (2 ꢁ CH3), 23.0 (2 ꢁ CH2), 24.6 (4 ꢁ CH3), 26.7
(2 ꢁ CH3), 30.4 (2 ꢁ CH), 31.2 (2 ꢁ CH2), 33.4 (2 ꢁ CH2), 34.2
(CH2), 34.9 (CH2), 42.8 (CH), 43.1 (CH), 46.2 (2 ꢁ CH), 61.6
(2 ꢁ C), 78.9 (CH), 79.3 (CH), 116.7 (CH), 116.7 (CH), 125.2 (CH),
125.3 (CH), 130.8 (2 ꢁ C), 138.7 (2 ꢁ C), 144.1 (2 ꢁ C), 158.2
5.1.8. General procedure for the preparation of 11a–11f
To a stirring solution of leubethanol (86 mg, 0.30 mmol) in 2 mL
of dry acetone, K2CO3 (0.80 mmol) was added. The solution was
stirred for 30 min and the corresponding alkyl halide (1 equiv)
was added and maintained for 6 h at room temperature. The crude
mixture was extracted with ethyl acetate, washed with water and
the organic layer dried over Na2SO4. The resulting products, ethers
11a–11f were purified by silica flash chromatography using hex-
ane/ethyl acetate (9:1) as eluent. Compound 12 resulted as a sec-
ondary product in the reaction to obtain 11f.
(2 ꢁ C). HRMS (ESI) for
C20H33NO2: calcd 319.4815; found
319.4932.
Reduction of azides 6a,b were performed similar to compounds
5a,b. To a stirring solution of 6a,b (0.11 mmol), NaBH4 (0.33 mmol)
and a catalytic amount of Pd/C in 1 mL of dry THF, dry methanol
(4 mL) drop by drop was added. The mixture was maintained at
room temperature for 12 h. The crude was purified by silica flash
chromatography using dichloromethane/methanol (95:5) as
eluent.
5.1.8.1. Leubethanol methyl ether (11a).
after chromatography); yellow oil. IR (
1578, 1271, 832 cmꢀ1
Yield: 63 mg (70%
m
max): 2952, 2925, 1610,
.
1H NMR (200 MHz, CDCl3) d (ppm) 1.02
(d, J = 6.8 Hz, 3H), 1.10–1.31 (m, 2H), 1.19 (d, J = 6.8 Hz, 3H),
1.50–1.97 (m, 5H), 1.60 (s, 3H), 1.71 (s, 3H), 1.82–2.01 (m, 2H),
2.35 (s, 3H), 2.61 (m, 1H), 3.18 (m, 1H), 3.85 (s, 3H), 5.04 (t,
J = 6.5 Hz, 1H), 6.55 (s, 1H), 6.65 (s, 1H); 13C NMR (50.3 MHz,
CDCl3) d (ppm) 17.7 (CH3), 18.9 (CH3), 19.5 (CH2), 21.5 (CH3),
21.7 (CH3), 25.8 (CH3), 26.4 (CH2), 26.6 (CH), 27.6 (CH2), 33.5
(CH2), 38.0 (CH), 42.5 (CH), 55.1 (CH3), 108.4 (CH), 122.2 (CH),
125.1 (CH), 128.7 (C), 131.1 (C), 134.7 (C), 140.5 (C), 157.1 (C).
HRMS (ESI) for C21H32O: calcd 300.2453 found: 300.3572.
5.1.6.2.
(8a,b).
14-Amino,15-hydroxy-14,15-dihydroleubethanol
Yield 15%: yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm) 0.96 (d, J = 6.8 Hz, 2 ꢁ 3H), 1.02 (s, 2 ꢁ 3H), 1.16 (s,
2 ꢁ 3H), 1.18 (d, J = 6.8 Hz, 2 ꢁ 3H), 1.42–1.98 (m, 16H), 2.21 (s,
2 ꢁ 3H), 2.43 (m, 2 ꢁ 1H), 2.59 (m, 2 ꢁ 1H), 3.07 (m, 4 ꢁ 1H),
3.44 (br s, NH2 + OH), 6.42 (s, 2 ꢁ 1H), 6.54 (s, 2 ꢁ 1H); 13C NMR
(50.3 MHz, CDCl3) d (ppm) 18.9 (CH3), 18.9 (CH3), 19.4 (2 ꢁ CH2),
21.2 (4 ꢁ CH3), 23.0 (2 ꢁ CH3), 26.6 (2 ꢁ CH), 27.3 CH3), 27.5
CH3), 27.5 (2 ꢁ CH2), 29.7 (CH2), 30.2 (CH2), 31.3 (CH2), 31.4
(CH2), 38.6 (CH), 39.2 (CH), 42.1 (2 ꢁ C), 60.2 (CH), 61.4 (CH),
71.7 (2 ꢁ C), 113.5 (C), 113.7 (C), 122.0 (CH), 122.4 (CH), 126.7
(2 ꢁ C), 135.2 (C), 135.4 (C), 140.6 (C), 140.8 (C), 153.4 (2 ꢁ C).
HRMS (ESI) for C20H33NO2: calcd 319.4815; found 319.45017.
5.1.8.2. Leubethanol n-butyl ether (11b).
Yield: 73 mg (71%
after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm) 0.91 (t, J = 6.6 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 1.18 (d,
J = 6.8 Hz, 3H), 1.20–1.99 (m, 13H), 1.58 (s, 3H), 1.68 (s, 3H), 2.29
(s, 3H), 2.58 (m, 1H), 3.15 (m, 1H), 3.92 (t, J = 6.6 Hz, 2H), 4.98 (t,
J = 6.5 Hz, 1H), 6.48 (s, 1H), 6.60 (s, 1H); 13CNMR (50.3 MHz, CDCl3)
d (ppm) 17.7 (CH3), 18.8 (CH3), 19.2 (CH3), 19.5 (CH2), 21.4 (CH3),
21.5 (CH3), 25.8 (CH2), 26.2 (CH2), 26.3 (CH3), 26.6 (CH), 27.6
(CH2), 31.4 (CH2), 33.5 (CH2), 38.3 (CH), 42.5 (CH), 67.5 (CH2),
109.2 (CH), 122.2 (CH), 125.1 (CH), 128.8 (C), 131.0 (C), 134.6 (C),
140.5 (C), 156.5 (C). HRMS (ESI) for C24H38O: calcd 342.2923 found:
342.3072.
5.1.7. Synthesis of compounds 9 and 10
To a stirring solution of leubethanol (200 mg, 0.70 mmol) in
2 mL dry ethanol, SeO2 (87 mg, 0.78 mmol) was added and the
mixture maintained at room temperature for 1 h. The solvent
was removed under vacuum and the solid mixture dissolved in
ethyl acetate, washed with brine and the organic layer dried over
Na2SO4. Elimination of the solvent gave a crude (200 mg) that
was purified by silica flash chromatography using n-hexane/ethyl
acetate (9:1) as eluent, to separate 15 mg of starting material,
129 mg (0.43 mmol, 61%) of the allylic alcohol (9) and 56 mg
5.1.8.3. Leubethanol n-hexyl ether (11c).
Yield: 74 mg (66%
after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm) 0.92 (t, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 1.16 (d,
J = 6.8 Hz, 3H), 1.19–1.97 (m, 17H), 1.57 (s, 3H), 1.66 (s, 3H), 2.30
(s, 3H), 2.56 (m, 1H), 3.17 (m, 1H), 3.93 (t, J = 6.5 Hz, 2H), 4.97 (t,
J = 6.5 Hz, 1H), 6.48 (s, 1H), 6.59 (s, 1H); 13C NMR (50.3 MHz,
CDCl3) d (ppm) 17.7 (CH3), 18.8 (CH3), 19.1 (CH3), 19.5 (CH2),
21.5 (CH3), 21.6 (CH3), 25.7 (CH2), 26.0 (CH2), 26.3 (CH3), 26.6
(CH), 27.6 (CH2), 29.5 (CH2), 30.9 (CH2), 31.6 (CH2), 33.5 (CH2),
38.0 (CH), 42.4 (CH), 67.5 (CH2), 109.0 (CH), 121.9 (CH), 125.1
(CH), 128.7 (C), 131.1 (C), 134.6 (C), 140.4 (C), 156.6 (C). HRMS
(ESI) for C26H42O: calcd 370.3236 found: 370.3559.
(0.19 mmol, 27%) of the
a,b-unsaturated aldehyde (10).
5.1.7.1. 16-Hydroxy leubethanol (9).
Yellow oil. 1H NMR
(200 MHz, CDCl3) d (ppm) 0.98 (d, J = 6.8 Hz, 3H), 1.12–1.52 (m,
4H), 1.20 (d, J = 6.8 Hz, 3H), 1.62 (s, 3H), 1.72–1.95 (m, 7H), 2.24
(s, 3H), 2.58 (m, 1H), 3.07 (m, 1H), 3.98 (s, 2H), 5.15 (br s, OH),
5.29 (1H, t, J = 6.5 Hz), 6.42 (s, 1H), 6.57 (s, 1H); 13C NMR
(50.3 MHz, CDCl3) d (ppm) 13.7 (CH3), 18.8 (CH3), 19.4(CH2), 21.2
(2 ꢁ CH3), 26.0 (CH2), 26.6 (CH), 27.5 (CH2), 33.1 (CH2), 38.3 (CH),
42.4 (CH), 69.2 (CH2), 113.3 (CH), 122.4 (CH), 128.6 (C), 127.0
(CH), 134.4 (C), 135.1 (C), 140.9 (C), 153.3 (C). HRMS (ESI) for
5.1.8.4. Leubethanol 4-chlorobenzyl ether (11d).
Yield:
121 mg (98% after chromatography); yellow oil. 1H NMR
(200 MHz, CDCl3) d (ppm) 1.02 (d, J = 6.8 Hz, 3H), 1.10–1.30 (m,
2H), 1.23 (d, J = 6.8 Hz, 3H), 1.50–2.03 (m, 7H), 1.61 (s, 3H), 1.71
(s, 3H), 2.35 (s, 3H), 2.63 (m, 1H), 3.25 (m, 1H), 5.05 (t, J = 6.4 Hz,
1H), 5.06 (s, 2H), 6.60 (s, 1H), 6.70 (s, 1H), 7.38 (d, J = 7.0 Hz, 2H),
7.66 (d, J = 7.0 Hz, 2H); 13CNMR (50.3 MHz, CDCl3) d (ppm) 17.7
(CH3), 18.9 (CH3), 19.4 (CH2), 21.6 (2 ꢁ CH3), 25.8 (CH3), 26.4
C20H30O2: calcd 302.4510; found 302.5312.
5.1.7.2. 16-Oxoleubethanol (10).
2951, 2868, 1703, 1675, 1580, 1248, 1059, 841 cmꢀ1
(200 MHz, CDCl3) (ppm) 1.04 (d, J = 6.8 Hz, 3H, 1.21 (d,
J = 6.8 Hz, 3H), 1.24–1.58 (m, 3H), 1.69–1.97 (m, 4H), 1.70 (s, 3H),
Yellow oil. IR (
m
): 3402,
.
1H NMR
d

J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
4161
(CH2), 26.7 (CH), 27.5 (CH2), 33.5 (CH2), 38.1 (CH), 42.5 (CH), 69.0
(CH2), 109.6 (CH), 122.8 (CH), 125.1 (CH), 128.4 (2 ꢁ CH), 128.7
(2 ꢁ CH), 129.0 (C), 131.2 (C), 133.4 (C), 134.8 (C), 136.4 (C),
140.8 (C), 156.0 (C). HRMS (ESI) for C27H35ClO: calcd 410.2376
found: 410.3746.
(CH3), 19.4 (CH2), 21.6 (2 ꢁ CH3), 25.8 (CH3), 26.3 (CH2), 26.6
(CH), 27.5 (CH2), 28.8 (CH2), 29.8 (CH2), 33.5 (CH2), 33.8 (CH2),
38.1 (CH), 42.4 (CH), 66.4 (CH2), 109.0 (CH), 122.3 (CH), 125.1
(CH), 128.7 (C), 131.2 (C), 134.7 (C), 140.6 (C), 156.2 (C). HRMS
(ESI) for C24H37BrO: calcd 420.2028; found: 420.2231.
5.1.9.1.1. Compound 11h. Yield: 56 mg (36% after chromato-
graphy); yellow oil. 1H NMR (200 MHz, CDCl3) d (ppm) 0.97 (d,
J = 6.8 Hz, 2 ꢁ 3H), 1.15 (d, J = 6.8 Hz, 2 ꢁ 3H), 1.30–2.09 (m,
2 ꢁ 9H), 1.56 (s, 2 ꢁ 3H), 1.66 (s, 2 ꢁ 3H), 2.02 (m, 2 ꢁ 2H), 2.29
(s, 2 ꢁ 3H), 2.56 (m, 2 ꢁ 1H), 3.16 (m, 2 ꢁ 1H), 3.95 (t, J = 6.8 Hz,
2 ꢁ 2H), 5.00 (t, J = 6.5 Hz, 2 ꢁ 1H), 6.49 (s, 2 ꢁ 1H), 6.59 (s,
2 ꢁ 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 17.8 (2 ꢁ CH3), 18.8
(2 ꢁ CH3), 19.4 (2 ꢁ CH2), 21.6 (2 ꢁ CH3), 21.7 (2 ꢁ CH3), 25.8
(2 ꢁ CH3), 26.1 (2 ꢁ CH2), 26.3 (2 ꢁ CH2), 26.6 (2 ꢁ CH), 27.6
(2 ꢁ CH2), 33.5 (2 ꢁ CH2), 38.0 (2 ꢁ CH), 42.5 (2 ꢁ CH), 67.1
(2 ꢁ CH2), 109.0 (2 ꢁ CH), 122.0 (2 ꢁ CH), 125.1 (2 ꢁ CH), 128.7
(2 ꢁ C), 131.2 (2 ꢁ C), 134.7 (2 ꢁ C), 140.5 (2 ꢁ C), 156.5 (2 ꢁ C).
HRMS (ESI) for C44H66O2: calcd 626.5063; found: 626.6128.
5.1.8.5. Leubethanol 4-nitrobenzyl ether (11e).
Yield: 52 mg
(41% after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3)
d (ppm), 0.98 (d, J = 6.8 Hz, 3H), 1.21 (d, J = 6.8 Hz, 3H), 1.1–1.4 (m,
2H), 1.51–2.07 (m, 7H), 1.56 (s, 3H), 1.66 (s, 3H), 2.30 (s, 3H), 2.60
(m, 1H), 3.25 (m, 1H), 4.99 (t, J = 6.5 Hz, 1H), 5.17 (s, 2H), 6.53 (s,
1H), 6.67 (s, 1H), 7.64 (d, J = 8.6, 2H), 8.27 (d, J = 8.6 Hz, 2H); 13C
NMR (50.3 MHz, CDCl3) d (ppm) 17.7 (CH3), 18.8 (CH3), 19.3
(CH2), 21.6 (2 ꢁ CH3), 25.8 (CH3), 26.3 (CH2), 26.7 (CH), 27.4
(CH2), 33.5 (CH2), 38.1 (CH), 42.4 (CH), 68.5 (CH2), 109.5 (CH),
123.1 (CH), 123.8 (2 ꢁ CH), 125.1 (CH), 127.3 (2 ꢁ CH), 129.0 (C),
131.2 (C), 134.9 (C), 141.1 (C), 145.4 (C), 147.6 (C), 155.6 (C). HRMS
(ESI) for C27H35NO3: calcd 421.2617 found: 421.3359.
5.1.8.6. Leubethanol 3,5-dinitrobenzyl ether (11f).
52 mg (36% after chromatography); yellow oil. IR ( ): 2926, 2868,
1609, 1544, 1343, 1269, 875 cmꢀ1 1H NMR (200 MHz, CDCl3) d
Yield:
5.1.9.2. Leubethanol 6-bromohexyl ether (11i).
Yield: 31 mg
m
(27% after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3)
d (ppm) 0.97 (d, J = 6.8 Hz, 3H), 1.10–1.30 (m, 2H), 1.15 (d,
J = 6.8 Hz, 3H), 1.50–2.07 (m, 7H), 1.56 (s, 3H), 1.66 (s, 3H), 1.88
(m, 8H), 2.30 (s, 3H), 2.57 (m, 1H), 3.15 (m, 1H), 3.43 (t,
J = 6.8 Hz, 2H), 3.96 (t, J = 6.8 Hz, 2H), 5.00 (t, J = 6.5 Hz, 1H), 6.48
(s, 1H), 6.60 (s, 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 17.7
(CH3), 18.8 (CH3), 19.5 (CH2), 21.6 (CH3), 21.7 (CH3), 25.6 (CH2),
25.8 (CH3), 26.4 (CH2), 26.6 (CH), 27.6 (CH2), 28.0 (CH2), 29.4
(CH2), 32.8 (CH2), 33.5 (CH2), 33.9 (CH2), 38.0 (CH), 42.4 (CH),
67.2 (CH2), 109.0 (CH), 122.1 (CH), 125.1 (CH), 128.7 (C), 131.1
(C), 134.7 (C), 140.5 (C), 156.4 (C). HRMS (ESI) for C26H41BrO: calcd
448.2341; found: 448.5296.
.
(ppm) 0.98 (d, J = 6.8 Hz, 3H), 1.10–1.30 (m, 2H), 1.23 (d,
J = 6.8 Hz, 3H), 1.50–2.03 (m, 7H), 1.60 (s, 3H), 1.65 (s, 3H), 2.31
(s, 3H), 2.60 (m, 1H), 3.28 (m, 1H), 4.98 (t, J = 6.4 Hz, 1H), 5.26 (s,
2H), 6.54 (s, 1H), 6.70 (s, 1H), 8.70 (d, J = 1.0 Hz, 2H), 9.02 (d,
J = 1.0 Hz, 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 17.7 (CH3),
18.8 (CH3), 19.3 (CH2), 21.6 (2 ꢁ CH3), 25.8 (CH3), 26.3 (CH2),
26.8 (CH), 27.3 (CH2), 33.5 (CH2), 38.1 (CH), 42.4 (CH), 67.1 (CH2),
109.1 (CH), 118.1 (CH), 123.7 (CH), 124.9 (CH), 126.8 (2 ꢁ CH),
129.1 (C), 131.3 (C), 135.0 (C), 141.4 (C), 142.6 (C), 148.8 (2 ꢁ C),
155.0 (C). HRMS (ESI) for C27H34N2O5: calcd 466.2468 found:
466.4132.
5.1.9.2.1. Compound 11j. Yield: 74 mg (45% after chromatogra-
phy); yellow oil. 1H NMR (200 MHz, CDCl3) d (ppm) 0.97 (d,
J = 6.8 Hz, 2 ꢁ 3H), 1.10–1.30 (m, 2 ꢁ 2H), 1.15 (d, J = 6.8 Hz,
2 ꢁ 3H), 1.50–2.09 (m, 2 ꢁ 7H), 1.56 (s, 2 ꢁ 3H), 1.66 (s, 2 ꢁ 3H),
1.88 (m, 8H), 2.29 (s, 2 ꢁ 3H), 2.56 (m, 2 ꢁ 1H), 3.16 (m, 2 ꢁ 1H),
3.95 (t, J = 6.8 Hz, 2 ꢁ 2H), 5.00 (t, J = 6.5 Hz, 2 ꢁ 1H), 6.49 (s,
2 ꢁ 1H), 6.59 (s, 2 ꢁ 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm)
17.8 (2 ꢁ CH3), 18.8 (2 ꢁ CH3), 19.4 (2 ꢁ CH2), 21.6 (2 ꢁ CH3),
21.7 (2 ꢁ CH3), 25.8 (2 ꢁ CH3), 26.1 (2 ꢁ CH2), 26.3 (2 ꢁ CH2),
26.6 (2 ꢁ CH), 27.6 (2 ꢁ CH2), 29.5 (2 ꢁ CH2), 33.5 (2 ꢁ CH2), 38.0
(2 ꢁ CH), 42.5 (2 ꢁ CH), 67.4 (2 ꢁ CH2), 109.0 (2 ꢁ CH), 122.0
(2 ꢁ CH), 125.1 (2 ꢁ CH), 128.7 (2 ꢁ C), 131.2 (2 ꢁ C), 134.7
(2 ꢁ C), 140.5 (2 ꢁ C), 156.5 (2 ꢁ C). HRMS (ESI) for C46H70O2: calcd
656.5533; found: 656.9119.
5.1.8.7. Leubethanol 5-(3,5-dinitrobenzyl) (12).
21 mg (15% after chromatography); yellow oil. IR ( ): 3400, 2926,
2873, 1591, 1541, 1452, 1344, 1241 cmꢀ1 1H NMR (200 MHz,
Yield:
m
.
CDCl3) d (ppm), 0.80 (d, J = 6.8 Hz, 3H), 1.1–1.2 (m, 2H), 1.24 (d,
J = 7.2 Hz, 3H), 1.35 (m, 2H), 1.5–1.7 (m, 4H), 1.52 (s, 3H), 1.64 (s,
3H), 1.88 (m, 1H), 2.05 (s, 3H), 2.64 (m, 1H), 3.20 (m, 1H), 4.09
(d, J = 17.1 Hz, 1H), 4.25 (d, J = 17.2 Hz, 1H), 4.76 (br s, OH), 4.92
(t, J = 6.5 Hz, 1H), 6.54 (s, 1H), 8.13 (d, J = 2.1, 2H), 8.34 (d,
J = 2.1 Hz, 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 17.7 (CH3),
18.1 (CH3), 20.2 (CH2), 20.5 (CH3), 22.4 (CH3), 25.4 (CH2), 25.8
(CH3), 26.7 (CH2), 26.8 (CH), 34.4 (CH2), 35.2 (CH2), 38.5 (CH),
39.9 (CH), 115.9 (CH), 116.6 (C), 124.5 (CH), 127.9 (2 ꢁ CH),
131.6 (2 ꢁ C), 134.8 (CH), 134.8 (C), 139.7 (C), 146.2 (C), 148.6
(2 ꢁ C), 154.2 (C). HRMS (ESI) for C27H34N2O5: calcd 466.2468;
found: 466.3596.
5.1.10. Preparation of 11k
To a cooled solution of leubethanol (0.25 mmol) in 1 mL of pyr-
idine, 1 mL of acetic anhydride was added and the mixture stirred
for 12 h on an ice bath. The product was extracted with ethyl acet-
ate, washed with 2 N HCl, with water to pH 7 and dried over
Na2SO4. The solvent was removed under vacuum and the crude
mixture purified by silica flash chromatography using hexane/
ethyl acetate (9:1) to obtain compound 11k.
5.1.9. General procedure for the preparation of 11g–11j
To a stirring solution of leubethanol (0.25 mmol) in 2 mL of dry
acetone, NaH (0.25 mmol) was added. Then, after 30 min
0.12 mmol of Br-(CH2)n-Br (n = 4 or 6) was added and maintained
for 6 h at room temperature. The crude mixture was extracted with
ethyl acetate, washed with water and the organic layer dried over
Na2SO4. The resulting products were purified by silica flash chro-
matography using hexane/toluene (9:1) as eluent.
5.1.10.1. Leubethanyl acetate (11k).
chromatography); yellow oil. IR (
1571, 1207 cmꢀ1
Yield: 70 mg (85% after
m
): 2954, 2926, 1765, 1619,
.
1H NMR (200 MHz, CDCl3) d (ppm) 0.99 (d,
5.1.9.1. Leubethanol 4-bromobutyl ether (11g).
Yield:
J = 6.8 Hz, 3H), 1.10–1.31 (m, 2H), 1.15 (d, J = 6.8 Hz, 3H), 1.50–
2.07 (m, 4H), 1.58 (s, 3H), 1.68 (s, 3H), 1.82–2.01 (m, 3H), 2.31 (s,
3H), 2.32 (s, 3H), 2.61 (m, 1H), 2.96 (m, 1H), 5.00 (t, J = 6.4 Hz,
1H), 6.71 (s, 1H), 6.89 (s, 1H); 13CNMR (50.3 MHz, CDCl3) d (ppm)
17.7 (CH3), 18.8 (CH3), 19.3 (CH2), 21.2 (3 ꢁ CH3), 25.8 (CH3),
26.3 (CH2), 27.2 (CH), 27.5 (CH2), 33.3 (CH2), 38.0 (CH), 42.4 (CH),
120.3 (CH), 124.9 (CH), 127.6 (CH), 131.2 (C), 131.6 (C), 135.1 (C),
22 mg (20% after chromatography); yellow oil. 1H NMR
(200 MHz, CDCl3) d (ppm) 0.97 (d, J = 6.8 Hz, 3H), 1.10–1.30 (m,
2H), 1.14 (d, J = 6.8 Hz, 3H), 1.50–2.07 (m, 11H), 1.56 (s, 3H), 1.66
(s, 3H), 2.21 (s, 3H), 2.57 (m, 1H), 3.14 (m, 1H), 3.52 (t, J = 6.5 Hz,
2H), 3.98 (t, J = 6.5 Hz, 2H), 4.99 (t, J = 6.5 Hz, 1H), 6.47 (s, 1H),
6.60 (s, 1H); 13C NMR (50.3 MHz, CDCl3) d (ppm) 17.7 (CH3), 18.8

4162
J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
141.2 (C), 148.7 (C), 169.8 (CO). HRMS (ESI) for C22H32O2: calcd
328.2402; found: 328.2591.
124.9 (CH), 127.9 (CH), 131.3 (C), 131.9 (C), 135.2 (C), 141.3 (C),
144.4 (C), 147.1 (CH), 148.1 (C), 157.2 (C). HRMS (ESI) calcd for
C25H32O3 380.2351; found: 380.3568.
5.1.11. General procedure for the preparation of 11m–11p
To a stirring solution of leubethanol (0.20 mmol) in 2 mL of dry
ethyl ether, NaH (0.20 mmol) was added. Then, after 30 min
0.20 mmol of the correspondent acyl halide was added and the
mixture maintained for 8 h at room temperature. The product
was extracted with ethyl acetate, washed with water and the or-
ganic layer dried over Na2SO4. The solvent was removed under va-
cuum and the crude mixture purified by silica flash
chromatography using hexane/ethyl acetate (9:1) as eluent, to ob-
tain esters 11m–11p.
5.2. Biochemical studies
The antimycobacterial activity was assessed against M. tuber-
culosis H37Rv ATTC 27294 susceptible to all five first-line anti-TB
drugs (streptomycin, isoniazid, rifampin, ethambutol, and pyrazi-
namide) in a modified Microplate Assay Blue Alamar.11,12 The
compounds for M. tuberculosis bioassays were prepared at a con-
centration of 1 mg/mL in 2.5% DMSO in Middlebrook 7H9 (Bec-
ton Dickinson and Co., Sparks MD, USA) broth. All solutions
were sterilized by filtration using 13 mm diameter PTFE acro-
5.1.11.1. Leubethanyl hexanoate (11m).
Yield: 53 mg (69%
discs (0.22
concentrations for organic compounds used ranged form
100 g/mL to 0.78 g/mL, results are reported as minimal inhibi-
tion concentration (MIC). Ethambutol (EMB) was used as positive
control. All biological assays were developed at least by
triplicate.
lm pore size, Millipore Co., Bedford, MA, USA): The
after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm) 0.93 (t, J = 7.1 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 1.10–1.30
(m, 2H), 1.14 (d, J = 6.8 Hz, 3H), 1.33 (m, 4H), 1.50–1.97 (m, 5H),
1.57 (s, 3H), 1.67 (s, 3H), 1.80–2.10 (m, 6H), 2.30 (s, 3H), 2.57 (t,
J = 7.2 Hz, 2H), 2.61 (m, 1H), 2.94 (m, 1H), 5.00 (t, J = 6.4 Hz, 1H),
6.68 (s, 1H), 6.86 (s, 1H); 13CNMR (50.3 MHz, CDCl3) d (ppm)
14.1 (CH3), 17.7 (CH3), 18.8 (CH3), 19.3 (CH2), 21.1 (CH3), 21.8
(CH3), 22.6 (CH2), 25.1 (CH2), 25.8 (CH3), 26.3 (CH2), 27.3 (CH2),
27.3 (CH), 29.0 (CH2), 31.6 (CH2), 33.3 (CH2), 34.6 (CH2), 38.0
(CH), 42.3 (CH), 120.4 (CH), 124.9 (CH), 127.6 (CH), 130.6 (C),
131.6 (C), 135.1 (C), 141.2 (C), 148.7 (C), 172.5 (CO). HRMS (ESI)
calcd for C27H42O2 398.3185; found: 398.6275.
l
l
5.3. Cytotoxicity assay
Cytotoxicity was determined according to the MTT method13
using African green monkey kidney epithelial cells (Vero cells)
grown in RPMI-1640 medium supplemented with penicillin (100
units/mL), streptomycin (100 lg/mL), and fetal bovine serum
(10%) at 37 °C under 5% CO2. In the confluence, 4000 cells per well
was applied to a 96-well microtitre plate. After incubation for 24 h
5.1.11.2. Leubethanyl benzoate (11n).
Yield: 63 mg (80%
after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm), 1.03 (d, J = 6.8 Hz, 3H), 1.21 (d, J = 6.6 Hz, 3H), 1.1–1.2 (m,
2H), 1.50–2.0 (m, 7H), 1.61 (s, 3H), 1.70 (s, 3H), 2.36 (s, 3H), 2.67
(m, 1H), 3.07 (m, 1H), 5.00 (t, J = 6.5 Hz, 1H), 6.88 (s, 1H), 6.95 (s,
1H), 7.54 (dd, J = 7.6; 7.5 Hz, 2H), 7.64 (dd, J = 7.5; 1.1 Hz, 1H),
8.36 (dd, J = 7.6; 1.1 Hz, 2H); 13CNMR (50.3 MHz, CDCl3) d (ppm)
17.7 (CH3), 18.8 (CH3), 19.4 (CH2), 21.2 (CH3), 22.1 (CH3), 25.8
(CH3), 26.3 (CH2), 27.3 (CH2), 27.3 (CH), 33.4 (CH2), 38.1 (CH),
40.4 (CH), 120.5 (CH), 125.0 (CH), 127.9 (CH), 128.7 (2 ꢁ CH),
130.1 (2 ꢁ CH), 131.3 (2 ꢁ C), 131.9 (C), 133.5 (CH), 135.2 (C),
141.3 (C), 148.9 (C), 165.3 (CO). HRMS (ESI) calcd for C27H34O2
390.2559; found: 390.6541.
(37 °C, 5% CO2), 100
ging from 500 to 0.5
added and incubated for 48 h. 25
l
l
L of a solution containing concentrations ran-
g/mL of the compound under evaluation was
L of MTT (4 mg/mL in PBS) was
added to each well and incubated for another 3 h. The solution in
each well was removed and DMSO (200 L) was then added to
l
l
each well. The absorbance was recorded on a microplate reader
at a wavelength of 575 nm. The CC50 value was calculated by linear
regression as the concentration of the compound inhibiting 50%
cellular viability.
Acknowledgements
Collaborative research performed under the auspices of Mexi-
can Network (PROMED) and CYTED-X.11: PIBATUB. R.E. thanks
the research contract financed by ISCIII-RICET. B.A. thanks the
Ph.D. fellowship received from CONACYT, Mexico.
5.1.11.3. Leubethanyl 4-nitrobenzoate (11o).
(46% after chromatography); yellow oil. IR ( ): 2959, 2926, 1741,
1609, 1530, 1348, 1250, 716 cmꢀ1 1H NMR (200 MHz, CDCl3) d
Yield: 40 mg
m
.
(ppm), 1.00 (d, J = 6.5 Hz, 3H), 1.16 (d, J = 6.8 Hz, 3H), 1.1–1.3 (m,
2H), 1.50–2.03 (m, 7H), 1.57 (s, 3H), 1.67 (s, 3H), 2.34 (s, 3H),
2.65 (m, 1H), 2.98 (m, 1H), 5.00 (t, J = 6.5 Hz, 1H), 6.85 (s, 1H),
6.95 (s, 1H), 8.34 (br s, 4H); 13C NMR (50.3 MHz, CDCl3) d (ppm)
17.7 (CH3), 18.8 (CH3), 19.2 (CH2), 21.2 (CH3), 22.1 (CH3), 25.8
(CH3), 26.3 (CH2), 26.3 (CH), 27.3 (CH2), 33.3 (CH2), 38.1 (CH),
42.3 (CH), 120.1 (CH), 123.8 (2 ꢁ CH), 124.9 (CH), 128.2 (CH),
131.2 (2 ꢁ CH), 131.6 (2 ꢁ C), 135.4 (2 ꢁ C), 141.6 (C), 148.5 (C),
150.9 (C), 163.5 (CO). HRMS (ESI) calcd for C27H33NO2 435.2410;
found: 435.2619.
References and notes
1. http://www.who.int/tb/publications/global_report/2010/en/index.html.
2. Andersen, P.; Doherty, T. M.; Pai, M.; Weldingh, K. Trends Mol. Med. 2007, 13,
175.
3. http://www.who.int/features/factfiles/tb_facts/en/index1.html.
4. http://www.who.int/tb/challenges/mdr/faqs/en/index.html.
5. Gandhi, N. R.; Moll, A.; Sturm, A. W.; Pawinski, R.; Govender, T.; Lalloo, U.;
Zeller, K.; Andrews, J.; Friedland, G. Lancet 2006, 368, 1575.
6. http://www.who.int/tb/challenges/xdr/en/index.html.
7. Mini-Rev. Med. Chem. 2008, 8, 1441.
8. Molina-Salinas, G. M.; Rivas-Galindo, V. M.; Said-Fernández, S.; Lankin, D. C.;
Muñoz, M. A.; Joseph-Nathan, P.; Pauli, G. F.; Waksman, N. J. Nat. Prod. 1842,
2011, 74.
9. Del Olmo, E.; Molina-Salinas, G. M.; Escarcena, R.; Alves, M.; Lopez-Perez, J. L.;
Hernandez-Pando, R.; Said-Fernández, S.; San Feliciano, M. Bioorg. Med. Chem.
Lett. 2009, 19, 5764.
10. Rodriguez, A. D.; Ramirez, C. J. Nat. Prod. 2001, 64, 100.
11. Dubrovska, A.; Kim, Ch.; Elliott, J.; Shen, W.; Kuo, T.-H.; Koo, D.-I.; Li, Ch.;
Tuntland, T.; Chang, J.; Groessl, T.; Wu, X.; Gorney, V.; Ramirez-Montagut, T.;
Spiegel, D.A.; Cho, Ch. Y.; Schultz, P.G. ACS Chem. Biol. 2011, doi: http://
dx.doi.org/10.1021/cb200222s.
12. Mycobaterial characterisation: The H37Rv MTB strain (ATCC cat. No. 27294),
resulted sensitive to all first-line anti-TB drugs, S, I, R, E and P. Using the
conventional BACTEC 460-radiometric system (Bactec 460).12
5.1.11.4. Leubethanyl 2-furoate (11p).
Yield: 62 mg (85%
after chromatography); yellow oil. 1H NMR (200 MHz, CDCl3) d
(ppm), 1.01 (d, J = 6.8 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H), 1.11–1.32
(m, 2H), 1.50–2.09 (m, 7H), 1.59 (s, 3H), 1.69 (s, 3H), 2.34 (s, 3H),
2.65 (m, 1H), 3.05 (m, 1H), 5.02 (t, J = 6.5 Hz, 1H), 6.61 (dd,
J = 3.4; 1.6 Hz, 1H), 6.66 (s, 1H); 6.93 (s, 1H), 7.38 (br d,
J = 3.4 Hz, 1H), 7.69 (dd, J = 1.6; 0.8 Hz, 1H); 13C NMR (50.3 MHz,
CDCl3) d (ppm) 17.7 (CH3), 18.8 (CH3), 19.3 (CH2), 21.2 (CH3),
22.0 (CH3), 25.8 (CH3), 26.3 (CH2), 27.3 (CH), 27.4 (CH2), 33.3
(CH2), 38.1 (CH), 42.4 (CH), 112.2 (CH), 119.0 (CH), 120.3 (CH),

J. Perez-Meseguer et al. / Bioorg. Med. Chem. 20 (2012) 4155–4163
4163
13. Assay: The modified Microplate Alamar Blue Assay (MABA) was used to
determine the anti-MTB activity. Summarily, solutions of compounds (2 mg/
mL, DMSO), standard drug, EMB.HCl (2 mg/mL, H2O), were sterilised by
the ranges: 1.0–32
mycobacterial growth control (100
included. After incubation (37 °C for 5–7 days in 5% CO2 atmosphere, into
plastic O2 permeable sealed bag), 32 L of freshly prepared 20:12 mixture
l
g/mL (EMB). A blank (100
l
L of medium), and a positive
lL of 1:50 bacterial suspension) were
filtration (PTFE acrodiscs, 0.22
lm pore size, Millipore Co., Bedford, MA, USA).
l
Std. drugs were divided in 0.5 mL aliquots, and stored at ꢀ70 °C until used,
Alamar Blue plus 10% Tween-80 (v/v), were added to each well. The plates
were re-incubated at 37 °C for 24 h. Pink and blue colors indicated
mycobacterial growth or its absence, respectively. The MIC value for a tested
compound, was assigned to that of the first blue well. Destruction of
mycobacteria in the first blue well was confirmed microscopically.
14. Plumb, J. A. Cell sensitivity assays: the MTT assay. In Cancer Cell Culture:
Methods and Protocols; Langdon, S. P., Ed.; Humana Press: Totowa, New Jersey,
USA, 2004; pp 165–169.
while the compounds were assayed immediately. Deionised sterile water
(200 lL) was dispensed in the perimeter wells to diminish evaporation. The
inner wells were divided in lanes of 6 to perform the assays. 100 lL of a 1:50
bacterial suspension (having about 6 ꢁ 106 CFU/mL, Mc Farland std. 1) in
Middlebrock 7H9-OADC enriched broth (Becton Dickinson and Co., Sparks, MD,
USA), were added to each testing well. In the 6-well lanes, compound and
standard drug solutions were two-step serially diluted with 7H9-OADC
medium (100 lL per well). Evaluations were performed on dilutions within