Simple dihydrosphyngosine analogues with potent activity against MDR-Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2009.07.147

Fifteen dihydrosphingosine analogues have been synthesized and tested in vitro against Mycobacterium tuberculosis (MTB). Two ether (3 and 4b) and one diamine (8b) derivatives have displayed high mycobactericidal potency, with similar MIC values of 1.25 μg/mL, against the virulent strain H37Rv, as well as against a clinical isolate resistant to the five first-line anti-TB drugs. The three compounds, tested on other eleven cultured MTB strains with different multi-drug-resistance (MDR) patterns, retained their MIC values for most strains, or even lowered it, as in the case of compound 4b, which, assayed on strain No. 332, also resistant to all first-line anti-TB drugs, attained the MIC value of 0.78 μg/mL.

Full text of DOI:10.1016/j.bmcl.2009.07.147

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5764–5768
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Simple dihydrosphyngosine analogues with potent activity
against MDR-Mycobacterium tuberculosis
b
a
a
a
Esther del Olmo a, , Gloria María Molina-Salinas , Ricardo Escarcena , Mario Alves , José L. López-Pérez ,
*
Rogelio Hernandez-Pando c, Salvador Said-Fernández b, , Arturo San Feliciano
a
*
a Departamento de Química Farmacéutica, Facultad de Farmacia, CIETUS, Universidad de Salamanca, Spain
b Laboratorio de Micobacteriología. Centro de Investigación Biomédica del Noreste, IMSS, Monterrey, Mexico
c Departamento de Patología. Instituto Nac. de Ciencias Médicas y Nutrición ‘Salvador Zubirán’, Tlalpan, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Fifteen dihydrosphingosine analogues have been synthesized and tested in vitro against Mycobacterium
tuberculosis (MTB). Two ether (3 and 4b) and one diamine (8b) derivatives have displayed high mycobac-
tericidal potency, with similar MIC values of 1.25 lg/mL, against the virulent strain H37Rv, as well as
against a clinical isolate resistant to the five first-line anti-TB drugs. The three compounds, tested on
other eleven cultured MTB strains with different multi-drug-resistance (MDR) patterns, retained their
MIC values for most strains, or even lowered it, as in the case of compound 4b, which, assayed on strain
Received 17 June 2009
Revised 29 July 2009
Accepted 30 July 2009
Available online 3 August 2009
Keywords:
Dihydrosphingosine
Ethambutol
No. 332, also resistant to all first-line anti-TB drugs, attained the MIC value of 0.78 lg/mL.
Ó 2009 Published by Elsevier Ltd.
Aminoalcohols
Diamines
Mycobactericidal
Mycobacterium tuberculosis
H37Rv strain
MDR strain
Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis
(MTB), has been widespread since ancient times and remains a
worldwide major health problem. TB morbidity, with 8–9 million
new infections per year, and its mortality, with 3 million deaths
annually,1 represents the largest numbers of incidence and of hu-
man deaths attributable to a single etiologic agent. Mycobacteria
are able to survive in a latent state in infected individuals, thereby
serving as a reservoir, waiting for the opportune reactivation.2
According to World Health Organisation (WHO) estimation, 2 bil-
lion people, almost one-third of the world’s population, are be-
lieved to be infected with MTB.3
After several decades of decline, TB is currently rising, mainly
due to co-infection with HIV (AIDS) in immunocompromised pa-
tients. It is estimated that 15 million people are simultaneously in-
fected with HIV and MTB, and around 0.5 million of the 1.36
million HIV-associated deaths in 2007 were directly attributable
to TB.4 An additional contributing factor is the continued emer-
gence of new MDR–MTB strains,5 often associated with poor com-
pliance to treatments and, since 2006, to the officially recognized
appearance of MTB strains with extended resistance (XDR-MTB),6
now spread to more than 50 countries.7 Furthermore, TB treatment
protocols are prolonged (six months), complex and rather expen-
sive and in the majority of cases end in the poor patient
compliance.
Due to the facts mentioned above and to TB latency new anti-TB
drugs and better therapeutic strategies against TB are urgently
needed. New drug candidates should shorten the standard treat-
ments and be sufficiently effective against MDR-TB. Though several
compounds are currently in advanced phases of clinical assay, dur-
ing some forty years no new compounds have been introduced in
the market for TB treatment. However, in recent years an empha-
sized research activity in the development of new TB drugs has
been produced. Some compounds are, at present, 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.8 Crossing the vast, heavy and most complex polypeptide–poly-
glycoside–polylipidic mycobacterial wall is one of the major prob-
lems these compounds have to overcome.9
Sphingosine (SPH, Fig 1) is a basic component of sphingolipids
and ceramides. It is biosynthesized from serine and palmitic acid
through its saturated precursor dihydrosphingosine (DSPH). Sphin-
gosine 1-phosphate (S1P), the activated form of SPH, is involved in
a wide spectrum of biological processes, including Ca2+ mobiliza-
tion, cell growth, differentiation, motility and cytoskeleton organi-
zation.10 Most interestingly it has been reported that the roles of
* Corresponding author. Fax: +34 923294515.
E-mail address: olmo@usal.es (E. del Olmo).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.07.147

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5764–5768
5765
For several years our research group has been working on the
synthesis of this type of family of compounds and has reported on
the results derived from several bioactivity tests, including phos-
pholipase A2 inhibition, anti-inflammatory and anti-parasitic eval-
uations.14a–e Further support for the hypothetical antimycobacterial
activity of these compounds were found in recent descriptions of
the potential interest of NSAID (through inhibition of prostaglandin
biosynthesis)15 and immune-modulation (through induction/
inhibition of selective cytokines)16 drugs for anti-TB therapy. Both
aspects, respectively represented by PLA2 inhibition,14a lymphopro-
liferation inhibition17 and vaccination adjuvation18 have been
documented previously by us for some substances belonging to
the series included in this research. Experimental results from such
diverse bioactivity evaluations reported in our previous papers, also
support the selection of the palmitic size (tetradecyl side-chain) as
the most promising for a better antimycobacterial activity of these
compounds, while facilitating comparisons on the influences due
to the range of functionalities implemented in the structures.
The synthesis of several series of lipidic aminoalcohols and
diamines has been reported previously by us.14 Here we include
only a brief description of the procedures followed to obtain those
compounds tested in this research (Scheme 1). In summary, the
Boc-protected aminoalcohol 1a, used as starting material, was
transformed into its benzyl ether 2. Boc-deprotection in acid con-
ditions gave the benzyl ether-amine 3. This intermediate 3, was
monoalkylated and transformed into the secondary amines 4a
and 4e, by treatment with either ethyl bromide or ethyl bromace-
tate, respectively. Amides 4c and 4d were obtained by acylation
with succinyl and glutaryl anhydrides, respectively. The tertiary
amine 4b resulted from dialkylation of 3 with ethyl bromide ex-
cess, while catalytic hydrogenolysis of the benzyl ether 4b led to
the free primary alcohol 5b.
Diamine derivatives were also prepared from compound 1a. The
hydroxyl group was first mesylated, then transformed into the cor-
responding azide and further reduced to the diamine 6a. Treat-
ment of 6a with either one or two equivalents of ethyl bromide
gave predominantly compounds 7a and 7b, respectively. The reac-
tion of 6a with glutaric anhydride yielded compound 7c, while its
alkylation with an excess of ethyl bromacetate led to the disubsti-
tuted amine 7d. Compound 7b was Boc-deprotected with acid to
yield the diamine 8b.
1-Aminohexadecan-2-ol derivatives were prepared from 1-
hexadecene (9), through epoxidation with meta-chloroperbenzoic
acid, followed by oxirane opening with cyclohexylamine or benzyl-
amine leading to aminoalcohols 11a and 11b, respectively, in good
yields (Scheme 2). The inclusion of only two compounds with in-
verted functionality in this study, as well as the selection of benzyl-
amine and cyclohexylamine, was due to several reasons. According
to the structure correlations shown in Figure 1 and to the fact that
the primary alcohol of SPH constitutes the actual activating phos-
phorylation target, the expectancy of activity for compounds con-
taining the inner fragment (III) of DSPH, with the secondary
alcohol, should be lower than that expected for those containing
the outer (I) fragment. The benzyl group as an N-substituent had
not been considered in compounds of types 4–8. It could easily
be removed and replaced by any other group, thus opening the
route to the needed or desired chemodiversity. Moreover, the
interest of the cyclohexyl fragment can be focused on its own sec-
ondary nature which could mimic the isopropyl group, also absent
in compounds 4–8 and, due to its potential free rotation around the
N-cyclohexyl bond, it could also mimic t-Bu, adamantanyl and
bulkier radicals.
Figure 1. The structural basis of this research.
SPH, S1P, ceramides and other lipid components on MTB containing
endosomes lead to lysosome activation in the MTB-infected cell.11
This observation could support the use of sphingoanalogues in the
configuration of effective drug regimes for attacking the human
MTB reservoir, the inveterate latent TB, affecting one third of the
planet’s population.
From the structural point of view, SPH and DSPH contain, as a
pseudo-symmetric functional arrangement, a duplicate aminoeth-
anol (hydroxyethylamine) fragment also present in duplicate
though separated by an ethylene bridge, in the structure of one
of the first-line anti-TB drugs, ethambutol (EMB, Fig. 1). In addition,
EMB contains the central ethylenediamine bridge, which is also
necessary for its antimycobacterial activity. Studies of the mecha-
nism of action of EMB have revealed its ability to interfere with the
integrity of the mycobacterial cell wall through the inhibition of
arabinogalactan biosynthesis,12 though other possible pathways
have also been proposed.13
These facts prompted us to consider the evaluation, against
MTB, of a selected number of linear aminoethanol and ethylenedi-
amine derivatives with the objective of discovering new potential
anti-TB agents. As can be seen in Fig. 1 the compounds selected
for evaluation (types I, II and III) contain fragments closely related
to DSPH and to EMB. In this preliminary in vitro evaluation we
have only included saturated and structurally simplified racemic
compounds, chemo-modulated mainly through common and sim-
ple alkylation/acylation of functional groups, in order to configure
false DSPH analogues containing either original or masked phar-
macophoric fragments of EMB to provide more polar and more sol-
uble or latentized forms.
Fifteen compounds of those represented in Schemes 1 and 2,
being four aminoalcohols, five aminoethers and six diamine deriva-
tives, were selected and submitted to the in vitro MABA anti-MTB as-
say.19–21 The MIC values found are shown in Table 1, along with the

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E. del Olmo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5764–5768
Scheme 1. Synthesis of some dihydrosphingoanalogues. Reagents and conditions: (i) BnCl/NaH/DMF; (ii) HCl/THF; (iii) EtBr/Et3N/DMF; (iv) glutaric anhydride/CH2Cl2; (v)
succinic anhydride/CH2Cl2; (vi) ethyl bromacetate/CH2Cl2; (vii) H2/Pd–C/AcOH; (viii) (a) MsCl/Et3N/CH2Cl2; (b) NaN3/DMF; Pd–C/THF/NaBH4/MeOH.
Table 1
MCPBA
Antimycobacterial effect of dihydrosphingoanalogues on sensitive (H37Rv) and MDR
O
(CIB99) MTB strainsa
H3C
85%
H3C
Type
Compd
H37Rv
M)
CIB99
M)
13
13
b
b
9
10
MIC(
l
PE
MIC(
l
PE
I 2-amino-1-alkanol
1a
1b
3
4a
4b
4c
4d
4e
5b
84
24
0.12
0.41
2.8
0.6
3.2
0.07
0.14
0.13
0.5
84
49
>5
RNH2 85%
>16
>43
>9.5
>50
>1.1
>2.3
>2.2
>3.9
NHR
OH
3.6
16.6
3.1
140
68
3.6
16.6
3.1
140
68
11
a
R
H3C
cyclohexyl
b
Bn
13
72
20
72
40
11
II 1,2-alkane-diamine
6a
6b
7a
7b
7c
7d
8b
30
12.6
32
15.2
133
59
0.3
0.8
0.3
0.65
0.07
0.17
2.5
30
12.6
32
15.2
133
59
>5.2
>12
>4.9
>10
>1.2
>2.7
>19
Scheme 2. Synthesis of 1-aminohexadecan-2-ol derivatives from 1-hexadecene.
value for EMB, for a close structure–activity comparison and the va-
lue for rifampicin (RMP) as reference of another first-line pharma-
ceutical, a very effective and potent anti-TB drug, which acts
through RNA-polymerase inhibition. Due to large differences in
molecular weight between compounds and standard drugs, MIC val-
4.0
8.0
III 1-amino-2-alkanol
Ref. drugs
11a
11b
33
36
0.30
0.28
11
72
>14
>2.2
ues are expressed in lM units rather than in lg/mL, most commonly
EMB
RMP
9.9
0.152
1
650
>158
>2.43
1
ꢀ65
found in the literature reporting screening anti-MTB results, to pro-
vide adequate comparisons of antimycobacterial potencies. Bold-
face numbers in each column represent MIC values lower than those
corresponding to EMB, the reference drug, structurally close to the
compounds evaluated. Antimicrobial potencies relative to EMB
(PE), calculated for both, sensitive and MDR strains by means of
CIB99 is a complete SIREP-resistant MTB strain.19
Anti-MTB potency, relative to that of eMB. [PE = MICE (lM)/MICCOMP (lM)].
a
b
being evaluated and to the differences in substituents with respect
to the other sub-series. Furthermore, compounds of Type I and
Type II seem to behave in an almost parallel manner, though with-
in all the results obtained only one direct comparison can be made.
the equation PE = MICE (lM) /MICCOMP(lM) are also included.
As can be observed the compounds of the three different series
were, in general, active against sensitive and MDR–MTB and better
against the MDR strain in comparison with the reference drug
EMB. It can also be seen that both Type I and Type II sub-series
contain representative compounds which were more potent than
EMB. Nevertheless, such considerations cannot be extended to
Type III derivatives due to the reduced number of compounds
In this case, the double primary 1,2-diamine 6b (12.6
one-dilution-level more potent than the free 2-aminopalmitol 1b
(24 M).
lM) resulted
l
One of the most interesting facts observed is related to the anal-
ogy of the relative anti-MTB potencies calculated for compounds 3

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5764–5768
5767
(PE value >43) and 4b (>50) and that estimated for the highly po-
tent anti-TB drug RMP (PE ꢀ65) in the case of the MDR strain. Other
PE bold-face values in the CIB99 column correspond to compounds
with antimycobacterial potency one-order or higher than that of
EMB against the MDR strain. Another observation relates to the
negative influence of the Boc-protecting group on the activity,
which can be observed either against the sensitive as the MDR
than to the other MDR strains included in Table 2. It is well known
that resistance to anti-mycobacterial agents is due to the presence
of mutations in one or more genes.23 Furthermore, within one gene
several mutations could appear which would produce different
grades of resistance against the same compound.24 Thus, possibly
one or more mutations could be present in CIB99, but not in the
other tested strains, making CIB99 more susceptible to 8b; or,
reciprocally, the other strains could have a particular mutation ab-
sent in CIB99 inducing resistance to the diamine 8b, but not affect-
ing the susceptibility to aminoalcohol derivatives 3 and 4b.
Nevertheless, this matter would need the characterization of the
complete genome of all the strains used in the present study.
In summary, representative compounds of the two main types
(I and II) of the dihydrosphingoanalogues tested in this study have
shown a fair in vitro anti-MTB activity against sensitive and MDR
strains and their potential utility as anti-TB drugs seems to merit
further consideration. On the basis of their behavior against MDR
strains compounds 3 and 4b have been sent for their acute toxicity
to be evaluated (LD50), before carrying out in vivo assays, necessary
to ascertain their real anti-TB effectiveness in infected mice.
Molecular modeling calculations, ADME and toxicity prediction,
involving compounds with some changes in functionalities and
side-chain sizes of analogues, as well as preparative synthetic
work, aiming to extend the evaluation to a larger number of com-
pounds, are being performed; particularly for compounds of type
III. Other studies to define the precise target(s) for these com-
pounds in the mycobacteria, oriented to establish the actual mech-
anism of action, are also being planned.
strain, for the pairs of compounds 1a/1b (84 vs 24
for H37Rv MICs), 6a/6b (30 vs 12.6 M) and 7b/8b (15.2 vs
4.0 M). On the other hand, the presence of the benzyl ether moi-
lM, respectively,
l
l
ety compares positively with its absence in the alcohol derivatives
tested, as can be seen for the pairs of compounds 3/1b (3.6 vs
24
l
M) and 4b/5b (3.1 vs 20
lM).
Relating to substituents attached to the amine groups, the neg-
ative influence mentioned above for N-Boc protection, can be ex-
tended to hemisuccinyl and hemiglutaryl amides belonging to
both the aminoalcohol and the diamine series, with respect to
unsubstituted, mono- or dialkylated amines. This can be seen for
Type I amides 4c, 4d (140, 68
compounds 3, 4a, 4b (3.6, 16.6, 3.1
l
M, respectively) in comparison with
M) and Type II amide 7c
M) as compared with compounds 6b, 7a, 7b (12.6, 32,
M). The analysis of the above values, in brackets, also dem-
l
(133
15.2
l
l
onstrates that unsubstituted and dialkylated amines are rather bet-
ter than those monosubstituted analogues. Finally, comparison of
anti-MTB potencies of ethyl (4a, 7b; 16/15.2
lM) or methoxycar-
bonylmethyl (4e, 7d; 72/59 M) alkylated amines give preference
l
for the first group. Nevertheless, more compounds with other sub-
stituents of different size and constitution should be prepared and
tested to attain a better definition of their influence on anti-MTB
activity.
Acknowledgements
Confirmatory assays were carried out with the most potent
antimycobacterial compounds 3, 4b and 8b22 on eleven MTB
strains with different MDR profiles. The results are reported in Ta-
ble 2 and confirmed the potentiality of two of these compounds
against multi-resistant MTB strains. As can be seen, while the dia-
mine derivative 8b showed a one-order reduced potency, com-
pounds 3 and 4b presented a sustained activity against most
MDR strains. Thus, compound 3 was similarly effective on strains
No.401 and No.366, resistant to four of the first-line drugs, and
on strain No. 411, resistant to all of them. Similarly, the diethyl-
amine derivative 4b, was effective on strain No. 363, I,E-resistant,
on strain No. 366, resistant to four TB drugs and on strain No.
535, resistant to all of them. Interestingly, compound 4b, with an
Collaborative work performed under the auspices of Programa
Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED),
Project X.11: PIBATUB. Financial support came from Spanish Min-
ister of Science (Grant: AGL-2005-02168 GAN). SSF and GMMS
wish to thank the Instituto Mexicano del Seguro Social for financial
support, which allowed the development of antituberculosis bioas-
says. We would like to thank Mr. G. H. Jenkins for his help with the
English version of the ms.
References and notes
1. http://www.who.int/tb/publications/global_report/2009/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. WHO report 2009. Global Tuberculosis Control: Epidemiology, Strategy, Financing,
Geneva, p 10.
MIC value of 1.9 lM, resulted even more effective against strain
No.332, resistant to all the first-line anti-TB drugs. It is interesting
to note that compound 8b was much more active against CIB99
5. http://www.who.int/tb/challenges/mdr/faqs/en/index.html
6. Gandhi, N. R.; Moll, A.; Sturm, A. W.; Pawinski, R.; Govender, T.; Lalloo, U.;
Zeller, K.; Andrews, J.; Friedland, G. Lancet 2006, 368, 1575.
7. http://www.who.int/tb/challenges/xdr/en/index.html
Table 2
Antimycobacterial profile of compounds 3, 4b and 8b on MDR–MTB strains
8. Ahirrao, P. Mini-Rev. Med. Chem. 2008, 8, 144.
MTB strain No.a
Resistance profile S I R E P
MIC (lM)
9. Minnikin, D. E.; Kremer, L.; Dover, L. G.; Besra, G. S. Chem. Biol. 2002, 9, 545.
10. Lynch, K. R.; Macdonald, T. L. Biochem. Biophys. Acta 2008, 1781, 508.
11. Russell, D. G. Nat. Cell Biol. 2003, 5, 776; Anes, E.; Kühnel, M. P.; Bos, E.; Moniz-
Pereira, J.; Haberman, A.; Griffiths, G. Nat. Cell Biol. 2003, 5, 793.
12. Takayama, K.; Kilburn, K. O. Antimicrob. Agents Chemother. 1989, 33, 1493;
Bhowruth, V.; Alderwick, L. J.; Brown, A. K.; Bhatt, A.; Besra, G. S. Biochem. Soc.
Trans. 2008, 36, 555.
13. (a) Cole, A.; May, P. M.; Williams, D. R. Agents Actions. 1981, 11, 296; (b) Solecki,
T. J.; Aviv, A.; Bogden, J. D. Toxicology 1984, 31, 207; (c) King, A. B.; Schwartz, R.
J. Nutri. 1987, 117, 704.
14. (a) Lucas, R.; Ubeda, A.; Paya, M.; Alves, M.; Olmo, E.; Lopez, J. L.; San Feliciano,
A. Bioorg. Med. Chem Lett. 2000, 10, 285; (b) Olmo, E.; Alves, M.; Lopez, J. L.;
Inchaustti, A.; Yaluff, G.; Rojas de Arias, A.; San Feliciano, A. Bioorg. Med. Chem
Lett. 2002, 12, 659; (c) Olmo, E.; Macho, M.; Alves, M.; Lopez, J. L.; Banaua, F.;
Muñoz, E.; San Feliciano, A. Bioorg. Med. Chem Lett. 2002, 12, 2621; (d) Lucas, R.;
Alves, M.; Olmo, E.; San Feliciano, A.; Paya, M. Biochem. Pharmacol. 2003, 65,
1539; (e) Lucas, R.; Villamon, E.; Paya, M.; Alves, M.; Olmo, E.; Gozalbo, D.; Gil,
M. L. FEMS Immunol. Med. Chem. 2004, 40, 239.
3
4b
8b
528
160
401
494
411
331
429
366
363
332
535
H37Rv
R R R R Rb
R R R R R
R R R S R
R R R S R
R R R R R
R R R R R
S R R R R
R R R R S
S R S R S
R R R R R
R R R R R
S S S S S
18
15.5
7.7
7.7
7.7
7.7
15.5
7.7
3.9
3.9
1.9
3.9
3.1
40
40
40
40
40
40
40
20
40
40
40
4.0
9.0
4.5
9.0
4.5
9.0
9.0
4.5
18
36
18
3.6
SIREP: Streptomycin, Isoniazid, Rifampicin, Ethambutol, Pyrazinamide.
a
Reference characterisation No. given at the CIBIN-IMSS Research Centre.
R: resistant and S: sensible, to the corresponding drug above.
15. Rangel, J.; Estrada, I.; Garcia, M. I.; Aguilar, D.; Marquez, R.; Hernández-Pando,
R. Immunology 2002, 106, 257.
b

5768
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5764–5768
16. Rook, G. A. W.; Lowrie, D. B.; Hernández-Pando, R. J. Infect. Dis. 2007, 196, 191.
17. Olmo, E.; Plaza, A.; Muro, A.; Martínez-Fernández, A. R.; Nogal-Ruiz, J. J.; López-
Pérez, J. L.; San Feliciano, A. Bioorg. Med. Chem. Lett. 2006, 16, 6091.
18. (a) Uribe, N.; Siles-Lucas, M.; López-Abán, J.; Esteban, A.; Suarez, L.; Martínez-
Fernandez, A. R.; Olmo, E. del.; Muro, A. Vaccine 2007, 25, 4533; (b) Siles-Lucas,
M.; Uribe, N.; López-Abán, J.; Vicente, B.; Orfao, A.; Nogal-Ruiz, J. J.; San
Feliciano, A.; Muro, A. Vaccine 2007, 25, 7217; (c) López-Abán, J.; Nogal-Ruiz, J.
J.; Vicente, B.; Diez-Baños, P.; Hillyer, G. V.; Martínez-Fernández, A. R.; San
Feliciano, A.; Muro, A. Vet. Parasitol. 2008, 153, 176.
re-incubated at 37 °C for 24 h. Pink and blue colours 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.
21. Molina-Salinas, G. M.; Ramos-Guerra, M. C.; Vargas-Villarreal, J.; Mata-
Cárdenas, B. D.; Becerril-Montes, P.; Said-Fernández, S. Arch. Med. Res. 2006,
37, 45.
22. Analytical data for compound 3: white crystal, mp: 72 °C. IR
mmax: 3379, 293,
1589, 1459, 1362, 1204, 900 and 736 cmÀ1 1H NMR (200 MHz, CDCl3): d (ppm)
.
19. 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. The CIBIN/
UMF28:099 (CIB99) strain was isolated from a patient with advanced lung TB
and characterised in the Mycobacteria laboratory of Centro de Investigaciones
Biomédicas del Noreste-IMSS, Monterrey, N.L., Mexico. This strain was resistant
to all the above mentioned drugs. Its MIC values for S, I, R and E, determined by
7.33 (5H, m, Ar); 4.53 (2H, s, CH2–Ar); 3.46 (1H, dd, J1 = 8.9, J2 = 3.6 Hz, H-1A);
3.23 (1H, dd, J1 = 8.9, J2 = 7.9 Hz, H-1B); 2.99 (1H, m, H-2); 1.25 (26H, m,
(CH2)13); 0.91, (3H, t, J1 = 6.1, CH3).). 13C NMR (50.3 MHz, CDCl3): d (ppm)
138.8, 128.4, 127.7, 75.8, 73.2, 51.1, 34.2, 31.7, 29.7, 29.4, 26.1, 22.7, 14.2.
HRMS: Calcd: 348.3244 (M++H); found: 348.3261. Anal. Calcd for C23H41NO: C,
79.48; H, 11.89; N, 4.03. Found: C, 79.46; H, 11.83; N, 4.05.
MABA, were respectively higher than 13.8, 15.0, 1.2 and 157
l
M. Using the
Analytical data for compound 4b: Yellow oil. IR
mmax: 2923, 2854, 1696, 1459,
conventional BACTEC 460-radiometric system (Bactec 460).25 This strain was
resistant to the critical antimicrobial concentration of S, I, R, E and P. Other
MDR–MTB strains (Table 2) were characterised similarly.
1371, 1205, 1107, 906 and 734 cmÀ1 1H NMR (200 MHz, CDCl3): d (ppm) 7.33
.
(5H, m, Ar); 4.51 (2H, s, CH2–Ar); 3.56 (1H, dd, J1 = 9.6, J2 = 5.7 Hz, H-1A); 3.35
(1H, dd, J1 = 9.6, J2 = 5.3 Hz, H-1B); 2.81 (1H, m, H-2); 2.53 [4H, m, (N–
CH2CH3)2]; 1.26 [26H, m, (CH2)13); 1.02 [6H, t, J = 7.1, (N–CH2CH3)2]; 0.88, (3H,
t, J1 = 6.8, CH3).). 13C NMR (50.3 MHz, CDCl3): d (ppm) 138.7, 128.4, 127.8, 73.2,
70.7, 59.2, 44.3, 32.0, 29.8, 29.4, 29.0, 27.1, 22.8, 14.2. HRMS: Calcd: 404.3906
(M++H); found: 404.3902. Anal. Calcd for C27H49NO: C, 80.33; H, 12.23; N, 3.47.
Found: C, 80.31; H, 12.19; N, 3.45.
20. Assay: The modified Microplate Alamar Blue Assay (MABA)21 was used to
determine the anti-MTB activity. Briefly, solutions of compounds (2 mg/mL,
DMSO), standard drugs, RMP (1
sterilised by filtration (PTFE acrodiscs, 0.22
l
g/mL, DMSO), EMBÁHCl (2 mg/mL, H2O), were
lm
pore size, Millipore Co.,
Bedford, MA, USA). Std drugs were divided in 0.5 mL aliquots, and stored at
À70 °C until used, while the compounds were assayed immediately. Deionised
Analytical data for compound 8b: Oil. IR
mmax: 3373, 2922, 2852, 1691, 1519,
sterile water (200
l
L) was dispensed in the perimeter wells to diminish
1389, 1251, 1173 and 1053 cmÀ1 1H NMR (200 MHz, CDCl3): d (ppm) 4.59 [1H,
.
evaporation. The inner wells were divided in lanes of 6 to perform the assays.
m, NHCOOC(CH3)3]; 3.52 (1H, m, H-2); 2.50 [4H, q, J = 7.1, (N–CH2CH3)2]; 2.34
(2H, d, J = 6.8, H-1); 1.24 [26H, m, (CH2)13); 1.44 [9H, s, NHCOOC(CH3)3]; 0.98
[6H, t, J = 7.1, (N–CH2CH3)2]; 0.87, (3H, t, J1 = 6.5, CH3). 13C NMR (50.3 MHz,
CDCl3): d (ppm) 156.1, 78.9, 57.2, 49.4, 47.4, 33.8, 31.9, 29.7, 29.4, 28.5 25.8,
22.7, 14.2, 11.8. HRMS: Calcd: 413.4086 (M+ + H); found: 413.4079. Anal. Calcd
for C25H52N2O2: C, 72.76; H, 12.70; N, 6.79. Found: C, 72.75; H, 12.68; N, 6.73.
23. Rattan, A.; Kalia, A.; Ahmad, N. Emerg. Infect. Dis. 1998, 4, 195.
24. Whelen, A. C.; Felmlee, T. A.; Hunt, J. M.; Williams, D. L.; Roberts, G. D.;
Stockman, L.; Persing, D. H. J Clin. Microbiol. 1995, 33, 556.
25. Siddiqi, S. BACTEC-460 TB System, product and procedure manual. Revision E.
1996. Becton Dickinson Mycrobioly Systems, Sparks, MD.
100
l
L of a 1:50 bacterial suspension (having about 6 Â 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
dilutions within the ranges: 1–100
(RMP) and 1.0–32 g/mL (EMB). A blank (100
mycobacterial growth control (100 of 1:50 bacterial suspension) were
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
Alamar Blue plus 10% Tween-80 (v/v) were added to each well. The plates were
l
L
per well). Evaluations were performed on
g/mL (compounds), 0.062–2.0 g/mL
L of medium), and a positive
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l
l
l
L
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