Synthesis and antimycobacterial activity of novel amino alcohols containing central core of the anti-HIV drugs lopinavir and ritonavir

Article abstract of DOI:10.1111/j.1747-0285.2011.01244.x

Eleven new amino alcohol derivatives have been synthesized from reactions of lopinavir intermediate and heteroaromatic aldehyde in good yields. These compounds, the antiretrovirals (lopinavir and ritonavir) and lopinavir key intermediate were evaluated as

Full text of DOI:10.1111/j.1747-0285.2011.01244.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01244.x
Chem Biol Drug Des 2011; 78: 1031–1034
Research Letter
Synthesis and Antimycobacterial Activity of
Novel Amino Alcohols Containing Central Core of
the Anti-HIV Drugs Lopinavir and Ritonavir
Claudia R. B. Gomes1, Marcele Moreth1,
emerged in the mid-1980s. The coinfected HIV-TB is a major world-
wide health problem, as the HIV-positive patients are more prone to
infection by M. tuberculosis, because the infection depends on state
of the immune system, which is completely impaired in these
Danielle Cardinot1, Valquiria Kopke1,
Wilson Cunico1,2, Maria Cristina da Silva
Lourenc¸o3 and Marcus V. N. de Souza1,*
patients.
1Instituto de Tecnologia em Fꢀrmacos – Farmanguinhos, FioCruz –
It is estimated that 15 million people are simultaneously infected
with HIV and M. tuberculosis, and around 0.5 million of the 1.36
million HIV-associated deaths in 2007 were directly attributable to
TB.a An additional contributing factor for the resurgence of TB is
the rapid spread of the multidrug-resistant TB because of inconsis-
tent or partial treatment and the lack of new drugs in the market.
Recently, another important problem is the advent of XDR-TB
(extensively drug-resistant TB), which is resistant to all first and
second line anti-TB drugs (3).b
Fundażo Oswaldo Cruz, R. Sizenando Nabuco, 100 Manguinhos,
21041-250 Rio de Janeiro, RJ, Brazil
2Centro de CiÞncias Quꢁmicas FarmacÞuticas e de Alimentos, UFPel,
Campus Universitꢀrio s ⁄ no, 96010-900 Pelotas, RS, Brazil
3Instituto de Pesquisas Clꢁnicas Evandro Chagas, Departamento de
Bacteriologia, FioCruz – Fundażo Oswaldo Cruz, Av. Brasil, 4365
Manguinhos, Rio de Janeiro, Brazil
*Corresponding author: Marcus V. N. de Souza,
marcos_souza@far.fiocruz.br
Eleven new amino alcohol derivatives have been
synthesized from reactions of lopinavir intermedi-
ate and heteroaromatic aldehyde in good yields.
These compounds, the antiretrovirals (lopinavir
and ritonavir) and lopinavir key intermediate were
evaluated as antibacterial agents against Myco-
bacterium tuberculosis H37Rv using the Alamar
Blue susceptibility test and their activity
expressed as the minimum inhibitory concentra-
tion (MIC) in lM. Ten amino alcohols evaluated dis-
played significant activity (MIC between 6.15 and
108.4 lM) when compared to first-line drug etham-
butol (MIC = 15.9 lM). Three of them showed more
activity than ethambutol (MIC = 6.15; 6.21 and
13.4 lM). The appreciable activity of these com-
pounds can be considered an important finding for
the rational design of new leads for anti-TB com-
pounds.
Nowadays, there is no treatment anti-TB specific for AIDS patients.
Thus, an effective single drug active against infections caused by
both HIV and M. tuberculosis is a very interesting approach in the
search for new drugs to treat coinfected patients.
In this context, amino alcohols come as a very important and versa-
tile class of compounds owing to its significant applications in
many fields, such as in synthetic and medicinal chemistry. Different
compounds containing the amino alcohol moiety have been synthe-
sized, evaluated, and used for the treatment of various diseases
(4,5), such as ethambutol (Figure 1), a first-line drug used in the
treatment of TB (6). Another example is hydroxyethylamine deriva-
tives, which have the capacity to inhibit aspartic protease enzymes
and are widely used in HIV treatment (7,8). This class of compounds
is also been investigated as antimalarial (9–13) and antileishmani-
ose (14) agents.
Key words: amino alcohol, drugs, lopinavir, ritonavir, tuberculosis
Another interesting class of compounds is Schiff base derivatives
based on amino alcohols, because of their functional similarities
with hydrazones, which exhibited remarkable anti-TB activity (15–
19). Besides, these compounds present significant lipophilicity,
which could facilitate their entry into the intracellular environment.
Received 24 March 2011, revised 23 August 2011 and accepted for
publication 11 September 2011
Considering the TB-HIV coinfection problem, and the importance
of amino alcohol derivatives in both diseases, the aim of this
article is to evaluate the in vitro antibacterial activity against
M. tuberculosis of several imine compounds of this class based
on the central core of the antiretrovirals lopinavir and ritonavir
(Figure 1).
Tuberculosis (TB) is a chronic bacterial infection transmitted through
the air. This disease is caused by the bacteria Mycobacterium tuber-
culosis and mainly affects the lungs (pulmonary TB), which is respon-
sible for more than 75% of cases (1,2). Different factors are
responsible for the resurgence of TB, such as AIDS epidemic, which
1031

Gomes et al.
H3C
CH3
Ph
H3C
CH3
Ph
O
O
O
O
H
H
N
O
CH3
N
S
N
H3C
H3C
HN
N
N
H
N
N
H
N
H
O
N
O
OH
CH3
O
OH
S
Ph
H3C
Ph
Ritonavir
MIC > 138.7 µM
Lopinavir
MIC > 159.0 µM
Ph
OH
HO
H2N
Ph
H
N
NH2
H3C
CH3
N
H
OH
Ethambutol
MIC = 15.9 µM
Central core of Lopinavir and Ritonavir
MIC = 88.0 µM
Figure 1: Lopinavir and ritonavir: anti-HIV drugs and ethambutol.
Table 1: Yields and selected physical properties of compounds
1–8, 1a–4a and the in vitro antimycobacterial activity of these
compounds and standard drugs ritonavir (RTV), lopinavir (LPV) and
ethambutol (EMB)
Materials and Methods
Chemicals
The synthesis of amino alcohol derivatives 2–4 involved the reac-
tion between four heteroaromatic aldehydes and the intermediate
of lopinavir 1 under reflux in ethanol for 6 h, leading to the desired
compounds in 69–74% yields.c Following that, products 2–4 plus
compound 1 were deprotected with trifluoroacetic acid in dichlo-
romethane at room temperature for 4 h affording the free amine
1a–4a (Scheme 1, Table 1).d
Product Heterocyclic (Het)
Yielda (%) LC-MS (m ⁄ z) MIC (lM) LogPb
1
–
–
384.4
523.2
462.3
507.2
549.4
461.3
578.3
462.7
307.2
432.3
362.2
407.3
–
130.1
6.21 7.582
13.4
6.15 6.940
5.016
2
5-nitro-thiophen-2-yl 69
imidazol-2-yl
5-nitro-furan-2-yl
4-(pyridine-2-yl)phenyl 71
pyrrol-2yl
thiophen-2-yl
furan-2-yl
–
3
70
74
5.828
4
5
91.0
8.233
6.755
7.499
6.858
1.273
3.839
2.085
3.197
7.506
5.259
0.350
6
67
68
73
65
108.4
104.5
108.1
88.0
7
8
Anti-mycobacterial activity
1a
2a
3a
4a
RTV
LPV
EMB
The antimycobacterial activities of compounds 1–8, 1a–4a and
standard drugs lopinavir, ritonavir, and ethambutol have been
assessed against M. tuberculosis ATTC 27294 using the microplate
Alamar Blue assay (20) (Table 1). This methodology is non-toxic,
uses a thermally-stable reagent, and shows good correlation with
proportional and BACTEC radiometric methods (21,22). The method
is described as follows: 200 mL of sterile deionized water was
added to all outer-perimeter wells of sterile 96-well plates (falcon,
3072; Becton Dickinson, Lincoln Park, NJ, USA) to minimize evapo-
ration of the medium in the test wells during incubation. A stock
solution of compounds 1–8, 1a–4a was prepared in DMSO at
100 lg ⁄ mL. The 96-well plates received 100 mL of the Middlebrook
5-nitro-thiophen-2-yl 68
imidazol-2-yl
5-nitro-furan-2-yl
–
–
–
59.1
63
65
–
>276.1
>245.5
>138.7
>159.0
15.9
–
–
–
–
aYields of isolated compounds; bLipophilicities were expressed as logP val-
ues (commercially available program http://www.molinspiration.com/cgi-bin/
properties).
7H9 broth (Difco laboratories, Detroit, MI, USA) and successive dilu-
tion of the compounds 2a–j and 3a–o was made directly on the
plate. The final drug concentrations tested were 0.01–20.0 lg ⁄ mL.
Plates were covered and sealed with parafilm and incubated at
37 ꢀC for 5 days. Twenty-five mililitre of a freshly prepared 1:1 mix-
ture of Alamar Blue (Accumed International, Westlake, OH, USA)
reagent and 10% Tween-80 were then added to the plate and incu-
bated for 24 h. A blue color in the well was interpreted as no bac-
terial growth, and a pink color was scored as growth. The minimal
inhibition concentration (MIC) was defined as the lowest drug con-
centration, which prevented a color change from blue to pink.
Ph
Ph
NH2
Ph
NH2
Ph
ii
H2N
BocHN
OH
OH
1
1a
i
Ph
Ph
N
Het
N
Het
ii
H2N
BocHN
OH
2a-4a
OH
Results and Discussion
Ph
Ph
2-8
All the compounds synthesized were identified by spectral data. In
the 1H NMR spectrum, the compounds 2–4 and 2a–4a showed
the imine proton (N=C-H) as a singlet at d 7.60–8.01 p.p.m. The
Scheme 1: Reaction conditions: i: HetCHO, EtOH reflux, 6 h; ii:
TFA ⁄ CH2Cl2 (1 ⁄ 3), r.t., 4 h.
1032
Chem Biol Drug Des 2011; 78: 1031–1034

Synthesis and Antimycobacterial Activity of Novel Amino Alcohols
13C NMR spectrum of substances 2–4 exhibit the C=O signals at d
151.1–155.8 p.p.m.
3. De Souza M.V.N. (2006) Current Status and future prospect for
new therapies for pulmonary tuberculosis. Curr Opin Pulm
Med;12:167–171.
These substances 2–4 and 1a–4a, the antiretrovirals (lopinavir
and ritonavir) and lopinavir intermediate (1) were evaluated as anti-
bacterial agents against M. tuberculosis H37Rv using the Alamar
Blue susceptibility test and their activity expressed as the MIC in
lM (Table 1).
4. Ferreira M.L., Vasconcelos T.R.A., de Carvalho E.M., LourenÅo
M.C.S., Wardell S.M.S.V., Wardell J.L., Ferreira V.F., de Souza
M.V.N. (2009) Synthesis and antitubercular activity of novel
Schiff bases derived from d-mannitol. Carbohyd Res;344:2042–
2047.
5. de Oliveira P.S.M., Ferreira V.F., de Souza M.V.N., de Carvalho
E.M. (2008) Synthesis of aminoalcohols from D-mannitol. Quꢀm
Nova;31:776–780.
6. Yendapally R., Lee R.E. (2008) Design, synthesis, and evaluation
of novel ethambutol analogues. Bioorg Med Chem Lett;18:1607–
1611.
7. Brik A., Wong C.-H. (2003) HIV-1 protease: mechanism and drug
discovery. Org Biomol Chem;1:5–14.
8. Ghosh A.K., Bilcer G., Schiltz G. (2001) Syntheses of FDA
approved HIV protease inhibitors. Synthesis;15:2203–2229.
9. Parikh S., Gut J., Istvan E., Goldberg D.E., Havlir D.V., Rosenthal
P.J. (2005) Antimalarial activity of human immunodeficiency virus
In the case of the anti-HIV lopinavir and ritonavir, both are inac-
tive against M. tuberculosis; however, its central core displayed a
MIC of 88.0 lM, indicating that modification into its structure
could lead an improvement in the biological activity. Our previous
work described compounds containing amino alcohol moiety with
better activity than compounds with Boc group (23); however, in
this work, the screening of the compounds synthesized containing
the Boc group 2–4 showed higher activity than the corresponding
free amines 1a–4a. Because of these results, we synthesized
more amino alcohols containing the Boc group, which showed
moderate to good antimycobacterial activity. The substances 2
(MIC = 6.21 lM), 3 (MIC = 13.4 lM), and 4 (MIC = 6.15 lM) dis-
played lowest values of MIC, being more active than the amino
alcohol first-line drug ethambutol (MIC = 15.9 lM). The lowest
MIC values of substances 2 and 4, when compared with com-
pounds 7 and 8 respectively, also demonstrate the importance of
the nitro group in heteroaromatic ring for the biological activity in
this class of compounds.
type
1
protease inhibitors. Antimicrob Agents Chemo-
ther;49:2983–2985.
10. Andrews K.T., Fairlie D.P., Madala P.K., Ray J., Wyatt D.M.,
Hilton P.M., Melville L.A., Beattie L., Gardiner D.L., Reid R.C.,
Stoermer M.J., Skinner-Adams T., Berry C., McCarthy J.S. (2006)
Potencies of human immunodeficiency virus protease inhibitors
in vitro against Plasmodium falciparum and in vivo against
murine malaria. Antimicrob Agents Chemother;50:639–648.
11. Cunico W., Ferreira M.L.G., Ferreira T.G., Penido C., Henriques
M.G.M.O., Krettli L.G., Varotti F.P., Krettli A.U. (2008) Synthesis
and Antimalarial Activity of Novel Hydroxyethylamines, Potential
Aspartyl Protease Inhibitors. Lett Drug Des Discov;5:178–181.
12. Cunico W., Gomes C.R.B., Moreth M., Manhanini D.P., Figueiredo
I.H., Penido C., Henriques M.G.M.O., Varotti F.P., Krettli A.U.
(2009) Synthesis and antimalarial activity of hydroxyethylpiper-
azine derivatives. Eur J Med Chem;44:1363–1368.
13. Cunico W., Gomes C.R.B., Facchinetti V., Moreth M., Penido C.,
Henriques M.G.M.O., Varotti F.P., Krettli L.G., Krettli A.U., da
Silva F.S., Caffarena E.R., de Magalh¼es C.S. (2009) Synthesis,
antimalarial evalution and molecular modeling studies of
hydroxyethylpiperazines, potential aspartyl protease inhibitors,
part 2. Eur J Med Chem;44:3816–3820.
Conclusion
In summary, we easily synthesized eleven amino alcohols deriva-
tives in moderate to good yields based on the central core of the
anti-HIV drugs lopinavir and ritonavir. Seven compounds (1, 5–8
and 1a–2a) exhibited a modest activity (MIC = 130.1; 91.0; 108.4;
104.5; 108.1; 88.0; 59.1 lM), respectively. The compound 3 pre-
sented MIC (13.4 lM) close to ethambutol (MIC = 15.9 lM), and 2
(MIC = 6.21 lM) and 4 (MIC = 6.15 lM) are more potent than eth-
ambutol. These compounds are not cytotoxic to host cells at the
concentrations effective in inhibiting M. tuberculosis infection. More
information regarding SAR and QSAR are currently under way in
our laboratory.
14. Savoia D., Allice T., Tovo P.-A. (2005) Antileishmanial activity of
HIV protease inhibitors. Int J Antimicrob Agents;26:92–94.
15. LourenÅo M.C.S., Ferreira M.L., De Souza M.V.N., Peralta M.A.,
Vasconcelos T.R.A., Henriques M.G.M.O. (2008) Synthesis and
anti-mycobacterial activity of (E)-N¢-(monosubstituted-benzylid-
ene)isonicotinohydrazide derivatives. Eur J Med Chem;43:1344–
1347.
16. Ferreira M.L., Cardoso L.N.F., GonÅalves R.S.B., Da Silva E.T.,
LourenÅo M.C.S., Vicente F.R., De Souza M.V.N. (2008) Synthesis
and antitubercular evaluation of N'-[(E) (hydroxy, methoxy and
ethoxy-substituted-phenyl) Methylidene]isonicotinohydrazide deriva-
tives. Lett Drug Des Discov;5:137–140.
Acknowledgments
The authors gratefully acknowledge Farmanguinhos and IPEC for the
financial support of the research. We also thank CNPq (V.K.) and
CIEE (D.C.).
References
1. De Souza M.V.N., Vasconcelos T.R.A. (2005) Drugs against tuber-
culose: past, present and future. Quim Nova;28:678–682.
2. De Souza M.V.N. (2006) Promising drugs against tuberculosis.
Recent Pat Anti-Infect Drug Discov;1:33–44.
17. LourenÅo M.C., De Souza M.V.N., Pinheiro A.C., Ferreira M.L.,
GonÅalves R.S.B., Nogueira T.C.M., Peralta M.A. (2007) Evalua-
tion of anti-tubercular activity of nicotinic and isoniazid ana-
logues. ARKIVOC;XV:181–191.
Chem Biol Drug Des 2011; 78: 1031–1034
1033

Gomes et al.
18. Taveira A.F., Le Hyriac M., Reis E.F.C., Araffljo D.P., Ferreira A.P.,
De Souza M.A., Alves L.L., LourenÅo M.C.S., Vicente F.R.C., De
Almeida M.V. (2007) Preparation and antitubercular activities of
alkylated amino alcohols and their glycosylated derivatives. Bio-
org Med Chem;15:7789–7794.
19. De Almeida M.A., Le Hyriac M., Amarante G.W., LourenÅo
M.C.S., Brand¼o M.L.L. (2007) Synthesis of amphiphilic galacto-
pyranosyl diamines and amino alcohols as antitubercular agents.
Eur. J. Med. Chem.;42:1076–1083.
20. Franzblau S.G., Witzig R.S., McLaughlin J.C., Torres P., Madico G.,
Hernandez A., Degnan M.T., Cook M.B., Quenzer V.K., Ferguson
R.M., Gilman R.H. (1998) Rapid, low-technology MIC determina-
tion with clinical mycobacterium tuberculosis isolates by using
the microplate Alamar blue assay. J Clin Microbiol;36:362–366.
21. Vanitha J.D., Paramasivan C.N. (2004) Evaluation of microplate
Alamar blue assay for drug susceptibility testing of Mycobacte-
rium avium complex isolates. Diagn Micr Infect Dis;49:179–182.
22. Reis R.S., Neves I. Jr, LourenÅo S.L.S., Fonseca L.S., LourenÅo
M.C.S. (2004) Comparison of flow cytometric and Alamar Blue
tests with the proportional method for testing susceptibility of
Mycobacterium tuberculosis to rifampin and isoniazid. J Clin
Microbiol;42:2247–2248.
Notes
aGlobal Tuberculosis Control: A short update to the 2009 report.
Available from World Health Organization 2009: http://www.who.
int/tb/publications/global_report/2009/update/tbu_9.pdf.
bTuberculosis (TB) Centers for Disease Control and Prevention. Avail-
able at: http://www.cdc.gov/tb/?404.
cGeneral procedure for the preparation of imine derivatives 2–8.
The amino alcohol derivatives 2–8 were prepared by reaction
between the appropriate heteroaromatic aldehydes (1.0 equiv.) with
intermediate of lopinavir 1 (1.0 equiv.) in ethanol (20 mL), under
nitrogen atmosphere. After stirring for 6 h at 80 ꢀC, the resulting
mixture was concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel, eluting with
hexane:ethyl acetate gradient (up to 100%) afforded the pure deriv-
atives 2–8.
dGeneral procedure for the preparation of compounds 1a–4a.
Trifluoracetic acid (1.5 mL, 20 mmol) was added to a solution of the
compounds 1–4 (2 mmol) in CH2Cl2 (6 mL). After 4 h, the solvent
was removed in high vacuum. The residue dissolved in EtOAc
(20 mL) was washed with 5% NaHCO3 aqueous solution, H2O, brine
and dried over MgSO4. The solvent was removed to afford the
compounds 1a–4a which was used without further purification.
23. Cunico W., Gomes C.R.B., Ferreira M.L.G., Ferreira T.G., Cardinot
D., de Souza M.V.N., LourenÅo M.C.S. (2011) Synthesis and
antimycobacterial activity of novel amino alcohol derivatives. Eur
J Med Chem;46:974–978.
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