Dual-protected amino acid derivatives as new antitubercular agents

Article abstract of DOI:10.1111/cbdd.13315

Tuberculosis is an infectious disease with high incidence and growing drug-resistant rates. In an attempt to develop new antitubercular agents, 35 compounds were synthesized, most of them bearing a carbamate and enantiopure amino acid moiety. These compounds had their activity evaluated toward a Mycobacterium tuberculosis strain (ATCC 27294) and cytotoxicity against fibroblast MRC-5 cells (ATCC CCL-171). Three of the prepared derivatives presented a good antimicrobial inhibition and two of them a moderate cytotoxicity. The lipophilicity seems to play a vital role in the cell growth activity, with best results for the derivatives with a higher logP.

Full text of DOI:10.1111/cbdd.13315

PROFESSOR GIOVANNI WILSON AMARANTE (Orcid ID : 0000-0003-1004-5395)
Article type : Research Letter
Dual-protected amino acid derivatives as new antitubercular agents
Pedro P. de Castro1, Débora L. Campos2, Fernando R. Pavan2 and Giovanni W. Amarante1
1Department of Chemistry, Federal University of Juiz de Fora, Campus Martelos, Juiz de Fora, Minas
Gerais 36036-900, Brazil
2 Department of Biological Sciences, School of Pharmaceutical Science, São Paulo State University,
Araraquara, São Paulo 14800-903, Brazil
Correspondence:
- Giovanni W. Amarante
E-mail: giovanni.amaranteufjf.edu.br
Abstract: Tuberculosis is an infectious disease with high incidence and growing drug-resistant rates.
In an attempt to develop new antitubercular agents, 35 compounds were synthesized, most of them
bearing a carbamate and enantiopure amino acid moiety. These compounds had their activity
evaluated towards a M. tuberculosis strain (ATCC 27294) and cytotoxicity against fibroblast MRC-5
cells (ATCC CCL-171). Three of the prepared derivatives presented a good antimicrobial inhibition
and two of them a moderate cytotoxicity. The lipophilicity seems to play a vital role in the cell
growth activity, with best results for the derivatives with a higher logP.
Keywords: Mycobacterium tuberculosis, cytotoxicity, carbamate, amino acid
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doi: 10.1111/cbdd.13315
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1. Introduction
Nowadays, tuberculosis is one of the most relevant diseases, being in the top 10 causes of
death worldwide.[1] The treatment of this pathology remains as a challenge, mainly due to the
emergence of multi-drug resistant M. tuberculosis strains, resulting in an alarmingly high incidence.[2]
In an attempt to increase the effectiveness of the treatment and avoid drug-resistance development,
even the standard first-line treatment consists in the concomitant administration of four drugs
(rifampicin, isoniazid, ethambutol and pyrazinamide). Nevertheless, a continuous increase in the
cases of drug-resistant and multidrug-resistant strains has lead tuberculosis as a major public health
problem.[3]
Furthermore, the conventional antitubercular chemotherapy has serious drawbacks, such as
a long treatment (that can last for 6-9 months), medication side-effects and in some cases the drug
administration route.[4] In this context, the development of new antitubercular drugs, preferentially
with new mechanisms of action, is essential for the treatment of multi-drug and extensively-drug
resistant M. tuberculosis and for an improvement in the pharmacotherapy.[5]
The carbamate moiety is present in several antimicrobial medicines with different activities,
such as antihelmintic (albendazole, mebendazole), antibacterial (linezolid, cefoxitina, novobiocin,
telithromycin, solithromycin and cethromycin) and antiviral (efavirenz, ritonavir, amprenavir,
atazanavir and darunavir), making it an important pharmacophore in the rational drug-design of new
antimicrobial drugs.[6–9] Besides, derivatives bearing a carbamate scaffold have been previously
prepared and investigated towards drug-resistant M. tuberculosis.[10]
Another interesting moiety for the design of candidates for new drugs consists of amino
acids. These cores have both carboxyl and an amine groups attached to a chiral center, allowing
functionalization of both sides of the amino acid scaffold.[11,12] Furthermore, an interesting aspect of
the use of amino acid in drug-design against bacteria strains consists in the presence of D-amino
acids in bacteria cells, especially in peptidoglycan[13–15]. Thus, the use of L-amino acids derivatives can
avoid metabolism by bacteria enzymes and present an enhanced activity.
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We envisioned that the coupling of a carbamate and an amino acid moiety would result in
new derivatives with potential applications as antitubercular candidates. Besides, we also thought
that functionalization of the amino acid carboxyl group employing a wide scope of amines would
lead to greater stability (especially if compared to the ester analogues, which can suffer enzymatic
degradation) and allow the control of the lipophilicity of these compounds.
2. Chemistry
2.1. Reagents and materials
All starting materials and chemicals were used as received, without further purification.
Solvents were dried following standard procedures. Analytical TLC was performed on TLC plates
(silica gel 60 F254) and visualized employing a ninhydrin (2,2-dihydroxyindane-1,3-dione) indicator.
All the tested compounds had satisfactory purity (≥95%), as verified by 1H and 13C NMR
spectroscopy, high resolution mass spectroscopy, infrared and chiral HPLC analysis.[16]
2.2. Synthesis of dual-protected amino acid derivatives
The dual-protected amino acid derivatives were prepared following a methodology
developed by our research group[16] (Scheme 1). Initially N-Boc protected amino acids were
synthesized by the following method: to an amino acid solution (L-alanine, L-valine, L-leucine, L-
isoleucine or L-phenylalanine - 10.0 mmol) in water/1,4-dioxane (3:2 v/v), 0.5 g of Na2CO3 was
added, followed by addition of Boc anhydride (15.0 mmol, 1.5 eq). The reaction mixture was stirred
for 18h and then purified following literature protocols.[17]
The N-Boc amino acids (1.0 mmol) were then added in a flask with dichloromethane and
EDC·HCl (1.1 mmol) was added. After stirring at 0 °C for half an hour, the crude reaction was washed
twice with distilled water and the dichloromethane immediately transferred to a flask containing the
amine nucleophile (2.0 mmol) and the (±)-camphorsulfonic acid (23 mg, 10 mol %) organocatalyst.
The reaction was kept stirring at room temperature for 24h and then concentrated in vacuo. The
desired compounds were then purified by recrystallization in hot EtOH/water, liquid−liquid
extraction, or column chromatography.
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For compound 7, the N-alkyldiamine 35 has been synthesized employing a literature
protocol[18] (Scheme 2) and then employed as a nucleophile as described above. Compound 35 has
also been evaluated towards its antitubercular activity, in an attempt to better compare the
influence of the incorporation of the amino acid moiety. Boc deprotection of the products 29 and 17
in the presence of trifluoroacetic acid has also been carried out, affording respectively the salts 33
and 34.
3. Biological evaluation
3.1. Minimum inhibitory concentration (MIC90) assay
The antitubercular activity of the compounds 1-35 was determined by the Resazurin
Microtiter Assay method (REMA).[19] Stock solutions of the tested compounds were prepared in
DMSO at 10 mg/mL and diluted in Middlebrook 7H9 broth (Difco) supplemented with oleic acid,
albumin, dextrose and catalase (OADC), to obtain the final drug concentration range of 0.09–25
µg/mL. Isoniazid, a first-line antimycobacterial drug, was dissolved in DMSO and used as standard
drug. A suspension of the M. tuberculosis H37Rv (ATCC 27294) was cultured in Middlebrook 7H9
broth supplemented with OADC. When the culture obtained a turbidity of 1 in McFarland standard,
it was adjusted to 3-5 × 105 colony-forming unities (CFU) per mL and then 100 µL of the inoculum
was added to each well of a 96-well microplate together with 100 µL of the compounds. Samples
were set up in triplicate. The plates were incubated for 7 days at 37 °C and 5% CO2. Resazurin
(solubilized in water) was added (30 µL of a 0.01% solution). The fluorescence of the wells was read
after 24 h with a Cytation 3 Imaging Reader (Biotek®). The MIC90 was defined as the lowest
concentration resulting in 90% inhibition of bacterial growth.
3.2. Citotoxicity against MRC-5 cells
Human fetal lung fibroblasts, MRC-5 cells (ATCC CCL-171), was used to determine the IC50 of
the compounds. The cells were cultured in DMEM medium (Vitrocell®), enriched with 10% fetal
bovine serum with gentamicin sulfate (50 mg/L) and amphotericin B (2 mg/L). Cells were cultured in
flasks and incubated at 37 °C with 5% CO2 until reaching cell confluence. The cells (2.5 x 105 cells/mL)
were seeded into a 96-well microplate that was incubated for 24 h at 37 ° C with 5% CO2 to allow cell
adhesion. After this time, the compounds were added to the cells in a dilution range of 0.39-100
μg/ml with 24 h incubation at the same conditions. After that, 50 μL of 0.01% resazurin (solubilized
in sterile and distilled water) was added and, after 3h, the fluorescence reading was performed using
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the Synergy H1 reader (Biotek®) at 530/590 nm. The IC50 value was defined as the lowest
concentration in which 50% of the cells remained viable.[20]
4. Results and discussion
The lipophilicity of a compound is one of the most important physicochemical properties,
that has direct consequences on the ability of the substance to penetrate in biological
membranes.[21] Thus, to measure this parameter the logP, defined as the partition coefficient
between 1-octanol and water, is usually employed. In an attempt to correlate the antitubercular
activity and the lipophilicity, the log P was calculated using ChemBioDraw Ultra 14.0 (CambridgeSoft)
(Table 1).[22]
As shown in Table 1, most of the compounds were inactive towards M. tuberculosis cells.
However, four of the products (4, 5, 7 and 35) showed considerable inhibition (below 40 μM) of the
cellular growth and three of them below 20 μM. Most interesting, a direct correlation of an increase
in the logP and an enhancement in the antitubercular activity could be observed, with better
inhibition for the most lipophilic compounds (logP > 6).
The importance of an increase in the lipophilicity can also be highlighted when performing a
comparison of compounds 1-5: when smaller side-chains (6-10 carbons) were employed, no
inhibition of cellular growth was detected; an increase of the side-chain to twelve carbon atoms
lowered the MIC90 to 39 μM; finally, the addition of two more carbon atoms lowered the MIC90 to
the best overall result, of 5 μM. The same analysis could be performed when comparing compounds
6, that had a lower lipophilicity and without antitubercular activity, and 7, in which a long side-chain
was added and showed a MIC90 of 6 μM. It is also worth mentioning that the cytotoxicity of
compound 5 in fibroblasts showed a value of 17 μM and a reasonable selectivity index of 3.4.
Another interesting observation is that the amino acid moiety seems to be important in the
activity. A comparison of the MIC90 of 35 (20 μM) and 7 (6 μM), showed a slightly enhancement in
the inhibition of M. tuberculosis cell growth by the addition of the Boc-amino acid group. This
scaffold also showed no influence on the cytotoxicity (Table 2), with the same result of 21 μM for
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both 7 and 35. The sum of the increase in the antitubercular activity, along with the same
cytotoxicity in fibroblasts, resulted in a better selectivity index of 3.5, when compared to only 1.1
(almost unselective) of the N-alkyldiamine 35.
Although isoniazid showed a greater activity than the synthesized compounds, it is worth
mention that this is an initial work that aims to develop new antitubercular agents based on a new
basic scaffold. Thus, the compounds do not possess a known pharmacophore present in clinical
antitubercular agents. Furthermore, the preparation of new derivatives based on the structure of
most active compounds and bearing similar physical-chemistry properties probably will lead to the
enhancement of the antitubercular activity and is under development by our research group.
5. Conclusion
In summary, a series of 35 compounds (including 32 dual-protected amino acid derivatives, 2
protected amino acid salts and an N-alkyldiamine) were synthesized and screened against a
Mycobacterium tuberculosis strain. Three of the prepared compounds exhibited good activities and
therefore are promising building blocks for new derivatives with enhanced activity. Since the most
active compounds bear a lipophilic side-chain and have the highest logP, it seems that these aspects
play an essential role in the increase of the activity. The N-alkyldiamine derivative (35) was less
active and had a lower selectivity index when compared to the compound bearing the amino acid
carbamate moiety (7), suggesting that these groups are important in the antitubercular activity and
in the increase of the selectivity towards bacteria cell. The most active compounds were assayed
towards their cytotoxicity in a fibroblast strain, with an IC50 between 17-21 μM and reasonable
selectivity index of up to 3.5. The preparation of new lipophilic derivatives based on the most
promising compounds is ongoing in our laboratory and will be reported in due course.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support from FAPEMIG, FAPESP, CAPES, CNPq and Rede
ineira de u mica. The authors thank Dr. Gustavo S. G. Carvalho for helpful suggestions.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
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ORCID
Pedro P. de Castro: 0000-0002-3018-3130
Giovanni W. Amarante: 0000-0003-1004-5395
List of figures and tables legends
Scheme 1. Preparation of compounds 1-34.
Scheme 2. Synthesis of product 35.
Table 1. Antitubercular activity against M. tuberculosis (ATCC 27294) and calculated logP for the
synthesized compounds.
Table 2. Cytotoxicity (IC50) towards pneumocytes and selectivity index for the most active
compounds.
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MIC90
(μM)
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
20
Compound
MIC90 (μM)
LogP
Compound
LogP
1
>67
>67
>67
39
3.46
4.30
5.13
5.96
6.80
0.59
6.15
3.12
1.81
3.49
3.05
2.70
3.05
3.88
2.92
2.91
2.30
0.99
19
0.99
1.88
2.23
2.67
1.22
-0.08
-0.08
0.80
1.15
1.59
1.94
0.63
1.52
2.31
ND
2
20
3
21
4
22
5
5
23
6
>67
6
24
7
25
8
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
>67
26
9
27
10
28
11
29
12
30
13
31
14
32
15
33
16
34
ND
17
35
4.11
ND
18
Isoniazid
0.2
* ND: not done.
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IC50
(μM)
17
Compound
Selectivity Index (SI)*
5
3.4
3.5
1.1
7
21
35
21
* Selectivity index calculated dividing IC50 by the MIC90 values.
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This article is protected by copyright. All rights reserved.