Synthesis, antimycobacterial and cytotoxic activity of α,β-unsaturated amides and 2,4-disubstituted oxazoline derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.01.024

The synthesis of six α,β,-unsaturated amides and six 2,4-disubstituted oxazolines derivatives and their evaluation against two Mycobacterium tuberculosis strains (sensitive H37Rv and a resistant clinical isolate) is reported. 2,4-Disubstituted oxazolines (S)-3b,d,e were the most active in the sensitive strain with a MIC of 14.2, 13.6 and 10.8?μM, respectively, and the compounds (S)-3d,f were the most active against resistant strain with a MIC of 6.8 and 7.4?μM. The ex-vivo evaluation of hepatotoxicity on precision-cut rat liver slices was also tested for the α,β-unsaturated amides (S)-2b and (S)-2d,f and for the oxazolines (S)-3b and (S)-3d,f at different concentrations (5, 15 and 30?μg/mL). The results indicate that these compounds possess promising antimycobacterial activity and at the same time are not hepatotoxic. These findings open the possibility for development of new drugs against tuberculosis.

Full text of DOI:10.1016/j.bmcl.2017.01.024

Accepted Manuscript
Synthesis, antimycobacterial and cytotoxic activity of α,β-unsaturated amides
and 2,4-disubstituted oxazoline derivatives
Francisco G. Avalos-Alanís, Eugenio Hernández-Fernández, Pilar Carranza-
Rosales, Susana López-Cortina, Jorge Hernández-Fernández, Mario Ordó ñez,
Nancy E. Guzmán-Delgado, Alejandro Morales-Vargas, Víctor M. Velázquez-
Moreno, María G. Santiago-Mauricio
PII:
S0960-894X(17)30037-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.024
BMCL 24600
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 November 2016
8 January 2017
9 January 2017
Please cite this article as: Avalos-Alanís, F.G., Hernández-Fernández, E., Carranza-Rosales, P., López-Cortina, S.,
Hernández-Fernández, J., Ordó ñez, M., Guzmán-Delgado, N.E., Morales-Vargas, A., Velázquez-Moreno, V.M.,
Santiago-Mauricio, M.G., Synthesis, antimycobacterial and cytotoxic activity of α,β-unsaturated amides and 2,4-
disubstituted oxazoline derivatives, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/
10.1016/j.bmcl.2017.01.024
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Synthesis, antimycobacterial and cytotoxic activity of
-unsaturated amides and 2,4-disubstituted oxazoline derivatives
Francisco G. Avalos-Alanísa, Eugenio Hernández-Fernándeza*, Pilar Carranza-Rosalesb*,
Susana López-Cortinaa, Jorge Hernández-Fernándeza, Mario Ordóñezc, Nancy E. Guzmán-
Delgadod, Alejandro Morales-Vargase, Víctor M. Velázquez-Morenoe, María G. Santiago-
Mauriciob
aUniversidad Autónoma de Nuevo León, Facultad de Ciencias Químicas, Pedro de Alba s/n, Ciudad Universitaria, 66400 San Nicolás de
los Garza, Nuevo León, México
bCentro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social. Monterrey, Nuevo León, México
cCentro de Investigaciones Químicas-IICBA, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001,
62209 Cuernavaca, Morelos, México
dUnidad Médica de Alta Especialidad 34. Instituto Mexicano del Seguro Social. 64730 Monterrey, Nuevo León, México
eLaboratorio Estatal de Salud Pública. Secretaría de Salud. 67180 Guadalupe, Nuevo León, México
Abstract: The synthesis of six ,-unsaturated amides and six 2,4-disubstituted oxazolines derivatives and their evaluation
against two Mycobacterium tuberculosis strains (sensitive H37Rv and a resistant clinical isolate) is reported. 2,4-Disubstituted
oxazolines (S)-3b,d,e were the most active in the sensitive strain with a MIC of 14.2, 13.6 and 10.8 µM, respectively, and the
compounds (S)-3d,f were the most active against resistant strain with a MIC of 6.8 and 7.4 µM. The ex-vivo evaluation of
hepatotoxicity on precision-cut rat liver slices was also tested for the ,-unsaturated amides (S)-2b and (S)-2d,f and for the
oxazolines (S)-3b and (S)-3d,f at different concentrations (5, 15 and 30 µg/mL). The results indicate that these compounds possess
promising antimycobacterial activity and at the same time are not hepatotoxic. These findings open the possibility for
development of new drugs against tuberculosis.
Keywords:
Organic synthesis
Unsaturated amides
,-2,4-Disubstituted oxazolines
Antimycobacterial activity
Hepatotoxic activity
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb)
that has become a serious global health problem. According to World Health Organization
(WHO) it is estimated that one-third of the world’s population is infected with Mtb.1,2
Patients infected with TB do not necessarily show any symptoms of the disease, but the
association of TB with human immunodeficiency virus (HIV) or its presence in patients
with diseases related to certain deficiencies in the immune system, would represent a high
risk of TB reactivation.3 Besides that, the effectiveness of anti-tuberculosis treatments is
affected by the emergence of multi- and extensively drug-resistant Mtb strains, along with
Corresponding author. Tel./Fax.: +52 (81) 8329 40 00, Ext 6293; E-mail: eugenio.hernandezfr@uanl.edu.mx
Corresponding author. Tel./Fax.: +52 (81) 8190 4036; E-mail: pilarcarranza@cibinmty.net

totally drug resistant strains.2,4 Therefore, exists a critical need and a growing interest for
novel intervention strategies related to anti-TB agents with enhanced activity, reduced
toxicity and good effect in the intracellular environment.5,6
In recent years, synthesis and biological evaluation of several compounds bearing amides
or oxazolines in their structures have shown a wide variety of interesting biological
activities, including antimycobacterial effect. Cinnamic acid-based drug derivatives
(amides, thioesters and hydrazides) display antioxidative, antitumor, anti-hepatitis C virus,
antimicrobial activity and several other biological properties.7 On the other hand, the
oxazoline based compounds have shown remarkable activity against Mtb with extremely
low toxicity, and therefore high therapeutic indexes, as well as activity against even the
more recalcitrant non-replicant forms of Mtb.8
Based on the above, the design of new compounds bearing these features, amide and
oxazoline groups, represents an important challenge given their potential application as
alternative anti-TB drugs. A goal of our research group during the last few years has been
the development of fast and effective synthesis of anti-TB agents.9 We herein report the
synthesis of α,β-unsaturated amides under microwave irradiation as an alternative
preparation method, which were converted into 2,4-disubstituted oxazolines. Additionally,
we also report the antimycobacterial and hepatotoxic activity of these α,β-unsaturated
amides and 2,4-disubstituted oxazolines.
For the synthesis of the target compounds 2 and 3, in the first step we carried out the
Horner-Wadsworth-Emmons reaction of the -phosphonoamide10 1 bearing L-
phenylalaninol group with commercially available aryl aldehydes such as benzaldehyde, 4-
fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-methoxybenzaldehyde, 4-(benzyloxy)-
benzaldehyde and 2-thiophenecarboxaldehyde, in the presence of K2CO3 and acetonitrile as
solvent under microwave irradiation at 100 ºC for 40 min, obtaining the α,β-unsaturated
amides (S)-2a-f in 51 to 86% yield. The second step was carried out following the
procedure recently described by our research group.9 Thus, the α,β-unsaturated amides (S)-
2a-f were reacted with SOCl2 in acetonitrile at reflux for 4 h in the presence of NaBr and

K2CO3, obtaining the 2,4-disubstituted oxazolines (S)-3a-f in 30 to 85% yield. The results
are shown in Table 1.
Table 1. Preparation of α,β-unsaturated amides (S)-2a-f and 2,4-disubstituted oxazolines
(S)-3a-f.
Entry
Product
Yield (%)a
Product
3a; Ar = C6H5
Yield (%)a
1
2
3
4
5
6
2a; Ar = C6H5
60
86
58
53
51
57
60
30
32
85
64
63
2b; Ar = 4-FC6H4
2c; Ar = 4-ClC6H4
2d; Ar = 4-MeOC6H4
2e; Ar = 4-BnOC6H4
2f; Ar = 2-thienyl
3b; Ar = 4-FC6H4
3c; Ar = 4-ClC6H4
3d; Ar = 4-MeOC6H4
3e; Ar = 4-BnOC6H4
3f; Ar = 2-thienyl
a Isolated after purification by column chromatography.
The most important contribution of the proposed method was the reduced reaction time of
the Horner-Wadsworth-Emmons step for the synthesis of the amide derivatives (S)-2a-f.
The synthesis was carried out in only 40 min by the microwave assisted method compared
to 4 h by the conventional reported method.9
It is also important to mention that the compounds (S)-2f and (S)-3f have not been
previously reported elsewhere, therefore they represent structural novelty derivatives.
1
The H NMR and 13C NMR data for the synthesized compounds correspond to those
previously reported by our research group confirming their structures. The data of the
newly synthesized compounds (S)-2f and (S)-3f also correspond to the expected.
In this work, we decided to evaluate the compounds (S)-2a-f and (S)-3a-f making a
comparison between amides and oxazolines ,-unsaturated with different groups only on
C-4 position in phenyl ring based on the results reported by others research groups,11 where
they explored the Structure-Activity-Relationship (SAR) and described that the substituents

on the position C-4 in the phenyl ring are more actives than compounds substituted on C-3
and C-2 position.
The antimycobacterial activity of the twelve synthetic compounds (S)-2a-f and (S)-3a-f,
and four pharmacological controls, Isoniazid (I), Rifampicin (R), Ethambutol (E) and
Ofloxacin (Ofx) were evaluated under the same experimental conditions using the
microplate Alamar blue assay.12 Chemical structures are shown in Figure 1.
Figure 1. Compounds evaluated against Mycobacterium tuberculosis strains.
The synthesized compounds (S)-2a-f and (S)-3a-f were evaluated against two
Mycobacterium tuberculosis strains, the H37Rv strain which is sensitive to the first-line
drugs Isoniazid, Rifampicin and Ethambutol; and a clinical isolate which is resistant to
treatment with Isoniazid, Rifampicin and Ethambutol drugs. The tested concentrations were
256, 128, 64, 32, 16, 8, 4, 2 and 1 µg/mL. In particular, the α,β-unsaturated amides (S)-2a,

(S)-2e and (S)-2f showed the lowest antitubercular activity with MIC = 56.8, 41.2 and 55.6
M, for the two tested strains (Table 2, entries 1, 5 and 6); whereas α,β-unsaturated amides
(S)-2b, (S)-2c and (S)-2d showed a better activity with MIC values of 26.7, 25.3 and 25.6
M for the H37Rv strain (Table 2, entries 2, 3 and 4), these values are similar to
Ethambutol drug (39.1 M) under the same study conditions.
On the other hand, oxazolines (S)-3a, (S)-3c and (S)-3f showed the highest MIC values
against the H37Rv strain (60.8, 26.9 and 29.7 M, respectively). However, a better activity
was observed with the oxazolines (S)-3b, (S)-3d and (S)-3e (14.2, 13.6 and 10.8 M,
respectively) for the same strain; these MIC values are similar to the reference drugs
Ethambutol and Rifampicin (39.1 and 4.8 M). It is important to mention that the
compounds (S)-3d and (S)-3f tested for the resistant strain (isolated clinical) were the most
active with MIC values of 6.8 and 7.4 M, these values are better than Isoniazid (58.3 M),
Rifampicin (19.4 M) and Ethambutol (>1,252.9 M).
These results seem to suggest that these molecules possess promising therapeutic potential,
especially against drug-resistant strains. MIC values for the anti-mycobacterial effect of all
tested compounds are summarized in Table 2.

Table 2. Antimycobacterial activity of α,β-unsaturated amides (S)-2a-f and oxazolines (S)-
3a-f.
MIC = µg/mL (µM)
Compound
MolWt
CLogPb
Sensitive Strainc
(H37Rv)
Resistant Straind
(clinical isolated)
16 (56.8)
281.34
299.33
315.79
311.37
387.47
287.37
263.33
281.32
297.78
293.36
369.46
269.36
137.14
820.96
204.31
361.14
3.071
3.214
3.784
2.99
16 (56.8)
2a
2b
2c
2d
2e
8 (26.7)
8 (25.3)
8 (25.6)
16 (41.2)
16 (55.6)
16 (60.8)
4 (14.2)
8 (26.9)
4 (13.6)
4 (10.8)
8 (29.7)
<1 (<7.2)
4 (4.8)
16 (53.4)
16 (50.6)
16 (51.3)
16 (41.2)
16 (55.6)
8 (30.4)
16 (56.9)
8 (26.9)
2 (6.8)
4.758
2.717
4.639
4.782
5.352
4.558
6.326
4.285
-0.668
NC
2f
3a
3b
3c
3d
3e
3f
Ia
Ra
Ea
Ofxa
8 (21.6)
2 (7.4)
8 (58.3)
16 (19.4)
>256 (>1,252.9)
2 (5.5)
0.1188
-0.507
8 (39.1)
<1 (<2.7)
aPharmacologic controls. I = Isoniazid, R = Rifampicin, E = Ethambutol, Ofx = Ofloxacin.
bCLogP was calculated using the Cambridge Soft ChemDraw Ultra 8.0 software.
cSensitive to the first-line drugs I, R, E. dResistant to the first-line drugs I, R, E.
To carry out the ex-vivo evaluation of hepatotoxicity on precision-cut rat liver slices, we
selected the ,-unsaturated amides (S)-2b, (S)-2d and (S)-2f and the oxazolines (S)-3b,
(S)-3d and (S)-3f for two reasons, the first one was to make a comparison between amides
and oxazolines containing the same type of substituent in its structure, and the secondly it
was because the majority of these compounds are the most active compounds against MTb
and therefore they were selected in order to evaluate their safety to hepatic tissue. In this
sense, we evaluated the hepatotoxic potential of these compounds by using precision-cut rat
liver slices, an ex-vivo-3D assay.13 Different concentrations (5, 15, and 30 µg/mL) were
chosen based on the results of Anti-MTb activity and Isoniazid was included as the
reference control. Because none of the active compounds were hepatotoxic, we show
representative images of liver slices incubated with oxazoline (S)-3d, one of the most
effective Anti-MTb compounds (Figure 2).

Figure 2. Representative photomicrographs of the cytotoxicity of compounds on precision-
cut liver slices. Light bright field microscopy. H&E staining (40x).
As it can be seen, liver slices cultivated with (a) culture medium only (control) and (b)
0.5% DMSO (the solvent used to dissolve the compounds, negative control), show the
characteristic histological architecture of normal hepatic tissue, with hepatocyte cords,
portal triads (PT) and central veins (CV) well preserved. In liver slices cultured in the
presence of 5, 15, and 30 µg/mL (c, d, and e, respectively) of compound 3d, which showed
the most relevant Anti-MTb activity, hepatotoxic effect was not observed; as in controls (a
and b) where well preserved hepatic cords, portal triads and central veins were observed.
By contrast, in liver slices incubated with 5, 15, and 30 µg/mL of Isoniazid (f, g, and h,
respectively) a marked histological damage was observed, being notable the presence of
periportal inflammatory infiltrate (arrows) and centrilobular necrosis (dotted lines).

Histopathological analysis of the liver slices incubated during 24 h in the presence of the
compounds possessing the most significant anti-MTb activity demonstrates that none of
them are hepatotoxic because liver parenchyma, sinusoidal spaces and portal triads are well
preserved in all the tested concentrations (Figure 2, c-e). Conversely, isoniazid (Figure 2, f-
h) induces balonoid degeneration of the hepatocytes, foci of inflammatory cells, and
cellular necrosis, especially at 15 and 30 µg/mL concentrations. Regarding to the last
findings, it has been reported that this drug causes direct hepatotoxic damage which is
characterized by centrilobulillar necrosis because throughout its metabolism in the liver it
induces the elevation of enzymatic activity and release of toxic secondary metabolites.14
In summary, the results of this study have demonstrated in a general way that oxazolines
(S)-3a-e are more active than the ,-unsaturated amides (S)-2a-e. In the series of ,-
unsaturated amides screened, the compounds bearing the substituent fluoro, chloro and
methoxy at C-4 position of the phenyl group [(S)-2b-d], were the most active compounds in
the sensitive strain (H37Rv) shown the MIC value of 26.7, 25.3 and 25.6 µM, respectively.
However, the antimycobacterial activity of these same compounds in the resistant strain
was not better. On the other hand, in the series of oxazoline derivatives, compounds (S)-3b,
(S)-3d and (S)-3e bearing the substituent fluoro, methoxy and benzyloxy at C-4 position of
the phenyl group, were the most active in the sensitive strain. It is important to mention that
oxazoline (S)-3d and (S)-3f derivatives were the most active in the resistant strain and even
were more active than Isoniazid, Rifampicin and Ethambutol and very similar to Ofloxacin.
The hepatotoxic activity using precision-cut rat liver slices for the most active compounds
(S)-2b, (S)-2d, (S)-2f, (S)-3b, (S)-3d and (S)-3f were evaluated and none of these
compounds were hepatotoxic. More detailed and complete studies of the biological activity
of these compounds are currently in progress as they are needed to explore the mechanism
of antimycobacterial activity.
Acknowledgments
The authors thank to CONACYT of México for the financial support via projects 101898
and 181816. We thank V. Labastida-Galván for the determination of the HRMS. F.G.A.A.
also thanks CONACYT for a Graduate Scholarship.

Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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Graphical Abstract
Synthesis, antimycobacterial and cytotoxic activity of -unsaturated amides and 2,4-
disubstituted oxazoline derivatives.
Francisco G. Avalos-Alanís a, Eugenio Hernández-Fernández a,*, Pilar Carranza-Rosales b,*, Susana
López-Cortinaa, Jorge Hernández-Fernández a, Mario Ordóñez c, Nancy Elena Guzmán-Delgado d,
Alejandro Morales-Vargas e, Víctor M. Velázquez-Moreno e, María Guadalupe Santiago-Mauriciob
aUniversidad Autónoma de Nuevo León, Facultad de Ciencias Químicas, Pedro de Alba s/n, Ciudad
Universitaria, 66400 San Nicolás de los Garza, Nuevo León, México
bCentro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social., Monterrey,
Nuevo León, México
cUniversidad Autónoma del Estado de Morelos, Centro de Investigaciones Químicas-IICBA, Av.
Universidad 1001, 62209 Cuernavaca, Morelos, México
dUnidad Médica de Alta Especialidad # 34. Instituto Mexicano del Seguro Social. Monterrey, NL,
CP 64730. México
eLaboratorio Estatal de Salud Pública. Secretaría de Salud. Guadalupe, NL, CP 67180. México
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