Synthesis and anticancer evaluation of new lipophilic 1,2,4 and 1,3,4-oxadiazoles

Article abstract of DOI:10.1016/j.ejmech.2019.01.001

A series of1,2,4- and 1,3,4-oxadiazole derivatives were synthesized and evaluated for their anticancer activity. Halogenated 1,2,4-oxadiazoles were obtained from benzonitrile and coupled either lipophilic amines or with aminoalcohols. Lipophilic 1,3,4-oxadiazole derivatives were obtained through the Mannich reactions between 5-(aryl)-1,3,4-oxadiazole-2-thiol and alkylated or acylated amines. The in vitro cytotoxic effects were evaluated against 4T1– mammary carcinoma and CT26 – colon cancer cells. The best results were obtained for the 1,3,4-oxadiazole coupled to alkylated piperazine with 10–14 carbon chain moiety, with IC₅₀ values ranging from 1.6 to 3.55μΜ for the 4T1 cell line, and from 1.6 to 3.9 μM for the CT26.WT cell line, and selectivity index up to 19. The most potent compounds were investigated with AnnexinV and PI staining as indicative of apoptosis induction.

Full text of DOI:10.1016/j.ejmech.2019.01.001

Accepted Manuscript
Synthesis and anticancer evaluation of new lipophilic 1,2,4 and 1,3,4-oxadiazoles
Wiliam Caneschi, Karine Braga Enes, Camille Carvalho de Mendonça, Fábio de
Souza Fernandes, Fabio Balbino Miguel, Jefferson da Silva Martins, Mireille Le
Hyaric, Roberto Rosas Pinho, Lucas Mattos Duarte, Marcone Augusto Leal de
Oliveira, Hélio F. Dos Santos, Miriam Teresa Paz Lopes, Dalton Dittz, Heveline Silva,
Mara Rubia Costa Couri
PII:
S0223-5234(19)30001-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.001
EJMECH 11008
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 August 2018
Revised Date: 30 December 2018
Accepted Date: 1 January 2019
Please cite this article as: W. Caneschi, K.B. Enes, C. Carvalho de Mendonça, Fá. de Souza Fernandes,
F.B. Miguel, J. da Silva Martins, M. Le Hyaric, R.R. Pinho, L.M. Duarte, M.A. Leal de Oliveira, Hé.F. Dos
Santos, M.T. Paz Lopes, D. Dittz, H. Silva, M.R. Costa Couri, Synthesis and anticancer evaluation of
new lipophilic 1,2,4 and 1,3,4-oxadiazoles, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.01.001.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and anticancer evaluation of new lipophilic 1,2,4 and 1,3,4-oxadiazoles
Wiliam Caneschia, Karine Braga Enesa, Camille Carvalho de Mendonçaa, Fábio de Souza Fernandesa,
Fabio Balbino Miguela, Jefferson da Silva Martinsb, Mireille Le Hyarica, Roberto Rosas Pinhob,
Lucas Mattos Duartea, Marcone Augusto Leal de Oliveiraa, Hélio F. Dos Santosa, Miriam Teresa Paz
Lopesc, Dalton Dittzc, Heveline Silvad and Mara Rubia Costa Couria*
aDepartamento de Química, ICE, Universidade Federal de Juiz de Fora, Campus Martelo,
CEP 36036-330, Juiz de Fora, MG-Brazil
bDepartamento de Física, ICE, Universidade Federal de Juiz de Fora, Campus Martelo, CEP
36036-330, Juiz de Fora, MG-Brazil
cDepartamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Pampulha,
CEP 31270-901, Belo Horizonte, MG-Brazil
dDepartamento de Química, Universidade Federal de Minas Gerais, Pampulha, CEP 31270-
901, Belo Horizonte, MG-Brazil
*Corresponding author: mara.rubia@ufjf.edu.br
Keywords: oxadiazoles, anticancer activity, Mannich bases, lipophilicity
Abstract:A series of1,2,4- and 1,3,4-oxadiazole derivatives were synthesized and
evaluated for their anticancer activity. Halogenated 1,2,4-oxadiazoles were obtained
from benzonitrile and coupled either lipophilic amines or with aminoalcohols.
Lipophilic 1,3,4-oxadiazole derivatives were obtained through the Mannich reactions
between 5-(aryl)-1,3,4-oxadiazole-2-thiol and alkylated or acylated amines. The in vitro
cytotoxic effects were evaluated against 4T1– mammary carcinoma and CT26 – colon
cancer cells. The best results were obtained for the 1,3,4-oxadiazole coupled to
alkylated piperazine with 10-14 carbon chain moiety, with IC50 values ranging from 1.6
to 3.55µΜ for the 4T1 cell line, and from 1.6 to 3.9 µM for the CT26.WT cell line, and
selectivity index up to 19. The most potent compounds were investigated with
AnnexinV and PI staining as indicative of apoptosis induction.
1. Introduction
The great challenge for chemists is the discovery of new efficient compounds
with minimal side effects and use of small doses, and many studies have been
conducted for the development of new selective drugs. Heterocyclicsystems play an
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important role in the discovery of new bioactive substances, since they are present in
various natural bioactive compounds [1].Due to the high number of deaths [2], cancer
treatment has attracted much attention from researchers and industries for the synthesis
of new,more selective anticancer agents. Undesirable effects, toxicity, drug resistance
and reduced bioavailability are known for current anticancer available agents [3].
Therefore, the discovery of new anticancer agents, more efficient and more selective is
urgent.
Over the past years, 1,2,4- and 1,3,4-oxadiazole derivatives have been widely
studied for theirantiviral [4], antibacterial [5], antioxidant [6], anti-inflammatory [7],
antifungal activity [8] and anticancer activities [9].The biological potential of these
heterocycles against cancer cells has been reported with different mechanisms of action,
such as inhibition of tubulin, blocking endothelin A receptor involved in apoptosis,
mitogenesis, angiogenesis and metastasis in tumors,focal adhesion kinase inhibition,
telomerase inhibition, interacting with several receptors involved in proliferation, cell
growth and DNA biosynthesis [10-19]. The presence of the azole group in their
structures makes them more lipophilic and, therefore, more susceptible to passage
through the cell membrane[20].
Moreover, interesting activities,such as antitubercular, immunosuppressive and
antiparasitic, associated with lipophilic structureshave been reported in the literature
[21-24]. Lipophilic molecules can interact with cell membranes, increasing their
permeability and consequently disturbing membrane-embedded proteins, inhibiting
respiration, altering the ion transport process or leading to cell lysis [25]. Small
lipophilic molecules bearing a moiety with potential anticancer activity can display
enhanced anticancer activity, as they are more susceptible to uptake by the cancer cells,
where they can interact with a specific binding site.
Considering the importance of oxadiazoles, we report herein the synthesis of
1,2,4 and 1,3,4- oxadiazolederivatives coupled to lipophilic amines and aminoalcohols
as promising antitumor agents. The anticancer activity of the compounds was evaluated
by colorimetric MTT assay against two different cancer cell lines (4T1 – mammary
carcinoma, CT26.WT – colon cancer cells) and one non-tumor cell line (BHK-21 –
baby hamster kidney).
2. Results and Discussion
2.1- Chemistry
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The synthesis of the 5-(bromomethyl)-3-phenyl-1,2,4-oxadiazole2 was initiated
by the reaction between benzonitrile 1 and hydroxylamine hydrochloride in basic
solution, generating an amidoxime in 87% yield. The latter was treated with
bromoacetyl bromide and submitted to heating, leading to the formation of the desired
heterocyclic compound 2(74% yield), which underwent an SN2 type reaction with 1-
methylpiperazine, 2-methylpiperazine and different lipophilic amines and
aminoalcohols according to the literature [26], generating the final compounds 3-18.
Furthermore, compound 16,containing a piperazine moiety, was reacted with different
long chain alkylandacyl chlorides, leading to compounds 19-26 (Scheme 1).
The synthesis of 5-(aryl)-1,3,4-oxadiazole-2-thiol derivatives 34-37 was initiated
by the esterification reaction [27] of the carboxylic acids 27-29 in ethanolic sulfuric acid
solution. The aromatic esters were then reacted with hydrazine (68% m/v) [27],
generating the corresponding hydrazides 31-33. The synthesized hydrazides and
isoniazid 30 were reacted with carbon disulfide in basic solution for the formation of the
desired heterocycles (Scheme 2). Finally, the alkylated and acylated piperazine
derivatives, previously prepared [28], were reacted separately with 1,3,4-oxadiazoles
34-37 and formaldehyde (37% v/v) through a Mannich type reaction [29], giving the
desired products 38-69 (Scheme 3).
Chemical data of the final compounds are reported in Supporting Information and
were characterized by melting point, ¹H NMR, ¹³C NMR and mass spectroscopy
(MALDI-TOF) and LC-MS.
3 R= -H (85%)
R
N
OH
4 R= -(CH2)7CH3 (51%)
5 R= -(CH2)9CH3 (67%)
6 R=- (CH2)11CH3 (79%)
7 R= -(CH2)13CH3 (95%)
O
N
N
N
N
8 R= -(CH2)3CH3 (90%)
9 R= -(CH2)7CH3 (88%)
10 R= -(CH2)9CH3 (85%)
11 R=- (CH2)11CH3 (86%)
12 R= -(CH2)13CH3 (91%)
13 R= -CH2CH2NH2 (31%)
14 R= -CH2CH2CH2NH2 (44%)
O
N
HN
R
CN
O
N
2
OH
OH
Br
N
d
a, b, c
O
N
HN
HO
R
19 R= -(CH2)7CH3 (72%)
20 R= -(CH2)9CH3 (86%)
21 R= -(CH2)11CH3 (77%)
22 R= -(CH2)13CH3 (76%)
1
O
N
N
N
N
N
N
15 (82%)
e
R'
O
R
O
N
N
N
O
N
23 R= -(CH2)7CH3 (50%)
24 R= -(CH2)9CH3 (76%)
25 R= -(CH2)11CH3 (78%)
26 R= -(CH2)13CH3 (86%)
R
N
N
f
16 R=R'= -H (86%)
17 R= -H; R'= -CH3 (55%)
18 R= -CH3; R'= -H (81%)
Scheme 1: Synthesis of 1,2,4-oxadiazole derivatives coupled to amines and
aminoalcohols. Reactions conditions: a) H2NOH.HCl (1.5 eq.), NaHCO3(1.6 eq.),
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MeOH, reflux, 6h; b) bromoacetyl bromide (1.35 eq.), K2CO3(1.35 eq.), CHCl3, r.t., 3h;
c) toluene, 110°C, 5h; d) amines (2.0 eq.), K2CO3(2.0 eq.), CH3CN, 70°C, 1-5h; e) alkyl
halides (1,5 eq.), K2CO3(1.5 eq.), CH3CN, 70°C, 1-5h; f) acyl chlorides (1.5 eq.),
K2CO3 (1.5 eq.), CH3CN, r.t., 1-72h.
Scheme 2: Synthesis of 5-(aryl)-1,3,4-oxadiazole-2-thiol. Reaction conditions: a)
H2SO4 conc., EtOH, reflux, 20h; b) H2NNH2 (68% m/v) (2.0 eq.), EtOH, reflux, 20h; c)
CS2 (4.0 eq.), KOH (3.0 eq.), EtOH, 55°C, 48h.
Scheme3: Synthesis of 1,3,4-oxadiazole derivatives from 1,3,4-oxadiazole.
Reactionconditions: a) alkylated amines (1.0 eq.), HCHO (1.5 eq.), MeOH, r.t.; b)
acylated amines (1.0 eq.), HCHO (1.5 eq.), MeOH, r.t.
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2.2. Cytotoxicity
In order to study the structure activity relationship, fifty-six different lipophilic
1,2,4- and 1,3,4-oxadiazole derivatives, differing from the group attached to the
heterocycle, were analyzed in vitro by MTT assay for their anticancer activity against
tumor cell lines (4T1 – mammary carcinoma and CT26.WT – colon cancer cell line)
and non-tumor cell line (BHK-21 – baby hamster kidney), with different embryonic
origins (epithelial and fibroblast) used to evaluate the selectivity index. The results are
summarized in Table 1. Although similar, the best activities were accessed for those
compounds containing an alkylated piperazine moiety. In general, 1,2,4-oxadiazole
derivatives did not show significant results for the cell lines tested, with the exception of
compounds 20 and 21 containing piperazine bearing a ten or twelve carbon chain with
IC50 values of 3.6±0.5µM and 8.1±1.6 µM respectively, and a great selectivity index for
4T1 cell (18.4 and 8.8, respectively). Besides, this less lipophilic structures such as 3
and 13-17, containing primary alcohols, primary orsecondary amines did not show
cytotoxicity even for the BHK-21 (non tumor cell line). In 2008, Silva and co-workers
[30] investigated the anticancer activity of some lipophilic complexes and found that the
most active compounds were those containing a twelve-carbon chain. Our group also
reportedthe importance of the lipophilic chain in the anticancer activity, where
carbohydrate derivatives with a twelve carbon chain were the most potent for the cells
tested [24]. All these results suggest that the alkyl chain linked to the piperazine is
important for the anticancer activity of these compounds, mainly for those with twelve
carbons.
In the case of 1,3,4-oxadiazoles derivatives, the results showed some
relationship between biological activity and lipophilicity. In most cases, the more
lipophilic structures showed higher levels of cytotoxicity for the cells used, being
optimal for those structures containing a twelve carbon chain (Figure 1). In general,
1,3,4-oxadiazoles derivatives 38-53 containing an alkylated piperazine (Figure 1a and
1b) showed better results than those containing an acylated piperazine 54-69 (Figure 1c
and 1d), especially compound 41, being 65 times more specific for the 4T1 cell line
compared to reference drug. Furthermore, comparing the heterocycles studied, 1,3,4-
oxadiazolederivatives were more active than the 1,2,4 regioisomers. In addition, for the
1,3,4-oxadiazole regioisomer, the compounds containing phenyl and pyridine ring
weremore active against the 4T1 cell line while the compounds containing 4-methoxy
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phenyl were the most active against the CT26.WT cell line.For both cell lines tested,
optimal results were shown for structures containingtwelve carbonalkyl chain. For the
acylated compounds, the best results were obtained for the structures containing
fourteen carbons.The alkylated compounds 39-45 showed the most relevant results
against the 4T1 cell line, with values ranging from 1.6±0.3µM to 17.5±0.1µM and high
selectivity index, varying from 4.3 to 19.6, showing that the presence of an alkyl chain
with ten or twelve carbons was optimal for the activity against this cell line.
Compound45 had an interesting IC50 (3.1±0.4µM) but poor selectivity index when
compared to the normal cell, BHK-21. In the case of the CT26 tumor cell line,
compounds 47-49 were the most active (IC50 ranging from 1.6±0.7 µM to 3.9±0.1µM)
with thehighest specificity of compound 48 bearing a twelve carbons chain. Acylated
structures showed a tendency toincrease the anticancer activity with the increase of the
alkyl chain highlighting the importance of this moiety for the cytotoxicity. In addition,
the analysis of the best results obtained for 1,2,4-oxadiazole derivatives 19-21 and
1,3,4-oxadiazoles 38-41, demonstrates the importance of the effect of the heterocycle on
the cytotoxicity against tumor cells, highlighting the high selectivity index of these
compounds exhibited, especially those alkylated structures with ten- carbon atoms. The
results show a decrease in the activity of alkylated 1,2,4-oxadiazolic derivatives, while
there was an increase in activity for the 1,3,4-oxadiazole regioisomer with concomitant
increase in lipophilicity of the same (Figure 2).
Table 1. Cytotoxic activities against cell lines. IC50 (µM± SD)
Tumor Cells IC50
CT26.WT
>100
Non-Tumor Cells
BHK-21
>100
Compounds
4T1
>100
SI
SI
-
-
3
4
>100
-
>100
-
-
>100
-
82.3 ± 5.0
>100
>100
5
-
1.1
-
1.0
1.6
-
84.3 ± 0.4
30.2 ± 3.0
>100
87.3± 3.0
48.3 ± 3.0
>100
>100
42.7 ± 4.0
>100
6
7
8
2.1
-
2.0
-
45.1 ± 0.4
>100
94.6 ± 4.0
>100
46.5
9
>100
10
11
12
13
>100
-
>100
-
>100
>100
-
>100
-
>100
-
-
92.3 ± 4.0
>100
>100
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>100
-
-
>100
>100
-
-
>100
>100
14
15
16
17
18
19
20
21
22
23
24
25
26
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
>100
>100
-
>100
-
>100
>100
-
>100
-
>100
>100
-
>100
-
>100
0.8
1.1
0.9
-
4.7
18.4
8.8
-
12.0 ± 4.1
3.6 ± 0.5
8.1 ± 1.6
26.4 ± 9.1
66.8 ± 1.9
62.2 ± 1.4
54.6 ± 5.9
29.6 ± 3.5
17.5 ± 0.1
3.55 ± 0.2
2.55 ± 0.1
3.2 ± 2.4
10.9 ± 2.8
1.9± 0.1
1.6 ± 0.3
3.1 ± 0.4
83.7 ± 3.6
34.7 ± 1.7
24.4 ± 2.4
22.4 ± 2.4
69.7 ± 2.3
59.5 ± 1.1
77.7 ± 4.2
>100
56.3 ± 1.8
66.3 ± 1.1
71.4 ± 2.0
>100
0.5
0.4
0.6
0.8
-
0.3
0.5
0.5
0.4
-
96.5 ± 2.1
54.2 ± 1.2
63.8 ± 2.3
55.7 ± 7.8
34.4 ± 4.3
28.6 ± 0.6
32.5 ± 1.9
22.7 ± 0.5
>100
>100
0.8
0.7
1.6
-
6.4
6.4
19.6
-
29.0 ± 1.8
22.1 ± 1.2
38.2 ± 0.8
15.4 ± 5.0
15.4 ± 0.4
6.3 ± 0.1
7.7 ± 0.2
19.4 ± 1.8
3.9 ± 0.1
1.6 ± 0.7
3.4 ± 0.2
>100
22.9 ± 1.0
16.4 ± 2.6
62.7 ± 3.4
>100
2.0
3.7
1.7
3.8
5.6
13.8
6.2
-
16.7
14.5
4.3
0.9
0.6
0.9
0.9
-
31.8 ± 0.2
23.3 ± 3.7
13.4 ± 8.4
74.0 ± 4.2
21.9 ± 2.6
22.1 ± 0.2
21.1 ± 2.1
69.5 ± 1.9
24.0 ± 1.6
6.1 ± 0.2
0.9 ± 0.1
>100
>100
1.1
0.7
0.1
-
1.0
0.2
0.0
-
21.1 ± 0.4
8.2 ± 0.1
6.7 ±0.9
>100
25.1 ± 1.3
24.8 ± 1.9
22.5 ± 1.7
93.3 ± 1.0
50.6 ± 1.6
21.9 ± 0.5
9.5 ± 0.5
>100
0.9
1.2
1.0
-
1.5
1.2
2.1
-
82.6 ± 4.0
21.8 ± 1.6
20.1 ± 1.3
>100
76.1 ± 3.1
27.2 ± 1.1
19.8 ± 0.3
>100
0.9
1.1
0.7
-
1.0
1.6
1.0
-
68.1 ± 0.1
17.1 ± 2.0
12.6 ± 1.0
>100
61.3 ± 4.0
12.0 ± 5.0
8.4 ± 0.8
>100
64.6 ± 2.0
18.9 ± 1.7
8.6 ± 0.6
>100
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0.7
0.9
0.5
-
-
82.6 ± 2.6
24.0 ± 0.8
22.3 ± 1.1
>100
60.3 ± 1.3
22.6 ± 0.5
11.6 ± 2.9
83.4 ± 3.4
58.6 ± 1.5
12.7 ± 0.8
2.2 ± 0.6
>100
63
0.8
0.5
-
28.1 ± 0.3
23.9 ± 4.6
>100
64
65
66
1.0
1.4
0.4
0.3
0.8
0.5
0.2
0.2
59.6 ± 1.1
8.9 ± 0.4
5.2 ± 0.3
0.3 ± 0.01
69.5 ± 1.9
24.6 ± 0.1
11.7 ± 1.3
0.42 ± 0.04
67
68
69
0.09± 0.01
Daunorubicin
Figure 1: Anticancer activity of 1,3,4-oxadiazolesversus lipophilicity: a and b)
alkylated structures; c and d) acylated structures; Blue: 5-(pyridine-4-yl)-1,3,4-
oxadiazole-3H-thione
derivatives;
Red:5-(4-phenyl)-1,3,4-oxadiazole-3H-thione
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derivatives;Green:5-(4-methoxyphenyl)-1,3,4-oxadiazole-3H-thione derivatives; Purple:
5-(4-nitrophenyl)-1,3,4-oxadiazole-3H-thione derivatives.
Figure 2:Comparison of the cytotoxic effect between 1,2,4 and 1,3,4-oxadiazole
heterocycles for 4T1 and CT26.WT cells
2.3. Pro-apoptotic activity
Considering that some chemotherapeutic agents promote apoptosis we investigated this
event by AnnexinV and PI staining analysis. According to the IC-50 values, compounds
44 and 48 were chosen to assess their pro-apoptotic activity in 4T1 and CT26.WT cell
lines, respectively. Firstly, subdiploid DNA (sub-G1 phase cells) was analyzed as
indicator of apoptosis as described in the literature [31]. Compound 44 increased DNA
fragmentation around 2 and 4 times at 5 µM and 10 µM, respectively in 4T1 cells,
compared to control. In CT26.WT, compound 48 increased 66 times subdiploid DNA at
50 µM, compared to control (Figure 3A). These increases in DNA fragmentation
indicates a potential pro-apoptotic activity which was confirmed analyzing Annexin V
positive cells. As shown in Figure 3B, 83.6% of 4T1 cells had apoptosis induced by
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compound 44 10 µM (49.1% early apoptosis and 34.5% late apoptosis). On the other
hand, compound 48 50 µM induced apoptosis in 99% of CT26.WT cells, most of them
(98%) in late stage. Even the experiment was carried out in the same time exposure of
MTT assay, the Annexin V/PI staining assay can show the difference of cell response
on the course of apoptosis. Both compounds belong to alkyl derivatives with the same
carbon chain length and differ only on para-benzyl substituent metoxyl. Although with
some differences in chain length and substitutes moieties, Khanam et al (2017) also
described an induction of apoptosis by potent antitumor thioalkyl-aryl-oxadiazole
derivatives [32].
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Figure 3: Pro-apoptotic activity of compounds 44 and 48 in 4T1 and CT26.WT cells.
(A) Representative histograms of cell cycle profile with subdiploid DNA indicated. (B)
Representative dotplot of Annexin V and Propidium iodide stained cells.
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3. Conclusion:
In conclusion,novel series of 1,2,4 and 1,3,4-oxadiazole derivatives containing a
lipophilic moiety were synthesized and evaluated for their antitumor activity. The
results showed that alkylated structures were more active thanthe acylated ones.
Although the compounds were less active when compared to the reference drug, they
displayed a higher SI. For the 1,2,4- oxadiazole derivatives, compounds20 and 21were
highly selective for the 4T1 cell line.In the case of 1,3,4-oxadiazole derivatives,
compounds containingphenyl and pyridine rings were themost active in the cytotoxic
assay, highlighting compounds 38-45. Less lipophilic structures did not show
cytotoxicity, suggesting that this feature is important forthe anticancer activity.The two
most active alkylated compounds 44 and 48 showed to be capable to induce apoptosis in
4T1 and CT26, a crucial cellular process for effective chemotherapeutic agents.
4. Experimental Protocols
4.1. Materials and experiments
Reagents and solvents were purchased as reagent grade and used without
purification. Thin layer chromatography (TLC) was performed on glass plates and silica
gel sheets (silica gel F254; Merck) visualized with iodine vapor, and /or ultraviolet light
264 nm. Column chromatography was carried out on silica-gel 60G 0.063-0.200mm
(70-230 mesh). Melting points were determined on a MQAPF-Microquimica apparatus.
IR spectra were acquired using a Bruker Alpha-E ATR (Attenuated total reflection)
spectrometer. NMR data were recorded on a Bruker 500 Advance spectrometer.
Chemical shifts of ¹H NMR and ¹³C NMR are given in ppm and constant coupling (J)
are given in Hz. TMS signal was used as internal reference. Mass spectra of the
compounds 3-13; 15-16; 19-26; 38-49; 54-56 and 58-65 were performed on Maldi-TOF
Shimadzu-Biotech Axima Performance spectrometer.The mass-to-charge (m/z)
characterization of the compounds 14; 17-18; 50-53; 57 and 66-69 was made with
6120Quadrupole LC/MS (Agilent Technologies, Singapore) equipped with anAPI-ES
ion source, coupled to the Capillary Electrophoresis instrument, Agilent CE 7100
(Agilent Technologies, Singapore), used to compounds infusion into the MS by flush
with methanol (MeOH). The sheathliquid (0.1% offormic aciddissolved in 1:1 (v/v)
methanol/water) was delivered at 0.01 mL/min flow using an LC isocratic pump (1260
Infinity, Agilent Technologies, Waldbronn, Germany).The MS was operated in the
positive ionization mode applying electrospray voltage of 3.0 kV. Nitrogen was used as
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drying gas at 250ºC, with a flowrate of 6 L/min; the pressure of the nebulizer gas was
set at 15 psig. The m/z characterization of the compounds was made byselected ion
monitoring (SIM) mode.In order to perform compounds infusion into the quadrupoleMS
by flush with MeOH, the fused-silica capillary (Polymicro Technologies, Phoenix,
USA) of 100 cm length and internal diameter of 50µm was used. The new capillary was
conditioned by flushing with 1.0mol L-1NaOH (10 min), water (10 min), and MeOH
(15 min). Samples were injected hydrodinamically at 50 mbar for 2.0 sec and cassette
temperature was maintained at 25º C. Among sample injections, a short preconditioning
in the capillary was performed by flushing with 1 M NaOH (1 min), water (1 min) and
MeOH (43 min).The data processing was performed with the Agilent ChemStation for
CE-MS version software.
4.2. General procedure for the synthesis of compound 2:
To a solution of benzonitrile (80 mmol) in MeOH (100 mL), H2NOH.HCl (1.5 eq.) and
NaHCO3 (1.6 eq.) were added at room temperature. The resulting mixture was allowed
to reflux for 5h. The reaction was cooled to room temperature, the solvent was removed
under reduced pressure and the resulting mixture was extracted with EtOAc and H2O.
The organic phase was washed with brine solution and concentrated under vacuum,
affording the amidoxime as a colorless oil in 83% yield. Then, bromoacetyl bromide
(1.35 eq.) was added dropwise to a solution of the amidoxime and K2CO3 (1.35 eq.) in
CHCl3 (60 mL). The reaction was stirred at room temperature for 3h. The solvent was
removed, the mixture was extracted with EtOAc and H2O and the organic phase was
washed with a brine solution, dried with Na2SO4, filtrated and concentrated under
reduced pressure. The resulting mixture was dissolved in toluene (50 mL) and heated at
100°C for 2h. The reaction was cooled and the solvent removed. The resulting mixture
was purified by chromatography on silica gel (eluent: EtOAc/hexane) which afforded
the desired product as white solid.
4.2.1- 5-(bromomethyl)-3-phenyl-1,2,4-oxadiazole 2
White solid; Yield: 74%; m.p.: 48-50°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.08 (2H;
3
4
dd; J= 8.0 Hz; J= 1.5 Hz), 7.51-7.46 (3H; m), 4.56(2H; s)[33].¹³C RMN (CDCl3; 125
MHz) δ ppm:174.7, 169.0, 131.6, 129.0, 127.6, 126.3, 16.6.
13

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4.3. General procedure for the synthesis of compounds 3-18:
To a solution of 2 (1.0 mmol) and K2CO3 (2.0 eq.) in CH3CN, amine (2.0 eq.) was
added at room temperature. The resulting mixture was allowed to reflux for 1 to 5h. The
solvent was removed under reduced pressure and the mixture was extracted with
CH2Cl2 and H2O and washed with brine solution. The organic layer was dried with
Na2SO4, filtrated and concentrated under reduced pressure. Then, the crude mixture was
purified by chromatography on silica gel (eluent: CH2Cl2/ MeOH) affording the desired
compounds.
4.3.1)5-[(2-hydroxyethyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 3
Oil colorless; Yield: 85%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.09-8.07 (2H, m); 7.52-
7.47 (3H, m), 4.16 (2H, s), 3.71 (2H, t, J= 5.0 Hz), 2.93 (2H, t, J= 5.0 Hz), 2.32 (2H,
bs). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.6, 168.4, 131.5, 129.0, 127.6, 126.6, 61.1,
51.0, 44.7.MALDI-TOF m/z: [M+H] calculated 220.1088; [M+H] found 220.1080.
4.3.2) 5-[(2-hydroxyethyl)(N-octyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 4
Brown oil, Yield: 51%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.09-8.07 (2H; m), 7.51-
7.47 (3H; m), 4.07 (2H; s), 3.66 (2H; d; J= 5.0 Hz), 3.03 (1H; bs), 2.85 (2H; d; J= 5.0
Hz), 2.65 (2H; t; J= 7.0 Hz), 1.54 (2H; m), 1.29-1.26 (10H; sl), 0.87 (3H; t; J= 7.0
Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm: 177.6, 168.3, 131.4, 129.0, 127.6, 126.7, 59.1,
56.5, 54.4, 48.8, 32.0-22.8, 14.2. MALDI-TOF m/z: [M+H] calculated 332.2340;
[M+H] found 332.2300.
4.3.3) 5-[(2-hydroxyethyl)(N-decyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 5
Colorless oil, Yield: 67%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.09 (2H; m), 7.52-7.48
(3H; m), 4.07 (2H; s), 3.66 (2H; d; J= 5.0 Hz), 3.03 (1H; bs), 2.85 (2H; d; J= 5.0 Hz),
2.65 (2H; t; J= 7.0 Hz), 1.54 (2H; m), 1.29-1.25 (14H; bs), 0.88 (3H; t; J= 7.0 Hz).¹³C
NMR (CDCl3; 125 MHz) δ ppm: 177.6, 168.3, 131.5, 129.1, 127.7, 126.8, 59.1, 56.5,
54.5, 48.8, 32.1-22.9, 14.3. MALDI-TOF m/z: [M+H] calculated 360.2653; [M+H]
found 360.2650.
4.3.4) 5-[(2-hydroxyethyl)(N-dodecyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 6
Colorless oil, Yield: 79%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.09 (2H; d; J= 7.5 Hz),
7.52-7.48 (3H; m), 4.07 (2H; s), 3.66 (2H; d; J= 5.0 Hz), 3.04 (1H; bs), 2.85 (2H; d; J=
5.0 Hz), 2.65 (2H; t; J= 7.0 Hz), 1.54 (2H; m), 1.29-1.25 (18H; bs), 0.87 (3H; t; J= 7.0
Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 177.6, 168.4, 131.5, 129.1, 127.7, 126.8,
14

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59.2, 56.6, 54.5, 48.8, 32.1-22.9, 14.3. MALDI-TOF m/z: [M+H] calculated 388.2966;
[M+H] found 388.2970.
4.3.5) 5-[(2-hydroxyethyl)(N-tetradecyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 7
Colorless oil, Yield: 95%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.09-8.07 (2H; m), 7.50-
7.45 (3H; m), 4.06 (2H; s), 3,65 (2H; d; J= 5.0 Hz), 3.10 (1H; bs), 2.83 (2H; d; J= 5.0
Hz), 2.64 (2H; t; J= 7.0 Hz), 1.52 (2H; m), 1.29-1.24 (22H; bs), 0.87 (3H; t; J= 7.0 Hz).
¹³C NMR (CDCl3; 125 MHz) δ ppm: 177.6, 168.3, 131.4, 129.0, 127.6, 126.7, 59.1,
56.5, 54.4, 48.8, 32.0-22.8, 14.3. MALDI-TOF m/z: [M+H] calculated 416.3279;
[M+H] found 416.3286.
4.3.6) 5-[(N-butyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 8
Colorless oil, Yield: 84%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.10-8.08 (2H; m), 7.50-
7.46 (3H; m), 4.13 (2H; s), 2.73 (2H; t; J= 7.0 Hz), 2.19 (1H, s), 1.53 (2H; m), 1.37
(2H; m), 0.92 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.4, 168.5,
131.4, 129.0, 127.6, 126.9, 49.2, 44.9, 32.0, 20.4, 14.0. MALDI-TOF m/z: [M+H]
calculated 232.1452; [M+H] found 232.1446.
4.3.7) 5-[(N-octyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 9
Colorless oil; Yield: 88%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.09-8.07 (2H; m), 7.50-
7.46 (3H; m), 4.11 (2H; s), 2.70 (2H; t; J= 7.0 Hz), 1.52 (3H; m), 1.28-1.26 (10H; bs),
0.87 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.7, 168.4, 131.3, 129.0,
127.6, 126.9, 49.6, 45.1, 31.9-22.8, 14.2. MALDI-TOF m/z: [M+H] calculated
288.2078; [M+H] found 288.2083.
4.3.8)5-[(N-decyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 10
Colorless oil; Yield: 85%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.10-8.08 (2H; m), 7.50-
7.46 (3H; m), 4.11 (2H; s), 2.70 (2H; t; J= 7.0 Hz), 1.71 (1H; s), 1.53 (2H; m), 1.32-
1.25 (22H; bs), 0.87 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.7,
168.4, 131.3, 129.0, 127.6, 126.9, 49.6, 45.1, 32.0-22.8, 14.2. MALDI-TOF m/z:
[M+H] calculated 316.2391; [M+H] found 316.2397.
4.3.9)5-[(N-dodecyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 11
Colorless oil; Yield: 86%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.10-8.08 (2H; m), 7.50-
7.45 (3H; m), 4.11 (2H; s), 2.70 (2H; t; J= 7.0 Hz), 1.51 (3H; m), 1.29-1.23 (18H; bs),
0.87 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.7, 168.4, 131.3, 129.0,
127.6, 126.9, 49.6, 45.1, 32.1-22.8, 14.2. MALDI-TOF m/z: [M+H] calculated
344.2704; [M+H] found 344.2702.
15

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4.3.10)5-[(N-tetradecyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 12
White solid; Yield: 91%; m.p.: 29-31°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.09 (2H;
m), 7.49 (3H; m), 4.11 (2H; s), 2.71 (2H; t; J= 7.0 Hz), 1.68 (1H; s), 1.53 (2H; m), 1.25
(22H; bs), 0.88 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.7, 168.4,
131.3, 129.0, 127.6, 126.9, 49.6, 45.1, 32.1-22.9, 14.3. MALDI-TOF m/z: [M+H]
calculated 372.3017; [M+H] found 372.3013.
4.3.11)5-[N-(2-aminoethyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 13
Brown oil; Yield: 33%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.06 (2H; d; J= 8.0 Hz),
7.49-7.43 (3H; m), 4.11 (2H; s), 2.82 (2H; t; J= 5.0 Hz), 2.76 (2H; t; J= 5.0 Hz), 1.65
(3H, bs). ¹³C NMR (CDCl3; 125 MHz) δppm:178.5, 168.4, 131.3, 128.9, 127.5, 126.7,
51.9, 44.9, 41.6. MALDI-TOF m/z: [M+H] calculated 219.1248; [M+H] found
219.1240; m/z [M+Na] calculated 241.1068; [M+Na] found 241.6152.
4.3.12)5-[N-(3-aminopropyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 14
Brown oil; Yield: 44%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.10-8.07 (2H; m); 7.52-
7.46 (3H; m); 5.04 (1H; s); 4.12 (2H; s); 2.85 (2H; t; J= 5.0 Hz); 2.81 (2H; t; J= 5.0
Hz); 2.67 (2H; s); 1.72 (2H; q; J= 5.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.5,
168.3, 131.2, 128.8, 127.4, 126.5, 47.2, 44.8, 40.0, 32.5. ESI-MS m/z: [M+H]
calculated 233.1; [M+H] found 233.0; m/z [M+Na] calculated 255.1; [M+Na] found
255.0.
4.3.13)5-[N-(tris hydroxymethyl)aminomethyl]-3-phenyl-1,2,4-oxadiazole 15
White solid; Yield: 82%; m.p.: 97-99°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.99-7.97
(2H; m), 7.57-7.52 (3H; m), 4.48 (2H; bs), 4.20 (2H; d; J= 7.0 Hz), 3.39 (6H; s), 2.27
(1H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm: 180.5, 167.6, 131.8, 129.5,
127.2, 126.4, 61.4, 60.3, 38.6. MALDI-TOF m/z: [M+H] calculated 280.1299; [M+H]
found 280.1307; [M+Na] calculated 302.1119; [M+Na] found 302.2585; [M+K]
calculated 318.2202; [M+K] found 318.0740.
4.3.14) 3-phenyl-5-(piperazin-1-ylmethyl)-1,2,4-oxadiazole 16
Brown oil; Yield: 86%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.08 (2H; m), 7.48-7.43
(3H; m), 3.88 (2H; s), 2.92 (4H; t; J= 5.0 Hz), 2.61-2.60 (4H; bs), 1.79 (1H; bs). ¹³C
NMR (CDCl3; 125 MHz) δ ppm: 176.3, 168.4, 131.3, 128.9, 127.6, 126.7, 54.2, 53.6,
45.9. MALDI-TOF m/z: [M+H] calculated 245.1404; [M+H] found 245.1410.
16

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4.3.15) (R)-5-[(3-methylpiperazin-1-yl)methyl]-3-phenyl-1,2,4-oxadiazole 17
Yellow oil; Yield: 55%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.02-8.01 (2H; m), 7.61-
3
4
7.55 (3H; m), 3.92 (2H; s), 2.81-2.65 (6H; m), 2.14-2.10 (1H; dt; J= 11.0 Hz; J= 3.5
Hz), 1,78 (1H; t; J= 10.0 Hz), 0.89 (2H; d; J=5.0 Hz). ¹³C NMR (CDCl3; 125 MHz)
δ ppm:177.0, 167.4, 131.6, 129.3, 127.0, 126.1, 60.1, 52.5, 50.0, 45.2. ESI-MS m/z:
[M+H] calculated 259.1; [M+H] found 259.0; [M+Na] calculated 281.1; [M+Na] found
281.0.
4.3.16) 5-[(4-methylpiperazin-1-yl)methy]-3-phenyl-1,2,4-oxadiazole 18
Yellowoil; Yield: 81%; ¹H NMR (DMSO-d6; 500 MHz) δ ppm: 8.03-8.02 (2H; m),
7.61-7.56 (3H; m), 3.96 (2H; s), 2.56 (4H; s), 2.34 (4H; s), 2.15 (3H, s). ¹³C NMR
(DMSO-d6; 125 MHz) δ ppm:177.4, 167.8, 131.9, 129.6, 127.4, 126.5, 54.9, 52.5, 46.1.
ESI-MS m/z: [M+Na] calculated 281.1; [M+Na] found 281.0.
4.4. General procedure for the synthesis of compounds 19-22:
To a solution of oxadiazole 16 in CH3CN and K2CO3 (1.5 eq.), alkyl chloride (1.5 eq.)
was added in ether solution. The reaction mixture was stirred at 70°C for 20h and
concentrated under reduced pressure. The crude mixture was extracted with CH2Cl2 and
H2O, the organic layer was washed with brine solution, dried with Na2SO4, filtrated and
concentrated under reduced pressure. Then, the mixture was purified by
chromatography on silica gel (eluent: CH2Cl2/MeOH) affording the desired compounds.
4.4.1) 5-[(4-octylpiperazin-1-yl)methy)]-3-phenyl-1,2,4-oxadiazole 19
Yellow oil; Yield: 72%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.08-8.06 (2H; m), 7.47-
7.42 (3H; m), 3.89 (2H; s), 2.70-2.32 (8H; m), 2.33 (2H; t; J= 8.0 Hz), 1.47 (2H; m);
1.25 (10H; bs); 0.85 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 500 MHz) δ ppm: 176.2,
168.4, 131.3, 128.9, 127.6, 126.8, 58.7, 53.1, 53.0, 52.8, 31.9-22.7, 14.2. MALDI-TOF
m/z: [M+H] calculated 357.2656; [M+H] found 357.2649.
4.4.2) 5[(4-decylpiperazin-1-yl)methyl]-3-phenyl-1,2,4-oxadiazole 20
Colorless oil; Yield: 86%; ¹H NMR (CDCl3; 500 MHz) δ ppm:8.06-8.05 (2H; m), 7.46-
7.41 (3H; m), 3.88 (2H; s), 2.68-2.51 (8H; m), 2.32 (2H; t; J= 7.0 Hz), 1.45 (2H; bs),
1.24-1.22 (14H; bs), 0.84 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:
176.2, 168.4, 131.3, 128.9, 127.6, 126.7, 58.7, 53.0, 52.9, 52.8, 31.9-22.7, 14.2.
MALDI-TOF m/z: [M+H] calculated 385.2969; [M+H] found 385.2973.
4.4.3) 5-[(4-dodecylpiperazin-1-yl)methyl]-3-phenyl-1,2,4-oxadiazole21
17

ACCEPTED MANUSCRIPT
White solid; Yield: 77%; m.p.: 47-49°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.10-8.08
(2H; m), 7.50-7.45 (3H; m), 3.93 (2H; s), 2.78-2.64 (8H; m), 2.42 (2H; t; J= 8.0 Hz),
1.53 (2H; m), 1.28-1.25 (18H; bs), 0.87 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125
MHz) δ ppm:176.2, 168.5, 131.4, 129.0, 127.7, 126.8, 58.6, 53.0, 52.9, 52.5, 32.0-22.8,
14.2. MALDI-TOF m/z: [M+H] calculated 413.3282; [M+H] found 413.3287.
4.4.4) 5[(4-tetradecylpiperazin-1-yl)methyl]-3-phenyl-1,2,4-oxadiazole 22
Brown solid; Yield: 76%; m.p.: 55-56°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.04-8.02
(2H; m), 7.42-7.83 (3H; m), 3.84 (2H; s), 2.64-2.47 (8H; m), 2.27 (2H; m), 1.41 (2H;
m), 1.21-1.19 (22H; sl), 0.82 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:
176.1, 168.3, 131.1, 128.8, 127.5, 126.7, 58.6, 52.9, 52.8, 31.9-22.7, 14.1. MALDI-TOF
m/z: [M+H] calculated 441.3595; [M+H] found 441.3603.
4.5. General procedure for the synthesis of compounds 23-26:
Carboxylic acids (caprylic acid, capric acid, lauric acid or myristic acid) were reacted
with SOCl2 (5 eq.) and heated at 75°C for 2h. The excess of SOCl2 was removed under
reduced pressure and the mixture was solubilized in dried CH2Cl2. Then, the acyl
chlorides (1.5 eq.) were added dropwise to a solution of the oxadiazole derivative 16 in
CH3CN and K2CO3 (1.5 eq.). The reaction mixture was stirred at room temperature for
30h. The solvent was removed under reduced pressure and the crude mixture was
extracted with CH2Cl2 and H2O and washed with brine solution. The organic layer was
dried with Na2SO4, filtrated and concentrated under vacuum. The mixture was purified
by chromatography on silica gel (eluent: CH2Cl2/ MeOH) affording the desired
compounds.
4.5.1) 5-{[4-(piperazin-1-yl)octan-1-one]methyl}-3-phenyl-1,2,4-oxadiazole 23
Brown oil; Yield: 50%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.05-8.03 (2H; m), 7.46-
7.41 (3H; m), 3.91 (2H; s), 3.67-3.49 (4H; m), 2.63-2.60 (4H; m), 2.26 (2H; t; J= 7.0
Hz); 1.57 (2H; m), 1.27-1.22 (8H; m), 0.82 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125
MHz) δ ppm: 175.6, 171.7, 168.4, 131.3, 128.9, 127.7, 126.5, 52.9, 52.5, 45.3, 41.3,
33.2-22.6, 14.1. MALDI-TOF m/z: [M+H] calculated 371.2449; [M+H] found
371.2455; [M+K] calculated 409.3352; [M+K] found 409.6732.
4.5.2) 5-{[4-(piperazin-1-yl)decan-1-one]methyl}-3-phenyl-1,2,4-oxadiazole 24
Brown solid; Yield: 76%; m.p.: 25-26°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.99-7.97
(2H; m), 7.38-7.34 (3H; m), 3.83 (2H; s), 3.58-3.40 (4H; m), 2.55-2.51 (4H; m), 2.18
18

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(2H; t; J= 7.0 Hz), 1.50 (2H; m), 1.19-1.14 (12H; m), 0.86 (3H; t; J= 7.0 Hz). ¹³C NMR
(CDCl3; 125 MHz) δ ppm: 175.6, 171.4, 168.1, 131.1, 128.7, 127.3, 126.4, 52.6, 52.4,
45.2, 41.1, 33.0-22.5, 13.9. MALDI-TOF m/z: [M+H] calculated 399.2702; [M+H]
found 399.2765; [M+Na] calculated 421.2582; [M+Na] found 421.2617.
4.5.3) 5{[4-(piperazin-1-yl)dodecan-1-one]methyl}-3-phenyl-1,2,4-oxadiazole 25
White solid; Yield: 78%; m.p.: 35-37°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.03-8.01
(2H; m), 7.44-7.39 (3H; m), 3.89 (2H; s), 3.63-3.46 (4H; m), 2.60-2.57 (4H; m), 2.24
(2H; t; J= 7.5 Hz), 1.54 (2H; m), 1.23-1.19 (16H; m); 0.81 (3H; t; J= 7.5 Hz). ¹³C NMR
(CDCl3; 125 MHz) δ ppm: 175.6, 171.7, 168.3, 131.3, 128.8, 127.4, 126.5, 52.8, 52.4,
45.3, 41.2, 33.2-22.6, 14.1. MALDI-TOF m/z: [M+H] calculated 427.3075; [M+H]
found 427.3069.
4.5.4) 5{[4-(piperazin-1-yl)tetradecan-1-one]methyl}-3-phenyl-1,2,4-oxadiazole26
White solid; Yield: 86%; m.p.: 41-43°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.04-8.01
(2H; m), 7.44-7.39 (3H; m), 3.89 (2H; s), 3.64-3.46 (4H; m), 2.60-2.59 (4H; bs), 2.24
(2H; t; J= 7.5 Hz), 1.55 (2H; m), 1.23-1.19 (20H; m), 0.81 (3H; t; J= 7.5 Hz). ¹³C NMR
(CDCl3; 125 MHz) δ ppm: 175.6, 171.6, 168.3, 131.3, 128.8, 127.4, 126.5, 52.8, 52.5,
45.3, 41.2, 33.2-22.6, 14.1. MALDI-TOF m/z: [M+H] calculated 455.3388; [M+H]
found 455.3394; [M+Na] calculated 477.3208; [M+Na] found 477.3342; [M+K]
calculated 493.4291; [M+K] found 493.4710.
4.6. General procedure for the synthesis of N-acylhydrazides 31-33:
Benzoic acid 27, p-methoxybenzoic acid 28 and p-nitrobenzoic acid 29 (40 mmol) were
treated with a sulfuric acid-ethanol solution. The resulting mixture was allowed to
reflux with stirring for 20h. Ethanol was removed under reduced pressure and the
mixture was extracted with Et2O and H2O and washed with saturated NaHCO3 solution.
The organic layer was dried with Na2SO4, filtrated and concentrated, furnishing the
esters derivatives. Then, a mixture of the appropriated aromatic esters and 2.0
equivalents of hydrazine hydrated (69% m/v) were reacted in an ethanol solution. The
resulting mixture was allowed under reflux with stirring for 20h. The reaction was
cooled and the precipitated formed was washed with ethanol and filtrated. The solid
obtained was recrystallized under ethanol solution affording the desired products.
4.6.1) Benzohydrazide 31
19

ACCEPTED MANUSCRIPT
Yield: 65%; ¹H NMR (DMSO-d6; 500 MHz) δ ppm: 9.77 (1H; s), 7.82 (2H; dd; 3J= 7.0
4
Hz; J= 1.5 Hz), 7.49 (1H; m), 7.43 (2H; t; J= 7.0 Hz), 4.41 (2H; bs). ¹³C NMR
(DMSO-d6; 125 MHz) δ ppm: 165.9, 133.3, 131.0, 128.3, 126.9.
4.6.2) 4-Methoxybenzohydrazide 32
Yield: 65%; ¹H NMR (DMSO-d6; 500 MHz) δ ppm: 9.63 (1H; s), 7.80 (2H; d; J= 9.0
Hz), 6.97 (2H; d; J= 9.0 Hz), 4.24 (2H; bs), 3.78 (3H; s). ¹³C NMR (DMSO-d6; 125
MHz) δ ppm: 165.6, 161.4, 128.7, 125.5, 113.5, 55.3.
4.6.3) 4-Nitrobenzohydrazide 33
Yield: 88%; ¹H NMR (DMSO-d6; 500 MHz) δ ppm: 10.12 (1H; s); 8.27 (2H; d; J= 9.0
Hz); 8.03 (2H; d; J= 9.0 Hz); 4.63 (2H; bs). ¹³C NMR (DMSO-d6; 125 MHz) δ ppm:
164.0, 148.9, 139.0, 128.5, 123.6.
4.7. General procedure for the synthesis of 1,3,4-oxadiazoles 34-37:
A mixture of isoniazid 30 and N-acylhydrazines31-33, CS2 (4.0 eq.) and KOH (3.0
equivalents) was reacted in ethanol. The resulting mixture were stirred at 55°C for 48h.
The excess of CS2 and ethanol was removed under reduced pressure and the crude
product was solubilized in a minimal quantity of H2O. The mixture was acidified with
HCl 4M until pH~1 and the resulting precipitated was filtered and washed with cold
water, affording the desired heterocycles.
4.7.1)5-(pyridin-4-yl)-1,3,4-oxadiazole-2-thiol 34
3
4
Yield: 68%; ¹H NMR (DMSO-d6; 500 MHz) δ ppm: 8.92 (2H; dd; J= 4.5 Hz, J= 1.5
Hz),7.83 (2H, dd, 3J= 4.5 Hz; 4J= 1.5 Hz) [34]. ¹³C NMR (DMSO-d6; 125 MHz) δ ppm:
177.9, 158.6, 150.6, 129.7, 119.5.
4.7.2) 5-Phenyl-1,3,4-oxadiazole-2-thiol 35
3
Yield: 70%; ¹H NMR (DMSO-d6; 500 MHz) δppm: 13.53 (1H; bs), 7.87 (2H; dd; J=
4
8.5 Hz; J= 1.5 Hz), 7.64-7.56 (3H; m)[35]. ¹³C NMR (DMSO-d6; 125 MHz) δ
ppm:177.4, 160.4, 132.1, 129.4, 126.0, 122.5.
4.7.3) 5-(4-Methoxyphenyl)-1,3,4-oxadiazole-2-thiol 36
Yield: 62%; ¹H NMR (DMSO-d6; 500 MHz) δppm: 14.64 (1H; bs), 7.80 (2H; d; J= 8.5
Hz), 7.11 (2H; d; J= 8.5 Hz), 3.83 (3H; s) [34]. ¹³C NMR (DMSO-d6; 125 MHz) δ
ppm:177.1, 162.2, 160.5, 127.9, 114.8, 114.7, 55.5.
4.7.4) 5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-thiol 37
20

ACCEPTED MANUSCRIPT
Yield: 67%; ¹H NMR (DMSO-d6; 500 MHz) δ ppm: 10.12 (1H; s); 8.28 (2H; dd; ³J=
4
4
7.0 Hz; J= 2.0 Hz); 8.04 (2H; dd; ³J= 7.0 Hz; J= 2.0 Hz) [36]. ¹³C NMR (DMSO-d6;
125 MHz) δ ppm: 163.9, 161.1, 148.9, 139.0, 128.4, 123.5.
4.8. General procedure for the synthesis of Mannich bases 38-69:
To a suspension of 1.0 mmol of 5-(aryl)-3H-1,3,4-oxadiazole-2-thione 1 in 5 mL of
absolute ethanol, piperazine derivatives (1.0 eq.).Formaldehyde 37% v/v (1.5 eq) was
added dropwise to the stirred suspensionand the reaction mixture was stirred at room
temperature for 16 h. After concentration under reduced pressure, the residue was
extracted with CH2Cl2 and H2O. The organic layer was dried with Na2SO4 and
concentrated, affording the Mannich bases.
4.8.1) 3-[(4-octylpiperazin-1-yl)methyl]-5-(piridin-4-yl)-1,3,4-oxadiazole-2(3H)-thione
38
Yellow solid; Yield: 83%; m.p.: 79-81 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm:8.65
(2H; d; J= 5.5 Hz), 7.60 (2H; d; J= 5.5 Hz), 4.97 (2H; s), 2.85-2.47 (8H; bs), 2.30 (2H;
t; J= 8.0 Hz), 1.36 (2H; m), 1.11 (10H; bs), 0.70 (3H; t; J= 7.0 Hz).¹³C NMR (CDCl3;
125 MHz) δ ppm:178.1, 156.5, 150.7, 129.3, 119.4, 70.2, 58.2, 52.6, 49.3, 31.5-22.4,
13.9. MALDI-TOF m/z: [M+H] calculated 390.2329; [M+H] found 390.2321.
4.8.2) 3-[(4-decylpiperazin-1-yl)methyl]-5-(pyridin-4-yl)-1,3,4-oxadiazole-2(3H)-thione
39
Orange solid; Yield: 77%; m.p.: 72-74 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm:8.57
(2H; d; J= 5.0 Hz), 7.52 (2H; d; J= 5.0 Hz), 4.91 (2H; s), 2.80-2.45 (8H; bs), 2.28 (2H;
t; J= 7.0 Hz), 1.32 (2H; m), 1.06-1.01 (14H; bs), 0.64 (3H; t; J= 7.0 Hz). ¹³C NMR
(CDCl3; 125 MHz) δ ppm:177.9, 156.3, 150.5, 129.1, 119.2, 69.9, 58.0, 52.3, 49.1,
31.5-22.3, 13.8. MALDI-TOF m/z: [M+H] calculated 418.6192; [M+H] found
418.6183.
4.8.3) 3-[(4-dodecylpiperazin-1-yl)methyl]-5-(pyridin-4-yl)-1,3,4-oxadiazole-2(3H)-
thione 40
Brown solid; Yield: 89%; m.p.: 75-77 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.65
3
4
(2H; d; J= 6.0 Hz), 7.59 (2H; dd; J= 6.0 Hz; J= 1.5 Hz), 4.96 (2H; s), 2.77 (4H; bs),
2.36 (4H; bs), 2.19 (2H; t; J= 7.5 Hz), 1.30 (2H; bs), 1.13-1.10 (18H; bs), 0.70 (3H; t;
J= 7.5 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:178.1, 156.4, 150.7, 129.3, 119.4, 70.3,
21

ACCEPTED MANUSCRIPT
58.4, 52.7, 49.7, 31.6-22.4, 13.9. MALDI-TOF m/z: [M+H] calculated 446.2955;
[M+H] found 446.2948.
4.8.4) 3-[(4-tetradecylpiperazin-1-yl)methyl]-5-(pyridin-4-yl)-1,3,4-oxadiazole-2(3H)-
thione 41
Brown solid; Yield: 88%; m.p.: 83-85 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm:8.71 (2H;
d; J= 6.0 Hz), 7.66 (2H; d; J= 6.0 Hz), 5.02 (2H; s), 2.84-2.43 (8H; bs), 2.26 (2H; t; J=
7.0 Hz), 1.37 (2H; m), 1.16-1.14 (22H; bs), 0.77 (3H; t; J= 7.0 Hz).¹³C NMR (CDCl3;
125 MHz) δ ppm: 178.3, 156.6, 150.8, 129.5, 119.5, 70.4, 58.5, 52.8, 49.8, 31.8-22.6,
14.1. MALDI-TOF m/z: [M+H] calculated 474.3268; [M+H] found 474.3266.
4.8.5) 3-[(4-octylpiperazin-1-yl)methyl]5-phenyl-1,3,4-oxadiazole-2(3H)-thione 42
Brown solid; Yield: 90%; m.p.: 56-58 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.87
3
4
(2H; d; J=7.0 Hz), 7.54 (1H; tt; J= 7.5 Hz; J= 1.0 Hz), 7.47 (2H; m), 5.07 (2H; s),
2.91-2.47 (8H; bs), 2.30 (2H; m), 1.43 (2H; m), 1.27-1.24 (12H; bs), 0.84 (3H; t; J= 7.5
Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.3, 159.0, 132.5, 129.2, 126.6, 122.5, 70.3,
58.8, 53.1, 50.2,31.9-22.7, 14.2.MALDI-TOF m/z: [M+H] calculated 389.2377; [M+H]
found 389.2380.
4.8.6) 3-[(4-decylpiperazin-1-yl)methyl]5-phenyl-1,3,4-oxadiazole-2(3H)-thione 43
White solid; Yield: 60%; m.p.: 65-67 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.87 (2H;
d; J=7.0 Hz), 7.50 (1H; bs), 7.44 (2H; bs), 5.04 (2H; s), 2.89-2.47 (8H; bs), 2.30 (2H; t;
J= 7.0 Hz), 1.42 (2H; bs), 1.19 (12H; bs), 0.82 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3;
125 MHz) δ ppm:178.2, 158.9, 132.3, 129.1, 126.5, 122.4, 70.2, 58.6, 53.0, 49.9,31.9-
22.7, 14.1.MALDI-TOF m/z: [M+H] calculated 417.2690; [M+H] found 417.2686.
4.8.7) 3-[(4-dodecylpiperazin-1-yl)methyl]5-phenyl-1,3,4-oxadiazole-2(3H)-thione 44
Brown solid; Yield: 79%; m.p.: 69-71 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.87
(2H; d; J=7.5 Hz), 7.50 (1H; t; J= 7.5 Hz), 7.44 (2H; t; J= 7.5 Hz), 5.05 (2H; s), 2.89-
2.45 (8H; bs), 2.83 (2H; t; J= 7.5 Hz), 1.41 (2H; m), 1.21-1.19 (18H; bs), 0.82 (3H; t; J=
7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:178.2, 158.9, 132.4, 129.1, 126.5, 122.4,
70.2, 58.7, 53.0, 50.1, 31.9-22.7, 14.2.MALDI-TOF m/z: [M+H] calculated 445.3003;
[M+H] found 445.3008.
4.8.8) 3-[(4-tetradecylpiperazin-1-yl)methyl]-5-phenyl-1,3,4-oxadiazole-2(3H)-thione
45
Brown solid; Yield: 83%; m.p.: 72-74 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.87
3
4
(2H; d; J= 7.0 Hz; J= 1.5 Hz), 7.50 (1H; t; J= 7.5 Hz), 7.44 (2H; t; J= 7.5 Hz), 5.04
22

ACCEPTED MANUSCRIPT
(2H; s), 2.88-2.44 (8H; bs), 2.27 (2H; t; J= 7.0 Hz), 1.40 (2H; bs), 1.19 (22H; bs), 0.83
(3H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:178.2,158.9, 132.3, 129.1, 126.5,
122.4, 70.2, 58.7, 53.1, 50.1, 31.9-22.7, 14.2.MALDI-TOF m/z: [M+H] calculated
473.3316; [M+H] found 473.3322.
4.8.9) 3-[(4-octylpiperazin-1-yl)methyl]-5-(4-methoxyphenyl)-1,3,4-oxadiazole-2(3H)-
thione 46
White solid; Yield: 85%; m.p.: 89-90 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.73 (2H;
3
4
3
4
dd; J= 7.0 Hz; J= 2.0 Hz), 6.87 (2H; dd; J= 7.0 Hz; J= 2.0 Hz), 4.96 (2H; s), 3.76
(3H; s), 2.83-2.40 (8H; bs), 2.23 (2H; t; J= 7.0 Hz), 1.36 (2H; m), 1.19-1.15 (14H; bs),
0.76 (3H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:177.8, 162.7, 158.8, 128.2,
114.6, 114.5, 69.9, 58.5, 55.4, 52.9, 49.9, 31.7-22.5, 14.0.MALDI-TOF m/z: [M+H]
calculated 419.2482; [M+H] found 419.2483.
4.8.10) 3-[(4-decylpiperazin-1-yl)methyl]-5-(4-methoxyphenyl)-1,3,4-oxadiazole-2(3H)-
thione 47
White solid; Yield: 71%; m.p.: 85-88 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.81 (2H;
3
4
3
4
dd; J= 7.0 Hz; J= 2.0 Hz), 6.94 (2H; dd; J= 7.0 Hz; J= 2.0 Hz), 5.04 (2H; s), 3.84
(3H; s), 2.99-2.61 (8H; bs), 2.43 (2H; t; J= 7.0 Hz), 1.50 (2H; bs); 1.24-1.21 (14H; bs);
0.84 (3H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:178.0, 162.9, 159.2, 128.5,
114.7, 114.7, 69.9, 58.5, 55.6, 52.8, 49.5, 31.9-22.7, 14.2.MALDI-TOF m/z: [M+H]
calculated 447.2795; [M+H] found 447.2791.
4.8.11) 3-[(4-dodecylpiperazin-1-yl)methyl]-5-(4-methoxyphenyl)-1,3,4-oxadiazole-
2(3H)-thione 48
White solid; Yield: 93%; m.p.: 88-90°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.84 (2H;
3
4
3
4
dd; J= 7.0 Hz; J= 2.0 Hz), 6.97 (2H; dd; J= 7.0 Hz; J= 2.0 Hz), 5.05 (2H; s), 3.87
(3H; s), 3.00-2.64 (8H; bs), 2.45 (2H; t; J= 7.0 Hz), 1.53 (2H; bs), 1.26-1.23 (18H; bs),
0.86 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.0, 163.0, 159.3, 128.6,
114.8, 114.7, 70.0, 58.6, 55.6, 52.9, 49.5,32.0-22.8, 14.3.MALDI-TOF m/z: [M+H]
calculated 475.3108; [M+H] found 475.3107.
4.8.12)3-[(4-tetradecylpiperazin-1-yl)methyl]-5-(4-methoxyphenyl)-1,3,4-oxadiazole-
2(3H)-thione 49
White solid; Yield: 79%; m.p.: 91-93 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.75 (2H;
3
4
3
4
dd; J= 7.0 Hz; J= 2.0 Hz), 6.88 (2H; dd; J= 7.0 Hz; J= 2.0 Hz), 4.97 (2H; s), 3.77
(3H; s), 2.85-2.42 (8H; bs), 2.25 (2H; t; J= 7.0 Hz), 1.38 (2H; m), 1.15 (22H; bs), 0.79
23

ACCEPTED MANUSCRIPT
(3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 177.8, 162.7, 158.8, 128.2,
114.6, 114.5, 69.9, 58.6, 55.4, 52.9, 49.9, 31.8-22.6, 14.1.MALDI-TOF m/z: [M+H]
calculated 503.3421; [M+H] found 503.3433.
4.8.13)
3-[(4-octylpiperazin-1-yl)methyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole-2(3H)-
thione 50
Yellow solid; Yield: 78%; m.p.: 86-87 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.34
(2H; d; J= 8.5 Hz); 8.08 (2H; d; J= 8.5 Hz); 5.12 (2H; s); 2.98 (4H; t; J= 4.5 Hz); 2.58
(4H; m); 2.41 (2H; t; J= 8.0 Hz); 1.48 (2H; bs); 1.25-1.23 (10H; bs); 0.84 (3H; t; J= 7.0
Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.5, 156.9, 149.9, 127.9, 127.5, 124.6,
70.6, 58.6, 52.9, 49.8, 31.9, 29.5, 29.3, 27.5, 26.6, 22.7, 14.2.ESI-MS: m/z433.2
calculated; m/z 433.0 found; [M+H] calculated 434.2; [M+H] found 434.0; [M+Na]
calculated 456.2; [M+Na] found 456.0.
.4.8.14) 3-[(4-decylpiperazin-1-yl)methyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole-2(3H)-
thione 51
Yellow solid; Yield: 87%; m.p.: 84-86 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.31
(2H; d; J= 8.5 Hz); 8.06 (2H; d; J= 8.5 Hz); 5.11 (2H; s); 3.02 (4H; t; J= 4.5 Hz); 2.67
(4H; bs); 2.49 (2H; t; J= 8.0 Hz); 1.53-1.52 (2H; bs); 1.25-1.21 (12H; bs); 0.83 (3H; t;
J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.4, 157.0, 127.8, 127.5, 124.5,
70.4, 58.5, 52.8, 49.4, 31.9, 29.6, 29.5, 29.3, 27.4, 26.2, 22.7, 14.2..ESI-MS: m/z461.2
calculated; m/z 461.0 found; [M+H] calculated 462.2; [M+H] found 462.0; [M+Na]
calculated 484.2; [M+Na] found 484.0.
4.8.15) 3-[(4-dodecylpiperazin-1-yl)methyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole-2(3H)-
thione 52
Yellow solid; Yield: 68%; m.p.: 91-93 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.32
(2H; d; J= 8.5 Hz); 8.07 (2H; d; J= 8.5 Hz); 5.09 (2H; s); 2.96 (4H; t; J= 4.5 Hz); 2.94
(4H; bs); 2.49 (2H; t; J= 7.0 Hz); 1.46 (2H; bs); 1.23-1.20 (18H; bs); 0.83 (3H; t; J= 7.0
Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.4, 156.9, 149.8, 127.9, 127.5, 124.5,
70.5, 58.6, 52.9, 49.7, 31.9, 29.7, 29.6, 29.5, 29.4, 27.5, 26.5, 22.7, 14.2.ESI-MS:
m/z489.3calculated; m/z found489.0; [M+H] calculated 490.3; [M+H] found 490.0;
[M+Na] calculated 512.3; [M+Na] found 512.0.
4.8.16)
3-[(4-tetradecylpiperazin-1-yl)methyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole-
2(3H)-thione 53
24

ACCEPTED MANUSCRIPT
Yellow solid; Yield: 71%; m.p.: 93-94 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.31
(2H; d; J= 9.0 Hz); 8.06 (2H; d; J= 9.0 Hz); 5.09 (2H; s); 2.92 (4H; bs); 2.52 (4H; bs);
2.34 (2H; t; J= 7.5 Hz); 1.44 (2H; bs); 1.22-1.19 (22H; bs); 0.83 (3H; t; J= 7.5 Hz). ¹³C
NMR (CDCl3; 125 MHz) δ ppm: 178.4, 156.8, 149.8, 127.8, 127.4, 124.5, 70.5, 58.6,
52.9, 49.9, 31.9, 29.7, 29.6, 29.4, 27.5, 26.6, 22.7, 14.2.ESI-MS: m/z 517.3 calculated;
m/z found 517.0; [M+H] calculated 518.3; [M+H] found 518.0; [M+Na] calculated
540.3; [M+Na] found 540.0.
4.8.17)
3-{[4-(piperazin-1-yl)octan-1-one]methyl}-5-(pyridin-4-yl)-1,3,4-oxadiazole-
2(3H)-thione 54
White solid; Yield: 85%; m.p.: 74-76 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm:8.75 (2H;
3
4
3
4
dd; J= 4.5 Hz; J= 2.0 Hz), 7.70 (2H; J= 4.5 Hz; J= 2.0 Hz), 5.06 (2H; s), 3.58-3.41
(4H; bs), 2.77 (4H; m), 2.20 (2H; t; J= 7.0 Hz), 1.50 (2H; m), 1.21-1.15 (8H; m), 0.77
(3H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.3, 171.6, 156.9, 150.9,
129.4, 119.6, 70.5, 50.5, 50.3, 45.4, 41.3, 33.2-22.5, 14.0. MALDI-TOF m/z: calculated
[M-H] 402.1962; [M-H] found 402.1948.
4.8.18) 3-{[4-(piperazin-1-yl)decan-1-one] methyl}- 5-(pyridin-4-yl)-1,3,4-oxadiazole-
2(3H)-thione 55
White solid; Yield: 80%; m.p.: 79-81 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.77 (2H;
3
4
3
4
dd; J= 7.0 Hz; J= 2.0 Hz), 7.72 (2H; J= 7.0 Hz; J= 2.0 Hz), 5.07 (2H; s), 3.60-3.43
(4H; bs), 2.78 (4H; m), 2.22 (2H; t; J= 7.0 Hz), 1.50 (2H; m); 1.21-1.17 (12H; m), 0.80
(3H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.4, 171.7, 157.0, 151.0,
129.5, 119.7, 70.6, 50.5, 50.1, 45.3, 41.4, 33.2-22.6, 14.1. MALDI-TOF m/z: [M+K]
calculated 470.3323; [M+K] found 470.2569.
4.8.19) 3-{[4-(piperazin-1-yl)dodecan-1-one]methyl}-5-(pyridin-4-yl)-1,3,4-oxadiazole-
2(3H)-thione 56
Yellow solid; Yield: 58%; m.p.: 81-83°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.80
(2H; dd; 3J= 4.5 Hz; 4J= 2.0 Hz), 7.75 (2H; 3J= 7.0 Hz; 4J= 2.0 Hz), 5.10 (2H; s), 3.62-
3.47 (4H; bs), 2.81 (4H; bs), 2.24 (2H; t; J= 7.0 Hz), 1.54 (2H; m), 1.24-1.20 (16H; m),
0.84 (3H; t; J= 7.0 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm: 178.5, 171.8, 157.1, 151.1,
129.6, 119.8, 70.6, 50.6, 50.1, 45.5, 41.5, 33.3-22.8, 14.2. MALDI-TOF m/z: [M-H]
calculated 458.2588; [M-H] found 458.2592.
4.8.20)
3-{[4-(piperazin-1-yl)tetradecan-1-one]methyl}-5-(pyridin-4-yl)-1,3,4-
oxadiazole-2(3H)-thione 57
25

ACCEPTED MANUSCRIPT
White solid; Yield: 72%; m.p.: 86-88 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.80 (2H;
d; J= 6.0 Hz), 7.76 (2H; J= 6.0 Hz), 5.11 (2H; s), 3.63-3.46 (4H; bs), 2.82 (4H; bs), 2.25
(2H; t; J= 7.5 Hz), 1.55 (2H; m), 1.25-1.21 (20H; m), 0.85 (3H; t; J= 7.5 Hz).¹³C NMR
(CDCl3; 125 MHz) δ ppm: 178.5, 171.8, 157.1, 151.1, 129.6, 119.8, 70.7, 50.7, 50.2,
45.6, 41.5, 33.9-22.8, 14.2. ESI-MS: m/z 487.7 calculated; m/z 487.0 found; [M+H]
calculated 488.7; [M+H] found 488.0; [M+Na] calculated 510.0; [M+Na] found 510.0.
4.8.21)3-{[4-(piperazin-1-yl)octan-1-one]methyl}-5-phenyl-1,3,4-oxadiazole-2(3H)-
thione 58
Brown solid; Yield: 80%; m.p.: 78-80°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.91 (2H;
m), 7.55 (2H; m), 7.49 (2H; m), 5.09 (2H; s), 3.62-3.46 (4H; bs), 2.83 (4H; bs), 2.25
(2H; t; J= 7.0 Hz), 1.56 (2H; m), 1.26-1.22 (8H; bs), 0.83 (3H; t; J= 7.0 Hz).¹³C NMR
(CDCl3; 125 MHz) δ ppm:178.3, 171.8, 159.2, 132.6, 129.3, 126.6, 122.3, 70.3, 50.6-
50.2, 45.6-41.5, 33.3-22.7, 14.2. MALDI-TOF m/z: [M+H] calculated 403.2169;
[M+H] found 403.2163.
4.8.22)3-{[4-(piperazin-1-yl) decan-1-one]methyl}-5-phenyl-1,3,4-oxadiazole-2(3H)-
thione 59
Brown oil; Yield: 63%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.89 (2H; m), 7.56 (2H;
m), 7.50 (2H; m), 5.07 (2H; s), 3.62-3.44 (4H; bs), 2.82 (4H; bs), 2.24 (2H; t; J= 7.0
Hz), 1.55 (2H; m); 1.25-1.19 (12H; bs), 0.82 (3H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125
MHz) δ ppm:178.3, 171.7, 159.1, 132.6, 129.3, 126.6, 122.3, 70.2, 50.6, 50.2, 45.5,
41.5,33.3-22.7, 14.1. MALDI-TOF m/z: [M+H] calculated 431.2482; [M+H] found
431.2483.
4.8.23) 3-{[4-(piperazin-1-yl)dodecan-1-one]methyl}-5-phenyl-1,3,4-oxadiazole-2(3H)-
thione 60
3
Colorless oil; Yield: 47%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.92 (2H; dd; J= 8.0
Hz; 4J= 1.5 Hz), 7.56 (2H; tt; 3J= 7.5 Hz; 4J= 1.0 Hz), 7.51-7.48 (2H; m), 5.10 (2H; s),
3.65-3.47 (4H; bs), 2.84 (4H; m), 2.26 (2H; t; J= 7.0 Hz), 1.57 (2H; m), 1.26-1.22 (16H;
bs), 0.86 (3H; t; J= 7.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:178.4, 171.8, 159.2,
132.7, 129.3, 126.7, 122.4, 70.3, 50.7, 50.2, 45.6, 41.5, 33.4-22.8, 14.3. MALDI-TOF
m/z: [M+H] calculated 459.2795; [M+H] found 459.2786.
4.8.24)3-{[4-(piperazin-1-yl)tetradecan-1-one]methyl}-5-phenyl-1,3,4-oxadiazole-
2(3H)-thione 61
26

ACCEPTED MANUSCRIPT
3
Brown oil; Yield: 83%; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.92 (2H; dd; J= 8.0 Hz;
4J= 1.5 Hz), 7.56 (2H; tt; 3J= 8.0 Hz; 4J= 1.5 Hz), 7.51-7.48 (2H; m), 5.10 (2H; s), 3.65-
3.48 (4H; bs), 2.84 (4H; m), 2.26 (2H; t; J= 7.5 Hz), 1.57 (2H; m), 1.24-1.22 (20H; bs),
0.86 (3H; t; J= 7.5 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.4, 171.9, 159.3, 132.7,
129.3, 126.7, 122.4, 70,3, 50.7, 50.2, 45.6, 41.5, 33.4-22.8, 14.3. MALDI-TOF m/z:
[M+H] calculated 487.3108; [M+H] found 487.3109.
4.8.25)3-{[4-(piperazin-1-yl)octan-1-one]methyl}-5-(4-methoxyphenyl)-1,3,4-
oxadiazole-2(3H)-thione 62
White solid; Yield: 83%; m.p.: 80-82 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.86 (2H;
3
4
3
4
dd; J= 9.0 Hz; J= 1.5 Hz), 6.99 (2H; d; J= 9.0 Hz; J= 1.5 Hz), 5.07 (2H; s), 3.87
(3H; s), 3.64-3.47 (4H; bs), 2.84 (4H; bs), 2.27 (2H; t; J= 7.0 Hz), 1.57 (2H; m), 1.27-
1.24 (8H; bs), 0.84 (3H; t; J= 7,0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:178.1, 171.8,
163.1, 159.4, 128.6, 114.8, 114.7, 70.2, 55.7, 50.7, 50.3, 45.6, 41.5, 33.4-22.7,
14.2.MALDI-TOF m/z: [M+H] calculated 433.2275; [M+H] found 433.2270.
4.8.26)
3-{[4-(piperazin-1-yl)decan-1-one]methyl}-5-(4-methoxyphenyl)-1,3,4-
oxadiazole-2(3H)-thione 63
White solid; Yield: 95%; m.p.: 83-85 °C; ¹H NMR (CDCl3; 500 MHz) δ ppm:7.85 (2H;
d; J= 9.0 Hz), 6.98 (2H; d; J= 9.0 Hz), 5.07 (2H; s), 3.87 (3H; s), 3.64-3.48 (4H; bs),
2.84 (4H; bs), 2.27 (2H; t; J= 8.0 Hz), 1.57 (2H; m), 1.26-1.23 (12H; bs), 0.85 (3H; t; J=
8.0 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:181.9, 171.9, 163.1, 159.4, 128.6, 114.8,
114.7, 70.2, 55.7, 50.7-50.3, 45.6, 41.6, 33.4-22.8, 14.2. MALDI-TOF m/z: [M+H]
calculated 461.2588; [M+H] found 461.2586.
4.8.27)3-{[4-(piperazin-1-yl)dodecan-1-one]methyl}-5-(4-methoxyphenyl)-1,3,4-
oxadiazole-2(3H)-thione 64
White solid; Yield: 65%; m.p.: 38-40°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 7.85 (2H;
m), 6.98 (2H; m), 5.05 (2H; s), 3.85 (3H; s), 3.62-3.46 (4H; bs), 2.82 (4H; bs), 2.25
(2H; t; J= 8.0 Hz), 1.57 (2H; bs), 1.26-1.23 (16H; bs); 0.85 (3H; t; J= 7.0 Hz).¹³C NMR
(CDCl3; 125 MHz) δ ppm:178.0, 171.8, 163.0, 159.3, 128.5, 114.7, 114.6, 70.2, 55.6,
50.6, 50.2, 45.6, 41.5, 29.6-22.7, 14.2. MALDI-TOF m/z: [M+H] calculated 489.2901;
[M+H] found 489.2897.
4.8.28)3-{[4-(piperazin-1-yl)tetradecan-1-one]methyl}-5-(4-methoxyphenyl)-1,3,4-
oxadiazole-2(3H)-thione 65
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ACCEPTED MANUSCRIPT
White solid; Yield: 65%; m.p.: 38-40°C; ¹H NMR (CDCl3; 500 MHz) δppm: 7.85 (2H;
m), 6.98 (2H; m), 5.05 (2H; s), 3.85 (3H; s), 3.62-3.46 (4H; bs), 2.82 (4H; bs), 2.25
(2H; t; J= 8.0 Hz), 1.57 (2H; bs), 1.26-1.23 (16H; bs); 0.85 (3H; t; J= 7.0 Hz).¹³C NMR
(CDCl3; 125 MHz) δ ppm:178.0, 171.8, 163.0, 159.3, 128.5, 114.7, 114.6, 70.2, 55.6,
50.6, 50.2, 45.6, 41.5, 29.6-22.7, 14.2. MALDI-TOF m/z: [M+H] calculated 517.3214;
[M+H] found 517.3217.
4.8.29) 3-{[4-(piperazin-1-yl)octan-1-one]methyl}-5-(4-nitrophenyl)-1,3,4-oxadiazole-
2(3H)-thione 66
Orange solid; Yield: 77%; m.p.: 60-61°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.34
(2H; dd; 3J= 9.0 Hz; 4J= 2.0 Hz); 8.09 (2H; d; J= 9.0 Hz); 5.10 (2H; s); 3.61 (2H; t; J=
5.0 Hz); 3.46 (2H; t; J= 5.0 Hz); 2.82 (4H; m); 2.22 (2H; t; J= 7.5 Hz); 1.50 (2H; m);
1.23-1.19 (8H; m); 0.79 (3H; t; J= 7.5 Hz).¹³C NMR (CDCl3; 125 MHz) δ ppm:178.4,
171.9, 157.0, 149.8, 127.7, 127.5, 124.5, 70.6, 50.6, 50.0, 45.5, 41.4, 33.2, 31.7, 29.4,
29.0, 25.3, 22.6, 14.1. ESI-MS: m/z calculated 447.2; m/z found 447.0; [M+H]
calculated 448.2; [M+H] found 448.0; [M+Na] calculated 470.3; [M+Na] found 470.0.
4.8.30) 3-{[4-(piperazin-1-yl)decan-1-one]methyl}-5-(4-nitrophenyl)-1,3,4-oxadiazole-
2(3H)-thione 67
Orange solid; Yield: 83%; m.p.: 62-63°C; ¹H NMR (CDCl3; 500 MHz) δ ppm: 8.37
(2H; d; J= 9.0 Hz); 8.12 (2H; d; J= 9.0 Hz); 5.13 (2H; s); 3.66 (2H; t; J= 5.0 Hz); 3.49
(2H; t; J= 5.0 Hz); 2.85 (4H; m); 2.27 (2H; t; J= 7.5 Hz); 1.56 (2H; m); 1.26-1.22 (12H;
m); 0.85 (3H; t; J= 7.5 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.5, 171.9, 157.,
150.0, 127.8, 127.6, 124.6, 70.7, 50.7, 50.2, 45.6, 41.5, 33.4, 32.0, 29.6, 29.5, 29.4,
25.4, 22.8, 14.2.ESI-MS: m/z calculated 475.2; m/z found 475.0; [M+H] calculated
476.2; [M+H] found 476.0; [M+Na] calculated 498.3; [M+Na] found 498.0.
4.8.31)
3-{[4-(piperazin-1-yl)dodecan-1-one]methyl}-5-(4-nitrophenyl)-1,3,4-
oxadiazole-2(3H)-thione 68
Orange solid; Yield: 79%; m.p.: 65-67°C; ¹H NMR (CDCl3; 500 MHz) δppm: 8.34 (2H;
d; J= 8.5 Hz); 8.12 (2H; d; J= 8.5 Hz); 5.11 (2H; s); 3.62 (2H; bs); 3.48 (2H; bs; H);
2.83-2.81 (4H; m); 2.25 (2H; t; J= 7.5 Hz); 1.53 (2H; m); 1.21-1.19 (12H; m); 0.83 (3H;
t; J= 7.5 Hz). ¹³C NMR (CDCl3; 125 MHz) δ ppm:178.4, 171.9, 157.0, 149.9, 127.7,
127.5, 124.5, 70.6, 50.6, 50.1, 45.5, 41.5, 33.3, 31.9, 29.6, 29.5, 29.4, 29.3, 25.3, 22.7,
14.2. ESI-MS: m/z calculated 503.2; m/z found 503.0; [M+H] calculated 504.2; [M+H]
found 504.0; [M+Na] calculated 526.2; [M+Na] found 526.0.
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