Synthesis of oxadiazoline and quinazolinone derivatives and their biological evaluation as nitric oxide synthase inhibitors

Article abstract of DOI:10.1007/s00044-016-1568-0

The synthesis of two new families of compounds with oxadiazoline and quinazolinone structures and their in vitro biological evaluation as inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS) are described. These derivatives have been obtained from cyclization of substituted benzohydrazides with acid anhydrides followed by reduction, using different synthetic procedures. Their structures were confirmed by high-resolution mass spectroscopy and ¹H and ¹³C nuclear magnetic resonance data. In general, the assayed compounds show better inhibition values of nNOS than of iNOS, being 7d the most active derivative with a quinazolinone scaffold, and 6t the best oxadiazoline one and the best nNOS inhibitor of all tested compounds. The structure–activity relationships are discussed in terms of the effects of the substituents on both 2- and 3-positions of the heterocyclic rings.

Full text of DOI:10.1007/s00044-016-1568-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2016) 25:1260–1273
DOI 10.1007/s00044-016-1568-0
ORIGINAL RESEARCH
Synthesis of oxadiazoline and quinazolinone derivatives
and their biological evaluation as nitric oxide synthase inhibitors
1
1
•
M. Jose Pineda de las Infantas M. Dora Carrion Mariem Chayah1
•
•
´
´
Luisa C. Lopez-Cara Miguel A. Gallo1 Darıo Acun˜a-Castroviejo
1
2
•
•
•
´
M. Encarnacion Camacho
´
1
´
Received: 10 March 2015 / Accepted: 1 April 2016 / Published online: 8 April 2016
Ó Springer Science+Business Media New York 2016
Abstract The synthesis of two new families of com-
pounds with oxadiazoline and quinazolinone structures and
their in vitro biological evaluation as inhibitors of both
neuronal and inducible nitric oxide synthases (nNOS and
iNOS) are described. These derivatives have been obtained
from cyclization of substituted benzohydrazides with acid
anhydrides followed by reduction, using different synthetic
procedures. Their structures were confirmed by high-reso-
lution mass spectroscopy and 1H and 13C nuclear magnetic
resonance data. In general, the assayed compounds show
better inhibition values of nNOS than of iNOS, being 7d
the most active derivative with a quinazolinone scaffold,
and 6t the best oxadiazoline one and the best nNOS inhi-
bitor of all tested compounds. The structure–activity rela-
tionships are discussed in terms of the effects of the
substituents on both 2- and 3-positions of the heterocyclic
rings.
Introduction
Nitric oxide (NO) is produced by oxidation of L-arginine
(Stuehr and Griffith 1992) in a NADPH- and O2-dependent
process catalyzed by nitric oxide synthase (NOS). Three
different isoforms of this enzyme have been identified
(neuronal or nNOS, endothelial or eNOS, and inducible or
iNOS) (Knowles et al., 1989; McCall et al., 1989). The
constitutive endothelial and neuronal isoforms are cal-
cium/calmodulin dependent and are physiologically acti-
vated by hormones or neurotransmitters that increase the
intracellular calcium concentration. However, the inducible
isoform is activated by basal intracellular calcium con-
centrations, and, once expressed, it remains activated,
yielding high NO concentrations. This mechanism is part
of the normal immune response against invading pathogens
and neoplastic cells (Pozo et al., 1998).
Overproduction of NO by nNOS has been associated
with several neurodegenerative disorders, such as Parkin-
son’s, Alzheimer’s, or Huntington’s diseases, as well as
migraine (Olesen, 2010; Aquilano et al., 2008; Villanueva
and Giulivi 2010), while NO overproduction by the indu-
cible isoform has been associated with tissue damage,
inflammation, rheumatoid arthritis, and inflammatory
bowel disease (Levy and Zochodne 2004; Vallance and
Leiper 2002; LaBuda et al., 2006; Ijuin et al., 2006). Also,
inflammatory reaction in Parkinson’s disease is associated
with iNOS (Dehmer et al., 2000). Since nNOS and iNOS
represent a therapeutic target, inhibition of these enzymes
by means of synthetic derivatives has become an interest-
ing objective in the potential treatment of these diseases
(Serafim et al., 2012).
Keywords Oxadiazolines Á Quinazolinones Á Inhibition Á
Neuronal nitric oxide synthase (nNOS) Á
Inducible nitric oxide synthase (iNOS)
´
& M. Encarnacion Camacho
ecamacho@ugr.es
1
´
´
´
Departamento de Quımica Farmaceutica y Organica,
Facultad de Farmacia, Universidad de Granada, Granada,
Spain
Melatonin 1, the hormone secreted by the pineal gland,
is a well-known NOS inhibitor (Bettahi et al., 1996; Leon
et al., 1998). In previous papers, we have described several
2
´
´
´
Centro de Investigacion Biomedica, Parque Tecnologico de
Ciencias de La Salud, Universidad de Granada, Granada,
Spain
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Med Chem Res (2016) 25:1260–1273
1261
compounds with kynurenamine 2 (Entrena et al., 2005) and
4,5-dihydro-1H-pyrazole 3 structures (Camacho et al.,
2004), as nNOS inhibitors. The last compounds are rigid
analogues of the main brain metabolite of melatonin,
the N-acetyl-5-methoxykynurenamine (2, R1 = OCH3,
R2 = CH3), which also inhibits the iNOS in sepsis asso-
ciated with Parkinson’s disease (Tapias et al., 2009). In
addition, we have published compounds with 5-phenyl-1H-
pyrrole-2-carboxamide structure 4 which show moderate
Results and discussion
Chemistry
Scheme 1 represents the general synthetic pathway of the
final aminophenyl-oxadiazoline derivatives 6, as well as
the cyclized quinazolinone derivatives 7 described in this
paper. The synthesis begins with the transformation of the
2-nitrobenzoic acid 8 into the corresponding ethyl 2-ni-
trobenzoate, followed by treatment with hydrazine in
ethanol to afford the 2-nitrobenzohydrazide 9 (Balo et al.,
2007). This compound was condensed to reflux with sev-
eral carbonyl compounds (aldehydes and ketones), to give
the benzohydrazides 10–15 (Raparti et al., 2009; Evans
et al., 1986), which were cyclized to the 1,3,4-oxadiazole
derivatives 16a–s by treatment to reflux with the corre-
sponding acid anhydrides (Chaaban et al., 2014; Joshi
et al., 2008). Finally, reduction of the nitro group belong-
ing to the aromatic ring of the aryloxadiazolines 16 leads
different types of compounds depending of the R1 and R2
substituents in C-2 of the heterocyclic ring. Thus, when this
position is monosubstituted (R1 = H), the reduction of the
nitro group belonging to derivatives 16a–l, by refluxing
with Fe/FeSO4 in water (Zhu et al., 1995), leads to the
opening of the oxadiazole ring and to the rearrangement of
the final quinazolinones 7a–l (Davidson, 1984). Never-
theless, when C-2 is disubstituted (R1 = Me), the reduction
of compounds 16m–p and 16r–t by the same procedure
yields the aminophenyl-oxadiazolines 6m–p and 6r–
t. Moreover, the reduction of 16p led not only to compound
6p, but also to 6q, due to the presence of a valeric anhy-
dride remainder. Finally, in order to obtain the
´
values of nNOS and iNOS inhibition (Lopez-Cara et al.,
2009). Finally, we have reported a group of 2,3-dihydro-
1,3,4-thiadiazoles 5, which exhibit good iNOS inhibition
´
values (Lopez-Cara et al., 2012) (Fig. 1).
In the present work, as an extension of the heterocyclic
compounds synthesized as NOS inhibitors, we report a new
group of derivatives with an 1,3,4-oxadiazoline scaffold 6,
where the sulfur atom of compounds 5 has been replaced
by its isosteric oxygen. In addition, under the last step of
the oxadiazolines’ synthesis we have found that, when the
C-2 of the heterocyclic ring is monosubstituted (R1 = H),
the molecules isomerize during the nitro group reduction to
yield the quinazolinones 7. These last derivatives have the
NH2 group forming a pyrimidine ring, and, consequently,
their conformational freedom is more restricted. As well as
the previously synthesized derivatives 2–5 and compounds
6 and 7 have been evaluated as in vitro inhibitors of both
neuronal and inducible NOS in order to find new deriva-
tives with interesting pharmacological properties. In this
way, oxadiazoles as well as quinazolines are considered to
be important chemical synthons with various therapeutic
uses (Soares de Oliveira et al., 2012; Daidonea et al.,
1999).
Fig. 1 Melatonin and some
NOS inhibitors synthesized by
our research group
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Med Chem Res (2016) 25:1260–1273
Scheme 1 General synthetic
pathway followed in the
preparation of derivatives 6a–i,
6m–t and 7a–l: i EtOH/H2SO4,
reflux, 24 h; ii NH2NH2, dry
EtOH, reflux, 5 h; iii R1COR2,
dry EtOH, reflux, 1.5–24 h; iv
(R3CO)2O, reflux, 2 h; v Fe/
FeSO4, H2O, reflux, 3 h; vi H2,
Pd–C (10 %), MeOH, 1 atm,
RT, 3 h
Table 1 Structural data and yields of the intermediate (10–16) and final (6, 7) compounds described
R1
R2
R3
R4
Comp.
Comp.
mg obtained (%Rto.)
Comp.
mg obtained (%Rto.)
Comp.
mg obtained (%Rto.)
H
Me
Me
Me
Me
Et
Me
Et
H
16a
16b
16c
16d
16e
16f
177 (71)
183 (70)
186 (67)
213 (73)
189 (72)
222(80)
198 (68)
232 (76)
196 (63)
234 (72)
200 (59)
227 (63)
168 (64)
233 (84)
236 (81)
238 (78)
6a
6b
6c
6d
6e
6f
85 (94)
87 (90)
92 (90)
102 (94)
89 (92)
97 (95)
99 (92)
103 (90)
107 (92)
7a
7b
7c
7d
7e
7f
72 (63)
82 (67)
85 (66)
90 (66)
89 (73)
83 (64)
93 (68)
71 (49)
80 (54)
73 (47)
65 (40)
127 (75)
H
H
10
H
Pr
H
H
Bu
Me
Et
H
H
H
H
Et
H
11
12
H
Et
Pr
H
16g
16h
16i
6g
6h
6i
7g
7h
7i
H
Et
Bu
Me
Et
H
H
Ph
Ph
Ph
Ph
Me
Me
Me
Me
Me
Et
H
H
H
16j
7j
H
Pr
H
16k
16l
7k
7l
H
Bu
Me
Et
H
Me
Me
Me
Me
Me
Me
Me
Me
H
16m
16n
16o
16p
6m
6n
6o
6p
6q
6r
6s
62 (51)
79 (61)
H
Pr
H
13
111 (81)
82 (57)
Bu
Bu
Me
Et
H
COBu
H
37 (27)
14
15
16r
16s
16t
208 (75)
230 (79)
267 (82)
107 (83)
107 (78)
124 (80)
Et
H
Ph
Me
H
6t
nNOS and iNOS inhibition
aminophenyl-1,3,4-oxadiazoline derivatives 6a–i with
R1 = H, we carried out the reduction of compounds 16a–
i by catalytic hydrogenation (Pd–C), with the purpose of
avoiding the formation of quinazoline derivatives. Table 1
shows the structural data of all the intermediate and final
compounds synthesized.
The biological activities of final compounds 6 and 7 as
inhibitors of both nNOS and iNOS have been evaluated by
means of in vitro assays using recombinant isoenzymes
(Bredt and Snyder 1990). In order to identify the most
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Med Chem Res (2016) 25:1260–1273
1263
potent compounds, a first assay using 1 mM concentration
of each compound was performed. Figure 2 shows the
percentages of residual activity of both isoforms in the
presence of each compound in relation to the control (c),
and Table 2 illustrates the inhibition percentages versus
iNOS and nNOS. The thiadiazolines 5a–e (5a: R1 = H,
R2 = Me; 5b: R1 = H, R2 = Et; 5c: R1 = H, R2 = Pr; 5d:
R1 = H, R2 = Bu; 5e: R1 = H, R2 = c-C3H5) (Fig. 1)
heterocyclic ring, with the exception of 6f (R3 = Et),
which is more potent than 6g and 6h, with less volume in
R3. Among the quinazolinones 7, there are not com-
pounds that exhibit a significant value of inhibition of this
isoform.
Table 2 also shows the nNOS inhibition values observed
in the presence of 1 mM concentration of final compounds
6 and 7. In this case, oxadiazolines have also better values
of inhibition than quinazolinones, since four compounds
display more than 40 % of inhibition versus only two
quinazolinones. Regarding oxadiazolines with the same
substituents R1 = R2 = Me (6m–q), the most potent
compounds have a methyl group (6m, 41.1 %) or a butyl
one (6p, 55.1 % and 6q, 48.8 %) in R3. In addition, 6t
(R1 = R3 = Me, R2 = Ph) is the most potent compound of
this group (74.0 % of inhibition) and the best nNOS inhi-
bitor of all the tested compounds. In quinazolinones,
compounds with R2 = Me (7a–d) show better inhibition
values than derivatives with Et (7e–h) or Ph (7i–l), being
the most active compounds 7a (R3 = Me, 43.1 %) and 7d
(R3 = Bu, 65.3 %), which is the most potent inhibitor of
this family.
´
previously described by our research group (Lopez-Cara
et al., 2012) and the well-known NOS inhibitor L-NAME
(Kilbourn and Griffith 1992) have been introduced as
controls to compare them with the new oxadiazolines and
quinazolinones.
In general, regarding the iNOS inhibition values, oxa-
diazolines 6 are better inhibitors than quinazolinones 7. In
derivatives with structure type 6, there are six compounds
which show more than 40 % of inhibition. Nevertheless,
some conclusions can be inferred from the experimental
data. From a qualitative point of view, the influence of R1
and R2 over the iNOS activity is variable. When R1 = H
(6a–i), compounds with R2 = Et (6e–h) have better values
of inhibitory activity than molecules with R2 = Me (6a–d)
or Ph (6i), being 6f, 6g, and 6h the best inhibitors of this
group with 43.5, 41.0, and 41.8 % of inhibition, respec-
tively. Furthermore, when R1 = Me (6m–t), derivatives
with R2 = Et or Ph show the best inhibition values, being
6s (R2 = R3 = Et, 52.9 %) and 6t (R2 = Ph, R3 = Me,
52.1 %) the most potent compounds of this group and the
best iNOS inhibitors of all the oxadiazoline derivatives
tested. On the other hand, improved inhibitory activity is
observed when the volume in R3 increases (from Me to
Bu), maintaining the same R1 and R2 substituents on the
Furthermore, Table 2 includes the inhibition values for
both isoenzymes of several thiadiazolines 5 previously
´
published (Lopez-Cara et al., 2012). Although some of
these compounds have demonstrated to be selective iNOS/
nNOS inhibitors, the comparison of compounds 5a–d and
6m–p wearing the same substituents in both the hetero-
cyclic ring and the acyl group indicates that oxadiazolines
have slightly higher values of inhibition than their isosteres
with a sulfur atom. In general, oxadiazolines have higher
potency versus nNOS than thiadiazolines, proving that the
Fig. 2 Percentage of iNOS and
nNOS residual activities in the
presence of 1 mM of the
oxadiazolines (compounds 6a–
t) and the quinazolinones
(compounds 7a–l) assayed as
compared to those of untreated
samples (c). Each value is the
mean of three experiments
performed in triplicate using
recombinant iNOS or nNOS
enzymes
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Med Chem Res (2016) 25:1260–1273
Table 2 In vitro nNOS and iNOS inhibition (%) observed in the presence of 1 mM concentration of final compounds 6a–t and 7a–l. Thia-
diazolines 5a–e and L-NAME are included as controls
Comp.
% iNOS
inhibitiona
% nNOS
inhibitiona
iNOS/nNOS
selectivity
Comp.
% iNOS
inhibitiona
% nNOS
inhibitiona
iNOS/nNOS
selectivity
6a
6b
6c
6d
6e
6f
26.15 0.70
32.78 2.36
32.96 0.53
33.51 0.86
35.23 0.59
43.55 1.40
41.04 2.11
41.76 0.76
33.13 2.66
28.41 1.76
31.38 0.23
37.63 1.33
40.28 1.89
34.98 2.32
32.20 0.45
52.85 1.58
52.10 0.46
20.56 2.82
18.98 0.87
25.59 1.21
12.24 1.85
19.93 2.21
24.03 0.71
2.36 2.51
0.09 1.20
11.85 0.67
41.08 3.12
36.50 2.04
28.01 0.32
55.11 1.25
48.78 0.21
13,11 2.03
13.32 1.23
73.95 0.49
1.27
2.73
1.29
2.74
1.77
1.81
17.39
464.0
2.80
0.69
0.86
1.34
0.73
0.72
2.46
3.97
0.71
7a
32.81 0.37
19.81 0.43
6.40 1.03
19.61 1.73
18.46 0.48
18.93 3.87
0.68 3.29
0.50 4.23
24.91 3.57
9.51 4.06
18.26 0.59
0.39 5.74
22.85 1.21
19.24 1.75
35.18 2.01
39.05 1.32
92.96 0.68
77.01 0.96
43.08 2.87
34.18 3.26
0.97 0.45
65.32 0.23
15.89 1.58
2.47 1.33
6.26 1.38
10.46 2.38
0.02 0.97
7.78 0.11
0.81 1.87
8.34 2.22
13.27 2.12
24.21 1.23
17.46 1.94
38.05 0.73
7.32 3.65
100.0 1.03
0.76
0.58
7b
7c
6.60
7d
0.30
7e
1.17
7f
7.66
6g
6h
6i
7g
0.11
7h
0.05
7i
1245.5
1.22
6m
6n
6o
6p
6q
6r
6s
7j
7k
22.54
0.05
7l
5ab
5bb
5cb
5db
5eb
L-NAMEc
1.72
0.79
2.01
1.03
6t
12.69
0.77
a
Data represent the mean SEM of the percentage of iNOS and nNOS inhibition produced by 1 mM concentration of each compound. Each
value is the mean of three experiments performed by triplicate using recombinant iNOS and nNOS enzymes
b
´
See Ref. Lopez-Cara et al. (2012)
c
See Ref. Kilbourn and Griffith (1992)
Conclusions
Table 3 IC50 values (mM) for the iNOS and nNOS inhibition by the
derivatives 6p, 6s, 6t, and 7d
In summary, in this paper we report the synthesis of 17
novel oxadiazole derivatives 6 structurally related to the
above-described thiadiazolines 5, as well as 12 new
quinazolinones 7. In this second family, the NH2 group of
the benzene ring, present in the previous inhibitors, is now
included in a pyrimidine ring, giving more conformation-
ally restricted molecules. In addition, we evaluate the
nNOS and iNOS activities of these new structures.
Although they mostly reveal moderate activity, several
compounds show promising values. In general, the tested
derivatives have better nNOS inhibition values than those
of iNOS, and the oxadiazolines are more potent than the
less flexible quinazolinones, showing that the inclusion of
the free amine group in a new ring is detrimental for the
inhibitory activity. Moreover, we have demonstrated that
the presence of an oxygen atom in the heterocyclic ring of
derivatives 6 leads to better inhibitory activity against
nNOS, in contrast to the higher potency of the isosteric
thiadiazolines 5 versus iNOS. Compound 7d is the most
potent nNOS inhibitor with a quinazolinone skeleton, and
6t is the best one with an oxadiazoline heterocycle, being
also the best nNOS inhibitor of all the tested compounds.
Compound
IC50 iNOSa
IC50 nNOSa
6p
6s
6t
7d
a
b
0.928
b
0.968
0.981
b
0.468
0.879
Data were obtained by measuring percentage of inhibition with at
least five concentrations of inhibitor
b
Not tested
presence of an oxygen atom in the heterocyclic ring
improves the nNOS inhibition.
Table 3 shows the IC50 values measured for the
nNOS and iNOS inhibition of the most interesting
compounds observed in the initial in vitro assay. The
oxadiazoline 6t and the quinazolinone 7d show the best
results versus nNOS, with IC50 values between 0.47 and
0.88 mM, respectively, confirming that 6t is the most
potent inhibitor of all synthesized compounds versus
nNOS. Respect to iNOS, IC50 values are around
0.97 mM.
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2-Methyl-5-(2-nitrophenyl)-3-propionyl-2,3-dihydro-1,3,4-
oxadiazole (16b) Yield 70 % (0.183 g) as a yellow oil.
1H NMR (400 MHz, CDCl3) d = 7.83 (2H, m, H-30, H-60),
7.64 (2H, m, H-40, H-50), 6.34 (1H, q, J = 4.0 Hz, H-2), 2.
60 (2H, q, J = 6.0 Hz, COCH2CH3), 1.66 (3H, d, J = 4.
0 Hz, CH3), 1.16 (3H, t, J = 6.0 Hz, COCH2CH3). 13C
NMR (100 MHz, CDCl3) d = 171.62 (C, C=O), 152.71
(C, C-5), 149.17 (C, C-20), 132.57 (CH, C-40), 132.28 (CH,
C-50), 130.81 (CH, C-60), 124.39 (CH, C-30), 119.30 (C,
C-10), 91.32 (CH, C-2), 27.37 (CH2, COCH2CH3), 20.16
(CH3, CH3), 8.82 (CH3, COCH2CH3).
Thus, 6t should be an interesting starting point in the
development of therapeutic agents for the treatment of NO-
related diseases, where both isoforms are implied, such as
Parkinson’s.
Experimental
Instruments and reagent
Melting points were determined using an Electrothermal-
1
1A-6301 apparatus and are uncorrected. H NMR and 13C
3-Butyryl-2-methyl-5-(2-nitrophenyl)-2,3-dihydro-1,3,4-oxa-
1
diazole (16c) Yield 67 % (0.186 g) as a yellow oil. H
NMR spectra were recorded on a Bruker AMX 300 spec-
1
trometer operating at 300.160 for H and 75.479 MHz for
13C, and on a Bruker ARX 400 spectrometer operating at
400.132 MHz for 1H and 100.623 MHz for 13C, in CDCl3,
(CD3)2CO, and (CD3)2SO at room temperature (rt). The
peaks are reported in ppm (d) and are referenced to the
residual solvent peak. High-resolution nano-assisted laser
desorption/ionization or electrospray ionization mass
spectra were carried out on a Bruker Autoflex or a Waters
LCT Premier mass spectrometer, respectively. Analytical
thin layer chromatography was performed using Merck
Kieselgel 60 F254 aluminum sheets, the spots being
developed with UV light (k = 254 nm). Flash chro-
matography was carried out using silica gel 60, 230–240
mesh (Merck), and the solvent mixture reported within
parentheses was used as eluent.
NMR (300 MHz, CDCl3) d = 7.86 (2H, m, H-30, H-60), 7.
64 (2H, m, H-40, H-50), 6.37 (1H, q, J = 6.0 Hz, H-2), 2.60
(2H, m, COCH2CH2CH3), 1.70 (5H, m, COCH2CH2CH3,
CH3), 1.01 (3H, t, J = 9.0 Hz, COCH2CH2CH3). 13C
NMR (75 MHz, CDCl3) d = 170.82 (C, C=O), 152.70 (C,
C-5), 150.85 (C, C-20), 132.57 (CH, C-40), 132.27 (CH,
C-50), 130.84 (CH, C-60), 124.41 (CH, C-30), 119.36 (C,
C-10), 91.31 (CH, C-2), 35.91 (CH2, COCH2CH2CH3), 20.
15 (CH3, CH3), 18.33 (CH2, COCH2CH2CH3), 14.06 (CH3,
COCH2CH2CH3).
2-Methyl-5-(2-nitrophenyl)-3-pentanoyl-2,3-dihydro-1,3,4-
oxadiazole (16d) Yield 73 % (0.213 g) as a yellow oil.
1H NMR (300 MHz, CDCl3) d = 7.86 (2H, m, H-30, H-60),
7.68 (2H, m, H-40, H-50), 6.36 (1H, q, J = 6.0 Hz, H-2), 2.61
(2H, m, COCH2CH2CH2CH3), 1.68 (5H, m, COCH2CH2
CH2CH3, CH3), 1.41 (2H, m, COCH2CH2CH2CH3), 0.95
(3H, t, J = 9.0 Hz, COCH2CH2CH2CH3). 13C NMR
(75 MHz, CDCl3) d = 170.99 (C, C=O), 152.69 (C, C-5),
149.17 (C, C-20), 132.58 (CH, C-40), 132.27 (CH, C-50), 130.
83 (CH, C-60), 124.39 (CH, C-30), 119.33 (C, C-10), 91.29
(CH, C-2), 33.74 (CH2, COCH2CH2CH2CH3), 26.94 (CH2,
COCH2CH2CH2CH3), 22.64 (CH2, COCH2CH2CH2CH3),
20.12 (CH3, CH3), 14.06 (CH3, COCH2CH2CH2CH3).
General synthetic procedure
General procedure for the preparation of compounds 16a–
p and 16r–t
A mixture of the appropriate benzohydrazides 10–15
(1.0 mmol) and the corresponding acid anhydride
(53.0 mmol) was heated under reflux for 2 h. The reaction
mixture was then concentrated under reduced pressure. The
residue was dissolved in ethyl acetate and washed with
saturated aqueous NaHCO3. The organic phase was dried
(Na2SO4) and filtered. Evaporation of the solvent rendered
a residue that was purified by flash chromatography (ethyl
acetate/hexane 1:1), to give 16.
3-Acetyl-2-ethyl-5-(2-nitrophenyl)-2,3-dihydro-1,3,4-oxadi-
azole (16e) Yield 72 % (0.189 g) as a yellow oil. 1H
NMR (300 MHz, CDCl3) d = 7.87 (2H, m, H-30, H-60), 7.
69 (2H, m, H-40, H-50), 6.28 (1H, q, J = 3.0 Hz, H-2), 2.31
(3H, s, COCH3), 2.07 (2H, m, CH2CH3), 1.02 (3H, t, J =
6.0 Hz, CH2CH3). 13C NMR (75 MHz, CDCl3) d = 168.
32 (C, C=O), 153.13 (C, C-5), 147.84 (C, C-20), 132.50
(CH, C-40), 132.30 (CH, C-50), 130.75 (CH, C-60), 124.36
(CH, C-30), 119.16 (C, C-10), 95.10 (CH, C-2), 26.68 (CH2,
CH2CH3), 21.64 (CH3, CH3), 7.12 (CH3, COCH3).
3-Acetyl-2-methyl-5-(2-nitrophenyl)-2,3-dihydro-1,3,4-oxa-
1
diazole (16a) Yield 71 % (0.171 g) as a yellow oil. H
NMR (300 MHz, CDCl3) d = 7.88 (2H, m, H-30, H-60), 7.
70 (2H, m, H-40, H-50), 6.39 (1H, q, J = 5.4 Hz, H-2), 2.30
(3H, s, COCH3), 1.71 (3H, d, J = 5.0 Hz, CH3). 13C NMR
(75 MHz, CDCl3) d = 168.04 (C, C=O), 152.92 (C, C-5),
146.22 (C, C-20), 132.65 (CH, C-40), 132.34 (CH, C-50),
130.88 (CH, C-60), 124.46 (CH, C-30), 119.32 (C, C-10), 91.
32 (CH, C-2), 21.61 (CH3, COCH3), 20.13 (CH3, CH3).
2-Ethyl-5-(2-nitrophenyl)-3-propionyl-2,3-dihydro-1,3,4-oxa-
1
diazole (16f) Yield 80 % (0.222 g) as a yellow oil. H
NMR (300 MHz, CDCl3) d = 7.75 (2H, m, H-30, H-60), 7.
123

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Med Chem Res (2016) 25:1260–1273
58 (2H, m, H-40, H-50), 6.16 (1H, q, J = 3.0 Hz, H-2), 2.55
(2H, m, COCH2CH3), 1.96 (2H, m, CH2CH3), 1.10 (3H, t,
J = 9.0 Hz, COCH2CH3), 0.90 (3H, t, J = 9.0 H, CH2
CH3). 13C NMR (75 MHz, CDCl3) d = 171.88 (C, C=O),
152.86 (C, C-5), 146.43 (C, C-20), 132.41 (CH, C-40), 132.
21 (CH, C-50), 130.65 (CH, C-60), 124.28 (CH, C-30), 119.
16 (C, C-10), 95.07 (CH, C-2), 27.44 (CH2, COCH2CH3),
26.69 (CH2, CH2CH3), 8.88 (CH3, COCH2CH3), 7.12
(CH3, CH2CH3).
NMR (300 MHz, CDCl3) d: 8.11–7.31 (9H, m, H-30–H-60,
H-200–H-600), 6.99 (1H, d, J = 6.0 Hz, H-2), 2.57 (2H, q,
J = 6.0 Hz, COCH2CH3), 1.08 (3H, t, J = 6.0 Hz,
COCH2CH3). 13C NMR (75 MHz, CDCl3) d: 172.06 (C,
C=O), 165.22 (C, C-5), 152.40 (C, C-20), 136.10 (C, C-100),
132.51 (CH, C-40), 132.38 (CH, C-50), 130.73 (CH, C-60),
129.07 (2CH, C-300, C-500), 127.39 (CH, C-400), 127.01
(2CH, C-200, C-600), 124.36 (CH, C-30), 118.85 (C, C-10),
93.81 (CH, C-2), 27.50 (CH2, COCH2CH3), 8.70 (CH3,
COCH2CH3).
3-Butyryl-2-ethyl-5-(2-nitrophenyl)-2,3-dihydro-1,3,4-oxa-
1
diazole (16g) Yield 68 % (0.198 g) as a yellow oil. H
3-Butyryl-5-(2-nitrophenyl)-2-phenyl-2,3-dihydro-1,3,4-oxa-
1
NMR (300 MHz, CDCl3) d = 7.86 (2H, m, H-30, H-60), 7.
68 (2H, m, H-40, H-50), 6.27 (1H, q, J = 3.0 Hz, H-2), 2.60
(2H, m, COCH2CH2CH3), 1.72 (4H, m, CH2CH3,
COCH2CH2CH3), 1.00 (6H, m, CH2CH3, COCH2CH2
CH3). 13C NMR (75 MHz, CDCl3) d = 171.15 (C, C=O),
152.98 (C, C-5), 146.43 (C, C-20), 132.45 (CH, C-40), 132.
26 (CH, C-50), 130.71 (CH, C-60), 124.31 (CH, C-30), 119.
16 (C, C-10), 95.05 (CH, C-2), 35.97 (CH2, COCH2CH2
CH3), 26.63 (CH2, CH2CH3), 18.41 (CH2, COCH2CH2
CH3), 14.06 (CH3, COCH2CH2CH3), 7.03 (CH3, CH2CH3).
diazole (16k) Yield 59 % (0.200 g) as a yellow oil. H
NMR (300 MHz, CDCl3) d = 8.19–7.40 (9H, m, H-30–H-
60, H-200–H-600), 7.12 (1H, d, J = 3.0 Hz, H-2), 2.30 (2H, t,
J = 6.0 Hz, COCH2CH2CH3), 1.66 (2H, m, COCH2CH2
CH3), 0.95 (3H, t, J = 6.0 Hz, COCH2CH2CH3). 13C
NMR (75 MHz, CDCl3) d = 173.49 (C, C=O), 163.97 (C,
C-5), 156.97 (C, C-20), 136.13 (C, C-100), 132.50 (CH,
C-40), 132.35 (CH, C-50), 130.68 (CH, C-60), 129.77 (2CH,
C-300, C-500), 127.40 (CH, C-400), 126.99 (2CH, C-200, C-600),
124.37 (CH, C-30), 118.12 (C, C-10), 93.80 (CH, C-2), 27.
63 (CH2, COCH2CH2CH3), 19.21 (CH2, COCH2CH2CH3),
13.65 (CH3, COCH2CH2CH3).
2-Ethyl-5-(2-nitrophenyl)-3-pentanoyl-2,3-dihydro-1,3,4-ox-
1
adiazole (16h) Yield 76 % (0.232 g) as a yellow oil. H
NMR (300 MHz, CDCl3) d = 7.82 (2H, m, H-30, H-60), 7.
64 (2H, m, H-40, H-50), 6.24 (1H, q, J = 3.0 Hz, H-2), 2.62
(2H, m, COCH2CH2CH2CH3), 1.63 (2H, m, COCH2CH2
CH2CH3), 1.37 (4H, m, CH2CH3, COCH2CH2CH2CH3), 0.
91 (6H, m, CH2CH3, COCH2CH2CH2CH3). 13C NMR
(75 MHz, CDCl3) d = 169.87 (C, C=O), 153.14 (C, C-5),
146.53 (C, C-20), 132.43 (CH, C-40), 132.24 (CH, C-50),
130.70 (CH, C-60), 124.31 (CH, C-30), 119.17 (C, C-10), 95.
06 (CH, C-2), 35.24 (CH2, COCH2CH2CH2CH3), 33.92
(CH2, COCH2CH2CH2CH3), 27.00 (CH2, CH2CH3), 22.52
(CH2, COCH2CH2CH2CH3), 14.06 (CH3, COCH2CH2
CH2CH3), 7.04 (CH3, CH2CH3).
5-(2-Nitrophenyl)-3-pentanoyl-2-phenyl-2,3-dihydro-1,3,4-
oxadiazole (16l) Yield 63 % (0.227 g) as a yellow oil. 1H
NMR (300 MHz, CDCl3) d = 8.19 (1H, d, J = 3 Hz,
H-60), 7.85 (2H, m, H-30, H-40), 7.65 (3H, m, H-50, H-200,
H-600), 7.25 (3H, m, J = 3.1 Hz, H-300–H-500), 7.05 (1H, d,
J = 3.0 Hz, H-2), 2.29 (2H, t, J = 6.0 Hz, COCH2CH2
CH2CH3), 1.61 (2H, m, COCH2CH2CH2CH3), 1.51 (2H,
m, COCH2CH2CH2CH3), 0.90 (3H, t, J = 6.0 Hz,
COCH2CH2CH2CH3). 13C NMR (75 MHz, CDCl3) d =
173.62 (C, C=O), 163.79 (C, C-5), 153.92 (C, C-20), 135.16
(C, C-100), 133.52 (CH, C-40), 132.85 (CH, C-50), 131.76
(CH, C-60), 128.99 (2CH, C-300, C-500), 127.32 (CH, C-400),
126.91 (2CH, C-200, C-600), 125.21 (CH, C-30), 118.27 (C,
C-10), 95.81 (CH, C-2), 40.17 (CH2, COCH2CH2CH2CH3),
26.63 (CH2, COCH2CH2CH2CH3), 25.65 (CH2, COCH2
CH2CH2CH3), 13.51 (CH3, COCH2CH2CH2CH3).
3-Acetyl-5-(2-nitrophenyl)-2-phenyl-2,3-dihydro-1,3,4-oxa-
1
diazole (16i) Yield 63 % (0.196 g) as a yellow oil. H
NMR (300 MHz, CDCl3) d = 7.31–7.80 (9H, m, H-30–H-
60, H-200–H-600), 6.99 (1H, d, 1H, J = 3.0 Hz, H-2), 1.90
(3H, s, COCH3). 13C NMR (75 MHz. CDCl3) d = 168.49
(C, C=O), 157.93 (C, C-5), 149.12 (C, C-20), 135.96 (C,
C-100), 132.59 (CH, C-40), 132.46 (CH, C-50), 130.79 (CH,
C-60), 129.17 (2CH, C-300, C-500), 127.01 (2CH, C-200,
C-600), 126.63 (CH, C-400), 124.41 (CH, C-30), 118.84 (C,
C-10), 93.77 (CH, C-2), 21.71 (CH3, COCH3).
3-Acetyl-2,2-dimethyl-5-(2-nitrophenyl)-2,3-dihydro-1,3,4-
oxadiazole (16m) Yield 64 % (0.168 g) as a yellow oil.
1H NMR (300 MHz, CDCl3) d = 7.86 (2H, m, H-30, H-60),
7.68 (2H, m, H-40, H-50), 2.28 (3H, s, COCH3), 1.85 (6H, s,
2CH3). 13C NMR (75 MHz, CDCl3) d = 167.53 (C, C=O),
151.16 (C, C-5), 149.13 (C, C-20), 132.57 (CH, C-40), 132.
13 (CH, C-50), 130.83 (CH, C-60), 124.38 (CH, C-30), 119.
62 (C, C-10), 102.11 (C, C-2), 24.78 (2CH3, 2CH3), 22.55
(CH3, COCH3).
5-(2-Nitrophenyl)-2-phenyl-3-propionyl-2,3-dihydro-1,3,4-
oxadiazole (16j) Yield 72 % (0.234 g) as a yellow oil. 1H
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Med Chem Res (2016) 25:1260–1273
1267
2,2-Dimethyl-5-(2-nitrophenyl)-3-propionyl-2,3-dihydro-
2-Ethyl-2-methyl-5-(2-nitrophenyl)-3-propionyl-2,3-dihy-
dro-1,3,4-oxadiazole (16s) Yield 79 % (0.230 g) as a
yellow oil. 1H NMR (300 MHz, CDCl3) d = 7.98 (1H, dd,
J = 1.9, 7.1 Hz, H-60), 7.86 (2H, m, H-30, H-40), 7.63 (1H,
m, H-50), 2.47 (2H, q, J = 8.0 Hz, COCH2CH3), 1.77 (2H,
m, CH2CH3), 1.63 (3H, s, CH3), 0.97 (3H, t, J = 7.6 Hz,
COCH2CH3), 0.79 (3H, t, J = 7.8, CH2CH3). 13C NMR
(75 MHz, CDCl3) d = 170.88 (C, C=O), 163.32 (C, C-5),
147.23 (C, C-20), 131.95 (CH, C-40), 126.13 (CH, C-50),
125.64 (CH, C-60), 123.60 (CH, C-30), 116.67 (C, C-10),
103.25 (C, C-2), 29.81 (CH2, COCH2CH3), 26.33 (CH2,
CH2CH3), 20.76 (CH3, CH3), 9.57 (CH3, CH2CH3), 8.75
(CH3, COCH2CH3).
1,3,4-oxadiazole (16n) Yield 84 % (0.233 g) as a yellow
1
oil. H NMR (300 MHz, CDCl3) d = 7.86 (2H, m, H-30,
H-60), 7.75 (2H, m, H-40, H-50), 2.62 (2H, q, J = 8.0 Hz,
COCH2CH3), 1.87 (6H, s, 2CH3), 1.16 (3H, t, J = 8.0 Hz,
COCH2CH3). 13C NMR (75 MHz, CDCl3) d = 171.20 (C,
C=O), 151.08 (C, C-5), 148.98 (C, C-20), 132.48 (CH,
C-40), 132.07 (CH, C-50), 130.75 (CH, C-60), 124.33 (CH,
C-30), 119.62 (C, C-10), 102.09 (C, C-2), 28.02 (CH2,
COCH2CH3), 24.78 (2CH3, 2CH3), 9.05 (CH3,
COCH2CH3).
3-Butyryl-2,2-dimethyl-5-(2-nitrophenyl)-2,3-dihydro-1,3,
4-oxadiazole (16o) Yield 81 % (0.236 g) as a yellow oil.
1H NMR (300 MHz, CDCl3) d = 7.82 (2H, m, H-30, H-60),
7.65 (2H, m, H-40, H-50), 2.24 (2H, t, J = 6.0 Hz,
COCH2CH2CH3), 1.85 (6H, s, 2CH3), 1.69 (2H, m,
COCH2CH2CH3), 0.97 (3H, t, J = 6.0 Hz, COCH2CH2
CH3). 13C NMR (75 MHz, CDCl3) d = 171.26 (C, C=O),
150.93 (C, C-5), 149.12 (C, C-20), 132.51 (CH, C-40), 132.
07 (CH, C-50), 130.76 (CH, C-60), 124.32 (CH, C-30), 119.
62 (C, C-10), 102.04 (C, C-2), 36.54 (CH2, COCH2CH2
CH3), 24.79 (2CH3, 2CH3), 18.36 (CH2, COCH2CH2CH3),
13.98 (CH3, COCH2CH2CH3).
3-Acetyl-2-methyl-5-(2-nitrophenyl)-2-phenyl-2,3-dihydro-
1,3,4-oxadiazole (16t) Yield 82 % (0.267 g) as a yellow
oil. 1H NMR (300 MHz, CDCl3) d = 7.99 (1H, dd, J = 2.
0, 7.3 Hz, H-60), 7.87 (2H, m, H-30, H-40), 7.78 (2H, m,
H-200, H-600), 7.64 (1H, m, H-50), 7.22 (3H, m, H-300–H-500),
2.18 (3H, s, COCH3), 2.09 (3H, s, CH3). 13C NMR
(75 MHz, CDCl3) d = 167.48 (C, C=O), 162.26 (C, C-5),
156.08 (C, C-20), 134.57 (C, C-100), 134.22 (CH, C-50), 130.
32 (CH, C-40), 129.51 (CH, C-60), 128.82 (2CH, C-300,
C-500), 127.42 (CH, C-400), 125.97 (2CH, C-200, C-600), 124.
39 (CH, C-30), 119.23 (C, C-10), 100.97 (C, C-2), 22.89
(CH3, COCH3), 22.35 (CH3, CH3,).
2,2-Dimethyl-5-(2-nitrophenyl)-3-pentanoyl-2,3-dihydro-1,
3,4-oxadiazole (16p) Yield 78 % (0.238 g) as a yellow
oil. 1H NMR (300 MHz, CDCl3) d = 7.98 (1H, dd, J = 2.
0, 7.2, H-60), 7.75 (2H, m, H-30, H-40), 7.62 (1H, m, H-50),
2.27 (2H, t, J = 7.5 Hz, COCH2CH2CH2CH3), 1.67 (2H,
m, COCH2CH2CH2CH3), 1.63 (3H, s, CH3), 1.59 (3H, s,
CH3), 1.53 (2H, m, COCH2CH2CH2CH3), 0.93 (3H, t, J =
7.5 Hz, COCH2CH2CH2CH3). 13C NMR (75 MHz,
CDCl3) d = 172.16 (C, C=O), 155.13 (C, C-5), 148.02 (C,
C-20), 131.91 (CH, C-40), 130.03 (CH, C-50), 127.83 (CH,
C-60), 116.82 (CH, C-30), 101.62 (C, C-2), 40.53 (CH2,
COCH2CH2CH2CH3), 26.56 (CH2, COCH2CH2CH2CH3),
23.49 (2CH3, 2CH3), 21.67 (CH2, COCH2CH2CH2CH3),
13.48 (CH3, COCH2CH2CH2CH3).
General procedure for the preparation of compounds 6a–i
A mixture of nitroarene 16a–i (0.414 mmol), palla-
dium/carbon (10 %, 10 mg), and methanol (20 mL) was
stirred at room temperature under a hydrogen atmosphere
(1 atm). After 3 h, the suspension was filtered through
Celite and evaporated. The residue was dissolved in CH2
Cl2, and this solution was washed with water, dried (Na2
SO4), filtered, and concentrated. The residue was purified
by flash chromatography (ethyl acetate/hexane 1:1), to
afford the corresponding aromatic amines 6a–i.
[2-(4-Acetyl-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)ph-
3-Acetyl-2-ethyl-2-methyl-5-(2-nitrophenyl)-2,3-dihydro-1,
enyl]amine (6a) Mp: 130–132 °C. Yield 94 % (0.085 g)
as a white solid. H NMR (400 MHz, CDCl3) d = 7.62
1
3,4-oxadiazole (16r) Yield 75 % (0.208 g) as a yellow
1
oil. H NMR (300 MHz, CDCl3) d = 8.02 (1H, m, H-60),
(1H, dd, J = 1.6, 8.0 Hz, H-3), 7.25 (1H, t, J = 8.0 Hz,
H-5), 6.73 (2H, m, H-4, H-6), 6.25 (1H, q, J = 5.4 Hz,
H-50), 5.50 (2H, bs, NH2), 2.30 (3H, s, COCH3), 1.65 (3H,
d, J = 4.0 Hz, CH3). 13C NMR (100 MHz, CD3Cl) d =
167.19 (C, C=O), 156.23 (C, C-20), 147.02 (C, C-1), 132.62
(CH, C-5), 128.68 (CH, C-3), 117.14 (CH, C-4), 116.06
(CH, C-6), 106.62 (C, C-2), 88.20 (CH, C-50), 21.78 (CH3,
COCH3), 20.31 (CH3, CH3). HRESIMS m/z (pos): 242.
0903 C11H13N3O2Na (calcd. 242.0905).
7.83 (2H, m, H-30, H-40), 7.72 (1H, m, H-50), 2.17 (3H, s,
COCH3), 1. 94 (2H, m, CH2CH3), 1.65 (3H, s, CH3), 0.87
(3H, t, J = 7.5 Hz, CH2CH3). 13C NMR (75 MHz, CDCl3)
d = 167.56 (C, C=O), 163.28 (C, C-5), 147.05 (C, C-20),
131.93 (CH, C-40), 126.23 (CH, C-50), 125.53 (CH, C-60),
123.62 (CH, C-30), 114.12 (C, C-10), 101.83 (C, C-2), 26.13
(CH2, CH2CH3), 23.56 (CH3, COCH3), 19.17 (CH3, CH3,),
9.45 (CH3, CH2CH3).
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Med Chem Res (2016) 25:1260–1273
(2H, m, H-4, H-6), 6.21 (1H, q, J = 2.8 Hz, H-50), 5.17
(2H, bs, NH2), 2.33 (3H, s, COCH3), 2.03 (2H, m, CH2
CH3), 0.99 (3H, t, J = 7.5, CH2CH3). 13C NMR
(100 MHz, CD3Cl) d = 167.24 (C, C=O), 156.25 (C,
C-20), 146.62 (C, C-1), 132.37 (CH, C-5), 128.44 (CH,
C-3), 117.39 (CH, C-4), 116.18 (CH, C-6), 107.51 (C,
C-2), 91.74 (CH, C-50), 26.43 (CH2, CH2CH3), 21.56 (CH3,
COCH3), 6.70 (CH3, CH2CH3). HRESIMS m/z (pos): 234.
1242 C12H16N3O2 (calcd. 234.1243).
[2-(5-Methyl-4-propionyl-4,5-dihydro-1,3,4-oxadiazol-2-
yl)phenyl]amine (6b) Mp 124–127 °C. Yield 90 % (0.
087 g) as a light yellow solid. 1H NMR (400 MHz, CDCl3)
d = 7.61 (1H, dd, J = 1.5, 8.0 Hz, H-3), 7.23 (1H, t, J =
8.0 Hz, H-5), 6.74 (2H, m, H-4, H-6), 6.25 (1H, q, J = 5.
3 Hz, H-50), 2.63 (2H, q, J = 7.6 Hz, COCH2CH3), 1.64
(3H, d, J = 5.3 Hz, CH3), 1.19 (3H, t, J = 7.6 Hz,
COCH2CH3). 13C NMR (100 MHz, CD3Cl) d = 170.45
(C, C=O), 155.76 (C, C-20), 146.25 (C, C-1), 132.31 (CH,
C-5), 128.42 (CH, C-3), 117.20 (CH, C-4), 116.02 (CH,
C-6), 106.84 (C, C-2), 88.15 (CH, C-50), 27.18 (CH2,
COCH2CH3), 19.34 (CH3, CH3), 8.52 (CH3, COCH2CH3).
HRESIMS m/z (pos): 234.1234 C12H16N3O2 (calcd. 234.
1243).
[2-(5-Ethyl-4-propionyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)ph-
enyl]amine (6f) Mp 122–125 °C. Yield 95 % (0.097 g) as
1
a white solid. H NMR (400 MHz, CDCl3) d = 7.65 (1H,
dd, J = 1.6, 8.0 Hz, H-3), 7.27 (1H, m, H-5), 6.77 (2H, m,
H-4, H-6), 6.20 (1H, q, J = 2.8 Hz, H-50), 5.15 (2H, bs,
NH2), 2.67 (2H, m, COCH2CH3), 2.02 (2H, m, CH2CH3),
1.22 (3H, t, J = 7.6, COCH2CH3), 0.99 (3H, t, J = 7.5,
CH2CH3). 13C NMR (100 MHz, CD3Cl) d = 170.73 (C,
C=O), 156.17 (C, C-20), 146.34 (C, C-1), 132.28 (CH, C-5),
128.40 (CH, C-3), 117.15 (CH, C-4), 115.98 (CH, C-6),
106.60 (C, C-2), 91.72 (CH, C-50), 27.24 (CH2, COCH2
CH3), 26.44 (CH2, CH2CH3), 8.59 (CH3, COCH2CH3), 6.
65 (CH3, CH2CH3). HRESIMS m/z (pos): 248.1392
C13H18N3O2 (calcd. 248.1399).
[2-(4-Butyryl-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)ph-
enyl]amine (6c) Mp 132–135 °C. Yield 90 % (0.092 g)
as a light yellow solid. 1H NMR (400 MHz, CDCl3) d = 7.
60 (1H, dd, J = 1.5, 8.0 Hz, H-3), 7.24 (1H, m, H-5), 7.15
(1H, m, H-4), 6.73 (1H, m, H-6), 6.26 (1H, q, J = 5.9 Hz,
H-50), 5.26 (2H, bs, NH2), 2.59 (2H, m, COCH2CH2CH3),
1.73 (2H, m, COCH2CH2CH3), 1.63 (3H, d, J = 4.0 Hz,
CH3), 0.99 (3H, t, J = 8.0 Hz, COCH2CH2CH3). 13C
NMR (100 MHz, CD3Cl) d = 169.64 (C, C=O), 155.78
(C, C-20), 146.53 (C, C-1), 132.30 (CH, C-5), 128.41 (CH,
C-3), 117.00 (CH, C-4), 115.87 (CH, C-6), 106.59 (C,
C-2), 88.08 (CH, C-50), 35.81 (CH2, COCH2CH2CH3, 20.
09 (CH3, CH3), 17.97 (CH2,COCH2CH2CH3, 13.92 (CH3,
COCH2CH2CH3). HRESIMS m/z (pos): 248.1395
C13H18N3O2 (calcd. 248.1399).
[2-(4-Butyryl-5-ethyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)ph-
enyl]amine (6g) Mp 129–131 °C. Yield 92 % (0.099 g)
1
as a white solid. H NMR (400 MHz, CDCl3) d = 7.81
(1H, dd, J = 1.0, 6.5 Hz, H-3), 7.67 (1H, m, H-5), 7.29
(1H, m, H-4), 6.80 (1H, m, H-6), 6.26 (1H, q, J = 2.8 Hz,
H-50), 2.66 (2H, m, COCH2CH2CH3), 2.06 (2H, m, CH2
CH3), 1.82 (2H, m, COCH2CH2CH3); 1.06 (3H, t, J = 7.
4 Hz, COCH2CH2CH3), 1.02 (3H, t, J = 7.5, CH2CH3).
13C NMR (100 MHz, CD3Cl) d = 169.73 (C, C=O), 155.
61 (C, C-20), 138.27 (C, C-1), 132.82 (CH, C-5), 128.14
(CH, C-3), 122.83 (CH, C-4), 120.06 (CH, C-6), 110.58 (C,
C-2), 92.39 (CH, C-50), 36.13 (CH2, COCH2CH2CH3), 19.
18 (CH2, COCH2CH2CH3), 18.08 (CH2, CH2CH3), 13.82
(CH3, COCH2CH2CH3), 6.58 (CH3, CH2CH3). HRESIMS
m/z (pos): 262.1548 C14H20N3O2 (calcd. 262.1556).
[2-(5-Methyl-4-pentanoyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)
phenyl]amine (6d) Mp 103–105 °C. Yield 94 % (0.
1
102 g) as a white solid. H NMR (400 MHz, CDCl3) d =
7.63 (1H, dd, J = 1.1, 8.6 Hz, H-3), 7.28 (1H, dd, J = 1.6,
8.0 Hz, H-5), 7.16 (1H, m, H-4); 6.78 (1H, m, H-6), 6.33
(1H, q, J = 5.3 Hz, H-50), 2.57 (2H, m, COCH2CH2CH2
CH3), 1.73 (2H, m, COCH2CH2CH2CH3), 1.70 (3H, d,
J = 5.3 Hz, CH3), 1.44 (2H, m, COCH2CH2CH2CH3), 0.
98 (3H, t, J = 7.3 Hz, COCH2CH2CH2CH3). 13C NMR
(100 MHz, CD3Cl) d = 169.61 (C, C=O), 155.19 (C,
C-20), 138.27 (C, C-1), 132.81 (CH, C-5), 128.17 (CH,
C-3), 122.79 (CH, C-4), 120.03 (CH, C-6), 110.65 (C,
C-2), 88.83 (CH, C-50), 38.53 (CH2, COCH2CH2CH2CH3),
27.72 (CH2, COCH2CH2CH2CH3), 22.52 (CH2, COCH2
CH2CH2CH3), 20.14 (CH3, CH3), 13.85 (CH3, COCH2
[2-(5-Ethyl-4-pentanoyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)ph-
enyl]amine (6h) Mp 126–129 °C. Yield 90 % (0.103 g)
1
as a white solid. H NMR (400 MHz, CDCl3) d = 7.74
(1H, dd, J = 1.1, 8.5 Hz, H-3), 7.60 (1H, dd, J = 1.6, 7.
0 Hz, H-5), 7.51 (1H, m, H-4), 7.14 (1H, m, H-6), 6.25
(1H, q, J = 2.8 Hz, H-50), 2.68 (2H, m, COCH2CH2CH2
CH3), 2.06 (2H, m, CH2CH3), 1.76 (2H, m, COCH2CH2
CH2CH3), 1.44 (2H, m, COCH2CH2CH2CH3), 0.98 (6H,
m, COCH2CH2CH2CH3, CH2CH3). 13C NMR (100 MHz,
CD3Cl) d = 169.86 (C, C=O), 155.60 (C, C-20), 138.28 (C,
C-1), 132.82 (CH, C-5), 128.14 (CH, C-3), 122.81 (CH,
C-4), 120.05 (CH, C-6), 110.58 (C, C-2), 92.39 (CH, C-50),
CH2CH2CH3).
HRESIMS
m/z
(pos):
262.1548
C14H20N3O2 (calcd. 262.1556).
[2-(4-Acetyl-5-ethyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)phe-
nyl]amine (6e) Mp 96–97 °C. Yield 92 % (0.089 g) as a
1
light yellow solid. H NMR (400 MHz, CDCl3) d = 7.66
(1H, dd, J = 1.6, 8.0 Hz, H-3), 7.27 (1H, m, H-5), 6.80
123

Med Chem Res (2016) 25:1260–1273
1269
38.53 (CH2, COCH2CH2CH2CH3), 33.84 (CH2, COCH2
CH2CH2CH3), 26.61 (CH2, COCH2CH2CH2CH3), 22.54
(CH2, CH2CH3), 13.85 (CH3, COCH2CH2CH2CH3), 6.59
(CH3, CH2CH3). HRESIMS m/z (pos): 276.1673
C15H22N3O2 (calcd. 276.1679).
76 (2H, m, H-4, H-6), 2.41 (2H, q, J = 6.0 Hz, COCH2-
CH3), 1.55 (3H, s, CH3), 1.52 (3H, s, CH3), 1.24 (t, 3H,
J = 6.0 Hz, COCH2CH3). 13C NMR (75 MHz, CD3CO)
d = 170.05 (C, C=O), 154.29 (C, C-20), 146.66 (C, C-1),
132.35 (CH, C-5), 128.59 (CH, C-3), 117.24 (CH, C-4),
116.14 (CH, C-6), 107.19 (C, C-2), 98.70 (C, C-50), 28.14
(CH2, COCH2CH3), 24.90 (CH3, CH3), 23.48 (CH3, CH3),
8.78 (CH3, COCH2CH3). HRESIMS m/z (pos): 248.1395
C13H18N3O2 (calcd. 248.1399).
[2-(5-Phenyl-4-acetyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)ph-
enyl]amine (6i) Mp 117–120 °C. Yield 92 % (0.107 g) as
1
a white solid. H NMR (400 MHz, CDCl3) d = 7.42–7.27
(7H, m, H-3, H-5, H-200–H-600), 6.69 (1H, d, J = 8.3 Hz,
H-4), 6.62 (1H, m, H-6), 6.00 (1H, s, H-50), 2.36 (3H, s,
COCH3). 13C NMR (100 MHz, CD3Cl) d = 168.74 (C, C=
O), 148.68 (C, C-20), 145.52 (C, C-1), 137.58 (C, C-100),
132.73 (CH, C-5), 129.07 (2CH, C-300, C-500), 128.57 (2CH,
C-200, C-600), 127.64 (CH, C-400), 126.89 (CH, C-3), 117.24
(CH, C-4), 116.74 (CH, C-6), 114.98 (C, C-2), 93.20 (CH,
C-50), 21.36 (CH3, COCH3). HRESIMS m/z (pos): 282.
1236 C16H16N3O2 (calcd. 282.1243).
[2-(4-Butyryl-5,5-dimethyl-4,5-dihydro-1,3,4-oxadiazol-2-
yl)phenyl]amine (6o) Mp 95–96 °C. Yield 81 % (0.
111 g) as a yellow solid. H NMR (300 MHz, (CD3)2CO)
1
d = 7.55 (1H, d, J = 9.0 Hz, H-3), 7.24 (1H, m, H-5), 6.83
(1H, m, H-6), 6.67 (1H, m, H-4), 2.64 (2H, t, J = 6.0 Hz,
COCH2CH2CH3), 1.87 (3H, s, CH3), 1.84 (3H, s, CH3), 1.
74 (2H, m, COCH2CH2CH3), 1.03 (3H, t, J = 9.0 Hz,
COCH2CH2CH3). 13C NMR (75 MHz, CD3CD) d = 169.
89 (C, C=O), 155.00 (C, C-20), 148.07 (C, C-1), 132.24
(CH, C-5), 127.95 (CH, C-3), 115.90 (CH, C-4), 115.54
(CH, C-6), 105.69 (C, C-2), 98.34 (C, C-50), 36.31 (CH2,
COCH2CH2CH3), 23.58 (CH3, CH3), 21.87 (CH3, CH3),
18.11 (CH2, COCH2CH2CH3), 12.97 (CH3, COCH2CH2
CH3). HRESIMS m/z (pos): 284.13735 C14H19N3O2Na
(calcd. 284.1375).
General procedure for the preparation of compounds 6m–t
Fe (5.24 mmol) and FeSO4 (0.524 mmol) were suspended
in water (11.0 mL), and the corresponding nitroarene 16m–
p and 16r–t (0.524 mmol) was added over the reaction
mixture and refluxed for 3 h. After cooling, the reaction
mixture was filtered through Celite and washed thoroughly
with CH2Cl2. The aqueous phase was extracted with
CH2Cl2 (3 9 15 mL) and ethyl acetate (3 9 15 mL). The
combined organic layers were washed with brine, dried
(Na2SO4), filtered, and concentrated to yield a residue that
was purified by flash chromatography (ethyl acetate/hexane
1:1), to give the corresponding aromatic amines 6m–p and
16r–t. Compound 6q was obtained along with 6p, when
16p was treated with Fe/FeSO4, due to the presence of a
valeric anhydride remainder.
[2-(5,5-Dimethyl-4-pentanoyl-4,5-dihydro-1,3,4-oxadia-
zol-2-yl)phenyl]amine (6p) Mp 132–135 °C. Yield
57 % (0.082 g) as a light yellow solid. 1H NMR
(400 MHz, CDCl3) d = 7.58 (1H, dd, J = 1.5, 7.8 Hz,
H-3), 7.23 (1H, m, H-5), 6.72 (2H, m, H-4, H-6), 2.59 (2H,
t, J = 7.5 Hz, COCH2CH2CH2CH3), 1.83 (6H, s, 2CH3), 1.
66 (2H, m, COCH2CH2CH2CH3), 1.39 (2H, m, COCH2
CH2CH2CH3), 0.93 (3H, t, J = 7.4 Hz, COCH2CH2CH2
CH3). 13C NMR (100 MHz, CD3Cl) d = 169.23 (C, C=O),
154.00 (C, C-20), 146.55 (C, C-1), 132.08 (CH, C-5), 128.
32 (CH, C-3), 116.86 (CH, C-4), 115.77 (CH, C-6), 106.83
(C, C-2), 98.44 (C, C-50), 34.35 (CH2, COCH2CH2CH2
CH3), 26.69 (CH2, COCH2CH2CH2CH3), 24.67 (CH3,
CH3), 24.65 (CH3, CH3), 22.49 (CH2, COCH2CH2CH2
CH3), 13.91 (CH3, COCH2CH2CH2CH3). HRESIMS m/z
(pos): 276.1711 C15H21N3O2 (calcd. 276.1712).
[2-(4-Acetyl-5,5-dimethyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)
phenyl]amine (6m) Mp 126–128 °C. Yield 51 % (0.
062 g) as a yellow solid. H NMR (300 MHz, (CD3)2CO)
1
d = 7.76 (1H, d, J = 7.7 Hz, H-3); 7.31 (1H, m, H-5), 6.
76 (2H, m, H-4, H-6), 6.11 (2H, bs, NH2), 2.37 (3H, s,
COCH3), 1.60 (3H, s, CH3), 1.57 (3H, s, CH3). 13C NMR
75 MHz, (CD3)2CO) d = 168.53 (C, C=O), 161.55 (C,
C-20), 157.55 (C, C-1), 133.71 (CH, C-5), 128.27 (CH,
C-3), 117.70 (CH, C-4), 114.83 (CH, C-6), 111.88 (C,
C-2), 98.85 (C, C-50), 23.48 (CH3, CH3), 22.53 (CH3,
CH3), 20.15 (CH3, COCH3). HRESIMS m/z (pos): 256.
1057 C12H15N3O2Na (calcd. 256.1062).
N-[2-(5,5-Dimethyl-4-pentanoyl-4,5-dihydro-1,3,4-oxadia-
zol-2-yl)phenyl]pentanamide (6q) Mp 96–98 °C. Yield
27 % (0.037 g) as a light yellow solid. 1H NMR
(400 MHz, CDCl3) d = 10.27 (1H, s, NH), 8.71 (1H, dd,
J = 1.1, 8.6 Hz, H-3), 7.73 (1H, dd, J = 1.6, 7.9 Hz, H-6),
7.46 (1H, m, H-5), 7.10 (1H, m, H-4), 2.63 (2H, t, J = 7.
5 Hz, NHCOCH2CH2CH2CH3), 2.42 (2H, t, J = 7.5 Hz,
COCH2CH2CH2CH3), 1.86 (6H, s, 2CH3), 1.72 (4H, m,
NHCOCH2CH2CH2CH3, COCH2CH2CH2CH3), 1.39 (4H,
m, NHCOCH2CH2CH2CH3, COCH2CH2CH2CH3), 0.95
[2-(5,5-Dimethyl-4-propionyl-4,5-dihydro-1,3,4-oxadiazol-
2-yl)phenyl]amine (6n) Mp 133–134 °C. Yield 61 % (0.
079 g) as a yellow solid. H NMR (300 MHz, (CD3)2CO)
1
d = 7.73 (1H, d, J = 6.0 Hz, H-3); 7.33 (1H, m, H-5), 6.
123

1270
Med Chem Res (2016) 25:1260–1273
(3H, t, J = 7.6 Hz, NHCOCH2CH2CH2CH3), 0.93 (3H, t,
J = 7.3 Hz, COCH2CH2CH2CH3). 13C NMR (100 MHz,
CD3Cl) d = 171.78 (C, NHC=O), 168.87 (C, C=O), 153.
41 (C, C-20), 138.19 (C, C-1), 132.58 (CH, C-5), 128.05
(CH, C-3), 122.68 (CH, C-6), 119.93 (CH, C-4), 110.86 (C,
C-2), 99.39 (C, C-50), 38.49 (CH2, COCH2CH2CH2CH3),
34.42 (CH2, NHCOCH2CH2CH2CH3), 27.70 (CH2,
COCH2CH2CH2CH3), 26.51 (CH2, NHCOCH2CH2CH2
CH3), 24.26 (CH3, CH3), 24.62 (CH3, CH3), 22.46 (CH2,
COCH2CH2CH2CH3), 22.41 (CH2, NHCOCH2CH2CH2
CH3), 13.88 (CH3, COCH2CH2CH2CH3), 13.77 (CH3,
NHCOCH2CH2CH2CH3). HRESIMS m/z (pos): 360.2292
C20H30N3O3 (calcd. 360.2287).
C-4), 115.80 (CH, C-6), 106.42 (C, C-2), 98.75 (C, C-50),
23.13 (CH3, CH3), 22.74 (CH3, COCH3). HRESIMS m/z
(pos): 296.1394 C17H18N3O2 (calcd. 296.1399).
General procedure for the preparation of compounds 7a–l
From the synthetic intermediates 16a–l, and following the
same procedure as for the preparation of compounds 6m–t,
the derivatives 7a–l were obtained. The final compounds
were purified by flash chromatography (ethyl acetate/hex-
ane 1:1).
N-(2-methyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)aceta-
mide (7a) Mp 130–132 °C. Yield 63 % (0.072 g) as a
white solid. 1H NMR (300 MHz, (CD3)2CO) d = 9.10
(1H, bs, NHCO), 7.79 (1H, d, J = 7.0 Hz, H-5), 7.33 (1H,
m, H-7), 6.80 (2H, m, H-6, H-8), 6.13 (1H, bs, H-1), 5.30
(1H, q, J = 6.0 Hz, H-2), 2.03 (3H, s, COCH3), 1.48 (3H,
d, J = 6.0 Hz, CH3). 13C NMR (75 MHz, (CD3)2CO) d =
169.06 (C, NHC=O), 163.08 (C, C-4), 148.15 (C, C-8a),
133.71 (CH, C-7), 128.55 (CH, C-5), 118.21 (CH, C-6),
115.16 (C, C-4a), 114.85 (CH, C-8), 68.34 (CH, C-2), 20.
00 (CH3, COCH3), 19.20 (CH3, CH3). HRESIMS m/z
(pos): 242.0911 C11H13N3O2Na (calcd. 242.0905).
[2-(4-Acetyl-5-ethyl-5-methyl-4,5-dihydro-1,3,4-oxadiazol-
2-yl)phenyl]amine (6r) Mp 132–135 °C. Yield 83 % (0.
107 g) as a yellow solid. 1H NMR (400 MHz, CDCl3) d =
7.65 (1H, dd, J = 1.5, 7.8 Hz, H-3), 7.26 (1H, m, H-5), 6.
80 (2H, m, H-4, H-6), 2.51 (3H, s, COCH3), 2.07 (2H, m,
CH2CH3), 2.06 (3H, s, CH3), 0.86 (3H, t, J = 7.4 Hz,
CH2CH3). 13C NMR (100 MHz, CD3Cl) d = 170.58 (C,
C=O), 155.72 (C, C-20), 148.66 (C, C-1), 133.37 (CH, C-5),
128.70 (CH, C-3), 120.56 (CH, C-4), 117.48 (CH, C-6),
114.95 (C, C-2), 109.94 (C, C-50), 23.60 (CH2, CH2CH3),
21.27 (CH3, COCH3), 20.98 (CH3, CH3), 8.29 (CH3,
CH2CH3). HRESIMS m/z (pos): 248.1395 C13H18N3O2
(calcd. 248.1399).
N-(2-methyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)propa-
namide (7b) Mp 148–150 °C. Yield 67 % (0.082 g) as a
white solid. 1H NMR (300 MHz, CDCl3) d = 8.33 (1H, bs,
NHCO), 7.88 (1H, d, J = 9.0 Hz, H-5), 7.29 (1H, m, H-7),
6.88 (1H, m, H-6), 6.72 (1H, d, J = 6.0 Hz, H-8), 5.31
(1H, q, J = 6.0 Hz, H-2), 3.48 (1H, bs, H-1), 2.39 (2H, q,
J = 6.0 Hz, COCH2CH3), 1.49 (3H, d, J = 6.0 Hz, CH3),
1.23 (3H, t, J = 6.0 Hz, COCH2CH3). 13C NMR (75 MHz,
CDCl3) d = 174.43 (C, NHC=O), 164.09 (C, C-4), 146.52
(C, C-8a), 134.43 (CH, C-7), 129.18 (CH, C-5), 119.89
(CH, C-6), 115.57 (CH, C-8), 115.43 (C, C-4a), 68.43 (CH,
C-2), 27.52 (CH2, COCH2CH3), 19.55 (CH3, CH3), 9.64
(CH3, COCH2CH3). HRESIMS m/z (pos): 256.1051 C12
H15N3O2Na (calcd. 256.1062).
[2-(5-Ethyl-5-methyl-4-propionyl-4,5-dihydro-1,3,4-oxadi-
azol-2-yl)phenyl]amine (6s) Mp 125–129 °C. Yield 78 %
(0.107 g) as a yellow solid. H NMR (400 MHz, CDCl3)
1
d = 7.59 (1H, d, J = 8.0 Hz, H-3), 7.22 (1H, m, H-5), 6.71
(2H, m, H-4, H-6), 2.64 (2H, q, J = 7.5 Hz, COCH2CH3),
2.24 (2H, m, CH2CH3), 1.80 (3H, s, CH3), 1.18 (3H, t, J =
7.5 Hz, COCH2CH3), 0.87 (3H, t, J = 7.5 Hz, CH2CH3).
13C NMR (100 MHz, CD3Cl) d = 169.78 (C, C=O), 154.
52 (C, C-20), 146.61 (C, C-1), 132.06 (CH, C-5), 128.28
(CH, C-3), 116.82 (CH, C-4), 115.76 (CH, C-6), 106.67 (C,
C-2), 100.96 (C, C-50), 29.89 (CH2, COCH2CH3), 27.89
(CH2, CH2CH3), 23.64 (CH3, CH3), 8.68 (CH3, COCH2
CH3), 7.16 (CH3, CH2CH3). HRESIMS m/z (pos): 262.
1557 C14H20N3O2 (calcd. 262.1556).
N-(2-methyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)butana-
mide (7c) Mp 153–155 °C. Yield 66 % (0.085 g) as a
white solid. 1H NMR (300 MHz, (CD3)2CO) d = 9.07
(1H, bs, NHCO); 7.78 (1H, d, J = 9.0 Hz, H-5), 7.32 (1H,
m, H-7), 6.80 (2H, m, H-6, H-8), 6.14 (1H, bs, H-1), 5.30
(1H, q, J = 6.0 Hz, H-2), 2.30 (2H, t, J = 6.0 Hz,
COCH2CH2CH3), 1.71 (2H, m, COCH2CH2CH3), 1.49
(3H, d, J = 6.0 Hz, CH3), 1.01 (3H, t, J = 6.0 Hz,
COCH2CH2CH3). 13C NMR (75 MHz, (CD3)2CO) d =
172.10 (C, NHC=O), 163.13 (C, C-4), 148.18 (C, C-8a),
133.68 (CH, C-7), 128.57 (CH, C-5), 118.22 (CH, C-6),
115.26 (C, C-4a), 114.84 (CH, C-8), 68.35 (CH, C-2), 35.
[2-(4-Acetyl-5-methyl-5-phenyl-4,5-dihydro-1,3,4-oxadia-
zol-2-yl)phenyl]amine (6t) Mp 137–139 °C. Yield 80 %
(0.124 g) as a light yellow solid. 1H NMR (400 MHz,
CDCl3) d = 7.65–7.24 (7H, H-3, H-5, H-200–H-600), 6.74
(2H, m, H-4, H-6), 2.30 (3H, s, COCH3), 2.27 (3H, s, CH3).
13C NMR (100 MHz, CD3Cl) d = 166.35 (C, C=O), 154.
22 (C, C-20), 146.72 (C, C-1), 139.32 (C, C-100), 132.30
(CH, C-5), 129.09 (CH, C-400), 128.56 (2CH, C-300, C-500),
128.39 (CH, C-3), 125.78 (2CH, C-200, C-600), 116.94 (CH,
123

Med Chem Res (2016) 25:1260–1273
1271
N-(2-ethyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)butana-
mide (7g) Mp 175–178 °C. Yield 68 % (0.093 g) as a
white solid. 1H NMR (300 MHz, (CD3)2SO) d = 9.95 (1H,
bs, NHCO), 7.52 (1H, d, J = 6.0 Hz, H-5), 7.20 (1H, t,
J = 9.0 Hz, H-7), 6.83 (1H, bs, H-1), 6.63 (2H, m, H-6,
H-8), 4.81 (1H, t, J = 6.0 Hz, H-2), 2.07 (2H, t, J = 6.
0 Hz, COCH2CH2CH3), 1.43–1.61 (4H, 2 m, COCH2
CH2CH3, CH2CH3), 0.84 (6H, m, CH2CH3, COCH2CH2
CH3). 13C NMR (75 MHz, (CD3)2SO) d = 172.15 (C,
NHCO), 163.00 (C, C-4), 148.32 (C, C-8a), 134.36 (CH,
C-7), 128.51 (CH, C-5), 117.72 (CH, C-6), 115.08 (CH,
C-8), 114.18 (C, C-4a), 72.58 (CH, C-2), 35.76 (CH2,
COCH2CH2CH3), 26.18 (CH2, CH2CH3), 19.17 (CH2,
COCH2CH2CH3), 14.24 (CH3, COCH2CH2CH3), 8.62
(CH3, CH2CH3). HRESIMS m/z (pos): 284.1374 C14H19
N3O2Na (calcd. 284.1375).
58 (CH2, COCH2CH2CH3), 19.23 (CH3, CH3), 18.87 (CH2,
COCH2CH2CH3), 13.38 (CH3, COCH2CH2CH3). HRE-
SIMS m/z (pos): 270.1214 C13H17N3O2Na (calcd. 270.
1218).
N-(2-methyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)pentana-
mide (7d) Mp 154–156 °C. Yield 66 % (0.090 g) as a
light yellow solid. 1H NMR (300 MHz, (CD3)2CO) d = 9.
08 (1H, bs, NHCO), 7.78 (1H, d, J = 9.0 Hz, H-5), 7.32
(1H, m, H-7), 6.80 (2H, m, H-6, H-8), 6.14 (1H, bs, H-1),
5.30 (1H, q, J = 6.0 Hz, H-2), 2.33 (2H, t, J = 6.0 Hz,
COCH2CH2CH2CH3), 1.68 (2H, m, COCH2CH2CH2CH3),
1.45 (5H, m, CH3, COCH2CH2CH2CH3), 0.95 (3H, t, J =
6.0 Hz, COCH2CH2CH2CH3), 13C NMR (75 MHz,
(CD3)2CO) d = 172.25 (C, NHC=O), 163.15 (C, C-4),
148.19 (C, C-8a), 133.69 (CH, C-7), 128.57 (CH, C-5),
118.22 (CH, C-6), 115.24 (C, C-4a), 114.85 (CH, C-8), 68.
35 (CH, C-2), 33.41 (CH2, COCH2CH2CH2CH3), 27.62
(CH2, COCH2CH2CH2CH3), 22.30 (CH2, COCH2CH2
CH2CH3), 19.23 (CH3, CH3), 13.48 (CH3, COCH2CH2
CH2CH3). HRESIMS m/z (pos): 284.1381 C14H19N3O2Na
(calcd. 284.1375).
N-(2-ethyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)pentana-
mide (7h) Mp 194–196 °C. Yield 49 % (0.071 g) as a
white solid. 1H NMR (300 MHz, (CD3)2SO) d = 10.04
(1H, bs, NHCO), 7.61 (1H, d, J = 6.0 Hz, H-5), 7.29 (1H,
t, J = 6.0 Hz, H-7), 6.91 (1H, bs, H-1), 6.72 (2H, m, H-6,
H-8), 4.90 (1H, t, J = 6.0 Hz, H-2), 2.19 (2H, t, J = 9.
0 Hz, COCH2CH2CH2CH3), 1.18–1.75 (6H, 3 m,
COCH2CH2CH2CH3, COCH2CH2CH2CH3, CH2CH3), 0.
90 (6H, m, CH2CH3, COCH2CH2CH2CH3). 13C NMR
(75 MHz, (CD3)2SO) d = 172.27 (C, NHCO), 163.01 (C,
C-4), 148.32 (C, C-8a), 134.36 (CH, C-7), 128.51 (CH,
C-5), 117.72 (CH, C-6), 115.08 (CH, C-8), 114.18 (C,
C-4a), 72.56 (CH, C-2), 33.54 (CH2, COCH2CH2CH2CH3),
27.83 (CH2, COCH2CH2CH2CH3), 26.18 (CH2, CH2CH3),
22.39 (CH2, COCH2CH2CH2CH3), 14.42 (CH3, COCH2
CH2CH2CH3), 8.62 (CH3, CH2CH3). HRESIMS m/z (pos):
298.1536 C15H21N3O2Na (calcd. 298.1531).
N-(2-ethyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)acetamide
(7e) Mp 186–188 °C. Yield 73 % (0.089 g) as a white
1
solid. H NMR (300 MHz, (CD3)2SO) d = 10.08 (1H, bs,
NHCO), 7.60 (1H, d, J = 9.0 Hz, H-5), 7.29 (1H, t, J = 9.
0 Hz, H-7), 6.93 (1H, bs, H-1), 6.72 (2H, m, H-6, H-8), 4.
88 (1H, t, J = 6.0 Hz, H-2), 1.92 (3H, s, COCH3), 1.70
(2H, m, CH2CH3), 0.92 (3H, t, J = 6.0 Hz, CH2CH3). 13C
NMR (75 MHz, (CD3)2SO) d = 169.23 (C, NHCO), 161.
92 (C, C-4), 148.25 (C, C-8a), 134.39 (CH, C-7), 128.49
(CH, C-5), 117.53 (CH, C-6), 115.08 (CH, C-8), 114.27 (C,
C-4a), 72.59 (CH, C-2), 26.20 (CH2, CH2CH3), 15.65
(CH3, COCH3), 8.67 (CH3, CH2CH3). HRESIMS m/z
(pos): 256.1067 C12H15N3O2Na (calcd. 256.1062).
N-(4-oxo-2-phenyl-1,4-dihydroquinazolin-3(2H)-yl)aceta-
mide (7i) Mp 146–148 °C. Yield 54 % (0.080 g) as a
white solid. 1H NMR (300 MHz, (CD3)2SO) d = 9.86 (1H,
bs, NHCO), 7.70 (1H, d, J = 9.0 Hz, H-5), 7.40 (6H, m,
H-7, H-20–H-60), 6.76 (2H, m, H-6, H-8), 6.00 (1H, s, H-2),
1.73 (3H, s, COCH3). 13C NMR (75 MHz, (CD3)2SO) d =
168.96 (C, NHCO), 163.27 (C, C-4), 148.24 (C, C-8a),
139.48 (C, C-10), 134.62 (CH, C-7) 129.78 (CH, C-5), 129.
08 (2CH, C-30, C-50), 128.63 (CH, C-40), 128.26 (2CH,
C-20, C-60), 118.29 (CH, C-6), 115.18 (CH, C-8), 114.23
(C, C-4a), 74.77 (CH, C-2), 20.96 (CH3, COCH3). HRE-
SIMS m/z (pos): 304.1061 C16H15N3O2Na (calcd. 304.
1062).
N-(2-ethyl-4-oxo-1,4-dihydroquinazolin-3(2H)-yl)propana-
mide (7f) Mp 154–156 °C. Yield 64 % (0.083 g) as a
white solid. 1H NMR (300 MHz, (CD3)2SO) d = 10.00
(1H, bs, NHCO), 7.60 (1H, d, J = 9.0 Hz, H-5), 7.29 (m,
1H, J = 9.0 Hz, H-7), 6.90 (1H, bs, H-1), 6.75 (2H, m,
H-6, H-8), 4.90 (1H, t, J = 6.0 Hz, H-2), 2.19 (2H, q, J =
9.0 Hz, COCH2CH3), 1.70 (2H, m, CH2CH3), 1.07 (3H, t,
J = 9.0 Hz, COCH2CH3), 0.93 (3H, t, J = 9.0 Hz, CH2
CH3). 13C NMR (75 MHz, (CD3)2SO) d = 173.05 (C,
NHCO), 163.02 (C, C-4), 148.32 (C, C-8a), 134.36 (CH,
C-7), 128.49 (CH, C-5), 117.72 (CH, C-6), 115.08 (CH,
C-8), 114.19 (C, C-4a), 72.57 (CH, C-2), 27.09 (CH2,
COCH2CH3), 26.18 (CH2, CH2CH3), 10.24 (CH3,
COCH2CH3), 8.64 (CH3, CH2CH3). HRESIMS m/z (pos):
270.1217 C13H17N3O2Na (calcd. 270.1218).
N-(4-oxo-2-phenyl-1,4-dihydroquinazolin-3(2H)-yl)propa-
namide (7j) Mp 153–157 °C. Yield 47 % (0.075 g) as a
white solid. 1H NMR (300 MHz, (CD3)2SO) d = 9.78 (1H,
123

1272
Med Chem Res (2016) 25:1260–1273
bs, NHCO), 7.70 (1H, d, J = 9.0 Hz, H-5), 7.40 (6H, m,
H-7, H-20–H-60), 6.77 (2H, m, H-6, H-8), 6.02 (1H, s, H-2),
1.96 (2H, m, COCH2CH3), 0.87 (3H, t, J = 6.0 Hz,
COCH2CH3). 13C NMR (75 MHz, (CD3)2SO) d = 172.68
(C, NHCO), 163.44 (C, C-4), 148.41 (C, C-8a), 139.15 (C,
C-10), 134.57 (CH, C-7), 129.83 (CH, C-5), 129.99 (2CH,
C-30, C-50), 128.65 (CH, C-40), 128.51 (2CH, C-20, C-60),
118.35 (CH, C-6), 115.22 (CH, C-8), 114.34 (C, C-4a), 74.
87 (CH, C-2), 20.93 (CH2, COCH2CH3), 10.26 (CH3,
COCH2CH3). HRESIMS m/z (pos): 318.1222 C17H17N3
O2Na (calcd. 318.1218).
bovine serum albumin (BSA), Dowex-50 W (50 9 8–200),
FAD, NADPH and 5,6,7,8-tetrahydro-L-biopterin dihydro-
cloride (H4-biopterin), tris-(hydroxymethyl)-aminomethane
(Tris–HCl), and calcium chloride were obtained from Sigma-
3
-1
´
Aldrich Quımica (Spain). L-[ H]-arginine (54 Ci mmol
)
was obtained from Amersham (Amersham Biosciences,
Spain).
Tris-(hydroxymethyl)-aminomethane (Tris–HCl) and
calcium chloride were obtained from Merck (Spain).
Calmodulin from bovine brain, and recombinant iNOS and
nNOS enzymes, were obtained from Alexis Biochemicals
(Enzo Life Sciences, Grupo Taper, Seville, Spain).
The nNOS activity was measured by the Bredt and
Snyder method (Bredt and Snyder 1990), monitoring the
conversion of L-[3H]-arginine to L-[3H]-citrulline. The
final incubation volume was 100 lL and consisted of 10 lL
of an aliquot of recombinant i/nNOS added to a buffer with
a final concentration of 25 mM Tris–HCl, 1 mM DTT,
4 lM H4-biopterin, 10 lM FAD, 0.5 mM inosine, 0.5 mg/
mL BSA, 0.1 mM CaCl2, 10 lM L-arginine, 10 lg/mL
calmodulin (only for nNOS) and 50 nM L-[3H]-arginine, at
pH 7.6 and 7.0 for iNOS and nNOS, respectively. The
reaction was started by the addition of 10 lL of 0.75 mM
NADPH and 10 lL of each 2,3-dihydro-1,3,4-oxadiazole
or 2,3-dihydroquinazolin-4(1H)-one derivative in ethanol
(20 %) to give a final concentration of 1 mM. The tubes
were vortex and incubated at 37 °C for 30 min. Control
incubations were performed by the omission of NADPH.
The reaction was halted by the addition of 400 lL of cold
0.1 M HEPES, 10 mM EGTA, and 0.175 mg/mL
L-citrulline, pH 5.5. The reaction mixture was decanted
into a 2-mL column packet with Dowex-50 W ion-ex-
change resin (Na? form) and eluted with 1.2 mL of water.
L-[3H]-citrulline was quantified by liquid scintillation
spectroscopy. The retention of L-[3H]-arginine in this
process was greater than 98 %. Specific enzyme activity
was determined by subtracting the control value, which
usually amounted less than 1 % of the radioactivity added.
The nNOS activity was expressed as picomoles of L-[3H]-
citrulline produced (/mg of protein/min).
N-(4-oxo-2-phenyl-1,4-dihydroquinazolin-3(2H)-yl)butana-
mide (7k) Mp 166–168 °C. Yield 40 % (0.065 g) as a
white solid. 1H NMR (300 MHz, (CD3)2SO) d = 9.79 (1H,
bs, NHCO) 7.69 (1H, d, J = 9.0 Hz, H-5); 7.40 (6H, m,
H-7, H-20–H-60), 6.77 (2H, m, H-6, H-8), 6.02 (1H, s, H-2),
1.93 (2H, m, COCH2CH2CH3), 1.38 (2H, m, COCH2
CH2CH3), 0.74 (3H, t, J = 6.0 Hz, COCH2CH2CH3). 13C
NMR (75 MHz, (CD3)2SO) d = 171.80 (C, NHCO), 163.
48 (C, C-4), 148.42 (C, C-8a), 139.12 (C, C-10), 134.57
(CH, C-7), 129.83 (CH, C-5), 128.98 (2CH, C-30, C-50),
128.67 (CH, C-40), 128.56 (2CH, C-20, C-60), 118.35 (CH,
C-6), 115.21 (CH, C-8), 114.35 (C, C-4a), 74.99 (CH, C-2),
35.55 (CH2, COCH2CH2CH3), 19.07 (CH2, COCH2CH2
CH3), 14.05 (CH3, COCH2CH2CH3). HRESIMS m/z (pos):
332.1380 C18H19N3O2Na (calcd. 332.1375).
N-(4-oxo-2-phenyl-1,4-dihydroquinazolin-3(2H)-yl)penta-
namide (7l) Mp 178–180 °C. Yield 75 % (0.127 g) as a
white solid. 1H NMR (300 MHz, (CD3)2SO) d = 9.78 (1H,
bs, NHCO), 7.69 (1H, d, J = 9.0 Hz, H-5), 7.40 (6H, m,
H-7, H-20–H-60), 6.77 (2H, m, H-6, H-8), 6.02 (1H, s, H-2),
1.95 (2H, m, COCH2CH2CH2CH3), 1.33 (2H, m,
COCH2CH2CH2CH3), 1.11 (2H, m, COCH2CH2CH2CH3),
0.77 (3H, t, J = 6.0 Hz, COCH2CH2CH2CH3). 13C NMR
(75 MH, (CD3)2SO) d = 171.89 (C, NHCO), 163.51 (C,
C-4), 148.44 (C, C-8a), 139.05 (C, C-10), 134.57 (CH, C-7),
129.81 (CH, C-5), 128.96 (2CH, C-30, C-50), 128.59 (3CH,
C-20, C-40, C-60), 118.36 (CH, C-6), 115.21 (CH, C-8), 114.
37 (C, C-4a), 74.99 (CH, C-2), 33.32 (CH2, COCH2CH2
CH2CH3), 27.75 (CH2, COCH2CH2CH2CH3), 22.11 (CH2,
COCH2CH2CH2CH3), 14.34 (CH3, COCH2CH2CH2CH3).
HRESIMS m/z (pos): 346.1485 C19H21N3O2Na (calcd.
346.1489).
Statistical analysis
Data are expressed as the mean SEM. Statistically sig-
nificant differences between groups were calculated by
Student’s t test for unpaired observations or for multiple
comparisons. An ANOVA followed by the Newmane-
Keuls multiple range test was used. A p \ 0.05 was con-
sidered statistically significant.
In vitro nNOS and iNOS activities determination
L-Arginine, L-citrulline, N-(2-hydroxymethyl)piperazine-N0-
(2-ethanesulfonic acid) (HEPES), DL-dithiothreitol (DTT),
hypoxanthine-9-b-D-ribofuranoside (inosine), ethylene glycol-
bis-(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA),
Acknowledgments This work was supported by Grants from the
´
Vicerrectorado de Polıtica Cientıfica e Investigacion de la Universi-
dad de Granada (GREIB.PYR_2010_03 and GENIL/GREIB 2011).
´
´
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Med Chem Res (2016) 25:1260–1273
1273
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