Quinazolinones, Quinazolinthiones, and Quinazolinimines as Nitric Oxide Synthase Inhibitors: Synthetic Study and Biological Evaluation

Article abstract of DOI:10.1002/ardp.201600020

The synthesis of different compounds with a quinazolinone, quinazolinthione, or quinazolinimine skeleton and their in vitro biological evaluation as inhibitors of inducible and neuronal nitric oxide synthase (iNOS and nNOS) isoforms are described. These d

Full text of DOI:10.1002/ardp.201600020

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–13
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Full Paper
Quinazolinones, Quinazolinthiones, and Quinazolinimines
as Nitric Oxide Synthase Inhibitors: Synthetic Study and
Biological Evaluation
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ꢀ
M. Encarnacion Camacho, Mariem Chayah, M. Esther Garcıa, Nerea Fernandez-Saez, Fabio Arias,
ꢀ
Miguel A. Gallo, and M. Dora Carrion
ꢀ
ꢀ
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Facultad de Farmacia, Departamento de Quımica Farmaceutica y Organica, Universidad de Granada,
Granada, Spain
The synthesis of different compounds with a quinazolinone, quinazolinthione, or quinazolinimine
skeleton and their in vitro biological evaluation as inhibitors of inducible and neuronal nitric oxide
synthase (iNOS and nNOS) isoforms are described. These derivatives were obtained from substituted
2-aminobenzylamines, using diverse cyclization procedures. Furthermore, the diamines were synthe-
sized by two routes: A conventional pathway and an efficient one-pot synthesis in a continuous-flow
1
hydrogenator. The structures of these heterocycles were confirmed by H and 13C nuclear magnetic
resonance and high-resolution mass spectroscopy data. The structure–activity relationships of the
target molecules are discussed in terms of the effects of both the R radical and the X heteroatom in
the 2-position. In general, the assayed compounds behave as better iNOS than nNOS inhibitors,
with the quinazolinone 11e being the most active inhibitor of all tested compounds and the most
iNOS/nNOS selective one.
Keywords: Inhibitors / Quinazoline / Synthesis
Received: January 22, 2016; Revised: May 23, 2016; Accepted: May 27, 2016
DOI 10.1002/ardp.201600020
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
NO production by nNOS takes part in neuronal signaling,
synaptic plasticity, modulation of pathways involved in
Introduction
Nitric oxide synthase (NOS) catalyzes the oxidation of a
L-arginine guanidinium nitrogen atom to nitric oxide (NO), a
potent biological signaling molecule that mediates a diverse
range of physiological processes within the cardiovascular,
immune, and nervous systems [1–3]. Three mammalian NOS
isoforms have been characterized so far. Two of them, the
endothelial (eNOS) and neuronal NOS (nNOS), are constitutive
and calcium-dependent. The third isoform, the inducible one
(iNOS), is formed in response to pathological challenges [4].
learning, and neurotoxicity; NO synthesized by eNOS partic-
ipates in regulation of vascular function, promotes vasodila-
tion, and modulates arterial pressure; and production of NO
by iNOS is part of non-specific immunity, it modulates the
immune system and acts as proinflammatory mediator [5]. An
overproduction of NO by nNOS produces reactive oxygen
species (ROS), reactive nitrogen species (RNS), oxidative and
nitrosative stress induction, thiole groups nitrosation, pheno-
lic residues nitration, mitochondrial damage, neuronal death,
and inflammation. Thus, it has been demonstrated the
implication of the nitrergic signaling dysregulation, nNOS
overexpression, and oxidative stress in neurodegenerative
disorders [6]. Meanwhile, astrocytes and activated glia
inflammatory mediators release more NO by iNOS, causing
neuronal damage and microglia increased activity [7].
ꢀ
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Correspondence: Prof. M. Dora Carrion, Quımica Farmaceutica y
ꢀ
Organica, C/Campus de Cartuja Facultad de Farmacia – Uni-
versidad de Granada, Granada 18071, Spain.
E-mail: dcarrion@ugr.es
Fax: þ34 958240728
While eNOS-generated NO plays a role in vascular regula-
tion, the potential therapeutic utility of NOS inhibitors is
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restricted to the inhibition of the neuronal or inducible
isoforms [8]. Overproduction of NO by nNOS is associated with
various diseases such as neurodegeneration, stroke, migraine,
Alzheimer, and Huntington diseases. On the other hand,
overproduction of NO by iNOS is related to hypotensive crises
during septic shock, arthritis, colitis, tissue damage, neuro-
pathic pain, and several kinds of inflammatory states [9–12].
Therefore, the use of substances with inhibitory properties of
the different NOS isoforms has great therapeutic potential for
the treatment of the above-mentioned pathologies [13, 14].
Consequently, over the last 20 years the design and synthesis
of NOS inhibitors have received much attention.
The best known inhibitors of NOS with a guanidine moiety
are amino acids related to the substrate L-arginine, for
example L-NA 1 (or its methyl esther L-NAME, 2) [15] (Fig. 1).
However, none of them is particularly selective. Other
inhibitors based on guanidines [16], isothioureas [17, 18],
and amidines [19–21] have been reported with different levels
of selectivity and potency in vitro. Thus, the acetamidine 3 [21]
is a potent iNOS inhibitor without affecting the endothelial
rutaecarpine, etc. isolated from a number of plant families,
animals, and from microorganisms [26].
Thus, quinazolinone scaffold has been incorporated into a
number of therapeutic agents such as anticancer (raltitrexed),
anticonvulsant (piriqualone), antihypertensive (ketanserin),
antitussive (chloroqualone), diuretic (metolazone and quine-
thazone), and sedative-hypnotic (methaqualone), among
others.
Nowadays, the quinazolinone ring system constitutes a
key structural component in modern medicinal chemistry.
Owing to its unique pharmaceutical significance, continuous
advancements in the synthesis of new quinazolinone deriv-
atives are an emerging source of research and development.
Hence, efficient and reliable methods for their synthesis
and fusion with many other bioactive molecules have led to
the consideration of quinazolinone as a pivotal moiety in
heterocyclic chemistry.
Previously, we have synthesized several heterocyclic fami-
lies of NOS inhibitors such as 2-aminophenylpyrazolines 9 [27]
and 2-aminophenylpyrroles 10 [28] (Fig. 1). In this paper, we
describe the synthesis of a new type of derivatives where the
free amine group belonging to the benzene ring is part of a
new cycle of quinazolinone 11 that restricts the conforma-
tional freedom. Furthermore, their isosteric compounds with
quinazolinthione 12 and quinazolinimine 13 structures have
also been synthesized (Fig. 2). These heterocycles carry a
guanidine moiety (existing in the NOS natural substrate
L-arginine), or an urea or thiourea isosteric fragment forming
part of a cycle. The synthetic route of these novel derivatives
have been made by different cyclization methods from
2-aminobenzylamines, which were synthesized using both a
traditional route and a flow hydrogenator. Also, we evaluate
their activities as inhibitors of nNOS and iNOS isoforms
in order to find new compounds with interesting pharmaco-
logical properties.
isoform, and the thiophene-2-carboximidamide
4 is an
interesting selective inhibitor of nNOS [22]. In addition, it
has been previously published the discovery of dihydroiso-
quinolines 5 [23] and thienopyridines 6 [24], by replacement
of the benzene ring by a thieno fused-ring that showed
potent activity against iNOS, although the best compound
produced hypotension in vivo. Moreover, Tinker et al. have
demonstrated potency, selectivity, and efficacy in the rat
adjuvant-induced arthritis model for 1,2-dihydro-4-quinazo-
linamine derivatives 7 and 8 [25] (Fig. 1).
Quinazoline and quinazolinone derivatives occur fre-
quently in natural and synthetic pharmaceutical products
which exhibit important biological properties. Quinazolinone
is a building block for approximately 150 naturally occurring
alkaloids such as glycosminine, echinozolinone, deoxyvascinone,
Figure 1. Examples of L-Arg analogs and other cyclic compounds as NOS inhibitors.
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the yields of both reactions are good and the overall yields
oscillate between 42 and 65% (Table 1).
While we performed the reductive amination of aldehydes
with primary amines in the two steps previously described, we
carried out the amine group reduction using a continuous
flow-reactor (H-Cube flow hydrogenator model HC-2.SS)
which employs a mixed hydrogen liquid flow stream. This
methodology enabled to reduce the reaction times and to
increase the yields (see Table 1). In the flow-reactor, the
hydrogen is internally generated by the electrolysis of water
and the gas-liquid mixture is pumped through a suitable
catalyst contained in an interchangeable metal cartridge.
To carry out the studies of reductive amination, we have
chosen the 2-nitrobenzaldehyde and the cyclopropylamine
to obtain 2-((cyclopropylamino)methyl)benzenamine 15e.
An important prerequisite for flow chemistry is the choice of
an appropriate solvent in order to avoid the precipitation
of both reagents and products, which may lead to a blockage
of the flow stream. In this case, out of the three solvents
examined (MeOH, DCM, and THF), THF gave the best results
under representative reaction conditions (10% Pd/C, full H2,
40°C, 0.05 M, flow rate 0.5 mL/min).
The optimization of the flow conditions for the direct
reductive amination and for the amino group reduction was
systematically investigated by varying the flow rate, pressure,
temperature, and the presence of acetic acid. The molar
relation between the aldehyde and amine reagents was
always 1:1.5 (Table 2).
The flow rate used was 1 or 0.5 mL/min. The increasing of
the flow rate (run 1) causes the decrease of the yield, as a
consequence of the residence time reduction in the flow-
reactor. In this case, the yield of 15e was low (10%) and we
Figure 2. General structure of derivatives 11, 12, and 13.
Results and discussion
Chemistry
The general transformation of amines from aldehydes is
an important method in organic synthesis because of their
versatile utility as intermediates for preparation of
pharmaceutical and agrochemical derivatives [29]. The
reductive amination is commonly used for this transfor-
mation. The procedure involves the initial formation of a
carbinolamine which dehydrates to give an imine. The
second step needs a weakly acidic pH and the imine is
protonated to form an iminium ion. Finally, subsequent
reduction of this iminium cation produces the correspond-
ing amino derivatives [30].
Scheme 1 shows the synthesis of derivatives 14a–i using this
methodology. The general procedure begins with the
reductive amination of 2-nitrobenzaldehyde with alkyl-,
cycloalkyl-, or benzyl-amines, via formation of the imino
derivatives with sodium sulfate, and then reduction by
sodium borohydride in methanol to yield the amino
derivatives 14a–i [31]. In a new stage, the reduction of the
nitro group belonging to the aromatic ring, using Fe/FeSO4 in
water [32], leads to the diamine derivatives 15a–i. In general,
mainly
isolated
2-aminobenzaldehyde
(50%)
and
(2-aminophenyl)methanol (10%). These products indicated
Scheme 1. Synthesis of final compounds with general structures 11, 12, and 13. (a) R-NH2/Na2SO4, MeOH, 2 h rt; (b) NaBH4/MeOH, 1 h
rt; (c) Fe/FeSO4, H2O, 3 h reflux; (d) R-NH2, 10% Pd/C, THF (flow hydrogenation); (e) (Cl3CO)2CO, Et3N, DCM, 0°C, then 1 h rt, or CDI,
THF, 3 h rt, then 18 h reflux; (f) Cl2CS, Et3N, Et2O, ꢀ78°C, then rt, or TCDI, THF, h rt, then 18 h reflux; (g) Me3OþBF4ꢀ, DCM, 12 h rt; (h)
ClNH4, MeOH, 3.5 h reflux; (i) Na2CO3.10H2O, H2O, 10 min rt; (j) Ac2O, Et3N, DCM, 3 h rt.
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Table 1. Yields of the nitrobenzylamines 14a–i, the diamines 15a–i obtained by conventional method, global yields of
both reactions, and yields of the diamines 15a–h by the H-Cube flow hydrogenator.
Flow
hydrogenator
yield (%)
Global yield
(%)
Compound
Yield (%)
Compound
Yield (%)
R
4a
63
70
85
73
51
70
73
74
80
15a
15b
15c
15d
15e
15f
15g
15h
15i
70
60
74
89
86
80
70
70
75
44
42
63
65
44
56
51
52
60
70
70
73
75
80
80
80
78
–
Me
Et
Pr
14b
14c
14d
14e
14f
14g
14h
14i
Bu
c-C3H5
c-C4H7
c-C5H9
tert-Bu
CH2Ph
that the imine derivative was formed in little amount and the
nitro and aldehyde groups were reduced to the amine and to
the primary alcohol, respectively. When we reduced the flow
rate to 0.5 mL/min (run 2), the yield of 15e was increased from
10 to 60%. This fact showed that the imine derivative was
obtained in better yield since the (2-aminophenyl)methanol
derivative was not isolated. Then, we decided to change the
full H2 by 20 bar (with this H-Cube model, it is not possible to
control the pressure in full H2 mode) but we did not isolate the
desired product (run 3). In another assay (run 4), we raised
the temperature to 60°C but the yield decreased. This
last result can be due to the partial evaporation of the
cyclopropylamine.
On the other hand, an adequate pH is crucial for the
formation of imine derivatives. The ideal pH is about 4.5.
The second step needs acid pH but if the solution is very acid
the amine is protonated, and as a consequence, the first step is
inhibited. Thus, to increase the yield, we used a drop (20 mL) of
glacial acetic acid with the aim of catalyzing the reaction (run
5). Before passing the reaction mixture through the H-Cube,
the pH was about 5, and in this occasion, the yield increased
up to 80%. Previously, Cho and Kang had published a
convenient procedure for reductive amination using sodium
borohydride activated by boric acid, p-toluenesulfonic acid
monohydrate, or benzoic acid as reducing agents [33]. These
acids, used to activate the reductive amination, are solid and
we could not use them in a continuous flow reactor. This way,
we decided to use acetic acid as an activator of the reductive
amination.
We applied the optimized reaction conditions to the
synthesis of the corresponding 2-aminobenzylamine deriva-
tives 15a–h. The compound 15i (R ¼ CH2Ph) could not be
synthesized by this methodology because of the loss of the
benzyl group in the H-Cube flow hydrogenator. The results
are shown in Table 1.
In all cases we obtained an optimum yield between 70 and
80%. In the experiments with methylamine and ethylamine,
we have used a 2 M solution in THF of the corresponding
amines. The smaller yields in these cases could be due to the
lower boiling point and basicity of these amines compared
to the rest of used amines.
3-Substituted-3,4-dihydroquinazolin-2(1H)-one derivatives
11a–i were synthesized via ring closure of the appropriate
diamines 15a–i, either with triphosgene and triethylamine [34]
or with 1,10-carbonyldiimidazole (CDI) [35] as shown in the
synthetic Scheme 1. The ring closure yield with triphosgene
was usually low (40–44%); in contrast, refluxing an equimolar
quantity of the diamine with CDI in THF gave the desired
quinazolinones with a good yield (74–83%).
Furthermore, the arylthiourea derivatives 12b–e were
obtained either by treatment of the corresponding diamines
15b–e with thiophosgene [36], or with 1,10-(thiocarbonyl)-
diimidazole (TCDI). The use of thiophosgene gave low yields
(35%); however, when the cyclation took place with TCDI, the
Table 2. Flow hydrogenation optimization results for compound 15e at 0.05 M concentration.
Flow rate
(mL/min)
Injection
volume (mL)
Temperature
Run
Acetic acid
Pressure
(ºC)
Yield (%)
1
2
3
4
5
–
1
20
20
20
20
20
Full H2
Full H2
20 bar
Full H2
Full H2
40
40
40
60
40
10
60
–
–
0.5
0.5
0.5
0.5
–
–
50
80
1 drop
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desired quinazolinthiones were obtained with better yields
(75–84%), being the first time that this reactive is used in the
synthesis of cyclic thioureas.
Finally, 3,4-dihydroquinazolin-2(1H)-imine derivatives 13a
and 13c–e were synthesized via the corresponding intermedi-
ate dihydromethoxyquinazolines 16a and 16c–e by addition
of trimethyloxonium tetrafluoroborate to the quinazolinones
11a and 11c–e, respectively, and then by treatment with
NH4Cl in methanol [37].
The derivatives 11j and 12j were synthesized by acylation of
(2-nitrophenyl)methanamine hydrochloride with acetic anhy-
dride [38], followed by reduction of the nitro group to give
the intermediate 15j, which was treated either with CDI in
THF to give 11j, or with TCDI to yield 12j (Scheme 1).
shows the inhibition percentages versus nNOS and iNOS.
The pyrrole 10a (R ¼ H, R’ ¼ c-C5H9) previously described [28]
and the known NOS inhibitor L-NAME [15] (Fig. 1) have been
included as controls.
In general, these compounds behave as weak inhibitors
against nNOS, since only three of them (11a, 12d, and 12h)
show a good percentage of inhibition at the concentration of
1 mM. Nevertheless, some conclusions can be inferred from
the experimental data. From a qualitative point of view, the
influence of R and X over the nNOS activity is variable. Most of
the compounds with linear or ramified alkyl radicals in
3-position are better inhibitors than those with cycloalkyl,
benzyl, or acyl ones, with some derivatives with methyl, butyl,
and tert-butyl substituents being the best inhibitors (11a,
67.9%, 12d, 60.4%, and 12h, 70.9%, respectively).
Biological results
Regarding the iNOS inhibition values, these compounds
behave as better inhibitors against this isoform, since eight
molecules show an inhibition percentage higher than 50%. In
general, derivatives with a quinazolinone heterocycle are
better inhibitors than those with quinazolinthione or
quinazolinimine moieties, since four quinazolinones (11a,
11c, 11e, and 11f) have good percentages of inhibition
The biological activities of the new heterocycles 11a–j, 12b–e,
12h, 12j, 13a, and 13c–e as inhibitors of both iNOS and nNOS
have been evaluated by means of in vitro assays using
recombinant isoenzymes. We have carried out the assays
using a 1 mM concentration of each compound in order to
identify the more potent and selective derivatives. Table 3
Table 3. In vitro nNOS and iNOS inhibition (%) observed in the presence of 1 mM concentration of compounds 11a–j,
12b–e, 12h, 12j, 13a, and 13c–e. L-NAME and pyrrole 10a have been included as controls.
Compound
X
R
% nNOS inhibitiona)
% iNOS inhibitiona)
L-NAMEb)
10c)
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
12b
100.0 ꢁ 1.03
5.36 ꢁ 3.19
67.91 ꢁ 0.45
17.37 ꢁ 0.16
2.21 ꢁ 1.33
32.41 ꢁ 2.45
0.93 ꢁ 3.22
32.02 ꢁ 2.38
12.50 ꢁ 0.77
6.55 ꢁ 1.62
11.49 ꢁ 3.08
9.66 ꢁ 1.23
6.06 ꢁ 3.81
11.91 ꢁ 0.96
60.35 ꢁ 1.80
0.38 ꢁ 1.5
77.01 ꢁ 0.96
52.79 ꢁ 1.70
58.86 ꢁ 1.93
41.27 ꢁ 2.20
58.65 ꢁ 3.45
34.27 ꢁ 3.08
72.74 ꢁ 2.47
57.44 ꢁ 1.23
26.22 ꢁ 3.78
36.45 ꢁ 0.87
26.79 ꢁ 1.79
22.01 ꢁ 3.87
20.38 ꢁ 1.77
25.69 ꢁ 3.48
59.83 ꢁ 2.73
10.16 ꢁ 1.47
55.35 ꢁ 3.61
51.96 ꢁ 2.48
57.53 ꢁ 3.29
27.19 ꢁ 2.33
32.99 ꢁ 3.62
25.98 ꢁ 0.67
O
O
O
O
O
O
O
O
O
O
S
Me
Et
Pr
Bu
c-C3H5
c-C4H7
c-C5H9
tert-Bu
CH2Ph
COMe
Et
Pr
Bu
c-C3H5
tert-Bu
COMe
Me
12c
S
S
S
S
12d
12e
12h
12j
13a
13c
70.85 ꢁ 3.55
14.42 ꢁ 1.98
45.02 ꢁ 1.76
0.73 ꢁ 2.38
11.83 ꢁ 1.95
11.53 ꢁ 0.85
S
NH
NH
NH
NH
Pr
Bu
c-C3H5
13d
13e
a) Data represent the percentage of nNOS and iNOS inhibition produced by 1 mM concentration of each compound. Each
value is the mean ꢁ SEM of three experiments performed in triplicate using recombinant iNOS and nNOS enzymes. b) See [15].
c)
See [31].
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(between 57.4 and 72.7%), being 11e (R ¼ c-C3H5) the best
inhibitor of this series and the most potent of all tested
compounds. In addition, among derivatives with a sulfur
atom, 12d (R ¼ Bu, 59.8%), 12h (R ¼ tert-Bu, 70.9%), and 12j
(R ¼ COMe, 52.0%) are the best ones. Finally, among the
quinazolinimines, 13a (R ¼ Me) is the best inhibitor of this
series (57.5% of inhibition). Comparing these results with
derivatives of general structure 10 previously described
(the shown pyrrole 10a is the best inhibitor of this family),
the quinazolinones 11c and 11e are more potent and selective
versus iNOS, demonstrating that the presence of an urea
fragment in these heterocycles, which could mimic the
guanidine moiety of the NOS natural substrate, is beneficial
for the inhibition.
In general, these compounds show better inhibition values
against iNOS than nNOS, what agrees with the published
biological activities for the quinazoline derivatives 7–8 (Fig. 1),
which are iNOS selective inhibitors. The most selective
compounds are 11c (iNOS/nNOS ¼ 26.5) and 11e (iNOS/nNOS
¼ 78.2), which should be an interesting starting point for
possible new alternatives in diseases where the iNOS is
increased, such as septic shock, arthritis, and various kinds of
inflammatory states.
spectra were recorded on a Bruker AMX 300 spectrometer
operating at 300.20 MHz for 1H and 75.49 MHz for 13C and on
a Bruker ARX 400 spectrometer operating at 400.17 MHz
1
for H and 125.69 MHz for 13C, in CDCl3 (at concentration of
ca. 27 mg/mL in all cases). The center of each peak of CDCl3
[7.26 ppm (1H) and 77.0 ppm (13C)] was used as internal
reference in a 5 mm 13C/1H dual probe (Wilmad, No. 528-PP).
The temperature of the sample was maintained at 297 K.
The peaks are reported in ppm (d). High-resolution mass
spectroscopy (HRMS) was carried out on a VG AutoSpec Q
high-resolution mass spectrometer (Fision Instruments). Flash
chromatography was carried out using silica gel 60, 230–
240 mesh (Merck), and the solvent mixture reported within
parentheses was used as eluent. The conventional organic
solvents were purchased from Fisher and all other chemicals
were purchased from Aldrich.
The InChI codes of the investigated compounds are
provided as Supporting Information.
General procedure for the synthesis of N-(2-nitrobenzyl)-
alkylamine derivatives 14a–i
To a cooled solution of 2-nitrobenzaldehyde (2 g, 13.23 mmol)
in methanol (20 mL) was added Na2SO4 (2 g, 15.88 mmol) and
the corresponding primary amine (1.2 mL, 14.55 mmol). After
stirring at room temperature for 2 h, the precipitates were
removed by filtration, and the solvent was evaporated. The
residue (imine) was dissolved in dry methanol (20 mL) and
treated with NaBH4 (0.25 g, 6.61 mmol) at room temperature
for 1 h. The mixture was concentrated, and the residue was
dissolved in DCM (30 mL), washed with water, dried (Na2SO4),
and concentrated to afford the crude mixture that was
purified by flash chromatography (AcOEt/hexane 1:3).
Finally, compounds 11a, 12d, and 12h, which are the best
nNOS inhibitors, also show good iNOS percentages of
inhibition, what could be useful in several illnesses where
nitric oxide is increased due to both nNOS and iNOS isoforms,
like Parkinson’s disease.
Conclusions
In summary, in the current study, we have reported different
strategies for the synthesis of a series of compounds which
have a skeleton of quinazolinone, quinazolinthione, and
quinazolinimine. We have demonstrated that the use of a
flow hydrogenator for the synthesis of the intermediate
2-aminobenzylamines reduces to one the reaction steps, and
the reductive amination activator acetic acid increases the
overall yields. In addition, the ring closure has been achieved
following two different methods in the quinazolinones as
well as in the quinazolinthiones, being the first time that TCDI
is used in the synthesis of this kind of cyclic compounds. Also,
we report the results on nNOS and iNOS inhibition of final
compounds. Among all derivatives, 12h is the best nNOS
inhibitor and 11e is the iNOS most potent one of all tested
compounds, and the most selective versus iNOS. This last
quinazolinone could be a reference to design new inhibitors
useful in inflammatory diseases such as septic shock or arthritis.
N-(2-Nitrobenzyl)methylamine (14a) [39, 40]
Yellow oil, yield 1385 mg (63%). 1H NMR (300.20 MHz, CDCl3)
0
0
0
0
0
d: 7.95 (dd, 1H, J3 -4 ¼ 8.1 Hz, J3 -5 ¼ 1.2 Hz, H-3 ), 7.60–7.53 (m,
2H, H-50, H-60), 7.43–7.39 (m, 1H, H-40), 4.91 (bs, 1H, –NH–), 4.10
(s, 2H, Ph–CH2–), 2.46 (s, 3H, H-1). 13C NMR (75.49 MHz, CDCl3)
d: 145.57 (C-20), 136.12 (C-10), 133.25 (C-50), 131.43 (C-60),
128.65 (C-40), 125.14 (C-30), 51.77 (Ph–CH2–), 35.36 (C-1).
N-(2-Nitrobenzyl)ethylamine (14b) [40]
Yellow oil, yield 1669 mg (70%). 1H NMR (400.17 MHz, CDCl3)
0
0
0
0
0
d: 7.90 (dd, 1H, J3 -4 ¼ 8.1 Hz, J3 -5 ¼ 1.3 Hz, H-3 ), 7.57–7.54 (m,
2H, H-50, H-60), 7.38–7.34 (m, 1H, H-40), 4.39 (bs, 1H, –NH–), 4.00
(s, 2H, Ph–CH2–), 2.66 (q, 2H, J1–2 ¼ 7.1 Hz, H-1), 1.08 (t, 3H,
J2–1 ¼ 7.1 Hz, H-2). 13C NMR (125.69 MHz, CDCl3) d: 149.05
(C-20), 134.72 (C-10), 133.27 (C-50), 131.51 (C-60), 128.17 (C-40),
124.75 (C-30), 50.13 (Ph–CH2–), 43.53 (C-1), 14.73 (C-2).
N-(2-Nitrobenzyl)propylamine (14c) [40]
Yellow oil, yield 2184 mg (85%). 1H NMR (300.20 MHz,0CDCl3)
Experimental
0
0
0
0
d: 7.99 (d, 1H, J3 -4 ¼ 8.1 Hz, H-3 ), 7.73–7.54 (m, 2H, H-5 , H-6 ),
7.50–7.36 (m, 1H, H-40), 4.08 (s, 2H, Ph–CH2–), 2.65 (t, 2H,
J1–2 ¼ 7.2 Hz, H-1), 2.47 (bs, 1H, –NH–), 1.58 (m, 2H, H-2), 0.95
(t, 3H, J3–2 ¼ 7.4 Hz, H-3). 13C NMR (75.49 MHz, CDCl3) d: 146.07
Chemistry
Melting points were determined using an Electrothermal-1A-
6301 apparatus and are uncorrected. 1H NMR and 13C NMR
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(C-20), 132.73 (C-10), 131.33 (C-50), 129.71 (C-60), 128.35 (C-40),
122.97 (C-30), 51.30 (C-1), 49.65 (Ph–CH2–), 23.20 (C-2), 11.58
(C-3).
131.88 (C-60), 129.20 (C-1), 128.71, 128.63 (C-2, C-3, C-5, C-6),
128.52 (C-40), 127.66 (C-4), 125.07 (C-30), 53.45 (Ph–CH2–), 50.04
(–CH2–Ph).
N-(2-Nitrobenzyl)butylamine (14d) [41]
General procedure for the preparation of N-(2-
aminobenzyl)alkylamine derivatives 15a–i
Yellow oil, yield 2011 mg (73%). 1H NMR (300.20 MHz, CDCl3)
0
0
0
0
0
d: 8.00 (dd, 1H, J3 -4 ¼ 8.1 Hz, J3 -5 ¼ 1.3 Hz, H-3 ), 7.71–7.59 (m,
2H, H-50, H-60), 7.48–7.42 (m, 1H, H-40), 4.09 (s, 2H, Ph–CH2–),
2.69 (t, 2H, J1–2 ¼ 7.2 Hz, H-1), 2.40 (bs, 1H, –NH–), 1.60–1.50
(m, 2H, H-2), 1.45–1.35 (m, 2H, H-3), 0.95 (t, 3H, J3–2 ¼ 7.3 Hz,
H-4). 13C NMR (75.49 MHz, CDCl3) d: 149.42 (C-20), 135.50 (C-10),
133.40 (C-50), 131.69 (C-60), 128.26 (C-40), 124.97 (C-30), 50.95
(Ph–CH2–), 32.30 (C-1), 29.93 (C-2), 20.58 (C-3), 14.09 (C-4).
To a suspension of the corresponding nitroarene (0.524 mmol)
in water, a mixture of Fe (5.24 mmol) and FeSO4 (0.524 mmol)
was added. The reaction was refluxed for 3 h, filtered
through celite and extracted with DCM (3 ꢂ 15 mL) and ethyl
acetate (3 ꢂ 15 mL). The combined organic layers were
washed with brine, dried (Na2SO4), and evaporated. The
residue was purified by flash chromatography (DCM/MeOH
9:1).
N-(2-Nitrobenzyl)cyclopropylamine (14e)
Yellow oil, yield 1297 mg (51%). 1H NMR (300.20 MHz,0CDCl3)
N-(2-Aminobenzyl)methylamine (15a) [40]
Yellow oil, yield 50 mg (70%). 1H NMR (300.20 MHz, CDCl3) d:
6.99–6.80 (m, 2H, H-40, H-60), 6.64–6.55 (m, 2H, H-30, H-50), 3.98
(s, 2H, Ph–CH2–), 3.51 (bs, 3H, –NH2, –NH–), 2.45 (s, 3H, H-1).
13C NMR (75.49 MHz, CDCl3) d: 146.50 (C-20), 129.94, 128.45
(C-40, C-60), 123.13 (C-10), 117.74, 115.68 (C-30, C-50), 49.50
(Ph–CH2–), 35.83 (C-1).
0
0
0
0
d: 7.95 (d, 1H, J3 -4 ¼ 8.1 Hz, H-3 ), 7.59–7.57 (m, 2H, H-5 , H-6 ),
7.45–7.39 (m, 1H, H-40), 4.10 (s, 2H, Ph–CH2–), 2.49 (bs, 1H,
–NH–), 2.16–2.11 (m, 1H, H-1), 0.47–0.40 (m, 4H, H-2, H-3).
13C NMR (75.49 MHz, CDCl3) d: 149.28 (C-20), 135.56 (C-10),
133.10 (C-50), 131.88 (C-60), 128.06 (C-40), 124.79 (C-30), 50.74
(Ph–CH2–), 30.07 (C-1), 6.56 (C-2, C-3).
N-(2-Nitrobenzyl)cyclobutylamine (14f)
N-(2-Aminobenzyl)ethylamine (15b) [40]
Yellow solid, yield 1910 mg (70%). m.p.: 72–74°C. 10H NMR
Yellow oil, yield 47 mg (60%). 1H NMR (300.20 MHz, CDCl3) d:
7.16–7.07 (m, 2H, H-40, H-60), 6.73–6.68 (m, 2H, H-30, H-50), 3.85
(s, 2H, Ph–CH2–), 2.73 (q, 2H, J1–2 ¼ 7.0 Hz, H-1), 1.17 (t, 3H,
J2–1 ¼ 7.0 Hz, H-2). 13C NMR (75.49 MHz, CDCl3) d: 147.03 (C-20),
130.38, 128.52 (C-40, C-60), 124.73 (C-10), 117.94, 116.02 (C-30,
C-50), 53.06 (Ph–CH2–), 44.03 (C-1), 15.61 (C-2).
0
0
(300.20 MHz, CDCl3) d: 7.96 (d, 1H, J3 -4 ¼ 8.3 Hz, H-3 ), 7.79–
7.63 (m, 2H, H-50, H-60), 7.52–7.46 (m, 1H, H-40), 3.99 (s, 2H,
Ph–CH2–), 3.42–3.23 (m, 1H, H-1), 2.70 (bs, 1H, –NH–), 2.18–
2.11, 1.75–1.69 (2m, 6H, H-2, H-3, H-4). 13C NMR (75.49 MHz,
CDCl3) d: 142.01 (C-20), 135.47 (C-10), 133.55 (C-50), 130.02
(C-60), 128.37 (C-40), 125.01 (C-30), 54.15 (C-1), 48.42 (Ph–CH2–),
31.05 (C-2, C-4), 15.10 (C-3).
N-(2-Aminobenzyl)propylamine (15c) [40]
Yellow oil, yield 64 mg (74%). 1H NMR (300.20 MHz, CDCl3)
d: 7.16–7.07 (m, 2H, H-40, H-60), 6.76–6.68 (m, 2H, H-30, H-50),
3.85 (s, 2H, Ph–CH2–), 3.73 (bs, 3H, –NH2, –NH–), 2.65 (t, 2H,
J1–2 ¼ 9.0 Hz, H-1), 1.64–1.52 (m, 2H, H-2), 0.97 (t, 3H,
J3–2 ¼ 9.0 Hz, H-3). 13C NMR (75.49 MHz, CDCl3) d: 147.18
(C-20), 130.17, 128.67 (C-40, C-60), 124.03 (C-10), 117.98, 116.07
(C-30, C-50), 52.93 (C-1), 51.17 (Ph–CH2–), 23.13 (C-2), 12.04
(C-3).
N-(2-Nitrobenzyl)cyclopentylamine (14g)
1
Yellow oil, yield 2127 mg (73%). H NMR 300.20 MHz,0CDCl3)
0
0
0
0
d: 7.95 (d, 1H, J3 -4 ¼ 8.1 Hz, H-3 ), 7.70–7.51 (m, 2H, H-5 , H-6 ),
7.48–7.34 (m, 1H, H-40), 4.03 (s, 2H, Ph–CH2–), 3.18–3.10 (m, 1H,
H-1), 1.89–1.70 (m, 4H, H-2, H-5), 1.59–1.37 (m, 4H, H-3, H-4).
13C NMR (75.49 MHz, CDCl3) d: 147.72 (C-20), 134.63 (C-10),
131.66 (C-50), 130.08 (C-60), 126.27 (C-40), 122.98 (C-30), 58.04
(C-1), 48.04 (Ph–CH2–), 31.57 (C-2, C-5), 22.45 (C-3, C-4).
N-(2-Aminobenzyl)butylamine (15d) [41]
N-(2-Nitrobenzyl)tert-butylamine (14h)
Yellow oil, yield 84 mg (89%). 1H NMR (300.20 MHz, CDCl3) d:
7.09–7.04 (m, 2H, H-40, H-60), 6.74–6.60 (m, 2H, H-30, H-50), 4.28
(s, 2H, Ph–CH2–), 2.70 (m, 1H, H-1), 1.70 (m, 2H, H-2), 1.28 (m,
2H, H-3), 0.70 (t, 3H, J4–3 ¼ 7.1 Hz, H-4). 13C NMR (75.49 MHz,
CDCl3) d: 147.18 (C-20), 133.16, 130.64 (C-40, C-60), 117.78,
117.19 (C-30, C-50), 115.23 (C-10), 47.57 (C-1), 45.09 (Ph–CH2–),
28.09 (C-2), 20.34 (C-3), 13.78 (C-4).
Yellow oil, yield 2039 mg (74%). 1H NMR (300.20 MHz, CDCl3)
d: 7.93 (d, 1H, J ¼ 8.1 Hz, H-30), 7.71–7.56 (m, 2H, H-50, H-60),
7.43–7.38 (m, 1H, H-40), 3.97 (s, 2H, Ph–CH2–), 1.20 (s, 9H,
13
C(CH3)3). C NMR (75.49 MHz, CDCl3) d: 145.62 (C-20), 135.29
(C-10), 133.38 (C-50), 129.76 (C-60), 128.49 (C-40), 124.67 (C-30),
51.17 (C-1), 45.71 (Ph–CH2–), 28.65 (C(CH3)3).
N-(2-Nitrobenzyl)benzylamine (14i) [40]
N-(2-Aminobenzyl)cyclopropylamine (15e)
Yellow oil, yield 2564 mg (80%). 1H NMR (300.20 MHz, CDCl3)
Yellow oil, yield 73 mg (86%). 1H NMR (300.20 MHz, CDCl3)
d: 7.16–7.10 (m, 2H, H-40, H-60), 6.77–6.65 (m, 2H, H-30, H-50),
3.89 (s, 2H, Ph–CH2–), 3.62 (bs, 3H, –NH2, –NH–), 2.21 (m, 1H,
H-1), 0.52–0.43 (m, 4H, H-2, H-3). 13C NMR (75.49 MHz, CDCl3)
d: 146.82 (C-20), 130.59, 128.98 (C-40, C-60), 123.11 (C-10),
0
0
0
0
0
d: 7.99 (dd, 1H, J3 -4 ¼ 8.1 Hz, J3 -5 ¼ 1.2 Hz, H-3 ), 7.71–7.58 (m,
2H, H-50, H-60), 7.48–7.29 (m, 6H, H-40, H-2–H-6), 4.12 (s, 2H,
Ph–CH2–), 3.88 (s, 2H, –CH2–Ph), 3.27 (bs, 1H, –NH–). 13C NMR
(75.49 MHz, CDCl3) d: 138.96 (C-20), 134.72 (C-10), 133.46 (C-50),
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118.26, 116.28 (C-30, C-50), 52.15 (Ph–CH2–), 30.59 (C-1), 6.01
(C-2, C-3).
Preparation of 3-alkyl- and 3-aryl-3,4-dihydroquinazolin-
2(1H)-ones 11a–i
Method A: A solution of triphosgene (0.624mmol) in dichloro-
methane (3 mL) was added dropwise to a solution of the
diamines 15a and 15c (1.248mmol) and Et3N (4.37 mmol) in
DCM (10 mL) at 0°C. The reaction mixture was allowed to
warm to room temperature and was stirred for 1 h and then
washed with H2O (30 mL), NaHCO3 saturated solution (30 mL),
and brine (30 mL). The organic phase was separated and
dried over Na2SO4. Filtration and evaporation of the solvent
gave the crude mixture which was purified by flash chroma-
tography (AcOEt/hexane 1:3) to yield the product.
N-(2-Aminobenzyl)cyclobutylamine (15f)
Yellow solid, yield 74 mg (80%). m.p.: 83–86°C. 1H NMR
(300.20 MHz, CDCl3) d: 7.06–6.97 (m, 2H, H-40, H-60), 6.65–6.58
(m, 2H, H-30, H-50), 4.16 (bs, 3H, –NH2, –NH–), 3.70 (s, 2H,
Ph–CH2–), 3.29–3.24 (m, 1H, H-1), 2.17–2.08, 1.86–1.57 (2m, 6H,
13
H-2, H-3, H-4). C NMR (75.49 MHz, CDCl3) d: 146.79 (C-20),
131.00, 129.74 (C-40, C-60), 120.97 (C-10), 118.11, 116.54 (C-30,
C-50), 52.47 (C-1), 48.07 (Ph–CH2–), 30.51, 29.50 (C-2, C-4), 15.75
(C-3).
Method B: To a stirred solution of the diamines 15b–i
(1.15 mmol) in 7.50 mL of THF was added CDI (1.43 mmol) and
the solution was stirred at room temperature for 3 h. After
18 h of reflux, the solution was cooled and the solvent was
evaporated. The crude was solved in DCM, washed with
water, and dried over Na2SO4. Filtration and evaporation of
the solvent gave the crude mixture which was purified by
flash chromatography (ethyl acetate/hexane 1:3) to yield
the product.
N-(2-Aminobenzyl)cyclopentylamine (15g)
Yellow oil, yield 70 mg (70%). 1H NMR (400.17 MHz, CDCl3)
d: 7.11–7.04 (m, 2H, H-40, H-60), 6.71–6.65 (m, 2H, H-30, H-50),
3.79 (s, 2H, Ph–CH2–), 3.14–3.11 (m, 1H, H-1), 1.88–1.38 (4m,
13
8H, H-2–H-5). C NMR (125.69 MHz, CDCl3) d: 146.90 (C-20),
129.69, 128.25 (C-40, C-60), 124.52 (C-10), 117.72, 115.70 (C-30,
C-50), 59.64 (C-1), 51.77 (Ph–CH2–), 33.20, 33.07 (C-2, C-5),
24.07, 23.99 (C-3, C-4).
N-(2-Aminobenzyl)tert-butylamine (15h)
3,4-Dihydro-3-methylquinazolin-2(1H)-one (11a) [43]
White solid. Method A: Yield 44 mg (44%). m.p.: 148–152°C.
1H NMR (400.17 MHz, CDCl3) d: 8.22 (bs, 1H, H-1), 7.17–7.13 (m,
1H, H-7), 7.01 (d, 1H, H-5, J ¼ 7.5 Hz), 6.93–6.89 (m, 1H, H-6),
6.75 (d, 1H, H-8, J ¼ 7.9 Hz), 4.44 (s, 2H, H-4), 3.04 (s, 3H, –CH3).
13C NMR (125.69 MHz, CDCl3) d: 154.82 (C-2), 137.19 (C-8a),
128.21 (C-7), 125.34 (C-5), 121.74 (C-6), 117.36 (C-4a), 113.85
(C-8), 50.92 (C-4), 34.56 (–CH3). MS (LSIMS): m/z 185.7904
[MþNa]þ, calcd. mass for C9H10N2ONa 185.7901.
Orange oil, yield 65 mg (70%). 1H NMR (400.17 MHz, CDCl3)
d: 7.12–7.07 (m, 2H, H-40, H-60), 6.74–6.67 (m, 2H, H-30, H-50),
3.81 (s, 2H, Ph–CH2–), 3.20 (bs, 3H, –NH2, –NH–), 1.22 (s, 9H,
C(CH3)3). 13C NMR (125.69 MHz, CDCl3) d: 146.89 (C-20), 129.86,
128.39 (C-40, C-60), 124.27 (C-10), 117.94, 116.01 (C-30, C-50),
51.26 (C-1), 45.62 (Ph–CH2–), 28.56 (C(CH3)3).
N-(2-Aminobenzyl)benzylamine (15i) [42]
Yellow solid, yield 83 mg (75%). m.p.: 122–125°C. 1H NMR
(300.20 MHz, CDCl3) d: 7.36–7.28 (m, 5H, H-2–H-6), 7.21–7.04
(m, 2H, C-40, C-60), 6.70–6.68 (m, 2H, C-30, C-50), 3.89, 3.84 (2s,
4H, 2 ꢂ Ph–CH2–). 13C NMR (75.49 MHz, CDCl3) d: 146.79 (C-20),
138.25 (C-1), 131.00 (C-60), 128.41, 128.20 (C-2, C-3, C-5, C-6),
128.37 (C-40), 126.61 (C-4), 123.52 (C-10), 117.11, 116.54 (C-30,
C-50), 52.69 (–CH2Ph), 48.90 (Ph–CH2–).
3-Ethyl-3,4-dihydroquinazolin-2(1H)-one (11b)
White solid. Method B: Yield 166 mg (82%). m.p.: 136–139°C.
1H NMR (300.20 MHz, CDCl3) d: 8.77 (bs, 1H, H-1), 7.17–7.14 (m,
1H, H-7), 7.03 (d, 1H, H-5, J ¼ 7.4 Hz), 6.95–6.90 (m, 1H, H-6),
6.81 (d, 1H, H-8, J ¼ 7.9 Hz), 4.46 (s, 2H, H-4), 3.54 (q, 2H,
–CH2–CH3, J ¼ 7.2 Hz), 1.25 (t, 3H, –CH2–CH3, J ¼ 7.2 Hz).
13C NMR (75.49 MHz, CDCl3) d: 154.98 (C-2), 137.62 (C-8a),
128.11 (C-7), 125.54 (C-5), 121.68 (C-6), 117.51 (C-4a), 114.46
(C-8), 48.26 (C-4), 42.15 (–CH2–CH3), 12.33 (–CH2–CH3). MS
(LSIMS): m/z 199.0799 [MþNa]þ, calcd. mass for C10H12N2ONa
199.0802.
General procedure for the preparation of 2-((alkylamino)-
methy)-lbenzenamine derivatives 15a–i using a flow
hydrogenator
In a flask were dissolved 1 mmol of 2-nitrobenzaldehyde,
1.5 mmol of the corresponding primary amine, and 57 mL of
the acetic acid glacial (synthesis grade) in 20 mL of dry THF.
This mixture was passed through the flow hydrogenator
(model HC-2.SS) using full H2, 40°C, flow rate 0.5 mL/min and a
cartridge of 10% Pd/C. Pre-packed cartridges (CatCart1)
containing catalyst, are available from Thales Nanotechnol-
ogy. In this study, 30 ꢂ 4 mm cartridges (CatCart130) were
used throughout. Then, the solvent was evaporated and the
residue was dissolved in DCM and washed with NaHCO3
saturated solution and brine. The product was dried (Na2SO4)
and concentrated to afford a residue that was purified by
chromatography (AcOEt/Hexane 1:1 and then DCM/MeOH
9:1). The yields of these compounds are given in Table 1.
3,4-Dihydro-3-propylquinazolin-2(1H)-one (11c) [35]
White solid. Method A: Yield 48 mg (40%). Method B: yield
164 mg (75%). m.p.: 111–113°C. 1H NMR (300.20 MHz, CDCl3)
d: 7.84 (bs, 1H, H-1), 7.11–7.06 (m, 1H, H-7), 6.96 (d, 1H, H-5,
J ¼ 7.6 Hz), 6.88–6.83 (m, 1H, H-6), 6.66 (d, 1H, H-8, J ¼ 7.9 Hz),
4.38 (s, 2H, H-4), 3.35 (t, 2H, –CH2–CH2–CH3, J ¼ 7.1 Hz), 1.63–
1.56 (m, 2H, –CH2–CH2–CH3), 0.89 (t, 3H, –CH2–CH2–CH3,
J ¼ 7.4 Hz). 13C NMR (75.49 MHz, CDCl3) d: 154.56 (C-2), 137.45
(C-8a), 128.17 (C-7), 125.40 (C-5), 121.89 (C-6), 117.63 (C-4a),
114.05 (C-8), 49.57, 48.57 (C-4, –CH2–CH2–CH3), 20.11
(–CH2–CH2–CH3), 11.05 (–CH2–CH2–CH3). MS (LSIMS): m/z
213.1008 [MþNa]þ, calcd. mass for C11H14N2ONa 213.1004.
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3-Butyl-3,4-dihydroquinazolin-2(1H)-one (11d)
C(CH3)3). 13C NMR (125.69 MHz, CDCl3) d: 156.25 (C-2), 137.76
(C-8a), 128.07 (C-7), 125.16 (C-5), 121.24 (C-6), 119.39 (C-4a),
112.76 (C-8), 56.33 (C-1tert-but), 44.70 (C-4), 28.48 (C(CH3)3). MS
(LSIMS): m/z 205.1331 [MþH]þ, calcd. mass for C12H17N2O
205.1341.
White solid. Method B: Yield 174 mg (74%). m.p.: 158–161°C.
1H (300.20 MHz, CDCl3) d: 7.76 (bs, 1H, H-1), 7.21–7.16
(m, 1H, H-7), 7.06 (d, 1H, H-5, J ¼ 7.4 Hz), 6.98–6.93 (m, 1H,
H-6), 6.75 (d, 1H, H-8, J ¼ 7.9 Hz), 4.48 (s, 2H, H-4), 3.48
(t, 2H, –CH2–CH2–CH2–CH3, J ¼ 7.4 Hz), 1.70–1.60 (m, 2H,
–CH2–CH2–CH2–CH3), 1.47–1.37 (m, 2H, –CH2–CH2–CH2–CH3),
0.99 (t, 3H, –CH2–CH2–CH2–CH3, J ¼ 7.3 Hz). 13C NMR
(75.49 MHz, CDCl3) d: 154.74 (C-2), 137.14 (C-8a), 128.45
(C-7),125.64(C-5),122.15(C-6),117.78(C-4a),113.94(C-8),48.81,
47.21 (C-4, –CH2–CH2–CH2–CH3), 29.28 (–CH2–CH2–CH2–CH3),
20.27 (–CH2–CH2–CH2–CH3), 14.12 (–CH2–CH2–CH2–CH3). MS
(LSIMS): m/z 227.1157 [MþNa]þ, calcd. mass for C12H16N2ONa
227.1160.
3-Benzyl-3,4-dihydroquinazolin-2(1H)-one (11i) [35]
White solid. Method B: Yield 227 mg (83%). m.p.: 155–158°C.
1H NMR (300.20 MHz, CDCl3) d: 7.99 (sa, 1H, H-1), 7.97–7.29
(2m, 9H, H-5–H-8, H-20–H-60), 4.10 (s, 2H, H-4), 3.86 (s, 2H,
–CH2Ph). 13C NMR (75.49 MHz, CDCl3) d: 154.76 (C-2), 136.99,
136.82 (C-8a, C-10), 128.76, 128.26, 128.14, 127.64, 125.58,
121.92 (C-5–C-7, C-20–C-60), 117.51 (C-4a), 113.85 (C-8), 50.52
(C-4), 48.17 (–CH2Ph). MS (LSIMS): m/z 261.1005 [MþNa]þ,
calcd. mass for C15H14N2ONa 261.1004.
3-Cyclopropyl-3,4-dihydroquinazolin-2(1H)-one (11e)
Pallid brown solid. Method B: Yield 177 mg (79%). m.p.: 143–
146°C. 1H NMR (300.20 MHz, CDCl3) d: 8.04 (bs, 1H, H-1), 7.21–
7.16 (m, 1H, H-7), 7.07 (d, 1H, H-5, J ¼ 7.4 Hz), 6.98–6.93 (m, 1H,
H-6), 6.78 (d, 1H, H-8, J ¼ 7.9 Hz), 4.44 (s, 2H, H-4), 2.71–2.65
(m, 1H, H-1cycloprop.), 0.94–0.70 (m, 4H, H-2–H-3cycloprop.).
13C NMR (75.49 MHz, CDCl3) d: 156.61 (C-2), 136.94 (C-8a),
128.35 (C-7), 125.53 (C-5), 120.81 (C-6), 118.75 (C-4a), 113.94
(C-8), 49.75 (C-4), 29.57 (C-1cycloprop.), 7.68 (C-2–C-3cycloprop.).
MS (LSIMS): m/z 211.0806 [MþNa]þ, calcd. mass for
Preparation of 3-alkyl-3,4-dihydroquinazolin-2(1H)-thione
derivatives 12b–e and 12h
Method A: A solution of the diamine 15b (2.0 mmol) and
0.642 mL (4.61 mmol) of Et3N was dissolved in 5 mL of dry
Et2O and was cooled to ꢀ78°C; to this was added, dropwise, a
solution of 0.266 g (2.31 mmol) of thiophosgene solved in
5 mL of dry Et2O, over a period of 15 min. The heterogeneous
mixture was allowed to warm to room temperature and was
filtered, and the solid was purified by chromatography
(AcOEt/hexane 1:4) to yield the product.
C
11H12N2ONa 211.0802.
Method B: To a stirred solution of the diamines 15b–e and
15 h (4.58 mmol) in 30 mL of THF, was added TCDI (5.73 mmol)
and the solution was stirred at room temperature for 3 h.
After 18 h of reflux, the solution was cooled and the solvent
was evaporated. The crude was solved in DCM, washed with
water, and dried over Na2SO4. Filtration and evaporation of
the solvent gave the crude mixture which was purified by
chromatography (AcOEt/hexane 1:4) to yield the product.
3-Cyclobutyl-3,4-dihydroquinazolin-2(1H)-one (11f)
White solid. Method B: Yield 186 mg (80%). m.p.: 162–164°C.
1H NMR (300.20 MHz, CDCl3) d: 7.35 (sa, 1H, H-1), 7.11–7.08 (m,
1H, H-7), 7.03 (d, 1H, H-5, J ¼ 7.5 Hz), 6.92–6.90 (m, 1H, H-6),
6.65 (d, 1H, H-8, J ¼ 7.9 Hz), 4.79–4.76 (m, 1H, H-1cyclobut.), 4.37
(s, 2H, H-4), 2.21–2.10 (m, 4H, H-2, H-4cyclobut.), 1.68–1.61 (m,
2H, H-3cyclobut.). 13C NMR (75.49 MHz, CDCl3) d: 154.85 (C-2),
137.21 (C-8a), 128.39 (C-7), 125.90 (C-5), 121.90 (C-6), 117.82
(C-4a), 113.34 (C-8), 49.33 (C-1cyclobut.), 43.79 (C-4), 27.21 (C-2,
C-4cyclobut.), 14.82 (C-3cyclobut.). MS (LSIMS): m/z 225.1008
[MþNa]þ, calcd. mass for C12H14N2ONa 225.1004.
3-Ethyl-3,4-dihydroquinazolin-2(1H)-thione (12b) [40]
White solid. Method A: Yield 134 mg (35%). Method B: Yield
660 mg (75%). m.p.: 123–126°C. 1H NMR (300.20 MHz, CDCl3)
d: 8.82 (bs, 1H, H-1), 7.23–7.17 (m, 1H, H-7), 7.05–6.98 (m, 2H,
H-6, H-8), 6.82 (d, 1H, H-5, J ¼ 8.0 Hz), 4.56 (s, 2H, H-4), 4.03 (q,
2H, –CH2–CH3, J ¼ 7.2 Hz), 1.34 (t, 3H, –CH2–CH3, J ¼ 7.2 Hz).
13C NMR (75.49 MHz, CDCl3) d: 175.94 (C-2), 134.81 (C-8a),
128.94 (C-7), 125.64 (C-8), 123.67 (C-6), 117.46 (C-4a), 113.62
(C-5), 49.07 (C-4), 29.93 (–CH2–CH3), 11.42 (–CH2–CH3). MS
(LSIMS): m/z 193.0798 [MþH]þ, calcd. mass for C10H13N2S
193.0799.
3-Cyclopentyl-3,4-dihydroquinazolin-2(1H)-one (11g) [44]
Pallid yellow solid. Method B: Yield 192 mg (77%). m.p.: 157–
160°C. 1H NMR (300.20 MHz, CDCl3) d: 8.75 (sa, 1H, H-1), 7.06–
6.95 (m, 1H, H-7), 6.93 (d, 1H, H-5, J ¼ 7.5 Hz), 6.92–6.90 (m, 1H,
H-6), 6.83 (d, 1H, H-8, J ¼ 7.9 Hz), 5.02–4.91 (m, 1H, H-
1
cyclopent.), 4.35 (s, 2H, H-4), 1.90–0.77 (m, 8H, H-2–H-5cyclopent.).
13C NMR (75.49 MHz, CDCl3) d: 156.89 (C-2), 138.76 (C-8a),
129.25 (C-7), 126.57 (C-5), 122.83 (C-6), 119.43 (C-4a), 115.29
(C-8), 56.23 (C-1cyclopent.), 44.07 (C-4), 28.95 (C-2, C-5cyclopent.),
25.88 (C-3, C-4cyclopent.). MS (LSIMS): m/z 239.1155 [MþNa]þ,
calcd. mass for C13H16N2ONa 239.1160.
3,4-Dihydro-3-propylquinazolin-2(1H)-thione (12c) [40]
White solid. Method B: Yield 765 mg (81%). m.p.: 122–124°C.
1H NMR (300.20 MHz, CDCl3) d: 8.42 (bs, 1H, H-1), 7.21–7.18 (m,
1H, H-7), 7.03–7.01 (m, 2H, H-6, H-8), 6.76 (d, 1H, H-5,
J ¼ 7.9 Hz), 4.54 (s, 2H, H-4), 3.91 (t, 2H, –CH2–CH2–CH3,
J ¼ 7.6 Hz), 1.83–1.78 (m, 2H, –CH2–CH2–CH3), 1.00 (t, 3H,
–CH2–CH2–CH3, J ¼ 7.4 Hz). 13C NMR (75.49 MHz, CDCl3) d:
175.44 (C-2), 134.53 (C-8a), 128.72 (C-7), 125.38 (C-8), 123.45
(C-6), 117.24 (C-4a), 113.24 (C-5), 55.43 (–CH2–CH2–CH3), 49.56
3-Tert-butyl-3,4-dihydroquinazolin-2(1H)-one (11h)
White solid. Method B: Yield 162 mg (69%). m.p.: 148–150°C.
1H NMR (400.17 MHz, CDCl3) d: 8.44 (bs, 1H, H-1), 7.17–7.13 (m,
1H, H-7), 7.04 (d, 1H, H-5, J ¼ 7.4 Hz), 6.91–6.88 (m, 1H, H-6),
6.74 (d, 1H, H-8, J ¼ 7.8 Hz), 4.39 (s, 2H, H-4), 1.51 (s, 9H,
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Archiv der Pharmazie
(C-4), 19.52 (–CH2–CH2–CH3), 11.15 (–CH2–CH2–CH3). MS
(LSIMS): m/z 207.0950 [MþH]þ, calcd. mass for C11H15N2S
207.0956.
(C-5–C-8), 121.24 (C-4a), 54.29 (–OCH3), 52.61 (C-4), 35.49
(–CH3).
3,4-Dihydro-2-methoxy-3-propylquinazoline (16c)
3-Butyl-3,4-dihydroquinazolin-2(1H)-thione (12d)
White solid, yield 569 mg (70%). m.p.: 116–119°C. 1H NMR
(300.20 MHz, CDCl3) d: 7.09–6.83 (3m, 4H, H-5–H-8), 4.45 (s, 2H,
H-4), 3.86 (s, 3H, –OCH3), 3.17 (t, 2H, –CH2–CH2–CH3, J ¼ 7.3
Hz), 1.57–1.51 (m, 2H, –CH2–CH2–CH3), 0.85 (t, 3H,
–CH2–CH2–CH3, J ¼ 7.5 Hz). 13C NMR (75.49 MHz, CDCl3)
d: 150.86, 149.92 (C-2, C-8a), 128.38, 124.94, 122.80, 122.67
(C-5–C-8), 122.30 (C-4a), 54.34 (–OCH3), 50.30, 50.09 (C-4,
–CH2–CH2–CH3), 20.46 (–CH2–CH2–CH3), 11.44 (–CH2–CH2–CH3).
Yellow solid. Method B: Yield 807 mg (80%). m.p.: 125–127°C.
1H NMR (300.20 MHz, CDCl3) d: 9.05 (bs, 1H, H-1), 7.11–7.05 (m,
1H, H-7), 6.93–6.86 (m, 2H, H-6, H-8), 6.77 (d, 1H, H-5,
J ¼ 7.8 Hz), 4.43 (s, 2H, H-4), 3.85 (t, 2H, –CH2–CH2–CH2–CH3,
J ¼ 7.7 Hz), 1.70–1.60 (m, 2H, –CH2–CH2–CH2–CH3), 1.38–1.26
(m, 2H, –CH2–CH2–CH2–CH3), 0.89 (t, 3H, –CH2–CH2–CH2–CH3,
J ¼ 7.3 Hz). 13C NMR (75.49 MHz, CDCl3) d: 176.24 (C-2), 135.10
(C-8a), 128.88 (C-7), 125.54 (C-8), 123.10 (C-6), 117.51 (C-4a),
113.68 (C-5), 53.77 (–CH2–CH2–CH2–CH3), 49.60 (C-4), 28.36
(–CH2–CH2–CH2–CH3), 20.26 (–CH2–CH2–CH2–CH3), 14.28
(–CH2–CH2–CH2–CH3). MS (LSIMS): m/z 221.1098 [MþH]þ,
calcd. mass for C12H17N2S 221.1102.
3-Butyl-3,4-dihydro-2-methoxyquinazoline (16d)
White solid, yield 634 mg (73%). m.p.: 120–123°C. 1H NMR
(300.20 MHz, CDCl3) d: 7.18–6.92 (3m, 4H, H-5–H-8), 4.55 (s, 2H,
H-4), 3.93 (s, 3H, –OCH3), 3.30 (t, 2H, –CH2–CH2–CH2–CH3,
J ¼ 7.3 Hz), 1.62–1.57 (m, 2H, –CH2–CH2–CH2–CH3), 1.41–1.31
(m, 2H, –CH2–CH2–CH2–CH3), 0.97 (t, 3H, –CH2–CH2–CH2–CH3,
J ¼ 7.3 Hz). 13C NMR (75.49 MHz, CDCl3) d: 156.67 (C-2), 144.08
(C-8a), 128.36, 124.92, 122.79, 122.66 (C-5–C-8), 121.10 (C-4a),
54.33 (–OCH3), 50.27, 48.14 (C-4, –CH2–CH2–CH2–CH3),
29.60 (–CH2–CH2–CH2–CH3), 20.20 (–CH2–CH2–CH2–CH3),
14.05 (–CH2–CH2–CH2–CH3).
3-Cyclopropyl-3,4-dihydroquinazolin-2(1H)-thione (12e)
Yellow solid. Method B: Yield 786 mg (84%). m.p.: 156–158°C.
1H NMR (300.20 MHz, CDCl3) d: 9.04 (bs, 1H, H-1), 7.19–7.16 (m,
1H, H-7), 7.03–6.97 (m, 2H, H-6, H-8), 6.83 (d, 1H, H-5,
J ¼ 7.8 Hz), 4.47 (s, 2H, H-4), 3.04–3.00 (m, 1H, H-1cycloprop.),
1.04–0.83 (2m, 4H, H-2, H-3cycloprop.). 13C NMR (75.49 MHz,
CDCl3) d: 179.85 (C-2), 134.37 (C-8a), 128.63 (C-7), 125.26 (C-8),
123.47 (C-6), 118.36 (C-4a), 113.31 (C-5), 50.46 (C-4), 34.81 (C-
3-Cyclopropyl-3,4-dihydro-2-methoxyquinazoline (16e)
Pallid brown solid, yield 724 mg (90%). m.p.: 114–117°C.
1H NMR (300.20 MHz, CDCl3) d: 7.24–6.95 (3m, 4H, H-5–H-8),
4.45 (s, 2H, H-4), 3.86 (s, 3H, –OCH3), 2.51–2.47 (m, 1H, H-
1
cycloprop.), 9.61 (C-2, C-3cycloprop.). MS (LSIMS): m/z 205.0796
[MþH]þ, calcd. mass for C11H13N2S 205.0799.
3-Tert-butyl-3,4-dihydroquinazolin-2(1H)-thione (12h)
Yellow solid. Method B: Yield 962 mg (72%). m.p.: 163–165°C.
1H NMR (400.17 MHz, CDCl3) d: 8.70 (bs, 1H, H-1), 7.24–7.22 (m,
1H, H-7), 7.12–7.04 (m, 2H, H-6, H-8), 6.82 (d, 1H, H-8,
J ¼ 7.8 Hz), 4.48 (s, 2H, H-4), 1.82 (s, 9H, C(CH3)3). 13C NMR
(125.69 MHz, CDCl3) d: 179.23 (C-2), 135.70 (C-8a), 128.61 (C-7),
124.77 (C-8), 123.18 (C-6), 120.10 (C-4a), 112.39 (C-5), 60.96
(C-1tert-but), 46.13 (C-4), 29.10 (C(CH3)3). MS (LSIMS): m/z
221.1109 [MþH]þ, calcd. mass for C12H17N2S 221.1112.
1
cycloprop.), 0.89–0.61 (2m, 4H, H-2, H-3cycloprop.). 13C NMR
(75.49 MHz, CDCl3) d: 158.51 (C-2), 143.78 (C-8a), 127.51,
124.59, 123.50, 122.93 (C-5–C-8), 122.85 (C-4a), 54.58 (–OCH3),
50.66 (C-4), 29.56 (H-1cycloprop.), 8.07 (H-2, H-3cycloprop.).
Preparation of 3-alkyl-3,4-dihydroquinazolin-2(1H)-imine
derivatives 13a and 13c–e
A
mixture of the dihydroquinazolines 16a and 16c–e
(1.43 mmol) and NH4Cl (0.16 g, 1.57 mmol) was solved in
methanol (20 mL) and was stirred under reflux for 3.5 h. After
this time, the solvent was evaporated. The crude mixture was
purified by chromatography (AcOEt/hexane 1:1) to yield the
product.
Preparation of 3-alkyl-3,4-dihydro-2-methoxyquinazoline
derivatives 16a and 16c–e
To a magnetically stirred slurry of trimethyloxonium tetra-
fluoroborate (4.77 mmol) in DCM (20 mL) under argon was
added the 3,4-dihydroquinazolin-2(1H)-ones 9a and 9c–e
(3.98 mmol). This mixture was stirred at room temperature for
12 h and then it was diluted with 15 mL of DCM, washed with
40 mL of NaHCO3 saturated solution, and dried (Na2SO4).
Filtration and evaporation of the solvent gave the crude
mixture which was purified by chromatography (AcOEt/
hexane 1:6) to yield the product.
3,4-Dihydro-3-methylquinazolin-2(1H)-imine (13a)
White solid, yield 150 mg (65%). m.p.: 193–195°C. 1H NMR
–
(300.20 MHz, CDCl3) d: 7.84 (bs, 2H, H-1, NH), 7.22–7.16 (m,
–
1H, H-7), 7.06 (d, 1H, H-5, J ¼ 7.5 Hz), 6.98–6.93 (m, 1H, H-6),
6.76 (d, 1H, H-8, J ¼ 7.9 Hz), 4.49 (s, 2H, H-4), 3.07 (s, 3H, ꢀCH3).
13C NMR (75.49 MHz, CDCl3) d: 137.25 (C-2), 136.35 (C-8a),
128.78 (C-7), 125.62 (C-5), 122.09 (C-6), 117.59 (C-4a), 114.00
(C-8), 51.13 (C-4), 34.80 (–CH3). MS (LSIMS): m/z 162.1097
[MþH]þ, calcd. mass for C9H12N3 162.1001.
3,4-Dihydro-2-methoxy-3-methylquinazoline (16a)
White oil, yield 583 mg (83%). 1H NMR (300.20 MHz, CDCl3)
d: 7.19–6.92 (3m, 4H, H-5–H-8), 4.48 (s, 2H, H-4), 3.92 (s, 3H,
–OCH3), 2.92 (s, 3H, –CH3). 13C NMR (75.49 MHz, CDCl3)
d: 156.97 (C-2), 144.29 (C-8a), 128.41, 124.91, 123.01, 122.69
3,4-Dihydro-3-propylquinazolin-2(1H)-imine (13c)
White solid, yield 168 mg (62%). m.p.: 194–197°C. 1H NMR
(300.20 MHz, CDCl3) d: 7.09–7.06 (m, 1H, H-7), 6.96 (d, 1H, H-5,
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Synthetic Quinazoline Derivatives as NOS Inhibitors
Archiv der Pharmazie
J ¼ 7.5 Hz), 6.88–6.83 (m, 1H, H-6), 6.59 (d, 1H, H-8, J ¼ 7.9 Hz),
4.37 (s, 2H, H-4), 3.33 (t, 2H, –CH2–CH2–CH3, J ¼ 7.0 Hz), 1.66–
1.55 (m, 2H, –CH2–CH2–CH3), 0.88 (t, 3H, –CH2–CH2–CH3,
J ¼ 7.0 Hz). 13C NMR (75.49 MHz, CDCl3) d: 137.25 (C-2), 136.20
(C-8a), 128.44, 125.71, 122.09 (C-5–C-7), 117.93 (C-4a), 113.66
N-(2-Aminobenzyl)acetamide (15j) [45]
Following the general method described for the compounds
15a–i it was obtained a residue which was purified by
chromatography (AcOEt/hexane 1:1) to yield the product.
White solid, yield 52 mg (60%). m.p.: 114–116°C. 1H NMR
(300.20 MHz, CDCl3) 7.16–7.04 (m, 2H, H-40, H-60), 6.73–6.68
(m, 2H, H-30, H-50), 5.92 (bs, 1H, –NH–), 4.38 (d, 2H, –CH2–,
J ¼ 6.2 Hz), 4.23 (bs, 2H, –NH2), 2.01 (s, 3H, H-2). 13C NMR
(75.49 MHz, CDCl3) d: 170.77 (C-1), 145.84 (C-20), 130.82, 129.54
(C-40, C-60), 122.13 (C-10), 117.98, 115.97 (C-30, C-50), 41.14
(–CH2–); 23.42 (C-2).
(C-8),
48.83
(C-4),
29.95
(–CH2–CH2–CH3),
20.44
(–CH2–CH2–CH3), 11.46 (–CH2–CH2–CH3). MS (LSIMS): m/z
190.1297 [MþH]þ, calcd. mass for C11H16N3 190.1302.
3-Butyl-3,4-dihydroquinazolin-2(1H)-imine (13d)
White solid, yield 198 mg (68%). m.p.: 194–197°C. 1H NMR
–
(400.17 MHz, CDCl ) d: 8.52 (s, 2H, H-1, NH), 7.15–7.12 (m, 1H,
–
3
H-7), 7.04(d, 1H,H-5, J¼ 7.4 Hz), 6.91–6.88(m, 1H, H-6), 6.76(d, 1H,
H-8, J ¼ 7.9 Hz), 4.43(s, 2H, H-4), 3.45(t, 2H, –CH2–CH2–CH2–CH3,
J ¼ 7.4 Hz), 1.65–1.59 (m, 2H, –CH2–CH2–CH2–CH3), 1.42–1.35
(m, 2H, –CH2–CH2–CH2–CH3), 0.96 (t, 3H, –CH2–CH2–CH2–CH3,
J ¼ 7.4 Hz). 13C NMR (125.69 MHz, CDCl3) d: 154.83 (C-2),
137.36 (C-8a), 128.05 (C-7), 125.24 (C-5), 121.51 (C-6), 117.52
(C-4a), 113.75 (C-8), 48.55 (C-4), 46.77 (–CH2–CH2–CH2–CH3),
29.06 (–CH2–CH2–CH2–CH3), 20.02 (–CH2–CH2–CH2–CH3), 13.88
(–CH2–CH2–CH2–CH3). MS (LSIMS): m/z 204.1502 [MþH]þ, calcd.
mass for C12H18N3 204.1500.
3-Acetyl-3,4-dihydroquinazolin-2(1H)-one (11j)
Following method B for the synthesis of compounds 11a–i,
and using the 2-aminobenzylacetamide 15j (0.204 g,
1.242 mmol), it was obtained a residue which was purified
by chromatography (AcOEt/hexane 1:4) to yield the product.
White solid, yield 178 mg (75%). m.p.: 150–153°C. 1H NMR
(300.20 MHz, CDCl3) d: 8.41 (bs, 1H, H-1), 7.30–7.21 (m, 2H, H-5,
H-7), 7.10–7.05 (m, 1H, H-6), 6.87 (d, 1H, H-8, J ¼ 7.8 Hz), 4.94
(s, 2H, H-4), 2.65 (s, 3H, –CH3). 13C NMR (75.49 MHz, CDCl3) d:
172.67 (–COCH3), 153.72 (C-2), 135.77 (C-8a), 128.85, 126.27,
123.66 (C-5–C-7), 119.82 (C-4a), 114.20 (C-8), 44.16 (C-4), 26.84
(–CH3). MS (LSIMS): m/z 213.0600 [MþNa]þ, calcd. mass for
3-Cyclopropyl-3,4-dihydroquinazolin-2(1H)-imine (13e)
Pallid brown solid, yield 174 mg (65%). m.p.: 194–197°C.
1H NMR (300.20 MHz, CDCl3) d: 7.18–7.13 (m, 1H, H-7), 7.05 (d,
1H, H-5, J ¼ 7.4 Hz), 6.95–6.91 (m, 1H, H-6), 6.68 (d, 1H, H-8,
J ¼ 7.6 Hz), 4.41 (s, 2H, H-4); 2.67–2.60 (m, 1H, H-1cycloprop.),
0.90–0.66 (2m, 4H, H-2, H-3cycloprop.). 13C NMR (75.49 MHz,
CDCl3) d: 156.26 (C-2), 136.23 (C-8a), 128.16 (C-7), 125.32 (C-5),
121.88 (C-6), 118.53 (C-4a), 113.56 (C-8), 49.49 (C-4), 29.30
(C-1cycloprop.), 7.48 (C-2, C-3cycloprop.). MS (LSIMS): m/z 188.1185
[MþH]þ, calcd. mass for C11H14N3 188.1188.
C
10H10N2O2Na 213.0603.
3-Acetyl-3,4-dihydroquinazolin-2(1H)-thione (12j)
Following method B for the synthesis of compounds 12b–e
and 12h, and using the 2-aminobenzylacetamide 15j (0.328 g,
2 mmol), it was obtained a crude product which was purified
by chromatography (AcOEt/hexane 1:4) to yield the product.
White solid, yield 289 mg (70%). m.p.: 123–125°C. 1H NMR
(400.17 MHz, CDCl3) d: 7.42–7.26 (2m, 4H, H-5–H-8), 6.06 (bs,
3H, H-1), 4.50 (s, 2H, H-4), 2.07 (s, 3H, –CH3). 13C NMR
(125.69 MHz, CDCl3) d: 175.23 (C-2), 172.81 (–COCH3), 136.69
(C-8a), 132.34, 131.39, 130.25, 129.49 (C-5–C-8), 125.51 (C-4a),
43.00 (C-4), 25.90 (–CH3). MS (LSIMS): m/z 229.0402 [MþNa]þ,
calcd. mass for C10H10N2OSNa 229.0411.
Preparation of 3-acetyl-3,4-dihydroquinazolin-2(1H)-one
11j, and 3-acetyl-3,4-dihydroquinazolin-2(1H)-thione 12j
N-(2-Nitrobenzyl)acetamide (14j) [38]
To a solution of (2-nitrophenyl)methanamine hydrochloride
(1.68 g, 8.91 mmol) in water (20 mL) was added
Na2CO3.10 H2O (5.94 g, 20.76 mmol), and was stirred while
10 min. The solution was washed with DCM, brine, and dried.
The crude (2-nitrophenyl)methanamine was solved in 20 mL
DCM, and added Et3N (2.05 mL, 11.59 mmol), and acetic
anhydride (1.40 mL, 11.59 mmol). The reaction mixture was
stirred to room temperature for 3 h, washed with 2 N HCl, 2 M
NaOH, H2O, and brine. The organic phase was dried (Na2SO4)
and filtered. Evaporation of the solvent rendered a residue
that was purified by flash chromatography (AcOEt/hexane
4:1) to yield the product. White solid, yield 1214 mg
(84%). m.p.: 99–101°C. 1H NMR (400.17 MHz, CDCl3) d: 8.05
(d, 1H, H-30, J ¼ 8.3 Hz); 7.67–7.59 (m, 2H, H-40, H-50); 7.47–7.43
(m, 1H, H-60); 6.43 (bs, 1H, –NH–); 4.65 (d, 2H, –CH2–, J ¼ 3.9 Hz);
1.99 (s, 3H, H-2). 13C NMR (125.69 MHz, CDCl3) d: 172.79 (C-1);
136.72 (C-20), 136.31 (C-10); 135.22 (C-50, C-60); 131.38, 127.67
(C-30, C-40); 43.88 (–CH2–); 25.88 (C-2).
In vitro nNOS and iNOS activities determination
L-Arginine, L-citrulline, N-(2-hydroxymethyl)piperazine-N’-(2-
ethanesulfonic acid) (HEPES), DL-dithiothreitol (DTT), hypo-
xantine-9-b-D-ribofuranosid (inosine), ethylene glycol-bis-(2-
aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), bovine
serum albumin (BSA), Dowex-50 W (50 ꢂ 8–200), FAD, NADPH,
and 5,6,7,8-tetrahydro-L-biopterin dihydrocloride (H4-bio-
ꢀ
pterin) were obtained from Sigma–Aldrich Quımica (Spain).
L-[3H]-Arginine (54 Ci/mmol) was obtained from Amersham
(Amersham Biosciences, Spain). Tris-(hydroxymethyl)-amino-
methane (Tris-HCl) and calcium chloride were obtained from
Merck (Spain). Calmodulin from bovine brain, and recombi-
nant iNOS and nNOS were obtained from Alexis Biochemicals
(Enzo Life Sciences, Grupo Taper, Seville, Spain).
The nNOS activity was measured by the Bredt and
Snyder [46] method, monitoring the conversion of L-[3H]-
arginine to L-[3H]-citrulline. The final incubation volume was
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100 mL and consisted of 10 mL of an aliquot of recombinant
nNOS added to a buffer with a final concentration of 25 mM
Tris-HCl, 1 mM DTT, 4 mM H4-biopterin, 10 mM FAD, 0.5 mM
inosine, 0.5 mg/mL BSA, 0.1 mM CaCl2, 10 mM L-arginine, and
50 nM L-[3H]-arginine, at pH 7.6. The reaction was started by
the addition of 10 mL of NADPH (0.75 mM final) and 10 mL
of each 3,4-dihydroquinazolin-2(1H)-one, -thione or -imine
derivative in ethanol 10% to give a final concentration of
1 mM. The tubes were vortexed 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 mL
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 packed with Dowex-50 W ion-exchange resin
(Naþ 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 to less than 1% of
the radioactivity added. The nNOS activity was expressed as
picomoles of L-[3H]-citrulline produced (/mg of protein/min).
For iNOS activity determination, the procedure was
essentially the same as for nNOS, excepting that an aliquot
of 10 mL recombinant iNOS was used instead of nNOS, and the
mixture was incubated in the absence of calmodulin.
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This work was supported by grants from the Vicerrectorado
A. Ammazzalorso, B. De Filippis, M. Di Mateo, S. Di
Silvestre, M. Fantacuzzi, L. Giampietro, M. A. Potenza,
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Granada (GREIB.PYR_2010_03 and GENIL/GREIB 2011).
The authors have declared no conflict of interest.
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