N, N ′-Disubstituted thiourea and urea derivatives: Design, synthesis, docking studies and biological evaluation against nitric oxide synthase

Article abstract of DOI:10.1039/c5md00477b

The synthesis and biological evaluation of new types of N,N′-disubstituted thiourea and urea derivatives as inhibitors of both neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) are described. These compounds have been designed by reduction of the carbonyl group in the thiourea and urea kynurenamine derivatives 3 previously synthesized by our research group. The synthetic route performed to this new family also allows us to obtain the molecules 3 with less synthetic steps and higher global yield. Regarding the biological results, in general, the new derivatives 4a-q inhibit the neuronal NOS isoform better than the inducible one. Furthermore, thioureas exhibit higher inhibition than ureas for both isoenzymes. Among all the tested compounds, 4g shows significant nNOS (80.6%) and iNOS (76.6%) inhibition values without inhibiting eNOS. This molecule could be an interesting starting point for the design of new inhibitors with application in neurological disorders where both isoenzymes are implicated such as Parkinson's disease.

Full text of DOI:10.1039/c5md00477b

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Page 1 of 21
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DOI: 10.1039/C5MD00477B
N,N’-disubstituted thiourea and urea derivatives: Design, synthesis, docking
studies and biological evaluation against nitric oxide synthase†
Mariem Chayaha, M. Encarnación Camachoa*, M. Dora Carrióna*,
Miguel A. Galloa, Miguel Romerob, Juan Duarteb.
aDepartamento de Química Farmacéutica y Orgánica, Facultad de Farmacia,
Universidad de Granada (Spain).
bDepartamento de Farmacología, Facultad de Farmacia, Universidad de Granada
(Spain).
*Correspondig authors: Dr. M. Encarnación Camacho. Tel.: +34ꢀ958ꢀ243844; Eꢀmail:
ecamacho@ugr.es and Dr. M. Dora Carrión. Tel.: +34ꢀ958ꢀ240728; Eꢀmail:
dcarrion@ugr.es
Abstract
The synthesis and biological evaluation of a new type of N,N’ꢀdisubstituted thiourea
and urea derivatives as inhibitors of both neuronal and inducible nitric oxide synthase
(nNOS and iNOS) are described. These compounds have been designed by reduction
of the carbonyl group in the thiourea and urea kynurenamine derivatives 3 previously
synthesized by our research group. The synthetic route performed to this new family
also allows us to obtain the molecules 3 with less synthetic steps and higher global
yield. Regarding the biological results, in general, the new derivatives 4a-q inhibit
better the neuronal NOS isoform than the inducible one. Furthermore, thioureas exhibit
higher inhibition than ureas for both isoenzymes. Among all the tested compounds, 4g
shows significant nNOS (80.6%) and iNOS (76.6%) inhibition values without inhibiting
eNOS. This molecule could be an interesting starting point for the design of new
inhibitors with application in neurological disorders where both isoenzymes are
implicated such as Parkinson disease.
Introduction
Nitric oxide synthase (NOS) is a family of isoenzymes that convert Lꢀarginine to Lꢀ
citrulline with nitric oxide (NO) release. There are three human NOS isoforms:
endothelial NOS (eNOS) which regulates blood pressure and flow, inducible NOS
(iNOS) involved in immune response, and neuronal NOS (nNOS) essential for
neurotransmission.1 Nonetheless, several studies demonstrated the implication of NOS
in many neurodegenerative,2ꢀ7 chronic inflammatory8,9 and cardiovascular10,11 diseases.
Indeed, NO overproduction, by nNOS or iNOS, leads to cellular and tissue damage
through nitrosative and oxidative stress.12ꢀ16 The underproduction of NO by eNOS
affects the vascular tone and blood flow controls and causes hypertension. Therefore
the inhibition of nNOS and iNOS but not eNOS is a viable therapeutic strategy to treat
and prevent disorders and pathologies as previously mentioned.
A comparison of the NOSs structures reveals a huge similarity especially in their
substrate binding sites.17 This fact represents a real challenge in designing selective
NOS inhibitors.
† The authors declare no compeꢀng interests.

MedChemComm
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In previous efforts to find selective and potent i/nNOS inhibitors, our research group
has synthesized and published several families of compounds. Figure 1 shows some of
them with analogous structures: the kynurenine 1,18 kynurenamine 2,19 and
kynurenamineꢀurea and thiourea 320 derivatives. These last ones have mostly better
inhibition results versus iNOS. Docking and molecular dynamic studies demonstrated
the urea group implication especially in the selectivity process. Also both, the aromatic
ring and the amine group, interact with the enzyme through πꢀcation interaction and
hydrogen bonds, respectively. However, any clear role was observed for the carbonyl
group. Therefore we decided to act at this point, changing this hydrogen bond acceptor
residue by another donor such as hydroxyl.
This way, herein we present a new series of compounds 4a-q where the carbonyl
group, present in the three families of the above derivatives, has been replaced by a
hydroxyl one, waiting increased interaction with the enzyme and improved inhibition. A
new synthetic pathway was developed to obtain this series of molecules. Subsequent
biological assays and docking studies were performed in this new family of compounds
to evaluate the results against NOSs’ isoforms.
In addition, compounds 2a-l previously published and evaluated as nNOS inhibitors19
were screened against iNOS and the inhibition data are presented and discussed in
this work. N,N’ꢀdisubstituted thioureas and ureas 4a-q were designed from the
kynurenamines with general skeleton 2, introducing two structural modifications:
replacement of the terminal acyl group by a thiourea or urea one, isosteric to the final
guanidine moiety of the NOS substrate (LꢀArg); and substitution of the carbonyl group
with a hydroxyl residue.
O
CO2H O
O
O
R1
R1
N
R2
R2
N
H
H
NH2
NH2
1
2
R1 = OMe, Cl
R2 = alkyl, cycloalkyl, phenyl
R1 = OMe
R2 = alkyl
OH
X
O
X
R1
R2
R1
R2
N
N
N
N
H
H
H
H
NH2
NH2
3
4a-q
R1 = H, OMe, Cl
R1 = H, OMe, Cl
R2 = alkyl
R2 = alkyl, phenyl
X = O, S
X = O, S
Figure 1. Some kynurenamine derivatives as NOS inhibitors synthesized by our
research group.
Results and discussion
Chemistry
† The authors declare no compeꢀng interests.

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The general synthetic pathway of all the final compounds 4a-q is represented in
Scheme 1. 5ꢀMethoxyꢀ2ꢀnitrobenzaldehyde 5b (synthesized from 5ꢀhydroxyꢀ2ꢀ
nitrobenzaldehyde by reaction with MeI in the presence of K2CO3/THF),21 the
commercially available 2ꢀnitrobenzaldehyde 5a and 5ꢀchloroꢀ2ꢀnitrobenzaldehyde 5c
were transformed into the βꢀhydroxynitriles 6a-c by treatment with BuLi and acetonitrile
in dry THF22 (70ꢀ90% yield). The nitrile group of these last intermediates was reduced
selectively with borane in THF23 to give the 3ꢀaminoꢀ1ꢀ(2ꢀnitrophenyl)propanꢀ1ꢀol
derivatives 7a-c. Nucleophilic addition of either alkyl or phenyl isothiocyanate or
isocyanate in situ, using microwave (MW) technique, gave the intermediates 8a-q with
good yields (70ꢀ85%). The application of the MW technique shortened the reaction time
from 18 h20,24 to 20 min, respect to the previously described synthesis. Finally,
reduction of the phenyl nitro group, with Pd/C in the flow hydrogenator, resulted in the
final derivatives 4a-q (75ꢀ91% yield).
On the other hand, this new synthetic route is useful to obtain urea and thiourea
kynurenamines 3. Thereby, as an example, we carried out the oxidation of the hydroxyl
group to a carbonyl one in the derivative 8o (R1 = Cl, R2 = Et, X = O), using the Jones
reagent followed by the nitro reduction to amino group20 (Scheme 1). This alternative
synthesis improves the previously described one20 by shortening the synthetic route
(from 8 to 5 steps) and doubling the global yield.
OH
OH
R1
CHO
NO2
R1
R1
b)
a)
N
NH2
NO2
NO2
5a-c
7a-c
6a-c
c)
OH
X
OH
X
R1
R2
R1
R2
d)
N
N
N
H
N
H
H
H
NH2
NO2
8a-q
4a-q
e) f)
R1 = H, OMe, Cl
O
X
R1
R2
R2 = Me, Et, Pr, Ph
X = O, S
N
H
N
H
NH2
3
Scheme 1. Synthesis of N,N’ꢀdisubstituted thiourea and urea derivatives 4a-q. a)
CH3CN, BuLi, THF, ꢀ78ºC, then RT; b) BH3ꢀTHF, 0ºC, then 4h RT; c) XCNR2, CH2Cl2,
20 min (MW); d) 10% Pd/C, MeOH (flow hydrogenation), 60ºC, 60 bar; e) CrO3,
CH3COCH3, H2SO4, 10 min; f) Fe/FeSO4, H2O, 95ºC, 3h.
Results and discussion
iNOS and nNOS inhibition
† The authors declare no compeꢀng interests.

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Compounds 4a-q were evaluated in vitro as inhibitors of iNOS and nNOS using
recombinant isoenzymes. The assays were made at 1mM concentration of each
compound in order to identify the more active and selective derivatives. Besides, IC50
values were measured for the most interesting compounds.
Table 1 illustrates the inhibition percentages versus iNOS and nNOS (the
kynurenamine derivatives 3a-b, previously described,20 were introduced as reference).
Table 1. In vitro iNOS and nNOS inhibition (%) observed in the
presence of 1 mM concentration of compounds 3a-b and 4a-q.
Compound
R1
R2
X
% iNOS
% nNOS
inhibitiona
inhibitiona
3ab
3bb
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
OMe Me
S
O
S
S
S
O
O
O
S
S
O
O
O
S
S
S
O
O
O
78.20 ± 2.43
78.63 ± 1.34
20.74 ± 1.15
19.36 ± 3.33
31.10 ± 0.55
18.34 ± 0.43
19.04 ± 1.45
27.86 ± 1.15
76.55 ± 0.33
25.39 ± 1.71
26.64 ± 2.05
14.66 ± 0.19
12.33 ± 1.30
22.13 ± 0.28
60.42 ± 2.31
54.98 ± 3.21
33.85 ± 1.86
19.61 ± 3.04
3.11 ± 2.36
46.04 ± 2.85
9.86 ± 3.17
70.17 ± 0.77
44.84 ± 0.02
51.74 ± 1.35
7.81 ± 3.06
7.00 ± 0.32
17.69 ± 1.32
80.55 ± 2.29
6.02 ± 1.61
43.02 ± 0.48
15.81 ± 1.03
12.10 ± 1.08
70.53 ± 4.60
70.61 ± 3.54
32.40 ± 2.24
15.22 ± 2.33
23.81 ± 1.34
38.62 ± 1.63
Cl
H
H
H
H
H
H
Et
Me
Et
Pr
Et
Pr
Ph
OMe Me
OMe Pr
OMe Et
OMe Pr
OMe Ph
Cl
Cl
Cl
Cl
Cl
Cl
Me
Et
Pr
Et
Pr
Ph
4m
4n
4o
4p
4q
aValues are 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. b3a-b were used as reference.20
In general, compounds 4a-q show better values of inhibition versus nNOS than iNOS,
since five compounds exhibit good nNOS percentage inhibition (4g, 80.6%; 4m, 70.6%;
4l, 70.5%, 4a, 70.2% and 4c, 51.7%) and only three compounds show good inhibitory
activity versus iNOS (4g, 76.6%, 4m, 60.4% and 4n, 55.0%). Regarding the R1 radical,
no clear structureꢀactivity relationship can be concluded. Independently of the
substituent nature ((R1 = H), electronꢀdonating (R1 = OCH3) or electron withdrawing (R1
= Cl)) different inhibition levels can be observed. It seems that the inhibitory activity
depends more on X and R2. Thus, derivatives with a thiourea residue (X = S) show
better inhibition data for both isoenzymes than those bearing urea (X = O). In addition,
compounds with methyl substituent in R2 produce higher inhibition for iNOS as 4g and
for nNOS such as 4a, 4g and 4l. It is noteworthy that compound 4m shows good
inhibition having a R2 = Et. Finally, 4g stands out as the best inhibitor of both isoforms.
Furthermore, Table 2 shows the iNOS inhibition values of kynurenamines 2a-l
previously synthesized (not published before), as well as their nNOS values already
described19 in order to compare them with the new N,N’ꢀdisubstituted thioureas and
ureas 4a-q.
† The authors declare no compeꢀng interests.

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The best nNOS inhibitors of derivatives 2a-l were 2a (R1 = OMe, R2 = Me, 65.4%), and
2b (R1 = OMe and R2 = Et, 50.9%); however, no compound with relevant iNOS
inhibition value was found, so they can be considered as selective inhibitors of nNOS
versus iNOS. Kynurenamine 2a, the N1ꢀmethylꢀ5ꢀmethoxykynurenamine, is the main
brain metabolite of melatonin hormone in mammalians, that intermediates some of the
actions of this indolamine. Both of them inhibited nNOS activity in vitro in a doseꢀ
related manner.25
If we compare the derivatives 4a-q with the kynurenamines 2a-l, we can see that both
series inhibit better nNOS than iNOS (compounds 4a-q having greater potency), unlike
the corresponding molecules 3 showing better iNOS inhibition values. Besides, the
best inhibitors of the two first families carry a methoxy group in R1 and a methyl in R2
(urea 4g and kynurenamine 2a, respectively), while the ureaꢀkynurenamine 3b which
has a chlorine in R1 and an ethyl in R2 is the best inhibitor of this family and the most
selective one. Finally, 4g has the highest percentage of inhibition of all the tested
derivatives with similar structures.
Table 2. In vitro iNOS and nNOS inhibition (%) observed in the
presence of 1 mM concentration of compounds 2a-l.
Compound
R1
R2
% iNOS
% nNOS
inhibitiona
inhibitionb
2a
2b
2c
2d
2e
2f
2g
2h
2i
OMe
OMe
OMe
OMe
OMe cꢀC3H5
OMe cꢀC4H7
OMe cꢀC5H9
OMe cꢀC6H11
OMe
Cl
Cl
Cl
Me
Et
Pr
30.74 ± 2.92
0.24 ± 1.95
7.35 ± 4.48
14.11 ± 0.89
2.66 ± 1.4
0.01 ± 1.52
0.61 ± 2.49
9.24 ± 2.1
10.86 ± 3.73
19.70 ± 0.57
5.37 ± 2.59
26.60 ± 4.52
65.36 ± 5.6
50.87 ± 1.9
42.81 ± 1.9
39.68 ± 1.17
40.44 ± 1.84
33.75 ± 1.39
45.04 ± 1.97
48.24 ± 2.42
46.47 ± 2.36
17.70 ± 1.13
9.66 ± 4.17
7.20 ± 1.29
Bu
Ph
Me
cꢀC3H5
Ph
2j
2k
2l
aValues are 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. bSee Ref.19
Table 3 includes the IC50 data of the most interesting derivatives 4a-q. Compound 4g
stands out the best values of inhibition, 130 and 180 ꢁM, versus nNOS and iNOS,
respectively, confirming the potency of this molecule.
Table 3. IC50 values (mM) for the inhibition of nNOS and iNOS activity
by the compounds 4a, 4c, 4g, 4l, 4m and 4n.
a
IC50
4a
4c
4g
4l
4m
4n
nNOS
0.35
0.88
0.13
0.71
0.77
>1
iNOS
>1
>1
0.18
>1
0.80
0.86
aData were obtained by measuring percentage of inhibition with at
least five concentrations of inhibitor.
eNOS inhibition
† The authors declare no compeꢀng interests.

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We carried out the eNOS inhibitory activity of compound 4g, using HUVECs incubated
with 100 ꢂmol/L of 4g or vehicle and measuring the NO production stimulated by the
known eNOS activator A23187. This agent increased in a time dependent manner NO
production. No significant differences (p>0.05) were observed in A23187ꢀstimulated
NO production in the presence of 4g, showing that this compound did not inhibit eNOS
(Figure 2a). Moreover, the endotheliumꢀdependent relaxation to acetylcholine was not
affected by 4g (Figure 2b), confirming the absence of eNOS inhibition of this
compound. However, LꢀNAME suppressed this NOꢀdependent relaxant response.
Figure 2. Effects of 4g on eNOS activity. a) A23187ꢀdependent NO production in
HUVECs incubated with DMSO or 4g (100 ꢂmol/L) for 30 min (n = 12) (c). b)
Acetylcholineꢀevoked relaxation in aortic rings with endothelium contracted with 1 ꢂM
noradrenaline in the presence of DMSO, 4g (100 ꢂmol/L), or LꢀNAME (100 ꢂmol/L) for
30 min (n = 5). Data are expressed as the mean ± SEM of n experiments.
Cell viability
We carried out the cell viability activity of compound 4g, using HUVECs. We found that
this compound has little toxicity, since only at concentrations >1mM weekly
(approximately 20 %) reduced cells viability (Figure 3).
Figure 3. Study of cell viability of 4g using MTT assay. *p<0.05 vs control (ctrl);
**p<0.01 vs ctrl.
Docking studies
† The authors declare no compeꢀng interests.

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Docking studies were performed to propose and understand the binding mode of N,N’ꢀ
disubstituted thiourea and urea derivatives 4a-q inside nNOS and iNOS.
Figure 4 illustrates the main poses obtained for these compounds in the nNOS (PDB
id: 1QW6)26 binding site. All the ligands interact with the Glu592 and a carboxylate
moiety of the heme group through the hydroxyl group and both thiourea/urea nitrogens,
respectively. Compound 4a (Figure 4a) shows additional interactions of the amino
group, forming two more hydrogen bonds, one with Trp587 and the other with Glu592.
Moreover 4a have a good orientation in the binding site: the phenyl ring points to the
lipophilic region (Phe584 and Pro565) being situated below the heme group
establishing a πꢀcation interaction, the aliphatic chain is directed to Val575 establishing
VdW interactions and the thiourea moiety is oriented to a polar pocket formed by three
arginines (Arg596, Arg603 and Arg481). In addition, more VdW interactions are
established with Gln478.
On the other hand, compound 4j (Figure 4b) shows no additional hydrogen bonds in
the binding site. Despite it presents a similar orientation to 4a, being longer adopts a
more constricted conformation which results more unstable and reduces the πꢀcation
interaction, due to the inclination of the aromatic ring respect to the heme group. This
fact can explain the loss of activity of some compounds.
Figure 4. Detailed view of the main poses obtained for compound 4a (a) and 4j (b) in
the nNOS binding site. Dotted lines indicate hydrogen bond interactions between the
ligand and the residues of the enzyme.
Figure 5 shows the most common poses obtained for hydroxypropyl derivatives in the
iNOS binding site. Compound 4g (Figure 5a) forms three hydrogen bonds, one with
Glu371 through the hydroxyl group and two with a heme propionate moiety through
both thiourea nitrogens. It is noteworthy that, although 4a has more favourable Hꢀbond
contribution inside nNOS, 4g seems to have higher VdW stabilising contribution inside
iNOS. This contribution is performed through the aliphatic chain which interacts with
the nearby residues Val346, Pro344 and Gln257, and especially through the thiourea
moiety which is oriented to Gln257 and to the arginines pocket.
† The authors declare no compeꢀng interests.

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Figure 5. Detailed view of the main poses obtained for compound 4g (a) and 4l (b) in
the iNOS binding site. Dotted lines indicate hydrogen bond interactions between the
ligand and the residues of the enzyme.
Other compounds such as 4l (Figure 5b) demonstrate weaker interaction with the iNOS
(PDB id: 3NW2)27 binding site. In this case, two hydrogen bonds are established, one
between the amino group and a propionate of the heme group, and the other between
a thiourea nitrogen and Glu371. Moreover, the conformation adopted by 4l moves
away the phenyl ring and the thiourea moiety reducing the πꢀcation interaction with the
heme group and the VdW interactions which decrease the stability and efficiency of the
binding process.
Conclusions
In summary, we have designed and successfully synthesized a series of novel N,N’ꢀ
disubstituted thiourea and urea derivatives. The synthetic pathway of these new
compounds can be used to prepare kynurenamineꢀthioureas and ureas easier, with
less synthetic steps and higher global yield than the previously described route. Their
biological evaluation was performed versus nNOS and iNOS isoforms for all
compounds and versus eNOS isoform for 4g. In general, compounds 4a-q inhibit better
the neuronal NOS isoform than the inducible one, exhibiting the thioureas higher
inhibition than the ureas. The derivatives 4a and 4g are the most active compounds
with IC50 values of 350 and 130 ꢁM, respectively, versus nNOS, and 180 ꢁM versus
iNOS for the second compound. In addition, 4g does not inhibit eNOS which is
necessary to avoid the hypertension. Moreover cell viability test demonstrated the
absence of cell toxicity in compound 4g at IC50 value. Both in silico and in vitro studies
reveal promising properties for these compounds which could be a reference to design
new nNOS and iNOS inhibitors without citotoxicity and adverse vascular effect, useful
in neurodegenerative disorders such as Parkinson disease, where both isoforms are
involved.
Experimental section
Chemistry
Melting points were determined using a capillary melting point apparatus and are
uncorrected. Analytical thin layer chromatography was performed using Merck
Kieselgel 60 F254 aluminum sheets and the spots were developed with UV light (λ =
254 nm). Flash chromatography was carried out using silica gel 60, 230ꢀ240 mesh
(Merck), and the eluents used are reported within parentheses. 1H NMR and 13C NMR
spectra were recorded on a Varian Inova Unity 300 spectrometer operating at 300.20
1
for H and 75.49 MHz for 13C, on a Varian direct drive 400 spectrometer operating at
400.57 MHz for 1H and 100.73 MHz for 13C, on a Varian Inova Unity 500 spectrometer
1
operating at 499.79 for H and 125.68 MHz for 13C and on a Varian direct drive 600
spectrometer operating at 600.25 MHz for 1H and 150.95 MHz for 13C in the deuterated
† The authors declare no compeꢀng interests.

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solvents indicated at ambient temperature. Chemical shifts are reported in ppm (δ) and
are referenced to the residual solvent peak. IR spectra were recorded on a Perkin
Elmer 782 spectrometer. Highꢀresolution mass spectrometry (HRMS) was carried out
on a Waters LCT Premier Mass Spectrometer. Elemental analyses were within ±0.4%
of the theoretical values. Small scale microwaveꢀassisted synthesis was carried out in
an Initiator 2.0 singleꢀmode microwave instrument producing controlled irradiation at
2.450 GHz (Biotage AB, Uppsala). Reaction time refers to hold time at 90ºC, not to
total irradiation time. The temperature was measured with an IR sensor outside the
reaction vessel. Anhydrous CH2Cl2 was used as solvent.
General
synthetic
method
of
3-hydroxy-3-(2-nitro-5-
substitutedphenyl)propanenitrile, 6a-c.
BuLi (1.6 M/hexane, 20.3 mL) was added to dry THF (37.5 mL) cooled to ꢀ78 ºC under
argon. Then, a solution of acetonitrile (1.7 mL) in dry THF (4.9 mL) was added
dropwise. The mixture was stirred at ꢀ78 ºC for 1 h. Afterward a solution of the
corresponding benzaldehyde 5a-c (16.16 mmol) in dry THF (4.9 mL) was added
dropwise. The mixture was stirred again at ꢀ78 ºC for 30 min and then warmed to RT.
The reaction was agitated for 15 min at RT and was quenched with cold water (25 mL),
diluted with diethyl ether (30 mL) and washed with 2% aqueous HCl (15 mL). The
aqueous layer was extracted with diethyl ether (3 x 15 mL). Finally the organic phase
was washed with NaHCO3 saturated solution (10 mL) and brine (15 mL), dried over
anhydrous Na2SO4, filtered and concentrated under vacuum. The crude mixture was
purified by flash chromatography (EtOAc/hexane, 1:2).
3-Hydroxy-3-(2-nitrophenyl)propanenitrile, 6a. Yellow solid (70%).28
3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propanenitrile, 6b. Yellow solid (90%); mp:
88 ꢀ 90 ºC; 1H NMR (300 MHz, CDCl3) δ 8.13 (d, 1H), 7.47 (d, 1H), 6.94 (dd, 1H), 5.81 ꢀ
5.70 (m, 1H), 3.94 (s, 3H), 3.07 (dd, 1H), 2.85 (dd, 1H); 13C NMR (125 MHz, CDCl3) δ
164.39, 140.30, 139.65, 127.97, 117.03, 114.49, 112.73, 65.50, 56.15, 27.27; HRMS
m/z 245.0540 [M + Na]+, calcd. mass for C10H10N2O4Na: 245.0538.
3-(5-Chloro-2-nitrophenyl)-3-hydroxypropanenitrile, 6c. Yellow solid (70%); mp: 63
ꢀ 64 ºC; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, 1H), 8.01 (d, 1H), 7.51 (dd, 1H), 5.69 (dd,
1H), 3.08 (dd, 1H), 2.87 (dd, 1H); 13C NMR (125 MHz, CDCl3) δ 145.07, 141.37,
138.95, 129.63, 128.61, 126.52, 116.64, 65.18, 27.41; HRMS m/z 249.0047 [M + Na]+,
calcd. mass for C9H7N2O3NaCl: 249.0045.
General synthetic method of 3-amino-1-(2-nitro-5-substitutedphenyl)propan-1-ol,
7a-c.
3ꢀHydroxyꢀ3ꢀ(2ꢀnitroꢀ5ꢀsubstitutedphenyl)propanenitrile derivatives 6a-c (1.7 mmol)
were treated dropwise with a solution of BH3 (12.3 mL) in THF at 0 ºC under argon.
The mixture was stirred during 4h at RT. Afterward the reaction mixture was cooled to
0 ºC and an iceꢀcold solution of 6 N HCl (7.5 mL) was added carefully. The THF was
evaporated and the aqueous phase was basified with 4 N NaOH to pH = 10, and
extracted with EtOAc (3 x 15 mL). The organic phase was washed with brine, dried
over Na2SO4 and concentrated. The crude was used for the next step without
purification.
General synthetic method of
N-alkyl or phenyl-N’-[3-Hydroxy-3-(2-nitro-5-
substituted phenyl)propyl]-thioureas and ureas, 8a-q.
† The authors declare no compeꢀng interests.

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DOI: 10.1039/C5MD00477B
Thioisocianate or isocianate (0.9 mmol) was added, under argon, to a solution of 3ꢀ
aminoꢀ1ꢀ(2ꢀnitroꢀ5ꢀsubstitutedphenyl)propanꢀ1ꢀol derivatives 7a-c (0.6 mmol) in dry
CH2Cl2. The reaction mixture was irradiated under microwave conditions at 90 ºC for
20 min. The crude mixture was purified by flash chromatography (EtOAc/hexane, 1:1).
N
-[3-Hydroxy-3-(2-nitrophenyl)propyl]-N’-methylthiourea, 8a. Yellow solid (0.42
mmol, 70%); mp: 40 ºC; 1H NMR (300 MHz, CDCl3) δ 7.98 ꢀ 7.90 (m, 2H), 7.70 ꢀ 7.63
(m, 1H), 7.44 ꢀ 7.35 (m, 1H), 5.37 (d, 1H), 4.36 (bs, 1H), 3.59 ꢀ 3.42 (m, 1H), 3.03 (s,
3H), 2.89 ꢀ 2.83 (m, 1H), 2.14 ꢀ 1.97 (m, 2H), 1.88 ꢀ 1.73 (m, 1H); 13C NMR (150 MHz,
CDCl3) δ 182.69, 146.98, 140.26, 134.13, 128.09, 128.26, 124.61, 66.09, 42.09, 38.92,
27.41; HRMS m/z 270.0916 [M + H]+, calcd. mass for C11H16N3O3S: 270.0912.
N
-Ethyl-N’-[3-hydroxy-3-(2-nitrophenyl)propyl]thiourea, 8b. Yellow oil (0.42 mmol,
1
70%); H NMR (300 MHz, CD3OD) δ 8.05 ꢀ 7.82 (m, 2H), 7.82 ꢀ 7.61 (m, 1H), 7.59 ꢀ
7.42 (m, 1H), 5.38 ꢀ 5.20 (m, 1H), 3.76 ꢀ 3.59 (m, 1H), 3.52 ꢀ 3.39 (m, 1H), 3.37 ꢀ 3.28
(m, 2H), 2.34 ꢀ 2.08 (m, 1H), 2.04 ꢀ 1.86 (m, 1H), 1.39 ꢀ 1.24 (m, 3H); 13C NMR (125
MHz, CD3OD) δ 185.83, 147.37, 140.47, 133.30, 128.00, 127.72, 123.87, 66.13, 44.33,
38.48, 36.70, 11.78; HRMS m/z 284.1064 [M + H]+, calcd. mass for C12H18N3O3S:
284.1069.
N
-[3-Hydroxy-3-(2-nitrophenyl)propyl]-N’-propylthiourea, 8c. Yellow oil (0.43 mmol,
1
73%); H NMR (500 MHz, CDCl3) δ 7.94 (dd, 1H), 7.90 (dd, 1H), 7.67 ꢀ 7.63 (m, 1H),
7.42 ꢀ 7.39 (m, 1H), 6.25 (bs, 1H), 6.15 (bs, 1H), 5.35 (d, 1H), 4.35 ꢀ 4.26 (m, 2H), 3.52
ꢀ 3.47 (m, 2H), 3.34 ꢀ 3.31 (m, 2H), 2.09 ꢀ 2.02 (m, 1H), 1.85 ꢀ 1.76 (m, 1H), 1.69 ꢀ 1.62
(m, 2H), 0.99 (t, 3H); 13C NMR (125 MHz, CD3OD) δ 181.91, 147.10, 140.18, 134.07,
128.24, 128.12, 124.60, 66.26, 45.65, 42.16, 38.83, 22.20, 11.55; HRMS m/z 282.1460
[M + H]+, calcd. mass for C13H20N3O4: 282.1454.
N
-Ethyl-N’-[3-hydroxy-3-(2-nitrophenyl)propyl]urea, 8d. Yellow oil (0.48 mmol,
81%); 1H NMR (600 MHz, CDCl3) δ 7.93 ꢀ 7.87 (m, 2H), 7.65 ꢀ 7.60 (m, 1H), 7.40 ꢀ 7.35
(m, 1H), 5.30 (dd, 1H), 3.82 ꢀ 3.75 (m, 1H), 3.24 ꢀ 3.14 (m, 3H), 2.01 ꢀ 1.94 (m, 1H),
1.68 ꢀ 1.61 (m, 1H), 1.14 (t, 3H); 13C NMR (150 MHz, CDCl3) δ 159.61, 147.06, 140.46,
133.70, 128.18, 127.65, 124.18, 65.91, 39.70, 36.96, 35.56, 15.27; HRMS m/z
268.1302 [M + H]+, calcd. mass for C12H18N3O4: 268.1297.
N
-[3-Hydroxy-3-(2-nitrophenyl)propyl]-N’-propylurea, 8e. Yellow oil (0.45 mmol,
1
76%); H NMR (500 MHz, CDCl3) δ 7.93 ꢀ 7.88 (m, 2H), 7.64 ꢀ 7.61 (m , 1H), 7.39 ꢀ
7.36 (m, 1H), 5.30 (dd, 1H), 4.78 (bs, 2H), 3.83 ꢀ 3.77 (m, 1H), 3.19 ꢀ 3.14 (m, 1H),
3.12 (t, 2H), 2.01 ꢀ 1.94 (m, 1H), 1.67 ꢀ 1.61 (m, 1H), 1.55 ꢀ 1.49 (m, 2H), 0.91 (t, 3H);
13C NMR (125 MHz, CDCl3) δ 159.87, 147.28, 140.61, 133.82, 128.35, 127.78, 124.32,
66.05, 42.67, 39.95, 37.11, 23.43, 11.42; HRMS m/z 298.1222 [M + H]+, calcd. mass
for C13H20N3O3S: 298.1225.
N
-[3-Hydroxy-3-(2-nitrophenyl)propyl]-N’-phenylurea, 8f. Yellow oil (0.42 mmol,
70%); 1H NMR (500 MHz, CDCl3) δ 7.93 ꢀ 7.86 (m, 2H), 7.63 ꢀ 7.60 (m, 1H), 7.51 ꢀ 7.44
(m, 1H), 7.39 ꢀ 7.36 (m, 2H), 7.25 ꢀ 7.20 (m, 2H), 7.04 ꢀ 6.99 (m, 1H), 5.34 (dd, 1H),
3.82 ꢀ 3.77 (m, 1H), 3.24 ꢀ 3.21 (m, 1H), 2.02 ꢀ 1.96 (m, 1H), 1.72 ꢀ 1.66 (m, 1H); 13C
NMR (125 MHz, CDCl3) δ 157.62, 147.07, 140.20, 137.84, 133.75, 129.36, 128.42,
127.82, 124.30, 121.81, 121.04, 66.39, 39.07, 37.20; HRMS m/z 316.1292 [M + H]+,
calcd. mass for C16H18N3O4: 316.1297.
N
-[3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl]-N’-methylthiourea, 8g. Yellow oil
(0.43 mmol, 72%); 1H NMR (500 MHz, CDCl3) δ 8.09 (d, 1H), 7.43 (d, 1H), 6.87 (dd,
1H), 5.51 (d, 1H), 4.39 (bs, 1H), 3.94 (s, 3H), 3.91 ꢀ 3.89 (m, 1H), 3.56 ꢀ 351 (m, 1H),
3.05 (s, 3H), 2.11 ꢀ 2.04 (m, 1H), 1.79 ꢀ 1.74 (m, 1H); 13C NMR (75 MHz, CDCl3) δ
† The authors declare no compeꢀng interests.

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DOI: 10.1039/C5MD00477B
181.77, 164.22, 144.04, 139.51, 127.70, 113.55, 111.97, 66.22, 55.98, 42.04, 38.62,
30.31; HRMS m/z 300.1021 [M + H]+, calcd. mass for C12H18N3O4S: 300.1018.
N
-[3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl]-N’-propylthiourea, 8h. Yellow oil
1
(0.46 mmol, 77%); H NMR (500 MHz, CDCl3) δ 8.07 (d, 1H), 7.40 (d, 1H), 6.84 (dd,
1H), 6.21 (bs, 1H), 6.14 (bs, 1H), 5.48 (d, 1H), 4.42 ꢀ 4.35 (m, 2H), 3.91 (s, 3H), 3.52 ꢀ
3.46 (m, 1H), 3.36 ꢀ 3.30 (m, 2H), 2.07 ꢀ 2.00 (m, 1H), 1.76 ꢀ 1.70 (m, 1H), 1.66 (q, 2H),
0.99 (t, 3H); 13C NMR (125 MHz, CDCl3) δ 182.16, 164.61, 144.37, 139.96, 128.09,
113.93, 112.37, 66.60, 56.36, 45.88, 42.41, 39.08, 22.46, 11.81; HRMS m/z 326.1159
[M + H]+, calcd. mass for C14H20N3O4S: 326.1175.
N
-ethyl-N’-[3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl]urea, 8i. Yellow oil (0.44
1
mmol, 74%); H NMR (500 MHz, CDCl3) δ 8.03 (d, 1H), 7.41 (d, 1H), 6.82 (dd, 1H),
5.45 (dd, 1H), 3.90 (s, 3H), 3.87 ꢀ 3.80 (m, 1H), 3.24 ꢀ 3.15 (m, 3H), 2.01 ꢀ 1.94 (m,
1H), 1.61 ꢀ 1.55 (m, 1H), 1.15 (t, 3H); 13C NMR (125 MHz, CDCl3) δ 164.23, 159.78,
144.63, 139.86, 127.57, 113.59, 112.02, 66.35, 56.06, 39.73, 37.17, 35.77, 15.43;
HRMS m/z 296.1254 [M + H]+, calcd. mass for C13H18N3O5: 296.1246.
N
-[3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl]-N’-propylurea, 8j. Yellow oil
1
(0.45 mmol, 75%); H NMR (500 MHz, CDCl3) δ 8.08 (d, 1H), 7.41 (d, 1H), 6.85 (dd,
1H), 5.45 (d, 1H), 4.73 (bs, 2H), 3.89 (s, 3H), 3.83 ꢀ 3.72 (m, 1H), 3.26 ꢀ 3.18 (m, 1H),
3.14 (t, 2H), 2.04 ꢀ 1.89 (m, 1H), 1.65 ꢀ 1.48 (m, 3H), 0.92 (t, 3H); 13C NMR (75 MHz,
CDCl3) δ 164.05, 159.77, 144.46, 139.70, 127.39, 113.38, 111.95, 66.39, 55.89, 42.61,
39.28, 37.29, 23.15, 11.23; HRMS m/z 312.1571 [M + H]+, calcd. mass for C14H22N3O5:
312.1559.
N
-[3-Hydroxy-3-(5-methoxy-2-nitrophenyl)propyl]-N’-phenylurea, 8k. Yellow solid
(0.48 mmol, 80%); mp: 77 ꢀ 78 ºC; 1H NMR (400 MHz, CDCl3) δ 8.02 (d, 1H), 7.38 (d,
1H), 7.34 ꢀ 7.24 (m, 4H), 7.09 ꢀ 7.04 (m, 1H), 6.80 (dd, 1H), 5.49 (dd, 1H), 3.87 (s, 3H),
3.84 ꢀ 3.77 (m, 1H), 3.23 ꢀ 3.17 (m, 1H), 2.01 ꢀ 1.93 (m, 1H), 1.65 ꢀ 1.56 (m, 1H); 13C
NMR (100 MHz, CDCl3) δ 164.10, 157.52, 144.30, 139.62, 138.15, 129.28, 127.52,
124.12, 121.52, 113.42, 111.91, 66.53, 55.89, 39.07, 37.04; HRMS m/z 368.1214 [M +
H]+, calcd. mass for C17H19N3O5Na: 368.1222.
N
-[3-(5-Chloro-2-nitrophenyl)-3-hydroxypropyl]-N’-methylthiourea, 8l. Yellow oil
1
(0.45 mmol, 75%); H NMR (300 MHz, CDCl3) δ 7.96 ꢀ 7.90 (m, 2H), 7.36 (dd, 1H),
6.33 (bs, 2H), 5.38 (dd, 1H), 4.36 (bs, 1H), 3.54 ꢀ 3.41 (m, 2H), 3.01 (s, 3H), 2.06 ꢀ 1.95
(m, 1H), 1.78 ꢀ 1.65 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 182.41, 144.83, 142.57,
140.79, 128.44, 128.07, 126.15, 66.67, 41.79, 38.84, 30.29; HRMS m/z 304.0522 [M +
H]+, calcd. mass for C11H15N3O3SCl: 304.0523.
N
-[3-(5-Chloro-2-nitrophenyl)-3-hydroxypropyl]-N’-ethylthiourea, 8m. Yellow oil
1
(0.42 mmol, 70%); H NMR (300 MHz, CDCl3) δ 7.99 ꢀ 7.82 (m, 2H), 7.35 (dd, 1H),
6.37 (bs, 2H), 5.38 (d, 1H), 4.34 (bs, 1H), 3.75 ꢀ 3.42 (m, 4H), 2.06 ꢀ 1.97 (m, 1H), 1.87
ꢀ 1.56 (m, 1H), 1.32 ꢀ 1.20 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 181.45, 144.90,
142.53, 140.71, 128.44, 128.02, 126.09, 66.69, 41.75, 38.80, 38.68, 13.99; HRMS m/z
318.0672 [M + H]+, calcd. mass for C12H17N3O3SCl: 318.0679.
N
-[3-(5-Chloro-2-nitrophenyl)-3-hydroxypropyl]-N’-propylthiourea, 8n. Yellow oil
1
(0.45 mmol, %); H NMR (300 MHz, CDCl3) δ 8.00 ꢀ 7.90 (m, 2H), 7.38 (dd, 1H), 6.36
(bs, 2H), 5.41 (d, 1H), 4.38 (bs,1H), 3.81 ꢀ 3.30 (m, 4H), 2.12 ꢀ 1.96 (m, 1H), 1.80 ꢀ
1.62 (m, 3H), 1.01 (t, 3H); 13C NMR (125 MHz, CDCl3) δ 181.57, 144.88, 142.58,
140.72, 128.46, 128.02, 126.10, 66.65, 45.67, 41.76, 38.89, 22.10, 11.41; HRMS m/z
332.0833 [M + H]+, calcd. mass for C13H19N3O3SCl: 332.0836.
† The authors declare no compeꢀng interests.

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DOI: 10.1039/C5MD00477B
N
-[3-(5-Chloro-2-nitrophenyl)-3-hydroxypropyl]-N’-ethylurea, 8o. Yellow oil (0.51
mmol, 85%); 1H NMR (500 MHz, CDCl3) δ 7.93 (d, 1H), 7.90 (d, 1H), 7.34 (dd,1H), 5.33
(dd, 1H), 4.94 (bs, 2H), 4.30 (bs, 1H), 3.85 ꢀ 3.79 (m, 1H), 3.24 ꢀ 3.15 (m, 3H), 1.99 ꢀ
1.92 (m, 1H), 1.61 ꢀ 1.55 (m, 1H), 1.15 (t, 3H); 13C NMR (125 MHz, CDCl3) δ 159.65,
145.07, 142.88, 140.53, 128.53, 127.80, 125.89, 65.66, 39.76, 36.86, 35.65, 15.25;
HRMS m/z 302.0903 [M + H]+, calcd. mass for C12H17N3O4Cl: 302.0908.
N
-[3-(5-Chloro-2-nitrophenyl)-3-hydroxypropyl]-N’-propylurea, 8p. Yellow oil (0.42
1
mmol, 70%); H NMR (500 MHz, CDCl3) δ 7.94 (d, 1H), 7.90 (d, 1H), 7.38 (dd, 1H),
5.32 (dd, 1H), 3.86 ꢀ 3.80 (m, 1H), 3.20 ꢀ 3.15 (m, 1H), 3.13 (t, 2H), 1.99 ꢀ 1.92 (m, 1H),
1.61 ꢀ 1.55 (m, 1H), 1.56 ꢀ 1.51 (m, 2H), 0.93 (t, 3H); 13C NMR (125 MHz, CDCl3) δ
159.75, 145.07, 142.85, 140.54, 128.52, 127.79, 125.89, 65.63, 42.60, 39.81, 36.86,
23.25, 11.27; HRMS m/z 316.1056 [M + H]+, calcd. mass for C13H19N3O4Cl: 316.1064.
N
-[3-(5-Chloro-2-nitrophenyl)-3-hydroxypropyl]-N’-phenylurea, 8q. Yellow solid
1
(0.42 mmol, 70%); mp: 53ºC; H NMR (400 MHz, CDCl3) δ 7.92 ꢀ 7.88 (m, 2H), 7.35 ꢀ
7.28 (m, 5H), 7.13 ꢀ 7.08 (m, 1H), 5.37 (dd, 1H), 3.87 ꢀ 3.80 (m, 1H), 3.20 ꢀ 3.15 (m,
1H), 1.98 ꢀ 1.90 (m, 1H), 1.64 ꢀ 1.56 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 157.65,
144.98, 142.67, 140.61, 137.76, 129.43, 128.44, 127.91, 126.00, 124.64, 122.09,
65.90, 39.32, 36.89; HRMS m/z 372.0722 [M + H]+, calcd. mass for C16H16N3O4NaCl:
372.0727.
General
synthetic
method
of
N-alkyl
or
phenyl-N’-[3-(2-amino-5-
substitutedphenyl)-3-hydroxypropyl]-thioureas and ureas, 4a-q.
A solution of each nitro precursor 8a-q (0.5 mmol) in MeOH (10 mL) was passed
through the Flowꢀhydrogenator under the following conditions: 60ºC, 60 bar, 0.2
mL/min flow rate and 10% Pd/C as catalyst. After evaporation of the solvent, the crude
mixture was purified by recrystallization (diethyl ether) or by Flash chromatography
(EtOAc or EtOAc/hexane, 1:1).
N
-[3-(2-Aminophenyl)-3-hydroxypropyl]-N’-methylthiourea, 4a. Yellow oil (0.37
mmol, 75%); 1H NMR (600 MHz, CD3OD) δ 7.06 (d, 1H), 6.96 ꢀ 6.93 (m, 1H), 6.66 (d,
1H), 6.63 ꢀ 6.60 (m, 1H), 4.72 (dd, 1H), 3.53 ꢀ 3.24 (m, 2H), 2.90 (s, 3H), 2.02 ꢀ 1.96
(m, 2H); 13C NMR (150 MHz, CD3OD) δ 181.71, 147.42, 130.83, 130.29, 129.13,
120.45, 119.16, 72.55, 42.10, 38.53, 32.02; HRMS m/z 240.1176 [M + H]+, calcd. mass
for C11H18N3OS: 240.1171; Anal. calcd for C11H17N3OS: C 55.20, H 7.16, N 17.56,
found: C 54.81, H 7.16, N 17.17.
N
-[3-(2-Aminophenyl)-3-hydroxypropyl]-N’-ethylthiourea, 4b. Yellow oil (0.37
mmol, 75%); 1H NMR (500 MHz, CDCl3) δ 7.09 ꢀ 7.04 (m, 2H), 6.73 ꢀ 6.70 (m, 1H), 6.64
(dd, 1H), 6.43 (bs, 1H), 6.15 (bs, 1H), 4.78 (dd, 1H), 3.92 (m, 1H), 3.49 ꢀ 3.33 (m, 1H),
3.35 ꢀ 3.30 (m, 2H), 2.21 ꢀ 2.15 (m, 1H), 1.98 ꢀ 1.91 (m, 1H), 1.19 (t, 3H); 13C NMR
(125 MHz, CDCl3) δ 181.25, 144.70, 128.67, 127.12, 127.08, 118.54, 116.96, 71.12,
42.09, 38.87, 34.69, 14.18; HRMS m/z 254.1175 [M + H]+, calcd. mass for C12H20N3OS:
254.1171; Anal. calcd for C12H19N3OS: C 56.89, H 7.56 , N 16.58, found: C 57.26, H
7.91, N 16.22.
N
-[3-(2-Aminophenyl)-3-hydroxypropyl]-N’-propylthiourea, 4c. Yellow solid (0.40
1
mmol, 80%); mp: 50 ºC; H NMR (500 MHz, CDCl3) δ 7.09 ꢀ 7.03 (m, 2H), 6.74 ꢀ 6.69
(m, 1H), 6.64 (d, 1H), 6.38 (bs, 1H), 6.15 (bs, 1H), 4.81 ꢀ 4.75 (m, 1H), 4.07 ꢀ 3.76 (m,
2H), 3.53 ꢀ 3.44 (m, 1H), 3.32 ꢀ 3.17 (m, 2H), 2.22 ꢀ 2.15 (m, 1H), 1.98 ꢀ 1.90 (m, 1H),
1.62 ꢀ 1.55 (m, 2H), 0.94 (t, 3H); 13C NMR (125 MHz, CDCl3) δ 181.61, 144.91, 128.83,
127.22, 127.18, 118.62, 117.06, 71.35, 46.03, 42.13, 34.82, 22.31, 11.55; HRMS m/z
† The authors declare no compeꢀng interests.

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DOI: 10.1039/C5MD00477B
268.1469 [M + H]+, calcd. mass for C13H22N3OS: 268.1484; Anal. calcd for C13H21N3OS:
C 58.39, H 7.92, N 15.71, found: C 58.14, H 8.12, N 15.41.
N
-[3-(2-Aminophenyl)-3-hydroxypropyl]-N’-ethylurea, 4d. White solid (0.41 mmol,
83%); mp: 115 ºC; 1H NMR (400 MHz, CD3OD) δ 7.09 (d, 1H), 7.03 ꢀ 6.98 (m, 1H), 6.71
(d, 1H), 6.69 ꢀ 6.65 (m, 1H), 4.75 (dd, 1H), 3.31 (bs, 1H), 3.29 ꢀ 3.18 (m, 2H), 3.13 (q,
2H), 2.03 ꢀ 1.88 (m, 2H), 1.09 (t, 3H); 13C NMR (150 MHz, CD3OD) δ 159.98, 144.89,
128.19, 127.56, 126.54, 117.67, 116.47, 70.07, 37.00, 35.90, 34.41, 14.34; IR (KBr) ν:
3608.79, 3583.37, 3351.29, 2929.21, 1699.47, 1624.22, 1565.96, 1495.79, 1456.51,
1261.70, 1157.66, 1069.88, 755.76, 665.51 cmꢀ1; HRMS m/z 238.1475 [M + H]+, calcd.
mass for C12H20N3O2: 238.1477; Anal. calcd for C12H19N3O2: C 60.74, H 8.07, N 17.71,
found: C 60.86, H 8.34, N 17.41.
N
-[3-(2-Aminophenyl)-3-hydroxypropyl]-N’-propylurea, 4e. Yellow solid (0.45 mmol,
90%); mp: 108 ꢀ 109 ºC; 1H NMR (600 MHz, CD3OD) δ 7.11 (d, 1H), 7.03 ꢀ 7.01 (m,
1H), 6.71 (d, 1H), 6.70 ꢀ 6.67 (m, 1H), 4.76 (dd, 1H), 3.31 ꢀ 3.26 (m, 1H), 3.24 ꢀ 3.19
(m, 1H), 3.08 (t, 2H), 2.04 ꢀ 1.98 (m, 1H), 1.97 ꢀ 1.91 (m, 1H), 1.53 ꢀ 1.48 (m, 2H), 0.93
(t, 3H); 13C NMR (150 MHz, CD3OD) δ 161.53, 146.32, 129.61, 128.99, 127.97, 119.09,
117.90, 71.52, 42.88, 38.43, 37.35, 24.47, 11.62; HRMS m/z 252.1701 [M + H]+, calcd.
mass for C13H22N3O2: 252.1712; Anal. calcd for C13H21N3O2: C 62.13, H 8.42, N 16.72,
found: C 62.23, H 8.44, N 16.41.
N
-[3-(2-Aminophenyl)-3-hydroxypropyl]-N’-phenylurea, 4f. White solid (0.35 mmol,
70%); mp: 95ºC; 1H NMR (500 MHz, CD3OD) δ 7.36 ꢀ 7.32 (m, 2H), 7.26 ꢀ 7.21 (m, 2H),
7.12 (d, 1H), 7.03 ꢀ 7.01 (m, 1H), 6.99 ꢀ 6.94 (m, 1H), 6.72 (dd, 1H), 6.69 ꢀ 6.66 (m,
1H), 4.80 (dd, 1H), 3.39 ꢀ 3.34 (m, 1H), 3.28 ꢀ 3.26 (m, 1H), 2.09 ꢀ 1.95 (m, 2H); 13C
NMR (125 MHz, CD3OD) δ 160.84, 148.58, 143.26, 132.04, 131.92, 131.30, 130.27,
125.67, 122.55, 121.42, 120.22, 74.02, 40.65, 39.40; IR (KBr) ν: 3608.66, 3583.14,
3352.13, 2927.61, 1645.83, 1624.22, 1597.47, 1554.11, 1498.40, 1456.94, 1440.69,
1312.68, 1238.51, 1071.53, 754.01, 665.86 cmꢀ1; HRMS m/z 286.1560 [M + H]+, calcd.
mass for C16H20N3O2: 286.1556; Anal. calcd for C16H19N3O2: C 67.35, H 6.71, N 14.73,
found: C 67.49, H 7.09, N 14.63.
N
-[3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl]-N’-methylthiourea, 4g. Yellow
solid (0.37 mmol, 75%); mp: 50 ºC; 1H NMR (300 MHz, CD3OD) δ 6.98 (d, 1H), 6.93 (d,
1H), 6.83 (dd, 1H), 4.92 (d, 1H), 3.82 (s, 3H), 3.80 ꢀ 3.78 (m, 2H), 2.98 (s, 3H), 2.12 ꢀ
2.06 (m, 2H); 13C NMR (150 MHz, CD3OD) δ 182.41, 155.80, 134.34, 131.26, 121.11,
115.91, 114.99, 72.27, 57.35, 38.38, 36.51, 31.05; HRMS m/z 270.1280 [M + H]+,
calcd. mass for C12H20N3O2S: 270.1276; Anal. calcd for C12H19N3O2S: C 53.51, H 7.11,
N 15.60, found: C 53.50, H 7.55, N 15.50.
N
-[3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl]-N’-propylthiourea, 4h. Yellow
solid (0.38 mmol, 76%); mp: 45 ºC; 1H NMR (500 MHz, CDCl3) δ 6.69 (d, 1H), 6.66 (dd,
1H), 6.62 (d, 1H), 6.50 (bs, 1H), 6.20 (bs, 1H), 4.77 (dd, 1H), 3.72 (s, 3H), 3.67 ꢀ 3.48
(m, 2H), 3.31 ꢀ 3.24 (m, 2H), 2.17 ꢀ 2.08 (m, 1H), 1.98 ꢀ 1.90 (m, 1H), 1.63 ꢀ 1.55 (m,
2H), 0.95 (t, 3H); 13C NMR (125 MHz, CDCl3) δ 181.63, 153.16, 137.56, 129.60,
118.71, 113.95, 113.07, 70.78, 55.92, 45.94, 42.21, 35.18, 22.33, 11.56; HRMS m/z
298.1580 (M + H)+, calcd. mass for C14H24N3O2S: 298.1589; Anal. calcd for
C14H23N3O2S: C 56.54, H 7.79, N 14.13, found: C 56.50, H 7.65, N 14.50.
N
-[3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl]-N’-ehylurea, 4i. Yellow solid
(0.39 mmol, 78%); mp: 128 ꢀ 129 ºC; 1H NMR (600 MHz, CD3OD) δ 6.79 (d, 1H), 6.71
(d, 1H), 6.67 (dd, 1H), 4.76 (dd, 1H), 3.73 (s, 3H), 3.32 ꢀ 3.27 (m, 1H), 3.25 ꢀ 3.20 (m,
† The authors declare no compeꢀng interests.

MedChemComm
Page 14 of 21
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DOI: 10.1039/C5MD00477B
1H), 3.15 (q, 2H), 1.98 ꢀ 1.91 (m, 2H), 1.11 (t, 3H); 13C NMR (150 MHz, CD3OD) δ
161.40, 154.32, 139.07, 131.79, 119.39, 114.52, 113.66, 70.94, 56.09, 38.39, 37.67,
35.85, 15.76; HRMS m/z 268.1647 (M + H)+, calcd. mass for C13H22N3O3: 268.1661;
Anal. calcd for C13H21N3O3: C 58.41, H 7.92, N 15.72, found: C 58.50, H 7.63, N 15.75.
N
-[3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl]-N’-propylurea, 4j. White solid
(0.37 mmol, 75%); mp: 130 ºC; 1H NMR (300 MHz, CD3OD) δ 6.83 (d, 1H), 6.76 (d,
1H), 6.71 (dd, 1H), 4.81 (dd, 1H), 3.78 (s, 3H), 3.36 ꢀ 3.25 (m, 2H), 3.13 (t, 2H), 2.04 ꢀ
1.95 (m, 2H), 1.61 ꢀ 1.50 (m, 2H), 0.98 (t, 3H); 13C NMR (100 MHz, CD3OD) δ 160.09,
152.88, 137.70, 130.33, 117.95, 113.13, 112.27, 69.55, 54.70, 41.47, 36.97, 36.26,
23.04, 10.19; IR (KBr)
ν: 3608.56, 3583.07, 3351.03, 2931.27, 2352.07, 1613.25,
1568.33, 1503.31, 1431.20, 1258.55, 1157.68, 1069.54, 1041.27, 840.60, 757.42,
665.87 cmꢀ1; HRMS m/z 282.1813 (M + H)+, calcd. mass for C14H24N3O3: 282.1818;
Anal. calcd for C14H23N3O3: C 59.77, H 8.24, N 14.94, found: C 59.79, H 8.40, N 14.75.
N
-[3-(2-Amino-5-methoxyphenyl)-3-hydroxypropyl]-N’-phenylurea, 4k. White solid
(0.37 mmol, 75%); mp: 157 ºC; 1H NMR (500 MHz, CD3OD) δ 7.36 ꢀ 7.31 (m, 2H), 7.27
ꢀ 7.21 (m, 2H), 6.98 ꢀ 6.95 (m, 1H), 6.79 (d, 1H), 6.70 (d, 1H), 6.65 (dd, 1H), 4.79 (dd,
1H), 3.41 ꢀ 3.35 (m, 1H), 3.40 ꢀ 3.27 (m, 1H), 3.31 (s, 3H), 2.03 ꢀ 1.94 (m, 2H); 13C
NMR (125 MHz, CD3OD) δ 157.13, 152.92, 139.55, 137.64, 130.37, 128.35, 121.99,
118.87, 118.02, 113.14, 112.25, 69.77, 54.67, 36.92, 36.01; IR (KBr)
ν: 3608.39,
3583.00, 3292.54, 2922.27, 1621.06, 1556.13, 1502.84, 1451.39, 1253.58, 1042.40,
757.87, 665.28 cmꢀ1; HRMS m/z 316.1667 [M + H]+, calcd. mass for C17H22N3O3:
316.1661; Anal. calcd for C17H21N3O3: C 64.74, H 6.71, N 13.32, found: C 64.72, H
6.57, N 13.52.
N
-[3-(2-Amino-5-chlorophenyl)-3-hydroxypropyl]-N’-methylthiourea, 4l. Yellow
solid (0.39 mmol, 79%); mp: 55 ºC; 1H NMR (400 MHz, CD3OD) δ 7.18 (d, 1H), 7.02
(dd, 1H), 6.72 (d, 1H), 4.80 (dd, 1H), 3.37 ꢀ 3.35 (m, 2H), 2.97 (s, 3H), 2.08 ꢀ 2.03 (m,
2H); 13C NMR (100 MHz, CD3OD) δ 181.67, 143.62, 129.94, 127.15, 126.02, 121.99,
117.36, 69.08, 41.16, 34.81, 29.30; HRMS m/z 274.0775 [M + H]+, calcd. mass for
C11H17N3OSCl: 274.0781; Anal. calcd for C11H16N3OSCl: C 48.26, H 5.89, N 15.35,
found: C 48.60, H 5.99, N 15.73.
N
-[3-(2-Amino-5-chlorophenyl)-3-hydroxypropyl]-N’-ethylthiourea, 4m. Yellow
solid (0.36 mmol, 72%); mp: 48 ºC; 1H NMR (500 MHz, CDCl3) δ 7.05 (d, 1H), 7.02 ꢀ
6.98 (m, 1H), 6.56 (d, 1H), 4.76 (dd, 1H), 4.55 ꢀ 4.35 (m, 1H), 4.34 ꢀ 4.25 (m, 1H), 4.24
ꢀ 4.17 (m, 2H), 2.15 ꢀ 2.05 (m, 1H), 1.96 ꢀ 1.87 (m, 1H), 1.28 (t, 3H); 13C NMR (125
MHz, CDCl3) δ 182.70, 142.94, 128.98, 128.17, 126.75, 123.14, 118.03, 69.25, 46.39,
40.64, 34.73, 14.10; HRMS m/z 288.0941 [M + H]+, calcd. mass for C12H19N3OSCl:
288.0937; Anal. calcd for C12H18N3OSCl: C 50.08, H 6.30, N 14.60, found: C 50.44, H
6.47, N 14.22.
N
-[3-(2-Amino-5-chlorophenyl)-3-hydroxypropyl]-N’-propylthiourea, 4n. White
solid (0.40 mmol, 80%); mp: 130 ºC; 1H NMR (400 MHz, CD3OD) δ 7.13 (d, 1H), 6.96
(dd, 1H), 6.67 (d, 1H), 4.74 (dd, 1H), 3.71 ꢀ 3.60 (m, 1H), 3.56 ꢀ 3.46 (m, 1H), 3.45 ꢀ
3.42 (m, 2H), 2.03 ꢀ 1.97 (m, 2H), 1.62 ꢀ 1.55 (m, 2H), 0.94 (t, 3H); 13C NMR (100 MHz,
CD3OD) δ 181.17, 143.63, 129.93, 127.14, 126.02, 121.98, 117.35, 69.15, 45.56,
41.24, 34.85, 21.98, 10.20; IR (KBr) ν: 3608.26, 3583.16, 3326.85, 2930.44, 1627.13,
1488.78, 1261.21, 1101.68, 808.25, 756.63, 665.93 cmꢀ1; HRMS m/z 302.1096 [M +
H]+, calcd. mass for C13H21N3OSCl: 302.1094; Anal. calcd for C13H20N3OSCl: C 51.73,
H 6.68, N 13.92, found: C 51.51, H 7.16, N 13.54.
† The authors declare no compeꢀng interests.

Page 15 of 21
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DOI: 10.1039/C5MD00477B
N
-[3-(2-Amino-5-chlorophenyl)-3-hydroxypropyl]-N’-ethylurea, 4o. Yellow solid
(0.45 mmol, 91%); mp: 48 ºC; 1H NMR (300 MHz, CD3OD) δ 7.10 (d, 1H), 6.97 (dd,
1H), 6.67 (d, 1H), 4.71 (dd, 1H), 3.33 ꢀ 3.24 (m, 2H), 3.23 ꢀ 3.17 (m, 2H), 1.95 ꢀ 1.87
(m, 2H), 1.09 (t, 3H); 13C NMR (100 MHz, CD3OD) δ 159.97, 143.45, 130.15, 127.10,
126.11, 122.11, 117.48, 69.22, 36.91, 35.94, 34.43, 14.33; IR (KBr)
ν: 3608.63,
3583.26, 3337.67, 2923.67, 1623.12, 1564.07, 1488.68, 1260.36, 1061.42, 755.82,
665.63 cmꢀ1; HRMS m/z 294.0982 [M + Na]+, calcd. mass for C12H18N3O2NaCl:
294.0985; Anal. calcd for C12H18N3O2NaCl: C 53.04, H 6.68, N 15.46, found: C 53.38, H
7.02, N 15.52.
N
-[3-(2-Amino-5-chlorophenyl)-3-hydroxypropyl]-N’-propylurea, 4p. Yellow solid
(0.41 mmol, 82%); mp: 137 ºC; 1H NMR (300 MHz, CD3OD) δ 7.03 (d, 1H), 6.96 (dd,
1H), 6.59 (d, 1H), 4.70 (dd, 1H), 3.39 ꢀ 3.03 (m, 4H), 1.93 ꢀ 1.71 (m, 2H), 1.53 ꢀ 1.46
(m, 2H), 0.92 (t, 3H); 13C NMR (100 MHz, CD3OD) δ 159.99, 143.54, 130.23, 127.15,
126.11, 122.15, 117.58, 69.32, 42.83, 36.92, 35.93, 22.58, 10.46; IR (KBr) ν: 3608.76,
3583.12, 3352.21, 2929.75, 1625.87, 1489.82, 1456.67, 1261.04, 1071.09, 756.29,
665.60 cmꢀ1; HRMS m/z 286.1318 [M + H]+, calcd. mass for C13H21N3O2Cl: 286.1322;
Anal. calcd for C13H20N3O2Cl: C 54.64, H 7.05, N 14.70, found: C 54.34, H 7.33, N
14.84.
N
-[3-(2-Amino-5-chlorophenyl)-3-hydroxypropyl]-N’-phenylurea, 4q. White solid
(0.35 mmol, 71%); mp: 105 ºC; 1H NMR (500 MHz, CD3OD) δ 7.36 ꢀ 7.32 (m, 2H), 7.26
ꢀ 7.22 (m, 2H), 7.13 (d, 1H), 6.99 ꢀ 6.94 (m, 2H), 6.67 (d, 1H), 4.76 (dd, 1H), 3.41 ꢀ 3.35
(m, 1H), 3.31 ꢀ 3.27 (m, 1H), 2.01 ꢀ 1.92 (m, 2H); 13C NMR (125 MHz, CD3OD) δ
157.14, 143.71, 139.52, 130.01, 128.36, 127.13, 126.10, 122.02, 121.94, 118.90,
117.38, 69.44, 36.89, 35.65; HRMS m/z 320.1168 [M + H]+, calcd. mass for
C16H19N3O2Cl: 320.1166; Anal. calcd for C16H18N3O2Cl: C 60.09, H 5.67, N 13.14,
found: C 60.21, H 5.96, N 13.38.
General synthetic method of
N-[3-(2-amino-5-chlorophenyl)-3-oxopropyl]-
N’-
ethylurea 3b from -[3-(5-chloro-2-nitrophenyl)-3-hydroxypropyl]-
N
N’-ethylurea 8o.
The Jones reagent (0.418 mL), freshly prepared, (a mixture of 2.67 g chromic
anhydride and 2.3 mL H2SO4 dissolved to 10 mL of water) was added to a solution of
8o (0.742 mmol) in acetone (3 mL). After 10 min stirring, the reaction was quenched
with iceꢀwater (25 mL) and saturated NaHSO4 solution (5 mL). The resulting mixture
was extracted with ethyl acetate, filtered, dried (Na2SO4) and concentrated under
vacuum. The crude mixture was purified by flash chromatography (EtOAc/hexane, 3:1).
The next step was the nitro group reduction to amino one using Fe/FeSO4 in water as
previously described.20
N
-[3-(5-Chloro-2-nitrophenyl)-3-oxopropyl]-N’-ethylurea. Following the procedure
described in this section, 0.67 mmol of a white solid was obtained (90%).
Spectroscopic data.20
N
-[3-(2-Amino-5-chlorophenyl)-3-oxopropyl]-
Biological Procedures
In vitro nNOS and iNOS activities determination
N
’-ethylurea 3b. Yellow solid (75%).20
LꢀArginine, Lꢀcitrulline, Nꢀ(2ꢀhydroxymethyl)piperazineꢀN’ꢀ(2ꢀethanesulfonic acid)
(HEPES), DLꢀdithiothreitol (DTT), hypoxantineꢀ9ꢀβꢀDꢀribofuranosid (inosine), ethylene
glycolꢀbisꢀ(2ꢀaminoethylether)ꢀN,N,N’,N’ꢀtetraacetic acid (EGTA), bovine serum
albumin (BSA), Dowexꢀ50W (50 x 8ꢀ200), FAD, NADPH and 5,6,7,8ꢀtetrahydroꢀLꢀ
† The authors declare no compeꢀng interests.

MedChemComm
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DOI: 10.1039/C5MD00477B
biopterin dihydrocloride (H4ꢀbiopterin), trisꢀ(hydroxymethyl)ꢀaminometane (TrisꢀHCl)
and calcium chloride were obtained from SigmaꢀAldrich Química (Spain). Lꢀ[3H]ꢀ
arginine (47.4 Ci/mmol) was obtained from Perkin Elmer (Spain). Calmodulin from
bovine brain, and recombinant iNOS and nNOS were obtained from Enzo Life
Sciences (Spain).
The i/nNOS activity was measured by the Bredt and Snyder method,29 monitoring the
conversion of Lꢀ[3H]ꢀarginine to Lꢀ[3H]ꢀcitrulline. The final incubation volume was 100
ꢂL and consisted of 10 ꢂL of an aliquot of recombinant i/nNOS added to a buffer with a
final concentration of 25 mM TrisꢀHCl, 1 mM DTT, 4 ꢂM H4ꢀbiopterin, 10 ꢂM FAD, 0.5
mM inosine, 0.5 mg/mL BSA, 0.1 mM CaCl2, 10 ꢂM Lꢀarginine, 10 ꢂg/mL calmodulin
(only for nNOS) and 50 nMLꢀ[3H]ꢀarginine, at pH 7.6. The reaction was started by the
addition of 10 ꢂL of 7.5 mM NADPH and 10 ꢂL of each derivative 4a-q in ethanol
(10%) to give a final concentration of 1 mM. The tubes were vortex and incubated at 37
ºC for 30 min. Control incubations were performed by the omission of NADPH. The
reaction was halted by the addition of 400 ꢂL of cold 0.1 M HEPES, 10 mM EGTA, and
0.175 mg/mL Lꢀcitrulline, pH 5.5. The reaction mixture was decanted into a 2 mL
column packet with Dowexꢀ50W ionꢀexchange resin (Na+ form) and eluted with 1.2 mL
of water. Lꢀ[3H]ꢀcitrulline was quantified by liquid scintillation spectroscopy. The
retention of Lꢀ[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 Lꢀ[3H]ꢀcitrulline produced (/mg of protein/min).
eNOS inhibition
Quantification of NO in human umbilical vein endothelial cells (HUVECs)
The investigation have been conducted according to the principles expressed in the
Declaration of Helsinki, and have been approved by the Ethics Committee for the
Human Investigation, at University of Granada, Granada, Spain (Approval no.
201302400001491). Subjects have been properly instructed and have indicated that
they consent to participate by signing the appropriate informed consent paperwork.
Endothelial cells were isolated from human umbilical cord veins using a previously
reported method with several modifications.30 The cells were cultured (Medium 199 +
20% fetal bovine serum + Penicillin/Streptomycin 2 mmol/L + Amphotericin B 2 mmol/L
+ Glutamine 2 mmol/L + HEPES 10 mmol/L + endothelial cell growth supplement 30
ꢂg/mL + Heparin 100 mg/mL) under 5% CO2 at 37°C. HUVECs were then used to
measure NO production by diaminofluoresceinꢀ2 (DAFꢀ2) fluorescence, as described
previously.30 Briefly, cells were incubated during 30 min in the presence of the 4g at the
concentration of 100 ꢂmol/L. After this period, cells were washed with PBS and then
were preꢀincubated with Lꢀarginine (100 ꢂmol/L in PBS, 5min, 37ºC). Subsequently,
DAFꢀ2 (0.1 ꢂmol/L) was incubated for 2 min and then the calcium ionophore calimycin
(A23187, 1 ꢂmol/L) was added for 30 min and cells were incubated in the dark at 37ºC.
Then the fluorescence (arbitrary units) was measured using a spectrofluorimeter
(Fluorostart, BMG Labtechnologies, Offenburg, Germany). The autoꢀfluorescence was
subtracted from each value. In some experiments, NGꢀnitroꢀLꢀarginine methyl ester (Lꢀ
NAME, 100 ꢂmol/L) was added 15 min before the addition of Lꢀarginine. The difference
between fluorescence signal without and with LꢀNAME was considered NO production.
Tissue preparation and measurement of tension
The investigation conforms to the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health (NIH Publication No. 85ꢀ23, revised
1996) and with the principles outlined in the Declaration of Helsinki and approved by
† The authors declare no compeꢀng interests.

Page 17 of 21
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DOI: 10.1039/C5MD00477B
the Ethics Committee for the welfare of experimental animals, at University of Granada,
Granada, Spain (Approval no. 459ꢀbisꢀCEEAꢀ2012). Male Wistar rats (250ꢀ300 g),
obtained from Harlam Laboratories SA (Barcelona, Spain), were euthanized by a quick
blow on the head followed by exsanguination. The descending thoracic aortic rings
were dissected, and rings were then mounted in organ chambers filled with Krebs
solution (composition in mmol/L: NaCl, 118; KCl, 4.75; NaHCO3, 25; MgSO4, 1.2;
CaCl2, 2; KH2PO4, 1.2; and glucose, 11) and were stretched to 2 g of resting tension by
means of two Lꢀshaped stainlessꢀsteel wires inserted into the lumen and attached to
the chamber and to an isometric forceꢀdisplacement transducer (UFꢀ1, Cibertec,
Madrid, Spain), and recorded in a recording and analysis system (MacLab AD
Instruments), as described previously.30 After equilibration, aortic rings with a functional
endothelium were incubated with vehicle (DMSO), 4g (100 ꢂmol/L), or LꢀNAME (100
ꢂmol/L) for 30 min, and contracted with phenylephrine (1ꢂmol/L). Once a plateau
contraction was reached, a concentrationꢀresponse curve was constructed by
cumulative addition of acetylcholine. Results are expressed as percentage of
phenylephrineꢀevoked contraction. Data are expressed as the mean ± SEM and n
reflects the number of aortic rings from different rats.
Cell viability tests
HUVECs in suspension were seeded at 1 × 104 cells per well in 96ꢀwell microtiter plate
and these cells were incubated at 37°C for up to 24 hours. The cell supernatants were
removed and replaced by fresh medium before MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide) analysis. Then the cells were exposed to serial dilutions
of 4g (10 ꢂM ꢀ 5 mM) for 30 minutes. At the chosen time, 20 ꢂL of 5 mg/mL MTT in
PBS was added to the cells and further incubated at 37 °C. After washing, 100 ꢂL of
DMSO were added in each well and absorbance at 570 nm was measured on a multiꢀ
well Plate Reader (Model 680XR, BIOꢀRAD) with subtraction of blank value at 630 nm,
and compared with control, untreated cells.
Statistical analysis
Data are expressed as the mean ± SEM. Statistically significant differences between
groups were calculated by Students´ t test for unpaired observations or for multiple
comparisons by an ANOVA followed by a Newman Keuls test. p<0.05 was considered
statistically significant.
Docking studies
The suite of programs Maestro (Schrödinger, LCC31) was used for the docking studies.
The Cartesian coordinates for the two proteins iNOS and nNOS were obtained from the
available Xꢀray structures with PDB id. 3NW2 and 1QW6, respectively (Protein Data
Bank). These receptors were prepared using the Protein Preparation Wizard
module32 implemented in Maestro. Charge and coordination sphere of the Fe(II) ion
within the heme prosthetic group were manually redefined before replacing and
minimizing all hydrogens in the receptor. LigPrep33 program was used to generate the
3Dꢀstructures of a set of conformers of 4a-q. Geometries of this set were optimized
using the MacroModel module. A rigid docking protocol was followed with Glide
program using a Standard Precision (SP) mode and taking into account the best 5
solutions. Figures were built using PyMOL (v1.3, Schrödinger, LLC).34
Acknowledgements
We are very grateful to Dr. Pedro A. SánchezꢀMurcia for his help. This work was
partially supported by the Instituto de Salud Carlos III through the grant FI11/00432 and
† The authors declare no compeꢀng interests.

MedChemComm
Page 18 of 21
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DOI: 10.1039/C5MD00477B
by Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (RIC
RD12/0042/0011).
Keywords
N,N’ꢀdisubstituted thiourea, N,N’ꢀdisubstituted urea, Inducible nitric oxide synthase
(iNOS), Neuronal nitric oxide synthase (nNOS), Oxide nitric synthase (NOS) inhibitors.
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† The authors declare no compeꢀng interests.

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N,N’-disubstituted thioureas and ureas as nNOS and iNOS inhibitors were synthesized.
Thiourea 4g was the best inhibitor without eNOS inhibition.