Development of urea and thiourea kynurenamine derivatives: Synthesis, molecular modeling, and biological evaluation as nitric oxide synthase inhibitors

Article abstract of DOI:10.1002/cmdc.201500007

Herein we describe the synthesis of a new family of kynurenamine derivatives with a urea or thiourea moiety, together with their in vitro biological evaluation as inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS, respectivel

Full text of DOI:10.1002/cmdc.201500007

DOI: 10.1002/cmdc.201500007
Full Papers
Development of Urea and Thiourea Kynurenamine
Derivatives: Synthesis, Molecular Modeling, and Biological
Evaluation as Nitric Oxide Synthase Inhibitors
Mariem Chayah,[a] M. Dora Carrión,[a] Miguel A. Gallo,[a] Rosario JimØnez,[b] Juan Duarte,[b] and
M. Encarnación Camacho*[a]
Herein we describe the synthesis of a new family of kynurena-
mine derivatives with a urea or thiourea moiety, together with
their in vitro biological evaluation as inhibitors of both neuro-
nal and inducible nitric oxide synthases (nNOS and iNOS, re-
spectively), enzymes responsible for the biogenesis of NO.
These compounds were synthesized from a 5-substituted-2-ni-
trophenyl vinyl ketone scaffold in a five-step procedure with
moderate to high chemical yields. In general, the assayed com-
pounds show greater inhibition of iNOS than of nNOS, with 1-
[3-(2-amino-5-chlorophenyl)-3-oxopropyl]-3-ethylurea
(com-
pound 5n) being the most potent iNOS inhibitor in the series
and the most iNOS/nNOS-selective compound. In this regard,
we performed molecular modeling studies to propose a bind-
ing mode for this family of compounds to both enzymes and,
thereby, to elucidate the differential molecular features that
could explain the observed selectivity between iNOS and
nNOS.
Introduction
Nitric oxide (NO) is an important second-messenger molecule
that regulates several physiological functions in the nervous,
immune, and cardiovascular systems.[1] A family of nitric oxide
synthase (NOS) enzymes catalyze the biosynthesis of NO using
l-arginine (l-Arg) as a substrate. Native NOS is a homodimeric
enzyme. Each monomer consists of an N-terminal oxygenase
domain with a catalytic heme active site and a cofactor tetra-
hydrobiopterin binding site, and a C-terminal electron-donat-
ing reductase domain binding flavin adenine dinucleotide
(FAD), flavin mononucleotide (FMN), and nicotinamide adenine
dinucleotide (NADPH).[2,3] The linker between the two function-
al domains is a calmodulin binding motif that enables electron
flow from the oxygenase domain to the reductase domain, cat-
alyzing the oxidation of l-Arg to l-citrulline with concomitant
production of NO.[4]
duces large toxic bursts of NO to fight pathogens, and is not
Ca2+-dependent.[6,7]
To exert appropriate functions, NO synthesis by the three
isozymes is under tight regulation. The overproduction of NO
by nNOS or iNOS and the underproduction by eNOS have
been associated with several pathological processes, including
neurodegenerative diseases such as Alzheimer’s,[8] Parkinson’s[9]
or Huntington’s,[10] as well as amyotrophic lateral sclerosis[11]
and chronic inflammatory[12] and autoimmune diseases such as
rheumatoid arthritis, multiple sclerosis, colitis,[13] hyperten-
sion,[14] and atherosclerosis.[15]
Accordingly, inhibition of nNOS or iNOS, but not of eNOS,
could provide an effective therapeutic approach. Moreover, se-
lective inhibitors could be useful pharmacological tools to in-
vestigate other biological functions of NO. The research objec-
tive of our group is to develop potent and selective nNOS or
iNOS inhibitors. To this end, we have carried out the publica-
tion of several families of inhibitors, some with good inhibition
and selectivity.
Three similar isoforms of NOS have been identified in mam-
mals.[5] Neuronal NOS (nNOS) and endothelial NOS (eNOS) are
constitutive and regulated by intracellular Ca2+/calmodulin.
They continually produce low levels of NO used for nerve func-
tion and blood regulation, respectively. Conversely, inducible
NOS (iNOS) is expressed by macrophages and microglia, pro-
In previous experiments, we demonstrated that melatonin 1,
the hormone secreted by the pineal gland, produces inhibitory
effects on the CNS in rats and humans.[16] These effects could
be interpreted as resulting from the inhibition of NOS. In par-
ticular, it has been shown that melatonin inhibits the nNOS iso-
[a] M. Chayah, Dr. M. D. Carrión, Prof. M. A. Gallo, Dr. M. E. Camacho
Departamento de Química FarmacØutica y Orgµnica
Facultad de Farmacia, Universidad de Granada (Spain)
E-mail: ecamacho@ugr.es
form in
manner.[17]
a
dose-dependent and calmodulin-dependent
In addition, we have described several compounds with ky-
nurenine 2,[18] kynurenamine 3,[19] and 4,5-dihydro-1H-pyrazole
4[20] structures as nNOS inhibitors. The last compounds are
rigid analogues of the main brain metabolite of melatonin, N-
acetyl-5-methoxykynurenamine (3, R1 =OCH3, R2 =CH3), which
also inhibits the iNOS in sepsis associated with Parkinson’s dis-
[b] Dr. R. JimØnez, Prof. J. Duarte
Departamento de Farmacología
Facultad de Farmacia, Universidad de Granada (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500007: characterization data for
compounds 9b–c, 9e, 9g–h, 9j, 9l–m, 9o, 10b–c, 10e, 10g--h, 10j,
10l–m, 10o, 5b–c, 5e, 5g–h, 5j, 5l--m, and 5o.
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toluenesulfonic acid yielded di-
oxolane derivatives 7a–c, re-
spectively. Reaction of these de-
rivatives with hydrazine opened
the phthalimide moiety to give
intermediates 2-(2-(5-substitut-
ed-2-nitrophenyl)-1,3-dioxolan-2-
yl)-ethanamines (8a–c). Nucleo-
philic addition of either alkyl iso-
cyanate or alkyl isothiocyanate[23]
to these derivatives gave inter-
Figure 1. Melatonin and NOS inhibitors synthesized by our research group.
mediates 9a–o (60–96% yield)
with an alkyl urea or thiourea
residue, which led to ketones
ease.[21] We have demonstrated that these pyrazole derivatives
behave as nNOS/iNOS-selective inhibitors[22] (Figure 1).
In this paper, we report the synthesis and biological evalua-
tion of a series of more flexible compounds (5). These products
were designed from kynurenamine derivatives 3, carrying
a urea or thiourea substituted group, isosteric to the final gua-
nidine moiety of the l-Arg, in order to find potent and selec-
tive inhibitors of NOS.
10a–o after acidification (61–94% yield). Finally, reduction of
the nitro group belonging to the aromatic ring with Fe/FeSO4
results in final kynurenamine derivatives 5a–o (64–95% yield).
Inhibition of iNOS and nNOS
The biological activity of new compounds 5a–o as inhibitors
of both iNOS and nNOS has been evaluated by means of in
vitro assays using recombinant isoenzymes. The assays were
performed with each compound at 1 mm to identify the most
potent and selective derivatives. Furthermore, the IC50 values
were measured for the most interesting compounds. Table 1 il-
lustrates the inhibition percentages versus iNOS and nNOS (ky-
nurenamine 3a, previously described,[19] was introduced as
a reference), and Figure 2 shows the percentages of residual
activity of both isoforms in the presence of each compound.
To interpret these biological results properly, we separately
studied the effect of each substituent (R1, R2, and X) on the po-
tency and/or selectivity of all resulting compounds.
Results and Discussion
Chemistry
The synthesis of derivatives 5a–o was carried out using the
general methodology shown in Scheme 1. Compounds 7a–
c were synthesized as previously reported[19] from 2-nitrophen-
yl, 5-methoxy-2-nitrophenyl, and 5-chloro-2-nitrophenyl vinyl
ketones 6a–c by treatment with phthalimide in the presence
of sodium methoxide to afford the N-(3-(2-nitro-5-substituted-
phenyl)-3-oxopropyl)phthalimides. Protection of the resultant
ketones by reaction with ethylene glycol in the presence of p-
In general, all compounds show better inhibition values for
iNOS than for nNOS, with the chlorinated series having the
most active compounds. Mole-
cules with neutral (R1 =H) or
electron-donating
(R1 =OCH3)
phenyl substituents have lower
inhibitory activity, indicating that
an electron withdrawing group
such as chloride is more suitable
for the inhibition of both iso-
forms. Derivatives with a methyl-
thiourea moiety (5a, 5 f, and 5k)
have, in general, good inhibition
versus both isoforms, independ-
ently of the R1 substituent.
When we compare urea and
thiourea derivatives with the
same R2 substituent, the iNOS in-
hibitory effect decreases when
the R2 volume increases in series
with R1 =H (5b–c and 5d–e)
and R1 =Cl (5l–m and 5n–o),
while in the series with R1 =
OMe, iNOS inhibition increases
Scheme 1. Synthesis of kynurenamine derivatives 5a–o. Reagents and conditions: a) Phthalimide, NaOMe, DMSO,
RT, 2 h; b) (CH2OH)2, p-TsOH, toluene, reflux, 10 h; c) NH2NH2, dry EtOH, reflux, 4.5 h; d) XCNR2, anhyd CH2Cl2, RT,
overnight; e) HCl, CH2Cl2, RT, 4 h; f) Fe/FeSO4, H2O, 958C, 3 h.
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Table 1. In vitro iNOS and nNOS inhibition observed in the presence of
compounds 5a–o.
Table 2. Inhibition of nNOS and iNOS activity by compounds 5a, 5 f, 5l,
5n, and 5o.
Compd
IC50 [mm][a]
nNOS
0.18
iNOS
5a
5 f
5l
5n
5o
0.58
0.11
0.59
0.10
0.86
[b]
–
0.59
[b]
–
Compd
R1
R2
X
Inhibition [%][a]
0.43
iNOS
nNOS
[a] Data obtained by measuring percent inhibition with at least five inhib-
itor concentrations. [b] Not tested.
3a
5a
5b
5c
5d
5e
5 f
5g
5h
5i
5j
5k
5l
5m
5n
5o
OMe
H
H
H
H
Me
Me
Et
Pr
Et
Pr
Me
Et
Pr
Et
Pr
Me
Et
Pr
Et
–
S
S
30.90Æ2.92
71.71Æ2.43
45.83Æ1.73
38.88Æ3.59
50.93Æ3.85
38.03Æ3.72
78.20Æ2.43
17.71Æ2.74
39.30Æ0.99
22.08Æ1.57
55.28Æ1.24
51.29Æ1.32
77.86Æ1.08
51.91Æ1.28
78.63Æ1.34
59.58Æ2.34
65.36Æ5.60[b]
77.67Æ1.02
12.93Æ3.45
47.48Æ2.97
11.80Æ0.39
18.25Æ0.87
46.04Æ2.85
40.30Æ0.32
47.21Æ1.23
0.47Æ3,34
S
O
O
S
S
S
O
O
S
S
S
H
except 5o. This effect can be observed clearly in compound
5n which, besides having very good inhibitory activity, had
eightfold selectivity for iNOS over nNOS. Table 2 shows the IC50
values of the most interesting synthesized kynurenamine deriv-
atives for both isoenzymes. Compounds 5 f and 5n had the
best results versus iNOS, confirming the potency and selectivi-
ty of 5n. With respect to nNOS, 5a showed the best IC50 value.
OMe
OMe
OMe
OMe
OMe
Cl
Cl
Cl
Cl
Cl
14.64Æ0.65
60.84Æ0.82
69.78Æ1.05
53.42Æ1.46
9.86Æ3.17
O
O
Pr
74.08Æ1.04
[a] Values are the mean ÆSEM of the percentage of iNOS and nNOS in-
hibition produced by each compound at 1 mm, determined from three
experiments performed in triplicate using recombinant iNOS and nNOS
enzymes. [b] Kynurenamine 3a was used as a reference; see ref. [19].
Effects of 5n on LPS-induced vascular hyporeactivity to
noradrenaline (NA) and eNOS inhibition
The most interesting compound in this family was 5n. To test
functional evidence and to ensure the iNOS selectivity of this
compound, we performed a pharmacological essay, using de-
scending thoracic aortic rings. LPS inhibited agonist-induced
contractions of isolated vascular preparations.[24] This effect is
related to NO overproduction from the induction of iNOS in
vascular smooth muscle. In fact, aminoguanidine, a preferential
inhibitor of iNOS, reverses the blunted phenylephrine-evoked
contraction of endothelium-denuded aortic rings from LPS-
treated rats or rings exposed to LPS in vitro. Aminoguanidine
did not impair the relaxation of aortic rings with endothelium
to acetylcholine, a known stimulator of eNOS.[25] As shown in
Figure 3a, incubation of endotoxin decreased the contractile
responses elicited by NA in aortic rings without endothelium.
Incubation of aortic rings with the NOS inhibitor l-NAME sig-
nificantly restored NA responses from the LPS group, showing
that NO is involved in the attenuated vascular reactivity to NA
induced by endotoxin (Figure 3b). Furthermore, the presence
of 5n partially prevented endotoxin-induced NA contractile hy-
poresponsiveness.
Figure 2. Residual activities of nNOS and iNOS in the presence of tested ky-
nurenamine derivatives (compounds 5a–o) at 1 mm, as compared with
those of untreated samples (C). Each value is the mean ÆSEM of three ex-
periments performed in triplicate using recombinant iNOS and nNOS en-
zymes.
We also carried out an eNOS inhibitory activity assay for 5n,
using human umbilical vein endothelial cells (HUVECs) incubat-
ed with 1 mm of 5n or vehicle and measuring the NO produc-
tion stimulated by the known eNOS activator A23187. This
agent increased NO production in a time-dependent manner.
No significant differences (p>0.05) were observed in A23187
stimulated NO production in the presence of 5n, showing that
this compound did not inhibit eNOS (Figure 3c). Moreover, the
endothelium-dependent relaxation by acetylcholine was not
affected by 5n (Figure 3d), confirming the absence of eNOS in-
hibition by this compound.
with the volume of R2 (5g–h and 5i–j). On the other hand,
nNOS inhibition values increase with the volume of the R2 sub-
stituent from an ethyl to a propyl group, with the exception of
5l–m, for which the opposite is true.
In this new family of kynurenamines, the urea residue plays
an important role in compound selectivity. All compounds
with a urea group inhibit iNOS to a greater extent than nNOS,
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tween the aromatic ring and the
heme group, together with the
hydrogen bonding network in-
volving the urea moiety. In par-
ticular, those compounds bear-
ing a phenyl group with an ori-
entation that is too inclined with
respect to the heme group lose
activity. In addition, the estab-
lishment of two hydrogen bonds
through the urea moiety reinfor-
ces the interaction and hence,
the activity.
Next, we performed molecular
dynamics (MD) simulations of
iNOS and nNOS in complex with
compound 5n to take into ac-
count the flexibility of the pro-
tein (Figure 5). The analysis of
the
trajectory
(Figure 5a)
showed that those interactions
described previously between
the NH2 group of 5n and
Glu371 from one side, and be-
tween the heme carboxylate
moiety with the 5n urea moiety
from the other side, were re-
tained during the MD simula-
tions. However, Figure 5b shows
new hydrogen bonds between
Figure 3. Effects of 5n on NA-induced contraction in LPS-treated rings and eNOS activation. NA-evoked contrac-
tion of rat aortic rings (n=6–8) without endothelium treated in vitro: a) with or without LPS (1 mgmLÀ1, 20 h), or
b) in the presence or absence of l-NAME (3”10À4 m), 5n (10À4 m), or DMSO, 30 min before NA. c) A23187-depen-
dent NO production in HUVECs incubated with DMSO or 5n (1 mm) for 30 min (n=12). d) Acetylcholine-evoked
relaxation in aortic rings with endothelium contracted with 1 mm NA in the presence of DMSO or 5n (1 mm) for
30 min (n=5). Data are expressed as the mean ÆSEM of n experiments; **p<0.01 versus LPS group.
Molecular modeling
the urea residue and the heme carboxylate moiety. Further-
more, the main differences between the MD simulations of
both isoforms with 5n were essentially the new additional sta-
bilizing interactions that 5n establishes with residues Gln257
and Arg260 (iNOS), located at the entrance of the binding site,
together with stacking with the heme group. In particular, the
conformation of Gln257 is controlled by the Arg260 side chain
orientation which, in turn, depends on the mutation of Thr277
in iNOS and Asn498 in nNOS.[28] These interactions, detected
from the simulation process, could explain the iNOS selectivity
shown by compounds like 5n and the good nNOS inhibition
observed for some compounds.
To propose a binding mode for these new kynurenamine deriv-
atives (5a–o) to i/nNOS, we performed docking and molecular
dynamics (MD) simulation studies. The main descriptive pose
obtained by docking for this family of compounds within iNOS
(PDB ID: 3NW2)[26] is illustrated in Figure 4a with compound
5n. According to this pose, interactions with the protein occur
in three ways: 1) each urea nitrogen is hydrogen bonded to
a carboxylate moiety of the heme group, 2) the 2“-NH2 group
of 5n interacts with the Glu371 side chain by means of two
hydrogen bonds, and 3) the phenyl ring is situated below the
heme group, establishing a p–cation interaction, and is sur-
rounded by hydrophobic residues such as Phe363, Val346, and
Pro344.
The binding of 5n to iNOS was more favorable than to
nNOS (À31.8Æ0.8 versus À27.3Æ2.2 kcalmolÀ1, respectively),
due to the great contribution of the p-stacking interaction
with the heme group. Finally, the binding energy per residue
(Table 3) observed for Gln478 and Arg481 (nNOS) with 5n was
less favorable than for the corresponding residues in iNOS
(Gln257 and Arg260).
On the other hand, Figure 4b shows the main pose adopted
by most compounds within nNOS (PDB ID: 1QW6).[27] In this
case, only one hydrogen bond is formed between the urea
amine group of 5n and the carboxylate group of Glu592. The
urea moiety is surrounded by a polar environment formed by
three arginine residues (Arg596, Arg603 and Arg481). It is
noteworthy that the phenyl is now located below the heme
group in a different position with respect to that observed in
iNOS, allowing the chloride atom of the phenyl group to inter-
act with Trp587.
Conclusions
Herein we report the synthesis of fifteen novel urea and thiour-
ea kynurenamine derivatives 5, each with different substituents
in the aromatic ring and the urea or thiourea moiety. In addi-
tion, we evaluate the nNOS and iNOS activity of these new
structures. In general, the compounds had better inhibitory ac-
With regard to the differences found within the series of
compounds 5a–o, the major variations observed in their inter-
action with the binding site are in the p–cation interaction be-
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Figure 4. Detailed view of the main poses obtained for compound 5n in the
a) iNOS and b) nNOS binding sites. Dotted lines indicate hydrogen bond in-
teractions between the ligand and residues of the enzyme.
Figure 5. Image of a) 5n–iNOS and b) 5n–nNOS complexes taken from both
average structures obtained from each MD simulation. Dotted lines indicate
hydrogen bond interactions between the ligand and the residues of the
enzyme.
Table 3. Calculated binding energy per residue of iNOS/nNOS with com-
pound 5n.[a]
quently, this derivative could be an interesting starting point
to find new therapeutic alternatives for inflammatory diseases.
iNOS
E [kcalmolÀ1
nNOS
E [kcalmolÀ1
]
Residue
]
Residue
Heme
À11.8Æ0.4
À2.5Æ0.1
À2.5Æ0.1
À2.5Æ0.1
À2.0Æ0.1
À1.3Æ0.1
À0.9Æ0.0
À0.7Æ0.1
À0.7Æ0.1
Heme
À6.2Æ0.5
À2.6Æ0.1
À6.5Æ0.2
À0.6Æ0.7
À1.3Æ0.1
À0.4Æ0.2
À1.2Æ0.1
À0.7Æ0.0
À0.9Æ0.0
Pro344
Glu371
Gln257
Val346
Arg260
Trp366
Gly365
Phe363
Pro565
Glu592
Gln478
Val567
Arg481
Trp587
Gly586
Phe584
Experimental Section
Chemistry
Melting points were determined using an Electrothermal-1 A-6301
apparatus and are uncorrected. Analytical thin layer chromatogra-
phy was performed using Merck Kieselgel 60 F254 aluminum sheets,
with the spots developed with UV light (l=254 nm). Flash chro-
matography was carried out using silica gel 60, 230–240 mesh
(Merck), and the solvent mixture reported within parentheses was
[a] Data are the mean ÆSD of the calculated binding energies along the
MD simulation (single run).
1
used as the eluent. H NMR and 13C NMR spectra were recorded on
a Varian Inova Unity 300 spectrometer operating at 300.20 MHz for
1H and 75.479 MHz for 13C, on a Varian direct drive 400 spectrome-
tivity against iNOS than nNOS, and derivatives with a withdraw-
ing substituent in the aromatic ring were the best inhibitors.
Thioureas similarly inhibited both isoenzymes, while ureas se-
lectively inhibited iNOS. The urea 5n was the most potent
iNOS inhibitor and the most iNOS/nNOS-selective. This was
confirmed by docking and MD simulations studies, which
showed the more favorable orientation of 5n in iNOS estab-
lishing good interactions with the enzyme. Also, this com-
pound did not inhibit eNOS, demonstrating the selectivity nec-
essary to avoid the adverse effects of hypertension. Conse-
1
ter operating at 400.17 MHz for H and 125.69 MHz for 13C, and on
a Varian direct drive 600 spectrometer operating at 600.24 MHz for
1H and 150.95 MHz for 13C in CDCl3, CD3OD, (CD3)2CO, and (CD3)2SO
at room temperature. The peaks are reported in ppm (d) and are
referenced to the residual solvent peak. High-resolution nano-as-
sisted laser desorption/ionization (HR-NALDI) or electrospray ioni-
zation mass spectra (ESI-MS) were carried out on a Bruker Autoflex
or a Waters LCT Premier mass spectrometer, respectively.
General method for the preparation of 1-(2-(2-(5-substituted-2-
nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-alkylthioureas and alkyl-
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ureas, 9a–o: Hydrazine (95%, 6.18 mmol) was added to a solution
of the corresponding phthalimide 7a–c (2.06 mmol) in dry EtOH
(60 mL). The mixture was held at reflux for 4.5 h, then cooled to
room temperature and filtered, and the solvent was removed by
evaporation. The crude residue was diluted with water (30 mL), ba-
sified with KOH, and extracted with EtOAc. The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated to give
amines 8a–c. These intermediates were dissolved in anhydrous
CH2Cl2 (9 mL) under argon atmosphere, and the corresponding al-
kylisocyanate or alkylisothiocyanate (4.12 mmol) was added drop-
wise. The reaction mixture was stirred overnight at room tempera-
ture. The crude mixture was concentrated and purified by flash
chromatography (EtOAc/hexane, 1:2).
A solution of concentrated HCl (13.5 mL, 12n) was added to a solu-
tion of the corresponding dioxolane precursor 9a–o (0.995 mmol)
in CH2Cl2 (6.8 mL). The mixture was stirred for 4 h at room temper-
ature and then quenched with an aqueous saturated solution of
KOH until achieving a basic pH. The former aqueous phase was ex-
tracted with CH2Cl2 (3”15 mL), and the organic layers were
washed with brine (1”15 mL), dried on anhydrous Na2SO4, filtered,
and concentrated under vacuum. The crude mixture was purified
by flash chromatography (EtOAc/hexane, 1:1).
1-(3-(2-Nitrophenyl)-3-oxopropyl)-3-methylthiourea, 10a: Yellow
oil (0.61 mmol, 61%); 1H NMR (400 MHz, CDCl3): d=2.94 (s, 3H),
3.16–3.23 (m, 2H), 4.01–4.09 (m, 2H), 6.51 (bs, 2H), 7.42 (d, 1H),
7.57–7.61 (m, 1H), 7.69–7.71 (m, 1H), 8.06 ppm (d, 1H); 13C NMR
(125 MHz, CDCl3): d=32.8, 39.5, 42.5, 124.5, 127.3, 131.1, 134.5,
137.2, 145.8, 181.5, 202.5 ppm; HRMS m/z [M+H]+ calcd for
C11H14N3O3S: 267.3049, found: 267.3052.
1-(2-(2-(2-Nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-methylthiourea,
9a: Yellow solid (1.96 mmol, 95%); mp: 122–1238C; 1H NMR
(600 MHz, CDCl3): d=2.48 (t, 2H), 2.95 (s, 3H), 3.64–3.70 (m, 4H),
3.99–4.06 (m, 2H), 7.42–7.43 (m, 2H), 7.51 (ddd,1H), 7.59 ppm (d,
1H); 13C NMR (150 MHz, CDCl3): d=30.3, 38.1, 40.0, 65.0, 109.7,
123.5, 128.4, 129.8, 131.6, 134.7, 149.5, 180.8 ppm; HRMS m/z [M+
H]+ calcd for C13H18N3O4S: 312.1018, found: 312.1016.
1-(3-(2-Nitrophenyl)-3-oxopropyl)-3-ethylurea, 10d: Yellow solid
1
(0.61 mmol, 61%); mp: 133–1348C; H NMR (300 MHz, CDCl3): d=
1.10 (t, 3H), 3.03 (t, 2H), 3.17 (q, 2H), 3.62 (t, 2H), 7.40 (dd, 1H),
7.55–7.61 (m, 1H), 7.67–7.73 (m, 1H), 8.07 ppm (d, 1H); 13C NMR
(75 MHz, CDCl3): d=15.6, 35.4, 35.6, 43.4, 124.7, 127.5, 131.0, 134.5,
137.6, 145.9, 158.5, 202.6 ppm; HRMS m/z [M+Na]+ calcd for
C12H15N3O4Na: 288.0954, found: 288.0960.
1-(2-(2-(2-Nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-ethylurea, 9d:
1
White solid (1.46 mmol, 71%); mp: 168–1698C; H NMR (300 MHz,
CDCl3): d=1.12 (t, 3H), 2.36 (t, 2H), 3.17 (q, 2H), 3.36 (t, 2H), 3.62–
3.67 (m, 2H), 3.96–4.02 (m, 2H), 7.37–7.42 (m, 2H), 7.45–7.52 (m,
1H), 7.55–7.60 ppm (m, 1H); 13C NMR (75 MHz, CDCl3): d=15.6,
35.7, 39.4, 65.1, 109.5, 123.4, 128.6, 129.7, 131.5, 135.2, 149.7,
158.5 ppm; HRMS m/z [M+H]+ calcd for C14H20N3O5: 332.1228,
found: 332.1222.
1-(3-(5-Methoxy-2-nitrophenyl)-3-oxopropyl)-3-methylthiourea,
10 f: Yellow solid (0.83 mmol, 84%); mp: 176–1808C; 1H NMR
(300 MHz, (CD3)2CO): d=2.92 (d, 3H), 3.15 (t, 2H), 3.87–3.93 (m,
2H), 3.96 (s, 3H), 6.91 (bs, 1H), 6.96 (bs, 1H), 7.06 (d, 1H), 7.16 (dd,
1H), 8.13 ppm (d, 1H); 13C NMR (75 MHz, (CD3)2CO): d=30.2, 39.4,
42.4, 56.4, 112.5, 115.6, 127.2, 138.6, 140.8, 164.7, 184.3, 201.3 ppm;
HRMS m/z [M+Na]+ calcd for C12H15N3O4SNa: 320.0680, found:
320.0681.
1-(2-(2-(5-Methoxy-2-nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-
methylthiourea, 9 f: Yellow oil (1.48 mmol, 72%); 1H NMR
(300 MHz, CDCl3): d=2.50–2.55 (m, 2H), 2.95 (d, 3H), 3.65–3.73 (m,
4H), 3.85 (s, 3H), 3.99–4.04 (m, 2H), 5.97 (bs), 6.43 (bs), 6.86 (dd,
1H), 7.06 (d, 1H), 7.48 ppm (d, 1H); 13C NMR (75 MHz, CDCl3): d=
30.5, 38.0, 40.4, 56.2, 65.1, 109.5, 113.4, 114.3, 126.2, 137.7, 143.0,
161.9, 182.2 ppm; HRMS m/z [M+Na]+ calcd for C14H19N3O5SNa:
364.0940, found: 364.0943.
1-(3-(5-Methoxy-2-nitrophenyl)-3-oxopropyl)-3-ethylurea,
10i:
1
Yellow solid (0.73 mmol, 73%); mp: 146–1478C; H NMR (300 MHz,
CDCl3): d=1.14 (t, 3H), 2.99 (t, 2H), 3.21 (q, 2H), 3.65 (t, 2H), 3.90
(s, 3H), 6.75 (d, 1H), 6.98 (dd, 1H), 8.13 ppm (d, 1H); 13C NMR
(75 MHz, CDCl3): d=15.4, 35.5, 35.9, 43.5, 56.6, 112.0, 115.4, 127.4,
138.2, 140.6, 158.7, 164.7, 202.6 ppm; HRMS m/z [M +Na]+ calcd
for C13H17N3O5Na: 318.1072, found: 318.1066.
1-(2-(2-(5-Methoxy-2-nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-eth-
1
ylurea, 9i: Yellow oil (1.38 mmol, 67%); H NMR (300 MHz, CDCl3):
d=0.92 (t, 3H), 2.72 (t, 2H), 3.30 (q, 2H), 3.36 (t, 2H), 3.63–3.67 (m,
1H), 3.84 (s, 3H), 4.15–4.20 (m, 2H), 6.48 (bs, 2H), 6.84 (dd, 1H),
7.13 (d, 1H), 7.46 ppm (d, 1H); 13C NMR (75 MHz, CDCl3): d=15.5,
35.6, 39.0, 41.8, 55.6, 64.8, 109.5, 113.2, 114.1, 125.7, 137.6, 144.9,
158.6, 161.4 ppm; HRMS m/z [M+H]+ calcd for C15H22N3O6:
340.1416, found: 340.1427.
1-(3-(5-Chloro-2-nitrophenyl)-3-oxopropyl)-3-methylthiourea,
10k: Yellow solid (0.61 mmol, 61%); mp: 129–1308C; 1H NMR
(300 MHz, CDCl3): d=2.96 (s, 3H), 3.17 (t, 2H), 4.07 (t, 2H), 7.36 (d,
1H), 7.56 (dd, 1H), 8.07 ppm (d, 1H); 13C NMR (75 MHz, CDCl3): d=
29.9, 39.7, 42.6, 126.4, 127.5, 131.1, 138.9, 141.7, 144.0, 181.8,
201.1 ppm; HRMS m/z [M+Na]+ calcd for C11H12N3O3SClNa:
324.0185, found: 324.0186.
1-(2-(2-(5-Chloro-2-nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-meth-
ylthiourea, 9k: Yellow solid (1.40 mmol, 68%); mp: 164–1658C;
1H NMR (300 MHz, CDCl3): d=2.45–2.49 (m, 2H), 2.95 (s, 3H), 3.67–
3.73 (m, 4H), 4.02–4.06 (m, 2H), 7.39–7.40 (m, 2H), 7.57–7.59 ppm
(m, 1H); 13C NMR (75 MHz, CDCl3): d=30.5, 38.1, 40.1, 65.3, 109.1,
125.2, 128.6, 130.0, 137.2, 137.9, 147.9, 181.7 ppm; HRMS m/z [M+
Na]+ calcd for C13H16N3O4SClNa: 346.0839, found: 346.0826.
1-(3-(5-Chloro-2-nitrophenyl)-3-oxopropyl)-3-ethylurea,
10n:
1
White solid (0.62 mmol, 62%); mp: 149–1508C; H NMR (300 MHz,
CDCl3): d=1.14 (t, 3H), 3.04 (t, 2H), 3.20 (q, 2H), 3.64 (t, 2H), 4.87
(bs, 2H), 7.36 (d, 1H), 7.54 (dd, 1H), 8.06 ppm (d, 1H); 13C NMR
(75 MHz, CDCl3): d=16.5, 36.7, 37.1, 44.5, 127.4, 128.8, 132.1, 142.8,
145.1, 160.0, 202.0; HRMS m/z [M+H]+ calcd for C12H15N3O4Cl:
300.0760, found: 300.0751.
1-(2-(2-(5-Chloro-2-nitrophenyl)-1,3-dioxolan-2-yl)ethyl)-3-ethyl-
urea, 9n: Yellow oil (1.26 mmol, 61%); 1H NMR (400 MHz, CDCl3):
d=1.10 (t, 3H), 2.35 (t, 2H), 3.17 (q, 2H), 3.29 (t, 2H), 3.65–3.67 (m,
2H), 4.02–4.04 (m, 2H), 7.36 À7.37 (m, 2H), 7.57–7.59 ppm (m,
1H); 13C NMR (125 MHz, CDCl3): d=15.3, 35.4, 35.4, 42.4, 64.9,
108.9, 124.7, 128.4, 129.4, 137.2, 137.3, 147.7, 158.8 ppm; HRMS
m/z [M+H]+ calcd for C14H19N3O5Cl: 344.7523, found: 344.7528.
General method for the preparation of 1-(3-(2-aminophenyl-5-
substituted)-3-oxopropyl)-3-alkylthioureas and alkylureas, 5a–o:
A mixture of nitro precursor 10a–o (0.93 mmol), iron powder
(9.3 mmol), and FeSO4 (0.93 mmol) in water (19.5 mL) was stirred
and heated for 3 h at 958C. Next, the suspension was filtered
through Celite and thoroughly washed with CH2Cl2. The aqueous
phase was extracted with CH2Cl2 (3”15 mL). The merged organic
layer was washed with brine (1”15 mL), dried (Na2SO4), filtered,
General method for the preparation of 1-(3-(5-substituted-2-ni-
trophenyl)-3-oxopropyl)-3-alkylthioureas and alkylureas, 10a–o:
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and evaporated under vacuum. The crude mixture was purified by
flash chromatography (EtOAc/hexane, 1:1).
Biological assays
In vitro nNOS and iNOS activity determination: l-Arginine, l-citrul-
line, N-(2-hydroxymethyl)piperazine-N’-(2-ethanesulfonic acid)
1-(3-(2-Aminophenyl)-3-oxopropyl)-3-methylthiourea, 5a: Yellow
solid (0.76 mmol, 82%); mp: 127–1288C; H NMR (300 MHz, CDCl3):
(HEPES), d,l-dithiothreitol (DTT), hypoxanthine-9-b-d-ribofurano-
side (inosine), (ethylene glycol)-bis-(2-aminoethylether)-N,N,N’,N’-
tetraacetic acid (EGTA), bovine serum albumin (BSA), Dowex-50W
(50”8–200), FAD, NADPH, and 5,6,7,8-tetrahydro-l-biopterin dihy-
1
d=2.96 (s, 3H), 3.35 (t, 2H), 3.98–4.04 (m, 2H), 5.30 (bs, 2H), 6.30
(bs, 1H), 6.51 (bs, 1H), 6.64–6.72 (m, 2H), 7.28–7.34 (m, 1H),
7.73 ppm (dd, 1H); 13C NMR (75 MHz, CDCl3): d=30.3, 38.5, 40.1,
116.3, 117.5, 117.6, 131.2, 135.0, 150.3, 182.2, 201.9 ppm; HRMS m/z
[M+Na]+ calcd mass for C11H15N3OSNa: 260.0834, found: 260.0838;
Anal. calcd for C11H15N3OS: C 55.67, H 6.37, N 17.71, found: C 55.35,
H 6.31, N 17.58.
drochloride
(H4-biopterin),
tris-(hydroxymethyl)aminomethane
(Tris·HCl), and calcium chloride were obtained from Sigma–Aldrich
Química (Spain). l-[3H]arginine (47.4 CimmolÀ1) was obtained from
PerkinElmer (Spain). Calmodulin from bovine brain and recombi-
nant iNOS and nNOS were obtained from Enzo Life Sciences
(Spain).
1-(3-(2-Aminophenyl)-3-oxopropyl)-3-ethylurea, 5d: White solid
(0.74 mmol, 80%); mp: 139–1418C; H NMR (300 MHz, CDCl3): d=
i/nNOS activity was measured by the Bredt and Snyder method,[29]
monitoring the conversion of l-[3H]arginine to l-[3H]citrulline. The
final incubation volume was 100 mL and consisted of 10 mL of an
aliquot of recombinant i/nNOS added to a buffer with a final con-
centration of 25 mm Tris·HCl, 1 mm DTT, 4 mm H4-biopterin, 10 mm
FAD, 0.5 mm inosine, 0.5 mgmLÀ1 BSA, 0.1 mm CaCl2, 10 mm l-argi-
nine, 10 mgmLÀ1 calmodulin (only for nNOS), and 50 nm l-
[3H]arginine, at pH 7.6 and 7.0 for iNOS and nNOS, respectively.
The reaction was started by the addition of 10 mL of 7.5 mm
NADPH and 10 mL of each kynurenamine derivative in EtOH (20%)
to give a final concentration of 1 mm. The tubes were vortexed
and incubated at 378C for 30 min. Control incubations were per-
formed by the omission of NADPH. The reaction was halted by the
addition of 400 mL of cold 0.1m HEPES, 10 mm EGTA, and
0.175 mgmLÀ1 l-citrulline, pH 5.5. The reaction mixture was deca-
nted into a 2 mL column packet with Dowex-50W ion-exchange
resin (Na+ form) and eluted with 1.2 mL 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 pmol l-[3H]citrulline produced per
mg protein per min.
1
1.09 (t, 3H), 3.11–3.19 (m, 4H), 3.57 (t, 2H), 4.77 (bs, 4H), 6.59–6.66
(m, 2H), 7.24 (ddd, 1H), 7.69 ppm (dd, 1H); 13C NMR (75 MHz,
CDCl3): d=15.4, 35.4, 35.5, 39.4, 116.1, 117.4, 117.9, 131.2, 134.6,
150.2, 158.8, 202.0 ppm; HRMS m/z [M+Na]+ calcd mass for
C12H17N3O2Na: 258.1218, found: 258.1227. Anal. calcd for
C12H17N3O2: C 61.26, H 7.28, N 17.86, found: C 61.53, H 7.55; N
17.46.
1-(3-(2-Amino-5-methoxyphenyl)-3-oxopropyl)-3-methylthiourea,
5 f: Yellow solid (0.87 mmol, 94%); mp: 125–1268C; 1H NMR
(300 MHz, CDCl3): d=2.90 (d, 3H), 3.27 (t, 2H), 3.74 (s, 3H), 3.93–
4.01(m, 2H), 5.95 (bs, 2H), 6.27 (bs, 1H), 6.47 (bs, 1H), 6.60 (d, 1H),
6.95 (dd, 1H), 7.12 ppm (d, 1H); 13C NMR (75 MHz, CDCl3): d=30.4,
38.9, 40.3, 56.3, 113.2, 117.5, 119.1, 124.6, 145.4, 150.5, 182.4,
201.6 ppm; HRMS m/z: [M+Na]+ calcd mass for C12H17N3O2SNa:
290.0939, found: 290.0940. Anal. calcd for C12H17N3O2S: C 53.91, H
6.41, N 15.72, S 11.99, found: C 53.55; H 6.79, N 15.11, S 11.73.
1-(3-(2-Amino-5-methoxyphenyl)-3-oxopropyl)-3-ethylurea, 5i:
1
Yellow solid (0.83 mmol, 89%); mp: 115–1178C; H NMR (300 MHz,
CDCl3): d=1.06 (t, 3H), 3.10–3.18 (m, 4H), 3.57 (t, 2H), 3.74 (s, 3H),
5.21 (bs, 4H), 6.73 (d, 1H), 6.95 (dd, 1H), 7.16 ppm (d, 1H);
13C NMR (75 MHz, CDCl3): d=15.6, 35.6, 35.6, 39.9, 56.2, 113.8,
119.3, 120.1, 123.5, 142.8, 151.5, 158.6, 201.9 ppm; HRMS m/z [M+
Na]+ calcd mass for C13H19N3O3Na: 288.1324, found: 288.1329.
Anal. calcd for C13H19N3O3: C 58.85, H 7.22, N 15.84, found: C 59.17,
H 7.55, N 15.57.
Tissue preparation and measurement of tension: This investigation
conformed to the Guide for the Care and Use of Laboratory Ani-
mals published by the U.S. National Institutes of Health (NIH Publi-
cation No. 85–23, revised 1996) and with the principles outlined in
the Declaration of Helsinki and approved by our institutional
review board. Male Wistar rats (250–300 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 the endothelium was re-
moved mechanically and incubated in Krebs solution (118 mm
NaCl, 4.75 mm KCl, 25 mm NaHCO3, 1.2 mm MgSO4, 2 mm CaCl2,
1.2 mm KH2PO4, and 11 mm glucose) at 378C and gassed with 95%
O2 and 5% CO2 for 20 h in a cell culture incubator in the absence
or presence of Escherichia coli 055:B5 lipopolysaccharide (LPS,
1 mgmLÀ1) (Sigma–Aldrich, Spain). Rings were then mounted in
organ chambers filled with Krebs solution and were stretched to
2 g of resting tension by means of two L-shaped stainless steel
wires inserted into the lumen and attached to the chamber and to
an isometric force-displacement transducer (UF-1, Cibertec, Spain)
and recorded in a recording and analysis system (MacLab AD In-
struments), as described previously.[30] After equilibration of aortic
1-(3-(2-Amino-5-chlorophenyl)-3-oxopropyl)-3-methylthiourea,
5k: Yellow solid (0.65 mmol, 70%); mp: 166–1688C; 1H NMR
(300 MHz, (CD3)2SO): d=2.76 (s, 3H), 3.16 (t, 2H), 3.61–3.63 (m,
2H), 6.76 (d, 1H), 7.24 (dd, 1H), 7.40 (bs, 2H), 7.75 ppm (d, 1H);
13C NMR (75 MHz, (CD3)2SO): d=31.1, 38.8, 40.0, 117.8, 119.7, 121.7,
130.8, 134.7, 150.5, 181.4, 200.6 ppm; HRMS m/z [M+H]+ calcd
mass for C11H15N3OSCl: 272.0624, found: 272.0609. Anal. calcd for
C11H14N3OS: C 48.61, H 5.19, N 15.46, found: C 53.63, H 6.20, N
12.57.
1-(3-(2-Amino-5-chlorophenyl)-3-oxopropyl)-3-ethylurea,
5n:
1
Yellow solid (0.70 mmol, 75%); mp: 157–1608C; H NMR (300 MHz,
CDCl3): d=1.10 (t, 3H), 3.13–3.21 (m, 4H), 3.58 (t, 2H), 4.59 (bs,
4H), 6.60 (d, 1H), 7.19 (dd, 1H), 7.63 ppm (d, 1H); 13C NMR
(75 MHz, CDCl3): d=15.5, 35.8, 35.8, 39.6, 118.6, 119.2, 120.8, 130.5,
135.0, 148.8, 158.6, 201.2 ppm; HRMS m/z [M+Na]+ calcd mass for
C12H16N3O2ClNa: 292.0829, found: 292.0835. Anal. calcd for
C12H16N3O2Cl: C 53.43, H 5.98, N 15.58 found: C 53.11, H 5.64, N
15.36.
rings, concentration–response curves for noradrenaline (NA, 10À9
–
10À5 m) were performed by increasing the concentration in the
organ chamber in cumulative increments after a steady-state re-
sponse was reached with each increment. In some experiments,
the non-selective NOS inhibitor, NG-nitro-arginine methyl ester (l-
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NAME, 3”10À4 m), compound 5n (10À4 m), or vehicle (DMSO) alone
were added to the organ chamber 30 min before the addition of
NA. The rings were then removed from the organ chambers, air-
dried for 24 h, and weighed. The contraction was expressed in
grams of force per milligram of dried tissue. The area under the
concentration–response curve (AUC) was measured.
Each enzyme–inhibitor complex was immersed in a cubic box of
TIP3P water molecules with a minimum distance of 12 Š from any
atom to the edge. The system was neutralized by incorporating
the necessary amount of sodium ions. Particle Mesh Ewald method
for long-range electrostatic interactions, together with standard
periodic boundary conditions, were used. The SHAKE algorithm
was applied to reach an integration step of 2.0 fs.
In another experiment, aortic rings with a functional endothelium
were incubated with vehicle or 5n (1 mm) for 30 min and contract-
ed with NA (1 mm). Once a plateau contraction was reached, a con-
centration–response curve was constructed by cumulative addition
of acetylcholine. Results are expressed as percentage of NA-evoked
contraction. n reflects the number of aortic rings from different
rats.
Before the simulation, the system was energetically minimized
with a protocol of: 2000 steps of steepest descent, followed by
3000 steps of conjugate gradient. In this protocol, hydrogen atoms
were first optimized, freezing the rest of the system, then only the
water molecules were allowed to move, and finally the whole
system was allowed to move. Next, the system was heated to
300 K in 20 ps at 1 atm using the Berendsen thermostat and a re-
straint constant on the alpha carbon of protein residues, ligands,
and cofactors. The restraint was then decreased slowly in the equi-
libration step. Finally, 2 ns of production simulation were per-
formed, collecting the coordinates of the system at each 1 ps. The
binding free energy was calculated using the MM-ISMSA model,[40]
and the estimated error was obtained with the block averaging
method implemented in the g analyse tool of the GROMACS
suite.[41]
Quantification of NO in human umbilical vein endothelial cells
(HUVECs): Endothelial cells were isolated from human umbilical
cord veins using a previously reported method with several modifi-
cations.31 The cells were cultured in Medium 199 with 20% fetal
bovine serum and penicillin/streptomycin (2 mm), amphotericin B
(2 mm), glutamine (2 mm), HEPES (10 mm), endothelial cell growth
supplement (30 mgmLÀ1), and heparin (100 mgmLÀ1) under 5%
CO2 at 378C. HUVECs were then used to measure NO production
by diaminofluorescein-2 (DAF-2) fluorescence, as described previ-
ously.[31] Briefly, cells were incubated for 30 min in the presence of
5n at a concentration of 1 mm. After this period, cells were
washed with PBS and then were pre-incubated with l-arginine
(100 mm in PBS for 5 min at 378C). Subsequently, DAF-2 (0.1 mm)
was incubated for 2 min, then the calcium ionophore calimycin
(A23187, 1 mm) was added for 30 min, and cells were incubated in
the dark at 378C. Fluorescence (arbitrary units) was then measured
using a spectrofluorimeter (Fluorostart, BMG Labtechnologies, Of-
fenburg, Germany). The auto-fluorescence was subtracted from
each value. The area under the time–fluorescence curve (AUC) was
measured.
Acknowledgements
This work was supported in part by the Instituto de Salud Carlo-
s III through grant FI11/00432 and project RIC RD12/0042/0011.
Keywords: biological activity · inhibitors · kynurenamines ·
molecular modeling · nitric oxide synthase
Statistical analysis: Data are expressed as the mean ÆSEM. Statisti-
cally significant differences between groups were calculated by
Students’ t test for unpaired observations or for multiple compari-
sons. ANOVA, followed by the Newmane–Keuls multiple range test,
was used. A p<0.05 was considered statistically significant.
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The Maestro suite of programs (Schrçdinger, LCC[32]) was used for
the docking studies. The Cartesian coordinates for the two pro-
teins, iNOS and nNOS, were obtained from the Protein Data Bank
and treated with the Protein Preparation Wizard module[33] of
Maestro. The 3D structures of the conformers of compounds 5a–o
were generated using the program LigPrep[34] after taking their
structures from fragment libraries and optimizing using the Macro-
model module. Rigid docking was performed using Glide with
a
Standard Precision (SP) protocol. Figure were built using
PyMOL.[35]
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software suite[36] in explicit solvent using the AMBER force field
leaprc.ff10. The PDB2PQR tool[37,38] was used to estimate the most
probable protonation states of titratable residues in the proteins at
pH 7.0 and also to add all missing hydrogen atoms. Parameters for
the prosthetic heme group were obtained from Giammona.[39] Pa-
rameters for compound 5n as well as for H4B were calculated
using the antechamber module and the generalized AMBER force
field with AM1-BCC charges.
[16] J. León, F. Vives, E. Crespo, E. Camacho, A. Espinosa, M. A. Gallo, G. Es-
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[19] A. Entrena, M. E. Camacho, M. D. Carrión, L. C. López-Cara, G. Velasco, J.
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Received: January 9, 2015
Published online on March 20, 2015
ChemMedChem 2015, 10, 874 – 882
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