Synthesis, characterization and no synthase inhibition testing of 2-aryl-5-aroyl-3,4,5,6-tetrahydropyrimidinium chlorides

Article abstract of DOI:10.1002/jhet.1925

Aryl methyl ketones can be easily converted to 1-aryl-2-dimethylaminomethylpropenones that are known as interesting lead structures for drug development. By reaction of these enone Mannich bases with benzamidines, a series of new 2-aryl-5-aroyl-3,4,5,6-te

Full text of DOI:10.1002/jhet.1925

24
Synthesis, Characterization and NO Synthase Inhibition Testing of 2-Aryl-
5-aroyl-3,4,5,6-tetrahydropyrimidinium Chlorides
Vol 52
a
b
a
a
a
*
Ullvi Bluhm, Jean-Luc Boucher, Bernd Clement, Ulrich Girreser, Dieter Heber,
Booma Ramassamy,b and Ulrich Wolschendorfa
aPharmaceutical Institute, Department of Pharmaceutical Chemistry, Christian-Albrechts–University of Kiel,
Gutenbergstraße 76, D-24118 Kiel, Germany
bLaboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, University Paris
Descartes, 45 Rue des Saints-Pères, 75270 Paris, Cedex 06, France
*
E-mail: bclement@pharmazie.uni-kiel.de
Received August 8, 2012
DOI 10.1002/jhet.1925
Published online 22 April 2014 in Wiley Online Library (wileyonlinelibrary.com).
Aryl methyl ketones can be easily converted to 1-aryl-2-dimethylaminomethylpropenones that are known
as interesting lead structures for drug development. By reaction of these enone Mannich bases with
benzamidines, a series of new 2-aryl-5-aroyl-3,4,5,6-tetrahydopyrimidines were synthesized. These struc-
tures were characterized according to their lipophilicity. Thirty five tetrahydropyrimidines were evaluated
as nitric oxide synthase (NOS) inhibitors in usual screening assays. Some interesting members of this class
of compounds were forwarded to more detailed tests determining mechanism of inhibition and inhibition of
NADH consumption. The investigated structures showed modest activity of NOS inhibition. However, some
new tetrahydropyrimidines bearing extended aromatic substituents such as the naphthyloyl or biphenyloyl
residue displayed some activity of neuronal NOS and endothelial NOS inhibition but without selectivity
for an isoform and should be of interest for further modifications.
J. Heterocyclic Chem., 52, 24 (2015).
INTRODUCTION
amine groups—drew the attention of pharmacologists, for
example, as analgetics [7]; and a leading series of
investigations of Dimmock and coworkers [8–14] on the
toxicological properties and the selective toxicity towards
malignant cancer cells has to be mentioned. Recently,
Zampieri et al. investigated EMBs 2 and bis-Mannich
bases according to their antifungal and antimycobacterial
activity [15], followed by another (re-)investigation of
antifungal activity by Mete et al. [16]. The group of
Lesieur reported on anti-microtubular activity and
inhibition of human platelet aggregation and some
antifungal potential [17–20]. The antifungal inhibition
power of the free bases of 2, with variations in the amino
part using cyclic aliphatic and aromatic amines, was also
investigated by Ogata et al. [21], and Loiseau and
Depreux investigated the interaction with microtubules
[22]. A major drawback of the suitability of 2 as a drug
is its high reactivity towards nucleophiles [26], which
has to be considered in any pharmacological study [7,12].
EMBs 2 (Table 1) were tested as epidermal growth factor
receptor inhibitors by Traxler et al. [23] and are intermedi-
ates in the formation of pyrido[2,3-d]pyrimidines [24,25],
which showed a promising potential for NO synthase inhibi-
tion. EMBs 2 are also versatile precursors to a large number
of interesting heterocycles [26–28]. We could demonstrate
the access to tetrahydropyrimidines (THPs) 4 starting from
EMBs 2, as shown in Scheme 1 [29].
1-Aryl-2-dimethylaminomethylprop-2-en-1-ones.
1-Aryl-
2-dimethylaminomethylprop-2-en-1-ones 2 are formed in
the reaction of activated aromatic methylketones 1 with
fomaldehyde and amines or preformed iminium salts.
First evidence for a double Mannich reaction of an
activated methylene group can already be found in the
work of Mannich and Baumroth as early as 1924 [1];
they found as follow-up product in the Mannich reaction of
pyruvic acid with formaldehyde and dimethylammonium
chloride the 4-dimethylaminomethyl-substituted 3-
hydroxy-2,5-dihydrofuran-2-one. In this reaction,
a
dimethylaminomethyl-prop-2-en-1-one can be anticipated
at least as a formal intermediate that reacts further
with intramolecular cyclization. The first report of a stable
and isolable 1-aryl-2-dimethylaminomethylpropenone was
given by Hunziker et al. in 1963, presenting NMR data in
D2O [2]. In the literature, the isolation of 1-aryl-2-
dimethylaminomethylprop-2-enone, also named acrylophenone,
has been reported in 1970 by Greenhill and Mehta [3] as
products in the Mannich reaction of an acetophenone
derivative in high boiling solvents; and at the same time,
some more reactions were reported by Back [4,5]. We were
also interested in this class of compounds and coined the
name enone Mannich bases (EMBs) for these propenones 2
[6]. These EMBs 2 (Scheme 1)—with different secondary
© 2014 HeteroCorporation

January 2015
Synthesis, Characterization and NO Synthase Inhibition Testing of
2-Aryl-5-aroyl-3,4,5,6-tetrahydropyrimidinium Chlorides
25
Scheme 1
have been identified. The constitutive neuronal NOS
(nNOS) and endothelial NOS (eNOS) are Ca++-dependent
enzymes that play key roles in the nervous and
cardiovascular systems, whereas the inducible nitric oxide
synthase (iNOS) produces large quantities of NO following
immunological challenge [37–39]. NOSs are homodimeric
enzymes, and each NOS subunit contains an NH2-terminal
oxygenase domain that bears binding sites for the substrate
L-arginine, the heme prosthetic group and the cofactor
(6R)-5,6,7,8-tetrahydro-L-biopterin (H4B), and a CO2H-
terminal reductase domain that contains binding sites for
the flavins, flavin mononucleotide, flavin adenine
dinucleotide, and the cofactor NADPH. These two domains
are fused by a Ca++-dependent calmodulin (CaM)-binding
sequence. CaM-binding to NOS activates both intradomain
as well as interdomain electron transfers and is required for
maximal NO-forming activity [37–39].
There is much evidence that NOS inhibitors could be
useful therapeutical agents in the treatment of diseases such
as diabetes, congestive heart failure, atherosclerosis,
migraine, asthma, cerebral ischemia and Parkinson’s
disease [30,31,40–43]. However, they must be selective
towards nNOS or iNOS to avoid interferences with the
vital functions of eNOS involved in the control of vascular
tone. Recent studies in the synthesis of new NOS inhibitors
have thus focused on the development of either nNOS or
iNOS selective inhibitors [44–47].
NOS inhibitors. Nitric oxide, NO, acts as a regulator of
the vascular tone, a neurotransmitter and a cytotoxic agent.
NO also plays important roles in the pathogenesis of
several diseases [30,31]. In particular, NO contributes to
inflammatory and autoimmune diseases such as rheumatoid
arthritis, osteoarthritis, inflammatory bowel disease, and
multiple sclerosis [32–36]. Therefore, nitric oxide
synthases (NOS), the enzymes that produce NO by
oxidation of L-arginine, are potential targets for new
therapeutic agents. In mammals, three isoforms of NOS
Several structurally diverse classes of NOS inhibitors
have been investigated. Analogs of the natural substrate
L-arginine, dipeptides, and peptidomimetics derived from
Table 1
Synthesis and properties of the enone Mannich bases 2a–n used in this work.
2
R1
Yield, %
Mp, ꢀC
logD(7.4)
References
a
b
c
d
e
H
4-Me
4-F
3-OMe
4-OMe
4-Cl
4-OEt
3,4-(CH═CH)2
3,4-(OMe)2
3,4-Cl2
36 [7]a
38 [7]a
36
158 [7]
160 [7]
169
138
153 [7]
154
162
159
165 [7]
177
180
1.25
1.52
1.00
1.22
1.25
1.70
1.85
2.26
1.05
2.40
2.82
1.82
1.71
[7,18]
[7]
[15], this work
[25], this work
[7,8]
20
44 [7]a
40
f
[18]
g
h
i
k
l
16
60
[25], this work
[25], this work
[7]
[7,8]
[6], this work
[7]
41 [7]a
26 [7]a
39
4-Ph
4-Br
3,4,5-(OMe)3
m
n
38 [7]a
40
181
188
[25], this work
aUsing method A in reference [7].
Journal of Heterocyclic Chemistry
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U. Bluhm, J-L. Boucher, B. Clement, U. Girreser, D. Heber, B. Ramassamy, and U. Wolschendorf
Vol 52
No-nitro-L-arginine (L-NNA) have been studied, some of
them exhibiting selectivities of up to three orders of magni-
tude over either eNOS or iNOS inhibition [48–53]. In the
meantime, a great number of non-amino acid compounds
have been tested as either nNOS or iNOS inhibitors. Substi-
tuted indazoles (with 7-nitroindazole, as the lead compound)
or imidazoles are potent NOS inhibitors but their selectivity
remains low, at least in vitro [54,55]. Potent non-amino acid
inhibitors with isothiourea and amidine functions have been
reported [56–64]. 2-Aminopyridines have been described as
NOS inhibitors, and selective nNOS inhibitors containing
this scaffold were recently reported [65,66]. However,
simple 2-amino-4-methylpyridine derivatives, which are
reported to be highly potent inhibitors of all three isoforms,
can exhibit high toxicity and should not be suitable for
further development [65]. The most recent developments in
the field of NO synthase inhibition have been summarized
in two reviews on new structures and new patents [67,68].
We were interested in the synthesis of new THPs 4 as
potential inhibitors for NOSs and considered them more suit-
able for drug development than the starting EMBs 2. These
THPs exhibit the typical phamacophoric centers found in
the EMBs 2, that is, the aroyl moiety and a cyclic aliphatic
spacer, and a basic cyclic amidine that could mimic the
guanidinium function of L-arginine. The presence of a
strongly basic moiety able to interact with a key-glutamate
residue of the active site of NOS is a pre-requisite for NOS
inhibition. Evans reported for 1,3,5,6-tetrahydropyrimidines,
which are substituted in 2-position, a pKa value of 13.0 in the
case of an aliphatic substituent and a value of 12.8 for an
aromatic substituent [69]. One can thus consider THPs 4 as
strong bases which are permanently charged under physio-
logical conditions. These compounds were evaluated as
inhibitors of the three purified NOS isoforms from various
species. Only two of them exhibited good inhibitory effects
with selectivity for one isoform of NOS thus opening the
way to further SAR studies.
heterocycles 4 in moderate yields (Table 2). All new
compounds were fully characterized by the usual analytical
methods. It has to be mentioned that the protonated
tetrahydropyrimidinium ring in 4 shows symmetry, that is,
for 4fb, one chemical shift of the nitrogen atom was
determined at ꢁ267.1 ppm relative to nitromethane by 15N
NMR. In all other cases, the NH-proton was very broad
and did not allow any inverse measurements. A large
number of benzamidines are commercially available or can
be synthesized in only one step via Pinner synthesis from
the corresponding nitriles. As starting moieties, we chose
the simple unsubstituted benzamidine 3a, benzamidines 3b
with an electron deficient nitro group in meta position, and
3c with an additional amide group in para position.
Determination of logD7.4
.
LogD values of the EMBs 2
and THPs 4 were determined experimentally by using a
gradient elution HPLC method and reference compounds
[70]. Under the test conditions (pH 7.4), the structures 2
and 4 are considered to be permanently charged. The results
are listed in Tables 1 and 2. Interestingly, quite similar
values were found for the EMBs and the corresponding
THPs formed with the unsubstituted benzamidine and with
the nitro group. Carboxamide substituted THPs show on
average a smaller value of about 0.5 logD units.
Biological testing – Griess assay.
In a first series of
experiments, NOS inhibition data were obtained for all
compounds introduced at 10, 100, and 1000 mM (final
concentration) using recombinant NOS isoforms from
different species and the colorimetric Griess assay for
nitrite determination [71]. From these results, IC50 values
were estimated by linear regression (Table 3). Experiments
including the known inhibitor of NOS No-nitro-L-arginine
(L-NNA) were performed for comparative purposes and are
also listed in Table 3. By using this assay, only a few of
the tested THPs 4 were found to be active, that is, the
chloro derivative 4fb, the naphthoyl derivatives 4ha–c, and
the benzoyl derivative 4la, all with a preference for eNOS
inhibition, in comparison with nNOS and iNOS.
Biological testing
–
hemoglobine assay.
We also
RESULTS AND DISCUSSION
determined the inhibitory effects of some selected THPs on
the activity of NOS using the classical spectrophotometric
hemoglobin assay for NO [72,73]. The results of this test
system are summarized in Table 4. All new compounds
were tested at a concentration of 1 mM, and the data are
based on the percentage of the complete system without
THP. This complementary test confirmed the results of the
Griess assay. Compounds 4fb, the 4-chloro benzoyl
derivative, and 4la, the biphenyl derivative, displayed
selectivity in inhibition of eNOS, whereas the naphthyl
derivative 4ha and the biphenyl derivative 4la
predominantly inhibited nNOS. However, none of the
tested compounds significantly inhibited iNOS.
Chemistry.
EMBs 2a–n are accessible through the
heating of aryl methyl ketones 1a–n and paraformaldehyde
and dimethylamine in N,N-dimethylformamide in moderate
yield (Scheme 1 and Table 1) [7]. The yields can be
improved using dimethyl(methylene)ammonium chloride
(Eschenmoser’s salt), a preformed iminium salt [7, method
B], no further investigations using other catalysts were
performed, as the starting materials are readily available in
bulk quantities. Tetrahydropyrimidines 4aa–4nc were
prepared by condensation of benzamidines 3a–c with
enone Mannich bases 2a–n in ethanol/water mixtures using
trimethyl amine as base [29]. After the addition of the
nucleophile to the enone structure, the dimethylamino
group was eliminated followed by ring closure to form the
For the more promising THPs, IC50 values were deter-
mined for nNOS inhibition in the presence of different
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Synthesis, Characterization and NO Synthase Inhibition Testing of
2-Aryl-5-aroyl-3,4,5,6-tetrahydropyrimidinium Chlorides
27
Table 2
Synthesis and characterization of tetrahydropyrimidines 4aa–4nc.
4
Structure
Yield, %
53 [29]
Mp, ꢀC
logD (7.4)
1.33
aa
>300 [29]
ab
ac
48 [29]
>300 [29]
1.31
0.78
51
299
bb
bc
42
15
>300
1.82
1.31
264
ca
39
>300
1.50
cb
cc
36
52
>300
1.47
0.88
288
(Continued)
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U. Bluhm, J-L. Boucher, B. Clement, U. Girreser, D. Heber, B. Ramassamy, and U. Wolschendorf
Vol 52
Table 2
(Continued)
4
Structure
Yield, %
40
Mp, ꢀC
204
logD (7.4)
db
dc
1.59
1.10
40
178
ea
49 [29]
280 [29]
1.60
eb
fa
40 [29]
299 [29]
1.57
2.00
44
>280
fb
40
32
280
298
1.96
2.13
ga
gb
32
>300
2.05
(Continued)
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Synthesis, Characterization and NO Synthase Inhibition Testing of
2-Aryl-5-aroyl-3,4,5,6-tetrahydropyrimidinium Chlorides
29
Table 2
(Continued)
4
Structure
Yield, %
34
Mp, ꢀC
259
logD (7.4)
1.61
gc
ha
31
>290
2.44
hb
hc
63
32
281
279
2.35
2.02
ib
ic
44
40
260
265
1.30
0.93
ka
kb
44 [29]
>300 [29]
2.52
2.55
45 [29]
297 [29]
(Continued)
Journal of Heterocyclic Chemistry
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U. Bluhm, J-L. Boucher, B. Clement, U. Girreser, D. Heber, B. Ramassamy, and U. Wolschendorf
Vol 52
Table 2
(Continued)
4
Structure
Yield, %
62
Mp, ꢀC
226
logD (7.4)
2.06
kc
la
30
283
2.84
lb
53
47
42
>280
2.88
2.06
1.60
mb
mc
230
272
na
nb
nc
30
32
26
278
269
263
1.61
1.59
1.15
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Synthesis, Characterization and NO Synthase Inhibition Testing of
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31
Table 3
Inhibition of nitrite formation by recombinant NOS isoforms in the presence of tetrahydropyrimidines 4aa–4nc and the reference compound L-NNA.a
IC50/mM
Compound
R1
R2
eNOS (rat)
iNOS (murine)
nNOS (bovine)
4aa
4ab
4ac
4bb
4bc
4ca
4cb
4cc
4db
4dc
4ea
4eb
4fa
H
H
H
4-Me
4-Me
4-F
4-F
4-F
3-OMe
3-OMe
4-OMe
4-OMe
4-Cl
4-Cl
4-OEt
4-OEt
4-OEt
3,4-(CH═CH)2
(human enzymes)
3,4-(CH═CH)2
(human enzymes)
3,4-(CH═CH)2
(human enzymes)
3,4-(OMe)2
3,4-(OMe)2
3,4-Cl2
H
>1
>1
>1
>1
>1
>1
0.92
>1
>1
>1
>1
>1
>1
0.34
>1
>1
>1
>1
0.73
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.90
>1
>1
>1
>1
>1
0.85
>1
>1
>1
0.96
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
>1
0.44
0.09
>1
>1
>1
>1
>1
>1
0.68
>1
0.81
0.68
>1
>1
>1
0.51
>1
0.82
>1
0.71
0.47
0.45
0.33
>1
0.86
>1
>1
3-NO2
4-CONH2
3-NO2
4-CONH2
H
3-NO2
4-CONH2
3-NO2
4-CONH2
H
3-NO2
H
3-NO2
H
3-NO2
4-CONH2
H
4fb
4ga
4gb
4gc
4ha
4ha
4hb
4hb
4hc
4hc
4ib
>1
>1
0.19
0.34
0.12
0.12
0.37
0.74
>1
>1
>1
>1
0.45
0.06
>1
0.94
0.86
>1
>1
>1
0.012
0.027
3-NO2
4-CONH2
3-NO2
4-CONH2
H
3-NO2
4-CONH2
H
3-NO2
3-NO2
4-CONH2
H
3-NO2
4-CONH2
4ic
4ka
4kb
4kc
4la
>1
3,4-Cl2
3,4-Cl2
4-Ph
4-Ph
4-Br
4-Br
3,4,5-(OMe)3
3,4,5-(OMe)3
3,4,5-(OMe)3
0.55
0.46
0.46
>1
>1
>1
>1
>1
>1
0.007
0.005
4lb
4mb
4mc
4na
4nb
4nc
L-NNA
L-NNA
(human enzymes)
NOS, nitric oxide synthase; eNOS, endothelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide synthase.
aAverage relative standard deviation 10%, n = 4.
amounts of L-arginine (Table 5). The significant increase of
the IC50 values when increasing L-arginine concentration
from 10 to 100 mM suggests that there was competition
between the substrate and the tested THPs.
information on their effects on the electron transfer from the
reductase domain to the heme domain and the possible
generation of superoxide anion and hydrogen peroxide by
nNOS74. The results are given in % of the complete system
without THP addition (Table 6). In the absence and in the
presence of a saturating concentration of substrate, 4ha and
4la were found to inhibit the NADPH oxidase activity of
nNOS; however, no clear effects were observed for 4lb and 4kb.
NADPH consumption.
In order to obtain further
information on the effects of some THPs on the activity of
NOSs, we have tested their effects on the NADPH
consumption catalyzed by nNOS. These data will give
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U. Bluhm, J-L. Boucher, B. Clement, U. Girreser, D. Heber, B. Ramassamy, and U. Wolschendorf
Vol 52
Table 4
CONCLUSIONS
Inhibitory effects of some THPs 4 on the activity of three NOS isoforms
measured by the hemoglobin assay.a
We have shown that a large number of 2,5-disubstituted tet-
rahydropyrimidines are accessible via two preparatively and
synthetically simple steps employing basic building blocks,
namely acetophenones 1 and benzamidines 3, which are com-
mercially available with large structural diversity. The THPs 4
thus available contain three typical pharmacological moieties,
the aromatic residue, the aliphatic chain, and a basic group,
here a cyclic N,N0-disubstituted amidinium ion. The struc-
tures might be of general interest because of their pharmaco-
logical profile, which still needs to be explored in detail.
We investigated the potential of these compounds to
inhibit NO synthases and found some of the more lipophilic
members (logD7.4 > 2) to be modest inhibitors of eNOS and
nNOS. The most potent compounds bear a large aromatic
group (biphenyl or naphthyl) and a small aromatic substituent
on the amidine residue, that is, 4ha and 4la. Our data suggest
that these compounds are competitive inhibitors with the
substrate L-arginine and could inhibit NADPH oxidation by
nNOS. Compared with other nonpeptoid NOs inhibitors,
the inhibition potency as well as the selectivity is moderate.
As lead compounds, however, they open the way to further
structural modifications to get more active compounds.
% activity of the complete systemb
Compound
eNOS
iNOS
nNOS
4aa
4ab
4ca
4cb
4cc
4dc
4ea
4eb
4fa
4fb
4ga
4gb
4gc
4ha
4ic
84
61
104
68
88
75
98
61
46
9
101
105
103
105
95
94
94
110
98
95
120
132
84
95
107
90
114
111
74
51
82
79
96
16
90
68
31
0
91
105
89
118
5
107
81
13
106
104
107
99
77
92
4ka
4kb
4la
71
0
4lb
24
93
10
THPs, tetrahydropyrimidines; NOS, nitric oxide synthase; eNOS, endo-
thelial nitric oxide synthase; nNOS, neuronal nitric oxide synthase; iNOS,
inducible nitric oxide synthase.
aResults are stated as % of the complete system. All compounds were
tested at 1 mM (final concentration).
EXPERIMENTAL
bEstimated relative error 20%, n = 2–3.
Lipophilicity. The logD values were determined by an HPLC
method with gradient elution. A Waters Terra X column C8 MS
was employed (4.6 ꢂ 50mm, 3.5 mm particle size) together with a
C8 Phenomenex precolumn (3ꢂ 2 mm). The solvent system
consisted of the following two mixtures: Solvent A 5% of
acetonitrile and 95% (by volume) of 25mM ammonium acetate,
pH 7.4. Solvent B consisted of 95% of acetonitrile and 5% of the
buffer, respectively. The solvents were eluted starting with 100%
of A, changing linearly within 50min to 100% of solvent B with
a flow rate of 0.5 mL. Detection was performed at 240 nm, and
the column was thermostated to 30ꢀC. The following reference
compounds were used (logP values in brackets [70]): acetanilide
(1.0), acetophenone (1.7), anisole (2.1), ethyl benzoate (2.4),
benzophenone (3.6), and diphenyl ether (4.2). The dead volume
was determined by using uracil, and the calculated k values were
correlated in a linear model with the reported logP values.
Table 5
Determination of IC50 values of selected THPs 4 on nNOS activity in the
presence of 10 or 100 mM L-arginine. The activity of nNOS was measured
by the hemoglobin assay.
Compounds
IC50/mMa
[L-Arg]/mM
4lb
4la
10
800
200
600
100
1100
450
4kb
800
L-Arg, L-arginine; THPs, tetrahydropyrimidines; nNOS, neuronal nitric
oxide synthase
aMeans from two experiments.
Chemistry.
General: Starting materials were of standard
quality and were employed as obtained from commercial suppliers
and were used without further purification. All NMR spectra were
recorded and evaluated in DMSO-d6 with a Bruker Avance III
Table 6
Effects of some THPs 4 on NADPH consumption by nNOS.a
1
300 spectrometer, at 300 K and at 300MHz for H, 75MHz for
Compounds
without L-Arg
with L-Arg (100 mM)
13C,1H-broadband-decoupled spectra and for the DEPT pulse
sequence (135ꢀ-tip angle), at 282 MHz for 19F, and at 30.5 MHz
for 15N. Chemical shifts d were calibrated with internal DMSO-d6
(1H at 2.50ppm for the center of the DMSO-d5 quintet, 13C at
39.5ppm for the DMSO-d6 septet) or with external CFCl3 in
DMSO-d6 (d(19F)= 0.0 ppm) or with internal nitro methane
(d(15N) = 0.0 ppm). Coupling constants J are given in Hertz. The
carbon resonances are given together with the phase of the signal
in the corresponding 135-DEPT 13C NMR spectrum. EI nominal
resolution mass spectra were recorded by using an HP5989 MS
4ha
4la
4lb
4kb
L-NNA
25 ꢃ 11
17 ꢃ 5
48
35 ꢃ 25
44 ꢃ 11
116 ꢃ 58
66 ꢃ 30
21
117 ꢃ 35
27
L-Arg, L-arginine; THPs, tetrahydropyrimidines; nNOS, neuronal nitric
oxide synthase.
aAll compounds were tested at 1 mM, final concentration. Results are
means from 2 to 3 experiments.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

January 2015
Synthesis, Characterization and NO Synthase Inhibition Testing of
2-Aryl-5-aroyl-3,4,5,6-tetrahydropyrimidinium Chlorides
33
engine A with ionization energy of 70 eV and a direct inlet probe
equipped with a tungsten wire. Elemental analyses were recorded
with a Heraeus C,H,N analyzer at the Institute of Inorganic
Chemistry at the University of Kiel, high resolution mass
spectra (HR-ESI(+)-MS) were recorded with electrospray
ionization with a Bruker Apex II FT ICR system at the Institute
of Analytical Chemistry at the University of Leipzig.
Generally, up to three peaks are analyzed: additionally to the
quasi-molecular ion [M + H]+, the value of the [M + 1 + H]+ or
[M + 2 + H]+, and, if available, of the dimeric cluster [2 M + H]+
is given.
1-(4-Chlorophenyl)-2-(dimethylaminomethyl)-prop-2-en-
1-one hydrochloride (2f) [18].
7.7 g (6.5mL, 50mmol) of
1f, 5.1g (170mmol) of paraformaldehyde and 9.0g (110mmol)
of dimethylammonium chloride in 25 mL of DMF afforded
3.4 g (13 mmol, 40%) of white crystals, mp 154ꢀC. 1H NMR
d: 2.77 (br s, 6H, NMe2), 4.09 (br s, NCH2), 6.20/6.80
(2 ꢂ s, 2 ꢂ 1H, ═CH2), 7.63 (d, 2H, J = 8.6, H-30/H-50), 7.82
(d, J = 8.6, 2H, H-20/60), 11.15 (br s, 1H, NH). 13C NMR d:
42.2 (+), 55.3 (ꢁ), 128.6 (+), 131.4 (+), 134.7, 136.8, 137.0
(br, ꢁ), 137.7, 194.2. MS m/z: 223 (M+, 4), 222 (9), 206
(67), 145 (10), 139 (22), 111 (15), 64 (9), 58 (100), 44 (15),
42 (17).
General procedure for the synthesis of EMBs 2.
About
50 mmol of the corresponding substituted acetophenone 1a–m
was heated to 130ꢀC for 1.5 h in 50 mL of DMF together with
2.2 equivalents of dimethylammonium chloride and 3.3
equivalents of paraformaldehyde. The solvent was then
evaporated in vacuo, and the residue was triturated for 30 min
with acetone at 0ꢀC. If necessary, the suspension was left
standing overnight at 6–8ꢀC for complete crystallization. The
solid was filtered off and dissolved in 100 mL of water. The pH
of this solution was adjusted to eight using sodium bicarbonate.
This solution was extracted three times with 100 mL of
dichloromethane, the organic layer was dried over sodium
sulfate, and then the solvent was evaporated. The oily remainder
was triturated with a mixture of 20 mL of ethanol and 10 mL of
concentrated hydrochloric acid. Finally, after twice adding and
evaporating 40 mL of toluene, the solid was recrystallized from
acetone/isopropanol mixtures.
2-(Dimethylaminomethyl)-1-(4-ethoxyphenyl)-prop-2-en-1-
one hydrochloride (2g).
8.2 g (50 mmol) of 1g and 5.1g
(170 mmol) of paraformaldehyde and 9.0 g (110 mmol) of
dimethylammonium chloride in 25 mL of DMF afforded 2.2 g
1
(8 mmol, 16%) of white crystals, mp 162ꢀC. H NMR d: 1.36
(t, J = 6.9, 3H, Me), 2.58 (br s, 6H, NMe2), 4.08 (br s, 2H,
NCH2), 4.14 (q, J = 6.9, 2H, OCH2), 6.12/6.66 (2 ꢂ s, 2 ꢂ 1 H,
═CH2), 7.07 (d, J = 8.7, 2H, H-30/H-50), 7.82 (d, J = 8.6, 2H, H-
20/60), 10.97 (br s, 1H, NH). 13C NMR d: 14.4 (+), 42.1
(+), 55.7 (ꢁ), 63.6 (ꢁ), 114.2 (+), 128.1, 132.1 (+), 134.8 (ꢁ),
136.9, 162.5, 193.7. MS m/z: 233 (M+, 12), 216 (73), 149 (15),
121 (19), 93 (10), 84 (11), 65 (15), 58 (100), 44 (19), 42
(26). HRꢁESI(+)-MS: Calcd for [C14H19NO2 + H]+ 234.1489,
found 234.1488; calcd for [13C12C13H19NO2 + H]+ 235.1523,
found 235.1522.
2-(Dimethylaminomethyl)-1-(20-naphthyl)-prop-2-en-1-one
Compounds.
The complete analytical characterization of
hydrochloride (2h).
8.5 g (50mmol) of 1h and 5.1g
the following enone Mannich bases is reported elsewhere [7]: 1-
Phenyl-2-(dimethylaminomethyl)-prop-2-en-1-one hydrochloride
(2a), 1-(4-methylphenyl)-2-(dimethylaminomethyl)-prop-2-en-1-one
hydrochloride (2b), 1-(4-methoxyphenyl)-2-(dimethylaminomethyl)-
prop-2-en-1-one hydrochloride (2e), 1-(3,4-dimethoxyphenyl)-
2-(dimethylaminomethyl)-prop-2-en-1-one hydrochloride (2i),
1-(3,4-dichlorophenyl)-2-(dimethylaminomethyl)-prop-2-en-1-one
hydrochloride (2k), 1-(4-bromophenyl)-2-(dimethylaminomethyl)-
prop-2-en-1-one hydrochloride (2m).
(170 mmol) of paraformaldehyde and 9.0 g (110mmol) of dimethy-
lammonium chloride in 25 mL of DMF afforded 8.2 g (30 mmol,
60%) of white crystals, mp 159ꢀC. Anal., %: calcd for C16H18NOCl
C 69.68, H 6.58, N 5.08; found C 69.58, H 6.65, N 4.95. 1H NMR
d: 2.83 (d, J = 4.4, 6H, NMe2), 4.17 (d, J = 4.8, 2H, NCH2), 6.30/
6.83 (2 ꢂ s, 2 ꢂ 1 H, ═CH2), 7.69 (m, 2H, ArH), 7.86 (dd, J = 8.5/
1.7, 1H, ArH), 8.02–7.17 (m, 3H, ArH), 8.48 (s, 1H, ArH) 10.91
(br s, 1H, NH). 13C NMR d: 42.4 (+), 56.0 (ꢁ), 125.0 (+), 127.0
(+), 127.6 (+), 128.3 (+), 128.6 (+), 129.5 (+), 131.4 (+), 131.8,
133.2, 134.8, 136.5 (br, ꢁ), 137.1, 195.3. MS m/z: 239 (M+, 7),
238 (7), 222 (19), 196 (5), 155 (7), 127 (15), 84 (7), 77 (6), 58
(100), 44 (31). HR–ESI(+)-MS: Calcd for [C16H17NO + H]+
240.1383, found 240.1384; calcd for [13C12C15H17NO + H]+
241.1417, found 241.1418.
2-(Dimethylaminomethyl)-1-(4-fluorophenyl)-prop-2-en-1-
one hydrochloride (2c) [15].
6.9 g (50 mmol) of 1c afforded
3.47 g (14mmol, 28%) of white crystals, mp 169ꢀC. Anal., %:
Calcd for C12H15NOFCl C 59.14, H, 6.21, N 5.75; found C
1
59.11, H 6.31, N 5.64. H-NMR d: 2.78 (d, J = 4.8, 6H, NMe2),
4.10 (d, J = 5.2, 2H, NCH2), 6.19/6.79 (2ꢂ s, 2 ꢂ 1H, ═CH2),
7.40 (m, 2H, H-30/50), 7.91 (m, 2H, H-20/60), 11.17 (br s,
1H, NH). 13C NMR d: 42.1 (+), 55.4 (ꢁ), 115.6 (+, JCF = 21.9),
132.5 (+, JCF = 9.5), 132.5, 132.6 (JCF = 2.2), 136.5 (ꢁ), 164.8
(JCF = 251.8), 193.9. 19F NMR d: ꢁ105.9 (tt, J = 9.2/5.1, 1F). MS
m/z: 207 (M+, 3), 190 (30), 123 (18), 112 (4), 95 (20), 84 (6), 75
(12), 58 (100), 44 (21), 42 (38).
1-(4-Biphenyl)-2-(dimethylaminomethyl)-prop-2-en-1-one
hydrochloride (21) [6].
9.8 g (50mmol) of 1l and 5.1g
(170 mmol) of paraformaldehyde and 9.0 g (110mmol) of dimethy-
lammonium chloride in 25 mL of DMF afforded 5.8 g
(19 mmol, 39%) of white crystals, mp 180ꢀC. 1H NMR d:
2.79 (br s, 6H, NMe2), 4.12 (br s, NCH2), 6.25/6.78 (2 ꢂ s,
21 ꢂ 1H, ═CH2), 7.44 (m, 1H, H-400), 7.52 (m, 2H, H-3000/
500), 7.77 (d, J = 7.0, 2H, H-200/600), 7.88 (m, 4H, H-20/30(50/
60), 11.00 (br s, 1H, NH). 13C NMR d: 42.4 (+), 55.6 (ꢁ),
126.7 (+),126.9 (+),128.4 (+),129.1 (+),130.3 (+), 134.7,
136.4 (br, ꢁ), 137.0, 144.4, 194.8. MS m/z: 265 (M+, 14),
264 (18), 248 (100), 181 (20), 152 (26), 112 (10), 84 (11),
58 (97), 44 (19), 42 (17).
2-(Dimethylaminomethyl)-1-(3-methoxyphenyl)-prop-2-en-
1-one hydrochloride (2d).
7.5 g (50 mmol) of 1d afforded
1
2.53 g (10 mmol, 20%) of white crystals, mp 138ꢀC. H NMR d:
2.78 (br s, 6H, NMe2), 3.83 (s, 3H, OMe), 4.11 (br s, 2H, NCH2),
6.21/6.79 (2 ꢂ s, 2 ꢂ 1H, ═CH2), 7.25 (ddd, J= 8.0/2.7/1.1, 1H,
H-6), 7.29 (dd, J= 2.7/1.6, 1H, H-2), 7.36 (ddd, J= 7.5/1.5/1.2, 1H,
H-4), 7.48 (dd, J= 7.7/7.7, 1H, H-5), 11.11 (br s, 1H, NH). 13C
NMR d: 42.1 (+), 55.3 (ꢁ), 55.4 (+), 114.2 (+), 118.7 (+), 121.9
(+), 129.6 (+), 136.87 (ꢁ), 136.91, 137.3, 159.1, 195.0. HR–ESI
(+)-MS: Calcd for [C13H17NO2 +H]+ 220.1332, found 220.1334;
calcd for [13C12C12H17NO2 +H]+ 221.1366, found 221.1367.
1-(3,4,5-Trimethoxyphenyl)-2-(dimethylaminomethyl)-
prop-2-en-1-one hydrochloride (2n).
4.2 g (50mmol) of
1n and 5.1 g (170 mmol) of paraformaldehyde and 9.0g
(110 mmol) of dimethylammonium chloride in 25mL of DMF
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

34
U. Bluhm, J-L. Boucher, B. Clement, U. Girreser, D. Heber, B. Ramassamy, and U. Wolschendorf
Vol 52
afforded 2.5 g (8 mmol, 40%) of yellowish crystals, mp 188ꢀC.
1H NMR d: 2.77 (d, J = 4.8, 6H, NMe2), 3.77/3.86 (2 ꢂ s,
1 ꢂ 3H, 1 ꢂ 6H, 3 ꢂ OMe), 4.12 (d, J = 5.0, 2H, NCH2), 6.25/
6.75 (2 ꢂ s, 2 ꢂ 1H, ═CH2), 7.13 (s, 2H, H-20/H-60) 11.22 (br
s, 1H, NH). 13C NMR d: 42.2 (+), 55.9 (ꢁ), 56.1 (+), 60.1 (+),
107.4 (+), 130.9 (+), 136.1, 136.7 (ꢁ), 141.8, 152.6, 194.2.
HR–ESI(+)-MS: Calcd for [C15H21NO4 + H]+ 280.1543, found
280.1543; calcd for [13C12C14H21NO4 + H]+ 281.1577, found
281.1578.
2-(40-Carbamoylpheny)-5-(400-methylbenzoyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4bc).
496 mg
(2.05 mmol) of 2b and 408 mg (2.04mmol) of 3c afforded 98mg
(0.55 mmol, 15%) of 4bc as white solid, mp 264ꢀC (dec.). 1H
NMR d: 2.41 (s, 3H, Me), 3.60 (dd, J = 13.5, 6.8, 2H, H-4/6),
3.79 (dd, J = 13.4, 4.6, 2H, H-4/6), 4.29 (m, 1H, H-5), 7.41 (d,
J = 8.0, 2H, H-300/500), 7.64/8.22 (2 ꢂ br s, 2 ꢂ 1H, CONH2), 7.83
(d, J = 8.4, 2H, H-20/60), 7.99 (d, J = 8.0, 2H, H-200/0600),
8.08 (2ꢂ d, J = 8.4, 2 ꢂ 2H, H-30/50), 10.42 (br s, 2H, NH). 13C
NMR d: 21.1 (+), 34.7 (+), 40.8 (ꢁ), 127.7 (+), 127.8 (+),128.6
(+), 129.5 (+), 130.2, 132.3, 136.5, 144.5, 158.1, 167.1, 197.4.
MS m/z: 321 (M+, 1), 164 (87), 148 (100), 120 (29), 103
(43), 76 (16), 65 (23), 51 (20), 44 (26). HR–ESI(+)-MS: Calcd
for [C19H19N3O2 + H]+ 322.1550, found 322.1548; calcd for
[13C12C18H19N3O2 + H]+ 323.1584, found 323.1583; calcd
for [(C19H19N3O2)2 + H]+ 643.3027, found 643.3034.
General procedure for the synthesis of the THP 4aa–
4nc.
1 mmol of EMB 2 and 1.1 mmol of the benzamidinium
hydrochloride 3 were dissolved in about 10 mL of a 1:1 mix-
ture of water and ethanol, then 0.6 mL of triethylamine was
added and the mixture was brought to reflux for 30 min. After
cooling, the solvents were evaporated in vacuum, and the re-
mainder was suspended and refluxed with 15 mL of isopropa-
nol. After filtration, the solid was washed with 5 mL of
isopropanol and dried in vacuo. The benzamidines 3a–c are
commercially available. The characterization of the following
THP 4 and their analytical data are reported elsewhere [29]: 5-
Benzoyl-2-phenyl-1,4,5,6-tetrahydropyrimidine hydrochloride
(4aa), 5-benzoyl-2-(3-nitrophenyl)-1,4,5,6-tetrahydropyrimidine
hydrochloride (4ab), 5-(4-methoxybenzoyl)-2-phenyl-1,4,5,6-
tetrahydropyrimidine hydrochloride (4ea), 5-(4-methoxybenzoyl)-
2-(3-nitrophenyl)-1,4,5,6-tetrahydropyrimidine hydrochloride
(4eb), 5-(3,4-dichlorobenzoyl)-2-phenyl-1,4,5,6-tetrahydropyrimidine
hydrochloride (4ka), 5-(3,4-dichlorobenzoyl)-2-(3-nitrophenyl)-
1,4,5,6-tetrahydropyrimidine hydrochloride (4kb).
5-(40-Fluorobenzoyl)-2-phenyl-1,4,5,6-tetrahydropyrimidine
hydrochloride (4ca).
243 mg (1.0 mmol) of 2c and 157 mg
(1.0 mmol) of 3a ꢂ H2O afforded 124 mg (0.39 mmol, 39%)
of 4ca as white solid, mp >300ꢀC. 1H NMR d: 3.60 (dd,
J = 13.3, 6.5, 2H, H-4/6), 3.80 (dd, J = 13.5, 4.0, 2H, H-4/6),
4.32 (m, 1H, H-5), 7.43 (t, J = 8.8, 2H, ArH), 7.62 (m, 2H,
ArH), 7.73 (m, 1H, ArH), 7.82 (d, J = 7.3, 2H, ArH), 8.19
(m, 2H, ArH), 10.52 (br s, 2H, NH). 13C NMR d: 34.9 (+),
40.7 (ꢁ), 116.0 (+, JCF = 22.0), 127.6 (+), 127.9, 129.0 (+),
131.6 (+, JCF = 9.8), 131.7 (JCF = 2.8), 133.2, 158.8, 165.4
(JCF = 253.0), 196.6. 19F NMR d: ꢁ104.4 (tt, J = 8.9/5.1,
1F). MS m/z: 282 (M+, 1), 159 (100), 123 (41), 117
(10), 104 (50), 95 (42), 77 (41), 75 (21), 56 (67), 51 (22).
HR–ESI(+)-MS: Calcd for [C17H15N2OF + H]+ 283.1241,
found 283.1239; calcd for [13C12C16H15N2OF + H]+
284.1275, found 284.1274.
5-Benzoyl-2-(40-carbamoylphenyl)-1,4,5,6-tetrahydropyrimidine
hydrochloride (4ac).
258 mg (1.14 mmol) of 2a and 248 mg
(1.23 mmol) of 3c afforded 201 mg (0.58mmol, 51%) of 4ac as
1
white solid, mp 299ꢀC. H NMR d: 3.61 (dd, J = 13.6, 6.7, 2H,
H-4/6), 3.80 (dd, J = 13.6, 4.4, 2H, H-4/6), 4.33 (m, 1H, H-5),
7.57/8.26 (2 ꢂ br s, 2 ꢂ 1H, CONH2), 7.61 (d, J = 7.8, 2H, H-
300/500), 7.71 (m, 1H, H-400), 7.84 (d, J = 8.3, 2H, H-30/50), 8.07
(2 ꢂ d, J = 8.4, 2 ꢂ 2H, H-20/60, H-200/H600), 10.50 (br s, 2H,
NH). 13C NMR d: 34.8 (+), 40.8 (ꢁ), 127.8 (+), 127.9 (+),
128.5 (+), 129.0 (+), 130.1, 133.9, 134.8 (+), 138.2, 158.2,
166.5, 198.0. MS m/z: 307 (M+, 1), 202 (100), 188 (6), 160 (7),
147 (24), 130 (8), 105 (44), 77 (53), 56 (61), 43 (14). HR–ESI
(+)-MS: Calcd for [C18H17N3O2 + H]+ 308.1394, found
308.1392; calcd for [13C12C17H17N3O2 + H]+ 309.1428,
found 309.1426; calcd for [(C18H17N3O2)2 + H]+ 615.2714,
found 615.2717.
5-(40-Fluorobenzoyl)-2-(30-nitrophenyl)-1,4,5,6-tetrahydropyri-
midine hydrochloride (4cb).
243 mg (1.0 mmol) of 2c and
166 mg (1.0 mmol) of 3b afforded 131 mg (0.36 mmol, 36%) of
1
4cb as white solid, mp >300ꢀC. H NMR d: 3.61 (dd, J = 13.5,
7.2, 2H, H-4/6), 3.81 (dd, J = 13.6, 4.5, 2H, H-4/6), 4.31 (m, 1H,
H-5), 7.45 (t, J = 8.8, 2H, H-300/500), 7.94 (t, J = 8.1, 1H, H-50),
8.16–8.22 (m, 3H, H-200/600, H-60), 8.56 (dd, J = 8.3, 2.2, 1H,
H-40), 8.62 (m, 1H, H-20), 10.71 (br s, 2H, NH). 13C NMR
d: 34.7 (+), 40.8 (ꢁ), 116.0 (+, JCF = 22.0), 123.0 (+), 127.6
(+), 130.7 (+), 131.6 (+, JCF = 9.9), 131.7 (JCF = 2.8), 134.3
(+), 147.5, 157.1, 165.4 (JCF = 253.0), 196.6. 19F NMR d: -
104.3 (tt, J= 9.1/5.2, 1F). MS m/z: 327 (M+, 1), 204 (95), 174 (9),
149 (15), 123 (55), 103 (27), 95 (27), 75 (28), 56 (100), 45
(16). HR–ESI(+)-MS: Calcd for [C17H14N3O3F+H]+ 328.1092,
found 328.1089; calcd for [13C12C16H14N3O3F + H]+ 329.1126,
found 329.1125; calcd for [(C17H14N3O3F)2 + H]+ 655.2111, found
655.2107.
5-(400-Methylbenzoyl)-2-(30-nitrophenyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4bb).
480 mg
(2.0 mmol) of 2b and 332 mg (2.0 mmol) of 3b afforded
304 mg (0.84 mmol, 42%) of 4bb as slightly yellowish solid,
1
mp >300ꢀC. H NMR d: 2.42 (s, 3H, Me), 3.60 (dd, J = 13.5,
7.2, 2H, H-4/6), 3.80 (dd, J = 13.5, 4.6, 2H, H-4/6), 4.29 (m,
1H, H-5), 7.41 (d, J = 8.0, 2H, H-300/500), 7.93 (t, J = 8.1, 1H, H-
50), 8.00 (d, J = 8.2, 2H, H-200/0600), 8.24 (d, J = 8.0, 1H, H-60),
8.55 (dd, J = 8.3, 2.2, 1H, H-40), 8.64 (t, J = 2.0, 1H, H-20),
10.81 (br s, 2H, NH). 13C NMR d: 21.1 (+), 34.6 (+), 40.9
(ꢁ), 123,1 (+), 127.5 (+), 128.6 (+), 129.3, 129.5 (+), 130.7
(+), 132.4, 134.3 (+), 144.5, 147.5, 157.1, 197.4. MS m/z:
323 (M+, 1), 204 (100), 174 (37), 131 (8), 119 (51), 103 (10),
91 (42), 65 (19), 56 (57), 44 (10). HR–ESI(+)-MS: Calcd
for [C18H17N3O3 + H]+ 324.1343, found 324.1341; calcd for
[13C12C17H17N3O3 + H]+ 325.1377, found 325.1375; calcd
for [(C18H17N3O3)2 + H]+ 647.2613, found 647.2616.
2-(40-Carbamoylphenyl)-5-(400-fluorobenzoyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4cc).
243 mg
(1.0 mmol) of 2c and 200 mg (1.0 mmol) of 3c afforded 192 mg
1
(0.53 mmol, 52%) of 4cc as white solid, mp 288ꢀC. H NMR d:
3.60 (dd, J = 13.4, 6.5, 2H, H-4/6), 3.80 (dd, J = 13.7, 4.5, 2H, H-
4/6), 4.32 (m, 1H, H-5), 7.43 (t, J = 8.7, 2H, ArH), 7.63/8.25 (2 ꢂ
br s, 2H, CONH2), 7.89 (m, 2H, ArH), 8.08 (d, J = 8.3, 2H, ArH),
8.19 (m, 2H, ArH), 10.60 (br s, 2H, NH). 13C NMR d: 34.8 (+),
40.7 (ꢁ), 116.0 (+, JCF = 22.0), 127.8 (+), 127.9 (+), 130.1, 131.6
(+, JCF = 2.8), 131.7 (JCF = 9.6), 138.3, 158.0, 165.5 (JCF = 253.1),
166.5, 196.6. 19F NMR d: ꢁ104.4 (tt, J = 8.6/5.2, 1F). MS m/z:
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

January 2015
Synthesis, Characterization and NO Synthase Inhibition Testing of
2-Aryl-5-aroyl-3,4,5,6-tetrahydropyrimidinium Chlorides
35
325 (M+, 2), 202 (100), 147 (27), 123 (54), 103 (24), 95
(44), 75 (22), 65 (12), 56 (96), 44 (39). HR–ESI(+)-
MS: Calcd for [C18H16N3O2F + H]+ 326.1299, found
326.1296; calcd for [13C12C17H16N3O2F + H]+ 327.1333,
found 327.1331; calcd for [(C18H16N3O2F)2 + H]+ 651.2526,
found 651.2520.
5-(400-Chlorobenzoyl)-2-(30-nitrophenyl)-1,4,5,6-tetra-
hydropyrimidine hydrochloride (4fb).
260 mg (1.0 mmol)
of 2f and 166 mg (1.0 mmol) of 3b afforded 141 mg (0.40 mmol,
40%) of 4fb as yellow solid, mp = 280ꢀC. 1H NMR d: 3.60 (dd,
J = 13.4, 6.8, 2H, H-4/6), 3.82 (dd, J = 13.4, 4.4, 2H, H-4/6),
4.33 (m, 1H, H-5), 7.68 (d, J = 8.4, 2H, H-300/500), 7.93
(t, J = 8.0, 1H, H-50), 8.11 (d, J = 8.5, 2H, H-200/600), 8.27 (d,
J = 7.7, 1H, H-60), 8.55 (d, J = 8.0, 1H, H-40), 8.66 (br S, 1H,
H-20), 10.93 (br s, 2H, NH). 13C NMR d: 34.8 (+), 40.7 (ꢁ),
123.1 (+), 127.6, 129.1 (+) ,129.3, 130.4 (+), 130.7 (+), 133.6,
134,3 (+), 138.9, 147.5, 157.1, 197.0. 15N NMR d: ꢁ13.9
(NO2), ꢁ267.1 (N1/3). MS m/z: 343 (M+, <1), 204 (100), 149
(7), 149 (16), 139 (21), 131 (7), 111 (14), 103 (14), 75 (9), 56
(39). HR–ESI(+)-MS: Calcd for [C17H14N3O335Cl + H]+
344.0796, found 344.0795; calcd for [13C12C16H14N3O335Cl +
5-(300-Methoxybenzoyl)-2-(30-nitrophenyl)-1,4,5,6-tetrahy-
dropyrimidine hydrochloride (4db).
256 mg (1.0 mmol) of
2d and 165 mg (1.0mmol) of 3b afforded 149mg (0.40 mmol,
40%) of 4db as slightly yellowish solid, mp 204ꢀC. Anal.,
%: calcd for C18H18N3O4Cl C 57.53, H 4.83, N 11.18; found
1
C 57.50, H 4.92, N 11.05. H NMR d: 3.61 (dd, J = 13.5, 7.1,
2H, H-4/6), 3.82 (dd, J = 13.5, 4.7, 2H, H-4/6), 3.85 (s, 3H,
OMe), 4.31 (m, 1H, H-5), 7.31 (ddd, J = 8.2, 2.7, 1.0, 1H,
H-400), 7.53 (t, J = 8.0, 1H, H-500), 7.56 (t, J = 2.0, 1H, H-200),
7.69 (ddd, J = 7.7, 1.6, 1.0, 1H, H-600), 7.94 (td, J = 8.0, 0.6,
1H, H-50), 8.23 (ddd, J = 8.0, 1.8, 1.0, 1H, H-60), 8.56 (ddd,
J = 8.3, 2.3, 1.0, 1H, H-40), 8.64 (t, J = 2.0, 1H, H-20), 10.70
(br s, 2H, NH). 13C NMR d: 34.8 (+), 40.9 (ꢁ), 55.4 (+),
113.2 (+),119.8 (+),120.9 (+),123.0 (+), 127.6 (+), 129.4,
130.2 (+), 130.8 (+), 134.3 (+), 136.3, 147.5, 157.1, 159.6,
197.8. MS m/z: 339 (M+, 1), 204 (100), 174 (20), 135
(21), 119 (8), 107 (17), 103 (11), 92 (14), 77 (23), 56 (57).
HR–ESI(+)-MS: Calcd for [C18H17N3O4 + H]+ 340.1292,
found 340.1288; calcd for [13C12C17H17N3O4 + H]+ 341.1326,
found 341.1323; calcd for [(C18H17N3O4)2 + H]+ 679.2511, found
679.2500.
H]
H]
+
345.0830, found 345.0829; calcd for [(C17H15N3O335Cl) +
687.1520, found 687.1532.
+
5-(400-Ethoxybenzoyl)-2-phenyl-1,4,5,6-tetrahydropyrimidine
hydrochloride (4ga).
270 mg (1.0 mmol) of 2 g and 157 mg
(1.0mmol) of 3aꢂ H2O afforded 110mg (0.32 mmol, 32%) of
1
4ga as white solid, mp= 298ꢀC. H NMR d: 1.36 (t, J = 6.9, 3H,
Me), 3.58 (dd, J = 13.3, 6.6, 2H, H-4/6), 3.76 (dd, J = 13.5, 4.0,
2H, H-4/6), 4.16 (q, J = 7.0, 2H, OCH2) 4.25 (m, 1H, H-5), 7.09
(d, J = 8.9, 2H, H-300/500), 7.62 (m, 2H, H-30/50), 7.73 (m, 1H,
H-40), 7.80 (d, J = 7.3, 2H, H-20/60), 8.06 (d, J = 8.9, 2H, H-200/600),
10.44 (br s, 2H, NH). 13C NMR d: 14.4 (+), 34.4 (+), 40.9 (ꢁ),
63.7 (ꢁ), 114.6 (+), 127.5 (+), 128.0, 128.9 (+), 130.9 (+), 133.2
(+), 158.6, 163.0, 196.2. MS m/z: 308 (M+, 1), 278 (3), 159
(100), 149 (12), 121 (20), 104 (17), 93 (8), 77 (13), 65 (11), 56
(26). HR–ESI(+)-MS: Calcd for [C19H20N2O2 + H]+ 309.1598,
found 309.1594; calcd for [13C12C18H20N2O2 + H]+ 310.1632,
found 310.1630; calcd for [(C19H20N2O2)2 + H]+ 617.3122, found
617.3115.
2-(40-Carbamoylphenyl)-5-(300-methoxybenzoyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4dc).
256 mg
(1.0mmol) of 2d and 200 mg (1.0 mmol) of 3c afforded 150 mg
(0.40 mmol, 40%) of 4dc as white solid, mp 178ꢀC. Anal., %:
calcd for C19H20N3O3Cl C 61.05, H 5.39, N 11.24; found C
61.00, H 5.31, N 11.13. 1H NMR d: 3.60 (dd, J = 13.6, 6.9, 2H,
H-4/6), 3.81 (m, 2H, H-4/6), 3.84 (s, 3H, OMe), 4.35 (m, 1H,
H-5), 7.30 (ddd, J = 8.1, 2.4, 1.0, 1H, H-400), 7.52 (t, J = 7.8,
1H, H-500), 7.56 (br s, 1H, H-200), 7.63/8.29 (2 ꢂ br s, 2 ꢂ 1H,
CONH2), 7.69 (ddd, J = 7.6, 1.5, 1.0, 1H, H-600), 7.93 (d,
J = 8.6, 2H, H-30/50), 8.09 (d, J = 8.5, 2H, H-20/H-60), 10.92 (br
s, 2H, NH). 13C NMR d: 34.9 (+), 40.8 (ꢁ), 55.5 (+), 113.1
(+), 119.9 (+), 121.0 (+), 127.8 (+), 127.9 (+), 130.1 (+), 130.2,
136.2, 138.3, 158.1, 159.6, 166.5, 197.9. MS m/z: 337 (M+, 2),
202 (100), 164 (14), 148 (24), 135 (30), 107 (20), 86 (59), 77
(27), 56 (46), 44 (42). HR–ESI(+)-MS: Calcd for [C19H19N3O3 +
H]+ 338.1499, found 338.1496; calcd for [13C12C18H19N3O3 +
H]+ 339.1533, found 339.1531; calcd for [(C19H19N3O3)
2 + H]+ 675.2926, found 675.2918.
5-(400-Ethoxybenzoyl)-2-(30-nitrophenyl)-1,4,5,6-tetrahy-
dropyrimidine hydrochloride (4gb).
270 mg (1.0mmol) of
2 g and 166 mg (1.0 mmol) of 3b afforded 125 mg (0.32 mmol,
1
32%) of 4gb as slightly yellowish solid, mp >300ꢀC. H NMR
d: 1.37 (t, J = 7.0, 3H, Me), 3.58 (dd, J = 13.4, 6.7,3H, H-4/6),
3.78 (dd, J = 13.5, 4.6, 2H, H-4/6), 4.16 (q, J = 7.0, 2H, CH2O),
4.24 (m, 1H, H-5), 7.10 (d, J = 8.9, 2H, 2H, H-300/500), 7.93 (t,
J = 8.1, 1H, H-50), 8.06 (d, J = 8.9, 2H, H-200/600), 8.21
(d, J = 8.4, 1H, H-60), 8.55 (m, 1H, H-40), 8.62 (m, 1H, H-20),
10.63 (br s, 2H, NH). 13C NMR d: 14.4 (+), 34.2 (+), 41.1 (ꢁ),
63.7 (ꢁ), 114.6 (+), 123.0 (+), 127.6 (+),129.5, 130.8 (+),
131.0 (two overlapping signals, +), 134.2 (+), 147.5, 157.2,
163.1, 196.1. MS m/z: 353 (M+, <1), 204 (100), 158 (4), 149
(16), 121 (23), 103 (8), 93 (9), 76 (5), 65 (11), 56 (25). HR–
ESI(+)-MS: Calcd for [C19H19N3O4 + H]+ 354.1448, found
354.1447; calcd for [13C12C18H19N3O4 + H]+ 355.1482,
found 355.1482; calcd for [(C19H19N3O4)2 + H]+ 707.2824,
found 707.2821.
5-(400-Chlorobenzoyl)-2-phenyl-1,4,5,6-tetrahydropyrimidine
hydrochloride (4fa).
260mg (1.0 mmol) of 2f and 157 mg
(1.0mmol) of 3aꢂ H2O afforded 148 mg (0.44 mmol, 44%) of
1
4fa as white solid, mp >280ꢀC. H NMR d: 3.55 (dd, J = 13.5,
7,2, 2H, H-4/6), 3.76 (dd, J = 13.4, 4.5, 2H, H-4/6), 4.18 (m, 1H,
H-5), 7.62 (m, 2H, H-30/50), 7.67 (d, J = 8.6, 2H, H-300/500), 7.73
(m, 1H, H-40), 7.81 (m, 2H, H-20/60), 8.11 (d, J = 8.6, 2H, H-200/
600), 10.06 (br s, 2H, NH). 13C NMR d: 35.0 (+), 40.7 (ꢁ), 127.6
(+), 128.0, 129.0 (+),129.1 (+), 130.4 (+), 133.2, 133.6, 138.8,
158.6, 197.1. MS m/z: 298 (M+, 3), 268 (6), 159 (100), 145 (10),
139 (18), 131 (7), 111 (11), 104 (25), 77 (16), 56 (29). HR–ESI
(+)-MS: Calcd for [C17H15N2O35Cl+ H]+ 299.0946, found
299.0945; calcd for [13C12C16H15N2O35Cl+ H]+ 300.0980,
found 300.0979; calcd for [C17H15N2O37Cl+ H]+ 301.0916, found
301.0914.
2-(40-Carbamoylphenyl)-5-(400-ethoxybenzoyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4gc).
270 mg
(1.0 mmol) of 2h and 200 mg (1.0 mmol) of 3c afforded
128 mg (0.34 mmol, 34%) of 4hc as white solid, mp 259ꢀC
(dec.). 1H NMR d: 1.36 (t, J = 6.9, 3H, Me), 3.42 (dd,
J = 13.2, 9.3, 2H, H-4/6), 3.63 (dd, J = 13.0, 4.1, 2H, H-4/6),
3.77 (m, 1H, H-5), 4.14 (q, J = 6.9, 2H, CH2O), 7.04 (d,
J = 8.9, 2H, H-300/500), 7.52/8.02 (2 ꢂ br s, 2 ꢂ 1H, CONH2),
7.84 (d, J = 8.5, 2H, H-20/60), 7.91 (d, J = 8.5, H-30/50), 8.01
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

36
U. Bluhm, J-L. Boucher, B. Clement, U. Girreser, D. Heber, B. Ramassamy, and U. Wolschendorf
Vol 52
(d, J = 8.9, 2H, H-200/600), NH-protons not observed. 13C NMR
d: 14.4 (+), 37.2 (+), 44.1 (ꢁ), 63.5 (ꢁ), 114.4 (+), 126.1 (+),
127.2 (+),128.5, 130.6 (+), 135.4, 137.3, 152.9, 162.6,
198.6. MS m/z: 351 (M+, 2), 304 (81), 202 (100), 184 (12),
149 (15), 103 (6), 93 (9), 65 (11), 56 (36), 44 (22). HR–
ESI(+)-MS: Calcd for [C20H21N3O3 + H]+ 352.1656, found
352.1653; calcd for [13C12C19H21N3O3 + H]+ 353.1690,
found 353.1690; calcd for [(C20H21N3O3)2 + H]+ 703.3239,
found 703.3228.
358.1546; calcd for [13C12C21H19N3O2 + H]+ 359.1584, found
359.1579; calcd for [(C22H19N3O3)2 + H]+ 715.3027, found
715.3014.
5-(300,400-Dimethoxybenzoyl)-2-(30-nitrophenyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4ib).
265 mg
(0.92 mmol) of 2i and 171 mg (1.03 mmol) of 3b afforded
163 mg (0.40 mmol, 44%) of 4ib as white solid, mp 260ꢀC,
dec. 1H NMR d: 3.61/3.79 (2 ꢂ m, 2 ꢂ 2H, 2 ꢂ H4/6) 3.84/
3.88 (2 ꢂ s, 2 ꢂ 3H, 2 ꢂ OMe), 4.31 (m, 1H, H-5), 7.15 (d,
J = 8.6, 1H, H-500), 7.53 (d, J = 2.0, 1H, H-200), 7.83 (dd,
J = 8.5, 2.0, 1H, H-600), 7.94 (dd, J = 8.1, 8.0, 1H, H-50),
8.22 (ddd, J = 7.9, 1.6, 1.0, 1H, H-60), 8.56 (ddd, J = 8.4,
2.2, 1.0, 1H, H-40), 8.63 (t, J = 2.0, 1H, H-20), 10.74 (br s,
2H, NH). 13C NMR d: 34.1 (ꢁ), 41.2 (+), 55.7 (+), 55.9
(+), 110.7 (+), 111.1 (+), 123.0, (+), 123.5 (+), 127.6 (+),
127.7, 129.5, 130.8 (+), 134.2 (+), 147.5, 148.9, 153.8,
157.1, 196.3. MS m/z: 369 (M+, 1), 204 (100), 174 (12),
165 (19), 158 (5), 149 (4), 137 (6), 103 (5), 77 (9), 56 (42).
HR–ESI(+)-MS: Calcd for [C19H19N3O5 + H]+ 370.1397,
found 370.1395; calcd for [13C12C18H19N3O5 + H]+ 371.1431,
found 371.1429; calcd for [(C19H19N3O5)2 + H]+ 739.2722, found
739.2728.
5-(200-Naphthoyl)-2-phenyl-1,4,5,6-tetrahydropyrimidine
hydrochloride (4ha).
255 mg (1.0 mmol) of 2h and
156 mg (1.0 mmol) of 3a ꢂ H2O afforded 110 mg (0.31 mmol,
31%) of 4ha as white solid, mp >290ꢀC. Anal., %: calcd for
C21H19N2OCl C 71.89, H 5.46, N 7.98; found C 71.85, H
1
5.54, N 7.82. H NMR d: 3.68 (dd, J = 13.6, 6.7, 2H, H-4/6),
3.87 (dd, J = 13.6, 4.5, 2H, H-4/6), 4.49 (m, 1H, H-5), 7.62–
7.77 (m, 5H, ArH), 7.82 (d, J = 7.3, 2H, ArH), 8.03–8.11 (m,
3H, ArH), 8.17 (d, J = 7.8, 1H, ArH), 8.90 (s, 1H, ArH), 10.46
(br s, 2H, NH). 13C NMR d: 34.9 (+), 41.0 (ꢁ), 123.7 (+),
127.1 (+), 127.6 (2 overlapping signals, 2 ꢂ +), 127.7 (+),
128.1, 128.6 (+), 129.0 (+), 129.6 (+), 130.8 (+), 132.1, 132.2,
133.3 (+), 135.3, 158.9, 197.9. MS m/z: 314 (M+, 2), 159
(100), 155 (16), 145 (9), 127 (29), 117 (5), 104 (15), 77 (20),
56 (42), 41 (7). HR–ESI(+)-MS: Calcd for [C21H18N2O + H]+
315.1492, found 315.1489; calcd for [13C12C20H18N2O + H]+
316.1526, found 316.1524; calcd for [(C21H18N2O)2 + H]+
629.2911, found 629.2904.
2-(4 0-Carbamoylphenyl)-5-(300,400-dimethoxybenzoyl)-
1,4,5,6-tetrahydropyrimidine hydrochloride (4ic).
285
mg (1.00 mmol) of 2i and 206 mg (1.03mmol) of 3c afforded
163 mg (0.40 mmol, 40%) of 4ic as white solid, mp 265ꢀC
(dec.). 1H NMR d: 3.60 (dd, J = 13.5, 6.7, 2H, H-4/6), 3.78 (dd,
J = 13.4, 4.7, 2H, H-4/6), 3.84/3.88 (2 ꢂ s, 2 ꢂ 3H, 2 ꢂ OMe),
4.30 (m, 1H, H-5), 7.14 (d, J = 8.6, 1H, H-500), 7.52 (d, J = 2.0,
1H, H-200), 7.61/8.20 (2 ꢂ br s, 2 ꢂ 1H, CONH2), 7.81 (dd,
J = 8.1, 2.0, 1H, H-600), 7.82 (d, J = 8.4, 2H, H-30/50), 8.09 (d,
J = 8.4, 2H, H-30/50), 10.35 (br s, 2H, NH). 13C NMR d: 34.3 (+),
41.0 (ꢁ), 55.7 (+), 55.9 (+), 110.7 (+),111.2 (+),123.6 (+), 127.7,
127.9 (2 overlapping signals, +), 130.1, 138.2, 148.9,
153.7, 157.8, 166.6, 196.4. MS m/z: 367 (M+, 1), 204 (100), 174
(12), 165 (19), 158 (5), 149 (4), 137 (6), 103 (5), 77 (9), 56 (42).
HR–ESI(+)-MS: Calcd for [C20H21N3O4 + H]+ 368.1605, found
368.1601; calcd for [13C12C19H21N3O4 + H]+ 369.1639,
found 369.1637; calcd for [(C20H21N3O4)2 + H]+ 735.3137,
found 735.3139.
5-(200-Naphthoyl)-2-(30-nitrophenyl)1,4,5,6-tetrahydro-
pyrimidine hydrochloride (4hb).
255 mg (1.0 mmol) of
2h and 166 mg (1.0 mmol) of 3b afforded 250 mg (0.63 mmol,
63%) of 4hb as pale yellowish solid, mp 281ꢀC. Anal., %: calcd
for C21H18N3O3Cl C 63.72, H 4.58, N 10.61; found C 63.63, H
1
4.49, N 10.54. H NMR d: 3.67 (dd, J = 13.4, 7.4, 2H, H-4/6),
3.81 (dd, J = 13.5, 4.5, 2H, H-4/6), 4.45 (m, 1H, H-5), 7.69
(m, 2H, Naphth-H), 7.92 (t, J = 8.0, 1H, H-50), 8.04/8.08/8.10/
8.16 (4 ꢂ m, 4H, Naphth-H), 8.23 (ddd, J = 7.8, 1.5, 0.9, 1H,
H-60), 8.56 (ddd, J = 8.1, 2.1, 0.9, 1H, H-40), 8.65 (t, J = 2,1,
1H, H-42), 8.87 (br s, 1H, H-100), 10.62 (br s, 2H, NH). 13C
NMR d: 34.9 (+), 41.3 (ꢁ), 122.9 (+), 123.7 (+), 127.1 (+),
127.4 (+), 127.7 (+) ,128.6 (+), 129.1 (+), 129.6 (+), 130.0,
130.7 (+), 130.8 (+), 132.2, 132.3, 134.1 (+), 135.3, 147.6,
156.9, 198.0. MS m/z: 359 (M+, 1), 204 (100), 174 (66), 155
(40), 127 (77), 119 (20), 92 (13), 77 (20), 56 (96), 44 (29).
HR–ESI(+)-MS: Calcd for [C21H17N3O3 + H]+ 360.1343, found
360.1340; calcd for [13C12C20H17N3O3 + H]+ 361.1377,
found 361.1375; calcd for [(C21H17N3O3)2 + H]+ 719.2613,
found 719.2607.
2-(40-Carbamoylphenyl)-5-(300,400-dichlorobenzoyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4kc).
170 mg
(0.57 mmol) of 2k and 161 mg (0.80 mmol) of 3c afforded
201 mg (0.49 mmol, 62%) of 4kc as white solid, mp 226ꢀC.
Anal., %: calcd for C18H16N3O2Cl3 C 52.39, H 3.91, N
10.18; found C 52.28, H 4.01, N 10.06. 1H NMR d: 3.60
(dd, J = 13.7, 4.7, 2H, H-4/6), 3.80 (dd, J = 13.5, 4.6, 2H,
H-4/6), 4.33 (m, 1H, H-5), 7.63/8.22 (2 ꢂ br s, 2 ꢂ 1H,
CONH2), 7.83 (d, J = 8.4, 2H, H-20/60), 7.92 (d, J = 8.4,
1H, H-500), 8.03 (dd, J = 8.4, 2.0, 2H, H-600), 8.08 (d, J = 8.4,
2H, H-30/50), 8.33 (d, J = 2.0, 1H, H-200), 10.45 (br s, 2H,
NH). 13C NMR d: 35.0 (+), 40.6 (–), 127.8 (+), 127.9 (+),
128.5 (+), 130.1, 130.5 (+), 131.3 (+), 132.1, 135.2, 136.7,
138.3, 158.1, 166.5, 197.3. MS m/z: 375 (M+, 1), 202
(100), 188 (11), 173 (16), 164 (50), 148 (79), 120 (26),
103 (49), 56 (67), 44 (48). HR–ESI(+)-MS: Calcd for
[C18H15N3O325Cl2 + H]+ 376.0614, found 376.0613; calcd for
[C18H15N3O325Cl37Cl + H]+ 378.0585, found 378.0583; calcd
for [C18H15N3O325Cl2 + C18H15N3O325Cl37Cl + H]+ 753.1126,
found 753.1138.
2-(40-Carbamoylphenyl)-5-(200-naphthoyl)-1,4,5,6-tetra-
hydropyrimidine hydrochloride (4hc).
255 mg (1.0 mmol)
of 2h and 200 mg (1.0 mmol) of 3c afforded 140 mg (0.32 mmol,
32%) of 4hc as white solid, mp 279ꢀC (dec.). 1H NMR d: 3.49/3.71
(2 ꢂ m, 2 ꢂ 2H, H, H-4/6), 3.96 (m, 1H, H-5), 7.39 (br s, 1H,
CONH2), 7.62 (m, 2H, ArH), 7.87 (m, 4H, ArH), 7.89–8.06
(m, 4H, 3 ꢂ ArH, CONH2), 8.16 (d, J = 7.7, 1H, ArH), 8.81 (s,
1H, ArH), NH not observed. 13C NMR d: 37.9 (+), 40.3 (ꢁ),
126.0 (+), 126.9 (+), 127.1 (+),127.6 (+),128.4 (+), 128.7 (+),
129.7 (+), 130.4 (+), 132.3, 133.1, 135.0, 135.2, 137.7, 152.6,
167.3, 200.5. MS m/z: 358 (M+, 1), 202 (100), 184 (16), 165
(9), 155 (23), 147 (13), 127 (38), 77 (10), 56 (62), 44 (37).
HR–ESI(+)-MS: Calcd for [C22H19N3O2 + H]+ 358.1550, found
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

January 2015
Synthesis, Characterization and NO Synthase Inhibition Testing of
2-Aryl-5-aroyl-3,4,5,6-tetrahydropyrimidinium Chlorides
37
13C NMR d: 34.9 (+), 40.7 (–), 127.7 (+), 127.9 (+), 128.1,
130.2, 132.0 (+), 133.9, 138.3, 158.1, 166.5, 197.3. MS m/z:
387/385 (M+, 1), 202 (100), 185/183 (15), 164 (24), 148 (34),
130 (13), 103 (13), 76 (31), 56 (66), 44 (34). HR–ESI(+)-MS:
Calcd for [C18H16N3O729Br + H]+ 386.0499, found 386.0498; calcd
for [C18H16N3O821Br+ H]+ 388.0478, found 388.0478; calcd
for [C18H16N3O729Br+ C18H16N3O281Br+ H]+ 773.0904, found
773.0916.
5-(400-Biphenyloyl)-2-phenyl-1,4,5,6-tetrahydropyrimidine
hydrochloride (4la).
302 mg (1.0 mmol) of 2l and 156 mg
(1.0 mmol) of 3a ꢂ H2O afforded 112 mg (0.30 mmol, 30%) of
4la as white solid, mp = 283ꢀC (dec.). 1H NMR d: 3.64 (dd,
J = 13.5/6.6, 2H, H-4/6), 3.82 (dd, J = 13.5/4.6, 2H, H-4/6),
4.36 (m, 1H, H-5), 7.43–7.57 (m, 3H, H-40/3000/5000), 7.63
(t, J = 7.8, 2H, H-30/50), 7.71–7.82 (m, 5H, ArH), 7.91 (d,
J = 8.3, 2H, H-300/500), 8.17 (d, J = 8.3, 2H, H-200/600), 10.43
(br s, 2H, NH). 10.06 (br s, 2H, NH). 13C NMR d: 34.9 (+),
40.8 (–), 127.0 (+), 127.1 (+), 127.6 (+), 128.0, 128.5 (+),
129.0 (+), 129.1 (+), 129.3 (+), 133.3 (+), 133.6, 138.6,
145.2, 158.8, 197.5. MS m/z: 340 (M+, 1), 310 (4), 181 (16),
159 (100), 152 (14), 145 (7), 117 (4), 104 (12), 77 (10), 56
(27). HR–ESI(+)-MS: Calcd for [C23H20N2O + H]+ 341.1648,
found 341.1646; calcd for [13C12C22H20N2O + H]+ 342.1682,
found 342.1679; calcd for [(C23H20N2O)2 + H]+ 681.3224,
found 681.3222.
2-Phenyl-5-(300,400,500-trimethoxybenzoyl)-1,4,5,6-tetra-
hydropyrimidine hydrochloride (4na).
631 mg (2.0 mmol)
of 2n and 313 mg (2.0 mmol) of 3a ꢂ H2O afforded 232 mg
1
(0.60 mmol, 30%) of 4na as white solid, mp 278ꢀC. H NMR
d: 3.59 (dd, J = 13.6, 6.7, 2H, H-4/6), 3.77/3.88 (2 ꢂ s, 3H/
6H, 3 ꢂ OMe), 3.79 (m, 2H, H-4/6), 4.41 (m, 1H, H-5), 7.37
(s, 2H, H-200/600), 7.63 (m, 2H, H-30/H-50), 7.74 (m, 1H, H-
40), 7.81 (d, J = 7.3, 2H, H-20/60), 10.41 (br s, 2H, NH). 13C
NMR d: 34.4 (+), 40.9 (–), 56.3 (+), 60.2 (+), 106.3 (+),
127.7, (+), 127.9, 129.0 (+), 130.1, 133.3 (+), 142.6, 153.0,
158.6, 197.0. MS m/z: 354 (M+, 1), 195 (6), 179 (2), 168
(2), 159 (100), 145 (5), 117 (3), 104 (9), 77 (7), 56 (23).
HR–ESI(+)-MS: Calcd for [C20H22N2O4 + H]+ 355.1652, found
355.1650; calcd for [13C12C19H22N2O4 + H]+ 356.1686,
found 356.1685; calcd for [(C20H22N2O4)2 + H]+ 709.3232,
found 709.3232.
5-(400-Biphenyloyl)-2-(30-nitrophenyl)-1,4,5,6-tetrahydro-
pyrimidine hydrochloride (4lb).
300 mg (1.0 mmol) of 2l
and 166 mg (1.0 mmol) of 3b afforded 224 mg (0.53 mmol, 53%)
1
of 4lb as pale yellow solid, mp >280ꢀC. H NMR d: 3.64 (dd,
J = 13.3, 7.2, 2H, H-4/6), 3.85 (dd, J = 13.6, 4.6, 2H, H-4/6),
4.37 (m, 1H, H-5), 7.42–7.55 (m, 3H, H-400/H-3000/5000), 7.79
(d, J = 7.3, 2H, H-2000/6000), 7.90–7.97 (m, 3H, H-50/300/500), 8.18
(d, J = 8.4, 2H, H-200/600), 8.23 (d, J = 7.9, 1H, H-60), 8.55 (d,
J = 8.4, 1H, H-40), 8.65 (br m, 1H, H-20), 10.77 (br s, 2H, NH).
13C NMR d: 34.9 (+), 41.0 (–), 123.0 (+), 127.0 (+), 127.1 (+),
127.4 (+), 128.5 (+), 129.0 (+), 129.3 (+), 129.7, 130.7 (+),
133.7, 134.2 (+), 138.6, 145.2, 147.5, 156.9, 197.6.MS m/z:
385 (M+, <1), 351 (7), 204 (100), 181 (29), 174 (22), 152 (31),
103 (8), 76 (9), 56 (66), 44 (34). HR–ESI(+)-MS: Calcd
for [C23H19N3O3 + H]+ 386.1499, found 386.1494; calcd for
[13C12C22H19N3O3 + H]+ 387.1533, found 387.1533; calcd for
[(C23H19N3O3)2 + H]+ 771.2926, found 771.2917.
2-(30-Nitrophenyl)-5-(300,400,500-trimethoxybenzoyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4nb).
631 mg
(2.0mmol) of 2m and 332 mg (2.0 mmol) of 3b afforded 275 mg
(0.63 mmol, 32%) of 4nb as slightly yellowish solid, mp
1
>269ꢀC (dec.). H NMR d: 3.60 (dd, J = 13.5, 6.7, 2H, H-4/6),
3.78/3.88 (2 ꢂ s, 3H/6H, 3 ꢂ OMe), 3.81 (dd, J = 13.5, 4.2, 2H,
H-4/6), 4.42 (m, 1H, H-5), 7.39 (s, 2H, H-200/600), 7.94 (t,
J = 8.0, 1H, H-50), 8.29 (d, J = 8.0, 1H, H-60), 8.55 (dd, J = 8.3,
2.2, 1H, H-40), 8.67 (m, 1H, H-20), 10.94 (br s, 2H, NH). 13C
NMR d: 34.1 (+), 41.0 (–), 56.3 (+), 60.2 (+), 106.4 (+), 123.1
(+), 127.6, (+), 129.3, 130.1, 130.7 (+), 134.4 (+), 142.7,
147.5, 153.0, 157.0, 197.0. MS m/z: 399 (M+, 1), 204 (100),
195 (11), 174 (21), 153 (7), 149 (4), 119 (4), 103 (6), 71 (5),
56 (22). HR–ESI(+)-MS: Calcd for [C20H21N3O6 + H]+ 400.1503,
found 400.1500; calcd for [13C12C19H21N3O6 + H]+ 401.1537,
found 401.1534; calcd for [(C20H22N2O4)2 + H]+ 799.2933, found
799.2936.
5-(400-Bromobenzoyl)-2-(30-nitrophenyl)-1,4,5,6-tetrahydro-
pyrimidine hydrochloride (4mb).
610 mg (2.0 mmol) of
2m and 332 mg (2.0 mmol) of 3b afforded 395 mg (0.93 mmol,
1
47%) of 4mb as yellowish solid, mp 230ꢀC. H NMR d: 3.60
(dd, J = 13.6, 7.0, 2H, H-4/6), 3.81 (dd, J = 13.6, 4.6, 2H, H-4/
6), 4.30 (m, 1H, H-5), 7.83 (d, J = 8.6, 2H, H-300/500), 7.93 (t,
J = 8.0, 1H, H-50), , 8.02 (d, J = 8.6, 2H, H-200/0600), 8.23 (d,
J = 8.4, 1H, H-60), 8.55 (dd, J = 8.3, 2.3, 1H, H-40), 8.61 (t,
J = 2.0, 1H, H-20), 10.81 (br s, 2H, NH). 13C NMR d: 34.8 (+),
40.8 (–), 123.0 (+), 127.6 (+), 128.1, 129.4, 130.5 (+), 130.7
(+), 132.0 (+), 133.9, 134.3 (+), 147.5, 157.1, 197.2. MS m/z:
387/385 (M+, 1), 204 (100), 185/183 (12), 174 (12), 157/155
(9), 131 (8), 119 (5), 103 (18), 76 (18), 56 (55). HR–ESI(+)-
MS: Calcd for [C17H14N3O739Br + H]+ 388.0291, found
388.0290; calcd for [C17H14N3O831Br + H]+ 390.0271, found
390.0268; calcd for [13C12C13H14N3O831Br + H]+ 391.0305,
found 391.0304.
2-(40-Carbamoylphenyl)-5-(300,400,500-trimethoxybenzoyl)-1,
4,5,6-tetrahydropyrimidine hydrochloride (4nc).
631 mg
(2.0 mmol) of 2n and 399 mg (2.0 mmol) of 3c afforded 230 mg
1
(0.52 mmol, 26%) of 4nc as white solid, mp 263ꢀC (dec.). H
NMR d: 3.60 (dd, J = 13.8, 6.9, 2H, H-4/6), 3.78/3.88 (2 ꢂ s,
3H/6H, 3 ꢂ OMe), 3.79 (m, 2H, H-4/6), 4.41 (m, 1H, H-5),
7.38 (s, 2H, H-200/600), 7.64/8.24 (2 ꢂ br s, 2 ꢂ 1H, CONH2),
7.87 (d, J = 8.4, 2H, H-20/60), 8.09 (d, J = 8.4, 2H, H-30/50),
10.52 (br s, 2H, NH). 13C NMR d: 34.3 (+), 41.0 (–), 56.3 (+),
60.2 (+), 106.3 (+), 127.7 (+), 127.9 (+), 130.1, 136.5, 138.3,
142.7, 153.0, 158.2, 167.2, 197.0. MS m/z: 397 (M+, 1), 202
(14), 164 (84), 148 (100), 120 (30), 103 (48), 76 (18), 65 (23),
51 (24), 44 (54). HR–ESI(+)-MS: Calcd for [C21H23N3O5 + H]+
398.1710, found 398.1708; calcd for [13C12C20H23N3O5 + H]+
399.1744, found 399.1742; calcd for [(C21H23N3O5)2 + H]+
795.3348, found 795.3356.
2-(40-Carbamoylphenyl)-5-(400-bromobenzoyl)-1,4,5,6-
tetrahydropyrimidine hydrochloride (4mc).
610 mg
(2.00 mmol) of 2a and 400 mg (2.00 mmol) of 3c afforded
364 mg (0.86 mmol, 42%) of 4mc as white solid, mp 272ꢀC
(dec.). 1H NMR d: 3.60 (dd, J = 13.6, 6.6, 2H, H-4/6), 3.79 (dd,
J = 13.4, 4.5, 2H, H-4/6), 4.29 (m, 1H, H-5), 7.63/8.24
(2 ꢂ br s, 2 ꢂ 1H, CONH2), 7.82 (d, J = 8.4, 2H, H-300/500),
7.85 (d, J = 8.3, 2H, H-20/60), 8.03 (d, J = 8.4, 2H, H-200/0600),
8.08 (2 ꢂ d, J = 8.3, 2 ꢂ 2H, H-30/50), 10.50 (br s, 2H, NH).
Biological testing.
L-Arginine, L-NNA, dithiothreitol,
hemoglobin, superoxide dismutase (SOD), catalase, human
iNOS, and porcine brain CaM were purchased from Sigma.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

38
U. Bluhm, J-L. Boucher, B. Clement, U. Girreser, D. Heber, B. Ramassamy, and U. Wolschendorf
Vol 52
(6R)-5,6,7,8-Tetrahydrobiopterin was purchased from Alexis
(Coger, France) and NADPH from Boehringer. Recombinant
nNOS was isolated and purified from the yeast Saccharomyces
cerevisiae transformed with a plasmid containing rat brain
nNOS [75]. Full length recombinant murin iNOS and bovine
eNOS were expressed in Escherichia coli and purified in the
absence of H4B and L-arginine as described previously [76,77].
They were estimated to be more than 95% pure by SDS–PAGE
electrophoresis.
coefficient of 6.2 mMꢁ1 ꢂ cmꢁ1. Cuvettes contained 150 mL of
50 mM Hepes, pH 7.4, 5 mM DTT, 100 mM CaCl2, 200 mM
NADPH, 10 mM H4B, 100 mM L-arginine, and the tested agents.
After preincubating the cofactor mixture and the inhibitor for
2 min at 37ꢀC, the nNOS sample (final concentration 15–50 nM)
was added, and the absorption at 340 nm was followed for
3 min. Control incubations were run in the presence of 1%
DMSO, in the absence of NADPH, or in the presence of 1 mM
L-NNA, a known inhibitor of NADPH consumption by NOS [74].
Nitrite determination (Griess assay).
The formation of
nitrite (stable end-product of NO) was followed by the
reaction with sulphanilamide and 1-naphthylethylenediamine
hydrochloride in hydrochloric acid. The absorption of the
resulting azo dye was measured at 543 nm on a Beckman
DU7500 UV/vis spectrophotometer (Beckman Coulter, Fullerton,
USA). Because NADPH can affect low values by reducing the
diazonium cation as well as the azo dye, it was necessary to
remove NADPH from the reaction using an additional incubation
with lactate dehydrogenase and sodium pyruvate [73].
Buffers contained 50 mM triethanolamine (iNOS and eNOS,
pH = 7.5; nNOS, pH = 7.0) and 50 mM triethanolamine, 1 mM
CHAPS, 10 mM 2-mercaptoethanol, and 0.5 mM EDTA. Typical
assay mixtures contained 0.5 mM Ca++ (nNOS and eNOS), 1 mM
Mg++ (iNOS), 5 mM flavine adenine dinucleotide, 5 mM flavine
mononucleotide, 0.5 mM NADPH, 10 mM H4B, 10 mg/mL
CaM, 0.5 mM (nNOS), or 1 mM L-arginine (iNOS and eNOS)
in a total volume of 80 mL. These mixtures were incubated at
37ꢀC (nNOS 30 min, iNOS, and eNOS 20 min) after the addition
of proteins. Then a solution of 1.6 mM pyruvic acid, sodium salt,
and 20 U/mL L-lactic dehydrogenase (20 ml) was added. These
preparations were incubated for 20 min at 37ꢀC, and the reactions
were stopped by the addition of 50 mL ice cold acetonitrile and
centrifugation (15,000 rpm, 4ꢀC). Then 12 mL each of a solution
of 5.8 mM naphthylethylendiamine dihydrochloride and 52 mM
sulfanilamide in 1 M HCl were added to 120 mL of the superna-
tants. Absorbances at 543 nm were measured after 5 min at
20ꢀC. All data were referred to 100% control experiments
containing 1% DMSO but without inhibitor. All experimental
data are means of four determinations.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet