Synthesis and evaluation of pyrido[1,2-a]pyrimidines as inhibitors of nitric oxide synthases

Article abstract of DOI:10.1016/j.ejmech.2008.12.007

A series of new 3-aroylpyrido[1,2-a]pyrimidines were synthesized from aryl methyl ketones in a simple two-step procedure and evaluated as nitric oxide synthases (NOS) inhibitors. In order to perform a structure-activity relationship study, different aroyl

Full text of DOI:10.1016/j.ejmech.2008.12.007

European Journal of Medicinal Chemistry 44 (2009) 2877–2887
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of pyrido[1,2-a]pyrimidines as inhibitors of
nitric oxide synthases
,
a
a
Ullvi Bluhm a, Jean-Luc Boucher b, Uwe Buss a, Bernd Clement a , Friederike Friedrich , Ulrich Girreser ,
*
Dieter Heber a, Thanh Lam a, Michel Lepoivre c, Mojgan Rostaie-Gerylowa, Ulrich Wolschendorf a
a Pharmaceutical Institute, Department of Pharmaceutical Chemistry, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany
b Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601, University Paris Descartes, 45 Rue des Saints-Pe`res, 75270 Paris Cedex 06, France
c CNRS URA 1116, University Paris Sud-Orsay, Bat 430, 91105 Orsay Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 3-aroylpyrido[1,2-a]pyrimidines were synthesized from aryl methyl ketones in a simple
two-step procedure and evaluated as nitric oxide synthases (NOS) inhibitors. In order to perform
a structure–activity relationship study, different aroyl groups were introduced in 3-position and methyl
groups were introduced at different positions of the pyrimidine heterocycle. Compounds with a biphe-
nyloyl, benzyloxybenzoyl or naphthoyl group displayed the highest inhibitory effects which were further
increased by introduction of a methyl group in position 8 of the pyrido[1,2-a]pyrimidine moiety. Some of
the compounds exhibited promising inhibitory effects with selectivity toward the purified inducible NOS
(iNOS) and were also active against iNOS expressed in stimulated RAW 264.7 cells.
Received 13 June 2008
Received in revised form
5 November 2008
Accepted 8 December 2008
Available online 16 December 2008
Keywords:
Nitric oxide synthase
Inhibitors
Ó 2008 Elsevier Masson SAS. All rights reserved.
Pyrido[1,2-a]pyrimidines
Isoform selectivity
1. Introduction
bears binding sites for the substrate
L-arginine, the heme prosthetic
group and the cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (H4B),
Nitric oxide, NO, is a recently identified mediator acting as
a regulator of the vascular tone, a neurotransmitter and a cytotoxic
agent. NO also plays important roles in the pathogenesis of several
diseases [1,2]. In particular, NO contributes to inflammatory and
autoimmune diseases such as rheumatoid arthritis, osteoarthritis,
inflammatory bowel disease and multiple sclerosis [3–7]. Therefore,
nitric oxide synthases (NOS), the enzymes that produce NO by
and a CO2H-terminal reductase domain which contains binding
sites for the flavins, FMN and FAD, and the cofactor NADPH. These
two domains are fused by a Caþþ-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 [8–10].
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
ischaemia and Parkinson’s disease [1,2,11–14]. However, they must
be selective towards n- 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 developmentof either nNOS or iNOS selective inhibitors [15–18].
Several structurally diverse classes of NOS inhibitors have been
oxidation of L-arginine, are potential targets for new therapeutic
agents. In mammals, three isoforms of NOS have been identified.
The constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS)
are Caþþ-dependent enzymes that play key roles in the nervous and
cardiovascular systems whereas the inducible nitric oxide syn-
thases (iNOS) produce large quantities of NOS following immuno-
logical challenge [8–10]. NOS are homodimeric enzymes and each
NOS subunit contains an NH2-terminal oxygenase domain which
investigated. Analogues of the natural substrate
L
-arginine, dipep-
tides and peptidomimetics derived from Nu-nitro-
L-arginine have
been studied, some of them exhibiting selectivities of up to three
orders of magnitude over either eNOS or iNOS inhibition [19–24]. In
the meantime, a great number of non-amino acid compounds have
been tested as either nNOS or iNOS inhibitors. Substituted inda-
zoles (with 7-nitroindazole, 7-NI, as the lead compound) or imid-
azoles, are potent NOS inhibitors but their selectivity remains low,
Abbreviations: n-NOS, neuronal nitric oxide synthase; i-NOS, inducible nitric
oxide synthase; eNOS, endothelial nitric oxide synthase; iNOSoxy
domain of the inducible NOS; 7-NI, 7-Nitro-1H-indazole;
-NNA, Nu-Nitro-
nine; -arginine; SEITU, S-Ethyl-iso-thiourea; 1400W, N-(3-
-NPA, Nu-Propyl-
,
Oxygenase
L
L-argi-
L
L
(aminomethyl)benzyl)acetamidine.
* Corresponding author. Tel.: þ49 0431 880 1126; fax: þ49 0431 880 1352.
E-mail address: bclement@pharmazie.uni-kiel.de (B. Clement).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.12.007

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U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
at least in vitro [25,26]. Potent non-amino acid inhibitors with
iso-thiourea and amidine functions have been reported [27–35]. 2-
Aminopyridines have been described as NOS inhibitors, and
selective nNOS inhibitors containing this scaffold were recently
reported [36,37]. However, 2-amino-4-methylpyridine derivatives,
which are reported to be highly potent inhibitors of all three iso-
forms, can exhibit high toxicity and should not be suitable for
further development [36].
3-benzoyl derivatives 8–22, five pyrido[1,2-a]pyrimidines 23–28
bearing a benzyloxybenzoyl substituent in position 3, nine naphthoyl
(28–30) and ten biphenyloyl substituted heterocycles (31–37) were
synthesized and evaluated as inhibitors of the three purified NOS
isoforms.
3. Biological results
Pyrido[1,2-a]pyrimidines are heterocyclic compounds that
include a 2-aminopyridine moiety and could thus appear as
potential inhibitors of NOS. Therefore we have synthesized a series
of new pyrido[1,2-a]pyrimidines bearing various aromatic substit-
uents and one or two methyl groups at different positions of the
pyrimidine ring.
3.1. Screening of the inhibitory effects of compounds 7–37 on the
activity of NOS using the Griess assay
In a first series of experiments, NOS inhibition data were
obtained for all compounds introduced at 100
mM (final concen-
tration) using recombinant NOS isoforms from different species
and the classical colorimetric Griess assay for nitrite determination
[40]. Experiments including the known inhibitors of NOS Nu-
These compounds were evaluated as inhibitors of the three
purified NOS isoforms from various species and of an iNOS
expressed in
a
murine macrophage cell line. Some of the
nitro-L-arginine (L L-arginine (L-NPA), S-ethyl-
-NNA), Nu-propyl-
compounds exhibited good inhibitory effects with some selectivity
for one isoform of NOS thus opening the way to further SAR studies.
iso-thiourea (SEITU), and N-(3-aminomethyl)benzylacetamidine
(1400W) were performed for comparative purposes (see Fig. 1 for
structures) [15–17]. As previously described, L-NNA and L-NPA more
efficiently inhibited nitrite formation catalyzed by n- and eNOS
whereas SEITU and 1400W were more efficient toward the induc-
ible NOS than the constitutive isoforms [15–17]. Using this
relatively insensitive assay, some of the tested pyrido[1,2-
a]pyrimidines were found as active as the known inhibitors.
However, no clear specificity for one isoform could be observed
under our experimental conditions (Table 1).
2. Chemistry
Pyrido[1,2-a]pyrimidines 8–37 were prepared by condensation of
2-aminopyridines7withenoneMannichbases4–6, whicharereadily
accessible through the heating of aryl methyl ketones 1–3 and N,N0-
dimethylmethyleniminium chloride in N,N-dimethylformamide [38]
(see Scheme 1 for reactions). After the addition of the nucleophile to
the enone structure, the dimethylamino group was eliminated fol-
lowed by ring closure to form anellated heterocycles [39]. Isolating
the resulting products as hydroperchlorates or as hydrochlorides
showed that an addition–elimination mechanism had taken place. In
the first step, the 2-aminopyridine adds to the double bond. Next
a new electrophilic center is generated by the elimination of N,N0-
dimethylammonium chloride to form a new enone moiety followed
by a second addition to the ring closed heterocycle (see Scheme 2 for
structures). The intermediates could not be isolated. The enones 4–6
were not characterized completely but immediately transformed to
the heterocycles 8–37. Details for the synthesis of some of these
enone Mannich bases have been published previously [38].
3.2. Inhibitory effects of some pyrido[1,2-a]pyrimidines on the
activity of NOS using a radioactive assay
Further experiments were performed to check the inhibitory
effects of a more limited number of compounds using the conver-
sion of [
assay to measure NOS activity [41]. This series of experiments was
performed in the presence of 100 M of each compound and 2.5
of substrate -arginine, a value close to the Km values of the various
g- L L-citrulline as a more sensitive
14C] -arginine to [14C]
m
mM
L
NOS isoforms [8–10]. The heterocyclic compound 7-NI was also
introduced as a known heterocyclic inhibitor [15–17,25].
As a general observation, all the tested compounds more effi-
ciently inhibited iNOS than nNOS or eNOS. Neuronal NOS was half-
The ring closure reaction was performed with 2-aminopyridine
(7a), 2-amino-3-methylpyridine (7b), 2-amino-4-methylpyridine (7c),
2-amino-6-methylpyridine (7d), and 2-amino-4,6-dimethylpyridine
(7e). Using phenyl, naphthyl, biphenyl, and benzyloxyphenyl
substituted enone Mannich bases 4–6 for the ring closure reaction, the
corresponding 3-aroylpyrido[1,2-a]pyrimidine derivatives 8–37 were
obtained in moderate (30%) to good (70%) yields from commercially
available aryl methyl ketones following simple procedures. Twenty six
inhibited by 100
mM of compounds 18b, 26c, 34c and 35c whereas
compounds 29e, 34c and 35c (all at 100
mM) were the most potent
inhibitorsofeNOS. Interestingly, compounds 35c and26c were found
more potent than 7-NI against nNOS and eNOS, respectively. Pyr-
ido[1,2-a]pyrimidines 26c, 29c, 29e, 34c, 35c, and 37c strongly
inhibited the inducible NOS, with compounds 29c and 35c being
more potent than
the most promising compounds were determined and compared to
those of -NNA and 7-NI. The studied compounds displayed IC50
value in the 10–100 M range and those of 35c (with iNOS) and 26c
(witheNOS) were close tothoseof -NNA forthese enzymes (Table 2).
L-NNA under those conditions. The IC50 values of
O
Ar = R1
L
Me
Ar
m
1
Ar =
R3
1-3
L
Ar =
R2
a,b
3.3. Mechanism of inhibition of iNOS by some pyrido[1,2-a]
pyrimidines
3
2
O
O
Further experiments were performed in order to better inves-
tigate the mechanism of the inhibition of iNOS by some of the most
promising pyrido[1,2-a]pyrimidines. In a first set of experiments,
the inhibitory effects of compounds 29e, 34c, 35c and 37c (0, 10, 25
R
R
+
c
NHMe2
Ar
N
Ar
+
Cl
N
NH2
N
H
+
ClO4_ or Cl_
and 100
concentrations of
[2,3,4-3H]
-arginine.
For compound 37c, plotting 1/activity as
-arginine] led to straight lines that intercepted the y-axis at the
mM) were determined in the presence of different
4-6
7
8-37
L
-arginine (0.25, 0.5, 1.0, 2.5, 5 and 25 M) and
m
L
Scheme 1. General method for the synthesis of pyrido[1,2-a]pyrimidines 8–37 from
acetophenones 1–3 and 2-amino-pyridines 7a–e. Reagents and conditions: (a)
H2C]NMe2Cl, DMF, 130 ꢀC, 2 h; (b) EtOH, reflux; (c) HClO4 or HCl, EtOH.
a
function of
2
1/[L

U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
2879
7
a
b
c
d
e
R
H
3-Me 4-Me 6-Me 4,6-Me2
Ar = C6H5
Compound
R1
H
4-Me 4-OH 4-F 4-CN 4-OMe 4-Cl 4-OEt
10 11 12 13 14 15
CO2H
a
8
9
16
Ar = C6H5
Compound
R1
3-F,4-OMe
3,4-(OMe)2
3,4-Cl2 4-Br cC6H11 3,4,5-(OMe)3
19 20 21
22a
17
18
Ar = C6H5 R1
Compound
OCH2Ph
OCH2C6H4CN
OCH2C6H4OMe
OCH2C6H4Cl
OCH2C6H4Br
23
24
25
26
27a
Ar = naphthyl R2
Compound
H
Me OMe OMe
28 29 30
30ea
Ar = biphenyl R3
Compound
H
OH
F
CN OMe CO2H Br
35 a 36a
37
31 32 a 33 34 a
Scheme 2. Structures of the compounds tested in this study. (a): Isolated as hydorchloride salts.
same point (constant Vm value), and the x-axis at different points
which correspond to different 1/apparent Km values (Fig. 2). These
results suggested that compound 37c inhibited iNOS in a competi-
3.4. Effects of some pyrido[1,2-a]pyrimidines on the spectra
of the heme
tive manner with
a function of the concentration of 37c led to a Ki value of 14
(Insert of Fig. 2). Almost identical results were observed with
compounds 29c and 35c which inhibited iNOS in a competitive
L
-arginine. Replot of the apparent Km values as
In order to check if pyrido[1,2-a]pyrimidines can interact with
the heme active site of NOS, we have performed UV–visible studies
on the oxygenase domain of iNOS (iNOSoxy) which displays iden-
tical spectrophotometric properties than the full length enzyme.
The UV–visible spectrum of native iNOSoxy in Hepes buffer pH 7.4
displayed a broad Soret peak with a maximum at 415 nm indi-
cating that it exists as a mixture of hexacoordinated heme-FeIII–
H2O (low spin) and pentacoordinated heme-FeIII states (high spin),
the former state being predominant, in agreement with literature
data [42]. Addition of 1 mM imidazole (ImH) was done to
completely convert iNOSoxy into a hexacoordinated heme-FeIII–
ImH complex absorbing at 430 nm. This method was very often
used to more easily follow the binding of substrates to the NOS
mM
manner and displayed Ki values of 23 and 15 mM, respectively (data
not shown). However, a more complex mechanism for inhibition of
iNOS by compound 34c was observed and further studies are
required to precisely determine its mode of action (data not
shown). In another series of experiments, iNOS (2 mM) was
preincubated for 15 min at 4 ꢀC in the presence of 1 mM of 37c
and aliquots of this mixture were diluted 100 fold in order to
perform the incubations under identical conditions as usual for the
[g- L-arginine assay. It was observed that the inhibition of iNOS
14C]
activity by compound 37c was abolished after dilution of the
sample.
These data indicated that the inhibition of iNOS by some of the
pyrimido[1,2-a]pyrimidines was competitive with the substrate
active site [43]. As already described, the addition of 100 mM L-NNA
or 7-NI to the iNOSoxy-FeIII–ImH complex gave rise to difference
spectra characterized by a trough at 425 nm and a peak at 396 nm
showing that the binding of these compounds close to the heme
shifted the iNOSoxy spin state equilibrium to the pentacoordinated
heme-FeIII-state corresponding to a displacement of bound ImH
L-arginine and reversible.
[43,44]. Under identical conditions, the addition of 100
pyrido[1,2-a]pyrimidines 17c, 19a, 29c, 29e, 34c, 35c and 37c led to
difference spectra almost identical to that obtained with -NNA
(peak close to 396–400 nm, Table 2). All the other derivatives
tested led to very weak, not significant, difference spectra after
addition either to native iNOSoxy or to iNOSoxy–ImH complex
(Table 2).
mM of
H
N
NH
NH
O2N NH NH
H3C
L
NH2
H3C
N
NH
NH
H
1400W
NH
OH
OH
H2N
H2N
3.5. Effects of compounds on the NADPH consumption by iNOS
O
O
H2N
S
CH3
L-NPA
L-NNA
In order to more deeply investigate the mechanism of inhibition
and to get further informations on the effects of some pyrido[1,2-
a]pyrimidines on the electron transfer in NOS, we have tested their
SEITU
Fig. 1.

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U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
Table 1
iNOS was very low in the absence of
increased about three times in the presence of
and completely abolished in the presence of
L
-arginine [45]. It was
-arginine (100 M)
-NNA [45]. All the
-arginine
M (final concentrations) of pyrido[1,2-a]pyrimidines.
Activity of various NOS isoforms incubated in the presence of 100
mM L-NNA, L-NPA,
L
m
SEITU, 1400W, 2-aminopyridines 7, and pyrido[1,2-a]pyrimidines 8–37a
L
Compd nNOS
Human Rat
iNOS
eNOS
Log D7.4
experiments were performed in the presence of 100
and 100
mM L
m
Human
Murine
Human
Bovine
Compounds 27c, 34c, 35c, 37c and 37e led to a significant inhibi-
tion of NADPH consumption by iNOS (Table 2). For compounds 34c
and 37c, IC50 values were determined to be 11 and 35 mM,
respectively, close to the values obtained for the inhibition of
citrulline formation. These data show that some of the pyrido[1,2-
a]pyrimidines inhibit the NADPH consumption and could reduce
the generation of reactive oxygen species such as hydrogen
peroxide by iNOS.
L
-NNA
-NPA
13 ꢁ 1
48 ꢁ 4
95 ꢁ 5
6 ꢁ 1
25 ꢁ 4
96 ꢁ 5
37 ꢁ 1
105 ꢁ 7
94 ꢁ 8
50 ꢁ 1
83 ꢁ 2
12 ꢁ 1
L
53 ꢁ 2
16 ꢁ 1
37 ꢁ 8
17 ꢁ 3
43 ꢁ 3
88 ꢁ 2
2 ꢁ 1
3 ꢁ 1
93 ꢁ 8
27 ꢁ 12
107 ꢁ 11
SEITU
1400W 73 ꢁ 3
5 ꢁ 1
7a
7c
7d
7e
8a
8b
8c
8d
72 ꢁ 2
21 ꢁ 4
48 ꢁ 1
10 ꢁ 1
95 ꢁ 8
29 ꢁ 8
47 ꢁ 15
7 ꢁ 3
96 ꢁ 1
92 ꢁ 2
102 ꢁ 1
103 ꢁ 10
16 ꢁ 1
91 ꢁ 18 1.4
92 ꢁ 2
78 ꢁ 1b 57 ꢁ 20
95 ꢁ 1
63 ꢁ 6
72 ꢁ 1
40 ꢁ 6
3.6. Inhibitory effects of some compounds on the activity of iNOS
expressed in RAW 264.7 cells
104 ꢁ 1
86 ꢁ 1
93 ꢁ 6
94 ꢁ 1b
93 ꢁ 3
1.5
9a
9b
9d
81 ꢁ 1
93 ꢁ 1
100 ꢁ 9
99 ꢁ 3
1.6
1.6
91 ꢁ 3
101 ꢁ 6
When activated by LPS and IFN-g, this cell line expresses an
inducible NOS [46]. We tested if some of our compounds could
inhibit the induction of iNOS by introducing them at the same time
10c
11a
11c
11d
12c
13a
14c
15c
16c
17c
18a
18b
18d
19a
20a
20c
20d
21c
22a
23c
24c
25c
26c
27c
28a
28c
28d
29c
29d
29e
30c
30d
30e
31c
31d
32c
33c
34c
35c
36c
37c
37d
37e
39 ꢁ 10
83 ꢁ 6
30 ꢁ 2
67 ꢁ 15
78 ꢁ 3
88 ꢁ 3
54 ꢁ 8
46 ꢁ 8
79 ꢁ 4
77 ꢁ 5
77 ꢁ 3
75 ꢁ 6
79 ꢁ 3
85 ꢁ 5
18 ꢁ 5
51 ꢁ 2
79 ꢁ 3
78 ꢁ 5
83 ꢁ 2
37 ꢁ 4
41 ꢁ 1
as LPS þ IFN-
inhibitory effects on the activity of this iNOS, 15 h after the addition
and washing of the cells. The effects of the
g. In another set of experiments, we tested their
90 ꢁ 1
2 ꢁ 2
100 ꢁ 8
28 ꢁ 8
97 ꢁ 1
90 ꢁ 2
20 ꢁ 2
87 ꢁ 11
1.5
1.4
of LPS þ IFN-
g
2 ꢁ 1b
50 ꢁ 6
7 ꢁ 1
compounds were measured using the Griess assay (nitrite forma-
tion). Identical results were obtained in both series of experiments,
suggesting that the compounds did not affect the induction of iNOS
88 ꢁ 7
69 ꢁ 7
89 ꢁ 5
93 ꢁ 6b
94 ꢁ 2
84 ꢁ 4
102 ꢁ 17
95 ꢁ 1
98 ꢁ 1
92 ꢁ 2
123 ꢁ 10
102 ꢁ 8
95 ꢁ 2
95 ꢁ 2
92 ꢁ 1
33 ꢁ 1
9 ꢁ 3
91 ꢁ 6
95 ꢁ 1
90 ꢁ 4
83 ꢁ 4
86 ꢁ 1
72 ꢁ 4
93 ꢁ 5
in this cell line. Furthermore, it clearly appears that 100 mM of
compounds 24c, 26c, 27c, 35c, 37c and 37d strongly inhibited
nitrite accumulation in the supernatants of the cells (Table 3).
Furthermore, the studied compounds were not toxic for these cells
at this concentration (data not shown).
1.5
1.8
1.7
49 ꢁ 16 81 ꢁ 1
105 ꢁ 7
110 ꢁ 5
69 ꢁ 1
93 ꢁ 2
76 ꢁ 1b
89 ꢁ 4
85 ꢁ 2
73 ꢁ 5
86 ꢁ 1
42 ꢁ 1
95 ꢁ 6
65 ꢁ 5
89 ꢁ 1
42 ꢁ 15
88 ꢁ 1
67 ꢁ 2
60 ꢁ 4
89 ꢁ 1b
105 ꢁ 2
86 ꢁ 3
4. Discussion of structure–activity relationships
81 ꢁ 3
85 ꢁ 14
90 ꢁ 13
15 ꢁ 4
39 ꢁ 1b
56 ꢁ 7
34 ꢁ 1
61 ꢁ 7
24 ꢁ 2
75 ꢁ 17
68 ꢁ 1
83 ꢁ 6
72 ꢁ 7
69 ꢁ 3
4 ꢁ 1
73 ꢁ 8
105 ꢁ 4
101 ꢁ 13
94 ꢁ 3
83 ꢁ 1b
93 ꢁ 5
47 ꢁ 2
75 ꢁ 1
78 ꢁ 4
46 ꢁ 4
8 ꢁ 2
Our preliminary data obtained by screening using the Griess
assay were reinforced by the experiments using the radioactive
assay for NOS. For comparison, the discussion will focus on the
inhibition data expressed as percentage of residual activity
obtained at an inhibitor concentration of 100 mM in all test systems.
These assays have shown that some of the studied pyrido[1,2-
16 ꢁ 5
60 ꢁ 7
60 ꢁ 4
75 ꢁ 3
11 ꢁ 1
75 ꢁ 8
86 ꢁ 7
98 ꢁ 1
6 ꢁ 1
16 ꢁ 5
15 ꢁ 1
39 ꢁ 10
97 ꢁ 5
28 ꢁ 1
75 ꢁ 3
90 ꢁ 5
80 ꢁ 11
50 ꢁ 12 1.7
75 ꢁ 2
49 ꢁ 6
2.1
89 ꢁ 2
103 ꢁ 8
88 ꢁ 10
83 ꢁ 1
88 ꢁ 1
80 ꢁ 14
95 ꢁ 10
17 ꢁ 2
a]pyrimidines were inhibitors of NOSs with potencies close to
those of compounds such as L-NNA, 1400W or 7-NI. No potent
2.2
selectivity for one isoform of NOS from any species could be
observed for this series of compounds. However, most of them
appear to be more potent against purified iNOS than against nNOS
and eNOS but the constitutive isoforms nNOS and eNOS were
inhibited in almost similar manners. Interestingly, the studied
compounds also inhibited iNOS expressed in intact cells without
apparent toxicity.
The selectivity of this class of compounds seems to be modu-
lated by the nature and position of the substituents on the aroyl
group. In nearly all cases, the pyrido[1,2-a]pyrimidines bearing
a methyl group in position 8 of the heterocyclic ring system
(synthesized from 2-amino-4-methylpyridine 7c), have a higher
potency compared to any other substitution pattern, i.e. corre-
sponding to structures a, b, d and e. In the structures under
investigation, the higher inhibition was observed for the pairs of
structures 8c/8d, 11c/11d, 20c/20d, 28c/28d, 29c/29d, 31c/31d, 37c/
37d, and only one pair, namely 30c/30d, showed higher inhibition
potency for the d isomer.
72 ꢁ 13
40 ꢁ 15
80 ꢁ 8
1.9
1.9
121 ꢁ 9
8 ꢁ 3
2 ꢁ 1
17 ꢁ 2
4 ꢁ 3
86 ꢁ 4
96 ꢁ 7
85 ꢁ 5
55 ꢁ 6
49 ꢁ 2
98 ꢁ 2
120 ꢁ 7
106 ꢁ 10
89 ꢁ 7
20 ꢁ 8
86 ꢁ 3
46 ꢁ 3
55 ꢁ 15
57 ꢁ 2
91 ꢁ 1
17 ꢁ 3
84 ꢁ 3
15 ꢁ 10
2.1
79 ꢁ 14
36 ꢁ 5
70 ꢁ 6
62 ꢁ 3
70 ꢁ 2
15 ꢁ 1
61 ꢁ 8
4 ꢁ 3
2.7
2.8
a
Results are expressed as % of residual activities of the enzymes following incu-
M of the studied compounds.
bations in the presence of 1% DMSO alone or 100
m
The activities were measured by the Griess assay for nitrite determination under
the conditions described in the Experimental. Results are means ꢁ S.D. from
5 experiments.
b
Means ꢁ S.D. from 3 experiments.
effects on the NADPH consumption catalyzed by iNOS. These data
will give informations on their effects on the electron transfer from
the reductase domain to the heme domain of iNOS and the possible
generation of superoxide anion and hydrogen peroxide by iNOS.
As previously described for this isoform, NADPH consumption by
The lipophilicity of some compounds has been evaluated at pH
7.4 using an HPLC method [47] and the log D7.4 values are given on
Table 1. The lipophilicity is clearly linked to the type and size of the
substituent at 3-position of the pyrido[1,2-a]pyrimidine. The
position and the number of methyl substituent on the pyrimidine

U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
2881
Table 2
Effects of L-NNA, 7-NI and some pyrido[1,2-a]pyrimidines on the activities of three purified NOS, on the NADPH consumption by iNOS, and on the visible spectrum of iNOSoxy.
Compd
Rat nNOS
% Activitya
% NADPH
Heme
Murine iNOS
Bovine eNOS
Oxidationb
spectra shiftc
L
-NNA
2 ꢁ 1
15 ꢁ 5
5 ꢁ 3
8
396
398
n.d.
400
400
398
n.d.
n.d.
398
n.d.
n.d.
n.d.
398
400
396
396
400
n.d.
7-NI
9a
9d
36 ꢁ 4 (27 ꢁ 5)d
92 ꢁ 8
25 (40 ꢁ 12)d
100 ꢁ 8
5 ꢁ 3 (41 ꢁ 15)d
100 ꢁ 12
n.d.e
91
94
87
73
89
92
100
77
64
100
70
72
40
40
64
72
76 ꢁ 8
66 ꢁ 8
84 ꢁ 10
11d
17c
18b
18d
19a
24c
26c
27c
29c
29e
34c
35c
37c
37e
83 ꢁ 6
66 ꢁ 10
71 ꢁ 12
69 ꢁ 8
45 ꢁ 8
59 ꢁ 10
48 ꢁ 8
96 ꢁ 10
94 ꢁ 14
100 ꢁ 5
91 ꢁ 10
81 ꢁ 14
87 ꢁ 4
85 ꢁ 8
83 ꢁ 10
60 ꢁ 8
30 ꢁ 6
44 ꢁ 8
49 ꢁ 10
25 ꢁ 8
2 ꢁ 2 (12 ꢁ 5)d
77 ꢁ 12
62 ꢁ 10
62 ꢁ 13
69 ꢁ 5
13 ꢁ 6 (29 ꢁ 9)d
28 ꢁ 8 (35 ꢁ 12)d
18 ꢁ 7 (50 ꢁ 12)d
13 ꢁ 8 (18 ꢁ 8)d
19 ꢁ 6 (28 ꢁ 10)d
35 ꢁ 10 (120 ꢁ 25)d
48 ꢁ 12
55 ꢁ 5 (60 ꢁ 15)d
17 ꢁ 8 (53 ꢁ 12)d
43 ꢁ 6 (128 ꢁ 15)d
18 ꢁ 8 (12 ꢁ 5) d
d
d
29 ꢁ 6 (87 ꢁ 14)
15 ꢁ 5 (60 ꢁ 15)
65 ꢁ 10
10 ꢁ 4
58 ꢁ 4
32 ꢁ 8 (188 ꢁ 25)d
Data are expressed as % of the initial activities measured in the presence of 1% DMSO.
a
Inhibition of purified recombinant n-, i- or eNOS was determined by the radioactive assay and are means ꢁ S.D. from 3 experiments. Incubations contained 2.5
mM [g- L-
14C]
arginine, 1% DMSO alone or 100 M of the studied compounds, and all the required cofactors as described in the Experimental.
m
b
Incubations were performed in the presence of 100 mM L-arginine, 10 mM H4B and 1% DMSO alone or 100 mM of the studied compounds, as described in the Experimental.
Data are expressed as % of the NADPH oxidase activity of iNOS containing 1% DMSO alone (580 ꢁ 110 nmol minꢂ1 mg protꢂ1).
c
Position of the maximum appearing in the difference spectrum obtained after addition of 100 mM of the studied compounds to a solution of 0.8–1.2 mM nNOSoxy containing
1 mM ImH. This position is mentioned only for compounds leading to a significant difference spectrum. n.d.: no appearance of a significant difference spectrum.
d
IC50 values (in
Not determined.
m
M) obtained from incubations performed in the presence of increasing concentrations of the studied compounds.
e
heterocycle seem to have a small effect on the log D7.4 values.
However, increasing the size of the aromatic part of the 3-aroyl
group increases the lipophilic character and the inhibition of NOS,
as shown with the chlorophenyl derivative 14c, the chloro
substituted benzyl ether 26c, and the 4-bromobiphenyl derivatives
37c and 37e. Considering the structures of molecules, compounds
which have a simple phenyl group on the C]O group are almost
inactive. Compounds 29c and 29e that contain a naphthyl group are
less potent than those containing a biphenyl (35c and 34c) or
a benzyloxy group (24c, 26c, 27c). A methoxy or a bromo substit-
uent on the biphenyl group reinforces the inhibitory effects. The
more lipophilic structures led to the highest inhibitory effects and
a slight preference for the inhibition of iNOS over n- and eNOS was
observed.
reduced NADPH consumption by iNOS suggesting that they could
interfere with the electron transfer between the reductase domain
and the heme domain and affect the generation of reactive oxygen
species by NOS. Furthermore, our UV–visible and mechanistic
studies suggest that some of the compounds could bind close to the
heme in an identical manner than
L-arginine and that they inhibit
iNOS by competition with -arginine binding at the active site. A
L
more precise mode of binding of the compounds will be obtained
with the help of crystallographic studies.
The synthetic approach to this class of heterocyclic compounds
is straightforward and would allow easy modifications of the pyr-
ido[1,2-a]pyrimidine backbone. Further SAR studies will help in the
design of more potent and selective compounds bearing this
scaffold.
For compounds 8a and 20a, a pKa value of 12.4 was determined
[48]. It can be assumed that substitution of the pyridine ring by one
or two methyl groups influenced this value only marginally. So the
basicity was not included as one of the factors of structure–activity
relationship.
5. Experimental
5.1. Chemistry
In this study, our primary aim was to check if the heterocyclic
pyrido[1,2-a]pyrimidines could constitute a new series of inhibitors
for NOS. The 2-aminopyridines 7a–e used as starting materials are
known NOS inhibitors, with the 4-methyl derivative described
a potent but non-selective inhibitor [36]. Careful HPLC analysis
showed no contamination of the tested pyridopyrimidines 8–37
with these aminopyridines, and no decomposition of 8–37 during
testing was noticeable. Our experiments show that some of the
pyrido[1,2-a]pyrimidines can efficiently inhibit NOS, sometimes
Melting points were determined with a Bu¨chi 510 melting point
apparatus and on a Thermowar microhotstage and are reported
uncorrected. 1H NMR spectra were obtained on a Bruker ARX 300
spectrometer at 300 K. All spectra were recorded in DMSO-d6 as
solvent. Chemical shifts (d values) are reported in ppm using TMS as
internal standard. All coupling constants (J values) are quoted in Hz.
The following NMR abbreviations are used: br (broad), s (singlet),
d (doublet), t (triplet), m (unresolved multiplet), Ar (aromatic
proton). Elemental analyses and high resolution mass spectra were
performed at the Institute of Organic Chemistry, University of Kiel.
All reagents for the synthesis of the pyrido[1,2-a]pyrimidines were
purchased from commercial sources and were used without further
purification.
High performance liquid chromatography for the determination
of purity of compounds was performed on a Waters System (Waters
600 controller with pump; Waters 2487 dual absorbance detector;
Waters WISP 710B autosampler; EZ ChromTM Elite chromatography
more efficiently than the well-known inhibitors 7-NI or
our experiments, the high IC50 values determined for the known
inhibitors could be linked to the high concentration of -arginine
used in the assays and to the fact that most of the studied
compounds could act as competitive inhibitors with -arginine, as
L-NNA. In
L
L
observed with 29c, 35c and 37c. Thus, it clearly appears that some
of these pyrido[1,2-a]pyrimidines are as potent as 7-NI or 1400W
when tested under identical conditions. Some of these compounds

2882
U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
0.5
0.4
0.3
0.2
0.1
0
pH 3/acetonitrile 85/15 v/v 10 mM tetramethylammoniumchloride)
and 0% solvent B (heptylsulfonate sodium salt 10 mM, pH 3/aceto-
nitrile 10/90 v/v 10 mM tetramethylammoniumchloride) within 10
min to 50% A, 50% B, hold for 15 min at 50% A, 50% B.’’ Sample elution
was detected by absorbance at 297 nm.
High performance liquid chromatography for the determination
of log D7.4 values of compounds [47] was performed on a Waters
System – Waters 1525 binary pump; Waters 486 tunable absor-
bance detector; Waters 717 Plus autosampler; Waters Breeze soft-
ware system; Kromasil 100 C18 5
m
m, 60 ꢂ 4.6 mm column. The
injection volume was 10 l. The mobile phase was a gradient from
m
0% solvent A (acetonitrile) and 100% solvent B (a mixture of aceto-
nitrile and So¨rensen phosphate buffer 11 mM) over 30 min to 29.5%
solvent A, 70.5% solvent B. Solvent B was a mixture of 30% aceto-
nitrile and 70% buffer for compounds 8–27, 37.5% acetonitrile and
62.5% buffer for compounds 28–36, and 45% acetonitrile and 55%
buffer for 37. As reference compounds aniline, benzamide, benzo-
nitrile, 4-ethylbenzaldehyde, 4-ethylbenzamide, 4-ethylbenzene,
4-ethylbenzonitrile, 4-methlybenzamide, o-cresol, phenol, n-
propylbenzene, toluene, cinnamaldehyde and uracil were used.
UV-detection was performed at 210 nm.
-0.4
0
0.4
0.8
1.2
1 / L-arginine (µM-1)
20
15
10
5
pKa values were determined according to the procedure of
Albert and Serjeant [48]. The pyrido[1,2-a]pyrimidines were dis-
solved in 10% of aqueous methanol. To 5 ml of these solutions were
added 5 ml of NaOH (concentration range of 0.002–6 M) and 1 M
y = 2.08 + 0.152x R = 0.999
HCl to make up a final concentration of 150
measured at a wavelength of 312 nm.
mM. The solutions were
5.1.1. General method for the preparation of pyrido[1,2-a]
pyrimidines
A solution of the 1-aryl-2-dimethylaminomethyl-2-propenone
(4 mmol) and 2-aminopyridine (4 mmol) in ethanol (50 ml) was
refluxed for 2–3 h. After evaporation of the solvent, the residue was
dissolved in 2-propanol (20 ml) followed by cooling and stirring in
an ice bath. Then perchloric acid (70% in water, 4 mmol) or
concentrated hydrochloric acid was added. The precipitate was
filtered, dried and recrystallised from methanol. The yields ranged
from 10 to 30% [38,39].
0
-20
0
20
40
60
80
100
120
Concentration of 37c (µM)
Fig. 2.
data system; LiChrospherÒ 100 RP-8 column; LiChroCARTÒ
125 ꢃ 4 mm cartridge, 5 m; guard column RP-Select B, 4 ꢃ 4 mm,
m). The injection volume was 10 l. ‘‘The mobile phase was
5.1.2. Preparation of compounds 8–37
m
3-Benzoyl-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine perchlorate
(8a), 3-benzoyl-3,4-dihydro-9-methyl-2H-pyrido[1,2-a]pyrimidine
perchlorate (8b), 3-benzoyl-3,4-dihydro-6-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (8d), 3,4-dihydro-3-(40-methylbenzoyl)-2H-
pyrido[1,2-a]pyrimidine perchlorate (9a), 3,4-dihydro-9-methyl-3-
(40-methylbenzoyl)-2H-pyrido[1,2-a]pyrimidine perchlorate (9b),
3,4-dihydro-6-methyl-3-(40-methylbenzoyl)-2H-pyrido[1,2-a]pyrim-
idine perchlorate (9d), 3,4-dihydro-3-(40-methoxybenzoyl)-2H-pyrido
[1,2-a]pyrimidine perchlorate (13a), 3,4-dihydro-3-(30,40-dimethoxy-
benzoyl)-2H-pyrido[1,2-a]pyrimidineperchlorate(18a), 3,4-dihydro-
3-(30,40-dimethoxybenzoyl)-9-methyl-2H-pyrido[1,2-a]pyrimidine
perchlorate (18b), 3,4-dihydro-3-(30,40-dimethoxybenzoyl)-6-methyl-
2H-pyrido[1,2-a]pyrimidine perchlorate (18d), 3-(30,40-dichloro-
benzoyl)-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine perchlorate (19a),
and 3-(40-bromobenzoyl)-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine
perchlorate (20a) were prepared following the general procedure
and their physico-chemical properties have been previously repor-
ted [39].
5
m
m
changed from 100% solvent A (heptylsulfonate sodium salt 10 mM,
Table 3
Inhibition of iNOS expressed in stimulated RAW 264.7 cells by some pyrido[1,2-
a]pyrimidinesa
Compd
% Inhibition
9a
9d
60 ꢁ 8
72 ꢁ 4
44 ꢁ 4
64 ꢁ 3
67 ꢁ 3
70 ꢁ 2
59 ꢁ 5
93 ꢁ 2
92 ꢁ 2
96 ꢁ 2
86 ꢁ 2
89 ꢁ 2
82 ꢁ 2
96 ꢁ 4
98 ꢁ 2
98 ꢁ 2
11d
17c
18b
18d
19a
24c
26c
27c
29c
29e
34c
35c
37c
37d
3-Benzoyl-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]pyrimidine
perchlorate (8c) was prepared from 2-(dimethylaminomethyl)-1-
phenylprop-2-en-1-one hydrochloride and 2-amino-4-methyl-
pyridine 7c according to the general method. Mp. > 290 ꢀC. 1H
a
LPS and
g
-interferon stimulated RAW 264.7 cells were incubated for 15 h at
NMR: d 2.29 (br s, 3H), 3.51 (m, 1H), 3.74 (d, 1H), 6.71 (dd, 1H, Ar-H,
37 ꢀC in the presence of 1% DMSO alone, or 100
mM of the studied compounds.
J ¼ 6.8 Hz), 6.79 (s, 1H, Ar-H), 7.57 (m, 2H, Ar-H), 7.70 (t, 1H, Ar-H,
Nitrite determination in the supernatants was performed as described in the
Experimental. Data are means ꢁ S.D. from 6 to 8 determinations.
J ¼ 7.4 Hz), 7.90 (d, 1H, Ar-H, J ¼ 6.8), 8.04 (d, 2H, J ¼ 7.3 Hz), 9.38 (br

U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
2883
s, 1H, NH). Anal. (C16H17N2O5Cl) calc C: 52.90, H: 4.25, N: 5.37;
found C: 53.20, H: 4.25, N: 5.31.
d
1.36 (t, 3H, J ¼ 6.9 Hz), 2.31 (s, 3H), 3.51 (m, 1H), 3.74 (d, 1H,
J ¼ 13.3 Hz), 4.18 (q, 2H, J ¼ 6.9 Hz), 4.33–4.52 (m, 3H), 6.71–6.75
(m, 2H, Ar-H), 7.08 (d, 2H, J ¼ 8.9 Hz), 7.90 (d, 1H, Ar-H, J ¼ 6.7 Hz),
8.03 (d, 2H, Ar-H, J ¼ 8.9 Hz), 9.15 (br s, 1H, NH). Anal.
(C18H21N2O6Cl) calc C: 54.48, H: 5.33, N: 7.06; found C: 54.39, H:
5.26, N: 6.96.
3,4-Dihydro-3-(40-hydroxybenzoyl)-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (10c) was prepared from 24c by ether
cleavage using 3 equiv. aluminium chloride and 4 equiv. anisole in
dichloromethane. The mixture was stirred for 40 min at room
temperature. After treatment with acetic acid and water the
aqueous solution was extracted with ethyl acetate and the organic
layer washed with NaHCO3-solution and brine. The solvent was
evaporated and the residue was crystallised from methanol. Mp.
3-(40-Carboxybenzoyl)-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine hydrochloride (16c) was prepared from 12c by adding
glacial acetic acid and water (1/1, v/v) and a few drops of ethanol
until the substance is dissolved. The solution was refluxed 3 h and
the solvent evaporated. The product was purified by crystallisation
146 ꢀC. 1H NMR:
d 2.32 (s, 3H), 3.49 (m, 1H), 3.70–3.74 (m, 1H),
4.30–4.35 (m, 2H), 4.45–4.48 (m,1H), 6.71 (d,1H, J ¼ 6.1 Hz), 6.80 (s,
1H), 6.91 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.89–7.95 (m, 3H, Ar-H), 9,39 (br s,
1H), 10.66 (s, 1H). Anal. (C16H16N2O2) HR-MS: calc for C16H16N2O2
268.1212; found 268.1212.
3-(40-Fluorobenzoyl)-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine
perchlorate (11a) was prepared from 2-(dimethylaminomethyl)-1-
(4-fluorophenyl)prop-2-en-1-one hydrochloride and 2-amino-
pyridine 7a according to the general method. Mp. 149 ꢀC. 1H NMR:
in methanol. Mp. 270 ꢀC. 1H NMR:
d 2.50 (s, 3H), 3.55 (m, 9H), 3.74–
3.79 (m, 1H), 4.38–4.55 (m, 3H), 6.73 (d, 1H, J ¼ 6.7 Hz), 6.84 (s, 1H),
7.93 (d, 1H. Ar-H, J ¼ 6.9 Hz), 8.10 (d, 2H, Ar-H, J ¼ 8.4 Hz), 8.16 (d,
2H, Ar-H, J ¼ 8.4 Hz), 9.52 (br s, 1H), 13.00–14.00 (br s, 1H). Anal.
(C17H17N2O3Cl) calc C: 61.35, H: 5.16, N: 8.42; found C: 60.98, H:
5.20, N: 8.30.
3-(30-Fluoro-40-methoxybenzoyl)-3,4-dihydro-8-methyl-2H-pyrido
[1,2-a]pyrimidine perchlorate (17c) was prepared from 1-(3-fluoro-
4-methoxyphenyl)-2-(dimethylaminomethyl)prop-2-en-1-one
hydrochloride and 7c according to the general method. Mp. 143 ꢀC.
d
3.54–3.61 (m, 1H), 3.79–3.85 (m, 1H), 4.41–4.60 (m, 3H), 7.06–7.10
(m, 1H, Ar-H), 7.44 (t, 2H, Ar-H, J ¼ 8.8 Hz), 8.15–8.21 (m, 2H, Ar-H),
8.57 (dd, 1H, Ar-H, J ¼ 2.1 Hz, J ¼ 6.6 Hz), 8.82 (dd, 1H, Ar-H,
J ¼ 2.1 Hz, J ¼ 4.3 Hz), 10.12 (br s, 1H, NH). Anal. (C15H14N2O5ClF)
calc C: 50.50, H: 3.96, N: 7.85; found C: 50.87, H: 4.29, N: 7.52.
3,4-Dihydro-3-(40-fluorobenzoyl)-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (11c) was prepared from 2-(dimethylami-
nomethyl)-1-(4-fluorophenyl)prop-2-en-1-one hydrochloride and
1H NMR:
d 2.32 (s, 3H), 3.41–3.55 (m,1H), 3.72–3.77 (m,1H), 3.96 (s,
3H), 4.33–4.53 (m, 3H), 6.72–6.76 (m, 2H, Ar-H), 7.36 (t, 1H, Ar-H,
J ¼ 8.5 Hz), 7.87–7.97 (m, 3H, Ar-H), 9.16 (br s, 1H, NH). Anal.
(C17H18N2O6FCl) calc C: 50.95, H: 4.53, N: 6.99; found C: 51.08, H:
4.53, N: 6.98.
3-(40-Bromobenzoyl)-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (20c) was prepared from 1-(4-bromo-
phenyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydrochloride
7c according to the general method. Mp. 146 ꢀC. 1H NMR:
d 2.31 (s,
3H, CH3), 3.50 (m, 1H, H-2/4), 3.73 (m, 1H, H-2/4), 4.28–4.52 (m, 3H,
H-3, H-2/4), 6.72–6.76 (m, 2H, H-7/9), 7.40 (m, 2H, Ar-H), 7.90 (m,
1H, H-6), 8.18 (m, 2H, Ar-H), 9.22 (br s, 1H, NH). Anal.
(C16H16N2O5ClF) calc C: 51.83, H: 4.35, N: 7.56; found C: 51.68, H:
4.49, N: 7.44.
and 7c according to the general method. Mp. 160 ꢀC. 1H NMR:
d 2.31
(s, 3H, CH3), 3.51 (m, 1H, H-2/4), 3.75 (d, 1H, H-2/4, J ¼ 12.6 Hz),
4.35–4.51 (m, 3H, H-3, H-2/4), 6.72–6.76 (m, 2H, H-7/9), 7.81 (d, 2H,
H-30/50, J ¼ 8.67 Hz), 7.91 (d, 1H, H-6, J ¼ 6.8 Hz), 7.98 (d, 2H, H-20/60,
J ¼ 8.7 Hz), 9.20 (br s, 1H, NH). Anal. (C16H16N2O5BrCl) calc C: 44.52,
H: 3.74, N: 6.49; found C: 44.48, H: 3.83, N: 6.39.
3-(40-Fluorobenzoyl)-3,4-dihydro-6-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (11d) was prepared from 2-(dimethylami-
nomethyl)-1-(4-fluorophenyl)prop-2-en-1-one hydrochloride and
2-amino-6-methyl-pyridine 7d according to the general method.
3-(40-Bromobenzoyl)-3,4-dihydro-6-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (20d) was prepared from 1-(4-bromo-
phenyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydrochloride
Mp. 149 ꢀC. 1H NMR:
d 2.52 (s, 3H), 3.43–3.53 (m, 1H), 3.77–3.81 (m,
1H), 4.26–4.48 (m, 3H), 6.81–6.89 (2d, 2H, Ar-H, J ¼ 7.1 Hz,
J ¼ 8.8 Hz), 7.43 (t, 2H, Ar-H, J ¼ 8.8 Hz), 7.71 (dd, 1H, Ar-H,
J ¼ 7.2 Hz, J ¼ 8.9 Hz), 8.19 (dd, 2H, Ar-H, J ¼ 5.5 Hz, J ¼ 8.9 Hz), 9.31
(br s, 1H, NH). Anal. (C16H16N2O5FCl) calc C: 51.83, H: 4.35, N: 7.56;
found C: 51.75, H: 4.29, N: 7.50.
and 7d according to the general method. Mp. 174 ꢀC. 1H NMR:
d 2.50
(br s, 3H), 3.48 (m, 1H), 3.76 (d, 1H, J ¼ 13.3 Hz), 4.29–4.50 (m, 3H),
6.81 (d, 1H, Ar-H, J ¼ 7.2 Hz), 6.87 (d, 1H, Ar-H, J ¼ 8.9 Hz), 7.70 (m,
1H, Ar-H), 7.82 (d, 2H, J ¼ 8.5 Hz), 8.02 (d, 2H, J ¼ 8.4), 9.39 (br s, 1H,
NH). Anal. (C16H16N2O5BrCl) calc C: 44.52, H: 3.74, N: 6.49; found C:
44.37, H: 3.83, N: 6.39.
3-(40-Cyclohexylbenzoyl)-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (21c) was prepared from 1-(40-cyclo-
hexylphenyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydro-
chloride and 7c according to the general method. Mp. 189 ꢀC. 1H
3-(40-Cyanobenzoyl)-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (12c) was prepared from 1-(4-cyano-
phenyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydrochloride
and 7c according to the general method. Mp. 149 ꢀC. 1H NMR:
d 2.32
(s, 3H), 3.49–3.55 (m, 1H), 3.74–3.78 (m, 1H), 4.39–4.54 (m, 3H),
6.74–6.76 (m, 2H), 7.92 (d, 1H, Ar-H, J ¼ 6.9 Hz), 8.08 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 8.21 (d, 2H, Ar-H, J ¼ 8.4 Hz), 9.17 (br s, 1H). Anal.
(C17H16N3O5Cl) calc C: 54.04, H: 4.28, N: 11.12; found C: 54.04, H:
4.32, N: 11.03.
3-(40-Chlorobenzoyl)-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (14c) was prepared from 1-(4-chlor-
ophenyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydrochlo-
ride and 7c according to the general method. Mp. 166 ꢀC. 1H NMR:
NMR: d 1.23–1.47 (m, 5H), 1.70–1.81 (m, 5H), 2.31 (s, 3H), 2.50–2.62
(m, 1H), 3.50–3.55 (m, 1H), 3.73–3.77 (m, 1H), 4.36–4.54 (m, 3H),
6.72–6.75 (m, 1H), 6.79 (s, 1H), 7.41 (d, 2H, Ar-H, J ¼ 8.2 Hz), 7.75 (d,
2H, Ar-H, J ¼ 8,2 Hz), 10.87 (br s, 1H). Anal. (C22H27N2O5Cl) calc C:
60.76, H: 6.26, N: 6.44; found C: 60.98, H: 6.35, N: 6.43.
3,4-Dihydro-3-(30,40,50-trimethoxybenzoyl)-2H-pyrido[1,2-a]
pyrimidine hydrochloride (22a) was prepared from 2-(dimethyla-
minomethyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one hydro-
chloride and 7a according to the general method. Mp. 207 ꢀC. 1H
d
2.31 (br s, 3H), 3.52 (m, 1H), 3.73–3.81 (m, 1H), 4.81 (m, 3H), 6.74
(d, 1H, Ar-H, J ¼ 6.9 Hz), 6.78 (s, 1H, Ar-H), 7.67 (d, 2H, Ar-H,
J ¼ 8.6 Hz), 7.90 (d, 1H, Ar-H, J ¼ 6.9 Hz), 8.08 (d, 2H, Ar-H,
J ¼ 8.6 Hz), 9.27 (br s, 1H, NH). Anal. (C16H16N2O5Cl2) calc C: 49.63,
H: 4.17, N: 7.23; found C: 49.77, H: 4.26, N: 7.11.
3-(40-Ethoxybenzoyl)-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (15c) was prepared from 1-(4-ethox-
yphenyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydrochlo-
ride and 7c according to the general method. Mp. 131 ꢀC. 1H NMR:
NMR: d 3.52 (m, 1H), 3.77 (s, 3H), 3.83 (m, 1H), 3.87 (s, 6H), 4.42 (m,
1H), 4.55–4.58 (m, 2H), 6.85 (t, 1H, Ar-H, J ¼ 6.4 Hz), 7.14 (d, 1H, Ar-
H, J ¼ 8.4 Hz), 7.36 (s, 2H, Ar-H), 7.80 (t, 1H, Ar-H, J ¼ 7.8 Hz), 8.04 (d,
1H, Ar-H, J ¼ 6.6 Hz), 9.90 (br s, 1H, NH). Anal. (C18H21N2O4Cl) calc
C: 59.26, H: 5.80, N: 7.68; found C: 59.18, H: 5.94, N: 7.52.
3-(40-Benzyloxybenzoyl)-3,4-dihydro-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (23c) was prepared from 1-(40-benzyloxy-
benzoyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydrochloride

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U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
and 7c according to the general method. Mp. 139 ꢀC. 1H NMR:
d
2.37
3,4-Dihydro-6-methyl-3-(2-naphthoyl)-2H-pyrido[1,2-a]pyrimidine
perchlorate (28d) was prepared from 2-(dimethylaminomethyl)-1-
(2-naphthyl)-prop-2-en-1-one hydrochloride and 7d according to
(s, 3H), 3.32–3.54 (m, 1H), 3.72–3.77 (m, 1H), 4.35–4.39 (m, 2H),
4.45–4.53 (m, 1H), 5.25 (s, 2H), 6.72–6.76 (m, 2H, Ar-H), 7.19 (d, 2H,
Ar-H, J ¼ 8.9 Hz), 7.33–7.49 (m, 5H), 7.91 (d, 1H, Ar-H, J ¼ 6.8 Hz),
8.05 (d, 2H, Ar-H, J ¼ 8.9 Hz), 9.15 (br s, 1H). Anal. (C23H23N2O6Cl)
calc C: 60.20, H: 5.05, N: 6.10; found C: 60.19, H: 5.15, N: 6.17.
the general method. Mp. 128 ꢀC. 1H NMR:
d 2.52 (br s, 3H), 3.56 (m,
1H), 3.85 (d, 1H, J ¼ 13.9 Hz), 4.38–4.62 (m, 3H), 6.82 (d, 1H, Ar-H,
J ¼ 7.0 Hz), 6.96 (d, 1H, Ar-H, J ¼ 8.8 Hz), 7.69 (m, 2H, Ar-H), 7.98–
8.16 (m, 5H, Ar-H), 8.90 (s, 1H, Ar-H), 9.59 (br s, 1H). Anal.
(C20H19N2O5Cl) calc C: 59.63, H: 4.75, N: 6.96; found C: 59.55, H:
4.75, N: 6.84.
3,4-Dihydro-8-methyl-3-[2-(6-methylnaphthoyl)]-2H-pyrido[1,2-a]
pyrimidine perchlorate (29c) was prepared from 2-(dimethylami-
nomethyl)-1-[2-(6-methylnaphthyl)]prop-2-en-1-one hydrochlo-
ride and 7c according to the general method. Mp. 128 ꢀC. 1H NMR:
5.1.3. 3-[40-(400-Cyanobenzyloxy)benzoyl]-3,4-dihydro-8-methyl-
2H-pyrido[1,2-a]pyrimidine
Perchlorate (24c) was prepared from 1-[40-(400-cyanobenzyloxy-
phenyl)]-2-(dimethylaminomethyl)prop-2-en-1-one hydrochloride
and 7c according to the general method. Mp. 285 ꢀC. 1H NMR:
d 2.33
(s, 3H), 3.52–3.58 (m, 1H), 3.75–3.83 (m, 4H), 4.38–4.55 (m, 3H),
6.74–6.77 (m, 2H), 7.08 (d, 2H, Ar-H, J ¼ 8.8 Hz), 7.75 (d, 2H, Ar-H,
J ¼ 8.8 Hz), 7.86 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.93 (d,1H, Ar-H, J ¼ 6.8 Hz),
8.11 (d, 2H, Ar-H, J ¼ 8.4 Hz), 9.17 (br s,1H). Anal. (C24H22N3O6Cl) calc
C: 59.57, H: 4.58, N: 8.68; found C: 59.18, H: 4.68, N: 8.67.
d
2.33 (s, 3H), 2.52 (s, 3H), 3.57–3.63 (m, 1H), 3.81–3.86 (m, 1H),
4.40–4.59 (m, 3H), 6.74–6.79 (m, 2H, Ar-H), 7.52 (d, 1H, Ar-H,
J ¼ 8.4 Hz), 7.81 (s, 1H, Ar-H), 7.95–7.97 (m, 3H, Ar-H), 8.04 (d, 1H,
Ar-H, J ¼ 8.4 Hz), 9.21 (br s, 1H). Anal. (C21H21N2O5Cl) calc C: 60.51,
H: 5.08, N: 6.72; found C: 60.17, H: 5.10, N: 6.88.
3,4-Dihydro-6-methyl-3-[2-(6-methylnaphthoyl)]-2H-pyrido[1,2-a]
pyrimidine perchlorate (29d) was prepared from 2-(dimethylami-
nomethyl)-1-[2-(6-methylnaphthyl)]prop-2-en-1-one hydrochlo-
ride and 7d according to the general method. Mp. 198 ꢀC. 1H NMR:
3,4-Dihydro-3-[40-(400-methoxy)benzyloxybenzoyl]-8-methyl-2H-
pyrido[1,2-a]pyrimidine perchlorate (25c) was prepared from 2-
(dimethylaminomethyl)-1-[40-(400-methoxybenzyloxyphenyl)]-prop-
2-en-1-one hydrochloride and 7c according to the general method.
Mp. 201 ꢀC. 1H NMR:
d 2.33 (s, 3H), 3.52–3.58 (m,1H), 3.75–3.83 (m,
4H), 4.38–4.55 (m, 3H), 6.74–6.77 (m, 2H), 7.08 (d, 2H, Ar-H,
J ¼ 8.8 Hz), 7.75 (d, 2H, Ar-H, J ¼ 8.8 Hz), 7.86 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 7.93 (d, 1H, Ar-H, J ¼ 6.8 Hz), 8.11 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 9.17 (br s, 1H). Anal. (C24H25N2O7Cl) calc C: 58.95, H:
5.15, N: 5.73; found C: 59.25, H: 5.48, N: 6.26.
d 2.53 (s, 6H), 3.56–3.59 (m, 1H), 3.83–3.87 (m, 1H), 4.35–4.56 (m,
3H), 6.83 (d, 1H, Ar-H, J ¼ 7.2 Hz), 6.90 (s, 1H, Ar-H, J ¼ 8.7 Hz), 7.51
(dd, 1H, Ar-H, J ¼ 1.4 Hz, J ¼ 8.4 Hz), 7.70–7.78 (m, 1H, Ar-H), 7.81 (s,
1H, Ar-H), 7.95–8.11 (m, 3H, Ar-H), 8.89 (s, 1H, Ar-H), 9.36 (br s, 1H).
Anal. (C21H21N2O5Cl) calc C: 60.51, H: 5.08, N: 6.72; found C: 61.07,
H: 5.12, N: 6.49.
3-[40-(400-Chlorobenzyloxy)benzoyl]-3,4-dihydro-8-methyl-2H-
pyrido[1,2-a]pyrimidine perchlorate (26c) was prepared from 1-
[40-(400-chlorobenzyloxyphenyl)]-2-(dimethylaminomethyl)prop-
2-en-1-one hydrochloride and 7c according to the general method.
3,4-Dihydro-6,8-dimethyl-3-[2-(6-methylnaphthoyl)]-2H-pyrido
[1,2-a]pyrimidine perchlorate (29e) was prepared from 2-(di-
methylaminomethyl)-1-[2-(6-methylnaphthyl)]prop-2-en-1-one
hydrochloride and 2-amino-4,6-dimethyl-pyridine 7e according to
Mp. 178 ꢀC. 1H NMR:
d 2.33 (s, 3H), 3.52–3.58 (m, 1H), 3.75–3.83 (m,
4H), 4.38–4.55 (m, 3H), 6.74–6.77 (m, 2H), 7.08 (d, 2H, Ar-H,
J ¼ 8.8 Hz), 7.75 (d, 2H, Ar-H, J ¼ 8.8 Hz), 7.86 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 7.93 (d, 1H, Ar-H, J ¼ 6.8 Hz), 8.11 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 9.17 (br s, 1H). Anal. (C23H22N2O6Cl2) calc C: 55.99, H:
4.49, N: 5.68; found C: 55.96, H: 4.59, N: 5.69.
the general method. Mp. 160 ꢀC. 1H NMR:
d 2.29 (s, 3H), 2.49 (s, 3H),
2.53 (s, 3H), 3.51–3.58 (m, 1H), 3.80–3.85 (m, 1H), 4.31–4.53 (m,
3H), 6.67 (s, 1H, Ar-H), 6.71 (s, 1H, Ar-H), 7.52 (dd, 1H, Ar-H,
J ¼ 1.5 Hz, J ¼ 8.4 Hz), 7.81 (s, 1H, Ar-H), 7.95–8.06 (m, 3H, Ar-H),
8.69 (s, 1H, Ar-H), 9.18 (br s, 1H). Anal. (C22H23N2O5Cl) calc C: 61.33,
H: 5.38, N: 6.50; found C: 61.57, H: 5.58, N: 6.78.
3,4-Dihydro-3-[2-(6-methoxynaphthoyl)]-8-methyl-2H-pyrido[1,2-a]
pyrimidine perchlorate (30c) was prepared from 2-(dimethyl-
aminomethyl)-1-[2-(6-methoxynaphthoyl)]prop-2-en-1-one hydro-
chloride and 7c according to the general method. Mp. 198 ꢀC. 1H
3-[40-(400-Bromobenzyloxy)benzoyl]-3,4-dihydro-8-methyl-2H-
pyrido[1,2-a]pyrimidine hydrochloride (27c) was prepared from 1-
[40-(400-bromobenzyloxyphenyl)]-2-(dimethylaminomethyl)prop-
2-en-1-one hydrochloride and 7c according to the general method.
Mp. 110 ꢀC. 1H NMR:
d 2.33 (s, 3H), 3.52–3.58 (m, 1H), 3.75–3.83 (m,
4H), 4.38–4.55 (m, 3H), 6.74–6.77 (m, 2H), 7.08 (d, 2H, Ar-H,
J ¼ 8.8 Hz), 7.75 (d, 2H, Ar-H, J ¼ 8.8 Hz), 7.86 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 7.93 (d,1H, Ar-H, J ¼ 6.8 Hz), 8.11 (d, 2H, Ar-H, J ¼ 8.4 Hz),
9.17 (br s, 1H). Anal. (C23H22N2O2BrCl) calc C: 58.31, H: 4.68, N: 5.91;
found C: 57.92, H: 4.95, N: 5.80.
NMR: d 2.33 (s, 3H), 3.56–3.85 (m, 1H), 3.93 (s, 3H), 4.40–4.60 (m,
3H), 6.74–6.79 (m, 2H, Ar-H), 7.30 (dd, 1H, Ar-H, J ¼ 2.5 Hz, J
¼ 8.9 Hz), 7.45 (d, 1H, Ar-H, J ¼ 2.4 Hz), 7.79–8.06 (m, 4H, Ar-H),
8.77 (s, 1H, Ar-H), 9.22 (s, 1H). Anal. (C21H21N2O6Cl) calc C: 58.27, H:
4.89, N: 6.47; found C: 58.14, H: 5.02, N: 6.43.
3,4-Dihydro-3-(2-naphthoyl)-2H-pyrido[1,2-a]pyrimidine per-
chlorate (28a) was prepared from 2-(dimethylaminomethyl)-1-(2-
naphthyl)prop-2-en-1-one hydrochloride and 7a according to the
3,4-Dihydro-3-[2-(6-methoxynaphthoyl)]-6-methyl-2H-pyrido
[1,2-a]pyrimidine perchlorate (30d) was prepared from 2-(di-
methylaminomethyl)-1-[2-(6-methoxynaphthyl)]prop-2-en-1-one
hydrochloride 7d according to the general method. Mp. 190 ꢀC. 1H
general method. Mp. 211 ꢀC. 1H NMR:
d 3.62 (m, 1H), 3.86 (d, 1H,
J ¼ 13.4 Hz), 4.46–4.67 (m, 3H, H-3), 6.83 (t, 1H, Ar-H, J ¼ 6.7 Hz),
7.05 (d, 1H, Ar-H, J ¼ 8.9 Hz), 7.70 (m, 2H, Ar-H), 7.82 (t, 1H, Ar-H,
J ¼ 7.2 Hz), 8.00–8.16 (m, 5H, Ar-H), 8.88 (s, 1H, Ar-H), 9.49 (br s,
1H). Anal. (C19H17N2O5Cl) calc C: 58.69, H: 4.41, N: 7.20; found C:
58.47, H: 4.72, N: 7.11.
NMR: d 2.53 (s, 3H), 3.52–3.58 (m, 1H), 3.93 (s, 3H), 4.33–4.55 (m,
2H), 6.82 (d, 1H, Ar-H, J ¼ 7.1 Hz), 6.90 (d, 1H, Ar-H, J ¼ 8.8 Hz), 7.31
(dd, 1H, Ar-H, J ¼ 2.5 Hz, J ¼ 9.0 Hz), 7.45 (d, 1H, Ar-H, J ¼ 2.5 Hz),
7.69–7.74 (m, 1H, Ar-H), 7.91–8.06 (m, 3H, Ar-H), 9.37 (br s, 1H).
Anal. (C21H20N2O2) HR-MS: calc for C21H20N2O2 332.1525; found
332.1525.
3,4-Dihydro-8-methyl-3-(2-naphthoyl)-2H-pyrido[1,2-a]pyrimidine
perchlorate (28c) was prepared from 2-(dimethylaminomethyl)-1-
(2-naphthyl)prop-2-en-1-one hydrochloride and 7c according to
3,4-Dihydro-3-[2-(6-methoxynaphthoyl)]-6,8-dimethyl-2H-pyrido
[1,2-a]pyrimidine hydrochloride (30e) was prepared from 2-(di-
methylaminomethyl)-1-[2-(6-methoxynaphthyl)]prop-2-en-1-one
hydrochloride and 7e according to the general method. Mp. 264 ꢀC.
the general method. Mp. 142 ꢀC. 1H NMR:
d 2.32 (br s, 3H), 3.62 (m,
1H), 3.84 (d, 1H, J ¼ 13.9 Hz), 4.41–4.61 (m, 3H), 6.74 (d, 1H, Ar-H,
J ¼ 6.9 Hz), 6.81 (s, 1H, Ar-H), 7.69 (m, 2H, Ar-H), 7.92 (d, 1H, Ar-H,
J ¼ 6.9 Hz), 7.99–8.15 (m, 5H, Ar-H), 8.86 (s,1H, Ar-H), 9.31 (br s,1H).
Anal. (C20H19N2O5Cl) calc C: 59.63, H: 4.75, N: 6.96; found C: 59.61,
H: 4.85, N: 6.90.
1H NMR:
d 2.28 (s, 3H), 2.49 (s, 3H), 3.50–3.56 (m, 1H), 3.80–3.83
(m, 1H), 3.93 (s, 3H), 4.30–4.49 (m, 3H), 6.73 (s, 2H, Ar-H), 7.30 (dd,
1H, Ar-H, J ¼ 2.5 Hz, J ¼ 9.0 Hz), 7.45 (d, 1H, Ar-H, J ¼ 2.4 Hz), 7.93–
8.07 (m, 3H, Ar-H), 8.81 (s, 1H, Ar-H), 9.62 (br s, 1H). Anal.

U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
2885
(C22H23N2O2Cl) calc C: 69.01, H: 6.05, N: 7.32; found C: 68.99, H:
6.14, N: 7.23.
7.86 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.93 (d, 1H, Ar-H, J ¼ 6.8 Hz), 8.11 (d,
2H, Ar-H, J ¼ 8.4 Hz), 9.17 (br s, 1H). Anal. (C23H21N2O3Cl) calc C:
67.56, H: 5.18, N: 6.85; found C: 67.65, H: 5.26, N: 6.74.
3-[40-(400-Bromophenyl)benzoyl]-3,4-dihydro-8-methyl-2H-pyrido
[1,2-a]pyrimidine perchlorate (37c) was prepared from 1-(4-
bromobiphenylyl)-2-(dimethylaminomethyl)-prop-2-en-1-one
hydrochloride and 7c according to the general method. Mp. 182 ꢀC.
3,4-Dihydro-8-methyl-3-(40-phenylbenzoyl)-2H-pyrido[1,2-a]
pyrimidine perchlorate (31c) was prepared from 1-(biphenylyl)-2-
(dimethylaminomethyl)prop-2-en-1-one hydrochloride and 7c
according to the general method. Mp. 111 ꢀC. 1H NMR:
d 2.33 (br s),
2.84–2.88 (m, 1H), 3.59 (m, 1H), 4.39–4.56 (m, 3H), 6.67–6.78 (m,
2H, Ar-H), 7.43–7.56 (m, 3H, Ar-H), 7.78 (d, 2H, Ar-H, J ¼ 7.0 Hz), 7.92
(d, 2H, Ar-H, J ¼ 8.5 Hz), 7.94 (d, 1H, Ar-H, J ¼ 6.8), 8.16 (d, 2H, Ar-H,
J ¼ 8.5 Hz), 9.20 (br s, 1H, NH). Anal. (C22H21N2O5Cl) calc C: 61.61, H:
4.93, N: 6.53; found C: 61.73, H: 5.11, N: 6.52.
1H NMR:
d
¼ 2.33 (s, 3H), 3.52–3.59 (m, 1H), 3.77–3.81 (m, 1H),
4.39–4.55 (m, 3H), 6.73–6.77 (m, 2H, Ar-H), 7.69–7.77 (m, 4H, Ar-H),
7.92 (t, 3H, Ar-H, J ¼ 7.8 Hz), 8.15 (d, 2H, Ar-H, J ¼ 8.4 Hz), 9.18 (br s,
1H, NH). Anal. (C22H20N2O5BrCl) calc C: 52.04, H: 3.97, N: 5.52;
found C: 52.03, H: 4.38, N: 5.70.
3,4-Dihydro-6-methyl-3-(40-phenylbenzoyl)-2H-pyrido[1,2-a]
pyrimidine perchlorate (31d) was prepared from 1-(biphenylyl)-2-
(dimethylaminomethyl)prop-2-en-1-one hydrochloride and 7d
3-[40-(400-Bromophenyl)benzoyl]-3,4-dihydro-6-methyl-2H-pyrido
[1,2-a]pyrimidine perchlorate (37d) was prepared from 1-(4-
bromobiphenylyl)-2-(dimethylaminomethyl)prop-2-en-1-one hydro-
chloride and 7d according to the general method. Mp. 235 ꢀC. 1H
according to the general method. Mp. 98 ꢀC. 1H NMR:
d 2.5 (br s,
3H), 3.51 (m, 1H), 3.79 (m, 1H), 4.35–4.55 (m, 3H), 6.81 (d, 2H, Ar-H,
J ¼ 7.0 Hz), 6.95 (d, 1H, Ar-H, J ¼ 9.0 Hz), 7.42–7.55 (m., 3H, Ar-H),
7.70 (m, 1H, Ar-H), 7.79 (d, 2H, Ar-H, J ¼ 7.0 Hz), 7.90 (d, 1H, Ar-H,
J ¼ 8.4 Hz), 8.18 (d, 2H, Ar-H, J ¼ 8.4 Hz), 9.60 (br s, 1H, NH). Anal.
(C22H21N2O5Cl) calc C: 61.61, H: 4.93, N: 6.53; found C: 61.82, H:
5.02, N: 6.38.
NMR:
d 2.53 (s, 3H), 3.29 (s, 2H), 3.49–3.55 (m, 1H), 3.79–3.83 (m,
1H), 4.33–4.50 (m, 3H), 6.82 (d, 1H, Ar-H, J ¼ 7.1 Hz), 6.92 (d, 1H, Ar-
H, J ¼ 9.0 Hz), 7.68–7.78 (m, 5H, Ar-H), 7.91 (d, 2H, Ar-H, J ¼ 8.5 Hz),
8.18 (d, 2H, Ar-H, J ¼ 8.5 Hz), 9.32 (s, 1H, NH). Anal.
(C22H20N2O5BrCl) calc C: 52.04, H: 3.97, N: 5.52; found C: 52.37, H:
4.05, N: 5.50.
3,4-Dihydro-3-[40-(400-hydroxyphenyl)benzoyl]-8-methyl-2H-pyrido
[1,2-a]pyrimidine hydrochloride (32c) was prepared from 2-
(dimethylaminomethyl)-1-(4-hydroxybiphenylyl)prop-2-en-1-one
hydrochloride and 7c according to the general method. Mp. 295 ꢀC.
3-[40-(400-Bromophenyl)benzoyl]-3,4-dihydro-6,8-dimethyl-2H-
pyrido[1,2-a]pyrimidine perchlorate (37e) was prepared from 1-
(40-bromobiphenyl)-2-(dimethylaminomethyl)prop-2-en-1-one
hydrochloride and 7e according to the general method. Mp. 302 ꢀC.
1H NMR:
d 2.32 (s, 3H), 3.53–3.56 (m, 1H), 3.75–3.79 (m, 1H), 4.34–
4.54 (m, 3H), 6.73 (d, 1H), 6.85 (s, 1H), 6.91 (d, 2H, Ar-H, J ¼ 8.5 Hz),
7.63 (d, 2H, Ar-H, J ¼ 8.6 Hz), 7.81 (d, 2H, Ar-H, J ¼ 7.9 Hz), 7.94 (d,
1H, Ar-H, J ¼ 6.5 Hz), 8.09 (d, 2H, Ar-H, J ¼ 8.2 Hz), 9.57 (s, 1H), 9.85
(br s, 1H). Anal. (C22H21N2O2Cl) calc C: 69.38, H: 5.56, N: 7.36; found
C: 69.81, H: 5.64, N: 7.32.
3-[40-(400-Fluorophenyl)benzoyl]-3,4-dihydro-8-methyl-2H-pyrido
[1,2-a]pyrimidine perchlorate (33c) was prepared from 2-(dime-
thylaminomethyl)-1-(4-fluorobiphenylyl)prop-2-en-1-one hydro-
chloride and 7c according to the general method. Mp. 144 ꢀC. 1H
1H NMR:
d 2.28 (s, 3H), 2.49 (s, 3H), 3.49–3.54 (m, 1H), 3.77–3.81
(m, 1H), 4.30–4.45 (m, 3H), 6.66 (s, 1H, Ar-H), 6.71 (s, 1H, Ar-H),
7.70–7.78 (m, 4H, Ar-H), 7.91 (d, 2H, Ar-H, J ¼ 8.5 Hz), 8.17 (d, 2H,
Ar-H, J ¼ 8.5 Hz), 9.12 (br s, 1H). Anal. (C23H21N2OBr) HR-MS: calc for
C23H21N2O79Br 420.0837; found 420.0837, calc for C23H21N2O81Br
422.0817; found 422.0819.
5.2. Biological assays
NMR:
d
2.33 (s, 3H), 3.55–3.59 (m, 1H), 3.76–3.81 (m, 1H), 4.39–4.55
5.2.1. Reagents and materials
(m, 3H), 6.74–6.78 (m, 2H), 7.36 (t, 2H, Ar-H, J ¼ 8.8 Hz), 7.81–7.95
(m, 5H, Ar-H), 8.14 (d, 2H, Ar-H, J ¼ 8.4 Hz), 9.21 (br s, 1H). Anal.
(C22H20N2O5FCl) calc C: 59.13, H: 4.51, N: 6.27; found C: 59.18, H:
4.70, N: 6.33.
3-[40-(400-Cyanophenyl)benzoyl]-3,4-dihydro-8-methyl-2H-pyrido
[1,2-a]pyrimidine hydrochloride (34c) was prepared from 1-(4-
cyanobiphenylyl)-2-(dimethylaminomethyl)-prop-2-en-1-one hydro-
chloride and 7c according to the general method. Mp. 285 ꢀC. 1H
N-(3-Aminomethyl)benzylacetamidine,
Nu-nitro-
L-arginine,
Nu-propyl-
L
-arginine, and tetrahydrobiopterine were purchased
from Alexis Biochemicals. Calcium chloride hexahydrate, NADPH,
DMSO, EDTA, sulfanilamide and 1-naphthylethylenediamine dihy-
drochloride were obtained from Merck. Tetramethylammonium
chloride and L-arginine were purchased from Fluka and Calmodu-
line from pig brain from Roche Diagnostics. Acetonitrile was
obtained from LGC Promochem. All other chemicals were obtained
from Sigma-Aldrich.
NMR: d 2.31 (s, 3H), 3.52–3.56 (m, 1H), 3.77–3.81 (m, 1H), 4.41–4.57
(m, 3H), 6.72 (d, 1H, J ¼ 6.1 Hz), 6.93 (s, 1H), 7.94–7.99 (m, 7H, Ar-H),
8.19 (d, 2H, Ar-H, J ¼ 8.3 Hz), 9.93 (br s, 1H). Anal. (C23H20N3OCl)
calc C: 70.85, H: 5.17, N: 10.78; found C: 70.57, H: 5.31, N: 10.68.
3-[40-(400-Methoxyphenyl)benzoyl]-3,4-dihydro-8-methyl-2H-
pyrido[1,2-a]pyrimidine hydrochloride (35c) was prepared from
1-(40-methoxybiphenyl)-2-(dimethylaminomethyl)prop-2-en-1-one
hydrochloride and 7c according to the general method. Mp. 201 ꢀC.
5.2.2. Origin of proteins
Isoforms of NOS used in the screening tests (Griess assay) were
rat brain nNOS (Alexis, recombinant from Sf9 insect cells, specific
activity 700 nmol/min ꢃ mg prot); human nNOS (B. Mayer,
University of Graz, from Pichia pastoris, specific activity 800 nmol/
min ꢃ mg prot); murine iNOS (Cayman Chemical, from Sf9 insect
cells, specific activity 6 nmol/min ꢃ mg prot); human iNOS (Alexis,
from Sf9 insect cells, specific activity 1 nmol/min ꢃ mg prot);
bovine eNOS (Cayman Chemical, from Sf9 cells, specific activity
3 nmol/min ꢃ mg prot); and human eNOS (B. Mayer, University of
Graz, from Pichia pastoris, specific activity 184 nmol/min ꢃ mg
prot).
Isoforms of NOS used in the radioactive experiments were as
follows: recombinant rat brain nNOS purified from the yeast
Saccharomyces cerevisiae (specific activity 290 nmol nmol/
min ꢃ mg prot); murine iNOS purified from Escherichia coli (specific
activity 340 nmol/min ꢃ mg prot); bovine eNOS from E. coli (specific
activity 40 nmol/min ꢃ mg prot), and iNOSoxy from E. coli were gifts
from D.J. Stuehr (Lerner Research Fundation, Cleveland, USA).
1H NMR:
d 2.33 (s, 3H), 3.52–3.58 (m, 1H), 3.75 (s, 3 H), 3.75–3.81
(m, 1H), 4.38–4.55 (m, 3H), 6.74–6.77 (m, 3H), 7.08/7.75 (2ꢃd,
2 ꢃ 2H, J ¼ 8.8 Hz), 7.86/8.11 (2ꢃd, 2 ꢃ 2H, J ¼ 8.4 Hz), 7.93 (d, 1H
J ¼ 6.8 Hz), 9.17 (bs 1H). Anal. (C23H22N2O2) HR-MS: calc for
C23H22N2O2 358.1681; found 358.1680.
3-[40-(400-Carboxyphenyl)benzoyl]-3,4-dihydro-8-methyl-2H-
pyrido[1,2-a]pyrimidine hydrochloride (36c) was prepared from
34c by adding 50% acetic acid in water and a few drops of ethanol
until the substance was dissolved. The solution was refluxed 3 h
and the solvent evaporated. The product was purified by crystal-
lisation from methanol. Mp. > 245 ꢀC. 1H NMR:
d 2.33 (s, 3H),
3.52–3.58 (m, 1H), 3.75–3.83 (m, 4H), 4.38–4.55 (m, 3H), 6.74–6.77
(m, 2H), 7.08 (d, 2H, Ar-H, J ¼ 8.8 Hz), 7.75 (d, 2H, Ar-H, J ¼ 8.8 Hz),

2886
U. Bluhm et al. / European Journal of Medicinal Chemistry 44 (2009) 2877–2887
Protein concentrations were determined by the method of
5.2.5. UV/visible interaction studies of NOSoxy domain with
pyrido[1,2-a]pyrimidines
Studies were carried out at room temperature in 1-cm path-
length cuvettes containing 0.8–1.0 M iNOSoxy in 50 mM Hepes, pH
Bradford using bovine serum albumin as a standard and the
Bradford reagent from Biorad [49]. They were estimated to be more
than 95% pure by SDS-PAGE electrophoresis. Their heme concen-
trations were determined optically from the [FeII–CO] – [FeII]
difference spectrum using a De445–480 nm of 74 mMꢂ1 ꢃ cmꢂ1 [50].
m
7.4. Assayed compounds were dissolved in DMSO, and equivalent
amounts of DMSO were added to the reference cuvette. At the
beginning, these experiments were done without the addition of
imidazole but no clear effect could be observed. This comes from
the fact that native iNOSoxy is an equilibrium of low- and high spin
heme-FeIII complexes, and that compounds modify only a small
part of the equilibrium. We then saturated the enzyme with an
excess of imidazole, a compound that binds to the iron of the heme
and completely forms a low spin complex absorbing at 425–
430 nm. Further additions of compounds that bind in an identical
5.2.3. Nitrite determination (Griess assay)
The formation of nitrite (stable end-product of NO) was
followed by the reaction with sulphanilamide and 1-naphthyl-
ethylenediamine hydrochloride in hydrochloric acid. The absorp-
tion of the resulting azo dye was measured at 543 nm on
a Beckmann DU7500 UV/vis spectrophotometer. Since 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 [40].
manner than
restore the high spin heme FeIII complex absorbing at 395–400 nm
[43]. Solutions of 0.8–1 M iNOSoxy in 50 mM Hepes buffer con-
taining 1 mM imidazole were transferred to each cuvette (150
total volume) and a baseline between 380 and 480 nm was recor-
ded. Then, successive additions of 1 l of the compounds (1 mM or
L-arginine can displace imidazole from the iron and
m
Buffers contained 50 mM triethanolamine (iNOS and eNOS,
pH ¼ 7.5, nNOS, pH ¼ 7.0), 1 mM CHAPS, 10 mM 2-mercaptoethanol
and 0.5 mM EDTA. Typical assay mixtures contained 0.5 mM Ca2þ
ml
m
(nNOS and eNOS), 1 mM Mg2þ (iNOS), 5
m
M flavine adenine dinu-
flavine mononucleotide (FMN), 0.5 mM
M H4B, 10 g/ml CaM, 0.5 mM (nNOS) or 1 mM
10 mM in DMSO) were done and the same volumes of DMSO were
added to the reference cuvette. After each addition, the spectra
were recorded.
cleotide (FAD),
NADPH, 10
5 mM
m
m
L
-arginine (iNOS and eNOS) in a total volume of 80 ml. These
mixtures were incubated at 37 ꢀC (nNOS 30 min, iNOS and eNOS
20 min) after the addition of proteins. Then a solution of 1.6 mM
5.2.6. Effects of pyrido[1,2-a]pyrimidines on NADPH consumption
by iNOS
pyruvic acid, sodium salt, and 20 U/ml
(20 l) was added. These preparations were incubated for 20 min at
37 ꢀC and the reactions were stopped by the addition of 50
l ice
cold acetonitrile and centrifugation (15,000 rpm, 4 ꢀC). Then 12
each of a solution of 5.8 mM naphthylethylendiamine dihydro-
chloride and 52 mM sulfanilamide were added to 120 l of the
supernatants. Absorbances at 543 nm were measured after 5 min at
20 ꢀC. All data were referred to 100% control experiments con-
taining 1% DMSO but without inhibitor. All experimental data are
means ꢁ of 3–5 determinations.
L
-lactic dehydrogenase
The initial rates of NADPH oxidation by iNOS were quantitated
spectrophotometrically at 340 nm using an extinction coefficient of
m
m
6.2 mMꢂ1 ꢃ cmꢂ1. Cuvettes contained 150
ml of 50 mM Hepes, pH
ml
7.4, 5 mM DTT,100
mM CaCl2, 200
mM NADPH,10
m
M H4B, -arginine
L
(100 mM) and the tested agents. After preincubating the cofactor
m
mixture and the inhibitor for 2 min at 37 ꢀC, the iNOS 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 pres-
ence of 1% DMSO, in the absence of NADPH, or in the presence of
1 mM L-NNA, a knowninhibitorof NADPH consumption byNOS [45].
5.2.4. Radioactive assays
5.2.7. Effects of pyrido[1,2-a]pyrimidines on NOS expressed in RAW
264.7 cells
NOS-dependent oxidation of
L-arginine to L-citrulline was
determined according to a previously described protocol [41].
Briefly, enzymatic reactions were conducted at 37 ꢀC for 5 min in
50 mM Hepes, pH 7.4, containing 5 mM DTT, 1 mM NADPH, 1 mM
The murine macrophage RAW 264.7 cells were plated at
100,000 cells/well in 96-well plates in 200 ml of RPMI 1640 culture
medium supplemented with 5% FCS and antibiotics, and incubated
for 24 h at 37 ꢀC under a 5% CO2 atmosphere. Fresh medium and
various concentrations of compounds were added 1 h before the
CaCl2, 10
200,000 cpm [
50 mCi/mmol, 2.5
pyrido[1,2-a]pyrimidines were added to the incubation mixtures as
l portions dissolved in DMSO. Final incubation volumes were
100 l. Control incubations contained similar amounts (1%) of
m
g/ml CaM, 10
14C]-
-arginine (Perkin–Elmer, specific activity
-arginine), and the tested compounds. The
mM H4B, 2 mM FAD, 2 mM FMN, about
g-
L
mM
L
addition of 40 U/ml
g-IFN and 50 ng/ml LPS. After incubation for
20 h at 37 ꢀC, 100
ml of the supernatants were analysed for nitrite
1
m
formation using the Griess’ reagent and absorbance at 550 nm was
measured on a Victor plate reader. Quantitation of nitrite formation
was performed with calibration curves using known amounts of
NaNO2 treated under the same conditions.
m
DMSO without inhibitors and values are expressed relative to the
DMSO controls. The reactions were started by the addition of
protein and terminated by the addition of 500
(20 mM sodium acetate, pH 5.5, 1 mM -citrulline, 2 mM EDTA, and
0.2 mM EGTA). Samples (500 l) were applied to small glass
columns containing 800
l of Dowex AG 50 W-X8 (Naþ form,
prepared from the Hþ form), pre-equilibrated with stop buffer and
a total of 1.5 ml of stop buffer was added to eluate [14C]
-citrulline.
Aliquots were then mixed with 1.5 ml Pico-Fluor 40 (Packard) and
counted on a Packard Tri-Carb 2300 liquid scintillation spectrom-
eter. Control sample without NOS are included for background
determinations. Incubations in the presence of iNOS were
performed similarly but CaCl2 and CaM were omitted. When
ml cold stop buffer
L
Acknowledgements
m
m
The authors thank B. Mayer (University of Graz, Austria) and D.J.
Stuehr (Lerner Research Fundation, Cleveland, USA) for the purified
NOS. They also acknowledge the excellent technical supports given
by M. Schu¨tt, M. and S. Wichmann (University of Kiel) and M.
Jaouen and B. Ramassamy (UMR 8601 CNRS, Paris).
L
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