Synthesis of alkyl 6-methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro- 2H-pyrimidine-2-one-5-carboxylates possessing a N-3 nitro substituent to determine calcium channel modulation structure-activity relationships

Article abstract of DOI:10.1002/jhet.5570440324

(Chemical Equation Presented) The Bigenelli acid catalyzed condensation of 2-trifluoromethylbenzaldehyde (1), urea (2) and an alkyl acetoacetate (3) afforded the respective alkyl (Me, Et, i-Pr, i-Bu) 6-methyl-4-(2- trifluoromethylphenyl)-1,2,3,4-tetrahydr

Full text of DOI:10.1002/jhet.5570440324

May-Jun 2007
669
Synthesis of Alkyl 6-Methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-
tetrahydro-2H-pyrimidine-2-one-5-carboxylates Possessing a N-3
Nitro Substituent to Determine Calcium Channel Modulation
Structure-Activity Relationships
Kuljeet Kaur and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
Received September 26, 2006
Me
F3C
C
C
R
O
O
F3C
H
NH2
Cu(NO3)2
Ac2O
Bigenelli
reaction
O2N
CO2R
Me
CO2R
Me
C
O
+
+
CF3
N
N
NH2
C
O
O
O
N
H
O
N
H
H
R = Me, Et, i-Pr, i-Bu
The Bigenelli acid catalyzed condensation of 2-trifluoromethylbenzaldehyde (1), urea (2) and an alkyl
acetoacetate (3) afforded the respective alkyl (Me, Et, i-Pr, i-Bu) 6-methyl-4-(2-trifluoromethylphenyl)-
1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylate (4-7). Subsequent N3-nitration of the alkyl esters (4-
7) using Cu(NO3)2•3H2O and Ac2O furnished the target alkyl 6-methyl-3-nitro-4-(2-trifluoromethyl-
phenyl)-1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylates (8-11). The N3-nitro compounds (8-11)
were less potent calcium channel antagonists (IC50 values in the 1.9 x 10-7 to 3.9 x 10-6 M range) on guinea
pig ileal longitudinal smooth muscle than the reference drug nifedipine (Adalat®, IC50 = 1.4 x 10-8 M). In
vitro calcium channel modulation studies on guinea pig left atrium (GPLA) showed that the methyl and
ethyl esters (8-9) induced a weak-to-modest positive inotropic (agonist) effect, and that the inactive
isopropyl (10) and isobutyl (11) esters did not alter the cardiac contractile force of GPLA.
J. Heterocyclic Chem., 44, 669 (2007).
groups are thought to be preferentially oriented in a plane
which intersects the plane of the dihydropyridine (DHP)
ring with an angle of between 30 and 60°. In the agonist
BAY K8644, the 3-nitro group is oriented in the plane of
the DHP ring [5].
INTRODUCTION
The calcium ion channel is an important drug design
receptor target which has specific binding sites for both
antagonist and agonist ligands that control the
respective closed and open conformational state of the
calcium channel. The closed and open states of the
channel have different affinities and/or access for drugs
which may result in quantitative and qualitative
differences in calcium channel modulation structure-
activity relationships [1]. Changes in the substituent
pattern at the C-3, C-4, and C-5 positions of the 1,4-
dihydropyridine ring alters potency, tissue selectivity and
the conformation of the boat-shaped 1,4-dihydropyridine
ring [2]. Hantzsch 1,4-dihydropyridines (see structures in
Figure 1) such as dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-
nitrophenyl) pyridine-3,5-dicarboxylate (nifedipine)
possessing C-3 and C-5 alkyl ester substitutents exhibit
calcium channel antagonist activity [3], whereas modified
CF3
NO2
CO2Me
CO2Me
O2N
Me
MeO2C
Me
N
H
Me
N
H
Me
Nifedipine
Bay K8644
Figure 1.
A number of N3-substituted-3,4-DHPMs (DHPMs =
dihydrodpyrimidines) with vasodilative and antihyper-
tensive activity [6], 2-hetero-substituted-4-aryl-1,4-
dihydro-6-methyl-5-pyrimidinecarboxylic acid esters
(active in vitro but inactive in vivo) [7], 3-substituted
(alkyl esters)-4-aryl-1,4-dihydro-6-methyl-5-pyrimidine-
carboxylic acid esters (inactive in vivo due to metabolism
to the 3-NH metabolite) [8], 3-carbamoyl-4-aryl-1,2,3,4-
tetrahydro-6-methyl-5-pyrimidinecarboxylic acid esters
(orally active and metabolically stable) [9], and
structurally related pyrimidine analogs [10-11] have been
reported. These 1,4-DHPMs, designed as mimics of
Hantzsch 1,4-dihydropyridines having
a C-3 nitro
substituent such as methyl 1,4-dihydro-2,6-dimethyl-3-
nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate
(Bay K8644) exhibit calcium channel agonist activity [4].
It has been proposed, based on MOPAC calculations, that
the important conformational feature which differentiates
agonist and antagonist activity is the orientation of the
ester (synperiplanar for antagonists) and nitro
(antiperiplanar for agonists). In antagonists both ester

670
K. Kaur and E. E. Knaus
Vol 44
DHPs, can adopt a conformation similar to DHPs [7], they
bind to the DHP CC receptor but with lower affinity [7],
the DHPM ring is more stable to oxidation than the DHP
ring [8], a N3-amido substituent on a 2-pyrimidinone ring
is metabolically stable, and the N3-H compounds are very
weak calcium channel antagonists [9].
Scheme I
Me
C
C
R
O
O
NH2
C
O
+
CF3
+
NH2
O
C
O
H
2
1
3
As part of our ongoing program to develop structure-
activity relationships for heterocyclic mimics of 1,4-
dihydropyridines with calcium channel modulating effects,
and for use as probes to study the structure-function
relationships of calcium channels, we now describe the
synthesis and calcium channel modulating effects for a
group of hitherto unknown N3-nitro derivatives of alkyl 6-
methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro-2H-
pyrimidine-2-one-5-carboxylates (8-11).
MeOH, HCl
85-87°, 8h
F3C
F3C
H
O2N
CO2R
Me
Cu(NO3)2.3H20
Ac2O, 25°, 5h
CO2R
N
N
O
N
H
O
N
H
Me
RESULTS AND DISCUSSION
8, R = Me
9, R = Et
4, R = Me
5, R = Et
A group of alkyl (Me, Et, i-Pr, i-Bu) 6-methyl-4-(2-
trifluoromethylphenyl)-1,2,3,4-tetrahydro-2H-pyrimidine-
2-one-5-carboxylates (4-7) were prepared using a classical
Biginelli reaction as illustrated in Scheme 1. Thus, the
acid catalyzed condensation of 2-trifluoromethyl-benz-
aldehyde (1), urea (2) and an alkyl acetoacetate (3) in
MeOH at 85-87° for 8 hours afforded the respective
product (4-7) in 27-50% yield. Nitration of the alkyl
esters (4-7) using Cu(NO3)2•3H2O and Ac2O [12],
following a procedure similar to that used for the
preparation of 1-methyl-3-nitrouracil [13], furnished the
respective alkyl 6-methyl-3-nitro-4-(2-trifluoromethyl-
phenyl)-1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carbox-
ylate (8-11) in 35-51% yield. 1H nmr spectral data
indicates that nitration occurs at the N-3, rather than the
N-1, position since the H-4 proton in the 3-nitro
compounds 8-11 was deshielded relative to the
corresponding H-4 proton in compounds 4-7, and the C-6
Me resonances in the two groups of compounds 4-7 and
8-11 were nearly identical. One plausible explanation for
N-3 nitration is that the N-1 nitrogen is deactivated due to
its enamine character.
The objective of this study was to determine whether
the alkyl 6-methyl-3-nitro-1,2,3,4-tetrahydro-2H-pyrimi-
dine-2-one-5-carboxylate moiety present in the target
compounds (8-11) was a bioisostere of the methyl 1,4-
dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylate moiety
present in the calcium channel agonist Bay K8644. In
vitro calcium channel modulation studies (see data in
Table 1) showed that compounds 8-11, unlike Bay K8644,
which induces a calcium channel agonist effect, exhibited
a calcium channel antagonist effect (IC50 values in the 1.9
x 10-7 to 3.9 x 10-6 M range) on guinea pig ileal
longitudinal smooth muscle (GPILSM). Compounds 8-11
are less potent calcium channel antagonists than the
reference drug nifedipine (Adalat®, IC50 = 1.4 x 10-8 M).
In vitro calcium channel modulation studies on guinea pig
left atrium (GPLA) showed that the methyl ester (8)
10, R = i-Pr
11, R = i-Bu
6, R = i-Pr
7, R = i-Bu
induced a modest positive inotropic calcium channel
agonist effect where cardiac contractile force was
increased by about 20% at a drug concentration of 1.61 x
10-6 M (see data in Table 1). In similar studies, the ethyl
ester (8) produced a weaker positive inotropic effect than
the methyl ester (9), and the inactive isopropyl (10) and
isobutyl (11) esters that did not change the contractile
force of GPLA. These structure-activity studies on GPLA
show that the alkyl 6-methyl-3-nitro-1,2,3,4-tetrahydro-
2H-pyrimidine-2-one-5-carboxylate moiety present in
compounds 8 (R = Me) and 9 (R = Et) is a partial
bioisostere of the methyl 1,4-dihydro-2,6-dimethyl-3-
nitropyridine-5-carboxylate moiety present in the calcium
channel agonist Bay K8644 with respect to a positive
inotropic effect on GPLA.
Table 1
In Vitro Calcium Channel Modulation Effects
Calcium Channel
Antagonist Effect
On GPILSM
Calcium Channel Agonist Effect
On GPLA[b]
Compound
(IC50)[a]
% Inotropic
Effect
Drug
Concentration (M)
1.61 x 10-6
1.61 x 10-6
1.61 x 10-6
1.61 x 10-6
—
8
9
10
11
3.9 ± 0.61 x 10-6
2.1 ± 0.12 x 10-6
1.9 ± 0.22 x 10-7
3.1 ± 1.20 x 10-6
1.4 ± 0.19 x 10-8
20.4 ± 6.0
10.6 ± 6.4
Inactive
Inactive
—
Nifedipine[c]
[a] The molar concentration of the test compound causing a 50%
decrease in the slow component or tonic contractile response (IC50
±
SEM, n = 3) in guinea pig ileal longitudinal muscle induced by the
muscarinic agonist carbachol (1.6 x 10-7 M) was determined graphically
from the dose-response curve. [b] The % increase in cardiac contractile
force (agonist effect, positive inotropic effect) exhibited by the test
compound on guinea pig left atrium was determined from the dose-
response curve at the highest molar concentration used (1.61 x 10-6.M).
[c] Nifedipine chemical name is dimethyl 2,6-dimethyl-4-(2-
nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate.

May-Jun 2007 Synthesis of Alkyl 6-methyl-3-nitro-4-aryl-1,2,3,4-tetrahydro-2H-pyrimidine-2-one-5-carboxylates
671
2.36 (s, 3H, C-6 CH3), 3.54 [d, Jgem = 10.6 of d, Jvic = 6.4 Hz,
1H, CHH’CH(CH3)2], 3.72 [d, Jgem = 10.6 of d, Jvic = 7.1 Hz,
1H, CHH’CH(CH3)2], 5.56 (s, 1H, H-4), 7.25 (s, 1H, N3-H),
7.42-7.51 (m, 2H, phenyl H-4, H-6), 7.62-7.70 (m, 2H, phenyl
H-3, H-5), 9.39 (s, 1H, N1-H). Anal. Calcd. for C17H19F3N2O3: C,
57.30; H, 5.37; N, 7.86. Found: C, 57.28; H, 5.11; N, 8.00.
General Procedure for the Synthesis of Alkyl 6-Methyl-3-
nitro-4-(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro-(2H)-
pyrimidine-2-ones (8-11). A mixture of Cu(NO3)2•3H2O (1.21
g, 5.0 mmoles) and acetic anhydride (9.0 ml, 12.1 mmoles) was
stirred at 25° for 1.5 hours, one of the alkyl 6-methyl-4-(2-
trifluoromethylphenyl)-1,2,3,4-tetrahydro-(2H)-pyrimidine-2-
ones (either 4, 5, 6 or 7) (5.0 mmoles) was added, and the
reaction was allowed to continue at 25° for 5 hours with stirring.
Methanol (50 ml) was added to quench the reaction, and the
mixture was stirred for a futher 30 minutes at 25°. The volatile
components were removed in vacuo, the residue was partitioned
between water (100 ml) and chloroform (100 ml), the aqueous
fraction was extracted with chloroform (4 x 100 ml), the
combined organic fractions were washed with water, and the
organic fraction was dried (MgSO4). Removal of the solvent
from the organic fraction in vacuo gave a residue that was
partially purified by silica gel column chromatography using
ethyl acetate:hexane (1:2, v/v) as eluant. Removal of the solvent
in vacuo gave a residue that was recrystallized from EtOH (98%,
v/v) to furnish the respective product 8, 9, 10 or 11. The physical
and spectral data for products 8-11 are listed below.
EXPERIMENTAL
Melting points were determined using a Thomas-Hoover
capillary apparatus and are uncorrected. H nmr spectra were
1
recorded on a Bruker AM-300 spectrometer. The assignment of
exchangeable protons (NH) was confirmed by the addition of
D2O. Silica gel column chromatography was carried out using
Silicyle (70-230 mesh) silica gel. Isopropyl acetoacetate was
purchased from the Lancaster Chemical Co. All other reagents
were purchased from the Aldrich Chemical Co. Elemental
analyses were performed for C, H and N (Micro-Analytical
Service Laboratory, Department of Chemistry, University of
Alberta). In vitro calcium channel antagonist and agonist
activities were determined using protocols approved by the
Health Sciences Animal Welfare Committee at the University of
Alberta.
General Procedure for the Synthesis of Alkyl 6-Methyl-4-
(2-trifluoromethylphenyl)-1,2,3,4-tetrahydro-(2H)-pyrimid-
ine-2-ones (4-7). Urea (2) (2.1 g, 40 mmoles) was added to a
solution of 2-trifluoromethylbenzaldehyde (1) (6.97 g, 40
mmoles) and an alkyl acetoacetate (3) (40 mmoles) with
strirring. Concentrated hydrochloric acid (4 drops of 37%, w/v)
was added, the reaction was allowed to proceed at 85-87° for 8
hours with stirring, the reaction mixture was cooled to 25° and
stirring was continued at 25° for 1 hour. Removal of volatile
components in vacuo gave an off-white residue that was washed
with cold water to remove urea and other water soluble
impurities. Recrystallization of this material from EtOH (98%,
v/v) afforded the respective product (4, R = Me; 5, R = Et; 6, R
= i-Pr; 7, R = i-Bu). The physical and spectral data for
compounds 4-7 are listed below.
Methyl 6-Methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-tetra-
hydro-(2H)-pyrimidine-2-one (4). Compound 4 was obtained
as a white solid in 27% yield, mp 210-212°; 1H nmr
(deuteriodimethylsulfoxide): ꢀ 2.33 (s, 3H, C-6 CH3), 3.38 (s,
3H, CO2CH3), 5.54 (s, 1H, H-4), 7.40 (s, 1H, N3-H), 7.42-7.51
(m, 2H, phenyl H-4, H-6), 7.61-7.70 (m, 2H, phenyl H-3, H-5),
9.37 (s, 1H, N1-H). Anal. Calcd. for C14H13F3N2O3: C, 53.51; H,
4.17; N, 8.91. Found: C, 53.53; H, 4.28; N, 8.67.
Ethyl 6-Methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-tetra-
hydro-(2H)-pyrimidine-2-one (5). Compound 5 was obtained
as a white solid in 50% yield, mp 200-202° (lit[14] mp 198-
200°); 1H nmr (deuteriodimethylsulfoxide): ꢀ 0.87 (t, J = 7.0 Hz,
3H, CH2CH3), 2.34 (s, 3H, C-6 CH3), 3.85 (q, J = 7.0 Hz, 2H,
CH2CH3), 5.56 (s, 1H, H-4), 7.30 (s, 1H, N3-H), 7.45-7.52 (m,
2H, phenyl H-4, H-6), 7.62-7.70 (m, 2H, phenyl H-3, H-5), 9.35
(s, 1H, N1-H). Anal. Calcd. for C15H15F3N2O3: C, 54.22; H, 4.69;
N, 8.43. Found: C, 54.15; H, 4.52; N, 8.52.
Methyl 6-Methyl-3-nitro-4-(2-trifluoromethylphenyl)-1,2,
3,4-tetrahydro-(2H)-pyrimidine-2-one (8). Compound 8 was
1
obtained as a white solid in 35% yield, mp 182-185°; H nmr
(deuteriodimethylsulfoxide): ꢀ 2.34 (s, 3H, C-6 CH3), 3.54 (s,
3H, CO2CH3), 7.02 (s, 1H, H-4), 7.50 (d, J = 8.0 Hz, 1H, phenyl
H-6), 7.60 (dd, J = 8.0, 7.6 Hz, 1H, phenyl H-4), 7.72 (dd, J =
8.0, 7.6 Hz, 1H, phenyl H-5), 7.78 (d, J = 8.0 Hz, 1H, phenyl H-
3), 10.89 (s, 1H, N1-H). Anal. Calcd. for C14H12F3N3O5•2/9H2O:
C, 46.29; H, 3.45; N, 11.57. Found: C, 46.21; H, 3.63; N, 11.87.
Ethyl 6-Methyl-3-nitro-4-(2-trifluoromethylphenyl)-1,2,3,4-
tetrahydro-(2H)-pyrimidine-2-one (9). Compound
obtained as a white solid in 36% yield, mp 164-166°; H nmr
(deuteriodimethylsulfoxide): ꢀ 1.06 (t, 7.0 Hz, 3H,
9 was
1
J
=
CH2CH3), 2.35 (s, 3H, C-6 CH3), 3.95-4.10 (m, 2H, CH2CH3),
7.06 (s, 1H, H-4), 7.51 (d, J = 8.0 Hz, 1H, phenyl H-6), 7.60 (dd,
J = 8.0, 7.6 Hz, 1H, phenyl H-4), 7.70 (dd, J = 8.0, 7.6 Hz, 1H,
phenyl H-5), 7.78 (d, J = 8.0 Hz, 1H, phenyl H-3), 10.89 (s, 1H,
N1-H). Anal. Calcd. for C15H14F3N3O5: C, 48.27; H, 3.78; N,
11.26. Found: C, 48.11; H, 3.71; N, 11.26.
Isopropyl 6-Methyl-3-nitro-4-(2-trifluoromethylphenyl)-1,2,
3,4-tetrahydro-(2H)-pyrimidine-2-one (10). Compound 10
1
Isopropyl
tetrahydro-(2H)-pyrimidine-2-one (6). Compound
6-Methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-
was
was obtained as a white solid in 46% yield, mp 145-146°; H
6
nmr (deuteriodimethylsulfoxide): ꢀ 0.92 and 1.15 [two d, J = 7.0
Hz, 3H each, CH(CH3)2], 1.78 (heptet, J = 7 Hz, 1H, CH(CH3)2],
2.36 (s, 3H, C-6 CH3), 7.06 (s, 1H, H-4), 7.50 (d, J = 8.0 Hz,
1H, phenyl H-6), 7.61 (dd, J = 8.0, 7.6 Hz, 1H, phenyl H-4),
7.71 (dd, J = 8.0, 7.6 Hz, 1H, phenyl H-5), 7.78 (d, J = 8.0 Hz,
1H, phenyl H-3), 10.98 (s, 1H, N1-H). Anal. Calcd. for
C16H16F3N3O5: C, 49.62; H, 4.16; N, 10.85. Found: C, 49.75; H,
4.16; N, 10.78.
1
obtained as a white solid in 40% yield, mp 193-195°; H nmr
(deuteriodimethylsulfoxide): ꢀ 0.64 and 1.03 [two d, J = 6.0 Hz,
3H each, CH(CH3)2], 2.33 (s, 3H, C-6 CH3), 4.73 [heptet, J = 6.0
Hz, 1H, CH(CH3)2], 5.55 (s, 1H, H-4), 7.21 (s, 1H, N3-H), 7.42-
7.51 (m, 2H, phenyl H-4, H-6), 7.63-7.70 (m, 2H, phenyl H-3, H-
5), 9.32 (s, 1H, N1-H). Anal. Calcd. for C16H17F3N2O3•1/9 H2O: C,
55.84; H, 5.04; N, 8.14. Found: C, 55.84; H, 5.02; N, 8.35.
Isobutyl 6-Methyl-4-(2-trifluoromethylphenyl)-1,2,3,4-
tetrahydro-(2H)-pyrimidine-2-one (7). Compound
obtained as a white solid in 37% yield, mp 173-175°; H nmr
(deuteriodimethylsulfoxide): ꢀ 0.51 and 0.68 [two d, J = 6.0 Hz,
3H each, CH2CH(CH3)2], 1.56-1.65 [m, 1H, CH2CH(CH3)2],
Isobutyl 6-Methyl-3-nitro-4-(2-trifluoromethylphenyl)-1,2,
3,4-tetrahydro-(2H)-pyrimidine-2-one (11). Compound 11
was obtained as a white solid in 51% yield, mp 124-126°; H
nmr (deuteriodimethylsulfoxide): ꢀ 0.68 and 0.71 [two d, J = 6.0
Hz, 3H each, CH2CH(CH3)2], 1.71-1.80 [m, 1H, CH2CH(CH3)2],
7 was
1
1

672
K. Kaur and E. E. Knaus
Vol 44
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2.39 (s, 3H, C-6 CH3), 3.78 [d, Jgem = 10.6 Hz, 2H,
CH2CH(CH3)2], 7.14 (s, 1H, H-4), 7.48 (d, J = 8.0 Hz, 1H,
phenyl H-6), 7.60 (dd, J = 8.0, 7.6 Hz, 1H, phenyl H-4), 7.71
(dd, J = 8.0, 7.6 Hz, 1H, phenyl H-5), 7.78 (d, J = 8.0 Hz, 1H,
phenyl H-3), 10.98 (s, 1H, N1-H). Anal. Calcd. for
C17H18F3N3O5: C, 50.88; H, 4.52; N, 10.47. Found: C, 50.64; H,
4.37; N, 10.22.
In Vitro Calcium Channel Antagonist and Agonist Assays.
Smooth muscle calcium channel antagonist activity was
determined as the micromolar (μM) concentration of the test
compound required to produce 50% inhibition of the muscarinic
receptor-mediated (carbachol, 0.167 μM) Ca+2-dependent
contraction (tonic response) of guinea pig ileum longitudinal
smooth muscle (GPILSM) using the procedure previously
reported[15]. The IC50 value (± SEM, n=3) was determined
graphically from the dose-response curve.
The cardiac calcium channel agonist effect was calculated as
the percentage increase (positive inotropic effect) in contractile
force of isolated guinea pig left atrium (GPLA) relative to its
basal contractile force in the absence of test compound. The
positive inotropic EC50 value (± SEM, n=3) was determined
graphically from the dose-response curve.
Acknowledgement We are grateful to the Canadian Institutes
of Health Research (CIHR) (MT-8892) for financial support of
this research.
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REFERENCES AND NOTES
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[15] Vo, D.; Matowe, W. C.; Ramesh, M.; Iqbal, N.; Wolowyk,
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* Correspondence to: E. E. Knaus, Email: eknaus@pharmacy.ualberta.ca
[1] Perez-Reyes, E.; Schneider, T. Drug Dev. Res. 1994, 33, 295.