Synthesis and cardiotonic activity of novel pyrimidine derivatives: Crystallographic and quantum chemical studies

Article abstract of DOI:10.1021/jm9508649

The synthesis of ethyl or methyl 4-substituted or unsubstituted 2- (dimethylamino)-5-pyrimidinecarboxylates 10-20, which is mainly carried out by reaction of ethyl or methyl 2-[(dimethylamino)methylene]-3-oxoalkanoates with 1,1-dimethylguanidine, is described. The above esters were hydrolyzed to the relative carboxylic acids 21-30, which were decarboxylated to the corresponding 2,4-disubstituted pyrimidines 31-40. All the new synthesized pyrimidines were evaluated in spontaneously beating and electrically driven atria from reserpine-treated guinea pigs. Their effects were compared to those induced by milrinone in both atria preparations. Compound 28 (4- benzyl-2-(dimethylamino)-5-pyrimidinecarboxylic acid) was the most effective positive inotropic agent, while the corresponding methyl ester 17 reduced both the contractile force and the frequency of guinea pig atria. An antagonism toward the negative influence exerted by endogenous adenosine on the heart seems to be involved in the contractile activity of compound 28. By contrast, compound 17 might be partial agonist at the purinergic inhibitory (A1) receptor. X-ray analysis carried out on 17 and 28 and molecular modeling investigations extended also to related derivatives allowed a possible rationalization between structure and inotropic activity for this series of compounds.

Full text of DOI:10.1021/jm9508649

J . Med. Chem. 1996, 39, 3671-3683
3671
Syn th esis a n d Ca r d ioton ic Activity of Novel P yr im id in e Der iva tives:
Cr ysta llogr a p h ic a n d Qu a n tu m Ch em ica l Stu d ies
Paola Dorigo,*,† Daniela Fraccarollo,† Giovanni Santostasi,† Ildebrando Maragno,† Maura Floreani,†
Pier Andrea Borea,‡ Luisa Mosti,| Laura Sansebastiano,| Paola Fossa,| Fulvia Orsini,§ Franco Benetollo,*, and
Gabriella Bombieri∇
Dipartimento di Farmacologia, Universita` di Padova, Largo E. Meneghetti 2, 35131 Padova, Italy, Istituto di Farmacologia,
Universita` di Ferrara, Via Fossato di Mortara 61 B, 44100 Ferrara, Italy, Istituto di Scienze Farmaceutiche, Universita` di
Genova, Viale Benedetto XV, 16132 Genova, Italy, Dipartimento di Chimica Organica e Industriale, Universita` di Milano, Via
Venezian 21, 20188 Milano, Italy, I.C.T.I.M.A. CNR, Corso Stati Uniti 4, 35100 Padova, Italy, and Istituto di Chimica
Farmaceutica, Universita` di Milano, Viale Abruzzi 42, 20131 Milano, Italy
Received November 27, 1995X
The synthesis of ethyl or methyl 4-substituted or unsubstituted 2-(dimethylamino)-5-pyrim-
idinecarboxylates 10-20, which is mainly carried out by reaction of ethyl or methyl
2-[(dimethylamino)methylene]-3-oxoalkanoates with 1,1-dimethylguanidine, is described. The
above esters were hydrolyzed to the relative carboxylic acids 21-30, which were decarboxylated
to the corresponding 2,4-disubstituted pyrimidines 31-40. All the new synthesized pyrimidines
were evaluated in spontaneously beating and electrically driven atria from reserpine-treated
guinea pigs. Their effects were compared to those induced by milrinone in both atria
preparations. Compound 28 (4-benzyl-2-(dimethylamino)-5-pyrimidinecarboxylic acid) was the
most effective positive inotropic agent, while the corresponding methyl ester 17 reduced both
the contractile force and the frequency of guinea pig atria. An antagonism toward the negative
influence exerted by endogenous adenosine on the heart seems to be involved in the contractile
activity of compound 28. By contrast, compound 17 might be partial agonist at the purinergic
inhibitory (A1) receptor. X-ray analysis carried out on 17 and 28 and molecular modeling
investigations extended also to related derivatives allowed a possible rationalization between
structure and inotropic activity for this series of compounds.
In tr od u ction
Although classified as PDE III inhibitors, the bipy-
ridine derivatives, such as amrinone and milrinone, at
elevated concentrations, displace endogenous adenosine
from its cardiac receptors.15-19 In order to obtain pure
adenosine antagonists, devoid of inhibitory influence on
PDE III, we had already synthesized a number of
milrinone analogues in which the 1,6-dihydro-6-oxopy-
ridine moiety of the parent drug has been variously
functionalized.20-22 Inotropic activity of some of these
compounds in the guinea pig atria was greater than that
of milrinone, and an inhibition of the negative influence
exerted by endogenous adenosine seems to be involved
in their contractile activity.23-25 Moreover, an X-ray
structural characterization and quantum chemical analy-
sis had been carried out on the above compounds.24,26,27
Theoretical data indicate that one of the most critical
factors for the different inotropic activities (positive or
negative) seems to be related to the steric and electronic
requirements of the substituent at position 2 in the 1,6-
dihydro-6-oxopyridine nucleus. On the other hand,
pyridinyl-2-pyrimidinamines were reported to be active
as cardiotonics by a Lesher U.S. patent.28 Since the
pyrimidine moiety represents an integral part of the
purine nucleoside adenosine, we utilized synthons 1-9
and synthesized a series of compounds (10-40) char-
acterized by the pyrimidine nucleus bearing the elec-
tronic donor dimethylamino group at position 2 and by
a variety of functionalizations at positions 4 and 5
(Scheme 1).
Antagonism of endogenous adenosine at A1 inhibitory
receptors in the heart represents an investigational area
which could offer a creative possibility in the treatment
of heart failure.1 Adenosine, which is released in high
concentrations during cardiac heart failure,2 may fur-
ther damage the heart by slowing conduction in the
sinoatrial and AV nodes and by reducing atrial
contractility.3-5 The negative effects exerted by ad-
enosine on the heart appear to be related to a reduction
of the duration of the action potential in response to a
direct inhibition of Ca2+ channels6-9 and/or to a reduc-
tion in Ca2+ flux into the cell due to an increased K+
conductance and hyperpolarization of cell membrane.7,10,11
Thus, an antagonism toward endogenous adenosine may
indirectly increase intracellular Ca2+ concentration and
activate the contractile system without increases in
cyclic AMP levels and without the related risk of
dangerous arrhythmias.12 In fact, all the phosphodi-
esterase III (PDE III) inhibitors, by increasing intrac-
ellular cAMP content, exert favorable hemodynamic
effects in patients with cardiac failure, but most of them
may exacerbate ventricular arrhythmias, provoke myo-
cardial ischemia, accelerate the progression of the
underlying disease, and increase the mortality rate.13,14
† Universita` di Padova.
‡ Universita` di Ferrara.
| Universita` di Genova.
§ Dipartimento di Chimica Organica e Industriale, Universita di
Milano.
The cardiac activity of these new compounds was
investigated by determining their effects on the con-
tractile force and the frequency rate of spontaneously
beating atria isolated from guinea pigs. The most
I.C.T.I.M.A. CNR.
∇ Istituto di Chimica Farmaceutica, Universita` di Milano.
X Abstract published in Advance ACS Abstracts, August 1, 1996.
S0022-2623(95)00864-8 CCC: $12.00 © 1996 American Chemical Society

3672 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Dorigo et al.
Sch em e 1a
a
(a) NaOEt/EtOH or NaOMe/MeOH at room temperature; (b) (1) KOH/EtOH or KOH/MeOH, reflux, (2) 6 M HCl; (c) heating over
their melting points.
interesting agents were further studied in electrically
driven left atrium. X-ray analysis and quantum chemi-
cal methods were also applied in order to gain additional
information on the possible structure-activity relation-
ships. The results have been related to the pharmaco-
logical data.
our compounds (Table 1), as well as spectral data,
unequivocally established the structure of pyrimidines
31, 32, 34, and 3735-37and consequently that of starting
esters 10, 11, 13, and 16.
Biologica l Activity: Isola ted Atr ia P r ep a r a tion s
All these new pyrimidine derivatives (1 µM-1 mM)
exerted qualitatively and/or quantitatively different
effects on both contractile force (Table 2) and frequency
(Table 3) of spontaneously beating atria from reserpine-
treated guinea pigs. Among the tested compounds, only
28 induced a marked increase in inotropism (Table 2).
The positive inotropic effect induced by 28 at the highest
concentration tested (1 mM), expressed as a percentage
variation with respect to the control, was greater than
that induced by higher active concentration of milrinone
(0.3 mM). Moreover the respective EC50 was not
determined since 28, in the range of concentrations
tested, did not reach its maximum inotropic effect. In
contrast to milrinone, 28 did not increase the beating
rate of the heart preparation (Table 3). Thus the acid
appears to be a more “force” than “frequency” specific
agent with a more advantageous pharmacological pro-
file. The positive inotropic activity of 28 was also
confirmed in electrically driven left atria (Table 4). On
the contrary, the related methyl ester, compound 17,
after a slight initial increase in the developed tension,
evoked marked negative inotropic and chronotropic
effects (Table 2 and 3).1 EC50 values4 for its negative
inotropic and chronotropic effect2 of 0.30 and 0.88 mM,
respectively, were calculated. The negative inotropic
activity of 17 was also confirmed in electrically driven
left atria (Table 4).
Ch em istr y
In recent papers29,30 we described the efficient reac-
tion of ethyl or methyl 2-[(dimethylamino)methylene]-
3-oxoalcanoates 1-9 with N-C-N dinucleophiles such
as guanidine, acetamidine, benzamidine, or 2-methyl-
isothiourea. This synthetic pathway afforded the rela-
tive esters of 4-substituted 2-amino-, 2-methyl, 2-phe-
nyl-, or 2-(methylthio)-5-pyrimidinecarboxylic acids,
respectively, in high yields.
Using our method, the methyl or ethyl esters of
4-substituted or unsubstituted 2-(dimethylamino)-5-
pyrimidinecarboxylic acids 10-20 (Scheme 1, Table 1)
were synthesized by reacting ethyl 2,2-diformylac-
etate,31 synthons 1-9,32,33 and ethyl-2-(ethoxymethyl-
ene)-4,4,4-trifluoro-3-oxobutanoate,34 respectively, with
2,2-dimethylguanidine sulfate in the presence of sodium
ethoxide or methoxide. Esters 10-20 were converted,
generally in high yields, to the corresponding 4-substi-
tuted or unsubstituted 2-(dimethylamino)-5-pyrimidin-
ecarboxylic acids 21-30 by the usual saponification
procedure (potassium hydroxide in boiling ethanol or
methanol) followed by acidification. Only in the case
of ester 19 did the above reaction afford a mixture of
dicarboxylic and monocarboxylic derivatives which proved
difficult to separate. Finally, decarboxylation of acids
21-30 by simply heating at temperatures above their
melting points led to 4-substituted or unsubstituted
2-(dimethylamino)pyrimidines 31-40 in high yields.
Some of these pyrimidines are already known, al-
though prepared by other routes. Therefore, the iden-
tity in general of their physical constants with those of
Mech a n ism of Action
The electrically driven left atrium was used to study
the mechanism of action of 17 and 28. Release of
endogenous catecholamines is not involved in the posi-

Synthesis and Cardiac Activity of New Pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3673
Ta ble 1. Physical Data of Compounds 10-40
heating
time (h)
mp (°C) or
bp (°C, mmHg)
compd
R
R′
solventa
yield
formula
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
H
CH3
C2H5
C2H5
CH3
C2H5
C2H5
C2H5
C2H5
CH3
60-61
A
B
A
A
B
59
87
70
85
86
82
72
41
86
41
70
70
66
77
99
89
96
78
95
99
82
95
73
83
89
83
86
81
62
90
88
C9H13N3O2
C10H15N3O2
C10H15N3O3
C11H17N3O2
C12H19N3O2
C12H19N3O2
C13H21N3O2
C15H17N3O2
C15H17N3O2
C12H17N3O4
C10H12F3N3O2
C7H9N3O2
C8H11N3O2
C9H13N3O2
C9H13N3O2
C10H15N3O2
C10H15N3O2
C11H17N3O2
C14H15N3O2
C13H13N3O2
C8H8F3N3O2
C6H9N3
C7H11N3
C8H13N3O
C8H13N3
C9H15N3
C9H15N3
C10H7N3
C13H15N3
C12H13N3
C7H8F3N3
56-58
CH2OCH3
CH2CH3
(CH2)2CH3
CH(CH3)2
C(CH3)3
CH2C6H5
C6H5
CO2C2H5
CF3
H
102-104
43-45
36-38
95-100 (0.4)
115-120 (0.9)
63-65
A
B
B
C2H5
C2H5
C2H5
37-39
67-69
95-100 (0.3)
263-264
C
D
D
D
D
D
D
D
C
E
CH3
193-195
CH2OCH3
CH2CH3
(CH2)2CH3
CH(CH3)2
C(CH3)3
CH2C6H5
C6H5
CF3
H
CH3
CH2OCH3
CH2CH3
(CH2)2CH3
CH(CH3)2
C(CH3)3
CH2C6H5
C6H5
190-192
178-180
162-164
152-154
167-169
183-185
236-238
188-190
1.5
5
5
5
7
7
5
20
5
20
85-90 (20) b
85-90 (13) c
102-106 (12)
91-92 (15) d
110-120 (13)
105-110 (15)
115-118 (14) e
120-125 (0.4)
48-49
A
CF3
85-90 (12)
a
Recrystallization solvents: A ) diethyl ether, B ) petroleum ether (bp 40-70 °C), C ) 95% ethanol, D ) ethyl acetate, E ) petroleum
ether-diethyl ether, 1:1. Lit.35 bp 85-86 °C (28); 88% yield. c Lit.36 bp 103-106 °C (40); 80% yield. Lit.36 bp 91-92 °C (15); 50% yield.
b
d
e Lit.37 bp 50-55 °C (0.1); 42% yield.
tive inotropic effect of 28 since the effect is evident in
atria isolated from reserpine-treated animals and thus
depleted in catecholamines. The involvement of endog-
enous adenosine in the action of the new compounds
was tested by treating left atria with adenosine deami-
nase, the enzyme which inactivates adenosine by me-
tabolizing it to inosine. In these conditions an increase
in developed tension occurred, which declined within
15-20 min, and the force of contraction reached a new
steady state which was 15% higher than the control. In
the presence of adenosine deaminase, the positive
inotropic activity of 28 was drastically reduced (Table
5). The positive inotropic effect of 28, not modified by
adenosine deaminase, was insensitive to â-blocker pro-
pranolol (0.1 µM), R-blocker prazosine (5 nM), and
histamine antagonists pyrilamine (0.1 µM) and raniti-
dine (10 µM) (data not shown). Thus the activation of
a catecholamine or histamine receptor is not involved
in the cardiac activity of 28, although an antagonism
toward endogenous adenosine seems to be present.
However, the nature of the component of the inotropic
effect not related to the adenosine antagonism remains
to be clarified.
In order to confirm the antagonism toward adenosine,
the effect of 28 on the left atrium was evaluated in the
presence of the potent agonist at A1 adenosine receptor,
(R)-PIA (N6-[(R)-phenylisopropyl]adenosine). Figure 1
shows that the concentration-effect curves for the
F igu r e 1. Cumulative concentration-effect curves for nega-
tive inotropic effect of (R)-PIA in the absence and presence of
increasing concentrations of compound 28. Schild regression
analysis is shown in the inset: (9) (R)-PIA, (O) (R)-PIA plus 3
× 10-6 M compound 28, (2) (R)-PIA plus 10-5 M compound
28, (3) (R)-PIA plus 3 × 10-5 M compound 28. Each point is
the mean ( SEM of four to six assays from six different
experiments.
negative inotropic effect exerted by (R)-PIA were shifted
parallel by increasing concentrations of 28, suggesting
a competitive antagonism between 28 and (R)-PIA. This
was confirmed by the analysis of the data according to

3674 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Dorigo et al.
Ta ble 2. Effect of Compounds 10-40 on the Contractile Force of Spontaneously Beating Atria from Reserpine-Treated Guinea Pigs:
Comparison with Amrinone and Milrinonea
developed tension (% variation from the control)
compd
10-6
M
3 × 10-6
M
10-5
M
3 × 10-5
M
10-4
M
3 × 10-4
M
10-3
M
amrinone
milrinone
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0.00 ( 0.00
0.00 ( 0.00
1.19 ( 0.06
2.79 ( 0.15
3.04 ( 0.21
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
1.73 ( 0.13
0.00 ( 0.00
7.15 ( 0.15
3.76 ( 0.12
0.00 ( 0.00
5.46 ( 0.17
0.00 ( 0.00
5.38 ( 0.02
1.79 ( 0.06
3.51 ( 0.14
3.52 ( 0.24
6.62 ( 0.81
2.25 ( 0.11
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
5.39 ( 0.15
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
6.43 ( 0.08
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
4.76 ( 0.03
7.12 ( 0.12
6.93 ( 0.15
0.00 ( 0.00
-5.29 ( 0.08
3.19 ( 0.13
2.08 ( 0.01
5.83 ( 0.09
10.24 ( 0.09
2.18 ( 0.03
11.26 ( 0.06
6.36 ( 0.06
3.77 ( 0.13
9.16 ( 0.15
2.44 ( 0.16
8.66 ( 0.71
4.06 ( 0.04
7.58 ( 0.81
9.37 ( 0.24
10.63 ( 0.71
6.15 ( 0.50
4.92 ( 0.15
2.27 ( 0.12
0.00 ( 0.00
9.91 ( 0.16
7.18 ( 0.13
0.00 ( 0.00
1.13 ( 0.09
10.71 ( 0.15
4.67 ( 0.08
2.47 ( 0.08
0.52 ( 0.01
16.31 ( 0.49
5.55 ( 0.14
9.91 ( 0.80
11.14 ( 0.34
-39.54 ( 0.91
-57.89 ( 0.65
1.06 ( 0.07
-12.52 ( 0.12
8.97 ( 0.57
12.05 ( 0.67
0.54 ( 0.06
10.67 ( 0.78
7.54 ( 0.13
5.18 ( 0.2
9.99 ( 0.53
4.47 ( 0.18
11.21 ( 0.15
7.13 ( 0.14
9.67 ( 0.09
15.24 ( 0.76
11.47 ( 0.79
9.45 ( 0.18
9.17 ( 0.14
8.90 ( 0.07
0.00 ( 0.00
15.05 ( 0.28
3.15 ( 0.06
-8.84 ( 0.07
-5.56 ( 0.04
9.18 ( 0.09
7.52 ( 0.06
-12.62 ( 0.11
4.50 ( 0.41
30.32 ( 0.31
11.04 ( 0.41
15.88 ( 0.31
14.95 ( 0.67
-67.44 ( 0.73
-100 ( 0.00
-34.22 ( 0.21
-50.08 ( 0.34
-6.54 ( 0.69
2.25 ( 0.18
-5.33 ( 0.13
-3.61 ( 0.11
10.48 ( 0.78
8.62 ( 0.56
9.72 ( 0.42
6.17 ( 0.31
15.72 ( 0.67
7.79 ( 0.19
12.51 ( 0.65
22.89 ( 0.87
12.11 ( 0.82
13.44 ( 0.44
11.63 ( 0.56
16.76 ( 0.74
-8.02 ( 0.05
9.21 ( 0.07
-10.63 ( 0.21
-28.95 ( 0.79
-24.35 ( 0.27
3.17 ( 0.06
5.57 ( 0.04
-30.94 ( 0.78
10.22 ( 0.85
37.77 ( 0.12
9.03 ( 0.04
-21.47 ( 0.51
16.95 ( 0.59
-100 ( 0.00
19.03 ( 0.72
45.62 ( 0.48
11.71 ( 0.08
-25.87 ( 0.09
18.95 ( 0.45
23.12 ( 1.42
31.09 ( 0.31
-59.61 ( 0.98
-49.52 ( 0.42
25.41 ( 0.83
-57.53 ( 0.47
-100 ( 0.00
-13.54 ( 0.98
-40.74 ( 0.87
-48.31 ( 0.96
-30.35 ( 0.14
9.39 ( 0.04
10.82 ( 0.67
8.24 ( 0.15
6.67 ( 0.21
18.35 ( 0.56
11.77 ( 0.43
13.23 ( 0.76
35.26 ( 0.93
12.75 ( 0.74
14.03 ( 0.75
15.08 ( 0.78
19.04 ( 0.18
-9.03 ( 0.07
-10.54 ( 0.66
-64.06 ( 0.69
-68.77 ( 0.97
-37.31 ( 0.77
-32.51 ( 0.73
-12.16 ( 0.85
-67.95 ( 0.64
-100 ( 0.00
-55.28 ( 0.07
-70.37 ( 0.95
-76.47 ( 0.89
-47.85 ( 0.78
8.34 ( 0.15
15.77 ( 0.17
8.19 ( 0.17
14.69 ( 0.67
19.73 ( 0.51
16.41 ( 0.59
14.21 ( 0.45
67.75 ( 0.98
14.22 ( 0.97
14.96 ( 0.81
37.73 ( 0.71
22.47 ( 0.07
-11.89 ( 0.09
-43.95 ( 0.24
-73.58 ( 0.71
-86.36 ( 0.99
-34.94 ( 0.61
-46.99 ( 0.42
-23.76 ( 0.44
-83.03 ( 0.89
-100 ( 0.00
-100 ( 0.00
-100 ( 0.00
-60.44 ( 0.45
9.64 ( 0.08
21.96 ( 0.95
5.92 ( 0.12
22.03 ( 0.15
23.61 ( 0.86
23.17 ( 0.74
18.64 ( 0.32
88.05 ( 0.99
18.20 ( 0.54
9.52 ( 0.09
41.66 ( 0.96
-36.67 ( 0.99
-20.93 ( 0.41
-84.45 ( 0.62
-91.86 ( 0.41
-100 ( 0.00
-35.14 ( 0.91
-57.09 ( 0.49
-100 ( 0.00
-100 ( 0.00
a
The effect of compound was defined by the difference between the force of contraction before and after its addition to the bathing fluid
and is expressed as percentage variation with respect to the basal force of contraction. In the absence of compounds, the force of contraction
of spontaneously beating atria was 3.3 ( 0.4 mN. Each value is the mean ( SEM of 6-10 assays from 10 different experiments. Negative
values indicate a negative inotropic effect.
Schild (insert of Figure 1), from which a slope of -0.98
and pA2 of 5.62 were calculated.
Taking into consideration all the above, these results
underline the ability of 28 to displace adenosine from
its A1 cardiac receptor. To substantiate this mechanism
of action, the influence of 28 on [3H]CHA (N6-cyclohexyl-
[3H]adenosine) binding to A1 receptor in the guinea pig
heart was studied. In the concentration range used to
evoke inotropic responses, compound 28 inhibited [3H]-
CHA binding to A1 adenosine receptors in guinea pig
atria showing an IC50 of 583.30 ( 16.7 µM (data not
shown). This concentration corresponds to the concen-
tration of compound 28 suitable in inducing 50% of the
maximum positive inotropic effect reached under our
experimental conditions.
Cardiac activity of compound 28 was studied in the
presence of carbachol to exclude any participation of
cAMP in its mechanism of action. This agent, in fact,
F igu r e 2. Effect of carbachol on 28-induced inotropism in
electrically driven left atrium from reserpine-treated guinea
pigs. Each data point is the mean ( SEM of five assays from
is known to abolish selectively the increase in heart
contractility induced either by adenylate cyclase stimu-
lation or by PDE III inhibition in different
preparations.38-40 Carbachol, at a concentration (0.3
µM) which almost completely inhibited the spontaneous
contractility and the inotropic response of the atria to
isoprenaline,24 enhanced the inotropic activity of 28
(Figure 2). While the latter effect may, in part, be due
to a suppressed base line, the lack of the antagonism
by carbachol suggests that an elevation of cAMP is
unlikely to mediate the cardiac effect of the pyrimidine
derivative. According to this observation, compound 28
five different experiments. P values were calculated versus
respective control (atrium incubated without carbachol). The
statistical significance of the changes induced by carbachol was
calculated by the Student’s test. *P < 0.001; CCH ) carbachol.
significantly inhibited cardiac PDE III activity only at
the highest concentration tested (0.1-1 mM) (Figure 3).
These results confirm our previous observation that a
lack of correlation exists between inhibition of PDE III
and an increase in inotropism induced in guinea pig
atria by amrinone,15 milrinone, and milrinone
analogues.23-25 This can be explained by the fact that

Synthesis and Cardiac Activity of New Pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3675
Ta ble 3. Effect of Compounds 10-40 on the Frequency Rate of Spontaneously Beating Atria from Reserpine-Treated Guinea Pigs:
Comparison with Amrinone and Milrinonea
frequency (% variation from the control)
compd
10-6
M
3 × 10-6
M
10-5
M
3 × 10-5
M
10-4
M
3 × 10-4
M
10-3
M
amrinone
milrinone
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
-3.57 ( 0.05
0.00 ( 0.00
-5.88 ( 0.20
0.00 ( 0.00
-3.85 ( 0.14
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
-6.25 ( 0.07
-6.06 ( 0.08
-2.94 ( 0.13
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
6.45 ( 0.11
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
-3.13 ( 0.13
3.33 ( 0.15
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
-5.29 ( 0.13
-5.15 ( 0.11
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
0.00 ( 0.00
15.22 ( 0.50
3.71 ( 0.04
0.00 ( 0.00
2.85 ( 0.04
-9.37 ( 0.21
-9.37 ( 0.17
-3.51 ( 0.08
4.76 ( 0.11
0.00 ( 0.00
3.70 ( 0.17
-4.76 ( 0.06
-3.23 ( 0.08
3.12 ( 0.13
9.67 ( 0.21
0.00 ( 0.00
0.00 ( 0.00
-2.85 ( 0.15
-3.13 ( 0.13
3.33 ( 0.15
0.00 ( 0.00
0.00 ( 0.00
-2.85 ( 0.18
1.33 ( 0.14
0.00 ( 0.00
-1.33 ( 0.18
-3.03 ( 0.08
-26.31 ( 0.16
-5.15 ( 0.11
-6.66 ( 0.04
3.03 ( 0.09
0.00 ( 0.00
-3.23 ( 0.01
1.50 ( 0.50
27.09 ( 0.24
7.43 ( 0.11
6.25 ( 1.35
34.16 ( 0.25
11.11 ( 0.14
7.14 ( 0.01
7.14 ( 0.19
-100 ( 0.00
7.20 ( 1.46
42.85 ( 0.33
14.81 ( 0.08
10.71 ( 0.12
7.14 ( 0.19
9.87 ( 0.46
37.20 ( 0.85
18.51 ( 0.38
-17.85 ( 0.22
11.43 ( 0.18
3.57 ( 0.21
3.57 ( 0.07
-14.76 ( 0.53
-100 ( 0.00
-0.91 ( 0.01
-7.64 ( 0.14
-8.03 ( 0.09
7.40 ( 0.08
-2.94 ( 0.07
-100 ( 0.00
-16.06 ( 0.08
-3.70 ( 0.07
-40.77 ( 0.16
-6.66 ( 0.14
3.44 ( 0.08
3.22 ( 0.06
-6.25 ( 0.15
1.11 ( 0.01
-100 ( 0.00
-55.15 ( 0.17
-14.81 ( 0.15
-55.55 ( 0.59
-6.66 ( 0.14
3.44 ( 0.08
-100 ( 0.00
-100 ( 0.00
-100 ( 0.00
-19.35 ( 0.25
6.25 ( 0.09
-7.41 ( 0.12
-3.61 ( 0.11
3.44 ( 0.08
3.22 ( 0.06
-3.12 ( 0.12
0.00 ( 0.00
6.45 ( 0.17
-3.12 ( 0.05
-9.37 ( 0.12
-7.69 ( 0.15
-4.28 ( 0.06
-28.57 ( 0.64
11.11 ( 0.12
-10.25 ( 0.36
-6.25 ( 0.14
-14.28 ( 0.09
25.17 ( 0.96
-17.34 ( 0.29
-9.33 ( 0.11
-42.41 ( 0.72
-52.63 ( 0.65
-100 ( 0.00
-40.00 ( 0.95
-12.12 ( 0.09
-100 ( 0.00
-100 ( 0.00
-6.25 ( 0.15
-4.79 ( 0.07
-5.71 ( 0.21
-17.58 ( 0.39
7.42 ( 0.25
-2.85 ( 0.15
-10.71 ( 0.19
3.33 ( 0.15
-2.85 ( 0.15
-14.28 ( 0.23
3.71 ( 0.16
-3.85 ( 0.06
0.00 ( 0.00
-5.02 ( 0.07
-3.12 ( 0.04
-3.03( 0.04
9.72 ( 0.06
-5.69 ( 0.08
-3.12 ( 0.04
-9.09 ( 0.21
17.44 ( 0.21
-6.25 ( 0.06
4.00 ( 0.09
-28.91 ( 0.24
-52.63 ( 0.65
-41.66 ( 0.37
-30.01 ( 0.61
-9.09 ( 0.18
-13.33 ( 0.14
-32.26 ( 0.39
-3.03 ( 0.04
3.72 ( 0.06
0.00 ( 0.00
0.00 ( 0.00
-4.00 ( 0.05
-4.77 ( 0.07
-31.58 ( 0.21
-17.26 ( 0.09
-13.33 ( 0.17
3.03 ( 0.09
-4.00 ( 0.05
-14.95 ( 0.36
-52.63 ( 0.65
-41.66 ( 0.37
-16.66 ( 0.07
-3.03 ( 0.03
0.00 ( 0.00
0.00 ( 0.00
-3.68 ( 0.08
-22.58 ( 0.64
a
The effect of compound was defined by the difference between the frequency of atria before and after its addition to the bathing fluid
and is expressed as percentage variation with respect to the spontaneous frequency of the atria. In the absence of compounds, the heart
rate was 161 ( 7 beats/min. Each value is the mean ( SEM of 6-10 assays from 10 different experiments. Negative values indicate a
negative chronotropic effect.
guinea pig atria, significantly inhibited cardiac PDE III
activity (Figure 3).
The negative influence exerted by 17 on heart con-
tractility was insensitive to the presence of atropine (1
µM) in the medium (data not shown), suggesting that
an acetylcholine-like action at the specific muscarinic
receptors must be excluded. In contrast, an action on
A1 adenosine receptors might be involved. Compound
17 reduced the positive inotropic effect induced by the
adenosine antagonist 8-phenyltheophylline (30 µM-0.1
mM) (Table 6). Unfortunately, because of its low
solubility, higher concentrations of xanthine were not
tested so that the nature of the inhibition exerted by
17 remains to be defined. However, in the concentration
range suitable to evoke negative influence of the atria
contractility, 17 inhibited [3H]CHA binding to A1 ad-
enosine receptors in the guinea pig heart showing an
IC50 of 21.30 ( 1.30 µM (data not shown). From the
comparison of the EC50s, compound 17 appears to be
more potent in inhibiting [3H]CHA binding to adenosine
receptors than in reducing cardiac inotropism, but the
difference might be due to the variable permeability of
intact tissue in comparison to isolate receptor prepara-
tion. Thus, an adenosine-like action at the specific
purinergic (A1) receptor could be involved in the nega-
tive inotropic effect of 17, suggesting that it behaves as
partial agonist. If this is true, we can conclude that the
esterification of the molecule is sufficient to modify the
biological activity of the compound from antagonist to
F igu r e 3. Concentration-effect curves for inhibition of
soluble PDE III isolated from calf heart by free 17 and 28.
Each point is the mean ( SEM from four duplicate experi-
ments. PDE III activity in the absence of 17 or 28 was 0.77 (
0.01 mmol/mg of protein/min.
PDEase III, the isoenzyme that is selectively inhibited
by amrinone and milrinone, in the guinea pig heart may
be either biochemically uncoupled from myocardial
contractile proteins or compartmentalized in the cytosol
so that increases in cAMP concentration do not neces-
sarily cause increases in contractility.41,42 The lack of
correlation between PDE III inhibition and increase in
cardiac contractility is further substained by present
results showing that compound 17, at a concentration
suitable to reduce spontaneous contractile activity in the

3676 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Dorigo et al.
Ta ble 4. Inotropic Effect of 17 and 28 in Electrically Driven Left Atrium from Reserpine-Treated Guinea Pigs: Comparison with
Milrinonea
developed tension (% variation from the control)
17
28
drug conctn (M)
milrinone
10-6
0.00 ( 0.00
9.31 ( 0.17
13.25 ( 0.21
22.81 ( 0.44
35.61 ( 0.34
45.61 ( 0.40
42.89 ( 0.33
0.00 ( 0.00
4.85 ( 0.07
ns
3.23 ( 0.10
9.70 ( 0.21
16.51 ( 0.66
24.74 ( 0.44
36.30 ( 0.83
52.95 ( 2.44
78.63 ( 1.77
P < 0.02
ns
P < 0.02
P < 0.05
ns
P < 0.001
P < 0.001
3 × 10-6
P < 0.01
P < 0.01
P < 0.01
P < 0.001
P < 0.001
P < 0.001
10-5
7.45 ( 0.76
3 × 10-5
10-4
-5.21 ( 0.37
-14.21 ( 0.89
-48.34 ( 0.09
-100.00 ( 0.00
3 × 10-4
10-3
a
The effect of compound was defined by the difference between the force of contraction before and after its addition to the bathing fluid
and is expressed as percentage variation with respect to the basal force of contraction. In the absence of compounds, the force of
concentration of electrically driven left atrium was 5.5 ( 0.6 mN. Each value is the mean ( SEM of six assays from six different
experiments. P values were determined versus milrinone-treated preparations. The statistical significance of the changes induced by 17
and 28 was calculated by the Student’s t-test.
Ta ble 5. Effect of Adenosine Deaminase on Inotropic Response
for 28 (see below). In the crystallographic cell are
to 28 in Electrically Driven Left Atrium from Reserpine-
present two molecules per asymmetric unit (the second
indicated by the letter A), which present some confor-
mational differences due mainly to the reciprocal ori-
Treated Guinea Pigsa
developed tension (% increase over the control)
entation of the pyrimidine and phenyl rings. The
dihedral angle between the respective mean planes is
74.3(1)° for one molecule and 70.6(1)° for the second (A).
The COOMe group is about coplanar with the pyrimi-
dine ring in both molecules, the dihedral angle between
the respective mean planes having 1.3(1)° for one
molecule and 2.1(2)° for molecule A, with the same
cisoid conformation in both molecules (the carbonyl
oxygen O(9) is oriented in the direction of the methylene
group C(11)). The heterocyclic moieties present a range
of the atom deviations from the best mean plane
calculated for the ring from -0.015(4) to 0.012(3) Å in
one molecule and from -0.009(4) to 0.007(3) Å in A, both
rings being nearly planar. In particular, molecular
stacking is observed between the centrosymmetric
related pyrimidine rings of the A molecule with a
distance of about 3.6 Å, in the b axis direction.
+ADA (2 U/mL)
28 (M)
-ADA
3 × 10-6
11.21 ( 0.22
27.41 ( 0.31
33.61 ( 0.86
44.95 ( 0.95
66.21 ( 0.99
88.35 ( 1.21
0.00 ( 0.00
P < 0.001
P < 0.001
P < 0.001
P < 0.001
P < 0.001
P < 0.001
10-5
4.76 ( 0.09
9.52 ( 0.08
19.05 ( 0.18
33.33 ( 0.21
57.24 ( 0.32
3 × 10-5
10-4
3 × 10-4
10-3
a
The effect of 28 was defined by the difference in force of
contraction before and after its addition to the bathing fluid and
is expressed as percentage variation with respect to the basal force
of contraction. In the experiments performed in the presence of
ADA, the basal force of concentration was that registered after
the addition of ADA itself. Each value is the mean ( SEM of six
assays from six different experiments. P values were determined
versus control (atrium incubated without adenosine deaminase).
The statistical significance of the changes induced by adenosine
deaminase was calculated by Student’s t-test. ADA ) adenosine
deaminase.
agonist, probably in view of a higher lipophilicity of the
ester, which could permit a different interaction with
the binding site within the receptor cavity.43
The present results indicate that compounds contain-
ing a pyrimidine ring behave as effective inotropic
agents, whose cardiac effects are not related to PDE III
inhibition. However, the search for a pure adenosine
antagonist, not affecting the cyclic AMP system, re-
mains to be pursued.
The N(3)-C(4)-C(11)-C(12) (τ2) torsion angle is
-92.2(4)° (-90.1(4)° in A), and C(4)-C(11)-C(12)-C(13)
(τ3) is -67.1(4)° (69.7(4)° in A). However, the intramo-
lecular contact distance between H(13) and O(9) is the
same (2.60(1) Å) in both molecules as well the H(110)‚‚‚O-
(9) contact of 2.30(1) and 2.28(1) Å in A. All of the other
corresponding bond distances and angles are equal in
both molecules. In compound 28, which differs from 17
by having the acid group unsubstituted, the molecular
conformation is different. An ORTEP view with the
atom-numbering scheme used is reported in Figure 5.
The proton on O(10) forms a strong hydrogen bond
interaction with the molecule centrosymmetric related
O(10)‚‚‚O(9)′ (2.631(2) Å), O(10)-H‚‚‚O(9)′ (1.815(2) Å),
and O(10)-H‚‚‚O(9)′ (173.5(2)°) (prime at -x,-y,-z)
Cr ysta llogr a p h ic Stu d ies
The molecular structures 17 are shown in Figure 4
as an ORTEP view with the atom-numbering scheme
used. Significant bond distances and angles are re-
ported in Table 7 together with the corresponding value
Ta ble 6. Effect of Compound 17 on Inotropic Response to 8-Phenyltheophylline in Electrically Driven Left Atrium from
Reserpine-Treated Guinea Pigsa
developed tension (% variation from the control)
3 × 10-5 M 17
10-4 M 17
3 × 10-4 M 17
8-Ph (M)
-
-
-
-6.66 ( 0.15
-9.37 ( 0.75
-33.52 ( 2.15
3 × 10-6
11.86 ( 0.97
14.08 ( 1.04
19.15 ( 0.75
29.87 ( 1.17
8.79 ( 0.25
10.96 ( 0.89
14.88 ( 0.78
20.22 ( 1.25
P < 0.05
P < 0.05
P < 0.05
P < 0.05
-4.34 ( 0.18
-3.33 ( 0.14
2.72 ( 1.15
P < 0.001
P < 0.001
P < 0.001
P < 0.05
-28.86 ( 1.17
-22.17 ( 1.08
-11.03 ( 1.06
-6.66 ( 0.89
P < 0.001
P < 0.001
P < 0.001
P < 0.001
10-5
3 × 10-5
10-4
19.02 ( 1.28
a
Cumulative concentration-effect curves for 8-phenyltheophylline were obtained in the absence and presence of different concentrations
of compound 17. The inotropic effect was determined as indicated under Table 4. Each data point is the mean ( SEM of four assays
from four different experiments. P values were determined versus control (atrium incubated with 8-Ph). The statistical significance of
the changes induced by compound 17 was calculated by Student’s t-test. 8-Ph ) 8-phenyltheophylline.

Synthesis and Cardiac Activity of New Pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3677
Ta ble 7. Sected Bond Lengths (Å) and Angles (Deg)
17
molecule 1
1A
28
N(1)-C(6)
N(1)-C(2)
N(3)-C(4)
N(3)-C(2)
N(7)-C(2)
N(7)-C(18)
N(7)-C(19)
O(9)-C(8)
O(10)-C(8)
O(10)-C(20)
C(4)-C(5)
C(4)-C(11)
C(5)-C(6)
C(5)-C(8)
C(11)-C(12)
C(12)-C(17)
C(12)-C(13)
C(17)-C(16)
C(16)-C(15)
C(15)-C(14)
C(14)-C(13)
1.314(5)
1.349(5)
1.336(5)
1.353(5)
1.342(5)
1.452(5)
1.455(5)
1.200(5)
1.327(5)
1.430(5)
1.404(5)
1.503(5)
1.396(5)
1.472(5)
1.518(5)
1.382(5)
1.379(5)
1.385(5)
1.370(6)
1.378(6)
1.390(5)
1.315(5)
1.350(5)
1.331(5)
1.354(5)
1.343(5)
1.442(5)
1.459(5)
1.196(5)
1.340(5)
1.426(5)
1.405(5)
1.501(5)
1.385(5)
1.457(5)
1.520(5)
1.383(5)
1.377(5)
1.379(5)
1.371(6)
1.368(6)
1.380(5)
1.321(3)
1.354(2)
1.333(3)
1.351(3)
1.341(3)
1.445(3)
1.455(3)
1.229(2)
1.309(2)
1.403(3)
1.501(3)
1.391(3)
1.457(3)
1.510(3)
1.379(3)
1.381(4)
1.384(5)
1.360(5)
1.358(5)
1.387(5)
N(3)-C(4)-C(11)
N(3)-C(4)-C(5)
N(3)-C(2)-N(7)
C(4)-N(3)-C(2)
N(1)-C(6)-C(5)
N(1)-C(2)-N(7)
N(1)-C(2)-N(3)
C(4)-C(5)-C(8)
C(4)-C(5)-C(6)
C(4)-C(11)-C(12)
C(5)-C(4)-C(11)
C(6)-C(5)-C(8)
C(6)-N(1)-C(2)
C(2)-N(7)-C(19)
C(2)-N(7)-C(18)
C(18)-N(7)-C(19)
C(8)-O(10)-C(20)
O(10)-C(8)-C(5)
O(9)-C(8)-C(5)
O(9)-C(8)-O(10)
C(11)-C(12)-C(13)
C(11)-C(12)-C(17)
114.8(3)
114.6(3)
121.2(3)
117.5(4)
117.7(3)
126.0(4)
116.8(3)
125.7(4)
124.0(3)
115.1(3)
112.3(3)
124.2(3)
120.9(3)
114.4(3)
121.1(3)
121.1(3)
117.8(3)
116.6(3)
127.5(4)
112.1(3)
120.4(4)
121.6(3)
120.1(3)
114.7(2)
120.5(2)
117.5(2)
117.9(2)
124.3(3)
116.8(2)
125.8(2)
124.0(2)
116.5(2)
115.6(2)
124.8(2)
119.5(3)
115.0(2)
120.5(2)
121.3(2)
118.2(2)
121.0(3)
117.4(3)
117.1(3)
125.1(4)
116.1(4)
126.5(3)
124.4(3)
115.8(3)
111.7(3)
124.1(3)
119.8(3)
114.5(4)
122.0(3)
120.6(3)
117.4(3)
115.6(3)
112.2(3)
125.7(4)
122.1(4)
121.9(3)
119.8(3)
121.8(2)
114.2(2)
121.8(2)
123.6(2)
119.0(2)
parallel, separated by about 3.4 Å, and shorter than in
the methyl-substituted 17.
Molecu la r Mod elin g Stu d y
In the present study the following procedure has been
used. (a) The structures reported in Figure 6 were built
from X-ray data when available (17 and 28) or using a
general 3D-builder.50 (b) The Monte Carlo-Metropolis
algorithm as implemented in MAD50 was used to
analyze the rotational space and obtain a conforma-
tional minimum which was further optimized using the
AM1 Hamiltonian51 as implemented in MOPAC.52 (c)
Starting from the AM1-optimized conformations, the
dihedral angles τ1, τ2, and τ3 were rotated from 0° to
360° by a 10° increment while relaxing all the other
geometrical parameters. AM1 was used to evaluate
energies and charge distributions. The results sum-
marized in Tables 9 and 10 illustrate some molecular
features of compounds listed in Figure 6. The prevously
reported positive inotropic agent 7,8-dihydro-7-methyl-
2,5-(1H,6H)-quinolinedione (SF 40)24 has been included
as a reference compound.
F igu r e 4. Molecular conformation of the two asymmetric
units of 17 (thermal ellipsoids are drawn at the 40% prob-
ability level).
which indicates dimer formation. The τ2 and τ3 torsion
angles are substantially changed with respect to the
corresponding methyl ester with values for τ2 of 101.4-
(2)° and for τ3 of 164.5(2)°. The dihedral angle between
the mean planes calculated on the pyrimidine and
phenyl rings is 89.15(7)°, while the acid group remains
coplanar with the pyrimidine moiety with the same
cisoid conformation as in 17. The H(110)‚‚‚O(9) contact
of 2.38(1) Å is longer than in 28, and no significant
contacts are present between the phenyl and the
heterocyclic moiety due to the orthogonal position of the
phenyl with respect to the heterocyclic moiety, which
is practically planar also in this molecule (the range of
deviations is -0.008(2) f 0.008(2) Å). Bond distances
and angles are in agreement with those of 17. The
molecular packing is again characterized by molecular
stacking with the centrosymmetric pyrimidine rings
In the most stable conformations of the series 11-30
(Figure 6, Table 9), when R ) methyl, benzyl, trifluo-
romethyl, the COR1 group (R1 ) H, C2H5) is coplanar

3678 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Dorigo et al.
F igu r e 6. Chemical structures of the compounds studied by
quantum chemical methods.
prevents a coplanar conformation so that the COOR1
group is rotated ≈60° and ≈-84°, respectively, from the
pyrimidine ring. On the basis of charge distribution
analysis (Table 10), three sets of compounds can be
considered. The first set includes 22, 23, 27, 28, and
30 and the reference compound SF 40. This set is
characterized by two areas with high electron density,
at 4-6 Å distance, illustrated as areas A and B in Figure
7a for compounds SF 40 and 28. Both areas include a
variable number (from one to three) of heteroatoms,
which can function simply as electron rich centers (also
with metal-complexing properties) (Lewis bases) or as
hydrogen bond acceptors (Bro¨nsted bases). Area B is
represented by the aminic or the pyrimidinic nitrogens
in 22-30 and by the carbonyl oxygen at C(8) in SF 40.
Area B is represented by the amidic oxygen of the
pyridone ring in SF 40 and by the carbonyl oxygen of
the carboalkoxy residue in 22-30. In addition to areas
A and B, a third area, herein defined as area C, consists
of a hydrogen donor moiety (Bro¨nsted acid) adjacent to
area B, the NH of the pyridone ring in SF 40, and the
OH of the carboxy group in 22-30.
F igu r e 5. Molecular conformation of 28 (thermal ellipsoids
are drawn at the 40% probability level).
Ta ble 8. Experimental Data for the Crystallographic Analyses
17
28
formula
mol wt
C15H17N3O2
271.3
C14H15N3O2
257.3
crystal size, mm 0.22 × 0.18 × 0.16
0.24 × 0.20 × 0.32
triclinic
P1h
crystal system
space group
a, Å
monoclinic
P21/c
9.235(3)
14.989(3)
20.481(4)
6.258(2)
9.023(2)
11.901(3)
104.09(2)
98.89(2)
91.13(2)
642.8(3)
2
b, Å
c, Å
R, deg
â, deg
91.14(3)
γ, deg
V, Å3
2835(1)
Z
8
Dc, g cm-3
F(000)
1.272
1152
1.329
272
2θ range, deg
radiation (λ, Å)
µ, cm-1
4-46
4-50
Mo KR (0.71069)
Mo KR (0.71069)
0.92
0.87
3802
no. reflections
collected
no. observed
[I g 2.5σ(I)]
weighting
scheme w
R(F)
1876
The second set includes compounds 11, 16, 17, 20, and
40. It is characterized by areas A and B only. The
former is represented by the aminic or pyrimidinic
nitrogens, the latter by the fluorine atoms in 20 and 40
or the carboalkoxy residue in 11, 16, and 20. In these
compounds the hydrogen donor moiety (area C) is
missing.
1695
1771
[σ2(Fo2) + (0.0902P*)2 [σ2(Fo2) + (0.0938P*)2
+ 2.10P]-1 + 0.17P]-1
0.037 0.049
0.101
0.86
Rw(F2)
goodness of fit
0.140
1.05
a
2
P ) max(Fo + 2Fc2)/3.
The third set includes compounds 32, 37, and 38.
Only area A is present, which can be further divided in
three subareas, at ≈2.5 Å distance, which can function
as hydrogen bond acceptors.
or nearly coplanar with respect to the C(4)-C(5) bond.
A cisoid conformation is preferred in 17 (R ) benzyl,
R1 ) methyl), 22 (R ) methyl, R1 ) H), 23 (R )
methoxymethyl, R1 ) H), and 28 (R ) benzyl, R1) H),
whereas a transoid one is more stable in 20, 30 and, to
a very light extent, 11. In all cases, however, the energy
difference between the cisoid and transoid conforma-
tions is <1.8 kcal/mol and the COOR1 rotational barrier
between τ1 ) 0° and τ2 ) 180° ranges from 1.8 (20) to
4.19 kcal/mol (23). It is therefore reasonable to assume
that, in solution, sufficient energy can be found to
accommodate the COOR1 group in a more convenient
conformation for the binding to the enzyme. In com-
pounds 16 and 17, the bulky tert-butyl substituent
A further differentiation for compound listed in Figure
6 is represented by the steric and electronic features of
the substituent (R) at position 4. Its nature can produce
different effects: (a) merely prevent a nearly coplanar
conformation of the COR1 residue and the pyrimidine
ring (as in 16 and 27), (b) create a lipophilic area, (c)
introduce an additional area with high electron density
which can act as a Lewis or Bro¨nsted base (as in 23
and 30), and (d) reduce the access to a certain part of
the molecule, in particular to area C (as indicated by
D1, R1 values in Table 9). (A limited access to the NH

Synthesis and Cardiac Activity of New Pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3679
Ta ble 9. Energy and Relevant Geometric Parameters for AM1 Selected Conformations of 11-30 and Reference Compound SF 40
∆Eb
(kcal/mol)
R1
d
a
a
a
d
compd
τ1 (deg)
τ2 (deg)
τ3 (deg)
Dc (Å)
Rd (deg)
D1c (Å)
(deg)
D2c (Å)
R2 (deg)
11
180.00
0.00
60.01
180.00
0.00
19.10
180.00
180.00
0.00
0.01
180.00
0.00
180.00
-84.82
180.00
0.00
0.00
0.13
0.00
3.99
2.10
0.00
0.92
0.00
1.65
0.00
0.69
0.00
0.97
0.00
7.40
6.00
0.00
1.70
0.00
0.11
1.85
2.43
2.44
2.74
2.70
2.70
3.32
3.32
2.82
2.82
2.45
2.43
3.24
3.18
2.74
2.72
2.64
3.36
3.35
2.78
2.76
2.83
3.41
86.31
93.19
51.96
49.35
49.31
49.68
55.71
63.48
63.48
93.23
93.23
47.20
41.95
51.41
57.80
48.73
62.67
58.88
57.45
56.68
63.93
54.47
16
17
20
22
23
27
-87.16
-72.80
76.98
-93.90
2.66
4.31
4.28
3.97
3.29
3.29
6.05
5.98
4.46
2.72
2.70
4.52
158.73
113.49
112.78
150.80
75.24
41.50
102.60
93.31
115.10
149.34
156.20
116.46
4.28
2.80
2.75
6.54
3.25
5.92
2.97
4.42
4.88
4.27
4.34
3.09
176.3
71.12
70.13
172.4
77.29
16.29
60.43
54.94
131.3
162.3
175.7
75.73
28
30
0.06
86.75
82.75
105.92
100.01
180.00
160.00
180.00
0.00
SF 40
177.00
a
The τ1, τ2, and τ3 torsion angles are defined as follows: τ1 ) O(9)-C(8)-C(5)-C(4); τ2 ) C(12)-C(11)-C(4)-N(3); τ3 ) C(13)-C(12)-
C(11)-C(4). ∆E is calculated with respect to the minimum energy conformation. c D, D1, and D2 are defined as depicted in Figure 6 and
b
d
are the distances corresponding to the minimum value of the R, R1, and R2 angles, respectively. The angles R, R1, and R2 are defined as
depicted in Figure 6 and are calculated with respect to each heavy atom of the R substituent.
Ta ble 10. Atomic Charges for Selected Atoms of 11-30 and
Reference Compound SF 40
compd
N(1)
N(3)
N(7)
O(9)
O(10)
Fa
11
-0.25
-0.25
-0.25
-0.26
-0.26
-0.25
-0.25
-0.25
-0.21
-0.22
-0.24
-0.24
-0.23
-0.23
-0.23
-0.24
-0.23
-0.22
-0.22
-0.22
-0.15
-0.22
-0.14
-0.12
-0.24
-0.24
-0.23
-0.23
-0.24
-0.24
-0.23
-0.24
-0.22
-0.22
-0.25
-0.25
-0.25
-0.25
-0.26
-0.25
-0.25
-0.21
-0.21
-0.22
-0.19
-0.20
-0.19
-0.14
0.28
-0.24
-0.25
-0.25
-0.25
-0.25
-0.25
-0.25
-0.25
-0.23
-0.23
-0.24
-0.24
-0.25
-0.24
-0.24
-0.24
-0.24
-0.24
-0.24
-0.24
-0.21
-0.24
-0.22
-0.21
-0.28
-0.29
-0.28
-0.27
-0.30
-0.29
-0.28
-0.29
-0.29
-0.29
-0.33
-0.32
-0.29
-0.29
-0.30
-0.33
-0.32
-0.31
-0.31
-0.33
-0.37
-0.37
-0.35
-0.38
-0.36
-0.37
-0.37
-0.37
-0.33
-0.33
-0.38
-0.38
-0.29
-0.32
-0.31
-0.38
-0.38
-0.36
-0.37
-0.34
16
17
20
22
27
-0.15
-0.15
28
30
-0.15
-0.15
-0.15
32
37
38
40
-0.15
SF 40
-0.30
-0.35
a
Average value.
F igu r e 7. (a) Overlay (manual) of the lowest energy confor-
mation of SF 40 (gray lines) with the transoid conformation
(τ1 ) 180°) of compound 28. (b) Overlay (manual) of the lowest
energy conformation of SF 40 (gray lines) with the cisoid
conformation (τ1 ) 0.06°) of compound 28.
moiety in 2-benzyl-R-pyridone derivatives was consid-
ered to depress the positive inotropic activity.24) This
obviously occurs when the R steric requirements are too
demanding.
Analysis of the compounds listed in Figure 6 reveals
that the first and the second of the requirements
proposed above for positive inotropic activity are fulfilled
in compounds 22, 23, 27, 28, and 30. Among these
compounds, however, 27 does not fulfill the third
requirement. Therefore, it should be expected to be the
least active. Furthermore, the R residue in 23 and 30
contains from one to three electron rich heteroatoms.
This could be at variance with the requirement of a
substituent which must fit to a lipophilic pocket (point
A previously suggested model for the positive inotro-
pic activity of SF 40 and related compounds was based
on the following requirements: (a) the presence of a
dipole and a nearby weakly acidic proton donor (the
HNCO group) (herein defined as areas B and C), (b) the
presence of a hydrogen bond acceptor represented by
the carbonyl oxygen (O(9), herein defined as area A),
(c) an overall planar topography containing areas A-C,
and (d) an alkyl substituent at C(2) which could fit to a
small lipophilic pocket.

3680 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Dorigo et al.
4) and could therefore depress the positive inotropic
activity.
The analysis of the distance between heteroatoms and
of their charge (sign and magnitude) suggests a reason-
able fitting of the pyrimidinic derivatives 22 and 28 to
the model proposed for SF 40. Possible overlays of the
lowest energy conformation of SF 40 with selected
conformations of 28 are illustrated in Figures 7 and 8.
In Figure 7a a rigid manual overlay of the minimum
energy conformation of SF 40 (Table 9) and the transoid
conformation of 28 (Table 9) is reported.53 The R-pyri-
done ring of SF 40 and the pyrimidinic ring of 28 are
kept on parallel planes; 28 has been moved as a rigid
group on top of SF 40 and oriented in such a way as to
bring areas A-C as near as possible. In Figure 7b
overlay of the minimum energy conformation of SF 40
and the cisoid conformation of 28 (Table 9) is reported.
In Figure 7, the areas A-C of the two compounds do
not fit exactly but are somewhat displaced. This,
however, would imply only a slight different conforma-
tion of enzyme groups which define the active site. In
Figure 7a, the benzyl group is directed toward the same
portion of space which accommodates the methylcyclo-
hexane residue of SF 40 but has, however, a greater
steric hindrance below the methylcyclohexane plane. A
lipophilic area with bulk tolerance should, therefore, be
expected in this part of the receptor.
The benzyl group is two carbons displaced from the
hydroxyl residue of the COOH group (area C) and does
not limit the access to it. In the more stable cisoid
conformation (Figure 7b), two lipophilic pockets should
be invoked on each side of the HXCO (X ) O, N) dipolar
moiety: one to accommodate the R substituent in SF 40
and related compounds, the other to accommodate the
benzylic group of 28. In Figure 8 alternate, computer-
generated54 alignments of the heteroatoms have been
represented. These, however, do not provide the same
level of match as that shown in Figure 7.
F igu r e 8. Computer-generated fit (MAD) of SF 40 lowest
energy conformation (gray lines) and cisoid conformation of
28 (black lines). (a) N(1), C(6), C(5), and C(4) atoms for SF 40
and O(10), C(8), C(5), and C(6) atoms for 28 were included in
the fitting procedure. (b) N(1), C(2), C(3), and C(8) atoms for
SF 40 and O(10), C(8), C(5), and C(4) atoms for 28 were
included in the fitting procedure.
pyrimidines 32-40 displayed generally negative ino-
tropic activity. The pyrimidine 31 showed a moderate
positive inotropic activity but only at high concentra-
tions.
Ester 17 and its related acid 28 (respectively negative
and positive inotropes), were selected for the study of
mechanism of action. An antagonism toward endog-
enous adenosine without variations in the cellular cyclic
AMP seems to be involved in the contractile activity of
the acid 28. The corresponding methyl ester 17, which
reduced both the contractile force and the frequency rate
of the atria, could be a partial agonist at the purinergic
inhibitor A1 receptor in the heart.
Crystallographic studies on 17 and 28 and molecular
modeling investigations performed on selected com-
pounds (Figure 6) added support to the model previously
proposed for positive inotropic activity founded on the
following requirements: (a) the presence of a dipole
(area B) and a nearby acidic proton donor (area C), the
COOH group, (b) the presence of a hydrogen bond
acceptor (area A), the pyrimidinic or aminic hydrogens,
and (c) an overall planar topography of areas A-C.
In these cases only two of the three different areas
(A-C) suggested above for positive inotropic activity fit.
In Figure 8a, areas B and C fit but not area A, which is
displaced by ≈2.81 Å. In Figure 8b there is a perfect
fit of one of the pyrimidine nitrogens in 28 with the
oxygen at C(8) in SF 40 (area A) and the OH of the
carboxy residue in 28 with the NH in SF 40 (area C).
However, area A does not overlay but is symmetrically
located with respect to the hydrogen bond donor (OH
in 28 or NH in SF 40). In addition the CO residue of
the COOH group in 28 would point where a lipophilic
pocket has been invoked for SF 40, a further feature
which disfavors this situation with respect to the other
overlays illustrated in Figures 7 and 8.
Con clu sion
A series of novel 2-(dimethylamino)-5-pyrımidines
variously functionalized at positions 4 and 5 was
synthesized. The synthesis took advantage of the use
of unsymmetrical 2-[(dimethylamino)methylene]1,3-di-
ones to obtain the pyrimidine derivatives through a
convenient way.
Cardiac effects of acids 21-30 tested on spontane-
ously beating and electrically driven atria from reser-
pine-treated guinea pigs showed the most significant
positive inotropic activity for 28 which was greater than
that of milrinone. In contrast, the esters 10-20 and
Among the examined compounds, only 22 and 28
fulfill the above stated requirements. The positive
inotropic activity of 28, higher than that of 22 but
comparable to that of SF 40, suggests the importance
of a properly sized lipophilic group like a phenyl (28) or
the substituted cyclohexane moiety (SF 40) to fit lipo-
philic pocket with bulk tolerance in the receptor site.
Previous studies27 indicate the presence of another small
pocket in the proximity of area B. The two lipophilic
areas would be located on the opposite sites with respect
to the pyrimidine 28 or the pyridone ring SF 40. The

Synthesis and Cardiac Activity of New Pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3681
with water (50 mL). The solution was washed with diethyl
ether and acidified with 6 N HCl (pH ∼1), and the white solid
which separated was filtered, washed with water, dried in a
vacuum oven at 100 °C, and purified by recrystallization from
a suitable solvent. The hydrolysis of ester 19 with the above
procedure gave a mixture of dicarboxylic and monocarboxylic
derivatives which proved difficult to separate.
methylated ester 17 in relation to the similarities with
the acid derivative 28 is missing the C area and the acid
proton which is responsible for hydrogen bond interac-
tions. This change could direct the molecule to a
different receptor site or simply produce a different
mode of binding, with respect to the positive inotropic
one, within the receptor cavity.
Compound 28 displayed the following spectral data: 1H-
NMR (DMSO-d6): δ 3.17 [s, 6 H, (CH3)2N], 4.41 (s, 2 H, CH2),
7.29 (s, 5 H, ar, C6H5), 8.81 (s, 1 H , ar, H-6), 12.70 (br s, 1 H,
CO2H, exchangeable proton); IR (KBr) 2900-2500, 1672, 1586,
Exp er im en ta l Section
1555, 1398, cm-1
.
Ch em istr y. All melting points were determined on a
Fisher-J ohns melting point apparatus and are uncorrected.
Microanalyses were performed with a Carlo Erba elemental
analyzer (model 1106) for C, H, N, and the results are within
(0.4% of theoretical values. The IR spectra were recorded
with a Perkin-Elmer 398 spectrophotometer and are expressed
in cm-1. The 1H-NMR spectra were obtained with a Hitachi
Perkin-Elmer R-600 instrument (60 MHz); chemical shifts are
reported in part per million (ppm) on the δ scale downfield
relative to tetramethylsilane (TMS) as internal standard;
coupling constants (J ) are reported in hertz (Hz). The follow-
ing abbreviations are used, s ) singlet, d ) doublet, t ) triplet,
q ) quartet, m ) multiplet, br ) broad, and ar ) aromatic
protons. The physical data of the newly synthesized com-
pounds are shown in Table 1.
Ma ter ia ls. Besides the commercially available starting
materials, the following products were prepared according to
reported methods: ethyl 2,2-diformylacetate31 and 2-(ethoxym-
ethylene)-4,4,4-trifluoro-3-oxobutanoate.34 The compounds used
in the biological experiments (and their sources of supply) were
as follows: milrinone (Sterling Winthrop), amrinone (Schiap-
parelli), adenosine deaminase, carbachol, propranolol, pra-
zosine, pyrilamine, ranitidine, 8-phenyltheophylline, tyramine,
cAMP, cGMP, DOWEX 1 × 2, DEAE cellulose, 5′-nucleotidase
from Crotalus Atrox (Sigma), (R)-PIA (RBI), [3H]CHA (NEN
Research Products), Instagel (Packard), and 8-[3H]cAMP (Am-
ersham). All the other chemicals and reagents were of analytic
grade.
Gen er a l P r oced u r e for N,N-Dim et h yl-2-p yr im id in -
a m in e (31) a n d 4-Su bstitu ted N,N-Dim eth yl-2-p yr im id i-
n a m in es 32-40. Acids 21-29 and 30 (about 10 mmol) were
decarboxylated by heating for a certain time at temperatures
above their melting points (Table 1) until evolution of carbon
dioxide subsided. The products were purified by distillation
in vacuo or recrystallization from a suitable solvent. Com-
pound 33 displayed the following spectral data: 1H-NMR
(CHCl3) δ 3.17 [s, 6 H, (CH3)2N], 3.45 (s, 3 H, CH3O), 4.35 (s,
2 H, CH2), 6.70 (d, J ) 5, 1 H, ar, H-5), 8.32 (d, J ) 5, 1 H, ar,
H-6); IR (CHCl3) 1583, 1560, 1407, cm-1
.
P h a r m a cology. Isola ted Atr ia P r ep a r a tion . Reserpine-
treated male guinea pigs (300-500 g) were killed by a blow to
the head followed by exanguination, and the atria were
separated from the ventricles and suspended vertically in a
bath containing 30 mL of physiologic salt solution of the
following composition (mM): NaCl, 120; KCl, 2.7; MgCl2, 0.09;
NaH2PO4 0.4; CaCl2, 1.37; NaHCO3, 11.9; and glucose, 5.5. The
solution was maintained at 29 °C and treated vigorously with
a mixture of 95% O2 and 5% CO2 which produced a pH of 7.5.
The resting tension was adjusted at 10 g, and the developed
tension was recorded isometrically by means of high-sensitivity
transducers (Basile type DYO for isolated auricles) and
registered by a writing oscillograph (Basile, Unirecord System,
model 7050). The basal developed tension ranged from 4.8 to
5.3 mN. Where indicated, the left atrium was mounted on
punctate electrodes with a load of 0.5 g and stimulated at a
frequency of 1.5 Hz by square-wave electrical pulse of 3 ms
duration and a voltage 10-20% greater than the threshold
value by a Grass Stimulator (model 24KR). The devel oped
tension was 3.28 ( 0.5 mN. The electrical stimulation was
performed in order to eliminate any influence on contractile
activity due to variations in frequency.
In otr op ic Activity. The experiments were performed on
spontaneously beating atria or an electrically driven left
atrium obtained from reserpine-treated guinea pigs. Reser-
pine (2 mg/kg, ip) was given 48 and 24 h before the animals
were killed in order to eliminate the influence of noradrenaline
which might be released from sympathetic nerve terminals.44
Noradrenaline depletion was determined by exposing isolated
atria to a single dose of tyramine (2 µg/mL) before starting
the experiments. Experiments were performed only in prepa-
rations not responding to tyramine. The drugs were added to
the perfusion fluid after 90 min of equilibration. All the
inotropic agents (amrinone, milrinone, and newly synthesized
compounds) were added cumulatively, and the inotropic effect
was recorded for 5 min before adding a higher concentration.
Where indicated, carbachol, propranolol, prazosine, pyril-
amine, and ranitidine were added to the perfusion medium
15 min before the inotropic agents. In the presence of
adenosine deaminase, the preincubation lasted 20 min. The
basal and stimulated contractile activity was determined by
measuring the amplitude of the developed tension. The data
are expressed as percent variation from control (atria incu-
bated without drugs). Compounds 17 and 28 were dissolved
in dimethyl sulfoxide (DMSO). The final concentration of
DMSO in the medium did not influence the basal activity of
the atrial preparation.
Gen er a l P r oced u r e for Eth yl 2-(Dim eth yla m in o)-5-
p yr im id in eca r boxyla te (10) a n d Ester s of 4-Su bstitu ted
2-(Dim eth yla m in o)-5-p yr im id in eca r boxylic Acid s 11-20.
1,1-Dimethylguanidine sulfate (1.4 g, 5 mmol) at room tem-
perature was added to a solution of sodium ethoxide or
methoxide (in the case of 12, 17) in ethanol or methanol,
prepared from sodium (0.23 g, 10 mmol) and anhydrous
ethanol or methanol, respectively (20 mL), followed by a
solution of ethyl 2,2-diformylacetate (10; 1.44 g, 10 mmol),
ethyl or methyl 2-[(dimethylamino)methylene]-3-oxoalcanoates
1-9 (11-19; 10 mmol), or 2-(ethoxymethylene)-4,4,4-trifluoro-
3-oxobutanoate (20; 2.40 g, 10 mmol). The mixture was boiled
for 1 (11-20) or 24 (10) h and evaporated under reduced
pressure. The residue was treated with water (100 mL). In
the case of 10, 12, and 17, a crystalline precipitate separated
and was filtered and recrystallized from a suitable solvent. In
all other cases, the resulting mixture was extracted thoroughly
with diethyl ether. The extracts were dried (MgSO4) and
evaporated to give a residue which was recrystallized from a
suitable solvent or purified by bulb-to-bulb distillation in
vacuo. Compounds 17 and 19 displayed the following spectral
data.
17: 1H-NMR (CHCl3) δ 3.20 [s, 6 H, (CH3)2N], 3.81 (s, 3 H,
CH3O), 4.41 (s, 2 H, CH2), 7.27 (m, 5 H, ar, C6H5), 8.84 (s, 1 H,
ar, H-6); IR (CHCl3) 1707, 1587, 1555, 1408 cm-1
.
19: 1H-NMR (CHCl3) δ 1.33 (t, J ) 7.2, 3 H, CH3), 1.38 (t,
J ) 7.2, 3 H, CH3), 3.25 [s, 6 H, (CH3)2N], 4.32 (q, J ) 7.2, 2
H, CH2), 4.44 (q, J ) 7.2, 2 H, CH2), 8.89 (s, 1 H, ar, H-6); IR
(CHCl3) 1736, 1707, 1586, 1564, 1410 cm-1
.
Gen er a l P r oced u r e for 2-(Dim eth yla m in o)-5-p yr im -
id in eca r boxylic Acid (21) a n d 4-Su bstitu ted 2-(Dim eth y-
la m in o)-5-p yr im id in eca r boxylic Acid s 22-30. Potassium
hydroxide (1.68 g, 30 mmol) dissolved in methanol or 95%
ethanol (20 mL) was added to a solution of the corresponding
ester 10 or 11-20 (10 mmol) in the same solvent (30 mL). The
resulting solution was boiled for 5 h, the solvent was evapo-
rated under reduced pressure, and the residue was dissolved
P r ep a r a tion of Solu ble P DE III fr om Ca lf Hea r t. PDE
III was isolated from calf hearts using the stepwise isolation
procedure described by Thompson et al.45 For each prepara-
tion, 10-20 g of bovine heart was placed in 10 vol of ice-cold
water and then minced and homogenized using a kinematic
PT-300 Polytron instrument with dispersion-aggregated PT-

3682 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Dorigo et al.
DA 3020/2P (three bursts of 10 s duration at 10 peripheral
speed) (m/s). The homogenate was then sonicated (30 s/mL
of homogenate) and centrifuged at 30000g for 20 min. The
resulting supernatant fraction was filtered through four layers
of gauze and applied to a DEAE cellulose column (30 × 3 cm)
prepared by cycling with 0.25 N NaOH, 0.25 N HCl, and 0.25
N NaOH and equilibrated with freshly prepared 70 mM
sodium-acetate and 5 mM 2-mercaptoethanol (pH 6.5). The
column was then washed with 2 bed vol of sodium acetate-
mercaptoethanol, after which the PDEs were eluted from the
column with step gradients of 220-350 and 700 mM sodium
acetate and 5 mM mercaptoethanol (pH 6.5), collecting frac-
tions of 7 mL each. Flow rate was 0.5-1 mL/min. The eluted
fractions containing proteins were pooled, dialyzed overnight
against 200 vol of ice-cold water, and assayed for protein
content according to Lowry et al.46 and for PDE activity. The
fraction eluting with 700 mM sodium acetate contained PDE
III. The PDE III from calf heart had Km for cyclic AMP of
0.67 ( 0.40 µM and Vmax of 0.73 ( 0.07 nmol/mg of protein/
min, respectively. When assayed at 0.4 µM cyclic AMP, the
activity was inhibited by 67 ( 7% by 4 µM cyclic GMP. No
significant changes in PDE III activity were observed with
rolipram. Aliquots of the fraction were iced and stored at -40
°C for several weeks without loss of activity.
Because of the low absorption coefficients, no absorption
correction was applied to the intensity data. The structures
were solved by using direct methods.55 Refinement was carried
out by full-matrix least-squares; the function minimized was
2
∑w(Fo - Fc2)2. All non-hydrogen atoms were refined with
anisotropic thermal parameters. The H atoms were placed
in calculated positions with fixed, isotropic thermal parameters
(1.2 Uequiv of the parent carbon atom). Structure refinement
was carried out with the SHELXL-9356 program and the
scattering factors enclosed therein; the drawings were pro-
duced with ORTEP II .57
Molecu la r Mod elin g Stu d ies. The structural modeling
was performed by using the semiempirical computer program
AM151 as implemented in the MOPAC52 package. The MAD50
molecular modeling system was used for molecular graphics
studies. Both quantum chemical and molecular graphics
investigations were performed on a IBM Risc System/6000
(model 520).
Ack n ow led gm en t. Financial support by Consiglio
Nazionale delle Ricerche (CNR-Roma), through Progetto
Finalizzato Chimica Fine II CNR-Milano, and MURST
(40%) are gratefully acknowledged.
P DE Activity Assa y. PDE activity was measured as
described by Thompson et al.47 in 0.4 mL of medium containing
40 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM EGTA-HCl (pH
8.0), 500 µg of bovine serum albumine, and 0.4 µM [3H]cyclic
AMP. Incubation time was 10 min at 30 °C, and the enzyme
levels did not utilize more than 15% of the substrate during
the incubation period. The reaction was stopped by freezing
the samples in liquid nitrogen (30 s) and then thawing them
in boiling water (90 s). The 5′-nucleotides were converted to
[3H]nucleosides with 0.3 U of 5′-nucleotidase. Radioactive
nucleosides were separated from labeled cyclic nucleotides
using Dowex 1 × 2 anion exchange resin in 2 vol of water.
The suspensions were centrifuged at 15000g for 10 min, and
the radioactivity was determined in aliquots of supernatant
by liquid scintillation spectroscopy.
Su p p or tin g In for m a tion Ava ila ble: Tables of atomic
coordinates and anisotropic thermal parameters (11 pages);
observed and calculated structure factors for compounds 17
and 28 (16 pages). Ordering information is given on any
current masthead page.
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48
CHA (13.5 Ci mmol-1
)
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