Efficient synthesis of 3,5-dicarbamoyl-1,4-dihydropyridines from pyridinium salts: Key molecules in understanding NAD(P)+/NAD(P)H pathways

Article abstract of DOI:10.1002/jhet.2031

3,5-Dicarbamoyl-1,4-dihydropyridines were prepared in high yields using a green protocol by reduction of the corresponding pyridinium salts in aqueous buffered sodium dithionite solutions. The pH value is a fundamental parameter for the reduction step and depends on the nature of substituent groups at positions 1, 3, and 5 of the pyridinium salts. These 3,5-dicarbamoyl dihydropyridines show a lower tendency towards oxidation and a higher stability than N-benzyl-3-carbamoyl-1,4-dihydropyridine at low pH values.

Full text of DOI:10.1002/jhet.2031

Month 2014
Efficient Synthesis of 3,5-Dicarbamoyl-1,4-dihydropyridines from
Pyridinium Salts: Key Molecules in Understanding NAD(P)+/NAD(P)H
Pathways
a
a
a
b
b
a
*
Paolo Mellini, Daniela De Vita, Barbara Di Rienzo, Salvatore La Rosa, Alessandro Padova, Luigi Scipione,
Silvano Tortorella,a and Laura Friggeria
aChimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
bSiena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
*
E-mail: daniela.devita@uniroma1.it
Received November 5, 2012
DOI 10.1002/jhet.2031
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).
3,5-Dicarbamoyl-1,4-dihydropyridines were prepared in high yields using a green protocol by reduction
of the corresponding pyridinium salts in aqueous buffered sodium dithionite solutions. The pH value is a
fundamental parameter for the reduction step and depends on the nature of substituent groups at positions
1, 3, and 5 of the pyridinium salts. These 3,5-dicarbamoyl dihydropyridines show a lower tendency towards
oxidation and a higher stability than N-benzyl-3-carbamoyl-1,4-dihydropyridine at low pH values.
J. Heterocyclic Chem., 000, 00 (2014).
INTRODUCTION
3,5-dicarboxylic acid by using oxalyl chloride, instead of
thionyl chloride for generating the acylchlorides [8,9] and
subsequent amination by gaseous ammonia or methylamine.
In our initial study, various conditions were used for the
synthesis of pyridinium salts such as the classical thermal
way, simply by dissolving the pyridine and the alkylating
agent in dimethyl sulfoxide (2g and h) and heating the
mixture for 5 h, or otherwise without solvents (2a and b)
when the alkylating agent was liquid. The best reaction
conditions for the synthesis of pyridinium salts were
obtained by microwave-assisted procedure, whenever the
thermal way did not give rise to significant yield of products
(2c–f). It should be pointed out that by using microwave
irradiation, some advantages have been demonstrated: (i)
desired products were obtained in high yields (84–93%);
(ii) the shorter reaction times (40 min); and (iii) decreased
amount of alkylating agent. To verify the advantages of the
microwave irradiation rather than conventional heating, we
have carried out the microwave procedure even if the thermal
heating has led to significant yields of product. Regarding
2a, microwave-assisted condition showed a drastic reduction
of both reaction time (40 min instead 8 h) and the used
amount of benzyl bromide (4eq instead of 8 eq) with a yield
improvement up to 80%.
Nicotinamide adenine dinucleotide cofactors, including
NAD+, NADP+, and the reduced forms (NADH and
NADPH), have long been known as key molecules in
energy metabolism and mitochondrial functions. More-
over, a rapidly growing body of information has suggested
that NAD(P)+ and NAD(P)H also play a crucial role in
various biological processes such as calcium homeostasis,
cellular oxidative balance, sirtuins modulation [1], gene
expression, immunological functions, and aging [2]. In this
context, 3-carbamoyl-1,4-dihydropyridines have always
been considered affordable NAD(P)H synthetic models.
Over the past few years, the chemistry of the 1-benzyl-3-
carbamoyl-1,4-dihydropyridine (4) and its derivatives have
been investigated [3,4], but the application in organic and me-
dicinal chemistry for these compounds is still limited because
of their tendency towards the oxidation as well as the poor sta-
bility at low pH value; because of enamine reactivity, they easily
add water across the 5,6-double bond [5–7]. In this scenario,
the identification of new carbamoyl-1,4-dihydropyridines with
a better applicability could be intriguing. Our interest in devel-
oping new NAD(P)H mimetic compounds prompted us in pre-
paring new series of dihydropyridines (DHP) stable towards
the oxidation and in acidic aqueous solutions, using efficient
and friendly procedures (Scheme 1).
Dihydropyridines 3a–h were obtained in high yields
from the corresponding pyridinium salts with a green
procedure, using a buffered solution under nitrogen
flow, and as reducing agent sodium dithionite, which is a
versatile and widely used in chemical and biochemical
research. It is well known that dithionite reduction appears
to be applicable to all pyridinium salts bearing electron-
RESULTS AND DISCUSSION
Pyridine-3,5-dicarboxamide (1a) and N,N0-dimethylpyridine-
3,5-dicarboxamide (1b) have been prepared from pyridine-
© 2014 HeteroCorporation

P. Mellini, D. De Vita, B. Di Rienzo, S. La Rosa, A. Padova, L. Scipione, S. Tortorella, and L. Friggeri Vol 000
Scheme 1. Reagents and conditions. a: CH2Cl2, (COCl)2, reflux, 10 h; b:
pH values to decompose: that is, pH = 7 for compound 3b
and pH = 6.5 for compounds 3a and c–h.
CH3CN, R1NH2(g), RT, 3–4 h; c: NaHCO3(aq) d: R2X, 120ꢂC, 8–10 h for
2a,b; CH3CN, R2X, microwaves, 120ꢂC, 40 min, (reactor setting: 200 psi
To evaluate the different stability of 3-carboxamide and
3,5-dicarboxamide-1,4-dihydropyridines towards the oxi-
dation, we have carried out a study of antioxidant activity
of selected representative compounds (4, 3a and 3h).
The antioxidant activity was evaluated using the 2,2-
diphenyl-2-picrylhydrazyl (DPPH) method according to
Brand-Williams et al. [12] based on the ability of antioxidant
max pressure, 300 W max power) for 2a,c–f ; DMSO, R2X 60ꢂC, 5 h
for 2g, reflux, 5 h for 2h; e: H2O, NaH2PO4/Na2HPO4, Na2S2O4 under N2
flow 30 min, pH = 6.5 for 3a,c–h, pH= 7.0 for 3b.
•
compounds to react with the stable radical DPPH. The EC50
was graphically extrapolated plotting the residual percentage
•
of DPPH concentration after 90min of reaction versus the
molar ratio [DHP]/[DPPH] (Fig. 1). The direct comparison
of the obtained results shows that 3a is not able to react with
DPPH radical and 3h instead seems having only a weak
antioxidant activity (EC50 of 109 mM). In contrast, 1-benzyl-
1,4-dihydropyridine-3-carboxamide 4 [13] has given a curve
with a pronounced slope, indicating an efficacious reaction
with DPPH radical with an EC50 of 14.4 mM. The different
behavior showed by 3,5-dicarbamoyl-1,4-dihydropyridines,
3h and particularly 3a, indicates that these compounds
possess higher stability towards oxidation with respect to
3-carbamoyl derivative 4.
The presence of the enamine moiety in the 1,4-
dihydropyridines makes them susceptible to the hydratation
of 5,6-double bond because of the protonation of carbon 5
and subsequent addition of water to carbon 6; this phenome-
non is strictly dependant on the H+ concentration [14]. The
electronic properties of substituents on the pyridine ring
may influence the sensitivity of the 1,4-dihydropyridine in
the acidic environment [7,15]. Therefore, we have decided
to test the stability of 1-benzyl-1,4-dihydropyridine-
3,5-dicarboxamide 3a and 1-benzyl-1,4-dihydropyridine-
3-carboxamide 4 in acidic media. The compounds 4 and 3a
were dissolved at 1.52ꢀ 10ꢁ3 M concentration in a buffer
HCl–glycine (pH = 3) and monitored with an UV/visible
spectrophotometer measuring the absorbance change among
220–460 nm. For compound 4, we have observed the imme-
diate disappearance of the absorption maximum at 354nm
(Fig. 2, red curve) due to the 5,6-double bond hydratation.
withdrawing substituents in the 3- or 3,5-positions, affor-
ding exclusively the corresponding 1,4-dihydropyridines,
as final products. In the course of the reduction, formed
intermediate adducts have been identified as sulfinate S-
anions [10]. Furthermore, these adducts are stable in
strong alkaline solutions, whereas at low alkaline pH
values, these were protonated to sulfinate, which promptly
decomposed into 1,4-dihydropyridines following SO2
elimination [11]. In this regard, we showed that the pH
value is a fundamental parameter for the reduction of
different pyridinium salts. Indeed, the 1,4-dihydropyridine
formation at pH= 8, which is described for 3-carbamoyl
derivatives [11], did not represent the best condition in the
preparation of 3,5-dicarboxamide derivatives that requires
lower pH values.
This different behavior could be related to the different
stability of the protonated sulfinate intermediate that
depends on the nature of substituent at positions 1, 3, and
5 of the pyridine ring; electron-withdrawing groups more
effectively stabilize the intermediate, which requires lower
•
Figure 1. Residual percentages of DPPH at the end of reaction with
dihydropyridine (DHP) plotted vs the molar ratio [DHP]/[DPPH].
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2014
Efficient Synthesis of 3,5-Dicarbamoyl-1,4-dihydropyridines from Pyridinium Salts:
Key Molecules in Understanding NAD(P)+/NAD(P)H Pathways
Melting points were determined on a Tottoli apparatus and are
uncorrected. IR spectra were recorded on a PerkinElmer
Spectrum-One spectrophotometer equipped with a ATR detector,
1
and band frequencies are given in wave number (cmꢁ1). H and
13C NMR spectra were acquired on a Bruker Avance 400
spectrometer operating at 400 and 100 MHz, respectively. Chem-
ical shift values are reported as d (ppm) relatively to Si(CH3)4 as
internal reference; coupling constant are given in Hz. Elemental
analyses were obtained by a PE 2400 (PerkinElmer) analyzer,
and the analytical results were within ꢃ0.4% of the theoretical
values. UPLC-MS analyses were run using a Acquity Waters
UPLC equipped with a Waters SQD (ES ionization) and Waters
Acquity PDA detector, using a column BEH C18 1.7 mm,
2.1 ꢀ 50 mm. Gradients were run using 0.05% formic acid
water/acetonitrile 95/5 and acetonitrile with a gradient 95/5
Figure 2. UV spectra of compound 4: dissolved in methanol (blue) and
to 100, flow: 0.8 mL/min over 3 or 5 min, temperature:
40ꢂC, and UV detection: 215 and 370 nm. ESI + detection in
the 80–1000 m/z range. HPLC-MS were run using a Waters
2795 separation module equipped with a Waters Micromass
ZQ (ES ionization) and Waters PDA 2996, using a X-Bridge
C18 3.5 mm 2.10 ꢀ 50 mm column, gradient: 0.1% formic acid/
water and 0.1% formic acid/acetonitrile with gradient 95/5 to
5/95 flow 0.8 mL/min over 10 min. Retention times (Rt) are
expressed in minutes, temperature at 40ꢂC, and UV detection at
215 and 370 nm. ESI+ detection is in the 80–1000 m/z range.
Microwave reactions were conducted using a CEM explorer 72
reactor.
immediately after dissolution in pH 3 buffer solution (red).
Differently, 3a (Fig. 3) resulted stable and does not
undergo the hydratation at the 5.6 double bond neither after
6 h at pH = 3; indeed, only little variations are evident in
the absorbance values.
In conclusion, it should be noted that the presence of
the second carboxamide group on C-5, because of its
strong withdrawal effect, makes the 1,4-dihydropyridine
system highly resistant towards the hydratation and the
oxidation. A friendly and efficient microwave procedure
has been developed to achieve pyridinium salts that are
obtained in very low yields by thermal way; furthermore,
3,5-dicarbamoyl-1,4-dihydropyridines were obtained in
high yields in aqueous buffer solution at adequate pH
values. These compounds are more stable than the corre-
sponding monocarbamoyl derivatives, thus suggesting a
high versatility in applications of organic and pharmaceuti-
cal chemistry.
Pyridine-3,5-dicarboxamide (1a).
Oxalyl chloride
(36.4 mmol) was dropwise added to the suspension of pyridine-
3,5-dicarboxylic acid (3.59 mmol) in dichloromethane (50 mL),
and the mixture was refluxed for 10 h. The obtained yellow
solution was evaporated under reduced pressure to give a crude
residue, which was treated with 30 mL of dry acetonitrile.
Through the obtained solution, gaseous ammonia was bubbled
for 3 h at room temperature. The obtained solid was filtered off,
washed with acetonitrile (3 ꢀ 5 mL), water (3 ꢀ 8 mL), and
treated at 70ꢂC with aqueous saturated NaHCO3 (35 mL) for
20 min. After cooling, a woolly precipitate was filtered off,
washed with water (3ꢀ 6 mL), and dried, to give pure 1a as a
brownish solid (yield 85%); mp 303–304ꢂC (lit. [8] 303–305ꢂC).
nmax (cmꢁ1) (neat): 3387, 3113, 1683, 1631, 1575. 1H NMR
(400 MHz, (CD3)2SO): d = 9.12 (s, 2H, H-2 and H-6), 8.62 (s, 1H,
H-4), 8.24 (s br, 2H, NHa), 7.68 (s br, 2H, NHb). 13C NMR
(100 MHz, (CD3)2SO): d = 166.4, 151.2, 134.2, 129.9. (Anal.
Calcd for C7H7N3O2: C, 50.91; H, 4.27; N, 25.44%; found C,
50.75; H, 3.98; N, 25.16%).
EXPERIMENTAL
All chemicals were of analytical grade and were purchased
from Sigma-Aldrich Chemical Co. (Milan, Italy).
N,N0-dimethylpyridine-3,5-dicarboxamide (1b).
Pyridine-
3,5-dicarbonyl dichloride (3.59 mmol), prepared as described for
1a, was dissolved in acetonitrile (30 mL), anhydrous methylamine
was bubbled, and the solution was stirred for 4 h at room temperature.
The solvent was removed under reduced pressure, and the
obtained crude residue was washed with water (4 ꢀ 10 mL) and
dried. The remaining solid was treated with aqueous saturated
K2CO3 (15 mL) and extracted with ethyl acetate (3 ꢀ 50 mL).
The combined organic extracts were dried over anhydrous
Na2SO4 and the solvent removed under reduced pressure. The
obtained product was purified on silica gel column chromatogra-
phy (ethylacetate–methanol, 7:3 v/v) to afford pure 1b as a
yellowish solid (yield 80%); mp 215–218ꢂC (lit. [9] 220–222ꢂC).
nmax (cmꢁ1) (neat): 3285, 3075,1634; 1542, 1296. 1H NMR
(400 MHz, (CD3)2SO): d = 9.07 (d, 2H, H-2 and H-6, J = 2.2),
8.73–8.78 (m, 2H, NHCH3), 8.56 (t, 1H, H-4), 2.81 (d, 6H,
Figure 3. UV spectra of compound 3a in pH 3 buffer solution, immedi-
ately after the dissolution (blue), after 3 h (red), and after 6 h (green).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

P. Mellini, D. De Vita, B. Di Rienzo, S. La Rosa, A. Padova, L. Scipione, S. Tortorella, and L. Friggeri Vol 000
NHCH3). 13C NMR (100 MHz, (CD3)2SO): d = 167.3, 151.1,
136.2, 130.4, 26,9. (Anal. Calcd for C9H11N3O2: C, 55.95; H,
3,5-Dicarbamoyl-1-[4-(trifluoromethyl)benzyl]pyridinium
bromide (2e). 2e was prepared using 4-(trifluoromethyl)benzyl
bromide (4.84 mmol) as alkylating agent, and it was obtained in
90% yield a as white solid. mp 269–271ꢂC. nmax (cmꢁ1) (neat):
3299, 3137, 1694, 1671. 1H NMR (400 MHz, (CD3)2SO):
d = 9.88 (s, 2H, H-2 and H-6), 9.54 (s, 1H, H-4), 8.70 (s br, 2H,
NHa), 8.28 (s br, 2H, NHb), 7.90 (d, 2H, J = 8.2, Ar), 7.81 (d,
2H, J = 8.2, Ar), 6.17 (s, 2H, CH2).13C NMR (100 MHz, (CD3)
2SO): 162.8, 147.0, 142.8, 138.5, 134.4, 130.1, 130.0 (q, JC–
F = 31 Hz), 126.4 (q, JC–F = 3 Hz), 125.7, 63.4. (Anal. Calcd for
C15H13BrF3N3O2: C, 44.57; H, 3.24; Br, 19.77; F, 14.10; N,
5.74; N, 21.75%; found C, 55.75; 5.35; N, 21.48%).
1-Benzyl-3,5-dicarbamoylpyridinium bromide (2a).
Method a. a mixture of pyridine-3,5-dicarboxamide 1a (6.05 mmol)
and benzyl bromide (48.48 mmol) was refluxed and maintained
under stirring for 8 h. The suspension was cooled at room
temperature, and the brown solid was filtered off, washed with
diethyl ether (7 ꢀ 10 mL), and dried to give pure 2a as a light
brown solid (yield 70%); mp > 300ꢂC (lit. [9], mp > 300ꢂC).
Method b. 2a was also prepared by microwave-assisted syn-
thesis using the same procedure described for 2c–f: (yield 80%).
10.40%; found: C, 44.73; H, 2.93; N, 10.59%.
1
nmax (cmꢁ1) (neat): 3358, 3158, 3056, 1674, 1598, 1443. H
3,5-Dicarbamoyl-1-(4-fluorobenzyl)pyridinium bromide (2f).
NMR (400 MHz, (CD3)2SO): d = 9.71 (d, 2H, J2,4 = J6,4 = 1.72,
H-2 and H-6), 9.40 (t, 1H, H-4), 8.63 (s br, 2H, NHa), 8.26
(s br, 2H, NHb), 7.63-7.44 (m, 5H, Ar), 5.97 (s, 2H, CH2).13C
NMR (100 MHz, (CD3)2SO): d = 162.8, 146.6, 142.6, 134.3,
134.0, 129.9, 129.7, 129.6, 64.2 (Anal. Calcd for C14H14BrN3O2:
C, 50.02; H, 4.20; Br, 23.77; N, 12.50%; found C, 50.19; H, 4.11;
2f was prepared using 4-fluorobenzyl bromide (4.84 mmol) as
alkylating agent, and it was obtained in 93% yield as a white
solid. mp 270–271ꢂC. nmax (cmꢁ1) (neat): 3291, 3133, 3064,
1673, 1624. 1H NMR (400 MHz, (CD3)2SO): d = 9.82 (s, 2H,
H-2 and H-6), 9.50 (s, 1H, H-4), 8.70 (s br, 2H, NHa), 8.28
(s br, 2H, NHb) 7.81–7.77 (m, 2H, Ar), 7.31–7.27 (m, 2H, Ar),
6.02 (s, 2H, CH2).
N, 12.32%).
13C NMR (100 MHz, (CD3)2SO): 163.1 (d, JC–F = 244.4), 162.9,
146.7, 142.6, 134.3, 132.4 (d, JC–F = 8), 130.3, 116.5 (d, JC–
F = 21.8), 63.4. (Anal. Calcd for C14H13BrFN3O2: C, 47.48; H, 3.70;
1-Benzyl-3,5-bis(methylcarbamoil)pyridinium bromide (2b).
2b was prepared using 1b as starting reagent (2.22 mmol)
following the procedure (method a) described for 2a. The brown
solid 2b, crystallized by EtOH, was obtained in 50% yield; mp
258–260ꢂC (lit. [9] 259–260ꢂC).
Br, 22.56; F, 5.36; N, 11.86%; found: C, 47.36; H, 3.51; N, 11.61%).
3,5-Dicarbamoyl-1-(2,6-dichlorobenzyl)pyridinium bromide (2g).
n
max (cmꢁ1) (neat): 3244, 3071, 2920, 1681, 1654, 1621, 1292.
A mixture of 1a (1.57 mmol) and dimethylsulfoxide (6.5 mL) was
heated until complete dissolution, and then, 2,6-dichlorobenzyl
bromide (3.17 mmol) was added. The obtained solution was heated
to 60ꢂC for 5 h, cooled at room temperature, and then water was
dropwise added until complete precipitation of the salt. The
precipitate was collected by filtration, washed with diethyl ether
(4 ꢀ 2mL), and dried under reduced pressure to give pure 2g
1H NMR (400 MHz, (CD3)2SO): d = 9.68 (s, 2H, H-2 and H-6),
9.34 (s, 1H, H-4), 9.20 (d, 2H, J = 4.64, NHCH3), 7.61-7.44
(m, 5H, Ar), 5.96 (s, 2H, CH2), 2.86 (d, 6H, NHCH3).13C NMR
(100 MHz, (CD3)2SO): d = 162.0, 146.7, 142.4, 134.7, 134.5,
130.4, 130.1, 64.7, 27.2. (Anal. Calcd for C16H18BrN3O2: C,
52.76; H, 4.98; Br, 21.94; N, 11.54%; found: C, 52.93; H, 4.76;
N, 11.35%).
(72% yield) as light yellow solid. mp 268–269ꢂC. nmax (cmꢁ1
)
(neat): 3334, 3140, 3061, 1675, 1610. 1H NMR (400 MHz,
(CD3)2SO): d = 9.44 (s, 1H, H-4), 9.36 (s, 2H, H-2 and H-6),
8.67 (s br, 2H, NHa), 8.28 (s br, 2H, NHb), 7.69–7.61 (m, 3H,
Ar), 6.25 (s, 2H, CH2). 13C NMR (100 MHz, (CD3)2SO):
162.7, 146.4, 142.9, 137.1, 134.1, 133.8, 130.0, 128.3,
59.9. (Anal. Calcd for C14H12BrCl2N3O2: C, 41.51; H,
2.99; Br, 19.73; Cl, 17.50; N, 10.37%; found: C, 41.27; H,
2.88; N, 10.11%.
General procedure for the synthesis of pyridinium salts 2c–f.
The appropriate 4-substituted benzyl bromide was added to a
suspension of 1a (1.21mmol) in acetonitrile (5 mL), and the
mixture was heated at 120ꢂC, (reactor setting: 300 W max power
and 200psi max pressure) for 40min in a microwave reactor. The
resulting solution was cooled at room temperature, 15 mL diethyl
ether were added, and the obtained precipitate was collected and
washed with diethyl ether (5 ꢀ 4 mL) to give pure salt as a solid.
3,5-Dicarbamoyl-1-(4-methylbenzyl)pyridinium bromide (2c).
3,5-Dicarbamoyl-1-methylpyridinium iodide (2h).
A
2c was prepared using 4-methylbenzyl bromide (4.86 mmol) as
alkylating agent, and it was obtained in 92% yield as a white solid.
mp 265–267ꢂC. nmax (cmꢁ1) (neat) 3332, 3161, 3060, 1678, 1610.
mixture of 1a (0.61 mmol) in dimethyl sulfoxide (3 mL) was
heated until completed dissolution, and then, iodomethane was
added (3.10mmol). The obtained solution was refluxed for 5 h,
cooled at room temperature, and then, water was added dropwise
until complete precipitation of the salt. The precipitate was
collected and washed with diethyl ether (4 ꢀ 2 mL), dried under
reduced pressure, and crystallized with ethanol to give pure 2h as
a yellow solid (49% yield). mp 275–279ꢂC. nmax (cmꢁ1) (neat):
3339, 3067, 1672, 1624. 1H NMR (400 MHz, (CD3)2SO):
d = 9.50 (s, 2H, H-2 and H-6), 9.28 (s, 1H, H-4), 8.53 (s br, 2H,
NHa), 8.21 (s br, 2H, NHb), 4.43 (s, 3H, CH3). 13C NMR
(100 MHz, (CD3)2SO): 163.0, 147.6, 141.7, 133.5, 49.1.(Anal.
Calcd for C8H10IN3O2: C, 31.29; H, 3.28; I, 41.33; N, 13.68%;
found: C, 31.61; H, 3.41; N, 13.51%).
1H NMR (400 MHz, (CD3)2SO): d = 9.74 (s, 2H, H-2 and
H-6), 9.46 (s, 1H, H-4), 8.67 (s br, 2H, NHa), 8.25 (s br, 2H,
NHb), 7.55 (d, 2H, J = 7.8, Ar), 7.26 (d, 2H, J = 7.8, Ar), 5.96
(s, 2H, CH2), 2.30 (s, 3H, CH3). 13C NMR (100 MHz, (CD3)
2SO): 162.9, 146.5, 142.5, 139.7, 134.3, 131.1, 130.2, 129.8,
64.1, 21.2. (Anal. Calcd for C15H16BrN3O2: C, 51.44; H, 4.60;
Br, 22.82; N, 12.00%; found: C, 51.17; H, 4.45; N, 11.69%.
3,5-Dicarbamoyl-1-(4-cyanobenzyl)pyridinium bromide (2d).
2d was prepared using 4-(bromomethyl)benzonitrile (4.85 mmol)
as alkylating agent, and it was obtained in 84% yield as a white
solid. mp 263–266ꢂC. nmax (cmꢁ1) (neat): 3369, 3203, 3144,
3055, 2239, 1682, 1614. 1H NMR (400 MHz, (CD3)2SO):
d = 9.85 (s, 2H, H-2 + H-6), 9.52 (s, 1H, H-4), 8.69 (s br, 2H,
NHa), 8.26 (s br, 2H, NHb), 7.93 (d, 2H, J = 8.1 Hz, Ar), 7.85
(d, 2H, J = 8.1 Hz, Ar), 6.15 (s, 2H, CH2). 13C NMR (100 MHz,
(CD3)2SO): 162.8, 147.0, 142.9, 139.1, 134.4, 133.4, 130.6,
118.8, 112.6, 63.4. (Anal. Calcd for C15H13BrN4O2: C, 49.88; H,
3.63; Br, 22.12; N, 15.51%; found: C, 49.76; H, 3.29; N, 15.32%).
1-Benzyl-1,4-dihydropyridine-3,5-dicarboxamide (3a).
A
solution of sodium dithionite (1.21 mmol) in NaH2PO4/Na2HPO4
buffer (pH = 6.5, 5.6 mL) under nitrogen atmosphere was cooled
in an ice bath. Then, a solution of 2a (0.60 mmol) dissolved in
7.0 mL of NaH2PO4/Na2HPO4 (pH = 6.5) was dropwise added.
The reaction was maintained under stirring for 40 min, and after
this time, the obtained yellow precipitate was collected by
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2014
Efficient Synthesis of 3,5-Dicarbamoyl-1,4-dihydropyridines from Pyridinium Salts:
Key Molecules in Understanding NAD(P)+/NAD(P)H Pathways
filtration, washed with cold water (3 ꢀ 1.5 mL), and dried under
reduced pressure, to give pure 3a (72% yield). mp 195–197ꢂC.
nmax (cmꢁ1) (neat): 3350, 3175, 1687, 1644, 1557. 1H NMR
(400 MHz, (CD3)2SO): d = 7.40–7.29 (m, 5H, Ar), 6.98 (s, 2H,
H-2 and H-6), 6.72 (s br, 4H, NH2), 4.44 (s, 2H, CH2), 3.07 (s,
2H, H-4 and H-40). 13C NMR (100 MHz, (CD3)2SO): 168.8,
138.5, 136.3, 129.2, 128.1, 127.8, 106.3, 56.7, 23.0. UPLC-MS
(3 min run): mass (ES) m/z: 258 (M + 1); Rt = 0.98 min (Anal.
Calcd for C14H15N3O2: C, 65.35; H, 5.88; N, 16.33%; found:
MS (10 min run): mass (ES) m/z: 326 (M + 1); Rt = 4.28 min
(Anal. Calcd for C15H14F3N3O2: C, 55.39; H, 4.34; F, 17.52;
N, 12.92%; found: C, 55.21; H, 4.08; N, 12.77%).
1-(4-Fluorobenzyl)-1,4-dihydropyridine-3,5-dicarboxamide
(3f). 3f was prepared following the same procedure described
for 3a, using 0.15 g of sodium dithionite (0.86 mmol) and
0.15 g of 2f (0.42 mmol). Pure 3f was obtained as a yellow
powder in 74% yield. mp 180–183ꢂC. nmax (cmꢁ1) (neat):
3342, 3172, 1687, 1644. 1H NMR (400 MHz, (CD3)2SO):
d = 7.36 (d, 2H, Ar), 7.22 (d, 2H, Ar), 6.98 (s, 2H, H-2 and
H-6), 6.73 (s br, 4H, NH2), 4.44 (s, 2H, CH2), 3.07 (s, 2H, H-4
and H-40). 13C NMR (100 MHz, (CD3)2SO): 168.8, 168.6
(d, JC–F = 258.0 HZ), 136.1, 134.7, 129.9 (d, JC–F = 8.8 Hz),
116.0 (d, JC–F = 21.0 Hz), 106.5, 55.9, 13.0. UPLC-MS (5 min
run): mass (ES) m/z: 276 (M + 1); Rt = 1.56 min (Anal. Calcd
for C14H14FN3O2: C, 61.08; H, 5.13; F, 6.90; N, 15.26%;
C, 65.01; H, 5.53; N, 16.15%).
1-Benzyl-N,N0-dimethyl-1,4-dihydropyridine-3,5-dicarbo-
xamide (3b). 3b was prepared following the same procedure
described for 3a, using 0.096 g of sodium dithionite
(0.55 mmol), 4.0 mL of NaH2PO4/Na2HPO4 buffer (pH = 7.0),
and 0.10 g of 2b (0.27 mmol) dissolved in 6.0 mL of
NaH2PO4/Na2HPO4 buffer (pH = 7.0). Pure 3b was obtained
as yellow powder in yield of 81%. mp 92–95ꢂC. nmax
(cmꢁ1) (neat): 3306, 1686, 1539. 1H NMR (400 MHz, (CD3)
2SO): d = 7.39–7.26 (m, 5H, Ar), 7.19 (d, 2H, J = 4.4 Hz,
NHCH3), 6.92 (s, 2H, H-2 and H-6), 4.45 (s, 2H, CH2), 3.09
(s, 2H, H-4 and H-40), 2.61 (d, 6H, NHCH3). 13C NMR
(100 MHz, (CD3)2SO): 167.4, 138.6, 135.5, 129.2, 128.0,
127.7, 106.2, 56.7, 26.4, 22.7. UPLC-MS (3 min run): mass
(ES) m/z: 286 (M + 1); Rt = 1.09 min (Anal. Calcd for
C16H19N3O2: C, 67.35; H, 6.71; N, 14.73%; found: C, 67.11;
found: C, 60.72; H, 4.94; N, 15.02).
1-(2,6-Dichlorobenzyl)-1,4-dihydropyridine-3,5-dicarbo-
xamide (3g). 3g was prepared following the same procedure
described for 3a, using 0.17 g of sodium dithionite (0.98 mmol)
and 0.20 g of 2g (0.49 mmol). Pure 3g was obtained as a yellow
powder in 80% yield. mp 201–204ꢂC. nmax (cmꢁ1) (neat):
3343, 3185, 1688, 1644, 1561. 1H NMR (400 MHz, (CD3)
2SO): d = 7.57–7.49 (m, 3H, Ar), 6.90 (s, 2H, H-2 and H-6),
6.73 (s br, 4H, NH2), 4.68 (s, 2H, CH2), 3.06 (s, 2H, H-4 and
H-40). 13C NMR (100 MHz, (CD3)2SO): 168.7, 137.1, 136.2,
135.7, 131.7, 129.6, 106.3, 51.9, 23.1. UPLC-MS (3 min run):
mass (ES) m/z: 326 (M + 1); Rt = 0.74 min (Anal. Calcd for
C14H13Cl2N3O2: C, 51.55; H, 4.02; Cl, 21.74; N, 12.88%;
H, 6.62; N, 14.63%).
1-(4-Methylbenzyl)-1,4-dihydropyridine-3,5-dicarboxamide
(3c). 3c was prepared following the same procedure described
for 3a, using 0.15 g of sodium dithionite (0.86 mmol) and 0.15 g
of 2c (0.43 mmol). Pure 3c was obtained as a yellow powder in
77% yield. mp 167–170ꢂC. nmax (cmꢁ1) (neat): 3343, 3179,
1688, 1640. 1H NMR (400 MHz, (CD3)2SO): d = 7.19 (s, 4H,
Ar), 6.96 (s, 2H, H-2 and H-6), 6.71 (s br, 4H, NH2), 4.39 (s, 2H,
CH2), 3.07 (s, 2H, H-4 and H-40), 2.29 (s, 3H, CH3). 13C NMR
(100 MHz, (CD3)2SO): 168.8, 137.3, 136.2, 135.4, 129.7, 127.8,
106.2, 56.5, 23.0, 21.1. UPLC-MS (5 min run): mass (ES) m/z:
272 (M + 1); Rt = 1.65 min (Anal. Calcd for C15H17N3O2: C,
found: C, 51.34; H, 3.71; N, 12.61%).
1-Methyl-1,4-dihydropyridine-3,5-dicarboxamide (3h).
3h was prepared following the same procedure described for
3a, using 0.25 g of sodium dithionite (1.44 mmol) and 0.20 g
of 2h (0.65 mmol). Pure 3h was obtained as a yellow powder
in 71% yield. mp 220–223ꢂC. nmax (cmꢁ1) (neat): 3415, 3163,
1704, 1651, 1558. 1H NMR (400 MHz, (CD3)2SO): d = 6.83
(s, 2H, H-2 and H-6), 6.69 (s br, 4H, NH2), 3.03 (s, 2H, H-4
and H-40), 2.99 (s, 3H, CH3). 13C NMR (100 MHz, (CD3)
2SO): 169.1, 137.0, 105.6, 40.8, 22.7.UPLC-MS (3 min run):
mass (ES) m/z: 182 (M+ 1); Rt = 0.19min (solvent front) (Anal.
Calcd for for C8H11N3O2: C, 53.03; H, 6.12; N, 23.19%; found:
66.40; H, 6.32; N, 15.49%; found: C, 66.13; H, 6.12; N, 15.15%).
1-(4-Cyanobenzyl)-1,4-dihydropyridine-3,5-dicarboxamide
(3d). 3d was prepared following the same procedure described
for 3a, using 0.14 g of sodium dithionite (0.80 mmol) and 0.15 g
of 2d (0.41 mmol). Pure 3d was obtained as a yellow powder in
80% yield. mp 110–112ꢂC. nmax (cmꢁ1) (neat): 3351, 3205,
2231, 1686, 1644. 1H NMR (400 MHz, (CD3)2SO): d = 7.87
(d, 2H, J = 8.31, Ar), 7.51 (d, 2H, J = 8.31, Ar), 6.98 (s, 2H, H-2
and H-6), 6.74 (s br, 4H, NH2), 4.57 (s, 2H, CH2), 3.08 (s, 2H,
H-4 and H-40). 13C NMR (100 MHz, (CD3)2SO): 168.7, 144.4,
136.1, 133.1, 129.0, 128.6, 110.8, 106.8, 56.1, 22.9. UPLC-MS
(5min run): mass (ES) m/z: 283 (M+ 1); Rt = 1.42min (Anal.
Calcd for C15H14N4O2: C, 63.82; H, 5.00; N, 19.85%; found: C,
C, 52.77; H, 5.99; N, 23.05%).
Evaluation of antioxidant activity. The antioxidant activity
of DHP was spectrophotometrically evaluated against stable
radical DPPH [12] (Sigma-Aldrich, Milano, Italia), measuring
the absorbance decrease at 515 nm by mean of UV/
visible spectrophotometer PerkinElmer (mod.Lambda 40).
Methanol was used as solvent. Standard polystyrene optical
cuvette (3 mL volume) were used, repeating each measure
at least in triplicate. Data were processed by mean of
Microsoft Excel 2010 software. A calibration curve with
DPPH solutions in the concentration range 20–180 mM
was obtained (r2 = 0.996). The decrease of the absorbance
of 60 mM DPPH solutions containing 30 mM 1-benzyl-3-
carbamoyl-1,4-dihydropyridine (4) was measured in a time
drive mode for 2 h; the final plateau absorbance values were
reached after 60 min. The absorbance of 60 mM DPPH
solution was measured, and thereafter, 30 mL of appropriate
stock DHP solution were added to obtain the desired
final concentration in cuvette. Each solution was stored at
25ꢂC in a dark box for 90 min, and then, the absorbance
was measured. The initial and final DPPH concentration
63.96; H, 4.83; N, 20.13%).
1-[4-(Trifluoromethyl)benzyl]-1,4-dihydropyridine-3,5-
dicarboxamide (3e). 3e was prepared following the same
procedure described for 3a, using 0.13 g of sodium dithionite
(0.74 mmol) and 0.15 g of 2e (0.37 mmol). Pure 3e was
obtained as a yellow powder in 84% yield. mp 147–150ꢂC.
nmax (cmꢁ1) (neat): 3365, 3192, 1687, 1620. 1H NMR
(400 MHz, (CD3)2SO): d = 7.77 (d, 2H, J = 8.08, Ar), 7.52
(d, 2H, J = 8.08, Ar), 7.00 (s, 2H, H-2 and H-6), 6.77 (s br,
4H, NH2), 4.58 (s, 2H, CH2), 3.09 (s, 2H, H-4 and H-40). 13C
NMR (100 MHz, (CD3)2SO): 168.7, 143.4, 136.1, 128.5,
126.1, 124.5 (q, JC–F = 270 Hz), 106.7, 56.1, 23.0. HPLC-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

P. Mellini, D. De Vita, B. Di Rienzo, S. La Rosa, A. Padova, L. Scipione, S. Tortorella, and L. Friggeri Vol 000
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for each cuvette were calculated using the DPPH calibration
curve equation. For each DHP, the percentages of residual
DPPH remaining after 90 min were plotted versus the molar
ratio [DHP]/[DPPH]. The molar ratio able to decrease by
50% the initial DPPH concentration was calculated from
these plots, and subsequently, the EC50 values were obtained
(DHP concentration needed to halve initial DPPH radical
concentration). The absorption of blank sample containing the
same amount of methanol and DPPH solution was prepared
and measured daily.
Acknowledgments. This study was financially supported by
Sapienza University of Rome and Siena Biotech S.p.A. The
authors thank MSc Letizia Fontana for her valuable contribution.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet