Synthesis and biological activity of 3- and 5-amino derivatives of pyridine-2-carboxaldehyde thiosemicarbazone

Article abstract of DOI:10.1021/jm9600454

A series of 3- and 5-alkylamino derivatives, as well as other structurally modified analogues of pyridine-2-carboxaldehyde thiosemicarbazone, have been synthesized and evaluated as inhibitors of CDP reductase activity and for their cytotoxicity in vitro and antineoplastic activity in vivo against the L1210 leukemia. Alkylation of 3- and 5-amino-2-(1,3-dioxolan-2-yl)pyridines (1, 2) resulted in corresponding 3-methylamino, 5-methylamino, 3-allylamino, 5-ethylamino, 5-allylamino, 5-propylamino, and 5-butylamino derivatives (5, 6, and 11-15), which were then condensed with thiosemicarbazide to yield the respective thiosemicarbazones (7, 8, and 1620). Oxidation of 3,5-dinitro-2- methylpyridine (21) with selenium dioxide, followed by treatment with ethylene glycol and p-toluenesulfonic acid, produced the cyclic ethylene acetal, 23. Oxidation of 2-(1,3-dioxolan-2-yl)-4-methyl-5-nitropyridine (26) with selenium dioxide, followed by sequential treatment with sodium borohydride, methanesulfonyl chloride, and morpholine afforded the morpholinomethyl derivative 30. Catalytic hydrogenation of 23 and 30 with Pd/C yielded the corresponding amino derivatives 24 and 31. Catalytic hydrogenation of 5-cyano-2-methylpyridine (33) with Raney nickel, followed by treatment with acetic anhydride, gave the amide derivative 35. N-Oxidation of 35, followed by rearrangement with acetic anhydride, produced the acetate derivative, 5-[(acetylamino)methyl]-2-(acetoxymethyl)pyridine (37). Repetition of the N-oxidation and rearrangement procedures with compound 37 yielded the diacetate derivative 39. Condensation of compounds 24, 31, and 39 with thiosemicarbazide afforded the respective 3,5-diaminopyridine-, 4-(4- morpholinylmethyl)-5-aminopyridine-, and 5-(aminomethyl)pyridine-2- carboxaldehyde thiosemicarbazones (25, 32, and 40). The most biologically active compounds synthesized were the 5-(methylamino)-, 5-(ethylamino)-, and 5-(allylamino)pyridine-2-carboxaldehyde thiosemicarbazones (8, 17, and 18), which were potent inhibitors of ribonucleotide reductase activity with corresponding IC₅₀ values of 1.3, 1.0, and 1.4 μM and which produced significant prolongation of the survival time of L1210 leukemia-bearing mice, with corresponding optimum % T/C values of 223, 204, and 215 being obtained when administered twice daily for six consecutive days at dosages of 60, 80, and 80 mg/kg, respectively.

Full text of DOI:10.1021/jm9600454

2
586
J . Med. Chem. 1996, 39, 2586-2593
Syn th esis a n d Biologica l Activity of 3- a n d 5-Am in o Der iva tives of
P yr id in e-2-ca r boxa ld eh yd e Th iosem ica r ba zon e
†
†
Mao-Chin Liu, Tai-Shun Lin, J oseph G. Cory, Ann H. Cory, and Alan C. Sartorelli*
Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine,
New Haven, Connecticut 06520-8066, and Department of Biochemistry, East Carolina University School of Medicine,
Greenville, North Carolina 27858
X
Received J anuary 16, 1996
A series of 3- and 5-alkylamino derivatives, as well as other structurally modified analogues
of pyridine-2-carboxaldehyde thiosemicarbazone, have been synthesized and evaluated as
inhibitors of CDP reductase activity and for their cytotoxicity in vitro and antineoplastic activity
in vivo against the L1210 leukemia. Alkylation of 3- and 5-amino-2-(1,3-dioxolan-2-yl)pyridines
(
1, 2) resulted in corresponding 3-methylamino, 5-methylamino, 3-allylamino, 5-ethylamino,
-allylamino, 5-propylamino, and 5-butylamino derivatives (5, 6, and 11-15), which were then
condensed with thiosemicarbazide to yield the respective thiosemicarbazones (7, 8, and 16-
0). Oxidation of 3,5-dinitro-2-methylpyridine (21) with selenium dioxide, followed by treatment
5
2
with ethylene glycol and p-toluenesulfonic acid, produced the cyclic ethylene acetal, 23.
Oxidation of 2-(1,3-dioxolan-2-yl)-4-methyl-5-nitropyridine (26) with selenium dioxide, followed
by sequential treatment with sodium borohydride, methanesulfonyl chloride, and morpholine
afforded the morpholinomethyl derivative 30. Catalytic hydrogenation of 23 and 30 with Pd/C
yielded the corresponding amino derivatives 24 and 31. Catalytic hydrogenation of 5-cyano-
2
-methylpyridine (33) with Raney nickel, followed by treatment with acetic anhydride, gave
the amide derivative 35. N-Oxidation of 35, followed by rearrangement with acetic anhydride,
produced the acetate derivative, 5-[(acetylamino)methyl]-2-(acetoxymethyl)pyridine (37).
Repetition of the N-oxidation and rearrangement procedures with compound 37 yielded the
diacetate derivative 39. Condensation of compounds 24, 31, and 39 with thiosemicarbazide
afforded the respective 3,5-diaminopyridine-, 4-(4-morpholinylmethyl)-5-aminopyridine-, and
5
-(aminomethyl)pyridine-2-carboxaldehyde thiosemicarbazones (25, 32, and 40). The most
biologically active compounds synthesized were the 5-(methylamino)-, 5-(ethylamino)-, and
-(allylamino)pyridine-2-carboxaldehyde thiosemicarbazones (8, 17, and 18), which were potent
inhibitors of ribonucleotide reductase activity with corresponding IC50 values of 1.3, 1.0, and
.4 µM and which produced significant prolongation of the survival time of L1210 leukemia-
5
1
bearing mice, with corresponding optimum % T/C values of 223, 204, and 215 being obtained
when administered twice daily for six consecutive days at dosages of 60, 80, and 80 mg/kg,
respectively.
Ribonucleoside diphosphate reductase is a critical
enzyme in the synthesis of the deoxyribonucleotide
precursors of DNA and, as such, is essential for cellular
replication. Thus, its presence and activity is closely
correlated with cellular growth rates.1 Since deoxyri-
bonucleoside triphosphates are present in extremely low
levels in mammalian cells, a strong inhibitor of ribo-
nucleoside diphosphate reductase would appear to be a
particularly useful weapon in the therapeutic arma-
mentarium against cancer, especially those neoplasms
that are rapidly proliferating.3 Several different classes
of agents have been shown to be inhibitors of ribo-
nucleoside diphosphate reductase. These include the
R-(N)-heterocyclic carboxaldehyde thiosemicarbazones
planted rodent neoplasms, as well as spontaneous
1
3,14
lymphomas of dogs.
Such broad spectrum activity
denotes clinical potential and suggests that a drug of
this class may well have utility in cancer therapy.
A variety of nucleoside analogues are also active as
inhibitors of ribonucleoside diphosphate reductase. These
,2
1
5-17
include 2,2′-difluoro-2′-deoxycytidine,
2′-fluoroad-
enine arabinoside, and 2-chloro-2′-deoxyadenosine.19
However, the 5′-triphosphate of each of these com-
pounds appears to be a more potent inhibitor of DNA
polymerase than that of ribonucleoside diphosphate
reductase, making DNA polymerase the more probable
primary target of these nucleoside analogues.1
18
8-20
One of the members of the HCT series, 5-hydroxypyr-
idine-2-carboxaldehyde thiosemicarbazone (5-HP), has
4
5
(
HCTs), hydroxyurea, N-hydroxy-N′-aminoguanidine
6
-8
9,10
21,22
derivatives,
and polyhydroxybenzohydroxamates.
received a phase I trial in patients with cancer.
The HCTs, as a class, are among the most potent known
inhibitors of ribonucleoside diphosphate reductase, de-
pending upon the HCT, being 80-5000 times more
These studies have shown that the tumor-inhibitory
activity of 5-HP in animal systems did not translate to
patients with cancer. The inactivity of 5-HP as an
antineoplastic agent in patients was attributed to a
relatively short biological half-life in humans, which was
due to the formation of an inactive O-glucuronide and
the rapid elimination of this conjugate.
potent than hydroxyurea, a clinically useful anticancer
agent.1
1,12
Members of the HCT class have shown
anticancer activity against a wide spectrum of trans-
†
Recently, we have reported the synthesis and evalu-
ation of several new HCTs that by virtue of their
East Carolina University School of Medicine.
Abstract published in Advance ACS Abstracts, May 15, 1996.
X
S0022-2623(96)00045-3 CCC: $12.00 © 1996 American Chemical Society

Pyridine-2-carboxaldehyde Thiosemicarbazones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2587
Sch em e 1
structures are resistant to O-glucuronidation.2
3-25
and 2 with sodium methoxide and paraformaldehyde
in methanol, followed by reduction of the resulting
methyleneamines (3 and 4) with NaBH4 and 1 N KOH,
gave the corresponding 3- and 5-(methylamino)-2-(1,3-
dioxolan-2-yl)pyridines (5 and 6), which were then
treated with thiosemicarbazide and concentrated hy-
drochloric acid in ethanol to afford the target products,
3- and 5-(methylamino)pyridine-2-carboxaldehyde thio-
semicarbazones (7 and 8). Acetylation of compounds 1
and 2 with acetyl anhydride in anhydrous pyridine at
room temperature gave the respective acetyl derivatives
9 and 10. Treatment of compounds 9 and 10 with NaH
in anhydrous THF, followed by an alkyl halide, afforded
the corresponding 3- and 5-alkyl-substituted derivatives
11-15. Direct condensation of compounds 11-15 with
thiosemicarbazide in the presence of hydrochloric acid
provided 3-(allylamino)-, 5-(ethylamino)-, 5-(allylami-
no)-, 5-(n-propylamino)-, and 5-(n-butylamino)pyridine-
2-carboxaldehyde thiosemicarbazones (16-20), respec-
tively.
Among these compounds, the 3- and 5-aminopyridine-
-carboxaldehyde thiosemicarbazones (3-AP and 5-AP)
2
showed significant antitumor activity in mice bearing
the L1210 leukemia. 3-AP appeared to be the more
promising, being 6 times more potent than 5-HP as an
inhibitor of CDP reductase activity, and 3 and 7.5 times
more potent than 5-HP as an inhibitor of the growth of
wild-type and hydroxyurea-resistant L1210 cells, re-
spectively.2
6,27
In contrast, 5-AP was slightly more
active than 5-HP as an inhibitor of CDP reductase
activity and of the growth of parental L1210 cells but
was 3 times more potent than 5-HP as an inhibitor of
the growth of hydroxyurea-resistant cells. Since acetyl-
amino derivatives of 3- and 5-AP were found to be
devoid of carcinostatic activity and N-acetylation is a
relatively ubiquitous metabolic reaction in vivo, we
designed and synthesized a series of 3- and 5-alkylamino
derivatives of pyridine-2-carboxaldehyde thiosemicar-
bazone. Alkylation of the amino groups on the pyridine
ring of 3- and 5-AP results in the conversion of these
compounds from primary amines to the corresponding
secondary amines and, therefore, would result in steric
hindrance to the enzymatic acetylation of the amino
function. Furthermore, introduction of an alkyl group
would increase lipid solubility, which might improve the
pharmacokinetic properties of the parent compounds.
In addition, to further characterize the structural
features required for optimal biological activity in the
HCT series, several other amino derivatives of pyridine-
3,5-Diaminopyridine-2-carboxaldehyde thiosemicar-
bazone (25) was synthesized as illustrated in Scheme
2. The starting material, 3,5-dinitro-2-methylpyridine
(21), was synthesized by methodology developed in our
2
9
laboratory. Oxidation of 21 with selenium dioxide by
the minor modification of a method reported by Achre-
3
0
mowicz yielded a mixture of 3,5-dinitro-2-pyridine
aldehyde (22a ) and its ethyl hemiacetal (22b) in a ratio
of 1:3 (estimated by NMR spectra), which, because of
sensitivity to air, was treated directly with ethylene
glycol and p-toluenesulfonic acid in toluene to form 3,5-
dinitro-2-(1,3-dioxalan-2-yl)pyridine (23). Catalytic hy-
drogenation of compound 23 with 10% Pd/C in ethanol
produced the corresponding diamino derivative 24,
which upon condensation with thiosemicarbazide under
acidic conditions gave 3,5-diaminopyridine-2-carboxal-
dehyde thiosemicarbazone (25).
2
-carboxaldehyde thiosemicarbazone were also synthe-
sized. The synthesized compounds were evaluated in
vitro as inhibitors of CDP reductase activity and of the
growth of L1210 cells and in vivo for antitumor activity
against the L1210 leukemia.
Ch em istr y
The synthesis of a variety of 3- and 5-mono(alkyl-
amino) derivatives of pyridine-2-carboxaldehyde thio-
semicarbazone is depicted in Scheme 1. Selective
monomethylation of 3- and 5-amino-2-(1,3-dioxolan-2-
yl)pyridines (1 and 2), which were prepared according
to the procedure previously reported by our laboratory,
was achieved by the methodology of Barluenga et al.,
with minor modifications. Treatment of compounds 1
The synthesis of 5-amino-4-(morpholinomethyl)pyri-
dine-2-carboxaldehyde thiosemicarbazone (32) is shown
in Scheme 3. Oxidation of 2-(1,3-dioxolan-2-yl)-4-meth-
yl-5-nitropyridine (26) with selenium dioxide in diox-
ane gave the corresponding aldehyde 27. Selective
reduction of the aldehyde function of 27 with sodium
borohydride in methanol yielded the 4-hydroxymethyl
derivative 28. Treatment of compound 28 with meth-
2
3
2
2
3
8

2
588 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Liu et al.
Sch em e 2
Sch em e 3
Sch em e 4
anesulfonyl chloride in anhydrous pyridine followed by
refluxing with morpholine produced the 4-morpholi-
nomethyl derivative 30 via the intermediate 29. Cata-
lytic hydrogenation of compound 30 over 10% Pd/C in
ethanol afforded the 5-amino derivative 31, which was
then converted to the target thiosemicarbazone 32 by
condensation with thiosemicarbazide in the presence of
hydrochloric acid. 5-(Aminomethyl)pyridine-2-carbox-
aldehyde thiosemicarbazone (40) was prepared as de-
34. The pressure of the hydrogen and the reaction time
are critical to the yield of the product. High hydrogen
pressure or a relatively long reaction time led to mostly
pyridine ring hydrogenated side products. Protection
of the amino function of 34 by reaction with acetic
anhydride in anhydrous pyridine yielded compound 35.
Treatment of 35 with 30% hydrogen peroxide in glacial
acetic acid produced the N-oxide 36, which was then
refluxed with a mixture of acetic acid and acetic
anhydride (1:2, v/v) to give the acetate 37. Repeating
the N-oxidation and rearrangement procedures with
compound 37 and the resulting N-oxide 38, respectively,
3
1
scribed in Scheme 4. Catalytic hydrogenation
-cyano-2-methylpyridine (33) in the presence of Raney
nickel in acetic acid gave the 5-aminomethyl derivative
of
5

Pyridine-2-carboxaldehyde Thiosemicarbazones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2589
Ta ble 1. Effects of Pyridine-2-carboxaldehyde
Thiosemicarbazone Derivatives on CDP Reductase Activity and
the Growth of L1210 Cells in Culture
Ta ble 2. Effects of Pyridine-2-carboxaldehyde
Thiosemicarbazone Derivatives on the Survival Time of Mice
Bearing the L1210 Leukemia
IC50 (µM)a
optimum
daily dosagea
(mg/kg)
av
av ∆
survivalc T/C × long-term
compd
CDP reductaseb
L1210 cell growth
b
100d
e
compd
wt (%)
(days)
survivors
3
5
7
8
1
1
1
1
2
2
3
4
-AP
-AP
0.82
2.7
1.0
1.3
1.7
1.0
1.4
1.4
2.5
1.4
2.5
1.5
3
5
7
8
1
1
1
1
2
2
3
4
-AP
-AP
20
10
40
60
80
80
60
60
40
60
40
80
-2.4
-4.3
-2.9
-4.4
-2.6
-3.4
-3.4
-5.2
-4.0
-17.1
-3.5
-2.4
18.0
15.5
11.8
17.8
10.1
16.3
17.2
11.7
9.2
225
194
148
223
126
204
215
146
115
135
125
168
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
2/5
0/5
1/5
2.4
6
7
8
9
0
5
2
0
.10
6
7
8
9
0
5
2
0
3.4
4.4
5.1
7.8
9.0
11.0
2.5
1.1
10.8
10.0
13.4
.10
2.1
a
The concentration (µM) required to inhibit CDP reductase
a
Drugs were administered in suspension by intraperitoneal
b
activity or L1210 cell growth by 50%. CDP reductase IC50 values
are the average of two separate determinations. The values varied
by less than 10%.
injection, given as two daily divided doses, beginning 24 h after
tumor implantation, for 6 consecutive days, with five mice per
group. bAverage change in body weight from onset to termination
c
of therapy. Average survival time includes only those mice that
produced the diacetate derivative 39. Treatment of 39
with thiosemicarbazide in the presence of hydrochloric
acid resulted in the desired compound 40.
died prior to day 60. dT/C × 100 represents the ratio of the survival
time of treated to control animals × 100. The average survival
time of the untreated tumor-bearing control animals was 8.0 days.
e
Long-term survivors are the number of mice that survived for
>60 days relative to the total number of treated mice.
Biologica l Resu lts a n d Discu ssion
The primary lesion created in cells by the HCTs is
interference with the biosynthesis of DNA, and this
action is primarily due to the potent inhibition of
ribonucleoside diphosphate reductase activity. For this
reason, the comparative effects of various 3- and 5-ami-
no derivatives of pyridine-2-carboxaldehyde thiosemi-
carbazone on CDP reductase activity and on the growth
of L1210 cells in culture were measured, and the results
are shown in Table 1. Introduction of an alkyl sub-
stituent onto the amino group on the pyridine ring had
different effects on the inhibitory activity of these
agents, depending upon whether the amino group was
in the 3- or the 5-position. Thus, alkylation of the amino
group of 3-AP with a methyl or an allyl group, com-
pounds 7 and 16, respectively, produced agents that
were slightly less active than 3-AP as inhibitors of CDP
reductase activity, with IC50 values of 1.0 and 1.7 µM
compared to an IC50 value of 0.82 µM for the parent
compound 3-AP. However, while the 3-methylamino
derivative, compound 7, showed almost equivalent
activity to 3-AP as an inhibitor of the growth of L1210
cells, with IC50 values of 1.5 and 1.4 µM, respectively,
the 3-allylamino derivative 16 was inactive as an
inhibitor of the growth of L1210 cells at up to 10 µM.
In contrast to the results with the 3-substituted amino
derivatives, alkylation of the amino group of 5-AP with
methyl, ethyl, allyl, and propyl groups, compounds 8,
with IC50 values of 3.4, 4.4, 5.1, and 7.8 µM, respectively.
Thus, it appears that the amino function in the 5-posi-
tion of the pyridine ring is more tolerant to the intro-
duction of an alkyl group than the corresponding amino
function in the 3-position for inhibitory activity on CDP
reductase activity and growth inhibition of L1210 cells.
The 3,5-diamino derivative, compound 25, which com-
bines the structural features of both 3-AP and 5-AP, was
much less active as an inhibitor of CDP reductase
activity than either monosubstituted agents. The rela-
tively potent inhibitory activity of compound 25 against
the growth of L1210 cells may well be the result of the
production of a metabolic lesion other than inhibition
of ribonucleoside diphosphate reductase. Introduction
of a bulky morpholinomethyl group onto the 4-position
of the pyridine ring of 5-AP resulted in an agent,
compound 32, that was much less active than the parent
compound, 5-AP. Insertion of a methylene substituent
between the 5-amino function and the pyridine ring of
5
-AP resulted in the production of compound 40, which
showed activity comparable to that of the parent
compound.
Since the various substituents would be expected to
impact on the pharmacokinetic properties of the syn-
thesized agents, the tumor-inhibitory properties of the
3- and 5-amino derivatives of pyridine-2-carboxaldehyde
thiosemicarbazone were determined by measuring their
ability to prolong the survival time of mice bearing the
L1210 leukemia. Compounds were administered in
suspension by intraperitoneal (ip) injection to groups
of five tumor-bearing mice, twice daily for 6 consecutive
1
7, 18, and 19, respectively, resulted in agents that were
more active as inhibitors of the reductase enzyme than
-AP, with IC50 values of 1.3, 1.0, 1.4, and 1.4 µM,
5
respectively, compared to an IC50 value of 2.7 µM for
the parent compound 5-AP. Substitution of the amino
group of 5-AP with the more bulky butyl group (com-
pound 20) showed activity similar to that of the parent
compound 5-AP, having an IC50 value of 2.5 µM.
However, while the 5-methylamino derivative, com-
pound 8, showed activity equivalent to that of 5-AP as
an inhibitor of the growth of L1210 cells, with IC50
values of 2.4 and 2.5 µM, respectively, the inhibitory
activity of compounds 17, 18, 19, and 20 was gradually
decreased as the size of the alkyl group was increased,
3
2
days, by methodology previously described.
The re-
sults of these tests are shown in Table 2. A wide range
of dosage levels of each compound of from 10 to 80 mg/
kg per day × 6 were tested, and the prolongation of life
produced by the optimum effective daily dose of each
compound is listed. For comparison, the effects of the
two parental compounds, 3-AP and 5-AP, are also
included. As shown in Table 2, the 5-methylamino,
5-ethylamino, and 5-allylamino derivatives, compounds

2
590 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
, 17, and 18, showed significant antitumor activity,
Liu et al.
8
(d, 3H, NCH
exchangeable), 5.72 (s, 1H, 2-CH), 6.82 (m, 1H, 4-H), 7.30 (d,
H, 3H), 7.90 (d, 1H, 6-H). Anal. (C ) C, H, N.
-(Meth yla m in o)pyr idin e-2-ca r boxa ldeh yd e Th iosem i-
3 2 2 2
), 4.05 (m, 4H, CH CH ), 4.10 (br s, 1H, NH, D O
producing corresponding % T/C values of 223, 204 and
15 at their optimum daily dosage levels. The antitu-
1
9 2
H12NO
2
3
mor activity of these compounds was comparable to that
of 3-AP, which had a maximum % T/C value of 225, and
slightly better than that of 5-AP, which had a maximum
ca r ba zon e (7). To a solution of 3-(methylamino)-2-(1,3-
dioxolan-2-yl)pyridine (5, 0.30 g, 1.6 mmol) in 5 mL of ethanol,
mL of water, and 1 mL of concentrated hydrochloric acid
was added 0.16 g (1.7 mmol) of thiosemicarbazide. The
mixture was stirred at room temperature overnight, refluxed
for 1 h, cooled, and filtered. The crude yellow hydrochloride
salt was dissolved in 20 mL of water and filtered. To the
filtrate was added 10 mL of 5% sodium bicarbonate solution.
The mixture was stirred at room temperature for 1 h, filtered,
and washed with water and ethanol to yield 0.23 g (70%) of
4
%
T/C value of 194. The 3-methylamino and 5-propyl-
amino derivatives, compounds 7 and 19, exhibited
moderate antitumor activity, and the 3-allylamino,
5
-butylamino, and 5-amino-4-morpholinomethyl deriva-
tives, compounds 16, 20, and 32, showed little or no
activity. In agreement with the effects of these agents
on CDP reductase activity and the growth of L1210 cells,
substitution of a bulky group such as a butyl group onto
the 5-amino function of 5-AP (20) or a 4-morpholino-
methyl group onto the pyridine ring of 5-AP (32) led to
a decrease or loss of activity both in vitro and in vivo.
The 3,5-diamino derivative, compound 25, gave a % T/C
value of 135 with 40% of the animals being 60-day long-
term survivors. This effect was obtained, however, at
the expense of a considerable loss in body weight (17.1%)
post drug treatment. The 5-aminomethyl derivative,
compound 40, was comparable to 5-AP as an antineo-
plastic agent, with a % T/C value of 168 along with one
of the five animals being a 60-day long-term survivor.
In summary, the 5-methylamino, 5-ethylamino, and
1
product as a yellow solid: mp 240-242 °C dec; H NMR (250
MHz, DMSO-d
D O exchangeable), 7.08 (d, 1H, 4-H, J
6
) δ 2.88 (d, 3H, NCH
3
), 6.92 (br s, 1H, 3-NH,
) 6 Hz), 7.22 (dd,
2
4,5
1H, 5-H, J 4,5 ) 6 Hz, J 5,6 ) 4 Hz), 7.86 (d, 1H, 6-H, J 5,6 ) 4
Hz), 8.24 and 8.36 (two s, 2H, CSNH , D O exchangeable), 8.32
s, 1H, 2-CH), 11.05 (s, 1H, NNH, D O exchangeable). Anal.
2
2
(
(
2
C
8
H
11
N
5
S‚0.2H
2
O) C, H, N.
5
-(Meth yla m in o)pyr idin e-2-ca r boxa ldeh yd e Th iosem i-
ca r ba zon e (8). This compound was prepared from 5-(meth-
ylamino)-2-(1,3-dioxolan-2-yl)pyridine (6, 0.50 g, 2.7 mmol) by
the procedure employed for the synthesis of 3-(methylamino)-
pyridine-2-carboxaldehyde thiosemicarbazone (7): yield 0.45
1
g (80%); mp 217-218 °C dec; H NMR (250 MHz, DMSO-d ) δ
6
3 2
2.75 (d, 3H, NCH ), 6.25 (br s, 1H, 3-NH, D O exchangeable),
6
J
7
1
0
.81 (dd, 1H, 4-H, J 3,4 ) 7 Hz, J 4,6 ) 2 Hz), 7.85 (d, 1H, 3-H,
3,4 ) 7 Hz), 7.90 (d, 1H, 6-H, J 4,6 ) 2 Hz), 7.95 (s, 1H, 2-CH),
5
-allylamino derivatives of pyridine-2-carboxaldehyde
.98 (s, 2H, CSNH
1.05 (s, 1H, NNH, D
.25H O) C, H, N.
-(Acetyla m in o)-2-(1,3-d ioxola n -2-yl)p yr id in e (9). To
2
, D
2
O exchangeable), 8.32 (s, 1H, 2-CH),
thiosemicarbazone (compounds 8, 17, and 18) were good
inhibitors of CDP reductase activity and showed sig-
nificant antitumor activity in vivo with acceptable
toxicity as indicated by loss in body weight, and there-
fore, further evaluation of these agents as potential
anticancer drugs appears to be merited.
2
O exchangeable). Anal. (C S‚
8
11 5
H N
2
3
a stirred solution of 1 (2.0 g, 12 mmol) in 10 mL of anhydrous
pyridine in an ice bath was added dropwise 2 mL of acetic
anhydride at 0-5 °C. The reaction mixture was stirred
overnight and evaporated in vacuo to dryness. The residue
was dissolved in CH
bicarbonate, brine, and water, and then dried (anhydrous
MgSO ). The solvent was removed, and the residue was
purified on a silica gel column (CH Cl /AcOEt, 1:4, v/v; R 0.27)
to produce 2.0 g (80%) of product: mp 92-93 °C; H NMR (90
MHz, CDCl ) δ 2.05 (s, 3H, CH ), 4.12 (m, 4H, CH CH ), 5.88
s, 1H, 2-CH), 7.22 (dd, 1H, 5-H, J 4,5 ) 4 Hz, J 5,6 ) 8 Hz), 8.25
d, 1H, 4-H, J 4,5 ) 4 Hz), 8.60 (d, 1H, 6-H, J 5,6 ) 8 Hz), 8.60
br s, 1H, NH, D O exchangeable). Anal. (C10 ) C, H,
2 2
Cl (30 mL), washed with 10% sodium
Exp er im en ta l Section
Melting points were determined with a Thomas-Hoover
Unimelt apparatus and are uncorrected. H NMR spectra
were recorded on a Varian EM-390 90 MHz NMR spectrometer
4
or a Bruker WM-250 250 MHz spectrometer with Me Si as
the internal reference. TLC was performed on EM precoated
silica gel sheets containing a fluorescent indicator. Elemental
analyses were carried out by Baron Consulting Co., Orange,
CT. Where analyses are indicated only by symbols of the
elements, the analytical results for those elements were within
4
1
2
2
f
1
3
3
2
2
(
(
(
2
12 2 3
H N O
N.
3
-(N-Acet yl-N-a llyla m in o)-2-(1,3-d ioxola n -2-yl)p yr i-
(
0.4% of the theoretical value.
d in e (11). To a solution of compound 9 (2.2 g, 11 mmol) in
anhydrous THF (80 mL) was added carefully a suspension of
NaH (0.64 g of 80% oil dispersion, 21.2 mmol) in 20 mL of
anhydrous THF. After effervescence ceased, the reaction
mixture was refluxed for 30 min and a solution of allyl bromide
3
-(Meth yla m in o)-2-(1,3-d ioxola n -2-yl)p yr id in e (5). So-
dium (0.6 g, 26 mmol) was added slowly to 15 mL of methanol.
Once the evolution of hydrogen had ceased, 3-amino-2-(1,3-
2
4
dioxolan-2-yl)pyridine (1 g, 6.0 mmol) and paraformaldehyde
0.42 g, 14 mmol) were added. The mixture was stirred at 50
C for 20 h, and then NaBH (0.4 g, 10.6 mmol) was added.
(
°
(2.6 g, 22 mmol) in 10 mL of anhydrous THF was added; the
4
mixture was then refluxed for another 2 h. The cooled reaction
mixture was evaporated in vacuo to dryness, and the oil
After the mixture was heated under reflux for 1 h, 10 mL of 1
N KOH was added and the resulting solution was refluxed
for 2 h. The reaction mixture was cooled, and the solvent was
removed under reduced pressure. The residue was partitioned
between methylene chloride and water, and the aqueous layer
was extracted with methylene chloride. The combined organic
layer was washed with brine and then water and dried over
residue was dissolved in CH
and dried (MgSO ). The solvent was evaporated, and the
residue was chromatographed on a silica gel column (CH Cl
EtOH, 20:1, v/v; R 0.40) to produce 2.3 g (87%) of product as
an oil: 1H NMR (90 MHz, CDCl
) δ 1.85 (s, 3H, COCH ), 3.60-
), 4.05-4.32 (m, 4H, OCH CH O), 4.85-
), 5.70-6.05 (m, 1H, dCH), 5.90 (s, 1H,
2 2
Cl (50 mL), washed with water,
4
2
2
/
f
3
3
3
5
2
.90 (m, 2H, NCH
.20 (m, 2H, dCH
2
2
2
MgSO
the residue was chromatographed on a silica gel (120 g) column
CH Cl /EtOH, 20:1, v/v; R 0.33). The product was obtained
as an oil (0.4 g, 37%): H NMR (90 MHz, CDCl ) δ 2.80 (d,
H, NCH ), 4.10 (m, 4H, CH CH ), 5.20 (br s, 1H, NH, D
exchangeable), 5.75 (s, 1H, 2-CH), 6.80-7.20 (m, 2H, 4-H and
-H), 7.75 (dd, 1H, 6-H). Anal. (C ) C, H, N.
-(Meth ylam in o)-2-(1,3-dioxolan -2-yl)pyr idin e (6). This
compound was prepared from 5-amino-2-(1,3-dioxolan-2-yl)-
4
. The filtrate was evaporated in vacuo to dryness, and
2
-CH), 7.40 (m, 2H, 4-H and 5-H), 8.75 (m, 1H, 6-H). Anal.
) C, H, N.
(
2
2
f
1
(C13
H
16
N
2
O
3
3
Compounds 12-15 were synthesized from 5-(acetylamino)-
3
3
2
2
2
O
2
-(1,3-dioxolan-2-yl)pyridine (10)24 and the appropriate allyl
5
9 2
H12NO
halide by methodology similar to that described for the
preparation of compound 11.
5
5-(N-Acet yl-N-et h yla m in o)-2-(1,3-d ioxola n -2-yl)p yr i-
2
4
pyridine (1 g, 6.0 mmol) by the procedure employed for the
d in e (12): isolated as an oil (0.34 g, 72%); TLC R
f
0.70 (CH
) δ 1.05-1.25
), 3.65-3.90 (q, 2H, NCH ),
2
-
1
synthesis of 3-(methylamino)-2-(1,3-dioxolan-2-yl)pyridine: yield
Cl
2
/EtOH, 10:1, v/v); H NMR (90 MHz, CDCl
), 1.90 (s, 3H, COCH
3
1
0
.7 g (63%); mp 85-87 °C; H NMR (90 MHz, CDCl
3
) δ 2.85
(t, 3H, CH
3
3
2

Pyridine-2-carboxaldehyde Thiosemicarbazones
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2591
4
2
.10-4.20 (m, 4H, OCH
2
CH
2
O), 5.80 (s, 1H, 2-CH), 7.62 (m,
) C,
precipitated black selenium. The filtrate was evaporated in
vacuo to dryness, and the residue was chromatographed on a
H, 3-H and 4-H), 8.50 (s, 1H, 6-H). Anal. (C12
16 2 3
H N O
H, N.
-(N-Acet yl-N-a llyla m in o)-2-(1,3-d ioxola n -2-yl)p yr i-
d in e (13): isolated as an oil (1.1 g, 84%); TLC R 0.63 (CH
) δ 1.90 (s, 3H,
O), 4.25 (d, 2H, NCH ),
), 5.75-5.90 (m, 1H, dCH), 5.88 (s,
H, 2-CH), 7.55 (m, 2H, 3-H and 4-H), 8.75 (m, 1H, 6-H). Anal.
‚0.25EtOH) C, H, N.
-(N-Acetyl-N-n -p r op yla m in o)-2-(1,3-d ioxola n -2-yl)p yr -
id in e (14): isolated as an oil (1.0 g, 76%); TLC R 0.65 (CH
) δ 0.90-1.05
), 1.92 (s, 3H, COCH ),
), 4.10-4.20 (m, 4H, OCH CH O), 5.85
s, 1H, 2-CH), 7.60 (m, 2H, 3-H and 4-H), 8.45 (d, 1H, 6-H).
Anal. (C14 ) C, H, N.
-(N-Acetyl-N-n -bu tyla m in o)-2-(1,3-d ioxola n -2-yl)p yr i-
d in e (15): isolated as an oil (1.1 g, 78%); TLC R 0.67 (CH
) δ 0.90 (t, 3H,
), 1.88 (s, 3H, COCH ), 3.72
CH O), 5.85 (s, 1H, 2-CH),
.55 (m, 2H, 3-H and 4-H), 8.35 (d, 1H, 6-H). Anal. (C13
‚0.25EtOH) C, H, N.
silica gel column (CH
2 2
Cl /AcOEt, 20:1, v/v) to afford 3.8 g of a
5
yellow syrup as a mixture of compounds 22a and 22b, which
was further refluxed with ethylene glycol (3 mL) and 50 mg
of p-toluenesulfonic acid monohydrate in toluene overnight.
The mixture was cooled and then washed with 20 mL of 10%
f
2
-
1
Cl
2
/EtOH, 10:1, v/v); H NMR (90 MHz, CDCl
), 4.10-4.20 (m, 4H, OCH CH
.00-5.20 (m, 2H, dCH
3
COCH
3
2
2
2
5
1
2
NaHCO
layer was dried over anhydrous MgSO
removed under reduced pressure. The residue was chromato-
graphed on a silica gel column (CH Cl /EtOAc, 30:1, v/v; R
0.36) to afford 3.0 g (79%) of product as a white solid: mp 109-
3
solution, followed by 25 mL of water. The toluene
4
, and the solvent was
13 16 2 3
(C H N O
5
2
2
f
f
2
-
1
1
Cl
t, 3H, CH
.62-3.85 (t, 2H, NCH
2
/EtOH, 10:1, v/v); H NMR (90 MHz, CDCl
3
110 °C; H NMR (90 MHz, CDCl
6.65 (s, 1H, 2-CH), 9.12 (m, 1H, 4-H), 9.70 (d, 1H, 6-H). Anal.
(C ) C, H, N.
3,5-Diam in o-2-(1,3-dioxolan -2-yl)pyr idin e (24). The dini-
3
) δ 4.10 (m, 4H, CH
2 2
CH ),
(
3
(
3
), 1.55-1.82 (m, 2H, CH
2
3
2
2
2
8 7 3 3
H N O
H
20
N
2
O
3
tro derivative 23 (0.50 g, 2.1 mmol) was dissolved in 120 mL
of ethanol and hydrogenated in a Parr apparatus under 50
psi of pressure in the presence of 10% Pd/C (70 mg) for 3 h.
After filtration, the filtrate was evaporated under reduced
pressure to give the product (0.33 g, 89%) as an off-white solid,
mp 60-62 °C, which was used for the next step in the reaction
5
f
2
-
1
Cl
2
/EtOH, 10:1, v/v); H NMR (90 MHz, CDCl
), 1.20-1.50 (m, 4H, CH CH
t, 2H, NCH ), 4.15 (m, 4H, OCH
3
CH
3
2
2
3
(
7
N
2
2
2
1
H
18
-
without further purification: ninhydrin positive; H NMR (90
2
O
3
MHz, DMSO-d
br s, 4H, 3- and 5-NH
6
) δ 3.85 (m, 4H, CH
2 2
CH ), 4.70 and 4.85 (two
, D O exchangeable), 5.40 (s, 1H, 2-CH),
Compounds 16-20 were synthesized from the corresponding
2
2
compounds 11-15 by methodology similar to that described
for the preparation of compound 7.
6.15 (d, 1H, 4-H), 7.05 (d, 1H, 6-H).
3,5-Dia m in op yr id in e-2-ca r boxa ld eh yd e Th iosem ica r -
ba zon e (25). This compound was prepared from 24 (0.22 g,
1.2 mmol) by the procedure employed for the synthesis of 7:
3
-(Allyla m in o)p yr id in e-2-ca r b oxa ld eh yd e t h iosem i-
1
ca r ba zon e (16): yield 1.9 g (77%); mp 215-217 °C dec; H
NMR (250 MHz, DMSO-d ) δ 4.00 (m, 2H, NCH ), 5.14 (m,
H, vinyl H), 5.90 (m, 1H, vinyl H), 6.72 (br s, 1H, 3-NH, D
exchangeable), 7.47 (d, 1H, 4-H, J 4,5 ) 6 Hz), 7.53 (dd, 1H,
1
6
2
yield 0.22 g (80%); mp 239-241 °C dec; H NMR (250 MHz,
2
2
O
DMSO-d
6
2 2
) δ 5.50 and 6.05 (two br s, 4H, 3- and 5-NH , D O
exchangeable), 6.15 (d, 1H, 4-H, J 4,6 ) 2 Hz), 7.30 (d, 1H, 6-H,
4,6 ) 2 Hz), 7.60 and 7.70 (two s, 2H, CSNH , D O exchange-
able), 8.10 (s, 1H, 2-CH), 10.90 (s, 1H, NNH, D O exchange-
able). Anal. (C S) C, H, N.
5
8
1
-H, J 4,5 ) 6 Hz, J 5,6 ) 4 Hz), 7.98 (d, 1H, 6-H, J 5,6 ) 4 Hz),
.43 (s, 2H, CSNH , D O exchangeable), 8.52 (s, 1H, 2-CH),
0.08 (s, 1H, NNH, D O exchangeable). Anal. (C10 S‚-
J
2
2
2
2
2
2
H
13
N
5
7 10 6
H N
HCl) C, H, N.
2-(1,3-Dioxola n -2-yl)-5-n it r op yr id in e -4-ca r b oxa ld e -
5
-(Eth yla m in o)p yr id in e-2-ca r boxa ld eh yd e th iosem i-
h yd e (27). A mixture of 2-(1,3-dioxolan-2-yl)-4-methyl-5-
nitropyridine (26, 6.5 g, 16 mmol) and selenium dioxide (10
1
23
ca r ba zon e (17): yield 0.34 g (72%); mp 197-198 °C dec; H
NMR (250 MHz, DMSO-d ) δ 1.15 (t, 3H, CH ), 3.05 (m, 2H,
NCH ), 6.12 (br s, 1H, 3-NH, D O exchangeable), 6.82 (dd, 1H,
-H, J 3,4 ) 7 Hz, J 4,6 ) 2 Hz), 7.76 (d, 1H, 3-H, J 3,4 ) 7 Hz),
.84 (d, 1H, 6-H, J 4,6 ) 2 Hz), 7.86 (s, 2H, CSNH
6
3
g, 90 mmol) was refluxed in anhydrous dioxane (100 mL) until
TLC showed that the reaction was complete (about 40 h). The
reaction mixture was evaporated in vacuo to dryness, and the
residue was stirred with 200 mL of methylene chloride for 1 h
and then filtered. The filtrate was concentrated to a small
2
2
4
7
2
, D
2
O
O
exchangeable), 7.95 (s, 1H, 2-CH), 11.08 (s, 1H, NNH, D
exchangeable). Anal. (C S) C, H, N.
-(Allyla m in o)p yr id in e-2-ca r b oxa ld eh yd e t h iosem i-
2
9
H
13
N
5
volume and chromatographed on a silica gel column (CH
EtOH, 25:1, v/v; R 0.56) to afford 4.8 g (66%) of product as an
off-white solid: mp 77-78 °C; H NMR (90 MHz, CDCl
4.15 (m, 4H, CH CH ), 5.90 (s, 1H, 2-CH), 7.95 (s, 1H, 3-H),
2
Cl
) δ
) C,
2
/
5
f
1
1
ca r ba zon e (18): yield 0.70 g (74%); mp 185-187 °C dec; H
NMR (250 MHz, DMSO-d ) δ 3.90 (m, 2H, NCH ), 5.20 (m,
H, vinyl H), 5.92 (m, 1H, vinyl H), 6.52 (br s, 1H, 3-NH, D
3
6
2
2
2
2
2
O
9.32 (s, 1H, 6-H), 10.50 (s, 1H, 4-CHO). Anal. (C
H, N.
9 8 2 5
H N O
exchangeable), 7.47 (d, 1H, 4-H, J 4,5 ) 6 Hz), 7.55 (dd, 1H,
5
8
-H, J 4,5 ) 6 Hz, J 5,6 ) 4 Hz), 7.98 (d, 1H, 6-H, J 5,6 ) 4 Hz),
.22 (s, 1H, 2-CH), 8.42 and 8.75 (two s, 2H, CSNH , D
O exchangeable). Anal.
2-(1,3-Dioxola n -2-yl)-4-(h yd r oxym et h yl)-5-n it r op yr i-
d in e (28). To a solution of compound 27 (4.6 g, 21 mmol) in
2
2
O
exchangeable), 10.05 (s, 1H, NNH, D
S) C, H, N.
-(n -P r opylam in o)pyr idin e-2-car boxaldeh yde th iosem i-
2
100 mL of methanol was added dropwise a solution of NaBH
(0.4 g) in 4 mL of water at 0-5 °C with stirring. After the
addition of the NaBH , the reaction mixture was stirred for
4
10 13 5
(C H N
5
4
1
ca r ba zon e (19): yield 0.56 g (80%); mp 192-194 °C dec; H
NMR (250 MHz, DMSO-d ) δ 1.05 (t, 3H, CH ), 1.50 (m, 2H,
CH ), 3.10 (m, 2H, NCH ), 6.90 (br s, 1H, 3-NH, D O exchange-
able), 7.10 (dd, 1H, 4-H, J 3,4 ) 6 Hz, J 4,6 ) 2 Hz), 7.86 (d, 1H,
an additional 30 min, and then evaporated to dryness. The
residue was stirred with 20 mL of water for 30 min and the
white solid that formed was collected by filtration and washed
with water to afford 3.4 g (77%) of product, which was used
for the next step in the reaction. An analytical sample was
6
3
2
2
2
3
2
1
1
-H, J 3,4 ) 6 Hz), 7.92 (d, 1H, 6-H, J 4,6 ) 2 Hz), 7.95 (s, 1H,
-CH), 8.12 and 8.25 (two s, 2H, CSNH , D O exchangeable),
1.08 (s, 1H, NNH, D O exchangeable). Anal. (C10 S‚
.2H O) C, H, N.
-(n -Bu tyla m in o)p yr id in e-2-ca r boxa ld eh yd e th iosem i-
2
2
obtained by purification of the crude product on a silica gel
1
2
H
13
N
5
column (CH
NMR (90 MHz, CDCl
4-CH ), 5.05 (t, 1H, OH, D
2
2
Cl
2
/AcOEt, 4:1, v/v, R
) δ 4.21 (m, 4H, CH
O exchangeable), 5.88 (s, 1H, 2-CH),
f
0.45): mp 119-120 °C; H
2
3
2 2
CH ), 5.00 (s, 2H,
5
2
1
ca r ba zon e (20): yield 0.7 g (74%); mp 179-180 °C dec; H
NMR (250 MHz, DMSO-d ) δ 0.90 (t, 3H, CH ), 1.32-1.52 (m,
H, CH CH ), 3.08 (m, 2H, NCH ), 6.30 (br s, 1H, 3-NH, D
8.10 (s, 1H, 3-H), 9.21 (s, 1H, 6-H). Anal. (C
N.
9 10 2 5
H N O ) C, H,
6
3
4
2
2
2
2
O
2-(1,3-Dioxola n -2-yl)-4-[[(m eth ylsu lfon yl)oxy]m eth yl]-
5-n itr op yr id in e (29). To a solution of compound 28 (0.50 g,
2.2 mmol) in 15 mL of methylene chloride was added dropwise
0.30 g (2.6 mmol) of methanesulfonyl chloride at 0-5 °C with
stirring, followed by 0.3 mL of triethylamine. The reaction
mixture was stirred for about 1 h, at which time TLC showed
that the reaction was complete. The mixture was washed with
exchangeable), 6.90 (dd, 1H, 4-H, J 3,4 ) 6 Hz, J 4,6 ) 1.5 Hz),
7
7
.86 (d, 1H, 3-H, J 3,4 ) 6 Hz), 7.90 (d, 1H, 6-H, J 4,6 ) 1.5 Hz),
.93 (s, 1H, 2-CH), 8.00 and 8.10 (two s, 2H, CSNH , D
O exchangeable). Anal.
2
2
O
exchangeable), 10.64 (s, 1H, NNH, D
S) C, H, N.
,5-Din itr o-2-(1,3-d ioxola n -2-yl)p yr id in e (23). A mix-
2
11 17 5
(C H N
3
ture of 2,4-dinitro-2-methylpyridine (21, 2.9 g, 16 mmol) and
selenium dioxide (2.9 g, 26 mmol) was refluxed in 20 mL of
water (2 × 5 mL), dried (MgSO
was evaporated to dryness, and the residue was purified by
silica gel column chromatography (CH Cl /AcOEt, 4:1, v/v, R
0.75) to give 0.5 g (75%) of product as white crystals: mp 126-
4
), and filtered. The filtrate
9
5% ethanol under an atmosphere of nitrogen for 20 h. The
2
2
f
reaction mixture was cooled and filtered to remove the

2
592 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Liu et al.
1
1
(
(
27 °C; H NMR (90 MHz, CDCl
3
) δ 3.15 (s, 3H, CH
), 5.82 (s, 2H, 4-CH ), 6.00 (s, 1H, 2-CH), 8.05
s, 1H, 3-H), 9.45 (s, 1H, 6-H). Anal. (C10 S) C, H, N.
-(1,3-Dioxola n -2-yl)-4-(4-m or p h olin ylm eth yl)-5-n itr o-
3
), 4.21
chromatography (CH
2
Cl
2
/AcOEt, 2:1, v/v, R
f
0.62) to yield 0.7
1
m, 4H, CH
2
CH
2
2
g (56%) of product as an oil: H NMR (90 MHz, CDCl
2.08, and 2.10 (three s, 9H, three CH ), 5.15 and 5.20 (s, 2H,
), 7.50 (d, 1H, 3-H), 7.65 (s, 1H, 2-CH), 7.77 (d, 1H, 4-H),
.75 (s, 1H, 6-H). Anal. (C13 ) C, H, N.
-(Am in om eth yl)pyr idin e-2-car boxaldeh yde Th iosem i-
3
) δ 2.05,
H
12
N
2
O
7
3
5
8
-CH
2
2
16 2 5
H N O
p yr id in e (30). A mixture of 29 (0.35 g, 1.2 mmol) and
morpholine (0.62 g, 7.2 mmol) in 15 mL of methylene chloride
was stirred until TLC showed that the reaction was complete
5
ca r ba zon e (40). This compound was prepared from 39 (0.60
g, 2.1 mmol) by the procedure employed for the synthesis of
(
∼2 d). The reaction mixture was evaporated to dryness, and
the residue was purified by silica gel column chromatography
CH Cl /AcOEt, 4:1, v/v, R 0.44) to afford 0.27 g (76%) of
product as an off-white solid: mp 98-99 °C; H NMR (90 MHz,
CDCl ) δ 2.40 [m, 4H, N(CH ], 3.62 [m, 4H, O(CH ], 3.75
s, 2H, 4-CH ), 4.10 [m, 4H, (OCH ], 5.75 (s, 1H, 2-CH), 7.72
s, 1H, 3-H), 8.95 (s, 1H, 6-H). Anal. (C13 ) C, H, N.
-(1,3-Dioxola n -2-yl)-4-(4-m or p h olin ylm et h yl)-5-a m i-
n op yr id in e (31). A solution of 30 (0.25 g, 0.85 mmol) in
ethanol (60 mL) was hydrogenated in a Parr apparatus at 50
psi of hydrogen in the presence of 10% Pd/C (0.1 g) for 3 h.
The reaction mixture was filtered through a Celite pad, and
the catalyst was washed with ethanol. The combined filtrate
and washings were evaporated in vacuo to dryness and
coevaporated with benzene. The remaining solid was recrys-
tallized from ethanol/ether to afford 0.21 g (95%) of product
as white crystals: mp 102-104 °C; H NMR (90 MHz, CDCl
δ 2.40 [m, 4H, N(CH
O(CH ], 4.05 [m, 4H, (OCH
exchangeable), 5.65 (s, 1H, 2-CH), 7.12 (s, 1H, 3-H), 8.00 (s,
H, 6-H). Anal. (C13 ) C, H, N.
-(4-Mor p h olin ylm eth yl)-5-a m in op yr id in e-2-ca r boxa l-
1
7
: yield 0.34 g (75%); mp 243-245 °C dec; H NMR (250 MHz,
DMSO-d
6
) δ 4.52 (s, 2H, CH
2 2 2
), 5.20 (br s, 2H, NH , D O
(
2
2
f
1
exchangeable), 7.95 (dd, 1H, 4-H, J 3,4 ) 6 Hz, J ) 1.5 Hz),
4,6
8
.04 (d, 1H, 6-H, J 4,6 ) 1.5 Hz), 8.10 and 8.16 (two s, 2H,
CSNH , D O exchangeable), 8.52 (s, 1H, 2-CH), 11.25 (s, 1H,
O exchangeable). Anal. (C S‚0.25H O) C, H,
3
2
)
2
2 2
)
(
(
2
2 2
)
2
2
NNH, D
N.
2
8
H
11
N
5
2
17 3 5
H N O
2
Assay of CDP Redu ctase Activity. Ribonucleoside diphos-
2
6
phate reductase was prepared as previously described and
assayed by the method of Steeper and Steuart.33 [14C]CDP
(3.75 nmol, 0.03 µCi; New England Nuclear Corp., Boston, MA)
was incubated with 150 mmol of ATP, 900 nmol of dithiothrei-
tol, and aliquots of the non-heme iron subunit and the effector-
binding subunit of ribonucleoside diphosphate reductase in a
volume of 0.15 mL for 30 min at 37 °C. The reaction was
terminated by heating in a boiling water bath for 4 min. After
snake venom treatment to convert nucleotides to the nucleo-
side level, deoxycytidine was separated from cytidine on a
Dowex-1-borate column.
1
3
)
2
)
2
], 3.45 (s, 2H, 4-CH
2
), 3.62 [m, 4H,
2
)
2
2
)
2
], 4.60 (br s, 2H, 5-NH
2
, D
2
O
Assa y of th e In h ibition of th e Gr ow th of L1210 Leu -
k em ia Cells. L1210 cell growth was assayed by the method
1
19 3 3
H N O
4
34
of Cory et al. Ninety-six well plates were seeded with 2000
d eh yd e Th iosem ica r ba zon e (32). This compound was
prepared from 31 (0.20 g, 0.75 mmol) by the procedure
cells/well, and test agents were added at various concentra-
tions 24 h later. After 72 h, 5-(3-(carboxymethoxy)phenyl)-2-
employed for the synthesis of 7: yield 0.21 g (95%); mp 248-
(4,5-dimethylthiazoyl)-3-(4-sulfophenyl)tetrazolium, inner salt
1
2
49 °C dec; H NMR (250 MHz, DMSO-d
6
) δ 3.32 [m, 4H,
], 4.50 (s, 2H, 4-CH ), 4.60 (br
O exchangeable), 6.35 (s, 1H, 3-H), 7.15 (s,
H, 6-H), 7.60 and 7.70 (two s, 2H, CSNH , D O exchangeable),
.90 (s, 1H, 2-CH), 10.80 (s, 1H, NNH, D O exchangeable).
S‚HCl) C, H, N.
-[(Acet yla m in o)m et h yl]-2-(a cet oxym et h yl)p yr id in e
37). A mixture of 5-cyano-2-picoline (33, 3.3 g, 28 mmol) and
and phenazine methosulfate were added, and plates were
incubated at 37 °C and read at 492 nm 2-3 h later as
previously described. IC50 values were determined using at
N(CH
2
)
2
], 3.85 [m, 4H, O(CH
, D
2
)
2
2
s, 2H, 5-NH
2
2
34
1
7
2
2
least five different concentrations for each agent.
2
Anal. (C12
18 6
H N
Ack n ow led gm en t. This research was supported by
the State of Connecticut’s Critical Technologies Program
in Drug Design and Public Health Service Grant CA-
5
(
active Raney Ni (3.2 g) in acetic acid (100 mL) was hydroge-
nated in a Parr apparatus at 5 psi of hydrogen for 4 h. The
catalyst was removed by filtration, and the filtrate was
evaporated in vacuo to dryness. The residue was purified by
silica gel column chromatography (CH
.52) to afford 34 (1.5 g, 44%) as an oil; H NMR (90 MHz,
CDCl ) δ 2.55 (s, 3H, 2-CH ), 4.70 (s, 2H, 5-CH ), 6.35 (s, 2H,
NH , D O exchangeable), 7.25 (d, 1H, 3-H), 7.75 (d, 1H, 4-H),
.45 (s, 1H, 6-H). The preceding product (34, 1.5 g) was
5
5540 from the National Cancer Institute. We wish to
thank Ms. Regina Loomis for her excellent technical
assistance.
2 2 f
Cl /EtOH, 10:1, v/v, R
1
0
Refer en ces
3
3
2
(
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J M9600454