Synthesis and Structure-Activity Relationships of Novel 7-Substituted 1,4-Dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylic Acids as Antitumor Agents. Part 2

Article abstract of DOI:10.1021/jm0304966

We have previously reported that a series of 7-substituted 6-fluoro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylic acids possess moderate cytotoxic activity. In a further attempt to find clinically useful antitumor agents, we investigated the structure-activity relationships (SARs) of a new series of compounds obtained by changing the C-6 position of the fluorine atom in addition to the C-5 and C-7 positions and evaluating their cytotoxic activity against several murine and human tumor cell lines. Our results showed that the 6-unsubstituted 1,8-naphthyridine structure had the most potent cytotoxic activity against murine P388 leukemia twice that of the 6-fluoro analogue. In addition, introduction of an amino group at the C-5 position did not have any substantial effect on the cytotoxic activity, while both the 5-chloro and 5-trifluoromethyl groups decreased the cytotoxic activity by 5- to 10-fold. Moreover, aminopyrrolidine derivatives at the C-7 position showed more potent cytotoxic activity than other amines or carbon derivatives. Among the 7-(3-aminopyrrolidinyl) derivatives, the trans-3-methoxy-4-methylaminopyrrolidinyl derivative (271) was determined to have potent cytotoxic activity in both in vitro and in vivo assays and high water solubility. Finally, the (S,S)-isomer (AG-7352, 3) of 271, with a cytotoxic activity against human tumor cell lines more potent than that of etoposide, was selected for further development.

Full text of DOI:10.1021/jm0304966

J . Med. Chem. 2004, 47, 2097-2109
2097
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of Novel 7-Su bstitu ted
1,4-Dih yd r o-4-oxo-1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid s a s
An titu m or Agen ts. P a r t 2
Yasunori Tsuzuki,* Kyoji Tomita, Koh-ichiro Shibamori, Yuji Sato, Shigeki Kashimoto, and Katsumi Chiba
Chemistry Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Enoki 33-94, Suita, Osaka 564-0053, J apan
Received October 3, 2003
We have previously reported that a series of 7-substituted 6-fluoro-1,4-dihydro-4-oxo-1-(2-
thiazolyl)-1,8-naphthyridine-3-carboxylic acids possess moderate cytotoxic activity. In a further
attempt to find clinically useful antitumor agents, we investigated the structure-activity
relationships (SARs) of a new series of compounds obtained by changing the C-6 position of
the fluorine atom in addition to the C-5 and C-7 positions and evaluating their cytotoxic activity
against several murine and human tumor cell lines. Our results showed that the 6-unsubstituted
1,8-naphthyridine structure had the most potent cytotoxic activity against murine P388
leukemia twice that of the 6-fluoro analogue. In addition, introduction of an amino group at
the C-5 position did not have any substantial effect on the cytotoxic activity, while both the
5-chloro and 5-trifluoromethyl groups decreased the cytotoxic activity by 5- to 10-fold. Moreover,
aminopyrrolidine derivatives at the C-7 position showed more potent cytotoxic activity than
other amines or carbon derivatives. Among the 7-(3-aminopyrrolidinyl) derivatives, the trans-
3-methoxy-4-methylaminopyrrolidinyl derivative (27l) was determined to have potent cytotoxic
activity in both in vitro and in vivo assays and high water solubility. Finally, the (S,S)-isomer
(AG-7352, 3) of 27l, with a cytotoxic activity against human tumor cell lines more potent than
that of etoposide, was selected for further development.
In tr od u ction
nucleus serves as a unique scaffold for finding new
clinically useful quinolones that have potent antitumor
activity and no cross-resistance with other agents.10
Thus, the 7-(3-aminopyrrolidinyl)-1,4-dihydro-4-oxo-1-
(2-thiazolyl)-1,8-naphthyridine-3-carboxylic acid (1) and
two of its close analogues have been shown to have good
antitumor activity in vitro against murine and human
tumor cell lines and in vivo test for mouse leukemia.
Additionally, the pyridopyrimidine analogue (2) as
another core structure has been demonstrated to be half
as active as the 6-fluoro-1,8-naphthyridine (1). In the
course of our search for more potent antitumor agents,
we focused our interest on modifications at the C-6
position of 1 to examine whether the trend of the
structure-activity relationships (SARs) for this position
would be the same as that for the C-6 position of
quinolone antibacterials. In the field of quinolone an-
tibacterials, a fluorine atom of this position has been
shown to be indispensable for enhanced antibacterial
activity.3 Thus, in this study, we investigated SARs of
a new series of compounds obtained by changing sub-
stituents at the C-6 position of the 1,8-naphthyridine
nucleus in addition to the C-5 and C-7 positions and
evaluating their cytotoxic activity against several mu-
rine and human tumor cell lines. Our findings led us to
a novel antitumor agent, AG-7352 (3), as a candidate
for further development. In this paper, we present these
findings and discuss the SARs and antitumor activity
that have been revealed during the study.
The properties of the quinolone class of antibacterials
have been known for over 40 years.1 Quinolones repre-
sent one of the first classes of antibacterial agents that
act by inhibiting DNA gyrase and topoisomerase IV,
bacterial topoisomerase II enzymes.2 Clearly, the most
significant modification of the quinolone antibacterials
for potent activity has proven to be the introduction of
a 6-fluoro substituent.3 On the other hand, mammalian
topoisomerase II, which possesses a mechanism of
action similar to that of DNA gyrase/topoisomerase IV,
has also been investigated, and accordingly, many
topoisomerase II inhibitors including etoposide, doxo-
rubicin, ellipticine, and amsacrine have been used in
preclinical studies and clinical treatments.4 Although
novel classes of quinolones as mammalian topoi-
somerase II inhibitors have been reported since 1992,
no compound reached clinical trial as an antitumor
agent so far to our knowledge. Generally, quinolone
topoisomerase II inhibitors are characterized by two
basic structures (Figure 1), i.e., 6,8-difluoroquinolone (A-
65282,5 CP-115,953,6 and WIN572947) and quinoben-
zoxazine (A-621768 and A-852269). These reported an-
titumor quinolones all have a fluorine atom at the C-6
position; however, the precise role of the fluorine atom
at the C-6 position has never been clearly defined in
the field of antitumor quinolones.
In a previous paper, we have reported that the
7-substituted 6-fluoro-1-(2-thiazolyl)-1,8-naphthyridine
Ch em istr y
* To whom correspondence should be addressed. Phone: +81-6-
6337-5900. Fax: +81-6-6338-7656. E-mail: yasunori-tuduki@dainippon-
pharm.co.jp.
To study the effect of various substituents at the C-6
position of the 1,8-naphthyridine ring, compounds 9,
10.1021/jm0304966 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/06/2004

2098 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Tsuzuki et al.
F igu r e 1. Structures of AG-7352 and reference compounds.
Sch em e 1a
Sch em e 2a
a
(a) (1) CDI, (2) EtOCOCH2COOK, MgCl2, Et3N; (b) (1)
(EtO)3CH, Ac2O, (2) 2-aminothiazole; (c) K2CO3; (d) R1R2NH; (e)
HCl.
16a , 16b, 16c, and 16d , which have H, NO2, NH2, OH,
and Cl at this position, respectively, were designed. A
synthetic route to the unsubstituted compound 9 is
illustrated in Scheme 1. The 2,6-dichloronicotinic acid
4 was converted to the nicotinoyl acetate 5 with ethyl
malonate potassium salt by means of 1,1′-carbonyldi-
imidazole (CDI). The nicotinoyl acetate 5 was treated
with ethyl orthoformate and acetic anhydride, followed
by reaction with 2-aminothiazole to produce the enami-
noester 6 as a mixture of keto and enol forms. The 1,8-
naphthyridine derivative 7 was obtained in good yield
through base-assisted cyclization reaction of 6. Coupling
reaction of 7 with 3-aminopyrrolidine was followed by
acid hydrolysis of the ester 8 formed to give the desired
1,8-naphthyridine-3-carboxylic acid 9. Synthesis of the
6-nitro derivative 16a , 6-amino derivative 16b, and
6-hydroxy derivative 16c is shown in Scheme 2. Because
attempts to introduce directly a NO2 sustitutent at the
C-6 position of 1,8-naphthyridine ring of 7 were unsuc-
cessful, we tried to prepare the 6-nitro derivative 16a
using the 2,6-dimethoxynicotinic acid 10 as starting
material. Nitration of 10 by means of HNO3 and acetic
a
(a) HNO3, Ac2O; (b) (1) CDI, (2) EtOCOCH2COOK, MgCl2,
Et3N; (c) (1) (EtO)3CH, Ac2O, (2) 2-aminothiazole; (d) K2CO3; (e)
3-(N-Boc-amino)pyrrolidine; (f) HCl-EtOH; (g) H2/Ra-Ni; (h) HCl;
(i) (1) NaNO2-HCl, (2) CuCl.
anhydride gave the 5-nitro compound 11. In a manner
similar to that described above, the naphthyridine ester
14 was prepared from the nicotinic acid 11 via the
ketoester 12 and the enaminoester 13. Reaction of
compound 14 with 3-[N-tert-butyloxycarbonyl(Boc)-ami-
no]pyrrolidine afforded the corresponding 6-nitro naph-
thyridine ester 15a . Reduction of 15a was effected by
hydrogenation in the presence of Ra-Ni to give the
amino derivative 15b. Acid hydrolysis of both com-
pounds 15a and 15b by HCl/EtOH afforded the desired
compounds 16a and 16b, respectively. Acid hydrolysis
of compound 15b at 100 °C for 1 week by concentrated
HCl furnished directly the 6-hydroxy derivative 16c.
Preparation of the 6-chloro derivative 16d by direct

7-Substituted Carboxylic Acids as Antitumor Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2099
Sch em e 3a
group in most cases. The overall yields of compounds
27a -x were calculated from 7 and are listed in Table 3
along with their in vitro cytotoxic activity data against
murine P388 leukemia cells.
The amines used in this study were purchased or
prepared following procedures in the literature except
for the new pyrrolidine derivatives 28-31. The designed
amine parts of 28, 29, 30, and 31, which afforded the
corresponding substituents of the naphthyridinecar-
boxylic acids 27c, 27f, 27m , and 27n , respectively, were
prepared by the methods shown in Scheme 4. The
pyrrolidine derivative 3211 was treated with Boc2O to
give 33, which was converted to 34 by reduction of the
Boc group with sodium bis(2-methoxyethoxy)aluminum
hydride and successive treatment with Boc2O. Deben-
zylation of 34 was achieved by hydrogenation in the
presence of Pd-C and produced the 3,3-disubstituted
pyrrolidine 28. N-Alkylation of the 3-(benzylamino)-
propionitrile 35 with epibromohydrine followed by a
ring-opening reaction of the epoxide using NaNH2
produced the pyrrolidine derivative 36. Treatment of 36
with thionyl chloride, followed by acid hydrolysis of the
cyanide formed, gave the amide 37, which was converted
to the aminopyrrolidine 38 by Hofmann rearrangement
and protection with Boc2O. Transformation of 38 to the
3,4-disubstituted pyrrolidine 29 was achieved by remov-
ing the N-benzyl substituent via reductive cleavage with
ammonium formate in the presence of Pd-C.20 The
pyrrolidine derivative 3922 was treated with acetic
anhydride to give 40, which was converted to 41 by
reduction of the acetyl group with sodium bis(2-meth-
oxyethoxy)aluminum hydride and successive treatment
with Boc2O. Removal of the benzyl group of 41 was
achieved by hydrogenation to give the desired pyrroli-
dine derivative 30. Addition of methanesulfenyl chloride
to the double bond of the 3-pyrroline 42 and successive
treatment with methylamine gave the 3,4-disubstituted
pyrrolidine 43. Acid-mediated removal of the Boc group
completed the synthesis of 31 as a dihydrochloride.
a
(a) (1) n-BuLi, (2) CO2; (b) SOCl2; (c) EtOCOCH2COOH,
MeMgBr; (d) (1) (EtO)3CH, Ac2O, (2) 2-aminothiazole; (e) K2CO3;
(f) benzylamine; (g) H2SO4-AcOH-H2O; (h) (1) H2SO4, AcOH (2)
H2O; (i) (1) 3-aminopyrrolidine, (2) HCl.
chlorination at the C-6 position of the 1,8-naphthyridine
ring of 7 was not successful. Therefore, the 6-chloro
derivative 18 was obtained in good yield by Sandmeyer
reaction of the 6-amino derivative 17, prepared from the
6-nitro derivative 14. Coupling reaction of compound 18
with 3-(N-Boc-amino)pyrrolidine in the presence of DBU
followed by acid hydrolysis of the resultant 15d afforded
the desired compound 16d .
To study the effects of various substituents at the C-5
position of the 1,8-naphthyridine ring, compounds 26a ,
26b, and 26c, which have Cl, CF3, and NH2 at this
position, respectively, were synthesized by the route
depicted in Scheme 3. Treatment of the 2,4,6-trichloro-
pyridine 19a and the 2,6-dichloro-4-trifluoromethyl-
pyridine 19b with n-BuLi at -78 °C followed by car-
boxylation of the resulting carbanion with CO2 gave the
nicotinic acids 20a and 20b, respectively. Reaction of
the nicotinoyl chlorides 21a and 21b, prepared from 20a
and 20b with SOCl2, with ethyl hydrogen malonate and
EtMgBr produced the ketoesters 22a and 22b. These
compounds (22a and 22b) were converted to the naph-
thyridine esters 24a and 24b via the enaminoesters 23a
and 23b according to the procedure described for 7 from
5. Coupling reaction of 24b with 3-aminopyrrolidine was
followed by acid hydrolysis to give the 5-trifluoromethyl
derivative 26b. Acid hydrolysis of 24a by means of
H2SO4-AcOH-H2O afforded the ester 25a . Reaction of
the 5,7-dichloro ester 24a with benzylamine in refluxing
toluene gave only the 5-benzylamino derivative 24d in
good yield. Debenzylation of 24d with H2SO4 was
followed by acid hydrolysis to give the 5-amino deriva-
tive 25c. The desired 5-chloro and 5-amino derivatives
(26a and 26c) were obtained by coupling reaction of the
naphthyridine-3-carboxylic acids 25a and 25c with
3-aminopyrrolidine.
The chiral pyrrolidine part of each isomer of 27l was
prepared by asymmetric synthesis using (+)-tartaric
acid.21 In addition, a large-scale preparation of the chiral
pyrrolidines was achieved through resolution of the
diastereoisomers.22 The absolute configurations of the
chiral pyrrolidines were determined as (S,S)/(R,R) by
X-ray crystallographic analysis of (S,S)-3-methoxy-4-
methylaminopyrrolidine.22
To study the cytotoxic activity of some analogues
possessing a C-C bond at position 7, compounds 45a -e
were synthesized by the route depicted in Scheme 5. The
overall yields of 45a -e were calculated from 7 and are
listed in Table 3 along with their in vitro cytotoxic
activity data against murine P388 leukemia cells. A
Stille-type palladium-catalyzed cross-coupling reaction
of the 7-chloronaphthyridine 7 with stannyl compounds
gave the 7-carbon derivatives 44a -d . Compound 44d
was prepared by coupling reaction of 7 with 1-tri-n-
butylstannyl-2-trimethylsilylacetylene, followed by depro-
tection of the silyl group. Finally, the acetylene deriva-
tive 44d was subjected to 1,3-dipolar addition reaction
with trimethylsilyldiazomethane to give the pyrazole
derivative 44e. Compounds 44a -e were converted to
the naphthyridine acids 45a -e by acid or base hydroly-
sis of the ester.
By a manner similar to that for the synthesis of 9,
various kinds of amines as shown Figure 2 were
incorporated into the C-7 position of the ester 7 to
provide the desired products 27a -x after hydrolysis of
the ester and the protecting group (Scheme 1). When
diamines were used in the coupling reaction with 7, one
of the nitrogens was protected with an acetyl or Boc

2100 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Tsuzuki et al.
F igu r e 2. R1R2N groups used in this study.
Sch em e 4a
Sch em e 5a
a
(a) Z-Sn(n-Bu)3 or Z-SnMe3, PdCl2(PPh3)2; (b) (1) 1-tri-n-
butylstannyl-2-trimethylsilylacetylene, PdCl2(PPh3)2, (2) KF; (c)
TMSCH2N2; (d) HCl; (e) NaOH.
quinolone and naphthyridone series.3 On the other
hand, in the field of antitumoral quinolones, the precise
role of the fluorine atom of the C-6 position has never
been clearly defined, although reported potent antitu-
moral quinolones shown in Figure 1 had the fluorine
atom of the C-6 position in all cases. We have previously
reported that the 6-fluoro-1,8-naphthyridone series have
good antitumor activity both in vitro and in vivo.10
Moreover, the pyridopyrimidine analogue (2), which has
very weak antibacterial activity, has been demonstra-
ted to be half as antitumor-active as 6-fluoro-1,8-naph-
thyridine (1). In a further attempt to find clinically
useful antitumor agents, we investigated SARs of a new
series of compounds obtained by changing substituents
at the C-6 position of the 1,8-naphthyridine nucleus in
addition to the C-5 and C-7 positions and evaluating
their cytotoxic activity against several murine and
human tumor cell lines.
a
(a) Boc2O; (b) (1) Na(OCH2CH2OMe)2AlH2, (2) Boc2O; (c) H2/
Pd-C; (d) (1) epibromohydrin, K2CO3, (2) NaNH2; (e) (1) SOCl2,
(2) concentrated H2SO4; (f) (1) NaOCl-MeOH, (2) concentrated
HCl, (3) Boc2O; (h) ammonium formate, Pd-C; (i) Ac2O; (j) (1)
MeSCl, (2) MeNH2; (k) 30% HCl-EtOH.
Resu lts a n d Discu ssion
As shown in Table 1, the 6-hydrogen compound 9 had
a cytotoxic activity (IC50) against murine P388 leukemia
cells twice that of the 6-fluorine counterpart (1). This
finding indicates a significant difference from what has
been reported in the literature; i.e., potent antitumor
quinolones have in all cases a 6-fluorine atom.5-9 With
In the field of quinolone antibacterials, introduction
of a fluorine atom into the C-6 position opened new era
of fluoroquinolone antibactrerials. It has also been
shown that the optimum substituent at the C-6 position
for antibacterial activity was a fluorine atom in both

7-Substituted Carboxylic Acids as Antitumor Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2101
Ta ble 1. Cytotoxic Activity (IC50) of C-6 Substituted
Compounds against Murine P388 Leukemia Cells
Ta ble 3. Physical Data and Cytotoxic Activity (IC50) of
1-(2-Thiazolyl)naphthyridine-3-carboxylic Acids 27 and 45
against Murine P388 Leukemia Cells
compd
mp, °C
% yielda
IC50,b µg/mL
9
27a
27b
27c
27d
27e
27f
27g
27h
27i
27j
27k
260-264 dec
280-282 dec
243-246
72
91
57
77
75
68
92
33
52
38
50
64
73
83
74
38
82
43
68
78
50
53
95
62
71
41
70
41
29
62
36
22
0.010
0.010
0.019
0.018
0.0096
0.012
0.024
0.11
0.094
0.020
0.017
0.018
0.010
0.0075
0.021
0.025
0.020
0.020
0.066
0.024
0.041
0.057
0.081
>1
297-299
269-271 dec
279-282 dec
255-258 dec
259-262 dec
296-299 dec
245-248
292-295 dec
275-278 dec
270-273
a
compd
X
IC50
,
µg/mL
1
9
16a
16b
16c
16d
F
H
NO2
NH2
OH
Cl
0.021
0.010
2.29
1.01
4.31
0.227
27l
a
Concentration of agent that reduces cell viability by 50%.
(S,S)-27l
(R,R)-27l
27m
27n
27o
27p
27q
27r
27s
27t
27u
27v
27w
27x
45a
45b
45c
45d
45e
278-282 dec
278-282 dec
294-296 dec
271-272
Ta ble 2. Cytotoxic Activity (IC50) of C-5 Substituted
Compounds against Murine P388 Leukemia Cells
263-269 dec
>300c
259-260 decc
275-277 decc
290-293 dec
290-295
263-265
a
compd
X
IC50
,
µg/mL
297-299 dec
298-300 decc
>300c
0.52
>10
9
H
Cl
CF3
NH2
0.010
0.049
0.098
0.0081
0.12
6.33
7.37
>1
2.3
4.34
26a
26b
26c
227-230 decc
272-275 dec
284-288 decc
>300c
a
Concentration of agent that reduces cell viability by 50%.
>300c
regard to other substituents, the 6-NO2, 6-NH2, and
6-OH compounds 16a -c were far less active than the
6-F counterpart (1). In addition, the 6-Cl compound 16d ,
which like compound 1 carries a halogen atom, exhibited
a cytotoxic activity against murine P388 leukemia 10-
fold less than that of compound 1. Generally, in this
group of substituents, the cytotoxic activity order was
as follows: H > F > Cl > NH2 > NO2 > OH. On the
other hand, the antibacterial activity order was as
follows: F > Cl > H > NH2 > NO2 ) OH, for example,
against Staphylococcus aureus 209P J C-1 (MIC50 ) 0.39,
1.56, 3.13, 50, >100, and >100 µg/mL, respectively) and
Escherichia coli NIHJ J C-2 (MIC50 ) 0.025, 0.2, 0.39,
1.56, >100, and >100 µg/mL, respectively). These
results indicate that SAR for the C-6 position of 1,8-
naphthyridones against cytotoxic activity is different
from that of antibacterial activity.
etoposide
doxorubicin
cisplatin
0.0085
0.004
0.011
a
b
Isolation yield based on 7. Concentration of agent that
reduces cell viability by 50%. c Prepared as a free form.
position of the 1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-
naphthyridone-3-carboxylic acid and tested for their
cytotoxic activity. Data for the synthesized compounds
27a -x are summarized in Table 3, along with those for
reference drugs, i.e., etoposide, doxorubicin, and cispla-
tin. In general, most pyrrolidine derivatives with or
without an amine group showed good cytotoxic activity.
The pyrrolidine itself (27p ) exhibited an activity 6-fold
less than the 3-aminopyrrolidine (9), which had the
same activity as cisplatin with an IC50 value of 0.011
µg/mL.
Since the 6-H compound 9 had a cytotoxic activity
twice that of the 6-F counterpart (1), further modifi-
cations at the C-5 and C-7 positions of the 1,8-naph-
thyridine nucleus were carried out using the 6-unsub-
stituted 1,8-naphthyridone series. As shown in Table
2, both the chloro and trifluoromethyl groups (com-
pounds 26a ,b) decreased the cytotoxic activity by 5- to
10-fold compared to the parent compound 9. However,
introduction of an amino group (compound 26c) had no
substantial effect on the activity of 9. With respect to
the 5-unsubtituted 9 and the 5-amino 26c activity, the
1,8-naphthyridine structure showed a SAR trend similar
to that reported for the quinobenzoxazines (A-62176 and
A-85226).8,9 In addition, the degree of activity change
among the C-5 modified compounds was smaller than
that among the C-6 modified compounds.
With regard to introduction of a methyl group on the
pyrrolidine ring, compound 27b, possessing a 3-methyl
group at the geminal position of the 3-amino group of
the parent compound 9, had slightly less activity than
9, while compound 27d , possessing a 4-methyl group
at the vicinal position, showed an activity almost similar
to that of 9. Dimethylation in the two positions resulted
in a little decreased activity (compound 27i). Similarly,
introduction of a 2-methyl group (27g) or 5-methyl group
(27h ) on the pyrrolidine ring resulted in much decreased
activity. Among the methylated compounds of 3-ami-
nopyrrolidine, the cytotoxic activity order was as fol-
lows: 4-methyl 27d > 3-methyl 27b ) 3,4-dimethyl 27i
> 2-methyl 27h ) 5-methyl 27g. Introduction of a
chlorine atom into the methyl group of 27d furnished
the vicinal chloromethyl derivative 27f, which had a
cytotoxic activity almost half that of 27d . On the other
On the basis of the results of C-6 modifications, many
kinds of cyclic amines were introduced into the C-7

2102 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Tsuzuki et al.
Ta ble 4. In Vivo Antitumor Activity of Selected Compounds
hand, the bicyclic aminopyrrolidine (27j) was slightly
less active than the monocylclic aminopyrrolidine (27d
or 27e).
against Murine P388 Leukemia Cella
compd
dose, mg/kg
T/C,b %
solubility,c mg/mL
0.0069
Introduction of a methoxy group on the pyrrolidine
ring of 9 produced a slight decrease in cytotoxic activity
with compound 27k having an IC50 value of 0.018 µg/
mL. Methylation of the amino group of 9 resulted in no
substantial effect on the activity (9 vs 27a , 27b vs 27c,
and 27d vs 27e) or slightly increased the activity (27k
vs 27l). Replacement of the N-methyl group of 27l with
an N-ethyl group, giving 27m , had a negative effect on
the activity. Also, replacement of the methoxy group of
27l with a methylthio group, giving 27n , resulted in a
decrease in the activity. With regard to the stereochem-
istry, the trans isomer 27l was twice as active as the
cis isomer 27o.
To compare the effect of ring size on the cytotoxic
activity, azetidine with a four-member ring and piperi-
dine with a six-member ring were investigated. The
3-methylaminoazetidine (27q) had slightly less activity
than the 3-methylaminopyrrolidine (27a ). Introduction
of a 2-methyl group (27r ) on this azetidine produced a
decrease in the cytotoxic activity. On the other hand,
the 3-methylaminopiperidine (27v) had much less activ-
ity than 27a . The piperazines (27s and 27t) with a cyclic
diamine showed decreased cytotoxic activity (IC50 values
of 0.057 and 0.081 µg/mL, respectively). Introduction of
a 2-methoxyphenyl group on the piperazine, giving 27u ,
deteriorated the cytotoxic activity. In comparison with
other rings, the 1,2,3,4- tetrahydroisoquinoline (27w )
was inactive, and the morpholine (27x) had weak
activity.
Next, analogues of the pyrrolidine derivatives pos-
sessing a C-C bond at the C-7 position were investi-
gated. A phenyl, 2,6-dimethyl-4-pyridinyl, vinyl, ethyn-
yl, or pyrazolyl group introduced at the C-7 position
(45a -e) regrettably resulted in much decreased cyto-
toxic activity compared to the pyrrolidine derivatives
(Table 3). With respect to the effect of C-C bond at the
C-7 position, the 1,8-naphthyridine structure obviously
showed a SAR trend dissimilar to that of the reported
6,8-difluoroquinolones (CP-115,953, WIN57294).6,7
An overall evaluation of substituents at the C-7
position of the 1,8-naphthyridine nucleus showed that
the 3-aminopyrrolidine derivatives have potent cytotoxic
activity with IC50 values similar to that of cisplatin.
With regard to substitution at the C-7 position, the
6-unsubstituted 1,8-naphthyridine series generally
showed a SAR trend similar to that of the 6-fluoro-1,8-
naphthyridine series. Thus, various 3-aminopyrrolidine
derivatives with specific substituents were found to have
potent in vitro cytotoxic activity with only small differ-
ences in IC50 values.
9
3.13
12.5
50
3.13
12.5
50
3.13
12.5
50
3.13
12.5
50
3.13
12.5
50
3.13
12.5
50
3.13
12.5
50
3.13
12.5
50
188
275
125
200
300
>375
150
213
138
163
225
113
175
200
288
188
288
75
163
238
338
200
250
>375
175
250
>375
27a
0.0080
0.0038
0.0072
0.017
27b
27c
27d
27e
0.0062
0.047
27k
27l
20.1
etoposide
3.13
12.5
50
a
b
See Experimental Section. (Median survival time of treated
mice)/(median survival time of controls) × 100. c Solubility mea-
sured in a pH 7.2 phosphate buffer.
mice (T) over that of untreated control mice (C) (T/C
(%); see Experimental Section). As shown in Table 4,
all test compounds were active even at the lowest dose
of 3.13 mg/kg; however, four compounds 9, 27b, 27c,
and 27e were considered to cause some degree of toxicity
at the highest dose of 50 mg/kg. The remaining com-
pounds 27a , 27d , 27k , and 27l displayed potent cyto-
toxic activity at the highest dose of 50 mg/kg with an
efficacy at all doses comparable to that of etoposide.
Regarding the solubility of these 1,8-naphthyridines,
while compounds 9, 27a -e, and 27k had very low water
solubility, compound 27l was highly soluble in water,
demonstrating a solubility in pH 7.2 buffer of 20.1 mg/
mL. Thus, the sole combination of a methoxy and
methylamino group in the trans position of 27l gave
remarkably high solubility. Considering an infusion
drug as a preferable formulation, this result encouraged
us to proceed to the next step of our investigation with
compound 27l.
Both optical isomers of the racemic compound 27l
were synthesized and evaluated for their in vitro and
in vivo cytotoxic activity and solubility in water. The in
vitro cytotoxic activity of the isomer (S,S)-27l was 3-fold
that of the corresponding (R,R)-27l and was superior
to that of cisplatin (IC50 ) 0.0075 vs 0.021 µg/mL and
IC50 ) 0.0075 vs 0.011 µg/mL, respectively; Table 3).
As for the in vivo cytotoxic activity, the (S,S)-isomer of
27l was more effective than the (R,R)-isomer at all
tested doses (Table 5). This in vivo activity of (S,S)-27l
is comparable to that of cisplatin, inferior to that of
doxorubicin, and superior to that of etoposide. Regarding
water solubility in pH 7.2 buffer, both isomers showed
almost the same solubility as the racemic 27l.
From the results above, selected compounds were
tested for their in vivo antitumor activity using mice
implanted with P388 leukemia cells. Data for the
selected compounds 9, 27a -e, 27k , and 27l are sum-
marized in Table 4, along with their water solubility
(pH 7.2 buffer). The in vivo test consisted of intraperi-
toneal (ip) implantation of tumor cells, followed 1 day
and 5 days later by ip treatment with each test
compound at doses of 3.13, 12.5, and 50 mg/kg. The end
point for response to treatment was taken as the relative
life span expressed as median survival time of treated
At this point we considered (S,S)-27l as one of the
promising antitumor agents and examined the physi-
cochemical properties of some of its salts and a free base.

7-Substituted Carboxylic Acids as Antitumor Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2103
Ta ble 5. In Vivo Antitumor Activity of Compound 27l and Its
tures in which the 6-fluorine atom has been shown to
be the most potent substituent for cytotoxic activity.
Second, introduction of an amino group (compound 26c)
at the C-5 position did not have any substantial effect
on the cytotoxic activity, while both the 5-chloro and
5-trifluoromethyl groups (compounds 26a ,b) decreased
the cytotoxic activity by 5- to 10-fold compared to the
parent compound 9. Third, overall evaluation of C-7
substituents showed that various 3-aminopyrrolidine
derivatives have potent in vitro cytotoxic activity against
murine P388 leukemia cells and that this activity is
similar to that of cisplatin.
As for the in vivo test, the 3-aminopyrrolidine deriva-
tives 27a , 27d , 27k , and 27l, having the best combina-
tion of substituents, displayed good cytotoxic activity
with an efficacy comparable to that of etoposide. Of
these compounds, only 27l had remarkably high water
solubility. Synthesis and in vitro/in vivo evaluation of
both optical isomers of the racemic compound 27l
revealed that (S,S)-27l has great cytotoxic activity
against murine and human tumor cell lines with high
water solubility. Finally the free base 3 of (S,S)-27l, with
its preferable physicochemical properties, was selected
as a drug candidate for further development.
Optical Isomers against Murine P388 Leukemia Cellsa
compd
27l
dose, mg/kg
T/C,b %
solubility,c mg/mL
20.1
0.78
1.56
3.13
6.25
12.5
25
0.78
1.56
3.13
6.25
12.5
25
0.78
1.56
3.13
6.25
12.5
25
0.78
1.56
3.13
6.25
12.5
25
150
163
200
225
250
300
150
188
225
275
>375
>375
125
125
163
188
238
300
138
150
175
200
250
>375
173
228
235
293
154
186
239
290
(S,S)-27l
(R,R)-27l
etoposide
23.7
19.8
Exp er im en ta l Section
Ch em istr y. All melting points were determined on
a
Yanagimoto micro-melting-point apparatus and are uncor-
rected. Infrared (IR) spectra were recorded on a Perkin-Elmer
1600 series FTIR spectrophotometer. 1H NMR sprctra were
taken at 200 MHz on a Varian Gemini-200 spectrometer.
Chemical shifts are expressed in ppm (δ) with tetramethyl-
silane as an internal standard. Mass spectra were obtained
on a Hitachi M-1000 or Hitachi M-80B spectrometer. The
spectral data for all compounds were consistent with the
assigned structures. All compounds that were stable solids
were analyzed for C, H, Cl, F, N, and S.
doxorubicin
cisplatin
0.78
1.56
3.13
6.25
0.78
1.56
3.13
6.25
a
b
See Experimental Section. (Median survival time of treated
mice)/(median survival time of controls) × 100. c Solubility mea-
sured in a pH 7.2 phosphate buffer.
3-Aminopyrrolidine, pyrrolidine, piperazine, 2,6-dimeth-
ylpiperazine, 1-(2-methoxyphenyl)piperazine, 1,2,3,4-tetrahy-
droisoquinoline, morpholine, and 2-aminothiazole were pur-
chased from commercial suppliers.
This led us to select the free base 3 (Figure 1) as a
candidate for development. Compound 3 was subjected
to assay against various types of human tumor cells.
In general, compound 3 displayed good cytotoxic activity
with an efficacy clearly higher than that of the reference
drug etoposide (Table 6). Detailed in vivo antitumor
efficacy of 3 will be reported elsewhere.
Additionally we have measured the inhibitory activi-
ties of 3 against topoisomerase II relaxation and cleav-
age assays.23 Compound 3 exhibited potent inhibitory
effect in topoisomerase II relaxation (IC50 ) 3.2 µg /mL),
whereas it did not induce topoisomerase II cleavage
(IC50 >1000 µg/mL).24 The IC50 values of etoposide were
>1000 and 1 µg /mL against topoisomerase II relaxation
and cleavage, respectively. The result implies that there
is a mechanistic difference between 3 and etoposide.
Eth yl 2,6-Dich lor on icotin oyla ceta te (5). To a solution
of of 2,6-dichloronicotinic acid 4 (401 g, 2.10 mol) in THF-
CH3CN (1:1, 1.4 L) was added CDI (379 g, 2.30 mol). The
resulting mixture was stirred at room temperature for 2.5 h.
This crude imidazolide solution was used without purification
in the next step. To a solution of ethyl malonate potassium
salt (376 g, 2.20 mol) in CH3CN (3.3 L) was added dropwise
MgCl2 (297 g, 3.10 mol) and Et3N (880 mL, 6.30 mol) under
ice-cooling. After the mixture was stirred at room temperature
for 5 h, the imidazolide prepared in the above was added. The
reaction mixture was stirred at room temperature for 15 h,
poured into ice-water, acidifed to pH 5-6 with concentrated
HCl, and then extracted with AcOEt. The organic layer was
dried over Na2SO4 and concentrated to dryness to give 513 g
(93%) of 5 as a mixture of keto and enol forms, bp 135-140
1
°C/2 mmHg. MS (m/z): 262 (MH+). IR (neat) cm-1: 1736. H
NMR (CDCl3) δ: 1.25 (t, 3 H × 1/2, J ) 7 Hz), 1.35 (t, 3 H ×
1/2, J ) 7 Hz), 4.09 (s, 2 H × 1/2), 4.20 (q, 2 H × 1/2, J ) 7
Hz), 4.30 (d, 1 H × 1/2, J ) 7 Hz), 5.73 (s, 1 H × 1/2), 7.36 (d,
1 H × 1/2, J ) 8.5 Hz), 7.40 (d, 1 H × 1/2, J ) 8.5 Hz), 7.92 (d,
1 H × 1/2, J ) 8.5 Hz), 7.98 (d, 1 H × 1/2, J ) 8.5 Hz), 12.54
(s, 1 H × 1/2).
Con clu sion s
We have designed and synthesized a novel type of
antitumor agent, 7-substituted 1,4-dihydro-4-oxo-1-(2-
thiazolyl)-1,8-naphthyridine-3-carboxylic acids. In the
course of the SAR study of this series of compounds, the
following findings were obtained. First, the 6-unsubsti-
tuted 1,8-naphthyridine 9 had an activity twice that of
the 6-fluoro-1,8-naphthyridine 1, indicating a significant
difference from conventional antitumor quinoline struc-
Eth yl 2-(2,6-Dich lor o-3-n icotin oyl)-3-(2-th ia zolyla m i-
n o)a cr yla te (6). A mixture of 5 (44.9 g, 171 mmol), ethyl
orthoformate (37.7 g, 255 mmol), and acetic anhydride (43.8
g, 429 mmol) was heated to reflux for 1 h at 140 °C, during
which period the resulting AcOEt was distilled off under
atmospheric pressure. After concentration under reduced
pressure, the residue was diluted with i-Pr2O (500 mL), and

2104 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Tsuzuki et al.
Ta ble 6. Cytotoxic Activity of Compound 3 against Human Tumor Cell Lines
a
IC50
,
µg/mL
compd
HL-60 leukemia
Hs746T stomach
PANC-1 pancreas
SBC-3 lung
SK-OV-3 ovary
SCaBER bladder
3
0.0498
0.150
0.194
3.29
0.388
1.60
0.0319
0.0676
0.266
1.06
0.121
0.276
etoposide
a
Concentration of agent that reduces cell viability by 50%. Each value is the mean of at least two independent experiments.
then 2-aminothiazole (20.0 g, 200 mmol) was added under ice-
cooling. After the mixture was stirred at room temperature
for 5 h, the resulting precipitates were collected by filtration,
washed with i-Pr2O, and then dried to give 52.8 g (83%) of 6
as a mixture of keto and enol forms, mp 119-122 °C. MS (m/
70 °C for 0.5 h. This crude imidazolide solution was used
without purification in the next step. To a suspension of ethyl
malonate potassium salt (18.6 g, 109 mmol) and MgCl2 (11.3
g, 118 mmol) in AcOEt (250 mL) was added dropwise Et3N
(28.0 g, 277 mmol) under ice-cooling. After the mixture was
stirred for 2.5 h at room temperature, the imidazolide prepared
above was added. The reaction mixture was stirred at 70 °C
for 1.5 h, poured into ice-water, and acidified to pH 5-6 with
concentrated HCl. The resulting precipitates were collected by
filtration and washed with AcOEt to give 8.6 g of 12. The
filtrate was extracted with AcOEt. The organic layer was dried
over Na2SO4 and concentrated to dryness to afford a crude
product, which was recrystallized from a mixture of AcOEt
and i-Pr2O to give 11.3 g of 12. Each product was combined to
give 19.9 g (82%) of 12. MS (m/z): 299 (MH+). 1H NMR
(DMSO-d6) δ: 1.19 (t, 3 H, J ) 7 Hz), 4.00 (s, 2 H), 4.10 (s, 3
H), 4.11 (q, 2 H, J ) 7 Hz), 4.15 (s, 3 H), 8.78 (s, 1 H).
Eth yl 2-(2,6-Dim eth oxy-5-n itr o-3-n icotin oyl)-3-(2-th i-
a zolyla m in o)a cr yla te (13). Following the procedure for 6,
13 was prepared from 12 in 74% yield, mp 166-170 °C. MS
(m/z): 377 (MH+ - MeOH). IR (KBr) cm-1: 1729, 1603. 1H
NMR (CDCl3) δ: 1.01 (t, 3 H × 1/4, J ) 7 Hz), 1.21 (t, 3 H ×
3/4, J ) 7 Hz), 4.00 (s, 3 H × 3/4), 4.02 (s, 3 H × 1/4), 4.08-
4.21 (m, 2 H), 4.14 (s, 3 H × 1/4), 4.16 (s, 3 H × 3/4), 6.95 (d,
1 H × 1/4, J ) 4 Hz), 7.02 (d, 1 H × 3/4, J ) 4 Hz), 7.48 (d, 1
H × 3/4, J ) 4 Hz), 7.78 (d, 1 H × 1/4, J ) 4 Hz), 8.49 (d, 1 H
× 1/4, J ) 13 Hz), 8.55 (s, 1 H × 3/4), 8.70 (s, 1 H × 1/4), 8.75
(d, 1 H × 3/4, J ) 13 Hz), 11.20 (d, 1 H × 1/4, J ) 13 Hz),
12.50 (d, 1 H × 3/4, J ) 13 Hz).
1
z): 373 (MH+). IR (KBr) cm-1: 1700, 1624. H NMR (CDCl3)
δ: 0.94 (t, 3 H × 1/3, J ) 7 Hz), 1.13 (t, 3 H × 2/3, J ) 7 Hz),
4.08 (q, 2 H × 1/3, J ) 7 Hz), 4.13 (q, 2 H × 2/3, J ) 7 Hz),
7.02 (d, 1 H × 1/3, J ) 3.8 Hz), 7.08 (d, 1 H × 2/3, J ) 3.7 Hz),
7.34 (d, 1 H, J ) 8 Hz), 7.48 (d, 1 H × 1/3, J ) 3.8 Hz), 7.53
(d, 1 H × 2/3, J ) 3.7 Hz), 7.58 (d, 1 H × 2/3, J ) 8 Hz), 7.68
(d, 1 H × 1/3, J ) 8 Hz), 8.81 (d, 1 H × 1/3, J ) 13 Hz), 8.94
(d, 1 H × 2/3, J ) 13 Hz), 11.62 (d, 1 H × 1/3, J ) 13 Hz),
12.80 (d, 1 H × 2/3, J ) 13 Hz). Anal. (C14H11Cl2N3O3S) C, H,
Cl, N, S.
E t h yl 7-Ch lor o-1,4-d ih yd r o-4-oxo-1-(2-t h ia zolyl)-1,8-
n a p h th yr id in e-3-ca r boxyla te (7). To a solution of 6 (51.7
g, 139 mmol) in dioxane (310 mL) was added K2CO3 (21.4 g,
155 mmol) at room temperature. The reaction mixture was
heated at 60 °C for 1 h and diluted with ice-water. The
resulting precipitates were collected by filtration, washed with
water, and then dried to afford a crude product, which was
recrystallized from a mixture of CHCl3 and i-Pr2O to give 44.8
g (96%) of 7, mp 176-177 °C. MS (m/z): 336 (MH+). IR (KBr)
cm-1: 1724, 1632. 1H NMR (CDCl3) δ: 1.43 (t, 3 H, J ) 6.5
Hz), 4.45 (q, 2 H, J ) 6.5 Hz), 7.38 (d, 1H, J ) 3.5 Hz), 7.52
(d, 1 H, J ) 8.5 Hz), 7.75 (d, 1H, J ) 3.5 Hz), 8.78 (d, 1 H, J
) 8.5 Hz), 10.00 (s, 1 H). Anal. (C14H10ClN3O3S) C, H, Cl, N,
S.
Eth yl 7-(3-Am in o-1-p yr r olid in yl)-1,4-d ih yd r o-4-oxo-1-
(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxyla te (8). To a
suspension of 7 (2.04 g, 6.10 mmol) in CH3CN (90 mL) was
added 3-aminopyrrolidine (1.56 g, 18.4 mmol) at room tem-
perature. The reaction mixture was stirred at the same
temperature for 1.5 h. The resulting precipitates were collected
by filtration, and recrystallized from CHCl3-MeOH to give
1.89 g (81%) of 8, mp 219-221 °C. MS (m/z): 386 (MH+). IR
E t h yl 1,4-Dih yd r o-7-m et h oxy-6-n it r o-4-oxo-1-(2-t h i-
a zolyl)-1,8-n a p h th yr id in e-3-ca r boxyla te (14). To a solution
of 13 (2.00 g, 4.90 mmol) in dioxane (90 mL) was added K2CO3
(1.00 g, 7.50 mmol) under ice-cooling. The reaction mixture
was heated at 60 °C for 3 h and then diluted with ice-water
and 20% HCl. The resulting precipitates were collected by
filtration, washed with water, EtOH, and i-Pr2O successively,
and dried to give 1.70 g (90%) of 14, mp 208-210 °C. MS (m/
1
1
(KBr) cm-1: 1727, 1635. H NMR (DMSO-d6) δ: 1.30 (t, 3 H,
z): 377 (MH+). IR (KBr) cm-1: 1739, 1648. H NMR (DMSO-
J ) 7 Hz), 1.75-1.95 (m, 1 H), 2.03-2.25 (m, 1 H), 3.10-4.00
(m, 7 H), 4.28 (q, 2 H, J ) 7 Hz), 6.72 (d, 1H, J ) 9 Hz), 7.72
(d, 1H, J ) 3.5 Hz), 7.79 (d, 1H, J ) 3.5 Hz), 8.21 (d, 1 H, J )
9 Hz), 9.63 (s, 1 H). Anal. (C18H19N5O3S‚0.2HCl) C, H, Cl, N,
S.
7-(3-Am in o-1-p yr r olid in yl)-1,4-d ih yd r o-4-oxo-1-(2-th ia -
zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid Hyd r och lo-
r id e (9). A suspension of 8 (1.02 g, 2.65 mmol) in 10% HCl
(15 mL) was heated to reflux for 5 h. After the mixture was
ice-cooled, the resulting precipitates were collected by filtra-
tion, washed with 0.5 N HCl and EtOH successively, and dried
to give 930 mg (89%) of 9, mp 260-264 °C (dec). MS (m/z):
358 (MH+). IR(KBr) cm-1: 1727, 1631. 1H NMR (DMSO-d6)
δ: 2.12-4.15 (m, 7 H), 6.99 (d, 1 H, J ) 9 Hz), 7.90 (m, 2 H),
8.40 (d, 1 H, J ) 9 Hz), 9.85 (s, 1 H), 14.1 (br s, 1 H). Anal.
(C16H15N5O3S‚HCl‚0.5H2O) C, H, Cl, N, S.
2,6-Dim eth oxy-5-n itr on icotin ic Acid (11). To a mixture
of concentrated HNO3 (5 mL) and acetic anhydride (60 mL)
was added dropwise 2,6-dimethoxynicotinic acid 10 (8.71 g,
47.6 mmol) under ice-cooling. The reaction mixture was stirred
at the same temperature for 3 h and then at room temperature
for 4 h. The resulting precipitates were collected by filtration,
washed with i-PrOH and i-Pr2O successively, and dried to give
6.95 g (64%) of 11, mp 227-230 °C. 1H NMR (DMSO-d6) δ:
4.03 (s, 3 H), 4.08 (s, 3 H), 8.71 (s, 1 H), 10.3 (br s, 1 H).
Eth yl 2,6-Dim eth oxy-5-n itr on icotin oyla ceta te (12). To
a solution of 11 (18.5 g, 81.3 mmol) in THF (150 mL) was added
CDI (14.8 g, 90.9 mmol). The resulting mixture was heated at
d6) δ: 1.31 (t, 3 H, J ) 7 Hz), 4.30 (s, 3 H), 4.33 (q, 2 H, J )
7 Hz), 7.85 (d, 1 H, J ) 4 Hz), 7.88 (d, 1 H, J ) 4 Hz), 9.08 (s,
1 H), 9.69 (s, 1 H).
Eth yl 7-[3-(N-ter t-Bu toxyca r bon yla m in o)-1-p yr r olid i-
n yl]-1,4-d ih yd r o-6-n it r o-4-oxo-1-(2-t h ia zolyl)-1,8-n a p h -
th yr id in e-3-ca r boxyla te (15a ). To a solution of 3-(N-Boc-
amino)pyrrolidine (5.90 g, 31.8 mmol) in DMF (150 mL) were
added 14 (10.3 g, 27.3 mmol) and K2CO3 (4.70 g, 34.1 mmol).
The reaction mixture was stirred at 60 °C for 2 h and cooled
to 0 °C. The resulting mixture was diluted with water and
extracted with AcOEt. The organic layer was dried over
Na2SO4 and concentrated to dryness to afford a crude product,
which was recrystallized from AcOEt-EtOH to give 11.4 g
(79%) of 15a , mp 204-205 °C. MS (m/z): 531 (MH+). IR (KBr)
1
cm-1: 1714, 1627. H NMR (DMSO-d6) δ: 1.32 (t, 3 H, J ) 7
Hz), 1.38 (s, 9 H), 2.0-2.5 (m, 2 H), 3.4-4.2 (m, 5 H), 4.30 (q,
2 H, J ) 7 Hz), 7.24 (m, 1H), 7.80 (d, 1 H, J ) 3.5 Hz), 7.84 (d,
1 H, J ) 3.5 Hz), 8.78 (s, 1 H), 9.65 (s, 1 H). Anal.
(C23H26N6O7S) C, H, N, S.
7-(3-Am in o-1-p yr r olid in yl)-1,4-d ih yd r o-6-n it r o-4-oxo-
1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid Hy-
d r och lor id e (16a ). A suspension of 15a (0.16 g, 0.30 mmol),
20% HCl (15 mL), and EtOH (1 mL) was heated at 100 °C for
3 h and cooled to 0 °C. The resulting precipitates were collected
by filtration, washed with 10% HCl and EtOH successively,
and then dried to give 60 mg (46%) of 16a , mp 255-257 °C
(dec). MS (m/z): 403 (MH+). IR (KBr) cm-1: 1734, 1634. 1H
NMR (DMSO-d6) δ: 2.1-2.5 (m, 2 H), 3.4-4.2 (m, 5 H), 7.90

7-Substituted Carboxylic Acids as Antitumor Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2105
(d, 1 H, J ) 3.5 Hz), 7.92 (d, 1 H, J ) 3.5 Hz), 8.98 (s, 1 H),
9.83 (s, 1 H), 14.0 (br s, 1 H). Anal. (C16H14N6O5S‚HCl‚0.25H2O)
C, H, Cl, N, S.
was chromatographed on silica gel with CHCl3-MeOH (100:
1) to give 70 mg (23%) of 15d , mp 105-110 °C. MS (m/z): 520
1
(MH+). IR (KBr) cm-1: 1719, 1625. H NMR (CDCl3) δ: 1.41
(t, 3 H, J ) 7 Hz), 1.48 (s, 9 H), 1.9-2.5 (m, 2 H), 3.8-4.3 (m,
5 H), 4.41 (q, 2 H, J ) 7 Hz), 4.8 (m, 1 H), 7.32 (d, 1 H, J ) 3.5
Hz), 7.70 (d, 1 H, J ) 3.5 Hz), 8.49 (s, 1 H), 9.20 (s, 1 H).
7-(3-Am in o-1-p yr r olid in yl)-6-ch lor o-1,4-d ih yd r o-4-oxo-
1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid Hy-
d r och lor id e (16d ). Following the procedure for 16a , 16d was
prepared from 15d in 73% yield, mp 288-291 °C (dec). MS
(m/z): 391 (M+). IR (KBr) cm-1: 1734, 1626. 1H NMR (DMSO-
d6) δ: 2.0-2.5 (m, 2 H), 3.9-4.3 (m, 5 H), 7.88 (s, 2 H), 8.40
(s, 1 H), 9.71 (s, 1 H). Anal. (C16H14ClN5O3S‚HCl‚0.5EtOH) C,
H, Cl, N, S.
2,4,6-Tr ich lor on icotin oic Acid (20a ). To a solution of 19a
(5.50 g, 30.4 mmol) in anhydrous THF (55 mL) was added of
n-BuLi (1.6 M in hexane, 20 mL, 32.0 mmol) at -78 °C under
a nitrogen atmosphere. After the mixture was stirred at the
same temperature for 1 h, excess amount of dry ice was added
to the reaction mixture. The whole was stirred at the same
temperature for 1 h, warmed to 0 °C, acidified with diluted
HCl, and extracted with AcOEt. The organic layer was dried
over Na2SO4 and concentrated to dryness to afford a crude
product, which was washed with i-Pr2O to give 6.5 g (75%) of
20a , mp 138-141 °C. IR (KBr) cm-1: 1715.
Eth yl 6-Am in o-7-[3-(N-ter t-bu toxyca r bon yla m in o)-1-
p yr r olid in yl]-1,4-d ih yd r o-4-oxo-1-(2-th ia zolyl)-1,8-n a p h -
th yr id in e-3-ca r boxyla te (15b). To a solution of 15a (1.00 g,
1.90 mmol) in EtOH-dioxane (2:1, 150 mL) was added Raney
nickel, which was washed with EtOH before use, under ice-
cooling. The resulting mixture was heated at 50 °C for 2.5 h
in an atmosphere of hydrogen gas. The resulting precipitates
were collected by filtration and dissolved in DMF under
heating. The catalyst was removed by filtration, and most of
the solvent was removed from the filtrate under reduced
pressure. The resulting precipitates were collected by filtration
and washed with EtOH to give 0.81 g (85%) of 15b, mp 270-
272 °C (dec). MS (m/z): 501 (MH+). IR (KBr) cm-1: 3342, 1731,
1682, 1621. 1H NMR (DMSO-d6) δ: 1.31 (t, 3 H, J ) 7 Hz),
1.41 (s, 9 H), 1.9-2.3 (m, 2 H), 3.6-4.2 (m, 5 H), 4.26 (q, 2 H,
J ) 7 Hz), 5.10 (br s, 2 H), 7.1-7.2 (m, 1H), 7.58 (s, 1 H), 7.71
(d, 1 H, J ) 3.5 Hz), 7.79 (d, 1 H, J ) 3.5 Hz), 9.51 (s, 1 H).
Anal. (C23H28N6O7S) C, H, N, S.
6-Am in o-7-(3-a m in o-1-p yr r olid in yl)-1,4-d ih yd r o-4-oxo-
1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid Hy-
d r och lor id e (16b). Following the procedure for 16a , 16b was
prepared from 15b in 76% yield, mp >300 °C. MS (m/z): 373
(M+). IR (KBr) cm-1: 1700, 1617. 1H NMR (DMSO-d6) δ: 2.0-
2.5 (m, 2 H), 3.8-4.3 (m, 5 H), 5.4 (br s, 2 H), 7.68 (s, 1 H),
7.85 (d, 1 H, J ) 2.5 Hz), 7.89 (d, 1 H, J ) 2.5 Hz), 8.3 (br s,
3 H), 9.68 (s, 1 H), 15.3 (br s, 1 H). Anal. (C16H16N6O3S‚HCl)
C, H, Cl, N, S.
7-(3-Am in o-1-p yr r olid in yl)-1,4-d ih yd r o-6-h yd r oxy-4-
oxo-1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid
Hyd r och lor id e (16c). A suspension of 15b (0.42 g, 0.84 mmol)
in 20% HCl (40 mL) was stirred at 100 °C for 1 week and cooled
to 0 °C. The resulting precipitates were collected by filtration,
dissolved in 5 mL of 1 N NaOH, and treated with active carbon
to remove insoluble materials. To this filtrate was added 20%
HCl (20 mL), and the resulting precipitates were collected by
filtration and washed with 10% HCl and EtOH successively
to give 50 mg (17%) of 16c, mp 275-279 °C (dec). MS (m/z):
374 (MH+). IR (KBr) cm-1: 3429, 1716, 1623. 1H NMR (DMSO-
d6) δ: 2.0-2.5(m, 2 H), 3.9-4.3 (m, 5 H), 7.63 (s, 1 H), 7.83 (d,
1 H, J ) 2.5 Hz), 7.88 (d, 1 H, J ) 2.5 Hz), 9.68 (s, 1 H). Anal.
(C16H15N5O4S‚HCl‚0.5H2O) C, H, Cl, N, S.
2,4,6-Tr ich lor on icotin oyl Ch lor id e (21a ). A mixture of
20a (6.50 g, 28.9 mmol) and SOCl2 (25 mL) was heated to
reflux for 3 h and cooled to room temperature. After excess
SOCl2 was removed under reduced pressure, the crude product
was distilled under reduced pressure to give 6.60 g (93%) of
21a , bp 93-95 °C/1 mmHg. IR (neat) cm-1: 1791.
Eth yl 2,4,6-Tr ich lor on icotin oyla ceta te (22a ). To a solu-
tion of ethyl hydrogen malonate (3.60 g, 27.3 mmol) in
anhydrous THF (30 mL) was added dropwise MeMgBr (3 M
in Et2O, 19 mL, 57.0 mmol) under ice-cooling. After being
stirred at room temperature for 1 h, a solution of 21a (6.60 g,
26.9 mmol) in anhydrous THF (30 mL) was added dropwise
at the same temperature. After the mixture was stirred at 60
°C for 1.5 h, most of the solvent was removed under reduced
pressure. The resulting residue was poured into ice-water,
acidified to pH 5-6 with concentrated HCl, and extracted with
CHCl3. The organic layer was dried over Na2SO4 and concen-
trated to dryness to afford a crude product, which was distilled
under reduced pressure to give 4.80 g (59%) of 22a , bp 160-
162 °C/2 mmHg. MS (m/z): 296 (MH+). IR (neat) cm-1: 1746.
1H NMR (CDCl3) δ: 1.26 (t, 3 H × 1/2, J ) 7 Hz), 1.34 (t, 3 H
× 1/2, J ) 7 Hz), 3.92 (s, 2 H × 1/2), 4.22 (q, 2 H × 1/2, J ) 7
Hz), 4.30 (d, 1 H × 1/2, J ) 7 Hz), 5.28 (s, 1 H × 1/2), 7.40 (s,
1 H × 1/2), 7.42 (s, 1 H × 1/2), 12.23 (s, 1 H × 1/2).
E t h yl 6-Am in o-1,4-d ih yd r o-7-m et h oxy-4-oxo-1-(2-t h i-
a zolyl)-1,8-n a p h th yr id in e-3-ca r boxyla te (17). Following
the procedure for 15b, 17 was prepared from 14 in 61% yield,
mp 272-275 °C (dec). MS (m/z): 347 (MH+) . IR (KBr) cm-1
3313, 1727, 1624. H NMR (DMSO-d6) δ: 1.32 (t, 3 H, J ) 7
Hz), 4.23 (s, 3 H), 4.28 (q, 2 H, J ) 7 Hz), 7.78 (d, 1H, J ) 3.5
Hz), 7.82 (d, 1 H, J ) 3.5 Hz), 8.47 (s, 1 H), 9.62 (s, 1 H).
:
1
E t h yl 2-(2,4,6-Tr ich lor o-3-n icot in oyl)-3-(2-t h ia zoly-
la m in o)a cr yla te (23a ). Following the procedure for 7, 23a
was prepared from 22a in 58% yield, mp 126-127 °C. MS (m/
z): 406 (MH+). IR (KBr) cm-1: 1691, 1636.
Eth yl 6-Ch lor o-1,4-d ih yd r o-7-m eth oxy-4-oxo-1-(2-th ia -
zolyl)-1,8-n a p h th yr id in e-3-ca r boxyla te (18). To a solution
of 17 (0.31 g, 0.90 mmol) in concentrated HCl (5 mL) was added
a solution of NaNO2 (0.12 g, 1.7 mmol) in water (2 mL) under
ice-cooling. After being stirred for 10 min, the reaction mixture
was added dropwise over 15 min to a mixture of CuCl (90 mg,
0.91 mmol) and concentrated HCl (2 mL). After being stirred
at 60 °C for 10 min, the resulting mixture was alkalized to
pH 8 with 20% NaOH and extracted with CHCl3. The organic
layer was dried over Na2SO4 and concentrated to dryness to
afford a crude product, which was recrystallized from CHCl3-
EtOH to give 0.27 g (82%) of 18, mp 219-221 °C. MS (m/z):
E t h yl 5,7-Dich lor o-1,4-d ih yd r o-4-oxo-1-(2-t h ia zolyl)-
1,8-n a p h th yr id in e-3-ca r boxyla te (24a ). To a suspension of
23a (3.80 g, 9.35 mmol) in AcOEt (40 mL) was added K2CO3
(1.5 g, 10.9 mmol) under ice-cooling. The reaction mixture was
heated at 60 °C for 1 h, and most of the solvent was removed
under reduced pressure. The resulting residue was treated
with ice-water and then extracted with CHCl3. The organic
layer was dried over Na2SO4 and concentrated to dryness to
afford a crude product, which was chromatographed on silica
gel with CHCl3-MeOH (200:1) and recrystallized from CHCl3
to give 2.50 g (72%) of 24a , mp 226-227 °C. MS (m/z): 370
(MH+). IR (KBr) cm-1: 1737, 1692.
1
366 (MH+). IR (KBr) cm-1: 1723, 1615. H NMR (DMSO-d6)
δ: 1.30 (t, 3 H, J ) 7 Hz), 4.18 (s, 3 H), 4.28 (q, 2 H, J ) 7
Hz), 7.65 (s, 1 H), 7.76 (d, 1H, J ) 3.5 Hz), 7.81 (d, 1 H, J )
3.5 Hz), 9.59 (s, 1 H).
Eth yl 7-[3-(N-ter t-Bu toxyca r bon yla m in o)-1-p yr r olid in -
yl]-6-chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-
ca r boxyla te (15d ). To a suspension of 18 (0.21 g, 0.58 mmol)
in CH3CN (15 mL) were added 3-(N-Boc-amino)pyrrolidine
(0.16 g, 0.86 mmol) and DBU (0.22 g, 1.45 mmol). After being
stirred at 70 °C for 3 days, the reaction mixture was concen-
trated under reduced pressure, and then the obtained residue
5,7-Dich lor o-1,4-dih ydr o-4-oxo-1-(2-th iazolyl)-1,8-n aph -
th yr id in e-3-ca r boxylic Acid (25a ). A solution of 24a (0.45
g, 1.22 mmol) in H2SO4-AcOH-H2O (1:8:6, 4.5 mL) was
heated to reflux for 15 h and cooled to room temperature. The
reaction mixture was diluted with ice-water. The resulting
precipitates were collected by filtration and washed with water
to give 0.35 g (84%) of 25a , mp 264-266 °C. MS (m/z): 342
1
(MH+). IR (KBr) cm-1: 1729. H NMR (DMSO-d6) δ: 7.92 (s,
2H), 8.12 (s, 1H), 9.82 (s, 1H).

2106 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Tsuzuki et al.
7-(3-Am in o-1-p yr r olid in yl)-5-ch lor o-1,4-d ih yd r o-4-oxo-
1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid Hy-
d r och lor id e (26a ). A mixture of 25a (0.35 g, 1.02 mmol), 3-(N-
Boc-amino)pyrrolidine (0.23 g, 1.24 mmol), Et3N (0.31 g, 3.07
mmol), and CH3CN (10 mL) was heated to reflux for 15 h. Most
of the solvent was removed under reduced pressure, and then
the resulting residual solid was washed with AcOEt and dried
to give 0.45 g (89%) of 7-[3-(N-Boc-amino)-1-pyrrolidinyl]-5-
chloro-1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-
carboxylic acid, mp >300 °C. MS (m/z): 492 (MH+). IR (KBr)
cm-1: 1732, 1677.
A mixture of the above compound (0.45 g, 0.92 mmol) and
20% HCl (10 mL) was heated to reflux for 1.5 h. The resulting
precipitates were collected by filtration, washed with 0.5 N
HCl and EtOH successively, and then dried to give 0.32 g
(89%) of 26a , mp >300 °C. MS (m/z): 392 (MH+). IR (KBr)
cm-1: 1726, 1626. 1H NMR (DMSO-d6) δ: 2.1-2.3 (m, 1H),
2.3-2.5 (m, 1 H), 3.4-4.0 (m, 5 H), 7.01 (s, 1 H), 7.80 (d, 1 H,
J ) 3.5 Hz), 7.85 (d, 1 H, J ) 3.5 Hz), 8.2 (br s, 3 H), 9.75 (s,
1 H), 14.3 (br s, 1 H). Anal. (C16H14ClN5O3S‚HCl‚0.75H2O‚
0.1EtOH) C, H, Cl, N, S.
3.5 Hz), 8.5 (br s, 3 H), 9.68 (s, 1 H). Anal. (C16H16N6O3S‚HCl)
C, H, Cl, N, S.
1,4-Dih yd r o-7-[(S,S)-3-m eth oxy-4-m eth yla m in o-1-p yr -
r olid in yl]-4-oxo-1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r -
boxylic Acid Hyd r och lor id e ((S,S)-27l). To a solution of
(S,S)-3-methoxy-4-methylaminopyrrolidine di-p-toluenesulfon-
ic acid21,22 (85.0 g, 179 mmol) in CH3CN (700 mL) was added
Et3N (100 mL, 719 mmol) at 0 °C. After the mixture was
stirred for 15 min, 7 (50.44 g, 150 mmol) was added. The
mixture was stirred for 1 day at room temperature, and then
the resultant precipitates were collected by filtration and
washed with CH3CN to give 52.4 g (87%) of ethyl 1,4-dihydro-
7-[(S,S)-3-methoxy-4-methylamino-1-pyrrolidinyl]-4-oxo-1-(2-
thiazolyl)-1,8-naphthyridine-3-carboxylate, mp 184-185 °C.
[R]28 -16.9° (c 0.50, CHCl3). MS (m/z): 430 (MH+). IR (KBr)
D
cm-1: 3350, 1730, 1630. H NMR (CDCl3) δ: 1.42 (t, 3 H, J )
1
6.8 Hz), 2.54 (s, 3 H), 3.44 (s, 3 H), 3.6-4.2 (m, 6 H), 4.41 (q,
2 H, J ) 6.8 Hz), 6.51 (d, 1 H, J ) 8.7 Hz), 7.27 (d, 1H, J ) 3.5
Hz), 7.69 (d, 1H, J ) 3.5 Hz), 8.46 (d, 1 H, J ) 8.7 Hz), 9.80 (s,
1 H). Anal. Calcd for C20H23N5O4S: C, 55.93; H, 5.40; N, 16.31;
S, 7.47. Found: C, 55.99; H, 5.33; N, 16.25; S, 7.28.
2,6-Dich lor o-4-tr iflu or om eth yln icotin oyl Ch lor ide (21b).
Following the procedure for 21a , 21b was prepared from 19b
A mixture of the above compound (28.0 g, 65.3 mmol), 0.5
N NaOH (170 mL, 85 mmol), and EtOH (10 mL) was stirred
at room temperature for 1 day. The reaction mixture was
acidified with 10% HCl. The resulting precipitates were
collected by filtration, washed with 0.5 N HCl and i-Pr2O
successively, and dried to give 10.9 g (97%) of (S,S)-27l, mp
278-284 °C (dec). [R]27D +50.4° (c 1.00, 1 N NaOH). MS (m/z):
in 59% yield via 20b, bp 77-78 °C/2 mmHg. IR (neat) cm-1
:
1797.
Eth yl 2,6-Dich lor o-4-tr iflu or om eth yln icotin oyla ceta te
(22b). Following the procedure for 22a , 22b was prepared from
21b in 33% yield. MS (m/z): 330 (MH+). IR (neat) cm-1: 1744,
1721.
1
402 (MH+). IR (KBr) cm-1: 1738, 1640. H NMR (DMSO-d6)
δ: 2.71 (s, 3 H), 3.42 (s, 3 H), 3.5-4.4 (m, 6 H), 7.03 (d, 1 H,
J ) 9 Hz), 7.84 (d, 1H, J ) 3.5 Hz), 7.90 (d, 1H, J ) 3.5 Hz),
8.45 (d, 1 H, J ) 9 Hz), 9.3 (br s, 2 H), 9.85 (s, 1 H). Anal.
(C18H19N5O4S‚HCl‚0.25H2O) C, H, Cl, N, S.
Eth yl 2-(2,6-Dich lor o-4-tr iflu or om eth yl-3-n icotin oyl)-
3-(2-th ia zolyla m in o)a cr yla te (23b). Following the procedure
for 7, 23b was prepared from 22b in 31% yield. MS (m/z): 440
(MH+). IR (neat) cm-1: 1713.
According to a similar procedure for 9 and (S,S)-27l,
compounds 27a -x and (R,R)-27l were prepared.
Eth yl 7-Ch lor o-1,4-d ih yd r o-4-oxo-1-(2-th ia zolyl)-5-tr i-
flu or om eth yl-1,8-n a p h th yr id in e-3-ca r boxyla te (24b). Fol-
lowing the procedure for 24a , 24b was prepared from 23b in
82% yield, mp 184-185 °C. IR (KBr) cm-1: 1736, 1703.
7-(3-Am in o-1-p yr r olid in yl)-1,4-d ih yd r o-4-oxo-1-(2-th ia -
zolyl)-5-t r iflu or om et h yl-1,8-n a p h t h yr id in e-3-ca r b oxyl-
ic Acid Hyd r och lor id e (26b). Following the procedure for
9, 0.14 g (96%) of 26b was prepared from 24b, mp 291-292
°C (dec). MS (m/z): 426 (MH+). IR (KBr) cm-1: 1746, 1631.
1H NMR (NaOD/D2O) δ: 1.6-1.9 (m, 1H), 2.0-2.3 (m, 1 H),
2.7-2.8 (m, 1 H), 2.9-3.1 (m, 1 H), 3.1-3.3 (m, 1 H), 3.3-3.6
(m, 2 H), 6.52 (d, 1 H, J ) 7.0 Hz). Anal. (C17H14F3N5O3S‚HCl‚
0.25H2O) C, H, Cl, F, N, S.
1-Be n zyl-3-(N -t er t -b u t oxyca r b on yl)m e t h yla m in o-3-
m eth ylp yr r olid in e (34). To a solution of 3-amino-1-benzyl-
3-methylpyrrolidine11 32 (20.0 g, 105 mmol) in CH2Cl2 (200
mL) was added Boc2O (25.2 g, 116 mmol) under ice-cooling.
The reaction mixture was stirred at room temperature for 2 h
and concentrated to dryness to afford a crude product, which
was chromatographed on silica gel with hexane-AcOEt (10:
1) to give 28.3 g (93%) of 33. MS (m/z): 291 (MH+). IR (neat)
cm-1: 3356, 1716, 1697.
To a solution of the above compound 33 (11.4 g, 39 mmol)
in toluene (150 mL) was added dropwise sodium bis(2-
methoxyethoxy)aluminum hydride (70% in toluene, 50 mL, 173
mmol) under ice-cooling. The reaction mixture was heated to
reflux for 2 h and then treated with EtOH and water under
ice-cooling. Insoluble material was removed by filtration, and
the filtrate was extracted with AcOEt. The organic layer was
dried over Na2SO4 and concentrated to dryness. The resulting
residue was diluted with CH2Cl2 (100 mL), and Boc2O (8.6 g,
39.4 mmol) in CH2Cl2 (5 mL) was added under ice-cooling. The
reaction mixture was stirred at room temperature for 2 h and
concentrated to dryness to afford a crude product, which was
chromatographed on silica gel with hexane-AcOEt (4:1) to give
7.5 g (63%) of 34. MS (m/z): 305 (MH+). IR (neat) cm-1: 1697.
3-(N-ter t-Bu toxyca r bon yl)m eth yla m in o-3-m eth ylp yr -
r olid in e (28). To a solution of 34 (7.50 g, 24.7 mmol) in EtOH
(35 mL) was added 10% Pd-C (0.83 g) under ice-cooling. The
resulting mixture was heated at 50 °C for 4 h in an atmosphere
of hydrogen gas. The catalyst was removed by filtration and
the filtrate was concentrated under reduced pressure to give
the product 28. MS (m/z): 215 (MH+). IR (neat) cm-1: 3337,
1697. This material was used without purification in the next
step.
Eth yl 5-Ben zyla m in o-7-ch lor o-1,4-d ih yd r o-4-oxo-1-(2-
th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxyla te (24d ). A mix-
ture of 24a (0.50 g, 1.35 mmol), benzylamine (0.14 g, 1.31
mmol), Et3N (0.28 g, 2.78 mmol), and toluene (15 mL) was
heated to reflux for 30 min. After most of the solvent was
removed under reduced pressure, the resulting residue was
recrystallized from AcOEt to give 0.51 g (86%) of 24d , mp 141-
1
143 °C. MS (m/z): 441 (MH+). IR (KBr) cm-1: 1733, 1635. H
NMR (CDCl3) δ: 1.42 (t, 3 H, J ) 7 Hz), 4.41 (q, 2 H, J ) 7
Hz), 4.49 (d, 1H, J ) 6.5 Hz), 6.47 (s, 1 H), 7.31 (d, 1 H, J )
3.5 Hz), 7.32-7.40 (m, 5 H), 7.70 (d, 1 H, J ) 3.5 Hz), 9.87 (s,
1 H), 11.2-11.7 (m, 1 H).
5-Am in o-7-ch lor o-1,4-d ih yd r o-4-oxo-1-(2-th ia zolyl)-1,8-
n a p h th yr id in e-3-ca r boxylic Acid (25c). A mixture of 24d
(500 mg, 1.14 mmol), H2SO4 (2 mL), and AcOH (8 mL) was
heated at 100 °C for 5 h and cooled to 0 °C. To the reaction
mixture was added H2O (8 mL), and the mixture was heated
at 100 °C for 1 h. After the mixture was ice-cooled, the
resulting precipitates were collected by filtration, washed with
H2O, and dried to give 350 mg (98%) of 25c, mp 264-265 °C.
MS (m/z): 322 (M+). IR (KBr) cm-1: 1727, 1636.
5-Am in o-7-(3-a m in o-1-p yr r olid in yl)-1,4-d ih yd r o-4-oxo-
1-(2-th ia zolyl)-1,8-n a p h th yr id in e-3-ca r boxylic Acid Hy-
d r och lor id e (26c). Following the procedure for 26a , 26c was
prepared from 25c in 47% yield, mp 261-263 °C (dec). MS
tr a n s-1-Ben zyl-3-cya n o-4-h yd r oxym et h ylp yr r olid in e
(36). A mixture of 3-(benzylamino)propionitrile 35 (50.0 g, 0.31
mmol), K2CO3 (86.0 g, 0.62 mmol), and epibromohydrine (27
mL, 0.32 mmol) in DMF (400 mL) was heated at 90 °C for 1 h,
during which period epibromohydrine was added twice [27 mL
(0.32 mmol) after 20 min and 30 mL (0.35 mmol) after 40 min].
After the mixture was ice-cooled, insoluble material was
(m/z): 373 (MH+). IR (KBr) cm-1
:
1712, 1628. 1H NMR
(DMSO-d6) δ: 2.1-2.3 (m, 1H), 2.3-2.5 (m, 1 H), 3.5-4.1 (m,
5 H), 5.64 (s, 1 H), 7.74 (d, 1 H, J ) 3.5 Hz), 7.81 (d, 1 H, J )

7-Substituted Carboxylic Acids as Antitumor Agents
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2107
filtered off. The filtrate was concentrated under reduced
pressure and dissolved in toluene (500 mL) and DMSO (200
mL). To the mixture was added sodium amide (25.0 g, 0.64
mmol), and the mixture was heated at 65 °C for 10 min, diluted
with ice-water, and extracted with toluene. The organic layer
was washed with water and 3% AcOH successively, dried over
MgSO4, and concentrated under reduced pressure to afford a
crude product, which was chromatographed on silica gel with
CHCl3 to give 9.0 g (13%) of 36. IR (neat) cm-1: 3417, 2241.
pressure to afford a crude product, which was chromato-
graphed on silica gel with CHCl3-MeOH (50:1) to give 1.30 g
of 40.
To a solution of the above compound 40 (1.30 g, 4.95 mmol)
in toluene (15 mL) was added dropwise sodium bis(2-meth-
oxyethoxy)aluminum hydride (70% in toluene, 3.60 mL, 12.5
mmol) under ice-cooling. The reaction mixture was heated at
90 °C for 1 day and then treated with water under ice-cooling.
Insoluble material was filtered off. The filtrate was extracted
with AcOEt, dried over Na2SO4, and concentrated to dryness
to afford a crude product, which was chromatographed on silica
gel with CHCl3-MeOH (50:1) to give 0.54 g (46%) of trans-1-
benzyl-3-(ethylamino)-4-methoxypyrrolidine. MS (m/z): 235
(MH+).
To a solution of the above compound (0.54 g, 2.3 mmol) in
MeOH (5 mL) was added Boc2O (0.75 g, 3.44 mmol) under ice-
cooling. The reaction mixture was stirred at room temperature
for 7 h and concentrated under reduced pressure. The resulting
residue was chromatographed on silica gel with CHCl3-MeOH
(100:1) to give 0.69 g (90%) of 41. MS (m/z): 335 (MH+).
tr a n s-3-(N-ter t-Bu t oxyca r b on yl)et h yla m in o-4-m et h -
oxyp yr r olid in e (30). Following the procedure for 28, 30 was
prepared from 41.
tr a n s-1-Ben zyl-4-ch lor om eth ylp yr r olid in e-3-ca r boxa -
m id e (37). A mixture of 36 (9.0 g, 42 mmol) and SOCl2 (7.6
mL, 104 mmol) in CHCl3 (120 mL) was heated to reflux for 4
h. The reaction mixture was treated with ice-water, alkalized
with 20% NaOH, and extracted with CHCl3. The organic layer
was washed with saturated brine, dried over MgSO4, and
concentrated under reduced pressure to afford a crude product,
which was chromatographed on silica gel with hexane-AcOEt
(3:1) to give 8.0 g (82%) of trans-1-benzyl-3-chloromethyl-4-
cyanopyrrolidine. MS (m/z): 234 (M+). IR (neat) cm-1: 2250.
To a solution of the above compound (8.0 g, 34 mmol) in
toluene (20 mL) was added a solution of concentrated H2SO4
(18 mL). The reaction mixture was stirred at room temperature
for 40 min and then heated at 60 °C for 30 min. After being
ice-cooled, the solution was alkalized with 50% NaOH and
extracted with AcOEt. The organic layer was washed with
saturated brine, dried over MgSO4, and concentrated under
reduced pressure to afford a crude product, which was recrys-
tallized from hexane-AcOEt to give 7.5 g (87%) of 37, mp 98-
99 °C. MS (m/z): 252 (M+). IR (KBr) cm-1: 3400, 3200, 1650,
1620.
t r a n s-1-N -t er t -B u t o x y c a r b o n y l-3-m e t h y la m in o -4-
m eth ylth iop yr r olid in e (43). To a solution of 1-Boc-pyrroline
42 (47.3 g, 0.28 mol) in CH2Cl2 (130 mL) was added dropwise
a solution of MeSCl (purity of 80%, 35.0 g, 0.42 mol) in CH2Cl2
(40 mL) under ice-cooling. The reaction mixture was stirred
at room temperature for 1 day and concentrated under reduced
pressure. To the resulting residue were added THF (270 mL)
and MeNH2 (40% aqueous solution, 240 mL, 2.8 mol) at room
temperature. The reaction mixture was heated at 70 °C for
15 h, treated with water under ice-cooling, and then extracted
with CH2Cl2. The organic layer was dried over Na2SO4 and
concentrated to dryness to afford a crude product, which was
chromatographed on silica gel with CHCl3-MeOH (50:1) to
tr a n s-1-Ben zyl-3-N-ter t-bu toxyca r bon yla m in o-4-ch lo-
r om eth ylp yr r olid in e (38). To a solution of 37 (7.0 g, 27.7
mmol) in MeOH (150 mL) was added dropwise NaOCl (8.5%
solution, 26.7 g, 30.5 mmol) under ice-cooling. The reaction
mixture was stirred at room temperature for 30 min and then
heated at 65 °C for 1.5 h. Most of the solvent was concentrated
under reduced pressure, and the obtained residue was ex-
tracted with CHCl3. The organic layer was dried over MgSO4
and concentrated under reduced pressure to afford a crude
product of 8.0 g of trans-1-benzyl-3-chloromethyl-4-methoxy-
give 53.1 g (77%) of 43. MS (m/z): 247 (MH+). IR (neat) cm-1
:
3322, 1693.
tr a n s-3-Meth yla m in o-4-m eth ylth iop yr r olid in e Dih y-
d r och lor id e (31). To a solution of 43 (24.7 g, 0.10 mol) in
EtOH (200 mL) was added 30% HCl-EtOH (42 mL). The
reaction mixture was stirred at room temperature for 1 day.
The resulting precipitates were collected by filtration, washed
with i-Pr2O, and then dried to give 20.3 g (92%) of 31, mp 193-
194 °C. MS (m/z): 147 (MH+). IR (KBr) cm-1: 3300.
carbonyaminopyrrolidine. MS (m/z): 282 (M+). IR (neat) cm-1
:
3310, 1720.
A mixture of the above compound (8.0 g) and concentrated
HCl (50 mL) was heated under reflux for 8 h and cooled to 0
°C. The reaction mixture was diluted with water, and activated
carbon was added. After filtration, the filtrate was alkalized
with 50% NaOH and extracted with AcOEt. The organic layer
was washed with saturated brine, dried over MgSO4, and
concentrated under reduced pressure. After the obtained
residue was dissolved in CHCl3 (50 mL), to the solution was
added a solution of Boc2O (4.4 g, 20 mmol) in CHCl3 (5 mL)
under ice-cooling. The reaction mixture was stirred at room
temperature for 3 h and then concentrated under reduced
pressure to afford a crude product, which was chromato-
graphed on silica gel with CHCl3 to give 4.8 g (53% from 37)
of 38, mp 87-88 °C. MS (m/z): 324 (M+). IR (KBr) cm-1: 3210,
1694.
E t h yl 1,4-Dih yd r o-4-oxo-7-p h en yl-1-(2-t h ia zolyl)-1,8-
n a p h th yr id in e-3-ca r boxyla te (44a ). To a mixture of 7 (1.00
g, 3.0 mmol) in toluene (40 mL) were added bis(triphenylphos-
phine)palladium dichloride (0.23 g, 0.33 mmol) and trimethyl-
(phenyl)tin (0.80 g, 3.3 mmol). The resulting mixture was
heated to reflux for 3 h. After ice-cooling, the reaction mixture
was poured into water and extracted with toluene. The organic
layer was dried over Na2SO4 and concentrated to dryness to
afford a crude product, which was chromatographed on silica
gel with CHCl3-MeOH (50:1) and recrystallized from EtOH
to give 0.74 g (66%) of 44a , mp 175-178 °C. MS (m/z): 378
(MH+). IR (KBr) cm-1: 1731, 1699. 1H NMR (DMSO-d6) δ: 1.33
(t, 3 H, J ) 7.0 Hz), 4.33 (q, 2 H, J ) 7.0 Hz), 7.50-7.70 (m,
3 H), 7.83 (d, 1 H, J ) 3.5 Hz), 7.87 (d,1 H, J ) 3.5 Hz), 8.26-
8.35 (m, 3 H), 8.72 (d, 1 H, J ) 8.0 Hz), 9.85 (s, 1 H).
1,4-Dih yd r o-4-oxo-7-p h en yl-1-(2-t h ia zolyl)-1,8-n a p h -
th yr id in e-3-ca r boxylic Acid (45a ). A mixture of 44a (0.54
g, 1.4 mmol), 20% HCl (55 mL), and EtOH (10 mL) was heated
at 100 °C for 10 h and cooled to 0 °C. The resulting precipitates
were collected by filtration and washed with water and EtOH
to afford a crude product, which was dissolved with NH4OH
and then acidified with 0.5 N HCl. The resulting precipitates
were collected by filtration, washed with water, EtOH, and
i-Pr2O, successively, and dried to give 0.31 g (62%) of 45a , mp
227-230 °C (dec). MS (m/z): 350 (MH+). IR (KBr) cm-1: 1739,
1615. 1H NMR (DMSO-d6) δ: 7.60-7.70 (m, 3 H), 7.93 (s, 2
H), 8.30-8.35 (m, 2 H), 8.39 (d, 1 H, J ) 8.0 Hz), 8.86 (d, 1 H,
J ) 8.0 Hz), 10.00 (s, 1 H). Anal. (C18H11N3O3S‚0.5H2O) C, H,
N, S.
tr a n s-3-N-ter t-Bu t oxyca r b on yla m in o-4-ch lor om et h -
ylp yr r olid in e (29). To a solution of 38 (1.03 g, 3.17 mmol),
5% Pd-C (0.24 g) and AcOH (0.70 mL, 12.2 mmol) in EtOH
(30 mL) was added ammonium formate (0.97 g, 15.4 mmol)
under ice-cooling. The reaction mixture was stirred at room
temperature for 1 h. The catalyst was removed by filtration
and the filtrate was concentrated under reduced pressure to
afford a crude product of 29. This material was used without
purification in the next step.
tr a n s-1-Ben zyl-3-(N-ter t-bu toxyca r bon yl)eth yla m in o-
4-m eth oxyp yr r olid in e (41). To a solution of trans-3-amino-
1-benzyl-4-methoxypyrrolidine22 39 (1.02 g, 4.95 mmol) in
CH2Cl2 (5 mL) was added acetic anhydride (5 mL, 52.9 mmol)
under ice-cooling. The reaction mixture was stirred at room
temperature for 4 h and then concentrated under reduced

2108 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Tsuzuki et al.
According to the similar procedure for 45a , compounds
45b,c were prepared.
medium (EMEM) supplemented with 10% fetal bovine serum
(FBS) and 50 units/mL of penicillin and 50 µg/mL of strepto-
mycin. HL-60 cells were grown in RPMI 1640 with 20% FBS.
Hs746T cells and PANC-1 cells were maintained in Dulbecco’s
modification of Eagle’s medium with 10% FBS. SBC-3 cells
were cultured in RPMI 1640 with 10% FBS. SKOV3 cells were
grown in RPMI 1640 with 10% heat-inactivated FBS. SCaBER
cells were maintained in EMEM supplemented with 10% FBS
and nonessential amino acids.
Eth yl 1,4-Dih yd r o-7-eth yn yl-4-oxo-1-(2-th ia zolyl)-1,8-
n a p h th yr id in e-3-ca r boxyla te (44d ). To a solution of 7 (3.01
g, 8.97 mmol) in toluene (110 mL) were added bis(triphen-
ylphosphine)palladium dichloride (0.70 g, 1.0 mmol) and 1-tri-
n-butylstannyl-2-trimethylsilylacetylene (3.94 g, 10.2 mmol).
The resulting mixture was heated to reflux for 5 h. After ice-
cooling, the reaction mixture was poured into water and
extracted with toluene. The organic layer was dried over
Na2SO4 and concentrated to dryness to afford a crude product,
which was chromatographed on silica gel with CHCl3 and
recrystallized from EtOH to give 2.49 g (70%) of ethyl 1,4-
dihydro-4-oxo-1-(2-thiazolyl)-7-(2-trimethylsilylethynyl)-1,8-
In Vitr o Assa y. Cells ((1-2) × 104 cells/mL) were put into
wells of a 96-well microtiter plate in the amount of 0.1 mL/
well, preincubated for 24 h except for P388 cells, and incubated
with various concentrations of a test compound in the 5% CO2
incubator at 37 °C for 72 h. After the culturing, 0.02 mL of a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) solution (5 mg/mL) was put in each well, and the cells
were cultured for a further 4 h. The medium was removed by
suction, and 0.2 mL of DMSO was put in each well to dissolve
the formed formazan. The absorbance was measured by
Multiskan Bichromatic (Labsystems, main wavelength 570
nm, subwavelength 690 nm). The IC50 was defined as the drug
concentration needed to produce a 50% reduction of absorbance
relative to the control.
In Vivo Assa y. Into BDF1 mice, 0.1 mL of diluted ascites
fluid containing 106 P388 leukemic cells was transplanted
intraperitoneally. Test compounds were suspended in 0.4%
CMC (carboxymethyl cellulose), and conventional antitumor
drugs were dissolved and diluted with distilled water and
administered ip on days 1 and 5 after tumor implantation.
Seven mice were used for each experimental group. Antitumor
activities were evaluated by determining the T/C (%), which
is (median survival time of treated group)/(median survival
time of control group) × 100.
1
naphthyridine-3-carboxylate. MS (m/z): 698 (MH+). H NMR
(CDCl3) δ: 0.32 (s, 9 H), 1.42 (t, 3 H, J ) 7.0 Hz), 4.45 (q, 2 H,
J ) 7.0 Hz), 7.37 (d, 1 H, J ) 3.5 Hz), 7.61 (d, 1 H, J ) 8.0
Hz), 7.72 (d, 1 H, J ) 3.5 Hz), 8.78 (d, 1 H, J ) 8.0 Hz), 10.05
(s, 1 H).
A mixture of the above compound (2.04 g, 6.28 mmol), KF
(1.05 g, 18.1 mmol), dioxane (50 mL), and EtOH (10 mL) was
stirred at room temperature for 3 h. The reaction mixture
concentrated to dryness, poured into water, and extracted with
CHCl3. The organic layer was dried over Na2SO4 and concen-
trated to dryness to afford a crude product, which was
chromatographed on silica gel with CHCl3 to give 1.10 g (54%)
1
of 44d , mp 228-231 °C (dec). MS (m/z): 326 (MH+). H NMR
(CDCl3) δ: 1.41 (t, 3 H, J ) 7.0 Hz), 3.33 (s, 1 H), 4.42 (q, 2 H,
J ) 7.0 Hz), 7.36 (d, 1 H, J ) 3.5 Hz), 7.66 (d, 1 H, J ) 8.0
Hz), 7.73 (d, 1 H, J ) 3.5 Hz), 8.80 (d, 1 H, J ) 8.0 Hz), 10.06
(s, 1 H).
1,4-Dih yd r o-7-et h yn yl-4-oxo-1-(2-t h ia zolyl)-1,8-n a p h -
th yr id in e-3-ca r boxylic Acid (45d ). Following the procedure
for 45a , 45d was prepared from 44d in 47% yield, mp >300
°C. MS (m/z): 298 (MH+). IR (KBr) cm-1: 1737, 1615. 1H NMR
(DMSO-d6) δ: 3.34 (s, 1 H), 7.85 (d, 1 H, J ) 3.7 Hz), 7.88 (d,
1 H, J ) 3.7 Hz), 7.93 (d, 1 H, J ) 8.2 Hz), 8.81 (d, 1 H, J )
8.2 Hz), 9.98 (s, 1 H). Anal. (C14H7N3O3S‚0.25H2O) C, H, N, S.
Eth yl 1,4-Dih yd r o-4-oxo-7-(3-p yr a zolyl)-1-(2-th ia zolyl)-
1,8-n a p h th yr id in e-3-ca r boxyla te (44e). To a solution of 44d
(0.34 g, 1.05 mmol) in CHCl3 (20 mL) was added dropwise
trimethylsilyldiazomethane (2 M in hexane, 1.05 mL, 2.10
mmol) at room temperature. After the mixture was stirred at
70 °C for 5 h, most of the solvent was concentrated to dryness
to afford a crude product, which was recrystallized from EtOH
to give 0.31 g (80%) of 44e, mp 285-287 °C (dec). MS (m/z):
Ack n ow led gm en t . We are grateful to Drs. T.
Sawayama and S. Mishio for their encouragement
throughout this work. Thanks are also due to members
of the Department of Physico-Chemical Analysis for
elemental analyses and spectral measurements.
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368 (MH+). H NMR (DMSO-d6) δ: 1.33 (t, 3 H, J ) 7.2 Hz),
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(KBr) cm-1: 1731, 1616. H NMR (DMSO-d6) δ: 7.28 (d, 1 H,
J ) 1.9 Hz), 7.88 (d, 1 H, J ) 3.5 Hz), 7.91 (d, 1 H, J ) 3.5
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