MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: Exploration of aromatic substituents

Article abstract of DOI:10.1016/j.bmc.2006.08.037

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone levo

Full text of DOI:10.1016/j.bmc.2006.08.037

Bioorganic & Medicinal Chemistry 14 (2006) 8506–8518
MexAB-OprM specific efflux pump inhibitors in Pseudomonas
aeruginosa. Part 6: Exploration of aromatic substituents
Ken-ichi Yoshida,a,* Kiyoshi Nakayama,a Yoshihiro Yokomizo,a Masami Ohtsuka,a
Makoto Takemura,a Kazuki Hoshino,b Hiroko Kanda,b Kenji Namba,b
Hironobu Nitanai,b Jason Z. Zhang,c Ving J. Leec and William J. Watkinsc
aMedicinal Chemistry Research Laboratory, Daiichi Pharmaceutical Co., Ltd., 16-13, Kita-Kasai 1-Chome,
Edogawa-ku, Tokyo 134-8630, Japan
bNew Product Research Laboratories I., Daiichi Pharmaceutical Co., Ltd., 16-13, Kita-Kasai 1-Chome,
Edogawa-ku, Tokyo 134-8630, Japan
cEssential Therapeutics, Inc., 850 Maude Avenue, Mountain View, CA 94043, USA
Received 18 July 2006; revised 23 August 2006; accepted 24 August 2006
Available online 18 September 2006
Abstract—A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were
synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone
levofloxacin (LVFX) and the anti-pseudomonas b-lactam aztreonam (AZT) in Pseudomonas aeruginosa. By incorporating hydro-
philic substituents onto the aryl nucleus, we found a morpholine analogue that possessed improved solubility, retained activity
in vitro, and displayed potentiation activity in vivo in a rat model of P. aeruginosa pneumonia.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
In a previous paper, we reported that several vinyl
stannanes, B-alkyl boranes, and boronic acids could be
reacted with an aryl chloride by palladium(0)-catalyzed
cross-coupling reactions, providing a powerful strategy
for derivatization at the C-2 position in a series of pyr-
idopyrimidines that inhibit MexAB-OprM.9 Although
the 2-carbon-linked variants, especially cyclic substitu-
ents, provided good potency, the high lipophilicity of
many of these analogues limited their aqueous solubility
and reduced their effectiveness in the presence of HSA.
Therefore, we extended our studies to include aromatic
substituents containing hydrophilic moieties.
In recent years, Pseudomonas aeruginosa has emerged as
a problematic pathogen due both to innate and expo-
sure-induced resistance to a variety of antibacterial
agents, and the significance of the phenomenon of efflux
has become apparent.1–3 Seven drug efflux pumps of P.
aeruginosa have been characterized thus far. The Mex-
AB-OprM system contributes to drug resistance of
wild-type strains and is commonly overexpressed in clin-
ical isolates.4 Therefore, we started research aimed at the
identification of a molecule for combined use with exist-
ing antibacterial agents that can restore their efficacy
through specific inhibition of drug efflux pumps, espe-
cially MexAB-OprM.5–9 The clinical impact of such a
drug combination, particularly in the treatment of
refractory infectious diseases such as those caused by
P. aeruginosa, would be significant.
Herein, we report the design, synthesis, and evaluation
of the resulting derivatives.
2. Chemistry
We planned to generate the desired analogues from the
appropriate boronic acids (2a–c, 2g–k) or pinacol esters
(2d–f) by typical Suzuki cross-coupling reactions10 fol-
lowed by hydrolysis of t-Bu ester moiety, as depicted
in Scheme 1. In practice, this proved to be a powerful
methodology, allowing for the incorporation of acidic,
Keywords: Efflux pump inhibitor; MexAB-OprM efflux pump; Pseu-
domonas aeruginosa; Drug resistance.
*
Corresponding author. Tel.: +81 3 5696 7331; fax: +81 3 5696
8772; e-mail: yoshi11v@daiichipharm.co.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.037

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
8507
Pd(Ph3P)4, Na2CO3
O
O
N
O
N
N
R2
N
O
R2
/ DME-H2O (4:1), reflux
hydrolysis
N
O
Cl
N
O
N
N
H
S
S
S
N
H
H
N
N
N
O
O
HO
HO
R2
or
B
R2
B
CO2tBu
CO2tBu
CO2H
4a-k
1
2a-c, 2g-k
2d-f
3a-k
CO2H
OH
HO
CO2H
OH
d
f
a
b
c
e
R2
=
N
N
N
OH
OH
g
i
j
k
h
Scheme 1. General procedure for synthesis of 2-Ar analogues.
neutral, and weakly basic hydrophilic functional groups
on phenyl and heteroaromatic substituents at the C-2
position.
methylamine (6n) and N-(2,2-difluoroethyl)-N-methyl-
amine (6o), which were not commercially available, were
prepared as shown in Scheme 4.
The benzylic alcohols 3g and 3h proved useful interme-
diates in the preparation of analogues incorporating
more basic benzylamines (Scheme 2). Reaction with
Ph3P and CBr4 afforded the benzyl bromides (5a, 5b).
Treatment with amines, with subsequent ester hydroly-
sis, gave the desired products (3l, 3m). This methodolo-
gy was extensively applied to survey derivatives of 5a, in
particular (Scheme 3). The isonipecotic acid analogue
(4u) was synthesized via the ethyl ester, with subsequent
hydrolysis with lithium hydroxide. N-Cyclopropyl-N-
The functionalized piperazine analogues (4x, 4y) were
synthesized by N-alkylation of the parent piperazine
(10), which itself was prepared via deprotection of the
N-allyl intermediate (Scheme 5).
3. Results and discussion
The compounds prepared in this study were tested for
in vitro potentiation of the activity of levofloxacin
Ph3P, CBr4
Me NH
O
O
O
N
N
N
2
3g
or
3h
/ THF
R2
/ CH2Cl2 -THF
N
R2
N
O
R2
HCl-dioxane
N
O
N
H
S
S
N
H
S
N
H
N
N
N
O
CO2H
CO2tBu
CO2tBu
Br
Br
N
N
N
5a: R2
5b: R2
=
=
3l: R2
=
4l: R2
=
3m: R2
=
4m: R2
=
N
Scheme 2. Introduction of benzylamine moiety.
amine 6n-6w
1) LiOH aq./THF (for 3u)
(Et3N for 6n, 6p, 6q)
O
R
N
N
O
R
2) TFA
/ THF
N
O
N
N
5a
S
N
N
H
S
H
N
O
CO2H
CO2tBu
4n-4w
3n-3w
F
F
CO2H
R =
N
N
N
N
N
N
OH
N
CO2H
N
O
N
N
N
qa
na
o
uc
pa,b
v
r
s
w
t
aHCl salt for 6
btBu ester for 6 and 3.
cEt ester for 6 and 3.
Scheme 3. Modification of benzylamine moiety.

8508
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
1) Tf2O, reflux
1) BnBr, Et3N / THF
2) BnNMe, Et3N / THF
3) H2, 5%Pd-C / MeOH
2) HCHO aq., NaBHCN / MeOH
3
H
F
F
3) H , 5%Pd-C / MeOH then HCl
N
H
2
NH2
N
OH
·HCl
6n
F
F
6o
7
8
Scheme 4. Preparation of secondary amines.
for 3x
Br(CH2)2OH, Et3N
/ THF, 50˚C
N
O
N
O
N
N
HN
NAllyl
N
O
N
N
N
5a
N
R
N
S
S
R
H
H
/ THF
N
for 3y
N
BrCH2CO2tBu, Et3N
/ THF
O
CO2tBu
R'
N,N'-dimethylbarbituric acid
Pd(Ph3P)4 / CH2Cl2
9: R = Allyl
10: R = H
3x: R = (CH2)2OH, R' = CO2tBu
4x: R = (CH2)2OH, R' = CO2H
HCl / THF
HCl / THF
3y: R = CH2CO2tBu, R' = CO2tBu
4y: R = CH2CO2H, R' = CO2H
Scheme 5. Modification of piperazine moiety.
(LVFX) and aztreonam (AZT) against PAM 1723, a
laboratory strain of P. aeruginosa in which the Mex-
AB-OprM pump is overexpressed, and the MexCD-
OprJ and MexEF-OprN pumps are disrupted.11 The re-
sults, expressed as the minimum concentration of inhib-
itor required to decrease (potentiate) the minimum
inhibitory concentration (MIC) of the combination
agent 8-fold (MPC8, lg/mL), together with aqueous sol-
ubilities (in pH 6.8 buffer solution, lg/mL) are presented
in Table 1. Antimicrobial assays were conducted in the
presence and absence of 0.125% human serum albumin
(HSA) in order to evaluate the effects of protein binding.
interesting feature of this series is that the migration of
the para-N,N-dimethylaminomethyl group to the meta
position led to a dramatic reduction in solubility (4l vs
4m).
The water-soluble benzylamine analogue (4l), which
showed moderate activity in vitro, had low potency
in vivo in combination with AZT and was found to be
acutely toxic in rats (data not shown). We therefore
embarked upon a program to modify the amine
substituents in the hopes of improving in vitro activity
and reducing the acute toxicity (Table 2).
The prototypical 2-Ph analogue (4a) showed encourag-
ing potency, but the reduced activity in the presence of
0.125% HSA and the limited aqueous solubility suggest-
ed that the lipophilicity might be a liability for the ser-
ies.9 In order to overcome this, analogues bearing
hydrophilic substituents were evaluated. Almost all
compounds bearing aromatic substituents at C-2 were
potent inhibitors, with 4-substituted analogues tending
to be more active than 3-substituted (e.g., 4b vs 4c).
Attachment of sp2-like carbon and electron-withdrawing
substituents, based on the hypothesis that acute toxicity
was the consequence of the strong basicity of the amine,
was examined (4n and 4o). Consistent with the results
for other less basic analogues (4i–4k), activity improved
but solubility was reduced. On the other hand, while
conversion to the a-amino acid (4p) improved solubility,
the potency was slightly reduced.
The 6-membered ring variant 4s was slightly more po-
tent than smaller congeners 4q and 4r. Attachment of
a hydroxyl group at the piperidine 4-position (4t) did
not increase solubility as much as expected, and the
isonipecotic acid derivative (4u) was less active. The
piperazine analogues (4v, 4x and 4y), which were synthe-
sized for the sake of ready derivatization, were equipo-
tent or slightly less active than the piperidine (4s).
Although the 4-benzoic acid derivative (4b) displayed
good activity which was not compromised in the pres-
ence of HSA, the solubility was still low. The introduc-
tion of hydroxyl groups was investigated in two ways.
Phenol analogues (4d–f) were less active potentiators
of AZT. On the other hand, the benzyl alcohol ana-
logues (4g, 4h) seemed to follow the same trend as that
observed for the benzoic acids and also possessed poor
solubility.
Overall, the morpholine derivative 4w showed the best
combination of in vitro potency (with and without
HSA) and solubility. In an acute toxicity assay, per-
formed by intravenous administration in rats, the lethal
dose was estimated to be above 100 mg/kg, consistent
with the hope that reduction in basicity of the amine
might prove beneficial.
From the above observations, the introduction of acidic
and neutral hydrophilic moieties had only incremental
effects on solubility. Thus attachment of basic functional
groups was next investigated. The conversion to N,N-
dimethylaniline (4i) and the switch from phenyl to pyridyl
substituents (4j, 4k) resulted in failure (low solubility and
low potency in the presence of HSA). However, a more ba-
sic N,N-dimethyl benzylamine analogue (4l) showed mod-
erate activity and high solubility (654 lg/mL). An
Because of its safety characteristics, compound 4w was
chosen as a prototypical example with which to perform
an exploratory in vivo experiment in combination with

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
Table 1. Incorporation of hydrophilic moiety on 2-Ar nucleia
8509
O
N
R2
N
O
N
S
H
N
CO2H
Compound
R2
MPC8 (LVFX) (lg/mL)
MPC8 (AZT) (lg/mL)
Sol (pH 6.8) (lg/mL)
Without HSA
With 0.125% HSA
4a
4b
4c
4d
4e
4f
4
16
0.5
2
2
CO2H
8
16
8
8
32
19
c
—
8
CO2H
OH
>16b
>32
>32
8
4
—
—
—
c
c
c
8
4
OH
HO
32
4
8
OH
4g
4h
4i
1
21
4
16
1
29
OH
N
c
—
4
16
2
N
4j
2
8
1
6.8
N
4k
4l
4
16
1
21
N
N
16
16
>32
>16b
4
654
4m
2
2.7
a All compounds lacked intrinsic antibacterial activity.
b The apparent lack of activity is attributable to precipitation of the compound.
c Not tested.
AZT. We evaluated its potentiation activity in a pneu-
monia model against P. aeruginosa PAM 1020, a wild-
type strain that exhibits basal levels of MexAB-OprM
and does not express MexCD-OprJ or MexEF-OprN.12
As shown in Table 3, when dosed at 10 mg/kg, 4w in
combination with 1000 mg/kg of AZT gave moderate
survival at the end of the study, whereas no obvious ef-
fects were observed on treatment with AZT alone.
stimulated the exploration of other substituents on ben-
zylic nitrogen atom and culminated in the morpholine
analogue, which showed potentiation activity in vivo
and a reasonable safety profile in an acute toxicity assay.
Additional research is required to further optimize the
series, which will be reported in due course.
5. Experimental
5.1. General
4. Conclusion
In summary, we have discovered a series of pyridopyr-
imidine derivatives, substituted at the 2-position with
carbon-linked aryl groups, that display in vitro inhibi-
tion of the MexAB-OprM efflux pump in P. aeruginosa.
The development of chemistry that introduces a variety
of aromatic rings onto scaffold was accomplished by
application of Suzuki cross-coupling methodology. Ben-
zyl amine analogues that exhibited improved solubility
Unless otherwise noted, materials were obtained from
commercial suppliers and used without further purifica-
tion. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-chloro-4-oxo-4H-pyrido[1,2-
a]pyrimidin-3-yl)acrylate (1), tert-butyl (2E)-3-(8-{[(4-
tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-2-
phenyl-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (3a)
and (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]car-

8510
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
Table 2. Modification of substituent of benzylic nitrogen atoma
O
N
R2
N
O
N
S
H
N
CO2H
Compound
R2
MPC8 (LVFX) (lg/mL)
MPC8 (AZT) (lg/mL)
Sol (pH 6.8) (lg/mL)
Without HSA
With 0.125% HSA
N
N
N
4n
4o
4p
4q
4r
8
32
4
2
66
14
F
4
16
16
16
8
16
32
F
CO2H
8
>830
130
N
N
32
4
c
32
4
—
N
N
N
c
4s
32
4
—
4t
8
16
4
74
OH
c
4u
4v
4w
4x
4y
32
8
>32
>8b
16
16
4
—
CO2H
N
12
N
N
N
4
2
140
O
N
c
8
16
4
—
OH
N
c
16
32
8
—
N
CO2H
a All compounds lacked intrinsic antibacterial activity.
b The apparent lack of activity is attributable to precipitation of the compound.
c Not tested.
Table 3. Efficacy of AZT (1000 mg/kg) combined with ABS (10 mg/kg)
by intravenous drip infusion in a model of P. aeruginosa pneumonia in
rats
Yanako MP-500D melting point apparatus and are
uncorrected. H NMR spectra were determined on a
JEOL JNM-EX400 spectrometer.
1
Conditions
Number of survival after infection
Chemical shifts are reported in parts per million relative
to tetramethylsilane as internal standard. Significant H
Day 0
Day 1
Day 3
Day 7
1
Non-treatment
AZT alone
AZT + 4w
8
8
8
2
8
8
0
1
4
0
1
3
NMR data are tabulated in the following order: number
of protons, multiplicity (s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; br, broad), coupling constant(s)
in hertz. Infrared (IR) spectra were obtained on a HOR-
IBA FT-720 spectrometer. High-resolution mass spectra
were obtained on a JEOL JMS-700 mass spectrometer
under electron impact ionization conditions (EI), or fast
bonyl}-4-oxo-2-phenyl-4H-pyrido[1,2-a]pyrimidin-3-
yl)acrylic acid (4a) were prepared according to the liter-
ature procedures.9 Melting points were taken on a

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
8511
atom bombardment ionization conditions (FAB). Ele-
MeOH = 98:2 ! 95:5
and
1:4 ! 1:2 ! 1:1) to afford 3d (35.3 mg) as an orange sol-
EtOAc/hexane = 1:9 !
mentary analyses were conducted at Research Technol-
ogy Center, Daiichi Pharmaceutical Co., Ltd. Column
chromatography refers to flash column chromatography
conducted on Merck silica gel 60, 230–400 mesh ASTM.
Thin-layer chromatography (TLC) was performed with
Merck silica gel 60 F254 TLC plates, and compound
visualization was effected with a 5% solution of phos-
phomolybdic acid in ethanol, UV lamp, or Wako ninhy-
drin spray. Unless otherwise specified, reactions were
carried out at ambient temperature. Combined organic
extracts were dried over anhydrous Na2SO4. Reagents
and solvents were removed from reaction mixture or
combined organic extracts by concentration in vacuo
using a rotary evaporator with bath at 35–45 ꢁC.
1
id. H NMR (CDCl3) d: 1.24 (9H, s), 1.37 (9H, s), 6.64
(1H, s), 7.02 (2H, d, J = 8.5 Hz), 7.40 (1H, d,
J = 15.6 Hz), 7.62 (2H, d, J = 8.5 Hz), 7.68 (1H, d,
J = 15.6 Hz), 7.70–7.74 (1H, m), 8.26 (1H, s), 9.18
(1H, d, J = 7.8 Hz). HRMS (FAB) Calcd for
C29H31N4O5S ([M+H]+): 547.2015. Found: 547.1993.
5.2.4. tert-Butyl (2E)-3-[8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-(3-hydroxyphenyl)-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-3-yl]acrylate (3e). Following the pro-
cedures as described for 3d, the title compound
(48.1 mg) was prepared from 1 (100 mg, 0.205 mmol)
1
and 2e (45.0 mg, 0.205 mmol) as an orange solid. H
NMR (CDCl3) d: 1.22 (9H, s), 1.24 (9H, s), 6.32 (1H,
s), 6.51–6.65 (1H, m), 6.76–6.89 (1H, m), 6.94–7.07
(1H, m), 7.08–7.17 (1H, m), 7.43 (1H, d, J = 15.6 Hz),
7.58–7.68 (1H, m), 7.75 (1H, d, J = 15.6 Hz), 8.60 (1H,
br s), 8.94–9.10 (1H, m). HRMS (FAB) Calcd for
C29H31N4O5S ([M+H]+): 547.2015. Found: 547.2026.
5.2. Synthesis
5.2.1. 4-(3-[(1E)-3-tert-Butoxy-3-oxoprop-1-en-1-yl]-8-
{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-
4H-pyrido[1,2-a]pyrimidin-2-yl)benzoic acid (3b). To a
solution of 1 (1.00 g, 2.05 mmol) in DME (20 mL)–
water (5 mL) were added 2b (339 mg, 2.05 mmol),
Na2CO3 (434 mg, 4.09 mmol), and Pd(Ph3P)4 (118 mg,
0.102 mmol), and refluxed for 23 h under Ar. The mix-
ture was concentrated, and the residue was diluted with
EtOAc and washed with brine. The organic layer was
dried and concentrated. The residue was purified by sil-
ica gel column chromatography (CHCl3 ! CHCl3/
EtOAc = 4:1 ! 2:1 ! CHCl3/MeOH = 9:1) to afford
5.2.5. tert-Butyl (2E)-3-[8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-(2-hydroxyphenyl)-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-3-yl]acrylate (3f). Following the pro-
cedures as described for 3d, the title compound
(21.9 mg) was prepared from 1 (100 mg, 0.205 mmol)
and 2f (45.0 mg, 0.205 mmol) as a yellow solid. 1H
NMR (CDCl3) d: 1.35 (9H, s), 1.46 (9H, s), 2.75 (1H,
dd, J = 14.8, 3.5 Hz), 2.87 (1H, dd, J = 14.8, 9.6 Hz),
6.14 (1H, dd, J = 9.8, 3.4 Hz), 6.64 (1H, s), 6.93–6.96
(1H, m), 7.07–7.12 (1H, m), 7.37–7.42 (1H, m), 7.58–
7.63 (1H, m), 8.19 (1H, dd, J = 7.8, 1.7 Hz), 8.22–8.26
(1H, m), 9.06 (1H, d, J = 7.3 Hz). HRMS (FAB) Calcd
for C29H31N4O5S ([M+H]+): 547.2015. Found:
547.1992.
1
3b (465 mg) as a yellow solid. H NMR (DMSO-d6) d:
1.31 (9H, s), 1.42 (9H, s), 6.87 (1H, s), 7.24 (1H, d,
J = 15.6 Hz), 7.40 (1H, d, J = 15.6 Hz), 7.71 (2H, d,
J = 8.3 Hz), 7.94 (1H, dd, J = 7.6, 1.2 Hz), 8.14 (2H, d,
J = 8.3 Hz), 8.47 (1H, s), 9.18 (1H, d, J = 7.6 Hz).
HRMS (FAB) Calcd for C30H31N4O6S ([M+H]+):
575.1964. Found: 575.1936.
5.2.6. tert-Butyl (2E)-3-{8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-[4-(hydroxymethyl)phenyl]-4-oxo-
4H-pyrido[1,2-a]pyrimidin-3-yl}acrylate (3g). Following
the procedures as described for 3b, the title compound
(815 mg) was prepared from 1 (1.00 g, 2.05 mmol) and
5.2.2. 3-(3-[(1E)-3-tert-Butoxy-3-oxoprop-1-en-1-yl]-8-
{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-
4H-pyrido[1,2-a]pyrimidin-2-yl)benzoic acid (3c). Follow-
ing the procedures as described for 3b, the title com-
1
pound (896 mg) was prepared from
2.05 mmol) and 2c (339 mg, 2.05 mmol) as a yellow sol-
1
(1.00 g,
2g (311 mg, 2.05 mmol) as an orange solid. H NMR
(CDCl3) d: 1.35 (9H, s), 1.50 (9H, s), 4.78 (2H, s), 6.56
(1H, s), 7.38 (1H, d, J = 15.6 Hz), 7.45 (2H, d,
J = 8.1 Hz), 7.55 (2H, d, J = 8.1 Hz), 7.60 (1H, d,
J = 15.6 Hz), 7.67 (1H, dd, J = 7.3, 1.6 Hz), 8.23 (1H,
d, J = 1.6 Hz), 9.13 (1H, d, J = 7.3 Hz). HRMS (FAB)
Calcd for C30H33N4O5S ([M+H]+): 561.2172. Found:
561.2212.
1
id. H NMR (DMSO-d6) d: 1.30 (9H, s), 1.41 (9H, s),
6.80 (1H, s), 7.23 (1H, d, J = 15.5 Hz), 7.45 (1H, d,
J = 15.5 Hz), 7.66 (1H, t, J = 7.6 Hz), 7.69–7.78 (1H,
m), 7.90–7.98 (1H, m), 8.15 (1H, d, J = 7.1 Hz), 8.21
(s, 1H), 8.44 (1H, s), 9.15 (1H, d, J = 7.3 Hz). HRMS
(FAB) Calcd for C30H31N4O6S ([M+H]+): 575.1964.
Found: 575.1936.
5.2.7. tert-Butyl (2E)-3-{8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-[3-(hydroxymethyl)phenyl]-4-oxo-
4H-pyrido[1,2-a]pyrimidin-3-yl}acrylate (3h). Following
the procedures as described for 3b, the title com-
5.2.3. tert-Butyl (2E)-3-[8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-(4-hydroxyphenyl)-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-3-yl]acrylate (3d). To a solution of 1
(100 mg, 0.205 mmol) in DME (4 mL)–water (1 mL)
were added 2d (45.0 mg, 0.205 mmol), Na2CO3
(43.4 mg, 0.409 mmol), and Pd(Ph3P)4 (11.8 mg,
10.2 lmol), and stirred at 80 ꢁC for 12.5 h under N2.
The mixture was diluted with 10% citric acid aq and
extracted with EtOAc (3·). The combined organic phas-
es were dried and concentrated. The residue was purified
by silica gel column chromatography (CHCl3 ! CHCl3/
pound (267 mg) was prepared from
1 (250 mg,
0.511 mmol) and 2h (77.7 mg, 0.511 mmol) as an or-
ange solid. 1H NMR (CDCl3) d: 1.37 (9H, s), 1.53
(9H, s), 4.76 (2H, s), 6.44 (1H, s), 7.13–7.28 (3H,
m), 7.32 (1H, d, J = 15.9 Hz), 7.47–4.67 (3H, m),
8.13 (1H, s), 8.90 (1H, d, J = 7.3 Hz). HRMS
(FAB) Calcd for C30H33N4O5S ([M+H]+): 561.2172.
Found: 561.2194.

8512
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
5.2.8. tert-Butyl (2E)-3-{8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-[4-(dimethylamino)phenyl]-4-oxo-
4H-pyrido[1,2-a]pyrimidin-3-yl}acrylate (3i). Following
the procedures as described for 3b, the title compound
(266 mg) was prepared from 1 (250 mg, 0.511 mmol)
1547, 1500, 1437, 1400, 1288, 1254, 1190, 1140, 1097, 1059.
1H NMR (DMSO-d6) d: 1.30 (9H, s), 6.88 (1H, s), 7.27 (1H,
d, J = 15.6 Hz), 7.44 (1H, d, J = 15.6 Hz), 7.71 (1H, t,
J = 7.8 Hz), 7.80–7.85 (1H, m), 7.88–7.93 (1H, m), 8.10–
8.15 (1H, m), 8.20 (1H, s), 8.50 (1H, s), 9.18 (1H, d,
J = 7.3 Hz). Anal. Calcd for C26H22N4O6SÆ0.5TFAÆ1.5-
H2O: C, 53.82; H, 4.27; N, 9.30; S, 5.32; F, 4.73. Found:
C, 53.82; H, 4.11; N, 9.03; S, 5.35; F, 5.23.
1
and 2i (84.4 mg, 0.511 mmol) as a red solid. H NMR
(CDCl3) d: 1.32 (9H, s), 1.52 (9H, s), 3.01 (6H, s), 6.49
(1H, s), 6.66 (2H, d, J = 9.0 Hz), 7.35 (1H, d,
J = 15.7 Hz), 7.47–7.54 (3H, m), 7.74 (1H, d,
J = 15.7 Hz), 8.12 (1H, d, J = 1.2 Hz), 8.97 (1H, d,
J = 7.3 Hz). HRMS (FAB) Calcd for C31H36N5O4S
([M+H]+): 574.2488. Found: 574.2460.
5.2.13. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(4-hydroxyphenyl)-4-oxo-4H-pyrido[1,2-a]-
pyrimidin-3-yl]acrylic acid (4d). Following the proce-
dures as described for 4b, the title compound
5.2.9. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-4-oxo-2-pyridin-4-yl-4H-pyrido[1,2-
a]pyrimidin-3-yl)acrylate (3j). Following the procedures
as described for 3b, the title compound (41.1 mg) was
(22.7 mg)
was
prepared
from
3d
(35.3 mg,
0.06458 mmol) as a yellow solid. Mp: 241–246 ꢁC
(dec). IR (ATR) cmꢀ1: 3111, 2964, 2871, 1666, 1604,
1560, 1500, 1452, 1410, 1365, 1311, 1275, 1220, 1171,
1
prepared from
1
(100 mg, 0.205 mmol) and 2j
1101, 1063. H NMR (DMSO-d6) d: 1.30 (9H, s), 6.88
(27.5 mg, 0.225 mmol) as a yellow solid. 1H NMR
(CDCl3) d: 1.36 (9H, s), 1.51 (9H, s), 6.53 (1H, s), 7.43
(1H, d, J = 15.6 Hz), 7.47–7.51 (2H, m), 7.54 (1H, d,
J = 15.6 Hz), 7.75 (1H, dd, J = 1.7, 7.4 Hz), 8.30–8.34
(1H, m), 8.75–8.81 (2H, m), 9.14 (1H, d, J = 7.4 Hz).
HRMS (FAB) Calcd for C28H30N5O4S ([M+H]+):
532.2019. Found: 532.1996.
(1H, s), 6.94 (2H, d, J = 8.5 Hz), 7.25 (1H, d,
J = 15.6 Hz), 7.49 (2H, d, J = 8.5 Hz), 7.57 (1H, d,
J = 15.6 Hz), 7.82–7.86 (1H, m), 8.41-8.44 (1H, m),
9.12 (1H, d, J = 7.3 Hz), 10.04 (1H, s). Anal. Calcd for
C25H22N4O5SÆ1.5H2O: C, 58.02; H, 4.87; N, 10.83; S,
6.20. Found: C, 58.25; H, 4.61; N, 10.47; S, 6.15.
5.2.14.
(2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)ami-
5.2.10. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-4-oxo-2-pyridin-3-yl-4H-pyrido[1,2-
a]pyrimidin-3-yl)acrylate (3k). Following the procedures
as described for 3b, the title compound (63.4 mg) was
no]carbonyl}-2-(3-hydroxyphenyl)-4-oxo-4H-pyrido[1,2-
a]pyrimidin-3-yl]acrylic acid (4e). Following the proce-
dures as described for 4b, the title compound (29.0 mg)
was prepared from 3e (48.5 mg, 0.08874 mmol) as a yel-
low solid. Mp: 289–295 ꢁC (dec). IR (ATR) cmꢀ1: 3099,
2964, 2870, 1658, 1610, 1560, 1510, 1477, 1412, 1367,
prepared from
1 (100 mg, 0.205 mmol) and 2k
(27.5 mg, 0.225 mmol) as a yellow solid. 1H NMR
(CDCl3) d: 1.35 (9H, s), 1.50 (9H, s), 6.51 (1H, s),
7.34–7.43 (1H, m), 7.39 (1H, d, J = 15.6 Hz), 7.51 (1H,
d, J = 15.6 Hz), 7.66–7.75 (1H, m), 7.83–7.91 (1H, m),
8.30 (1H, s), 8.71–8.85 (2H, m), 9.08 (1H, d,
J = 7.3 Hz). HRMS (FAB) Calcd for C28H30N5O4S
([M+H]+): 532.2019. Found: 532.1989.
1
1311, 1282, 1242, 1203, 1103, 1066. H NMR (DMSO-
d6) d: 1.30 (9H, s), 6.86–7.00 (4H, m), 7.26 (1H, d,
J = 15.6 Hz), 7.36 (1H, t, J = 7.8 Hz), 7.52 (1H, d,
J = 15.6 Hz), 7.87–7.91 (1H, m), 8.41–8.44 (1H, m), 9.16
(1H, d, J = 7.3 Hz), 9.77 (1H, s). Anal. Calcd for
C25H22N4O5SÆH2O: C, 59.04; H, 4.76; N, 11.02; S, 6.31.
Found: C, 58.83; H, 4.69; N, 11.01; S, 6.39.
5.2.11.
4-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]car-
bonyl}-3-[(E)-2-carboxyvinyl]-4-oxo-4H-pyrido[1,2-a]pyr-
imidin-2-yl}benzoic acid (4b). To TFA (10 mL) was added
3b (250 mg, 0.435 mmol) and stirred for 1 h. The mixture
was concentrated, and the residue was diluted with Et2O.
The precipitated solid was collected by filtration and
washed with Et2O to afford 4b (203 mg) as a yellow solid.
Mp: 322–327 ꢁC (dec). IR (ATR) cmꢀ1: 3180, 3095, 2964,
2871, 2800, 1678, 1604, 1568, 1547, 1508, 1448, 1396,
1367, 1311, 1292, 1242, 1205, 1153, 1103, 1061, 1018. 1H
NMR (DMSO-d6) d: 1.31 (9H, s), 6.89 (1H, s), 7.28 (1H,
d, J = 15.5 Hz), 7.43 (1H, d, J = 15.5 Hz), 7.72 (2H, d,
J = 7.9 Hz), 7.93 (1H, dd, J = 1.7, 7.6 Hz), 8.14 (2H, d,
J = 7.9 Hz), 8.46–8.49 (1H, m), 9.20 (1H, d, J = 7.6 Hz),
12.92 (1H, br). Anal. Calcd for C26H22N4O6SÆ0.5H2O:
C, 59.19; H, 4.39; N, 10.62; S, 6.08. Found: C, 58.95; H,
4.38; N, 10.51; S, 6.15.
5.2.15. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(2-hydroxyphenyl)-4-oxo-4H-pyrido[1,2-a]-
pyrimidin-3-yl]acrylic acid (4f). Following the proce-
dures as described for 4b, the title compound
(17.4 mg)
was
prepared
from
3f
(21.9 mg,
0.04003 mmol) as a yellow solid. Mp: 304–309 ꢁC
(dec.). IR (ATR) cmꢀ1: 2968, 1670, 1608, 1556, 1522,
1485, 1446, 1427, 1362, 1313, 1281, 1234, 1203, 1173,
1
1101, 1068, 1043. H NMR (DMSO-d6) d: 1.31 (9H,
s), 2.62 (1H, dd, J = 15.4, 3.2 Hz), 2.70 (1H, dd,
J = 15.4, 10.0 Hz), 5.97 (1H, dd, J = 10.0, 3.2 Hz), 6.88
(1H, s), 6.96 (1H, d, J = 8.3 Hz), 7.14–7.20 (1H, m),
7.44–7.50 (1H, m), 7.76 (1H, dd, J = 7.6, 1.7 Hz), 8.17
(1H, dd, J = 7.6, 1.7 Hz), 8.47 (1H, s), 8.99 (1H, d,
J = 7.3 Hz), 12.76 (1H, br s). Anal. Calcd for
C25H22N4O5SÆ0.75H2O: C, 59.57; H, 4.70; N, 11.12; S,
6.36. Found: C, 59.58; H, 4.45; N, 11.11; S, 6.28.
5.2.12.
3-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]car-
bonyl}-3-[(E)-2-carboxyvinyl]-4-oxo-4H-pyrido[1,2-a]pyr-
imidin-2-yl}benzoic acid (4c). Following the procedures as
described for 4b, the title compound (24.4 mg) was prepared
from 3c (28.4 mg, 0.0494 mmol) as a yellow solid. Mp: 234–
240 ꢁC (dec). IR (ATR) cmꢀ1: 3062, 2962, 2870, 1678, 1583,
5.2.16.
(2E)-3-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)ami-
no]carbonyl}-2-[4-(hydroxymethyl)phenyl]-4-oxo-4H-pyr-
ido[1,2-a]pyrimidin-3-yl}acrylic acid (4g). Following the
procedures as described for 4b, the title compound
(189 mg) was prepared from 3g (250 mg, 0.446 mmol)

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
8513
as a yellow solid. Mp: 282–295 ꢁC (dec.). IR (ATR)
cmꢀ1: 3103, 3053, 2960, 2871, 1670, 1639, 1603, 1560,
1549, 1500, 1442, 1402, 1363, 1311, 1267, 1201, 1147,
C, 50.93; H, 4.99; N, 12.37; S, 5.67. Found: C, 50.90; H,
4.85; N, 12.25; S, 5.84.
1
1101, 1057. H NMR (DMSO-d6) d: 1.31 (9H, s), 4.62,
5.2.20. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-4-oxo-2-pyridin-3-yl-4H-pyrido[1,2-a]pyri-
midin-3-yl)acrylic acid (4k). Following the procedures as
described for 4i, the title compound (41.9 mg) was pre-
pared from 3k (63.4 mg, 119 lmol) as an orange solid.
Mp: 266–271 ꢁC (dec). IR (ATR) cmꢀ1: 3371, 3055,
2964, 2085, 1682, 1635, 1601, 1543, 1493, 1466, 1454,
1414, 1363, 1306, 1279, 1228, 1186, 1142, 1101, 1059.
1H NMR (DMSO-d6) d: 1.31 (9H, s), 6.89 (1H, s),
7.29 (1H, d, J = 15.4 Hz), 7.41 (1H, d, J = 15.4 Hz),
7.76 (1H, dd, J = 5.1, 7.8 Hz), 7.95 (1H, dd, J = 2.0,
7.3 Hz), 8.18 (1H, d, J = 7.8 Hz), 8.49 (1H, s), 8.82–
8.86 (1H, m), 8.87 (1H, d, J = 2.0 Hz), 9.21 (1H, d,
J = 7.3 Hz). Anal. Calcd for C24H21N5O4SÆ0.9HClÆ2.4-
H2O: C, 52.26; H, 4.88; N, 12.70; S, 5.81; Cl, 5.78.
Found: C, 52.38; H, 4.99; N, 12.49; S, 5.83; Cl, 5.66.
4.63 (total 2H, 2s), 5.36 (1H, t, J = 5.7 Hz), 6.86 (1H,
s), 7.28 (1H, d, J = 15.6 Hz), 7.49–7.60 (5H, m), 7.88–
7.93 (1H, m), 8.42–8.46 (1H, m), 9.16 (1H, d,
J = 7.6 Hz). Anal. Calcd for C26H24N4O5SÆ2.25H2O:
C, 57.29; H, 5.27; N, 10.28; S, 5.88. Found: C, 57.29;
H, 4.80; N, 10.23; S, 6.02.
5.2.17. (2E)-3-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-[3-(hydroxymethyl)phenyl]-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-3-yl}acrylic acid (4h). Following the
procedures as described for 4b, the title compound
(27.0 mg) was prepared from 3h (64.2 mg, 0.115 mmol)
as a yellow solid. Mp: 246–252 ꢁC (dec). IR (ATR)
cmꢀ1: 2962, 1672, 1599, 1560, 1546, 1504, 1452, 1406,
1365, 1311, 1292, 1277, 1223, 1203, 1101, 1061. 1H
NMR (DMSO-d6) d: 1.29 (9H, s), 4.56–4.64 (2H, m),
5.33 (1H, t, J = 5.7 Hz), 6.81 (1H, s), 7.27 (1H, d,
J = 15.6 Hz), 7.37–7.43 (1H, m), 7.45–7.53 (3H, m),
7.59 (1H, s), 7.92 (1H, d, J = 7.6 Hz), 8.42 (1H, s),
9.16 (1H, d, J = 7.6 Hz). Anal. Calcd for C26H24N4O5-
SÆ1.25H2O: C, 59.25; H, 5.07; N, 10.63; S, 6.08. Found:
C, 59.18; H, 4.78; N, 10.52; S, 6.19.
5.2.21. tert-Butyl (2E)-3-(2-[4-(bromomethyl)phenyl]-8-
{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-
4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (5a). To a solu-
tion of 3g (615 mg, 1.10 mmol) in CH2Cl2 (6 mL)–
THF (6 mL) were added CBr4 (728 mg, 2.19 mmol)
and Ph3P (719 mg, 2.74 mmol) at 0 ꢁC, and stirred for
30 min at 0 ꢁC. The mixture was concentrated, and the
residue was purified by silica gel column chromatogra-
phy (EtOAc/hexane = 1:4) to afford 5a (649 mg) as an
5.2.18. (2E)-3-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-[4-(dimethylamino)phenyl]-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-3-yl}acrylic acid (4i). To a solution of
3i (266 mg, 0.463 mmol) in 4 N HCl solution in dioxane
(5 mL) was added concentrated HCl (0.5 mL) and stir-
red for 1.5 h. The mixture was concentrated, and the
residue was purified by silica gel column chromatogra-
phy (CHCl3 ! CHCl3/MeOH = 95:5 ! 10:1). The com-
bined fractions were concentrated and the residue was
solidified with Et2O. The precipitated solid was collect-
ed by filtration and washed with Et2O to afford 4i
(153 mg) as an orange solid. Mp: 288–294 ꢁC (dec).
IR (ATR) cmꢀ1: 2964, 2860, 2798, 1672, 1599, 1558,
1508, 1441, 1406, 1362, 1311, 1292, 1255, 1228, 1196,
1
orange solid. H NMR (CDCl3) d: 1.33 (9H, s), 1.55
(9H, s), 4.46 (2H, s), 6.40 (1H, s), 7.389 (2H, d,
J = 8.3 Hz), 7.392 (1H, d, J = 15.6 Hz), 7.46 (2H, d,
J = 8.3 Hz), 7.52–7.58 (1H, m), 7.62 (1H, d, J = 15.6
Hz), 8.15 (1H, d, J = 1.0 Hz), 8.93–9.00 (1H, m).
5.2.22. tert-Butyl (2E)-3-(2-[3-(bromomethyl)phenyl]-8-
{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-
4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (5b). Following
the procedures as described for 5a, the title compound
(261 mg) was prepared from 3h (267 mg, 476 lmol) as
an orange foam. 1H NMR (CDCl3) d: 1.37 (9H, s),
1.53 (9H, s), 4.76 (2H, s), 6.44 (1H, s), 7.13–7.28 (3H,
m), 7.32 (1H, d, J = 15.9 Hz), 7.47–4.67 (3H, m), 8.13
(1H, s), 8.90 (1H, d, J = 7.3 Hz).
1
1151, 1124, 1101, 1063, 1005. H NMR (DMSO-d6) d:
1.31 (9H, s), 3.04 (6H, s), 6.85–6.90 (3H, m), 7.27
(1H, d, J = 15.6 Hz), 7.56 (2H, d, J = 8.8 Hz), 7.65
(1H, d, J = 15.6 Hz), 7.80 (1H, dd, J = 1.8, 7.6 Hz),
8.40–8.43 (1H, m), 9.08 (1H, d, J = 7.6 Hz). Anal.
Calcd for C27H27N5O4SÆ0.5H2O: C, 61.58; H, 5.36; N,
13.30; S, 6.09. Found: C, 61.58; H, 5.16; N, 13.33; S,
6.33.
5.2.23. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-{4-[(dimethylamino)methyl]phenyl}-
4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (3l). To a
solution of 5a (250 mg, 0.401 mmol) in THF (5 mL)
was added Me2NH (2.0 M THF solution, 401 lL,
0.802 mmol) at 0 ꢁC and stirred for 64 h. The mixture
was diluted with satd. NaHCO3 aq and extracted
with CHCl3 (5·). The combined organic phases
were dried and concentrated. The residue was purified
by silica gel column chromatography (CHCl3/
MeOH = 99:1 ! 98:2) to afford 3l (214 mg) as a yellow
5.2.19. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-4-oxo-2-pyridin-4-yl-4H-pyrido[1,2-a]pyrim-
idin-3-yl)acrylic acid (4j). Following the procedures as de-
scribed for 4i, the title compound (22.7 mg) was prepared
from 3j (41.1 mg, 77.3 lmol) as a yellow solid. Mp: 294–
304 ꢁC (dec). IR (ATR) cmꢀ1: 3435, 3373, 2968, 1691,
1637, 1603, 1556, 1495, 1462, 1414, 1365, 1336, 1288,
1
solid. H NMR (CDCl3) d: 1.33 (9H, s), 1.50 (9H, s),
2.27 (6H, s), 3.50 (2H, s), 6.49 (1H, s), 7.36 (1H, d,
J = 15.7 Hz), 7.41 (2H, d, J = 7.9 Hz), 7.51 (2H, d,
J = 7.9 Hz), 7.58–7.63 (1H, m), 7.63 (1H, d,
J = 15.7 Hz), 8.10 (1H, s), 9.05 (1H, d, J = 7.3 Hz).
HRMS (FAB) Calcd for C32H38N5O4S ([M+H]+):
588.2645. Found: 588.2628.
1
1230, 1192, 1144, 1107, 1061. H NMR (DMSO-d6) d:
1.31 (9H, s), 6.89 (1H, s), 7.28 (1H, d, J = 15.6 Hz), 7.34
(1H, d, J = 15.6 Hz), 7.77 (2H, d, J = 5.6 Hz), 7.94–8.00
(1H, m), 8.48 (1H, s), 8.91 (2H, d, J = 5.6 Hz), 9.23 (1H,
d, J = 7.3 Hz). Anal. Calcd for C24H21N5O4SÆHClÆ3H2O:

8514
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
5.2.24. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-{3-[(dimethylamino)methyl]phenyl}-
4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (3m). Fol-
lowing the procedures as described for 3l, the title com-
pound (198 mg) was prepared from 5b (261 mg,
7.39 (1H, d, J = 15.7 Hz), 7.47 (2H, d, J = 7.8 Hz),
7.59 (1H, d, J = 7.3 Hz), 7.66 (1H, d, J = 15.7 Hz),
8.20 (1H, br s), 9.04 (1H, d, J = 7.3 Hz). HRMS
(FAB) Calcd for C34H40N5O4S ([M+H]+): 614.2801.
Found: 614.2783.
1
418 lmol) as a yellow solid. H NMR (CDCl3) d: 1.32
(9H, s), 1.47 (9H, s), 2.29 (6H, s), 3.57 (2H, s), 6.54
(1H, s), 7.25–7.70 (5H, m), 7.31 (1H, d, J = 15.7 Hz),
7.54 (1H, d, J = 15.7 Hz), 8.11 (1H, s), 8.98–9.12 (1H,
m). HRMS (FAB) Calcd for C32H38N5O4S ([M+H]+):
588.2645. Found: 588.2660.
5.2.28. tert-Butyl (2E)-3-[8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-(4-{[(2,2-difluoroethyl)(methyl)ami-
no]methyl}phenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-
yl]acrylate (3o). Following the procedures as described
for 3l, the title compound (249 mg) was prepared from
1
5a (250 mg, 0.401 mmol) and 6o as a yellow solid. H
5.2.25. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-{4-[(dimethylamino)methyl]phenyl}-4-oxo-4H-
pyrido[1,2-a]pyrimidin-3-yl)acrylic acid (4l). Following
the procedures as described for 4i, the title compound
(190 mg) was prepared from 3l (214 mg, 0.364 mmol)
NMR (CDCl3) d: 1.32 (9H, s), 1.53 (9H, s), 2.36 (3H,
s), 2.81 (2H, dt, J = 4.4, 14.9 Hz), 3.63 (2H, s), 5.88
(1H, tt, J = 4.4, 56.0 Hz), 6.43 (1H, s), 7.36 (2H, d,
J = 7.9 Hz), 7.39 (1H, d, J = 15.6 Hz), 7.49 (1H, d,
J = 7.9 Hz), 7.59 (1H, dd, J = 1.7, 7.3 Hz), 7.65 (1H, d,
J = 15.6 Hz), 8.20 (1H, d, J = 1.2 Hz), 9.01 (1H, d,
J = 7.3 Hz), 11.10 (1H, br). HRMS (FAB) Calcd
for C33H38F2N5O4S ([M+H]+): 638.2613. Found:
638.2597.
as a red solid. Mp: 216–223 ꢁC (dec). IR (ATR) cmꢀ1
:
2962, 2868, 2750, 2683, 2584, 1739, 1693, 1645, 1593,
1566, 1491, 1390, 1342, 1304, 1221, 1182, 1101, 1057.
1H NMR (DMSO-d6) d: 1.30 (9H, s), 2.491, 2.494 (total
3H, 2s), 2.75, 2.76 (total 2H, 2s), 6.88 (1H, s), 7.27 (1H,
d, J = 15.6 Hz), 7.44 (1H, d, J = 15.6 Hz), 7.68 (2H, d,
J = 8.0 Hz), 7.73–7.79 (2H, m), 7.90–7.95 (1H, m),
8.40–8.43 (1H, m), 9.18 (1H, d, J = 7.6 Hz). Anal. Calcd
for C28H29N5O4SÆ2.25HClÆ1.75H2O: C, 52.12; H, 5.43;
N, 10.85; S, 4.97; Cl, 12.36. Found: C, 52.32; H, 5.50;
N, 10.81; S, 4.99; Cl, 12.06.
5.2.29. tert-Butyl (2E)-3-(2-(4-{[(2-tert-butoxy-2-oxoeth-
yl)(methyl)amino]methyl}phenyl)-8-{[(4-tert-butyl-1,3-
thiazol-2-yl)amino]carbonyl}-4-oxo-4H-pyrido[1,2-a]pyri-
midin-3-yl)acrylate (3p). Following the procedures as
described for 3n, the title compound (220 mg) was pre-
pared from 5a (200 mg, 0.321 mmol) and 6p (175 mg,
0.963 mmol) as a yellow oil. 1H NMR (CD3OD) d:
1.33 (9H, s), 1.44 (9H, s), 1.50 (9H, s), 2.39 (3H, s),
3.21 (2H, s), 3.77 (2H, s), 6.64 (1H, s), 7.13 (1H, d,
J = 15.6 Hz), 7.46 (1H, d, J = 15.6 Hz), 7.51 (2H, d,
J = 8.3 Hz), 7.56 (2H, d, J = 8.3 Hz), 7.75 (1H, d,
J = 7.3 Hz), 8.14 (1H, s), 9.07 (1H, d, J = 7.3 Hz).
HRMS (FAB) Calcd for C37H46N5O6S ([M+H]+):
688.3169. Found: 688.3157.
5.2.26. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-{3-[(dimethylamino)methyl]phenyl}-4-oxo-4H-
pyrido[1,2-a]pyrimidin-3-yl)acrylic acid (4m). Following
the procedures as described for 4i, the title compound
(185 mg) was prepared from 3m (198 mg, 0.337 mmol)
as an orange solid. Mp: 275–283 ꢁC (dec). IR (ATR)
cmꢀ1: 3464, 3128, 2964, 1730, 1674, 1649, 1579, 1500,
1475, 1346, 1311, 1250, 1205, 1167, 1149, 1097, 1061,
1
1014. H NMR (DMSO-d6) d: 1.31 (9H, s), 2.74, 2.76
5.2.30. tert-Butyl (2E)-3-(2-[4-(azetidin-1-ylmethyl)phen-
yl]-8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-
oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (3q). Fol-
lowing the procedures as described for 3n, the title com-
pound (173 mg) was prepared from 5a (250 mg,
0.401 mmol) and 6q (75.0 mg, 0.802 mmol) as a yellow
(total 6H, each s), 4.39, 4.40 (total 2H, each s), 6.89
(1H, s), 7.27 (1H, d, J = 15.6 Hz), 7.46 (1H, d,
J = 15.6 Hz), 7.68–7.79 (3H, m), 7.81–7.86 (1H, m),
7.95 (1H, dd, J = 1.7, 7.3 Hz), 8.46 (1H, d, J = 1.7 Hz),
9.20 (1H, d, J = 7.3 Hz), 10.77–10.87 (1H, m). Anal.
Calcd for C28H29N5O4SÆ2.4HClÆ2.5H2O: C, 50.63; H,
5.52; N, 10.54; S, 4.83; Cl, 12.81. Found: C, 50.69; H,
5.51; N, 10.50; S, 4.90; Cl, 12.43.
1
solid. H NMR (CDCl3) d: 1.34 (9H, s), 1.47 (9H, s),
2.01 (2H, quintet, J = 6.9 Hz), 3.32 (4H, t, J = 6.9 Hz),
3.80 (2H, s), 6.52 (1H, s), 7.34 (1H, d, J = 15.6 Hz),
7.42 (2H, d, J = 8.0 Hz), 7.51 (2H, d, J = 8.0 Hz), 7.59
(1H, d, J = 15.6 Hz), 7.61 (1H, d, J = 7.3 Hz), 7.95
(1H, d, J = 1.2 Hz), 9.05 (1H, d, J = 7.3 Hz). HRMS
(FAB) Calcd for C33H38N5O4S ([M+H]+): 600.2645.
Found: 600.2672.
5.2.27. tert-Butyl (2E)-3-[8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-(4-{[cyclopropyl(methyl)amino]meth-
yl}phenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]acrylate
(3n). To a solution of N-cyclopropyl-N-methylamine
hydrochloride 6n (122 mg, 1.13 mmol) in THF (5 mL)
was added Et3N (166 lL, 1.19 mmol) and stirred for
10 min. To the mixture was added 5a (200 mg,
0.321 mmol) and stirred for 17 h. The mixture was con-
centrated, and the residue was diluted with satd NaH-
CO3 aq and extracted with CHCl3. The organic layer
was washed with brine. The organic layer was dried
and concentrated. The residue was purified by PTLC
(CHCl3/MeOH = 10:1) to afford 3n (176 mg, 90%) as a
yellow oil. 1H NMR (CDCl3) d: 0.38–0.56 (4H, m),
1.33 (9H, s), 1.53 (9H, s), 1.70–1.80 (1H, m), 2.25 (3H,
s), 3.68 (2H, s), 6.45 (1H, s), 7.32 (2H, d, J = 7.8 Hz),
5.2.31. tert-Butyl (2E)-3-{8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-4-oxo-2- [4-(pyrrolidin-1-ylmethyl)phen-
yl]-4H-pyrido[1,2-a]pyrimidin-3-yl}acrylate (3r). Follow-
ing the procedures as described for 3l, the title com-
pound (227 mg) was prepared from 5a (250 mg,
0.401 mmol) and 6r (66.9 lL, 0.802 mmol) as a yellow
1
solid. H NMR (CDCl3) d: 1.35 (9H, s), 1.49 (9H, s),
1.66 (4H, br), 2.60 (4H, br), 3.78 (2H, s), 6.52 (1H, s),
7.35 (1H, d, J = 15.6 Hz), 7.49 (2H, d, J = 8.1 Hz),
7.54 (2H, d, J = 8.1 Hz), 7.57 (1H, dd, J = 1.7, 7.3 Hz),
7.62 (1H, d, J = 15.6 Hz), 7.89 (1H, s), 9.04 (1H, d,

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
8515
J = 7.3 Hz). HRMS (FAB) Calcd for C34H40N5O4S
5.2.36. tert-Butyl (2E)-3-{8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-[4-(morpholin-4-ylmethyl)phenyl]-4-
oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}acrylate (3w). Fol-
lowing the procedures as described for 3l, the title com-
pound (176 mg) was prepared from 5a (300 mg,
0.481 mmol) and 6w (83.9 lL, 0.962 mmol) as a yellow
([M+H]+): 614.2801. Found: 614.2792.
5.2.32. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-{4-[(piperidin-1-yl)methyl]phenyl}-
4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (3s). Fol-
lowing the procedures as described for 3l, the title com-
pound (226 mg) was prepared from 5a (250 mg,
0.401 mmol) and 6s (79.4 lL, 0.802 mmol) as a yellow
solid. 1H NMR (CDCl3) d: 1.33 (9H, s), 1.38–1.47
(2H, m), 1.49–1.58 (4H, m), 1.52 (9H, s), 2.42 (4H,
br), 3.51 (2H, s), 6.45 (1H, s), 7.37 (1H, d,
J = 15.6 Hz), 7.38 (2H, d, J = 8.1 Hz), 7.47 (2H, d,
J = 8.1 Hz), 7.57 (1H, dd, J = 1.7, 7.3 Hz), 7.66 (1H,
d, J = 15.6 Hz), 8.10 (1H, d, J = 1.7 Hz), 9.02 (1H, d,
J = 7.1Hz). HRMS (FAB) Calcd for C35H42N5O4S
([M+H]+): 628.2958. Found: 628.2947.
1
solid. H NMR (CDCl3) d: 1.34 (9H, s), 1.50 (9H, s),
2.50 (4H, m), 3.56 (2H, s), 3.74 (4H, t, J = 4.5 Hz),
6.56 (1H, s), 7.41 (1H, d, J = 15.6 Hz), 7.47 (2H, d,
J = 8.1 Hz), 7.57 (2H, d, J = 8.1 Hz), 7.65 (1H, d,
J = 15.6 Hz), 7.68 (1H, dd, J = 1.7, 7.3 Hz), 8.24 (1H,
s), 9.16 (1H, d, J = 7.6 Hz). HRMS (FAB) Calcd for
C34H40N5O5S ([M+H]+): 630.2750. Found: 630.2778.
5.2.37. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(4-{[cyclopropyl(methyl)amino]methyl}phen-
yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]acrylic acid (4n).
Following the procedures as described for 4i, the title
compound (151 mg) was prepared from 3n (176 mg,
0.287 mmol) as an orange solid. Mp: 227–232 ꢁC (dec).
IR (ATR) cmꢀ1: 2962, 2871, 2501, 1691, 1643, 1565,
1494, 1446, 1419. 1H NMR (DMSO-d6) d: 0.70–0.85
(3H, m), 0.94–1.05 (1H, m) 1.31 (9H, s), 2.85 (3H, d,
J = 3.9 Hz), 2.82–2.95 (1H, m), 3.62–3.75 (1H, m), 4.54
(2H, s), 6.90 (1H, s), 7.27 (1H, d, J = 15.6 Hz), 7.44
(1H, d, J = 15.6 Hz), 7.67 (2H, d, J = 8.2 Hz), 7.78
(2H, d, J = 8.2 Hz), 7.94 (1H, dd, J = 7.3, 1.5 Hz), 8.45
(1H, br s), 9.20 (1H, d, J = 7.3 Hz), 10.21 (1H, br s).
Anal. Calcd for C30H31N5O4SÆHClÆ1.9H2O: C,
54.20; H, 5.58; N, 10.53. Found: C, 54.70; H, 5.84; N,
10.00.
5.2.33. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-{4-[ (4-hydroxypiperidin-1-yl)meth-
yl]phenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate
(3t). Following the procedures as described for 3l, the ti-
tle compound (184 mg) was prepared from 5a (200 mg,
0.321 mmol) and 6t (163 mg, 1.61 mmol) as a yellow sol-
1
id. H NMR (CDCl3) d: 9.16 (1H, d, J = 7.59 Hz), 8.24
(1H, d, J = 1.47 Hz), 7.70–7.63 (2H, m), 7.56 (2H, d,
J = 8.32 Hz), 7.45 (2H, d, J = 8.08 Hz), 7.40 (1H, d,
J = 15.91 Hz), 6.57 (1H, s), 3.76 (1H, s), 3.57 (2H, s),
2.80 (2H, s), 2.21 (2H, s), 1.91 (2H, s), 1.64 (2H, q,
J = 9.30 Hz), 1.49 (9H, s), 1.34 (9H, s). HRMS (FAB)
Calcd for C35H42N5O5S ([M+H]+): 644.2907. Found:
644.2932.
5.2.38. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(4-{[(2,2-difluoroethyl)(methyl)amino]meth-
yl}phenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]acrylic
acid (4o). Following the procedures as described for 4i,
the title compound (229 mg) was prepared from 3o
(249 mg, 0.390 mmol) as an orange solid. Mp: 253–
256 ꢁC (dec). IR (ATR) cmꢀ1: 3032, 2966, 2873, 1965,
1603, 1562, 1493, 1448, 1417, 1358, 1323, 1282, 1194,
5.2.34. Ethyl 1-[4-(3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-
1-yl]-8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-
oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)benzyl]piperidine-4-
carboxylate (3u). Following the procedures as de-
scribed for 3l, the title compound (47.6 mg) was pre-
pared from 5a (50.0 mg, 0.0802 mmol) and 6u
(57.5 mg, 0.366 mmol) as a yellow solid. 1H NMR
(CDCl3) d: 1.26 (3H, t, J = 7.10 Hz), 1.34 (9H, s),
1.49 (9H, s), 1.77–2.07 (4H, m), 2.30 (1H, m), 2.89–
2.91 (2H, m), 3.55 (2H, s), 4.14 (2H, q, J = 7.18 Hz),
6.57 (1H, s), 7.41 (1H, d, J = 15.66 Hz), 7.46 (2H, d,
J = 8.08 Hz), 7.57 (2H, d, J = 8.08 Hz), 7.64–7.68
(2H, m), 8.24 (1H, s), 9.17 (1H, d, J = 7.34 Hz).
HRMS (FAB) Calcd for C38H46N5O6S ([M+H]+):
700.3169. Found: 700.3159.
1
1136, 1090, 1053. H NMR (DMSO-d6) d: 1.31 (9H,
s), 2.80 (2H, s), 4.48 (2H, br), 6.65 (1H, br t,
J = 53.0 Hz), 6.89 (1H, s), 7.29 (1H, d, J = 15.6 Hz),
7.46 (1H, d, J = 15.6 Hz), 7.70 (2H, d, J = 8.3 Hz),
7.75–7.82 (2H, m), 7.94 (1H, dd, J = 1.7, 7.3 Hz), 8.42
(1H, s), 9.20 (1H, d, J = 7.6 Hz). Anal. Calcd for
C29H29F2N5O4SÆ1.6HClÆ1.4H2O: C, 52.36; H, 5.06; N,
10.53; Cl, 8.53; F, 5.71; S, 4.82. Found: C, 52.30; H,
4.80; N, 10.46; Cl, 8.37; F, 5.53; S, 4.89.
5.2.35. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-{4-[ (4-methylpiperazin-1-yl)meth-
yl]phenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate
(3v). Following the procedures as described for 3l, the ti-
tle compound (217 mg) was prepared from 5a (250 mg,
0.401 mmol) and 6v (88.3 lL, 0.802 mmol) as a yellow
5.2.39. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(4-{[(carboxymethyl)(methyl)amino]methyl}-
phenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]acrylic acid
(4p). Following the procedures as described for 4i, the
title compound (188 mg) was prepared from 3p
(220 mg, 0.319 mmol) as an orange solid. Mp: 204–
220 ꢁC (dec). IR (ATR) cmꢀ1: 2962, 2873, 1724, 1685,
1
solid. H NMR (CDCl3) d: 1.33 (9H, s), 1.52 (9H, s),
2.30 (3H, s), 2.48 (8H, br), 3.51 (2H, s), 6.46 (1H, s),
7.38 (2H, d, J = 8.2 Hz), 7.39 (1H, d, J = 15.6 Hz),
7.48 (2H, d, J = 8.2 Hz), 7.59 (1H, dd, J = 1.7, 7.3 Hz),
7.65 (1H, d, J = 15.6 Hz), 8.16 (1H, d, J = 1.7 Hz),
9.04 (1H, d, J = 7.3 Hz). HRMS (FAB) Calcd
for C35H43N6O4S ([M+H]+): 643.3067. Found:
643.3073.
1
1565, 1496, 1446. H NMR (DMSO-d6) d: 1.31 (9H,
s), 2.85 (3H, s), 3.60–3.75 (2H, m), 4.17 (2H, s), 6.90
(1H, s), 7.31 (1H, d, J = 15.6 Hz), 7.47 (1H, d,
J = 15.6 Hz), 7.72 (2H, d, J = 8.6 Hz), 7.75 (2H, d,
J = 8.6 Hz), 7.95 (1H, d, J = 7.2 Hz), 8.42 (1H, s), 9.20
(1H, d, J = 7.2 Hz). Anal. Calcd for C29H29N5O6SÆ

8516
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
2.2HClÆ1.9H2O: C, 50.47; H, 5.11; N, 10.15. Found: C,
51.19; H, 5.31; N, 9.37.
NMR (DMSO-d6) d: 1.31 (9H, s), 1.45–1.43 (2H, m),
1.75–1.73 (2H, m), 2.14–2.11 (2H, m), 2.73 (2H, br s),
3.49 (1H, br s), 3.58 (2H, s), 4.56 (1H, s), 6.85 (1H, s),
7.28 (1H, d, J = 15.62 Hz), 7.56–7.52 (5H, m), 7.92
(1H, dd, J = 7.45, 1.83 Hz), 8.42 (1H, s), 9.17 (1H, d,
J = 7.57 Hz). Anal. Calcd for C31H33N5O5SÆ1.5H-
ClÆ0.25H2O: C, 56.00; H, 5.61; N, 10.53. Found: C,
56.38; H, 5.24; N, 10.13.
5.2.40. (2E)-3-(2-[4-(Azetidin-1-ylmethyl)phenyl]-8-{[(4-
tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-4H-
pyrido[1,2-a]pyrimidin-3-yl)acrylic acid (4q). Following
the procedures as described for 4i, the title compound
(139 mg) was prepared from 3q (173 mg, 0.289 mmol)
as an orange solid. Mp: 271–274 ꢁC (dec). IR (ATR)
cmꢀ1: 2968, 2787, 2636, 2434, 1695, 1637, 1603, 1547,
1493, 1448, 1415, 1358, 1321, 1281, 1201, 1140, 1099,
5.2.44. 1-(4-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]car-
bonyl}-3-[(E)-2-carboxyvinyl]-4-oxo-4H-pyrido[1,2-a]pyr-
imidin-2-yl}benzyl)piperidine-4-carboxylic acid (4u). To a
solution of 3u (45.0 mg, 0.0643 mmol) in THF (10 mL)
was added a solution of lithium hydroxide monohydrate
(8.00 mg, 0.191 mmol) in H2O (5 mL) and stirred for
16 h. To the mixture was added lithium hydroxide
monohydrate (13.4 mg, 0.319 mmol) and stirred for
6 h. The mixture was acidified with 1 N HCl and con-
centrated. The residue was purified by PTLC (CHCl3/
MeOH/H2O = 7:3:1, organic phase) to afford 1-[4-(3-
[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-yl]-8-{[(4-tert-bu-
tyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-2-yl)benzyl]piperidine-4-carboxylic
acid (39.3 mg, 91%) as a yellow solid. Following the pro-
cedures as described for 4b, the title compound (127 mg)
was prepared from 1-[4-(3-[(1E)-3-tert-butoxy-3-oxo-
prop-1-en-1-yl]-8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]car-
bonyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)benzyl]pi-
peridine-4-carboxylic acid (125 mg, 0.186 mmol) as a
yellow solid. Mp: 121–127 ꢁC. IR (ATR) cmꢀ1: 2966,
1668, 1566, 1502, 1448, 1414, 1290, 1182, 1134, 1061.
1H NMR (CD3OD) d: 1.35 (9H, s), 1.86–1.99 (2H, m),
2.27–2.31 (2H, m), 2.65–2.69 (1H, m), 3.10–3.16 (2H,
m), 3.60–3.65 (2H, m), 4.45 (2H, s), 6.76 (1H, br s),
7.35 (1H, br s), 7.50–7.58 (1H, m), 7.72 (2H, d,
J = 8.2 Hz), 7.77 (2H, d, J = 8.2 Hz), 7.94 (1H, br s),
8.33 (1H, br s), 9.26 (1H, s, br s). Anal. Calcd for
C32H33N5O6SÆ1.5TFAÆ1.5H2O: C, 51.66; H, 4.64; N,
8.61. Found: C, 51.57; H, 4.59; N, 8.46.
1
1057. H NMR (DMSO-d6) d: 1.31 (9H, s), 2.31–2.45
(2H, m), 4.00–4.19 (4H, m), 4.48 (2H, d, J = 6.1 Hz),
7.29 (1H, d, J = 15.5 Hz), 7.45 (1H, d, J = 15.5 Hz),
7.67 (2H, d, J = 8.3 Hz), 7.70 (2H, d, J = 8.3 Hz), 7.94
(1H, d, J = 8.3 Hz), 8.42 (1H, s), 9.20 (1H, d,
J = 7.6 Hz), 10.7 (1H, br). Anal. Calcd for C29H29N5O4-
SÆ1.5HClÆ1.7H2O: C, 55.38; H, 5.43; N, 11.13; S, 5.10; Cl,
8.46. Found: C, 55.30; H, 5.24; N, 11.04; S, 5.17; Cl, 8.32.
5.2.41. (2E)-3-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-4-oxo-2-[4-(pyrrolidin-1-ylmethyl)phenyl]-4H-
pyrido[1,2-a]pyrimidin-3-yl}acrylic acid (4i). Following
the procedures as described for 4b, the title compound
(196 mg) was prepared from 3r (227 mg, 0.370 mmol)
as an orange solid. Mp: 219–226 ꢁC (dec). IR (ATR)
cmꢀ1: 2964, 2873, 2488, 1682, 1601, 1568, 1495, 1446,
1406, 1360, 1325, 1290, 1267, 1207, 1149, 1099, 1057.
1H NMR (DMSO-d6) d: 1.30 (9H, s), 1.84–2.11 (4H,
m), 3.06–3.17 (2H, m), 3.32–3.40 (2H, m), 4.46 (1H, d,
J = 5.9 Hz), 6.88 (1H, s), 7.27 (1H, d, J = 15.6 Hz),
7.45 (1H, d, J = 15.6 Hz), 7.67, (2H, d, J = 8.0 Hz),
7.79 (2H, d, J = 8.0 Hz), 7.92 (1H, dd, J = 1.7, 7.6 Hz),
8.41 (1H, s), 9.18 (1H, d, J = 7.6 Hz), 10.8 (1H, br).
Anal. Calcd for C30H31N5O4SÆ1.5HClÆ2H2O: C, 56.84;
H, 5.55; N, 11.05; S, 5.06; Cl, 8.39. Found: C, 56.91;
H, 5.60; N, 10.81; S, 5.03; Cl, 8.35.
5.2.42. (2E)-3-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-4-oxo-2-[4-(piperidin-1-ylmethyl)phenyl]-4H-
pyrido[1,2-a]pyrimidin-3-yl}acrylic acid (4s). Following
the procedures as described for 4i, the title compound
(207 mg) was prepared from 3s (226 mg, 0.361 mmol)
as an orange solid. Mp: 234–243 ꢁC (dec). IR (ATR)
cmꢀ1: 2966, 2871, 2650, 2557, 1699, 1635, 1604, 1547,
1493, 1446, 1415, 1354, 1323, 1282, 1194, 1149, 1092,
5.2.45. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-{4-[(4-methylpiperazin-1-yl)methyl]phenyl}-
4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylic acid (4v).
Following the procedures as described for 4i, the title
compound (204 mg) was prepared from 3v (217 mg,
0.337 mmol) as an orange solid. Mp: 209–214 ꢁC (dec).
IR (ATR) cmꢀ1: 2964, 2436, 1685, 1637, 1603, 1560,
1495, 1446, 1414, 1360, 1321, 1279, 1178, 1151, 1099,
1
1055. H NMR (DMSO-d6) d: 1.25–1.46 (2H, m), 1.30
(9H, s), 1.65–1.88 (6H, m), 2.85–2.98 (2H, m), 4.38
(2H, d, J = 4.9 Hz), 6.88 (1H, s), 7.26 (1H, dd, J = 1.2,
15.5 Hz), 7.42 (1H, dd, J = 1.2, 15.5 Hz), 7.67 (2H, d,
J = 7.7 Hz), 7.78 (2H, d, J = 7.7 Hz), 7.92 (1H, d,
J = 7.6 Hz), 8.39 (1H, s), 9.18 (1H, d, J = 7.3 Hz),
10.26 (1H, br). Anal. Calcd for C31H33N5O4SÆ1.5H-
ClÆ0.5H2O: C, 58.60; H, 5.63; N, 11.02; S, 5.05; Cl.
8.37. Found: C, 58.48; H, 5.63; N, 10.84; S, 4.98; Cl, 8.54.
1
1059, 1016. H NMR (DMSO-d6) d: 1.32 (9H, s), 2.51
(4H, m), 2.84 (3H, s), 3.65 (4H, m), 4.47 (2H, br), 6.90
(1H, s), 7.30 (1H, d, J = 15.6 Hz), 7.48 (1H, d,
J = 15.6 Hz), 7.69 (2H, d, J = 7.7 Hz), 7.85 (2H, d,
J = 7.7 Hz), 7.94 (1H, dd, J = 1.7, 7.6 Hz), 8.42 (1H,
s), 9.20 (1H, d, J = 7.3 Hz). Anal. Calcd for
C31H34N6O4SÆ2.3HClÆ2.1H2O: C, 52.56; H, 5.76; N,
11.86; S, 4.53; Cl, 11.51. Found: C, 52.53; H, 5.78; N,
11.76; S, 4.63; Cl, 11.58.
5.2.43. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-{4-[(4-hydroxypiperidin-1-yl)methyl]phenyl}-
4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylic acid (4t).
Following the procedures as described for 4i, the title
compound (88.0 mg) was prepared from 3t (184 mg,
0.286 mmol) as a yellow solid. Mp: 274 ꢁC. IR (ATR)
cmꢀ1: 1697, 1604, 1552, 1493, 1448, 1311, 1053. 1H
5.2.46. (2E)-3-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-[4-(morpholin-4-ylmethyl)phenyl]-4-oxo-4H-
pyrido[1,2-a]pyrimidin-3-yl}acrylic acid (4w). Following
the procedures as described for 4i, the title compound
(153 mg) was prepared from 3w (176 mg, 0.280 mmol)

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
8517
as an orange solid. Mp: 269–282 ꢁC. IR (ATR) cmꢀ1
:
amine (677 mg, 3.67 mmol) in MeOH (6 mL) was added
5% Pd-C (200 mg, containing 50% water), and the mix-
ture was stirred under atmospheric pressure of hydrogen
for 14.5 h. The catalyst was filtered off and washed with
MeOH to afford a solution of 6o in MeOH (25 mL, ca.
0.146 mol/L). This compound was utilized for the next
reaction without further purification.
3109, 2964, 2873, 2611, 2569, 2480, 1697, 1635, 1603,
1562, 1493, 1446, 1415, 1358, 1323, 1302, 1188, 1151,
1
1128, 1084, 1055. H NMR (DMSO-d6) d: 1.31 (9H,
s), 3.11–3.37 (4H, m), 3.71–3.82 (2H, m), 3.94–4.02
(2H, m), 4.48 (2H, d, J = 4.4 Hz), 6.90 (1H, s), 7.30
(1H, d, J = 15.6 Hz), 7.47 (1H, d, J = 15.6 Hz), 7.71
(2H, d, J = 8.2 Hz), 7.80 (2H, d, J = 8.2 Hz), 7.95 (1H,
dd, J = 1.7, 7.5 Hz), 8.41 (1H, s), 9.20 (1H, d,
J = 7.6 Hz), 10.82 (1H, br). Anal. Calcd for
C30H31N5O5SÆ1.5HClÆ1.4H2O: C, 55.13; H, 5.44; N,
10.72; S, 4.91; Cl, 8.14. Found: C, 55.13; H, 5.15; N,
10.68; S, 5.00; Cl, 7.97.
5.2.49.
tert-Butyl
(2E)-3-(2-{4-[(4-allylpiperazin-1-
yl)methyl]phenyl}-8-{[(4-tert-butyl-1,3-thiazol-2-yl)ami-
no]carbonyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acry-
late (9). Following the procedures as described for
3l, the title compound (34.0 mg) was prepared from
5a (50.0 mg, 0.0802 mmol) and 1-allylpiperazine
(46.7 lL, 0.321 mmol) as a yellow solid. 1H NMR
(CDCl3) d: 1.32 (9H, s), 1.52 (9H, s), 2.51 (8H,
br), 3.01 (2H, d, J = 6.6 Hz), 3.51 (2H, s), 5.11–
5.24 (2H, m), 5.87 (1H, ddt, 17.1, 10.2, 6.6 Hz),
6.44 (1H, s), 7.37 (2H, d, J = 7.7 Hz), 7.38 (1H,
d, J = 15.6 Hz), 7.47 (2H, d, J = 7.7 Hz), 7.58
(1H, d, J = 6.8 Hz), 7.65 (1H, d, J = 15.6 Hz),
8.15 (1H, s), 9.02 (1H, d, J = 7.3 Hz). HRMS
5.2.47. N-Cyclopropyl-N-methylamine hydrochloride
(6n). To
a solution of cyclopropylamine (2.65 g,
46.4 mmol) and Et3N (9.71 mL, 69.9 mmol) in THF
(100 mL) was added dropwise benzyl bromide
(6.62 mL, 55.7 mmol) and stirred for 17 h. The mix-
ture was diluted with satd NaHCO3 aq and extracted
with EtOAc. The organic layer was washed with
brine, dried, and concentrated. The residue was puri-
fied
by
silica
gel
column
chromatography
(FAB)
Calcd
for
669.3223. Found: 669.3244.
C37H45N6O4S
([M+H]+):
(CHCl3 ! CHCl3/MeOH = 50:1) to afford N-benzyl-
N-cyclopropylamine (3.27 g) as a colorless oil. To a
solution of N-benzyl-N-cyclopropylamine (3.27 g,
22.2 mmol) in MeOH (60 mL) was added dropwise
formalin (1.43 mL, 18.9 mmol) at 0 ꢁC. To the mixture
was added NaBH3CN (1.01 g, 15.2 mmol) and stirred
for 20 min at 0 ꢁC. After the mixture was stirred for
19 h, it was diluted with satd NaHCO3 aq, and
MeOH was removed by concentration. The residue
was extracted with CHCl3 and the combined organic
layers were washed with brine, dried, and concentrat-
ed. The residue was purified by silica gel column chro-
matography (CHCl3 ! CHCl3/MeOH = 98:1) to afford
N-benzyl-N-cyclopropyl-N-methylamine (1.80 g) as a
colorless oil. To a solution of N-benzyl-N-cyclopro-
pyl-N-methylamine (1.80 g, 11.2 mmol) in MeOH
(50 mL) was added 5% Pd-C (0.18 g, containing 50%
water), and the mixture was stirred under atmospheric
pressure of hydrogen for 16 h. The catalyst was fil-
tered off and washed with MeOH. The combined fil-
trate and washings were concentrated. To the residue
was added 4 N HCl–dioxane (20 mL) and concentrat-
5.2.50. tert-Butyl (2E)-3-{8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-4-oxo-2-[4-(piperazin-1-ylmethyl)-
phenyl]-4H-pyrido[1,2-a]pyrimidin-3-yl}acrylate (10). To
a solution of 9 (185 mg, 0.276 mmol) in CH2Cl2
(4 mL) were added N,N0-dimethylbarbituric acid
(129 mg, 0.829 mmol) and Pd(PPh3)4 (16.0 mg,
13.8 lmol), and stirred for 4 h under Ar. The mixture
was concentrated, and the residue was diluted with
EtOAc and washed with satd Na2CO3 aq (3·). The com-
bined organic layers were dried and concentrated to af-
ford 10 (210 mg) as inseparable mixture, which was
utilized for the next reaction without further
purification.
5.2.51. tert-Butyl (2i)-3-[8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-(4-{[4-(2-hydroxyethyl)piperazin-1-
yl]methyl}phenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-
yl]acrylate (3x). To a solution of crude 10 (210 mg) in
THF (5 mL) were added Et3N (154 lL, 1.11 mmol)
and 2-bromoethanol (39.2 lL, 0.553 mmol) and stirred
for 14 h. After the mixture was further stirred for 9.5 h
at 50 ꢁC, Et3N (154 lL, 1.11 mmol) and 2-bromoetha-
nol (39.2 lL, 0.553 mmol) were added and stirred for
13 h at 50 ꢁC. The mixture was concentrated, and the
residue was diluted with satd NaHCO3 aq and extracted
with EtOAc (3·). The combined organic layers were
dried and concentrated. The residue was purified by sil-
1
ed to afford 6n (1.18 g) as a pale yellow oil. H NMR
(CD3OD) d: 0.80–0.94 (4H, m), 2.70–2.80 (1H, br s),
2.77 (3H, s), 3.55–3.77 (1H, m).
5.2.48. N-(2,2-Difluoroethyl)-N-methylamine (6o). To a
solution of N-methylbenzylamine (1.00 mL, 7.76 mmol)
in THF (5 mL) was added 2,2-difluoroethyl trifluorom-
ethanesulfonate13 (830 mg, 3.88 mmol) and stirred for
21 h. To the mixture were added Et3N (2.16 mL,
15.5 mmol) and a solution of 2,2-difluoroethyl trifluo-
romethanesulfonate (830 mg, 3.88 mmol) in THF
(2 mL) and stirred for 16.5 h. The mixture was concen-
trated and the residue was purified by silica gel column
chromatography (CH2Cl2 ! CH2Cl2/MeOH = 95:5 !
90:10) to afford N-benzyl-N-(2,2-difluoroethyl)-N-
methylamine (677 mg) as a pale yellow oil. To a
solution of N-benzyl-N-(2,2-difluoroethyl)-N-methyl-
ica
gel
column
chromatography
(CHCl3/
MeOH = 99:1 ! 95:5) to afford 3x (129 mg) as a yellow
1
film. H NMR (CDCl3) d: 1.33 (9H, s), 1.50 (9H, s),
2.42–2.62 (8H, m), 2.57 (2H, t, J = 5.4 Hz), 3.53 (2H,
s), 3.65 (2H, t, J = 5.4 Hz), 6.51 (1H, s), 7.395 (1H, d,
J = 15.6 Hz), 7.404 (2H, d, J = 8.1 Hz), 7.52 (2H, d,
J = 8.1 Hz), 7.62–7.66 (1H, m), 7.65 (1H, d,
J = 15.6 Hz), 8.21–8.24 (1H, m), 9.10 (1H, d,
J = 7.6 Hz). HRMS (FAB) Calcd for C36H45N6O5S
([M+H]+): 673.3172. Found: 673.3185.

8518
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 8506–8518
5.2.52. tert-Butyl (2E)-3-(2-(4-{[4-(2-tert-butoxy-2-oxo-
ethyl)piperazin-1-yl]methyl}phenyl)- 8-{[(4-tert-butyl-1,3-
thiazol-2-yl)amino]carbonyl}-4-oxo-4H-pyrido[1,2-a]pyr-
imidin-3-yl)acrylate (3y). To a solution of crude 10
(211 mg) in THF (5 mL) were added Et3N (150 lL,
1.08 mmol) and bromoacetic acid tert-butyl ester
(79.5 lL, 0.538 mmol) and stirred for 13 h. The mixture
was concentrated, and the residue was diluted with satd
NaHCO3 aq and extracted with EtOAc. The organic
layer was dried and concentrated. The residue was puri-
fied by silica gel column chromatography (CHCl3/
MeOH = 10:1) to afford 3y (183 mg) as a yellow oil.
1H NMR (CDCl3) d: 1.33 (9H, s), 1.46 (9H, s), 1.50
(9H, s), 2.53 (4H, br), 2.60 (4H, br), 3.14 (2H, s), 3.52
(2H, s), 6.50 (1H, s), 7.391 (1H, d, J = 15.7 Hz), 7.394
(2H, d, J = 8.2 Hz), 7.50 (2H, d, J = 8.2 Hz), 7.64 (1H,
dd, J = 1.7, 7.3 Hz), 7.65 (1H, d, J = 15.7 Hz), 8.22
(1H, d, J = 1.7 Hz), 9.09 (1H, d, J = 7.3 Hz). HRMS
(FAB) Calcd for C40H51N6O6S ([M+H]+): 743.3591.
Found: 743.3628.
lated at 1 · 106 CFU/mL and incubated at 37 ꢁC for
18 h. MICs were determined by visual observation of
growth.
5.4. In vivo efficacy
Pulmonary infection of SD rats by P. aeruginosa
PAM1020 was instilled by intratracheal inoculation of
bacteria enmeshed in agar beads. Two hours after bacte-
rial challenge, rats were treated with AZT (1000 mg/kg/
2 h) or AZT+ABS-EPI (ABS-EPI were dosed 10 mg/kg/
2 h) by intravenous drip infusion.
Acknowledgments
Members of the Research Technology Center, Daiichi
Pharmaceutical Co., Ltd. are gratefully acknowledged
for analytical data.
References and notes
5.2.53. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(4-{[4-(2-hydroxyethyl)piperazin-1-yl]meth-
yl}phenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]acrylic
acid (4x). Following the procedures as described for 4i,
the title compound (105 mg) was prepared from 3x
(129 mg, 0.191 mmol) as an orange solid. Mp: 209–
216 ꢁC. IR (ATR) cmꢀ1: 2966, 1691, 1637, 1603, 1568,
1495, 1446, 1414, 1360, 1327, 1277, 1207, 1176, 1151,
1097, 1059. 1H NMR (DMSO-d6) d: 1.32 (9H, s),
3.02–4.06 (14H, m), 6.90 (1H, s), 7.30 (1H, d,
J = 15.7 Hz), 1.49 (1H, d, J = 15.7 Hz), 7.69 (2H, d,
J = 7.6 Hz), 7.80 (2H, br), 7.94 (1H, d, J = 7.1 Hz),
8.41 (1H, s), 9.20 (1H, d, J = 7.6 Hz). Anal. Calcd for
C32H36N6O5SÆ2.5HClÆ2.5H2O: C, 51.05; H, 5.82; N,
11.16; S, 4.26; Cl; 11.77. Found: C, 51.04; H, 5.66; N,
10.99; S, 4.28; Cl; 11.63.
1. Zhanel, G. G.; Hoban, D. J.; Schurek, K.; Karlowsky, J.
A. Int. J. Antimicrob. Agents 2004, 24, 529.
2. Poole, K. J. Antimicrob. Chemother. 2005, 56, 20.
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4. Poole, K.; Krebes, K.; McNally, C.; Neshat, S. J.
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5. Nakayama, K.; Ishida, Y.; Ohtsuka, M.; Kawato, H.;
Yoshida, K.; Yokomizo, Y.; Hosono, S.; Ohta, T.;
Hoshino, K.; Ishida, H.; Yoshida, K.; Renau, T. E.;
´
Leger, R.; Zhang, J. Z.; Lee, V. J.; Watkins, W. J. Bioorg.
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Yoshida, K.; Yokomizo, Y.; Ohta, T.; Hoshino, K.; Otani,
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5.2.54. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(4-{[4-(carboxymethyl)piperazin-1-yl]meth-
yl}phenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]acrylic
acid (4y). Following the procedures as described for 4i,
the title compound (164 mg) was prepared from 3y
(191 mg, 0.257 mmol) as an orange solid. Mp: 212–
222 ꢁC (dec). IR (ATR) cmꢀ1: 3014, 2978, 2936, 2536,
2441, 1718, 1685, 1651, 1583, 1558, 1498, 1444, 1429,
1373, 1344, 1277, 1230, 1196, 1169, 1099, 1061, 1030.
1H NMR (DMSO-d6) d: 1.30 (9H, s), 3.32–3.80 (8H,
m), 4.07 (2H, s), 4.47 (2H, s), 6.88 (1H, s), 7.28 (1H,
d, J = 15.6 Hz), 7.46 (1H, d, J = 15.6 Hz), 7.69 (2H, d,
J = 8.2 Hz), 7.82 (2H, d, J = 8.2 Hz), 7.93 (1H, dd,
J = 2.0, 7.3 Hz), 8.38–8.40 (1H, m), 9.18 (1H, d,
J = 7.3 Hz). Anal. Calcd for C32H34N6O6SÆ3.5HClÆ2.5-
H2O: C, 47.84; H, 5.33; N, 10.46; S, 3.99; Cl, 15.45.
Found: C, 47.94; H, 5.17; N, 10.26; S, 3.94; Cl, 15.54.
7. Nakayama, K.; Kawato, H.; Watanabe, J.; Ohtsuka, M.;
Yoshida, K.; Yokomizo, Y.; Sakamoto, A.; Kuru, N.;
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Bioorg. Med. Chem. Lett. 2004, 14, 475.
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Sakamoto, A.; Kawato, H.; Yoshida, K.; Ohta, T.;
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Bioorg. Med. Chem. Lett. 2004, 14, 2493.
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5.3. In vitro potentiation activity
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MIC assays against P. aeruginosa utilized Mueller–Hin-
ton broth, following the broth microdilution methodol-
ogy outlined by the National Committee for Clinical
Laboratory Standards (NCCLS). Bacteria were inocu-