MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 5: Carbon-substituted analogues at the C-2 position

Article abstract of DOI:10.1016/j.bmc.2005.10.043

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with carbon-linked substituents, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas β-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Palladium-catalyzed cross-coupling methods were applied for the incorporation of aliphatic and aromatic substituents.

Full text of DOI:10.1016/j.bmc.2005.10.043

Bioorganic & Medicinal Chemistry 14 (2006) 1993–2004
MexAB-OprM specific efflux pump inhibitors
in Pseudomonas aeruginosa. Part 5: Carbon-substituted
analogues at the C-2 position
Ken-ichi Yoshida,a,* Kiyoshi Nakayama,a Noriko Kuru,a Shozo Kobayashi,a
Masami Ohtsuka,a Makoto Takemura,a Kazuki Hoshino,b Hiroko Kanda,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 4 October 2005; revised 25 October 2005; accepted 25 October 2005
Available online 15 November 2005
Abstract—A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with carbon-linked substituents,
were synthesized 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. Palladium-catalyzed cross-coupling methods were
applied for the incorporation of aliphatic and aromatic substituents.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
use of a pharmacophore model.6–9 This strategy fur-
nished pyridopyrimidine-based inhibitors possessing
nitrogen-linked C-2 substituents that play a critical role
for potency (Fig. 1). The pharmacophore model also in-
spired us to incorporate some carbon-linked C-2 substit-
uents to compare the potency and the opportunity for
the improvement of physicochemical properties (Fig. 2).
A particularly problematic pathogen in the clinical set-
ting is Pseudomonas aeruginosa, an opportunistic Gram
negative bacterium characterized by intrinsic resistance
to a wide variety of antimicrobial agents.1–3 This prop-
erty has been attributed both to the impermeability of
the outer membrane as well as to the activity of several
efflux systems of the resistance-nodulation-division
(RND) superfamily, five of which have been identified
to date as: MexAB-OprM, MexCD-OprJ, MexEF-
OprN, MexXY-OprM, and MexJK-OprM.1–5 The resis-
tance might be overcome by the development of efflux
pump inhibitors (EPIs) that could thereby enhance or
restore the potency of anti-pseudomonas agents.
Herein, we report the design, synthesis, and evaluation
of the resulting derivatives.
2. Chemistry
We envisioned that an sp2 carbon with p-electrons would
mimic the nitrogen-linked leads, and thus investigated
synthetic methodology to obtain vinyl or aryl substituents
at C-2. The starting materials for the cross-coupling reac-
tions were prepared as depicted in Scheme 1.
We recently reported the discovery of the first example
of a MexAB-OprM specific efflux pump inhibitor and
the early strategy for lead optimization through the
We explored the details of several palladium-mediated
cross-coupling reactions (Stille,10–12 Suzuki–Miya-
ura,13,14 etc.) as shown in Scheme 2. The conditions
depicted therein were found to be optimal for each type
of reaction.
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 ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.043

1994
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
Figure 1. Structures of the first generation of MexAB-OprM specific efflux pump inhibitors and pharmacophore model generated by CATALYSTTM
compounds 1 (red) and 2 (green). Light blue spheres are hydrophobic sites, and the deep blue sphere represents an acidic ionizable site.
;
Figure 2. Novel MexAB-OprM specific efflux pump inhibitor and the design of 2-carbon-linked analogues.
Scheme 1. Preparation of ArCl for the cross-coupling reactions.
Scheme 2. Optimal reaction conditions for various reagents.

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
1995
Scheme 3. Introduction of 2-carba substituent onto scaffold.
Thus, utilizing the Stille protocol, we initially generated
the vinyl variant (21) (Scheme 3). In addition, ethyl (29)
and 2-chloroethyl (20) variants were obtained via reduc-
tion and an unexpected chlorination, respectively; the fi-
nal step for the deprotection of the tert-butyl ester using
HCl afforded the latter via conjugate addition of chlo-
ride to the activated olefin.
shown in Scheme 4. In addition to the optimized condi-
tions for each reagent described above, the choice of
substrate (15 and 22) was based on coupling efficiency.
A Suzuki–Miyaura reaction, catalyzed by Pd(Ph3P)4
with Na2CO3 as base in DME–H2O, smoothly intro-
duced a phenyl group into the C-2 position of the pyri-
dopyrimidine nucleus (for 32). Cyclohexenyl variants
(for 37) were incorporated by Stille coupling with appro-
priate organostannanes, which were prepared according
to the literature.15,16 The substrate possessing a Boc-
protected nitrogen (for 44) required the more powerful
coupling conditions reported by Littke and Fu17 (i.e.,
In the case of nitrogen-linked analogues, variants
containing a six-membered ring (piperidines) provided
optimal potency.8 Thus phenyl, cyclohexenyl, and
1,2,3,6-tetrahydropyridin-4-yl C-2 substituents were ex-
plored next. The syntheses of these derivatives are
Scheme 4. Incorporation of six-membered ring at C-2 position.

1996
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
Scheme 5. Synthesis of H-analogue.
Scheme 6. Functionalization on heterocycle.
Scheme 7. Water-soluble quaternary ammonium salt analogue.
Pd2(dba)3ÆCHCl3, CsF, and t-Bu3P). Coupling reactions
with B-alkyl boranes18 (sp3–sp2 bond formation) were
accomplished by a Suzuki–Miyaura reaction using
Pd(dppf)Cl2 as catalyst (for 51). In addition, the unsub-
stituted (2-H) analogue 55, which was synthesized from
12 (Scheme 5), served as useful comparator for analysis
of the SAR of the novel 2-carba analogues.
tion, lg/mL) are presented in Table 1. Antimicrobial as-
says were conducted in the presence and absence of
0.125% human serum albumin (HSA) in order to evalu-
ate the effects of protein binding.
Introduction of the ethyl substituent increased the
potency 4-fold by comparison with the corresponding
2-vinyl analogue or unsubstituted analogue (29 vs 21
and 55). The influence of the addition of HSA was some-
what greater than in the case of the 2-N-substituted
compounds (e.g., the activity of the corresponding
piperidine derivatives was reduced no more than 2-fold
by the addition of HSA9). Introduction of the methy-
lene-linked saturated piperidine (51) resulted in de-
creased activity. On the other hand, attachment of
cyclic substituents directly at C-2 position, whether aryl
or alkenyl, maintained activity (32, 37, and 44). The
successful incorporation of a secondary amine (giving
a zwitterionic analogue) with retention of good activity
is particularly notable. Further functionalization of
the nitrogen atom of the piperidine moiety successfully
increased the solubility (62: 160 lg/mL and 65:
1200 lg/mL at pH 6.8), although the activity was slight-
ly compromised (62 and 65 vs 58).
In order to increase solubility, further functionalization
of the tetrahydropyridine moiety by incorporation of an
amino acid was also conducted. In addition, the N-Ac
analogue 58 was synthesized as a comparator for SAR
analysis (Scheme 6). Highly water-soluble derivative
was synthesized by condensation of the tetrahydropyri-
dine intermediate with sarcosine, followed by quatern-
ization with bromoacetic acid ester and deprotection
to afford ammonium analogue such as 65 (Scheme 7).
3. Results and discussion
The series of 2-carbon-linked pyridopyrimidines pre-
pared in this study was tested for in vitro potentiation
of the activity of LVFX and AZT against PAM
1723,19 a laboratory strain of P. aeruginosa in which
the MexAB-OprM pump is over-expressed and the
MexCD-OprJ and MexEF-OprN pumps are disrupted.
The in vitro potencies, expressed as the minimum con-
centration of inhibitor required to decrease (potentiate)
the MIC of the antibacterial agent 8-fold (MPC8, lg/
mL), and the aqueous solubilities (in pH 6.8 buffer solu-
4. Conclusions
In summary, we synthesized a series of 2-substituted
pyridopyrimidine derivatives through the use of palladi-
um-mediated cross-coupling reactions and explored

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
1997
Table 1. Chemical structures, potentiation activity, and aqueous solubility of 2-carba analoguesa
Compound
R2
MPC8 (LVFX) (lg/mL)
Without serum With 0.125% HSA
MPC8 (AZT) (lg/mL)
Sol (pH 6.8) (lg/mL)
55
20
21
29
8
4
8
2
32
16
32
8
2
2
0.5
2
1.8
2.6
b
—
0.5
51
32
37
44
58
62
65
16
4
>32
16
4
4
0.5
1
37
2
b
—
1
4
16
4
2
31
2
2
410
160
16
16
32
32
16
8
1200
a All compounds lacked intrinsic antibacterial activity.
b Not tested.
their potential as potentiators of the anti-pseudomonal
activity of LVFX and AZT. Like the 2-nitrogen-linked
analogues,8,9 2-carbon-linked variants, especially those
with cyclic substituents, provided good potency. The
high lipophilicity of many of these analogues limited
their aqueous solubility and limited their effectiveness
in the presence of HSA. Further work was stimulated
by the observation that activity could be retained in
more hydrophilic examples such as 44, 62, and 65, and
the results of these efforts will be reported in due course.
ylmethyl)-1-(tert-butoxycarbonyl)piperidine (45)18 were
prepared according to the literature procedures. Melting
points were taken with a Yanako MP-500D melting point
1
apparatus and are uncorrected. H NMR spectra were
determined on a JEOL JNM-EX400 spectrometer.
Chemical shifts are reported in parts per million relative
to tetramethylsilane as internal standard. Significant H
1
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 HORI-
BA FT-720 spectrometer. High-resolution mass spectra
were obtained on a JEOL JMS-700 mass spectrometer
under electron impact ionization conditions (EI) or fast
atom bombardment ionization conditions (FAB).
Elementary analyses were conducted at Research Tech-
nology 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
5. Experimental
5.1. General
Unless otherwise noted, materials were obtained from
commercial suppliers and used without further purifica-
tion. TCPM (9),20 1-tri-n-butylstannylcyclohexene (33),15
1-(tert-butoxycarbonyl)-4-tri-n-butylstannyl-1,2,3,6-tetra-
hydropyridine (38),21 and 4-(9-borabicyclo[3.3.1]non-9-

1998
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
visualization was effected with a 5% solution of phospho-
molybdic acid in ethanol, UV lamp, or Wako ninhydrin
spray. Unless otherwise specified, reactions were carried
out at ambient temperature. Combined organic extracts
were dried over anhydrous Na2SO4. Reagents and sol-
vents were removed from reaction mixture or combined
organic extracts by concentration in vacuo using a rotary
evaporator with bath at 35–45 ꢁC.
solid. 1H NMR (CD3OD) d: 1.56 (9H, s), 7.34 (1H,
d, J = 15.7 Hz), 7.84 (1H, dd, J = 1.7, 7.3 Hz), 7.89 (1H,
d, J = 15.7 Hz), 8.23–8.28 (1H, m), 9.16 (1H, d,
J = 7.3 Hz). HRMS (FAB) calcd for C16H1635ClN2O5S
([M+H]+): 351.0748. Found: 351.0725.
5.2.5. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-yl)ami-
no]carbonyl}-2-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-
acrylate (15). To a solution of 14 (86.4 mg, 0.553 mmol) and
13 (194 mg, 0.553 mmol) in CH2Cl2 (5 mL) were added
diisopropylethylamine (289 lL, 1.66 mmol) and N,N-bis-
(2-oxo-3-oxazolidinyl)phosphinic acid chloride (211 mg,
0.829 mmol), and stirred for 12.5 h. The mixture was con-
centrated, and the residue was diluted with CHCl3. The
organic layer was washed successively with 10% citric acid
aq, satd NaHCO3 aq, and brine. The organic layer was
dried and concentrated. The residue was purified by silica
gel column chromatography (CHCl3) to afford 15
(205 mg, 76%) as an orange solid. 1H NMR (CDCl3)
d: 1.35 (9H, s), 1.57 (9H, s), 6.51 (1H, s), 7.35 (1H,
d, J = 15.7 Hz), 7.79–7.87 (1H, m), 7.83 (1H, d,
J = 15.7 Hz), 8.22 (1H, d, J = 1.5 Hz), 9.02 (1H, d,
J = 7.3 Hz), 11.0 (1H, br). HRMS (FAB) calcd for
C23H2635ClN4O4S ([M+H]+): 489.1363. Found: 489.1323.
5.2. Synthesis
5.2.1. Methyl 2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-
8-carboxylate (10). To a suspension of 8 (2.77 g,
18.2 mmol) in toluene (50 mL) was added TCPM (9)
(9.27 g, 20.2 mmol), and the mixture was refluxed for
1 h. The mixture was cooled and concentrated. The residue
was diluted with Et2O, and precipitated solid was collected
by filtration, and washed with Et2O to afford 9 (2.05 g,
1
51%) as a yellow solid. H NMR (CD3OD) d: 4.04 (3H,
s), 5.63 (1H, s), 7.64 (1H, d, J = 7.3 Hz), 8.05 (1H, s),
9.08 (1H, d, J = 7.3 Hz). HRMS (FAB) calcd for
C10H9N2O4 ([M+H]+): 221.0562. Found: 221.0557.
5.2.2. Methyl 2-chloro-3-formyl-4-oxo-4H-pyrido[1,2-a]py-
rimidine-8-carboxylate (11). To DMF (10 mL) was added
phosphoryl chloride (1.27 mL, 13.6 mmol) at 0 ꢁC. After
the mixture was stirred at 0 ꢁC for 40 min, a solution of
10 (1.00 g, 4.54 mmol) in DMF (10 mL) was added and
stirred at 80 ꢁC for 1 h. The mixture was cooled and con-
centrated in vacuo, and the residue was diluted with water
and extracted with CHCl3 (3·). The combined organic
layers were washed with brine, dried, and concentrated.
The residue was purified by silica gel column chromatog-
raphy (CH2Cl2/MeOH = 9:1) to afford 11 (995 mg, 82%)
5.2.6. Methyl 3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-yl]-
4-oxo-2-vinyl-4H-pyrido[1,2-a]pyrimidine-8-carboxylate
(17). To a solution of 12 (100 mg, 0.274 mmol) in DMF
(2 mL) were added catalytic amount of 2,6-di-tert-butyl-
p-cresol, tri-n-butylvinyltin (88.1 lL, 0.303 mmol) and
Pd(Ph3P)4 (31.7 mg, 27.4 lmol), and stirred at 50 ꢁC
for 1 h and further at 80 ꢁC for 3 h under Ar. The mix-
ture was concentrated and the residue was purified by
silica gel column chromatography (EtOAc/hexane = 1:5)
to afford 17 (83.8 mg) as an inseparable mixture. This
compound was utilized for the next reaction without
1
as a pale yellow solid. H NMR (CDCl3) d: 4.06 (3H,
s), 7.86 (1H, dd, J = 1.7, 7.3 Hz), 8.34 (1H, dd, J = 0.7,
1.7 Hz), 9.25 (1H, dd, J = 0.7, 7.3 Hz), 10.45 (1H, s).
HRMS (EI) Calcd for C11H735ClN2O4 (M+): 266.0094.
Found: 266.0084.
1
further purification. H NMR (CDCl3) d: 1.54 (9H, s),
4.02 (3H, s), 5.83 (1H, dd, J = 2.0, 10.8 Hz), 6.67 (1H,
dd, J = 2.0, 16.6 Hz), 7.29 (1H, dd, J = 10.8, 16.6 Hz),
7.29 (1H, d, J = 15.4 Hz), 7.55 (1H, dd, J = 1.7,
7.3 Hz), 7.88 (1H, d, J = 15.4 Hz), 8.24 (1H, dd,
J = 0.7, 1.7 Hz), 9.03 (1H, dd, J = 0.7, 7.3 Hz). HRMS
(EI) Calcd for C19H20N2O5 (M+): 356.1372. Found:
356.1368.
5.2.3. Methyl 3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-yl]-
2-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylate
(12). To a solution of 11 (995 mg, 3.73 mmol) in THF
(40 mL)–DMF (10 mL) was added (tert-butoxycarbon-
ylmethylene)triphenylphosphorane (2.11 g, 5.60 mmol).
After stirred for 47 h, the mixture was concentrated.
The residue was purified by silica gel column chroma-
tography (EtOAc/hexane = 1:2) to afford 12 (1.26 g,
5.2.7. 3-[(1E)-3-tert-Butoxy-3-oxoprop-1-en-1-yl]-4-oxo-2-
vinyl-4H-pyrido[1,2-a]pyrimidine-8-carboxylic acid (18).
Following the procedures as described for 13, the title
compound (220 mg) was prepared in 55% yield from 17
(418 mg, 1.17 mmol) as a yellow solid. 1H NMR (CDCl3)
d: 1.54 (9H, s), 5.85 (1H, dd, J = 1.7, 10.5 Hz), 6.69 (1H,
dd, J = 1.7, 16.6 Hz), 7.19–7.35 (2H, m), 7.54 (1H,
dd, J = 1.7, 7.6 Hz), 7.9 (1H, d, J = 15.6 Hz), 8.31
(1H, m), 9.07 (1H, d, J = 7.6 Hz). HRMS (EI) Calcd
for C18H18N2O5 (M+): 342.1216. Found: 342.1194.
1
93%) as a yellow solid. H NMR (CDCl3) d: 1.55 (9H,
s), 4.04 (3H, s), 7.40 (1H, d, J = 15.7 Hz), 7.74 (1H,
dd, J = 1.8, 7.4 Hz), 7.92 (1H, d, J = 15.7 Hz), 8.27
(1H, dd, J = 0.7, 1.8 Hz), 9.13 (1H, dd, J = 0.7,
7.4 Hz). HRMS (EI) Calcd for C17H1735ClN2O5 (M+):
364.0826. Found: 364.0830.
5.2.4. 3-[(1E)-3-tert-Butoxy-3-oxoprop-1-en-1-yl]-2-chloro-
4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylic acid (13).
To a solution of 12 (216 mg, 0.592 mmol) in THF
(2 mL) was added 1 N NaOH aq (1.19 mL, 1.19 mmol)
and stirred for 30 min. The mixture was acidified to pH
4 with 1 N HCl aq, diluted with water, and extracted with
CHCl3 (5·). The combined organic layers were dried and
concentrated to afford 13 (194 mg, 93%) as an orange
5.2.8. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-yl)ami-
no]carbonyl}-4-oxo-2-vinyl-4H-pyrido[1,2-a]pyrimidin-3-yl)-
acrylate (19). Following the procedures as described for 15,
the title compound (274 mg) was prepared in 89% yield
from 14 (100 mg, 0.640 mmol) and 18 (220 mg,
0.643 mmol) as a yellow foam. 1H NMR (CDCl3) d:
1.33 (9H, s), 1.55 (9H, s), 5.77–5.83 (1H, m), 6.59 (1H, s),

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
1999
1
6.59–6.68 (1H, m), 7.15–7.29 (2H, m), 7.60 (1H, dd,
J = 1.8, 7.3 Hz), 7.82 (1H, dd, J = 3.9, 15.7 Hz), 8.14
(1H, m), 9.03 (1H, dd, J = 2.9, 7.3 Hz), 10.33 (1H, br).
HRMS (EI) Calcd for C25H28N4O4S (M+): 480.1831.
Found: 480.1828.
1.61 mmol) as a yellow solid. H NMR (CDCl3) d: 4.01
(3H, s), 4.04–4.09 (2H, m), 4.24–4.30 (2H, m), 5.72 (1H,
dd, J = 10.5, 2.2 Hz), 6.40 (1H, s), 6.67 (1H, dd,
J = 16.8, 2.2 Hz), 7.29 (1H, dd, J = 16.8, 10.5 Hz), 7.53
(1H, dd, J = 7.4, 1.7 Hz), 8.24 (1H, dd, J = 1.7, 0.7 Hz),
8.98 (1H, dd, J = 7.4, 0.7 Hz). HRMS (EI) Calcd for
C15H14N2O5 (M+): 302.0903. Found: 302.0882.
5.2.9.
(2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(2-chloroethyl)-4-oxo-4H-pyrido[1,2-a]pyrimi-
din-3-yl]acrylic acid (20). To 4 N HCl in 1,4-dioxane
(4 mL) was added 19 (28.0 mg, 58.3 lmol) and stirred
for 15 h. The mixture was concentrated, and the residue
was diluted with Et2O. Precipitated solid was collected
by filtration and washed with Et2O to afford 20
(16.7 mg, 62%) as a yellow solid. Mp: 241–250 ꢁC
(dec). IR (ATR) cmꢀ1: 3105, 2962, 2870, 1674, 1603,
1560, 1504, 1456, 1414, 1367, 1309, 1292, 1240, 1203,
5.2.13. Methyl 3-(dimethoxymethyl)-2-ethyl-4-oxo-4H-
pyrido[1,2-a]pyrimidine-8-carboxylate (24). To a solution
of 23 (100 mg, 0.331 mmol) in MeOH (6 mL) was added
10% Pd–C (100 mg, containing 50% water), and the mix-
ture was stirred under atmospheric pressure of hydrogen
for 3 h. The catalyst was filtered off and washed with
MeOH. The combined filtrate and washings were con-
centrated to afford 24 (98.9 mg, 98%) as a yellow oil,
which was utilized for the next reaction without further
purification. 1H NMR (CDCl3) d: 1.30 (3H, t,
J = 7.5 Hz), 3.05 (2H, q, J = 7.5 Hz), 3.49 (6H, s), 4.01
(3H, s), 5.88 (1H, s), 7.55 (1H, dd, J = 7.4, 2.0 Hz),
8.24 (1H, dd, J = 2.0, 0.7 Hz), 9.01 (1H, dd, J = 7.4,
0.7 Hz). HRMS (FAB) calcd for C15H19N2O5
([M+H]+): 307.1294. Found: 307.1277.
1
1174, 1099, 1059. H NMR (DMSO-d6) d: 1.31 (9H, s),
3.45 (2H, t, J = 6.6 Hz), 4.13 (2H, t, J = 6.6 Hz), 6.88
(1H, s), 7.28 (1H, d, J = 15.5 Hz), 7.75 (1H, d,
J = 15.5 Hz), 7.88 (1H, d, J = 7.3 Hz), 8.44 (1H, s),
9.13 (1H, d, J = 7.3 Hz). Anal. Calcd for C21H12N4O4Cl-
SÆ0.5H2O: C, 53.67; H, 4.72; N, 11.92; S, 6.82. Found: C,
53.40; H, 4.47; N, 11.65; S, 6.72.
5.2.10. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]car-
bonyl}-4-oxo-2-vinyl-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylic
acid (21). To TFA (2 mL) was added 19 (100 mg,
0.208 mmol) and stirred for 30 min. The mixture was con-
centrated, and the residue was diluted with Et2O. The pre-
cipitated solid was collected by filtration and washed with
Et2O to afford 21 (73.0 mg, 83%) as a yellow solid. Mp:
224–235 ꢁC (dec). IR (ATR) cmꢀ1: 3111, 2962, 2908,
2870, 1678, 1599, 1564, 1504, 1446, 1367, 1311, 1292,
5.2.14. Methyl 2-ethyl-3-formyl-4-oxo-4H-pyrido[1,2-a]-
pyrimidine-8-carboxylate (25). To formic acid (5 mL)
was added 24 (231 mg) and stirred for 70 min. The mix-
ture was concentrated to afford 25 (201 mg, 80% for
two steps) as an orange oil, which was utilized for the next
1
reaction without further purification. H NMR (CDCl3)
d: 0.92 (3H, t, J = 7.3 Hz), 3.22 (2H, q, J = 7.3 Hz), 4.05
(3H, s), 7.73 (1H, dd, J = 1.7, 7.3 Hz), 8.29–8.33 (1H,
m), 9.15–9.20 (1H, m), 10.59 (1H, s). HRMS (EI) Calcd
for C13H12N2O4 (M+): 260.0797. Found: 260.0815.
1
1240, 1201, 1176, 1101, 1059, 1032. H NMR (DMSO-
d6) d: 1.32 (9H, s), 5.90 (1H, dd, J = 2.2, 10.7 Hz), 6.62
(1H, dd, J = 2.0, 16.3 Hz), 6.88 (1H, s), 7.23 (1H, d,
J = 15.4 Hz), 7.33 (1H, dd, J = 10.7, 16.3 Hz), 7.77–7.82
(1H, m), 7.86 (1H, d, J = 15.4 Hz), 8.43 (1H, s), 9.05
(1H, d, J = 7.6 Hz). Anal. Calcd for C21H20N4O4SÆ0.25-
H2O: C, 58.80; H, 4.82; N, 13.06; S, 7.48. Found: C,
58.78; H, 4.70; N, 12.98; S, 7.53.
5.2.15. Methyl 3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-yl]-
2-ethyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylate (26).
Following the procedures as described for 12, the title com-
pound (111 mg) was prepared in 40% yield from 25
(201 mg, 0.772 mmol) as a yellow solid. 1H NMR (CDCl3)
d: 1.35 (3H, t, J = 7.3 Hz), 1.54 (9H, s), 3.01 (2H, q,
J = 7.3 Hz), 4.02 (3H, s), 7.39 (1H, d, J = 15.5 Hz), 7.61
(1H, d, J = 7.3 Hz), 7.78 (1H, d, J = 15.5 Hz), 8.25 (1H,
s), 9.10 (1H, d, J = 7.3 Hz). HRMS (EI) Calcd for
C19H22N2O5 (M+): 358.1529. Found: 358.1514.
5.2.11. Methyl 2-chloro-3-(1,3-dioxolan-2-yl)-4-oxo-4H-
pyrido[1,2-a]pyrimidine-8-carboxylate (22). To a suspen-
sion of 11 (1.16 g, 4.19 mmol) in toluene (20 mL) were
added ethylene glycol (5 mL) and Amberlystꢂ 15
(120 mg), and refluxed for 2 h. The mixture was cooled
and insoluble materials were filtered off and washed with
CHCl3. The combined filtrate and washings were con-
centrated. The residue was diluted with CHCl3 and
washed with water (3·). The combined organic layers
were dried and concentrated to afford 22 (1.32 g, quan-
5.2.16. 3-[(1E)-3-tert-Butoxy-3-oxoprop-1-en-1-yl]-2-ethyl-
4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylic acid (27).
Following the procedures as described for 13, the title
compound (110 mg) was prepared from 26 (111 mg,
1
0.310 mmol) as a yellow solid. H NMR (CD3OD) d:
1.34 (3H, t, J = 7.3 Hz), 1.56 (9H, s), 3.02 (2H, q,
J = 7.3 Hz), 7.32 (1H, d, J = 15.7 Hz), 7.72 (1H, dd,
J = 1.2, 7.3 Hz), 7.77 (1H, d, J = 15.7 Hz), 8.25 (1H,
s), 9.11 (1H, d, J = 7.3 Hz). HRMS (EI) Calcd for
C18H20N2O5 (M+): 344.1372. Found: 344.1385.
1
titative) as a yellow solid. H NMR (400 MHz, CDCl3)
d: 4.00–4.16 (2H, m), 4.02 (3H, s), 4.32–4.41 (2H, m),
6.43 (1H, s), 7.68 (1H, dd, J = 2.0, 7.3 Hz), 8.21–8.25
(1H, m), 9.05 (1H, d, J = 7.3 Hz). HRMS (FAB) calcd
for C13H1235ClN2O5 ([M+H]+): 311.0435. Found:
311.0435.
5.2.17. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-ethyl-4-oxo-4H-pyrido[1,2-a]pyrim-
idin-3-yl)acrylate (28). Following the procedures as de-
scribed for 15, the title compound (122 mg) was
prepared in 79% yield for two steps from 14 (48.6 mg,
0.311 mmol) and crude 27 (110 mg) as a yellow foam.
5.2.12. Methyl 3-(1,3-dioxolan-2-yl)-4-oxo-2-vinyl-4H-
pyrido[1,2-a]pyrimidine-8-carboxylate (23). Following the
procedures as described for 17, the title compound
(214 mg) was prepared in 44% yield from 22 (500 mg,

2000
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
1
1H NMR (CDCl3) d: 1.30–1.38 (3H, m), 1.32 (9H, s),
1.54 (9H, s), 2.93–3.02 (2H, m), 6.60 (1H, s), 7.35 (1H,
dd, J = 2.2, 15.6 Hz), 7.66 (1H, dd, J = 1.7, 7.6 Hz),
7.75 (1H, d, J = 2.2, 15.6 Hz), 8.12–8.19 (1H, m), 9.13
(1H, d, J = 7.6 Hz), 10.09 (1H, br). HRMS (EI) Calcd
for C25H30N4O4S (M+): 482.1988. Found: 482.1985.
tion. H NMR (CDCl3) d: 1.53 (9H, s), 1.70–1.89 (4H,
m), 2.27–2.34 (2H, m), 2.41–2.48 (2H, m), 4.01 (3H, s),
5.94–5.99 (1H, m), 7.35 (1H, d, J = 15.7 Hz), 7.60 (1H,
dd, J = 1.7, 7.6 Hz), 7.77 (1H, d, J = 15.7 Hz), 8.27–
8.30 (1H, m), 9.10 (1H, d, J = 0.7, 7.6 Hz). HRMS (EI)
Calcd for C23H26N2O5: 410.1842. Found: 410.1857.
5.2.18.
(2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
5.2.22. 3-[(1E)-3-tert-Butoxy-3-oxoprop-1-en-1-yl]-2-cyclo-
hex-1-en-1-yl-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylic
acid (35). Following the procedures as described for 13, the
title compound (67.4 mg) was prepared in 25% for two
steps from 34 (153 mg) as a colorless solid, which was uti-
carbonyl}-2-ethyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-
acrylic acid (29). Following the procedures as described
for 21, the title compound (88.8 mg) was prepared in
83% yield from 28 (122 mg, 0.252 mmol) as a yellow sol-
id. Mp: 285–289 ꢁC (dec). IR (ATR) cmꢀ1: 3107, 3059,
2962, 2871, 2166, 1678, 1597, 1566, 1500, 1452, 1412,
1365, 1311, 1294, 1240, 1217, 1201, 1176, 1099, 1063.
1H NMR (DMSO-d6) d: 1.30 (3H, t, J = 7.3 Hz), 1.32
(9H, s), 2.97 (2H, q, J = 7.3 Hz), 6.88 (1H, s), 7.28
(1H, d, J = 15.4 Hz), 7.76 (1H, d, J = 15.4 Hz), 7.82–
7.89 (1H, m), 8.40 (1H, s), 9.11 (1H, d, J = 7.3 Hz).
Anal. Calcd for C21H22N4O4SÆ0.5H2O: C, 57.91; H,
5.32; N, 12.87; S, 7.36. Found: C, 58.00; H, 5.27; N,
12.84; S, 7.42.
1
lized for the next reaction without further purification. H
NMR (CD3OD) d: 1.54 (9H, s), 1.74–1.91 (4H, m), 2.28–
2.35 (2H, m), 2.40–2.47 (2H, m), 5.91–5.95 (1H, m), 7.23
(1H, d, J = 15.7 Hz), 7.71 (1H, dd, J = 1.1, 7.4 Hz), 7.76
(1H, d, J = 15.7 Hz), 8.21–8.24 (1H, m), 9.09–9.14 (1H,
m). HRMS (EI) Calcd for C22H24N2O5 (M+) : 396.1685.
Found: 396.1681.
5.2.23. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-yl)-
amino]carbonyl}-2-cyclohex-1-en-1-yl-4-oxo-4H-pyrido[1,
2-a]pyrimidin-3-yl)acrylate (36). Following the proce-
dures as described for 15, the title compound (76.5 mg)
was prepared in 84% yield from 14 (26.6 mg,
0.170 mmol) and 35 (67.4 mg, 0.170 mmol) as a yellow
5.2.19. 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 (31). To a solution of 15 (50.0 mg, 0.102 mmol)
in DME (4 mL)–water (1 mL) were added phenylboronic
acid (12.5 mg, 0.102 mmol), Na2CO3 (21.7 mg, 0.205
mmol), and Pd(Ph3P)4 (5.9 mg, 5.11 lmol), and refluxed
for 3.5 h under Ar. The mixture 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 silica gel column chromatography
(EtOAc/hexane = 1:3) to afford 31 (52.9 mg) as inseparable
mixture, which was utilized for the next reaction without
1
foam. H NMR (DMSO-d6) d: 1.34 (9H, s), 1.54 (9H,
s), 1.66–1.85 (4H, m), 2.21–2.32 (2H, m), 2.37–2.46
(2H, m), 5.91–5.97 (1H, m), 6.57 (1H, s), 7.30 (1H, d,
J = 15.8 Hz), 7.61 (1H, dd, J = 2.0, 7.3 Hz), 7.76 (1H,
d, J = 15.8 Hz), 8.12–8.16 (1H, m), 9.08 (1H, d,
J = 7.3 Hz). HRMS (FAB) calcd for C29H35N4O4S
([M+H]+): 535.2379. Found: 535.2348.
5.2.24. (2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-cyclohex-1-en-1-yl-4-oxo-4H-pyrido[1,2-a]pyrimi-
din-3-yl)acrylic acid (37). Following the procedures as
described for 21, the title compound (49.6 mg) was prepared
in 73% yield from 36 (76.5 mg, 0.143 mmol) as a yellow sol-
id. Mp: 225–233 ꢁC (dec). IR (ATR) cmꢀ1: 3176, 3109,
2931, 2859, 2657, 1673, 1564, 1504, 1435, 1365, 1311,
1
further purification. H NMR (CDCl3–CD3OD) d: 1.37
(9H, s), 1.48 (9H, s), 6.65 (1H, s), 7.32 (1H, d,
J = 15.6 Hz), 7.54–7.67 (5H, m), 7.57 (1H, d, J = 15.6 Hz),
7.90–7.96 (1H, m), 8.37 (1H, m), 9.23 (1H, d, J = 7.6 Hz).
HRMS (FAB) calcd for C29H31N4O4S: 531.2066. Found:
531.2044.
1
1286, 1203, 1147, 1097, 1061. H NMR (DMSO-d6) d:
5.2.20. (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)-
acrylic acid (32). Following the procedures as described
for 21, the title compound (32.9 mg) was prepared in
68% for two steps from 32 (52.9 mg) as a yellow solid.
Mp: 300–305 ꢁC (dec). IR (ATR) cmꢀ1: 3102, 3051,
2962, 2871, 1687, 1666, 1601, 1549, 1506, 1454, 1414,
1365, 1311, 1292, 1271, 1223, 1196, 1099, 1057. 1H
NMR (DMSO-d6) d: 1.31 (9H, s), 6.89 (1H, br), 7.28
(1H, d, J = 15.6 Hz), 7.49 (1H, d, J = 15.6 Hz), 7.56–
7.63 (5H, m), 7.88–7.93 (1H, m), 8.45 (1H, br), 9.18
(1H, d, J = 7.3 Hz). Anal. Calcd for C25H22N4O4SÆH2O:
C, 60.96; H, 4.91; N, 11.37; S, 6.51. Found: C, 60.85; H,
4.54; N, 11.37; S, 6.64.
1.31 (9H, s), 1.66–1.82 (4H, m), 2.21–2.28 (2H, m), 2.40–
2.48 (2H, m), 5.89 (1H, br), 6.88 (1H, s), 7.24 (1H, d,
J = 15.7 Hz), 7.73 (1H, d, J = 15.7 Hz), 7.81–7.86 (1H, m),
8.38 (1H, s), 9.09 (1H, d, J = 7.3 Hz). Anal. Calcd for
C25H26N4O4SÆ0.4H2O: C, 61.81; H, 5.56; N, 11.53; S, 6.60.
Found: C, 61.89; H, 5.35; N, 11.49; S, 6.60.
5.2.25. Methyl 2-[1-(tert-butoxycarbonyl)-1,2,3,6-tetra-
hydropyridin-4-yl]-3-(1,3-dioxolan-2-yl)-4-oxo-4H-pyrido[1,2-
a]pyrimidine-8-carboxylate (39). To a suspension of 38
(7.98 g, 16.9 mmol) and 22 (5.00 g, 16.1 mmol) in 1,4-diox-
ane (100 mL) were added CsF (5.38 g, 35.4 mmol), t-Bu3P
(212 mg, 0.966 mmol), and Pd2(dba)3ÆCHCl3 (250 mg,
0.241 mmol). The mixture was stirred at 80 ꢁC for 14.5 h un-
der Ar. To the mixture were added 38 (2.00 g, 4.23 mmol),
CsF (1.35 g, 8.85 mmol), t-Bu3P (212 mg, 0.966 mmol), and
Pd2(dba)3ÆCHCl3 (250 mg, 0.241 mmol), and further stirred
at 80 ꢁC for 7 h under Ar. The mixture was cooled, and
insoluble materials were filtered off through Celite, and
washed with CHCl3. The combined filtrate and washings
were diluted with water and extracted with CHCl3 (5·).
5.2.21. Methyl 3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-yl]-
2-cyclohex-1-en-1-yl-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-
carboxylate (34). Following the procedures as described
for 17, the title compound (153 mg) was prepared from
12 (250 mg, 0.685 mmol) as inseparable mixture, which
was utilized for the next reaction without further purifica-

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
2001
The combined organic layers were dried and concentrated.
The residue was purified by silica gel column chromatogra-
phy (CHCl3/EtOAc = 4:1 ! 3:1 ! 2:1) to afford 39 (5.51 g,
75%) as an orange foam. 1H NMR (CDCl3) d: 1.50 (9H, s),
2.59 (2H, br), 3.63–3.71 (2H, m), 3.98–4.14 (4H, m), 4.00
(3H, s), 4.31–4.37 (2H, m), 5.96 (1H, br), 6.16 (1H, s),
7.55 (1H, dd, J = 1.7, 7.6 Hz), 8.23 (1H, dd, J = 0.7,
1.7 Hz), 9.04 (1H, dd, J = 0.7, 7.6 Hz). HRMS (EI) Calcd
for C23H27N3O7 (M+): 457.1849. Found: 457.1867.
7.34 (1H, d, J = 15.7 Hz), 7.67 (1H, d, J = 7.3 Hz), 7.73
(1H, d, J = 15.7 Hz), 8.16 (1H, s), 9.08 (1H, d,
J = 7.3 Hz), 10.59 (1H, br). HRMS (EI) Calcd for
C33H41N5O6S (M+): 635.2778. Found: 635.2775.
5.2.30.
(2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-4-oxo-2-(1,2,3,6-tetrahydropyridin-4-yl)-4H-pyr-
ido- [1,2-a]pyrimidin-3-yl]acrylic acid (44). Following the
procedures as described for 21, the title compound
(73.7 mg) was prepared in 81% yield from 43 (86.2 mg,
0.136 mmol) as a yellow solid. Mp: 181–198 ꢁC (dec). IR
(ATR) cmꢀ1: 3062, 2968, 1684, 1604, 1552, 1508, 1446,
1415, 1360, 1313, 1284, 1186, 1138, 1099, 1059. 1H NMR
(CD3OD) d: 1.36 (9H, s), 2.86–2.93 (2H, m), 3.52–3.59
(2H, m), 3.94–3.99 (2H, m), 6.01–6.60 (1H, m), 6.74–6.77
(1H, m), 7.38–7.45 (1H, m), 7.78–7.85 (1H, m), 7.89–7.94
(1H, m), 8.26–8.30 (1H, m), 9.19–9.23 (1H, m). Anal. Calcd
for C24H25N5O4SÆ1.5TFAÆH2O:C, 48.50;H, 4.30;N,10.47;
S 4.80. Found: C, 48.49; H, 4.16; N, 10.38; S, 4.86.
5.2.26. Methyl 2-[1-(tert-butoxycarbonyl)-1,2,3,6-tetra-
hydropyridin-4-yl]-3-formyl-4-oxo-4H-pyrido[1,2-a]py-
rimidine-8-carboxylate (40). To
a solution of 39
(64.9 mg, 0.142 mmol) in acetone (2 mL)–H2O (2 mL)
was added AcOH (2 mL) at 0 ꢁC, and the mixture was
stirred for 1 h. The mixture was concentrated, and the
residue was diluted with EtOAc, washed with satd NaH-
CO3 aq and brine. The organic layer was dried and con-
centrated. The residue was purified by silica gel column
chromatography (EtOAc/hexane = 1:2 ! 1:1) to afford
1
40 (33.6 mg, 57%) as a yellow oil. H NMR (CDCl3)
5.2.31. Methyl 2-{[1-(tert-butoxycarbonyl)piperidin-4-yl]-
methyl}-3-(1,3-dioxolan-2-yl)-4-oxo-4H-pyrido[1,2-a]pyrim-
idine-8-carboxylate (46). To a solution of 45 (95.2 mg,
0.4828 mmol) in THF (2 mL) was added 9-BBN (0.5 M
solution in THF, 966 lL, 0.483 mmol) under Ar, and the
mixture was refluxed for 1 h. To the mixture were added
a solution of 22 (100 mg, 0.322 mmol) and Pd(dppf)
Cl2ÆCH2Cl2 (13.1 mg, 0.0161 mmol) in DMF (3 mL) and
K2CO3 (89.0 mg, 0.644 mmol) at room temperature, and
the mixture was stirred at 60 ꢁC for 4 h. The mixture
was cooled and diluted with satd NH4Cl aq and extracted
with CHCl3. The combined organic layers were dried and
concentrated. The residue was purified by silica gel column
chromatography (EtOAc/hexane = 1:2 ! 1:1 ! 3:2) to af-
d: 1.50 (9H, s), 2.57 (2H, s), 3.67–3.77 (2H, m), 4.05
(3H, s), 4.14–4.19 (2H, m), 5.89–6.11 (1H, m), 7.75
(1H, dd, J = 7.3, 1.7 Hz), 8.33–8.36 (1H, m), 9.22 (1H,
d, J = 7.3 Hz), 10.30 (1H, s). HRMS (EI) Calcd for
C21H23N3O6 (M+): 413.1587. Found: 413.1573.
5.2.27. Methyl 2-[1-(tert-butoxycarbonyl)-1,2,3,6-tetra-
hydropyridin-4-yl]-3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-
yl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylate (41).
Following the procedures as described for 12, the title
compound (29.6 mg, 57.9 lmol) was prepared in 71% yield
1
from 40 (33.6 mg, 81.2 lmol) as a yellow oil. H NMR
(CDCl3) d: 1.51 (9H, s), 1.53 (9H, s), 2.62 (2H, s), 3.65–
3.76 (2H, m), 4.02 (3H, s), 4.17 (2H, s), 5.96 (1H, br s),
7.36 (1H, d, J = 15.6 Hz), 7.62 (1H, dd, J = 7.3, 1.7 Hz),
7.73 (1H, d, J = 15.6 Hz), 8.27 (1H, dd, J = 1.7, 0.7 Hz),
9.11 (1H, dd, J = 7.3, 0.7 Hz). HRMS (EI) Calcd for
C27H33N3O7 (M+) : 511.2319. Found: 511.2327.
1
ford 46 (149 mg, 98%) as a yellow oil. H NMR (CDCl3)
d: 1.19–1.34 (2H, m), 1.46 (9H, s), 1.59–1.74 (2H, m), 2.08–
2.21 (1H, m), 2.62–2.76 (2H, m), 2.85 (2H, d, J = 7.1 Hz),
3.99–4.15 (4H, m), 4.01 (3H, s), 4.22–4.29 (2H, m),
6.32 (1H, s), 7.53–7.57 (1H, m), 8.19–8.22 (1H, m), 9.00–
9.04 (1H, m). HRMS (FAB) calcd for C24H32N3O7
([M+H]+): 474.2240. Found: 474.2237.
5.2.28. 2-[1-(tert-Butoxycarbonyl)-1,2,3,6-tetrahydropyri-
din-4-yl]-3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-yl]-4-oxo-
4H-pyrido[1,2-a]pyrimidine-8-carboxylic acid (42). Follow-
ing the procedures as described for 13, the title compound
(29.0 mg) was prepared in quantitative yield from 41
(29.6 mg) as a yellow solid, which was utilized for the next
reaction without further purification. 1H NMR (CD3OD–
CDCl3) d: 1.52 (9H, s), 1.54 (9H, s), 2.63 (2H, br), 4.18
(2H, br), 4.43 (2H, br), 5.98 (1H, br), 7.32 (1H, d,
J = 15.8 Hz), 7.68–7.79 (1H, m), 7.23 (1H, d,
J = 15.8 Hz), 8.29 (1H, s), 9.12 (1H, d, J = 7.3 Hz). HRMS
(EI) Calcd for C26H32N3O7 (M+): 498.2240. Found:
498.2215.
5.2.32. 2-{[1-(tert-Butoxycarbonyl)piperidin-4-yl]methyl}-
3-(1,3-dioxolan-2-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-
8-carboxylic acid (47). Following the procedures as
described for 13, the title compound (120 mg) was pre-
pared in 83% yield from 46 (149 mg, 0.314 mmol) as a
1
creamy solid. H NMR (CD3OD–CDCl3) d: 1.22–1.35
(2H, m), 1.46 (9H, s), 1.64–1.77 (2H, m), 2.10–2.23
(1H, m), 2.65–2.80 (2H, m), 2.88 (2H, d, J = 7.1 Hz),
4.01–4.12 (2H, m), 4.23–4.29 (2H, m), 4.52–4.63 (2H,
m), 6.31 (1H, s), 7.55–7.58 (1H, m), 7.67–7.72 (1H, m),
9.02 (1H, d, J = 7.3 Hz). HRMS (FAB) calcd for
C23H30N3O7 ([M+H]+): 460.2084. Found: 460.2080.
5.2.29. tert-Butyl 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)-3,6-dihydropyridine-1(2H)-
carboxylate (43). Following the procedures as described
for 15, the title compound (86.2 mg) was prepared in
84% yield from 14 (38.9 mg, 0.249 mmol) and 42
(124 mg, 0.249 mmol) as a yellow solid. 1H NMR (CDCl3)
d: 1.36 (9H, s), 1.52 (9H, s), 1.54 (9H, s), 2.57 (2H, br),
3.66 (2H, br), 4.14 (2H, br), 5.94 (1H, br), 6.56 (1H, s),
5.2.33. tert-Butyl 4-{[8-{[(4-tert-butyl-1,3-thiazol-2-yl)-
amino]carbonyl}-3-(1,3-dioxolan-2-yl)-4-oxo-4H-pyrido[1,2-
a]pyrimidin-2-yl]methyl}piperidine-1-carboxylate (48). Follow-
ing the procedures as described for 15, the title compound
(102 mg) was prepared in 65% yield from 14 (40.7 mg,
0.261 mmol) and 47 (120 mg, 0.261 mmol) as a yellow
1
foam. H NMR (CDCl3) d: 1.20–1.35 (2H, m), 1.34 (9H,
s), 1.47 (9H, s), 1.60–1.72 (2H, m), 2.07–2.10 (1H, m),

2002
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
2.62–2.76 (2H, m), 2.78–2.88 (2H, m), 4.02–4.16 (4H, m),
4.24–4.31 (2H, m), 6.32 (1H, s), 6.60 (1H, s), 7.58 (1H, dd,
J = 1.7, 7.4 Hz), 8.09 (1H, s), 8.97 (1H, d, J = 7.4 Hz).
HRMS (FAB) calcd for C30H40N5O6S ([M+H]+):
598.2699. Found: 598.2687.
J = 1.7, 7.4 Hz), 8.30–8.31 (1H, m), 8.52 (1H, s), 9.18
(1H, dd, J = 0.7, 7.4 Hz). HRMS (EI) Calcd for
C17H18N2O5 (M+): 330.1216. Found: 330.1208.
5.2.38. tert-Butyl (2E)-3-(8-{[(4-tert-butyl-1,3-thiazol-2-yl)-
amino]carbonyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-
acrylate (54). To a solution of 52 (149 mg, 0.452 mmol)
in THF (10 mL) was added 1 N NaOH aq (4.13 mL,
4.13 mmol) and stirred for 2.5 h. The mixture was acid-
ified to pH 3 with 1 N HCl aq and concentrated. The
residue was partitioned between water and CHCl3,
and the aqueous layer was extracted with CHCl3 (5·).
The combined organic layers were concentrated. To a
solution of the residue in CH2Cl2 (40 mL)–DMF
(40 mL) were added 14 (77.7 mg, 0.497 mmol), DMAP
(110 mg, 0.904 mmol), HOBt (30.5 mg, 0.226 mmol),
and WSCDÆHCl (173 mg, 0.904 mmol). After
stirring overnight, the mixture was concentrated. The
residue was purified by silica gel column chromatogra-
phy (CH2Cl2/MeOH = 20:1) to afford 54 (205 mg,
5.2.34. tert-Butyl 4-[(8-{[(4-tert-butyl-1,3-thiazol-2-yl)-
amino]carbonyl}-3-formyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-
2-yl)methyl]piperidine-1-carboxylate (49). Following the
procedures as described for 40, the title compound
(93.4 mg) was prepared in 99% yield from 48 (102 mg,
1
0.170 mmol) as an orange foam. H NMR (CDCl3) d:
1.28–1.37 (2H, m), 1.38 (9H, s), 1.49 (9H, s), 1.61–1.72
(2H, m), 1.60–1.75 (2H, m), 1.90–2.04 (1H, m), 2.59–2.76
(1H, m), 2.84–3.30 (2H, m), 4.00–4.14 (2H, m), 6.51 (1H,
s), 7.94 (1H, d, J = 7.3 Hz), 8.39 (1H, s), 9.19 (1H, d,
J = 7.3 Hz), 10.58 (1H, s). HRMS (FAB) calcd for
C28H36N5O5S ([M+H]+): 554.2437. Found: 554.2453.
5.2.35. tert-Butyl 4-[(3-[(1E)-3-tert-butoxy-3-oxoprop-1-
en-1-yl]-8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbon-
yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)methyl]piperidine-
1-carboxylate (50). Following the procedures as described
for 12, the title compound (96.1 mg) was prepared in 87%
yield from 49 (93.4 mg, 0.169 mmol) as a yellow foam. 1H
NMR (CDCl3) d: 1.31–1.37 (2H, m), 1.34 (9H, s), 1.47
(9H, s), 1.54 (9H, s), 1.62–1.71 (2H, m), 2.05 (1H, s),
2.62–2.76 (2H, m), 2.89 (2H, d, J = 6.8 Hz), 4.01–4.18
(2H, m), 6.58 (1H, s), 7.38 (1H, d, J = 15.5 Hz), 7.70
(1H, d, J = 1.7 Hz), 7.74 (1H, d, J = 15.5 Hz), 8.21 (1H,
s), 9.12 (1H, d, J = 7.3 Hz), 10.45 (1H, br). HRMS
(FAB) calcd for C34H46N5O6S ([M+H]+): 652.3169.
Found: 652.3173.
1
quantitative) as a yellow powder. H NMR (CDCl3)
d: 1.35 (9H, s), 1.54 (9H, s), 6.61 (1H, s), 7.16 (1H,
d, J = 15.9 Hz), 7.58 (1H, d, J = 15.9 Hz), 7.80 (1H, d,
J = 7.3 Hz), 8.30 (1H, s), 8.52 (1H, s), 9.23 (1H, d,
J = 7.3 Hz). HRMS (EI) Calcd for C23H26N4O4S (M+):
454.1675. Found: 454.1674.
5.2.39.
(2E)-3-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylic acid
(55). Following the procedures as described for 21, the title
compound (142 mg) was prepared in 79% yield from 54
(205 mg, 0.451 mmol) as a yellow foam. Mp: 302–
305 ꢁC. IR (ATR) cmꢀ1: 1689, 1670, 1608, 1576, 1481,
1323, 1286, 1234, 1051, 989, 737, 482. 1H NMR (CD3OD)
d: 1.31 (9H, s), 6.87 (1H, s), 7.08 (1H, d, J = 15.6 Hz), 7.64
(1H, d, J = 15.6 Hz), 7.90 (1H, dd, J = 1.8, 7.5 Hz), 8.45
(1H, s), 8.79 (1H, s), 9.16 (1H, d, J = 7.5 Hz). Anal. Calcd
for C19H18N4O4SÆ0.75H2O: C, 55.40; H, 4.77; N, 13.60.
Found: C, 55.42; H, 4.46; N, 13.35.
5.2.36. (2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]car-
bonyl}-4-oxo-2-(piperidin-4-ylmethyl)-4H-pyrido[1,2-a]pyr-
imidin-3-yl]acrylic acid (51). Following the procedures as
described for 21, the title compound (61.8 mg) was pre-
pared in 65% yield from 50 (96.1 mg, 0.147 mmol) as a yel-
low solid. Mp: 154–161 ꢁC. IR (ATR) cmꢀ1: 3045, 2966,
2866, 2754, 2517, 1670, 1604, 1549, 1502, 1458, 1408,
1360, 1300, 1198, 1130, 1061. 1H NMR (CD3OD) d: 1.36
(9H, s), 1.54–1.66 (2H, m), 1.97–2.07 (2H, m), 2.23–2.36
(1H, m), 2.94–3.07 (4H, m), 3.35–3.44 (2H, m), 6.76 (1H,
s), 7.43 (1H, d, J = 15.4 Hz), 7.85–7.92 (2H, m), 8.26 (1H,
s), 9.18 (1H, d, J = 7.3 Hz). Anal. Calcd for C25H29N5O4-
SÆTFAÆ2H2O: C, 50.23; H, 5.31; N, 10.85; S, 4.97. Found:
C, 50.20; H, 5.06; N, 10.58; S, 4.71.
5.2.40. tert-Butyl (2E)-3-[8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-4-oxo-2-(1,2,3,6-tetrahydropyridin-4-
yl)-4H-pyrido[1,2-a]pyrimidin-3-yl]acrylate (56). To a
solution of 43 (2.14 g, 3.37 mmol) in CH2Cl2 (40 mL)
was added TFA (4 mL) at 0 ꢁC, and the mixture was stir-
red for 15 h at 0 ꢁC. Additionally, TFA (4 mL) was added
to the mixture at 0 ꢁC and stirred further for 4.5 h at 0 ꢁC.
The mixture was diluted with satd NaHCO3 aq at 0 ꢁC
and extracted with CHCl3 (3·). The combined organic
layers were dried and concentrated to afford 56 (1.23 g,
5.2.37. Methyl 3-[(1E)-3-tert-butoxy-3-oxoprop-1-en-1-
yl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylate (52).
To a solution of 12 (1.16 g, 3.19 mmol) in DMF (30 mL)
were added Ph3P (42.0 mg, 0.160 mmol), Pd(OAc)2
(14.3 mg, 0.0638 mmol), Et3N (1.36 mL, 9.76 mmol),
and formic acid (241 lL, 6.38 mmol), and stirred at
70 ꢁC for 24 h. The mixture was concentrated, and the res-
idue was diluted with CHCl3. Insoluble materials were re-
moved by filtration and washed with CHCl3. The filtrate
and washings were concentrated. The residue was purified
by silica gel column chromatography (EtOAc/hex-
ane = 1:2) to afford 52 (149 mg, 14%) as a yellow solid.
1H NMR (CDCl3) d: 1.54 (9H, s), 4.03 (3H, s), 7.16 (1H,
d, J = 15.7 Hz), 7.57 (1H, d, J = 15.7 Hz), 7.68 (1H, dd,
1
68%) as a yellow solid. H NMR (CD3OD) d: 1.36 (9H,
s), 1.54 (9H, s), 2.82 (2H, br), 3.43–3.51 (2H, m), 3.85–
3.92 (2H, m), 5.97–6.05 (1H, m), 6.72 (1H, s), 7.30 (1H,
d, J = 15.7 Hz), 7.74 (1H, d, J = 15.7 Hz), 7.84–7.90
(1H, m), 8.20–8.25 (1H, m), 9.11–9.18 (1H, m). HRMS
(FAB) calcd for C28H34N5O4S ([M+H]+): 536.2332.
Found: 536.2310.
5.2.41. tert-Butyl (2E)-3-(2-(1-acetyl-1,2,3,6-tetrahydro-
pyridin-4-yl)-8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbon-
yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)acrylate (57). To
a solution of 56 (200 mg, 0.373 mmol) in CH2Cl2 (5 mL)
were added Et3N (78.1 lL, 0.560 mmol) and acetyl

K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
2003
chloride (29.2 lL, 0.411 mmol) at 0 ꢁC, and stirred for
3.5 h. The mixture was concentrated, and the residue
was diluted with EtOAc, and washed with 10% citric acid
aq, satd NaHCO3 aq and brine. The organic layer was
dried and concentrated to afford 57 (172 mg, 80%) as a
yellow solid. 1H NMR (CDCl3) d: 1.36 (9H, s), 1.52
(9H, s), 2.29, 2.33 (total 3H, 2s), 2.65–2.73 (2H, m),
3.73–3.79, 3.97–4.03 (total 2H, 2m), 4.24–4.28, 4.35–4.39
(total 2H, 2m), 5.95, 6.04 (total 1H, 2s), 6.59 (1H, s),
7.35, 7.39 (total 1H, 2d, J = 11.5 Hz for each), 7.74, 7.70
(total 1H, 2d, J = 11.5 Hz for each), 7.70–7.74, 7.76–7.82
(total 1H, 2m), 8.36, 8.47 (total 1H, each s), 9.12, 9.14
(total 1H, 2d, J = 7.6 Hz for each). HRMS (FAB) calcd
for C30H36N5O5S ([M+H]+): 578.2437. Found: 578.2419.
5.2.45. tert-Butyl (2E)-3-{8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-2-[1-(N,N-dimethylglycyl)-1,2,3,6-tetra-
hydropyridin-4-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-
acrylate (63). To a solution of 56 (200 mg, 0.373 mmol)
and Sar-OH (42.4 mg, 0.411 mmol) in CH2Cl2 (2 mL)–
DMF (2 mL) were added DMAP (91.2 mg, 0.747 mmol),
HOBt (55.5 mg, 0.411 mmol), and WSCDÆHCl (78.7 mg,
0.411 mmol) at 0 ꢁC and stirred for 13 h. The mixture
was concentrated, and the residue was diluted with
CHCl3, and washed with 10% citric acid aq, satd NaH-
CO3 aq, and brine. The organic layer was dried and con-
centrated. The residue was purified by PTLC (CHCl3/
MeOH = 10:1) to afford 63 (89.5 mg, 39%) as a yellow
solid. 1H NMR (CD3OD) d: 1.35 (9H, s), 1.52, 1.53
(total 9H, 2s), 2.456, 2.465 (total 6H, 2s), 2.61–2.74
(2H, m), 3.49 (2H, s), 3.82–3.92 (2H, m), 4.30–4.38
(2H, m), 5.96–6.04 (1H, m), 6.73 (1H, s), 7.25 (1H, d,
J = 15.6 Hz), 7.74 (1H, d, J = 15.6 Hz), 7.86 (1H, dd,
J = 1.7, 7.3 Hz), 8.24 (1H, s), 9.14–9.18 (1H, m). HRMS
(FAB) calcd for C32H41N6O5S ([M+H]+): 621.2859.
Found: 621.2882.
5.2.42. (2E)-3-(2-(1-Acetyl-1,2,3,6-tetrahydropyridin-4-yl)-
8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-4H-
pyrido[1,2-a]pyrimidin-3-yl)acrylic acid (58). Following the
procedures as described for 21, the title compound
(121 mg) was prepared in 76% yield from 57 (172 mg,
0.297 mmol) as a yellow solid. Mp: 200–210 ꢁC (dec). IR
(ATR) cmꢀ1: 3049, 2960, 2873, 2789, 1682, 1597, 1493,
1446, 1414, 1365, 1288, 1238, 1203, 1144, 1099, 1061.
1H NMR (DMSO-d6) d: 1.31 (9H, s), 2.08, 2.12 (total
3H, 2s), 2.49–2.69 (2H, m), 3.67–3.75 (2H, m), 4.14–4.25
(2H, m), 5.98 (1H, m), 6.89 (1H, s), 7.26 (1H, d,
J = 15.6 Hz), 7.71 (1H, d, J = 15.6 Hz), 7.84–7.88 (1H,
m), 8.40 (1H, s), 9.11 (1H, d, J = 7.6 Hz). Anal. Calcd
for C26H27N5O5SÆ0.75H2O: C, 58.36; H, 5.37; N, 13.09;
S, 5.99. Found: C, 58.28; H, 5.29; N, 12.83; S, 6.02.
5.2.46. [{2-[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)-3,6-dihydropyridin-1(2H)-yl]-2-
oxoethyl}(dimethyl)ammonio]acetate (65). To a solution of
63 (89.5 mg, 0.144 mmol) in DMF (4 mL) was added tert-
butyl bromoacetate (106 lL, 0.721 mmol) and stirred in
the dark for 12.5 h. Additionally, tert-butyl bromoacetate
(106 lL, 0.721 mmol) was added and stirred in the dark
for 6.5 h. The mixture was concentrated to afford 64
(108 mg) as inseparable mixture, which was utilized for
the next reaction without further purification. Following
the procedures as described for 21, the title compound
(46.6 mg) was prepared in 45% yield for two steps from
64 (108 mg) as a yellow solid. Mp: 198–207 ꢁC (dec). IR
(ATR) cmꢀ1: 2962, 2868, 1635, 1547, 1502, 1441, 1387,
1311, 1273, 1236, 1200, 1130, 1101, 1061. 1H NMR
(CD3OD) d: 1.35 (9H, s), 2.62–2.79 (2H, m), 3.46 (3H,
s), 3.47 (3H, s), 3.74–3.80, 3.88–3.94 (total 2H, 2m),
4.18–4.36 (4H, m), 4.86–4.98 (2H, m), 5.95–6.06 (1H,
m), 6.74 (1H, s), 7.37 (1H, d, J = 15.7 Hz), 7.73 (1H, d,
J = 15.7 Hz), 7.81–7.86 (1H, m), 8.22–8.26 (1H, m),
9.14–9.19 (1H, m). Anal. Calcd for C29H32N6O7SÆ0.5TFAÆ
2H2O: C, 52.02; H, 5.42; N, 11.74; S 4.48. Found: C, 51.78;
H, 5.49; N, 11.58; S, 4.50.
5.2.43. [{2-[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)-3,6-dihydropyridin-1(2H)-yl]-2-
oxoethyl}(methyl)amino]acetic acid (61). To 59 (100 mg,
0.680 mmol) was added acetic anhydride and refluxed
for 4 h. The mixture was concentrated to afford crude
60, which was utilized for the next reaction without further
purification. To a solution of crude 60 in toluene (2 mL)–
pyridine (0.5 mL) was added 56 and refluxed for 1 h. The
mixture was concentrated and the residue was purified by
silica gel column chromatography (CHCl3 ! CHCl3/
MeOH = 10:1 ! CHCl3/MeOH/H2O = 8:3:0.5) to afford
61 (233 mg) as a brown solid. This compound was utilized
for the next reaction without further purification. HRMS
(FAB) calcd for C33H41N6O7S ([M+H]+): 665.2757.
Found: 665.2764.
5.3. In vitro Potentiation Activity
5.2.44.
(2E)-3-[8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]-
carbonyl}-2-(1-{[(carboxymethyl)(methyl)amino]acetyl}-
1,2,3,6-tetrahydropyridin-4-yl)-4-oxo-4H-pyrido[1,2-a]pyr-
imidin-3-yl]acrylic acid (62). Following the procedures as
described for 21, the title compound (152 mg) was prepared
in 51% yield for two steps from 56 (233 mg) as a yellow sol-
id. Mp: 222–236 ꢁC (dec). IR (ATR) cmꢀ1: 2962, 2929,
2871, 1674, 1603, 1547, 1500, 1444, 1369, 1309, 1282,
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-
lated at 1 · 106 CFU/mL and incubated at 37 ꢁC for
18 h. MICs were determined by visual observation of
growth.
1
1238, 1201, 1140, 1101, 1063. H NMR (DMSO-d6) d:
1.27, 1.29 (total 9H, 2s), 2.30–2.41 (3H, m), 2.57–2.67
(2H, m), 3.03–4.46 (8H, m), 5.82–6.07 (1H, m), 6.48–6.67
(1H, m), 6.95–7.09 (1H, m), 7.38 (br, 1H), 7.63–7.80 (1H,
m), 7.92–8.13 (1H, m), 8.78–8.91 (1H, m). Anal. Calcd
for C29H32N6O7SÆTFAÆ4H2O: C, 46.85; H, 5.20; N,
10.57; S 4.03. Found: C, 46.89; H, 5.43; N, 10.84; S, 4.15.
Acknowledgment
Members of the Research Technology Center, Daiichi
Pharmaceutical Co., Ltd. are gratefully acknowledged
for analytical data.

2004
K. Yoshida et al. / Bioorg. Med. Chem. 14 (2006) 1993–2004
9. Nakayama, K.; Kuru, N.; Ohtsuka, M.; Yokomizo, Y.;
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