MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: Highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate

Article abstract of DOI:10.1016/j.bmc.2007.07.039

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, substituted at the 2-position with piperidines bearing quaternary ammonium salt side chains, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the β-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Attachment of the charged entity using an N-ethylcarbamoyloxy linker led to the discovery of the highly soluble compound 22 (D13-9001), which maintained good potency in vitro and displayed excellent activity in vivo in a rat pneumonia model of P. aeruginosa.

Full text of DOI:10.1016/j.bmc.2007.07.039

Bioorganic & Medicinal Chemistry 15 (2007) 7087–7097
MexAB-OprM specific efflux pump inhibitors in Pseudomonas
aeruginosa. Part 7: Highly soluble and in vivo active quaternary
ammonium analogue D13-9001, a potential preclinical candidate
Ken-ichi Yoshida,a,* Kiyoshi Nakayama,a Masami Ohtsuka,a Noriko Kuru,a
Yoshihiro Yokomizo,a Atsunobu Sakamoto,a Makoto Takemura,a Kazuki Hoshino,b
Hiroko Kanda,b Hironobu Nitanai,b Kenji Namba,b Kumi Yoshida,c Yuichiro Imamura,c
Jason Z. Zhang,d Ving J. Leed and William J. Watkinsd
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
cDrug Metabolism & Physicochemistry Research Laboratory, Daiichi Pharmaceutical Co., Ltd, 16-13, Kita-Kasai 1-Chome,
Edogawa-ku, Tokyo 134-8630, Japan
dEssential Therapeutics, Inc., 850 Maude Avenue, Mountain View, CA 94043, USA
Received 19 June 2007; revised 11 July 2007; accepted 14 July 2007
Available online 22 August 2007
Abstract—A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, substituted at the 2-position with piperidines bearing quater-
nary ammonium salt side chains, were synthesized and evaluated for their ability to potentiate the activity of the fluoroquinolone
levofloxacin (LVFX) and the b-lactam aztreonam (AZT) in Pseudomonas aeruginosa. Attachment of the charged entity using an
N-ethylcarbamoyloxy linker led to the discovery of the highly soluble compound 22 (D13-9001), which maintained good potency
in vitro and displayed excellent activity in vivo in a rat pneumonia model of P. aeruginosa.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Among these, the MexAB-OprM system is constitutive
and commonly overexpressed in clinical isolates.5 There-
fore, we targeted a MexAB-OprM specific efflux pump
inhibitor (EPI) for use as a potentiator of existing anti-
bacterial agents through combinational use. This kind
of agent would be useful in the treatment of the increas-
ingly common clinically refractory infections caused by
P. aeruginosa.
The emergence of drug resistance in many organisms is a
serious public health issue and, therefore, has attracted
much attention in recent years. In particular, reports of
multidrug resistance in Gram-negative organisms such
as Pseudomonas aeruginosa have increased significantly.1
A major component of this problem, and of intrinsic
resistance, is attributable to the phenomenon of efflux,
whereby a differentially low concentration of the antibi-
otic is maintained inside the bacterium. At least seven
RND (resistance-nodulation-cell division) superfamily
efflux pumps are known to exist in P. aeruginosa.2–5
In previous papers, we reported two types of pyridopyr-
imidine-based EPI: (i) vinyl tetrazoles with piperidine
2-substituents such as 1 and (ii) acrylic acid analogues
such as 2 (Fig. 1) bearing aryl residues in place of the
piperidine, but each class bore liabilities as clinical can-
didates.6–11 Compound 1 showed excellent potency
in vitro, but the solubility was very poor for intravenous
use. On the other hand, the more soluble compound 2
possessed a reasonable safety profile in an acute toxic as-
say, but unfortunately showed only moderate potentia-
tion activity in vivo. Thus we set out to improve the
Keywords: Efflux pump inhibitor; MexAB-OprM efflux pump;
Pseudomonas aeruginosa; Drug resistance.
*
Corresponding author at present address: Daiichi Sankyo Co., Ltd,
16-13, Kita-Kasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan.
Tel.: +81 3 3680 0151; fax: +81 3 5696 8772; e-mail: yoshida.kenichi.
rs@daiichisankyo.co.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.039

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K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
O
O
NH2
O
O
N
N
N
N
O
2
N
N
O
O
N
N
S
S
H
H
N
N
H
N
CO2H
N
1
N
2
N
Figure 1. Structure of the pyridopyrimidine-based MexAB-OprM specific efflux pump inhibitors.
solubility of the strong potentiator 1 by incorporating
highly water-soluble moieties such as quaternary ammo-
nium salts, directed by our cumulative knowledge of the
SAR in both series.
deprotection of tetrazole moiety (and ester hydrolysis,
where necessary), gave the desired derivatives (20–23).
A compound in which the carbamate linker was re-
placed by amide was synthesized as shown in Scheme
3. After attachment of 3-piperidineacetic acid ester
(24) to the pyridopyrimidine nucleus, ester hydrolysis,
EDC-mediated amide formation with N,N-dimethyle-
thylenediamine, quaternization, and treatment with
TFA provided the piperidine acetic acid derivative (26).
Herein, we report the design, the synthesis, and evalua-
tion of analogues resulting from this effort.
2. Chemistry
We planned to synthesize the quaternary ammonium
salts from the appropriate intermediate (11) by attach-
ment of the linker moiety, quaternization with a suitable
alkyl halide, and finally by deprotection of p-methoxyb-
enzyl group, as depicted in Scheme 2. A series of substi-
tuted piperidines was introduced via nucleophilic
displacement of a diphenyl phosphonate at the C-2 po-
sition. The primary carbamate 1 was synthesized via
an N-trichloroacetyl protected intermediate as displayed
in Scheme 1. The stereochemistry of the 3-hydroxyl
group on the piperidine was fixed as (R) since the (S)-
isomer exhibited less potency.9 The appropriate alcohol
(11) was reacted with 1,10-carbonyldiimidazole and then
with x-dimethylaminoalkylamine to provide a series of
N,N-dimethylamino intermediates (13, 14), which were
useful for the preparation of various quaternary ammo-
nium salts. Treatment with alkyl halides, followed by
In an attempt to remove the chiral center, two avenues
were explored. The asymmetric carbonate linker was re-
placed with an acrylamide as illustrated in Scheme 4.
The acrylic acid moiety was generated by Horner–
Wadsworth–Emmons reaction on the 3-oxo-piperidine
(27), and subsequent steps analogous to earlier ana-
logues afforded the desired compound (31).
Finally, an achiral 4-piperidinyl variant (34) was synthe-
sized using the same route as that described for 22
(Scheme 5).
3. Results and discussion
The compounds prepared in this study were tested for
in vitro potentiation of the activity of levofloxacin
1) SOCl2, 80˚C
HO2C
NHAc
2) 4, pyridine /CH2Cl2
HCl / EtOH, 80˚C
O
O
N
N
NHAc
NH2
N
N
N
S
S
H
H
N
N
N
3
5
6
NH2
O
S
4
O
N
1) (COCl), DMF / CH2Cl2
2
N
N
O
OH
2) 8, piperidine, pyridine, reflux
TCPM* / xylene, reflux
Cl
N
S
N
O
OH
H
N
S
N
PMB
N
Cl
H
PMB
N
N
O
O
HO2C
N
N
N
N
O
O
N
Cl
Cl
Cl
Cl
N
9
7
8
O
*TCPM
OH
O
NH2
(PhO)2P(O)Cl, iPr2NEt
/ DMF, 0˚C
O
N
1) Cl3CCONCO / EtOAc
O
N
N
N
2) HCO Na / MeOH, H2O
N
H
2
then 10, 80˚C
S
N
N
N
S
N
H
OH
N
PMB
R
O
N
O
N
N
HN
HCl·
N
11
N
N
N
N
12: R = PMB
1: R = H
10
TFA, anisole, 60˚C
Scheme 1. Synthesis of 2-piperidine attached vinyl tetrazole analogue 1.

K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
7089
O
O
+ R'
( )n
N
( )n
O
N
N
O
N
H
H
CDI/ CH2Cl2
O
O
N
N
then Me2N(CH )nNH2
R'-X / DMF
2
N
O
N
N
O
N
N
N
11
S
S
H
H
N
N
R
N
R
N
N
N
N
N
N
N
13: n = 2, R = PMB
14: n = 3, R = PMB
15: n = 2, R = H
16: n = 2, R = PMB, R' = Me
17: n = 2, R = PMB, R' = CH2CONH
20: n = 2, R = H, R' = Me
TFA, anisole, 60˚C
21: n = 2, R = H, R' = CH2CONH
2
2
22 (D13-9001): n = 2, R = H, R' = CH2CO2-
23: n = 3, R = H, R' = CH2CO2-
18: n = 2, R = PMB, R' = CH2CO2tBu
19: n = 3, R = PMB, R' = CH2CO2tBu
1) 4N HCl-dioxane (if necessary)
2) TFA, anisole, 60˚C
Scheme 2. Synthesis of quaternary ammonium salt derivatives.
1) NaOHaq. / THF-MeOH
2) H2N(CH )2N(CH )2, EDC
O
2
3
CO2Me
N+ CO2
-
HOBt / CH2Cl2-DMF
N
H
3) BrCH CO2tBu / DMF
1) (PhO) P(O)Cl, iPr NEt
2
2
2
O
O
N
N
4) 4N HCl-dioxane
/ DMF-MeCN, 0˚C
2) 24, 80˚C
N
O
N
N
O
N
5) TFA, anisole, 80˚C
N
N
S
S
9
H
H
N
N
CO2Me
PMB
H
N
N
N
N
N
25
26
N
N
HN
HCl·
N
24
Scheme 3. Synthesis of piperidineacetic acid linker analogue 26.
CO2Et
EtO2C
1) (EtO)2P(O)CH CO2Et, NaH / THF
O
2
2) 4N HCl-dioxane
+
BOCN
27
HN
HCl·
HCl·HN
29
28
1) NaOHaq. / THF-MeOH
O
2) H2N(CH )2N(CH )2, EDC
2
3
CO2Et
PMB
N
N
N
N+ CO2
-
HOBt / CH2Cl2-DMF
N
H
1) (PhO) P(O)Cl, iPr2NEt
3) BrCH CO2tBu / DMF
2
2
O
O
N
N
/ DMF-MeCN, 0˚C
4) 4N HCl-dioxane
N
N
N
O
N
2) 28, 80˚C
N
N
5) TFA, anisole, 80˚C
S
S
9
H
H
N
N
H
N
O
N
30
31
N
N
N
Scheme 4. Synthesis of acrylamide linker analogue 31.
1) CDI/ CH2Cl2 then Me2(CH )2NH2
2
H
2) BrCH CO2tBu / DMF
(PhO) P(O)Cl, iPr2NEt
OH
2
O
N
2
N+
CO2
-
O
N
O
N
3) 4N HCl-dioxane
/ DMF, 0˚C
N
O
N
N
N
O
4) TFA, anisole, 80˚C
then 32, 80˚C
N
N
S
S
H
9
H
N
N
PMB
HN
OH
H
N
O
N
N
32
N
N
33
34
N
N
N
Scheme 5. Synthesis of 4-hydroxypiperidine linker analogue 34.
(LVFX) and aztreonam (AZT) against PAM1723, a
laboratory strain of P. aeruginosa in which the Mex-
AB-OprM pump is over-expressed and the MexCD-
OprJ and MexEF-OprN pumps are disrupted.12 The
results, expressed as the minimum concentration of
inhibitor required to decrease (potentiate) the mini-
mum inhibitory concentration (MIC) of the combina-
tion agent 8-fold (MPC8, lg/mL), together with
aqueous solubilities (in pH 6.8 buffer solution, lg/mL),
are presented in Table 1. Antimicrobial assays were

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K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
Table 1. Chemical structures and potentiation activity of quaternary ammonium analoguesa
O
N
N
O
R2
N
S
H
N
H
N
N
N
N
Compound
R2
MPC8 (LVFX) (lg/mL)
MPC8 (AZT) (lg/mL)
Sol (pH 6.8) (lg/mL)
Without HSA
With 0.125% HSA
O
O
O
O
O
O
O
O
NH2
O
1
0.5
2
60.25
9.2
N
N
N
N
N
H
O
O
O
O
O
c
—
15
2
2
4
4
4
4
4
8
1
2
4
2
2
4
8
4
N+
N+
N+
N
H
20
—c (547)d
O
O
NH2
N
H
21
4
95
N
N
N
N
O
N
H
22 (D13-9001)
2
747
O
N+
N
H
23
26
31
34
4
610
O
O
O
O
N+
N
H
4
860
N+
O
N
H
16
>16b
insolublee
N
N
H
O
O
N
O
N+
8
16
170
O
a All compounds lacked intrinsic antibacterial activity.
b The apparent lack of activity is attributable to precipitation of the compound.
c Not tested.
d The solubility of its enantiomer.
e Precipitation was observed and the solubility was not quantitated.
conducted in the presence and absence of 0.125%
human serum albumin (HSA) in order to evaluate
the effects of protein binding. The previously reported
3-(R)-carbamoyloxy piperidine derivative (1) showed
excellent potency. However, this compound did not
possess adequate solubility for intravenous use (empir-
ically, ca. >100 lg/mL). In order to solve this prob-
lem, the attachment of a quaternary ammonium

K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
7091
moiety through a variety of tethers was explored.
Although the introduction of a dimethylaminoethyl
substituent onto the carbamate slightly reduced the
potency, we chose the ethylene linker for this study
because the compound 15 still displayed good activity
as potentiator of AZT. Moreover, the influence of the
addition of HSA was lower than 1.
8
6
4
2
0
non treatment
AZT 1000 mg/kg
+D13-9001 1.25 mg/kg
+D13-9001 5 mg/kg
+D13-9001 20 mg/kg
The trimethylammonium analogue (20) showed almost
the same activity as 15. This compound, when dosed
intravenously, proved lethal to 0/3 and 2/3 mice at 50
and 100 mg/kg, respectively. More hydrophilic quater-
nary compounds were also tested. The acetamide ana-
logue (21) displayed an improved safety profile (no
deaths at 100 mg/kg), but was less potent than 20,
and its rather low solubility was disappointing. On
the other hand, the acetic acid analogue 22 (D13-
9001) retained good activity in vitro and was not lethal
to mice at 100 mg/kg; moreover, its solubility profile
was excellent.
0
20
40
60
80
100 120 140 160
Hours after infection
Figure 2. Efficacy of AZT combined with 22 (D13-9001) by intrave-
nous infusion in a model of P. aeruginosa pneumonia in rats.
100
1.25 mg/kg
5 mg/kg
20 mg/kg
The length of the linker was extended in 23. The potency
and solubility were slightly reduced.
10
1
Varying the nature and position of the carbamate also
changed the biological profile of the analogues. The car-
ba analogues 26 and 31 displayed rather reduced po-
tency, and the introduction of the double bond in 31
reduced the solubility dramatically.
0.1
0.01
0.001
The migration of the carbamate from the 3-position to
the 4-position of the piperidine, generating an achiral
compound, reduced the activity by 2- to 4-fold. Interest-
ingly, this transformation also led to significant lower
solubility (22 vs 34).
0
1
2
3
4
Time (h)
Figure 3. Serum concentration of 22 (D13-9001) after intravenous
infusion to rats.
Because of its high solubility and improved safety char-
acteristics, we chose compound 22 (D13-9001) as a pro-
totypical EPI to perform in vivo profiling in
combination with AZT, evaluating its potentiation
activity in a lethal pneumonia model in rats using the
P. aeruginosa strain PAM1020.13 As shown in Figure
2, the combination of 1.25, 5, and 20 mg/kg of 22
(D13-9001) with 1000 mg/kg of AZT gave improved sur-
vival rates at the end of day seven, whereas no obvious
effect was observed on treatment with AZT alone.
4. Conclusions
In summary, we have discovered a series of pyridopyr-
imidine derivatives, substituted at the 2-position with
piperidines bearing quaternary ammonium salt side
chains, that are highly water soluble. Of these, ammo-
nium acetic acid analogue 22 (D13-9001) exhibited po-
tent efficacy in vivo, high solubility, and a good safety
profile in an acute toxicity assay. Additional research
is in progress in order to extend to a clinically effec-
tive efflux pump inhibitor versus Pseudomonas
aeruginosa.
Figure 3 displays the serum concentration-time curve
observed in rats after the administration of 22 (D13-
9001) with 1000 mg/kg of AZT by intravenous infusion
over 2 h (dose levels 1.25, 5, and 20 mg/kg). Pharmaco-
kinetic parameters are shown in Table 2. The compound
exhibits reasonably linear PK characteristics in the dose
range from 5 to 20 mg/kg and therapeutic efficacy
in vivo was clearly demonstrated at these exposures.
The serum levels of AZT were the same with and with-
out co-administration of the EPI (data not shown). In
addition, the PK parameters of 22 (D13-9001) in mon-
keys are presented in Table 3. Here again reasonable
dose linearity was observed, with serum levels at 5 mg/
kg exceeding those in the rat at the same dose. Hence
we expect that in vivo efficacy would be obtainable at
a lower doses in mammals.
5. Experimental
5.1. General
Unless otherwise noted, materials were obtained from
commercial suppliers and used without further purifica-
tion. [1-(4-Methoxybenzyl)-1H-tetrazol-5-yl]acetic acid
(8) was prepared according to the literature proce-
dures.14 Melting points were taken on a Yanako MP-
500D melting point apparatus and are uncorrected.

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K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
Table 2. PK parameters of 22 (D13-9001) after intravenous infusion to rats
Dose (mg/kg)
C2h (lg/mL)
AUC (lgÆh/mL)
MRT (h)
1
CL (mL/min/kg)
1
Vdss1 (L/kg)
t1/2 (t = 2.25ꢀ3.0) (h)
1
1.25
5
0.262
1.836
8.040
0.486
3.529
14.213
1.186
1.209
1.215
42.84
23.61
23.45
3.049
1.712
1.710
0.23
0.18
0.25
20
Linear extrapolation to time 0. Approximated concentration was used in extrapolation to time 1.
Table 3. PK parameters of 22 (D13-9001) after intravenous infusion to monkeys
Dose (mg/kg)
C1h (lg/mL)
AUC (lgÆh/mL)
MRT (h)
1
CL (mL/min/kg)
1
Vdss1 (L/kg)
t1/2 (h)
1
1
5
1.224
7.635
1.491
7.891
0.614
0.625
11.23
10.70
0.414
0.400
0.28 (t = 1.25–2.0)
0.41 (t = 1.5–3.0)
Linear extrapolation to time 0. Approximated concentration was used in extrapolation to time 1.
Optical rotations were measured in a 0.5-dm cell at ca.
25 ꢁC at 589 nm with a HORIBA SEPA-300 polarime-
ter. 1H NMR spectra were determined on a JEOL
JNM-EX400 spectrometer. Chemical shifts are re-
ported in parts per million relative to tetramethylsilane
br), 8.72 (1H, s). HRMS (ESI) Calcd for C15H19N4O2S
([M + H]+): 319.1229. Found: 319.1191.
5.2.2. 2-Amino-N-(4-tert-butyl-1,3-thiazol-2-yl)isonicoti-
namide (6). To a solution of 5 (546 mg, 1.71 mmol) in
EtOH (12 mL) was added dropwise conc. HCl
(1.2 mL), and the mixture was stirred at 80–90 ꢁC for
1 h. The mixture was concentrated, neutralized with
1 N NaOH aq and extracted with CHCl3. The organic
layer was dried and concentrated. The residue was puri-
1
as internal standard. Significant H NMR data are tab-
ulated in the following order: number of protons, mul-
tiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad), coupling constant(s) in hertz.
Infrared (IR) spectra were obtained on a HORIBA
FT-720 spectrometer. High-resolution mass spectra
(HRMS) were obtained on a JEOL JMS-T100LP mass
spectrometer under electron spray ionization condi-
tions (ESI). Elementary analyses were conducted at Re-
search Technology 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 phosphomolybdic acid in etha-
nol, UV lamp, or Wako ninhydrin spray. Unless other-
wise 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 evap-
orator with bath at 35–45 ꢁC.
fied
by
silica
gel
column
chromatography
(CHCl3 ! CHCl3/MeOH = 60:1 ! 20:1) to afford 6
1
(247 mg) as a white solid. H NMR (CD3OD) d: 1.33
(9H, s), 6.63 (1H, d, J = 0.7 Hz), 7.05 (1H, t,
J = 0.7 Hz), 7.09 (1H, ddd, J = 5.4, 1.5, 0.7 Hz), 8.13
(1H, dd, J = 5.4, 0.7 Hz).
5.2.3. N-(4-tert-Butyl-1,3-thiazol-2-yl)-2-hydroxy-4-oxo-
4H-pyrido[1,2-a]pyrimidine-8-carboxamide (7). To a sus-
pension of 6 (200 mg, 0.724 mmol) in xylene (20 mL)
was added TCPM (370 mg, 0.799 mmol), and the mix-
ture was stirred at 130 ꢁC for 1 h. The mixture was
cooled and concentrated. The residue was diluted with
CHCl3, and the precipitated solid was collected by filtra-
tion, and washed with CHCl3 to afford 7 (209 mg) as an
1
orange solid. H NMR (DMSO-d6) d: 1.31 (9H, s), 3.45
(2H, m), 6.88 (1H, s), 7.77 (1H, d, J = 7.1 Hz), 8.02 (1H,
s), 8.99 (1H, d, J = 7.1 Hz). HRMS (ESI) Calcd for
C16H17N4O3S ([M + H]+): 345.1021. Found: 345.1013.
5.2. Synthesis
5.2.4. N-(4-tert-Butyl-1,3-thiazol-2-yl)-2-hydroxy-3-{(E)-
2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-
4H-pyrido[1,2-a]pyrimidine-8-carboxamide (9). (i) To a
solution of DMF (16.9 mL, 218 mmol) in CH2Cl2
(500 mL) was added dropwise oxalyl chloride
(18.6 mL, 218 mmol) at 0 ꢁC. After the mixture was stir-
red at 0 ꢁC for 15 min, 7 (25.0 g, 72.6 mmol) was added
at 0 ꢁC and stirring was continued for 2 h. The mixture
was diluted with satd NaHCO3 aq at 0 ꢁC and acidified
to pH 3 with 1 N HCl aq at 0 ꢁC. The precipitated solid
was collected by filtration and washed with water to
afford N-(4-tert-butyl-1,3-thiazol-2-yl)-3-formyl-2-hydroxy-
4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide (15.5 g)
5.2.1. 2-(Acetylamino)-N-(4-tert-butyl-1,3-thiazol-2-yl)iso-
nicotinamide (5). To 3 (500 mg, 2.79 mmol) was added
dropwise thionyl chloride (5 mL, 68.5 mmol), and the
mixture was stirred at 80 ꢁC for 30 min. The mixture
was concentrated, and the residue was added to a solu-
tion of 4-tert-butyl-1,3-thiazol-2-amine (525 mg,
3.34 mmol) in pyridine (0.25 mL)-CH2Cl2 (5 mL) at
0 ꢁC. The mixture was stirred at 0 ꢁC for 30 min and fur-
ther at ambient temperature for 2.5 h. The mixture was
diluted with water and extracted with CHCl3. The or-
ganic layer was dried and concentrated. The residue
was purified by silica gel column chromatography
(CHCl3 ! CHCl3/MeOH = 80:1 ! 50:1) to afford 5
1
as a yellow solid. H NMR (DMSO-d6) d: 1.31 (9H,
1
(546 mg) as a white solid. H NMR (CDCl3) d: 1.31
s), 6.88 (1H, br s), 7.88–7.92 (2H, m), 9.07 (1H, d,
J = 7.8 Hz), 10.06 (1H, s). HRMS (ESI) Calcd for
C17H17N4O4S ([M + H]+): 373.0971. Found: 373.0998.
(9H, s), 2.26 (3H, s), 6.61 (1H, s), 7.63 (1H, dd,
J = 5.1, 0.5 Hz), 8.44 (1H, d, J = 5.1 Hz), 8.51 (1H,

K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
7093
ii) N-(4-tert-Butyl-1,3-thiazol-2-yl)-3-formyl-2-hydroxy-4-
oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide (11.6 g,
28.4 mmol) and 8 (10.0 g, 40.3 mmol) were dissolved in
pyridine (200 mL)-piperidine (40 mL), and the mixture
was refluxed for 3 h. The mixture was cooled and con-
centrated, and the residue was diluted with 1 N HCl
aq and EtOAc. The precipitated solid was collected by
filtration, and washed with water and EtOAc to afford
1.010, CHCl3). HRMS (ESI) Calcd for C33H37N10O5S
([M + H]+): 685.2669. Found: 685.2661.
5.2.7. (3R)-1-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]carb-
onyl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-
a]pyrimidin-2-yl}piperidin-3-yl carbamate (1). To 12
(76.7 mg, 0.112 mmol) were added TFA (1 mL) and ani-
sole (50 lL), and the mixture was stirred at 60 ꢁC for
6 h. The mixture was concentrated and co-evaporated
with toluene and the residue was purified by PTLC
(CHCl3/MeOH/H2O = 8:3:0.5) and solidified from
EtOH-Et2O to afford 1 (40.0 mg) as an orange solid.
Mp 216–218 ꢁC. IR (ATR) cmꢀ1: 2958, 2866, 1664,
1637, 1608, 1541, 1512, 1439, 1402, 1350, 1311, 1259,
1228, 1103, 1049. 1H NMR (CD3OD/CDCl3) d: 1.36
(9H, s), 1.71 (1H, m), 1.97 (3H, m), 3.60 (1H, m),
3.76 (2H, m), 3.89 (1H, m) , 4.87 (1H, s), 6.63 (1H,
s), 7.49 (1H, s), 7.62 (1H, d, J = 7.3 Hz), 7.68 (1H, d,
1
9 (11.3 g) as a red solid. H NMR (DMSO-d6) d: 1.30
(9H, s), 3.71 (3H, s), 5.57 (2H, s), 6.86 (1H, d,
J = 12.0 Hz), 6.92 (1H, d, J = 8.6 Hz), 7.22 (1H, d,
J = 8.6 Hz), 7.70 (1H, d, J = 15.9 Hz), 7.93 (1H, d,
J = 7.3 Hz), 7.95 (1H, s), 8.05 (1H, d, J = 15.9 Hz),
9.14 (1H, d, J = 7.3 Hz). HRMS (ESI) Calcd for
C27H27N8O4S ([M + H]+): 559.1876. Found: 559.1928.
5.2.5. N-(4-tert-Butyl-1,3-thiazol-2-yl)-2-[(3R)-3-hydroxy-
piperidin-1-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetra-
zol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-car-
boxamide (11). To a suspension of 9 (10.8 g, 19.3 mmol)
in MeCN (130 mL)-DMF (65 mL) were added diisopro-
pylethylamine (10.0 mL, 57.4 mmol) and diphenyl
chlorophosphate (4.20 mL, 20.3 mmol) at 0 ꢁC. After
stirring at 0 ꢁC for 50 min under Ar, 10 (2.00 g,
19.8 mmol) was added at 0 ꢁC, and the mixture was stir-
red at 80 ꢁC for 1 h. After concentration, the residue was
diluted with EtOAc and washed with 1 N HCl aq and
satd NaHCO3 aq. The organic layer was dried and
concentrated. The residue was purified by silica gel
column chromatography (CH2Cl2 ! CHCl3/EtOAc =
2:1 ! 1:1) to afford 11 (10.8 g) as a red amorphous
J = 15.6 Hz), 7.86 (1H, d, J = 15.6 Hz), 8.12 (1H, s),
24:9
D
8.98 (1H, d, J = 7.3 Hz). ½aꢁ
+8.3ꢁ (c 1.011, DMSO).
Anal. Calcd for C25H28N10O4SÆ0.25Et2OÆ2.25H2O: C,
50.07; H, 5.66; N, 22.46; S, 5.14. Found: C, 49.91; H,
5.04; N, 22.34; S, 5.15.
5.2.8. (3R)-1-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]carb-
onyl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-
oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl[2-(di-
methylamino)ethyl]carbamate (13). To a solution of 11
(100 mg, 0.156 mmol) in CH2Cl2 was added 1,10-car-
bonylbis-1H-imidazole (50.5 mg, 0.312 mmol) and the
mixture was stirred for 3 h. N,N-dimethylethylenedi-
amine (85.5 lL, 0.779 mmol) was added and stirring
was continued for 68 h. The mixture was diluted with
water and extracted with CHCl3 (3·). The organic
layer was dried and concentrated. The residue was
purified by silica gel column chromatography
(CHCl3/MeOH = 98:2 ! 95:5) to afford 13 (110 mg)
as an orange solid. 1H NMR (CDCl3) d: 1.35 (9H,
s), 1.62–2.32 (5H, m), 2.22 (6H, s), 2.43–2.57 (2H,
m), 3.25–3.48 (3H, m), 3.57–3.86 (3H, m), 3.77 (3H,
s), 4.84 (1H, br s), 5.50 (2H, s), 5.99 (1H, br s),
6.58 (1H, s), 6.89 (2H, d, J = 8.5 Hz), 7.35 (2H, d,
1
material. H NMR (CDCl3) d: 1.15–1.60 (2H, m), 1.39
(9H, s), 1.67–2.06 (3H, m), 3.36–4.15 (5H, m), 3.77
(3H, s), 5.50 (2H, s), 6.47 (1H, s), 6.90 (2H, d,
J = 8.8 Hz), 7.35 (2H, d, J = 8.8 Hz), 7.39–7.45 (1H,
m), 7.77 (2H, s), 7.99 (1H, br), 8.80 (1H, d,
ꢀ12.4ꢁ (c 1.019, CHCl3). HRMS
24:7
D
J = 7.3 Hz). ½aꢁ
(ESI) Calcd for C32H36N9O4S ([M + H]+): 642.2611.
Found: 642.2569.
5.2.6. (3R)-1-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]carb-
onyl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-
oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl carba-
mate (12). To a solution of 11 (100 mg, 0.156 mmol) in
EtOAc (3 mL) was added trichloroacetyl isocyanate
(74.3 lL, 0.312 mmol) at 0 ꢁC, and the mixture was stir-
red for 45 min. After concentration, the residue was dis-
solved in MeOH (2 mL)-H2O (1 mL) and sodium
formate (100 mg, 1.47 mmol) was added. After stirring
for 2 h, more sodium formate (100 mg, 1.47 mmol)
was added and stirring was continued for 18.5 h. After
the mixture was concentrated, the residue was diluted
with water and extracted with CHCl3 (3·). The organic
layer was dried and concentrated. The residue was puri-
fied by PTLC (CHCl3/EtOAc = 1:1) to afford 12
(76.7 mg) as a red solid. 1H NMR (CDCl3) d: 1.32
(9H, s), 1.64–1.91 (3H, m), 2.04–2.18 (3H, m), 3.16–
3.28 (1H, m), 3.45 (1H, d, J = 13.4 Hz), 3.78 (3H, s),
3.82–3.93 (1H, m), 4.00–4.10 (1H, m), 4.95–5.02 (1H,
m), 5.51 (2H, s), 6.10 (2H, br s), 6.61 (1H, s), 6.89
(2H, d, J = 8.8 Hz), 7.36 (2H, d, J = 8.8 Hz), 7.57 (1H,
J = 8.5 Hz), 7.47 (1H, d, J = 7.6 Hz), 7.80–7.97 (3H,
m), 8.96 (1H, d, J = 7.6 Hz). ½aꢁ
24:8
D
+102.4ꢁ (c 1.038,
CHCl3). HRMS (ESI) Calcd for C37H46N11O5S
([M + H]+): 756.3404. Found: 756.3360.
5.2.9. (3R)-1-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]carb-
onyl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-
oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl[3-(di-
methylamino)propyl]carbamate (14). Following the pro-
cedure described for 13, the title compound (101 mg)
was prepared from 11 (100 mg, 0.159 mmol) as an or-
ange solid. 1H NMR (CDCl3) d: 1.36 (9H, s), 1.62–
2.09 (6H, m), 2.16–2.32 (1H, m), 2.22 (6H, s), 2.39
(2H, t, J = 6.7 Hz), 3.20–3.45 (3H, m), 3.60–3.85
(3H, m), 3.77 (3H, s), 4.82 (1H, br s), 5.50 (2H, s),
6.25–6.37 (1H, m), 6.89 (2H, d, J = 8.8 Hz), 7.23
(1H, br s), 7.35 (2H, d, J = 8.8 Hz), 7.51 (1H, d,
J = 7.3 Hz), 7.88–8.01 (3H, m), 8.97 (1H, d,
ꢀ78.9ꢁ (c 1.008, CHCl3). HRMS
24:9
D
J = 7.3 Hz). ½aꢁ
dd, J = 7.3, 2.0 Hz), 7.96 (2H, s), 8.09 (1H, d,
J = 1.0 Hz), 8.99 (1H, d, J = 7.3 Hz). ½aꢁ
(ESI) Calcd for C38H48N11O5S ([M + H]+): 770.3561.
Found: 770.3512.
24:7
D
+46.2ꢁ (c

7094
K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
5.2.10.
(3R)-1-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]
C31H40N12O5SÆTFAÆ2H2O: C, 50.24; H, 6.13; N, 21.31.
Found: C, 50.40; H, 6.10; N, 21.06.
carbonyl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyr-
ido[1,2-a]pyrimidin-2-yl}piperidin-3-yl [2-(dimethylamino)
ethyl]carbamate (15). Following the procedure described
for 1, the title compound (43.8 mg) was prepared from 13
(270 mg, 0.357 mmol) as an orange solid. Mp 194–
200 ꢁC. IR (ATR) cmꢀ1: 2954, 2860, 1711, 1658, 1635,
1618, 1512, 1425, 1379, 1309, 1248, 1225, 1151, 1101,
5.2.13. [[2-({[((3R)-1-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)
amino]carbonyl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-
4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)oxy]carbo-
nyl}amino)ethyl](dimethyl)ammonio]acetate (22 = D13-
9001). To a solution of 13 (405 mg, 0.536 mmol) in
DMF (8 mL) was added bromoacetic acid tert-butyl es-
ter (0.396 mL, 2.67 mmol). The solution was stirred for
16 h and concentrated to afford crude 18. To the obtained
18 was added 4 N HCl-dioxane (10 mL) and stirring was
continued for 9 h. After the mixture was concentrated, the
residue was purified with PTLC (CHCl3/MeOH/H2O =
8:3:1). The fraction was dissolved with TFA (10 mL)-ani-
sole (0.5 mL) and stirred at 60 ꢁC for 2 h. After the mix-
ture was concentrated, the residue was purified by
PTLC (CHCl3/MeOH/H2O = 8:3:1). The obtained
fraction was dissolved with MeOH-H2O and basified to
pH = 8 by 0.1 N NaOH aq. The mixture was purified
by HPLC (SHISEIDO CAPCELL PAK C18, SG-120A,
30 · 250 mm, MeOH:H2O = 60:40) to afford 22
(103 mg) as an orange solid. Mp 220–233 ꢁC. IR (ATR)
cmꢀ1: 2958, 1700, 1650, 1625, 1511, 1423, 1247, 1103.
1H NMR (CD3OD) d: 1.29 (9H, s), 1.69 (1H, m), 1.94
(2H, m), 2.03 (1H, m), 3.26 (6H, s), 3.44–3.71 (6H, m),
3.84 (2H, s), 3.92 (1H, m), 4.06 (1H, m), 6.71 (1H, s),
7.45 (1H, d, J = 15.9 Hz), 7.64 (1H, dd, J = 1.7, 7.6 Hz),
7.94 (1H, d, J = 15.9 Hz), 8.04 (1H, d, J = 1.7 Hz), 8.96
1
1068. H NMR (CD3OD) d: 1.35 (9H, s), 1.71 (1H, br
s), 1.88–1.92 (1H, m), 2.00 (2H, br s), 2.76 (6H, s),
3.06–3.07 (2H, m), 3.30–3.56 (5H, m), 3.88–3.91 (2H,
m), 6.74 (1H, s), 7.55–7.59 (2H, m), 7.97 (1H, d,
J = 16.4 Hz), 8.02 (1H, s), 8.97 (1H, d, J = 7.6 Hz).
½aꢁ
24:9
D
ꢀ210.1ꢁ (c 1.025, DMSO). Anal. Calcd for
C29H37N11O4SÆ0.5Et2OÆ2.75H2O: C, 51.54; H, 6.63; N,
21.33; S, 4.44. Found: C, 51.66; H, 6.10; N, 21.35; S, 4.43.
5.2.11.
5-[(E)-2-(8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]
carbonyl}-4-oxo-2-{(3R)-3-[({[2-(trimethylammonio)ethyl]
amino}carbonyl)oxy]piperidin-1-yl}-4H-pyrido[1,2-a]pyr-
imidin-3-yl)vinyl]tetrazol-1-ide (20). To a solution of 13
(100 mg, 0.1323 mmol) in DMF (5 mL) was added
MeI (500 lL) and the solution was stirred at 5 ꢁC for
67 h. After the mixture was concentrated, the residue
was purified by PTLC (CHCl3/MeOH/H2O = 8:3:0.5)
to afford crude 16. To this were added TFA (1 mL)
and anisole (50 lL), and the solution was stirred at
60 ꢁC for 2.5 h. After the mixture was concentrated,
the residue was purified by PTLC (CHCl3/MeOH/
H2O = 8:3:0.5) and solidified from Et2O to afford 20
(14.7 mg) as an orange solid. Mp 159–169 ꢁC. IR
(ATR) cmꢀ1: 2962, 1664, 1637, 1512, 1423, 1365, 1309,
(1H, d, J = 7.6Hz). ½aꢁ24:9 +55.6ꢁ (c 1.012, DMSO). Anal.
D
Calcd for C31H39N11O6SÆ6H2O: C, 46.43; H, 6.41; N,
19.21. Found: C, 46.48; H, 5.86; N, 18.66.
1
1250, 1201, 1174, 1124, 1080, 1043. H NMR (CD3OD)
d: 1.35 (9H, s), 1.70 (1H, br), 1.97 (3H, br), 3.16 (9H, s),
3.42 (3H, m), 3.55 (3H, m), 3.93 (2H, m), 4.89 (1H, s),
6.75 (1H, s), 7.56 (1H, d, J = 16.1 Hz), 7.60 (1H, dd,
5.2.14. [[3-({[((3R)-1-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)
amino]carbonyl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-
4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)oxy]carbo-
nyl}amino)propyl](dimethyl)ammonio]acetate (23). Fol-
lowing the procedure described for 22, the title
compound (232 mg) was prepared from 14 (277 mg,
0.360 mmol) as an orange solid. Mp 157–163 ꢁC. IR
(ATR) cmꢀ1: 2970, 2868, 1674, 1637, 1541, 1508, 1439,
J = 7.3, 1.2 Hz), 7.96 (1H, d, J = 16.1 Hz), 8.03 (1H,
25:0
D
s), 8.99 (1H, d, J = 7.3 Hz). ½aꢁ
ꢀ211.0ꢁ (c 1.018,
DMSO). Anal. Calcd for C30H39N11O4SÆ1.2CF3COO-
HÆ2.9H2O: C, 46.39; H, 5.53; N, 18.37; F, 8.15; S,
3.82. Found: C, 46.70; H, 5.19; N, 18.00; F, 7.92; S, 3.82.
1
1252, 1205, 1178, 1128, 1039. H NMR (DMSO-d6) d:
5.2.12. 5-[(E)-2-(2-((3R)-3-{[({2-[(2-Amino-2-oxoethyl)
(dimethyl)ammonio]ethyl}amino)carbonyl]oxy}piperidin-
1-yl)-8-{[(4-tert-butyl-1,3-thiazol-2-yl)amino]carbonyl}-4-
oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)vinyl]tetrazol-1-ide
(21). To a solution of 13 (3.60 g, 4.76 mmol) in DMF
(30 mL) was added 2-iodoacetamide (1.00 g, 5.41 mmol)
and the mixture was stirred for 17 h and concentrated to
afford crude 17. To the obtained 17 were added TFA
(160 mL) and anisole (5 mL), and the solution was stir-
red at 60 ꢁC for 3 h. After the mixture was concentrated,
the residue was purified by silica gel column chromatog-
raphy (CHCl3/MeOH/H2O = 70:26:4) to afford 21
(825 mg) as an orange solid. Mp 185–193 ꢁC. IR
(ATR) cmꢀ1: 3186, 2958, 1695, 1660, 1637, 1624, 1542,
1516, 1425, 1379, 1311, 1250, 1227, 1153, 1103. 1H
NMR (CD3OD) d: 1.35 (9H, s), 1.71 (1H, m), 1.98
(3H, m), 3.32 (6H, s), 3.63 (6H, m), 3.89 (2H, m), 4.17
(2H, m), 4.80 (1H, m), 6.73 (1H, s), 7.52 (1H, d,
J = 16.1 Hz), 7.57 (1H, d, J = 7.3 Hz), 7.94 (1H, d,
1.31 (9H, s), 1.66–1.93 (6H, m), 2.94–3.74 (16H, m),
4.65 (1H, m), 6.85 (1H, s), 7.47 (1H, d, J = 16.3 Hz),
7.62 (1H, d, J = 7.3 Hz), 7.76 (1H, br), 7.87 (1H, d,
J = 16.3 Hz), 8.24 (1H, s), 8.96 (1H, d, J = 7.3 Hz).
½aꢁ
25:0
D
ꢀ33.5ꢁ (c 1.039, DMSO). Anal. Calcd for
C32H41N11O6SÆ6H2OÆ3CF3COOH: C, 39.41; H, 4.87;
N, 13.31. Found: C, 39.12; H, 4.43; N, 13.51.
5.2.15. Methyl [(3S)-1-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-
tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-
yl)piperidin-3-yl]acetate (25). To a solution of 9 (5.00 g,
8.95 mmol) in MeCN (60 mL)-DMF(30 mL) were
added diisopropylethylamine (6.24 mL, 35.8 mmol)
and diphenyl chlorophosphate (2.05 mL, 9.85 mmol) at
0 ꢁC. After stirring at 0 ꢁC for 1 h under Ar, 24
(2.25 g, 11.6 mmol) was added at 0 ꢁC, and stirring
was continued at 80 ꢁC for 1 h. After the mixture was
concentrated, the residue was diluted with CHCl3 and
washed with 5% citric acid aq satd NaHCO3 aq and
brine. The organic layer was dried and concentrated.
J = 16.1 Hz), 8.00 (1H, s), 8.96 (1H, d, J = 7.3 Hz).
ꢀ150.9ꢁ (c 1.005, DMSO). Anal. Calcd for
24:9
D
½aꢁ

K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
7095
The residue was purified by silica gel column chroma-
tography (CHCl3 ! CHCl3/MeOH = 100:1 ! 50:1) to
(1H, dd, J = 13.1, 9.4 Hz), 3.13–3.25 (1H, m), 3.38–
3.54 (2H, m), 3.77 (3H, s), 3.98 (1H, d, J = 12.9 Hz),
4.13 (1H, d, J = 12.9 Hz), 5.51 (2H, s), 6.56 (1H, s),
6.85–6.92 (1H, m), 6.89 (2H, d, J = 8.8 Hz), 7.35 (2H,
d, J = 8.8 Hz), 7.49 (1H, dd, J = 7.3, 1.6 Hz), 7.67 (1H,
1
afford 25 (5.25 g) as a red solid. H NMR (CDCl3) d:
1.17–1.29 (1H, m), 1.40 (9H, s), 1.51–1.73 (2H, m),
1.81–1.92 (1H, m), 2.01–2.35 (3H, m), 2.74–2.87 (1H,
m), 2.95–3.09 (1H, m), 3.58 (3H, s), 3.79 (3H, s), 3.85–
3.94 (1H, m), 3.99–4.10 (1H, m), 5.52 (2H, s), 6.56
(1H, s), 6.91 (2H, d, J = 8.8 Hz), 7.30–7.40 (1H, m),
7.38 (2H, d, J = 8.8 Hz), 7.74 (2H, d, J = 15.4 Hz),
d, J = 15.4 Hz), 7.84 (1H, d, J = 15.4 Hz), 7.88 (2H, d,
+18.7ꢁ (c
24:9
D
J = 1.6 Hz), 8.92 (1H, d, J = 7.3 Hz). ½aꢁ
1.028, CHCl3). HRMS (ESI) Calcd for C38H48N11O4S
([M + H]+): 754.3611. Found: 754.3565.
7.76–7.79 (1H, m), 7.89 (1H, d, J = 15.4 Hz), 8.81 (1H,
d, J = 6.1 Hz). ½aꢁ
24:8
D
+16.5ꢁ (c 1.014, CHCl3). HRMS
(iii) Following the procedure described for 22, the title
compound (48.6 mg) was prepared from N-(4-tert-butyl-
1,3-thiazol-2-yl)-2-[(3S)-3-(2-{[2-(dimethylamino) ethyl]-
amino}-2-oxoethyl)piperidin-1-yl]-3-{(E)-2-[1-(4-methoxy-
benzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]
pyrimidine-8-carboxamide (550 mg, 0.730 mmol) as an
orange solid. Mp 225–230 ꢁC. IR (ATR) cmꢀ1: 2960,
1631, 1514, 1439, 1377, 1309, 1254, 1227, 1101. 1H
NMR (CD3OD) d: 1.34 (9H, s), 1.75–1.80 (2H, m),
1.92 (1H, br), 2.19–2.23 (3H, m), 2.91–2.97 (1H, m),
3.18–3.34 (8H, m), 3.54 (2H, br), 3.70–3.71 (2H, m),
3.87 (2H, br), 4.06–4.09 (2H, m), 6.72 (1H, s), 7.47
(1H, d, J = 15.7 Hz), 7.56 (1H, d, J = 7.2 Hz), 7.82
(ESI) Calcd for C35H40N9O5S ([M + H]+): 698.2873.
Found: 689.2859.
5.2.16.
[(2-{[((3S)-1-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)
amino]carbonyl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-
4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetyl]ami-
no}ethyl)(dimethyl)ammonio]acetate (26). (i) To a solu-
tion of 25 (5.25 g, 7.52 mmol) in THF (23 mL)-MeOH
(23 mL) was added 1 N NaOH aq (22.6 mL, 22.6 mmol)
and the mixture was stirred for 80 min. After concentra-
tion, the residue was diluted with water. The aqueous
phase was acidified to pH 1–2 with 1 N HCl aq and
extracted with CHCl3. The organic layer was dried
and concentrated. The residue was purified by silica
gel column chromatography (CHCl3! CHCl3/MeOH =
100:1 ! 50:1) to afford [(3S)-1-(8-{[(4-tert-butyl-1,3-
thiazol-2-yl)amino]carbonyl}-3-{(E)-2-[1-(4- methoxyb-
enzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2- a]pyr-
imidin-2-yl)piperidin-3-yl]acetic acid (4.20 g) as a red
solid. 1H NMR (CDCl3) d: 1.19–1.43 (1H, m), 1.38
(9H, s), 1.59–1.77 (1H, m), 1.90 (1H, d, J = 13.4 Hz),
2.07 (1H, d, J = 12.0 Hz), 2.27 (1H, dd, J = 13.4,
12.0 Hz), 2.47–2.61 (1H, m), 2.68 (1H, dd, J = 13.7,
3.2 Hz), 2.87–3.01 (2H, m), 3.77 (3H, s), 4.28 (1H, d,
J = 12.0 Hz), 4.42 (1H, d, J = 13.7 Hz), 5.11 (1H, d,
J = 15.1 Hz), 5.32 (1H, d, J = 15.1 Hz), 6.63 (1H, s),
6.85 (2H, d, J = 8.5 Hz), 7.22 (2H, d, J = 8.5 Hz), 7.32
(1H, d, J = 15.4 Hz), 7.48 (1H, d, J = 15.4 Hz), 7.53
(1H, d, J = 15.7 Hz), 7.96 (1H, s), 8.92 (1H, d,
24:8
D
J = 7.2 Hz). ½aꢁ ꢀ27.2ꢁ (c 0.264, DMSO). Anal. Calcd
for C32H41N11O5SÆ0.5HCO2HÆH2O: C, 53.27; H, 6.05;
N, 21.02. Found: C, 53.25; H, 6.36; N, 20.63.
5.2.17. (3E)-3-(2-Ethoxy-2-oxoethylidene)piperidine hydro-
chloride (28). (i) To a suspension of NaH (55% in liquid
paraffin, 2.59 g, 64.9 mmol) in THF (300 mL) was added
ethyl diethylphosphonoacetate (11.8 mL, 59.5 mmol) at
0 ꢁC under N2, and the mixture was stirred for 20 min.
To it was added a solution of 27 (15.0 g, 54.1 mmol) in
THF (100 mL) at 0 ꢁC and stirring was continued for
45 min. The mixture was diluted with water and concen-
trated to remove the THF. The residue was diluted with
EtOAc and washed with water and brine. The organic
layer was dried and concentrated. The residue was puri-
fied by silica gel column chromatography (EtOAc/hex-
ane = 10:1 ! 4:1) to afford tert-butyl (3Z)-3-(2-ethoxy-
2-oxoethylidene)piperidine-1-carboxylate (5.36 g) as a
white solid and tert-butyl (3E)-3-(2-ethoxy-2-oxoethylid-
(1H, dd, J = 7.3, 1.7 Hz), 8.00 (1H, d, J = 1.7 Hz), 9.01
(1H, d, J = 7.3 Hz). ½aꢁ
24:8
D
+773.9ꢁ (c 1.008, CHCl3).
HRMS (ESI) Calcd for C34H38N9O5S ([M + H]+):
684.2717. Found: 684.2746.
ene)piperidine-1-carboxylate (8.86 g) as
oil. tert-Butyl (3Z)-3-(2-ethoxy-2-oxoethylidene)piperi-
dine-1-carboxylate: H NMR (CDCl3) d: 1.29 (1H, t,
a colorless
(ii) To a solution of [(3S)-1-(8-{[(4-tert-butyl-1,3-thia-
zol-2-yl)amino]carbonyl}-3-{(E)-2-[1-(4-methoxybenzyl)
-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimi-
din-2-yl)piperidin-3-yl]acetic acid (2.00 g, 2.92 mmol)
and N,N-ethylenediamine (482 lL, 4.39 mmol) in
CH2Cl2 (15 mL)-DMF (15 mL) were added HOBt
(790 mg, 5.85 mmol) and EDCÆHCl (1.12 g, 5.85 mmol)
at 0 ꢁC and stirred for 18.5 h. The mixture was diluted
with satd NaHCO3 aq and extracted with CHCl3 (3·).
The organic layer was dried and concentrated. The res-
idue was purified by silica gel column chromatography
(CHCl3/MeOH = 95:5 ! 90:10) to afford N-(4-tert-bu-
tyl-1,3-thiazol-2-yl)-2-[(3S)-3-(2-{[2-(dimethylamino)ethyl]-
amino}-2-oxoethyl)piperidin-1-yl]-3-{(E)-2-[1-(4-meth-
oxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-
a]pyrimidine-8-carboxamide (2.09 mg) as an orange
solid. 1H NMR (CDCl3) d: 1.17–1.44 (1H, m), 1.35
(9H, s), 1.54–1.78 (2H, m), 1.89–2.00 (1H, m), 2.11–
2.35 (3H, m), 2.25 (6H, s), 2.53–2.60 (2H, m), 2.96
1
J = 7.2 Hz), 1.45 (9H, s), 1.71–1.76 (2H, m), 2.32–2.36
(2H, m), 3.49 (2H, d, J = 5.7 Hz), 4.17 (2H, q,
J = 7.2 Hz), 4.62 (2H, s), 5.66 (1H, s). HRMS (ESI) Calcd
for C14H24NO4 ([M + H]+): 270.1705. Found: 270.1686.
tert-Butyl (3E)-3-(2-ethoxy-2-oxoethylidene)piperidine-1-
carboxylate: 1H NMR (CDCl3) d: 1.28 (1H, t,
J = 7.1 Hz), 1.45 (9H, s), 1.67–1.73 (2H, m), 2.92–2.96
(2H, m), 3.48 (2H, d, J = 5.6 Hz), 3.94 (2H, br), 4.16
(2H, q, J = 7.1 Hz), 5.74 (1H, br). HRMS (ESI) Calcd
for C14H24NO4 ([M + H]+): 270.1705. Found: 270.1688.
(ii) To tert-butyl (3E)-3-(2-ethoxy-2-oxoethylidene)
piperidine-1-carboxylate (8.86 g, 32.9 mmol) was added
4 N HCl-dioxane (165 mL) and the mixture was stirred
for 1 h prior to concentration and co-evaporation with
1
toluene to afford 28 (6.27 g) as white solid. H NMR
(CDCl3) d: 1.28 (1H, t, J = 7.2 Hz), 2.00–2.05 (2H, m),

7096
K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
3.03 (2H, t, J = 6.1 Hz), 3.30 (2H, br), 3.72 (2H, s), 4.17
(2H, q, J = 7.2 Hz), 5.95 (1H, s). HRMS (ESI) Calcd for
C9H16NO2 ([M + H]+): 170.1181. Found: 170.1168.
title compound (368 mg) was prepared from 9 (500 mg,
0.895 mmol) as an orange solid. H NMR (CDCl3) d:
1
1.34 (9H, s), 1.55–1.90 (3H, m), 2.06–2.20 (2H, m),
3.33–3.61 (2H, m), 3.78 (3H, s), 3.91–4.25 (3H, m), 5.53
(2H, s), 6.60 (1H, s), 6.90 (2H, d, J = 8.8 Hz), 7.25–7.27
(1H, m), 7.37 (2H, d, J = 8.8 Hz), 7.51 (1H, d,
J = 7.3 Hz), 7.75 (1H, d, J = 15.4 Hz), 7.88–7.90 (1H,
m), 7.92 (1H, d, J = 15.4 Hz), 8.96 (1H, d, J = 7.3 Hz).
HRMS (ESI) Calcd for C32H36N9O4S ([M + H]+):
642.2611. Found: 642.2584.
5.2.18. [(2-{[(2E)-2-(1-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)
amino]carbonyl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-
pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-ylidene)acetyl]amino}
ethyl)(dimethyl)ammonio]acetate (31). (i) Following the
procedure described for 25 and 26, (2E)-[1-(8-{[(4-tert-
butyl-1,3-thiazol-2-yl)amino]carbonyl}-3-{(E)-2-[1-(4-
methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-2-yl)piperidin-3-ylidene]acetic acid
5.2.20. [[2-({[(1-{8-{[(4-tert-Butyl-1,3-thiazol-2-yl)amino]
carbonyl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyri-
do[1,2-a]pyrimidin-2-yl}piperidin-4-yl)oxy]carbonyl}amino)
ethyl](dimethyl)ammonio]acetate (34). (i) Following the
procedure described for 13, 1-(8-{[(4-tert-butyl-1,3-thia-
zol-2-yl)amino]carbonyl}-3-{(E)-2-[1-(4-methoxybenzyl)
-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimi-
din-2-yl)piperidin-4-yl [2-(dimethylamino)ethyl]carba-
mate (103 mg) was prepared from 33 (100 mg, 0.159
1
(1.41 g) was prepared from 9 (5.00 g) as a red solid. H
NMR (CDCl3) d: 1.37 (9H, s), 1.94 (2H, br s), 2.32
(2H, br s), 3.25 (2H, br s), 3.75 (3H, s), 3.85 (2H, br s),
5.44 (2H, s), 6.65 (1H, s), 6.86 (2H, d, J = 8.5 Hz), 7.02
(1H, s), 7.25–7.37 (1H, m), 7.30 (2H, d, J = 8.5 Hz),
7.54 (1H, d, J = 7.3 Hz), 7.77 (2H, s), 8.31 (1H, s), 8.91
(1H, d, J = 7.3 Hz). HRMS (ESI) Calcd for
C34H36N9O5S ([M + H]+): 682.2560. Found: 682.2522.
1
mmol) as an orange solid. H NMR (CDCl3) d: 1.40
(ii) Following the procedure described for 26, N-(4-tert-
butyl-1,3-thiazol-2-yl)-2-[(3E)-3-(2-{[2-(dimethylamino)
ethyl]amino}-2-oxoethylidene)piperidin-1-yl]-3-{(E)-2-
[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-
pyrido[1,2-a]pyrimidine-8-carboxamide (1.36 g) was pre-
(9H, s), 1.64–1.78 (2H, m), 1.87–2.02 (2H, m), 2.24 (6H,
s), 2.43 (2H, t, J = 5.5 Hz), 3.28 (2H, q, J = 5.5 Hz),
3.37–3.48 (2H, m), 3.71-3.82 (2H, m), 3.79 (3H, s), 4.81–
4.99 (1H, m), 5.29–5.39 (1H, m), 5.53 (2H, s), 6.59 (1H,
s), 6.92 (2H, d, J = 8.5 Hz), 7.16 (1H, s), 7.35–7.42 (1H,
m), 7.38 (2H, d, J = 8.5 Hz), 7.75 (1H, d, J = 15.6 Hz),
7.87–7.90 (1H, m), 7.91 (1H, d, J = 15.6 Hz), 8.85 (1H,
d, J = 7.3 Hz). HRMS (ESI) Calcd for C37H46N11O5S
([M + H]+): 756.3404. Found: 756.3374.
pared
from
(2E)-[1-(8-{[(4-tert-butyl-1,3-thiazol-2-
yl)amino]carbonyl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-
tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-
yl)piperidin-3-ylidene]acetic acid (1.41 g, 2.07 mmol) as
an orange solid. 1H NMR (CDCl3) d: 1.36 (9H, s),
1.89–2.01 (2H, m), 2.14–2.31 (3H, m), 2.24 (6H, s),
2.52 (2H, t, J = 6.3 Hz), 2.99 (2H, s), 3.43–3.58 (2H,
m), 3.71–3.79 (2H, m), 3.77 (3H, s), 5.51 (2H, s), 6.57
(1H, s), 6.65 (1H, br s), 6.89 (2H, d, J = 8.8 Hz), 7.03–
7.11 (1H, m), 7.36 (2H, d, J = 8.8 Hz), 7.47 (1H, dd,
J = 7.3, 2.0 Hz), 7.81–7.90 (3H, m), 8.84–8.91 (1H, m).
HRMS (ESI) Calcd for C38H46N11O4S ([M + H]+):
7523455. Found: 752.3437.
(ii) Following the procedure described for 22, the title
compound (122 mg) was prepared from 1-(8-{[(4-tert-
butyl-1,3-thiazol-2-yl)amino]carbonyl}-3-{(E)-2-[1-(4-
methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyri-
do[1,2-a]pyrimidin-2-yl)piperidin-4-yl[2-(dimethyl-
amino)ethyl]carbamate (495 mg, 0.655 mmol) as an
orange solid. Mp 211–227 ꢁC. IR (ATR) cmꢀ1: 2960,
2862, 1701, 1655, 1630, 1514, 1444, 1421, 1383, 1309,
1
1263, 1220, 1149, 1101, 1030. H NMR (CD3OD) d:
(iii) Following the procedure described for 26, 31
(490 mg) was prepared from N-(4-tert-butyl-1,3-thia-
zol-2-yl)-2-[(3E)-3-(2-{[2-(dimethylamino)ethyl]amino}-
2-oxoethylidene)piperidin-1-yl]-3-{(E)-2-[1-(4-methoxy-
benzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]
pyrimidine-8-carboxamide (1.36 g, 1.81 mmol) as an or-
ange solid. Mp 225–230 ꢁC. IR (ATR) cmꢀ1: 3354, 3334,
2958, 1660, 1624, 1585, 1506, 1462, 1423, 1387, 1333,
1.35 (9H, s), 1.87 (2H, br), 2.07 (2H, br), 3.29 (6H, s),
3.57 (4H, m), 3.73 (2H, m), 3.87 (4H, m), 4.91 (1H, br),
6.70 (1H, s), 7.40 (1H, d, J = 16.1 Hz), 7.64 (1H, dd,
J = 7.3, 1.7 Hz), 7.93 (1H, d, J = 16.1 Hz), 8.04 (1H, s),
8.96 (1H, d, J = 7.3 Hz). Anal. Calcd for C31H39N11O6-
SÆ1.75H2OÆ0.25Et2O: C, 51.67; H,6.10; N,20.71; S, 4.31.
Found: C, 51.50; H, 5.81; N, 20.66; S, 4.31.
1
1296, 1234, 1205, 1103, 1049. H NMR (DMSO-d6) d:
5.3. In vitro potentiation activity
1.31 (9H, s), 2.04 (2H, m), 2.43 (2H, m), 3.16–3.17
(2H, m), 3.24 (2H, s), 3.53–3.55 (2H, m), 3.69 (3H, s),
3.81 (2H, m), 3.92 (2H, m), 4.08 (2H, s), 5.93 (1H, s),
6.87 (1H, s), 7.56 (1H, d, J = 9.1 Hz), 7.82 (1H, d,
J = 15.7 Hz), 8.05 (1H, br), 8.11–8.14 (2H, m), 8.94
(1H, d, J = 7.4 Hz). Anal. Calcd for C32H39N11O5-
SÆ0.1HClÆ0.1CF3CO2HÆ3.5H2O: C, 50.37; H, 6.06; Cl,
0.46; F, 0.74; N, 20.06; S, 4.18. Found: C, 50.42; H,
5.67; Cl, 0.50; F, 0.71; N, 19.83; S, 4.25.
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; now CLSI). Bacteria
were inoculated at 1 · 106 CFU/mL and incubated at
37 ꢁC for 18 h. MICs were determined by visual obser-
vation of growth.
5.4. In vivo efficacy
5.2.19. N-(4-tert-Butyl-1,3-thiazol-2-yl)-2-(4-hydroxypi-
peridin-1-yl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-
5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carbox-
amide (33). Following the procedure described for 11, the
Pulmonary infection of SD rats by P. aeruginosa
PAM1020 was instilled by intratracheal inoculation of
bacteria enmeshed in agar beads. Two hours after bacte-

K. Yoshida et al. / Bioorg. Med. Chem. 15 (2007) 7087–7097
7097
rial challenge, rats were treated with AZT (1000 mg/kg/
2 h) or AZT+ABS-EPI (ABS-EPI were dosed 1.25, 5
and 20 mg/kg/2 h) by intravenous drip infusion.
References and notes
1. Driscoll, J. A.; Brody, S. L.; Kollef, M. H. Drugs 2007, 67, 351.
2. Zhanel, G. G.; Hoban, D. J.; Schurek, K.; Karlowsky,
J. A. Int. J. Antimicrob. Agents 2004, 24, 529.
3. Poole, K. J. Antimicrob. Chemother. 2005, 56, 20.
4. Kumar, A.; Schweizer, H. P. Adv. Drug Delivery Rev.
2005, 57, 1486.
5. Poole, K.; Krebes, K.; McNally, C.; Neshat, S. J.
Bacteriol. 1993, 175, 7363.
6. 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.;
5.5. Pharmacokinetic studies in rats
Male Sprague–Dawley rats (Slc:SD, 6–8 weeks of age,
Japan SLC, Inc., Shizuoka, Japan) were used. Each
group consisted of three animals. ABS-EPI and AZT
were administered by intravenous infusion at doses of
1.25, 5, and 20 mg/kg and 1000 mg/kg in 40 mL/kg
0.0005 N NaOH-saline solution over 2 h. Blood samples
(0.25 mL) were collected at 40, 80, and 120 min after the
beginning of the infusion and at 0.25, 0.5, 1, 2, and 4 h
after the end of the infusion. Blood samples were centri-
fuged (10,000 rpm, 10 min, 4 ꢁC) and the plasma sam-
ples were subsequently stored at ꢀ20 ꢁC until
analyzed. The mixture of the plasma (100 lL) and pro-
pranolol solution (10 lL, 500 ng/mL in H2O, used as
internal standard) was extracted with MeCN (400 lL)
followed by centrifugation (12,000 rpm, 10 min, 4 ꢁC).
The supernatant was evaporated to dryness under nitro-
gen and the dried extract was re-dissolved in MeOH/
H2O (100 lL, 30/70, v/v). The mixture was re-centri-
fuged (12,000 rpm, 10 min, 4 ꢁC) and the supernatant
was analyzed by LC/MS/MS.
´
Leger, R.; Zhang, J. Z.; Lee, V. J.; Watkins, W. J. Bioorg.
Med. Chem. Lett. 2003, 13, 4201.
7. Nakayama, K.; Ishida, Y.; Ohtsuka, M.; Kawato, H.;
Yoshida, K.; Yokomizo, Y.; Ohta, T.; Hoshino, K.; Otani,
T.; Kurosaka, Y.; Yoshida, K.; Ishida, H.; Lee, V. J.;
Renau, T. E.; Watkins, W. J. Bioorg. Med. Chem. Lett.
2003, 13, 4205.
8. Nakayama, K.; Kawato, H.; Watanabe, J.; Ohtsuka, M.;
Yoshida, K.; Yokomizo, Y.; Sakamoto, A.; Kuru, N.;
Ohta, T.; Hoshino, K.; Yoshida, K.; Ishida, H.; Cho, A.;
Palme, M. H.; Zhang, J. Z.; Lee, V. J.; Watkins, W. J.
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Sakamoto, A.; Kawato, H.; Yoshida, K.; Ohta, T.;
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Takemura, M.; Hoshino, K.; Kanda, H.; Namba, K.;
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5.6. Pharmacokinetic studies in monkeys
Female cynomolgus monkeys (4–5 years age, Toyota
Tsusho Co., Tokyo, Japan) were used. Each group con-
sisted of 2–3 animals. ABS-EPI was administered by
intravenous infusion at doses of 1 and 5 mg/kg in
10 mL/kg saline solution over 1 h. Blood samples
(1 mL) were collected at 0.25, 0.5, 0.75, 1, 1.083, 1.25,
1.5, 2, 3, and 5 h after the administration. These analyt-
ical samples were prepared and analyzed according to
the procedure detailed above.
13. Lomovskaya, O.; Lee, A.; Hoshino, K.; Ishida, H.; Mistry,
A.; Warren, M. S.; Boyer, E.; Chamberland, S.; Lee, V. J.
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Acknowledgment
Members of the Research Technology Center, Daiichi
Pharmaceutical Co., Ltd. are gratefully acknowledged
for analytical data.