Quinolone derivatives containing strained spirocycle as orally active glycogen synthase kinase 3β (GSK-3β) inhibitors for type 2 diabetics

Article abstract of DOI:10.1016/j.bmc.2011.12.046

The design, synthesis, and evaluation of 6-6-7 tricyclic quinolones containing the strained spirocycle moiety aiming at the GSK-3β inhibitor were described. Among the synthesized compounds, 44, having a cyclobutane ring on a spirocycle, showed excellent GSK-3β inhibitory activity in both cell-free and cell-based assays (IC₅₀ = 36 nM, EC₅₀ = 3.2 μM, respectively). Additionally, 44 decreased the plasma glucose concentration dose-dependently after an oral glucose tolerance test in mice.

Full text of DOI:10.1016/j.bmc.2011.12.046

Bioorganic & Medicinal Chemistry 20 (2012) 1188–1200
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quinolone derivatives containing strained spirocycle as orally active
glycogen synthase kinase 3b (GSK-3b) inhibitors for type 2 diabetics
Shigeki Seto a, , Kazuhiko Yumoto a, Kyoko Okada a, Yoshikazu Asahina a, Aya Iwane a, Maki Iwago a,
⇑
Reiko Terasawa a, Kevin R. Shreder b, Koji Murakami a, Yasushi Kohno a
a Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd, 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
b ActivX Biosciences, Inc., 11025 N. Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and evaluation of 6-6-7 tricyclic quinolones containing the strained spirocycle moi-
ety aiming at the GSK-3b inhibitor were described. Among the synthesized compounds, 44, having a
cyclobutane ring on a spirocycle, showed excellent GSK-3b inhibitory activity in both cell-free and
Received 11 November 2011
Revised 21 December 2011
Accepted 21 December 2011
Available online 30 December 2011
cell-based assays (IC50 = 36 nM, EC50 = 3.2
cose concentration dose-dependently after an oral glucose tolerance test in mice.
Ó 2011 Elsevier Ltd. All rights reserved.
lM, respectively). Additionally, 44 decreased the plasma glu-
Keywords:
Tricyclic quinolones
Spirocycle
GSK-3b inhibitor
1. Introduction
NH2
O
O
N
N
F
CO2H
Glycogen synthase kinase 3 (GSK-3) is a serine/threonine
protein kinase having two isoforms, GSK-3
H
N
N
H
N
a
and GSK-3b.1 GSK-3
N
H
N
N
H
N
N
H
N
phosphorylates and inactivates glycogen synthase (GS), the late-
limiting enzyme of glycogen formation.2 Insulin has been demon-
strated to cause inactivation of GSK-3, resulting in GS activation
and the subsequent formation of glycogen.3 In type 2 diabetics, li-
ver and muscle glycogen synthesis is decreased, while GSK-3
expression and activity are increased.4 Hence, the identification
of small molecules aimed at GSK-3 inhibitors has become a focus
of research for pharmaceutical companies during the past decade.
For example, CHIR-99021 (1) displayed potent GSK-3 inhibitory
activity and in vivo effects in Zucker diabetic fatty rats.5
NC
Cl
Cl
1 (CHIR99021)
: IC50 = 30 nM
2: IC50 = 12 nM
Figure 1. Structures of known GSK-3b inhibitors.
In a previous communication, we described the discovery of
quinolone derivative 2, which shows not only potent GSK-3b inhib-
itory activity in a cell-free assay (IC50 = 12 nM), but also has selec-
tive inhibitory activity for GSK-3b against 90 kinases (Fig. 1).6 To
further biologically evaluate the series derivatives, we evaluated
the cellular effect of 2 by measuring glycogen synthesis activity in
Hep G2 cells and found that 2 is less effective than 1 (EC50 = 20
and 1.5 lM, respectively) in cell-based assays, perhaps because of
insufficient cell permeability. We therefore focused next on an
exploration of a potent compound in both cell-free and cell-based
assays by structural modification of 2. As an initial step, we tried
to fit together compounds 1 and 2 as shown in Figure 27, and we
⇑
Corresponding author.
E-mail address: shigeki.seto@mb.kyorin-pharm.co.jp (S. Seto).
Figure 2. Structural comparison between 1 and 2. Yellow shows 1 and green shows 2.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.046

S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
1189
6-6-6 tricyclic ring system
6-6-7 tricyclic ring system
(New compounds)
6-6-7 ring system is less active than compound 2 having the 6-6-
6 ring system in the cell-free assay (IC50 = 250 and 12 nM, respec-
tively), whereas both compounds show almost the same activity
NH2
O
NH2
O
N
F
CO2H
F
CO2H
in the cell-based assay (EC50 = 13 and 20 lM, respectively). This re-
H
N
H
N
N
N
sult led us to perform a further optimization study using derivatives
having the 6-6-7 ring system with spirocycles to obtain more cellu-
lar efficacious compounds, as shown in Figure 4. In addition, the
synthesis of the spirocycle containing 6-6-7 tricyclic quinolones
has not been reported as far as we know.
Herein, we report the synthesis and biological evaluation of no-
vel 6-6-7 tricyclic quinolones with the strained spirocycles. Addi-
tionally, we show the in vivo result of a selected compound for
an antidiabetic agent.
N
H
N
N
H
O
O
2
3
IC50 = 12 nM
IC50 = 250 nM
Cell-free
µ
M
µ
M
Cell-based
EC50 = 20
EC50 = 13
Figure 3. Comparison between 6-6-6 and 6-6-7 tricyclic ring system.
NH2
O
F
CO2H
2. Chemistry
H
N
N
N
H
N
X
The synthesis of spirocycles 3, 43–47 is shown in Scheme 1.
Grohe-heiter reaction8,9 was used for the synthesis of the tetracy-
clic intermediates 17–22. Condensation of ketoester 4 with triethyl-
orthoformate, followed by treatment with aminoalcohols 5–9 or
diamine 10, afforded cyclization precursors 11–16. The one-pot
tandem cyclization reactions of 11–16 proceeded well except for
that of 13. Treatment of the precursors 11, 12, 14–16 with sodium
hydride in DMF at 90 °C afforded spirocycles 17, 18, 20–22 in good
to moderate yield. However, in the case of 13, bicyclic compound 24
was the only product that was produced by the loss of cyclopentyl-
ethanol moiety when one-pot tandem cyclization reaction was
(CH2)n
Rb
Ra
Figure 4. Design of 6-6-7 tricyclic quinolones having the strained spirocycle.
hypothesized that the bulkiness of the spirocycle moiety of 2, which
can overlap with the 2,4-dichlorophenyl moiety of 1, could affect
the GSK-3b inhibitory activity and cell permeability. As a prelimin-
ary study, we synthesized 3 having the 6-6-7 tricyclic ring system
to compare with 2 as shown in Figure 3. Compound 3 having the
O
O
O
F
F
CO2Et
NH
F
F
CO2Et
F
F
CO2Et
a
b
F
N
F
NH
F
Ra
X
F
Ra
(CH2)n
Ra
(CH2)n
Rb
4
(CH2)n
Rb
Rb
HX
HX
11 16
17 22
-
-
5 10
-
23
13
)
19
23
from )
c (for
from
d (for
O
O
F
F
CO2Et
F
CO2Et
N
F
N
H
F
HO
F
24
23
NH2
O
N
NH2
O
N
NO2
O
N
F
F
CO2H
F
F
CO2Et
g or h
F
F
CO2Et
e
f
X
X
X
(CH2)n
Ra
(CH2)n
Ra
(CH2)n
Ra
Rb
Rb
Rb
25 30
-
31 36
-
37 42
-
NH2
O
F
CO2H
i
5,11,17,25,31,37,43: n = 1, Ra = Rb = H, X = O
6,12,18,26,32,38,44: n = 2, Ra = Rb = H, X = O
7,13,19,27,33,39,3: n = 3, Ra = Rb = H, X = O
H
N
N
N
H
N
X
(CH2)n
Ra
,
,
,
,
,
,
: n = 2, Ra = Me, Rb = H,
8 14 20 28 34 40 45
Rb
X = O
9 15 21 29 35 41 46
,
,
,
,
,
,
: n = 2, Ra = H, Rb = Me,
3, 43 47
-
X = O
10,16,22: n = 2, Ra = Rb = H, X = NBoc
30,36,42,47: n = 2, Ra = Rb = H, X = NH
Scheme 1. Reagent and conditions: (a) (1) HC(OEt)3, AcO, 120 °C, 3 h. (2) 5–10, rt, 2 h, 68% (for 11), 61% (for 12), 75% (for 13), 67% (for 14), 83% (for 15), 93% (for 16); (b) NaH,
DMF, rt, 1 h, then 90 °C, 1 or 0.5 h, 61% (for 17), 26% (for 18), 80% (for 20), 67% (for 21), 68% (for 22); (c) NaH, DMF, rt, 1 h, 65% (d) NaH, DMF, 80 °C, 0.5 h, 31%; (e) KNO3, H2SO4,
0 °C, 1 h, 41% (for 25), 79% (for 26), 46% (for 27), 82% (for 28), 93% (for 29), 68% (for 30); (f) 10% Pd-C, H2, DMF, 50 °C, 1.5 h, 77% (for 31), 79% (for 32), 28% (for 33), 91% (for 34),
79% (for 35), 88% (for 36); (g) H2SO4, AcOH, H2O, 100 °C, 1 h, 93% (for 37), 90% (for 38), 96% (for 39), 98% (for 40), 99% (for 41); (h) NaOH, EtOH, 50 °C, 3 h, 83%; (i) N-2-
pyridinyl-1,2-ethanediamine, Et3N, DMSO, 100 °C, 2 h, 47% (for 43), 66% (for 44), 69% (for 3), 44% (for 45), 59% (for 46), 64% (for 47).

1190
S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
(CH2)n
(CH2)n
R3
CO2Et
(CH2)n
R3
CO2Et
As the spirocycles 17–22 were in hand, the nitration and subse-
a
b
quent hydrogenation of the nitrogroup and the hydrolysis of the
ester group afforded carboxylic acid 37–42. Finally, the nucleo-
philic displacement reaction of 37–42 with N-2-pyridinyl-1,2-eth-
anediamine afforded the target compounds 3, 43–47.
HO
HN
Ph
O
O
48: n = 2
50: n = 2, R3 = H
53: n = 2, R3 = H
: n = 3, R3 = H
: n = 3, R3 = H
49
: n = 3
51
52
54
55
: n = 2, R3 = Me
: n = 2, R3 = Me
Scheme 2 shows the preparation of the intermediate aminoal-
cohols 6–9 and N-Boc-protected diamine 10. Reformatsky reac-
(CH2)n
R3
OH
(CH2)n
R3
OH
c
d
tion10 of ketones 48 and 49 with
a-bromoacetates provided
HN
H2N
b-hydroxy esters 50–52. Subsequent Ritter reaction11 of 50–52
with cyanobenzene gave N-benzoyl-3-amino acid esters 53–55.
Reduction of 53–55 with LiAlH4, followed by hydrogenation with
Pd–C, afforded corresponding aminoalcohols 6–8.
Ph
56: n = 2, R3 = H
57
58
6: n = 2, R3 = H
: n = 3, R3 = H
: n = 3, R3 = H
7
8
: n = 2, R3 = Me
: n = 2, R3 = Me
The aminoalcohol 8 was prepared in four steps from the interme-
diate 53. The methyl group was elongated via the reduction of 53
using DIBAL-H and subsequent methylation of aldehyde 59 with
methyl magnesium chloride. Treatment of 60 in the same procedure
with the synthesis of 6–8 afforded the desired aminoalcohol 9.
The intermediate 53 was also used to prepare N-Boc-protected
diamine 10. N-benzyl-protected diamine 64 was formed in three
steps via conversion to amide 63 though carboxylic acid 62, fol-
lowed by the reduction of two amide groups of 63. Finally, N-Boc
protection of 64 and subsequent debenzylation afforded N-Boc-
protected diamine 10.
e
i
f
j
g
h
53
HN
HN
Ph
HN
H2N
Me
Me
OH
CHO
Ph
O
O
Ph Me
OH
OH
59
60
61
9
k
53
HN
Ph
HN
Ph
HN
Ph
CO2H
CONH2
O
NH2
O
62
63
64
l
m
HN
Ph
H2N
3. Results and discussion
NHBoc
NHBoc
65
10
3.1. GSK-3b inhibitory activity in cell-free and cell-based assays
Scheme 2. Reagent and conditions: (a) BrCH2CO2Et or BrCH(Me)CO2Et, Zn, Et2O, rt,
1 h, 54% (for 50), 99% (for 51), 100% (for 52); (b) PhCN, c-H2SO4, 80 °C, 1 h, 51% (for 53),
42% (for 54), 46% (for 55); (c) LiAlH4, THF, reflux, 5 h, 99% (for 56), 91% (for 57), 96% (for
58); (d) 10% Pd–C, H2, EtOH, 5 kgf/cm2, rt, 5 h, 73% (for 6), 77% (for 7), 74% (for 8); (e)
DIBAL-H, THF, ꢀ78 °C, 0.5 h, 32%; (f) MeMgCl, THF, rt, 5 h, 87%; (g) LiAlH4, THF, reflux,
0.5 h, 91%; (h) 10% Pd-C, H2, EtOH, 5 kgf/cm2, rt, 5 h, 94%; (i) 3 N KOH, EtOH, rt, 1 h,
91%; (j) (1) SOCl2, rt, 1 h. (2) 30% aq NH3, THF, rt, 1 h, 93%; (k) LiAlH4, THF, reflux, 5 h,
99%; (l) Boc2O, 0 °C, 0.5 h, 54%; (m) 10% Pd-C, H2, EtOH, 5 kgf/cm2, rt, 5 h, 92%.
Table 1 shows the results of the effects of newly synthesized
compounds 3, 43–47 on cell-free GSK-3b kinase assay using re-
combinant full-length human GSK-3b and a cell-based assay using
Hep G2 cells, together with the control compound 1. A cell-free
GSK-3b kinase assay detects inhibitors that interact directly with
the polypeptide GSK-3b, while a cell-based assay can detect the
ability on glycogen synthesis. Table 1 also shows anti-microbial
activity to select a compound with minimal anti-bacterial activity
that may not be desirable for GSK-3b inhibitors.6
attempted. To diminish the yield of 24, we tried a stepwise proce-
dure. The intermediate 23 was isolated after the first cyclization
reaction of 13 and then treated with sodium hydride in DMF at
80 °C in a short time to afford 19 with a minimum amount of
undesired 24.
To examine the optimal size of the ring connected with a tricy-
clic core, we examined the potency in terms of cell-free and
cell-based assays of compounds 3, 43, 44. Compared to compound
Table 1
Spirocycles SAR
NH2
O
F
CO2H
H
N
N
N
H
N
R1
R2
X
R4
R3
Compd
R1
R2
R3
R4
X
GSK-3b
Cell-based assay
Anti-bacterial activity: MIC (lM)
Cell-free assay
IC50 (nM)
a
EC50
(lM)
Efficacyb (%)
E. coli
S. aureus
3
44
43
45
46
47
–CH2CH2CH2CH2–
–CH2CH2CH2–
–CH2CH2–
–CH2CH2CH2–
–CH2CH2CH2–
–CH2CH2CH2–
H
H
H
Me
H
H
H
H
H
Me
H
O
O
O
O
O
NH
250
36
37
13
85
65
42
48
94
12
100
>34
>34
9
>34
18
5
3.2
c
—
10
1.4
2.1
>34
>34
>140
>34
>34
>140
250
550
30
H
d
d
CHIR99021 (1)
1.5
—
—
a
b
c
EC50 values were calculated when the efficacy showed more than 50. EC50 values were the molar concentration of the test compounds that cause 50% of the efficacy. n = 3.
Efficacy values were calculated as percentage of 1 (CHIR99021) at the dosage of 30 lM.
Not certain.
Not tested.
d

S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
1191
3 with a cyclopentane ring, compounds 44 and 43, having a cyclo-
Control
50 mg/kg
100 mg/kg
150 mg/kg
300 mg/kg
700
600
500
400
300
200
100
0
butane ring and a cyclopropane ring, respectively, showed potent
activity in the cell-free assay (3, IC50 = 250 nM; 44, IC50 = 36 nM;
43, IC50 = 37 nM). As expected, in the cell-based assay, 44 showed
more potent activity than 3.
*
*
*
*
*
Next, we investigated the influence of the substitution of a
methyl group on the seven-membered ring using compounds 45
and 46, and the replacement of oxygen with nitrogen using 47.
Compared to the parent compound 44, one regioisomer 45 showed
2- to 3-fold improved potency in both the cell-free and cell-based
assays, while another regioisomer, 46, exhibited weak potency in
the cell-free assay. The replacement of oxygen with nitrogen was
also disfavored, as demonstrated by comparing 44 and 47.
With regard to antibacterial activity, the ring size of the spiro-
cycle seems to be important. Compound 43, having a cyclopropane
*
*
*
*
- 60
- 30
0
30
60
90
120
150
Time (min)
Figure 5. OGTT results. ICR mice were fasted overnight and treated with vehicle
control or the indicated dosage of compound 44, 30 min prior to the oral glucose
tolerance test, using 5 g/kg body wt glucose feeding. Data are shown as means S.E.
ring, showed antibacterial activity with MIC values of 9
lM and
5
l
M (Escherichia coli and Staphylococcus aureus, respectively). In
contrast, other derivatives that had larger ring sizes than the cyclo-
and
R
Glucose30–60 inhibition %. n = 5-6. ⁄p <0.05 versus control.
propane ring showed no antibacterial activity.
Thus, we selected the cyclobutane derivatives 44 (cell-free,
pounds, 44, having a cyclobutane ring on the spirocycle, showed
excellent GSK-3b inhibitory activity in both cell-free and cell-based
assays. Additionally, 44 showed a great reducing effect of plasma
glucose concentration dose-dependently after the oral glucose tol-
erance test (OGTT). Further biological data on 44 will be reported
elsewhere.
IC50 = 36 nM; cell-based, EC50 = 3.2
lM) and 45 (cell-free, IC50 =
10 nM; cell-based, EC50 = 1.4 M) for further evaluation of their
l
pharmacokinetic and pharmacological properties.
3.2. Pharmacokinetic and pharmacological study
The pharmacokinetic (PK) characteristics of 44, 45, and 1 at a
dose of 300 mg/kg in ICR mice are shown in Table 2. The area under
the curve (AUC) of 44 was 32-fold greater in liver than in plasma
5. Experimental section
5.1. Chemistry
(760 and 24
l
M h, respectively). A similar ratio was observed in
M h, respectively),
the liver and plasma AUC of 45 (217 and 6.8
l
although these values were 3.5-fold smaller in 45 than in 44. In
contrast, 1 showed very large AUC in plasma, but the hepatic up-
take of 1 was extremely low, 3.8 of the liver/plasma concentration
ratio. Consequently, the AUC in liver of both 44 and 1 were almost
Melting points were determined with a Yamato MP-500 melt-
ing point apparatus and are uncorrected. 1H NMR spectra were
measured in CDCl3 or DMSO-d6 with TMS and the solvent peak
as internal standards, on a JEOL ECA-400 (400 MHz) spectrometer.
Mass spectra (MS) were obtained on a Hitachi M-2000 mass spec-
trometer. Column chromatography was carried out on Merck silica
gel 60. Analytical thin-layer chromatography (TLC) was performed
on Merck precoated silica gel 60F254 plates, and the compounds
were visualized by UV illumination (254 nm) or by heating after
spraying with phosphomolybdic acid in ethanol. The data for ele-
mental analysis were within 0.4% of the theoretical values and
were determined by a Yanaco CHN corder MT-5.
the same (760 and 820 lM h, respectively).
Table 2 also shows the reducing effects on plasma glucose con-
centration after the oral glucose tolerance test (OGTT) at a dose of
300 mg/kg in ICR mice. Interestingly, the inhibition of the plasma
glucose concentration was accompanied by the increase in liver
AUC. Both 44 and 1 displayed very high liver AUC, and they also
showed significantly lower plasma glucose concentrations than
in control animals after the OGTT (31% and 38%, respectively).
Figure 5 shows the dose effects of 44 on OGTT. Compound 44
reduced the plasma glucose level dose-dependently by 20%, 32%,
39%, and 38% when 44 was treated at doses of 50, 100, 150, and
300 mg, respectively.
5.1.1. Ethyl 3-{[1-(2-hydroxyethyl)cyclopropyl]amino}-2-(2,3,4,
5,-tetrafluorobenzoyl)acrylate (11)
A solution of 4 (3.09 g, 11.7 mmol), Ac2O (6.7 mL, 70.9 mmol),
and triethyl orthoformate (3.90 mL, 23.5 mmol) was heated at
120 °C for 3 h. The mixture was concentrated in vacuo and dried
under high vacuum. The crude product was dissolved in anhydrous
toluene (40 mL), and 512 (1.82 g, 11.7 mmol) was added very
slowly at 0 °C. The reaction mixture was stirred at room tempera-
ture for 5 h and diluted with toluene. The organic layer was
4. Conclusions
We successfully designed and synthesized a series of novel 6-6-
7 tricyclic quinolones containing the strained spirocycle moiety
aiming at the GSK-3b inhibitor. Among the synthesized com-
Table 2
PK profile and in vivo screening
Compd
PK profile poa
OGTTa
AUC0–25 h
(l
M h)
Liver/plasma concentration ratiob
Liver AUCc
(l
M h)
Glucose inhibition (%)
44
45
24
6.8
32
32
760
217
31
13
1 (CHIR99021)
210
3.8
820
38
a
b
c
ICR mice were treated with 300 mg/kg of the tested compound. Data are shown as means. n = 5–6.
Time point: 4 h.
Liver AUC was calculated based on AUC in plasma and the concentration ratio of liver to plasma at 4 h after po administration.

1192
S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
washed with water and brine, and then was dried. The solvent was
removed by evaporation, and the crude material was purified by
flash column chromatography on silica gel (hexane:EtOAc = 2:1
to 1:1) to give 11 as a yellow solid (2.99 g, 68%).
(2H, m), 4.02 (0.2H, q, J = 7.3 Hz), 4.07 (0.8H, q, J = 7.3 Hz), 6.97–
7.13 (1H, m), 8.24 (0.8H, d, J = 15.3 Hz), 8.25 (0.2H, d, J = 15.3 Hz),
10.03 (0.2H, d, J = 14.1 Hz), 11.40 (0.8H, d, J = 14.1 Hz). HRMS
(ESI+) for C19H22F4NO4 [M+H]+: calcd, 404.14850; found,
404.14862. Anal. calcd for C19H21F4NO4: C, 56.57; H, 5.25; N, 3.47.
Found: C, 56.29; H, 5.12; N, 3.41.
Mp: 75–78 °C. 1H NMR (400 MHz, CDCl3)d 0.85–0.92 (2H, m),
0.94–1.11 (5H, m), 1.85–1.91 (2H, m), 3.85–3.89 (2H, m), 3.99–
4.09 (2H, m), 6.95–7.13 (1H, m), 8.19 (1H, d, J = 14.1 Hz), 9.84
and 11.2 (total 1H, each d, J = 13.4 Hz). HRMS (EI) for C17H17F4NO4
(M+): calcd, 375.1094; found, 375.1094.
5.1.5. Ethyl 3-{[1-(2- hydroxypropane-1-yl)cyclobutyl]amino}-
2-(2,3,4,5-tetrafluorobenzoyl)acrylate (15)
A stirred solution of 4 (2.45 g, 9.27 mmol), Ac2O (5.30 mL,
56.1 mmol), and triethyl orthoformate (3.10 mL, 18.6 mmol) was
heated at 120 °C for 3 h. The mixture was concentrated in vacuo
and dried under high vacuum. To the mixture of the residue in anhy-
drous toluene (30 mL), was added 9 (1.20 g, 9.29 mmol) in anhydrous
toluene (10 mL) very slowly at 0 °C and stirred at room temperature
for 2 h. The solvent was removed by evaporation. The crude material
was purified by flash column chromatography on silica gel
(AcOEt:hexane = 1:1) to give 15 as a pale yellow solid (3.10 g, 83%).
Mp: 145–146 °C. 1H NMR (400 MHz, CDCl3) d 0.95 (0.6H, t,
J = 7.3 Hz), 1.09 (2.4H, t, J = 7.3 Hz), 1.29 (3H, d, J = 6.1 Hz), 1.51
(1H, br s), 1.83–2.05 (4H, m), 2.18–2.35 (3H, m), 2.41–2.53 (1H,
m), 3.95–4.15 (3H, m), 6.96–7.13 (1H, m), 8.27 (0.8H, d,
J = 14.7 Hz), 8.31 (0.2H, d, J = 14.7 Hz), 10.30 (0.2H, d, J = 13.4 Hz),
11.56 (0.8H, d, J = 13.4 Hz). HRMS (ESI+) for C19H22F4NO4 [M+H]+:
calcd, 404.14850; found, 404.14759. Anal. calcd for C19H21F4NO4:
C, 56.57; H, 5.25; N, 3.47. Found: C, 56.36; H, 5.04; N, 3.38.
5.1.2. Ethyl 3-{[1-(2-hydroxyethyl)cyclobutyl]amino}-2-(2,3,4,5-
tetrafluorobenzoyl)acrylate (12)
A stirred solution of 4 (3.67 g, 13.9 mmol), Ac2O (7.89 mL,
83.5 mmol), and triethyl orthoformate (4.63 mL, 27.8 mmol) was
heated at 120 °C for 3 h. The mixture was concentrated in vacuo
and dried under high vacuum. To the mixture of the residue in
anhydrous toluene (50 mL), was added 6 (1.60 g, 13.9 mmol) in
anhydrous toluene (20 mL) very slowly at 0 °C and stirred at room
temperature for 2 h. The solvent was removed by evaporation. The
crude material was purified by flash column chromatography on
silica gel (AcOEt:hexane = 1:1) to give 12 as a pale yellow solid
(3.30 g, 61%).
Mp: 90–93 °C. 1H NMR (400 MHz, CDCl3) d 0.96 (0.6H, t,
J = 7.3 Hz), 1.09 (2.4H, t, J = 7.3 Hz), 1.85–2.03 (12H, m), 2.06–
2.13 (2H, m), 2.20–2.30 (2H, m), 2.32–2.43 (2H, m), 3.82–3.88
(2H, m), 4.02 (0.4H, q, J = 7.3 Hz), 4.07 (1.6H, q, J = 7.3 Hz), 6.94–
7.03 (0.2H, m), 7.05–7.13 (0.8H, m), 8.23–8.30 (1H, m), 10.05–
10.17 (0.2H, m), 11.36–11.50 (0.8H, m). HRMS (EI) for C18H19F4NO4
(M+): calcd, 389.1250; found, 389.1291.
5.1.6. Ethyl 3-{[1-(2-(tert-butoxycarbonylamino)ethyl)cyclobu
tyl]amino}-2-(2,3,4,5-tetrafluorobenzoyl)acrylate (16)
A stirred solution of 4 (5.28 g, 20.0 mmol), Ac2O (11.4 mL,
0.121 mol), and triethyl orthoformate (6.66 mL, 40.0 mmol) was
heated at 120 °C for 3 h. The mixture was concentrated in vacuo
and dried under high vacuum. To the mixture of the residue in
anhydrous toluene (70 mL), was added 10 (4.29 g, 20.0 mmol) in
anhydrous toluene (30 mL) very slowly at 0 °C and stirred at room
temperature for 1 h. The solvent was removed by evaporation. The
crude material was purified by flash column chromatography on
silica gel (AcOEt:hexane = 2:1) to give 16 as a pale yellow oil
(9.10 g, 93%).
5.1.3. Ethyl 3-{[1-(2-hydroxyethyl)cyclopentyl]amino}-2-(2,3,4,
5-tetrafluorobenzoyl)acrylate (13)
A stirred solution of 4 (20.3 g, 76.8 mmol), Ac2O (44.0 mL,
0.465 mol), and triethyl orthoformate (25.6 mL, 0.154 mol) was
heated at 120 °C for 3 h. The mixture was concentrated in vacuo
and dried under high vacuum. To the mixture of the residue in
anhydrous toluene (200 mL), was added 7 (9.94 g, 76.9 mmol) in
anhydrous toluene (50 mL) very slowly at 0 °C and stirred at room
temperature for 2 h. The solvent was removed by evaporation. The
crude material was purified by flash column chromatography on
silica gel (AcOEt:hexane = 2:1) to give 13 as a colorless solid
(23.2 g, 75%).
1H NMR (400 MHz, CDCl3) d 0.98 (0.7H, t, J = 7.3 Hz), 1.10 (2.3H,
t, J = 7.3 Hz), 1.43 (9H, s), 1.90–2.07 (4H, m), 2.17–2.38 (4H, m),
3.12–3.23 (2H, m), 4.00–4.15 (2H, m), 4.53–4.65 (1H, br), 6.95–
7.03 (0.7H, m), 7.05–7.13 (0.3H, m), 8.13 (1H, d, J = 14.1 Hz), 9.88
(0.3H, d, J = 13.5 Hz), 11.29 (0.7H, d, J = 13.5 Hz). HRMS (ESI+) for
Mp: 67–70 °C. 1H NMR (400 MHz, CDCl3) d 0.96 (0.6H, t,
J = 7.3 Hz), 1.09 (2.4H, t, J = 7.3 Hz), 1.48–1.54 (1H, m), 1.74–1.90
(6H, m), 1.97–2.06 (4H, m), 3.78–3.85 (2H, m), 3.98–4.10 (2H,
m), 6.95–7.12 (1H, m), 8.17–8.24 (1H, m), 9.81–9.94 (0.3H, m),
11.20–11.35 (0.7H, m). HRMS (ESI+) for C19H22F4NO4 [M+H]+: calcd,
404.14850; found, 404.14870. Anal. calcd for C19H21F4NO4 0.2H2O:
C, 56.07; H, 5.20; N, 3.44. Found: C, 55.92; H, 5.05; N, 3.37.
C
18H19F4NO4 [M+H]+: calcd, 489.20126; found, 489.20200.
5.1.7. Ethyl 100,110-difluoro-20,30-dihydro-80-oxo-spiro[cyclopro
pane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-70-carbox
ylate (17)
A solution of NaH (617 mg, 15.4 mmol, 60% in oil) in DMF
(30 mL) was cooled to 0 °C and treated dropwise with 11 (2.63 g,
7.01 mmol) in DMF (4 mL). The reaction mixture was stirred at
room temperature for 1 h and for an additional hour at 80 °C.
The reaction mixture was poured into ice water, and the resulting
precipitate was removed by filtration and washed with water. The
resulting solid was dissolved in 100 mL of EtOH and filtered. The
filtrate was concentrated to 50 mL, and the resulting precipitate
was removed by filtration, washed with EtOH, and then dried to
give 17 as a pale brown solid (1.43 g, 61%).
5.1.4. Ethyl 3-{[1-(1- hydroxypropane-2-yl)cyclobutyl]amino}-
2-(2,3,4,5-tetrafluorobenzoyl)acrylate (14)
A stirred solution of 4 (6.13 g, 23.2 mmol), Ac2O (13.2 mL,
0.140 mol), and triethyl orthoformate (7.80 mL, 46.8 mmol) was
heated at 120 °C for 3 h. The mixture was concentrated in vacuo
and dried under high vacuum. To the mixture of the residue in
anhydrous toluene (100 mL), was added 8 (3.00 g, 23.2 mmol) in
anhydrous toluene (40 mL) very slowly at 0 °C and stirred at room
temperature for 2 h. The solvent was removed by evaporation. The
crude material was purified by flash column chromatography on
silica gel (AcOEt:hexane = 1:1) to give 14 as a colorless solid
(6.23 g, 67%).
Mp: 236–238 °C. 1H NMR (400 MHz, DMSO-d6)d 1.03 (2H, br),
1.18 (2H, br), 1.26 (3H, t, J = 7.3 Hz), 2.03–2.73 (2H, br), 4.21 (2H,
q, J = 7.3 Hz), 7.66 (1H, dd, J = 10.4, 7.9 Hz), 8.51 (1H, s). HRMS
(EI) for C17H15F2NO4 (M+): calcd, 335.0969; found, 335.0989. Anal
calcd for C17H15F2NO4: C, 60.89; H, 4.51; N, 4.18; found: C, 60.83;
H, 4.53; N, 4.16.
Mp: 105–106 °C. 1H NMR (400 MHz, CDCl3) d 0.95 (0.6H, t,
J = 7.3 Hz), 0.97 (3H, d, J = 7.3 Hz), 1.09 (2.4H, t, J = 7.3 Hz), 1.54
(1H, t, J = 4.9 Hz), 1.85–2.15 (3H, m), 2.19–2.50 (4H, m), 3.62–3.75

S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
1193
5.1.8. Ethyl 100,110-difluoro-20,30-dihydro-80-oxo-spiro[cyclobut
ane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-70-carbox
ylate (18)
To an ice-cold solution of 12 (2.80 g, 7.19 mmol) in DMF
(30 mL) was added NaH (350 mg, 8.75 mmol) and the mixture
was stirred at room temperature for 0.5 h and the mixture was
heated at 80 °C for 0.5 h. The mixture was treated portionwise at
0 °C with water and the resulting precipitate was combined by fil-
tration, washed successively with water and then dried to give 18
as a colorless solid (655 mg, 26%).
was combined by filtration, washed successively with water and
diisopropyl ether and then dried to give 22 as a pale yellow solid
(312 mg, 68%).
Mp: 237–238 °C. 1H NMR1H NMR (400 MHz, DMSO-d6) d 1.28
(3H, t, J = 7.3 Hz), 1.42 (5H, s), 1.54 (4H, s), 1.67–1.80 (1H, m),
1.80–1.95 (1H, m), 2.10–2.22 (1H, m), 2.22–2.37 (2H, m), 2.40–
2.56 (1H, m), 2.65–2.80 (2H, m), 4.17–4.37 (3H, m), 7.93–8.01
(1H, m), 8.47 (0.44H, s), 8.51 (0.56H, s). HRMS (ESI+) for
C
23H27F2N2O5 [M+H]+: calcd, 449.18880; found, 449.18905. Anal.
calcd for C23H26F2N2O5: C, 61.60; H, 5.84; N, 6.25. Found: C,
61.61; H, 5.93; N, 6.21.
Mp: 224–226 °C. 1H NMR (400 MHz, DMSO-d6) d 1.27 (3H, t,
J = 7.3 Hz), 1.67–1.79 (1H, m), 1.91–2.04 (1H, m), 2.42–2.53 (4H,
m), 2.67 (2H, t, J = 6.7 Hz), 4.24 (2H, q, J = 7.3 Hz), 4.55 (2H, t,
J = 6.7 Hz), 7.72 (1H, dd, J = 10.4 and 7.9 Hz), 8.41 (1H, s). HRMS
(ESI+) for C18H18F2NO4 [M+H]+: calcd, 350.12039; found,
350.12110. Anal. calcd for C18H17F2NO4: C, 61.89; H, 4.91; N,
4.01. Found: C, 61.77; H, 4.94; N, 3.90.
5.1.12. Ethyl 1,4-dihydro-1-[1-(2-hydroxyethyl)cyclopropyl]-4-
oxo-6,7,8-trifluoro-3-quinolinecarboxylate (23)
To an ice-cold solution of 13 (22.7 g, 56.3 mmol) in THF
(200 mL) was added NaH (3.60 g, 90.0 mmol). The mixture was
stirred at room temperature for 1 h. The mixture was treated por-
tionwise at 0 °C with water. The resulting mixture was extracted
with ethyl acetate. The combined extracts were concentrated in
vacuo. The crude material was purified by flash column chroma-
tography on silica gel (AcOEt:hexane = 2:1) to give 23 as a colorless
solid (14.1 g, 65%).
5.1.9. Ethyl 100,110-difluoro-20,30-dihydro-30-methyl-80-oxo-spir
o[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylate (20)
To an ice-cold solution of 14 (404 mg, 1.00 mmol) in DMF
(4 mL) was added NaH (40 mg). After stirring at 0 °C for 1 h, the
mixture was added NaH (40 mg). The mixture was stirred at 0 °C
for 1 h, at room temperature for 1 h, at 70 °C for 1 h. The mixture
was treated portionwise at 0 °C with water and the resulting pre-
cipitate was combined by filtration, washed successively with
water and ethyl acetate, and then dried to give 20 as a pale yellow
solid (289 mg, 80%).
Mp: 230–233 °C. 1H NMR (400 MHz, DMSO-d6) d 1.27 (3H, t,
J = 7.3 Hz), 1.53–1.75 (4H, m), 2.10–2.26 (4H, m), 2.31–2.44 (2H,
m), 4.23 (2H, q, J = 7.3 Hz), 4.55–4.60 (1H, m), 7.99–8.07 (1H, m),
8.80 (1H, s). HRMS (ESI+) for C19H21F3NO4 [M+H]+: calcd,
384.14227; found, 384.14170. Anal. calcd for
C19H20F3NO4
0.1H2O: C, 59.25; H, 5.23; N, 3.64. Found: C, 59.17; H, 5.20; N, 3.61.
5.1.13. Ethyl 100,110-difluoro-20,30-dihydro-80-oxo-spiro[cyclo
pentane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-70-
carboxylate (19)
To a solution of 23 (3.84 g, 10.0 mmol) in DMF (40 mL) was added
NaH (480 mg, 12.0 mmol), and the mixture was heated at 80 °C for
0.5 h. The mixture was treated portionwise at 0 °C with water. The
resulting mixture was extracted with ethyl acetate. The combined
extracts were concentrated in vacuo. The crude material was puri-
fied by flash column chromatography on silica gel (AcOEt:hex-
ane = 2:1) to give 19 as a colorless solid (1.12 g, 31%).
Mp: 225–228 °C. 1H NMR (400 MHz, DMSO-d6) d 0.93 (3H, d,
J = 6.1 Hz), 1.27 (3H, t, J = 7.3 Hz), 1.66–1.77 (1H, m), 1.85–2.00
(1H, m), 2.35 (1H, q, J = 10.4 Hz), 2.53–2.69 (3H, m), 2.96–3.05
(1H, m), 3.82 (1H, t, J = 11.6 Hz), 4.24 (2H, qd, J = 7.3 and 2.4 Hz),
4.77 (1H, dd, J = 12.9 and 8.9 Hz), 7.73 (1H, dd, J = 10.4 and
8.6 Hz), 8.25 (1H, s). HRMS (ESI+) for C19H20F2NO4 [M+H]+: calcd,
364.13604; found, 364.13681. Anal. calcd for C19H19F2NO4 0.8
H2O: C, 60.41; H, 5.07; N, 3.71. Found: C, 60.16; H, 5.11; N, 3.69.
5.1.10. Ethyl 100,110-difluoro-20,30-dihydro-20-methyl-80-oxo-spi
ro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]
-70-carboxylate (21)
To an ice-cold solution of 15 (404 mg, 1.00 mmol) in DMF
(4 mL) was added NaH (40 mg). After stirring at 0 °C for 1 h, the
mixture was added NaH (40 mg). The mixture was stirred at 0 °C
for 1 h, at room temperature for 1 h, at 80 °C for 1 h. The mixture
was treated portionwise at 0 °C with water and the resulting pre-
cipitate was combined by filtration, washed successively with
water and iPr2O, and then dried to give 21 as a colorless solid
(244 mg, 67%).
Mp: 185–187 °C. 1H NMR (400 MHz, DMSO-d6) d 1.42 (3H, t,
J = 7.3 Hz), 1.52–1.64 (2H, m), 1.75–1.88 (2H, m), 2.11–2.21 (2H,
m), 2.27–2.37 (2H, m), 2.47 (2H, t, J = 6.7 Hz), 4.39 (2H, q,
J = 7.3 Hz), 4.47 (2H, t, J = 6.7 Hz), 7.98 (1H, dd, J = 9.8 and
7.9 Hz), 8.67 (1H, s). HRMS (ESI+) for C19H20F2NO4 [M+H]+: calcd,
364.13604; found, 364.13274. Anal. calcd for C19H19F2NO4: C,
62.80; H, 5.27; N, 3.85. Found: C, 62.52; H, 5.23; N, 3.83.
5.1.14. Ethyl 100,110-difluoro-20,30-dihydro-90-nitro-80-oxo-spiro
[cyclopropane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylate (25)
Mp: 211–213 °C. 1H NMR (400 MHz, DMSO-d6) d 1.27 (3H, t,
J = 7.3 Hz), 1.43 (3H, d, J = 6.1 Hz), 1.72 (1H, q, J = 10.4 Hz), 1.90–
2.04 (1H, m), 2.24 (1H, q, J = 10.4 Hz), 2.34–2.43 (1H, m), 2.53–
2.74 (4H, m), 4.24 (2H, q, J = 7.3 Hz), 4.48–4.58 (1H, m), 7.72 (1H,
qd, J = 10.4 and 7.9 Hz), 8.40 (1H, s). HRMS (ESI+) for C19H20F2NO4
[M+H]+: calcd, 364.13604; found, 364.13588. Anal. calcd for
A solution of 17 (1.2 g, 3.58 mmol) in concentrated H2SO4
(15 mL) was treated portionwise at 0 °C with solid KNO3
(510 mg, 5.04 mmol). After stirring at 0 °C for 2 h, the reaction mix-
ture was poured into ice-water and the resulting precipitate was
removed by filtration. The resulting solid was washed with EtOH
and dried to give 25 as a brown solid (554 mg, 41%).
Mp: 310–313 °C. 1H NMR (400 MHz, DMSO-d6)d 1.02 (2H, br),
1.10–1.27 (5H, m), 1.91–2.86 (2H, br), 4.21 (2H, q, J = 7.3 Hz), 4.61
(2H, br), 4.52 (2H, br), 8.58 (1H, s). HRMS (EI) for C17H14F2N2O6
(M+): calcd, 380.0820; found, 380.0856. Anal calcd for C17H14F2N2O6:
C, 53.69; H, 3.71; N, 7.37; found: C, 53.58; H, 3.57; N, 7.36.
C19H19F2NO4 0.3H2O: C, 61.88; H, 5.19; N, 3.80. Found: C, 61.87;
H, 5.11; N, 3.85.
5.1.11. Ethyl 10-(tert-butoxycarbonyl)-100,110-difluoro-20,30-
dihydro-80-oxo-spiro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef
][1,4]benzodiazepine]-70-carboxylate (22)
To an ice-cold solution of 16 (500 mg, 1.02 mmol) in DMF
(5 mL) was added NaH (82.0 mg, 2.05 mmol) under iced-water
cooling and the mixture was stirred on iced-water bath for
30 min and then at room temperature for 2 h. The mixture was
treated portionwise at 0 °C with water and the resulting precipitate
5.1.15. Ethyl 100,110-difluoro-20,30-dihydro-90-nitro-80-oxo-spiro
[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylate (26)
A solution of 18 (925 mg, 2.65 mmol) in concentrated H2SO4
(11 mL) was treated portionwise at 0 °C with solid KNO3

1194
S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
(363 mg, 3.59 mmol). After stirring at 0 °C for 2 h, the reaction mix-
ture was poured into ice-water and the resulting precipitate was
combined by filtration, washed with water. The crude material
was purified by flash column chromatography on silica gel (CH2Cl2:
MeOH = 10: 1) to give 26 as a yellow solid (822 mg, 79%).
5.1.19. Ethyl 100,110-difluoro-20,30-dihydro-90-nitro-80-oxo-spiro
[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzodiazepine]-
70-carboxylate (30)
A solution of 22 (5.50 g, 12.3 mmol) in concentrated H2SO4
(60 mL) was treated portionwise at 0 °C with solid KNO3 (1.37 g,
13.6 mmol). After stirring at 0 °C for 1 h, the reaction mixture
was poured into ice-water and the resulting precipitate was com-
bined by filtration, washed with water. The crude material was
purified by flash column chromatography on silica gel (CH2Cl2:
AcOEt = 1: 1) to give 30 as a yellow solid (3.28 g, 68%).
Mp: 340–345 °C. 1H NMR (400 MHz, DMSO-d6) d 1.27 (3H, t,
J = 7.3 Hz), 1.68–1.78 (1H, m), 1.90–2.03 (1H, m), 2.40–2.58 (4H,
m), 2.69 (2H, t, J = 6.7 Hz), 4.24 (2H, q, J = 7.3 Hz), 4.63 (2H, t,
J = 6.7 Hz), 8.44 (1H, s HRMS (ESI+) for C18H17F2N2O6 [M+H]+: calcd,
395.10547; found, 395.10611. Anal. calcd for
C18H16F2N2O6
0.1H2O: C, 54.81; H, 4.07; N, 7.07. Found: C, 54.31; H, 3.95; N, 6.96.
Mp: 245–248 °C. 1H NMR (400 MHz, DMSO-d6) d 1.26 (3H, t,
J = 7.3 Hz), 1.63–1.75 (1H, m), 1.85–1.98 (1H, m), 2.35–2.57 (6H,
m), 3.47–3.55 (2H, m), 4.22 (2H, q, J = 7.3 Hz), 6.96–7.02 (1H, m),
8.32 (1H, s). HRMS (ESI+) for C18H18F2N3O5 [M+H]+: calcd,
394.12145; found, 394.12213. Anal. calcd for C18H17F2N3O5: C,
54.96; H, 4.36; N, 10.68. Found: C, 55.07; H, 4.39; N, 10.48.
5.1.16. Ethyl 100,110-difluoro-20,30-dihydro-90-nitro-80-oxo-spiro
[cyclopentane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylate (27)
A solution of 19 (50.0 g, 0.138 mmol) in concentrated H2SO4
(1 mL) was treated portionwise at 0 °C with solid KNO3 (20.0 mg,
0.199 mmol). After stirring at 0 °C for 1 h, the reaction mixture
was poured into ice-water and the resulting precipitate was com-
bined by filtration, washed with water and dissolved in CH2Cl2-
MeOH (5:1 v/v). The combined extracts were concentrated in
vacuo. The crude material was purified by flash column chroma-
tography on silica gel (CH2Cl2: MeOH = 10: 1) to give 27 as a yellow
solid (26.0 mg, 46%).
5.1.20. Ethyl 90-amino-100,110-difluoro-20,30-dihydro-80-oxo-spir
o[cyclopropane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]
-70-carboxylate (31)
A solution of 25 (500 mg, 1,31 mmol) and 10% Pd/C (100 mg) in
DMF (30 mL) was stirred under a hydrogen atmosphere at 50 °C for
1.5 h. The catalyst was removed by filtration over Celite, and the
filtrate was concentrated in vacuo. The resulting solid was dis-
solved in CH2Cl2 and EtOH (3:1, 65 mL), and filtered. After the
CH2Cl2 was removed, the precipitate was isolated by filtration,
washed with EtOH, and dried to give 31 as a brown solid
(355 mg, 77%).
Mp: 243–246 °C. 1H NMR (400 MHz, DMSO-d6) d 1.37 (3H, t,
J = 7.3 Hz), 1.52–1.66 (2H, m), 1.78–1.90 (2H, m), 2.15–2.30 (4H,
m), 2.50 (2H, t, J = 6.7 Hz), 4.36 (2H, q, J = 7.3 Hz), 4.55 (2H, t,
J = 6.7 Hz), 8.69 (1H, s). HRMS (ESI+) for C19H19F2N2O6 [M+H]+:
calcd, 409.12112; found, 409.12385. Anal. calcd for C19H18F2N2O6
0.2H2O: C, 55.40; H, 4.40; N, 6.80. Found: C, 55.34; H, 4.56; N, 6.51.
Mp: 274–277 °C. 1H NMR (400 MHz, DMSO-d6)d 0.99 (2H, br),
1.13 (2H, br), 1.25 (3H, t, J = 7.3 Hz), 1.64–2.91 (2H, br), 4.18 (2H,
q, J = 7.3 Hz), 4.35 (2H, br), 7.46 (2H, br), 8.35 (1H, s). HRMS (EI)
for C17H16F2N2O4 (M+): calcd, 350.1078; found, 350.1033. Anal
calcd for C17H16F2N2O4: C, 58.28; H, 4.60; N, 8.00; found: C,
58.21; H, 4.47; N, 7.87.
5.1.17. Ethyl 100,110-difluoro-20,30-dihydro-30-methyl-90-nitro-
80-oxo-spiro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]
benzoxazepine]-70-carboxylate (28)
A solution of 20 (1.00 g, 2.75 mmol) in concentrated H2SO4
(12 mL) was treated portionwise at 0 °C with solid KNO3
(377 mg, 3.73 mmol). After stirring at 0 °C for 2 h, the reaction mix-
ture was poured into ice-water and the resulting precipitate was
combined by filtration, washed with water. The crude material
was purified by recrystalization of the cake from DMF (20 mL) to
give 28 as a yellow solid (923 mg, 82%).
5.1.21. Ethyl 90-amino-100,110-difluoro-20,30-dihydro-80-oxo-spir
o[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylate (32)
A solution of 26 (770 mg, 1.95 mmol) in DMF (70 mL) was
hydrogenated under atmospheric pressure over 10% Pd/C
(200 mg) at 50 °C for 1 h. The catalyst was removed by filtration
over Celite and the filtrate was concentrated in vacuo. The crude
material was purified by flash column chromatography on silica
Mp: 330–333 °C. 1H NMR (400 MHz, DMSO-d6) d 0.94 (3H, d,
J = 6.1 Hz), 1.26 (3H, t, J = 7.3 Hz), 1.67–1.78 (1H, m), 1.83–1.98
(1H, m), 2.40–2.64 (4H, m), 2.92–3.05 (1H, m), 3.98 (1H, t,
J = 11.6 Hz), 4.24 (2H, t, J = 7.3 Hz), 4.80 (1H, dd, J = 11.6 and
7.9 Hz), 8.25 (1H, s). HRMS (ESI+) for C19H19F2N2O6 [M+H]+: calcd,
409.12112; found, 409.12181. Anal. calcd for C19H18F2N2O6: C,
55.88; H, 4.44; N, 6.86. Found: C, 55.75; H, 4.37; N, 6.91.
gel (CH2Cl2: MeOH = 10: 1) to give 32 as
a colorless solid
(559 mg, 79%).
Mp: 257–260 °C. 1H NMR (400 MHz, DMSO-d6) d 1.26 (3H, t,
J = 7.3 Hz), 1.65–1.77 (1H, m), 1.87–2.01 (1H, m), 2.35–2.62 (6H,
m), 4.21 (2H, q, J = 7.3 Hz), 4.31–4.42 (2H, brt), 7.40–7.60 (2H, br
s), 8.23 (1H, s). HRMS (ESI+) for C18H19F2N2O4 [M+H]+: calcd,
365.13129; found, 365.13127. Anal. calcd for C18H18F2N2O4
0.2H2O: C, 58.76; H, 4.93; N, 7.61. Found: C, 58.70; H, 4.84; N, 7.40.
5.1.18. Ethyl 100,110-difluoro-20,30-dihydro-20-methyl-90-nitro-
80-oxo-spiro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]
benzoxazepine]-70-carboxylate (29)
A solution of 21 (182 mg, 0.501 mmol) in concentrated H2SO4
(2 mL) was treated portionwise at 0 °C with solid KNO3 (72.0 mg,
0.712 mmol). After stirring at 0 °C for 2 h, the reaction mixture
was poured into ice-water and the resulting precipitate was com-
bined by filtration, washed with water and then dried to give 29 as
a pale yellow solid (190 mg, 93%).
5.1.22. Ethyl 90-amino-100,110-difluoro-20,30-dihydro-80-oxo-spiro
[cyclopentane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylate (33)
A solution of 27 (2.00 g, 4.90 mmol) in DMF (120 mL) was
hydrogenated under atmospheric pressure over 10% Pd/C
(400 mg) at 50 °C for 1.5 h. The catalyst was removed by filtration
over Celite and the filtrate was concentrated in vacuo. The crude
material was purified by flash column chromatography on silica
gel (CH2Cl2: MeOH = 10: 1) to give 33 as a yellow solid (527 mg,
28%).
Mp: 300–303 °C. 1H NMR (400 MHz, DMSO-d6) d 1.27 (3H, t,
J = 7.3 Hz), 1.45 (3H, d, J = 6.1 Hz), 1.61–1.77 (1H, m), 1.90–2.03
(1H, m), 2.29–2.48 (3H, m), 2.57–2.72 (3H, m), 4.24 (2H, q,
J = 7.3 Hz), 4.62–4.73 (1H, m), 8.43 (1H, s). HRMS (ESI+) for
C
19H19F2N2O6 [M+H]+: calcd, 409.12112; found, 409.12140. Anal.
Mp: 144–146 °C. 1H NMR (400 MHz, DMSO-d6) d 1.26 (3H, t,
J = 7.3 Hz), 1.37–1.50 (2H, m), 1.65–1.77 (2H, m), 2.06–2.25 (4H,
m), 2.36 (2H, t, J = 6.7 Hz), 4.20 (2H, q, J = 7.3 Hz), 4.30 (2H, t,
calcd for C19H18F2N2O6: C, 55.88; H, 4.44; N, 6.86. Found: C,
55.68; H, 4.29; N, 6.78.

S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
1195
J = 6.7 Hz), 7.53 (2H, br s), 8.47 (1H, s). HRMS (ESI+) for
19H21F2N2O4 [M+H]+: calcd, 379.14694; found, 379.15131. Anal.
calcd for C19H20F2N2O4: C, 60.31; H, 5.33; N, 7.40. Found: C,
60.06; H, 5.23; N, 7.40.
14.5 (1H, s). HRMS (EI) for C15H12F2N2O4 (M+): calcd, 322.0765;
found, 322.0773. Anal calcd for C15H12F2N2O4: C, 55.90; H, 3.75;
N, 8.69; found: C, 55.52; H, 3.71; N, 8.36.
C
5.1.27. 90-Amino-100,110-difluoro-20,30-dihydro-80-oxo-spiro
[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylic acid (38)
5.1.23. Ethyl 90-amino-100,110-difluoro-20,30-dihydro-30-methyl-
80-oxo-spiro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4] ben
zoxazepine]-70-carboxylate (34)
To a mixture of 32 (513 mg, 1.41 mmol) in EtOH (14 mL) was
added 2 N NaOH (7.0 mL, 14.0 mmol) at room temperature and
the mixture was heated at 50 °C for 3 h. The reaction mixture
was added 2 N HCl (7.0 mL) and water. A precipitate formed and
was collected by filtration, washed successively with water and
then dried to give 38 as a colorless solid (427 mg, 90%).
Mp: 330–333 °C. 1H NMR (400 MHz, DMSO-d6) d 1.64–1.77 (1H,
m), 1.89–2.02 (1H, m), 2.56–2.70 (2H, br), 4.30–4.50 (2H, br), 7.35–
7.52 (2H, br s), 8.47 (1H, s), 14.60 (1H, s). HRMS (ESI+) for
C16H15F2N2O4 [M+H]+: calcd, 337.09999; found, 337.10033. Anal.
calcd for C16H14F2N2O4 0.1 H2O: C, 56.84; H, 4.17; N, 8.29. Found:
C, 56.91; H, 4.25; N, 7.99.
A solution of 28 (900 mg, 2.20 mmol) in DMF (55 mL) was
hydrogenated under atmospheric pressure over 10% Pd/C
(180 mg) at 50 °C for 3 h. The catalyst was removed by filtration
over Celite and the filtrate was concentrated in vacuo. The residue
was washed with EtOH and collected by filtration and then dried to
give 34 as a pale yellow solid (756 mg, 91%).
Mp: 248–250 °C. 1H NMR (400 MHz, DMSO-d6) d 0.89 (3H, br s),
1.26 (3H, t, J = 7.3 Hz), 1.65–1.77 (1H, m), 1.80–1.95 (1H, m), 2.30–
2.67 (4H, m), 2.82–2.94 (1H, m), 3.52–3.66 (1H, m), 4.15–4.28 (2H,
m), 4.58–4.70 (1H, m), 7.40–7.65 (2H, br), 8.05 (1H, s). HRMS (ESI+)
for C19H21F2N2O4 [M+H]+: calcd, 379.14694; found, 379.14659.
5.1.24. Ethyl 90-amino-100,110-difluoro-20,30-dihydro-20-methyl-
80-oxo-spiro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4] ben
zoxazepine]-70-carboxylate (35)
5.1.28. 90-Amino-100,110-difluoro-20,30-dihydro-80-oxo-spiro
[cyclopentane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-
70-carboxylic acid (39)
A solution of 29 (1.13 g, 2.77 mmol) in DMF (70 mL) was hydro-
genated under atmospheric pressure over 10% Pd/C (230 mg) at
50 °C for 2 h. The catalyst was removed by filtration over Celite
and the filtrate was concentrated in vacuo. The residue was washed
with EtOH and collected by filtration and then dried to give 35 as a
pale yellow solid (824 mg, 79%).
To a mixture of 33 (482 mg, 1.27 mmol) in EtOH (13 mL) was
added 2 N NaOH (6.5 mL, 13.0 mmol) at room temperature and
the mixture was heated at 50 °C for 3 h. The reaction mixture
was added 2 N HCl (6.5 mL) and water. A precipitate formed and
was collected by filtration, washed successively with water and
then dried to give 39 as a colorless solid (425 mg, 96%).
Mp: 242–245 °C. 1H NMR (400 MHz, DMSO-d6) d 1.35–1.52 (2H,
m), 1.65–1.75 (2H, m), 2.10–2.30 (4H, m), 2.43 (2H, t, J = 6.7 Hz),
4.34 (2H, t, J = 6.7 Hz), 7.50 (2H, br s), 8.67 (1H, s), 14.5 (1H, br
s). HRMS (ESI+) for C17H17F2N2O4 [M+H]+: calcd, 351.11564; found,
351.11965. Anal. calcd for C17H16F2N2O4: C, 58.28; H, 4.60; N, 8.00.
Found: C, 58.21; H, 4.60; N, 7.77.
Mp: 211–213 °C. 1H NMR (400 MHz, DMSO-d6) d 1.26 (3H, t,
J = 7.3 Hz), 1.36 (3H, d, J = 6.1 Hz), 1.70 (1H, q, J = 10.4 Hz), 1.86–
2.00 (1H, m), 2.25 (1H, q, J = 10.4 Hz), 2.30–2.39 (1H, m), 2.42–
2.63 (4H, m), 4.14–4.27 (2H, m), 4.27–4.37 (1H, m),7.49 (2H, br
s), 8.22 (1H, s). HRMS (ESI+) for C19H21F2N2O4 [M+H]+: calcd,
379.14694; found, 379.14645. Anal. calcd for C19H20F2N2O4: C,
60.31; H, 5.33; N, 7.40. Found: C, 60.34; H, 5.23; N, 7.22.
5.1.29. 90-Amino-100,110-difluoro-20,30-dihydro-30-methyl-80-oxo
-spiro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxaze
pine]-70-carboxylic acid (40)
5.1.25. Ethyl 90-amino-100,110-difluoro-20,30-dihydro-80-oxo-spi
ro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzodiaze pi
ne]-70-carboxylate (36)
To a mixture of 34 (720 mg, 1.90 mmol) in EtOH (20 mL) was
added 2 N NaOH (10.0 mL) at room temperature and the mixture
was heated at 50 °C for 3 h. The reaction mixture was added 2 N
HCl (10.0 mL) and water. A precipitate formed and was collected
by filtration, washed successively with water and then dried to
give 40 as a yellow solid (654 mg, 98%).
A solution of 22 (1.23 g, 3.13 mmol) in DMF (77 mL) was hydro-
genated under atmospheric pressure over 10% Pd/C (250 mg) at
50 °C for 1 h. The catalyst was removed by filtration over Celite
and the filtrate was concentrated in vacuo . The crude material
was purified by flash column chromatography on silica gel (CH2Cl2:
MeOH = 2: 1) to give 36 as a yellow solid (1.00 g, 88%).
Mp: 305–308 °C. 1H NMR (400 MHz, DMSO-d6) d 0.90 (3H, d,
J = 5.5 Hz), 1.64–1.76 (1H, m), 1.81–1.96 (1H, m), 2.35–2.72 (4H,
m), 2.88–3.02 (1H, m), 3.64 (1H, t, J = 11.6 Hz), 4.60–4.75 (1H,
m), 7.46 (2H, br s), 8.26 (1H, s), 14.60 (1H, br s). HRMS (ESI+) for
C17H17F2N2O4 [M+H]+: calcd, 351.11564; found, 351.11536.
Mp: 176–178 °C. 1H NMR (400 MHz, DMSO-d6) d 1.25 (3H, t,
J = 7.3 Hz), 1.62–1.73 (1H, m), 1.82–1.96 (1H, m), 2.24–2.41 (4H,
m), 2.45–2.60 (2H, m), 3.27–3.38 (2H, m), 4.19 (2H, q, J = 7.3 Hz),
5.28–5.34 (1H, m), 7.10 (2H, br s), 8.12 (1H, s). HRMS (ESI+) for
C
18H20F2N3O3 [M+H]+: calcd, 364.14727; found, 364.14797. Anal.
5.1.30. 90-Amino-100,110-difluoro-20,30-dihydro-20-methyl-80-
oxo-spiro[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]ben
zoxazepine]-70-carboxylic acid (41)
calcd for C18H19F2N3O3 0.2H2O: C, 58.91; H, 5.22; N, 11.45. Found:
C, 58.91; H, 5.20; N, 11.06.
To a mixture of 35 (768 mg, 2.03 mmol) in EtOH (20 mL) was
added 2 N NaOH (10.0 mL) at room temperature and the mixture
was heated at 50 °C for 3 h. The reaction mixture was added 2 N
HCl (10.0 mL) and water. A precipitate formed and was collected
by filtration, washed successively with water and then dried to
give 41 as a yellow solid (703 mg, 99%).
5.1.26. 90-Amino-100,110-difluoro-20,30-dihydro-80-oxo-spiro[cycl
opropane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzoxazepine]-70-
carboxylic acid (37)
A solution of 31 (320 mg, 0.913 mmol) in mixture of AcOH-H2O-
H2SO4 (6:4:1 v/v, 5.3 mL) was heated at reflux for 1 h. The reaction
mixture was poured into ice water and a precipirtate was collected
by filtration, washed with water and then dried to give the title
compound 37 as a yellow solid (274 mg, 93%).
Mp: 297–300 °C. 1H NMR (400 MHz, DMSO-d6) d 1.37 (3H, d,
J = 6.1 Hz), 1.70 (1H, q, J = 11.0 Hz), 1.88–2.02 (1H, m), 2.31 (1H,
q, J = 11.0 Hz), 2.37–2.69 (5H, m), 4.30–4.42 (1H, m), 7.43 (1H, br
s), 8.47 (1H, s), 14.61 (1H, br s). HRMS (ESI+) for C17H17F2N2O4
[M+H]+: calcd, 351.11564; found, 351.11474. Anal. calcd for
Mp: 356–365 °C. 1H NMR (400 MHz, DMSO-d6)d 0.63–1.55 (4H,
br), 1.90–3.11 (2H, br), 3,78–4.96 (2H, br), 7.40 (2H, s), 8.64 (1H, s),

1196
S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
C17H16F2N2O4 0.15H2O: C, 57.84; H, 4.57; N, 7.94. Found: C, 58.10;
H, 4.52; N, 7.65.
stirred at 100 °C for 4 h. The reaction mixture was added portion-
wise at 0 °C with ice-water and the resulting precipitate was com-
bined by filtration, washed with water and iPr2O. The cake washed
with EtOH and collected by filtration and then dried to give 3 as a
pale yellow solid (185 mg, 69%).
5.1.31. 90-Amino-100,110-difluoro-20,30-dihydro-80-oxo-spiro
[cyclobutane-1,40-[4H,8H]pyrido[1,2,3-ef][1,4]benzodiazepine]-
70-carboxylic acid (42)
Mp: 180–183 °C. 1H NMR (400 MHz, DMSO-d6) d 1.34–1.49 (2H,
m), 1.60–1.75 (2H, m), 2.03–2.23 (4H, m), 2.34 (2H, t, J = 6.7 Hz),
3.40–3.55 (2H, m), 3.55–3.64 (2H, m), 4.17 (2H, t, J = 6.7 Hz),
6.25–6.33 (1H, br), 6.43–6.51 (2H, m), 6.69 (1H, t, J = 5.5 Hz), 7.06
(2H, br), 7.35 (1H, ddd, J = 6.7, 6.7 and 1.8 Hz), 8.86 (1H, dd,
J = 4.9 and 1.2 Hz), 8.49 (1H, s), 15.1 (1H, br s). HRMS (ESI+) for
To a mixture of 36 (515 mg, 1.41 mmol) in EtOH (15 mL) was
added 2 N NaOH (7.0 mL, 14.0 mmol) at room temperature and
the mixture was heated at 50 °C for 3 h. The reaction mixture
was added 2 N HCl (7.0 mL) and water. A precipitate formed and
was collected by filtration, washed successively with water and
then dried to give 42 as a yellow solid (397 mg, 83%).
Mp: 220–225 °C. 1H NMR (400 MHz, DMSO-d6) d 1.61–1.73 (1H,
m), 1.82–1.97 (1H, m), 2.30–2.44 (4H, m), 2.55–2.69 (2H, m), 3.25–
3.50 (2H, m), 5.53–5.59 (1H, br), 7.03 (2H, br s), 8.35 (1H, s), 14.75
(1H, s). HRMS (ESI+) for C16H15F2N3O3 [M+H]+: calcd, 336.11597;
found, 336.11648.
C
24H27FN5O4 [M+H]+: calcd, 468.20471; found, 4678.20118. Anal.
calcd for C24H26FN5O4 0.1H2O: C, 61.42; H, 5.58; N, 14.92. Found:
C, 61.32; H, 5.75; N, 14.63.
5.1.35. 90-Amino-100-fluoro-20,30-dihydro-30-methyl-80-oxo-110-
[2-(2-pyridylamino)ethylamino]spiro[cyclobutane-1,40-[8H]pyr
ido[1,2,3-ef][1,4]benzoxazepine]-70-carboxyic acid (45)
Anal. calcd for C16H15F2N3O3 0.1 H2O: C, 57.01; H, 4.48; N, 12.46.
Found: C, 57.22; H, 4.51; N, 12.08.
A
solution of 40 (177 mg, 1.29 mmol) and triethylamine
(180
lL) in DMSO (4 mL) was stirred at 120 °C for 5 h. The reaction
5.1.32. 90-Amino-100-fluoro-20,30-dihydro-80-oxo-110-[2-(2-
pyridylamino)ethylamino]spiro[cyclopropane-1,40-[4H,8H]
pyrido[1,2,3-ef][1,4] benzoxazepine]-70-carboxylic acid (43)
mixture was added portionwise at 0 °C with ice-water and the
mixture was extracted with CH2Cl2. The combined extracts were
dried over anhydrous Na2SO4, filtered, and then concentrated in
vacuo. The crude material was purified by flash column chroma-
tography on silica gel (CH2Cl2: MeOH = 10: 1) to give 45 as a yellow
amorphous solid (177 mg, 44%).
A
solution of 37 (150 mg, 0.465 mmol), triethylamine
(0.100 mL, 0.717 mmol) and N-2-(pyridinyl)-1,2-ethanediamine
(95.5 mg, 0.696 mmol) in DMSO (2 mL) was stirred at 100 °C for
3 h. The reaction mixture was poured into ice water and the result-
ing precipitate was removed by filtration and washed with ethanol.
The resulting solid was dissolved in DMF and filtered. The filtrate
was poured into water and the resulting precipitate was removed
by filtration, washed with water and dried to give 43 as a dark yel-
low solid (95.6 mg, 47%).
Mp: 201–204 °C. 1H NMR (400 MHz, DMSO-d6) d 0.86 (3H, d,
J = 5.5 Hz), 1.62–1.73 (1H, m), 1.79–1.93 (1H, m), 2.25–2.38 (1H,
m), 2.38–2.64 (3H, m), 2.80–2.93 (1H, m), 3.27–3.50 (3H, m),
3.52–3.14 (2H, m), 4.50–4.60 (1H, m), 6.28 (1H, br s), 6.42–6.52
(2H, m), 6.69 (1H, t, J = 5.5 Hz), 7.01 (2H, br s), 7.32–7.40 (1H, m),
7.96 (1H, dd, J = 4.9 and 1.2 Hz), 8.07 (1H, s), 15.24 (1H, s). HRMS
(ESI+) for C24H27FN5O4 [M+H]+: calcd, 468.20471; found,
468.20923. Anal. calcd for C24H26FN5O4: C, 61.61; H, 5.61; N,
14.98. Found: C, 61.39; H, 5.51; N, 14.70.
Mp: 237–239 °C 1H NMR (400 MHz, DMSO-d6) d 0.81–1.50 (4H,
m), 3.40–3.51 (2H, m), 3.55–3.66 (2H, m), 3.80–4.60 (4H, m), 6.32
(1H, br s), 6.40–6.50 (2H, m), 6.70 (1H, t, J = 5.5 Hz), 9.97 (2H, br s),
7.30–7.40 (1H, m), 7.90–8.00 (1H, m), 15.14 (1H, s). HRMS (ESI) for
C
22H23FN5O4 [M+H]+: calcd, 440.17340; found, 440.17315. Anal.
5.1.36. 90-Amino-100-fluoro-20,30-dihydro-20-methyl-80-oxo-110-
[2-(2-pyridylamino)ethylamino]spiro[cyclobutane-1,40-[8H]
pyrido[1,2,3-ef][1,4]benzoxazepine]-70-carboxyic acid (46)
A solution of 41 (351 mg, 1.00 mmol), N-2-pyridinyl-1,2-eth-
anediamine (210 mg, 1.53 mmol) and triethylamine (0.22 mL) in
DMSO (5 mL) was stirred at 120 °C for 5 h. The reaction mixture
was added portionwise at 0 °C with ice-water and the mixture
was extracted with CH2Cl2. The combined extracts were dried over
anhydrous Na2SO4, filtered, and then concentrated in vacuo. The
crude material was purified by flash column chromatography on
silica gel (CH2Cl2: MeOH = 10: 1) to gave 46 as a yellow amorphous
solid (278 mg, 59%).
Calcd for C22H22FN5O4, 0.2H2O: C, 59.64; H, 5.10; N, 15.81. Found:
C, 59.44; H, 5.14; N, 15.61.
5.1.33. 90-Amino-100-fluoro-20,30-dihydro-80-oxo-110-[2-(2-pyrid
ylamino)ethylamino]spiro[cyclobutane-1,40-[8H]pyrido [1,2,3-
ef][1,4]benzoxazepine]-70-carboxyic acid (44)
A solution of 38 (186 mg, 0.553 mmol), Jiwen (Jim) Liu, Amgen
Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080,
USA2-pyridinyl-1,2-ethanediamine (115 mg, 0.838 mmol) and tri-
ethylamine (117
lL) in DMSO (2.5 mL) was stirred at 120 °C for
4 h. The reaction mixture was added portionwise at 0 °C with
ice-water and the mixture was added 2 N HCl (3 drops). The result-
ing precipitate was combined by filtration. The cake washed with
EtOH and collected by filtration and then dried to give 44 as a yel-
low solid (165 mg, 66%).
Mp: 106–108 °C. 1H NMR (400 MHz, DMSO-d6) d 1.29 (3H, d,
J = 6.1 Hz), 1.67 (1H, q, J = 10.4 Hz), 1.84–1.99 (1H, m), 2.21 (1H,
q, J = 10.4 Hz), 2.30–2.40 (1H, m), 2.40–2.62 (4H, m), 3.40–3.50
(2H, m), 3.53–3.67 (2H, m), 4.08–4.20 (1H, m), 5.91 (1H, br s),
6.42–6.50 (2H, m), 6.65 (1H, t, J = 5.5 Hz), 7.00 (2H, br s), 7.32–
7.38 (1H, m), 7.94 (1H, dd, J = 4.9 and 1.2 Hz), 8.25 (1H, s), 15.24
(1H, s). HRMS (ESI+) for C24H27FN5O4 [M+H]+: calcd, 467.20471;
found, 467.20453. Anal. calcd for C24H26FN5O4 0.4H2O: C, 60.72;
H, 5.52; N, 14.75. Found: C, 60.61; H, 5.49; N, 14.67.
Mp: 173–175 °C. 1H NMR (400 MHz, DMSO-d6) d 1.61–1.74 (1H,
m), 1.84–2.00 (1H, m), 2.30–2.65 (6H, m), 3.40–3.50 (2H, m), 3.52–
3.62 (2H, m), 4.10–4.33 (2H, br), 6.20–6.30 (1H, m), 6.42–6.51 (2H,
m), 6.69 (1H, t, J = 5.5 Hz), 6.90–7.08 (2H, br), 7.32–7.40 (1H, m),
7.97 (1H, dd, J = 4.3 and 1.2 Hz), 8.26 (1H, s), 15.23 (1H, s). HRMS
(ESI+) for C23H25FN5O4 [M+H]+: calcd, 454.18906; found,
454.18677. Anal. calcd for C23H24FN5O4 0.2H2O: C, 60.44; H, 5.29;
N, 15.32. Found: C, 60.56; H, 5.35; N, 15.07.
5.1.37. 90-Amino-100-fluoro-20,30-dihydro-80-oxo-110-[2-(2-
pyridylamino)ethylamino]spiro[cyclobutane-1,40-[8H]pyr
ido[1,2,3-ef][1,4]benzodiazepine]-70-carboxyic acid (47)
A solution of 42 (300 mg, 0.895 mmol), N-2-pyridinyl-1,2-eth-
anediamine (185 mg, 1.35 mmol) and triethylamine (0.23 mL) in
DMSO (4 mL) was stirred at 120 °C for 5 h. The reaction mixture
was added portionwise at 0 °C with iced-water and the mixture
was extracted with CH2CH2. The organic layer was dried over
5.1.34. 90-Amino-100-fluoro-20,30-dihydro-80-oxo-110-[2-(2-pyrid
ylamino)ethylamino]spiro[cyclopentane-1,40-[8H]pyrido[1,2,3-
ef][1,4]benzoxazepine]-70-carboxyic acid (3)
A solution of 39 (200 mg, 0.571 mmol) and N-2-pyridinyl-1,2-
ethanediamine (118 mg, 0.860 mmol) in DMSO (2.5 mL) was

S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
1197
anhydrous Na2SO4, and concentrated in vacuo. The crude material
was purified by flash column chromatography on silica gel (CH2Cl2:
MeOH = 10: 1) to give 47 as a yellow solid (259 mg, 64%).
1H NMR (400 MHz, CDCl3) d 1.21 (3H, d, J = 6.8 Hz), 1.29 (3H, t,
J = 7.3 Hz), 1.51–1.64 (1H, m), 1.80–1.90 (1H, m), 1.96–2.17 (4H,
m), 2.70 (1H, q, J = 7.3 Hz), 3.44 (1H, s), 4.18 (2H, qd, J = 7.3 and
1.2 Hz). HRMS (EI) for C9H16O3 (M+): calcd, 172.1099; found,
172.1133.
Mp: 110–115 °C. 1H NMR (400 MHz, DMSO-d6) d 1.58–1.70 (1H,
m), 1.78–1.92 (1H, m), 2.21–2.38 (4H, m), 2.43–2.60 (2H, m), 3.19–
3.37 (2H, m), 3.37–3.51 (4H, m), 4.50 (1H, t, J = 5.5 Hz), 5.50–5.58
(1H, br), 6.42–6.51 (2H, m), 6.65 (1H, t, J = 5.5 Hz), 6.82 (2H, br
s), 7.36 (1H, td, J = 6.7 and 1.8 Hz), 7.95 (1H, dd, J = 4.9 and
1.2 Hz), 8.18 (1H, s), 15.28 (1H, s). HRMS (ESI+) for C23H26FN6O3
[M+H]+: calcd, 453.20504; found, 453.20466. Anal. calcd for
5.1.41. Ethyl 1-(benzoylamino)cyclobutylacetate (53)
To a mixture of 50 (6.45 g, 40.8 mmol) and benzonitrile (40 mL,
0.392 mol), H2SO4 (2.20 mL, 41.3 mmol) was slowly added at room
temperature. The mixture was stirred for 1 h at room temperature
and then at 80 °C for 1 h. The mixture was cooled on iced water, to
which was added 2 N NaOH solution until pH reached 7. The mix-
ture was extracted with ethyl acetate. The combined extracts were
dried over anhydrous Na2SO4, filtered, and then concentrated in
vacuo. The crude material was purified by flash column chroma-
tography on silica gel (AcOEt:hexane = 5:1) to give 53 as a colorless
solid (5.40 g, 51%).
C23H25FN6O3 0.3H2O: C, 60.33; H, 5.50; N, 18.35. Found: C, 60.47;
H, 5.58; N, 18.06.
5.1.38. Ethyl 1-hydroxycyclobutylacetate (50)
Trimethylchlorosilane (1.14 mL, 8.92 mmol) was added from a
syringe to a suspension of zinc powder (7.97 g, 0.122 mol) in
Et2O (200 mL). The mixture was stirred for 15 min at room temper-
ature and then heated to reflux. The heating was stopped, and ethyl
bromoacetate (10.3 mL, 92.9 mmol) was added at such a rate that
the ether boiled gently. After being heated to reflux for 1 h, the
mixture was stirred for 1 h at room temperature. A solution of 48
(6.00 g, 75.9 mmol) in Et2O (30 mL) was added while the tempera-
ture of the mixture was maintained at 19-20 °C by intermittent
cooling. After being stirred for 1 h at room temperature, the mix-
ture was poured into iced 25% ammonia (400 mL). The aqueous
phase was extracted with ether, the combined phases were dried
on K2CO3 and evaporated, and 50 was obtained as a colorless oil
(6.50 g, 54%).
Mp: 44–46 °C. 1H NMR (400 MHz, CDCl3)
d 1.22 (3H, t,
J = 7.3 Hz), 1.84–2.07 (2H, m), 2.22–2.32 (2H, m), 2.44–2.55 (2H,
m), 3.05 (2H, s), 4.11 (2H, q, J = 7.3 Hz), 6.73 (1H, s), 7.39–7.52
(3H, m), 7.72–7.79 (2H, m). HRMS (EI) for C15H19NO3 (M+): calcd,
261.1365; found, 261.1360. Anal. calcd for C15H19NO3 0.1H2O: C,
68.47; H, 7.28; N, 5.32. Found: C, 68.75; H, 7.28; N, 5.03.
5.1.42. Ethyl 1-(benzoylamino)cyclopentylacetate (54)
To
a mixture of 51 (17.3 g, 0.100 mol) and benzonitrile
(200 mL, 1.96 mol), H2SO4 (5.40 mL, 0.101 mol) was slowly added
at room temperature. The mixture was stirred for 1 h at room
temperature, and then at 80 °C for 1 h. The mixture was cooled
on iced water, to which was added 2 N NaOH solution until
pH reached 7. The mixture was extracted with ethyl acetate.
The combined extracts were dried over anhydrous Na2SO4, fil-
tered, and then concentrated in vacuo. The crude material was
purified by flash column chromatography on silica gel
(AcOEt:hexane = 5:1) to give 54 as a colorless amorphous mass
(11.65 g, 42%).
1H NMR (400 MHz, CDCl3) d 1.29 (3H, t, J = 7.3 Hz), 1.47–1.64
(1H, m), 1.76–1.87 (1H, m), 1.93–2.06 (2H, m), 2.12–2.22 (2H,
m), 2.67 (2H, s), 3.70 (1H, s), 4.19 (2H, q, J = 7.3 Hz). HRMS (CI+)
for C8H15O3 [M+H]+: calcd, 159.1021; found, 159.0994.
5.1.39. Ethyl 1-hydroxycyclopentylacetate (51)
Trimethylchlorosilane (3.60 mL, 28.2 mmol) was added from a
syringe to a suspension of zinc powder (25.2 g, 0.385 mol) in Et2O
(500 mL). The mixture was stirred for 15 min at room temperature
and then heated to reflux. The heating was stopped, and ethyl bro-
moacetate (32.4 mL, 0.292 mol) was added at such a rate that the
ether boiled gently. After being heated to reflux for 1 h, the mixture
was stirred for 1 h at room temperature. A solution of 49 (20.2 g,
0.240 mol) in Et2O (30 mL) was added while the temperature of
the mixture was maintained at 19-21 °C by intermittent cooling.
After being stirred for 1 h at room temperature, the mixture was
poured into iced 25% ammonia (500 mL). The aqueous phase was ex-
tracted with ether, the combined phases were dried on K2CO3 and
evaporated, and 51 was obtained as a colorless oil (41.0 g, 99%).
1H NMR (400 MHz, CDCl3) d 1.13 (3H, t, J = 7.3 Hz), 1.34–1.52
(4H, m), 1.61–1.75 (4H, m), 2.45 (2H, s), 3.22 (1H, s), 4.03 (2H, q,
J = 7.3 Hz). HRMS (CI+) for C9H17O3 [M+H]+: calcd, 173.1178; found,
173.1181.
1H NMR (400 MHz, CDCl3) d 1.20 (3H, t, J = 7.3 Hz), 1.65–1.87
(6H, m), 2.23–2.33 (2H, m), 2.97 (2H, s), 4.10 (2H, q, J = 7.3 Hz),
6.45 (1H, s), 7.38–7.50 (3H, m), 7.72–7.76 (2H, m). HRMS (ESI+)
for C16H22NO3 [M+H]+: calcd, 276.15997; found, 276.15872. Anal.
calcd for C16H21NO3 0.1H2O: C, 69.34; H, 7.64; N, 5.05. Found: C,
69.36; H, 7.59; N, 4.88.
5.1.43. Ethyl 2-[1-(benzoylamino)cyclobutyl]propionate (55)
To a mixture of 52 (12.5 g, 72.6 mmol) and benzonitrile (75 mL),
H2SO4 (3.90 mL, 73.2 mmol) was slowly added at room tempera-
ture. The mixture was stirred for 1 h at room temperature, and
then at 80 °C for 1 h. The mixture was cooled on iced water, to
which was added 2 N NaOH solution until pH reached 7. The mix-
ture was extracted with ethyl acetate. The combined extracts were
dried over anhydrous Na2SO4, filtered, and then concentrated in
vacuo. The crude material was purified by flash column chroma-
tography on silica gel (AcOEt:hexane = 5:1) to give 55 as a pale yel-
low solid (9.24 g, 46%).
5.1.40. Ethyl 2-(1-hydroxycyclobutyl)propionate (52)
Trimethylchlorosilane (950 lL, 7.43 mmol) was added from a syr-
inge to a suspension of zinc powder (6.65 g, 0.102 mol) in abs. Et2O
(170 mL). The mixture was stirred for 15 min at room temperature
and then heated to reflux. The heating was stopped, and ethyl 2-
bromopropionate (10.1 mL, 77.8 mmol) was added at such a rate that
the ether boiled gently. After being heated to reflux for 1 h, the mixture
was stirred for 1 h at room temperature. A solution of 48 (5.00 g,
63.2 mmol) in Et2O (10 mL) was added while the temperature of the
mixture was maintained at 19-20 °C by intermittent cooling. After
being stirred for 1 h at room temperature, the mixture was poured into
iced 25% ammonia (400 mL). The aqueous phase was extracted with
ether, the combined phases were dried on K2CO3 and evaporated,
and 52 was obtained as a colorless oil (12.9 g, 100%).
Mp: 55–58 °C. 1H NMR (400 MHz, CDCl3)
d 1.25 (3H, t,
J = 7.3 Hz), 1.30 (3H, d, J = 7.3 Hz), 1.73–1.87 (1H, m), 1.98–2.09
(2H, m), 2.21–2.32 (1H, m), 2.53–2.63 (1H, m), 2.84–2.94 (1H,
m), 3.11 (1H, q, J = 7.3 Hz), 4.08–4.20 (2H, m), 6.88 (1H, br s),
7.40–7.52 (3H, m), 7.75–7.80 (2H, m). HRMS (APCI+) for
C16H22NO3 [M+H]+: calcd, 276.15997; found, 276.15976. Anal.
calcd for C16H21NO3 0.3 H2O: C, 68.45; H, 7.54; N, 4.49. Found: C,
68.45; H, 7.77; N, 4.80.
5.1.44. 1-(Benzylamino)-1-(2-hydroxyethyl)cyclobutane (56)
To a solution of 53 (5.30 g, 20.3 mmol) in THF (100 mL) was
added LiAlH4 (3.88 g, 0.102 mol) at room temperature. The mixture

1198
S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
was stirred for 1 h, then refluxed for 1 h. The mixture was cooled
on ice-water bath, to which was dropped a little water. The mix-
ture was diluted with ethyl acetate, then dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The crude material
was purified by distillation to give 56 as a colorless oil (2.53 g,
61%).
and the filtrate was concentrated in vacuo. The crude material
was purified by distillation to give 8 as a colorless oil (2.96 g, 74%).
1H NMR (400 MHz, CDCl3) d 1.03 (3H, d, J = 7.3 Hz), 1.63–1.74
(3H, m), 1.78–1.94 (2H, m), 2.10–2.28 (2H, m), 3.50 (1H, dd,
J = 11.0 and 4.3 Hz), 3.91 (1H, dd, J = 11.0 and 3.1 Hz). HRMS (CI+)
for C7H16NO [M+H]+: calcd, 130.1232; found, 130.1254.
bp 177-178 °C (12 Torr). 1H NMR (400 MHz, CDCl3) d 1.69–1.86
(2H, m), 1.88 (2H, t, J = 5.5 Hz), 1.92–2.09 (4H, m), 3.73 (2H, s), 3.87
(2H, t, J = 5.5 Hz), 7.22–7.35 (5H, m). HRMS (CI+) for C13H20NO
[M+H]+: calcd, 206.1545; found, 206.1510.
5.1.50. 1-(Benzoylamino)cyclobutylacetaldehyde (59)
To a stirred solution of 53 (6.86 g, 26.3 mmol) in THF (130 mL),
DIBAL (40.0 mL, 1 M solution in toluene) was added dropwise at
ꢀ78 °C for 0.5 h. After stirring at ꢀ78 °C for 6 h, to the mixture
was added dropwise MeOH (10 mL) at ꢀ78 °C for 0.5 h, to which
was added saturated aqueous NH4Cl (20 mL). The mixture was stir-
red at room temperature for 1 h and then extracted with Et2O. The
combined extracts were dried over anhydrous Na2SO4, filtered, and
then concentrated in vacuo. The crude material was purified by
flash column chromatography on silica gel (hexane:AcOEt = 2:1)
to give 59 as a colorless solid (1.82 g, 32%).
5.1.45. 1-(Benzylamino)-1-(2-hydroxyethyl)cyclopentane (57)
To a solution of 54 (11.6 g, 42.1 mmol) in THF (200 mL) was
added LiAlH4 (8.00 g, 0.211 mol) at room temperature and the mix-
ture was stirred for 0.5 h, then, refluxed 1 h. The mixture was
cooled on ice-water bath, to which was dropped a little water.
The mixture was diluted with ethyl acetate, then dried over anhy-
drous Na2SO4, filtered, and then concentrated in vacuo. The crude
material was purified by distillation to give 57 as a colorless oil
(8.41 g, 91%).
Mp: 87–89 °C. 1H NMR (400 MHz, CDCl3) d 1.87–2.07 (2H, m),
2.25–2.34 (2H, m), 2.36–2.45 (2H, m), 3.28 (2H, s), 6.57 (1H, br
s), 7.39–7.45 (2H, m), 7.46–7.53 (1H, m), 7.70–7.76 (2H, m), 9.79
(1H, s). HRMS (APCI+) for C13H16N2O2 [M+H]+: calcd, 218.11810;
found, 218.11823. Anal. calcd for C13H15N2O2: C, 71.87; H, 6.96;
N, 6.45. Found: C, 71.77; H, 7.16; N, 6.40.
bp 184-185 °C (9.2-9.0 Torr). 1H NMR (400 MHz, CDCl3) d 1.57–
1.85 (10H, m), 3.76 (2H, s), 3.87–3.92 (2H, m), 7.22–7.35 (5H, m).
HRMS (EI) for C14H21NO (M+): calcd, 219.1623; found, 219.1623.
5.1.46. 1-(Benzylamino)-1-(1-hydroxypropane-2-yl)cyclobutane
(58)
5.1.51. 1-(Benzoylamino)-1-(2-hydroxypropane-1-
To a solution of 55 (9.07 g, 32.9 mmol) in THF (160 mL) was
added LiAlH4 (6.30 g, 0.166 mol) at room temperature and the mix-
ture was stirred for 1 h, then, refluxed 1 h. The mixture was cooled
on ice-water bath, to which was dropped a little water. The mix-
ture was diluted with ethyl acetate, then dried over anhydrous
Na2SO4, filtered, and then concentrated in vacuo. The crude mate-
rial was purified by distillation to give 58 as a pale yellow oil
(6.92 g, 96%).
yl)cyclobutane (60)
To a stirred solution of 59 (2.82 g, 13.0 mmol) in THF (30 mL),
methylmagnesium chloride (14.0 mL, 3 M solution in THF) was
added at 0 °C, and then the mixture was stirred at room tempera-
ture for 5 h. To this mixture was added saturated aqueous NH4Cl
(10 mL) at 0 °C, and then extracted with ethyl acetate. The com-
bined extracts were dried over anhydrous Na2SO4, filtered, and
then concentrated in vacuo. The crude material was purified by
flash column chromatography on silica gel (hexane:AcOEt = 2: 1)
to give 60 as a colorless oil (2.65 g, 87%).
1H NMR (400 MHz, CDCl3) d 1.00 (3H, d, J = 6.7 Hz), 1.66–1.77
(1H, m), 1.81–1.97 (2H, m), 2.02–2.10 (1H, m), 2.10–2.24 (3 H,
m), 3.59 (1H, dd, J = 11.0 and 7.9 Hz), 3.71 (1H, d, J = 11.6 Hz),
3.74 (1H, dd, J = 11.0 and 3.7 Hz), 3.89 (1H, d, J = 11.6 Hz), 7.23–
7.36 (5H, m). HRMS (CI+) for C14H22NO [M+H]+: calcd, 220.1701;
found, 220.1670.
1H NMR (400 MHz, CDCl3) d 1.25 (3H, d, J = 6.1 Hz), 1.77–2.12
(5H, m), 2.28–2.40 (1H, m), 2.52 (1H, q, J = 10.4 Hz), 2.75–2.85
(1H, m), 3.26 (1H, d, J = 3.7 Hz), 3.97–4.07 (1H, m), 6.91 (1H, br
s), 7.39–7.46 (2H, m), 7.46–7.53 (1H, m), 7.75–7.82 (2H, m).
HRMS (APCI-) for C14H18NO2 [M-H]+: calcd, 232.11375; found,
232.13421.
5.1.47. 1-Amino-1-(2-hydroxyethyl)cyclobutane (6)
To a solution of 56 (4.00 g, 19.5 mmol) in EtOH (100 mL) was
added 10% Pd-C (500 mg). The mixture was stirred under H2 gas,
0.5 MPa, at room temperature for 6 h. The mixture was filtered,
and the filtrate was concentrated in vacuo. The crude material
was purified by distillation to give 6 as a colorless oil (1.65 g, 73%).
bp 71-72 °C (2.3 Torr). 1H NMR (400 MHz, CDCl3) d 1.60–1.72
(2H, m), 1.75 (2H, t, J = 5.5 Hz), 1.77–1.87 (2H, m), 2.03–2.13 (2H,
m), 3.81 (2H, q, J = 5.5 Hz). HRMS (CI+) for C6H14NO [M+H]+: calcd,
116.1075; found, 116.1032.
5.1.52. 1-(Benzylamino)-1-(2-hydroxypropane-1-yl)cyclobutane
(61)
To a solution of 60 (2.64 g, 11.3 mmol) in THF (40 mL) was
added LiAlH4 (1.20 g, 31.6 mmol) at room temperature and the
mixture was stirred for 1 h, then, refluxed 0.5 h. The mixture was
cooled on ice-water bath, to which was dropped a little water.
The mixture was diluted with ethyl acetate, then dried over anhy-
drous Na2SO4, filtered, and then concentrated in vacuo. The crude
material was purified by flash column chromatography on silica
gel (AcOEt) to give 61 as a colorless oil (2.25 g, 91%).
5.1.48. 1-Amino-1-(2-hydroxyethyl)cyclopentane (7)
To a solution of 57 (8.40 g, 38.3 mmol) in EtOH (150 mL) was
added 10% Pd-C (1.70 g). The mixture was stirred under H2 gas
5 kgf/cm2 at room temperature for 2 h. The mixture was filtered,
and the filtrate was concentrated in vacuo. The crude material
was purified by distillation to give 7 as a colorless oil (3.82 g, 77%).
bp 99-102 °C (6.2-5.8 Torr). 1H NMR (400 MHz, CDCl3) d 1.48–
1.82 (10H, m), 2.00–3.00 (2H, br), 3.82–3.89 (2H, m). HRMS (CI+)
for C7H16NO [M+H]+: calcd, 130.1232; found, 130.1203.
1H NMR (400 MHz, CDCl3) d 1.21 (3H, d, J = 6.1 Hz), 1.63 (1H, dd,
J = 13.7 and 2.4 Hz), 1.73–1.90 (5H, m), 2.04–2.13 (1H, m), 2.18–
2.27 (1H, m), 3.59 (1H, d, J = 11.6 Hz), 3.81 (1H, d, J = 11.6 Hz),
4.07–4.16 (1H, m), 7.22–7.35 (5H, m). HRMS (CI+) for C14H22NO
[M+H]+: calcd, 220.1701; found, 220.1693.
5.1.53. 1-Amino-1-(2-hydroxypropane-1-yl)cyclobutane (9)
To a solution of 61 (2.24 g, 10.2 mmol) in EtOH (50 mL) was
added 10% Pd-C (300 mg) and the mixture was stirred under H2
gas 5 kgf/cm2 at room temperature for 10 h. The mixture was fil-
tered, and the filtrate was concentrated in vacuo. The crude mate-
rial was purified by distillation to give 9 as a pale yellow oil (1.24 g,
94%).
5.1.49. 1-Amino-1-(1-hydroxypropane-2-yl)cyclobutane (8)
To a solution of 58 (6.81 g, 31.1 mmol) in EtOH (150 mL) was
added 10% Pd-C (700 mg). The mixture was stirred under H2 gas
5 kgf/cm2 at room temperature for 6 h. The mixture was filtered,

S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
1199
1H NMR (400 MHz, CDCl3) d 1.17 (3H, d, J = 6.1 Hz), 1.43 (1H,
ddd, J = 14.7, 10.4 and 1.2 Hz), 1.61–1.69 (1H, m), 1.72 (1H, dd,
J = 14.7 and 1.8 Hz), 1.75–1.90 (3H, m), 1.93–2.03 (1H, m), 2.12–
2.20 (1H, m), 4.00–4.08 (1H, m). HRMS (CI+) for C7H16NO [M+H]+:
calcd, 130.1232; found, 130.1227.
C18H28N2O2 0.1H2O: C, 70.60; H, 9.22; N, 9.15. Found: C, 70.38;
H, 9.06; N, 8.92.
5.1.58. 1-Amino-1-[2-(tert-
butoxycarbonylamino)ethyl]cyclobutane (10)
To a solution of 65 (117 mg, 0.384 mmol) in EtOH (5 mL) was
added 10% Pd-C (35 mg) and the mixture was stirred under H2
gas 5 kgf/cm2 at room temperature for 5 h. The mixture was fil-
tered, and the filtrate was concentrated in vacuo. The crude mate-
rial was purified by distillation to give 10 as a colorless solid
(76.0 mg, 92%).
5.1.54. 1-(Benzoylamino)cyclobutylacetic acid (62)
To a solution of 53 (3.87 g, 14.8 mmol) in EtOH (30 mL) was
added 3 N KOH (10 mL, 30 mmol) at room temperature. The mix-
ture was stirred at room temperature for 1 h. The reaction mixture
was concentrated in vacuo and added to 6 N HCl (5.0 mL). The pre-
cipitate was collected by filtration, washed successively with water
and diisopropyl ether, and then dried to give 62 as a colorless solid
(3.15 g, 91%).
Mp: 53–55 °C. 1H NMR (400 MHz, DMSO-d6) d 1.37 (9H, s),
1.49–1.58 (2H, m), 1.62–1.77 (4H, m), 1.82–1.91 (2H, m), 2.92–
3.01 (2H, m), 6.78 (1H, t, J = 5.5 Hz). HRMS (CI+) for C11H23N2O2
[M+H]+: calcd, 215.1760; found, 215.1747. Anal. calcd for
Mp: 183–185 °C. 1H NMR (400 MHz, DMSO-d6) d 1.76–1.89 (2H,
m), 2.16–2.26 (2H, m), 2.28–2.40 (2H, m), 2.90 (2H, s), 7.43 (2H, t,
J = 6.7 Hz), 7.50 (1H, t, J = 6.7 Hz), 7.83 (2H, d, J = 6.7 Hz), 8.45 (1H,
s), 12.00 (1H, br s). HRMS (CI+) for C13H16NO3 [M+H]+: calcd,
234.1130; found, 234.1115. Anal. calcd for C13H15NO3: C, 66.94;
H, 6.48; N, 6.00. Found: C, 66.84; H, 6.52; N, 6.01.
C11H22N2O2: C, 61.65; H, 10.35; N, 13.07. Found: C, 61.53; H,
10.29; N, 12.98.
5.2. Biology
5.2.1. GSK-3b inhibitory assay13
5.1.55. 1-(Benzoylamino)cyclobutylacetamide (63)
First, 10–25 ng of recombinant full-length human GSK-3b is
incubated in the presence or absence of the compound at varying
concentrations for 1 h at 30 °C in 20 mM MOPS, pH 7.0, 10 mM
magnesium acetate, 0.2 mM EDTA, 2 mM EGTA, 30 mM magne-
The mixture of 62 (1.00 g, 4.29 mmol) and thionyl chloride
(3.20 mL, 43.9 mmol) was stirred at room temperature for 1 h.
The mixture was concentrated in vacuo. The residue in THF
(30 mL) was added to 25% aqueous ammonia (30 mL) cooled on
ice water. The mixture was stirred at room temperature for 1 h.
The mixture was diluted with AcOEt–MeOH (3:1 v/v) and then
washed with water. The organic layer was dried over anhydrous
Na2SO4, filtered, and then concentrated in vacuo to give 63 as a col-
orless solid (925 mg, 93%).
sium chloride, 62.5
lM phospho-glycogen synthase peptide-2,
5
lM ATP, 10 mM b-glycerol phosphate, 1 mM sodium orthovana-
date, and 1 mM dithiothreitol. Proceed to KinaseGlo luciferase
reaction (Promega), after which an equal volume of KinaseGlo
luciferase reagent (Promega) is added. The luminescence is then
read using a luminescence plate reader within 5–10 min. Com-
pound activity is expressed as a percentage of inhibition relative
to the maximal inhibition observed at the maximal dose. IC50 val-
ues are calculated from the resulting curve using curve-fitting soft-
ware (GraphPad Prizm).
Mp: 215–217 °C. 1H NMR (400 MHz, DMSO-d6) d 1.75–1.87 (2H,
m), 2.14–2.23 (2H, m), 2.37–2.47 (2H, m), 2.70 (2H, s), 6.80 (1H, s),
7.29 (1H, s), 7.43 (2H, t, J = 6.7 Hz), 7.50 (1H, t, J = 6.7 Hz), 7.82 (2H,
d, J = 6.7 Hz), 8.39 (1H, s). HRMS (CI+) for C13H17N2O2 [M+H]+:
calcd, 233.1290; found, 233.1265. Anal. calcd for C13H16N2O2
0.5H2O: C, 64.71; H, 6.68; N, 11.61. Found: C, 64.42; H, 6.69; N,
11.53.
5.2.2. Glycogen synthesis assay in Hep G2 cells
Hep G2 cells were obtained from the Japanese Collection of
Research Bioresources and were grown in standard culture med-
ium: a low-glucose Dulbecco0s modified Eagle0s medium (DMEM),
containing 10% fetal calf serum supplemented with 100 U/mL pen-
5.1.56. 1-(2-Aminoethyl)-1-(benzylamino)cyclobutane (64)
To a solution of 63 (687 mg, 3.00 mmol) in THF (30 mL) was
added portionwise LiAlH4 (570 mg, 15.0 mmol) at room tempera-
ture for 30 min and the mixture was heated to reflux for 5 h. The
mixture was cooled on ice-water bath, to which was dropped
aqueous THF and a little water. The mixture was filtered on celite,
and the solution dried over anhydrous Na2SO4. The solution was
concentrated in vacuo to give 64 as a pale yellow oil (609 mg,
99%).
icillin and 100 lg/mL streptomycin, in a humidified and 5% CO2
atmosphere kept at 37 °C. The Hep G2 cells were harvested with
0.25% trypsin solution containing 1 mM EDTA and were seeded
on 12-well plates at 1 ꢁ 105 cells per well. Following a culture
for 3 days, the cells were washed once with phosphate-buffered
saline (PBS), and were incubated with serum-free low-glucose
DMEM supplemented with 100 U/mL penicillin and 100
streptomycin. Following a culture for 3 h, the compounds provided
herein at various concentrations and 2.5 Ci/mL
-[2-3H]glucose
lg/mL
1H NMR (400 MHz, CDCl3) d 1.65–1.80 (2H, m), 1.82 (2H, t,
J = 7.3 Hz), 1.86–2.02 (4H, m), 2.82 (2H, t, J = 7.3 Hz), 3.65 (2H, s),
7.20–7.38 (5H, m). HRMS (CI+) for C13H21N2 [M+H]+: calcd,
205.1705; found, 205.1743.
l
D
(PerkinElmer, Boston, MA, USA) were added to the serum-free
low-glucose DMEM. A vehicle control of DMSO (0.3%, final concen-
tration) was also used. The total volume per well of the reaction
medium was 1.0 mL of serum-free low-glucose DMEM. After incu-
bation at 37 °C for 3 h, the medium was aspirated and the cells
were washed twice with PBS, after which 0.25 mL of 1 N KOH con-
taining 0.4 mg/mL carrier glycogen was added. After incubation at
37 °C for 30 min, 0.25 mL of 48.8% (w/v) KOH was added to each
well for cell lysis. After incubation at 95 °C for 30 min, 1.5 mL of
95% (v/v) ethanol was added to the cell lysate. Total glycogen
was precipitated overnight at –20 °C. Glycogen precipitates were
recovered by centrifugation at 19,000 x g for 30 min at 4 °C. Precip-
itates were washed once with 1 mL of 70% (v/v) ethanol, and were
re-suspended in 0.5 mL water. [3H]Glucose incorporation into gly-
cogen was assessed using a liquid scintillation counter (Packard
Instrument, Meriden, CT, USA). The EC50 values of the tested
5.1.57. 1-(Benzylamino)-1-[2-(tert-
butoxycarbonylamino)ethyl]cyclobutane (65)
To a solution of 64 (205 mg, 1.00 mmol) in THF (1 mL) was
added Boc2O (230 mg, 1.05 mmol) under ice-water cooling. The
mixture was stirred in an ice-water bath for 30 min. The mixture
was then concentrated in vacuo. The crude material was purified
by flash column chromatography on silica gel (AcOEt:hexane = 2:1)
to give 65 as a colorless solid (164 mg, 54%).
Mp: 67–70 °C. 1H NMR (400 MHz, DMSO-d6) d 1.36 (9H, s),
1.60–1.84 (6H, m), 1.84–1.94 (2H, m), 2.92–3.04 (2H, m), 3.53
(2H, s), 6.83 (1H, t, J = 5.5 Hz), 7.21 (1H, t, J = 7.3 Hz), 7.29 (2H, t,
J = 7.3 Hz), 7.35 (2H, d J = 7.3 Hz). HRMS (CI+) for C18H29N2O2
[M+H]+: calcd, 305.2229; found, 305.2228. Anal. calcd for

1200
S. Seto et al. / Bioorg. Med. Chem. 20 (2012) 1188–1200
compounds were derived from the curve fitting using the Prism
program (GraphPad Software, San Diego, CA). The standard refer-
at 2,500ꢁg for 5 min, and separated plasma was kept on ice and
analyzed on the same day. Plasma glucose levels were determined
using the glucose C II-test (Wako Pure Chemical Industries, Osaka,
Japan). The sum of plasma glucose levels at 0.5 and 1 h after glu-
cose load was compared to that of vehicle treatment, and the re-
sults were presented as a percentage decrease.
ence compound is 1 (CHIR99021) (EC50 = 1.5
activation at 30 M).
lM, 100% maximum
l
5.2.3. Anti-bacterial activity
Anti-bacterial activity was measured by determining MIC val-
ues using standard microdilution methods recommended by the
Clinical and Laboratory Standards Institute with Mueller–Hinton
Broth (Becton Dickinson, Cockeysville, MD). The MIC was defined
as the lowest concentration of the compound that inhibited visible
growth after incubation for 18 h at 35 °C. See: Wikler, M. A.;
Cockerill, F. R., III; Bush, K.; Dudley, M. N.; Eliopoulos, G. M.; Hardy,
D. J.; Hindler, J. F.; Patel, J. B.; Powell, M.; Turnidge, J. D.; Weinstein,
M. P.; Zimmer, B. L.; Ferraro, M. J.; Swenson, J.M. in Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically; Approved Standard-Eighth Edition. Clinical and
Laboratory Standards Institute, 2009, M07-A8.
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study. Test compounds were suspended in 0.3% carboxymethyl-
cellulose sodium salt (CMC-Na; Sigma, St. Louis, MO). After fasting
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