Discovery of potent and orally active tricyclic-based FBPase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.05.041

With the aim of exploring the effect of tricyclic-based FBPase inhibitors in cells and in vivo, a series of prodrugs of tricyclic phosphonates was designed and synthesized. Introducing prodrug moieties into tricyclic-based phosphonates led to the discover

Full text of DOI:10.1016/j.bmc.2010.05.041

Bioorganic & Medicinal Chemistry 18 (2010) 5346–5351
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent and orally active tricyclic-based FBPase inhibitors
Tomoharu Tsukada a, Osamu Kanno a, Takahiro Yamane a, Jun Tanaka b, Taishi Yoshida b, Akira Okuno b,
Takeshi Shiiki c, Mizuki Takahashi d, Takahide Nishi a,
*
a Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Biological Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Exploratory Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo, 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim of exploring the effect of tricyclic-based FBPase inhibitors in cells and in vivo, a series of
prodrugs of tricyclic phosphonates was designed and synthesized. Introducing prodrug moieties into tri-
cyclic-based phosphonates led to the discovery of prodrug 15c, which strongly inhibited glucose produc-
tion in monkey hepatocytes. Furthermore, prodrug 15c lowered blood glucose levels in fasted
cynomolgus monkeys.
Received 30 March 2010
Revised 13 May 2010
Accepted 14 May 2010
Available online 20 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Fructose-1,6-bisphosphatase
FBPase inhibitors
Prodrug
1. Introduction
In the previous paper, we described the design and synthesis of
tricyclic thiazoles as FBPase inhibitors, and a series of SAR studies
Type 2 diabetes mellitus (T2DM), which accounts for more than
90% of all diabetics, is characterized by fasting hyperglycemia and
an excessive rise in the plasma glucose concentration after glucose
or meal ingestion. The global incidence of this disease is predicted
to rise to more than 366 million by the year 2030.1 T2DM usually
leads to complications such as retinopathy, nephropathy, and neu-
ropathy. Clinical studies have suggested that fasting hyperglyce-
mia in T2DM is associated with excessive glucose production
through gluconeogenesis.2 Thus, the inhibition of gluconeogenesis
is a useful approach in reducing increased blood glucose levels in
patients with T2DM.
led to the identification of phosphate 3 and difluoromethylene -
phosphonate
4 exhibiting potent FBPase inhibitory activities
(IC50 = 13, 47 nM, respectively) (Fig. 2).11 In addition, in order to im-
prove metabolic stability and enhance FBPase inhibitory activity, we
further developed tricyclic-based FBPase inhibitors with the aid of
NH2
S
NH2
S
O
P
O
P
NH
N
N
HO
HO
EtO
O
O
N
H
O
Fructose-1,6-bisphosphatase (FBPase) is one of the rate-limiting
enzymes of hepatic gluconeogenesis.3 FBPase is predominantly ex-
pressed in the liver and the kidney and catalyzes the hydrolysis of
fructose-1,6-bisphosphate to fructose-6-phosphate. FBPase inhibi-
tors would lower blood glucose levels by reducing hepatic glucose
output and are expected to be a novel class of drugs for the treat-
ment of T2DM. There are several small-molecule inhibitors of
FBPase,4–9 in which AMP mimetic MB05032 (1) exhibited high
inhibitory activity (Fig. 1). The prodrug of MB05032 (CS-917, 2)
lowered blood glucose levels in animal models and was in clinical
development.10
EtO
O
1
(MB05032)
2 (CS-917)
Figure 1. Structures of known FBPase inhibitors.
NH2
O
(HO)2P
O
N
N
S
X
(HO)2P
S
O
O
NH2
5
3
X = O
X = CF2
4
* Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: nishi.takahide.xw@daiichisankyo.co.jp (T. Nishi).
Figure 2. Structures of tricyclic-based FBPase inhibitors.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.041

T. Tsukada et al. / Bioorg. Med. Chem. 18 (2010) 5346–5351
5347
(EtO)2P
O
O
(EtO)2P
O
O
OH
O
O
d, e
a, b, c
Br
Br
COOAllyl
6
7
8
O
P
NH
EtO
N
(EtO)2㪧
O
O
H
N
N
S
O
EtO
f, g
h, i, j
㪦
S
O
COOAllyl
COOAllyl
9
10
O
P
NH
EtO
O
N
H
O
N
k, l
S
EtO
O
CONHR4
11a-c
Scheme 1. Synthesis of prodrugs 11a–c. Reagents and conditions: (a) ClCH2CH2COCl,
pyridine, CH2Cl2, 0 °C to rt, 85%; (b) AlCl3, 180 °C, 36%; (c) (EtO)2P(O)CH2OTs, K2CO3,
DMF, 80 °C, 67%; (d) Pd(OAc)2, dppp, CO, Et3N, TMSCH2CH2OH, DMF, 70 °C, 55%; (e)
TBAF, allyl bromide, Et3N, 93%; (f) CuBr2, EtOH, 60 °C, 91%; (g) P2S5, HCONH2, THF,
reflux, 71%; (h) TMSBr, CH2Cl2; (i) (COCl)2, DMF, CH2Cl2; (j) Ala-OEt, DIPEA, CH2Cl2,
À20 °C to rt, 69% over three steps; (k) Pd(PPh3)4, PPh3, pyrrolidine, MeCN, 50 °C, 92%;
(l) amine, DIPEA, WSC, HOBt, DMF, 56–88%.
Figure 3. X-ray crystal structures of human liver FBPase in complex with 5.
structure-based drug design, which led to the discovery of phospho-
nate 5, exhibiting more potent FBPase inhibitory activity
(IC50 = 1 nM).12 The key feature of phosphonate 5 was that this com-
pound possessed no amino group which might be the cause of met-
abolic instability. The X-ray co-crystal structure of 5 suggested that
high affinity was achieved by hydrophobic interaction which com-
pensated for the loss of the amino group, and also by a hydrogen-
bonding network involving the amide side chain of 5 (Fig. 3).
This paper describes our continuingefforts to explore theeffect of
the tricyclic phosphonates in cells and in vivo. Our phosphonate
compounds seemed to have low membrane permeability due to
the high negative charge of phosphonate moieties, which prompted
us to convert the phosphonate compounds into corresponding pro-
drugs. There are many classes of phosphonate prodrugs,13 from
which we selected phosphonic diamides as prodrugs in consider-
ation of the advantage that the cleavage byproducts are nontoxic
amino acids. In addition, the significant glucose-lowering effect of
diamide prodrug CS-917 in animal models encouraged us to explore
this type of prodrug. In order to investigate the cellular activity and
in vivo efficacy of our tricyclic-based inhibitors, we focused our
attentionondeveloping diamideprodrugsoftricyclic phosphonates.
OH
(EtO)2P
O
O
O
(EtO)2㪧
O
N
a, b, c
d, e
㪦
S
R2
R2
R2
R1
R1
R1
12a-c
13a-c
14a-c
(R3NH)2P
O
N
f, g, h
㪦
S
R2
R1
15a-e
Scheme 2. Synthesis of prodrugs 15a–e. Reagents and conditions: (a) ClCH2CH2COCl,
pyridine, CH2Cl2, 0 °C to rt, 56–72%; (b) AlCl3, 180 °C, 32–90%; (c) (EtO)2P(O)CH2OTs,
K2CO3, DMF, 80 °C, 78–98%; (d) CuBr2, EtOH, 60 °C, 90–99%; (e) P2S5, HCONH2, THF,
reflux, 57–66%; (f) TMSBr, CH2Cl2; (g) (COCl)2, DMF, CH2Cl2; (h) amino acid ester,
DIPEA, CH2Cl2, À20 °C to rt, 55–65% over three steps.
12a–c as starting materials (Scheme 2). The prodrugs 15a–e were
obtained by condensation with the corresponding amino acid es-
ters in the final step.
2. Results and discussion
The inhibitory effects on glucose production in monkey hepato-
cytes varied widely with the side chains of the parent phospho-
nates (Table 1). With the intent to rigorously evaluate the in vivo
potential of prodrugs, this cell assay was performed under severe
conditions wherein the hepatocytes were preincubated with pro-
drugs for only a short time (2 min). Initially, phosphonate 5, which
possesses an amide side chain and demonstrated potent inhibitory
activities against human and monkey FBPase (IC50 = 1, 22 nM,
The prodrugs of the tricyclic phosphonates were synthesized
according to Schemes 1 and 2. The prodrugs containing amide side
chains (11a–c) were prepared from commercially available 4-bro-
mophenol 6 (Scheme 1). Acylation of 6 followed by a Fries rear-
rangement and the introduction of a diethyl phosphonate unit
resulted in diethyl phosphonate 7. CO insertion of 7 followed by
a transesterification reaction afforded allyl ester 8, which was
transformed into tricyclic thiazole 9 via bromination and cycliza-
tion with thioformamide (in situ generation).
10 was obtained by cleaving the phosphonate ethyl groups of 9,
dichlorination, and subsequent condensation with -alanine ethyl
esters. Cleaving an allyl group of 10 and amidation with corre-
sponding amines led to prodrugs 11a–c. Prodrugs containing alkyl
side chains were synthesized by methods similar to those de-
scribed for prodrugs 11a–c, using side chains containing phenols
respectively), was converted to L-alanine diamide 11a. However,
prodrug 11a showed only a modest inhibitory effect on glucose
production, about ninefold less potent than CS-917. Similarly, pro-
drug 11b and 11c containing another amide side chain showed lit-
tle effect. In contrast, our efforts to convert the phosphonates
containing alkyl side chains to prodrugs resulted in a major in-
crease in inhibitory activities in the cell assays. Prodrug 15a con-
taining a methyl side chain exhibited high inhibitory activity
L-Alanine diamide
L
(IC50 = 7.4 lM), whereas prodrug 15b containing a ethyl side chain

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T. Tsukada et al. / Bioorg. Med. Chem. 18 (2010) 5346–5351
Table 1
The inhibitory effects on glucose production in hepatocytes
(R3NH)2P
O
O
N
S
R2
R1
R1
R2
R3
Human FBPase IC50 (nM)
Monkey FBPase IC50 (nM)
Hepatocytes IC50 (lM)
a
b
c
Compound
11a
11b
11c
15a
15b
15c
15d
15e
CS-917
CONH2
H
H
H
H
(S)-CH(Me)CO2Et
(S)-CH(Me)CO2Et
(S)-CH(Me)CO2Et
(S)-CH(Me)CO2Et
(S)-CH(Me)CO2Et
(S)-CH(Me)CO2Et
(S)-CH(Me)CO2iPr
CH2CO2Et
1
2
11
8
11
10
10
10
10
22
19
31
24
23
34
34
34
30
94
CONHMe
>100
>100
7.4
23
6.8
9.0
9.4
10
CONHCH2tBu
Me
Et
Me
Me
Me
—
H
Me
Me
Me
—
—
a
b
c
Inhibition of human liver FBPase (corresponding parent phosphonates).
Inhibition of monkey liver FBPase (corresponding parent phosphonates).
Inhibition of glucose production in primary monkey hepatocytes.
was about threefold less potent (IC50 = 23
tion, prodrug 15c containing two methyl side chains exhibited
inhibitory activity (IC50 = 6.8 M) similar to that of prodrug 15a.
Prodrugs derived from other amino acid esters such as -alanine
isopropyl esters (15d) and glycine ethyl esters (15e) were slightly
less potent (IC50 = 9.0, 9.4 M, respectively) than -alanine diamide
15c. Large differences in the inhibitory activity between prodrugs
11a–c and 15a–e suggested that a judicious combination of tricy-
clic-based phosphonate and prodrug moiety was required to exhi-
bit high inhibitory activity in cell assays.
lM) than 15a. In addi-
70
60
50
40
l
L
l
L
*
Vehicle
15c
The physicochemical properties of the prodrugs provided
important clues for understanding a broad range of inhibitory
activities in the cell assays (Table 2). Some physicochemical prop-
erties are useful in predicting drug absorption. Considering Lipin-
0
2
4
6
8
10
12
Time (h)
ski’s rules14 and the polar surface area (PSA),15
L-alanine diamide
Figure 4. Oral glucose-lowering effect of prodrug 15c in fasted cynomolgus
monkeys (20 mg/kg). Data were the mean S.E.M (n = 7, P <0.05 compared to
*
prodrugs 11a–c and 15a–c appear to have different physicochem-
ical parameters with respect to cLog P and PSA. In particular, com-
pared to prodrugs 15a–c, which possess alkyl side chains and
exhibited potent inhibitory effects in the cell assays, prodrugs
11a–c have high PSA values due to the presence of amide side
chains. PSA is defined as the sum of the surfaces of the polar atoms
in a molecule and correlate well with cell membrane permeability.
A short preincubation of hepatocytes with prodrugs in the cell as-
says resulted in placing emphasis on the cell membrane perme-
ability of the prodrugs. It was suggested that prodrugs 15a–c
exhibited high inhibitory activities in the cell assays, presumably
due to low PSA values and relatively high membrane permeability.
Prodrug 15c exhibiting high inhibitory activity in the cell assays
also demonstrated in vivo efficacy in an animal model (Fig. 4). In
vehicle group, paired t-test).
order to investigate the in vivo efficacy of the prodrugs, fasted
cynomolgus monkeys were used as an animal model, considering
that the fasting plasma glucose level correlates with the hepatic
glucose production originating in gluconeogenesis. Prodrug 15c
was administered orally at 20 mg/kg to fasted cynomolgus mon-
keys. The blood glucose levels of the monkeys were significantly
decreased 4 h after dosing compared to the vehicle-treated ani-
mals. Hypoglycemia, a safety concern of gluconeogenesis inhibi-
tion, was not observed. In addition, the plasma concentration of
prodrug 15c and corresponding phosphonate 16 in this in vivo
study showed that prodrug 15c disappeared rapidly and the corre-
Table 2
Physicochemical properties of L-alanine diamide prodrugsa
HB-Db
HB-Ac
cLog P
PSAd
Hepatocytes IC50
(l
M)
e
Compound
MW
11a
11b
11c
15a
15b
15c
524.53
538.55
594.66
495.53
509.56
509.56
4
3
3
2
2
2
11
11
11
9
9
9
0.69
0.9
2.66
2.47
3
196.99
183
183
153.9
153.9
153.9
94
>100
>100
7.4
23
6.8
2.93
a
b
c
Calculated using ACD/labs version 9.0 (Advanced Chemistry Development, Inc.).
Hydrogen bond donors.
Hydrogen bond acceptors.
d
e
Polar surface area (Å2).
Inhibition of glucose production in primary monkey hepatocytes (experimental values).

T. Tsukada et al. / Bioorg. Med. Chem. 18 (2010) 5346–5351
5349
lyzer (i-STAT Corp., Princeton, NJ). The concentrations of prodrug
15c and phosphonate 16 were also measured by using LC–MS/MS
analysis (Quattro micro, Micromass Ltd).
10
1
16
15c
(HO)
O
2P
O
N
S
4.2. Chemistry
16
0.1
NMR spectra were recorded on a Varian Mercury 400 or 500 spec-
trometer with tetramethylsilane as an internal reference. Infrared
spectra were recorded on a Jasco FT/IR-830 spectrophotometer.
Mass spectra were recorded on a JEOL JMS-AX505H. TLC analysis
was performed on 60F254 plates. Column chromatography was per-
formed on Silica gel 60 (Merck, 230–400).
O
P
NH
EtO
N
O
H
0.01
0.001
N
O
S
EtO
O
15c
0
2
4
6
8
10
12
Time (h)
4.2.1. Diethyl {[(6,7-dimethyl-3-oxo-2,3-dihydro-1H-inden-4-
yl)oxy]methyl}phosphonate (13c)
Figure 5. Plasma concentration of prodrug 15c and corresponding parent phos-
phonate 16 in cynomolgus monkeys. Data were the mean SD (n = 7).
A solution of 3,4-dimethylphenol 12c (20.0 g, 164 mmol) in
CH2Cl2 (328 mL) was cooled to 0 °C. After the addition of pyridine
(19.9 mL, 246 mmol) and 3-chloropropanoyl chloride (20.4 mL,
213 mmol) at 0 °C, the mixture was stirred at room temperature
for 2 h. A saturated solution of NH4Cl was added and the aqueous
layer was extracted with CH2Cl2. The organic layer was washed with
a saturated solution of NaCl, dried over Na2SO4 and evaporated. The
residue was purified by column chromatography (hexane/EtOAc) to
afford 22.0 g of 3,4-dimethylphenyl 3-chloropropanoate (63% yield).
A mixture of 3,4-dimethylphenyl 3-chloropropanoate (22.0 g,
103 mmol) and aluminum chloride (55.2 g, 414 mmol) was heated
at180 °C for2 h. Aftercooling, ice, concentratedHClandCH2Cl2 were
added and the aqueous layer was extracted with CH2Cl2. The com-
bined organic layers were washed with H2O and a saturated solution
of NaCl, dried over Na2SO4 and evaporated. The remaining solid was
collected by filtration and washed with EtOAc to afford 14.1 g of 7-
hydroxy-4,5-dimethylindan-1-one (78% yield).
sponding parent phosphonate 16 remained for a long time at rela-
tively high concentrations (Fig. 5). This result suggested that pro-
drug 15c was rapidly converted to corresponding phosphonate
16 in the body and L-alanine diamide prodrug moiety functioned
as a prodrug. Reliable evidence of the in vivo efficacy of prodrug
15c has provided the possibility of developing new drugs for the
treatment of T2DM.
3. Conclusion
In summary, we developed prodrugs of tricyclic-based FBPase
inhibitors in order to investigate their effects in cells and in vivo.
In contrast to prodrugs containing amide side chains, prodrugs
containing alkyl side chains potently inhibited glucose production
in monkey hepatocytes. The physicochemical properties of pro-
drugs suggested that low PSA values and relatively high membrane
permeability was probably critical to the exhibition of high inhib-
itory activity in the cell assays. The in vivo study in fasted cyno-
molgus monkeys revealed that prodrug 15c lowered blood
glucose levels and was rapidly converted to corresponding phos-
phonate 16. These results have provided the possibility of develop-
ing new drugs for the treatment of T2DM.
A
mixture of 7-hydroxy-4,5-dimethylindan-1-one (5.00 g,
28.4 mmol), K2CO3 (5.89 g, 42.6 mmol) and diethoxyphosphorylm-
ethyl tosylate (11.0 g, 34.1 mmol) in DMF (95 mL) was stirred at
80 °C for 4 h. After cooling, a saturated solution of NH4Cl was added.
DMF was evaporated and the remaining aqueous solution was ex-
tracted with EtOAc. The organic layer was washed with a saturated
solution of NaCl, dried over Na2SO4 and evaporated. The residue
was purified by column chromatography (hexane/EtOAc) to afford
7.26 g of 13c (78% yield). 1H NMR (400 MHz, CDCl3): d 1.36 (6H, t,
J = 7.1 Hz), 2.16 (3H, s), 2.33 (3H, s), 2.62–2.66 (2H, m), 2.92–2.97
(2H, m), 4.32 (4H, dq, J = 7.1, 7.1 Hz), 4.40 (2H, d, J = 9.4 Hz), 6.67
(1H, s). IR (KBr): 1705, 1244, 1029 cmÀ1. MS (FAB): m/z 327 (M+H)+ .
4. Experimental
4.1. Biological assays
4.1.1. Gluconeogenesis inhibition in monkey hepatocytes
4.2.2. Diethyl {[(6,7-dimethyl-8H-indeno[1,2-d][1,3]thiazol-4-
Primary monkey hepatocytes (KAC, Kyoto, Japan) were incu-
yl)oxy]methyl}phosphonate (14c)
bated overnight in DMEM containing 5% FBS, 1
lM dexametha-
A
mixture of 13c (7.26 g, 22.2 mmol) and CuBr2 (9.92 g,
sone, and 100 M dbcAMP before being washed with Dulbecco’s
l
44.4 mmol) in EtOH (74 mL) was heated at 60 °C for 2 h. After cool-
ing, asaturatedsolutionofNaHCO3 wasadded. EtOHwasevaporated
and the remaining aqueous solution was extracted with EtOAc. The
organic layer was washed with a saturated solution of NH4Cl, H2O
and a saturated solution of NaCl, dried over Na2SO4 and evaporated
to afford 9.00 g of diethyl {[(2-bromo-6,7-dimethyl-3-oxo-2,3-dihy-
dro-1H-inden-4-yl)oxy]methyl}phosphonate (99% yield).
Formamide (2.65 mL, 66.6 mmol) was added dropwise to a sus-
pension of P2S5 (2.96 g, 13.3 mmol) in THF (89 mL). The mixture
was stirred at room temperature for 4 h and filtered. The filtrate
was added to a solution of diethyl {[(2-bromo-6,7-dimethyl-3-
oxo-2,3-dihydro-1H-inden-4-yl)oxy]methyl}phosphonate (9.00 g,
22.2 mmol) in THF (22 mL) and the mixture was refluxed for 2 h.
After cooling, a saturated solution of NaHCO3 was added. THF
was evaporated and the remaining aqueous solution was extracted
with CH2Cl2. The organic layer was washed with a saturated solu-
tion of NaCl, dried over Na2SO4 and evaporated. The residue was
purified by column chromatography (EtOAc/MeOH). Recrystalliza-
phosphate buffered saline containing 25 mM sodium bicarbonate,
25 mM HEPES and 1% BSA (test medium). The hepatocytes were
preincubated in the test medium with test compounds for 2 min,
followed by subsequent incubation for 4 h with gluconeogenic
substrates (10 mM lactate and 1 mM pyruvate) after washing out
the test compounds. The glucose concentration in the medium
was measured by Glucose CII-test Wako (Wako Pure Chemical
Industries, Ltd, Osaka, Japan).
4.1.2. In vivo efficacy in cynomolgus monkeys
Vehicle (50% PEG 400, 0.5% MC, 0.5% Tween 80) or prodrug 15c
at 20 mg/kg (dissolved in the vehicle) was orally administered to
overnight-fasted cynomolgus monkeys (n = 7, 5–8 years old). The
same monkeys were used for each regimen after a washout period
of at least 2 weeks. Blood samples were collected just before dosing
and 1, 2, 4, 6, 8, 10, and 12 h after dosing. The plasma glucose levels
in each sample were measured using an i-STAT 300F blood ana-

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T. Tsukada et al. / Bioorg. Med. Chem. 18 (2010) 5346–5351
tion from EtOAc–hexane produced 4.70 g of 14c (58% yield). 1H
NMR (400 MHz, CDCl3): d 1.35 (6H, t, J = 7.0 Hz), 2.24 (3H, s),
2.34 (3H, s), 3.74 (2H, s), 4.31 (4H, dq, J = 7.0, 7.0 Hz), 4.63 (2H, d,
J = 8.6 Hz), 6.88 (1H, s), 8.79 (1H, s). IR (KBr): 2980, 1236, 1106,
1047, 1024 cmÀ1. MS (FAB): m/z 368 (M+H)+.
4.2.7. Ethyl 4-{[(6,7-dimethyl-8H-indeno[1,2-d][1,3]thiazol-4-
yl)oxy]methyl}-7-oxo-8-oxa-3,5-diaza-4-phosphadecan-1-oate
4-oxide (15e)
The title compound was prepared according to the procedure
for 15c utilizing ethyl glycinate hydrochloride instead of ethyl L-
alaninate methanesulfonate. 1H NMR (400 MHz, CDCl3): d 1.24
(6H, t, J = 7.3 Hz), 2.25 (3H, s), 2.33 (3H, s), 3.76 (2H, s), 3.81–4.00
(4H, m), 4.17 (4H, q, J = 7.3 Hz), 4.43 (2H, d, J = 9.4 Hz), 4.66–4.76
(2H, m), 6.89 (1H, s), 8.88 (1H, s). MS (FAB): m/z 482 (M+H)+.
4.2.3. Ethyl (2S,6S)-4-{[(6,7-dimethyl-8H-indeno[1,2-d][1,3]thia
zol-4-yl)oxy]methyl}-2,6-dimethyl-7-oxo-8-oxa-3,5-diaza-4-
phosphadecan-1-oate 4-oxide (15c)
Bromotrimethylsilane (3.59 mL, 27.2 mmol) was added to a solu-
tion of 14c (2.00 g, 5.44 mmol) in CH2Cl2 (54 mL). The mixture was
stirred at room temperature for 16 h and the volatiles were
evaporated.
The remaining residue was dissolved in CH2Cl2 (54 mL). After the
addition of oxalyl chloride (2.37 mL, 27.2 mmol) and DMF (one
drop), the mixture was stirred at room temperature for 1 h. The reac-
tion mixture was concentrated and azeotroped with toluene.
The remaining residue was dissolved in CH2Cl2 (54 mL) and
4.2.8. Diethyl {[(7-bromo-3-oxo-2,3-dihydro-1H-inden-4-yl)oxy
]methyl}phosphonate (7)
The title compound was prepared according to the procedure
for 13c from 4-bromophenol 6 instead of 3,4-dimethylphenol
12c. 1H NMR (500 MHz, CDCl3): d 1.37 (6H, t, J = 7.3 Hz), 2.66–
2.72 (2H, m), 2.99–3.05 (2H, m), 4.33 (4H, dq, J = 7.3, 7.3 Hz),
4.42 (2H, d, J = 8.8 Hz), 6.81 (1H, d, J = 8.8 Hz), 7.66 (1H, d,
J = 8.8 Hz). MS (FAB): m/z 377 (M+H)+.
cooled to À20 °C. After the addition of ethyl
L-alaninate methanesul-
4.2.9. Allyl 7-[(diethoxyphosphoryl)methoxy]-1-oxoindane-4-
carboxylate (8)
fonate (5.80 g, 27.2 mmol) and N,N-diisopropylethylamine (9.48
mL, 54.4 mmol) at À20 °C, the mixturewas allowed to warm to room
temperature and stirred for 4 h. A saturated solution of NaHCO3 was
added and the aqueous phase was extracted with CH2Cl2. The organ-
ic layer was washed with a saturated solution of NaCl, dried over
Na2SO4 and evaporated. The residue was purified by column chro-
matography (EtOAc/MeOH) to afford 1.51 g of 15c (55% yield). 1H
NMR (400 MHz, CDCl3): d 1.22 (3H, t, J = 7.0 Hz), 1.23 (3H, t,
J = 7.0 Hz), 1.42 (3H, d, J = 7.0 Hz), 1.48 (3H, d, J = 7.0 Hz), 2.24 (3H,
s), 2.32 (3H, s), 3.75 (2H, s), 4.07–4.23 (6H, m), 4.30 (1H, dd,
J = 12.5, 9.6 Hz), 4.37–4.45 (1H, m), 4.38 (1H, dd, J = 12.5, 9.0 Hz),
4.95–5.07 (1H, m), 6.84 (1H, s), 8.86 (1H, s). IR (KBr): 3199, 1748,
1193, 1159 cmÀ1. MS (FAB): m/z 510 (M+H)+.
A mixture of 7 (20.1 g, 53.3 mmol), 2-(trimethylsilyl)ethanol
(75.0 mL, 523 mmol), triethylamine (25.0 mL, 180 mmol), 1,3-
bis(diphenylphosphino)propane (6.60 g, 16.0 mmol) and palla-
dium(II) acetate (3.59 g, 16.0 mmol) in DMF (80 mL) was purged
with carbon monoxide gas and heated at 70 °C for 18 h. After cool-
ing, the mixture was filtered, concentrated and diluted with EtOAc
and H2O. The organic layer was washed with H2O and a saturated
solution of NaCl, dried over Na2SO4 and evaporated. The residue
was purified by column chromatography (hexane/EtOAc) to afford
13.0 g of 2-(trimethylsilyl)ethyl 7-[(diethoxyphosphoryl)methoxy]-
1-oxoindane-4-carboxylate (55% yield).
To a solution of 2-(trimethylsilyl)ethyl 7-[(diethoxyphospho-
ryl)methoxy]-1-oxoindane-4-carboxylate (13.0 g, 29.4 mmol) in
THF (250 mL) was added a 1.0 M TBAF solution in THF (44.0 mL,
44.0 mmol). After stirring at room temperature for 15 h, triethyl-
amine (12.2 mL, 87.5 mmol) and allyl bromide (7.60 mL, 87.8 mmol)
were added. The mixture was stirred at room temperature for 4 h,
concentrated and diluted with EtOAc and H2O. The organic layer
was washed with H2O and a saturated solution of NaCl, dried over
Na2SO4 and evaporated. The residue was purified by column chro-
matography (hexane/EtOAc) to afford 10.5 g of 8 (93% yield). 1H
NMR (400 MHz, CDCl3): d 1.38 (6H, t, J = 7.0 Hz), 2.64–2.70 (2H, m),
3.43–3.48 (2H, m), 4.34 (2H, q, J = 7.0 Hz), 4.36 (2H, q, J = 7.0 Hz),
4.48 (2H, d, J = 9.0 Hz), 4.83 (2H, dt, J = 5.5, 1.2 Hz), 5.31 (1H, dd,
J = 10.6, 1.2 Hz), 5.41 (1H, dd, J = 17.2, 1.2 Hz), 5.99–6.11 (1H, m),
6.93 (1H, d, J = 8.6 Hz), 8.28 (1H, d, J = 8.6 Hz). MS (FAB): m/z 383
(M+H)+.
4.2.4. Ethyl (2S,6S)-2,6-dimethyl-4-{[(7-methyl-8H-indeno[1,2-
d][1,3]thiazol-4-yl)oxy]methyl}-7-oxo-8-oxa-3,5-diaza-4-
phosphadecan-1-oate 4-oxide (15a)
The title compound was prepared according to the procedure
for 15c from 4-methylphenol 12a as a starting material instead
of 3,4-dimethylphenol 12c. 1H NMR (400 MHz, CDCl3): d 1.20–
1.26 (6H, m), 1.43 (3H, d, J = 7.0 Hz), 1.48 (3H, d, J = 7.0 Hz), 2.36
(3H, s), 3.75 (2H, s), 4.06–4.26 (6H, m), 4.29–4.46 (3H, m), 4.95–
5.07 (1H, m), 6.96 (1H, d, J = 8.2 Hz), 7.03 (1H, d, J = 8.2 Hz), 8.91
(1H, s). MS (FAB): m/z 496 (M+H)+.
4.2.5. Ethyl (2S,6S)-4-{[(7-ethyl-8H-indeno[1,2-d][1,3]thiazol-4-
yl)oxy]methyl}-2,6-dimethyl-7-oxo-8-oxa-3,5-diaza-4-
phosphadecan-1-oate 4-oxide (15b)
The title compound was prepared according to the procedure
for 15c from 4-ethylphenol 12b as a starting material instead of
3,4-dimethylphenol 12c. 1H NMR (400 MHz, CDCl3): d 1.20–1.25
(6H, m), 1.28 (3H, t, J = 7.4 Hz), 1.43 (3H, d, J = 7.0 Hz), 1.48 (3H,
d, J = 7.0 Hz), 2.70 (2H, q, J = 7.4 Hz), 3.79 (2H, s), 4.05–4.24 (6H,
m), 4.29–4.44 (3H, m), 4.95–5.06 (1H, m), 6.97 (1H, d, J = 8.6 Hz),
7.05 (1H, d, J = 8.6 Hz), 8.88 (1H, s). MS (FAB): m/z 510 (M+H)+.
4.2.10. Allyl 4-[(diethoxyphosphoryl)methoxy]-8H-indeno[1,2-
d][1,3]thiazole-7-carboxylate (9)
The title compound was prepared according to the procedure
for 14c from 8 instead of 13c. 1H NMR (400 MHz, CDCl3): d 1.35
(6H, t, J = 6.8 Hz), 4.25 (2H, s), 4.33 (2H, q, J = 6.8 Hz), 4.35 (2H, q,
J = 6.8 Hz), 4.67 (2H, d, J = 8.8 Hz), 4.86 (2H, dt, J = 5.9, 1.5 Hz),
5.32 (1H, dd, J = 10.3, 1.5 Hz), 5.44 (1H, ddd, J = 17.1, 2.9, 1.2 Hz),
6.03–6.13 (1H, m), 7.11 (1H, d, J = 8.8 Hz), 8.01 (1H, d, J = 8.8 Hz),
8.85 (1H, s). MS (FAB): m/z 424 (M+H)+
4.2.6. Isopropyl (2S,6S)-4-{[(6,7-dimethyl-8H-indeno[1,2-d][1,3]
thiazol-4-yl)oxy]methyl}-2,6,9-trimethyl-7-oxo-8-oxa-3,5-diaza
-4-phosphadecan-1-oate 4-oxide (15d)
The title compound was prepared according to the procedure
4.2.11. Allyl 4-[(bis{[(1S)-2-ethoxy-1-methyl-2-oxoethyl]amino}
phosphoryl)methoxy]-8H-indeno[1,2-d][1,3]thiazole-7-carboxylate
(10)
for 15c utilizing isopropyl
L-alaninate hydrochloride instead of
ethyl
L
-alaninate methanesulfonate. 1H NMR (500 MHz, CDCl3): d
1.17–1.25 (12H, m), 1.42 (3H, d, J = 6.8 Hz), 1.47 (3H, d,
J = 7.3 Hz), 2.25 (3H, s), 2.33 (3H, s), 3.76 (2H, s), 4.10–4.21 (2H,
m), 4.27–4.44 (3H, m), 4.96–5.09 (3H, m), 6.86 (1H, s), 8.89 (1H,
s). MS (FAB): m/z 538 (M+H)+.
The title compound was prepared according to the procedure
for 15c from 9 instead of 14c. 1H NMR (400 MHz,, CDCl3): d 1.20
(3H, t, J = 7.0 Hz), 1.21 (3H, t, J = 7.0 Hz), 1.43 (3H, d, J = 7.4 Hz),
1.47 (3H, d, J = 7.4 Hz), 4.02–4.22 (7H, m), 4.24 (2H, s), 4.42 (1H,

T. Tsukada et al. / Bioorg. Med. Chem. 18 (2010) 5346–5351
5351
dd, J = 12.1, 9.8 Hz), 4.51 (1H, dd, J = 12.1, 9.4 Hz), 4.82–4.95 (3H,
m), 5.29–5.34 (1H, m), 5.39–5.46 (1H, m), 6.00–6.12 (1H, m),
7.03 (1H, d, J = 8.6 Hz), 7.98 (1H, d, J = 8.6 Hz), 8.85 (1H, s). MS
(FAB): m/z 566 (M+H)+.
1H NMR (400 MHz, CDCl3): d 1.01 (9H, s), 1.18–1.24 (6H, m), 1.43
(3H, d, J = 6.3 Hz), 1.47 (3H, d, J = 7.0 Hz), 3.29 (2H, d, J = 6.7 Hz),
4.03–4.22 (7H, m), 4.23 (2H, s), 4.39 (1H, dd, J = 12.1, 9.4 Hz),
4.47 (1H, dd, J = 12.1, 9.4 Hz), 4.85–5.05 (1H, m), 6.05–6.13 (1H,
m), 7.02 (1H, d, J = 8.6 Hz), 7.40 (1H, d, J = 8.6 Hz), 8.86 (1H, s).
MS (FAB): m/z 595 (M+H)+.
4.2.12. Ethyl (2S,6S)-4-{[(7-carbamoyl-8H-indeno[1,2-d][1,3]
thiazol-4-yl)oxy]methyl}-2,6-dimethyl-7-oxo-8-oxa-3,5-diaza-
4-phosphadecan-1-oate 4-oxide (11a)
References and notes
A mixture of 10 (7.76 g, 13.7 mmol), tetrakis(triphenylphos-
phine)palladium(0) (793 mg, 0.686 mmol), triphenylphosphine
(720 mg, 2.74 mmol) and pyrrolidine (2.29 mL, 27.4 mmol) in aceto-
nitrile(150 mL)was heated at50 °C for 2 h. Themixturewas concen-
trated and diluted with EtOAc and a solution of NaHCO3. The
aqueous layer was washed with EtOAc. After the addition of 1 N
hydrochloric acid, the aqueous layer was extracted with EtOAc and
the organic layer was washed with saturated solution of NaCl, dried
over Na2SO4 and evaporated. The residue was purified by column
chromatography (EtOAc/MeOH) to afford 6.62 g of 4-[(bis{[(1S)-2-
ethoxy-1-methyl-2-oxoethyl]amino}phosphoryl)methoxy]-8H-in-
deno[1,2-d][1,3]thiazole-7-carboxylic acid (92% yield).
A mixture of 4-[(bis{[(1S)-2-ethoxy-1-methyl-2-oxoethyl]amino
}phosphoryl)methoxy]-8H-indeno[1,2-d][1,3]thiazole-7-carboxylic
acid (1.65 g, 3.14 mmol), ammonium chloride (840 mg, 15.7 mmol),
diisopropylethylamine (2.73 mL, 15.7 mmol), 1-ethyl-3-(3-dimeth-
ylaminopropyl)carbodiimide hydrochloride (1.20 g, 6.28 mmol) and
1-hydroxybenzotriazole (849 mg, 6.28 mmol) in DMF (25 mL) was
stirred at room temperature for 4 h. The mixture was concentrated
and the remaining solid was collected by filtration and washed with
H2O and acetone. Recrystallization from EtOH produced 926 mg of
11a (56% yield). 1H NMR (500 MHz, DMSO-d6): d 1.07–1.13 (6H, m),
1.29 (3H, d, J = 7.3 Hz), 1.30 (3H, d, J = 7.3 Hz), 3.90–4.16 (6H, m),
4.22 (2H, s), 4.32 (1H, dd, J = 12.2, 8.8 Hz), 4.40 (1H, dd, J = 12.2,
8.8 Hz), 4.94 (1H, t, J = 11.2 Hz), 5.04 (1H, t, J = 11.2 Hz), 7.17 (1H, d,
J = 8.8 Hz), 7.27–7.34 (1H, m), 7.69 (1H, d, J = 8.8 Hz), 7.82–7.88 (1H,
m), 9.14 (1H, s). MS (FAB): m/z 525 (M+H)+.
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4.2.13. Ethyl (2S,6S)-2,6-dimethyl-4-({[7-(methylcarbamoyl)-
8H-indeno[1,2-d][1,3]thiazol-4-yl]oxy}methyl)-7-oxo-8-oxa-
3,5-diaza-4-phosphadecan-1-oate 4-oxide (11b)
The title compound was prepared according to the procedure
for 11a utilizing methylamine hydrochloride instead of ammonium
chloride. 1H NMR (400 MHz, CDCl3): d 1.14–1.26 (6H, m), 1.43 (3H,
d, J = 7.0 Hz), 1.47 (3H, d, J = 7.0 Hz), 3.03 (3H, d, J = 5.1 Hz),
4.03–4.24 (7H, m), 4.25 (2H, s), 4.36 (1H, dd, J = 12.1, 9.4 Hz),
4.45 (1H, dd, J = 12.1, 9.4 Hz), 4.87–4.98 (1H, m), 6.22–6.31 (1H,
m), 7.00 (1H, d, J = 8.6 Hz), 7.42 (1H, d, J = 8.6 Hz), 8.88 (1H, s).
MS (FAB): m/z 539 (M+H)+.
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4.2.14. Ethyl (2S,6S)-4-[({7-[(2,2-dimethylpropyl)carbamoyl]-
8H-indeno[1,2-d][1,3]thiazol-4-yl}oxy)methyl]-2,6-dimethyl-7-
oxo-8-oxa-3,5-diaza-4-phosphadecan-1-oate 4-oxide (11c)
The title compound was prepared according to the procedure
for 11a utilizing neopentylamine instead of ammonium chloride.