Synthesis of novel 2-benzothiopyran and 3-benzothiepin derivatives and their stimulatory effect on bone formation

Article abstract of DOI:10.1021/jm980583b

In a search for therapeutic agents for the treatment of osteoporosis and bone fracture, we found that 2-benzothiopyran-1-carboxamide derivatives 1, derived from ipriflavone as a lead compound, increase cellular alkaline phosphatase activity in cultures of rat bone marrow stromal cells. Further modification of 1 has led to the discovery of more potent 3-benzothiepin-2- carboxamide derivatives 2. Of these, 3-benzothiepin derivatives bearing a 4- (dialkoxyphosphorylmethyl)phenyl group on the 2-carboxamide moiety such as 2h and 2q exhibited significant improvement of activity compared to ipriflavone. Asymmetric synthesis of 2h and 2q revealed that the (-)-isomers possessed activities superior to those of the (+)-isomers. Further evaluation of these compounds using the mouse osteoblastic cell line MC3T3-E1 revealed that (-)- 2q enhanced the effect of bone morphogenetic protein. In addition, application of a sustained-release agent containing 2q increased the area of newly formed bone in a rat skull defect model. Based on these findings, (-)- 2q was selected for further investigation as a new drug stimulating bone formation. Synthesis and structure-activity relationships for this novel series of 2-benzothiopyran and 3-benzothiepin derivatives are detailed.

Full text of DOI:10.1021/jm980583b

J . Med. Chem. 1999, 42, 751-760
751
Syn th esis of Novel 2-Ben zoth iop yr a n a n d 3-Ben zoth iep in Der iva tives a n d Th eir
Stim u la tor y Effect on Bon e F or m a tion 1
Tsuneo Oda, Kohei Notoya, Masayuki Gotoh, Shigehisa Taketomi, Yukio Fujisawa, Haruhiko Makino, and
Takashi Sohda*
Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., 17-85, J usohonmachi 2-chome, Yodogawa-ku,
Osaka 532-8686, J apan
Received October 13, 1998
In a search for therapeutic agents for the treatment of osteoporosis and bone fracture, we found
that 2-benzothiopyran-1-carboxamide derivatives 1, derived from ipriflavone as a lead
compound, increase cellular alkaline phosphatase activity in cultures of rat bone marrow
stromal cells. Further modification of 1 has led to the discovery of more potent 3-benzothiepin-
2-carboxamide derivatives 2. Of these, 3-benzothiepin derivatives bearing a 4-(dialkoxyphos-
phorylmethyl)phenyl group on the 2-carboxamide moiety such as 2h and 2q exhibited significant
improvement of activity compared to ipriflavone. Asymmetric synthesis of 2h and 2q revealed
that the (-)-isomers possessed activities superior to those of the (+)-isomers. Further evaluation
of these compounds using the mouse osteoblastic cell line MC3T3-E1 revealed that (-)-2q
enhanced the effect of bone morphogenetic protein. In addition, application of a sustained-
release agent containing 2q increased the area of newly formed bone in a rat skull defect model.
Based on these findings, (-)-2q was selected for further investigation as a new drug stimulating
bone formation. Synthesis and structure-activity relationships for this novel series of
2-benzothiopyran and 3-benzothiepin derivatives are detailed.
Ch a r t 1
In tr od u ction
Metabolic bone diseases are disorders representative
of the aging process. The associated decrease in bone
mass frequently results in fractures in aged individuals
who require prolonged periods of hospitalization.2 Often
this results in significant inactivity and morbidity,
leading not only to a reduction in the overall physical
and mental well-being of patients but also to negative
economic effects on society.
repair.9 However, they are polypeptides, and have
problems associated with their route of administration,
cost of production, and systemic and local toxicity.
Therefore, their clinical application may be limited.
In the mature skeleton, bone mass is maintained by
a process of bone remodeling in which old bone is
removed and then replaced by new bone. This process
persists throughout life as a delicate balance between
bone resorption by osteoclasts and bone formation by
osteoblasts. An increment in osteoclastic activity and/
or a decrease in osteoblastic activity can readily con-
tribute to steady and progressive bone loss and conse-
quent increase in risk of fracture.2 Present therapeutic
strategies are focused on preventing further bone loss
using antiresorptive drugs, such as calcitonins,3 bispho-
sphonates,4 estrogens,5 and selective estrogen receptor
modulators (SERMs).6 On the other hand, there is no
satisfactory bone anabolic agent which restores lost bone
mass and stimulates fracture healing. Although fluo-
ride7 and parathyroid hormone (PTH)8 are known to
increase bone mass, these drug therapies need special
care of administration because of their possible side
effects (clinical trials are ongoing with sustained-release
agent of fluoride and with intermittent low-dose admin-
istration of PTH). In recent years, local growth factors
including bone morphogenetic proteins (BMPs) have
attracted particular interest in the field of fracture
We previously reported that ipriflavone (7-isopro-
poxyisoflavone, Chart 1) enhanced bone-like tissue
formation in vitro due to stimulation of differentiation
of rat bone marrow stromal cells into osteoblasts.10 This
finding led us to search for compounds with more potent
effects on bone formation than ipriflavone. Among
compounds designed based on the chemical structure
of ipriflavone, 2-benzothiopyran-1-carboxamide deriva-
tives (1, Chart 1) were found to increase cellular alkaline
phosphatase (ALP) activity, one of the markers char-
acteristic of the osteoblast phenotype, in cultures of rat
bone marrow stromal cells. Various 2-benzothiopyran-
1-carboxamide derivatives and the ring-expanded 3-ben-
zothiepin-2-carboxamide derivatives (2, Chart 1) have
been synthesized and evaluated for activity. In this
article, we report the synthesis of this novel series of
2-benzothiopyran (1) and 3-benzothiepin (2) derivatives
and structure-activity relationships (SARs) with regard
to stimulation of ALP activity. The osteogenic activity
of the active compounds including the in vivo effect in
a rat skull defect model is also discussed.
* Correspondence should be addressed to this author. Phone:
+81-6-6300-6308. Fax: +81-6-6300-6306.
10.1021/jm980583b CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/06/1999

752 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Oda et al.
Sch em e 1a
a
Reagents: (a) HSCH(R2)CO2H, K2CO3 or Et3N; (b) AcSK; (c) morpholine; (d) BrCH(R2)CO2H, K2CO3; (e) (COCl)2, DMF or SOCl2,
pyridine; (f) AlCl3 or SnCl4; (g) NaOR (R2 * H); (h) 2 N KOH; (i) R3NH2, DEPC, Et3N; (j) R3NH2, Et3N.
Sch em e 2a
a
Reagents: (a) methyl (R)-(+)-lactate, WSC, DMAP; (b) methyl (R)-(-)-mandelate, WSC, DMAP; (c) methyl (S)-(+)-mandelate, WSC,
DMAP.
Ch em istr y
successfully resolved to two diastereomers (11a , 11b)12
using methyl (R)-(+)-lactate. The optical isomers of 10q
were similarly obtained. Esterification of (()-10q with
methyl (R)-(-)- and (S)-(+)-mandelate gave (2R,4S)-
(-)-12 and (2S,4R)-(+)-12, respectively.12 Acid hydroly-
sis of the esters gave the corresponding carboxylic acids.
These carboxylic acids were readily converted to the
amides by coupling with diethyl 4-aminobenzylphos-
phonate using 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (WSC) and 1-hydroxybenzotria-
zole (HOBt).
2-Benzothiopyran-1-carboxamide derivatives (1) and
3-benzothiepin-2-carboxamide derivatives (2) were syn-
thesized starting from R-haloesters (3, 4) as shown in
Scheme 1. The sulfides (5, 6) were prepared generally
by coupling of 3 or 4 with R-mercaptocarboxylic acids
in the presence of a base. An alternative three-step
sequence involving conversion of the halides to thiols
was used for variation of the substituent R2. The sulfides
(5, 6) were converted into acyl chlorides and cyclized
by intramolecular Friedel-Crafts reaction to give esters
(7, 8), which were then hydrolyzed to provide carboxylic
acids (9, 10). The amides (1, 2) were prepared by
straightforward coupling reactions of 9 or 10 with
amines (R3NH2).11 The intramolecular cyclization of the
sulfides (5, 6: when R2 * H) gave a mixture of cis and
trans products, which were treated with alkoxide to
afford the more stable trans form as a single product.
Optical isomers of 10h and 10q were obtained through
esterification with optically active glycolates (Scheme
2) followed by acid hydrolysis. The racemic 10h was
Resu lts a n d Discu ssion
The structures and physical constants of the 2-ben-
zothiopyran-1-carboxamide and 3-benzothiepin-2-car-
boxamide derivatives synthesized are listed in Tables
1-4. The effects of the compounds on cellular alkaline
phosphatase (ALP) activity, one of the markers char-
acteristic of the osteoblast phenotype, were evaluated
using stromal cells isolated from the femoral bone
marrow of young rats.10 The cells and the compounds

Synthesis of Novel 2-Benzothiopyran Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 753
Ta ble 1. Physical Data, Yield, and ALP Stimulatory Activity of 2-Benzothiopyran-1-carboxamide Derivatives 1
compd
R1
R2
R3
C6H4(4-Cl)
yielda (%)
mp (°C)
formula
anal.b
ALP (10-5 M)c
ipriflavone
1.6**d
2.0**
1.7**
2.3**
2.4**
2.0**
1.7**
1.3
1.3**
1.5**
1.5**
1.3**
1.1
1a
1b
1c
1d
1e
1f
1g
1h
1i
6-cyclohexyl
6-cyclohexyl
6-cyclohexyl
6-cyclohexyl
6-cyclohexylmethyl
6-C6H4(4′-Cl)
6,7-Me2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
60
87
52
87
78
54
61
52
85
91
80
71
74
77
65
42
48
176-177 C22H22NO2SCl
181-182 C23H25NO2S
117-118 C26H31NO4S
171-172 C27H34NO5SP
167-168 C28H36NO5SP
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C6H4(4-Me)
C6H3[2,5-(EtO)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
3-pyridyl
188-189 C27H27NO5SPCl C, H, N
198-199 C17H16N2O2S
228-229 C16H15N3O2S
219-220 C22H26NO5SP
194-195 C23H28NO5SP
205-206 C19H16NO2SCl
187-188 C23H26NO5SP
218-219 C18H16NO4SCl
173-174 C23H28NO7SP
203-204 C22H24NO7SP
131-132 C21H16N2O4S2
178-179 C23H26NO7SP
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
6,7-Me2
6,7-Me2
6,7-Me2
pyrazinyl
C6H4[4-P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4(4-Cl)
C6H4[4-P(O)(OEt)2]
C6H4(4-Cl)
1j
1k
1l
6,7-(CH2)3-
6,7-(CH2)3-
6,7-(MeO)2
6,7-(MeO)2
6,7-OCH2O-
6,7-OCH2O-
6,7-OCH2O-
1m
1n
1o
1p
1q
1.3*
2.5**
1.2
1.3**
1.2
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
Me 4-phenyl-2-thiazolyl
Me C6H4[4-CH2P(O)(OEt)2]
a
b
Yield from 9. All compounds gave satisfactory results ((0.4%). c The effect of compounds (10-5 M) on ALP activity in the culture of
rat bone marrow stromal cells was evaluated according to the method of Maniatopoulos et al.10,13 (see Biological Procedures) and expressed
as the ratio value compared to the control group (n ) 5-10). Statistically significant at *p < 0.05 and **p < 0.01 (vs control), by Student’s
t-test.
d
Ta ble 2. Physical Data, Yield, and ALP Stimulatory Activity of 6-Cyclohexyl-2-benzothiopyran-1-carboxamide Derivatives 1
compd
R′
yielda (%)
mp (°C)
formula
anal.b
ALP (10-7 M)c
1d
1r
1s
1t
1u
1v
1w
1x
1y
1z
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-P(O)(OMe)2]
C6H4[4-P(O)(OEt)2]
C6H4[4-P(O)(On-Pr)2]
C6H4[4-CH2P(O)(OMe)2]
C6H4[4-CH2P(O)(Oi-Pr)2]
C6H4[4-CH2P(O)(On-Bu)2]
C6H4[2-CH2P(O)(OEt)2]
C6H4[4-CH2CH2P(O)(OEt)2]
P(O)(OEt)2
87
74
77
71
64
80
80
84
75
36
171-172
210-211
195-196
174-175
219-220
179-180
133-134
119-120
181-182
163-164
C27H34NO5SP
C24H28NO5SP
C26H32NO5SP
C28H36NO5SP
C25H30NO5SP
C29H38NO5SP
C31H42NO5SP
C27H34NO5SP
C28H36NO5SP
C20H28NO5SP
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
1.7**d
1.5**
1.6**
1.5**
1.7**
2.0**
1.7**
1.5**
1.5**
1.1*
a
b
d
Yield from 9. All compounds gave satisfactory results ((0.4%). c See the footnote of Table 1. Statistically significant at *p < 0.05
and **p < 0.01 (vs control), by Student’s t-test.
(10-5 M or 10-7 M) were cultured in the presence of
â-glycerophosphate and dexamethasone according to the
method of Maniatopoulos et al.13 The results are shown
in Tables 1-4, and the potency is expressed as fold
increase of cellular ALP activity compared to the control
value.
Our search for novel potent bone formation stimulants
began with chemical modification of ipriflavone (Chart
1). Variation of the 1-benzopyran structure led to the
finding of 2-benzothiopyran-1-carboxamide as a promis-
ing skeleton. As shown in Table 1, the 2-benzothiopyran-
1-carboxamide derivatives with a phenyl moiety as the
N-substituent (R3) exhibited considerable ALP stimu-
latory activity equipotent to that of ipriflavone. No
significant structure-activity relationships (SARs) were
observed with regard to the substituent(s) on the
benzene ring (R1). However, the activity of compounds
1d , 1e, and 1n suggests that a phosphonate moiety on
the phenyl moiety is advantageous. The phosphonate
moiety was introduced in order to increase the affinity
of the molecule for the inorganic substrate of bone.14
Although the possible affinity may be unrelated to their
efficacy in the bone marrow cell culture assay involving
no bone matrix, the phosphonate derivatives possessed
the favorable activity. The effects of the dialkoxy func-
tion in the phosphonate moiety were examined with
compounds in a series of 6-cyclohexyl-2-benzothiopyrans
(Table 2). While most of these derivatives prepared in
this analogue program had potency equivalent to that
of 1d , no significant improvement in activity was
achieved. The methylene linker between the phenyl and
the phosphonate moieties could be shortened (1d vs 1s),
lengthened (1d vs 1y), or shifted to the ortho position
of the phenyl moiety without affecting the activity (1d
vs 1x). Removal of the phenylene moiety resulted in a
compound with decreased efficacy (1d vs 1z).

754 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Oda et al.
Ta ble 3. Physical Data, Yield, and ALP Stimulatory Activity of 3-Benzothiepin-2-carboxamide Derivatives 2
compd
R1
R2
R3
yielda (%) mp (°C)
formula
anal.b
ALP (10-5 M)c
ipriflavone
1.6**d
1.5**
1.1
2a
2b
2c
2d
2e
2f
H
H
H
H
Me
Ph
H
H
H
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-P(O)(OEt)2]
42
58
89
79
56
65
51
79
221-222 C22H26NO5SP
C, H, N
7-Cl
7-Me
7-Me
7-Me
7,8-Me2
7,8-(MeO)2
7,8-(MeO)2
240-241 C22H25NO5SPCl C, H, N
232-233 C23H28NO5SP
228-229 C24H30NO5SP
198-199 C29H32NO5SP
203-204 C23H28NO5SP
171-172 C23H28NO7SP
217-218 C24H30NO7SP
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
1.1
1.7**
1.2**
2.2**
2.6**
4.3**
2g
2h
C6H4[4-P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
2i
7,8-(MeO)2
H
H
27
218-219 C24H28NO7SP
C, H, N
2.7**
2j
7,8-(MeO)2
80
145-146 C24H28NO7SP
C, H, N
2.8**
2k
2l
7,8-(MeO)2
7,8-(MeO)2
7,8-(MeO)2
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2O-
Me
Et
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
76
44
61
81
60
65
82
69
64
66
74
231-232 C25H32NO7SP
230-231 C26H34NO7SP
239-240 C27H36NO4SCl
192-193 C23H26NO7SP
185-186 C23H26NO7SP
218-219 C22H24NO7SP
220-221 C24H28NO7SP
165-166 C24H28NO7SP
151-152 C24H28NO7SP
212-213 C26H32NO7SP
171-172 C25H30NO7SP
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
2.3**
1.5**
1.4**
2.9**
1.8**
7.2**
5.5**
1.1
2m
2n
2o
2p
2q
2r
2s
2t
2u
i-Pr C6H4[4-CH2P(O)(OEt)2]
H
C6H4[4-CH2P(O)(OEt)2]
C6H4[4-P(O)(OEt)2]
Me
Me
Me
Me
Me
Me
Me
C6H4[4-CH2P(O)(OMe)2]
C6H4[4-CH2P(O)(OEt)2]
C6H4[2-CH2P(O)(OEt)2]
C6H4[3-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(Oi-Pr)2]
C6H4[4-CH2CH2P(O)(OEt)2]
1.8**
3.9**
1.9**
2v
7,8-OCH2O-
Me
33
203-204 C24H26NO7SP
C, H, N
3.0**
2w
2x
2y
2z
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2O-
7,8-OCH2CH2O-
Me
Et
CH2CH2P(O)(OEt)2
C6H4[4-CH2P(O)(OEt)2]
68
63
53
93
139-140 C19H26NO7SP
193-194 C25H30NO7SP
202-203 C26H32NO7SP
245-246 C24H28NO7SP
C, H, N
C, H, N
C, H, N
C, H, N
1.1
4.6**
3.3**
1.2
i-Pr C6H4[4-CH2P(O)(OEt)2]
C6H4[4-CH2P(O)(OEt)2]
H
a
b
d
Yield from 10. All compounds gave satisfactory results ((0.4%). c See the footnote of Table 1. Statistically significant at **p <
0.01 (vs control), by Student’s t-test.
Ta ble 4. ALP Stimulatory Activity of 3-Benzothiepin-2-carboxamide Derivatives 2h , 2q, and Their Optical Isomers in Rat Bone
Marrow Stromal Cells
ALP activity (nmol/min/well)b
compd
configa
10-7
M
10-6
M
10-5
M
(()-2h
(-)-2h
(+)-2h
racemic
2R
2S
1912.8 ( 96.2**c
2094.0 ( 94.7**
770.1 ( 39.3**
(control: 408.6 ( 45.5)
(()-2q
(-)-2q
(+)-2q
racemic
2R, 4S
2S, 4R
185.4 ( 27.4**
192.6 ( 19.5**
165.6 ( 13.2**
411.0 ( 54.9**
682.5 ( 123.2**
159.6 ( 10.4**
1212.6 ( 88.6**
1636.5 ( 169.7**
566.0 ( 74.9**
(control: 113.7 ( 8.1)
a
b
Determined by X-ray crystallographic analysis.12 The effect of compounds on ALP activity in the culture of rat bone marrow stromal
cells was evaluated according to the method of Maniatopoulos et al.10,13 (see Biological Procedures) and expressed as mean ( SE (n )
5-10). c Statistically significant at **p < 0.01 (vs control), by Student’s t-test.
Considering the SARs mentioned above, attempts
were made to further improve activity by expanding the
six-membered 2-benzothiopyran ring to a seven-mem-
bered 3-benzothiepin ring. The results are presented in
Table 3. In general, activity could be maintained in this
series of 3-benzothiepin-2-carboxamides. A sharp in-
crease in activity was observed with compounds 2h , 2p ,
and 2q. We therefore proceeded to explore the SARs
around 2h , 2p , and 2q, further introducing fine modi-
fication of the dialkoxy group of the phosphonate moiety
and the substituent at the 4-position of the thiepin ring.
The effect of the substituent at the 4-position (R2) was
studied for 7-methyl- (2c, 2d , and 2e), 7,8-dimethoxy-
(2h , 2k , 2l, and 2m ), and 7,8-methylenedioxy- (2n , 2q,
2x, and 2y) 3-benzothiepin derivatives. In general,
introduction of a methyl substituent at the 4-position
increased activity (2d vs 2c; 2q vs 2n ), although the
unsubstituted compound (2h vs 2k , 2l, 2m ) was the
most active in the 7,8-dimethoxy series. The X-ray
analysis of (-)-12 revealed that 2,4-trans form was a
stable diastereomer of the 4-methyl-3-benzothiepin
ring.12 Increase of steric hindrance at the 4-position
reduced activity (2d vs 2e; 2k vs 2l, 2m ; 2q vs 2x, 2y).
These findings suggest that the substituent at the

Synthesis of Novel 2-Benzothiopyran Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 755
Ta ble 5. ALP Stimulatory Activity of Optical Isomers of 2h
and 2q in MC3T3-E1 Cells
a
ALP activity (A405
)
compd
dose (M)
+BMP
-BMP
(()-2h
(-)-2h
(+)-2h
(()-2q
10-5
10-5
10-5
10-6
10-5
10-6
10-5
10-5
(0)
0.169 ( 0.002**
0.199 ( 0.006**
0.131 ( 0.002
0.143 ( 0.003††
0.164 ( 0.004††
0.120 ( 0.001
0.133 ( 0.002††
0.178 ( 0.004††
0.154 ( 0.003††
0.195 ( 0.008††
0.115 ( 0.001
0.118 ( 0.001
0.162 ( 0.008**
0.221 ( 0.008**
0.191 ( 0.003**
0.284 ( 0.010**
0.131 ( 0.001
(-)-2q
(+)-2q
control
0.134 ( 0.002
a
The effect of the compounds was assessed by determining ALP
activity in the culture of MC3T3-E1 in the presence (3 ng/mL,
“+BMP”) or absence (“-BMP”) of BMP-4/7 heterodimer (see
Biological Procedures) and expressed as the absorbance at 405 nm.
Mean ( SE (n ) 6). Statistically significant at **p < 0.01 (vs
control, +BMP) or ††p < 0.01 (vs control, -BMP), by Dunnett’s
test.
4-position of the benzothiepin ring might play an
important role in exerting activity through a steric
constraint of the thiepin ring. In studies of variation of
the dialkoxy part of the phosphonate moiety, cyclic
phosphonates did not potentiate activity (2h vs 2i, 2j;
2q vs 2v). Small alkoxies such as methoxy and ethoxy
moieties appear to be best for activity.
From among the compounds with favorable activity
obtained above, 2h and 2q were selected as candidates
for further evaluation based on their chemical and
biological profile. Comparison of the activities of their
optical isomers was performed (Table 4). The ALP
activity of the compound-treated cells increased signifi-
cantly more than that of the control cells. In both cases,
the (-)-isomers were superior to the (+)-isomers, and
the potency of the (-)-isomers was much higher than
that of ipriflavone.10
Several important observations were obtained in
additional experiments using cultures of rat bone mar-
row stromal cells. The compounds 2h and 2q stimulated
other markers characteristic of osteoblast phenotype
besides ALP activity, including collagen and osteocalcin
secretion, implying stimulation of bone matrix deposi-
tion.15 Furthermore, continuous treatment with these
compounds for 1-21 days resulted in an increase in the
area of bone-like mineralized tissue.15 These findings
demonstrate that the compounds 2h and 2q have
biological profiles similar to ipriflavone10,16 with regard
to osteogenic activity.
To clarify the relationship between the stimulatory
effects of compounds 2h and 2q and the presence of
dexamethasone in culture medium, we used the mouse
osteoblastic cell line MC3T3-E1, which spontaneously
exhibits osteoblast phenotype without steroid. Under
the conditions of culture, the compounds (()-2h , (-)-
2h , (()-2q, and (-)-2q also stimulated cellular ALP
activity (Table 5). The (-)-isomers [(-)-2h and (-)-2q]
were more potent than the corresponding (+)-isomers
[(+)-2h and (+)-2q]. These findings were similar to
those for rat bone marrow stromal cells, suggesting that
the compound acts on committed osteoblast lineage
cells, rather than modulating the effects of dexametha-
sone. Recent studies suggest that autocrine/paracrine
factors including bone morphogenetic proteins (BMPs)
or other growth factors regulate cellular growth and
differentiation of osteoblasts. For this reason, the activi-
F igu r e 1. Effect of (()-2q on Bone Formation in vivo. (A) A
rat skull defect model (see Biological Procedures). (B) Area of
newly formed bone in defect was calculated according to the
following formula: [area of the initial defect created by
trephination (12.6 mm2)] - [area of the remaining defect].
Statistically significant at **p < 0.01 (vs control), by Student’s
t-test.
ties of these compounds were evaluated in the presence
of BMP using MC3T3-E1 cells (Table 5). The BMP-4/7
heterodimer alone at a concentration of 3 ng/mL stimu-
lated ALP activity of these cells.17 The compounds (-)-
2h and (-)-2q further enhanced the stimulatory effect
of BMP. Similar results were also observed in rat bone
marrow stromal cells (data not shown). These findings
suggest that the enhancement of BMP activity by
compound (-)-2q may have contributed to the stimu-
latory effect of these compounds on bone formation.
The in vivo effect of compound 2q was tested using a
rat skull defect model.18 An ethylene vinyl acetate (EVA)
copolymer pellet containing 1 mg of 2q was prepared.
The pellet permits sustained release of various mol-
ecules19 and allows evaluation of new bone formation
with local application of test compound (Figure 1). The
area of newly formed bone in the model was increased
significantly by treatment with the sustained release
pellet containing 2q for 4 weeks, compared with that
in the control group. Histological studies showed that
the structure of calcified new bone formed in the defect
was similar to that of the original bone.15
In conclusion, we found that a novel series of 3-ben-
zothiepin-2-carboxamides with a N-[4-(dialkoxyphos-
phorylmethyl)phenyl] moiety designed based on the
chemical structure of ipriflavone possessed potent ALP
stimulatory activity. These derivatives were proven to
have biological features similar to those of iprifla-
vone,10,16 in addition to significantly improved efficacy.
One of the active derivatives, 2q, exhibited a promising
effect in the rat skull defect model, suggesting the
potential therapeutic application of the compound for
treatment of osteoporosis and bone fracture. Compound
(-)-2q (TAK-778) was selected as a candidate for further
pharmacological evaluation.15

756 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Oda et al.
yl)p r op ion a te (6h ). A solution of 4h (23.2 g, 77 mmol) in N,N-
dimethylformamide (DMF) (20 mL) was added dropwise to a
solution of mercaptoacetic acid (7.8 g, 84 mmol) and trieth-
ylamine (17.0 g, 168 mmol) in DMF (100 mL) at 0 °C. After
being stirred at 0 °C for 1 h, the reaction mixture was poured
into H2O (350 mL) and washed with Et2O. The aqueous layer
was acidified with concentrated HCl and extracted with
AcOEt. The AcOEt extract was washed with H2O and brine,
dried over MgSO4, and concentrated in vacuo to give 6h as an
oil (22.1 g, 92%): 1H NMR (CDCl3) δ 2.94 (1H, dd, J ) 14.0,
6.4 Hz), 3.17 (1H, dd, J ) 14.0, 9.0 Hz), 3.35 (1H, d, J ) 16.0
Hz), 3.49 (1H, d, J ) 16.0 Hz), 3.68 (3H, s), 3.66-3.75 (1H,
m), 3.85 (3H, s), 3.86 (3H, s), 6.72-6.83 (3H, m), 8.05 (1H, br
s).
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Yanagim-
oto micro melting point apparatus and are uncorrected. Optical
rotations of CHCl3, MeOH, or dimethyl sulfoxide (DMSO)
solutions were measured on a J asco DIP-370 digital polarim-
eter. 1H NMR spectra of deuteriochloroform (CDCl3) or DMSO-
d6 solutions (tetramethylsilane as an internal standard, δ 0)
were recorded on a Varian Gemini-200 or a Varian EM-390
spectrometer, and the following abbreviations are used: s )
singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet,
br s ) broad singlet. IR spectra were recorded on a J asco IR-
810 spectrophotometer. Elemental analysis (C, H, and N) was
carried out in Takeda Analytical Research Laboratories, Ltd.
All compounds exhibited 1H NMR, IR, and analytical data
consistent with the proposed structures. Silica gel column
chromatography was performed on E. Merck silica gel 60
(0.063-0.200 mm). A Hitachi 655A liquid chromatography
system equipped with a Chiralcel OD column (Daicel Chemical
Industries, Tokyo, J apan) was employed for chiral stationary
phase HPLC.
Gen er a l P r oced u r e for Eth yl r-Ch lor op h en yla ceta tes
(3). Eth yl r-Ch lor o-4-cycloh exylp h en yla ceta te (3d ). Ethyl
4′-cyclohexylmandelate20 (52.0 g, 198 mmol) was dissolved in
thionyl chloride (100 mL) and refluxed for 1 h. The reaction
mixture was concentrated in vacuo, diluted with H2O (500 mL)
and extracted with Et2O. The extract was washed with H2O
and brine, dried over MgSO4, and concentrated in vacuo to
give 3d as an oil (56.0 g, quant.): 1H NMR (CDCl3) δ 1.23 (3H,
t, J ) 7.1 Hz), 1.23-1.87 (10H, m), 2.48-2.52 (1H, m), 4.21
(2H, q, J ) 7.1 Hz), 5.30 (1H, s), 7.18 (2H, d, J ) 8.8 Hz), 7.40
(2H, d, J ) 8.8 Hz).
Gen er a l P r oced u r e for Meth yl 2-Br om o-3-p h en ylp r o-
p ion a tes (4). Meth yl 2-Br om o-3-(3,4-d im eth oxyp h en yl)-
p r op ion a te (4h ). A solution of NaNO2 (12.6 g, 183 mol) in
H2O (20 mL) was added dropwise to a solution of 3,4-
dimethoxyaniline (25.4 g, 166 mol) and 47% aqueous HBr (85.8
g, 498 mmol) in acetone (250 mL) at 5-10 °C. After the
mixture was stirred at 5 °C for 30 min, methyl acrylate (85.8
g, 997 mmol) was added and the temperature was raised to
15 °C. Then Cu2O (ca. 0.5 g) was added portionwise with
vigorous stirring. An exothermic reaction occurred and the
temperature was kept below 40 °C. After being stirred at
ambient temperature for 1.5 h, the reaction mixture was
concentrated in vacuo, diluted with H2O (300 mL) and
extracted with Et2O. The extract was washed with H2O and
brine, dried over MgSO4, and concentrated in vacuo. The
residual oil was distilled to give 4h as an oil (23.2 g, 46%): bp
155-157 °C (0.37 mmHg); 1H NMR (CDCl3) δ 3.18 (1H, dd, J
) 14.0, 6.8 Hz), 3.42 (1H, dd, J ) 14.0, 8.6 Hz), 3.73 (3H, s),
3.86 (3H, s), 3.87 (3H, s), 4.37 (1H, dd, J ) 8.6, 6.8 Hz), 6.73
(1H, s), 6.77-6.85 (2H, m).
Met h yl
2-(1-Ca r b oxyet h ylt h io)-3-(3,4-m et h ylen ed i-
oxyp h en yl)p r op ion a te (6q). The title compound was pre-
pared according to the procedure described for 6h in 99% yield
as an oil: 1H NMR (CDCl3) major diastereomer δ 1.44 (3H, d,
J ) 7.2 Hz), 2.84-3.16 (2H, m), 3.70 (3H, s), 3.57-3.85 (2H,
m), 5.92 (2H, s), 6.66-6.71 (3H, m); minor diastereomer δ 1.47
(3H, d, J ) 7.2 Hz), 2.88-3.20 (2H, m), 3.45-3.85 (2H, m),
3.67 (3H, s), 5.93 (2H, s), 6.66-6.71 (3H, m).
Gen er a l P r oced u r e for Meth yl 2-(Ca r boxya lk ylth io)-
3-p h en ylp r op ion a t es (6). Met h yl 2-(1-Ca r b oxyp r op yl-
th io)-3-(3,4-d im eth oxyp h en yl)p r op ion a te (6l). A solution
of 4h (25.0 g, 85 mmol) in DMF (20 mL) was added dropwise
to a mixture of potassium thioacetate (10.4 g, 91 mmol) and
DMF (100 mL) at 0 °C. After being stirred at 0 °C for 1.5 h,
the reaction mixture was poured into H2O (500 mL) and
extracted with Et2O. The extract was washed with H2O and
brine, dried over MgSO4, and concentrated in vacuo to give
methyl 2-acetylthio-3-(3,4-dimethoxyphenyl)propionate as an
oil (23.7 g, 97%): 1H NMR (CDCl3) δ 2.33 (3H, s), 2.96 (1H,
dd, J ) 14.0, 7.2 Hz), 3.20 (1H, dd, J ) 14.0, 8.2 Hz), 3.68
(3H, s), 3.86 (3H, s), 3.87 (3H, s), 4.43 (1H, dd, J ) 8.2, 7.2
Hz), 6.73-6.82 (3H, m).
Morpholine (27.7 g, 318 mmol) was added dropwise to a
solution of methyl 2-acetylthio-3-(3,4-dimethoxyphenyl)propi-
onate (23.7 g, 80 mmol) in MeOH (110 mL) at room temper-
ature. After being stirred at room temperature for 1 h, the
reaction mixture was poured into H2O (250 mL) and extracted
with AcOEt. The extract was successively washed with H2O,
1 N HCl, H2O, and brine, dried over MgSO4, and concentrated
in vacuo. The residual oil was chromatographed on SiO2 (270
g) with AcOEt-hexane (1:4 to 1:2, v/v) to give methyl 2-mer-
capto-3-(3,4-dimethoxyphenyl)propionate as an oil (15.2 g,
75%): 1H NMR (CDCl3) δ 2.12 (1H, d, J ) 9.0 Hz), 2.97 (1H,
dd, J ) 14.0, 6.8 Hz), 3.20 (1H, dd, J ) 14.0, 8.6 Hz), 3.58
(1H, dt, J ) 6.8, 8.8 Hz), 3.70 (3H, s), 3.86 (3H, s), 3.87 (3H,
s), 6.72 (1H, d, J ) 1.8 Hz), 6.74 (1H, dd, J ) 8.4, 1.8 Hz),
6.81 (1H, d, J ) 8.4 Hz).
K2CO3 (6.0 g, 43 mmol) was added to a solution of methyl
2-mercapto-3-(3,4-dimethoxyphenyl)propionate (5.1 g, 20 mmol)
and 2-bromobutyric acid (3.3 g, 20 mmol) in DMF (30 mL) at
room temperature. After being stirred at room temperature
for 1.5 h, the reaction mixture was poured into H2O (200 mL)
and washed with Et2O. The aqueous layer was acidified with
concentrated HCl and extracted with AcOEt. The AcOEt
extract was washed with H2O and brine, dried over MgSO4,
and concentrated in vacuo to give 6l as an oil (5.8 g, 86%): 1H
NMR (CDCl3) major diastereomer δ 1.02 (3H, t, J ) 7.2 Hz),
1.61-1.98 (2H, m), 2.98 (1H, dd, J ) 6.6, 4.8 Hz), 3.12 (1H, t,
J ) 9.0 Hz), 3.41 (1H, t, J ) 7.4 Hz), 3.68 (3H, s), 3.68-3.83
(1H, m), 3.85 (3H, s), 3.86 (3H, s), 6.72-6.77 (3H, m); minor
diastereomer δ 0.99 (3H, t, J ) 7.2 Hz), 1.61-1.98 (2H, m),
2.91 (1H, dd, J ) 6.2, 4.8 Hz), 3.19 (1H, t, J ) 9.0 Hz), 3.29
(1H, t, J ) 7.4 Hz), 3.66 (3H, s), 3.68-3.83 (1H, m), 3.85 (3H,
s), 3.86 (3H, s), 6.72-6.77 (3H, m).
Meth yl 2-Br om o-3-(3,4-m eth ylen ed ioxyp h en yl)p r op i-
on a te (4q). The title compound was prepared according to the
procedure described for 4h in 62% yield as an oil: bp 146-
148 °C (0.4 mmHg); 1H NMR (CDCl3) δ 3.15 (1H, dd, J ) 14.2,
7.0 Hz), 3.38 (1H, dd, J ) 14.2, 8.6 Hz), 3.74 (3H, s), 4.34 (1H,
dd, J ) 8.6, 7.0 Hz), 5.94 (2H, s), 6.66 (1H, dd, J ) 8.0, 1.6
Hz), 6.69 (1H, d, J ) 1.6 Hz), 6.75 (1H, d, J ) 8.0 Hz).
Gen er a l P r oced u r e for Eth yl r-(Ca r boxym eth ylth io)-
p h en yla ceta tes (5) a n d Meth yl 2-(Ca r boxym eth ylth io)-
3-p h en ylp r op ion a tes (6). Eth yl r-Ca r boxym eth ylth io-4-
cycloh exylp h en yla ceta te (5d ). A mixture of 3d (5.6 g, 20
mmol), mercaptoacetic acid (1.8 g, 20 mmol), K2CO3 (5.5 g, 40
mmol) and 2-butanone (70 mL) was refluxed for 5 h and
concentrated in vacuo. The residue was diluted in H2O (100
mL) and washed with Et2O. The aqueous layer was acidified
with concentrated HCl and extracted with AcOEt. The AcOEt
extract was washed with H2O and brine, dried over MgSO4,
and concentrated in vacuo to give 5d as an oil (5.7 g, 85%):
1H NMR (CDCl3) δ 1.23 (3H, t, J ) 7.1 Hz), 1.30-2.00 (10H,
m), 2.48-2.52 (1H, m), 3.07 (1H, d, J ) 15.4 Hz), 3.30 (1H, d,
J ) 15.4 Hz), 4.16 (2H, q, J ) 7.1 Hz), 4.83 (1H, s), 7.17 (2H,
d, J ) 8.8 Hz), 7.38 (2H, d, J ) 8.8 Hz).
Gen er a l P r oced u r e for Eth yl 3,4-Dih yd r o-4-oxo-1H-2-
ben zoth iop yr a n -1-ca r boxyla tes (7) a n d Meth yl 1,2,4,5-
Tetr ah ydr o-5-oxo-3-ben zoth iepin -2-car boxylates (8). Eth -
yl 6-Cycloh exyl-3,4-d ih yd r o-4-oxo-1H-2-ben zoth iop yr a n -
1-ca r boxyla te (7d ). A mixture of 5d (45.0 g, 134 mmol),
thionyl chloride (23.9 g, 200 mmol), pyridine (5 drops), and
Met h yl 2-Ca r b oxym et h ylt h io-3-(3,4-d im et h oxyp h en -

Synthesis of Novel 2-Benzothiopyran Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 757
Et2O (300 mL) was stirred at room temperature for 2 h and
refluxed for 20 min. The solvent and excess thionyl chloride
were evaporated off. The residual oil was dissolved in CH2Cl2
(50 mL), and the solution was added dropwise to a mixture of
AlCl3 (37.5 g, 281 mmol) and CH2Cl2 (400 mL) at 0 °C. After
being stirred at 0 °C for 3 h, the reaction mixture was poured
onto ice-water (600 mL) followed by addition of AcOEt (60
mL). After the mixture was stirred at room temperature for 1
h, the organic layer was separated and the aqueous layer was
extracted with CH2Cl2. The combined organic layer was
washed with H2O and brine, dried over MgSO4, and concen-
trated in vacuo. The residual oil was chromatographed on SiO2
(400 g) with AcOEt-hexane (1:10, v/v) to give 7d as an oil
(36.0 g, 84%): 1H NMR (CDCl3) δ 1.31 (3H, t, J ) 7.2 Hz),
1.35-1.46 (5H, m), 1.84-1.88 (5H, m), 2.52-2.56 (1H, m), 3.26
(1H, dd, J ) 16.4, 1.0 Hz), 4.24 (2H, q, J ) 7.2 Hz), 4.27 (1H,
d, J ) 16.4 Hz), 4.41 (1H, s), 7.16 (1H, d, J ) 8.0 Hz), 7.34
(1H, dd, J ) 8.0, 2.0 Hz), 7.97 (1H, d, J ) 2.0 Hz).
Meth yl 1,2,4,5-Tetr a h yd r o-7,8-d im eth oxy-5-oxo-3-ben -
zoth iep in -2-ca r boxyla te (8h ). Oxalyl chloride (10.7 g, 84
mmol) was added dropwise to a solution of 6h (22.1 g, 70 mmol)
and DMF (1 drop) in tetrahydrofuran (THF) (100 mL) at room
temperature. After the mixture was stirred at room temper-
ature for 3 h, the solvent and excess oxalyl chloride were
evaporated off. The residual oil was dissolved in CH2Cl2 (200
mL). SnCl4 (40.2 g, 154 mmol) was added dropwise to the ice-
cooled solution. After being stirred at 0 °C for 1 h and at room
temperature for 1 h, the reaction mixture was poured onto ice-
water (300 mL) followed by addition of AcOEt (30 mL). After
the mixture was stirred at room temperature for 1 h, the
organic layer was separated and the aqueous layer was
extracted with CH2Cl2. The combined organic layer was
washed with H2O and brine, dried over MgSO4, and concen-
trated in vacuo. The residual oil was chromatographed on SiO2
(330 g) with AcOEt-hexane (1:3 to 1:1, v/v) to give crystals.
Recrystallization from AcOEt-hexane gave 8h as colorless
prisms (12.1 g, 58%): mp 143-144 °C; 1H NMR (CDCl3) δ 3.18
(1H, dd, J ) 14.6, 5.0 Hz), 3.41 (1H, d, J ) 17.6 Hz), 3.47 (1H,
dd, J ) 14.6, 11.6 Hz), 3.70 (1H, dd, J ) 11.6, 5.0 Hz), 3.81
(3H, s), 3.93 (3H, s), 3.95 (3H, s), 3.98 (1H, d, J ) 17.6 Hz),
6.71 (1H, s), 7.51 (1H, s). Anal. (C14H16O5S) C, H.
Meth yl tr a n s-1,2,4,5-Tetr a h yd r o-4-m eth yl-7,8-m eth yl-
en ed ioxy-5-oxo-3-ben zoth iep in -2-ca r boxyla te (8q). Oxalyl
chloride (39.5 g, 311 mmol) was added dropwise to a solution
of 6q (80.9 g, 259 mmol) and DMF (3 drops) in THF (320 mL)
at room temperature. After the mixture was stirred at room
temperature for 3 h, the solvent and excess oxalyl chloride
were evaporated off. The residual oil was dissolved in CH2Cl2
(700 mL). SnCl4 (148.5 g, 570 mmol) was added dropwise to
the ice-cooled solution. After being stirred at 0 °C for 1 h and
at room temperature for 1 h, the reaction mixture was poured
onto ice-water (1100 mL) followed by addition of AcOEt (110
mL). After the mixture was stirred at room temperature for 1
h, the organic layer was separated and the aqueous layer was
extracted with CH2Cl2. The combined organic layer was
washed with H2O and brine, dried over MgSO4, and concen-
trated in vacuo. The residue was suspended in MeOH (800
mL) followed by addition of a solution of sodium methoxide in
MeOH (28%, 33 mL). The mixture was stirred at room
temperature for 1 h and poured into 2 N HCl (800 mL). After
the mixture was stirred at room temperature for 10 min, the
crystals were collected by filtration and successively washed
with H2O, EtOH, and i-Pr2O. Recrystallization from AcOEt
gave 8q as colorless prisms (57.0 g, 75%): mp 170-171 °C;
1H NMR (CDCl3) δ 1.51 (3H, d, J ) 7.0 Hz), 3.17 (1H, dd, J )
14.0, 4.2 Hz), 3.44 (1H, dd, J ) 14.0, 12.4 Hz), 3.57 (1H, dd, J
) 12.4, 4.2 Hz), 3.82 (3H, s), 4.05 (1H, q, J ) 7.0 Hz), 6.03
(2H, dd, J ) 2.3, 1.3 Hz), 6.68 (1H, s), 7.53 (1H, s). Anal.
(C14H14O5S) C, H.
was added to a solution of 7d (15.0 g, 47 mmol) in MeOH (80
mL) at room temperature. After being stirred at room tem-
perature for 1 h, the reaction mixture was diluted with H2O
(100 mL) and washed with Et2O. The aqueous layer was
acidified with concentrated HCl and extracted with AcOEt.
The AcOEt extract was washed with H2O and brine, dried over
MgSO4, and concentrated in vacuo to give crystals. Recrystal-
lization from AcOEt-hexane gave 9d as colorless prisms (10.4
1
g, 77%): mp 171-172 °C; H NMR (CDCl3) δ 1.30-1.52 (5H,
m), 1.77-1.92 (5H, m), 2.53-2.57 (1H, m), 3.26 (1H, d, J )
16.6 Hz), 4.24 (1H, d, J ) 16.6 Hz), 4.43 (1H, s), 6.51 (1H, br
s), 7.18 (1H, d, J ) 8.0 Hz), 7.37 (1H, dd, J ) 8.0, 2.0 Hz),
7.99 (1H, d, J ) 2.0 Hz). Anal. (C16H18O3S) C, H.
1,2,4,5-Tetr ah ydr o-7,8-dim eth oxy-5-oxo-3-ben zoth iepin -
2-ca r boxylic Acid (10h ). The title compound was prepared
according to the procedure described for 9d in 94% yield as
1
colorless prisms: mp 245-246 °C (AcOEt); H NMR (DMSO-
d6) δ 3.23-3.40 (2H, m), 3.41 (1H, d, J ) 17.0 Hz), 3.66 (1H,
dd, J ) 10.2, 6.0 Hz), 3.79 (3H, s), 3.84 (3H, s), 3.98 (1H, d, J
) 17.0 Hz), 7.02 (1H, s), 7.36 (1H, s). Anal. (C13H14O5S) C, H.
tr a n s-1,2,4,5-Tetr ah ydr o-4-m eth yl-7,8-m eth ylen edioxy-
5-oxo-3-ben zoth iep in -2-ca r boxylic Acid (10q). The title
compound was prepared according to the procedure described
for 9d in 79% yield as colorless needles: mp 209-210 °C
(MeOH); 1H NMR (CDCl3) δ 1.54 (3H, d, J ) 7.0 Hz), 3.22 (1H,
dd, J ) 14.4, 5.0 Hz), 3.42 (1H, dd, J ) 14.4, 12.2 Hz), 3.60
(1H, dd, J ) 12.2, 5.0 Hz), 4.05 (1H, q, J ) 7.0 Hz), 6.04 (2H,
dd, J ) 2.4, 1.0 Hz), 6.69 (1H, s), 7.53 (1H, s). Anal. (C13H12O5S)
C, H.
Gen er a l P r oced u r e for 3,4-Dih yd r o-4-oxo-1H-2-ben -
zoth iop yr a n -1-ca r boxa m id es (1). N-[4-(Dieth oxyp h os-
p h or ylm et h yl)p h en yl]-6-cycloh exyl-3,4-d ih yd r o-4-oxo-
1H-2-ben zoth iop yr a n -1-ca r boxa m id e (1d ). Diethyl phos-
phorocyanidate (DEPC) (538 mg, 3.3 mmol) was added to a
solution of 9d (871 mg, 3.0 mmol) in DMF (10 mL) at 0 °C.
After the mixture was stirred at 0 °C for 30 min, diethyl
4-aminobenzylphosphonate (803 mg, 3.3 mmol) and triethy-
lamine (334 mg, 3.3 mmol) were added in that order. The
reaction mixture was stirred at 0 °C for 1 h, poured into H2O
(100 mL), and extracted with AcOEt. The extract was washed
with H2O and brine, dried over MgSO4, and concentrated in
vacuo to give crystals. Recrystallization from EtOH gave 1d
as colorless prisms (1.3 g, 87%): mp 171-172 °C; 1H NMR
(CDCl3) δ 1.26 (6H, t, J ) 7.1 Hz), 1.37-1.85 (10H, m), 2.50-
2.54 (1H, m), 3.12 (2H, d, J ) 21.4 Hz), 3.26 (1H, d, J ) 16.2
Hz), 3.94-4.10 (4H, m), 4.27 (1H, d, J ) 16.2 Hz), 4.67 (1H,
s), 7.10-7.43 (6H, m), 7.97 (1H, d, J ) 2.0 Hz), 9.62 (1H, s).
Anal. (C27H34NO5SP) C, H, N.
Gen er a l P r oced u r e for 1,2,4,5-Tet r a h yd r o-5-oxo-3-
ben zoth iep in -2-ca r boxa m id es (2). N-[4-(Dieth oxyp h os-
p h or ylm eth yl)p h en yl]-1,2,4,5-tetr a h yd r o-7,8-d im eth oxy-
5-oxo-3-ben zoth iep in -2-ca r boxa m id e (2h ). Oxalyl chloride
(1.9 g, 15 mmol) was added to a solution of 10h (3.4 g, 12 mmol)
in THF (60 mL) at room temperature followed by addition of
DMF (1 drop). The mixture was stirred at room temperature
for 2 h, and the solvent and excess oxalyl chloride were
evaporated off. The residue was dissolved in THF (25 mL),
and the solution was added dropwise to a solution of diethyl
4-aminobenzylphosphonate (3.2 g, 13 mmol) and triethylamine
(1.4 g, 13 mmol) in THF (90 mL) at room temperature. After
being stirred at room temperature for 1 h, the reaction mixture
was poured into H2O (500 mL) and extracted with AcOEt. The
extract was successively washed with 1 N HCl, H2O, saturated
aqueous NaHCO3, H2O, and brine, dried over MgSO4 and
concentrated in vacuo to give crystals. Recrystallization from
CHCl3-EtOH gave 2h as colorless prisms (4.85 g, 79%): mp
217-218 °C; 1H NMR (CDCl3) δ 1.17 (3H, t, J ) 7.0 Hz), 1.21
(3H, t, J ) 7.0 Hz), 3.11 (2H, d, J ) 21.6 Hz), 3.33-3.62 (3H,
m), 3.93 (3H, s), 3.94 (3H, s), 3.91-4.04 (6H, m), 6.76 (1H, s),
7.20 (2H, dd, J ) 8.4, 2.6 Hz), 7.39 (2H, d, J ) 8.2 Hz), 7.56
(1H, s), 9.12 (1H, s). Anal. (C24H30NO7SP) C, H, N.
Gen er a l P r oced u r e for 3,4-Dih yd r o-4-oxo-1H-2-ben -
zoth iop yr a n -1-ca r boxylic Acid s (9) a n d 1,2,4,5-Tetr a h y-
d r o-5-oxo-3-ben zoth iep in -2-ca r boxylic Acid s (10). 6-Cy-
c lo h e x y l-3,4-d ih y d r o -4-o x o -1H -2-b e n zo t h io p y r a n -1-
ca r boxylic Acid (9d ). 2 N Aqueous KOH (35 mL, 70 mmol)
tr a n s-N-[4-(Dieth oxyph osph or ylm eth yl)ph en yl]-1,2,4,5-
t et r a h yd r o-4-m et h yl-7,8-m et h ylen ed ioxy-5-oxo-3-b en -
zoth iep in -2-ca r boxa m id e (2q). The title compound was

758 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Oda et al.
prepared according to the procedure described for 2h in 82%
yield as colorless prisms: mp 220-221 °C (CHCl3-EtOH); 1H
NMR (CDCl3) δ 1.17 (3H, t, J ) 7.0 Hz), 1.20 (3H, t, J ) 7.0
Hz), 1.55 (3H, d, J ) 7.0 Hz), 3.11 (2H, d, J ) 21.2 Hz), 3.33
(1H, dd, J ) 14.8, 5.0 Hz), 3.53 (1H, dd, J ) 14.8, 12.2 Hz),
3.82 (1H, dd, J ) 5.0, 12.2 Hz), 3.88-4.04 (4H, m), 4.16 (1H,
q, J ) 7.0 Hz), 6.04 (2H, s), 6.74 (1H, s), 7.19 (2H, dd, J ) 8.6,
2.4 Hz), 7.43 (2H, d, J ) 8.4 Hz), 7.56 (1H, s), 9.41 (1H, s).
Anal. (C24H28NO7SP) C, H, N.
Gen er a l P r oced u r e for Cyclic P h osp h on a tes. 2-(4-
Am in oben zyl)-1,3,2-d ioxa p h osp h ep a n e-2-oxid e. A mix-
ture of 4-nitrobenzylphosphonic acid11c (54.2 g, 250 mmol),
thionyl chloride (210 mL), and DMF (7 drops) was refluxed
for 5 h and concentrated in vacuo. The residual oil was
dissolved in THF (500 mL). A solution of 1,4-butanediol (22.5
g, 250 mmol) in acetonitrile (75 mL) was added dropwise to
the solution at -78 °C. Pyridine (41.4 g, 524 mmol) was added
dropwise to the mixture at the same temperature. After the
mixture was stirred at room temperature for 15 h, the
precipitate was removed by filtration. The filtrate was con-
centrated in vacuo, and the residue was chromatographed on
SiO2 (750 g) with AcOEt-CHCl3-MeOH (10:10:1, v/v) to give
crystals. Recrystallization from EtOH-hexane gave 2-(4-
nitrobenzyl)-1,3,2-dioxaphosphepane-2-oxide as colorless needles
(39.9 g, 59%): mp 136-137 °C; 1H NMR (CDCl3) δ 1.79-1.99
(4H, m), 3.33 (2H, d, J ) 22.4 Hz), 3.79-3.94 (2H, m), 4.20-
4.38 (2H, m), 7.51 (2H, dd, J ) 8.8, 2.4 Hz), 8.20 (2H, d, J )
8.8 Hz). Anal. (C11H14NO5P) C, H, N.
Tet r a h yd r o-4-m et h yl-7,8-m et h ylen ed ioxy-5-oxo-3-b en -
zoth iep in -2-ca r boxyla te [(-)-12]. A solution of WSC (12.6
g, 66 mmol) in CH2Cl2 (200 mL) was added to a solution of
(()-10q (15.3 g, 55 mmol) and methyl (R)-(-)-mandelate (18.2
g, 109 mmol) in DMF (200 mL) at 0 °C followed by addition of
DMAP (3.3 g, 27 mmol). After being stirred at 0 °C for 1 h
and at room temperature for 15 h, the reaction mixture was
poured into H2O (600 mL) and extracted with CHCl3. The
extract was washed with H2O and brine, dried over MgSO4,
and concentrated in vacuo. The residual oil was dissolved in
AcOEt (600 mL), and the solution was washed with H2O and
brine, dried over MgSO4, and concentrated in vacuo to give
crystals. Recrystallization from AcOEt-hexane gave (-)-12 as
colorless needles (4.1 g, 17%): mp 140-141 °C; [R]23D -244.2°
(c ) 0.50, CHCl3); 1H NMR (CDCl3) δ 1.50 (3H, d, J ) 7.2 Hz),
3.28 (1H, dd, J ) 14.9, 5.6 Hz), 3.45 (1H, dd, J ) 14.9, 12.1
Hz), 3.73 (1H, dd, J ) 12.1, 5.6 Hz), 3.78 (3H, s), 4.07 (1H, q,
J ) 7.2 Hz), 6.04 (3H, s), 6.72 (1H, s), 7.40-7.49 (5H, m), 7.52
(1H, s). Anal. (C22H20O7S) C, H. The absolute configuration of
(-)-12 was confirmed as (2R,4S)-form by the X-ray crystal-
lographic analysis.12
(r′S)-r′-Met h oxyca r b on ylb en zyl (2S,4R)-(+)-1,2,4,5-
Tet r a h yd r o-4-m et h yl-7,8-m et h ylen ed ioxy-5-oxo-3-b en -
zoth iep in -2-ca r boxyla te [(+)-12]. The title compound was
prepared from (()-10q and methyl (S)-(+)-mandelate accord-
ing to the procedure described for (-)-12 in 20% yield as
colorless needles: mp 141-142 °C (AcOEt-hexane); [R]23
D
1
+239.7° (c ) 0.50, CHCl3); H NMR (CDCl3) δ 1.50 (3H, d, J
A solution of 2-(4-nitrobenzyl)-1,3,2-dioxaphosphepane-2-
oxide (39.4 g, 145 mmol) in MeOH (900 mL) was hydrogenated
in the presence of 5% Pd-C (50% wet) (14.0 g) at room
temperature under ordinary pressure. After removal of the
catalyst by filtration, the filtrate was concentrated in vacuo
to give crystals. Recrystallization from EtOH-hexane gave
2-(4-aminobenzyl)-1,3,2-dioxaphosphepane-2-oxide as colorless
prisms (33.6 g, 96%): mp 128-129 °C; 1H NMR (CDCl3) δ
1.68-1.91 (4H, m), 3.14 (2H, d, J ) 21.2 Hz), 3.59-3.82 (4H,
m), 4.10-4.29 (2H, m), 6.64 (2H, d, J ) 8.0 Hz), 7.09 (2H, dd,
J ) 8.7, 2.5 Hz). Anal. (C11H16NO3P) C, H, N.
) 7.2 Hz), 3.28 (1H, dd, J ) 14.9, 5.6 Hz), 3.44 (1H, dd, J )
14.9, 12.1 Hz), 3.73 (1H, dd, J ) 12.1, 5.6 Hz), 3.77 (3H, s),
4.06 (1H, q, J ) 7.2 Hz), 6.04 (3H, s), 6.71 (1H, s), 7.39-7.47
(5H, m), 7.51 (1H, s). Anal. (C22H20O7S) C, H.
(2R)-(-)-1,2,4,5-Tetr a h yd r o-7,8-d im eth oxy-5-oxo-3-ben -
zoth iep in -2-ca r boxylic Acid [(-)-10h ]. A mixture of 11a
(0.50 g, 1.4 mmol), AcOH (2.5 mL), and concentrated HCl (2.5
mL) was refluxed for 30 min. The reaction mixture was poured
into H2O (50 mL) to give crystals, which were collected by
filtration and successively washed with H2O, EtOH, and Et2O
to give (-)-10h as colorless needles (0.20 g, 53%): mp 223-
(1′R)-1′-Meth oxycar bon yleth yl (2R)-(-)-1,2,4,5-Tetr ah y-
d r o-7,8-d im et h oxy-5-oxo-3-b en zot h iep in -2-ca r b oxyla t e
(11a ) a n d (1′R)-1′-Meth oxyca r bon yleth yl (2S)-(+)-1,2,4,5-
Tet r a h yd r o-7,8-d im et h oxy-5-oxo-3-b en zot h iep in -2-ca r -
boxyla te (11b). A solution of 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (WSC) (4.9 g, 26 mmol) in
CH2Cl2 (80 mL) was added to a solution of (()-10h (6.0 g, 21
mmol) and methyl (R)-(+)-lactate (4.4 g, 43 mmol) in DMF (80
mL) at 0 °C followed by addition of 4-(dimethylamino)pyridine
(DMAP) (1.3 g, 11 mmol). After being stirred at 0 °C for 1 h
and at room temperature for 15 h, the reaction mixture was
concentrated in vacuo, and the residue was dissolved in
AcOEt-H2O (1:1, 1 L). The organic layer separated was
washed with H2O and brine, dried over MgSO4, and concen-
trated in vacuo. The crystals were collected by filtration,
washed with Et2O, and recrystallized from AcOEt-hexane to
give 11a as pale yellow needles (2.2 g, 28%): mp 161-162 °C;
224 °C; [R]23 -190.0° (c ) 0.50, DMSO); 1H NMR (DMSO-d6)
D
δ 3.23-3.39 (2H, m), 3.41 (1H, d, J ) 17.0 Hz), 3.67 (1H, dd,
J ) 10.2, 5.8 Hz), 3.79 (3H, s), 3.85 (3H, s), 3.98 (1H, d, J )
17.0 Hz), 7.02 (1H, s), 7.37 (1H, s). Anal. (C13H14O5S) C, H.
(2S)-(+)-1,2,4,5-Tetr a h yd r o-7,8-d im eth oxy-5-oxo-3-ben -
zoth iep in -2-ca r boxylic Acid [(+)-10h ]. The title compound
was prepared according to the procedure described for (-)-
10h in 56% yield as colorless needles: mp 223-224 °C; [R]22
D
1
+196.7° (c ) 0.50, DMSO); H NMR (DMSO-d6) δ 3.23-3.39
(2H, m), 3.41 (1H, d, J ) 17.0 Hz), 3.67 (1H, dd, J ) 10.2, 5.8
Hz), 3.79 (3H, s), 3.85 (3H, s), 3.98 (1H, d, J ) 17.0 Hz), 7.02
(1H, s), 7.37 (1H, s). Anal. (C13H14O5S) C, H.
(2R,4S)-(-)-1,2,4,5-Tetr a h yd r o-4-m eth yl-7,8-m eth ylen e-
d ioxy-5-oxo-3-ben zoth iep in -2-ca r boxylic Acid [(-)-10q].
A mixture of (-)-12 (4.2 g, 9.8 mmol), AcOH (45 mL) and
concentrated HCl (30 mL) was refluxed for 30 min. The
reaction mixture was poured into H2O (800 mL) to give
crystals, which were collected by filtration and dissolved in
AcOEt (150 mL). The solution was washed with brine, dried
over MgSO4, and concentrated in vacuo to give crystals.
Recrystallization from AcOEt-hexane gave (-)-10q as color-
[R]16 -194.1° (c ) 0.50, CHCl3); 1H NMR (CDCl3) δ 1.56 (3H,
D
d, J ) 7.0 Hz), 3.26 (1H, dd, J ) 15.0, 5.4 Hz), 3.42 (1H, d, J
) 17.6 Hz), 3.47 (1H, dd, J ) 15.0, 11.4 Hz), 3.78 (1H, dd, J )
11.4, 5.4 Hz), 3.80 (3H, s), 3.94 (3H, s), 3.96 (3H, s), 3.99 (1H,
d, J ) 17.6 Hz), 5.21 (1H, q, J ) 7.0 Hz), 6.76 (1H, s), 7.51
(1H, s). Anal. (C17H20O7S) C, H.
less needles (1.62 g, 59%): mp 194-195 °C; [R]23 -210.8° (c
D
1
) 0.50, MeOH); H NMR (CDCl3) δ 1.54 (3H, d, J ) 7.0 Hz),
The filtrate was concentrated in vacuo to give crystals,
which were collected by filtration and recrystallized from
AcOEt-hexane to give 11b as pale orange plates (1.6 g, 20%):
3.22 (1H, dd, J ) 14.6, 5.1 Hz), 3.41 (1H, dd, J ) 14.6, 12.1
Hz), 3.60 (1H, dd, J ) 12.1, 5.1 Hz), 4.05 (1H, q, J ) 7.0 Hz),
6.05 (2H, dd, J ) 2.4, 1.4 Hz), 6.69 (1H, s), 7.52 (1H, s). Anal.
(C13H12O5S) C, H.
mp 121-122 °C; [R]16 +234.3° (c ) 0.50, CHCl3); 1H NMR
D
(CDCl3) δ 1.57 (3H, d, J ) 7.0 Hz), 3.26 (1H, dd, J ) 14.8, 5.2
Hz), 3.44 (1H, d, J ) 17.6 Hz), 3.49 (1H, dd, J ) 14.8, 11.6
Hz), 3.74 (1H, dd, J ) 11.6, 5.2 Hz), 3.79 (3H, s), 3.94 (3H, s),
3.96 (3H, s), 4.00 (1H, d, J ) 17.6 Hz), 5.22 (1H, q, J ) 7.0
Hz), 6.73 (1H, s), 7.52 (1H, s). Anal. (C17H20O7S) C, H. The
absolute configuration of 11b was confirmed as (2S)-form by
the X-ray crystallographic analysis.12
(2S,4R)-(+)-1,2,4,5-Tetr a h yd r o-4-m eth yl-7,8-m eth ylen e-
d ioxy-5-oxo-3-ben zoth iep in -2-ca r boxylic Acid [(+)-10q].
The title compound was prepared according to the procedure
described for (-)-10q in 48% yield as colorless needles: mp
192-193 °C (AcOEt-hexane); [R]23D +212.9° (c ) 0.50, MeOH);
1H NMR (CDCl3) δ 1.54 (3H, d, J ) 7.0 Hz), 3.22 (1H, dd, J )
14.6, 5.1 Hz), 3.41 (1H, dd, J ) 14.6, 12.1 Hz), 3.60 (1H, dd, J
(r′R)-r′-Met h oxyca r b on ylb en zyl (2R,4S)-(-)-1,2,4,5-

Synthesis of Novel 2-Benzothiopyran Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 759
) 12.1, 5.1 Hz), 4.06 (1H, q, J ) 7.0 Hz), 6.04 (2H, dd, J ) 2.4,
P h osp h a ta se (ALP ) Activity. (1) Ra t Bon e Ma r r ow Str o-
m a l Cells.10 Bone marrow stromal cells were obtained from
the femoral bone marrow of 7-week-old male Sprague-Dawley
rats (J apan Charles River, Tokyo, J apan).13 The standard
culture medium consisting of R-minimum essential medium
(MEM) containing 10 mM HEPES (pH 7.0) was used. It
contained 15% fetal bovine serum (FBS), 2 mM glutamine, 50
1.4 Hz), 6.69 (1H, s), 7.52 (1H, s). Anal. (C13H12O5S) C, H.
(2R)-(-)-N -[4-(Die t h oxyp h osp h or ylm e t h yl)p h e n yl]-
1,2,4,5-tetr a h yd r o-7,8-d im eth oxy-5-oxo-3-ben zoth iep in -
2-ca r boxa m id e [(-)-2h ]. A solution of WSC (149 mg, 0.78
mmol) in CH2Cl2 (3 mL) was added to a solution of (-)-10h
(183 mg, 0.65 mmol) and diethyl 4-aminobenzylphosphonate
(158 mg, 0.65 mmol) in DMF (3 mL) at 0 °C followed by
addition of 1-hydroxybenzotriazole (HOBt) (109 mg, 0.71
mmol). After being stirred at 0 °C for 1 h and at room
temperature for 15 h, the reaction mixture was poured into
H2O (30 mL) and extracted with CHCl3. The extract was
washed with H2O and brine, dried over MgSO4, and concen-
trated in vacuo. The residual oil was chromatographed on SiO2
(10 g) with AcOEt-CHCl3-MeOH (1:1:0 to 15:15:1, v/v) to give
µg/mL of ascorbic acid, 10 mM â-glycerophosphate, 10-7
M
dexamethasone, and antibiotics (80 µg/mL of gentamicin and
100 µg/mL of kanamycin). The cells were seeded in 100 mm
culture dishes (Falcon 3003: Becton Dickinson and Co.,
Lincoln Park, NJ ) containing 10 mL of culture medium and
cultured at 37 °C in an atmosphere of 5% CO2 in humidified
air. After 1 week, confluent cells in the primary culture were
harvested after treatment with 0.25% trypsin and 0.2% EDTA
and then subcultured in a 6-well plate (Falcon 3046: Becton
Dickinson and Co., Lincoln Park, NJ ) at a cell density of 4 ×
104 cells/well (day 0). The test compound was dissolved in a
solution of EtOH-DMSO (1:1, v/v) or DMF at a concentration
of 10 mM before use, diluted with culture medium to the
designated concentrations, and added to cultures from day 1
to the end of the experiment (day 10-14). Culture medium
was changed every other day. The test compound was freshly
dissolved and diluted with culture medium at each medium
change. The cellular ALP activity was determined by the
method of Lowry et al.21 using the supernatant of the cell
lysate as previously described.10
(2) MC3T3-E1 cells. MC3T3-E1 cells were seeded in a 96-
well plate (NUNCLON: Nunc, Roskilide, Denmark) at a cell
density of 3000 cells/well and were cultured in R-MEM
containing 10% FBS. Three days later, the medium was
replaced with R-MEM containing 1% FBS and designated
concentrations of the test compound with or without 3 ng/mL
recombinant (Xenopus) BMP-4/7 heterodimer,17 followed by
further cultivation for 3 days. The ALP activity in the
supernatant of the cell lysate was assayed.
(-)-2h as a colorless amorphous solid (136 mg, 41%): [R]23
D
-155.0° (c ) 0.50, CHCl3); 1H NMR (CDCl3) δ 1.18 (3H, t, J )
7.0 Hz), 1.19 (3H, t, J ) 7.1 Hz), 3.11 (2H, dd, J ) 21.5, 2.1
Hz), 3.38 (1H, dd, J ) 15.0, 5.2 Hz), 3.52 (1H, d, J ) 17.0 Hz),
3.56 (1H, dd, J ) 15.0, 12.0 Hz), 3.93 (3H, s), 3.94 (3H, s),
3.83-4.06 (6H, m), 6.76 (1H, s), 7.22 (2H, dd, J ) 8.7, 2.5 Hz),
7.40 (2H, d, J ) 8.2 Hz), 7.56 (1H, s), 8.98 (1H, s). Anal. (C24H30
NO7SP) C, H, N. The optical purity of (-)-2h was found to be
>99% ee by chiral stationary phase HPLC.
-
(2S )-(+)-N -[4-(Die t h oxyp h osp h or ylm e t h yl)p h e n yl]-
1,2,4,5-tetr a h yd r o-7,8-d im eth oxy-5-oxo-3-ben zoth iep in -
2-ca r boxa m id e [(+)-2h ]. The title compound was prepared
according to the procedure described for (-)-2h in 40% yield
as a colorless amorphous solid: [R]23 +155.3° (c ) 0.50,
D
CHCl3); 1H NMR (CDCl3) δ 1.18 (3H, t, J ) 7.0 Hz), 1.19 (3H,
t, J ) 7.1 Hz), 3.11 (2H, dd, J ) 21.5, 2.1 Hz), 3.38 (1H, dd, J
) 15.0, 5.2 Hz), 3.52 (1H, d, J ) 17.0 Hz), 3.56 (1H, dd, J )
15.0, 12.0 Hz), 3.92 (3H, s), 3.94 (3H, s), 3.86-4.03 (6H, m),
6.75 (1H, s), 7.20 (2H, dd, J ) 8.7, 2.5 Hz), 7.38 (2H, d, J )
8.2 Hz), 7.55 (1H, s), 9.10 (1H, s). Anal. (C24H30NO7SP) C, H,
N. The optical purity of (+)-2h was found to be >99% ee by
chiral stationary phase HPLC.
2. Effect on a Ra t Sk u ll Defect Mod el. An EVA copoly-
mer pellet containing the compound was prepared according
to the procedure described by Wakamatsu et al.19 Release of
the compound from the pellet was confirmed by the increase
of ALP activity in culture of rat bone marrow stromal cells.19
Six-week-old male Splague-Dawley rats (J apan Charles River,
Tokyo, J apan) were used.22 With the rats under anesthesia
by intraperitoneal injection (0.1 mL/100 g body weight) of 50
mg/mL pentobarbital sodium (Abott Laboratories, North Chi-
cago, IL), an incision was made in the skin over the sagittal
suture, and the parietal bones were exposed. A trephine defect,
4 mm in diameter, was drilled with a 4 mm sterile trephine
under a continuous saline buffer irrigation using a low-speed
dental drill. An EVA copolymer pellet containing the compound
or an empty EVA copolymer pellet was placed onto the
adjacent nondefected skull as illustrated in Figure 1 and the
skin flaps were sutured. Four weeks after the operation, the
animals were killed by carbon dioxide inhalation. The skulls
were removed and the new bone formation was evaluated
radiographically. Area of newly formed bone in defect was
calculated according to the following formula: [area of the
initial defect created by trephination (12.6 mm2)] - [area of
the remaining defect].
(2R,4S)-(-)-N-[4-(Dieth oxyp h osp h or ylm eth yl)p h en yl]-
1,2,4,5-tetr a h yd r o-4-m eth yl-7,8-m eth ylen ed ioxy-5-oxo-3-
ben zoth iep in -2-ca r boxa m id e [(-)-2q]. A solution of WSC
(0.39 g, 2.0 mmol) in CH2Cl2 (7 mL) was added to a solution of
(-)-10q (0.47 g, 1.7 mmol) and diethyl 4-aminobenzylphos-
phonate (0.41 g, 1.7 mmol) in DMF (7 mL) at 0 °C followed by
addition of HOBt (0.28 g, 1.8 mmol). After being stirred at 0
°C for 1 h and at room temperature for 15 h, the reaction
mixture was poured into H2O (50 mL) and extracted with
CHCl3. The extract was washed with H2O and brine, dried over
MgSO4, and concentrated in vacuo to give crystals. Recrystal-
lization from MeOH-hexane gave (-)-2q as colorless prisms
(0.37 g, 44%): mp 181-182 °C; [R]23 -187.4° (c ) 0.50,
D
CHCl3); 1H NMR (CDCl3) δ 1.17 (3H, t, J ) 7.0 Hz), 1.18 (3H,
t, J ) 7.1 Hz), 1.54 (3H, d, J ) 7.2 Hz), 3.11 (2H, dd, J ) 21.5,
3.9 Hz), 3.31 (1H, dd, J ) 14.8, 5.0 Hz), 3.54 (1H, dd, J ) 14.8,
12.4 Hz), 3.83 (1H, dd, J ) 12.4, 5.0 Hz), 3.83-4.02 (4H, m),
4.17 (1H, q, J ) 7.2 Hz), 6.04 (2H, s), 6.73 (1H, s), 7.18 (2H,
dd, J ) 8.4, 2.6 Hz), 7.41 (2H, d, J ) 8.0 Hz), 7.55 (1H, s),
9.51 (1H, s). Anal. (C24H28NO7SP) C, H, N. The optical purity
of (-)-2o was found to be >99% ee by chiral stationary phase
HPLC.
3. Sta tistica l An a lysis. All results are expressed as means
( standard errors of the mean (SE). Intergroup differences in
means were statistically analyzed using Dunnett’s multiple
comparison method or Student’s t-test. A p-value of less than
0.05 was considered significant.
(2S,4R)-(+)-N-[4-(Dieth oxyp h osp h or ylm eth yl)p h en yl]-
1,2,4,5-tetr a h yd r o-4-m eth yl-7,8-m eth ylen ed ioxy-5-oxo-3-
ben zoth iep in -2-ca r boxa m id e [(+)-2q]. The title compound
was prepared according to the procedure described for (-)-2q
in 41% yield as colorless prisms: mp 183-184 °C (MeOH-
hexane); [R]23 +190.5° (c ) 0.50, CHCl3); 1H NMR (CDCl3) δ
D
1.17 (3H, t, J ) 7.0 Hz), 1.18 (3H, t, J ) 7.1 Hz), 1.54 (3H, d,
J ) 7.2 Hz), 3.11 (2H, dd, J ) 21.5, 3.9 Hz), 3.31 (1H, dd, J )
14.8, 5.0 Hz), 3.54 (1H, dd, J ) 14.8, 12.4 Hz), 3.82 (1H, dd, J
) 12.4, 5.0 Hz), 3.83-4.02 (4H, m), 4.16 (1H, q, J ) 7.2 Hz),
6.03 (2H, s), 6.73 (1H, s), 7.17 (2H, dd, J ) 8.4, 2.6 Hz), 7.41
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NO7SP) C, H, N. The optical purity of (+)-2q was found to be
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Biologica l P r oced u r es. 1. Assa y of Cellu la r Alk a lin e

760 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
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Institutional Experimental Animal Care and Use Committee.
J M980583B