Synthesis and biological activity of 4-methyl-3,5-dioxane derivatives as thromboxane A2 receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(99)00204-7

The synthesis and biological activity of novel 4-methyl-3,5-dioxane analogues are described. All compounds were produced through modification of the substituent formally corresponding to the ω-octenol side chain of thromboxane A₂ (TXA₂

Full text of DOI:10.1016/S0968-0896(99)00204-7

Bioorganic & Medicinal Chemistry 7 (1999) 2635±2645
Synthesis and Biological Activity of 4-Methyl-3,5-dioxane
Derivatives as Thromboxane A2 Receptor Antagonists
Hiroshi Marusawa, Hiroyuki Setoi,* Akio Kuroda, Akihiko Sawada, Jiro Seki,
Yukio Motoyama and Hirokazu Tanaka
Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 5-2-3 Tokodai, Tsukuba-shi, Ibaraki 300-2698, Japan
Received 23 April 1999; accepted 21 July 1999
AbstractÐThe synthesis and biological activity of novel 4-methyl-3,5-dioxane analogues are described. All compounds were pro-
duced through modi®cation of the substituent formally corresponding to the o-octenol side chain of thromboxane A2 (TXA2), in
reference to the structure of SQ29548. Several compounds were found to be potent TXA2 receptor antagonists. Compound 8b was
the most e€ective inhibitor of 9,11-epoxymethano PGH2 (U-46619)-induced human platelet aggregation (IC50=7.4 nM). # 1999
Elsevier Science Ltd. All rights reserved.
Introduction
Thromboxane A2 (TXA2) 11,2 is a potent stimulator of
platelet aggregation3,4 and also induces contraction of
smooth muscle.5 Blocking the action of TXA2 is becoming
an established method for treating asthma and various
cardiovascular diseases in the clinic.6±10 Several drugs and
development candidates with prostanoid structures hav-
ing a semicarbazide or a semicarbazone moiety, such as
SQ29548 2,11,12 SQ27825 313 and EP045 414 or dioxane
derivatives, such as ICI180080 5a,15 ICI185282 5b,16
ICI192605 5c17,18 and with non-prostanoid structures,
such as Sulotroban 619±21 have been discovered and con-
®rmed as selective TXA2 receptor antagonists.
After investigation of conformational similarities
between SQ29548 and TXA2, we identi®ed a 4-methyl-
3,5-dioxane skeleton, which includes two oxygen atoms
in its ring system like TXA2, as a new structurally stable
lead for TXA2 receptor antagonists. In addition, based
on earlier studies22 on the side chain stereochemistry of
the ring system, we chose the cis-substituted compound
7a, which has similar activity to SQ29548, as a useful
prototype for a novel series of TXA2 receptor antago-
nists. In order to improve TXA2 receptor antagonistic
activity, we maintained certain structural features of
TXA2, particularly the 1,3-dioxane ring system and the
a-heptenoic acid chain, and chose to alter the nature of
the substituent formally corresponding to the o-octenol
side chain. In the course of these studies, we discovered
(Z) - 7- {(1S,2R,4R) - 2 - [2 - aza - 2(((phenyamino)thioxo -
methyl)amino)vinyl]-4-methyl-3,5-dioxanyl}hept-5-enoic
acid 8a, which possesses speci®c and potent TXA2
receptor antagonistic activity. The synthesis and biolo-
gical properties of 8a and 8b and certain structural
analogues are described below.
* Corresponding author.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00204-7

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H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
(R1 and R2) shown in Scheme 2, was accomplished by
coupling 20 with the appropriate amine, followed by
hydrolysis with 1 N NaOH/MeOH, and provided the
target compounds 7a, 8a, 21±25. For compounds 7a and
8a, sodium salts 7b and 8b were also synthesized.
Sodium cyanoborohydride (NaBH3CN) reduction of
the imines gave the corresponding amines, which were
then treated with 1 N NaOH/MeOH to a€ord the
desired compounds 27±29. Compound 26 was obtained
from 7a by treatment with NaBH3CN.
Chemistry
The synthesis of the key intermediate 19 for the 4-
methyl-3,5-dioxane analogues proceeded from commer-
cially available glucose derivative 9 and is outlined in
Scheme 1. 1,3-Dioxane diol 11 was obtained by NaIO4
oxidation23,24 of 9, followed by reaction of the inter-
mediate aldehyde 10 with NaBH4 in EtOH, as described
in the literature.25 Protection of the primary hydroxy
group in 11 with t-butyldimethylsilyl (TBDMS) chloride,
followed by Mo€att oxidation provided the ketone 13.
Wittig reaction of 13 with ethyl (triphenylphosphorany-
lidene)acetate a€orded the ester 14 (E/Z mixture), which
was then subjected to catalytic reduction on 10% Pd-C
in EtOH, followed by silica gel chromatography (n-
hexane±AcOEt) to a€ord the desired ester 15 (55.7%
from 14, major product). Diisobutylaluminium hydride
(DIBAL) reduction of 15 gave aldehyde 16, which was
then treated with the Wittig reagent prepared from (4-
carboxybutyl) triphenylphosphonium bromide and
dimsyl sodium in DMSO, to yield acid 17. The acid was
methylated with CH3I and the TBDMS group in the
resulting ester 18 easily removed by treatment with n-
Bu4NF, to a€ord the desired intermediate alcohol 19.
Conversion of 19 to the phthalimide 30 was accomplished
by displacement of the corresponding tosylate with
potassium phthalimide. Treatment of 30 with hydrazine
hydrate a€orded the amine 31, which was then condensed
with the appropriate isocyanate or acid, followed by
alkaline hydrolysis or was treated with 1 N NaOH/MeOH
followed by condensation with the appropriate isocya-
nate, to provide the desired compounds 32±34.
Analogues in which an oxygen atom was located at the
14-position were synthesized as outlined in Scheme 4.
Treatment of 19 with phenylisocyanate or 2-picolyl
chloride a€orded urethane 35 and ether 37, respectively,
which were then treated with 1 N NaOH in aqueous
methanol, to yield target compounds 36 and 38.
Results and Discussion
We assumed that the semicarbazide and semicarbazone
structures corresponding to the o-octenol side chain
were very important for TXA2 receptor antagonistic
activity, as shown by the activity of SQ29548, SQ27825
and EP045. In addition, we thought that pyridine deri-
vatives and the like were their equivalents, so we syn-
thesized one group of analogues which possess a stable
Analogues in which a nitrogen atom was located at the
14-position were synthesized as outlined in Schemes 2
and 3. Oxidation of the alcohol 19 with Collins' reagent
gave the aldehyde 20. Conversion of 20 to imines (for
example, 39±42), where R represents the substituents
Scheme 1. (a) NaIO4/H2O, 5ꢀC; (b) NaBH4/EtOH, 5ꢀC; (c) TBDMSCl/imidazole/DMF, rt; (d) DMSO/DCC/TFA/pyridine/benzene, rt; (e)
Ph3P=CHCOOEt/THF, rt; (f) 10%Pd-C/H2(3atm)/EtOH, rt then silica gel column; (g) DIBAL-H/toluene, 78ꢀC; (h) NaH/DMSO, 75ꢀC then
(Ph3PC4H9COOH)+Br /DMSO, rt; (i) CH3I/K2CO3/DMF, rt; (j) n-Bu4NF/THF, rt.

H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
2637
.
Scheme 2. (a) CrO3 2pyridine/CH2Cl2, rt; (b) R1NH2 or R2NH2/AcOH/MeOH, rt; (c) 1 N NaOH/MeOH, rt; (d) NaBH3CN/AcOH/MeOH, rt; (e) 1
N NaOH/MeOH, rt then treated at pH>10.
ꢀ
.
Scheme 3. (a) TsCl/pyridine, rt then potassium pthalimide/DMSO, 100 C; (b) H2NNH2 H2O/EtOH, rt; (c) PhNCO/pyridine, rt or PhCH2NCO/
pyridine, rt or PhCH2COCOOH/DCC/EtOAc, rt; (d) 1 N NaOH/MeOH, rt.
.
Scheme 4. (a) PhNCO/pyridine, rt; (b) 1 N NaOH/MeOH, rt; (c) 2-picolylchloride HCl/NaH/DMF, rt.
imine functionality (7a, 8a, 21±25) at the 14 position
and another group of analogues which possess a stable
amine functionality (26, 27±29, 32±34) at the 14 posi-
tion, in order to ®nd a novel potent TXA2 receptor
antagonist and to extract the important attributes in the
case of 4-methyl-3,5-dioxane analogues.
signi®cant loss in TXA2 receptor antagonistic activity.
Therefore, we con®rmed the importance of the NH
group at the 15 position on the o side chain. Replace-
ment of the anilino group next to the carbonyl group at
the 16 position of 7a with a pyridy 1 group, as in 22
signi®cantly decreased the potency (IC50>100 mM).
This tendency was apparent for the reduced compound
26 and the thienyl compound 28 as well. In addition,
comparison of compounds 22 and 23 showed that dele-
tion of the carbonyl group leads to an increase in activ-
ity. This suggests that the NH group at the 15 position
does not behave as an acidic proton in its biological
activity.
The target compounds described in Schemes 2 and 3
were evaluated for their ability to inhibit platelet aggre-
gation of rabbit platelet-rich plasma (PRP) induced by
26,27
9, 11-azo PGH2
(5 mL, ®nal concentration 1 mM) or
adenosine diphosphate (ADP, 5 mL, ®nal concentration
2.5 mM). The concentrations (IC50) that caused 50%
inhibition of platelet aggregation are shown in Table 1.
Consistent with their mechanism of action, none of the
compounds was e€ective in inhibiting ADP induced
platelet aggregation.
Comparison between compounds 7a and 26 demon-
strates that reduction of the imine functionality leads to
a decrease in potency, in contrast with the result28,29 for
SQ29548, but consistent with the result for a series of
norbornane semicarbazones.30 Up to the present date,
the cloning of a human TXA2 receptor has been done31
As shown from the results for both groups in Table 1,
replacement of the NH group at the 15 position led to a

2638
H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
and a detailed analysis of the TXA2 receptor±TXA2
receptor antagonist interaction has been reported.32
Furthermore, estimation of intramolecular hydrogen
bonding33 in TXA2 receptor antagonists and proposal
of a common spatial pharmacophore34 for TXA2
receptor antagonists has also been reported. However,
we believe that it is very di®cult to explain such a dif-
ference in activity clearly using the above information.
In this series of 4-methyl-3,5-dioxane analogues, it is
assumed that the imine functionality is more suitable
than the amine one from the results shown in Table 1.
From these results, we concluded that the optimal new
o side chain was a 2-aza-2{[(phenylamino)thioxo-
methyl]amino}vinyl group from this study. In order to
clarify this and to further evaluate activity with a view
to selecting a potential drug candidate, we then synthe-
sized 8b, the sodium salt of 8a, together with 7b, the
sodium salt of 7a, and examined additional biological
activities, as shown in Tables 2±5. In a radioligand
binding assay with guinea-pig platelets, we con®rmed
that 7b and 8b inhibited the binding of U-4661935,36
7
8
with IC50 values of 1.6Â10
M and 7.7Â10
M,
respectively and possessed high anity for the TXA2
receptor (Table 2).
In an attempt to explore further possibilities for the o
side chain of the 4-methyl-3,5-dioxane skeleton, we
synthesized ether derivatives (36, 38) at the 14 position,
as described in Scheme 4, but TXA2 receptor antag-
onistic activity was not observed (Table 1). These results
also demonstrated the requirement for the NH group at
the 15 position to maintain antagonistic activity.
We selected compound 8b through these experiments,
and next evaluated in vitro inhibitory activities against
U-46619- and collagen-induced platelet aggregation in
various species. As shown in Table 3, 8b displayed
highly speci®c inhibitory activity, especially against
human and monkey platelets. In addition, we evaluated
inhibitory activity against ex vivo platelet aggregation
induced by U-46619, following oral administration of
8b to guinea-pigs, and con®rmed the activity, as shown
in Table 4. Furthermore, compound 8b was e€ective in
the arachidonic acid-induced pulmonary infraction
mouse model, which is well known for evaluating TXA2
receptor antagonists (Table 5). The stability of these 4-
methyl-3,5-dioxane skeleton was also demonstrated
through the e€ectiveness of these oral administration
experiments as we expected.
Table 1. In vitro activity of 4-methyl-3,5-dioxane analogues
Compound
R
Inhibition of rabbit
platelet aggregation
(IC50, mMa)
9,11-azo PGH2
ADP
7a
8a
21
CH=NNHCONHPh
CH=NNHCSNHPh
CH=NNHCOOtBu
0.26
0.055
53
>100
>100
>100
Conclusion
We have investigated various modi®cations of the sub-
stituent formally corresponding to the o-octenol side
chain of TXA2, and concluded that the NH group at the
15 position is of importance for the activity of 4-methyl-
3,5-dioxane analogues. Through evaluation of these deri-
vatives, compound 8b was found to be the most potent
22
23
24
>100
36
>100
>100
>100
35
Table 2. Potencies of 7b and 8b for inhibition of [3H]-U46619 bind-
ing to washed guinea-pig platelets
25
26
CH=N OCHPh2
CH2NHNHCONHPh
>100
16
>100
>100
Compound
7b
8b
27
52
>100
1.6Â10 7 M
7.7Â10
M
a
8
IC50
a
IC50 values were calculated by regression analyses from ®ve dose
groups, using three di€erent reparations.
28
29
>100
>100
>100
>100
Table 3. In vitro activities of compound 8b in various species
32
33
34
36
CH2NHCONHPh >100
CH2NHCONHCH2Ph >100
>100
>100
>100
>100
Species
Human
Agonist
IC50, Ma
9
CH2NHCOCOCH2Ph
CH2OCONHPh
>100
>100
U-46619
Collagen
U-46619
Collagen
U-46619
Collagen
Collagen
7.4Â10
5.0Â10
5.6Â10
2.0Â10
5.2Â10
7.5Â10
2.2Â10
9
9
7
8
8
7
Monkey
Dog
Guinea-pig
38
>100
0.23
>100
>100
SQ 29, 548
Rat
a
IC50 values were calculated by regression analyses from three dose
groups, using three di€erent preparations.
a
IC50 values were calculated by regression analyses from three dose
groups, using four di€erent preparations.

H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
2639
Table 4. Inhibition of ex vivo platelet aggregation by oral adminis-
tration of compound 8b in guinea-pig
48.0mmol) under cooling in an ice bath and the mixture
was stirred at room temperature for 3 h. The resulting
solution was added to a mixture of ethyl acetate (50 mL)
and water (30 mL), and stirred for 30min. After removal
of insoluble urea by ®ltration, the organic layer was sepa-
rated and washed successively with water and brine. The
solution was dried over magnesium sulfate and the solvent
was evaporated in vacuo to give a crude oil. The oil was
puri®ed by a silica gel column (50 g; n-hexane:ethyl ace-
tate=10:1) to give 3.22g (77.3%) of 13 as a pale yellow
oil: 1H NMR (200 MHz, CDCl3) d 0.07 (3H, s), 0.09 (3H,
s), 0.89 (9H, s), 1.47 (3H, d, J=5.5 Hz), 3.98 (2H, d,
J=3.5Hz), 4.3±4.5 (3 H, in), 5.11 (1H, q, J=5.5Hz). ESI-
MS m/z: 261 (M+H)+.
8b (mg/kg)
Inhibition (%)a
U-46619
Collagen
1.0
3.2
1.0
3.2
45.1
98.3**
60.5*
87.5**
a
*P<0.05 and **P<0.01, compared with control group. Data
represents the average percent of inhibition in a group of 5 animals.
Table 5. E€ects of 8b in the arachidonic acid-induced pulmonary
infraction mice model
Compound
8b
Dose (mg/kg, po)
Inhibition (%)a
3.2
30
10
50
32
100
Ethyl (2R,4R)-{4-methyl-2-[(1,1,2,2-tetramethyl-1-silapro-
poxy)methyl]- 3,5 - dioxanylidene}acetate (14). A mixture
of 13 (2.50 g, 9.60 mmol) and ethyl (triphenylphosphor-
anylidene)acetate (4.00 g, 12.0 mmol) in tetrahydrofuran
(25 mL) was stirred at room temperature for 24h and the
solvent was evaporated in vacuo. The residue was chro-
matographed on a silica gel column (50 g) with a mixture
of n-hexane and ethyl acetate (10:1) as eluent to give
2.09 g (65.9%) of 14 as an oil: 1H NMR (200 MHz,
CDCl3) d 0.08 (6H, s), 0.90 (9H, s), 1.38 (3H, t, J=7.5 Hz),
1.47 (1H, d, J=5.0 Hz), 3.83 (1H, dd, J=9, 11Hz), 3.87
(1H, dd, J=9, 11 Hz), 4.17 (1H, q, J=7.5 Hz), 4.30 (1H,
m), 4.56 (1H, dd, J=17, 2 Hz), 4.93 (1H, q, J=5Hz), 5.44
(1H, d, J=17 Hz), 5.89 (1H, m). ESI-MS m/z: 331
(M+H)+.
a
Data represents the survival percent in a group of 10 mice.
and speci®c in inhibiting U-46619 or collagen induced
9
9
human platelet aggregation (7.4Â10 M, 5.0Â10 M,
respectively). Our further studies on optimizing this
activity will be the subject of subsequent papers.
Experimental
Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker AM-200 spectrometer using
tetramethylsilane (TMS) or 3-(trimethylsilyl)propionic
acid -d4 sodium salt (TSP-d4) as an internal reference. The
ESI mass spectra (MS) were recorded on a Plateform LC-
MS spectrometer (Micromass). Column chromatography
was carried out on silica gel 60 (E. Merck, 70±200 mesh).
Thin-layer chromatography (TLC) was performed on
0.5 or 2 mm precoated silica gel plates from E. Merk
(Kieselgel 60F254).
Ethyl (1S,2R,4R)-{4-methyl-2-[(1,1,2,2-tetramethyl-1-si-
lapropoxy)methyl]-3,5-dioxanyl}acetate (15). A solution
of 12 (17.0 g, 51.4 mmol) in ethanol (170mL) was shaken
under hydrogen (3 atm) with 10% palladium on carbon at
room temperature for 1.5 h. After removal of the catalyst
by ®ltration, the solvent was evaporated in vacuo and the
residue was chromatographed on a silica gel column
(500g) with a mixture of n-hexane and ethyl acetate (20:1)
as eluent to give 9.52g (55.7%) of 15 as a pale yellow oil:
1H NMR (200MHz, CDCl3) d 0.07 (6H, s), 0.89 (9H, s),
1.26 (3H, t, J=7Hz), 1.31 (3H, d, J=5.0 Hz), 2.06 (1H,
m), 2.43 (1H, m), 2.71 (1H, dd, J=10, 16Hz), 3.52 (1H,
dd, J=7, 11 Hz), 3.68 (1H, dd, J=7, 11Hz), 3.83 (1H, dt,
J=2, 11Hz), 3.89 (1H, dt, J=3, 7 Hz), 4.06 (1H, d,
J=12 Hz), 4.14 (2H, q, J=7Hz), 4.72 (1H, q, J=5Hz).
ESI-MS m/z: 333 (M+H)+.
(2R,4S,5R)-2-Methyl-4-[(1,1,2,2-tetramethyl-1-silapro-
poxy)methyl]-1,3-dioxan-5-ol (12). A mixture of (2R,4S,
5R)-4-hydroxymethyl-2-methyl-1,3-dioxan-5-ol 11 (13.0 g,
87.7 mmol), tert-butylchlorodimethylsilane (14.5 g,
133 mmol) and imidazole (13.1 g, 192 mmol) in N,N-
dimethylformamide (130 mL) was stirred at room tem-
perature for 2 h and the mixture was diluted with ethyl
acetate (500mL). The solution was washed successively
with water, diluted hydrochloric acid, saturated aqueous
sodium hydrogen carbonate and brine, and dried over
magnesium sulfate. The solvent was evaporated in vacuo
(1S,2R,4R)-{4-Methyl-2-[(1,1,2,2-tetramethyl-1-silapro-
poxy)methyl] - 3,5 - dioxanyl}ethanal (16). A solution of
15 (9.3 g, 28.0 mmol) in toluene (93 mL) was cooled in a
dry ice acetone bath and to the solution was added
dropwise diisobutyl aluminum hydride (1.5 M solution
in toluene, 26.4 mL, 39.6 mmol). The mixture was stir-
red at the same temperature for 1 h. After quenching the
mixture with saturated aqueous anunonium chloride, to
the solution was added a mixture of ethyl acetate
(300 mL) and water (300 mL). Insoluble materials were
®ltered o€. The ®ltrate was extracted with ethyl acetate
and the organic layer was washed with brine and dried
over magnesium sulfate. The solvent was evaporated in
vacuo and the residue was chromatographed on a silica
gel column (250 g) with a mixture of n-hexane and ethyl
1
to give 24.2g (100%) of 12 as a colorless oil: H NMR
(200MHz, CDCl3) d 0.10 (3H, s), 0.12 (3H, s), 0.91 (9H,
s), 1.32 (3H, d, J=5 Hz), 3.3±3.6 (3H, m), 3.7±3.8 (2H, m),
3.94 (1H, dd, J=4, 9 Hz), 4.13 (1H, dd, J=5, 9 Hz), 4.70
(1H, q, J=5 Hz). ESI-MS m/z: 263 (M+H)+.
(2R,4S)-2-Methyl-4-[(1,1,2,2-tetramethyl-1-silapropoxy)-
methyl]-1,3-dioxan-5-one (13). A solution of 12 (4.2 g,
16.0 mmol) in a mixture of benzene (30 mL) and dime-
thylsulfoxide (5.7mL, 73.6 mmol) was added to pyridine
(1.3 mL, 16.1mmol), tri¯uoroacetic acid (0.62 mL,
8.05mmol) and N,N0-dicyclohexylcarbodiimide (9.90 g,

2640
H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
acetate (10:1) as eluent to live 6.61 g (81.9%) of 16 as a
1
(3H, m), 2.3±2.6 (3H, m), 3.68 (3H, s), 3.7±3.9 (3H, m),
3.93 (1H, m), 4.00 (1H, dd, J=2, 12.5 Hz), 4.76 (1H, q,
J=5.0Hz), 5.3±5.6 (2H, m). ESI-MS m/z: 273 (M+H)+.
colorless oil: H NMR (200 MHz, CDCl3) d 0.08 (6H,
s), 0.88 (9H, s), 1.31 (3H, d, J=5.5 Hz), 2.19 (1H, m),
2.63 (1H, dd, J=5, 17 Hz), 2.88 (1H, dd, J=9, 17 Hz),
3.50 (1H, dd, J=10, 11 Hz), 3.68 (1H, dd, J=7, 11 Hz),
3.8±4.1 (2H, m), 4.74 (1H, q, J=5.5 Hz). ESI-MS m/z:
289 (M+H)+.
Methyl (Z)-7-((1S,2R,4R)-2-formyl-4-methyl-3,5-dioxa-
nyl)hept-5-enoate(20). To a solution of pyridine (1.64
mL, 20.3 mmol) in dichloromethane (45 mL) was added
chromium trioxide (1.07 g, 10.7mmol) at 10ꢀC and the
solution was stirred at room temperature for 1 h. The
solution was cooled in an ice bath and a solution of 19
(500mg, 1.84mmol) in dichloromethane (3mL) added.
After 2 h, the solution was diluted with ether (100mL) and
passed through a silica gel column. The eluate was eva-
porated in vacuo and the residue was chromatographed
on a silica gel column (20 g) with a mixture of n-hexane
and ethyl acetate (1:1) as eluent to give 336 mg (67.7%) of
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[(1,1,2,2-tetramethyl-1-si-
lapropoxy)methyl]-3,5-dioxanyl}hept-5-enoic acid (17).
A suspension of sodium hydride (3.49 g, 60% in oil,
87.3 mmol) in dimethylsulfoxide (75 mL) was heated at
75ꢀC for 1 h and the resulting solution was cooled to
room temperature. To the solution was added dropwise
(4-carboxybutyl)triphenylphosphonium bromide (32.2 g,
72.6 mmol) in dimethylsulfoxide (100 mL). After stirring
at room temperature for 15 min, to the mixture was
added 16 (6.3 g, 21.8 mmol) in dimethylsulfoxide
(10 mL) and the solution was stirred at room tempera-
ture for 1.5 h. To the reaction mixture was added aque-
ous ammonium chloride (100mL) and the mixture
adjusted to pH 4 with oxalic acid. The mixture was
extracted with ethyl acetate and the organic layer washed
successively with water and brine, and dried over magne-
sium sulfate. The solvent was evaporated in vacuo and the
residue was chromatographed on a silica gel column
(150g) with a mixture of n-hexane and ethyl acetate (10:1±
1:1) as eluent to give 5.50g (67.7%) of 17 as a colorless oil:
1H NMR (200MHz, CDCl3) d 0.07 (6H, s), 0.89 (9H, s),
1.31 (3H, d, J=5 Hz), 1.50 (1H, m), 1.6±1.8 (2H, m), 2.0±
2.2 (3H, m), 2.3±2.6 (3H, m), 3.5±3.8 (3H, m), 3.89 (1H,
m), 4.02 (1H, d, J=11 Hz), 4.73 (1H, q, J=5Hz), 5.3±5.6
(2H, m). ESI-MS m/z: 371 (M H) .
1
20 as a colorless oil: H NMR (200MHz, CDCl3) d 1.43
(3H, d, J=5.5 Hz), 1.5±1.8 (3H, m), 1.91 (1H, m), 2.0±2.2
(2H, m), 2.3±2.4 (2H, m), 2.55 (1H, m), 3.69 (3H, s), 3.78
(1H, m), 4.03 (1H, dd, J=2, 11Hz), 4.27 (1H, d, J=2Hz),
4.80 (1H, q, J=5.5 Hz), 5.3±5.6 (2H, m), 9.62 (1H, s). ESI-
MS m/z: 271 (M+H)+.
Methyl (Z)-7-{(1S,2R,4R)-2-[2-aza-2-(((phenyamino)-
thioxomethyl)amino)vinyl]-4-methyl-3,5-dioxanyl}hept-5-
enoate (40). To a mixture of 20 (62 mg, 0.229 mmol)
and hydrazino(phenylamino)methan-l-thione (46 mg, 0.275
mmol) in ethanol (2 mL) was added acetic acid (1 drop)
and the solution was stirred at room temperature for
4 h. The mixture was diluted with chloroform (15 mL)
and washed with brine and dried over magnesium sul-
fate. The solvent was evaporated in vacuo to give 110 mg
1
(100%) of 40 as an oil: H NMR (200 MHz, CDCl3) d
1.38 (3H, d, J=5 Hz), 1.5±1.8 (3H, m), 2.0±2.2 (3H, m),
2.2±2.4 (2H, m), 2.53 (1H, m), 3.69 (3H, s), 3.85 (1H, m),
4.05 (1H, m), 4.53 (1H, dd, J=3, 4.5 Hz), 4.82 (1H, m),
5.3±5.6 (2H, m), 7.2±7.5 (4H, m), 7.62 (3H, m), 9.04 (1H,
s), 9.73 (1H, s). ESI-MS m/z: 420 (M+H)+.
Methyl (Z)-7-{(1S,2R,4R)-4-methyl-2-[(1,1,2,2-tetrame-
thyl-1-silapropoxy)methyl]-3,5-dioxanyl}hept-5-enoate
(18). To a solution of 17 (4.75 g, 12.7 mmol) in N,N-
dimethylformamide (50 mL) was added potassium car-
bonate (1.76 g, 12.7 mmol) and methyl iodide (1.62 mL,
26.0 mmol), and the mixture stirred at room tempera-
ture for 5 h. The solution was poured into water and
extracted with ether. The organic layer was washed suc-
cessively with water and brine, and dried over magnesium
sulfate. The solvent was evaporated in vacuo and the
residue was chromatographed on a silica gel column (75 g)
with a mixture of n-hexane and ethyl acetate (20:1) as
(Z)-7-{(1S,2R,4R)-2-[2-Aza-2-(((phenyamino)thioxome-
thyl)amino)vinyl] - 4 - methyl - 3,5 - dioxanyl}hept - 5 - enoic
acid (8a). A solution of 40 (110 mg, 0.262 mmol) in a
mixture of methanol (2 mL) and 1 N sodium hydroxide
(1.00 mL, 1.00 mmol) was stirred at room temperature
for 2 h and the reaction mixture adjusted to pH 7 with 1
N hydrochloric acid. Solvent was evaporated in vacuo
and the residue was dissolved in a mixture of chloro-
form and methanol (3:1, 10 mL). The solution was dried
over magnesium sulfate and the solvent was evaporated in
vacuo to give a crude oil. The crude product was puri®ed
by preparative TLC to give 65mg (61.1%) of 8a as an oil:
1H NMR (200MHz, CDCl3) d 1.42 (3H, d, J=5Hz), 1.5±
1.8 (3H, m), 2.0±2.2 (2H, m), 2.3±2.6 (4H, m), 3.88 (1H,
dd, J=11.7, 2 Hz), 4.10 (1H, d, J=11.5 Hz), 4.56 (1H, dd,
J=2.3, 5.2 Hz), 4.83 (1H, q, J=5Hz), 5.55 (2H, m), 7.2±
7.7 (6H, m), 9.07 (1H, s), 10.8 (1H, br s). ESI-MS m/z: 404
(M H) .
1
eluent to give 4.17g (67.6%) of 18 as an oil: H NMR
(200MHz, CDCl3) d 0.07 (6H, s), 0.96 (9H, s), 1.31 (3H, d,
J=5Hz), 1.48 (1H, m), 1.6±1.8 (2H, m), 2.0±2.2 (3H, m),
2.3±2.6 (3H, m), 3.5±3.7 (3H, m), 3.68 (3H, s), 3.89 (1H,
m), 4.00 (1H, d, J=11 Hz), 4.72 (1H, q, J=5Hz), 5.3±5.6
(2H, m). ESI-MS m/z: 387 (M+H)+.
Methyl (Z)-7-((1S,2R,4R)-2-hydroxymethyl-4-methyl-
3,5-dioxanyl)hept-5-enoate (19). A mixture of 18 (4.00 g,
10.3 mmol) and tetra-n-butyl ammonium ¯uoride (15 mL
of 1 M solution, 15mmol) in tetrahydrofuran (40 mL) was
stirred at room temperature for 3 h and the solvent was
evaporated in vacuo. The residue was chromatographed
on a silica gel column (80 g) with a mixture of n-hexane
and ethyl acetate (20:15) as eluent to give 2.88g (100%) of
Sodium (Z)-7-{(1S,2R,4R)-2-[2-aza-2-(((phenyamino)-
thioxomethyl)amino)vinyl]-4-methyl-3,5-dioxanyl}hept-5-
enoate (8b). A solution of 8a (1.39 g, 3.43 mmol) in a
mixture of methanol (10 mL) and 1 N sodium hydroxide
(3.43 mL, 3.43 mmol) was stirred at room temperature
1
19 as a colorless oil: H NMR (200MHz, CDCl3) d 1.35
(3H, d, J=5.0 Hz), 1.43 (1H, m), 1.6±1.8 (2H, m), 1.9±2.2

H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
2641
for 2 h and the solvent then evaporated in vacuo. The
residue was dissolved in water (50 mL) and the solution
applied to a column of Diaion HP-20 (trademark, sold
by Mitsubishi Chemical Industries Ltd.) (200mL). The
column was washed with water (500mL) and the object
compound was eluted with a mixture of water and
methanol (1:1, 1 L). The organic solvent was removed in
vacuo and the aqueous residue was lypholyzed to give
sodium hydroxide (1.00 mL, 1.00 mmol) gave 79.8 mg
1
(65.9%) of 24 as an oil: H NMR (200MHz, CDCl3) d
1.45 (3H, d, J=5.5 Hz), 1.6±1.9 (3H, m), 2.0±2.2 (3H, m),
2.3±2.6 (3H, m), 3.90 (1H, dd, J=2, 11Hz), 4.18 (1H, d,
J=11 Hz), 4.70 (1H, dd, J=3, 4 Hz), 4.88 (1H, q,
J=5.5Hz), 5.4±5.7 (2H, m), 7.56 (1H, m), 7.6±7.8 (2H,
m), 7.90 (1H, m), 7.98 (1H, m), 8.41 (1H, m). ESI-MS m/z:
397 (M H) .
1
1.77g (100%) of 8b as a pale yellow powder: H NMR
(200MHz, D2O) d 1.39 (3H, d, J=5.5Hz), 1.5±1.7 (3H,
m), 1.9±2.1 (3H, m), 2.1±2.3 (4H, m), 2.37 (1H, m), 4.05
(1H, s), 4.98 (1H, q, J=5.5Hz), 4.8±5.1 (2H, m), 7.3±7.5
(6H, m). ESI-MS m/z: 404 (M H) .
(Z)-7-{(1S,2R,4R)-2-[2-Aza-2-((tert-butoxy)carbonylami-
no)vinyl]-4-methyl-3,5-dioxanyl}hept-5-enoic acid (21).
By the procedure used for 40 and 8a, 20 (270mg,
1.00 mmol), tert-butoxy(hydrazino)methan-1-one (160 mg,
1.20 mmol) and 1 N sodium hydroxide (2.00 mL,
2.00 mmol) gave 94.6 mg (28.1% from 20) of 21 as an
oil: 1H NMR (200 MHz, CDCl3) d 1.2±1.5 (1H, m), 1.35
(3H, d, J=5 Hz), 1.50 (9H, s), 1.6±1.8 (3H, m), 2.0±2.2
(2H, m), 2.35 (2H, d, J=6.5 Hz), 2.46 (1H, m), 3.77
(1H, m), 4.04 (1H, d, J=11 Hz), 4.57 (1H, m), 4.80 (1H,
m), 5.3±5.5 (2H, m), 7.21 (1H, m), 7.26 (1H, s). ESI-MS
m/z: 369 (M H) .
Methyl (Z)-7-{(1S,2R,4R)-2-(2-aza-2-((N-phenylcarba-
moyl)amino)vinyl]-4-methyl-3,5-dioxanyl)hept-5-enoate
(39). By the procedure used for 40, 20 (300mg, 1.11
mmol) and hydrazino(phenylamino)methan-l-one (227
mg, 1.50mmol) gave 403 mg (90.0%) of 39 as a pale yellow
oil: 1H NMR (200MHz, CDCl3) d 1.40 (3H, d, J=5.5Hz),
1.6±1.8 (3H, m), 2.0±2.2 (3H, m), 2.2±2.4 (2H, m), 2.55
(1H, m), 3.69 (3H, s), 3.85 (1H, m), 4.04 (1H, m), 4.57 (1H,
dd, J=3, 4.5 Hz), 4.85 (1H, m), 5.3±5.6 (2H, m), 7±09 (1H,
t, J=7Hz), 7.18 (1H, d, J=5Hz), 7.35 (2H, m), 7.50 (3H,
m), 7.95 (1H, s), 8.53 (1H, s). ESI-MS m/z: 404 (M+H)+.
(Z)-7-{(1S,2R,4R)-2-[2-Aza-2-((2-pyridyl)carbonylami-
no)vinyl]-4-methyl-3,5-dioxanyl}hept-5-enoic acid (22).
By the procedure used for 40 and 8a, 20 (270mg,
1.00mmol), hydrazino(2-pyridyl)methan-1-one (165mg,
1.20mmol) and 1 N sodium hydroxide (2.00 mL, 2.00
(Z)-7-{(1S,2R,4R)-2-[2-Aza-2-((N-phenylcarbamoyl)ami-
no)vinyl]-4-methyl-3,5-dioxanyl)hept-5-enoic acid (7a).
By the procedure used for 8a, 39 (403mg, 0.999 mmol)
and 1 N sodium hydroxide (5.00 mL, 5.00mmol) gave
1
mmol) gave 175 mg (46.6% from 20) of 22 as an oil: H
NMR (200 MHz, DMSO-d6) d 1.26 (3H, d, J=5Hz),
1.45±1.65 (3H, m), 2.0±2.2 (4H, m), 2.3±2.5 (2H, m) 3.86
(2H, m), 4.6 (1H, m), 4.82 (1H, q, J=5Hz), 5.42 (1H, m),
7.54 (1H, dd, J=5, 2 Hz), 7.78 (1H, d, J=5Hz), 8.32 (1H,
dd, J=5, 2 Hz), 8.74 (1H, d, J=5Hz), 9.08 (1H, s). ESI-
MS m/z: 374 (M H) .
1
203 mg (52.2%) of 7a as a pale yellow oil: H NMR
(200MHz, CDCl3) d 1.39 (3H, d, J=5.5 Hz), 1.5±1.8 (3H,
m), 2.0±2.2 (2H, m), 2.3±2.6 (3H, m), 3.87 (1H, dd, J=2,
13Hz), 4.09 (1H, d, J=13 Hz), 4.55 (1H, dd, J=2, 5 Hz),
4.83 (1H, q, J=5.5 Hz), 5.4±5.6 (2H, m), 7.10 (1H, t,
J=7.5Hz), 7.2±7.4 (4H, m), 7.50 (2H, m), 8.05 (1H, s),
9.40 (1H, s). ESI-MS m/z: 388 (M H) .
(Z)-7-{(1S,2R,4R)-2-[2-Aza-2-(2-pyridylamino)vinyl]-4-
methyl-3,5-dioxanyl}hept-5-enoic acid (23). By the pro-
cedure used for 40 and 8a, 20 (40 mg, 0.148 mmol), 2-
hydrazinopyridine (20 mg, 0.183 mmol) and 1 N sodium
hydroxide (1.00 mL, 1.00 mmol) gave 51.4 mg (100%
Sodium (Z)-7-{(1S,2R,4R)-2-[2-aza-2-((N-phenylearba-
moyl)amino)vinyl]-4-methyl-3,5-dioxanyl}hept-5-enoate
(7b). By the procedure used for 8b, 7a (117mg, 0.300
mmol) and 1 N sodium hydroxide (0.300mL, 0.300 mmol)
gave 123 mg (100%) of 7b as a powder: 1H NMR
(200MHz, D2O) d 1.41 (3H, d, J=5.5Hz), 1.5±1.7 (3H,
m), 1.8±2.4 (8H, m), 4.02 (1H, s), 4.95 (1H, q, J=5.5Hz),
5.3±5.6 (2H, m), 7.21 (1H, m), 7.4±7.5 (5H, m). ESI-MS
m/z: 388 (M H) .
1
from 20) of 23 as an oil: H NMR (200 MHz, CDCl3) d
1.41 (3H, d, J=5 Hz), 1.53 (1H, m), 1.6±1.9 (2H, m),
2.0±2.2 (2H, m), 2.35±2.55 (4H, m), 3.90 (1H, dd,
J=11.3, 2.5 Hz), 4.08 (1H, d, J=11.3 Hz), 4.55 (1H, dd,
J=5, 3 Hz), 4.83 (1H, q, J=5 Hz), 5.4±5.6 (2H, m), 6.76
(1H, br t, J=6 Hz), 7.25 (1H, d, J=9 Hz), 7.40 (1H, d,
J=5.5 Hz), 7.62 (1H, m), 7.93 (1H, br d, J=5.5 Hz).
ESI-MS m/z: 346 (M H) .
Methyl (Z)-7-{(1S,2R,4R)-2-[2-aza-2-(1-phthalazinylami-
no)vinyl]-4-methyl-3,5-dioxanyl}hept-5-enoate (41). By
the procedure used for 40, 20 (500mg, 1.84mmol) and 1-
hydrazinophthalazine hydrochloride (393mg, 2.00mmol)
gave 481 mg (63.5%) of 41 as an oil: 1H NMR (200MHz,
CDCl3) d 1.33 (3H, d, J=5.5 Hz), 1.6±1.8 (3H, m), 2.0±2.2
(2H, m), 2.43 (2H, t, J=8Hz), 2.67 (1H, m), 3.67 (1H, m),
3.85 (1H, d, J=12 Hz), 4.05 (1H, d, J=12 Hz), 4.88 (1H,
dd, J=3, 4 Hz), 4.87 (1H, q, J=5.5Hz), 5.3±5.6 (2H, m),
7.51 (1H, m), 7.6±7.7 (2H, m), 7.88 (1H, m), 7.36 (1H, m),
10.48 (1H, broad). ESI-MS m/z: 413 (M+H)+.
Methyl (Z)-7-{(1S,2R,4R)-2-[(E)-2-aza-2-(diphenylme-
thoxy)vinyl]-4-methyl-3,5-dioxanyl}hept-5-enoate (42). A
mixture of 20 (326 mg, 1.21 mmol), hydroxylamine hy-
drochloride (374 mg, 5.38 mmol) and sodium hydrogen
carbonate (374 mg, 4.45 mmol) in a mixture of methanol
(6 mL) and water (3 mL) was stirred at room tempera-
ture for 3 h. To the solution was added chloroform and
brine and the organic layer separated, dried over mag-
nesium sulfate, and evaporated in vacuo. The residue
was chromatographed on a silica gel column (15 g) with
a mixture of n-hexane and ethyl acetate (5:1) as eluent.
Methyl (Z)-7-{(1S,2R,4R)-2-[(Z)-2-aza-2hydroxyvinyl]-
4-methyl-3,5-dioxanyl}hept-5-enoate (100 mg, 29.1%)
was obtained from the ®rst fractions as an oil: 1H NMR
(Z)-7-{(1S,2R,4R)-2-[2-Aza-2-(1-phthalazinylamino)vinyl]-
4-methyl-3,5- dioxanyl}hept-5-enoic acid (24). By the
procedure used for 8a, 41 (125mg, 0.303 mmol) and 1 N

2642
H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
(200MHz, CDCl3) d 1.35 (3H, d, J=5 Hz), 1.6±1.8 (3H,
m), 1.9±2.2 (4H, m), 2.34 (2H, d, J=7.5Hz), 2.57 (1H, m),
3.69 (3H, s), 3.81 (1H, d, J=11.5 Hz), 3.98 (1H, d,
J=11.5 Hz), 4.79 (1H, q, J=5 Hz), 4.98 (1H, dd, J=3.5,
4.5 Hz), 5.4±5.6 (2H, m), 6.78 (1H, d, J=3.5 Hz), 8.00 (1H,
s). ESI-MS m/z: 286 (M+H)+. Methyl (Z)-7-{(1S,2R,4R)-
2-[(E)-2-aza-2-hydroxyvinyl]-4-methyl-3,5-dioxanyl}hept-5-
enoate (142 mg, 41.3%) was obtained from the second
J=5 Hz), 5.4±5.6 (2H, m), 7.04 (1H, t, J=7.3 Hz), 7.30
(2H, m), 7.45 (2H, d, J=7.5 Hz), 8.28 (1H, s). ESI-MS
m/z: 390 (M H) .
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[2-((1-phthalazinylamino)
amino)methyl]-3,5-dioxanyl}hept-5-enoic acid (27). To a
solution of 41 (450mg, 1.09mmol) in a mixture of metha-
nol (4mL) and acetic acid (2 mL) was added sodium cy-
anoborohydride (125mg, 1.99mmol), and the mixture
stirred at room temperature for 2 h. The solution was
adjusted to pH 7 with saturated aqueous sodium hydrogen
carbonate and the resulting mixture diluted with chloro-
form. The solution was washed successively with water
and brine, dried over magnesium sulfate, and solvent eva-
porated in vacuo to give methyl (Z)-7-{(1S,2R,4R)-4-
methyl-2-[2-((1-phthalazinylamino)amino)methyl]-3,5-di-
oxanyl}hept-5-enoate that was dissolved in a mixture of
methanol (5mL) and 1 N aqueous sodium hydroxide
(2.00 mL, 2.00mmol). After stirring at room temperature
overnight, the mixture was neutralized with 1 N hydro-
chloric acid and the resulting solution evaporated to dry-
ness in vacuo. The residue was puri®ed by preparative thin
layer chromatography with a mixture of chloroform and
methanol (10:1) as eluent to give 96.3 mg (22.0% from 41)
of 27 as a pale yellow oil: 1H NMR (200MHz, CDCl3) d
1.44 (1H, d, J=5Hz), 1.5±1.9 (3H, m), 1.9±2.2 (3H, m),
2.3±2.6 (3H, m), 3.88 (1H, d. J=12.5 Hz), 4.15 (1H, d,
J=12.5 Hz), 4.70 (1H, m), 4.87 (1H, q, J=5Hz), 5.4±5.7
(2H, m), 7.57 (1H, m), 7.6±7.8 (2H, m), 7.9±8.1 (2H, m),
8.41 (1H, m), 9.60 (1H, s). ESI-MS m/z: 399 (M H) .
1
fractions as an oil: H NMR (200 MHz, CDCl3) d 1.37
(3H, d, J=5 Hz), 1.58 (1H, m), 1.6±1.8 (2H, m), 2.0±2.2
(2H, m), 2.27 (1H, m), 2.55 (1H, m), 2.85 (2H, t,
J=7.5 Hz), 3.69 (3H, s), 3.80 (1H, d, J=11.5Hz), 4.03
(1H, d, J=11.5Hz), 4.55 (1H, dd, J=2, 4.5Hz), 4.80 (1H,
q, J=5Hz), 5.3±5.6 (2H, m), 7.43 (1H, d, J=4.5 Hz), 7.83
(1H, s). ESI-MS m/z: 286 (M+H)+.
To a solution of methyl (Z)-7-{(1S,2R,4R)-2-[(E)-2-aza-
2-hydroxyvinyl]-4-methyl-3,5-dioxanyl}hept-5-enoate
(100mg, 0.350 mmol) in N,N-dimethylformamide (5 mL)
was added sodium hydride (14 mg, 60% in oil, 0.350
mmol) at 5ꢀC. After being stirred at the same temperature
for 30 min, bromodiphenylmethane (86.6 mg, 0.350 mmol)
was added and the mixture stirred in an ice bath for an
additional 2 h. The solution was diluted with ethyl acetate
and washed successively with water and brine, and dried
over magnesium sulfate. The solvent was evaporated in
vacuo and the crude residue was puri®ed by preparative
TLC with a mixture of n-hexane and ethyl acetate (4:1) as
1
eluent to give 130 mg (82.1%) of 42 as an oil: H NMR
(200MHz, CDCl3) d 1.34 (3 H, d, J=5 Hz), 1.49 (1H, m),
1.6±1.8 (3H, m), 2.0±2.2 (3H, m), 2.32 (2H, t, J=7.5 Hz),
2.55 (1H, m), 3.68 (3H, s), 3.70 (1H, d, J=11.5 Hz), 3.96
(1H, d, J=11.5 Hz), 4.49 (1H, dd, J=2, 5.5 Hz), 4.74 (1H,
q, J=5 Hz), 5.2±5.5 (2H, m), 6.23 (1H, s), 7.2±7.4 (11H,
m), 7.59 (1H, d, J=5.5 Hz). ESI-MS m/z: 452 (M+H)+.
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[((2-thienylearbonyl)ami-
no)methyl]-3,5-dioxanyl}hept-5-enoic acid (28). By the
procedure used for 40 and 27, 20 (47.0 mg, 0.174 mmol),
hydrazino(2-thienyl)methan-1-one (27.0 mg, 0.171 mmol)
and sodium cyanoborohydride (20.0 mg, 0.318 mmol), 1
N sodium hydroxide (0.40 mL, 0.40mmol) gave 55.0mg
(Z)-7-{(1S,2R,4R)-2-[(E)-2-Aza-2-(diphenylmethoxy)vi-
nyl]-4-methyl-3,5-dioxanyl}hept-5-enoic acid (25). By the
procedure used for 8a, 42 (95 mg, 0.210 mmol) and 1 N
sodium hydroxide (1.00 mL, 1.00mmol) gave 62.0 mg
(67.5%) of 25 as an oil: 1H NMR (200MHz, CDCl3) d 1.34
(3H, d, J=5 Hz), 1.50 (1H, m), 1.6±1.8 (3H, m), 2.0±2.2
(3H, m), 2.85 (2H, t, J=7.5Hz), 2.59 (1H, m), 3.77 (1H,
m), 3.96 (1H, d, J=11.5 Hz), 4.50 (1H, dd, J=2.5, 5.5 Hz),
4.74 (1H, m), 5.00 (1H, dd, J=2.5, 4.5 Hz), 5.2±5.6 (2H,
m), 6.22 (1H, s), 6.78 (1H, d, J=4.5 Hz), 7.2±7.4 (11H, m),
7.56 (1H, d, J=5.5Hz). ESI-MS m/z: 436 (M H) .
1
(85.9% from 20) of 28 as an oil: H NMR (200MHz,
CDCl3) d 1.34 (3H, d, J=5Hz), 1.5±1.9 (2H, m), 2.0±2.5
(4H, m), 2.36 (2H, t, J=7Hz), 3.03 (1H, dd, J=3.8,
12.5Hz), 3.18 (1H, dd, J=8.1, 12.5Hz), 3.74 (2H, m), 4.04
(3H, m), 4.75 (1H, q, J=5Hz), 5.3±5.6 (2H, m), 7.10 (1H,
m), 7.55 (2H, m). ESI-MS m/z: 367 (M H) .
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[(2-pyridylmethylamino)-
methyl]-3,5-dioxanyl}hept-5-enoic acid (29). By the pro-
cedure used for 40 and 27, 20 (200 mg, 0.740 mmol), 2-
(aminomethyl)pyridine (0.150 mL, 1.46 mmol) and
sodium cyanoborohydride (100mg, 1.59mmol), and 1 N
sodium hydroxide (3.00 mL, 3.00mmol) gave 135 mg
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[(((N-phenycarbamoyl)
amino)amino)methyl]-3,5-dioxanyl}hept-5-enoic acid (26).
To a solution of 7a (48 mg, 0.123 mmol) in ethanol (2 mL)
was added sodium cyanoborohydride (15 mg,
0.239 mmol) and acetic acid (1 drop), and the mixture
stirred at room temperature for 2 h. The solvent was eva-
porated in vacuo and the residue was extracted with
chloroform at pH 3. The organic layer were combined and
dried over magnesium sulfate. Solvent was evaporated in
vacuo and the crude product puri®ed by preparative TLC
with ethyl acetate to give 22mg (45.6%) of 26 as an oil: 1H
NMR (200MHz, CDCl3) d 1.32 (3H, d, J=5Hz), 1.6±1.9
(2H, m), 2.0±2.2 (4H, m), 2.36 (2H, t, J=7Hz), 2.4±2.6
(1H, m), 2.85 (1H, dd, J=2.6, 12.8 Hz), 3.05 (1H, dd,
J=9.7, 12.8 Hz), 3.72 (1H, m), 4.01 (1H, m), 4.70 (1H, q,
1
(57.6% from 20) of 29 as an oil: H NMR (200MHz,
CDCl3) d 1.27 (1H, m), 1.30 (1H, d, J=5Hz), 1.45 (1H,
m), 1.5±1.8 (2H, m), 2.05 (1H, m), 2.2±2.4 (4H, m), 2.8±3.0
(2H, m), 3.72 (1H, dd, J=1.5, 11.5Hz), 3.9±4.2 (4H, m),
4.73 (1H, q, J=5Hz), 5.3±5.6 (2H, m), 5.94 (1H, broad),
7.2±7.4 (2H, m), 7.72 (1H, dt, J=1.5, 10Hz), 8.65 (1H, d,
J=5Hz). ESI-MS m/z: 347 (M H) .
Methyl (Z)-7-{(1S,2R,4R)-2-[(1,3-dioxoisoindolin-2-yl)-
methyl]-4-methyl-3,5-dioxanyl}hept-5-enoate (30). To a
solution of 19 (456 mg, 1.64 mmol) in pyridine (2 mL) was
added p-toluenesulfonyl chloride (640mg, 3.36mmol) and
the mixture was stirred at room temperature for 2 h. The

H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
2643
reaction mixture was quenched with water (10 mL) and
extracted with ethyl acetate. The organic layers were com-
bined and washed with aqueous sodium bicarbonate
(5 mL) and brine (5 mL), and dried over sodium sulfate.
The solvent was evaporated in vacuo and the crude pro-
duct was dissolved in dimethylsulfoxide (2 mL). To the
solution was added potassium phthalimide (550mg
2.97 mmol) and the mixture was stirred at 100ꢀC for 6 h.
The reaction mixture was cooled, added to water, and
extracted with ether. The organic layers were combined
and dried over anhydrous sodium sulfate. Solvent was
evaporated in vacuo and the crude product was puri®ed by
preparative TLC to give 400mg (59.5%) of 30 as an oil: 1H
NMR (200 MHz, CDCl3) d 1.28 (3H, d, J=5 Hz), 1.3±1.9
(4H, m), 2.0±2.5 (5H, m), 2.63 (1H, m), 3.69 (3H, s), 3.6±3.8
(2H, m), 4.00 (2H, m), 4.18 (1H, m), 4.63 (1H, q, J=5 Hz),
5.3±5.6 (2H, m), 7.7±7.9 (4H, m). ESI-MS m/z: 402
(M+H)+.
20 mg (100% from 31) of 33 as an oil: 1H NMR
(200 MHz, CDCl3) d 1.22 (3H, d, J=5 Hz), 1.68 (2H,
m), 1.9±2.3 (3H, m), 2.32 (2H, t, J=6.5 Hz), 3.18 (1H,
m), 3.38 (1H, m), 3.6±4.1 (3H, m), 4.2±4.4 (2H, m), 4.63
(1H, q, J=5 Hz), 5.3±5.7 (3H, m), 7.1±7.4 (5H, m). ESI-
MS m/z: 389 (M H) .
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[(2-oxo-3-phenylpropan-
oylamino)methyl]-3,5-dioxanyl}hept-5-enoic acid (34).
To a solution of 31 (35 mg, 0.129 mmol) in ethyl acetate
(2.4 mL) was added N,N0-dicyclohexylcarbodiimide
(36 mg, 0.163 mmol) and 2-oxo-3-phenylpropanoic acid
(27 mg, 0.164 mmol), and the mixture stirred at room
temperature overnight. The reaction mixture was ®ltered
and the ®ltrate was evaporated in vacuo to give crude
methyl (Z)-7-{(1S,2R,4R)-4-methyl-2-[((phenylcarbonyl)
carbonylamino)methyl]-3,5-dioxanyl}hept-5-enoate. By the
procedure used for 8a, this residue and 1 N sodium hy-
droxide (0.500 mL, 0.500 mmol) gave 12 mg (23.1%
from 31) of 34 as an oil: 1H NMR (200 MHz, CDC13) d
1.30 (3H, d, J=5 Hz), 1.5±1.8 (2H, m), 2.0±2.2 (2H, m),
2.36 (2H, t, J=7 Hz), 2.47 (1H, m), 3.21 (1H, m), 3.5±
4.0 (4H, m), 4.22 (2H, s), 4.67 (1H, q, J=5 Hz), 5.3±5.6
(2H, m), 7.1±7.4 (5H, m). ESI-MS m/z: 402 (M H) .
Methyl (Z)-7-((1S,2R,4R)-2-aminomethy-4-methyl-3,5-
dioxanyl)hept-5-enoate (31). To a solution of 30 (54 mg,
0.135mmol) in ethanol (2 mL) was added hydrazine
monohydrate (0.007 mL, 0.144 mmol) and the mixture
stirred at room temperature overnight. Solvent was eva-
porated in vacuo and the crude product puri®ed by pre-
parative TLC (chloroform:methanol:concd aqueous
ammonia=85:15:0.1) to give 13mg (35.6%) of 31 as an oil:
1H NMR (200 MHz, CDCl3) d 1.33 (3H, d, J=5 Hz), 1.69
(2H, m), 1.9±2.2 (3H, m), 2.32 (2H, t, J=7.6 Hz), 2.3±2.6
(2H, m), 2.71 (1H, m), 2.94 (1H, dd, J=9, 13Hz), 3.68 (3H,
s), 3.65±3.85 (2H, m), 3.98 (1H, d, J=11.2 Hz), 4.73 (1H, q,
J=5Hz), 5.3±5.6 (2H, m). ESI-MS m/z: 272 (M+H)+.
Methyl (Z)-7-{(1S,2R,4R)-4-methyl-2-[(N-phenylcarba-
moyloxy)methyl]-3,5-dioxanyl}hept-5-enoate (35). To a
solution of 19 (108 mg, 0.397 mmol) in pyridine (0.5 mL)
was added phenyl isocyanate (0.100 mL, 0.920 mmol) and
the mixture was stirred at room temperature for 30 min.
Water (1 mL) was added, and the solution extracted with
ether. The organic layers were combined and dried over
anhydrous sodium sulfate. Solvent was evaporated in
vacuo and the crude product was puri®ed by preparative
TLC (n-hexane:ether=1:1) give 140 mg (90.2%) of 35 as an
oil: 1H NMR (200 MHz, CDCl3) d 1.36 (3H, d, J=5 Hz),
1.4±1.8 (3H, m), 2.0±2.3 (3H, m), 2.33 (2 H, t, J=7 Hz),
2.4±2.6 (1H, m), 3.67 (3H, s), 3.72 (1H, m), 3.95±4.25 (4H,
m), 4.75 (1H, q, J=5 Hz), 5.3±5.6 (2H, m), 6.94 (1H, br s),
7.07 (1H, m), 7.3±7.5 (4H, m). ESI-MS m/z: 392 (M+H)+.
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[((N-phenylcarbamoyl)
amino)methyl]-3,5-dioxanyl}hept-5-enoic acid (32). To a
solution of (Z)-7-((1S,2R,4R)-2-aminomethy-4-methyl-
3,5-dioxanyl)hept-5-enoic acid (50 mg, 0.194 mmol),
which was prepared from 31 as described for the prepara-
tion of 8a, in pyridine (0.5 mL) was added phenyl isocya-
nate (0.10 mL, 0.920 mmol) and stirred at room
temperature for 1 h. The mixture was diluted with ethyl
acetate and the organic layer washed with 1 N hydro-
chloric acid. The organic layer was evaporated in vacuo
and the crude product puri®ed by preparative TLC to give
27mg (36.9% from 31) of 32 as an oil: 1H NMR
(200MHz, CDCl3) d 1.32 (3H, d, J=5Hz), 1.45 (1H, m),
1.5±1.8 (2H, m), 2.0±2.4 (4H, m), 2.37 (2H, t, J=6.5Hz),
3.2±3.5 (2H, m), 3.68 (1H, dd, J=11.3, 2 Hz), 3.94 (1H,
m), 4.03 (1H, d, J=11.3 Hz), 4.71 (1H, q, J=5Hz), 5.4±
5.6 (2H, m), 5.74 (1H, br t, J=6Hz), 7.02 (1H, m), 7.1±7.4
(4H, m), 7.55 (1H, br s). ESI-MS m/z: 375 (M H) .
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[(N-phenylcarbamoyloxy)
methyl]-3,5-dioxanyl)hept-5-enoic acid (36). By the pro-
cedure used for 8a, 35 (140 mg, 0.358mmol) and 1 N
sodium hydroxide (1.00 mL, 1.00 mmol) gave 115 mg
(85.2%) of 36 as an oil: 1H NMR (200 MHz, CDCl3) d 1.35
(3H, d, J=5Hz), 1.4±1.8 (3H, m), 2.0±2.3 (3H, m), 2.38
(2H, t, J=7Hz), 2.4±2.6 (1H, m), 3.73 (1H, m), 4.0±4.4
(4H, m), 4.75 (1H, q, J=5 Hz), 5.3±5.6 (2H, m), 6.88 (1H,
br s), 7.0±7.15 (1H, m), 7.3±7.5 (4H, m). ESI-MS m/z: 376
(M H) .
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[((benzylamino)carbonyl-
amino)methyl]-3,5-dioxanyl)hept-5-enoic acid (33). To a
solution of 31 (13 mg and 0.0479mmol) in pyridine
(0.3 mL) was added benzyl isocyanate (0.020 mL,
0.162 mmol) and the mixture stirred at room temperature
for 60min. Water (0.1 mL) was added to the reaction
mixture, and the solvent was evaporated in vacuo to
give methyl (Z)-7-{(1S,2R,4R)-4-methyl-2[((benzylamino)-
carbonylarnino)methyl]-3,5-dioxanyl}hept-5-enoate as a
crude residue. By the procedure used for 8a, this residue
and 1 N sodium hydroxide (0.300 mL, 0.300 mmol) gave
Methyl (Z)-7-{(1S,2R,4R)-4-methyl-2-[(2-pyridylme-
thoxy)methyl]-3,5- dioxanyl}hept-5-enoate (37). To a
solution of 19 (183 mg, 0.671 mmol) in N,N-dimethyl-
formamide (10 mL) was added sodium hydride (53.6 mg,
60% in oil, 1.34 mmol) and 2-(chloromethyl)pyridine
hydrochloride (110mg, 0.671 mmol) and the mixture stir-
red at room temperature overnight. The reaction mixture
was quenched with saturated aqueous ammonium chlo-
ride and the resulting solution extracted with ethyl acetate.
The organic layer was washed with brine and dried over
magnesium sulfate. Solvent was evaporated in vacuo and

2644
H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
the residue was puri®ed by preparative thin layer chro-
matography with ethyl acetate as eluent to give 201 mg
(82.5%) of 37 as an oil: H NMR (200 MHz, CDCl3) d
1.0Â109 cells. Aliquots of platelet suspension (1.0 mL)
were incubated with approximately 5 nM (®nal con-
centration)
[3H]-U46619
([15 3H(N)]-9,11-epoxy-
1
1.35 (3H, d, J=5.5 Hz), 1.47 (1H, m), 1.6±1.8 (3H, m),
1.9±2.2 (3H, m), 2.2±2.4 (2H, m), 2.50 (1H, m), 3.57
(1H, dd, J=5, 10 Hz), 3.67 (3H, s), 3.74 (1H, m), 4.00
(1H, d, J=12 Hz), 4.13 (1H, m), 4.70 (2H, ABq,
J=13 Hz), 4.78 (1H, q, J=5.5 Hz), 5.3±5.6 (2H, m),
7.19 (1H, dd, J=5, 7 Hz), 7.45 (1H, d, J=8 Hz), 7.71
(1H, dt, J=1.5, 8 Hz), 8.55 (1H, d, J=5 Hz). ESI-MS
m/z: 364 (M+H)+.
methano PGH2, 22.4 Ci/mmole, NEN) plus various
concentrations of the compound 7b or 8b for 5 min at
37ꢀC with stirring, In order to block U-46619-induced
platelet activation, the cells were treated with PGI2
(®nal concentration 250 nM) for 45 s prior to mixing
them with drugs. After mixing, a 0.4 mL aliquot of the
binding assay mixture was transferred to a 1.5 mL
Eppendorf tube, and the incubation was terminated at
the appropriate time by centrifugation at 15 000 rpm for
1 min in a Eppendorf 5414 centrifuge. The supernatant
was quickly removed by aspiration, and the inside of the
Eppendorf tubes, as well as the surface of the pellet
was rinsed twice with 0.4 mL of ice cold resuspension
bu€er, which was immediately removed by aspiration.
The tip of the tube was cut o€, and placed in a poly-
ethylene vial (Wheaton) including 0.4 mL of tissue
solubilizer (NCS, Amersham). The mixture was incu-
bated for 3±4 h at 50ꢀC, then 5 mL of toluene scinti-
lator (OMNIFLUOR 4 g/L toluene) was added to the
mixture. The radioactivity was measured in a liquid
scintillation counter.
(Z)-7-{(1S,2R,4R)-4-Methyl-2-[(2-pyridylmethoxy)me-
thyl]-3,5-dioxanyl}hept-5-enoic acid (38). By the proce-
dure used for 7a, 37 (190mg, 0.520 mmol) and 1 N sodium
hydroxide (2.00 mL, 2.00 mmol) gave 145 mg (78.1%) of
38 as an oil: 1H NMR (200MHz, CDCl3) d 1.33 (3H, d,
J=5Hz), 1.49 (1H, m), 1.6±1.9 (3H, m), 2.0±2.3 (4H, m),
2.38 (2H, t, J=7 Hz), 3.6±3.8 (2H, m), 4.0±4.1 (2H, m),
4.70 (2H, ABq, J=13 Hz), 4.77 (1H, q, J=5Hz), 5.4±5.6
(2H, m), 7.33 (1H, dd, J=5, 7 Hz), 7.48 (1H, d, J=8Hz),
7.80 (1H, dt, J=1.5, 8 Hz), 8.69 (1H, d, J=5Hz). ESI-MS
m/z: 348 (M H) .
Biological methods
Inhibitory e€ect of 8b on U-46619- or collagen-induced
platelet aggregation in human, monkey, dog, guinea-pig
or rat. These tests were performed by a similar proce-
dure to the test for platelet aggregation in rabbits. In this
case, the ®nal concentration of U-46619 (Cyman Chemi-
cal) was 1 mM (Human), 2 mM (Monkey) or 0.5 mM
(Guinea-pig), and that of collagen (Horm) was 0.5 mg/mL
(human, guinea-pig or rat) or 20 mg/mL (dog). The con-
centration of PRP was adjusted to 3.0Â105 cells/mL in
the case of human, monkey and dog.
Inhibitory e€ects on 9,11-azo PGH2-induced platelet ag-
gregation in rabbit. Blood was collected from the car-
otid artery of male rabbits into plastic vessels containing
0.1 volume of 3.8% aqueous sodium citrate. Platelet-
rich plasma (PRP) was then prepared by centrifugation
at 150Âg for 15 min. Platelet-poor plasma (PPP) was
prepared from the remaining blood after removing
PRP, by centrifugation at 1500Âg for 10 min. PRP
was then adjusted to a concentration of 6.0Â105 cells/
mL by using PPP. Platelet aggregation was investigated
using the turbidometric method with an aggregometer
(NKK HEMATRACER 1 Model 4A). To 225 mL of
PRP, 25 mL of the test compound solution or vehicle
solution was added, and the whole stirred at 1000 rpm
for 2 min at 37ꢀC. Next, 5 mL of 9,11-azo PGH2 (®nal
concentration 1.0 mM) or ADP (®nal concentration
2.5 mM, Sigma) was added as an aggregating inducer.
The concentration which caused 50% inhibition (IC50)
of 9,11-azo PGH2-induced maximum aggregation was
obtained by regression analysis of the concentration
versus inhibition of aggregation curve.
Inhibitory e€ect of 8b on ex vivo platelet aggregation in
guinea-pig. Male Hartley strain guinea-pigs weighing
about 300 g were used after overnight fasting. Blood was
collected from the abdominal artery at 1 h after oral
administration of 8b or vehicle. PRP was prepared as
described before, and platelet aggregation was induced
by adding 5 mL of U-46619 (®nal concentration, 0.5 mM)
or collagen (fmal concentration, 0.5 mg/mL) to 250 mL of
PRP.
Protective e€ects of 8b against arachidonic acid-induced
pulmonary infraction in mice. Male ddY mice weighing
about 30 g were used after overnight fasting. Compound
8b was orally administered in saline at doses from 3.2 to
32 mg/kg. Control animals were given only saline. At 1 h
after administration of 8b or saline, arachidonic acid
(90 mg/kg, Sigma) was injected intravenously for 20 s.
The e€ect of 8b was evaluated by measuring the inci-
dence of death within 5 min of injection. Results are
expressed as percent survival. Each group comprised of
10 mice.
3
Inhibitory e€ects of 7b and 8b on H-U46619 binding to
washed guinea-pig platelets. This was carried out
according to the modi®ed method of Kattelman et al.37
PRP was prepared as described above from male Hart-
ley strain guinea-pigs and treated with aspirin (®nal
concentration 1 mM), then centrifuged at 150Âg for
15 min to remove residual contaminating red blood
cells. PGI2 (®nal concentration 40 nM, Funakoshi) was
added to aid in resuspension of the platelets. The PRP
was then spun at 1100Âg for 15 min to pellet the cells.
The platelet free plasma was discarded, and the platelet
pellets were gently resuspended in bu€er (sodium chlo-
ride (138 mM), potassium chloride (5 mM), magnesium
chloride (5 mM), glucose (5.5 mM) and Tris±HCl
(25 mM), pH 7.4) to a ®nal cell count of approximately
Acknowledgements
We are grateful to Dr. Teruo Oku and Dr. Masashi
Hashimoto for their pertinent advice.

H. Marusawa et al. / Bioorg. Med. Chem. 7 (1999) 2635±2645
2645
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