Synthesis and evaluation of 1,4-diphenylbutadiene derivatives as inhibitors of plasminogen activator inhibitor-1 (PAI-1) production

Article abstract of DOI:10.1016/j.bmc.2010.01.032

Butadiene-imide 1 (T-686) derivatives were synthesized and evaluated for their inhibitory activity against PAI-1 production and their ADMET (DMPK and toxicology) profiles. Among these derivatives, compound 15k (T-2639) showed good antithrombotic activity in two rat thrombosis models without affecting bleeding time, indicating reduction of haemorrhagic risk. We also describe in this report a practical synthesis of 15k suitable for scale-up using Z,E-selective Stobbe condensation.

Full text of DOI:10.1016/j.bmc.2010.01.032

Bioorganic & Medicinal Chemistry 18 (2010) 1968–1979
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of 1,4-diphenylbutadiene derivatives as inhibitors
of plasminogen activator inhibitor-1 (PAI-1) production
a
a
b
b
a
Hiroshi Miyazaki a, , Hiroshi Sai , Hiroshi Ohmizu , Jun Murakami , Akio Ohtani , Tsuyoshi Ogiku
*
a Medicinal Chemistry Laboratory, Mitsubishi Tanabe Pharma Co., Ltd, 3-16-89, Kashima, Yodogawa, Osaka 532-8505, Japan
b Pharmacology Laboratory, Mitsubishi Tanabe Pharma Co., Ltd, 2-2-50, Kawagishi, Toda, Saitama 335-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Butadiene-imide 1 (T-686) derivatives were synthesized and evaluated for their inhibitory activity
against PAI-1 production and their ADMET (DMPK and toxicology) profiles. Among these derivatives,
compound 15k (T-2639) showed good antithrombotic activity in two rat thrombosis models without
affecting bleeding time, indicating reduction of haemorrhagic risk. We also describe in this report a prac-
tical synthesis of 15k suitable for scale-up using Z,E-selective Stobbe condensation.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 16 December 2009
Revised 12 January 2010
Accepted 13 January 2010
Available online 18 January 2010
Keywords:
Plasminogen activator inhibitor-1
Inhibitor of PAI-1 production
T-2639
1. Introduction
thrombus growth in animal models of venous thrombosis5 and
arterial thrombosis.6 To date, small molecules have been reported
The first step in the healing of a bleeding wound is a natural
physiologic response that causes blood at the injured site to clot.
This response is controlled by a homeostatic balance between
procoagulant forces and anticoagulant/fibrinolytic forces. How-
ever, under disease state ‘thrombotic disorders’, this balance is
tipped in favor of coagulation, leading to the pathologic formation
of thrombi in veins, arteries, or heart chambers. Thrombi can
obstruct blood flow at the site of formation, causing atrial fibrilla-
tion, heart attack, myocardial infarction, unstable angina, deep vein
thrombosis (DVT), pulmonary embolism, or acute ischemic stroke.
Plasminogen activator inhibitor-1 (PAI-1), a specific inhibitor of
both tissue-type plasminogen activator (tPA) and urokinase-type
plasminogen activator (uPA), is a key negative regulator of the
fibrinolytic system.1 High levels of PAI-1 reduce fibrinolytic poten-
tial and contribute to the development of thrombosis. Indeed, ele-
vated levels of PAI-1 in plasma have been observed in patients with
deep vein thrombosis2 and unstable angina.3 Furthermore, a num-
ber of animal studies have shown that high levels of PAI-1 interfere
with fibrinolytic activity in thrombotic and prethrombotic states.4
Thus, inhibition of PAI-1 activity or reduction of its production may
shift the balance between thrombogenesis and thrombolysis to-
wards thrombolysis, leading to beneficial therapeutic effects for a
number of thrombotic disorders. In fact, it has been reported that
an antibody against PAI-1 can enhance clot lysis and decrease
to inhibit PAI-17 and show in vivo efficacy in animal thrombosis
models.8,9
Among such small molecules, butadiene-imide compound 1
(T-686, Fig. 1),8a an inhibitor of PAI-1 production that prevents
fibrinolysis shutdown,10,11 was previously shown to exhibit anti-
thrombotic activity in animal models.12 Although compound 1
was believed to represent a new class of orally active antithrom-
botics, it showed several drawbacks, including low water
solubility (9 lg/ml), poor oral bioavailability (<3% in dog), and
chromosomal aberration toxicity. Further studies of butadiene-
imide derivatives8b led us to the discovery of 1,4-diphenylbutadi-
ene T-2639 as a strong in vitro and in vivo PAI-1 inhibitor with
good ADMET profile. In this paper, we report our work on the
discovery of T-2639 (drug design, synthesis, and evaluation of
1,4-diphenylbutadiene derivatives) and examine the beneficial
effects of this compound in two rat thrombosis models. Moreover,
we describe a practical synthesis of T-2639 using Z,E-selective
Stobbe condensation.13
* Corresponding author.
E-mail address: miyazaki.hiroshi@me.mt-pharma.co.jp (H. Miyazaki).
Figure 1. 1 (T-686) and T-2639.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.032

H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
1969
2. Results and discussion
2.1. Synthesis
We have previously reported syntheses of the butadiene deriv-
atives (1–4, Fig. 2).8a The butadiene analogues 8, 9, 11, 12 were
synthesized by conventional Stobbe condensation14 as noted in a
previous report8b (Scheme 1). In the case of Stobbe condensation
between succinates and ketones, the condensation proceeded
unselectively, leading to a mixture of two isomers. Due to difficulty
to directly separate the two isomers as carboxylic acids, we pro-
ceeded with their separation after conversion into the correspond-
ing methyl esters (6, 7) or amides (11, 12). Products 6 and 7 were
transformed by Stobbe condensation and amidation into 8 and 9.
Syntheses of 15a–n are shown in Scheme 2. After conversion of
Stobbe condensation product into the corresponding methoxy-
methyl (MOM) ester and separation of the two isomers by chroma-
tography, the methoxymethyl (MOM) ester 13 was afforded.
Treatment of 13 with HCl provided the key intermediate (2Z,3E)-
14, whose stereochemistry was confirmed by X-ray crystallogra-
phy (Fig. 3). (2Z,3E)-14 was transformed into the desired amides
and hydrazones 15a–l using a conventional method, and the imide
compounds 15m,n were afforded by treatment of 15a,b with so-
dium hydroxide solution followed by treatment with HCl.
The synthetic route of compound 21a,b is shown in Scheme 3.
Stobbe reaction between dimethyl succinate and 30,50-dimethoxy-
acetophenone proceeded selectively, and esterification of the crude
product of this condensation predominantly provided the (E)-dies-
ter 17. Isomerization of 17 by photo-irradiation and separation of
the formed isomers by chromatography afforded the (Z)-diester
18, which was transformed into 21a,b in several conventional
steps.
Scheme 1. Reagents and conditions: (a) dimethyl succinate, t-BuOK, t-BuOH, rt,
1 h; (b) SOCl2, MeOH, rt, 16 h; (c) 3,4,5-trimethoxybenzaldehyde, t-BuOK, t-BuOH,
THF, rt, 1 h; (d) SOCl2, CHCl3, reflux, 30 min; (e) 28% NH4OH, 0 °C to rt, 30 min; (f)
30,40,50-trimethoxyacetophenone, t-BuOK, t-BuOH, THF, rt, 1 h.
ity in dog blood. Although 8, 9, and 11 showed decreased activity,
2–4 and 12 were found to possess moderate to high inhibitory
activity of PAI-1 production. Compounds 2 and 4 showed a largely
decreased stability in dog blood compared to 1, and their ester
moiety seemed to be unstable for hydrolysis (0% remains in dog
blood). On the other hand, compounds 3 and 12 exhibited a largely
increased stability in dog blood compared with 2 and 4 (3, 75%; 12,
96%). It is believed that ester hydrolysis in these compounds is ste-
rically blocked by the (Z)-positioned 3,4,5-trimethoxyphenyl ring.
As expected, 12 exhibited improved solubility in water compared
The E or Z configuration of the products in Scheme 1–3 could be
confirmed by 1H NMR. Assignments of their stereochemistry are in
concordance with the previous reports.15
2.2. Design and evaluation of butadiene-imide 1 (T-686)
derivatives
with 1 (12, 106 lg/ml; 1, 9 lg/ml, Table 3) and enhanced bioavail-
ability (12, 9%; 1, 3% in dog). Moreover, 12 was negative in chromo-
somal aberration test.
We first started our design of butadiene-imide 1 derivatives
with optimization of the butadiene scaffold. The relative inhibitory
activity against lipopolysaccharide (LPS)-induced PAI-1 production
in cultured vascular endothelial cells (compound concentration:
We next intensified our efforts in optimizing compound 12. We
first considered metabolic stability, and thus tried to remove one of
the three methoxy groups as these groups are generally known to
be typical metabolic targets leading to demethylated phenol
metabolite16 (Segment A, Fig. 4). We also tried to replace the phe-
nyl ring with a pyridine ring to enhance water solubility (Segment
B). Moreover, we considered introducing other amide moieties that
would form a salt, and thereby improve solubility (Segment C). Fi-
nally, we cycled the amide-ester part into an imide and introduced
a pyridine ring to improve solubility (Segment D).
10 lM) and the stability in dog blood are summarized in Table 1.
As the good crystalline properties of the imide are believed to be
responsible for low water solubility and poor bioavailability, the
amide-ester type compounds 2–4 and compounds 8, 9, 11, and
12, where a methyl group was introduced, were synthesized and
evaluated for their PAI-1 production inhibitory activity and stabil-
The relative inhibitory activity against LPS-induced and trans-
forming growth factor (TGF)-b-induced PAI-1 production in cul-
tured vascular endothelial cells is shown in Table 2 (LPS-induced:
compound concentration 1
centration 2 M). Among the amide-ester type compounds, com-
pounds 15a–d, having pyridine ring, and the hydrazone
lM, TGF-b-induced: compound con-
l
a
compound 15k revealed equal or enhanced inhibitory activity
against LPS-induced PAI-1 production compared with 12. However,
other amides (15e–j) and the hydrazone 15l exhibited in general
weak inhibitory activity. Replacement of the phenyl ring in the seg-
ment B with a 4-pyridyl ring (21a) led to an inhibitory activity
equal to that of 12, while the use of a 3-pyridyl ring (21b) led to
decreased inhibitory activity. Finally, the imide type compounds
15m,n, having a pyridine ring like 15a,b, exhibited good inhibitory
activity against LPS-induced PAI-1 production.
Figure 2. Compound 1 and its derivatives (2–4).

1970
H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
Scheme 2. Reagents and conditions: (a) 30,50-dimethoxyacetophenone 16, t-BuOK, t-BuOH, THF, 50 °C, 4 h; (b) MOMCl, i-Pr2NEt, CH2Cl2, rt, 0.5 h; (c) concd HCl, THF, rt, 3 h;
(d) CDI, CH2Cl2, rt, 2 h, then 3- or 4-picolylamine, rt, 8 h; (e) (COCl)2, DMF (cat.), CH2Cl2, rt, 1 h, then HNR1R2, (Et3N or pyridine), THF, rt; (f) 4N HCl/EtOAc, rt; (g) mCPBA,
CH2Cl2, ꢀ50 °C to rt, 1 h; (h) NaOH aq, THF, rt, 30 min.
moderate to good inhibitory activity against TGF-b-induced PAI-1
production.
Next, 15a–d,k,m,n, and 21a were evaluated for their physico-
chemical and pharmacokinetic properties (Table 3). The solubility
of amide-ester type compounds was largely enhanced compared
to 1, and stability of most compounds in dog blood was improved.
This improvement in solubility and stability led to improved bio-
availability with compounds 15a–c,k, and 21a satisfying our crite-
rion (>20%). On the other hand, the imide type compounds 15m,n
exhibited a solubility lower than that of the amide-ester type com-
pounds and their photostability proved to be remarkably scarce
compared with that of 15a,b. As the photostability of 1 is also poor,
it is believed that imide type compounds are inherently unstable.
Finally, we examined compounds 15a–c,k, and 21a for chromo-
somal aberration, antithrombotic activity, and toxicity (300 mg/kg
po) in male rats. Among the five compounds, only compound 15k
showed a good profile in all studies. Thus, we selected 15k (T-
2639) as the best compound.
2.3. Scale-up synthesis of 15k (T-2639)
In the synthetic route of 15k in Scheme 2, Stobbe condensation
between compound 10 and 30,50-dimethoxyacetphenone 16 pro-
ceeded unselectively, resulting in a mixture of two isomers
(2Z,3E)- and (2E,3E)-14, which were difficult to separate. In order
to improve selectivity in this Stobbe condensation, we examined
the reaction conditions (Scheme 4).
The results of a previous report13 have indicated that Stobbe
condensation provides only a small amount of (2Z,3E)-14 Na salt
as crystals when 24% w/w NaOMe solution in MeOH18 is used as
Figure 3. Perspective drawing of X-ray structure of (2Z,3E)-14.
a base and toluene as a solvent. This means that the crystallinity
of (2Z,3E)-14 Na salt in toluene is higher than that of (2E,3E)-14
Na salt.19 We have also confirmed that isomerization from
As for the relative inhibitory activity against TGF-b-induced
PAI-1 production, similar results as those obtained with inhibition
of LPS-induced PAI-1 production were observed, with only few
exceptions. Specifically, compounds 15a–d,k,m,n, and 21a showed
(2E,3E)-14 to (2Z,3E)-14 occurs under basic conditions. Indeed,
when 330 g of (2E,3E)-14 was treated with t-BuOK (1.1–1.2 equiv)
in t-BuOH at 50–60 °C for 10 min, 132 g (42%) of (2Z,3E)-14 and
102 g (27%) of (2E,3E)-14 were obtained. From these results, we

H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
1971
Scheme 3. Reagents and conditions: (a) dimethyl succinate, t-BuOK, t-BuOH, 50 °C, 4 h; (b) MOMCl, i-Pr2NEt, CH2Cl2, rt, 16 h; (c) h
m
, MeCN, rt, 8 h; (d) LDA, THF, ꢀ78 °C, 0.5 h,
then 3- or 4-pyridinecarboxaldehyde, THF, ꢀ100 °C, 20 min; (e) MsCl, Et3N, CH2Cl2, 0 °C, 0.5 h, then DBU, CH2Cl2, rt, 16 h; (f) concd HCl, THF, rt, 1 h; (g) CDI, THF, CH2Cl2, rt,
16 h, then 28% NH4OH, 0 °C, 2 h; (h) 4N HCl/EtOAc, rt.
Table 1
Table 2
Inhibitory activity on PAI-1 production and stability in dog blood
Inhibitory activity on LPS- or TGF-b-induced PAI-1 production in cultured vascular
endothelial cells
Compound Relative inhibitory
activitya
Stability in dog blood % of remainb
Compound
Relative inhibitory activitya
LPS-induced TGF-b-induced
1 (T-686)
2
3
4
8
9
11
12
1.0
0.9
0.7
0.7
0.2
0.3
0.3
1.0
79
nd
75
nd
nt
nt
nt
96
1 (T-686)
12
(2Z,3E)-14
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
21a
1.0
1.5
0.4
1.3
2.1
1.8
2.2
0.1
0.1
0.4
0.6
0.3
0.8
1.5
0.2
1.0
1.8
1.5
0.8
1.0
0.7
0.3
1.8
1.9
1.1
1.1
nt
nd: not detected.
nt: not tested.
nt
nt
nt
a
Relative inhibitory activity on LPS-induced PAI-1 production in cultured vas-
cular endothelial cells (compound concentration: 10 lM). Compound 1 (T-686) is
used as reference.8a
0.3
<0.1
0.9
nt
1.4
1.4
1.1
1.2
b
% of remains shown in dog blood after 1 h at 37 °C.
anticipated that (2Z,3E)-14 Na salt could predominantly be ob-
tained if isomerization of (2E,3E)-14 to (2Z,3E)-14 takes place
and the generated (2Z,3E)-14 is separated as a solid from the reac-
tion mixture under reaction conditions (Scheme 5).
Predominant precipitation and in situ isomerization were final-
ly achieved by a combination of seeding (2Z,3E)-14 Na salt
(0.01 equiv) into the reaction mixture and removal of MeOH,
which originated from NaOMe solution, from the Stobbe reaction
system to precipitate the desired (2Z,3E)-14 salt. As a result,
(2Z,3E)-14 was selectively obtained in 82% yield with 91:9
selectivity.13
21b
nt: not tested.
a
Relative inhibitory activity on LPS- or TGF-b-induced PAI-1 production in cul-
M, TGF-
M). Compound 1 (T-686) is used as a ref-
erence. The IC50 values of 1 for LPS- or TGF-b-induced PAI-1 production were
0.9 M or 4.3 M, respectively.
tured vascular endothelial cells (LPS-induced: compound concentration 1
b-induced: compound concentration 2
l
l
l
l
As described above, we could selectively prepare (2Z,3E)-14.
This optimized method was also applicable to a scale-up synthesis
of 15k. In the synthesis of 15k, the undesired isomer was removed
by recrystallization in the final step. Four hundred and twenty
gram of pure 15k could be provided without chromatography in
all steps. (Scheme 7).
As for the isomerization between (2Z,3E)-14 and (2E,3E)-14, we
speculate that the mechanism for this isomerization is conjugate
addition of base (t-butoxide or methoxide) to the a,b-unsaturated
ester group and isomerization of the resulting anion followed by
re-elimination of the base as reported previously (Scheme 6).15
Figure 4. Optimization of 12.

1972
H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
Table 3
Water solubility, stability in dog blood, bioavailability, photostability, geno-toxicity, antithrombotic activity, and toxicity in rats of selected compounds
Compound Water
solubility (
Stability in dog
Bioavailability Photostability% Chromosomal
Antithrombotic activity %
Toxicity in male rats
at 300 mg/kg po
l
g/ blood % remaina
(%) (dog)
remainb
aberration test
decrease in thrombus weightc
ml)
1 (T-686)
12
15a
9
106
1600
79
96
87
3
9
21
12
91
90
+
nt
nt
53
nt
nt
ꢀ
ꢀ
Decrease in locomotor
activity
15b
15c
>2000
>3000
76
87
28
52
96
85
+
ꢀ
nt
58
nt
Decrease in locomotor
activity
15d
15k
15m
15n
21a
>2000
>4000
38
111
>4000
83
89
65
60
88
17
49
nt
18
78
88
91
47
38
97
nt
ꢀ
nt
ꢀ
+
nt
50
nt
nt
nt
nt
No toxicity
nt
nt
nt
nt: not tested.
a
% of remains shown in dog blood after 1 h at 37 °C.
% of remains shown in 0.1N HCl solution under room light after 24 h.
Antithrombotic effects in rat venous thrombosis model (3 mg/kg/day ꢁ 3 days). See Ref. 17.
b
c
Scheme 6. Mechanism of isomerization between (2Z,3E)-14 and (2E,3E)-14.
Scheme 4.
Scheme 7. Scale-up synthesis of 15k.
ment with 15k (10 mg/kg/day) for eight consecutive days
inhibited thrombus formation by 22% (Fig. 5).
Haemorrhagic risk of 15k and Warfarin was evaluated using
ED50 value of antithrombotic effects in the rat venous thrombosis
model and ED100 value of bleeding effects in the rat tail incision
bleeding time test12 (Fig. 6, Table 4). Compound 15k did not pro-
long bleeding time in the rat tail even at 125 times its effective
dose in the venous thrombosis model, whereas Warfarin prolonged
bleeding time at or near its antithrombotic dose.
Scheme 5. Selective preparation of (2Z,3E)-14 by predominant precipitation of
(2Z,3E)-14 Na salt and in situ isomerization of (2E,3E)-14 to (2Z,3E)-14.
3. Conclusion
2.4. Antithrombotic activity of 15k (T-2639)
We examined the structure–activity relationships of a series
butadiene-imide 1 (T-686) derivatives and found 15k (T-2639) as
an orally active inhibitor of PAI-1 production. The synthesis of
15k also exhibited antithrombotic activity in a rat arterio-ve-
nous shunt model. In the rat arterio-venous shunt model,12 treat-

H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
1973
Table 4
Antithrombotic activity versus haemorrhagic risk in rats treated with 15k or Warfarin
for 8 days
15k (T-2639)
Warfarin
ED50VT (venous thrombosis)
ED100BT (bleeding time)
ED100BT/ED50VT
0.8 mg/kg/day
>100 mg/kg/day
>125.0
0.1 mg/kg/day
0.2 mg/kg/day
2.0
Relative risk
1.0
>62.5
Elmer PARAGON1000. 1H and 13C NMR spectra were recorded on
a Bruker AC-200 spectrometer, Bruker AVANCE 400 spectrometer,
or Varian UNITY INOVA500 with Me4Si as an internal standard.
Mass spectra were obtained on a Hitachi M-2000A spectrometer,
ThermoQuest LCQ Advantage, ThermoFisher FINNIGAN LXQ or a
Q-TOF Ultima API mass spectrometer. Elemental analyses were ob-
tained on a Perkin-Elmer 2400 II (C, H, N) and Dionex DX-320 (Cl).
4.1.1. Dimethyl 2-[1-phenyl-(E)-ethylidene]succinate (6)
Acetophenone (15.5 g, 0.129 mol) and dimethyl succinate
(27.1 g, 0.185 mol) were added dropwise to a suspension of t-BuOK
(14.5 g, 0.129 mol) in t-BuOH (100 ml) below 40 °C, and the reac-
tion mixture was stirred at room temperature for 1 h. Water was
then added to the mixture and the resulting mixture was washed
with i-Pr2O. After the aqueous layer was acidified with concd HCl
(pH 1), AcOEt was added. The organic layer was separated, washed
with water and brine, dried over MgSO4, and concentrated in va-
cuo. The resulting oil was dissolved with MeOH (150 ml), and thio-
nyl chloride (12.5 ml, 0.194 mol) was slowly added at 0 °C. The
reaction mixture was stirred at room temperature for 16 h, and
then concentrated in vacuo. After the resulting oil was dissolved
with AcOEt, water was added to the mixture. The organic layer
was separated, washed with sat. NaHCO3 aq and brine, dried over
MgSO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel to give 620 (18.6 g, 59%) and 720 (6.2 g, 20%)
as an oil.
Figure 5. Antithrombotic effect of 15k in the rat arterio-venous shunt model. Data
are given as the mean SE of nine rats. p <0.01 compared with the control.
**
15k was improved for pilot scale-up, and the biological effects of
15k in rat arterio-venous shunt model and rat venous thrombosis
model were evaluated. As a result, 15k was proved not to possess
the drawbacks seen in 1 and to show antithrombotic activity in the
two experimental models without affecting bleeding time. These
results suggest that 15k, or its derivatives, might provide a safe
treatment for thrombosis. Further research on the antithrombotic
effects of 15k is ongoing.
4. Experimental
4.1. Chemistry
1H NMR (200 MHz, CDCl3) d: 2.41 (3H, s), 3.20 (2H, s), 3.65 (3H,
s), 3.79 (3H, s), 7.10–7.20 (2H, m), 7.25–7.40 (3H, m). IR (film)
cmꢀ1: 2970, 2940, 2860, 1745, 1440, 1270, 1200, 1180, 1135,
1060, 770, 705. MS m/z: 248 ([M]+).
Melting points were measured using a Büchi 535 capillary melt-
ing point apparatus and are uncorrected. IR spectra were recorded
on a Perkin–Elmer Spectrum One FT-IR spectrometer or Perkin-
**
Figure 6. Antithrombotic effects of 15k and Warfarin in the rat venous thrombosis model (A) and rat tail incision bleeding time test (B). p <0.01 compared with the control.
a5 of 7 rats died under the test.

1974
H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
4.1.2. Dimethyl 2-[1-phenyl-(Z)-ethylidene]succinate (7)
1H NMR (200 MHz, CDCl3) d: 2.14 (3H, s), 3.39 (3H, s), 3.54 (2H,
s), 3.72 (3H, s), 7.10–7.20 (2H, m), 7.25–7.40 (3H, m). IR (film)
cmꢀ1: 2960, 2920, 2850, 1740, 1710, 1440, 1320, 1250, 1195,
1170, 1140, 765, 700. MS m/z: 248 ([M]+).
7.24 (2H, m), 7.27–7.34 (3H, m), 7.38 (1H, br). IR (Nujol) cmꢀ1
:
3438, 3183, 2925, 2854, 1719, 1664, 1581, 1464, 1231, 1129. MS
APCI m/z: 412 ([M+H]+). Anal. Calcd for C23H25NO6: C, 67.14; H,
6.12; N, 3.40. Found: C, 67.00; H, 5.73; N, 3.41.
4.1.6. Methyl (Z)-2-((E)-1-carbamoyl-2-phenylvinyl)-3-(30,40,50-
trimethoxyphenyl)-2-butenoate (12)
4.1.3. Methyl (E)-3-carbamoyl-4-phenyl-2-[1-(30,40,50-trimethoxy-
phenyl)-(E)-ethylidene]-3-pentenoate (8)
1H NMR (500 MHz, DMSO-d6) d: 1.72 (3H, s), 3.39 (3H, s), 3.66
(3H, s), 3.76 (6H, s), 6.50 (2H, s), 7.26–7.37 (3H, m), 7.37–7.44
(2H, m), 7.52–7.59 (3H, m). IR (Nujol) cmꢀ1: 3426, 3322, 2924,
2853, 1709, 1581, 1459, 1129. MS APCI m/z: 412 ([M+H]+). Anal.
Calcd for C23H25NO6: C, 67.14; H, 6.12; N, 3.40. Found: C, 67.06;
H, 6.07; N, 3.38.
A solution of 6 (5.0 g, 20.1 mmol) and 3,4,5-trimethoxybenzal-
dehyde (4.0 g, 20.1 mmol) in THF (20 ml) was added dropwise to
a suspension of t-BuOK (2.26 g, 20.1 mmol) in t-BuOH (20 ml) be-
low 40 °C, and the reaction mixture was stirred at room tempera-
ture for 1 h. Water was added to the mixture, and the resulting
mixture was washed with i-Pr2O. After the aqueous layer was acid-
ified with concd HCl (pH 1), AcOEt was added. The organic layer
was separated, dried over MgSO4, and concentrated in vacuo. The
resulting residue was chromatographed on silica gel. After the ob-
tained carboxylic acid (6.50 g, 15.8 mmol) was dissolved in CHCl3
(30 ml), thionyl chloride (1.15 ml, 15.8 mmol) and DMF (three
drops) were added, and the reaction mixture was stirred at reflux
for 30 min. Twenty-eight percent NH3 aq. (20 ml) was next added
to the mixture at 0 °C, and the reaction mixture was stirred at room
temperature for 30 min. After water and CHCl3 were added to the
mixture, the organic layer was separated, dried over MgSO4, and
concentrated in vacuo. The resulting oil was chromatographed on
silica gel to give 8 (4.7 g, 57%) as crystals.
4.1.7. 1-Methyl 2-[1-(30,50-dimethoxyphenyl)-(Z)-ethylidene]-3-
[1-phenyl-(E)-methylidene]-4-succinic Acid ((2Z,3E)-14)
A solution of 30,50-dimethoxyacetophenone (16) (4.8 g, 26.6
mmol) and 10 (6.3 g, 26.78 mmol) in THF (25 ml) was added drop-
wise to a suspension of t-BuOK (3.3 g, 29.4 mmol) in t-BuOH
(20 ml) below 40 °C, and the reaction mixture was stirred at
50 °C for 4 h. Water was added to the mixture, and the resulting
mixture was washed with i-Pr2O. After the aqueous layer was acid-
ified with concd HCl (pH 1), AcOEt was added. The organic layer
was next separated, washed with brine, dried over MgSO4, and
concentrated in vacuo. The resulting residue was dissolved in CH2Cl2
(100 ml), and N,N-diisopropylethylamine (5.18 g, 40.0 mmol) and
methoxymethyl chloride (2.59 g, 32.2 mmol) were slowly added to
the mixture. The reaction mixture was stirred at room temperature
for 30 min. Water and CHCl3 were then added to the mixture. The or-
ganic layer was separated, washedwith brine, driedover MgSO4, and
concentrated in vacuo. The residue was chromatographed on silica
gel to give 13 (2.50 g, 22%) as an oil.
1H NMR (500 MHz, DMSO-d6) d: 2.25 (3H, s), 3.62 (3H, s), 3.71
(3H, s), 3.76 (6H, s), 6.81 (2H, dd, J = 6.4, 2.9 Hz), 7.05 (2H, s),
7.06 (1H, br), 7.13–7.16 (3H, m), 7.24 (1H, br), 7.29 (1H, s). IR (Nu-
jol) cmꢀ1: 3416, 3194, 2924, 2854, 1712, 1657, 1464, 1243, 1121.
MS APCI m/z: 412 ([M+H]+). Anal. Calcd for C23H25NO6: C, 67.14;
H, 6.12; N, 3.40. Found: C, 67.06; H, 6.13; N, 3.28.
1H NMR (200 MHz, CDCl3) d: 1.75 (3H, s), 3.51 (3H, s), 3.51 (3H,
s), 3.79 (6H, s), 5.40 (2H, s), 6.31 (2H, d, J = 2.2 Hz), 6.39 (1H, t,
J = 2.2 Hz), 7.25–7.45 (3H, m), 7.50–7.70 (2H, m), 7.95 (1H, s). MS
m/z: 426 ([M]+).
Concd HCl (2.5 ml) was added dropwise to a solution of 13
(3.9 g, 9.1 mmol) in THF (25 ml). After the mixture was stirred at
room temperature for 3 h, it was neutralized by adding 2N NaOH
aq. After water and AcOEt were added to the mixture, the organic
layer was separated, dried over MgSO4, and concentrated in vacuo.
The resulting residue (2Z,3E)-14 (foam, 3.5 g, quant.) was used for
the next step without purification.
4.1.4. Methyl (Z)-3-carbamoyl-4-phenyl-2-[1-(30,40,50-trimeth-
oxyphenyl)-(E)-ethylidene]-3-pentenoate (9)
This compound was prepared from 7 as described in the synthe-
sis of 8.
1H NMR (500 MHz, DMSO-d6) d: 1.74 (3H, s), 3.68 (3H, s), 3.77
(6H, s), 3.78 (3H, s), 6.58 (1H, br), 7.05 (1H, br), 7.23–7.38 (7H,
m), 7.75 (1H, s). IR (Nujol) cmꢀ1: 3416, 2924, 2854, 1693, 1654,
1459, 1243, 1132. MS APCI m/z: 412 ([M+H]+). Anal. Calcd for
C23H25NO6: C, 67.14; H, 6.12; N, 3.40. Found: C, 67.12; H, 5.90;
N, 3.43.
1H NMR (CDCl3, 400 MHz) d: 1.78 (3H, s), 3.53 (3H, s), 3.80 (6H,
s), 6.33 (2H, d, J = 2.3 Hz), 6.40 (1H, t, J = 2.3 Hz), 7.30–7.45 (3H, m),
7.60–7.70 (2H, m), 8.00 (1H, s). 13C NMR (CDCl3, 100 MHz) d: 23.6,
51.9, 55.3, 99.4, 104.6, 124.8, 128.0, 128.7, 129.8, 130.0, 134.7,
144.6, 144.9, 150.2, 160.5, 167.6, 172.1. IR (Nujol) cmꢀ1: 2924,
2853, 1710, 1685, 1603, 1589, 1451, 1424, 1274, 1206. MS ESI m/
z: 381 ([MꢀH]ꢀ). HR-MS ESI calcd for C22H23O6 ([M+H]+):
383.1495. Found: 383.1485.
4.1.5. Methyl (E)-2-((E)-1-carbamoyl-2-phenylvinyl)-3-(30,40,50-
trimethoxyphenyl)-2-butenoate (11)
A solution of 108a (22.5 g, 119 mmol) and 30,40,50-trimethoxy-
acetophenone (25.0 g, 119 mmol) in THF (100 ml) was added drop-
wise to a suspension of t-BuOK (12.0 g, 119 mmol) in t-BuOH
(125 ml) below 40 °C, and the reaction mixture was stirred at room
temperature for 1 h. Water was added to the mixture, and the
resulting mixture was washed with i-Pr2O. After the aqueous layer
was acidified with concd HCl (pH 1), AcOEt was added. The organic
layer was separated, washed with water and brine, dried over
MgSO4, and concentrated in vacuo. The resulting residue was chro-
matographed on silica gel. After the obtained carboxylic acids were
dissolved in CHCl3 (100 ml), thionyl chloride (7.5 ml, 103 mmol)
was added to the mixture. The reaction mixture was then stirred
at reflux for 30 min. Twenty-eight percent NH3 aq (50 ml) was
added to the mixture at 0 °C, and the reaction mixture was stirred
at room temperature for 30 min. After water and CHCl3 were added
to the mixture, the organic layer was separated, dried over MgSO4,
and concentrated in vacuo. The resulting oil was chromatographed
on silica gel to give 11 (13.2 g, 27%) and 12 (10.3 g, 21%) as crystals.
1H NMR (500 MHz, DMSO-d6) d: 2.32 (3H, s), 3.46 (6H, s), 3.58
(3H, s), 3.66 (3H, s), 6.05 (2H, s), 7.07 (1H, s), 7.12 (1H, br), 7.18–
5.1.7.1. X-ray crystallographic data of (2Z,3E)-14. The com-
pound crystallized in the monoclinic system, space group P21/c
(No. 14), a = 12.967 (2), b = 12.0214 (2), c = 13.360 (1) Å, b = 101.12
(1)0, V = 2043.4 (5) Å3, Z = 4, Dcalc = 1.243 mg mꢀ3
,
l
= 0.747 mmꢀ1
scan technique using
radiation (k = 1.54184 Å) on a Rig-
.
The intensity data were collected by 2h ꢀ
x
graphite monochromated Cu-K
a
aku AFC5R diffractometer. The structure was solved by direct meth-
od using SHELXS-9721 and subsequent difference Fourier method.
The refinement of atomic parameters was carried out using SHEL-
XL-9721 with anisotropic thermal parameters for non-H atoms. All
hydrogen atoms were located geometrically and fixed. The final R
factors converged to R = 0.046, wR = 0.165. All calculations were per-
formed using the CrystalStructure software package. Detailed crys-
tallographic results (atomic coordinates, bond lengths, bond angles

H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
1975
and thermal parameters) have been deposited with the Cambridge
4.1.11. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-2-phenyl-
Crystallographic Data Centre, UK, CCDC-705024.
1-(pyridin-3-ylcarbamoyl)vinyl]-2-butenoate hydrochloride
(15d)
4.1.8. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-{(E)-2-phenyl-1-
[(pyridin-3-ylmethyl)carbamoyl]vinyl}-2-butenoate hydrochloride
(15a)
This compound was prepared from (2Z,3E)-14 as described in
the synthesis of 15b.
This compound was prepared from (2Z,3E)-14 as described in
the synthesis of 15c.
1H NMR (500 MHz, DMSO-d6) d: 1.72 (3H, s), 3.38 (3H, s), 3.74
(6H, s), 6.30 (2H, d, J = 2.1 Hz), 6.45 (1H, t, J = 2.1 Hz), 7.37–7.44
(1H, m), 7.46 (2H, t, J = 7.4 Hz), 7.64 (2H, d, J = 7.4 Hz), 7.72 (1H,
br), 7.81 (1H, br), 8.53 (2H, br), 9.18 (1H, br), 10.84 (1H, br). IR (Nu-
jol) cmꢀ1: 2921, 2852, 1706, 1671, 1589, 1545, 1458, 1375, 1204,
1155. MS APCI m/z: 459 ([M+H]+). HR-MS ESI calcd for
C27H26N2O5 ([M+H]+): 459.1920. Found: 459.1900.
1H NMR (500 MHz, DMSO-d6) d: 1.71 (3H, s), 3.38 (3H, s), 3.74
(6H, s), 4.55 (2H, d, J = 5.7 Hz), 6.36 (2H, d, J = 2.0 Hz), 6.45 (1H, t,
J = 2.2 Hz), 7.25–7.38 (1H, m), 7.38–7.44 (2H, m), 7.56 (1H, s),
7.58 (2H, s), 7.90 (1H, br), 8.27 (1H, br), 8.61 (1H, br), 8.75 (2H,
s). IR (ATR) cmꢀ1: 3266, 2947, 2839, 2555, 2087, 1720, 1649,
1589, 1506, 1422, 1243, 1203, 1153, 1047, 683. MS APCI m/z:
473 ([M+H]+). HR-MS ESI calcd for C28H28N2O5 ([M+H]+):
473.2076. Found: 473.2070.
4.1.12. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-2-phenyl-
1-(pyrimidin-2-ylcarbamoyl)vinyl]-2-butenoate hydrochloride
(15e)
Oxalyl chloride (780 mg, 6.15 mmol) and N,N-dimethylformam-
ide (100 ll) were added to a solution of (2Z,3E)-14 (2.0 g,
4.1.9. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-{(E)-2-phenyl-1-
[(pyridin-4-ylmethyl)carbamoyl]vinyl}-2-butenoate hydrochloride
(15b)
5.24 mmol) in CH2Cl2 (20 ml) at 0 °C. The reaction mixture was
then stirred at room temperature for 1 h and concentrated in va-
cuo. The resulting residue was dissolved in THF (50 ml), and the
mixture was slowly added to 2-aminopyrimidine (600 mg, 6.31
mmol) and pyridine (1 ml) in THF (20 ml) at 0 °C. Next, the reaction
mixture was stirred at room temperature for 30 min, and AcOEt
and water were added. The organic layer was separated, dried over
MgSO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel. 4N HCl in AcOEt (0.7 ml) was added to a solu-
tion of the obtained amide in CHCl3 (20 ml), and the mixture was
concentrated in vacuo. The residue was diluted with Et2O and
filtered to give 15e (250 mg, 10%).
1,10-Carbonyldiimidazole (700 mg, 4.32 mmol) was added to a
solution of (2Z,3E)-14 (1.5 g, 3.93 mmol) in CH2Cl2 (10 ml), and
the reaction mixture was stirred at room temperature for 2 h. 4-
Picolylamine (509 mg, 4.71 mmol) was then added at 0 °C to the
mixture, and the reaction mixture was stirred for 8 h at room tem-
perature. After AcOEt and water were added to the mixture, the or-
ganic layer was separated, dried over MgSO4, and concentrated in
vacuo. The residue was chromatographed on silica gel. 4N HCl in
AcOEt (0.15 ml) was added to a solution of the obtained amide in
AcOEt (10 ml), and the mixture was concentrated in vacuo. The
residue was diluted with Et2O, and filtered to give 15b (440 mg,
22%).
1H NMR (200 MHz, CDCl3) d: 1.95 (3H, s), 3.55 (3H, s), 3.81 (6H,
s), 6.40 (2H, d, J = 2.1 Hz), 6.40–6.45 (1H, m), 7.04 (1H, t, J = 4.9 Hz),
7.25–7.50 (3H, m), 7.63–7.80 (2H, m), 7.94 (1H, s), 8.68 (2H, d,
J = 4.9 Hz), 9.41 (1H, br). IR (ATR) cmꢀ1: 2948, 2840, 1725, 1687,
1572, 1533, 1201, 1154, 759, 696. MS APCI m/z: 460 ([M+H]+).
HR-MS ESI calcd for C26H25N3O5 ([M+H]+): 460.1872. Found:
460.1856.
1H NMR (500 MHz, DMSO-d6) d: 1.75 (3H, s), 3.43 (3H, s), 3.74
(6H, s), 4.64 (2H, d, J = 4.3 Hz), 6.37 (2H, d, J = 2.1 Hz), 6.45 (1H, t,
J = 2.1 Hz), 7.34–7.39 (1H, m), 7.43 (2H, t, J = 7.5 Hz), 7.56–7.64
(3H, m), 7.78 (2H, br), 8.64 (1H, br), 8.82 (2H, br). IR (ATR)
cmꢀ1: 3366, 2947, 2839, 2599, 2088, 1708, 1639, 1589, 1505,
1243, 1203, 1153, 1048, 695. MS APCI m/z: 473 ([M+H]+). HR-
MS ESI calcd for C28H28N2O5 ([M+H]+): 473.2076. Found:
473.2072.
4.1.13. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-2-phenyl-
1-(tetrazol-5-ylcarbamoyl)vinyl]-2-butenoate (15f)
Oxalyl chloride (390 mg, 3.07 mmol) and N,N-dimethylformam-
ide (one drop) were added to a solution of (2Z,3E)-14 (1.0 g,
2.61 mmol) in CH2Cl2 (10 ml) at 0 °C. The reaction mixture was
then stirred at room temperature for 1 h and concentrated in va-
cuo. The resulting residue was dissolved in THF (10 ml) and the
mixture was slowly added to 5-aminotetrazol (232 mg, 2.73 mmol)
and Et3N (0.76 ml) in THF (10 ml) at 0 °C. Next, the reaction mix-
ture was stirred at room temperature for 30 min, and AcOEt and
water were added. The organic layer was separated, dried over
MgSO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel and triturated with Et2O to give 15f
(590 mg, 50%).
4.1.10. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-2-phenyl-
1-(pyridin-4-ylcarbamoyl)vinyl]-2-butenoate hydrochloride (15c)
Oxalyl chloride (2.0 g, 15.8 mmol) was added dropwise to a
solution of (2Z,3E)-14 (5.0 g, 13.0 mmol) and N,N-dimethylform-
amide (one drop) in CH2Cl2 (20 ml) at 0 °C. The reaction mixture
was then stirred at room temperature for 1 h and concentrated
in vacuo. The resulting residue was dissolved in THF (10 ml) and
the mixture was added dropwise to 4-aminopyridine (3.0 g,
32 mmol) in THF (20 ml) at 0 °C. Next, the reaction mixture was
stirred at room temperature for 2 h, concentrated in vacuo, and
AcOEt and sat NaHCO3 aq were added to the residue. The organic
layer was separated, washed with brine, dried over MgSO4, and
concentrated in vacuo. The resulting residue was chromatographed
on silica gel. 4N HCl in AcOEt (2.7 ml) was added to a solution of
the obtained amide in CHCl3 (10 ml), and the mixture was concen-
trated in vacuo. The residue was diluted with i-Pr2O and filtered to
give 15c (2.7 g, 42%).
1H NMR (400 MHz, DMSO-d6) d: 1.72 (3H, s), 3.39 (3H, s), 3.74
(6H, s), 6.32 (2H, s), 6.44 (1H, s), 7.39 (1H, dd, J = 14.3, 7.4 Hz),
7.45 (2H, t, J = 7.4 Hz), 7.62 (2H, d, J = 7.4 Hz), 7.78 (1H, s), 11.42
(1H, br). IR (ATR) cmꢀ1: 2949, 2840, 1663, 1587, 1204, 1154,
1043, 767, 694. MS APCI m/z: 450 ([M+H]+). HR-MS ESI calcd for
C23H23N5O5 ([M+H]+): 450.1777. Found: 450.1775.
1H NMR (500 MHz, DMSO-d6) d: 1.71 (3H, s), 3.39 (3H, s), 3.74
(6H, s), 6.29 (2H, d, J = 2.1 Hz), 6.45 (1H, t, J = 1.9 Hz), 7.38–7.45
(1H, m), 7.48 (2H, t, J = 7.6 Hz), 7.66 (2H, d, J = 7.6 Hz), 7.81 (1H,
br), 8.21 (2H, br), 8.73 (2H, d, J = 6.9 Hz), 11.54 (1H, br). IR (Nujol)
cmꢀ1: 2921, 2631, 1686, 1634, 1590, 1505, 1465, 1321, 1192, 1154.
MS APCI m/z: 459 ([M+H]+). HR-MS ESI calcd for C27H26N2O5
([M+H]+): 459.1920. Found: 459.1905.
4.1.14. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-2-phenyl-
1-(quinolin-8-ylcarbamoyl)vinyl]-2-butenoate hydrochloride
(15g)
This compound was prepared from (2Z,3E)-14 as described in
the synthesis of 15i.
1H NMR (400 MHz, DMSO-d6) d: 2.01 (3H, s), 3.37 (3H, s), 3.83
(6H, s), 6.59 (1H, t, J = 2.1 Hz), 6.73 (2H, d, J = 2.1 Hz), 7.38–7.50

1976
H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
(3H, m), 7.63–7.77 (5H, m), 7.90 (1H, s), 8.48 (1H, dd, J = 8.4,
1.5 Hz), 8.83 (1H, dd, J = 3.4, 1.5 Hz), 8.85 (1H, d, J = 1.5 Hz), 10.80
(1H, s). IR (ATR) cmꢀ1: 2943, 2588, 1698, 1652, 1592, 1294,
1153, 829, 696, 458. MS APCI m/z: 509 ([M+H]+). HR-MS ESI calcd
for C31H28N2O5 ([M+H]+): 509.2076. Found: 509.2091.
(2H, d, J = 1.8 Hz), 6.43 (1H, s), 7.30–7.50 (4H, m), 7.54 (2H, d,
J = 7.2 Hz), 9.48 (1H, s), 10.11 (1H, br). 13C NMR (CDCl3, 100 MHz)
d: 22.2, 43.2, 51.3, 52.4, 53.3, 55.5, 99.9, 104.7, 125.1, 128.6,
128.6, 129.5, 129.7, 134.7, 140.9, 143.5, 149.0, 160.8, 164.9,
168.9. IR (Nujol) cmꢀ1: 3196, 2924, 2854, 1698, 1662, 1595,
1464, 1262. MS APCI m/z: 480 ([M+H]+). Anal. Calcd for
C27H34ClN3O5: C, 62.84; H, 6.64; N, 8.14; Cl, 6.87. Found: C,
62.61; H, 6.71; N, 8.09; Cl, 6.79.
4.1.15. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-2-phenyl-
1-([1,2,4]triazol-4-ylcarbamoyl)vinyl]-2-butenoate
hydrochloride (15h)
This compound was prepared from (2Z,3E)-14 as described in
the synthesis of 15c.
4.1.19. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-1-(4-methyl-
4-oxy-piperazin-1-ylcarbamoyl)-2-phenylvinyl]-2-butenoate (15l)
Eighty percent mCPBA (248 mg, 1.15 mmol) was added at -
50 °C to a free form solution of 15k (500 mg, 1.05 mmol) in CH2Cl2
(25 ml),. The reaction mixture was stirred at room temperature for
1 h and concentrated in vacuo. The residue was chromatographed
on silica gel to give 15l (296 mg, 57%). 1H NMR (400 MHz, CDCl3) d:
3.10 (2H, d, J = 10.8 Hz), 3.26 (2H, d, J = 10.8 Hz), 3.28 (3H, s), 3.51
(3H, s), 3.53–3.78 (4H, m), 3.81 (6H, s), 6.33 (2H, d, J = 2.3 Hz), 6.43
(1H, t, J = 2.3 Hz), 7.30–7.45 (3H, m), 7.57 (1H, s), 7.64 (2H, d,
J = 6.9 Hz), 7.83 (1H, s). IR (ATR) cmꢀ1: 2949, 1715, 1655, 1590,
1204, 1154, 1049, 749, 696. MS APCI m/z: 496 ([M+H]+). HR-MS
ESI calcd for C27H33N3O6 ([M+H]+): 496.2448. Found: 496.2445.
1H NMR (400 MHz, DMSO-d6) d: 1.78 (3H, s), 3.43 (3H, s), 3.74
(6H, s), 6.36 (2H, d, J = 2.0 Hz), 6.45 (1H, t, J = 2.2 Hz), 7.38–7.50
(3H, m), 7.61 (2H, d, J = 7.2 Hz), 7.74 (1H, s), 8.84 (2H, s), 11.79 (1H,
br). IR (ATR) cmꢀ1: 3105, 2952, 2301, 1871, 1727, 1680, 1595,
1249, 1209, 1191, 1152, 693. MS APCI m/z: 449 ([M+H]+). HR-MS
ESI calcd for C24H24N4O5 ([M+H]+): 449.1825. Found: 449.1809.
4.1.16. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-1-(4-methyl-
piperazin-1-carbonyl)-2-phenylvinyl]-2-butenoate hydrochloride
(15i)
Oxalyl chloride (2.0 g, 15.7 mmol) was added dropwise to a solu-
tion of (2Z,3E)-14 (5.0 g, 13.0 mmol) and N,N-dimethylformamide
(one drop) in CH2Cl2 (50 ml) at 0 °C. The reaction mixture was then
stirred at room temperature for 1 h and concentrated in vacuo. The
resulting residue was dissolved in THF (10 ml), and the mixture
was added dropwise to N-methylpiperazine (1.57 g, 15.7 mmol)
and Et3N (2.21 ml, 15.7 mmol) in THF (50 ml) at 0 °C. Next, the reac-
tion mixture was stirred at room temperature for 2 h. After the mix-
ture was concentrated in vacuo, AcOEt and sat NaHCO3 aq were
added to the residue. The organic layer was separated, washed with
brine, dried over MgSO4, and concentrated in vacuo. The resulting
residue was chromatographed on silica gel. 4N HCl in AcOEt
(2.7 ml) was added to a solution of the obtained amide in CHCl3
(20 ml), and the mixture was concentrated in vacuo. The residue
was diluted with Et2O and filtered to give 15i (3.9 g, 60%).
Mp: >219 °C (dec.). 1H NMR (500 MHz, DMSO-d6) d: 1.74 (3H, s),
2.82 (3H, s), 3.05 (2H, br), 3.43 (3H, s), 3.25–3.50 (4H, br), 3.73 (6H,
s), 4.39 (2H, br), 6.20 (2H, d, J = 2.0 Hz), 6.44 (1H, br), 6.99 (1H, s),
7.33–7.38 (1H, m), 7.42 (2H, t, J = 7.4 Hz), 7.54 (2H, d, J = 7.4 Hz),
10.76 (1H, br). IR (Nujol) cmꢀ1: 2924, 2854, 2345, 1708, 1606,
1460, 1208, 1160. MS APCI m/z: 465 ([M+H]+). Anal. Calcd for
C27H33ClN2O5: C, 64.73; H, 6.64; Cl, 7.08; N, 5.59. Found: C,
64.56; H, 6.75; Cl, 7.01; N, 5.57.
4.1.20. 3-[1-(30,50-Dimethoxyphenyl)-(Z)-ethylidene]-4-[1-phenyl-
(E)-methylidene]-1-pyridin-3-ylmethylpyrrolidine-2,5-dione
hydrochloride (15m)
2N NaOH aq (2 ml, 4 mmol) was added dropwise to a free form
(1.1 g, 2.33 mmol) solution of 15a in THF (20 ml), and the mixture
was stirred for 30 min at room temperature. After the mixture was
neutralized with 2N HCl aq, AcOEt was added. The organic layer
was separated, washed with brine, dried over MgSO4, and concen-
trated in vacuo. The resulting residue was next chromatographed
on silica gel. 4N HCl in AcOEt (2 ml) was added to a solution of
the obtained imide in AcOEt (50 ml), and the mixture was concen-
trated in vacuo. The residue was diluted with Et2O, and filtered to
give 15m (720 mg, 70%). 1H NMR (400 MHz, DMSO-d6) d: 1.70 (3H,
s), 3.74 (6H. s), 4.83 (2H, s), 6.51 (1H, t, J = 2.0 Hz), 6.54 (2H, d,
J = 2.0 Hz), 7.35–7.44 (1H, m), 7.45–7.52 (2H, m), 7.57–7.64 (3H,
m), 7.87 (1H, dd, J = 7.9, 5.4 Hz), 8.28 (1H, d, J = 7.9 Hz), 8.76 (1H,
dd, J = 5.4, 1.4 Hz), 8.79 (1H, d, J = 1.4 Hz). IR (ATR) cmꢀ1: 3406,
2939, 2410, 2087, 1754, 1698, 1587, 1421, 1396, 1333, 1203,
1153, 1037, 768, 693, 586, 503, 477. MS APCI m/z: 441 ([M+H]+).
HR-MS ESI calcd for C27H24N2O4 ([M+H]+): 441.1814. Found:
441.1805.
4.1.17. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-1-(1-methyl-
piperizin-4-ylcarbamoyl)-2-phenylvinyl]-2-butenoate hydrochlo-
ride (15j)
4.1.21. 3-[1-(30,50-Dimethoxyphenyl)-(Z)-ethylidene]-4-[1-phenyl-
(E)-methylidene]-1-pyridin-4-ylmethylpyrrolidine-2,5-dione
hydrochloride (15n)
This compound was prepared from (2Z,3E)-14 as described in
the synthesis of 15i.
This compound was prepared from the free form of 15b as de-
scribed in the synthesis of 15m.
Mp: >184 °C (dec.). 1H NMR (500 MHz, DMSO-d6) d: 1.62 (3H, s),
1.81 (2H, br), 1.97 (2H, br), 2.74 (3H, s), 3.05 (2H, br), 3.36 (3H, s),
3.43 (2H, br), 3.73 (6H, s), 3.87 (1H, br), 6.28 (2H, d, J = 2.0 Hz), 6.43
(1H, br), 7.30–7.37 (1H, m), 7.37–7.44 (2H, m), 7.55 (2H, d,
J = 7.3 Hz), 8.15 (1H, br), 9.93 (1H, br). IR (Nujol) cmꢀ1: 3273,
2923, 2853, 1695, 1641, 1595, 1463, 1376, 1153. MS APCI m/z:
479 ([M+H]+). HR-MS ESI calcd for C28H34N2O5 ([M+H]+):
479.2546. Found: 479.2543.
Mp: 127 °C. 1H NMR (400 MHz, DMSO-d6) d: 1.73 (3H, s), 3.74
(6H. s), 4.91 (2H, s), 6.51 (1H, t, J = 2.3 Hz), 6.55 (2H, d,
J = 2.3 Hz), 7.38–7.45 (1H, m), 7.45–7.53 (2H, m), 7.59–7.67 (3H,
m), 7.83 (2H, d, J = 6.4 Hz), 8.80 (2H, d, J = 6.4 Hz). IR (ATR) cmꢀ1
:
2960, 2314, 2093, 2030, 1987, 1758, 1704, 1629, 1605, 1583,
1411, 1389, 1335, 1156, 1041, 924, 770, 699, 476, 467. MS APCI
m/z: 441 ([M+H]+). HR-MS ESI calcd for C27H24N2O4 ([M+H]+):
441.1814. Found: 441.1801.
4.1.18. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-1-(4-
methylpiperazin-1-ylcarbamoyl)-2-phenylvinyl]-2-butenoate
hydrochloride (15k, T-2369)
4.1.22. 4-Methoxymethyl 1-methyl 2-[1-(30,50-dimethoxyphenyl)-
(Z)-ethylidene]-3-[1-pyridin-4-yl-(E)-methylidene]succinate (20a)
A solution of 30,50-dimethoxyacetophenone (16) (30 g, 166
mmol) and dimethyl succinate (29.1 g, 199 mmol) in t-BuOH
(100 ml) was added to a suspension of t-BuOK (18.7 g, 166 mmol)
in t-BuOH (200 ml) below 40 °C and the reaction mixture was stir-
red at 50 °C for 4 h. Water was then added to the mixture, and the
This compound was prepared from (2Z,3E)-14 as described in
the synthesis of 15i.
Mp: 235–240 °C. 1H NMR (DMSO-d6, 500 MHz) d: 1.65 (3H, s),
2.79 (3H, s), 3.00–3.50 (8H, m), 3.37 (3H, s), 3.73 (6H, s), 6.29

H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
1977
resulting mixture was washed with i-Pr2O. After the aqueous layer
was acidified with concd HCl (pH 1), AcOEt was added. The organic
layer was separated, washed with water and brine, dried over
MgSO4, and concentrated in vacuo. The resulting residue was
diluted with CH2Cl2 (500 ml), and N,N-diisopropylethylamine
(30 ml, 169 mmol) and methoxymethyl chloride (16 ml, 172
mmol) were added to the mixture. The reaction mixture was then
stirred overnight at room temperature and concentrated in vacuo.
After AcOEt and water were added to the mixture, the organic layer
was separated, washed with water and brine, dried over MgSO4,
and concentrated in vacuo. The residue was chromatographed on
silica gel to give 17. Compound 17 was dissolved in acetonitrile
(500 ml) and the solution was illuminated with a mercury lamp
for 8 h. The solution was then concentrated in vacuo and the resi-
due was chromatographed on silica gel to give 18 (20.2 g, 36%).
(30.3 g of 17 was recovered (54%).)
trated in vacuo. The residue was diluted with CHCl3, filtered and
washed with Et2O to give 21a (2.58 g, 44%) as pale yellow crystals.
1H NMR (500 MHz, DMSO-d6) d: 1.73 (3H, s), 3.40 (3H, s), 3.74
(6H, s), 6.35 (2H, d, J = 2.1 Hz), 6.44 (1H, t, J = 2.1 Hz), 7.53–7.65
(3H, m), 7.87 (2H, br), 8.83 (2H, br). IR (Nujol) cmꢀ1: 2921, 2853,
1699, 1592, 1461, 1373, 1153. MS APCI m/z: 383 ([M+H]+). HR-
MS ESI Calcd for C21H22N2O5 ([M+H]+): 383.1607. Found: 383.1605.
4.1.24. 4-Methoxymethyl 1-methyl 2-[1-(30,50-dimethoxyphenyl)-
(Z)-ethylidene]-3-[1-pyridin-3-yl-(E)-methylidene]succinate (20b)
This compound was prepared from 18 as described in the syn-
thesis of 20a.
1H NMR (200 MHz, CDCl3) d: 1.78 (3H, s), 3.51 (6H, s), 3.79 (6H,
s), 5.41 (2H, s), 6.29 (2H, d, J = 2.2 Hz), 6.40 (1H, t, J = 2.2 Hz), 7.35
(1H, dd, J = 7.9, 4.8 Hz), 7.92 (1H, S), 8.02 (1H, d, J = 7.9 Hz), 8.58
(1H, d, J = 3.4 Hz), 8.81 (1H, s). IR (film) cmꢀ1: 2952, 1713, 1592,
1424, 1158, 1051, 928, 709. MS m/z: 427 ([M]+).
1H NMR (200 MHz, CDCl3) d: 2.13 (3H, s), 3.45 (3H, s), 3.48 (3H,
s), 3.56 (2H, s), 3.77 (6H, s), 5.28 (2H, s), 6.30 (2H, d, J = 2.2 Hz), 6.38
(1H, t, J = 2.2 Hz). IR (film) cmꢀ1: 2950, 1739, 1593, 1424, 1156,
1092, 929. MS m/z: 338 ([M]+).
4.1.25. Methyl (Z)-2-((E)-1-carbamoyl-2-pyridin-3-ylvinyl)-3-
(30,50-dimethoxyphenyl)-2-butenoate hydrochloride (21b)
This compound was prepared from 20b as described in the syn-
thesis of 21a.
1.6 M n-BuLi in hexane (17.7 ml, 28.4 mmol) was added below
ꢀ70 °C to a solution of diisopropylamine (4.0 ml, 28.4 mmol) in
THF (60 ml). After the mixture was stirred at ꢀ78 °C for 30 min,
18 (8.0 g, 23.6 mmol) in THF (80 ml) was added dropwise to the
solution below ꢀ70 °C. The reaction mixture was again stirred at
ꢀ78 °C for 30 min, and then a solution of 4-pyridinecarboxalde-
hyde (3.0 g, 28.4 mmol) in THF (30 ml) was added dropwise to
the mixture at ꢀ100 °C. After the mixture was stirred at ꢀ100 °C
for 20 min, water and AcOEt were added to the mixture. The organ-
ic layer was separated, washed with water and brine, dried over
MgSO4, and concentrated in vacuo. The resulting residue was dis-
solved in CH2Cl2 (60 ml), and triethylamine (4.0 ml, 28.4 mmol)
and methanesulfonyl chloride (2.2 ml, 28.4 mmol) were added to
the mixture at 0 °C. After the reaction mixture was stirred for
30 min at 0 °C, DBU (4.2 ml, 24.8 mmol) was added to the mixture.
The mixture was then stirred overnight at room temperature. After
AcOEt and water were added to the mixture, the organic layer was
separated, washed with water and brine, dried over MgSO4, and
concentrated in vacuo. The residue was chromatographed on silica
gel to give 20a (6.0 g, 59%).
1H NMR (500 MHz, DMSO-d6) d: 1.73 (3H, s), 3.39 (3H, s), 3.74
(6H, s), 6.36 (2H, d, J = 2.1 Hz), 6.43 (1H, t, J = 2.1 Hz), 7.47 (2H,
br), 7.59 (1H, s), 7.75 (1H, br), 8.26 (1H, br), 8.67 (1H, br), 8.85
(1H, br). IR (Nujol) cmꢀ1: 2918, 1743, 1699, 1671, 1589, 1523,
1462, 1204, 1155, 1037. MS APCI m/z: 383 ([M+H]+). Anal. Calcd
for C21H23ClN2O5: C, 60.22; H, 5.53; Cl, 8.46; N, 6.69. Found: C,
60.23; H, 5.40; Cl, 8.44; N, 6.66.
4.2. Scale-up synthesis of 15k (T-2639)
4.2.1. 1-Methyl 2-[1-(30,50-dimethoxyphenyl)-(Z)-ethylidene]-3-
[1-phenyl-(E)-methylidene]-4-succinic acid ((2Z,3E)-14): Z,E-
selective Stobbe condensation
Twenty-four percent w/w NaOMe solution in MeOH (20.5 g,
91.5 mmol) was added to a solution of 10 (17.1 g, 73 mmol) in
toluene (50 ml), and the mixture was stirred at 30–40 °C for
30 min. After 30,50-dimethoxyacetophenone (16) (11.0 g, 61 mmol)
was added to the reaction mixture at 30–40 °C, powder of
(2Z,3E)-14 Na salts (233 mg, 0.61 mmol) was added as seeds to
the reaction mixture, and the resulting mixture was stirred at re-
flux for 1 h. After the salts were precipitated, the mixture was
concentrated in vacuo until the volume was about 22 ml (2 v/w
of 30,50-dimethoxyacetophenone (16)). The mixture was again
stirred at reflux for 1 h. Acetic acid (2.9 g, 49 mmol) was then
added to the mixture below 15 °C, and ice-water was poured into
the mixture. The organic and aqueous layers were separated, and
the organic layer was washed with 5% aqueous NaHCO3 solution
(100 ml). AcOEt (250 ml) was added to the aqueous layer, and the
mixture was acidified with concd HCl below 15 °C (pH 2). The or-
ganic and aqueous layers were again separated, and the organic
layer was washed with 10% brine (100 ml) and saturated brine
(100 ml) and dried over MgSO4. After activated charcoal was
added to the solution, the suspension was filtered, and the filtrate
was concentrated in vacuo. The residue was diluted with i-Pr2O
and filtered to give 14 (19.1 g, 82%, (2Z,3E)-14:(2E,3E)-14 = 91:9)
as a solid.
1H NMR (200 MHz, CDCl3) d: 1.76 (3H, s), 3.51 (3H, s), 3.52 (3H,
s), 3.79 (6H, s), 5.41 (2H, s), 6.25 (2H, d, J = 2.3 Hz), 6.40 (1H, t,
J = 2.3 Hz), 7.50 (2H, dd, J = 4.6, 1.3 Hz), 7.85 (1H, s), 8.67 (2H, dd,
J = 4.6, 1.6 Hz). IR (film) cmꢀ1: 2952, 1724, 1592, 1423, 1157,
1050, 928, 821, 698, 538. MS m/z: 427 ([M]+).
4.1.23. Methyl (Z)-2-((E)-1-carbamoyl-2-pyridin-4-ylvinyl)-3-
(30,50-dimethoxyphenyl)-2-butenoate hydrochloride (21a)
Concd HCl (6.0 ml) was added to a solution of 20a (6.0 g,
14 mmol) in THF (60 ml). The mixture was stirred at room temper-
ature for 1 h, diluted with water, and neutralized with 2N NaOH aq
After AcOEt was added to the mixture, the organic layer was sepa-
rated, dried over MgSO4, and concentrated in vacuo. The residue
was chromatographed on silica gel. The obtained carboxylic acid
was dissolved in THF (300 ml), and 1,10-carbonyldiimidazole
(3.4 g, 21.0 mmol) was added to the mixture. After the reaction
mixture was stirred at room temperature for 2 h, THF (200 ml),
CH2Cl2 (50 ml), and 1,10-carbonyldiimidazole (1.7 g, 10.5 mmol)
were added again. The mixture was then stirred overnight at room
temperature. Twenty-eight percent NH3 aq (5.0 ml) was added to
the mixture at 0 °C and the resulting mixture was stirred at 0 °C
for 2 h. After water and CHCl3 were added to the mixture, the or-
ganic layer was separated, dried over MgSO4, and concentrated in
vacuo. The resulting residue was chromatographed on silica gel.
Next, 4N HCl in AcOEt (1.5 ml) was added to a solution of the ob-
tained amide in CHCl3 (20 ml), and then the mixture was concen-
4.2.2. 1-Methyl 2-[1-(30,50-dimethoxyphenyl)-(E)-ethylidene]-3-
[1-phenyl-(E)-methylidene]-4-succinic Acid ((2E,3E)-14)
1H NMR (CDCl3, 400 MHz) d: 2.44 (3H, s), 3.54 (6H, s), 3.79 (3H,
s), 5.85 (2H, d, J = 2.3 Hz), 6.22 (1H, t, J = 2.3 Hz), 7.25–7.40 (5H, m),
7.50 (1H, s). 13C NMR (CDCl3, 100 MHz) d: 23.1, 52.0, 55.1, 100.4,
103.9, 124.0, 128.3, 129.2, 129.4, 129.9, 135.0, 143.4, 144.8,
154.7, 160.1, 167.5, 172.6. IR (ATR) cmꢀ1: 2841, 2524, 1711,

1978
H. Miyazaki et al. / Bioorg. Med. Chem. 18 (2010) 1968–1979
1658, 1592, 1426, 1316, 1205, 1157. MS ESI m/z: 381 ([M-H]ꢀ).
HR-MS ESI calcd for C22H23O6 ([M+H]+): 383.1495. Found:
383.1485.
4.5. Antithrombotic activity of 15k (T-2639) and Warfarin in a
rat venous thrombosis model
4.5.1. Administration of 15k and Warfarin
4.2.3. Methyl (Z)-3-(30,50-dimethoxyphenyl)-2-[(E)-1-(4-methyl-
piperazin-1-ylcarbamoyl)-2-phenylvinyl]-2-butenoate hydro-
chloride (15k, T-2369)
15k (0.3, 1, and 3 mg/kg) or Warfarin (0.06, 0.2, and 0.6 mg/kg)
were orally administered to rats once a day for 8 days. Vehicle was
administered to the control group.
Oxalyl chloride (194 g, 133.3 ml, 1.52 mol) was added dropwise
over 20 min to (2Z,3E)-14 (487 g, 1.27 mol, containing <10% of
(2E,3E)-14) solution in CH2Cl2 (1 l) and DMF (1 l). The reaction mix-
ture was then stirred for 2 h and concentrated in vacuo. After the
residue was dissolved in THF (4 l), a mixture of triethylamine
(214 ml, 1.52 mol) and 1-amino-4-methylpiperazine (168 ml,
1.52 mol) was added dropwise to the mixture while maintaining
the temperature below 20 °C. After 2 h, the reaction mixture was
diluted with AcOEt (4 l) and washed with water (3 l) twice and
brine (2 l). The organic layer was dried over MgSO4 and concen-
trated in vacuo. The resulting oil was diluted with CH2Cl2 (3 l),
and i-Pr2O (1 l) was added. The mixture was next concentrated in
vacuo until the volume was about 1.5 l. After the mixture was stir-
red for 5 h at room temperature, the crystals were collected by fil-
tration and washed with i-Pr2O. The crystals obtained were next
dissolved in EtOH (4.9 l), and concd HCl (86 ml) was added drop-
wise at room temperature. The reaction mixture was left at room
temperature for 1 h and then cooled in a refrigerator (ꢀ25 °C) for
14 h. Crystals were collected by filtration and washed with Et2O
(1 l) to provide 420 g of 15k (64%).
4.5.2. Thrombus formation
Rats were anesthetized with NembutalÒ (Abbot, 50 mg/kg ip)
and the abdomen was surgically opened to expose the vena cava.
All branches of the vena cava between the left renal vein and the
bifurcation were ligated with a medical suture (Matsuda Sutures,
Tokyo, Japan). Two hours after the last administration of each com-
pound or its vehicle, 0.1 ml of diluted rabbit brain thromboplastin
(1:10, SIGMA Chemicals, St. Louise, MO, USA) was injected into the
dorsal vein of the penis. Ten seconds after injection of thrombo-
plastin, the vena cava was ligated beneath the left renal vein. The
abdominal cavity was provisionally closed. After 15 min of stasis,
the abdominal cavity was reopened and the vena cava was ligated
near the bifurcation (2 cm beneath the left renal vein) and was lon-
gitudinally opened by a surgical blade. The formed thrombus was
removed and its dry weight was measured using Sartorius Super-
micro S4 (Sartorius).
4.6. Effects of 15k (T-2639) and Warfarin in the rat tail incision
bleeding time test
4.6.1. Administration of 15k and Warfarin
4.3. Inhibitory effect on elevation of PAI-1 antigen induced by
15k (10, 30, and 100 mg/kg) or Warfarin (0.1, 0.2, and 0.4 mg/
kg) were orally administered to rats once a day for 8 days. Vehicle
was administered to the control group.
LPS or TGF-b in bovine endothelial cells (BCaEs)
BCaEs (passages 8 and 9) were grown to confluence on a 24-
well culture plate in E’MEM supplemented with 10% fetal calf ser-
um (FCS). The cells were exposed to test compounds (1 or 2
4.6.2. Determinations of bleeding time
lM) in
Rats were anesthetized with NembutalÒ (Abbot, 50 mg/kg ip).
Two hours after the last administration of each compound or its
vehicle, an incision 2 mm in depth was made with a surgical blade
3.5 cm from the tip of the tail. Blood was blotted in filter paper (No.
2) every 30 s and bleeding time was determined by measuring the
time until no blood was seen on the filter paper.
the presence or absence of LPS (1 g/ml) or TGF-b (0.3 ng/ml) after
l
serum starvation for 24 h. Twenty-four fours after incubation with
test compounds, the conditioned medium was collected and cen-
trifuged at 3000 rpm for 10 min at 4 °C to remove cellular debris.
PAI-1 antigen in the conditioned medium was determined using
a specific enzyme-linked immunosorbent assay (ELISA) kit ob-
tained from Biopool AB (Umea, Sweden).
4.7. Stability in dog blood
4.4. Antithrombotic activity of 15k (T-2639) in a rat arterio-
venous shunt model
One ml of stock solution (100 lg/ml in acetonitrile) was diluted
with saline to a final volume of 10 ml. Next, 0.3 ml of the solution
was added to 3 ml of a heparinized dog blood, which was pre-incu-
bated at 37 °C, and the whole was incubated at 37 °C. After 1 h, the
plasma was separated with diethylether by centrifugation and test
compounds plasma concentrations were determined by HPLC.
4.4.1. Administration of 15k
Compound 15k was orally administered at a dose of 10 mg/kg
once a day for 8 days. Vehicle was administered to the control
group.
4.8. Photostability
4.4.2. Thrombus formation
Rats were anesthetized with NembutalÒ (Abbot, 50 mg/kg ip). A
cervical incision was made in the mid-line to expose the right car-
otid artery and left jugular vein. A 24-cm-long polyethylene tube
with 6-cm-long silk thread fixed in the lumen was filled with saline
and one end of the tube was inserted into the left jugular vein and
tied. The proximal side of the right carotid artery was clamped to
block blood flow temporarily, while the free end of the tube was
inserted into the artery and tied.
Two hours after the last administration of the test compound or
its vehicle, the clamp was removed and the tube was perfused with
arterial blood. After 30 min perfusion, the silk thread covered with
thrombus was removed from the tube, and the wet weight of
thrombus was immediately determined using a Sartorius A120S
(Sartorius, Germany).
To 0.1 ml of stock solution (100 lg/ml in acetonitrile) was added
0.9 ml of 0.1N HCl aq. The solution was stored at room temperature
for 24 h with or without aluminum foil cover to protect from light.
The covered and uncovered solutions were analyzed by HPLC.
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demethylated to phenol metabolite in the 3,4,5-trimethoxyphenyl group
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days. Two hours after the last administration, thrombi were induced and their
dry weights were measured. See Ref. 12.
18. NaOMe solution was used instead of powder NaOMe because it was easy to
handle in consideration of application to process chemistry. The reaction will
take place similarly even if powder NaOMe is used.
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may be related to precipitation.
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