Structure-activity relationships of new N-acylanthranilic acid derivatives as plasminogen activator inhibitor-1 inhibitors

Article abstract of DOI:10.1248/cpb.59.215

Novel anthranilic acid derivatives having substituted N-acyl side chains were designed and synthesized for evaluation as plasminogen activator inhibitor-1 (PAI-1) inhibitors. Compounds with a 4-diphenylmethyl-1-piperazinyl moiety on the acyl side chains in general exhibited potent in vitro PAI-1 inhibitory activity and good pharmacokinetic profiles after oral administration in rats. Compound 16f (TM5275) was identified as a promising candidate for further pharmacological evaluation.

Full text of DOI:10.1248/cpb.59.215

February 2011
Regular Article
Chem. Pharm. Bull. 59(2) 215—224 (2011)
215
Structure–Activity Relationships of New N-Acylanthranilic Acid
Derivatives as Plasminogen Activator Inhibitor-1 Inhibitors
a
Nagahisa YAMAOKA, ,a Hidehiko KODAMA,a Yuko IZUHARA,b Toshio MIYATA,b and Kanji MEGURO
*
a CT Laboratory, Hamari Chemicals, Ltd.; 1–4–29 Kunijima, Higashiyodogawa-ku, Osaka 533–0024, Japan: and b Center
for Translational and Advanced Research, Tohoku University School of Medicine; 2–1 Seiryo-Machi, Aoba-ku, Sendai
980–8575, Japan. Received September 1, 2010; accepted November 8, 2010; published online November 9, 2010
Novel anthranilic acid derivatives having substituted N-acyl side chains were designed and synthesized for
evaluation as plasminogen activator inhibitor-1 (PAI-1) inhibitors. Compounds with a 4-diphenylmethyl-1-piper-
azinyl moiety on the acyl side chains in general exhibited potent in vitro PAI-1 inhibitory activity and good phar-
macokinetic profiles after oral administration in rats. Compound 16f (TM5275) was identified as a promising
candidate for further pharmacological evaluation.
Key words plasminogen activator inhibitor-1; inhibitor; N-acylanthranilic acid derivative; structure–activity relationship;
TM5275
Elevated levels of plasminogen activator inhibitor-1 (PAI- 2, respectively, in rats given 50 mg/kg. We therefore contin-
1), a serine protease which inhibits tissue-type and uroki- ued our search to identify compounds with both potent PAI-1
nase-type plasminogen activators (tPA and uPA, respec- inhibitory activity and improved oral PK profiles by design-
tively), is thought to be a cause of a variety of diseases,1) ing more drug-like structures than the previously reported
such as thrombotic diseases (e.g., brain infarction, deep vein thiophene dimers (TM5001, 1, 2). Our major efforts were di-
thrombosis, etc.), tissue fibrotic diseases (e.g., lung thrombo- rected toward the syntheses of novel compounds without the
sis, renal thrombosis, liver thrombosis etc.), arteriosclerosis, dimer structure, possessing as low molecular weight as possi-
obesity and cancer, due to insufficient activation of a prote- ble, and with lower ClogP9) compared to those of TM5001
olytic enzyme plasmin. Furthermore, recent reports suggest (5.79), 1 (5.32) and 2 (7.06).
that PAI-1 inhibitors may be useful to treat Alzheimer’s dis-
One of the most important findings in the thiophene dimer
ease,2) glaucoma,3) and retinopathy.4) Thus, a number of syn- series which we have reported was that the dicarboxyl com-
thetic small molecule PAI-1 inhibitors, e.g., PAI-039 (tiplax- pound 1 and the corresponding mono-ester compound 2 were
tinin),5) PAI-749 (diaplasinin),6) PAZ-4172) have been re- almost equipotent in in vitro PAI-1 inhibition, suggesting that
ported to date targeted towards the development of new the symmetrical dimer structure was not always necessary
drugs to modify above-mentioned diseases, although the and one carboxyl moiety was sufficient to elicit PAI-1 in-
clinical benefits of these inhibitors are still not known.
hibitory property.8) Based upon these findings, we investi-
We have also previously reported novel PAI-1 inhibitors gated further structure-optimization as briefly illustrated in
TM5001,7) TM50077) and their related compounds such as 1 Fig. 2. This paper describes structure-activity relationship
and 28) (Fig. 1) with an acylaminothiophene-carboxylic acid (SAR) studies to identify a novel PAI-1 inhibitor, TM527510)
dimer structure to which lipophilic or/and bulky groups are (Fig. 3) as an orally bioavailable, promising drug candidate.
attached as the aromatic ring substituents. Although these
Chemistry General synthetic routes to various novel tar-
compounds had potent in vitro PAI-1 inhibitory activity, they get compounds are outlined in Charts 1 and 2.
exhibited rather undesirable pharmacokinetic (PK) property
N-Acylanthranilic acid derivatives (8a, aꢀ, 13) were first
(e.g., slow and insufficient absorption, too long duration of prepared as shown in Chart 1 in order to demonstrate if one
the plasma level) when administered orally; plasma Cmax
,
or both of the thiophene rings of the lead compounds,
Tmax and T1/2 were 32 mM, 18 h, and 54 h for TM5001,7) TM5001, 1 and 2 could be converted to the possibly bioisos-
5.8 mM, 18 h, and ꢀ174 h for 1, 2.32 mM, 48 h, and >48 h for teric benzene ring(s). tert-Butyl 2-amino-4-phenylthiophene-
Fig. 1. Modifications of N-Acylaminothiophene Dimers
Fig. 2. Schematic Design for Lead Optimization
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: nagahisa-yamaoka@hamari.co.jp

216
Vol. 59, No. 2
3-carboxylate (3) and p-chloroaniline (9) were acylated with
methyl adipoyl chloride, followed by alkaline hydrolysis to
obtain the corresponding adipic acid mono-amide derivatives
5 and 11, respectively. Compounds 5 and 11 were condensed
with the methyl anthranilates (6a, aꢀ) to afford 7a, aꢀ and 12,
which were then hydrolyzed to furnish N-acylanthranilic acid
Fig. 3. Structure of TM5275 (16f)
Reagents: (a) MeOCO(CH2)4COCl, (b) OHꢁ; (c) (i) i-BuOCOCl, Et3N, (ii) 6a; (d) (i) MsCl, Et3N, (ii) 6aꢀ; (e) (i) SOCl2, (ii) 6a.
Chart 1. Synthesis of N-Acylanthranilic Acid Derivatives (8a, 8aꢀ, 13)
Reagents: (a)
(X C, O, S, MeN,
ꢂ
); (b) (i) (COCl)2/DMF, SOCl2, or WSC/HOBt (ii) amines (R5H) ; (c) OHꢁ;
(d) R6-boronic acids or R6-boronic acid esters/base/Pd(PPh3)4 or R6ZnBr; (e) NaH, MeI.
* For substituents R5 see Table 2.
Chart 2. Synthesis of N-Acylanthranilic Acid Derivatives (16, 18, 20)

February 2011
217
derivatives, with additional thiophenamide (8a, aꢀ) and tPA : PAI-1ꢂ1 : 2.5). Method B provides a more sensitive
anilide (13) structures incorporated in the other side of the assay than Method A to evaluate the PAI-1 activity at lower
molecule, respectively.
concentrations.
Chart 2 illustrates the syntheses of a variety of N-acylan-
The biological property of compounds with a benzoic acid
thranilic acid derivatives (16, 18, 20) with hydrophobic car- moiety in place of a thiophene-carboxylic acid structure in
bamoyl moieties as well as various –CH2–X–CH2– groups the compound 2 was first evaluated utilizing compounds
(XꢂCH2, O, S, MeN, 1,1-cyclohexylene) as the linker L 8a, aꢀ (Table 1). Although compound 8aꢀ without any sub-
(Fig. 2). The ester-carboxylic acids (14) were prepared from stituent on the benzene ring showed considerably decreased
6 by reaction with the corresponding acid anhydrides. Then PAI-1 inhibitory activity, 8a with a chlorobenzoic acid struc-
compounds 14 were condensed with hydrophobic amines ture showed activity comparable to TM5001. The results ob-
(R5H) via the acid chlorides or the activated esters to give the tained here suggest that a phenyl-substituted thiophene-car-
corresponding amides (15).
boxylic acid moiety as in 2 can be replaced by a chloroben-
Compounds 15 where R4ꢂBr were successfully converted zoic acid moiety without significant loss of activity. In addi-
to the alkyl-, aralkyl- or aryl-substituted anthranilic acid ester tion, it was to be noted that compound 13 possessing an-
derivatives (17) under Suzuki–Miyaura or Negishi cross-cou- thranilic acid structure with an anilide moiety retained PAI-1
pling reaction conditions.11,12) The target N-acylanthranilic inhibitory property comparable to 8aꢀ, despite the facts that
acid derivatives (16a—q, 18a—d, Table 2) were obtained no thiophene ring was incorporated in the molecule of 13,
from these esters (15, 17) by alkaline hydrolysis in good and there was a considerable difference between the ClogP
yields.
values of the two compounds 8aꢀ and 13. While appropriate
The N-methyl derivative of 16f was also prepared to inves- lipophilic or/and bulky substituents may be necessary for
tigate the effect of the N-hydrogen atom at this position on these compounds to elicit potent in vitro effects, as reported
the PAI-1 inhibitory activity. Methylation of 15f with methyl previously,8) ClogP value of the whole molecule does not
iodide in the presence of sodium hydride followed by hydrol- seem to affect much the activity. However, it was considered
ysis furnished 20.
worthwhile to decrease ClogP to improve oral PK profile of
this series.
Results and Discussion
Based upon these observations and concepts, our efforts
PAI-1 inhibitory activities of newly synthesized com- were then focused, as shown in Table 2, on the syntheses of
pounds are shown in Tables 1 and 2 together with those of compounds possessing a substituted-anthranilic acid struc-
previously reported compounds (TM5001, 1, 2). The in vitro ture as A part illustrated in Fig. 2, with a variety of hy-
PAI-1 activity is expressed as the remaining activity in per- drophobic amide moieties R5 on the other end (B part) of the
cent (%) after incubation of PAI-1 with test compounds as molecule and with different linkers L, taking ClogP of the
described in the experimental section. Method A and Method molecule in consideration as well.
B differ with the relative molar ratio of PAI-1 and tPA added
Next we focused on the structure–activity relationships
to the medium (Method A; tPA : PAI-1ꢂ1 : 6.9, Method B; (SAR) of the B part (Fig. 2, Table 2). While substituents such
Table 1. PAI-1 Inhibitory Activity of Anthranilic Acid Derivatives
PAI-1 activity (%)
(method A)b)
Rat PK
(50 mg/kg, p.o.)
Compounds
R4
R5
ClogPa)
100 mM
50 mM
Cmax (mM)
Tmax (h)
T1/2 (h)
8ac)
Cl
H
7.48d)
6.65d)
12.5ꢃ7.6
12.1ꢃ5.3
42.5ꢃ5.4
ND
ND
ND
8aꢀc)
97.8ꢃ1.2
29.8ꢃ9.7
2
9
13
Cl
5.16
5.79
37.7ꢃ11.3
15.9ꢃ8.3
99.9ꢃ0.1
51.4ꢃ5.9
ND
ND
18
ND
54
TM5001
31.9ꢃ9.8
1
5.32
7.06
7.9ꢃ3.9e)
15.5ꢃ3.6
12.7ꢃ4.0
5.8ꢃ1.2
18
48
ꢀ174
ꢀ48
2 f )
ND
2.32g)
Data are expressed as meanꢃS.D., ND: no data, a) calculated by ChemDraw 10.0, b) see Experimental, c) evaluated as sodium salt, d) calculated for the free carboxilic acid,
e) tPA is slightly inhibited in the test, f ) tested by method B, g) data of Nꢂ1.

218
Vol. 59, No. 2
Table 2. PAI-1 Inhibitory Activity and Rat PK of Anthranilic Acid Derivatives
PAI-1 activity (%)
(method B)b)
Rat PK
(50 mg/kg, p.o.)
R4 or R6
(position)
Compounds
R5
R7
H
X
O
ClogPa)
4.29
50 mM
20 mM
Cmax (mM)
Tmax (h)
T1/2 (h)
16ac)
5-Cl
53.7ꢃ7.7
100.0ꢃ0.5
ND
ND
ND
16bd)
16c
5-Cl
5-Cl
H
H
O
O
4.50
4.69
77.8ꢃ7.8
47.5ꢃ7.1
99.8ꢃ0.3
98.4ꢃ2.0
ND
ND
1.0
ND
1.0
89.2ꢃ17.0
16d
5-Cl
5-Cl
5-Cl
H
H
H
O
O
O
4.22
5.77
3.37
89.1ꢃ8.7
7.6ꢃ2.8
6.0ꢃ1.4
98.8ꢃ1.0
81.0ꢃ13.7
79.0ꢃ8.4
ND
11.6e)
ND
2.0
2.0
ND
2.6
2.5
16ed)
16fd, f )
34.2ꢃ2.6
16g
5-Cl
5-Cl
5-F
H
H
H
H
O
O
O
O
3.66
4.27
2.80
3.52
15.5ꢃ0.4
26.8ꢃ7.1
50.4ꢃ2.3
12.2ꢃ6.2
98.8ꢃ1.8
86.1ꢃ5.4
92.3ꢃ5.7
88.9ꢃ0.7
ND
ND
ND
ND
ND
2.0
ND
ND
ND
7.0
16h
16i
ND
16jc,d)
5-Br
22.5ꢃ4.0
16kd)
16lc)
4-Br
5-Ph
H
H
O
O
3.52
4.43
10.2ꢃ3.5
16.8ꢃ2.7
79.8ꢃ11.7
83.8ꢃ5.2
ND
ND
1.0
ND
1.5
4.5ꢃ0.4
16md)
16n
4-Cl
5-Cl
5-Cl
5-Cl
5-Cl
H
H
H
H
H
O
CH2
S
3.37
4.07
3.75
3.93
6.18
13.3ꢃ2.5
16.4ꢃ4.5
15.9ꢃ3.8
29.2ꢃ3.8
6.5ꢃ4.6
91.3ꢃ9.7
85.1ꢃ8.7
71.2ꢃ13.0
92.8ꢃ7.0
35.1ꢃ4.2
8.3ꢃ2.0
69.7ꢃ23.1
97.4ꢃ14.5
49.4ꢃ15.8
1.8ꢃ0.4
1.0
2.0
1.0
2.0
1.0
0.8
2.4
2.3
1.5
1.6
16og)
16pc)
16qg)
MeN
18ad)
5-i-Pr
H
O
3.97
9.4ꢃ1.7
91.6ꢃ10.6
15.4ꢃ4.4
2.0
1.6
18bd)
18c
5-Bn
5-
H
H
O
O
4.61
3.06
9.5ꢃ3.2
98.9ꢃ1.4
86.1ꢃ0.2
2.5ꢃ0.8
2.9ꢃ0.2
1.0
1.0
0.5
1.3
11.8ꢃ1.6
18d
20
4-
H
O
O
3.06
4.08
9.6ꢃ4.0
48.6ꢃ14.2
27.3ꢃ0.5h)
0.2ꢃ0.1
1.0
0.9
5-Cl
Me
ND
ND
ND
Data are expressed as meanꢃS.D., ND: no data, a) ClogP was calculated for free carboxylic acid/amine by ChemDraw 10.0, b) see Experimental, c) tested by method A, d)
evaluated as sodium salt, e) data of Nꢂ1, f ) PAI-1 activities (%) of 16f tested by method A are 23.3ꢃ3.2 and 89.6ꢃ6.5 at 50 mM and 20 mM, respectively, g) evaluated as hy-
drochloride salt, h) PAI-1 activity (%) of 150 mM is shown.
as diphenylamino (16b), 1,1-diphenylmethylamino (16c), ety (A part) were evaluated with compounds 16i—18d. Al-
and 4-phenyl-1-piperidinyl group (16d) were not satisfactory though introduction of a fluorine atom to the 5-position
to potentiate the activity, compounds with 4,4-diphenyl- resulted in a slight decrease in activity, compounds with
1-piperidinyl (16e) and 4-(diphenylmethyl)-1-piperazinyl bulkier or more hydrophobic substituents such as bromine
groups (16f) showed activities superior to 16a. Although atom (16j), isopropyl (18a) and benzyl (18b) groups tended
there was little difference between 16e and 16f in in vitro ac- to retain the activity. 4-Bromo, 5-(3-pyridyl) and 4-(4-
tivity, we decided to concentrate in 16f type of compounds, pyridyl) derivatives (16k, 18c, 18d) also exhibited potent in-
because 16f showed a smaller ClogP and a better PK profile hibitory activity. The difference in the position of these sub-
than 16e, and also diphenylmethylpiperazinyl derivatives stituents (4- or 5-position) did not seem to significantly affect
could be prepared relatively easily compared to diphenyl- the potency of in vitro activity, suggesting that steric bulki-
piperidinyl ones. Introduction of N-bis(4-fluorophenyl)- ness adjacent to the phenyl ring appears to be more important
methyl (16g) and N-9H-fluorenyl (16h) substituents in piper- than the position of the substitution as has been observed
azine moiety also resulted in a slight decrease in the in vitro with the thiophene-carboxylic acid series.8) Variation of the
activity compared to 16f. Therefore, we fixed the B part (Fig. linker L by introducing a methylene group, sulfur and nitro-
2) as the 4-diphenylmethyl-1-piperazinyl group to conduct gen atoms as X (Fig. 2) as in 16n—16q (Table 2), indicated
further SAR studies.
The positions of the substituents on the benzoic acid moi-
that the activity observed with 16f was retained or improved.
N-Methylation of 16f, i.e., compound 20, resulted in a

February 2011
219
(THF) (100 ml) was added 1 N aqueous NaOH solution (104 ml) and the
mixture was stirred for 2.5 h at 60 °C. After evaporation of organic solvent,
the aqueous solution was washed with ethyl acetate (EtOAc). After 1 N aque-
ous HCl was added to the aqueous solution, the mixture was extracted with
EtOAc three times. The combined organic layers were washed with brine,
dried (Na2SO4), and concentrated in vacuo. The residue was purified by re-
crystallization from EtOAc/n-hexane to afford 20.2 g (yield 72%) of 5 as a
solid, H-NMR (CDCl3) d: 1.19 (9H, s), 1.66—1.94 (4H, m), 2.43 (2H, t,
Jꢂ7.1 Hz), 2.56 (2H, t, Jꢂ7.1 Hz), 6.55 (1H, s), 7.19—7.40 (5H, m), 11.3
(1H, s).
tert-Butyl 2-[(6-{[4-Chloro-2-(methoxycarbonyl)phenyl]amino}-6-oxo-
hexanoyl)amino]-4-phenylthiophene-3-carboxylate (7a) To a mixture of
5 (1.0 g, 2.47 mmol) and triethylamine (Et3N) (326 mg, 3.22 mmol) in THF
(19 ml) was added isobutyl chloroformate (406 mg, 2.97 mmol) at 0 °C and
the mixture was stirred for 1 h. A solution of methyl 2-amino-5-chloroben-
zoate (6a) (460 mg, 2.47 mmol) in THF (1 ml) was added to the reaction
mixture at 0 °C. The mixture was stirred at room temperature for 16 h. Water
drastic loss in activity indicating that the NH group at this
position seems to play a very important role in the N-acylan-
thranilic acid derivatives to elicit potent PAI-1 inhibitory ac-
tivity (Table 2).
In order to identify a good orally-bioavailable compound
for further in vivo studies, pharmacokinetic (PK) profiles of
the representative compounds were compared in rats by oral
administration (Table 2; see Experimental in detail). Al-
though no direct correlation was seen between ClogP and
PK, compounds showing good PK had ClogPꢄ5.0 as shown
in Table 2. Compounds 18c and 18d with a pyridyl group at
the 4- or 5-position had a low ClogP (3.06) but exhibited
only poor PK profiles, while the reason is not known at pres-
ent. From these results compound 16f (TM5275)10) possess-
1
ing both potent in vitro PAI-1 inhibitory activity and a good was added to the reaction mixture and the whole mixture was extracted with
EtOAc three times. The combined organic layers were washed with brine,
dried (Na2SO4) and concentrated in vacuo. The residue was purified by col-
PK profile, e.g., high maximum plasma concentration (Cmax),
was selected as a candidate for future development.
umn chromatography on silica gel (EtOAc/n-hexane 1/6) to afford 650 mg
1
(yield 46%) of 7a as a solid, H-NMR (CDCl3) d: 1.19 (9H, s), 1.81—1.93
Conclusion
(4H, m), 2.46—2.64 (4H, m), 3.94 (3H, s), 6.54 (1H, d, Jꢂ0.9 Hz), 7.20—
7.39 (5H, m), 7.48 (1H, dd, Jꢂ9.2, 2.6 Hz), 7.99 (1H, d, Jꢂ2.6 Hz), 8.72
(1H, d, Jꢂ9.2 Hz), 11.0 (1H, s), 11.3 (1H, s).
Based upon the basic structure of 2-acylamino-3-thio-
phenecarboxylic acids (TM5001, 1, 2) which we have previ-
ously reported,7,8) novel N-acylanthranilic acid derivatives, in
particular N-acyl-5-chloroanthranilic acid derivatives were
designed, synthesized and evaluated for in vitro PAI-1 in-
hibitory activity and oral PK profiles in rats. It was found
that both PAI-1 inhibition and oral bioavailability were much
improved when a 4-diphenylmethyl-1-piperazinyl group was
introduced on the acyl side chain. Thus compound 16f
(TM5275)10) was finally identified as a promising candidate
for further pharmacological evaluation.
tert-Butyl 2-[(6-{[2-(Methoxycarbonyl)phenyl]amino}-6-oxohexanoyl)-
amino]-4-phenylthiophene-3-carboxylate (7aꢀ) To a mixture of 5 (1.0 g,
2.47 mmol) and Et3N (326 mg, 3.22 mmol) in THF (9 ml) was added
methanesulfonyl chloride (369 mg, 322 mmol) at 0 °C and the mixture was
stirred for 1 h. A solution of methyl anthranilate (6aꢀ) (487 mg, 3.22 mmol)
in THF (1 ml) was added to the reaction mixture at 0 °C. The mixture was
stirred at room temperature for 16 h. Water was added to the reaction mix-
ture and the whole mixture was extracted with EtOAc three times. The com-
bined organic layers were washed with brine, dried (Na2SO4) and concen-
trated in vacuo. The residue was purified by column chromatography on sil-
ica gel (acetone/n-hexane 1/5) to afford 730 mg (yield 55%) of 7aꢀ as a
solid, 1H-NMR (CDCl3) d: 1.19 (9H, s), 1.80—1.94 (4H, m), 2.43—2.66
(4H, m), 3.93 (3H, s), 6.54 (1H, s), 7.03—7.13 (1H, m), 7.20—7.40 (5H,
m), 7.49—7.58 (1H, m), 8.00—8.06 (1H, m), 8.69—8.76 (1H, m), 11.1 (1H,
s), 11.3 (1H, s).
TM5275 may provide not only a useful tool for a better
understanding of the pathophysiology of PAI-1 but also a
potential new treatment for diseases where PAI-1 has been
implicated.
Sodium 2-[(6-{[3-(tert-Butoxycarbonyl)-4-phenylthiophen-2-yl]amino}-
6-oxohexanoyl)amino]-5-chlorobenzoate (8a) To a solution of 7a (650
mg, 1.13 mmol) in THF (10 ml) was added 1 N aqueous NaOH solution
(2.3 ml) and the mixture was stirred for 2 h at 60 °C. After cooling, the reac-
Experimental
General Melting points (mp) were determined on METTLER TOLEDO
MP70 Melting Point Systems and are uncorrected. 1H-NMR spectra were tion mixture was evaporated in vacuo to remove THF and water was added.
recorded on a Varian Gemini-200 (200 MHz) spectrometer, with tetram-
ethylsilane as an internal standard. TLC analyses were carried out on
MERCK Silica gel 60 F254 plates. Elemental analyses were carried out by
Takeda Analytical Laboratories, Ltd., and are within ꢃ0.4% of the theoreti-
cal values unless otherwise noted. Chromatographic purification was carried
out on silica gel columns (MERCK Silica gel 60 0.040—0.063 mm) unless
otherwise noted. The yields reported are not optimized. Analytical HPLC
was conducted on a Puresil C18 column (5 mm, 4.4ꢅ150 mm i.d.) eluted with
0.1% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.08% (v/v)
TFA in acetonitrile (MeCN)/water (80%/20% (v/v)) (solvent B), according
to the following elution gradient: 0—20 min, 50—80% B; 20—45 min, 80%
B at a flow rate of 0.8 ml/min. LC/MS spectra were recorded on an Alliance
HPLC system using a ULTORON VX-ODS C18 column (5 mm, 4.6ꢅ150
mm i.d.) coupled with an micromass ZQ as MS detector in the mode of elec-
trospray ionization (ESI) and a gradient of 5—100% B over 20 min was used
with a flow rate of 1.0 ml/min. Formic acid 0.1% was added to solvents A
and B (water (A) and MeCN (B)).
The precipitate was collected by filtration, washed with water and diiso-
propyl ether (IPE), and dried in vacuo to afford 507 mg (yield 77%) of 8a as
1
an amorphous solid, H-NMR (DMSO-d6) d: 1.17 (9H, s), 1.60—1.75 (4H,
m), 2.25—2.40 (2H, m), 2.51—2.64 (2H, m), 6.83 (1H, s), 7.20—7.44 (6H,
m), 7.92 (1H, d, Jꢂ2.7 Hz), 8.49 (1H, d, Jꢂ8.8 Hz), 11.0 (1H, s), 14.3 (1H,
s), HPLC (250 nm) 99.4% (tRꢂ42.0 min), LC/MS (ESI) m/z: 557 (MꢆH)ꢆ,
555 (MꢁH)ꢁ.
Sodium 2-[(6-{[3-(tert-Butoxycarbonyl)-4-phenylthiophen-2-yl]amino}-
6-oxohexanoyl)amino]benzoate (8aꢀ) This compound was prepared in
33% yield as an amorphous solid from 7aꢀ and 1 N aqueous NaOH solution
following a procedure similar to that described for the preparation of 8a, 1H-
NMR (DMSO-d6) d: 1.17 (9H, s), 1.53—1.80 (4H, m), 2.25—2.40 (2H, m),
2.55—2.67 (2H, m), 6.84 (1H, s), 6.86—6.96 (1H, m), 7.17—7.43 (6H, m),
7.94—8.00 (1H, m), 8.42—8.50 (1H, m), 11.0 (1H, s), 14.3 (1H, s), HPLC
(250 nm) 98.7% (tRꢂ32.1 min), LC/MS (ESI) m/z: 523 (MꢆH)ꢆ, 521
(MꢁH)ꢁ.
Methyl 6-[(4-Chlorophenyl)amino]-6-oxohexanoate (10) Methyl
adipoyl chloride (8.6 g, 48 mmol) was added to a solution of 4-chloroaniline
(9) (6.7 g, 53 mmol) in DMA at 0 °C, and the mixture was stirred at room
temperature for 17 h. Aqueous NaHCO3 solution was added to the reaction
mixture and the resulting precipitate was collected by filtration, washed with
water and IPE, and dried in vacuo to afford 11.0 g (yield 85%) of 10 as a
solid, 1H-NMR (DMSO-d6) d: 1.51—1.59 (4H, m), 2.28—2.37 (4H, m),
3.58 (3H, s), 7.30—7.38 (2H, m), 7.58—7.65 (2H, m), 10.0 (1H, s).
6-[(4-Chlorophenyl)amino]-6-oxohexanoic Acid (11) To a solution of
10 (12.0 g, 44 mmol) in THF (445 ml) was added 1 N aqueous NaOH solu-
tion (61.2 ml) and the mixture was stirred for 2 h at 60 °C. After cooling,
THF was removed in vacuo and 1 N aqueous HCl solution was added to the
residue. The precipitate was collected by filtration, washed with EtOAc,
tert-Butyl 2-[(6-Methoxy-6-oxohexanoyl)amino]-4-phenylthiophene-3-
carboxylate (4) Methyl adipoyl chloride (15.8 g, 88.2 mmol) was added
to a solution of tert-butyl 2-amino-4-phenylthiophene-3-carboxylate (3)
(24.3 g, 88.2 mmol) in dimethylacetamide (DMA) (70 ml) at 0 °C, and the
mixture was stirred at room temperature for 1 h. Aqueous NaHCO3 solution
was added to the reaction mixture and the resulting precipitate was collected
by suction filtration, washed with water and n-hexane, and dried in vacuo to
1
afford 30.3 g (yield 82%) of 4 as a solid, H-NMR (CDCl3) d: 1.19 (9H, s),
1.64—1.92 (4H, m), 2.39 (2H, t, Jꢂ7.1 Hz), 2.55 (2H, t, Jꢂ7.1 Hz), 3.68
(3H, s), 6.55 (1H, d, Jꢂ0.7 Hz), 7.21—7.39 (5H, m), 11.3 (1H, s).
6-{[3-(tert-Butoxycarbonyl)-4-phenylthiophen-2-yl]amino}-6-oxohexa-
noic Acid (5) To a solution of 4 (29.0 g, 69.4 mmol) in tetrahydrofuran

220
Vol. 59, No. 2
water and IPE, and dried in vacuo to afford 10.3 g (yield 86%) of 11 as a
the preparation of 14a, 1H-NMR (DMSO-d6) d: 1.82 (2H, q, Jꢂ7.3 Hz),
solid, 1H-NMR (DMSO-d6) d: 1.50—1.67 (4H, m), 2.21—2.35 (4H, m), 2.30 (2H, t, Jꢂ7.3 Hz), 2.42 (2H, t, Jꢂ7.3 Hz), 3.85 (3H, s), 7.66 (1H, dd,
7.30—7.38 (2H, m), 7.59—7.66 (2H, m), 10.0 (1H, s), 12.0 (1H, bs).
Methyl 5-Chloro-2-({6-[(4-chlorophenyl)amino]-6-oxohexanoyl}-
amino)benzoate (12) Thionyl chloride (2.2 ml, 30 mmol) was added to 11
(770 mg, 3.0 mmol) at 0 °C, and the mixture was stirred at room temperature
Jꢂ9.0, 2.6 Hz), 7.83 (1H, d, Jꢂ2.6 Hz), 8.18 (1H, d, Jꢂ9.0 Hz), 10.5 (1H,
s), 12.1 (1H, br).
[(2-{[4-Chloro-2-(methoxycarbonyl)phenyl]amino}-2-oxoethyl)sul-
fanyl]acetic Acid (14h) This compound was prepared in 84% yield as a
for 1 h. The solvent was removed in vacuo. A solution of 6a (610 mg, solid from 6a and 1,4-oxathiane-2,6-dione following a procedure similar to
3.3 mmol) in DMA (10 ml) was add to the acid chloride at room temperature
for 17 h. Aqueous NaHCO3 solution was added to the reaction mixture and
the whole mixture was extracted with EtOAc three times. The combined or-
ganic layers were washed with brine, dried (Na2SO4) and concentrated in
vacuo. IPE was added to the residue and the resulting crystal was collected
that described for the preparation of 14a, 1H-NMR (DMSO-d6) d: 3.38 (2H,
s), 3.56 (2H, s), 3.88 (3H, s), 7.68 (1H, dd, Jꢂ9.0, 2.7 Hz), 7.87 (1H, d,
Jꢂ2.7 Hz), 8.33 (1H, d, Jꢂ9.0 Hz), 11.1 (1H, s), 12.7 (1H, br).
[(2-{[4-Chloro-2-(methoxycarbonyl)phenyl]amino}-2-oxoethyl)-
(methyl)amino]acetic Acid (14i) A mixture of N-methyliminodiacetic
acid (3.2 g, 21.5 mmol) and acetic anhydride (16 ml) was refluxed for 0.5 h.
by filtration, and dried in vacuo to afford 610 mg (yield 48%) of 12 as a
1
solid, H-NMR (CDCl3) d: 1.80—1.87 (4H, m), 2.39—2.56 (4H, m), 3.93 After evaporation of acetic anhydride, a solution of 6a (4.0 g, 21.6 mmol) in
(3H, s), 7.23—7.30 (2H, m), 7.47—7.54 (3H, m), 7.69 (1H, s), 8.00 (1H, d, THF (20 ml) was added to this residue and the mixture was refluxed for
Jꢂ2.7 Hz), 8.70 (1H, d, Jꢂ9.2 Hz), 11.0 (1H, s).
1.5 h. After evaporation of organic solvent, the residue was purified by col-
umn chromatography on silica gel (CHCl3/methanol (MeOH)ꢂ5/1) to afford
5.0 g (yield 73%) of 14i as a solid, 1H-NMR (DMSO-d6) d: 2.46 (3H, s),
3.38 (2H, s), 3.46 (2H, s), 3.89 (3H, s), 7.68 (1H, dd, Jꢂ9.0, 2.6 Hz), 7.91
(1H, d, Jꢂ2.4 Hz), 8.64 (1H, d, Jꢂ9.0 Hz), 11.6 (1H, s).
5-Chloro-2-({6-[(4-chlorophenyl)amino]-6-oxohexanoyl}amino)benzoic
Acid (13) To a solution of 12 (570 mg, 1.3 mmol) in THF (13 ml) was
added 1 N aqueous NaOH solution (2.0 ml) and the mixture was stirred for
3 h at 60 °C. After cooling, THF was removed in vacuo and 1 N aqueous HCl
solution was added to the residue. The precipitate was collected by filtration,
[1-(2-{[4-Chloro-2-(methoxycarbonyl)phenyl]amino}-2-oxoethyl)cy-
washed with water and IPE, and dried in vacuo to afford 490 mg (yield 91%) clohexyl]acetic Acid (14j) To a solution of 6a (9.3 g, 49.9 mmol) in THF
of 13 as a solid, mp 232—235 °C, 1H-NMR (DMSO-d6) d: 1.53—1.72 (4H, (60 ml) was added 3-oxaspiro[5.5]undecane-2,4-dione (10.0 g, 49.9 mmol)
m), 2.27—2.38 (4H, m), 7.27—7.40 (2H, m), 7.53—7.67 (2H, m), 7.60 (1H, and the mixture was refluxed for 35 h. After cooling, solvent was removed in
dd, Jꢂ9.0, 2.7 Hz), 7.91, (1H, d, Jꢂ2.7 Hz), 8.50 (1H, d, Jꢂ9.0 Hz), 10.1
vacuo to give a crude product which was recrystallized from EtOAc to af-
(1H, s), 11.5 (1H, s), HPLC (250 nm) 99.4% (tRꢂ16.1 min), LC/MS (ESI) ford 10.9 g (yield 59%) of 14j as a solid, 1H-NMR (DMSO-d6) d: 1.30—
m/z: 409 (MꢆH)ꢆ, 407 (MꢁH)ꢁ.
1.40 (10H, m), 2.43 (2H, s), 2.56 (2H, s), 3.85 (3H, s), 7.65 (1H, dd, Jꢂ9.0,
2.6 Hz), 7.83 (1H, d, Jꢂ2.6 Hz), 8.23 (1H, d, Jꢂ9.0 Hz), 10.5 (1H, s), 12.1
(1H, br).
Methyl 5-Chloro-2-[({2-[(4-chlorophenyl)amino]-2-oxoethoxy}acetyl)-
amino]benzoate (15a) To a mixture of 14a (500 mg 1.66 mmol) and N,N-
dimethylformamide (DMF) (cat. amount) in THF (5 ml) was added oxalyl
dichloride (414 mg,1.99 mmol) at 0 °C, and the mixture was stirred at room
(2-{[4-Chloro-2-(methoxycarbonyl)phenyl]amino}-2-oxoethoxy)acetic
Acid (14a) To a solution of 6a (10 g, 53.9 mmol) in THF (135 ml) was
added diglycolic anhydride (6.9 g, 59.3 mmol) and the mixture was refluxed
for 7.5 h. After cooling, solvent was removed in vacuo and IPE was added to
the residue. The resulting precipitate was collected by filtration, washed with
1
IPE, and dried in vacuo to afford 15.7 g (yield 97%) of 14a as a solid, H-
NMR (DMSO-d6) d: 3.90 (3H, s), 4.22 (2H, s), 4.28 (2H, s), 7.71 (1H, dd, temperature for 1 h. After evaporation of organic solvent, a solution of 4-
Jꢂ9.0, 2.7 Hz), 7.93 (1H, d, Jꢂ2.7 Hz), 8.62 (1H, d, Jꢂ8.8 Hz), 11.4 (1H, s)
12.9 (1H, br).
(2-{[4-Fluoro-2-(methoxycarbonyl)phenyl]amino}-2-oxoethoxy)acetic
Acid (14b) This compound was prepared in 97% yield as a solid from
methyl 2-amino-5-fluorobenzoate (6b) and diglycolic anhydride following a
chloroaniline (211 mg, 1.66 mmol) in DMA (5 ml) was added to the residue
at 0 °C, and the mixture was stirred at room temperature for 1 h. Aqueous
NaHCO3 solution was added to the reaction mixture and the resulting pre-
cipitate was collected by suction filtration and washed with water to afford
345 mg (yield 51%) of 15a as a solid, 1H-NMR (CDCl3) d: 3.85 (3H, s),
procedure similar to that described for the preparation of 14a, 1H-NMR 4.26 (2H, s), 4.27 (2H, s), 7.26—7.35 (2H, m), 7.55 (1H, dd, Jꢂ9.2, 2.6 Hz),
(DMSO-d6) d: 3.89 (3H, s), 4.20 (2H, s), 4.27 (2H, s), 7.55 (1H, dddd, 7.65—7.75 (2H, m), 8.05 (1H, d, Jꢂ2.6 Hz), 8.78 (1H, d, Jꢂ9.2 Hz), 8.83
Jꢂ14.6, 5.1, 3.1, 1.4 Hz), 7.73 (1H, dd, Jꢂ9.3, 3.1 Hz), 8.60 (1H, dddd, (1H, s), 11.9 (1H, s).
Jꢂ14.6, 9.3, 5.1, 3.1 Hz), 11.3 (1H, s).
Methyl 5-Chloro-2-({[2-(diphenylamino)-2-oxoethoxy]acetyl}amino)-
benzoate (15b) To a mixture of 14a (1.0 g, 3.31 mmol) and DMF (cat.
amount) in THF (10 ml) was added oxalyl dichloride (505 mg, 3.98 mmol) at
0 °C, and the mixture was stirred at room temperature for 1 h. After evapora-
tion of organic solvent, a solution of diphenylamine (617 mg, 3.65 mmol) in
DMA (10 ml) was added to the residue at 0 °C, and the mixture was stirred
(2-{[4-Bromo-2-(methoxycarbonyl)phenyl]amino}-2-oxoethoxy)acetic
Acid (14c) This compound was prepared in 92% yield as a solid from
methyl 2-amino-5-bromobenzoate (6c) and diglycolic anhydride following a
procedure similar to that described for the preparation of 14a, 1H-NMR
(DMSO-d6) d: 3.89 (3H, s), 4.20 (2H, s), 4.26 (2H, s), 7.85 (1H, dd, Jꢂ9.0,
3.0 Hz), 8.08 (1H, d, Jꢂ3.0 Hz), 8.57 (1H, d, Jꢂ9.0 Hz), 11.4 (1H, s), 12.9 at room temperature for 1.5 h. Aqueous NaHCO3 solution was added to the
(1H, br).
reaction mixture and the solution was extracted with EtOAc three times. The
(2-{[3-(Methoxycarbonyl)biphenyl-4-yl]amino}-2-oxoethoxy)acetic combined organic layers were washed with brine, dried (Na2SO4) and con-
Acid (14d) This compound was prepared in 89% yield as a solid from
centrated in vacuo. The residue was purified by column chromatography on
methyl 4-aminobiphenyl-3-carboxylate (6d)13) and diglycolic anhydride fol-
silica gel (EtOAc/n-hexaneꢂ1/1) to afford 717 mg (yield 48%) of 15b as a
1
1
lowing a procedure similar to that described for the preparation of 14a, H- solid, H-NMR (CDCl3) d: 3.92 (3H, s), 4.22 (2H, s), 4.25 (2H, s), 7.24—
NMR (DMSO-d6) d: 3.93 (3H, s), 4.23 (2H, s), 4.29 (2H, s), 7.35—7.71 7.48 (10H, m), 7.48 (1H, dd, Jꢂ9.0, 2.6 Hz), 8.00 (1H, d, Jꢂ2.6 Hz), 8.72
(5H, m), 7.98 (1H, dd, Jꢂ8.8, 2.4 Hz), 8.24 (1H, d, Jꢂ2.4 Hz), 8.71 (1H, d, (1H, d, Jꢂ9.0 Hz), 11.6 (1H, s).
Jꢂ8.8 Hz), 11.5 (1H, s), 12.9 (1H, br).
Methyl 5-Chloro-2-[({2-[(diphenylmethyl)amino]-2-oxoethoxy}acetyl)-
amino]benzoate (15c) To a mixture of 14a (0.81 g, 2.68 mmol) and DMF
(cat. amount) in THF (15 ml) was added oxalyl dichloride (411 mg, 3.24
mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h.
(2-{[5-Bromo-2-(methoxycarbonyl)phenyl]amino}-2-oxoethoxy)acetic
Acid (14e) This compound was prepared in 89% yield as a solid from
methyl 2-amino-4-bromobenzoate (6e) and diglycolic anhydride following a
procedure similar to that described for the preparation of 14a, 1H-NMR After evaporation of organic solvent, a solution of benzhydrylamine (594
(DMSO-d6) d: 3.89 (3H, s), 4.22 (2H, s), 4.28 (2H, s), 7.42 (1H, dd, Jꢂ8.6, mg, 3.24 mmol) in DMA (15 ml) was added to the residue at 0 °C, and the
2.2 Hz), 7.92 (1H, d, Jꢂ8.6 Hz), 8.86 (1H, d, Jꢂ2.2 Hz), 11.5 (1H, s), 12.9 mixture was stirred at room temperature for 14 h. Aqueous NaHCO3 solu-
(1H, br).
(2-{[5-Chloro-2-(methoxycarbonyl)phenyl]amino}-2-oxoethoxy)acetic
Acid (14f) This compound was prepared in 94% yield as a solid from
methyl 2-amino-4-chlorobenzoate (6f) and diglycolic anhydride following a
procedure similar to that described for the preparation of 14a, 1H-NMR
tion was added to the reaction mixture and the solution was extracted with
EtOAc three times. The combined organic layers were washed with brine,
dried (Na2SO4) and concentrated in vacuo. IPE was added to the residue and
the resulting crystal was collected by filtration, and dried in vacuo to afford
610 mg (yield 49%) of 15c as a solid, 1H-NMR (CDCl3) d: 3.32 (3H, s),
(DMSO-d6) d: 3.89 (3H, s), 4.23 (2H, s), 4.28 (2H, s), 7.29 (1H, dd, Jꢂ8.6, 4.21 (2H, s), 4.25 (2H, s), 6.48 (1H, d, Jꢂ9.2 Hz), 7.25—7.36 (10H, m),
2.0 Hz), 8.00 (1H, d, Jꢂ8.6 Hz), 8.71 (1H, d, Jꢂ2.0 Hz), 11.5 (1H, s), 12.9 7.52 (1H, dd, Jꢂ9.0, 2.6 Hz), 7.90 (1H, d, Jꢂ9.2 Hz), 7.98 (1H, d,
(1H, br).
5-{[4-Chloro-2-(methoxycarbonyl)phenyl]amino}-5-oxopentanoic
Jꢂ2.6 Hz), 8.76 (1H, d, Jꢂ9.0 Hz), 11.9 (1H, s).
Methyl 5-Chloro-2-({[2-oxo-2-(4-phenylpiperidin-1-yl)ethoxy]acetyl}-
Acid (14g) This compound was prepared in 61% yield as a solid from 6a amino)benzoate (15d) This compound was prepared in 85% yield as a
and glutaric anhydride following a procedure similar to that described for solid from 14a, oxalyl dichloride, DMF and 4-phenylpiperidine following a

February 2011
221
procedure similar to that described for the preparation of 15b, 1H-NMR amino]biphenyl-3-carboxylate (15l) This compound was prepared in
(CDCl3) d: 1.50—1.80 (2H, m), 1.80—2.00 (2H, m), 2.60—2.90 (2H, m), 61% yield as a solid from 14d, oxalyl dichloride, DMF and 1-benzhydryl-
3.10—3.30 (1H, m), 3.85—3.05 (1H, m), 3.92 (3H, s), 4.28 (2H, s), 4.44 piperazine following a procedure similar to that described for the prepara-
1
(2H, d, Jꢂ2.0 Hz), 4.70—4.85 (1H, m), 7.15—7.40 (5H, m), 7.45 (1H, dd, tion of 15b, H-NMR (CDCl3) d: 2.38—2.44 (4H, m), 3.54—3.64 (4H, m),
Jꢂ9.1, 2.7 Hz), 8.02 (1H, d, Jꢂ2.7 Hz), 8.77 (1H, d, Jꢂ9.1 Hz), 11.7 (1H, 3.83 (3H, s), 4.22 (1H, s), 4.24 (2H, s), 4.37 (2H, s), 7.14—7.62 (15H, m),
br).
Methyl 5-Chloro-2-({[2-(4,4-diphenylpiperidin-1-yl)-2-oxoethoxy]-
7.79 (1H, dd, Jꢂ8.7, 2.2 Hz), 8.27 (1H, d, Jꢂ2.2 Hz), 8.83 (1H, d,
Jꢂ8.7 Hz), 11.7 (1H, s).
acetyl}amino)benzoate (15e) To a mixture of 14a (1.0 g, 3.31 mmol) and
DMF (cat. amount) in THF (20 ml) was added oxalyl dichloride (505 mg,
3.98 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h.
After evaporation of organic solvent, a mixture of 4,4-diphenylpiperidine
hydrochloride (998 mg, 3.65 mg) and Et3N (369 mg, 3.65 mmol) in DMA
(20 ml) was added to the residue at 0 °C, and the mixture was stirred at room
temperature for 1.5 h. Aqueous NaHCO3 solution was added to the reaction
mixture and the solution was extracted with EtOAc three times. The com-
Methyl 4-Chloro-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (15m) This compound was prepared in
96% yield as a solid from 14f, WSC, HOBt and 1-benzhydrylpiperazine fol-
1
lowing a procedure similar to that described for the preparation of 15k, H-
NMR (CDCl3) d: 2.39—2.48 (4H, m), 3.52—3.65 (4H, m), 3.80 (3H, s),
4.22 (2H, s), 4.22 (1H, s), 4.34 (2H, s), 7.08 (1H, dd, Jꢂ8.6, 2.0 Hz), 7.14—
7.42 (10H, m), 7.95 (1H, d, Jꢂ8.6 Hz), 8.86 (1H, d, Jꢂ2.0 Hz), 11.8 (1H, s).
Methyl 5-Chloro-2-({5-[4-(diphenylmethyl)piperazin-1-yl]-5-oxopen-
bined organic layers were washed with brine, dried (Na2SO4) and concen- tanoyl}amino)benzoate (15n) This compound was prepared in 88% yield
trated in vacuo. The residue was purified by column chromatography on sil-
as a solid from 14g, WSC, HOBt and 1-benzhydrylpiperazine following a
ica gel (EtOAc/n-hexaneꢂ1/1) to afford 850 mg (yield 49%) of 15e as a
procedure similar to that described for the preparation of 15k, 1H-NMR
1
solid, H-NMR (CDCl3) d: 2.40—2.52 (4H, m), 3.46—3.77 (4H, m), 3.81 (CDCl3) d: 2.06 (2H, q, Jꢂ7.1 Hz), 2.34—2.56 (8H, m), 3.45—3.64 (4H,
(3H, s), 4.23 (2H, s), 4.38 (2H, s), 7.10—7.37 (10H, m), 7.49 (1H, dd, m), 3.92 (3H, s), 4.21 (1H, s), 7.14—7.43 (10H, m), 7.47 (1H, dd, Jꢂ9.0,
Jꢂ9.2, 2.6 Hz), 8.00 (1H, d, Jꢂ2.6 Hz), 8.75 (1H, d, Jꢂ9.2 Hz), 11.7 (1H, 2.6 Hz), 7.99 (1H, d, Jꢂ2.6 Hz), 8.67 (1H, d, Jꢂ9.0 Hz), 11.0 (1H, s).
s).
Methyl 5-Chloro-2-{[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
Methyl 5-Chloro-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethyl}sulfanyl)acetyl]amino}benzoate (15o) To a mixture of 14h (4.88 g,
ethoxy}acetyl)amino]benzoate (15f) This compound was prepared in 15.4 mmol) in DMA (66 ml) were added 1-benzhydrylpiperazine (3.88 g,
81% yield as a solid from 14a, oxalyl dichloride, DMF and 1-benz- 15.4 mmol), WSC (3.53 g, 18.4 mmol) and HOBt (2.49 g, 18.4 mmol) at
hydrylpiperazine following a procedure similar to that described for the 0 °C, and the mixture was stirred at room temperature for 6 h. Aqueous
preparation of 15b, 1H-NMR (CDCl3) d: 2.40—2.50 (4H, m), 3.50—3.66 NaHCO3 solution was added to the reaction mixture and the solution was
(4H, m), 3.81 (3H, s), 4.21 (2H, s), 4.21 (1H, s), 4.34 (2H, s), 7.14—7.41 extracted with EtOAc three times. The combined organic layers were washed
(10H, m), 7.49 (1H, dd, Jꢂ9.0, 2.7 Hz), 7.99 (1H, d, Jꢂ2.7 Hz), 8.75 (1H, d, with brine, dried (Na2SO4) and concentrated in vacuo. The residue was puri-
Jꢂ9.0 Hz), 11.7 (1H, s).
Methyl 2-{[(2-{4-[Bis(4-fluorophenyl)methyl]piperazin-1-yl}-2-oxo-
ethoxy)acetyl]amino}-5-chlorobenzoate (15g) This compound was pre- (4H, m), 3.43—3.63 (4H, m), 3.46 (2H, s), 3.52 (2H, s), 3.90 (3H, s), 4.18
fied by column chromatography on silica gel (EtOAc/n-hexaneꢂ1/1) to af-
ford 6.55 g (yield 77%) of 15o as a solid, H-NMR (CDCl3) d: 2.28—2.46
1
pared in 97% yield as a solid from 14a, oxalyl dichloride, DMF and 1-
(1H, s), 7.13—7.43 (10H, m), 7.49 (1H, dd, Jꢂ9.0, 2.7 Hz), 8.01 (1H, d,
[bis(4-fluorophenyl)methyl]piperazine following a procedure similar to that Jꢂ2.7 Hz), 8.69 (1H, Jꢂ9.0 Hz), 11.5 (1H, s).
1
described for the preparation of 15b, H-NMR (CDCl3) d: 2.35—2.40 (4H,
Methyl 5-Chloro-2-({[{2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
m), 3.50—3.66 (4H, m), 3.83 (3H, s), 4.22 (2H, s), 4.22 (1H, s), 4.35 (2H, ethyl}(methyl)amino]acetyl}amino)benzoate (15p) This compound was
s), 6.93—7.02 (4H, m), 7.26—7.36 (4H, m), 7.51 (1H, dd, Jꢂ9.1, 2.6 Hz), prepared in 44% yield as a solid from 14i, methanesulfonyl chloride and
8.00 (1H, d, Jꢂ2.6 Hz), 8.75 (1H, d, Jꢂ9.1 Hz), 11.7 (1H, s).
Et3N following a procedure similar to that described for the preparation of
6a, 1H-NMR (CDCl3) d: 2.29—2.43 (4H, m), 2.48 (3H, s), 3.35 (2H, s),
3.44 (2H, s), 3.54—3.66 (4H, m), 3.71 (3H, s), 4.15 (1H, s), 7.10—7.40
(10H, m), 7.50 (1H, dd, Jꢂ9.2, 2.6 Hz), 7.99 (1H, d, Jꢂ2.6 Hz), 8.78 (1H, d,
Jꢂ9.2 Hz), 11.8 (1H, s).
Methyl 5-Chloro-2-[({2-[4-(9H-fluoren-9-yl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (15h) This compound was prepared in
95% yield as a solid from 14a, oxalyl dichloride, DMF and 1-[bis(4-fluo-
rophenyl)methyl]piperazine following a procedure similar to that described
for the preparation of 15a, 1H-NMR (CDCl3) d: 2.45—2.47 (4H, m), 2.75—
Methyl 5-Chloro-2-{[(1-{2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
2.80 (2H, m), 3.41—3.43 (2H, m), 3.62—3.67 (2H, m), 3.80 (3H, s), 4.19 ethyl}cyclohexyl)acetyl]amino}benzoate (15q) This compound was pre-
(2H, s), 4.31 (2H, s), 4.85 (1H, s), 7.21—7.68 (9H, m), 7.98 (1H, d, pared quantitatively as a solid from 14j, WSC, HOBt and 1-benzhydryl-
Jꢂ2.6 Hz), 8.73 (1H, d, Jꢂ9.1 Hz), 11.6 (1H, s).
piperazine following a procedure similar to that described for the prepara-
tion of 15k, 1H-NMR (CDCl3) d: 1.20—1.60 (10H, m), 2.20—2.40 (4H, m),
2.49 (2H, s), 2.73 (2H, s), 3.45—3.70 (4H, m), 3.92 (3H, s), 4.08 (1H, s),
7.12—7.45 (10H, m), 7.46 (1H, dd, Jꢂ9.0, 2.7 Hz), 7.99 (1H, d, Jꢂ2.7 Hz),
8.62 (1H, d, Jꢂ9.0 Hz), 10.9 (1H, s).
Methyl 2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)-
amino]-5-fluorobenzoate (15i) This compound was prepared in 90%
yield as a solid from 14b, oxalyl dichloride, DMF and 1-benzhydrylpiper-
azine following a procedure similar to that described for the preparation of
15b, 1H-NMR (CDCl3) d: 2.28—2.42 (4H, m), 3.51—3.66 (4H, m), 3.79
5-Chloro-2-[({2-[(4-chlorophenyl)amino]-2-oxoethoxy}acetyl)amino]-
(3H, s), 4.22 (2H, s), 4.22 (1H, s), 4.35 (2H, s), 7.15—7.42 (11H, m), 7.70 benzoic Acid (16a) To a solution of 15a (345 mg, 0.84 mmol) in THF
(1H, dd, Jꢂ9.2, 3.3 Hz), 8.76 (1H, dd, Jꢂ9.2, 5.2 Hz), 11.6 (1H, s).
(5 ml) was added 1 N aqueous NaOH solution (1.3 ml) and the mixture was
stirred for 0.5 h at 60 °C. The reaction solution was acidified with 1 N aque-
ous HCl solution and the organic solvent was removed in vacuo. The result-
ing precipitate was collected by filtration and washed with water and IPE,
and dried in vacuo to afford 309 mg (yield 93%) of 16a as a solid, mp 128—
Methyl 5-Bromo-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (15j) This compound was prepared in
91% yield as a solid from 14c, oxalyl dichloride, DMF and 1-benz-
hydrylpiperazine following a procedure similar to that described for the
preparation of 15b, 1H-NMR (CDCl3) d: 2.31—2.48 (4H, m), 3.50—3.66 130 °C, 1H-NMR (DMSO-d6) d: 4.28 (2H, s), 4.31 (2H, s), 7.32—7.43 (2H,
(4H, m), 3.81 (3H, s), 4.21 (2H, s), 4.22 (1H, s), 4.34 (2H, s), 7.14—7.41 m), 7.68—7.78 (2H, m), 7.70 (1H, dd, Jꢂ9.0, 2.7 Hz), 7.98 (1H, d,
(10H, m), 7.64 (1H, dd, Jꢂ9.2, 2.6 Hz), 8.15 (1H, d, Jꢂ2.6 Hz), 8.68 (1H, d, Jꢂ9.0 Hz), 8.69 (1H, d, Jꢂ9.0 Hz), 9.88 (1H, s), 11.9 (1H, s), HPLC
Jꢂ9.2 Hz), 11.6 (1H, s).
Methyl 4-Bromo-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo- (MꢁH)ꢁ.
ethoxy}acetyl)amino]benzoate (15k) To mixture of 14e (4.0 g,
(250 nm): 97.2% (tRꢂ16.8 min), LC/MS (ESI) m/z: 397 (MꢆH)ꢆ, 395
a
Sodium 5-Chloro-2-({[2-(diphenylamino)-2-oxoethoxy]acetyl}amino)-
benzoate (16b) To a solution of 15b (717 mg, 1.6 mmol) in THF (13 ml)
was added 1 N aqueous NaOH solution (4.3 ml) and the mixture was stirred
11.6 mmol) in DMA (28 ml) were added 1-benzhydrylpiperazine (2.93 g,
11.6 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(WSC) (2.67 g, 13.9 mmol) and 1-hydroxy-1H-benzotriazole (HOBt) (1.88 at 60 °C for 7 h. After cooling and evaporation of THF in vacuo, the result-
g, 13.9 mmol) at 0 °C, and the mixture was stirred at room temperature for
2 h. Aqueous NaHCO3 solution was added to the reaction mixture and the
ing precipitate was collected by suction filtration and washed with water to
give a crude product which was recrystallized from a mixed solvent of
resulting precipitate was collected by filtration and washed with water to af-
ford ‘quantitative’ yield of 15k as a solid, H-NMR (CDCl3) d: 2.37—2.45 ꢀ300 °C (MeOH/H2O), H-NMR (DMSO-d6) d: 4.12 (2H, s), 4.14 (2H, s),
(4H, m), 3.50—3.68 (4H, m), 3.80 (3H, s), 4.21 (2H, s), 4.34 (1H, s), 4.34 7.15—7.60 (10H, m), 7.29 (1H, dd, Jꢂ8.9, 2.8 Hz), 7.92 (1H, d, Jꢂ2.8 Hz),
MeOH and water to afford 508 mg (yield 70%) of 16b as a solid, mp
1
1
(2H, s), 7.14—7.41 (11H, m), 7.87 (1H, d, Jꢂ8.6 Hz), 9.02 (1H, d, Jꢂ
2.0 Hz), 11.7 (1H, s).
8.51 (1H, d, Jꢂ8.9 Hz), 14.3 (1H, s), HPLC (250 nm): ꢀ99.9% (tRꢂ16.6
min), LC/MS (ESI) m/z: 439 (MꢆH)ꢆ, 437 (MꢁH)ꢁ.
Methyl 4-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)-
5-Chloro-2-[({2-[(diphenylmethyl)amino]-2-oxoethoxy}acetyl)amino]-

222
Vol. 59, No. 2
benzoic Acid (16c) This compound was prepared in 92% yield as a solid
from 15c following a procedure similar to that described for the preparation
of 16a, mp 208—210 °C, 1H-NMR (DMSO-d6) d: 4.23 (2H, s), 4.25 (2H, s),
6.20 (1H, d, Jꢂ8.5 Hz), 7.22—7.35 (10H, m), 7.67 (1H, dd, Jꢂ9.0, 2.6 Hz),
7.96 (1H, d, Jꢂ2.6 Hz), 8.66 (1H, d, Jꢂ9.0 Hz), 8.81 (1H, d, Jꢂ8.5 Hz),
12.0 (1H, s), HPLC (250 nm): 99.4% (tRꢂ18.9 min), LC/MS (ESI) m/z: 453
(MꢆH)ꢆ, 451 (MꢁH)ꢁ.
5-Chloro-2-({[2-oxo-2-(4-phenylpiperidin-1-yl)ethoxy]acetyl}amino)-
benzoic Acid (16d) This compound was prepared in 85% yield as a solid
from 15d following a procedure similar to that described for the preparation
of 16a, mp 178—179 °C, 1H-NMR (DMSO-d6) d: 1.40—1.90 (4H, m),
2.60—2.85 (2H, m), 2.95—3.10 (1H, m), 3.70—3.90 (1H, m), 4.19 (2H, s),
4.40—4.60 (1H, m), 4.43 (2H, d, Jꢂ2.7 Hz), 7.10—7.35 (5H, m), 7.67 (1H,
dd, Jꢂ8.9, 2.7 Hz), 7.96 (1H, d, Jꢂ2.7 Hz), 8.69 (1H, d, Jꢂ8.9 Hz), 12.1
(1H, s), HPLC (250 nm): 97.7% (tRꢂ15.0 min), LC/MS (ESI) m/z: 431
(MꢆH)ꢆ, 429 (MꢁH)ꢁ.
Sodium 5-Chloro-2-({[2-(4,4-diphenylpiperidin-1-yl)-2-oxoethoxy]-
acetyl}amino)benzoate (16e) This compound was prepared in 88% yield
as an amorphous solid from 15e following a procedure similar to that de-
scribed for the preparation of 16b, 1H-NMR (DMSO-d6) d: 2.16—2.50 (4H,
m), 3.30—3.52 (4H, m), 4.08 (2H, s), 4.35 (2H, s), 7.00—7.40 (11H, m),
7.96 (1H, d, Jꢂ2.7 Hz), 8.54 (1H, d, Jꢂ8.8 Hz), 14.3 (1H, s), HPLC
(250 nm): 98.9% (tRꢂ20.2 min), LC/MS (ESI) m/z: 507 (MꢆH)ꢆ, 505
(MꢁH)ꢁ.
(2H, s), 7.13—7.44 (11H, m), 7.92 (1H, d, Jꢂ8.3 Hz), 8.74 (1H, d,
Jꢂ2.2 Hz), 14.6 (1H, s), HPLC (250 nm): ꢀ99.9% (tRꢂ6.9 min), LC/MS
(ESI) m/z: 566 (MꢆH)ꢆ, 564 (MꢁH)ꢁ.
4-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)amino]-
biphenyl-3-carboxylic Acid (16l) This compound was prepared in 88%
yield as an amorphous solid from 15l following a procedure similar to that
described for the preparation of 16g, 1H-NMR (DMSO-d6) d: 2.23—2.40
(4H, m), 3.30—3.58 (4H, m), 4.14 (2H, s), 4.28 (1H, s), 4.36 (2H, s), 7.13—
7.66 (15H, m), 7.85 (1H, dd, Jꢂ8.5, 2.2 Hz), 8.30 (1H, d, Jꢂ2.2 Hz), 8.72
(1H, d, Jꢂ8.5 Hz), 12.6 (1H, s), HPLC (250 nm): 99.2% (tRꢂ10.6 min),
LC/MS (ESI) m/z: 564 (MꢆH)ꢆ, 562 (MꢁH)ꢁ.
Sodium 4-Chloro-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (16m) This compound was prepared in
68% yield as a solid from 15m following a procedure similar to that de-
1
scribed for the preparation of 16b, mp 240 °C (dec.), H-NMR (DMSO-d6)
d: 2.28—2.38 (4H, m), 3.37—3.52 (4H, m), 4.08 (2H, s), 4.30 (2H, s), 4.30
(1H, s), 7.00 (1H, dd, Jꢂ8.3, 2.1 Hz), 7.18—7.44 (10H, m), 7.98 (1H, d,
Jꢂ8.3 Hz), 8.59 (1H, d, Jꢂ2.1 Hz), 14.6 (1H, s), HPLC (250 nm): 98.6%
(tRꢂ7.2 min), LC/MS (ESI) m/z: 522 (MꢆH)ꢆ, 520 (MꢁH)ꢁ.
5-Chloro-2-({5-[4-(diphenylmethyl)piperazin-1-yl]-5-oxopentanoyl}-
amino)benzoic Acid (16n) This compound was prepared in 93% yield as
an amorphous solid from 15n following a procedure similar to that de-
scribed for the preparation of 16g, 1H-NMR (DMSO-d6) d: 1.76—1.92 (2H,
m), 2.34—2.54 (8H, m), 3.41—3.62 (4H, m), 4.43 (1H, s), 7.17—7.48
(10H, m), 7.63 (1H, dd, Jꢂ9.0, 2.7 Hz), 7.92 (1H, d, Jꢂ2.7 Hz), 8.48 (1H, d,
Jꢂ9.0 Hz), 11.1 (1H, s), HPLC (250 nm): 97.0% (tRꢂ6.5 min), LC/MS
(ESI) m/z: 520 (MꢆH)ꢆ, 518 (MꢁH)ꢁ.
5-Chloro-2-{[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxoethyl}sul-
fanyl)acetyl]amino}benzoic Acid Hydrochloride (16o) This compound
was prepared in 90% yield as a solid from 15o following a procedure similar
to that described for the preparation of 16a, mp 205 °C (dec.), 1H-NMR
(DMSO-d6) d: 2.60—4.20 (8H, m), 3.56 (2H, s), 3.60 (2H, s), 5.30 (1H, br),
7.26—7.90 (10H, m), 7.68 (1H, dd, Jꢂ9.0, 2.7 Hz), 7.94 (1H, d, Jꢂ2.7 Hz),
8.51 (1H, d, Jꢂ9.0 Hz), 11.5 (1H, s), HPLC (250 nm): 94.2% (tRꢂ7.1 min),
MS (ESI) m/z 538 (MꢆH)ꢆ, 536 (MꢁH)ꢁ.
Sodium 5-Chloro-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (16f) This compound was prepared in
83% yield as a solid from 15f following a procedure similar to that de-
1
scribed for the preparation of 16b, mp 240 °C (dec.), H-NMR (DMSO-d6)
d: 2.28—2.48 (4H, m), 3.34—3.44 (4H, m), 4.06 (2H, s), 4.29 (2H, s), 4.32
(1H, s), 7.14—7.45 (11H, m), 7.93 (1H, d, Jꢂ2.7 Hz), 8.52 (1H, d,
Jꢂ8.8 Hz), 14.4 (1H, s), Anal. Calcd for C28H27ClN3NaO5: C, 61.82; H,
5.00; N, 7.72. Found: C, 61.64; H, 5.01; N, 7.68, HPLC (250 nm): 99.5%
(tRꢂ6.3 min), LC/MS (ESI) m/z: 522 (MꢆH)ꢆ, 520 (MꢁH)ꢁ.
2-{[(2-{4-[Bis(4-fluorophenyl)methyl]piperazin-1-yl}-2-oxoethoxy)-
acetyl]amino}-5-chlorobenzoic Acid (16g) To a solution of 15g (1.47 g,
2.56 mmol) in THF (26 ml) was added 1 N aqueous NaOH solution (3.8 ml)
and the mixture was stirred at 60 °C for 1 h. The reaction solution was neu-
tralized with 1 N aqueous HCl solution and the organic solvent was removed
in vacuo. The resulting precipitate was collected by filtration and washed
with water and IPE, and dried in vacuo to afford 1.33 g (yield 93%) of 16g
as an amorphous solid, 1H-NMR (DMSO-d6) d: 2.20—2.38 (4H, m), 3.35—
3.57 (4H, m), 4.13 (2H, s), 4.35 (2H, s), 4.40 (1H, s), 7.08—7.46 (8H, m),
7.65 (1H, dd, Jꢂ9.0, 2.4 Hz), 7.95 (1H, d, Jꢂ2.4 Hz), 8.67 (1H, d,
Jꢂ9.0 Hz), 12.1 (1H, s), HPLC (250 nm): 95.5% (tRꢂ8.6 min), LC/MS
(ESI) m/z: 558 (MꢆH)ꢆ, 556 (MꢁH)ꢁ.
5-Chloro-2-[({2-[4-(9H-fluoren-9-yl)piperazin-1-yl]-2-oxoethoxy}-
acetyl)amino]benzoic Acid (16h) This compound was prepared in 96%
yield as a solid from 15h following a procedure similar to that described for
the preparation of 16g, mp 224 °C (dec.), 1H-NMR (DMSO-d6) d: 2.42—
2.53 (2H, m), 3.24—3.57 (4H, m), 4.11 (2H, s), 4.34 (2H, s), 4.97 (1H, s),
7.25—7.80 (8H, m), 7.67 (1H, dd, Jꢂ9.1, 2.6 Hz), 7.94 (1H, d, Jꢂ2.6 Hz),
8.66 (1H, d, Jꢂ9.1 Hz), 11.9 (1H, s), HPLC (250 nm): 95.0% (tRꢂ5.9 min),
LC/MS (ESI) m/z: 520 (MꢆH)ꢆ, 518 (MꢁH)ꢁ.
2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)amino]-
5-fluorobenzoic Acid (16i) This compound was prepared in 93% yield as
an amorphous solid from 15i following a procedure similar to that described
for the preparation of 16g, 1H-NMR (DMSO-d6) d: 2.35—2.59 (4H, m),
3.49—3.60 (4H, m), 4.14 (2H, s), 4.37 (2H, s), 4.53 (1H, s), 7.19—7.60
(11H, m), 7.73 (1H, dd, Jꢂ9.3, 2.9 Hz), 8.68 (1H, dd, Jꢂ9.3, 5.1 Hz), 11.7
(1H, s), HPLC (250 nm) ꢀ99.9% (tRꢂ2.3 min), LC/MS (ESI) m/z: 506
(MꢆH)ꢆ, 504 (MꢁH)ꢁ.
5-Chloro-2-({[{2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxoethyl}-
(methyl)amino]acetyl}amino)benzoic Acid (16p) To a solution of 15p
(760 mg, 1.38 mmol) in THF (10 ml) was added 1 N aqueous NaOH solution
(2.1 ml) and the mixture was stirred for 1 h at 40 °C. The mixture was neu-
tralized with 1 N aqueous HCl solution, and the mixture was concentrated in
vacuo. The residue was dissolved in IPE and the solution was dried
(Na2SO4) and concentrated to give oil. EtOAc and n-hexane was added to
residue and the resulting precipitate was collected by filtration to afford
1
517 mg (yield 70%) of 16p as a solid, mp 179—181 °C, H-NMR (DMSO-
d6) d: 2.18—2.32 (4H, m), 2.42 (3H, s), 3.34 (2H, s), 3.49 (2H, s), 3.35—
3.60 (4H, m), 4.25 (1H, s), 7.10—7.45 (10H, m), 7.66 (1H, dd, Jꢂ9.0,
2.7 Hz), 7.94 (1H, d, Jꢂ2.6 Hz), 8.64 (1H, d, Jꢂ9.0 Hz), 12.1 (1H, s), HPLC
(250 nm): 97.3% (tRꢂ2.9 min), LC/MS (ESI) m/z: 535 (MꢆH)ꢆ, 533
(MꢁH)ꢁ.
5-Chloro-2-{[(1-{2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxoethyl}cy-
clohexyl)acetyl]amino}benzoic Acid Hydrochloride (16q) This com-
pound was prepared in 59% yield as a solid from 15q following a procedure
similar to that described for the preparation of 16a, mp 190—192 °C, 1H-
NMR (DMSO-d6) d: 1.10—1.70 (10H, m), 2.04—2.20 (4H, m), 2.40 (2H,
s), 2.60 (2H, s), 3.35—3.52 (4H, m), 4.03 (1H, s), 7.13—7.43 (10H, m),
7.67 (1H, dd, Jꢂ9.0, 2.6 Hz), 7.96 (1H, d, Jꢂ2.6 Hz), 8.49 (1H, d,
Jꢂ9.0 Hz), 11.0 (1H, s), HPLC (250 nm): ꢀ99.9% (tRꢂ13.4 min), LC/MS
(ESI) m/z: 588 (MꢆH)ꢆ, 586 (MꢁH)ꢁ.
Methyl 2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)-
amino]-5-(2-methylpropyl)benzoate (17a) Under argon atmosphere, a
solution of 0.5 M isobutylzinc bromide in THF (16 ml, 8.0 mmol) was added
to a solution of 15j (1.16 g, 2.0 mmol) in THF (20 ml) and tetrakis(triph-
enylphosphine)palladium (Pd(PPh3)4) (0.23 g, 0.2 mmol), and the mixture
was refluxed for 9 h. Aqueous NH4Cl solution was added to the reaction
mixture and the solution was extracted with EtOAc three times. The com-
bined organic layers were washed with brine, dried (Na2SO4) and concen-
trated in vacuo. The residue was purified by column chromatography on sil-
ica gel (EtOAc/n-hexaneꢂ1/3) to afford 0.32 g (yield 25%) of 17a as a solid,
1H-NMR (CDCl3) d: 0.89 (3H, s), 0.92 (3H, s), 1.74—1.98 (1H, m), 2.34—
2.41 (4H, m), 2.46 (2H, d, Jꢂ7.1 Hz), 3.53—3.67 (4H, m), 3.78 (3H, s),
4.21 (2H, s), 4.22 (1H, s), 4.35 (2H, s), 7.14—7.23 (11H, m), 7.79 (1H, d,
Jꢂ2.2 Hz), 8.64 (1H, d, Jꢂ8.6 Hz), 11.6 (1H, s).
Sodium 5-Bromo-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (16j) This compound was prepared in
77% yield as a solid from 15j following a procedure similar to that de-
1
scribed for the preparation of 16b, mp 240 °C (dec.), H-NMR (DMSO-d6)
d: 2.20—2.39 (4H, m), 3.30—3.59 (4H, m), 4.08 (2H, s), 4.30 (1H, s), 4.30
(2H, s), 7.14—7.44 (10H, m), 7.53 (1H, dd, Jꢂ9.0, 2.7 Hz), 8.09 (1H, d,
Jꢂ2.7 Hz), 8.51 (1H, d, Jꢂ9.0 Hz), 13.7 (1H, s), HPLC (250 nm): 97.5%
(tRꢂ6.2 min), LC/MS (ESI) m/z: 566 (MꢆH)ꢆ, 564 (MꢁH)ꢁ.
Sodium 4-Bromo-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (16k) This compound was prepared in
79% yield as a solid from 15k following a procedure similar to that de-
1
scribed for the preparation of 16b, mp 240 °C (dec.), H-NMR (DMSO-d6)
Methyl 5-Benzyl-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (17b) This compound was prepared in
d: 2.27—2.39 (4H, m), 3.30—3.51 (4H, m), 4.08 (2H, s), 4.30 (1H, s), 4.30

February 2011
223
47% yield as a solid from 15j, Pd(PPh3)4 and benzylzinc bromide following
was refluxed for 7 h. After cooling and evaporation of THF in vacuo, aque-
a procedure similar to that described for the preparation of 17a, 1H-NMR ous sodium thiosulfate solution was added to the residue and the whole mix-
(CDCl3) d: 2.34—2.52 (4H, m), 3.55—3.73 (4H, m), 3.76 (3H, s), 3.96 (2H, ture was extracted with EtOAc three times. The combined organic layers
s), 4.20 (2H, s), 4.21 (1H, s), 4.33 (2H, s), 7.12—7.40 (16H, m), 7.86 (1H, d, were washed with brine, water, dried (Na2SO4) and concentrated in vacuo.
Jꢂ2.2 Hz), 8.65 (1H, d, Jꢂ8.6 Hz), 11.6 (1H, s).
The residue was purified by column chromatography on silica gel (EtOAc/n-
hexane/MeOHꢂ2/2/0.2 to 2/3/0.3) to afford 1.10 g (yield 50%) of 19 as a
solid, 1H-NMR (CDCl3) d: 2.30—2.45 (4H, m), 3.14 (3H, s), 3.50—3.65
(4H, m), 3.78 (1H, d, Jꢂ15.2 Hz), 3.88 (3H, s), 3.89 (1H, d, Jꢂ15.2 Hz),
4.14 (1H, s), 4.22 (2H, s), 7.10—7.45 (11H, m), 7.55 (1H, dd, Jꢂ8.2,
2.6 Hz), 7.97 (1H, d, Jꢂ2.6 Hz).
5-Chloro-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}-
acetyl)(methyl)amino]benzoic Acid (20) To a solution of 19 (1.08 g,
1.96 mmol) in THF (20 ml) was added 1 N aqueous NaOH solution (2.9 ml)
and the mixture was stirred at 40 °C for 1 h. The mixture was neutralized
with 1 N aqueous HCl solution. The precipitate was collected by suction fil-
Methyl 2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)-
amino]-5-(pyridin-3-yl)benzoate (17c) Under argon atmosphere, 3-
pyridineboronic acid (0.34 g, 3.8 mmol) was added to a mixture of 15j
(1.16 g, 2.0 mmol), Pd(PPh3)4 (69.3 mg, 0.06 mmol) in a mixture of toluene
(6.7 ml), MeOH (1.6 ml), and 2 N Na2CO3 aqueous solution (5.6 ml), and the
resulting mixture was refluxed for 15 h. Water was added to the reaction
mixture and the mixture was extracted with EtOAc three times. The com-
bined organic layers were washed with brine, dried (Na2SO4) and concen-
trated in vacuo. The residue was purified by column chromatography on sil-
ica gel (EtOAc/n-hexane/MeOHꢂ2/2/0.3 to 2/2/0.5) to afford 0.92 g (yield
80%) of 17c as a solid, 1H-NMR (CDCl3) d: 2.38—2.43 (4H, m), 3.53— tration and washed with water. The resulting residue was purified by column
3.67 (4H, m), 3.85 (3H, s), 4.25 (1H, s), 4.29 (2H, s), 4.38 (2H, s), 7.15— chromatography (CHCl3/MeOHꢂ9/1) and recrystallized form EtOAc/n-
7.42 (11H, m), 7.78 (1H, dd, Jꢂ8.6, 2.4 Hz), 7.86—7.91 (1H, m), 8.27 (1H, hexane to afford 510 mg (yield 49%) of 20 as an amorphous solid, 1H-NMR
d, Jꢂ2.4 Hz), 8.61 (1H, dd, Jꢂ5.0, 1.7 Hz), 8.85—8.91 (2H, m), 11.8 (1H, (DMSO-d6) d: 2.10—2.35 (4H, brs), 3.01 (3H, s), 3.25—3.55 (4H, br s),
s).
3.79 (2H, s), 4.05 (2H, s), 4.30 (1H, s), 7.10—7.55 (12H, m), 7.66 (1H, d,
Jꢂ2.4 Hz), HPLC (250 nm): ꢀ99.9% (tRꢂ4.0 min), LC/MS (ESI) m/z: 536
(MꢆH)ꢆ, 534 (MꢁH)ꢁ.
Biological Procedures. Method A and Method B PAI-1 activity was
evaluated by two different methods. For the screening of newly synthesized
compounds for their PAI-1 inhibitory activities, we used a biological assay
utilizing a synthetic substrate for tPA. In brief, human PAI-1 (Molecular In-
novations, Southfield, MI, U.S.A.) was incubated at 37 °C for 15 min in the
reaction buffer containing 100 mM Tris HCl, pH 8, 0.1% Tween 80 in the
presence or absence of the tested compounds in a 96-well polystyrene plate.
Methyl 2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)-
amino]-4-(pyridin-4-yl)benzoate (17d) Under argon atmosphere, 4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (0.62 g, 3.0 mmol)
was added to a mixture of 15k (1.16 g, 2.0 mmol), Cs2CO3 (0.98 g,
3.0 mmol) and Pd(PPh3)4 (0.23 g, 0.2 mmol) in THF (20 ml) and the mixture
was refluxed for 8 h. After evaporation of organic solvent, water was added
to the mixture and the aqueous solution was extracted with EtOAc three
times. The combined organic layers were washed with brine, dried (Na2SO4)
and concentrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (EtOAc/MeOHꢂ4/1) to afford ‘quantitative’ yield of 17d The mixture was subsequently incubated for 15 min with human tPA (Amer-
as a solid, 1H-NMR (CDCl3) d: 2.38—2.43 (4H, m), 3.41—3.65 (4H, m), ican Diagnostica Inc., Stanford, CT, U.S.A.), and eventually fortified with a
3.85 (3H, s), 4.23 (1H, s), 4.26 (2H, s), 4.38 (2H, s), 7.15—7.73 (13H, m), chromogenic substrate, S-2288 (Chromogenix, Milano, Italy). The final mix-
8.13 (1H, d, Jꢂ8.2 Hz), 8.67—8.71 (2H, m), 9.14 (1H, d, Jꢂ1.9 Hz), 11.8
(1H, s).
Sodium 2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}-
acetyl)amino]-5-(2-methylpropyl)benzoate (18a) This compound was
ture contained 100 mM Tris–HCl, pH 8, 30 mM NaCl, 1% DMSO, 0.1%
Tween 80, 67 nM PAI-1 (Method A) or 24.5 nM (Method B), 9.8 nM tPA,
1 mM S-2288, and tested compounds at various concentrations (20, 50,
100 mM). Kinetics of p-nitroaniline release during peptide cleavage was
prepared in 54% yield as a solid from 17a following a procedure similar to monitored with a spectrophotometer at 405 nm. The residual inhibitory ac-
that described for the preparation of 16b, mp 230 °C (dec.), 1H-NMR tivity of PAI-1 was expressed as the percentage of the initial activity.
(DMSO-d6) d: 0.83 (3H, s), 0.86 (3H, s), 1.71—1.86 (1H, m), 2.18—2.38
Pharmacokinetics Compounds (50 mg/kg) were given by gavage to
(4H, m), 2.39 (2H, d, Jꢂ6.8 Hz), 3.21—3.63 (4H, m), 4.06 (2H, s), 4.27 male Wistar rats. Heparinized blood samples were collected from the vein
(1H, s), 4.34 (2H, s), 7.16—7.42 (11H, m), 7.79 (1H, d, Jꢂ1.9 Hz), 8.45 before (0 h) and at 1, 2, 6, 18, 24 48 h after administration. Drug concentra-
(1H, d, Jꢂ8.5 Hz), 13.6 (1H, s), HPLC (250 nm): 98.9% (tRꢂ11.6 min), tion was determined in the plasma by reverse-phase HPLC. Maximum drug
LC/MS (ESI) m/z: 544 (MꢆH)ꢆ, 542 (MꢁH)ꢁ.
concentration (Cmax) and drug half-life (T1/2) were calculated.
Sodium 5-Benzyl-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)amino]benzoate (18b) This compound was prepared in
Acknowledgments We thank Dr. Takashi Dan and Ms. Hiroko Yuzawa
54% yield as an amorphous solid from 17b following a procedure similar to for their assistance in in vitro test, Drs. Takao Takaya and Shigehisa Take-
tomi for helpful discussions, and Dr. Tatsunori Sato and Mr. Kenji Murano
that described for the preparation of 16b, 1H-NMR (DMSO-d6) d: 2.18—
2.36 (4H, m), 3.21—3.53 (4H, m), 3.90 (2H, s), 4.07 (2H, s), 4.26 (1H, s), for reviewing the manuscript.
4.32 (2H, s), 7.14—7.41 (16H, m), 7.86 (1H, d, Jꢂ2.2 Hz), 8.49 (1H, d,
Jꢂ8.3 Hz), 13.0 (1H, s), HPLC (250 nm): ꢀ99.9% (tRꢂ10.8 min), LC/MS
References and Notes
(ESI) m/z: 578 (MꢆH)ꢆ, 576 (MꢁH)ꢁ.
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2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)amino]-
5-(pyridin-3-yl)benzoic Acid (18c) This compound was prepared in 66%
yield as an amorphous solid from 17c following a procedure similar to that
described for the preparation of 16g, 1H-NMR (DMSO-d6) d: 2.28—2.40
(4H, m), 3.38—3.59 (4H, m), 4.17 (2H, s), 4.34 (1H, s), 4.38 (2H, s), 7.15—
7.54 (11H, m), 8.00—8.13 (2H, m), 8.30 (1H, d, Jꢂ2.4 Hz), 8.59 (1H, d,
Jꢂ4.9 Hz), 8.79 (1H, d, Jꢂ8.8 Hz), 8.92 (1H, d, Jꢂ2.4 Hz), 11.9 (1H, s),
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2-[({2-[4-(Diphenylmethyl)piperazin-1-yl]-2-oxoethoxy}acetyl)amino]-
4-(pyridin-4-yl)benzoic Acid (18d) This compound was prepared in 67%
yield as a solid from 17d following a procedure similar to that described for
the preparation of 16g, mp 190 °C (dec.), 1H-NMR (DMSO-d6) d: 2.29—
2.38 (4H, m), 3.38—3.57 (4H, m), 4.17 (2H, s), 4.31 (1H, s), 4.38 (2H, s),
7.14—7.43 (10H, m), 7.58 (1H, dd, Jꢂ8.3, 1.7 Hz), 7.68 (2H, d, Jꢂ6.1 Hz),
8.13 (1H, d, Jꢂ8.3 Hz), 8.70 (1H, d, Jꢂ6.1 Hz), 9.07 (1H, d, Jꢂ1.7 Hz),
12.1 (1H, s), HPLC (250 nm): 96.4% (tRꢂ2.0min), LC/MS (ESI) m/z: 565
(MꢆH)ꢆ, 563 (MꢁH)ꢁ.
Methyl 5-Chloro-2-[({2-[4-(diphenylmethyl)piperazin-1-yl]-2-oxo-
ethoxy}acetyl)(methyl)amino]benzoate (19) To a suspension of sodium
hydride (60% purity, 230 mg, 6.0 mmol) in THF (35 ml) was added 15f
(2.14 g, 4.0 mmol) at 0 °C, and stirred at 0 °C for 0.5 h under an argon atmo-
sphere. Iodomethane (2.48 ml, 40 mmol) was then added and the mixture
9) ClogP was calculated by utilizing the ChemDraw Ultra software, ver-
sion 10.0.

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