Synthesis and structure-activity relationships of potent and orally active sulfonamide ETB selective antagonists

Article abstract of DOI:10.1016/S0968-0896(00)00305-9

The synthesis and structure-activity relationships of a series of N-pyrimidinyl benzenesulfonamides as ET_B selective antagonists are described. N-Isoxazolyl benzenesulfonamide 1a, previously reported, was selected as a lead compound, and isosteric replacement of the isoxazole ring of 1a with a pyrimidine ring led to the discovery of the highly potent ET_B selective antagonist 6e with oral bioavailability. Modification of the terminal aldehyde group at the 6-position of the pyrimidine ring was investigated, and malonate 15b and acylhydrazone 16f were found to be equipped to aldehyde 6e. Compound 6e showed ET_B antagonistic activity on in vivo evaluation. Copyright

Full text of DOI:10.1016/S0968-0896(00)00305-9

Bioorganic & Medicinal Chemistry 9 (2001) 897±907
Synthesis and Structure±Activity Relationships of Potent and
Orally Active Sulfonamide ETB Selective Antagonists
Yasuhiko Kanda,a,* Yasuyuki Kawanishi,a Katsuo Oda,a Teruo Sakata,a
Shin-ichi Mihara,a Kenji Asakura,b Toshiyuki Kanemasa,b Mitsuyoshi Ninomiya,b
Masafumi Fujimotoa and Toshiro Konoikec,*
aShionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553±0002, Japan
bShionogi Research Laboratories, Shionogi & Co., Ltd., Toyonaka, Osaka 561±0825, Japan
cShionogi Research Laboratories, Shionogi & Co., Ltd., Amagasaki, Hyogo 660±0813, Japan
Received 28 August 2000; accepted 10 November 2000
AbstractÐThe synthesis and structure±activity relationships of a series of N-pyrimidinyl benzenesulfonamides as ETB selective
antagonists are described. N-Isoxazolyl benzenesulfonamide 1a, previously reported,1 was selected as a lead compound, and isos-
teric replacement of the isoxazole ring of 1a with a pyrimidine ring led to the discovery of the highly potent ETB selective antagonist
6e with oral bioavailability. Modi®cation of the terminal aldehyde group at the 6-position of the pyrimidine ring was investigated,
and malonate 15b and acylhydrazone 16f were found to be equipotent to aldehyde 6e. Compound 6e showed ETB antagonistic
activity on in vivo evaluation. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
receptor in the pathological conditions. However, only
recently have non-peptide ETB selective antagonists
been reported,9 and the roles of the ETB receptor in
both normal physiological and pathological conditions
are poorly understood.
Endothelins (ET-1, ET-2, ET-3), 21-amino acid bicyclic
peptides, are the most potent known vasoconstrictors.2,3
Two distinct G-protein coupled receptors, ETA and ETB,
were cloned and characterized with respect to their anity
for each endothelin.4,5 The ETA receptor on vascular
smooth muscle cells has high anity for ET-1 and ET-2,
and mediates vasoconstriction and smooth muscle cell
proliferation. The ETB receptor on vascular endothelial
and smooth muscle cells has high anities for all three
endothelins. It mediates both vasodilatation and vaso-
constriction. As these two subtypes of receptors are widely
distributed in human tissues, selective or non-selective
endothelin receptor antagonists have been envisioned to
be useful for the treatment of various vascular diseases,
including hypertension, congestive heart failure, myo-
cardial infarction, vasospasm, and renal failure.6
Recently, we have described the identi®cation of non-
peptide ETB selective antagonists aldehyde 1a and
oxime 1b1 by modi®cation of sulfamethoxazole10 and its
iodo derivative. Although isoxazole aldehyde 1a showed
sucient activity and high selectivity for the ETB
receptor, it had low oral bioavailability. Therefore, in
order to elucidate the role of the ETB receptor, ETB
antagonists with better pharmacokinetic pro®les had
been required. Since almost all the sulfonamide ET
antagonists reported at that time had an isoxazole or
pyrimidine ring as the heterocycle part,7a,8a,11 we
replaced the isoxazole ring of 1a with a pyrimidine ring
and conducted modi®cation to ®nd better ETB selective
antagonists. Our modi®cation strategy is shown in
Scheme 1. First, we investigated the e€ect of heterocycle
parts and optimized the substituents on the pyrimidine
ring. Second, we sought other functional groups to
replace the terminal aldehyde group. Substituents at the
5 and 6 position of the pyrimidine ring were tethered via
the oxygen atom in a same manner as Ro 47-0203
(Bosentan).8a
A number of groups have reported the discovery of
non-peptide endothelin antagonists since 1994. Most of
them were ETA selective or non-selective antagonists,7,8
which were valuable in exploring the role of the ETA
*Corresponding author. Fax: +81-6-6458-0987; e-mails: yasuhiko.
kanda@shionogi.co.jp (Y. Kanda), toshiro.konoike@shionogi.co.jp
(T. Konoike)
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00305-9

898
Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
Scheme 1. Modi®cation strategy of N-pyrimidinyl benzenesulfonamide derivatives.
In this paper, we describe the synthesis and structure±
activity relationships of this class of compounds and the
discovery of orally active ETB selective antagonists 6b
and 6e with higher activity and selectivity than those of
1a. The pharmacokinetic and pharmacological pro®les
of these compounds are also presented.
aldehyde 6e. 5-Unsubstituted compound 8 was also
synthesized from commercially available 4,6-dichloro-
pyrimidine 7 by a similar procedure. Bromination of the
5-position in the pyrimidine ring of 8 with NBS gave 5-
bromo derivative 9.
Compounds 12±14 (Table 1) bearing various nitrogen
spacers at the 4-position of the pyrimidine ring were pre-
pared as follows (Scheme 3). Displacement of the chloride
at the 6-position of the pyrimidine ring of 4 with alkoxide
of g-hydroxy butyraldehyde acetal 1013 gave 11. Next,
chloride 11 was converted to the corresponding benza-
mide or benzylamine derivative, whose acetal groups were
hydrolyzed in the aqueous formic acid solution to give
aldehyde 12 and 13, respectively. Methylation of the
sulfonamide nitrogen of 6e with iodomethane gave 14.
Chemistry
N-Pyrimidinyl benzenesulfonamide derivatives 6a±g, 8, 9
(Table 1) were synthesized as shown in Scheme 2. As a
typical example of 6a±g, the synthesis of 6e is discussed.
Alkylation of 3-methoxyphenol 2 with dimethyl chloro-
malonate and subsequent condensation with formamidine
acetate gave 4,6-pyrimidinedione 3. It was chlorinated
with phosphorous oxychloride in the presence of collidine
to produce dichloride 4. Condensation of 4 with potas-
sium 4-tert-butylbenzenesulfonamide gave sulfonamide
5.12 Chloride displacement of 5 with alkoxide of 1,4-
butanediol followed by oxidation with PCC a€orded
Compounds 15a±e (Table 2) were synthesized by con-
densation of aldehyde 6e with malonic acid, dimethyl and
di-tert-butyl malonate, acetylacetone or ethyl cyanoace-
tate. Compounds 16a±g were also prepared similarly from
Scheme 2. Synthesis of N-pyrimidinyl benzenesulfonamide derivatives 6e, 8, 9. (a) Dimethyl chloromalonate, NaOMe, MeOH, rt; (b) ꢀHC(=NH)-
ꢀ
ꢀ
ꢀ
.
NH2 AcOH, NaOMe, MeOH, 0 C; (c) POCl3, collidine, 135 C; (d) 4-t-Bu-PhSO2NHK, DMSO, 120 C; (e) NaH, HO(CH2)4OH, 100 C; (f) PCC,
CH2Cl2, rt; (g) NBS, DMF, 0 ꢀC.

Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
899
Scheme 3. Synthesis of pyrimidine derivatives 12±14. (a) NaH, THF, re¯ux; (b) 4-t-BuPhCONH2, NaH, DMF, 100 ꢀC; (c) HCO2Na, HCO2H,
70 ꢀC; (d) 4-t-BuPhCH2NH2, 120 ꢀC; (e) MeI, K2CO3, DMF, rt.
Scheme 4. Synthesis of pyrimidine derivatives 15 and 16. (a) R1CH2R2, piperidine, EtOH, (b) R3NH2, EtOH.
6e by treatment with hydroxylamine, O-ethylhydroxyl-
amine or acylhydrazines (Scheme 4).
unsubstituted derivative 8 and 5-bromo derivative 9 had
extremely lower binding anity for the ETB receptor
compared with 6b, indicating that introduction of a
large hydrophobic aromatic substituent is necessary for
the ETB receptor binding. The binding anity for the
ETA receptor also increased by introducing aromatic
substituent at the 5-position of the pyrimidine ring.
Compounds 6d and 6e were prepared to optimize the
position of the methoxy group in the phenoxy group of 6b.
Among them, ortho-substituted 6b showed the highest
ETB selectivity (6100), and meta-substituted 6e showed
the highest anity for the ETB receptor (0.06 nM). In
fact, 6e is the most potent non-peptide ETB receptor
antagonist reported so far. Introduction of the sub-
stituent R2 at the 2-position of the pyrimidine ring
slightly decreased the ETB anity (6f, 6g). The sulfona-
mide group at the 4-position of the pyrimidine ring was
important for the ETB receptor binding as well as ETA
receptor binding.14 Replacement of the sulfonamide
group of 6e with carboxamide group greatly decreased
the ETB anity (12). The more basic benzylamine deri-
vative 13 and N-methylated sulfonamide derivative 14
showed diminished activity. This implies that the acidic
NH proton of the sulfonamide group is necessary for
the high anity binding to the ETB receptor.
Results and Discussion
Structure±activity relationships are discussed using IC50
values obtained from radioreceptor binding studies
(Tables 1 and 2). IC50 is the concentration of the antago-
nist required to cause 50% inhibition of [125I]ET-1 binding
to the ETA receptor and [125I]ET-3 binding to the ETB
receptor. Selectivity for the ETB receptor was shown as
the ratio of the IC50 value for ETA over that for ETB.
The substitution e€ect on the pyrimidine ring is sum-
marized in Table 1. We designed pyrimidine aldehyde
6a±c and optimized the alkyl chain length (n=2±4) at
the 6-position of the pyrimidine ring as shown in
Scheme 1. Among these compounds, 6b (n=3) showed
highly ETB selective antagonistic activity. The binding
anity of 6b was in the subnanomolar range (0.15 nM),
which was 13-fold potent than isoxazole aldehyde 1a.
ETB selectivity also increased 7-fold as compared to 1a.
This demonstrates that pyrimidine derivatives also have
potent binding anity for the ETB receptor and that the
aldehyde group at the 6-position of the pyrimidine ring
plays a crucial role in the ETB receptor binding as with
the isoxazole derivative 1a.
A conformation of 6e in a crystalline form was deter-
mined by X-ray crystallography. Figure 1 shows a
superimposition of the crystal structure of 6e on the
modeled 1a. Key functional groups of 1a and 6e such as
the aromatic ring, sulfonamide group and aldehyde
We next investigated the in¯uence of other substituents
in the pyrimidine ring. As for R1 at the 5-position,

900
Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
Table 1. Structure±activity relationships of the pyrimidine derivatives
IC50 (nM)
ETB selectivity
(IC50 ETA/IC50 ETB)
Compound
n
R1
R2
X
ETA
3000
910
590
21,000
>100,000
2300
ETB
680
6a
6b
6c
8
2
3
4
3
3
3
3
3
3
3
3
3
2-MeO-PhO
2-MeO-PhO
2-MeO-PhO
H
H
H
H
H
H
H
H
Me
Ph
H
ÀSO2NHÀ
ÀSO2NHÀ
ÀSO2NHÀ
ÀSO2NHÀ
ÀSO2NHÀ
ÀSO2NHÀ
ÀSO2NHÀ
ÀSO2NHÀ
ÀSO2NHÀ
ÀCONHÀ
ÀCH2NHÀ
ÀSO2NMeÀ
4.4
6100
11
6.4
>670
500
3000
340
1400
>20
Ð
0.15
42
3300
150
9
Br
6d
6e
6f
6g
12
13
14
4-MeO-PhO
3-MeO-PhO
3-MeO-PhO
3-MeO-PhO
3-MeO-PhO
3-MeO-PhO
3-MeO-PhO
4.6
180
190
880
0.06
0.56
0.64
>10,000
>10,000
>1000
510
>10,000
>1000
H
H
Ð
Table 2. E€ects of modifying the aldehyde group in the 6-position of the pyrimidine ring of 6e
IC50 (nM)
ETB selectivity
(IC50 ETA/IC50 ETB)
Compound
X
R1
R2
ETA
ETB
15a
15b
15c
15d
15e
16a
16b
16c
16d
16e
16f
16g
C
C
C
C
C
N
N
N
N
N
N
N
CO2H
CO2Me
CO2LtBu
COMe
CN
CO2H
CO2Me
11,000
430
2400
160
260
71
71
0.32
3.7
0.26
0.68
8.0
56
1.8
1.0
0.29
0.15
0.22
160
1300
650
620
380
8.9
5.0
89
730
1300
2300
2000
CO2tBu
COMe
CO2Et
Ð
Ð
Ð
Ð
Ð
Ð
Ð
OHa
OEt
280
160
730
390
340
440
NHCONH2
NHCOMe
NHCOPh
NHCO-4-pyridyl
NHSO2Ph
aMixture of E- and Z-isomers (1:1).
group occupy similar spatial positions, thus supporting
the e€ectiveness of our ®rst modi®cation strategy.
butyl ester decreased the activity (15c). Acetylacetone
derivative 15d and cyano acetic acid derivative 15e were
equipotent to 15b although ETB selectivity decreased.
Having discovered the potent ETB selective antagonist
6e, we further modi®ed the aldehyde group at the 6-
position of the pyrimidine ring (Table 2). Since the
electrophilic carbonyl carbon seemed to play an impor-
tant role in interacting with the ETB receptor,1 we
introduced other electrophilic functional groups. We
designed malonic acid derivatives (15a±c), which were
synthesized from aldehyde 6e. Malonic acid 15a showed
greatly reduced binding anity for the ETB receptor
compared with 6e. However, the dimethyl ester of 15a
had potent binding anity in the subnanomolar range
(15b, 0.32 nM). The introduction of a bulkier di-tert-
Oxime 16a was designed as a pyrimidine analogue of
isoxazole oxime 1b. Although 16a had better binding
anity for the ETB receptor (8.0 nM) than 1b, ETB
selectivity was low. Oxime ether 16b showed decreased
anity for ETA and ETB receptors than 16a. This indi-
cates that the hydrogen bond-donating oxime hydroxyl
group is e€ective for the ETB receptor binding. Therefore,
we synthesized derivatives which had other hydrogen
bond-donating groups. For this purpose, acylhydrazone
derivatives (16c±g) were tested because a variety of
compounds were easily prepared from acylhydrazine

Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
901
Figure 1. Stereo-view of a superimposition of the crystal structure of 6e (colored by atom type) on the modeled 1a (magenta).
and aldehyde 6e. Semicarbazone 16c had a potent ETB
anity, but ETB selectivity was insucient. Then, the
amino group at the terminal part of the acylhydrazone
group of 16c was replaced with more hydrophobic
methyl group to give 16d, which had better anity and
selectivity for the ETB receptor than 16c. Introduction
of larger phenyl and pyridyl groups further enhanced
ETB anity to the subnanomolar range (16e, 0.29 nM
and 16f, 0.15 nM) while the orders of magnitude of ETA
anity were maintained. Sulfonylhydranone 16g was
equipotent to 16f. This result suggests that there is a
hydrophobic binding site on the ETB receptor which is
occupied by the terminal part of the substituent in the 6-
position of the pyrimidine ring.
biphasic response consisting of a sharp and transient
decrease in blood pressure followed by a sustained pressor
response.9c On the other hand, after 6e treatment, the
depressor response disappeared and the subsequent
pressor response was enhanced, which resulted from
removal of the early depressor component (the chart at
1 h after administration of 6e is shown). Because the
depressor response is mediated via ETB-induced release
of nitric oxide and the pressor response is mediated via
the ETA receptor,9c this result suggests that 6e has ETB
selective antagonistic activity in vivo. From the chart as
shown in Figure 2a, the depressor and pressor responses
were quanti®ed as the maximum change of mean artery
blood pressure (MABP) relative to the baseline blood
pressure before ET-1 treatment (Fig. 2b). Before the
treatment with 6e, the depressor and pressor responses
induced by ET-1 were À11.7Æ 1.7 mmHg and
+11.7Æ1.7 mmHg, respectively (Fig. 2b, open and solid
circles at 0 h). After the oral administration of 6e, the
depressor response was completely blocked (Fig. 2b,
open circles at 1 to 5 h), and the pressor response was
enhanced 4- to 5-fold (Fig. 2b, solid circles at 1 to 5 h).
These e€ects were sustained for at least 5 h, but dis-
appeared at 24 h after administration of 6e (data not
shown). Based on the above discussion, we conclude
that 6e is a potent and orally active ETB selective
antagonist.
Pharmacokinetic pro®les of the selected compounds 6b
and 6e were evaluated in rats (Table 3). Plasma con-
centrations of 6b and 6e after intravenous (5 mg/kg) or
oral (30 mg/kg) administration were measured by HPLC
analysis of the plasma samples taken over time. As an iv
dose, 6b and 6e had similar half-life time (T1=2) and AUC
values. As an oral dose, 6b showed more rapid absorp-
tion and higher peak plasma concentration (Cmax) of
12.7 mg/mL than 6e, resulting in a higher AUC value.
The calculated oral bioavailabilities (B.A.) of 6b and 6e
were 57% and 39%, respectively.
Compound 6e was evaluated for the ETB antagonistic
activity in vivo. Figure 2 shows the change of blood
pressure induced by ET-1 administration (0.1 nmol/kg)
at 1-h intervals before and after the administration of 6e
(30 mg/kg, po) in conscious rats. As shown in Figure 2a,
an intravenous bolus injection of ET-1 induced a
Conclusion
We discovered a highly potent ETB selective antagonist 6e
with subnanomolar IC50 value by modifying the amino
heterocycle part of 1a. Modi®cation of the terminal alde-
hyde group at the 6-position of the pyrimidine ring led to
the discovery of malonic acid derivatives and acylhy-
drazone derivatives equipotent to aldehyde 6e. Compound
6e had an oral bioavailability of 39% and displayed ETB
antagonistic activity in vivo. This compound is being eval-
uated as a potential therapeutic reagent and useful tool
for understanding the role of the ETB receptor in
pathological conditions.
Table 3. Pharmacokinetic pro®les of compounds 6b and 6e in rats
iv (5mg/kg)
po (30 mg/kg)
Compound T1=2 (h)
AUC
Cmax/Tmax
AUC
BA
(mg-h/mL) (mg/mL; h) (mg-h/mL) (%)
6b
6e
0.58
0.87
11.4
11.1
12.7/0.8
7.7/1.3
38.9
26.2
57
39

902
Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
Figure 2. (a) E€ects on mean artery blood pressure (MABP) of the ET-1 administered (0.1 nmol/kg) at 1-h intervals in conscious rats (data before
and at 1 h after administration of 6e is shown). After the ®rst treatment of ET-1, compound 6e (30 mg/kg) was administered orally. (b) E€ects on the
depressor and pressor responses induced by ET-1 administration of 6e. The depressor (open circles) and pressor (solid circles) responses were
quanti®ed as the maximum change of MABP relative to the baseline blood pressure before ET-1 treatment. Values expressed as meanÆS.E.M.
Experimental
concentrated. Water was added to the mixture, and the
aqueous layer was extracted with toluene. The organic
layer was washed with 1% aqueous NaOH solution and
saturated NaCl, dried and concentrated. The residue
was distilled under reduced pressure (bp. 155 ꢀC/
0.5 mmHg) to give crude dimethyl-3-methoxyphenox-
ymalonate (67.3 g, 67%), which was used in a following
Chemistry
Reactions were carried out under a nitrogen atmosphere
in anhydrous solvents (dried over molecular sievses type
4A). Organic extracts were dried over anhydrous MgSO4.
Solvent removal was accomplished under aspirator
pressure using a rotary evaporator. TLC was performed
with Merck precoated TLC plates silica gel 60 F254, and
compound visualization was made by UV light. Silica gel
chromatography was done with Merck silica gel 60 (70±
230 mesh). 1H NMRand 13C NMRwere determined as
CDCl3 solution at 300 and 75.5 MHz, respectively. J
values are given in hertz.
1
reaction without further puri®cation: H NMR d 3.78
(3H, s), 3.85 (3H, s), 6.46±6.63 (3H, m), 7.19 (1H, t,
J=8.0). Formamidine acetate (8.6 g, 82.6 mmol) and the
malonate (14.0 g, 55.1 mmol) from the preceding reac-
tion were added to a sodium methoxide solution (1.0 M
methanol solution, 165 mL) at 0 ꢀC. The reaction mix-
ture was stirred for 2.5 h at rt and concentrated. Water
was added to the mixture, and the aqueous layer was
extracted with toluene. The aqueous layer was acidi®ed
with 1 N HCl. The resulting precipitate was collected,
washed with water and dried to give 3 (10.8 g, 84%): mp
5-(3-Methoxyphenoxy)-1H-pyrimidin-4,6-dione (3). m-
Methoxyphenol (49.0 g, 0.40 mol) was added dropwise
over 15 min to a sodium methoxide solution (1.0 M
methanol solution, 395 mL) at 0 ꢀC. After being stirred
for 15 min, dimethyl chloromalonate (75.0 g, 0.45 mol)
was added dropwise over 15 min at the same tempera-
ture. The reaction mixture was stirred for 20 h at rt and
>300 ꢀC; H NMR(DMSO- d6) d 3.71 (3H, s), 6.38±
1
6.41 (2H, m), 6.54±6.57 (1H, m), 7.11±7.17 (1H, m), 8.01
(1H, s). Anal. calcd for C11H10N2O4 0.1 H2O: C, 55.98;
H, 4.36; N, 11.87. Found: C, 56.08; H, 4.36; N, 11.83.

Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
903
4,6-Dichloro-5-(3-methoxyphenoxy)pyrimidin (4). To a
mixture of 3 (10.8 g, 46.1 mmol) and collidine (15.0 mL,
113.5 mmol) was added phosphorous oxychloride (62.6
mL, 6.7 mol) portionwise at 0 ꢀC. The reaction mixture
was stirred at 135 ꢀC for 4 h. The cooled reaction mix-
ture was poured into ice-water and extracted with
AcOEt. The organic layer was washed with aqueous
NaHCO3 solution and saturated NaCl solution, dried
and concentrated. The residue was puri®ed by silica gel
chromatography eluting with hexane/AcOEt (4/1 to 2/1)
to give 4 (9.7 g, 78%): mp 113±114 ꢀC (hexane/AcOEt);
1H NMR d 3.81 (3H, s), 6.33±6.38 (2H, m), 6.45 (1H, t,
J=2.4), 6.66±6.71 (1H, m), 7.23 (1H, t, J=8.2), 8.69
(1H, s). Anal. calcd for C11H8Cl2N2O2 0.1 H2O: C,
48.41; H, 3.03; Cl, 25.98; N, 10.26. Found: C, 48.37; H,
2.94; Cl, 25.89; N, 10.27.
t, J=1.2); 13C NMR d 21.26, 31.06, 35.26, 39.88, 55.49,
65.96, 102.27, 107.38, 108.97, 121.55, 125.83, 128.34,
130.22, 136.21, 150.29, 152.91, 157.43, 157.50, 160.87,
161.04, 201.01. Anal. calcd for C25H29N3O6S: C, 60.10;
H, 5.85; N, 8.41; S, 6.41. Found: C, 59.90; H, 5.95; N,
8.66; S, 6.41.
The following compounds were prepared using a similar
procedure described for 6e.
4-tert-Butyl-N-[5-(2-methoxyphenoxy)-6-(4-oxopropoxy)
pyrimidin-4-yl]benzenesulfonamide (6a). Mp 130±132 ꢀC
1
(hexane/AcOEt); H NMR d 1.33 (9H, s), 2.77 (2H, dt,
J=1.5, 6.0), 3.95 (3H, s), 4.66 (2H, t, J=6.0), 6.82±6.90
(1H, m), 6.95±7.02 (2H, m), 7.08±7.16 (1H, m), 7.48,
8.00 (4H, A2B2, J=9.0), 8.24 (1H, s), 8.77 (1H, s), 9.65
(1H, t, J=1.5). Anal. calcd for C24H27N3O6S: C, 59.37;
H, 5.60; N, 8.65; S, 6.60. Found: C, 59.38; H, 5.64; N,
8.64; S, 6.85.
4-tert-Butyl-N-[6-chloro-5-(3-methoxyphenoxy)pyrimidin-
4-yl]benzenesulfonamide (5). A solution of 4 (5.3 g, 19.5
mmol) and potassium 4-tert-butyl-benzenesulfonate
(9.8 g, 40.0 mmol) in DMSO (25 mL) was stirred at
120 ꢀC for 30 min. 1 N HCl was added, and the mix-
ture was extracted with AcOEt. The organic layer
was washed with water, dried and concentrated. The
residue was crystallized with MeOH to give 5 (7.5 g,
4-tert-Butyl-N-[5-(2-methoxyphenoxy)-6-(4-oxobutoxy)-
pyrimidin-4-yl]benzenesulfonamide (6b). TLC (hexane/
AcOEt (1/1)) Rf 0.43; mp 88±89ꢀC (hexane/AcOEt); IR
(Nujol) 3201, 1722, 1711, 1585, 1499, 1170, 1078 cmÀ1; 1H
NMR d 1.33 (9H, s), 1.88±2.01 (2H, m), 2.33 (2H, dt, J=
0.9, 7.2), 3.94 (3H, s), 4.34 (2H, t, J=6.3), 6.80±6.90 (1H,
m), 6.93±7.02 (2H, m), 7.08±7.16 (1H, m), 7.49, 8.01 (4H,
A2B2, J=9.0), 8.23 (1H, s), 8.60 (1H, s), 9.64 (1H, t,
J=0.9); 13C NMR d 21.35, 31.05, 35.21, 40.03, 56.03,
65.97, 112.47, 119.00, 121.17, 124.21, 125.18, 125.74,
128.21, 136.47, 145.86, 149.79, 151.04, 152.43, 157.27,
160.91, 200.98. Anal. calcd for C25H29N3O6S: C, 60.10; H,
5.85; N, 8.41; S, 6.41. Found: C, 59.90; H, 6.01; N, 8.20; S,
6.14.
ꢀ
1
77%): mp 152 C (MeOH); H NMR d 1.34 (9H, s),
3.78 (3H, s), 6.29±6.39 (1H, m), 6.40±6.42 (1H, m),
6.66±6.71 (1H, m), 7.20 (1H, t, J=8.2), 7.54, 8.03 (4H,
A2B2, J=8.2), 8.49 (1H, s). Anal. calcd for
C21H22ClN3O4S: C, 56.31; H, 4.95; Cl, 7.91; N, 9.38;
S, 7.16. Found: C, 56.25; H, 4.99; Cl, 7.52; N, 9.29; S,
7.13.
4-tert-Butyl-N-[5-(3-methoxyphenoxy)-6-(4-oxobutoxy)-
pyrimidin-4-yl]benzenesulfonamide (6e). Sodium hydride
(60% dispertion in mineral oil, 2.0 g, 50.0 mmol) was
added to 1,4-butandiol (40 mL), and the mixture was
stirred at rt for 30 min. Compound 5 (7.5 g, 16.7 mmol)
was added, and the reaction mixture was stirred at
100 ꢀC for 6.5 h. 1 N HCl was added, and the mixture
was extracted with AcOEt. The organic layer was
washed with water and saturated NaCl solution, dried
and concentrated. The residue was puri®ed by silica gel
chromatography eluting with hexane/AcOEt (1/2 to 1/3)
to give 4-tert-butyl-N-[6-(4-hydroxybutoxy)-5-(3-meth-
oxy-phenoxy)-pyrimidin-4-yl]-benzenesulfonamide (6.0 g,
71%), which was immediately used in a following reac-
4-tert-Butyl-N-[5-(2-methoxyphenoxy)-6-(4-oxopentoxy)-
pyrimidin-4-yl]benzenesulfonamide (6c). Obtained as oil,
1H NMR d 1.33 (9H, s), 1.50±1.74 (2H, m), 2.37 (2H,
dt, J=1.5, 7.2), 3.96 (3H, s), 4.33 (2H, t, J=6.0), 6.82±
6.89 (1H, m), 6.96±7.02 (4H, m), 7.07±7.14 (1H, m),
7.49, 8.01 (4H, A2B2, J=8.7), 8.24 (1H, s), 8.67 (1H, s),
9.68 (1H, t, J=1.5). Anal. calcd for C26H31N3O6S: C,
60.80; H, 6.08; N, 8.18; S, 6.24. Found: C, 60.52; H,
6.15; N, 8.14; S, 6.41.
4-tert-Butyl-N-[5-(4-methoxyphenoxy)-6-(4-oxobutoxy)-
pyrimidin-4-yl]benzenesulfonamide (6d). Mp 125 ꢀC
(hexane/AcOEt); 1H NMR d 1.34 (9H, s), 1.82±1.90 (2H,
m), 2.17 (2H, t, J=8.0), 3.78 (3H, s), 4.27 (2H, t, J=6.0),
6.71±6.83 (4H, m), 7.52, 8.05 (4H, A2B2, J=8.8), 8.27
(1H, s), 9.54 (1H, s). Anal. calcd for C25H29N3O6S: C,
60.10; H, 5.85; N, 8.41; S, 6.41. Found: C, 59.95; H, 5.69;
N, 8.39; S, 6.44.
1
tion: H NMR d 1.32 (9H, s), 1.59±1.68 (4H, m), 3.34±
3.48 (2H, m), 3.73 (3H, s), 4.24±4.48 (2H, m), 6.34±6.41
(2H, m), 6.61±6.66 (1H, m), 7.17 (1H, t, J=8.2), 7.52, 8.04
(4H, A2B2, J=8.6,), 8.29 (1H, s). To a solution of the
alcohol (6.0 g, 12.0 mmol) from the preceding reaction in
CH2Cl2 (120mL) was added PCC (3.0 g, 13.9 mmol) at
0 ꢀC, and the mixture was stirred at rt for 2 h. The reaction
mixture was concentrated, and the residue was puri®ed
by silica gel chromatography eluting with hexane/AcOEt
(1/1) to give 6e (5.0 g, 84%): TLC (hexane/AcOEt (1/1))
Rf 0.53; mp 110±111 ꢀC (hexane/AcOEt); IR(Nujol)
4-tert-Butyl-N-[5-(3-methoxyphenoxy)-2-methyl-6-(4-oxo-
butoxy)pyrimidin-4-yl]benzenesulfonamide (6f). Mp 127 ꢀC
(hexane/AcOEt); 1H NMR d 1.34 (9H, s), 1.78±1.91
(2H, m), 2.19 (2H, t, J=7.0), 2.47 (3H, s), 3.77 (3H, s),
4.27 (2H, t, J=6.2), 6.32±6.41 (2H, m), 6.60±6.65 (1H,
m), 7.15 (1H, d, J=8.0), 7.51 (2H, d, J=8.8), 8.05 (2H,
t, J=8.6), 9.54 (1H, s). Anal. calcd for C26H31N3O6S: C,
60.80; H, 6.08; N, 8.18; S, 6.24. Found: C, 60.77; H,
6.16; N, 8.14; S, 6.17.
3267, 1717, 1581, 1490, 1453, 1341, 1079 cmÀ1 1H
;
NMR d 1.34 (9H, s), 1.82±1.90 (2H, m), 2.18 (2H,
dt, J=1.2, 7.2), 3.77 (3H, s), 4.29 (2H, t, J=6.0), 6.32±
6.41 (2H, m), 6.62±6.66 (1H, m), 7.16 (1H, t, J=8.1),
7.52, 8.03 (4H, A2B2, J=8.4), 8.28 (1H, s), 9.53 (1H,

904
Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
4-tert-Butyl-N-[5-(3-methoxyphenoxy)-6-(4-oxobutoxy)-
2-phenylpyrimidin-4-yl]benzenesulfonamide (6g). Mp
washed with water, dried and concentrated. The residue
was puri®ed by silica gel chromatography eluting with
hexane/AcOEt (2/1) to give benzamide (34 mg, 4.9%),
ꢀ
1
124 C (hexane/AcOEt); H NMR d 1.31 (9H, s), 1.84±
1.98 (2H, m), 2.23 (2H, t, J=7.0), 3.78 (3H, s), 4.42 (2H,
t, J=6.0), 6.62±6.68 (1H, m), 7.18 (1H, t, J=8.0), 7.43±
7.47 (3H, m), 7.51, 8.08 (4H, A2B2, J=8.8), 8.22±8.27
(2H, m), 9.56 (1H, s). Anal. calcd for C31H33N3O6S: C,
64.68; H, 5.78; N, 7.30; S, 5.57. Found: C, 64.52; H,
5.76; N, 7.36; S, 5.50.
1
which was immediately used in a following reaction: H
NMR d 0.70 (3H, s), 1.16 (3H, s), 1.47±1.57 (2H, m), 1.70±
1.81 (2H, m), 3.36 (2H, d, J=11.4), 3.56 (2H, d, J=11.4),
3.77 (3H, s), 4.34 (1H, t, J=4.8), 4.38 (2H, t, J=6.6),
6.45±6.54 (2H, m), 6.61±6.68 (1H, m), 7.19 (1H, t, J=8.1),
7.45, 7.70 (4H, A2B2, J=8.7), 8.41 (1H, s), 8.55 (1H, s). To
a solution of benzamide (34 mg, 0.06mmol) from the
preceding reaction in formic acid (0.7 mL) was added
sodium formate (21 mg, 0.31 mmol), and the mixture
was stirred at 70 ꢀC for 1.5 h. Water was added, and the
mixture was extracted with AcOEt. The organic layer
was washed with water, dried and concentrated. The
residue was puri®ed by silica gel chromatography elut-
ing with hexane/AcOEt (2/1 to 1/1) to give 12 as oil
4-tert-Butyl-N-[6-(4-oxobutoxy)pyrimidin-4-yl]benzene-
sulfonamide (8). Mp 185±186 ꢀC (hexane/AcOEt); 1H
NMR d 1.33 (9H, s), 2.07±2.15 (2H, m), 2.62 (2H, t,
J=6.9), 4.42 (2H, t, J=6.3), 7.53, 8.03 (4H, A2B2, J=9.0),
7.86 (1H, s), 9.82 (1H, s). Anal. calcd for C18H23N3O4S: C,
57.28; H, 6.14; N, 11.13; S, 8.50. Found: C, 57.10; H,
6.10; N, 11.01; S, 8.43.
1
(19 mg, 65%): H NMR d 1.32 (9H, s), 1.89±1.98 (2H,
N-[5-Bromo-6-(4-oxobutoxy)pyrimidin-4-yl]-4-tert-butyl-
benzenesulfonamide (9). To a solution of 8 (2.0 g, 5.3
mmol) in DMF (10 mL) was added N-bromosuccinimide
(1.23 g, 6.9 mmol) at 0 ꢀC, and the mixture was stirred for
30min at the same temperature. Aqueous Na2SO3 solu-
tion was added, and the mixture was extracted with
AcOEt. The organic layer was washed with water, dried
and concentrated. The residue was puri®ed by silica gel
chromatography eluting with hexane/AcOEt (2/1) to give
9 (1.33 g, 55%): mp 164±165 ꢀC (hexane/AcOEt); 1H
NMR d 1.34 (9H, s), 2.05±2.15 (2H, m), 2.63 (2H, t,
J=7.5), 4.38 (2H, t, J=6.3), 6.66 (s, 1H), 7.53, 7.85
(4H, A2B2, J=8.4), 8.63 (1H, s), 9.84 (1H, s). Anal.
calcd for C18H22BrN3O4S 0.2H2O 0.1AcOEt: C, 47.14;
H, 4.99; Br, 17.05; N, 8.96; S, 6.84. Found: C, 47.23; H,
4.81; Br, 16.90; N, 8.98; S, 7.01.
m), 2.25 (2H, t, J=6.9), 3.77 (3H, s), 4.39 (2H, t, J=6.0),
6.46±6.53 (2H, m), 6.61±6.66 (1H, m), 7.20 (1H, t,
J=8.1), 7.46, 7.72 (4H, A2B2, J=8.1), 8.43 (1H, brs),
8.55 (1H, brs), 9.53 (1H, s). Anal. calcd for C26H29N3O5
0.2 H2O: C, 66.85; H, 6.34; N, 9.00. Found: C, 66.86; H,
6.26; N, 8.97.
4-[6-(4-tert-Butylbenzylamino)-5-(3-methoxyphenoxy)-
pyrimidin-4-yloxy]butyraldehyde (13). This compound
was prepared as oil using a similar procedure described
for 12. 1H NMR d 1.30 (9H, s), 1.87±1.96 (2H, m), 2.26
(2H, t, J=7.2), 3.76 (3H, s), 4.32 (2H, t, J=6.0), 4.64
(2H, d, J=5.7), 6.37±6.61 (3H, m), 7.13±7.22 (3H, m),
7.30±7.35 (2H, m), 8.19 (1H, s), 9.58 (1H, s). Anal. calcd
for C26H31N3O4 0.3 H2O: C, 68.64; H, 7.00; N, 9.35.
Found: C, 68.58; H, 7.12; N, 9.26.
4-Chloro-6-[3-(5,5-dimethyl-1,3-dioxinan-2-yl-propoxy]-
5-(3-methoxyphenoxy)pyrimidine (11). Sodium hydride
(60% dispersion in mineral oil, 4.65g, 0.12mol) was
added to a solution of 1012 (20.3 g, 0.12mol) in THF
(300mL) at 0 ꢀC, and the mixture was re¯uxed for 30min.
After cooling, 4 (30.0 g, 0.11mol) was added and the
reaction mixture was re¯uxed for 3 h. Water was added,
and the reaction mixture was extracted with toluene. The
organic layer was washed with water, dried and con-
centrated. The residue was puri®ed by silica gel chro-
matography eluting with hexane/AcOEt (4/1 to 2/1) to
give 11 as oil (40.2 g, 89%): 1H NMR d 0.70 (3H, s), 1.16
(3H, s), 1.50±1.60 (2H, m), 1.72±1.86 (2H, m), 3.35 (2H,
d, J=10.6), 3.55 (2H, d, J=10.6), 3.78 (3H, s), 4.33±4.43
(3H, m), 6.36±6.41 (1H, m), 6.47 (1H, t, J=2.2), 6.60±
6.65 (1H, m), 7.17 (1H, t, J=8.2), 8.41 (1H, s). Anal.
calcd for C20H25ClN2O5: C, 58.75; H, 6.16; Cl, 8.67; N,
6.85. Found: C, 58.71; H, 6.16; Cl, 8.59; N, 6.93.
4-tert-Butyl-N-[5-(3-methoxyphenoxy)-6-(4-oxobutoxy)-
pyrimidin-4-yl]-N-methylbenzenesulfonamide (14). To a
solution of 6e (20 mg, 0.04 mmol) in DMF (0.3 mL) was
added K2CO3 (6.6 mg, 0.048 mmol) and iodomethane
(25 mL, 0.40 mmol), and the mixture was stirred at rt for
12 h. Ice-water and 1 N HCl were added, and the mix-
ture was extracted with AcOEt. The organic layer was
washed with water, dried and concentrated. The residue
was puri®ed by silica gel chromatography eluting with
hexane/AcOEt (2/1 to 1/1) to give 14 as oil (19 mg,
1
93%): H NMR d 1.34 (9H, s), 1.85±1.98 (2H, m), 2.23
(2H, t, J=7.0), 3.15 (3H, s), 3.78 (3H, s), 4.37 (2H, t,
J=6.2), 6.41±6.50 (2H, m), 6.60±6.65 (1H, m), 7.17 (1H,
t, J=8.2), 7.50, 7.89 (4H, A2B2, J=8.6), 8.43 (1H, s),
9.59 (1H, s). Anal. calcd for C26H31N3O6S 0.1 H2O: C,
60.59; H, 6.10; N, 8.15; S, 6.22. Found: C, 60.44; H,
6.12; N, 8.03; S, 5.98.
2-{4-[6-(4-tert-Butylbenzenesulfonylamino)-5-(3-methoxy-
phenoxy)pyrimidin-4-yloxy]butylidene}malonic acid dime-
thyl ester (15b). To a solution of 6e (142 mg, 0.28 mmol)
in EtOH (3 mL) was added dimethy malonate (113 mg,
0.86 mmol) and piperidine (14 mL, 0.14 mmol) at rt. The
reaction mixture was stirred at 90 ꢀC for 2 h. Ice-water
and 1 N HCl were added, and the mixture was extracted
with AcOEt. The organic layer was washed with water,
dried and concentrated. The residue was puri®ed by
silica gel chromatography eluting with hexane/AcOEt
4-tert-Butyl-N-[5-(3-methoxyphenoxy)-6-(4-oxobutoxy)-
pyrimidin-4-yl]benzamide (12). Sodium hydride (60%
dispersion in mineral oil, 148 mg, 3.70 mmol) was added
to a solution of 4-tert-butyl-benzamide (654 mg, 3.70
mmol) in DMF (6.5 mL) at 0 ꢀC, and the mixture was
stirred at rt for 30 min. Compound 11 (503 mg, 1.23 mmol)
in DMF (0.5mL) was added, and the reaction mixture was
stirred at 100ꢀC for 4h. Ice-water was added, and the
mixture was extracted with AcOEt. The organic layer was

Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
905
1
(2/1) to give 15b as oil (46 mg, 26%): H NMR d 1.34
(9H, s), 1.65±1.79 (2H, m), 2.05±2.18 (2H, m), 3.75±3.78
(9H, m), 4.27 (2H, t, J=6.4), 6.31±6.41 (2H, m), 6.59±
6.65 (1H, m), 6.86 (1H, t, J=7.8), 7.15 (1H, t, J=8.2),
7.51, 8.03 (4H, A2B2, J=8.6), 7.72 (1H, brs), 8.29 (1H,
s). Anal. calcd for C30H35N3O9S: C, 58.71; H, 5.75; N,
6.85; S, 5.23. Found: C, 58.43; H, 5.77; N, 6.87; S, 4.94.
6.42 (1H, t, J=2.4), 6.52 (t, J=5.7), 6.60±6.68 (1H, m),
7.17 (t, J=5.7), 7.13±7.14 (1H, m), 7.61, 7.91 (4H, A2B2,
J=8.4), 8.27 (1H, s). Anal. calcd for C25H30N4O6S 0.2
H2O: C, 58.09; H, 5.96; N, 10.75; S, 6.15. Found: C,
57.96; H, 5.84; N, 10.64; S, 6.04.
4-tert-Butyl-N-[6-(4-ethoxyiminobutoxy)-5-(3-methoxy-
phenoxy)pyrimidin-4-yl]benzenesulfonamide (16b). This
compound was prepared as oil using a similar procedure
The following compounds were prepared using a similar
procedure described for 15b.
1
described for 16a. H NMR d 1.19 (3H, t, J=7.2), 1.34
(9H, s), 1.67±1.78 (2H, m), 2.14 (2H, dt, J=5.4, 8.1), 3.76
(3H, s), 4.04 (2H, q, J=7.2), 4.28 (2H, t, J=6.3), 6.35 (1H,
dt, J=2.4,8.4), 6.41 (1H, t, J=2.4), 6.47 (1H, t, J=5.4),
6.63 (1H, dt, J=2.4, 8.4), 7.16 (1H, t, J=8.4), 7.52, 8.04
(4H, A2B2, J=9.0), 7.66 (1H, s), 8.29 (1H, s). Anal. calcd
for C27H34N4O6S 0.1 H2O: C, 59.56; H, 6.33; N, 10.29; S,
5.89. Found: C, 59.45; H, 6.36; N, 10.26; S, 5.94.
2-{4-[6-(4-tert-Butylbenzenesulfonylamino)-5-(3-methoxy-
phenoxy)pyrimidin-4-yloxy]butylidene}malonic acid (15a).
Obtained as oil, H NMR d 1.33 (9H, s), 1.60±1.80 (2H,
m), 2.48±2.68 (2H, m), 3.72 (3H, s), 4.21±4.34 (2H, m),
6.28±6.45 (2H, m), 6.52±6.64 (1H, m), 7.12 (1H, t, J=7.8),
7.25 (1H, brs), 7.52, 7.90 (4H, A2B2, J=8.6), 8.11 (1H, s).
Anal. calcd for C28H31N3O9S: C, 57.43; H, 5.34; N, 7.18;
S, 5.48. Found: C, 57.55; H, 5.66; N, 7.29; S, 5.40.
1
4-tert-Butyl-N-(5-(3-methoxyphenoxy)-6-{4-[(1-phenyl-
methanoyl)hydrazono]butoxy}pyrimidin-4-yl)benzenesul-
fonamide (16e). To a solution of 6e (567 mg, 1.13 mmol)
in EtOH (9 mL) was added benzoylhydrazine (186 mg,
1.36 mmol) at rt, and the reaction mixture was stirred at
90 ꢀC for 1 h. Ice-water and 1 N HCl were added, and
the mixture was extracted with AcOEt. The organic
layer was washed with water, dried and concentrated.
The residue was puri®ed by silica gel chromatography
eluting with CHCl3/MeOH (20/1) to give 16e (110 mg,
26%): mp 155±156 ꢀC (AcOEt); 1H NMR d 1.33 (9H, s),
1.74±1.88 (2H, m), 2.10±2.24 (2H, m), 3.72 (3H, s), 4.32
(2H, t, J=6.3), 6.30±6.39 (2H, m), 6.57±6.62 (1H, m),
7.12 (1H, t, J=8.1), 7.41 (2H, t, J=6.9), 7.45±7.55 (1H,
m), 7.50, 8.01 (4H, A2B2, J=8.4), 7.77 (2H, d, J=7.2),
8.26 (1H, s), 9.12 (1H, s). Anal. calcd for C32H35N5O6S
0.5 H2O: C, 61.33; H, 5.79; N, 11.17; S, 5.12. Found: C,
61.44; H, 5.74; N, 11.04; S, 5.09.
2-{4-[6-(4-tert-Butylbenzenesulfonylamino)-5-(3-methoxy-
phenoxy)pyrimidin-4-yloxy]butylidene}malonic acid di-tert-
butyl ester (15c). Obtained as oil, H NMR d 1.34 (9H,
s), 1.47 (18H, s), 1.63±1.76 (2H, m), 2.02±2.13 (2H, m),
3.78 (3H, s), 4.27 (2H, t, J=6.3), 6.31±6.41 (2H, m),
6.60±6.66 (1H, m), 6.61 (1H, t, J=7.8), 7.16 (1H, t,
J=8.1), 7.51, 8.03 (4H, A2B2, J=8.7), 8.29 (1H, s). Anal.
calcd for C36H47N3O9S: C, 61.96; H, 6.79; N, 6.02; S,
4.60. Found: C, 61.86; H, 6.72; N, 6.12; S, 4.66.
1
N-[6-(5-Acetyl-6-oxohept-4-enyloxy)-5-(3-methoxyphen-
oxy)pyrimidin-4-yl]-4-tert-butylbenzenesulfonamide (15d).
1
Obtained as oil, H NMR d 1.33 (9H, s), 1.70±1.79 (2H,
m), 2.02±2.09 (2H, m), 2.22 (3H, s), 2.26 (3H, s), 3.77 (3H,
s), 4.29 (2H, t, J=5.7), 6.32±6.40 (2H, m), 6.50 (1H, t,
J=7.8), 6.61±6.65 (1H, m), 7.16 (1H, t, J=8.1), 7.72 (1H,
brs), 7.51, 8.03 (4H, A2B2, J=8.7), 8.29 (1H, s). Anal.
calcd for C30H35N3O7S 0.3 H2O: C, 61.38; H, 6.11; N,
7.16; S, 5.46. Found: C, 61.34; H, 6.20; N, 7.16; S, 5.33.
The following compounds were prepared using a similar
procedure described for 16e.
{4-[6-(4-tert-Butylbenzenesulfonylamino)-5-(3-methoxy-
phenoxy)pyrimidin-4-yloxy]butylidene}-2-cyanoacetic acid
4-tert-Butyl-N-[5-(3-methoxyphenoxy)-6-(4-semicabazono-
butoxy)pyrimidin-4-yl]benzenesulfonamide (16c). Obtained
as oil, 1H NMR d 1.33 (9H, s), 1.70±1.79 (2H, m), 1.95±
2.04 (2H, m), 3.74 (3H, s), 4.29 (2H, t, J=6.0), 6.33±
6.36 (1H, m), 6.40±6.42 (1H, m), 6.59±6.62 (1H, m), 6.90
(1H, t, J=4.8), 7.13 (1H, t, J=8.4), 7.50, 8.02 (4H,
A2B2, J=9.0), 8.26 (1H, s), 9.16 (1H, s). Anal. calcd for
C26H32N6O6S 0.3 H2O: C, 55.56; H, 5.85; N, 14.95; S,
5.71. Found: C, 55.62; H, 5.55; N, 14.76; S, 5.76.
ethyl ester (15e). Mp 124 ꢀC; H NMR d 1.30±1.39 (3H,
1
m), 1.35 (9H, s), 1.75±1.85 (2H, m), 2.29±2.37 (2H, m),
3.79 (3H, s), 4.27±4.34 (4H, m), 6.35±6.43 (2H, m), 6.62±
6.67 (1H, m), 7.18 (1H, t, J=8.1), 7.46 (1H, t, J=9.0),
7.66 (1H, s), 7.53, 8.05 (4H, A2B2, J=9.0), 8.30 (1H, s).
Anal. calcd for C30H34N4O7S: C, 60.59; H, 5.76; N, 9.42;
S, 5.39. Found: C, 60.45; H, 5.80; N, 9.32; S, 5.36.
4-tert-Butyl-N-[6-(4-hydroxyiminobutoxy)-5-(3-methoxy-
phenoxy)pyrimidin-4-yl]benzenesulfonamide (16a). To a
solution of 6e (200 mg, 0.40 mmol) and hydroxylamine
hydrochloride (36 mg, 0.52 mmol) in EtOH (1 mL) was
added pyridine (42 mL, 0.52 mmol) at 0 ꢀC. The reaction
mixture was stirred at the same temperature for 30 min.
Ice-water and 1 N HCl were added, and the mixture was
extracted with AcOEt. The organic layer was washed
with water, dried and concentrated. The residue was
puri®ed by silica gel chromatography eluting with hex-
N-{6-[4-(Acetylhydrazono)butoxy]-5-(3-methoxyphenoxy)
pyrimidin - 4 - yl} - 4 - tert - butylbenzenesulfonamide (16d).
Obtained as oil, H NMR d 1.34 (9H, s), 1.78±1.86 (2H,
m), 2.00±2.10 (2H, m), 2.17 (3H, s), 3.78 (3H, s), 4.33 (2H,
t, J=6.3), 6.34±6.41 (2H, m), 6.62±6.66 (1H, m), 6.83 (1H,
t, J=5.4), 7.18 (1H, t, J=8.1), 7.52, 8.04 (4H, A2B2,
J=8.7), 8.17 (1H, s), 8.29 (1H, s). Anal. calcd for
C27H33N5O6S 0.3 H2O: C, 57.80; H, 6.04; N, 12.48; S,
5.72. Found: C, 57.67; H, 5.92; N, 12.31; S, 5.68.
1
1
ane/AcOEt (1/1) to give 16a as oil (77 mg, 38%): H
NMR d 1.29 (9H, s), 1.55±1.70 (2H, m), 1.86±2.12 (2H,
m), 3.72 (3H, s), 4.25 (2H, t, J=6.3), 6.28±6.36 (1H, m),
4-tert-Butyl-N-(5-(3-methoxyphenoxy)-6-{4-[(1-pyridin-
4-ylmethanoyl)hydrazono]butoxy}pyrimidin-4-yl)benzene
sulfonamide (16f). Mp 118±119 ꢀC; 1H NMR d 1.33

906
Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
(9H, s), 1.70±1.90 (2H, m), 2.02±2.24 (2H, m), 3.72 (3H,
s), 4.30 (2H, t, J=5.6), 6.26±6.38 (2H, m), 6.54±6.63 (1H,
m), 7.12 (1H, t, J=8.2), 7.51±7.72 (3H, m), 7.50, 8.00 (4H,
A2B2, J=8.6), 8.25 (1H, s), 8.67 (1H, s). Anal. calcd for
C31H34N6O6S 0.3 H2O: C, 59.66; H, 5.59; N, 13.47; S,
5.14. Found: C, 59.87; H, 5.61; N, 13.16; S, 4.97.
solution (pH 7.4), each well was incubated with 25pM
125I]endothelin-3 in 0.1 mL of HEPES-bu€ered Hanks'
[
solution containing protease inhibitors in the absence
and presence of various concentrations of compounds.
Equilibrium binding studies were performed at 37ꢀC for
60min. The incubation was terminated by ®ltering
through Whatman GF/C glass ®ber ®lters. The ®lters were
washed 4 times with 2.5 mL each of 50mM Tris±HCl
(pH 7.4), and the radioactivity was counted. Non-
speci®c binding was determined in the presence of
0.1 mM endothelin-3.
N-{6-[4-(Benzenesulfonylhydrazono)butoxy]-5-(3-methoxy-
phenoxy)pyrimidin-4-yl}-4-tert-butylbenzenesulfonamide
1
(16g). Obtained as oil, H NMR d 1.33 (9H, s), 1.65±
1.76 (2H, m), 1.93±2.04 (2H, m), 3.74 (3H, s), 4.16 (2H,
t, J=5.7), 6.27±6.35 (2H, m), 6.58±6.62 (1H, m), 6.87
(1H, t, J=5.1), 7.12 (1H, t, J=8.1), 7.36±7.90 (6H,
m), 7.52, 8.04 (4H, A2B2, J=8.7), 8.25 (1H, s). Anal.
calcd for C31H35N5O7S2 0.2 H2O: C, 56.64; H, 5.43; N,
10.65; S, 9.76. Found: C, 56.61; H, 5.41; N, 10.53; S,
9.56.
Pharmacokinetic analysis
The pharmacokinetics of 6b and 6e were evaluated in
Jcl-Sprague±Dawley rats. The test compound was for-
mulated as a 2.5 mg/mL solution in an N,N-dimethyla-
cetamide : PEG400 : saline (10:50:40, by volume) for the
intravenous route and as a 15.0 mg/mL solution/sus-
pension in 0.5% methyl cellulose/saline for the oral
route. Three individual rats were dosed by slow bolus
injection into the jugular vein at 5 mg/kg (2 mL/kg) or
by gavage at 30 mg/kg (2 mL/kg). Serial blood samples
(200 mL) were taken from the jugular vein using a
heparin-coated syringe at 0.03, 0.17, 0.5, 1, 2, 4, 8, and
24 h postdose for the intravenous groups and at 0.25, 0.5,
1, 2, 3, 4, 6, 8, and 24 h postdose for the oral groups.
Plasma samples obtained from blood by centrifugation
were analyzed by reverse-phase HPLC following
methanol precipitation of the plasma proteins. Assum-
ing dose proportionality and correcting for the di€er-
ence in dosing, comparison of the area under the curve
(AUC) value after an oral dosing with that obtained
after an intravenous dose provided an estimate of the
bioavailability (BA).
Molecular modeling
Molecular modeling and other graphical manipulation
were performed using the SYBYL 6.6.1 software pack-
age.15 A three-dimensional model of compound 1a was
built from the crystal structure of hydroxymethyl deri-
vative of 1a (R=CH2OH in Scheme 1) by replacing the
terminal hydroxymethyl group with an aldehyde group.
The points used for ®tting the modeled 1a on the crystal
structure of 6e were carbon 4 in the phenyl ring of the
benzenesulfonamide group, the N and H atoms of the
sulfonamide group, and the C and O atoms of the alde-
hyde group.
Receptor binding studies. ETA: Rat aortic smooth muscle
A7r5 cells expressing only ETA receptors were obtained
from Dainippon Seiyaku (Osaka) and cultured in Dul-
becco's modi®ed Eagle's medium (GIBCO, Grand Island,
NY) supplemented with 10% fetal calf serum (GIBCO),
10mM HEPES bu€er (pH 7.4), 50mg/mL of streptomy-
cin, and 50U/mL of penicillin G (GIBCO) in a 5% CO2-
95% air incubator at 37 ꢀC in 48-well culture plates. After
3 to 5 days, the culture medium was aspirated and the cells
were washed twice with ice-cold HEPES (20 mM)-buf-
fered Hanks' solution (pH 7.4). Each well was incubated
with 8.3 pM [125I]endothelin-1 in 0.2 mL of ice-cold
HEPES-bu€ered Hanks' solution containing 0.1 mM
phenylmethylsulfonyl ¯uoride (PMSF), 10 mg/mL apro-
tonin, 10 mg/mL leupeptin, pepstatatin A, 250 mg/mL
bacitracin, and 10 mg/mL soybean trypsin inhibitor in the
absence and presence of various concentrations of com-
pounds. Equilibrium binding studies were performed at
37 ꢀC for 60 min. The incubation was terminated by rapid
removal of the incubation medium and addition of
0.25 mL of ice-cold HEPES-bu€ered Hanks' solution.
Free ligand was removed by washing the intact attached
cells with ice-cold HEPES-bu€ered Hanks' solution.
The cells were then resolved in 0.1 N NaOH and trans-
ferred to a test tube, then the radioactivity was counted.
Nonspeci®c binding was determined in the presence of
0.1 mM endothelin-1.
In vivo antagonistic activities to ET-1 induced responses
Male Wistar rats were anesthetized with 2% halothane.
Polyethylene catheters (SP31; Natsume Seisakusho,
Japan) were implanted in the femoral artery and vein,
which were passed subcutaneously and held at the back
of the neck. After the rats were allowed to recover
overnight, blood pressure (mm Hg) was measured under
conscious conditions via a catheter inserted into the
femoral artery by connection to a pressure transducer
(TP-200T; Nihon Kohden). ET-1 (0.1 nmol/kg) was
administered via the femoral vein after the blood pres-
sure had been stable for about 1 h. We ®rst con®rmed
the e€ects of the ET-1 treatment on mean artery blood
pressure (MABP) in conscious rats. After the ET-1
treatment, compound 6e (30 mg/kg) was administered
orally. Next, ET-1 (0.1 nmol/kg) was administered from
1 to 5 h at 1-h intervals, and the ET-1 induced responses
on MABP were monitored repeatedly.
Acknowledgements
The authors thank Dr. Masanori Nishiuchi for evaluat-
ing the pharmacokinetic pro®les of 6b and 6e. We also
thank Mr. Hiroshi Nakai for performing the X-ray
crystallographic analyses.
ETB: for ETB receptor binding assay, we employed COS-7
cells transfected with porcine ETB receptors. After the
cells were washed with HEPES (20 mM)-bu€ered Hanks'

Y. Kanda et al. / Bioorg. Med. Chem. 9 (2001) 897±907
907
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