Inhibition of restriction enzymes EcoRI, BamHI and HindIII by phenethylphenylphthalimides derived from thalidomide

Article abstract of DOI:10.1248/cpb.59.880

We discovered inhibitors of the restriction enzymes EcoRI, BamHI and HindIII by screening our library of compounds with a phenethylphenylphthalimide skeleton, based on α-glucosidase inhibitors and liver X receptor antagonists derived from thalidomide. Str

Full text of DOI:10.1248/cpb.59.880

880
Note
Chem. Pharm. Bull. 59(7) 880—884 (2011)
Vol. 59, No. 7
Inhibition of Restriction Enzymes EcoRI, BamHI and HindIII by
Phenethylphenylphthalimides Derived from Thalidomide
Kazunori MOTOSHIMA, Minoru ISHIKAWA, Yuichi HASHIMOTO,* and Kazuyuki SUGITA
Institute of Molecular & Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan.
Received February 17, 2011; accepted April 19, 2011; published online April 25, 2011
We discovered inhibitors of the restriction enzymes EcoRI, BamHI and HindIII by screening our library of
compounds with a phenethylphenylphthalimide skeleton, based on a-glucosidase inhibitors and liver X receptor
antagonists derived from thalidomide. Structural development afforded the potent restriction enzyme inhibitors
25 and 26.
Key words restriction enzyme; endonuclease; enzyme inhibitor; phthalimide
We have been engaged in the creation of bioactive com- found to inhibit the growth of influenza A.27) We also investi-
pounds based on the multi-template approach utilizing gated the mechanism of this growth inhibition and found that
thalidomide (1).1—6) The basic idea of the multi-template ap- these compounds inhibit influenza virus PA endonuclease.
proach is that the number of three-dimensional spatial struc- So, we speculated that various inhibitors toward other DNA
tures (fold structures) of human proteins is only approxi- recognizing enzymes might be possible to generate by modi-
mately 1000, which is much smaller than the number of fication of phenethylphenylphthalimide derivatives. Thus, we
human proteins, estimated to be 50000—70000.7—9) There- screened our library of these derivatives for inhibitory activ-
fore, ignoring physical/chemical interactions, a template/ ity towards restriction enzymes EcoRI, BamHI and HindIII,
scaffold structure which is spatially complementary to one which resemble endonucleases and are frequently utilized in
fold structure might serve as a multi-template for structural biological experiments to proof our hypothesis. EcoRI, iso-
development of ligands that would interact specifically with lated from strains of Escherichia (E.) coli, recognizes the fol-
at least 50—70 different human proteins. We have focused lowing palindromic DNA sequence: 5ꢀ-GAATTC-3ꢀ
on thalidomide (1) as a candidate multi-template structure. 3ꢀ-CTTAAG-5ꢀ. BamHI, derived from Bacillus amylolique-
Thalidomide (1) was launched as a hypnotic/sedative drug in faciens, recognizes the palindromic sequence: 5ꢀ-GGATCC-
the 1950’s, but was withdrawn from the market in the 1960’s 3ꢀ 3ꢀ-CCTAGG-5ꢀ. HindIII, derived from Haemophilus influ-
because of severe teratogenicity. But, subsequently, thalido- enzae, recognizes the palindromic sequence 5ꢀ-AAGCTT-3ꢀ
mide (1) has been established to be useful for the treatment 3ꢀ-TTCGAA-5ꢀ.
of Hansen’s disease and multiple myeloma. Further, many re-
ports have appeared on its therapeutic potential for the treat- Results and Discussion
ment of a range of diseases, including cancers, rheumatoid
EcoRI, BamHI and HindIII were selected as target en-
arthritis and diabetes.1—6,10) Using our multi-template ap- zymes and pBR322 was selected as the assay substrate. At
proach, we have developed many biological response modi- the initial screening of phenethylphenylphthalimide deriva-
fiers, including tumor necrosis factor-a (TNF-a) production tives, compound 19 showed EcoRI-inhibitory activity with
regulators,11,12) nitric oxide synthase (NOS) inhibitors,13,14) the IC50 value of 70 mM (Table 1). Compound 16 also showed
cyclooxygenase (COX) inhibitors,15—17) liver X receptor weak EcoRI-inhibitory activity. Since compounds 16 and 19
(LXR) antagonists,18—21) a-glucosidase inhibitors21—24) and possess a tetrachlorophthalimide skeletons and a dihydroxy-
glycogen phosphorylase inhibitors.25) On the other hand, phenyl group, these might be important for the EcoRI-in-
based on a result that thalidomide inhibits the replication of hibitory activity. Next, we tried to improve the inhibitory ac-
human immunodeficiency virus type 1 (HIV-1),26) we specu- tivity of compounds 16 and 19. Based on the results of struc-
lated that thalidomide and its derivatives might inhibit the ture–activity relationship studies, phenethylphenylphthalim-
growth of other viruses. We focused on inhibitors of the in- ides bearing trihydroxy substituents were designed as candi-
fluenza virus A, and phenethylphenylphthalimide derivatives dates for more potent EcoRI inhibitors.
2, 3 and 4, which were derived from thalidomide-related a-
These analogs were synthesized as shown in Chart 1.
glucosidase inhibitors and liver X receptors (Fig. 1), were 3,4,5-Trimethoxybenzyl alcohol was allowed to react with
PBr3 to generate 3,4,5-trimethoxybenzyl bromide. Reaction
of the intermediate with triphenylphosphine provided triph-
enylphosphonium salt 30. Diphenylethene derivatives 31a—c
were prepared as E/Z mixtures by Wittig reaction of ni-
trobenzaldehyde with 30. After simultaneous reduction of the
nitro group and olefin moiety of compounds 31a—c,
phenethylphenylphthalimides 33a—c and 34a—c were ob-
tained by condensation with phthalic or tetrachlorophthalic
anhydride, respectively. The conversion of the methoxy
groups to hydroxyl groups using BBr3 was conducted to give
Fig. 1. Chemical Structures of Thalidomide (1) and Compounds 2—4
trihydroxy analogs 20—22 and 24—26. Dihydroxyl analogs
∗ To whom correspondence should be addressed. e-mail: hashimot@iam.u-tokyo.ac.jp
© 2011 Pharmaceutical Society of Japan

July 2011
881
23 and 27—29 were also generated in the reaction and puri- Hence, a hydroxyl group at the 5ꢁ position might be impor-
fied by silica gel column chromatography. tant for EcoRI-inhibitory activity. Since compounds with a
Introduction of a hydroxyl group at the 5ꢁ position of 16 hydroxyl group at the 5ꢁ position of the non-substituted ph-
and 19, i.e., compounds 25 and 26, resulted in a drastic in- thalimide skeleton (20—22) had no apparent activity, the
crease of EcoRI-inhibitory activity (Table 2). On the other tetrachloro substitution of the phthalimide skeleton might
hand, the potency of analogs possessing a methoxy group in- also be important for EcoRI-inhibitory activity.
stead of a hydroxyl group, i.e., compounds 28 and 29, was
Subsequently, we evaluated the BamHI- and HindIII-in-
decreased compared with that of 25 and 26, respectively. hibitory activities of the phenethylphenylphthalimide deriva-
tives possessing EcoRI-inhibitory activity (Table 3). Com-
Table 1. EcoRI-Inhibitory Activity of Phenethylphenylphthalimide Deriv-
atives (2—19)
pounds 25 and 26 showed BamHI- and HindIII-inhibitory ac-
tivities. Compounds 16 and 23, which possess weak EcoRI-
inhibitory activity, also showed weak BamHI-inhibitory ac-
Table 2. EcoRI-Inhibitory Activity of Trisubstituted Phenethylphenyl-
phthalimide Derivatives (20—29)
Inhibitory ratio at 100 mM
Compound
X
Position
R
(IC50 [mM])
5
6
2
7
8
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
2ꢀ
2ꢀ
2ꢀ
3ꢀ
3ꢀ
3ꢀ
4ꢀ
4ꢀ
4ꢀ
2ꢀ
2ꢀ
2ꢀ
3ꢀ
3ꢀ
3ꢀ
4ꢀ
4ꢀ
4ꢀ
H
OMe
OH
H
OMe
OH
H
OMe
OH
H
OMe
OH
H
0
0
0
0
0
0
0
0
0
0
0
0
0
0
29%
0
Inhibitory ratio at 100 mM
(IC50 [mM])
Compound
X
Position
R
3
9
20
21
22
—
—
23
24
25
26
27
28
29
H
H
H
H
H
2ꢀ
3ꢀ
4ꢀ
2ꢀ
3ꢀ
4ꢀ
2ꢀ
3ꢀ
4ꢀ
2ꢀ
3ꢀ
4ꢀ
OH
OH
OH
OMe
OMe
OMe
OH
OH
OH
OMe
OMe
OMe
0
0
0
10
11
12
13
4
14
15
16
17
18
19
—
—
H
17
0
Cl
Cl
Cl
Cl
Cl
Cl
100% (4.2)
97% (5.3)
2
4
OMe
OH
H
OMe
OH
0
85% (45)
68% (70)
Reagents and conditions: (a) PBr3, CH2Cl2, 0 °C; (b) PPh3, toluene, reflux; (c) K2CO3, 18-crown-6, CH2Cl2, reflux; (d) H2, Pd/C, EtOAc, rt; (e) phthalic or tetra-
chlorophthalic anhydride; (f) BBr3, CH2Cl2, 0 °C.
Chart 1

882
Vol. 59, No. 7
for 30 min. The precipitate was filtered to give the target compound (4.52 g,
86% for 2 steps) as a white solid. 1H-NMR (500 MHz, CDCl3) d: 7.79—
7.75 (m, 9H), 7.65—7.61 (m, 6H), 6.47 (d, 2H, Jꢃ2.4 Hz), 5.40 (d, 2H,
Jꢃ14.0 Hz), 3.77 (s, 3H), 3.52 (s, 6H). FAB-MS m/z: 443 [MꢄBr]ꢅ.
1,2,3-Trimethoxy-5-[2-(2-nitrophenyl)vinyl]benzene (31a) This com-
pound was prepared from 2-nitrobenzaldehyde and 30 by means of GP-A.
Compound 31a was obtained in 96% yield as a yellow solid. 1H-NMR
(500 MHz, CDCl3) 31aZ d: 8.09 (dd, 1H, Jꢃ8.5, 1.2 Hz), 7.46 (dd, 1H,
Jꢃ7.3, 1.2 Hz), 7.40 (t, 2H, Jꢃ7.9 Hz), 6.87 (d, 1H, Jꢃ12.2 Hz), 6.67 (d,
1H, Jꢃ12.2 Hz), 6.25 (s, 2H), 3.92 (s, 3H), 3.59 (s, 6H). 31aE d: 7.97 (dd,
1H, Jꢃ8.5, 1.2 Hz), 7.75 (d, 1H, Jꢃ7.9 Hz), 7.60 (td, 1H, Jꢃ7.3, 1.2 Hz),
7.50 (d, 1H, Jꢃ15.9 Hz), 7.35 (d, 1H, Jꢃ7.9 Hz), 7.02 (d, 1H, Jꢃ15.9 Hz),
6.76 (s, 2H), 3.88 (s, 3H), 3.80 (s, 6H). FAB-MS m/z: 315 [M]ꢅ, 316
[MꢅH]ꢅ.
Table 3. EcoRI-, BamHI- and HindIII-Inhibitory Activities of Phenethyl-
phenylphthalimide Derivatives (16, 19, 23, 25, 26, 29)
Inhibitory ratio at 100 mM
(IC50 [mM])
Compound
X
Position
R
EcoRI
BamHI HindIII
1,2,3-Trimethoxy-5-[2-(3-nitrophenyl)vinyl]benzene (31b) This com-
pound was prepared from 3-nitrobenzaldehyde and 30 by means of GP-A.
Compound 31b was obtained in 100% yield as a yellow solid. 1H-NMR
(500 MHz, CDCl3) 31bZ d: 8.16 (s, 1H), 8.05 (dd, 1H, Jꢃ8.5, 2.4 Hz), 7.60
(d, 1H, Jꢃ7.9 Hz), 7.40 (t, 1H, Jꢃ7.9 Hz), 6.69 (d, 1H, Jꢃ12.2 Hz), 6.58 (d,
1H, Jꢃ11.6 Hz), 6.43 (s, 2H), 3.85 (s, 3H), 3.67 (s, 6H). 31bE d: 8.37 (t,
1H, Jꢃ1.8 Hz), 8.10 (dd, 1H, Jꢃ7.9, 1.2 Hz), 7.79 (d, 1H, Jꢃ7.9 Hz), 7.53
(t, 1H, Jꢃ7.9 Hz), 7.18 (d, 1H, Jꢃ16.5 Hz), 7.05 (d, 1H, Jꢃ15.9 Hz), 6.78
(s, 2H), 3.94 (s, 6H), 3.89 (s, 3H). FAB-MS m/z: 315 [M]ꢅ, 316 [MꢅH]ꢅ.
1,2,3-Trimethoxy-5-[2-(4-nitrophenyl)vinyl]benzene (31c) This com-
pound was prepared from 4-nitrobenzaldehyde and 30 by means of GP-A.
Compound 31c was obtained in 97% yield as a yellow solid. 1H-NMR
(500 MHz, CDCl3) 31cZ d: 8.12 (dt, 2H, Jꢃ8.5, 2.4 Hz), 7.44 (d, 2H,
Jꢃ8.5 Hz), 6.73 (d, 1H, Jꢃ12.2 Hz), 6.58 (d, 1H, Jꢃ11.6 Hz), 6.43 (s, 2H),
3.86 (s, 3H), 3.68 (s, 6H). 31cE d: 8.23 (d, 2H, Jꢃ9.2 Hz), 7.63 (d, 2H,
Jꢃ9.2 Hz), 7.20 (d, 1H, Jꢃ16.5 Hz), 7.05 (d, 1H, Jꢃ15.9 Hz), 6.78 (s, 2H),
3.93 (s, 6H), 3.89 (s, 3H). FAB-MS m/z: 315 [M]ꢅ, 316 [MꢅH]ꢅ.
16
19
23
25
26
29
Cl
Cl
H
Cl
Cl
Cl
3ꢀ
4ꢀ
4ꢀ
3ꢀ
4ꢀ
4ꢀ
H
H
OMe
OH
OH
OMe
29%
68% (70)
17%
100% (4.2)
97% (5.3)
85% (45)
40%
41%
23%
56%
64%
56%
51%
74%
25%
67%
78%
41%
tivity and modest HindIII-inhibitory activity.
In conclusion, we discovered restriction enzyme inhibitors
among our library of phenethylphenylphthalimide deriva-
tives. Compounds 25 and 26 showed potent EcoRI-inhibitory
activity with the IC50 values of 4.2 and 5.3 mM, respectively,
together with modest BamHI- and HindIII-inhibitory activi-
ties. These results indicate that the phenethylphenylphthalim-
ide skeleton can be used as a multi-template for endonucle-
ase inhibitors.
2-[2-(3,4,5-Trimethoxyphenyl)ethyl]phenylamine (32a) This com-
pound was prepared from 31a by means of GP-B. Compound 32a was ob-
tained in 93% yield as a colorless oil. 1H-NMR (500 MHz, CDCl3) d:
7.07—7.03 (m, 2H), 6.74 (t, 1H, Jꢃ7.3 Hz), 6.68 (d, 1H, Jꢃ7.3 Hz), 6.37 (s,
2H), 3.83 (s, 3H), 3.81 (s, 6H), 3.49 (br s, 2H), 2.89—2.86 (m, 2H), 2.80—
2.77 (m, 2H). FAB-MS m/z: 287 [M]ꢅ, 288 [MꢅH]ꢅ.
Experimental
General Melting points were determined by using a Yanagimoto hot-
stage melting point apparatus and are uncorrected. 1H-NMR spectra were
recorded on a JEOL JNM-GX500 (500 MHz) spectrometer. Chemical shifts
are expressed in parts per million relative to tetramethylsilane. Mass spectra
were recorded on a JEOL JMS-DX303 spectrometer.
General Procedure A (GP-A) To a solution of nitrobenzaldehyde in
dehydrated CH2Cl2 were added benzyltriphenylphosphonium salt (1.0 eq),
potassium carbonate (1.1 eq) and 18-crown-6 (0.18 eq) and the mixture was
refluxed, then filtered and concentrated. The residue was purified by silica
gel column chromatography to give the target compound as an E/Z mixture.
General Procedure B (GP-B) Substituted nitrobenzene was dissolved
in EtOAc and hydrogenated with 10% Pd/C (catalytic amount). The mixture
was filtered through a pad of Celite, and the filtrate was evaporated under re-
duced pressure. The residue was purified by silica gel columnn chromatogra-
phy to give the target compound.
General Procedure C (GP-C) A mixture of phthalic anhydride and
substituted aniline (1.0 eq) was heated at 160 or 200 °C for 1 h. After the re-
action was completed, the residue was purified by silica gel column chro-
matography to give the target compound.
General Procedure D (GP-D) To a solution of substituted methoxy-
benzene in dehydrated dichloromethane was added dropwise boron tribro-
mide (1.0 M solution in dichloromethane) (10 eq) at 0 °C under an argon
3-[2-(3,4,5-Trimethoxyphenyl)ethyl]phenylamine (32b) This com-
pound was prepared from 31b by means of GP-B. Compound 32b was ob-
1
tained in 91% yield as a white solid. H-NMR (500 MHz, CDCl3) d: 7.08
(dt, 1H, Jꢃ7.9, 1.2 Hz), 6.61 (d, 1H, Jꢃ7.3 Hz), 6.54 (d, 1H, Jꢃ6.7 Hz),
6.54 (s, 1H), 6.38 (s, 2H), 3.83 (s, 6H), 3.83 (s, 3H), 2.86—2.83 (m, 2H),
2.83—2.80 (m, 2H). FAB-MS m/z: 287 [M]ꢅ, 288 [MꢅH]ꢅ.
4-[2-(3,4,5-Trimethoxyphenyl)ethyl]phenylamine (32c) This com-
pound was prepared from 31c by means of GP-B. Compound 32c was ob-
1
tained in 90% yield as a white solid. H-NMR (500 MHz, CDCl3) d: 6.98
(dt, 2H, Jꢃ8.5, 2.4 Hz), 6.64 (dt, 2H, Jꢃ8.5, 2.4 Hz), 6.37 (s, 2H), 3.83 (s,
6H), 2.80 (s, 3H). FAB-MS m/z: 287 [M]ꢅ, 288 [MꢅH]ꢅ.
2-{2-[2-(3,4,5-Trimethoxyphenyl)ethyl]phenyl}isoindole-1,3-dione
(33a) This compound was prepared from phthalic anhydride and 32a by
means of GP-C. Compound 33a was obtained in 87% yield as a white solid.
1H-NMR (500 MHz, CDCl3) d: 7.96 (dd, 2H, Jꢃ5.5, 2.4 Hz), 7.81 (dd, 2H,
Jꢃ5.5, 2.4 Hz), 7.41 (td, 1H, Jꢃ7.9, 1.8 Hz), 7.36 (td, 1H, Jꢃ7.9, 1.8 Hz),
7.33 (dd, 1H, Jꢃ7.9, 1.8 Hz), 7.21 (dd, 1H, Jꢃ7.9, 1.8 Hz), 6.21 (s, 2H),
3.78 (s, 3H), 3.68 (s, 6H), 2.82—2.79 (m, 2H), 2.79—2.76 (m, 2H). FAB-
MS m/z: 417 [M]ꢅ, 418 [MꢅH]ꢅ.
2-{3-[2-(3,4,5-Trimethoxyphenyl)ethyl]phenyl}isoindole-1,3-dione
(33b) This compound was prepared from phthalic anhydride and 32b by
atmosphere. The mixture was stirred for 5—60 min, then poured into water means of GP-C. Compound 33b was obtained in 94% yield as a white solid.
and extracted with CH2Cl2. The organic layer was washed with H2O and
dried over MgSO4. The solvent was removed under reduced pressure. The
residue was purified by silica gel column chromatography to give the target
compound.
1H-NMR (500 MHz, CDCl3) d: 7.96 (dd, 2H, Jꢃ5.5, 2.4 Hz), 7.80 (dd, 2H,
Jꢃ5.5, 2.4 Hz), 7.42 (t, 1H, Jꢃ7.9 Hz), 7.27—7.26 (m, 2H), 7.21 (d, 1H,
Jꢃ7.9 Hz), 6.37 (s, 2H), 3.82 (s, 6H), 3.82 (s, 3H), 3.00—2.97 (m, 2H),
2.92—2.89 (m, 2H). FAB-MS m/z: 417 [M]ꢅ, 418 [MꢅH]ꢅ.
Triphenyl-(3,4,5-trimethoxybenzyl)phosphonium Bromide (30) To a
solution of 3,4,5-trimethoxybenzyl alcohol (1.98 g, 10 mmol) in
dichloromethane (15 ml) was added dropwise phosphorus tribromide
(658 ml, 7 mmol) at 0 °C under an argon atmosphere. The mixture was
2-{4-[2-(3,4,5-Trimethoxyphenyl)ethyl]phenyl}isoindole-1,3-dione
(33c) This compound was prepared from phthalic anhydride and 32c by
means of GP-C. Compound 33c was obtained in 54% yield as a white solid.
1H-NMR (500 MHz, CDCl3) d: 7.96 (dd, 2H, Jꢃ5.5, 2.4 Hz), 7.80 (dd, 2H,
stirred for 1 h, then poured into ice water (50 ml) and alkalized with sat. Jꢃ5.5, 2.4 Hz), 7.34 (d, 2H, Jꢃ8.5 Hz), 7.32 (d, 2H, Jꢃ8.5 Hz), 6.37 (s,
NaHCO3 (50 ml). The aqueous mixture was extracted with CH2Cl2 (20 mlꢂ
3). The organic layer was washed with H2O (20 mlꢂ2) and then dried over
MgSO4. The solvent was removed under reduced pressure. The residue was
purified by silica gel column chromatography to give an intermediate. To a
solution of the intermediate (2.48 g, 9.50 mmol) in toluene (30 ml) was
added triphenylphosphine (3.74 g, 14.25 mmol). The mixture was refluxed
2H), 3.84 (s, 6H), 3.83 (s, 3H), 2.98—2.96 (m, 2H), 2.91—2.88 (m, 2H).
FAB-MS m/z: 417 [M]ꢅ, 418 [MꢅH]ꢅ.
4,5,6,7-Tetrachloro-2-{2-[2-(3,4,5-trimethoxyphenyl)ethyl]phenyl}-
isoindole-1,3-dione (34a) This compound was prepared from tetra-
chlorophthalic anhydride and 32a by means of GP-C. Compound 34a was
obtained in 77% yield as a yellow solid. 1H-NMR (500 MHz, CDCl3) d:

July 2011
883
4,5,6,7-Tetrachloro-2-{3-[2-(3,4,5-trihydroxyphenyl)ethyl]phenyl}-
isoindole-1,3-dione (25) and 4,5,6,7-Tetrachloro-2-{3-[2-(3,4-dihydroxy-
5-methoxyphenyl)ethyl]phenyl}isoindole-1,3-dione (28) These com-
pounds were prepared from 34b by means of GP-D. Compounds 25 and 28
was obtained in 13% yield as colorless needles after recrystallization from
EtOAc/n-hexane and in 10% yield as yellow needles after recrystallization
from EtOAc/n-hexane, respectively.
7.42 (td, 1H, Jꢃ7.3, 1.2 Hz), 7.36 (td, 1H, Jꢃ7.3, 1.2 Hz), 7.31 (dd, 1H,
Jꢃ7.3, 1.2 Hz), 7.17 (dd, 1H, Jꢃ7.3, 1.2 Hz), 6.26 (s, 2H), 3.78 (s, 3H), 3.75
(s, 6H), 2.82—2.80 (m, 2H), 2.79—2.77 (m, 2H). FAB-MS m/z: 553 [M]ꢅ,
554 [MꢅH]ꢅ, 555 [Mꢅ2]ꢅ, 556 [Mꢅ3]ꢅ, 557 [Mꢅ4]ꢅ, 558 [Mꢅ5]ꢅ.
4,5,6,7-Tetrachloro-2-{3-[2-(3,4,5-trimethoxyphenyl)ethyl]phenyl}-
isoindole-1,3-dione (34b) This compound was prepared from tetra-
chlorophthalic anhydride and 32b by means of GP-C. Compound 34b was
obtained in 84% yield as a yellow solid. 1H-NMR (500 MHz, CDCl3) d:
7.43 (m, 1H), 7.25—7.22 (m, 3H), 6.35 (s, 2H), 3.82 (s, 9H), 2.99—2.96 (m,
2H), 2.91—2.87 (m, 2H). FAB-MS m/z: 553 [M]ꢅ, 554 [MꢅH]ꢅ, 556
[Mꢅ3]ꢅ, 558 [Mꢅ5]ꢅ.
4,5,6,7-Tetrachloro-2-{4-[2-(3,4,5-trimethoxyphenyl)ethyl]phenyl}-
isoindole-1,3-dione (35c) This compound was prepared from tetra-
chlorophthalic anhydride and 32c by means of GP-C. Compound 35c was
obtained in 98% yield as a yellow solid. 1H-NMR (500 MHz, CDCl3) d:
7.31 (s, 4H), 6.36 (s, 2H), 3.83 (s, 3H), 3.83 (s, 6H), 2.99—2.96 (m, 2H),
2.91—2.88 (m, 2H). FAB-MS m/z: 553 [M]ꢅ, 554 [MꢅH]ꢅ, 555 [Mꢅ2]ꢅ,
556 [Mꢅ3]ꢅ, 557 [Mꢅ4]ꢅ, 558 [Mꢅ5]ꢅ.
25: mp 249.0—250.5 °C. 1H-NMR (500 MHz, DMSO-d6) d: 8.64 (br s,
2H), 7.85 (br s, 1H), 7.44 (t, 1H, Jꢃ7.9 Hz), 7.33—7.30 (m, 2H), 7.23 (dt,
1H, Jꢃ8.5, 1.8 Hz), 6.15 (s, 2H), 2.84—2.81 (m, 2H), 2.65—2.62 (m, 2H).
FAB-MS m/z: 511 [M]ꢅ, 512 [MꢅH]ꢅ, 513 [Mꢅ2]ꢅ, 514 [Mꢅ3]ꢅ, 515
[Mꢅ4]ꢅ, 516 [Mꢅ5]ꢅ. Anal. Calcd for C22H13Cl4NO5: C, 51.49, H, 2.55, N,
2.73. Found: C, 51.31, H, 2.73, N, 2.66.
28: mp 208.5—210.0 °C. 1H-NMR (500 MHz, DMSO-d6) d: 8.67 (s, 1H),
7.98 (s, 1H), 7.44 (t, 1H, Jꢃ7.9 Hz), 7.32 (d, 2H, Jꢃ1.8 Hz), 7.32—7.31 (m,
1H), 7.23 (d, 1H, Jꢃ7.9 Hz), 6.30—6.29 (m, 2H), 3.69 (s, 3H), 2.89—2.85
(m, 2H), 2.73—2.70 (m, 2H). FAB-MS m/z: 525 [M]ꢅ, 526 [MꢅH]ꢅ, 527
[Mꢅ2]ꢅ, 528 [Mꢅ3]ꢅ, 529 [Mꢅ4]ꢅ, 530 [Mꢅ5]ꢅ. Anal. Calcd for
C23H15Cl4NO5: C, 52.40, H, 2.87, N, 2.66. Found: C, 52.16, H, 2.97, N, 2.61.
4,5,6,7-Tetrachloro-2-{4-[2-(3,4,5-trihydroxyphenyl)ethyl]phenyl}-
isoindole-1,3-dione (26) and 4,5,6,7-Tetrachloro-2-{4-[2-(3,4-dihydroxy-
5-methoxyphenyl)ethyl]phenyl}isoindole-1,3-dione (29) These com-
pounds were prepared from 34c by means of GP-D. Compounds 26 and 29
was obtained in 4% yield as a white powder after recrystallization from
EtOAc/n-hexane and in 23% yield as colorless needles after recrystallization
from EtOAc/n-hexane, respectively.
2-{2-[2-(3,4,5-Trihydroxyphenyl)ethyl]phenyl}isoindole-1,3-dione (20)
This compound was prepared from 33a by means of GP-D. Compound 20
1
was obtained in 75% yield as a white solid. mp 104.0—108.0 °C. H-NMR
(500 MHz, CDCl3) d: 7.94 (dd, 2H, Jꢃ5.5, 1.8 Hz), 7.80 (dd, 2H, Jꢃ5.5,
2.4 Hz), 7.41—7.38 (m, 1H), 7.35—7.32 (m, 2H), 7.20—7.17 (m, 1H), 6.15
(s, 2H), 5.17 (br s, 2H), 5.03 (br s, 1H), 2.78—2.75 (m, 2H), 2.73—2.70 (m,
2H). FAB-MS m/z: 375 [M]ꢅ, 376 [MꢅH]ꢅ. Anal. Calcd for
C22H17NO5·1/2H2O: C, 68.74, H, 4.72, N, 3.64. Found: C, 68.63, H, 4.88, N,
3.53.
2-{3-[2-(3,4,5-Trihydroxyphenyl)ethyl]phenyl}isoindole-1,3-dione (21)
This compound was prepared from 21 by means of GP-D. Compound 33b
was obtained in 17% yield as a white powder after recrystallization from
EtOAc/n-hexane. mp 155.5—158.5 °C. 1H-NMR (500 MHz, DMSO-d6) d:
8.64 (br s, 2H), 7.96 (dd, 2H, Jꢃ5.5, 2.4 Hz), 7.90 (dd, 2H, Jꢃ5.5, 2.4 Hz),
7.84 (br s, 1H), 7.41 (t, 1H, Jꢃ7.9 Hz), 7.33 (s, 1H), 7.29 (d, 1H, Jꢃ7.9 Hz),
7.25 (d, 1H, Jꢃ7.9 Hz), 6.16 (s, 2H), 2.83—2.80 (m, 2H), 2.65—2.62 (m,
2H). FAB-MS m/z: 375 [M]ꢅ, 376 [MꢅH]ꢅ. Anal. Calcd for
C22H17NO5·1/2H2O: C, 68.74, H, 4.72, N, 3.64. Found: C, 68.45, H, 4.82, N,
3.60.
26: mp 259.0—262.0 °C. 1H-NMR (500 MHz, DMSO-d6) d: 8.65 (s, 2H),
7.85 (s, 1H), 7.39 (d, 2H, Jꢃ7.9 Hz), 7.31 (d, 2H, Jꢃ8.5 Hz), 6.17 (s, 2H),
2.86—2.83 (m, 2H), 2.69—2.65 (m, 2H). FAB-MS m/z: 511 [M]ꢅ, 512
[MꢅH]ꢅ, 513 [Mꢅ2]ꢅ, 514 [Mꢅ3]ꢅ, 515 [Mꢅ4]ꢅ, 516 [Mꢅ5]ꢅ. Anal.
Calcd for C22H13Cl4NO5·1/3H2O: C, 51.04, H, 2.63, N, 2.71. Found: C,
51.02, H, 2.80, N, 2.55.
29: mp 215.5—218.0 °C. 1H-NMR (500 MHz, DMSO-d6) d: 8.68 (s, 1H),
7.99 (s, 1H), 7.39 (d, 2H, Jꢃ7.9 Hz), 7.32 (d, 2H, Jꢃ7.9 Hz), 6.32 (d, 1H,
Jꢃ1.8 Hz), 6.29 (d, 1H, Jꢃ1.8 Hz), 3.70 (s, 3H), 2.90—2.87 (m, 2H),
2.76—2.73 (m, 2H). FAB-MS m/z: 525 [M]ꢅ, 526 [MꢅH]ꢅ, 527 [Mꢅ2]ꢅ,
528 [Mꢅ3]ꢅ, 529 [Mꢅ4]ꢅ, 530 [Mꢅ5]ꢅ. Anal. Calcd for
C23H15Cl4NO5·1/3H2O: C, 51.81, H, 2.96, N, 2.63. Found: C, 51.98, H,
2.94, N, 2.62.
Restriction Enzyme-Inhibitory Activity EcoRI, BamHI and HindIII
were purchased from Fermentas. pBR322 was purchased from Nippon
Gene, Japan. Restriction enzyme (0.2 unit) in Milli Q water (16 ml) and
10ꢂbuffer (2 ml) in an Eppendorf tube were incubated for 3 min at 37 °C,
then 2 ml pBR322 solution (final concentration 0.01 mg/ml) was added. The
mixture was incubated at 37 °C for 30 min, 37 °C for 30 min or 37 °C for
15 min in the inhibitory assays for EcoRI, BamHI or HindIII, respectively.
Then EcoRI, BamHI or HindIII was inactivated by incubation at 75 °C for
15 min, 85 °C for 20 min or 85 °C for 20 min. pBR322 was analyzed by
agarose electrophoresis and stained with ethidium bromide. Quantitative
analysis was performed by measuring the amount of pBR322 using FLA-
7000 (GE Healthcare, U.K.).
2-{4-[2-(3,4,5-Trihydroxyphenyl)ethyl]phenyl}isoindole-1,3-dione (22)
and 2-{4-[2-(3,4-Dihydroxy-5-methoxyphenyl)ethyl]phenyl}isoindole-1,3-
dione (23) These compounds were prepared from 33c by means of GP-D.
Compounds 22 and 23 was obtained in 22% yield as a yellow powder after
recrystallization from EtOAc/n-hexane and in 9% yield as colorless needles
after recrystallization from EtOAc/n-hexane, respectively.
22: mp 218.5—221.0 °C. 1H-NMR (500 MHz, DMSO-d6) d: 8.65 (s, 2H),
7.96 (dd, 2H, Jꢃ5.5, 2.4 Hz), 7.90 (dd, 2H, Jꢃ5.5, 2.4 Hz), 7.84 (s, 1H),
7.35 (d, 2H, Jꢃ8.5 Hz), 7.34 (d, 2H, Jꢃ8.5 Hz), 6.17 (s, 2H), 2.86—2.82
(m, 2H), 2.68—2.65 (m, 2H). FAB-MS m/z: 375 [M]ꢅ, 376 [MꢅH]ꢅ. Anal.
Calcd for C23H19NO5·1/3H2O: C, 69.28, H, 4.67, N, 3.67. Found: C, 68.52,
H, 4.59, N, 3.57.
23: mp 215.0—216.5 °C. 1H-NMR (500 MHz, DMSO-d6) d: 8.68 (s, 1H),
7.98 (s, 1H), 7.96 (dd, 2H, Jꢃ5.5, 2.4 Hz), 7.90 (dd, 2H, Jꢃ5.5, 2.4 Hz),
7.36 (d, 2H, Jꢃ8.5 Hz), 7.34 (d, 2H, Jꢃ8.5 Hz), 6.32 (d, 1H, Jꢃ1.8 Hz),
6.29 (d, 1H, Jꢃ1.8 Hz), 2.90—2.86 (m, 2H), 2.76—2.72 (m, 2H). FAB-MS
m/z: 389 [M]ꢅ, 390 [MꢅH]ꢅ. Anal. Calcd for C23H19NO5·1/3H2O: C, 68.96,
H, 5.01, N, 3.54. Found: C, 69.81, H, 5.02, N, 3.39.
Acknowledgment The work described in this paper was partially sup-
ported by Grants-in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan, and the Japan Soci-
ety for the Promotion of Science.
4,5,6,7-Tetrachloro-2-{2-[2-(3,4,5-trihydroxyphenyl)ethyl]phenyl}-
isoindole-1,3-dione (24) and 4,5,6,7-Tetrachloro-2-{2-[2-(3,4-dihydroxy-
5-methoxyphenyl)ethyl]phenyl}isoindole-1,3-dione (27) These com-
pounds were prepared from 34a by means of GP-D. Compounds 24 and 27
was obtained in 21% yield as a yellow powder after recrystallization from
EtOAc/n-hexane and in 31% yield as a yellow solid, respectively.
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