Specific nonpeptide inhibitors of puromycin-sensitive aminopeptidase with a 2,4(1H,3H)-quinazolinedione skeleton.

Article abstract of DOI:10.1248/cpb.51.1273

Potent, specific, chemically stable and non-peptide/small-molecular inhibitors of puromycin-sensitive aminopeptidase, such as 3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (PAQ-22, 5), were prepared by the structural development of a potent PSA inhibitor, 2-(2,6-diethylphenyl)-1,2,3,4-tetrahydroisoquinoline-1,3-dione (PIQ-22, 4). The design was carried out partly by applying electrostatic potential field information obtained from PIQ-22 (4) and its derivatives based on thalidomide (2). This information revealed that a positive electrostatic potential field around the benzylic methylene in the tetrahydroisoquinoline ring is necessary for potent activity. Lineweaver-Burk plot analysis showed that PAQ-22 (5) and its derivatives inhibit puromycin-sensitive aminopeptidase (PSA) in a non-competitive manner. These potent and specific PSA inhibitors showed dose-dependent cell invasion-inhibitory activity in a Matrigel assay using mouse melanoma B16F10/L5 cells, in spite of their low cell toxicity.

Full text of DOI:10.1248/cpb.51.1273

November 2003
Chem. Pharm. Bull. 51(11) 1273—1282 (2003)
1273
Specific Nonpeptide Inhibitors of Puromycin-Sensitive Aminopeptidase
with a 2,4(1H,3H)-Quinazolinedione Skeleton
*
Hiroki KAKUTA, Aya TANATANI, Kazuo NAGASAWA, and Yuichi HASHIMOTO
Institute of Molecular & Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan.
Received June 13, 2003; accepted July 26, 2003
Potent, specific, chemically stable and non-peptide/small-molecular inhibitors of puromycin-sensitive
aminopeptidase, such as 3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (PAQ-22, 5), were prepared by the
structural development of a potent PSA inhibitor, 2-(2,6-diethylphenyl)-1,2,3,4-tetrahydroisoquinoline-1,3-dione
(PIQ-22, 4). The design was carried out partly by applying electrostatic potential field information obtained from
PIQ-22 (4) and its derivatives based on thalidomide (2). This information revealed that a positive electrostatic po-
tential field around the benzylic methylene in the tetrahydroisoquinoline ring is necessary for potent activity.
Lineweaver–Burk plot analysis showed that PAQ-22 (5) and its derivatives inhibit puromycin-sensitive aminopep-
tidase (PSA) in a non-competitive manner. These potent and specific PSA inhibitors showed dose-dependent cell
invasion-inhibitory activity in a Matrigel assay using mouse melanoma B16F10/L5 cells, in spite of their low cell
toxicity.
Key words puromycin-sensitive aminopeptidase; inhibitor; quinazolinedione; structure–activity relationship
and impaired pain response.35) On the other hand, the degra-
dation of enkephalins was not detected in these mice.35)
Thus, the physiological function of PSA is unclear. In addi-
tion, the lack of specific inhibitors has made the elucidation
of the physiological function of PSA difficult,36) and there-
fore, development of PSA-specific inhibitors which can be
used as biological/biochemical probes is important.
Though PIQ-22 (4) is a potent and specific inhibitor of
PSA, it is chemically labile and easily oxidized at the ben-
zylic methylene position in the tetrahydroisoquinoline ring to
give an inactive tricarbonyl derivative,20,26,27) which makes 4
unsuitable for use as a biological/biochemical tool. There-
fore, we designed and developed chemically stable PSA spe-
cific inhibitors, 3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazo-
linedione (5: PAQ-22) and N-(2,6-diethylphenyl)-2-amino-
4H-3,1-benzoxazin-4-one (6: PAZOX-22) (Fig. 1), partly
based on electrostatic potential field considerations.37) We
also developed fluorescent analogs of 5 which can be used as
probes to visualize PSA in living cells, i.e., 3-(9-anthra-
cenyl)-2,4(1H,3H)-quinazolinedione (7: ANTAQ) and 3-
(2,6-diethyl-4-dansylaminophenyl)-1-methyl-2,4(1H,3H)-
quinazolinedione (8: DAMPAQ-22) (Fig. 1).38)
Neutral alanine-aminopeptidases (NAPs), which degrade
tissue barriers and regulate cell adhesion and mobility, are
thought to play important roles in the malignant metastatic
cascade, including the tumor cell invasion step, i.e., the pas-
sage of tumor cells through connective tissue barriers, which
consist of various adhesive molecules such as fibronectin,
laminin, and so on.1—15) In the course of our structural devel-
opment studies of thalidomide,16—20) we reported the prepara-
tion and structure–activity relationships of novel small-mole-
cular nonpeptide aminopeptidase inhibitors with a cyclic
imide skeleton.20—27) Among them, 2-(2,6-diethylphenyl)-
1,2,3,4-tetrahydro-isoquinoline-1,3-dione (4: PIQ-22) (Fig.
1)20,21,23—27) showed specific and more potent NAP-inhibitory
activity than bestatin (3)13) (Fig. 1) in a well-established
assay system for aminopeptidase N-inhibitory activity (APN:
EC 3.4.11.2/CD13), i.e., measuring 7-amino-4-methyl-
coumarin (AMC) liberated from L-methylcoumarylamide
(Ala-AMC)12,13) with intact human acute lymphoblastic
leukemia MOLT-4 cells.11,23—27) This enzyme is thought to
play a crucial role in matrix degradation and invasion by
tumor cells. However, our previous studies revealed that 4
does not inhibit the activity of authentic APN, and that the
true target enzyme of 4 is puromycin-sensitive aminopepti-
dase (PSA: EC 3.4.11.14).20,21,26,27)
PSA, which was purified as a candidate enkephalinase by
Hersh and McKelvy in 1981,28) is a single chain protein of 99
kDa9,28) that hydrolyzes N-terminal amino acids with a pref-
erence for basic and hydrophobic residues.29—32) This en-
zyme is an exopeptidase containing a Zn2+ ion in its catalytic
site.28—30) It is distributed in the brain (especially the hip-
pocampus), the central nervous system (CNS), the heart,
skeletal muscles, kidney, placenta, and testicles.29,30) In cells,
PSA is localized both to the nucleus and to the cyto-
plasm.30,33,34) The activity of PSA is inhibited by puromycin
(1) (Fig. 1) at a low concentration,28) but 1 is not a specific
inhibitor of PSA. This is because the amino acid sequences
recognized by 1, i.e., the catalytic site for hydrolysis and the
substrate-binding site, are similar among various NAPs.35)
Recently, it was reported that PSA gene-deficient mice ob-
tained by a gene-trapping method show increased anxiety
Fig. 1. Structures of Puromycin (1), Thalidomide (2), Bestatin (3), PSA-
Specific Inhibitors (PIQ-22: 4, PAQ-22: 5, PAZOX-22: 6), and Fluorescent
Probes for Visualization of PSA (ANTAQ: 7, DAMPAQ-22: 8)
To whom correspondence should be addressed. e-mail: hashimot@iam.u-tokyo.ac.jp
© 2003 Pharmaceutical Society of Japan

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Vol. 51, No. 11
Here, we describe in detail the design of PAQ-22 (5) and pearance of the PSA-inhibitory activity. However, introduc-
its derivatives, their structure–activity relationship, and their tion of an electron-donating group such as an amino (10, 11)
inhibitory activity towards cell invasion.
or a hydroxyl (12, 13) group into the phthalimide moiety of
the inactive compound 14 resulted in the reappearance of the
activity. On the other hand, introduction of an electron-with-
Results and Discussion
Design of PAQ-22 (5): Structure–Activity Relationship drawing nitro group (15, 16) had no effect on the activity.
Studies of N-2,6-Diethylphenyl(homo)phthalimides with The tricarbonyl derivative of PIQ-22 (4), i.e., 17, which is
Electrostatic Potential Field Maps Typical previous re- also an auto-oxidation product of 4 in a protic solvent under
sults regarding the structure–activity relationship are illus- air with exposure to daylight, is completely inactive toward
trated in Fig. 2.20,26,27) The ring reduction of PIQ-22 (4), i.e., PSA. The difluorinated analog of 4, i.e., 18, which was syn-
N-2,6-diethylphenylphthalimide (14), resulted in the disap- thesized in order to prevent auto-oxidization at the benzylic
Fig. 2. PSA-Inhibitory Activitiy of N-Phenyl(homo)phthalimide Derivatives and Electrostatic Potential Field Maps of Selected Compounds
Positions indicated by arrows are designated as the south-western part in this paper.
Fig. 6. Electrostatic Potential Field Maps of PAQ-22 (5) and Its Derivatives

November 2003
1275
methylene moiety in the isoquinoline ring, is also completely of 5 was performed through reaction of the amide 24 and
inactive.
triphosgene in 1,2-dichloroethene in 79% yield.45) N-Alky-
Comparison of the structures of active compounds with lated PAQ-22s (22a—e) were prepared by treatment with
those of inactive/weakly active compounds, i.e., amino/hy- sodium hydride in dimethylformamide, followed by reaction
droxyl-containing arylphthalimide analogs (10—13) versus of the resulting salt with an alkyl halide at room temperature.
inactive derivatives (14—17), led us to guess that the active
A structural isomer of PAQ-22 (5), N-(2,6-diethylphenyl)-
compounds possess a common feature. We focused on the 2-amino-4H-3,1-benzoxazinone (6: PAZOX-22), which can
electrostatic potential around the benzylic methylene moiety have a benzylic proton at the corresponding position through
of the homophthalimide analogs (4, 17, 18: colored moieties protonation or tautomerization from the imine form to the
in structural formulae in the upper panels in Fig. 2, and re- amine form, was prepared from the ureido intermediate 21b
gions indicated by arrows in the lower part of Fig. 2) and the under acidic conditions (Fig. 4).37,45)
corresponding parts of phthalimide analogs (10—16: also
A derivative oxygenated at the corresponding benzylic NH
colored or indicated by arrows in Fig. 2). These parts appear position, 3-(2,6-diethylphenyl)-2H-1,3-benzoxazine-2,4(3H)-
to be located at roughly similar positions in relation to the quinazolinedione (26: POQ-22), was prepared according to
fixed N-2,6-diethylphenyl group, i.e., the bottom left, when Brown et al.46) Briefly, a solution of N-(2,6-diethylphenyl)-2-
the electrostatic potential field maps of the compounds are hydroxybenzamide 25 in pyridine at 0 °C was treated with
drawn as presented in the lower part of Fig. 2. In this paper, ethyl chloroformate, then the mixture was allowed to warm
the position is designated as the “southwestern part” (Fig. 2). to room temperature and heated to reflux (Fig. 5).
The electrostatic potential surfaces of these molecules39—43)
The structures of the prepared compounds were confirmed
generated by means of computer calculations43) are shown in by 1H-NMR, mass spectroscopy, and elemental analysis,
the lower part of Fig. 2.
which gave appropriate analytical values.
All of the active compounds listed in Fig. 2 can be re-
Inhibition of PSA by the compounds was assessed by mea-
garded as possessing a positive electrostatic field around the suring 7-amino-4-methylcoumarin (AMC) liberated from L-
southwestern part of the molecules. On the other hand, all of methylcoumarylamide (Ala-AMC) using intact human acute
the inactive compounds listed in Fig. 2 possess a negative lymphoblastic leukemia MOLT-4 cells.11,26,27,37) In order to
electrostatic field at the corresponding location.
examine the specificity of PSA-inhibitory activity, inhibition
The results suggested that a positive electrostatic field of another aminopeptidase, APN, by the compounds was also
around the benzylic methylene of the tetrahydroisoquinoline assessed by measuring AMC liberated from Ala-AMC with
ring, at which auto-oxidation occurs in the case of PIQ-22 human promyelocytic leukemia HL-60 cells. All experiments
(4), is necessary for potent PSA-inhibitory activity. Based on were performed at least in duplicate.
this consideration, we designed N-2,6-diethylphenyl-2,4-
(1H,3H)-quinazolinedione (PAQ-22, 5) (Fig. 3).
Syntheses and PSA-Inhibitory Activity of PAQ-22 (5)
and Related Analogs PAQ-22 (5) was prepared by the
condensation of 2,6-diethylaniline (19) and methyl anthrani-
late in the presence of triphosgene by the method previously
reported, as shown in Fig. 4.37,45) An alternative preparation
Fig. 3. Design of PAQ-22 (5)
(a) Triphosgene, triethylamine, 1,2-dichloroethane (q.y.). (b) Methyl anthranilate, Et2O. (c) Anthranilic acid, THF. (d) NaOHaq, EtOH. (e) HClaq (65%, 3 steps). (f) NaH, DMF,
alkyl halide (62%-q.y.). (g) H2SO4aq, NaHCO3 (40%, 2 steps). (h) 2-Nitrobenzoyl chloride, pyridine, CHCl3 (84%). (i) H2, 10% Pd/C, EtOAc (q.y.). (j) Triphosgene, triethylamine,
1,2-dichloroethane (79%).
Fig. 4. Preparation of PAQ-22 (5) and Its Derivatives

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Vol. 51, No. 11
(a) Ethyl chloroformate, pyridine (39%).
Fig. 5. Preparation of POQ-22 (26)
Table 1. Aminopeptidase-Inhibitory Activity of PAQ-22 Derivatives
PSA IC50
APN IC50
R
(mM)
(a) CS2, DBU, DMF (78%). (b) NaOH, formalin, EtOH, 60 °C (90%). (c) P4S10, xy-
lene (88%). (d) 2,6-Diethylaniline, K2CO3, DMF. (e) FeO(OH), NH2NH2, MeOH 60 °C
(98%, 2 steps). (f) (CCl3O)2CO, Et3N, 1,2-dichloroethane, reflux (83%).
5: PAQ-22
22a: PAQ-22M
22b: PAQ-22E
22c: PAQ-22P
22d: PAQ-22B
22e: PAQ-22Bn
26: POQ-22
NH
NMe
NEt
NPr
NBu
NBn
O
3.8
3.4
0.7
0.6
0.9
1.1
53.4
9.8
0.6
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Fig. 7. Synthesis of Analogs Related to PAQ-22 (5)
6: PAZOX-22
1: Puromycin
–
–
4.8
The inhibitory activities are listed as IC50 values in Table
1. PAQ-22 (5) and N-alkylated PAQ-22(s) (22a—e) have po-
tent inhibitory activity towards PSA, but no inhibitory activ-
ity towards APN. PAZOX-22 (6) showed a similar level of
activity to PAQ-22, whereas POQ-22 (26) had only low in-
hibitory activity towards PSA.
Electrostatic potential surfaces of these molecules were
generated by means of computer calculations.44) The results
are shown in Fig. 6. POQ-22 (26), whose PSA-inhibitory ac-
tivity is low, shows a negative electrostatic field at the south-
(a) 2,6-Diethylaniline, EDC, HOBt, DMF (65—80%). (b) (CCl3O)2CO, Et3N, 1,2-
dichloroethane, reflux (70—80%). (c) ClCO2Et, Et3N, THF, Ϫ20 °C, (d) NaN3/PhCH3,
reflux. (e) 2,6-Diethylaniline. (f) NaOH, H2O/EtOH; HCl/H2O (85%, 4 steps). (g) H2,
Pd-C, EtOH (80%-q.y.).
Fig. 8. Synthesis of Nitro/Amino-Substituted PAQ-22 Derivatives
western part of the molecule. On the other hand, PAQ-22 (5), dihydro derivative, 28, was prepared by treatment of 24 with
N-alkylated PAQ-22(s) and PAZOX-22 (6), which have po- formalin and sodium hydroxide in ethanol (Fig. 7-i). The
tent PSA-inhibitory activity, show positive electrostatic fields preparation of the 4-thiocarbonyl derivative, 29, was
around the corresponding region. The results are consistent achieved by treatment of PAQ-22 (5) with phosphorus sulfide
with our hypothesis, i.e., the requirement of a positive elec- in xylene under reflux for 10 h (Fig. 7-ii). The 4-dihydro ana-
trostatic field at the southwestern part for inhibitory activity. log, 30, was prepared by treatment of 2-amino-N-(2,6-di-
Moreover, PAQ-22 (5), and N-alkylated PAQ-22(s) (22a—e) ethylphenyl)benzenemethanamine (32), which was prepared
were chemically quite stable under conditions where PIQ-22 from 2-nitrobenzylbromide (31) and 2,6-diethylaniline (19),
(4) was completely auto-oxidized.
with triphosgene in 1,2-dichloroethane (Fig. 7-iii). The nitro-
Structural Development of PAQ-22 (5): Effects of De- substituted derivatives of PAQ-22 (5), 35a and b, were pre-
rivatization of the Quinazolinedione Moiety and N- pared by the treatment of 34 with triphosgene in 1,2-
Phenyl Moiety To reach a better understanding of the dichloroethane (Fig. 8). The regio-isomer, 35c, was prepared
structure–activity relationships of PAQ-22-type PSA in- by the method described by Canonne et al.47) The resulting
hibitors, we prepared other PAQ-22 derivatives. First, we re- compounds were reduced by catalytic hydrogenation over
placed the carbonyl group of PAQ-22 (5) with a thiocarbonyl Pd/C in ethanol to give amino derivatives, 36a—c (Fig. 8).
group or a methylene group (Fig. 7, Table 2). Next we intro- Compounds 38—45 were obtained by the method used for
duced amino/nitro groups into the quinazolinedione aromatic the preparation of PAQ-22 (5), with the corresponding ani-
ring of PAQ-22 (5) (Fig. 8, Table 3). The role of the 2,6-di- line analog(s). The PSA-inhibitory activity of prepared com-
ethyl groups on the side aromatic ring of the PAQ-22 (5) was pounds is shown in terms of IC50 values in Tables 2—4.
also examined (Table 4).
As shown in Table 2, among compounds 27—31, only 27
Preparation of 27, which has a thiocarbonyl group at the 2- showed potent PSA-inhibitory activity, which is comparable
position, was achieved by treatment of the amide compound to that of PAQ-22 (5). All of the other compounds (28—31)
24 with carbon disulfide and DBU in DMF (Fig. 7-i). The 2- are less potent than PAQ-22 (5) or inactive, suggesting that

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Table 2. Aminopeptidase (PSA and APN)-Inhibitory Activity of PAQ-22 Table 4. Aminopeptidase (PSA and APN)-Inhibitory Activity of 38—45
(5) and Related Compounds
PSA IC50 APN IC50
PSA IC50
APN IC50
X
Y
(mM)
(mM)
5: PAQ-22
CO
CS
CO
CH2
CO
CO
CO
CS
CO
CH2
3.8
3.8
25.3
26.7
Ͼ100
Ͼ100
Ͼ100
Ͼ100
Ͼ100
PN-22 (38)
39
0.8
>100
>100
>100
27
29
28
30
Ͼ100
Table 3. Aminopeptidase (PSA and APN)-Inhibitory Activity of PAQ-22
Derivatives
PSA IC50
APN IC50
R
(mM)
40: PAQ-00
41: PAQ-11
5: PAQ-22
42: PAQ-33
43
H
Me
Et
iPr
MeO
>100
78.3
3.8
>100
>100
>100
>100
>100
PSA IC50
APN IC50
R
(mM)
7.0
4.5
5: PAQ-22
35a
H
3.8
76.8
33.4
14.6
10.0
6.6
>100
>100
>100
>100
>100
>100
>100
6-NO2
7-NO2
8-NO2
6-NH2
7-NH2
8-NH2
35b
35c
36a
36b
36c
0.5
PSA IC50
APN IC50
the carbonyl groups are important for potent PSA-inhibitory
activity. The compounds with a carbonyl group at the 4-posi-
tion (27, 28) are more potent inhibitors than the correspond-
ing compounds possessing a carbonyl group at the 2-position
(29, 30, respectively).
RЈ
(mM)
44
45
H
OMe
37.0
1.0
>100
>100
Concerning the effects of a substituent on the aromatic
ring of the quinazolinedione moiety, amino derivatives
(36a—c) possess more potent PSA-inhibitory activity than
the corresponding nitro derivatives (35a—c) (Table 3).
Among the compounds listed in Table 3, 36c shows the most
potent activity. This result suggests that a positive electrosta-
tic field near the 2-carbonyl group is important for potent in-
hibitory activity. This information led us to design and syn-
thesize a ring-closure derivative, 38, which also showed more
potent PSA-inhibitory activity than PAQ-22 (5) (Table 4).
The aromatic ring on the side chain seems to be necessary
for potent activity, since 39 showed no inhibitory activity
(Table 4).
Concerning the effects of 2,6-dialkyl substituents on the
N-phenyl group, the non-alkylated derivative, 40, is almost
inactive (Table 4). Introduction of alkyl or methoxyl groups
at the ortho positions (5, 41—43) resulted in the appearance
of PSA-inhibitory activity. The activity decreased in the
order of: PAQ-22 (5)Ͼ43Ͼ42Ͼ41Ͼ40, indicating the supe-
riority of an ethyl group over other alkyl and methoxyl
groups, as was the case for PIQ-22 (4) derivatives reported
previously (Table 4).23—27) These results suggest that moder-
ately bulky substitution and a hydrophobic environment
around the nitrogen atom in the quinazolinedione moiety and
perpendicular orientation of the N-aryl group with respect to
the plane of the quinazolinedione moiety are critical factors
for potent PSA-inhibitory activity. The information concern-
ing the structure–activity relationship of PAQ-22 derivatives
described above is summarized in Fig. 9.
PSA Inhibition Mode of PAQ-22 and Its Derivatives
Because there is no obvious structural relationship between
PAQ-22 (5) and puromycin (1), the mode of inhibition
elicited by these two inhibitors, as well as bestatin (3), was
analyzed by Lineweaver–Burk plot analysis.20,26,27) The
analysis clearly indicates that PAQ-22 (5) and its derivatives
inhibit PSA in a non-competitive manner, while puromycin
(1) and bestatin (3) inhibit PSA in a competitive manner. The
results suggest that PAQ-22 (5) and its derivatives do not act

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Vol. 51, No. 11
As shown in Fig. 10, PIQ-22 (4), PAQ-22 (5), PAQ-22M
(22a) and PN-22 (38) exhibited potent and dose-dependent
cell invasion-inhibiting activity. Compared with PIQ-22 (4),
the PSA inhibitors PAQ-22 (5), PAQ-22M (22a) and PN-22
(38), which are all more potent than PIQ-22 (4), showed
more potent cell invasion-inhibitory activity than 4. Thus,
potent cell invasion-inhibiting activity of the compounds co-
incides with potent PSA inhibition, suggesting that PSA
plays a role in cell mobility.
Fig. 9. Important Factors for Potent and Specific PSA Inhibitors
Conclusion
In conclusion, we created potent, specific, chemically sta-
ble and non-peptide/small-molecular PSA inhibitors, such as
PAQ-22 (5), by structural development of a PSA inhibitor,
PIQ-22 (4). Design was based on electrostatic potential field
information, gathered from PIQ-22 (4) and its derivatives.
The new PSA inhibitors showed potent and dose-dependent
tumor cell invasion-inhibitory activity, despite their low cell
toxicity. Our PSA inhibitors should be useful tools for inves-
tigation of the molecular/cell level mechanism(s) of action
and the physiological roles of PSA.
Experimental
Abbreviations AcOH, acetic acid; AcONa, sodium acetate; DMF, N,N-
dimethylformamide; BINAP, 2,2Ј-bis(diphenylphosphino)-1,1Ј-binaphthyl;
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMSO, dimethyl sulfoxide;
EDC,
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride;
Fig. 10. Effects of PSA Inhibitors on Invasion of B16F10 Cells into
Matrigel
EtOH, ethanol; EtOAc, ethyl acetate; Et3N, triethylamine; Hex, n-hexane;
HOBt, 1-hydroxybenzotriazole; MeOH, methanol.
General Comments Melting points were determined with a Yanagi-
moto hot-stage melting point apparatus and are uncorrected. Elemental
analyses were carried out in the Microanalytical Laboratory, Faculty of Phar-
maceutical Sciences, University of Tokyo, and results were within Ϯ0.3% of
the theoretical values. NMR spectra were recorded on a JEOL JNM-a-500
(500 MHz) spectrometer.
as substrate-mimics, but bind at a specific site of PSA which
is not the substrate-binding site, while puromycin (1) and
bestatin (3) act as substrate-mimics. Identification of the
binding site of PAQ-22 (5) and its derivatives is in progress.
Cell Invasion-Inhibiting Activity of PAQ-22 and Its De-
rivatives First we examined the cytotoxicity of our PSA in-
hibitors. Since puromycin (1) induces apoptosis, PSA is
2-(2,6-Diethylphenyl)-1,2,3,4-tetrahydroisoquinoline-1,3,4-trione (17)
1
mp 163—165 °C; H-NMR (500 MHz, CDCl3) d: 8.41 (dd, 1H, Jϭ7.5, 1.0
Hz), 8.34 (dd, 1H, Jϭ7.5, 1.0 Hz), 7.97 (td, 1H, Jϭ7.5, 1.0 Hz), 7.92 (td, 1H,
Jϭ7.5, 1.0 Hz), 7.42 (t, 1H, Jϭ7.5 Hz), 7.26 (d, 2H, Jϭ7.5 Hz), 2.41 (q, 4H,
thought to participate in proteolytic events that are essential Jϭ7.5 Hz), 1.14 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 308 (MϩH)ϩ; IR (KBr,
cmϪ1) 1680, 1280, 1260; Anal. Calcd for C19H17NO3: C, 74.24; H, 5.59; N,
for cell growth and viability.48) The cell proliferation-in-
hibitory activity of our PSA inhibitors were examined using
MOLT-4 cells with WST-1. However, none of the inhibitors
4.56. Found: C, 74.32; H, 5.84; N, 4.43.
2-(2,6-Diethylphenyl)-4,4-difluoro-1,2,3,4-tetrahydroisoquinoline-1,3-
1
dione (18) mp 122—123 °C; H-NMR (500 MHz, CDCl3) d: 8.32 (d, 1H,
examined showed significant cell proliferation-inhibitory ac-
tivity at the concentration of 10 mM. Only slight proliferation
inhibitory activity was observed for several compounds at the
concentration of 100 mM (data not shown). On the other hand,
puromycin (1) showed potent cell proliferation-inhibitory ac-
tivity even at the concentration of 1 mM: the efficacy was 48%
at 1 mM, and 98% at 10 mM. The results suggest that PSA is
not closely associated with proliferation, and the cell prolif-
eration-inhibitory activity elicited by puromycin (1) can be
attributed to the characteristic inhibition of protein synthesis
in ribosomes by puromycin, because of the structural similar-
ity of 1 to aminoacyl-tRNA.
Next, we examined the cell invasion-inhibiting activity of
our PSA-inhibitors. As already reported, PSA has been pro-
posed to be a target molecule of cell invasion inhibitors.23—27)
Tumor cell invasion inhibition was assessed by counting
mouse metastatic tumor cells (B16F10/L5 mouse melanoma
cells) that invaded into Matrigel-coated filters, as described
by Albini et al.49) Each sample was tested in triplicate, and
the mean values for selected compounds are shown in Fig.
10.
Jϭ7.5 Hz), 8.00 (d, 1H, Jϭ7.5 Hz), 7.90 (t, 1H, Jϭ7.5 Hz), 7.81 (t, 1H, Jϭ
7.5 Hz), 7.42 (t, 1H, Jϭ7.5 Hz), 7.26 (d, 2H, Jϭ7.5 Hz), 2.40 (q, 4H, Jϭ7.5
Hz), 1.14 (t, 6H, Jϭ7.5 Hz); Anal. Calcd for C19H17NO2F2: C, 69.30; H,
5.17; N, 4.25. Found: C, 69.02; H, 5.11; N, 4.46.
General Procedure for the Synthesis of 3-Substituted 2,4(1H,3H)-
Quinazolinediones from Amines To a mixture of amine (1.0 mmol) and
Et3N (1.0 mmol) in 1,2-dichloroethane (10 ml) was added triphosgene (0.40
mmol), and the resulting solution was heated at reflux until the starting
amine disappeared (for ca. 2 h). Next, methyl anthranilate (1.0 mmol) was
added, and the resulting mixture was stirred at reflux for 2 h. The solvent
was removed under reduced pressure, and to the residue was added EtOH (2
ml) and 2 N NaOH solution (1 ml). The resulting solution was then heated in
a steam bath until a clear solution was obtained. This solution was cooled,
diluted with water, acidified with 2 N HCl (ca. 2 ml), and extracted with
EtOAc. The organic layer was washed with water and brine, dried over
MgSO4 and concentrated under reduced pressure. Purification via silica gel
column chromatography (eluent: EtOAc/Hex) gave the 3-substituted
2,4(1H,3H)-quinazolinedione.
PAQ-22: (3-(2,6-Diethylphenyl)-2,4(1H,3H)-quinazolinedione) (5)
According to the general procedure, 5 was obtained in 65% yield as color-
less crystalline cubes after the recrystallization from EtOAc. mp 226 °C; 1H-
NMR (500 MHz, CDCl3) d: 9.19 (br s, 1H), 8.17 (d, 1H, Jϭ8.0 Hz), 7.59 (td,
1H, Jϭ8.5, 2.0 Hz), 7.42 (t, 1H, Jϭ8.0 Hz), 7.27 (d, 2H, Jϭ8.0 Hz), 7.25 (t,
1H, Jϭ8.0 Hz), 6.94 (d, 1H, Jϭ8.0 Hz), 2.47 (q, 4H, Jϭ8.0 Hz), 1.17 (t, 6H,
Jϭ7.5 Hz); high reslution (HR)-MS (FAB) Calcd for C18H18N2O2ϩH:

November 2003
1279
295.1447, Found: 295.1463. IR (KBr, cmϪ1) 3200, 1720, 1660; Anal. Calcd
for C18H18N2O2: C, 73.45; H, 6.16; N, 9.52. Found: C, 73.55; H, 6.41; N,
9.26.
7.99. Found: C, 75.28; H, 7.50; N, 8.00.
PAQ-22Bn: (1-Benzyl-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazoline-
dione) (22e) To a solution of NaH (7.0 mg, 0.17 mmol (60% in mineral
oil, washed with Hex)) in DMF (1 ml) was added a solution of PAQ-22 (5)
(45 mg, 0.15 mmol) in DMF (1 ml), and the mixture was stirred at room tem-
perature for 10 min. To the resulting mixture was added benzyl bromide (29
mg, 0.17 mmol), and the whole was stirred for 1 h. Water was added, and or-
ganics were extracted with EtOAc. The extract was washed with brine, dried
over Na2SO4 and concentrated under reduced pressure. The residue was pu-
rified by silica gel column chromatography (eluent: EtOAc/Hex: 1/10—1/4)
to give PAQ-22Bn (22e) (38 mg, 65%) as colorless cubes, which were re-
N-(2,6-Diethylphenyl)-2-nitrobenzamide (23) To a solution of 2-ni-
trobenzoyl chloride (1.71 g, 10 mmol) and pyridine (0.5 ml) in CH2Cl2 (5 ml)
was added 2,6-diethylaniline (19) (1.49 g, 10 mmol). After having been
stirred for 30 min at room temperature, the reaction mixture was poured into
water and extracted with CH2Cl2. The organic layer was washed with water
and brine, dried over Na2SO4, and concentrated under reduced pressure. Pu-
rification via silica gel column chromatography (eluent: EtOAc/Hex: 1/2)
gave N-(2,6-diethylphenyl)-2-nitrobenzamide (23) (2.51 g, 84%). Colorless
needles from EtOAc/Hex; mp 151—153 °C; 1H-NMR (500 MHz, CDCl3) d:
8.11 (d, 1H, Jϭ8.0 Hz ), 7.76 (td, 1H, Jϭ8.0, 1.0 Hz), 7.73 (dd, 1H, Jϭ8.0,
1.0 Hz), 7.65 (td, 1H, Jϭ8.0, 1.0 Hz), 7.29 (t, 1H, Jϭ8.0 Hz), 7.19 (d, 2H,
Jϭ8.0 Hz), 7.09 (br s, 1H), 2.81 (q, 4H, Jϭ7.5 Hz), 1.29 (t, 6H, Jϭ7.5 Hz);
FAB-MS m/z: 299 (MϩH)ϩ; Anal. Calcd for C17H18N2O3: C, 68.44; H, 6.08;
N, 9.39. Found: C, 68.35; H, 6.17; N, 9.33.
2-Amino-N-(2,6-diethylphenyl)benzamide (24) N-(2,6-Diethylphenyl)-
2-nitrobenzamide (23) (1.98 g, 6.65 mmol) was dissolved in EtOH (20 ml)
and hydrogenated (1 bar H2) over 10% palladium on charcoal. The mixture
was filtered through a pad of Celite, and the filtrate was evaporated in vacuo
to give 2-amino-N-(2,6-diethylphenyl)benzamide (24) (1.86 g, quantitative
yield). Colorless needles from EtOAc/Hex; mp 123—124 °C; 1H-NMR (500
MHz, CDCl3) d: 7.56 (d, 1H, Jϭ7.5 Hz), 7.28 (td, 1H, Jϭ7.5, 2.0 Hz), 7.25
(d, 1H, Jϭ7.5 Hz), 7.17 (d, 2H, Jϭ7.5 Hz), 6.74 (d, 1H, Jϭ7.5 Hz), 6.73 (t,
1H, Jϭ7.5 Hz), 5.57 (br s, 2H), 2.66 (q, 4H, Jϭ7.0 Hz), 1.22 (t, 6H, Jϭ7.0
Hz); FAB-MS m/z: 269 (MϩH)ϩ; Anal. Calcd for C17H20N2O: C, 76.09; H,
7.51; N, 10.44. Found: C, 75.87; H, 7.52; N, 10.27.
1
crystallized from MeOH; mp 188—190 °C; H-NMR (500 MHz, CDCl3) d:
8.28 (dd, 1H, Jϭ8.0, 1.0 Hz ), 7.61 (td, 1H, Jϭ8.0, 1.0 Hz), 7.38 (t, 1H,
Jϭ8.0 Hz), 7.35 (t, 1H, Jϭ8.0 Hz), 7.31 (d, 2H, Jϭ8.0 Hz), 7.27 (t, 1H,
Jϭ8.0 Hz), 7.26 (t, 1H, Jϭ8.0 Hz), 7.25 (d, 2H, Jϭ8.0 Hz), 7.22 (d, 2H,
Jϭ8.0 Hz), 5.43 (br s, 2H), 2.48 (q, 4H, Jϭ8.0 Hz), 1.18 (t, 6H, Jϭ8.0 Hz);
FAB-MS m/z: 385 (MϩH)ϩ; Anal. Calcd for C25H24N2O2: C, 78.10; H, 6.29;
N, 7.29. Found: C, 78.11; H, 6.47; N, 7.29.
PAZOX-22: N-(2,6-Diethylphenyl)-3,1-benzoxazin-4-one (6) To
a
mixture of 2,6-diethylaniline (19) (149 mg, 1.0 mmol) and Et3N (100 mg, 1.0
mmol) in 1,2-dichloroethane (10 ml) was added triphosgene (119 mg, 0.40
mmol), and the mixture was heated at reflux until the amine was consumed
(for ca. 2 h). The reaction mixture was concentrated in vacuo, and to the
residue was added a solution of anthranilic acid (137 mg, 1.0 mmol) in THF
(1 ml). This mixture was stirred at 60 °C for 20 h, then diluted with water,
and the organics were extracted with EtOAc. The extract was washed with
water and brine, dried over MgSO4 and concentrated under reduced pressure
to give 2-[3-(2,6-diethylphenylureido)]benzoic acid (21b). The acid 21b was
dissolved in conc. H2SO4 (3 ml), and the mixture was stirred at room
temperature for 1.5 h. To the reaction mixture was added ice-cold water,
and after neutralization with 2 N NaOH, the organics were extracted with
CH2Cl2. The organic solution was dried over MgSO4 and concentrated under
reduced pressure, and the residue was purified by recrystallization from
CH2Cl2/Hex to give 6 (119 mg, 40%) as a white powder; mp 185—187 °C;
1H-NMR (500 MHz, CDCl3) d: 8.06 (dd, 1H, Jϭ8.5, 1.5 Hz), 7.61 (t, 1H,
Jϭ8.0 Hz), 7.31 (t, 1H, Jϭ8.0 Hz), 7.22 (td, 1H, Jϭ8.0, 1.5 Hz), 7.20 (d, 1H,
Jϭ7.5 Hz), 7.19 (d, 2H, Jϭ8.0 Hz), 2.67 (q, 4H, Jϭ7.5 Hz), 1.22 (t, 6H,
Jϭ8.0 Hz); HR-MS (FAB) Calcd for C18H18N2O2ϩH 295.1447; Found
295.1461; IR (KBr, cmϪ1) 3000, 1780, 1670. Anal. Calcd for C18H18N2O2:
C, 73.45; H, 6.16; N, 9.52, Found: C, 73.15; H, 6.15; N, 9.37.
General Procedure for Synthesis of N-Alkylated PAQ-22 To a solu-
tion of NaH (13 mg, 0.33 mmol) (60% in mineral oil, washed with Hex) in
DMF (2 ml) was added a solution of PAQ-22 (5) (89 mg, 0.30 mmol) in
DMF (2 ml), then the mixture was stirred for 10 min at room temperature. To
the resulting mixture was added alkyl iodide (0.30 mmol), then the mixture
was stirred for another 1 h at room temperature. To the reaction mixture was
added water, and the organics were extracted with EtOAc. The extract was
washed with water and brine, dried over Na2SO4, and concentrated under re-
duced pressure. Purification via silica gel column chromatography (eluent:
EtOAc/Hex) gave N-alkylated PAQ-22 (22).
PAQ-22M: (1-Methyl-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazoline-
dione) (22a) According to the general procedure, 22a was obtained in
81% yield as colorless crystalline cubes from EtOAc/Hex. mp 126—127 °C;
1H-NMR (500 MHz, CDCl3) d: 8.28 (dd, 1H, Jϭ8.5, 2.0 Hz ), 7.76 (td, 1H,
Jϭ8.5, 2.0 Hz), 7.37 (t, 1H, Jϭ7.5 Hz), 7.31(d, 1H, Jϭ8.5 Hz), 7.30 (d, 1H,
Jϭ8.0 Hz), 7.24 (d, 2H, Jϭ7.5 Hz), 3.67 (s, 3H), 2.42 (q, 4H, Jϭ8.0 Hz),
1.15 (t, 6H, Jϭ8.0 Hz); FAB-MS m/z: 309 (MϩH)ϩ; HR-MS (FAB) Calcd
for C19H20N2O2ϩH: 309.1603, Found: 309.1597; Anal. Calcd for
C19H20N2O2: C, 74.00; H, 6.54; N, 9.08. Found: C, 74.06; H, 6.66; N, 9.07.
PAQ-22E: (1-Ethyl-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazoline-
dione) (22b) According to the general procedure, 22b was obtained quan-
titatively as colorless needles, which were recrystallized from EtOAc; mp
N-(2,6-Diethylphenyl)-2-hydroxybenzamide (25) A solution of 2,6-di-
ethylaniline (19) (1.49 g, 10 mmol) and salicylic acid (1.38 g, 10 mmol) in
xylene (20 ml) was treated with PCl3 (0.87 ml, 10 mmol) at room tempera-
ture, and the mixture was heated to reflux for 3 h. The reaction mixture was
cooled, and water was added. The organics were extracted with EtOAc, and
the extract was washed with water and brine, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was purified by silica gel col-
umn chromatography (eluent: EtOAc/Hex: 1/4) to give N-(2,6-di-
ethylphenyl)-2-hydroxybenzamide (25) (2.31 g, 86%), as colorless crys-
1
talline cubes from diisopropyl ether; H-NMR (500 MHz, CDCl3) d: 12.16
1
(s, 1H), 7.59 (dd, 1H, Jϭ7.5, 1.0 Hz), 7.51 (br s, 1H), 7.48 (td, 1H, Jϭ7.5,
1.0 Hz), 7.30 (t, 1H, Jϭ7.5 Hz), 7.19 (d, 2H, Jϭ7.5 Hz), 7.06 (d, 1H, Jϭ7.5
Hz), 6.95 (t, 1H, Jϭ7.5 Hz), 2.65 (q, 4H, Jϭ7.5 Hz), 1.21 (t, 6H, Jϭ7.5 Hz);
FAB-MS m/z: 270 (MϩH)ϩ; Anal. Calcd for C17H19NO: C, 75.81; H, 7.11;
N, 5.20. Found: C, 75.73; H, 7.10; N, 5.05.
123—124 °C; H-NMR (500 MHz, CDCl3) d: 8.29 (dd, 1H, Jϭ8.0, 2.0 Hz),
7.74 (td, 1H, Jϭ8.0, 2.0 Hz), 7.36 (t, 1H, Jϭ8.0 Hz), 7.31(d, 1H, Jϭ8.0 Hz),
7.29 (t, 1H, Jϭ8.0 Hz), 7.23 (d, 2H, Jϭ8.0 Hz), 4.25 (q, 2H, Jϭ7.5 Hz), 2.41
(q, 4H, Jϭ7.5 Hz), 1.38 (t, 3H, Jϭ7.5 Hz), 1.15 (t, 6H, Jϭ7.5 Hz); FAB-MS
m/z: 323 (MϩH)ϩ; Anal. Calcd for C20H22N2O2: C, 74.51; H, 6.88; N, 8.69.
Found: C, 74.80; H, 7.03; N, 8.75.
POQ-22: 3-(2,6-Diisopropylphenyl)-2H-1,3-benzoxazine-2,4(3H)-
quinazolinedione (26) A solution of N-(2,6-diethylphenyl)-2-hydroxybenz
-
PAQ-22P: (1-Propyl-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazoline-
dione) (22c) According to the general procedure, 22c was obtained in 70%
yield as colorless needles, which were recrystallized from EtOAc/n-pentane;
amide (25) (135 mg, 0.5 mmol) in pyridine (2 ml) at 0 °C was treated with
ethyl chloroformate (109 mg, 1.0 mmol), allowed to warm to room tempera-
ture, then the mixture was heated to reflux for 2 h. To the reaction mixture
was added water, and after neutralization with 2 N HCl, the organics were
extracted with EtOAc. The extract was washed with water and brine, dried
over Na2SO4, and concentrated under reduced pressure. Purification by silica
gel column chromatography (eluent: EtOAc/Hex: 1/10—1/4) gave POQ-22
(26) (57 mg, 37%) as colorless needles from EtOH/Hex; mp 119—120 °C;
1H-NMR (500 MHz, CDCl3) d: 8.14 (dd, 1H, Jϭ8.0, 1.5 Hz), 7.77 (td, 1H,
Jϭ8.0, 1.5 Hz), 7.42 (t, 1H, Jϭ8.0 Hz), 7.41 (t, 1H, Jϭ8.0 Hz), 7.38 (d, 1H,
Jϭ8.0 Hz), 7.26 (d, 2H, Jϭ8.0 Hz), 2.47 (q, 2H, Jϭ8.0 Hz), 2.46 (q, 2H,
Jϭ8.0 Hz), 1.18 (t, 6H, Jϭ8.0 Hz); FAB-MS m/z: 296 (MϩH)ϩ; Anal. Calcd
for C18H17NO3: C, 73.20; H, 5.80; N, 4.74. Found: C, 73.07; H, 5.86; N,
4.73.
1
mp 131.5—132 °C; H-NMR (500 MHz, CDCl3) d: 8.29 (d, 1H, Jϭ8.0 Hz),
7.73 (t, 1H, Jϭ8.0 Hz), 7.36 (t, 1H, Jϭ8.0 Hz), 7.28 (d, 1H, Jϭ8.0 Hz), 7.28
(t, 1H, Jϭ8.0 Hz), 7.23 (d, 2H, Jϭ8.0 Hz), 4.16 (q, 2H, Jϭ8.0 Hz), 2.41 (q,
4H, Jϭ8.0 Hz), 1.82 (qt, 2H, Jϭ8.0, 8.0 Hz), 1.15 (t, 6H, Jϭ8.0 Hz), 1.03 (t,
3H, Jϭ8.0 Hz); FAB-MS m/z: 337 (MϩH)ϩ; Anal. Calcd for C21H24N2O2:
C, 74.97; H, 7.19; N, 8.33. Found: C, 75.01; H, 7.20; N, 8.33.
PAQ-22B: (1-Butyl-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazoline-
dione) (22d) According to the general procedure, 22d was obtained in
95% yield as colorless needles, which were recrystallized from EtOH/n-pen-
tane; mp 84.5—85 °C; 1H-NMR (500 MHz, CDCl3) d: 8.29 (dd, 1H, Jϭ8.0,
2.0 Hz ), 7.74 (td, 1H, Jϭ8.0, 2.0 Hz), 7.36 (t, 1H, Jϭ8.0 Hz), 7.29 (d, 1H,
Jϭ8.0 Hz), 7.28 (t, 1H, Jϭ8.0 Hz), 7.23 (d, 2H, Jϭ8.0 Hz), 4.19 (t, 2H,
Jϭ8.0 Hz), 2.41 (q, 4H, Jϭ7.5 Hz), 1.76 (tt, 2H, Jϭ7.5, 7.5 Hz), 1.46 (qt,
2H, Jϭ7.5, 7.5 Hz), 1.15 (t, 6H, Jϭ7.5 Hz), 0.99 (t, 3H, Jϭ7.5 Hz); FAB-
MS m/z: 351 (MϩH)ϩ; Anal. Calcd for C22H26N2O2: C, 75.40; H, 7.48; N,
3-(2,6-Diethylphenyl)-2-thio-2,4(1H,3H)-quinazolinedione (27)
A
mixture of carbon disulfide (0.4 ml, 6.74 mmol), DBU (0.05 ml, 0.337 mmol)
and 2-amino-N-(2,6-diethylphenyl)benzamide (24) (90 mg, 0.337 mmol) in

1280
Vol. 51, No. 11
DMF (1 ml) was heated at 50 °C for 3 h under argon. To the reaction mixture 20 h. The reaction mixture was concentrated in vacuo, and the residue was
was added water, and the organics were extracted with EtOAc. The extract purified by silica gel column chromatography (eluent: EtOAc/Hex: 1/4) to
was washed with brine, dried over MgSO4, filtered and concentrated in give 34. A mixture of the amide compound 34 (627 mg, 2.0 mmol),
vacuo. The residue was purified by silica gel column chromatography (elu- Et3N (223 mg, 2.2 mmol) and triphosgene (237 mg, 0.80 mmol) in 1,2-
ent: EtOAc/Hex: 1/4) to give 27 (81 mg, 78%) as colorless plates from dichloroethane (20 ml) was heated at reflux for 30 min. The reaction mixture
1
EtOAc/Hex; mp 248—254 °C; H-NMR (500 MHz, CDCl3) d: 11.45 (br s,
was cooled, and the organics were extracted with EtOAc. The extract was
1H), 8.19 (d, 1H, Jϭ8.0 Hz), 7.64 (t, 1H, Jϭ8.0 Hz), 7.46 (t, 1H, Jϭ8.0 Hz), washed with water and brine, dried over MgSO4 and concentrated under re-
7.33 (t, 1H, Jϭ7.5 Hz), 7.30 (d, 2H, Jϭ7.5 Hz), 7.22 (d, 1H, Jϭ8.0 Hz), duced pressure. The residue was purified by silica gel column chromatogra-
2.56—2.39 (m, 4H), 1.21 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 311 (MϩH)ϩ; phy (eluent: EtOAc/Hex) to give the nitro-substituted PAQ-22 (35).
Anal. Calcd for C18H18N2OS: C, 69.65; H, 5.84; N, 9.02. Found: C, 69.76;
6-Nitro-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (35a)
According to the general procedure, 35a was obtained in 32% yield (2 steps)
H, 5.99; N, 8.96.
3-(2,6-Diethylphenyl)-2,3-dihydro-4(1H)-quinazolinone (28) To a so- as pale yellow plates from EtOAc/Hex; mp 268—272 °C; 1H-NMR (500
lution of 2-amino-N-(2,6-diethylphenyl)benzamide (24) (135 mg, 0.50 MHz, CDCl3) d: 10.25 (br s, 1H), 9.01 (d, 1H, Jϭ2.5 Hz), 8.26 (dd, 1H,
mmol) and NaOH (20 mg, 0.50 mmol) in EtOH (5 ml) was added formalin
Jϭ9.0, 2.5 Hz), 7.56 (t, 1H, Jϭ7.5 Hz), 7.36 (d, 2H, Jϭ7.5 Hz), 6.68 (d, 1H,
(37%, 1 ml), then the mixture was stirred for 30 min at 60 °C. The reaction Jϭ9.0 Hz), 2.46 (q, 4H, Jϭ8.0 Hz), 1.17 (t, 6H, Jϭ8.0 Hz); FAB-MS m/z:
mixture was cooled and diluted with water, and the organics were extracted 340 (MϩH)ϩ; Anal. Calcd for C18H17N3O4: C, 63.71; H, 5.05; N, 12.38: C,
with EtOAc. The extract was washed with water and brine and concentrated 63.57, H, 5.18; N, 12.18.
under reduced pressure. Purification by silica gel column chromatography
7-Nitro-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (35b)
(eluent: EtOAc/Hex: 1/2) gave 28 (127 mg, 90%) as colorless needles from According to the general procedure, 35b was obtained in 65% yield (2 steps)
1
1
EtOAc/Hex; mp 182—184 °C; H-NMR (500 MHz, CDCl3) d: 7.98 (d, 1H, as yellow needles from EtOAc/Hex; mp 204—209 °C; H-NMR (500 MHz,
Jϭ7.5 Hz), 7.31 (td, 1H, Jϭ7.5, 1.0 Hz), 7.24 (t, 1H, Jϭ7.5 Hz), 7.16 (d, 2H, CDCl3) d: 9.73 (br s, 1H), 8.36 (d, 1H, Jϭ8.5 Hz), 8.04 (dd, 1H, Jϭ8.5, 2.5
Jϭ7.5 Hz), 6.89 (t, 1H, Jϭ7.5 Hz), 6.70 (d, 1H, Jϭ7.5 Hz), 4.68 (s, 2H), Hz), 7.87 (d, 1H, Jϭ2.0 Hz), 7.43 (t, 1H, Jϭ8.0 Hz), 7.27 (d, 2H, Jϭ8.0 Hz),
2.72—2.57 (m, 4H), 1.19 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 281 (MϩH)ϩ;
Anal. Calcd for C18H20N2O: C, 77.11; H, 7.19; N, 9.99 Found: C, 77.09; H,
7.28; N, 9.93.
2.45 (q, 4H, Jϭ7.5 Hz), 1.18 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 340
(MϩH)ϩ; Anal. Calcd for C18H17N3O4: C, 63.71; H, 5.05; N, 12.38: C,
63.56, H, 5.16; N, 12.24.
3-(2,6-Diethylphenyl)-4-thio-2,4(1H,3H)-quinazolinedione (29) A so-
8-Nitro-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (35c) To
lution of PAQ-22 (5) (295 mg, 1.0 mmol) and P4S10 (267 mg, 0.60 mmol) in a mixture of 37 (1.25 g, 5.55 mmol) and Et3N (1.55 ml, 11.11 mmol) in THF
xylene (10 ml) was stirred at 130 °C for 10 h, then concentrated in vacuo, (15 ml) was added ethyl chloroformate (0.8 ml, 8.33 mmol), dropwise, at
and the residue was purified by silica gel column chromatography (eluent: Ϫ10 °C, and the resulting mixture was stirred for 1 h. TLC analysis showed
EtOAc/Hex: 1/4) to give 29 (274 mg, 88%) as orange needles from complete conversion to a very non-polar material. To the mixture was added
EtOAc/Hex; mp 189 °C; 1H-NMR (500 MHz, CDCl3) d: 10.10 (br s, 1H), dropwise a solution of NaN3 (0.9 g, 13.87 mmol) in water (5.4 ml), and the
8.56 (d, 1H, Jϭ8.0 Hz), 7.51 (td, 1H, Jϭ8.0, 1.0 Hz), 7.44 (t, 1H, Jϭ7.5 Hz), mixture was stirred for another 1 h. The reaction mixture was diluted with
7.31 (d, 2H, Jϭ7.5 Hz), 7.20 (td, 1H, Jϭ8.0, 1.0 Hz), 6.73 (d, 1H, Jϭ8.0 water and extracted with toluene (30 ml). The combined organic extract was
Hz), 2.44 (q, 4H, Jϭ7.5 Hz), 1.19 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 311 washed with brine, dried over Na2SO4, filtered, and concentrated to ca. half
(MϩH)ϩ; Anal. Calcd for C18H18N2OS: C, 69.65; H, 5.84; N, 9.02. Found: volume in vacuo. The toluene solution was then slowly brought to reflux.
C, 69.44; H, 6.06; N, 8.62.
2-Amino-N-(2,6-diethylphenyl)benzenemethanamine (32) To a mix- added. After having been stirred for 30 min, the solution was acidified with
ture of 2,6-diethylaniline (0.82 g, 5.5 mmol) and K2CO3 (0.83 g, 6.0 mmol) 2 N HCl and extracted with EtOAc. The organic phase was washed
After 1.5 h, the solution became yellow, and then methyl anthranilate was
in DMF (5 ml) was added 2-nitrobenzyl bromide (31) (1.08 g, 5.0 mmol), with water and brine, dried over MgSO4, and concentrated under reduced
and the mixture was stirred at 60 °C for 6 h. The reaction mixture was di- pressure. Purification by silica gel column chromatography (eluent:
luted with water and extracted with EtOAc. The extract was washed with EtOAc/Hexϭ1/1) gave 35c (1.48 g, 79%) as white needles from EtOAc/Hex;
1
water and brine, and concentrated under reduced pressure. Purification by mp 239—241 °C; H-NMR (500 MHz, CDCl3) d: 10.09 (br s, 1H), 7.56 (t,
silica gel column chromatography (eluent: EtOAc/Hex: 1/10) gave N-2,6- 1H, Jϭ8.0 Hz), 7.45 (t, 1H, Jϭ8.0 Hz), 7.28 (d, 2H, Jϭ8.0 Hz), 7.22 (d, 1H,
diethylphenyl-2-nitrobenzylamine (1.02 g, 72%). A mixture of N-2,6-di- Jϭ8.0 Hz), 6.86 (d, 1H, Jϭ8.0 Hz), 2.45 (q, 4H, Jϭ7.5 Hz), 1.17 (t, 6H,
ethylphenyl-2-nitrobenzylamine (143 mg, 0.50 mol), iron(III) oxide hydrox- Jϭ7.5 Hz); FAB-MS m/z: 340 (MϩH)ϩ; Anal. Calcd for C18H17N3O4: C,
ide (100 mg), and MeOH (85 ml) was stirred at 60 °C for 10 min. To the re-
sulting mixture was added hydrazine hydrate (100%, 0.1 ml), dropwise, then
63.71; H, 5.05; N, 12.38: C, 63.65, H, 5.13; N, 12.34.
General Procedure for Synthesis of Amino-Substituted PAQ-22 from
the mixture was stirred at 60 °C for another 4 h. The reaction mixture was Nitro-Substituted PAQ-22 Nitro-substituted PAQ-22 (35) was dissolved
cooled to room temperature, diluted with EtOAc, filtered, and concentrated. in EtOH and hydrogenated (1 bar H2) over 10% palladium on charcoal. The
Purification via silica gel column chromatography (eluent: EtOAc/Hex: mixture was filtered through a pad of Celite. The filtrate was evaporated to
1/8—1/4) gave 2-amino-N-(2,6-diethylphenyl)benzenemethanamine (32) give the amino-substituted PA-22 (36).
(125 mg, 98%) as a yellow oil. 1H-NMR (500 MHz, CDCl3) d: 7.14 (td, 1H,
Jϭ8.0, 1.0 Hz), 7.12 (dd, 1H, Jϭ8.0, 1.0 Hz), 7.10 (s, 1H), 7.08 (s, 2H), 7.03
(d, 1H, Jϭ8.0 Hz), 7.02 (td, 1H, Jϭ8.0, 1.0 Hz), 6.74 (td, 1H, Jϭ8.0, 1.0
6-Amino-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (36a)
According to the general procedure, 36a was obtained in 79% yield as white
needles from EtOAc; mp 258 °C; 1H-NMR (500 MHz, CDCl3) d: 8.56 (br s,
Hz), 6.73 (d, 2H, Jϭ8.0 Hz), 4.02 (s, 2H), 2.73 (q, 4H, Jϭ7.5 Hz), 1.26 (t, 1H), 7.42 (d, 1H, Jϭ3.0 Hz), 7.39 (t, 1H, Jϭ8.0 Hz), 7.25 (d, 2H, Jϭ8.0 Hz),
6H, Jϭ7.5 Hz).
6.99 (dd, 1H, Jϭ8.0, 3.0 Hz), 6.84 (d, 1H, Jϭ8.0 Hz), 3.76 (s, 2H), 2.45 (q,
3-(2,6-Diethylphenyl)-3,4-dihydro-2(1H)-quinazolinone (30) To a so- 4H, Jϭ8.0 Hz), 1.16 (t, 6H, Jϭ8.0 Hz); FAB-MS m/z: 310 (MϩH)ϩ; Anal.
lution of 2-amino-N-(2,6-diethylphenyl)benzenemethanamine (32) (55 mg, Calcd for C18H19N3O2: C, 69.88; H, 6.19; N, 13.58: C, 69.63, H, 6.30; N,
0.21 mmol) and triethylamine (0.03 ml, 0.21 mmol) in 1,2-dichloroethane (2 13.32.
ml) was added triphosgene (24 mg, 0.08 mmol), and the resulting mixture
7-Amino-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (36b)
was heated at reflux for 2 h. To the resulting mixture was added methyl an- According to the general procedure, 36b was obtained in quantitative yield
1
thranilate (1.0 mmol), and stirring was continued at reflux for another 2 h. as white needles from EtOAc; mp 275 °C; H-NMR (500 MHz, CDCl3) d:
The reaction mixture was concentrated in vacuo, and the residue was puri- 8.89 (br s, 1H), 7.93 (d, 1H, Jϭ8.5 Hz), 7.38 (t, 1H, Jϭ8.0 Hz), 7.24 (d, 2H,
fied by silica gel column chromatography (eluent: EtOAc/Hex: 1/4) to give Jϭ8.0 Hz), 6.49 (d, 1H, Jϭ8.5 Hz), 6.13 (br s, 1H), 2.46 (q, 4H, Jϭ7.5 Hz),
1
30 (48 mg, 83%) as colorless cubes from EtOAc/Hex; mp 191 °C; H-NMR 1.16 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 310 (MϩH)ϩ; Anal. Calcd for
(500 MHz, CDCl3) d: 7.57 (br s, 1H), 7.29 (t, 1H, Jϭ8.0 Hz), 7.20 (d, 2H, C18H19N3O2: C, 69.88; H, 6.19; N, 13.58: C, 69.73, H, 6.11; N, 13.81.
Jϭ8.0 Hz), 7.18 (t, 1H, Jϭ8.0 Hz), 7.02 (d, 1H, Jϭ8.0 Hz), 6.95 (t, 1H,
8-Amino-3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione (36c)
Jϭ8.0 Hz), 6.71 (d, 1H, Jϭ8.0 Hz), 4.62 (s, 2H), 2.73—2.55 (m, 4H), 1.23 According to the general procedure, 36c was obtained in 95% yield as color-
(t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 281 (MϩH)ϩ; Anal. Calcd for C18H20N2O: less needles from EtOAc/Hex; mp 245—250 °C; 1H-NMR (500 MHz,
C, 77.11; H, 7.19; N, 9.99 Found: C, 77.13; H, 7.30; N, 9.95.
General Procedure for the Synthesis of Nitro-Substituted PAQ-22 7.23 (t, 1H, Jϭ8.0 Hz), 6.36 (d, 1H, Jϭ7.5 Hz), 6.13 (d, 1H, Jϭ7.5 Hz), 2.48
from Nitroanthranilic Acid A mixture of nitro-substituted anthranilic (qd, 4H, Jϭ7.5, 1.5 Hz), 1.18 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 310
acid (33) (910 mg, 5 mmol), 2,6-diethylaniline (19) (750 mg, 5 mmol), HOBt
(740 mg, 5.5 mmol) and EDC (1.15 g, 6 mmol) in DMF (5 ml) was stirred for 69.75, H, 6.22; N, 13.41.
CDCl3) d: 9.05 (br s, 1H), 7.40 (t, 1H, Jϭ7.5 Hz), 7.26 (d, 2H, Jϭ8.0 Hz),
(MϩH)ϩ; Anal. Calcd for C18H19N3O2: C, 69.88; H, 6.19; N, 13.58: C,

November 2003
1281
PN-22: 1-(2,6-Diethylphenyl) benzo[de]isoquinoline-1,3-dione (38)
A
Hz), 7.54 (d, 1H, Jϭ8.0 Hz), 7.44 (td, 1H, Jϭ8.0, 2.0 Hz), 7.40 (td, 1H,
Jϭ8.0, 2.0 Hz), 7.29 (d, 1H, Jϭ8.0 Hz), 7.26 (t, 1H, Jϭ8.0 Hz), 3.88 (s, 3H);
FAB-MS m/z: 319 (MϩH)ϩ; Anal. Calcd for C19H14N2O3: C, 71.69; H, 4.43;
N, 8.80. Found: C, 71.39; H, 4.63; N, 8.66.
Cells MOLT-4 cells were maintained in an RPMI1640 medium supple-
mented with 10% v/v fetal bovine serum at 37 °C under a 5% CO2 atmos-
phere.
mixture of 1,8-naphthalic anhydride (198 mg, 1 mmol) and 2,6-diethylani-
line (19) (149 mg, 1 mmol) in AcOH (10 ml) was heated at 130 °C for 20 h.
The reaction mixture was poured onto ice and this aqueous mixture was ex-
tracted with CHCl3. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated under reduced pressure. Purification by
silica gel column chromatography (eluent: EtOAc/Hex: 1/1—2/1) gave 38
(217 mg, 66%) as colorless crystalline cubes from EtOAc; mp 235—237 °C;
1H-NMR (500 MHz, CDCl3) d: 8.66 (d, 2H, Jϭ7.5 Hz), 8.29 (d , 2H, Jϭ7.5
Hz), 7.81 (t, 1H, Jϭ7.5 Hz), 7.41 (t, 2H, Jϭ7.5 Hz), 7.28 (d, 2H, Jϭ7.5 Hz),
2.47 (q, 4H, Jϭ7.5 Hz), 1.14 (t, 6H, Jϭ7.5 Hz); FAB-MS m/z: 330
(MϩH)ϩ; Anal. Calcd for C22H19NO3: C, 80.22; H, 5.81; N, 4.25: C, 80.11,
H, 5.97; N, 4.20.
BAQ-33: 3-(4-Heptyl)-2,4(1H,3H)-quinazolinedione (39) To a mix-
ture of 4-heptylamine (116 mg, 1.0 mmol) and Et3N (1.0 mmol) in 1,2-
dichloroethane (10 ml) was added triphosgene (0.40 mmol), and the resulting
solution was heated at reflux for 30 min. To the resulting mixture was added
methyl anthranilate (152 mg, 1.0 mmol), and the mixture was stirred at re-
flux for another 1 h. The solvent was removed under reduced pressure and
the residue was dissolved in 1,4-dioxane (5 ml) and 2 N NaOH solution (1
ml). The resulting solution was heated at 50 °C for 12 h. The reaction mix-
ture was cooled, diluted with water, acidified with 2 N HCl (ca. 2 ml) and ex-
tracted with EtOAc. The organic layer was washed with water and brine,
dried over MgSO4, and concentrated under reduced pressure. Purification by
silica gel column chromatography (eluent: EtOAc/Hex: 1/4) gave 39 (74 mg,
28%) as pale pink needles from EtOH; mp 112—114 °C; 1H-NMR (500
MHz, CDCl3, 55 °C) d: 10.28 (br s, 1H), 8.04 (d, 1H, Jϭ8.0 Hz), 7.50 (td,
1H, Jϭ8.0, 1.0 Hz), 7.12 (t, 1H, Jϭ8.0 Hz), 6.99 (d, 1H, Jϭ8.0 Hz), 5.03 (br
m, 1H), 2.20—2.12 (m, 2H), 1.73—1.65 (m, 2H), 1.30—1.18 (m, 4H), 0.85
(t, 6H, Jϭ8.0 Hz); FAB-MS m/z: 261 (MϩH)ϩ; Anal. Calcd for C15H20N2O2:
C, 69.20; H, 7.74; N, 10.76. Found: C, 69.22; H, 7.74; N, 10.64.
Assay of Enzyme Activity Neutral aminopeptidase activity was evalu-
ated in the usual assay by measuring 7-amino-4-methylcoumarin (AMC) lib-
erated from L-methylcoumarylamide (Ala-AMC).12,13) Briefly, to a solution
of 800 ml of 50 mM Tris–HCl (pH 7.4), in the presence or absence of a test
inhibitor (various concentrations), was added intact cell suspension (2ϫ106
cells/ml) at 37 °C for exactly 10 min for pre-incubation. To this solution was
added 1 ml of Ala-AMC (1 mM) at 37 °C for exactly 30 min. Then, 3 ml of
1 M AcONa–AcOH (pH 4.0) was added to stop the enzymatic reaction. The
amounts of liberated AMC were measured in terms of fluorescence intensity
(excitation at 380 nm, emission at 420 nm). The assay was performed at least
in duplicate, and the mean value was taken. Though the values deviated
from experiment to experiment, the results (order of potency) were basically
reproducible, and a typical set of data is shown in the tables.
Cytotoxicity Assay Test compounds were dissolved in DMSO at 20, 2
and 0.2 mM. A 5 ml aliquot of each solution was added to 100 ml of MOLT-4
cell suspension and 400 ml of RPMI1640 in 24-well plates, in which the cell
concentration was about 1ϫ105 cells/ml; the final DMSO concentration was
kept below 0.5%. Control samples received the same volume of DMSO
alone. The cells were incubated for 3 d at 37 °C under a 5% CO2 atmos-
phere. Cytotoxicity was evaluated in terms of the amounts of lactose dehy-
drogenase (LDH) produced by cyto Tox 96TM. The results are shown as rela-
tive values of LDH to the control (DMSO only; taken as 1).
Proliferation-Inhibitory Activity Conditions for assay of the prolifera-
tion-inhibitory activity were the same as described above. Proliferation-in-
hibitory activity was evaluated by counting cells with WST-1. The results are
shown as relative values of numbers of cells to that of a control (DMSO
only; taken as 1).
PAQ-00: (3-Phenyl-2,4(1H,3H)-quinazolinedione (40) According to
the general procedure, 40 was obtained in 94% yield as white cubes from
1
EtOH/CH2Cl2; mp 242—245 °C; H-NMR (500 MHz, CDCl3) d: 8.82 (br s,
1H), 8.16 (d, 1H, Jϭ8.0 Hz), 7.62 (t, 1H, Jϭ8.0 Hz), 7.54 (t, 2H, Jϭ7.5 Hz),
7.48 (t, 1H, Jϭ7.5 Hz), 7.31 (d, 2H, Jϭ7.5 Hz), 7.25 (t, 1H, Jϭ8.0 Hz), 6.99
(d, 1H, Jϭ8.0 Hz); FAB-MS m/z : 239 (MϩH); Anal. Calcd for C14H10N2O2:
C, 70.58; H, 4.23; N, 11.76. Found: C, 70.41; H, 4.46; N, 11.71.
PAQ-11: 3-(2,6-Dimethylphenyl)-2,4(1H,3H)-quinazolinedione (41)
According to the general procedure, 41 was obtained quantitatively as color-
less crystalline cubes from EtOH; mp 211—214 °C; 1H-NMR (500 MHz,
CDCl3) d: 10.09 (br s, 1H), 8.14 (dd, 1H, Jϭ7.5, 1.0 Hz), 7.46 (td, 1H,
Jϭ7.5, 1.0 Hz), 7.32 (t, 1H, Jϭ7.5 Hz), 7.23 (d, 2H, Jϭ8.0 Hz), 7.21 (t, 1H,
Jϭ7.5 Hz), 6.68 (d, 1H, Jϭ8.0 Hz), 2.17 (s, 6H): FAB-MS m/z: 267
(MϩH)ϩ; Anal. Calcd for C16H14N2O2: C, 72.16; H, 5.30; N, 10.52. Found:
C, 72.28; H, 5.49; N, 10.49.
PAQ-33: 3-(2,6-Diisopropylphenyl)-2,4(1H,3H)-quinazolinedione (42)
According to the general procedure, 42 was obtained in 97% yield as color-
less needles from EtOH/H2O; mp 245—248 °C; 1H-NMR (500 MHz,
CDCl3) d: 8.36 (br s, 1H), 8.19 (d, 1H, Jϭ8.0 Hz), 7.65 (t, 1H, Jϭ8.0 Hz),
7.46 (t, 1H, Jϭ8.0 Hz), 7.30 (d, 2H, Jϭ8.0 Hz), 7.27 (t, 1H, Jϭ8.0 Hz), 7.03
(d, 1H, Jϭ8.0 Hz), 2.70 (qq, 2H, Jϭ7.0, 7.0 Hz), 1.19 (d, 6H, Jϭ7.0 Hz),
1.16 (d, 6H, Jϭ7.0 Hz); FAB-MS m/z: 323 (MϩH)ϩ; Anal. Calcd for
C20H22N2O2: C, 74.51; H, 6.88; N, 8.69. Found: C, 74.41; H, 7.00; N, 8.72.
3-(2,6-Dimethoxyphenyl)-2,4(1H,3H)-quinazolinedione (43) Accord-
ing to the general procedure, 43 was obtained as colorless crystalline cubes
from EtOAc/Hex; mp Ͼ300 °C; 1H-NMR (500 MHz, CDCl3) d: 8.15 (d, 1H,
Jϭ8.0 Hz), 7.88 (br d, 1H), 7.60 (t, 1H, Jϭ8.0 Hz), 7.35 (t, 1H, Jϭ8.0 Hz),
7.22 (t, 1H, Jϭ8.0 Hz), 6.99 (d, 2H, Jϭ8.0 Hz), 3.77 (br s, 6H); FAB-MS
m/z: 299 (MϩH)ϩ; Anal. Calcd for C16H14N2O4: C, 64.42; H, 4.73; N, 9.39.
Found: C, 64.31; H, 4.86; N, 9.37.
3-(1-Naphthyl)-2,4(1H,3H)-quinazolinedione (44) According to the
general procedure, 44 was obtained in 73% yield as colorless plates from
MeOH/EtOAc; mp 280—283 °C; 1H-NMR (500 MHz, CDCl3) d: 9.37 (s,
1H), 8.15 (d, 1H, Jϭ8.0 Hz), 8.01 (d, 1H, Jϭ8.0 Hz), 7.96 (d, 1H, Jϭ8.0
Hz), 7.63 (t, 1H, Jϭ8.0 Hz), 7.62 (d, 1H, Jϭ8.0 Hz), 7.54—7.46 (m, 4H),
7.24 (t, 1H, Jϭ8.0 Hz), 6.82 (d, 1H, Jϭ8.0 Hz); FAB-MS m/z: 289 (MϩH)ϩ;
Anal. Calcd for C18H12N2O2: C, 74.99; H, 4.20; N, 9.72. Found: C, 74.89; H,
4.50; N, 9,89.
Cell Invasion Assay Tumor cell invasion was assessed by counting
mouse metastatic tumor cells (B16F10/L5 mouse melanoma cells) that in-
vaded Matrigel-coated filters, according to Albini et al.49) Briefly, an inva-
sion chamber, whose upper filter surface is coated with Matrigel (Becton
Dickinson), was soaked in a 12-well plate having 500 ml/well of RPMI cell-
conditioned medium containing 10% BSA and human plasma fibronectin 20
mg/ml. B16F10/L5 cells (2.0ϫ105 cells/ml in 200 ml of RPMI containing
0.1% BSA ) were added to the upper part of the chamber. The cells were in-
cubated for 24 h at 37 °C under a 5% CO2 atmosphere in the presence or ab-
sence of the test compounds. After incubation, cells present in the lower part
of the cell chamber and on the lower surface of the filter (stained with Crys-
tal violet after careful removal of the cells on the upper surface of the filter
by wiping with cotton swabs) were counted manually under a microscope.
Each sample was tested in triplicate, and the mean values are shown.
Acknowledgment The work described in this paper was partially sup-
ported by Grants-in-Aid for Scientific Research from The Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan.
References and Notes
1) Filder I. J., Gersten D. M., Hart I. R., Adv. Cancer Res., 28, 149—250
(1978).
2) Liotta l. A., Tryggvason K., Garbisa S., Hart I., Foltz C. M., Shafie S.,
Nature (London), 284, 67—68 (1980).
3) Liotta l. A., Goldfarb R. H., Brundage R., Siegel G. P., Terenova V.,
Garbisa S., Cancer Res., 41, 4629—4636 (1981).
4) Sloane B. F., Honn K. V., Sadler J. G., Cancer Res., 42, 980—986
(1982).
5) Nagaoka I., Tamashita T., Biochim. Biophys. Acta, 598, 169—172
(1987).
6) Yoneda J., Saiki I., Fujii H., Abe F., Kojima Y., Azuma I., Clin. Exp.
Metastasis, 10, 49—59 (1992).
7) Tsuruo T., Nagamura K., Iida H., Yamori T., Tsukagoshi S., Sakurai
Y., J. Antibiot., 34, 1206—1209 (1981).
8) Saiki I., Murata J., Watanabe K., Fujii H., Abe F., Azuma I., Jpn. J.
Cancer Res., 80, 873—878 (1989).
3-(2-Methoxy-1-naphthyl)-2,4(1H,3H)-quinazolinedione (45) Ac-
cording to the general procedure, 45 was obtained in 45% yield as a pale
pink powder from EtOAc; mp 275—278 °C; 1H-NMR (500 MHz, d6-
DMSO) d: 11.65 (s, 1H), 8.08 (d, 1H, Jϭ8.0 Hz), 7.96 (d, 1H, Jϭ8.0 Hz),
7.94 (d, 1H, Jϭ8.0 Hz), 7.75 (td, 1H, Jϭ8.0, 1.0 Hz), 7.59 (d, 1H, Jϭ8.0
9) Look A. T., Ashmun R. A., Shapiro L. H., Peiper S. C., J. Clin. Invest.,
83, 1299—1307 (1989).
10) Bowes M. A., Kenny A. J., J. Immunol., 60, 247—253 (1987).
11) Saiki I., Fujii H., Yoneda J., Abe F., Nakajima M., Tsuruo T., Azuma

1282
Vol. 51, No. 11
I., Int. J. Cancer, 54, 137—143 (1993).
12) Fujii H., Nakajima M., Aoyagi T., Tsuruo T., Biol. Pharm. Bull., 19,
6—10 (1996).
13) Umezawa H., Aoyagi T., Suda H., Hamada M., Takeuchi T., J.
Antibiot., 29, 97—99 (1976).
14) Nicolson G. L., Cancer Res., 47, 1473—1487 (1987).
15) Moscattelli D., Rifkin D. B., Biochim. Biophys. Acta, 948, 67—85
(1988).
33) McLellan S., Dyer S. H., Rodriguenz G., Hersh L.B., J. Neurochem.,
51, 1552—1559 (1988).
34) Constam D. B., Tobler A. R., Rensing-Ehl A., Kemler I., Hersh L. B.,
Fontana A., J. Biol. Chem., 270, 26931—26939 (1995).
35) de Gandarias J. M., Irazusta J., Gil J., Fernandez D., Varona A., Casis
L., Brain Res. Bull., 50, 283—290 (1999).
36) Osada T., Ikegami S., Takiguchi-Hayashi K., Yamazaki Y., Katoh-
Fukui Y., Higashinakagawa T., Sasaki Y., Takeuchi Y., J. Neurochem.
19, 6068—6078 (1999).
16) Hashimoto Y., Curr. Med. Chem., 5, 163—178 (1998).
17) Miyachi H., Azuma A., Kitamoto T., Hayashi K., Kato S., Koga M., 37) Kakuta H., Koiso Y., Takahashi H., Nagasawa K., Hashimoto Y., Hete-
Sato B., Hashimoto Y., Bioorg. Med. Chem. Lett., 7, 1483—1488
(1997).
18) Miyachi H., Azuma A., Hashimoto Y., Yakugaku Zasshi, 117, 91—107
(1997).
19) Hashimoto Y., Recent Res. Develop. Org. Chem., 4, 87—120 (2000).
20) Kakuta H., Takahashi H., Sou S., Kita T., Nagasawa K., Hashimoto Y.,
Recent Res. Devel. Medicinal Chem., 1, 189—211 (2001).
21) Kagechika H., Komoda M., Fujimoto Y., Koiso Y., Takayama H.,
rocycles, 55, 1433—1438 (2001).
38) Kakuta H., Koiso Y., Nagasawa K., Hashimoto Y., Bioorg. Med. Chem.
Lett., 13, 83—86 (2003).
39) Mallamo J. P., Pilling G. M., Wetzel J. R., Kowalczik P. J., Bell M. R.,
Kullnig R. K., Batzold F. H., Juniewiecz P. E., Winnecker R. C., Luss
H. R., J. Med. Chem., 35, 1663—1670 (1992).
40) Kollman P., McKelvey J., Johansson A., Rothenberg S., J. Amer. Chem.
Soc., 97, 955—965 (1975).
Kadoya S., Miyata K., Kato F., Kato M., Hashimoto Y., Biol. Pharm. 41) Weiner P. K., Langridge R., Blaney J. M., Schaefer R., Kollman P. A.,
Bull., 22, 1010—1012 (1999). Proc. Natl. Acad. Sci. U.S.A., 79, 3754—3758 (1982).
22) Shimazawa R., Miyachi H., Takayama H., Kuroda K., Kato F., Kato 42) Naray-Szabo G., J. Mol. Graphics, 7, 76—81 (1989).
M., Hashimoto Y., Biol. Pharm. Bull., 22, 224—226 (1999).
23) Miyachi H., Kato M., Kato F., Hashimoto Y., J. Med. Chem., 41,
263—265 (1998).
24) Shimazawa R., Takayama H., Hashimoto Y., Jpn. J. Synth. Org. Chem.,
57, 92—104 (1999).
25) Shimazawa R., Takayama H., Fujimoto Y., Komoda M., Dodo K.,
Yamasaki R., Shirai R., Koiso Y., Miyata K., Kato F., Kato M., Miya-
chi H., Hashimoto Y., J. Enzyme Inhibit., 14, 259—275 (1999).
43) Rashkin M. J., Waters M. L., J. Amer. Chem. Soc., 124, 1860—1861
(2002).
44) Electrostatic potential surfaces were generated by mapping PM3 elec-
trostatic potentials onto the surfaces of molecular electron density
using Spartan (Wavefunction, Inc. Irvine, CA, U.S.A.). Potential en-
ergy range is Ϫ50—50 kcal/mol for all surfaces shown. Red indicates
areas of greater electron density and blue indicates areas of less elec-
tron density.
26) Takahashi H., Komoda M., Kakuta H., Hashimoto Y., Yakugaku 45) Papadopoulas E. P., Torres C. D., J. Heterocycl. Chem., 19, 269—272
Zasshi, 120, 909—921 (2000). (1982).
27) Komoda M., Kakuta H., Takahashi H., Fujimoto Y., Kadoya S., Kato 46) Brown F. J., Bernstain P. R., Cronk L. A., Dosset D. L., Hebbel K. C.,
F., Hashimoto Y., Bioorg. Med. Chem., 9, 121—131 (2001).
Maduskuie T. P., Jr., Shapiro H. S., Vacek E. P., Yee Y. K., Willard A.
28) Hersh L. B., McKelvy J. F., J. Neurochem., 36, 171—178 (1981).
K., Krell R. D., Snyder D. W., J. Med. Chem., 32, 807—826 (1989).
29) Bauer W. O., Nanda I., Beck G., Schmid M., Jacob F., Cytogenet. Cell 47) Canonne P., Akssira M., Dahdouh A., Kasmi H., Boumzebra M., Hete-
Genet., 92, 221—224 (2001). rocycles, 36, 1305—1313 (1993).
30) Tobler A. R., Constam D. B., Schmitt-Graff A., Malipiero U., 48) Sekine K., Fujii H., Abe F., Nishikawa K., Int. J. Cancer, 94, 485—
Schlabach R., Fontana A., J. Neurochem., 68, 889—897 (1997).
31) Johnson G. D., Hersh L. B., Arch. Biochem. Biophys., 276, 305—309
(1990).
491 (2001).
49) Albini A., Iwamoto Y., Kleinman H. K., Martin G. R., Aaronson S. A.,
Kozlowski J. M., McEwan R. N., Cancer Res., 47, 3239—3245
(1997).
32) Rawlings N. D., Barrett A. J., Biochem. J., 290, 205—218 (1993).