Novel biological response modifiers: Phthalimides with tumor necrosis factor-α production-regulating activity

Article abstract of DOI:10.1021/jm970109q

Novel N-substituted phthalimides (2-substituted 1H-isoindole-1,3- diones) were prepared, and their effects on tumor necrosis factor-α (TNF- α) production by human leukemia cell line HL-60 stimulated with 12-O- tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) were examined. A structure-activity relationship study of the N-phenylphthalimides and N- benzylphthalimides revealed that their enhancing effect on TPA-induced TNF- α production by HL-60 cells and their inhibiting effect on OA-induced TNF- α production by HL-60 cells are only partially correlated.

Full text of DOI:10.1021/jm970109q

2858
J . Med. Chem. 1997, 40, 2858-2865
Novel Biologica l Resp on se Mod ifier s: P h th a lim id es w ith Tu m or Necr osis
F a ctor -r P r od u ction -Regu la tin g Activity
Hiroyuki Miyachi, Akihiko Azuma, Asuka Ogasawara, Eiji Uchimura,† Naoko Watanabe,† Yoshiro Kobayashi,†
Fuminori Kato,‡ Masanari Kato,‡ and Yuichi Hashimoto*
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, J apan,
Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274, J apan,
R & D Group of Pharmaceuticals, Central Research Institute, Ishihara Sangyo Kaisha, Ltd., 2-3-1 Nishi-shibukawa,
Kusatsu, Shiga 525, J apan
Received February 24, 1997X
Novel N-substituted phthalimides (2-substituted 1H-isoindole-1,3-diones) were prepared, and
their effects on tumor necrosis factor-R (TNF-R) production by human leukemia cell line HL-
60 stimulated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) were
examined. A structure-activity relationship study of the N-phenylphthalimides and N-
benzylphthalimides revealed that their enhancing effect on TPA-induced TNF-R production
by HL-60 cells and their inhibiting effect on OA-induced TNF-R production by HL-60 cells are
only partially correlated.
In tr od u ction
Tumor necrosis factor-R (TNF-R), an important cy-
tokine produced by activated monocytes/macrophages,
was originally identified as an endotoxin-induced serum
factor that causes hemorragic necrosis of transplanted
solid tumors.1 Because TNF-R exhibits striking cyto-
toxicity selectively against various tumor cells, it has
attracted attention as a potential antitumor drug.2
Investigation of the biological effects elicited by TNF-R
has revealed that it is a pleiotropic cytokine with num-
erous effects on mammalian cells, and its actions are
initiated by binding to high-affinity receptors.3 Though
TNF-R plays a critical role in certain physiological im-
mune systems, it causes severe damage to the host when
produced in large excess. Therefore, TNF-R can be re-
F igu r e 1. Structures of thalidomide, (S)-methylthalidomide,
garded as possessing both favorable and unfavorable ef-
fects. The favorable effects include direct tumor-killing
effect,2 stimulation of the host’s immune system,4 and
action as a growth factor for normal B-cells.5 The un-
favorable effects include induction of tissue inflamma-
tion,6 tumor-promoting action,7 stimulation of human
immunodeficiency virus (HIV) replication,8 and induc-
tion of insulin resistance.9 These pleiotropic effects of
TNF-R indicate that TNF-R production enhancers in
some cases and production inhibitors in other cases
would be useful as biological response modifiers (BRMs)
under various circumstances.10 Moreover, tissue- and/
or cell-type-specific regulation of TNF-R production
would be useful, since TNF-R is rapidly cleared from
the circulation.
A possible lead compound for the above purpose is
thalidomide [N-R-phthalimidoglutarimide (10) (Figure
1)]. Thalidomide was developed as a hypnotic/sedative
agent but had to be withdrawn from the market because
of its teratogenicity. In spite of this, there has been a
resurgance of interest in the drug in recent years due
to its potential usefulness for the treatment of acquired
immunodeficiency syndrome (AIDS),8 graft-versus-host
disease (GVHD),11 leprosy,12 and other related dis-
and (R)-methylthalidomide.
eases.13 The effectiveness of the drug in these diseases
has been attributed to its specific inhibitory activity on
TNF-R production.14 However, we found recently that
the regulation by thalidomide of TNF-R production is
specific to cell type and to inducer, i.e., (1) thalidomide
enhances TPA-induced TNF-R production by human
leukemia HL-60 cells, while it inhibits TPA-induced
TNF-R production by another human leukemia cell line,
THP-1, and (2) it inhibits TNF-R production by both HL-
60 and THP-1 cells when the cells are stimulated with
okadaic acid (OA).15
On the basis of these findings, we have been engaged
in structural modification of thalidomide with the aim
of creating superior regulators of TNF-R production. For
the evaluation of the compounds obtained, TNF-R pro-
duction-enhancing activity was assayed using HL-60
stimulated with TPA, and TNF-R production-inhibiting
activity was assayed using HL-60 stimulated with OA.
Studies on structural simplification of the glutarimide
moiety of thalidomide afforded potent bidirectional
TNF-R production regulators, such as 2-(2,6-diisopro-
pylphenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3-dione
[FPP-33 (30)] (Table 5) and 2-(2,6-diisopropylphenyl)-
1-oxo-3-thioxo-1H-isoindole [PPS-33 (18)] (Table 3).16
Both compounds enhanced TPA-induced TNF-R produc-
tion in the nanomolar/micromolar concentration range,
* To whom correspondence should be addressed.
† Toho University.
‡ Central Research Institute.
X Abstract published in Advance ACS Abstracts, August 15, 1997.
S0022-2623(97)00109-X CCC: $14.00 © 1997 American Chemical Society

Novel Biological Response Modifiers
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2859
Ta ble 1. Effects of Monosubstituted N-Phenylphthalimides on
TNF-R Production
while they completely inhibited OA-induced TNF-R
production in the same concentration range.16
O
More recently, we also found a clear enantiodepen-
dence of the inducer-specific bidirectional TNF-R pro-
duction-regulating activity. In the case of optically ac-
tive thalidomide analogues, (S)- and (R)-R-methylthal-
idomide (Figure 1), only the (S)-form shows TNF-R
production-enhancing activity in the TPA/HL-60 assay
system. In contrast, the (R)-form shows much more
potent TNF-R production-inhibiting activity than the
(S)-form in the OA/HL-60 assay system.17 On the basis
of this observation, we anticipated that separation of
the bidirectional TNF-R production-regulating activities
could be achieved by the use of optically active phthal-
imides. We found that optically active tetrafluorophthal-
imides, (R)-FPTP (33) and (R)-FPTN (35) (Table 6),
exhibited potent TNF-R production-inhibiting activity
in the nanomolar concentration range without concomi-
tant TNF-R production-enhancing activity.18
N
R
O
amount of
TNF-R (%)a
code
(30 µM)
TPA/
OA/
cytotoxicityb
compd
R
HL-60 HL-60 IC50 (µg/mL)
1
2
3
4
5
6
7
8
9
PP-00
PP-10
H
100
100
151
158
262
95
89
158
139
61
68
35
>100
>100
>100
>100
>100
>100
>100
>100
>100
2-Me
PP-n50
PP-30
PP-ph0
PP-M0
PP-N0
PP-03
2-nPen
2-iPr
105
40
2-Ph
2-MeO
2-NO2
3-iPr
21
53
105
33
PP-003
4-iPr
10
thalidomide
135
58
>100
In this paper we would like to describe the structure-
activity relationships of novel N-phenyl- and N-ben-
zylphthalimides, based on assays of TPA-induced TNF-R
production-enhancing activity and OA-induced TNF-R
production-inhibiting activity.
a
HL-60 cells were treated with 10 nM TPA or 50 nM OA in
the presence of 30 µM test compound. The amount of TNF-R
secreted in the culture medium was measured by ELISA. The
amount of TNF-R production in the presence of 10 nM TPA alone
or 50 nM OA alone was defined as 100%. b WI-38 cells were treated
with test compounds, and cell viability was determined by the
WST-1 method as described in the text.
Resu lts
Assa y System s. The HL-60 cells used produce no
detectable amount of TNF-R under the usual culture
conditions, but begin to produce TNF-R after treatment
with TPA or OA. The induction of TNF-R by TPA and
OA was dose-dependent, and we chose TPA and OA
concentrations of 10 and 50 nM respectively, because
the effects of the test compounds were clearly apparent
at these concentrations of TPA/OA. The amount of
TNF-R produced by HL-60 cells under our standard
experimental conditions (5 × 105 cells/mL, incubated in
the presence of 10 nM TPA or 50 nM OA for 16 h) was
150-180 or 1000-1200 pg/mL, respectively, and the
value separately determined in each set of experiments
was taken as 100%. The effect of a compound was
represented as the amount (%) of TNF-R produced by
HL-60 cells in the co-presence of TPA or OA and the
test compound. The amount of TNF-R produced by
TPA/OA-stimulated HL-60 cells in the presence of test
compounds showed some variation from experiment to
experiment. However, the order of efficacy of the test
compounds was reproducible. Therefore, typical data
obtained in experiments performed at the same time are
presented. In each set of experiments, the assay was
performed in duplicate or triplicate (the mean value was
taken) and at least three times. A typical set of data
obtained at the same time is presented in each table
and figure. None of the phthalimides described in this
paper induced TNF-R production by themselves in the
concentration range investigated. Cell numbers were
counted at the time when the amount of TNF-R was
measured. Almost no difference in the cell numbers
between incubation mixtures in the presence and ab-
sence of the test compound was observed in the concen-
tration range investigated.
IC50 values determined are presented in Tables 1-6.
As shown in the tables, only a poor correlation was
observed between the TNF-R production-regulating
activity (especially TNF-R production-inhibiting activity)
and the cell toxicity of the compounds. For example,
both PP-33 (17) and 4APP-33 (25) are potent TNF-R
production inhibitors on OA-stimulated HL-60 cells
(21%, Table 4), but the latter is much more toxic (IC50
) 25 µM) than the former (IC50 ) >100 µg/mL, corre-
sponding to >322 µM) (Table 4). In addition, FPP-33
(30), which is highly toxic (IC50 ) 2.3 µM, Table 5), and
PPS-33 (18), which is less toxic (IC50 ) 95 µM, Table
3), both showed a potent TNF-R production-enhancing
effect on TPA-stimulated HL-60 cells. These results
indicate that the TNF-R production-regulating activity
of these compounds is independent of their toxic effect,
at least in part.
Effects of Or th o Su bstitu en ts a t th e N-P h en yl
Gr ou p . First, we investigated the effects of ortho sub-
stituents introduced at the N-phenyl moiety of N-phen-
ylphthalimide on TPA- and OA-induced TNF-R produc-
tion by HL-60 cells (Table 1).20 Our preliminary experi-
ments revealed that several active compounds showed
activity at the concentration range 1-100 µM. There-
fore, all of the compounds were assessed at 30 µM con-
centration. As previously reported, unsubstituted N-
phenylphthalimide (PP-00: 1) lacks TNF-R production-
enhancing activity, but introduction of alkyl substitu-
ents at the ortho position of the phenyl moiety causes
the appearance of the activity. In a series of ortho-
substituted compounds, the enhancing activity in-
creased in the order of H (PP-00: 1, 100%) ) Me (PP-
10: 2, 100%) < n-Pen (PP-n50: 3, 151%) < i-Pr (PP-30:
4, 158%) < Ph (PP-ph0: 5, 262%), indicating that the
steric bulk of the substituents plays a critical role in
the TNF-R production-enhancing activity. On the other
hand, the electronic factor of the substituent had little
or no effect: the MeO (PP-M0: 6) and NO2 (PP-N0: 7)
substituted compounds did not exhibit TNF-R produc-
tion-enhancing activity.
To assess the cell toxicity of the compounds, human
embryonic lung fibroblast WI-38 cells were treated with
various concentrations of test compounds at 37 °C for
48 h. After the incubation, the viability of the treated
cells was measured by testing the succinate-tetrazo-
lium reductase system using the WST-1 method.19 The

2860 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Miyachi et al.
F igu r e 2. Dose-response curves of typical phthalimide derivatives. Horizontal scale: concentration of added test compound.
Vertical scale: amount of TNF-R (%, vide infra). (a and c) TPA-induced TNF-R production-enhancing activity. HL-60 cells were
treated with 10 nM TPA in the presence of a test compound. The amount of TNF-R produced by HL-60 cells stimulated with 10
nM TPA alone was defined as 100%. (b and d) OA-induced TNF-R production-inhibiting activity. HL-60 cells were treated with
50 nM OA in the presence of a test compound. The amount of TNF-R produced by HL-60 cells stimulated with 50 nM OA alone
was defined as 100%. Part a: (9) thalidomide, (0) PP-00 (1), (2) PP-11 (11), (two concentric circles) PP-33 (17), (O) PPS-33 (18),
(3) 4NPP-33 (23), (b) FPP-33 (30). Part b: (9) thalidomide, (0) PP-00 (1), (2) PP-11 (11), (two concentric circles) PP-33 (17), (O)
PPS-33 (18), (b) FPP-33 (30). Part c: (b) FPP-33 (30), (two concentric circles) PP-33 (17), (O) ClPP-33 (31). Part d: (b) (R)-FPTP
(33), (O) (R)-FPTN (35), (two concentric circles) (R)-FPTH (37), (9) (R)-methylthalidomide, (0) (S)-methylthalidomide.
As for TNF-R production-inhibiting activity, PP-00 (1)
exhibited moderate activity (61%), comparable to that
of thalidomide (10) (58%) (Table 1). In a series of ortho-
substituted compounds, we could not find any clear
substituent effect on the TNF-R production-inhibiting
activity. The dose-response curves for the enhancing
and inhibiting activities of typical compounds on TNF-R
production are shown in Figure 2a,b.
Effects of Dia lk yl Su bstitu en ts a t th e N-P h en yl
Gr ou p . Because TNF-R production-enhancing activity
was suspected to depend on the steric factor of the sub-
stituents at the N-phenyl moiety, we next investigated
N-(dialkyl-substituted phenyl)phthalimides at the con-
centration of 30 µM (Table 2).20 As previously reported,
o,o′-dimethylation (PP-11: 11) caused the appearance
of the TNF-R production-enhancing activity (161%). The
ortho positions seem to be the best at which to introduce
two methyl groups to obtain potent enhancing activity,
i.e., PP-11 (11) was more active than the m,m′-dimethy-
lated analog (PP-0101: 12, 136%), although the latter
compound was moderately active. As was the case
among ortho-monoalkylated compounds, the steric bulk
of the two alkyl groups introduced seems to be critical,
and the o,o′-diisopropyl derivative (PP-33: 17) exhibited
potent TNF-R production-enhancing activity amounting
to ca. 600 % at 30 µM in this experiment (Table 2).
Interestingly, the TNF-R production-inhibiting activ-
ity showed similar but not identical structure-activity
relationships. PP-33 (17) showed the strongest inhibi-
tory activity on OA-induced TNF-R production HL-60
cells (21%) among the o,o′-dialkylated analogues listed
in Table 2. Though the inhibitory activity decreased in
the order of PP-33 (17, 21%) > PP-11 (11, 40%) > PP-
00 (1, 61%) (Table 2: the same order as that of the
enhancing activity on TPA-induced TNF-R production
by the same cell line), PP-22 (14) exceptionally showed
inhibitory activity (53%) slightly weaker than that of
PP-11 (11, 40%). The effective concentration ranges of
these compounds are roughly the same for the TPA-
induced HL-60 and OA-induced HL-60 assay systems
(1-100 µM, Figure 2a,b).
Effects of Va r ia tion of th e Su ccin im id e Moiety.
Next, we investigated the effect of modifying the suc-
cinimide moiety.21 For this purpose, the 2,6-diisopro-
pylphenyl group was used as the nitrogen substituent
throughout (Table 3).
Replacement of the succinimide moiety of thalidomide
with either cyclopentanedione or lactam structure has

Novel Biological Response Modifiers
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2861
Ta ble 2. Effects of Disubstituted N-Phenylphthalimides on
TNF-R Production
Ta ble 4. Effects of Substituents at the Phthaloyl Moiety of
PP-33 on TNF-R Production Regulation
O
O
N
R1
R
N
O
O
R2
amount of
amount of
TNF-R (%)a
TNF-R (%)a
code
compd (30 µM)
TPA/
OA/
cytotoxicityb
code
(30 µM)
TPA/ OA/
HL-60 HL-60
cytotoxicityb
IC50 (µg/mL)
R1
R2
HL-60 HL-60 IC50 (µg/mL)
compd
R
1
11
12
13
14
15
16
17
PP-00
PP-11
PP-0101 3-Me 5-Me
PP-21
PP-22
PP-31
PP-32
PP-33
H
H
100
161
136
188
453
234
255
602
61
40
89
82
53
33
26
21
>100
17
23
24
25
26
27
28
29
PP-33
4NPP-33 4-NO2
5NPP-33 5-NO2
4APP-33
5APP-33
4HPP-33 4-OH
5HPP-33 5-OH
NAP-33c
H
602
792
683
248
242
239
153
144
21
54
54
21
28
64
7
>100
2-Me 6-Me
>100
93.1 (264 µM)
42.7 (121 µM)
8.1 (25 µM)
17.4 (53 µM)
>100
>100
2-Et 6-Me
2-Et 6-Et
2-iPr 6-Me
2-iPr 6-Et
2-iPr 6-iPr
26.2 (99 µM)
38.0 (136 µM)
28.1 (97 µM)
6.0 (20 µM)
>100
4-NH2
5-NH2
12.7 (39 µM)
>100
48
a,b
a,b
See Table 1.
See Table 1. c NAP-33 ) 2-(2,6-diisopropylphenyl)-1H-naph-
thoisoindole-1,3-dione.
Ta ble 3. Effects of variation of the Succinimide Moiety of
PP-33 on TNF-R Production Regulation
to more than 1200% and 1600% at 30 and 100 µM,
respectively (Figure 2a), while it completely inhibited
OA-induced TNF-R production at 30 µM (Figure 2b).
Effects of Su bstitu en ts on th e Con d en sed Ben -
zen e Rin g. We next investigated the effects of sub-
stituents introduced at the condensed benzene ring of
the phthalimide moiety (Table 4).20 As shown in the
table, introduction of an electron-donating group such
as a hydroxyl (4HPP-33: 27, 5HPP-33: 28) or an amino
group (4APP-33: 25, 5APP-33: 26) decreased the TPA-
induced TNF-R production-enhancing activity (150-
250%) compared to PP-33 (17, 602% in this experiment)
(Table 4). The effects elicited by introduction of these
two groups are similar. In both cases, the 4-substituted
analogs (25 and 27) show higher activity than the
corresponding 5-substituted analogs (26 and 28, respec-
tively) (although 25 and 26 showed almost the same
efficacy). In contrast to the introduction of electron-
donating groups, introduction of the electron-withdraw-
ing nitro group (4NPP-33: 23, 5NPP-33: 24) increased
the enhancing activity (Table 4 and Figure 2a). In this
case also, the TNF-R production-enhancing activity of
the 4-nitro derivative (23, 792%) was higher than that
of the 5-nitro derivative (24, 683%).
B
amount of
TNF-R (%)a
code
TPA/
OA/
cytotoxicityb
compd
B
(30 µM) HL-60 HL-60
IC50 (µg/mL)
17
O
PP-33
602
21
0
>100
N
O
S
18
PPS-33
1252
30.8 (95 µM)
N
O
O
19
20
IP-33
214
268
40
50
43.1 (148 µM)
>100
N
O
PIQ-33
N
In contrast, introduction of the electron-withdrawing
nitro group (23, 24) decreased OA-induced TNF-R
production-inhibiting activity (54%) compared to PP-33
(17, 21%). To our surprise, the electron-donating amino
group (25, 26) did not affect the activity (21-28%), and
a 5-hydroxyl group (5HPP-33: 28) enhanced the inhibit-
ing activity (7%). It is curious that 4HPP-33 (27)
showed less potent inhibiting activity (64%) than that
of the nonsubstituted analogue PP-33 (17, 21%), despite
the presence of the electron-donating hydroxyl group
(Table 4). We speculated that a hydrogen bonding inter-
action between the 4-OH group and the neighboring
imide carbonyl oxygen might be related to the decrease
in inhibiting activity.
Effects of Ha logen a tion of th e Con d en sed Ben -
zen e Rin g. It is of great interest that introduction of
four fluorine atoms into the phthalimide moiety (FPP-
33: 30) greatly lowered the concentration of the com-
pound which is necessary to elicit both TNF-R produc-
tion-enhancing and -inhibiting activity (Table 5; the
activity of FPP-33 (30) was assessed at 0.3 µM be-
O
21
22
O
MIP-33
EIP-33
134
138
37
75
NTc
NTc
N
NMe
O
N
NEt
See Table 1. c NT ) not tested.
a,b
been reported to enhance the teratogenicity,22 but the
effect on TNF-R production has not been reported so far.
Table 3 shows the effects of structural variation at the
succinimide moiety of the potent TNF-R production
regulator PP-33 (17). As shown in the table, all of the
compounds (except PPS-33: 18) showed TNF-R produc-
tion-regulating activity weaker than that of PP-33 (17)
(Figure 2a,b). The monothiocarbonyl derivative (PPS-
33: 18) showed activity superior to that of PP-33 (17)
(Table 3). It enhanced TPA-induced TNF-R production

2862 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Miyachi et al.
Ta ble 5. Effects of Halogenation at the Phthaloyl Moiety of
PP-33 on TNF-R Production Regulation
X
O
X
N
X
O
X
amount of
TNF-R (%)a
concn
TPA/
OA/
cytotoxicityb
compd (µM)
code
X
HL-60 HL-60 IC50 (µg/mL)
17
30
31
32
30
0.3
30
30
PP-33
FPP-33
ClPP-33 Cl
BrPP-33 Br
H
F
602
375
250
235
21
0
19
3
>100
0.9 (2.3 µM)
>100
F igu r e 3. Effects of FPP-33 on LPS-induced TNF-R produc-
tion inhibition. Horizontal scale: concentration of added test
compound. Vertical scale: amount of TNF-R (%, vide infra).
J 774.1 cells were treated with 1 µg/mL of LPS in the presence
of various concentrations of FPP-33. The amount of TNF-R
secreted in the culture medium was measured by ELISA. The
amount of TNF-R production in the presence 1 µg/mL of LPS
alone was defined as 100%.
NTc
a
HL-60 cells were treated with 10 nM TPA or 50 nM OA in
the presence of 30 µM (except FPP-33, 300 nM) test compound.
The amount of TNF-R secreted in the culture medium was
measured by ELISA. The amount of TNF-R production in the
presence of 10 nM TPA alone or 50 nM OA alone was defined as
100%. See Table 1. c NT ) not tested.
b
Str u ctu r e-Activity Rela tion sh ip s of th e Novel
Ser ies of N-Ben zylp h th a lim id es. As already re-
ported, optically active phthalimide derivatives showed
enantiodependent regulation of TNF-R production by
HL-60 cells.18 That is (S)-methylthalidomide (Figure
1), (S)-FPTP (34), and (S)-FPTH (38) (Table 6) exhibited
an enhancing effect on TPA-induced TNF-R production
by HL-60 cells, and the corresponding (R)-enantiomers
((R)-methylthalidomide (Figure 1), 33, and 37 (Table 6),
respectively) were completely inactive, while (R)-meth-
ylthalidomide, (R)-FPTP (33), and (R)-RPTH (37) showed
more potent inhibiting activity than the corresponding
(S)-enantiomers on OA-induced TNF-R production by
the same cell line.
To examine the generality of this phenomenon and
to study the structure-activity relationship of com-
pounds of this type, further chemical modification of
both enantiomers of FPTP (33, 34) was performed.
Because tetrafluorination dramatically lowered the ef-
fective concentration of the compounds as stated above,
we focused on the tetrafluorophthalimide skeleton and
assayed the compounds at 0.3 µM. The results are
summarized in Tables 6 and 7. Elongation of the side
chain methyl group of (S)-FPTP (34) to an ethyl group
[(S)-FPEP (40)] enhanced the TPA-induced TNF-R
production, but this modification greatly decreased OA-
induced TNF-R production (Table 6). Cyclization of the
side chain of (S)-FPTP (34) [(S)-FP13P (42)] greatly in-
creased the TNF-R production-enhancing activity (Table
6). It is of interest that the antipodal (R)-enantiomer
[(R)-FP13P (41)] also showed TNF-R production-enhanc-
ing activity. Cyclization of the side chain (41-43)
decreased OA-induced TNF-R production (Table 6).
FP22P (43), a positional isomer of FP13P, lacked
enhancing activity, but it showed moderate inhibiting
activity. This means that not the length, but the
width of the substituent introduced at the imide nitro-
gen is critical for potent TNF-R production-enhancing
activity. In contrast, a sterically bulky substituent at
the imide nitrogen is not necessary for potent TNF-R
production-inhibiting activity. These results indicated
a distinct structural requirement for the succinimide
moiety of N-phenylphthalimide for potent TNF-R pro-
duction enhancement, while the requirement in the case
of TNF-R production inhibition was considerably less
strict.
cause it showed the maximum activity at this concen-
tration, while the other compounds were assessed at
30 µM).20 As previously reported, FPP-33 (30) exhibited
TNF-R production-enhancing activity amounting to
more than 300% at 0.1 µM (Figure 2a). On the other
hand, the tetrachloro (ClPP-33: 31) and tetrabromo
(BrPP-33: 32) analogues are much less active than
FPP-33 (30), both as enhancers and inhibitors. Both
ClPP-33 (31) and BrPP-33 (32) were less active than
0.3 µM FPP-33 (30) even at 100 times higher concentra-
tion (30 µM) (Table 5). These observations suggest that
the increase in activity is specific to tetrafluorination
of the phthaloyl group. Recently, Liu and co-workers
also reported this unique effect of tetrafluorination.23
They synthesized tetrafluorothalidomide and found that
the compound is over 500-fold more potent than thali-
domide. This promising effect with respect to TNF-R
production regulation cannot be explained only in terms
of the electronegative character of the fluorine substit-
uents; the reasons remain unknown. The dose-
response curves for the enhancing activity of the halo-
genated compounds on TNF-R production are shown in
Figure 2a-c.
Because FPP-33 (30) showed potent TNF-R produc-
tion-regulating activity, we attempted to assay the
TNF-R production-regulating activity of the compound
in normal human peripheral blood cells. However, the
results were not reproducible: sometimes enhancement
was observed and sometimes inhibition. Normal pe-
ripheral blood cells used in our experiments might be
heterogeneous in their responses to cell stimulators and
to our compounds. In relation to this phenomenon, we
previously reported cell-type-dependent bidirectional-
regulation of TNF-R production with our compounds.17
As a cell stimulator/inducer of TNF-R production, li-
popolysaccharide (LPS) is considered to play a central
role in the pathogenesis of septic shock syndromes.
Unfortunately, HL-60 is not induced by LPS to produce
TNF-R protein. Liu and co-workers reported that one
of our compounds, FPP-33 (30), strongly inhibited LPS-
induced TNF-R production by vitamin D3-treated THP-1
cells.23 We also observed potent inhibitory activity of
FPP-33 (30) on LPS-induced TNF-R production using a
mouse macrophage-like cell line, J 774.1,24 with an IC50
of 250 nM (Figure 3).

Novel Biological Response Modifiers
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2863
Ta ble 6. Effects of N-Substituted Phthalimides on TNF-R
Production
Ta ble 7. Effects of N-Benzylphthalimides on TNF-R
Production
F
F
R
F
O
O
F
F
R1
F
F
R1
N
N
R2
O
O
F
amount of
R3
TNF-R (%)a
amount of
code
TPA/ OA/ cytotoxicityb
HL-60 HL-60 IC50 (µg/mL)
TNF-R (%)a
compd (0.3 µM)
R1
R2
code
(0.3 µM)
TPA/
HL-60
OA/
HL-0
33
34
35
36
37
38
39
40
(R)-FPTP CH3
(S)-FPTP CH3
(R)-FPTN CH3
(S)-FPTN CH3
(R)-FPTH CH3
(S)-FPTH CH3
(R)-FPEP C2H5
(S)-FPEP C2H5
97
143
100
101
98
2
12.8 (40 µM)
compd
R1
R3
33
34
44
45
46
47
48
49
(R)-FPTP
(S)-FPTP
CH3
CH3
CH3 NO2
CH3 NO2
CH3 NH2
CH3 NH2
H
H
97
143
239
268
241
262
220
268
2
70
27
32
27
38
55
80
70 26.8 (83 µM)
(R)-FPTP-00N
(S)-FPTP-00N
(R)-FPTP-00A
(S)-FPT-00A
(R)-FPTP-93
(S)-FPTP-93
2
2.8 (8 µM)
CH3 NHCOCH3
CH3 NHCOCH3
a
See Table 6.
101 2.8 (8 µM)
10 >100
65 >100
67 NTc
duction of a substituent at the para position of the
benzyl moiety of (R)/(S)-FPTP (44-49) generally in-
creased the enhancing activity on TPA-induced TNF-R
production. On the other hand, introduction of a para
substituent decreased the inhibiting activity on OA-
induced TNF-R production for (R)-isomers (compared
with (R)-FPTP:33) and increased it for (S)-isomers
(except (S)-FPTP-93 (49), which showed almost the
same activity as (S)-FPTP (compared with (S)-FPTP:
34) (Table 7)). Therefore, the degree of enantiodepen-
dence was decreased by the introduction of substituents
at the para position of the benzyl moiety of FPTP. To
summarize the structure-activity relationships of the
N-benzylphthalimide derivatives, elongation of the side
chain, cyclization of the side chain, and introduction of
substituents at the para position of the benzyl moiety
enhanced the TPA-induced TNF-R production by HL-
60 cells, while these structural modifications decreased
the OA-induced TNF-R production-inhibiting activity of
(R)-FPTP (33). The degree of enantiodependence was
decreased by these structural modifications. The dose-
response curves for the inhibiting activity of optically
active phthalimide derivatives on TNF-R production are
shown in Figure 2d.
124
111
235
87 NTc
41
42
(R)-FP13P
233
392
104
43 30.3 (87 µM)
98 >100
R =
(S)-FP13P
R =
43
FP22P
43 0.9 (2.6 µM)
R =
Discu ssion
a
HL-60 cells were treated with 10 nM TPA or 50 nM OA in
the presence of 0.3 µM test compound. The amount of TNF-R
secreted in the cuture medium was measured by ELISA. The
amount of TNF-R production in the presence of 10 nM TPA alone
The TNF-R production-regulating activity and struc-
ture-activity relationships of N-substituted phthalim-
ides were investigated. On the basis of our experiments
using HL-60 stimulated with TPA or OA, the structural
requirements for the TNF-R production-enhancing ac-
tivity are the presence of a bulky, hydrophobic substitu-
ent at the imide nitrogen moiety, phthalimide, or
monothiophthalimide structure, and the introduction of
electron-withdrawing groups at the phthalimide moiety.
In contrast, structural requirements for the TNF-R
production-inhibiting activity are the presence of a
bulky hydrophobic substituent at the imide nitrogen
moiety, and the introduction of electron-donating groups
at the phthalimide moiety. Phthalimide structure is not
always required for potent TNF-R production-inhibiting
activity. Substitution of the four hydrogen atoms at the
phthalimide moiety with fluorine greatly decreased the
concentration of the compound which is necessary to
elicit TNF-R production-regulating activity.
or 50 nM OA alone was defined as 100%. See Table 1. c NT )
not tested.
b
In the series of N-benzylphthalimide derivatives (44-
49, Table 7), introduction of a substituent at the para
position of the benzyl moiety enhanced TPA-induced
TNF-R production. Compounds 44-49 listed in Table
7 showed TNF-R production-enhancing activity amount-
ing to >200 at 0.3 µM, while the activities of the mother
compounds (FPTPs: 33 and 34) were 97% and 143%.
In this case, both enantiomers exhibited equipotent
enhancing activity (compounds 44-49). The enhancing
activity does not depend on the electronic effect of the
substituent, since the compounds with the electron-
withdrawing nitro group (44, 45) or the electron-
donating amino group (46, 47) showed equipotent TPA-
induced TNF-R production-enhancing activity. Intro-

2864 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Miyachi et al.
a microscope, and the cells were collected by centrifugation
(2000 rpm × 10 min). The amount of TNF-R in the superna-
tant was measured by the use of a human TNF-R ELISA
system (Amersham Co.) according to the supplier’s protocol.
The amount of TNF-R produced in the presence of inducer
alone was defined as 100%.
Tetrafluorophthalimide derivatives show activity at
concentrations of the order of 10-8 M. The presence of
compounds which are effective at such low concentra-
tions and the well-defined structutre-activity relation-
ships suggests that there is a distinct specific target
molecule(s) [binding protein(s)] of phthalimide-type
TNF-R production regulators. Though the molecular
mechanism(s) of these compounds eliciting inducer/cell
type-specific bidirectional regulatory activity on TNF-R
production is not clear at this stage, we previously
proposed the existence of two distinct target molecules
of the TNF-R production regulators, of which one plays
a role in enhancement of TNF-R production and the
other transmits the inhibitory effect on TNF-R produc-
tion.16 In fact, we have found (R)-form-specific and (S)-
form-specific binding proteins in HL-60 cells, while the
nature of these binding proteins is not clear at this stage
(details will be published elsewhere). Of course, other
explanations are possible. Our phthalimides, especially
FPP-33 (30) (bidirectional TNF-R production-regulator),
(R)-FPTN (35) (selective TNF-R production-inhibitor)
and (S)-FP13P (42) (selective TNF-R production-en-
hancer) should be useful tools to investigate TNF-R
production-regulatory mechanisms, and they also rep-
resent lead compounds for the development of superior
thalidomidal BRMs targeting TNF-R.
Concerning the spectrum/specificity of the phthalim-
ide derivatives described in this paper (compounds 1-9,
11-49), these compounds were found to inhibit TNF-R
production in both TPA-induced and OA-induced THP-1
cells (details will be published elsewhere). Analysis of
the difference between THP-1 and HL-60 cells at the
molecular level, in relation to the different TNF-R
production-regulatory responses to our compounds, is
in progress.
In conclusion, though interpretation of the cell type
specificity and inducer specificity of our compounds at
the molecular level must await further investigation,
we believe that several of these compounds, especially
FPP-33 (R)-FPTP, and (R)-FPTN, have potential for
development as novel BRMs to control TNF-R produc-
tion. Pharmacological evaluation of these compounds
is in progress.
J 774.1 cell line was kindly provided by Dr. Nishijima and
maintained in RPMI 1640 containing 10% FCS (GIBCO). The
cells at a concentration of 5 × 105 cells/mL in RPMI1640
containing 10% FCS were cultured in the presence or absence
of 1 × 10-6 g/mL of LPS at 37 °C for 24 h with or without a
test compound. The amount of TNF-R in the supernatant was
measured by the use of a mouse TNF-R ELISA system
(Amersham Co.) according to the supplier’s protocol. To assess
the cell toxicity of the compounds, human embryonic lung
fibroblast WI-38 cells (Dainippon Pharmaceutical Co.) were
plated (5 × 103 cells/well) in 150 µL of RPMI1640 medium
supplemented with 10% v/v FCS in 96-well microtiter plates.
Various concentrations of test compounds in 50 µL of culture
medium were added to the wells, and the plates were incu-
bated at 37 °C for 48 h. After the incubation, viable cell
number was assessed by WST-1 assay.19 Briefly, WSR-1
solution (10 µL, 3.3 mg/mL) containing 1-methoxy PMS (70
µg/mL) was added to each well, and the plate was incubated
at 37 °C for 2 h. Then the absorbance at 450 nm (red
formazan) was measured.
Ch em ica ls. Most of the compounds listed in Tables 1-6
were prepared (yield; 34%-98%) by condensation of phthalic
anhydride (or its derivatives) with appropriate amines without
any solvent at 160-180 °C for 2 h. The product was separated
by silica gel column chromatography and recrystallized from
an appropriate solvent(s).
Physicochemical properties of PP-00 (1), PP-10 (2), PP-n50
(3), PP-30 (4), PP-M0 (6), PP-N0 (7), PP-11 (11), PP-0101 (12),
PP-21 (13), PP-22 (14), PP-31 (16), PP-33 (17), PPS-33 (18),
4NPP-33 (23), 5NPP-33 (24), 4APP-33 (25), 5APP-33 (26),
4HPP-33 (27), 5HPP-33 (28), FPP-33 (30), ClPP-33 (31), (R)-
FPTP (33), (S)-FTP (34), (R)-FPTN (35), (S)-FPTN (36), (R)-
FPTH (37) and (S)-FPTH (38) were described previously.16-18,20
2-(2-Bip h en ylyl)-1H-isoin d ole-1,3-d ion e (P P -p h 0: 5)
(yield, 72%): mp 163-164 °C (from n-hexane/ethyl acetate);
1H-NMR (500 MHz, CDCl3) δ 7.20-7.34 (5H, m), 7.50-7.54
(4H, m), 7.70 (2H, dd, J ) 5.50, 3.05 Hz), 7.81 (2H, dd, J )
5.50, 3.05 Hz); MS m/z 371 (M+). Anal. (C20H13NO2) C, H, N.
2-(2,6-Diisop r op ylp h en yl)-1H-n a p h th oisoin d ole-1,3-d i-
on e (NAP -33: 29) (yield, 34%): mp 251-252.3 °C (from
n-hexane/ethyl acetate); 1H-NMR (500 MHz, CDCl3) δ 1.18
(12H, d, J ) 6.84 Hz), 2.77 (2H, hept, J ) 6.84 Hz), 7.32 (2H,
d, J ) 7.73 Hz), 7.48 (1H, t, J ) 7.73 Hz), 7.72-7.76 (2H, m),
8.09-8.13 (2H, m), 8.49 (2H, s); MS m/z 357 (M+). Anal.
(C24H23NO2) C, H, N.
Exp er im en ta l Section
(R)-2-[1-(4-Nit r op h en yl)et h yl]-4,5,6,7-t et r a flu or o-1H -
isoin d ole-1,3-d ion e [(R)-F P TP -00N: 44] (yield, 98%): mp
Gen er a l. Melting points were determined on a Yanagimoto
micro melting point apparatus and are uncorrected. 1H-NMR
spectra were measured in CDCl3 with TMS and the solvent
peak as internal standards, on a J EOL J MN-A500 spectrom-
eter. Mass spectra (MS) were obtained on a J EOL J MS-HX110
spectrometer. Optical rotations were measured on a J ASCO
DIP-100 digital polarimeter. Column chromatography was
carried out on Merck silica gel 60. Analytical thin-layer
chromatography (TLC) was performed on Merck precoated
silica gel 60F254 plates, and the compounds were visualized
by UV illumination (254 nm) or by heating 150 °C after
spraying with phosphomolybdic acid in ethanol. Elemental
analysis was performed in the microanalytical laboratory of
the Institute of Molecular and Cellular Biosciences, The
University of Tokyo, or at Kyorin Pharmaceutical Co., Ltd.
Cells a n d Mea su r em en t of TNF -r. HL-60 cells were
maintained as previously described.20 The exponentially
growing cells in RPMI1640 medium supplemented with 10%
v/v fetal bovine serum (2.5 × 105 cells in 0.5 mL) were treated
or not treated with TPA or OA for 16 h at 37 °C using 24-well
multidish plates. For testing the effects of compounds, cells
were treated with TPA (10 nM) or OA (50 nM) as an inducer
in the presence or absence of a sample compound at the
concentrations indicated in the tables. Then the number of
cells was counted, the cellular morphology was checked under
158-160 °C (from n-hexane/ethyl acetate). [R]20 +40.4° (c
D
1
0.365, CH3CO2C2H5); H-NMR (500 MHz, CDCl3) δ 1.95 (3H,
d, J ) 7.33 Hz), 5.60 (1H, q, J ) 7.33 Hz), 7.65 (2H, d, J )
8.85 Hz), 8.21 (2H, d, J ) 8.85 Hz); MS m/z 368 (M+). Anal.
(C16H8F4N2O4) C, H, N.
(S)-2-[1-(4-Nit r op h en yl)et h yl-4,5,6,7-t et r a flu or o-1H -
isoin d ole-1,3-d ion e [(S)-F P TP -00N: 45] (yield, 78%): mp
158-159.5 °C (from n-hexane/ethyl acetate); [R]20 -43.3° (c
D
0.21, CH3CO2C2H5); 1H-NMR (500 MHz, CDCl3) δ 1.95 (3H,
d, J ) 7.33 Hz), 5.60 (1H, q, J ) 7.33 Hz), 7.65 (2H, d, J )
8.85 Hz), 8.20 (2H, d, J ) 8.85 Hz); MS m/z 368 (M+). Anal.
(C16H8F4N2O4) C, H, N.
2-(2,6-Diisop r op ylp h en yl)-3-(m et h ylim in o)-1-oxo-1H -
isoin d ole (MIP -33: 21). A mixture of 18 (500 mg, 1.55
mmol), methylamine hydrochloride (190 mg, 1.63 mmol),
ethanol (20 mL), and 0.1 N NaOH solution (15 mL) was
refluxed for 10 h, and the reaction mixture was evaporated.
The residue was purified by flash column chromatography on
silica gel, eluting with CH2Cl2/methanol (20:1 v/v). The crude
product was recrystallized from n-hexane/ethyl acetate to
give 160 mg (yield, 32%) of colorless needles (5:1 mixture of
regioisomers): mp 114-115 °C (from n-hexane/ethyl acetate);
1H-NMR (500 MHz, CDCl3) major isomer δ 1.13-1.58 (12H,
m), 2.87 (2H, quint, J ) 7.02 Hz), 3.71 (3H, s), 7.26-7.28 (2H,

Novel Biological Response Modifiers
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2865
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m), 7.41-7.45 (1H, m), 7.71-7.78 (2H, m), 8.04 (1H, dd, J )
7.01, 0.9 Hz), 8.12 (1H, d, J ) 7.32 Hz); minor isomer δ 1.13-
1.58 (12H, m), 2.87 (2H, quint, J ) 7.02 Hz), 2.90 (3H, s), 7.26-
7.28 (2H, m), 7.41-7.45 (1H, m), 7.66 (1H, t, J ) 7.60 Hz),
7.71-7.78 (2H, m), 7.91 (1H, dt, J ) 7.01, 0.9 Hz); MS m/z
320 (M+). Anal. C21H24N2O) C, H, N.
2-(2,6-Diisopr opylph en yl)-3-(eth ylim in o)-1-oxo-1H-isoin -
d ole (EIP -33: 22). A procedure similar to that described for
21, utilizing 18, base, and ethylamine hydrochloride, afforded
22 (5:1 mixture of regioisomers) (yield, 35%): mp 132-133 °C
(from n-hexane/ethyl acetate); 1H-NMR (CDCl3) major isomer
δ 1.09-1.19 (12H, m), 1.30 (3H, t, J ) 7.02 Hz), 2.71 (2H,
quint, J ) 7.02 Hz), 4.04 (2H, quint, J ) 7.32 Hz), 7.24-7.27
(2H, m), 7.41-7.45 (1H, m) 7.69-7.76 (2H, m), 8.04-8.05 (2H,
m), 8.12 (1H, d, J ) 7.32 Hz); minor isomer δ 1.09-1.19 (15H,
m), 2.85 (2H, quint, J ) 7.02 Hz), 3.03 (2H, quint, J ) 7.32
Hz), 7.24-7.27 (2H, m), 7.41-7.45 (1H, m), 7.65 (1H, t, J )
7.02 Hz), 7.69-7.76 (1H, m), 7.90 (1H, d, J ) 7.32 Hz), 7.96
(1H, d, J ) 7.32 Hz); MS m/z 334 (M+). Anal. (C22H26N2O) C,
H, N.
(R)-2-[1-(4-Am in op h en yl)eth yl]-4,5,6,7-tetr a flu or o-1H-
isoin d ole-1,3-d ion e [(R)-F P TP -00A: 46]. A mixture of 44
(150 mg, 0.407 mmol), 10% Pd-C (50 mg), and ethyl acetate
(100 mL) was hydrogenated for 1 h. The catalyst was removed
by filtration, and the filtrate was evaporated. The residue was
purified by flash column chromatography on silica gel, eluting
with CH2Cl2/methanol (20:1 v/v). The crude product was
recrystallized from n-hexane/ethyl acetate to give 100 mg of
colorless powder (yield, 73%): mp 175-177 °C (from n-hexane/
ethyl acetate); [R]20 +67.4° (c 0.161, CH3CO2C2H5); 1H-NMR
D
(500 MHz, CDCl3) δ 1.86 (3H, d, J ) 7.32 Hz), 3.67 (2H, br s),
5.43 (1H, q, J ) 7.32 Hz), 6.63 (2H, d, J ) 8.54 Hz), 7.29 (2H,
d, J ) 8.54 Hz); MS m/z 338 (M+). Anal. (C16H10F4N2O2) C,
H, N.
(S)-2-[1-(4-Am in op h en yl)eth yl]-4,5,6,7-tetr a flu or o-1H-
isoin d ole-1,3-d ion e [(S)-F P TP -00A: 47]. A procedure simi-
lar to that described for 46, utilizing 45, 10% Pd-C, and ethyl
acetate, afforded [(S)-FPTP-00A: 47] (yield, 75%): mp 175-
177 °C (from n-hexane/ethyl acetate); [R]20 -66.4° (c 0.135,
D
1
CH3CO2C2H5); H-NMR (500 MHz, CDCl3) δ 1.86 (3H, d, J )
7.32 Hz), 3.67 (2H, br s), 5.43 (1H, q, J ) 7.32 Hz), 6.63 (2H,
d, J ) 8.54 Hz), 7.29 (2H, d, J ) 8.54 Hz); MS m/z 338 (M+).
Anal. (C16H10F4N2O2) C, H, N.
(R)-2-[1-[4-(Acetyla m in o)p h en yl]eth yl]-4,5,6,7-tetr a flu -
or o-1H-isoin dole-1,3-dion e [(R)-FP TP -93: 48]. Acetyl chlo-
ride (78.5 mg, 1.00 mmol) was added dropwise to a solution of
46 (338 mg, 1.00 mmol), triethylamine (101 mg, 1.00 mmol),
and CHCl3 (20 mL) at 0 °C. The solution was stirred overnight
at room temperature, and the reaction was quenched by the
addition of water (100 mL). The organic phase was washed
with 0.1 N NaOH and 0.1 N HCl and then evaporated. The
residue was purified by flash column chromatography on silica
gel, eluting with CH2Cl2/methanol (20:1 v/v). The crude
product was recrystallized from n-hexane/ethyl acetate to give
210 mg of yellow powder (yield, 55%): mp 140-143 °C (from
n-hexane/ethyl acetate); [R]20D +47.8° (c 0.298, CH3CO2C2H5);
1H-NMR (500 MHz, CDCl3) δ 1.89 (3H, d, J ) 7.32 Hz), 2.16
(3H, s), 5.49 (1H, q, J ) 7.32 Hz), 7.13 (1H, br s), 7.44-7.45
(4H, m); MS m/z 380 (M+). Anal. (C18H12F4N2O3) C, H, N.
(S)-2-[1-[4-(Acetyla m in o)p h en yl]eth yl]-4,5,6,7-tetr a flu -
or o-1H-isoin d ole-1,3-d ion e [(S)-F P TP -93: 49]. A proce-
dure similar to that described for 48, using 47, base, and acetyl
chloride, afforded 49 (yield, 60%): mp 140-143 °C (from
n-hexane/ethyl acetate); [R]20D -45.3° (c 0.788, CH3CO2C2H5);
1H-NMR (500 MHz, CDCl3) δ 1.88 (3H, d, J ) 7.32 Hz), 2.15
(3H, s), 5.48 (1H, q, J ) 7.32 Hz), 7.17 (1H, br s), 7.42-7.46
(4H, m); MS m/z 380 (M+). Anal. (C18H12F4N2O3) C, H, N.
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