Tumor necrosis factor-alpha production-regulating activity of phthalimide derivatives in genetically modified murine melanoma cells B78H1

Article abstract of DOI:10.1016/S0014-827X(03)00049-1

The effect of imides, monothioimides, trimellitimides, as well as 5′-deoxy-5′-phthaloylamino-derivatives of azidothymidine on tumor necrosis factor-alpha (TNF-α) production by genetically modified murine B78H1 melanoma cells transduced with the gene for human TNF-α (B78/TNF) was investigated. It was found that N-(adamant-1-yl)monothiophthalimide (1e) and N-(adamant-2-yl)-monothiophthalimide (1f) showed over 200% enhancing of TNF-α production while some of imides were inhibitors.

Full text of DOI:10.1016/S0014-827X(03)00049-1

Il Farmaco 58 (2003) 371Á376
/
www.elsevier.com/locate/farmac
Tumor necrosis factor-alpha production-regulating activity of
phthalimide derivatives in genetically modified murine melanoma
cells B78H1
a,
b
b
b
´
Andrzej Orzeszko *, Witold Lasek , Tomasz Switaj , Magdalena Stoksik ,
Beata Kamin´ska a
a Institute of Chemistry, Agricultural University, ul. Nowoursynowska 159c, 02-787 Warsaw, Poland
b Department of Immunology, Centre of Biostructure, Medical University, ul. Cha lbin´skiego 5, 02-004 Warsaw, Poland
Received 11 October 2002; accepted 10 January 2003
Abstract
The effect of imides, monothioimides, trimellitimides, as well as 5?-deoxy-5?-phthaloylamino-derivatives of azidothymidine on
tumor necrosis factor-alpha (TNF-a) production by genetically modified murine B78H1 melanoma cells transduced with the gene
for human TNF-a (B78/TNF) was investigated. It was found that N-(adamant-1-yl)monothiophthalimide (1e) and N-(adamant-2-
yl)-monothiophthalimide (1f) showed over 200% enhancing of TNF-a production while some of imides were inhibitors.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Imide; Thioimide; TNF-a
1. Introduction
production enhancers in some cases and production
inhibitors in other cases would be useful as biological
response modifiers under various circumstances. In this
context, it is interesting to search for new drugs working
as stimulators or inhibitors of TNF-a production.
Usually, for such biological evaluations human leu-
kaemia cell lines (HL-60, K592, U937) are used [2,3,5].
These cells do not secrete TNF-a without stimulation,
but they are beginning to produce the cytokine in
response to stimulators including two types of tumor
promotors 12-O-tetradecanoylphorbol-13-acetate or
okadaic acid. It was found that many chemicals exert
TNF-a production-enhancing activity and production-
inhibiting activity in such systems. There are two main
groups of compounds showing TNF-a production-
regulating properties including ‘thalidomide like’ phtha-
Tumor necrosis factor-alpha (TNF-a) is a pleiotropic
cytokine produced by numerous cell types among which
monocytes/macrophages play a major role. TNF-a
exerts its biological activity by binding to TNF-RI
(p55) or TNF-RII (p75) receptors and activating several
signaling pathways. TNF-RI mediates mainly cell death
(proapoptotic) signals, whereas TNF-RII primary in-
duces cell growth and survival pathways [1]. Investiga-
tion of the biological properties of TNF-a, in vitro as
well as in vivo models, has revealed that this cytokine
has both beneficial and unfavorable effects [2,3]. The
beneficial effects include direct antitumor activity be-
cause it exhibits cytotoxicity selectively against various
tumor cells. On the other hand, the overproduction of
TNF-a has been strongly implicated in the pathogenesis
of such diseases as septic shock, rheumatoid arthritis,
inflammatory bowel disease, multiple sclerosis [4]. These
pleiotropic effects of TNF-a indicate that TNF-a
limide derivatives and aminoheterocycles [2Á8]. Re-
/
cently, also we have found that some of imide
derivatives caring adamantane or nucleoside moieties
were biologically active as antibacterial and anticancer
agents [9Á12]. Now, we would like to check an influence
/
of these and similar compounds on TNF-a production.
Recently, we have presented the studies on cultures of
genetically modified mouse melanoma cells B78H1
* Corresponding author.
E-mail address: orzeszkoa@delta.sggw.waw.pl (A. Orzeszko).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00049-1

372
A. Orzeszko et al. / Il Farmaco 58 (2003) 371Á376
/
secreting TNF-a at a constant rate without stimulators
(B78/TNF) and by this reason useful for testing the
effects of various chemicals on TNF-a production. We
have found that some adamantylaminopyridines en-
hance TNF-a production several times in B78/TNF
melanoma cells [7,8].
In the presented paper we have synthesized a series of
imides, thioimides, trimellitimides as well as 5?-deoxy-5?-
phthaloylaminoderivatives of azidothymidine (AZT).
Then, these compounds were studied as TNF-a produc-
tion-regulating agents.
2.2. General procedure for the synthesis of imides 1a
/
d
Á
The synthesis of N-adamantyl substituted phthali-
mides and trimellitimides (see Scheme 1) were performed
according to the well-known method from proper
anhydrides and 1-aminoadamantane or 2-aminoada-
mantane in boiling DMF, respectively [9]. The crude
products were crystallized from ethanol/water (70:30)
system.
2.3. General procedure for the synthesis of
monothioimides 1e,f
Both monothioimides were synthesized from proper
imides using Lawesson’s reagent in boiling toluene
according to the procedure described previously [13].
Detailed spectroscopic and crystal data for these com-
pounds were given also in cited publication (Table 1,
Scheme 1).
2. Experimental
2.1. General methods
Melting points (m.p.) were taken in open capillary
tubes on a Gallenkamp 5 m.p. apparatus and were
uncorrected. The structures of products were confirmed
by elemental analysis, FTIR and 1H NMR spectro-
scopy. The NMR spectra were measured on a Varian
Gemini 200 MHz spectrometer in CDCl3 solutions.
FTIR spectra were recorded on a Perkin Elmer 2000
apparatus using KBr pellet method. Analyses indicated
2.4. General procedure for the synthesis of imides 2a
/
f
Á
f were
2-Substituted 1,3-dioxo-2,3-dihydro-1H-isoindole-5-
carboxylic acid adamantan-1-ylmethyl esters 2a
the same as described in our previous papers [9Á
order to achieve better solubility in water, we have
converted these compounds into sodium salts using
cation exchange cellulose Cellex CM according to the
standard procedure (see Scheme 2).
/
Á
/
11]. In
by symbols were within 90.4% of theoretical values.
/
Scheme 1.

A. Orzeszko et al. / Il Farmaco 58 (2003) 371Á
/376
373
Table 1
Synthesis of N-substituted imides and thioimides 1a
cm3 of dry DMF and were refluxed over 3 h. Then,
mixture was purred into aqueous 5% HCl. Crude
product was crystallized from ethanol. M.p. 151 8C;
yields 60%.
/
f studied
Á
FTIR (in cmꢀ1): 3300Á
1725 CÄOimide,acid
/
2500 OHacid, 1780 CÄ/Oimide,
/
.
2.5.2. 2-(2-chloroethyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carboxylic acid adamantan-1-ylmethyl ester
(2g) and 2-(2-chloroethyl)-1,3-dioxo-2,3-dihydro-1H-
isoindole-5-carboxylic acid adamantan-1-yl ester (2h)
A total of 2.54 g (10 mmol) of N-(2-chloroethyl)tri-
mellitimide and 10 mmol of 1-adamantanemethanol or
1-adamantanol, respectively, were dissolved in dry
methylene chloride. Next, 2.06 g (10 mmol) of DCC
was added. The mixture was stirred over 6 h. Then, after
filtration, the solvent was evaporated and crude pro-
ducts were crystallized from ethanol/H2O system. Re-
presentative spectroscopic data are reported for 2h.
1H NMR (in ppm): 1.22Á
2H), 3.93 (m, 2H), 8.00Á8.31 (m, 3H).
FTIR (in cmꢀ1): 2918, 2856 (CHadam.), 1779 (CÄ
Oimide), 1725 (CÄOimide,ester) (Table 2, Scheme 2).
/
2.50 (m, 15Hadam.), 3.86 (m,
/
/
/
2.5. The synthesis of imides 2g,h
2.5.1. N-(2-chloroethyl)trimellitimide
2.6. Synthesis of AZT derivatives 3a
/
c
Á
4-dihydro-2H-pyrimidin-1-yl)-tetrahydrofuran-2-yl-
A total of 1.92 g (10 mmol) trimellitic anhydride, 1.16
g of 2-chloroethylamine hydrochloride (10 mmol) and
2.02 g (20 mmol) of triethylamine were dissolved in 20
The synthesis of 2-[3-azido-5-(5-methyl-2,4-dioxo-3,
Scheme 2.

374
A. Orzeszko et al. / Il Farmaco 58 (2003) 371Á376
/
Table 2
Synthesis of N-substituted trimellitimides 2a
Table 3
Synthesis of AZT derivatives [12]
/
h studied [9Á11]
/
Á
3
X1 X2 X3 X4 X5 X6 M.p. (8C)
Yield (%)
a
a
b
c
O
O
S
H
Br Br Br Br
H
H
O
O
S
135
240
160
65
32
35
H
H
H
H
a
.
1H NMR (in ppm): 1.68 (s, 3H, CH3), 2.37 (m, 2H, H-
2?), 2.58 (m, 1H, H-3?), 3.98 (m, 2H, H-5?), 4,46 (m, 1H,
H-4?), 6.05 (t, 1H, H-1?), 7.45 (s, 1H, 6-H), 11.29 (s, 1H,
NH).
FTIR (in cmꢀ1): 3690 (NH), 2105 (CN), 1769 (CÄ
/
O),
1703(CÄ
/
O), 1606 (CÄ/C) (Table 3, Scheme 3).
2.7. Biological evaluation
The ability of the imide derivatives to stimulate TNF-
a production described above was studied in cultures of
B78H1 murine melanoma cells that had been transduced
with the gene for human TNF-a (clone 9, hereafter
named B78/TNF) [14]. The compounds were incubated
with melanoma cells (2ꢁ
105 cells/cm3) for 24 h as
/
methyl]-1,3-dioxo-1H-isoindole derivatives 3a,b and 2-
[3-azido-5-(5-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-tetrahydrofuran-2-yl-methyl]-3-thioxo-2,3-di-
hydroisoindol-1-one 3c were perform according to the
general synthetic pathway via Mitsunobu reaction given
in Scheme 3, [14]. A THF solution of mixture of 3?-AZT
(10 mmol), proper imides (10 mmol), triphenyl phos-
phine (10 mmol) and diethyl azodicarboxylate (10
mmol) was stirred in room temperature for 12 h. Then
THF was evaporated and solid crude products were
purified by means of flash-column chromatography
(SiO2) using ethyl acetate/hexane (2:1) as an eluent
system. The structures were confirmed also using 1H
NMR and FTIR spectroscopy. For example, spectro-
scopic data for 3b:
described elsewhere [7]. The concentration of TNF-a in
culture supernatant was measured using an enzyme-
linked immunosorbent assay. The concentration of
compounds tested was 100 mM. TNF-a stimulatory
activity of imide compounds were expressed as percent
of TNF-a level measured in control cultures (ꢂ
/100%)
[7,8].
3. Results and discussion
The ability of the compounds studied to stimulate
TNF-a production has been expressed as diagrams (Fig.
1). It should be noticed that N-(adamant-1-yl)phthali-
mide (1a) was examined by Hashimoto as enhancer of
Scheme 3.

A. Orzeszko et al. / Il Farmaco 58 (2003) 371Á
/376
375
Fig. 1. TNF-a production-regulating activity of phthalimide derivatives expressed as percent of TNF-a level in control cultures (incubated without
tested compounds (ꢂ100%).
/
TNF-a production on human leukaemia cell line HL-60
in presence of 12-O-tetradecanoylphorbol-13-acetate
and showed 500% overproduction of the cytokine [15].
It is interesting that in our evaluations this compound
showed rather weak activity. Among imides studied only
1b, 1e and 1f showed significant activity as enhancers of
TNF-a production (at least 200% of the control). It is
worth to notice that also in other papers sulfur-contain-
ing agents showed strong activity [2,8].
Acknowledgements
This study was supported in part by grant 1M19/W2
from the Medical University of Warsaw, Poland.
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