Aza analogues of thalidomidesynthesis and evaluation as inhibitors of tumor necrosisfactor-α production in vitro

Article abstract of DOI:10.1016/S0968-0896(00)00323-0

A synthetic entry to derivatives of the new classes of 5-phthalimidouracils and 5-phthalimidobarbituric acids is reported. These 5-phthalimidopyramines as well as phthalimido-2,4-difluorobenzenes were designed as analogues of thalidomide, a well known inhibitor of TNF-α production. A preliminary in vitro investigation of the compounds as inhibitors of the TNF-α production was performed. Among the compounds of the present series, (5-ethyl-1-phenyl-5- tetrafluorophthalimido)barbituric acid and (2- 2,4-difluorophenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3 2H)-dione were proved to be potent inhibitors. Both compounds showed inhibitory activity in the lower micromolar range of the LPS-induced TNF-α production in human monocytes. Copyright

Full text of DOI:10.1016/S0968-0896(00)00323-0

Bioorganic & Medicinal Chemistry 9 (2001) 1059±1065
Aza Analogues of Thalidomide:
Synthesis and Evaluation as Inhibitors of Tumor Necrosis
Factor-ꢀ Production In Vitro
Michael Gutschow,a,* Thomas Hecker,a Andrea Thiele,b Sunna Hauschildtb
and Kurt Egera
aInstitute of Pharmacy, Pharmaceutical Chemistry, University of Leipzig, D-04103 Leipzig, Germany
bInstitute of Zoology, Department of Immunobiology, University of Leipzig, D-04103 Leipzig, Germany
Received 18 October 2000; revised 21 November 2000; accepted 28 November 2000
AbstractÐA synthetic entry to derivatives of the new classes of 5-phthalimidouracils and 5-phthalimidobarbituric acids is reported.
These 5-phthalimidopyrimidines as well as phthalimido-2,4-di¯uorobenzenes were designed as analogues of thalidomide, a well
known inhibitor of TNF-a production. A preliminary in vitro investigation of the compounds as inhibitors of the TNF-a produc-
tion was performed. Among the compounds of the present series, 5-ethyl-1-phenyl-5-(tetra¯uorophthalimido)barbituric acid and
2-(2,4-di¯uorophenyl)-4,5,6,7-tetra¯uoro-1H-isoindole-1,3(2H)-dione were proved to be potent inhibitors. Both compounds
showed inhibitory activity in the lower micromolar range on the LPS-induced TNF-a production in human monocytes. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
lidomide and some of its derivatives is bidirectional and
speci®c to cell type and to inducer.6 Both thalidomide
enantiomers racemize with relatively high velocity under
physiological conditions;4,7 therefore, enantio-dependence
of the TNF-a production-regulating activity was also
investigated with nonracemizable analogues, for
example, 30-methylthalidomide.6,8 Modi®cations of the
thalidomide structure led to several analogues with
improved anti-TNF-a activities. Examples include amino
substitution at the phthaloyl moiety,9 phenyl substitution
at 50-position (e.g., 2),10 and phthaloyl b-amino amide
derivatives;11 the latter compounds were designed as
analogues of hydrolysis products of 1. Several tetra-
¯uorophthalimides were found to be superior to non-
¯uorinated analogues in blocking TNF-a production in
LPS-stimulated monocytes with inhibitor 3 being the
most potent.12
Tumor necrosis factor-a (TNF-a) is a pleiotropic cyto-
kine produced by numerous cell types among which
monocytes/macrophages play a major role. Over-
production of TNF-a is associated with a wide range of
pathological conditions.1 This has led to much e€ort in
®nding ways to downregulate its production. Attention
has focused on the use of low molecular weight agents
as TNF-a inhibitors, such as thalidomide (1, Fig. 1 )
which was reported to be a selective inhibitor of TNF-a
production in monocytes stimulated with bacterial lipo-
polysaccharide (LPS).2 Despite the teratogenicity of
thalidomide, there is a remarkable resurgence of interest
in the drug in recent years due to its usefulness for the
treatment of erythema nodosum leprosum and other
in¯ammatory and autoimmune disease states such as
chronic graft-versus-host disease, Behcet's disease,
rheumatoid arthritis, HIV-associated aphthous ulcera-
tion and wasting syndrome.3À5 The e€ectiveness of the
drug in these diseases has mostly been attributed to its
speci®c inhibitory activity on TNF-a production. It was
found that the regulation of TNF-a production by tha-
In the present paper, we report our initial results on the
preparation of novel 5-phthalimidopyrimidines and
their e€ects on TNF-a production. This approach is
based on replacing the ±CH±CH2±CH2± unit in 1 by
±C=CH±NH±, resulting in 5-phthalimidouracil (15,
Scheme 4). In contrast to 1, the dehydrogenated aza
analogue 15 contains no chiral centre. With respect to
structure 2, 5-phthalimidouracils with a phenyl substituent
*Corresponding author. Tel.: +49-341-973-6810; fax: +49-341-973-
6749; e-mail: guetscho@uni-leipzig.de
0968-0896/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00323-0

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M. Gutschow et al. / Bioorg. Med. Chem. 9 (2001) 1059±1065
È
Figure 1. Phthalimide derived inhibitors of TNF-a production.
Scheme 2. (a) Br2, acetic acid, room temperature; (b) NaN3, acetone-
H2O, re¯ux; (c) H2, Pd/C, acetic acid, room temperature.
Scheme 1. (a) NaH, dioxane, re¯ux, 4 N H2SO4, re¯ux; (b) Br2, acetic
acid, room temperature; (c) NH3, MeOH, 120 ꢀC.
at NÀ1are included in the present study. Furthermore,
the uracil unit of 5-phthalimidouracil was replaced by
the 2,4-di¯uorophenyl substituent thus leading to a
nonpolar isostere.13 Another structural modi®cation
included the introduction of a barbituric acid moiety.
For these derivatives, 5-alkyl substitution was applied in
order to prevent a labile hydrogen at the chiral carbon.
Scheme 3.
barbituric acids 8 when subjected to the conditions of
the azidolysis18 produce azido derivatives 9 (R=Ph,
cyclohexyl), conversion of the corresponding 5-methyl
derivative 11 (Scheme 3) to the desired azido compound
12 was not successful. The urea 13 was obtained, prob-
ably as a result of hydrolytic ring opening of the inter-
mediate 12 followed by decarboxylation. The synthesis
of the ®nal 5-phthalimidopyrimidines 15±22 is depicted
in Scheme 4. Phthalimidouracils 15±18 were prepared
by reacting phthalic anhydrides 14 with 5-aminouracil
or 6, respectively, in glacial acetic acid. Accordingly,
aminobarbituric acids 10 were transformed to phthali-
midobarbituric acids 19±22, and 2,4-di¯uoroaniline was
converted to the corresponding phthaloyl derivatives
23±25.
Results and Discussion
Chemistry
The synthesis of the uracil precursor 6 is shown in
Scheme 1. Methyl 3,3-dimethoxypropanoate was con-
densed with phenylurea to 1-phenyluracil (4).14 The
1-phenyl substitution was con®rmed by MS, considering
the reverse Diels-Alder fragmentation (M+±HNCO)
and the absence of the (M+±PhNCO) fragment, and 1H
NMR: On irradiation of the 6-H resonance, a NOE
enhancement in the multiplet of the phenyl protons was
observed. Selective bromination in the 5-position was
accomplished on treatment of 4 with bromine in acetic
acid by a procedure that turned out to be advantageous
compared to the described photolytic15 or oxidative16
bromination of uracils. Attempts to convert the bro-
mouracil 5 to the desired primary amine 6 by Gabriel
synthesis, or via the corresponding 5-azido derivative
were not successful, since 5 failed to react both with
potassium phthalimide or sodium azide. However, the
aminouracil 6 could be prepared from 5 with a solution
of ammonia in methanol on performing the reaction in a
closed vessel at 120 ꢀC for 20 h. The synthesis of 5-amino-
barbituric acids 10 (Scheme 2) followed a route that has
recently been described.17 Whereas 5-ethyl-5-bromo-
Inhibition of the TNF-ꢀ production
The inhibitory potential of 5-phthalimidopyrimidines
(15±22) and 2,4-di¯uorophenyl phthalimides (23±25)
was assessed by determining the TNF-a production of
LPS-stimulated monocytes (Table 1). 5-Phthalimido-
uracil (15), whose structure resembled that of thalido-
mide most compared to the other compounds of this
series, did show only weak activity (19% inhibition at
50 mM). Introduction of a phenyl, or cyclohexyl residue
at position 1 of the pyrimidine moiety resulted in a loss

M. Gutschow et al. / Bioorg. Med. Chem. 9 (2001) 1059±1065
È
1061
Table 1. Inhibition of LPS-induced TNF-a production in human
monocytes
% Inhibitiona (and cell viabilityb)
compd
at 50 mM
at 25 mM
at 5 mM
at 0.5 mM
1 (thalidomide)
15 (98)
19 (99)
<10
15
16
17
18
19
20
21
22
23
24
25
<10 (91)
<10 (95)
<10 (95)
<10 (90)
100 (68)
16 (68)
<10 (77)
<10 (87)
100 (78)
<10 (74)
<10
<10
<10
100 (73)
88 (80)
84 (95)
12 (87)
100 (88)
<10 (92)
aHuman monocytes (1Â106/mL) were incubated in the presence of
di€erent concentrations of the indicated compounds for 15 min before
addition of LPS (100 ng/mL). After 4 h, TNF-a concentration in the
culture supernatants was determined by ELISA. Percentage of inhibi-
tion refers to the amount of TNF-a produced in the presence of LPS
alone (100% activation). Values represent results of three independent
experiments.
bCytotoxic e€ects on LPS-treated monocytes are indicated in par-
entheses as percentage of surviving cells. Values represent results of
three independent experiments.
highly potent inhibitors.11 Compared to thalidomide,
which was reported to inhibit 50±60% of the LPS-
induced TNF-a release at 200 mM,6,13 IC50 values of
approximately 3 mM for the analogues 20 and 24 could
be estimated. These compounds showed complete inhi-
bition at 25 mM. However, at this concentration cell
viability was slightly a€ected by both compounds
(Table 1).
The mechanisms underlying the action of compounds
20 and 24 are not clear. Potential intervention sites in
TNF-a production have been reviewed (for example, see
ref 1a) and include both the induction of TNF expression,
as well as TNF synthesis, processing and release. Similar
to thalidomide,19,20 the compounds may exert their e€ect
by intervening with the transcriptional regulation of the
TNF-a gene. Another target of thalidomide has been
identi®ed as a1-acid glycoprotein (AGP).21 However,
tetra¯uoro thalidomide derivatives did not compete for
the thalidomide binding site on AGP and additional
pieces of evidence also support the conclusion that
¯uorinated and non¯uorinated phthalimides might
interact with distinct target proteins to modulate TNF-a
production.21 Moreover, inhibition of cAMP phospho-
diesterases (PDEs) is one mechanism by which TNF-a
production may be reduced. PDE4 is the major PDE
isoenzyme in monocytes and macrophages. Among
thalidomide analogues with TNF-a inhibitory activity,
certain inhibitors indeed acted as PDE4 inhibitors,22,23
but others, including thalidomide itself, failed to inhibit
PDE4.9,23
Scheme 4. (a) 5-Aminouracil or 6, acetic acid, re¯ux or 130 ꢀC; (b) 10
(R=Ph or cyclohexyl), acetic acid, re¯ux or 170 ꢀC; (c) 2,4-di¯uoro-
aniline, acetic acid, re¯ux.
of activity, provided that the phthalimido portion was
unsubstituted (17, 19, 22) or 30-nitro substituted (18,
21). The tetra¯uoro phthalmidopyrimidine 20 exhibited
strong inhibitory activity. Among the three di¯uoro-
phenyl phthalimides (23±25), only 24 was active. Thus,
in the present series, tetra¯uoro substitution at the
phthalimide unit was necessary for strong inhibitory
activity (20 versus 19; 24 versus 23), but not sucient,
since 16 was found to be inactive. The replacement of
the unsubstituted uracil moiety by a di¯uorophenyl rest
(16 versus 24) remarkably increased the inhibitory
potential. The di¯uorobenzene derivative 24 can be
regarded as a closed steric mimic13 for the uracil deri-
vative 16. Thus, the increased activity of 24 might be
attributed to the enhanced lipophilicity and the loss of
hydrogen bond donor capability, respectively, that
results from the isosteric replacement. Noteworthy, in a
series of phthaloyl b-amino amides, the TNF-a lowering
ability required a lipophilic group within the side chain,
and 3-phenylpropionic acid derivatives proved to be
In summary, we have developed a synthetic entry to
certain derivatives of the new classes of 5-phthalimido-
uracils and 5-phthalimidobarbituric acids. The synthetic
routes reported will be applicable to prepare various

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M. Gutschow et al. / Bioorg. Med. Chem. 9 (2001) 1059±1065
È
analogues in order to obtain structural diversity of
potential inhibitors of TNF-a production. Since, as
shown within the present series, high activity depended
on the presence of a tetra¯uorinated phthalimide unit,
combination of the ®xed tetra¯uorophthalimide portion
with various pyrimidinetrione and di¯uorophenyl sub-
stituents appears as a promising concept to develop
potent phthalimide derived inhibitors of TNF-a pro-
duction. For such syntheses, 5-substituted 5-amino-
barbituric acids can be utilized and these precursors can
be separated to non-racemizable enantiomers by stan-
dard procedures.24 We are currently investigating this
approach.
Detection of TNF-ꢀ in culture supernatants
The content of TNF-a in the cell culture supernatants
was determined by Enzyme-Linked Immunosorbent
Assay (ELISA). The primary anti-TNF-a antibody as
well as the secondary puri®ed rabbit polyclonal anti-
TNF-a antibody were kindly provided by W. Buurmann
(Maastricht University, Maastricht, The Netherlands).
The peroxidase labelled goat-anti-rabbit antibody was
from Sigma Aldrich, Deisenhofen, Germany.
1-Phenylpyrimidine-2,4(1H,3H)-dione (4). Phenylurea
(2.72 g, 20 mmol) was added in small portions to a mix-
ture of sodium hydride (675 mg of a 80% suspension in
oil, 22.5 mmol) and anhydrous dioxane (33 mL) at
argon atmosphere. When the hydrogen evolution has
ceased, methyl 3,3-dimethoxypropanoate (3.3 g,
22.5 mmol) was added. The mixture was re¯uxed for
90 min, poured into 4 N sulfuric acid (200 mL) and
re¯uxed again for 15 min, cooled and extracted with
CH2Cl2 (3Â200 mL). The organic layer was dried
(Na2SO4), evaporated to dryness under reduced pres-
sure and the residue was recrystallized from EtOH to
obtain 4 (1.4 g, 37%): mp 244±246 ꢀC (lit.26 mp 247 ꢀC);
1H NMR (DMSO-d6) d 5.68 (dd, 1H, J=7.9, 2.3 Hz),
7.40±7.52 (m, 5H), 7.70 (d, 1H, J=7.9 Hz), 11.42 (s,
1H); 13C NMR (DMSO-d6) d 101.60, 126.79, 128.20,
129.10, 138.88, 145.49, 150.36, 163.67; MS (EI) m/z (rel
intensity) 188 (M+, 100), 145 (M+ÀHCNO, 78).
Experimental
Chemistry
Melting points were determined on a Boetius apparatus
and are not corrected. Thin-layer chromatography was
performed on Merck aluminium sheets, silica gel 60
F254.
13C NMR spectra (75 MHz), 1H NMR spectra
(300 MHz), and 19F NMR spectra (188 MHz) were
recorded on a Varian Gemini 300. IR spectra were
measured with a Perkin-Elmer 16 PC FTIR spectro-
meter. Mass spectra (70 eV) were obtained using a Varian
MAT CH6 spectrometer. 5-Aminouracil, tetra¯uoro-
phthalic anhydride, 3-nitrophthalic anhydride, and 2,4-
di¯uoroaniline were purchased from Aldrich (Steinheim,
Germany). 5-Aminobarbituric acids 10, and 5-bromo-5-
methyl-1-phenylbarbituric acid (11) were prepared as
described.17
5-Bromo-1-phenylpyrimidine-2,4(1H,3H)-dione (5).
A
solution of bromine (2.4 g, 15 mmol) in glacial acetic
acid (10 mL) was added dropwise over 2 h to a solution
of 4 (2.82 g, 15 mmol) in glacial acetic acid (150 mL).
The mixture was stirred at room temperature overnight
and evaporated to dryness under reduced pressure. The
residue was washed withꢀ H2O and dried to give 5
(3.61g, 90%): mp 273±275 C (lit.15 mp 271±273 ꢀC); 1H
NMR (DMSO-d6) d 7.40±7.55 (m, 5H), 8.26 (s, 1H),
11.96 (s, 1H); 13C NMR (DMSO-d6) d 95.81, 126.79,
128.48, 129.05, 138.36, 144.93, 149.85, 159.76; MS (EI)
m/z (rel intensity) 268, 266 (M+, 45), 225, 223
(M+ÀHNCO, 44), 77 (100).
Cell separation and stimulation
Peripheral blood mononuclear cells (PBMCs) from
healthy donors were isolated by centrifugation over a
Ficoll-Paque (Pharmacia, Freiburg, Germany) density
gradient. After repeated washing in phosphate-bu€ered
saline containing 0.3 mM EDTA (PBS/EDTA) the PBMCs
were further separated by counter¯ow centrifugation using
the J6-MC elutriator system (Beckmann Instruments,
Palo Alto, USA) as described previously.25 Monocytes
of >90% purity were incubated at a density of 1Â106/mL
overnight in RPMI 1640 (Sigma Aldrich, Deisenhofen,
Germany), containing 10% FCS (Sigma Aldrich,
Deisenhofen, Germany), 2 mM glutamine (Biochrom
KG, Berlin, Germany), 100 U/mL penicillin (Bio-
chrom KG, Berlin, Germany) and 100 mg/mL strepto-
mycin (Biochrom KG, Berlin, Germany) in 96 well-
plates (Greiner, Trickenhausen, Germany). Inhibitor
stock solutions were prepared in DMSO. Dilutions were
done with RPMI 1640 containing 10% FCS to a ®nal
concentration of 0.05% DMSO in the cell culture. For
solvent controls, DMSO was added to obtain the same
®nal concentration as in the inhibitor-treated cultures.
After preincubation of the monocytes with the inhibi-
tors for 15 min, LPS (100 ng/mL) (Sigma Aldrich,
Deisenhofen, Germany) was added, and after 4 h
supernatants were collected and analyzed for TNF-a
concentrations. Cell viability was assayed by Trypan
blue exclusion.
5-Amino-1-phenylpyrimidine-2,4(1H,3H)-dione (6). Com-
pound 5 (1g, 3.74 mmol) was dissolved in a 20%
methanolic solution of NH3 (30 mL) and heated for 20 h
in a sealed tube at 120 ꢀC. The solution was evaporated
to dryness, redissolved in EtOH (100 mL), treated with
charcoal and ®ltrated. The ®ltrate was concentrated,
cooled, and the precipitate was ®ltered o€ to obtain 6
(290 mg, 38%): mp 223±225 ꢀC; 1H NMR (DMSO-d6) d
4.19 (s, 2H), 6.85 (s, 1H), 7.35±7.50 (m, 5H), 11.34 (s,
1H); 13C NMR (DMSO-d6) d 120.04, 123.26, 126.55,
127.61, 129.05, 139.73, 148.55, 161.11; MS (EI) m/z (rel
intensity) 203 (M+, 100), 133 (35), 105 (69). Anal. calcd
for C10H9N3O2: C, 59.11; H, 4.46; N, 20.68. Found: C,
58.81; H, 4.40; N, 20.35.
1-(2-Azidopropionyl)-3-phenylurea (13). Compound 11
was reacted with sodium azide in 50% aqueous acetone
as described18 to obtain compound 13: mp 177±180 ꢀC;
1H NMR (DMSO-d6) d 1.45 (d, 3H, J=6.9 Hz), 4.13 (q,

M. Gutschow et al. / Bioorg. Med. Chem. 9 (2001) 1059±1065
È
1063
1H, J=6.9 Hz), 7.07±7.13 (m, 1H), 7.30±7.40 (m, 2H),
7.50±7.60 (m, 2H), 10.21 (s, 1H), 10.90 (s, 1H); 13C
NMR (DMSO-d6) d 16.36, 57.09, 119.85, 123.86,
128.90, 137.32, 150.25, 172.98; IR (KBr, cmÀ1) 2104
(N3), 1702 (CO); MS (EI) m/z (rel intensity) 233 (M+,
22), 119 (100). Anal. calcd for C10H11N5O2: C, 51.50; H,
4.75; N, 30.03. Found: C, 51.30; H, 4.69; N, 30.30.
44 (100). Anal. calcd for C18H10N4O6: C, 57.15; H, 2.66;
N, 14.81. Found: C, 57.01; H, 2.55; N, 14.46.
5-Ethyl-1-phenyl-5-phthalimidobarbituric acid (19). A
mixture of 5-amino-5-ethyl-1-phenylbarbituric acid
(1.24 g, 5 mmol), phthalic anhydride (814 mg, 5.5 mmol)
and glacial acetic acid (60 mL) was re¯uxed for 5 h. The
solvent was evaporated under reduced pressure and the
residue was recrystallized from EtOH to give 19 (1.2 g,
5-Phthalimidopyrimidine-2,4(1H,3H)-dione (15). A mixture
of phthalic anhydride (592 mg, 4 mmol), 5-aminouracil
(508 mg, 4 mmol) and glacial acetic acid (25 mL) was
re¯uxed for 2 h. After cooling, the precipitate was ®l-
tered o€, washed with H2O and dried to give 15
(820 mg, 80%): mp >320 ꢀC, dec. (lit.27 mp >300 ꢀC);
1H NMR (DMSO-d6) d 7.90 (s, 1H), 7.91±8.03 (m, 4H),
11.41 (s, br, 1H), 11.64 (s, 1H); 13C NMR (DMSO-d6) d
105.34, 123.61, 131.33, 135.02, 143.44, 150.69, 160.70,
166.83; MS (EI) m/z (rel intensity) 257 (45, M+), 104
(60), 44 (100). Anal. calcd for C12H7N3O4: C, 56.04; H,
2.74; N, 16.34. Found: C, 55.70; H, 3.06; N, 16.00.
ꢀ
1
64%): mp 212±214 C; H NMR (DMSO-d6) d 1.11 (t,
3H, J=7.3 Hz), 2.89 (q, 2H, J=7.3 Hz), 7.21±7.38 (m,
2H), 7.42±7.59 (m, 3H), 7.95 (s, 4H), 12.43 (s, 1H); 13C
NMR (DMSO-d6) d 9.41, 27.02, 67.66, 123.84, 128.56,
128.99, 129.26, 130.30, 134.04, 135.64), 149.08, 167.54,
167.84, 167.90; MS (EI) m/z (rel intensity) 377 (M+,
31), 230 (50), 104 (100). Anal. calcd for C20H15N3O5: C,
63.66; H, 4.01; N, 11.14. Found: C, 63.33; H, 4.01; N,
11.01.
5-Ethyl-1-phenyl-5-(tetra¯uorophthalimido)barbituric acid
(20). A mixture of 5-amino-5-ethyl-1-phenylbarbituric
acid (250 mg, 1mmol), tetra¯uorophthalic anhydride
(260 mg, 1.2 mmol) and acetic acid (7 mL) was re¯uxed
for 3 h. The solvent was evaporated to dryness under
reduced pressure. The residue was recrystallized from
EtOH to give 20 (270 mg, 60%): mp 218±220 ꢀC; 1H
NMR (DMSO-d6) d 1.09 (t, 3H, J=7.2 Hz), 2.86 (q,
2H, J=7.3 Hz), 7.20±7.32 (m, 2H), 7.45±7.59 (m, 3H);
13C NMR (DMSO-d6) d 9.36, 26.90, 68.10, 112.20,
112.65, 128.05, 128.56, 129.27, 133.91, 141.10, 147.05,
148.88, 162.51, 166.85, 167.18; MS (EI) m/z (rel inten-
sity) 449 (M+, 62), 421(M +, 20), 230 (39), 176 (70), 119
(100). Anal. calcd for C20H11N3O5F4: C, 53.46; H, 2.47;
N, 9.35. Found: C, 53.26; H, 2.78; N, 9.04.
5-(Tetra¯uorophthalimido)pyrimidine-2,4(1H,3H)-dione
(16).
A mixture of tetra¯uorophthalic anhydride
(440 mg, 2 mmol), 5-aminouracil (254 mg, 2 mmol) and
glacial acetic acid (50 mL) was re¯uxed for 6 h. After
cooling, the precipitate was ®ltered o€, washed with
H2O and dried to give 16 (260 mg, 40%): mp 350±
355 ꢀC; 1H NMR (DMSO-d6) d 7.89 (d, 1H, J=6.2 Hz),
11.47 (d, 1H, J=6.2 Hz), 11.72 (s, 1H); 19F NMR
(DMSO-d6/CFCl3) d À142.0 (m, 2F), À136.5 (m, 2F);
MS (EI) m/z (rel intensity) 329 (M+, 50), 176 (100).
Anal. calcd for C12H3N3O4F4: C, 43.79; H, 0.92; N,
12.77. Found: C, 43.60; H, 1.16; N, 12.50.
1-Phenyl-5-phthalimidopyrimidine-2,4(1H,3H)-dione (17).
A mixture of compound 6 (203 mg, 1mmol), phthalic
anhydride (163 mg, 1.1 mmol) and glacial acetic acid
(10 mL) was re¯uxed for 90 min. The solvent was evap-
orated under reduced pressure and the residue was
recrystallized twice from EtOH to give 17 (200 mg,
5-Ethyl-5-(3-nitrophthalimido)-1-phenylbarbituric acid (21).
A mixture of 5-amino-5-ethyl-1-phenylbarbituric acid
(990 mg, 4 mmol), 3-nitrophthalic anhydride (772 mg,
4 mmol) and glacial acetic acid (40 mL) was heated in a
sealed tube at 170 ꢀC for 15 h. The solvent was evapo-
rated to dryness under reduced pressure. The residue
was recrystallized from ethyl acetate to give 21 (390 mg,
60%): mp 262±270 ꢀC; H NMR (DMSO-d6) d 7.48±
1
7.54 (m, 5H), 7.92±8.01 (m, 4H), 8.28 (s, 1H), 12.04 (s,
1H); 13C NMR (DMSO-d6) d 106.56, 123.68, 126.71,
128.65, 129.28, 131.27, 135.10, 138.29, 146.26, 149.66,
160.24, 166.66; MS (EI) m/z (rel intensity) 333 (58,
M+), 104 (100). Anal. calcd for C18H11N3O4: C, 64.86;
H, 3.33; N, 12.61. Found: C, 64.59; H, 3.22; N, 12.22.
23%): mp 214±216 ꢀC; H NMR (DMSO-d6) d 1.11 (t,
1
3H, J=7.3 Hz), 2.90 (q, 2H, J=7.3 Hz), 7.27±7.33 (m,
2H), 7.46±7.55 (m, 3H), 8.13±8.19 (m, 1H), 8.26±8.30
(m, 1H), 8.39±8.43 (m, 1H), 12.50 (s, 1H); 13C NMR
(DMSO-d6) d 9.40, 27.08, 68.06, 121.49, 127.93, 128.57,
129.03, 129.26, 129.81, 132.34, 133.94, 137.55, 144.46,
148.96, 163.49, 165.55, 167.04, 167.46; MS (EI) m/z (rel
intensity) 422 (M+, 35), 289 (30), 230 (32), 177 (30), 119
(100). Anal. calcd for C20H14N4O7: C, 56.88; H, 3.34, N,
13.27. Found: C, 56.90; H, 3.33; N, 12.90.
5-(3-Nitrophthalimido)-1-phenylpyrimidine-2,4(1H,3H)-
A mixture of compound 6 (508 mg,
dione (18).
2.5 mmol), 3-nitrophthalic anhydride (580 mg, 3 mmol)
and glacial acetic acid (40 mL) was heated in a sealed
tube at 130 ꢀC for 17 h. The solvent was evaporated
under reduced pressure and the oily residue was tritu-
rated with EtOH (10 mL) to obtain a precipitate which
was ®ltered o€. The crude product was recrystallized
from EtOH with charcoal to give 18 (650 mg, 69%): mp
1-Cyclohexyl-5-ethyl-5-phthalimidobarbituric acid (22).
A mixture of 5-amino-1-cyclohexyl-5-ethylbarbituric
acid (500 mg, 2 mmol), phthalic anhydride (300 mg,
2 mmol) and glacial acetic acid (20 mL) was re¯uxed for
5 h. The mixture was cooled, the precipitate was ®ltered
o€, washed with H2O and dried to give 22 (470 mg,
232±235 ꢀC; H NMR (DMSO-d6) d 7.47±7.55 (m, 5H),
1
8.13±8.20 (m, 1H), 8.24 (s, 1H), 8.29±8.32 (m, 1H),
8.38±8.42 (m, 1H), 12.09 (s, 1H); 13C NMR (DMSO-d6)
d 105.98, 122.37, 126.67, 127.54, 128.74, 129.06, 129.31,
132.89, 137.11, 138.26, 144.57, 146.49, 149.58, 160.01,
162.03, 164.62; MS (EI) m/z (rel intensity) 378 (M+, 4),
61%): mp 248±253 ꢀC; H NMR (DMSO-d6) d 0.99 (t,
1
3H, J=7.4 Hz), 1.01±2.21 (m, 10H), 2.68 (q, 2H,
J=7.4 Hz), 4.13±4.29 (m, 1H), 7.90 (s, 4H), 12.09 (s,
1H); 13C NMR (DMSO-d6) d 9.10, 24.83, 25.62, 25.71,

1064
M. Gutschow et al. / Bioorg. Med. Chem. 9 (2001) 1059±1065
È
27.23, 27.92, 29.05, 54.66, 67.83, 123.74, 130.32, 135.56,
149.24, 167.20, 167.79, 168.28; MS (EI) m/z (rel intensity)
383 (M+, 5), 302 (M+, 100), 105 (52). Anal. calcd for
C20H21N3O5: C, 62.65; H, 5.52; N, 10.96. Found: C,
62.30; H, 5.85; N, 10.89.
259 (81). Anal. calcd for C16H11NO2F2: C, 66.90; H,
3.86; N, 4.88. Found: C, 67.20; H, 3.77; N, 4.59.
Acknowledgements
2-(2,4-Di¯uorophenyl)-1H-isoindole-1,3(2H)-dione (23).
A mixture of 2,4-di¯uoroaniline (2 g, 15.5 mmol),
phthalic anhydride (2.07 g, 14 mmol) and acetic acid
(100 mL) was re¯uxed for 3.5 h. The solvent was evapo-
rated to dryness under reduced pressure. The residue
was dissolved in CH2Cl2 (150 mL), washed with 0.1 M
HCl (3Â50 mL) and H2O (50 mL), and dried (Na2SO4).
After removal of the solvent, the residue was recrys-
tallized twice from EtOH to give 23 (2.8 g, 77%): mp
The authors wish to thank R. Zahn for excellent technical
assistance. M. G. and K. E. would like to thank the
`Fonds der Chemischen Industrie' for ®nancial support.
References
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ꢀ
1
167±168 C; H NMR (DMSO-d6) d 7.25±7.33 (m, 1H),
7.48±7.56 (m, 1H), 7.62±7.71 (m, 1H), 7.90±8.03 (m,
4H); 13C NMR (DMSO-d6) d 104.98 (dd, 2J=26.8,
0
0
2
4
24.0 Hz, C-3 ), 112.17 (dd, J=22.7, J=3.5 Hz, C-5 ),
115.83 (dd, J=13.0, J=3.9 Hz, C-10), 123.70 (C-5, C-
2
4
6), 131.38 (C-3a, C-7a), 131.94 (C-60), 134.95 (C-4, C-7),
157.73 and 162.23 (dd, J=251.1, J=13.28 Hz and dd,
1J=247.7, J=11.7 Hz, C-2 and C-4 ), 166.17 (C-1, C-
3); MS (EI) m/z (rel intensity) 259 (M+, 100). Anal.
calcd for C14H7NO2F2: C, 64.87; H, 2.72; N, 5.40.
Found: C, 65.20; H, 2.61; N, 5.22.
1
4
0
0
3
2-(2,4-Di¯uorophenyl)-4,5,6,7-tetra¯uoro-1H-isoindole-
1,3(2H)-dione (24). A mixture of 2,4-di¯uoroaniline
(1g, 7.75 mmol), tetra¯uorophthalic anhydride (1.54 g,
7 mmol) and glacial acetic acid (50 mL) was re¯uxed for
3.5 h. The solvent was evaporated to dryness under
reduced pressure. The residue was dissolved in CH2Cl2
(75 mL), washed with 0.1M HCl (3 Â25 mL) and H2O
(2Â25 mL), and dried (Na2SO4). After removal of the
solvent, the residue was recrystallized from EtOH to
give 24 (980 mg, 42%): mp 145±146 ꢀC; 1H NMR
(DMSO-d6) d 7.22±7.38 (m, 1H), 7.50±7.66 (m, 2H); 19
F
NMR (DMSO-d6/CFCl3) d À142.3 (m, 2F), À136.9 (m,
2F), À113.8 (m, 1F), À105.6 (m, 1F); MS (EI) m/z (rel
intensity) 331(M +, 65), 287 (68), 148 (100). Anal. calcd
for C14H3NO2F6: C, 50.78; H, 0.91; N, 4.23. Found: C,
50.60; H, 0.83; N, 3.95.
2-(2,4-Di¯uorophenyl)-4,7-dimethyl-1H-isoindole-1,3(2H)-
dione (25).
A mixture of 2,4-di¯uoroaniline (2 g,
15.5 mmol), 3,6-dimethylphthalic anhydride28 (2.46 g,
14 mmol) and glacial acetic acid (100 mL) was re¯uxed
for 3.5 h. The solvent was evaporated to dryness under
reduced pressure. The residue was dissolved in CH2Cl2
(150 mL), washed with 0.1 M HCl (3Â50 mL) and H2O
(2Â50 mL), and dried (Na2SO4). After removal of the
solvent, the residue was recrystallized from EtOH to
give 25 (1.27 g, 32%): mp 212±212.5 ꢀC; 1H NMR
(DMSO-d6) d 2.59 (s, 6H), 7.24±7.32 (m, 1H), 7.46±7.56
(m, 1H), 7.54 (s, 2H) 7.56±7.66 (m, 1H); 13C NMR
(DMSO-d6) d 16.87 (CH3), 104.93 (dd, 2J=27.1,
20. Moreira, A. L.; Sampaio, E. P.; Zmudzinas, A.; Frindt, P.;
Smith, K. A.; Kaplan, G. J. Exp. Med. 1993, 177, 1675.
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H.; Hashimoto, Y. Bioorg. Med. Chem. Lett. 1998, 8, 1071.
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Patterson, R. T.; Chen, Y.; Stirling, D. I. Bioorg. Med. Chem.
Lett. 1998, 8, 2669.
24.2 Hz, C-30), 112.08 (dd, J=22.6, 0 J=3.6 Hz, C-50),
2
4
2
4
115.93 (dd, J=13.2, J=3.9 Hz, C-1 ), 128.15 (C-4, C-
7), 132.07 (dd, J=10.1, 2.1 Hz, C-60) 135.37 (C-3a, C-
3
7a), 136.62 (C-5, C-6), 157.81 and 162.08 (d,
J=264.1Hz and d, J=235.6 Hz, C-20 and C-40), 166.64
(C-1, C-3); MS (EI) m/z (rel intensity) 287 (M+, 100),

M. Gutschow et al. / Bioorg. Med. Chem. 9 (2001) 1059±1065
È
1065
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