N-pyridinyl(alkyl)polyhalogenobenzamides acting as TNF-α production inhibitors

Article abstract of DOI:10.1211/146080899128734659

A series of N-pyridinyl(methyl)fluorobenzamides issued from 2,4-dimethyl-6-aminopyridine and 3-aminomethylpyridine were synthesized and evaluated as inhibitors of TNF-α production. Although less active than the corresponding phthalimides, several pentaflu

Full text of DOI:10.1211/146080899128734659

Pharm. Pharmacol. Commun. 1999, 5: 233±238
# 1999 Pharm. Pharmacol. Commun.
N-Pyridinyl(alkyl)polyhalogenobenzamides Acting as TNF-a
Production Inhibitors
X. COLLIN, J. -M. ROBERT, S. ROBERT, G. LE BAUT, C. BOBIN-DUBIGEON*, N. GRIMAUD*,
F. LANG* AND J. -Y. PETIT*
Laboratoires de Chimie Organique et de Chimie Th e rapeutique and *Laboratoire de Pharmacologie,
Facult e de Pharmacie, 1 rue Gaston Veil, 44035 Nantes, France
Abstract
A series of N-pyridinyl(methyl)¯uorobenzamides issued from 2,4-dimethyl-6-amino-
pyridine and 3-aminomethylpyridine were synthesized and evaluated as inhibitors of
TNF-a production. Although less active than the corresponding phthalimides, several
penta¯uorobenzamides exhibited signi®cant activity at 10 mM. N-(4,6-dimethylpyridin-2-
yl)penta¯uorobenzamide was selected for a preliminary in-vivo assay. Although its
inhibitory activity against carrageenan-induced rat paw oedema was moderate, it induced
a signi®cant reduction in ear thickness in the PMA-induced mouse ear-swelling test
�
1
(
49Æ 6% inhibition after a dose of 0Á1 mM kg , p.o.).
Tumour necrosis factor-a (TNF-a) is produced by
immunologically competent cells during the host
response to infection, injury or in¯ammation, and
mediates metabolic and biochemical changes in
responding cells when present at low concentra-
tions (Tracey & Cerami 1993; Eigler et al 1997).
However, overproduction of TNF-a can cause a
destructive immune response associated with many
in¯ammatory diseases including rheumatoid
arthritis, osteoarthritis, multiple sclerosis, lupus,
asthma, in¯ammatory bowel disease (Badger &
Adams 1998), septic shock (Beutler 1992) or brain
injury and in¯ammation following stroke (Trembly
patients require repeated parenteral treatments.
Moreover most develop human antibodies which can
lead to tachyphylaxis. Thus, there is a need to develop
moreeconomicandorallyactivechemicalsubstances.
Over the past few years, studies have shown that
in addition to its known hypnotic or sedative and
teratogenic effects, thalidomide (1) (Figure 1)
inhibits TNF-a production in lipopolysaccharide-
stimulated human monocytes (Sampaio et al 1991).
(Poly)¯uorine and amino substitution of thalido-
mide and other N-aryl-phthalimides (2, 3) results in
analogues with TNF-a inhibition potency
approaching 200-times that of thalidomide (Muller
et al 1996; Niwayama et al 1996). Formation of the
arene oxide metabolite of thalidomide, which is
thought to be responsible for its teratogenic effect,
could be prevented by tetra¯uorine substitution
(Niwayama et al 1996).
We recently synthesized and tested a series of
N-azaaryl(alkyl)phthalimides incorporating ami-
no(alkyl)pyridines (Collin et al 1998). N-(4,6-di-
methylpyridin-2-yl)tetra¯uorophthalimide (4) was
found to be a potent TNF-a production inhibitor
(IC50 ˆ 10 mM). We also studied the TNF-a inhibi-
tory activity of N-(4,6-dimethylpyridin-2-yl)heter-
oarylcarboxamides (Lang et al 1995; Vernhet et al
1997). The furan-2-carboxamide 5 (Robert et al
1995) was found to have greater activity than thali-
domide (IC50: 70 and 200 mM approx., respectively).
We describe here the synthesis and the evaluation
of in-vitro TNF-a production inhibitory activity of
&
Slikker 1995).
Non-steroidal anti-in¯ammatory drugs (NSAIDs)
act by inhibiting prostaglandin synthesis, but have
low ef®cacy in these type of diseases, at con-
centrations compatible with their gastrointestinal
and renal toxicity. TNF-a production or maturation
inhibitors could represent an interesting alternative
to NSAIDs.
Neutralizing anti-TNF-a monoclonal antibodies
suchasin¯iximab)haveprovidedthe®rstevidenceof
(
the bene®cial effect in rheumatoid arthritis and
Crohn's disease (Elliot et al 1994); TNF-a-soluble
receptors are now undergoing clinical trials for rheu-
matoid arthritis (Sander et al 1996). However, pro-
duction costs for biological agents are high and
Correspondence: G. Le Baut, Laboratoires de Chimie Orga-
nique et de Chimie Th e rapeutique, Facult e de Pharmacie, 1 rue
Gaston Veil, 44035 Nantes, France.

2
34
X. COLLIN ET AL
room temperature led to compound 6 (Mariella &
Belcher1952;Fox&Threlfall1964). Yield:78%;mp
ꢀ
ꢀ
� 1
1
3
43 C (lit. 145±146 C). IR (KBr), n (cm ) 3415,
1
300, 3160 (n NH), 840 (n C� Br). H NMR (CDCl )
3
d 2Á28 (s, 3H, 4-CH ), 2Á50 (s, 3H, 6-CH ), 4Á32 (s,
3
3
2
H, NH ), 6Á25 (s, 1H, Pyr-3H).
2
6-Amino-3,5-dibromo-2,4-lutidine (7). Method b.
Dibromination of 6-amino-2,4-lutidine was carried
out using the same protocol as for monobromina-
tion but adding two equivalents of bromine. Yield:
ꢀ
ꢀ
3
(
8%; mp 135 C (lit. 136±137 C). IR (KBr), n
�
1
1
cm ) 3130 (n NH), 670 (n C� Br). H NMR
Figure 1. Chemical structures of phthalimides 1±4 and
furan-2 carvoxamide 5 inhibiting TNF-a production.
(CDCl ) d 2Á49 (s, 3H, 4-CH ), 2Á53 (s, 3H, 6-
3
3
CH ), 4Á92 (s, 2H, NH ).
3
2
6
-Amino-3-bromo-5-nitro-2,4-lutidine (8). Method
structurally-related compounds, N-pyridinyl(alk-
yl)¯uorobenzamides.
c. This compound was synthesized as described
by Graboyes & Day (1957). Nitric acid was added
dropwise to a solution of monobrominated amine 6
in sulphuric acid and the mixture was stirred at
Materials and Methods
ꢀ
room temperature. Yield: 61%; mp 169 C (lit.
ꢀ
� 1
1
69±170 C). IR (KBr), n (cm ) 3400, 3180
Chemistry
(
(
n NH ), 1620 (n C ˆ N), 1500 (n NO ), 1320
2
as
2
Melting points were determined on a Tottoli-B uÈ chi
apparatus and are uncorrected. Structures were
supported by IR, H NMR and mass spectra. IR
1
n NO ). H NMR (CDCl ) d 2Á37 (s, 3H, 4-
s
2
3
CH ), 2Á49 (s, 3H, 6-CH ), 7Á06 (s, 2H, NH ).
1
3
3
2
spectra were run with KBr pellets on a Perkin
Elmer-Paragon PC 1000 infrared spectro-
photometer. H NMR spectra were recorded on a
5
,6-Diamino-3-bromo-2,4-lutidine (9). Method d.
Reduction of compound 8 by SnCl in concentrated
hydrochloric acid according to Graboyes & Day
1
2
Bruker AC 250 spectrometer (250 MHz), using
CDCl or d -(CH ) SO as solvent. Chemical shifts
ꢀ
(
1
1957) gave 9. Yield: 92%; mp 169 C (lit. 169±
ꢀ
� 1
3
6
3 2
70 C). IR (KBr), n (cm ) 3400, 3200 (n NH ),
645 (n C ˆ N). H NMR (CDCl ) d 2Á16 (s, 3H, 4-
2
(
d ppm) are reported down®eld from tetra-
1
1
3
methylsilane, the internal standard; coupling con-
stants are in Hz. Mass spectra were recorded on a
double beam Varian Mat 112 spectrometer; ioni-
zation energy 70 eV. Analytical TLC was per-
formed on precoated silica-gel aluminium plates
CH ), 2Á30 (s, 3H, 6-CH ), 4Á57 (s, 2H, NH ), 5Á52
3
3
2
(
s, 2H, NH ).
2
(
0Á2 mm, GF254, E Merck). Spots were located by
Synthesis of benzamides
UV illumination. Evaporation (rotating evaporator)
was done in-vacuo. Sodium sulphate or phosphorus
pentoxide were used as the drying agents. Most of
the crude products were passed through short col-
umns of silica gel (silica gel 60, 70±230 mesh, E
Merck) with an appropriate mixture of dichlor-
omethane and ethanol.
Commercially available solvents and chemicals
were used for syntheses, with the exception of the
following polyfunctional amines which were pre-
pared using methods reported in the literature.
N-(4,6-Dimethylpyridin-2-yl)-2,4-di¯uorobenzamide
(11). Method e. A solution of 2,4-di¯uorobenzoic
acid (0Á9 g, 5Á7 mmol), 2-amino-4,6-dimethylpyridine
(0Á69g, 5Á7 mmol) and triethylamine (2Á4 mL,
17Á1 mmol) in dry 1,2-dichloroethane (60mL) was
cooled in an ice bath. Phenyl dichlorophosphate
(0Á85mL,5Á7 mmol)wasaddeddropwise.Themixture
was stirred at room temperature for 24 h. The solvent
wasremovedunderreducedpressure.Theresiduewas
puri®ed by column chromatography using
dichloromethane±ethanol (95:5) as eluent. After
recrystallization from ethanol, 0Á91g pure product
ꢀ
Synthesis of intermediary amines
was obtained. Yield: 61%; mp 116 C. IR (KBr), n
�
1
(
cm ) 3220 (n NH), 1700 (n C ˆ O), 1500 (d NH),
1
6
bromination of 6-amino-2,4-lutidine in acetic acid at
-Amino-3-bromo-2,4-lutidine (6). Method a. Mono-
1290 (combined NH=CN). H NMR (CDCl ) d 2Á36
3
(s, 3H, 4-CH ), 2Á45 (s, 3H, 6-CH ), 6Á80 (s, 1H, Pyr-
3
3

TNF-a PRODUCTION INHIBITORS
235
5
H),6Á95(m,1H,Ar-3H),7Á06(m,1H,Ar-5H),8Á02(s,
H, Pyr-3H), 8Á13 (m, 1H, Ar-6H), 9Á11 (s, 1H,
1330 (combined NH=CN). EI-MS m=z (%) 302 (M,
1
82), 195 (100), 167 (47), 117 (25). H NMR (CDCl )
1
CONH).
3
d 4Á62 (d, 2H, CH , J ˆ 5Á7), 7Á18 (m 1H, NH), 7Á28
2
(
Pyr-5H, Pyr-6H).
m, 1H, Pyr-2H), 7Á70 (m, 1H, Pyr-4H), 8Á45 (m, 2H,
N-(4,6-Dimethylpyridin-2-yl)-3-¯uorobenzamide
(
10) (Robert et al 1994). Method e. Yield: 68%;
ꢀ
ꢀ
� 1
N-(5-Bromo-4,6-dimethylpyridin-2-yl)penta¯uoro-
ꢀ
mp 119 C (lit. 115±116 C). IR (KBr), n (cm )
360 (n NH), 1675 (n C ˆ O), 1550 (d NH), 1280
benzamide (16). Method e. Yield: 47%; mp 186 C.
3
�
1
1
IR (KBr), n (cm ) 3250 (n NH), 1705 (n C ˆ O),
575 (d NH), 1270 (combined NH=CN). EI-MS
m=z (%) 397 (M ‡ 2, 20), 395 (M, 19), 195
(
3
5
combined NH=CN). H NMR (CDCl ) d 2Á34 (s,
3
1
H, 4-CH ), 2Á36 (s, 3H, 6-CH ), 6Á76 (s, 1H, Pyr-
3
3
H), 6Á93 (m, 4H, Ar-H), 8Á03 (s, 1H, Pyr-3H), 8Á92
1
(
2
1
100), 167 (50), 117 (20). H NMR (CDCl ) d
(s, 1H, CONH).
3
Á47 (s, 3H, 4-CH3), 2Á55 (s, 3H, 6-CH ), 8Á06 (s,
3
H, Pyr-3H), 8Á43 (s, 1H, CONH).
N-(4,6-Dimethylpyridin-2-yl)-2,3,6-tri¯uorobenz-
ꢀ
amide (12). Method e. Yield: 69%; mp 182 C. IR
�
1
N-(3,5-Dibromo-4,6-dimethylpyridin-2-yl)penta-
¯uorobenzamide (17). Method e. Yield: 49%; mp
(
(
(
KBr), n (cm ) 3440 (n NH), 1690 (n C ˆ O), 1620
1
d NH), 1240 (combined NH=CN). H NMR
� 1
ꢀ
1
(
19 C. IR (KBr), n (cm ) 3250 (n NH), 1705
n C ˆ O), 1575 (d NH), 1270 (combined
NH=CN). EI-MS m=z (%) 195 (100), 167 (25),
CDCl ) d 2Á37 (s, 3H, 4-CH ), 2Á38 (s, 3H, 6-
3
3
CH ), 6Á80 (s, 1H, Pyr-5H), 6Á93 (m, 1H, Ar-5H),
3
7
1
Á25 (m, 1H, Ar-4H), 8Á00 (s, 1H, Pyr-3H), 8Á50 (s,
1
1
2
17 (9). H NMR (CDCl ) d 2Á51 (s, 3H, 4-CH ),
3
3
H, CONH).
Á68 (s, 3H, 6-CH ), 6Á98 (s, 1H, CONH).
3
N-(4,6-Dimethylpyridin-2-yl)-2-chloro-4,5-di¯uoro-
ꢀ
N-(5-Bromo-3-nitro-4,6-dimethylpyridin-2-yl)-
penta¯uorobenzamide (18). Method e. Yield: 45%;
benzamide (13). Method e. Yield: 72%; mp 123 C. IR
�
1
ꢀ
�
1
(
KBr), n (cm ) 3205 (n NH), 1690 (n C ˆ O), 1565 (d
mp 121 C. IR (KBr), n (cm ) 3450 (n NH), 1665
(n C ˆ O), 1510 (d NH), 1525 (n NO ), 1370 (ns
1
NH), 1305 (combined NH=CN). H NMR (CDCl ) d
2
3
as
2
Á37 (s, 3H, 4-CH ), 2Á38 (s, 3H, 6-CH ), 6Á79 (s, 1H,
3
3
NO ), 1225 (combined NH=CN). EI-MS m=z (%)
2
Pyr-5H), 7Á29 (dd, 1H, Ar-3H, J ˆ 9Á5, 6Á8), 7Á58 (dd,
4
1
42 (M ‡ 2, 5), 440 (M, 4), 195 (100), 167 (21),
1
1
H, Ar-6H, J ˆ 9Á5, 8Á2), 7Á97 (s, 1H, Pyr-3H).
17 (7). H NMR (CDCl ) d 2Á53 (s, 3H, 4-CH ),
3
3
2Á62 (s, 3H, 6-CH ), 9Á29 (s,1H, CONH).
3
N-(4,6-Dimethylpyridin-2-yl)penta¯uorobenz-
amide (14). Method e. Yield: 72%; mp 139±140 C.
ꢀ
N-(2-Amino-5-bromo-4,6-dimethylpyridin-3-yl)
penta¯uorobenzamide (19). Method e. Yield: 47%;
�
1
IR (KBr), n (cm ) 3205 (n NH), 1705 (n C ˆ O),
495 (d NH), 1230 (combined NH=CN). EI-MS
m=z (%) 316 (M, 15), 269 (100), 195 (57), 167
ꢀ
� 1
1
mp 255 C. IR (KBr), n (cm ) 3470 (n NH), 1675
(n C ˆ O), 1520 (d NH), 1330 (combined NH=CN).
EI-MS m=z (%) 412 (M ‡ 2, 12), 410 (M, 13), 195
1
(
45), 117 (17). H NMR (CDCl ) d 2Á28 (s, 3H, 4-
3
1
CH ), 2Á37 (s, 3H, 6-CH ), 6Á78 (s, 1H, Pyr-5H),
3 3
(67), 167 (34), 135 (100), 117 (21). H NMR
7
Á96 (s, 1H, Pyr-3H), 9Á22 (s, 1H, CONH).
(CDCl ) d 2Á23 (s, 3H, 4-CH ), 2Á45 (s, 3H, 6-
3
3
CH ), 5Á86 (s, 2H, NH ), 10Á26 (s, 1H, CONH).
3
2
Method f. 1,1-Carbonyldiimidazole (1Á03 g, 5Á6
mmol) was added portionwise to a solution of
penta¯uorobenzoic acid (1Á2 g, 5Á6 mmol) in dry
tetrahydrofuran (40 mL). The mixture was stirred
at room temperature for 1 h. 2-Amino-4,6-
dimethylpyridine (0Á68 g, 5Á6 mmol) was then
added. The solution obtained was stirred at room
temperature for 24 h. The solvent was removed
under reduced pressure. The residue was puri®ed
by column chromatography using dichloro-
methane±ethanol (95:5) as eluent. Recrystalliza-
tion from ethanol afforded 1Á11 g pure compound.
Yield: 62%.
Pharmacology
TNF-a inhibitory activity
The effect of the different compounds on the pro-
duction of TNF-a, by in-vitro activated peritoneal
macrophages, was quanti®ed as previously descri-
bed (Lang et al 1995). Adult male Swiss CF mice,
18±25 g, were obtained from CREJ, France.
Brie¯y, thioglycollate-elicited mouse macrophages
were isolated by peritoneal washing with Ca-Mg
phosphate-buffered saline (PBS), after cervical
6
dislocation. The cell suspension (1±2 6 10 cells=
well) was incubated in 24-well culture plate in
ꢀ
CO . After three washes with PBS, mouse macro-
phages were pre-incubated with the different
N-(Pyridin-3-ylmethyl))penta¯uorobenzamide (15).
ꢀ
RPMI 10% foetal calf serum for 2 h at 37 C, 5%
Method e. Yield: 82%; mp 120±121 C. IR (KBr), n
2
�
1
(
cm ) 3180 (n NH), 1690 (n C ˆ O), 1505 (d NH),

2
36
X. COLLIN ET AL
compounds solubilized in ethanol (maximum ®nal
concentration of ethanol: 0Á5%) or in dimethyl-
sulphoxide (maximum ®nal concentration: 0Á2%) at
1
0 mM. Macrophages were then stimulated with
1
�
bacterial lipopolysaccharides or LPS (0Á5 mg mL )
from Escherichia coli (Sigma) for 4 h. Supernatants
were collected and assessed for TNF-a content
using Wehi 164 clone 13 cytotoxic assay (Espevik
&
Nissen-Meyer 1986).
Acute phorbol ester-induced mouse ear-swelling
test
In the acute mouse ear-swelling test (Carlson et al
1
985), 2Á5 mg phorbol-12- O-myristate-13-acetate
(PMA) (Sigma) in 10 mL ethanol±water (8:2, v=v)
was applied topically in a single dose to the inner
and outer surfaces of the right ear of mice. The
mice were randomly divided into seven groups, ®ve
Figure 2. Synthesis of intermediary amines. Reagents: i. Br
(1 equiv.), AcOH, r.t., 3 h; ii. Br
2
(2 equiv.) AcOH, r.t., 3 h; iii.
2
, 60 C, 20 min; iv. SnCl
ꢀ
ꢀ
conc HNO
0 min.
3
, H
2
SO
4
2
, conc HCl, 80 C,
3
�
1
mice per group: vehicle, 0Á1, 0Á2 and 0Á4 mM kg
compound 14 and the reference compound, ibu-
profen. The appropriate doses of 14 and ibuprofen
were orally administered 1 h before PMA applica-
tion. The left ear of each animal received only
vehicle. Ear thickness (mm) as an index of
in¯ammation was measured after 3Á5 h, using a
Dyer model micrometer gauge (Dyer Co. Inc.,
Lancaster, PA). The percentage inhibition of the
in¯ammatory reaction was determined by compar-
ison with controls. Results are expressed as
meanÆ s.e.
Figure 3. Synthesis of benzamides. Reagents: i. phenyl
dichlorophosphate, TEA, 1,2-dichloroethane, r.t., 24 h; ii.
,1-carbonyldiimidazole, THF, r.t., 24 h.
1
it was adopted for the synthesis of the other ben-
zamides (10±13, 15±19). The variability in yields
obtained (45±82%) by this method could be
explained by the difference in nucleophilicity of the
starting amines; for instance, b-picolylamine
afforded compound 15 in a 82% yield whereas 6-
amino-3-bromo-5-nitro-2,4-lutidine gave com-
pound 18 in only 45% yield.
Carrageenan-induced rat paw oedema
The inhibitory activity of 14 and ibuprofen on carra-
geenan-induced rat paw oedema was determined
according to the method of Winter et al (1962), with
slight modi®cations (Du¯os et al 1998).
Pharmacology
Results and Discussion
The effect of the test compounds on the production
of TNF-a was quanti®ed in in-vitro LPS-stimulated
peritoneal macrophages (Table 1). Although 3-
¯uorobenzamide 10 was one of the most ef®cient
N-lutidinylcarboxamides studied in the rat paw
oedema inhibition, it did not exert a signi®cant
inhibitory effect on TNF-a production, at least at
10 mM. Even di- and tri-halogenobenzamides 11±
13 were inef®cient at this concentration.
Nevertheless, as previously observed in the
phthalimide series (Collin et al 1998), per¯uorina-
tion of benzamides issued from 6-amino-2,4-luti-
dine and b-picolylamine allowed emergence of
inhibitory activity in compounds 14 and 15 (33%
approx.). Pharmacomodulation at the level of the
pyridinyl carbons 3 and 5 in the lutidinyl sub-ser-
ies, was carried out by introducing a bromo, nitro
Chemistry
The synthesis of intermediary amines was carried
out as shown in Figure 2. The preparation of ben-
zamides 10±19 was carried out as shown in Figure
3
. Corresponding substituted benzoic acids were
condensed with appropriate amines using the
methods which afforded the best results in previous
studies: phenyl dichlorophosphate activation of the
carboxylic acid in the presence of triethylamine in
1
,2-dichloroethane at room temperature (Arrieta et
al 1985) or 1,1-carbonyldiimidazole activation in
tetrahydrofuran at room temperature (Paul &
Anderson 1960). The dichloro phosphate method
provided compound 14 in better yield (72%) than
the 1,1-carbonyldiimidazole method (62%) and so

TNF-a PRODUCTION INHIBITORS
237
Table 1. TNF-a inhibitory activity of 10 mM N-pyridinyl Table 2. Inhibition of phorbol myristate acetate-induced
(
alkyl)¯uorobenzamides. mouse ear oedema after oral administration of compound 14.
Compound
Inhibition (%)
�
1
)
Dose (mM kg
Compound
X
R
Inhibition (%)
0Á1
0Á2
0Á4
1
Ibuprofen
4
49Æ 6
35Æ 8
65Æ 9
56Æ 4
69Æ 4
86Æ 6
10
11
12
13
14
15
16
17
3-F
<10
2, 4-diF
<10
<10
<10
33
derivative 19, afforded the most effective com-
pound of the series (40% at 10 mM).
Of the four active penta¯uorobenzamides (14, 15,
2, 3, 6-triF
17 and 19), compound 14, an analogue of the N-
lutidinyl tetra¯uorophthalimide 4, was selected for
preliminary evaluation of in-vivo anti-in¯amma-
tory activity. Its effect in the acute PMA-induced
mouse ear-swelling test, was measured after oral
administration (Table 2). It exerted a signi®cant
reduction in ear thickness and its level of activity at
2-Cl, 4, 5-diF
�
1
a concentration of 0Á1 mM kg , compared favour-
2, 3, 4, 5, 6-pentaF
2, 3, 4, 5, 6-pentaF
2, 3, 4, 5, 6-pentaF
2, 3, 4, 5, 6-pentaF
ably with ibuprofen.
This benzamide was also tested against carra-
geenan-induced rat paw oedema; its inhibitory
�
1
35
effect after oral administration of 0Á2 mM kg
remained moderate (31Æ 14%), and no activity
�
1
increase was observed at a dose of 0Á4 mM kg . At
the same doses, percentage inhibition by ibuprofen
was 50Æ 1% and 57Æ 2%, respectively.
<10
25
The multiple PMA-induced model of chronic
in¯ammation is considered to be a relevant model
of human psoriasis; we wish to evaluate the most
potent poly¯uorophthalimides and benzamides in
the subchronic mouse ear-swelling test (Alford et al
1992).
1
9
2, 3, 4, 5, 6-pentaF
40
Acknowledgements
Partial ®nancial support for this work was gener-
ously provided by the AGISMED Foundation,
C.H.R.U. of Nantes, France.
Inhibition is expressed as the mean of six values obtained in
two separate experiments. Individual values differed from the
mean by less than 5%. Because of its very low solubility in the
assay solvent system, compound 18 could not be tested.
References
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67
(
25%) was observed after 3,5-dibromination. The
2
corresponding 3-nitro compound 18 could not be
tested due to insolubility in the solvent system used
for the in-vitro assay; trials to increase its aqueous
solubility, by maleate salt formation, failed.
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2
38
X. COLLIN ET AL
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