4-(2',4'-DIFLUOROBIPHENYL-4-YL)-2-METHYL-4-OXOBUTANOIC ACID AND ITS DERIVATIVES

Article abstract of DOI:10.1135/cccc19942705

Friedel-Crafts reaction of itaconic anhydride (III) with 2,4-difluorobiphenyl (II) afforded unsaturated acid IV which was hydrogenated to give 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acod (I).A series of functional derivatives and salts of the acid I has been prepared.The antiinflammatory effect of these compounds was compared with that of acid I using selected experimental inflammation models.The analgesic activity in the intraperitoneal iritation test was also evaluated.In the case of (R)-(+)-1-phenylethylamide Vf the ratio of the diastereisomers was determined by HPLC.

Full text of DOI:10.1135/cccc19942705

Functional Derivatives of Flobufen
2705
4-(2′,4′-DIFLUOROBIPHENYL-4-YL)-2-METHYL-4-OXOBUTANOIC ACID
AND ITS DERIVATIVES
Miroslav KUCHAR, Marie POPPOVA, Hana ZUNOVA, Eva KNEZOVA, Vaclav VOSATKA
and Marek PRIHODA
Research Institute for Pharmacy and Biochemistry,
130 60 Prague 3, The Czech Republic
Received June 23, 1994
Accepted September 20, 1994
Friedel–Crafts reaction of itaconic anhydride (III) with 2,4-difluorobiphenyl (II) afforded unsaturated
acid IV which was hydrogenated to give 4-(2′,4′-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid
(I). A series of functional derivatives and salts of the acid I has been prepared. The antiinflammatory
effect of these compounds was compared with that of acid I using selected experimental inflamma-
tion models. The analgesic activity in the intraperitoneal irritation test was also evaluated. In the case
of (R)-(+)-1-phenylethylamide Vf the ratio of the diastereoisomers was determined by HPLC.
In the second half of the eighties we have developed1 a new antiinflammatory com-
pound, 4-(2′,4′-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (flobufen, I). Its
discovery is the result of our investigation of antiinflammatory activity of aryloxoalka-
noic acids2 in which the structure of the studied compounds was optimized using the
regression analysis of structure–activity relationships3,4. In accord with the proposed
biotransformation pathway of compounds of this type, the antiinflammatory activity of
the acid I was markedly protracted4. During the preclinical evaluation it has been
found5 that the inhibition of prostaglandin biosynthesis is not decisive for the
mechanism of the antiinflammatory effect of this compound. The IC50 value toward
cyclooxygenase from rat PMN cells for the antiinflammatory drugs based on arylalka-
noic acids is up to 1 000 times lower than for flobufen. A significant immunomodula-
tory effect of compound I has been found6 which probably plays an important role in
the mechanism of its antiinflammatory activity, particularly in a model of adjuvant
arthritis, by decreasing lymphocyte proliferation induced by PPD (purified protein) or
ConA (concanavalin A). In the mechanism of the antiinflammatory action of acid I the
inhibition of biosynthesis of leukotriene B4 plays a non-negligible role because this acid
inhibits more 5-lipoxygenase than cyclooxygenase5. Flobufen I now undergoes the II. – III.
phase of clinical trials.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

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Kuchar, Poppova, Zunova, Knezova, Vosatka, Prihoda:
EXPERIMENTAL
Melting points were determined on a Kofler block Boetius M. 1H NMR spectra were measured in
deuteriochloroform (6% solution) with tetramethylsilane as internal standard on a BS 487s-80 MHz
Tesla spectrometer. The presence of the 3-methyl isomer in compound I was evaluated by gas liquid
chromatography on a Fractovap 2450 chromatograph (Carlo Erba) on a fused silica capillary column
(25 m, i.d. 0.22 µm) coated with SE-54 (thickness 0.2 µm). Prior to the measurements, compound I
was converted into its methyl ester by treatment with diazomethane. The presence of the 3-methylene
isomer in compound IV was determined by HPLC on a 3.9 × 300 mm µ-Bondapak C18 column
(Waters) in an acetonitrile–phosphate buffer pH 3.5 (50 : 50 v/v), using a liquid chromatograph con-
sisting of an LC-6A (Shimadzu) pump, a Rheodyne 7125 injector and an SPD-6AV (Shimadzu) de-
tector. The content of diastereoisomers of (R)-(+)-1-phenylethylamide VIf was determined on the
same liquid chromatograph on a 3.9 × 300 mm µ-Bondapak-CN column (Waters) in heptane–dichloro-
methane (80 : 20 v/v, 1 ml of acetic acid was added to 1 000 ml of the mixture). Similar results were
obtained with a β-Cyclodextrin–Daltosil column (4 µm, 4.5 × 250 mm, Serva) with methanol–water
(50 : 50 v/v) as the mobile phase.
Itaconic Anhydride (III)
A mixture of itaconic acid ( 131.4 g, 0.1 mol) and acetic anhydride (280 ml) was heated at 80 °C for
4 h. After removal of acetic acid and acetic anhydride by distillation at diminished pressure
(0.4 kPa), the distillation residue was mixed with dichloromethane (360 ml) and hexane (100 ml) and
the deposited itaconic acid was filtered off. The filtrate was concentrated and the residue diluted with
hexane (50 ml) at 50 – 60 °C. The crystalline material was collected; yield 103.7 g (92%) of the
anhydride III, 98.5% purity (HPLC), m.p. 64 – 67 °C (reported7 m.p. 66 – 68 °C).
4-(2′,4′-Difluorobiphenyl-4-yl)-2-methylene-4-oxobutanoic Acid (IV)
A solution of 2,4-difluorobiphenyl8 (76.7 g, 0.4 mol) and anhydride III (45.1 g, 0.4 mol) in dichloro-
methane (260 ml) was added dropwise at 0 °C to a suspension of aluminium chloride (106.7 g,
0.8 mol) in dichloromethane (60 ml). The mixture was stirred at 10 °C for 4 h and at 20 °C for 2 h
and then poured into a mixture of ice (800 g) and concentrated hydrochloric acid (260 ml). The pre-
cipitate was filtered, washed with dichloromethane and suspended in 80% methanol (620 ml). After
brief boiling and subsequent cooling to 20 °C, the solid was filtered and washed with 80% methanol.
This procedure reduced the content of the 3-methylene isomer from 2% to 0.5%. Yield 75.6 g (63%)
of IV, m.p. 172 – 174 °C, purity 99.2% (HPLC).
4-(2′,4′-Difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic Acid (I)
Compound IV (15.1 g, 0.05 mol) was hydrogenated over a Pd/C catalyst (1.0 g, Hereaus K.0224, 3%
of Pd in dry material) in dioxane (100 ml) at 20 °C and 0.5 MPa in an autoclave. After the hydroge-
nation had ended, the catalyst was filtered off and the filtrate was concentrated under diminished
pressure (0.4 kPa). The residue was dissolved in acetic acid (45 ml) at 90 °C, cooled and the crude
product was collected by filtration. Further crystallization from 90% acetic acid afforded 11.3 g
(74%) of compound I, m.p. 160 – 161 °C. For C17H14F2O3 (304.3) calculated: 67.10% C, 4.64% H,
12.49% F; found: 67.15% C, 4.82% H, 12.41% F.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Functional Derivatives of Flobufen
2707
General Method for Preparation of Amides Va – Vf
1-Ethylpiperidine (2.7 g, 24 mmol) and ethyl chloroformate (2.6 g, 24 mmol) were added at −15 °C
to a solution of acid I (5.0 g, 16 mmol) in a mixture of N,N-dimethylformamide (35 ml) and
dichloromethane (150 ml). After stirring for 30 min, the mixture was cooled to −30 °C and a solution
of the corresponding amine (16 mmol) in dichloromethane (10 ml) was added. The mixture was
stirred at 20 °C for 2 h and then washed with 5% NaHCO3 (3 × 100 ml), water (100 ml) and 1 M HCl
(3 × 100 ml). The organic phase was separated, the solvent evaporated and the residue crystallized
from an appropriate solvent (for melting points, solvents and analyses see Table I). The amides were
obtained in the following yields: Va 67%, Vb 73%, Vc 72%, Vd 66%, Ve 38%, and Vf 40%.
R-(+)-1-Phenylethylamide of 4-(2′,4′-Difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic Acid
(Vf, sample 19d)
Amide Vf (4.5 g, sample 17, Table I), prepared from racemic acid I and (R)-(+)-1-phenylethylamine
as described in the preceding experiment, was dissolved in boiling mixture of methanol and water
(4 : 1, 150 ml). After cooling to 20 °C, the crystalline portion (sample 19a) was collected and recry-
stallized from a minimum amount of methanol–water (4 : 1). Two more crystallizations afforded a
product (sample 19d) which was homogeneous according to HPLC (for analytical details see the first
part of Experimental, for results Table II).
General Method for Preparation of Esters Vg and Vh
Thionyl chloride (1.8 ml, 27 mmol) was added to ethanol (37.5 ml, 0.64 mol) at −15 °C.
After 30 min, acid I (6.1 g, 20 mmol) was added, the mixture was stirred for further 30 min at
−15 °C and then heated at 40 – 45 °C for 2 h. The mixture was cooled, concentrated in vacuo and
the residue was coevaporated with benzene (2 × 30 ml). Crystallization from acetone afforded 4.6 g
(69%) of ethyl ester Vg.
Isobutyl ester Vh was prepared in 91% yield by the same procedure from butanol, thionyl chloride
and acid I.
For melting points and analytical data see Table I.
Potassium 4-(2′,4′-Difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoate (VIa)
A solution of potassium hydroxide (1.0 g, 18 mmol) in methanol (50 ml) was added to a solution of
acid I (6.1 g, 20 mmol) in methanol (60 ml). The mixture was concentrated in vacuo and the viscous
residue was stirred with ether (50 ml). The crystalline portion was collected on filter and again mixed
with ether (50 ml) to give 4.72 g (65%) of monohydrate of salt VIa.
Calcium 4-(2′,4′-Difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoate (VIb)
A solution of calcium chloride (1.1 g, 10 mmol) in water (20 ml) was added at 70 °C to a solution
of sodium salt of I (prepared by dissolving acid I (3.05 g, 10 mmol) in 5 M NaOH (55 ml)). The
precipitate that deposited after cooling was collected; yield 2.7 g (84%) of calcium salt VIb; content
of acid I: 99.4% of theoretical amount (determined potentiometrically).
General Method for Preparation of Salts VIc, VIe and VIf
A solution of the corresponding amine (10 mmol) in acetone–ether (2 : 1, 20 ml) was added to a
solution of acid I (3.05 g, 10 mmol) in acetone–ether (2 : 1, 25 ml). After standing for 1 h, the
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2708
Kuchar, Poppova, Zunova, Knezova, Vosatka, Prihoda:
TABLE I
Physicochemical characteristics of compounds Va – Vh and VIa –VIf
Calculated/Found
M.p., °C
Formula
(M.w.)
Compound
X (B+)
Solventa
% C % H % F % N
Va
Vb
Vc
Vd
Ve
Vf
NHCH2COOC2H5
NHC2H5
95 – 96 C21H21F2NO4
64.77 5.44
64.84 5.35
9.76 3.60
9.66 3.34
A
(389.4)
128
A
C19H19F2NO2
(331.4)
68.87 5.78 11.47 4.23
68.49 5.88 11.26 4.14
NH(CH2)3CH3
N(CH2CH2)2O
NHCH2C6H5
NHCH(CH3)C6H5
OC2H5
114 – 115 C21H23F2NO2
(359.4)
95 – 97 C21H21F2NO3
A/H 4 : 1 (373.4)
123 – 125 C24H21F2NO2
M/W 4 : 1 (393.4)
115 – 130 C25H23F2NO2
M/W 4 : 1 (407.5)
67 – 68 C19H18F2O3
(332.4)
50 – 52 C21H22F2O3
(360.4)
70.18 6.45 10.57 3.90
70.40 6.60 10.44 3.89
A
67.55 5.67 10.18 3.75
67.36 5.81 10.29 3.66
73.27 5.38
72.99 5.44
9.66 3.56
9.68 3.36
73.69 5.69
73.22 5.86
9.33 3.44
8.99 3.34
Vg
Vh
VIab
68.67 5.46 11.43
68.41 5.41 11.37
–
–
–
A
OCH2CH(CH3)2
K+
69.99 6.15 10.54
69.78 6.16 10.28
A
173 – 174 C17H13F2KO3 . 56.66 4.20 10.54
. H2O
56.71 4.04 10.28
–
(360.4)
VIbc
VIc
Ca2+
155 – 157 C34H26CaF4O6 63.15 4.05 11.75
–
d
d
d
–
(646.7)
139 – 140 C23H27F2NO3
(403.5)
c-C6H11NH+3
68.47 6.75
68.18 6.74
9.42 3.47
9.25 3.42
–
VId
VIe
S-(+)-
165 – 167 C23H28F2N2O5 61.32 6.26
8.43 6.22
8.34 6.01
HOOCCH(NH2)(CH2)4NH3+
–
(450.5)
123 – 125 C24H23F2NO3
(411.5)
115 – 125 C25H25F2NO3
61.13 6.43
C6H5CH2NH+3
70.06 5.63
69.75 5.75
9.23 3.40
9.14 3.36
–
VIf
R-(+)-C6H5CH(CH3)NH3+
70.57 5.92
70.65 6.02
8.93 3.29
8.73 3.70
–
(425.5)
a
b
c
Solvents: A acetone, H hexane, M methanol, W water. Monohydrate. Calculated: 6.20% Ca;
d
found: 6.42% Ca. Not determined.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Functional Derivatives of Flobufen
2709
deposited salt was filtered and washed with acetone (2 × 10 ml). The respective yields of the salt VIc
and VIe were 86% and 72%.
The (R)-(+)-1-phenylethylammonium salt VIf, [α]2D0 +0.72° (c 0.2, 50% methanol), was prepared in
97% yield by a similar procedure using only ether as solvent. The acid I, liberated from a sample of
this salt, had m.p. 158 – 160 °C and [α]2D0 0° (c 0.4, 50% methanol).
(S)-(+)-Lysine Salt of 4-(2′,4′-Difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic Acid (VId)
An aqueous solution (57%) of (S)-(+)-lysine (15.2 g, 0.104 mol) was added to a stirred solution of
acid I (35.0 g, 0.115 mol) in dimethyl sulfoxide (150 ml). After dilution with acetone (1 050 ml), the
deposited product was filtered, yield 44.5 g (95%) of the salt VId, [α]2D0 +3.21° (c 0.2, 50% methanol).
The acid I, liberated from a sample of this salt, had m.p. 158 – 160 °C and [α]2D0 −0.94° (corresponds
to 97% content of racemate).
Biological Assays
Inhibition of carrageenan-induced rat paw edema was evaluated by the method of Winter9; the ex-
perimental conditions are described elsewhere10. Inhibition of rat paw adjuvant edema was assayed
using the method of Pearson11 described in ref.12. In both cases, the effect was expressed in per cent
of edema inhibition compared with an untreated control.
Inhibition of experimental pleuritis was evaluated by the method of Hidaka13 in a group of Wistar
Han female rats pre-treated with 0.5% carrageenan in saline (intrapleural injection). The tested com-
pounds, in suspension with gum arabic, were applied orally in a single dose 1 h before the carragee-
nan injection. The volume of the exudate in the pleural cavity was compared with that of untreated
animals; the total cell number and cellularity (determined by Sysmex cell counter) were also com-
pared.
Adjuvant arthritis was studied with male rats by the method of Pearson11. The effect on the devel-
opment of arthritis was assayed by body weight growth, swelling of hind footpads, mobility and grip
strength, and bone and joint damages as compared with both arthritic and naive controls.
Analgetic effect was evaluated in male mice using the test14 based on intraperitoneal irritation with
0.7% acetic acid. The tested compounds were applied orally 30 min before the application of acetic
acid. The lysine salt VId was applied also parenterally in saline. The pain inhibition was expressed in
per cent as compared with the untreated group.
TABLE II
Optical resolution of R-(+)-1-phenylethylamide Vf diastereoisomers
Sample
1st peaka
2nd peaka
[α]2D0, °
M.p., °C
17
51.3
60.1
48.7
39.9
24.3
15.3
0
+78.84
+76.03
+69.63
+66.10
+57.74
115 – 130
115 – 130
135 – 145
135 – 150
154 – 157
19a
19b
19c
19d
75.7
84.7
100.0
a
HPLC was performed on µ-Bondapak CN (cf. Experimental).
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

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Kuchar, Poppova, Zunova, Knezova, Vosatka, Prihoda:
Immunosuppressive activity was evaluated by the GVHR method (graft versus host reaction15) in
which local GVHR was evoked by transfer of spleen cells into the left hind foot of A/PHaxC57BL10
hybrid mice. The GVHR was expressed as the weight difference between the left and right popliteal
lymph nodes (L − R). After GVHR, the compound was elicitation of administered once daily for five
days.
RESULTS AND DISCUSSION
Originally, 2-methyl-4-oxobutanoic acids were synthesized3,4 by reaction of methylsuc-
cinic anhydride with the corresponding aromatic compounds. With 2,4-difluorobiphe-
nyl (II), however, this reaction gives more than 10% of the undesired isomeric 3-methyl
derivative. We therefore studied other, potentially more regioselective, approaches to
the acid I. The method of choice is based on the reaction of itaconic anhydride (III)
with 2,4-difluorobiphenyl (II). The obtained 4-(2′,4′-difluorobiphenyl-4-yl)-2-
methylene-4-oxobutanoic acid (IV) was hydrogenated over Pd/C to give the desired
product16 (Scheme 1) containing usually less than 0.3% of the 3-methyl isomer. In the
crude product IV, the content of the 3-methylene isomer, determined by TLC densito-
metry, did not exceed 2%. This finding agrees with the results described17,18 for the
preparation of Itanoxon, i.e. 4-(2′-chlorobiphenyl-4-yl)-2-methylene-4-oxobutanoic
acid.
Because of the hitherto promising results obtained with flobufen I, we prepared a
series of its functional derivatives Va – Vh and salts VIa – VIf (ref.19) whose physico-
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

Functional Derivatives of Flobufen
2711
TABLE III
Biological properties of compounds Va – Vh and VIa – VIf a
Pleuritise, %
Analgesic
activity
%
Mortalityb
%
Inhibition CEc Inhibition FAd
Compound
%
%
A
B
C
Va
0
0
40
42
33
38
42
35
49
45
39
44
47
46g
68
74
46
51
50
27
f
62
f
50
f
41
30
Vb
f
f
f
Vc
0
f
44
f
33
f
Vd
20
12
35
60
47
53
f
f
f
f
Ve
20
0
Vf
7
f
32
f
28
f
Vg
0
55
48
50
51
53
38
42
47
35
58
Vh
10
20
30
0
28
30
40
56
27
40
VIa
VIb
VIc
VId
VIe
VIf
35
f
62
f
43
f
0
58
f
57
16
34
46
73
33
63
61
39
37
45
42
77h
f
20
20
50
f
f
Flobufen
65
79
a
The antiinflammatory and analgesic activities were compared with those of flobufen (in equimolar
doses); doses of flobufen 20 mg/kg in antiinflammatory assays and 100 mg/kg in analgesic activity
b
tests. Acute oral toxicity in female mice after single 500 mg/kg doses; % of mortality 7 days after
c
d
e
application. Carrageenan edema. Freund’s adjuvans-induced edema.
A volume of the edema, B
f
g
total cell number, C cellularity in volume unit. Not evaluated. 40% CE inhibition after parenteral
dose of 10 mg/kg. 48% inhibition of pain after parenteral dose of 80 mg/kg.
h
Collect. Czech. Chem. Commun. (Vol. 59) (1994)

2712
Kuchar, Poppova, Zunova, Knezova, Vosatka, Prihoda:
chemical characteristics are summarized in Table I. In the preparation of (R)-(+)-1-phenyl-
ethylamide of flobufen (Vf) we obtained a mixture of two diastereoisomers, the ratio
of which was determined by HPLC on µ-Bondapak, Separon SGX and β-Cyclodextrin–
Daltosil (see Experimental). The isomers were very well separated on all these station-
ary phases; the obtained results correspond to the measured optical rotations (Table II).
For the biological assays we used a sample (No. 17, Table II) that contained the dia-
stereoisomers in the ratio 51 : 49 (i.e. amide of the (R)-(+)-amine with racemic acid I).
A practically pure diastereoisomer derived from (−)-flobufen was obtained by crystal-
lization from 80% methanol.
The synthesized derivatives Va – Vh and salts VIa – VIf have shown an analogous
antiinflammatory activity as flobufen: none of the compounds was significantly more
active than the starting acid I. The results of the assays, including the analgesic acti-
vities, are summarized in Table III. Some of the compounds exhibit an enhanced solu-
bility in water which extends potentialities of their therapeutical utilization. For this
reason, we paid particular attention to the (S)-(+)-lysine salt of flobufen (VId) whose
antiarthritic and immunomodulatory activity was compared with the parent flobufen I.
The results, summarized in Table IV, confirm that the acid I and its lysine salt VId have
analogous pharmacodynamic profiles. The solubility of the salt VId in water enabled its
parenteral application in aqueous solution. Also this salt is a mixture of two diastereo-
isomers which can be separated by crystallization. We prepared this salt under condi-
tions (see Experimental) leading to the (S)-(+)-lysine salt of racemic flobufen; with this
substance we also performed the biological assays.
TABLE IV
Antiarthritic and immunosuppressive activity of compound VId (for details see Experimental)
Adjuvant arthritisa
GVHR
L − R, mg %
Conditions
b
A
B
C
D
Application of VId
Naive control
8.13
23.13
−6.88
0.08
0
0.75
0
43.0
60.0
16.1
1.89
–
65.4
–
Arthritic control
1.37
2.75
2.89
100.0
a
A body weight growth, B size of edema, C mobility and grip strength, D bone and joint damage.
Compared with untreated control.
b
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Functional Derivatives of Flobufen
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This work was financially supported by the Galena company, Opava, The Czech Republic.
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Translated by M. Tichy.
Collect. Czech. Chem. Commun. (Vol. 59) (1994)