Synthesis and Selective Inhibitory Activity of 1-Acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole Derivatives against Monoamine Oxidase

Article abstract of DOI:10.1021/jm031042b

A novel series of 1-acetyl-3-(4-hydroxy- and 2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-pyrazole derivatives 1 - 12 have been synthesized and investigated for the ability to selectively inhibit the activity of the A and B isoforms of monoamine oxidase

Full text of DOI:10.1021/jm031042b

J . Med. Chem. 2004, 47, 2071-2074
2071
Syn th esis a n d Selective In h ibitor y Activity of
1-Acetyl-3,5-d ip h en yl-4,5-d ih yd r o-(1H)-p yr a zole Der iva tives a ga in st Mon oa m in e
Oxid a se
Franco Chimenti,‡ Adriana Bolasco,*,‡ Fedele Manna,‡ Daniela Secci,‡ Paola Chimenti,‡ Olivia Befani,§
Paola Turini,§ Valentina Giovannini,§ Bruno Mondov`ı,§ Roberto Cirilli,| and Francesco La Torre|
Dipartimento di Studi di Chimica e Tossicologia delle Sostanze Biologicamente Attive, Universita` degli Studi di Roma “La
Sapienza”, P.le A.Moro 5, 00185 Roma, Italy, Dipartimento di Scienze Biochimiche “A.Rossi Fanelli” and Centro di Biologia
Molecolare del CNR, Universita` degli Studi di Roma “La Sapienza”, P.le A.Moro 5, 00185 Roma, Italy, and Istituto Superiore
di Sanita`, Laboratorio di Chimica del Farmaco, Viale Regina Elena 299, 00161 Rome, Italy
Received September 23, 2003
A novel series of 1-acetyl-3-(4-hydroxy- and 2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-
pyrazole derivatives 1-12 have been synthesized and investigated for the ability to selectively
inhibit the activity of the A and B isoforms of monoamine oxidase (MAO). The new synthesized
compounds 1-12 proved to be more reversible, potent, and selective inhibitors of MAO-A than
of MAO-B. Knowing that stereochemistry may be an important modulator of biological activity,
we performed the semipreparative chromatographic enantioseparation of the most potent,
selective, and chiral compounds, 6 and 11. The separated enantiomers were then submitted to
in vitro biological evaluation while increasing their inhibitory activity and A selectivity. The
(-)-6 enantiomer shows Ki(MAO-A) ) 2 nM and SI ) 165 000, (+)-6 shows Ki(MAO-A) ) 6 nM and
SI ) 166 666, (-)-11 shows Ki(MAO-A) ) 4 nM and SI ) 80 000, and (+)-11 shows Ki(MAO-A) ) 7
nM and SI ) 38 571.
In tr od u ction
Amine oxidases (AOs) are widely distributed enzymes
among all living organisms with important oxidatively
deaminated biological functions, such as biogenic amine
metabolism.1 They are divided into two classes: AOs
containing flavin adenine dinucleotide as a cofactor
(FAD-AOs), and semicarbazide-sensitive AOs (ssAOs)
containing copper(II)-2,4,5-trihydroxyphenylalanine
F igu r e 1. Structures of our previously published pyrazoline
quinone as a cofactor (TPQ-Cu AOs). FAD-AOs, located
in the outer membrane of the mitochondria, are named
MAOs and exist as two isoforms, MAO-A and MAO-B,
which differ in substrate specificity, sensitivity to
inhibitors, and amino acid sequence.2-4 In recent years,
there has been considerable renewed interest in MAOs
on account of the key role played by the two MAO
isoforms in the metabolism of neurotransmitters. As a
result, MAO inhibitors (MAOI) are useful in the treat-
ment of several psychiatric and neurological diseases.5-7
Reversible, selective MAO-A inhibitors are used as
antidepressant and antianxiety drugs,8 and selective
MAO-B inhibitors are coadjuvant in the treatment of
Parkinson’s disease and perhaps also in Alzheimer’s
disease.9,10
derivatives active as AO inhibitors.
been possible because the three-dimensional structures
of the catalytic site of the enzyme are still unknown. A
few studies reported in the literature12-14 have contrib-
uted to a better understanding of the factors responsible
for MAOI activity and MAO-A/B selectivity. Such stud-
ies report that the factors affecting selectivity are
fundamentally different. To inhibit the active site of
MAO-B, molecule lipophilicity is fundamental, while to
inhibit the MAO-A site, a fundamental element is the
N5 charge transfer bond of the isoalloxazine nucleus of
FAD. Besides these, other equally essential factors are
the hydrogen bonds that stabilize the enzyme-inhibitor
complex between the polar groups on the inhibitor, the
amino acidic residues of the active site of the enzyme,
and finally the planar structure of the inhibitor that can
best adapt to the receptor pocket.
Even though the experimental crystal structure of
human MAO-B was deposited in the RCSB Protein Data
Bank in late 2001,11 the rational design of potent
reversible pharmacophores of MAO inhibitors has not
It is known that 1,3,5-triphenylpyrazolines show
monoamine oxidase inhibitory properties unrelated to
tranquillizing, muscle relaxant, psychoanaleptic, or
anticonvulsant activities.15-17 In a previous work we
synthesized a series of substituted 1,3,5-triphenyl-4,5-
dihydro-(1H)-pyrazoles (Figure 1, a ) which showed
reversible and selective inhibitory activity on TPQ-Cu
dependent AOs but were inactive on MAOs.18 In a
* To whom correspondence should be addressed. A.B.: tel and fax,
+39 06 4991 3763; e-mail, adriana.bolasco@uniroma1.it.
‡ Dipartimento di Studi di Chimica e Tossicologia delle Sostanze
Biologicamente Attive, Universita` degli Studi di Roma “La Sapienza”.
§ Dipartimento di Scienze Biochimiche “A.Rossi Fanelli” and Centro
di Biologia Molecolare del CNR, Universita` degli Studi di Roma “La
Sapienza”.
| Istituto Superiore di Sanita`.
10.1021/jm031042b CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/16/2004

2072 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Ta ble 1. Chemicophysical Data of Derivatives 1-12
Chimenti et al.
Sch em e 1
compound
R
R′
2-Cl
3-Cl
4-Cl
3-CH3
4-CH3
2-OCH3
4-OCH3
2,4-OCH3
4-Cl
4-CH3
2-OCH3
4-OCH3
yield %
mp, °C
1
2
3
4
5
6
7
8
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
82.7
57.0
73.0
57.2
67.0
93.5
90.0
70.0
75.6
70.0
82.5
70.0
214-5
216-8
259-6
218-2
235-7
183-4
217-8
262-4
253-5
256-8
237-8
253-5
9
10
11
12
subsequent work by the authors,19 the substitution of
the aromatic ring on N1 of the pyrazoline nucleus with
an acetyl group (Figure 1, b) favored inhibitory activity
toward the MAOs (FAD-dependent AOs). As a matter
of fact, this substitution reduces the steric hindrance
of the molecules, raising the positive charge of N1 of
the heterocycle, which strengthens the charge-transfer
bond with the isoalloxazine nucleus of FAD. Though
interesting, the inhibitory activity data toward MAOs
reported in this work (in the order of 10-8) do not show
any selectivity toward the two isoforms. For this reason,
we synthesized a new series of 1-acetyl-3-(4-hydroxy-
phenyl)-5-phenyl-4,5-dihydro-(1H)-pyrazole and 1-acetyl-
3-(2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-pyra-
zole derivatives whose structure contains polar groups
capable of forming hydrogen bonds, which as stated
earlier, stabilize the bond with FAD while contributing
to selectivity toward MAO-A.
aldehyde to form the protected chalcone, which is
subsequently freed by hydrolysis (Route 2 in Scheme
1).
Ch r om a togr a p h ic (HP LC) Resolu tion of Ra ce-
m ic Sa m p les 6 a n d 11. The enantiomers of racemic
compounds 6 and 11 were separated both on the
analytical and the semipreparative scale by enantiose-
lective HPLC on a chiral Chiralcel OD column packed
with cellulose carbamate derivative/silica gel.20 Baseline
enantioseparations were achieved within 20 min using
n-hexane containing 30% vol of ethanol as a mobile
phase at a temperature of 25 °C. Flow rates of 1.0 and
3.0 mL min-1 for analytical and semipreparative sepa-
rations, respectively, were used. For the semiprepara-
tive runs, 5 mg of racemic sample 6 (dissolved in 500
µL of ethanol) were injected onto a 10 mm I.D. Chiralcel
OD chiral stationary phase (CSP). The very poor
solubility (<1 mg/mL) of 11 in ethanol and n-hexane-
alcohol (ethanol or 2-propanol) mixtures precluded
injection of more than 0.4 mg (dissolved in 1 mL of
ethanol) of racemic 11. The sign of the optical rotation
of the two enantiomers of 6 and 11 was determined by
on-line polarimetric detection. The first eluting enan-
tiomers of 6 and 11 were levorotatory at a wavelength
of 365 and 589 nm. After semipreparative separations,
the collected fractions were analyzed by an analytical
Chiralcel OD column (250 × 4.6 mm I.D.) to determine
their enantiomeric excess (ee). The first and second
eluting enantiomers of 6 had ee ) 99.0% and ee )
99.8%, respectively. The enantiomers of 11 were ob-
tained with ee ) 99.0% (less retained enantiomer) and
ee ) 99.8% (more retained enantiomer).
Because of the presence of a chiral center at the C5
position of pyrazole moiety, knowing that stereochem-
istry may be an important modulator of biological
activity, we performed the semipreparative chromato-
graphic enantioseparation of the most potent, selective,
and active chiral compounds.
Ch em istr y
Starting from chalcones A-N, the new 1-acetyl-3,5-
diphenyl-4,5-dihydro-(1H)-pyrazole derivatives 1-12
(Table 1) were obtained by addition of hydrazine hydrate
in acetic acid according to a previous method19 (Scheme
1).
The 4-hydroxy chalcones A-H were obtained by direct
Claisen-Schmidt condensation between the aromatic
aldehydes and the substituted acetophenone, using 20%
potassium hydroxide as catalyst in ethanol (Route 1 in
Scheme 1).
Resu lts a n d Discu ssion
Since the potassium salts precipitated because of the
highly alkaline reaction environment due to the syn-
thesis of the 2,4-dihydroxy chalcones I-N, the hydroxyls
had to be protected with 3,4-dihydro-R-pyran before the
condensation reaction. Of the two hydroxyls only the
one in position 4 is protected, while the one in position
2 is not available, as it is involved in an intramolecular
hydrogen bond with the oxygen atom of the carbonyl
group. The acetophenone thus protected reacts with the
All compounds were first evaluated for their ability
to inhibit MAO in the presence of kynuramine as a
substrate (Table 2). It is interesting to note that all
compounds act through the reversible mode, as shown
by dialysis performed over 24 h in a cold room against
a 0.1 M potassium phosphate buffer (pH 7.2) capable of
restoring 90-100% of the activity of the enzyme. After
a first assessment of their inhibitory ability on the MAO,
the compounds were tested to determine their activity

1-Acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole MAO Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2073
Ta ble 2. Monoamine Oxidase Inhibitory Activity of Derivatives 1-12a
compound
MAO IC50
MAO-A IC50
MAO-B IC50
SIb selectivity
1
2
3
4
5
6
7
8
5.0 × 10-5 ( 0.05
1.2 × 10-5 ( 0.02
4.0 × 10-5 ( 0.09
9.5 × 10-6 ( 1.00
2.0 × 10-7 ( 0.05
3.0 × 10-5 ( 0.05
3.6 × 10-6 ( 0.03
1.0 × 10-5 ( 0.01
2.0 × 10-5 ( 0.06
9.5 × 10-6 ( 0.08
4.0 × 10-5 ( 0.02
1.0 × 10-5 ( 0.04
8.6 × 10-9 ( 0.02
1.0 × 10-8 ( 0.01
1.0 × 10-7 ( 0.09
8.0 × 10-9 ( 0.05
1.0 × 10-7 ( 0.04
8.8 × 10-9 ( 0.01
9.0 × 10-9 ( 0.08
1.0 × 10-8 ( 0.25
1.0 × 10-8 ( 0.18
9.0 × 10-9 ( 0.02
8.0 × 10-9 ( 0.01
9.0 × 10-9 ( 0.04
8.9 × 0-5 ( 0.03
2.5 × 10-5 ( 0.06
4.0 × 10-5 ( 0.40
1.9 × 10-5 ( 0.03
3.0 × 10-5 ( 0.05
1.0 × 10-4 ( 0.06
7.2 × 10-6 ( 0.28
2.0 × 10-5 ( 0.30
1.2 × 10-4 ( 0.55
4.2 × 10-5 ( 0.07
1.3 × 10-4 ( 0.03
8.3 × 10-5 ( 0.05
10349
2500
400
2375
300
11363
800
2000
12000
4666
16250
9222
9
10
11
12
a
b
Data represent mean values of at least of three separate experiments. SI: selectivity index ) IC50 (MAO-B)/IC50 (MAO-A).
Ta ble 3. Monoamine Oxidase Inhibitory Activity of Racemate
6, Enantiomers (-)- and (+)-6, Racemate 11, and Enantiomers
(-)- and (+)-11a
Among all compounds, 6 and 11 afford the highest anti-
MAO-A activity with maximum A/selectivity especially
for the single enantiomer (-)- and (+)- of the compound
6. The biological results agree with our preliminary
hypothesis of a correlation between the presence of polar
group/s and high anti-MAO-A activity.
These findings will be taken into account when
planning novel, selective MAO-A and MAO-B inhibitors,
structurally related to the synthesized compounds 1-12,
with the aim of developing a SAR model by a rational
design in order to explore in depth the nature of the
interactions with the active site.
compound
Ki MAO-A
Ki MAO-B
SIb selectivity
6
9.0 × 10-9 ( 0.010 7.6 × 10-4 ( 0.02
2.0 × 10-9 ( 0.015 3.3 × 10-4 ( 0.05
6.0 × 10-9 ( 0.020 1.0 × 10-3 ( 0.03
8.0 × 10-9 ( 0.050 6.0 × 10-4 ( 0.04
4.0 × 10-9 ( 0.016 3.2 × 10-4 ( 0.04
7.0 × 10-9 ( 0.020 2.7 × 10-4 ( 0.07
84444
165000
166666
75000
80000
38571
(-)-6
(+)-6
11
(-)-11
(+)-11
a
Data represent mean values of at least of three separate
b
experiments. SI: selectivity index ) Ki( MAO-B)/Ki(MAO-A).
toward MAO-A and MAO-B selectively in the presence
of the specific substrates, serotonin and benzylamine,
respectively (Table 2). This test better shows that some
compounds link high inhibitory activity and high A
selectivity expressed as IC50 (MAO-B)/IC50 (MAO-A)
ratio in Table 2 (SI ) selectivity index).
These biological results indicate not only the influence
of the para-substituted hydroxyl group on the aromatic
ring bonded to C3 of the pyrazoline ring, but also that
of the ortho-substituted methoxyl group on the aromatic
ring bonded to C5 of the pyrazoline nucleus.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Bu¨chi 510
apparatus and are uncorrected. IR spectra were recorded on
a Perkin-Elmer 1605 FT spectrophotometer on potassium
1
bromide mulls. H NMR spectra were performed on a Bruker
AM-400 (400 MHz) FT spectrometer in CDCl3, and the
chemical shifts were reported in ppm referring to the solvent
peak. Elemental analyses for C, H, and N were performed on
a Carlo Erba model 1106 elemental analyzer, and the analyti-
cal results were within (0.4% of the theoretical values.
Gen er a l P r oced u r e for th e P r ep a r a tion of 4-Hyd r oxy-
p h en yl Ch a lcon es A-H (Rou te 1). An aqueous solution of
potassium hydroxide (20%, 10 mL) was added under stirring
overnight to a solution of the appropriate aryl aldehyde (30
mmol) and 4-hydroxyacetophenone (30 mmol) in 96% ethanol
(75 mL) at room temperature. The reaction mixture was then
poured into water (100 mL), and after neutralization with
hydrochloric acid (5%), a yellow solid was recrystallized from
ethanol and identified by observing the formation of the CHd
CH at 7.60 and 7.90 ppm in the 1H NMR spectra and the
disappearance of the aldehyde proton at 9.87 ppm.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2,4-Dih y-
d r oxyp h en yl Ch a lcon es I-N (Rou te 2). A solution of 3,4-
dihydro-R-pyran (89.68 mmol) in methylene chloride (50 mL)
was added dropwise to a suspension of 2,4-dihydroxyacetophe-
none (30 mmol) and pyridinium p-toluenesulfonate (PPTS)
(0.72 mmol) in methylene chloride (150 mL) and stirred at
room temperature for 12 h. The reaction mixture was washed
with water, and the organic layer dried (Na2SO4) and concen-
trated in vacuo to obtain the protected acetophenone. A
solution of 2,4-dihydroxy-4-(tetrahydropyran-2-yloxy)acetophe-
none (5 mmol) and the appropriate benzaldehyde (5 mmol) in
ethanol (12 mL) was slowly added to a solution of barium
hydroxide octahydrate (7.43 mmol) in ethanol (60 mL) and
stirred for 24 h at 40 °C. The reaction mixture was concen-
trated in vacuo, washed with water (100 mL), neutralized with
1 M HCl, and extracted with ethyl acetate. The organic layer
was dried (Na2SO4) and concentrated in vacuo, yielding the
crude 2,4-dihydroxy-4-(tetrahydropyran-2-yloxy) chalcone. The
compound was suspended in ethanol (30 mL), and p-toluene-
sulfonic acid (0.12 mmol) was added. The reaction mixture was
stirred for 12 h at room temperature, diluted with water (30
mL), neutralized with Na2CO3, and extracted with ethyl
The data reported in Table 2 show that compounds
1, 4, 6, 7, 10, 11, and 12 all have a high inhibitory
activity on MAO-A in the range 8.0 × 10-9 to 9.0 × 10-9
.
Interestingly, in particular, compared to others, 6 and
11 are the only compounds that have both the hydroxyl
group in para and the methoxyl group in ortho positions.
Moreover, these compounds have a high IC50 g 10-9
value that is associated with a high A-selectivity (SI )
11 363 and 16 250, respectively).
Furthermore, because of the presence of a chiral
center at the C5 position of the pyrazole moiety, semi-
preparative chromatographic enantioseparation of the
most potent, selective, and active chiral compounds 6
(Ki(MAO-A)) 9 nM; SI ) 84 444) and 11 (Ki(MAO-A)) 8 nM;
SI ) 75 000) was performed. The separated enantiomers
were then submitted to in vitro biological evaluation
(Table 3). From the results of these experiments it has
been possible to point out a difference between the
racemic mix and the individual enantiomers in inhibit-
ing the two isoforms selectively. This is particularly
evident in compound 6, for which anti-MAO-A activity
varies slightly, while selectivity doubles for the enan-
tiomeric pair, (-)- and (+)-, reaching 165 000 and
166 666, respectively.
In conclusion, we prepared new 1-acetyl-3,5-diphenyl-
4,5-dihydro-(1H)-pyrazole derivatives 1-12, which have
been shown to be potent and selective MAO-A inhibitors.

2074 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Chimenti et al.
acetate. The organic layer was dried (Na2SO4) and evaporated
in vacuo to give the chalcones I-N.
reported in Table 3. The data are the means of three or more
experiments performed in duplicate.
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-Acetyl-
3-(4-h yd r oxy- a n d 2,4-d ih yd r oxyp h en yl)-5-p h en yl-4,5-
d ih yd r o-(1H)-p yr a zoles 1-12. A solution of chalcones A-N
(5 mmol) in 30 mL of acetic acid was added dropwise to 0.6
mL of hydrazine hydrate (12.5 mmol) and kept under stirring
at 120 °C for 24 h. The mixture was then poured in ice-water,
obtaining the crude pyrazole derivatives 1-12, which were
crystallized from ethanol.
Ack n ow led gm en t. This work was supported by
grants from MURST.
Refer en ces
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1H NMR a n d IR Sp ectr a l Da ta of Der iva tives 1-12. For
all the synthesized compounds, the diagnostic infrared absorp-
tions were 3450-3100 cm-1 (OH), 1618 cm-1 (CdO), and 1590
1
cm-1 (CdN). As regards H NMR spectra, we always detected
the following typical peaks for the pyrazoline nucleus: 5.53-
5.47 (q, 1H, H5), 3.89-3.79 (m, 1H, H4′), and 3.28-3.20 (dd,
1H, H4). For the 2,4-dihydroxy derivatives 9-12, two different
peaks for the hydroxyl groups were observed as broad singlets
at 10.26 and 10.04 ppm, which disappeared upon treatment
with D2O. For the 4-hydroxy derivatives 1-8, only a broad
singlet at 10.20 ppm, which disappears with D2O, was ob-
served. For all compounds, 1-12, the aromatic protons appear
as multiplets in the range 7.60-6.40 ppm. The methyl group
of the N1-acetyl was always observed at 2.03 ppm, while for
mono- and dimethoxy derivatives 6-8 and 12, a singlet
centered at 3.73 ppm was observed. Finally, the methyl-
substituted compounds 4, 5, and 11 show an additional peak
at 2.35 ppm.
(6) Youdim, M. B. H.; Finberg, J . P. M. New directions in monoam-
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Ch r om a t ogr a p h ic (H P LC) R esolu t ion of R a cem ic
Sa m p les 6 a n d 11. Chiral HPLC of 6 and 11 was performed
by stainless steel Chiralcel OD columns (250 × 4.6 mm I.D.
and 250 × 10 mm I.D.) (Daicel Chemical Industries, Tokyo,
J apan). HPLC-grade solvents were supplied by Carlo Erba
(Milan, Italy).
Chiral HPLC was performed using a Waters (Milford, MA)
510 pump equipped with a Rheodyne (Cotati, CA) injector, a
1 mL sample loop, an HPLC Perkin-Elmer (Norwalk, CT) oven,
and a Waters Model 996 diode array detector (DAD).
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The sign of the optical rotation of the two enantiomers of 6
and 11 was measured on-line at a wavelength of 365 and 589
nm by a Perkin-Elmer polarimeter model 241 equipped with
Hg/Na lamps and a 40 µL flow-cell. The system was kept at a
constant temperature of 25 °C. The output signal was acquired
and processed by Millenium 2010 software.
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George, P.; Durant, F. A reversible monoamine oxidase
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Inhibition of monoamine oxidase-B by condensed pyridazines
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relationships. J . Med. Chem. 1998, 41, 3812-3820.
Bioch em istr y. All chemicals were commercial reagents of
analytical grade and were used without purification. Bovine
brain mitochondria were isolated according to Basford.21 In
all experiments the AO activities of the beef brain mitochon-
dria were determined by a fluorimetric method, according to
Matsumoto et al.22 using kinuramine as a substrate at four
different final concentrations ranging from 5 µM to 0.1 mM.
Briefly, the incubation mixtures contained: 0.1 mL of 0.25 M
potassium phosphate buffer (pH 7.4), mitochondria (6 mg/mL),
and drug solutions with final concentration ranging from 0 to
10-3 µM.
The solutions were incubated at 38 °C for 30 min. Addition
of perchloric acid ended the reaction. The samples were
centrifuged at 10 000 g for 5 min, and the supernatant was
added to 2.7 mL of 0.1 N NaOH. The pyrazole derivatives were
dissolved in dimethyl sulfoxide (DMSO) and added to the
reaction mixture from 0 to 10-3 µM. To study the inhibition of
pyrazole derivatives on the activities of both MAO-A and -B
separately, the mitochondrial fractions were preincubated at
38 °C for 30 min before adding the specific inhibitors
(L-deprenyl 0.5 µM to estimate MAO-A activity, and clorgyline
0.05 µM to assay the isoform B), considering that MAO-A is
irreversibly inhibited by a low concentration of clorgyline, but
is unaffected by a low concentration of L-deprenyl, which is
used in the MAO-B form. Fluorimetric measurements were
recorded with a Perkin-Elmer LS 50B Spectrofluorimeter. The
protein concentration was determined according to Bradford.23
The results are reported in Table 2. Dixon plots were used to
estimate the inhibition constant (Ki) of the inhibitors; data are
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