close

Вход

Забыли?

вход по аккаунту

?

Geometrically Constrained Analogues of N-Benzylindolylglyoxylylamides[1 2 4]Triazino[4 3-a]benzimidazol-410H-one Derivatives as Potential New Ligands at the Benzodiazepine Receptor.

код для вставкиСкачать
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
a
b
c
Dipartimento di Scienze
Farmaceutiche,
Università di Pisa, Pisa, Italy
Dipartimento di Chimica
Farmaceutica e
Tossicologica,
Università di Napoli
“Federico II”, Napoli, Italy
Dipartimento di Psichiatria,
Neurobiologia,
Farmacologia e
Biotecnologie,
Università di Pisa, Pisa, Italy
413
Geometrically Constrained Analogues of
N-Benzylindolylglyoxylylamides:
[1,2,4]Triazino[4,3-a]benzimidazol-4(10H)-one
Derivatives as Potential New Ligands at the
Benzodiazepine Receptor
A series of 3-benzylamino- and 3-arylalkylaminocarbonyl[1,2,4]triazino[4,3-a]benzimidazoles 1–12 were synthesized and biologically assayed as geometrically constrained analogues of N-benzylindolylglyoxylylamides II, which are high affinity ligands at the benzodiazepine receptor (BzR). The intermediate 3-ethoxycarbonyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 14 and its N(10)-methyl analogue 15, closely related to 3-alkoxycarbonyl-β-carbolines I, were also investigated.The title compounds exhibited a lower affinity compared with the corresponding
indolylglyoxylylamide derivatives II. Attempts were made to rationalize these results
taking into account the possible tautomeric equilibria involving these ligands.
Keywords: Triazinobenzimidazole; Indolylglyoxylylamides; Benzodiazepine receptor
Received: February 10, 2003; Accepted: April 9, 2003 [FP788]
DOI 10.1002/ardp.200300788
Introduction
γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system
(CNS), where it decreases neuronal excitability by increasing membrane chloride conductance [1].
GABA elicits its physiological effects through interaction
with three major classes of receptors: two types of ligand-operated chloride channels, GABAA and GABAC receptors, and GABAB receptors, which are coupled to Gproteins [2].
um from a Bz-like (full agonist) to a Bz-contrary action
(inverse agonist) [5–8].
We have recently reported a new class of potent BzR ligands, the N-(arylalkyl)indolylglyoxylylamides II (Chart
1); these are hypothesized to assume a pseudoplanar
arrangement mimicking the β-carbolines I (Chart 1) [8],
which are high affinity ligands at the BzR [9, 10]. The
most active indole derivatives are those featuring a benzylamine residue (n = 1) in the side chain, with Ki values
in the nanomolar range [10]. In search for rigid analogues of II, we have disclosed a series of potent BzR
The GABAA receptors are membrane-bound heteropentameric proteins that are made up of five subunits out
of the 8 classes which have so far been cloned and sequenced (α, β, γ, δ, ε, π, θ, and ρ). Most GABAA receptors are composed of α-, β- and γ-subunits.The so-called
benzodiazepine receptor (BzR) is located between the
α- and γ-subunits, and its occupation by a ligand can allosterically modulate the affinity of the GABA neurotransmitter for its specific binding site [3, 4].
Several substances with a chemical structure different
from benzodiazepines (Bz) bind with high affinity at the
BzR on the GABA receptor complex; they exhibit a wide
variety of pharmacological actions, ranging in a continuCorrespondence: Giampaolo Primofiore, Dipartimento di
Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6,
Pisa, Italy. Phone: +390 50 500209, Fax: +390 50 40517,
e-mail: primofiore@farm.unipi.it.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Giampaolo Primofiorea,
Federico Da Settimoa,
Sabrina Taliania,
Anna Maria Marinia,
Francesca Simorinia,
Ettore Novellinob,
Giovanni Grecob,
Letizia Trincavellic,
Claudia Martinic
Potential Ligands of the Benzodiazepine Receptor
414
Primofiore et al.
ligands: the 3-aryl[1,2,4]triazino[4,3-a]benzimidazol4(10H)-ones III (ATBIs) (Chart 1) [11].The triazinobenzimidazole (TBI) nucleus represents a geometrically constrained system with respect to the indolylglyoxylyl moiety of compounds II, the oxalyl CO(2) oxygen of II being
replaced by the N(2) of TBI.
With the aim of optimizing the potency of TBI derivatives
at the BzR, we recently took into consideration different
side chains in position 3 of this tricyclic system. Here we
report the results of our research, describing the synthesis and the affinity data of a series of 3-benzylamino- and
3-arylalkylaminocarbonyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones 1-12 (Chart 2).
While these compounds were under investigation, it was
realized that the ester derivative 14, which is the intermediate for the synthesis of products 4–12, is structurally
related to the 3-alkoxycarbonyl-β-carbolines I. Therefore, compound 14 and its N(10)-methyl analogue 15
were assayed for their affinity at the BzR as well.
Chemistry
The benzylamino derivatives 13 were prepared by a
silylation-amination reaction [12]: a mixture of
Scheme 1.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
[1,2,4]triazino[4,3-a]benzimidazol-3,4-dione 13 [13], the
appropriate benzylamine and hexamethyldisilazane
(HMDS) was heated at 140 °C in the presence of a catalytic quantity of p-toluenesulfonic acid. At the end of the
reaction (TLC analysis), the mixture obtained was treated with hot toluene to give a solid residue made up of the
crude products 1–3 (Scheme 1).
The synthetic procedure employed to prepare the triazinobenzimidazole derivatives 14, 15 involved the reaction of the appropriate 2-hydrazinobenzimidazole 16, 17
[14, 15] with diethylketomalonate in refluxing absolute
ethanol. For the synthesis of amide derivatives 4–12, the
ester intermediate 14 was allowed to react with the appropriate amine in refluxing xylene. Products 4–12 were
then isolated by filtration of the hot suspension (Scheme
2).
When compound 14 was allowed to react with methyl iodide in DMF in the presence of sodium hydride at room
temperature, only the 1-methyl derivative 18 was obtained (Scheme 3). The structure of compound 18 was
confirmed by comparing its physical and spectral data
with those of the 10-methyl isomer 15 and previously described similar products [11]. Actually, in the 1H-NMR 18
showed a singlet relative to the N(1)-CH3 at 4.10 δ iden-
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
Potential Ligands of the Benzodiazepine Receptor
415
Scheme 2.
Scheme 3.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
416
Primofiore et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
Table 1. Physical properties of [1,2,4]triazino[4,3-a]benzimidazole derivatives 1–12 and 14–15 and their inhibition of
[3H]flumazenil specific binding in bovine brain membranes.
Compound
R
R1
1
2
3
4
5
6
7
8
9
10
11
12
14
15
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
NHCH2C6H5
NHCH2C6H4-4-Cl
NHCH2C6H4-4-F
CONHC6H5
CONHC6H4-4-Cl
CONHC6H4-4-OCH3
CONHCH2C6H5
CONHCH2C6H4-4-Cl
CONHCH2C6H4-4-OCH3
CONH(CH2)2C6H5
CONH(CH2)2C6H4-4-Cl
CONH(CH2)2C6H4-4-OCH3
CO2C2H5
CO2C2H5
a
b
c
d
Mp
(°C)
Formulaa
Inhibitionb
(%)
(10 µM)
Kic (µM)
GABA
ratiod
259–261
275–277
232–234
>300
>300
298–300
>300
285–287
265–266
>300
292–294
248–250
229–231
233–235
C16H13N5O
C16H12ClN5O
C16H12FN5O
C16H11N5O2
C16H10ClN5O2
C17H13N5O3
C17H13N5O2
C17H12ClN5O2
C18H15N5O3
C18H15N5O2
C18H14ClN5O2
C19H17N5O3
C12H10N4O3
C13H12N4O3
84.5
55.2
61.0
82.0
42.4
60.0
72.6
62.0
61.8
45.0
3.0
14.0
52.0
40.0
1.0 ± 0.10
1.2
1.7 ± 0.17
1.4
1.6 ± 0.12
0.88
Yield Cryst.
(%) solvent
40
60
60
80
61
86
63
63
80
65
60
70
72
75
toluene
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
EtOH
EtOH
Elemental analyses for C, H, N, were within ± 0.4 % of the calculated values.
Percentages of inhibition of specific [3H]flumazenil binding at 10 µM compound concentration are means ± SEM of five determinations.
Ki values are means ± SEM of three determinations.
GABA ratio = Ki without GABA/Ki with GABA.
tical to that displayed by the N(1)-CH3 ATBI derivatives,
and different from 3.98 δ of compound 15 and
3.80–3.90 δ of N(10)-CH3 ATBIs.
All products were purified by recrystallization from the
appropriate solvent, after filtration, when necessary,
through a silica gel column, and their structure was confirmed by IR, 1H-NMR, MS, and elemental analysis
(Tables 1 and 2).
ously [16, 17]. The Ki values were calculated only for
those compounds inhibiting radioligand binding by more
than 70 % at a fixed concentration of 10 µM. The in vitro
efficacy of active compounds was measured by the
GABA ratio, which predicts the pharmacological profile
of a BzR ligand [18–20].The binding data for compounds
1–12 and 14–15 are reported in Table 1.
Results and discussion
Biochemistry
The binding affinity of each of the newly synthesized TBI
derivatives at the BzR in bovine brain membranes was
determined by competition experiments against the radiolabelled antagonist [3H]flumazenil, as described previ-
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All the compounds 1–12 and 14–15 showed moderate or
no ability to inhibit specific [3H]flumazenil binding in bovine cortical membranes. The benzylamine derivatives
1–3 demonstrated a potency lower than that of the corresponding N-benzylindolylglyoxylylamides II, which
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
Potential Ligands of the Benzodiazepine Receptor
417
Table 2. Spectral data of [1,2,4]triazino[4,3-a]benzimidazole derivatives 1–12 and 14–15.
R1
IR
(cm–1)
1
Compound
R
H-NMR
(ppm)
MS
m/e (%)
1
H
NHCH2C6H5
3370, 1680,
1630, 1575,
1505, 740.
4.46 (d, 2 H, J = 0.64 Hz, CH2); 7.13–
7.41 (m, 8 H, Ar-H); 8.18 (dd, 1 H, J =
0.72, 0.16 Hz, 6-H).
[M+] = 291
(34); 91
(100).
2
H
NHCH2C6H4-4-F
3375, 1670,
1630, 1570,
1505, 740.
4.43 (d, 2 H, J = 0.56 Hz, CH2); 6.94–
7.36 (m, 7 H, Ar-H); 7.96 (bt, 1 H,
NHCH2, exch. with D2O); 8.23 (dd, 1 H, J
= 0.80, 0.08 Hz, 6-H); 12.63 (bs, 1 H, 10NH, exch. with D2O).
[M+] = 309
(22); 109
(100).
3
H
NHCH2C6H4-4-Cl
3370, 1680,
1630, 1580,
1500, 740.
4.45 (d, 2 H, J = 0.64 Hz, CH2); 7.03–
7.47 (m, 7 H, Ar-H); 7.90 (bt, 1 H,
NHCH2, exch. with D2O), 8.20 (dd, 1 H,
J = 0.80, 0.08 Hz, 6-H); 12.65 (bs, 1 H,
10-NH, exch. with D2O).
[M+] = 325
(22); 125
(100).
4
H
CONHC6H5
3250,
1700,
1510,
1130,
3150,
1590,
1220,
740.
7.10–7.81 (m, 8 H, Ar-H); 8.10 (bs, 1 H,
NHPh, exch. with D2O); 8.40 (dd, 1 H,
J = 0.72, 0.08 Hz, 6-H); 10.67 (s, 1 H,
10-NH, exch. with D2O).
[M+] = 305
(15); 93
(100).
5
H
CONHC6H4-4-Cl
3100, 1680,
1600, 1550,
1450, 1100,
750.
7.30–7.84 (m, 7 H, Ar-H); 8.20 (bs, 1 H,
NHPh, exch. with D2O); 8.39 (dd, 1 H,
J = 0.80, 0.16 Hz, 6-H); 10.73 (s, 1 H,
10-NH, exch. with D2O).
[M+] = 339
(14); 127
(100).
6
H
CONHC6H4-4-OCH3
3100, 1680,
1610, 1510,
1450, 1240,
780.
3.75 (s, 3 H, CH3); 6.93 (d, 2 H, J =
0.88 Hz, 3⬘,5⬘-H); 7.37–7.74 (m, 5 H,
Ar-H); 8.36 (dd, 1 H, J = 0.64, 0.16 Hz,
6-H); 10.50 (s, 1 H, 10-NH, exch. with
D2O).
[M+] = 335
(2); 122
(100).
7
H
CONHCH2C6H5
3250, 3150,
1650, 1630,
1500, 1160,
700.
4.56 (d, 2 H, J = 0.56 Hz, CH2); 7.20–
7.73 (m, 8 H, Ar-H); 8.35 (dd, 1 H, J =
0.72, 0.08 Hz, 6-H); 9.21 (bt, 1 H, NHCH2, exch. with D2O).
[M+] = 106
(100).
8
H
CONHCH2C6H4-4-Cl
3250, 1680,
1630, 1580,
1490, 1160,
760.
4.56 (d, 2 H, J = 0.56 Hz, CH2); 7.35–
7.72 (m, 7 H, Ar-H); 8.37 (dd, 1 H, J =
0.72, 0.16 Hz, 6-H); 9.34 (bt, 1 H, NHCH2, exch. with D2O).
[M+] = 353
(14); 140
(100).
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
418
Primofiore et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
Table 2. (contiuned).
R1
IR
(cm–1)
1
Compound
R
H-NMR
(ppm)
MS
m/e (%)
9
H
CONHCH2C6H4-4-OCH3
3250, 1660,
1630, 1610,
1260, 1150,
760.3.
73 (s, 3 H, CH3); 4.50 (d, 2 H, J =
0.56 Hz, CH2); 6.85 (d, 2 H, J = 0.80
Hz, 3⬘,5⬘-H); 7.23–7.72 (m, 5 H, Ar-H);
8.36 (dd, 1 H, J = 0.80, 0.08 Hz, 6-H);
9.21 (bt, 1 H, NH-CH2, exch. with D2O).
[M+] = 349
(23); 136
(100).
10
H
CONH(CH2)2C6H5
3100, 1650,
1510, 1440,
1280, 740.
2.87 (t, 2 H, J = 0.69 Hz, CH2Ph);
3.40–3.52 (m, 2 H, NHCH2); 7.04–7.76
(m, 8 H, Ar-H); 8.36 (dd, 1 H, J = 0.78,
0.08 Hz, 6-H); 8.90 (bt, 1 H, NHCH2,
exch. with D2O); 10.75 (bs, 1 H, 10-NH,
exch. with D2O).
[M+] = 333
(10); 213
(100).
11
H
CONH(CH2)2C6H4-4-Cl
3250, 1650,
1620, 1600,
1160, 1140,
760.
2.89 (t, 2 H, J = 0.72 Hz, CH2Ph);
3.50–3.66 (m, 2 H, NHCH2); 7.29–7.62
(m, 7 H, Ar-H); 8.36 (dd, 1 H, J = 0.72,
0.16 Hz, 6-H); 8.90 (bt, 1 H, NH-CH2,
exch. with D2O).
[M+] = 367
(6); 213
(100).
12
H
CONH(CH2)2C6H4-4-OCH3
3275, 3075,
1660, 1510,
1250, 1140,
760.
2.81 (t, 2 H, J = 0.80 Hz, CH2Ph); 3.48– [M+] = 363
3.63 (m, 2 H, NHCH2); 3.72 (s, 3 H, CH3); (100); 134
6.83 (d, 2 H, J = 0.88 Hz, 3⬘,5⬘-H);
(100).
7.12–7.65 (m, 5 H, Ar-H); 8.38 (dd, 1 H,
J = 0.64, 0.16 Hz, 6-H); 8.89 (bt, 1 H,
NH-CH2, exch. with D2O).
14
H
CO2C2H5
3130, 3095,
1710, 1580,
1410, 1100.
1.39 (t, 3 H, J = 0.72 Hz, CH3); 4.39 (q,
2 H, J = 0.72 Hz, CH2); 7.36–7.67 (m,
3 H, Ar-H); 8.32 (dd, 1 H, J = 0.72, 0.20
Hz, 6-H).
[M+] = 258
(100).
15
CH3
CO2C2H5
1710, 1550,
1400, 1300,
1100, 765.
1.33 (t, 3 H, J = 0.68 Hz, CH2CH3); 3.98
(s, 3 H, N-CH3); 4.34 (q, 2 H, J = 0.72
Hz, CH2); 7.46–7.81 (m, 3 H, Ar-H);
8.43 (dd, 1 H, J = 0.80, 0.08 Hz, 6-H).
[M+] = 272
(12); 118
(100).
present Ki values ranging from 52 to 120 nM [10]. The
most potent compound was 1, characterized by an unsubstituted phenyl on the side chain, which showed a Ki
value of 1 µM. Insertion of a small atom such as fluorine
or chlorine in the para position of the side phenyl ring
(compounds 2 or 3) had an extremely negative influence
on the affinity.
The low potency of the newly synthesized compounds
cannot be related to an excessive steric bulk, as the indolylglyoxylylamides II, similar in size, exhibited Ki values in the nanomolar range. This also holds true for the
scarcely active compound 14, which has dimensions
similar to the highly potent 3-ethoxycarbonyl-β-carboline.
Insertion of a second carbonyl group within the side
chain left the situation unchanged, since all the amide
derivatives 4–12 showed a very low potency. Also in this
case, the more active compounds are those unsubstituted on the side phenyl ring (4: Ki = 1.7 µM; 7: Ki = 1.6 µM).
It should be noted that these TBI derivatives may exist in
the three tautomeric forms A, B and C (Scheme 4).
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
We have previously hypothesized that for these types of
products, the tautomeric form A is the active one, since it
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
Potential Ligands of the Benzodiazepine Receptor
419
Scheme 4.
In conclusion, for these types of structures, equilibria between different tautomeric forms must be taken into consideration, since only one tautomer is involved in the interaction with the receptor site. Moreover, these results
unequivocally demonstrated that for TBI derivatives,
N(10)-H represents one of the necessary groups for interaction with the receptor site, as previously hypothesized by us [11].
Acknowledgement
This work was financially supported by MIUR (cofin.
2001, ex 40 %, prot. 2001037552-004).
Figure 1. Hypothesized interaction of TBI derivatives at
the BzR binding cleft in agreement with Cook’s pharmacophore model [24]. H1, H2 are hydrogen bond donor
sites and A2 is a hydrogen bond acceptor site. L1 and L2
are lipophilic pockets.
features the N(10)-H group which, according to our hypothesis, donates a hydrogen bond to the A2 site of the
BzR (see the pharmacophoric scheme in Figure 1) [11].
The tautomeric form B likewise features a hydrogen
bond donor NH group, but not at the right distance from
the hydrogen bond acceptor groups N2 and C4=O.
Therefore, we thought it possible that the low affinity displayed by the newly synthesized products could be due
to the stabilization of a tautomeric form different from the
active one, A.
To investigate this possibility, a methylation reaction was
carried out on compound 14 with methyl iodide in DMF, in
the presence of sodium hydride, at room temperature.
Only the 1-methyl derivative 18 was obtained by this reaction. Certainly, the product of a chemical reaction depends not only on thermodynamic but also on kinetic factors, anyway the result of this methylation seemed to
suggest that these derivatives mainly exist in the “inactive” tautomeric form B.
Experimental
Chemistry
Melting points were determined using a Reichert Köfler hotstage apparatus (C. Reichert, Vienna, Austria) and are uncorrected. Infrared spectra were recorded with a PYE/UNICAM Infracord Model PU 9516 spectrophotometer (Pye Unicam Ltd.
Cambridge, England) in Nujol mulls. Routine nuclear magnetic
resonance spectra were recorded in DMSO-d6 solution on a
Varian CFT 20 spectrometer operating at 80 MHz (Varian Inc.,
Palo Alto, CA, USA), using tetramethylsilane (TMS) as the internal standard. Mass spectra were obtained on a HewlettPackard 5988 A spectrometer (Hewlett Packard, Palo Alto, CA,
USA) using a direct injection probe and an electron beam energy of 70 eV. Evaporation was performed in vacuo (rotary evaporator). Analytical TLC was carried out on Merck 0.2 mm precoated silica gel aluminum sheets (60 F-254) (Merck, Darmstadt, Germany). Elemental analyses were performed by our
Analytical Laboratory and agreed with theoretical values to
within ± 0.4 %.
The following compounds were prepared in accordance with
reported
procedures: [1,2,4]triazino[4,3-a]benzimidazol3,4(10H)-dione 13 [13, 15]; 2-hydrazino benzimidazole 16 [14,
15]; 1-methyl-2-hydrazinobenzimidazole 17 [14, 15].
3-(4-Substituted benzylamino)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones 1–3
A mixture of 0.404 g (0.002 mol) of [1,2,4]triazino[4,3-a]benzimidazol-3,4(10H)-dione 13, 0.01 mol of the appropriate amine,
0.038 g (0.0002 mol) of p-toluenesulfonic acid and 1.47 mL
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
420
Primofiore et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
(0.007 mol) of HMDS was heated at 140 °C for 24 h. After cooling, the semisolid mixture obtained was solidified by treatment
with hot toluene.The crude product 1–3 was collected and then
purified by recrystallization from the appropriate solvent, if necessary after silica-gel filtration (Tables 1 and 2).
3-Ethoxycarbonyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one
14 and 3-Ethoxycarbonyl-10-methyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 15
A solution of 2-hydrazinobenzimidazole 16 or 2-hydrazino-1methylbenzimidazole 17 (0.01 mol) and diethylketomalonate
(1.68 mL, 0.011 mol) in 50 mL of absolute ethanol was refluxed
for 7–12 h, monitoring the reaction by TLC analysis. After cooling, the precipitate which formed was collected and washed
with absolute ethanol. The ethanol solution was concentrated
and the separated precipitate was collected to yield an additional amount of crude product. The quantities of ester derivatives obtained from the initial insoluble precipitate or from the
ethanol solution were variable, depending upon the solubility of
the various compounds. The crude products 14 and 15 were
purified by recrystallization from the appropriate solvent (Tables 1 and 2).
3-(4-Substituted arylamino)carbonyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones 4-12
A suspension of the ester derivative 14 (0.516 g, 0.002 mol)
and 0.0044 mol of the appropriate amine in 15 mL of anhydrous
xylene was refluxed for 12–48 h, monitoring the reaction by
TLC analysis. The hot suspension was then filtered to yield the
crude product 4–12, which was purified by recrystallization
from DMF (Tables 1 and 2).
Reaction of 3-Ethoxycarbonyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 14 with methyl iodide
Sodium hydride (1.2 mmol, 50 % dispersion in mineral oil) was
added portionwise, under a nitrogen atmosphere, to an icecooled solution of the ester derivative 14 (0.258 g, 1 mmol) in
2 mL of freshly distilled DMF. Once hydrogen evolution had
ceased, a solution of methyl iodide (0.075 mL, 1.2 mmol) in
0.5 mL of the same solvent, was added dropwise, and the reaction mixture was maintained at room temperature for 15 h while
stirring constantly. The solution was then slowly poured onto
crushed ice and the solid precipitate was collected and purified
by recrystallization from ethanol to give 0.226 g (yield 83 %) of
pure 18, mp 189–190 °C. Anal. Calcd. for C13H12N4O3.
IR, cm–1: 1727, 1700, 1563, 1506, 1450, 1230, 754.
H-NMR, DMSO-d6, δ: 1.33 (t, 3 H, J = 0.67 Hz, COOCH2CH3);
4.10 (s, 3 H, NCH3); 4.36 (q, 2 H, J = 0.71 Hz, COOCH2CH3);
7.44–7.83 (m, 3 H, Ar-H); 8.34 (dd, 1 H, J = 0.70, 0.08 Hz, 6-H).
1
+
MS, m/e (relative intensity): 272 (M , 12.8); 118 (100).
Binding studies
[3H]Flumazenil (specific activity 70.8 Ci/mmol) was obtained
from NEN Life Sciences Products (Boston, MA, USA). All other
chemicals were of reagent grade and were obtained from commercial suppliers.
Bovine cerebral cortex membranes were prepared in accordance with [21]. The membrane preparations were subjected to
a freeze-thaw cycle, washed by suspension and centrifugation
in 50 mM tris-citrate buffer pH 7.4 (T1), and then used in the
binding assay. Protein concentration was assayed by the method of Lowry et al. [22].
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[3H]Flumazenil binding studies were performed as reported
previously [16, 17]. At least six different concentrations spanning 3 orders of magnitude were used to calculate the IC50
value of each compound. IC50 values, computer-generated using a non-linear regression formula on a computer program
(Graph-Pad San Diego, CA, USA), were converted to Ki values,
using the Cheng and Prusoff equation [23].
The potencies of the new compounds to inhibit [3H]flumazenil
binding in the presence and absence of GABA were compared.
The differences obtained were expressed as the GABA ratio,
namely the ratios of the Ki values obtained in the absence of
GABA over the Ki values obtained in the presence of GABA.
References
[1] W. Sieghart, Pharmacol. Rev. 1995, 47, 181–234.
[2] M. Chebib, G. A. R. Johnston, J. Med. Chem. 2000, 43,
1427–1447.
[3] V. Tretter, E. Noosha, K. Fuchs, W. Sieghart, J. Neurosci.
1997, 17, 2728–2737.
[4] P. B.Wingrove, S. A.Thompson, K. A.Wafford, P. J.Whiting,
Mol. Pharmacol. 1997, 52, 874–881.
[5] N.Yokoyama, B. Ritter, A. D. Neubert, J. Med. Chem. 1982,
25, 337–339.
[6] H. Shindo, S. Takada, S. Murata, M. Eigyo, A. Matsushita,
J. Med. Chem. 1989, 32, 1213–1217.
[7] M. L. Trudell, S. L. Lifer, Y. C. Tan, M. J. Martin, L. Deng, P.
Skolnick, J. M. Cook, J. Med. Chem. 1990, 33, 2412–2420.
[8] M. S. Allen, T. J. Hagen, M. L.Trudell, P.W. Codding, P. Skolnick, J. M. Cook, J. Med. Chem. 1988, 31, 1854–1861.
[9] A. M. Bianucci, A. Da Settimo, F. Da Settimo, G. Primofiore,
C. Martini, G. Giannaccini, A. Lucacchini, J. Med. Chem.
1992, 35, 2214–2220.
[10] A. Da Settimo, G. Primofiore, F. Da Settimo, A. M. Marini,
E. Novellino, G. Greco, C. Martini, G. Giannaccini, A. Lucacchini, J. Med. Chem. 1996, 39, 5083–5091.
[11] G. Primofiore, F. Da Settimo, S. Taliani, A. M. Marini, C. La
Motta, E. Novellino, G. Greco, M. Gesi, L. Trincavelli, C.
Martini, J. Med. Chem. 2000, 43, 96–102.
[12] H. Vorbrüeggen, K. Krulikiewicz, Chem. Ber. 1984, 117,
1523–1541.
[13] M. Z. A. Badr, A. M. Mahmoud, S. A. Mahgoub, Z. A. Hozien, Bull. Chem. Soc. Jpn. 1988, 61, 1339–1344.
[14] N. P. Bednyagina, I. Ya. Postovskii, Zhur. Obschei. Khim.
1960, 30, 1431–1437, Chem. Abstr. 1961, 55, 1586h.
[15] F. Da Settimo, G. Primofiore, A. Da Settimo, C. La Motta, S.
Taliani, F. Simorini, E. Novellino, G. Greco, A. Lavecchia,
E. Boldrini, J. Med. Chem. 2001, 44, 4359–4369.
[16] G. Primofiore, A. M. Marini, F. Da Settimo, C. Martini, A.
Bardellini, G. Giannaccini, A. Lucacchini, J. Med. Chem.
1989, 32, 2514–2518.
[17] G. Primofiore, F. Da Settimo, S. Taliani, A. M. Marini, E.
Novellino, G. Greco, A. Lavecchia, F. Besnard, L.Trincavelli, B. Costa, C. Martini, J. Med. Chem. 2001, 44, 2286–
2297.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 413–421
Potential Ligands of the Benzodiazepine Receptor
421
[18] C. Braestrup, M. Nielsen, T. Honoré, L. H. Jensen, E. M. Petersen, Neuropharmacology 1983, 22, 1451–1457.
[22] O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall, J.
Biol. Chem. 1951, 193, 265–275.
[19] C. Braestrup, M. Nielsen in Handbook Psychopharmacology (Eds.: L. L. Iversen, S. D. Iversen, S. H. Snyder),
Plenum Press, New York, 1983, Vol. 17, pp 285–384.
[23] Y. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22,
3099–3108.
[20] R. I. Fryer, R. Rios, P. Zhang, Z. Q. Gu, G. Wong, A. S. Basile, P. Skolnick, Med. Chem. Res. 1993, 3, 122–130.
[24] W. Zhang, K. F. Koehler, P. Zhang, J. M. Cook, Drug Design
Discovery 1995, 12, 193–248.
[21] C. Martini, A. Lucacchini, G. Ronca, S. Hrelia, C. A. Rossi,
J. Neurochem. 1982, 38, 15–19.
Alles zur Biopharmazie
GERT FRICKER
Universität Heidelberg,
PETER LANGGUTH
Universität Mainz,
HEIDI WUNDERLI-ALLENSPACH,
ETH Zürich, Schweiz
2003. Ca. 450 Seiten.
Gebunden.
ISBN 3-527-30455-X
Ca. € 47,90* / sFr 72,-
Mit der jüngsten Änderung der
Approbationsordnung für
Apotheker ist die Biopharmazie
als Forschungsbereich, Lehr- und
Studienfach weiter aufgewertet
worden. Dies ist das erste vollständig neu konzipierte Lehr- und
Handbuch, das sämtliche Themen
der Biopharmazie aktuell und
übersichtlich darstellt.
* Der Euro-Preis ist ausschließlich
gültig für Deutschland.
Register now for the free
WILEY-VCH Newsletter!
www.wiley-vch.de/home/pas
Pharmazeuten in Wissenschaft
und Industrie werden die
Erfahrung des renommierten
Autorenteams schätzen, für
Studenten hilfreich sind prüfungsund praxisrelevante
Übungsaufgaben sowie ein
umfangreiches Glossar und
Symbolverzeichnis.
66913072_kn
Biopharmazie
WILEY-VCH • Postfach 10 11 61 • D-69451 Weinheim
Fax: +49 (0) 62 01 - 60 61 84
e-Mail: service@wiley-vch.de • http://www.wiley-vch.de
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Документ
Категория
Без категории
Просмотров
4
Размер файла
126 Кб
Теги
constraint, triazine, new, derivatives, ligand, analogues, potential, geometrical, benzylindolylglyoxylylamides, one, benzodiazepine, receptov, 410h, benzimidazole
1/--страниц
Пожаловаться на содержимое документа