Potential metabolites of a condensed 2,3-benzodiazepine derivative

Article abstract of DOI:10.1016/j.bmcl.2005.07.080

Putative metabolites of an AMPA antagonist imidazo-2,3-benzodiazepine (2) were synthesized and compared to constituents formed from the parent compound by a rat liver perfusion method. As metabolic transformations, hydroxylation of the 2-methyl group and

Full text of DOI:10.1016/j.bmcl.2005.07.080

0
Bioorganic & Medicinal Chemistry Letters 15 (2005) 4662–4665
Potential metabolites of a condensed 2,3-benzodiazepine derivative
a
a
Emese Csuzdi, Katalin Migleczi, Istvan Hazai, Pal Berzsenyi, Istvan Pallagi,
a
a
a
´
Gyula Horvath, Gyo¨rgy Lengyel and Sandor Solyom
´
´
´
a
b
a,
*
´
´
´
aIVAX Drug Research Institute Ltd., H-1045 Budapest, Berlini u. 47-49, Hungary
bInstitute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, H-1025 Budapest,
Pusztaszeri u. 59-67, Hungary
Received 10 June 2005; revised 25 July 2005; accepted 29 July 2005
Available online 8 September 2005
Abstract—Putative metabolites of an AMPA antagonist imidazo-2,3-benzodiazepine (2) were synthesized and compared to constit-
uents formed from the parent compound by a rat liver perfusion method. As metabolic transformations, hydroxylation of the
2-methyl group and N-acetylation of the amino functionality in parent compound (2) were registered. The hydroxylated analogue
12 of 2 exhibits a weak AMPA antagonist activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1
N
AMPA (2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)pro-
CH3
CH3
pionic acid) antagonists possess a significant role among
the ionotropic glutamate receptor ligands and have the
capacity among others to serve as anticonvulsant and
neuroprotective drugs.1,2
O
O
O
N
N
8
N
CH3 Cl
N
6
5
Talampanel (1) (Fig. 1) is a non-competive AMPA
antagonist 2,3-benzodiazepine (BDZ) that is now under
advanced Phase II clinical investigation.3–5 Broad struc-
ture activity relationship studies have revealed several
different structural features related to a BDZ ring system
that greatly influences the APMA antagonist activity,
consequently, some successful substitutes for the func-
tions of molecule 1 were found.6 One of the major recog-
nitions was that BDZs with a condensed azole ring,
attached to atoms 3,4 of the BDZ skeleton, possess a
high AMPA antagonist activity.7–9 As a representative
of this condensed BDZ derivative 2 (GYKI-47261) is
exemplified, which showed a broad spectrum anticon-
vulsant activity against seizures evoked by electroshock
and different chemoconvulsive agents; however, its neu-
roprotective effects in a focal ischemia test and a MPTP-
induced Parkinson model were more pronounced.8 The
unique structure of BDZs with an attached azole ring
from among the different AMPA antagonists of the
NHR
NH2
(R = H)
(R = -COCH3)
2
3
1
Figure 1. Structures of talampanel and a representative of condensed
BDZs.
BDZ type was also corroborated by the finding that
their corresponding pharmacophore model differed only
in one donor site attaching point from that of other
BDZ structures and the biological consequences of
which are still to be clarified.10
Remarkable neuroprotective potency of 2 has paved the
way for becoming a potential drug candidate, necessitat-
ing the study of its metabolism.11 A widely accepted
method of research on metabolism is to employ radiola-
beled compounds in animal experiments. This way the
metabolites can be detected in biological samples
(blood, excreta, bile, etc.) using selective detection by
means of radiochromatography. A drawback of this
Keywords: AMPA antagonist; Imidazole[2,3]benzodiazepine; Metabo-
lite; HPLC/MS analysis.
*
Corresponding author. Tel.: +36 1 399 3346; fax: +36 1 399 3356;
e-mail: Sandor-Solyom@idri.hu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.080

E. Csuzdi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4662–4665
4663
procedure, however, lies in the fact that a tedious sample
cleanup procedure is usually required for a structure
elucidation. In this study, we used a reverse approach,
namely the potential metabolites were synthesized and
compared to the constituents formed from the parent
compound. An ex vivo technique was applied to obtain
samples containing a far less amount of endogenous
constituents than the samples of in vivo experiments.12
Additionally, by this method radiolabeling was dis-
pensed with. Therefore, on the basis of different consid-
erations, several metabolite-like derivatives of 2 have
been synthesized and analyzed for their identity with
main metabolites of 2.
solution. Alkylation of 5 with ethyl bromopyruvate
resulted in a product which showed no keto-carbonyl
1
group in the IR spectrum and which according to H
NMR investigations can be characterized by the hemia-
minal structure 6. Latter, when treated with p-toluene-
sulfonic acid, it gave a 2:1 mixture of 7 and 8,
respectively. The formation of hydrolysis product 8 is
a proof that alkylation with ethyl bromopyruvate takes
place at the ring nitrogen atom. Compound 7 served as
the starting material for all the potential metabolite-like
analogues of 2. Transfer hydrogenation gave amino-es-
ter 9 and hydrolysis of the latter resulted in carboxylic
acid 10, whereas a lithium aluminum hydride reduction
of 9 gave 2-hydroxymethyl derivative 12. The necessary
N-acetyl derivatives were prepared by simple acetylation
of 10 and 12 to provide 11 and 13, respectively (for phys-
ical and spectroscopic data, see Ref. 14).
As a possible metabolic transformation of 2, except that
of acetylation of the amino function,13 an oxidation of
the 2-methyl group was envisaged, resulting in the for-
mation of derivatives with different oxygenated groups
at position 2. Therefore, 2-carboxylic acid and related
derivatives of 2 were prepared, according to Scheme ꢁ1.
Thus, thio-oxo compound 48 was reacted in a Teflon -
coated autoclave with a saturated ammonia–THF
solution in the presence of mercury chloride to give an
amidine, possessing the tautomeric structure 5 in DMSO
Liver perfusion was performed, according to standard
procedure,12 and the perfusate was analyzed, as given
in Ref. 15.
In Table 1 chromatographic and mass spectrometric
characteristics of the synthesized compounds to those
OH
S
NH2
N
CO2C2H5
c
a
b
NH
N
N
N
N
N
Cl
Cl
Cl
R1
R1
R1
6
5
4
N
O
CO2C2H5
N
N
N
N
+
CO2C2H5
Cl
Cl
R1
O
R1
8
7
d
9 (with R2)
f
e
N
CO2H
N
OH
N
N
N
N
Cl
Cl
R2
10
R2
12
g
h
(with R3)
(with R3)
13
11
R1 =
R2 =
R3 =
NO2
NH2
NHCOCH3
Scheme 1. Reagents and conditions: (a) NH3/THF/HgCl2, autoclave, 70 ꢂC, 81%; (b) ethyl bromopyruvate, DMF, K2CO3, rt, 65%; (c) cat. p-TsOH,
EtOH/CHCl3, 58% (7), 28% (8); (d) RaNi, NH2NH2monohydrate, CH2Cl2/MeOH, 87%; (e) NaOH, EtOH, reflux, 63%; (f) LiAlH4, THF, 59%;
(g) Ac2O, CH2Cl2, rt, 79%; (h) AcCl, pyridine, rt, 41%.

4664
E. Csuzdi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4662–4665
Table 1. Comparison of chromatographic and mass spectrometric properties of synthesized standards and metabolites
Compounds
Synthesized standards
Rat liver perfusate
Retention
time (min)
Molecular
mass
Major fragments
Retention
time (min)
Molecular
mass
Major fragments
2
3
23.81
21.57
323
365
282 (60%), 217 (40%)
350 (15%), 338 (15%),
324 (55%), 217 (15%)
335 (100%)
23.84
21.61
323
365
282 (60%), 217 (40%)
350 (15%), 338 (15%),
324 (55%), 217 (15%)
n.d.
10
11
12
13
8.23
8.41
353
395
339
381
n.d.
n.d.
n.d.
n.d.
339
381
377 (100%)
321 (100%)
363 (100%)
n.d.
321 (100%)
363 (100%)
13.31
12.08
13.42
12.06
n.d.: not detected.
Table 2. Screening results with some new BDZs
Compounds
Behavioural changes
(100 mg/kg ip; 200 mg/kg po)
MES test ED50
mg/kg po
,
Inclined screen test
ED50, mg/kg ip
Retinal spreading depression test IC50 (lM)
or inhibition (%) in 20 lM
1 (referent)
2 (referent)
Loss of righting reflex
Loss of righting reflex
Decrease of SMA,b ataxy
Decrease of SMAb
8.6 (7.0–10.6)
24.0 (17.9–32.1)
56.0
13.4 (11.2–16.0)
36.5 (29.4–45.2)
>200c
1.7
7.3
3a
>20b
10–20
55%
33%
5.0
9
>100c
>200c
10
11
12
Slight decrease of SMAb
>100c
>100c
25–50
>200c
>200c
100–200
Decrease of SMA,b ataxia,
decrease of muscle tone
n.t.d
13
>100b
n.td
n.tc
a See also Ref. 8.
b Spontaneous motor activity.
c Highest test concentration.
d Not tested.
of metabolites present in liver perfusate are compared. It
is seen that apart from the parent compound three peaks
eluted with retention identical to those of synthetic stan-
dards. In addition, these compounds exhibited mass
spectra identical to those of standards. Thus, these
metabolites were identified as the N-acetylated analogue
3 of the parent compound 2, 12 (a hydroxylated ana-
logue of the parent compound 2), and 13 (N-acetylated
derivative of 12). As a consequence, the above findings
show that the main metabolic transformations of the
compound under study (2) are those of N-acetylation,
as well as hydroxylation of the side-chain methyl group.
A similar metabolic hydroxylation of an analogous
methyl-imidazo-BDZ was noticed recently.9
observed in vivo in the given test system up to the test
concentrations used. The only compound that possesses
a weak AMPA antagonist activity in all of the tests was
the hydroxymethyl derivative 12, which was also detect-
ed among the metabolites formed from 2 under ex vivo
conditions. As a result, a conclusion can be drawn that
out of five putative metabolites actually three com-
pounds formed under ex vivo conditions; however, none
of them exhibited any strong AMPA antagonist activity.
References and notes
1. Parsons, G. C.; Danysz, W.; Quack, G. Drug News
Perspect. 1988, 11, 523.
2. De Sarro, G.; Gitto, R.; Russo, E.; Ibbadu, G. F.; Bareca,
M. L.; Chimirri, A. Curr. Top. Med. Chem. 2005, 5, 31.
3. Tarnawa, I.; Vizi, E. S. Restor. Neurol. Neurosci. 1998, 13,
41.
4. Chappel, A. S.; Sandler, J. W.; Brodie, M. J.; Chadwick,
D.; Lledo, A.; Zhang, D.; Bjerke, J.; Kiesler, G. M.;
Arroyo, S. Neurology 2002, 58, 1680.
5. Langan, Y. M.; Lucas, R.; Jewell, H.; Toublanc, N.;
Schaefer, H.; Sander, J. W.; Patsalos, P. N. Epilepsia 2003,
44, 46.
In further experiments, the new aminophenyl BDZs (9,
10, 11, 12, 13) were subjected to in vitro screening for
inhibition of AMPA (5 lM) evoked spreading depres-
sion in isolated retina prepared from young chicken.16
In addition, these molecules were also tested for anticon-
vulsant activity using the maximal electroshock seizure
model,17 as well as for muscle relaxant activity, using
the inclined screen test in mice.18 The gross behavioral
changes were evaluated in mice according to Irwin19
the results of which are shown in Table 2.
´
6. Solyom, S.; Tarnawa, I. Curr. Pharm. Design 2002, 8, 913.
7. Csuzdi, E.; Hamori, T.; Abraham, G.; Solyom, S.;
´
´
´
´
´
Tarnawa, I.; Berzsenyi, P.; Andrasi, F.; Ling, I.; Simay,
A.; Gal, M.; Horvath, K.; Szentkuti, E.; Szo¨llosy, M.;
Data in Table 2 demonstrate the fact that certain com-
pounds possessing an oxygenated side chain on the
imidazole moiety show a slight effect however, this fact
can be demonstrated in the in vitro retinal spreading
depression test only (e.g., 9–11) and could not be
}
´
´
Pallagi, I. PCT Int. Appl. WO 97, 28, 63; Chem. Abstr.
1997, 127, 205597n.
´
8. Abraham, G.; Solyom, S.; Csuzdi, E.; Berzsenyi, P.; Ling,
´
I.; Tarnawa, I.; Hamori, T.; Pallagi, I.; Horvath, K.;
´
´
´

E. Csuzdi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4662–4665
4665
´
Patthy, M.; Horvath, G. Bioorg. Med. Chem. 2000, 8, 2127.
9. Elger, B.; Huth, A.; Neuhaus, R.; Ottow, E.; Schneider,
H.; Seilheimer, B.; Turski, L. J. Med. Chem. 2005, 48,
4618.
´
´
Andrasi, F.; Kapus, G.; Harsing, L. G. J.; Kiraly, I.;
´
CI-MS: 351 [MÀCO2+H]+ (100), 350 [MÀCO2]+ (74);
compound 12: mp: >260 ꢂC, 1H NMR (DMSO-d6,
T = 300K) d 7.63 (dd, J1 = 8.2 Hz, J2 = 2.3 Hz, 1H), 7.56
(d, J = 8.2 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.20 (d,
J = 2.3 Hz, 1H), 7.20 (s, 1H), 6.64 (d, J = 8.4 Hz, 2H),
5.81 (s, 2H, exchangeable), 4.81 (t, J = 5.5 Hz, 1H,
exchangeable), 4.28 (d, J = 5.5 Hz, 2H), 3.84 (br s, 2H);
compound 13: mp: >260 ꢂC, EI-MS: m/z: 380, 382 [M]+Å
(100,34), 337 (28), 218 (43).
´
10. Rezessy, B.; Solyom, S. Lett. Drug Des. Dis. 2004, 1, 217.
11. For preliminary results see: Migleczi, K.; Hazai, I.;
´
´
Jemnitz, K.; Patfalusi, M. J. Plan. Chrom. 2001, 14, 226.
12. Kukan, M. In Handbook of Drug Metabolism; Woolf, T.
F., Ed.; Marcel Dekker, Inc.: New York, 1999; pp 425–
442.
13. Eckstein, J. A.; Swanson, S. P. J. Chromatogr., B 1995,
668, 153.
15. Wistar male rat liver was cannulated into vena portae
and washed with HanksÕ balanced salt perfusion solution
saturated with O2/CO2 (95/5) for about 10 min. This
solution was spiked with 1 mg/ml of 2 in DMSO to give
a final concentration of 10 g/ml. Perfusion was carried
out at 37 ꢂC for 2 h. The flow rate was 40 ml/min.
Sample cleanup of perfusate solution was done using C18
solid-phase extraction cartridges, as described earlier.11
Methanol eluate was evaporated to dryness and was
reconstituted in a mixture of ethanol/0.05 M ammonium-
acetate, pH 8.5 buffer (1/1) and subjected to HPLC/MS
analysis. HPLC conditions: Purospher RP18 5 lm
250 · 4.0 mm column with 4 · 4.0 mm guard column,
flow rate: 1 ml/min, eluents: (A) acetonitrile/ammonium-
acetate buffer pH 8.5 (10/90), (B) acetonitrile/ammonium-
acetate buffer pH 8.5 (90/10). Gradient program: 0–
2 min: 10% B, 2–4 min: 10–20% B, 4–6 min: 20–30% B,
6–16 min: 30–40% B, 16–27 min: 40–80% B, 27–30 min:
80–10% B, and 30–35 min: 10% B. IT/MS conditions:
positive electrospray ionization, flow rate: 0.3 ml/min,
sheath gas: 20, spray voltage: 4.5 kV, capillary temper-
ature: 200 ꢂC, capillary voltage: 7. Selected ion monitor-
ing: 0–10 min: m/z 350–400, 10–12.3 min: m/z 380–390,
12.3–15.5 min: m/z 338–345, 15.5–17.6 min: m/z 380–390,
17.6–20.9 min: m/z 338–345, 20.9–23.4 min: m/z 364–369,
23.4–35 min: m/z 320–330. MS/MS experiment: normal-
ized collision energy: 50.
14. Physical and spectroscopic data of new compounds.
Compound 6: mp: 188–191 ꢂC, 1H NMR (DMSO-d6,
T = 25 ꢂC) d 8.32 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.5 Hz,
2H), 7.65 (dd, J1 = 8.3 Hz, J2 = 1.9 Hz, 1H), 7.60 (d,
J = 8.3 Hz, 1H), 7.15 (d, J = 1.9 Hz, 1H), 6.72 (s, 1H,
exchangeable), 4.25 (d, J = 10.8 Hz, 1H), 4.10 (q,
J = 7.2 Hz, 2H), 3.85 (s, 2H), 3.75 (d, J = 10.8 Hz, 1H),
1.13 (t, J = 7.2 Hz, 3H); 13C NMR d 170.7 (s), 166.9 (s),
154.3 (s), 147.8 (s), 143.1 (s), 136.1 (s), 133.4 (s), 131.6 (s),
131.3 (d), 130.6 (d), 129.8 (d), 128.4 (d), 123.6 (d), 93.6 (s),
62.7 (t), 61.0 (t), 33.0 (t) 13.9 (q); IR (KBr past.)
1754 cmÀ1
;
compound 7: mp: >260 ꢂC, 1H NMR
(DMSO-d6, T = 25 ꢂC) d 8.42 (d, J = 8.8 Hz, 2H), 8.23
(s, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.76 (dd, J1 = 8.4 Hz,
J2 = 2.0 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.22 (d,
J = 2.0 Hz, 1H), 4.25 (s, 2H), 4.23 (q, J = 6.9 Hz, 2H),
1.36 (t, J = 6.9 Hz, 3H); compound 8: mp: 151–153 ꢂC, 1H
NMR (DMSO-d6, T = 27 ꢂC) d 8.33 (d, J = 8.8 Hz, 2H),
7.83 (d, J = 8.8 Hz, 2H), 7.73 (dd, J1 = 8.4 Hz,
J2 = 2.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.23 (d,
J = 2.0 Hz, 1H), 5.10 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H),
3.17 (br s, 2H), 1.23 (t, J = 7.2 Hz, 3H), 13C NMR d 187.9
(s), 167.9 (s), 159.4 (s), 158.2 (s), 148.3 (s), 142.7 (s), 135.5
(s), 132.3 (d), 131.7 (s), 130.6 (d), 130.1 (d), 128.0 (d), 123.6
(d), 62.0 (t), 57.1 (t), 40.3 (t), 13.7 (q); IR (KBr past.)
16. Sheardown, M. J. Brain Res. 1993, 607, 189, Compounds
were tested first at 20 lM in six retina preparations.
Compounds showing less than 40% inhibition were
designed as IC50 > 20 lM.
17. Swinyard, B. A. J. Pharmacol. Exp. Ther. 1952, 106, 319
(10 mice/dose and 3–4 doses were used; 60 min pretreat-
ment po).
1
1734 cmÀ1, 1668 cmÀ1; compound 9: mp: 250–253 ꢂC, H
NMR (DMSO-d6, T = 298 K) d 8.00 (s, 1H), 7.67 (dd,
J1 = 8.3 Hz, J2 = 1.8 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H),
7.40 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 1.8 Hz, 1H), 6.63 (d,
J
= 8.5 Hz, 2H), 5.95 (s, 2H, exchangeable), 4.17 (q
J = 7.0 Hz, 2H), 4.05 (br s, 2H), 1.25 (t, J = 7.0 Hz, 3H);
compound 10: mp: >260 ꢂC, EI-MS: m/z:352, 354 [M]+Å
(44,17) 308, 310 (100,33), 268 (14), CI-MS: 353, 355
[M+H] (100,42); compound 11: mp: >260 ꢂC, EI-MS: m/z:
394, 396 [M]+Å (44,15), 350, 352, (100,48), 349, 351 (28,33),
18. Randall, L. O.; Schallek, W.; Heise, G. A.; Keth, E. F.;
Bagdon, R. E. J. Pharmacol. Exp. Ther. 1960, 129, 163 (10
mice/dose and 3–4 doses were used; 30 min pretreatment
ip).
19. Irwin, S. Psychopharmacologia 1968, 13, 222.