G. J. Pacofsky et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3219–3222
3221
The lead benzodiazepinones1 were quite potent when
Table 2. Comparative data for BZDs
evaluated in our receptor-binding assay, however, the
amidine-containing analogues of 1 generally displayed
reduced potencies (Table 1). A modest preference was
noted for smaller appendages in the series of hydro-
phobic analogues. For example, compounds 5a and 5b
were somewhat less potent than 1 while incorporation
of a phenyl group (5c) or a branched alkyl (5d) into the
amidine decreased binding affinity dramatically. This
trend was evident with hydrophilic analogues as well.
Compounds 5e and 5f were markedly weaker binders
than 1, but, again, the bulkier heterocycle-containing 5g
and 5h were considerably less potent than 1. As satis-
factory performance in the receptor binding assay was a
prerequisite for continued investigation, only simply
substituted amidines offered an alternative to the lactam
1 and amidine 5a emerged as the lead in this series.
Receptor affinity for 5a had decreased by 20-fold rela-
tive to 1, but 5a now displayed an improved solubility
profile (13 mg/mL at pH 3) relative to both midazolam
(2, ꢃ8 mg/mL at pH 3) and 1 (<3 mg/mL at pH 3
(estimated value) ). Furthermore, this amidine elicited a
full agonism response in the rat loss-of-righting reflex
(LRR)14 assessment (Table 2) to confirm that structural
changes had not altered pharmacology. Also note-
worthy are the recovery times (initial=15 min,
total=22 min) observed with this compound in the
LRR. An identical response was observed when 5a was
dosed in water adjusted to pH3. These data positioned
the amidines, specifically 5a, as a potential replacement
for 1 and broadly supported the hypothesis of lactam-
to-amidine modification.
Compd
Solubilitya
Doseb
LRR Recoveryc
Initial
Total
1
2
5a
<3d
ꢃ8
13
25
30
25
50
25
25
25
25
25
25
25
9
23
15
9
17
6
23
7
13
9
24
121
22
16
23
21
33
10
15
11
24
10
1
16a
16b
16c
17a
17b
17c
18a
ND
ND
<1
ND
2
13
ND
20
aDetermined at pH 3;15 mg/mL; ND=not determined.
bBolus injection, mg/kg; vehicle=50% PEG/25% EtOH/25% saline.
cRecovery time in min. A compound was identified as inactive in this
model if LRR was not observed within 5 min following injection.
dEstimated value.
retained. While the recovery times (initial=9 min,
total=16 min) observed in the LRR were noteworthy and
provided a significant advantage over both 1 and 5a, the
unanticipated drop in its pH-dependent solubility (1 mg/
mL at pH 3) was disappointing. Interestingly, combining
the amidine and C5 replacement strategies in the form of
11 offered no advantage in terms of receptor affinity.
Both amidine- and C5-pyridyl-containing BZDs dis-
played reasonable pharmacological profiles, but neither
series provided entities that combined these properties
with aqueous solubility. We speculated that incorpora-
tion of an additional heterocycle into the BZD scaffold
might improve solubility without impacting other attri-
butes. Towards that end, 16a–c, 17a–d, and 18a and b
were synthesized and evaluated. Receptor binding affi-
nities paralleled those of the respective parent BZDs,
and the aforementioned preference1 for the (S)-stereo-
isomer prevailed with the midazolam-derived esters, 18a
and b, as well. Moderate selectivity (i.e., receptor
potency of ester versus acid) was observed with these
series, so compounds were progressed to the in vivo
LRR assessment to confirm their full agonism profiles.
Importantly, total recovery times observed with each of
these compounds was significantly shorter than the
recovery time observed in animals dosed with 2. The
decreased aqueous solubility of 16c, as compared to 2,
suggested that related methyl group positional isomers in
the 20-haloaryl series, especially 18a, would possess solu-
bility profiles similar to 16c. This observation together
with the synthetic protocol required to deliver 18a promp-
ted its removal from further consideration despite its per-
formance in the LRR assessment. In contrast, compound
17c presented a level of in vivo efficacy and aqueous solu-
bility commensurate with our objectives. However, like
compound 5a, its relatively weak receptor affinity would
likely necessitate administration of inconveniently higher
doses, and dose volumes, and reduce its clinical utility.
Concurrently, we explored the effects of replacing the
C5-haloaryl moiety with a C5-pyridyl appendage. Bio-
logical evaluation of 10 showed a significant drop in
binding affinity as well as selectivity, relative to the
parent C5-haloaryl BZD, but a full agonism profile was
Table 1. Receptor binding affinity of BZDs
Compd
R
R1
Kia
Selectivityb
984
1
2
7
2
3
5a
5b
5c
5d
5e
5f
5g
438
96
CH3
CH2CH3
CH2Ph
156
146
875
1012
303
208
792
1230
139
1075
7
7
8
79
60
98
130
11
CH2CH(CH3)2
CH2CH2OH
(CH2)2-4-imidazolyl
(CH2)2-4-pyridyl
CH2-4-pyridyl
5h
10
11
46
16a
16b
16c
17a
17b
17c
17d
18a
18b
H
H
CH3
H
H
CH3
CH3
H
CH3
H
H
CH3
H
64
156
74
176
156
147
40
CH3
226
28
Improving the physical properties of our lead benzodia-
zepinones was achieved through various manipulations
of the BZD scaffold. The three strategies of lactam-to-
amidine modification, C5-haloaryl replacement, and
3572
aRat BZD receptor, nM.
bSelectivity=Ki (acid)/Ki (ester).