H. Watanabe et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127911
(Figure 2).15
As shown in Table 1, compounds 1–3 did not have clear M1 agonistic
activity up to 10
(EC50; 4.7
μM, whereas its analogue, compound 4, was active
μ
M, Emax; 64 ± 4%). Comparing the structure of 2 and 4, we
thought the hydrogen atom should be placed as an R3 substituent to
yield M1 agonistic activity. Thus, we explored the structure and activity
relationship (SAR) study based on compound 4 to identify compounds
with ideal profiles.
The synthetic routes for compounds 4–14 are shown in scheme 1.
The chloride intermediate (2, 8-disubstituted 11-chloro-5H-dibenzo[b,
e][1,4]diazepine) was prepared according to the previously reported
method.16 The substituted 2-fluoronitrobenzene was reacted with
substituted anthranilic acids by heating in DMF in the presence of
Cs2CO3 (A1-A7), the iron-catalyzed hydrogenation of the nitro group
(B1-B7), followed by reaction with WSC to produce a tricyclic inter-
mediate. The intermediate was treated with phosphorous oxychloride
under reflux in toluene in the presence of N,N-dimethylaniline to form a
chloride intermediate (C1-C7). Suzuki-Miyaura coupling of the chloride
intermediate and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-1,2,3,6-tetrahydropyridine gave compound 4, and 7–12. The
coupling of the chloride intermediate (C1) and 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyridine, alkylation of nitrogen atom at pyridyl
group by ethyl iodide or isopropyl iodide, then reduction by sodium
borohydride gave compounds 5 and 6, respectively. In the case of
compound 13, coupling of the C1 with BOC-protected boronate and then
the de-BOC group by trifluoroacetic acid yielded target compound 13.
Compound 14 was prepared according to a similar method to 13.
The SAR of the test compounds on M1 agonism is summarized in
Table 2. Replacement of the methyl group at R4 in 4 with ethyl (5) and i-
propyl (6) resulted in the loss of M1 agonistic activity. The results of
compound 2, 5, and 6 suggested that a sterically large substituent
around a basic nitrogen atom is unacceptable. Compound 7, in which
the fluorine atom at R2 in 4 was replaced with hydrogen, demonstrated
> 10-fold stronger M1 agonistic activity, but unfavorable M3 agonistic
activity accompanied. Its potency was the same as that of NDMC. M3
receptors are expressed on salivary glands and their agonist causes
salivation. Such hypersalivation was noted in a phase II clinical study of
NDMC.17 Thus, compound 7 can be considered to have a hypersalivation
risk. Next, we replaced the chlorine atom at R1 in 4 with a methyl group;
the molecular size of a methyl group is similar to that of a chlorine atom,
but the electronic behavior is opposite. The EC50 of compound 8 was
Fig. 1. Antagonist-like behavior of clozapine on NDMC-induced muscarinic
M
1 agonism.
Fig. 2. Structures of antipsychotic drugs and compounds 1–4.
Table 1
1 agonistic activity of reference drugs and compounds 1–4.
M
Compound
M1 agonism
EC50 M)
(
μ
Emax
Olanzapine
>100
>10
>10
0.048
>10
>10
>10
4.7
<5%
Quetiapine
<5%
0.31 μM, over > 10-fold stronger than compound 4. The hypersalivation
Clozapine
26 ± 4%
75 ± 3%
13%
risk of 8 was considered to be lower than that of clozapine because 8 did
not exhibit M3 agonism. Based on 8, we replaced the R2 substituent, and
the EC50 of methyl (9), ethyl (10), and methoxy (11) groups was over 10
NDMC
1
2
3
4
42%
28%
μ
M, whereas the Emax was reduced according to the substituent size,
64 ± 4%
suggesting a sterically small substituent to be preferable as R2. Swapping
the R1 and R2 substituents of 4 diminished the activity (12). Compounds
13 and 14 were desmethylated analogues of compounds 4 and 8,
Emax values are shown as the mean ± SE of two or more experiments
respectively. Of note, their M1 agonistic activity was diminished (EC50
;
HPLC analysis. The amounts of conjugate in compounds 4–14 were 25-
fold lower than that in clozapine (Table 2). The reactive metabolite
formation in clozapine was initiated by the activation of a nitrenium ion
(nitrogen atom at the 11th position of clozapine), but the nitrogen atom
was replaced with carbon in the present series of compounds; therefore,
we hypothesized that reactive metabolite formation was suppressed.18
This suggested that our compounds have a low risk in terms of agran-
ulocytosis due to reactive metabolites. Considering all results, we
selected compound 8 for further evaluation.
>10 μM), differing from clozapine versus NDMC. Thus, a hydrogen atom
as R4 was not acceptable in the present series of compounds. These re-
sults suggested that the SAR on M1 agonism is narrow, and a methyl
group as R1 and R4, and a fluorine atom as R2 were revealed to be a
favorable combination for strong and selective M1 agonistic activity.
Furthermore, compound 8 exhibited binding affinity toward D1 and D2
receptors, and its Ki values were similar to those of clozapine and NDMC.
In CHO cells stably expressing human D1 or D2 receptors, compound 8
antagonized dopamine-stimulated Ca2+ accumulation to a similar de-
gree as clozapine.
We evaluated the binding affinity of 8 toward various GPCRs. As
shown in Table 3, the affinity of 8 was similar with those of clozapine
and NDMC. As dopamine D1/D2 and 5HT2A are important targets to
exert antipsychotic activity, we consider compound 8 to be a “serotonin-
dopamine antagonist”. We performed a mouse PK study of 8 by oral
administration, and the blood and brain sample were collected one hour
We previously reported that compounds 1–3 were superior to clo-
zapine in terms of reactive metabolite formation.15 Dansylated gluta-
thione (dGSH) was used as the trapping agent for the quantitative
estimation of reactive metabolites. Test compounds were incubated with
dGSH and human hepatocyte microsome fraction, and the amount of test
compound-dGSH conjugate was measured by fluorescence detector in
after dosing. Compound
8
displayed good brain penetration
2
Horiguchi, Masakuni
