Synthesis and pharmacological evaluation of 11-(1,6-dimethyl-1,2,3,6-tetrahydropyridin-4-yl)-5H-dibenzo[b,e][1,4]diazepines with clozapine-like receptor occupancy at dopamine D1/D2 receptor

Article abstract of DOI:10.1016/j.bmcl.2020.127563

Clozapine-like compound without agranulocytosis risk is need to cure the treatment resistant schizophrenia (TRS). We discovered (S)-3 as Clozapine-like dopamine D₂/D₁ receptor selectivity and improved reactive metabolites formation profile by the modification of piperazine moiety in Clozapine. The optimization of (S)-3 gave compound 5 to be best compound (approximately 10-fold stronger affinity for D₂/D₁ receptor and similar D₂/D₁ selectivity ratio with Clozapine). Clozapine-like D₂/D₁ receptor occupancy profile was proved by in vivo evaluation. In addition, the reactive metabolites derived agranulocytosis risk of compound 5 was considered to be lower than Clozapine. The pharmacology detail of compound 5 is being investigated to develop it for TRS treatment.

Full text of DOI:10.1016/j.bmcl.2020.127563

Bioorganic&MedicinalChemistryLetters30(2020)127563
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological evaluation of 11-(1,6-dimethyl-1,2,3,6-
tetrahydropyridin-4-yl)-5H-dibenzo[b,e][1,4]diazepines with clozapine-like
receptor occupancy at dopamine D1/D2 receptor
Hitoshi Watanabe, Kyoji Ishida, Masanori Yamamoto, Masakuni Horiguchi, Yoshiaki Isobe⁎
Drug Research Division, Sumitomo Dainippon Pharma. Co., Ltd. 3-1-98, Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Clozapine-like compound without agranulocytosis risk is need to cure the treatment resistant schizophrenia
(TRS). We discovered (S)-3 as Clozapine-like dopamine D2/D1 receptor selectivity and improved reactive me-
tabolites formation profile by the modification of piperazine moiety in Clozapine. The optimization of (S)-3 gave
compound 5 to be best compound (approximately 10-fold stronger affinity for D2/D1 receptor and similar D2/D1
selectivity ratio with Clozapine). Clozapine-like D2/D1 receptor occupancy profile was proved by in vivo eva-
luation. In addition, the reactive metabolites derived agranulocytosis risk of compound 5 was considered to be
lower than Clozapine. The pharmacology detail of compound 5 is being investigated to develop it for TRS
treatment.
Treatment resistant schizophrenia
Dopamine
Clozapine
Agranulocytosis
Reactive metabolites
Antipsychotics
Schizophrenia is a heterogeneous and severe neuropsychiatric dis-
order that affects nearly 1% of the population worldwide. Antipsychotic
drugs are the mainstay of treatment, but 30% of patients are categor-
ized as treatment-resistant schizophrenia (TRS).1,2 TRS is defined as a
non-response to at least two therapies of antipsychotic drugs. Clozapine
is an effective medication for TRS with 60–70% of those treated
showing a response, but its mechanism of action has not completely
clarified.3 Several hypotheses have been proposed. (1) Active metabo-
lite: N-desmethylclozapine (NDMC) is a main metabolite of Clozapine,
but plasma Clozapine/NDMC ratios were consistent in Clozapine-re-
sponders and non-responders. In addition, NDMC did not show a clear
efficacy in a phase II clinical trial.4 (2) Muscarinic receptor agonistic
activity: Clozapine has an agonistic activity toward muscarinic M1 re-
ceptor, while the structurally similar Olanzapine has no agonistic ac-
tivity. M1 agonistic activity of NDMC was stronger than Clozapine, but
the clinical trial failed.5 (3) Dopamine D1 receptor antagonistic activity:
it has been reported that D1 receptor alleles predict the clinical response
to Clozapine.6 Selective D1 antagonist, SCH-39116, showed efficacy in
experimental animal models, but had no effect in a clinical trial.7
Therefore, the development of Clozapine-like compounds have not been
successful to date, since the mechanism of action of Clozapine was
unclear (Fig. 1).
schizophrenia and antipsychotic drugs help patients by occupying D2
receptors, though too much D2 antagonism induce side effects like ex-
trapyramidal symptom and neuroleptic dysphoria.9,10 Abi-dargham
et al. reported that significant upregulation in D1 receptor binding in the
dorsolateral prefrontal cortex in patients with schizophrenia.11 Mild
blockade of D1 receptor signaling would be needed as D1 receptor sig-
naling generally follows an inverted U-shape dose response curve.12
Thus, moderate D1 and D2 antagonism would be the important key for
adequate antipsychotic effect. It is noteworthy that the equivalent oc-
cupancy of striatal dopamine D1 and D2 receptors was demonstrated by
Clozapine in a human PET study at the clinical dosing regimen (55% for
D1, 61% for D2), whereas the occupation of other existing antipsychotic
drugs did not show such behavior (Olanzapine, D1: 43%, D2: 79%;
Quetiapine, D1: 12%, D2: 30%; Risperidone, D1: 25%, D2: 81%).13 Si-
milar clinical results were reported in other papers.14,15 These studies
suggest that this distinctive effect on D1 and D2 receptors may be re-
sponsible for the unique effectiveness of Clozapine in TRS. Recently,
Lundbeck developed Zicronapine (D1 > D2 antagonist) for TRS, but
the development of Zicronapine was terminated in a phase II clinical
study. We consider that Zicronapine did not achieve an equivalent oc-
cupancy of D2/D1 receptor in its clinical study. In the current study, we
tested the binding affinity of several compounds toward D2/D1 re-
ceptors, and the results are summarized in Table 1. D2/D1 receptor
selectivity ratio of Clozapine was calculated to be 8.8, whereas there
Patients with schizophrenia, when psychotic, show a heightened
synthesis of dopamine and hyperdopaminergic state.8 The increase in
D2 receptor stimulation is thought to be responsible of psychosis of
was
a lower value for Olanzapine (1.2) and higher value for
⁎ Corresponding author.
E-mail address: yoshiaki-isobe@ds-pharma.co.jp (Y. Isobe).
https://doi.org/10.1016/j.bmcl.2020.127563
Received 29 July 2020; Received in revised form 4 September 2020; Accepted 16 September 2020
Availableonline22September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

H. Watanabe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127563
Fig. 1. Structures of antipsychotic drugs.
compound-dGSH conjugate was measured by LC/MS/MS analysis. As
shown in Table 1, the dGSH-conjugate formation of Clozapine was
much higher than that of Olanzapine, a clinically safer antipsychotic.
The amount of conjugate in compounds 1–3 were lower than Clozapine
(1/6–1/34), suggesting that our strategy was successful. In particular,
(S)-enantiomer of 3 ((S)-3) showed a similar D2/D1 receptor selectivity
ratio with Clozapine. Wood et al. reported a structure and activity re-
lationship (SAR) information of a Clozapine analogue, in particular,
focusing on the substituent at the 2nd and 8th positions, although the
substitutes were limited to hydrogen and chlorine atoms.19 Thus, we
initiated an optimization study based on compound (S)-3 to identify the
Clozapine-like compound for clinical development.
Table 1
SAR exploration of reference drugs and compounds 1–3.
Compound
D1 (ki; nM)
D2 (ki; nM)
D2/D1 ratio
dGSH (mmol/L)
Clozapine
Olanzapine
Zicronapine
SCH39166
1
44
389
8.8
1.2
215
1,380
3.6
21.4
9.4
–
7.5
47
58
0.077
0
0.13
0.9
380
55
28
1,240
1,380
1,180
479
nt
0.21
0.99
1.3
2
(S)-3
51
(R)-3
780
> 1,400
0.84
nt; not tested.
Common intermediate (8,11-dichloro-5H-dibenzo[b,e][1,4]diaze-
pine) was prepared according to the reported method (Scheme 1).20
The halogenated nitrobenzene was substituted with anthranilic acid by
heating in DMF in the presence of Cs2CO3, the iron-catalyzed hydro-
genation of the nitro group, followed by reaction with WSC to produce
a cyclized intermediate. The intermediate was treated with phos-
phorous oxychloride under reflux in toluene in the presence of N,N-
dimetheylaniline to form a chloride intermediate. This intermediate
was further converted to target compounds 1–3 by treatment with ap-
propriate boronate reagents (Suzuki-coupling). We used chirally pure
(S)- or (R)-1,6-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1,2,3,6-tetrahydropyridine for the synthesis of (S)-3 or (R)-3.
Compounds 4–20 were (S)-enatiomers and prepared by a similar
method to that of compounds 1–3 using appropriate substituted-an-
thranilic acid and halogenated nitrobenzene.
Zicronapine (215). Based on the background clinical information, we
speculate that Clozapine-like D2/D1 receptor selectivity ratio is the
Clozapine-like D2/D1 receptor selectivity ratio is critical for demon-
strating the efficacy in TRS.
Agranulocytosis is a life-threatening side effect of Clozapine, which
limits the broader application of this highly effective drug.16 The re-
active metabolite formation has been proposed as a potential me-
chanism of the agranulocytosis caused by Clozapine, and was initiated
by the activation of a nitrenium ion (illustrated in Fig. 2).17,18 In order
to identify the compounds that have Clozapine-like D2/D1 receptor
selectivity ratio without agranulocytosis risk, we designed carbon atom-
linked Clozapine analogues, i.e. piperazine was replaced with a piper-
idine ring to avoid nitrenium ion generation. Dansylated glutathione
(dGSH) was used as the trapping agent for the quantitative estimation
of reactive metabolites. Test compound was incubated with dGSH and
human hepatocyte microsome fraction, and the amount of test
Firstly, we introduced various substituents at the 2nd position (R2)
of compound (S)-3. The results are summarized in Table 2. The
Fig. 2. Proposed route of Clozapine-GSH conjugation formation and our designed compounds 1–3.
2

H. Watanabe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127563
Scheme 1. Synthetic route for compounds 1–20 Reagents and conditions: (i) anthranilic acid, Cs2CO3, DMF, 120 °C. (ii) Fe, NH4Cl, EtOH, reflux. (iii) HOBt, WSC,
DMF, rt. (iv) POCl3, N,N-dimethylaniline, toluene, reflux. (v) Pd(PPh3)4, Na2CO3, THF, reflux.
(R1) of (S)-3 was replaced with a methyl group. The molecular size of
Table 2
SAR exploration of compounds 3–20.
the methyl group was similar to that of the chlorine atom, but the
electronic behavior was opposite, so we prepared and examined the D1/
D2 receptor affinity of compounds 12–17. As shown in Table 1, the
affinity on the D2 receptor showed an opposite trend to the 8-Cl ana-
logues, i.e. the electron-donating groups (13–15) resulted in a loss or
decreased the affinity toward the D2 receptor. Interestingly, the affinity
for the D2 receptor was still demonstrated in medium molecular size
electron-withdrawing groups (17). These results suggested that the
electron-rich benzene ring did not fit well with the D2 receptor. In the
cases of hydrogen atom as the R1 substituent (18–20), similar trends
were seen with those of the 8-Me analogues. The results of compounds
(S)-3 and 20 showed similar trends with those of Clozapine versus
Isoclozapine.18 Among the compounds prepared, both compounds 5
and 7 displayed similar D2/D1 receptor selectivity ratio with Clozapine,
and the potency was approximately 10-fold stronger than that of Clo-
zapine. In CHO cells stably expressing human D1 or D2 receptor, both
compounds 5 and 7 antagonized a dopamine-stimulated Ca2+ accu-
mulation as well as Clozapine. Clozapine is categorized as a serotonine-
dopamine antagonist, and 5HT2A antagonism is also an important me-
chanism of action; Clozapine, compound 5 and 7 showed strong affinity
for the 5HT2A receptor, and Ki values were 1.3 nM, 0.094 nM and
0.091 nM, respectively. These results indicated that the potency of
compound 5, as well as the affinity for dopamine D2/D1 receptor, was
stronger than that of Clozapine by over 10-fold.
Compound R1 R2
Ki (nM)
D2/
D1
5HT2A
hERG
dGSH
(µmol/
L)
(Ki; nM) (IC50
µM)
;
D1
D2
ratio
Clozapine
44
47
51
2.3
1.4
2
389
8.8
1.2
9.4
28
1.4
0.42
nt
3.2
nt
7.5
Olanzapine
58
0.077
1.3
(S)-3
4
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
H
479
nt
Me
Et
63
0.094
0.14
nt
0.8
1.1
1.5
0.9
1.9
1.2
0.9
0.6
nt
0.35
0.53
0.89
0.77
0.58
0.24
0.72
0.49
nt
5
28
20
6
OMe
OEt
OCF3
F
69
35
7
3.7
2
47
13
0.091
0.14
nt
8
110
55
9
0.2
0.9
1.1
nt
127
635
118
123
–
10
11
12
13
14
15
16
17
18
19
20
Cl
106
nt
CN
H
135
nt
> 1,400
> 1,400
> 1,400
1218
453
nt
Me Me
nt
–
nt
nt
nt
Me OMe nt
Me OCF3 2.4
–
nt
nt
nt
508
82
nt
nt
nt
Me
F
5.5
nt
1.6
1.1
nt
0.25
0.38
0.85
0.86
0.67
Me Cl
0.48 98
204
104
42
nt
As shown in Table 2, the amounts of dGSH-conjugation of all test
compounds including compound 5 were lower than that of Clozapine
by about 1/10-fold. This result suggested that our compounds may be
considered to be low risk in term of the agranulocytosis derived from
reactive metabolites. In addition, hERG/D2 selectivity of compound 5
was larger than that of 7; thus, compound 5 may be considered to be a
safer compound from the viewpoint of cardiovascular toxicity, and we
selected compound 5 for future evaluation.
H
H
H
Me
7.9
4
819
nt
Et
Cl
167.5
124
nt
0.6
0.6
1.6
78
nt
nt; not tested.
introduction of the substituent was efficacious in significantly im-
proving the affinity toward the D1 receptor, regardless of the elec-
tronic/steric nature of the substituents. The electron-donating groups
(4–8) showed slightly stronger affinity on the D2 receptor than the
electron-withdrawing groups (9–11), such that the D2/D1 receptor se-
lectivity ratios of compounds 9–11 were much higher than that of
Clozapine. Among the electron-donating groups, compounds 5 and 7
appeared to be preferable profiles in terms of the D2/D1 receptor affi-
nity and selectivity ratio. Next, the chlorine atom at the 8th position
Compound 5, Clozapine, Olanzapine, and Zicronapine were selected
for in vivo D2/D1 receptor occupancy (RO) evaluation. The test com-
pounds were subcutaneously administered to male rats 1 h before ra-
cropride (a D2 antagonist) or SCH-23390 (a D1 antagonist) adminis-
tration. D1 or D2 receptor occupancy in the brain striatum was
evaluated by measuring racropride or SCH-23390 concentration by LC/
MS/MS analysis. The test compounds bound the D1 and D2 receptor
3

H. Watanabe, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127563
Table 3
Declaration of Competing Interest
D2/D1 receptor occupancy profiles of reference drugs and compound 5.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Compound
RO50 plasma free conc. (nM)
D2/D1 ratio D2/D1 ratio (in
RO50
vitro)
D1
D2
Clozapine
73.4
480
24.2
3.8
23
0.33
0.0079
3.5
8.8
1.2
215
20
Appendix A. Supplementary data
Olanzapine
Zicronapine 6.6
5
6
1
0.17
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127563.
dose-dependently, and the corresponding free plasma concentrations of
RO50 and the D2/D1 selectivity ratio are summarized in Table 3. In the
case of Clozapine, RO50 values of D2 and D1 were 24.2 and 73.4 nM,
respectively, and the resulting D2/D1 selectivity ratio was calculated to
be 0.33. Moreover, D2/D1 selectivity ratio of Olanzapine and Zicrona-
pine were calculated to be 0.0079 and 3.5, respectively. The rank order
of the ratio was well consistent with that of the in vitro results. The
selectivity ratio of compound 5 was 0.17, which is close to that of
Clozapine. These results indicated that the in vivo D2/D1 ratio of com-
pound 5 was comparable to that of Clozapine, whereas those of Olan-
zapine and Zicronapine were not.
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