The first structure-activity relationship studies for designer receptors exclusively activated by designer drugs

Article abstract of DOI:10.1021/cn500325v

Over the past decade, two independent technologies have emerged and been widely adopted by the neuroscience community for remotely controlling neuronal activity: optogenetics which utilize engineered channelrhodopsin and other opsins, and chemogenetics which utilize engineered G protein-coupled receptors (Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)) and other orthologous ligand-receptor pairs. Using directed molecular evolution, two types of DREADDs derived from human muscarinic acetylcholine receptors have been developed: hM3Dq which activates neuronal firing, and hM4Di which inhibits neuronal firing. Importantly, these DREADDs were not activated by the native ligand acetylcholine (ACh), but selectively activated by clozapine N-oxide (CNO), a pharmacologically inert ligand. CNO has been used extensively in rodent models to activate DREADDs, and although CNO is not subject to significant metabolic transformation in mice, a small fraction of CNO is apparently metabolized to clozapine in humans and guinea pigs, lessening the translational potential of DREADDs. To effectively translate the DREADD technology, the next generation of DREADD agonists are needed and a thorough understanding of structure-activity relationships (SARs) of DREADDs is required for developing such ligands. We therefore conducted the first SAR studies of hM3Dq. We explored multiple regions of the scaffold represented by CNO, identified interesting SAR trends, and discovered several compounds that are very potent hM3Dq agonists but do not activate the native human M3 receptor (hM3). We also discovered that the approved drug perlapine is a novel hM3Dq agonist with >10000-fold selectivity for hM3Dq over hM3.

Full text of DOI:10.1021/cn500325v

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Article
The First Structure-activity Relationship Studies for Designer
Receptors Exclusively Activated by Designer Drugs (DREADDs)
Xin Chen, Hyunah Choo, Xi-Ping Huang, Xiaobao Yang, Orrin Stone, Bryan L Roth, and Jian Jin
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/cn500325v • Publication Date (Web): 14 Jan 2015
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The First Structure-activity Relationship Studies for Designer
Receptors Exclusively Activated by Designer Drugs (DREADDs)
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‡
,†
§,¶,†
§
‡
§
,§
Xin Chen, Hyunah Choo, Xi-Ping Huang, Xiaobao Yang, Orrin Stone, Bryan L. Roth,*
,
‡
Jian Jin *
‡
Departments of Structural and Chemical Biology, Oncological Sciences, and Pharmacology and
Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York 10029,
United States
§
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, United States
¶
Center for Neuro-Medicine, Korea Institute of Science and Technology, Seongbuk-gu, Seoul
1
36-791, Republic of Korea
RECEIVED DATE (to be automatically inserted after your manuscript is accepted)
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Abstract
Over the past decade, two independent technologies have emerged and been widely adopted by
the neuroscience community for remotely controlling neuronal activity: optogenetics which
utilize engineered channelrhodopsin and other opsins, and chemogenetics which utilize
engineered G protein-coupled receptors (Designer Receptors Exclusively Activated by Designer
Drugs (DREADDs)) and other orthologous ligand-receptor pairs. Using directed molecular
evolution, two types of DREADDs derived from human muscarinic acetylcholine receptors have
been developed: hM3Dq which activates neuronal firing, and hM4Di which inhibits neuronal
firing. Importantly, these DREADDs were not activated by the native ligand acetylcholine, but
selectively activated by clozapine N-oxide (CNO), a pharmacologically inert ligand. CNO has
been used extensively in rodent models to activate DREADDs and although CNO is not subject
to significant metabolic transformation in mice, a small fraction of CNO is apparently
metabolized to clozapine in humans and guinea pigs, lessening the translational potential of
DREADDs. To effectively translate the DREADD technology, the next generation of DREADD
agonists are needed and a thorough understanding of structure – activity relationships (SAR) of
DREADDs is required for developing such ligands. We therefore conducted the first SAR
studies of hM3Dq. We explored multiple regions of the scaffold represented by CNO, identified
interesting SAR trends, and discovered several compounds that are very potent hM3Dq agonists
but do not activate the native human M3 receptor (hM3). We also discovered that the approved
drug perlapine is a novel hM3Dq agonist with >10,000-fold selectivity for hM3Dq over hM3.
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Introduction
To elucidate how neuronal ensembles interactively encode higher brain processes, new
and improved methods for both recording and manipulating neuronal activity will be
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required. The ability to selectively modulate the activity of defined neuronal populations and
to elucidate the behavioral consequences of this selective neuronal modulation affords powerful
approaches for studying mammalian brain function in health and disease. Historically, important
methods include Wilder Penfield's pioneering studies of focal electrical stimulation of the human
3
cortex. The development of the optogenetics technology pioneered by Diesseroth and colleagues
to visualize and activate neuronal activity with exquisite temporal resolution using engineered
4
,5
6
channelrhodopsin and other opsins has provided an expanding toolbox for decoding the
4
, 6-10
neuronal correlates of brain function.
More recently, Designer Receptors Exclusively
Activated by Designer Drugs (DREADDs) have been developed as a powerful chemogenetics
1
1, 12
technology for remotely controlling neuronal activity
and have been widely adopted by the
1
3-18
neuroscience and greater biological communities.
5
DREADDs, first revealed in 2005, were developed using directed molecular evolution of
1
1, 12
human muscarinic acetylcholine receptors.
After multiple rounds of random mutagenesis,
DREADDs derived from the human muscarinic acetylcholine M3 receptors (hM3Dq) to be
insensitive to the endogenous ligand acetylcholine but potently and selectively activated by the
pharmacologically inert clozapine N-oxide (CNO) were discovered. Importantly, CNO lacks
11, 19,
appreciable affinity (K
i
> 1 µM) for all relevant native CNS (central nervous system) targets
2
0
11
The DREADDs have no detectable constitutive activity in vitro and, thus, provide an
attractive orthologous receptor-effector chemogenetic platform for modifying neuronal activity
remotely with minimal invasiveness. In addition to hM3Dq, which activates neuronal firing upon
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the CNO stimulation in part by depolarization and elevation of intracellular calcium levels,
hM4Di was developed from human muscarinic acetylcholine M4 receptors for inhibiting
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neuronal firing via activation of G-protein inwardly rectifying potassium (GIRK) channels.
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Since the introduction of the DREADD technology, a large number of papers have independently
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validated the utility of excitatory and inhibitory DREADDs.
to the ectopic expression of hM3Dq or hM4Di has been observed.
In addition to being pharmacologically inert, CNO, the “chemical switch” of this
In addition, no effect related
3
5, 36, 21, 37
chemogenetic approach, is orally bioavailability and CNS penetrant
and is not subject
to significant metabolic transformation in mice and rats. However, a small fraction of CNO is
3
8-40, 41
apparently metabolized to clozapine in humans, non-human primates and guinea pigs.
4
2
Because clozapine modulates the activity of many native CNS receptors, thus interfering with
the selective activation of the DREADDs in defined neuronal populations, the ‘back-metabolism’
issue presents a hurdle for translating the DREADD technology forward. To ultimately develop
the next generation of DREADD ligands that can selectively activate defined neuronal
populations in primates including human, a thorough understanding of structure – activity
relationships (SAR) of DREADDs is needed. To date, no SAR studies have been reported for
any DREADDs including hM3Dq and hM4Di.
Here, we report the first SAR studies of hM3Dq. We extensively explored multiple
regions of the scaffold represented by CNO, which resulted in the discovery of compounds 13
and 21 that are very potent hM3Dq agonists but do not activate the native human M3 receptor
(
hM3). We describe the design, synthesis, and pharmacological evaluation of new CNO analogs
and discuss the interesting SAR trends revealed from the studies. We also report the discovery
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that perlapine, a hypnotic agent first reported in 1966,
is a novel, potent, and selective
agonist of hM3Dq (> 10,000-fold selective for hM3Dq over hM3).
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Results and Discussion
Design and Synthesis. To understand the SAR of CNO analogs as hM3Dq agonists, we
1
explored several regions of the CNO scaffold. In particular, we focused on investigating the R
2
and R substituents as well as modifications to the piperazine ring (highlighted in red in Fig. 1).
3
We also studied whether the chloro group (R , highlighted in blue in Fig. 1) on the tricyclic core
is required and whether a different tricyclic core (e.g., the core of the perlapine scaffold, see
below) can be tolerated.
Figure 1. SAR studies of the CNO scaffold.
1
We first explored the size of the alkyl group (R ) on the N4’ position of the CNO scaffold
and synthesized a set of close analogs as outlined in Scheme 1. The commercially available 2-
((2-amino-4-chlorophenyl)amino)benzoic acid (1) was refluxed in xylene for 48 hours to give the
3
cyclized compound 2, which was then treated with POCl and N,N-dimethylaniline in toluene at
o
9
5 C for 2 hours to afford the chloride 3. Compound 3 was reacted with various N-
o
alkylpiperazines in toluene at 120 C for 2 hours to yield compounds 4a to 4d, which were then
converted to their corresponding N-oxides 5a to 5d by treating with meta-chloroperoxybenzoic
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acid (mCPBA) at room temperature in CH
2
Cl
2
for 10 minutes. The synthetic route to compounds
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4
a – 4d was described previously.
a
Scheme 1. Synthesis of N4’-alkyl substituted CNO analogs
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Reagents and conditions: a) xylene, reflux, 48 h, 95% yield; b) POCl , N,N-dimethylaniline,
o
o
toluene, 95 C, 2 h, 67% yield; c) N-alkylpiperazines, toluene, 120 C, 2 h, 69 – 80% yield; and d)
mCPBA, CH Cl , rt, 10 min, 65 - 75% yield.
2
2
We next synthesized the analogs outlined in Scheme 2 to determine whether the positive
charge of CNO is required for activating hM3Dq. For example, compound 6, which contains a
quaternary ammonium moiety, has a permanent positive charge while compounds 7, 9, 12, 14
and 15 do not possess a basic amino group, therefore do not contain a positive charged group. As
illustrated in Scheme 2, compound 4a (clozapine) was converted to the quaternary ammonium
3
iodide 6 by stirring overnight with CH I in acetone at room temperature. Compound 3 was
o
treated with piperazin-2-one at 99 C overnight in the 1:1 mixture of 1,4-dioxane and ethanol to
give compound 7. Similarly, compound 9 was produced by treating compound 3 with
commercially available 1,3,8-triazaspiro[4.5]decane-2,4-dione (8) in the 2:1 mixture of 1,4-
o
dioxane and N,N-dimethylformamide (DMF) at 130 C for 24 hours. Hydrolysis of compound 9
using 0.5 N aqueous NaOH solution in 1,2-dimethoxyethane under microwave irradiation
afforded compound 10. Likewise, compound 11 was prepared from compound 3 and piperazine
o
o
2 2
in toluene at 120 C for 2 hours. Acetylation of compound 11 by AcCl in CH Cl at 0 C in the
presence of triethylamine (TEA) yielded compound 12, which was subsequently reduced to the
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deuterated compound 13 using LiAlD under reflux conditions, followed by quenching with
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CD
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OD at 0 C. In addition, compound 11 was reacted with MsCl in CH
2
Cl
2
at 0 C in the
presence of diisopropylethylamine (DIPEA) to give the methylsulfonamide 14.
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a
Scheme 2. Synthesis of compounds 6, 7, and 9 – 14
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a
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Reagents and conditions: a) CH I, acetone, rt, overnight, 55% yield; b) 2-oxypiperazine, 1,4-
o
dioxane/ethanol 1:1, 99 C, overnight, 65% yield; c)ꢀ1,3,8-triazaspiro[4.5]decane-2,4-dione (8),
o
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,4-dioxane/DMF (2:1), 130 C, 24 h, 66% yield; d) 1,2-dimethoxyethane, 0.5 N NaOH,
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, 0 C, 1 h, 86% yield; g) (1) LiAlD
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2 3
, THF, N , reflux, 2h, (2) CD OD, 0 C, (3)
o
o
NH
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OH, 0 C, 84% yield; h) MsCl, DIPEA, CH Cl
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, 0 C, 1 h, 93% yield.
To determine whether the 8-Cl group on the tricyclic core is required to activate hM3Dq,
4
8
we prepared compounds 21 – 23 according to the synthetic route outlined in Scheme 3. The
commercially available 2-aminobenzoic acid (15) and 2-nitrophenyl iodide (16) were subjected
4
9
to Ullmann coupling conditions to afford the aniline 17. Reduction of the nitro moiety of
compound 17 yielded compound 18, which was refluxed in xylene to generate the
3
benzodiazepine 19. Treatment of compound 19 with POCl provided the chloride 20, which was
then displaced with piperazine to afford compound 21. Similarly, compound 22 was prepared by
the displacement reaction of the chloride 20 with 1-ethylpiperazine in toluene under reflux
conditions. In addition, the oxidation of compound 22 by mCPBA in CH
2 2
Cl afforded the N-
oxide 23.
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a
Scheme 3. Synthesis of compounds 22 – 24
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a
Reagents and conditions: a) K
2
2 4
CO3, Cu, 3-methylbutan-1-ol, reflux, 4 h; b) NaS O ,
o
NH
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OH/H
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O 3:2, 80 C, 30 min, 75% yield in two steps; c) xylene, reflux, Dean-Stark
conditions, 48 h, 96% yield; d) POCl
piperazine, toluene, reflux, overnight, 63% yield; f) 1-ethylpiperazine, toluene, reflux, 2 h, 72%
yield; and g) mCPBA, CH Cl , rt, 10 min, 78% yield.
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, N,N-dimethylaniline, toluene, reflux, 3 h, 52% yield; e)
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Biological Evaluation. The newly synthesized compounds were evaluated in the hM3Dq
2
+
TETRA
and hM3 Ca mobilization fluorometric imaging plate reader (FLIPR
) assays according to
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the protocols reported previously.
Agonist activities of these compounds in the hM3Dq
and hM3 functional assays are summarized in Table 1.
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Table 1. Agonist activities of new compounds in hM3Dq and hM3 FLIPR assays.*
hM3Dq
hM3
E
max
Compound
E
max
EC50 (nM)
EC50 (nM)
(relative
acetylcholine)
to
(relative to CNO)
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>30,000
>30,000
>30,000
2.1
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>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
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>30,000
>30,000
>30,000
490
NA
NA
NA
NA
NA
NA
NA
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NA
NA
NA
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b
c
d
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100
NA
NA
NA
95
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>30,000
9.6
>30,000
1.7
1.3
220
NA
86
NA
100
81
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
NA
NA
NA
NA
NA
NA
1
1
2
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1
22
2
3
59
*
EC50 values are the average of at least 2 duplicate experiments with standard deviations (SD) values that
are 3-fold less than the average. NA: not applicable.
For the size of the N-alkyl group in compounds 4a – 4d and 5a – 5d, we observed a clear
trend showing that the longer and/or bulkier the N-alkyl group, the weaker the compounds’
potency for hM3Dq. The replacement of the methyl group in compounds 4a and 5a with the n-
propyl group in compounds 4d and 5d resulted in a potency decrease of approximately 70- and
1
00-fold, respectively. In addition to the loss in potency, the compounds with a longer or bulkier
N-alkyl group (e.g., compounds 4b – 4d and 5b – 5d) in general displayed lower agonist efficacy
for hM3Dq and became partial agonists of hM3Dq rather than full agonists as seen for
compounds 4a and 5a. Interestingly, compounds 4c and 5c, which contain an i-propyl group,
were more potent than compounds 4d and 5d, which contain a n-propyl group, suggesting that
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the length of the N-alkyl group plays a more significant role than the bulkiness of the N-alkyl
group in reducing agonist potency. We were also pleased to find that compounds 4b – 4d and 5b
–
5d did not display any agonist activity (EC50 > 30,000 nM) for the native human M3 receptor
hM3), in contrast to compound 4a (clozapine), which was a hM3 agonist with sub-µM potency.
In addition, compounds 4a – 4d were in general more potent than their corresponding N-oxides
a – 5d at activating hM3Dq, suggesting that the negative charge on the N-oxides is not only not
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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36
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
5
required for activating hM3Dq, but also reduces agonist potency.
The quaternary ammonium salt 6 was an extremely potent full agonist of hM3Dq with an
EC50 value of 69 pM and about 15-fold more potent than compound 4a (clozapine). However,
compound 6 was also a potent full agonist of hM3 (EC50 = 9.5 nM, Emax = 92) even though it
achieves >100-fold higher potency for hM3Dq over hM3. On the other hand, compounds 7, 12,
and 14, which do not contain a basic amino group or a group with permanent positive charge, did
not display any agonist activity for hM3Dq. As expected, these compounds did not activate hM3
either. Taken together, these results suggest that either a basic amino group as in compounds 4a
and 4b or a group with permanent positive charge as in compounds 5a and 6 is required to retain
hM3Dq agonist activity. In addition, compound 9 that contains a hydantoin moiety and
compound 10 that contains an amino acid moiety in this region did not activate hM3Dq and
hM3. On the other hand, compound 11, which is the des-methyl clozapine, showed similar
potency and efficacy for hM3Dq and hM3 as clozapine, suggesting that the N-methyl group is
not required for activating hM3Dq. Interestingly, compound 13, which is a deuterated analog of
compound 4b, exhibited similar potency and efficacy (EC50 = 9.6 nM, Emax = 86%) for hM3Dq
as compounds 4b and 5a (CNO) (Fig. 2). Importantly, compound 13 did not display any agonist
activity for hM3. Because compound 13 contains an α,α-di-deutero ethyl group, it is likely that
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the N-de-alkylation, the major metabolic pathway that converts clozapine to des-methyl
3
9, 40
clozapine,
will be significantly reduced on the basis of the well-documented primary kinetic
5
2
53, 54
isotope effect in similar systems.
0
1
2
3
4
5
6
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8
9
0
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8
9
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2
3
4
5
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7
8
9
0
Figure 2. Compounds 13 and 21 are potent hM3Dq agonists and do not activate hM3 being
similar to compound 5a (CNO). The endogenous ligand acetylcholine (ACh), on the other hand,
is a potent hM3 agonist and does not activate hM3Dq.
We were also pleased to find that the 8-chloro group was not required to maintain high
agonist potency and efficacy for hM3Dq. In particular, compound 21 was a potent full agonist
(EC50 = 1.7 nM, Emax = 100%) of hM3Dq (Fig. 2). In contrast to compound 11, a full hM3
agonist with sub-µM potency, compound 21 did not display any agonist activity for hM3 (EC50
>
30,000 nM). In addition, compound 22 was found to be a potent hM3Dq agonist (EC50 = 1.3 nM,
E
=
max = 81%), which was more potent than the corresponding chloro analog, compound 4b (EC50
7.0 nM, Emax = 91%). On the other hand, the N-oxide 23 (EC50 = 220 nM, Emax = 59%) was
about 10-fold less potent for hM3Dq than compound 5b, the corresponding chloro analog (EC50
19 nM, Emax = 50%). Similar to compound 21, both compounds 22 and 23 did not exhibit any
agonist activity for hM3.
=
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We next selected a subset of the above hM3Dq agonists that are inactive against hM3 and
assessed their binding affinities to other aminergic GPCRs. Because compound 4a (clozapine)
showed high binding affinities to 5HT2A and 5HT2C serotonin, α1A adrenergic, and H
1
histamine
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11
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receptors with K values of 5.4, 9.4, 1.6, and 1.1 nM, respectively (Table 2), we tested
i
1
compounds 4b, 4c, 5b, 5c, 13 and 21 in 5HT2A, 5HT2C, α1A, and H radioligand binding assays.
The assay results are summarized in Table 2.
Table 2. Binding affinities of selected hM3Dq agonists to other GPCRs.*
K
i
(nM)
Compound
5HT2A
5.4
29
5HT2C
9.4
24
H1
α
1.6
46
37
>10,000
320
67
1A
4
4
4
5
a
b
c
1.1
1.9
4.6
160
6200
5.0
6.0
16
17
b
1900
5200
71
5100
6700
280
170
5c
1
2
3
1
66
280
*
K
i
values are the average of at least 2 duplicate experiments with standard deviation (SD) values that are
3-fold less than the average.
Compounds 4b and 4c had reduced binding affinities to 5HT2A, 5HT2C, and α1A (K
46 nM) compared with compound 4a (clozapine), but retained high binding affinities to H
< 5.0 nM). On the other hand, the N-oxide 5b displayed weak binding affinities for 5HT2A
> 1,000 nM) and was about 8-fold selective for hM3Dq over H
oxide 5c displayed poor binding affinities to 5HT2A, 5HT2C, and H (K > 5,000 nM) but was
i
= 16
–
1
(K
i
,
5
HT2C, and α1A (K
i
1
while the N-
1
i
only about 2-fold selective for hM3Dq over α1A. Interestingly, compound 13, a deuterated analog
of compound 4b, exhibited reduced binding affinities to all four receptors compared with
compound 4b. Compound 13 was selective for hM3Dq over 5HT2A (7-fold), 5HT2C (29-fold),
1
and α1A (7-fold), but was not selective over H . We were pleased to find that compound 21
displayed much improved selectivity compared with compound 4a (clozapine). In addition to
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being inactive at hM3, compound 21, a potent full agonist of hM3Dq (EC50 = 1.7 nM), was 40-
fold selective over 5HT2A, 100-fold selective over 5HT2C, and 165-fold selective over α1A
Although it was only 3.5-fold selective for hM3Dq over H , the overall selectivity profile of
compound 21 is significantly better than compound 4a (clozapine).
.
1
0
1
2
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9
0
Lastly, to identify an alternative compound that might activate hM3Dq, we conducted a
screen of the commercially available Library Of Pharmaceutically Active Compounds (LOPAC;
N = 1,280 compounds) and Prestwick Chemical Library (N = 1,280 compounds) using the
hM3Dq FLIPR assay. From this screen, we discovered perlapine as a novel, potent agonist of
hM3Dq (Fig. 3). Importantly, perlapine was > 10,000-fold selective for hM3Dq over hM3.
Interestingly, perlapine contains a different tricyclic core in comparison with CNO. The high
hM3Dq potency of perlapine suggests that the benzodiazepine tricyclic core of the CNO
(compound 5a) scaffold is not required for maintaining high hM3Dq agonist activity.
Figure 3. Perlapine is a potent full agonist of hM3Dq and does not activate hM3. CNO
(compound 5a) was used as a positive control in the hM3Dq FLIPR assay and acetylcholine
(ACh) was used as a positive control in the hM3 FLIPR assay.
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Conclusion
In summary, we conducted the first SAR studies for hM3Dq, a chemogenetic platform
for activating neuronal firing, by the design, synthesis, and pharmacological evaluation of new
CNO analogs. We explored multiple regions of the CNO scaffold and observed the following
interesting SAR trends: (1) a longer or bulkier N-alkyl group as in compounds 4c, 4d, 5c, and 5d
reduces both potency and efficacy for hM3Dq; (2) a basic amino group as in compounds 4a and
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4
b or a permanent positive charge group as in compounds 5a and 6 is required to retain hM3Dq
agonist activity; (3) the negative change on the N-oxides such as 5a – 5d reduces hM3Dq agonist
potency; (4) the 8-chloro group is not required to maintain high agonist potency and efficacy for
hM3Dq; and (5) modifications to the benzodiazepine tricyclic core of CNO is tolerated. From
these SAR studies, we discovered several compounds such as 13 and 21, which are very potent
full agonists of hM3Dq but do not activate the native human M3 receptor (hM3). In addition, the
selectivity of compound 21 against a number of aminergic GPCRs is significantly improved
compared with clozapine. Furthermore, we discovered perlapine as a novel, potent hM3Dq
agonist, which is > 10,000-fold selective for hM3Dq over hM3. These SAR studies lay the
foundation for developing the next generation of DREADD ligands that can selectively activate
defined neuronal populations in primates.
Methods
Chemistry. General Methods. HPLC spectra of all compounds were acquired from an Agilent
6
110 Series system with UV detector set to at 220 nm. Samples were injected (5 µL) onto an
Agilent Eclipse Plus 4.6 x 50 mm, 1.8 µM, C18 column at room temperature. A linear gradient
from 10% to 100% B (MeOH + 0.1% Acetic Acid) in 5.0 min was followed by pumping 100% B
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4
4
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5
5
5
5
5
5
5
5
5
6
for another 2 minutes with A being H
2
O + 0.1% acetic acid. The flow rate was 1.0
mL/min. Mass spectra (MS) data were acquired in positive ion mode using an Agilent 6110
single quadrupole mass spectrometer with an electrospray ionization (ESI) source. HRMS
analysis was conducted on an Agilent Technologies G1969A high-resolution API-TOF mass
spectrometer attached to an Agilent Technologies 1200 HPLC system. Samples were ionized by
electrospray ionization (ESI) in positive mode. Nuclear Magnetic Resonance (NMR) spectra
0
1
2
3
4
5
6
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8
9
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9
0
1
2
3
4
5
6
7
8
9
0
1
were recorded at Varian Mercury spectrometer with 400 MHz for proton ( H NMR) and 100
1
3
MHz for carbon ( C NMR); chemical shifts are reported in ppm (δ). Preparative HPLC was
performed on Agilent Prep 1200 series with UV detector set to at 220 nm. Samples were injected
onto a Phenomenex Luna 75 x 30 mm, 5 µΜ, C18 column at room temperature. The flow rate
2
was 30 mL/min. Different linear gradient for different compounds were used with A being H O
+
0.5% TFA and B being MeOH.
4
6, 47
46, 47
46, 47
46
55, 56
Compounds 2,
3,
4a,
4b, and 5a
were prepared according to the procedures
described previously.
-Chloro-11-(4-isopropylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine (4c)
8
A solution of 8,11-dichloro-5H-dibenzo[b,e][1,4]diazepine (3, 0.397 g, 1.44 mmol) and 1-
o
isopropylpiperazine (1 g, 7.799 mmol) in 1,4-dioxane (20 mL) was stirred overnight at 120 C.
After cooling down, the reaction mixture was concentrated and the residue was dissolved with 50
mL of EtOAc. The resulting solution was washed with 30 mL of aqueous NaHCO
3
. The organic
layer was dried over Na SO , and the filtrate was concentrated and the residue was purified by
2
4
2 2
flash column chromatography with 5-10% MeOH in CH Cl to give the desired product 4c
1
(
0.410 g) in 80% yield: H NMR (400 MHz, CDCl
3
) δ 7.38 – 7.16 (m, 2H), 7.04 (d, J = 1.5 Hz,
1
H), 6.99 (t, J = 7.5 Hz, 1H), 6.88 – 6.72 (m, 2H), 6.58 (d, J = 8.3 Hz, 1H), 4.86 (s, 1H), 3.46
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9
13
(
br, s, 4H), 2.70 (quin, J = 6.5 Hz, 1H), 2.58 (br s, 4H), 1.06 (d, J = 6.5 Hz, 6H); C NMR (101
MHz, , CDCl ) δ 162.84, 152.90, 142.13, 140.59, 132.00, 130.55, 129.24, 126.94, 123.69,
23.18, 123.13, 120.25, 120.15, 54.80 (2C), 48.95 (2C), 47.77, 18.80 (2C); HPLC purity 100%,
3
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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33
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39
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
+
+
RT 4.099 min; MS (ESI) 355.2 [M+H] ; HRMS (ESI) calcd for C20
4
H24ClN [M+H] :ꢀ355.1689,
Found: 355.1693.
8
-Chloro-11-(4-propylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine (4d)
Compound 4d (0.380 g, 70% yield) was prepared via the same procedure as preparing compound
4c from 8,11-dichloro-5H-dibenzo[b,e][1,4]diazepine (3, 0.400 g, 1.52 mmol), 1-
propylpiperazine HCl salt (0.500 g, 3.04 mmol), and DIPEA (2 mL, 11.48 mmol) in 1,4-dioxane
1
(
20 mL). H NMR (400 MHz, CDCl
3
) δ 7.31 – 7.19 (m, 2H), 7.04 (d, J = 2.3 Hz, 1H), 6.99 (t, J
= 7.5 Hz, 1H), 6.79 (dd, J = 8.2, 2.0 Hz, 2H), 6.58 (d, J = 8.3 Hz, 1H), 4.86 (s, 1H), 3.45 (br s,
4
3
H), 2.50 (br s, 4H), 2.33 (t, J = 7.6 Hz, 2H), 1.52 (sixtet, J = 7.6 Hz, 2H), 0.90 (t, J = 7.3 Hz,
1
3
3
H); C NMR (101 MHz, CDCl ) δ 162.93, 152.90, 142.08, 140.59, 132.03, 130.52, 129.25,
126.96, 123.69, 123.21, 123.20, 120.26, 120.16, 60.93 (2C), 53.40 (2C), 47.47, 20.21, 12.18;
+
HPLC purity 100%, RT 4.113 min; MS (ESI) 355.2 [M+H] ; HRMS (ESI) calcd for
+
+
C
20
H24ClN
4
[M+H] :ꢀ355.1689, Found: 355.1687.
-(8-Chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)-1-ethylpiperazine N-oxide (5b)
A solution of compound 4b (0.100 g, 0.293 mmol) in CH Cl (5 mL) was treated with mCPBA
0.064 g, 0.371 mmol) at room temperature. After 10 min, the reaction was completed. The
resulting mixture was concentrated and purified by flash column chromatography with 5-15% C
4
2
2
(
(
2% NH
4
OH in MeOH) in CH
2
Cl
2
to give the desired N-oxide compound 5b (0.073 g,) in 70%
) δ 7.39 – 7.34 (m, 1H), 7.32 (dd, J = 7.8, 1.2 Hz, 1H), 7.05
1
yield: H NMR (400 MHz, MeOH-d
4
(
t, J = 7.5 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.87 (dd, J = 8.4, 2.4 Hz,
1
H), 6.81 (d, J = 8.4 Hz, 1H), 3.88 (br s, 2H), 3.74 (t, J = 12.1 Hz, 2H), 3.55 – 3.43 (m, 2H),
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
3
.367 (q, J = 7.2 Hz, 2H), 3.19-3.09 (m, 2H), 1.38 (t, J = 7.2 Hz, 3H); C NMR (101 MHz,
CDCl ) δ 162.06, 153.18, 141.54, 140.62, 132.57, 130.29, 129.35, 126.98, 123.90, 123.56,
22.96, 120.45, 120.43, 66.73 (2C), 63.23 (2C), 42.23, 7.69; HPLC purity 100%, RT 4.394 min;
3
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
MS (ESI) 357.2 [M+H] ; HRMS (ESI) calcd for C19
4
H22ClN O [M+H] :ꢀ 357.1482, Found:
357.1481.
4
-(8-Chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)-1-isopropylpiperazine
N-oxide
(5c)
Compound 5c (0.068 g, 65% yield) was prepared similarly as 5b from compound 4c (0.100 g,
1
0.282 mmol) and mCPBA (0.063 g, 0.365 mmol) in CH
2
Cl
2
(5 mL). H NMR (400 MHz,
MeOH-d
4
) δ 7.39 – 7.30 (m, 2H), 7.05 (t, J = 7.5 Hz, 1H), 7.01 (d, J = 7.9 Hz, 1H), 6.97 (d, J =
2
1
.3 Hz, 1H), 6.87 (dd, J = 8.4, 2.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 3.90 (br s, 2H), 3.73 (t, J =
1
3
1.9 Hz, 1H), 3.57 – 3.40 (m, 3H), 3.19-3.12 (m, 2H), 1.38 (d, J = 6.5 Hz, 3H); C NMR (101
) δ 162.02, 153.15, 141.64, 140.61, 132.51, 130.31, 129.38, 126.97, 123.81, 123.55,
23.01, 120.43, 120.39, 70.99 (2C), 60.41 (2C), 42.27, 16.54 (2C); HPLC purity 100%, RT
MHz, CDCl
3
1
4
+
+
+
4
.305 min; MS (ESI) 371.2 [M+H] ; HRMS (ESI) calcd for C20H24ClN O [M+H] :ꢀ371.1639,
Found: 371.1642.
4
-(8-Chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)-1-propylpiperazine N-oxide (5d)
Compound 5d (0.075 g, 72% yield) was prepared using the same procedure as preparing 5b from
1
compound 4d (0.100 g, 0.282 mmol) and mCPBA (0.063 g, 0.365 mmol) in CH
NMR (400 MHz, MeOH-d
7.5 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.87 (dd, J = 8.4, 2.4 Hz, 1H),
2
Cl
2
(5 mL) H
4
) δ 7.39 – 7.34 (m, 1H), 7.32 (dd, J = 7.8, 1.2 Hz, 1H), 7.05 (t, J =
6
3
.81 (d, J = 8.4 Hz, 1H), 3.86 (br s, 2H), 3.74 (t, J = 11.9 Hz, 3H), 3.57 – 3.44 (m, 2H), 3.29 –
1
3
.21 (m, 2H), 3.20-3.10 (m, 2H), 2.00 – 1.84 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H); C NMR (101
MHz, MeOH-d
4
) δ 164.31, 155.62, 143.36, 142.96, 133.85, 131.29, 129.72, 127.45, 124.99,
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24.29, 124.04, 121.68, 121.53, 73.69 (2C), 64.26 (2C), 43.23, 16.42, 11.37; HPLC purity 100%,
+
+
+
4
RT 4.439 min; MS (ESI) 371.2 [M+H] ; HRMS (ESI) calcd for C20H24ClN O [M+H] :ꢀ
3
71.1639, Found: 371.1640.
-(8-Chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)-1,1-dimethylpiperazin-1-ium iodide (6)
10
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60
4
A solution of 4a (0.100 g, 0.306 mmol) in acetone (5 mL) was treated with methyl iodide (21
µL, 0.337 mmol) at room temperature. After overnight, the resulting mixture was concentrated,
dissolved in 1 mL of MeOH, and diluted with 10 mL of distilled water. The aqueous solution
was washed with EtOAc (2 X 10 mL) and concentrated. The residue was purified by preparative
1
HPLC to give the compound 6 (0.079 g) in 55% yield: H NMR (400 MHz, MeOH-d
4
) δ 7.57 (t,
J = 7.7 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 2.3 Hz, 1H), 7.27-7.13 (m, 3H), 7.00 (d, J
1
3
=
4
8.5 Hz, 1H), 4.03 (br s, 4H), 3.72 (br s, 4H), 3.33 (s, 6H); C NMR (101 MHz, MeOH-d ) δ
1
1
66.47, 156.55, 145.82, 136.58, 135.26, 132.80, 130.24, 128.46, 126.86, 125.08, 122.75, 122.70,
19.64, 61.86 (2C), 52.46 (2C), 44.44 (2C); HPLC purity 100%, RT 3.893 min; MS (ESI) 341.2
+
[
M+H] .
4
-(8-Chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)piperazin-2-one (7)
A solution of compound 3 (0.200 g, 0.760 mmol) and 2-oxypiperazine (0.152 g, 1.52 mmol) in a
o
1
:1 mixture of 1,4-dioxane and ethanol (15 mL) was stirred overnight at 99 C. After
concentration, the residue was diluted with EtOAc (50 mL) and the solution was washed with 20
mL of aqueous NaHCO . The organic layer was dried over Na SO and the filtrate was
3
2
4
concentrated. The residue was purified by flash column chromatography with 30-50% EtOAc in
1
hexanes to afford the desired product (0.162 g) in 65% yield: H NMR (400 MHz, CDCl
3
) δ 7.30
(t, J = 7.6 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.05 (s, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.84 (d, J =
8
2
.3 Hz, 1H), 6.81 (d, J = 7.9 Hz, 1H), 6.60 (d, J = 8.3 Hz, 1H), 6.03 (s, 1H), 4.90 (s, 1H), 4.10 (s,
1
3
3
H), 3.67 (br s, 2H), 3.50 (br s, 2H); C NMR (101 MHz, CDCl ) δ 168.89, 161.84, 153.15,
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41.39, 140.49, 132.63, 130.04, 129.37, 127.19, 124.02, 123.52, 122.99, 120.48, 120.39, 51.47,
+
4
4.12, 41.15; HPLC purity 100%, RT 4.681 min; MS (ESI) 327.1 [M+H] ; HRMS (ESI) calcd
+
+
4
for C17H16ClN O [M+H] :ꢀ327.1007, Found: 327.1006.
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8
9
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1
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8
9
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9
0
8
-(8-Chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)-1,3,8-triazaspiro[4.5]decane-2,4-dione (9)
A mixture of compound 3 (0.250 g, 0.951 mmol) and commercially available compound 8 (0.200
o
g, 1.18 mmol) in 20 mL of a mixture of 1,4-dioxane and DMF (2:1) was heated to 130 C for 24
h. The reaction mixture was cooled down to room temperature and concentrated. The residue
2 2
was purified by flash column chromatography with 0-10% MeOH in CH Cl to give the desired
1
product 9 (0.250 g) in 66% yield: H NMR (400 MHz, CDCl
3
) δ 8.62 (s, 1H), 7.37 – 7.17 (m,
2
H), 7.10 – 7.03 (m, 2H), 7.01 (t, J = 7.5 Hz, 1H), 6.85 – 6.76 (m, 2H), 6.62 (d, J = 8.3 Hz, 1H),
1
3
5.06 (s, 1H), 3.90 (br s, 2H), 3.20 (br s, 2H), 2.18 – 2.05 (m, 2H), 1.74 (s, 2H); C NMR (101
MHz, CDCl ) δ 176.84, 163.26, 156.74, 152.90, 141.62, 140.70, 132.47, 130.29, 129.42, 126.97,
23.82, 123.59, 123.43, 120.48, 62.15 (2C), 43.28, 33.19 (2C); HPLC purity 100%, RT 4.289
3
1
+
+
+
5 2
min; MS (ESI) 396.1 [M+H] ; HRMS (ESI) calcd for C20H19ClN O [M+H] :ꢀ396.1222, Found:
3
96.1221.
-Amino-1-(8-chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)piperidine-4-carboxylic acid (10)
4
A solution of compound 9 (0.100 g, 0.253 mmol) in 1,2-dimethoxyethane (5 mL) was treated
with 5 mL of 0.5 N NaOH at room temperature. The resulting mixture was heated under
o
microwave for 10 min (max power 100 W, max temperature 150 C, max pressure 17.0 bar).
After cooling down to room temperature, the reaction mixture was quenched with 10% citric
acid and filtered. The filtrated was purified with preparative HPLC to afford the desired product
1
(0.015 g) in 16% yield: H NMR (400 MHz, MeOH-d
4
) δ 7.63 (t, J = 7.7 Hz, 1H), 7.54 (d, J =
8
3
.0 Hz, 1H), 7.34 (d, J = 19.4 Hz, 1H), 7.25 (t, J = 7.9 Hz, 3H), 7.08 (d, J = 8.6 Hz, 1H), 4.36-
.59 (m, 4H), 2.70-2.40 (m, 2H), 2.40-2.01 (m, 2H); HPLC purity 100%, RT 3.585 min; MS
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+
+
+
(
ESI) 371.2 [M+H] ; HRMS (ESI) calcd for C19
71.1264.
Compound 11 was prepared according to the previously published procedures.
-(4-(8-Chloro-5H-dibenzo[b,e][1,4]diazepin-11-yl)piperazin-1-yl)ethanone (12)
To the solution of compound 10 (0.400 g, 1.28 mmol) and TEA (0.27 mL, 2.0 mmol) in CH
H
20ClN
4
O
2
[M+H] :ꢀ 371.1269, Found:
3
4
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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33
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46
47
48
49
50
51
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53
54
55
56
57
58
59
60
1
2
Cl
2
o
o
was added AcCl (0.10 mL, 1.4 mmol) at 0 C. The resulting mixture was then stirred at 0 C for 1
h. After removing the solvents, the residue was purified by flash column chromatography with 0-
1
5% MeOH in CH
400 MHz, CDCl
.02 (td, J = 7.6, 1.0 Hz, 1H), 6.86 – 6.77 (m, J = 2.4 Hz, 2H), 6.60 (d, J = 8.3 Hz, 1H), 4.89 (s,
2
Cl
2
to give the desired product 12 (0.390 g, 1.10 mmol) in 86% yield: H NMR
(
3
) δ 7.30 (td, J = 7.7, 1.5 Hz, 1H), 7.26 – 7.22 (m, 1H), 7.04 (d, J = 2.4 Hz, 1H),
7
1
3
1H), 3.67 (br s, 2H), 3.59 – 3.46 (m, 4H), 3.34 (br s, 2H), 2.11 (s, J = 11.5 Hz, 3H); C NMR
101 MHz, CDCl ) δ 169.41, 162.90, 153.15, 141.57, 140.65, 132.40, 130.20, 129.25, 126.97,
23.75, 123.39, 123.32, 120.42, 120.34, 47.80, 47.26, 46.13, 41.50, 21.62; HPLC purity 100%,
(
3
1
+
+
+
4
RT 4.919 min; MS (ESI) 355.2 [M+H] ; HRMS (ESI) calcd for C19H20ClN O [M+H] :ꢀ
3
55.1320, Found: 355.1315.
-Chloro-11-[4-(1,1-dideutrioethyl)piperazin-1-yl]-5H-dibenzo[b,e][1,4]diazepine (13)
8
To the solution of compound 12 (0.100 g, 0.282 mmol) in 15 mL of anhydrous THF was added
LiAlD (0.024 g, 0.572 mmol) at room temperature under N atmosphere. The reaction mixture
was heated under reflux conditions for 2 h. The reaction was quenched with 0.1 mL of CD OD at
OH at 0 C and filtered through celite
4
2
3
o
o
0 C. The resulting mixture was treated with 0.5 mL of NH
4
and the filtrate was concentrated. The residue was purified by flash column chromatography with
1
0
-10% MeOH in CH
MHz, CDCl
dd, J = 8.3, 2.4 Hz, 2H), 6.60 (d, J = 8.3 Hz, 1H), 4.88 (s, 1H), 3.49 (br, s, 4H), 2.54 (br, s, 4H),
2
Cl
2
to give the desired product 13 (0.081 g) in 84% yield: H NMR (400
3
) δ 7.33 – 7.23 (m, 3H), 7.06 (d, J = 2.4 Hz, 1H), 7.01 (td, J = 7.6, 1.0 Hz, 1H), 6.81
(
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2
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13
1
1
3
.10 (s, 3H); C NMR (101 MHz, CDCl ) δ 162.90, 152.87, 142.03, 140.59, 132.02, 130.46,
29.17, 126.91, 123.60, 123.17, 120.24, 120.16, 52.87 (2C), 51.78 (q, J = 20.0 Hz), 47.44, 11.86
+
(
2C); HPLC purity 100%, RT 4.072 min; MS (ESI) 343.2 [M+1] ; HRMS (ESI) calcd for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
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7
8
9
0
1
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7
8
9
0
1
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3
4
5
6
7
8
9
0
+
+
2 4
C19H20D ClN [M+H] :ꢀ343.1653, Found: 343.1653.
8-Chloro-11-(4-(methylsulfonyl)piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine (14)
To a solution of compound 11 (0.102 g, 0.326 mmol) and DIPEA (87 µl, 0.907 mmol) in 10 mL
o
of CH
The reaction mixture was diluted with 50 mL of CH
NaHCO . The organic layer was dried over Na SO , and the filtrate was concentrated and the
2
Cl
2
, MsCl (27.8 µl, 0.359 mmol) was added at 0 C. After 1 h, the reaction was completed.
2
2
Cl and washed with 10 mL of aqueous
3
2
4
residue was purified by flash column chromatography with 50% EtOAc in hexanes to give the
1
desired product 14 (0.118 g) in 93% yield: H NMR (400 MHz, CDCl
3
) δ 7.31 (t, J = 7.7 Hz,
1
4
1
1
H), 7.22 (d, J = 7.8 Hz, 1H), 7.08 – 6.96 (m, 2H), 6.87 – 6.78 (m, 2H), 6.61 (d, J = 8.3 Hz, 1H),
1
3
6
.90 (s, 1H), 3.57 (br s, 4H), 3.29 (br s, 4H), 2.79 (s, 3H); C NMR (101 MHz, DMSO-d ) δ
62.44, 154.11, 142.23, 141.48, 132.24, 129.80, 126.69, 125.58, 122.88, 122.64, 122.40, 120.69,
20.40, 46.33, 45.14 (2C), 33.85 (2C); HPLC purity 100%, RT 4.752 min; MS (ESI) 391.1
+
+
+
[M+H] ; HRMS (ESI) calcd for C18
4 2
H20ClN O S [M+H] :ꢀ391.0990, Found: 391.0994.
Compounds 17, 18, 19, 20, and 21 were prepared according to the previously reported
4
8
procedures.
1
11-(Piperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine (21) H NMR (400 MHz, MeOH-d4) δ
7
2
1
.39 – 7.24 (m, 1H), 7.06 – 6.95 (m, 3H), 6.94 – 6.82 (m, 3H), 3.34 (br s, 1H), 2.96 – 2.85 (m,
1
3
H); C NMR (101 MHz, MeOH-d4) δ 165.39, 156.04, 145.04, 141.55, 133.31, 131.47, 127.85,
25.34, 124.88, 124.66, 123.78, 121.26, 120.71, 46.47; HPLC purity 99%, RT 2.772 min; MS
+
+
+
(
19 4
ESI) 279.2 [M+1] ; HRMS (ESI) calcd for C17H N [M+H] :ꢀ279.1604, Found: 279.1610.
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1-(4-Ethylpiperazin-1-yl)-5H-dibenzo[b,e][1,4]diazepine (22)
A solution of compound 20 (0.420 g, 1.84 mmol) and 1-ethylpiperazine (1.5 mL, 11.81 mmol) in
toluene (20 mL) was heated under reflux conditions for 2 h. After cooling down to room
10
11
12
13
14
15
16
17
18
19
20
21
22
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43
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47
48
49
50
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53
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59
60
temperature and concentration, the residue was purified by flash column chromatography with 0-
1
1
0% MeOH in CH
MHz, CDCl
J = 7.3, 3.7 Hz, 1H), 6.80 (dd, J = 7.4, 1.1 Hz, 1H), 6.67 (dd, J = 7.7, 1.4 Hz, 1H), 4.89 (s, 1H),
2
Cl
2
to give the desired product 22 (0.409 g) in 72% yield: H NMR (400
3
) δ 7.30 – 7.18 (m, 2H), 7.06 (dd, J = 7.8, 1.5 Hz, 1H), 7.00 – 6.90 (m, 2H), 6.84 (tt,
1
3
3.45 (br s, 4H), 2.53 (br s, 4H), 2.46 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); C NMR (101
MHz, CDCl ) δ 162.52, 153.35, 141.97, 140.73, 131.82, 130.54, 127.40, 124.46, 123.86, 123.82,
22.93, 120.16, 119.48, 53.06 (2C), 52.65 (2C), 47.56, 12.18; HPLC purity 100%, RT 3.122
3
1
+
+
+
23 4
min; MS (ESI) 307.2 [M+H] ; HRMS (ESI) calcd for C19H N [M+H] :ꢀ 307.1917, Found:
3
07.1918.
-(5H-Dibenzo[b,e][1,4]diazepin-11-yl)-1-ethylpiperazine N-oxide (23)
Cl (10 mL) was treated with mCPBA
0.069 g) at room temperature. After 10 min, the reaction mixture was concentrated and the
4
A solution of compound 22 (0.095 g, 0.31 mmol) in CH
2
2
(
residue was purified with 0-10% C (5% NH
4
OH in MeOH) in CH
2 2
Cl to afford the desired
1
product 23 (0.078 g) in 78% yield: H NMR (400 MHz, MeOH-d
4
) δ 7.39 – 7.29 (m, 2H), 7.07 –
6
3
.97 (m, 3H), 6.96 – 6.82 (m, 3H), 3.84 (br s, 2H), 3.79 – 3.66 (m, 2H), 3.56 – 3.43 (m, 2H),
1
3
.35 (q, J = 7.0 Hz, 2H), 3.18 – 3.07 (m, 2H), 1.37 (t, J = 7.1 Hz, 3H); C NMR (101 MHz,
) δ 163.66, 155.93, 144.50, 141.44, 133.64, 131.23, 127.98, 125.69, 125.05, 124.16,
24.13, 121.44, 120.80, 66.90 (2C), 63.79 (2C), 43.32, 7.70; HPLC purity 100%, RT 3.312 min;
MeOH-d
4
1
+
+
+
MS (ESI) 323.2 [M+H] ; HRMS (ESI) calcd for C19
23 4
H N O [M+H] :ꢀ 323.1866, Found:
3
23.1863.
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Biological Assays. hM3Dq and hM3 FLIPR assays were performed according to the protocols
1
1, 50, 51
reported previously.
1
Protocols for 5-HT2A, 5-HT2C, α1A and H radioligand binding assays
are available at the National Institute of Mental Health – Psychoactive Drug Screening Program
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website (http://pdsp.med.unc.edu/UNC−CH%20Protocol%20Book.pdf).
AUTHOR INFORMATION
Corresponding Authors: *J.J.: Phone:ꢀ 212-659-8699. E-mail:ꢀ jian.jin@mssm.edu. B.L.R.:
Phone:ꢀ919-966-7535. E-mail:ꢀbryan_roth@med.unc.edu
Author Contributions: X.C., H.C., X.P.H., X.Y., and O.S. performed the experiments. J.J. and
B.L.R. designed the studies. J.J., B.L.R., and X.C. wrote the manuscript.
†
These authors contributed equally to this work.
Funding: This work was supported by the NIH grant U01MH105892 (to B.L.R. and J.J.).
Notes: The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. H. Ümit Kaniskan for checking synthetic procedures and NMR spectra of novel
compounds.
REFERENCES
1
. Sternson, S. M., and Roth, B. L. (2014) Chemogenetic Tools to Interrogate Brain Functions,
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. Urban, D. J., and Roth, B. L. (2015) DREADDs (Designer Receptors Exclusively Activated
by Designer Drugs): Chemogenetic Tools with Therapeutic Utility, Annu. Rev.
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. Armbruster, B. N., and Roth, B. L. (2005) Mining the receptorome, J. Biol. Chem. 280, 5129-
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