N-heterocyclic (4-phenylpiperazin-1-yl)methanones derived from phenoxazine and phenothiazine as highly potent inhibitors of tubulin polymerization

Article abstract of DOI:10.1021/acs.jmedchem.6b01591

We report here a series of 27 10-(4-phenylpiperazin-1-yl)methanones derived from tricyclic heterocycles which were screened for effects on tumor cell growth, inhibition of tubulin polymerization, and induction of cell cycle arrest. Several analogues, among them the 10-(4-(3-methoxyphenyl)piperazine-1-carbonyl)-10H-phenoxazine-3-carbonitrile (16o), showed excellent antiproliferative properties, with low nanomolar GI₅₀ values (16o, mean GI₅₀ of 3.3 nM) against a large number (93) of cancer cell lines. Fifteen compounds potently inhibited tubulin polymerization. Analysis of cell cycle by flow cytometry revealed that inhibition of tumor cell growth was related to an induction of G2/M phase cell cycle blockade. Western blotting and molecular docking studies suggested that these compounds bind efficiently to β-tubulin at the colchicine binding site. Our studies demonstrate the suitability of the phenoxazine and phenothiazine core and also of the phenylpiperazine moiety for the development of novel and potent tubulin polymerization inhibitors.

Full text of DOI:10.1021/acs.jmedchem.6b01591

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Article
N-Heterocyclic (4-Phenylpiperazin-1-yl)methanones Derived from Phenoxazine
and Phenothiazine as Highly Potent Inhibitors of Tubulin Polymerization
Helge Prinz, Ann-Kathrin Ridder, Kirsten Vogel, Konrad Joachim Böhm,
Igor Ivanov, Jahan B. Ghasemi, Elham Aghaee, and Klaus Müller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01591 • Publication Date (Web): 03 Jan 2017
Downloaded from http://pubs.acs.org on January 3, 2017
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NꢀHeterocyclic (4ꢀPhenylpiperazinꢀ1ꢀyl)methanones Derived from
Phenoxazine and Phenothiazine as Highly Potent Inhibitors of
Tubulin Polymerization
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#
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Helge Prinz* , AnnꢀKathrin Ridder , Kirsten Vogel , Konrad J. Böhm , Igor Ivanov , Jahan
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B. Ghasemi , Elham Aghaee , Klaus Müller
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Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-University,
ƒ
Corrensstraße 48, D-48149 Münster, Germany, Leibniz Institute on Aging – Fritz Lipmann
‡
Institute (FLI), Beutenbergstrasse 11, D-07745 Jena, Germany, Oncolead GmbH & Co. KG,
§
Zugspitzstraße 5, D-85757 Karlsfeld, Germany, Drug Design in Silico Lab, Chemistry
Faculty, School of Sciences, University of Tehran, Teheran, Iran
#
Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelmsꢀ
University
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Leibniz Institute on Aging – Fritz Lipmann Institute (FLI)
‡
Oncolead GmbH & Co. KG
§
Drug Design in Silico Lab, University of Tehran
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Abstract
We report here a series of twentyꢀseven 10ꢀ(4ꢀphenylpiperazinꢀ1ꢀyl)methanones derived from
tricyclic heterocycles which were screened for effects on tumor cell growth, inhibition of
tubulin polymerization, and induction of cell cycle arrest. Several analogs, among them the
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0ꢀ(4ꢀ(3ꢀmethoxyphenyl)piperazineꢀ1ꢀcarbonyl)ꢀ10Hꢀphenoxazineꢀ3ꢀcarbonitrile
(16o),
showed excellent antiproliferative properties, with low nanomolar GI50 values (16o, mean
GI of 3.3 nM) against a large number (93) of cancer cell lines. Fifteen compounds potently
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inhibited tubulin polymerization. Analysis of cell cycle by flow cytometry revealed that
inhibition of tumor cell growth was related to an induction of G2/M phase cell cycle
blockade. Western blotting and molecular docking studies suggested that these compounds
bind efficiently to βꢀtubulin at the colchicine binding site. Our studies demonstrate the
suitability of the phenoxazine and phenothiazine core and also of the phenylpiperazine moiety
for the development of novel and potent tubulin polymerization inhibitors.
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Introduction
Microtubules are key components of the cytoskeleton and play vital roles in essential
eukaryotic cellular processes, such as organizing the spatial distribution of organelles
throughout interphase, cell motility, vesicle transport and segregation of chromosomes during
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cell division. Cancer cells grow and divide in an uncontrolled manner. Assembly of tubulin
leads to the formation of the mitotic spindle apparatus, which is one of the most important
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targets in cancer chemotherapy. The encouraging prospects of microtubuleꢀtargeted drugs
for anticancer therapy stimulated intensive investigations aimed at the development of novel
smallꢀmolecular tubulin binders by an integrative approach. This is not only based on the
largeꢀscale screening of compound libraries but also on the identification of active ingredients
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ꢀ6
from natural materials, such as plant extracts, marine sources or traditional remedies. Many
cytotoxic agents exert their effects by inhibition of tubulin polymerization or by stabilizing
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microtubules. As a result, cell division is inhibited by arresting cells specifically in the
G2/M phase of the cell cycle, leading to cell death and the induction of apoptosis.
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Tricyclic heterocycles are important scaffolds for a variety of different drugs. The most
common tricyclic ring is the phenothiazine (1a) as the core structure of the phenothiazin
antipsychotics, with chlorpromazine (2) being the most prominent representative.
Phenothiazines (1a, Chart 1) are related to a wide variety of pharmacological effects, such as
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psychotropic, anticancer, antihelminthic and other properties. We recently reported the
discovery of Nꢀbenzoylated phenothiazineꢀ and phenoxazine (3a) derivatives as tubulin
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polymerization inhibitors with potent in vitro antitumor activities. Within the series, we
identified 4 (Chart 1) as a highly active analogue. In continuation of this work, we here focus
on the synthesis and biological testing for (4ꢀphenylpiperazinꢀ1ꢀyl)methanones derived from
phenothiazine, phenoxazine and structurally related analogs. The arylpiperazine scaffold has
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been classified as a privileged structure for lead discovery and optimization and functions as
an important and often recurring cyclic component of numerous drugs across a wide spectrum
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of pharmacological properties.
Findings from recent literature prove the importance of the
phenylpiperazine structural element for the development of tubulin polymerization inhibitors.
About a decade ago, a series of ketopiperazides prepared from arylꢀ or heteroaryl carboxylic
acids and diverse piperazines were described as potent, smallꢀmolecule tubulin
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polymerization inhibitors (5, Chart 2).
It is only recently that Ishii et al. revealed the
phenylpiperazineꢀbased α1ꢀadrenoceptor (AR) antagonist naftopidil (6) as a tubulinꢀbinding
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drug. Also, chlorophenylpiperazine AK301 (7) has been identified as an inhibitor of tubulin
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polymerization and an effective sensitizer of cancer cells to apoptotic ligands. Furthermore,
(E)ꢀ3ꢀ(3,4ꢀdihydroxyphenyl)acrylylpiperazine derivatives as exemplified by 8 have been
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described by Yin et al. as potent tubulin polymerization inhibitors.
Inspired by the above literature findings and on basis of the encouraging data set obtained
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with our Nꢀbenzoylated tubulin polymerization inhibitors, we considered the synthesis and
evaluation of phenothiazine– and phenoxazine–derived (4ꢀphenylpiperazinꢀ1ꢀyl)methanones
particularly interesting. The most active compounds of this work exerted strong growth
inhibitory potencies in the low submicromolar range across a wide range of tumor cell lines,
closely related to potent inhibition of tubulin polymerization and concentrationꢀdependent
induction of cell cycle arrest. Notably, SAR results were distinct from our previously
characterized Nꢀbenzoylated analogs. The most potent of the newly synthesized compounds
had effects comparable or superior to those of known inhibitors, such as nocodazole,
podophyllotoxin and colchicine.
Chemistry
The Nꢀheterocyclic (4ꢀphenylpiperazinꢀ1ꢀyl)methanones were prepared according to the
method depicted in Scheme 1. Phenothiazines 1a and 1b, phenoxazines 3aꢀ3d, 1ꢀ
phenylpiperazines, 5Hꢀdibenzo[b,f]azepine 9, 10,11ꢀdihydroꢀ5Hꢀdibenz[b,f]azepine 10,
acridone 11, diphenylamine 12, 2ꢀphenoxyaniline 13, anilines, and benzylamine precursors
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for 22aꢀ22b were purchased or synthesized according to literature methods. The starting 2ꢀ
chloroꢀ10Hꢀphenoxazine 3b and 2,8ꢀdichloroꢀ10Hꢀphenoxazine 3c were prepared in five
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steps starting via a 2ꢀaminoꢀ2´ꢀchlorodiaryl ether according to literature procedures. The
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0Hꢀphenoxazineꢀ3ꢀcarbonitrile 3d was prepared as described by Eastmond et al. In general,
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synthesis of various intermediate carbamoyl chlorides 14 was accomplished without isolating
them by chlorocarbonylation of Nꢀheterocycles or secondary amines with bis(trichloromethyl)
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carbonate in 1,2ꢀdichloroethane and pyridine, as illustrated in Scheme 1. Then, the desired
4ꢀphenylpiperazinꢀ1ꢀyl)methanones 15aꢀ15h, 16aꢀ16p, 17aꢀ17b and 18 were successfully
(
obtained by carbamoylation of the appropriate 1ꢀphenylpiperazines. In a similar fashion, Nꢀ
phenylꢀ10Hꢀphenoxazineꢀ10ꢀcarboxamide 21 as well as Nꢀbenzylꢀ10Hꢀphenoxazineꢀ10ꢀ
carboxamides 22aꢀ22b (Scheme 1) were prepared by reaction of phenoxazineꢀderived
carbamoyl chlorides with diverse anilines or benzylamines, respectively. The synthesis of 1ꢀ
(
10Hꢀphenoxazinꢀ10ꢀyl)ꢀ2ꢀ(4ꢀphenylpiperazinꢀ1ꢀyl)ethanꢀ1ꢀones 24aꢀ24b (Scheme 2) was
accomplished via the 2ꢀchloroꢀ1ꢀ(10Hꢀphenoxazinꢀ10ꢀyl)ethanꢀ1ꢀone (23), which was
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obtained by initial chloroacylation of 3a with chloroacetyl chloride under reflux in toluene,
and subsequent reaction with 1ꢀ(methoxyphenyl)piperazines. Despite a promising literature
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protocol, several attempts to further modify the tricyclic heterocycle by carbamoylation of
acridone 11 did not give any desired 10ꢀ(4ꢀphenylpiperazineꢀ1ꢀcarbonyl)acridinꢀ9(10H)ꢀone
such as 26 (Scheme 3). Repeated attempts clearly revealed the formation of
phenylpiperazinylacridines 27aꢀ27f instead. In this context, acylation of acridone to yield 10ꢀ
benzoylacridinꢀ9(10H)ꢀone 25, also failed (Scheme 3). Reaction of acridone 11 with benzoyl
chloride gave the phenolic ester 28 instead of amide 25.
Biological Results and Discussion
In Vitro Cell Growth Inhibition Assay
We preliminarily evaluated the cytotoxicity of the compounds towards K562 cells (human
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chronic myelogenous leukemia, DSMZ ACCꢀ10). After 48 h of treatment, cell proliferation
was quantified by counting the cells using a Neubauer hemocytometer. We routinely use this
cell line as a suitable in vitro model, since treatment with antiꢀtubulin drugs characteristically
alters the roundꢀshaped cells and induces the formation of elongated angular forms within 1ꢀ2
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hours.
Antiproliferative activities against K562 are expressed in Tables 1, 2 and 3,
together with the data for the inhibition of tubulin polymerization (see below). Data for the
positive controls are also included. Our nhibitory data cover a wide range of activities, with a
high information content. Regarding the phenothiazine– and phenoxazine derivatives, ten
compounds (15bꢀ15c, 15g, 16a, 16cꢀ16d, 16jꢀ16k, 16n and 16o) showed excellent growth
inhibition in the nanomolar range (IC50 values ≤ 30 nM). This finding could be confirmed on
the panel screening (see below). In contrast to its inactive (IC50 K562 > 30 µM) Nꢀ
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benzoylated analog, phenothiazineꢀbased (4ꢀphenylpiperazinꢀ1ꢀyl)methanone 15a, also
being devoid of any ring substitutents – showed potent K562 cell growth inhibitory properties
(IC50 0.13 µM). Likewise, similar but more distinctive antiproliferative potencies were
observed for phenoxazineꢀderived 16a (IC50 0.03 µM), being nearly equipotent with methoxyꢀ
substituted analogs 16c and 16d. In general, the introduction of a paraꢀmethoxy group in the
terminal phenyl ring caused a marked loss in antiproliferative potency (e.g., 15bꢀ15c, IC50
0
.02 µM vs. 15d, IC50 3.70 µM; 16cꢀ16d, IC50 0.02 µM vs. 16e, IC50 3.13 µM; 16o, IC50
0.007 µM vs. 16p, IC50 3.70 µM ). On the other hand, methoxy groups located orthoꢀ or meta
strongly promoted potency. This result runs contrary to our findings among the Nꢀbenzoylated
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analogs, where a paraꢀmethoxyꢀsubstitution turned out to be optimal. Consistent with the
pattern in the phenothiazine series, phenoxazine analogues bearing orthoꢀ or metaꢀmethoxy
substitutents in the phenylpiperazine scaffold (16c, IC50 0.02 µM and 16d, IC50 0.02 µM),
respectively) were most active, confirming that a paraꢀmethoxy substitution is less favorable
in the terminal phenyl ring. Cell growth inhibitory activities of 16cꢀ16d proved identical to
those of the corresponding phenothiazines 15bꢀ15c.
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In the phenothiazine series, the introduction of additional substituents into the tricyclic
nucleus did not contribute to improvement of antiproliferative potencies. Chloroꢀsubstituted
phenothiazine analogue 15f (IC50 K562 0.09 µM), for example, appeared nearly 5ꢀfold less
active than 15b (IC50 0.02 µM), whereas in case of the corresponding phenoxazine 16j (IC50
K562 0.03 µM) potency was retained in comparison with 16c (IC50 K562 0.02 µM).
Moreover, the dichloroꢀsubstituted phenoxazines 16l (IC K562 IC 0.13 µM) and 16m (IC
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K562 IC50 0.18 µM) were potent submicromolar inhibitors but were less active than the
monochloro analogs, demonstrating that the 2,7ꢀdisubstitution doesn´t entail any additional
advantage in terms of antiproliferative potencies. In good agreement with our previous
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observations, outstanding antiproliferative potencies in the phenoxazine series could be
attributed analogs bearing a a cyano functional group at the 3ꢀposition of the phenoxazine.
Overall, the 10ꢀ(4ꢀ(3ꢀmethoxyphenyl)piperazineꢀ1ꢀcarbonyl)ꢀ10Hꢀphenoxazineꢀ3ꢀcarbonitrile
(16o) was the most active compound, which inhibited K562 cell growth with an IC50 of 7 nM.
In this context, several aspects concerning the biological function of the nitrile group
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reviewed by Fleming et al., might be of interest.
In general, electronꢀwithdrawing paraꢀsubstituents in the terminal phenyl ring, such as
trifluoromethyl (16g, IC K562 > 60 µM), nitro (16h, IC K562 > 70 µM), or cyano (16i)
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were considered weak to inactive and showed strongly reduced antiproliferative activities
versus the most active compounds. Another interesting compound is 16f. Given the fact, that
chloro analog 16f showed good activities and proved 5ꢀfold more active than paraꢀmethoxyꢀ
substituted 16e against K562 cells, the synthesis of orthoꢀ or metaꢀchloro analogs – metaꢀ
chloro is also present in the structure of 7 (Chart 2) ꢀ should also be a very promising
approach.
The comparison of the IC50 values obtained for phenylpiperazines 15d or 16e with those
obtained for Nꢀphenyl substituted compound 21 (Scheme 1, K562 IC50 > 80 µM) clearly
demonstrated that the piperazine ring is a key structural element and contributes to potency.
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As an interesting subsidiary aspect in terms of SAR, the result obtained with 21 demonstrates
at the same time that the amide group shown in 4 (Chart 1), a representative and active Nꢀ
benzoylated analog, can obviously not be successfully replaced by a urea structural element.
The mere presence of a urea structural element, lacking the piperazine, obviously leads to
weakly active or inactive compounds. This result is backed up by the replacement of the
phenylpiperazine by a benzylamine (22a, IC 6 µM; 22b, 19 µM, Table 3, Chart 3), which
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also resulted in low potencies in comparison with 16c and 16d (Table 2). Incorporating a
bridging methylene as seen with 24aꢀ24b caused a dramatic loss of antiproliferative potency
(
24a, IC50 9 µM, and 24b, IC50 5 µM, respectively, vs. 16c, IC50 0.02 µM). This result
confirms, once again, that the intact (4ꢀphenylpiperazinꢀ1ꢀyl)methanone is particularly
important for antiproliferative activity.
When testing alternative scaffolds, ringꢀopened diphenylamine analog 19 proved considerably
less effective compared to the most active analogs (19, IC50 K562 18 µM vs. 15c, IC50 K562
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.02 µM or 16d, IC50 K562 0.02 µM), indicating that rigidifying the diphenylamine is
important for target binding. The same observations were made on ringꢀopened 2ꢀ
phenoxyanilineꢀbased analogues 20aꢀ20c (Scheme 1, Table 3), which were not active.
Interestingly, decreasing the ring size to obtain carbazoles (6ꢀ5ꢀ6 fused) induced strong
potencies (< 100 nM for the most active analogs, manuscript in preparation). Also, the ringꢀ
expanded (6ꢀ7ꢀ6 fused) 5Hꢀdibenzo[b,f]azepines 17aꢀ17b (17a, IC50 K562 0.45 µM and 17b,
IC50 K562 0.94 µM, respectively) as well as the dihydro analog 18 (IC50 K562 0.84 µM) were
submicromolar, potent inhibitors of K562 cell growth, but proved clearly less active than the
most active analogs. To complement this, 9ꢀ(4ꢀ(phenyl)piperazinꢀ1ꢀyl)acridines 27aꢀ27f,
lacking the carbonyl compared with 5 (Chart 2), were inactive or only weakly active (K562
IC50: 27a, 4.5 µM; 27b 8.8 µM, 27cꢀ27f > 10 µM).
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Cell Panel Screen
Cellꢀbased cancer screening panels in combination with data analysis and interpretation can
be of great value for assigning a molecular target to test compound action and allows hit
prioritization for additional studies. Therefore, we explored the potencies of twelve selected
compounds (15aꢀ15c, 15gꢀ15h; 16a, 16c, 16e, 16jꢀ16k, 16nꢀ16o) against a broad range (93, +
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resting, nonꢀproliferating PBMCs) of tumor cell lines. Visualization of results by mean
graphs provides a compact way to mirror profiles of relative sensitivity and resistance of
cellular parameters in response to treatment of mutiple cell lines with potential anticancer
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drugs. Our screening included a large panel of tumor cell lines (93), covering multiple tumor
3
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types. In addition, resting, nonꢀproliferating peripheral blood mononuclear cells (PBMC)
were included in the studies. For quantification of cell proliferation, cells were stained with
fluorescent dye sulforhodamine B (SRB).
Our results from the panel screening proved the outstanding overall antiproliferative potencies
(GI50, the molar concentration causing 50% cell growth inhibition in relation to the initial cell
seeding number) of cyanoꢀsubstituted analogs 16n and 16o, evidenced by extremely low
nanomolar mean GI values over all tumour cell lines (93) tested (16n, mean GI of 4.1 nM
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and 16o, mean GI of 3.3 nM, respectively). Compounds 15aꢀ15c and 15g as well as 16a,
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6c, and 16jꢀ16k showed also strong potencies with mean GI50 data in the range of 14ꢀ35 nM
(see SI for details). Among the analogs tested the panel screen, as expected, 15h and 16e were
the least active, with GI50 data of 1.57 and 1.97 µM, respectively. Noteworthy, when
13
comparing the GI50 values for 16n and 16o with previously described Nꢀbenzoylated 4
(Chart 1), some divergences became quite striking. On the one hand, GI data of 16n, 16o
50
and 4 (83 cell line screening, same method) against the most sensitive cell lines were quite
similar (≤ 10 nM), whereas GI50 data observed for the eight most resistant cell lines strongly
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differed, covering a wide range in the case of 4 (60 nM up to 8.5 µM) but retaining high
nanomolar potencies in a narrow span for 16n (GI 7.0ꢀ15.6 nM) and 16o (GI 5.9ꢀ7.9 nM),
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respectively. This finding clearly indicates more distinct antiproliferative properties of the (4ꢀ
phenylpiperazinꢀ1ꢀyl)methanones in overall cell lines compared to the Nꢀbenzoylated
analogs. We used a mean graph display format for visualization of the 8 most sensitive and
most resistant cell lines, or, alternatively, a boxꢀplot graph (Table 4, Fig. 1 A, B). Briefly,
horizontal bars extending to the right from the zero value refer to less sensitive cell lines, bars
extending to the left indicate more resistant cell lines. Each bar, therefore, represents the
relative activity of the compound in the given cell lines deviating from the mean in all cell
lines (GI50). No activity could be detected in quiescent peripheral blood mononuclear cells
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(PBMC), suggesting that antiproliferative activities are related to actively cycling cells.
In VitroꢀTubulin Polymerization Assays
Next, we explored the effect of the compounds on tubulin polymerization. Colchicine,
podophyllotoxin, nocodazole, and vinblastine were used as reference compounds (Table 1).
Selfꢀassembly of αβꢀtubulin is usually followed by an increase in turbidity at 340ꢀ360 nm
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over a 45 min period at 37 °C. This is based on the fact, that light scattering by microtubules
is proportional to the amount of microtubule polymer. Usually, typical sigmoidal tubulin
polymerization curves are obtained, with a plateau reached with the steady state equilibrium
level. Typically, in response to a microtubule destabilizer, the level of steady state turbidity
decreases in a dose–dependent manner, as exemplified for 16o (Figure 2, A).
Figure 2 should be inserted here
Thirteen compounds (15aꢀ15c, 15fꢀ15g, 16cꢀ16d, 16f, 16jꢀ16k, 16mꢀ16o) proved to be potent
tubulin assembly inhibitors (IC50 ≤ 1 µM), with potencies comparable or superior to the
reference antiꢀtubulin drugs. These strong potencies, in particular those of 16n and 16o, were
in excellent correlation with the data from the cellular proliferation assay. The most active
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inhibitors of tumor cell growth were also the most effective inhibitors of tubulin
polymerization. The 2-methoxyꢀ as well as the 3ꢀmethoxyꢀsubstituted substituted compounds
showed the greatest potency. Additionally, in good agreement with the cellular assays, strong
tubulin polymerization inhibiting properties can also be found with 15a as well as 16a, both
being unsubstituted phenylpiperazine scaffold. Moreover, compound 16l was slightly more
potent than colchicine, whereas analogous 17aꢀ17b were moderate inhibitors of tubulin
polymerization. In general, compounds having IC values in the range of ≥ 10 µM showed no
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appreciable activity as an inhibitor of tubulin polymerization, which applied to phenothiazineꢀ
derived 15h and phenoxazines 16gꢀ16i (Table 1, 2) and several structurally related analogues
(Table 3). An overview of the resulting SAR from the cellꢀbased assay and the tubulin
assembly assay is depicted in Fig. 5.
EBI Competition Assay
Microtubuleꢀdestabilizing as well as destabilizing drugs are known to interact with tubulin
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through different binding sites. To explore whether compounds 15b and 16o directly interact
with tubulin at the colchicineꢀbinding site, we performed a competition assay with N,N′ꢀ
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ethyleneꢀbis(iodoacetamide) (EBI) in K562 cells.
As an alkylating agent, EBI has the
property to crossꢀlink the Cys239 and the Cys354 residues of βꢀtubulin involved in the
colchicineꢀbinding site. The EBIꢀassay is based on the fact, that – in the absence of a potential
colchicine binding agent ꢀ the EBI/βꢀtubulin adduct can be detected as an immunoreactive βꢀ
tubulin band that can be seen below the βꢀtubulin band in the Western blot. So, if EBI is
added to cells previously treated with a colchicineꢀsite binder, the binding site is already
occupied and the EBI adduct cannot be observed. Thus, compounds that bind to the
colchicineꢀbinding site in βꢀtubulin prevent the formation of the EBI:βꢀtubulin adduct.
Colchicine (5 ꢁM) as well as 15b (20 ꢁM and 40 µM, respectively) and 16o (14 ꢁM and 7
µM, respectively) prevented the binding of EBI to βꢀtubulin in living K562 cells (Fig. 3),
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resulting in the absence of the adduct band. Therefore, we consider it very likely that the
novel compounds exert their biological activities by binding to the colchicineꢀbinding site of
βꢀtubulin.
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Effect on CellꢀCycle Progression
Tubulin polymerization inhibitors suppress microtubule formation and induce cell cycle arrest
during interphase at G2/M, a hallmark of tubulin polymerization inhibitors. As a result,
38,39
chromosome separation is seriously impaired.
Doseꢀdependent effects on the cell cycle
were studied in K562 cells (15c, 16cꢀ16d, 16o) and percentage distribution of cells on cellꢀ
cycle phases was measured (see SI). It became evident that phenoxazine–derived 16o
accumulated cells at G2/M at concentrations down to 30 nM (Fig. 4), comparable to
colchicine, which served as positive G2/M arrest control. A normal cell cycle distribution was
observed in the vehicleꢀtreated control cells.
Figure 4 should be inserted here
Our findings indicate that the compounds in this paper, as represented by 16n, can be
classified as potent antimitotic drugs that arrest proliferating cells at G2/M.
Figure 5 should be inserted here
Binding model of compounds 16n with tubulin
In recent years, 3Dꢀquantitative structureꢀactivity relationship (3DꢀQSAR) and docking
studies have widely been used to predict inhibitory activities of new tubulin polymerization
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inhibitors.
To gain insight on the possible binding mode the novel 10ꢀ(4ꢀphenylpiperazinꢀ
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ꢀyl)methanones and to create predictive 3DꢀQSAR models, CoMFA and CoMSIA analyses
were performed. Due to its high tubulin binding activity, compound 16n was used as a
template.
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The comparative molecular field analysis (CoMFA) uses data from known active molecules
and is an important 3DꢀQSAR method which focuses on nonꢀbonding interactions between
the protein and the ligand. It provides a common way to display electrostatic (Coulombic) and
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steric (LennardꢀJones) fields of the regions important for biological activity. An alternative
computational approach is the comparative molecular similarity indices analysis (CoMSIA),
which employs a distanceꢀbased Gaussianꢀtype function to evaluate five molecular fields of
different physicoꢀchemical properties (i.e. steric, electrostatic, hydrophobic, and hydrogen
44,45
bonding donor and acceptor).
Docking results
Molecular docking was used as a computational tool to gain insight into the binding mode of
the novel 10ꢀ(4ꢀphenylpiperazinꢀ1ꢀyl)methanones. As a parameter of docking accuracy, we
calculated a highꢀlevel rootꢀmeanꢀsquare distance (RMSD) value of 0.774 Å between bound
ligand and reꢀdocked colchicine. This value documents the reliability of GOLD for the
reproduction and identification of the correct binding mode of a lead compound. Based on our
docking studies, there is a hydrogen bond between the methanone C=O group and Val181
from the α chain. Furthermore, when focusing on the binding mode of the best docked pose of
compound 16n, two cationꢀπ interactions between the phenyl rings and Lys352 and Lys254 of
tubulin β chain were identified (Fig. 6).
Fig. 6 should be inserted here.
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CoMFA and CoMSIA results
The results from the 3DꢀQSAR studies are summarized in Tables 1ꢀ3 (see SI). Partial least
squares (PLS) regression analysis of the compounds in training set showed a CoMFAꢀregion
focusing (CoMFAꢀRF) QSAR model (grid spacing = 1) with an excellent q2 value of 0.829 (3
components), indicating significant predictive properties. The non crossꢀvalidated PLS
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analysis revealed a high squared correlation coefficient r value of 0.957 and a high predictive
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correlation coefficient (r pred) of 0.759. Fig. 7. presents the relationship between the
experimental and predicted pIC50 (Table 1, SI).
Fig. 7 should be inserted here.
The CoMFAꢀRF steric and electrostatic fields from the final best non crossꢀvalidated analysis
4
were plotted as 3D colored contour maps (Fig. 8). A large region of green colour near R and
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R (general structure Table 2) in the 3D coloured contour map suggested that the introduction
of bulky substituents such as methoxy would lead to potent analogs. This is confirmed by
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different activities of compounds 16c (R = OCH , pIC = 6.194) and 16d (R = OCH , pIC
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=
6.161) compared with 16a (R = R = H, pIC = 5.860).
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Fig. 8 should be inserted here.
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A red contour placed near R and R substituents indicates that electronegative groups such as
methoxy group contribute to an increase in antiproliferative potencies. Also, a small red
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contour near the R substituent demonstrates that electronegative groups such as CN are
favorable and increase the potency. This is the reason why cyanoꢀsubstituted compounds 16n
and 16o are among the most active analogs of all compounds tested. Moreover, a blue region
near R6 indicates that electropositive substituents such as H would increase the potency.
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CoMSIA as an analysis tool was performed using a lattice box with a grid spacing of 2 Å and
the combination of fields was systematically varied to obtain the best results. CoMSIA PLS
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analysis yielded a good q value of 0.729, a high r value of 0.958 and gave a high predictive
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correlation coefficient (r pred) of 0.813 with 3 PLS components by using the combination of
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five fields (steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor). When
comparing contour maps obtained from both the CoMSIA and the CoMFA approach, some
similarities became obvious (Fig. 9). The visual display of the CoMSIA steric contour maps
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shows, as indicated by the yellow colour, that less steric groups near substituents R and R
would increase antiproliferative potencies. On the other hand, the large green contour reveals
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that more bulky substituents such as methoxy near R and R would promote the formation
more potent analogs. With reference to the hydrophobic contour map of CoMSIA, the white
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contour near the R and R substituents suggests that more hydrophilic groups at this position
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would increase the activity. A yellow contour near R emphasises the need for hydrophobic
groups in this area to promote tubulinꢀinhibitory properties.
Fig. 9 should be inserted here.
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The magenta contours near the R and C=O substituents point towards regions where
hydrogen bond acceptor groups are favored, whereas red contours mark regions where
hydrogen bond acceptor groups are less favorable for binding. Obviously, there is a hydrogen
bond donor (Val181) which effectively complements the C=O in the receptor. Also, the
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electrostatic red contour at the R region and the magenta contour near the R substituent
complement each other.
Conclusion
Synthesis and focused SAR studies for phenylpiperazinylmethanones derived from a
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phenothiazine and phenoxazine structural scaffold as novel antiꢀtubulin agents were
performed. The novel compounds showed excellent potencies as inhibitors of tumour cell
growth and tubulin polymerization. The most promising analogs, represented by 10ꢀ(4ꢀ(3ꢀ
methoxyphenyl)piperazineꢀ1ꢀcarbonyl)ꢀ10Hꢀphenoxazineꢀ3ꢀcarbonitrile (16o), inhibited
tubulin polymerization in the range of the positive controls and proved highly active in
comprehensive cellꢀbased cancer screening panels, as evidenced by exceptionally low
nanomolar mean GI values over all tumour cell lines (93) tested (16n, mean GI of 4.1 nM
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and 16o, mean GI50 of 3.3 nM, respectively). The most active analogs showed two essential
moieties. One is the tricyclic (6ꢀ6ꢀ6) phenothiazine or phenoxazine scaffold. The second is the
intact phenylpiperazinylmethanone structure. Taken together, our findings highlight not only
the phenoxazine and phenothiazine but also the phenylpiperazine moiety as important
4,5
structural elements for inhibition of tubulin polymerization. The presence of orthoꢀ or metaꢀ
methoxy groups in the phenylpiperazine or – somewhat surprising – an unsubstituted phenyl
turned out to be crucial for growth inhibitory potencies and impairment of tubulin assembly
into microtubules. We conclude that the growth inhibitory properties are most likely mediated
through an impairment of tubulin polymerization, as documented by the EBI assay and the
fact that the most potent inhibitors of cancer cell growth turned out to be the most efficacous
tubulin assembly inhibitors. Closely related to this, key representatives such as 16o blocked
the cell cycle at the G2/M phase at concentrations down to 30 nM. CoMFA and CoMSIA
approaches provided useful insights into the binding of the analogs in tubulin. We could once
again confirm that a nitrile functional group placed at the 3ꢀposition of the phenoxazine
scaffold significantly contributes to antiproliferative properties.
In summary, we envision that Nꢀheterocyclic (4ꢀphenylpiperazinꢀ1ꢀyl)methanones hold great
potential for further structural modifications and we believe that our studies provide some
valuable information for the design of effective antiꢀcancer drugs. Additional studies on the
synthesis and biological activities of further analogues are in progress and the results are
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being published in due course.
Experimental Section
1
Melting points were determined with a Kofler melting point apparatus and are uncorrected. H
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NMR (600 MHz, 400 MHz) and C NMR (151 MHz, 100 MHz) were obtained on an Agilent
600ꢀMR, Agilent 400ꢀMR, and Mercury Plus AS 400 NMR spectrometer (Varian); δ in ppm
related to tetramethylsilane. Fourierꢀtransform IR spectra were recorded on a Jasco FT/IRꢀ
4
100 (attenuated total reflection, ATR) spectrometer by applying ATR correction. Mass
spectra were obtained on Finnigan MAT GCQ and LCQ apparatuses applying electron beam
ionization (EI) and electrospray ionization (ESI). Atmospheric pressure chemical ionization
(
APCI) method was performed with a micrOTOFꢀQII apparatus (Bruker Daltonics´, Bremen,
Germany) and APCI IIꢀinterface or ESIꢀinterface (Bruker Daltonics´, Bremen, Germany). The
purity of all target compounds was determined by reversed phase HPLC at 254 nm.
Compound purity, as determined by analytical reversedꢀphase HPLC for all target
compounds, is ≥ 95%. The HPLC system applied a C18 phase (Nucleosil, 3 ꢁm, 4.0 × 125
mm, CSꢀChromatographie Service GmbH, Langerwehe, Germany) eluting the compounds
2
with an acetonitrile/H O gradient at a flow rate of 0.40 mL/min. All organic solvents were
appropriately dried or purified prior to use. Purification by chromatography refers to column
chromatography on silica gel (MachereyꢀNagel, 70ꢀ230 mesh). In most cases, the
concentrated pure fractions obtained by chromatography using the indicated eluants were
treated with a small amount of nꢀhexane to induce precipitation. All new compounds
1
13
displayed H NMR, C NMR and MS spectra consistent with the assigned structure. Yields
have not been optimized. Analytical TLC was done on Merck silica 60 F254 alumina coated
plates (E. Merck, Darmstadt).
2
ꢀChloroꢀ10Hꢀphenoxazine (3b).
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The title compound was prepared according to the literature protocol.
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2,8ꢀDichloroꢀ10Hꢀphenoxazine (3c).
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The title compound was prepared according to the literature protocol.
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0Hꢀphenoxazineꢀ3ꢀcarbonitrile (3d).
The title compound was prepared according to the literature protocol.
10HꢀPhenothiazinꢀ10ꢀyl)(4ꢀphenylpiperazinꢀ1ꢀyl)methanone (15a). The title compound
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(
was prepared from 1a (2 mmol, 0.40 g) and 1ꢀphenylpiperazine (0.32 g, 2 mmol) in 1,2ꢀ
dichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described for 15g.
Purification by chromatography (ethyl acetate / petroleum ether, 3:7) afforded 15a as a white
ꢀ1
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powder (0.27 g, 35%). mp 141ꢀ142 °C; FTIR: 1647 cm (C=O); H NMR (400 MHz, CDCl )
δ 7.69 (dd, J = 8.1, 1.3 Hz, 2H), 7.32 (dd, J = 7.7, 1.5 Hz, 2H), 7.27 – 7.20 (m, 4H), 7.11 (td,
J = 7.5, 1.3 Hz, 2H), 6.87 (q, J = 6.4, 4.9 Hz, 3H), 3.53 (t, J = 5.1 Hz, 4H), 3.11 – 2.98 (m,
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H); C NMR (101 MHz, CDCl ) 157.19, 141.34, 129.20, 128.94, 127.69, 127.56, 127.30,
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+
25.23, 122.25, 120.40, 116.49, 48.94, 45.64; MS (APCI) calcd for C23
88.1484; found 388.1467; purity (HPLC): 99.48 %.
21 3
H N OS [M+H]
(4ꢀ(2ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)(10Hꢀphenothiazinꢀ10ꢀyl)methanone (15b). The title
compound was prepared from 1a (2 mmol, 0.40 g) and 1ꢀ(2ꢀmethoxyphenyl)piperazine (0.38
g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7)
ꢀ
1
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afforded 15b as a white powder (0.23 g, 28%). mp 163 °C; FTIR: 1659 cm (C=O); H NMR
(400 MHz, CDCl
3
) δ 7.68 (dd, J = 8.1, 0.9 Hz, 2H), 7.30 (dd, J = 7.7, 1.3 Hz, 2H), 7.27 – 7.21
(m, 2H), 7.10 (td, J = 7.6, 1.2 Hz, 2H), 7.03ꢀ6.98 (m, 1H), 6.92 – 6.82 (m, 3H), 3.83 (s, 3H),
13
3.57 (s, br, 4H), 2.93 (s, br, 4H); C NMR (101 MHz, CDCl ) 157.06, 152.14, 141.43,
3
128.60, 127.64, 127.56, 125.10, 123.42, 121.96, 121.01, 118.32, 111.25, 77.32, 77.20, 77.00,
76.68, 55.38, 50.25, 45.87; MS (EI, 70 eV) m/z (%) 417 (26), 219 (100); purity (HPLC): 98
%
.
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(4ꢀ(3ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)(10Hꢀphenothiazinꢀ10ꢀyl)methanone (15c). The title
compound was prepared from 1a (2 mmol, 0.40 g) and 1ꢀ(3ꢀmethoxyphenyl)piperazine (0.38
g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7)
10
11
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14
15
16
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60
ꢀ1
1
afforded 15c as a white powder (0.35 g, 42%). mp 105 °C; FTIR: 1663 cm (C=O); H NMR
(600 MHz, CDCl ) δ 7.68 (dd, J = 8.2, 1.2 Hz, 2H), 7.32 (dd, J = 7.7, 1.5 Hz, 2H), 7.25 – 7.21
3
(m, 2H), 7.17 – 7.13 (m, 1H), 7.11 (td, J = 7.5, 1.3 Hz, 2H), 6.50 – 6.35 (m, 3H), 3.76 (s, 3H),
13
3
.52 (s, br, 4H), 3.04 (s, 4H); C NMR (151 MHz, CDCl
3
) 233.89, 160.58, 160.56, 157.22,
152.15, 141.33, 129.90, 129.00, 127.71, 127.58, 127.49, 125.27, 122.31, 109.15, 105.06,
102.90, 55.21, 48.81, 45.59; MS (EI, 70 eV) m/z (%) 417 (28), 219 (100); purity (HPLC):
9.75 %.
4ꢀ(4ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)(10Hꢀphenothiazinꢀ10ꢀyl)methanone (15d). The title
9
(
compound was prepared from 1a (2 mmol, 0.40 g) and 1ꢀ(4ꢀmethoxyphenyl)piperazine (0.38
g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7)
ꢀ1
1
afforded 15d as a white powder (0.32 g, 38%). mp 116 °C; FTIR: 1670 cm (C=O); H NMR
(
600 MHz, CDCl ) δ 7.68 (dd, J = 8.1, 1.2 Hz, 2H), 7.31 (dd, J = 7.7, 1.5 Hz, 2H), 7.24 (d, J
3
=
1.3 Hz, 2H), 7.11 (dd, J = 7.5, 1.3 Hz, 2H), 6.81 (s, 4H), 3.75 (s, 3H), 3.52 (s, br, 4H), 2.91
13
(s, br, 4H); C NMR (151 MHz, CDCl
3
) δ 157.13, 141.36, 128.81, 127.66, 127.54, 125.17,
1
1
4
22.14, 118.68, 114.47, 60.37, 55.52, 50.40, 45.81; MS (EI, 70 eV) m/z (%); purity (HPLC):
00 %.
ꢀ(4ꢀ(10HꢀPhenothiazineꢀ10ꢀcarbonyl)piperazinꢀ1ꢀyl)benzonitrile
(15e).
The
title
compound was prepared from 1a (2 mmol, 0.40 g) and 1ꢀ(4ꢀcyanophenyl)piperazine (0.37 g,
2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described
for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7) afforded 15e as
ꢀ1
1
a white powder (0.34 g, 41%). mp 217 °C; FTIR: 2222 (CN), 1647 cm (C=O); H NMR (400
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MHz, CDCl ) δ 7.70 (dd, J = 8.1, 1.3 Hz, 2H), 7.50 – 7.43 (m, 2H), 7.34 (dd, J = 7.7, 1.5 Hz,
3
2
4
1
H), 7.28 – 7.22 (m, 2H), 7.14 (dd, J = 7.6, 1.3 Hz, 2H), 6.81 – 6.74 (m, 2H), 3.53 – 3.45 (m,
13
H), 3.24 – 3.16 (m, 4H); C NMR (101 MHz, CDCl
3
) δ 157.36, 152.66, 141.13, 133.53,
29.50, 127.79, 127.60, 125.48, 122.67, 119.69, 114.39, 101.14, 46.69, 45.15; MS (APCI)
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
9
0
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7
8
9
0
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4
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7
8
9
0
+
calcd for C24
H
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N
4
OS [M+H] 413.1436; found 413.1471; purity (HPLC): 99.25 %.
(2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀyl)(4ꢀ(2ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone
(
15f). The title compound was prepared from 1b (2 mmol, 0.47 g) and 1ꢀ(2ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 3:7) afforded 15f as a white powder (0.33 g, 37%). mp 151 °C; FTIR: 1651
ꢀ1
1
cm (C=O); H NMR (400 MHz, CDCl ) δ 7.88 (d, J = 2.1 Hz, 1H), 7.47 (dd, J = 8.1, 1.2 Hz,
3
1
H), 7.29 (dd, J = 7.7, 1.5 Hz, 1H), 7.27 – 7.21 (m, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.14 – 7.05
(m, 2H), 7.05 – 6.98 (m, 1H), 6.93 – 6.81 (m, 3H), 3.83 (s, 3H), 3.60 (s, br, 4H), 2.94 (s, 4H);
1
3
3
C NMR (101 MHz, CDCl ) 156.46, 152.16, 142.31, 141.17, 133.48, 128.04, 127.90, 127.82,
127.77, 126.44, 125.40, 124.99, 123.54, 121.85, 121.54, 121.03, 118.39, 111.26, 77.32, 77.20,
7.00, 76.68, 55.40, 50.25, 45.82. MS (EI, 70 eV) m/z (%) 451 (10), 219 (100); purity
7
(HPLC): 99.41 %.
(2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀyl)(4ꢀ(3ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone
(15g). In a typical procedure, a stirred mixture of 2ꢀchlorophenothiazine 1b (0.47 g, 2 mmol),
bis(trichloromethyl) carbonate (0.356 g, 1.2 mmol), pyridine (1 mL) and 1,2ꢀdichlororethane
(20 mL) was heated under reflux (N atmosphere, oil bath, 75 °C). After 3 h, formation of the
2
intermediate carbamoyl chloride was completed (TLC control, dichloromethane / nꢀhexane,
/1). Then, 1ꢀ(3ꢀmethoxyphenyl)piperazine (0.38 g, 2 mmol) was added in one portion.
Stirring was continued after formation of the product appeared to be complete (2ꢀ3 h, TLC
control, ethyl acetate / petroleum ether 40ꢀ60 °C, 3:7). The mixture was poured into H O (200
mL)/6M HCl (10 mL) and extracted with CH Cl (3 × 30 mL). The combined organic layers
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were washed with water and dried over Na SO . Then, the drying agent was removed by
2
4
filtration and the solvent was evaporated in vacuum. The residue was then purified by silica
gel chromatography (ethyl acetate/petroleum ether 40ꢀ60 °C, 3/7) to afford 15g as a paleꢀ
ꢀ1
1
yellow solid (0.40 g, 44 %). mp 118ꢀ119 °C; FTIR: 1663 cm (C=O); H NMR (600 MHz,
CDCl ) δ 7.90 (d, J = 2.1 Hz, 1H), 7.45 (dd, J = 8.2, 1.2 Hz, 1H), 7.31 (dd, J = 7.7, 1.5 Hz,
1H), 7.25 – 7.21 (m, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.18 – 7.07 (m, 3H), 6.51 – 6.38 (m, 3H),
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58
59
60
3
1
3
3
1
1
.77 (s, 3H), 3.57 – 3.50 (m, 4H), 3.06 (t, J = 5.2 Hz, 4H); C NMR (151 MHz, CDCl3)
60.60, 156.62, 142.16, 141.10, 133.50, 129.95, 128.35, 128.10, 128.09, 127.86, 126.82,
25.59, 125.17, 122.21, 121.87, 109.28, 105.24, 103.07, 60.40, 55.22, 48.96, 45.53, 14.21.
MS (EI, 70 eV) m/z (%) 451 (29), 219 (100); purity (HPLC): 98.61 %.
(
2ꢀChloroꢀ10Hꢀphenothiazinꢀ10ꢀyl)(4ꢀ(4ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone
15h). The title compound was prepared from 1b (2 mmol, 0.47 g) and 1ꢀ(4ꢀ
(
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 3:7) afforded 15h as a beige powder (0.23 g, 22%). mp 108ꢀ109 °C; FTIR:
ꢀ1
1
1
659 cm (C=O); H NMR (600 MHz, CDCl ) δ 7.89 (d, J = 2.2 Hz, 1H), 7.47 – 7.43 (m,
3
1H), 7.30 (dd, J = 7.7, 1.5 Hz, 1H), 7.25 – 7.21 (m, 1H), 7.25 – 7.22 (m, 1H), 7.19 (d, J = 8.3
Hz, 1H), 7.14 – 7.05 (m, 2H), 6.85 – 6.79 (m, 3H), 3.75 (s, 3H), 3.59 – 3.50 (m, 4H), 2.95 –
13
2.90 (m, 4H); C NMR (151 MHz, CDCl
3
) δ 156.55, 154.35, 145.14, 142.24, 141.14, 133.49,
1
1
4
8
28.67, 128.42, 128.14, 128.08, 127.83, 127.77, 127.63, 127.26, 126.81, 126.64, 126.56,
25.50, 125.09, 122.11, 122.04, 121.72, 118.83, 114.50, 114.16, 113.01, 55.54, 51.28, 50.50,
5.81, 31.60, 22.66, 14.13; MS (EI, 70 eV) m/z (%) 451 (24), 219 (100); purity (HPLC):
7.48 %.
(10HꢀPhenoxazinꢀ10ꢀyl)(4ꢀphenylpiperazinꢀ1ꢀyl)methanone (16a). The title compound
was prepared from 3a (2 mmol, 0.36 g) and 1ꢀphenylpiperazine (0.32 g, 2 mmol) in 1,2ꢀ
dichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described for 15g.
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Purification by chromatography (ethyl acetate / petroleum ether 3:7) afforded 16a as a white
ꢀ1
1
powder (0.36 g, 49%). mp 101ꢀ102 °C; FTIR: 1666 cm (C=O); H NMR (600 MHz, CDCl )
3
δ 7.31 – 7.24 (m, 2H), 7.05 – 6.98 (m, 2H), 6.94 – 6.84 (m, 9H), 3.75 (t, J = 5.1 Hz, 4H), 3.22
13
–
3.14 (m, 4H); C NMR (151 MHz, CDCl
3
) δ 218.62, 154.27, 150.65, 145.71, 130.34,
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29.30, 123.92, 123.91, 123.80, 120.77, 116.75, 116.50, 116.49, 116.14, 49.53, 45.21; MS
(EI, 70 eV) m/z (%) 371 (20), 189 (100); purity (HPLC): 99.71 %.
10HꢀPhenoxazinꢀ10ꢀyl)(4ꢀ(methylphenyl)piperazinꢀ1ꢀyl)methanone (16b). The title
compound was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(4ꢀmethylphenyl)piperazine (0.35 g,
mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described
for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7) afforded 16b as
(
2
ꢀ1
1
a white powder (0.41 g, 54 %). mp 125ꢀ126 °C; FTIR: 1686 cm (C=O); H NMR (600 MHz,
CDCl ) δ 7.08 (d, J = 8.2 Hz, 2H), 7.02 – 6.97 (m, 2H), 6.92 – 6.85 (m, 6H), 6.84 – 6.79 (m,
3
13
2
H), 3.74 (t, J = 5.1 Hz, 4H), 3.12 (t, J = 5.1 Hz, 4H), 2.27 (s, 3H); C NMR (151 MHz,
CDCl ) δ 218.64, 154.23, 148.58, 145.64, 130.34, 129.81, 123.91, 123.75, 123.64, 117.07,
116.48, 116.45, 116.06, 115.87, 51.38, 50.05, 45.60, 45.26, 31.60, 22.67, 20.46, 14.14; MS
3
(
EI, 70 eV) m/z (%) 385 (21), 204 (15), 203 (100), 182 (16); purity (HPLC): 99.72 %.
(
4ꢀ(2ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)(10Hꢀphenoxazinꢀ10ꢀyl)methanone (16c). The title
compound was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(2ꢀmethoxylphenyl)piperazine (0.38
g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7)
ꢀ1
1
afforded 16c as a white powder (0.37 g, 46 %). mp 123 °C; FTIR: 1663 cm (C=O); H NMR
(
600 MHz, CDCl ) δ 7.06 – 7.01 (m, 1H), 7.00 – 6.96 (m, 2H), 6.94 – 6.84 (m, 9H), 3.86 (s,
3
1
3
3
3
H), 3.82 – 3.74 (m, 4H), 3.09 – 3.01 (m, 4H) ); C NMR (151 MHz, CDCl ) δ 233.91,
154.16, 152.19, 152.18, 145.48, 140.42, 130.34, 123.90, 123.68, 123.60, 121.05, 118.42,
16.42, 115.87, 111.32, 55.43, 50.63, 45.49; MS (EI, 70 eV) m/z (%) 401 (24), 219 (100), 191
14), 182 (17); purity (HPLC): 99.63 %.
1
(
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(4ꢀ(3ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)(10Hꢀphenoxazinꢀ10ꢀyl)methanone (16d). The title
compound was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(3ꢀmethoxylphenyl)piperazine (0.38
g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7)
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
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49
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59
60
ꢀ1
1
afforded 16d as a pale yellow powder (0.54 g, 67 %). mp 122 °C; FTIR: 1661 cm (C=O); H
NMR (600 MHz, CDCl ) δ 7.18 (t, J = 8.1 Hz, 1H), 7.04 – 6.98 (m, 2H), 6.93 – 6.84 (m, 6H),
3
1
3
6
.47 (dd, J = 7.9, 2.2 Hz, 3H), 3.78 (s, 4H), 3.73 (t, J = 10.4 Hz, 3H), 3.21 – 3.15 (m, 4H); C
NMR (151 MHz, CDCl ) δ 160.63, 154.28, 151.91, 145.75, 130.34, 130.01, 123.93, 123.91,
23.83, 116.51, 116.19, 109.39, 105.45, 103.26, 55.24, 49.44, 45.12; MS (EI, 70 eV) m/z (%)
401 (22), 219 (100), 191 (14), 182 (16), 149 (20); purity (HPLC): 99.39 %.
4ꢀ(4ꢀMethoxyphenyl)piperazinꢀ1ꢀyl)(10Hꢀphenoxazinꢀ10ꢀyl)methanone (16e). The title
3
1
(
compound was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(4ꢀmethoxylphenyl)piperazine (0.38
g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7)
ꢀ1
1
afforded 16e as a white powder (0.44 g, 55 %). mp 118ꢀ119 °C; FTIR: 1670 cm (C=O); H
NMR (600 MHz, CDCl ) δ 7.03 – 7.00 (m, 2H), 6.94 – 6.84 (m, 10H), 3.78 (s, 3H), 3.76 (t, J
3
13
=
5.0 Hz, 4H), 3.07 (t, J = 5.0 Hz, 4H); C NMR (151 MHz, CDCl ) δ 154.49, 154.21,
3
1
45.61, 145.01, 130.34, 123.91, 123.73, 118.95, 116.48, 116.46, 116.03, 114.55, 55.56, 50.97,
45.38; MS (EI, 70 eV) m/z (%) 401 (28), 182 (17), 149 (69); purity (HPLC): 98.57 %.
4ꢀ(4ꢀChlorophenyl)piperazinꢀ1ꢀyl)(10Hꢀphenoxazinꢀ10ꢀyl)methanone (16f). The title
compound was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(4ꢀchlorophenyl)piperazine (0.39 g,
mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described
for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7) afforded 16f as
(
2
ꢀ1
1
a pale yellow powder (0.62 g, 77 %). mp 158ꢀ160 °C; FTIR: 1690 cm (C=O); H NMR (400
MHz, CDCl
3
) δ 7.24 – 7.18 (m, 2H), 7.03 (ddd, J = 5.9, 3.5, 0.6 Hz, 2H), 6.94 – 6.86 (m, 6H),
13
6
.84 – 6.79 (m, 2H), 3.77 – 3.66 (m, 4H), 3.20 – 3.06 (m, 4H); C NMR (101 MHz, CDCl ) δ
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154.32, 149.18, 145.85, 130.34, 129.15, 125.76, 123.92, 123.89, 117.95, 116.53, 116.30,
+
49.49, 45.07; MS (APCI) calcd for C H ClN O [M+H] 406.1322; found 406.1423; purity
23
20
3
2
HPLC): 99.76 %.
(4ꢀ(4ꢀ(Trifluoromethyl)phenyl)piperazinꢀ1ꢀyl)(10Hꢀphenoxazinꢀ10ꢀyl)methanone (16g).
0
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The title compound was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(4ꢀ
trifluoromethylphenyl)piperazine (0.46 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and
pyridine (1 mL) in a similar manner as described for 15g. Purification by chromatography
(ethyl acetate / petroleum ether, 3:7) afforded 16g as a white powder (0.16 g, 18 %). mp 201ꢀ
ꢀ1
1
3
202 °C; FTIR: 1686 cm (C=O); H NMR (400 MHz, CDCl ) δ 7.49 (d, J = 8.7 Hz, 2H), 7.10
1
3
– 7.02 (m, 2H), 6.96 – 6.86 (m, 8H), 3.76 – 3.69 (m, 4H), 3.32 – 3.24 (m, 4H); C NMR (101
MHz, CDCl ) δ 154.42, 152.62, 146.01, 130.35, 128.53, 126.60, 126.56, 126.52, 126.48,
3
1
25.83, 124.01, 123.94, 123.14, 121.79, 121.46, 121.13, 116.58, 116.47, 115.15, 48.16, 44.90;
MS (EI, 70 eV) m/z (%) 439 (18), 257 (100), 215 (12), 214 (10), 183 (12), 182 (51), 172 (12),
45 (15), 70 (36); purity (HPLC): 98.05 %.
1
(4ꢀ(4ꢀNitrophenyl)piperazinꢀ1ꢀyl)(10Hꢀphenoxazinꢀ10ꢀyl)methanone (16h). The title
compound was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(4ꢀnitrophenyl)piperazine (0.41 g, 2
mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described
for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 3:7) afforded 16h as
ꢀ1
1
a white powder (0.14 g, 17 %). mp 210ꢀ211 °C; FTIR: 1674 cm (C=O); H NMR (400 MHz,
CDCl
3
) δ 8.20 – 8.05 (m, 2H), 7.18 – 7.08 (m, 2H), 6.99 – 6.87 (m, 6H), 6.84 – 6.74 (m, 2H),
13
3
1
1
.75 – 3.65 (m, 4H), 3.47 – 3.40 (m, 4H); C NMR (101 MHz, CDCl ) δ 154.62, 154.21,
3
46.36, 139.22, 130.34, 126.03, 125.92, 124.26, 124.07, 123.99, 116.85, 116.69, 113.08,
+
12.32, 46.84, 46.12, 44.65, 29.68; MS (APCI) calcd for C23
H
21
N
4
O
4
[M+H] 417.1653;
found 417.1502; purity (HPLC): 95.43 %.
ꢀ(4ꢀ(10HꢀPhenoxazineꢀ10ꢀcarbonyl)piperazinꢀ1ꢀyl)benzonitrile (16i). The title compound
4
was prepared from 3a (2 mmol, 0.36 g) and 1ꢀ(4ꢀcyanophenyl)piperazine (0.37 g, 2 mmol) in
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1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described for 15g.
Purification by chromatography (ethyl acetate / petroleum ether, 3:7) afforded 16i as a white
ꢀ1
1
powder (0.44 g, 55 %). mp 197 °C; FTIR: 2207 (CN), 1690 cm (C=O); H NMR (400 MHz,
CDCl ) δ 7.54 – 7.45 (m, 2H), 7.13 – 7.05 (m, 2H), 6.96 – 6.88 (m, 6H), 6.86 – 6.79 (m, 2H),
.75 – 3.63 (m, 4H), 3.38 – 3.26 (m, 4H); C NMR (101 MHz, CDCl
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
3
3
) δ 154.52, 152.73,
146.20, 133.88, 133.59, 130.34, 124.15, 123.96, 119.61, 116.99, 116.68, 116.64, 114.65,
+
1
09.99, 101.47, 47.13, 44.73; MS (APCI) calcd for C H N O [M+H] 397.1665; found
2
4
21
4
2
397.1592; purity (HPLC): 100.00 %.
(2ꢀChloroꢀ10Hꢀphenoxazinꢀ10ꢀyl)(4ꢀ(2ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone (16j).
The title compound was prepared from 3b (2 mmol, 0.43 g) and 1ꢀ(2ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 3:7) afforded 16j as a white powder (0.49 g, 56%). mp 112 °C; FTIR: 1682
ꢀ1
1
cm (C=O); H NMR (600 MHz, CDCl
3
) δ 7.05 (t, J = 4.6 Hz, 2H), 6.95 – 6.81 (m, 8H), 6.76
1
3
(d, J = 8.5 Hz, 1H), 3.86 (s, 3H), 3.80 (s, 4H), 3.08 (s, 4H); C NMR (151 MHz, CDCl
3
) δ
53.47, 152.22, 144.99, 143.94, 140.31, 131.21, 129.62, 128.80, 124.14, 123.98, 123.78,
23.09, 121.08, 118.51, 117.13, 116.54, 115.75, 111.33, 55.45, 50.61, 45.50; MS (EI, 70 eV)
1
1
m/z (%) 220 (14), 219 (100), 191 (11), 134 (12); purity (HPLC): 98.99 %.
(2ꢀChloroꢀ10Hꢀphenoxazinꢀ10ꢀyl)(4ꢀ(3ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone (16k).
The title compound was prepared from 3b (2 mmol, 0.43 g) and 1ꢀ(3ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
ꢀ1
petroleum ether, 3:7) afforded 16k as a white powder (0.20 g, 23%). 78 °C; FTIR: 1670 cm
1
(C=O); H NMR (600 MHz, CDCl
3
) δ 7.19 (t, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.93 – 6.89 (m,
2H), 6.89 – 6.83 (m, 3H), 6.78 (d, J = 8.5 Hz, 1H), 6.58 – 6.53 (m, 1H), 6.49 (d, J = 8.3 Hz,
13
2
H), 3.79 (s, 3H), 3.76 (t, J = 5.2 Hz, 4H), 3.20 (t, J = 5.2 Hz, 4H); C NMR (151 MHz,
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2
2
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3
3
3
3
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4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
CDCl ) δ 160.65, 153.61, 145.27, 144.22, 131.19, 130.07, 129.62, 128.83, 124.23, 124.19,
3
1
23.33, 117.21, 117.20, 116.66, 116.09, 116.04, 109.55, 105.82, 103.46, 77.23, 77.02, 76.81,
5
5.27, 49.63, 45.07; MS (EI, 70 eV) m/z (%) 435 (10), 220 (13), 219 (100), 216 (11), 150
(10), 149 (14), 1345 (5), 134 (7) ; purity (HPLC): 100 %.
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
(2,8ꢀDichloroꢀ10Hꢀphenoxazinꢀ10ꢀyl)(4ꢀ(2ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone
(16l). The title compound was prepared from 3c (2 mmol, 0.50 g) and 1ꢀ(2ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 3:7) afforded 16l as a white powder (0.28 g, 30%). mp 136ꢀ137 °C; FTIR:
ꢀ1
1
1668 cm (C=O); H NMR (400 MHz, CDCl
8.5, 2.3 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 3.95 – 3.81 (m, 7H), 3.18 (s, br, 4H); C NMR
101 MHz, CDCl ) δ 152.87, 152.26, 143.51, 130.43, 129.07, 123.60, 121.25, 117.32, 115.72,
3
) δ 7.10 (s, 2H), 6.99 – 6.89 (m, 4H), 6.85 (dd, J
1
3
=
(
3
1
11.61, 55.55, 50.73, 45.17; MS (EI, 70 eV) m/z (%) 469 (4), 220 (15), 219 (100), 134 (11),
150 (10), 149 (14); purity (HPLC): 98.77 %.
(2,8ꢀDichloroꢀ10Hꢀphenoxazinꢀ10ꢀyl)(4ꢀ(3ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone
16m). The title compound was prepared from 3c (2 mmol, 0.47 g) and 1ꢀ(3ꢀ
(
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 3:7) afforded 16m as a white powder (0.59 g, 63%). mp 131 °C; FTIR: 1659
ꢀ1
1
cm (C=O); H NMR (400 MHz, CDCl
3
) δ 7.24 – 7.16 (m, 1H), 6.95 (d, J = 2.3 Hz, 2H), 6.86
(dd, J = 8.5, 2.3 Hz, 2H), 6.77 (d, J = 8.5 Hz, 2H), 6.61 – 6.47 (m, 3H), 3.82 – 3.71 (m, 7H;
1
3
three of them to s, 3.79, 3H), 3.25 – 3.19 (m, 4H); C NMR (101 MHz, CDCl ) δ 160.66,
3
160.65, 152.93, 143.70, 130.45, 130.11, 129.09, 123.72, 117.37, 115.92, 109.70, 106.31,
103.66, 55.27, 49.81, 45.06, 43.43; MS (EI, 70 eV) m/z (%) 471 (5), 470 (2), 469 (7), 220
(16), 219 (100), 150 (12), 149 (18), 148 (9); purity (HPLC): 99.50 %.
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10ꢀ(4ꢀ(2ꢀMethoxyphenyl)piperazineꢀ1ꢀcarbonyl)ꢀ10Hꢀphenoxazineꢀ3ꢀcarbonitrile (16n).
The title compound was prepared from 3d (2 mmol, 0.47 g) and 1ꢀ(2ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 3:7) afforded 16n as a white powder (0.52 g, 61%). mp 188 °C; FTIR: 2214
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ1
1
(CN), 1686 cm (C=O); H NMR (400 MHz, CDCl ) δ 7.28 – 7.23 (m, 1H), 7.17 (dd, J = 8.4,
3
1.9 Hz, 1H), 7.10 – 7.04 (m, 1H), 7.03 (d, J = 1.8 Hz, 1H), 6.96 – 6.87 (m, 5H), 6.85 – 6.80
13
(
m, 1H), 6.75 – 6.69 (m, 1H), 3.90 – 3.79 (m, 7H), 3.11 (s, 4H); C NMR (101 MHz, CDCl
3
)
δ 152.38, 152.19, 144.69, 143.92, 134.52, 128.79, 128.69, 124.56, 124.46, 121.11, 119.31,
118.39, 116.67, 114.97, 114.89, 111.46, 105.94, 55.46, 50.67, 45.40; MS (EI, 70 eV) m/z (%)
4
9
1
27 (3), 426 (8), 220 (14), 219 (100), 134 (17), 120 (14), 86 (11), 84 (15); purity (HPLC):
8.89 %.
0ꢀ(4ꢀ(3ꢀMethoxyphenyl)piperazineꢀ1ꢀcarbonyl)ꢀ10Hꢀphenoxazineꢀ3ꢀcarbonitrile (16o).
The title compound was prepared from 3d (2 mmol, 0.41 g) and 1ꢀ(3ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 3:7) afforded 16o as a white powder (0.44 g, 52%). mp 167 °C; FTIR: 2222
ꢀ1
1
(
CN), 1682 cm (C=O); H NMR (600 MHz, CDCl
3
) δ 7.19 (td, J = 8.2, 0.7 Hz, 1H), 7.17
(ddd, J = 8.4, 1.9, 0.8 Hz, 1H), 7.04 (dd, J = 1.9, 0.7 Hz, 1H), 6.94 – 6.88 (m, 3H), 6.86 – 6.82
(m, 1H), 6.73 – 6.69 (m, 1H), 6.52 (dd, J = 8.3, 2.3 Hz, 1H), 6.49 (dd, J = 8.1, 2.3 Hz, 1H),
13
6
.45 (d, J = 2.4 Hz, 1H), 3.83 – 3.74 (m, 7H), 3.21 (t, J = 5.2 Hz, 4H); C NMR (151 MHz,
CDCl ) 160.66, 152.42, 151.69, 144.83, 144.04, 134.51, 130.09, 128.81, 128.71, 128.70,
3
124.60, 124.58, 119.39, 118.37, 116.77, 115.08, 115.04, 109.47, 106.11, 105.69, 103.46,
+
55.26, 49.57, 45.11; MS (APCI) calcd for C25
purity (HPLC): 99.40 %.
H
23
N
O
4 3
[M+H] 427.1770; found 427.1786;
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2
2
2
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3
3
3
3
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10ꢀ(4ꢀ(4ꢀMethoxyphenyl)piperazineꢀ1ꢀcarbonyl)ꢀ10Hꢀphenoxazineꢀ3ꢀcarbonitrile (16p).
The title compound was prepared from 3d (2 mmol, 0.42 g) and 1ꢀ(4ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 1:1) afforded 16p as a white powder (0.23 g, 27%). mp 171 °C; FTIR: 1678
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
ꢀ1
1
cm (C=O); H NMR (400 MHz, CDCl ) δ 7.17 (ddd, J = 8.4, 1.9, 0.5 Hz, 1H), 7.04 (dd, J =
3
1
4
1
.8, 0.6 Hz, 1H), 6.94 – 6.88 (m, 5H), 6.87 – 6.82 (m, 3H), 6.74 – 6.68 (m, 1H), 3.81 (t, J =
13
.9 Hz, 4H), 3.77 (s, 3H), 3.09 (t, J = 5.1 Hz, 4H); C NMR (101 MHz, CDCl
3
) δ 152.39,
44.78, 144.00, 134.51, 128.79, 128.70, 124.57, 124.54, 119.36, 119.15, 118.36, 116.72,
23 4 3
115.04, 114.61, 109.99, 106.05, 55.55, 51.17, 45.31; MS (APCI) calcd for C25H N O
+
[
M+H] 427.1770; found 427.1811; purity (HPLC): 99.40 %.
(
5HꢀDibenzo[b,f]azepinꢀ5ꢀyl)(4ꢀ(2ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone (17a). The
title compound was prepared from 9 (2 mmol, 0.47 g) and 1ꢀ(2ꢀmethoxyphenyl)piperazine
0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 1:1)
(
ꢀ1
1
afforded 17a as a white powder (0.30 g, 37%). mp 188ꢀ189 °C; FTIR: 1647 cm (C=O); H
NMR (600 MHz, CDCl ) δ 7.59 (dd, J = 8.1, 1.2 Hz, 2H), 7.38 (ddd, J = 8.3, 7.2, 1.7 Hz, 2H),
3
7
.28 – 7.23 (m, 2H), 7.22 (td, J = 7.4, 1.3 Hz, 2H), 7.00 – 6.96 (m, 1H), 6.95 (s, 2H), 6.87 (td,
J = 7.6, 1.4 Hz, 1H), 6.84 – 6.77 (m, 2H), 3.81 (s, 3H), 3.36 – 3.23 (m, 4H), 2.88 – 2.60 (m,
1
3
4
3
H); C NMR (151 MHz, CDCl ) δ 218.62, 159.13, 152.13, 142.78, 140.95, 134.08, 131.32,
1
31.32, 129.04, 128.87, 127.63, 126.34, 123.16, 120.94, 118.22, 111.18, 55.36, 50.18, 46.15;
+
MS (APCI) calcd for C H N O [M+H] 412.2025; found 412.2060; purity (HPLC): 100.00
25
26
3
3
%
.
(5HꢀDibenzo[b,f]azepinꢀ5ꢀyl)(4ꢀ(3ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methanone (17b). The
title compound was prepared from 9 (2 mmol, 0.39 g) and 1ꢀ(3ꢀmethoxyphenyl)piperazine
(
0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as
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7
8
9
described for 15g. Purification by chromatography (ethyl acetate / petroleum ether, 1:1)
ꢀ
1
1
afforded 17b as a white powder (0.18 g, 22 %). mp 112ꢀ113 °C; FTIR: 1647 cm (C=O); H
NMR (400 MHz, CDCl ) δ 7.57 (dd, J = 8.0, 1.2 Hz, 2H), 7.38 (ddd, J = 7.8, 7.0, 1.8 Hz, 2H),
.30 – 7.19 (m, 4H), 7.13 (s, 1H), 6.96 (s, 2H), 6.41 (s, 3H), 3.76 (s, 3H), 3.27 (s, br, 4H),
3
7
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
3
.90 (s, br, 4H); C NMR (101 MHz, CDCl ) δ 160.53, 159.11, 142.54, 134.06, 131.28,
129.79, 129.08, 128.89, 127.63, 126.45, 108.89, 104.65, 102.60, 55.18, 48.55, 45.79, 31.58,
4.11; MS (EI, 70 eV) m/z (%) 412 (20), 411 (63), 263 (5), 262 (19), 250 (5), 259 (23), 221
6), 220 (36), 219 (72), 194 (9), 193 (45), 192 (100), 191 (37), 190 (212), 189 (5), 177 (9),
76 (11), 175 (42), 165 (21), 164 (10), 163 (47), 162 (45); purity (HPLC): 100.00 %.
1
(
1
(10,11ꢀDihydroꢀ5Hꢀdibenzo[b,f]azepinꢀ5ꢀyl)(4ꢀ(2ꢀmethoxyphenyl)piperazinꢀ1ꢀ
yl)methanone (18). The title compound was prepared from 10 (2 mmol, 0.39 g) and 1ꢀ(2ꢀ
methoxyphenyl)piperazine (0.38 g, 2 mmol) in 1,2ꢀdichloroethane (20 mL) and pyridine (1
mL) in a similar manner as described for 15g. Purification by chromatography (ethyl acetate /
petroleum ether, 1:1) afforded 18 as a white powder (0.22 g, 27%). mp 188ꢀ189 °C; FTIR:
ꢀ1
1
1643 cm (C=O); H NMR (400 MHz, CDCl
3
) δ 7.47 (s, 2H), 7.21 – 7.09 (m, 6H), 7.03 –
6
.95 (m, 1H), 6.93 – 6.81 (m, 3H), 3.83 (s, 3H), 3.52 (s, br, 4H), 3.17 (s, 4H), 2.90 (s, br, 4H);
13
C NMR (101 MHz, CDCl ) δ 158.66, 152.16, 143.04, 134.98, 129.85, 127.52, 126.69,
3
126.59, 123.22, 121.00, 118.27, 111.25, 55.37, 50.30, 46.22, 31.02; MS (EI, 70 eV) m/z (%)
414 (27), 413 (93), 219 (100), 195 (26), 194 (59), 193 (22), 1902 (13), 191 (21), 134 (49);
purity (HPLC): 100.00 %.
4ꢀ(3ꢀMethoxyphenyl)ꢀN,Nꢀdiphenylpiperazineꢀ1ꢀcarboxamide (19). The title compound
was prepared from 12 (2 mmol, 0.34 g) and 1ꢀ(3ꢀmethoxyphenyl)piperazine (0.38 g, 2 mmol)
in 1,2ꢀdichloroethane (20 mL) and pyridine (1 mL) in a similar manner as described for 15g.
Purification by chromatography (ethyl acetate / petroleum ether, 3:7) afforded 19 as a white
ꢀ1
1
powder (46 mg, 6 %). mp 97ꢀ98 °C; FTIR: 1655 cm (C=O); H NMR (400 MHz, CDCl
3
) δ
7
.36 – 7.28 (m, 4H), 7.20 – 7.11 (m, 3H), 7.10 – 7.03 (m, 4H), 6.52 – 6.40 (m, 3H), 3.78 (s,
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