Synthesis and biochemical characterization of new phenothiazines and related drugs as MDR reversal agents

Article abstract of DOI:10.1002/ardp.200800115

Chemotherapy is one of the most important methods in the treatment of cancer. However, development of drug resistance during chemotherapy is the leading cause of treatment failure and decreased survival in cancer patients. Multidrug resistance (MDR) is one of the extensively studied forms of drug resistance for more than 30 years. The members of ATP-binding cassette protein family are responsible for multidrug resistance with P-glycoprotein as most representative transporter. To overcome multidrug resistance, pharmacological modulation of the transporters by efflux pump inhibitors seem to be the first choice, but preclinical studies did not lead to clinical applications. Therefore, a systematical research for pharmacophor structures is a promising strategy to increase the efficacy of those drugs still influencing multidrug resistance. In this study a range of phenothiazine derivatives was synthesizied with systematical variation of three molecule domains. The biochemical determination of multidrug resistance reversal activity was achieved with the crystalviolet assay on LLC-PK1/MDR1 cells. The results will be discussed considering of hypotheses in the literature directed to new structure-acitivity relationships to overcome drug resistance in the future.

Full text of DOI:10.1002/ardp.200800115

624
Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
Full Paper
Synthesis and Biochemical Characterization of New
Phenothiazines and Related Drugs as MDR Reversal Agents
Matthias Schmidt1, Marlen Teitge1, Marianela E. Castillo1, Tobias Brandt2, Bodo Dobner2, and
Andreas Langner2
1 Department of Medicinal Chemistry, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle
(Saale), Germany
2 Department of Biochemical Pharmacy, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg,
Halle (Saale), Germany
Chemotherapy is one of the most important methods in the treatment of cancer. However,
development of drug resistance during chemotherapy is the leading cause of treatment failure
and decreased survival in cancer patients. Multidrug resistance (MDR) is one of the extensively
studied forms of drug resistance for more than 30 years. The members of ATP-binding cassette
protein family are responsible for multidrug resistance with P-glycoprotein as most representa-
tive transporter. To overcome multidrug resistance, pharmacological modulation of the trans-
porters by efflux pump inhibitors seem to be the first choice, but preclinical studies did not lead
to clinical applications. Therefore, a systematical research for pharmacophor structures is a
promising strategy to increase the efficacy of those drugs still influencing multidrug resistance.
In this study a range of phenothiazine derivatives was synthesizied with systematical variation
of three molecule domains. The biochemical determination of multidrug resistance reversal
activity was achieved with the crystalviolet assay on LLC-PK1/MDR1 cells. The results will be dis-
cussed considering of hypotheses in the literature directed to new structure-acitivity relation-
ships to overcome drug resistance in the future.
Keywords: Chemotherapy / Crystalviolet assay / LLC-PK1/MDR1 cells / P-Glycoprotein / Structure-acitivity relation-
ships /
Received: June 12, 2008; accepted: July 15, 2008
DOI 10.1002/ardp.200800115
gp are presently unknown. A range of clinically used
Introduction
drugs, e. g. calcium channel blockers, anti arrhythmics,
neuroleptics, and antidepressants are known to be able
to reverse MDR [1]. But the original pharmacological
effect of these substances turns into an unrequested side
effect. The last decades are characterized by the develop-
ment of the second (e. g. valspodar) and third (e. g. elacri-
dar and tariquidar) generation of potential modulators
but, actually, there is no drug with MDR indication in
clinical application at all [2]. An optimal chemosensitizer
for overcoming of multidrug resistance should inhibit
the transport protein, e. g. p-gp, without noteworthy side
effects and tolerable cytotoxicity [3]. Series of phenothia-
zines, thioxanthenes, and structurally related com-
pounds which are representative for the well studied
class of neuroleptics were investigated by CoMFA (Com-
Multidrug resistance (MDR) is one of the most important
reasons of failure in cancer chemotherapy. Responsible
for stagnation of the cytotoxic process is an evident drug
efflux mainly caused by the transmembrane transport P-
glycoprotein (p-gp). P-gp is a membrane-integrated trans-
port protein from the family of ATP-binding casette (ABC)
proteins. The direct structure-function relationships of p-
Correspondence: Matthias Schmidt, Department of Medicinal Chemis-
try, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg,
06120 Halle (Saale), Wolfgang-Langenbeck-Str. 4, Germany.
E-mail: matthias.schmidt@pharmazie.uni-halle.de
Fax: 0049 345 552-7018
Abbreviations: ATP-binding casette (ABC); modulator quotient (MQ);
multidrug resistance (MDR); p-glycoprotein (p-gp)
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
New Phenothiazines and Related Drugs as MDR Reversal Agents
625
parative Molecular Field Analysis) and CoMSIA (Compara- ided in various domains undergoing H-bond interactions
tive Molecular Similarity Indices Analysis). The dominant and hydrophobic interactions in different ways. Another
role of the hydrophobic and hydrogen-bond acceptor pharmacophor model was developed for Hoechst 33342
fields for MDR reversing activity of the investigated com- [18] containing five aromatic centers, four points for H-
pounds was demonstrated [4, 5]. The structural regions bond acceptors and three points for H-bond donors. The
responsible for differences in anti-MDR activity were ana- nitrogen can act as a donor or acceptor depending on
lyzed with respect to their hydrophobic, hydrogen-bond protonation. Generally, the results of all published mod-
acceptor, and steric nature [6].
els allow to hazard the guess that p-gp has multiple bind-
A key role of p-gp modulators is its high lipophilicity ing sites and can bind and release substrates in multiple
and the presence of two or more aromatic rings. Further- pathways [19].
more, at physiological pH, they have a positive charge
As result of the research concerning calmodulin antag-
caused by the basic part of the molecule resulting in an onists of first generation modulators, a range of drugs
amphiphilic character [7]. Tertiary amine structures (e. g. trifluoperazine, chlorpromazine, trifluopromazine,
show a better efficiency compared to primary or secon- flupenthixol) reversed MDR significantly at concentra-
dary. Additionally, the integration of tertiary amines tions ranging from 1 to 10 lM [20, 21, 4]. In continuation
into cyclic structures, e. g. piperazine and piperidine of our work to new potential modulators of MDR [22], we
derivatives, is beneficial [8]. Pearce et al. postulated two synthesized new phenothiazines and related drugs fol-
aromatic domains and a basic nitrogen atom, connected lowed by biochemical characterization to contribute to a
by an aliphatic linker as essential structural feature of better understanding of multidrug-resistance phenom-
MDR modulators [9]. Hait and Aftab published a model enon.
for a phenothiazine binding site on p-gp [10]. Thereby,
the lipophilic aromatic core structure of phenothiazines
interacts with two phenylalanine residues by p-electron
interactions. The N-containing protonable part of the
Chemistry
molecule interacts with a hydrophilic binding domain Phenothiazines and structurally related drugs are known
consisting of three glutamic acid residues. Not only the to possess antidopaminergic, anticalmodulin, and anti-
presence of aromatic ring systems, but also their steric tumor properties. Miller et al. also demonstrated that
orientation plays an important role in the modulatoric these compounds potentiate the clinical activity of cyto-
potential of the compounds. Suzuki et al. found the angle static drugs in carcinoma patients [23]. Several studies
of 90–1058 between the aromatics to be optimal, and the have been performed to correlate the in-vitro MDR rever-
distance between the protonable nitrogen and the center sal activity and the structure of this class of compounds.
of the hydrophobic domaine should be at least 5 ꢀ [11]. The results obtained show the role of hydrophobicity as a
Seelig postulated the existance of two or three electron- space-oriented molecular property to explain the rela-
donor groups with a defined steric distance as essential tionship between structure and activity [24, 25].
characteristic. Simultaneously, the strength of the bind-
Among the class of MDR-reversing agents, phenothia-
ing to p-gp correlates with the number and strength of zines and related compounds are known to sensitize
the electron-donor groups [12]. De facto, the transmem- MDR by interacting with ABC-transporters like p-gp.
branal domains 4–6 and 11–12 possess different amino There exists a range of general structure-acitivity rela-
acids with electron-acceptor groups [13]. Most of these tionships [26]. Zamora et al. emphasized the importance
transmembranal domains are exactly the areas responsi- of aromatic rings in the hydrophobic moiety of the drugs
ble for substrate binding and transport [14]. Based on the and also the relative ring position [7]. The compounds
analysis of various biological test systems, Ekins et al. with phenyl rings deviating from the plane show a
developed a pharmacophor model consisting of an H- higher activity than planar ring systems. In investiga-
bond acceptor, an aromatic ring, and two hydrophobic tions to chemosensitizers against chloroquine-resistant
molecule areas [15]. All models described overlap only in Plasmodium falciparum, a range of analogous compounds
parts, permiting the hypothesis that different substrate was designed and synthesized. Various aromatic amine
binding sites (3–4) exist in P-glycoprotein. A general ring systems, cyclic or noncyclic amino groups, and
pharmacophor model was created by Pajeva et al. [16] hydrophilic linkers were examined [27]. Variations in the
based on investigations to the affinity of modulators to “butterfly” angle between the two aromatic rings and ser-
the verapamil binding site [17]. It contains two lipophilic ies of N-10 alkyl amides of phenothiazines were looked at
domains, three H-bond acceptors, and a H-bond donor in in relation to their potency and selectivity toward choli-
stericly defined orientation. The binding site can be div- nesterase inhibition [28].
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626
M. Schmidt et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
For the synthesis of phenothiazines, three general aromatic domain, linker, and basic moiety. All final com-
methods are known. Bernthsen primarily described the pounds were purified by flash chromatography and con-
reaction of diphenylamine derivatives with sulfur in the verted into the maleic acid salts to have a water-soluble
presence of iodine as catalyst [29]. Phenothiazines with form for biochemical tests. Table 1 shows the synthesized
substituents in position 2, 3, and 4 can be achieved via final compounds. Group R1 represents the variable basic
the analogous {2-[(2-chlorophenyl)thio]phenyl}amine moiety, R2 –R5 substituents at the core structure, and n
derivatives [30]. An often used method for preparation of means the length of the linker. Some of the synthesized
2-substituted compounds comprises the reaction of 2- derivatives have been described elsewhere. In this study,
aminobenzenethiol with 2-chloro-1-nitrobenzene deriv- all the final compounds were fully characterized by 1H-
atives followed by acetylation and Smiles rearrangement NMR, ESI-MS, and elemental analysis.
[31, 32].
Various phenothiazines with functionalities at the
ring system in combination with modification of the N-
alkyl side chain were already synthesized by Golinski et
Bioevaluation
al. [33]. These phenothiazines were evaluated for their Preliminary for in-vitro cell tests of the synthesized com-
ability to inhibit the calmodulin-mediated inhibition of pounds, the crystalviolet assay was performed on LLC-
phosphodiesterase. The synthesis of the 2-phenothiazinyl PK1 cells. This method determines the viability of cells.
ketones was achieved by Friedel–Crafts method starting Consequently, it is possible to quantify the antiprolifera-
from 10-acetylphenothiazine [34].
tive effect of the cytostatic drug, and the cytostatic drug
Due to the structural similarity to the class of pheno- in combination with the test substance which is a param-
thiazines, a range of alternative structures were investi- eter of the MDR-reversal activity of the test substances.
gated regarding their MDR-reversing activity. This core The cytotoxicity of the compounds can also be analyzed
structures are well-known as components of drugs with by incubation of cells with different concentrations of
different indications – not yet in connection with MDR. the test substances and determination of the number of
Analysis of general structure-activity relationships let surviving cells. The crystalviolet test is based on the
assume a good MDR reversing effect for the structures: assessment of the crystalviolet-dye uptake by living cells.
5H-dibenzo[b,f]azepine, 10,11-dihydro-5H-dibenzo[b,f]aze- The absorption of the dye determined at a wavelength of
pine, 6,7-dihydro-5H-dibenzo[c,e]azepine, 1,2,3,9-tetra- 620 nm correlates with the number of living cells [38].
methoxy-6,7-dihydro-5H-dibenzo[c,e]azepine, 9H-carba- The tests were performed at a porcine kidney epithelial
zol, 5,5-diphenylimidazolidin-2,4-dione. Synthesis of 6,7- cell line (LLC-PK1) [39]. In this cell line, human P-glycopro-
dihydro-5H-dibenzo[c,e]azepine was achieved by the tein was overexpressed to warrant a high content of the
modified method of Hawkins and Fu [35] by direct reac- transporter (LLC-MDR1) which was approved by western
tion of 2,29-bis(bromomethyl)-1.19-biphenyl with the blot analysis [40]. LLC-MDR1 cells (0.56104 cells/mL) were
sodium salt of the trifluoroacetamid. After cyclization, seeded in 96-well microtiter plates and preincubated for
the alkaline hydrolysis of the amide led to the expected 5 h at 378C and 5% CO2. For the resistant cells posses a
compound. All further structures were commercially resistance against vincristine, we determined an IC50
available.
value for vincristine of 2.12 lM with a quite high stand-
One possibility for the insertion of the linkers is the ard deviation especially in the high concentration inter-
reaction in strongly alkaline medium created by the addi- val which is founded by the resistance phenomenon.
tion of sodiumhydrid [36]. Under these conditions, the According to clinical practices, we decided to use a con-
deprotonation of the nitrogen from weakly NH-acid com- stant molar concentration of the cytotoxic drug vincris-
pounds, e. g. R and S (abbreviations see Table 1), also suc- tine of 640 nM as standard. Therefore, cells were incu-
ceeded. The reaction was arranged with DMSO as solvent bated with vincristine (640 nM), vincristine (640 nM) +
under argon atmosphere resulting in a methylsulfinyl- references (0.1–400 lM), and finally, vincristine (640 nM)
carbanion [37]. Addition of the secondary amine + test substance (0.1–400 lM) for 68 h at 378C. The test
occurred after formation of the Na-dimsyl followed by for cytotoxicity was carried out treating of cells only with
deprotonation and finally adding the alkylhalogenides.
The last reaction step in our synthetic scheme is the N- experiment was performed 3–5 times at different days.
alkylation of the basic residue with the alkylated core For the evaluation of this test, the IC values were deter-
structures as intermediates leading to the desired final mined by the GraphPadPrism and Origin 7G program.
compounds. All final products are characterized by the To determine the modulator potential of the investi-
essential molecular structure consisting of lipophilic, gated substances, different values were calculated
the test substance in decreasing concentrations. Every
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Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
New Phenothiazines and Related Drugs as MDR Reversal Agents
627
Table 1. Scheme of the final compounds with activity.
Compound
n
R1
R2
R3
R4
R5
IC50mod.
pIC50mod. IC50tox.
pIC50tox. MQ
(lM l SD)
(lM l SD)
P3MP
P4MP
P5MP
P6MP
P7MP
P8MP
P10MP
P11MP
2
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
-O(CH2)2MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
MP
DPh1P
DPh2P
DPh3P
DPh4P
DPh5P
DPh1A
DPh2A
DPh3A
Cl-Ph.Ph1P
DFPh1P
H
H
H
H
H
H
H
H
H
=O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Ac
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
tBu
H
H
H
H
H
H
H
H
H
H
H
H
H
tBu
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.96 l 0.47 5.71
1.09 l 0.19 5.96
1.27 l 0.25 5.90
2.26 l 0.42 5.65
3.15 l 0.67 5.50
2.52 l 0.21 5.60
4.87 l 1.27 5.31
7.56 l 1.27 5.12
6.25 l 1.10 5.20
7.64 l 0.67 5.12
4.32 l 1.06 5.37
1.73 l 0.19 5.76
1.50 l 0.06 5.82
0.49 l 0.08 6.31
0.72 l 0.02 6.14
0.22 l 0.04 6.66
0.91 l 0.22 6.04
0.46 l 0.18 6.34
1.24 l 0.39 5.91
0.40 l 0.14 6.40
0.41 l 0.13 6.39
4.40 l 0.73 5.36
3.07 l 0.35 5.51
1.88 l 0.26 5.73
0.76 l 0.11 6.12
0.36 l 0.01 6.44
0.20 l 0.01 6.70
9.82 l 1.42 5.01
5.17 l 0.97 5.29
5.99 l 0.55 5.22
3.06 l 0.36 6.51
5.42 l 1.55 5.27
1.97 l 0.40 5.71
1.17 l 0.17 5.93
1.89 l 0.39 5.72
0.51 l 0.04 6.29
0.62 l 0.14 6.21
2.29 l 0.46 5.64
1.22 l 0.13 5.91
1.99 l 0.14 5.70
1.68 l 0.17 5.78
1.72 l 0.20 5.76
0.58 l 0.07 6.24
0.70 l 0.08 6.16
5.32 l 0.24 5.27
2.62 l 0.26 5.58
2.39 l 0.89 5.62
5.25 l 0.65 5.28
4.77 l 0.31 5.32
28.22 l 3.71 4.55
8.91 l 1.24 5.05
4.54
4.09
6.17
7.32
5.62
3,01
2.46
3.12
2.43
3
4
5
6
7
9
10
11
2
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
5
7
7
7
3
3
3
3
3
3
3
3
3
3
3
4.44 l 1.03 5.35
7.83 l 0.49 5.11
16.55 l 2.53 4.78
17.72 l 0.71 4.75
7.57 l 0.91 5.12
12.00 l 3.81 4.92
23.57 l 2.02 4.63
15.21 l 3.26 4.82
119.67 l 8.21 3.92
17.58 l 1.88 4.76
14.87 l 0.26 4.83
25.41 l 5.94 4.60
13.18 l 2.06 4.88
15.77 l 1.30 4.80
6.31 l 0.39 5.20
14.25 l 0.62 4.85
7.64 l 0.39 5.12
6.13 l 1.05 5.21
7.91 l 1.08 5.10
8.33 l 1.85 5.08
22.65 l 3.12 4.65
15.73 l 0.75 4.80
22.47 l 5.58 4.65
11.53 l 3.40 4.94
7.32 l 0.18 5.14
7.66 l 0.58 5.12
47.43 l 3.05 4.32
14.66 l 0.66 4.83
21.78 l 2.98 4.66
13.32 l 1.21 4.88
21.44 l 3.16 4.67
5.42 l 0.58 5.27
10.32 l 2.33 4.99
10.24 l 2.05 4.99
6.21 l 0.43 5.21
8.01 l 0.74 5.10
27.74 l 1.55 4.56
8.43 l 0.62 5.07
24.09 l 2.04 4.62
24.04 l 1.68 4.62
22.82 l 2.44 4.64
9.17 l 1.06 5.04
10.55 l 0.88 4.98
308 l 18.21 3.51
19.28 l 2.72 4.72
12.58 l 1.19 4.90
43.33 l 1.44 4.36
57.07 l 2.91 4.24
62.41 l 2.95 4.21
P12MP
P3acMP
P2O2MP
15.67
4.07
8.60
2MeP3MP
2AcP3MP
2Prop(ac)P3MP
2Bu(ac)P3MP
2BzlP3MP
3MeOP3MP
3EtOP3MP
DtBuP3MP
[a]BnP3MP
[c]BnP3MP
2MeOP4MP
2MeSP4MP
2AcP4MP
2Prop(ac)P4MP
2Bu(ac)P4MP
2BzlP4MP
3MeP4MP
3BuP4MP
3iBuOP4MP
3PhP4MP
tBuP4MP
DtBuP4MP
[a]BnP4MP
[c]BnP4MP
2BzlP6MP
2Prop(ac)P8MP
2Bu(ac)P8MP
2BzlP8MP
P4DPh1P
P4DPh2P
P4DPh3P
P4DPh4P
P4DPh5P
P4DPh1A
P4DPh2A
P4DPh3A
P4Cl-Ph.Ph1P
P4DFPh1P
P4DPhOH1PIP
16.97
26.89
21.88
28,79
15.71
16.53
4.95
19.93
20,31
5.15
Prop(ac) H
Bu(ac)
Bzl
H
H
H
[a]Bn
H
MeO
MeS
Ac
H
H
MeO
EtO
tBu
H
[c]Bn
H
H
H
5.12
11.92
15.16
20.19
38.37
4.83
2.84
3.63
4.35
3.96
Prop(ac) H
Bu(ac)
Bzl
H
H
H
H
H
H
[a]Bn
H
Bzl
Prop(ac) H
Bu(ac)
Bzl
H
H
H
H
H
H
H
Me
Bu
iBuO
Ph
tBu
tBu
H
2.75
8.85
5.40
[c]Bn
H
12.18
12.92
12.10
6.92
12.14
14.28
13.26
15.79
15.16
8.73
7.36
5.24
8.26
11.96
2.21
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
DPhOH1PIP H
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628
M. Schmidt et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
Table 1. Continued.
Compound
n
R1
R2
R3
R4
R5
IC50mod.
pIC50mod. IC50tox.
pIC50tox. MQ
(lM l SD)
(lM l SD)
MTC4MP
MTC4DPP
Ib4MP
Ib4DPP
Is4MP
3
MP
DPh5P
MP
DPh5P
MP
DPh5P
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4.53 l 0.44 5.34
0.63 l 0.06 6.20
41.44 l 5.48 4.38
1.09 l 0.11 5.96
1.12 l 0.23 5.95
5.96 l 1.08 5.23
14.81 l 0.44 4.83
3.27
21.70
8.72
13.88
13.9
3
3
3
3
3
13.64 l 0.47 4.87
361.47 l 7.89 3.44
15.13 l 2.74 4.82
15.65 l 1.29 4.81
53.83 l 8.49 4.27
CH2-CH2
CH2-CH2
CH=CH
CH=CH
Is4DPP
9.03
R4DPP
S4DPP
3
3
DPh5P
DPh5P
H
OCH3
H
OCH3
H
OCH3
–
–
2.85 l 0.25 5.55
4.59 l 0.49 5.34
8.38 l 1.47 5.08
15.03 l 1.38 4.82
2.94
3.27
MTP4MP
MTP4DPP
3
3
MP
DPh5P
–
–
–
–
–
–
–
–
45.48 l 3.27 4.34
1.99 l 0.15 5.70
130.93 l 8.45 3.88
6.61 l 0.89 5.18
2.88
3.32
trifluoperazine
flupentixol
fluphenazine
propafenone
verapamil
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.20 l 0.48 5.92
0.45 l 0.12 6.35
1.22 l 0.13 5.91
1.30 l 0.27 5.89
1.30 l 0.24 5.89
9.41 l 1.23 5.03
9.67 l 0.56 5.02
10.58 l 1.11 4.98
25.10 l 2.4 4.60
65.03 l 10.33 4.19
7.84
21.71
8.68
19.28
50.00
–
–
–
Abbreviations: MP = methylpiperazine; DPh1P = diphenylmethylpiperazine; DPh2P = diphenylethylpiperazine; DPh3P = diphenyl-
propylpiperazine; DPh4P = diphenylbutylpiperazine; DPh5P = diphenylpentylpiperazine, DPh1A = diphenylmethylamine; DPh2A =
diphenylethylamine; DPh3A = diphenylpropylamine; Cl-Ph,Ph1P = (4-chlorophenyl)phenylmethylpiperazine; DFPh1P = bis(4-fluoro-
phenyl)methylpiperazine; DPhOH1PIP = 4-[hydroxy(diphenyl)methyl]piperidine; Prop(ac) = propionyl residue; Bu(ac) = butyryl resi-
due; IC50mod. = molar concentration (lM) of the modulator that inhibits the growth of cells by 50% in presence of a constant concen-
tration of 640 nM vincristine, IC50tox. = molar inhibitory concentration (lM) of the modulator without vincristine; MQ = modulator
quotient.
(Table 1). IC50tox. represents the molar concentration of a is the ratio of IC50mod. to IC50tox. and is deemed to be the
substance with a viability of the cells of 50%. It is a quan- measurement of the modulator activity of substances
tity of the toxicity of the investigated compound. IC50mod. depending on their toxicity.
represents the molar concentration of the substance
MQ = IC50tox. / IC50mod.
with a viability of 50% in the presence of a constant con-
centration of cytostatic drugs. During the experiments,
we chose 640 nM vincristine sulfate which was well toler-
ated by the LLC-MDR1 cells. The modulator quotient MQ
It was assumed that an inhibition of the transport by
modulators leads to an increase of the toxic effect of cyto-
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New Phenothiazines and Related Drugs as MDR Reversal Agents
629
Figure 1. Comparison of IC50mod. values (dark-colored) with variable phenothiazine core structures, constant
linker length (C3) and 4-methylpiperazine residue (control substances light-colored).
static drug and, consequently, to a decrease of the viabil- 4-methylpiperazine residue based on the structure of tri-
ity of cells. fluperazine as variance comparison. It was shown that
In conclusion, this method allows fast quantification the benzoyl, propionyl, and butyryl residues in the 2-posi-
of the interaction of drugs with p-gp and has the poten- tion are the most effective substituents. Good results
tial to serve as a high-throughput screening to detect were obtained with compounds containing a condensed
compounds prone to p-gp-mediated transport.
benzene ring in [a] and [c] position, or ethoxy and
methoxy groups in the 3-position. Less effective were sub-
stances with voluminous tert.–butyl-residues or hydro-
phobic alkyl residues in positions 3 and 7.
Results and discussion
One of the main problems of MDR is the fact that the
Based on the results of the cell experiments and besides original pharmacological effect of drugs, e. g. phenothia-
general structure-activity relationships, conclusions zines (blockade of dopamine, muscarinic or histamine
should also be drawn on the effectiveness of separate receptors) can limit the use as MDR modulator. We tried
structures of the compounds by systematic variation and to weaken the neuroleptic effects by modifying the
combination of molecule domains (lipophilic core struc- length of the linker. First step was the elongation of the
ture, linker, basic residue). In the following schemata, linker to four carbon atoms. As a result of this test, com-
the determined values of selected compounds will be pounds with a C4-linker also show the same tendency in
illustrated, each with two identical, constant, and one substitution resulting in substance 2BzlP4MP with the
variable molecule domain. The IC50mod. values of the lowest IC50mod. value (0.20 lM) of all synthesized com-
respective substances will be compared. We used these pounds. Generally, it should be noted that the extension
values, and not MQ, because the drawback of this factor of the linker leads at most to a marginal reduction of the
is that the quotient of high scores can sham a good MQ- modulator activity of the compounds tolerable by expect-
value with very low toxicity and minor modulator acitiv- ant loss of side effects (Fig. 2).
ity. Therefore, in analysis and discussion, the IC50mod. was
chosen as parameter to make conclusions about struc- alkyl chains on the modulator activity of phenothiazine
ture-activity-relationships. derivatives with constant core structure and a 4-methyl-
Figure 3 shows the influence of different linkers and
Figure 1 shows the IC50mod. values with variable pheno- piperazine residue. Introduction of a carbonyl or ethyl-
thiazine core structures, constant linker length (C3), and ene group leads to reduction of the activity as well as the
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630
M. Schmidt et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
Figure 2. Comparison of IC50mod. values (dark-colored) with variable phenothiazine core structures, con-
stant linker length (C4) and 4-methylpiperazine residue (control substances light-colored).
Figure 3. Comparison of IC50mod. values (dark-colored) with variable linker, constant phenothiazine
core structure and 4-methylpiperazine residue (control substances light-colored).
elongation of the alkyl chain to about six carbon atoms. residue and piperazine structure (C1–C3), introduction
Linker length between C3 and C5 do not seem to differ of a hydroxyl group or H-bond donator groups (Cl-Ph,
significantly in their activity considering the SD-values.
Ph1P and DFPh1P), and loss of an N-containing ring sys-
Figure 4 shows the IC50mod. values of compounds with tem (DPh1A, DPh2A, DPh3A) lead to reduction of basicity
variable basic residue and a constant 10-butyl-phenothia- of the piperazine and piperidine structures, respectively,
zine residue. As a result of these investigations, the diphe- other basic moieties. Generally, the combination of N-
nylalkylpiperazine residues with butyl and pentyl chains containing ring systems with high basicity and a defined
show better activity than the 4-methylpiperazine residue distance between piperazine and the hydrophobic
as standard. Contraction of the linker between diphenyl domains is favorably for modulator activity.
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New Phenothiazines and Related Drugs as MDR Reversal Agents
631
Figure 4. Comparison of IC50mod. values (dark-colored) with variable basic residue and constant 10-butyl-
phenothiazine residue (control substances light-colored).
Figure 5. Comparison of IC50mod. values (dark-colored) with variable non phenothiazine core struc-
tures and constant diphenylpentylpiperazine residue with C4 linker (control substances light-colored).
Comparison of the other hydrophobic core structures Ib4DPP show IC50mod. values comparable with the best
was realized using substances with constant C4 linker phenothazine derivatives (Fig. 5). These results are aston-
and diphenylpentylpiperazine residue as most efficient ishing because carbazole is a planar tricyclic ringsystem
basic moiety resulting from the analysis (Fig. 4). Gener- (1808) and 5H-dibenzo[b,f]azepine also differs with an
ally, most of the tested compounds have a lower poten- angle of 1208 from the hypothesis of Suzuki et al.
tial to reverse MDR in this assay. Only the carbazole deriv-
In this comparison, as in the other comparisons, the
ative MTC4DPP and the 5H-dibenzo[b,f]azepine derivative IC50mod values of clinical drugs with known modulatoric
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632
M. Schmidt et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
lett-Packard HP 5890 II / MS: 5971 A (Hewlett-Packard, Palo Alto,
CA, USA). Elemental analyses were performed on a CHNS-932
microanalyzer (LECO-Corporation, St. Joseph, MI, USA). H-NMR
effect on p-gp were used as controll substances. The best
modulator activity was determined for the thioxanthene
derivative cis-flupentixol. The phenothiazine derivatives
trifluoperazine and fluphenazine, the calcium antago-
nist verapamil, and the anti-arrhythmic propafenon
show approximately identical values. Considering the
toxicity, the most potent efflux-pump inhibitor is verapa-
mil as result of evaluation of the modulator quotient val-
ues (MQ).
1
spectra were obtained with a Varian Gemini 2000 spectrometer
(Varian Inc., Palo Alto, CA, USA) operating at 400 MHz; all values
are reported in ppm (d) downfield from solvent. Polarimetric
measurements were accomplished by an Eloptron/Polartronic E
(Fa. Schmid + Haensch GmbH & Co, Germany). Flash chromatog-
raphy was performed on silica gel (Kieselgel 60, 40–63 mesh;
Merck, Darmstadt, Germany). TLC was carried out on silica gel
plates (E. Merck 60 F254); zones were detected visually by ultravio-
let irradiation (254 nm). All reagents were used as purchased
unless otherwise stated. Solvents were dried, according to stand-
ard procedures. All reactions were carried out under an atmos-
phere of dry argon. All chemicals were purchased from Sigma-
Aldrich Chemie GmbH (Munich, Germany).
Conclusions
The aim of this study was the systematical modification
of phenothiazine derivatives in order to find new struc-
tures suitable as MDR-reversal agents. Generally, the
majority of our newly synthezised compounds has
shown significant modulatoric activity on p-gp. The in-
vitro tests were realized with the crystalviolet assay on
LLC-PK1/MDR1 cells to determine IC50 values of the modu-
latoric activity, toxicity, and modulator quotient, respec-
tively.
Chemistry
5,7-Dihydro-6H-dibenz[c,e]azepin R
Synthesis of R was achieved by a modified method of Hawkins
and Fu [26]. Experimental data corresponds with the literature
[41].
25: Yield 51%; Anal. C14H13N requires: C, 86.12; H, 6.71; N 7.17;
found: C, 86.19; H, 6.78; N, 7.08; MS (ES+) m/z: 195.3 [M + H].
General procedure for the final compounds
In particular, the influence of substituents in positions
2, 3, and 7 at the phenothiazine core structure was inves-
tigated with the result that the 2-benzoyl residue is the
most effective group. Investigations of various linker
lengths and structures revealed that the butyl chain is
the optimal linker with the benefit of side effect reversal.
Amongst compounds with different basic moieties, the
diphenylpentylpiperazine residue is the most promising
group.
The data generated in this project enable suggestions
to be made for further structure-activity relationships.
Future studies will be directed to design compounds
with optimal combination of several molecule domains,
and to computer-aided methods involving new, more
efficient QSAR methods, which will hopefully lead to the
directed prediction of simplified, even more active and
less toxic substances for further development.
10 mmol of the compounds core structure and 10 mmol sodium
hydrid were dissolved in 25 mL DMSO and stirred for 1 h at RT
under argon atmosphere. To the mixture, 10 mmol dibromoal-
kane was added and stirred for 12 h at RT. Once the reaction was
finished, the mixture was hydrolyzed with water. The organic
layer was diluted with 100 mL AcOEt, washed twice with 30 mL
saturated sodium bicarbonate solution, dried (Na2SO4) and
evaporated. The crude residue was analyzed by MS (ES+). The
intermediates were used without futher purification.
Method A: 10 mmol of the hydrophobic structure / linker was
dissolved in 30 mmol of the basic moiety and heated for 3 h at
1208C. Then, 50 mL water and 50 mL AcOEt were given to the
mixture. The organic layer was separated, washed with water,
dried (Na2SO4), and evaporated in vacuo. The residue was purified
by flash chromatography (CHCl3 / MeOH 9 : 1) to get the product
as base which was converted into the maleic acid salts.
Method B: 10 mmol sodium hydrid was dissolved in 30 mL
DMSO and stirred for 1 h at room temperature. 10 mmol of the
basic moiety was given to the mixture and stirred for another
hour at room temperature. Then 10 mmol of the hydrophobic
structure / linker was given to the mixture and stirred for 12 h at
room temperature. Once the reaction was finished, it was hydro-
lyzed with water. The mixture was extracted with AcOEt. The
organic layer was dried (Na2SO4) and evaporated in vacuo. The res-
idue was purified by flash chromatography (CHCl3 / MeOH 9 : 1)
to get the product as base which was converted into the maleic
acid salts.
This work was supported by research funds of “Kultusministe-
rium des Landes Sachsen-Anhalt”.
The authors have declared no conflict of interest.
Experimental section
General method for synthesis of the maleic acid salts
To a solution of the free base in Et2O was dropped a saturated sol-
ution of maleic acid in Et2O. Once the precipitation was finished,
the salt was filtered off in vacuo and recrystallized from MeCN to
get the salts as piperidine hydrogen maleate and piperazine bis-
(hydrogen maleate).
Materials and general methods
Mass spectra (MS) were recorded on a Finnigan MAT-710C spec-
trometer (Thermo Electron Corporation, Bremen, Germany). Gas
chromatography-mass spectra (GC-MS) were recorded on a Hew-
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New Phenothiazines and Related Drugs as MDR Reversal Agents
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10-[3-(4-Methylpiperazino)propyl]phenothiazine bis-
(hydrogen maleate) P3MP
10-[10-(4-Methylpiperazino)decyl]phenothiazine bis-
(hydrogene maleate) P10MP
Yield 67%; 1H-NMR (CD3OD)
d
[ppm]: 1.93–2.03 (qd, 2H,
Yield 55%; 1H-NMR (CD3OD) d [ppm]: 1.22–2.13 (m, 16H,
-CH2(CH2)8CH2-), 2.62 (s, 3H, -CH3) 3.00–3.20 (m, 10H, -NCH2-), 4.21
(t, 2H, -CH2(CH2)8CH2-), 6.26 (s, 4H, maleate), 7.23–7.89 (m, 8H,
aromat.); Anal. C35H47N3O8S requires: C, 62.76; H, 7.07; N, 6.27; S,
4.79; found: C, 62.68; H, 7.14; N, 6.24; S, 4.63; MS (ES+) m/z: 438.7
[M + H].
-CH2CH2CH2-), 2.66–2.75 (m, 9H, -CH3, -NCH2), 3.03 (m, 4H,
-CH2CH2CH2-, -NCH2-), 4.04 (t, 2H, -CH2CH2CH2-), 6.27 (s, 4H, mal-
eate), 6.79–7.02 (m, 4H, aromat.), 7.10-7.22 (m, 4H, aromat.);
Anal. C28H33N3O8S requires: C, 58.83; H, 5.82; N, 7.35; S 5,61;
found: C, 58.62; H, 5.89; N, 7.38; S, 5.52; MS (ES+) m/z: 340.5 [M +
H].
10-[11-(4-Methylpiperazino)undecyl]phenothiazine bis-
(hydrogen maleate) P11MP
10-[4-(4-Methylpiperazino)butyl]phenothiazine bis-
(hydrogene maleate) P4MP
Yield 48%; 1H-NMR (CD3OD) d [ppm]: 1.25–2.11 (m, 18H,
-CH2(CH2)9CH2-), 2.61 (s, 3H, -CH3) 3.02–3.18 (m, 10H, -NCH2-), 4.18
(t, 2H, -CH2(CH2)9CH2-), 6.24 (s, 4H, maleate), 7.18–7.85 (m, 8H,
aromat.); Anal. C36H49N3O8S requires: C, 63.23; H, 7.22; N, 6.14; S,
4.69; found: C, 63.18; H, 7.15; N, 6.22; S, 4.56; MS (ES+) m/z: 452.7
[M + H].
Yield 69%; 1H-NMR (CD3OD)
d [ppm]: 1.69–1.88 (m, 4H,
-CH2CH2CH2CH2-), 2.63–2.92 (m, 13H, -CH3, -NCH2-), 4.03 (t, 2H,
-CH2-(CH2)3-), 6.27 (s, 4H, maleate), 6.83–7.01 (m, 4H, aromat.),
7.11–7.23 (m, 4H, aromat.); Anal. C29H35N3O8S requires: C, 59.47;
H, 6.02; N, 7.17; S, 5.47; found C, 59.71; H, 6.17; N, 7.24; S, 5.53;
MS (ES+) m/z: 354.5 [M + H].
10-[12-(4-Methylpiperazino)dodecyl]phenothiazine bis-
(hydrogen maleate) P12MP
Yield 45%; 1H-NMR (CD3OD) d [ppm]: 1.22–2.12 (m, 20H,
-CH2(CH2)10CH2-), 2.58 (s, 3H, -CH3) 3.01–3.23 (m, 10H, -NCH2-),
4.19 (t, 2H, -CH2(CH2)10CH2-), 6.24 (s, 4H, maleate), 7.05–7.81 (m,
8H, aromat.); Anal. C37H51N3O8S requires: C, 63.68; H, 7.37; N,
6.02; S, 4.59; found: C, 63.57; H, 7.21; N, 6.11; S, 4.53; MS (ES+) m/
z: 466.7 [M + H].
10-[5-(4-Methylpiperazino)pentyl]phenothiazine bis-
(hydrogen maleate) P5MP
Yield 57%; 1H-NMR (CD3OD)
d [ppm]: 1.52–1.85 (m, 6H,
-CH2(CH2)3CH2-), 2.61–2.91 (m, 13H, -CH3, -NCH2-), 3.95 (t, 2H,
-CH2(CH2)4-), 6.27 (s, 4H, maleate), 6.87–6.98 (m, 4H, aromat.),
7.08–7.21 (m, 4H, aromat.); Anal. C30H37N3O8S requires: C, 60.09;
H, 6.22; N, 7.01; S, 5.35; found: C, 60.18; H, 6.32; N, 7.12; S, 5.27;
MS (ES+) m/z: 368.5 [M + H].
12-[3-(4-Methylpiperazino)propyl]benzo[a]phenothiazine
bis-(hydrogene maleate) Bn[a]P3MP
1
Yield 53%; H-NMR (CD3OD) d [ppm]: 1.81 (m, 2H, -CH2CH2CH2-),
2.15 (s, 3H, -CH3), 2.58–2.77 (m, 10H, -NCH2-), 4.07 (m, 2H,
-CH2CH2CH2-), 6.26 (s, 4H, maleate), 6.97–7.76 (m, 9H, aromat.),
8.04 (d, 1H, C1 aromat.); Anal. C32H35N3O8S requires: C, 61.82; H,
5.67; N, 6.76; S, 5.16; found: C, 61.75; H, 5.75; N, 6.68; S, 5.09; MS
(ES+) m/z: 390.6 [M + H].
10-[6-(4-Methylpiperazino)hexyl]phenothiazine bis-
(hydrogen maleate) P6MP
Yield 58%; 1H-NMR (CD3OD)
d [ppm]: 1.54–1.89 (m, 8H,
-CH2(CH2)4-), 2.60-2.93 (m, 13H, -CH3, -NCH2-), 3.99 (t, 2H,
-CH2(CH2)5-), 6.31 (s, 4H, maleate), 6.75–6.99 (m, 4H, aromat.),
7.07–7.24 (m, 4H, aromat.); Anal. C31H39N3O8S requires: C, 60.67;
H, 6.41; N, 6.85; S, 5.22; found: C, 60.58; H, 6.37; N, 6.92; S, 5.29;
MS (ES+) m/z: 382.6 [M + H].
7-[3-(4-Methylpiperazino)propyl]benzo[c]phenothiazine
bis-(hydrogen maleate) Bn[c]P3MP
1
Yield 57%; H-NMR (CD3OD) d [ppm]: 1.82 (m, 2H, -CH2CH2CH2-),
2.28 (s, 3H, -CH3), 2.52–2.80 (m, 10H, -NCH2-), 4.05 (m, 2H,
-CH2CH2CH2-), 6.25 (s, 4H, maleate), 6.96–7.84 (m, 9H, aromat.),
8.05 (d, 1H, C6 aromat.); Anal. C32H35N3O8S requires: C, 61.82; H,
5.67; N, 6.76; S, 5.16; found: C, 61.78; H, 5.71; N, 6.69; S, 5.11; MS
(ES+) m/z: 390.6 [M + H].
10-[7-(4-Methylpiperazino)heptyl]phenothiazine bis-
(hydrogen maleate) P7MP
Yield 53%; 1H-NMR (CD3OD) d [ppm]: 1.55–1.92 (m, 10H,
-CH2(CH2)5-), 2.59–2.91 (m, 13H, -CH3, -NCH2-), 3.95 (t, 2H,
-CH2(CH2)6-), 6.33 (s, 4H, maleate), 6.76–6.98 (m, 4H, aromat.),
7.04–7.24 (m, 4H, aromat.); Anal. C32H41N3O8S requires: C, 61.23;
H, 6.58; N, 6.69; S, 5.11; found: C, 61.31; H, 6.61; N, 6.62; S, 5.07;
MS (ES+) m/z: 396.6 [M + H].
3,7-Di-tert-butyl-10-[3-(4-methylpiperazino)propyl]-
phenothiazine bis-(hydrogen maleate) DTBuP3MP
Yield 52%; 1H-NMR (CD3OD) d [ppm]: 1.36 (s, 18H, -CCH3), 1.94–
1.98 (m, 2H, -CH2CH2CH2-), 2.53–3.19 (m, 13H, -CH3, -NCH2), 3.59
(t, 2H, -CH2CH2CH2-), 6.26 (s, 4H, maleate), 7.09–7.82 (m, 6H, aro-
mat.); Anal. C36H49N3O8S requires: C, 63.23; H, 7.22; N, 6.14; S,
4.69; found: C, 63.32; H, 7.27; N, 6.07; S, 4.63; MS (ES+) m/z: 452.7
[M + H].
10-[8-(4-Methylpiperazino)octyl]phenothiazine bis-
(hydrogen maleate) P8MP
Yield 56%; 1H-NMR (CD3OD) d [ppm]: 1.30–1.89 (m, 12H,
-CH2(CH2)6CH2-), 2.60–2.96 (m, 13H, -CH3, -NCH2-), 3.93 (t, 2H,
-CH2(CH2)6CH2-), 6.27 (s, 4H, maleate), 6.89–6.95 (m, 4H, aromat.),
7.06–7.15 (m, 4H, aromat.); Anal. C33H43N3O8S requires: C, 61.76;
H, 6.75; N, 6.85; S, 5.00; found: C, 61.68; H, 6.81; N, 6.81; S, 4.93;
MS (ES+) m/z: 410.6 [M + H].
10-[3-(4-Methylpiperazino)propanoyl]phenothiazine bis-
(hydrogen maleate) P3acMP
To a solution of 25 mmol phenothiazine dissolved in 25 mL tol-
uene 30 mmol 3-chloropropanoyl chloride was given dropwise.
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M. Schmidt et al.
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The mixture was refluxed for 5 h. When the reaction was fin-
ished the mixture was evaporated and recrystallized from EtOH.
The intermediate was processed according to Method A.
10-[4-(4-(5,5-Diphenylpentyl)piperazino)butyl]-
phenothiazine bis-(hydrogen maleate) P4DPh5P
Yield 54%; 1H-NMR (CD3OD) d [ppm]: 1.38–2.05 (m, 10H, -CH2-),
2.61–3.12 (m, 12H, -NCH2-), 3.37 (t, 2H, -CH2CH2CH2CH2-P), 3.78 (t,
1H, -CH-), 6.24 (s, 4H, maleate), 6.92–7.33 (m, 18H, aromat.);
Anal. C45H51N3O8S requires: C, 68.07; H, 6.47; N, 5.29; S, 4.04;
found: C, 67.95; H, 6.37; N, 5.21; S, 4.01; MS (ES+) m/z: 562.8 [M +
H].
Yield 72%; 1H-NMR (CD3OD) d [ppm]: 2.25 (t, 2H, -COCH2-), 2.39
(s, 3H, -CH3), 3.13–3.71 (m, 10H, -NCH2-), 6.24 (s, 4H, maleate),
7.16–7.34 (m, 4H, aromat.), 7.40–7.52 (m, 4H, aromat.); Anal.
C28H31N3O9S requires: C, 57.43; H, 5.34; N, 7.18; S, 5.47; found: C,
57.37; H, 5.39; N, 7.08; S, 5.36; MS (ES+) m/z: 354.5 [M + H].
10-[(4-Methylpiperazino)ethoxyethyl]phenothiazine bis-
10-[4-(Diphenylmethylamino)butyl]phenothiazine
hydrogen maleate P4DPh1A
Yield 18%; H-NMR (CD3OD) d [ppm]: 1.76–1.83 (m, 4H, -CH2-),
2.95–3.03 (t, 2H, -CH2CH2CH2CH2NH-), 3.83 (t, 2H,
-CH2CH2CH2CH2NH-), 4.18–4.26 (t, 1H, -CH-), 6.24 (s, 2H, maleate),
6.91–7.22 (m, 18H, aromat.); Anal. C33H32N2O4S requires: C,
71.72; H, 5.84; N, 5.07; S, 5.80; found: C, 71.76; H, 5.81; N, 5.03; S,
5.75; MS (ES+) m/z: 437.6 [M + H].
(hydrogen maleate) P2O2MP
Yield 79%; 1H-NMR (CD3OD) d [ppm]: = 2.67 (s, 3H, -CH3), 2.78–
2.96 (m, 10H, -OCH2CH2-, -NCH2-), 3.63 (t, 2H, -OCH2CH2-), 3.79 (t,
2H, -CH2CH2O-), 4.14 (t, 2H, -CH2CH2O-), 6.27 (s, 4H, maleate),
6.90–7.01 (m, 4H, aromat.), 7.10–7.23 (m, 4H, aromat.); Anal.
C29H35N3O9S requires: C, 57.89; H, 5.86; N, 6.98; S, 5.33; found: C,
57.68; H, 5.75; N, 6.82; S, 5.18; MS (ES+) m/z: 370.5 [M + H].
1
10-[4-(4-Diphenylmethyl)piperazino)butyl]phenothiazine
bis-(hydrogen maleate) P4DPh1P
10-[4-(2,2-Diphenylethylamino)butyl]phenothiazine
hydrogen maleate P4DPh2A
Yield 24%; H-NMR (CD3OD) d [ppm]: 1.78–1.82 (m, 4H, -CH2-),
Yield 42%; 1H-NMR (CD3OD)
d [ppm]: 1.61–1.78 (m, 4H,
1
-CH2CH2CH2CH2-), 2.73–3.12 (m, 12H, -NCH2-), 3.98 (t, 2H,
-CH2CH2CH2CH2-), 4.21 (s, 1H, -CH-), 6.25 (s, 4H, maleate), 6.83–
7.01 (m, 4H, aromat.), 7.15–7.29 (m, 14H, aromat.); Anal.
C41H43N3O8S requires: C, 66.74; H, 5.87; N, 5.69; S, 4.34; found: C,
66.82; H, 5.81; N, 5.75; S, 4.28; MS (ES+) m/z: 506.7 [M + H].
2.96–3.04 (t, 2H, -CH2CH2CH2CH2NH-), 3.59–3.63 (d, 2H, -NHCH2-
), 3.97–4.04 (t, 2H, -CH2CH2CH2CH2NH-), 4.21–4.29 (t, 1H, -CH-),
6.23 (s, 2H, maleate), 6.88–7.39 (m, 18H, aromat.); Anal.
C34H34N2O4S requires: C, 72.06; H, 6.05; N, 4.94; S, 5.66; found: C,
72.09; H, 6.03; N, 4.93; S, 5.45; MS (ES+) m/z: 451.6 [M + H].
10-[4-(4-(2,2-Diphenylethyl)piperazino)butyl]-
phenothiazine bis-(hydrogen maleate) P4DPh2P
10-[4-(3,3-Diphenylpropylamino)butyl]phenothiazine
hydrogen maleate P4DPh3A
Yield 36%; H-NMR (CD3OD) d [ppm]: 1.72–1.87 (m, 4H, -CH2-),
Yield 47%; 1H-NMR (CD3OD)
d
[ppm]: 1.62–1.80 (m, 4H,
1
-CH2CH2CH2CH2-), 2.74–3.10 (m, 12H, -NCH2-), 3.99 (t, 2H,
-CH2CH2CH2CH2-), 4.22 (t, 1H, -CH-), 6.27 (s, 4H, maleate), 6.89–
7.00 (m, 4H, aromat.), 7.10–7.27 (m, 14H, aromat.); Anal.
C42H45N3O8S requires: C, 67.09; H, 6.03; N, 5.59; S, 4.26; found: C,
67.16; H, 6.12; N, 5.47; S, 4.18; MS (ES+) m/z: 520.7 [M + H].
2.26–2.38 (q, 2H, -NHCH2CH2-), 2.79–2.95 (m, 4H, -CH2NHCH2-),
3.91–4.01 (m, 3H, -NCH2-, -CH-), 6.23 (s, 2H, maleate), 6.88–7.33
(m, 18H, aromat.); Anal. C35H36N2O4S requires: C, 72.39; H, 6.25;
N, 4.82; S, 5.52; found: C, 72.42; H, 6.23; N, 4.79; S, 5.49; MS (ES+)
m/z: 465.7 [M + H].
rac-10-[4-(4-(4-chlorophenyl)phenylmethyl)piperazino)-
butyl]phenothiazine bis-(hydrogene maleate)
P4PhpClPh1P
10-[4-(4-(3,3-Diphenylpropyl)piperazino)butyl]-
phenothiazine bis-(hydrogen maleate) P4DPh3P
Yield 54%; 1H-NMR (CD3OD)
d [ppm]: 1.52–1.58 (m, 2H,
Yield 39%; 1H-NMR (CD3OD)
d [ppm]: 1.62–1.79 (m, 4H,
-CH2CH2CH2CH2-P), 1.78–1.85 (m, 2H, -CH2CH2CH2CH2-P), 2.04–
2.26 (m, 4H, -CH2CH2CH-), 2.58–2.83 (m, 10H, -NCH2-), 3.71 (t, 1H,
-CH-), 3.98 (t, 2H, -CH2CH2CH2CH2-P), 6.25 (s, 4H, maleate), 6.87–
7.28 (m, 18H, aromat.); Anal. C43H47N3O8S requires: C, 67.43; H,
6.19; N, 5.49; S, 4.19; found: C, 67.31; H, 6.31; N, 5.42; S, 4.12; MS
(ES+) m/z: 534.8 [M + H].
-CH2CH2CH2CH2-), 2.71–3.17 (m, 12H, -NCH2-), 4.01 (t, 2H,
-CH2CH2CH2CH2-), 4.26 (s, 1H, -CH-), 6.24 (s, 4H, maleate), 6.81–
7.03 (m, 8H, aromat.), 7.19–7.45 (m, 9H, aromat.); Anal.
C41H42ClN3O8S requires: C, 63.76; H, 5.48; Cl, 4.59; N, 5.44; S, 5.94;
found: C, 63.81; H, 5.45; Cl, 4.54; N, 5.41; S, 5.89; MS (ES+) m/z:
541.2 [M + H].
10-[4-(4-(4,4-Diphenylbutyl)piperazino)butyl]-
phenothiazine bis-(hydrogen maleate) P4DPh4P
10-[4-(4-(Bis-(4-fluorophenyl)methyl)piperazino)butyl]-
phenothiazine bis-(hydrogene maleate) P4DpFPh1P
Yield 57%; 1H-NMR (CD3OD)
d [ppm]: 1.52-1.59 (m, 2H,
-CH2CH2CH2CH2-P), 1.70–1.75 (m, 2H, -CH2CH2CH2CH-), 1.83–1.85
(m, 2H, -CH2CH2CH2CH2-P), 2.08–2.16 (m, 2H, -CH2CH2CH2CH-),
2.62–2.86 (m, 12H, -NCH2-), 3.46 (t, 1H, -CH-), 3.96 (t, 2H,
-CH2CH2CH2CH2-P), 6.27 (s, 4H, maleate), 6.96–7.31 (m, 18H, aro-
mat.); Anal. C44H49N3O8S requires: C, 67.76; H, 6.33; N, 5.39; S,
4.11; found: C, 67.65; H, 6.27; N, 5.28; S, 4.02; MS (ES+) m/z: 548.8
[M + H].
Yield 43%; 1H-NMR (CD3OD)
d [ppm]: 1.58–1.82 (m, 4H,
-CH2CH2CH2CH2-), 2.69–3.12 (m, 12H, -NCH2-), 3.98 (t, 2H,
-CH2CH2CH2CH2-), 4.27 (s, 1H, -CH-), 6.25 (s, 4H, maleate), 6.85–
7.07 (m, 8H, aromat.), 7.25–7.78 (m, 8H, aromat.); Anal.
C41H41F2N3O8S requires: C, 63.64; H, 5.34; F, 4.91; N, 7.76; S, 5.92;
found: C, 63.58; H, 5.41; F, 4.84; N, 7.68; S, 5.88; MS (ES+) m/z:
542.7 [M + H].
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Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
New Phenothiazines and Related Drugs as MDR Reversal Agents
635
18H, aromat.); Anal. C39H49N3O10 requires: C, 65.07; H, 6.86; N,
5.84; found: C, 65.12; H, 6.91; N, 5.82; MS (ES+) m/z: 530.8 [M + H].
10-[4-(4-(Diphenylhydroxymethyl)piperidin-1-
yl)butyl]phenothiazine P4DPhMeOH1P
Yield 32%; 1H-NMR (CD3OD) d [ppm]: = 1.63–1.93 (m, 9H,
-CH2CH2CH2CH2-, -CH-, -CH2-), 2.15 (s, 1H, -OH), 2.67–2.93 (m, 6H,
-NCH2-), 3.60 (t, 2H, -CH2CH2CH2CH2-), 7.12-7.54 (m, 18H, aromat.);
Anal. C34H36N2OS requires: C, 78.42; H, 6.97; N, 5.38; S, 6.16;
found: C, 78.51; H, 5.42; N, 5.35; S, 6.12; MS (ES+) m/z: 521.7 [M +
H].
3-[4-(4-Methylpiperazinyl)butyl]-5,5-diphenyl-
imidazolidine-2,4-dion bis-(hydrogen maleate) MTP4MP
Yield 92%; 1H-NMR (CDCl3) d [ppm]: 1.38–1.68 (m, 4H, -CH2-), 2.23
(s, 3H, -CH3), 2.30–2.60 (m, 10H, -Npip.CH2-), 3.56-3.60 (m, 2H,
-Nimi.CH2-), 6.26 (s, 4H, maleate), 7.29–7.38 (m, 10H, aromat.);
Anal. C32H38N4O10 requires: C, 60.18; H, 6.00; N, 8.77; found: C,
60.22; H, 6.04; N, 8.79; MS (ES+) m/z: 407.5 [M + H].
5-(4-[4-Methylpiperazinyl]butyl)-10,11-dihydro-5H-
dibenz[b,f]azepine bis-(hydrogene maleate) Ib4MP
Yield 83%; 1H-NMR (CD3OD) d [ppm]: 1.61–1.66 (m, 4H, -CH2-CH2-
CH2-CH2-), 2.62 (s, 3H, -CH3), 2.73–2.81 (m, 10H, -Npip.CH2-), 3.11–
3.12 (m, 2H, -Naz.CH2-), 3.50–3.67 (m, 4H, -Caz.H2-), 6.27 (s, 4H, mal-
eate), 7.25–7.33 (m, 8H, aromat.); Anal. C31H39N3O8 requires: C,
64.01; H, 6.76; N, 7.2; found: C, 64.35; H, 7.03; N,7.53; MS (ES+)
m/z: 350.5 [M + H].
3-(4-(4-(5,5-Diphenylpentyl)piperazinyl)butyl)-5,5-
diphenylimidazolidine-2,4-dion bis-(hydrogen maleate)
MTP4DPP
1
Yield 81%; H-NMR (CD3OD) d [ppm]: 1.33–2.14 (m, 10H, -CH2-),
2.63–3.14 (m, 12H, -Npip.CH2-), 3.60 (t, 2H, -Nimi.CH2-), 3.91 (t, 1H,
-CH-), 6.27 (s, 4H, maleate), 7.12–7.43 (m, 20H, aromat.); Anal.
C48H54N4O10 requires: C, 68.07; H, 6.43; N, 6.61; found: C, 68.32;
H, 6.48; N, 6.70; MS (ES+) m/z: 615.8 [M + H].
5-(4-[4-(5,5-Diphenylpentyl)piperazinyl]butyl)-10,11-
dihydro-5H-dibenz[b,f]azepine bis-(hydrogene oxalate)
trihydrate Ib4DPP
6-(4-[4-(5,5-Diphenylpentyl)piperazinyl]butyl)-6,7-
dihydro-5H-dibenz[c,e]azepine bis-(hydrogen oxalate)
dihydrate R4DPP
Yield 65%; 1H-NMR (CD3OD) d [ppm]: 1.34–2.15 (m, 10H, 2[-CH2-
CH2-CH2-CH2-], -CH2-CH-), 3.04–3.26 (m, 12H, -Npip.CH2-), 3.64–3.70
(m, 4H, -Caz.H2-), 3.92 (t, 1H, -CH-), 4.13-4.18 (m, 2H, -Naz.CH2-),
7.07–7.26 (m, 18H, aromat.); Anal. C43H57N3O11 requires: C,
65.22; H, 7.25; N, 5.31; found: C, 65.21; H, 7.05; N, 5.57; MS (ES+)
m/z: 558.2 [M + H].
1
Yield 58%; H-NMR (CD3OD) d [ppm]: 1.27–1.64 (m, 8H, -CH2-]),
2.05–2.11 (m, 2H, -CH2-CH-), 2.56-2.60 (t, 2H, -Naz.CH2-CH2-), 3.23–
3.30 (m, 12H, -Npip.CH2-), 3.32–3.51 (dd, 4H, -CH2Naz.CH2-), 3.89 (t,
1H, -CH-), 7.09–7.63 (m, 18H, aromat.); Anal. C43H55N3O10
requires: C, 67.08; H, 6.68; N, 5.46; found: C, 67.02; H, 6.74; N,
5.39; MS (ES+) m/z: 558.8 [M + H].
5-(4-[4-(5,5-Diphenylpentyl)piperazinyl]butyl)-5H-
dibenz[b,f]azepine bis-(hydrogen oxalate) dihydrate
Is4DPP
1
6-(4-[4-(4,4-Diphenylpentyl)piperazinyl]butyl)-1,2,3,9-
tetramethoxy-6,7-dihydro-5H-dibenz[c,e]azepine bis-
(hydrogen maleate) S4DPP
Yield 68%; H-NMR (CD3OD) d [ppm]: 1.24–2.15 (m, 10H, 2[-CH2-
CH2-CH2-CH2-], -CH2-CH-), 3.29–3.31 (m, 15H, -Npip.CH2-, -Naz.CH2-),
3.92 (t, 1H, -CH-), 6.70 (s, 2H, =CH-), 7.21–7.80 (m, 18H, aromat.);
Anal. C43H53N3O10 requires: C, 66.91; H, 6.92; N, 5.44; found: C,
66.78; H, 6.89; N, 5.40; MS (ES+) m/z: 556.8 [M + H].
1
Yield 46%; H-NMR (CD3OD) d [ppm]: 1.47–1.51 (m, 8H, -CH2-),
2.07-2.13 (m, 12H, -Npip.CH2-), 2.61–2.66 (m, 2H, -Naz.CH2-CH2-),
3.18–3.20 (m, 3H, -OCH3-), 3.73 (t, 1H, -CH-), 3.85–3.97 (m, 13H,
-OCH3-, -CH2Naz.CH2-]), 6.27 (s, 2H, maleate), 7.10–7.27 (m, 14H,
aromat.); Anal. C50H61N3O12 requires: C, 67.02; H, 6.86; N, 4.69;
found: C, 66.92; H, 6.99; N, 4.58; MS (ES+) m/z: 664.9 [M + H].
5-(4-[4-Methylpiperazinyl]butyl)-5H-dibenz[b,f]azepine
bis-(hydrogen maleate) Is4MP
Yield 92%; 1H-NMR (CD3OD) d [ppm]: 1.54–1.57 (m, 4H, -CH2-CH2-
CH2-CH2-), 2.17–2.69 (m, 13H, -Npip.CH2-, -CH3), 3.71 (t, 2H,
-Naz.CH2-), 6.26 (s, 4H, maleate), 6.70 (s, 2H, =CH-), 6.98–7.78 (m,
8H, aromat.); Anal. C31H37N3O8 requires: C, 64.24; H, 6.43; N, 7.25;
found: C, 64.29; H, 6.52; N, 7.21; MS (ES+) m/z: 348.5 [M + H].
2-Acetyl-10-[3-(4-methylpiperazino)propyl]phenothiazin
2AcP3MP
Yield 63%; 1H-NMR (CD3OD)
d [ppm]: 1.94–2.01 (qd, 2H,
-CH2CH2CH2-), 2.58 (s, 3H, -COCH3), 2.79 (s, 3H, -NCH3), 2.89–3.17
(m, 10H, -NCH2-), 4.03 (t, 2H, -NPTACH2-), 6.27 (s, 4H, maleate),
6.85–7.07 (m, 4H, aromat.), 7.28–7.65 (m, 3H, aromat.); Anal.
C30H35N3O9S requires: C, 58.72; H, 5.75; N, 6.85; S 5.22; found: C,
58.65; H, 5.86; N, 6.81; S, 5.15; MS (ES+) m/z: 382.5 [M + H].
9-(4-[4-Methylpiperazinyl]butyl)-9H-carbazole bis-
(hydrogen oxalate) hydrate MTC4MP
Yield 86%; 1H-NMR (CDCl3) d [ppm]: 1.53–1.61 (m, 2H, -CH2-),
1.86–1.93 (m, 2H, -CH2-), 2.27 (s, 3H, -N-CH3), 2.33–2.43 (m, 10H,
-Npip.CH2-), 4.31 (t, 2H, -Ncarb.CH2-), 7.18–8.09 (m, 8H, aromat.);
Anal. C25H33N3O9 requires: C, 57.80; H, 6.40; N, 8.09; found: C,
57.79; H, 6.42; N, 8.05; MS (ES+) m/z: 322.5 [M + H].
2-Propionyl-10-[3-(4-methylpiperazino)propyl]-
phenothiazine bis-(hydrogen maleate) 2Prop(ac)P3MP
Yield 57%; 1H-NMR (CD3OD) d [ppm]: 1.31 (t, 3H, -CH2CH3), 1.93–
2.03 (qd, 2H, -CH2CH2CH2-), 2.84–3.31 (m, 15H, -NCH2-, -NCH3,
-COCH2-), 4.00 (t, 2H, -NPTACH2-), 6.26 (s, 4H, maleate), 6.88–7.04
(m, 4H, aromat.), 7.17–7.54 (m, 3H, aromat.); Anal. C31H37N3O9S
requires: C, 59.32; H, 5.94; N, 6.69; S 5.11; found: C, 59.41; H,
5.98; N, 6.61; S, 5.05; MS (ES+) m/z: 396.6 [M + H].
9-[4-(4-[5,5-Diphenylpentyl]piperazinyl)butyl]-9H-
carbazole bis-(hydrogen oxalate) dihydrate MTC4DPP
Yield 78%; 1H-NMR (CDCl3) d [ppm]: 1.43–1.60 (m, 6H, 3[-CH2-]),
1.85–1.98 (m, 4H, -CH2-), 2.19–2.56 (m, 12H, -Npip.CH2-), 4.08–
4.13 (m, 1H, -CH-), 4.29–4.33 (t, 2H, Ncarb.CH2-), 7.12–8.09 (m,
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636
M. Schmidt et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
2-Butyryl-10-[3-(4-methylpiperazino)propyl]-
phenothiazine bis-(hydrogen maleate) (P-4)
2Bu(ac)P3MP
3-Butyl-10-[4-(4-methylpiperazino)butyl]phenothiazine
bis-(hydrogen maleate) 3BuP4MP
Yield 58%; 1H-NMR (CD3OD) d [ppm]: 0.91 (t, 3H, -CH2CH3), 1.32–
1.98 (m, 8H, -CH2CH2-), 2.37–2.46 (m, 2H, Ar-CH2-), 2.74–3.25 (m,
13H, -CH3, -NCH2-), 4.01 (t, 2H, -NPTACH2-), 6.27 (s, 4H, maleate),
6.68–7.27 (m, 7H, aromat.); Anal. C33H43N3O8S requires: C, 61.76;
H, 6.75; N, 6.55; S, 5.00; found: C, 61.71; H, 6.81; N, 6.48; S, 4.97;
MS (ES+) m/z: 410.6 [M + H].
Yield 60%; 1H-NMR (CD3OD) d [ppm]: 1.17 (t, 3H, -CH2CH3), 1.62–
1.75 (m, 2H, -CH2CH3), 2.01–2.07 (qd, 2H, -CH2CH2CH2-), 2.84–
3.27 (m, 15H, -NCH2-, -NCH3, -COCH2-), 3.97 (t, 2H, -NPTACH2-), 6.26
(s, 4H, maleate), 6.98–7.81 (m, 7H, aromat.); Anal. C32H39N3O9S
requires: C, 59.89; H, 6.13; N, 6.55; S 5.00; found: C, 59.82; H,
6.18; N, 6.49; S, 4.95; MS (ES+) m/z: 410.6 [M + H].
2-Methylthio-10-[4-(4-methylpiperazino)butyl]-
phenothiazine bis-(hydrogen maleate) 2MeSP4MP
2-Benzoyl-10-[3-(4-methylpiperazino)propyl]-
phenothiazine bis-(hydrogen maleate) 2BzlP3MP
Yield 52%; H-NMR (CD3OD) d [ppm]: 1.93–1.97 (m, 2H, -CH2-),
2.63–2.81 (m, 13H, -CH3, -NCH2), 3.99 (t, 2H, -NPTACH2-), 6.26 (s, 4H,
maleate), 6.88–7.72 (m, 12H, aromat.); Anal. C35H37N3O9S
requires: C, 62.21; H, 5.52; N, 6.22; S, 4.74; found: C, 62.28; H,
5.59; N, 6.13; S, 4.62; MS (ES+) m/z: 444.6 [M + H].
Yield 56%; 1H-NMR (CD3OD)
d [ppm]: 1.65–1.97 (m, 4H,
-CH2CH2CH2CH2-), 2.32 (s, 3H, Ar-SCH3), 2.84–3.32 (m, 13H, -CH3,
-NCH2-), 4.00 (t, 2H, -NPTACH2-), 6.27 (s, 4H, maleate), 6.68–7.67 (m,
7H, aromat.); Anal. C30H37N3O8S2 requires: C, 57.04; H, 5.90; N,
6.65; S, 10.15; found: C, 56.99; H, 5.97; N, 6.58; S, 10.03; MS (ES+)
m/z: 400.6 [M + H].
1
2-Methoxy-10-[4-(4-methylpiperazino)butyl]-
phenothiazine bis-(hydrogen maleate) 2MeOP4MP
3-Methoxy-10-[3-(4-methylpiperazino)propyl]-
Yield 62%; 1H-NMR (CD3OD)
d [ppm]: 1.68–1.99 (m, 4H,
phenothiazine bis-(hydrogen maleate) 3MeOP3MP
Yield 30%; 1H-NMR (CD3OD) d [ppm]: 1.98 (qd, 2H, -CH2-CH2-CH2-),
2.63–2.91 (m, 13H, -Npip.CH2-, -CH3), 3.73 (s, 3H, -OCH3), 3.91–3.97
(t, 2H, -NPTACH2-), 6.27 (s, 4H, maleate), 6.74–6.79 (m, 2H, aro-
mat.), 6.86–6.97 (m, 3H, aromat.), 7.09–7.22 (m, 2H, aromat.);
Anal. C29H35N3O9S requires: C, 57.89; H, 5.86; N, 6.98; S, 5.33;
found: C, 57.92; H, 5.96; N, 6.85; S, 5.27; MS (ES+) m/z: 602.7 [M +
H].
-CH2CH2CH2CH2-), 2.81–3.27 m, 13H, -CH3, -NCH2-), 3.78 (s, 3H,
-OCH3), 4.02 (t, 2H, -NPTACH2-), 6.26 (s, 4H, maleate), 6.58–7.37 (m,
7H, aromat.); Anal. C30H37N3O9S requires: C, 58.52; H, 6.06; N,
6.82; S, 5.21; found: C, 58.49; H, 6.17; N, 6.85; S, 5.13; MS (ES+)
m/z: 384.6 [M + H].
3-Butoxy-10-[4-(4-methylpiperazino)butyl]phenothiazine
bis-(hydrogen maleate) 3BuOP4MP
Yield 53%; 1H-NMR (CD3OD) d [ppm]: 0.96 (t, 3H, -CH2CH3), 1.43–
2.02 (m, 8H, -CH2CH2-), 2.77–3.32 (m, 13H, -NCH3, -NCH2-), 3.96–
4.04 (m, 4H, -OCH2-, -NPTACH2-), 6.26 (s, 4H, maleate), 6.72–7.26
(m, 7H, aromat.); Anal. C33H43N3O9S requires: C, 60.26; H, 6.59; N,
6.39; S, 4.87; found: C, 60.17; H, 6.68; N, 6.34; S, 4.76; MS (ES+)
m/z: 426.6 [M + H].
3-Ethoxy-10-[3-(4-
methylpiperazino)propyl]phenothiazine bis-(hydrogen
maleate) 3EtOP3MP
Yield 54%;1H-NMR (CD3OD) d [ppm]: 1.33 (t, 3H, -OCH2CH3), 1.91–
2.01 (qd, 2H, -CH2-CH2-CH2-), 2.67–2.74 (m, 9H, -Npip.CH2-, -CH3),
2.92–3.06 (m, 4H, -Npip.CH2-), 3.90–4.01 (m, 4H, -OCH2CH3,
-NPTACH2-), 6.26 (s, 4H, maleate), 6.71–7.21 (m, 7H, aromat.); Anal.
C30H37N3O9S requires: C, 58.52; H, 6.06; N, 6.82; S, 5.21; found: C,
58.36; H, 5.79; N, 6.91; S, 5.38; MS (ES+) m/z: 616.7 [M + H].
3-Isobutoxy-10-[4-(4-methylpiperazino)butyl]-
phenothiazine bis-(hydrogen maleate) 3isoBuOP4MP
Yield 57%;1H-NMR (CD3OD) d [ppm]: 1.12 (d, 6H, -CH(CH3)2), 1.64–
1.96 (m, 4H, -CH2CH2-), 2.24–2.36 (m, 1H, -CH-), 2.84–3.36 (m,
13H, -NCH3, -NCH2-), 3.76–3.78 (m, 2H, -OCH2-), 4.01 (t, 2H,
-NPTACH2-), 6.27 (s, 4H, maleate), 6.74–7.42 (m, 7H, aromat.); Anal.
C33H43N3O9S requires: C, 60.26; H, 6.59; N, 6.39; S, 4.87; found: C,
60.15; H, 6.46; N, 6.31; S, 4.79; MS (ES+) m/z: 426.6 [M + H].
2-Methyl-10-[3-(4-methylpiperazino)propyl]phenothiazine
bis-(hydrogen maleate) 2MeP3MP
Yield 61%; 1H-NMR (CD3OD)
d [ppm]: 1.95–2.04 (qd, 2H,
-CH2CH2CH2-), 2.22 (s, 3H, Ar-CH3), 2.86 (s, 3H, -NCH3), 3.02–3.28
(m, 10H, -NCH2-), 4.03 (t, 2H, -NPTACH2-), 6.27 (s, 4H, maleate),
6.98–7.58 (m, 7H, aromat.); Anal. C29H35N3O8S requires: C, 59.47;
H, 6.02; N, 7.17; S 5,47; found: C, 59.42; H, 6.09; N, 7.11; S, 5.42;
MS (ES+) m/z: 354.5 [M + H].
3-Phenyl-10-[4-(4-methylpiperazino)butyl]phenothiazine
bis-(hydrogen maleate) 3PhP4MP
Yield 59%; 1H-NMR (CD3OD)
d [ppm]: 1.72–1.98 (m, 4H,
-CH2CH2CH2CH2-), 2.76–2.92 (m, 13H, -CH3, -NCH2-), 4.02 (t, 2H,
-NPTACH2-), 6.27 (s, 4H, maleate), 6.64–7.58 (m, 12H, aromat.);
Anal. C35H39N3O8S requires: C, 63.52; H, 5.94; N, 6.35; S, 4.84;
found: C, 63.47; H, 5.97; N, 6.27; S, 4.73; MS (ES+) m/z: 430.6 [M +
H].
3-Methyl-10-[4-(4-methylpiperazino)butyl]phenothiazine
bis-(hydrogen maleate) 3MeP4MP
Yield 64%; 1H-NMR (CD3OD) d [ppm]: 1.67–1.96 (m, 4H, -6.2.3.29
CH2CH2CH2CH2-), 2.18 (s, 3H, Ar-CH3), 2.67–3.24 (m, 13H, -CH3,
-NCH2-), 4.03 (t, 2H, -NPTACH2-), 6.26 (s, 4H, maleate), 6.67–7.25 (m,
7H, aromat.); Anal. C30H37N3O8S requires: C, 60.09; H, 6.22; N,
7.01; S, 5.35; found: C, 59.99; H, 6.17; N, 6.98; S, 5.31; MS (ES+)
m/z: 368.6 [M + H].
10-[4-(4-Methylpiperazino)butyl]benzo[c]phenothiazine
bis-(hydrogen maleate) Benz[c]P4MP
Yield 63%; 1H-NMR (CD3OD)
d [ppm]: 1.67–1.85 (m, 4H,
-CH2CH2CH2CH2-), 2.61–2.97 (m, 13H, -CH3, -N-CH2-), 4.02 (t, 2H,
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Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
New Phenothiazines and Related Drugs as MDR Reversal Agents
637
-CH2-(CH2)3-), 6.27 (s, 4H, maleate), 6.92 (d, 2H, aromat.), 7.12–
7.21 (m, 3H, aromat.), 7.32 (t, 1H, aromat.), 7.46 (t, 1H, aromat.),
7.63 (d, 1H, aromat.), 7.71 (d,1H, aromat.), 8.10 (d, 1H, aromat.);
Anal. C33H37N3O8S requires: C, 62.35; H, 5.87; N, 6.61; S, 5.04;
found: C, 62.31; H, 5.91; N, 6.52; S, 5.03; MS (ES+) m/z: 404.6 [M +
H].
2-Benzoyl-10-[4-(4-methylpiperazino)butyl]phenothiazine
bis-(hydrogen maleate) 2BzlP4MP
Yield 45%; H-NMR (CD3OD) d [ppm]: 1.35–1.38 (m, 2H, -CH2-),
1.93–1.97 (m, 2H, -CH2-), 2.64–2.83 (m, 13H, -CH3, -Npip.CH2), 4.00
(t, 2H, -NPTACH2-), 6.26 (s, 4H, maleate), 6.82–8.01 (m, 12H, aro-
mat.); Anal. C36H39N3O9S requires: C, 62.69; H, 5.70; N, 6.09; S,
4.65; found: C, 62.54; H, 5.81; N, 6.21; S, 4.53; MS (ES+) m/z: 458.6
[M + H].
1
10-[4-(4-Methylpiperazino)butyl]benzo[a]phenothiazine
bis-(hydrogen maleate) Benz[a]P4MP
Yield 47%; 1H-NMR (CD3OD)
d [ppm]: 1.66–1.92 (m, 4H,
2-Propionyl-10-[8-(4-methylpiperazino)octyl]-
-CH2CH2CH2CH2-), 2.74–3.14 (m, 13H, -CH3, -NCH2-), 4.02 (t, 2H,
-NPTACH2-), 6.26 (s, 4H, maleate), 6.98 (d, 2H, aromat.), 7.08–7.16
(m, 3H, aromat.), 7.36 (t, 1H, aromat.), 7.52 (t, 1H, aromat.), 7.64
(d, 1H, aromat.), 7.78 (d,1H, aromat.), 8.14 (d, 1H, aromat.); Anal.
C33H37N3O8S requires: C, 62.35; H, 5.87; N, 6.61; S, 5.04; found: C,
62.42; H, 5.85; N, 6.49; S, 5.01; MS (ES+) m/z: 404.6 [M + H].
phenothiazine bis-(hydrogen maleate) 2Prop(ac)P8MP
Yield 53%; 1H-NMR (CD3OD) d [ppm]: 1.24–1.98 (m, 15H, -CH2CH3,
-CH2(CH2)6CH2-), 2.74–3.32 (m, 15H, -NCH2-, -NCH3, -COCH2-), 4.01
(t, 2H, -NPTACH2-), 6.26 (s, 4H, maleate), 6.86–7.04 (m, 4H, aro-
mat.), 7.16–7.48 (m, 3H, aromat.); Anal. C36H47N3O9S requires: C,
61.96; H, 6.79; N, 6.02; S 4.59; found: C, 61.88; H, 6.84; N, 5.98; S,
4.51; MS (ES+) m/z: 466.7 [M + H].
3,7-Di-tert-butyl-10-[4-(4-methylpiperazino)butyl]-
phenothiazine bis-(hydrogen oxalate) 3,7DTBuP4MP
Yield 48%; H-NMR (CD3OD) d [ppm]: 1.38 (s, 18H, -CCH3), 1.82–
2.15 (m, 4H, -CH2-), 2.53–3.34 (m, 13H, -CH3, -Npip.CH2), 3.58 (t, 2H,
-NPTACH2-), 6.89–7.62 (m, 6H, aromat.); Anal. C33H47N3O8S
requires: C, 61.37; H, 7.34; N, 6.51; S, 4.96; found: C, 61.24; H,
7.38; N, 6.37; S, 4.87; MS (ES+) m/z: 466.7 [M + H].
2-Butyryl-10-[8-(4-methylpiperazino)octyl]phenothiazine
1
bis-(hydrogen maleate) 2Bu(ac)P8MP
Yield 57%; 1H-NMR (CD3OD) d [ppm]: 1.04 (t, 3H, -CH2CH3), 1.28–
2.06 (m, 14H, -CH2CH3, -CH2(CH2)6CH2-), 2.78–3.26 (m, 15H, -NCH2-
, -NCH3, -COCH2-), 3.96 (t, 2H, -NPTACH2-), 6.27 (s, 4H, maleate),
6.86–7.63 (m, 7H, aromat.); Anal. C37H49N3O9S requires: C, 62.43;
H, 6.94; N, 5.90; S 4.50; found: C, 62.35; H, 6.98; N, 5.86; S, 4.43;
MS (ES+) m/z: 480.7 [M + H].
3-tert-Butyl-10-[4-(4-methylpiperazino)butyl]phenothiazin
bis-(hydrogen maleate) 3TBuP4MP
1
Yield 52%; H-NMR (CD3OD) d [ppm]: 1.32 (s, 9H, -CCH3), 1.76–
2-Benzoyl-10-[6-(4-methylpiperazino)hexyl]-
2.04 (m, 4H, -CH2-), 2.78–3.32 (m, 13H, -CH3, -Npip.CH2), 3.60 (t, 2H,
-NPTACH2-), 6.86–7.32 (m, 6H, aromat.); Anal. C33H43N3O8S
requires: C, 61.76; H, 6.75; N, 6.55; S, 5.00; found: C, 61.64; H,
6.81; N, 6.47; S, 4.97; MS (ES+) m/z: 410.6 [M + H].
phenothiazine bis-(hydrogen maleate) 2BzlP6MP
1
Yield 37%; H-NMR (CD3OD) d [ppm]: 1.38–1.87 (m, 8H, -CH2-),
2.61–2.88 (m, 13H, -CH3, -Npip.CH2), 3.99 (t, 2H, -NPTACH2-), 6.28 (s,
4H, maleate), 6.78–7.98 (m, 12H, aromat.); Anal. C38H43N3O9S
requires: C, 63.58; H, 6.04; N, 5.85; S, 4.47; found: C, 63.52; H,
6.11; N, 5.72; S, 4.41; MS (ES+) m/z: 486.7 [M + H].
2-Acetyl-10-[4-(4-methylpiperazino)butyl]phenothiazine
bis-(hydrogen maleate) 2AcP4MP
ield 63%; 1H-NMR (CD3OD) d [ppm]: 1.68–2.00 (m, 4H, -CH2-), 2.54
(s, 3H, -COCH3), 2.76 (s, 3H, -NCH3), 2.92–3.24 (m, 10H, -NCH2-),
4.01 (t, 2H, -NPTACH2-), 6.26 (s, 4H, maleate), 6.84–7.14 (m, 4H, aro-
mat.), 7.26–7.48 (m, 3H, aromat.); Anal. C31H37N3O9S requires: C,
59.32; H, 5.94; N, 6.69; S 5.11; found: C, 59.26; H, 5.98; N, 6.61; S,
5.07; MS (ES+) m/z: 396.6 [M + H].
2-Benzoyl-10-[8-(4-methylpiperazino)octyl]phenothiazine
bis-(hydrogen maleate) 2BzlP8MP
Yield 32%; H-NMR (CD3OD) d [ppm]: 1.28–1.97 (m, 12H, -CH2-),
2.62–2.87 (m, 13H, -CH3, -Npip.CH2), 3.99 (t, 2H, -NPTACH2-), 6.27 (s,
4H, maleate), 6.82–8.03 (m, 12H, aromat.); Anal. C40H47N3O9S
requires: C, 64.41; H, 6.35; N, 5.63; S, 4.30; found: C, 64.45; H,
6.41; N, 5.57; S, 4.21; MS (ES+) m/z: 514.7 [M + H].
1
2-Propionyl-10-[4-(4-methylpiperazino)butyl]-
phenothiazine bis-(hydrogen maleate) 2Prop(ac)P4MP
Yield 54%; 1H-NMR (CD3OD) d [ppm]: 1.28 (t, 3H, -CH2CH3), 1.67–
2.02 (m, 4H, -CH2-), 2.78–3.34 (m, 15H, -NCH2-, -NCH3, -COCH2-),
4.03 (t, 2H, -NPTACH2-), 6.27 (s, 4H, maleate), 6.84–7.08 (m, 4H, aro-
mat.), 7.18–7.46 (m, 3H, aromat.); Anal. C32H39N3O9S requires: C,
59.89; H, 6.13; N, 6.55; S 5.00; found: C, 59.83; H, 6.19; N, 6.52; S,
4.98; MS (ES+) m/z: 410.6 [M + H].
Biological evaluation
Cell culture and drugs
LLC-PK1 cell line, which was obtained from American Type Cul-
ture Collection (ATCC), Rockville, MD, USA (passage 36) was kept
under standard culture conditions (Dulbecco's medium 199,
10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin
and 100 lg/mL streptomycin) at 378C in the presence of 5% CO2.
Medium for LLC-MDR cells aditionally contained 640 nM vincris-
tine sulfate to abide p-gp expression. Cells were subcultured by
trypsinization every week and the medium was replaced twice a
week. The resistant lines were obtained by stepwise selection in
10 nM vincristine containing medium. Cell culture reagents
were obtained from Gibco BRL (Invitrogen, Karlsruhe, Germany).
Vincristine was obtained from Universitꢁtsapotheke Klinikum
Krꢂllwitz (Halle). References were obtained from Sigma-Aldrich
Chemie GmbH (Germany).
2-Butyryl-10-[4-(4-methylpiperazino)butyl]phenothiazine
bis-(hydrogen maleate) 2Bu(ac)P4MP
Yield 56%; 1H-NMR (CD3OD) d [ppm]: 1.06 (t, 3H, -CH2CH3), 1.62–
2.06 (m, 6H, -CH2CH3, -CH2-), 2.81–3.24 (m, 15H, -NCH2-, -NCH3,
-COCH2-), 3.99 (t, 2H, -NPTACH2-), 6.26 (s, 4H, maleate), 6.86–7.68
(m, 7H, aromat.); Anal. C33H41N3O9S requires: C, 60.44; H, 6.30; N,
6.41; S 4.89; found: C, 60.32; H, 6.35; N, 6.38; S, 4.83; MS (ES+) m/z:
424.6 [M + H].
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

638
M. Schmidt et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 624–638
[17] S. Dꢂppenschmitt, H. Spahn-Langguth, C. Regꢄrdh, P.
Crystalviolet assay
Langguth, Pharm. Res. 1998, 1001–1006.
Cells were seeded in 96-well plates (Millipore, Eschborn, Ger-
many) at a density of 56103 cells per well. After incubation at
378C and 5% CO2 for 4 h, cells were left unloaded (control, 0.1%
DMSO) or treated with vincristine (1 lM); vincristine (1 lM) + tri-
fluoperazine (0.1–400 lM) and vincristine + test substance (0.1–
400 lM) for 68 h at 378C. After the completion of drug exposure,
the supernatant of dead cells was removed. Residue with living
cells was denaturated with 100 lL MeOH for 10 min, fixed, and
washed. Then, cells were dyed with 100 lL of 0.1% aqueous solu-
tion of crystalviolet for 10 min. After washing, the absorbed dye
was dissolved with 0.1 M ethanolic solution of sodium citrate.
The absorbance was measured at k = 620 nm on a Polarstar Gal-
axy plate reader (BMG LabTechnologies GmbH, Offenburg, Ger-
many). The percentage of viable cells was calculated to get IC50-
values.
[18] I. K. Pajeva, C. Globisch, M. Wiese, J. Med. Chem. 2004, 47,
2523–2533.
[19] C. Globisch, I. Pajewa, M. Wiese, Chem.Med.Chem. 2008, 3,
280–295.
[20] T. Tsuruo, H. Iida, S. Tsukagoshi, Y. Sakurai, Cancer Res.
1982, 42, 4730–4733.
[21] R. Ganapathi, D. Grabowski, R. Turinic, R. Valenzuela, Eur.
J. Cancer Clin. Oncol. 1984, 20, 799–806.
[22] M. Schmidt, J. Ungvꢅri, J. Glꢂde, B. Dobner, A. Langner,
Bioorg. Med. Chem. 2007, 15, 2283–2297.
[23] R. L. Miller, R. M. Bukowski, G. T Budd, J. Clin. Oncol. 1988,
6, 880–888.
[24] A. Ramu, N. Ramu, Cancer Chemother. Pharmacol. 1992, 30,
165–173.
[25] I. Pajeva, M. Wiese, Quant. Struct.-Act. Relat. 1997, 16, 1–10.
[26] G. Klopman, L. M. Shi, A. Ramu, Mol. Pharmacol. 1997, 52,
References
323–334.
[27] J. Guan, D. E. Kyle, L. Gerena, Q. Zhang, et al., J. Med. Chem.
2002, 45, 2741–2748.
[1] C. Avendano, J. Menꢃndez, Curr. Med. Chem. 2002, 9, 159–
193.
[28] S. Darvesh, R. S. McDonald, A. Penwell, S. Conrad, et al.,
Bioorg. Med. Chem. 2005, 13, 211–222.
[2] R. Krishna, L. D. Mayer, Eur. J. Pharm. Sci. 2000, 11, 265–
283.
[29] A. Bernthsen, Ber. Dtsch. Chem. Ges. 1883, 16, 2896–2904.
[30] Rhꢆne-Poulenc, Belg. P. 556475, C. 1960, 16863.
[31] H. L. Yale, JACS, 1955, 77, 2270–2272.
[3] J. Robert, C. Jarry, J. Med. Chem. 2003, 23, 4805–4817.
[4] J. M. Ford, E. P. Briggeman, I. Pastan, M. Gottesman, W. N.
Hait, Cancer Res. 1990, 50, 1748–1756.
[32] W. J. Evans, S. Smiles, J. Chem. Soc. 1935, 181, 1263–1265.
[5] I. Pajeva, M. Wiese, J. Med. Chem. 1998, 41, 1815–1826.
[33] M. Golinski, P. J. DelaLuz, R. Floresca, T. J. Delcamp, et al.,
Bioconjugate Chem. 1995, 6, 549–557.
[6] J. M. Ford, W. C. Prozialeck, W. N. Hait, Mol. Pharmacol.
1989, 35, 105–115.
[34] S. P. Massie, I. Cooke, W. A. Hills, J. Org. Chem. 1956, 21,
[7] J. M. Zamora, H. L. Pearce, W. T. Beck, Mol. Pharmacol. 1988,
33, 454465.
1006–1008.
[35] J. M. Hawkins, G. C. Fu, J. Org. Chem. 1986, 51, 2820–2822.
[8] M. Wiese, I. K. Pajeva, Current Med. Chem. 2001, 8, 685–713.
[36] L. Adams, C. Gray, R. Williams, Tetrahedron Letters 2004, 45,
[9] H. Pearce, A. Safa, N. Bach, M. Winter, et al., Proc. Natl.
Acad. Sci. U. S. A 1989, 5128–5132.
4489–4493.
[37] O. Kimura, T. Osawa, K. Mochida, Jpn. Kokai Tokkyo Koho,
[10] W. Hait, D. Aftab, Biochem. Pharmacol. 1992, 43, 103–107.
1989, 7–11.
[11] T. Suzuki, N. Fukazawa, K. San-nohe, W. Sato, et al., J. Med.
Chem. 1997, 2047–2052.
[38] D. A. Flick, G. E. Gifford, J. Immunol. Methods 1984, 167–
175.
[12] A. Seelig, Eur. J. Biochem. 1997, 252–261.
[39] Y. Tanigawara, N. Okamura, M. Hirai, M. Yasuhara, et al., J.
Pharmacol. Exp. Ther. 1992, 263, 840–845.
[13] A. Seelig, E. Landwojtowicz, Eur. J. Pharm. Sci. 2000, 31–40.
[14] T. W. Loo, D. M. Clarke, J. Membrane Biol. 2005, 173–185.
[40] A. H. Schinkel, E. Wagenaar, L. van Deemter, C. A. Mol, P.
[15] S. Ekins, R. B. Kim, B. F. Leake, A. H. Dantzig, et al., Mol.
Pharmacol. 2002, 974–981.
Borst, J. Clin. Invest. 1995, 1698–1705.
[41] P. Scafato, G. Cunsolo, S. Labano, C. Rosini, Tetrahedron
2004, 40, 8801–8806.
[16] I. K. Pajeva, M. Wiese, J. Med. Chem. 2002, 45, 5671–5686.
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com