Antimalarial and antitrypanosomal activity of a series of amide and sulfonamide derivatives of a 2,5-diaminobenzophenone

Article abstract of DOI:10.1016/j.bmc.2009.09.043

Here, we describe a series of readily obtainable benzophenone derivatives with antimalarial and antitrypanosomal activity. The most active compounds display submicromolar activity against Plasmodium falciparum. Micromolar activity is obtained against Trypanosoma brucei. Main problem of the compounds is low selectivity. However, there are indications that separation of antimalarial and cytotoxic activity might by possible. In addition, some compounds inhibit human ABC transporter with nanomolar activity.

Full text of DOI:10.1016/j.bmc.2009.09.043

Bioorganic & Medicinal Chemistry 17 (2009) 7690–7697
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimalarial and antitrypanosomal activity of a series of amide
and sulfonamide derivatives of a 2,5-diaminobenzophenone
Mirko Altenkämper a, Benjamin Bechem a, Johann Perruchon a, Swetlana Heinrich a, Andrea Mädel a,
Regina Ortmann a, Hans-Martin Dahse b, Ellen Freunscht c, Yulin Wang c, Jennifer Rath d, August Stich d,
Manuela Hitzler e, Peter Chiba e, Michael Lanzer c, Martin Schlitzer a,
*
a Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35032 Marburg, Germany
b Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e.V. Hans-Knöll-Institut, Beutenbergstrasse 11a, 07745 Jena, Germany
c Institut für Parastitologie, Universität Heidelberg, Im Neuenheimer Feld 24, 69120 Heidelberg, Germany
d Tropenmedizinische Abteilung, Missionsärztliche Klinik GmbH, Salvatorstr. 7, 97074 Würzburg, Germany
e Center for Physiology, Pathophysiology and Immunology, Institute of Medical Chemistry, Medical University of Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2009
Revised 18 September 2009
Accepted 21 September 2009
Available online 25 September 2009
Here, we describe a series of readily obtainable benzophenone derivatives with antimalarial and antitry-
panosomal activity. The most active compounds display submicromolar activity against Plasmodium fal-
ciparum. Micromolar activity is obtained against Trypanosoma brucei. Main problem of the compounds is
low selectivity. However, there are indications that separation of antimalarial and cytotoxic activity
might by possible. In addition, some compounds inhibit human ABC transporter with nanomolar activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Malaria
Sleeping sickness
Plasmodium falciparum
Trypanosoma brucei
Multidrug resistance transporter
Drug development
1. Introduction
benzophenones has been replaced by simple aryloyl residues,
which in a related series of compounds significantly reduced farne-
Malaria is one of the most important infectious diseases affect-
ing almost half of the world’s population, especially in the develop-
ing countries. The amatory for the fight against malaria is rather
limited and continuously threatened by the development of resis-
tance.1,2 Although far less people are affected by African sleeping
sickness, here the situation is even worse. Untreated this disease
leads inevitably to death. Currently, there are only five drugs avail-
able for the treatment. Their use is limited by severe drug toxicity
and resistance.3–5 Therefore, there is an urgent need for the devel-
opment of novel agents against these diseases preferably with
hitherto unexploited mechanisms of action.
We have previously investigated the class of 2,5-diaminobenz-
ophenones as farnesyltransferase inhibitors with antimalarial and
antitrypanosomal activity.6–8 Some of these compounds had an
additional weak inhibitory effect on human ABC transporters
(unpublished results). This effect was more pronounced, when
the arylfurylacryloyl residue of our farnesyltransferase inhibiting
syltransferase inhibitory activity.9
Here, we investigated the effect of our novel benzophenone
derivatives on the growth of cultured Plasmodium falciparum and
Trypanosoma brucei parasites as well as their ability to inhibit mul-
tidrug resistance proteins.
2. Results and discussion
2.1. Chemistry
Synthesis (Scheme 1) of the target compounds followed the
route described previously6 for this type of compounds. It started
with the acylation of commercially available 2-amino-5-nitrobenz-
ophenone (1) which was acylated using
a-chlorophenylacetic acid
chloride or -chloroacetic acid chloride yielding the acyl deriva-
a
tives 2a and 2b. After replacement of the chlorine by N-methylpi-
perazine, the nitro group of the resulting amino acid derivative 3
was reduced to obtain the amino derivative 4. The sequence is
completed by the acylation of the amino group yielding the target
compounds 5 and 6, respectively.
* Corresponding author. Tel.: +49 6421 28 25896.
E-mail address: schlitzer@staff.uni-marburg.de (M. Schlitzer).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.043

M. Altenkämper et al. / Bioorg. Med. Chem. 17 (2009) 7690–7697
7691
R2
Cl
an IC50 value of 110 nM the highest antimalarial activity in this ser-
ies. This value falls within the range of the reference compounds and
is well below the threshold of 1 lM. Compounds with in vitro activ-
ities below this threshold are normally regarded as being worth fur-
ther investigations.
O
O2N
NH2
O
O2N
NH
O
a
b
To further explore structure activity relationships, the phenyl
residue at the acyl moiety on the 2-amino group has been omitted
in compounds 6a–6d. In general, this markedly reduced antimalar-
ial activity resulting in several inactive compounds.
2 a: R2 = Ph
b: R2 = H
1
O
R2
N
O
R2
O2N
NH
O
H2N
NH
O
N
c
d
2.3. Antitrypanosomal activity
N
N
In vitro antitrypanosomal activity of the compounds was deter-
mined against the Trypanosoma brucei brucei TC 221 laboratory
strain, cultured under standard conditions.11 Parasite viability
was assessed using the Alamar Blue assay. In living parasites, the
dye is reduced by redox equivalents stemming from the glycolytic
pathway. A linear correlation has been found to exist between the
fluorescence signal of the dye and the concentration of living
trypanosomes.12
3 a: R2 = Ph
b: R2 = H
4 a: R2 = Ph
b: R2 = H
O
R2
N
R1
NH
O
N
Activity against T. brucei brucei followed mainly the trends seen
in antimalarial activity, but did not reach submicromolar levels
(Table 1). However, in vitro activities of several compounds (5d–
5f, 5j–5l, and 6e) are similar to that of nifurtimox (IC50 = 3.4 l
M),13
5 a-n: R2 = Ph
6 a-e: R2 = H
which has originally been developed and used against American
Trypanosomias (Chaga’s Disease), but now is also evaluated in
combination with other antitrypanosomal drugs for the treatment
of sleeping sickness14 although it is in vitro markedly less active
than currently used antitrypanosomal agents (IC50’s: melarsoprol
2.6 nM, pentamidine 2.9 nM, suramin 310 nM).13
Scheme 1. Synthesis of acylbenzophenone derivatives: (a) 2-chloro-2-phenylacetyl
chloride or 2-chloroacetyl chloride, toluene, reflux; (b) 1-methylpiperazine, aceto-
nitrile, reflux; (c) SnCl2 (dihydrate), ethyl acetate, reflux; (d) acyl chloride or
carbamoyl chloride, toluene, reflux.
2.4. Cytotoxicity assay
2.2. Antimalarial activity
Cytotoxicity of selected compounds was evaluated against HeLa
cells. Viability of the cells was determined after a 72 h incubation
period using methylene blue staining and photometric evaluation
(Table 1).
Major problem of these compounds is the high cytotoxicity and
accordingly the low selectivity toward the malaria parasites. How-
ever, there is one exemption which is the most active compound,
the di-isopropyl urea derivative 5n, which because of its high anti-
malarial activity displays some selectivity toward malaria para-
sites. The selectivity index of this compound of 36 is still far to
low but it indicates that it might be possible to further separate
antimalarial from cytotoxic activity.
We first investigated the in vitro antiplasmodial activity of the
compounds, using a standard cell proliferation assay in which syn-
chronized blood stage cultures of the P. falciparum clone Dd2 were
incubated with different concentrations of the compound for 72 h
before the half maximal growth inhibition coefficient (IC50 value)
was determined. IC50 values ranging from 110 20 nM (compound
5n) to >16 lM (compounds 5m, 6b, and 6c) were obtained (Table
1). In parallel, we investigated the IC50 value of the Dd2 strain for
the established antimalarial drugs chloroquine and quinine
(137 11 nM and 284 18 nM, respectively). The chloroquine
and quinine IC50 values were consistent with published data.10
In the series of the alkylacyl derivatives antimalarial activity in-
crease slightly with increasing size of the alkyl residue from
2.5. Inhibition of P-glycoproteins
9.72
ative 5c. Replacement of the alkyl residues by phenyl resulted in a
slightly more active derivative 5d (IC50 = 1.61 M), while further
lM of the acetyl derivative 5a to 3.04 lM of the pivaloyl deriv-
ATP-dependent multidrug efflux pumps have been recognized
as a major cause for the failure of anticancer therapy but its role
in restricting the effectiveness of antimicrobial therapy is also
increasingly recognized.15 Multidrug efflux pumps use the energy
provided by ATP-binding and hydrolysis to prevent toxic com-
pounds from entering cells. In its functional form ATP-binding cas-
sette (ABC) transporters consist of two nucleotide binding domains
and two membrane spanning substrate (solute) binding domains.
Because of the structural similarity of the benzophenones with
known inhibitors of multidrug resistance transporters we have
tested our compounds for possible activity against these transport-
ers using the daunomycin efflux assay.
l
insertion of a methylene and a ethylene spacer (5e and 5f, respec-
tively) did not further increased antimalarial activity as did the
replacement of the phenyl moiety by a 3-pyridyl residue (5g). When
the carbonyl group was replaced by a sulfonyl moiety (compounds
5h–5j), no significant influence on antimalarial activity could be re-
corded. However, submicromolar activity was obtained with the
naphthyl derivatives 5k and 5l. Interestingly, the position of connec-
tion between the naphthyl residue and the carbonyl makes a notable
difference in antimalarial activity. While the 1-naphthyl derivative
5k displays an IC50 of 930 nM, the 2-naphthyl isomer 5l is nearly
fourfold more active (IC50 = 240 nM). This marked dependence of
antimalarial activity on the precise orientation of the naphthyl resi-
due argues for well-defined geometric requirements in the putative
target structure. The same holds true for the pair of urea derivatives
5m and 5n. While the 4-methylpiperidyl derivative 5m is virtually
The daunomycin efflux assay16 is a direct and accurate func-
tional method to measure inhibition of PGP-mediated transmem-
brane transport. The resistant human T-lymphoblast cell line
CCRF VCR 1000 overexpressing the ABCB1 transporter was used
in these studies. The time dependent decrease in mean cellular
fluorescence is determined in the presence of various concentra-
inactive (IC50 >16 lM), the di-isopropyl derivative 5n displays with

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M. Altenkämper et al. / Bioorg. Med. Chem. 17 (2009) 7690–7697
Table 1
Structure and antiparasitic activity of target compounds 5 and 6
Cmpd
R1/R2
IC50 P. falciparum (
lM)
CC50
105
(lM)
SIb
11
IC50 T. brucei bruceic
(lM)
EC50 (lM)
a
d
5a
MeCONH/Ph
9.72 1.16
9.05 2.69
6.59 1.11
16.73 2.00
24.62 7.02
11.42 0.25
4.67 1.61
3.28 0.13
3.28 0.24
3.64 0.65
3.25 0.10
3.27 1.18
3.02 0.21
5.06 2.25
7.02 1.73
5.58 2.66
5.84 2.60
5.38 0.78
2.92 1.18
3.01 0.61
3.12 0.06
1.93 0.33
2.68 0.18
1.98 0.88
2.64 0.35
nd
6.29 1.11
3.30 0.74
0.39 0.04
0.24 0.19
0.36 0.12
0.06 0.01
5.12 1.45
15.52 1.37
0.89 0.36
0.04 0.01
0.23 0.04
0.04 0.01
5b
5c
5d
5e
5f
EtCONH/Ph
7.19 1.21
3.04 0.31
1.61 0.28
2.00 0.26
1.80 0.26
2.26 0.56
10.62 3.27
0.91 0.46
1.24 0.72
0.93 0.16
0.24 0.05
84
45
16
16
8
12
15
10
8
t-BuCONH/Ph
PhCONH/Ph
Ph-CH2–CONH/Ph
Ph-(CH2)2–CONH/Ph
3-Pyridyl-CONH/Ph
MeSO2NH/Ph
4
5g
5h
5i
37
99
26
12
4
16
9
PhSO2NH/Ph
29
10
4
5j
(PhSO2)2NH/Ph
1-Naphthyl-CONH/Ph
2-Naphthyl-CONH/Ph
5k
5l
4
17
5m
5n
6a
N-Methyl-1-piperidyl CONH/Ph
(iPr)2NCONH/Ph
MeCONH/H
>16
0.11 0.02
9.45 0.34
nd
4
53
nd
36
6
4.97 2.63
0.46 0.13
nd
nd
23.05 7.12
22.91 4.56
53.43 18.05
71.49 14.07
49.43 3.59
83.18 17.94
13.92 0.65
21.89 1.19
0.81 0.33
2.01 0.44
6b
6c
6d
6e
EtCONH/H
>16
>122
>109
80
>8
>7
12
5
nd
PhCONH/H
>16
nd
Ph-(CH2)2–CONH/H
2-Naphthyl-CONH/H
6.72 0.23
2.01 0.30
2.23 0.23
0.48 0.05
9
a
b
c
Cytotoxicity (HeLa cells).
SI = selectivity index = CC50 (HeLa)/IC50 (P. falciparum).
48 h/72 h.
d
EC50 refers to daunomycin efflux-test, nd = not determined.
tions of modifier and the first-order rate constants (Vmax/Km) are
calculated by nonlinear regression analysis. A correction for simple
diffusion is achieved by subtracting the efflux rates observed in the
parental line. EC50 values of modifiers are calculated from dose–re-
sponse curves of Vmax/Km versus modifier concentration. These
studies were performed according to previously published meth-
ods,17,18 slightly adjusted to our requirements.19
shown). So, currently we do not have firm clue on the mechanism
of action of these compounds against malaria parasites.
3. Conclusion
In the series of readily obtainable benzophenone derivatives de-
scribed here we saw mainly micromolar activity against P. falcipa-
rum and T. brucei brucei. However, two compounds of this series
displayed considerable antimalarial activity with IC50 values of
240 and 110 nM, respectively, which merit further attention to this
class of compounds. Although selectivity is generally low and even
with the most active compound far from being satisfactory, this
compound demonstrates that it has not necessary be a fruitless at-
tempt to try to dissociate antimalarial from cytotoxic activity.
Further investigations will be directed toward the improvement
of antimalarial activity with concurrent reduction of cytotoxicity
and thereby enhancement of selectivity against malaria parasites.
Activity of the benzophenones against the ABCB1 transporter
(Table 1) ranges between 15.5 lM for the methylsulfonyl deriva-
tive 5h and 40 nM for the 2-naphthoyl derivative 5l and the bis-
(phenylsulfonyl) derivative 5j with several other derivatives being
also in the submicromolar range. Two general trends can be out-
lined. Sulfonamides are less active than the corresponding carbon-
amides and more spacious lipophilic acyl residues tend to produce
higher activity in the daunorubicin efflux assay.
An analogue of human ABC transporters is the P. falciparum
P-glycoprotein homologue Pgh-1 (or multidrug resistance protein
1 (PfMDR 1)), which is located in the membrane of the digestive
vacuole and has been shown to transport solutes, including anti-
malarial drugs, into the digestive vacuole.12,20 Copy number of
the pfmdr1 gen and polymorphism alters parasite sensitivity to
arylaminoalcohols.21 Furthermore, the theory has been put for-
ward that Pgh-1 might be the target of quinine and other aryl-
aminoalcohols.12 At present, we have no data suggesting that our
compounds interfere with Pgh-1. At micromolar concentrations,
none of the compounds inhibited Pgh-1-mediated accumulation
of fluorochromes into the parasite’s digestive vacuole (data not
4. Experimental
4.1. Chemistry
1H NMR and 13C NMRspectra were recorded on a Jeol Lambda 500
delta, a Jeol JNM-GX-400, a Jeol Eclipse 500 and a Jeol Eclipse 400
spectrometer. Massspectra wereobtainedwith a VacuumGenerator
VG 7070H using a Vector 1 data acquisition system from Teknivent,

M. Altenkämper et al. / Bioorg. Med. Chem. 17 (2009) 7690–7697
7693
an AutoSpec mass spectrometer from Micromass, an API 2000 LC–
MS–MS system of PE SCIEX using Analyst 1.2 of Applied Biosys-
tems/MDS SCIEX and on a MStation JMS 700 from Jeol using Jeol
Mass Data System MS-MP9021D 2.30. IR spectra were recorded on
a Nicolet 510PFTIR spectrometer, a Jasco FT/IR 410 FTIR, a Perkin–El-
mer Paragon 1000 FT-IR or a Brucker alpha-P spectrometer. Microa-
nalyses were obtained with a CH analyzer according to Dr. Salzer
from Labormatic, a Hewlett–Packard CHN analyzer type 185, and a
Vario EL from Elementar. Melting points were obtained with a Reic-
hert Austria microscope and are uncorrected. Column-chromatogra-
phy was carried out using Silica Gel 60 (0.062–0.200 mm) from
Machery-Nagel and Silica Gel 60 (0.040–0.063) from Merck.
10.0 mmol). Purification: recrystallization from ethanol to give a
white crystalline solid: yield 3.06 g (96%). Mp 176 °C; 1H NMR
(500 MHz, CDCl3) dH (ppm) = 4.25 (s, 2H), 7.53–7.59 (m, 2H), 7.66–
7.72 (m, 1H), 7.72–7.77 (m, 2H), 8.44–8.46 (m, 1H), 8.51 (m, 1H),
8.91 (m, 1H), 11.92 (s, 1H); 13C NMR (125 MHz, CDCl3) dC
(ppm) = 45.05, 121.62, 123.55, 128.60, 128.86, 128.87, 129.98,
133.67, 137.04, 142.14, 144.52, 165.96, 197.49; IR (KBr):
m = 3241,
1689, 1641, 1617, 1597, 1587, 1549, 1511, 1447, 1414, 1400, 1345,
1320, 1305, 1284, 1225, 1172, 1126, 1093, 1080, 969, 915, 855,
805, 762, 702, 654; MS (ESI): m/z 288 (8), 341 (82) [35Cl, M+Na]+,
659 (100) [2 (35Cl, M+Na)]+; HRMS (ESI): calcd: 341.0305, found:
341.0302 [35Cl, M+Na]+.
4.1.1. General procedure 1: activation of various acids as acid
chlorides and reaction with aromatic amines
4.1.3.3. (R,S)-N-(2-Benzoyl-4-nitrophenyl)-2-(4-methyl-1-piper-
azinyl)-2-phenylacetamide (3a). According to general procedure
2 from (R,S)-N-(2-benzoyl-4-nitrophenyl)-2-chloro-2-phenylacet-
amide (1.97 g, 5.0 mmol) and N-methylpiperazine (1.66 mL,
15.0 mmol). Purification: column-chromatography, silica gel, ethyl
acetate/isohexane (1:3): yield 1.54 g (67%). Mp 101 °C; 1H NMR
(500 MHz, CDCl3) dH (ppm) = 2.32 (s, 3H), 2.59 (s, 8H), 4.04 (s,
1H), 7.29–7.42 (m, 5H), 7.55–7.61 (m, 3H), 7.71 (m, 1H), 7.79–
7.82 (m, 2H), 8.37 (m, 1H), 8.46 (m, 1H), 12.13 (s, 1H); 13C NMR
(125 MHz, CDCl3) dC (ppm) = 45.60, 54.62, 76.80, 121.40, 123.79,
127.89, 128.16, 128.56, 128.61, 128.87, 128.90, 130.00, 133.57,
134.67, 137.27, 141.46, 145.03, 171.72, 196.74; IR (KBr):
The various carboxylic acids were dissolved in dichloromethane
and 0.2 mL oxalylchloride per mmol acid was added. The mixture
was stirred for 2 h and the volatiles were evaporated in vacuo.
The resulting acyl chlorides were dissolved in dioxane (approx.
30 mL) and added to a solution of the appropriate aromatic amine
in hot toluene (approx. 50 mL). The mixtures were heated under
reflux for 2 h. After the solvent was removed in vacuo, the crude
products were purified by recrystallization.
4.1.2. General procedure 2: substitution of the halogen of 2-
chloro-(phenyl)alkylcarboxylic acid derivatives with amines
The 2-chloro-(phenyl)alkylcarboxylic acid derivative was dis-
solved in freshly distilled acetonitrile, 3 equiv of the amine were
added and the mixture was heated under reflux for 24–48 h. After
removing the acetonitrile under reduced pressure, the obtained so-
lid was dissolved in ethyl acetate and was purified either by col-
umn-chromatography or by washing with a saturated solution of
K2CO3, drying over Na2SO4 and removing the solvent under re-
duced pressure. In case further purification was necessary, the
crude product was recrystallized, or chromatographed on silica gel.
m
= 2936, 2797, 1700, 1647, 1577, 1494, 1336, 1275, 1256, 1135,
1092, 1008, 697, 648; MS (ESI): m/z 189 (23), 459 (100) [35Cl,
M+H]+; HRMS (ESI): calcd: 459.2032, found: 459.2031.
4.1.3.4. N-(2-Benzoyl-4-nitrophenyl)-2-(4-methyl-1-piperazi-
nyl)-acetamide (3b). According to general procedure 2 from N-
(2-benzoyl-4-nitrophenyl)-2-chloroacetamide (2.9 g, 9.0 mmol)
and N-methylpiperazine (3.0 mL, 27.0 mmol). Purification: precip-
itation from dichloromethane solution with n-pentane: yield
1.99 g (58%). Mp 178 °C; 1H NMR (500 MHz, CDCl3) dH
(ppm) = 2.49 (s, 3H), 2.79 (s, br, 8H), 3.25 (s, 2H), 7.51–7.59 (m,
2H), 7.67–7.72 (m, 1H), 7.74–7.77 (m, 2H), 8.39–8.45 (m, 2H),
8.93–8.97 (m, 1H), 11.94 (s, 1H); 13C NMR (125 MHz, CDCl3) dC
(ppm) = 45.46, 52.71, 54.48, 61.96, 121.36, 124.02, 127.98,
128.48, 128.84, 130.02, 133.60, 137.20, 141.54, 144.54, 170.52,
4.1.3. General procedure 3: reduction of aromatic nitro
compounds
Aromatic nitro compounds were dissolved in ethyl acetate (50–
100 mL) and SnCl2 ꢀ 2H2O (1.125 g per mmol nitro compound)
was added. The mixture was heated under reflux for 2 h. Then,
NaHCO3-solution was added until pH 7–8 was reached and the or-
ganic layer was separated. The aqueous layer was extracted three
times with ethyl acetate. The combined organic layers were
washed with brine and dried over MgSO4. Then, the solvent was re-
moved in vacuo to obtain the crude products.
196.31; IR (KBr):
m = 3426, 3203, 2937, 2820, 1696, 1653, 1609,
1597, 1577, 1560, 1522, 1499, 1449, 1414, 1374, 1341, 1279,
1255, 1172, 1140, 1096, 1011, 985, 966, 917, 874; MS (EI): m/z
33 (22), 41 (22), 58 (28), 70 (54), 71 (24), 86 (23), 98 (21), 99
(29), 113 (100), 114 (58), 157 (62), 382 (58) [M]+, 383 (13)
[M+1]+; HRMS (ESI): calcd: 382.1641, found: 382.1673.
4.1.3.1. (R,S)-N-(2-Benzoyl-4-nitrophenyl)-2-chloro-2-phenyl-
acetamide (2a). According to general procedure 1 from (R,S)-2-
chloro-2-phenylacetyl chloride (1.46 mL, 10.0 mmol) and 2-ami-
no-5-nitrobenzophenone (2.42 g, 10.0 mmol). Purification: recrys-
tallization from ethanol to give a yellowish solid: yield 3.63 g
(92%). Mp 134 °C; 1H NMR (500 MHz, CDCl3) dH (ppm) = 5.52 (s,
1H), 7.38–7.43 (m, 3H), 7.55–7.59 (m, 4H), 7.70 (m, 1H), 7.73–
7.75 (m, 2H), 8.42 (m, 1H), 8.52 (m, 1H), 8.86 (m, 1H), 12.12 (s,
1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 62.11, 121.50, 123.26,
127.75, 128.76, 128.87, 128.94, 129.10, 129.45, 129.95, 133.59,
135.85, 137.10, 142.02, 144.96, 167.46, 197.68; IR (KBr):
4.1.3.5. (R,S)-N-(4-Amino-2-benzoylphenyl)-2-(4-methyl-1-pip-
erazinyl)-2-phenylacetamide (4a). According to general proce-
dure
3 from (R,S)-N-(2-benzoyl-4-nitrophenyl)-2-(4-methyl-1-
piperazinyl)-2-phenylacetamide (1.10 g, 2.5 mmol) and tin(II)
chloride (2.80 g, 12.3 mmol). Purification: column-chromatogra-
phy, silica gel, ethyl acetate/methanol (1:1): yield 907 mg (86%).
Mp 148 °C; 1H NMR (500 MHz, DMSO-d6) dH (ppm) = 2.14 (s, 3H),
2.17–2.25 (m, 4H), 2.27–2.40 (m, 4H), 3.83 (s, 1H), 5.21 (s, 2H),
6.33 (m, 1H), 6.74 (m, 1H), 7.25–7.31 (m, 5H), 7.51–7.58 (m, 3H),
7.63–7.67 (m, 1H), 7.72 (m, 2H), 10.38 (s, 1H); 13C NMR
(125 MHz, DMSO-d6) dC (ppm) = 45.60, 54.62, 75.15, 117.20,
121.89, 123.79, 123.85, 125.87, 127.89, 128.14, 128.36, 128.73,
129.54, 132.63, 136.35, 137.64, 144.97, 168.90, 196.70; IR (KBr):
m
= 3113, 2960, 2362, 1841, 1718, 1690, 1642, 1616, 1596, 1581,
1535, 1509, 1350, 1277, 1249, 1152, 742; MS (ESI): m/z 417
(100) [35Cl, M+Na]+, 419 (35) [37Cl, M+Na]+; HRMS (ESI): calcd:
417.0618, found: 417.0615.
m
= 3455, 3361, 3274, 1671, 1632, 1606, 1595, 1577, 1512, 1446,
1434, 1329, 1289, 1251, 1178, 1158, 1139, 1010, 964, 868, 700;
MS (EI): m/z 70 (13), 91 (12), 105 (25), 189 (100), 190 (59), 195
(21), 211 (33), 212 (42), 224 (18), 238 (13), 239 (18), 240 (21),
271 (7), 328 (7), 330 (10), 428 (30) [M]+; HRMS (EI): calcd:
428.2212, found: 428.2213.
4.1.3.2. N-(2-Benzoyl-4-nitrophenyl)-2-chloroacetamide (2b).
According to general procedure 1 from 2-chloroacetyl chloride
(0.80 mL, 10.0 mmol) and 2-amino-5-nitrobenzophenone (2.42 g,

7694
M. Altenkämper et al. / Bioorg. Med. Chem. 17 (2009) 7690–7697
4.1.3.6. N-(4-Amino-2-benzoylphenyl)-2-(4-methyl-1-piperazi-
nyl)acetamide (4b). According to general procedure 3 from N-(2-
benzoyl-4-nitrophenyl)-2-(4-methyl-1-piperazinyl)acetamide (2.0
g, 5.0 mmol) and tin(II) chloride (5.6 g, 26.0 mmol). Purification: col-
umn-chromatography, silica gel, ethyl acetate/methanol (3:2): yield
605 mg (34%). Mp 74 °C; 1H NMR (500 MHz, CDCl3) dH (ppm) = 2.36
(s, 3H), 2.60(s, br, 8H), 3.10(s, 2H), 3.64 (s, br, 2H), 6.77(m, 1H), 6.87–
6.91 (m, 1H), 7.46–7.49 (m, 2H), 7.57–7.60 (m, 1H), 7.77–7.80 (m,
2H), 8.27–8.29 (m, 1H), 10.93 (s, 1H,) 13C NMR (125 MHz, CDCl3) dC
(ppm) = 45.88, 53.39, 54.82, 62.11, 117.70, 119.67, 123.68, 127.33,
128.28, 130.03, 130.11, 132.60, 138.21, 141.53, 169.28, 197.43; IR
123.75, 124.93, 125.69, 128.23, 128.48, 128.65, 128.91, 130.22,
132.63, 132.96, 135.33, 135.55, 137.96, 170.76, 176.55, 197.69; IR
(KBr):
m = 3340, 2967, 2801, 1685, 1534, 1507, 1400, 1291, 1241,
1162, 702; MS (EI): m/z 56 (18), 91 (15), 105 (8), 189 (100), 290
(71), 211 (14), 296 (38), 322 (20), 414 (12), 512 (39) [M]+; HRMS
(EI): calcd: 512.2787, found: 512.2784.
4.1.3.10. (R,S)-N-(2-Benzoyl-4-benzoylaminophenyl)-2-(4-meth-
yl-piperazin-1-yl)-2-phenylacetamide (5d). According to general
procedure 1 from (R,S)-N-(4-amino-2-benzoylphenyl)-2-(4-methyl-
1-piperazinyl)-2-phenylacetamide (4a) (0.43 g, 1.0 mmol) and ben-
zoyl chloride (0.17 g, 1.2 mmol). Purification: column-chromatogra-
phy, silica gel, methanol/diethyl ether/isohexane (8:2:1): yield
474 mg (89%). Mp 135 °C; 1H NMR (500 MHz, CDCl3) dH (ppm) 2.42
(s, 3H), 2.62 (s, br, 4H), 2.74 (s, br, 4H), 3.98 (s, 1H), 7.27–7.32 (m,
3H), 7.35–7.38 (m, 2H), 7.42–7.46 (m, 2H), 7.50–7.55 (m, 3H), 7.59–
7.64 (m, 2H), 7.80–7.85 (m, 4H), 8.01 (s, 1H), 8.05–8.10 (m, 1H),
8.54 (m, 1H), 11.64 (s, 1H); 13C NMR (125 MHz, CDCl3) dC
(ppm) = 45.05, 54.39, 76.49, 122.34, 124.66, 125.22, 125.49, 127.06,
128.48, 128.76, 128.81, 128.87, 129.05, 130.16, 132.03, 132.62,
132.98, 134.36, 135.26, 135.77, 138.04, 165.73, 170.54, 198.12; IR
(KBr):
m
(cmꢁ1): = 3429, 2937, 2816, 1670, 1596, 1577, 1616, 1437,
1374, 1328, 1290, 1245, 1169, 1012, 975, 830, 746, 707, 479; MS
(EI, 70 eV): m/z 70 (45), 99 (21), 113 (90), 114 (53), 212 (81), 225
(24), 282 (25), 295 (47), 296 (24), 309 (22), 352 (100) [M]+, 353
(29) [M+1]+; MS (EI-HRMS): calcd: 352.1899, found: 352.1910.
4.1.3.7. (R,S)-N-(4-Acetylamino-2-benzoylphenyl)-2-(4-methyl-
1-piperazinyl)-2-phenylacetamide (5a). According to general
procedure
1
from (R,S)-N-(4-amino-2-benzoylphenyl)-2-(4-
methyl-1-piperazinyl)-2-phenylacetamide (4a) (0.43 g, 1.0 mmol)
and acetic acid chloride (0.09 g, 1.2 mmol). Purification: column-
chromatography, silica gel, ethyl acetate/methanol (1:2): yield
132 mg (28%). Mp 85 °C; 1H NMR (500 MHz, CDCl3) dH (ppm)
2.09 (s, 3H), 2.29 (s, 3H), 2.53 (s, br, 8H), 3.94 (s, 1H), 7.27 (s,
1H), 7.25–7.31 (m, 2H), 7.34–7.39 (m, 3H), 7.43 (m, 1H), 7.49–
7.53 (m, 2H), 7.59–7.63 (m, 1H), 7.80–7.83 (m, 2H), 7.90 (m, 1H),
8.48 (m, 1H), 11.56 (s, 1H); 13C NMR (125 MHz, CDCl3) dC
(ppm) = 24.31, 45.65, 54.67, 76.84, 122.33, 124.16, 124.77,
125.31, 125.39, 128.26, 128.40, 128.66, 128.84, 130.11, 132.38,
132.78, 135.47, 137.95, 168.30, 170.90, 197.80; IR (KBr):
(KBr):
m = 3282, 2937, 2804, 1654, 1596, 1507, 1401, 1290, 1139,
1009, 980, 703; MS (EI): m/z 70 (12), 77 (5), 91 (17), 105 (39), 106
(8), 146 (6), 187 (6), 189 (100), 190 (72), 191 (10), 211 (7), 212 (4),
216 (8), 316 (34), 317 (9), 342 (33), 343 (15), 434 (13), 530 (10),
532 (31) [M]+; HRMS (EI): calcd: 532.2474, found: 532.2478.
4.1.3.11. (R,S)-N-(2-Benzoyl-4-phenylacetylaminophenyl)-2-(4-
methyl-piperazin-1-yl)-2-phenylacetamide (5e). According to
general procedure
2-(4-methyl-1-piperazinyl)-2-phenylacetamide
1
from (R,S)-N-(4-amino-2-benzoylphenyl)-
(4a) (0.43 g,
m
= 3276, 2933, 2846, 2669, 1550, 1508, 1453, 1402, 1292, 1140,
1.0 mmol) and phenyl acetyl chloride (0.2 mL, 1.5 mmol). Purifica-
tion: column-chromatography, silica gel, methanol/diethyl ether
(4:1): yield 233 mg (43%). Mp 190 °C; 1H NMR (500 MHz, CDCl3)
dH (ppm) = 2.27 (s, 3H), 2.49 (s, br, 8H), 3.65 (s, 2H), 3.92 (s, 1H),
7.28 (s, 1H), 7.21–7.43 (m, 11H), 7.50 (m, 2H), 7.61 (m, 1H), 7.80
(m, 2H), 7.92 (m, 1H), 8.43 (m, 1H), 11.49 (s, 1H); 13C NMR
(125 MHz, CDCl3) dC (ppm) = 44.53, 45.78, 54.75, 76.91, 122.22,
123.92, 124.66, 125.51, 127.70, 128.22, 128.41, 128.63, 128.92,
129.21, 129.44, 130.20, 132.19, 132.93, 134.09, 135.51, 135.55,
1009, 702; MS (EI): m/z 70 (6), 91 (8), 105 (6), 146 (4), 189
(100), 190 (43), 211 (6), 212 (7), 254 (14), 280 (10), 372 (5), 470
(14) [M]+; HRMS (EI): calcd: 470.2317, found: 470.2320.
4.1.3.8. (R,S)-N-(2-Benzoyl-4-propanoylaminophenyl)-2-(4-meth-
yl-piperazin-1-yl)-2-phenylacetamide (5b). According to general
procedure 1 from (R,S)-N-(4-amino-2-benzoylphenyl)-2-(4-methyl-
1-piperazinyl)-2-phenylacetamide (4a) (0.43 g, 1.0 mmol) and propio-
nyl chloride (0.11 g, 1.2 mmol). Purification: column-chromatography,
silica gel, methanol/diethyl ether (4:1): yield 300 mg (62%). Mp 80 °C;
1H NMR (500 MHz, CDCl3) dH (ppm) = 1.18 (t, 3H), 2.29 (s, 3H), 2.31 (q,
2H), 2.53 (s, br, 8H), 3.94 (s, 1H), 7.24–7.29 (m, 3H), 7.29 (s, 1H), 7.36–
7.40(m, 2H), 7.45(m, 1H), 7.51 (m, 2H), 7.62 (m, 1H), 7.82 (m, 2H), 7.91
(m, 1H), 8.47 (m, 1H), 11.51 (s, 1H); 13C NMR (125 MHz, CDCl3) dC
(ppm) = 9.37, 30.09, 45.70, 54.69, 76.78, 122.33, 123.72, 124.52,
125.77, 128.20, 128.29, 128.55, 128.87, 130.03, 132.78, 133.17,
137.88, 169.12, 170.93, 197.61; IR (KBr):
m = 3328, 3029, 2938,
2838, 2795, 1695, 1658, 1545, 1504, 1453, 1400, 1322, 1288,
1247, 1139, 1013, 846, 700; MS (EI): m/z 70 (10), 91 (44), 105
(10), 119 (11), 136 (20), 189 (100), 190 (70), 212 (23), 238 (24),
330 (25), 356 (16), 428 (17), 546 (11) [M]+; HRMS (EI): calcd:
546.2631, found: 546.2650 [M]+.
4.1.3.12. (R,S)-N-[2-Benzoyl-4-(3-phenylpropionyl)aminophenyl]-
2-(4-methylpiperazin-1-yl)-2-phenylacetamide (5f). According
to general procedure 1 from (R,S)-N-(4-amino-2-benzoylphenyl)-
134.61, 135.35, 137.80, 170.96, 172.38, 197.58; IR (KBr):
m = 3276,
3062, 2939, 2804, 1669, 1548, 1506, 1453, 1403, 1291, 1243, 1009;
MS (EI): m/z 70 (16), 91 (20), 98 (12), 105 (10), 146 (8), 187 (9), 190
(72), 211 (12), 216 (11), 238 (22), 268 (24), 394 (15), 386 (10), 484
(49) [M]+; HRMS (EI): calcd: 484.2474, found: 484.2477.
2-(4-methyl-1-piperazinyl)-2-phenylacetamide
(4a)
(0.43 g,
1.0 mmol) and 3-phenylpropionic acid chloride (0.2 mL, 1.5 mmol).
Purification: column-chromatography, silica gel, methanol/ethyl
acetate (1:4): yield 393 mg (70%). Mp 65 °C; 1H NMR (500 MHz,
3
4.1.3.9. (R,S)-N-(2-Benzoyl-4-pivaloylaminophenyl)-2-(4-meth-
yl-piperazin-1-yl)-2-phenylacetamide (5c). According to gen-
eral procedure 1 from (R,S)-N-(4-amino-2-benzoylphenyl)-2-(4-
methyl-1-piperazinyl)-2-phenylacetamide (4a) (0.30 g, 0.7 mmol)
and pivalic acid chloride (0.11 mL, 0.8 mmol). Purification: col-
umn-chromatography, silica gel, ethyl acetate/methanol (1:2):
yield 215 mg (60%). Mp 90 °C; 1H NMR (500 MHz, CDCl3) dH
(ppm) 1.27 (s, 9H), 2.29 (s, 3H), 2.52 (s, br, 8H), 3.94 (s, 1H), 7.26
(s, 1H), 7.27–7.30 (m, 3H), 7.34–7.39 (m, 2H), 7.47–7.49 (m, 1H),
7.50–7.55 (m, 2H), 7.60–7.63 (m, 1H), 7.82–7.85 (m, 2H), 7.87–
7.89 (m, 1H), 8.49 (m, 1H), 11.44 (s, 1H); 13C NMR (125 MHz,
CDCl3) dC (ppm) = 27.56, 39.52, 45.58, 54.61, 76.86, 122.31,
CDCl3) dH (ppm) = 2.32 (s, 3H), 2.58 (t, 2H, JH–H = 7.6 Hz), 2.61 (s,
3
br, 8H), 2.98 (t, 2H, JH–H = 7.6 H), 3.94 (s, 1H), 7.13–7.24 (m, 4H),
7.24–7.28 (m, 4H), 7.29 (s, 1H), 7.32–7.34 (m, 1H), 7.35–7.38 (m,
2H), 7.52 (m, 2H), 7.62–7.64 (m, 1H), 7.74–7.83 (m, 3H), 8.44 (m,
1H), 11.55 (s, 1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 31.48,
39.27, 45.27, 54.39, 77.20, 122.30, 124.24, 124.99, 125.29, 126.42,
128.30, 128.33, 128.43, 128.63, 128.70, 128.86, 130.14, 132.30,
132.90, 135.43, 135.47, 137.98, 140.43, 170.42, 170.80, 197.80; IR
(KBr):
m = 3274, 3061, 3027, 2937, 2823, 1667, 1506, 1454, 1403,
1290, 1140, 1076, 1010, 749, 699; MS (EI): m/z 91 (12), 105 (6),
189 (100), 190 (44), 212 (11), 238 (12), 344 (12), 560 (9) [M]+;
HRMS (EI): calcd: 560.2787, found: 560.2785 [M]+.

M. Altenkämper et al. / Bioorg. Med. Chem. 17 (2009) 7690–7697
7695
4.1.3.13. (R,S)-N-(2-Benzoyl-4-nicotinoylaminophenyl)-2-(4-me-
thyl-piperazin-1-yl)-2-phenylacetamide (5g). According to
2-benzoylphenyl)-2-(4-methyl-1-piperazinyl)-2-phenylacetamide
(4a) (0.43 g, 1.0 mmol) and benzene sulfonyl chloride (0.2 mL,
2.0 mmol). Purification: column-chromatography, silica gel, meth-
anol/diethyl ether (2:1): yield 300 mg (72%). Mp 175 °C; 1H NMR
(500 MHz, CDCl3) dH (ppm) = 2.32 (s, 3H), 2.57 (s, br, 8H), 3.98 (s,
1H), 7.11 (m, 1H), 7.27–7.33 (m, 4H), 7.40 (m, 4H), 7.47 (m, 2H),
7.48–7.54 (m, 4H), 7.64–7.72 (m, 4H), 7.90 (m, 3H), 8.66 (m, 1H),
11.96 (s, 1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 45.72, 54.71,
77.16, 121.87, 124.54, 127.21, 127.53, 128.43, 128.46, 128.51,
128.70, 128.79, 128.87, 129.13, 129.16, 130.03, 132.96, 132.98,
133.09, 134.10, 135.26, 135.98, 136.44, 137.60, 141.29, 171.44,
general procedure
2-(4-methyl-1-piperazinyl)-2-phenylacetamide
1
from (R,S)-N-(4-amino-2-benzoylphenyl)-
(4a) (0.43 g,
1.0 mmol) and nicotinic acid chloride hydrochloride (0.27 g,
1.5 mmol) under addition of triethylamine (0.3 mL, 2.0 mmol).
Purification: column-chromatography, silica gel, methanol/diethyl
ether/isohexane (8:2:2): yield 350 mg (66%). Mp 95 °C; 1H NMR
(500 MHz, CDCl3) dH (ppm) = 2.29 (s, 3H), 2.52 (s, br, 8H), 3.93 (s,
1H), 7.25–7.29 (m, 3H), 7.32–7.39 (m, 3H), 7.50 (m, 2H), 7.58–
7.62 (m, 2H), 7.81–7.85 (m, 2H), 7.93 (m, 1H), 8.14 (m, 1H), 8.45
(s, 1H), 8.46 (m, 1H), 8.69–8.72 (m, 1H), 9.03–9.07 (m, 1H), 11.58
(s, 1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 45.72, 54.72,
76.84, 122.51, 123.56, 124.71, 125.56, 125.64, 128.32, 128.47,
128.68, 128.91, 130.16, 130.22, 132.22, 132.99, 135.33, 135.42,
135.87, 137.87, 148.04, 152.52, 163.91, 171.15, 197.59; IR (KBr):
197.00; IR (KBr):
m = 3202, 3059, 2935, 2796, 1692, 1508, 1448,
1385, 1285, 1260, 1170, 936, 881, 723; MS (EI): m/z 77 (19), 91
(11), 105 (11), 142 (10), 189 (100), 190 (68), 211 (30), 219 (11),
237 (18), 351 (13), 378 (24), 470 (9); MS (ESI): 569 (20), 709
(100) [M+H]+, 1417 (8) [2 (M+H)]+; m/z HRMS (ESI): calcd:
709.2155, found: 709.2146 [M+H]+.
m
= 3259, 2938, 2804, 1673, 1545, 1506, 1452, 1402, 1288, 1139,
1010, 729, 702; MS (EI): m/z 70 (14), 91 (21), 106 (39), 189
(100), 190 (69), 211 (16), 317 (52), 343 (58), 435 (19), 533 (25)
[M]+; HRMS (EI): calcd: 533.2427, found: 533.2434 [M]+.
4.1.3.17. (R,S)-N-(2-Benzoyl-4-(1-naphthoylamino)phenyl)-2-(4-
methyl-piperazin-1-yl)-2-phenylacetamide (5k). According to
general procedure
2-(4-methyl-1-piperazinyl)-2-phenylacetamide
1
from (R,S)-N-(4-amino-2-benzoylphenyl)-
(4a) (0.43 g,
4.1.3.14. (R,S)-N-[2-Benzoyl-4-(methylsulfamoyl)phenyl]-2-(4-me-
thyl-piperazin-1-yl)-2-phenylacetamide (5h). According to general
procedure 1 from (R,S)-N-(4-amino-2-benzoylphenyl)-2-(4-methyl-1-
piperazinyl)-2-phenylacetamide (4a) (0.43 g, 1.0 mmol) and methylsul-
fonyl chloride (0.17 g, 1.5 mmol). Purification: column-chromatogra-
phy, silica gel, methanol/diethyl ether (2:1): yield 368 mg (73%). Mp
95 °C; 1H NMR (500 MHz, CDCl3) dH (ppm) = 2.28 (s, 3H), 2.51 (s, br,
8H), 2.91 (s, 3H), 3.95 (s, 1H), 7.25–7.34 (m, 6H), 7.27 (s, 1H), 7.46 (m,
1H), 7.48–7.52 (m, 2H), 7.61–7.63 (m, 1H), 7.77–7.81 (m, 2H), 8.53 (m,
1H), 11.57 (s, 1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 39.45, 45.75,
54.73, 76.81, 122.96, 126.00, 126.47, 127.07, 128.32, 128.54, 128.69,
128.91, 130.15, 130.81, 133.17, 135.32, 137.09, 137.61, 171.18,
1.0 mmol) and 1-naphthoyl chloride (0.19 g, 1.2 mmol). Purifica-
tion: column-chromatography, silica gel, methanol/ethyl acetate
(2:1): yield 430 mg (74%). Mp 100 °C; 1H NMR (500 MHz, CDCl3)
dH (ppm) 2.29 (s, 3H), 2.52 (s, br, 8H), 3.91 (s, 1H), 7.28–7.32 (m,
3H), 7.36–7.39 (m, 2H), 7.41–7.45 (m, 1H), 7.50–7.55 (m, 4H),
7.58–7.68 (m, 3H), 7.84–7.92 (m, 4H), 7.94 (s, 1H), 8.05–8.10 (m,
1H), 8.25–8.29 (m, 1H), 8.54 (m, 1H), 11.58 (s, 1H); 13C NMR
(125 MHz, CDCl3) dC (ppm) = 45.59, 54.66, 60.35, 76.80, 122.39,
124.12, 124.58, 125.04, 125.12, 125.20, 125.43, 126.55, 127.34,
128.28, 128.39, 128.45, 128.66, 128.89, 129.99, 130.16, 131.14,
132.70, 132.90, 133.66, 133.87, 135.46, 135.71, 137.95, 167.54,
197.21; IR (KBr):
m
= 3242, 2937, 2807, 1685, 1508, 1448, 1396, 1322,
170.83, 197.74; IR (KBr): m = 3258, 3059, 2937, 2800, 1649, 1503,
1289, 1236, 1151, 985, 702; MS (EI): m/z 70 (17), 91 (22), 98 (11), 146
(10), 189 (100), 190 (72), 211 (23), 216 (12), 237 (12), 316 (15), 408
(7), 506 (24) [M]+; HRMS (EI): calcd: 506.1988, found: 506.1999 [M]+.
1450, 1404, 1288, 1139, 1009, 781, 702; MS (EI): m/z 70 (15), 91
(21), 98 (12), 99 (12), 127 (33), 146 (10), 155 (64), 156 (27), 172
(13), 188 (13), 189 (100), 190 (73), 191 (15), 216 (12), 366 (64),
367 (20), 392 (40), 393 (20), 484 (15), 485 (7), 582 (23) [M]+; HRMS
(EI): calcd: 582.2631, found: 582.2633.
4.1.3.15. (R,S)-N-[2-Benzoyl-4-(phenylsulfamoyl)phenyl]-2-(4-
methylpiperazin-1-yl)-2-phenylacetylamide (5i). Similar to
general procedure 1 from (R,S)-N-(4-amino-2-benzoylphenyl)-2-(4-
methyl-1-piperazinyl)-2-phenylacetamide (4a) (0.43 g, 1.0 mmol)
dilutedindichloromethaneandcooleddownto0 °C.Benzenesulfonyl
chloride (0.1 mL, 1.0 mmol) was added and stirred for further 3 h.
After that the reaction mixture was washed with saturated aqueous
sodiumhydrogencarbonate solution (20 mL) and saturated aqueous
sodiumchloride solution (20 mL). The combined organic phases were
dried over anhydrous sodiumsulfate. The solvent was removed in va-
cuo. Purification:column-chromatography, silicagel, methanol/ethyl
acetate (1:2): yield 100 mg (18%). Mp 73 °C; 1H NMR (500 MHz,
CDCl3) dH (ppm) = 2.30 (s, 3H), 2.53 (s, br, 8H), 3.94 (s, 1H), 7.14 (m,
1H), 7.27 (s, 1H), 7.28–7.30 (m, 4H), 7.33–7.36 (m, 2H), 7.38–7.48
(m, 4H), 7.57 (m, 1H), 7.58–7.64 (m, 3H), 7.67–7.70 (m, 2H), 8.45
(m, 1H), 11.60 (s, 1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 45.58,
54.61, 76.85, 113.75, 122.53, 125.34, 126.93, 127.13, 128.00,
128.28, 128.46, 128.65, 128.70, 128.89, 129.16, 130.73, 132.94,
4.1.3.18. (R,S)-N-(2-Benzoyl-4-[2-naphthoylamino)phenyl]-2-(4-
methyl-piperazin-1-yl)-2-phenylacetamide (5l). According to
general procedure
2-(4-methyl-1-piperazinyl)-2-phenylacetamide
1
from (R,S)-N-(4-amino-2-benzoylphenyl)-
(4a) (0.43 g,
1.0 mmol) and 2-naphthoyl chloride (0.29 g, 1.5 mmol). Purifica-
tion: column-chromatography, silica gel, methanol/ethyl acetate
(1:5): yield 340 mg (58%). Mp 110 °C; 1H NMR (500 MHz, CDCl3)
dH (ppm) = 2.31 (s, 3H), 2.54 (s, br, 8H), 3.90 (s, 1H), 7.23–7.29
(m, 3H), 7.31–7.36 (m, 2H), 7.44–7.56 (m, 5H), 7.61 (m, 1H),
7.76–7.87 (m, 6H), 8.07–8.12 (m, 1H), 8.30 (m 1H), 8.48 (m, 1H),
8.52 (s, 1H), 11.59 (s, 1H); 13C NMR (125 MHz, CDCl3) dC
(ppm) = 45.47, 54.54, 76.91, 122.36, 123.53, 124.53, 125.38,
125.47, 126.86, 127.72, 127.75, 127.90, 128.26, 128.41, 128.55,
128.64, 128.87, 128.89, 130.13, 131.57, 132.46, 132.83, 132.86,
134.81, 135.44, 135.53, 137.95, 165.90, 170.92, 197.76; IR (KBr):
m
= 3377, 3059, 2936, 2799, 1648, 1597, 1539, 1505, 1401, 1319,
135.31, 136.97, 137.73, 139.06, 171.00, 197.35; IR (KBr):
m
= 3231,
1288, 1239, 1138, 1010, 775, 702; MS (EI): m/z 70 (20), 91 (27),
98 (19), 105 (11), 127 (32), 146 (14), 154 (64), 189 (100), 190
(76), 216 (16), 366 (70), 392 (63), 484 (35), 582 (38) [M]+; HRMS
(EI): calcd: 582.2631, found: 582.2644 [M]+.
3061, 2937, 2825, 1685, 1646, 1596, 1578, 1512, 1448, 1320, 1290,
1164, 1091, 1010, 984, 721, 690, 583; MS (EI): m/z 70 (18), 91 (24),
98 (14), 105 (16), 146 (11), 189 (100), 190 (70), 211 (16), 216 (15),
219 (14), 237 (22), 352 (10), 378 (34), 470 (12), 568 (21) [M]+; HRMS
(EI): calcd: 568.2144, found: 568.2141 [M]+.
4.1.3.19. (R,S)-N-[2-Benzoyl-4-(N0,N0-diisopropylureido)phenyl]-2-
(4-methylpiperazin-1-yl)-2-phenylacetamide (5m). According to
general procedure 1 from (R,S)-N-(4-amino-2-benzoylphenyl)-2-(4-
methyl-1-piperazinyl)-2-phenylacetamide (4a) (0.30 g, 0.7 mmol)
and N,N-diisopropylcarbamoyl chloride (0.17 g, 1.1 mmol).
4.1.3.16. (R,S)-N-[2-Benzoyl-4-[N-(phenylsulfonyl)phenyl-sulfa-
moyl]phenyl]-2-(4-methyl-piperazin-1-yl)-2-phenylacetamide
(5j).
According to general procedure 1 from (R,S)-N-(4-amino-

7696
M. Altenkämper et al. / Bioorg. Med. Chem. 17 (2009) 7690–7697
Purification: column-chromatography, silica gel, methanol/ethyl ace-
4.1.3.23. N-(2-Benzoyl-4-phenylacetylaminophenyl)-2-(4-meth-
ylpiperazin-1-yl)-2-phenylacetamide (6d). According to general
procedure 1 from N-(4-amino-2-benzoylphenyl)-2-(4-methyl-1-
piperazinyl)acetamide (4b) (176 mg, 0.5 mmol) and 3-phenylpro-
pionic acid chloride (0.1 mL, 0.8 mmol). Purification: column-chro-
matography, silica gel, methanol; yield 94 mg (39%). Mp 57 °C; 1H
NMR (500 MHz, DMSO-d6) dH (ppm) = 2.51–2.60 (m, 8H), 2.79–
2.90 (m, 3H), 3.08 (s, 2H), 7.14–7.29 (m, 5H), 7.54–7.58 (m, 2H),
7.66–7.84 (m, 5H), 8.09–8.14 (m, 1H), 10.10 (m 1H), 10.77 (s,
1H); 13C NMR (125 MHz, DMSO-d6) dC (ppm) = 31.29, 35.80,
38.42, 51.14, 53.88, 61.12, 120.01, 122.31, 123.16, 123.80, 126.5,
128.77, 128.84, 128.87, 129.06, 130.28, 133.42, 138.09, 141.45,
tate (1:1): yield 138 mg (36%). Mp 150 °C; 1H NMR (500 MHz, CDCl3)
3
dH (ppm) = 1.21 (d, 12H, JH–H = 6.6 Hz), 2.10 (s, 3H), 2.24 (s, br, 8H),
3.78 (s, 1H), 3.88 (q, 2H, 3JH–H = 6.5 Hz), 7.25–7.35 (m, 4H), 7.54–7.58
(m, 4H), 7.65–7.70 (m, 2H), 7.73–7.78 (m, 2H), 7.82 (m, 1H), 8.19 (s,
1H), 10.68 (s, 1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 21.02,
45.85, 54.88, 59.63, 75.01, 121.43, 122.22, 123.52, 126.74, 127.76,
128.12, 128.44, 128.75, 129.72, 130.52, 132.82, 135.93, 136.65,
137.38, 153.52, 169.28, 196.28; IR (KBr):
m = 3307, 2967, 2934, 2798,
1666, 1597, 1505, 1452, 1319, 1292, 1237, 1142, 1010, 702; MS (EI):
m/z 70 (29), 86 (72), 87 (34), 91 (18), 101 (46), 105 (42), 128 (35),
146 (8), 189 (100), 190 (76), 238 (28), 264 (71), 296 (12), 365 (18),
454 (17), 555 (5) [M]+; HRMS (EI): calcd: 555.3209, found: 555.3208
[M]+.
141.65, 171.07, 174.26, 197.06; IR (KBr):
m
(cmꢁ1): = 3425, 1685,
1507, 1403, 1289, 701; MS (EI, 70 eV): m/z 70 (35), 113 (78), 114
(61), 344 (75), 357 (35), 427 (52), 428 (45), 441 (36), 482 (27),
484 (100) [M]+, 485 (35) [M+1]+; MS (EI-HRMS): calcd: 484.2474,
found: 484.2475.
4.1.3.20. N-(4-Acetylamino-2-benzoylphenyl)-2-(4-methyl-1-
piperazinyl)acetamide (6a).
According to general procedure
1
from N-(4-amino-2-benzoylphenyl)-2-(4-methyl-1-piperazi-
nyl)acetamide (4b) (353 mg, 1.0 mmol) and acetic acid chloride
(0.9 g, 1.2 mmol). Purification: column-chromatography, silica
gel, ethyl acetate/methanol (1:2): yield 296 mg (75%). Mp 165 °C;
1H NMR (500 MHz, CDCl3) dH (ppm) 2.11 (s, 3H), 2.33 (s, 3H),
2.57 (s, br, 8H), 3.13 (s, 2H), 7.48 (m, 4H), 7.58 (m, 1H), 7.80 (m,
2H), 7.85 (m, 1H), 8.56 (m, 1H), 11.30 (s, 1H); 13C NMR
(125 MHz, CDCl3) dC (ppm) = 24.33, 45.97, 53.48, 54.82, 62.20,
122.45, 123.79, 124.52, 125.89, 128.35, 130.20, 132.58, 132.85,
4.1.3.24. N-[2-Benzoyl-4-(2-naphthoylamino)phenyl]-2-(4-me-
thylpiperazin-1-yl)-2-phenylacetamide (6e). According to gen-
eral procedure
1
from N-(4-amino-2-benzoylphenyl)-2-(4-
methyl-1-piperazinyl)acetamide (4b) (176 mg, 0.5 mmol) and 2-
naphthoyl chloride (142 mg, 0.8 mmol). Purification: column-chro-
matography, silica gel, methanol/ethyl acetate (1:3): yield 121 mg
(48%). Mp 68 °C; 1H NMR (500 MHz, CDCl3) dH (ppm) = 2.35 (s, 3H),
2.63 (s, br, 8H), 3.13 (s, 2H), 7.47–7.59 (m, 5H), 7.66–7.72 (m, 1H),
7.80–7.91 (m, 6H), 8.03 (m, 1H), 8.09 (m, 1H), 8.33 (m, 1H), 8.61 (m
1H), 11.38 (s, 1H); 13C NMR (125 MHz, CDCl3) dC (ppm) = 45.45,
52.96, 54.45, 62.06, 120.49, 123.44, 124.16, 125.09, 125.83,
127.00, 127.67, 127.81, 128.02, 128.46, 128.75, 128.94, 130.29,
131.63, 132.56, 132.69, 132.94, 134.91, 135.33, 137.95, 165.77,
134.95, 137.88, 168.35, 169.91, 197.06; IR (KBr):
m
(cmꢁ1) = 3370,
2950, 2811, 1667, 1653, 1640, 1594, 1558, 1539, 1506, 1446,
1401, 1329, 1279, 1015, 831, 707; MS (EI, 70 eV): m/z 70 (37), 99
(29), 113 (71), 140 (33), 254 (55), 267 (41), 337 (54), 351 (41),
394 (100) [M]+; MS (EI-HRMS): calcd: 394.2005, found: 394.2019.
169.79, 197.25; IR (KBr):
m
(cmꢁ1): = 3855, 3752, 3745, 3676,
4.1.3.21. N-(2-Benzoyl-4-propanoylaminophenyl)-2-(4-methyl-
piperazin-1-yl)acetamide (6b). According to general procedure 1
from N-(4-amino-2-benzoylphenyl)-2-(4-methyl-1-piperazinyl)-
acetamide (4b) (353 mg, 1.0 mmol) and propionyl chloride (0.1 g,
1.2 mmol). Purification: column-chromatography, silica gel, metha-
nol/diethyl ether (4:1): yield 317 mg (78%). Mp 120 °C; 1H NMR
3425, 1735, 1701, 1685, 1654, 1648, 1560, 1507, 1400, 1284; MS
(EI, 70 eV): m/z 70 (40), 113 (100), 114 (60), 155 (46), 366 (74),
367 (24), 379 (24), 449 (36), 450 (28), 504 (25), 506 (82) [M]+,
507 (31) [M+1]+; MS (EI-HRMS): calcd: 506.2318, found: 506.2289.
4.2. Antimalarial activity
3
(500 MHz, CDCl3) dH (ppm) = 1.19 (t, 3H, JH–H = 7.5 Hz), 2.33 (q,
3
2H, JH–H = 7.5 Hz), 2.57 (s, br, 8H), 3.12 (s, 2H), 7.36 (m, 4H), 7.50
The multidrug resistant P. falciparum clone Dd222 was main-
tained in continuous culture as described23 and synchronized
using the sorbitol method.24 Drug assays based on [3H]-hypoxan-
thine incorporation were done25 and the percent inhibition was
determined as described.26 Each assay was repeated at least three
independent times and the mean SEM was calculated. IC50 values
were calculated from the sigmoidal dose–response curves using a
Hill function (SigmaPlot, SPSS).
(m, 1H), 7.80 (m, 2H), 7.86 (m, 1H), 8.51 (m, 1H), 11.26 (s, 1H); 13C
NMR (125 MHz, CDCl3) dC (ppm) = 9.48, 30.49, 45.93, 53.45, 54.79,
62.18, 122.47, 123.48, 124.38, 126.00, 128.38, 130.21, 132.69,
132.88, 134.78, 137.87, 169.85, 171.98, 197.07; IR (KBr):
m
(cmꢁ1) = 2937, 2811, 1668, 1596, 1546, 1508, 1460, 1401, 1294,
1013, 835, 706; MS (EI, 70 eV): m/z 43 (28), 57 (42), 70 (34), 84
(32), 113 (100), 205 (52), 268 (47), 322 (30), 351 (30), 408 (78)
[M]+; MS (EI-HRMS): calcd: 408.2161, found: 408.2160.
4.3. Antitrypanosomal activity
4.1.3.22. N-(2-Benzoyl-4-benzoylaminophenyl)-2-(4-methyl-pi-
perazin-1-yl)-2-phenylacetamide (6c). According to general
procedure 1 from N-(4-amino-2-benzoylphenyl)-2-(4-methyl-1-
piperazinyl)acetamide (4b) (353 mg, 1.0 mmol) and benzoyl chlo-
ride (0.9 g, 1.2 mmol). Purification: column-chromatography, silica
gel, methanol/diethyl ether/isohexane (8:2:1): yield 289 mg (63%).
Mp 185 °C; 1H NMR (500 MHz, CDCl3) dH (ppm): 2.34 (s, 3H), 2.59
(s, br, 8H), 3.14 (s, 2H), 7.50 (m, 5H), 7.60 (m, 1H), 7.68 (m, 1H),
7.83 (m, 4H), 8.00 (m, 1H), 8.59 (m, 1H), 11.34 (s, 1H); 13C NMR
(125 MHz, CDCl3) dC (ppm) = 45.93, 53.44, 54.80, 62.18, 122.53,
124.02, 125.00, 125.99, 127.04, 128.42, 128.74, 130.23, 131.98,
132.65, 132.90, 134.46, 135.18, 137.88, 165.73, 169.92, 197.06; IR
4.3.1. Parasite culture
Trypomastigote forms of T. brucei brucei laboratory strain TC
221 were cultured in Baltz medium according to standard
conditions.11
4.3.2. In vitro cytotoxicity assays
The test compounds were dissolved in DMSO or 0.1 M NaOH. A
defined number of parasites (104 trypanosomes per mL were ex-
posed in test chambers of 96-well plates to various concentrations
of the test substances in a final volume of 200 lL. Positive (trypan-
osomes in culture medium) and negative controls (test substance
without trypanosomes) were run with each plate. The plates were
then incubated at 37 °C in an atmosphere of 5% CO2 for a total time
period of 72 h. A reading was done at 48 h. The effect of test sub-
stances was quantified in ED50 values by linear interpolation27 of
three different measurements. The activity of the test substances
(KBr):
m
(cmꢁ1): = 3312, 2930, 2797, 1689, 1668, 1616, 1596,
1513, 1445, 1396, 1332, 1293, 1253, 1166, 1013, 895, 836, 703;
MS (EI, 70 eV): m/z 70 (40), 105 (42), 113 (80), 316 (78), 329
(30), 400 (51), 414 (35), 456 (6) [M]+, 457 (100) [M+1]+; MS (EI-
HRMS): calcd: 456.2161, found: 456.2165.

M. Altenkämper et al. / Bioorg. Med. Chem. 17 (2009) 7690–7697
7697
was measured by light absorption in an MR 700 Microplate Reader
at a wave length of 550 nm with a reference wave length of
630 nm, using the Alamar BlueÒ.12
containing 10% fetal bovine serum and 3
l
M daunomycin. Eight dif-
ferent concentrations of the compound were added to aliquots of
the cell suspension and one aliquot was prepared without compound.
Mixtures were incubated for 30 min at 37 °C. Tubes were chilled on
ice and washed once with ice-cold medium at a density of 1 ꢀ 106/
mL. Cell pellets were resuspended in ice-cold stop solution and were
measured within 1 h on a FACScalibur flow cytometer (BD Biosci-
ences, Heidelberg, Germany). Five thousand viable cells were gated
and accumulated for the determination of mean fluorescence values.
4.4. Cytotoxic assay
HeLa (DSM ACC 57) cells were grown in RPMI 1640 culture
medium (GIBCO BRL 21875-034) supplemented with 25 lg/mL
gentamicin sulfate (BioWhittaker 17-528Z), and 10% heat inacti-
vated fetal bovine serum (GIBCO BRL 10500-064) at 37 °C in high
density polyethylene flasks (NUNC 156340). The test substances
were dissolved in DMSO (10 mg/mL) before being diluted in the
cell culture medium (1:200). The adherent HeLa cells were har-
vested at the logarithmic growth phase after soft trypsinization,
using 0.25% trypsin in PBS containing 0.02% EDTA (Biochrom KG
L2163). For each experiment approximately 10,000 cells were
seeded with 0.1 mL RPMI 1640 (GIBCO BRL 21875-034), containing
4.5.4. Calculation of EC50 values
For efflux experiments, first-order rate constants were calculated
from the time depended linear decrease in mean fluorescence by
nonlinear regression analysis. EC50 values were determined from
dose–response curves of modifier concentration versus initial efflux
rates. EC50 values from steady state accumulation experiments were
obtainedbydose–response curvesof daunomycinsteadystatelevels
versus modifier concentration using a hyperbolic curve fitting pro-
cedure. At least two independently performed experiments were
used to calculate the average EC50 value of a compound.
25 lg/mL gentamicin sulfate (BioWhittaker 17-528Z), but without
HEPES, per well of the 96-well microplates (NUNC 167008). For the
cytotoxic assay HeLa cells were preincubated for 48 h without the
test substances. The dilutions of the test substances were carried
out carefully on the monolayers of HeLa cells after the preincuba-
tion time. The HeLa cells were further incubated for 72 h at 37 °C in
a humidified atmosphere and 5% CO2. The adherent HeLa cells
were fixed with 25% glutaraldehyde and stained with a 0.05% solu-
tion of methylene blue for 15 min. After gently washing the stain
was eluted with 0.2 mL of 0.33 M HCl per well. The optical densi-
ties were measured at 660 nm in SUNRISE microplate reader (TE-
CAN). For data analysis the Magellan software (TECAN) was used.
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CCRF-CEM vcr1000 was provided by V. Gekeler (Byk Gulden, Kon-
stanz, Germany). Cells were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum under standard culture
conditions. The resistant cell line was cultured in the presence of
1000 ng/mL vincristine. The selective agent was washed out one
week before experiments.
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Cells were pelleted, the supernatant was removed and the cells
were resuspended at a density of 1 ꢀ 106/mL in RPMI1640 medium
containing 10% fetal calf serum and 3 lM daunomycin. Cell sus-
pensions were incubated at 37 °C for 30 min. Tubes were chilled
on ice and pelleted at 500 g. Supernatant were removed and cells
were washed once with ice-cold medium at a density of 1 ꢀ 106
cells/mL. Subsequently, cells were resuspended in medium pre-
warmed to 37 °C, containing either no compound or various con-
centrations of the compound.
Eight concentrations (serial dilution of 1:2, 5) were tested for
each substance. After 1–4 min, aliquots of the incubation mixture
were transferred to tubes containing an equal volume of ice-cold
19. Chiba, P.; Ecker, G.; Schmid, D.; Drach, J.; Tell, B.; Gekeler, V. Mol. Pharmacol.
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stop solution (medium plus 100 lM verapamil). Samples were mea-
sured within 1 h on a FACScalibur flow cytometer (BD Biosciences,
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4.5.3. Steady state drug accumulation protocol
Cells were pelleted, the supernatant was removed and the cells
were resuspended at a density of 1 ꢀ 106/mL in RPMI1640 medium