Substituted 4-acylpyrazoles and 4-acylpyrazolones: Synthesis and multidrug resistance-modulating activity

Article abstract of DOI:10.1021/jm980121y

A series of 4-acyl-3-pyrazolone derivatives with a 3-substituted 2- hydroxy-3-aminopropyl chain attached to pyrazole N-1 (7-20) as well as isomeric 4-acyl-5-(3-substituted 3-amino-2-hydroxypropoxy)pyrazole derivatives (5, 6) were synthesized, and their mu

Full text of DOI:10.1021/jm980121y

J . Med. Chem. 1998, 41, 4001-4011
4001
Su bstitu ted 4-Acylp yr a zoles a n d 4-Acylp yr a zolon es: Syn th esis a n d Mu ltid r u g
Resista n ce-Mod u la tin g Activity†
Peter Chiba,§ Wolfgang Holzer,*,‡ Marion Landau,‡ Gerhard Bechmann,‡ Karin Lorenz,‡ Brigitte Plagens,‡
Manuela Hitzler,§ Elisabeth Richter,§ and Gerhard Ecker*,‡
Institute of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Wien, Austria, and
Institute of Medical Chemistry, University of Vienna, Waehringerstrasse 10, A-1090 Wien, Austria
Received February 23, 1998
A series of 4-acyl-3-pyrazolone derivatives with a 3-substituted 2-hydroxy-3-aminopropyl chain
attached to pyrazole N-1 (7-20) as well as isomeric 4-acyl-5-(3-substituted 3-amino-2-
hydroxypropoxy)pyrazole derivatives (5, 6) were synthesized, and their multidrug resistance
(MDR)-modulating activity was measured using the daunomycin efflux assay. Reaction of N1-
substituted 4-acyl-3-pyrazolones (tautomer to 4-acyl-5-hydroxypyrazoles) with excessive
epichlorohydrin and successive treatment with an appropriate amine resulted in N-alkylation
and thus afforded the target pyrazolone derivatives 7-20. In contrast, O-alkylation occurred
upon reaction with 1 equiv of epichlorohydrin and subsequent treatment with amine leading
to the corresponding 4-acyl-5-pyrazolyl ethers 5 and 6. QSAR studies showed a good correlation
of MDR-modulating activity with lipophilicity of the compounds. Inclusion of hydrogen bond
acceptor strength and steric parameters as descriptors led to a QSAR equation with remarkably
increased predictive power (r2cv ) 0.92). Additionally, ortho substitution of the propanolamine
side chain and the acyl moiety is favorable. Detailed NMR spectroscopic investigations were
carried out with the title compounds.
In tr od u ction
to restore drug sensitivity in multidrug-resistant CCRF-
CEM vcr100 cells.16 Structure-activity relationship
studies showed that modifications in the phenone
moiety such as reduction of the carbonyl group or
different substitution patterns on the central aromatic
ring decrease modulatory potency.17 To gain further
insights in the structural requirements necessary for
interaction with PGP, we synthesized a series of struc-
turally related pyrazole derivatives (5-20) and tested
their ability to inhibit PGP-mediated efflux of dauno-
mycin.
Development of unspecific mechanisms of resistance
represents an increasing problem in cancer and anti-
microbial therapy. The phenomenon that malignant
cells acquire cross-resistance to a broad panel of drugs
when exposed to only one of these agents has been
termed multidrug resistance (MDR).1 In most cases,
MDR has been shown to be accompanied by a decrease
in drug accumulation of resistant cells.2 It seems to be
widely accepted that one of the reasons of this decrease
in intracellular drug levels is active efflux caused by
P-glycoprotein (PGP), a membrane-bound protein of the
ABC-transporter family.3 PGP functions as an ATP-
driven efflux pump, transporting a great variety of
structurally and functionally diverse drugs, such as
anthracyclines, epipodophyllotoxins, vinca alkaloids,
colchicine, actinomycin D, and taxol.4 Within the past
decade several inhibitors of PGP-mediated drug efflux
have been identified.5 These so-called modulators of
MDR lead to resensitization of multidrug-resistant
tumor cells and include ion channel blockers such as
verapamil,6 dexniguldipine,7 and amiodarone,8 steroids,9
cyclosporines,10 phenothiazines,11 and thioxanthenes12
as well as the triazinoaminopiperidine S 978813 and the
acridone carboxamide GF 120918.14 Additionally, a
large number of compounds are described in the patent
literature.15 We recently identified the class Ic antiar-
rhythmic agent propafenone (Figure 1) as being capable
Ch em istr y a n d Str u ctu r a l Assign m en t
The synthesis of the compounds investigated in this
study is outlined in Schemes 1 and 2, respectively.
Precursors 1-4 can exist in several tautomeric forms,
among them the OH (form A) and the NH (form B)
isomers (Scheme 1).18-20 The alkylation of such ambi-
dent species can lead to different substitution products,
whereby the regioselectivity of the reaction strongly
depends on the substrate, the alkylating agent, and the
reaction conditions. Frequently, mixtures of regioiso-
meric reaction products are obtained. We found that
reaction of 1-4 (after transformation into the corre-
sponding sodium salts) with excessive epichlorohydrin
and successive treatment of the intermediate epoxides
with appropriate amines afforded the N-substituted
pyrazolones 7-20. In contrast, the use of only 1 equiv
of epichlorohydrin in DMF and subsequent treatment
of the intermediate epoxides 21 and 22 with amine led
to the formation of O-alkyl products 5 and 6. A possible
explanation for this reaction behavior might be the fact
that under equilibrium conditions (excessive epichloro-
† Dedicated to Prof. Dr. Gottfried Heinisch with best personal wishes
on the occasion of his 60th anniversary.
* To whom correspondence should be addressed. Tel: +43-1-31336-
8023 (W.H.), -8571 (G.E.). Fax: +43-1-31336-771. E-mail: holzer@
merian.pch.univie.ac.at or ecker@speedy.pch.univie.ac.at.
‡ Institute of Pharmaceutical Chemistry.
§ Institute of Medical Chemistry.
S0022-2623(98)00121-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/12/1998

4002 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Chiba et al.
F igu r e 1. Chemical structures of selected MDR modulators.
Sch em e 1a
a
(a) NaOMe/MeOH; (b) 2,3-epoxypropanol/diethyl azodicarboxylate/Ph3P/THF (Mitsunobu conditions); (c) epichlorohydrin (excessive);
(d) epichlorohydrin (1 equiv)/DMF; (e) R2R3NH.
hydrin) the reaction yields the more stable alkylation
product. Semiempirical MO calculations (AM1)21 re-
vealed the primary N-alkylation products to be ther-
modynamically more stable than their corresponding
O-isomers of type 21 and 22, whereas under kinetic
control obviously O-alkylation takes place.

Substituted 4-Acylpyrazoles and -pyrazolones
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4003
Sch em e 2a
a
2,3-Epoxypropanol/diethyl azodicarboxylate/Ph3P/THF (Mitsunobu conditions); (b) NaOMe/MeOH; (c) epichlorohydrin (1 equiv)/DMF;
(d) n-propylamine; (e) N-(o-tolyl)piperazine.
1
Ch a r t 1. NOEs and Long-Range H,13C Correlations
An alternative, selective approach to 21 and 22 was
Used for the Structural Assignment of Compounds
also possible via reaction of 2 and 4, respectively, with
7-20, 5, and 6
2,3-epoxypropanol under Mitsunobu conditions accord-
ing to ref 22 (Scheme 2). Surprisingly, this synthetic
route did not permit access to the corresponding n-
propylamino derivatives 23 and 24 as treatment of 21
and 22 with n-propylaminesunder formal loss of the
2,3-epoxypropoxy groupsled to the 5-(propylamino)-
pyrazoles 25 and 26. The latter compounds were not
available from treatment of precursors 1 and 4 with
n-propylamine, as this reaction afforded 4-[(propylami-
no)methylene]pyrazoles 27 and 28, which formally are
structural isomers to 25 and 26. However, besides
and selective long-range INEPT experiments (detection
traces of N-substitution product 12, 4-(aminomethyl-
of the long-range couplings 3J (C-5,N1-CH2) for 7-20
ene)pyrazole 29 was isolated as the main component of
3
and J (C-5,5-OCH2) for 5 and 6, Chart 1). Z-Configu-
the reaction mixture resulting from treatment of the
sodium salt of 2 with 1 equiv of epichlorohydrin and
successive heating with o-tolylpiperazine.
The structure of all novel compounds was confirmed
ration (regarding the exocyclic CdC bond) of compounds
27-29 follows from NOEs beween 5-Me and Ph H-2′′,6′′
(27-29) and CH2-CH2Ph (28). The large chemical shift
(δ > 11 ppm) of the NH proton in compounds 27 and
by IR, MS, and mainly NMR spectroscopic methods.
28 provides a hint that these compounds are stabilized
Complete and unambiguous assignments for all 1H and
by an intramolecular hydrogen bond between NH and
13C resonances could be achieved on the basis of chemi-
cal shift considerations, coupling information (APT23
and gated decoupled 13C NMR spectra), and NOE
difference,24 COSY-45,25 HMQC,26 and 1D-TOCSY27
spectra as well as on 1D-HETCOR28 and long-range
INEPT experiments29 with selective DANTE excitation
(Chart 1).
pyrazolone CdO.18
Ca lcu la tion of P h ysicoch em ica l P a r a m eter s
The log P values were calculated according to the
method of Ghose and Crippen30 using the software
package MOLGEN.31 Molecules were generated using
the builder function and energetically minimized with
the optimization function. Conformationally indepen-
dent lipophilicity values were calculated.
The hydrogen bond acceptor strength of the exocyclic
carbonyl oxygen was calculated using the software
package HYBOT.32 The corresponding Ca values were
taken as a measure of the hydrogen bond acceptor
strength.
Discrimination between N-substitution products 7-20
and O-substitution products 5 and 6 was achieved
considering 13C chemical shifts (7-20, δ N1-CH2- ∼ 50
ppm; corresponding δ C5-OCH2- of 5, 6 ∼ 78 ppm), NOE
difference experiments (positive NOEs on signals of
protons due to the amino alcohol chain upon irradiation
of the 5-Me resonance with compounds 7-20, Chart 2),

4004 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Chiba et al.
Ch a r t 2. Numbering of Nuclei Used for the Assignment of NMR Spectra
Ta ble 1. Physicochemical Parameters and MDR-Modulating Activity of Compounds 5-20
EC50 (µM; (SD)
daunomycina rhodamine 123b
ndc
2.22 ((0.34)
nd
no.
calcd log P
Ca
Ch
L
5
6
7
8
2.62
4.28
1.20
1.99
1.82
2.86
2.04
3.86
1.59
1.01
1.22
2.45
2.28
2.86
2.50
4.32
1.68
1.68
1.68
1.68
1.68
1.68
1.68
1.68
1.68
1.68
1.68
1.73
1.73
1.73
1.73
1.73
-0.402
-0.402
-0.402
-0.402
-0.402
-0.402
-0.402
-0.402
-0.409
-0.409
-0.409
-0.412
-0.412
-0.412
-0.412
-0.412
3.00
6.28
3.00
6.28
6.28
6.28
6.28
6.28
6.28
6.28
6.28
6.28
6.28
6.28
6.28
6.28
1.21 ((0.12)
1.06 ((0.15)
24.12 ((7.31)
28.21 ((9.89)
83.18 ((8.11)
12.59 ((0.78)
81.28 ((10.28)
0.89 ((0.16)
72.44 ((7.31)
123.03 ((7.93)
72.44 ((8.98)
6.31 ((0.54)
11.75 ((1.20)
2.69 ((0.20)
7.76 ((0.61
64.55 ((23.25)
225.51 ((45.50)
21.91 ((0.97)
167.52 (( 52.50)
2.43 ((0.06)
173.61 ((112.60)
648.05 ((176.45)
94.74 ((11.43)
15.19 ((0.50)
34.19 ((10.41)
4.02 ((0.70)
17.84 ((2.71)
nd
9
10
11
12
13
14
15
16
17
18
19
20
0.21 ((0.05)
0.32 (( 0.04)
0.12 (( 0.03)
0.06 ((0.01)
propafenone
verapamil
niguldipine
nd
nd
nd
a
b
Inhibition of daunomycin efflux from multidrug-resistant CEM vcr1000 human T-lymphoblasts. Inhibition of rhodamine 123 efflux
from mdr1 transfectant L5178Y VMDR1 C.06 mouse lymphoma cells. c nd, not determined.,
The partial charge of the exocyclic carbonyl oxygen
was calculated using the molecular modeling software
package SYBYL.33 Thus, the compounds were gener-
ated and minimized with the Powell algorithm (1000
iterations) using the Tripos force field and Gasteiger-
Hu¨ckel charges. Charges were calculated using the
Gasteiger-Hu¨ckel option.
rection for simple diffusion was achieved by subtracting
the efflux rates observed in the parental line. EC50
values of modifiers were calculated from dose-response
curves of Vmax/Km vs modifier concentration. Values are
given in Table 1 and represent the mean of at least five
independently performed experiments. Generally, in-
terexperimental variation was below 20%.
Multiple linear regression analysis was performed
using an in-house software package developed by K.-J .
Schaper, Borstel, Germany. Values in parentheses
brackets generally correspond to the 95% confidence
interval.
Resu lts a n d Discu ssion
A series of phenylpyrazolone analogous propafenone
derivatives were synthesized and tested for their MDR-
modulating activity. Table 1 summarizes the EC50
values for inhibition of PGP-mediated rhodamine 123
efflux in transfectant L5178YVMDR1 C.06 cells. These
values are highly correlated with those obtained for
daunomycin efflux in CEM vcr1000 cells, which were
selected stepwise in vincristine-containing medium
(Figure 2). This demonstrates that the EC50 values are
a measure of modulator activity and are both toxin- and
cell line-independent. In addition, resistance in the
vincristine-selected cell line is due to PGP expression.
The most active compound tested is the arylpiperazine
12 followed by the diisopropyl derivative 6. Similar
results were found for propafenones with the analogous
nitrogen substituents.16 In general, activities of phe-
nylpyrazolones are lower than those observed for cor-
MDR-Mod u la tin g Activity
The efflux assay is a direct and accurate functional
method to measure inhibition of PGP-mediated trans-
membrane transport.17 Both inhibition of daunomycin
efflux in the resistant human T-lymphoblast cell line
CEM vcr100034 as well as inhibition of rhodamine 123
efflux in the transfectant mouse lymphoma line L5178Y
VMDR1 C.0617 were used to characterize the MDR-
modulating activity of our compounds. The time-
dependent decrease in mean cellular fluorescence was
determined in the presence of various concentrations
of modifier, and the first-order rate constants (Vmax/Km)
were calculated by nonlinear regression analysis. Cor-

Substituted 4-Acylpyrazoles and -pyrazolones
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4005
F igu r e 2. Plot of log(1/EC50) values of compounds 5-19
obtained in rhodamine 123 efflux studies from transfectant
L5178Y VMDR1 C.06 mouse lymphoma cells vs those obtained
in daunomycin efflux studies from CEM vcr1000 human
F igu r e 3. Correlation of MDR-modulating activity of com-
pounds 5-19 (expressed as log(1/EC50) values of daunomycin
efflux inhibition) vs lipophilicity of the molecules: (9) benzoyl
derivatives 6 and 8-12, (b) phenylpropionyl (16-19) and
thienoyl (13-15) derivatives, (2) N-methyl analogues 5 and
7.
T-lymphoblast cells. An excellent correlation (log(1/EC50
)
Rh123
) 1.04 log(1/EC50
)
- 0.31; r ) 0.98, n ) 13) is obtained.
dauno
L36 to describe the structural difference of the methyl
and phenyl derivatives (eq 2). The regression coefficient
is negative, which provides additional evidence for steric
hindrance. Nevertheless, using an indicator variable
for the N-methyl analogues 5 and 7 (I ) 1, else I ) 0)
would lead to an equation with identical predictiveness,
since only two different values for L are present in the
data set.
responding propafenones. Benzophenone derivatives
5-12 were obtained by shortening the distance between
the two aromatic systems. In comparison to phenyl-
propionyl derivatives 16-19, this modification results
in a decrease of activity by a factor of 5-10 (8 vs 16, 9
vs 17, and 11 vs 19). Replacement of the benzoyl
residue by 2-thienoyl (15 vs 11, 14 vs 9, and 13 vs 8)
leads to a further decrease of potency. Replacement of
the N-phenyl group by a methyl group does not remark-
ably influence activity (5 vs 6 and 7 vs 10). A pairwise
comparison of 10 and 6 on one hand and 7 and 5 on the
other hand indicates that the pyrazolyloxypropanola-
mines 5 and 6 (benzoyl substituent in ortho position to
the ether oxygen) are more active by at least an order
of magnitude than the isomeric pyrazolones 7 and 10
(benzoyl substituent and propanolamine side chain in
meta position). The requirement for both an ether
oxygen linker group between the heteroaromatic ring
and the propanolamine side chain or the different
substitution pattern on the heteroaromatic ring could
account for this result. For propafenones the latter has
been shown to lead to decreased activity of meta and
para derivatives.17
Overall lipophilicity was shown to be a major predic-
tive parameter for activity of propafenone type modula-
tors.35 Figure 3 shows a plot of calculated log P values
of pyrazoles vs biological activity. Although a trend of
increased activity with increased lipophilicity is appar-
ent (eq 1), the whole set of compounds seems to fall into
three different groups. The first contains the benzoyl
derivatives 6 and 8-12. A second group of compounds
comprises the phenylpropionyl derivatives 16-19 and
the thienyl compounds 13-15, whereas the N-methyl
derivatives 5 and 7 show markedly higher activity/
lipophilicity ratios.
log(1/EC50) ) 0.74((0.18) log P - 0.24((0.14)L -
1.43((0.87) (2)
n ) 15, r ) 0.94, s ) 0.28, F ) 45.06; r2 ) 0.83
cv
As shown previously for propafenones, hydrogen bond
acceptor strength of the phenone carbonyl oxygen seems
to be an additional and independent predictive param-
eter for activity.17 Thus, we calculated the hydrogen
bond acceptor strength (Ca) of the exocyclic acyl carbonyl
oxygen using the software package HYBOT. Neverthe-
less, the calculation of Ca values for the carbonyl group
in the benzoyl and thienoyl series leads to identical
results, which seems rather unlikely. Since hydrogen
bond acceptor strength is correlated with the charge of
the corresponding atoms, the charges (Ch) of the acyl
carbonyl oxygens were calculated using the Gasteiger-
Hu¨ckel algorithm. By including this additional param-
eter into a multiple linear regression analysis, we
obtained an improved equation with good predictive
power (eq 3; Figure 4):
log(1/EC50) ) 0.82((0.11) log P -
50.24((24.12)Ch - 0.32((0.10)L -
21.52((9.66) (3)
n ) 15, r ) 0.98, s ) 0.17, F ) 87.17; r2 ) 0.92
log(1/EC50) ) 0.69((0.24) log P - 2.72((0.60) (1)
cv
n ) 15, r ) 0.86, s ) 0.39, F ) 38.54; r2 ) 0.68
Scaling of the x- and y-parameters, which reflects the
contribution of each regression coefficient in a normal-
ized way, yielded eq 4:
cv
The decrease in the log P/log potency ratio for phenyl
analogues might be due to steric hindrance. To test this
hypothesis we introduced the STERIMOL parameter
log(1/EC50) ) 1.02 log P - 0.31Ch - 0.50L (4)

4006 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Chiba et al.
70 eV). NMR spectra were recorded on a Bruker AC80
spectrometer (80 MHz for 1H, 20 MHz for 13C) or a Varian
1
UnityPlus spectrometer (300 MHz for H, 75 MHz for 13C) from
CDCl3 solutions at 28 °C. The center of the solvent signal was
used as an internal standard which was related to TMS with
δ 7.26 ppm (1H) and δ 77.0 ppm (13C). The numbering of nuclei
used for the assignments of 1H and 13C NMR spectra is given
in Chart 2. Column chromatographic separations were per-
formed on Merck Kieselgel 60 (70-230 mesh). Light petro-
leum refers to the fraction of bp 50-70 °C. Yields given below
are not optimized and refer to analytically pure material.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Alk oxy-
1H-p yr a zole Der iva tives 5 a n d 6. Sodium (230 mg, 10
mmol) was dissolved in 10 mL of dry MeOH with stirring. After
the evolution of hydrogen had ceased, 10 mmol of educt (1,37
2.16 g; 2,38 2.78 g) was added, and the resulting solution was
evaporated to dryness under reduced pressure. The residue
was dissolved in 10 mL of dry DMF, then 925 mg (10 mmol)
of epichlorohydrin was added, and the mixture was refluxed
(110 °C) for 4 h. After filtration, the solvents were removed
under reduced pressure, the residue was treated with 10 mL
(71 mmol) of diisopropylamine, and the mixture was refluxed
for 24 h. After filtering, the excessive amine was evaporated,
and the residue was purified as described below.
F igu r e 4. Plot of observed vs predicted MDR-modulating
activity (expressed as log(1/EC50) values using the daunomycin
efflux assay) for compounds 5-19 (9) using eq 6. Predicted
values were obtained with a leave-one-out cross-validation
procedure. Compound 20 also is shown (1).
Ta ble 2. Calculated log P Values and MDR-Modulating
Activity of Byproducts 25-29
[5-(3-(Diisop r op yla m in o)-2-h yd r oxyp r op oxy)-1,3-d im -
eth yl-1H-pyr azol-4-yl]ph en ylm eth an on e (5). Column chro-
matography (eluent: EtOAc-MeOH, 5:1) afforded a tan oil:
yield 31%; MS m/z 373 (M+, <1), 216 (78), 215 (74), 139 (52),
138 (63), 137 (14), 115 (33), 114 (100), 105 (61), 86 (22), 77
(52), 73 (10), 72 (69), 70 (29), 69 (18), 67 (29), 60 (27), 56 (25);
1H NMR (300 MHz) δ 0.92 (d, 3J ) 6.6 Hz, 6H, Me of i-Pr),
no.
calcd log P
EC50 (µM; (SD)a
25
26
27
28
29
3.95
4.32
1.73
4.18
5.27
11.03 ((2.65)
8.38 ((0.99)
272.50 ((37.50)
3.79 ((0.48)
0.68 ((0.05)
3
0.97 (d, J ) 6.6 Hz, 6H, Me of i-Pr), 2.11 (m, 1H, Hc), 2.18 (s,
3H, 5-Me), 2.25 (m, 1H, Hc), 2.93 (m, 2H, CH of i-Pr), 3.48 (m,
1H, Hb), 3.64 (m, 1H, Ha), 3.69 (s, 3H, N-Me), 3.75 (m, 1H,
Ha), 7.42 (m, 2H, H-3′′,5′′), 7.52 (m, 1H, H-4′′), 7.74 (m, 2H,
H-2′′,6′′); 13C NMR (75 MHz) δ 14.8 (3-Me, 1J ) 128.5 Hz),
19.4 and 22.1 (Me of i-Pr), 33.8 (N-Me, 1J ) 140.8 Hz), 45.8
(Cc), 48.2 (CH of i-Pr), 65.3 (Cb), 78.3 (Ca), 105.5 (C-4, 3J C-4,3-Me
) 2.5 Hz), 128.2 (C-3′′,5′′), 129.0 (C-2′′,6′′), 132.1 (C-4′′), 139.6
(C-1′′), 149.2 (C-3, 2J C-3,3-Me ) 6.8 Hz), 154.6 (C-5), 190.3 (Cd
O). Anal. (C21H31N3O3) C, H, N.
a
Inhibition of daunomycin efflux from multidrug-resistant CEM
vcr1000 human T-lymphoblasts.
To further test this hypothesis, the phenylpropionyl
analogue (20) of compound 12, which should show
remarkably high MDR-modulating activity, was syn-
thesized and tested in the daunomycin efflux assay
(Table 1). Indeed, the observed EC50 value (EC50 ) 0.21)
is in excellent agreement to that calculated using eq 3
(EC50 ) 0.19).
In addition, the PGP-inhibitory activity of the byprod-
ucts 25-29 was tested. The corresponding values are
given in Table 2. All compounds showed lower log P/log
potency ratios than those obtained for compounds 5-20.
This might be due to the fact, that the “basic” nitrogen
atom in 25-29 has to be considered as a vinylogous
amide nitrogen rather than an alkylamine.
[5-(3-(Diisop r op yla m in o)-2-h yd r oxyp r op oxy)-3-m eth -
yl-1-p h en yl-1H-p yr a zol-4-yl]p h en ylm eth a n on e (6). Col-
umn chromatography (eluent: EtOAc) afforded a yellowish
oil: yield 22%; MS m/z 435 (M+, <1), 322 (12), 279 (13), 278
(61), 277 (56), 200 (21), 115 (69), 114 (100), 105 (73), 100 (19),
1
91 (23), 77 (81), 72 (85), 70 (23), 67 (25), 57 (29), 56 (51); H
3
NMR (300 MHz) δ 0.88 (d, J ) 6.6 Hz, 6H, Me of i-Pr), 0.92
(d, 3J ) 6.6 Hz, 6H, Me of i-Pr), 2.11 (m, 1H, Hc), 2.26 (m, 1H,
Hc), 2.27 (s, 3H, 3-Me), 2.88 (m, 2H, CH of i-Pr), 3.45 (m, 1H,
Hb), 3.78 (m, 2H, Ha), 7.32 (m, 1H, H-4′), 7.45 (m, 2H, H-3′,5′),
7.48 (m, 2H, H-3′′,5′′), 7.57 (m, 1 H, H-4′′), 7.75 (m, 2 H,
H-2′,6′), 7.86 (m, 2 H, H-2′′,6′′); 13C NMR (75 MHz) δ 14.9 (3-
Me), 19.4 and 21.7 (Me of i-Pr), 46.8 (Cc), 48.6 (CH of i-Pr),
65.3 (Cb), 78.2 (Ca), 106.7 (C-4), 123.2 (C-2′,6′), 127.4 (C-4′),
128.4 (C-3′′,5′′), 129.0 (C-3′,5′), 129.2 (C-2′′,6′′), 132.5 (C-4′′),
137.7 (C-1′), 139.3 (C-1′′), 150.2 (C-3), 154.3 (C-5), 190.5 (Cd
O). Anal. (C26H33N3O3) C, H, N.
Con clu sion s
Phenylpyrazoles of type 5-19 show a dependence of
potency on lipophilicity of the compounds similar to the
group of propafenones, whereby high lipophilicity is
associated with high activity. Ortho substitution of the
propanolamine side chain and the acyl moiety is favor-
able. The QSAR equation with the highest predictive
power is obtained when hydrogen bond acceptor strength
and steric parameters are included as descriptors.
Using this equation, the activity of the additionally
synthesized derivative 20 was predicted with excellent
precision.
Gen er a l P r oced u r e for th e P r ep a r a tion of P yr a zolo-
n es 7-11 a n d 13-19. Sodium (230 mg, 10 mmol) was
dissolved in 10 mL of dry MeOH with stirring. After the
evolution of hydrogen had ceased, 10 mmol of educt (1,37 2.16
g; 2,38 2.78 g; 3,39 2.84 g; 4,40 3.06 g) was added, and the
resulting solution was evaporated to dryness under reduced
pressure. To the residue was added 10 mL (128 mmol) of
epichlorohydrin, and the mixture was refluxed (110 °C) for 4
h. Then the mixture was filtered and evaporated under
reduced pressure. The residue was treated with 20 mL of the
appropriate amine, and the resulting mixture was then
refluxed for 4 h (reactions with n-propylamine) or 24 h
(reactions with isopropylamine, n-butylamine, or tert-buty-
lamine), respectively. After evaporation of excessive amine
the obtained raw products were purified as described below.
Ma ter ia ls a n d Meth od s
Ch em istr y. Melting points were determined on a Reichert-
Kofler hot-stage microscope and are uncorrected. Elemental
analyses were performed by Mikroanalytisches Laboratorium,
Institute of Physical Chemistry, University of Vienna. Mass
spectra were obtained on a Varian MAT 311A instrument (EI,

Substituted 4-Acylpyrazoles and -pyrazolones
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4007
4-Ben zoyl-1-(3-(d iisop r op yla m in o)-2-h yd r oxyp r op yl)-
2,5-d im eth yl-1,2-d ih yd r op yr a zol-3-on e (7). The raw mate-
rial was taken up in 50 mL of dichloromethane and was
washed five times with 40 mL of water. After evaporation of
the solvent the residue was purified by column chromatogra-
phy (eluent: EtOAc-MeOH, 5:1); subsequent recrystallization
from diisopropyl ether-MeOH afforded colorless crystals of
mp 140 °C: yield 32%; MS m/z 373 (M+, <1), 144 (20), 114
(100), 105 (24), 77 (23), 72 (39), 56 (16); 1H NMR (300 MHz) δ
1.00 (d, J ) 6.6 Hz, 6H, Me of i-Pr), 1.02 (d, J ) 6.6 Hz, 6H,
Me of i-Pr), 2.30 (m, 1H, Hc), 2.53 (m, 1H, Hc), 2.53 (s, 3H,
5-Me), 3.00 (m, 2H, CH of i-Pr), 3.36 (s, 3H, N-Me), 3.69 (m,
1H, Hb), 3.79 (m, 1H, Ha), 3.92 (m, 1H, Ha), 7.38 (m, 2H,
H-3′′,5′′), 7.46 (m, 1H, H-4′′), 7.83 (m, 2H, H-2′′,6′′); 13C NMR
(75 MHz) δ 12.2 (5-Me), 20.0 and 21.6 (Me of i-Pr), 29.1 (N-
Me), 47.9 (Cc), 48.4 (CH of i-Pr), 49.8 (Ca), 66.2 (Cb), 106.2 (C-
4), 127.6 (C-3′′,5′′), 129.3 (C-2′′,6′′), 131.8 (C-4′′), 138.9 (C-1′′),
153.5 (C-5), 163.0 (C-3), 190.5 (CdO). Anal. (C21H31N3O3) C,
H, N.
Hc), 2.65 (s, 3H, 5-Me), 3.55 (m, 1H, Hb), 3.69 (m, 1H, Ha), 3.86
(m, 1H, Ha), 7.28 (m, 2H, H-2′,6′), 7.33 (m, 1H, H-4′), 7.36 (m,
2H, H-3′′,5′′), 7.41 (m, 1H, H-4′′), 7.44 (m, 2H, H-3′,5′), 7.86
(m, 2H, H-2′′,6′′); 13C NMR (75 MHz) δ 13.0 (5-Me, 1J ) 130.9
Hz), 29.0 (Me of t-Bu, 1J ) 125.0 Hz), 45.3 (Cc), 50.1 (Ca), 50.2
3
(CMe3), 68.1 (Cb), 106.6 (C-4, J C-4,5-Me ) 2.9 Hz), 126.3 (C-
2′,6′), 127.6 (C-3′′,5′′), 128.1 (C-4′), 129.3 (C-2′′,6′′), 129.4 (C-
3′,5′), 131.7 (C-4′′), 134.6 (C-1′), 138.9 (C-1′′), 159.5 (C-5,
2J C-5,5-Me ) 6.7 Hz), 164.1 (C-3), 190.5 (CdO). Anal. (C24H29
N3O3) C, H, N.
-
3
3
1-(2-Hydr oxy-3-(pr opylam in o)pr opyl)-5-m eth yl-2-ph en -
yl-4-(2-th ien oyl)-1,2-d ih yd r op yr a zol-3-on e (13). Column
chromatography (eluent: EtOAc-NEt3-EtOH, 8:1:1) and
subsequent recrystallization from EtOAc-light petroleum
afforded nearly colorless crystals of mp 140-143 °C: yield 26%;
MS m/z 399 (M+, <1), 200 (12), 111 (26), 102 (12), 72 (100), 43
(36); 1H NMR (80 MHz) δ 0.84 (t, 3J ) 6.7 Hz, 3H, Me of n-Pr),
1.33 (m, 2H, -CH2-Me of n-Pr), 2.16-2.51 (m, 6H, NCH2 of
n-Pr, Hc, OH, NH), 2.68 (s, 3H, 5-Me), 3.61-4.07 (m, 3H, Ha,
3
4-Ben zoyl-1-(2-h ydr oxy-3-(pr opylam in o)pr opyl)-5-m eth -
yl-2-p h en yl-1,2-d ih yd r op yr a zol-3-on e (8). Column chro-
matography (eluent: CH2Cl2-MeOH, 1:1) afforded a viscous,
yellow-orange oil which solidified upon standing: yield 40%;
MS m/z 393 (M+, 2), 321 (12), 278 (22), 200 (24), 196 (15), 105
Hb), 7.06 (dd, J ) 3.9 and 4.9 Hz, 1H, H-4′′), 7.23-7.50 (m,
5H, H-2′-6′), 7.56 (dd, 3J ) 4.9 Hz, 4J ) 1.1 Hz, 1H, H-5′′),
8.40 (dd, 3J ) 3.9 Hz, 4J ) 1.1 Hz, 1H, H-3′′); 13C NMR (20
MHz) δ 11.4 (Me of n-Pr), 13.0 (5-Me), 22.7 (CH2-Me of n-Pr),
50.0 (Ca), 51.1 (NCH2 of n-Pr), 52.3 (Cc), 67.0 (Cb), 105.8 (C-4),
126.5 (C-2′,6′), 127.6 (C-4′′), 128.3 (C-4′), 129.3 (C-3′,5′), 132.8
(C-5′′), 133.9 (C-1′), 134.2 (C-3′′), 145.1 (C-2′′), 158.6 (C-5),
163.3 (C-3), 180.9 (CdO). Anal. (C21H25N3O3S) C, H, N.
1
3
(75), 77 (34), 72 (100); H NMR (80 MHz) δ 0.85 (t, J ) 6.7
Hz, 3H, Me of n-Pr), 1.34 (m, 2H, -CH2-Me of n-Pr), 2.35-
2.53 (m, 6H, NCH2 of n-Pr, Hc, OH, NH), 2.64 (s, 3H, 5-Me),
3.61-3.99 (m, 3H, Ha, Hb), 7.26-7.56 (m, 8H, H-2′-6′ and
H-3′′-5′′), 7.79-7.91 (m, 2H, H-2′′,6′′); 13C NMR (20 MHz) δ
11.3 (Me of n-Pr), 12.9 (5-Me), 22.5 (CH2-Me of n-Pr), 49.9
(Ca), 51.1 (NCH2 of n-Pr), 52.1 (Cc), 66.8 (Cb), 105.9 (C-4), 126.4
(C-2′,6′), 127.5 (C-3′′,5′′), 128.2 (C-4′), 129.1 (C-2′′,6′′), 129.3
(C-3′,5′), 131.7 (C-4′′), 133.9 (C-1′), 138.4 (C-1′′), 158.6 (C-5),
163.8 (C-3), 190.3 (CdO). Anal. (C23H27N3O3‚H2O) C, H, N.
1-(2-H yd r oxy-3-(isop r op yla m in o)p r op yl)-5-m et h yl-2-
p h en yl-4-(2-th ien oyl)-1,2-d ih yd r op yr a zol-3-on e (14). The
raw product was taken up in 25 mL of CH2Cl2 and washed
several times with water. After drying (Na2SO4) the solvent
was evaporated, and the residue was recrystallized from
diisopropyl ether-EtOH to give yellowish crystals of mp 127-
134 °C: yield 20%; MS m/z 399 (M+, <1), 200 (13), 111 (23),
72 (100), 60 (20), 45 (62), 43 (48); 1H NMR (80 MHz) δ 0.94 (d,
4-Ben zoyl-1-(2-h yd r oxy-3-(isop r op yla m in o)p r op yl)-5-
m et h yl-2-p h en yl-1,2-d ih yd r op yr a zol-3-on e (9). Column
chromatography (eluent: EtOAc-NEt3-EtOH, 8:1:1) and
subsequent crystallization from EtOAc afforded yellowish
crystals of mp 141-142 °C: yield 27%; MS m/z 393 (M+, <1),
200 (10), 105 (39), 102 (18), 77 (21), 72 (100); 1H NMR (80 MHz)
3
3J ) 6.7 Hz, 3H, Me of i-Pr), 0.95 (d, J ) 6.7 Hz, 3H, Me of
i-Pr), 2.12-2.63 (m, 3 H, CH of i-Pr, Hc), 2.68 (s, 3H, 5-Me),
3
3.60-3.97 (m, 3H, Ha, Hb), 7.06 (dd, J ) 3.9 and 4.9 Hz, 1H,
H-4′′), 7.23-7.51 (m, 5H, H-2′-6′), 7.57 (dd, 3J ) 4.9 Hz, 4J )
1.1 Hz, 1H, H-5′′), 8.40 (dd, 3J ) 3.9 Hz, 4J ) 1.1 Hz, 1H, H-3′′);
13C NMR (20 MHz) δ 13.0 (5-Me), 22.3 and 22.6 (Me of i-Pr),
48.3 (CH of i-Pr), 49.9 (Ca and Cc), 67.3 (Cb), 105.9 (4), 126.5
(C-2′,6′), 127.6 (C-4′′), 128.3 (C-4′), 129.3 (C-3′,5′), 132.8 (C-
5′′), 133.9 (C-1′), 134.2 (C-3′′), 145.1 (C-2′′), 158.7 (C-5), 163.3
(C-3), 180.9 (CdO). Anal. (C21H25N3O3S) C, H, N.
3
3
δ 0.95 (d, J ) 6.7 Hz, 3H, Me of i-Pr), 0.96 (d, J ) 6.7 Hz,
3H, Me of i-Pr), 2.26-2.62 (m, 5H, CH of i-Pr, Hc,OH, NH),
2.66 (s, 3H, 5-Me), 3.56-3.79 (m, 3H, Ha, Hb), 7.23-7.48 (m,
8H, H-2′-6′ and H-3′′-5′′), 7.80-7.92 (m, 2H, H-2′′,6′′); 13C
NMR (20 MHz) δ 12.9 (5-Me), 22.2 and 22.4 (Me of i-Pr), 48.3
(CH of i-Pr), 49.7 (Ca), 49.9 (Cc), 67.1 (Cb), 105.8 (C-4), 126.3
(C-2′,6′), 127.4 (C-3′′,5′′), 128.2 (C-4′), 129.1 (C-2′′,6′′ and
C-3′,5′), 131.6 (C-4′′), 133.9 (C-1′), 138.6 (C-1′′), 158.7 (C-5),
163.7 (C-3), 190.3 (CdO). Anal. (C23H27N3O3‚H2O) C, H, N.
1-(2-H yd r oxy-3-(ter t-b u t yla m in o)p r op yl)-5-m et h yl-2-
p h en yl-4-(2-th ien oyl)-1,2-d ih yd r op yr a zol-3-on e (15). The
raw product was taken up in 25 mL of CH2Cl2 and washed
several times with water. After drying (Na2SO4) the solvent
was evaporated, and the residue was recrystallized from
EtOAc-EtOH to give colorless crystals of mp 195-196 °C:
yield 25%; MS m/z 413 (M+, <1), 298 (10), 116 (46), 111 (28),
4-Ben zoyl-1-(3-(d iisop r op yla m in o)-2-h yd r oxyp r op yl)-
5-m eth yl-2-p h en yl-1,2-d ih yd r op yr a zol-3-on e (10). Col-
umn chromatography (eluent: EtOAc-MeOH, 4:1) and sub-
sequent recrystallization from diisopropyl ether-MeOH afforded
colorless crystals of mp 163 °C: yield 25%; MS m/z 435 (M+,
1
86 (100), 60 (57), 57 (42), 43 (25); H NMR (300 MHz) δ 0.98
(s, 9H, t-Bu), 2.20 (m, 1H, Hc), 2.35 (m, 1H, Hc), 2.69 (s, 3H,
5-Me), 3.58 (m, 1H, Hb), 3.71 (m, 1H, Ha), 3.89 (m, 1H, Ha),
7.07 (dd, 3J ) 3.9 and 4.9 Hz, 1H, H-4′′), 7.30 (m, 2H, H-2′,6′),
7.41 (m, 1H, H-4′), 7.45 (m, 2H, H-3′,5′), 7.57 (dd, 3J ) 4.9 Hz,
4J ) 1.1 Hz, 1H, H-5′′), 8.41 (dd, 3J ) 3.9 Hz, 4J ) 1.1 Hz, 1H,
H-3′′); 13C NMR (75 MHz) δ 13.2 (5-Me), 28.8 (Me of t-Bu),
45.3 (Cc), 50.1 (CMe3 and Ca), 67.9 (Cb), 106.3 (C-4), 126.5 (C-
2′,6′), 127.7 (C-4′′), 128.3 (C-4′), 129.4 (C-3′,5′), 132.8 (C-5′′),
134.2 (C-1′), 134.4 (C-3′′), 145.2 (C-2′′), 159.1 (C-5), 163.5 (C-
3), 181.1 (CdO). Anal. (C22H27N3O3S) C, H, N.
1
<1), 114 (100), 105 (23), 77 (21), 72 (23); H NMR (300 MHz)
3
3
δ 0.89 (d, J ) 6.6 Hz, 6H, Me of i-Pr), 0.94 (d, J ) 6.6 Hz,
6H, Me of i-Pr), 2.11-2.28 (m, 2H, Hc), 2.69 (s, 3H, 5-Me), 2.89
(m, 2H, CH of i-Pr), 3.60 (m, 1H, Hb), 3.77 (m, 2H, Ha), 7.28
(m, 2H, H-2′,6′), 7.33 (m, 1H, H-4′), 7.38 (m, 2H, H-3′′,5′′), 7.44
(m, 2H, H-3′,5′), 7.46 (m, 1H, H-4′′), 7.88 (m, 2H, H-2′′,6′′);13C
NMR (75 MHz) δ 13.0 (5-Me), 19.6 and 21.8 (Me of i-Pr), 47.7
(Cc), 48.3 (CH of i-Pr), 50.4 (Ca), 65.4 (Cb), 106.7 (C-4), 126.2
(C-2′,6′), 127.6 (C-3′′,5′′), 128.0 (C-4′), 129.3 (C-2′′,6′′ and
C-3′,5′), 131.8 (C-4′′), 134.1 (C-1′), 138.8 (C-1′′), 159.7 (C-5),
164.1 (C-3), 190.6 (CdO). Anal. (C26H33N3O3) C, H, N.
1-(2-Hydr oxy-3-(pr opylam in o)pr opyl)-5-m eth yl-2-ph en -
yl-4-(3-p h en ylp r op ion yl)-1,2-d ih yd r op yr a zol-3-on e (16).
The raw product was taken up in 25 mL of CH2Cl2 and washed
several times with water. After drying (Na2SO4) the solvent
was evaporated, and the residue was recrystallized from
EtOAc-light petroleum to give colorless crystals of mp 120-
124 °C: yield 49%; MS m/z 421 (M+, 9), 307 (12), 228 (10),
201 (12), 175 (14), 116 (16), 115 (18), 102 (17), 98 (21), 93 (23),
4-Ben zoyl-1-(2-h yd r oxy-3-(ter t-bu tyla m in o)p r op yl)-5-
m eth yl-2-p h en yl-1,2-d ih yd r op yr a zol-3-on e (11). The raw
product was taken up in 25 mL of CH2Cl2 and washed several
times with water. After drying (Na2SO4), the solvent was
evaporated, and the residue was recrystallized from diisopropyl
ether-EtOAc to give colorless crystals of mp 163-165 °C:
yield 42%; MS m/z 407 (M+, 1), 292 (21), 278 (12), 200 (17),
169 (13), 116 (47), 105 (55), 86 (100), 77 (20), 60 (40); 1H NMR
(300 MHz) δ 0.97 (s, 9H, t-Bu), 2.19 (m, 1H, Hc), 2.38 (m, 1H,
1
3
91 (25), 86 (20), 72 (100); H NMR (300 MHz) δ 0.83 (t, J )
6.7 Hz, 3H, Me of n-Pr), 1.36 (m, 2H, -CH2-Me of n-Pr), 2.30
(m, 1H, Hc, NH*), 2.40 (m, 2H, NCH2 of n-Pr), 2.41 (m, 1H,

4008 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Chiba et al.
Hc), 2.70 (s, 3H, 5-Me), 2.90 (br s, 1H, OH*), 2.96 (m, 2H, CH2-
Ph), 3.22 (m, 1H, CH2-CO), 3.34 (m, 1H, CH2-CO), 3.66 (m,
1H, Hb), 3.67 (m, 1H, Ha), 3.85 (m, 1H, Ha), 7.09-7.48 (m, 10H,
H-2′-6′ and H-2′′-6′′); 13C NMR (75 MHz) δ 11.4 (Me of n-Pr),
13.2 (5-Me), 22.7 (CH2-Me of n-Pr), 30.0 (CH2-Ph), 43.0
(CH2-CO), 49.7 (Ca), 51.3 (NCH2 of n-Pr), 52.2 (Cc), 67.2 (Cb),
277 (14), 219 (17), 189 (81), 187 (63), 156 (41), 118 (44), 105
(100), 91 (39), 77 (88), 71 (18), 70 (94), 67 (26), 57 (33), 56 (38),
55 (24); H NMR (300 MHz) δ 2.18 (m, 2H, Hc), 2.27 (s, 3H,
1
Me of tolyl), 2.30 and 2.53 (m, each 2H, H-2,6 of piperazine),
2.68 (s, 3H, 5-Me), 2.77 (m, 4H, H-3,5 of piperazine), 3.74-
3.86 (m, 3H, Ha, Hb), 6.96 (m, 1H, H-6 of tolyl), 6.99 (m, 1H,
H-4 of tolyl), 7.17 (m, 1H, H-5 of tolyl), 7.18 (m, 1H, H-3 of
tolyl), 7.30 (m, 2H, H-2′,6′), 7.35 (m, 1H, H-4′), 7.40 (m, 2H,
H-3′′,5′′), 7.47 (m, 2H, H-3′,5′), 7.48 (m, 1H, H-4′′), 7.90 (m,
2H, H-2′′,6′′); 13C NMR (75 MHz) δ 13.1 (5-Me), 17.7 (Me of
tolyl), 50.4 (Ca), 51.6 (C-3,5 of piperazine), 53.6 (C-2,6 of
piperazine), 61.1 (Cc), 65.0 (Cb), 106.6 (C-4), 118.9 (C-6 of tolyl),
123.2 (C-4 of tolyl), 126.3 (C-2′,6′), 126.5 (C-5 of tolyl), 127.7
(C-3′′,5′′), 128.1 (C-4′), 129.4 (C-2′′,6′′ and C-3′,5′), 131.0 (C-3
of tolyl), 131.9 (C-4′′), 132.5 (C-2 of tolyl), 134.4 (C-1′), 138.7
(C-1′′), 151.1 (C-1 of tolyl), 159.6 (C-5), 164.0 (C-3), 190.5 (Cd
O). Anal. (C31H34N4O3) C, H, N.
3
105.9 (C-4, J C-4,5-Me ) 2.5 Hz), 125.6 (C-4′′), 126.4 (C-2′,6′),
128.1 (C-3′′,5′′), 128.5 (C-4′ and C-2′′,6′′), 129.5 (C-3′,5′), 134.4
(C-1′), 141.9 (C-1′′), 158.5 (C-5), 165.1 (C-3), 196.0 (CdO).
Anal. (C25H31N3O3) C, H, N.
1-(2-H yd r oxy-3-(isop r op yla m in o)p r op yl)-5-m et h yl-2-
p h e n yl-4-(3-p h e n ylp r op ion yl)-1,2-d ih yd r op yr a zol-3-
on e (17). The raw product was taken up in 25 mL of CH2Cl2
and washed several times with water. After drying (Na2SO4)
the solvent was evaporated, and the residue was recrystallized
from EtOAc-light petroleum to give colorless crystals of mp
146-149 °C: yield 42%; MS m/z 421 (M+, 16), 307 (11), 228
(11), 116 (13), 115 (17), 102 (23), 93 (16), 91 (17), 72 (100); 1H
NMR (80 MHz) δ 0.92 (d, 3J ) 6.3 Hz, 3H, Me of i-Pr), 0.94 (d,
3J ) 6.3 Hz, 3H, Me of i-Pr), 2.25-2.68 (m, 3H, CH of i-Pr,
Hc), 2.72 (s, 3H, 5-Me), 2.87-3.87 (m, 7H, Ha, Hb, CO-CH2-
CH2-), 7.14-7.51 (m, 10H, H-2′-6′ and H-2′′-6′′);13C NMR
(20 MHz) δ 13.1 (5-Me), 22.4 and 22.7 (Me of i-Pr), 29.8 (CH2-
Ph), 42.9 (CH2-CO), 48.4 (CH of i-Pr), 49.5 (Cc), 49.9 (Ca), 67.3
(Cb), 105.3 (C-4), 125.4 (C-4′′), 126.3 (C-2′,6′), 128.0 (C-3′′,5′′),
128.3 (C-4′ and C-2′′,6′′), 129.3 (C-3′,5′), 133.9 (C-1′), 141.6 (C-
1′′), 158.1 (C-5), 164.8 (C-3), 195.8 (CdO). Anal. (C25H31N3O3)
C, H, N.
1-(2-Hyd r oxy-3-(bu tyla m in o)p r op yl)-5-m eth yl-2-p h en -
yl-4-(3-p h en ylp r op ion yl)-1,2-d ih yd r op yr a zol-3-on e (18).
The raw product was taken up in 25 mL of CH2Cl2 and washed
several times with water. After drying (Na2SO4) the solvent
was evaporated, and the residue was recrystallized from
EtOAc-light petroleum to give colorless crystals of mp 95-
97 °C: yield 21%; MS m/z 435 (M+, 7), 349 (10), 307 (11), 229
(10), 228 (11), 201 (11), 175 (12), 130 (15), 116 (23), 93 (21), 91
(24), 86 (100), 44 (32); 1H NMR (80 MHz) δ 0.87 (m, 3H, Me of
n-Bu), 1.23-1.32 (m, 4H, Me-CH2-CH2- of n-Bu), 2.28-2.51
(m, 6H, N-CH2 of n-Bu, Hc, OH, NH), 2.72 (s, 3H, 5-Me), 2.84-
3.77 (m, 7H, Ha, Hb, CH2-CH2-Ph), 7.19-7.51 (m, 10H, H-2′-
6′ and H-2′′-6′′); 13C NMR (20 MHz) δ 13.1 (5-Me), 13.7 (Me
of n-Bu), 20.0 (Me-CH2 of n-Bu), 29.8 (CH2-Ph), 31.8 (CH2-
CH2-N of n-Bu), 42.9 (CH2-CO), 49.1 (CH2-N of n-Bu), 49.5
(Ca), 52.3 (Cc), 67.0 (Cb), 105.2 (C-4), 125.4 (C-4′′), 126.3 (C-
2′,6′), 128.0 (C-3′′,5′′), 128.3 (C-4′ and C-2′′,6′′), 129.3 (C-3′,5′),
133.8 (C-1′), 141.6 (C-1′′), 158.0 (C-5), 164.8 (C-3), 195.8 (Cd
O). Anal. (C26H33N3O3) C, H, N.
1-(2-H yd r oxy-3-(ter t-b u t yla m in o)p r op yl)-5-m et h yl-2-
p h e n yl-4-(3-p h e n ylp r op ion yl)-1,2-d ih yd r op yr a zol-3-
on e (19). The raw product was recrystallized from diisopropyl
ether-EtOH to afford colorless crystals of mp 178-180 °C:
yield 52%; MS m/z 435 (M+, 9), 129 (18), 116 (35), 114 (33), 86
(100), 72 (29), 60 (64), 57 (48), 56 (26), 45 (67), 43 (24); 1H NMR
(80 MHz) δ 0.99 (s, 9H, Me of t-Bu), 2.16-2.52 (m, 4H, Hc,
OH, NH), 2.73 (s, 3H, 5-Me), 2.93-3.87 (m, 7H, Ha, Hb, CH2-
CH2-Ph), 7.17-7.52 (m, 10H, H-2′-6′ and H-2′′-6′′); 13C NMR
(20 MHz) δ 13.1 (5-Me), 28.6 (Me of t-Bu), 29.8 (CH2-Ph), 42.9
(CH2-CO), 45.2 (Cc), 49.5 (Ca), 50.1 (CMe3 of t-Bu), 67.6 (Cb),
105.3 (C-4), 125.4 (C-4′′), 126.3 (C-2′,6′), 128.0 (C-3′′,5′′), 128.3
(C-4′ and C-2′′,6′′), 129.4 (C-3′,5′), 133.9 (C-1′), 141.7 (C-1′′),
158.1 (C-5), 164.8 (C-3), 195.9 (CdO). Anal. (C26H33N3O3) C,
H, N.
4-Ben zoyl-1-[2-h yd r oxy-3-(4-o-tolylp ip er a zin -1-yl)p r o-
p yl]-5-m eth yl-2-p h en yl-1,2-d ih yd r op yr a zol-3-on e (12). As
described for the synthesis of compounds 7-11, and 13-19,
crude epoxide product was prepared from pyrazolone 2 (2.78
g, 10 mmol) and epichlorohydrin (10 mL, 128 mmol). After
evaporation of excessive epichlorohydrin, the residue was
treated with 1.76 g (10 mmol) of o-tolylpiperazine in 75 mL of
MeOH, and the mixture was heated to reflux for 8 h. After
evaporation in vacuo the residue was subjected to column
chromatography (eluent: EtOAc); subsequent recrystallization
from 2-propanol-EtOAc gave colorless crystals of mp 175 °C:
yield 15%; MS m/z 510 (M+, 23), 417 (13), 364 (19), 278 (15),
1-[2-Hydr oxy-3-(4-o-tolylpiper azin -1-yl)pr opyl]-5-m eth -
yl-2-p h en yl-4-(3-p h en ylp r op ion yl)-1,2-d ih yd r op yr a zol-3-
on e (20). Starting from pyrazolone 4, compound 20 was
prepared similarly as described for the preparation of 12.
Repeated column chromatography of the raw product (elu-
ents: 1. gradient CH2Cl2-EtOAc-NH3, 10:1:0.1, to CH2Cl2-
MeOH-NH3, 10:1:0.1; 2. EtOAc) and subsequent recrystalli-
zation from EtOAc gave colorless crystals of mp 188 °C: yield
14%; MS m/z 538 (M+, 41), 445 (12), 392 (16), 201 (21), 189
(99), 187 (61), 174 (20), 146 (38), 131 (32), 118 (46), 105 (26),
91 (100), 77 (52), 70 (61), 67 (37), 65 (24), 57 (26), 56 (26), 42
(35); 1H NMR (300 MHz) δ 2.10 (m, 2H, Hc), 2.17 (s, 3H, Me of
tolyl), 2.27 and 2.49 (m, each 2H, H-2,6 of piperazine), 2.67 (s,
3H, 5-Me), 2.70 (m, 4H, H-3,5 of piperazine), 2.90 (m, 2H,
CH2-Ph), 3.12-3.41 (m, 2H, CH2-CO), 3.65-3.80 (m, 3H, Ha,
Hb), 6.87 (m, 1H, H-6 of tolyl), 6.92 (m, 1H, H-4 of tolyl), 7.05-
7.45 (m, 12H, H-2′-6′, H-2′′-6′′, H-3 and H-5 of tolyl); 13C
NMR (75 MHz) δ 13.3 (5-Me), 17.7 (Me of tolyl), 29.9 (CH2-
Ph), 43.1 (CH2-CO), 49.8 (Ca), 51.5 (C-3,5 of piperazine), 53.5
(C-2,6 of piperazine), 61.0 (Cc), 64.9 (Cb), 106.0 (C-4), 118.8
(C-6 of tolyl), 123.3 (C-4 of tolyl), 125.6 (C-4′′), 126.2 (C-2′,6′),
126.5 (C-5 of tolyl), 128.1 (C-3′′,5′′), 128.4 (C-4′), 128.5 (C-2′′,6′′),
129.6 (C-3′,5′), 131.0 (C-3 of tolyl), 132.5 (C-2 of tolyl), 134.2
(C-1′), 141.8 (C-1′′), 151.0 (C-1 of tolyl), 158.7 (C-5), 164.9 (C-
3), 196.2 (CdO). Anal. (C33H38N4O3) C, H, N.
1-[5-(2,3-Epoxypr opoxy)-3-m eth yl-1-ph en yl-1H-pyr azol-
4-yl]-3-p h en ylp r op a n -1-on e (22). To a solution of 306 mg
(1 mmol) of pyrazolone 4, 393 mg (1.5 mmol) of Ph3P, and 93
mg (1.25 mmol) of 2,3-epoxypropanol in 20 mL of THF was
added slowly 261 mg (1.5 mmol) of diethyl azodicarboxylate,
and the mixture was stirred for 20 h at room temperature.
Then 1 mL of MeOH was added; the mixture was poured onto
20 mL of water and was then exhaustively extracted with
ether. The combined organic phases were washed with 2 M
NaOH, water (several times), and brine, dried over anhydrous
Na2SO4, and evaporated in vacuo. Column chromatography
(eluent: CH2Cl2-EtOAc-light petroleum, 3:1:1) gave a nearly
colorless oil: yield 50%; MS m/z 362 (M+, 78), 279 (15), 277
(20), 257 (27), 231 (21), 201 (90), 200 (21), 174 (29), 167 (34149
(82), 117 (43), 105 (29), 100 (13), 92 (20), 91 (89), 83 (24), 77
(70), 71 (37), 70 (24), 69 (32), 67 (26), 65 (21), 60 (21), 57 (100),
1
56 (18), 55 (41), 51 (16); H NMR (300 MHz) δ 2.43 (m, 1H,
CH2 of oxirane), 2.50 (s, 3H, 3-Me), 2.70 (m, 1H, CH2 of
oxirane), 3.06 (m, 2H, CH2-Ph), 3.08 (m, 1H, CH of oxirane),
3.18 (m, 2H, CH2-CO), 3.75 (m, 1H, OCH2), 4.13 (m, 1H,
OCH2), 7.19 (m, 1H, H-4′′), 7.27 (m, 4H, H-2′′,3′′,5′′,6′′), 7.37
(m, 1H, H-4′), 7.48 (m, 2H, H-3′,5′), 7.65 (m, 2H, H-2′,6′); 13C
NMR (75 MHz) δ 15.9 (3-Me), 29.9 (CH2-Ph), 43.0 (CH2-CO),
44.3 (CH2 of oxirane), 49.3 (CH of oxirane), 76.5 (OCH2), 109.0
(C-4), 123.3 (C-2′,6′), 126.0 (C-4′′), 127.9 (C-4′), 128.4 (C-
2′′,3′′,5′′,6′′), 129.3 (C-3′,5′), 137.5 (C-1′), 141.5 (C-1′′), 150.3
(C-3), 153.9 (C-5), 194.2 (CdO). Anal. (C22H22N2O3) C, H, N.
(3-Meth yl-1-p h en yl-5-(p r op yla m in o)-1H-p yr a zol-4-yl)-
p h en ylm eth a n on e (25). Epoxide 2122 (334 mg, 1 mmol) was
treated with 414 mg (7 mmol) of n-propylamine, and the
mixture was stirred for 2 h at 50-60 °C. Then the excessive
amine was evaporated, and the residue was taken up in

Substituted 4-Acylpyrazoles and -pyrazolones
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4009
CH2Cl2. The organic phase was washed several times with
water, dried, and evaporated. Column chromatography (elu-
ent: EtOAc-MeOH, 9:1) afforded nearly colorless crystals of
mp 65-66 °C: yield 30%; MS m/z 320 (M+ + 1, 42), 319 (M+,
100), 318 (M+ - 1, 55), 291 (17), 290 (72), 200 (28), 105 (61),
91 (18), 77 (58); 1H NMR (300 MHz) δ 0.79 (t, 3J ) 7.4 Hz,
3H, Me of n-Pr), 1.44 (m, 2H, CH2-Me), 1.86 (s, 3H, 3-Me),
2.79 (q, J ) 6.7 Hz, 2H, N-CH2), 7.36-7. 58 (m, 10H, H-2′-
139.0 (C-1′′), 139.2 (C-1′), 145.9 (C-5, 3J C-5,5-Me ) 6.7 Hz), 166.1
(C-3), 167.4 (CdC-N). Anal. (C22H25N3O) C, H, N.
5-Meth yl-2-p h en yl-4-[(Z)-p h en yl(4-o-tolylp ip er a zin -1-
yl)m eth ylen e]-2,4-d ih yd r op yr a zol-3-on e (29). Educt
2
(2.78 g, 10 mmol) was successively treated with 1 equiv of
NaOMe in MeOH and 1 equiv of epichlorohydrin as described
for the preaparation of 6. Then 1.76 g (10 mmol) of o-
tolylpiperazine was added, and the mixture was refluxed for
30 h. After filtration, the excessive amine was removed in
vacuo, and the residue was taken up in CH2Cl2. The organic
layer was washed several times with water, dried, and
evaporated. The residue was subjected to column chromatog-
raphy (eluent: EtOAc) to affordsbesides 3% of 12scompound
29, which was crystallized from MeOH-EtOAc, 1:1, to afford
20% of yellow crystals of mp 230 °C: MS m/z 437 (M+ + 1,
13), 436 (M+, 40), 277 (30), 146 (100), 144 (15), 132 (34), 131
3
6′ and H-2′′-6′′), 7.61 (t, J ) 6.7 Hz, 1H, NH);13C NMR (75
MHz) δ 11.1 (Me of n-Pr), 15.3 (3-Me), 23.4 (CH2-Me), 46.7
(NCH2), 105.9 (C-4), 125.6 (C-2′,6′), 127.5 (C-3′′,5′′), 128.2 (C-
2′′,6′′), 128.3 (C-4′), 129.1 (C-3′,5′), 130.5 (C-4′′), 139.6 (C-1′),
141.3 (C-1′′), 149.6 (C-3), 153.7 (C-5), 192.2 (CdO). Anal.
(C20H21N3O) C, H, N.
1-(3-Met h yl-1-p h en yl-5-(p r op yla m in o)-1H -p yr a zol-4-
yl)-3-p h en ylp r op a n -1-on e (26). Compound 26 was prepared
from raw epoxide 22 (achieved from 306 mg (1 mmol) of
pyrazolone 4) in a similar fashion as described for 25. Column
chromatography (eluent: light petroleum-EtOAc, 3:1) af-
fordedsbesides 12% of 28scompound 26, which after crystal-
lization from EtOH afforded tan crystals of mp 97 °C: yield
45% (regarding 4); MS m/z 348 (M+ + 1, 27), 347 (M+, 100),
319 (18), 318 (20), 242 (90), 226 (21), 217 (19), 216 (29), 215
(48), 200 (18), 186 (22), 173 (19), 105 (17), 92 (15), 91 (71), 77
1
(19), 130 (15), 118 (44), 91 (50), 77 (33), 65 (15), 56 (18); H
NMR (300 MHz) δ 1.35 (s, 3H, 5-Me), 2.34 (s, 3H, Me of tolyl),
3.07 and 3.21 (each m, 2H, piperazine H-3 and H-5), 3.62 and
4.19 (each m, 2H, piperazine H-2 and H-6), 7.03 (m, 1H, H-4
of tolyl), 7.06 (m, 1H, H-6 of tolyl), 7.11 (m, 1H, H-4′), 7.20 (m,
1H, H-5 of tolyl), 7.21 (m, 1H, H-3 of tolyl), 7.38 (m, 2H,
H-3′,5′), 7.48 (m, 2H, H-2′′,6′′), 7.54 (m, 2H, H-3′′,5′′), 7.61 (m,
1H, H-4′′), 8.05 (m, 2H, H-2′,6′); 13C NMR (75 MHz) δ 15.5
(5-Me, 1J ) 128.2 Hz), 17.8 (Me of tolyl), 51.6 and 55.7
(piperazine C-2 and C-6), 52.8 (piperazine C-3,5), 103.0 (C-4,
3J C-4,5-Me ) 2.5 Hz), 119.1 (C-6 of tolyl), 119.1 (C-2′,6′), 123.7
(C-4′), 124.0 (C-4 of tolyl), 126.6 (C-5 of tolyl), 128.5 (C-3′,5′),
129.0 (C-3′′,5′′), 130.5 (C-2′′,6′′), 131.3 (C-3 of tolyl), 131.8 (C-
4′′), 132.8 (C-2 of tolyl), 135.2 (C-1′′), 139.6 (C-1′), 149.4 (C-5,
2J C-5,5-Me ) 6.9 Hz), 150.2 (C-1 of tolyl), 161.9 (C-3), 166.2 (Cd
C-N). Anal. (C28H28N4O) C, H, N.
P h a r m a cology. 1. Cell Lin es. The CCRF-CEM T-
lymphoblast cell line, as well as the resistant lines, were
obtained as described previously.17,34 Cells were kept in
RPMI1640 medium supplemented with 10% fetal calf serum
under standard culture conditions. The resistant CCRF
vcr1000 cell line was kept in medium containing 1000 ng/mL
vincristine. The selecting agent was washed out at least 1
week prior to the experiments. The cell line used in our
studies was selected in the presence of increasing doses of
vincristine without prior mutagenization.34 This cell line has
been chosen on grounds of distinct PGP expression and does
not show the mutation at codon 185.41 In addition, no
significant contribution of other factors to MDR was observed.
The mouse lymphoma cell line L5178Y was infected by a
retroviral vector carrying the human mdr1 gene. The vector
was grown in, and encapsidated by, the packaging cell line
PA12MDR1/A-1. The viral RNA is from a replication-defective,
amphotropic virus that is extruded by the cells into surround-
ing medium.42 Polybrene was added to a final concentration
of 2 µg/mL to this filtered supernatant which was then diluted
1:5 with growth medium. Here, too, Polybrene was added to
a final concentration of 2 µg/mL. The cells to be infected were
plated in medium containing the virus and Polybrene. After
2 days, this medium was replaced with McCoy’s 5A medium
containing 10% horse serum, to which 60 ng/mL colchicine was
added to select resistant cells.43 These resistant cells were
termed L5178Y VMDR C.06. The line was maintained in
colchicine-containing culture medium.
2. Rh od a m in e 123 a n d Da u n om ycin Efflu x Stu d ies.
Rhodamine efflux studies were performed using modified
published methods.17 Cells were pelleted, the supernatant was
removed by aspiration, and the cells were resuspended at a
density of 1 × 106/mL in RPMI1640 medium containing
rhodamine 123 (Sigma Chemical Co., St. Louis, MO) at a final
concentration of 0.2 µg/mL (0.53 µmol/L). Cell suspensions
were incubated at 37 °C for 15 min. Tubes were chilled on ice
and pelleted at 500g in an Eppendorf 5403 centrifuge (Eppen-
dorf, Germany). Supernatants were removed, and the cell
pellet was resuspended in medium which was prewarmed to
37 °C and contained either no modulator or chemosensitizer
at various concentrations ranging from 3 nM to 500 µM,
depending on solubility and the expected potency of the
1
3
(51), 66 (16), 65 (16); H NMR (300 MHz) δ 0.77 (t, J ) 7.4
Hz, 3H, Me of n-Pr), 1.41 (m, 2H, CH2-Me), 2.44 (s, 3H, 3-Me),
3
2.73 (q, J ) 6.6 Hz, 2H, N-CH2), 3.06 (“s”, 4H, CH2-CH2-
Ph), 7.18-7. 52 (m, 10H, H-2′-6′ and H-2′′-6′′), 7.91 (t, 3J ∼
6.0 Hz, 1H, NH); 13C NMR (75 MHz) δ 11.0 (Me of n-Pr), 16.3
(3-Me), 23.4 (CH2-Me), 30.4 (CH2-Ph), 42.4 (CH2-CO), 46.5
(NCH2), 105.5 (C-4), 125.6 (C-2′,6′), 126.0 (C-4′′), 128.2 (C-4′),
128.3 (C-2′′,6′′), 128.4 (C-3′′,5′′), 129.0 (C-3′,5′), 139.6 (C-1′),
2
141.5 (C-1′′), 148.6 (C-3, J C-3,3-Me ) 6.6 Hz), 153.6 (C-5,
3J C-5,NCH2 ) 3.2 Hz), 194.5 (CdO). Anal. (C22H25N3O) C, H,
N.
2,5-Dim eth yl-4-[(Z)-p h en yl(p r op yla m in o)m eth ylen e]-
2,4-d ih yd r op yr a zol-3-on e (27). Educt 1 (216 mg, 1 mmol)
was taken up in 5 mL of n-propylamine, and the mixture was
refluxed for 4 h. Excessive amine was removed in vacuo, and
the residue was subjected to column chromatography (eluent:
EtOAc-MeOH, 5:1). Recrystallization from diisopropyl ether
gave a tan powder of mp 67-68 °C: yield 38%; MS m/z 258
(M+ + 1, 24), 257 (M+, 100), 256 (M+ - 1, 36), 214 (33), 199
(34), 125 (28), 104 (27), 77 (28), 66 (25), 58 (18); 1H NMR (300
MHz) δ 0.88 (t, 3J ) 7.4 Hz, 3H, Me of n-Pr), 1.32 (s, 3H, 5-Me),
1.54 (m, 2H, CH2-Me), 3.04 (m, 2H, N-CH2), 3.39 (s, 1H,
2-Me), 7.24 (m, 2H, H-2′′,6′′), 7.47 (m, 1H, H-4′′), 7.48 (m, 2H,
H-3′′,5′′), 11.17 (s, 1H, NH); 13C NMR (75 MHz) δ 11.0 (Me of
1
1
n-Pr, J ) 126.0 Hz), 15.0 (5-Me, J ) 128.2 Hz), 23.4 (CH2-
Me, 1J ) 125.4 Hz), 30.6 (2-Me, 1J ) 138.7 Hz), 45.8 (N-CH2),
98.5 (C-4, 3J C-4 5-Me
) 2.3 Hz), 127.2 (C-2′′,6′′), 128.8 (C-3′′,5′′),
,
130.0 (C-4′′), 131.5 (C-1′′), 145.9 (C-5, 2J C-5,5-Me ) 7.0 Hz), 165.4
3
3
(C)C-N, J C)C-N,NCH2 ) 3.7 Hz), 166.0 (C-3, J C-3,2-Me ) 2.1
Hz). Anal. (C15H19N3O) C, H, N.
5-Meth yl-2-ph en yl-4-[(Z)-3-ph en yl-1-(pr opylam in o)pr o-
p ylid en e]-2,4-d ih yd r op yr a zol-3-on e (28). Educt 4 (306 mg,
1 mmol) was taken up in 4 mL (49 mmol) of n-propylamine,
and the mixture was refluxed for 5 h. Then excessive n-
propylamine was removed in vacuo, the residue was taken up
in CH2Cl2, and the organic layer was washed with 2 N NaOH
and water, dried, and evaporated. Recrystallization from
EtOH afforded 24% of colorless crystals of mp 106 °C: MS
m/z 348 (M+ + 1, 24), 347 (M+, 100), 330 (11), 200 (18), 199
(30), 97 (16), 91 (47), 77 (19), 69 (22), 58 (31), 57 (38), 55 (28);
1H NMR (300 MHz) δ 1.01 (t, 3J ) 7.4 Hz, 3H, Me of n-Pr),
1.68 (m, 2H, CH2-Me), 2.43 (s, 3H, 5-Me), 2.95 (“s“, 4H, CH2-
CH2-Ph), 3.22 (m, 2H, N-CH2), 7.13 (m, 1H, H-4′), 7.21 (m,
2H, H-2′′,6′′), 7.29 (m, 1H, H-4′′), 7.31 (m, 2H, H-3′′,5′′), 7.39
(m, 2H, H-3′,5′), 8.02 (m, 2H, H-2′,6′), 11.60 (s, 1H, NH); 13C
NMR (75 MHz) δ 11.3 (Me of n-Pr), 17.0 (5-Me, 1J ) 127.8
Hz), 22.8 (CH2-Me), 30.4 (CH2-CH2-Ph), 34.4 (CH2-CH2-
Ph), 44.7 (N-CH2), 97.9 (C-4), 119.2 (C-2′,6′), 124.1 (C-4′),
127.0 (C-4′′), 128.1 (C-2′′,6′′), 128.6 (C-3′,5′), 128.9 (C-3′′,5′′),

4010 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Chiba et al.
(14) Dodic, N.; Dumaitre, B.; Daugan, A.; Pianetti, P. Synthesis and
Activity Against Multidrug Resistance in Chinese Hamster
Ovary Cells of New Acridone-4-carboxamides. J . Med. Chem.
1995, 38, 2418-2426.
(15) Ecker, G.; Chiba, P. Recent Developments in Overcoming
Tumour Cell Multi-Drug Resistance. Exp. Opin. Ther. Patents
1997, 7, 589-599.
(16) Chiba, P.; Burghofer, S.; Richter, E.; Tell, B.; Moser, A.; Ecker,
G. Synthesis, Pharmacologic Activity and Structure-Activity-
Relationships of a Series of Propafenone-Related Modulators of
Multi-Drug-Resistance. J . Med. Chem. 1995, 38, 2789-2793.
(17) Chiba, P.; Ecker, G.; Schmid, D.; Drach, J .; Tell, B.; Gekeler, V.
Structural Requirements for Activity of Propafenone Type
Modulators in PGP-Mediated Multidrug Resistance. Mol. Phar-
macol. 1996, 49, 1122-1130.
(18) Elguero, J .; Marzin, C.; Katritzky, A. R.; Linda, P. The Tautom-
erism of Heterocycles. In Advances in Heterocyclic Chemistry,
Supplement 1; Katritzky, A. R., Boulten, A. J ., Eds.; Academic
Press: New York, San Francisco, London, 1976; pp 313-336 and
references therein.
(19) Schofield, K.; Grimmett, M. R.; Keene, B. T. R. Heteroaromatic
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(21) Dewar, M. J . S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J . J . P.
AM1: A New General Purpose Quantum Mechanical Molecular
Model. J . Am. Chem. Soc. 1985, 107, 3902-3909. AMPAC 5.0,
1994; Semichem, 12715 W. 66th Terrace, Shawnee, KS 66216.
(22) Holzer, W.; Plagens, B.; Lorenz, K. Alkylation of Pyrazolones
via the Mitsunobu Reaction. Heterocycles 1997, 45, 309-314.
(23) Patt, S. L.; Shoolery, J . N. Attached Proton Test for Carbon-13
NMR. J . Magn. Reson. 1982, 46, 535-539.
modifier. Eight concentrations (serial dilution 1:2.5) were
tested for each modulator. After 30, 60, 90, and 120 s, aliquots
of the incubation mixture were transferred to tubes containing
an equal volume of ice-cold stop solution (RPMI1640 medium
containing verapamil at a final concentration of 10 µg/mL).
Zero time points were done by immediately pipetting rhodamine
123-preloaded cells into ice-cold stop solution. Non-PGP-
expressing parental CCRF-CEM cells were used as controls
for simple plasma membrane diffusion, whereby initial
rhodamine 123 fluorescence levels were adjusted to be equal
to initial levels observed in resistant cells. Samples drawn at
the respective time points were kept in an ice water bath and
measured within 1 h on a Becton Dickinson FACSCALIBUR
flow cytometer (Becton Dickinson, Vienna, Austria). Viable
cells were gated on the basis of forward and side scatter. The
excitation wavelength was 488 nm, and the emission was
measured in the FL1 channel (520-550 nm); 5000 gated
events were accumulated for the determination of mean
fluorescence values. Time points were fitted by an exponential
curve, and the first-order rate constant (Vmax/Km) was deter-
mined as the slope of the curve at the zero time point.
For daunomycin efflux an analogous experimental protocol
was used, whereby the daunomycin concentration was 3.2
µmol/L and the preloading time was 30 min. Time points were
60, 120, 180, and 240 s. The excitation wavelength was 488
nm, and the emission was measured in the FL3 channel (650-
780 nm). Otherwise, conditions were identical to those
outlined for the rhodamine 123 efflux experiments.
(24) Neuhaus, D.; Williamson, M. P. The Nuclear Overhauser Effect
in Structural and Conformational Analysis; VCH Publishers:
New York, Weinheim, Cambridge, 1989; pp 211-252.
(25) Bax, A.; Freeman, R. Investigation of Complex Networks of
Spin-Spin Coupling by Two-Dimensional NMR. J . Magn. Reson.
1981, 44, 542-561.
Ack n ow led gm en t. We gratefully acknowledge the
financial support provided by the Austrian Science Fund
(Grant P11760-MOB) and the J ubilaeumsfond of the
Austrian National Bank (Grant 6899). We thank Prof.
Dr. M. Begtrup for helpful discussions.
(26) Bax, A.; Subramanian, S. Sensitivity-Enhanced Two-Dimen-
sional Heteronuclear Shift Correlation NMR Spectroscopy. J .
Magn. Reson. 1986, 67, 565-569.
(27) Davis, D. G.; Bax, A. Simplification of 1H NMR Spectra by
Selective Excitation of Experimental Subspectra. J . Am. Chem.
Soc. 1985, 107, 7197-7198.
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