Lipophilicity and membrane interactions of cationic-amphiphilic ...

European Journal of Pharmaceutical Sciences 14 (2001) 167–175
www.elsevier.nl/locate/ejps
Lipophilicity and membrane interactions of cationic-amphiphilic
compounds: syntheses and structure–property relationships
Christian D.P. Klein
*, Gerald F. Tabeteh , Alice V. Laguna , Ulrike Holzgrabe , Klaus Mohr
a, b b c b
a
Pharmaceutical Chemistry, ETH Zurich, ¨ Winterthurerstr. 190, CH-8057 Zurich, ¨ Switzerland
b
Department of Pharmacology and Toxicology, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
c
Institute of Pharmacy and Food Chemistry, University of Wurzburg, ¨ Am Hubland, D-97074 Wurzburg, ¨ Germany
Received 22 December 2000; received in revised form 18 June 2001; accepted 18 June 2001
Abstract
This study was performed to elucidate the relationship between steric factors, lipophilicity, and the potency of cationic-amphiphilic
compounds to displace calcium ions from phosphatidylserine monolayers. The latter property is considered to be a substance/
phospholipid affinity measure. A series of cationic-amphiphilic 3-phenyl-N,N-dimethylpropylamine derivatives with systematic structural
variations was synthesized. Lipophilicity values were determined by chromatographic (RP-HPLC, log D
7.4), shake-flask (log P), and
theoretical (CLOGP) techniques. The potency of the compounds to displace calcium ions from phosphatidylserine monolayers was
45 21
determined using a radiotracer technique, employing the isotope Ca . The experimental lipophilicity values of several isomeric
biphenyl- and diphenyl-congeners differ more than could be expected from the CLOGP-calculations and show a good correlation to the
calculated molecular surface areas. Although the affinity of the substances to the phospholipid monolayer tends to increase with
lipophilicity, no general interrelation between the two properties could be found. Surprisingly, the assay system (a phospholipid
monolayer) was quite sensitive towards small steric changes at the ‘ligand’ molecules. Stereochemical factors have a considerable
influence on the interaction of solutes with phospholipid membranes. It must be questioned whether lipophilicity measures alone, without
taking other molecular features into account, can meaningfully be used to explain or predict the influence of solutes on membrane-related
processes and properties. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Phosphatidylserine; Solute-membrane interactions; Lipophilicity; Log P; Cationic-amphiphilic; Phospholipid monolayer
1. Introduction interactions. If cationic-amphiphilic substances (see, for
example, the general formula shown in Table 1) are added
The interaction of drugs with biological membranes is of to this system, the lipophilic parts of the molecules are
major importance for their pharmacokinetic and pharmaco- incorporated into the hydrocarbon chain region of the
dynamic properties. The essential structural feature of phospholipid monolayer. The cationic parts of the combiomembranes
is the phospholipid bilayer, and artificial pounds are located within the phospholipid headgroup
phospholipid preparations are often used as model systems region or at the phospholipid–water interface and displace
for the probing of drug/biomembrane interactions. We calcium ions from their ionic ‘binding sites’. This process
have established an assay system in which the adsorption is depicted in Fig. 1. The potency of cationic amphiphilic
21
of Ca -ions to a phosphatidylserine monolayer, formed at compounds to displace calcium ions depends on their
an air-buffer interface, can be measured by means of a affinity to the phosphatidylserine monolayer (Hauser and
radiotracer method (e.g., Girke et al., 1989). The addition Dawson, 1968; Scheufler et al., 1990; Vogelgesang and
of cationic-amphiphilic substances to the buffer solution Scheufler, 1990). It is usually expressed as the concen-
21
leads to a decrease in adsorbed Ca . The displacement of
21
tration which leads to a 50% decrease in adsorbed Ca ,
21
Ca is due to an ion-exchanging effect: The negatively i.e. the IC50
value.
charged phosphatidylserine headgroups bind cations from We would like to present a dataset in which a common,
the aqueous buffer, for example calcium ions, via ionic cationic-amphiphilic structure (N,N-dimethyl-3-phenylpropylamine,
cpd. 1 of Table 1) has been systematically
*Corresponding author. Tel.: 141-1-635-6072; fax: 141-1-635-6884. extended. In particular, we intended to clarify the relation-
E-mail address: cklein@pharma.ethz.ch (C.D.P. Klein).
ships between lipophilicity, steric factors, and the affinity
0928-0987/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S0928-0987(01)00170-1

168 C.D.P. Klein et al. / European Journal of Pharmaceutical Sciences 14 (2001) 167 –175
Table 1
Structures, log P and log D7.4 values, and 2log IC50
values of the dataset of cationic-amphiphilic compounds. All compounds were assayed as
hydrochlorides
a
Cpd. log D7.4 log P CLOGP 2 log IC50
(RP-HPLC, pH 7.4) (shake-flask, pH 12.5) (IC in mol/l)
c c c
50
b
1 R1–55H 1.50 n.d. 2.49 3.00
2 R 5CH 2.00 n.d. 2.99 3.25
1 3
3 R 5C H 2.45 n.d. 3.52 3.45
1 2 5
4 R 5n-C H 2.88 n.d. 4.05 4.31
1 3 7
5 R 5i-C H 2.74 n.d. 3.92 3.60
1 3 7
6 R 5(C H ) CH 3.67 n.d. 4.98 4.92
1 2 5 2
7 R 5Phenyl 3.15 6.08 4.38 5.12
1
8 R 5Phenyl 3.02 6.20 4.38 4.70
2
9 R 5Phenyl 2.71 5.53 4.08 4.23
3
10 R 5Phenyl 2.34 5.40 3.93 3.93
4
11 R 5Phenyl 2.26 5.22 3.93 3.42
5
12 R 5OCH 1.48 n.d. 2.41 2.76
1 3
13 R 54-OCH –phenyl, 2.33 n.d. 3.85 3.79
1 3
R 5Phenyl
4
14 R 54-OH–phenyl, 1.06 n.d. 3.26 3.87
1
R 5Phenyl
4
15 R154-OCH 3–phenyl, 2.15 n.d. 3.85 3.29
R55Phenyl
16 3.19 n.d. 4.07 3.95
17 2.13 n.d. 3.10 3.98
a
unless otherwise stated, all R5H.
b
n.d.: not determined.
c
n53–5 experiments, standard errors for the given values are smaller than 0.15 log units.
Fig. 1. Cationic-amphiphilic compounds displace calcium ions from their ‘binding sites’ at the aqueous side of the phosphatidylserine monolayer.
Phosphatidylserine of natural origin, which was used in the experiments, carries variable fatty acid chains and not necessarily palmitoyl chains as shown
here.

C.D.P. Klein et al. / European Journal of Pharmaceutical Sciences 14 (2001) 167 –175 169
to phosphatidylserine monolayers. The dataset shown here compounds. The CLOGP values were calculated for the
is partially identical with a dataset given previously (Klein non-ionized species of the compounds.
et al., 1999; Klein and Hopfinger, 1998). We would like to
point out that all compounds which were also part of the
previous dataset have been re-assayed, which accounts for 2.3. Experimental determination of partition coefficients
minor differences between the former values and the ones
presented here. It was necessary to re-determine these
values because the employed phospholipid (phosphatidyl- 2.3.1. Shake-flask octanol/water partitioning
serine) is of biological origin (bovine brain) and therefore Shake-flask octanol–water partition coefficients (Fujita
the IC50
values have a certain, though small, batch-to- et al., 1964) were determined for several isomeric bibatch
variability. All values given here were determined phenyl- and diphenyl-propylamines (7–11). In order to
using the same batch of phosphatidylserine.
eliminate the ionized, cationic species of the compounds,
For the previous dataset that also included other pharma- the partitioning experiments were carried out using a
cological endpoints (antiarrhythmic activity and depression strongly basic (pH 12.5) aqueous buffer. We have previof
a liposome phase transition temperature), we have tried ously measured pKa
values of 9.5 to 10 for several close
to develop Quantitative Structure–Activity Relationships analogs (Klein et al., 1999). Therefore, it can be assumed
using intramolecular and intermolecular descriptors. that the cationic species was present in a concentration of
Whereas we succeeded in developing good QSAR models less than 1%.
for the antiarrhythmic activity, the biophysical properties 1-Octanol of the best commercially available quality
were more difficult to explain in a quantitative manner. was used for the shake-flask octanol/water partitioning
The major drawback of the previous dataset was its experiments. The aqueous buffer consisted of trisodium
fragmentation–the
45 21
Ca -displacing potency, for exam- phosphate trihydrate (0.2 M, 76.02 g/l) and potassium
ple, was measured for only a few compounds of a hydrogen phosphate (0.1 M, 17.42 g/l), dissolved in
congeneric series. In contrast, the present dataset allows a distilled water. The calculated pH of this buffer was 12.6,
systematic study of steric and lipophilic effects.
and a pH of 12.5 was measured. Buffer and octanol were
saturated with each other and filtered before use. A diode
array spectrophotometer (HP 8452A) with a 1 cm-cell was
2. Material and methods used for the absorption measurements of the aqueous phase
after partitioning, and the spectral data was stored and
2.1. Displacement of
45 21
Ca from phospholipid evaluated using a computer (HP89532A UV–Vis softmonolayers
ware). All experiments were run in triplicate and at room
temperature (258C).
A detailed description of the experimental procedure Plastic vials proved to be unsuitable for the partitioning
®
was given by Girke et al. (1989). In short, a teflon experiments, probably because the high lipophilicity of the
planchette of 4.5 cm diameter was filled with 5 ml of compounds caused their adsorption at the container sur-
®
buffer (0.01 mM CaCl2
supplemented with trace amounts face. Instead, glass vials with teflon seals were used.
45
of CaCl
2, 5 mM NaCl, 2 mM TES, 2 mM histidine; pH All test compounds were dissolved (as hydrochloride
7.5). Radioactivity was detected by a Geiger-Muller ¨ count- salts) in distilled water at a concentration of 2 mg/ml to
ing tube (Frieseke & Hoepfner, Erlangen, Germany) give stock solutions. The amounts of sample, buffer and
positioned above the teflon dish. 3 nmol of phospha- octanol were chosen so that absorptions of 0.35 to 0.6 were
tidylserine (from bovine brain; purity 98–99%, Sigma reached after partitioning. In case of the very lipophilic
Chemical) dissolved in 1 ml of chloroform were carefully compounds 7 and 8, an absorption of only 0.17 and 0.25,
applied to the surface of the buffer to form a phospholipid respectively, was measured. A typical partitioning experi-
45 21
monolayer. Adsorption of Ca to the phospholipid was ment was performed with 700 ml of sample stock solution
indicated by a twofold elevation of the counting rate under (corresponding to 1.4 mg of sample), 700 ml of buffer and
control conditions. Test compounds were dissolved in the 20 ml of octanol. All substances were pipetted into the
buffer at the appropriate concentrations. An experiment vials, the octanol was added by microliter syringe, the vials
included measurement of a range of concentrations in were shaked in a rotating shaking machine at 120 rpm for
order to obtain a concentration/effect-curve for the dis- 60 min, centrifuged at 5000 rpm for 5 min to afford
placement of
45 21
Ca by a given test compound. Experi- complete phase separation, and the octanol was removed.
ments were repeated three to five times.
The absorption of the aqueous phase was determined. In
most cases, absorptions were measured at the maximum of
2.2. Calculation of partition coefficients the B-band. The concentration in the octanol layer was
determined by difference. Calibration was done in exactly
The ‘CLOGP for Windows V. 4.0.0’ program (Biobyte the same manner as the partitioning, except that no octanol
Corp.) was used to calculate CLOGP values for the target and smaller amounts of sample were used.

170 C.D.P. Klein et al. / European Journal of Pharmaceutical Sciences 14 (2001) 167 –175
2.3.2. RP-HPLC recorded using KBr tablets (Perkin-Elmer spectrometer,
The distribution coefficients (log D
7.4) of all target model 298). Combustion analyses (C, H, N) for the target
compounds were determined by RP-chromatography with compounds gave results within60.4% of the calculated
methanol/aqueous buffer (60/40 and 30/70) mobile values. All starting compounds were used as received
phases (cf. Kaliszan (1990) for a review of log P de- (Merck, Fluka, Sigma-Aldrich). An overview of the syntermination
by HPLC). The chromatographic systems were thetic pathways is given in Fig. 2.
calibrated with solutes for which an experimentally de-
13
C NMR spectral data will be provided upon request.
termined octanol/water partition-coefficient was available 1–5, 7, 10, 12: Synthesized as described previously
(Hansch et al., 1995) and which are not charged at pH 7.4. (Klein et al., 1999).
The capacity factors of the reference substances were 6, N,N-Dimethyl-3-(4-[3-pentyl]-phenyl)-propylamine
correlated against the experimental octanol/water log P hydrochloride: 4-(3-Pentyl)-acetophenone (6a) was synvalues,
and the correlation equation, thus obtained, was thesized from propiophenone in a two-step synthesis
used to calculate lipophilicity values (now log D, because according to Cram (1952). Purified by distillation in vacuo.
D 1
the test compounds are ionized at pH 7.4) for the test 6a: bp 150–1538C [22 hPa], n20 1.5171, H NMR (CDCl
3,
2 6
compounds. d ): 7.88 (d, 2 H, J58.4 Hz, H , H aro), 7.20 (d, 2 H,
3 5
The RP-HPLC system for the determination of log D7.4 J58.3 Hz, H , H aro), 2.55 (s, 3 H, COCH
3), 2.38 (m, 1
21
values consisted of a Kontron Instruments HPLC pump H), 1.45–1.75 (m, 4 H), 0.73 (m, 6 H); IR [cm ] (film):
‘420’, a Rheodyne injection valve with 50 ml sample loop, 2960, 1680, 1605, 1270. 6a (0.01 mol, 1.9 g) was
a GromSil ODS-2 5 mm ‘fully endcapped’ 12534 mm converted to the corresponding chloropropeniminium salt
column, and a Perkin Elmer LC-480 Autoscan diode array (3-chloro-3-(4-[3-pentyl]-phenyl) - prop - 2 - ene - 1 - ylidenedetector.
The chromatographic data were stored and evalu- N,N-dimethyliminium perchlorate, 6b) in 60% yield by the
ated on a PC (LabControl LC-DESplus software). A previously described method (Klein et al. (1999), Method
1
methanol/buffer (60/40 or 30/70) mobile phase was used B). 6b: yellow crystals, mp 1608C. H NMR (CD3CN, d ):
at a flow rate of 2 ml/min. The buffer was a pH 7.4 8.73 (d, 1 H, J510 Hz, CH–CH5N), 7.96 (d, 2 H, J58.6
phosphate buffer, obtained by mixing a 0.2 M potassium Hz, H aro), 7.39 (d, 2 H, J58.5 Hz, H aro), 7.31 (d, 1 H,
dihydrogen phosphate solution (1000 ml) and 0.1 M J510 Hz, CH–CH5N), 3.69 (s, 3 H, E–CH5N(CH )),
3 2
sodium hydroxide (1573.6 ml). N,N-Dimethylhexylamin 3.57 (s, 3 H, Z–CH5N(CH ) ), 2.45–2.55 (m, 1 H,
3 2
(0.02%) was added to the mobile phase to shield unreacted CH(CH2CH 3) 2), 1.50–1.80 (m, 4 H, CH(CH2CH 3) 2),
21
silanol groups and minimize peak tailing (Gill et al., 0.75 (m, 6 H, CH(CH CH ) ); IR [cm ]: 2940, 1640,
2 3 2
1982). The column and the mobile phase were thermo- 1075. 6b was hydrogenated (H
2: 300–400 kPa) in methastated
at 308C (water bath, Haake Thermostat D1).
nol solution over Pd/C (10%) catalyst, followed by the
Aromatic compounds with known octanol/water parti- usual workup procedures, to give the desired 6 in quantita-
1
tion coefficients (Hansch et al., 1995) were used for the tive yield. 6: White crystals, mp 1388C. H NMR (CD3OD,
calibration of the system (benzene, chlorobenzene, toluene, d ): 7.15 (d, 2 H, J57.1 Hz, H aro), 7.09 (d, 2 H, J57.1
ethylbenzene, cumene, phenylethanol, biphenyl, anth- Hz, H aro), 3.14 (m, 2 H, CH
2–N(CH 3) 2), 2.87 (s, 6 H,
3
racene, N,N-dimethylaniline). All analytes were dissolved N(CH ) ), 2.69 (m, 2 H, C H ), 2.25–2.30 (m, 1 H,
3 2 2
2
2 3 2 2
in methanol at a concentration of 300 mg/ml. Experiments CH(CH CH ) ), 2.00–2.10 (m, 2 H, C H ), 1.49–1.70
were run in triplicate, and peak maxima were determined (m, 4 H, CH(CH2CH 3) 2), 0.75 (m, 6 H, CH(CH2CH 3)).
2
21
at the respective absorption maximum. Capacity ratios IR [cm ]: 2950, 1460.
were determined as k95(t 2t )/t , where t is the average 8, 3-(39-Biphenylyl)-N,N-dimethylpropylamine hydror
0 0 r
retention time of the analyte and t0
is the ‘retention’ time chloride: 3-Phenylbenzaldehyde (8a) (Shoppee, 1933) was
of a non-retained compound (thiourea). A linear regression synthesized in a 4-step synthesis from 3-nitroaniline. 3-
analysis was performed for the log k9/log P data of the Nitroaniline was converted to 3-nitrobiphenyl following
reference compounds, and the regression equations were the procedure described by Elks et al. (1940). This nitro
used to calculate the log D7.4
of the compounds. The compound was reduced to give 3-aminobiphenyl, which,
equations were: (MeOH/H2O 60/40) log D7.452.04 log upon diazotation and reaction with potassium iodide,
2
k911.37, n59, R 50.99; (MeOH/H O 30/70) log D 5 afforded 3-iodobiphenyl (Campaigne and Reid, 1946).
2
2 7.4
2.21 log k9 20.59, n54, R 50.98. Conversion to the Grignard reagent and reaction with
N-formylpiperidine (following the procedure of Olah and
2.4. Syntheses and analytical data Arvanaghi, 1981) gave 8a in 60% yield. Purification by
column chromatography (silica gel, petrol ether/ethyl
1 D 1
20 3
NMR spectra were recorded at 299.956 MHz ( H) or acetate 90/10). 8a: n 1.6277; H NMR (CDCl , d ):
13
75.443 MHz ( C) on a Varian XL 300 FT-NMR spec- 10.10 (s, 1 H, Ar–CHO), 7.38–8.12 (m, 9 H, H aro); IR
trometer, with the solvent proton/carbon signals being
21
[cm ] (film): 1690 b, 1590, 1180, 750. To a stirred
used as internal standard for the calibration of the fre- suspension of N,N-dimethylaminoethyltriphenylphosquency
scale. Unless otherwise stated, all IR spectra were phonium bromide (0.00321 mol, 1.34 g) (prepared accord-

C.D.P. Klein et al. / European Journal of Pharmaceutical Sciences 14 (2001) 167 –175 171
Fig. 2. Overview of synthetic pathways.
ing to Marxer and Leutert, 1978) in THF (15 ml), an give the desired 3-(39-biphenylyl)-N,N-dimethylpropylequimolar
solution of 1.4 M n-butyllithium in n-hexane amine hydrochloride (8) in quantitative yield. 8: mp
1
was added under nitrogen at 08C. After 30 min a solution 1458C; H NMR (CDCl
3, d ): 7.63–7.11 (m, 9 H, H aro),
1
of 8a (0.00321 mol, 0.584 g) in THF (7 ml) was slowly 3.02–2.96 (t, 2 H, J57.5 Hz, C H), 2.79 (t, 2 H, J57.7
3
added, and the reaction mixture was stirred overnight at Hz, C H), 2.77 (s, 6 H, N(CH
3) 2), 2.21–2.27 (m (b), 2 H,
2 21
608C. The solution was acidified with 3 N hydrochloric C H); IR [cm ]: 2490, 1480, 760.
acid (10 ml) and the tetrahydrofuran was removed in 9, 3-(29-Biphenylyl)-N,N-dimethylpropylamine hydrovacuo.
After extraction with toluene (2310 ml) the water chloride: 2-Aminobiphenyl was converted to 2-
phase was made alkaline with 2 N sodium hydroxide and iodobiphenyl following the procedure given by Gilman et
extracted with diethyl ether (3320 ml). The N,N-di- al. (1929). From 2-iodobiphenyl, 2-phenylbenzaldehyde
methyl-39-phenylcinnamylamine base was obtained by (Zaheer and Faseeh, 1944) (9a) was prepared in 80% yield
stripping the dried (K2CO 3) extracts of solvent in vacuo, using the method of Olah and Arvanaghi (1981). Purified
D
and converted to the hydrochloride by the addition of by distillation in vacuo. 9a: bp 1378C (7 hPa), n20
1.6210;
1
ethereal hydrochloric acid. The hydrochloride salt (8b, H NMR (CDCl
3, d ): 10.00 (d, 1 H, J50.9 Hz, carbonyl–
yield: 50%) was recrystallized from ethanol/diethyl ether. H), 8.07 (d, 1 H, J57.9 Hz, H aro), 7.0–7.7 (m, 9 H, H
1 21
8b: mp1648C. H NMR (CDCl
3, d ): 7.61–7.26 (m, 9 H, aro); IR [cm ] (film): 3040, 2820, 1680. This compound
3
J515.9 Hz, H aro), 6.82 (d, 1 H, C H), 6.50 (dt, 1 H, was converted to N,N-dimethyl-29-phenylcinnamylamine
2 1
J515.9, 7.3 Hz, C H), 3.79 (d, 2 H, J57.3 Hz, C H), hydrochloride (9b) by the procedure given above for 8a
21 1
2.82 (s, 6 H, N(CH
3) 2); IR [cm ]: 2680, 2080, 1480, 970, (yield: 30%). 9b: mp.1908C. H NMR (CDCl
3, d ): 7.45–
760. 8b was hydrogenated as described above for 6b, to 7.28 (m, 8 H, H aro), 7.14 (d, 1 H, J57.6 Hz, H aro), 6.93

172 C.D.P. Klein et al. / European Journal of Pharmaceutical Sciences 14 (2001) 167 –175
3
(d, 1 H, J511.5 Hz, C H), 6.00 (dt, 1 H, J511.5, 7.1 was obtained in 47% yield with respect to 13a. The
2 1
Hz,C H), 3.60 (d, 2 H, J57.0 Hz, C H), 2.61 (s, 6 H, compound is strongly hygroscopic; therefore, no meaning-
21 1
N(CH
3) 2); IR [cm ]: 2640, 1465, 735. Catalytic hydro- ful melting point could be determined. 13: H NMR (D2O,
2 6
genation (see 6b) afforded to desired 3-(29-biphenylyl)- d ): 7.40 (m, 5 H, H aro), 7.33 (d, 2 H, J58.6 Hz, H , H
N,N-dimethylpropylamine hydrochloride (9) in quantita-
3 5
aro), 6.95 (d, 2 H, J58.6 Hz, H , H aro), 4.00 (t, 1 H,
1 3
tive yield. 9: mp 1158C. H NMR (CDCl31 CD3OD, d ): C H), 3.76 (s, 3 H, Ar–OCH
3), 3.00 (m, 2 H, CH
2–
1
7.16–7.42 (m, 9 H, H aro), 2.70–2.80 (m, 2 H, C H), 2.59 N(CH
3) 2), 2.88 (s, 6 H, N(CH
3) 2), 2.38 (m, 2 H, CH–
3 21
(s, 6 H, N(CH
3) 2), 2.56 (m, 2 H, C H), 1.84 (m, 2 H, CH
2–CH 2–N); IR [cm ]: 1610, 1250, 1030.
2 21
C H); IR [cm ]: 2650 b, 1460, 750.
14, N,N-Dimethyl-3-(4-hydroxyphenyl)-3-phenylpropyl-
11, N,N-Dimethyl-2,3-diphenyl-propylamine hydrochloin
amine hydrochloride: A solution of 13 (3.16 g, 0.01 mol)
ride (Wawzonek and Nagler, 1955): 2,3-Diphenylreflux
concentrated hydrobromic acid (40 ml) was heated to
propionitrile was prepared as described by Abe and
for 2 h. After cooling, water (100 ml) was added
Miyano (1972). 0.02 mol (4.15 g) of this compound was and the resulting mixture was alkalized with conc. amdissolved
in ethanol (20 ml). A 33% solution of dithree
monia to give pH 9. The resulting solution was extracted
methylamine in ethanol (0.3 mol, 40 g) and 10% palladium
times with 75 ml of diethyl ether. The combined
on barium sulfate (2 g) were added. The reaction mixture organic layers were washed twice with water, dried over
was then shaked under hydrogen (300–400 kPa) until the K2CO 3, and about 120 ml of the solvent were evaporated.
theoretical uptake of hydrogen had occurred. The catalyst Addition of ethereal hydrochloric acid gave the crude
was removed by filtration and the filtrate evaporated to phenol hydrochloride, which was recrystallized from ace-
dryness. The residue was dissolved in water (50 ml) and
1
tone/diethyl ether. Yield: 70%. 14: mp 2128C. H NMR
brought to pH 11 by adding 2 N sodium hydroxide.
Subsequent extraction with diethyl ether (3350 ml),
(CD3OD, d ): 7.28 (m, 5 H, CH aro), 7.13 (d, 2 H, J58.5
Hz, H
2
, H
6
aro), 6.73 (d, 2 H, J58.6 Hz, H
3
, H
5
aro), 3.93
drying of the pooled extracts (K2CO 3), and evaporation of (t, 1 H, CH), 3.00 (m, 2 H, CH
2–N(CH 3) 2), 2.82 (s, 6 H,
21
the solvent under vacuum yielded the crude base, which N(CH
3) 2), 2.46 (m, 2 H, CH–CH
2–CH 2); IR [cm ]:
was separated from the primary amine by-product by 2960, 1515, 1260, 1220, 705.
flash-chromatograpy (silica gel, mobile phase: ethyl aceylamine
15, N,N-Dimethyl-2-phenyl-3-(4-methoxyphenyl)-prop-
tate/petroleum ether/conc. ammonia 900/500/10). The
hydrochloride (Kindler et al., 1948): 2-Phenyl-3-
N,N-dimethyl-2,3-diphenyl-propylamine hydrochloride (4-methoxyphenyl)-propionitrile (0.02 mol, 4.75 g), syn-
(11) was prepared in the usual manner by precipitation thesized according to the procedure of Abe and Miyano
with ethereal hydrochloric acid and was recrystallized from (1972), was treated with H
2
/Pd–C/dimethylamine as
1
acetone/diethyl ether. Yield: 70%. 11: mp 1698C; H described above for cpd. 11. The same workup procedure
NMR (CDCl
3, d ): 6.98–7.29 (m, 10 H, H aro), 3.5–3.6 as for cpd. 11 was employed, giving a yield of 85%
2 1
(m, 1 H, C H), 3.25–3.45 (m, 2 H, C H), 3.07 (dd, 1 H, N,N-dimethyl-2-phenyl-3-(4-methoxyphenyl)-propylamine
3
1
J56.7, 13.6 Hz, C H), 2.89 (dd, 1 H, J58.3, 13.6 Hz, hydrochloride (15). 15: mp 1428C. H NMR (CD3OD, d ):
3
3 5
C H), 2.60 (d, 3 H, J54.9 Hz, N(CH
3) 2), 2.49 (d, 3 H, 7.15–7.30 (m, 5 H, H aro), 6.91 (d, 2 H, J58.7 Hz, H , H
21
2 6
J54.9 Hz, N(CH
3) 2); IR [cm ]: 2450, 1440, 690. aro), 6.69 (d, 2 H, J58.7 Hz, H , H aro), 3.70 (s, 3 H,
2 1
13, N,N-Dimethyl-3-(4-methoxyphenyl)-3-phenylpropyl- Ar–OCH
3), 3.50 (m, 1 H, C H), 3.35 (m, 2 H, C H), 3.00
3 3
amine hydrochloride: 4-Methoxyacetophenone was treated (dd, 1 H, C H), 2.85 (dd, 1 H, C H), 2.56 b (s, 6 H,
21
with phenylmagnesiumbromide, affording 1-(4-methox- N(CH
3) 2); IR [cm ]: 3490, 1505, 1240.
yphenyl)-1-phenylethanol (Novak and Protiva, 1959) 16, N,N-Dimethyl-3-(2-tetralyl)-propylamine hydrochlo-
(13a). 0.1 Mol of the carbinol 13a were dissolved in ride: 2-Acetyltetralin (Smith and Lo, 1948) (16a) was
anhydrous N,N-dimethylformamide (0.65 mol, 47.5 g, 50.0 synthesized from tetralin using the method described by
ml). The mixture was cooled in an ice bath, and phosphormethoxynaphthalene.
Arsenijevic et al. (1988) for the synthesis of 2-acetyl-6-
us oxychloride (0.2 mol, 30 g, 17.9 ml) was added
16a (0.0115 mol, 2 g) was converted
dropwise, while the internal temperature was kept below to the corresponding chloropropeniminium salt (3-chloro-
108C. Stirring was continued for 24 h at ambient temperaperchlorate,
3-(2-tetralyl)-prop-2-ene-1-ylidene-N,N-dimethyliminium
ture. Afterwards, the mixture was poured into an ice-cold
16b) in 55% yield by the previously described
methanol/water (3/2) mixture, and 70% perchloric acid method (Klein et al. (1999), Method B). 16b: mp 1658C.
(25 ml) was added to precipitate the perchlorate salt. This 1 H NMR (CD3CN, d ): 8.69 (d, 1 H, J510.3 Hz, CH–
caused the precipitation of a yellowish, semi-solid mass, CH5N), 7.70 (m, 2 H, H aro), 7.25 (m, 2 H, CH–CH5N/
which was removed manually. The crude 3-(4-methoxy- H aro), 3.68 (s, 3 H, E–CH5N(CH
3) 2), 3.56 (s, 3 H,
phenyl) - 3 - phenylprop - 2 - ene - 1 - ylidene-N,N - dimethyl- Z–CH5N(CH
3) 2), 2.83 (s (b), 4 H, Ar(CH
2) 2(CH 2) 2),
21
iminium perchlorate (13b) could be hydrogenated without 1.80 (m, 4 H, Ar(CH
2) 2(CH 2) 2); IR [cm ]: 2920, 1650,
further purification (see description for 6b). The crude 1570, 1090. Catalytic hydrogenation of 16b (see cpd. 6a)
product base was distilled in vacuo. N,N-Dimethyl-3-(4- afforded N,N-dimethyl-3-(2-tetralyl)-propylamine hydromethoxyphenyl)-3-phenylpropylamine
hydrochloride (13)
1
chloride (16) in quantitative yield. 16: mp 1728C. H NMR

C.D.P. Klein et al. / European Journal of Pharmaceutical Sciences 14 (2001) 167 –175 173
(D2O, d ): 7.01–7.07 (m, 3 H, H aro), 3.13–3.18 (m, 2 H, lipophilicity and devised a new method for the calculation
CH
2–N(CH 3) 2), 2.92 (s, 6 H, N(CH
3) 2), 2.65–2.74 (m, 6 of log P values. We built analogs 7–11 with the di-
H, Ar(CH
2)(CH));CH 2 2 2 2–CH 2–CH 2–N), 1.99–2.09 (m, methylaminopropyl side chain in all-trans conformation,
2 H, CH
2–CH 2–CH 2–N), 1.76 (m, 4 H, performed a geometry optimization using the AM1
21
Ar(CH
2 )
2 (CH
2 )
2 ); IR [cm ]: 2910, 1250, 790.
semiempirical hamiltonian (MOPAC 6.0 (Stewart, 1990)),
17: Synthesized according to Marxer and Leutert determined their Connolly surface areas (probe radius 1.4
(1978). Å) (Cerius2, v.3.0) and correlated it to their log D7.4
(RP-HPLC) values. The results are shown in Fig. 3.
Although the statistical significance of a dataset with n55
is limited, we think that there is a clear dependency
3. Results and discussion between surface area and log P or log D
7.4. Bulky
compounds, such as 10 and 11, expose less of their
All experimental results are given in Table 1, along with aromatic, lipophilic surface areas than the elongated comthe
calculated log P values. Overall, the CLOGP program pounds (7, 8). This is reflected by higher log P/log D 7.4
performed quite well in predicting the relative lipo- values in the latter case. An attempt to correlate the surface
philicities of the compounds in the dataset.
area of the other compounds with their log D values failed.
Initially, we did not intend to determine any log P values This could be due to the different number of aromatic rings
by the shake-flask method, and wanted to rely solely on the in the compounds.
RP-HPLC method. However, the chromatographic log D7.4
We would now like to focus on the interaction of the
values for the isomeric compounds 7–11 were more analogs with phosphatidylserine monolayers. Over all,
different than we expected. Whereas the calculated log P there certainly is a dependency between the lipophilicity
values (CLOGP) vary from 3.93 to 4.38, the chromato- and the calcium-displacing potency of the compounds (see
graphic method gave log D7.4
values from 2.26 to 3.15. It Fig. 4). However, a more detailed consideration of the data
should be noted that molecular modeling programs which in Table 1 gives a picture that is less clear.
calculate log P values by means of an atom-based ap- The calcium-displacing potency, expressed as 2log
proach predicted an even smaller difference in lipo- IC
50, of the isomers 7–11 increases with lipophilicity, and
2
philicities than CLOGP. The majority of commercial one finds a good, linear correlation (r 0.94; log D7.4
from
modeling programs uses atom-based approaches for the RP-HPLC) for the two properties — for this data-subset.
calculation of log P values. Of course, some steric in- If, however, other compounds are considered, the relationfluence
could be expected, but we began to doubt the ship between lipophilicity and membrane interaction bevalidity
of the HPLC method and decided to perform comes much more vague. For example, compounds 7 and
‘classical’ shake-flask experiments for cpds. 7–11. Be- 16 are practically iso-lipophilic, whereas their potency to
21
cause the shake-flask technique can, due to experimental displace Ca -ions from phosphatidylserine monolayers
problems (formation of micelles, accumulation of the differs by more than an order of magnitude. The same
compounds at the octanol–water interface), not be per- observation, although less pronounced, can be made for
fomed with amphiphilic compounds (5surfactants), it was compounds 9 and 5. In both cases, the bi-aromatic analogs
necessary to use a strongly basic, aqueous buffer, in which (7, 9) are distinctly more active than the iso-lipophilic
the ionized, amphiphilic species is practically eliminated.
Therefore, a phosphate/hydrogen phosphate buffer was
used for the partitioning experiments. Not surprisingly,
compounds 7–11 are three orders of magnitude more
lipophilic (as determined by the shake-flask method) at pH
12.5 than at pH 7.4 (RP-HPLC). Nevertheless, the correlation
between shake-flask- and RP-HPLC-values is good,
and both methods give values for cpds. 7–11 that differ
(‘within’ each method) by about one order of magnitude.
What is the reason for the large variation of the log P
values for cpds. 7–11? It appeared obvious to determine
the molecular surface areas of these isomers and see if
there was any interrelation with the log P values. Correlations
between log P and surface area are not a new
concept. Yalkowski and Valvani (1976) used the solventaccessible
surface area of hydrocarbons to calculate their
lipophilicity. These studies were later extended by Camilleri
and colleagues (1988), who correlated the Connolly Fig. 3. Correlation of Connolly surface area versus log P for compounds
(1983) surface areas of benzene derivatives to their 7–11. A probe radius of 1.4 A˚
was used.

174 C.D.P. Klein et al. / European Journal of Pharmaceutical Sciences 14 (2001) 167 –175
45 21
Fig. 4. Correlation plot of 2log IC
50
(displacement of Ca from
phosphatidylserine monolayers) versus log D
7.4
of all compounds.
for the remaining dataset becomes 0.75. This observation
shows once more that small structural changes can have a
large influence on the interaction of solutes with phospholipid
membranes. The mechanistic background for the
strong activity of compound 14 can only be hypothesized.
One possible explanation could be that the phenolic moiety
of compound 14 represents an additional ‘binding partner’
for the monolayer/water interface, thereby increasing the
compound’s affinity.
The main conclusion that can be drawn from this work
is that the interaction of phospholipid assemblies with
solute molecules can have a remarkably high steric selectivity.
The effect observed here, i.e. displacement of
45 21
Ca , reflects binding of the cationic model amphiphilic
compounds. The octanol/water partition coefficient (log P)
of the solutes might be a useful parameter for the prediction
or explanation of general trends of binding affinity.
However, other factors appear to be involved and minor
steric variations may have a pronounced impact on the
adsorption, absorption, and penetration behavior of small
molecules.
alkyl-aromatic compounds (5, 16) This is probably due to
the rigidity of the biphenyl moieties, which aids their
‘intercalation’ between the fatty acid chains of the phospholipids.
The four compounds 2, 11, 15, and 17 have similar
lipophilicities (log D : 2.00–2.26). The calcium-displac-
Acknowledgements
7.4
ing potency of cpds. 2, 11, and 15 is about the same (2log
D. Heber and M. Klingmuller ¨
IC around 3.3), whereas 17 is distinctly more active
of the University of Kiel,
50
(2log IC : 3.98). Furthermore, the calcium-displacing
Pharmaceutical Institute, kindly provided cpds. 2, 3, and 4.
50
potency does not increase with lipophilicity. 17, the most
C.K. appreciates financial support by the Konrad-
active compound in this sub-group, is less lipophilic than
Adenauer-Foundation.
11 and 15. Compound 2, carrying a methyl group in the
4-position, is nearly iso-lipophilic with cpd. 17 that has a
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