Chem -B~oL Interactions, 75 (1990) 93--104
93
Elsevier Scientific Pubhshers Ireland Ltd.
LYSIS OF EGG PHOSPHATIDYLCHOLINE TRICYCLIC CARBOXAMIDE ANTITUMOR
VESICLES AGENTS
BY
CHARMIAN J O'CONNOR', DIANE P EMERY', HOLGER TANK', WILLIAM A DENNYb and JUNZO SUNAMOTO° "Department of Chemistry and bCancer Research Laboratory, School of Medw~ne, Unzvers~ty of Auckland, Pmvate Bag, Auckland (New Zealand) and ~Laboratory of Matemals Science of Polymers and Art~fw~al Cell Technology, Department of Polymer Chemistry, Kyoto University, Kyoto 606 (Japan)
(Received October 27th, 1989) (RevImonreceived February 5th, 1990) (Accepted February 5th, 1990)
SUMMARY T h e stability of small u n i l a m e l l a r vesicles f o r m e d by e g g phosphatidylcholine has b e e n e x a m i n e d in the p r e s e n c e of 38 tricyclic c a r b o x a m i d e D N A - i n t e r c a l a t i n g a g e n t s (19 p h e n y l q u i n o h n e s , 17 phenylbenzimidazoles, an acridine and an anthracene). L y s i s of the v e s i c u l a r m e m b r a n e is time-depend e n t and also d e p e n d e n t on the c o n c e n t r a t i o n of t h e cytotoxic agent. T h e r e l a t i v e c o n c e n t r a t i o n of a g e n t to cause a fixed d e g r e e of lysis in a fixed time, as m e a s u r e d by the r e l e a s e of e n c a p s u l a t e d 6-carboxyfluorescein, is directly r e l a t e d to the r e l a t i v e h y d r o p h o b i c i t y of t h e a g e n t s .
Key
w o r d s : Tricyclic c a r b o x a m i d e s -- P h e n y l q u i n o l i n e s
dazoles -
- PhenylbenzimlM e m b r a n e d a m a g e -- L i p o s o m e s -- P h o s p h a t i d y l c h o l i n e
INTRODUCTION T h e r e is m u c h i n t e r e s t in the use of liposomes as d r u g d e l i v e r y s y s t e m s , p a r t i c u l a r l y in c a n c e r c h e m o t h e r a p y w h e r e t r e a t m e n t is n e a r l y a l w a y s limited by toxic side-effects. D r u g encapsulation within a site-specific d r u g carrier, such as t u m o r cell t a r g e t e d liposomes, p e r m i t s s m a l l e r q u a n t i t m s to be a d m i n i s t e r e d , r e d u c i n g g e n e r a l side effects. S e v e r a l studies h a v e shown liposomal doxorubicin to be at l e a s t as effective as the free drug, but much less cardiotoxm in t u m o r - b e a r i n g r o d e n t s and dogs [1]. F o r d r u g s such as C y t a r a b i n e whmh are r e q u i r e d to be g i v e n f r e q u e n t l y , liposome encapsulation has b e e n s h o w n to p r o v i d e an effective slow-release f o r m of t h e d r u g [2]. 0009-2797/90/$03 50 © 1990 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland
94 Liposomes can also be used to target specific tissues or cell types [3,4]. Their ability to be sequestered by the liver is thought to be the reason why liposoreal doxorubicin has been shown in several studies to be significantly more effective than free drug against liver metastases of a variety of tumor types [5]. Hydrophobic drugs such as cytarabine esters can be distributed among the hydrocarbon bilayers or, alternatively, anchored by a long chain terminating in a polar headgroup. The tricyclic carboxamides are a new class of cationic DNA-intercalating agents which, like doxorubicin, appear to act as antitumor agents via inhibition of the function of the enzyme DNA topoisomerase II [6], but which lack the redox-cycling capabilities that are considered responsible for the cardiotoxic effects of the anthracyclines [7]. Interest in the class was sparked by the very high in vivo antitumor activity found [8] for certain compounds such as the fused linear tricyclic acridinecarboxamide (1). Later work [9--11] aimed at the preparation of less aromatic and therefore more weakly DNAbinding compounds focused on '2-1' chromophores of the 2-phenylquinoline (e.g., 3) and 2-phenylbenzimidazole (e.g., 22) types. These compounds were shown to bind weakly to DNA by intercalation, while retaining good in vivo antitumor activity.
CONH(CH2)2N(CH3)2 1
C
2
0
~
CONH(CH2)2N(CH3)2
OH COCH20H
H
39
~
"°'OH
NH2
40
95 We were interested in studying the effects of liposome encapsulation on the biological activity of these compounds, but attempts [12] to encapsulate the acridine derivative (1) into large unilamellar egg phosphatidylcholine vesicles (LUVs) by the reverse phase encapsulation method failed, with only approx. 0.2o/o of the drug being incorporated, despite its very high watersolubility (17 mg/ml) for the monohydrochloride. Further studies [12] showed that addition of (1) or the anthracene (2) (also a DNA-intercalating agent [11]) to small unilamellar vesicles (SUVs) containing carboxyfluorescein (CF) incorporated into the encapsulated aqueous phase as a hydrophilic fluorescent probe caused a concentration- and time-dependent release of fluorescent label, indicating that these drugs cause lysis of the liposomal membrane. The methodology for using liposomes as drug carrier systems has been relatively well-established, as have their physicochemical properties and fate in biological systems [13], and drugs which induce damage of the liposomal membrane have been shown to be unsuitable for encapsulation. In order to explore further the possibility of encapsulating the tricyclic carboxamides, a study was undertaken of the relative abilities of a series of analogues to lyse egg phosphatidylcholine SUVs, and these results are reported here. MATERIALS AND METHODS
Materials
Syntheses and characterization of all the tricychc carboxamides used have been reported [6--9]. Two fused tricyclic compounds were studied, the acridine (1) and the anthracene (2). However, most of the compounds were '2-1' tricyclics, 19 2-phenylquinolines (3--21) and 17 2-phenylbenzimidazoles (22-38) (Tables I and II). In each series, the compounds were selected to provide the largest possible range of lipophilic and electronic properties. Egg phosphatidylcholine was prepared and formulated into small unilamellar vesicles containing 6-carboxyfluorescein (CF) by published methods [12,14,15]. Fluorescence spectra were measured with a Shimadzu RF540 spectrofluorophotometer. Me thods
The CF-containlng liposomes were purified by gel chromatography, using a 2 × 40 cm column of Sepharose 4B. The column was equilibrated with a buffer of tris (20 raM) and NaC1 (200 rev./mm), adjusted to pH 8.6 with HC1, and the liposome suspension was then chromatographed in the same buffer. Aliquots of 3.6 ml were collected, and monitored by absorption at 360 nm. The approprlate aliquots were pooled, and the concentration of egg phosphatidylcholine was determined by phosphate analysis [16]. The volume was then adjusted to bring the concentration to 0.1 mM phosphatidylcholine. Fluorescence cuvettes containing the liposomal suspension (3 ml) were preincubated in the spectrofluorimeter for 10--15 rain at 36.5°C, and an initial reading of fluorescence intensity,/o, was recorded at an emission wavelength
96
100
= ~. = = . . . . .
~. . . . . .
J
80-
3.0x10-3M
1.0xlO-3M 80xlOJ'M
//
~60-
"6 o~ ~o
6 0x10-%
30xlO-"M 2O
t O0 i 5
I0
15
20
25
30
t i m e (rain) Fig 1. Time-dependent plots for percent release of 6-carboxyfluorescem (CF) from small umlamellar vesmles (phosphatldylchohne 0.1 mM) at 36.5°C m the presence of varying concentrahons of the phenylbenmmldazole 22.
of 520 nm and excitation w a v e l e n g t h of 470 nm. A d r u g solution of known concentration was t h e n injected into the c u v e t t e , and fluorescence intensity was m o n i t o r e d for an a p p r o p r i a t e period, to give plots of the t y p e shown in Fig. 1. F r o m t h e s e plots, a s t a n d a r d time of 5 min was selected for use in assays to q u a n t i t a t e the r e l a t i v e ability of the compounds to effect lysis. The percentage of CF r e l e a s e d a f t e r 5 min was calculated from the e x p r e s s i o n measured
% CF released
= [(/t -
/o)/(/of -
/o)] x
100
w h e r e Inf was m e a s u r e d following addition of the s u r f a c t a n t T r i t o n X-100 (40 ~l, 10O/o v/v), which is known [15] to d e s t r o y completely liposomal membranes. In m a n y cases the fluorescent p r o p e r t i e s of the drugs i n t e r f e r e d with the fluorescence of the r e l e a s e d carboxyfluorescein probe, and it was
97 n e c e s s a r y to c o r r e c t for this by dividing t h e / f value d e t e r m i n e d above by t h e / [ value m e a s u r e d s e p a r a t e l y in the absence of drug. C o r r e c t e d values w e r e t h e n given by the e x p r e s s i o n c o r r e c t e d % CF r e l e a s e d = /nf
m e a s u r e d % CF released x
(absence of drug)
/,nf (presence of drug)
and w e r e used for all studies. Full c o n c e n t r a t i o n / r e l e a s e profiles w e r e determined for all compounds, and r e p r e s e n t a t i v e plots are shown in Fig. 2. RESULTS AND DISCUSSION The concentrations of d r u g r e q u i r e d to release both 10% and 500/0 of the CF from the vesicles w e r e d e t e r m i n e d from the concentration/CF release plots (Fig. 2), and t h e s e are r e c o r d e d in Tables I and II as log(1/(lysis%)) values. E i g h t of the compounds (17--21 and 36--38) w e r e not able to achieve
100 m
, - 80 O 0
60
° ~0 u~
/
,
L--
LL- 20 (..)
0
~
~. ~
i 7 a 94n_~
'
2
j
4
s i 7 ~9
IU
Edrug]
10-3
2
3
4
(M)
Fig. 2 Concentration-dependent plots for percent release of 6-carboxfluorescem (CF) after 5 mm from small undamellar vesmles (phosphahdylchohne 0.1 raM) at 36.5°C m the presence of the phenylbenzimidazoles 22 (A), 23 (e), 24 (ram);25 ( . )
98 TABLE I 2-PHENYLQUINOLINE-4-CARBOXAMIDES
R ~ N H ( C H 2 ) 2 N ( C H 3 ) 2 No. R 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
1og(1/50%)~
log(I/10%)b
4'-OH 4'-OCH3 4'-F 4'-I 4'-C1 4'-Br 4'-CF3 4'-C8H5 3',4'-benz 3'-C1 H: f H:~
4.00 3 85 4.25 4.17 4.60 4.64 4.80 4.70 5.42 5.04 4 60 374 3.89
4.49 4.41 4.89 4 68 5.18 5.18 5 22 5.10 6.00 5.55 524 436 4.46
- 0.01 - 0.33 - 0 16 - 0 04 0.13 0 05 0 15 0.15 0.33 0.10 0.05 -0.05 0 06
H: h
396
448
-003
3 30 3 00 2.52 2.40 230
- 0 38 - 0.58 - 0 46 - 0.74 - 0 87
H
4'-NHSO2CH 3 3'-aza 4'-NHCOCH 8 4'-S02CH 3 4'-aza
R~
r~
oa
0 - 0.67 - 0.02 0.14 1.12 0.71 0 86 0.88 1.96
0 - 0.37 - 0 27 0.06 0.18 0 23 0.23 0 54 - 0 01
0 71
0 37
1 18 11 0 97 1 03 11
0.03 0 71 00 0 72 0 78
-
•Log of the reverse of the concentration (M) of drug to achmve 50% release of CF from vesicles after 5 mm at 35 5°C bSlmllar value for 10% lyres ~Rmvalues are taken from Refs. 8-- 10 de~ and o values taken from Ref 17 f-- CONH(CH~)zN(CH3)2 sldechaln - CONH(CH2)2NH(CHz)20H sldecham. h_CONH(CHe)2 N 0 X__/
sldeeham
50% lysis of the vesicles at any concentration, lines and benzimidazoles, trations
there
but for the remaining
was a good correlation
between
quino-
the concen-
n e e d e d t o a t t a i n 1 0 % a n d 5 0 % l y s i s ( E q n . 1).
l o g ( 1 / 5 0 % ) = 0.93 l o g ( 1 / 1 0 % ) n = 28
r = 0.93
s = 0.22
(1)
0.15 F1,26 =
170
P < 0.0001
Structure-activity relationships were then studied tuted phenylquinolines ( 3 - - 1 1 , 13 a n d 17--21),
for the where
set of 4'-substiboth Hammett
99 TABLE II 2-PHENYLBENZIMIDAZOLE-4-CARBOXAMIDES 3"
2"
CONH(CH2)2N(CH3)2 No R
log(1/50%~
1og(1/10%)b
R~
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
3.24 3.23 3.55 4.07 3.21 3.37 3.37 3.43 3.74 4.17 3.54 3.72 4 11 4.68
354 3.80 4 21 4.51 3 80 3 85 3 85 3.96 4.26 4.66 4.04 4 22 4.49 4.12 2.05 2 10 2.30
-026 - 0.42 - 0.18 - 0.08 - 0 40 - 0.20 - 0.14 - 0 31 - 0.10 - 0 01 - 0.23 - 0 14 - 0.11 - 0 27 - 0.95 - 0.98 - 1.32
H 4'-0CH 3 4'-CH 8 4'-C1 2'-0CH 8 2'-CH 8 2'-Cl 3'-0CH s 3'-CH 8 3'-Cl 7'-CH 8 6'-CH 3 2',3'-benz 3',4'-benz 4'-aza 3'-aza 2'-aza
"~See Table I footnotes
electronic and Hansch lipophilic parameters are available for the substituent g r o u p s [17]. F o r t h e s e 1 5 c o m p o u n d s t h e r e w a s a g o o d c o r r e l a t i o n b e t w e e n l o g 1/(10O/o l y s i s ) a n d t h e l i p o p h i l i c i t y p a r a m e t e r ~ ( E q n . 2), w i t h t h e m o r e lipophilic compounds being more efficient lysing agents. l o g ( I / 1 0 % ) = 1.08 n + 4 . 8 4 n = 15 r = 0.93 s = 0.46 In contrast, there was a much e l e c t r o n i c p r o p e r t i e s ( E q n . 3).
(2) F1,13 = 6 8 poorer
P
93
Elsevier Scientific Pubhshers Ireland Ltd.
LYSIS OF EGG PHOSPHATIDYLCHOLINE TRICYCLIC CARBOXAMIDE ANTITUMOR
VESICLES AGENTS
BY
CHARMIAN J O'CONNOR', DIANE P EMERY', HOLGER TANK', WILLIAM A DENNYb and JUNZO SUNAMOTO° "Department of Chemistry and bCancer Research Laboratory, School of Medw~ne, Unzvers~ty of Auckland, Pmvate Bag, Auckland (New Zealand) and ~Laboratory of Matemals Science of Polymers and Art~fw~al Cell Technology, Department of Polymer Chemistry, Kyoto University, Kyoto 606 (Japan)
(Received October 27th, 1989) (RevImonreceived February 5th, 1990) (Accepted February 5th, 1990)
SUMMARY T h e stability of small u n i l a m e l l a r vesicles f o r m e d by e g g phosphatidylcholine has b e e n e x a m i n e d in the p r e s e n c e of 38 tricyclic c a r b o x a m i d e D N A - i n t e r c a l a t i n g a g e n t s (19 p h e n y l q u i n o h n e s , 17 phenylbenzimidazoles, an acridine and an anthracene). L y s i s of the v e s i c u l a r m e m b r a n e is time-depend e n t and also d e p e n d e n t on the c o n c e n t r a t i o n of t h e cytotoxic agent. T h e r e l a t i v e c o n c e n t r a t i o n of a g e n t to cause a fixed d e g r e e of lysis in a fixed time, as m e a s u r e d by the r e l e a s e of e n c a p s u l a t e d 6-carboxyfluorescein, is directly r e l a t e d to the r e l a t i v e h y d r o p h o b i c i t y of t h e a g e n t s .
Key
w o r d s : Tricyclic c a r b o x a m i d e s -- P h e n y l q u i n o l i n e s
dazoles -
- PhenylbenzimlM e m b r a n e d a m a g e -- L i p o s o m e s -- P h o s p h a t i d y l c h o l i n e
INTRODUCTION T h e r e is m u c h i n t e r e s t in the use of liposomes as d r u g d e l i v e r y s y s t e m s , p a r t i c u l a r l y in c a n c e r c h e m o t h e r a p y w h e r e t r e a t m e n t is n e a r l y a l w a y s limited by toxic side-effects. D r u g encapsulation within a site-specific d r u g carrier, such as t u m o r cell t a r g e t e d liposomes, p e r m i t s s m a l l e r q u a n t i t m s to be a d m i n i s t e r e d , r e d u c i n g g e n e r a l side effects. S e v e r a l studies h a v e shown liposomal doxorubicin to be at l e a s t as effective as the free drug, but much less cardiotoxm in t u m o r - b e a r i n g r o d e n t s and dogs [1]. F o r d r u g s such as C y t a r a b i n e whmh are r e q u i r e d to be g i v e n f r e q u e n t l y , liposome encapsulation has b e e n s h o w n to p r o v i d e an effective slow-release f o r m of t h e d r u g [2]. 0009-2797/90/$03 50 © 1990 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland
94 Liposomes can also be used to target specific tissues or cell types [3,4]. Their ability to be sequestered by the liver is thought to be the reason why liposoreal doxorubicin has been shown in several studies to be significantly more effective than free drug against liver metastases of a variety of tumor types [5]. Hydrophobic drugs such as cytarabine esters can be distributed among the hydrocarbon bilayers or, alternatively, anchored by a long chain terminating in a polar headgroup. The tricyclic carboxamides are a new class of cationic DNA-intercalating agents which, like doxorubicin, appear to act as antitumor agents via inhibition of the function of the enzyme DNA topoisomerase II [6], but which lack the redox-cycling capabilities that are considered responsible for the cardiotoxic effects of the anthracyclines [7]. Interest in the class was sparked by the very high in vivo antitumor activity found [8] for certain compounds such as the fused linear tricyclic acridinecarboxamide (1). Later work [9--11] aimed at the preparation of less aromatic and therefore more weakly DNAbinding compounds focused on '2-1' chromophores of the 2-phenylquinoline (e.g., 3) and 2-phenylbenzimidazole (e.g., 22) types. These compounds were shown to bind weakly to DNA by intercalation, while retaining good in vivo antitumor activity.
CONH(CH2)2N(CH3)2 1
C
2
0
~
CONH(CH2)2N(CH3)2
OH COCH20H
H
39
~
"°'OH
NH2
40
95 We were interested in studying the effects of liposome encapsulation on the biological activity of these compounds, but attempts [12] to encapsulate the acridine derivative (1) into large unilamellar egg phosphatidylcholine vesicles (LUVs) by the reverse phase encapsulation method failed, with only approx. 0.2o/o of the drug being incorporated, despite its very high watersolubility (17 mg/ml) for the monohydrochloride. Further studies [12] showed that addition of (1) or the anthracene (2) (also a DNA-intercalating agent [11]) to small unilamellar vesicles (SUVs) containing carboxyfluorescein (CF) incorporated into the encapsulated aqueous phase as a hydrophilic fluorescent probe caused a concentration- and time-dependent release of fluorescent label, indicating that these drugs cause lysis of the liposomal membrane. The methodology for using liposomes as drug carrier systems has been relatively well-established, as have their physicochemical properties and fate in biological systems [13], and drugs which induce damage of the liposomal membrane have been shown to be unsuitable for encapsulation. In order to explore further the possibility of encapsulating the tricyclic carboxamides, a study was undertaken of the relative abilities of a series of analogues to lyse egg phosphatidylcholine SUVs, and these results are reported here. MATERIALS AND METHODS
Materials
Syntheses and characterization of all the tricychc carboxamides used have been reported [6--9]. Two fused tricyclic compounds were studied, the acridine (1) and the anthracene (2). However, most of the compounds were '2-1' tricyclics, 19 2-phenylquinolines (3--21) and 17 2-phenylbenzimidazoles (22-38) (Tables I and II). In each series, the compounds were selected to provide the largest possible range of lipophilic and electronic properties. Egg phosphatidylcholine was prepared and formulated into small unilamellar vesicles containing 6-carboxyfluorescein (CF) by published methods [12,14,15]. Fluorescence spectra were measured with a Shimadzu RF540 spectrofluorophotometer. Me thods
The CF-containlng liposomes were purified by gel chromatography, using a 2 × 40 cm column of Sepharose 4B. The column was equilibrated with a buffer of tris (20 raM) and NaC1 (200 rev./mm), adjusted to pH 8.6 with HC1, and the liposome suspension was then chromatographed in the same buffer. Aliquots of 3.6 ml were collected, and monitored by absorption at 360 nm. The approprlate aliquots were pooled, and the concentration of egg phosphatidylcholine was determined by phosphate analysis [16]. The volume was then adjusted to bring the concentration to 0.1 mM phosphatidylcholine. Fluorescence cuvettes containing the liposomal suspension (3 ml) were preincubated in the spectrofluorimeter for 10--15 rain at 36.5°C, and an initial reading of fluorescence intensity,/o, was recorded at an emission wavelength
96
100
= ~. = = . . . . .
~. . . . . .
J
80-
3.0x10-3M
1.0xlO-3M 80xlOJ'M
//
~60-
"6 o~ ~o
6 0x10-%
30xlO-"M 2O
t O0 i 5
I0
15
20
25
30
t i m e (rain) Fig 1. Time-dependent plots for percent release of 6-carboxyfluorescem (CF) from small umlamellar vesmles (phosphatldylchohne 0.1 mM) at 36.5°C m the presence of varying concentrahons of the phenylbenmmldazole 22.
of 520 nm and excitation w a v e l e n g t h of 470 nm. A d r u g solution of known concentration was t h e n injected into the c u v e t t e , and fluorescence intensity was m o n i t o r e d for an a p p r o p r i a t e period, to give plots of the t y p e shown in Fig. 1. F r o m t h e s e plots, a s t a n d a r d time of 5 min was selected for use in assays to q u a n t i t a t e the r e l a t i v e ability of the compounds to effect lysis. The percentage of CF r e l e a s e d a f t e r 5 min was calculated from the e x p r e s s i o n measured
% CF released
= [(/t -
/o)/(/of -
/o)] x
100
w h e r e Inf was m e a s u r e d following addition of the s u r f a c t a n t T r i t o n X-100 (40 ~l, 10O/o v/v), which is known [15] to d e s t r o y completely liposomal membranes. In m a n y cases the fluorescent p r o p e r t i e s of the drugs i n t e r f e r e d with the fluorescence of the r e l e a s e d carboxyfluorescein probe, and it was
97 n e c e s s a r y to c o r r e c t for this by dividing t h e / f value d e t e r m i n e d above by t h e / [ value m e a s u r e d s e p a r a t e l y in the absence of drug. C o r r e c t e d values w e r e t h e n given by the e x p r e s s i o n c o r r e c t e d % CF r e l e a s e d = /nf
m e a s u r e d % CF released x
(absence of drug)
/,nf (presence of drug)
and w e r e used for all studies. Full c o n c e n t r a t i o n / r e l e a s e profiles w e r e determined for all compounds, and r e p r e s e n t a t i v e plots are shown in Fig. 2. RESULTS AND DISCUSSION The concentrations of d r u g r e q u i r e d to release both 10% and 500/0 of the CF from the vesicles w e r e d e t e r m i n e d from the concentration/CF release plots (Fig. 2), and t h e s e are r e c o r d e d in Tables I and II as log(1/(lysis%)) values. E i g h t of the compounds (17--21 and 36--38) w e r e not able to achieve
100 m
, - 80 O 0
60
° ~0 u~
/
,
L--
LL- 20 (..)
0
~
~. ~
i 7 a 94n_~
'
2
j
4
s i 7 ~9
IU
Edrug]
10-3
2
3
4
(M)
Fig. 2 Concentration-dependent plots for percent release of 6-carboxfluorescem (CF) after 5 mm from small undamellar vesmles (phosphahdylchohne 0.1 raM) at 36.5°C m the presence of the phenylbenzimidazoles 22 (A), 23 (e), 24 (ram);25 ( . )
98 TABLE I 2-PHENYLQUINOLINE-4-CARBOXAMIDES
R ~ N H ( C H 2 ) 2 N ( C H 3 ) 2 No. R 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
1og(1/50%)~
log(I/10%)b
4'-OH 4'-OCH3 4'-F 4'-I 4'-C1 4'-Br 4'-CF3 4'-C8H5 3',4'-benz 3'-C1 H: f H:~
4.00 3 85 4.25 4.17 4.60 4.64 4.80 4.70 5.42 5.04 4 60 374 3.89
4.49 4.41 4.89 4 68 5.18 5.18 5 22 5.10 6.00 5.55 524 436 4.46
- 0.01 - 0.33 - 0 16 - 0 04 0.13 0 05 0 15 0.15 0.33 0.10 0.05 -0.05 0 06
H: h
396
448
-003
3 30 3 00 2.52 2.40 230
- 0 38 - 0.58 - 0 46 - 0.74 - 0 87
H
4'-NHSO2CH 3 3'-aza 4'-NHCOCH 8 4'-S02CH 3 4'-aza
R~
r~
oa
0 - 0.67 - 0.02 0.14 1.12 0.71 0 86 0.88 1.96
0 - 0.37 - 0 27 0.06 0.18 0 23 0.23 0 54 - 0 01
0 71
0 37
1 18 11 0 97 1 03 11
0.03 0 71 00 0 72 0 78
-
•Log of the reverse of the concentration (M) of drug to achmve 50% release of CF from vesicles after 5 mm at 35 5°C bSlmllar value for 10% lyres ~Rmvalues are taken from Refs. 8-- 10 de~ and o values taken from Ref 17 f-- CONH(CH~)zN(CH3)2 sldechaln - CONH(CH2)2NH(CHz)20H sldecham. h_CONH(CHe)2 N 0 X__/
sldeeham
50% lysis of the vesicles at any concentration, lines and benzimidazoles, trations
there
but for the remaining
was a good correlation
between
quino-
the concen-
n e e d e d t o a t t a i n 1 0 % a n d 5 0 % l y s i s ( E q n . 1).
l o g ( 1 / 5 0 % ) = 0.93 l o g ( 1 / 1 0 % ) n = 28
r = 0.93
s = 0.22
(1)
0.15 F1,26 =
170
P < 0.0001
Structure-activity relationships were then studied tuted phenylquinolines ( 3 - - 1 1 , 13 a n d 17--21),
for the where
set of 4'-substiboth Hammett
99 TABLE II 2-PHENYLBENZIMIDAZOLE-4-CARBOXAMIDES 3"
2"
CONH(CH2)2N(CH3)2 No R
log(1/50%~
1og(1/10%)b
R~
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
3.24 3.23 3.55 4.07 3.21 3.37 3.37 3.43 3.74 4.17 3.54 3.72 4 11 4.68
354 3.80 4 21 4.51 3 80 3 85 3 85 3.96 4.26 4.66 4.04 4 22 4.49 4.12 2.05 2 10 2.30
-026 - 0.42 - 0.18 - 0.08 - 0 40 - 0.20 - 0.14 - 0 31 - 0.10 - 0 01 - 0.23 - 0 14 - 0.11 - 0 27 - 0.95 - 0.98 - 1.32
H 4'-0CH 3 4'-CH 8 4'-C1 2'-0CH 8 2'-CH 8 2'-Cl 3'-0CH s 3'-CH 8 3'-Cl 7'-CH 8 6'-CH 3 2',3'-benz 3',4'-benz 4'-aza 3'-aza 2'-aza
"~See Table I footnotes
electronic and Hansch lipophilic parameters are available for the substituent g r o u p s [17]. F o r t h e s e 1 5 c o m p o u n d s t h e r e w a s a g o o d c o r r e l a t i o n b e t w e e n l o g 1/(10O/o l y s i s ) a n d t h e l i p o p h i l i c i t y p a r a m e t e r ~ ( E q n . 2), w i t h t h e m o r e lipophilic compounds being more efficient lysing agents. l o g ( I / 1 0 % ) = 1.08 n + 4 . 8 4 n = 15 r = 0.93 s = 0.46 In contrast, there was a much e l e c t r o n i c p r o p e r t i e s ( E q n . 3).
(2) F1,13 = 6 8 poorer
P
