quantitative structure–activity and structure–toxicity relationships of 4-aminodiphenyl sulphone...

Quantitative Structure±Activity and Structure±ToxicityRelationships of 4-Aminodiphenyl Sulphone Derivatives withAntiin¯ammatory Activity

Joachim K. Seydel1*, Hanna BuÈrger1, Anil K. Saxena1**, Michael D. Coleman2, Stephen N. Smith2 and Alan D. Perris2

1Borstel Research Center, Center for Medicine and Biosciences, D-23845 Borstel, Germany2Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK

Dedicated to Prof. Corwin Hansch on the occasion of his 80th birthday

Abstract

It has been shown that dapsone and its derivatives are not

only potential inhibitors of bacterial and plasmodial folate

synthesis but also possess antiin¯ammatory activity. The

application of dapsone is, however, limited by its toxic side

effects at higher dose manifested especially by its potential

to produce methaemoglobin. We have analyzed the

structural dependence of this property on a set of 29

derivatives. A highly signi®cant nonlinear dependence on

the lipophilicity could be derived. Two exceptions to the

general relation were found and the mechanism for these

could be derived. It was also shown that the bilinear

dependence is not due to lipophilicity mediated diffusion

into erythrocytes. It was found that the diffusion is linearly

dependent on lipophilicity. Principal Component (PC)-

Analysis revealed that the antiin¯ammatory activities,

determined as inhibition of zymosane stimulated cell burst

and cell adhesion are orthogonal to methaemoglobin

formation and cell death potential of the studied derivatives.

The antiin¯ammatory activity seems to depend on elec-

tronic and steric effects of the 2 0-substituents. The ortho-

gonality of substituent in¯uences on the toxic and wanted

effects opens up the possibility to further optimize the

selectivity of aminodiphenylsulphones.

Quant. Struct.-Act. Relat., 18 (1999) # WILEY-VCH Verlag GmbH, D-69469 Weinheim 0931-8771/99/0510-0043 $17.50+.50/0 43

Quantitative Structure±Activity and Structure±Toxicity Relationships QSAR

1 Introduction

4-Aminodiphenyl sulphones and especially 4,4 0-Diamino-

diphenylsulphone (Dapsone) are known and used in the

therapy of bacterial infections, malaria, pneumocystis

carinii and toxoplasmosis. They act as inhibitors of

dihydropteroic acid synthase in the de novo synthesis of

folate [1±3] and combined with dihydrofolate reductase

inhibitors such as trimethoprim (TMP) lead to synergistic

effects in the inhibition of parasitic growth [4]. Dapsone is

the drug of choice for the treatment of dermatitis

herpetiformis and related in¯ammatory conditions which

are characterized by tissue neutrophil in®ltration [5, 6].

Dapsone inhibits neutrophil adhesion, chemotaxis, lipoxy-

genase activity and the ability of cells to generate oxidative

species [7±11].

Unfortunately the applications of dapsone are dose-limited

due to its hepatic phase I activation to haemotologically

toxic hydroxylamines [12, 13].

Clinically, dermatitis herpetiformis patient responses vary

and dosages can range from 50±300 mg dayÿ1 [14]. At the

higher dose, hydroxylamine metabolites of dapsone can

cause signi®cant methaemoglobinaemia and haemolysis

[14, 15]. Short-term efforts to reduce dapsone toxicity and

improve patients' tolerance involving co-administration of

cimetidine have been moderately successful [12, 14].

* To receive all correspondence

** A. v. Humboldt-Fellow from the Central Drug Research Institute,

Lucknow, India

Key words: 2 04 0-substituted 4-aminodiphenyl sulphones, anti-

in¯ammatory activity, cell burst, cell adhesion, methaemoglobin

formation, diffusion into erythrocytes, PC-and multiple regression

analysis.

Abbreviations: TMP, trimethoprim; DDS, dapsone; RBC, red

blood cell; PBS, phosphate buffer salin; QDDS, effect of drug at

10 mM drug/effect of DDS at 10 mM; Derythr., partitioning of drug

into erythrocytes; cellbr, inhibition of neutrophil zymosan-

mediated respiratory burst; celladh, inhibition of neutrophil

interleukin-1 stimulated cell adhesion.

However, a number of potentially less toxic sulphones

which display increased antibacterial activity have been

synthesized in our laboratories [3, 16].

QSAR-studies to explain the observed variation in anti-

bacterial activity have led to the conclusion that electronic

and steric substituent effects are decisive for this activity

and that sulphones and sulfonamides bind to the same

receptor site [2, 3, 17]. Preliminary animal and in vitro

studies [16, 18] have indicated that a number of these

analogues are far less toxic than dapsone. In consequence a

series of these dapsone analogues have been toxicologically

tested using human and rat tissues and their inhibitory

effects on neutrophil function determined in vitro [19, 20].

The aim of this study was to derive quantitative structure±

toxicity and structure±activity relationships which could

allow further optimization of the antiin¯ammatory activity

and selectivity of sulphones.

2 Material and Methods

Chemicals

The synthesis of the studied dapsone analogues has been

described in detail [16]. Dapsone was obtained from

Aldrich (Poole, UK). The compounds structures are listed

in Table 1.

Experimental

Methaemoglobin Formation

a) 1-Compartment System

The ®nal reaction mixture contained: 1 ml erythrocyte

suspension, 0.25 ml NADPH; 0.2 ml 0.3 M nicotinamide;

0.3 ml 0.1 M MgCl2; 0.1 ml 5% glucose; 2.05 ml phosphate

buffer (pH 7.4); 0.1 ml ethanolyH2O solution of the

sulphone derivative; 1 ml of rat liver microsome prepara-

tion. The reaction mixture was incubated for 2 hrs at 37�C,

then placed in ice water and methaemoglobin readings were

taken according to Havemann and BuÈrger [18, 21]. Read-

ings were taken with an Eppendorf photometer at 650 nm.

As a measure of methaemoglobin formation the percent

methaemoglobin produced by 10 mM of the drug is de®ned

[18]. In a later paper in another laboratory the following

2-compartment system has been used [19].

b) 2-Compartment System

Rat (male Sprague-Dawley) microsomal preparations were

prepared according to the method of [22]. The assay

involved a two-compartment in vitro system which consists

of two Te¯on compartments separated by a cellulose

(molecular weight cut-off 5000 Da) membrane [23]. Com-

partment A contained 2 mg rat liver microsomes, 1 mM

NADPH and 5 ml DMSO solution of a test analogue.(®nal

concentration 100 mM). This concentration was applied in

previous analogue studies [24]. Vehicle controls contained

1% DMSO and buffer and microsomal controls contained

all the above except for NADPH. Compartment B contained

500 mL washed (50% haematocrit) human erythrocytes.

Incubations were carried out in triplicate. In all cases, no

methaemoglobin was generated in NADPH-free incubations

prior to determination of methaemoglobin formation using

an IL-482 CO-oximeter.

Partitioning into Erythrocytes

The partition of the compounds in the system erythrocyte

(RBC's)ybuffer was determined in RBC suspensions in pH

7.4 phosphate buffer at room temperature (21�C) essentially

according to our previously reported method [25] except

that the concentration of free drug before and after

equilibration was determined in the buffer phase by HPLC

instead of using the Bratton and Marshall photometric

method. The partition coef®cient D is de®ned:

D � ��AT�y�AB�� 1yH� ÿ 1yH� 1

H� hematocrit; VB true� true volume of buffer; VT true �true volume of whole blood; VRBC true� true volume of red

Table 1. List of compounds used in derivation of structure±toxicity and structure±activity relationships of 2 0,4 0 substituted 4-aminodiphenylsulphones

Nr Substitution Nr. Substitution

1 (1±7) 4 0-amino (dapsone) 24 (2±4,6) 2 0-OCH3; 4 0-NHC2H5

2 (1,4,5) 4 0-H 25 (4) 2 0-OCH3; 4 0-NHC3H7

3 (1,4,5) 4 0-NHCH3 26 (2±4,7) 3 0-OCH3; 4 0-NHC2H5

4 (4) 4 0-NHC2H5 27 (4) 3 0-OCH3; 4 0-NHC3H7

5 (2,3) 4 0-N(CH3)2 28 (4) 2 0-CH3; 4 0-NH2

6 (1±3) 4 0-NHCH2CH2OH 29 (2,3,7) 2 0-CH3; 4 0-NHCH3

7 (1±3) 4 0-NHCH2COOH 30 (2±4,7) 2 0-CH3; 4 0-NHC2H5

8 (1,4,5) 4 0-NHCH2CONHNH2 31 (3, 4) 2 0-CH3; 4 0-NHC3H7

9 (1,4,5) 4 0-OH 32 (1±3,6) 2 0-CH3, 4 0-NHC5H9

10 (1,4,5) 4 0-OCH3 33 (2,3,6) 2 0-CH3; 4 0-NHC6H13

11 (1,4,5) 4-CH3 34 (2,3,7) 2 0-CH3; 4 0-N(CH3)2

12 (1,5) 4 0-COOH 35 (1±3,6) 2 0-CH2OH; 4 0-NHC2H5

13 4 0-COOCH3 36 (1±3,6) 2 0-CF3; 4 0-NH2

13a 4 0-COOC3H7

14 (1±5) 4 0-CONHNH2 37 (1±3,6,7) 2 0-CF3; 4 0-NHC2H5

15 (1) 4 0-NO2 38 (2,3,7) 2 0-COCH3;4 0-NH2

16 (1) 4 0-CL 39 (2,3,7) 2 0-CONH2; 4 0-NH2

17 (1,4,5) 4 0-Br 40 (2,3,7) 2 0-COOCH3; 4 0-NH2

18 (2,5,7) 2 0-NH2 4 0-H 41 (2,3,7) 2 0-CL; 4 0-NH2

19 (6) 2 0-NH2 ; 4 0-NH2 42 (2,3,7) 2 0-Br; 4 0-NH2

20 (2,3,7) 2 0-NHCH3; 4 0-NHC3H7 43 (2,3) 2 0-NO2; 4 0-NH2

21 (1±3,6) 2 0-OH; 4 0-NH2 44 (2,3,7) 2 0-CN; 4 0-NHC2H5

22 (2±4,7) 2 0-OH; 4 0-NHC2H5 45 4,4 0-CH3NH-

23 (2±4,6) 2 0-OH; 4 0-NHC3H7

The numbers in parentheses indicate the regression Eqs. where this

derivative is included.

QSAR Joachim K. Seydel, Hanna BuÈrger, Anil K. Saxena, Michael D. Coleman, Stephen N. Smith and Alan D. Perris

44 Quant. Struct.-Act. Relat., 18 (1999)

blood cells. H or VB trueyVT true� 1-H; AT� total amount in

the synthetic drug; AB� amount of drug in buffer.

Inhibition of Zymosan-mediated Human Neutrophil

Respiratory Burst

Whole blood was obtained by venepuncture from healthy

volunteers. The ability of neutrophils to generate a

respiratory burst was measured using whole blood, whereas

adhesion to endothelial cells was measured using puri®ed

neutrophil preparations. Neutrophil respiratory burst was

elicited by means of an opsonized zymosan preparation.

The respiratory burst was assessed by means of lucigenin-

enhanced chemiluminescence in a Bio-Orbit 1523 lumin-

ometer (Labtech International, Sussex, UK). Chemilumi-

nescence readings were taken in intervals. Before analysis

samples of puri®ed neutrophiles or whole blood were

incubated with the compounds (0.5, 1.0, or 1.5 mM) for

30 min at 37�C. For details see [20].

Inhibition of Interleukin-1-stimulated neutrophil adhesion

Isolation of neutrophils before measurement of adhesion

were performed according to Afford [26] with some

modi®cations. Neutrophil adherence studies were per-

formed using transformed human umbilical vein cells

(ECV 304) grown to con¯uence in 96-well plates. The

cells were then incubated in a 95 : 5% air-CO2 mixture at

37�C for 4 h during which time up-regulation of adhesion

molecules occurs on the endothelial cell surface. The

medium was then removed and puri®ed isolated neutrophils

suspension pretreated with dapsone or analogues; (100 mL)

were added. The neutrophils were left for 30 min to adhere,

then unadhered neutrophils were removed by washing with

PBS. The adherent neutrophils were then assessed by means

of the method of Junger [27]. The neutrophils were lyzed

with Triton X-100 in PBS and the liberated myeloperox-

idase measured using hydrogen peroxide (0.02%) as

substrate and O-dianisidine dihydrochloride (0.34 mM) as

chromogen at pH 5. Absorbance was measured at 405 nM.

The concentration range 0.5±1.5 mM was chosen on the

basis of in vitro pilot studies which showed complete

inhibition of both neutrophil responses by dapsone at 1 mM.

The twelve candidate analogues were compared with the

parent dapsone for their ability to inhibit adhesion and

respiratory burst completely in this concentration range.

All statistical analysis was performed by Student's t-test.

Where more than one comparison was made with data, the

Bonferroni correction was employed where the acceptable

level of signi®cance was reduced to 0.05yk (where k is the

number of tests) to compensate for the increased testing

likelihood of reaching P< 0.05 during multiple testing [28].

Physicochemical Parameters

HPLC-capacitor factors, in form of log k 0, determined on a

octanol coated column have been used as descriptors of

lipophilic substituent effects [3]. They are signi®cantly

correlated with p-values (r2 � 0:91; n � 25; F � 227;

Q2 � 0:89).

The van der Waals volume, VW, the parameters for molar

polarizability, MR, and s-Hammett values and Swain-

Lupton parameters used in this paper for substituents in

o- and p-position have been taken from compilations

[29, 30].

Correlation Analysis

Linear and nonlinear regression analysis as well as Principal

Component-Analysis were carried out using programmes

locally written.

3 Results and Discussion

Methaemoglobin formation in microsomal preparations of

rat liver

It is highly likely that the structural analogues of dapsone

under study in this report are also oxidized as the parent

drug is, to hydroxylamines, which in turn may be cytotoxic

as well as generators of methaemoglobin [12, 24]

Methaemoglobin formation is the most common side-effect

which limits dapsone's effectiveness in patients [15], and is

thus valuable in the study of sulphone analogue toxicity

estimations [23]. In general, sulphone-derived hydroxyl-

amines react with oxyhaemoglobin, which is thought to

result in the formation of both methaemoglobin as well as of

short-lived nitroso derivative [31].

From previous studies in vitro using a simple 1-compart-

ment test system and almost solely 4 0-substituted 4-

aminodiphenylsulphones, a bilinear correlation has been

observed between the logarithm of the % methaemoglobin

formation at a constant drug concentration of 10 mM divided

by the amount produced by dapsone (QDDS) and log k 0 ([18]

Eq. 1). The methaemoglobin production increases with

increasing lipophilicity up to an optimum log k 0 of about 1.6

and then levels of or decreases.

log QDDS�0:74��0:10� log k 0ÿ0:94��0:31� log�0:08 k 0�1�ÿ 0:48��0:13� �1�

n � 18 r2 � 0:92 s � 0:23 log k 0max � 1:63 F � 27:8

Three derivatives did not follow this general relationship

and were excluded from the regression (Eq. 1). These are

the two carbonic esters (Table 1, #13, 13a) and the 4,4 0


Quant. Struct.-Act. Relat., 18 (1999) 45

methylamino derivative (Table 1, # 45), the latter possesses

no primary amino group. It was found that the two carbonic

acid esters are rapidly hydrolyzed by the microsomal liver

preparation to the corresponding acids. After 15 minutes of

incubation no ester can be detected. In the methaemoglobin

assay the readings for methaemoglobin formation are

however, taken after 2 hrs. It is therefore not surprising

that a very low amount of methaemoglobin is produced by

these derivatives corresponding to the low amount formed

by the hydrophilic acid (#12). In experiments using

haemolyzed erythrocytes it was checked if permeation

problems are responsible for the low amount of methae-

moglobin formed by the carbonic acids. It was found that

there was no difference in methaemoglobin formation using

haemolyzed or intact erythrocytes so that permeation

problems are not responsible but the lipophilicity was

found to be low in accordance with the general correlation.

Detailed studies were also performed to ®nd the reason for

the low production of methaemoglobin by the 4,4 0-methylamino derivative. It was known that N-alkylated

derivatives can lead to methaemoglobin formation or can be

N-oxidized, respectively, [32, 33]. Also in our experiments

methaemoglobin formation did occur. At higher substrate

concentration, however, a reduction in the formation of

methaemoglobin was observed. (Figure 1). This could have

been due to the low solubility of the compound. Another

explanation could be substrate or product inhibition. To

evaluate this possibility three series of concentrations with

three different microsome dilutions, i.e. with different

substrateyreceptor ratios were incubated simmultaneously.

In case of substrate- or product autoinhibition a shift of the

maximum of methaemoglobin formation to lower substrate

concentration at reduced enzymeysubstrate ratio should be

observable. This was indeed seen (maximum at approxi-

mately 36, 20, 10, respectively, Figure 2). In a further

experiment we wanted to evaluate if the inhibition was

restricted to the metabolism of the 4,4 0-methylamino

derivative or if the presence of this derivative also inhibitedthe metabolism of other derivatives. Figure 3 shows the

dose-response curves for the metabolism (methaemoglobin

formation for DDS and 4,4 0-methylamino diphenylsulphone

alone and for the mixture of both. At higher dapsone

concentrations the methaemoglobin formation is inhibited

by the presence of the 4,4 0-methylamino derivative. This

could indicate that the metabolism of the primary amino

group of DDS and the metabolism of the secondary amino

group of the 4,4 0-methylamino derivative are catalyzed by

the same enzyme.

The two examples underline the power of QSAR to not only

detect structure activity relationships but also to ®nd

signi®cant exceptions to these relationships and to shed

light on the mechanism of action of the exceptions.

We have extended these studies on methaemoglobin

formation to a larger group with additional substitution in

Figure 1. Methaemoglobin production by DDS and 4,4 0-methyl-amino-diphenylsulphone at increasing drug concentrations (stan-dardized microsome test, 1-compartment system (BuÈrger 1990)).

Figure 2. Methaemoglobin production by 4,4 0-methylamino-diphenylsulphone at different microsome dilutions. Drug concen-trations as indicated on the ordinate. The microsome dilutions 1 : 2;1 : 6 and 1 : 12 correspond to a ®nal dilution of the microsomes inthe reaction mixture (rat liver weight) of 1 : 60; 1 : 30 and 1 : 10,respectively.

Figure 3. Concentration dependent methaemoglobin productionby DDS and 4,4 0-methylamino-diphenylsulphone alone and incombination. Ratio in the mixture of DDS plus 4,4 0-amino-diphenylsulphone is 1 : 1.



2-position. These derivatives are of interest with respect to

their toxic properties because of their increased antibacterial

and antiin¯ammatory activities [3, 19, 20]. The two

compartment system as described in the experimental

section was used to determine the % methaemoglobin

produced by 100 mM of the test substances.

A highly signi®cant correlation could be derived showing

again a bilinear dependence of the methaemoglobin

formation on log k 0 (Figure 4). The predictive power was

tested by two derivatives not included in the test set (Eq. 2),

derivatives #8 and # 31 (Table 1). The calculated and found

formation of methaemoglobin were 22.2% calc. 22.9% obs.

and 67.9% calc., 64.6% obs., respectively. The physico-

chemical descriptor used to derive quantitative structure-

toxicity relationships and the observed and calculated % of

methaemoglobin formation using the most signi®cant

regression equation derived are given in Table 2 (DDS

effect� 100%).

log %methaemoglobin � 0:528��0:094� log k 0

ÿ 0:608��0:088� log�0:391��0:29�k 0 � 1�� 1:765��0:117�

n � 27 r2 � 0:85 s � 0:12 F � 31:6 �2�

including #8 and #31:

log %methaemoglobin �0:528��0:089� log k 0 ÿ 0:608��0:084�

log�0:387��0:26�k 0 � 1� � 1:763��0:105�

n � 29 r2 � 0:86 s � 0:11 F � 37:4 �3�

The results are in excellent agreement with the previously

derived structure toxicity relationships of a different set of

sulphones using a simpler test system and lower drug

concentrations (Eq. 1) [18].

The k 0-values have been determined at the pH of the test

system so that the in¯uence of ionizable substituents like

22OH and 22COOH is considered.

In contrast, studies performed on some derivatives in vivo in

cats demonstrated that more lipophilic derivatives showed

less formation of methaemoglobin (Table 3). The same was

observed when serum albumin was added to the in vitro

experiments, indicating a competitive protecting function of

the albumin (Figure 5). The more lipophilic compounds are

more tightly bound to serum albumin.

To produce methaemoglobin in the erythrocytes, the

sulphones must partition into the erythrocytes; this is also

a necessary precondition for their antimalarial activity. To

evaluate if the partitioning might be the rate limiting step

we have studied the partitioning of a series of 20 sulphones

into erythrocytes at pH 7.4. The results are listed in Table 2

(log Derythrocytes). Regression analysis leads to a highly

signi®cant linear correlation with log k 0 (Eq.4), despite the

fact that the log k 0 values are exceeding the value of 1.6

found as optimum value for methaemoglobin production for

a similar data set. It supports the argument that competition

with protein binding is reponsible for the nonlinear relation

found for methaemoglobin formation and the observed

lower toxicity of the highly lipophilic derivatives in

experimental animal studies (Table 3) and in in vitro

studies where serum albumin has been added to the reaction

mixture (Fig. 5).

One referee suggested to ®t the data to a nonlinear function,

log Derythr.� log(A6k 0B� 1)�C which describes better

the highly hydrophilic derivative 12, but then derivatives 8,

26, 27 are deviating more from the general regression than

12 before in the linear ®t (Eq. 4, deviation was less than

2 sd).

Figure 4. Bilinear dependence of methaemoglobin production byvarious substituted 4-amino-diphenylsulphones (Table 1, 2) as afunction of lipophilicity (log k 0).

Figure 5. Methaemoglobin production by DDS and 4-aminoÿ4 0-ethylamino-diphenylsulphone in the absence -d- and in thepresence -s- of serum albumin (4gy100 mL). Time of incubation2.5 h, dilution of microsome preparation 1 : 50 (BuÈrger 1991).



It can also be shown that the methaemoglobin formation for

10 derivatives studied in both systems correlates with the

partitioning into erythrocytes (Eq. 5, Table 2).

log Derythr: � 0:164��0:015� log k 0 � 0:511��0:024� �4�n � 20 r2 � 0:87 s � 0:07 F � 126 Q2 � 0:78

log QDDS � 10:17��3:65��log D�2erythrocytes

ÿ 6:34��3:19� log Derythrocytes

ÿ 3:94��0:96� �5�n � 10 r2 � 0:95 s � 0:20 F � 66 Q2 � 0:90

PC-analysis

The antiin¯ammatory activities, expressed as inhibition of

the neutrophil zymosan-mediated respiratory burst (cellbr),

the inhibition of neutrophil interleukin-1 stimulated cell

adhesion (celladh) and the methaemoglobin formation of

sulphones have been studied on a smaller set of 11 deri-

vatives [20]. First a PC-analysis has been performed to

analyze for possible multiple intercorrelations amongst the

three test systems.The result shows that with 2 PC's 89% of

the total information can be extracted. The ®rst PC after

varimax rotation is loaded by the cellbr- and celladh-data, the

second with the methaemoglobin formation ability (Table 4).

The total orthogonality of the methaemoglobin formation

ability, the cell toxicity and the properties of the compounds

to exert antiin¯ammatory activities suggests great promise

for the process of further optimization of the antiin¯amma-

tory effect of the sulphones.

More than 12 physicochemical parameters describing

electronic steric and lipophilic substituent effects in o-

and p-position have thus been analyzed for their ability to

explain the observed variance in the biological activity data.

Ortho and related proximate substituent effects can be

separated according to Charton [34] into inductive,

resonance and steric contributions.

log k � asI � bsR �CrV � h

Table 2. Methaemoglobin formation and partitioning into ery-throcytes by 4-aminodiphenylsulphones Structure±toxicity rela-tionships

log QDDS log % Methaemgl. log DErythrocytes

1-compartment 2-compartment

obs. calc. obs. calc. obs. calc.

Nr log k 0 eq. 1 eq. 2 eq. 4

1 0.339 0.00 ÿ0.29 2.00 1.78 0.59 0.57

2 1.375 0.21 0.09 Ð Ð 0.67 0.74

3 1.20 0.02 0.06 Ð Ð 0.66 0.71

4 1.646 Ð Ð Ð Ð 0.71 0.78

5 1.770 Ð Ð 1.74 1.86 Ð Ð

6 0.155 ÿ0.14 ÿ0.41 1.83 1.83 Ð Ð

7 ÿ1.716 ÿ2.00 ÿ1.75 1.04 0.86 Ð Ð

8 ÿ0.705 ÿ1.30 ÿ1.01 Ð Ð 0.29 0.39

9 0.965 0.08 0.00 Ð Ð 0.72 0.67

10 1.506 0.06 0.10 Ð Ð 0.68 0.76

11 1.890 ÿ0.04 0.09 Ð Ð 0.81 0.82

12 ÿ2.471 ÿ2.00 ÿ2.31 Ð Ð 0.23 0.11

14 ÿ0.197 ÿ1.09 ÿ0.64 1.49 1.60 0.42 0.48

15 1.589 ÿ0.03 0.10 Ð Ð Ð Ð

16 2.27 0.08 0.05 Ð Ð Ð Ð

17 2.38 0.07 0.03 Ð Ð 0.94 0.90

18 1.08 Ð Ð 1.97 1.88 Ð Ð

20 1.86 Ð Ð 1.75 1.86 Ð Ð

21 ÿ0.009 ÿ0.37 ÿ0.51 1.56 1.68 Ð Ð

22 1.258 Ð Ð 1.82 1.88 0.75 0.72

23 1.932 Ð Ð 1.72 1.85 0.82 0.81

24 1.277 Ð Ð 2.01 1.88 0.69 0.72

25 1.850 Ð Ð Ð Ð 0.86 0.82

26 2.03 Ð Ð 1.94 1.85 0.71 0.84

27 2.604 Ð Ð Ð Ð 1.09 0.94

28 0.833 Ð Ð Ð Ð 0.67 0.65

29 1.659 Ð Ð 1.96 1.87 Ð Ð

30 2.15 Ð Ð 1.95 1.84 0.88 0.86

31 2.22 Ð Ð Ð Ð 0.95 0.88

32 3.75 ÿ0.03 ÿ0.06 1.74 1.71 Ð Ð

33 4.25 Ð Ð 1.72 1.67 Ð Ð

34 3.03 Ð Ð 1.77 1.77 Ð Ð

35 0.89 Ð Ð 1.96 1.87 Ð Ð

36 1.41 0.13 0.09 1.91 1.88 Ð Ð

37 2.45 0.11 0.02 1.79 1.82 Ð Ð

38 0.192 Ð Ð 1.76 1.74 Ð Ð

39 ÿ1.534 Ð Ð 0.78 0.95 Ð Ð

40 1.625 Ð Ð 1.75 1.87 Ð Ð

41 1.058 Ð Ð 1.90 1.88 Ð Ð

42 1.32 Ð Ð 1.78 1.88 Ð Ð

43 1.31 Ð Ð 1.92 1.88 Ð Ð

44 1.86 Ð Ð 1.70 1.85 Ð Ð

45 2.05 ÿ0.25 Ð Ð Ð Ð Ð

QDDS� effect of drugyeffect of DDS at 10mM.

log Derythrocytes�Partitioning into Erythrocytes.

Table 3. Methaemoglobin-formation (%) in cats 6 hrs and 24 hrsafter iv administration of the indicated sulphones (200 mgykg) andthe corresponding log k 0-values

Substance

% methaemo-

globin

6 hrs after

administration

24 hrs after

administration

dapsone (1) 6.09 4.4 0.34

28 0.7 1.2 0.83

31 0.0 0.0 0.00

Table 4. Loadings of the original scaled activity variables onprincipal components after Varimax rotation

PC 1 PC 2

I log % Methaem. formation 0.013 0.992

II log Cell burst inhibition 0.902 0.178

III log Cell adhesion inhibition 0.909 0.147

n� 10, derivative 35 omitted, no effects in cell adhesion test, 2PCs contain

89% of the information of the 4 original variables.



Similarly a treatment of o-substituent effects based on

Swain-Lupton treatment [35] has been proposed by Fujita

and Nishioka [36].

log k � ff� rR � dEos � h

A highly signi®cant correlation was derived for the

inhibition of zymosan-mediated respiratory burst. The most

signi®cant correlation was found with sI The activity

increases with increasing sI-effect of the substituents in o-

position.(log Cellbr.� log effectDDSylog effect drug at

1 mM).

log cellbr: � 0:860��0:276�sI � 0:043��0:084� �6a�n � 11 r2 � 0:85 s � 0:081 F � 50 Q2 � 0:80

Alternatively the ®eld effect f has been used (Eq. 6b). The

intercorrelation sIyf in this data set is r2� 0.80.

log cellbr: � 0:762��0:286�f� 0:086��0:085� �6b�n � 11 r2 � 0:80 s � 0:092 F � 36 Q2 � 0:73

The inclusion of the resonance effect, R, in Eqs. 6a,b led to

a slight increase in r2 (0.86, 0.81 respectively) was,

however, not signi®cant. The inclusion of a steric parameter

(MR2) led also to an increase in r2 (0.85) and was signi®cant

on the 82% level.

Later this series was extended using a smaller drug

concentration of 0.1 mM instead of 1 mM.

Again a highly signi®cant correlation with sI is found but in

addition the MR2-value of the o-substituents plays a role in

explaining the observed variance. In this extended data set

substituents with larger steric demands are included (see

Tables 1, 5).

log 1y%cellbr � 0:133��0:071�sI ÿ 0:0117��0:004�MR2

ÿ 1:701��0:032� �7a�n � 15 r2 � 0:77 s � 0:002 F � 21 Q2 � 0:67

log 1y%cellbr � 0:094��0:066�fÿ 0:0123�0:005�MR2

� 1:697�0:036� �7b�n � 15 r2 � 0:75 s � 0:025 F � 18 Q2 � 0:64

Table 5. Observed and calculated antiin¯ammatory activities and descriptors used in the derivation of the regression equations

log cellbra log 1y%cellbrb

Eq. 6a Eq.7a log

cell

Nr. obs. calc. obs. calc. adhcc sI f MR2 R logk 0

1 0.00 0.04 ÿ1.72 ÿ1.71 0.00 0.00 0.00 1.03 0.00 0.34

18 Ð Ð ÿ1.73 ÿ1.74 Ð 0.17 0.02 5.42 ÿ0.68 1.08

19 0.176 0.189 Ð Ð 0.129 0.17 0.02 5.42 ÿ0.68 ÿ0.096

20 Ð Ð ÿ1.82 ÿ1.80 Ð 0.13 ÿ0.11 10.33 ÿ0.74 Ð

21 0.163 0.249 Ð Ð 0.00 0.24 0.29 2.85 ÿ0.64 ÿ0.009

22 Ð Ð ÿ1.71 ÿ1.70 Ð 0.24 0.29 2.85 ÿ0.64 1.26

23 0.419 0.249 Ð Ð 0.280 0.24 0.29 2.85 ÿ0.64 1.93

24 0.335 0.307 ÿ1.72 ÿ1.75 0.501 0.30 0.26 7.87 ÿ0.51 1.77

26 Ð Ð 0.00 0.00 1.03 0.00 2.03

29 Ð Ð ÿ1.73 ÿ1.77 Ð ÿ0.01 ÿ0.04 5.65 ÿ0.13 1.66

30 Ð Ð ÿ1.76 ÿ1.77 Ð ÿ0.01 ÿ0.04 5.65 ÿ0.13 2.15

32 0.016 0.034 Ð Ð 0.155 ÿ0.01 ÿ0.04 5.65 ÿ0.13 3.75

33 0.034 0.035 Ð Ð 0.083 ÿ0.01 ÿ0.04 5.65 ÿ0.13 4.25d

34 Ð Ð ÿ1.79 ÿ1.77 Ð ÿ0.01 ÿ0.04 5.65 ÿ0.13 3.03

35 0.187 0.138 Ð Ð Ð 0.11 0.00 7.19 0.00 0.89

36 0.415 0.387 Ð Ð 0.063 0.40 0.38 5.02 0.19 1.41

37 0.260 0.387 ÿ1.71 ÿ1.71 0.063 0.40 0.38 5.02 0.19 2.45

38 Ð Ð ÿ1.79 ÿ1.79 ± 0.30 0.32 11.18 0.20 0.192

39 Ð Ð ÿ1.79 ÿ1.78 ± 0.28 0.24 9.81 0.14 ÿ1.53

40 Ð Ð ÿ1.82 ÿ1.81 ± 0.32 0.33 12.87 0.15 1.63

41 Ð Ð ÿ1.73 ÿ1.71 ± 0.47 0.41 6.03 ÿ0.15 1.06

42 Ð Ð ÿ1.71 ÿ1.74 ± 0.47 0.44 8.88 ÿ0.17 1.32

43 0.629 0.619 Ð Ð 0.266 0.67 0.67 7.63 0.16 1.31

44 Ð Ð ÿ1.70 ÿ1.70 ± 0.63 0.51 6.33 0.19 1.86

(a) log cellbr� log (effect DDSyeffect drug) at 1 mM [20].

(b) log %cellbr at 0.1 mM of the drugs.

(c) log celladh� log effect DDSyeffect drug at 1 mM [20].

(d) calc.



As the descriptors used in Eqs. 7a and 7b have very

different values (size), it is dif®cult to judge their relative

importance. All parameters have therefore been scaled:

1ylog %cellbr � 0:592��0:316�sI ÿ 0:867��0:316�MR2

n � 15 r2 � 0:77 s � 0:53 F � 21 Q2 � 0:67 �8a�

1ylog %cellbr: � 0:556��0:310�fÿ 0:835��0:323�MR2

n � 15 r2 � 0:75 s � 0:56 F � 18 Q2 � 0:64 �8b�

Intercorrelation MR2ysI, r2 � 0:10

Inclusion of the resonance effect in Eqs. 7ay7b did slightly

improve r2 but the contribution was again not signi®cant.

The QSAR results on inhibition of respiratory cell burst

using drug concentrations of only 0.1 mM have, however, to

be considered with caution because of the small range in

biological activities (Eqs. 7ayb). This also may be the

reason for the lack of signi®cant correlations for the data on

inhibition of cell adhesion. The best equation obtained was

with sI in accordance with the results of the PC-analysis.

The scores of PC 2 which contain the information extracted

from the original variables cellbr and celladh correlate with

sI:

PC2 � 3:88��2:36�sI ÿ 0:931��0:75�n � 10 r2 � 0:64 s � 0:67 F � 14:0 Q2 � 0:53 �9�

The two most deviating derivatives are #23 and 24 (Table

1). A reason for this cannot be given.

4 Conclusion

The toxic effect (methaemoglobin formation) of 2-sub-

stituted 4-aminodiphenylsulphones depends solely on log k 0

in a nonlinear function.In contrast, the antiin¯ammatory

activity, expressed as inhibition of cell burst and cell

adhesion, depends on electronic and steric substituent

effects of the substituent in 2 0-position as found previously

also for their antibacterial effect. No in¯uence on changes

in lipophilicity occurred with the exception that under in

vivo conditions a further decrease in methaemoglobin

formation with increasing lipophilicity was observed. This

can also be demonstrated in vitro upon addition of serum

albumin. The orthogenality in the in¯uence of physico-

chemical properties on the toxic effects compared to the

wanted antiin¯ammatory and antibacterial effects can lead

to an optimization in the selectivity of the sulphones.

In summary, we have used a wide range of biological data

including cytotoxicity, methaemoglobin formation, cell

burst and cell adhesion assays and physicochemical

properties in a mathematical analysis to derive QSAR

relationships aimed at optimizing the selectivity and

minimizing the toxicity of antibacterial and antiin¯amma-

tory sulphone drugs.

References

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Received on May 25, 1998; accepted on August 31, 1998

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