ANTICANCER RESEARCH J9, 3895-3900 (1999)

ElectronlC Structure and CytOtOXlc Activity of 6(Half-Mustard Type"

PhenothiaZines by MM3 and PM3 MethOds TERUO KURIHARAl, KAZUMICHI NOJIMAI , HIROSHI.SAKAGAM12> NOBORU MOTOHASH13

and JOSEPH MOLNAR4

IFaculty of Science, Josai University, Sakaclo, Saitama ' 2Meikai Universty School ofDentistly, Sakado, Saitalna'

-?Mc'lji Phalmaceutical University, Kiyosc', Tokyo, Japan' 4Albel't S'-ent-G)*d'~gyi Medical University, Szc'gecl, Hungaly

Abstract. Among 54 cance/' cell lin,es, colon-can,cer cells

we,'e the most sc'nsitiye to six "haJf-mustard type" phen,o-

th,iazines f7-12J, followed by leukemia, melanoma, pl'ostate-,

CNS-, b7'east-, Iung-, ,'enal and ova/'ian cancei' cells. The

distlibution of electrostatic potential (ESP) of "half-musta'~;/

type" phen,oth,iazines f7-12J suggests that the ESP su'face of

urea site is impol'tant fol' th,e inte,'action between. "half-

musta/~i type" phen,othiazines f7-12J and talget cance,' celJ

structu/"es (Ol' DNA base sequence). Actually, the u"ea site of

"h,alf-musta/~l type" phenothiazines displayed extensive

l)ariability in the enel~y of lone pai/' 07hita! and net atomic

chalges of NJ, O and N3 atoms.

Previous studies have shown a possible relationship between

antitumor activity and dipole moments of "half-mustard type"

phenothiazines (1, 2, 3, 4). NCI-Information Intensive-

Approach (5) demonstrated that six "half-mustard type"

phenothiazines [7･12] were more cytotoxic than six phthalimide compounds [1-6]. Anrong 54 tumor cell lines,

colon-cancer cell was the most sensitive to six "half-mustard

type" phenothiazines [7-12], followed by leukemia, melanoma, prostate-, CNS-, breast-, Iung-, renal and ovarian

cancer ~ cells. Based on the experimental results, the

significance of urea site on the electronically flexible

phenothiazine framework was predicted. Therefore, the purpose of the present study was to investigate the possible

relationship between cytotoxic activity and electronic

structure of the particular urea site. In a plausible approximation, the role of the unshared electron pair on the

urea site in half-mustard phenothiazines [7-12] was estimated

by multiple regression analysis.

Conlespondence to: Dr. Teruo Kurihara, Department of Chemistry, Faculty of Science, Josai University, 1-1 Keyakidai,

Sakado, Saitama 350-0295, Japan. Tel: (+81)-492-71-79. 59; Fax:

(+81 )-492-71-7985. E-mail: <Tkuri@josai.ac.jp>

ICc'y Words, PM3 method. MM3 method, cytotoxic activity, "half-

mustard type" phenothiazines, multiple regression analysis.

Materials and Methods

Synthesis of che,nicals. All rclated phenothiazines [1-12] were prepared as descrlbed previously (6).

Assay f'ol' ai7titumor acti~*ity. The 50(~/c' grov~'th inhibitory concentration

(GI50), 1000~7c, tumor growth inhibitory concentratlon (TGI) ',md 500/(~

lethal concentration (LC50) values of six "half-mustard type" phenothiazines [7-12] were determined by dose-response curve, using

4 Ieukemias, 9 non-srnall ceH Iung carcinomas, 7 COIOn cancers, 5

CNS-cancers, 8 melanomas, 6 ovarian cancers, 8 renal cancers, 1 prostate cancer, and 6 breast cancer ce]1 Iines (5). The mean v'alues

of Gl_~-0, TGI and LC_~~o were calculated in each tumor group for the

six tested compounds [7-12]. Their logl.o ¥'alues were converted to

molar concentration using the previous data for compounds [7-12] (T',ible I).

~fheoretica.1 ca!cu!ations. About 30 conformations each for six

compounds were processed to geometric optimization by the Molecular Mechanics 3 (MM3) method. Among about 30 optimized structures for each molecule, two typical structures with both

maximum ',ind minimum dipo]e moments were selected. The moiecular orbital for these two structures were calculated by the

Parametric Method 3 (PM3). MM3 and PM3 calculations were performed with the application of Alchemy 2000 Program (7) and

winMOPAC (8), respectively. For this calculation, 'an IBM Aptiva

E47 personai computer was used.

Results and Discussion

Relation,ship between cytotoxic activity and dipole lnom,ents.

Table I shows cytotoxic activity (as measured by GI50, TGI

and LC50) of "half-mustard type" phenothiazines [7-12]

against 9 cell lines (2, 9, 10). The dipole moment (~L) was

used to determine the interaction between two molecules

vvith different dipoles. Therr u values of "half mustard

type'> phenothiazines [7-12] were calculated by the PM3

method. The ma**nitude of u ranged from 1.05 to 3.71 D in

[7]; 0.72 to 4.22 D in [8]; 0.85 to 5.55 D in [9]; 1.54 to 2.78

D in [10]; 2.89 to 6.56 D in [1l]; and 1.52 to 5.77 D in [12],

respectively (Table II). The maximum u ranged from 2.78

to 6.56 D, and thc minimum u ranged from O.72 to 2.89 D,

respectively (Table II). Interestingly, among six compounds

0250-7005/99 $2.00 + .40 3895

ANTICANCER RESEARCH 19.- 3895-3900 (1999)

Table I. I inesl , 2)

Cytotoxic activty of sl~ 1-(2-chlo,vethyl)-3-(2-substituted-1()H-p/?enothiazin-10lyl)a!/vlulleas f7-12J against nine dlffe/'ent gl'oups of cancer ce[l

Compd Cytotoxic activity

Leukemia Non-small (4) cell lung

(9)

Colon-

cancer (7)

CNS-cancer

(5)

Melanoma (8)

Ovarian

cancer (6)

Rena]

cancer (8)

Prostate Breast-

cancer cancer (1 ) (6)

7

8

9

lO

11

12

1 .06

O.48

O.19

O. 1 6

O.19

O. 1 6

1 .44

O.76

O.40

O.25

O._35

O.29

l .06

0.40

O.2O

O.14

O.21

O.17

Gl50 (x lO~)M)

i .37

O.87

O.~_6

O.?_()

0.14

O . 20

1 .3~)_

O.59

O.20

O.17

O.24

O. 1 8

l.39

1 .02

O.56

O.31

O.69_

O.39

1.11

O.74

O.47

0.9_8

0.49

O.?_9

1.07

O.54

O.25

O.21

0.26

0.25

1.46

O.60

O.37

O.21

0.26

O.26

7

8

9

10

11

12

2.91

l.77

O.45

0.42

O.52

O.43

2.8 l

~_.1 1

l.07

O.64

0.97

O.79

2.39

1.13

O.46

0.28

0.48

0.36

TGI (x 10~~M)

?~.72

'_.21

O.80

O,_52

O.8_5~

O.5(,*

2.64

1 .76

0.40

O.32

O.57

O.33

2.72

2.39

1.81

l.18

l.93

i .50

1.07

?_.02

1.30

O.71

1.37

0.95

2.24

1 .82

1.20

O.81

1.35

1.20

2.77

1.74

0.85

O.50

0.81

0.71

7

8

9

10

11

12

7.59

5.96

1 .41

2. 1 1

4.04

1 .40

5,52

4,85

2,83

2,04

2.62

2.53

5.03

2.75

1.03

O.57

1.14

O.83

LC50 (x lO~)M)

5,37

4.92

2.47

1 .86

3.09

1 .99

5 . 22

4.10

O.92

O.60

1 .45

0.69

5,33

5,15

4,54

4.25

4,88

5.1 1

5.11

4,60

3 , 23

2,07

3,42

3,17

4.68

4,27

3,55

3,02

3,80

3,63

5,68

4,83

2,68

1,39

2,40

2.08

l) ref 2 2) Transformation' 10 x 10 - IB' in order to evaluate the outcome of the drug combination fractional inhibitory concentration (FIC)

indices were calculated as FICA + FICB, when FICA and FICB represent the minimum concentrations that drugs A and B inhibited the organism

growth, respectively (10, 1 1)

[7-12j, I).

compound [1l] showed the highest Lt value (Table

Relationship between cytotoxic activity and, 7r-HOMO ol' fr-

LUMO enel~y. If the cytotoxic activity of the compounds

used in this study depends on the electron donor or

electron acceptor property, then ~-HOMO energy can be

used as a measure of electron donating capacity. By this

assumption, first, the Jc-HOMO energy of six compounds

[7-12] was calculated: (-7.93 eV) [9], (-7.95 eV) [8], (-8.02

eV) [7], (-8.12 eV) [10], (-8.24 eV) [1~_] and (-8.27 eV) [1l],

respectively (Table II). Second, ~-LUMO energies of six

compounds [7-12] characterized by the electron acceptor

property were calculated as follows: (-0.95 eV) [12], (-0.77

eV) [1l], (-0.54 eV) [10], (-0.37 eV) [9], (-0.30 eV) [7] and

(-0.24 eV) [8], respectively (Table II).

Assuming that the compound with the lower ~-HOMO binds more easily to cancer cell DNA, compounds [11, 12]

3896

Kurihara et aL PM3 Study of "Half-Mustard Type" Phenothiazmes

Table ll. Dipole molnent, molc'cl'tla;' o,j)ital enelgy a,rd net atoll7ic cha'ge of "half-mustard type"phenothiazines f 7-12j, calculated by PM3 mc'thod.

O

~~ lN l

(CH2)n l; l lO f~},N,~~/~IO* ~ R

7C/~:511SO(U¥~141 ~ 3

5

[1] R=H,

[2] R=H,

[3] R=Cl,

[4] R=Cl,

f:5] R=CF3,

[6], R=CF3,

n=:3

Il =: 4

n=3 n:::4

n:::3

n::::4

(3) (1)

HN N l ¥(C2Vl H (CH2)n ll I

Q

/9~¥~;9laN~Y~~(~~lO, ~ R

7 ~ _1 10 .1 ~ ~L5a S5~i4a 3

[7] R=H,

18] R=H,

[9] R=Cl,

[10] R=Cl,

[1l] R=CF3,

[12], R=CF3,

n=3 n=4 n=3 n=4 n=3 n=4

Cl

Compd's

No Dipole moment

(in D)

Molecular orbital energy

(in eV)

Net atomic charge

umin ~) um2Lxb) ~: HOMO J~-LUMO AEc) d) / Za

nbe) / 7 o f )

C(=0) O( C)

7

8

9

In

ll

12

l .05

O.72

O,85

l .54

9~,89

1.52

3.71

4.22

~5.55

2.78

6.56

5.77

-8.02

~7.95

_7.c)3

-8. 1 2

-8.27

~8.24

-o.30

-o.24

-o.37

-o.54

-o.77

-o.95

7.7･~

7.71

7.56

7.58

7.50

7.29

-9*94

-10.02

~9,98

-9.96

-10.03

-9*99

-9 , 72

-9,87

~9.65

*9.66

-9,84

~9.67

-11.10

-il.17

- 1 1 .05

-1 1 .09

-11.18

-11.12

o.22

o.22

0.23

o.22

o.22

o.22

-O.40

-O.40

-O.41

-O.4i

-O.39

-O.40

a)

b)

~t~nin: minimum dipole moment. ~tmax: maximum dipole moment. c) AE: energy gaps (HOMO-LUMO). 'rtoms llb' Ione pair orbital of N1 and N3 atoms O no' Ione pair orbital of O atom. * . . e) . < < . .

d) n,,: Ione pair orbital of N1, O and N3

should have higher cytotoxic activity against these cells

(Tables I and ll). Actually, compound [12] (~c-HOMO: -

8.24 eV; ~-LUMO: -0.95 eV), which had the lowest ~-

HOMO and JC-LUMO energies, showed the highest cytotoxic activity against 9. cancer cell strains (Tables I and

II). This suggests a possible electrochemical interaction

between "half-mustard type" phenothiazines and cancer

cell DNA.

Relcltionship betM)een cytotoxic activity and olbital ene,gies of

lone pai7' of electl'on, on u/'ea site. The na orbital represents

an unpaired e]ectron orbital of N1, O and N3 atoms on

urea site. The nb and no orbitals represent an unpaired

electron orbital at N1 and N3 atoms, and O atom on urea

site, respectively (Tab]e II).

Multiple /'egression clnalysis between cytotoxic actil)ity an,d

elect7'onic st/'Ltctu,'e in, u/'ea site. Among nine types of cancer

ce]Is, Ieukemia and colon cancer cells were the most sensitive to "half-mustard type" phenothiazines, based on

GI50 values, possibly due to intra-molecular synergy between the antiproliferative property of phenothiazine

and the leukemia specificity of "half-mustard type" phenothiazines. On the other hand, non-small cell lung,

ovarian cancer and renal 6ancers were relatively resistant.

In order to obtain a more quantitative correlation between

cytotoxic activity and electronic prope2rty on urea site, the

multiple correlation coefficient (r ) and distribution

function of F value between cytotoxic activity and various

factors described above were calculated. First, the multiple correlation coefficient (r2) between

GI50 values and fQur parameters of three orbital energies

(na, nb, no) and one net atomic charge of O atom in urea

site were 0.91 for CNS-cancer cells and 0.98-0.99 for other

8 cancer cells. The F value (76.6) for colon-cancer was the

highest, followed by renal cancer (F: 41.5), breast cancer

(F: 22.9), melanoma (F: 19.6), non-small cell lung (F:

11.9), prostate cancer cells (F: 11.7), ovarian cancer cells

(F: 10.9) and CNS cancer (F: 2.5). An acceptable correlation was not established, since F values of nine

cancer cells for this model were much smaller than the five

percent critical value of F (4, l; 0.05) = 225. Second, the multiple correlation coefficients (r2)

between GI50 values and four electronic parameters such

3897

ANTICANCER RESEARCH 19: 3895-3900 (1999)

'f ',1: ", .:

: .::: ~.i.1;t' :.r ":~.r'..:V' I~ .:,1~ t

" :' ! :; 'J.1:4 ': ' ::.'}:.:~.

tl.lr J ' . ,,~.'-･ ' a;~ . 'l~ ,!'t.~::~. 1 ~E.

- ~.. 'l "' '.~';.1;?' ~ '

"':r ' ~ L?;;',. .; ,'1':, . j"b

' : 1't .~i~ ; .U :: ." It':: ' L~~ ' (""" :. ~ ;I~ : C b. ; :~..~rl' "': ' '~ ':':':~ :: &~ :r.

::: i~" :1:1~; ""~ : ';. t " Ie#.. ' ~~ i' '*:' .' ..,(. . L:~!Sj "'1::: 'L" :' :1 ' ~ VI' :S'I"L

IL~ I :1' ~:.~.:i!･,i: '.' !."':i ' :':: Ia' t.. ' :.. :1'L::~:1~~fl'l ;~ :!h't S' *f ~' ' s ~ T. '.' !' If'~!" ~': ._ .. : ~ _-1 ' t . . 1!' '? '*

oompound [7] compQund [8] Figure I Dist,thulion of eiectrostatic potentiai of "haif ,nus'ard VPe"phenothieJzfnes [7-9J

*~~

compound [9]

' :,: 1 ' ~s~

.':1.'JL : ,

't:1'LI･,Xtll, I-I ':~･ .!111.. ~7 f-

. t I Il' I. 111:I t' .~5 t IFI .: ･ l.' ~ :,･f,Ll

.'.1 L

Gompound [1Ql Gompound [111 compound [12]

Figure I (cont ) Dist,fbuiion of e!ectrostatic potentia! af "halfmustard type"phenothiazines !10-12]

as orbital energies (n,~ no) and net atomic charges of C and

O atoms on urea site were 0.98 for CNS-callcer cells and 0.99 for other 8 c~ncer cells. The F value fo'breast cancer

cell (360683.6) was the highest, followed by melanoma

(65965.5), Ieukemia (10632.5), ovarian cancer cell (1421 O),

non-small cell lung (1362 3), prostate cancer (221 6), colon

cancer (208 1), renal cancer (36 8) and CNS cancer cells

(13 2) Thus, acceptable correlation was established, since

the high F values of most of the cells were higher than the

5% critical value of F (4, 1; O.05) = 225

3898

玉く1篶riharaε‘oZ．・ PM3Stωy　of“Haif－M淑ard　Type”Pheηo倣aziηes

　　Third，based　oηTGI　va1泌es，co王oトcancer棚d　me旦a篶o幻αa

w釘e㎜ore　seηsi芝ive　than　ov肌ian　c＆ncer，re蝸1caηc飢棚d

pros主ate　cancer　ce1旦s．The㎜u旦tip互e　corre1磁ioηcoefficie耐

（・2）b榊・バG1・・h1…心・w・1・・舳i・p跡・m舳・

s辻1chasorbiω㎝ergies（η。，η。）a皿“etatomicchargesof

Ca三｝dO＆tomsinはreas三tewereO．98f帆renaic棚cerceuaI三d0．99for　other8caΩcer　ce1旦s．　The　F　va王びe　for　co工oη一

cancer　c釧（五ユ80ユ。3）was　the　highest，fo玉丑owed　by　ovarian

cancer（662．6），狐elaηoma（61．6），breas言cancer（5！．7），

leuke㎜ね（39．9），no阯sma豆1cel〕uηg（33．5），CNS－caηcer

（25．O），Pro舳eca㏄er（24．4）a双dre篶alca㏄eてce1ls（ユ3．6）．

○がy　thc　F　vah蛇of　co王o双caηcer　was　si即ifica畝1y　higher

tha孤5％critical　va1ue　of　F（4，主；O．05）肥225．From　the　　　　　　　　　　　　　　　　　　　　　2＆bove　ca1culatioηs　of　r　aΩd　F　va1泌cs，coloΩcaひcer　was

foはnd　to　be　the　mosモseηsitive　among　g　ca巫cer　ce11s乏o

“half－mびst肌d　type”phenoth主azines［7－12］。The　iηhib｛tioη

ofthcdmgtralユspor廠pr㈱iHesponsib1efordmgc舳xmighta㏄辻㎜山telhe“half一肌1stardph㎝othiazines”inthe

ca㏄er㏄llsヨProd㏄i㎎thecytotox1cactivityagainsいhem舳idmg　resista耐（MDR）ca㏄cr　or　co1on　ca㏄er（6，9）．

D｛8エγめ〃主oη　oグ　肋ε　εZεαγoδ施此　ραθ1切〃8．　The　ESP

cal㎝1aむ㎝of“ha1f－mustardtype”Pheむo伽azi舵s17・121

w㈹performed　by　PM3㎜ethod　wれh　ESP　a1gorithm（10ラ！！）．　The　ESP　surface　iηtheし汲ea　site幻a｛ght　have　aむ

㎞porta耐rokfo〔hei耐eractio1三betweeパhalf－m耐ard

typc”Phenothiazines口・121aηdca㏄ercellsurfa㏄sorDNA（a旦ky王atio玉】or　i耐ぴcalatio互ユ）（Fig以rcユ）．Acωany，＆

泌reaportiolNf“ha1f珊耐ardtype”Phe篶othiazineshas＆

c㎝siderablyvariable㎝ergyofthe1㎝epairorbiω詠nd舵tato㎜icch鮒gesatN五，OandN3ato狐s（Tab1eH）（Figure1）、Theregi㎝s泌rro泌ndi㎎carbo篶y1oxygena主d幻α

has王肥gative　charge（一：symbo王in　bh旦e　of別g泌re1）ラwhereas

the　environme耐肌oUnd　the　urea’s　NH　at　the　oppos｛te　side

haspositivecharge（斗：sy㎜boいHedofH脾e！）。Thesepositive　aM　篶egative　ch鮒ges　might　co鮒ribリte　to　丞

co㎜p1ex　fom珊tion〃αthe　dipo亘e－dipo亘e　iΩ芝eractioη，wheη

a　DNA　compo鵬耐（ε．g．，cytosi雪三e）c狐be鮒acked　by　the

リreas1teofa“half一狐泌stardtype”Phe篶othi＆zi附

Refere烈ces

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　　伽o伽1bac廠ialactivity狐いheore重1c豊！cal㎝1象tiolioパ‘h轟1f－

　　mむs芝ard　type”pheno伽azjnes、伽＝Non　Andbiotics：A11ew　cl＆ss　of

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