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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
王Motohash三N,Kuriharaγ,Sわa狐pa G,Das室i幽r SGラChakr曲arti
AラCh婁krab鮒ty AN a双d Mo正納T J-Re茎a言io欄hip be重ween伽〃ro一加
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
u研㏄ognized a耐imicrobics(Chakrab&rty AN,Moln益r J,Dε}st1d肌S
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mびst鮒d芝ype”pheηo舳azi烈es.An芝icancer Res/9二iηpress,王999.
3 Motohashi N享K鮒ih欲aγ,Saka酔m三洲aD(量M〇三n益r J:M〇三ec山段r
orbi言al of cy言otoxic“ha1雲一㎜洲t狐d type”帥e…?oth…azi鵬s,A鮎i-
caηcerRes/9■リress,至999.
4 Kuζ三h鮒a T,Motohashi N,Sakaga醐{篶aηd Mo正Mr J:Re王ationship
betweeηcy辻o室ox三c登c迂{v1ty and dipo…e mo㎜e玉1言for phthaljmido一
棚d c三葦1oroe宣hy1-pheno伽azi舵s,An芝icancer Res/9{}press,三999.
5Wuonola MA,Pa附ey㎜an MG,Motohash川ラKawase M,Gab&y S,Nacs&J aηd Mo]n益r J:γhe primary fη一’”1’0a…廿it讐moすsc茎‘eeni訟9
of“half-mus辻葦…rd type”ph㈱o舳az1脱s.A柵caηcer Res/7’3409-
3423ラ至997.
6 Motoha曲i N,Kawase M,Kuriha顯丁亭汽ev6r A,N窪gy S,Oscovski
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(2-ch至croethy至)一3_(2-sむbstit独室ed一呈0〃_pheno芝…三jazin_!0-y])a]ky,1」rca
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7 Tripos,Inc:A玉che㎜y2000Program,工996.
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D,Nacsa J,Ya㎜&n級&W,Keζim A&nd Mo!n益r J:Dmgresist狐ce
reveぎs餐1, an芝i-mutagenicity 搬d antiretrov{ral effect of
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reversa1on r汀dr tun〕oζce】ls of dihydropyrinines.Prepara室ion for
Arznei醐一Forscわ言ヱ999.
至玉Be此r B狩,M欽z KM鯛d Kolhηan PA:Atomic charges derived
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ノ{ocερ犯6〃oツ24.1999
3899
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