Application of Molecular Orbital Calculations To Estimate the Active

Chapter 30 Application of Molecular Orbital Calculations To Estimate the Active Conformation of Azole Compounds

Downloaded by UNIV LAVAL on May 9, 2016 | http://pubs.acs.org Publication Date: November 3, 1987 | doi: 10.1021/bk-1987-0355.ch030

Toshiyuki Katagi, Nobuyoshi Mikami, Tadashi Matsuda, and Junshi Miyamoto Laboratory of Biochemistry and Toxicology, Takarazuka Research Center, Sumitomo Chemical Company, 4-2-1 Takatsukasa, Takarazuka, Hyogo 665, Japan Molecular orbital calculations were introduced to estimate the active conformations of azole compounds at an enzyme active site. The computed data were discussed referring to the spectroscopic information and utilized for the steric fit evaluation. The a c t i v e c o n f o r m a t i o n of a b i o l o g i c a l l y active compound bound t o the t a r g e t s i t e ( s ) a f f o r d s a v a l u a b l e i n f o r m a t i o n to d i s c u s s i t s e f f i c a c y or t o x i c i t y at the molecular level. The t h r e e - d i m e n s i o n a l s t r u c t u r e o f enzyme a c t i v e s i t e ( s ) o r b i n d i n g s i t e ( s ) o f r e c e p t o r , u s u a l l y o b t a i n e d from X - r a y a n a l y s i s , makes i t e a s i e r t o estimate the a c t i v e conformation of the chemical. However, such i n f o r m a t i o n and even t h e p h y s i c o - c h e m i c a l p r o p e r t i e s of these macromolecules are not a v a i l a b l e i n many c a s e s . Under these c i r c u m s t a n c e s , i t i s i n e v i t a b l e to estimate the a c t i v e conformation o f a c h e m i c a l by another approach. The q u a n t i t a t i v e structure-actiνity r e l a t i o n s h i p ( Q S A R ) (1_) i s o n e o f t h e i m p o r t a n t approaches, p a r t i c u l a r l y when t h e t a r g e t s i t e o f a b i o l o g i c a l l y a c t i v e compound i s unknown. Although X-ray c r y s t a l l o g r a p h y i s a l s o h e l p f u l to e s t i m a t e the a c t i v e conformation, i t provides the c o n f o r m a t i o n a l i n f o r m a t i o n in a s o l i d phase. More i m p o r t a n t i s the c o n f o r m a t i o n o f a chemical i n s o l u t i o n , w h i c h c a n be a s s i g n e d i n p a r t b y spectroscopic studies. Nuclear magnetic resonance (NMR) s p e c t r o s c o p y has been u t i l i z e d to estimate the r e l a t i v e o r i e n t a t i o n o f e a c h atom i n a m o l e c u l e (_2~j5). Infra-red (IR) s p e c t r o s c o p y i s sometimes a u s e f u l t o o l , especially when h y d r o g e n b o n d s a r e p r e s e n t (6). Recently,

0097-6156/87/0355-0340$06.00/0 © 1987 American Chemical Society

Baker et al.; Synthesis and Chemistry of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


SO.

ΚΑΤ AGI ET AL.

Active Conformation of Azole Compounds

341

r e s o n a n c e raman s p e c t r o s c o p y has been i n t r o d u c e d to the s t r u c t u r a l s t u d y o f m a c r o m o l e c u l e s s u c h as porphyrin d e r i v a t i v e s (7_). Furthermore, fluorescence spectroscopy c a n be u s e d t o d e t e r m i n e t h e c o n f o r m a t i o n s o f a m o l e c u l e (8.,9.) a n d the p o l a r i t y of m i c r o e n v i r o n m e n t (10). However, these s p e c t r o s c o p i c methods d e a l with various low e n e r g y c o n f o r m a t i o n s o f m o l e c u l e s i n s o l u t i o n or c r y s t a l s t a t e and i t is d i f f i c u l t to seek the a c t i v e c o n f o r m a t i o n o n l y from the s p e c t r o s c o p i c studies. Recently, the r e l a t i v e e n e r g i e s of various c o n f o r m a t i o n s of a c h e m i c a l have been e s t i m a t e d by molecular orbital calculations. Semi-empirical methods s u c h as P C I L O ( H , L 2 ) , CNDO/2 (13 ) , a n d MNDO U 4 ) are u t i l i z e d because of t h e i r a v a i l a b i l i t y w i t h short computation time. However, s i n c e the t h e o r e t i c a l c o m p u t a t i o n s are u s u a l l y c a r r i e d out f o r the 'isolated' m o l e c u l e , i t i s n o t so s i g n i f i c a n t t o d i s c u s s the e f f i c a c y or t o x i c i t y of a c h e m i c a l by u s i n g the calculated configuration. M o r e o v e r , i t i s not easy to d e c i d e which c o n f o r m e r ( s ) are i n v o l v e d i n the i n t e r m o l e c u l a r i n t e r a c t i o n w i t h an a c t i v e s i t e , since t h e b i n d i n g c o n f o r m e r may n o t be t h e g l o b a l e n e r g y m i n imum. A l t h o u g h each of the methods s t a t e d above i s a l r e a d y known to s t u d y the c o n f o r m a t i o n s o f m o l e c u l e s , an i n h e r e n t d e f e c t i n e a c h m e t h o d m a k e s i t d i f f i c u l t to estimate an a c t i v e c o n f o r m e r . We combined each method as s u m m a r i z e d i n F i g u r e 1 and a p p l i e d t h i s s t r a t e g y to the a z o l e compounds, d i n i c o n a z o l e (ER p u r e ) ( I ) and uniconazole ( E S p u r e ) ( I I ) as e x a m p l e s . We estimated t h e i r a c t i v e c o n f o r m e r s by t h e t h e o r e t i c a l c a l c u l a t i o n s combined w i t h the s p e c t r o s c o p i c a n a l y s e s , followed by s t e r i c f i t e v a l u a t i o n with the a i d of computer graphics. P r i o r to s t e r i c f i t e v a l u a t i o n , the s u b s t r a t e binding a s s a y u s i n g t h e m i c r o s o m a l e n z y m e s was c a r r i e d out to c l a r i f y the i n t e r m o l e c u l a r i n t e r a c t i o n s between the a c t i v e s i t e ( s ) and a z o l e compounds. Mode

of

act ion

Two a z o l e compounds ( I ) and ( I I ) , shown i n F i g u r e 2, h a v e b e e n d e v e l o p e d by S u m i t o m o C h e m i c a l C o . , Ltd. (15). ( I ) s h o w s a s i g n i f i c a n t f u n g i t o x i c i t y by inhibiting o x i d a t i v e C14 demethy l a t i o n of the intermediate l a n o s t e r o l i n the b i o s y n t h e s i s of e r g o s t e r o l i n f u n g i (_H>). T h i s r e a c t i o n i s k n o w n t o be c a t a l y z e d by fungal c y t o c h r o m e P - 4 5 0 e n z y m e s ( i 7_, ). In c o n t r a s t , (II) possessing a s i m i l a r chemical s t r u c t u r e to ( I ) i s a p l a n t g r o w t h r e g u l a t o r and r e t a r d s a s t e m e l o n g a t i o n in h i g h e r p l a n t s at a recommended d o s a g e . I z u m i e__t a ^ . 09) has r e p o r t e d t h a t ( I I ) i n h i b i t s the successive metabolic o x i d a t i o n o f t h e C19 m e t h y l g r o u p o f intermediate (-)-kaur-16-ene i n the b i o s y n t h e t i c pathways of g i b b e r e l l i n . This reaction is considered to be c a t a l y z e d by t h e m i x e d f u n c t i o n o x i d a s e s in plants


SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS

342

which a r e s p e c t r o p h o t o m e t r i c a 1 l y analogous mammals ( _2 0^, _2 ) . The s i t e s o f i n h i b i t i o n c o m p o u n d s a r e s h o w n i n F i g u r e 3. Binding

to those o f b y two a z o l e

assay

I t i s n o t so easy t o p r e p a r e t h e f u n g a l m i c r o s o m a l f r a c t i o n and the mixed f u n c t i o n oxidases i n higher plants. Therefore, the microsomal f r a c t i o n of r a t l i v e r ( 2 ^ ) was u s e d i n s t e a d f o r t h e s u b s t r a t e b i n d i n g a s s a y t o a s c e r t a i n t h e i n t e r a c t i o n o f ( I ) and ( I I ) with c y t o c h r o m e P-450 e n z y m e s . Both ( I ) , ( I I ) , and t h e i r r a c e m i c m i x t u r e s showed t h e Type I I s u b s t r a t e d i f f e r e n c e s p e c t r a (2_3 24) w i t h s t o i c h i o m e t r i c b i n d i n g t o c y t o c h r o m e P-450 e n z y m e s . These r e s u l t s strongly s u g g e s t that ( I ) and ( I I )c o - o r d i n a t e t o t h e i r o n atom o f t h e p o r p h y r i n m o i e t y i n c y t o c h r o m e P-450 e n z y m e s v i a the n i t r o g e n atom a t t h e 4 - p o s i t i o n (N4) o f t h e 1,2,4-triazoly1 moiety. In c o n t r a s t , the Z-isomer o f (I) (designated a s ( I I I ) ) shows a n o t h e r t y p e o f s p e c t r a (Type I) which i n d i c a t e t h e l o o s e b i n d i n g o f ( I I I ) t o the c y t o c h r o m e P-450 e n z y m e s . The d i f f e r e n c e between T y p e I a n d I I s p e c t r a i s s h o w n i n F i g u r e 4.


):

E s t ima t i o n ο ξ . t h e c o n f o r m a t i o n s s o l u t i o n by s p e c t r o s c o p y

of azole

compounds i n

To e s t i m a t e t h e c o n f o r m a t i o n s o f ( I ) and ( I I ) a t t h e e n z y m e a c t i v e s i t e o f f u n g i o r p l a n t s , IR a n d H-NMR s p e c t r a o f t h e a z o l e compounds i n s o l u t i o n s were measured. F r o m t h e r e s u l t s o f mode o f a c t i o n a n d b i n d i n g assay, ( I ) and ( I I ) a r e c o n s i d e r e d to locate i n the c l o s e p r o x i m i t y to t h e p r o s t h e t i c p o r p h y r i n group o f c y t o c h r o m e P-450 e n z y m e s . The p o l a r i t y o f macromo1 e c u 1es c l o s e t o t h e p o r p h y r i n m o i e t y o f apohemoglob i n has been d e t e r m i n e d by f l u o r e s c e n c e study t o be s i m i l a r t o t h a t o f n - o c t a n o l 00)· In our study, carbon t e t r a c h l o r i d e and deuterioch1orοform o f which p o l a r i t i e s were s i m i l a r t o t h a t o f n - o c t a n o l were u s e d . B o t h c o m p o u n d s s h o w e d a b r o a d IR a b s o r p t i o n a t c a . 3 , 4 6 0 cm-1 a t c o n c e n t r a t i o n s approaching the s o l u b i l i t y l i m i t i n carbon t e t r a c h l o r i d e . T h e IR a b s o r p t i o n s p e c t r a o f ( I ) a r e shown i n F i g u r e 5 a . F u r t h e r m o r e , t h e s m a l l t e m p e r a t u r e d e p e n d e n c e o f t h e H-NMR c h e m i c a l shift o w i n g t o t h e h y d r o x y p r o t o n was o b s e r v e d a t t h e c o n c e n t r a t i o n o f 0.006 m o l e / l i t e r i n d e u t e r i o c h 1 o r ο f o r m a t -50 C t o 25 C, a s s h o w n i n F i g u r e 6. In the case o f ( I ) a t 0.06 m o l e / l i t e r , t h e h y d r o x y p r o t o n showed broad s i g n a l s with a s i g n i f i c a n t shift to the l o w - f i e l d at t h e t e m p e r a t u r e below 0 C. These observations s t r o n g l y s u g g e s t t h a t t h e i n t r a m o l e c u l a r h y d r o g e n bond is formed a t a lower concentration. Which m o i e t y o f ( I ) formed t h e i n t r a m o l e c u l a r h y d r o g e n b o n d w i t h t h e h y d r o x y p r o t o n was e s t i m a t e d a s follows. The r a c e m i c d e r i v a t i v e s o f ( I ) , l a c k i n g t h e


50. K A T A G I E T A L .

YES

Biologically active compounds;

Are" ^ X-ray data available w

Is target site known2^ " YES

NO

QSAR study

\

••^Structural studyI I Γ Theoretical Spectroscopic calculation measurements (MO cal.) (NMR, IR) Estimation of active conformation


343

Active Conformation of Azole Compounds

Steric fit evaluation

Mode of action la t a molecular level

Figure 1 The s t r a t e g y to estimate the a c t i v e c o n f o r m a t i o n of b i o l o g i c a l l y a c t i v e compounds. CI H

(ι)

OH

N

Figure

2

Chemical

^

(.")

N

structures

of azole

compounds,

H

CH OH — • C H O - > : ^ 2

D iηicο ηa ζ ο Ie(ΕR

CH,-^CH OH—•CHO 2

19

3

Ο

2

4^ο

Unico ηa ζ ο Ie (E S

E r g o s t e r o l

pure)

— • - • - • G i b b e r e Min pure)

Figure 3 The s i t e s o f i n h i b i t i o n i n t h e e r g o s t e r o l and g i b b e r e l l i n

by a z o l e compounds biosynthesis.


344

SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS •0.04

•0.02

LU 2

< m

s m

Application of Molecular Orbital Calculations To Estimate the Active

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