N-NMR Studies of Antitumor Complexes - American Chemical Society

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Pt- and N-NMR Studies of Antitumor Complexes

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.ch008

ISMAIL M. ISMAIL and PETER J. SADLER Birkbeck College, Department of Chemistry, University of London, Malet Street, London WC1E 7HX England

195

15

Pt and N NMR studies at 4.7 and 9.4 Τ are used to characterise new Pt(II) and Pt(IV) anti-tumour drugs, to study the interaction of inosine (I) and 5 -adenosine monophosphate (AMP) with cisplatin, and release of amines induced by binding to methionine sulphur of peptides and proteins. Drug characteri sation is aided by the predictability of Pt shifts and Pt - N coupling constants. Broaden ing through scalar coupling to N can sometimes be overcome by raising the temperature. Contributions to Pt relaxation from chemical shift anisotropy, not only make it difficult to observe signals from Pt bound to macromolecules, but can also lead to broadening of Pt satellites of H, C, and N resonances. N7-06 chelation was not detected for I bound to cis-Pt( NH )Cl at acidic pH levels, but may occur for the diaquo complex. Several previously undetected I and AMP species are reported. Finally, facile release of both NH and a chelated diamine (ethylenediamine) was observed on reaction with N-Ac-Met, Gly-Met and RNase A in solution near neutral pH and it is suggested that such reactions may play an important part in the mechanism of action of Pt anti-tumour drugs. 1

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195

15

14

195

195

1

13

15

15

3

2

3

0097-6156/83/0209-0171 $06.00/0 © 1983 American Chemical Society

Lippard; Platinum, Gold, and Other Metal Chemotherapeutic Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

METAL CHEMOTHERAPEUTIC AGENTS

172

In t h i s paper we study three problems: ( i ) the c h a r a c t e r i s a t i o n o f P t ( I I ) and P t ( I V ) anti-tumour drugs i n s o l u t i o n by P t NMR, ( i i ) t h e i n t e r a c t i o n o f two n u c l e i c a c i d bases i n o s i n e ( I ) and adenosine 5^monophosphate (AMP) w i t h N - l a b e l l e d c i s - P t ( N H ) C l and c i s - P t ( N H ) ( H 0 ) by N and P t NMR, and ( i i i ) the r e l e a s e o f amines from c i s - P t ( N H ) C l and P t ( e n ) C l on b i n d i n g t o methionine-containing peptides and p r o t e i n s , u s i n g p r i m a r i l y N NMR. M u l t i n u c l e a r NMR i s p o t e n t i a l l y a very powerful technique f o r d e f i n i n g f u l l y the c o o r d i n a t i o n sphere of platinum anti-tumour drugs i n s o l u t i o n and upon i n t e r a c t i o n w i t h biomolecules. The p r o p e r t i e s of some u s e f u l n u c l e i a r e l i s t e d i n Table I . I n our s t u d i e s we seek i n f o r m a t i o n about the o x i d a t i o n s t a t e o f platinum usually Pt(II) or Pt(IV), 1 9 5

1 5

3

2 +

3

2

2

1 5

a

1 9 5

2

3

2

2


1 5

Table I P r o p e r t i e s o f some NMR n u c l e i u s e f u l i n the study o f Pt anti-tumour drugs

Isotope 1

H 13 N 35 19 5

I h h h

C

1 5

C1

3/2

p t

h

Abund.(%) 99.98 1.11 0.37 75.53 33.8

Freq.(MHz)

n.O.e. _

200 50.29 20.27 39.19 42.82

Recept. 1.0 Ι.βχΙΟ" * 3.9xl03.6x103.4x101

+3.0 -3.9

6

-

3

3

+3.3

the number and types of coordinated l i g a n d s , t h e i r stereochemical arrangements, and dynamic processes i n c l u d i n g the r a t e s o f l i g a n d s u b s t i t u t i o n . Pt-induced l i g a n d a c t i v a t i o n s (e.g. lowered pK s ) are sometimes a l s o d e t e c t a b l e . A l l o f these f e a t u r e s appear to c o n t r i b u t e t o the b i o l o g i c a l a c t i v i t y o f platinum complexes as e x e m p l i f i e d by the f o l l o w i n g : c i s but not t r a n s P t ( I I ) diamine complexes a r e a c t i v e anti-tumour drugs ( 1 ) , P t C l * and P t C l ~ are potent a l l e r g e n s ( 2 ) , whereas P t ( e n ) i s a neurotoxin (3). f

2 -

2

6

l f +

3


2

8. ISMAIL AND SADLER

5

15

Pt- and N-NMR Studies

173

*H NMR has the advantage o f h i g h s e n s i t i v i t y , and P t b i n d i n g can o f t e n be detected v i a s p i n - s p i n couplings t o l i g a n d n u c l e i up to 4 bonds away from P t . These couplings can provide stereochem i c a l i n f o r m a t i o n ( 4 ) . However, P t s a t e l l i t e s o f H, C and N resonances can be very broad and d i f f i c u l t t o a s s i g n a t h i g h f i e l d s (5., 6) where s e n s i t i v i t y i s g r e a t e s t f o r work on biomolecules. C 1 NMR i s u s e f u l f o r studying C I " r e l e a s e (7), but s i g n a l s f o r Pt-bound c h l o r i d e are too broad t o observe. We were a t t r a c t e d t o P t NMR by i t s l a r g e , p r e d i c t a b l e chem i c a l s h i f t range o f ca. 15,000 ppm. (8). P t - * N one-bond c o u p l i n g constants are p a r t i c u l a r l y s e n s i t i v e t o the nature o f t r a n s l i g a n d C9,10,11,12). Sometimes P t c o u p l i n g t o quadr u p o l a r N i s r e s o l v a b l e but, i n g e n e r a l , we have r e s o r t e d t o enrichment o f P t diamines w i t h N . This has the advantage o f making d i r e c t o b s e r v a t i o n o f N NMR s i g n a l s p o s s i b l e . Most o f the work d e s c r i b e d here has only become p o s s i b l e i n the l a s t few years w i t h the r o u t i n e a v a i l a b i l i t y o f high f i e l d (4.7 - 9.4 T) F o u r i e r transform spectrometers w i t h l a r g e sample tubes (10 - 15 mm diameter). 1 9 5

X

1 3

15

35

1 9 5

1 9 5

t h e

5

1 9 5


llf

1 5

1 5

Experimental 1 9 5

1 5

P t and N NMR s p e c t r a were recorded on V a r i a n XL 200 (4.7 T ) , Bruker WM200 (4.7 T) o r Bruker WH400 (9.4 T) spectrometers i n 10 o r 15 mm diameter tubes. H 0 was used as s o l v e n t , w i t h 510% D 0 added o r D 0 i n a c o n c e n t r i c tube f o r l o c k . This avoided s i g n i f i c a n t d e u t e r a t i o n o f amine l i g a n d s . References were 1 M Na PtCl i n D 0 for P t , and 2.4 M NH*C1 i n 1 M HC1 f o r N . Most N s p e c t r a were obtained from overnight accumulations u s i n g 30°-60° p u l s e s , 3.6s pulse r e p e t i t i o n r a t e s and a data p o i n t r e s o l u t i o n o f 0.6 Hz/point. I n view o f p o s s i b l e negative n.O.e.'s s e v e r a l s p e c t r a were checked a g a i n s t a phase r e f e r e n c e . The h i g h frequency - p o s i t i v e s h i f t s i g n convention i s used. 2

2

2

1 9 5

2

6

15

1 5

2

1 5

1 9 5

P t Chemical S h i f t Range

Most P t complexes have l o w - l y i n g e x c i t e d e l e c t r o n i c s t a t e s , and P t NMR s h i f t s are dominated by the paramagnetic term i n Ramsey's equation ( 8 ) . The P t ( I V ) h a l i d e s alone span some 12,500 ppm ( 8 ) : 1 9 5

2

PtF ~ 7326 6

δ/ppm

PtCl 0

2 6

"

2

PtBr " -1870 6

2

Ptl "*" -5121 6

The c a l c u l a t e d s h i f t o f P t ( 0 ) i s -10,427 ppm ( 8 ) , and removal o f e l e c t r o n s t o g i v e P t ( I I ) leads t o d e s h i e l d i n g and high-frequency s h i f t s . P t ( I V ) resonances tend t o be f u r t h e s t t o h i g h frequency (low f i e l d ) , but there i s c o n s i d e r a b l e o v e r l a p .


174


Ligand exchange r e a c t i o n s of P t ( I I ) and (even more so) P t ( I V ) are u s u a l l y slow enough on the NMR t i m e s c a l e f o r separate reson ances to be observable f o r each d i s t i n c t Pt complex i n s o l u t i o n . I n t e r e s t i n g exceptions are P t ( I I ) aquo-hydroxo complexes which t i t r a t e as a s i n g l e , averaged resonance (13), and exchange of amine w i t h t r a n s - P t ( e t h e n e ) ( a m i n e ) C l which i s r a p i d i n CDC1 (14). The l a t t e r may be r e l e v a n t to the behavior of anti-tumour drugs i n membranes· S u b s t i t u t i o n s h i f t s u s u a l l y show r e g u l a r , incremental p a t t e r n s (15). For s u b s t i t u t i o n of B r " i n t o P t C l " and P t C l ~ , the s h i f t increments (Δ6) vary w i t h s u b s t i t u t i o n s t e p , n, according to (16): 2

3

z

z


4

6

- Δ ό ( Ι Ι ) - 203 + 23.5n ppm - Δ δ ( ΐ ν ) - 275 + 11.7n ppm Even c i s - t r a n s and mer-fac isomers are u s u a l l y w e l l - s e p a r a t e d i n the spectrum: 10 ppm f o r the isomers of P t C l B r " " , 208 ppm f o r P t ( N H ) ( H 0 ) , and 310 ppm f o r P t ( N 0 ) i , C l - (12). S u b s t i t u t i o n s h i f t s are s m a l l e s t f o r s u b s t i t u t i o n s t r a n s to l i g a n d s w i t h h i g h t r a n s i n f l u e n c e s . Thus, i n P t ( I V ) n i t r i t e complexes, a Br"* f o r CI"" s u b s t i t u t i o n i s ca. 50 ppm l a r g e r when t r a n s to C I " compared to t r a n s to N0 ~ (12). The term "trans i n f l u e n c e " i s used to denote the dependence of NMR (ground-state) parameters on the nature of the t r a n s l i g a n d . This i s d i s t i n c t from the "trans e f f e c t " which i s r e l a t e d to bond l a b i l i z a t i o n as measured by ligand substitution rates. At very h i g h f i e l d s (> 4.7 Τ ) , C 1 / C 1 and B r / B r i s o t o p omers are r e s o l v a b l e (17) and, i n p r i n c i p l e , the number of h a l i d e ions bound to Pt can be deduced from the isotopomer s p l i t t i n g p a t t e r n . In p r a c t i c e , the s h i f t s (0.167 ppm f o r C I , 0.028 ppm f o r Br) are too s m a l l i n comparison w i t h the temperature dependence of P t s h i f t s (0.3-1 ppm per °C r i s e i n temp.) and n a t u r a l l i n e widths of many complexes. Temperature g r a d i e n t s across samples are p a r t i c u l a r l y marked when l a r g e sample tubes and H decoupling are used. Representative P t s h i f t s f o r anti-tumour and r e l a t e d com plexes are given i n Table I I . Figure 1 i l l u s t r a t e s the r e g u l a r p a t t e r n of NH - H 0 s u b s t i t u t i o n s h i f t s i n P t ( I I ) complexes. A s i m i l a r graph i s obtained f o r the NH - C l ~ system, g i v i n g the f o l l o w i n g incremental s h i f t s : 2

2

2 +

3

2

2

2

2

2

2

2

2

3 5

3 7

7 9

8 1

1 9 5

X

1 9 5

3

2

3

Δδ(NH by 0H ) = + 590 Δ6(ΝΗ by CI") = + 270 3

2

3

ppm ppm

1 9 5

The s e n s i t i v i t y of P t NMR s h i f t s to s m a l l changes i n the types of bound l i g a n d s i s very u s e f u l f o r the c h a r a c t e r i s a t i o n of new drugs. For example, the c o o r d i n a t i o n of b i s - c a r b o x y l a t e s i s o f t e n not c o n v i n c i n g l y d e t e c t a b l e by H or C NMR, but Table I I shows t h a t replacement of two H 0 l i g a n d s by c y c l o b u t a n e d i c a r b o x y l a t e g i v e s a low frequency s h i f t of 133 ppm (18). X

13

2



N and

1 9 5

3

2

2

7

2

2

4

2

2

2

3

2

2

2

2

2

2

-30.5

-145

-1723

Pt(NH ) (CBDCA)

2

2

3

2

c_-Pt(NH ) Cl

2

2

3

2

2

Pt(en)(H 0)

3

3

2 +

2

2

2 +

2

2

H0

312 -2168

2

2

DMSO

DMF

2

H0

2

H0

2

H0

2

H0

2

2

H0

H0

2

H0

2

H 0/H 0 2

2

D0

2

D0

312

302

411

360

366

387

339

342

247

275

2

D 0/H 0

Solvent

-2097

-2048

-51.9

-83.7

-1694

Pt(NH ) (Etmal)

3

-1914

-89.0

-81.7

-37.9

+860

-1590

2

c-Pt(NH ) (H 0)

3

[Pt(NH ) (0H)] +

3

χ5

Ρί- N)/Hz

266

195

+881

-79.1

2

-1499

[Pt(NH ) (OH)] +

Pt(II)

3

3

ç,t-?t(NH ) C1 (OH)2

7

c-Pt(NH ) CU

ç,

3

c , c , t - P t (C H NH ) C 1 (OH)

3

c-Pt(C H NH ) (0H)

^(

249

15

6( N)/ppm

+1521

+1570

Ô( Pt)/ppm

195

P t NMR s h i f t s and one-bond c o u p l i n g constants f o r P t ( I I ) and P t ( I V ) diamines

jc, t-Pt(NH ) (mal) (OH)

Pt(IV)

Complex

1 5

Table I I



176


Pt(H)(NH ) ( H / » _ 3

1 9 5

P t Chemical shift

x

4

1 9 5

x

1 5

P t - N Coupling

m

m

15

Figure 1. Plots of the variation in Pt chemical shift (left) and Pt- N one-bond coupling constants with the number of NH ligands in the complexes [Pt(NH ) (H 0) . ] * (right). The values for tiam-Pt(NH ) (H O) + are those reported in Ref. 12. S

s

2

%

k x

2

s

g

g

2


x

m

ISMAIL AND SADLER

8. 19 5

_

p t

ltf

15

N >

N

C

1 9 5

Pt-

o

u

p

l

l

15

and

n

g

N-NMR Studies

177

s

15

One bond P t - N c o u p l i n g constants depend on the s c h a r a c t e r of the Pt o r b i t a l s used i n bonding to N, and are expected to be s m a l l e r f o r a Ν l i g a n d t r a n s to a l i g a n d which has a l a r g e t r a n s i n f l u e n c e s i n c e t h i s tends to weaken the t r a n s bond (9,10,11). The remarkable r e g u l a r i t y of c o u p l i n g s i n P t ( I I ) N H / H 0 complexes i s shown i n F i g u r e 1, where i t can be seen t h a t s m a l l c i s e f f e c t s a l s o occur. For c i s - P t ( N H ) 2 complexes, the order of t r a n s i n f l u e n c e s based on J i s : 3

2

a

l


ligand

: :

1

J/Uz

H0 390 2

N-NMR Studies of Antitumor Complexes - American Chemical Society

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