Inorganic Chemistry in Biology and Medicine - ACS Publications

17 The Design of Chelating Agents for the Treatment of Iron Overload Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 10, 2017 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch017

COLIN G. PITT Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC 27709 ARTHUR E. MARTELL Department of Chemistry, Texas A&M University, College Station, TX 77843

Iron is an essential component of man's biochemistry but, in common with other elements, becomes toxic when in excess (1,2). This arises in part because of the tendency of iron(III) to separate in tissues as very insoluble hydroxide and phosphate salts at the physiological pH and higher unless bound to trans ferrin, the iron transport protein, or to ferritin, the iron storage protein. Iron absorption via the diet is physiologically controlled, but the body has no regulatory mechanisms for elim inating a toxic excess introduced by accidental overdose or by multiple transfusions. Cooley's anemia and its treatment provide an example of the difficulties of correcting deficient iron metabolism (3 ,4). Cooley's anemia is a genetic disease originating from errant bio synthesis of the β-chain of hemoglobin which can only be treated by an interminable transfusion regimen. The increased iron input (20-25 mg/day) exceeds the capacity of transferrin and ferritin, resulting in separation of insoluble iron in critical tissues, e.g. the heart, liver, pancreas. In p r i n c i p l e , t h i s u l t i m a t e l y f a t a l c o n d i t i o n can be t r e a t e d by a d m i n i s t r a t i o n of an i r o n che l a t i n g agent which promotes r e m o b i l i z a t i o n and e x c r e t i o n of the deposited i r o n . In p r a c t i c e , n e i t h e r desferrioxamine Β (DFB), the most commonly used i r o n c h e l a t o r , nor any other agent c l i n i c a l l y evaluated to date has been able to do more than r e t a r d the c o n d i t i o n (5). I t has been stated (5) that the i d e a l i r o n c h e l a t i n g agent should be inexpensive and o r a l l y a c t i v e . I t should have a high and s e l e c t i v e a b i l i t y to bind i r o n ( I I I ) r a p i d l y under p h y s i o l o g i c a l c o n d i t i o n s , r e l a t i v e to f e r r i t i n and t r a n s f e r r i n , while not i n t e r f e r i n g with hemoglobin, myoglobin, the cytochromes, and normal i r o n biochemistry. I t should be f r e e of both acute and chronic side e f f e c t s , and r e s i s t a n t to metabolic changes which impair i t s a b i l i t y to bind i r o n . These requirements may be achieved with some degree of p r e d i c t a b i l i t y and are now discussed together with the r e s u l t s of animal screens of p o t e n t i a l i r o n ( I I I ) c h e l a t o r s which have been reported by d i f f e r e n t l a b o r a t o r i e s (6-9). 0-8412-0588-4/80/47-140-279$08.50/0 ©

1980 A m e r i c a n Chemical Society

Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Coordination of F e r r i c

BIOLOGY A N D

MEDICINE

Iron

The b a s i c requirement of an i r o n c h e l a t i n g drug i s that i t have a high and s e l e c t i v e a f f i n i t y to bind i r o n a v i d l y under p h y s i o l o g i c a l c o n d i t i o n s . The t r i p o s i t i v e F e ( I I I ) i o n i s a hard a c i d and consequently i s bound most s t r o n g l y by hard bases, the most e f f e c t i v e of which are oxyanions, such as hydroxide, phenoxide, carboxylate, hydroxamate and phosphonate ( 1 0 ) . The coord i n a t i o n number i s u s u a l l y s i x , although some seven-coordinate complexes are known. The most f a v o r a b l e geometry i s an o c t a h e d r a l arrangement of donor atoms, p e r m i t t i n g the maximum p o s s i b l e d i s t a n c e between t h e i r formal or p a r t i a l negative charges. Charge n e u t r a l i z a t i o n i s an important f a c t o r , and i s optimum when the t o t a l charge of the s i x donor atoms i s - 3 , as f o r the bidentate hydroxamate and tropolonate l i g a n d s . S t e r i c e f f e c t s can be im portant and, i n the case of the EDTA complex of F e ( I I I ) , prevent the formation of the optimal octahedral arrangement of the s i x donor atoms; here binding to a solvent molecule or an a d d i t i o n a l oxyanion and formation of a seven coordinate species i s p r e f e r r e d (11).

S t a b i l i t y Constants as a Measure of A f f i n i t y f o r Iron The a f f i n i t y of a l i g a n d f o r i r o n ( I I I ) may be defined q u a n t i t a t i v e l y i n terms of the thermodynamic constants of the e q u i l i b r i a i n v o l v e d between the aquo metal i o n and the deprotonated l i g a n d L: FeL + L s f = ^ F e L

FeL

2

+ L

2

FeL^

The formation or s t a b i l i t y constants K l i b r i a are defined by equation ( 1 ) . [FeL] Κ

[FeL ]

These i n turn may where

=

3

=

K

l

_ 2

[Fe] [L]

h

[FeLJ

9

Κ

= 1

f o r t h i s s e r i e s of e q u i

n

z

=

Η

K

1

K

2

Κ

= n

[FeL][L]

be expressed

n

Κ

as the s t a b i l i t y products,

=

K

1

K

(1)

[FeL^HL]

2

K

n

3.,

(

2

)

and t

11

F

e

L

J S [Fe][L]

(3) n

S t a b i l i t y constants are g e n e r a l l y a p p l i c a b l e to experimental c o n d i t i o n s which are most f a v o r a b l e f o r c h e l a t e formation, i . e . , i n the absence of competing ions and at the optimum pH. I t has been estimated ( 1 2 ) that under these c o n d i t i o n s the s t a b i l i t y pro-


17.

Chelating

Ρ ί τ τ AND MARTELL

281

Agents

duct (3 of t r a n s f e r r i n , the body's i r o n t r a n s p o r t p r o t e i n , i s approximately 1 0 . This i n d i c a t e s a very high order of s t a b i l ity. Classes of s y n t h e t i c c h e l a t i n g agents which have a compar able o r greater a f f i n i t y f o r i r o n ( I I I ) under optimum i n v i t r o c o n d i t i o n s are shown i n Figure 1. The e f f i c a c y of a c h e l a t i n g drug i n v i v o i s u s u a l l y reduced s u b s t a n t i a l l y by competing ions present i n the b i o l o g i c a l m i l i e u (13). Calcium ions (10~" Μ ) , hydrogen ions (10~ Μ ) , and hydrox ide ions ( H T M) a r e the most s e r i o u s i n t e r f e r e n c e s which com pete with e i t h e r the drug f o r i r o n ( I I I ) (OH"), or with the i r o n ( I I I ) f o r the c h e l a t i n g drug ( C a , H ) . For example, the greater the b a s i c i t y of the c h e l a t o r , the greater i t s a f f i n i t y f o r i r o n ( I I I ) , but t h i s e f f e c t i s p a r a l l e l e d by a higher a f f i n i t y f o r protons. When the pK of the c h e l a t o r i s s u b s t a n t i a l l y greater than the p h y s i o l o g i c a l pH, proton competition w i l l g r e a t l y decrease the c o n c e n t r a t i o n of the b a s i c form of c h e l a t o r , thus reducing the metal i o n b i n d i n g . 3 6

3

7

7


2 +

+

Computer s i m u l a t i o n of such m e t a l - l i g a n d e q u i l i b r i a i n b i o f l u i d s has been reported with the object of c h e l a t i o n therapy. In one paper (14) the d i s t r i b u t i o n of Ca , M g , Fe . , C u , Z n , and P b amongst 5000 complexes formed with 40 l i g a n d s was computed. However, i n c o n s i d e r i n g l i g a n d design i t i s o f t e n more h e l p f u l to estimate i n t e r f e r e n c e s by use of an i n t e r f e r e n c e term α (13,15,16). For example, i n the case of proton i n t e r f e r e n c e , represents the f r a c t i o n of l i g a n d i n i t s completely deproton ated form. I f T i s the t o t a l c o n c e n t r a t i o n of the uncomplexed drug i n the medium, then equation (4) may be d e r i v e d . 2+

2 +

2+

2

2

2 +

L

m

[L -] = T a L

(4)

L

where a

L

-

(1+

[ H

+

]

E

;

[Η ] β« +

+

[HV^)"

2

+

1

and 3 i s the a p p r o p r i a t e s t a b i l i t y product of LH. formation, i.e., [H L]/[lT^] [L]. The corresponding expressions f o r calcium i o n and hydroxide ion i n t e r f e r e n c e a r e equations (5) and (6), r e s p e c t i v e l y , and the e f f e c t i v e b i n d i n g constant, K f f , of the i r o n ( I I I ) - d r u g complex i s defined by equation (7) 1

i

1

i

e

(5) CaL a

= (1 + [ O H i e f + [ O H ]

F e

log K

e f f

= log 6

F e L

- nlog(a^

2 6

[OHlXJV

f +

1

L

+ a" ) - log ( a ^ )


1

(6)

(7)

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AND

.. Chelate

η

Log β ° n

Log Κ ° ef f

FeL

1

25.0

FeL

1

28.0

MEDICINE

Τ,-0.001 L

Τ,-1.00 L

8.10

-3.81

-3.81

10.96

-0.95

-0.95

3 - I s o p r o p y l t r o p o l o n e , HL

Acetohydroxamic

acid,

HL

C

H

^

j NH

c

\ ^ Ν

CH C00H 2

CH.COOH CH COOH 2

Ethylenediaminetetraacetic

HOOCCH. N—CH.CH-—Ν

Dlethylenetriamlnepenta-

HOOCCH.

\

CH.COOH

I

2

CH.COOH

/

2

2

^NCH CH NCH CH N^ 2

Figure 1.

2

2

2

Iron(III) affinities of chelating agents in terms of standard and effective stability constants


Chelating

ρ ι τ τ AND MARTELL

Agents

Log β

HOOCCH.

Trlethylenetetraminehexaa c e t l c a c i d , H^L

/CH,C00H\

CCH. \

/CH-COOHv (


Log Κ ,.

Τ -0.001 Τ -1.00

FeL

1

26.8

10.61

-1.30

FeL~

1

39.7

20.78

8.87

-1.30

[I ]/ NCH,CH,1-NCH,CH,4N

2

2

J.

\

HOOCCH,

N,N'-Bls(o-hydroxybenzyl) ethylenediamlne-N,Ν *d l a c e t l c a c i d , H.L

CH.COOH

\

2

_

h\ CH.COOH

u

2h

cn

y

/

c

X

Ν

8.87'

2

Ν

CH,

Ethylenebls-N,Ν'-(2-ohydroxypheny1)glycln< H.L

\

CO Chelate 3

FeL "

η 1

Log β

Log Κ

52

25

Τ -0.001 Τ -1.00

13

13

r

( Ψ"^> \ H

OH CO \

N

/ /

NH

0 D e s f e r r i o x a m i n e B, H.L

° ^ C 0 _

/C(

OH

0

II I

OH

0

OH

I

II

I

C H , — C — N — (CH ) N H C 0 ( C H ) ~ C — N — (CH,) ^NHCO(CH,) , — C — N — (CH,) ^NH. 2

5

2

2

FeL

Standard data used i n these c a l c u l a t i o n s a r e : l o g log 6 f

e 0 H

- 24.4; - l o g Κ

s[

e0H

-

1

30.6

16.34

4.44

4.44

0

11.09; l o g

β**" -

21.96; - l o g K

s p

Fe(0H> -

- 13.795; pH - 7.40.


3

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Equation (6) only takes i n t o c o n s i d e r a t i o n the formation of s o l uble mononuclear hydroxy complexes o f i r o n . Since a p has a f i x e d value of 1 0 · a t pH 7.4, and i s independent of other l i g a n d s , the r e l a t i v e e f f i c a c i e s o f d i f f e r e n t c h e l a t i n g drugs may be compared by taking only calcium and hydrogen i o n i n t e r f e r e n c e s i n t o a c count. T r a n s f e r r i n i s b e l i e v e d (17) t o bind i r o n ( I I I ) with three phenolate ( t y r o s i n e ) r e s i d u e s and, being weakly a c i d i c , proton i n t e r f e r e n c e i s r e s p o n s i b l e f o r the s i g n i f i c a n t d i f f e r e n c e between 3 ( 1 0 ) and K f f ( 1 0 ) . The phenolate group i s a l s o present i n seven of the fourteen s t r u c t u r e s i n Figure 1, which i s testimony to the p a r t i c u l a r l y strong and s e l e c t i v e a f f i n i t y of t h i s group f o r i r o n ( I I I ) . Proton i n t e r f e r e n c e increases with the number of phenolate groups and i n the case of the t r i s complex of 2,3-dihydroxynaphthalene-6-sulfonic a c i d , proton i n t e r f e r e n c e reduces 33 by a f a c t o r o f 1 0 . Hydroxamic a c i d s a r e stronger a c i d s (pK 8-9) and, consequ e n t l y , proton i n t e r f e r e n c e i n v i v o i s l e s s s e r i o u s . T h i s may be one reason why DFB, a hexadentate hydroxamic a c i d (Figure 2), has been of some u t i l i t y i n the treatment of i r o n overlaod. DFB i s competitive with t r a n s f e r r i n f o r i r o n ( I I I ) on the b a s i s o f t h e i r K f f values and model i n v i t r o s t u d i e s , and the main drawbacks of DFB appear to be poor a b s o r p t i o n when administered o r a l l y p l u s a s u s c e p t a b i l i t y to r a p i d metabolism and degradation i n plasma (18). Tropolones ought to be a most promising c l a s s of compounds f o r study. The p K of the pseudo-phenolic group i s about 7 and, consequently, there i s v i r t u a l l y no proton i n t e r f e r e n c e . Calcium ion i n t e r f e r e n c e i s minor and i n the case of 3-isopropyltropolone the value of log K f f a t pH 7.4 i s 21.9 compared with a l o g 33 value of 32. T h i s value of l o g K f f i s the highest of the group of bidentate c h e l a t o r s i n Figure 1, and 1 0 times greater than the K f f of t r a n s f e r r i n . A plasma c o n c e n t r a t i o n of 5 χ Ι Ο " M (6 yg/ml) i s c a l c u l a t e d to be s u f f i c i e n t to sequester 10% of t r a n s ferrin-bound i r o n , i n s p i t e of the f a c t that t h i s tropolone i s only b i d e n t a t e . The major disadvantages of tropolones a r e expense, s y n t h e t i c i n a c c e s s i b i l i t y , some CNS t o x i c i t y , and d i f f i c u l t y i n c r e a t i n g hexadentate forms. R e l i a b l e e q u i l i b r i u m constants a r e not a v a i l a b l e f o r any phosphonic a c i d s i n the form of bidentate c h e l a t i n g agents, but comparison of the data f o r hexadentate agents shown i n Table I demonstrates that the phosphonate group has a higher a f f i n i t y f o r i r o n ( I I I ) than the carboxylate group. e

9

3

3 6

1 6


e

2 5

a

e

a

e

e

6

5

e

F e r r i c Ion H y d r o l y s i s and F e r r i c Hydroxide S o l u b i l i z a t i o n The most s e r i o u s i n t e r f e r e n c e to F e ( I I I ) b i n d i n g and excre t i o n i s h y d r o l y s i s to produce aquo complexes and, i n the most extreme case, to form a p r e c i p i t a t e of f e r r i c hydroxide. The f i r s t stage i n the c o n d i t i o n o f i r o n overload i n v i v o occurs when the plasma i r o n l e v e l exceeds that of f e r r i t i n and t r a n s f e r r i n .


17.

Chelating

P U T AND MARTELL

285

Agents

TABLE I C h e l a t i n g Tendencies of N,N -bis(o-hydroxybenzyl-Ν,Ν -ethylenediaminedi(methylenephosphonic) a c i d (HBEDPO) f

1

0 H O ^ I I

PCH

0

0 IU0H CH P ^ 0 H 0

/

2

N—CH CH —Ν


2

2

>H HO

.n+ Cu Ni Co Ca Mg Fe

2+ 2+ 2+ 2+

EDTA

HBED

HBEDPO

18.70

21.38

24.00

18.52

19.31

17.91

16.26

19.89

18.02

10.61

9.29

8.36

8.83

10.51

2+ 3+

7.95

39.68

25.0

>40

At t h i s p o i n t , excess i r o n i n the plasma i s bound only weakly as n o n - s p e c i f i c p r o t e i n complexes. As the i r o n concentration i n c r e a s e s , the i r o n begins to separate i n the form of i n s o l u b l e phosphate or hydroxide complexes. An i r o n c h e l a t i n g drug must be capable of r e m o b i l i z i n g t h i s form of i r o n . Assuming the worst case, where i r o n i s separated as the l e s s s o l u b l e (a) form of Fe(OH)^, the i r o n c o n c e n t r a t i o n i s given by equation (8) (19,20).

v

ιη

K

-41

= 10

s p

ΓτΓ

3+

= [Fe

ir

-,3

][0H ]

[Fe =

J

3+ -42 ]10

I

(8)

LH J i.e.,

K

Sol

o

[Fe

J

] = 10[H ]

One may then determine the e f f e c t i v e s o l u b i l i z i n g constant c h e l a t i n g agent, which i s the r a t i o of bound s o l u b l e f

t

h

e


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i r o n (FeL ) to the uncoordinated l i g a n d i n s o l u t i o n , T L - Combin ing equations (4), (5), (8), and (9) and n e g l e c t i n g i n t e r f e r e n c e by other metal ions one may d e r i v e equation (10). n

(9)

3 +

FeL

[Fe ]

[FeL ] η

6


Sol

+

FeL ( a

i.e.,

log K

S o l

= log e

3

10[H ] T n L . -1 _,_ - l . n L Ca +

a

( n _ 1 ) T

(10)

}

1

+ 1 - 3 p H - n l o g ( a " + a"*) η + (η - l ) l o g Τ p e L

A v a l u e of l o g Κ$ ± equal to one or higher i s i n d i c a t i v e of a s i g n i f i c a n t a b i l i t y to d i s s o l v e Fe(0H)3, the tendency i n c r e a s i n g r a p i d l y as the v a l u e of l o g K g ^ i n c r e a s e s i n magnitude. As an example, EDTA has a l o g 3 value of 25 and at pH 7.4 i n the presence of 10~ M calcium i o n the i n t e r f e r e n c e term OIL ^ 1 0 " · The c a l c u l a t e d value of l o g i s -5.1 and f e r r i c hydroxide i s not expected to d i s s o l v e i n t h i s system. The values of l o g K g i of the bidentate c h e l a t i n g agents shown i n F i g u r e 1 i n d i c a t e s a l i c y l i c a c i d and r e l a t e d compounds w i l l not d i s s o l v e Fe(0H)3 while hydroxamic a c i d s w i l l be m a r g i n a l l y e f f e c t i v e . The n a t u r a l sexadentate hydroxamic a c i d , desferrioxamine B, however, i s pre dicted to be f a i r l y e f f e c t i v e . Sexadentate l i g a n d s c o n t a i n i n g two phenolic groups, EHPG and HBED, are seen to be h i g h l y e f f e c t i v e i n s o l u b i l i z i n g f e r r i c hydroxide, and the sexadentate t r i s catecholate l i g a n d e n t e r o b a c t i n i s without doubt the most e f f e c t i v e l i g a n d l i s t e d i n t h i s paper. The a b i l i t y to bind and s o l u b i l i z e i r o n ( I I I ) i n a l k a l i n e medium i s not e n t i r e l y p r e d i c t a b l e on the b a s i s of the above con s i d e r a t i o n s . For example, the three hydroxyethyl analogs of NTA i n F i g u r e 3 show an a b i l i t y to sequester i r o n ( I I I ) i n a l k a l i n e s o l u t i o n i n the order TEA > DHG > HIMDA. As the number of hydroxy e t h y l groups i n c r e a s e s , the r e s i s t a n c e of the F e ( I I I ) complex to d i s p o r p o r t i o n a t i o n v i a f e r r i c hydroxide p r e c i p i t a t i o n extends i n c r e a s i n g l y i n t o the a l k a l i n e r e g i o n . In the case of the highest member of the s e r i e s , triethanolamine, s o l u b l e , c o l o r l e s s F e ( I I I ) chelates are formed a t pH 14 and above, and even i n s o l i d a l k a l i hydroxides. I t has been demonstrated (21) that above pH 13, i n c r e a s i n g [0H~] increases the e f f e c t i v e n e s s of triethanolamine i n formation of s t a b l e complexes having the general formula Fe (0H)b(H_ L) 3a-b-nc, whereby b and the r a t i o a/c becomes much greater than u n i t y at very high pH. While members of t h i s s e r i e s of compounds become more e f f e c t i v e f o r F e as the pH i n c r e a s e s , they become i n c r e a s i n g l y l e s s e f f e c t i v e at low pH, so that mix0

0

F e L

3

s

1 2

0

a

n

c

3 +



17.

Chelating

Ρίττ A N D M A R T E L L

NH \ (CH ) 2

CO / (£H,)

5

0" 0C-(CH )gC-0

287

Agents

9

Ο

Fe *"0-N-(CH ) -NH 3

2

(CH ) 2

2

5

CH,

5

Figure 2.

NH,

HIMDA

Iron(III) complex of desferrioxamine Β

DHG ^CH2C00H

^CH2CH2OH

HOCH2CH2—Ν

HOOCCH 2 —Ν

VCH C00H

^CHgCHgOH

2

TEA

HEDTA HOCH ? CH ?

Η. /

HOOCCH2

Figure 3.

/ N-CH.CrL-N 2

2

^CH2CH2OH

ChLCOOH 2

H 0 C H 2 C H 2 - NI

X CH2COOH

Hydroxy ethyl derivatives of NT A and

^CHgCHgOH

EDTA


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tures of these l i g a n d s have been employed i n i n d u s t r y to achieve e f f e c t i v e n e s s of F e ( I I I ) complexing over a broad pH range. Fre quently i n d u s t r i a l sequestering agent preparations are composed of mixtures of these l i g a n d s with EDTA. The replacement of one acetate f u n c t i o n of EDTA by a hydroxye t h y l group, to give HEDTA, a l s o r e s u l t s i n the formation of a compound which extends the u s e f u l F e ( I I I ) - s e q u e s t e r i n g range of EDTA to higher pH (the EDTA-Fe(III) c h e l a t e system decomposes to p r e c i p i t a t e Fe(0H)3 around pH 8, depending on c o n d i t i o n s and the c o n c e n t r a t i o n of excess EDTA). Since s i m i l a r v a r i a t i o n of the NTA s t r u c t u r e provides F e ( I I I ) - s p e c i f i c " complexing agents through d i s s o c i a t i o n of the hydroxy group to negative a l k o x i d e donors, i t was f i r s t thought that HEDTA f u n c t i o n s i n a s i m i l a r way, by the formation of c h e l a t e s such as FeH-iL" (where H3L represents HEDTA). While such i s c e r t a i n l y the case f o r Th^ ", which forms a unique p o l y n u c l e a r complex with HEDTA (22), evidence has been presented to show that the hydroxyethyl group remains i n t a c t i n F e ( I I I ) complexes, even a t pH values high enough to produce hydroxo complexes and the corresponding μ-οχο dimer (23). This c o n c l u s i o n i s f u r t h e r supported by the c r y s t a l s t r u c t u r e of Fe(III)-HEDTA μ-οχο dimer (11). I f one accepts s t r u c t u r e s f o r aqueous s o l u t i o n s i m i l a r t o T h o s e found i n the s o l i d s t a t e i n


f t

4

which the hydroxyethyl group i s not coordinated, one i s s t i l l l e f t with the problem of e x p l a i n i n g the experimental f a c t that HEDTAF e ( I I I ) complexes are more s t a b l e than those of EDTA with respect to Fe(0H)3 p r e c i p i t a t i o n , even though the s t a b i l i t y constant of the normal c h e l a t e (FeL" f o r EDTA and FeL f o r HEDTA) i s much greater i n magnitude f o r EDTA. Under these circumstances, and i n view of the x-ray data, the only reasonable explanation l i e s i n the greater s t a b i l i t i e s of the h y d r o l y t i c forms of the i r o n ( I I I ) HEDTA c h e l a t e , f o r which e q u i l i b r i u m data are a v a i l a b l e (10), and which are i l l u s t r a t e d s c h e m a t i c a l l y i n F i g u r e 4. Apparently the species FeLOH" and FeL(OH)\~ are r e l a t i v e l y r e s i s t a n t to d i s p r o p o r t i o n a t i o n to f e r r i c hydroxide, and the hydroxyethyl group must somehow be i n v o l v e d (e.g., through s o l v a t i o n ) i n the achievement of t h i s e f f e c t . L i m i t a t i o n s of the Use of S t a b i l i t y

Constants

While values of 3, K , and K provide a q u a n t i t a t i v e assessment of the a b i l i t y of a c h e l a t o r to bind i r o n , i t must be recognized that they a r e e q u i l i b r i u m constants and provide no information about the r a t e a t which e q u i l i b r i a a r e e s t a b l i s h e d . This has been c l e a r l y shown by i n v i t r o measurements of the r a t e s of removal of i r o n ( I I I ) from t r a n s f e r r i n by EDTA, c i t r a t e , and NTA (24). The percent i r o n t r a n s f e r r e d a t e q u i l i b r i u m was found to f o l l o w the order EDTA > c i t r a t e * NTA, which i s the order pre d i c t e d from the K f f v a l u e s of the c h e l a t e s ; however, e q u i l i b r i u m was e s t a b l i s h e d more r a p i d l y with c i t r a t e and NTA (1 hr) than with EDTA (>12 hr under the s p e c i f i c experimental c o n d i t i o n s ) . e f f

S o l

e



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Obviously, the advantage of using a drug with a high K f f value w i l l be l a r g e l y negated i f the time r e q u i r e d to e s t a b l i s h e q u i l i brium i s s u b s t a n t i a l l y g r e a t e r than the i n v i v o l i f e t i m e o f the drug. This may be one reason why DFB i s not a more e f f e c t i v e i r o n c h e l a t i n g drug, f o r i t i s r a p i d l y metabolized (18) by plasma enzymes y e t i s unable to remove i r o n from t r a n s f e r r i n a t a s i g n i f i c a n t r a t e i n v i t r o or jLn v i v o . Recently, i t has been shown that DFB w i l l remove i r o n r a p i d l y (hrs) from t r a n s f e r r i n i n the presence of a t h i r d , l e s s e f f e c t i v e l i g a n d such as NTA, c i t r a t e , ATP, and 2,3-diphosphoglycerate (25,26,27). This confirms the k i n e t i c nature of the problem, and suggests t h a t an optimum i r o n c h e l a t i n g drug should i n c o r p o r a t e some molecular f e a t u r e which promotes exchange. Unfortunately, there i s not a good understanding of what t h i s f u n c t i o n a l group should be. A p o s s i b l e explanation f o r t h i s e f f e c t i s c a t a l y s i s or t r a n s f e r of the metal i o n from one sexadentate l i g a n d to another through the formation of mixed l i g and intermediate ternary complexes, as described by Margerum (28) f o r analogous r e a c t i o n s i n non-biochemical systems.


e

E q u i l i b r i u m constants are p o s s i b l y even l e s s r e l i a b l e when p r e d i c t i n g the a b i l i t y of c h e l a t o r s to m o b i l i z e f e r r i t i n - b o u n d i r o n f o r i t has been found that the percent i r o n removed i n a given time f o r c r y s t a l l i n e and n o n - c r y s t a l l i n e f e r r i t i n by the above three c h e l a t o r s i s NTA >> EDTA > c i t r a t e (29). T h i s order shows no r e l a t i o n s h i p to the c a l c u l a t e d 3, eff» * S o l values of the c h e l a t i n g agents and some other f a c t o r , e.g., the s t e r i c a c c e s s i b i l i t y of the f e r r i t i n core (30) and a s s o c i a t e d k i n e t i c f a c t o r s must be i n v o l v e d . Redox mechanisms by which i r o n i s m o b i l i z e d from f e r r i t i n s t o r e s have been proposed and i t has been shown that 2 , 2 ' - b i p y r i d y l can m o b i l i z e s e v e r a l hundred i r o n atoms from f e r r i t i n i n the absence of any other reducing agent with concomitant formation of 2 , 2 - b i p y r i d y l - N - o x i d e (31). Thus, one of the mechanisms f o r m o b i l i z i n g i r o n i n v o l v e s r e d u c t i o n o f F e ( I I I ) to F e ( I I ) . The importance of k i n e t i c e f f e c t s and s p e c i f i c biochemical mechanisms of i r o n t r a n s p o r t do not negate the use of s t a b i l i t y constants as guides to the design of new i r o n c h e l a t i n g drugs, but they do i n d i c a t e the need to consider the bulk of the chel a t i n g agent and i t s p o s s i b l e e f f e c t on the k i n e t i c s of i r o n mobilization. I t i s q u i t e conceivable that i n some cases the advantages of a compact b i d e n t a t e drug may o f f s e t the advantages of the c h e l a t e e f f e c t a s s o c i a t e d with the use of a hexadentate drug, or that the e f f i c a c y of a hexadentate drug may be enhanced by c o - a d m i n i s t r a t i o n of a smaller, k i n e t i c a l l y more l a b i l e i r o n chelator. K

a n c

K

f

The Chelate E f f e c t S i x coordinate i r o n ( I I I ) may be bound by e i t h e r one molecule of a hexadentate drug, two molecules of a t r i d e n t a t e drug, three molecules of a bidentate drug, s i x molecules o f a monodentate


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drug, or some combination thereof. The choice becomes important because of the e x t r a s t a b i l i t y a s s o c i a t e d with the chelate e f f e c t , which f a v o r s the use of compact multidentate l i g a n d s . The chelate e f f e c t a r i s e s because of (a) f a v o r a b l e s t a b i l i t y constants a s s o c i ated with the formation of compact multidentate s t r u c t u r e s , and (b) a concentration f a c t o r , which becomes dominant a t the concen t r a t i o n s l i k e l y to be encountered i n v i v o . The former can be i l l u s t r a t e d by c o n s i d e r i n g the e q u i l i b r i u m between the i r o n ( I I I ) chelates of DFB, a hexadentate l i g a n d (Figure 2), and i t s bidentate analog, acetohydroxamic a c i d : -NH DFB

Fe(DFB) + 3MeC0NH0H

+

(12)

=CMe [Fe(DFB)]

K[DFB]

[Fe(MeCONHO) ]

(13)

[MeCONHOH]^

3

2

2

From s t a b i l i t y constant measurements, l o g Κ = 2.3 mol -£, (32,33). The e q u i l i b r i u m therefore favors the hexadentate chelate f o r a l l but extreme l i g a n d concentrations. The second component of the c h e l a t e e f f e c t , the concentration f a c t o r , can be i l l u s t r a t e d by c o n s i d e r i n g the a b i l i t y of a drug (D) to sequester transferrin-bound i r o n : TF-Fe + nD

^=

[TF-Fe] i.e. [D -Fe] n

(14)

TF + D -Fe η TF efft l D ^, η K [D] K

T F

Tr

(15)

r

e f f

For a bidentate drug n= 3, while f o r a hexadentate drug n= 1. Given a plasma concentration of 4 χ ΙΟ"" M f o r the t r a n s f e r r i n i r o n complex, and assuming that the same plasma concentration of the drug can be a t t a i n e d , one may c a l c u l a t e the s t a b i l i t y constant of the drug ( K § ) which i s r e q u i r e d i f 50% of the t r a n s f e r r i n bound i r o n i s to be sequestered by the drug. I f the drug i s hexa dentate, t h i s i s achieved when K ^ f f = K | f f . However, i f the drugs are t r i d e n t a t e or b i d e n t a t e , t h e i r s t a b i l i t y constants must be 1 0 and 10 times that of K ^ f , r e s p e c t i v e l y . The same property i s i l l u s t r a t e d by the data i n Table I I which compares the degrees of d i s s o c i a t i o n of t e t r a c o o r d i n a t e com plexes with 0, 2, and 3 c h e l a t e r i n g s . A c h e l a t e e f f e c t of 1 0 per c h e l a t e r i n g i s assumed as the b a s i s f o r the a r b i t r a r y s t a b i l i t y constants l i s t e d . The s u p e r i o r p r o p e r t i e s of the metal chelate i n d i l u t e s o l u t i o n are d r a m a t i c a l l y i l l u s t r a t e d by a simple c a l c u l a t i o n of the degrees of d i s s o c i a t o n i n 1.0 molar and 5

f f

4

8

f

2


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TABLE I I Degrees of D i s s o c i a t i o n of Complexes and Chelates i n D i l u t e S o l u t i o n

No. of chelate rings

Formation Constants

Value 10


[M][A]

18

1 χ 10

-5

1 χ 10

-3

1 χ 10

-4

10

4

[MB~]

Ο

_2

10 [Μ][Β]

-3 1.0x10 M Complexes % DissoFree [M] ciation

1.0 M Complexes % DissoFree [M] ciation

3x10

O 3x10

_Q 5x10

Λ 5x10

2

ΙΟ

2 4

1χ

10"

1 2

10"

1 0

3 χ ΙΟ"

1 4

3 χ

ΙΟ"

9

[M][L]

J

1.0 χ 10 molar s o l u t i o n s . I t has been pointed out by Adamson (34) and others (35,36) that the e n t r o p y - r e l a t e d c h e l a t e e f f e c t , as manifested i n the s t a b i l i t y constants, disappears when u n i t mole f r a c t i o n r e p l a c e s u n i t m o l a l i t y as the standard s t a t e of s o l u t e s i n aqueous sys tems. On t h i s b a s i s the s t a b i l i t y constants assumed f o r the model compounds i n Table I I (20) would have to be equivalent i n magnitude r e g a r d l e s s of the number of c h e l a t e r i n g s formed. On the other hand the r e l a t i v e degrees of d i s s o c i a t i o n of the model compounds i n Table I I remain an experimental f a c t , with the l a r g e r c o n c e n t r a t i o n u n i t g i v i n g smaller numerical concentrations f o r the s o l u t i o n s i l l u s t r a t e d , thus compensating f o r the d i s a p pearance of the c h e l a t e e f f e c t i n the numerical values of the s t a b i l i t y constants. Some other f a c t o r s which must be taken i n t o account i n designing l i g a n d s with maximum s t a b i l i t y and s e l e c t i v i t y are summarized i n Table I I I (20) and a s p e c i f i c example of the ad verse e f f e c t of i n c r e a s i n g r i n g s i z e on ΔΗ and AS i s shown i n Table IV (37). Mutual coulombic r e p u l s i o n s between donor groups i n the metal c h e l a t e are important, and the extent to which these r e p u l s i o n s are p a r t i a l l y overcome i n the f r e e c h e l a t i n g l i g a n d r e l a t i v e to analogous unidentate l i g a n d s i s a manifesta t i o n of the enthalpy-based c h e l a t e e f f e c t . T h i s property, which g r e a t l y i n c r e a s e s s t a b i l i t y constants, i s developed to an even higher degree i n m a c r o c y c l i c and c r y p t a t e l i g a n d s that hold the donor groups at geometric p o s i t i o n s r e l a t i v e l y c l o s e to the p o s i t i o n s that they would assume i n the c h e l a t e . Thus s t a b i l i t y and s p e c i f i c i t y would be increased i n a l l types of multidentate l i g a n d s by s y n t h e s i z i n g s t r u c t u r e s i n which the freedom of the donor groups to move away from each other i s decreased as much as


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TABLE I I I Factors I n f l u e n c i n g S o l u t i o n S t a b i l i t i e s Enthalpy E f f e c t s V a r i a t i o n of bond strength with e l e c t r o n e g a t i v i t i e s of metal ions and l i g a n d donor atoms


Ligand

of Complexes

Entropy E f f e c t s Number of chelate r i n g s Size of the chelate r i n g Arrangement of chelate r i n g s

f i e l d effects

Enthalpy e f f e c t s r e l a t e d to the conformation of the uncoordin ated l i g a n d S t e r i c and e l e c t r o s t a t i c r e p u l s i o n s between l i g a n d donor groups i n the complex Other coulombic f o r c e s involved i n chelate r i n g formation

Changes of s o l v a t i o n on complex formation Entropy v a r i a t i o n s i n uncoord inated ligands E f f e c t s r e s u l t i n g from d i f f e r ences i n configurâtional entrop i e s of the f r e e l i g a n d and the l i g a n d i n complex compounds

TABLE IV V a r i a t i o n of Thermodynamic Constants as a of Chelate Ring Size 2+

Ca (aq) +

(""OOCCHJ N- (CH„) -N(CH C00") = Ζ Ζ ζ η Ζ ζ 0

Κ = 2

10.7

o

-ΔΗ°(kcal/mole)

o

Function

[Ca

chelate]

AS°(cal/degree

6.55

26.6

3

7.28

1.74

27.4

4

5.66

0.9

29.7

5

5.2

-

-

8

4.6

_

-

p o s s i b l e . One of the obvious ways to achieve t h i s o b j e c t i v e i s to synthesize organic ligands having r i g i d molecular frameworks, as may be achieved by use of unsaturated linkages and aromatic r i n g s i n the b r i d g i n g groups of the l i g a n d s . Increase i n r i g i d i t y of the l i g a n d would a l s o minimize or remove completely the unfavor able entropy e f f e c t s r e l a t e d to the decrease of v i b r a t i o n a l and r o t a t i o n a l freedom of l i g a n d atoms that g e n e r a l l y occurs i n metal ion c o o r d i n a t i o n . I t i s obvious that many of the f a c t o r s l i s t e d i n Table I I I (e.g., number of r i n g s , chelate r i n g shape) are not s e n s i t i v e to p r o p e r t i e s of the metal i o n and therefore w i l l not provide d i f f e r -



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ences i n metal-ligand i n t e r a c t i o n . S e l e c t i v i t y i n metal complex formation, as measured by d i f f e r e n c e s i n s t a b i l i t y constants, r e quires the use of f a c t o r s that are very s e n s i t i v e to the nature of the metal i o n . The most e f f e c t i v e ways to achieve s e l e c t i v i t y i n chelate formation with multidentate ligands i s to change the nature of the donor group i n such a way as to change the degree of covalency of the metal l i g a n d bonds formed. Thus the matching of the a and b character o f the metal i o n and l i g a n d donor atoms would take advantage of d i f f e r e n c e s i n e l e c t r o n e g a t i v i t y and p o l a r i z a b i l i t y of the metal ions under c o n s i d e r a t i o n . Those f a c t o r s that are s e n s i t i v e to only the s i z e of the metal ion (e.g., s i z e of the c h e l a t e r i n g s ) would probably be l e s s e f f e c t i v e i n achieving s e l e c t i v i t y f o r open-structured l i g a n d s , but may be much more e f f e c t i v e f o r macrocyclic anc c r y p t a t e l i g a n d s . For metal ions d i f f e r i n g i n i o n i c charge and/or c o o r d i n a t i o n number there i s no d i f f i c u l t y i n achieving high degrees of s e l e c t i v i t y , even with r e l a t i v e l y simple c h e l a t i n g l i g a n d s . Geometric C o n s t r a i n t s of Chelate Design There are o f t e n geometric r e s t r i c t i o n s on the c o n s t r u c t i o n of compact, multidentate c h e l a t i n g agents which prevent the most e f f i c i e n t u t i l i z a t i o n of donor groups or atoms. Several authors (38,39) have discussed the geometric c o n s t r a i n t s and pointed out the unique a b i l i t y of the n i t r o g e n atom (and the other Group V elements) to a c t as both a donor atom and as a l i n e a r or b i f u r cate l i n k between other c o o r d i n a t i n g groups, so p e r m i t t i n g the c o n s t r u c t i o n of compact c h e l a t e s t r u c t u r e s . EDTA and i t s analogs are c l a s s i c examples of b i f u r c a t e c h e l a t o r s . Unfortunately, oxygen does not perform t h i s dual f u n c t i o n and DFB i s an example of an oxygen based c h e l a t o r where c r e a t i o n of the optimal octahedral geometry of a hexadentate complex can only be achieved a t the expense of i n t r o d u c t i n g two e s s e n t i a l l y superfluous, elevencarbon chains (Figure 2). Therefore, i f one i s to u t i l i z e the strong a f f i n i t y of oxygen ligands f o r i r o n ( I I I ) , i t i s necessary e i t h e r to incorporate n i t r o g e n i n t o the chelate system or to mimic DFB by using l a r g e and superfluous chain l i n k s . Three i n t e r e s t i n g examples of s y n t h e t i c hexadentate c h e l a t o r s based on c a t e c h o l and designed to mimic the n a t u r a l m i c r o b i a l agent e n t e r o b a c t i n are shown i n Figure 5 (9^,40,^1,^2,^3) . B i o a v a i l a b i l i t y and B i o s t a b i l i t y The e f f i c a c y of an i r o n c h e l a t i n g drug which i s administered o r a l l y w i l l be dependent on the extent to which i t i s absorbed i n the g a s t r o i n t e s t i n a l t r a c t . In man the g a s t r o i n t e s t i n a l pH v a r i e s from ca. 2 i n the stomach, to ca. 6.5 i n the small i n t e s t i n e and 8 i n the colon, while the pH of blood plasma i s 7.4. Passage through the g a s t r o i n t e s t i n a l e p i t h e l i u m i s , f o r most drugs, a passive d i f f u s i o n process which i s more f a c i l e f o r the more l i p o -



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p h i l i c drug. Thus, a drug which i s f u l l y i o n i z e d i n the pH range of the g a s t r o i n t e s t i n a l t r a c t w i l l not pass through the e p i t h e l i a l membrane unless some s p e c i f i c c a r r i e r i s a v a i l a b l e . A drug which i s p a r t i a l l y i o n i z e d w i l l pass through t h i s l i p o - p r o t e i n membrane i n i t s unionized form and w i l l be p a r t i t i o n e d between the plasma and the g a s t r o i n t e s t i n a l t r a c t to an extent which i s dependent on i t s pK and the g a s t r o i n t e s t i n a l pH (44,45). In the case of weak acids


a

[Drue] [Drug]' GI

=

7

4

(l+10 - -

p K

a)(l 10 +

p H

-

p K

a)

(16)

For these reasons, a b s o r p t i o n of an i r o n c h e l a t i n g drug w i l l be favored i f (a) the drug i s l i p o p h i l i c , and (b) the pK of any a c i d i c group(s) i n the molecule i s >3 and the pK of any b a s i c group(s) i n the molecule i f by i n t r o d u c t i o n of long chain a l k y l s u b s t i t u e n t s . The pK i s l e s s e a s i l y manipulated because i t i s g e n e r a l l y the a c i d i c or b a s i c group which i s i n v o l v e d i n i r o n binding; m o d i f i c a t i o n of the pK may then d i m i n i s h i r o n binding c a p a c i t y . For t h i s reason i t i s probably d e s i r a b l e to p r o t e c t s t r o n g l y a c i d i c and b a s i c groups by temporary conversion to d e r i v a t i v e s which regenerate the a c t i v e f u n c t i o n a l group a f t e r the a b s o r p t i o n process. For example, the use of an a l k y l e s t e r of a strong a c i d w i l l e l i m i n ate i o n i z a t i o n and increase l i p o p h i l i c i t y , and subsequent a c t i o n of plasma esterases w i l l serve to regenerate the f r e e a c i d . Various examples of t h i s technique, know as "drug l a t e n t i a t i o n " , have been documented by Harper (46) and more r e c e n t l y by Sinkula and Yalkowsky (47). I n t e r e s t i n g l y , Catsch (48) has suggested that the improved e f f i c a c y of c e r t a i n EDTA-like c h e l a t i n g agents i n promoting heavy metal e x c r e t i o n may be r e l a t e d to t h e i r a b i l i t y to form i n t e r n a l e s t e r s with enhanced b i o a v a i l a b i l i t y ; e.g., a

a

e

a

a

OOCCH„

HOOCCH

2

CH.COO

CH CH OH 2

2

OOCCH„

HOOCCH

2

CH„—C*^

CH —CH 2

2

The o r a l e f f i c a c y of at l e a s t one i r o n c h e l a t i n g drug, EHPG (see Figure 1), i s markedly enhanced by temporarily b l o c k i n g the c a r b o x y l i c a c i d groups as the a l k y l e s t e r . Following i t s a b s o r p t i o n from the g a s t r o i n t e s t i n a l t r a c t , an i r o n c h e l a t i n g drug w i l l be transported to the l i v e r v i a the p o r t a l v e i n . Here the drug must s u r v i v e " d e t o x i f i c a t i o n by 11



17.

Ρίττ ANDMARTELL

Chelating

297

Agents

microsomal o x i d a t i o n , conjugation ( i n the biochemical sense), and e x c r e t i o n , p r i o r to entering the p e r i p h e r a l c i r c u l a t o r y system. For example, a number of p o t e n t i a l l y u s e f u l i r o n - c h e l a t i n g drugs are d e r i v a t i v e s of phenols, c a r b o x y l i c a c i d s , and amines. Carb o x y l i c a c i d s , being a metabolic end product, are s t a b l e to microsomal o x i d a t i o n but a r e subject to conjugation. Phenols are s i m i l a r l y subject to conjugation and a l s o to microsomal hydroxylat i o n . Amines are subject to conjugation ( i f primary or secondary), and to o x i d a t i o n and N - d e a l k y l a t i o n . Conjugation may be blocked to some degree by the same proced ure employed to enhance absorption, i . e . , a d m i n i s t r a t i o n of the i r o n c h e l a t i n g drug i n the form of a d e r i v a t i v e which has t r a n s i t ory i n v i v o s t a b i l i t y and r e v e r t s to the f r e e drug i n the c i r c u l a t o r y system (44). The l o s s of drug due to microsomal metabolism may a l s o be reduced by molecular m o d i f i c a t i o n of the drug. However, i t i s more d i f f i c u l t to accomplish because of the d i f f i c u l t y i n pre d i c t i n g a. p r i o r i what the major metabolic pathway of a s p e c i f i c drug w i l l be. I f t h i s can be determined experimentally, s u b s t i tuents can often be introduced (or removed) to block an u n d e r s i r able metabolic process. The blockage of microsomal h y d r o y x l a t i o n of s t e r o i d s at C-6 by i n t r o d u c t i o n of a s u b s t i t u e n t a t C-6, or a t C-16 by i n t r o d u c t i o n of a s u b s t i t u e n t at C-17, are cases i n point (49). Studies of the metabolism of three i r o n c h e l a t i n g drugs, EHPG, HBED and N-methyl-N-(2-hydroxybenzyl)glycine, are i n pro gress and w i l l h o p e f u l l y provide some information on what f a c t o r s determine t h e i r r e l a t i v e e f f i c a c y i n v i v o , and how t h e i r e f f i c a c y may be improved (50). HOOC

COOH

HBED

HOOC

N-methyl-N-(2-hydroxybenzyl)glycine

The requirement that the c h e l a t i n g agent and i t s i r o n ( I I I ) chelate not i n t e r f e r e with c e l l u l a r biochemistry must be consider ed. Drugs which can pass through the g a s t r o i n t e s t i n a l e p i t h e l i u m are a l s o l i k e l y to penetrate other c e l l membranes. A drug which i s s u i t a b l e f o r o r a l a d m i n i s t r a t i o n may t h e r e f o r e have undesirabel s i d e e f f e c t s unless i t s l i p o p h i l i c character i s r a p i d l y



298

INORGANIC

CHEMISTRY

IN

BIOLOGY

AND

MEDICINE

reduced i n v i v o . This may be p o s s i b l e i f i t s l i p o p h i l i c i t y i s a s s o c i a t e d with an e s t e r f u n c t i o n which i s r a p i d l y cleaved by plasma esterases, and i f the drug acquires i o n i c charge on binding i r o n . In some cases i t i s p o s s i b l e to c o n t r o l the t i s s u e d i s t r i b u t i o n of the c h e l a t i n g agent DFB has been d e r i v a t i z e d as the long chain f a t t y a c y l amide at the terminal amino group and t h i s m o d i f i c a t i o n has been shown to enhance b i l i a r y e x c r e t i o n of the drug (51). The p r i n c i p a l of enterohepatic c h e l a t i n g agents has been proposed (52). Here the b a s i c features which must be exp l o i t e d i n the design of such agents are those which (1) make the c h e l a t i n g agent large and s u f f i c i e n t l y non-polar to prevent r e n a l f i l t r a t i o n and/or (2) provide i t with a s t r u c t u r e which permits i t to p a r t i c i p a t e i n the enterohepatic c i r c u l a t i o n of the b i l e a c i d s and t h e i r d e r i v a t i v e s . The c i r c u l a t i o n ceases when the c h e l a t o r acquires charge and greater h y d r o p h i l i c i t y on binding F e ( I I I ) , and reabsorption i n the i n t e s t i n e i s no longer f a v o r a b l e . Advantages of t h i s approach are the use of c h e l a t i n g agents more l i k e l y to be o r a l l y a c t i v e , avoidance of n e p h r o t o x i c i t y , and more e f f e c t i v e l o c a l i z a t i o n i n the l i v e r , an organ where i r o n overload can be acute. Cholylhydroxamic a c i d (53) may be an example of an i r o n c h e l a t i n g agent which f u n c t i o n s by t h i s mechanism. Some long chain a l k y l d e r i v a t i v e s of c h e l a t i n g agents have been prepared with the idea of promoting l o c a l i z a t i o n i n the myocardium (54). This has obvious relevence to i r o n c h e l a t i o n therapy because of the f a t a l s u s c e p t i b i l i t y of the heart to i r o n overload. Liposomal encapsulation of the c h e l a t i n g agent p r i o r to i n j e c t i o n i s an a l t e r n a t i v e means of l o c a l i z i n g i t i n the l i v e r and spleen, an approach which i s already being u t i l i z e d s u c c e s s f u l l y f o r the a d m i n i s t r a t i o n of drugs to t r e a t l e i c h m a n i a s i s (55). DTPA has been encapsulated with liposomes and i n t h i s form i s reported to be more e f f e c t i v e than the non-encapsulated c h e l a t o r i n removing plutonium i n mice (56). The attachment of i r o n c h e l a t i n g l i g a n d s to polymers i s an a l t e r n a t i v e means of modifying b i o a v a i l a b i l i t y . DFB has been c o v a l e n t l y bonded to p o l y ( a c r o l e i n ) and other s y n t h e t i c polymers and shown to have some p o t e n t i a l f o r use i n e x t r a c o r p o r e a l detoxi f i c a t i o n of acute i r o n overloaded plasma (57). Poly(N-methacrylo y l - 3 - a l a n i n e hydroxamic a c i d ) , a polydentate polymer obtained by d e r i v a t i z a t i o n of p o l y ( a c r y l i c acid) with pendant hydroxamic a c i d groups, has shown s i g n i f i c a n t i r o n c h e l a t i o n a c t i v i t y i n v i v o (58), a r e s u l t which i s p o s s i b l y r e l a t e d to the longer r e t e n t i o n of polymeric species i n the c i r c u l a t o r y system. F i n a l l y , i t should be mentioned that continuous i n f u s i o n of DFB has proven f a r more e f f e c t i v e than s i n g l e i n j e c t i o n s f o r the same dose (59,60). This may be because DFB i s only able to remove i r o n from c e r t a i n body pools, which are r e p l e n i s h e d r e l a t i v e l y slowly by i r o n from other body pools. Continuous i n f u s i o n serves to maintain an e f f e c t i v e plasma l e v e l of DFB, which would other-


17.

Chelating

ριττ A N D M A R T E L L

Agents

299

wise be reduced by the r a p i d metabolism of the drug.


E v a l u a t i o n of Iron Chelating Agents Using Animal Models of Overload

Iron

A s i g n i f i c a n t amount of information on the r e l a t i v e e f f i c e n c i e s of i r o n c h e l a t i n g drugs i n v i v o has been c o l l e c t e d r e c e n t l y using two animal models to simulate the c o n d i t i o n of i r o n over load (7,61,62,63). Both animal models u t i l i z e i p i n j e c t i o n s of heat damaged red c e l l s to achieve overload. In one screen r a t i s the t e s t animal (7,61), drug a d m i n i s t r a t i o n and t r a n s f u s i o n s are concurrent, and e f f i c a c y i s measured by the i r o n excreted i n the urine and feces. The second screen uses the mouse (62,63), drug a d m i n i s t r a t i o n i s i n i t i a t e d two days a f t e r t r a n s f u s i o n s are com p l e t e , and e f f i c a c y i s based on percent i r o n depleted from the l i v e r and spleen, plus u r i n a r y i r o n e x c r e t i o n . Both screens can be extended to i n c l u d e e v a l u a t i o n of other t i s s u e s . Published r e s u l t s (53,61,63,64) obtained using these animal screens are shown i n Tables V-VII, and DFB serves as a u s e f u l standard i n both screens. I t i s apparent that there are some d i f f e r e n c e s i n the r e s u l t s of the two screens; i n p a r t i c u l a r , 2,3dihydroxybenzoic a c i d and r h o d o t o r u l i c a c i d are a c t i v e i n the r a t screen but not i n the mouse screen. This discrepancy p o s s i b l y a r i s e s because of the d i f f e r e n c e i n the transfusion-drug adminis t r a t i o n sequence i n the two screens, f o r i t i s known that i r o n becomes p r o g r e s s i v e l y l e s s a c c e s s i b l e to c h e l a t i o n as the time between t r a n s f u s i o n and drug a d m i n i s t r a t i o n i n c r e a s e s . Most of the compounds l i s t e d i n Tables V-VII are known or can be expected to have a high and s e l e c t i v e a f f i n i t y f o r i r o n ( I I I ) , with 3 and K j f values of at l e a s t 1 0 and 1 0 , r e s p e c t i v e l y . Despite t h i s , many of the compounds tested are only weakly a c t i v e while others show r e l a t i v e a c t i v i t i e s not p r e d i c t e d by t h e i r s t a b i l i t y constants. Of the compounds shown i n Figure 1, 3 - i s o p r o p y l tropolone, 1,8-dihydroxynaphthalene and acetohydroxamic a c i d are e s s e n t i a l l y i n a c t i v e , while 2,3-dihydroxynaphthalene-6-sulfonic a c i d and 8-hydroxyquinoline are a c t i v e i n both screens. EHPG (β = 1 0 ' , K e f f = 1 0 · ) i s more e f f e c t i v e than HBED (3 = 1 0 · , K f f = 1 0 ' ) which i s the reverse order of t h e i r i r o n ( I I I ) s t a b i l i t y constants. These i r r e g u l a r i t i e s must be a t t r i b u t e d to the importance of f a c t o r s already r e f e r r e d to, such as b i o a v a i l a b i l i t y , b i o s t a b i l i t y , and the k i n e t i c s of i r o n c h e l a t i o n . The increase i n the o r a l e f f i c a c y of EHPG on conversion to the dimethyl ester (63) has already been noted. The l o s s of a c t i v i t y on s u l f o n a t i o n of 8-hydroxyquinoline and 1,8-dihydroxynaphthalene i s another i l l u s t r a t i o n of the importance of the i o n i c form of the c h e l a t o r . Hydroxamic a c i d s r e p r e s e n t a t i v e of a l l of the major s t r u c t u r a l f a m i l i e s of m i c r o b i a l i r o n chelates (65) have been evaluated. With the p o s s i b l e exception of r h o d o t o r u l i c a c i d (vide supra) and t r i a c e t y l f u s a r i n i n e C, none show a c t i v i t y approaching that of DFB. Several bidentate hydroxamic a c i d s increase i r o n l e v e l s i n the 2 8

20

e

3 3

9

1 6

2 0

3

8

e


3 9

7

INORGANIC

300

Q) rH Ο

μ U £2

O C ^ N f O O N N o M

INORGANIC CHEMISTRY IN BIOLOGY A N D

312

40. 41. 42. 43. 44.


45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

63. 64. 65.

MEDICINE

Collins, D.J.; Lewis, G.; Swan, J.M. Aust. J. Chem., 1974, 27, 2593. Corey, E.J.; Hurt, S.D. Tetrahedron Lett., 1977, 3923. Venuti, M.C.; Rastesster, W.H.; Neilands, J.B. J. Med. Chem. 1979, 22, 123. Weitl, F.L.; Raymond, K.N. J. Am. Chem. Soc., 1979, 101, 2728. Schanker, L.S.; Tocco, D.J.; Brodie, B.B.; Hogben, C.A. J. Pharmacol. Exp. Ther., 1958, 123, 81. Goldstein, Α.; Aronow, L.; Kalman, S.K. "Principles of Drug Action", Wiley, New York, 1974. Harper, N.J. Progr. Drug. Res., 1962, 4, 221. Sinkula, A.A.; Yalkowsky, S.F. J. Pharm. Sci., 1975, 64, 181. Catsch, A. Fed. Proc., 1961, 20, Suppl.10, pt. II, 206. Deghenghi, R.; Munson, A.J. in "Medicinal Chemistry" Burger, A. Ed., Wiley-Interscience, New York, 1970, p.900. Cook C.E. Research Triangle Institute, unpublished studies. Meyer-Brunot, H.G.; Keberle, H. Amer. J. Physiol, 1968, 214, 1193. Jones, M.M.; Pratt, T.H.; Mitchell, W.G.; Harbison, R.D.; McDonald, J.S. J. Inorg. Nucl. Chem., 1976, 38, 613. Grady, R.W.; Graziano, J.H.; White, C.P.; Jacobs, Α.; Cerami, A. J. Pharmacol. Exp. Ther., 1978, 205, 757. Eckelman, W.C.; Karesh, S.M.; Reba, R.C. J. Pharm. Sci., 1975, 64, 704. Alving, C.R.; Steck, E.A.; Hanson, W.L.; Loizeaux, P.S.; Chapman, Jr., W.L.; Waits, V.B. Life Sciences, 1978, 22, 1021. Rahman, Y.E.; Rosenthal, M.W.; Cerny, A.E. Science, 1973, 180, 300. Ramirez, R.S.; Andrade, J.D. J. Macromol. Sci. Chem., 1976, A10, 309. Winston, Α.; McLaughlin, G.R. J. Poly. Sci., 1976, 14, 2155. Copper, B.; Bunn, H.F.; Propper, R.D.; Nathan, D.G.; Rosenthal, D.S.; Maloney, W.C. Amer. J. Med., 1977, 63, 958. Propper, R.D.; Cooper, B.; Rudo, R.R. N. Engl. J. Med., 1977, 297, 418. Graziano, J.H.; Grady, R.W.; Cerami, A. J. Pharmacol. Exp. Ther., 1974, 190, 570. Gralla, E.J. in "Symposium on the Development of Iron Chelators for Clinical Use", Anderson, W.F.; Hiller, M.C. Eds., DHEW Publication No. (NIH) 77-994, Bethesda, Md., 1975, pp.229-254. Pitt, C. G.; Gupta, G.; Estes, W.E. et al. J. Pharmacol. Exp. Ther., 1979, 208, 12. Grady, R. W.; Graziano, J.H.; Akers, H.A.; Cerami, J. Pharmaco1. Exp. Ther., 1976, 196, 478. Neilands, J.B. 178th National Meeting, American Chemical Society, Washington, D.C., Sept.9-14, 1979, Paper No. Inorg. 36.

RECEIVED May 22,

1980.


Inorganic Chemistry in Biology and Medicine - ACS Publications

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