Biochemistry and Pharmacology of Ring-Fluorinated Imidazoles - ACS

2 Biochemistry and Pharmacology of Ring-Fluorinated Imidazoles

Downloaded by UNIV OF PITTSBURGH on October 11, 2013 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0028.ch002

KENNETH L. KIRK and LOUIS A. COHEN Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Md. 20014

Medicinal chemists and pharmacologists have long recognized the value of fluorine in the design of analogues of metabolically significant molecules (1, 2). The high electronegativity of fluo rine can effect a marked alteration in electron-density d i s t r i b u tion, pK, conformation, etc.; simultaneously, this atom, by virtue of i t s small van der Waals radius (1.35 A), offers minimal steric interference to binding of the analogue at a specific macromolec ular s i t e . The effective size of fluorine attached to an sp carbon may be considered to fall between those of hydrogen and the hydroxyl group, while fluorine on an sp carbon is probably some what smaller — the result of lone pair overlap with the adjacent pi system (3, 4). Effective size undoubtedly varies, also, with solvation effects and specific lone pair interactions. 3

2

C = C

―

C ― C =

+

Ring-fluorinated analogues of a variety of aromatic and heteroaromatic biomolecules have been synthesized and evaluated as agonists and antagonists of their natural relatives. We realized, some years ago, that the imidazole ring, one of the most ubiquit ous and important of natural heteroaromatic systems, could claim no documented fluoro derivatives, and initiated an effort to fill this gap. After we had exhausted the classical synthetic routes (5, 6), abandonment of the effort seemed inevitable. In other studies, we had found that imidazolediazonium ions, which are un usually stable to heat, are transformed photochemically to highly reactive species, possibly carbonium ions or carbenes (with expul sion of molecular nitrogen) (7). It seemed reasonable that such reactive species might capture fluoride and other poorly nucleo p h i l i c anions. Indeed, the f i r s t ring-fluorinated imidazole was obtained in 1971 by photolysis of ethyl 4-diazoniumimidazole-5carboxylate in 50% aqueous fluoroboric acid (8, 9). Subsequently, a wide variety of both 2- and 4-fluoroimidazoles were prepared for chemical and biological studies, of which many are still in pro gress. 23 In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

24

BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS


S y n t h e t i c Methods The most general procedure f o r the s y n t h e s i s of 4 - f l u o r o imidazoles i s i l l u s t r a t e d i n F i g u r e 1 (10, 11), using aminoimidazole precursors. Unless they have the c a p a b i l i t y f o r resonance overlap with an e l e c t r o n s i n k ( n i t r o , cyano, carboxylate, etc.) at C-5, 4-aminoimidazoles show an i n s t a b i l i t y resembling that of v i n y l a m i n e s , and cannot normally be i s o l a t e d without p a r t i a l decomposition. C a t a l y t i c hydrogénation of 4 - n i t r o i m i d a z o l e s proved u n s a t i s f a c t o r y as a source of n o n s t a b i l i z e d 4-aminoimidazoles; however, z i n c dust r e d u c t i o n of the n i t r o group i n 50% f l u o r o b o r i c a c i d , which proceeded r a p i d l y and q u a n t i t a t i v e l y at low temperat u r e , became the method of choice. Immediately upon completion of r e d u c t i o n , the aminoimidazole i s d i a z o t i z e d w i t h sodium n i t r i t e i n s i t u , and the r e s u l t i n g diazonium i o n i s subjected to p h o t o l y s i s , again i n s i t u . A f t e r n e u t r a l i z a t i o n of the f l u o r o b o r i c a c i d medium, the product i s u s u a l l y recovered by e t h y l acetate e x t r a c t i o n , o v e r a l l y i e l d s (based on n i t r o i m i d a z o l e ) ranging from 20-40%. Since the other products of p h o t o l y s i s are g e n e r a l l y h i g h l y p o l a r , hydroxylated imidazoles, they are not removed by the e x t r a c t i o n s o l v e n t and do not i n t e r f e r e with p u r i f i c a t i o n . In c o n t r a s t to the 4-amino s e r i e s , 2-aminoimidazoles show the s t a b i l i t y to be expected of a l k y l a t e d guanidines. These compounds are generated by c a t a l y t i c hydrogénation of 2-arylazoimidazoles which, i n t u r n , are obtained by coupling of the imidazole w i t h aryldiazonium i o n (Figure 2 ) . Although such coupling occurs predominantly at C-2, smaller q u a n t i t i e s of the 4- and 2,4-bisa r y l a z o d e r i v a t i v e s are formed (12). I t i s e s s e n t i a l that the d e s i r e d isomer be freed of these contaminants ( u s u a l l y by column chromatography) p r i o r to hydrogénation, s i n c e p u r i f i c a t i o n of the r e s u l t a n t 2-aminoimidazole has proved extremely l a b o r i o u s . The pure 2-aminoimidazole i s then d i a z o t i z e d and the diazonium i o n i s photolyzed i n s i t u (13). This photochemical method has proved i t s e l f of v a l u e , w i t h respect to r a p i d i t y , convenience, and y i e l d , i n the synthesis of r i n g - f l u o r i n a t e d d e r i v a t i v e s of s e n s i t i v e or complex aromatic systems (£.£., a l k a l o i d s (14) and catecholamines (15)), as w e l l as other heteroaromatic systems, such as t h i a z o l e (7) and p y r r o l e (16). Special

Properties

A s i n g l e f l u o r o s u b s t i t u e n t , at C-2 or C-4, reduces the basi c i t y of the imidazole r i n g by 5-6 pK u n i t s and increases i t s a c i d i t y (NH—*N~) by 4-5 u n i t s (17) . The magnitudes of these e f f e c t s are s i g n i f i c a n t l y greater than those of the other halogens and i n d i c a t e that, i n c o n t r a s t to the fluorobenzenes, the i n d u c t i v e e f f e c t of f l u o r i n e on the imidazole r i n g overwhelms any e l e c t r o n - r e l e a s i n g e f f e c t due to resonance. I n c o n s i s t e n c i e s i n the l ^ F nmr s i g n a l s f o r the f l u o r o i m i d a z o l e s i n d i c a t e f u r t h e r that

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

2.

KIRK AND COHEN

/=(

Ring-Fluorinated

0 N

£H CH NHAc 2

2

2

25

Imidazoles

^CH^NHAc

N

HN0

3

Zn 50%HBF -10

4


e

7\

CH2CH2NHAC NaN02 2. h*. -10"

N^NH

CH CH NHAc 2

2

=

N^NH

Figure 1

ArN + pH 8-9

ArN^

2

.R

ArN ^

N^NH

1

2

NyNH

NaAr

NeAr

Pt.Hî AcOH

Mutt be removed before hydrogenolysn + ArNH

2

N

y

N

^R

H

NM

2

Figure 2


26



these compounds cannot be t r e a t e d i n p a r a l l e l w i t h the f l u o r o benzenes (17). The f l u o r i n e atom a t C - 4 , unless a c t i v a t e d by an e l e c t r o n s i n k a t C-5, has shown no evidence of r e a c t i v i t y toward nucleop h i l e s ; i n c o n t r a s t , 2 - f l u o r o i m i d a z o l e s are moderately r e a c t i v e toward displacement, p a r t i c u l a r l y i n the ring-protonated form (13), while the f l u o r o i m i d a z o l e anion i s s i g n i f i c a n t l y more s t a b l e (Figure 3 ) . Thus, c t - N - t r i f l u o r o a c e t y l - 2 - f l u o r o h i s t i d i n e cannot be deacylated by a c i d h y d r o l y s i s without l o s s of the f l u o r i n e atom, but the compound i s r e a d i l y deacylated i n m i l d base. Another consequence of the r e a c t i v i t y of 2 - f l u o r o i m i d a z o l e s i s the f a c i l e condensation to c y c l i c t r i m e r s (Figure 4), which appear t o be l a r g e r i n g , heteroannular aromatic systems. Since imidazole i t s e l f has been found to undergo f a c i l e hydrogen-isotope exchange a t C-2 but not a t C-4 (18), we expected an even more f a c i l e exchange w i t h 4 - f l u o r o i m i d a z o l e s . Surprisingl y , the l a t t e r compounds show no tendency t o exchange a t C-2 under a wide v a r i e t y of c o n d i t i o n s ; i n c o n t r a s t , 2 - f l u o r o i m i d a z o l e s exchange r a p i d l y a t C-4 above pH 10, but much more slowly a t n e u t r a l pH. Thus, a route became a v a i l a b l e f o r the s p e c i f i c t r i t i u m l a b e l l i n g of 2 - f l u o r o h i s t i d i n e and 2-fluorohistamine, compounds which subsequently proved t o have extensive biochemical u t i l i t y . The a b i l i t y o f F-2 t o a c t i v a t e H-4 i s q u i t e unique; t o date, no other s u b s t i t u e n t a t C-2, whether more o r l e s s e l e c t r o n e g a t i v e than f l u o r i n e , ^ h a s demonstrated a c a p a b i l i t y of such magnitude. A route t o 2-[ H ] - 4 - f l u o r o h i s t i d i n e was developed r e c e n t l y , based on our observation that these exchange processes a r e subject to strong b u f f e r c a t a l y s i s . B i o l o g i c a l Properties Several d e t a i l e d r e p o r t s o f s t u d i e s w i t h f l u o r o i m i d a z o l e s have already been published or a r e i n press; the m a j o r i t y , however, a r e s t i l l i n t h e i r e x p l o r a t o r y stages. The f o l l o w i n g d i s c u s s i o n does not represent a comprehensive l i s t i n g of these studi e s ; r a t h e r , i t presents a sampling, intended t o demonstrate the v a r i e t y and scope o f the b i o l o g i c a l a p p l i c a t i o n s of f l u o r o i m i d a z o l e s and, h o p e f u l l y , t o suggest d i r e c t i o n s f o r f u r t h e r a p p l i c a tion. 2 - F l u o r o h i s t i d i n e . When t r i t i a t e d 2 - f l u o r o - L - h i s t i d i n e i s administered t o mice subcutaneously, the amino a c i d i s r a p i d l y d i s t r i b u t e d throughout the animal. Ten minutes a f t e r a d m i n i s t r a t i o n , the p r i n c i p a l organs ( i n c l u d i n g b r a i n ) show t r i t i u m l e v e l s 2-7 times that o f the blood. A f t e r 72 h r , two-thirds of the o r i g i n a l t r i t i u m count i s s t i l l w i t h i n the animal, and h a l f o f t h i s amount i s found i n i n s o l u b l e p r o t e i n f r a c t i o n s (19). Since we had shown that replacement of the 2 - f l u o r o group by any other s u b s t i t u e n t prevents exchange o f isotope a t C - 4 , and s i n c e a l l the t r i t i u m could be back exchanged from the p r e c i p i t a t e d p r o t e i n


2.

KIRK AND COHEN

Ν

Π η

+

R

S

H

27

Imidazoles

•

H^piH

+

R

S

F


F

Ring-Fluorinated


"


28

BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS

f r a c t i o n s at pH 11, the analogue must have entered i n t o de novo p r o t e i n synthesis i n place of h i s t i d i n e ; s i n c e the 2-fluorοimid azole moiety i s i n t a c t , covalent attachment of the f l u o r o h i s t i d i n e must have occurred at the amino a c i d s i d e chain. Such i n c o r p o r a t i o n i n t o the mouse i s blocked by cycloheximide and, i n i s o l a t e d l i v e r ribosomes, by actinomycin, both drugs being i n h i b i t o r s of protein synthesis. Studies with r a t p i n e a l gland have s u p p l i e d f u r t h e r evidence f o r the i n c o r p o r a t i o n of 2 - f l u o r o h i s t i d i n e i n t o newly synthesized p r o t e i n (20). H i s t i d i n e and i t s 2 - f l u o r o analo gue are u t i l i z e d c o m p e t i t i v e l y , s i n c e a d m i n i s t r a t i o n of an excess of h i s t i d i n e reduces f l u o r o h i s t i d i n e i n c o r p o r a t i o n . Presumably, the h i s t i d i n e analogue i s incorporated i n t o a l l newly synthesized pro t e i n without d i s c r i m i n a t i o n ; y e t , i t i s conceivable that c e r t a i n f l u o r i n e - c o n t a i n i n g enzymes may r e t a i n a c t i v i t y . S i n g l e doses of 2 - f l u o r o h i s t i d i n e (up to 250 mg per k i l o ) give no evidence of t o x i c i t y , organ degeneration, or r e t a r d a t i o n of growth of the mouse over a 30-day p e r i o d . ^ Concentrations of 2 - f l u o r o h i s t i d i n e as low as 10 M are b a c t e r i o s t a t i c ; i n h i b i t i o n of growth of E. c o l i (wild) i s essen t i a l l y complete i n 3 hr at 37°, but the i n h i b i t i o n i s reversed by a d d i t i o n of h i s t i d i n e (20 yg/ml) (21). During the course of the i n h i b i t i o n , the mass of the c u l t u r e increases about t h r e e f o l d , but the number of v i a b l e c e l l s increases about n i n e f o l d . These data suggest that the b a c t e r i a , which contain two to three copies of t h e i r chromosome during the normal growth phase, are unable to c a r r y out f u r t h e r chromosomal r e p l i c a t i o n i n the presence of the drug but are able to undergo c e l l u l a r d i v i s i o n . As shown i n Table I, 2 - f l u o r o h i s t i d i n e i s the most e f f e c t i v e b a c t e r i o s t a t i c agent of the s e v e r a l f l u o r o h e t e r o c y c l e s t e s t e d to date. The preceding s t u d i e s suggest that 2 - f l u o r o h i s t i d i n e may be u s e f u l wherever p r o t e i n synthesis i n a l i e n organisms or c e l l s occurs more r a p i d l y than i n the host s p e c i e s . Thus, the amino a c i d shows a n t i v i r a l behavior i n various i n f e c t e d c e l l c u l t u r e s (Table I I ) , at concentrations s i g n i f i c a n t l y below those needed to produce any v i s i b l e e f f e c t on the host c e l l s (22). To our know ledge, t h i s i s the f i r s t amino a c i d analogue to show a n t i v i r a l p r o p e r t i e s . Although i t s mechanism of a c t i o n has yet to be e l u c i dated, b i o s y n t h e s i s of ' f a l s e ' phosphorylases or v i r u s - c o a t p r o t e i n s are l o g i c a l p o s s i b i l i t i e s . Small peptides c o n t a i n i n g f l u o r i n e are more r e a d i l y obtained by t o t a l synthesis than by b i o s y n t h e s i s . Thus, t h y r o t r o p i n - r e l e a s i n g f a c t o r (TRF) and l u t e i n i z i n g hormone-releasing f a c t o r (LHRF) have been synthesized with 2- and 4 - f l u o r o h i s t i d i n e as r e p l a c e ments f o r the s i n g l e h i s t i d i n e residues (23). While the 4 - f l u o r o analogues are i n a c t i v e , those c o n t a i n i n g 2 - f l u o r o h i s t i d i n e show 20-30% r e l e a s i n g a c t i v i t y . In view of the marked l o s s i n b a s i c i t y


2.

KIRK AND COHEN

Table I .

Ring-Fluorinated

E f f e c t of F l u o r o Compounds on E. c o l l

O.D. Compound added


29

Imidazoles

(wild)

a f t e r 18 h r a t 37° (Klett units)

None 2-Fluorohistidine 4-Fluorohistidine 4-Fluoroimidazole 2-Fluorourocanic a c i d 2-Fluoro-4-hydroxyethylthiazole Same + thiamine (2 yg/ml)

375 8 375 370 370 63 270

In minimal glucose medium; e s s e n t i a l l y the same r e s u l t s were obtained with 10*3, 10""*, or 10~ M i n h i b itor. 5

Table I I .

A n t i v i r a l A c t i v i t i e s of

Fluorohistidines

Minimal i n h i b i t o r y Cell culture

Virus

d

VSV HSV-1 Vaccinia VSV Coxs. B4 Polio I Measles Coxs. B4

Required

d

PRK PRK PRK HeLa HeLa HeLa VERO VERO

2-Fluorohistidine

30 30 30 30 30 30 10 30

4-Fluoro- , histidine

100 >100 >100 >100 >100 >100 >100 >100

e

cone.

(pg/ml)

a

Ribavirin

C

_

-10 10 30 7 30

to reduce v i r u s - i n d u c e d c y t o p a t h o g e n i c i t y by 50%.

E s s e n t i a l l y s i m i l a r r e s u l t s were obtained f o r p - f l u o r o phenylalanine. An e s t a b l i s h e d a n t i v i r a l agent, Ι-β-Dribofuranosyl-1,2,4-triazole-3-carboxamide. Abbreviations are i d e n t i f i e d i n r e f . 32. At concentrations of 100 ug/ml, morphological a l t e r a t i o n of the host c e l l s was apparent only a f t e r 3 days. e


30


p-Glu-His-Pro-NH

2

(TRF)

p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH

2

(LHRF)


of the imidazole r i n g f o l l o w i n g i n t r o d u c t i o n of f l u o r i n e , the e x i s t e n c e of any a c t i v i t y i s s u r p r i s i n g , and these r e s u l t s suggest that r e c o g n i t i o n of the peptide by i t s receptor may depend more on o v e r a l l conformation than on imidazole b a s i c i t y . A - F l u o r o h i s t i d i n e . Despite the minimal s t e r i c consequences of f l u o r i n e s u b s t i t u t i o n and the s i m i l a r i t y i n r i n g b a s i c i t i e s of the isomers, 2 - f l u o r o - and A - f l u o r o h i s t i d i n e are r e a d i l y d i f f e r e n t i a t e d by the h i s t i d i n e t-RNA systems. To date, the evidence i n d i c a t e s that A - f l u o r o h i s t i d i n e does not s u b s t i t u t e f o r h i s t i d i n e i n p r o t e i n b i o s y n t h e s i s , nor does t h i s analogue show s i g n i f i c a n t b a c t e r i o s t a t i c or a n t i v i r a l a c t i v i t y (Tables I and I I ) . Nmr s t u d i e s suggest that the isomers have d i f f e r e n t conformations (17), f l u o r i n e at C-A being i n v o l v e d , perhaps, i n an intramolec u l a r hydrogen bond. Yet, the A - f l u o r o isomer i s a s u b s t r a t e f o r b a c t e r i a l h i s t i d i n e decarboxylase (but not f o r the mammalian enzyme), while both isomers are modest substrates f o r h i s t i d i n e ammonia-lyase (Table I I I ) . The a b i l i t y of A - f l u o r o h i s t i d i n e to f u n c t i o n both as a weak s u b s t r a t e and a strong competitive i n h i b i t o r f o r the l a t t e r enzyme prompted a r e i n v e s t i g a t i o n of i t s mechTable I I I . E f f e c t s of F l u o r o h i s t i d i n e s on H i s t i d i n e Ammonia-Lyase (pH 9, 25°) Κ Compound

or (mM)

m

L-Histidine

2.7

A-F-L-Histidine

1.25

2-F-L-Histidine

170

K. 1

V (uM/mg/min)

30 0.85 1-2

anism of a c t i o n (2A). A n a l y s i s of k i n e t i c data, r e v e r s i b i l i t y , i s o t o p e i n c o r p o r a t i o n , and isotope e f f e c t s demonstrates that the r a t e - l i m i t i n g step f o r t h i s enzyme i s not the breakdown of an intermediate aminoenzyme (as p r e v i o u s l y supposed), but the almost concerted l o s s of a 3-hydrogen atom and the c o v a l e n t l y bound amino group. While the isomers l o s e ammonia at comparable r a t e s , Af l u o r o h i s t i d i n e i s bound much more e f f e c t i v e l y . Urocanase, the enzyme f o l l o w i n g the ammonia-lyase i n the c a t a b o l i c sequence f o r h i s t i d i n e (Figure 5), promotes the h y d r a t i o n rearrangement of urocanic a c i d (25). While n e i t h e r f l u o r o u r o c a n i c a c i d i s s i g n i f i c a n t as a s u b s t r a t e (Table IV), the 2 - f l u o r o isomer


2.

KIRK AND COHEN

Ring-F luorinated

31

Imidazoles

Table IV. E f f e c t s of F l u o r o u r o c a n i c A c i d s on Urocanase (pH 7.4, 25°)

7

10 K Compound


Urocanic a c i d

m

or (M)

K

±

V (units/ml)

400

1.2

-

4-F-Urocanic

acid

-

2-F-Urocanic

acid

1

0.008

measurable a c t i v i t y as i n h i b i t o r or substrate.

i s now a very potent i n h i b i t o r (26). We are not aware of any enzyme capable of d i r e c t removal of f l u o r i n e from the imidazole r i n g . The enzymes which degrade the imidazole r i n g of h i s t i d i n e , e v e n t u a l l y to glutamic a c i d , can a l s o operate on the f l u o r o h i s t i d i n e s , a l b e i t very slowly. Thus f a r , we have found no evidence f o r metabolites of these analogues i n v i v o . As already mentioned, the f l u o r o h i s t i d i n e s can be i n c o r p o r a t e d i n t o a p o l y p e p t i d e sequence v i a t o t a l s y n t h e s i s . Thus, the peptide fragment, r i b o n u c l e a s e - S - ( l - 1 5 ) , has been s y n t h e s i z e d with 4- f l u o r o - L - h i s t i d i n e r e p l a c i n g h i s t i d i n e - 1 2 (27). T h i s f l u o r i n e c o n t a i n i n g peptide (4-F-His-RNase-(l-15)) a s s o c i a t e s w i t h RNase-S(21-124) as w e l l as, and even more s t r o n g l y than, RNase-S-(l-20). A v a r i e t y of c r i t e r i a suggest that the f l u o r o h i s t i d i n e - c o n t a i n i n g aggregate has a three-dimensional s t r u c t u r e very s i m i l a r to that of the complex, RNase-S'. The noncovalent recombination of RNase5- (l-15) or -(1-20) w i t h RNase-S-(21-124) r e s t o r e s p r a c t i c a l l y a l l the enzymatic a c t i v i t y of the o r i g i n a l enzyme, RNase-A; the a n a l ogous complex with 4-F-His-RNase-(l-15) i s t o t a l l y devoid of a c t i v i t y , although i t i s probably s t i l l capable of b i n d i n g s u b s t r a t e . The r e s u l t s provide strong support f o r the proposal that His-12 f u n c t i o n s as a general a c i d - g e n e r a l base c a t a l y s t i n the enzymatic process — a r e d u c t i o n of 5 u n i t s i n the pK of the r i n g being more than ample to remove t h i s c a t a l y t i c c a p a b i l i t y . Fluorohistamines. Two types of r e c e p t o r , termed HI and H2, have been i d e n t i f i e d as mediators of histamine's b i o l o g i c a l a c t i o n s . C h a r a c t e r i z a t i o n of these r e c e p t o r s , and of histamine's b i o l o g i c a l r o l e s , have been hampered by l a c k of agents which s e l e c t i v e l y b i n d one type. Recently, i t has been found that v a r i o u s s u b s t i t u t i o n s at C-2 or C-4 of the histamine r i n g s e l e c t i v e l y decrease b i n d i n g f o r H2 or HI r e c e p t o r s , r e s p e c t i v e l y . Thus, 2phenylhistamine r e t a i n s some 20% of histamine's potency at HI r e c e p t o r s , but i s l e s s than 0.1% as e f f e c t i v e at H2 r e c e p t o r s . In a p r e l i m i n a r y study, 2-fluorohistamine was found (28) to have


32

BIOCHEMISTRY

INVOLVING C A R B O N - F L U O R I N E BONDS

/CH -CHC00H

CH=CHCOOH

2

NH

Lyase

2


-NH3 Urocanase +H2Q CL H00CCHCH2CH C00H 2

Hydrolase

NH

£H CH C00H 2

2

N ^ N H

CH= NH Figure 5

-do HO-CHl

H0-CH2

ON

OH

5-FICAR 5-Fluoro-L β-D-ribofuranosylimidazole4-carboxamide

ribavirin 1 -β-D-ribofuranosyîl 2,4-triazoh-3carboxamide y

5-AICAR 5-amino-l -β-Ό-rihofuranosuUmidazole4-carboxamide

Figure 6


2.

ΚΓΑΚ AND COHEN

Ring-Fluonnated

Imidazoles

33


equal or greater a f f i n i t y f o r HI r e c e p t o r s ( i n guinea p i g ileum) than the p r e v i o u s l y most e f f e c t i v e HI a g o n i s t , 2-aminoethylthiaz o l e . By analogy, 4-fluorohistamine might act as a s e l e c t i v e agon i s t f o r H2 r e c e p t o r s , a p o s s i b i l i t y now under i n v e s t i g a t i o n . 2-Fluorohistamine was a l s o found to be an e f f e c t i v e s t i m u l a t o r of c y c l i c AMP formation i n b r a i n s l i c e s , which c o n t a i n both HI and H2 receptors (29). Fluoroimidazole Ribosides. R e l a t i v e l y simple imidazole d e r i v a t i v e s occupy key r o l e s as intermediates i n the b i o s y n t h e s i s of the purine n u c l e o s i d e s . A major approach to the design of a n t i v i r a l agents i s based on analogues of Imidazole species which might i n t e r f e r e with the b i o s y n t h e s i s of n u c l e o s i d e s or of n u c l e i c a c i d s (Figure 6 ) . Thus, r i b a v i r i n , a t r i a z o l e analogue of 5-AICAR, commands s e r i o u s a t t e n t i o n as a broad-spectrum a n t i v i r a l agent (30) . We have synthesized 5-FICAR, a f l u o r o analogue of 5-AICAR (31) ; t h i s compound shows a p a t t e r n of a n t i v i r a l a c t i v i t y q u i t e s i m i l a r to that of r i b a v i r i n (32), and both compounds were found to b l o c k the b i o s y n t h e s i s of both DNA and RNA. R i b a v i r i n has been shown to act by i n h i b i t i n g the enzyme, IMP dehydrogenase (33), and we assume, t e n t a t i v e l y , that 5-FICAR f u n c t i o n s at the same p o i n t i n the b i o s y n t h e t i c pathway. Future

Plans

The r e s u l t s of these and other s t u d i e s i n f l u o r o i m i d a z o l e chemistry and biochemistry have r a i s e d i n t e r e s t i n g questions f o r the f u t u r e . Some aspects of the p h y s i c a l and chemical p r o p e r t i e s of f l u o r o i m i d a z o l e s do not conform to expectations based on data f o r other imidazole systems and suggest, » that some sub s t i t u t e d imidazoles a r e , at b e s t , b o r d e r l i n e aromatic systems. Further understanding may be provided by a study of d i f l u o r o imidazoles: 4 , 5 - d i f l u o r o i m i d a z o l e has been synthesized and i s , indeed, found to have anomalous p r o p e r t i e s ; d e s p i t e a r a t h e r ex t e n s i v e e f f o r t , however, the 2 , 4 - d i f l u o r o isomer has not y e t been prepared. On the b i o l o g i c a l s i d e , we may ask, , why the isomeric f l u o r o h i s t i d i n e s show such marked d i f f e r e n c e s i n b i n d i n g and r e sponse to h i s t i d i n e - s p e c i f i c enzymes; whether the replacement of h i s t i d i n e by f l u o r o h i s t i d i n e i n an enzyme sequence i n v a r i a b l y leads to l o s s of a c t i v i t y ; whether the 2 - f l u o r o i m i d a z o l e s can be made s u f f i c i e n t l y r e a c t i v e to f u n c t i o n as covalent a f f i n i t y l a b e l s f o r receptor s i t e s . H o p e f u l l y , these and other questions w i l l have been answered before the next F l u o r i n e Symposium.



34


Literature Cited (1) Goldman, Peter, Science (1969), 164, 1123. (2) Ciba Foundation, "Carbon-Fluorine Compounds," Elsevier, Amsterdam, 1972. (3) Reference 2, p. 211. (4) Belsham, M. G., Muir, A. R., Kinns, Michael, Phillips, Lawrence, and Twanmoh, Li-Ming, J. Chem. Soc., Perkin Trans. 2 (1974), 119. (5) Pavlath, A. E. and Leffler, A. J., "Aromatic Fluorine Com pounds," Reinhold, New York, 1962. (6) Hudlicky, Miklos, "Organic Fluorine Chemistry," Plenum Press, New York, 1970. (7) Kirk, K. L. and Cohen, L. Α., unpublished observations. (8) Kirk, K. L. and Cohen, L. Α., Symposium on Fluorine in Medic inal Chemistry, 162nd National Meeting of the American Chem ical Society, Washington, D. C., Sept., 1971, FLUO 18. (9) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1971), 93, 3060. (10) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1973), 95, 4619. (11) Kirk, K. L. and Cohen, L. Α., J. Org. Chem. (1973), 38, 3647. (12) Nagai, Wakatu, Kirk, K. L., and Cohen, L. Α., J_. Org. Chem. (1973),38,1971. (13) Kirk, K. L., Nagai, Wakatu, and Cohen, L. Α., J. Amer. Chem. Soc. (1973),95,8389. (14) Lousberg, R. J. J. Ch. and Weiss, Ulrich, Experientia (1974), 30, 1440. (15) Kirk, K. L., manuscript in preparation. (16) Lowenbach, W. A. and King, M. M., unpublished data. (17) Yeh, H. J. C., Kirk, K. L., Cohen, L. Α., and Cohen, J. S., J. Chem. Soc., Perkin Trans. 2 (1975), 928. (18) Wong, J. L. and Keck, J. H., Jr., J. Org. Chem. (1974), 39 2398, and earlier references cited therein. (19) Kirk, K. L., McNeal, Elizabeth, Cohen, L. Α., and Creveling, C. R., manuscript in preparation. (20) Klein, D. C., Kirk, K. L., Weller, J. L., and Parfitt, A. G., Mol. Pharmacol., in press. (21) Furano, Α. V., Kirk, K. L., and Cohen, L. Α., unpublished data. (22) De Clercq, Erik, Kirk, K. L., and Cohen, L. Α., unpublished data. (23) Monahan, Μ. Α., Vale, Wylie, Kirk, K. L., and Cohen, L. Α., unpublished data. (24) Klee, C. Β., Kirk, K. L., Cohen, L. Α., and McPhie, Peter, J. Biol. Chem. (1975), 250, 5033. (25) Kaeppeli, Franz, and Retey, Janos, Eur. J. Biochem. (1971), 23, 198, and earlier references cited therein. (26) Klee, C. B., Kirk, K. L., and Cohen, L. Α., unpublished data.



2.

KIRK AND COHEN

Ring-Fluorinated

Imidazoles

35

(27) Dunn, Β. Μ., DiBello, Carlo, Kirk, K. L., Cohen, L. Α., and Chaiken, I. M., J. Biol. Chem. (1974), 249, 6295. (28) Dismukes, R. Κ., unpublished data. (29) Dismukes, R. K., Rogers, Michael, and Daly, J. W., Neurochemistry, in press. (30) Khare, G. P., Sidwell, R. W., Witkowski, J. T., Simon, L. N., and Robins, R. K., Antimicrob. Ag. Chemother. (1973), 3, 517. (31) Reepmeyer, J. C., Kirk, K. L., and Cohen, L. Α., Tetrahedron Letters, in press. (32) De Clercq, Erik, Luczak, Miroslav, Reepmeyer, J. C., Kirk, K. L., and Cohen, L. Α., Life Sciences (1975), 17, 187. (33) Streeter, D. G., Witkowski, J. T., Khare, G. P., Sidwell, R. W., Bauer, R. J., Robins, R. Κ., and Simon, L. Ν., Proc. Nat. Acad. Sci. USA (1973), 70, 1174. Discussion Q. A.

Has there been a check of monitoring on whether C-F cleavage occurs? I assume you r e f e r to s t a b i l i t y i n b i o l o g i c a l systems. The normal pathway f o r h i s t i d i n e degradation i n v o l v e s conversion to urocanic a c i d , and, u l t i m a t e l y , to glutamic a c i d . As I had i n d i c a t e d , 2 - f l u o r o h i s t i d i n e i s a very poor s u b s t r a t e f o r the f i r s t two enzymes i n t h i s pathway; a t the t h i r d s t e p , f l u o r i d e i o n would be r e l e a s e d , together with innocuous degrada t i o n products. 4 - F l u o r o h i s t i d i n e can be transformed slowly to 4 - f l u o r o u r o c a n i c a c i d , which i s probably a dead end. Except f o r t h i s data, we have found no i n d i c a t i o n s f o r enzymatic removal of f l u o r i n e .

Q.

Have you t e s t e d the b i o l o g i c a l a c t i v i t y of f l u o r o a l k y l i m i d a -

A.

We have not made any C F ^ - s u b s t i t u t e d i m i d a z o l e s .

Q. A.

As f a r as you know, are any of them known? Yes. A s e r i e s of 4 - t r i f l u o r o m e t h y l i m i d a z o l e s have been reported by Baldwin and h i s colleagues a t Merck Sharp & Dohme [ c f . J . Med. Chem. (1975), l g , 895] and 2 - t r i f l u o r o m e t h y l imidazoles by Lombardino and Wiseman [ J . Med. Chem. (1974), 17, 1182.]

Q.

What do you think would be the long-term s t a b i l i t y of the C-F bond i n 2-fluoroimidazoles? Simple compounds, such as 2 - f l u o r o i m i d a z o l e and 2 - f l u o r o - 4 methylimidazole can be kept i n d e f i n i t e l y i n the s o l i d s t a t e at -80°, but t r i m e r i z e i n a month or two a t 0 ° . In c o n t r a s t , 2 - f l u o r o h i s t i d i n e appears to be i n d e f i n i t e l y s t a b l e a t room temperature. D i l u t e s o l u t i o n s of 2 - f l u o r o h i s t i d i n e i n phos phate b u f f e r (pH 7) have been kept at 0° f o r months without evidence of d e t e r i o r a t i o n .

z o l e s - r e p l a c i n g F by

A.

CF3?


36

Q. A.

Q.


A.

Q.

A.

BIOCHEMISTRY INVOLVING

C A R B O N - F L U O R I N E BONDS

Can you say anything about the a n t i - a r t h r i t i c a c t i v i t y of the fluorohistidines ? T e s t i n g has not yet been done. We are aware of the p o s s i b i l i t i e s i n t h i s d i r e c t i o n and hope to get such a study going soon. I'd likÇgto ask whether j g u ever considered the use of r a d i o active F as a probe? F has a h a l f - l i f j g O f 110 minutes. Yes, we have. Studies on the s y n t h e s i s of F imidazoles were i n i t i a t e d some time ago at the Atomic Energy Commission i n Bucharest. You're acquaintecjgwith the work of A l Wolf and others a t Brookhaven with F fluor©phenylalanine and other b i o l o g i c a l l y i n t e r e s t i n g molecules? ^g Yes, I am. A l Wolf i s aware of our a c t i v i t i e s with F. A p o i n t of p a r t i c u l a r i n t e r e s t i s that 2 - f l u o r o h i s t i d i n e passes the b l o o d - b r a i n b a r r i e r very q u i c k l y , and so there i s c o n s i d e r a b l e i n t e r e s t i n t h i s compound f o r b r a i n s c i n t i g r a p h y .


Biochemistry and Pharmacology of Ring-Fluorinated Imidazoles - ACS

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