Discovery of Drugs from Microbiological Sources - American Chemical

3 Discovery of Drugs from Microbiological Sources LLOYD H. CONOVER

Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

Pfizer Medical Research Laboratories, Groton, Conn. 06340

Demonstration

of the safety and therapeutic

cillin

with

coupled

mycin,

and

the

streptothricin

antibiotic

discovery

prototypes

of virtually

now

important

discovery

cal sources dropped (notably

proved biological discovery cillins

sharply

properties

eries in the future.

increased

and detection

new im

A

landmark

techniques,

peni

understand

chemical

for antibiotic activity

of

having

of semisynthetic

antibiotic

of previously

1959,

microbiologi

by increased

important

of the

antibiotics After

synthesis

sharply.

mechanisms,

Examination

types of biological

cant new

Guided

will provide

genera of microorganisms new culturing

discovered. partial

era

this period,

and tetracyclines)

was the first preparation

ing of action and resistance

diverse

while

β-lactams

actino-

of antibacterial

were

of peni

halcyon

useful new drugs from

by Sheehan (1958).

of new congeners

the

During

all families

in medicine

value

of tyrothricin,

initiated

(1940-1959).

of medically

antibiotics

discoveries

synthesis discov

little

studied

elaboration,

use of

and testing for more

will also provide

signifi

discoveries.

' " p h e m y c e l i a of the f u n g u s Calviceps •*· r y e w a s u s e d f o r centuries

purpurea

w h i c h infects

b y the practitioners

flowering

of E u r o p e a n f o l k

m e d i c i n e . A b o o k p u b l i s h e d i n 1582 r e c o r d e d the use of sclerotia f r o m Secale cornutum

to c o n t r o l p o s t p a r t u m h e m o r r h a g e .

I n 1918 S t o l l crys

t a l l i z e d the a l k a l o i d ergotamine, s m a l l doses of w h i c h e l i c i t e d r a p i d a n d l o n g l a s t i n g uterine contractions. T h e m e d i c i n a l use of materials of m i c r o b i o l o g i c a l o r i g i n is thus v e r y o l d . I n contrast, significant use of the pres e n t l y most i m p o r t a n t d r u g s of m i c r o b i o l o g i c a l o r i g i n — t h e a n t i b i o t i c s — extends b a c k a scant 30 years. 33

In Drug Discovery; Bloom, B., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1971.

34

DRUG

DISCOVERY

It seems at first glance that w i t h the i s o l a t i o n of t y r o t h r i c i n b y D u b o s (I),

a c t i n o m y c i n , s t r e p t o t h r i c i n , a n d s t r e p t o m y c i n b y W a k s m a n et

(2, 3, 4) a n d p e n i c i l l i n b y F l o r e y , C h a i n et al. (5),

al.

the era of a n t i b i o t i c

discoveries was f u l l y l a u n c h e d w i t h l i t t l e scientific p r e c e d e n t save F l e m ing's n o w c e l e b r a t e d c h a n c e o b s e r v a t i o n (6).

I n r e a l i t y these w e r e the

c u l m i n a t i n g discoveries that t r a n s f o r m e d i n v e s t i g a t i o n of m i c r o b i a l ant a g o n i s m a n d a n t i b i o t i c substances f r o m a n obscure e r r a t i c a l l y p u r s u e d a c a d e m i c e n d e a v o r to a h i g h l y o r g a n i z e d a p p l i e d science.

The discovery

of antibiotics w i d e l y u s e f u l i n m e d i c i n e w a s i n fact presaged b y m a n y


p r e g n a n t observations.

F a s c i n a t i n g r e v i e w s of these early

Table I. Substance or Preparation

findings

Some Early Observations of Antimicrobial

Microbial

Organism( Inhibited Killed

Source

s) or

fungus of roasted green corn

bacteria

fungus

bacteria

M o l d y bread L i q u i d culture Mycophenolic acid (crystalline)

Penicillia Pénicillium species Pénicillium brevicompactum

bacteria bacteria

L i q u i d culture K o j i c acid

Pénicillium Aspergillus

glaucum oryzae

M y c e l i a l extract Penicillic acid (crystalline)

Aspergillus

fumigatus

f o w l plague bacteria, fungi Mycobacterium tuberculosis

Pénicillium

puberulum

Escherichia

A g a r culture m e d i u m

actinomycete

Bacillus Bacillus

A g a r culture m e d i u m

actinomycete

bacteria

Sparassol (crystalline)

Sparassis

fungi

L i q u i d culture medium

actinomycetes

Gliotoxin

Trichoderma

lignorum

fungi

A c t i n o m y c e t i n (protein precipitate)

Streptomyces

albus

bacteria

"Cuxum" Muscus ex huniano

have

cranio

Bacillus

ramosa

anthracis

coli mycoides vulgatus

gram-positive, gramnegative b a c t e r i a

" T h e work of W e l s c h w i t h a c t i n o m y c e t i n was tions of G r a t i a .

a c o n t i n u a t i o n of the


investiga-

3.

Microbiological

CONOVER

35

Sources

b e e n w r i t t e n b y W a k s m a n ( 7 ) a n d the O x f o r d g r o u p ( 8 ) .

A representa-

tive selection of these observations is s u m m a r i z e d i n T a b l e s I a n d II. T h e f o l l o w i n g are w o r t h y of s p e c i a l m e n t i o n :

(1)

use of m o l d s to

c o m b a t s u p e r f i c i a l infections was a p a r t of E u r o p e a n a n d M a y a n f o l k medicine; (2)

G r a t i a a n d D a t h ( 1 9 2 6 ) consciously u n d e r t o o k t o isolate

a c t i n o m y c e t e a n d f u n g a l cultures that p r o d u c e d substances

antagonistic

to b a c t e r i a ; their sources w e r e m u d , tap water, a n d air; ( 3 ) L o u i s Pasteur w a s one of the first to r e c o r d ( 1 8 7 7 ) the p h e n o m e n o n of m i c r o b i a l antagonism;

(4)

the b a s i c m e t h o d o l o g y n o w u s e d to detect

antibiotics


b o t h i n l i q u i d a n d s o l i d g r o w t h m e d i a was e v o l v e d b y a n u m b e r of early

A c t i v i t y Produced by Fungi, Molds, and Actinomycetes Therapeutic or Other Application

Discoverer or Recorder of Antimicrobial Action

Year

infections of s k i n a n d intestines

M a y a n Indians

wounds

J. Parkinson

1640

wounds —

E u r o p e a n peasants, etc. Tyndall

— 1876

—

Gosio

1896

—

Tartakovski

1904

—

Saito

1907

Vaudremer

1913

—

Alsberg & Black

1913

—

Grieg-Smith

1917

—

Lieske

1921

—

Falck

1923

immunization with b a c t e r i a l lysates

Gratia & Dath

1926

p l a n t fungus infections

Weindling & Emerson

1936

immunization with b a c t e r i a l lysates

Welsch"

1937

h u m a n tuberculosis

Pre-Columbian period


36

DRUG

Table II.


Substance or Preparation

Microbial

Source

DISCOVERY

Some E a r l y Observations of

Organism (s) Inhibited or Killed

L i q u i d culture

aerobic b a c t e r i a

Bacillus


bacteria

bacteria

G e l a t i n culture medium

Staphylococci

Bacillus

G e l a t i n culture medium (containing diffusable secret o r y products)

Bacillus

Staphylococcus

G e l a t i n or a g a r c u l ture m e d i u m (zones of i n h i b i tion)

cocci

Bacillus

"Pyocyanase" precipitate)

Pseudomonas aeruginosa

bacteria

Agar and liquid culture m e d i u m (cont a i n i n g diffusable inhibitory m a terial)

Micrococcus tetragenus

Bacillus anthracis Staphylococcus aureus

Agar and liquid culture media

bacteria

bacteria


Bacillus


Bacilli

(crude

L i q u i d and agar culture medium

fluorescens

subtilis

mesentericus

bacteria


Bacillus

anthracis

aureus

anthracis

bacteria Mycobacterium tuberculosis

Bacillus

" S e n t o c y m " (bact e r i a l lysates)

anthracis

Proteus, Meningococcus, Cory neb acterium diphtheriae

bacteria scaber

Bacillus Vibrio

anthracis, cholerae


3.

CONOVER

Microbiological

37

Sources

Antimicrobial A c t i v i t y Produced by Bacteria


Therapeutic or Other Application


Year

Pasteur & Joubert

1877

Soyka

1885

Babes

1885

Garré

1887

Doehle

1889

human meningitis, diphther i a , g r i p p e , l o c a l infections (commercially produced 1901-1935)

Emmerich & Low

1899

(not therapeutic i n a n i m a l s )

Lode

1903

Frost

1904

Nicolle

1907

guinea p i g tuberculosis

Rappin

1912

human upper respiratory i n fections (local a p p l i c a t i o n )

Pringsheim

1920

human dysentary, typhoid fever, u r i n a r y t r a c t infections

Much Rosenthal

1925 1926


38

DRUG

DISCOVERY

Table


Substance or Preparation

Microbial

Source

Organism (s) Inhibited or Killed

Pyocyanine (crystalline)


bacteria

Prodigiosin

Serratia

trypanosomes, fungi

Hemopyocyanine (crystalline)


bacteria, fungi

Iodinin

Chromobacterium iodinum

bacteria

marcescens

II.

investigators, a m o n g w h o m F r o s t ( 1 9 0 4 ) was n o t a b l e ; ( 5 )

"Pyocyanase"

m u s t be c o n s i d e r e d the first c o m m e r c i a l a n t i b i o t i c p r o d u c t ; it w a s p r o d u c e d i n G e r m a n y b e t w e e n 1901 a n d 1935, a n d w h e n p r o p e r l y p r e p a r e d , it h a d a t h e r a p e u t i c effect against b a c t e r i a l infections i n m a n a n d animals. I n retrospect, it seems that the d i s c o v e r y a n d b r o a d a p p l i c a t i o n of a n t i b i o t i c s i n m e d i c i n e w a s o v e r d u e w h e n it c a m e to pass. I n the p r e c e d i n g decades scientists p r i m a r i l y interested i n c o n t r o l l i n g infectious diseases w e r e engrossed i n the i m m u n o l o g i c a l a p p r o a c h , h a d b e e n d i s i l l u s i o n e d w i t h chemotherapy

b y the f a i l u r e of disinfectants

to c o n t r o l

systemic

infections a n d finally w e r e e n c o u r a g e d b y the success of the s u l f o n a m i d e s to seek a d d i t i o n a l synthetic

antimetabolites.

The Golden Era" of Antibacterial Microbial Metabolite Discoveries, 1940-1959 tf

O n e of the r e m a r k a b l e aspects of the era that f o l l o w e d the d i s c o v eries of D u b o s , F l o r e y , C h a i n , a n d W a k s m a n w a s the r a p i d i t y w i t h w h i c h m a j o r drugs w e r e d i s c o v e r e d a n d p u t to p r a c t i c a l use.

The long induc-

t i o n p e r i o d w h i c h p r e c e d e d e x p l o i t a t i o n of m i c r o b i a l sources of a n t i b a c t e r i a l d r u g s p e r m i t t e d this e x p l o i t a t i o n to be r a p i d , once b e g u n . B y 1940, b a s i c k n o w l e d g e a n d e x p e r i m e n t a l t e c h n i q u e s w e r e i n h a n d w h i c h permitted: (1)

f a c i l e c o l l e c t i o n , i s o l a t i o n , a n d g r o w t h of cultures of f u n g i ,

m o l d s , b a c t e r i a , a n d actinomycetes; fication,

( 2 ) detection, b i o l o g i c a l assay, p u r i -

isolation, a n d structure p r o o f of c o m p l e x , unstable

having antimicrobial activity; (3)

e v a l u a t i o n of the

metabolites

chemotherapeutic

efficacy a n d safety of a n t i b a c t e r i a l drugs i n l a b o r a t o r y animals a n d m a n ; ( 4 ) a r t i f i c i a l m u t a t i o n of a n t i b i o t i c - p r o d u c i n g m i c r o o r g a n i s m s w i t h select i o n of mutants h a v i n g i m p r o v e d p r o d u c t i v i t y ; a n d ( 5 )

d e v e l o p m e n t of

i n d u s t r i a l - s c a l e s u b m e r g e d , aerated fermentations, a n d of r e c o v e r y p r o c -


3.

CONOVER

Microbiological

39

Sources

Continued


Therapeutic or Other Application


Year

h u m a n d i p h t h e r i a carriers (upper r e s p i r a t o r y disinfection)

Hettche

1932 (isolated i n 1860)

t r y p a n o s o m i a s i s of m i c e

Masera, Fischl

1934-1935

Kramer

1935 ( p o s s i b l y i s o l a t e d i n 1863)

Mcllwain

1941

esses f o r t h e a n t i b i o t i c s p r o d u c e d thereby.

T h e requisite knowledge,

methods, a n d techniques were d r a w n f r o m mycology, bacteriology, plant, a n d s o i l m i c r o b i o l o g y , m i c r o b i a l genetics, c h r o m a t o g r a p h y , e x p e r i m e n t a l chemotherapy, chemical engineering, a n d industrial fermentation

tech-

nology. A c o i n c i d e n c e of k e y t e c h n i c a l d e v e l o p m e n t s a n d external influences a c c e l e r a t e d d e v e l o p m e n t s i n t h e field, once its potentialities w e r e r e c o g nized.

T h u s , t h e d e v e l o p m e n t of p r a c t i c a l m a n u f a c t u r i n g processes f o r

the n e w a n t i b i o t i c s d i s c o v e r e d after 1944 w a s g r e a t l y f a c i l i t a t e d b y t h e existence of t h e b a s i c t e c h n o l o g y p e r f e c t e d i n t h e w a r t i m e " c r a s h " effort d e v o t e d to p e n i c i l l i n p r o d u c t i o n . T h e d i s c o v e r y of p a p e r c h r o m a t o g r a p h y i n 1944 w a s e x c e p t i o n a l l y t i m e l y ( 9 ) .

This technique, b y providing a

s i m p l e a n d sensitive a n a l y t i c a l m e t h o d f o r associating a specific c h e m i c a l entity w i t h in vitro

a n t i m i c r o b i a l a c t i v i t y , m a d e possible t h e s c r e e n i n g

of large n u m b e r s of m i c r o b i a l cultures f o r s m a l l concentrations of n e w , active entities. T h e years b e t w e e n 1940 a n d 1959 h a v e b e e n justly c a l l e d t h e g o l d e n era of a n t i b i o t i c d i s c o v e r y . D u r i n g this p e r i o d e v e r y i m p o r t a n t class of a n t i b a c t e r i a l a n t i b i o t i c n o w k n o w n w a s r e c o g n i z e d ( T a b l e I I I ). I n d e e d , m a n y specific drugs ( e.g., b e n z y l p e n i c i l l i n , s t r e p t o m y c i n , o x y t e t r a c y c l i n e , chloramphenicol, neomycin, a n d erythromycin) w h i c h presently occupy major places i n t h e r a p e u t i c p r a c t i c e w e r e d i s c o v e r e d d u r i n g that p e r i o d .

The Decline in Antibacterial Microbial Metabolite Discoveries, 1960-1970 Since 1959 r e l a t i v e l y f e w n e w l y d i s c o v e r e d m i c r o b i a l metabolites h a v e r e a c h e d g e n e r a l use i n h u m a n or v e t e r i n a r y m e d i c i n e ( T a b l e I V ) ; most of t h e major discoveries ( t h e penicillinase-resistant p e n i c i l l i n s , t h e


40

DRUG

Table III.


Year of First Literature Report

1939 1940 1943 1944 1947 1948 1950 1953 1955 1956

DISCOVERY

Year of Discovery of Structural Classes of Antibacterial Antibiotics

Cfass c y c l i c peptide penicillin steroid aminoglycoside chloramphenicol tetracycline macrolide virginiamycin lincomycin cycloserine novobiocin cephalosporin

First Generally Useful Member

First Discovered Member tyrothricin p e n i c i l l i n F , G , etc, helvolic acid streptomycin chloramphenicol chlortetracycline picromycin streptogramin celesticetin cycloserine novobiocin cephalosporin C 6

Polymyxin penicillin G f u s i d i c a c i d (1962)° streptomycin chloramphenicol chlortetracycline e r y t h r o m y c i n (1952) v i r g i n i a m y c i n (1955) l i n c o m y c i n (1962) cycloserine" novobiocin" cephalothin, c e p h a l o r i d i n e (1962) vancomycin" r i f a m y c i n S V (1961) 0

c

1957 α b c

vancomycin ansamacrolide

vancomycin streptovaricins

c

Only useful member to date. PA-114 was the first member to be recognized as a synergistic mixture (119). Semisynthetic.

broad-spectrum

penicillins, doxycycline, the new

cephalosporins,

and

r i f a m p i c i n ) h a v e b e e n m a d e b y c h e m i c a l m o d i f i c a t i o n o f existing a n t i biotics. T h i s shift w i t h t i m e i n the source of major discoveries is s h o w n g r a p h i c a l l y i n F i g u r e 1. T h e relationships of l i n c o m y c i n (10,

11,12,13),

f u s i d i c a c i d (14, 15, 16, 17, 18, 19, 20, 21), g e n t a m i c i n (22, 23, 24, 25), a n d c a p r e o m y c i n (26, 27, 28, 29) ( w h i c h w e r e d i s c o v e r e d after 1959) t o t h e i r s t r u c t u r a l antecedents are s h o w n i n F i g u r e 2. C l e a r l y , the felicitous c o m b i n a t i o n o f t e c h n i c a l factors w h i c h f a c i l i t a t e d the i n i t i a l burst of m i c r o b i a l m e t a b o l i t e d r u g discoveries n o longer operates w i t h the same effect. T h i s has l e d to a j u d g m e n t o n the p a r t of some that n o i m p o r t a n t n e w a n t i b a c t e r i a l drugs or d r u g classes w i l l b e d e r i v e d d i r e c t l y f r o m m i c r o b i o l o g i c a l sources i n the f u t u r e . T h e v a l i d i t y o f this j u d g m e n t o b v i o u s l y w i l l not b e k n o w n u n t i l a n u m b e r o f years h a v e passed.

T h o s e presently s e e k i n g t o d i s c o v e r su

p e r i o r n e w a n t i b a c t e r i a l drugs must, h o w e v e r , m a k e a n assessment b a s e d u p o n available evidence.

A n y r a t i o n a l attempt t o m a k e this assessment

b r i n g s t o m i n d a c o m p l e x o f s u b s i d i a r y questions—e.g., c a n the causes f o r the decrease i n m i c r o b i a l m e t a b o l i t e

d r u g discoveries d e p i c t e d i n

F i g u r e 2 b e i d e n t i f i e d ? D o these suggest possibilities f o r i n c r e a s i n g the d i s c o v e r y rate? T h e s e a n d other questions c o n c e r n i n g the f u t u r e course a n d nature o f d r u g discoveries f r o m m i c r o b i o l o g i c a l sources w e r e i n c o r -


3.

Microbiological

CONOVER

Table I V . Year of First Literature Report

41

Sources

T i m i n g and Source of Significant Antibacterial Antibiotic Discoveries 1939-1969

From Structural

Modification

From Structural Modification by Bio- or Chemical Synthesis

tyrothricin [tyrocidin, gramicidin] penicillin, actinomycins" streptomycin bacitracin

1939


α

1940 1944 1945 1946 1947 1948 1949 1950 1951 1952 1958 1954 1955

6

dihydrostreptomycin chloramphenicol, polymyxins chlortetracycline neomycin oxytetracycline viomycin erythromycin leucomycins oleandomycin spiramycin, virginiamycin/ cycloserine, cephalo sporin C novobiocin, vancomycin, mikamycins" kanamycins, 6-demethyltetracycline

phenoxymethylpenicillin

chloramphenicol tetracycline

c

palmitate

a

1956 1957 1958

rifamycins, paromomycin, tylosin

1959

triacetyloleandomycin proprionylerythromycin, pyrrolidinomethyltetracycline phenethicillin, propicillin

d

1960 1961 1962

lincomycin, fusidic acid

1963 1965

gentamicin,

capreomycin rifamide, dicloxacillin, cephaloglycine cephalexin, r i f a m p i c i n , carbenicillin clindamycin

1967 1968 1970

methicillin ampicillin, nafcillin, oxacillin, methacycline, r i f a m y c i n S V cephalothin, cephaloridine, doxycycline, cloxacillin, lysinomethyltetracycline, phenbenicillin

e

Historically important ; not used or not now important as an antibacterial drug. Report on therapeutic efficacy of crude penicillin. "Produced by "biosynthetic" methods; not utilized until 1953. Used primarily or exclusively in animals. Newer discoveries whose utility has not yet been established have been omitted.

a

b

d e


42

DRUG


Legend

1939-1944

^

Microbial Metabolites

^

Chemically or Biochemically Modified Microbial Metabolites

1945-1949

1950-1954

1955-1959

1960-1964

DISCOVERY

1965-1969

Period Figure 1.

Timing and source of significant antibacterial discoveries, 1939-1969

antibiotic

p o r a t e d into a q u e s t i o n n a i r e sent t o some 120 scientists i n i n d u s t r i a l , a c a d e m i c , a n d g o v e r n m e n t laboratories; c o m p l e t e d questionnaires w e r e r e c e i v e d f r o m 70 i n d i v i d u a l s . r e s p o n s i b l e b o t h f o r past

T h e p a n e l o f respondents i n c l u d e s m e n

a n t i b i o t i c discoveries a n d f o r p r e s e n t - d a y

m i c r o b i a l m e t a b o l i t e a n d a n t i b a c t e r i a l d r u g research.

T h e questionnaire

sought t o o b t a i n t h e same g l o b a l j u d g m e n t s , synthesis of v i e w s , a n d d e f i n i t i o n o f p r o b l e m s a n d o p p o r t u n i t i e s that w o u l d h a v e e m e r g e d h a d first-hand

discussions b e e n possible w i t h a l l o f t h e respondents.

Much

of the r e m a i n d e r o f this p a p e r is d e v o t e d t o a s u m m a r y a n d i n t e r p r e t a t i o n of t h e responses t o t h e q u e s t i o n n a i r e . T h e first section of the q u e s t i o n n a i r e p r o b e d t h e causes for t h e d r o p i n t h e d i s c o v e r y rate o f u s e f u l n e w a n t i b a c t e r i a l m i c r o b i a l metabolites, t h e n sought j u d g m e n t s as t o w h e t h e r , a n d i f so h o w , this t r e n d c o u l d b e reversed. F i n a l l y i t asked the respondents t o rate the potentialities o f five d i s c o v e r y approaches f o r p r o v i d i n g u s e f u l n e w a n t i b a c t e r i a l d r u g s over the next d e c a d e .


3.

Microbiological

CONOVER

43

Sources

The N a t u r e and Causes of the Decline in Discovery Rate of A n t i bacterial Microbial Metabolites.

T h e p a n e l does not s u p p o r t the h y

pothesis that v i r t u a l l y a l l of the p o t e n t i a l l y u s e f u l s t r u c t u r a l classes of a n t i b a c t e r i a l a n t i b i o t i c s h a v e b e e n d i s c o v e r e d . T h e t h r u s t of the m a j o r i t y opinion (83%

a g r e e m e n t ) is that the search f o r a n t i b a c t e r i a l m i c r o b i a l

metabolites, massive a n d p r o l o n g e d as it has b e e n , has n o t d r a w n u p o n the t o t a l reservoir of a n t i b i o t i c - p r o d u c i n g organisms a n d t h e i r s e c o n d a r y m e t a b o l i t e s ; the same sector of the t o t a l m i c r o b i a l p o p u l a t i o n has b e e n repeatedly sampled a n d examined for antibiotic elaboration b y techniques w h i c h h a v e c h a n g e d l i t t l e i n 30 years. P o t e n t i a l l y v a l u a b l e n e w a n t i b i o t i c s Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

h a v e b e e n m i s s e d because the p r o d u c i n g o r g a n i s m w a s not i s o l a t e d , d i d not g r o w , or d i d n o t p r o d u c e a sufficient q u a n t i t y of a n t i b i o t i c to b e d e t e c t e d u n d e r the c o n d i t i o n s e m p l o y e d . that changes

S o m e respondents

reported

i n m e d i a a n d f e r m e n t a t i o n c o n d i t i o n s o n the one h a n d

a n d / o r a p p l i c a t i o n of n e w , m o r e sensitive d e t e c t i o n m e t h o d s o n the other h a v e b e e n r e s p o n s i b l e for discoveries i n t h e i r o w n laboratories.

The

c o m p o s i t i o n or t e m p e r a t u r e of the c u l t u r e m e d i u m or t h e d u r a t i o n of the f e r m e n t a t i o n m a y d e t e r m i n e w h e t h e r a n a n t i b i o t i c is d e t e c t e d or, i n some cases, w h a t a n t i b i o t i c is detected.

Antibiotics produced in minor

amounts, e s p e c i a l l y i n the presence of other easily d e t e c t e d a n t i b i o t i c s , h a v e u n d o u b t e d l y b e e n o v e r l o o k e d i n the past.

( C o n v e r s e l y , some s u c h

a n t i b i o t i c s o w e t h e i r d i s c o v e r y to the f a c t that t h e y w e r e present i n broths w h i c h w e r e b e i n g i n v e s t i g a t e d i n t e n s i v e l y because of other c o m ponents present.

C e p h a l o s p o r i n C , f o r e x a m p l e , is so w e a k l y active that

h a d it not b e e n a c c o m p a n i e d b y p e n i c i l l i n Ν a n d c e p h a l o s p o r i n P , i t m i g h t easily h a v e b e e n o v e r l o o k e d . ) T h e r e are genera a n d species of a n t i b i o t i c - p r o d u c i n g m i c r o o r g a n i s m s that, r e l a t i v e l y s p e a k i n g , h a v e b e e n n e g l e c t e d f o r the past 20 years. F r o m a b o u t 1950 o n w a r d , Streptomyces

species o b t a i n e d f r o m s o i l h a v e re

c e i v e d b y f a r the greatest s t u d y ( 3 0 ) .

I n the e a r l y years, there w a s g o o d

reason f o r t h i s : t h e y c o u l d b e o b t a i n e d easily i n s e e m i n g l y endless v a riety, a n d they w e r e a r i c h source of n e w a n t i b i o t i c s . years k n o w n metabolites of Streptomyces edly.

The

discoveries

of

In more

phosphonomycin, negamycin,

actinonin, showdomycin, and kasugamycin ( F i g u r e 3) respondents

as e v i d e n c e that

recent

h a v e b e e n r e d i s c o v e r e d repeat

e v e n so, Streptomyces

sparsomycin,

were cited by species

are

still

c a p a b l e of p r o v i d i n g a n t i b a c t e r i a l a n t i b i o t i c s of n o v e l structure. T h e m a j o r i t y of respondents

(69%)

f e e l that the spectacular

suc

cesses a c h i e v e d b y c h e m i c a l m o d i f i c a t i o n of existing a n t i b i o t i c s i n the late 1950's a n d e a r l y 1960's c a u s e d a shift of research effort a w a y f r o m s o i l s a m p l e screening a n d that this w a s i n p a r t r e s p o n s i b l e for the smaller n u m b e r of significant f e r m e n t a t i o n - a n t i b i o t i c discoveries.

A substantial

m i n o r i t y ( 2 3 % ) h o l d , h o w e v e r , that i n absolute terms the effort a p p l i e d


44

DRUG

DISCOVERY

to d i s c o v e r y o f m i c r o b i a l metabolites has not d e c r e a s e d ; o n l y t h e p r o p o r t i o n of the t o t a l d i s c o v e r y effort so a p p l i e d has decreased

as

the

s e m i s y n t h e t i c efforts h a v e g r o w n q u i t e large. P u b l i s h e d statistics w h i c h reflect the l e v e l of f e r m e n t a t i o n - a n t i b i o t i c d i s c o v e r y effort o v e r t h e years d o suggest that there has b e e n n o r e a l c u r t a i l m e n t of this a c t i v i t y . B e r d y a n d M a g y a r ' s t a b u l a t i o n (30)

of a n t i b i o t i c discoveries ( T a b l e V ) shows

a n absolute increase i n the n u m b e r of n e w a n t i b i o t i c s r e p o r t e d over e a c h successive (31)

five-year

p e r i o d f r o m 1940 t h r o u g h 1965.

P e r l m a n s analysis

of y e a r - b y - y e a r totals ( 1 9 4 0 - 1 9 6 8 ) shows a steady increase

until

1961, t h e n a l e v e l i n g off at a rate w e l l a b o v e the average for t h e p r e v i o u s Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

t w o decades.

Lincomycin

CH -CH -CH

Celesticetin

H

2

2

3

Figure 2.

3-ketoA*

Cephalosporin P,

oc-OH

Fusidic Acid

oc-OH

Rehtionship

CH

CH

R Helvolic Acid

H 3

3

CHe-CHg-O-C.^

R,

R

2

*3

^tf-OAc

=0

Η

ac-OAc

/-OH

Η

Η

Η

oc-OH

of significant antibacterial microbial metabochemistry shown for the


3.

CONOVER

Microbiological

45

Sources

T h e s e statistics p o i n t u p a f a c t that m a y not b e g e n e r a l l y a p p r e c i a t e d : w h i l e the d i s c o v e r y rate for a n t i b a c t e r i a l m i c r o b i a l metabolites r e a c h i n g g e n e r a l use i n h u m a n or v e t e r i n a r y m e d i c i n e has i n d e e d d r o p p e d s h a r p l y , the t o t a l n u m b e r of a n t i b i o t i c s d i s c o v e r e d a n n u a l l y has not decreased. U n q u e s t i o n a b l y , m a n y of the n e w a n t i b i o t i c s i s o l a t e d i n the p e r i o d 1960-1969 w o u l d h a v e c o n s t i t u t e d significant discoveries i n the 1940's w h e n a n t i b i o t i c s w e r e filling a v i r t u a l c h e m o t h e r a p e u t i c v a c u u m . S t a n d ards f o r m e d i c a l a n d c o m m e r c i a l a c c e p t a n c e h a v e r i s e n c o n t i n u o u s l y as h a v e r e q u i r e m e n t s f o r r e g u l a t o r y a p p r o v a l of n e w a n t i b i o t i c s . I n the past d e c a d e these influences p r o p e r l y d i s c o u r a g e d the d e v e l o p m e n t a n d c o m Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

m e r c i a l i z a t i o n of n e w a n t i b i o t i c s not possessing significant

advantages

OH

Kanamycin A

Genfomicins

R

C,

CH

C,a

Viomycin

3

H

CH

3

H

2

CH

antecedents.

The

C

Viomycidine

Ri

3

H

Capreomycidine

Capreomycin

lite discoveries 1959-1970, to structural gentamicins is tentative ( 1 4 0 ) .

stereo-


46

DRUG

DISCOVERY


H

Figure 3.

Sporsomycin

Phosphonomycin

Actinonin

Negamycin

Recent antibacterial

antibiotic

discoveries of novel structure

over existing drugs. O n e c a n b e q u i t e sure, h o w e v e r , that some n e w a n t i biotics h a v e b e e n rejected w i t h o u t benefit of the extensive c o m p a r i s o n s w i t h existing d r u g s that w o u l d h a v e r e v e a l e d u n i q u e superiorities.

A

great i n v e s t m e n t i n t i m e a n d resources is u s u a l l y r e q u i r e d to o b t a i n the quantities of a p u r i f i e d n e w l y d i s c o v e r e d a n t i b i o t i c n e e d e d f o r a d e f i n i t i v e assessment. W h i l e i n most cases this investment proves fruitless, v a l u a b l e properties c a n o n l y be d i s c o v e r e d i f t h e y are tested f o r . Table V .

Cumulative Totals of N e w Antibiotics from Three Major Sources 1940-1965

Schizomycetes Year

Actinomycetales

1940 1945 1950 1955 1960 1965 a

6 14 82 363 812 1266 Fungi

imperjecti,

Eubacteriales 14 26 100 141 187 222

~, Other Classes of Fungi

Totals

19 52 143 220 282 374

39 92 325 724 1281 1862

a

b a s i d i o m y c e t e s , ascomycetes,

phycomycetes.


bive-Year Increase 53 233 399 557 581

Library American Chemical Society 3.

CONOVER

Microbiological

47

Sources

Proposals Designed to Increase the Discovery Rate of Antibacterial Microbial Metabolites. T h e q u e s t i o n n n a i r e c i t e d five specific approaches that h a v e b e e n a d v o c a t e d as means o f i n c r e a s i n g t h e rate at w h i c h d i s coveries o f significant n e w a n t i b a c t e r i a l a n t i b i o t i c s a r e m a d e . T h e p a n e l was a s k e d to evaluate t h e v a l i d i t y of e a c h a p p r o a c h separately, t h e n to r a n k these five approaches

" i n o r d e r of t h e i r p r a c t i c a l p o t e n t i a l i t y f o r

i n c r e a s i n g t h e rate o f d i s c o v e r y . . . over t h e next d e c a d e . " lists these approaches a n d s u m m a r i z e s the responses.


Table V I .

Table V I

T h e t w o approaches

Ranking of Approaches to Increased Antibacterial Microbial Metabolite Discovery 1970—1979 Approach

Rank

a

Percent Approach

Judging Fruitful

b

A p p l i c a t i o n of n e w techniques f o r c o l l e c t i n g , s t o r i n g , a n d processing soil samples, f o r isolating and growing potential antibioticp r o d u c i n g m i c r o o r g a n i s m s , a n d for detect i n g new a n t i b i o t i c s

1

67

E x a m i n a t i o n of genera of m i c r o o r g a n i s m s t h a t have received r e l a t i v e l y l i t t l e a t t e n t i o n thus f a r i n the search for a n t i b i o t i c s

2

58

E x a m i n a t i o n of m a r i n e m i c r o o r g a n i s m s

3

53

E x a m i n a t i o n of t e r r e s t r i a l m i c r o o r g a n i s m s t h a t grow under u n u s u a l or extreme e n v i ronmental conditions

3

42

E x a m i n a t i o n of m i c r o o r g a n i s m s t h a t grow i n the presence of pathogens

4

33

The over-all rank was derived from individual rankings by use of a weighted scoring system. Respondents were asked whether a given discovery approach could be expected to be fruitful for the discovery of useful new antibacterial antibiotics in the next decade. α

b

g i v e n strongest s u p p o r t ( a p p l i c a t i o n of n e w techniques a n d e x a m i n a t i o n of n e g l e c t e d genera of m i c r o o r g a n i s m s ) w e r e forecast b y t h e analysis of the causes o f t h e h i s t o r i c a l d e c l i n e i n d i s c o v e r y fate.

M a r i n e microor

ganisms are not expected to p r o v i d e a c o r n u c o p i a of n e w a n t i b i o t i c s ; they constitute o n e category of little s t u d i e d organisms w h i c h

deserves

s t u d y b u t w h i c h m a y pose s p e c i a l p r o b l e m s i n terms of c o l l e c t i o n , iso l a t i o n , a n d g r o w t h . T h e q u e s t i o n n a i r e r e c o n f i r m e d the g e n e r a l l y a c c e p t e d c o n c l u s i o n that antibiotics d o n o t p l a y a n e c o l o g i c a l role, a n d thus there is n o reason to expect a h i g h e r p r o p o r t i o n of a n t i b i o t i c p r o d u c e r s i n a n e n v i r o n m e n t w h e r e pathogens a b o u n d . I s h o u l d l i k e to consider i n some d e t a i l t h e basis f o r t h e expectation that s t r u c t u r a l l y n o v e l antibiotics w i l l b e d i s c o v e r e d i f efforts are c o n -


48

DRUG

c e n t r a t e d u p o n species

w i t h i n "neglected" genera

DISCOVERY

of m i c r o o r g a n i s m s .

F i r s t , this e x p e c t a t i o n is s u p p o r t e d b y past e x p e r i e n c e ; s e c o n d l y , i t rests u p o n the p r o p o s i t i o n that t h e structures of m i c r o b i a l s e c o n d a r y m e t a b o lites are a n expression of t h e genetic i n d i v i d u a l i t y of t h e e l a b o r a t i n g species.

T o t h e extent that c l a s s i c a l t a x o n o m y reflects t h e m a g n i t u d e

of genetic difference b e t w e e n m i c r o o r g a n i s m s , t a x o n o m i c a l l y w i d e l y separ a t e d organisms s h o u l d elaborate a n t i b i o t i c s w h i c h d i f f e r w i d e l y i n struct u r e w h i l e c l o s e l y r e l a t e d organisms m a y p r o d u c e t h e same o r closely related antibiotics. F i g u r e 4 shows i n s i m p l i f i e d f o r m t h e t a x o n o m i c locations of m i -


c r o b i a l classes, orders, a n d f a m i l i e s w h i c h p r o d u c e t h e k n o w n s t r u c t u r a l classes of u s e f u l a n t i b a c t e r i a l a n t i b i o t i c s .

N o n e of t h e u s e f u l s t r u c t u r a l

classes o r i g i n a l l y d i s c o v e r e d as p r o d u c t s of S c h i z o m y c e t e s has b e e n isol a t e d f r o m a species o f t h e Fungi imperfecta; r e p o r t (32)

that a Streptomyces

there is b u t one p r e l i m i n a r y

species has b e e n i s o l a t e d w h i c h p r o d u c e s

p e n i c i l l i n N , o t h e r w i s e k n o w n e x c l u s i v e l y as a p r o d u c t of Fungi perfecti.

im-

E v e n e l a b o r a t i o n of t h e same a n t i b i o t i c b y organisms b e l o n g i n g

to different orders of t h e same class is rare. t h e s i z e d b y several Streptomyces

C y c l o s e r i n e w h i c h is s y n -

species a n d b y Pseudomonas

fluorescens

constitutes one of the f e w k n o w n examples.

(33)

T h e most c o m m o n

finding

closely r e l a t e d substances

is t h e e l a b o r a t i o n of t h e same o r v e r y

b y different strains of the same

Division

species or

I FUNGI I

Class

FUNGI IMPERFECT!!

Order

MONILIALES

I I

Family

1 M0NIL1ACEÂÊ]

Classes of Useful Antibacterial Antibiotics

Penicillin Cephalosporin-C Steroid

I SCHIZOMYCETES I

ι

1

IACTINOMYCETALESI

I EUBACTERIALESl

I STREPTOMYCETACEAEl

| BACILLACEAÎ]

Aminoglycoside Chloramphenicol Tetracycline Macrolide

Polymyxin * Cycloserine

Ansa macrolide

Lincomycin * Cycloserine Novobiocin

Figure 4. Taxonomic location of microbial orders and families producing useful antibacterial antibiotics. Other classes of fungi known to produce antibiotics are basidiomycetes, ascomycetes, and phycomycetes. For simplicity, the older classification of Schizomycetes as a class of fungi has been used. Schizomycetes and blue-green algae are generally now placed in a kingdom distinct from that of fungi. This classification in no way alters the argument that Fungi imperfecti and schizomycetes, being very widely separated taxonomically, may be expected to produce structurally distinct secondary metabolites.


3.

Microbiological

CONOVER

49

Sources

different species of the same genus. Species b e l o n g i n g to different genera of the same f a m i l y m a y p r o d u c e the same a n t i b i o t i c , b u t i n notable i n stances they p r o d u c e s t r u c t u r a l l y d i s t i n c t i v e analogs. this p o i n t w i t h the ^-lactams macrolides.

P e n i c i l l i n Ν is e l a b o r a t e d

a n d a Paecilomyces

sporium,

a n d some of the

a n d Trichophyton

F i g u r e 5 illustrates

aminoglycosides

b y several species of

species ( a n d p e r h a p s b y some

and

CephaloAspergillus

s p e c i e s — a l l m e m b e r s of the M o n i l i a c e a e f a m i l y ). T h e

other " n a t u r a l " p e n i c i l l i n s are o n l y p r o d u c e d b y Penicillia

w h i l e cephalo

s p o r i n C , a s t r u c t u r a l relative of p e n i c i l l i n N , has not b e e n f o u n d as a m e t a b o l i t e of a Pénicillium.

( P e n i c i l l i n Ν a n d c e p h a l o s p o r i n C h a v e also


b e e n isolated f r o m the species Emericellopsis class A s c o m y c e t e s . Cephalosporium,

Since these organisms

w h i c h falls i n the

terricola

are the p e r f e c t stage of

the t a x o n o m i c separation is not c o n s i d e r e d

a

pertinent

to the present discussion.) Family

IM0NIL1ACEAË1

Genus |PENICILLIUM| |CEPHALOSPORIUM| |PAECILOMYCES| |ASPERGILLUS] |TRICHOPHYTON] Penicillins ( F, Dihdro F, G) Κ,Μ,Χ etc.

Penicillin Ν Cephalosporin - C

Family

Penicillin Ν

"Penicillin"

Penicillin like substance

ISTREPTOMYCETACEAE1

Genus

iSTREPTOMYCËSl

|MICROMONOSPORA|

Kanamycin A Erythromycin

Figure 5.

Gentamicins ( C, , C, , C ) Megalomicin A a

t

Taxonomic location of genera-producing related β-lactam, glycoside, and macrolide antibiotics

amino

A m o n g the a m i n o g l y c o s i d e s , the g e n t a m i c i n C f a m i l y has thus far o n l y been

obtained

f r o m Micromonospora

e l a b o r a t e d o n l y b y Streptomyces b y a Micromonospora

w h i l e the k a n a m y c i n s

species. M e g a l o m i c i n A (34)

are

produced

species represents a n o v e l a n a l o g of e r y t h r o m y c i n

C , a p r o d u c t of Streptomyces

species ( F i g u r e 6 ).

A d e t a i l e d c o n s i d e r a t i o n of the n e g l e c t e d genera is b e y o n d the scope of this discussion. (35)

T h i s subject has r e c e i v e d attention f r o m W a k s m a n

a n d other authors.

It does a p p e a r that s t r u c t u r a l l y n o v e l congeners

of a g i v e n a n t i b i o t i c m a y b e f o u n d b y e x a m i n i n g organisms f a l l i n g i n a genus closely r e l a t e d to that of the k n o w n p r o d u c e r . T h i s w o u l d suggest e x a m i n a t i o n of genera of S t r e p t o m y c e t a c e a e other t h a n a n d of M o n i l i a c e a e other t h a n Pénicillium.

Streptomyces,

T h e fact that t h r o u g h

m o r e antibiotics h a d b e e n r e p o r t e d f r o m the Fungi

imperfecti,


1955

basidio-


50

DRUG

R

=

H

DISCOVERY

Erythromycin C

Megalomycin A

Figure 6. mycetes, ascomycetes,

Erythromycin

and megalomycin A

a n d p h y c o m y c e t e s t h a n f r o m the s c h i z o m y c e t e

o r d e r actinomycetales, suggests that classes o f f u n g i other t h a n s c h i z o mycetes s h o u l d b e e x a m i n e d m o r e t h o r o u g h l y (see T a b l e V a n d F i g u r e 7).

Just as a l l d r u g research is b e c o m i n g m o r e difficult a n d t i m e c o n -

s u m i n g , some o f t h e n e g l e c t e d genera w i l l p r o v e m o r e difficult t o collect, isolate, a n d g r o w t h a n t h e Streptomyces,

a n d t h e p r o p o r t i o n of active

cultures they p r o v i d e m a y b e l o w e r . T h e c h a n c e that their s e c o n d a r y metabolites, once d e t e c t e d , w i l l b e n e w s h o u l d b e greater, h o w e v e r .

Evaluation of Proposed Approaches to Antibacterial Drug Discovery, 1970-1980 It is germane to c o n s i d e r not o n l y means b y w h i c h discoveries of u s e f u l a n t i b a c t e r i a l m i c r o b i a l metabolites

m i g h t b e i n c r e a s e d i n the

f u t u r e b u t also to w e i g h t h e r e l a t i v e potentialities o f a l l a p p r o a c h e s t o antibacterial drug discovery. w o u l d b e greater elsewhere.

P e r h a p s the r e w a r d f o r effort e x p e n d e d T h e q u e s t i o n n a i r e r e q u e s t e d a r a n k i n g of

the f o l l o w i n g five approaches i n terms of their " p r a c t i c a l p o t e n t i a l i t y f o r p r o v i d i n g u s e f u l n e w a n t i b a c t e r i a l drugs over t h e next d e c a d e " : ( 1 ) isol a t i o n o f n e w m i c r o b i a l metabolites; ( 2 ) p r e p a r a t i o n o f s t r u c t u r a l analogs of existing u s e f u l a n t i b i o t i c s b y c h e m i c a l or other means; ( 3 ) p r e p a r a t i o n


3.

CONOVER

Microbiological

51

Sources

of s t r u c t u r a l analogs of existing toxic or p o o r l y efficacious a n t i b i o t i c s b y c h e m i c a l or other means; ( 4 ) e m p i r i c a l screening of o r g a n i c c o m p o u n d s u n r e l a t e d to existing a n t i b i o t i c s ; ( 5 ) d i r e c t e d synthesis of o r g a n i c c o m p o u n d s b a s e d u p o n a b i o c h e m i c a l rationale. T h e successes a c h i e v e d i n the past d e c a d e w i t h c h e m i c a l l y m o d i f i e d p e n i c i l l i n s , tetracyclines,

and lincomycin, undoubtedly influenced

the

j u d g m e n t of the p a n e l that p r e p a r a t i o n of s t r u c t u r a l analogs of u s e f u l a n t i b i o t i c s b y c h e m i c a l or other means s h o u l d b e r a n k e d as one of the two

most p r o m i s i n g d i s c o v e r y approaches for the next decade.

Before

p r o c e e d i n g f u r t h e r w i t h the e v a l u a t i o n m a d e b y the respondents of the Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

five approaches

(Table

V I I ) , some aspects of the h i s t o r y a n d

nature

of the s t r u c t u r a l m o d i f i c a t i o n a p p r o a c h are e x a m i n e d .

Division

Class

FUNGI

Fungi Imperfecti

Bosidiomycetes

Ascomycetes Phycomycetes| SchizomycetesI

Order

Actinomycetqles|

Genus

| Streptomyces |

Figure 7.

Cfosses of fungi

Structural Modification of Antibiotics.

HISTORICAL

REVIEW.

Writ-

i n g i n 1945, W a k s m a n ( 7 ) set f o r t h i n p r o p h e t i c terms the r o l e that the m e d i c i n a l chemist was to p l a y i n the a n t i b i o t i c

field.

. . . the d i s c o v e r y of n e w c h e m i c a l agents possessing a n t i b a c t e r i a l or a n t i f u n g a l properties offers the chemist m a n y n e w m o d e l s to d r a w u p o n f o r v a r i e d types of syntheses. A l t h o u g h o n l y v e r y f e w a n t i b i o t i c agents h a v e so f a r b e e n isolated, a n d e v e n f e w e r c r y s t a l l i z e d , it is a l r e a d y w e l l established that w e are d e a l i n g here w i t h a great v a r i e t y of c h e m i c a l c o m p o u n d s . . . . M a n y a chemist is a w a i t i n g the s o l u t i o n of the p r o b l e m of the c h e m i c a l n a t u r e of p e n i c i l l i n b e f o r e b e g i n n i n g n e w syntheses. D o u b t l e s s most of the c o m p o u n d s that p r o v e to b e u s e f u l as c h e m o t h e r a p e u t i c agents w i l l sooner or later b e s y n t h e s i z e d . T h e c o n t r i b u t i o n of the bacteriologist m a y be a l l b u t forgotten i n the l i g h t of f o r t h c o m i n g c h e m i c a l d e v e l o p m e n t s , b u t e v e n the bacteriologist w i l l b e g r a t e f u l f o r n e w tools to h e l p c o m b a t d i s e a s e - p r o d u c i n g agents. . . . It is fitting that W a k s m a n ' s o w n d i s c o v e r y , s t r e p t o m y c i n , p r o v i d e d the v e h i c l e for m a k i n g the first u s e f u l semisynthetic a n t i b i o t i c — d i h y d r o s t r e p t o m y c i n (36,

37).

It is interesting that d i h y d r o s t r e p t o m y c i n w a s

s u b e q u e n t l y f o u n d as a m i c r o b i a l m e t a b o l i t e

(38).


52

DRUG

The

DISCOVERY

massive w a r t i m e B r i t i s h - A m e r i c a n i n v e s t i g a t i o n of p e n i c i l l i n

c h e m i s t r y i n c l u d e d attempts t o

. . m o d i f y penicillin chemically i n the

hope o f obtaining n e w compounds w h i c h might differ qualitatively or q u a n t i t a t i v e l y i n their b i o l o g i c a l a c t i v i t y , s t a b i l i t y o r rate of e x c r e t i o n . . . there w a s a l w a y s t h e h o p e that a c h e m i c a l m o d i f i c a t i o n of t h e m o l e c u l e m i g h t so alter t h e specificity of t h e d r u g as to greatly b r o a d e n its field of a p p l i c a t i o n " (39).

T h e thiazolidine carboxyl of benzylpenicillin a n d

the a c t i v a t e d o r t h o positions o f p - h y d r o x y b e n z y l p e n i c i l l i n ( F i g u r e

8)

p r o v e d amenable to chemical modification, b u t no n e w penicillins superior i n chemical stability o r chemotherapeutic

action were identified


i n this w o r k . M o s t of t h e c h e m i c a l reactions of p e n i c i l l i n s o b s e r v e d i n e a r l y studies d e s t r o y e d or r e d u c e d b i o l o g i c a l a c t i v i t y .

R - C - N - T

/?-Hydroxybenzy I penicillin (X)

Benzylpenicillin ( G)

Figure

8.

Early substrates for chemical fication of penicillins

modi-

F r o m t h e p o i n t of v i e w of i m p o r t a n c e a n d c h e m i c a l f e a s i b i l i t y , c h l o r amphenicol modification.

(Figure

9)

presented

a n excellent

subject f o r s t r u c t u r a l

I t w a s t h e first t r u l y b r o a d - s p e c t r u m a n t i b i o t i c isolated,

a n d its structure a n d t o t a l synthesis w e r e b o t h r e p o r t e d t w o years after the d i s c o v e r y w a s a n n o u n c e d (40, 41, 42).

T h e synthesis o f c h l o r a m -

p h e n i c o l analogs p r o v e d to b e one of t h e great d i s a p p o i n t m e n t s of early c h e m i c a l research i n t h e a n t i b i o t i c

field.

H u n d r e d s o f analogs w e r e s y n -

t h e s i z e d , b u t n o n e w a s f o u n d s u p e r i o r to t h e p a r e n t d r u g i n terms either of a n t i m i c r o b i a l a c t i v i t y or t h e r a p e u t i c i n d e x (43).

T h e palmitate a n d

h e m i s u c c i n a t e esters h a v e p r o v i d e d s u p e r i o r dosage f o r m s f o r o r a l a n d p a r e n t e r a l use. O n e synthetic a n a l o g , t h i a m p h e n i c o l (44)

has a c h i e v e d

l i m i t e d use i n h u m a n a n d v e t e r i n a r y m e d i c i n e . B e c a u s e e a r l y experience

w i t h the penicillins, streptomycin, a n d

c h l o r a m p h e n i c o l d i d n o t f u l f i l l W a k s m a n ' s o p t i m i s t i c p r e d i c t i o n , there f o l l o w e d a p e r i o d of s k e p t i c i s m r e g a r d i n g t h e p o t e n t i a l v a l u e of c h e m i c a l modification of antibacterial

substances d e r i v e d f r o m m i c r o b i o l o g i c a l

sources; i t w a s a r g u e d i n some quarters that a n t i b i o t i c s represented t h e


3.

Microbiological

CONOVER

53

Sources

I

H n H H ^ C ^ N — C —C—Cl

I

h

CH OR Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

2

R= - H

Chloramphenicol

R= - C - ( C H ) , - C H 2

4

Chloramphenicol Palmitate

3

9

fl h R= - C - C H - C H - C - 0 2

2

Figure 9.

m

Na

Chloramphenicol Hemisuccinate

Chemical modification of

chloramphenicol

c u l m i n a t i o n of a n e v o l u t i o n a r y process d e s i g n e d to g i v e the e l a b o r a t i n g o r g a n i s m the o p t i m u m c h e m i c a l w e a p o n i n the c o m p e t i t i o n f o r s u r v i v a l . R o b i n s o n expressed this s k e p t i c i s m (45)

w h e n he w r o t e i n

1953:

. . i n d e e d one of the d i s a p p o i n t m e n t s i n a n t i b i o t i c w o r k is that it seems i m p o s s i b l e to m o d i f y the m o l e c u l e w i t h o u t r e d u c i n g or e l i m i n a t i n g its antimicrobial activity . .

T h e discoveries e a r l y i n the 1950's of tetra-

c y c l i n e a n d p h e n o x y m e t h y l p e n i c i l l i n established b e y o n d d o u b t , h o w e v e r , that m o d i f i c a t i o n of a n t i b i o t i c s b y c h e m i c a l or b i o s y n t h e t i c means c o u l d y i e l d superior drugs. SEMISYNTHETIC

(46, 47, 48)

TETRACYCLINES.

T e t r a c y c l i n e ( F i g u r e 10, T a b l e

w a s the first major s e m i s y n t h e t i c a n t i b i o t i c d i s c o v e r e d .

IV) This

p r o d u c t w a s d i s c o v e r e d i n d e p e n d e n t l y i n m y l a b o r a t o r y a n d that

of

B o o t h e at L e d e r l e after the structures of o x y t e t r a c y c l i n e a n d c h l o r t e t r a c y c l i n e h a d b e e n d e t e r m i n e d b y the P f i z e r g r o u p i n c o l l a b o r a t i o n w i t h W o o d w a r d (49, 50, 51,52). of c h l o r t e t r a c y c l i n e .

It w a s f o r m e d b y the c a t a l y t i c h y d r o g e n o l y s i s

T e t r a c y c l i n e w a s f o u n d to b e a p o t e n t

broad-spec-

t r u m a n t i b i o t i c w h i c h w a s m o r e stable a n d better tolerated t h a n its fermentation-produced progenitor.

I n a f e w years i t almost

displaced chlortetracycline f r o m m e d i c a l practice.

completely

Interestingly, as h a p -

p e n e d i n the case of d i h y d r o s t r e p t o m y c i n , t e t r a c y c l i n e w a s f o u n d as a m i c r o b i a l m e t a b o l i t e after its c h e m i c a l synthesis h a d b e e n a c c o m p l i s h e d (53).

F u r t h e r selective transformations of t e t r a c y c l i n e a n t i b i o t i c s a f f o r d -

i n g u s e f u l n e w d r u g s w e r e s l o w i n c o m i n g . It was to b e 10 years b e f o r e


54

DRUG

CI

CH

OH

3

CH

CH

^CH,

5

DISCOVERY

CH

3

CH

3

3

OH

Catalyst OH

~

H

,

•

Chlortetracycline CH


OH Hz

B

, 6 « I ,

Tetracycline CHs

S

N

c

r

R

X 2

"3 .CH N ^

3

HP

Catalyst

Oxytetracycline

R, - CH ,

R - OH

Tetracycline

R, - CH ,

R

6-DémethyItetracycline

3

3

R/ =

Figure 10.

6-Deoxy tetracyclines

2

2

H , R

2

C0NH

=H =H

Tetracycline

and

6-deoxytetracyclines

Stephens, v o n W i t t e n a u , B l a c k w o o d , a n d c o - w o r k e r s o f P f i z e r r e p o r t e d the discoveries o f m e t h a c y c l i n e a n d d o x y c y c l i n e (54, 55). W o o d w a r d has c h a r a c t e r i z e d t h e m o l e c u l e o f o x y t e t r a c y c l i n e as a " d i a b o l i c a l c o n c a t e n a t i o n of r e a c t i v e g r o u p i n g s " ( 5 6 ) .

Indeed, the complexity a n d labil-

i t y o f t h e p a r e n t tetracyclines p r o v e d t o b e a great obstacle t o d r u g d i s c o v e r y . A t the outset the tetracyclines, l i k e a n u m b e r o f other a n t i b i o t i c classes (/^-lactams, m a c r o l i d e s , a m i n o g l y c o s i d e s ) , w e r e not a m e n a b l e to f a c i l e m o l e c u l a r m o d i f i c a t i o n f o r e x p l o r i n g r e l a t i o n s h i p s structure a n d b i o l o g i c a l properties.

between

T h e most r e a c t i v e f u n c t i o n a l i t i e s

p r o v e d , i n general, to b e r e q u i r e d f o r b i o l o g i c a l a c t i v i t y . I n t h e e n d , c o n t r o l o f the c h e m i s t r y o f the C 6 h y d r o x y l f u n c t i o n p r o v i d e d t h e k e y to n e w d r u g d i s c o v e r y . I n 1958, the P f i z e r g r o u p r e p o r t e d the successful h y d r o g e n o l y s i s o f the C 6 h y d r o x y l o f o x y t e t r a c y c l i n e , t e t r a c y c l i n e , a n d 6-demethyltetrac y c l i n e ( F i g u r e 10) (57). S i m i l a r w o r k w a s r e p o r t e d later b y M c C o r m i c k et al. of L e d e r l e (58). T h e r e a c t i o n p r o d u c t s w e r e o f interest i n that t h e y w e r e b i o l o g i c a l l y a c t i v e a n d also because t h e y w e r e stable t o t h e c o n d i t i o n s of e l e c t r o p h i l i c a r o m a t i c s u b s t i t u t i o n reactions.

It was n o w

p o s s i b l e t o p r e p a r e a large v a r i e t y o f D r i n g - s u b s t i t u t e d tetracyclines f o r b i o l o g i c a l s t u d y . F r o m s u c h studies c a m e the d i s c o v e r y of m i n o c y c l i n e b y M a r t e l l a n d B o o t h e o f L e d e r l e (59). T h i s c o m p o u n d is u n i q u e i n its in vivo efficacy against some infections c a u s e d b y tetracycline-resistant pathogens.


2

3.

CONOVER

Microbiological

55

Sources

Insights w e r e sought at P f i z e r b y w h i c h m o l e c u l a r structure a n d shape, e l e c t r o n i c p r o p e r t i e s , a c i d strength, c h e l a t i n g a b i l i t y , a n d l i p o p h i l i c i t y m i g h t b e r e l a t e d to p o t e n c y o r r a n g e of a n t i b a c t e r i a l a c t i v i t y . T h e s e studies w e r e c o m p l i c a t e d b y t h e f a c t that large differences o b s e r v e d in vitro w e r e o f t e n r e d u c e d o r c o m p l e t e l y n u l l i f i e d in vivo.

Such observa-

tions f o c u s e d a t t e n t i o n u p o n t h e i n t e r p l a y o f s t r u c t u r a l a n d p h a r m a c o k i n e t i c p r o p e r t i e s o f tetracyclines.

CH ^CH 3

Oral Absorption Dogs 3


OH

~I00% OH

O

OH

0

6-Dl METHYL-6-DEOXYTETRACYCLINE

40% C0NH

2

C0NH

2

~

15%

15% OH

0

OH

Ô

C0NH

2

6-DEMETHYLCHLORTETRACYCLINE

9% C0NH

pH 4.0

Figure

11.

pH 5.5

2

pH 7.0 pHZ.O

Relationship of chloroform-water distribution constants and oral absorption in dogs for some tetracyclines


56

DRUG

DISCOVERY

Studies b y v o n W i t t e n a u a n d c o - w o r k e r s d e m o n s t r a t e d that f o r most t e t r a c y c l i n e a n t i b i o t i c s s t u d i e d , in vivo b e h a v i o r ( s u c h as

completeness

of o r a l a b s o r p t i o n , degree of s e r u m p r o t e i n - b i n d i n g , tissue affinity, rate of u r i n a r y excretion, a n d b i o l o g i c a l h a l f - l i f e ) i m p o r t a n t to d r u g efficacy c o u l d b e r e l a t e d to d r u g l i p o p h i l i c i t y as reflected b y c h l o r o f o r m - b u f f e r d i s t r i b u t i o n constants (60,

61, 62, 6 3 ) .

T h e principles w h i c h

emerged

b a s e d o n studies i n dogs p r o v e d a p p l i c a b l e i n m a n . F i g u r e 11 indicates the r e l a t i o n s h i p b e t w e e n o r a l a b s o r p t i o n i n dogs a n d chloroform—water d i s t r i b u t i o n constants f o r a g r o u p of tetracyclines. T h e 6-deoxytetracyclines p r o v i d e d a series i n w h i c h the b i o l o g i c a l Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

consequences of r e m o v i n g the 6 - h y d r o x y l g r o u p , of m o d u l a t i n g l i p o p h i l i c i t y , a n d of a l t e r i n g c o n f i g u r a t i o n at C 6 c o u l d b e s t u d i e d .

Studies

w i t h 6 - d e m e t h y l - 6 - d e o x y t e t r a c y c l i n e i n d i c a t e d that a l t h o u g h this a n a l o g retains essentially the same in vitro

s p e c t r u m as t e t r a c y c l i n e , is c h e m o -

t h e r a p e u t i c a l l y effective, is efficiently a b s o r b e d a n d affords a l o n g s e r u m h a l f - l i f e , its great tissue affinity is reflected i n i n c r e a s e d t o x i c i t y . W h i l e the in vitro p o t e n c y of this c o m p o u n d against g r a m - p o s i t i v e organisms is g e n e r a l l y e n h a n c e d c o m p a r e d w i t h its parent, c a t a l y t i c h y d r o g e n o l y s i s of o x y t e t r a c y c l i n e gives a 6-deoxy c o m p o u n d h a v i n g l o w e r in vitro

po

tency. T h e e x p l a n a t i o n f o r this a n o m a l y lies i n the f a c t that d u r i n g the h y d r o g e n o l y s i s of o x y t e t r a c y c l i n e i n v e r s i o n occurs at C 6 , p r e s u m a b l y b e c a u s e the a face of t h e m o l e c u l e is less h i n d e r e d ( F i g u r e 12 ).

Figure 12.

Formation of β-6-deoxyS-hydr

oxytetracycline


3.

CONOVER

Microbiological

57

Sources

6 - D e o x y - 5 - h y d r o x y t e t r a c y c l i n e h a v i n g the n o r m a l α - m e t h y l c o n f i g u r a t i o n at C 6 h a d b e e n sought i n c h e m i c a l studies starting as e a r l y as 1952.

D i s c o v e r y a n d d e v e l o p m e n t of this c o m p o u n d ( w h i c h p r o v e d to

h a v e s o m e w h a t e n h a n c e d in vitro

a n t i b a c t e r i a l a c t i v i t y , l o w e r affinity

f o r c a l c i u m , a l o w e r effect o n the gastrointestinal flora, a n d near i d e a l p h a r m a c o k i n e t i c properties ) w a s m a d e possible b y the m a s t e r y of f u r t h e r n e w c h e m i s t r y at C 6 . I n the e n d , t w o stereoselective routes to the e l u s i v e c o m p o u n d w e r e d i s c o v e r e d (54,

55).

T h e k e y reactions are s h o w n i n

F i g u r e 13. N o t e that success i n the d i r e c t hydrogénation r o u t e r e q u i r e d r e v e r s a l of the u s u a l preference f o r c a t a l y t i c hydrogénation to o c c u r at


the least h i n d e r e d face of a m o l e c u l e .

oC- 6-Deoxy - 5- hydroxytetracycline ( Doxycycline )

Figure 13.

Stereoselective synthesis of

SEMISYNTHETIC

PENICILLINS.

a-6-deoxy-5-hydroxytetracycline

Just as the i n d e p e n d e n t lines of i n q u i r y

of D u b o s , W a k s m a n , a n d the O x f o r d g r o u p c o n v e r g e d to o p e n t h e a n t i b i o t i c era, the p e r i o d of s e m i s y n t h e t i c p e n i c i l l i n discoveries w a s i n i t i a t e d b y a s i m i l a r convergence.

A s a n o u t g r o w t h of the early o b s e r v a t i o n that

the c h e m i c a l n a t u r e of the p e n i c i l l i n s p r o d u c e d b y f e r m e n t a t i o n w a s i n fluenced

b y the c o m p o s i t i o n of the g r o w t h m e d i u m , the p r e p a r a t i o n of

"biosynthetic" penicillins was accomplished b y a d d i n g substituted p h e n y l acetic a c i d d e r i v a t i v e s ( a n d r e l a t e d structures)

to p e n i c i l l i n

fermenta-

tions. B y this m e t h o d B e h r e n s a n d c o - w o r k e r s at the E l i L i l l y C o . h a d b y 1948 p r e p a r e d some 30 p e n i c i l l i n s m o d i f i e d i n the a c y l m o i e t y


(64).

58

DRUG

DISCOVERY

I n p r i n c i p l e , o n l y the l i m i t a t i o n s i n substrate structure i m p o s e d b y the specificities of the e n z y m e or e n z y m e s i n v o l v e d i n the a c t i v a t i o n a n d c o u p l i n g reactions p r e v e n t e d this a n d other b i o s y n t h e t i c investigations f r o m a n t i c i p a t i n g the m a j o r discoveries m a d e a d e c a d e a n d m o r e later b y the s e m i s y n t h e t i c a p p r o a c h . A s it w a s , it a p p e a r e d i n i t i a l l y that the o n l y u s e f u l a d v a n t a g e possessed b y a b i o s y n t h e t i c p e n i c i l l i n was l o w e r allergenicity.

T h i s p r o p e r t y w h i c h was a t t r i b u t e d to p e n i c i l l i n 0 ( a l l y l m e r -


c a p t o m e t h y l p e n i c i l l i n ) ( F i g u r e 14) p r o v e d i l l u s o r y .

COMMON

NAME

CHEMICAL

NAME

R

PENICILLIN

ν

Phenoxymethylpenicillin

PENICILLIN

ο

AUylmercaptomethylpenicillin Figure 14.

Biosynthetic

C H O-CH 6

5

2

CH =CH-CH -S-CH 2

2

2

penicillins

I n 1954, h o w e v e r , B r a n d i a n d M a r g r e i t e r (65)

r e p o r t e d that b i o

synthetic p h e n o x y m e t h y l p e n i c i l l i n ( p e n i c i l l i n V ) w a s s u p e r i o r to b e n z y l p e n i c i l l i n i n terms of a c i d s t a b i l i t y . S i n c e this c o m p o u n d retains g o o d a c t i v i t y against

penicillin-sensitive gram-positive bacteria

penicillin

V

q u i c k l y g a i n e d a c c e p t a n c e as the o n l y r e l i a b l e p e n i c i l l i n for o r a l a d m i n istration. The

fledgling

B e e c h a m p e n i c i l l i n research t e a m w a s s t i m u l a t e d b y

the a d v e n t of p h e n o x y m e t h y l p e n i c i l l i n to seek a d d i t i o n a l s u p e r i o r p e n i c i l l i n s m o d i f i e d i n the a c y l m o i e t y (66).

T h e general approach envisioned

was the c o n v e r s i o n of one p e n i c i l l i n b e a r i n g a reactive f u n c t i o n a l i t y to a v a r i e t y of n e w m o d i f i e d p e n i c i l l i n s ; s p e c i f i c a l l y b i o s y n t h e t i c p - a m i n o b e n z y l p e n i c i l l i n w a s selected f o r m o d i f i c a t i o n via a c y l a t i o n of the a m i n e function (Figure

15).

D u r i n g the same p e r i o d , S h e e h a n was w o r k i n g t o w a r d a t o t a l s y n thesis of p e n i c i l l i n s . I n 1958, he a n n o u n c e d the synthesis of 6-aminopenicillanic acid (6-ΑΡΑ)

a n d its u t i l i t y for the p r e p a r a t i o n of

p e n i c i l l i n s b y a c y l a t i o n (67, 68).

new

( A l m o s t 10 years earlier, this substance

h a d b e e n p o s t u l a t e d to b e a n i n t e r m e d i a t e i n the biosynthesis of p e n i c i l l i n s (69, 70).

P r i o r Japanese literature also c o n t a i n e d clear suggestions

that it h a d b e e n f o r m e d b y e n z y m a t i c h y d r o l y s i s of b e n z y l p e n i c i l l i n ( 71 ) a n d i n fermentations c a r r i e d out i n the absence of side c h a i n precursors


3.

CONOVER

Microbiological

Sources

0

C0 H

59

>-N—k

2

p - Aminobenzylpenicillin

0

2

Λ

^C0 H 2

Ν

'\

6-Aminopenicillanic Acid


Figure 15. (72,

73).

Preparation of semisynthetic

penicillins

T h e significance of these p r e g n a n t observations h a d

escaped

scientists engaged i n a n t i b i o t i c research at the t i m e . ) M e a n w h i l e , the B e e c h a m g r o u p d i s c o v e r e d 6 - A P A i n d e p e n d e n t l y i n the course of s t u d y i n g the biosynthesis of p - a m i n o b e n z y l p e n i c i l l i n . T h i s d i s c o v e r y also f o l l o w e d f r o m the o b s e r v a t i o n that i n p r e c u r s o r - s t a r v e d fermentations a m a t e r i a l w a s f o r m e d w h i c h c o n t a i n e d the ^ - l a c t a m f u n c t i o n b u t w h i c h w a s not b i o l o g i c a l l y active (74). t a t i o n m e t h o d p r o v i d e d the quantities of 6 - A P A .

first

The Beecham

p r a c t i c a l means

fermen

for o b t a i n i n g

large

A l t h o u g h it h a d p r e v i o u s l y b e e n a r g u e d b y some

that this structure w a s too l a b i l e to be isolable, once the existence of 6 - A P A was g e n e r a l l y r e c o g n i z e d , e n z y m a t i c m e t h o d s f o r its p r e p a r a t i o n w e r e q u i c k l y p e r f e c t e d i n a n u m b e r of laboratories ( 75, 76, 77, 78, 79 ). T h e d i s c o v e r y of 6 - a m i n o p e n i c i l l a n i c a c i d p r e s e n t e d the m e d i c i n a l chemist w i t h a n e x c e p t i o n a l o p p o r t u n i t y . It was n o w possible to v a r y the a c y l m o i e t y of p e n i c i l l i n s at w i l l to i d e n t i f y a n d elaborate those struc t u r a l a n d p h y s i c a l characteristics

w h i c h c o n t r o l l e d t h e r a p e u t i c a l l y rele

vant properties s u c h as a c i d s t a b i l i t y , o r a l a b s o r p t i o n , s e r u m p r o t e i n b i n d i n g , p e n i c i l l i n a s e resistance,

and gram-negative

activity.

Virtually

every n e w a c y l p e n i c i l l a n i c a c i d s y n t h e s i z e d r e t a i n e d some b i o l o g i c a l ac tivity.

A c i d stable h o m o l o g s of p h e n o x y m e t h y l p e n i c i l l i n w e r e the

first

of the semisynthetic p e n i c i l l i n s to b e r e p o r t e d a n d to r e a c h c l i n i c a l use ( F i g u r e 16)

(80).

It w a s later s h o w n that the a c i d s t a b i l i t y of p e n i c i l l i n s

c a n be c o r r e l a t e d w i t h the strength of the a c i d c o r r e s p o n d i n g to the a c y l moiety (81),

a

finding

consistent w i t h A b r a h a m ' s p o s t u l a t i o n that

the

electronic properties of the p h e n o x y substituent are r e s p o n s i b l e for the a c i d s t a b i l i t y of p h e n o x y m e t h y l p e n i c i l l i n

(82).

Since, i n general, o n l y m o n o s u b s t i t u t e d acetic a c i d d e r i v a t i v e s served as p e n i c i l l i n b i o s y n t h e t i c precursors, one of the p r i m e s t r u c t u r a l v a r i a tions m a d e feasible f o r the first t i m e b y p a r t i a l synthesis w a s that of d i a n d t r i s u b s t i t u t i o n at the «-position of the a c y l substituent.

A l l of the

semisynthetic p e n i c i l l i n s w h i c h h a v e b e c o m e i m p o r t a n t i n m e d i c a l p r a c tice are i n fact d i s u b s t i t u t e d at the c a r b o n a to the a m i d e c a r b o n y l . S u c h


60

DRUG

DISCOVERY

c o m p o u n d s p r o v i d e d i m p o r t a n t insights r e l a t i n g structure t o a n u m b e r of b i o l o g i c a l l y i m p o r t a n t properties.

A s the c h a i n l e n g t h o f α - a l k y l sub-

stituents o n p h e n o x y m e t h y l p e n i c i l l i n w a s increased, so w a s the efficiency of o r a l a b s o r p t i o n , the s e r u m h a l f - l i f e , a n d the d e g r e e o f s e r u m b i n d i n g . In

potency

vitro

toward penicillin-sensitive bacteria

was n o t altered

g r e a t l y i n the s i m p l e h o m o l o g s ; h o w e v e r , i t w a s o b s e r v e d that as the b u l k of t h e «-substituent

was i n c r e a s e d , this e n d o w e d t h e m o l e c u l e w i t h a

s m a l l b u t significant degree o f resistance t o d e s t r u c t i o n b y b e n z y l p e n i c i l l i n a s e — t h e e n z y m e r e s p o n s i b l e f o r t h e resistance o f m a n y

Staphylo

coccus aureus strains to b e n z y l p e n i c i l l i n . a - P h e n o x y i s o b u t y l p e n i c i l l i n was


a b l e t o protect m i c e against a n i n f e c t i o n c a u s e d b y a b e n z y l p e n i c i l l i n resistant S. aureus.

R

GENERIC

H-

PENICILLIN

CH 3

CH -CH 3

2

CH,

NAME V

CHEMICAL NAME

cx -Phenoxymethylpenicillin

PHENETHICILLIN

cx-Phenoxy ethyl penicillin

PROPICILLIN

oc - Phenoxypropylpenicillin

H_

_

Figure 16.

oc-Phenoxyisobutylpenicillin Acid-stable

penicillins

R e c o g n i t i o n that the o c c u r r e n c e o f i n c r e a s e d p e n i c i l l i n a s e resistance a c c o m p a n i e d i n c r e a s e d steric b u l k a b o u t the «-position p r o v i d e d a p o w e r f u l r a t i o n a l e f o r d i r e c t e d synthesis: insights g a i n e d f r o m studies o f steric h i n d r a n c e o f o r g a n i c reactions w e r e d i r e c t l y a p p l i c a b l e t o d r u g d e s i g n . T h e e n d result w a s a q u a l i t a t i v e c h a n g e i n efficacy s p e c t r u m c o m p a r e d w i t h b e n z y l p e n i c i l l i n (83).

A n a l o g synthesis progressed t h r o u g h t r i s u b -

s t i t u t e d m e t h y l p e n i c i l l i n s , s u c h as t r i p h e n y l m e t h y l p e n i c i l l i n (84) to t h e d i s u b s t i t u t e d a r y l a n d h e t e r o a r y l p e n i c i l l i n s s u c h as m e t h i c i l l i n a n d oxa c i l l i n ( F i g u r e 1 7 ) ( 8 5 , 86).

M e t h i c i l l i n a n d the oxacillin family have

b e c o m e the mainstays o f the c l i n i c a l a r m a m e n t a r i u m u s e d against p e n i c i l l i n a s e - p r o d u c i n g S. aureus. S e v e r a l factors m u s t h a v e suggested t h e p r e p a r a t i o n o f a - a m i n o b e n z y l p e n i c i l l i n ( a m p i c i l l i n ) (87) to the B e e c h a m g r o u p .

I t w a s a-substi-


3.

Microbiological

CONOVER

61

Sources

8

R

- C -

CH

3

,CeH R =

ÇeH C H -Ç6

Penicillinase Resistant

5

C H E

CH

6

3

OCH

Q -

C H6

3

6

Methicillin


H Ç H E

3

Penicillinase Sensitive

6

R = C H -C— E

6

6

5

0CH

H

CH

3

CH 0

3

• * - 0

3

Figure 17.

Steric hindrance and penicillinase

resistance

t u t e d , i t c o u l d b e e x p e c t e d t o b e a c i d stable, a n d e v i d e n c e w a s a l r e a d y i n h a n d that a m i n o s u b s t i t u t e d p e n i c i l l i n s ( p - a m i n o b e n z y l p e n i c i l l i n , p e n i c i l l i n N ) possessed

e n h a n c e d a c t i v i t y against

gram-negative

bacteria

( F i g u r e 1 8 ) . A m p i c i l l i n e x t e n d e d the range of c h e m o t h e r a p e u t i c efficacy of the p e n i c i l l i n s to m a n y of the g r a m - n e g a t i v e b a c t e r i a . T h i s q u a l i t a t i v e i m p r o v e m e n t i n in vivo p e r f o r m a n c e d e p e n d e d u p o n h i g h q u a n t i t a t i v e i m p r o v e m e n t over the w e a k in vitro

a c t i v i t y o f b e n z y l p e n i c i l l i n against

the same organisms. I t n o w appears that the p h y s i c a l p r o p e r t i e s of a m p i c i l l i n f a c i l i t a t e its passage r e l a t i v e to that o f b e n z y l p e n i c i l l i n , t h r o u g h

? H R-C-N-

ay I

Ν

CH

3

CH

3

k C0 H %

2

C0 H I R= N H - C H - ( C H ) -

Penicillin Ν

R= NHo

yP-Aminobenzylpenicillin

2 2

2

2

3

cx-Aminobenzylpenicillin

NH

( Ampicillin )

2

Figure

18.

Amino-substituted

penicillins


62

DRUG

t h e c e l l w a l l of Escherichia

DISCOVERY

coli, f o r b o t h are potent i n h i b i t o r s of the iso

l a t e d t r a n s p e p t i d a s e e n z y m e — w h i c h is a major site of p e n i c i l l i n a c t i o n (88). F u r t h e r studies of α - s u b s t i t u t e d b e n z y l p e n i c i l l i n s l e d to the i n d e p e n d e n t d i s c o v e r y b y P f i z e r a n d B e e c h a m chemists of c a r b e n i c i l l i n (a-carb o x y b e n z y l p e n i c i l l i n ) ( F i g u r e 19)

(89,

i n w h i c h the g r a m - n e g a t i v e

90),

s p e c t r u m is e x t e n d e d s t i l l f u r t h e r (to Pseudomonas


a m p i c i l l i n - i n s e n s i t i v e i n d o l e - p o s i t i v e Proteus

Figure 19.

a n d the

aeruginosa

species).

Carbenicillin

Structural Modification by Biosynthetic Methods. T h e structure of u s e f u l a n t i b i o t i c s c a n be a l t e r e d b y means other t h a n c h e m i c a l synthesis and transformation.

It has a l r e a d y b e e n p o i n t e d o u t that the first struc

t u r a l l y m o d i f i e d p e n i c i l l i n to a c h i e v e w i d e s p r e a d c l i n i c a l use w a s

the

b i o s y n t h e t i c p h e n o x y m e t h y l p e n i c i l l i n . A m a j o r i t y ( 6 7 % ) of the r e s p o n d ents b e l i e v e that m o d i f i c a t i o n of the structures of p r e s e n t l y u s e d a n t i biotics

a

fruitful

a p p r o a c h to d i s c o v e r y of n e w a n t i b a c t e r i a l a n t i b i o t i c s i n the

b y m a n i p u l a t i o n of

coming

decade.

biosynthetic

pathways

will

be

A m o n g the p o s s i b l e v a r i a t i o n s of this a p p r o a c h a r e :

simple

u t i l i z a t i o n of precursors b y a n t i b i o t i c - p r o d u c i n g m i c r o o r g a n i s m s (as i n the case of p h e n o x y m e t h y l p e n i c i l l i n ) , u t i l i z a t i o n of precursors b y m u tants h a v i n g specific m e t a b o l i c b l o c k s ( as i n the p r e p a r a t i o n of m o d i f i e d n e o m y c i n s b y Shier, R i n e h a r t , a n d G o t t l i e b )

use of i n h i b i t o r s of

(91),

specific e n z y m a t i c reactions ( as i n the f o r m a t i o n of 6 - d e m e t h y l c h l o r t e t r a c y c l i n e b y a c h l o r t e t r a c y c l i n e p r o d u c e r i n the presence of e t h i o n i n e )

(92),

use of v a r i o u s b i o l o g i c a l means of c o m b i n i n g genetic m a t e r i a l of different organisms at least one of w h i c h is a n a n t i b i o t i c p r o d u c e r (e.g., s y n c y t i c recombination, transformation, transduction a n d lysogenic a n d m u t a t i o n of a n t i b i o t i c p r o d u c e r s

(93),

6-demethylchlortetracycline

(94)

(as

conversion)

i n the p r e p a r a t i o n of

and rifamycin S V

(95).

I n p r i n c i p l e , b i o s y n t h e t i c m e t h o d s c a n p r o v i d e s t r u c t u r a l variants that are inaccessible or v e r y d i f f i c u l t l y accessible b y c h e m i c a l m e t h o d s ; o n the other h a n d , except w h e r e i n c o r p o r a t i o n of a w i d e v a r i e t y of p r e cursors is p o s s i b l e these m e t h o d s d o not n o w p r o v i d e a means f o r d i r e c t l y m a k i n g pre-selected changes i n structure.

E n z y m a t i c or m i c r o b i o l o g i c a l

t r a n s f o r m a t i o n of a n t i b i o t i c s is a r e l a t e d a p p r o a c h that i n p r i n c i p l e c a n bring

about

selective

a n d specific

s t r u c t u r a l changes.

Although

this

m e t h o d was u s e d w i t h success i n the s t e r o i d field, it has not yet p r o v i d e d


3.

CONOVER

Microbiological

63

Sources

significant d r u g discoveries i n the a n t i b i o t i c

field.

It has r e c e i v e d r e l a -

t i v e l y l i t t l e attention. Structural Modification of Toxic or Poorly Efficacious Antibiotics. M o d i f i c a t i o n of toxic o r p o o r l y efficacious a n t i b i o t i c s does not q u i t e g a i n m a j o r i t y s u p p o r t of the p a n e l as a f r u i t f u l d i s c o v e r y a p p r o a c h f o r t h e next d e c a d e

(Table V I I ) .

T h e a u t h o r joins the m i n o r i t y ( — 4 0 % )

of

respondents o n this q u e s t i o n . If a d r u g fails to meet present d a y standards because of l o w in

vitro

p o t e n c y , m e t a b o l i c , or c h e m i c a l i n s t a b i l i t y , p o o r o r a l a b s o r p t i o n o r h i g h degree of s e r u m a n d tissue b i n d i n g , experience teaches that the prospects


f o r i m p r o v e m e n t via s t r u c t u r a l m o d i f i c a t i o n are g o o d — i f systematic struct u r a l m o d i f i c a t i o n w i t h r e t e n t i o n of b i o l o g i c a l a c t i v i t y is feasible.

The

clinically

and

established

semisynthetic

cephalosporins,

rifamycin SV

r i f a m p i c i n represent p r e c i s e l y this k i n d of i m p r o v e m e n t , w h i l e l a b o r a t o r y d a t a i n d i c a t e that i t has also b e e n a c h i e v e d i n the c o u m e r m y c i n series as w e l l (96,

97).

Table VII.

Ranking of Discovery Potential of Approaches to Antibacterial D r u g Discovery Approach

Rank"

Percent Approach

Judging Fruitful

P r e p a r a t i o n of s t r u c t u r a l analogs of u s e f u l a n t i b i o t i c s b y c h e m i c a l or other means

1

83

I s o l a t i o n of new m i c r o b i a l metabolites

1

92

P r e p a r a t i o n of s t r u c t u r a l analogs of toxic or p o o r l y efficacious a n t i b i o t i c s b y c h e m i c a l or other means

2

42

D i r e c t e d synthesis of organic c o m p o u n d s based u p o n a b i o c h e m i c a l r a t i o n a l e

3

h

E m p i r i c a l screening of organic compounds u n related to e x i s t i n g a n t i b i o t i c s

4

B

The over-all rank was derived from individual rankings by use of a weighted scoring system. Appropriate question was not asked; however, 65% of the respondents judged that the search for new microbial metabolites will represent a better approach to the discovery of antibacterial drugs having novel structure, mode of action, and range of efficacy. a

b

SEMISYNTHETIC CEPHALOSPORINS.

C b e c a m e k n o w n (98), analogs

O n c e the structure of c e p h a l o s p o r i n

attempts w e r e m a d e to find c h e m i c a l l y m o d i f i e d

h a v i n g superior biological properties.

This

was

successfully

a c h i e v e d w i t h the synthesis of c e p h a l o t h i n , c e p h a l o r i d i n e , c e p h a l o g l y cine, a n d cephalexin ( F i g u r e 2 0 ) .

I n the first t w o , in vitro p o t e n c y w a s

e n h a n c e d as m u c h as 10,000 f o l d ( 9 9 ) , i n the latter t w o o r a l a b s o r p t i o n



64

DRUG

Cephaloglycine

Cephalexin

Figure 20. was achieved

(100).

DISCOVERY

Semisynthetic

cephalosporins

T h e starting p o i n t f o r this research

was quite

different f r o m that f o r the semisynthetic p e n i c i l l i n s . T h e p a r e n t

com-

p o u n d w a s too w e a k l y active to m e r i t c l i n i c a l a p p l i c a t i o n a l t h o u g h q u a l i t a t i v e l y , its b i o l o g i c a l properties e l i c i t e d interest; i t w a s active both

g r a m - p o s i t i v e a n d gram-negative

bacteria

against

a n d w a s resistant

to

benzylpenicillinases. H e r e as w i t h the tetracyclines, t h e s o l u t i o n of d i f f i c u l t c h e m i c a l p r o b lems w a s a p r e r e q u i s i t e to successful n e w d r u g d i s c o v e r y .

A t first t h e

c o u n t e r p a r t of 6 - a m i n o p e n i c i l l a n i c a c i d c o u l d b e m a d e o n l y i n v e r y l o w y i e l d s b y c h e m i c a l h y d r o l y s i s . A p r a c t i c a l e n z y m a t i c h y d r o l y s i s of c e p h a l o s p o r i n C to 7 - a m i n o c e p h a l o s p o r a n i c

acid ( 7 - A C A ) was not found.

R . B . M o r i n a n d c o - w o r k e r s p r o v i d e d t h e elegant s o l u t i o n ( F i g u r e 2 1 ) (101),

w h i c h m a d e the p r e p a r a t i o n of 7 - A C A a n d semisynthetic c e p h a l o -

sporins possible o n a p r a c t i c a l scale.

T h e i m p e t u s to persevere i n this

C0 H 2

7- Aminocephalosporanic Acid ( 7ACA)

Figure 21.

7-Aminocephalosporanic

acid


3.

Microbiological

CONOVER

65

Sources

e n d e a v o r was i n d u b i t a b l y p r o v i d e d b y the k n o w l e d g e that s u p e r i o r semi synthetic p e n i c i l l i n s h a d b e e n d e r i v e d f r o m 6 - A P A .

If c e p h a l o s p o r i n C

h a d b e e n the o n l y ^ - l a c t a m a n t i b i o t i c i s o l a t e d f r o m m i c r o b i a l sources, it is q u e s t i o n a b l e

w h e t h e r β-lactam a n t i b i o t i c s of either

s e m i s y n t h e t i c o r i g i n w o u l d b e i n c l i n i c a l use t o d a y . whether

there exist

neglected

antibiotics

m i c r o b i a l or

O n e m u s t n o w ask

w h i c h are

as

p r o m i s i n g as

c e p h a l o s p o r i n C as s t a r t i n g p o i n t s f o r s t r u c t u r a l m o d i f i c a t i o n a n d w h e t h e r , b y the c r i t e r i a n o w u s e d to evaluate n e w l y d i s c o v e r e d a n t i b i o t i c s , p o t e n t i a l v a l u e of a c e p h a l o s p o r i n C

(discovered i n isolation)

the

would

be r e c o g n i z e d .


SEMISYNTHETIC

RIFAMYCINS.

T h e degree a n d v a r i e t y of the i m p r o v e

ments that c a n be m a d e i n a n a n t i b i o t i c that has no c l i n i c a l u t i l i t y per are w e l l i l l u s t r a t e d b y the s e m i s y n t h e t i c r i f a m y c i n s ( F i g u r e 22) T h e m i c r o b i a l m e t a b o l i t e , r i f a m y c i n B , is u n s t a b l e i n aqueous

se

(102). solutions

exposed to o x y g e n a n d owes most, i f not a l l , of its a p p a r e n t a c t i v i t y to the c o r r e s p o n d i n g 1 , 4 - q u i n o n e , r i f a m y c i n S.

R e d u c t i o n of this q u i n o n e

C H , — C —

CH 0 3

R RIFAMYCIN

Β

-CH -C0 H

-H

RIFAMYCIN

SV

-H

-H

2

2

0 -CH -C-N:

RIFAMIDE

2

RIFAMPICIN

C2H5

-H

Figure 22.

The

2

-H Η Γ Λ -C*N-N N-CH

rifamycins


3

66

DRUG

DISCOVERY

p r o v i d e d r i f a m y c i n S V , a p a r e n t e r a l d r u g w h i c h is c l i n i c a l l y u s e d p r i m a r i l y against g r a m - p o s i t i v e a n d b i l i a r y tract infections.

The biological

consequences of a w i d e v a r i e t y of a d d i t i o n a l s t r u c t u r a l changes w e r e s u r v e y e d , a n d active

structures

capable

of systematic

variation

were

identified. s h o w e d that,

generally

s p e a k i n g , i n c r e a s e d d r u g l i p o p h i l i c i t y is a c c o m p a n i e d b y a

Study

of

the

Ν,Ν-dialkylrifamycinamides

decreased

rate of b i l i a r y excretion,

increased

increased

biological half-life and

c h e m o t h e r a p e u t i c efficacy after o r a l a d m i n i s t r a t i o n , r e l a t i v e to p a r e n t e r a l . R i f a m i d e ( r i f a m y c i n Β Ν,Ν-diethylamide)

w h i c h has a c h i e v e d significant


c l i n i c a l use has a t h e r a p e u t i c i n d e x s u p e r i o r to that of r i f a m y c i n S V .

The

3-formylrifamycin S V hydrazones provided compounds exhibiting good o r a l a b s o r p t i o n , l o n g b i o l o g i c a l h a l f - l i f e , l o w b i l i a r y excretion rate, a n d excellent c h e m o t h e r a p e u t i c

e x p e r i m e n t a l infections.

tuberculosis member

a c t i o n vs. g r a m - p o s i t i v e a n d

of the

series,

represents

Mycobacterium

R i f a m p i c i n , w h i c h is the o p t i m u m an important new

a d d i t i o n to

the

c a n s p e l l the difference

be

a r m a m e n t a r i u m of a n t i t u b e r c u l a r drugs. T h a t s t r u c t u r a l v a r i a t i o n of congeners

t w e e n drugs of l i t t l e or no c l i n i c a l u t i l i t y a n d drugs of w i d e a p p l i c a b i l i t y c a n also be seen b y c o m p a r i n g the properties of v a r i o u s m e m b e r s of a n t i b i o t i c classes isolated solely f r o m m i c r o b i o l o g i c a l sources. of these cases are s u m m a r i z e d i n T a b l e T h e deoxystreptamine momycin),

Some

VIII.

a m i n o g l y c o s i d e antibiotics c a t e n u l i n

neomycin, kanamycin, and gentamicin

qualitatively

ototoxicity, n e p h r o t o x i c i t y , a n d a n t i b a c t e r i a l

a c t i v i t y ; the

h o w e v e r is u s e d o n l y f o r intestinal infections

(amoebic

first

and

(paro share named

bacterial),

the second is u s e d p r i m a r i l y for l o c a l a n d i n t e s t i n a l infections w h i l e k a n a m y c i n w h i c h combines lower nephro- a n d ototoxicity w i t h good activity Table VIII. Congeners of Antibacterial Antibiotics with Improved Biological Properties Antibiotic

Class

Inferior Member

Superior Congener

Improvement

polymyxins A , C,D catenulin (paro momycin neomycin kanamycin

polymyxins Β, Ε

safety

neomycin

efficacy

kanamycin gentamicin

safety

Macrolides

picromycin

safety, efficacy

Steroids

helvolic acid, c e p h a l o s p o r i n Pi

erythromycin, oleandomycin fusidic acid

Lincomycins

celesticetin

Polymyxins Aminoglycosides

lincomycin

s p e c t r u m , efficacy

efficacy safety


3.

Microbiological

CONOVER

vs. E. coli, Proteus

67

Sources

sp. a n d S. aureus is a u s e f u l p a r e n t e r a l d r u g . I n t u r n ,

g e n t a m i c i n b y v i r t u e of increased p o t e n c y vs. g r a m - p o s i t i v e a n d g r a m negative

bacteria

(especially

Pseudomonas

aeruginosa),

has

i m p r o v e d t h e r a p e u t i c i n d e x a n d s p e c t r u m of in vivo efficacy with kanamycin.

both

an

compared

I n the c y c l i c p o l y p e p t i d e class, p o l y m y x i n s Β a n d

Ε

are s u b s t a n t i a l l y less n e p h r o t o x i c t h a n the congeners A , C , a n d D w h i c h are not u s e d c l i n i c a l l y . E r y t h r o m y c i n , l i n c o m y c i n a n d f u s i d i c a c i d f o l l o w e d congeners

that never r e a c h e d m e d i c a l use.

The

improvements

e m b o d i e d i n the later d i s c o v e r e d analogs are i n d i c a t e d i n T a b l e

VIII.

I n p r i n c i p l e , the same sorts of i m p r o v e m e n t s s h o u l d b e a c h i e v a b l e

by


c h e m i c a l m o d i f i c a t i o n of some p r e s e n t l y k n o w n b u t u n u s e d a n t i b i o t i c s . O n e o b v i o u s reservation s h o u l d b e p o i n t e d out; i m p r o v e m e n t of therapeutic

i n d e x of a n a n t i b i o t i c w i t h retention

of a c t i v i t y m a y

the be

i m p o s s i b l e w h e r e the m e c h a n i s m of t o x i c i t y is r e l a t e d to the m e c h a n i s m of a c t i o n . T h i s is true, f o r e x a m p l e , of the a c t i n o m y c i n s . Guiding Principles for Structural Modification of Existing A n t i biotics.

Some g e n e r a l p r i n c i p l e s emerge f r o m the f o r e g o i n g d i s c u s s i o n

of d r u g d i s c o v e r y via antibiotics

s t r u c t u r a l m o d i f i c a t i o n of e x i s t i n g

( p r e s e n t l y u s e f u l or

antibacterial

otherwise).

( 1 ) D r u g d i s c o v e r y u s u a l l y requires p r i o r mastery of the c h e m i s t r y of the p a r e n t a n t i b i o t i c s t r u c t u r e : reasonably f a c i l e m e t h o d s for m a k i n g selective a n d systematic s t r u c t u r a l m o d i f i c a t i o n s m u s t b e p e r f e c t e d . A versatile i n t e r m e d i a t e f r o m w h i c h a v a r i e t y of congeners m a y b e s y n t h e s i z e d is e x c e e d i n g l y u s e f u l i n this r e g a r d . ( 2 ) T h e testing of n e w congeners m u s t take i m a g i n a t i v e c o g n i z a n c e b o t h of the o b v i o u s shortcomings of the p r o t o t y p e d r u g a n d its thera p e u t i c a l l y m a r g i n a l b u t i n t r i n s i c a l l y d e s i r a b l e properties (e.g., the w e a k in vitro a c t i v i t y of b e n z y l p e n i c i l l i n vs. g r a m - n e g a t i v e s ) . Quantitative enhancement of s u c h properties c a n effect a q u a l i t a t i v e change i n the range of t h e r a p e u t i c efficacy. P o t e n t i a l l y i m p o r t a n t properties m a y be o v e r l o o k e d i n the absence of s u c h testing. ( 3 ) C h a n g e s i n t h e r a p e u t i c a l l y r e l e v a n t properties s u c h as resistance to c h e m i c a l or e n z y m a t i c i n a c t i v a t i o n , in vitro p o t e n c y , degree of s e r u m p r o t e i n b i n d i n g , rate of excretion, etc. c a n u s u a l l y be c o r r e l a t e d w i t h changes i n specific s t r u c t u r a l a n d / o r p h y s i c a l properties. S u c h c o r r e l a tions p r o v i d e the best g u i d a n c e p r e s e n t l y a v a i l a b l e to the m e d i c i n a l chemist for the d e s i g n of congeners i n w h i c h o p t i m i z a t i o n of a p a r t i c u l a r b i o l o g i c a l p r o p e r t y is sought. (4) S u b s t a n t i a l i m p r o v e m e n t of t h e r a p e u t i c i n d e x is often either b y e n h a n c e m e n t of t h e r a p e u t i c p o t e n c y , r e d u c t i o n of t o x i c i t y , or b o t h . If the m o l e c u l a r m e c h a n i s m s of t o x i c i t y a n d are the same the prospect f o r i m p r o v e m e n t i n t h e r a p e u t i c i n d e x A n u n d e r s t a n d i n g of these m e c h a n i s m s is thus i m p o r t a n t .

possible intrinsic activity is p o o r .

Isolation of N e w Microbial Metabolites. S u r p r i s i n g l y , this d i s c o v e r y a p p r o a c h is p l a c e d o n a p a r w i t h c h e m i c a l m o d i f i c a t i o n of u s e f u l a n t i biotics as the most p r o m i s i n g for the next d e c a d e ( T a b l e V I I ) .


T h i s is

68

DRUG

DISCOVERY

the j u d g m e n t o f t h e p a n e l despite t h e d i s p a r a t e records of t h e t w o a p proaches

o v e r t h e past d e c a d e

r e c o g n i t i o n that

improvements

(Figure 1).

This judgment implies a

i n b i o l o g i c a l properties

structural

via

modification of a given type of antibiotic d o have practical limits.

Micro-

b i a l m e t a b o l i s m is s t i l l v i e w e d as t h e best p o t e n t i a l source o f s t r u c t u r a l l y a n d m e c h a n i s t i c a l l y n o v e l a n t i b a c t e r i a l agents ( 6 5 %

agreement).

T h e r a t i o n a l bases f o r t h e e x p e c t a t i o n that e x a m i n a t i o n o f m i c r o b i a l fermentations

can provide important n e w antibacterial

antibiotic

dis-

coveries h a v e a l r e a d y b e e n e x p l o r e d . F o r some o f t h e respondents there is also a n element of f a i t h i n v o l v e d . W h a t is e x p e c t e d is n o t r e b i r t h of Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

the e r a of p r o l i f i c discoveries b u t t h e i s o l a t i o n a n d r e c o g n i t i o n i n t h e next d e c a d e

of a f e w s i g n i f i c a n t l y i m p r o v e d congeners

of a n t i b i o t i c s

a l r e a d y i n use, a f e w a n t i b i o t i c s b e l o n g i n g to n e w s t r u c t u r a l classes which

will

have

important

p r o t o t y p e structures

therapeutic

advantages,

and a few new

w o r t h y of chemical modification.

Synthesis of Organic Compounds Structurally Unrelated to A n t i biotics.

T h e m a j o r i t y of respondents

r a n k e d d i r e c t e d synthesis

based

u p o n a b i o c h e m i c a l r a t i o n a l e as a r e l a t i v e l y u n p r o m i s i n g d i s c o v e r y a p p r o a c h . I b e l i e v e , h o w e v e r , that t h e i n c r e a s i n g l y d e t a i l e d insights ( w h i c h h a v e c o m e i n large p a r t t h r o u g h t h e s t u d y of a n t i b i o t i c m e c h a n i s m s o f action) and

c o n c e r n i n g s u c h basic processes as b a c t e r i a l c e l l w a l l , p r o t e i n ,

n u c l e i c a c i d synthesis w i l l p r o v i d e g u i d a n c e f o r a n t i b i o t i c a n a l o g

d e s i g n that is d i s t i n c t f r o m a n d c o m p l e m e n t s that d e r i v e d s t r i c t l y f r o m structure-activity

correlations.

E m p i r i c a l screening

of o r g a n i c

p o u n d s w a s r a t e d t h e least p r o m i s i n g o f t h e five approaches

com-

considered.

N o approaches d i s t i n c t l y different f r o m those l i s t e d i n T a b l e V I I w e r e p r o p o s e d b y respondents.

Projection of Antibacterial Drug Discoveries, 1970—1980 A s a l o g i c a l s e q u e l to the r a n k i n g of t h e d i s c o v e r y p o t e n t i a l o f t h e five

approaches

to a n t i b i o t i c d r u g d i s c o v e r y l i s t e d i n T a b l e V I I , t h e

p a n e l w a s a s k e d to p r e d i c t i n w h a t d r u g categories discoveries of major, substantial, or m a r g i n a l i m p o r t a n c e w o u l d b e m a d e i n t h e next

decade.

G e n e r a l l y , t h e expectations s u m m a r i z e d i n T a b l e I X are consistent the p r e v i o u s l y discussed judgments o f a p p r o a c h .

with

T h u s 9 3 % of those

( a p p r o x i m a t e l y 60 persons) w i l l i n g t o assume t h e r o l e of oracle, expect that n e w drugs s t r u c t u r a l l y r e l a t e d to a n t i b a c t e r i a l a n t i b i o t i c s n o w i n use w i l l constitute discoveries of m a j o r o r s u b s t a n t i a l i m p o r t a n c e . r a n k i n g of specific classes is as f o l l o w s : ( 1 ) c e p h a l o s p o r i n s ,

The

(2) peni-

c i l l i n s , ( 3 ) arwamacrolides, a n d ( 4 ) a m i n o g l y c o s i d e s . A m a j o r i t y of t h e respondents

expect i m p o r t a n t discoveries to b e m a d e i n e a c h o f these

classes.


3.

CONOVER

Microbiological

Table IX.

69

Sources

Predicted Importance of Antibacterial D r u g Discoveries 1970-1980"


Substantial or Major Discovery

No Significant Discovery

Marginal Discovery

P r e s e n t l y used a n t i b a c t e r i a l antibiotics cephalosporins penicillins ansamacrolides (rifamycins) aminoglycosides macrolides lincomycins tetracyclines polymyxins

93 68 61 54 52 44 35 29 10

5 20 18 32 29 40 43 34 20

2 12 21 14 19 16 22 37 70

P r e s e n t l y u n k n o w n class of m i c r o b i a l metabolites

80

7

13

A synthetic drug unrelated i n structure to e x i s t i n g a n t i b i o t i c s

49

16

35

P r e s e n t l y k n o w n t o x i c or m a r g i n a l l y effective class

39

34

27

a

Numbers represent percentage of respondents making designation. E i g h t y p e r c e n t of respondents p r e d i c t that p r e s e n t l y u n k n o w n classes

of m i c r o b i a l metabolites w i l l p r o v i d e discoveries of m a j o r or s u b s t a n t i a l importance, w h i l e only 3 9 %

expect

that a p r e s e n t l y k n o w n t o x i c or

m a r g i n a l l y effective class w i l l d o so.

F o r t y - n i n e percent expect t h a t a

synthetic d r u g u n r e l a t e d i n s t r u c t u r e to existing a n t i b i o t i c s w i l l constitute an important discovery.

T h i s p r e d i c t i o n is s o m e w h a t at v a r i a n c e w i t h

the j u d g m e n t ( a l r e a d y d i s c u s s e d ) that m o d i f i c a t i o n of toxic or m a r g i n a l l y effective

antibiotics

is s u p e r i o r as

a research

a p p r o a c h to e m p i r i c a l

s c r e e n i n g of o r g a n i c c o m p o u n d s a n d b i o c h e m i c a l l y b a s e d d i r e c t e d s y n thesis.

Microbial Metabolite Drugs of Diverse Application, 1939—1970 T o this p o i n t , t h e d i s c u s s i o n has b e e n c o n c e r n e d e x c l u s i v e l y w i t h antibacterial antibiotics.

H i s t o r i c a l l y , these w e r e the first to g a i n w i d e -

s p r e a d use, t h e y h a v e b e e n the m o s t s t u d i e d , a n d t h e y are the numerous.

most

G e n e r a l p r i n c i p l e s of d r u g d i s c o v e r y d e r i v e d f r o m a c o n -

s i d e r a t i o n of this class s h o u l d b e a p p l i c a b l e to other classes as w e l l . T h e selective t o x i c i t y of a n t i b i o t i c s is, of course, not c o n f i n e d to b a c t e r i a l pathogens.

S o m e of the earliest d i s c o v e r e d a n t i b i o t i c s possessed

other types of a c t i v i t y . T h u s g r i s e o f u l v i n ( 1 9 3 9 ) a n d n y s t a t i n ( 1 9 5 1 ) are a n t i f u n g a l agents, a n d the a c t i n o m y c i n s ( 1 9 4 0 ) are

antineoplastic


70

DRUG

as w e l l as a n t i b a c t e r i a l .

DISCOVERY

T h e a c t i o n of t h e b r o a d - s p e c t r u m a n t i b i o t i c s

( c h l o r a m p h e n i c o l a n d the tetracyclines ) extends t o some p r o t o z o a , m y c o p l a s m a , r i c k e t t s i a , spirochetes, lymphogranuloma type.

a n d t h e s o - c a l l e d l a r g e viruses o f t h e

A n t i b i o t i c s are presently t h e d o m i n a n t agents

u s e d f o r t h e r a p y of diseases c a u s e d b y f u n g i a n d a l l of t h e organisms just m e n t i o n e d save p r o t o z o a ; t h e y are b e c o m i n g a n i n c r e a s i n g l y i m p o r tant factor i n cancer c h e m o t h e r a p y .

I n the animal health

m y c i n (103)

are a n t h e l m i n t i c agents f o r s w i n e

a n d d e s t o m y c i n (J04)

field,

hygro-

a n d c h i c k e n s , a n d m o n e n s i c a c i d ( 1 0 5 ) is u n d e r d e v e l o p m e n t as a n a n t i p r o t o z o a l ( c o c c i d i o s t a t i c ) agent f o r use i n c h i c k e n s . Downloaded by MONASH UNIV on October 2, 2013 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch003

(106),

v i r g i n i a m y c i n (107),

t h e m i k a m y c i n s (108)

Moenomycin

a n d zeranol (109)

are b e i n g u s e d as a n i m a l g r o w t h p r o m o t a n t s . A s t h e d i s c o v e r y rate of c l i n i c a l l y a c c e p t e d a n t i b a c t e r i a l m i c r o b i a l metabolites has f a l l e n , that f o r m i c r o b i a l metabolites h a v i n g other types of u s e f u l b i o l o g i c a l a c t i v i t y has r e m a i n e d at a f a i r l y constant l e v e l ( T a b l e X).

E x a m i n a t i o n of t h e structures of t h e m i c r o b i a l metabolites

whose

p r i m a r y use is n o t as c h e m o t h e r a p e u t i c a n t i b a c t e r i a l agents shows that most d o n o t b e l o n g to a n y of t h e major s t r u c t u r a l classes of a n t i b a c t e r i a l a n t i b i o t i c s . F u r t h e r m o r e , representatives o f u s e f u l n e w classes h a v e b e e n d i s c o v e r e d w i t h i n the past d e c a d e .

N e w structures w i l l b e f o u n d w h e n

m i c r o b i a l fermentations are tested f o r n e w types of b i o l o g i c a l a c t i v i t y . Table X .

Metabolite

Microbial Metabolites with Useful Antifungal, Antiparasitic, or Antineoplastic A c t i v i t y Year of First Literature Report Biological Activity

Griseofulvin Fumagillin Nystatin Paromomycin Trichomycin Candicidin Sarkomycin Dactinomycin Amphotericin Β Mitomycin C Hygromycin Β Pimaricin Chromomycin A Hamycin Mithramycin Daunomycin Pyrrolnitrin Destomycin Bleomycin Monensic acid Adriamycin

3

antifungal antiprotozoal antifungal antiprotozoal antifungal/antiprotozoal antifungal antineoplastic antineoplastic antifungal antineoplastic anthelmintic antifungal antineoplastic antifungal antineoplastic antineoplastic antifungal anthelmintic antineoplastic antiprotozoal antineoplastic

1939 1949 1950 1952 1952 1953 1953 1954 1956 1957 1958 1958 1960 1960 1962 1964 1964 1965 1966 1968 1969


3.

Microbiological

CONOVER

Clearly,

71

Sources

microbial metabolism should be

source of d r u g s f o r a v a r i e t y of uses.

considered a

potential

O n e q u e s t i o n t h a t arises i n this

c o n n e c t i o n concerns the r e l a t i v e f r e q u e n c y w i t h w h i c h different t y p e s of a c t i v i t y o c c u r .

A f e w authors h a v e a p p r o a c h e d this q u e s t i o n

by

a n a l y z i n g the types of b i o l o g i c a l a c t i v i t y r e p o r t e d f o r k n o w n a n t i b i o t i c s . D a t a a b s t r a c t e d f r o m K u r y l o w i c z (110)

a n d B e r d y a n d M a g y a r (30)

are

shown i n Table X I . Table X I .

Reported Incidence of Inhibitory Action of Known Antibiotics


Γ

IP

Gram-positive bacteria Gram-negative bacteria A c i d fast bacteria F u n g i a n d yeasts

37% 15% 16% 20%

Protozoa Viruses Tumors'

4% 3% 21%

α b c

Staphylococcus aureus Escherichia coli Mycobacterium sp. Candida albicans dermatophyta protozoa viruses tumors helminths

60.5% 29.2% 25% 25% 16% 11% 7% 4% 0.2%

W. Kurylowicz {109). J. Berdy and K. Magyar (80). Reported as cytostatic activity. T h e m a j o r i t y of the p a n e l believes, h o w e v e r , that the f r e q u e n c y w i t h

w h i c h a p a r t i c u l a r t y p e of b i o l o g i c a l a c t i v i t y has b e e n d i s c o v e r e d has b e e n d e t e r m i n e d to a n i m p o r t a n t extent b y the f r e q u e n c y w i t h w h i c h effective tests h a v e b e e n m a d e f o r s u c h a c t i v i t y ; ( 9 0 %

agreement).

In

t u r n , this f r e q u e n c y has b e e n d e t e r m i n e d l a r g e l y b y the a v a i l a b i l i t y of (1)

a h i g h c a p a c i t y r e p r o d u c i b l e test system s u i t a b l e f o r use w i t h fer

m e n t a t i o n broths a n d ( 2 )

r a p i d , sensitive m e t h o d s f o r d e t e c t i n g a n d

a s s a y i n g b i o l o g i c a l l y active m a t e r i a l s a n d thus f o r g u i d i n g p u r i f i c a t i o n and isolation ( 9 6 % been

tested

agreement).

I n g e n e r a l , m i c r o b i a l metabolites h a v e

narrowly for biological activity ( 8 4 %

agreement),

pre

s u m a b l y because specific laboratories h a v e c o n c e n t r a t e d o n specific types of a c t i v i t y w h i c h w e r e c o n s i d e r e d i m p o r t a n t a n d w h i c h c o u l d b e d e t e c t e d b y a v a i l a b l e in vitro

methods.

B e c a u s e of this, u s e f u l d r u g s h a v e u n

d o u b t e d l y b e e n m i s s e d . I d o n o t h i n k the p o s i t i o n of t h e p a n e l is that a l l types of b i o l o g i c a l a c t i v i t y o c c u r w i t h e q u a l f r e q u e n c y .

Indeed, the

p u b l i s h e d studies (e.g., those of A v r a a m o v a et al. a n d of B u r k h o l d e r ) i n w h i c h large n u m b e r s of isolates h a v e b e e n tested f o r different types of a c t i v i t y i n d i c a t e o t h e r w i s e (111,

112).

U n t i l , however, large numbers

of isolates are tested i n v a r i o u s m e d i a b y e q u a l l y sensitive d e t e c t i o n m e t h o d s f o r a v a r i e t y of b i o l o g i c a l activities, n o accurate estimate of the intrinsic frequencies

can be

made.


72

DRUG

DISCOVERY

The Future Role of Microbial Metabolites in Drug Research T h e p a n e l a g r e e d ( 8 1 % ) that i n t h e next d e c a d e m i c r o b i a l m e t a b o lites w i l l c o n t i n u e to p r o v i d e significant discoveries n o t o n l y i n areas of h u m a n m e d i c i n e a n d a n i m a l h e a l t h i n w h i c h d r u g s of m i c r o b i o l o g i c a l o r i g i n h a v e b e e n p r o m i n e n t i n the past 30 years b u t also i n n e w fields. T a b l e X I I tabulates the j u d g m e n t s of t h e p a n e l c o n c e r n i n g t h e i m p o r t a n c e that s e c o n d a r y m i c r o b i a l metabolites w i l l h a v e a m o n g n e w d r u g discoveries i n m a j o r fields of c h e m o t h e r a p y a n d i n t h e r e a l m of p h y s i o l o g i c a l l y a c t i v e d r u g s . T h e panel's o v e r v i e w of t h e r o l e w h i c h m i c r o b i a l


metabolites w i l l p l a y i n t h e n e w d r u g discoveries of t h e next d e c a d e i s : among antibacterial a n d antifungal drugs, a continuing major role; among antiprotozoal a n d animal g r o w t h stimulating drugs, a n increasingly i m portant role; among antineoplastic, antiviral a n d anthelmintic drugs, a significant b u t n o t major role.

M o s t i n t e r e s t i n g is the v i e w of 4 6 % of

t h e respondents that m i c r o b i a l metabolites w i l l b e a m o n g t h e s u b s t a n t i a l or most i m p o r t a n t discoveries of d r u g s h a v i n g p h y s i o l o g i c a l actions. W h e n the t i m e p e r i o d c o n s i d e r e d is 20 years, this percentage 74%

rises to

a n d exceeds that f o r a n t i n e o p l a s t i c a n d a n t i v i r a l d r u g s . ( A l t h o u g h

this subject is b e y o n d t h e scope of this p a p e r , i t is e x p e c t e d that m i c r o b i a l metabolites w i l l also p r o v i d e n e w h e r b i c i d e s , insecticides, f u n g i c i d e s , a n d p l a n t g r o w t h regulators i n the f u t u r e . ) W h a t is t h e basis f o r t h e v i e w that m i c r o b i a l m e t a b o l i t e d r u g s w i l l l e a p t h e b o u n d s of i n f e c t i o u s disease a n d cancer c h e m o t h e r a p y to w h i c h t h e y s e e m i n g l y h a v e b e e n c o n f i n e d since 1940? Just as i t w a s clear p r i o r to 1940 that m i c r o o r g a n i s m s p r o d u c e a v a r i e t y of a n t i b a c t e r i a l a n d a n t i Table XII. Predicted Importance of Microbial Metabolite Discoveries 1970-1980 by D r u g Category

Drug

Category

Antibacterial Antiprotozoal Antifungal Antiviral Antineoplastic A n i m a l growth stimulant Anthelmintic Drugs having physiological actions"

Among Most Important Discoveries

Among Substantial Discoveries

Among Minor Discoveries

Not Among Significant Discoveries

39 14 20 31 27

61 54 54 18 18

0 12 16 13 16

0 20 10 38 39

22 10

55 35

7 20

16 35

22

24

3

51

"For example, drugs acting on the central nervous system, cardiovascular system, respiratory system, diabetes, rheumatoid arthritis, etc.


3.

Microbiological

CONOVER

73

Sources

f u n g a l substances, t o d a y it is clear that m i c r o o r g a n i s m s p r o d u c e a v a r i e t y of metabolites ( i n a d d i t i o n to v i t a m i n s a n d ergot a l k a l o i d s ) w h i c h affect o r h a v e the p o t e n t i a l to affect p h y s i o l o g i c a l processes i n m a n a n d a n i mals.

( T h i s subject has r e c e n t l y b e e n r e v i e w e d b y P e r l m a n a n d P e r u z -

z o t t i (113).)

T h e examples c i t e d i n T a b l e X I I I demonstrate this p o i n t .

T h e s i t u a t i o n is, of course, not strictly p a r a l l e l to that of 1940 b e c a u s e p h y s i o l o g i c a l l y active substances d i s c o v e r e d t o d a y w i l l not enter a thera p e u t i c v a c u u m as d i d n e w a n t i b a c t e r i a l d r u g s 30 years ago.

Chemical

m o d i f i c a t i o n of n e w l e a d structures m a y b e r e q u i r e d b e f o r e n e w d r u g s


actually

emerge.

Table XIII.

Microbial Metabolites having Demonstrated or Potential Physiological A c t i v i t y Type of Biological

Compound Fusaric acid Colisan Monorden Muscarine Slaframine Serotonin Psilocybin and Psilocin HO-2135 Nigrifactin Zeranol Mycophenolic acid EJrgosterol Leupeptins

Pepstatin Chymostatin Desferrioxamine-B

Activity

hypotensive, dopamine-βhydroxylase inhibition antispasmodic sedative parasympathomimetic parasympathomimetic biogenic a m i n e hallucinogenic serotonin a n t a g o n i s m antihistaminic, hypotensive estrogenic, a n a b o l i c immunosuppressive pro-vitamin D plasmin and t r y p s i n proteolysis inhibition, thrombokinase inhibition pepsin i n h i b i t i o n chymotrypsin inhibition t h e r a p e u t i c for bronze diabetes, hemochromatosis, a n d acute i r o n p o i s o n i n g (via i r o n chelation)

Reference (120) (121) (122-4) (125) (126,127) (128,129) (128,130) (131) (132) (109) (133) (184)

(135) (136,137) (138)

(139)

Japanese w o r k e r s are at the f o r e f r o n t i n the search f o r p h y s i o l o g i c a l l y active m i c r o b i a l metabolies.

T h e i r d e t e c t i o n m e t h o d s are w o r t h y

of n o t e for l a c k of satisfactory testing m e t h o d s has l o n g h a m p e r e d this search.

U m e z a w a a n d his c o - w o r k e r s u s e d in vitro

enzyme inhibition

tests to detect f u s a r i c a c i d , p e p s t a t i n , c h y m o s t a t i n , a n d the l e u p e p t i n s . N i g r i f a c t i n w a s d i s c o v e r e d b y T e r a s h i m a a n d c o - w o r k e r s b y seeking i n experimental

fermentations,

materials

h a v i n g the

chemical

properties

of a l k a l o i d s . F u r t h e r examples of the o c c u r r e n c e of u s e f u l or p o t e n t i a l l y u s e f u l p h y s i o l o g i c a l a c t i v i t y a m o n g m i c r o b i a l metabolites

are e m e r g i n g f r o m


74

DRUG

DISCOVERY

observations of s e c o n d a r y b i o l o g i c a l properties of antibiotics. T h u s , some of the a n t i f u n g a l p o l y e n e m a c r o l i d e s ( c a n d i c i d i n , a m p h o t e r i c i n B , filipin ) have

been

r e p o r t e d to r e d u c e

g l a n d v o l u m e i n dogs (114,

115).

a n t i - i n f l a m m a t o r y a c t i v i t y (116,

s e r u m cholesterol

levels

and

prostate

G r i s e o f u l v i n has b e e n r e p o r t e d to have 117,

118)

and mithramycin, in addition

to its b e n e f i c i a l effect u p o n testicular t u m o r s , is a n t i h y p e r c a l c e m i c .

It is

interesting that the p o l y e n e m a c r o l i d e s deplete cholesterol, m i t h r a m y c i n depletes c a l c i u m , a n d d e s f e r r i o x a m i n e Β ( T a b l e X I I I ) depletes i r o n , i n v i e w of the fact that these m i c r o b i a l metabolites


the same materials in

f o r m complexes

with

vitro.

Conclusion F o r the foreseeable f u t u r e , i m p o r t a n t n e w d r u g discoveries w i l l be d e r i v e d f r o m p r o d u c t s of m i c r o b i a l m e t a b o l i s m .

If the f u l l potentialities

of this d i s c o v e r y source are to be r e a l i z e d , d i s c o v e r y processes w i l l h a v e to be r e f i n e d a n d b r o a d e n e d i n scope. and

T h e s e are some of the

o p p o r t u n i t i e s that I see for the d e c a d e

challenges

ahead:

( 1 ) T h e standards for acceptance of n e w a n t i b a c t e r i a l drugs are n o w e x c e e d i n g l y h i g h a n d w i l l b e c o m e e v e n m o r e d e m a n d i n g . T h e re m a i n i n g unsatisfied t h e r a p e u t i c needs constitute d i f f i c u l t targets for d r u g t h e r a p y . I n v i e w of this, r e l a t i v e l y f e w n e w l y d i s c o v e r e d m i c r o b i a l metabolites w i l l q u a l i f y per se for c o m m e r c i a l i z a t i o n a n d general c l i n i c a l a p p l i c a t i o n i n this field. P r e l i m i n a r y testing of n e w m i c r o b i a l metabolites s h o u l d t h e n i d e n t i f y b o t h p o t e n t i a l d r u g s a n d p r o t o t y p e structures w h o s e b i o l o g i c a l a n d c h e m i c a l properties justify s t r u c t u r a l m o d i f i c a t i o n d e s i g n e d to u p g r a d e b i o l o g i c a l p e r f o r m a n c e . T h e roster of p r e v i o u s l y d i s c o v e r e d , s t r u c t u r a l l y n o v e l b u t u n u s e d antibiotics s h o u l d be r e e x a m i n e d for sub stances of the latter t y p e . A p p l i c a t i o n of b i o c h e m i c a l tests for m e c h a n i s m a n d selectivity of a c t i o n s h o u l d h e l p d e t e r m i n e w h i c h prototypes h o l d the most p r o m i s e . ( 2 ) A n t i b i o t i c s p r o v i d e some of the best o p p o r t u n i t i e s for u n d e r standing d r u g action i n intimate detail. Research directed toward i m p r o v e m e n t of a t h e r a p e u t i c a l l y i m p o r t a n t p r o p e r t y of a n a n t i m i c r o b i a l agent s h o u l d seek an u n d e r s t a n d i n g of the c r i t i c a l d e t e r m i n a n t ( s ) of that p r o p e r t y . T h u s to increase in vitro p o t e n c y one m a y n e e d to increase resistance to e n z y m a t i c d e s t r u c t i o n , increase rate of p e n e t r a t i o n of the c e l l w a l l or m e m b r a n e , or increase i n t r i n s i c p o t e n c y at the m o l e c u l a r site of a c t i o n . W h e r e a n t i m i c r o b i a l d r u g a c t i o n c a n b e s t u d i e d at the m o l e c u l a r l e v e l , the o p p o r t u n i t y s h o u l d b e g r a s p e d to l e a r n as m u c h as possible a b o u t the effect of changes i n e l e c t r i c a l , g e o m e t r i c a l , a n d c h e m i c a l properties o n this a c t i o n . W i t h o u t a f u l l u n d e r s t a n d i n g of the n a t u r e of a n d r e q u i r e m e n t s f o r f r u i t f u l d r u g - r e c e p t o r interactions, n e i t h e r basic b i o c h e m i c a l studies nor x-ray v i s u a l i z a t i o n of receptors w i l l p r o v i d e the k e y to r a t i o n a l d e s i g n of drugs. ( 3 ) T h e systematic s t r u c t u r a l v a r i a t i o n n e e d e d f o r d r u g d i s c o v e r y studies w i l l often be i m p o s s i b l e w i t h o u t mastery of c o m p l e x c h e m i s t r y


3.

CONOVER

Microbiological

75

Sources


a n d w i t h o u t the p e r f e c t i o n of s i m p l e a n d versatile synthetic m e t h o d s a p p l i c a b l e to i m p o r t a n t d r u g types. W e r e s u c h syntheses n o w a v a i l a b l e for β-lactams, m a c r o l i d e s , a n d a m i n o g l y c o s i d e s , they c o u l d p r o v i d e pres e n t l y inaccessible s t r u c t u r a l variants for b i o l o g i c a l s t u d y . T h i s is a c h a l l e n g e to w h i c h a c a d e m i c o r g a n i c chemists m i g h t w e l l r e s p o n d . ( 4 ) M i c r o o r g a n i s m s that are m o r e d i f f i c u l t to collect, isolate, a n d g r o w s h o u l d b e e x a m i n e d , i n c l u d i n g those that h a v e resisted a r t i f i c i a l c u l t i v a t i o n i n the past. E x a m i n a t i o n of e x p e r i m e n t a l m i c r o b i a l f e r m e n tations m u s t b e c o m e m o r e t h o r o u g h a n d i m a g i n a t i v e to detect m i n o r active components, substances f o r m e d o n l y u n d e r u n u s u a l c o n d i t i o n s , a n d substances h a v i n g diverse b i o l o g i c a l activities. M e d i a a n d c o n d i t i o n s that are o p t i m a l for f o r m a t i o n of a n a n t i b a c t e r i a l m e t a b o l i t e m a y w e l l not be o p t i m a l f o r f o r m a t i o n of substances h a v i n g other types of a c t i v i t y . Sensitive a n d specific in vitro tests i n d i c a t i v e of p o t e n t i a l p h y s i o l o g i c a l a c t i v i t y s h o u l d b e p e r f e c t e d a n d a p p l i e d as screening tools. N e w tech n o l o g y s u c h as h i g h pressure l i q u i d c h r o m a t o g r a p h y s h o u l d be a d a p t e d to the r a p i d i s o l a t i o n of r e l a t i v e l y p u r e c o m p o n e n t s f r o m m i c r o b i a l fer mentations thus f a c i l i t a t i n g the i s o l a t i o n a n d in vivo testing of p o t e n t i a l p h y s i o l o g i c a l l y active c o m p o n e n t s . C o u p l i n g of the most r a p i d a n d efficient separation a n d i d e n t i f i c a t i o n t e c h n i q u e s w i t h c o m p u t e r analysis of d a t a w i l l be r e q u i r e d . I n c o n c l u s i o n , a s i m p l e t r u t h bears r e p e t i t i o n w h i c h has b e e n a m p l y d e m o n s t r a t e d i n the d i s c o v e r y endeavors r e v i e w e d i n this p a p e r . w h o m a k e l a n d m a r k discoveries h a v e m i n d s r e c e p t i v e to the

Those

precedent-

b r e a k i n g significance o c c a s i o n a l l y c o n t a i n e d i n u n c o n v e n t i o n a l , obscure, a n o m a l o u s , or n e g l e c t e d

observations,

interpretations,

and

hypotheses.

T h e y b r e a k the i n t e l l e c t u a l b o n d s i m p o s e d o n most of us b y i m m e r s i o n i n i m m e d i a t e t e c h n i c a l p r o b l e m s a n d b y a c c e p t a n c e of c u r r e n t scientific r a t i o n a l i z a t i o n s , fashions, a n d d o g m a s . I n c o n s i d e r i n g the d e v e l o p m e n t s c i t e d here that h a v e a p p e a r e d so o b v i o u s after the fact (e.g., the r e c o g n i t i o n of u s e f u l a n t i b i o t i c s a n d the i s o l a t i o n a n d use of 6 - a m i n o p e n i c i l l a n i c a c i d ) , w h a t i m p o r t a n t

advances

n o w lie b e f o r e a l l of us, u n r e c o g n i z e d b u t easily attainable if o n l y w e c o u l d r e m o v e the scales f r o m o u r eyes?

F o r the present, I j o i n L o u i s

Pasteur i n his l a m e n t " M e s s i e u r s , c'est les m i c r o b e s q u i a u r o n t le d e r n i e r mot."

Acknowledgment T h e a u t h o r expresses his a p p r e c i a t i o n to a l l w h o so e n t h u s i a s t i c a l l y a n d p a i n s t a k i n g l y r e p l i e d to the questionnaire, to his colleagues, W a l t e r Celmer, F r a n k Sciavolino, John Routien, Kenneth Butler, and M a x M i l l e r for the ideas, i n f o r m a t i o n , a n d a d v i c e w h i c h t h e y generously c o n t r i b u t e d d u r i n g the p r e p a r a t i o n of this m a n u s c r i p t , a n d finally to B l a n c h e B r a l i c h , R a y m o n d Sumner,

a n d D a n i e l G i l l e n for their i n v a l u a b l e a i d i n the

c o m p i l a t i o n of q u e s t i o n n a i r e replies, v a l i d a t i o n of references, a n d p r e p a r a t i o n of tables a n d

figures.


76

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DISCOVERY


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