University of Southern California Dissertations and Theses

Pyrimidine nucleoside antagonists

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Pyrimidine nucleoside antagonists

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Content PYRIMIDINE NUCLEOSIDE ANTAGONISTS
A Thesis
Presented to
the Faculty of the Department of Biochemistry
and Nutrition
The University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
By
T. Kay Fukuhara
June 1952
UMI Number: EP41320
Ail rights reserved
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UMI EP41320
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e,o 'si f?«
This thesis, written by
..... T....MI...1OTHABA.......
under the guidance of h&£...Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fullfiH-
ment of the requirements for the degree of
MASTER OF SCIENCE
Date mY.lkv._1952
Faculty Committee
Chairman
TABLE OF CONTENTS
I
I
I
i
i
INTRODUCTION ........................................
Nucleic Acids ........ .... ...............
Biosynthesis and Metabolism.............. . . .
Significance of Nucleic Acids ...........
Biological Antagonism .........................
Practical Consideration ........ . ...........
Organisms for Screening Nucleic Acid
Antagonists ..... .......................
Antimetabolites of Nucleic Acid Biosynthesis
STATEMENT OF THE PROBLEM AND PLAN OF ATTACK . . . .
EXPERIMENTAL METHODS .............'..................
Synthesis of 5-Chlorouridine .................
Synthesis of 5-Bromouridine ...................
Preparation of 5-Bromouracil .................
Preparation of 5-Chlorouracil .................
Microbiological Determinations ...............
Neurospora.......... . . .
Streptococcus faeoalis ...................
Mycobacterium tuberculosis...............
I Biological Determination .....................
i
EXPERIMENTAL RESULTS ................................
i Neurospora.. .....................................
iv
PAGE
Inhibitions and Reversals . .............. 59
Specificity of Inhibitions . . .......... 62
Other Microorganisms........................... 6k
Streptococcus faecalis .................... 6k
Mycobacterium tuberculosis ............... 6k
Animal Experiment ...................... 6**
DISCUSS I O H ....................................... 68
SUMMARY AID CONCLUSIONS.................. 7k
BIBLIOGRAPHY ........................ ?6
LIST OF TABLES
TABLE
I.
II.
III.
Basal Medium Formula For Ifeurosuora........
Growth Inhibition of Neurosnora Ho. 1298 by
5-Halogenomicleosides .....................
Independent Inhibition of Uridine and Cytidine
Utilization by Ghlorouridine .............
PAGE
52
61
69
LIST OF FIGURES
I
*
figure
1. Inhibition of the Growth of Neurospora
Ho. 1298 by 5-Halogenonucleosid.es in the
Presence of Uridine or Cytidine as a Growth
Requirement ..... ........... ...........
2. Effects on the Growth of Heurosnora No. 1298
of Chlorouridine in the Presence of Uracil
3. Inhibition Reversals by the Metabolites . . . .
PAGE
60
63
65
INTRODUCTION
Nucleic Acids
Nucleic acids (1, 2, 3? *0 as components of cells
have been known since their discovery in 1869 by
F. Miescher. The chemistry of nucleic acids originated
with the works of Kossel in 1891 and was followed by mani
fold works on the isolation, the structure, and the
synthesis of nucleic acid derivatives (1, 6).
Two types of nueleic acids are known; namely, the
ribonucleic acid (ENA) and the desoxyribonucleic acid (DNA),
which are characterized by the inclusion of two specific
sugars, B-ribose and D-2-desoxyribose, respectively. The
RNA is present in the cytoplasm and nucleus, and the DNA
occurs in the chromosomes of the nucleus (*+, 8). Nucleic
acids are polynucleotide polymers of high molecular weight
and acidity. In the cell nucleic acids are associated
with a basic protein which results in a characteristic
cellular protein, the nucleoproteins. The theory of
tetranucleotides as building units for the nucleie acids
has been abandoned due to the discrepant results obtained
from physical-chemical determintions (3, 6, 7). The
structure of the nucleie acids is still unsettled.
Hydrolysis of nucleic acids can proceed enzymatic-
2
ally by the catalytic action of nuclease, nucleotidase, and
nucleosidase or chemically by acids and alkali (1). The
ultimate products of hydrolysis of ENA are the nitrogen-
containing components of nucleic acids, namely, the purines,
adenine and guanine, and the pyrimidines, cytosine and
uracil. The hydrolysis of DNA produces the purines, adenine
and guanine, and the pyrimidines, cytosine, thymine and
5-methylcytosine (8).
Nucleosides may be described as N-pentosyl deriva
tives of the heterocyclic bases. The purine ribonucleo-
sides include adenosine and guanosine. Desoxyadenosine
and desoxyguanosine comprise the purine desoxyribonucleo-
0=CX ,GH
W
H
Adenine Guanine 5-MethyIcytosine
0
I I
HN CH
I I I I I
0=C CH
Uracil Cytosine Thymine
3
sides. The shortened desoxyadenosine for desoxyribosyl-
i
!adenine proposed by Brady (9) has wide acceptance. Inosine
originates from the deamination of adenosine. Uric acid
riboside has been found in liver cells (10). The naturally-
occurring pyrimidine ribonueleosides and desoxynueleosides
are uridine and cytidine, and desoxycytidine and thymidine,
respectively. Desoxyuridine has not been found in nucleic
acids (3)* The sugar, ribose or desoxyribose, is attached
at position Ho. 9 of the purine (11) and on the nitrogen
Ho. 1 of the pyrimidine nucleus (12). The beta-linkage
of the sugar with the purines and pyrimidines (13) exists
at the ribosyl center. The ring structure of ribose is the
furanose form as proven by methylation (I1 *) and tritylation
(15). A similar structure for desoxyribose of thymidine is
demonstrated by tritylation (16).
The final proof of structure of the nucleosides thus
far positively confirmed by chemical degradation and synthe
sis are synthetic 1-beta-D-ribofuranosyluraciI (13), 1-beta-
D-ribofuranosylcytosine (17)> and 9-beta-D-ribofuranosyl-
adenine (18) which are identical to naturally-occurring uri
dine, cytidine, and adenosine, respectively. !
I
b
X2
r c-Na.
I I I >H
HCV C-r /O ,H
^ xc^ ^C-jCH20H
h ' '
OH
N C-ll
H H
Adenosine
0
i i
HM^CH
| IK ^CH
HV % / ° ^ h 2oh
H\|J/
6 6
H H
Inosine
0
A
HN G-CH.
0-C.
HOH'
5 . A . /
V s
H 0=C. .CH
7T
uridine
HOHoC-b^ >C
W *
6 H
H
Thymidine
The sugar imparts solubility to the nucleosides and
serves as the connecting link for the phosphates. The
nucleotides are pentosyl phosphoesters of the nucleosides.
The ribonucleotides derived from the nucleic acids include
adenylic acid, guanylic acid, uridylic acid, and eytidylie
acid. Desoxyadenylie, desoxyguanylic, desoxyeytidylie,
thymidylic acids and , recently, desoxy-5-siethyleytidylic
acid (19) constitute the desoxynucleotides. The location of
the ribonucleotide phosphoester has been found to be at the
5
earbon No. 3 of the carbohydrate moiety (16); however, due
"to the existence of isomeric ribonucleotides, a question
rises for the occurrence of a C-21-phosphoric acid ester
also (20). Enzymatic evidence for a C-3'-phosphate ester of
desoxynueleotides has been presented (21). This is in con
formity with the observation that a hydroxyl group is not
attached at C-2‘ and a trityl derivative of thymidylic acid
is obtained (16). These findings exclude carbon 2‘ and 5*
of the desoxyribose as the location of the phosphoester.
Adenylic acid of the muscle and coenzyme nucleotide is found
to be adenosine-5*-phosphate (22).
It is thought that the purine and pyrimidine nucleo
tides are interlinked through the ribose or the desoxyribose
by covalent bonding of phosphate groups and built into
larger aggregates of nucleic acids (2, 3> **•)•
Biosynthesis and Metabolism
Purines. Mass speetrographic analyses of the labeled
uric acid which is excreted after the administration of N^-^-
labeled ammonium citrate (23)? N^^-labeled glycine (2*f),
C^-labeled formate (25)» aud C^3-iabeled carbon dioxide
(26) gave evidence for the following incorporations. These
results indicated the precursors of adenine and guanine.
6
formate
glycine
formate
n*h3
Uric Acid
Elwyn and Sprinson have found that the N1^ Df serine
entered position 7 of the purines in the pigeon (27), and
the beta-carbon of serine and the methylene carbon of
glycine were extensively incorporated into the ureide
carbons of RNA and DNA purines of the rat (28). This in
corporation was explained from the observations that the
biological precursors of the “formate* 1 can be derived from
serine (29), glycine (30), ot the methyl group of choline
(31). Studies with C^-labeled formate on folic acid de
ficient chicks and rats gave evidence that folic acid is
concerned with the one-carbon metabolic intermediate (32).
Of the preformed purine precursors of nucleic acids,
lebeled adenine is incorporated into adenine and guanine
of RNA and DNA of the rat (33)• labeled guanine is not
incorporated into the rat tissues (33), but the purine
derivative, 2,6-diaminopurine (3* 0 , has been found to be a
7
precursor of nucleic acid guanine and not adenine in the
rat. Tagged xanthine, hypoxanthine, and isoguanine were
excreted as labeled uric acid (35)«
It has been reported that S-C-^-labeled adenine was
utilized by the yeast in the formation of its ENA adenine
and guanine. Guanine was efficiently utilized also, but it
was incorporated into RNA guanine only. The presence of
preformed guanine inhibited the interconversion of the
adenine into guanine (3&). Lactobacillus casei can readily
transform adenine into guanine and vice versa (37)*
Pyrimidines. The precursors of the pyrimidines have
not been completely established, however, the following
works have been reported. Some incorporation of N^-^-
labeled ammonia was found in positions 1 and 3 of cytosine
and thymine (25)* Berstrand, gt al obtained glycine-N1^
incorporation in the uridine moleeule of the rat (3^), and
Reichard found a significant isotope concentration in the
amino group of cytidine from the 1 #5-glycine injected rat
(39). Heinrich and Wilson have shown that Cll+-labeled CO2
was incorporated into the carbon 2 of uracil in the rat,
but they were unable to determine the radioactivity of the
other carbons individually (**0). Lagerkvist has isolated,
and degraded the pyrimidine nucleosides from a rat which
was injected with labeled bicarbonate and has found that
g
the carbon 2 and k- of the pyrimidines were radioactive ( * * - 1).
Extensive incorporation of the beta-C-^1 - of serine and alpha-
carbon of glycine into the methyl group of thymine was
observed (27). Recent work with Cil+-labeled formate gave
evidence that this one-carbon fragment was a precursor of
the methyl group of thymine, the process requiring folic
acid (32).
t
i
H*H-> j C*Q?
^ i r""' gly©ine-N
____________
C^Oo O s s G *> ! s G — i-GHo fo rm ate
2 | , ,J|-f---3------ -
N*Hg
Pyrimidine
Metabolic studies with dogs, which were fed pre
formed substances, have shown that the utilization of
dietary pyrimidine nucleosides, nucleotides and nucleic
acids were favored over the pyrimidines, uracil and cyto
sine, which were largely excreted unchanged (^2). Uracil
fed orally to rabbits was quantitatively exereted, but its
nucleoside or nucleic acid was metabolized (V3). Emerson
and Cerecedo found that uridine and cytidine were metabo-
; lized to urea. Cytosine was partly exereted unchanged and
partly deaminated to uracil (M+).
9
More recent studies with stable and radioactive iso
topes have confirmed these earlier findings. When labeled
thymine , uracil (**5), cytosine (*+6), and uridine and
cytidine (*+7) were administered to rats, the nucleosides
and not the pyrimidines were found to be the effective
precursors of ENA and DNA in rats. 2,*4— Diaminopyrimidine
is not incorporated into rat tissues (**8).
Eeichard and Estborn have studied the distribution
of the injected biosynthetically labeled N^-desoxyribosyl-
cytosine, thymine, and hypoxanthine in the rat. Desoxy-
cytidine is utilized for the synthesis of thymine and cyto
sine of DNA but not ENA. Thymidine is incorporated into
DNA thymine, but de soXyhypoxanthine is not utilized for the
synthesis of the polynucleotides 0+9)* It has been shown
previously that labeled cytidine was incorporated into ENA
and into the desoxycytidine of DNA This reaction
evidently is not reversible (*+9) •
Loring and Pierce (50) have shown that the mutant
Neurosnora No. 1298 was able to metabolize uridine or
uridylic acid 10 to 60 times more actively than uracil.
This effect was more striking with cytosine which is in
active, whereas cytidine was as active as uridine.
Mitchell and Houlahan (51) have investigated the activity
of some compounds as possible precursors of uracil which is
10
required, for growth by the Neurosoora mutant Ho. 1298 and
found that oxaloacetate and aminofumarie acid amide were
moderately active in stimulating the growth of this mold.
The authors proposed that the two compounds may be precur
sors of uridine (51)•
Orotic acid, a 6-carboxyl derivative of uracil, was
able to replace the folic acid requirement of certain bac
terial organisms (52) indicating a possible role of this
orotic acid was found to be a biologieal precursor of BHA
and DHA pyrimidines of the rat (53)> and isotope analysis
showed that the incorporation of this acid into cytidine was
not necessarily through the amination of uridine (39)•
Of the various compounds microbiologically tested
with Lactobacillus bulearis 09 which specifically requires
orotic acid for growth (5* 0 ? only ureidosuccinie acid and
HO
0=0
\
G-H h-hn js-h
/
H2S^G
COOH
Oxaloacetic
acid
Aminofumarie
acid amide
Orotie acid
acid in the biosynthesis of the nucleic acids. H-^^-labeled
11
5(carboxymethylidene)-hydantoin partially replaced the
orotic acid requirement and were suggested as possible
precursors of the pyrimidines (5*+a).
Bergstrom et al have found that two of the intermediates in
5-acetyl-hydantoin and 5-(carboxymethylidene)-hydantoin,
were not biological precursors of polynucleotide pyrimi
dines in the rat (55)•
The precursors of ribose have been suggested to be a
combination of glycol aldehyde with a triose to form a
pentose based on the evidence obtained by Meyerhof and
Lohmann with aldolase (3). Low has found a significant
incorporation of C^+OOH-labeled glycine in the nucleic acid
ribose of the rat and concluded that synthesis of ribose
can occur from glycine (56). Radioactivity was found in
the ribose moiety of the nucleosides isolated from the
animal given C*^-labeled formate (32).
The problem of ribose and desoxyribose formation in
connection with virus infection led Scott and Cohen (57)
Q=C
CHo
Ureido-succinic
acid
m -C=GH—GOGH
5-(Garboxymethylidene)-
hydantoin
the chemical synthesis of orotie acid, namely, ^-labeled
12
to reinvestigate the oxidation end-products of 6-phospho-
gluconate which is formed by the action of glucose phos
phate dehydrogenase and TPN on glucose-6-phosphate (Warburg
and Christian, 1933) an& found that ribose and.a pentose
enediol were formed by the action of the yeast enzyme.
Horeeker, Smyriniotis, and Seegmiller (58) obtained as oxi
dation end-products of the added sugar ribulose-5-phosphate
and a 70-80 per cent yield of ribose-5-phosphate, which is
formed by the action of pentose-phosphate-isomerase in the
yeast enzyme preparation. These investigators obtained
ribulose-5-phosphate and ribose-5-phosphate from the oxida
tion of 6-phosphogluconate by the liver and bone marrow
enzymes also (58a).
A highly active ribose-l-phosphate which can react
with hypoxanthine to produce inosine and phophate has been
demonstrated by Kalckar (59)• Since ribose-l-phosphate was
unstable, Schlenk and Waldvogel have shown a possibility of
rapid stabilization by enzymatic rearrangement to the
stable ribose-5-phosphate (60). A crystalline desoxyribose-
Ribulose-5- Ribose-5-
phosphate
13
1-phosphate has bean isolated from enzymatic phosphorolysis
of guanine desoxyriboside (61).
Significance of Nucleic Acids
The living protoplasm which is contained in the cell
of simple to complex plants and animals is a highly organ
ized colloidal system in dynamic equilibrium (62). By-
controlled interrelated metabolic processes of synthesis
and breakdown, the structure of the protoplasm is maintained
by continuous rejuvenation (63).
Nucleic acids are concerned with the anabolic activi
ties— particularly, the synthesis of specific proteins
necessary for cellular growth, blood, defense, hormones,
and other activities such as contractility, motility, con
ductivity, permeability, etc. As energy is required for the
functioning of life processes (61 *), it is assumed that the
nucleic acids are closely associated with the enzymes and
other energy-yielding activities which produce this energy
(^, 63).
The self-duplicating power, a phenomenon character
istic of living systems, is found in the ribonucleic acid
containing chromidia of the cytoplasm and the desoxyribo
nucleic acid-containing nuclear chromosomes under genetic
control (63, 65). The cytoplasm with capabilities of
lb
anabolic and catabolic activities can survive independently
of the nucleus; however, the function of the genes and
chromosomes of the nucleus are required for reproduction.
Conversely, the nucleus is dependent upon the cytoplasm for
energy and raw materials.
Growth, the ultimate manifestation of life process,
is a predetermined, multiple process encompassing the
stages of initiation, proliferation, differentiation,
organization, and anabolic growth which includes the con
structive increase in mass or substance as well as non
growth activities and maintenance (page 13). Cell division
is preceded by dedifferentiation of the parent cell follow
ed by complete reconstitution of the specialized organiza
tion in the new cells. It is thought that cancer rises
from an incomplete reconstitution caused by a specific
mutation of the chromidia (63). During the induction of
hepatic tumor in rats by * 4 — diaminoazobenzene, an accumula
tion of an aminozao dye-protein complex is formed in the
pretumorous region of the liver, evidently due to slower
rate of removal of the dye complex (67). Continual de
pletion or alteration of the liver protein by feeding the
azo dye probably affects one or more specific synthetic
mechanism controlling normal growth, and the initial tumor
cell is formed. The liver tumor cells do not form protein-
15
bound dye complex (67).
Nucleic acid production is vital to such entities
as virus and bacteria which are dependent on rapid pro
liferation for survival. A crystalline tobacco mosaic
virus has been found to consist of ribonucleic acid (68).
A Tl^ bacteriophage, viral parasite of E. coli. is 99 per
cent desoxynucleoprotein and dependent on the host for the
synthesis of its constituents (63, 69). Ultraviolet
microspectroscopic studies reveal viruses which proliferate
in the cytoplasm, others which grow in the nucleus, and
some which infect the nucleolus (70).
The evident upset in the synthesis of nueleoprotein
in malignant tumor led Brues, Tracy, and Cohn to investi
gate the rate of nucleic acid synthesis in the rat. The
result shov7ed a turnover ratio of 5*2:1 for the RNA:DIJA
phosphorus in the liver three hours after the administra
tion of p32(71). This finding proved the existence of a
dynamic state for the nucleic acids. The rate of nucleic
acid synthesis was significantly higher for the regenerat
ing liver and hepatoma (71)- In a similar type of experi
ment, Hammarsten and Hevesy found a turnover ratio of 33:1
for the RNA:DNA phosphorus six hours after the administra
tion of radioactive P32 indicating a relative stability of
the DNA (72).
16
Berstrand et al determined the rate of glycine-H-*-?
incorporation into RHA and DRA of the rat liver and obtain
ed a ratio of 2.6si renewal in the non-growing liver. The
regenerating liver RRA-R^sBNA-N-^ was i.^.x with an 11
fold increase in the BRA-H^ incorporation (38). LePage
and Heidelberger found approximately similar glycine-2-C-^
activity in the adenine and guanine samples of ERA and DRA
from the rat liver. The ratio of the turnover rate of
ERA:DRA was 1.7*1 for the non-growing liver (73)*
Brown, Peterman, and Furst obtained a ratio.of 29:1
for the?* dietary adenine-N-^ incorporation into ERA and DRA
purines of the total rat viscera (7*+)« The adenine-R-1 -^
distribution in the non-growing liver was 73*1 for ERAsDRA
purines, indicating a decided stability of the DRA. The
rate of incorporation was higher for the regenerating
liver (7^-a). The turnover ratio of ERAsDRA cytosine was
about 2:1 following the administration of labeled cytidine
as a precursor of the nucleic acids (^7)* The simultaneous
administration of N15-glycine and 8-C1**-adenine to the rat,
a procedure which eliminated variable experimental con
ditions, resulted in duplication of the activity ratios
obtained by earlier investigators. It was concluded that,
even in non-growing tissues, there is a continuous forma
tion of DRA as well as ERA purines from known precursor
17
substances though a negligible formation of DNA purines
from dietary adenine was obtained (7*tb).
The rate of nucleic acid synthesis of parasite
tissues of all kinds whether of rickettsiae, viruses,
protozoa, or neoplasms are apparently more rapid than host
tissue (75} 10?). Perhaps this differential metabolic rate
may be utilized advantageously for chemotherapy (10?).
Biological Antagonism
The concept of biological antagonism produced by
structurally-related metabolite analogues was first hy
pothesized by Woods (76) following his investigation of the
mode of sulfanilamide inhibition on the growth of Strepto
coccus haemolvticus. He discovered that p-aminobenzoie
acid (PAB) counteracted this inhibition and undoubtedly was
a metabolite for the bacteria. Because of the structural
similarity of p-aminobenzoic acid and sulfanilamide in
which the C00H group of the former is replaced by the
sulfonamide group on the latter, Woods postulated that the
bacteriostatic action of sulfanilamide was a competitive
inhibition of the metabolite by a structurally related
analogue (76).
Though Woods is credited with the postulation of this
type of biological antagonism, there were other contributing
ideas anri observations which were used by Woods to formulate!
18
his concept. Some of the reported works are given as fol
lows: (a) the idea of Quastel and Wooldridge that there is
an active enzyme center which is specific for enzyme action
(77), (h) the malonate inhibition of succinic acid oxidation
(78), (c) Glares hypothesis for the mechanism of drug ac
tion as a formation of a reversible complex with the cell
receptors (79)? (d) studies by Woolley with pyridine deriva
tives as substitutes for nicotinic acid (80), (e) other
earlier works on sulfanilamide inhibition (81, 82),,and (f)
Fildes' proposal that there may be an interference with an
‘ ‘essential metabolite”. He defined “essential metabolite”
to be an organic substance without which metabolism cannot
proceed to the extent required by growth (83). Subsequent
ly, biological antagonism as produced by structurally re
lated analogues has been demonstrated repeatedly for
vitamins, hormones, amino acids, and nucleic acids (8V, 85?
86, 87).
Biological antagonism involving the synthesis or
utilization of the metabolite may be caused by one of the
following mechanism: (a) the formation of an inactive salt
or complex with the enzyme, (b) a chemical reaction forming
an inactive product as with SH groups (88, 89), (e) the
oxidation of a metabolite which is active in the reduced
form or the reduction of a metabolite required in the
19
oxidized form, (d) or the competitive inhibition between the
structural analogue and the metabolite for the sameenzyme
(89, 90).
It is proposed that the essential metabolites which
have catalytic action may have a “combining group” and a
“functional group”. The alteration of the “combining group”
which associates with the enzyme would result in an inactive
or less active compound. Alteration of the “functional
structure”, which is the active catalytic center, may yield
antagonists (86).
Most of the active antagonists have been found to be
the competitive type (91)• The effectiveness of the antag
onism depends on the ratio of the inhibitor and the meta
bolite concentration. The mechanism (92) of action by which
the inhibitor (analogue), (I), competes with the substrate
(metabolite), (S), for the enzyme is illustrated. P repre
sents the product of the reaction, and ES and El represent
the enzyme-substrate and enzyme-inhibitor complex, res
pectively.
E + S--- ^ ES ---1 + P
E + I --- =» El
20
By mass action,
(E)(S) , , KsCES)
(1) Kg - (is) or ® = (f)
x . (E)(1)
(2) Kj _ (El) OI> ”*
Ks is the dissociation constant of the enzyme-substrate
complex, and Kj is the dissociation constant of the enzyme-
inhibitor complex. By equating (1) and (2), the following
relationship is obtained:
KS(ES) _ Ki(EI)
(3)
(**■)
(S) (I)
(I) _ KjCEI)
(S) “ Kg(ES)
The simplest method of applying these equations for a given
biological system is to determine the relationship between
the inhibitor and substrate on the growth of microorganisms
under limited conditions of defined basal medium, size of
inoculum, time, temperature of incubation, etc. Under these
conditions, the rate, r, of growth of the organism will be
directly proportional to (ES), thus
(5) r = k(ES)
In the presence of a metabolite antagonist competing with
the substrate for the enzyme the total enzyme concentration,
(E t)» both fre e and combined may be d efin e d as:
(6) (Et) = (E ) + (El) + (ES)
If the (Et) of the cell is assumed to be constant through
out the experiment, and sufficiently high (I) and (S) con
centration approaching enzyme saturation is used so that
the (E) approaches zero, the rate determining factors
become the effective (ES) concentration of the cell, (CEg),
and the proportionate amount of (El) formed, (Cgj). Sub
stitution of (CEg) and (CEj) info equation (V) results in:
(7) (I) K-^Cgj)
(S) “ KS(GES)
where I/S is the molar ratio of analogue to metabolite in
the biological system neeessary for inhibition and is
directly related to the growth response of the organism.
The effect of the substrate (metabolite) on the synthesis
and stability of the enzyme is predetermined by growth
determination without.the inhibitor.
The inhibition index (89, 92) has been defined as
the molar ratio of the inhibitor and the substrate which
produces half-maximum microbial growth. The smaller
indices indicate a more active inhibitor. In order to
confirm a competitive antagonism it is necessary to
22
produce constant effects with constant ratios of inhibitor
to metabolite, and the inhibitions must be completely re
versed or suppressed by an excess of metabolite or inhi
bitor, respectively.
Some generalization of the structural modifications
of the metabolite to produce inhibitory compounds have been
formulated by Woolley (8*4-, 85). They are as follows:
I. Replacement of the COOH group with
some other more or less acidic grouping.
Example: The sulfonamide group of
sulfanilamide, the sulfonic, acid group
of pyridine-3-sulfonic acid, the R-CO-
group of p-amino acetophenone.
II. Exchange of one or more atoms in a
ring system of a cyclic metabolite for some other
atom.
Example: -C=C- for S of pyrithiamine,
Hfor C of benzimidazole, H for C of
8-azaguanine, 0 for € of dicoumarol.
III. Replacement of a substituent on an
aromatic nucleus.
Example: Halogen for an alkyl side-
chain of 2,3-diehloronaphthoquinone
and 6,7-dichloro-9-ribitylisoalloxa-
23
zine, WH2 group for OH of aminopterin.
IV. Miscellaneous.
Example: Addition of extra C as in
glucoaseorbic acid and galactoflavin,
decrease in C as in pteroylaspartic
acid, elimination of an atom in a ring
to produce open-chained compound as
desthiohiotin.
There is apparently no unique manner in which the structure
of the metabolite must be altered. However, the antagonist
usually must be closely related to the metabolite for the
active group to fit the enzyme yet sufficiently unrelated
to be devoid of essential metabolic activity (90). These
modifications of known metabolite have been /suggested as a
rational approach toward possible chemotherapeutic agents
(85, 90, 93, 9*0.
Practical Considerations
The effect of I/S as previously described (page 21)
can be demonstrated and studied by the use of a living
system. In a microbiological system the effects may be de
creased rate or complete inhibition of growth. The inhibi
tion analysis method of Shive (92) can be applied. It is
based upon the assumption that an antagonist blocks a
2b
specific reaction which is essential for growth. If the
product of the inhibited reaction is added, the inhibition
should he relieved. In some cases, the analogue inhibition
of an essential metabolite ean be reversed by several sub
stances. For example, if the inhibitor, (I), inhibits
metabolite, (S), which produces several products, P-j_, P2,
by finding the products which will reverse the toxicity of
(I), it is possible to demonstrate the metabolic relation
ship of the metabolite (S) to its products (92, 95) •
With animal work, the criteria of antagonism may be
measured by the growth rate, survival time, or time re
quired to develop deficiency symptoms (91? 95) • hue to the
complexity of the interrelationship of the metabolites, it
is difficult to establish competitive antagonism in animals.
The antagconism may or may not be reversed, nevertheless,
the manifestation of deficiency symptoms indicates an
interference with one or more metabolic process (91). This
effect may be demonstrated schematically:
2?
E* ---:&*-
(c)
y
— A + B ----> C + D ^ E x
(a) (b)
Beaction A to E represents a sequence in a biochemical
synthesis of a complex metabolite E (for example, uridine).
The incorporation of A to form C is competitively inhibited
by an antagonist at (a). There seems to be a number of
ways to overcome this inhibition. Due to a deficit in C,
the enzyme (e) may be stimulated to synthesize more A.
B and D, to some extent, might tend to overcome the de
ficiency in C and lessen the inhibition at (a). Addition
of C will remove the inhibition. There is a possibility of
another metabolite, E1, capable of replacing E; the
presence of E or E1 will nullify the G requirement alto
gether (89). The effects of these secondary reversing
agents may range from an increased inhibition index to
complete removal of the inhibition (95). In order to pre
vent the formation of E in the presence of E1, a combina
tion of two antagonists to inhibit at (b) and (c) would
be required.
In many cases the ability of a structural analogue
to inhibit has been related to the specific requirement of
the metabolite by the organism (85). For example, thio-
26
panic acid inhibits the growth of some bacterial species
which require pantothenic acid as growth supplement and is
not toxic to several species which synthesize their own
(96); the same is true of pyrithiamine, a thiamine analogue
(97). In exception is benzimidazole which is inhibitory to
growth irrespective of requirement (98). Sulfanilamide also
inhibits certain microbial growth irregardless of the PAB
requirement; most of the inhibitions are reversed by PAB,
some are not (99)•
Actually, in a living system, a very complex and close
relationship exists among the metabolites. By inhibiting
one reaction, the living organism will exhaust all possi
bilities in overcoming this inhibition (as above). For
investigative purpose, it is usually advisable to choose an
organism with a specific metabolite requirement which
structurally resembles the potential anti-metabolite in
order to demonstrate direct competitive antagonism and,
preferably, a strain of microorganism which can grow on a
completely synthetic media (100).
Heurosnora is a microorganism which is adaptable for
inhibition studies. The mold can be grown under aseptic
conditions on a simple medium consisting of inorganic salts
as a souree of nitrogen, glucose or sucrose as the carbon
source, and biotin as the only specific growth factor (100).
27
This ascomyeete Neurospora (100) is a form of a bread mold
which thrives in the tropics* Near the turn of the last
century Professor Went, a Dutch botanist, returned from Java
with this mold and domesticated it for laboratory use. In
1927 it was introduced as genus Neurospora for use in
genetic studies. After its life cycle was clarified, mutant
strains were developed, and subsequently adopted as a bio
chemical tool (101). The heterothallic species, Neurospora
crassa and Neurospora sitophila. are used.
The mold growth is determined by the amount of
myeelia produced by the rapid elongation and branching of
the individual hyphae. The extent of mycelial growth may be
determined in several ways, namely, by the dry weight method
the tube growth method, and the conidial growth method (102)i
The dry weight method, which is simple, precise and relia
ble, is the one most commonly used. The mold is grown in
liquid medium allowing addenda to be supplied in similar
form. The myeelia can be handled easily prior to weighing,
and the growth response of the mold is unaffected by com
paratively wide range of pH and temperature variatipns, size
of inoculum, light, increase of carbon and nitrogen sources,
quantity of medium, concentration of the metabolite-require-
ment, etc. The tube method requires the measurement at time
intervals of the progressing mycelial frontier on an agar
28
slant. In the eonidial growth method, a microscope is used
to determine the germinating time of the conidia.
After several days growth, a chain of multinueleated
asexual spores (conidia) are cut off at the tips of the
aerial hyphae. The nuclei are haploid, each containing
seven chromosomes. The conidia are propagated by placing
together the spores or hyphae of two sexes on a suitable
medium and allowing fusion to occur with the formation of
the fruiting bodies. Within these bodies are spore sacs
(asci) where fusion of the nuclei occurs resulting in
diploid zygote nucleus which undergoes two meiotic and
finally a mitotic division along the ascus axis resulting
in a row of eight nuclei each with seven chromosomes. Fur-
thur mitotic division leaves a dinucleate ascospore at
maturity (65).
On the assumption that specific genes control all
enzymatically catalyzed reactions (103), it has been possi
ble to produce gene mutation at random and select mutant
strains of Neurospora•in which specific biochemical reac
tions are blocked (65). These mutants are produced by
X-ray, gamma ray, neutron, nitrogen mustard, or ultraviolet
radiation of the conidia. The treated conidia are.crossed
to produce ascospores, and a single ascospore is cultured.
Since mutation is produced prior to meiosis, the descendant
29
ascospores are genetically homogeneous. Mutants ean he
detected and characterized hy schematic series of tests run
on minimal media. Genetic analysis is made hy further
cultivation of the mutant spore (65).
Strains with separate and specific requirement for
each of the accepted B vitamins with the exception of folic
acid, for each of the amino acids, and for the purines and
pyrimidines have been developed (65, 100).
Organisms for Screening Nucleic Acid Antagonists
With increased interest in the development of nucleic
acid inhibitors, screening agents for the potential antago
nists are desirable. Preliminary tests are carried out on
the growth response of microorganisms with growth require
ments which have been thoroughly investigated.
Lactobacillus casei, which can grow on a medium con
taining thymine plus adenine or folic acid plus adenine
(10*+), is used for testing the activity of purines,.pyrimi
dines, and folic acid analogues as possible nucleic acid
antagonists (105)-
thymine 4 adenine
----------------------- ^ nucleic acid
or folic acid 4 adenine
Streptococcus faecalis. with a more specific require
ment for folic acid in place of thymine plus adenine or
30
guanine (106), is used for the inhibition analysis of folic
acid analogues (107).
folic acid
— ^ nucleic acid
or thymine + adenine
guanine
A strain of Tetrahvmena geleii. an animal micro
organism, which requires guanine for growth has been used
for screening purine derivatives (108). Another strain of
Tetrahvmena geleii. which requires uracil or cytidylic acid
only as supplement, has been used to study the effects of
uracil analogues (109).
There is a uracil-requiring Neurosnora mutant No.
1298 which has been found to utilize uridine or cytidine as
growth factors (50). Scheme of biosynthesis is presented
as follows (51)i nucleic acid
I V
block block
^ x ^ uridine
h .
orotic
dcid uracil
^ ^ ^ dine
* : ^ 7 uridylic acid cytidylic
acid
This mutant has been used to test the activity of several
synthetic nucleosides (110).
Compounds which proved to inhibit nucleic acid
biosynthesis of microorganisms are further screened using
more complex biological systems such as the laboratory mouse
(105, 1 W , rat (123, 156, 161), and chick (156, 160, 161).
Since it is logical that nucleic acid inhibitors may be
valuable agents for the treatment of cancer and leukemia,
such compounds usually have been tested for this possibili
ty. In order to investigate the effects of depressed
nucleic acid synthesis, mice which are genetically suscepti
ble to various types of cancer closely related to the human
types have been developed by selection and high-inbreeding
(111). C57 black mice bearing acute myelogenous leukemia
GlH-98 and mice with Ik1 * strain of transplanted acute
lymphoid leukemia have been used to screen a number of
possible inhibitors by determining any increase in the sur
vival time of the treated animal over the controls (139,
1^0, Ibl, l¥f, 158, 162).
Antimetabolites of Nucleic Acid Biosynthesis
Nutritional studies using microorganisms have helped
in the discovery and isolation of new vitamins such as
biotin, pantothenic acid, pyridoxal, pyridoxamine, and gave
evidence for the metabloic importance of previously known
vitamins, e.g. inositol and PAB (112). Similarily, though
deficiency symptoms of folic acid and vitamin Bj_2 have been
observed in animals, it was the independent discovery of a
.growth factor in liver extract which was capable of re_____
32
placing thymine and purines as the growth requirement of
both Lactobacillus casei (lO^f) and Streptococcus faecalis
(106) and stimulated the growth of Lactobacillus lactis
Dorner (113) Leuconostoc citrovorum (ll1! - ) which led the
way to the isolation and identification of folic acid (115)»
the isolation of crystalline vitamin B12 (116), and.the
synthesis and isolation of folinic acid or citrovorum factor
(CF) (117, 118), respectively.
Thymidine was found to replace the vitamin B12 re
quirement for the growth of Lactobacillus lactis Dorner
(119), and the vitamin Bj2 involvement in the biosynthesis
of nucleic acid purines and thymidine (120) and the metabo
lism of choline (121) was demonstrated.
A substance which was more active than folic acid
was found in hog liver concentrates and named folinic acid
(122). The ability of liver slices to convert folic acid
into folinic acid in the presence of ascorbic acid and the
higher activity of folinic acid in overcoming the aminop-
terin toxicity in rats (123) suggested that the CF is
either the eatalytieally active form of folic acid or is
more closely related to this form than folic acid.
Intracellularly, vitamin B^2 activity is concentrated
in the mitochondria while the folinic acid activity is dis
tributed throughout the cell suggesting that the role of
33
the former vitamin may be more specific whereas the folic
acid functions in diverse enzyme systems (121 +).
A great deal of evidence is now available to indi
cate that folic acid (or CF) is required, either directly
or indirectly, for the metabolism of serine (12?), thymine
(or thymidine) (32), the purines (126, 12?) and hence the
nucleic acids (112). Pteroylglutamic acid (PGA) was shown
to influence the biosynthesis of nucleic acids in Lacto-
bacillus easei, and a marked decrease in DNA synthesis of
the PGA deficient bacteria was observed although its BUA
remained unaffected (128). This effect was contrasted to
the stimulated RNA and DNA synthesis in the biotin and
riboflavin deficient bacteria (128).
A possible relationship of folic acid to cancer was
suspected as a result of finding a high folic acid content
in tumor tissues. The striking difference between the
behavior of folic acid from the liver of normal and cancer-
bearing rats further supported this conjecture (129).
H
✓N\ /W\,
H2N-er Ncr
V' 2 6 ^ j L
6e h6=o ch:
Folinic Acid
Results of initial studies with folic acid on some sponta
neous tumor was promising; however, continued investigations
of this effect proved its inefficacy for the anemia associ
ated with leukemia (130), spontaneous hreast cancer in mice
(131), and leukemia (129) which was aggravated by excess
folic acid in mice (132). Thymine had no curative or pre
ventive effect on the folic acid deficient rat (133)*
However, the nutritional anemia in man responded favorably
to thymine and folic aeid, but vitamin B-j _ 2 was by far the
most potent anti-anemic substance (13H-).
The major impetus in the development of antimetabo
lites of the purine and pyrimidine series resulted from the
experimental evidence of the folic acid involvement in the
nucleic acid biosynthesis of Lactobacillus easel and Strep
tococcus faecalis.
Pyrimidine derivatives. Among the first compounds
studied were the pyrimidine analogues of thymine. Their
effect on the growth of Lactobacillus casei and Strepto
coccus faecalis were studied. Of these compounds, 5-bromo-
uracil and 5-chlorouraeil were found to be good antimetabo
lites of thymine. 5-^itrouracil inhibited the growth stimu
lation produced by folic acid, and J-^i^ouracil and 5-
hydroxyuracil inhibited the growth stimulated by thymine or
PGA (135)* Reversal studies (136) of these inhibitions
35
showed that bromo- and chloronracil were interfering direct
ly with thymine -utilization, and 5-nitrouracil was a com
petitive inhibitor of folic acid. The antagonism by 5-amino
/uracil was reversible by folie acid in a manner which was
not competitive, whereas thymine competitively reversed this
inhibition. The inhibition of growth produced by 5rhydroxy-
uracil in thymine or folic acid-containing medium was re
versed by uracil.
The growth response of Lactobacillus casei with PGA
as the requirement in the absence of purines was inhibited
by nearly all of the 2,k— diaminopyrimidines, most of the
2-aminopyrimidine derivatives; several 2-amino-lt —hydroxy-
pyrimidines; 2, *+-diamino-6,7-dimethylpteridines; and 2,6-
diaminopurine. Studies on the reversal of these inhibitions
showed that several enzyme systems were involved (137).
Studies of several nucleic acid antimetabolites on
the growth of Lactobacillus casei grown in varied media of
folic acid with adenine or thymine gave the following
results. 2,^Diamino-5-chlorophenoxypyrimidine (87, 105)
was antagonistic to the folic acid growth effects of Lacto
bacillus casei and also possessed anti-malarial activity ap
proximating quinine against Plasmodium gallinaceum; this
activity was partially reversed by folic acid in vivo. It
inhibited the growth of Tetrahvmena geleii in the presence
36
of excess folic acid, and the inhibition was reversed by
uracil. Ho evidence of antifolic acid activity was obtain
ed on administration of the compound to the mouse or chick.
2,U--Diamino-5,6-dimethylpyrimidine had antifolic acid effect
on the growth of Lactobacillus casei. but it was inactive
for complex systems. Since the biological activity of 2,1 +-
diamino-5-formamido-6-hydroxypyrimidine was similar to .that
of adenine, it was identified as a purine precursor (105).
1-Methyluracil, 3-methyluracil, 5-bromouracil, and 5-
nitrouracil were found to be the most active inhibitors of
the growth of the uracil-requiring Tetrahvmena geleii (109).
The effects of various substituents on the uracil molecule
are summarized as follows:
No. 1: reduced the activity
0
A
ffl? sCH
' 1 I I
0=C* , «CH
W
w
2: S or NH2 was inhibitory
1 1
reduced the activity
w
hi 0 destroyed, GH^ reduced activity
H
t i
5:
N02 or Br reduced, CH3 destroyed activity
TJracil
t !
6: destroyed activity
The site of substitution which destroys the activity of the
metabolite may be the point of attachment of the enzyme as
observed by Fildes (138) with indole and tryptophane inhibi
tors.
•37
Despite the fact that simple pyrimidines antagonized
the bacterial growth in the presence of PGA, all were in
effective in prolonging the life of the leukemic mouse (139?
1*40, 1*4-1).
Purine derivatives. Benzimidazole, an antagonist of
adenine and guanine, was inhibitory to the growth of sever
al yeasts and bacteria. The anesthesia produced in the mice
following the injection of the compound was not prevented
by the simultaneous administration of adenine (98)* Benzi
midazole had no anti-leukemic activity for the mice (1*4-0).
guanine (1*4-2) and a PGA antagonist of Lactobacillus casei
(137)? is toxic to rats, mice, and dogs (1*4-3) * Beversal
studies (l*+5) of the 2,6-diaminopurine inhibition of the
growth of Lactobacillus casei showed the existence of a
competitive inhibition with folic acid at low concentra
tions but a non-competitive type of inhibition at higher
levels of the inhibitor. The antagonism produced in the
H
l
1 I >
Benzimidazole 2,6-Diaminopurine
2,6-Biaminopurine, a precursor of polynucleotide
38
medium containing folic acid pins adenine was relieved by
the addition of adenine. The ribonneleoside and ribonucleo
tide growth effects were abolished by 2,6-diaminopurine
whereas purine alone stimulated, suggesting that the utili
zation of purine ribonucleoside or nucleotides was inhibi
ted. In increase in the survival time of mice with Akk-
strain of leukemia was observed (lMf).
Some 52 substituted purines (l*f6) were tested as
possible replacements of the obligate purine requirement for
Lactobacillus casei grown with thymine in the medium. It
was found that adenine, hypoxanthine, guanine, and xanthine
were equally effective growth promoters. It appeared that
ribonucleosides and ribonucleotides were degraded to purines
before utilization. Various C-methyl and N-methyl purines,
except 1-methyl derivative, were inactive. ^-Aminor5-form-
amidopyrimidine, an intermediate in the Traube's synthesis
of natural purines, was also inactive.
8-Aryl substituted guanine derivatives inhibited the
growth of Lactobacillus casei. These inhibitions were not
relieved by adenine or folic acid. 2,6-Alkylaminopurines
had a lower activity than diaminopurines, but 8-alky1-2,6-
diaminopurine was a more active inhibitor than the unalkyl
ated form (I1*?) •
5’ -Amino-7-hydroxy-lH-v-triazolo(d)pyrimidine (also
39
8-azaguahine or guanazolo), was found to be a powerful com
petitive antimetabolite of guanine for a strain of Tetra
hvmena geleii (108). It also strongly inhibited the growth
coli and Staph. aureus in the presence of purines
(1^8). 0H
i
IT C---
i n
8-Azaguanine
This compound showed an inhibitory effect on two transplant
able leukemias in mice (1^-9), but it did not affect the
growth of influenza virus or Lansing strain of poliomyelitis
virus and was toxic to the chick embryo (150). The,radio
activity of S-azaguanine-a-C-*-1 * was excreted in the urine
2h hours after administration to mice, and no evidence for
the preferential accumulation, fixation, or incorporation
of the labeled inhibitor in the tumor tissue was found
except that there was a generalized distribution in all
tissues (151)- The radioactivity of guanazolo-2-C-1 - lf was
i concentrated in the nucleic acid fraction of the viscera and
tumor tissues probably as an enzyme-inhibitor complex since
no incorporation into adenine or guanine was found (lj?la).
Pteridine derivatives. Following the observation
_that_sulfanilamide.-interfer.ed_in_the_f.ormation_of_folic__acid|
ko
from PAB (152), Daniel et al (153) investigated the effects
of pterins on the growth of several folic acid-requiring and
non-requiring bacteria and found 2,*f-diamino-6,7-diphenyl-
pteridine consistently inhibitory to growth. As the 2,*+-
diamino group appeared essential for activity, it was con
cluded that the 2-NH2 group was the site of attachment of
the apoenzyme since it is common to pterins and folic acid
pteridine. The 3 +-MH2 group of the pterin may be responsible
for the inhibitory effect.
The antagonistic effects of the 2,lf-diamino substi
tuted pteridines were confirmed by Elion and Hitchings (15*0
who found the effects paralleled the activites of 2,^-di-
aminopyrimidines. The existence of some enzymatic system
for the combination of 2-amino-1! —hydroxypteridine deriva
tives and p-aminobenzoylglutamic acid to form new inhibitors
of folic acid was noted. Although many pteridine deriva
tives inhibited the growth of microorganisms, all of these
compounds were chemotherapeutically inactive on transplant
ed mouse leukemia (139)•
Pterovlglutamic acid derivatives. The amino -
pteroylglutamic acid (M—HHg-PGA or aminopterin), a folic
acid analogue, was found to strongly inhibit the growth of
Streptococcus faecalis (155) and extremely toxic to rats and
chicks in an uncompetitive way (156). Snell and Cravens
bl
found that thymine, h$rp oxan thine, folic acid, vitamin B^,
or concentrated CF, alone or in various combinations, were •
ineffective in counteracting the 1 f-lH2~PGA inhibition is
chick embryo (157)* but Nichol and Welch were able to
prevent the lethal effect of aminopterin in rats by the
daily administration of folinic acid (123). effects on
the survival time of leukemic mice indicate aminopterin to
be a promising metabolite antagonist (158).
Derivatives of N-(p-aminobenzoyl)-glutamic acid moiety of
^-aminopteroylglutamic acid were devoid of anti-leukemic
activity in mice (159)*
Pteroylaspartie acid, another PGA analogue, also in
hibited the PGA growth stimulation of Streptococcus faeca
lis. This compound inhibited the growth of chieks but was
nontoxic to the rat (160).
The inhibition of growth produced by M-aminopteroyl-
H2N-cf^NC/8^CH
i I I i
00H !
. —_____j
Pteridine N-(b-methylaminobenzoyl)-glutamic
Pteroylglutamie acid ‘
b2
aspartic acid which interferes with the utilization of PGA
in Streptococcus faecalis was completely reversed with PGA,
thymine, or thymidine. It was 50 times more active than
pteroylaspartic acid in the chick hnt was much inferior
to ! +-NH2“PGA as an inhibitor in the rat (161). It xms
concluded that the *f-HH2 group of the pteridine was not
the sole determining factor for these inhibitions, b-Amino-
pteroylaspartic acid and aminopteroylthreonine showed
moderate anti-leukemic activity in mice with AKH- leukemia
and its use in the treatment of acute leukemia of children
and adults has been reported (I63),
Woolley and Pringle have isolated *f-amino-5-carbox-
amidoimidazole from the medium of E. coli grown in the
presence of aminopterin (16*+) and amethopterin (lb^a).
V-lmino-5-carboxamidoimidazole
The same substance was obtained by Stetten and Fox (165)
from the culture medium of E. coli grown under limited
( 162) .
Amethopterin, amino-H^-methyl-pteroylglutamie
acid, has a protective effect on the leukemic mouse (lM*)
H2N-C=Q
1 + 3
purine synthesis with sulfonamides and identified hy Shive,
Set al as ^^-amino-M^-carboxamidoiisidazole (166).
Glycine and threonine stimulated this formation (I67).
Other types of nucleic acid antagonists such as 8-azaguanine,
nitrogen-mustard, 2,6-diaminopurine, and urethane did not
result in the formation of the diazotizable amine (l6*fa).
Since insignificant incorporation of labeled M5)-amino-
5(}+)-carboxamidoimidazole was found in the polynucleotide
purines of the rat, it was concluded that this compound is
not formed as a normal intermediate for this animal (168).
It seems that the role of folic acid or folinic acid
is involved in the completion of the purine skeleton. Per
haps aminopterin or amethopterin compete for this center to
prevent the formation of a folic acid complex to allow for
purine synthesis or directly inhibit the synthesis of PGA
(I69). The resultant effect has been manifest in the
ability to inhibit the production of leukemic white blood
cells (170, 171).
STATEMENT OP THE PROBLEM AND PLAN OF ATTACK
Though many substituted purines, pyrimidines, pteri-
dines, and folic acid analogues have been investigated for
antagonistic effect on the nucleic acid synthesis, com
paratively few substituted nucleosides have been studied.
The synthetic nucleosides of uracil containing un
natural sugars (172) as possible uridine antagonists have
been synthesized following the observation of the antago
nistic effect produced by the replacement of the ribityl
group of riboflavin by a duleityl group. Nucleosides of
ehlorouracil and bromouracil (172) containing pyranose
sugars and thymine nucleosides also containing pyranose
sugars (173) were synthesized and their activity tested on
various microorganisms.
The results of microbiological studies (110) showed
that 5-ehlorouracil was slightly stimulatory to growth of
a uracil-requiring ]2. coli mutant. The other nucleosides
were neither inhibitory nor stimulatory to growth of this
bacteria. With Neurospora crassa mutant No. 1298, the
effects were similar to those obtained with B. coli. Some
of the thymine nucleosides were able to replace the thymine
requirement of Lactobacillus casei and Streptococcus faeca-
lis, but others were inactive without the thymine supple-
|~ ”” “ *?
ment. The results of these studies indicated that the
nucleosides containing pyranose sugars were not biological
ly active compounds.
Other nucleoside derivatives reported were the
methylated purine nucleosides, which were synthesized and
studied for diuretic and blood pressure effects on some
animals (17*0 •
The purpose of this investigation was to find a spe
cific antagonist for the utilization of pyrimidine nucleo
sides since experimental evidence showed that nucleosides
containing a furanose sugar were polynucleotide precursors
(page 9)(110). Such a compound would aid in the study of
nucleic acid synthesis and might have some therapeutic
application.
Since uridine in which the sugar moiety retains its
natural furanose configuration was commercially available,
it was suggested that compounds in which the hydrogen in
position 3 or 5 of the uracil nucleus is replaced by other
groups might be interesting antimetabolites of pyrimidine
nucleosides. Furthermore, methods for the synthesis of
this type of compounds have been reported, for example 5-
bromouridine (175)) pyranosyl-5-ehlorouridine (172),
pyranosyl-5-bromouridine (172), and N-methyluridine (12).
The preparation of these compounds showed that halogenations
can be effected directly and positions Bo. 3 and 5 of the
pyrimidine nucleus were reactive.
Accordingly, a natural uridine derivative in which
chlorine was substituted for the hydrogen on carbon Ho. 5
of the uracil moiety and 5-kromouridine (175) were synthe
sized and tested for growth with the uridine-requiring
Neurosnora mutant Mo. 1298 (63) using the Byan and Brand
modification (1?6) of the basal medium of Horowitz and
Beadle (177)•
The activities of the antagonist on the growth of
Streptococcus faeealis. which requires thymine or folic acid
for growth, and Mycobacterium tuberculosis. whieh was found
to contain 5-methylcytosine (1), were also studied.. Bio
logical activity studies on leukemic mice were suggested
if the preliminary results warranted further investigation.
1
EXPERIMENTAL METHODS
Synthesis of 5-Chlorouridine
Five grams of powdered uridine, sup. 166-166.5° C.,
were partially dissolved in 300 ml. of anhydrous acetic acid
at room temperature, and a 10 per cent excess of dry chlor
ine dissolved in cold anhydrous carbon tetrachloride (172)
was added at room temperature. The suspension cleared in
three or four hours and was allowed to stand overnight at
room temperature. On the following day, the solvent was
removed by lengthy vacuum distillation by means of a pump
at temperature not exceeding 35° C. Evidently, the nucleo
side is hydrolyzed by the HC1 present if excess heat is
applied to encourage distillation. Adopting the method of
lyophilization for removing the acetic acid greatly simpli
fied and facilitated this process.
The white residue obtained after the removal,of
i
acetic acid was readily soluble in alcohol and was crys
tallized from hot water, forming fine, white needles, m.p.
170-171.2° C. The analytical data indicated that the eom-
ipouhd might be a diaeetate of 5-chlorouridine.
C13H15°8N2C1 Calculated N 7-72, Cl 9.78,
(362.73) Found N 7.92, Cl 10.12. I
* + ■ 8
The ester was hydrolyzed with 0.5 gm. dry HG1 gas in
100 cc. of absolute methanol at room temperature overnight,
and the alcohol was removed under reduced pressure. The
white, semi-crystalline residue was readily dissolved in
water, and the acidic solution was passed through a column
of Amberlite IBAAOO anion-exchange resin (178). The water
solution, pH 6-7, was lyophilized, and the remaining white
residue of 5-cblorouridine was crystallized from absolute
ethanol in white needles, m.p. 217*0-217■•5° C. The yield
was 3.7 gm. (6*fA per cent).
GoHiiG.MpCl Calcu- C 38.71, H 3.98, N 10.06, Cl 12.73,
0 lated
(278.66) Found C 38.91, H 3*98, H 10.03, Cl 12.63.
(Note: Uridine was purchased from Nutritional Biochemical
Laboratories, Cleveland, Ohio. Amberlite IBA-Voo was ob
tained from Bohm and Haas Company, Philadelphia, Pennsyl
vania. It was regenerated with per cent NaOH.)
Synthesis of 5-Bromouridine
This compound was prepared following the procedure
of Levene and LaForge (175)* 2wo grams of uridine were
treated with bromine water until the red color of bromine
no longer disappeared immediately. The solution was evapo
rated to a syrup under reduced pressure. The residue was
taken up in absolute alcohol and evaporated on the water-__
1 + 9
"bath with stirring until the compound crystallized. The
precipitate was rubbed with absolute alcohol and then re
crystallized from 5 parts of 95 per cent alcohol. Fine,
white crystals were obtained, m.p. 179*9-180.5° C. The
yield was 1.25 gm* 0*7•5 P©r cent).
C9HllN2°6Br Calculated N 8.6?,
(323.12) Found H 8.55,*
Preparation of 5-Bromouracil
Bromouracil was prepared according to the procedure
of Wheeler and Johnson (179)* Two gm. of uracil were sus
pended in 8 ec. of water and 6 gm. of bromine were added.
The uracil dissolved completely on warming, and upon cool
ing, a crystalline mass of dibromo-oxyhydrouraci1 sepa
rated. On prolonged boiling in aqueous solution, the di-
bromoxyhydrouracil decomposed to the 5-bromouracil. 5-
Bromouraeil does not form a purple-colored precipitate with
saturated barium hydroxide (180). A characteristic blue-
green flame test for halogen was obtained with the copper
wire. 5-Bromouraeil was crystallized from hot alcohol.
The crystals sublime without melting. The yield was 1.5 gsi*
(*+6 per cent).
C^NgOgBr Calculated N l*f.67,
(190.98) Found N 1^.79*
50
Preparation of 5-Chlorouracil
This compound was prepared following the procedure
of Barrett, Goodman, 'and Dittmer (181) for the synthesis of
5-halogenothiouracil. Ten gm. of uraeil were dissolved in
an excess of glacial acetic acid containing 5 per cent
acetic anhydride to remove all moistnre. A trace of ferric
chloride was added as catalyst. The solution was treated
with a 20 per cent excess of chlorine in carbon tetra
chloride. The solntion became warm, and the temperature
was maintained at 50° to 60° C. until most of the HC1 fumes
were evolved. After cooling, the precipitate of 5-chloro-
uracil formed. More was obtained on concentrating the
filtrate. The crude ehlorouracil was decolorized with
norite and purified by reerystallizations from hot absolute
ethanol. 5-Ohlorouracil sublimes without melting. The
yield was 8.7 gm. (62.7 per cent).
C ^ N 202C1 Calculated N 19.06,
(1^6.52) Pound N 18.95*
Nitrogen Determination
The nitrogen analysis for these compounds was made
by the semi-micro Kjeldahl method, using 10 mg. sample of
the compounds (182).
51
Microbiological Determinations
Neurospora. The pyrimidine-requiring mutant strain
of Neurospora Ho. 1298 and Neurospora Abbot A (wild type)
were used in this investigation. The stock culturesof these
Neurospora were obtained through the courtesy of Dr. H. K.
Mitchell, California Institute of Technology, Pasadena,
California.
The formula for the Ryan and Brand modification (176)
of the basal medium is given in Table I. The medium was
made up in double strength without sucrose and biotin, auto-
claved and stored in a liter flask. The sucrose and biotin
were added before the medium was used. The solution should
be almost colorless and clear. The pH is about 5»5»
The stock cultures of Neurospora No. 1298 were kept
on agar slants of 10 cc. of basal medium containing 2 per
cent Bactoagar, 0.05 per cent casein hydrolysate, and b mg.
of uracil per test tube. Similar tubes were prepared for
the wild type of Neurospora except that uracil was not add
ed. The slants were inoculated with spores from the stock
culture tube, grown for 6-8 days at room temperature, and
stored in the refrigerator.
The spore suspension (inoculum) for inoculation is
prepared by suspending the spores from a stock eulture tube
in about 7 cc. of sterile water and pipetting this suspen-
52
TABLE I
Basal Medium Formula For Heurosnora
(Double Strength)
Ammonium tartrate 1G.G gm.
Ammonium nitrate 2.0 gm.
Potassium acid phosphate 2.0 gm.
Magnesium sulfate, anhydrous 0.b8Q gm.
Sodium chloride 0.2 gm.
Calcium chloride * 2H2O 0.266 gm.
Sucrose 30.0 gm.
Biotin 8 gamma
*Traee element solution 2.0 ml.
Water to make 1 liter
*Trace element solution
Boric acid 57 mg.
Ferric chloride * 6H2O 96 mg.
Ammonium molybdate 6b mg.
Zinc chloride *+20 mg.
Copper sulfate 251 mg.
Manganese sulfate •
158 mg.
Water to make 1 liter
53
sion into 50 ml* of sterile water. One drop of this inocu
lum was added to each reaction flask prior to incubation.
The reaction flasks were 50 ce. Erlenmeyer flasks containing
a total volume of 10 ml. of reaction media consisting of 5
ml. of double strength basal medium plus 5 ml. of growth
supplement, and/or inhibitor and water.
The determination proceeded as follows; Sucrose and
biotin were added to the double strength medium to complete
the basal medium. Five ml. of the basal medium was added to
each 50 ee. reaction flask and diluted to 10 ml. with the
addenda. For each determination the addenda contained
uracil, uridine, eytidine, or uridine plus eytidine as the
growth supplement for the mutant mold. The flasks were
stoppered with non-absorbent cotton and sterilized for 10
minutes at 15 lb. pressure. Upon cooling, each flask re
ceived aseptically a drop of the prepared inoculum and were
incubated at 25° C. for y J2 hours.
A white mat of mycelia was produced following a lag
period of about 36 hours, and this formation continued till
maximum growth is reached at 72 hours after which sporula-
tion begins. The preliminary lag period (102) is believed
to be governed by the time necessary to build up a steady
state of assimilation. The age and condition of the conidia
will influence the period somewhat. Since the mycelial
5b
weight was used as the criteria of growth, the experiment
was terminated at 72 hours. Incubation temperature of 25°
C. decreases the frequency of adaptation of the mutant
(102). The adaptive growth response is not considered in
the statistical analyses (102). The appearance of the myce-
lia may range from a thick mat to mottled patches indicating
unrestricted growth to inhibited growth, respectively. The
contents of each flask were filtered by suction on a small
filter paper (Whatman Ho. 1), washed with distilled water
and then with acetone. The filtered mycelia were removed
and placed in the depressions of a spot plate, dried at
70° C. overnight, and weighed. The activity of the halogen-
ated nucleosides and pyrimidines were determined by the
growth response produced in the presence of one of.the
growth requirements.
Streptococcus faecalis. The effect of ehlorouridine
on the growth of Streptococcus faecalis was studied with
thymine or folic acid as one of the requirement in the
medium according to the procedure of Teply and Elve^'hem
(183). The inoculated test tubes were incubated at 37° C.
for 18 hours, and the extent of bacterial proliferation was
measured with the Klett photometer. (This experiment was
conducted by Dr. Martin Roberts).
Mycobacterium tuberculosis. The bacteriostatic
55
studies of chlorouridine on Mycobacterium tuberculosis
human strain 88 was carried out on Proskauer and Beck syn
thetic medium containing potassium acid phosphate, magnesium
citrate, potassium sulfate, asparagine, and glyeerol. The
method was as follows: 0.^ Cc. of the solution of the com
pound in ten times the final dilution was pipetted into *+.5
ec. of Proskauer and Beck liquid medium (Youman’s modifica
tion) and planted with 0.1 mg. of Mycobacterium tuberculosis
human strain 88. The reaction mixture was incubated at 37°
C. for 6 weeks. (This experiment was conducted by
Miss Hisako Nishihara of the Barlow Sanatorium).
Biological Determination
Since it was found that 5-chlorouridine inhibited
the growth of Neurospora No. 1298, the effect of this anta
gonist on the survival time of the C57 black mice bearing
the myelogenous leukemic tumor 01^98 was investigated. The
C57 black mice bearing the 01* 4-98 tumor and C5>7 stock mice
were obtained from Roscoe B. Jackson Memorial Laboratory,
Bar Harbor, Maine. The initial shipment of three tumor-
bearing mice was used for the preliminary studies. One
mouse was used as a control, one mouse was treated with
chlorouridine, and a third mouse, used as a source of tumor
transplant, was also treated after recovery from the opera
56
tion. Changes in weight and blood picture were noted after
treatment with chlorouridine.
A nine day old tumor was used as the source of
transplant. For this operation a blood transfusion needle
fitted with a plunger was used, and the tumor tissue was
implanted subcutaneously in the axillary region of the C57
stock mice (1111. A small lump of the growing tumor beeame
palpable about 5 days following inoculation, and the mice
succumb to this acute leukemia about the 12th day after the
transplant. Mice transplanted with the tumor in this manner
were used for toxicity studies to determine the maximum
tolerated and the lethal doses (181 *, 185)- A concentrated
aqueous solution of chlorouridine was used for the intra-
peri toneal injections, and an equivalent volume of normal
saline was given to the controls.
For further investigation with ehlorouridine, mice
bearing the 01^98 tumor which was transplanted at Bar
Harbor, Maine were used. The effects of variation in dose
and time of injections on the survival time were studied,
and attempts were made to duplicate the results of the pre
liminary experiment. The mice were maintained on Purina
Chow diet with a weekly supplement of rolled oats.
The leukemic state, which is produced by the ClM^
tumor, is characterized by an abnormal rise in the circula-
51
ting white blood cells, particularly the immature granulo
cytes (186, 187), caused by a flushing of leukemic cells
from the tumor into the systemic circulation with infiltra
tion of these cells to the bone marrow, lymph gland, liver,
spleen, and other organs (111). This state is usually
accompanied by hemoconcentration and a decrease in the
number of red blood cells (130, 188). The effect of chloro
uridine on the blood picture of the leukemic mice was
followed using the simplified blood counting procedure of
Wroblewski, Weiner, and Shapiro (189) who utilized the cali
brated capillary tubes for blood sampling. The peripheral
blood was taken from the tail of the mouse, and the total
red and white cell counts were made from the 10th day while
the animals survived. The normal blood cell count was taksi
on the ?th day after transplant. Blood for the red cell
count was taken up in capillary tubes prepared and calibrat
ed with mercury to eontain an amount of blood equal to the
0,5 mark of the Thoma blood cell pipette. The blood-filled
capillary tube was dropped into a small test tube (75 x 10
mm.) containing the diluting fluid (Hayem!s solution) which
was drawn up to the 101 mark of the standard blood cell
pipette , mixed by shaking, and the resulting cell suspen
sion was transferred by a glass rod or applicator stick to
the counting chamber and counted in the usual manner.
58
An identical method was used for the white cell counts
except that calibrated white cell capillary tubes and di
luting fluid of 2 per cent acetic acid were used. Normally,
the white cell pipette dilutes the blood Is20. The Is21
dilution resulting by this capillary method was corrected by
withdrawing an equivalent excess volume from the delivered
diluting fluid.
EXPERIMENTAL RESULTS
Neurospora
Inhibitions and reversals. Since molar equivalents
of uridine or eytidine give similar growth curves (190),
the maximum growth obtained with 0.6 mg. of uridine or 0.6
mg. of eytidine per flask was chosen for testing the inhi
bitions.
In the presence of uridine the growth of the mold may
be completely inhibited by chlorouridine, giving an inhibi
tion index of 3*05 (Fig. 1; (a) and (b), Table II). This
inhibition is reversible over a considerable range, since
constant ratios of O.87, 1.71 *) 2*61, and 3.*+8 mg. of chloro
uridine to 0.25) 0.50, 0.75) and 1.00 mg. of uridine gave
similar submaximum mycelial growths of 9«*+) 8.3) 7*3) and
8.2 mg., respectively.
The Inhibition index is lower (0.58) if eytidine is
substituted for uridine in the medium (Fig. 1). The inhibi
tion is reversible, since varying concentrations of cyti-
dine require a proportionate amount of chlorouridine to give
the same amount of growth ((e) and (f), Table II). At con
stant ratios of 0.17, 0.31 +, 0.51) and 0.68 mg. of chloro
uridine to 0.25) 0.5) 0.75) and 1.0 mg. of eytidine, similar
inhibited growths of l.1 *, 2.^, 1.6, and 1.8 mg. of mycelia,
30
28
26
24
22
c r 20
( B ) \(C) ( D ) ( A )
50
3.0 4.0 I .0 2.0 5.0 6.0 7.0
MG. OF CHLOROURIDINE OR BROM OUR I Dl NE
Fig. 1. Inhibition of the growth of Neurosnora
No. 1298 by 5-kalogenonucleosides in the presence of uri
dine or eytidine as a growth requirement.
Curve A, 0.6 mg, of eytidine per 10 ml. of medium
with varying amounts of chlorouridine.
Curve B, 0.3 mg. of uridine and 0.3 mg, of eytidine
per 10 ml. of medium with varying amounts of chlorouridine.
Curve C, 0.6 mg. of uridine per 10 ml. of medium
with varying amounts of chlorouridine.
Curve D, 0.6 mg. of uridine per 10 ml. of medium
with varying amounts of bromouridine.
TABLE II
61
Growth Inhibition of Neurospora No. 1298
by 5-Halogenonucleosides^/
Mg. per 10 Mg. per 10p/ Inhibition
Metabolite ml. medium Inhibitor ml. medium*' Index3/
(a) Uridine
i
0.6 Chlorouridine 2.1 3.00
0>)
i t
0.5
i t
l.8o
3.09
(c)
t i
0.6 Bromouridine
3.3
*+.08
Cd)
i t
0.75
t i
*f.O V .00
(e) Gytidine 0.6 Chlorouridine O A 0.58
(f)
t i
1A9
t i
1.0
0.59
(g) Uridine 0.3
*
+ Chlorouridine 1.2 5 1.80
Cytidine 0.3
•i/ Bata from growth inhibition curves obtained by the
addition of varying amounts of inhibitor to a medium con
taining a fixed amount of nucleoside metabolite.
To reduce growth to half maximum.
^ Molar ratio of inhibitor to metabolite to produce
half-maximum growth.
62
respectively, are obtained.
Chlorouridine reversibly inhibits the growth of the
mold in the presence of a mixture of 0.3 mg. of uridine and
0.3 mg. of eytidine, and an inhibition index of 1.80 is ob
tained (Fig. 1; (g), Table II). Bromouridine reversibly in
hibits the growth in the presence of uridine. The inhibi
tion index is H-.08 (Fig. 1; (c) and (d), Table II).
in insignificant growth increase is produced with 2.0
mg. of uracil and varying amounts of chlorouridine (Fig. 2).
In the presence of 3.6 mg. of uracil a growth stimulation is
produced with increasing amounts of chlorouridine, and l*f
mg. of the inhibitor did not reverse this growth effect
(Fig. 2). Another observation is that the maximum growth
response obtained with 0.5 mg. of uracil and 0.3 mg. of uri
dine is completely inhibited with 8.0 mg. of chlorouridine
(Fig. 2). 0.5 Mg. of uracil and 0.3 mg. of uridine produce
imperceptible and half-maximum mold growth, respectively.
The wild strain of Neurospora Abbot A is uninhibited
at concentrations of 6-8 mg. of chlorouridine, bromouridine,
ehlorouracil, or bromouraeil.
Specificity of inhibitions. Some indications con
cerning the site of the inhibition were obtained by a study
of the growth responses of the mold upon the addition of
varying amounts of eytidine to a medium containing both uri-
26
24
22
20
I
H
*
O
oc
©
_ l
<
U1
CJ
>-
2
L l
o
o
2
2.0 4.0 6.0 8.0
MG. OF CHLOROURIDINE
Fig. 2. Effects on the growth of Neurosoora No. 1298
of chlorouridine in the presence of uracil.
Curve A, 2.0 mg. uracil per 10 ml. medium with
varying amounts of chlorouridine.
Curve B, 3*6 mg. of uracil per 10 ml. medium with
varying amounts of chlorouridine.
Curve C, 0.5 mg. of uracil plus 0,3 mg. uridine per
10 ml. medium with varying amounts of chlorouridine.
6b
dine and sufficient chlorouridine to inhibit the growth com
pletely (Fig. 3, Curve A). Similar growth curves were ob
tained by the addition of varying amounts of uridine to a
medium containing eytidine and chlorouridine (Fig. 3? Curve
B). The results are summarized in Table III, and the impli
cations of the results are given in the discussion.
Other Microorganisms
Streptococcus faecalis. Though a great excess of
chlorouridine was used, no significant stimulation or inhi
bition of growth of Streptococcus faecalis was observed with
either thymine or folic acid as the growth supplement.
Mycobacterium tuberculosis. The presence of chloro
uridine, bromouridine, bromouracil, or uridine in 1:10^,
1:10^, Is10^ dilutions did not produce inhibition of growth
of Mycobacterium tuberculosis human strain 88. A partial to
complete inhibition was observed with a lslcA dilution of
ehlorouracil; higher dilutions were ineffective.
Animal Experiment
It was found that C57 black mice were able to toler
ate a single intraperitoneal injection of 50-65 mg. of
chlorouridine. A single injection of 100 mg. was lethal to
the animals. The normal white count ranged between 8,700 to
17,900 per cmm. of blood, and the red cell count ranged from'
26
24
!00
22
*
O
DC
o
<
_i
ui
o
>-
2
(A) (B)
50 2
t i .
o
10
25
(A) 10 6 8 2 4
0.2 0.4 0.6 0.8 1.0 (B)
MG. OF CYTIDINE (A) OR URIDINE (B)
Fig. 3* Inhibition reversals by the metabolites.
Curve A, inhibitory mixture of 0.6 mg. uridine plus
5.0 mg. of chlorouridine per 10 ml. medium with varying
amounts of eytidine.
Curve B, inhibitory mixture of 0.6 mg. eytidine plus
3.0 mg. of chlorouridine per 10 ml. medium with varying
amounts of uridine.
66
9,070,000 to 1^,200,000 per emm* The leukemic white count
prior to death was from 27,100 up to 69,750 per emm. or
higher.
Of the 33 leukemic mice treated with chlorouridine in
varying doses and time of injections, one mouse survived 58
per eent longer than the controls. The white count for this
mouse dropped from 30,000 per cmm. on the eleventh day after
transplant to 21,^50 per cmm. on the twelfth day following
injections of 25 mg. and 50 mg. of chlorouridine. This
decline continued giving a count of 10,500 per cmm. on the
eighteenth day of survival. The red cell count of 11,880,
000 per cmm. on the seventh day after transplant decreased
to 6,250,000 per emm. on the eighteenth day. The weights of
the liver and spleen of this mouse and a control are com
pared as follows:
Tumorous mice
Survival
time
Body wt. Liver Spleen
Control
Treated
12 days
19 days
18.3 gm.
2*+.0 gm.
1.95 gm.
l.J+8 gm.
0.35 gm.
0.26 gm.
Attempts to transplant the tumor were unsuccessful
sinee many negative takes and decrease in virulence of the
transplanted tumor resulted. The reason for these results
•was later fonnd to be due to an improper choice of tumor
sections for transplanting purpose.
DISCUSSION
Chlorouridine or bromouridine inhibits the growth of
Neurospora No. 1298, whether uridine or eytidine supplies
the pyrimidine requirements of this organism. Growth is
more readily inhibited when eytidine, rather than uridine,
is present, but the inhibition may be completely reversed
by the addition of sufficient amounts of either nucleoside.
Pierce and Loring (191) have suggested that at least
two reactions may be involved in the growth inhibition of
Neurospora No. 1298, as produced by the purine nucleoside
or nucleotidesj namely, the deamination of eytidine to uri
dine and the utilization of uridine for the ribonueleic acid
synthesis In the mold. This suggestion implies that the
interconversion of the pyrimidine nucleosides is required
when only one of the nucleosides is supplied.
Since the growth response of the mold is the same
with molar equivalents of eytidine or uridine in the absence
of an inhibitor (190), and since chlorouridine reversibly
inhibits the growth in either case, the growth curves ob
tained in the presence of uridine, eytidine and chlorouri
dine gave an indication of the site of inhibition. These
data (Table III) indicate that utilization of each nucleo
side is inhibited independently. This is assumed from the
TABLE III
Independent Inhibition of Uridine and Cytidine
Utilization by Chloro uridine
Constant additions
per 10 ml. medium^/
Variable additions
per 10 ml. medium^/
Moles per liter
Cl-uridine3/
NUcleoside
Inhibitor,
Cl-uridine
Nucleoside
Gale,
x 10-4
Found
x 10"4
(a) Cytidine
( 1.3 x io-4)l/
mg.
0.3
mg.
1*25 Uridine
(1.3 x 10-4)1/
mg.
0.3 4.7 4*5
(b) Cytidine
(1.3: x 10*4)
0.3 0.6 Uridine
(4.0 x 10”4)
0.1 2.0 2.2
\
t
(c) Cytidine
( 2.5 x 10-4)
0.6 3*0 Uridine
(2.9 x 10-4)
0.71 1.03 1.08
(d) Uridine
( 2.5 x 10-4)
0.6 5*0 Cytidine
(1.73 x 10-4)
4.3 1*77 1.79
i/ftucleoside and inhibitor eoncentration such that no growth of Neurospora
No. 1298 occurs.
of nucleoside required to peimit half-maximum growth in the presence of
the constant additions.
^/Calculated from the inhibition index of uridine and cytidine (Table II) as
necessary to reduce growth to half maximum compared to the value experimentally found.
1/Molea per liter.
70
observation that in the medium containing both uridine and
cytidine the amount of chlorouridine experimentally required
to inhibit the growth to half-maximum approximately equals
the sum of the amounts calculated as necessary from the
individual concentrations of uridine and cytidine and the
inhibition index with uridine alone and cytidine alone. For
example; from (c) (Table III) when 2.5 x 10_lf mole of cyti
dine and 1.08 x 10“^ mole of chlorouridine are present, 2.9
x 10_if mole of uridine is required to stimulate to half-
maximum growth. The calculated amount of chlorouridine re
quired is 2.5 x K r lf x 0.58 plus 2.9 x 10"L | ' x 3.05 = 1.03 x
10-lf mole. The amount of chlorouridine calculated for
several other concentrations of uridine and cytidine is, with
in the limits of error, the same as that experimentally re
quired .
Since both uridine and cytidine occur as components
of nucleic acids, conversion of some cytidine to uridine (or
vice versa) must occur at some stage in the nucleie acid
£
biosynthesis to permit growth when only one of these nucleo
sides is supplied. If intereonversion of the nucleoside was
the process being inhibited by chlorouridine, the inhibitor
should be less effective or ineffective when a mixture of j
the two nucleosides is supplied. Since this is not the
case, both cytidine and uridine appear to be utilized as
71
such, and the amination or deamination required for the
polynucleotide synthesis must occur at a subsequent stage of
nucleic acid synthesis.
The stimulation of growth by chlorouridine in the
presence of uracil was unexpected. This result may.be
partially explained by assuming that uracil is not converted
into free uridine but to a ribosyl intermediate ”xM prior to
incorporation into nucleic acids or that the ribose moiety
of chlorouridine is transferred by some nucleosidase action
to a pyrimidine precursor to form more ribosyl intermediate
"x". Chlorouracil or bromouraeil neither appreciably pro
mote nor retard the growth of the mold in the presence of
uridine (110) or uracil.
It is difficult to explain the observation that con
siderably more 5-chlorouridine is required to inhibit a mix
ture of 0.5 mg. uracil and 0.3 mg. uridine than would be
required for complete inhibition of growth in the presence
of uridine alone. Since 0.5 mg. of uracil produces no
perceptible growth, it would be expected that no growth
would oecur when utilization of the added uridine is com
pletely blocked by 5-chlorouridine, Perhaps chlorouridine
inhibits the utilization of uridine, and at the same time
the growth is affected by the presence of uracil in a manner
similar to that suggested to explain the stimulation of
72
growth produced by chlorouridine in the presence of uracil.
Although it has been shown that chlorouracil strong
ly inhibited the growth stimulation produced by thymine
(135) in Streptococcus faecalis (136), the effect of the
addition of ribofuranose to chlorouracil eliminated the
inhibitory nature of the halogenopyrimidine. Pyranosyl-5-
chlorouracil was also ineffective (110). It has been shown
that pyranosyl nucleoside derivatives were inactive in pro
moting or inhibiting the growth of the uridine-requiring
Neurospora mutant (110). The 5-chloro and 5-bromo deriva
tives of natural uridine containing the furanose sugar con
figuration were found to inhibit competitively the utiliza
tion of the metabolite in the same Neurospora. The results
of these studies seem to indicate that the natural sugar
configuration of the metabolite is required by the anti
metabolite for a biologically active pyrimidine nucleoside.
The results of anti-leukemic studies with chlorouri
dine indicate that this antagonist is inactive in prolong
ing the survival time of the C57 black mice bearing myelo
genic leukemic tumor C1lj -98 under these conditions.
The author is greatly indebted to Dr. D. W. Visser
for his direction of this research program. We also ack
nowledge the assistance given by Dr. M. Roberts in carrying
i
out the screening tests with Streptococcus faecalis and
Miss H. Nishikara for conducting the anti-tuber culosis
studies.
SUMMARY AND CONCLUSIONS
A method for the partial synthesis of 5-chlorouridine
has been presented. It has been found that chlorouridine or
bromouridine reversibly inhibits the growth of Neurospora
No. 1298 in a medium containing either uridine or cytidine
as a growth requirement. The molar ratios of chlorouridine
to the metabolite to give half-maximum growth are 0.58 and
3.05, respectively, for cytidine and uridine. The inhibi
tion index for bromouridine in the presence of uridine is
Evidence is presented that chlorouridine inhibits the
utilization of uridine and cytidine independently and that
the inhibition does not involve cytidine deamination.
Chlorouridine stimulates the growth of the mold in
the presence of submaximal amount of uracil. It would
1
therefore appear that uracil is not converted to Uridine but
may condense with ribose from chlorouridine to form a ribo
syl intermediate prior to incorporation into nucleic
acids. The growth of the wild strain of Neurospora is not
inhibited by chlorouridine, bromouridine, chlorouracil, or
bromouracil.
The growth of Streptococcus faecalis is unaffeeted by
chlorouridine in a medium containing either folic acid or
thymine as the growth supplement. Some inhibitory effect on
75
the growth of Mycobacterium tuberculosis human strain 88 was
obtained with chlorouracil; chlorouridine and bromouridine
were inactive. Chlorouridine was also inactive in prolong
ing the life of the leukemic C57 black mice bearing myelo
genic leukemic tumor 01^98.
Since 5-chlorouridine has been shown to be inhibitory
to the growth of Neurospora No. 1298? other uridine and
cytidine derivatives may prove to be of additional interest.
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Studies of fatty acids: Effects on nutritional status and tissue cholesterol levels of rats

Asset Metadata

Creator Fukuhara, T. Kay (author)

Core Title Pyrimidine nucleoside antagonists

Degree Master of Science

Degree Program Biochemistry and Nutrition

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-777446

Unique identifier UC11349085

Identifier EP41320.pdf (filename),usctheses-c17-777446 (legacy record id)

Legacy Identifier EP41320.pdf

Dmrecord 777446

Document Type Thesis

Rights Fukuhara, T. Kay

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA