The Influence of Chemical Constitution on Antibacterial Activity. Part III: A Study of 8 ...

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THE BRITISH JOURN-,A-L OF
EXPERIMENTAL PATHOLOGY
V-OL. XXVIII APRIL. 194A7 NO. )
THE INFLUENCE OF CHEIC-AL CONSTTUTION ON ANTI-
BACTERIAL ACTIVITY. PART III: A STUDY OF
8-HYDROXYQUINOLINE (OXINE) AND
RELATED COMPOUNDS.
A. ALBERT. S. D. RUBBO, R. J. GOLDACRE AND B. G. BALFOUR.
From the Department of Organic Chemistry, University of Sydney, and the School
of Bacteriology, University of Melbourne.
Receivedforpublication December 30, 1946.
AT various times hvdroxvquinolines have been tested for antibacterial
activitv (von Oettingen. 1933), but never under conditions where their relative
potencies couldbeassessed. Thebestknownofthesevenisomeridesis 8-hvdroxv-
quinoline, also known as oxine. andfor brevitvwe propose to use the latter name.
The familiar Chinosol of commerce is a mixture ofoxine sulphate and potassium
sulphate.
Since oxine is known to be active against bacteria and fungi in very high
dilution, it probably acts by interfering with particular metabolic processes
essential for the growth ofthese organisms. That this specific action might well
take the form of a combination between the drug and trace metals. essential in
bacterial metabolism, was put forward by one of us (Albert, 1944). This sug-
gestion was based onthefactthat oxinereadilycheates, thatis to say. it combines
with polvalent metals to give feebly dissociated complexes in which the metal
forms part of a heterocyclic ring system. Those compounds with groups which
are so placed and are of such a nature that they can bind a metal in this manner
were termed chelate" by Morgan and Drew (1920) from analogy with chela,
the crab's claw.
The discovery that 1-hvdroxyacridine has an antibacterial activity greater
than could be accounted for on the basis of kationic ionization (Albert, Rubbo,
Goldacre. Davey and Stone, 1945) stimulated the present investigation, since
it was realized that the hydroxvl group in 1-hvdroxyacridine is situated. relative
to the nitrogen atom, exactly as in oxine (Fig. 1). Hence it was thought
desirable to determine whether the same specificity for the position of this
hvdroxyl group obtained in the quinoline series as in the acridine series.
Whilst the present studies were proceeding along these lines, Zentmyer (1943)
suggested that oxine may act on fungi through chelation with essential metals,
and later (1944) he demonstrated a reversal bv zinc.
The results of our investigations show that oxine, which is the onlv mono-
hvdroxyquinoline capable ofchelation, isthe only one ofseven possible isomerides
6
70 A. ALBERT, S. D. RUBBO, R. J. GOLDACRE AND B. G. BALFOUR.
to possess antibacterial properties. Further, it is shown that not only oxine, but
also structurally related substances that chelate equally well, are particularly
active against Gram-positive types. Whilst chelation is revealed as the essential
attribute determining the activity of this series of drugs, several other types of
chelate compounds are shown to be without a like antibacterial activitv. Thus
the architecture of the drug molecule is no less important than its potentialities
for chelation.
OH
FIG. I.-(a) I-Hydroxyacridine. (b) 8-Hydroxyquinoline(oxine).
An important extension ofthe present work will be concerned with the nature
of the metabolically essential metal or metals that are inactivated by oxine.
Preliminary experiments reported here indicate that cobalt may have an impor-
tance not previously ascribed to it in nutritional studies of micro-organisms.
eHEMISTRY.
Mlany metals. in the form of their salts, react with appropriately constituted
organic compounds to give ring structures having totally new properties. The
chelate complexes thus formed have stabilities which varv. but are often ofa high
order. Such complexes are usually only sparingly soluble in water and display
an increased solubility in organic solvents. Their metallic salt-like nature is
masked, and they resemble organic, rather than inorganic. compounds.
The present work is concerned principally with those chelate complexes in
which a metal forms a new ring by linking simultaneously with a phenolic oxygen
(through a primarv valency) andwith nitrogen (by co-ordination). For example,
two molecules of oxine react with one molecule of a zinc salt to give a chelate
complex Zn(C9HI6,ON)2, as in Fig. 2,and in this way an aqueous solution ofa zinc
salt can be largely deprived of its zinc ions. Oxine also chelates with trivalent
metals as in Fe (Cg9H60N)3; molybdenum and allied metals give complexes of
the type M002(C9H660N)2.
+Zn
FIG. 2.-Reaction between two oxineanions and one zinc ion to give oxine-metal chelate complex.
8-HYDROXYQUINOLINE (OxnE) AND RELATED COMPOUTNDS. 71
As will be seen, oxine chelates with metals to form five-membered rings in
which the co-ordinate bond between metal and nitrogen consists oftwo electrons
which aresupplied bythe latter (this is indicated bythe useofanarrowpointing
to the metal). Furthermore, it is most likely that the hydroxyl group in oxine
must ionize as an anion before it can contribute to the formation of a chelate
ring.* Therefore, any factors that interfere with the availability ofthe electrons
from the nitrogen atom orwith theionization ofthehydroxyl group will interfere
withchelation. Suchfactorsinclude (I)thesaturationofthelonepairofelectrons
by an alkyl group (as in N-methylation) or by a hydrogen ion (as in internal
salt formation), and ((H) repression of ionization of the hydroxyl group either
totally, by etherification, or partially through the adverse influence of electron-
repelling substituents. The influence of these factors on antibacterial activity
hasbeen investigated in the present work, and in general the connectionbetween
chelation and activity is well maintained.
Although it is intended here to deal principally with the quinoline series,
similar hydroxy compounds from related fmilies such as phenazines andphenan-
throlines have been included. In addition, a large number of miscellaneous
organic reagents used in chemical analysis for the determination of traces of
metals have been examined biologically.
In collateral publications (Albert and Gledhill, 1947; Albert and Magrath,
1947) the chelate powers ofall these substances are compared by a semi-quanti-
tative technique, using the degree of precipitation as a measure of chelation.
This method was especially designed to test chelate strength at the physiological
pH of 7-3, so that the bacteriological findings of the present work could be cor-
related with chelation. The degree of chelation is indicated in the following
tables by setting the chemical symbols in two columns. "Strong" connotes a
pronounced reading either in the form ofa heavy precipitate at standard dilution
(X/I000) or as a slight, but definite, precipitate at a tenfold further dilution.
" Less Strong " implies a slight but definite precipitate at the standard dilution
only. Where the symbol is not cited, no chelation took place under these con-
ditions; for instance, in no case did calcium or magnesium react. The metals
investigated were chosen because of their biological interest, namely, calcium,
magnesium, manganese, zinc, iron, -cadmium, cobalt, lead and copper. Ferrous
and ferric iron behaved similarly under aerobic conditions.
It is important to note that the firmness with which a chelate compound can
holdametalvarieswiththetypeofchelategroups. Groupsofthetypenowunder
consideration are known to confer a high degree of stability on the complex.
Nevertheless, even these metal complexes exist in equilibrium with small pro-
portions offree metallic ions, a fact which makes reversal possible under carefully
controlled conditions. Amino acids present in the medium are also capable of
chelation, but it seems likelv that the metallic complexes thus formed are meta-
bolized by the bacterium. An exact measure of the equilibrium between each
chelate complex and its constituents would be afforded by determination ofthe
stability constant:
[metal oxine complex]
K -
[metal] [oxine]
Unfortunately no satisfactory method is available.
* The anionic ionization of oxine at 370 C. and pH 7-3 is approximately 1-5 per cent.
72 A. ALBERT, S. D. RUBBO, R. J. GOLDACRE AND B. G. BALFOUR.
The preparation of the compounds used is described in a collateral paper
(Albert and Mfagrath. 1947). For the purpose of the present study every care
was taken to exclude traces of heavy metals in the finished product.
BACTERIOLOGY.
The same general technique for testing was followed as in Parts I and II of
thisseries.except thatthemediumused wasameatinfusion brothpreparedaccord-
ingtoWright's method (1933). plus 0 0O per cent glucose when usingstreptococci.
and adjusted to pH 7-2-7-4 (glass electrode). Plain broth was chosen in this
work in preference to serum broth. because it was anticipated that serum might
be an interfering factor in subsequent metal reversal experiments. Serum broth
(10 per cent). however, was used fortesting the activity ofa number ofsubstances
of possible clinical interest (Table VIII). No attempt was made to exclude
trace elements during preparation of the media.
The broth medium was distributed aseptically by means of an automatic
25 ml. pipette into sterile six-ounce bottles and incubated for 48 hours at 37D C.
before use. Each derivative was dissolved in sterile water to give an Mi 814
solution with the aid of 1.5 equivalents of hydrochloric acid. except where the
substances were strongly acidic (-COOH. -S3H. -Cl and -N.O2 derivatives).
in which cases Ia equivalents of sodium hydroxide were used instead. The
first dilution was usually prepared by adding a ml. of xi 80 solution to 45 ml. of
broth. thus givinganinitial concentration equal toMi 800. Geometrical (twofold)
dilutions were made by transferring 25 ml. volumes to each successive dilution
bottle. The broth dilutions were then transferred to test-tubes. each bottle
yielding twelve 2 ml. quantities. which sufficed to test six organisms in duplicate.
The organisms used in this work were the stock strains used in Parts I and
II. plus a pigment-producing strain of Pseudomonas pyocyanea. The inoculum.
as before, was standardized as 0-03 ml. ofa 24-hour culture. The highest dilution
which completely prevented visible growth after 48 hours incubation at 37' C.
was taken as the end-point of bacteriostasis. The concentrations required to
achieve this result are given in all tables. In comparing two substances, a
difference of more than twofold in duplicate tests was considered significant.
RESULTS.
The material has been classified into four categories (Tables I to IV) on the
basis of molecular structure.
(1) Quinoline and the Seren JIono-hydroxyquinolines.
In this group of compounds (Table I) the strong positive correlation existing
between chelating power and antibacterial activitv is plainly seen. Of the
compoundslistedinTableIonlythe 8-hydroxyisomeride, oxine (No. 8). iscapable
of chelation. and it alone is antibacterial. The bacteriostatic index against the
Gram-positive organisms is especially high. The figures for Cl. wdehii. Str.
pyogenes and Staph. aureus (M/100,000) correspond to a dilution of one part in
680,000 approximately, a titre considerably higher than the best of the acridine
drugs. e.g. 5-aminoacridine (No. 9). This superiority, however. is reversed in
favour of the acridines where Gram-negative organisms are concerned.
This series of isomeric compounds is an outstanding example of biological
specificity in so far as only one of the seven isomerides manifests definite anti-
8-IYDROXYQUINOLIN-E (OXIN) AND REATED COMPOUNDS. 73
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76 A. ALBERT, S. D. RU-BBO, R. J. GOLDACRE AND B. G. BALFOUR.
bacterial activitv. It may be compared with the aminobenzene-sulphonamides
and the mono-amino-acridines where one out of three, and two out offive, iso-
merides respectively are definitely antibacterial.
Some years ago an attempt was made to introduce 3-hydroxyquinoline (No.
3) as a powerful antiseptic on the grounds that it would kill Staph. aureus in
one second at a dilution of 1: 4000 (Davin and Schlauch, 1932). However, we
find that this dilution will not inhibit growth ofthe organism in broth.
(2) Substituted 8-Hydroxyquinolines (Oxineq).
Table II is concerned with the simpler derivatives ofoxine. Among these are
some in which certain factors which modify chelation (see chemistry section) have
been introduced. As already stated in the chemical section of this paper, the
chelating power ofoxine istotallysuppressedwhentheionization ofthe hydroxyl
group is prevented or the nitrogen atom made quaternary. Examples of this
are shown in Table II, No. 10 and 12, where the ionizable hydroxyl of oxine
(No. 8) has been blocked by methylation, and again in No. 13 where the nitrogen
of oxine has been made quaternary. It will be seen that in all these cases not
only is chelation suppressed, but also antibacterial activity.
The remaining compounds in Table II (No. 14 to 31 inclusive) are variously
substituted derivatives of oxine. Although in these -substances, the -OH and
=N- are free and hence permit of chelation, the extent to which the latter
occurs will depend on the deformation exerted by each substituent on the elec-
tronic distribution ofthemolecule. Thisisdiscussed inthe collateralpublication.
What is important to note here is that antibacterial action is well maintained
in those compounds in which chelation is ofthe same order as in oxine. However,
attention is drawn to the unexpectedly low antibacterial activity of 2-methyl-
oxine (No. 11) compared with its isomeride, No. 14, an observation which we
consider sheds some light on the mode of attack of these drugs on possible
bacterial enzymes and which will be discussed later.
(3) Compound8 Related to Oxine.
In this section we have classified as best we could a number of substances
whichdisplav(a) the same type ofchelation as in oxine,but in a differentnucleus,
or (b) a different type of chelation in the same (i.e. the quinoline) nucleus.
It has already been pointed out that all the structural elements responsible
for chelation in the oxine molecule are also present in 1-hydroxyacridine (see
Fig. 1). Reference to Table III below shows that 1-hydroxyacridine (No. 32)
has strong chelating properties, and, as it is a poorly ionized acridine, chelation
apparently determines its antibacterial activity. That this is so is confirmed by
theinactivity of I-methoxyacridine (No. 33), which is also poorly ionized, andin
which no -OH is available for chelation. Similar remarks apply to 1-hydroxy-
phenazine (No. 34) and its methyl ether (No. 35).
It is interesting to note that 1-hydroxyacridine and 1-hydroxvphenazine do
not achieve an antibacterial potency comparable to that of oxine in spite of
effective chelation. -This is attributed to a steric hindrance to adsorption of
these molecules at the metal-bearing surface. The significance of this steric
factor, which probably operates against full activity in 2-methyl-oxine (No. 11),
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78 A. ALBERT, S. D. RUBBO, R. J. GOLDACRE AND B. G. BALFOUR.
will be discussed later. It should be noted that benzo- and pyrido-derivatives
of oxine have an antibacterial action comparable to their chelate ability (cf.
5: 6-benzo-oxine and 6-hydroxy-m-phenanthroline, No. 37 and 38 respectively)
butintheseexamples theextraringis situatedat somedistancefromthechelating
portion of the molecule.
FIG. 3.-(a) 1-Hydroxyphenazine. (b) 6-Hydroxy-mn-phenanthroline.
The sulphur analogue of oxine, 8-mercaptoquinoline (No. 39), was difficult
to examine because ofits poor solubility and ease ofoxidation, but it will be seen
that its antibacterial activity is of a high order, as would be expected from its
chelate properties. Conversely 8-aminoquinoline (No. 40),which does not chelate
appreciably, has no antibacterial properties.
Two interesting derivatives of quinoline which form chelate oomplexes that
differ in type from those of oxine will now be discussed. They are quinaldinic
acid and quinoline-8-carboxylic acid (No. 41 and 43, Fig. 4; cf. Fig. 1).
===O
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FIG. 4.-Chelatecomplexeswithadivalentmetal (M). (a) Quinoline-8-carboxylic acid. (b) Quino-
line-2-carboxylic acid.
These differences in chelate ring structure are reflected in the altered chelating
properties (quinolhne-8-carboxylic acid (No. 43) being practically a specific agent
for copper) and in the absence of any antibacterial activity.
Cinchoninic acid(No.42),anisomerideofNo.41and43,hasnochelatingpropertiesandisdxevoid
ofbacteriogtatic qualities. Xanthurenic acid (No. 44), aproductoftryptophanmetabolism appear-
ingintheurineofratsfedahighproteindiet,deficientinpyridoxine,containsthechelatesystemsof
both oxine and quinaldinic acid. It gives colour changes with metals but no precipitates and is
not antibacterial.