Note: Descriptions are shown in the official language in which they were submitted.
WO91/0~61 2 0 719 8 2 PCT/GB90/01997
`''J --1
NOVEL PRODUC.S
The present invention relates to a novel microorganism and
to parasiticidal compounds obtainable therefrom and
s synthetically.
Compounds known as marcfortines are disclosed by Polonsky et
al in J.Chem.Soc.Chem.Comm (1980) pp 601-602 (marcfortine A)
and Tet. Letters (1981) Vol 22 pp 1977-1980 (marcfortine B
10 and C). The compounds are metabolites of the fungus
Penicillium roqueforti.
Structurally related to the marcfortines is a compound known
as paraherquamide, disclosed by Yamazaki et al in Tet.
15 Letters (1981) Vol 22 pp 135-136. Paraherquamide is a
metabolite of the fungus Penicillium Paraherquei. Also
disclosed is its dihydro derivative, obtained by catalytic
hydrogenation.
20 ~S-A-4~66060 describes the use of marcofortines A, B, C as
anti-parasltic agents. US-A-4923867 discloses derivatives
of the marcfortines and their use as antiparasitic agents.
EP-A-0301742 describes the use of paraherquamide and
2s dihydroparaherquamide as antiparasitic agents. EP-A-
0322937, EP-A-0354615 and EP-A-0390532 dlsclose derivatives
of paraherquamide and their use as antiparasitic agents.
We have now discovered a novel paraherquamide producing
30 fungus. Compounds produced as metabolites in addition to
paraherquamide also have parasiticidal properties, and
therefore are of use in the treatment of parasitic
infestations in humans and animals.
35 We have also discovered a novel modification to the basic
SUBSTITUTE SHEET
.
.
- ` . .
'. .
`: . : ~ ` - . .
~0~71982
WO91/0~61 PCT/GB9~/01997
--2--
marcfortine/paraherquamide structure which provides
compounds with parasiticidal properties.
A first aspect of the invention provides Penicillium SD. IMI
5 332995, or a mutant thereof, more particularly in
biologically pure form.
A second aspect of the invention provides compounds of
formula (I):
(O)r~;~ R~
~ ~ ~'~C~/3
l S C~'3 >~_ X H
c,~ yJ ~I)
wherein: X is oxygen or a bond, Y is a single bond or a
double bond, Rl is hydrogen or methyl, R2 is hydrogen or
20 hydroxy; and n is 0 or l; but excluding paraherquamide and
dihydroparaherquamide.
A third aspect of the invention provides a compound of
formula ~II):
~ H
~II)
wherein: p is 0 or l.
SUPSTITUTE SHEET
~ WO 91/0996l 2 0 71 9 8 2 PCI/GB90/01997
nond ~lc Uon ~o: PCT~GB90 / 01997
. __ _ _
MICROORGA~ISMS
o~ 5_ ~ c~oo~ t~
. ID~ nC~ a~ro~r ~
O -
_ _
C.A.B. International Mycological Institute
Ferry Lane, Kew, Surrey TW9 3AF, United Kingdom.
D~ ~ ~cc_ _
31st May 1989 ¦ IMI 332995
~, ~ODlnO~L I~DlCAnO~ n t~t ~. Tl~ ~,t ~ ~ ~.~1- ~c~ t O
In respect of those designations in which a European patent is sought, a
sample of the deposited microorganism will be made available until the
publication of the mention of the grant of the European patent or until
the date on which the application has been refused or withdrawn or is
deemed to be withdrawn, only by the issue of such a sample to an expert
nominated by the person requesting the sample. (Rule Z8~4)EPC)
e. O~U--~T~O T~T~--~O~ WI~ICII 1- oicAno~c ~T ~.o~ ~- t- ~1-~ - ~_-- ~1
._ I
o. ~ T~ ~UI~ O~ OIC~T O~ ~ t~ _ I
~ q
o~-
~ d Ol~
lo~m PCT'RO`~I IJ-~u~ 0
SUBS~l-rl)TE SHEE~
~ . 20719~32
WO9~/0~6l - PCT/GB90/01997
--4--
This compound is believed to be a bio-precursor of
paraherquamide and may be used as an interm~diate in the
synthesis of paraherquamide or
derivatives thereof.
s
A fourth aspect of the invention provides a marcfortine or
paraherquamide derivative of partial formula (III):
O ~(Z~
N .2 ,7~ ~
~/\~/~0
I s ~III)
~
wherein Z is CH2 and m is 0 or 1, R3 and R4 are selected
from hydrogen, hydroxy, Cl_6 alkyl, C1_6 alkoxy, C1_6
alkenyl, C1_6 alkenyl-C1_6 alkoxY, Cl_6 alkynyl Cl_6
C1_6 alkanoyloxy, poly Cl_6 alkoxy-Cl_6 alkoxy, phenyl,
20 phenyl-Cl_6 alkyl, tri-Cl_6 alkylsilyloxy,
diphenylphosphoryloxy and halogen, or R3 and R4 together
form ~ or = CH2, and R5 is hydrogen or Cl_6 alkyl.
In effect, this aspect of the invention provides an N~12)
2s oxide of a marcfortine or parherquamide or a derivative
thereof (when the usual numbering for these compounds is
used). Substitution at other positions is optional, but in
the partial formula preferably occurs on the carbon atom at
position 14, or the amide nitrogen atom.
The marcfortine derivative may be any of those disclosed in
US-A-4923867, the disclosure of whlch is incorporated herein
by reference. The paraherquamide derivative may be any of
those disclosed in EP-A-0301742, EP-A-0322937, EP-A-0354615
35 or EP-A-0390532, the disclosures of which are incorporated
herein by reference.
SUBSTITUTE SHEET
`
.
`:
.
.. ~ .. .
'- !2b7~98~
WO 9l/0996l ~ pcr/GB9o/ol997
~,
_.5_
The preferred r~(12)oxide is VM55556, the N~12)oxide of
paraherquamide itself.
Compounds of formula (I) in which Y is a double bond
5 obtainable as metabolites of IMI 332995 are set out in Table
I below:
Table I
10 CompoundI~ASS X Rl ¦ R2
VM 29919 493 ~~ CH3OH absent
(Paraherquamide)
VM 55596 509 ~~ CH3OH present
(Paraherquamide
N(12~-oxide)
VM 54159 477 ~~ CH3 H absent
VM 54158 477 ~ CH3OH absent
VM 55594 461 ~ CH3 H absent
VM 55595 447 _ H H absent
_
The compound of formula (II) in which P is 0 (VMS5599) is
30 also obtalnable as a metabolite of IMI 332995.
A further aspect of the invention provides a process for the
production of compounds of formula (I) or formula ~II), or a
derivative thereof, which comprises cultivating Penicillium
35 sP. IMI 332995, or a mutant thereof, and subsequently
isolating the compound or derivative thereof from the
culture.
SUESTITUTE SHEET
~q~982
WO91/0~61 PCT/~90/01997
--6--
The present in~ention also provides a process for the
production of a compound of the invention or a derivative
thereof, which comprises cultivating a producing
microorganism, and subsequently isolating the compound or
5 derivative thereof from the culture.
The present invention furthermore provides a process for the
preparation of a compound of the invention or derivative
thereof, which comprises chromatographically separating the
O compound or derivative thereof from a solution thereof in
admixture with other substances into a fraction comprising
the compound or d~rivative thereof and other fractions.
The term 'cultivation' ~and derivatives of that term) as
5 used herein means the deliberate aerobic growth of an
organism in the presence of assimilable sources of carbon,
nitrogen, sulphur and mineral salts. Such aerobic growth
may take place in a solid or semi-solid nutritive medium, or
in a liquld medium in which the nutrients are dissolved or
20 suspended. The cultivation may take place on an aerobic
surface or by submerged culture. The nutritive medium may
be composed of complex nutrients or may be chemically
defined.
2s It has been found that suitable microorganisms for use in
the cultivation process according to the invention for the
production of VM 55595 andJor VM 55596 and/or VM 55599
include strains of fungi belonglng to the genus Penicillium
that are capable of elaborating compounds according to the
30 invention. It has further been found that Penicilllum sP.
IMI 332995, which has been isolated from soil, and also
mutants and natural variants thereof, is particularly
suitable for this purpose.
35 The term 'mutant' as used herein includes any mutant strain
which arises spontaneously or through the effect of an
external agent whether that agent is applied deliberately or
otherwise. Suitable methods of producing mutant strains
SU~;STI I-UTE SHEET
. .
:, , - . . . -:
, . ~ . . .
.' ' ' ` ' ' . ' - : ~
,-..
W091/0~6l -~ 2 0~ 8 2 PCT/GB9o/0l997
~ 7
include those outlined by H.I.Adler in 'Techniques for the
Development of Micro- or~anisms' in ''Radiation and
Radioisotopes for Industrial Microorganisms'', Proceedings
of a Symposium, Vienna, 1973, page 241, International Atomic
s Energy Authority, and these inc~ude:
(i) Ionizing radiation ~e.q. X-rays and ~-rays), u.v.
light, u.v. light plus a photosensitizing agent (e.q.
8-methoxypsoralen), nitrous acid, hydroxylamine,
0 pyrimidine base analogues ~e.~. S-bromouracil),
acridines, alkylating agents (e.q. mustard gas,
ethyl-methane sulphonate), hydrogen peroxide, phenols,
formaldehyde, nitrosoguanidine, heat, and
1S ~ii) Genetic techniques, including, for example,
recombination, transformation, transduction,
lysogenisation, lysogenic conversion, protoplast
fusion, and selective techniques for spontaneous
mutants.
Penicillium sP. IMI 332995 is believed to comprise a
previously unreported strain in the genus Penicillium. It
has been deposited in the C.A.8. }nternational Mycological
Institute, Ferry Lane, Kew, Surrey, TW9 3AF, England, the
2s deposit (IMI 332995; filing date 31st May, 1989) being made
under the terms of the Budapest Treaty on the International
Recognitlon of the Deposit of Micro-organisms for the
Purposes of Patent Procedure.
30 The characteristics of Penicillium sP. IMI 332~95 were as
follows:
After being grown for seven days on Czapek Dox agar medium
at 28C, Penicillium sP. IMI 332995 had produced colonies
35 which were 20-30mm diameter. Margins of the colonies were
low and velutinous, central areas were floccose; mycelium
SLiE~T~TUTE SHEET
:, . ' ,, :
WO91/0996l ~ Qq i~ ~ ~ pcT/GBso/
-8-
was mostly white in peripheral areas, at the centres yellow
green to grey green. A yellow exudate was produced over the
colony; reverse colour of colony was yellow brown.
5 Conidiophores were borne from subsurface or surface hyphae,
stipes 100-300 x 2.2-3.0~m, smooth walled,
characteristically terminating in well defined verticils of
3-5 divergent metulae. Some subterminal or intercalary
metulae were produced also. Metulae were usually of uniform
lo length ~12-18 x 2.2-2.5~m), phialides were in compact
verticils of 8-12, ampulliform usually with short collula;
conidia were spheroidal to subspheroidal, 2.2-3.0~m diam,
with walls smooth or finely roughened, typically borne in
long well defined columns, one per metula.
The fermentation medium for cultivating the producing
organism may be solid, semisolid or liquid and suitably
contains sources of assimilable carbon and assimilable
nitrogen together with inorganic salts. Suitable sources of
20 nitrogen include yeast extract, soybean flour, meat extract,
cottonseed flour, malt, distillers dried solubles, amino
acids, protein hydrolysates an~ ammonium and nitrate
nitrogen. Suitable carbon sources include glucose, sucrose,
lactose, maltose, starch and glycerol. Suitably the culture
2s medium also includes alkali metal ions (for example,
sodium), alkaline earth metal lons (for example, magnesium),
halogen ions (for example, chloride)~ and trace elements
(for example,iron).
30 The cultlvation may suitably be effected at a temperature of
about 20 to 35C, advantageously 27 to 28C, and the culture
may suitably be harvested after 2 to 35 days, advantageously
about 5 to 20 days, after the initiation of fermentation in
order to give an optimum yield of the desired compound.
SUBS~17 UTE SHEE~
` ''
:
WO 91/09961 ` '~ J~ Y ~ ~, PCI/GB90/01997
_g_
The desired compound or derivative thereof may then be
isolated from the culture medium and worked up and purified
using conventional techniques.
s The desired product may be obtained from either the mycelial
growth or from the culture medium. It may therefore be
convenient for the first isolation step to involve the
separation of the mycelium from the culture medium by, for
example, filtration or centrifugation of fermentation broth,
o to give a clarified culture filtrate and solid material.
Alternatively, the whole medium including the mycelium can
be extracted directly.
It may be convenient to include an organic solvent
15 extraction step in the isolation or purification procedure,
suitably using a solvent such as acetone or chloroform.
Further isolation of the desired compound may conveniently
be effected by chromatographic techniques. The extract may
20 contain additional substances, and therefore chromatoqraphic
separatlon may result in a plurality of fractions, of which
the desired fraction or fractions is or are the fraction(s)
comprising the desired compound or a derivative thereof.
2s The desired fraction~s) may readily be identified in a
routine manner by testing for anthelmintic activity and/or
by monitoring each fraction chromatographically. The
desired fraction(s) is/are that/those identified by such
procedures as containing the desired compound or a
30 derivative thereof.
If necessary, repeated chromatographic separation may be
carried out in a routine manner. At each stage of the
separation procedure, the fractions containing the desired
35 compound or a derivative thereof may be combined and then
SUBSTIlUTE SllE~T
W091/0996l 2 ~ 7 I 9 ~ 2 PCT/GB90/01~7
--10--
subjected to further purification steps. In the initial
separation steps, it may be convenient to identify the
desired fractions merely as those having anthelmintic
activity and to combine all such fr~ctions. In later stages
5 of the separation, it may be necessary to identify the
desired fraction or fractions more precisely in order to
separate the desired compound or a derivative thereof from
any other substances that may be present. Separation may
advantageously be continued in order to give one or more
lo fractions consisting essentially of the desired compound or
a derivative thereof.
The expression 'fraction consisting essentially of the
desired compound or a derivative thereof' means a fraction
15 contalning the desired compound or a derivative thereof as
the sole component present in that fraction, or as the major
component ~whether other components are active or are
inactlve impurities) present in that fractlon. The
expression 'major component' means the component that is
20 present in the greatest amount relative to other indlvidual
components ~exclusive of solvent). Suitably, the major
component is present in an amount greater than the sum of
the amounts of all other components (excluding solvent).
More suitably, the major component is present in an amount
2s of at least 60%, advantageously at least 70%, preferably at
least 80%, especially from 90% to 100~, by weight, relative
to the total amount of active material, or relative to the
total amount of material whether active or inactive
~exclusive of solvent), as the case may be, present in the
30 fraction. Typically, the compounds of formula (I) are
produced in admixture with one another, so that fractions
may be obtained which consist essentially of a mixture of
two or more compounds of formula ~I).
35 It has been found convenient to carry out chromatographic
separation using HPLC. Two or more chromatographic
separation steps may be carried out successively.
S~JBSTi T UTE SHEEr
?
" ~ .
wo gl/0~61 - 2 0 71 9 8 2 PCT/GB90/01997
,,. ., ,, - .
In a further aspect of the invention VM 55596 may be
prepared by the N-oxidation of paraherquamide. A suitable
procedure ls described in J. Org. Chem. [1970] 35, 1721 et
seq. The same procedure may be used to prepare the compound
5 of formula (II) in which p is 0 by ~-oxidation of VM 55599.
Similarly compounds of partial formula (III~ can be obtained
by N-oxidation of the derivatives disclosed in the patents
incorporated herein by reference.
10 Accordingly the present invention provides a method for the
production of a compoun~ of partial formula (III) which
comprises reacting a marcfortine or paraherquamide on
derivative thereof of partial formula (IV)
lS
~\r~)/~\O
I S ~IV)
in which Z, m, R3, R4 and R5 are as defined for formula
~III), with a N-oxidising agent.
2s General procedures for the preparation of heteroaromatic
N-oxides can be found in Chapter II of "Chemistry of the
Heterocyclic N-Oxides", A.R.Katritzky and J.M.Lagowski,
published 1971 Academic Press (Vol.l9 of ORGANIC CHEMISTRY -
A Series of Monographs). Typically the N-oxide is formed by
30 reaction with a percarboxylic acid in an appropriate
solvent. Most suitably an aromatic peracid in a non-polar
solvent is used, since the reaction may usually be carried
out at room temperature. Suitable aromatic peracids include
perbenzoic acid, chloroperbenzoic acid perphthalic acid.
SUB--TITUIE SHEET
WO91t~9961 ~ i i , , PCT/~B90/01997
Preferably the reaction is c~rried out in chloroform using
m-chloroper~enzoic acid. of the non-aromatic peracids,
peracetic acid is the most common reagent, but is less
suitable in view of the more adverse conditions, such as
5 heating the heterocyclic compound with glacial acetic acid
and hydrogen peroxide at temperatures from 20 to 90C.
Compounds of formula (I) that are not obtainable as
metabolites of IMI 332995, or by N-oxidation as described
lO above, may be prepared by chemical modification of compounds
that are obtainable as metabolites, using substitution
techniques that are well known in organic chemistry, for
example for a alkylation of amide nitrogens.
lS The dihydro derivatives of the compounds of formula ~I)
~where Y is a single bond) may be prepared from the
compounds of formula ~I) in which Y is a double bond using
the procedure described in Example ll of EP-A-0 301742,
referred to above.
The compound or mixture of compounds according to the
invention is suitably provided in substantially pure form,
for example at least 50% pure, suitably at least 60% pure,
advantageously at least 75% pure, preferably at least 85%
pure, more preferably at least 95% pure, especially at least
98% pure, all percentages being calculated as weight/weight.
An impure or less pure form of a compound according to the
inventlon may, for example, be used in the preparation of a
30 more pure form of the same compound or of a related compound
(for example a corresponding derivatlve) suitable for
pharmaceutical use.
The compounds of the invention have parasiticidal
35 properties, for example against nematodes such as
SUBSTITUTE SHEE~
: ,
- . ' ~` - ~ : :
.
WO91/0~61 2 0 ~19 8 2 PCT/GB9OJ01997
13-
Trichostronqylus colubriformis, and are useful for the
treatment of helminthiasis in animals such as mammals,
including humans and domesticated animals (including farm
animals).
Accordingly the present invention also provides a compound
according to the invention, for use in the treatment of the
human or animal body, especially for treating endo- and
ectoparasitic infestations and particularly for treating
0 helminthiasis of domestic and farm animals.
The term helminthiasis encompasses those diseases of man and
animals caused by infestation with parasitic worms such as
Strongyles, Ascarids, hookworms lungworms, filarial worms,
15 whipworms, and liver flukes. The compound may also be used
against nematodes occurring in the soil or parasitic to
plants.
The compounds of the invention are also active against
20 Arthropods. The phylum Arthropoda comprises insects - such
as biting flies, lice, bugs, beetles and fleas - and
arachnids - such as mites and ticks.
Thus, a broad aspect of the invention provides a method of
25 eradlcating arthropod or nematode infestations, which method
comprises applying a compound according to the invention or
a derivative thereof to the arthropods or nematodes or to
their environment.
30 The present invention thus provides a pesticidal compositlon
comprising a compound according to the invention or a
derivative thereof together with a suitable carrier or
excipient, such as an aerosol formulation.
35 The present invention also provides a pharmaceutical or
veterinary composition comprising a compound according to
the invention or a pharmaceutically acceptable derivative
SUBSTI~UTE SHEET
.
2 0 ~ ~ 9 ~ ~ ,
W091/0~61 PCT/GB90/01997
-14-
thereof together with a pharmaceutically or veterinarily
acceptable carrier or excipient.
The present invention also provides a method of treatment or
5 prophylaxis of endo- and ectoparasitic infestations,
especially helminthiasis, of animals, especially humans and
domesticated mammals, which comprises administering an
effective non-toxic amount of a compound according to the
invention or a pharmaceutically acceptable derivative
O thereof, or a composition according to the invention, to a
patient in need thereof.
The composition according to the invention may be formulated
for administration in any convenient way for use in human or
s veterinary medicine, by analogy with other anthelmintics.
In suitable formulations the drug may be administered to
animals orally (as a paste, drench, bolus, capsule or
tablet), parenterally, percutaneously, as a food additive
20 ~eg granules, pellets or powder), or may be prepared as an
aerosol spray formulation.
The compounds of the invention may be formulated as a
mixture with each other and/or with other anthelmintics,
25 insecticides, acaricides or other pharmacologically active
substances.
Suitably the composition consists of sufficient material to
provide a dose of from O.Ol to 50mg of active ingredient per
30 kg of animal body weight per dose, more suitably O.l to
lOmg/kg per dose.
A composltion according to the invention may suitably
contain from O.OOl to 99% by weight of the compound
35 according to the invention ~based on the total weight of the
composition), depending on the method of administration.
SUBS~I~UTE SHEEl^
..
.
: ' `
WO91~09961 2 ~ ~ 9 8 2 PCT/GB90/01997
-15-
In certain circumstances the crude fermentation broth may be
administered, for example by incorporating the freeze-dried
fermentation broth into the feed of the animal.
s It will be appreciated that, in some cases, it will be
advisable to repeat the dosing of the infected or
potentially infected human or animal with the compound of
the invention according to conventional dosage regimes used
with anthelmintics.
The following Examples illustrate the invention.
SUBSTITUTE SHEET
-
.
.
.
. - .
WO91/09961 2 Oi7 19 8 2 PCT/GB90/01997 ~
-l6-
Example l
A conidial suspension of Penicillium sp. IMI 332995 was
prepared in phosphate buffered saline containing 10%
s glycerol and 1% Tween 80 ~PBSGT) from a well grown potato
dextrose agar* plate culture,
2-3mls of suspension were spread onto Czapek Dox agar in
large bioassay dishes (245mm2) and incubated at 28C. The
10 culture and agar was harvested after 20 days growth into
large glass beakers.
5 Czapek Dox Agar
g/L
Sucrose ~BDH Analar) 30
NaNO3 3
MgSOq.7H2o 0.5
KCl o 5
2s
FeS04 . 7H20 0 . 01
X2HP04 1 . O
Technical Agar (Oxoid) l8g
Deionised water l000ml
* Potato Dextrose Agar (Oxoid Ltd. Basingstoke, UX)
SUBSTI~UTE SHEET
. . . -
-- . .
.
wosl/~6l Zo 7i 982 PCT/GB9~/0l997
Extraction and Purification of VM 29919 VM 54148 VM 54159,
VM 55594 and VM 55595
Culture IMI 332995, grown for 20 days on Czapek Dox agar
s (5L) was macerated in acetone ~6L) (Waring commercial
blendor, Dynamics Corporation of America) centrifuged and
the solids further extracted with 2 washes of acetone (6L
per wash, the solids being separated by centrifugation).
The acetone was evaporated under vacuum yielding an aqueous
lO residue which was then extracted three times with 2 volumes
of CHC13. The organic phase was evaporated under vacuum
leaving ~.23g of solid extract.
The primary purification was by preparative reverse phase
15 HPLC (Dynamax - 60A C-18 column 41.4 x 250mm, Rainin
Instrument Co., USA) eluted with methanol- water (75:25) at
20 mls/min. The entire extract was suspended in methanol,
after allowing to stand the soluble portion was injected on
to the column via a Sml sample loop (4 consecutive
20 in~ections). (Subsequent weighing of the insoluble residue
showed that 3.34g of extract was loaded on to the column at
this stage).
The flow from the column was continually collected lnto a
2s fractlon collector in 0.8mln. ~16ml) fractions. lml
allquots were taken from alternate fractions, dried under
vacuum, redissol~ed ln 0.5ml CHC13 and examined by TLC
(Plastic backed Silica -60 plates, E. Merck, Darmstadt, FRG
(TYPE 5735) developed with CHC13: methanol:glaclal acetic
30 acid 89:10:1) Spots were visualised by spraying with
Ehrlichs reagent (1% 4-Dimethylaminobenzaldehyde in 1:3
concentrated HCl:Methanol) followed by heating. TLC of the
starting material showed three major spots glving a plnk
colour with Ehrlichs reagent. The largest at Rf 0.55 and
35 two smaller spots at Rf 0.6 and 0.5.
SUBSTI~UTE SHEET
2071~82.
W0~l~0996l ; ~ PCT/GB90tO1997
-18-
The TLC of column fractions showed that the Rf 0.55 spot
eluted from the column first (fractions 10-27) followed by
the other two spots together in fractions 28-56. These
fractions were therefore separately pooled to give bulk
5 fractions 'A' (Fractions 10-27) and 'B'(Fractions 28-56).
The insoluble material not loaded onto the inital column was
dried and weighed (0.89g). It was almost completely soluble
in a larger volume (200ml) of methanol. This was filtered
0 and re concentrated under vacuum to 20mls. This was
chromatographed under essentially identical conditions to
those previously described and similar bulk fractions A and
B were obtained.
15 [Fractions A and B from each column were pooled and weighed
(A:0.93g, B:0.18g).
Further purification of both fractions A and B was by
preparative Silica HPLC (Dynamax - 60A Si column
20 21.4 x 250mm. Rainin Instrument Co USA) eluted with
CH2Cl2:methanol gradients at 8ml/min. Fractions were
examined directly by TLC developed with
90:10: CHCl3:methanol. The metabolites were visualized as
pink spots after spraying with Ehrlichs reagent.
Sample A was dissolved in CH2C12 and injected onto the
silica column which was eluted with a linear gradient of
CH2Cl2:methanol, 100:0 to 90:10 over 120 mins. Fractions
were collected throughout. By reference to TLC results it
30 was found that the material of lnterest ~the mos~ abundant
Ehrlich positive pink spot) eluted between 60 and 71 minutes
~at 97:3 CH2C12:methanol). Fractions containing this
material were pooled and dried to give 353 mq of pale yellow
solid.
This sample was divided into two and each portion
chromatographed by Silica HPLC as before except that in
SUBSTITUTE SHEET
- ,
- ` . . . - - -
~U (1~
~, WO 91/09961 !, PCT/GB90/01997
1 9--
addition the column eluent was monitored by W at 235 and
290nm tlOOOS Diode Array Detector, Applied Biosystems Ltd.
Warrington, UK). It was found that, in each run, the
material eluted as before except that two distinct peaks
s were discernable by W monitoring. Each was collected
separately and corresponding material from each column run
was pooled and dried down on a rotary evaporator. The
faster eluting peak yielded 117mg of VM 29919.
0 Characterisation Data for VM 29919
W ~max 225nm ~in MeOH)
Mass (FAB, NOBAlNa) [MH+] = 494
~13C (CDCL3) 183.1, 171.6, 146.2, 139.1, 135.4,
132.7, 125.1, 120.3, 117.3, 1~5.0, 79.8, 78.1,
71.4, 65.3, 63.2, 59.2, 51.8, 51.6, 46.4, 38.2,
37.1, 29.94, 29.85, 25.9, 23.7, 22.2, 20.4, 19.2
This was found to be identical to Paraherquamide
20 ~Yamazaki and Okuyama 1981, Tetrahedron Letters 22 135-136)
Fractions containing the slower peak yielded 34mg of VM
54158.
25 Characterisation Data for V~ 54158
W ~max 244mm. ~in MeOH)
Mass (FAB, NOBA~Na) lMH+] = 478
~13C (CDC13 184.3, 153.1, 137.1, 131.3, 125.6,
121.3, 116.1, 109.6, 105.3, 78.0, 76.4, 71.4,
65.3, 62.6, 59.2, 51.9, 51.7, 46.4, 38.1, 37.1,
27.9, 27.8, 25.9, 22.2, 22.2, 20.4, 19.1
SUBSTITUTE SHEET
W091/0~6l ~ O ~ 1 9 g 2 PCT/GB90/01997 ~
-20-
Sample B ~from the reverse phase separation) was dissolved
in 4mls CH2Cl2 and injected onto a silica column eluted with
a CH2Cl2:methanol gradient from 100:0 to 97:3 over 30 min
(linear) followed by isocratic elution at 97:3. The column
s was monitored by TLC and W (235, 244 and 320nm) monitoring
with spectral scanning (190-375nm) at 0.5min intervals (AB1
1000S diode array detector). Fractions were collected
throughout.
0 VM 54159 and VM 55594 eluted as adjacent peaks (43-44 mins
and 45-49 mins respectively) being distinguished on the
basis of W ~max of the spectral scans (VM 54159 ~max 226nm,
VM 55594 ~max 244nm).
S VMS5595 eluted as a discrete peak at 59-65 mins (~max 244nm)
obtained after evaporation of sol~ent.
The yields of essentially pure material from the appropriate
fractions were: VM 54159, 10.7mg; VM 55599, 16.3mg;
20 VM 55S95, 9.6mg.
Characterisation Data
VM 54159
2s
W~ max 225nm (in MeOH)
Mass (FAB, NOBA/Na) [MH+]: 478
~13C (CDCl3) 182.S, 172.2, 146.0, 139.0, 135.2,
132.3, 125.4, 120.S, 117.3, llS.1, 79.8, 67.7,
6S.2, 62.9, 60.1, 53.4, 52.1, 46.3, 40.3, 37.S,
30.2, 30.0, 29.8, 27.7, 25.S, 24.0, 20.8, 13.1
SUBSTITUTE SHEET
.. ` -
. W091/09961 2 0 7 ~ `9 8 2 PCT/GB90/01997
~,--,.
- -21-
~M 55594
W ~max 245nm (in MeOH)
Mass (FAB, NOBA/Na) ~MH+] = 462
~13C (CDC13) 185.7, 172.4, 153.0, 137.5, 131.2,
125.5, 121.6, 116.3, 109.5, 105.6, 76.4, 67.7,
65.2, 62.8, 60.0, 53.3, 52.4, 46.2, 40.3, 37.3,
30.3, 27.9, 27.8, 27.7, 25.5, 23.9, 20.6, 13.2.
10 VM 55595
W ~max 245nm (in MeOH)
Mass (FAB, NOBA/Na) [MH+] 448
~13C (CDCl3) 184.9, 174.3, 153.0, 137.5, 131.2,
125.6, 121.8, 116.3, 109.4, 105.4, 76.3, 68.2,
62.5, 61.8, 60.2, 53.4, 52.8, 46.2, 40.0, 39.9,
30.3, 28.0, 27.8, 27.6, 24.1, 20.6, 13.1
EXAMPLE 2
EXTRACTION AND PURIFICATION OF VM 55596
Culture IMI 332995, grown for 24 days on 10L Czapek Dox agar
(Example 1), was harvested into acetone and extracted into
2s chloroform, as described in Example 1, to produce 5.1 g of
extract.
This extract was divided into 2 aliquots, I
and II(containing approximately 4 and 1 g respectively).
30 Each was chromatographed by preparative reverse phase HPLC
(Dynamax - 60A C-18 41.4 x 250 mm, Rainin Instrument Co.,
USA), eluted with methanol:water (75:25) at 15 ml~min.
15 ml fractions were collected throughout and an aliquot of
each was examined by TLC (Merck glass-backed silica gel
SUBSTI~UTE SHEET
WO9i/09961 2 0 7 ~ 9 8 2 Pcr/GBgo/o1997f~
-2~
60 plates, 20 x 20 cm, developed with 90:l0
chloroform:methanol, visualised by spraying with Ehrlichs
reagent, and heating). Fractions containing the major
Ehrlichs positive component in each run (corresponding to
5 fraction A in Example l) were combined (fractions 9-24 in
run I and 7-14 in run II) and concentrated to dryness under
vacuum to yield l.93 g.
Thls material was divided into 2 equal aliquots and each was
0 chromatographed by Silica HPLC (Dynamax-60A Si 21.4 x
250 mm, Rainin Instrument Co. USA) with CH2Cl2:methanol
gradient elution (8 ml/min). In one run, the gradient was
as follows:
Time CH2Cl2:MeOH
0-20 minutes l00:0 to 97:3 linear gradient
20-40 minutes 97:3 isocratic
2040-80 minutes 97:3 to 90:l0 linear gradient
80 minutes onwards 90:l0 isocratic
_
8 ml fractions were collected throughout.
2s
The column was monitored by W absorbance (235 and 244 nm,
l,000S Diode Array Detector, Applied Biosystems Limited,
Warrington, UK), and each fraction examined by TLC as
before. Paraherquamide-rich material eluted in
30 fractions 34-37. Fractions ll0-llS, corresponding to a peak
in W absorbance and an Ehrlichs positive spot on TLC
~f 0.19) were pooled and combined with the corresponding
fractions~by TLC1 from the other column run (which was
identical except that the 97:3 isocratic elution was
35 continued for 60 minutes). After evaporating the solvent
l9 mg of material was recovered and was purified by a final
silica column (Dynamax - 60A, l0 x 250 mm) eluted with a
linear gradient of CH2Cl3:methanol, 98:2 to 88:12 over
SUBSTITUTE SHEET
.
:'. ' ~ ' ' , ' ~
. ~ .
W091/0~6l ~ 0 7~1~ 8 2 PCT/~B90/01997
23-
90 minutes at 4 ml/min. Monitoring was by ~J absorbance at
235 and 244 nm with spectral scanning every 0.5 minutes ~AB1
1000S Diode Array Detector). 4 ml fractions were collected
throughout. A major peak of W absorbance occurred between
s fractions 28-47. W spectra indicated that the early
fractions were impure. Therefore fractions 37-47 were
pooled and dried under vacuum. This yielded 10.0 mg of
VM 55596.
0 Characterisation_of VM 55596
W max. 225 nm ~in MeOH) Mass (FAB, Thioglycerol) [MH]+ =
510 with a major ion at 494 =[M~-16]+.
15 ~13~ (CDCl3) 182.2, 167.7, 146.4, 13B.9, 135.4, 132.4,
124.1, 120.5, 117.7, 115.2, 83.8, 7g.9, 77.9, 76.2, 69.4,
62.9, 62.8, 51.0, 46.8, 39.1, 36.0, 29.9, 29.7, 27.1, 23.3,
22.2, 21.1, 14.5
20 Further evidence that VM 55596 was the N-oxide derivative of
paraherquamide came from the demonstration that
de-oxygenation with triphenylphosphene ~TPP) produced
paraherquamide ~0.5 mg VM 55596 in 0.5 ml dioxane was mixed
with 3 mg TPP in 0.5 ml dioxane. After heating to 60C for
25 one hour, the product was examined by TLC ~as above) and
HPLC ~Microsorb C-18 4.2 x 250 mm column, 90:10
methanol:water at 1 ml/min, Waters 990 Diode Array Detector)
and found to be indistinguishable from paraherquamide).
30 Example 3
A conidlal suspension of Penicillium sP. IMI 332995 was
prepared in phosphate buffered saline containing 10~
glycerol and 1% Tween 80 (PBSGT) from a well grown culture
35 on a potato dextrose agar ~Oxoid Ltd. Basingstoke, UK)
plate.
SUBSTITUTE SHEET
WO91/0~61 2 ~ 9 g2 PCT/GB90/01997
-24-
2-3mls of suspension were spread onto Czapek Dox agar plates
and incubated at 28C.
Czapek Dox Agar g~L
s
Sucrose (BDH Analar) 30
NaNO3 3
MgSO4.7H2o 0-5
KCl o 5
FeS04 . 7H20 0 . 01
K2HPO4 1.0
Technical Agar (Oxoid) 18g
Deionised water 1000ml
Two batches were grown on 10 and 20 L of agar for 27 and 26
days respectively. Each batch was separately extracted into
acetone by macerating the agar with 1-2 volumes of solvent
and removing the solids by centrifugation and filtration
25 after which the agar was twice more extracted with acetone.
The acetone was evaporated under vacuum yielding an aqueous
residue which was then extracted, four times, each with 2.S
volumes of chloroform. The organic phase was separated and
30 evaporated under vacuum to leave 3.64 g and 11.15 g of
yellow oil from the two batches, which were then combined.
SUBSTITUTE SHEET
.
- . .
.
`, -: ` ,:
2971282
;,. WO 91tO9961 - . PCr/GB90/01997
i -,, . _ ~
-25-
The combined extract was suspended in approximately 200 ml
of methanol which caused a white precipitate to form. This
was filtered off and rinsed with 50 mi of methanol. The
precipitated material was resuspended in approximately
5 200 ml of warm (30CJ methanol and again filtered. The
precipitate was discarded and the methanolic solutions
pooled and the solvent evaporated to leave 9.44 q of
material.
10 A column 10 cm diameter x 25 cm was packed with HP20-SS
~Mitsubishi Chemicals, Japan), well washed with acetone and
equilibrated in 7:3 methanol/water.
The sample was suspended in 50 ml of 7:3 methanol/water and
15 applied to the top of the column which was then eluted as
follows, 500 ml fractions were collected throughout.
6.5 L 70:30methanol:water, Fractions 1-13
3.5 L 75:25'' '' '' 14-20
3.5 L 80:20'' '' '' 21-27
6.5 L 85:15'' '' '' 28-40
8.0 L 90:10'' '' '' 41-56
A 10 ml aliquot of each fraction was dried under vacuum and
2s redissolved in 0.5 ml of chloroform and examined by TLC
~10 x 10 cm HPTLC plates, Silica gel 60 F 254 (E. Merck)
developed with 9:1 CHCl3~MeOH, spots were visualized under
W llght (254 nm) and by Spraying with Ehrlichs reagent
(1% 4-Dimethylaminobenzaldehyde in 1:3 concentrated
30 HCl:methanol) followed by heating).
On the basis of this data the individual fractions showing
similar spots on TLC were pooled. Thus fractions 40-46 were
combined and designated as group 'G'
SUBSTITUTE SHEET
WO91/~51 2 0 7 1 g 8 2 PCTtGB90/01997 ~
--26-
When pooled and the solvent evaporated under vacuum group
'G' yielded 0.297 g of material. TLC showed this to contain
a number of Ehrlichs positive components. This was further
purified by normal phase ~PLC using a Dynamax 60A Si column
5 41.4 x 250 mm (Rainin Instrument Co. USA) eluted with
CH2Cl2/Methanol, initially as a linear gradient ~100~ CH2Cl2
to 96:4 CH2CL2/MeOH over 60 mins) and then isocratically at
96:4 (CH2CL2/MeOH), all at 20 ml/min. 18 ml fractions were
collected from the start of the isocratic phase. Each
0 fraction was examined by TLC (20 x 20 cm TLC plates, Silica
gel 60 F254 (E. Merck) developed and visualized as above).
Tubes 29-31 were found to show a single Ehrlichs positive
spot of Rf 0.7 so were combined. After evaporation of the
solvent this sample ~designated 'Gl') weighed 15.1 mg.
Sample 'G1' was further chromatographed by normal phase HPLC
using a Dynaxax 60A Si column 10 x 250 mm eluted with
hexane/propan-2-ol pumped at 4 ml/min. The solvent
composition was as follows: 0-18 min Isocratic, 98:2
20 hexane/propan-2-ol; 18-88 min linear gradient 98:2 to
90:10hexane/propan-2-ol; over 60 mins but held at 95:5 for
10 mins as the main peak eluted. 4 ml fractions were
collected throughout. The column was monitored by W
detection at 226 and 205 nm, and by ~LC. The main peak
25 detected by W eluted in fractions 42-53. TLC indlcated
that each of these fractions contained the same, pure,
material corresponding to the Ehrlichs positive spot seen in
sample 'G1'. Thus fractions 42-53 were pooled and the
solvent evaporated to yield 5.0 mg of product (VM 55599).
The product VM 55599 was characterized as follows:-
W max (MeOH) 226, 282 nm
Mass spectrometry (El and FAB) indicated a molecular weight
35 of 349.13C (CDCl3) 174.8, 141.2, 136.5, 126.8, 121.3, 119.0, 117.7,
110.6, 104.0, 66.3, 58.8, 55.6, 53.5, 46.7, 34.2, 33.0,
SUr~ . i I I.'TE SHEET
.. . .. . ..
:
W091/0~6l ~q ~ 1~ 8 2 PCT/GB90/01997
30.5, 30.2, 30.~, 26.8, 23.9, 17.4.
Example 4
5 Synthesis of Paraherquamide N-_xide (VM 55596)
To a solution of paraherquamide (2S mg) in chloroform ~1.5
ml) at room temperature was added a solution of m-
chloroperbenzoic acid (10.4 mg 85%) in chloroform (1 ml) and
0 the mixture stirred for 2h. The solvent was evaporated and
the product purified by preparative TLC (silica eluted with
5~ methanol in dichloromethane).
SUBSTITUTE SHEET
` . - . ` , .
,. , - . , .
~ ` - ` :" `' :
