This invention concerns herbicidal compounds, some of which are new,
a process for their preparation, and compositions containing them.
We have found that the 3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3]diazepin-8-ol
derivatives of the formula:
and sugar conjugates thereof, are herbicidally active.
As is conventional, the dotted line in the formula indicates that
the bond between the two carbon atoms may be either a single bond or a double bond.
Those compounds where there is a single bond between the relevant carbon atoms
are novel, and in one aspect this invention provides them per se.
The sugar conjugates of the compounds of formula I as that term is
used herein are those where one or more of the -OH groups or the -NH group in the
molecule is replaced with a group -OR or -NR respectively, where R is a sugar
moiety, especially a hexose moiety, and particularly a glucose moiety. It is preferred
that just a single sugar group is present in the sugar conjugates. It is also
preferred that this is where the group -OR replaces the -OH in the -CH&sub2;OH
group in formula I.
The term 'compounds of formula I' is used hereinafter to include
The compounds of formula I are herbicidally-active against a range
of broad-leaved and grassy weeds. They may thus be of use as herbicides, either
as total herbicides, or possibly as selective herbicides, particularly in the
control of a range of weeds in cereals or other crops, eg wheat, rice, barley,
maize, soya beans, oilseed rape, cotton or sugar beet.
In another aspect, the invention provides the use of one or more
compounds of formula I as a herbicide, and also a herbicidal composition which
comprises one or more compounds of formula I in association with a suitable carrier
and/or surface active agent.
The compounds of formula I each have a number of optical centres
and thus a number of optical isomers. This invention is not limited in any way
to specific optical isomers, but as is usual in such compounds, some optical isomers
may well exhibit greater activity in certain respects than others.
Preferred compounds of the invention include 3-[2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol
(hereinafter referred to as 'Compound A'), 3-[2,3-dihydroxy-4-(β-D-glucosyloxymethyl)cyclopentyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol
(ie a glucose conjugate of Compound A), and 3-[4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]-diazepin-8-ol
(hereinafter referred to as 'Compound B'). The preferred optical isomers of these
compounds are believed to be 8R-3-[(1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol
(hereinafter referred to as 'Compound A1'), the corresponding 4-(β-D-glucosyloxymethyl)
derivative thereof (hereinafter referred to as 'Compound A2'), 8R-3-[(1R,2S,3R,4S)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol
(hereinafter referred to as 'Compound A3'), and 8R-3-[(1R,4R,5S)-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]-diazepin-8-ol
(hereinafter referred to as 'Compound B1').
The compositions usually contain from 0.01 to 99% by weight of the
present compounds, and are normally produced initially as concentrates containing
from 0.5 to 99%, preferably from 0.5 to 85%, and especially from 10 to 50% by
weight thereof. Such concentrates are diluted if necessary before application to
the locus to be treated such that the active ingredients comprise from 0.01 to
5% by weight of the formulation applied.
The carrier may be water, in which case an organic solvent may also
be present, though this is not usually employed.
The carrier may alternatively be a water immiscible organic solvent
in which the compounds are dissolved or suspended. An emulsifiable concentrate
containing a water immiscible solvent may be formed with a surface active agent
so that the concentrate acts as a self-emulsifiable oil on admixture with water.
The carrier may alternatively be a water-miscible organic solvent
eg 2-methoxyethanol, methanol, propylene glycol, diethylene glycol, diethylene
glycol monoethyl ether, methylformamide or dimethylformamide.
The carrier may alternatively be a solid, which may be finely divided
or granular. Examples of suitable solids are limestone, clays, sand, mica, chalk,
attapulgite, diatomite, perlite, sepiolite, silicas, silicates, lignosulphonates
and solid fertilizers. The carrier can be of natural or synthetic origin or can
be modified natural material.
Wettable powders soluble or dispersible in water may be formed by
admixing the compound in particulate form with a particulate carrier or spraying
molten compound on to the particulate carrier, admixing a wetting agent and a
dispersing agent and finely grinding the whole powder mixture.
An aerosol composition may be formed by admixing the present compounds
with a propellant, eg a polyhalogenated alkane such as dichlorofluoromethane, and
suitably also with a solvent.
The term 'surface active agent' is used in the broad sense to include
materials variously called emulsifying agents, dispersing agents and wetting agents.
Such agents are well known in the art.
The surface active agents used may comprise anionic surface active
agents, for example mono- or di-esters of phosphoric acid with a fatty alcohol
ethoxylate, or salts of such esters, fatty alcohol sulphates such as sodium dodecyl
sulphate, ethoxylated fatty alcohol sulphates, ethoxylated alkylphenol sulphates,
lignin sulphates, petroleum sulphonates, alkylaryl sulphonates such as alkyl-benzene
sulphonates or lower alkylnaphthalene sulphonates, salts of sulphonated naphthaleneformaldehyde
condensates, salts of sulphonated phenolformaldehyde condensates, or more complex
sulphonates such as the amide sulphonates, eg the sulphonated condensation product
of oleic acid and N-methyl taurine or the dialkyl sulphosuccinates eg the sodium
sulphonate of dioctyl succinate.
The surface active agents may also comprise non-ionic agents, for
example condensation products or fatty acid esters, fatty alcohols, fatty acid
amides or alkyl-substituted phenols with ethylene oxide, fatty esters of polyhydric
alcohol ethers eg sorbitan fatty acid esters, condensation products of such esters
with ethylene oxide eg polyoxyethylene sorbitan fatty acid esters, block copolymers
of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetramethyl-5-decyn-4,7-diol,
or ethoxylated acetylenic glycols.
The surface active agents may also comprise cationic agents, for
example alkyl- and/or aryl-substituted quaternary ammonium compounds such as cetyl
trimethylammonium bromide, or ethoxylated tertiary fatty amines.
Preferred surface active agents include ethoxylated fatty alcohol
sulphates, lignin sulphonates, alkyl-aryl sulphonates, salts of sulphonated naphthaleneformaldehyde
condensates, salts of sulphonated phenolformaldehyde condensates, sodium oleoyl
N-methyltauride, dialkyl sulphosuccinates, alkyl phenol ethoxylates, and fatty
The present active compounds may be admixed with inorganic compounds,
eg (NH&sub4;)&sub2;SO&sub4;, an oil, or another pesticide, eg a herbicide, fungicide
or insecticide, or a plant growth regulator, particularly another herbicide. Suitable
further herbicides include trietazine, linuron, MCPA, dichlorprop, isoxaben, diflufenican,
metolachlor, fluometuron, oxyfluorfen, fomesafen, bentazone, prometryne, norflurazon,
chlomazone, EPTC, imazaquin, and especially glyphosate, metsulfuron methyl, sulfometuron,
isoproturon, methabenzthiazuron, trifluralin, ioxynil, bromoxynil, benazolin,
mecoprop, fluroxypyr, alachlor, acifluorfen, lactofen, metribuzin, pendimethalin,
ethofumesate, benfuresate, and phenmedipham.
The present compounds may be applied to plants, the soil, land or
aquatic areas, and particularly to a locus at which a crop is growing. The compounds
are particularly active post-emergence. They may be applied at rates of from 0.02
to 2 kg/ha, especially from 0.1 to 1 kg/ha.
The compounds of the invention may be prepared by the processes discussed
Thus according to a further aspect of the invention we provide a
process for the production of a compound of formula I which comprises the step
of cultivating a microorganism capable of producing the compound of formula I,
and if desired isolating said compound therefrom.
Microorganisms capable of producing the compounds of the invention
may readily be identified by using a small scale test and analysing a test sample
obtained from fermentation of the microorganism by high performance liquid chromatography.
In particular the microorganism to be used in the process according
to the invention is a previously undescribed strain of microorganism deposited
on 13th April 1989 in the permanent culture collection of the National Collection
of Industrial and Marine Bacteria, Torry Research Station, 135 Abbey Road, Aberdeen,
Scotland under accession no NCIMB 40131. NCIMB 40131 is an actinomycete characterised
as Amycolatopsis spp (Lechevier et al Int J Syst. Bacteriol 36, 29-37 (1986))
on the basis of the following taxonomic markers:
- wall chemotype IV, containing meso-diaminopimelic acid, arabinose and
galactose as diagnostic sugars (whole cell sugar pattern A);
- no mycolic acids present
- phospholipid pattern II, thus containing phosopatidyl ethanolamine as diagnostic
The generic status of the organisms was also confirmed using actinophage
specific for Amycolatopsis
species (Prauser W2, W4, W7, W11).
The characteristics of NCIMB 40131 are given in Example 6 below.
The invention provides in a further aspect the microorganism NCIMB
40131 per se and mutants thereof.
Mutants of the above strain may arise spontaneously or may be produced
by a variety of methods including those outlined in Techniques for the Development
of Microorganisms by H I Adler in "Radiation and Radioisotopes for Industrial Microorganisms",
Proceedings of the Symposium, Vienna 1973, p241, International Atomic Energy Authority.
Such methods include ionising radiation, chemical methods eg treatment with N-methyl-N&min;-nitro-N-nitrosoguanidine
(NTG), heat, genetic techniques, such as recombination, transduction, transformation,
lysogenisation and lysogenic conversion, and selective techniques for spontaneous
According to a still further aspect of the invention we provide the
genetic material of NCIMB 40131 and mutants thereof that participates in the synthesis
of the compounds of formula I. Such material may be obtained using conventional
genetic engineering techniques including those outlined by D A Hopwood in "Cloning
Genes for Antibiotic Biosynthesis in Streptomyces Spp : Production of a Hybrid
Antibiotic" p 409-413 in Microbiology 1985, Ed L Lieve, American Society of Microbiology,
Washington DC 1985. Such techniques may be used in a similar manner to that described
previously for cloning antibiotic biosynthetic genes, including the biosynthetic
genes for actinorhodin (Malpartida, F and Hopwood, D A 1984, Nature 309, p 462-464),
erythromycin (Stanzak, R et al, 1986, Biotechnology, 4, p 229-232) and an important
enzyme involved in penicillin and cephalosporin production in Acremonium chrysogenum
S M et al, 1985) Nature, 318, p 191-194). The genetic material so obtained may
be used, for example, for strain improvement, for production of biosynthetic enzymes
for in vitro applications, or for generating novel herbicides by introduction
of such material into organisms other than NCIMB 40131.
The production of the compounds of the invention by fermentation
of a suitable organism may be effected by conventional means, ie by culturing the
organism in the presence of assimilable sources of carbon, nitrogen and mineral
Assimilable sources of carbon, nitrogen and minerals may be provided
by either simple or complex nutrients. Sources of carbon will generally include
glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose,
carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils.
Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation
Sources of nitrogen will generally include soya bean meal, corn steep
liquors, distillers solubles, yeast extracts, cottonseed meal, peptones, ground
nut meal, malt extract, molasses, casein, amino acid mixtures, ammonia (gas or
solution), ammonium salts or nitrates. Urea and other amides may also be used.
Sources of nitrogen will generally comprise from 0.1 to 10% by weight of the fermentation
Nutrient mineral salts which may be incorporated into the culture
medium include the generally used salts capable of yielding sodium, potassium,
ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium,
calcium, copper, molybdenum, borate, phosphate, sulphate, chloride and carbonate
Cultivation of the organism will generally be effected at a temperature
of from 20 to 40°C preferably from 25 to 35°C, especially around 28°C, and will
desirably take place with aeration and agitation eg by shaking or stirring. The
medium may initially be inoculated with a small quantity of a suspension of the
sporulated microorganism but in order to avoid a growth lag a vegetative inoculum
of the organism may be prepared by inoculating a small quantity of the culture
medium with the spore form of the organism, and the vegetative inoculum obtained
may be transferred to the fermentation medium, or more preferably to one or more
seed stages where further growth takes place before transfer to the principal
fermentation medium. The fermentation will generally be carried out in the pH range
of 5.5 to 8.5, preferably 5.5 to 7.5. It may be necessary to add a base or an
acid to the fermentation medium to keep the pH within the desired range. Suitable
bases which may be added include alkali metal hydroxides such as aqueous sodium
hydroxide. Suitable acids include mineral acids such as hydrochloric or sulphuric
The fermentation may be carried out for a period of 2-10 days, eg
about 5 days. An antifoam may be present to control excessive foaming and added
at intervals as required.
The compounds according to the invention are predominantly contained
in the fermentation broth. The mycelia may conveniently be removed from the broth
by filtration or centrifugation.
For use as agricultural herbicides it may not be necessary to separate
the compounds from the fermentation medium in which they are produced.
Where it is desired to separate the compounds of the invention from
the whole fermentation this may be carried out by conventional isolation and separation
techniques. The isolation techniques may also be applied to the fermentation broth
either before or after clarification. It will be appreciated that the choice of
isolation techniques may be varied widely.
The compounds of the invention may be isolated and separated by a
variety of fractionation techniques, for example adsorption-elution, precipitation,
fractional crystallisation, solvent extraction and liquid-liquid partition which
may be combined in various ways.
Chromatography on a suitable support in the form of a bed or, more
preferably, packed into a column, has been found to be particularly suitable for
isolating and separating the compounds of the invention.
Purification and/or separation of the compounds of the invention
from the fermentation broth may be conveniently effected by chromatography (including
high performance liquid chromatography) on a suitable support such as silica;
a non-functional macroreticular adsorption resin for example cross-linked styrene
divinyl benzene polymer resins such as Amberlite XAD-2, XAD-4, XAD-16 or XAD-1180
resins (Rohm & Haas Ltd) or Kastell S112 (Montedison); a substituted styrene-divinyl
benzene polymer, for example a halogenated (eg brominated) styrene divinyl benzene
polymer such as Diaion SP207 (Mitsubishi); an organic solvent-compatible cross-linked
dextran such as Sephadex LH20 (Pharmacia UK Ltd), or on reverse phase supports
such as hydrocarbon linked silica eg C&sub1;&sub8;-linked silica.
Suitable solvents/eluents for the chromatographic purification/separation
of the compounds of the invention will, of course, depend on the nature of the
column support. When using column supports such as Amberlite XAD-2 and C&sub1;&sub8;-linked
silica we have found alcohols such as methanol to be particularly suitable, especially
when combined with a polar solvent such as water.
The presence of the compounds of the invention during the extraction/isolation
procedures may be monitored by conventional techniques such as high performance
liquid chromatography or UV spectroscopy or by utilising the properties of the
compounds described hereinafter.
Where a compound of the invention is obtained in the form of a solution
in an organic solvent, for example after purification by chromatography, the solvent
may be removed by conventional procedures, eg by evaporation, to yield the compound
in a solid or crystalline form. If desired, the compounds of the invention may
be further purified by the aforementioned chromatographic techniques and/or recrystallisation.
By a suitable combination of the foregoing procedures the compounds
of the invention have been isolated as solids. It will be appreciated that the
order in which the above purification steps are carried out and the choice of
those which are used may be varied widely.
The invention is illustrated by the following Examples.
Spores of actinomycete NCIMB 40131 were inoculated onto agar slants
made up of the following ingredients.
g/lYeast extract (Oxoid L21)0.5 Malt extract (Oxoid L39)30.0 Mycological peptone (Oxoid L40)5.0 Agar No 3 (Oxoid L13)15.0
Distilled water to 1 litre
pH approximately 5.4
and were incubated at 28°C for 10 days.
The mature slant was then covered with 6 ml of a 10% glycerol solution
and scraped with a sterile tool to loosen the spores and mycelium. 0.4 ml aliquots
of the resulting spore suspension were transferred to sterile polypropylene straws
which were then heat-sealed and stored in liquid nitrogen vapour until required.
The contents of a single straw were used to inoculate two 50 ml aliquots
of a seed medium (A) as follows:
Distilled water to 1 litre
pH adjusted to 6.5 with 5N NaOH
The flasks were grown, with shaking, at 28°C for 5 days.
The cells and culture fluid were separated by centrifugation.
50 ml of seed medium (A) were placed in each of eight 250 ml Erlenmeyer
flasks, and the pH was adjusted from an initial value of 6.7 to 7.0 with aqueous
sodium hydroxide. After autoclaving, the pH was 7.3. The flasks were each inoculated
with 0.2 ml of the spore suspension taken from straws and prepared according to
the method described in Example 1 above.
The flasks were incubated at 28°C for 3 days on a shaker rotating
at 250 rpm with a 50 mm diameter orbital motion.
The contents of the eight flasks were pooled and used to inoculate
a 20-litre fermenter vessel containing 12 litres of medium (B), the pH being adjusted
to 6.5 with 5N NaOH before autoclaving.
The inoculated medium was agitated with conventional impellers rotating
at 800 rpm. Aeration of the culture was achieved by dispensing sterile air through
the medium at a rate of 0.5 volume of air per volume of culture medium per minute.
Temperature was controlled at 28°C and excessive foaming overcome
by the addition of silicone antifoam. The culture was harvested after 5 days growth
and processed as described in Example 1.
100g of Amberlite XAD-2 resin (Rohm and Haas Limited) was added to
2 litres of aqueous supernatant from the above fermentation, and the mixture was
stirred for 20 hours at room temperature. The resin was filtered off and then
washed with 250ml portions of 10% aqueous methanol, fractions of approximately
250ml being collected. 5µl Aliquots of each fraction were applied to the growing
tips of a number of Polygonum lapathifolium plants, which were then grown
on in a controlled environment room for 7 days, after which time the plants were
assessed for herbicidal effect. Fractions exhibiting herbicidal activity were
combined and loaded onto a column of C-18-linked silica (5cm x 2cm) packed in water.
The column was then washed with 98:2 water:methanol, fractions of approximately
250ml being collected. Fractions exhibiting herbicidal activity in a repetition
of the above test were combined, evaporated and subjected to preparative hplc on
Dynamax C-18 (250mm x 21mm, Rainin Instruments) using a gradient system of water
and methanol. Material eluting from the column was monitored by UV spectroscopy
at 280nm. The biologically-active fractions were analysed by hplc on Dynamax C-18
(250mm x 4.6mm, Rainin Instruments) using water as the eluting phase at a flow
rate of 1ml/min, and those fractions containing similar components (retention
times of compounds B1, A3, A1 and A2 being approximately 10 minutes, 17 minutes,
21 minutes and 23 minutes respectively) were combined, evaporated and subjected
to further preparative hplc on a Zorbax TMS (250mm x 10mm) column, monitoring
the column eluant at 280nm. Evaporation of the biologically-active fractions yielded
compounds A and B and the glucose conjugates of each (where the glucose moiety
replaces the hydrogen atom of the -OH group in the group -CH&sub2;OH) as solids.
Their structures were confirmed by UV, nmr and mass spectroscopy,
the characteristic peaks of the main compounds being as follows: Compound B1 (retention time approx 10 mins)
UV (methanol): 279nm
Mass Spectrum (Thermospray): 281 (M+H&spplus;)
NMR (300MHz, D&sub2;O): δ7.25 (1H,s), 7.05 (1H,s), 5.80 (1H,d),
5.22 (1H,d), 5.05 (1H,d), 4.55 (1H,d),
4.20 (2H,s), 4.10 (1H,m), 3.35 (1H,d),
3.20 (1H,d). Compound A1 (retention time approx 21 mins)
UV (methanol): 282nm
Mass Spectrum (Thermospray): 283 (M+H&spplus;)
NMR (300MHz, D&sub2;O): δ7.50 (1H,s), 7.05 (1H,s), 5.02 (1H,d),
4.50 (1H,m), 4.20 (1H,dd),
3.90 (1H,dd), 3.55 (2H,d),
3.40 (1H,dd), 3.30 (1H,d),
2.30 (1H,m), 2.10 (1H,m), 1.48 (1H,m) Compound A2 (retention time approx 23 mins)
UV (methanol): 281nm
Mass Spectrum (Fast atom bombardment, thioglycerol):
NMR (300MHz, D&sub2;O): δ7.40 (1H,s), 7.05 (1H,s), 5.05 (1H,d),
4.55 (1H,m), 4.41 (1H,d),
4.20 (1H,dd), 4.05 (1H,m),
3.75 (1H,d), 3.60 (1H,dd),
3.50 (2H,m), 3.2-3.4 (6H,m),
2.30 (2H,m), 1.45 (1H,m). Compound A3 (retention time approx 17 mins)
UV (methanol): 280nm
Mass Spectrum (Thermospray): 283 (M+H&spplus;)
NMR (300MHz, D&sub2;O): δ7.50 (1H,s), 7.00 (1H,s), 5.00 (1H,d),
4.61 (1H,m), 4.25 (1H,dd),
4.10 (1H,dd), 3.50 (2H,m),
3.35 (1H,dd), 3.25 (1H,dd),
2.45 (1H,m), 2.00 (1H,m), 1.75 (1H,m)
The procedures of Examples 1 and 2 were repeated, but replacing medium
(B) with the following medium:
g/lGlycerol23.0 L-proline11.5 MOPS (3-(N-morpholino)propane sulphonic acid21.0 EDTA0.25 NaCl0.5 MgSO&sub4;.7H&sub2;O0.49 CaCl&sub2;.2H&sub2;O0.029 K&sub2;HPO&sub4;0.52 Trace salts0.5 ml pH6.5 The trace salts contained: H&sub2;SO&sub4; (1M)10 ml ZnSO&sub4;.4H&sub2;O8.6g MnSO&sub4;.4H&sub2;O2.23g H&sub3;BO&sub3;0.62g CuSO&sub4;.5H&sub2;O1.25g Na&sub2;MoO&sub4;.2H&sub2;O0.48g CoCl&sub2;.6H&sub2;O0.48g FeSO&sub4;.7H&sub2;O18.0g KI0.83g
Distilled water to 1 litre.
The ingredients were dissolved in the distilled water in the order shown.
The crops and weeds listed in the table below were grown in sterilised
loam in controlled environment rooms at 25°C (non-temperate species) or 21°C (temperate
species). The plants were sprayed at an early growth stage. The compounds produced
as in Example 2 and as listed below were each formulated in 25% methanol in distilled
water, with 0.5% Tween 20 and 0.05% Pluronic L61 as wetters. The volume of the
spray application was 2000 litres per hectare, giving an application rate of active
ingredient of between 0.2 and 0.5 kg/ha. Treated plants were either returned to
the controlled environment rooms or placed in glasshouses and assessed after 2
weeks, on a scale where 0 indicates no effect, 1 indicates slight damage, 2 indicates
moderate damage, 3 indicates good control, and 4 indicates complete kill. In the
following table, the compounds A1, A2, A3 and B1 are as identified hereinbefore.
1. A herbicidal composition which comprises from 0.01 to 99% by weight of one
or more 3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3]diazepin-8-ol derivatives of the
where the dotted line indicates that the bond between the two carbon atoms may
be either a single or a double bond, or sugar conjugates thereof, in association
with a suitable carrier and/or surface active agent.
2. The use of one or more compounds of formula I as defined in claim 1, or
sugar conjugates thereof, as a herbicide.