WO2022103280A1 - Methanogen inhibitors - Google Patents

Methanogen inhibitors Download PDF

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Publication number
WO2022103280A1
WO2022103280A1 PCT/NZ2021/050199 NZ2021050199W WO2022103280A1 WO 2022103280 A1 WO2022103280 A1 WO 2022103280A1 NZ 2021050199 W NZ2021050199 W NZ 2021050199W WO 2022103280 A1 WO2022103280 A1 WO 2022103280A1
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alkyl
compound
formula
mmol
nmr
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PCT/NZ2021/050199
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French (fr)
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David Rennison
Ian Kenneth Boddy
Margaret Brimble
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Pastoral Greenhouse Gas Research Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/111—Aromatic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants

Definitions

  • the present invention relates to a new class of methanogen inhibitors for ruminants, in particular new pyromellitic diimide derivatives for ruminants.
  • the invention also extends to the use of such derivatives in ruminants to reduce methane production in the rumen and/or to enhance productivity in the ruminant.
  • Methane is produced as a natural consequence of digestion of feed by bacteria, fungi and protozoa in ruminant animals. This fermentation leads to the production of volatile fatty acids and peptides for the host. Fermentation also produces copious quantities of CO2 and H2 which are used by methanogenic archaea within the rumen to produce methane, which is ultimately released from the rumen, mostly through eructation.
  • methane forming methanogens are members of the Archaea, and are quite different in a number of features compared to bacteria, fungi and protozoa. Methanogens have a number of unusual and archaeal-specific features, including cell wall structures, lipids, cofactors, and amino acid synthesis pathways, as well as their signature energy metabolism that is linked to methane production. They represent only about 1-4% of the rumen microbial community. It is known that, due to the unique metabolic pathways of methanogens and their low numbers, methanogen- specific inhibitors can be developed that do not adversely affect the fermentation of feed.
  • methane loss represents a loss of energy for the ruminant. It has been previously recognised that if one could inhibit or reduce the release of methane from ruminants that the impact of methane on the environment and atmosphere would be reduced and productivity gains might be achieved in the ruminants.
  • the present disclosure provides a compound of Formula I Formula I wherein R 1 is selected from -H, -alkyl, -alkyl-ONO2, or -ONO2; wherein X is different to R 1 and is selected from -H, -alkyl- ONO2, -ONO2 or a substituent of Formula II, Formula II wherein each n is independently selected from 0, 1, 2, 3, 4, 5 or 6 and when n is 0, 1, 2, 3, 4, 5, 6 any adjacent carbons are optionally unsaturated to form a double or triple bond, wherein R 2 is selected from -H, -alkyl, - alkyl-ONO2, -aryl, -heteroaryl or -heterocyclyl, - CO2R 3 ; wherein -R 1 and -R 2 together form an optionally substituted aliphatic ring which is optionally saturated; wherein -Z is selected from -H, -alkyl, -aryl, -heteroaryl,
  • Z is a substituent of Formula III Formula III wherein m is 0, 1, 2, 3 or 4, and when m is 0, 1, 2, 3 or 4, any adjacent carbons are optionally unsaturated to form a double or triple bond;
  • R 4 when present is selected from -H, -alkyl, alkyl-OH or -alkyl-ONO2;
  • the compound of Formula I includes at least one -ONO2 substituent.
  • the compound of Formula I Ri is -H, X is -ONO2 and Z is a compound of Formula III.
  • R 4 is selected from -H or - alkyl-ONO2 and R 5 is selected from -H, -O-alkyl, -ONO2, -CO2-alkyl or aryl.
  • Z is selected from -H, -alkyl, -aryl, -heteroaryl, -heterocyclyl, -CO2R 3 ,
  • p is selected from 0, or 1 ;
  • R 4 when present is selected from -H, -alkyl-OH, or -alkyl-ONO 2 ;
  • R 1 is -H
  • X is -ONO 2
  • Z is -H, -alkyl or a substituent of Formula III.
  • n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2.
  • R 1 is -H
  • X is Formula II
  • n is 0, 1 or 2
  • Z is a substituent of Formula III and m is 0, 1 or 2.
  • R 1 is -H
  • X is Formula II
  • R 2 is - CH 2 ONO 2
  • n is 0, 1 or 2
  • Z is a substituent of Formula III and m is 0, 1 or 2.
  • the compound of Formula I is or a salt thereof.
  • the compound of Formula I is or a salt thereof.
  • the compound of Formula I is or a salt thereof. In one embodiment the compound of Formula I is or a salt thereof.
  • the compound of Formula I is or a salt thereof. In one embodiment the compound of Formula I is
  • the compound of Formula I is or a salt thereof. In one embodiment the compound of Formula I is o r a salt thereof.
  • the compound of Formula I is or a salt thereof.
  • the present invention provides the compound of Formula I as defined above for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.
  • a compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants.
  • a compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 10%.
  • a compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 15%.
  • the compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 20%.
  • the present invention further provides a method for reducing the production of methane emanating from a ruminant and/or for improving ruminant animal performance, comprising administering orally to the ruminant an effective amount of at least one compound of Formula I or a salt thereof to the ruminant.
  • Oral administration is to be understood as a route involving drenching, addition to feed, water source or pasture; or manual administration of a bolus or a capsule.
  • the effective amount of at least one compound of Formula I or a salt thereof is administered at least once-daily to the ruminant.
  • the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 10% per day. In one embodiment the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 15% per day.
  • the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 20% per day.
  • composition for oral administration comprising at least one compound of Formula I or a salt thereof for reducing the production of methane emanating from a ruminant, and the composition further including at least one agriculturally and orally acceptable excipient.
  • composition is adapted for use as a feed additive.
  • composition is adapted for use as a water additive.
  • composition is adapted for use as a ruminant lick.
  • composition is adapted for use as an oral drench.
  • composition is adapted for use as a rumen bolus or capsule.
  • composition is adapted to reduce the production of methane emanating from the ruminant by at least 10% per day.
  • composition is adapted to reduce the production of methane emanating from the ruminant by at least 15% per day.
  • composition is adapted to reduce the production of methane emanating from the ruminant by at least 20% per day.
  • the excipient includes one or more minerals and/or one or more vitamins.
  • the excipient includes one or more vitamins selected from vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3, vitamin B 12, biotin and choline, vitamin B l, vitamin B2, vitamin B6, niacin, folic acid or the like.
  • the excipient includes one or more minerals selected from calcium, phosphorus, sodium, manganese, zinc, iron, copper, chlorine, sulphur, magnesium, iodine, selenium, and cobalt or the like.
  • composition further includes sunflower oil, electrolytes such as ammonium chloride, calcium carbonates, starch, proteins or the like.
  • the compounds of the present invention have potential for use in a ruminant to reduce the formation of methane without affecting microbial fermentation in a way that would be detrimental to the ruminant.
  • ruminant as used herein is a mammal that is able to acquire nutrients from plantbased food by fermenting it in a specialized foregut (the rumen) prior to digestion, principally through microbial activity.
  • Representative examples of ruminants and other foregut fermenters include cattle, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, and antelope.
  • an effective amount refers to an amount of at least one compound of Formula I or a salt thereof that either reduces the production of methane emanating from the ruminant or improves ruminant performance.
  • ruminant performance refers to improving the productivity of the ruminant, such as increased weight gain, milk yield or quality, wool growth or quality, surviving offspring per parturition, or the like.
  • halo refers to a halogen atom selected from Cl, Br, or F.
  • aliphatic ring is a cyclic organic compound containing carbon atoms joined to form a non-aromatic ring.
  • the aliphatic ring is optionally substituted with alkyl, hydroxy, halo, -ONO2 or the like.
  • alkyl refers to a straight or branched chain, saturated hydrocarbon having from 1 to 6 carbon atoms.
  • Representative C 1 -C 6 alkyl groups include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (eg n- pentyl), hexyl and the like.
  • the C 1 -C 6 alkyl group may be further substituted with, hydroxy or aryl.
  • aryl refers to “aryl”, unless specifically limited, denotes a C6-12 aryl group, suitably a C 6 - 10 aryl group, more suitably a C6-8 aryl group.
  • Aryl groups will contain at least one aromatic ring (e.g. one, two or three rings). Examples of a typical aryl group with one aromatic ring is phenyl. An example of a typical aryl group with two aromatic rings is naphthyl.
  • heteroaryl refers to ”, unless specifically limited, denotes an aryl residue, wherein one or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or 3) ring atoms are replaced by heteroatoms selected from N, S and O, or else a 5-membered aromatic ring containing one or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or 3) ring atoms selected from N, S and O.
  • Exemplary monocyclic heteroaryl groups having one heteroatom include: five membered rings (e.g., pyrrole, furan, thiophene); and six membered rings (e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl).
  • five membered rings e.g., pyrrole, furan, thiophene
  • six membered rings e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl.
  • Exemplary monocyclic heteroaryl groups having two heteroatoms include: five membered rings (e.g., pyrazole, oxazole, isoxazole, thiazole, isothiazole, imidazole, such as imidazol-l-yl, imidazol-2-yl imidazol-4-yl); six membered rings (e.g., pyridazine, pyrimidine, pyrazine).
  • five membered rings e.g., pyrazole, oxazole, isoxazole, thiazole, isothiazole, imidazole, such as imidazol-l-yl, imidazol-2-yl imidazol-4-yl
  • six membered rings e.g., pyridazine, pyrimidine, pyrazine.
  • Exemplary monocyclic heteroaryl groups having three heteroatoms include: 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4- oxadizole, 1,2,5-oxadizole and 1,3,4 oxadiazole, 1,2,3-thiadazole, 1,2,4-thiadazole, 1,2,5- thiadazole and 1,3,4-thiadazole.
  • Exemplary monocyclic heteroaryl groups having four heteroatoms include tetrazole and optionally substituted tetrazole.
  • Exemplary bicyclic heteroaryl groups include: indole (e.g., indol-6-yl), benzofuran, benzothiophene, quinoline, isoquinoline, indazole, benzimidazole, benzthiazole, quinazoline and purine.
  • the heteroaryl groups may be substituted with alkyl or alkyl-aryl (such as benzyl) or aryl (such as phenyl).
  • heterocyclyl as used herein includes a “carbocycle” as defined herein, wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with a heteroatom (e.g. O, N, or S).
  • heterocycle or “heterocyclyl” includes saturated rings (i.e., heterocycloalkyls), and partially unsaturated rings.
  • Substituted heterocyclyls include, for example, heterocyclic rings substituted with any of the substituents disclosed herein including carbonyl groups.
  • heterocycles also include by way of example and not limitation sugars such as glucose, sucrose, fructose, galactose, lactose, maltose, ribose and deoxyribose.
  • Some of the crystalline forms of the compounds may exist in more than one polymorphic form and as such all forms are intended to be included in the present disclosure.
  • some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this disclosure.
  • the compounds, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.
  • the present disclosure further includes within its scope prodrugs of the compounds described herein.
  • prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired active compound.
  • the term “administering” shall encompass the treatment of the various disorders described with prodrug versions of one or more of the claimed compounds, but which converts to the above specified compound in vivo after administration to the subject.
  • composition is intended to encompass a product comprising a claimed compound(s) in a therapeutically effective amount, as well as any product which results, directly or indirectly, from combinations of the claimed compounds.
  • a salt thereof is a salt of an acid or a basic nitrogen atom.
  • Illustrative salts include, but are not limited, to sodium salt, potassium salt, lithium salt, calcium salt, ammonium salt, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, and the like.
  • the resultant crude solid was taken up in 1,2 -dichloroethane (25 mL) and was added urea (1.5 mg, 0.03 mmol), 70% nitric acid (0.07 mL, 1.11 mmol) and acetic anhydride (0.20 mL, 2.12 mmol), then heated under reflux for 4 h. After cooling to room temperature, the reaction mixture was diluted with dichloromethane (20 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo.
  • the resultant crude solid was taken up in 1,2 -dichloroethane (10 mL) and was added urea (0.8 mg, 0.01 mmol), 70% nitric acid (0.10 mL, 1.31 mmol) and acetic anhydride (0.25 mL, 2.63 mmol) in 1,2-dichloroethane (10 mL), then heated under reflux for 2 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo, diluted with ethyl acetate (20 mL) and washed with water (20 mL).
  • 3.4-f]isoindol-2(1H)- yl)acetamide A stirred mixture of pyromellitic imide anhydride (1.00 g, 4.61 mmol) and 2-amino- N,N- dimethylacetamide (0.56 g, 5.53 mmol) in DMF (20 mL) was heated at 100 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture.
  • the compounds of the present invention were initially screened for activity using an in vitro fully automated incubation system for the measurement of total gas production and gas composition (methane and hydrogen).
  • the system used is described in detail in the paper by Muetzel et al. (2014; Animal Feed Science and Technology 196, 1-11).
  • the system in this specification is referred to as the rumen in vitro system or RIV.
  • the RIV system provides a relatively simple tool for determining the rate and production of methane and also whether the rate and production of methane is inhibited or reduced by a particular compound or feed.
  • Rumen in vitro assays use rumen fluid from donor animals (typically either sheep or cattle) which is typically combined with a buffer and then incubated in sealed fermentation vessels at 39 °C for either 24 or 48 h. These assays systems have been well-characterised and used by the scientific community over the last two decades (Rymer et al. 2005). The system that has been used here to characterise the effects of inhibitors on methane production and gas production, and the formation of H 2 , was described by Muetzel et al. (2014).
  • Rumen in vitro assay systems reflect what can occur in the rumen in vivo, but only short-term, due to their short incubation times (typically not exceeding 48 h) which is limited by its buffering capacity and the fact that it is a closed system.
  • Stock solutions of inhibitors were re-suspended in dimethylformamide (DMF) at concentrations 1000-fold higher than the highest used for the assays. Any further dilutions were also prepared using DMF.
  • the total amount of inhibitor solution added to the rumen fluidbuffer mixture (60 mL) was 60 pl.
  • Incubations used 60 ml of medium containing 12 ml of filtered rumen fluid and 48 ml of buffer in serum bottles for 24-48 h, essentially as described by Muetzel et al. (2014). Two fistulated cattle were used as donor animals for rumen fluid and treatments were incubated in duplicate bottles. Sets of duplicate incubation vials that contained rye grass (with rumen fluid -buffer mixture), and rye grass with 30 pM bromoethanesulfonate (BES) were also incubated as negative and positive controls, respectively.
  • BES bromoethanesulfonate
  • One experiment consists of 2 replicates of an inhibitor at any one concentration run at the same time, and a run can contain up to 32 bottle, including positive (30 pM BES) and negative (no inhibitor added) controls.
  • Compound 12 was tested using the 60 mL rumen in vitro assay system at 100 pM and 2 pM final concentrations and compared to a positive control with 30 pM BES. At 30 pM and 2 pM, inhibition levels were approximately 86.4 % and 16.6 % at 18 h, respectively.
  • mice testing used two groups with three mice (female Swiss mice, non-pregnant), all at approximately 8-9 weeks of age. On the day of testing, food was withdrawn 1-2 hours prior to dosing. Following fasting, the animals were weighed and the test substances administered. Access to food was resumed 1-2 hours after administration of the compounds. The compounds were administered in a single dose by oral gavage in corn oil (maximum of 0.3 ml per dose) at the selected dose and the time recorded. The mice were observed individually after dosing at least once during the first 30 minutes, periodically over the first 24 hours, with special attention given to the first 4 hours, and then daily thereafter for a total of 14 days.
  • mice were weighed every seven days (0, 7 and 14 days). All observations were systematically recorded for each individual animal. These observations included (if present): tremors, convulsions, salivation, diarrhoea, lethargy, sleep, coma, and changes in fur, skin, eyes, mucus membranes, respiration, motor activity and behaviour. No adverse effects were noted for Compound 12 at 300 mg/kg.
  • the animals were adapted to GP diet for 14 days prior to entering the methane measurement chambers.
  • Table 1 Data for 2-day dosing with Compound 12. P2 values are the average of the first and second days of dosing.

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Abstract

The present invention relates to a new class of methanogen inhibitors for ruminants, in particular new pyromellitic diimide derivatives for ruminants. The invention also extends to the use of such derivatives in ruminants to reduce methane production in the rumen and/or to enhance productivity in the ruminant.

Description

METHANOGEN INHIBITORS
FIELD OF INVENTION
The present invention relates to a new class of methanogen inhibitors for ruminants, in particular new pyromellitic diimide derivatives for ruminants. The invention also extends to the use of such derivatives in ruminants to reduce methane production in the rumen and/or to enhance productivity in the ruminant.
BACKGROUND OF INVENTION
Methane is produced as a natural consequence of digestion of feed by bacteria, fungi and protozoa in ruminant animals. This fermentation leads to the production of volatile fatty acids and peptides for the host. Fermentation also produces copious quantities of CO2 and H2 which are used by methanogenic archaea within the rumen to produce methane, which is ultimately released from the rumen, mostly through eructation.
The methane forming methanogens are members of the Archaea, and are quite different in a number of features compared to bacteria, fungi and protozoa. Methanogens have a number of unusual and archaeal-specific features, including cell wall structures, lipids, cofactors, and amino acid synthesis pathways, as well as their signature energy metabolism that is linked to methane production. They represent only about 1-4% of the rumen microbial community. It is known that, due to the unique metabolic pathways of methanogens and their low numbers, methanogen- specific inhibitors can be developed that do not adversely affect the fermentation of feed.
It has been recognised that the release of such methane is deleterious for two reasons. One is that methane is a greenhouse gas and the other is that the methane loss represents a loss of energy for the ruminant. It has been previously recognised that if one could inhibit or reduce the release of methane from ruminants that the impact of methane on the environment and atmosphere would be reduced and productivity gains might be achieved in the ruminants.
It is therefore an object of the present invention to overcome the above mentioned difficulties or to at least provide the public with a useful alternative.
SUMMARY OF INVENTION
In one aspect the present disclosure provides a compound of Formula I
Figure imgf000003_0001
Formula I wherein R1 is selected from -H, -alkyl, -alkyl-ONO2, or -ONO2; wherein X is different to R1 and is selected from -H, -alkyl- ONO2, -ONO2 or a substituent of Formula II, Formula II
Figure imgf000003_0002
wherein each n is independently selected from 0, 1, 2, 3, 4, 5 or 6 and when n is 0, 1, 2, 3, 4, 5, 6 any adjacent carbons are optionally unsaturated to form a double or triple bond, wherein R2 is selected from -H, -alkyl, - alkyl-ONO2, -aryl, -heteroaryl or -heterocyclyl, - CO2R3; wherein -R1 and -R2 together form an optionally substituted aliphatic ring which is optionally saturated; wherein -Z is selected from -H, -alkyl, -aryl, -heteroaryl, -heterocyclyl, -CO2R3, -C(=O)R3, - alkyl-OH, -(alkyl-OH)-alkyl-OH, -alkyl-(alkyl-OH)2, -alkyl-(alkyl-OH)3,
-(alkyl-O-alkyl)k, -(alkyl-O)k-L; -alkyl-(C=O)O-(alkyl-O)k-L; -alkyl-SH, -alkyl-S-alkyl, - alkyl-(S=O)-alkyl, -alkyl-(SO2)-alkyl; -alkyl-(SO2)-OH; -alkyl-(SO2)-NH2; -alkyl-(SO2)- NHalkyl, -alkyl-(SO2)-N(alkyl)2; -alkyl-(SO2)-NH(alkyl-O)k-L; -alkyl-(C=O)-NH2; -alkyl- (C=O)-NH(alkyl); -alkyl-(C=O)-N(alkyl)2; -alkyl-(C=O)-NH(alkyl-O)k-L; -alkyl heterocyclyl, wherein k isl, 2, 3, 4, 5 or 6; and wherein L is -H, -alkyl; wherein R3 is selected from -H, -alkyl, -NHalkyl, -N(alkyl)2 or -aryl with the proviso that R3 is only selected from -H, -alkyl, or aryl for -CO2R3; or
Z is a substituent of Formula III
Figure imgf000004_0001
Formula III wherein m is 0, 1, 2, 3 or 4, and when m is 0, 1, 2, 3 or 4, any adjacent carbons are optionally unsaturated to form a double or triple bond; Y is selected from -CH2-, -O-, -C(=O)- -S-, -S(=O)- , -SO2-, -NH- or N(alkyl)- p is selected from 0, or 1 ;
R4 when present is selected from -H, -alkyl, alkyl-OH or -alkyl-ONO2;
R5 when present is selected from -H, -OH, -O-alkyl, -NH2, -alkyl, -NH(alkyl), -N(alkyl)2, - ONO2, -alkyl-ONO2, -aryl, -heteroaryl, -heterocyclyl, -C(=O)R3 or - CO2R3; or or a salt thereof.
In one embodiment the compound of Formula I includes at least one -ONO2 substituent.
In one embodiment the compound of Formula I, Ri is -H, X is -ONO2 and Z is a compound of Formula III. In one embodiment in the compound of Formula III, R4 is selected from -H or - alkyl-ONO2 and R5 is selected from -H, -O-alkyl, -ONO2, -CO2-alkyl or aryl.
In one embodiment, Z is selected from -H, -alkyl, -aryl, -heteroaryl, -heterocyclyl, -CO2R3,
-C(=O)R3; -(alkyl-O)k-L, wherein k is 1, 2, 3, 4, 5 or 6; and wherein L is -H, -alkyl; or wherein R3 is selected from H, alkyl or aryl; or Z is a substituent of Formula III
Formula III
Figure imgf000004_0002
wherein m is 0, 1, 2, 3 or 4, and when m is 0, 1, 2, 3 or 4, any adjacent carbons are optionally unsaturated to form a double or triple bond;
Y is selected from -CH2-, -O-, -(C=O) -S-, -S(=O)-,
-SO2-, -N(alkyl)-; p is selected from 0, or 1 ;
R4 when present is selected from -H, -alkyl-OH, or -alkyl-ONO2;
R5 when present is selected from -H, -OH, -NH2, -alkyl, -NH(alkyl), -N(alkyl)2, -ONO2, -- alkyl-ONO2, -aryl, -heteroaryl, -heterocyclyl, -C(=O)R3 or - CO2R3.
In one embodiment Z is selected from -H, -alkyl, -aryl, -heteroaryl, -heterocyclyl, - CO2R3, -C(=O)R3 -(CH2CH2O)k-L, wherein R3 can be selected from H, alkyl or aryl, wherein k is 1, 2, 3, 4, 5 or 6; and wherein L is -H, -alkyl.
In one embodiment, R1 is -H, X is -ONO2; Z is -H, -alkyl or a substituent of Formula III.
In one embodiment of the compound of Formula I, n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2.
In one embodiment of the compound of Formula I, R1 is -H, X is Formula II, n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2.
In one embodiment of the compound of Formula I, R1 is -H, X is Formula II, R2 is - CH2ONO2, n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2.
In one embodiment the compound of Formula I or a salt thereof is selected from one or more of the following:
Figure imgf000005_0001
Figure imgf000006_0001
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0005
In one embodiment the compound of Formula I is
Figure imgf000009_0001
In one embodiment the compound of Formula I is or a salt thereof.
Figure imgf000009_0002
In one embodiment the compound of Formula I is or a salt thereof.
Figure imgf000009_0003
In one embodiment the compound of Formula I is
Figure imgf000009_0004
or a salt thereof. In one embodiment the compound of Formula I is or a salt thereof.
Figure imgf000010_0001
In one embodiment the compound of Formula I is or a salt thereof.
Figure imgf000010_0002
In one embodiment the compound of Formula I is
Figure imgf000010_0003
In one embodiment the compound of Formula I is
Figure imgf000010_0004
or a salt thereof. In one embodiment the compound of Formula I is or a salt thereof.
Figure imgf000010_0005
In one embodiment the compound of Formula I is
Figure imgf000011_0001
or a salt thereof.
In another aspect, the present invention provides the compound of Formula I as defined above for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.
In one embodiment a compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants.
In one embodiment a compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 10%.
In another embodiment of a compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 15%.
In yet another embodiment the compound of Formula I as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 20%.
In one aspect the present invention further provides a method for reducing the production of methane emanating from a ruminant and/or for improving ruminant animal performance, comprising administering orally to the ruminant an effective amount of at least one compound of Formula I or a salt thereof to the ruminant. Oral administration is to be understood as a route involving drenching, addition to feed, water source or pasture; or manual administration of a bolus or a capsule.
In one embodiment the effective amount of at least one compound of Formula I or a salt thereof is administered at least once-daily to the ruminant.
In one embodiment the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 10% per day. In one embodiment the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 15% per day.
In one embodiment the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 20% per day.
In a further aspect of the present invention there is provided a composition for oral administration comprising at least one compound of Formula I or a salt thereof for reducing the production of methane emanating from a ruminant, and the composition further including at least one agriculturally and orally acceptable excipient.
In one embodiment the composition is adapted for use as a feed additive.
In one embodiment the composition is adapted for use as a water additive.
In another embodiment the composition is adapted for use as a ruminant lick.
In one embodiment the composition is adapted for use as an oral drench.
In another embodiment the composition is adapted for use as a rumen bolus or capsule.
In one embodiment the composition is adapted to reduce the production of methane emanating from the ruminant by at least 10% per day.
In one embodiment the composition is adapted to reduce the production of methane emanating from the ruminant by at least 15% per day.
In one embodiment the composition is adapted to reduce the production of methane emanating from the ruminant by at least 20% per day.
In one embodiment the excipient includes one or more minerals and/or one or more vitamins.
In one embodiment the excipient includes one or more vitamins selected from vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3, vitamin B 12, biotin and choline, vitamin B l, vitamin B2, vitamin B6, niacin, folic acid or the like. In one embodiment the excipient includes one or more minerals selected from calcium, phosphorus, sodium, manganese, zinc, iron, copper, chlorine, sulphur, magnesium, iodine, selenium, and cobalt or the like.
In one embodiment the composition further includes sunflower oil, electrolytes such as ammonium chloride, calcium carbonates, starch, proteins or the like.
The compounds of the present invention have potential for use in a ruminant to reduce the formation of methane without affecting microbial fermentation in a way that would be detrimental to the ruminant.
DETAILED DESCRIPTION OF INVENTION
DEFINTIONS
The term “ruminant” as used herein is a mammal that is able to acquire nutrients from plantbased food by fermenting it in a specialized foregut (the rumen) prior to digestion, principally through microbial activity. Representative examples of ruminants and other foregut fermenters include cattle, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, and antelope.
The term “effective amount” as used herein refers to an amount of at least one compound of Formula I or a salt thereof that either reduces the production of methane emanating from the ruminant or improves ruminant performance.
The term “ruminant performance” as used herein refers to improving the productivity of the ruminant, such as increased weight gain, milk yield or quality, wool growth or quality, surviving offspring per parturition, or the like.
The term “halo” as used herein refers to a halogen atom selected from Cl, Br, or F.
The term “aliphatic ring” is a cyclic organic compound containing carbon atoms joined to form a non-aromatic ring. The aliphatic ring is optionally substituted with alkyl, hydroxy, halo, -ONO2 or the like.
The term “alkyl” as used herein refers to a straight or branched chain, saturated hydrocarbon having from 1 to 6 carbon atoms. Representative C1-C6 alkyl groups include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl (eg n- pentyl), hexyl and the like. The C1-C6 alkyl group may be further substituted with, hydroxy or aryl.
The term “aryl” as used herein refers to “aryl”, unless specifically limited, denotes a C6-12 aryl group, suitably a C6-10 aryl group, more suitably a C6-8 aryl group. Aryl groups will contain at least one aromatic ring (e.g. one, two or three rings). Examples of a typical aryl group with one aromatic ring is phenyl. An example of a typical aryl group with two aromatic rings is naphthyl.
The term “heteroaryl” as used herein refers to ”, unless specifically limited, denotes an aryl residue, wherein one or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or 3) ring atoms are replaced by heteroatoms selected from N, S and O, or else a 5-membered aromatic ring containing one or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or 3) ring atoms selected from N, S and O. Exemplary monocyclic heteroaryl groups having one heteroatom include: five membered rings (e.g., pyrrole, furan, thiophene); and six membered rings (e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl). Exemplary monocyclic heteroaryl groups having two heteroatoms include: five membered rings (e.g., pyrazole, oxazole, isoxazole, thiazole, isothiazole, imidazole, such as imidazol-l-yl, imidazol-2-yl imidazol-4-yl); six membered rings (e.g., pyridazine, pyrimidine, pyrazine). Exemplary monocyclic heteroaryl groups having three heteroatoms include: 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4- oxadizole, 1,2,5-oxadizole and 1,3,4 oxadiazole, 1,2,3-thiadazole, 1,2,4-thiadazole, 1,2,5- thiadazole and 1,3,4-thiadazole. Exemplary monocyclic heteroaryl groups having four heteroatoms include tetrazole and optionally substituted tetrazole. Exemplary bicyclic heteroaryl groups include: indole (e.g., indol-6-yl), benzofuran, benzothiophene, quinoline, isoquinoline, indazole, benzimidazole, benzthiazole, quinazoline and purine. The heteroaryl groups may be substituted with alkyl or alkyl-aryl (such as benzyl) or aryl (such as phenyl).
The term “heterocyclyl” as used herein includes a “carbocycle” as defined herein, wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with a heteroatom (e.g. O, N, or S). The terms “heterocycle” or “heterocyclyl” includes saturated rings (i.e., heterocycloalkyls), and partially unsaturated rings. Substituted heterocyclyls include, for example, heterocyclic rings substituted with any of the substituents disclosed herein including carbonyl groups. Examples of heterocycles also include by way of example and not limitation sugars such as glucose, sucrose, fructose, galactose, lactose, maltose, ribose and deoxyribose.
All possible stereoisomers of the claimed compounds are included in the present disclosure. Where a compound described herein has at least one chiral center, it may accordingly exist as enantiomers. Where a compound possesses two or more chiral centers it may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present disclosure.
Some of the crystalline forms of the compounds may exist in more than one polymorphic form and as such all forms are intended to be included in the present disclosure. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this disclosure. The compounds, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.
The present disclosure further includes within its scope prodrugs of the compounds described herein. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired active compound. Thus, in these cases, the methods of treatment of the present invention, the term “administering” shall encompass the treatment of the various disorders described with prodrug versions of one or more of the claimed compounds, but which converts to the above specified compound in vivo after administration to the subject.
As used herein, the term “composition” is intended to encompass a product comprising a claimed compound(s) in a therapeutically effective amount, as well as any product which results, directly or indirectly, from combinations of the claimed compounds.
The term “or a salt thereof,” as used herein, is a salt of an acid or a basic nitrogen atom. Illustrative salts include, but are not limited, to sodium salt, potassium salt, lithium salt, calcium salt, ammonium salt, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, and the like.
Methods of making the Compounds of Formula I
The following compounds were prepared as follows: (l,3,5,7-Tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate
(Compound 1)
Figure imgf000016_0001
To a stirred mixture of pyromellitic diimide (1.00 g, 4.63 mmol) in DMF (100 mL) was added paraformaldehyde (0.11 g, 3.70 mmol), and the reaction mixture heated at 80 °C for 18 h, before evaporating to dryness to afford crude 2-(hydroxymethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone, which was used directly in the next step. The resultant crude solid was taken up in 1,2-dichloroethane (10 mL) and was added urea (1.8 mg, 0.03 mmol), 70% nitric acid (0.10 mL, 1.52 mmol) and acetic anhydride (0.29 mL, 3.05 mmol) in 1,2- dichloroethane (10 mL), then heated under reflux for 4 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. Purification by flash column chromatography (CH2C12/acetone, 19:1) afforded Compound 1 as a colourless solid (0.16 g, 0.53 mmol, 11% over two steps). 1H NMR (500 MHz, d6-DMSO) δ 6.03 (2H, s), 8.24 (2H, s), 11.89 (1H, s); 13C NMR (125 MHz, d6-DMSO) δ 67.6 (CH2), 117.8 (CH), 136.6 (C), 138.3 (C), 164.8 (C), 167.5 (C).
(6-Methyl-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate (Compound 2)
Figure imgf000016_0002
A stirred mixture of 2-(hydroxymethyl)-6-methylpyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)- tetraone (0.41 g, 1.58 mmol), urea (5.0 mg, 0.83 mmol), 70% nitric acid (0.25 mL, 3.95 mmol) and acetic anhydride (0.75 mL, 7.95 mmol) in 1,2 -dichloroethane (20 mL) was heated under reflux for 3 h. After cooling to room temperature, the solids were collected by filtration, washed with CH2CI2 (20 mL) and water (20 mL), then dried in vacuo to afford Compound 2 as a colourless solid (0.23 g, 0.75 mmol, 48%). 1H NMR (400 MHz, d6-DMSO) δ 3.10 (3H, s), 6.04 (2H, s), 8.28 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 24.2 (CH3), 67.6 (CH2), 117.6 (CH), 136.6 (C), 137.5 (C), 164.7 (C), 166.3 (C). (6-Allyl-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate
(Compound 3)
Figure imgf000017_0001
To a stirred mixture of 2-allyl-6-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.16 g, 0.85 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was then stirred at room temperature for 18 h, before evaporating to dryness to afford crude 2-allyl-6-(hydroxymethyl)pyrrolo[3,4- /]isoindolc-L3,5,7(2H,6H)-tctraonc, which was used directly in the next step. The resultant crude solid was taken up in 1,2 -dichloroethane (25 mL) and was added urea (1.5 mg, 0.03 mmol), 70% nitric acid (0.07 mL, 1.11 mmol) and acetic anhydride (0.20 mL, 2.12 mmol), then heated under reflux for 4 h. After cooling to room temperature, the reaction mixture was diluted with dichloromethane (20 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded Compound 3 as a colourless solid (44.0 mg, 0.13 mmol, 32% over two steps). 1H NMR (400 MHz, CDC13) 5 4.37 (2H, dt, J= 5.9 and 1.4 Hz), 5.25-5.33 (2H, m), 5.85-5.95 (1H, m), 6.09 (2H, s), 8.39 (2H, s); 13C NMR (100 MHz, CDCI3) 5 41.0 (CH2), 66.6 (CH2), 119.2 (CH), 119.5 (CH2), 130.7 (CH), 136.6 (C), 138.1 (C), 164.2 (C), 165.4 (C).
(l,3,5,7-Tetraoxo-6-phenyl-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate (Compound 5)
Figure imgf000017_0002
A stirred mixture of 2-(hydroxymethyl)-6-phenylpyrrolo[3,4-f|isoindole-l, 3, 5, 7(2H,6H)- tetraone (0.20 g, 0.62 mmol), urea (2.0 mg, 0.03 mmol), 70% nitric acid (0.10 mL, 1.58 mmol) and acetic anhydride (0.29 mL, 3.07 mmol) in 1,2 -dichloroethane (40 mL) was heated under reflux for 18 h. After cooling to room temperature, the solids were collected by filtration, washed with CH2C12 (20 mL) and water (20 mL), then dried in vacuo to afford Compound 5 as a pale-yellow solid (0.11 g, 0.29 mmol, 48%). 1H NMR (400 MHz, d6-DMSO) δ 6.06 (2H, s), 7.47-7.59 (5H, m), 8.39 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 67.6 (CH2), 118.1 (CH), 127.2 (CH), 128.4 (CH), 128.9 (CH), 131.5 (C), 136.8 (C), 137.3 (C), 164.7 (C), 165.3 (C).
(l,3,5,7-Tetraoxo-6-(pyridin-2-yl)-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)methyl nitrate (Compound 6)
Figure imgf000018_0001
A stirred mixture of 2-(hydroxymethyl)-6-(pyridin-2-yl)pyrrolo[3,4-f|isoindole- 1,3,5,7(2H,6H)-tetraone (0.25 g, 0.77 mmol), urea (1.2 mg, 0.02 mmol), 70% nitric acid (0.15 mL, 1.93 mmol) and acetic anhydride (0.37 mL, 3.87 mmol) in 1,2-dichloroethane (20 mL) was heated under reflux for 3 h. Additional 70% nitric acid (0.15 mL, 1.93 mmol) and acetic anhydride (0.37 mL, 3.87 mmol) was added, and the reaction mixture was then heated under reflux for a further 2 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo and water (20 mL) was added. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford Compound 6 as a paleyellow solid (0.15 g, 0.40 mmol, 53%). 1H NMR (500 MHz, d6-DMSO) δ 6.06 (2H, s), 7.57 (1H, ddd, 7 = 7.5, 4.6 and 0.8 Hz), 7.61 (1H, d, 7 = 7.9 Hz), 8.09 (lH, td, 7 = 7.7 and 1.9 Hz), 8.42 (2H, s), 8.68 (1H, dd, 7 = 4.8 and 1.1 Hz); 13C NMR (125 MHz, d6-DMSO) δ 67.6 (CH2), 118.5 (CH), 122.9 (CH), 124.5 (CH), 137.0 (C), 137.1 (C), 138.9 (CH), 145.5 (C), 149.5 (CH), 164.7 (C), 164.8 (C).
2-(Acetoxymethyl)-6-(6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7- tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Compound 7)
Figure imgf000018_0002
A stirred mixture of 2-(acetoxymethyl)-6-(6-(hydroxymethyl)-l, 3,5, 7-tetraoxo-3, 5,6,7 - tetrahydropyrrolo[3,4-f|isoindol-2(1H)-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (0.38 g, 0.67 mmol), urea (2.3 mg, 0.04 mmol), 70% nitric acid (0.11 mL, 1.74 mmol) and acetic anhydride (0.32 mL, 3.39 mmol) in 1,2-dichloroethane (25 mL) was heated under reflux for 3 h. After cooling to room temperature, the reaction mixture was diluted with dichloromethane (30 mL) and washed with water (30 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded Compound 7 as pale-yellow solid (0.24 g, 0.39 mmol, 65%). 1H NMR (400 MHz, CDC13) 5 1.88 (3H, s), 2.04 (3H, s), 2.07 (3H, s), 2.08 (3H, s), 3.88 (1H, dt, J = 9.9 and 3.6 Hz), 4.21-4.22 (2H, m), 5.25-5.38 (2H, m), 5.47 (1H, d, J = 9.4 Hz), 5.96 (1H, t, J = 9.3 Hz), 6.09 (2H, s), 8.43 (2H, s). 13C NMR (100 MHz, CDCI3) 5 20.5 (CH3), 20.7 (CH3), 20.8 (CH3), 61.8 (CH2), 66.6 (CH2), 67.9 (CH), 68.5 (CH), 73.4 (CH), 75.0 (CH), 78.4 (CH), 120.2 (CH), 137.0 (C), 163.9 (C), 169.5 (C), 169.6 (C), 170.2 (C), 170.7 (C).
(6-Acetyl-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate Compound 9
Figure imgf000019_0001
To a stirred mixture of crude 2-acetyl-6-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4- f]isoindole-1 ,3,5,7(2H,6H)-tctraonc (0.50 g) in dichloromethane (12 mL) was added trifluoroacetic acid (3 mL). The reaction mixture was then stirred at room temperature for 18 h, before evaporating to dryness to afford crude 2-acetyl-6-(hydroxymethyl)pyrrolo[3,4- f]isoindole-1 ,3,5,7(2H,6H)-tctraonc, which was used directly in the next step. The resultant crude solid was taken up in 1,2 -dichloroethane (15 mL) and was added urea (1.6 mg, 0.03 mmol), 70% nitric acid (0.20 mL, 2.60 mmol) and acetic anhydride (0.49 mL, 5.20 mmol) in 1,2-dichloroethane (15 mL), then heated under reflux for 2 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 7:3) afforded Compound 9 as a colourless solid (0.09 g, 0.26 mmol, 20% over two steps). 1H NMR (400 MHz, d6-DMSO) δ 2.58 (3H, s), 6.04 (CH2), 8.40 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 26.5 (CH3), 67.6 (CH2), 118.8 (CH), 136.5 (C), 137.4 (C), 163.9 (C), 164.5 (C), 168.3 (C). Ethyl 6-((nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindole-
2(1H) -carboxylate Compound 10
Figure imgf000020_0001
A stirred mixture of ethyl 6-(hydroxymethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.30 g, 0.94 mmol), urea (1.4 mg, 0.02 mmol), 70% nitric acid (0.19 mL, 2.36 mmol) and acetic anhydride (0.46 mL, 4.71 mmol) in 1,2-dichloroethane (15 mL) was heated under reflux for 2 h. Additional 70% nitric acid (0.19 mL, 2.36 mmol) and acetic anhydride (0.46 mL, 4.71 mmol) was added, and the reaction mixture was then heated under reflux for a further 2 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo, then diluted with ethyl acetate (30 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo to afford Compound 10 as a colourless solid (0.26 g, 0.72 mmol, 76%). 1H NMR (400 MHz, d6- DMSO) δ 1.34 (3H, t, J = 7.1 Hz), 4.41 (2H, q, J = 7.1 Hz), 6.04 (2H, s), 8.37 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 13.9 (CH3), 63.5 (CH2), 67.6 (CH2), 118.7 (CH), 136.6 (C), 137.2 (C), 147.6 (C), 162.2 (C), 164.5 (C).
3-(6-((Nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)propane-l,2-diyl dinitrate (Compound 12)
Figure imgf000020_0002
A stirred mixture of 2-(2,3-dihydroxypropyl)-6-(hydroxymethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.23 g, 0.72 mmol), urea (3.2 mg, 0.05 mmol), 70% nitric acid (0.42 mL, 5.39 mmol) and acetic anhydride (1.02 mL, 10.8 mmol) in 1,2-dichloroethane (20 mL) was heated under reflux for 2 h. Additional 70% nitric acid (0.21 mL, 2.70 mmol) and acetic anhydride (0.51 mL, 5.39 mmol) was added, and the reaction mixture was then heated under reflux for a further 2 h. After cooling to room temperature, the precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford Compound 12 as a colourless solid (0.23 g, 0.56 mmol, 78%). 1H NMR (400 MHz, d6-DMSO) δ 4.07- 4.09 (2H, m), 4.90 (1H, dd, J = 12.5 and 6.2 Hz), 4.98 (1H, dd, J = 12.7 and 3.0 Hz), 5.62 (1H, brs), 6.04 (2H, s), 8.34 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 36.4 (CH2), 67.6 (CH2), 70.1 (CH2), 77.3 (CH), 118.0 (CH), 136.7 (C), 137.3 (C), 164.7 (C), 166.1 (C).
2-(6-((Nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)propane-l,3-diyl dinitrate (Compound 13)
Figure imgf000021_0001
A stirred mixture of 2-(l,3-dihydroxypropan-2-yl)-6-(hydroxymethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.30 g, 0.94 mmol), urea (8.4 mg, 0.14 mmol), 70% nitric acid (0.45 mL, 7.03 mmol) and acetic anhydride (1.33 mL, 14.1 mmol) in 1,2-dichloroethane (15 mL) was heated under reflux for 3 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo, then diluted with ethyl acetate (20 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 7:3) afforded Compound 13 as a colourless foam (0.16 g, 0.35 mmol, 38%). 1H NMR (500 MHz, d6-DMSO) 5 4.82-4.89 (1H, m), 4.98-5.07 (4H, m), 6.05 (2H, s), 8.37 (2H, s); 13C NMR (125 MHz, d6- DMSO) δ 45.9 (CH), 67.6 (CH2), 69.3 (CH2), 118.3 (CH), 136.7 (C), 137.0 (C), 164.6 (C), 165.6 (C).
Methyl 3-(nitrooxy)-2-(6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7- tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)propanoate (Compound 14)
Figure imgf000021_0002
A stirred mixture of methyl 3-hydroxy-2-(6-(hydroxymethyl)-l, 3,5, 7-tetraoxo-3, 5,6,7 - tetrahydropyrrolo[3,4-f|isoindol-2(1H)-yl)propanoate (0.28 g, 0.80 mmol), urea (4.8 mg, 0.08 mmol), 70% nitric acid (0.26 mL, 4.02 mmol) and acetic anhydride (0.76 mL, 8.04 mmol) in 1,2-dichloroethane (10 mL) was heated under reflux for 2 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. Purification by flash column chromatography (CH2Cl2/MeOH, 99:1) afforded Compound 14 as a colourless foam (0.16 g, 0.36 mmol, 44%). 1H NMR (400 MHz, d6-DMSO) δ 3.72 (3H, s), 4.99 (1H, dd, J = 12.0 and 8.2 Hz), 5.15 (1H, dd, J = 11.8 and 4.4 Hz), 5.54 (1H, dd, J = 8.5 and 4.4 Hz), 6.06 (2H, s), 8.42 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 49.0 (CH), 53.2 (CH3), 67.6 (CH2), 69.2 (CH2), 118.7 (CH), 119.0 (CH), 136.4 (C), 136.5 (C), 137.3 (C), 137.3 (C), 164.5 (C), 164.9 (C), 164.9 (C), 165.0 (C), 166.2 (C).
(6-(2-Hydroxyethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)methyl nitrate (Compound 15)
Figure imgf000022_0001
To a stirred solution of ethyl 6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7 - tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.10 g, 0.28 mmol) in THF (6 mL) was added dropwise ethanolamine (17.0 mg, 0.28 mmol) in THF (2 mL). The reaction mixture was then stirred at room temperature for 1 h before being concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:4) afforded Compound 15 as a colourless solid (44 mg, 0.13 mmol, 48%). 1H NMR (400 MHz, d6-DMSO) δ 3.60-3.64 (2H, m), 3.69-3.72 (2H, m), 4.87 (1H, t, J = 6.0 Hz), 6.04 (2H, s), 8.29 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 41.0 (CH2), 57.8 (CH2), 67.6 (CH2), 117.7 (CH), 136.6 (C), 137.4 (C), 164.7 (C), 166.2 (C).
(6-(2-Methoxyethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)methyl nitrate (Compound 16)
Figure imgf000022_0002
To a stirred solution of 2-(2-methoxyethyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.28 g, 1.02 mmol) in DMF (7.5 mL) was added paraformaldehyde (61.3 mg, 2.04 mmol), and the reaction mixture heated at 80 °C for 18 h, before evaporating to dryness to afford crude 2- (hydroxymethyl)-6-(2-methoxyethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone, which was used directly in the next step. The resultant crude solid was taken up in 1,2 -dichloroethane (10 mL) and was added urea (0.8 mg, 0.01 mmol), 70% nitric acid (0.10 mL, 1.31 mmol) and acetic anhydride (0.25 mL, 2.63 mmol) in 1,2-dichloroethane (10 mL), then heated under reflux for 2 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo, diluted with ethyl acetate (20 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo to afford Compound 16 as a yellow solid (0.12 g, 0.34 mmol, 33% over two step). 1H NMR (400 MHz, d6-DMSO) δ 3.24 (3H, s), 3.57 (2H, t, J = 5.6 Hz), 3.81 (2H, t, J = 5.6 Hz), 6.04 (2H, s), 8.30 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 37.5 (CH2), 57.8 (CH3), 67.6 (CH2), 68.4 (CH2), 117.9 (CH), 136.8 (C), 137.2 (C), 165.8 (C), 166.0 (C).
6-(2-(Methylthio)ethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)methyl nitrate (Compound 17)
Figure imgf000023_0001
A mixture of ethyl 6-((nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- |isoindole-2(1H)-carboxylate (0.1 g, 0.275 mmol) and 2-(methylthio)ethylamine (27.6 mg, 0.303 mmol) in tetrahydrofuran (5 mL) was stirred at 50 °C for 18 h. The reaction mixture was concentrated in vacuo then diluted with ethyl acetate (20 mL) and washed with water (30 mL). The aqueous layer was repeatedly extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 7:3) afforded Compound 17 as a pale yellow solid (36 mg, 0.099 mmol, 36%). 1H NMR (400 MHz, d6- DMSO) δ 2.10 (3H, s), 2.78 (2H, t, J = 6.9 Hz), 3.85 (2H, t, J = 6.9 Hz), 6.04 (2H, s), 8.32 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 14.3 (CH3), 31.0 (CH2), 36.7 (CH2), 67.6 (CH2), 118.0 (CH), 136.9 (C), 137.1 (C), 164.7 (C), 166.0 (C).
(6-(2-(Methylsulfinyl)ethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol- 2(lH)-yl)methyl nitrate (Compound 18)
Figure imgf000024_0001
A stirred mixture of 2-(hydroxymethyl)-6-(2-(methylthio)ethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.40 g, 1.25 mmol), urea (1.9 mg, 0.03 mmol), 70% nitric acid (0.25 mL, 3.12 mmol) and acetic anhydride (0.59 mL, 6.24 mmol) in 1,2-dichloroethane (15 mL) was heated under reflux for 2 h. Additional 70% nitric acid (0.25 mL, 3.12 mmol) and acetic anhydride (0.59 mL, 6.24 mmol) was added, and the reaction mixture was then heated under reflux for a further 2 h. After cooling to room temperature, the precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford Compound 18 as a pale-yellow solid (0.16 g, 0.42 mmol, 34%). 1H NMR (400 MHz, d6-DMSO) δ 2.59 (3H, s), 2.96-3.02 (1H, m), 3.13-3.20 (1H, m), 3.99-4.09 (2H, m), 6.04 (2H, s), 8.31 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 32.4 (CH2), 38.1 (CH2), 51.0 (CH3), 67.6 (CH2), 117.9 (CH), 136.7 (C), 137.3 (C), 164.7 (C), 165.8 (C).
(6-(2-(Methylsulfonyl)ethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-
2(lH)-yl)methyl nitrate (Compound 19)
Figure imgf000024_0002
A stirred mixture of 2-(hydroxymethyl)-6-(2-(methylsulfonyl)ethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.70 g, 1.99 mmol), urea (3.0 mg, 0.05 mmol), 70% nitric acid (0.39 mL, 4.97 mmol) and acetic anhydride (0.94 mL, 9.93 mmol) in 1,2-dichloroethane (20 mL) was heated under reflux for 3 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. Purification by flash column chromatography (CH2Cl2/MeOH, 49:1) afforded Compound 19 as a colourless solid (0.23 g, 0.58 mmol, 29%). 1H NMR (400 MHz, d6-DMSO) δ 3.08 (3H, s), 3.53 (2H, t, J = 6.8 Hz), 4.08 (2H, t, J = 6.8 Hz), 6.04 (2H, s), 8.33 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 31.7 (CH2), 40.4 (CH3), 50.5 (CH2), 67.6 (CH2), 118.0 (CH), 136.9 (C), 167.2 (C), 164.7 (C), 165.7 (C). (6-(2-(Dimethylamino)ethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-
2( l/7)-yl)methyl nitrate, as its nitric acid salt (Compound 20)
Figure imgf000025_0001
To a stirred solution of 2-(2-(dimethylamino)ethyl)-6-((2- (trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole-1,3,5,7(27/,67/)-tetraone (0.30 g, 0.72 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (2.5 mL). The reaction mixture was then stirred at room temperature for 18 h, before evaporating to dryness to afford crude 2-(2-(dimethylamino)ethyl)-6-(hydroxymethyl)pyrrolo[3,4-f|isoindole-1,3,5,7(2H,6H)- tetraone, which was used directly in the next step. The resultant crude residue was taken up in 1,2-dichloroethane (40 mL) and was added urea (2.2 mg, 0.033 mmol), 70% nitric acid (0.12 mL, 1.90 mmol) and acetic anhydride (0.34 mL, 3.60 mmol), then heated under reflux for 18 h. After cooling to room temperature, the solids were collected by filtration and washed with dichloromethane (20 mL). Purification by RP-HPLC afforded Compound 20 as its nitric acid salt (colourless solid; 53.0 mg, 0.12 mmol, 17% over two steps). 1H NMR (400 MHz, d6- DMSO ) 5 2.88 (3H, s), 2.89 (3H, s), 3.40-3.44 (2H, m), 4.01 (2H, t, J = 5.7 Hz), 6.05 (2H, s), 8.36 (2H, s), 9.36 (1H, brs); 13C NMR (100 MHz, d6-DMSO) δ 33.4 (CH2), 42.3 (CH3), 54.4 (CH2), 67.6 (CH2), 117.9 (CH), 136.8 (C), 137.4 (C), 164.7 (C), 166.2 (C).
(6-Benzyl-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate (Compound 21)
Figure imgf000025_0002
A stirred mixture of 2-benzyl-6-(hydroxymethyl)pyrrolo[3,4-f|isoindole-l,3,5,7(27/,67/)- tetraone (0.25 g, 0.74 mmol), urea (2.3 mg, 0.04 mmol), 70% nitric acid (0.12 mL, 1.90 mmol) and acetic anhydride (0.35 mL, 3.71 mmol) in 1,2-dichloroethane (40 mL) was heated under reflux for 18 h. After cooling to room temperature, the solids were collected by filtration, washed with CH2C12 (20 mL) and water (20 mL), then dried in vacuo to afford Compound 21 as a colourless solid (0.13 g, 0.34 mmol, 46%). 1H NMR (400 MHz, d6-DMSO) δ 4.83 (2H, s), 6.04 (2H, s), 7.27-7.37 (5H, m), 8.32 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 41.5 (CH2), 67.6 (CH2), 118.0 (CH), 127.5 (CH), 127.6 (CH), 128.6 (CH), 136.1 (C), 136.7 (C), 137.3 (C), 164.7 (C), 166.0 (C).
(l,3,5,7-Tetraoxo-6-(pyridin-2-ylmethyl)-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)methyl nitrate (Compound 22)
Figure imgf000026_0001
A stirred mixture of 2-(hydroxymethyl)-6-(pyridin-2-ylmethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.20 g, 0.59 mmol), urea (0.9 mg, 0.01 mmol), 70% nitric acid (0.12 mL, 1.48 mmol) and acetic anhydride (0.28 mL, 2.96 mmol) in 1,2-dichloroethane (5 mL) was heated under reflux for 2 h. After cooling to room temperature, the solids were collected by filtration, washed with (20 mL), then dried in vacuo to afford Compound 22 as a colourless solid (0.16 g, 0.42 mmol, 71%). 1H NMR (400 MHz, d6-DMSO) δ 5.03 (2H, s), 6.06 (2H, s), 7.55 (1H, t, J = 5.7 Hz), 7.73 (1H, d, J = 8.7 Hz), 8.08 (1H, t, J = 7.5 Hz), 8.32 (2H, s), 8.61 (1H, d, J = 4.6 Hz); 13C NMR (100 MHz, d6-DMSO) δ 41.4 (CH2), 67.6 (CH2), 117.9 (CH), 132.2 (CH), 133.9 (CH), 137.0 (C), 137.3 (C), 146.6 (CH), 153.2 (C), 165.8 (C), 166.0 (C).
2-(6-((Nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)acetic add (Compound 24)
Figure imgf000026_0002
A stirred mixture of 2-(6-(hydroxymethyl)-l, 3,5,7 -tetraoxo-3, 5,6,7 -tetrahydropyrrole [3, 4- |isoindol-2(1H)-yl)acetic acid (0.15 g, 0.49 mmol), urea (0.7 mg, 0.01 mmol), 70% nitric acid (0.10 mL, 1.23 mmol) and acetic anhydride (0.23 mL, 2.47 mmol) in 1,2-dichloroethane (10 mL) was heated under reflux for 5 h. After cooling to room temperature, the precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford Compound 24 as a colourless solid (0.08 g, 0.23 mmol, 47%). 1H NMR (500 MHz, d6-DMSO) 5 4.40 (2H, s), 6.05 (2H, s), 8.38 (2H, s), 13.39 (1H, brs); 13C NMR (125 MHz, d6-DMSO) δ
39.2 (CH2), 67.6 (CH2), 118.3 (CH), 136.9 (C), 137.1 (C), 164.7 (C), 165.5 (C), 168.5 (C).
Methyl 2-(6-((nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-
2(lH)-yl)acetate (Compound 25)
Figure imgf000027_0001
A stirred mixture of methyl 2-(6-(hydroxymethyl)-l, 3,5, 7-tetraoxo-3, 5,6,7- tetrahydropyrrolo[3,4-f|isoindol-2(1H)-yl)acetate (0.11 g, 0.35 mmol), urea (0.5 mg, 0.01 mmol), 70% nitric acid (0.07 mL, 0.86 mmol) and acetic anhydride (0.16 mL, 1.73 mmol) in 1,2-dichloroethane (10 mL) was heated under reflux for 2 h. Aftercooling to room temperature, the reaction mixture was diluted with dichloromethane (20 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo to afford Compound 25 as a colourless solid (0.12 g, 0.32 mmol, 93%). 1H NMR (500 MHz, d6-DMSO) δ 3.71 (3H, s), 4.53 (2H, s), 6.05 (2H, s), 8.39 (2H, s); 13C NMR (125 MHz, d6-DMSO) δ 39.2 (CH2), 52.6 (CH3), 67.6 (CH2), 118.4 (CH), 136.8 (C), 137.2 (C), 164.6 (C), 165.3 (C), 167.7 (C).
(6-(2-(Dimethylamino)-2-oxoethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- f]isoindol-2(lH)-yl)methyl nitrate (Compound 26)
Figure imgf000027_0002
A stirred mixture of 2-(6-(hydroxymethyl)-l, 3,5,7 -tetraoxo-3, 5,6,7 -tetrahydropyrrolo [3, 4- f|isoindol-2(1H)-yl)-N,N-dimethylacetamide (0.25 g, 0.75 mmol), urea (1.1 mg, 0.02 mmol), 70% nitric acid (0.15 mL, 1.89 mmol) and acetic anhydride (0.36 mL, 3.77 mmol) in 1,2- dichloroethane (15 mL) was heated under reflux for 3 h. After cooling to room temperature, the precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford Compound 26 as a colourless solid (0.15 g, 0.40 mmol, 53%). 1H NMR (500 MHz, d6-DMSO) δ 2.85 (3H, s), 3.09 (3H, s), 4.58 (2H, s), 6.05 (2H, s), 8.36 (2H, s); 13C NMR (125 MHz, d6-DMSO) δ 35.3 (CH3), 35.8 (CH3), 67.6 (CH2), 118.2 (CH), 137.1 (C), 164.7 (C), 164.9 (C), 165.8 (C).
(6-(2, 5, 8,11, 14-Pentaoxahexadecan-16-yl)-l, 3,5, 7-tetraoxo-3, 5,6,7- tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate (Compound 27)
Figure imgf000028_0001
A stirred mixture of 2-(2,5,8,l l,14-pentaoxahexadecan-16-yl)-6-(hydroxymethyl)pyrrolo[3,4- |isoindole-l,3,5,7(2H,6H)-tetraone (0.35 g, 0.73 mmol), urea (1.1 mg, 0.02 mmol), 70% nitric acid (0.14 mL, 1.82 mmol) and acetic anhydride (0.34 mL, 3.64 mmol) in 1,2-dichloroethane (20 mL) was heated under reflux for 3 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. Purification by flash column chromatography (CH2Cl2/MeOH, 49:1) afforded Compound 27 as a yellow oil (0.31 g, 0.59 mmol, 81%). 1H NMR (400 MHz, d6-DMSO) δ 3.23 (3H, s), 3.40-3.53 (16H, m), 3.66 (2H, t, J = 5.8 Hz), 3.81 (2H, t, J = 5.8 Hz), 6.04 (2H, s), 8.30 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 37.7 (CH2), 58.0 (CH3), 66.8 (CH2), 67.6 (CH2), 69.5 (CH2), 69.6 (CH2), 69.7 (CH2), 71.2 (CH2), 117.9 (CH), 136.7 (C), 137.2 (C), 164.7 (C), 166.0 (C).
2-(l,3,5,7-Tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)ethyl nitrate (Compound 28)
Figure imgf000028_0002
A stirred solution of 2-(2-hydroxyethyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.20 g, 0.77 mmol), urea (2.3 mg, 0.04 mmol), 70% nitric acid (0.12 mL, 1.90 mmol) and acetic anhydride (0.36 mL, 3.82 mmol) in 1,2-dichloroethane (30 mL) was heated under reflux for 18 h. After cooling to room temperature, the reaction mixture was diluted with dichloromethane (20 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (CH2Ch/MeOH, 9:1) afforded Compound 28 as a colourless solid (68.0 mg, 0.22 mmol, 29%). 1H NMR (400 MHz, d6-DMSO) δ 4.00 (2H, t, J = 5.1 Hz), 4.73 (2H, t, J = 5.1 Hz), 8.18 (2H, s), 11.84 (1H, brs); 13C NMR (400 MHz, d6-DMSO) δ 35.3 (CH2), 70.7 (CH2), 117.4 (CH), 136.8 (C), 138.1 (C), 166.1 (C), 167.5 (C).
(6-Ethyl-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)methyl nitrate (Compound 29)
Figure imgf000029_0001
To a stirred mixture of ethyl 6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7 - tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.10 g, 0.28 mmol) in THF (10 mL) was added dropwise ethylamine (0.14 mL, 2 M in THF, 0.28 mmol). The reaction mixture was then stirred at room temperature for 18 h before being concentrated in vacuo to about ~3 mL in volume, and the residue triturated with Et2O (30 mL). The resulting solids were collected by filtration, washed with Et2O and dried in vacuo to afford Compound 29 as a beige solid (71.0 mg, 0.22 mmol, 81%). 1H NMR (400 MHz, d6-DMSO) δ 1.21 (3H, t, J = 7.2 Hz), 3.67 (2H, q, J = 7.2 Hz), 6.04 (2H, s), 8.28 (2H, s).
(6-(l,3-Dihydroxypropan-2-yl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-
2(lH)-yl)methyl nitrate (Compound 30)
Figure imgf000029_0002
A mixture of ethyl 6-((nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- |isoindole-2(1H)-carboxylate (0.1 g, 0.275 mmol) and serinol (2-amino-l,3-propanediol) (27.6 mg, 0.303 mmol) in tetrahydrofuran (3 mL) was stirred at room temperature for 5.5 h. The reaction mixture was concentrated in vacuo then diluted with ethyl acetate (20 mL) and washed with water (30 mL). The aqueous layer was repeatedly extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (CH2Cl2/acetone, 8:2) afforded Compound 30 as a colourless solid (39 mg, 0.107 mmol, 39%). 1H NMR (400 MHz, d6-DMSO) δ 3.68 (2H, m), 3.83 (2H, t, J = 10.6 Hz), 4.29 (1H, m), 4.93 (2H, brs), 6.05 (2H, s), 8.29 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 57.4 (CH), 58.0 (CH2), 67.6 (CH2), 117.7 (CH), 136.7 (C), 137.2 (C), 164.8 (C), 166.6 (C).
(6-(2,3-Dihydroxypropyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol- 2(lH)-yl)methyl nitrate (Compound 31)
Figure imgf000030_0001
A mixture of ethyl 6-((nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- |isoindole-2(1H)-carboxylate (0.1 g, 0.275 mmol) and 3-amino-l,2-propanediol (0.023 mL, 0.302 mmol) in tetrahydrofuran (3 mL) was stirred at room temperature for 1.5 h. The reaction mixture was concentrated in vacuo then diluted with ethyl acetate (20 mL) and washed with water (30 mL). The aqueous layer was repeatedly extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 3:7) afforded Compound 31 as a colourless solid (27 mg, 0.074 mmol, 27%). 1H NMR (400 MHz, d6- DMSO) δ 3.34-3.45 (2H, m), 3.64 (2H, d, J = 6.3 Hz), 3.81-3.87 (1H, m), 4.70 (1H, t, J = 5.3 Hz), 4.98 (1H, d, J = 5.3 Hz), 6.04 (2H, s), 8.29 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 42.4 (CH2), 64.1 (CH2), 67.6 (CH2), 68.3 (CH), 117.7 (CH), 136.6 (C), 137.4 (C), 164.8 (C), 166.3 (C).
(6-(2-(2-(2-Methoxyethoxy)ethoxy)ethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- /|isoindol-2( 1H)-yl)methyl nitrate (Compound 32)
Figure imgf000031_0001
To a stirred solution of ethyl 6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7 - tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.10 g, 0.28 mmol) in THF (6 mL) was added dropwise 2-(2-(2-methoxyethoxy)ethoxy)ethanamine (45.0 mg, 0.28 mmol) in THF (2 mL). The reaction mixture was stirred at room temperature for 18 h before being diluted with EtOAc (15 mL) and washed with H2O (10 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:4) afforded Compound 32 as a beige solid (72.0 mg, 0.16 mmol, 60%). 1H NMR (400 MHz, d6-DMSO) δ 3.18 (3H, s), 3.33-3.35 (2H, m), 3.42-3.46 (4H, m), 3.51-3.53 (2H, m), 3.64-3.67 (2H, m), 3.79-3.82 (2H, m), 6.04 (2H, s), 8.30 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 37.7 (CH2), 58.0 (CH3), 66.8 (CH2), 67.6 (CH2), 69.47 (CH2), 69.53 (CH2), 69.6 (CH2), 71.2 (CH2), 117.9 (CH), 136.7 (C), 137.2 (C), 164.7 (C), 166.0 (C).
((6-(2-Amino-2-oxoethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-
2(lH)-yl)methyl nitrate (Compound 33)
Figure imgf000031_0002
To a solution of 2-aminoacetamide hydrochloride (33.5 mg, 0.303 mmol) in tetrahydrofuran (2 mL) was added triethylamine (0.042 mL, 0.303 mmol), and the mixture stirred at room temperature for 10 minutes. To this mixture was added ethyl 6-((nitrooxy)methyl)-l,3,5,7- tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.1 g, 0.275 mmol) in tetrahydrofuran (5 mL), and the mixture stirred at room temperature for a further 18 h. The reaction mixture was concentrated in vacuo then diluted with ethyl acetate (20 mL) and washed with water (30 mL). The aqueous layer was repeatedly extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The resulting solids were washed with ether (3 x 10 mL) to afford the Compound 33 as a colourless solid (50 mg, 0.096 mmol, 52%). 1H NMR (400 MHz, d6- DMSO) δ 4.21 (2H, s), 6.05 (2H, s), 7.33 (1H, brs), 7.75 (1H, brs), 8.35 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 40.5 (CH2), 67.6 (CH2), 118.1 (CH), 136.9 (C), 137.3 (C), 164.7 (C), 165.8
(C), 167.6 (C).
(6-(2-Mercaptoethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)methyl nitrate (Compound 34)
Figure imgf000032_0001
A mixture of ethyl 6-((nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- |isoindole-2(1H)-carboxylate (0.1 g, 0.275 mmol) and 2-aminoethanethiol (23.4 mg, 0.303 mmol) in tetrahydrofuran (5 mL) was stirred at room temperature for 1.5 h. The reaction mixture was concentrated in vacuo then diluted with ethyl acetate (20 mL) and washed with water (30 mL). The aqueous layer was repeatedly extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The resulting solids were washed with ether (3 x 10 mL) to afford Compound 34 as a colourless solid (65 mg, 0.097 mmol, 67%). 1H NMR (400 MHz, d6-DMSO) δ 2.77 (2H, t, J = 4.8 Hz), 3.78-3.81 (2H, m), 6.04 (2H, s), 8.31 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 21.9 (CH2), 41.0 (CH2), 67.6 (CH2), 117.9 (CH), 136.7 (C), 137.3 (C), 164.7 (C), 166.0 (C).
(l,3,5,7-Tetraoxo-6-(2-sulfamoylethyl)-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(1H)- yl)methyl nitrate (Compound 35)
Figure imgf000032_0002
To a stirred solution of ethyl 6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7- tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.10 g, 0.28 mmol) in THF (6 mL) was added 2-aminoethanesulfonamide (35.0 mg, 0.28 mmol). The reaction mixture was then stirred at room temperature for 18 h before adding H2O (10 mL). The resulting precipitated solids were then collected by filtration, washed with H2O and dried in vacuo to afford Compound 35 as a beige solid (42 mg, 0.11 mmol, 38%). 1H NMR (400 MHz, d6-DMSO) δ 3.36-3.40 (2H, m), 4.00-4.04 (2H, m), 6.04 (2H, s), 7.07 (2H, s), 8.31 (2H, s); 13C NMR (100 MHz, d6-DMSO)
5 33.1 (CH2), 51.4 (CH2), 67.6 (CH2), 117.8 (CH), 136.7 (C), 137.3 (C), 164.7 (C), 165.7 (C).
6-((l-Methyl-lH-tetrazol-5-yl)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- f]isoindol-2(lH)-yl)methyl nitrate (Compound 36)
Figure imgf000033_0001
To a stirred solution of ethyl 6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7- tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.10 g, 0.28 mmol) in THF (6 mL) was added dropwise (l-methyl-1H-tetrazol-5-yl)methanamine (32.0 mg, 0.28 mmol) in THF (2 mL). The reaction mixture was then stirred at room temperature for 18 h before being concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:4) afforded Compound 36 as a beige solid (23.0 mg, 0.06 mmol, 22%). 1H NMR (400 MHz, d6-DMSO) δ 4.15 (3H, s), 5.23 (2H, s), 6.05 (2H, s), 8.37 (2H, s); 13C NMR (100 MHz, d6- DMSO) δ 30.9 (CH3), 33.9 (CH2), 67.6 (CH2), 118.3 (CH), 136.99 (C), 137.04 (C), 151.1 (C), 164.7 (C), 165.4 (C).
(6-((l-Ethyl-lH-tetrazol-5-yl)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- f]isoindol-2(lH)-yl)methyl nitrate (Compound 37)
Figure imgf000033_0002
To a stirred solution of ethyl 6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7 - tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.10 g, 0.28 mmol) in THF (6 mL) was added dropwise (l-ethyl-1H-tetrazol-5-yl)methanamine (35.0 mg, 0.28 mmol) in THF (2 mL). The reaction mixture was then stirred at room temperature for 18 h before being concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:4) afforded Compound 37 as a beige solid (23.0 mg, 0.06 mmol, 22%). 1H NMR (400 MHz, d6-DMSO) 5 1.50 (3H, t, J = 7.3 Hz), 4.53 (2H, q, J = 7.3 Hz), 5.24 (2H, s), 6.05 (2H, s), 8.38 (2H, s). (6-((l-Benzyl-lH-tetrazol-5-yl)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- f]isoindol-2(1H)-yl)methyl nitrate (Compound 38)
Figure imgf000034_0001
To a stirred solution of ethyl 6-((nitrooxy)methyl)-l, 3,5, 7-tetraoxo-3, 5,6,7- tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.10 g, 0.28 mmol) in THF (6 mL) was added dropwise (l-benzyl-1H-tetrazol-5-yl)methanamine (53.0 mg, 0.28 mmol) in THF (2 mL). The reaction mixture was then stirred at room temperature for 18 h before being concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:2) afforded Compound 38 as a beige solid (64.0 mg, 0.14 mmol, 50%). 1H NMR (400 MHz, d6-DMSO) δ 5.23 (2H, s), 5.78 (2H, s), 6.05 (2H, s), 7.20-7.34 (5H, m), 8.31 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 31.3 (CH2), 50.2 (CH2), 67.6 (CH2), 118.3 (CH), 127.4 (CH), 128.3 (CH), 128.8 (CH), 134.0 (C), 136.8 (C), 136.9 (C), 151.0 (C), 164.6 (C), 165.2 (C).
2-(6-((Nitrooxy)methyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)ethyl nitrate (Compound 39)
Figure imgf000034_0002
A stirred mixture of 2-(2-hydroxyethyl)-6-(hydroxymethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.41 g, 1.41 mmol), urea (8.4 mg, 0.14 mmol), 70% nitric acid (0.45 mL, 7.11 mmol) and acetic anhydride (1.40 mL, 14.8 mmol) in 1,2-dichloroethane (20 mL) was heated under reflux for 3 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo and the solids were washed with CH2C12 (20 mL) and water (20 mL), then dried in vacuo to afford Compound 39 as a colourless solid (0.23 g, 0.60 mmol, 43%). 1H NMR (400 MHz, d6-DMSO) δ 4.01 (2H, t, J = 5.0 Hz), 4.74 (2H, t, J = 5.0 Hz), 6.04 (2H, s), 8.33 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 35.4 (CH2), 67.6 (CH2), 70.7 (CH2), 118.0 (CH), 136.8 (C), 137.2 (C), 164.7 (C), 165.9 (C). 3-(6-(2-(Nitrooxy)ethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)propane-l,2-diyl dinitrate (Compound 40)
Figure imgf000035_0001
A stirred mixture of 2-(2,3-dihydroxypropyl)-6-(2-hydroxyethyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (100 mg, 0.299 mmol), urea (2.7 mg, 0.045 mmol), 70% nitric acid (0.14 mL, 2.24 mmol) and acetic anhydride (0.42 mL, 4.49 mmol) in 1,2 -dichloroethane (5 mL) was heated under reflux for 0.5 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo and ice water (30 mL) was added. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford Compound 40 as a colourless solid (130 mg, 0.277 mmol, 93%). 1H NMR (400 MHz, d6-DMSO) δ 4.01 (2H, t, J = 5.1 Hz), 4.07 (1H, d, J = 2.3 Hz), 4.08 (1H, d, J = 1.5 Hz), 4.74 (2H, t, J = 4.7 Hz), 4.89 (1H, dd, J = 12.6 and 6.3 Hz), 4.98 (1H, dd, J = 12.5 and 3.0 Hz), 5.60-5.65 (1H, m), 8.29 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 35.4 (CH2), 36.3 (CH2), 70.1 (CH2), 70.7 (CH2), 77.3 (CH), 117.5 (CH), 136.9 (C), 137.1 (C), 166.0 (C), 166.2 (C).
3-(6-(l-(Nitrooxy)propan-2-yl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-
2(lH)-yl)propane-l,2-diyl dinitrate (Compound 41)
Figure imgf000035_0002
A stirred mixture of 2-(2,3-dihydroxypropyl)-6-(l-hydroxypropan-2-yl)pyrrolo[3,4- |isoindole-l,3,5,7(2H,6H)-tetraone (40 mg, 0.115 mmol), urea (1.0 mg, 0.017 mmol), 70% nitric acid (0.05 mL, 0.861 mmol) and acetic anhydride (0.16 mL, 1.72 mmol) in 1,2- dichloroethane (5 mL) was heated under reflux for 0.5 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo and ice water (30 mL) was added. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded Compound 41as a colourless foam (29 mg, 0.059 mmol, 52%). 1H NMR (400 MHz, d6- DMSO) δ 1.51 (3H, d, J = 7.1 Hz), 4.07-4.09 (2H, m), 4.61-4.65 (1H, m), 4.81 (1H, ddd, J = 11.4, 4.5 and 0.7 Hz), 4.87-4.93 (2H, m), 4.98 (1H, d, J = 12.7 and 3.1 Hz), 5.60-5.64 (1H, m), 8.27 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 14.3 (CH3), 36.4 (CH2), 44.3 (CH), 70.1 (CH2), 72.7 (CH2), 77.3 (CH), 117.6 (CH), 136.6 (C), 137.1 (C), 166.0 (C), 166.2 (C).
3-(6-(3-(Nitrooxy)butan-2-yl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol- 2(lH)-yl)propane-l,2-diyl dinitrate (Compound 42)
Figure imgf000036_0001
To a solution of pyromellitic dianhydride (1.00 g, 4.58 mmol) in DMF (50 mL) was added slowly 3-amino-2-butanol (409 mg, 4.58 mmol) in DMF (5 mL) and 3 -aminopropane- 1,2-diol (418 mg, 4.58 mmol) in DMF (5 mL). The reaction mixture was then heated at 100 °C for 18 h, before being diluted with water (100 mL). The precipitated solids were collected by filtration, washed with water (3 x 50 mL), then dried in vacuo. The resulting residue (90 mg) was taken up in 1,2-dichloroethane (5 mL) and to this was added urea (2.2 mg, 0.037 mmol), acetic anhydride (0.35 mL, 3.73 mmol) and 70% nitric acid (0.12 mL, 1.86 mmol), and the mixture heated at reflux for 2 h. Following cooling to room temperature, the reaction mixture was first concentrated in vacuo, then diluted with ethyl acetate (20 mL) and washed with water (20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 7:3) afforded Compound 42 as an inseparable 1:1 mixture of diastereomers (colourless foam, 42 mg, 0.084 mmol, 2% over two steps). 1H NMR (400 MHz, d6-DMSO) δ 1.33 (3H, d, J = 6.5 Hz), 1.55 (3H, d, J = 7.3 Hz), 4.03-4.11 (2H, m), 4.53 (1H, qd, J = 7.1 and 5.4 Hz), 4.88 (2H, ddd, J = 12.7, 6.4 and 2.2 Hz), 5.49 (1H, qd, J = 6.5 and 5.6 Hz), 5.59-5.65 (1H, m), 8.27 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 13.1 (CH3), 14.4 (CH3), 36.3 (CH2), 48.9 (CH), 70.1 (CH2), 77.3 (CH), 80.8 (CH), 117.58 (CH), 136.6 (C), 137.13 (C), 166.0 (C), 166.23 (C). Other diastereomer: 1H NMR (400 MHz, d6-DMSO) δ 1.43 (3H, d, J = 6.4 Hz), 1.50 (3H, d, J = 7.2 Hz), 4.03-4.11 (2H, m), 4.44 (1H, dq, J = 9.1 and 7.2 Hz), 4.98 (2H, dt, J = 12.6 and 3.0 Hz), 5.59-5.65 (2H, m), 8.26 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 14.6 (CH3), 16.1 (CH3), 36.4 (CH2), 48.8 (CH), 70.1 (CH2), 77.3 (CH), 81.6 (CH), 117.6 (CH), 136.7 (C), 137.07 (C), 166.22 (C), 166.4 (C).
The following precursor or intermediate compounds referred to above were made as follows:
2-(Hydroxymethyl)-6-methylpyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000037_0001
A mixture of 2-methylpyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.54 g, 2.35 mmol) and paraformaldehyde (0.11 g, 3.66 mmol) in DMF (15 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (2 x 20 mL), then dried in vacuo to afford the title compound as a colourless solid (0.41 g, 1.58 mmol, 67%). 1H NMR (400 MHz, d6-DMSO) δ 3.10 (3H, s), 5.01 (2H, d, J = 7.1 Hz), 6.52 (1H, t, J = 7.1 Hz), 8.24 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 24.2 (CH3), 60.6 (CH2), 117.7 (CH), 136.7 (C), 137.5 (C), 165.8 (C), 166.4 (C).
2-Methylpyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000037_0002
To a stirred solution of pyromellitic imide anhydride (5.00 g, 23.0 mmol) in acetone (150 mL) at 0 °C was added methylamine (7.20 mL, 33 wt. % in EtOH, 58.4 mmol) dropwise. The reaction mixture was stirred at room temperature for 30 min and concentrated in vacuo. Water (25 mL) was then added and the insoluble solids filtered off. The filtrate was then acidified to pH 1 with 2 M aq. hydrochloric acid solution and the mixture stirred at room temperature for 18 h. The precipitated solids were collected by filtration, washed with water (20 mL) then dried in vacuo to afford the title compound as a colourless solid (1.53 g, 6.65 mmol, 29%). 1H NMR (400 MHz, d6-DMSO) δ 3.09 (3H, s), 8.13 (2H, s), 11.81 (1H, brs). 2-Allyl-6-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)- tetraone
Figure imgf000038_0001
To a stirred solution of 2-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.37 g, 1.07 mmol) in DMF (10 mL) at room temperature was added sodium hydride (51.0 mg, 60% dispersion in mineral oil, 1.28 mmol), and the reaction mixture was stirred for 30 min. Allyl bromide (0.11 mL, 1.27 mmol) was then added dropwise and the reaction mixture stirred at room temperature for a further 18 h, then quenched with water (20 mL). The resulting solids were filtered, washed with water (10 mL), then dried in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded the title compound as a colourless solid (0.33 g, 0.85 mmol, 80%). 1H NMR (400 MHz, CDCL) 6 -0.00 (9H, s), 0.94-0.98 (2H, m), 3.65-3.69 (2H, m), 4.36 (2H, dt, J = 5.9 and 1.4 Hz), 5.16 (2H, s), 5.24-5.33 (2H, m), 5.85-5.94 (1H, m), 8.35 (2H, s); 13C NMR (400 MHz, CDCI3) 6 -1.29 (CH3), 18.1 (CH2), 40.9 (CH2), 67.4 (CH2), 67.8 (CH2), 119.0 (CH), 130.8 (CH2). 137.2 (C), 137.7 (C), 165.8 (C), 166.0 (C).
2-((2-(Trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000038_0002
To a stirred solution of pyromellitic diimide (2.00 g, 9.25 mmol) in DMF (200 mL) was added sodium hydride (0.24 g, 60% dispersion in mineral oil, 10.2 mmol). The reaction mixture was stirred at room temperature for 30 min before 2-(triemethylsilyl)ethoxymethyl chloride (1.80 mL, 10.2 mmol) was added dropwise. After a further 1 h, the reaction mixture was quenched with water (200 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were then washed with brine (3 x 200 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 3:1) afforded the title compound as a colourless solid (0.64 g, 1.85 mmol, 20%). 1H NMR (400 MHz, d6-DMSO) δ -0.03 (9H, s), 0.87 (2H, dd, J = 8.9 and 7.8 Hz), 3.61 (2H, dd, J = 8.9 and 7.9 Hz), 5.01 (2H, s), 8.35 (2H, s), 11.86 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ -1.4 (CH3), 17.3 (CH2), 66.3 (CH2), 66.9 (CH2), 117.7 (CH), 136.6 (C), 138.2 (C), 166.2 (C), 167.5 (C); ESI-MS: m/z calcd for C16H18N2O5Si: 346.0985; found [M+Na]+: 369.0867.
2-(Hydroxymethyl)-6-phenylpyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000039_0001
A stirred solution of 2-phenylpyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.50 g, 1.71 mmol) and paraformaldehyde (77.0 mg, 2.56 mmol) in DMF (15 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (40 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (2 x 20 mL), then dried in vacuo to afford the title compound as a pale-yellow solid (0.25 g, 0.78 mmol, 45%). 1H NMR (400 MHz, d6-DMSO) δ 5.04 (2H, d, J = 7.1 Hz), 6.55 (1H, t, J = 7.1 Hz), 7.46-7.59 (5H, m), 8.35 (2H, s); 13C NMR (400 MHz, d6-DMSO) δ 60.7 (CH2), 117.9 (CH), 127.3 (CH), 128.4 (CH), 129.0 (CH), 131.5 (C), 136.9 (C), 137.2 (C), 165.4 (C), 165.8 (C).
2-Phenylpyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000039_0002
To a stirred solution of pyromellitic imide anhydride (1.00 g, 4.61 mmol) in DMF (15 mL) at 0 °C was added aniline (0.42 mL, 4.60 mmol) dropwise, then the reaction mixture was heated at 100 °C for 18 h. After cooling to room temperature, the insoluble by-products were filtered off, and water (40 mL) was added to the filtrate. The precipitated solids were collected by filtration, washed with water (20 mL), then dried in vacuo to afford the title compound as a pale-yellow solid (0.70 g, 2.40 mmol, 52%). 1H NMR (400 MHz, d6-DMSO) δ 7.45-7.58 (5H, m), 8.24 (2H, s), 11.88 (1H, brs); 13C NMR (400 MHz, d6-DMSO) δ 117.5 (CH), 127.3 (CH), 128.4 (CH), 128.9 (CH), 131.5 (C), 136.8 (C), 138.1 (C), 165.5 (C), 167.5 (C). 2-(Hydroxymethyl)-6-(pyridin-2-yl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000040_0001
A stirred mixture of 2-(pyridin-2-yl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.40 g, 1.36 mmol) and paraformaldehyde (81.9 mg, 2.73 mmol) in DMF (5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a pale-yellow solid (0.42 g, 1.30 mmol, 95%). 1H NMR (400 MHz, d6-DMSO) δ 5.04 (2H, d, J = 7.1 Hz), 6.56 (1H, t, J = 7.1 Hz), 7.57 (1H, ddd, J = 7.5, 4.9 and 1.0 Hz), 7.60 (1H, d, J = 7.9 Hz), 8.08 (1H, td, J = 11.6 and 1.9 Hz), 8.37 (2H, s), 8.68 (1H, ddd, J = 4.8, 1.9 and 0.7 Hz); 13C NMR (100 MHz, d6-DMSO) δ 60.7 (CH2), 118.3 (CH), 122.9 (CH), 124.4 (CH), 137.0 (C), 137.2 (C), 138.9 (CH), 145.5 (C), 149.5 (CH), 164.8 (C), 165.7 (C).
2-(Pyridin-2-yl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000040_0002
A stirred mixture of pyromellitic imide anhydride (0.50 g, 2.30 mmol) and pyridine-2-amine (0.26 g, 2.76 mmol) in DMF (5 mL) was heated at 100 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), and dried in vacuo. Purification by flash column chromatography (CH2Cl2/acetone, 9:1) afforded the title compound as a pale-yellow solid (0.48 g, 1.64 mmol, 71%). 1H NMR (400 MHz, d6-DMSO) δ 7.56 (1H, ddd, J = 7.5, 4.9 and 1.1 Hz), 7.59 (1H, dt, J = 8.0 and 0.9 Hz), 8.07 (1H, td, J = 11.5 and 1.9 Hz), 8.27 (2H, s), 8.67 (1H, ddd, J = 4.8, 1.9 and 0.8 Hz), 11.90 (1H, brs); 13C NMR (100 MHz, d6-DMSO) δ
117.9 (CH), 122.9 (CH), 124.4 (CH), 136.6 (C), 138.3 (C), 138.9 (CH), 145.5 (C), 149.5 (CH),
164.9 (C), 167.5 (C). 2-(Acetoxymethyl)-6-(6-(hydroxymethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- f]isoindol-2(lH)-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000041_0001
To a stirred solution of 2-(acetoxymethyl)-6-(l,3,5,7-tetraoxo-6-((2- (trimethylsilyl)ethoxy)methyl)-3,5,6,7-tetrahydropyrrolo[3,4-f|isoindol-2(1H)-yl)tetrahydro- 2 H-pyran-3,4,5-triyl triacetate (0.45 g, 0.66 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (2 mL), and the reaction mixture stirred at room temperature for 18 h. The reaction was then diluted with dichloromethane (15 mL) and washed with sat. aq. sodium bicarbonate solution (2 x 10 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuo to afford the title compound as a pale-yellow solid (0.38 g, 2.05 mmol, 99%). 1H NMR (400 MHz, CDC13) 6 1.89 (3H, s), 2.04 (3H, s), 2.07 (3H, s), 2.08 (3H, s), 3.23 (1H, t, J = 8.1 Hz), 3.88 (1H, dt, J = 9.9 and 3.6 Hz), 4.21-4.22 (2H, m), 5.25-5.38 (4H, m), 5.48 (1H, d, J = 9.4 Hz), 5.98 (1H, t, J = 9.3 Hz), 8.38 (2H, s); 13C NMR (400 MHz, CDCI3) 6 20.5 (CH3), 20.7 (CH3), 20.8 (CH3), 61.8 (CH2), 62.3 (CH2), 67.9 (CH), 68.5 (CH), 73.4 (CH), 75.0 (CH), 78.4 (CH), 119.7 (CH), 137.6 (C), 165.5 (C), 169.5 (C), 169.6 (C), 170.2 (C), 170.8 (C).
2-(Acetoxymethyl)-6-(l, 3,5, 7-tetraoxo-6-((2-(trimethylsilyl)ethoxy)methyl)-3, 5,6,7- tetrahydropyrrolo[3,4-f]isoindol-2(1H)-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
Figure imgf000041_0002
To a stirred solution of 2-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.60 g, 1.73 mmol) in DMF (10 mL) at room temperature was added sodium hydride (76.0 mg, 60% dispersion in mineral oil, 1.90 mmol), and the reaction mixture stirred for 30 min. 2-(Acetoxymethyl)-6-bromotetrahydro-1H-pyran-3,4,5-triyl triacetate (0.79 g, 1.92 mmol) in DMF (5 mL) was then added dropwise and the reaction mixture stirred at room temperature for a further 18 h, then quenched with water (30 mL). The resulting solids were filtered, washed with water (10 mL) and dried in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded the title compound as a pale-yellow solid (0.46 g, 0.68 mmol, 39%). 1H NMR (400 MHz, CDC13) 6 0.00 (9H, s), 0.94-0.98 (2H, m), 1.89 (3H, s), 2.04 (3H, s), 2.07 (3H, s), 2.08 (3H, s), 3.64-3.68 (2H, m), 3.88 (1H, dt, J = 9.9 and 3.6 Hz), 4.22 (2H, d, J = 3.6 Hz), 5.16 (2H, s), 5.25-5.38 (2H, m), 5.47 (1H, d, J = 9.4 Hz), 5.98 (1H, t, J = 9.3 Hz), 8.38 (2H, s); 13C NMR (400 MHz, CDCI3) 6 -1.3 (CH3), 18.1 (CH2), 20.5 (CH3), 20.7 (CH3), 20.8 (CH3), 61.8 (CH2), 67.5 (CH2), 67.8 (CH2), 67.9 (CH), 68.5 (CH), 73.4 (CH), 74.9 (CH), 78.3 (CH), 119.6 (CH), 137.6 (C), 165.7 (C), 169.5 (C), 169.6 (C), 170.2 (C), 170.7 (C).
2-Acetyl-6-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f]isoindole-1,3,5,7(2H,6H)- tetraone
Figure imgf000042_0001
To a stirred solution of 2-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.50 g, 1.44 mmol) in DMF (10 mL) was added sodium hydride (58.9 mg, 60% dispersion in mineral oil, 1.73 mmol). The reaction mixture was stirred at room temperature for 30 min before acetyl chloride (0.12 mL, 1.73 mmol) was added dropwise. After a further 30 min, the reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo to give the title compound as a ca. 3: 1 mixture (0.50 g) of product and 2-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole-l,3,5,7(27/,67/)- tetraone which was used without further purification. 1H NMR (400 MHz, d6-DMSO) δ -0.03 (9H, s), 0.88 (2H, dd, J = 8.6 and 7.8 Hz), 2.58 (3H, s), 3.62 (2H, dd, J = 8.6 and 7.8 Hz), 5.02 (2H, s), 8.38 (2H, s).
Ethyl 6-( hydroxy methyl)- 1,3.5, 7-tetraoxo-3,5, 6,7-tetrahydropyrrolo[3,4f]isoindole-
2( 1H) -carboxylate
Figure imgf000043_0001
To a stirred solution of ethyl l,3,5,7-tetraoxo-6-((2-(trimethylsilyl)ethoxy)methyl)-3,5,6,7- tctrahydropyrrolo[3,4;/]isoindolc-2( l /7)-carboxylatc (0.60 g, 1.43 mmol) in dichloromethane (16 mL) was added trifluoroacetic acid (4 mL). The reaction mixture was stirred at room temperature for 6 h, then concentrated in vacuo to afford the title compound as a colourless solid (0.44 g, 1.37 mmol, 96%). 1H NMR (400 MHz, d6-DMSO) δ 1.34 (3H, t, J = 7.1 Hz), 4.41 (2H, q, J = 7.1 Hz), 5.02 (2H, s), 8.34 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 13.9 (CH3), 60.8 (CH2), 63.6 (CH2), 118.6 (CH), 136.6 (C), 137.3 (C), 147.6 (C), 162.3 (C), 165.5 (C).
Ethyl l,3,5,7-tetraoxo-6-((2-(trimethylsilyl)ethoxy)methyl)-3,5,6,7- tetrahydropyrrolo[3,4-f]isoindole-2(1H)-carboxylate
Figure imgf000043_0002
To a stirred solution of 2-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.70 g, 2.02 mmol) in DMF (10 mL) was added sodium hydride (82.4 mg, 60% dispersion in mineral oil, 2.42 mmol). The reaction mixture was stirred at room temperature for 30 min before ethyl chloroformate (0.23 mL, 2.42 mmol) was added dropwise. After a further 1 h, the reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo to afford the title compound as a colourless solid (0.69 g, 1.65 mmol, 82%). 1H NMR (400 MHz, d6-DMSO) δ -0.03 (9H, s), 0.88 (2H, dd, J = 8.9 and 7.3 Hz), 1.34 (3H, t, J = 7.1 Hz), 3.62 (2H, dd, J = 8.9 and 7.3 Hz), 4.41 (2H, q, J = 7.1 Hz), 5.01 (2H, s), 8.35 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ -1.4 (CH3), 13.9 (CH3), 17.3 (CH2), 63.4 (CH2), 66.3 (CH2), 67.0 (CH2), 118.7 (CH), 136.5 (C), 137.2 (C), 147.6 (C), 162.2 (C), 165.9 (C); ESI-MS: m/z calcd for Ci9H22N2O7Si: 418.1196; found [M+Na]+: 441.1073. 2-(2,3-Dihydroxypropyl)-6-(hydroxymethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)- tetraone (via Scheme 1)
Figure imgf000044_0001
To a stirred solution of 2-(2,3-dihydroxypropyl)-6-((2- (trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.09 g, 0.21 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (1 mL). The reaction mixture was stirred at room temperature for 18 h, then diluted with dichloromethane (10 mL). The organic layer was washed with sat. aq. sodium bicarbonate solution (10 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo to afford the title compound as a colourless solid (0.06 g, 0.19 mmol, 88%). 1H NMR (400 MHz d6-DMSO) δ 3.35-3.44 (2H, m), 3.64 (2H, d, J = 6.4 Hz), 3.82-3.84 (1H, m), 4.69 (1H, brs), 4.96 (1H, d, J = 4.7 Hz), 5.01 (2H, d, J = 6.8 Hz), 6.53 (1H, t, J = 7.0 Hz), 8.25 (2H, m); 13C NMR (100 MHz, d6-DMSO) δ 42.3 (CH2), 60.7 (CH2), 64.0 (CH2), 68.3 (CH), 117.5 (CH), 136.7 (C), 137.3 (C), 165.8 (C), 166.4 (C); ESI-MS: m/z calcd for CI4HI2N2O7: 320.0645; found [M+Na]+: 343.0542.
2-(2,3-Dihydroxypropyl)-6-(hydroxymethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)- tetraone (via Scheme 2)
Figure imgf000044_0002
A stirred mixture of 2-(2,3-dihydroxypropyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.20 g, 0.69 mmol) and paraformaldehyde (41.4 mg, 1.38 mmol) in DMF (5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture, and was then left to stand at room temperature for 18 h. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford compound the title compound as a colourless solid (0.13 g, 0.39 mmol, 57%). 1H NMR (400 MHz d6-DMSO) 5 3.35-3.44 (2H, m), 3.64 (2H, d, J = 6.4 Hz), 3.82-3.84 (1H, m), 4.69 (1H, brs), 4.96 (1H, d, J = 4.7 Hz), 5.01 (2H, d, J = 6.8 Hz), 6.53 (1H, t, J = 7.0 Hz), 8.25 (2H, m); 13C NMR (100 MHz, d6-DMSO) δ 42.3 (CH2), 60.7 (CH2), 64.0 (CH2), 68.3 (CH), 117.5 (CH), 136.7 (C), 137.3 (C), 165.8 (C), 166.4 (C); ESI-MS: m/z calcd for CI4HI2N2O7: 320.0645; found [M+Na]+: 343.0542.
2-(2,3-Dihydroxypropyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000045_0001
A stirred mixture of pyromellitic imide anhydride (1.00 g, 4.61 mmol) and 3-aminopropane- 1,2-diol (0.39 mL, 5.07 mmol) in DMF (15 mL) was heated at 50 °C for 42 h. After cooling to room temperature, water (100 mL) was added to the reaction mixture, and was then left to stand at room temperature for 48 h. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a colourless solid (0.19 g, 0.67 mmol, 15%). 1H NMR (400 MHz d6-DMSO) δ 3.38 (1H, dd, J = 11.0 and 6.0 Hz), 3.44 (1H, dd, J = 11.0 and 5.3 Hz), 3.66 (2H, d, J = 6.4 Hz), 3.85 (1H, pentet, J = 6.0 Hz), 4.70 (1H, brs), 4.98 (1H, brs), 8.16 (2H, s), 11.84 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 42.2 (CH2), 64.1 (CH2), 68.3 (CH), 117.1 (CH), 137.0 (C), 137.9 (C), 166.4 (C), 167.6 (C); ESI-MS: m/z calcd for CI3HI0N2O6: 290.0539; found [M+Na]+: 313.0432.
2-(2,3-Dihydroxypropyl)-6-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3, 4-f] isoindole-
1,3, 5, 7 (2H,6H ) -tetraone
Figure imgf000045_0002
A mixture of ethyl 1,3, 5,7 -tetraoxo-6-((2-(trimethylsilyl)ethoxy)methyl)-3, 5,6,7- tetrahydropyrrolo[3,4-f|isoindole-2(177)-carboxylate (0.13 g, 0.31 mmol) and 3- aminopropane-l,2-diol (0.037 mL, 0.47 mmol) in tetrahydrofuran (5 mL) was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo then diluted with ethyl acetate (20 mL) and washed with water (30 mL). The aqueous layer was repeatedly extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (EtOAc, neat) afforded the title compound as a colourless solid (0.06 g, 0.14 mmol, 46%). 1H NMR (400 MHz, d6-DMSO) δ 0.00 (9H, s), 0.88 (2H, dd, J = 8.3 and 7.3 Hz), 3.34-3.46 (2H, m), 3.61 (1H, d, J = 8.0 Hz), 3.64 (1H, d, J = 6.6 Hz), 3.83 (1H, pentet, J = 5.9 Hz), 4.69 (1H, t, J = 5.7 Hz), 4.96 (1H, d, J = 5.1 Hz), 5.01 (2H, s), 8.27 (2H, s); 13C NMR (100 MHz, d6- DMSO) δ -1.4 (CH3), 17.3 (CH2), 42.3 (CH2), 64.1 (CH2), 66.3 (CH2), 67.0 (CH2), 68.3 (CH), 117.6 (CH), 136.6 (C), 137.4 (C), 166.2 (C), 166.3 (C); ESI-MS: m/z calcd for Ci9H24N2O7Si: 420.1353; found [M+Na]+: 443.1245.
2-(l,3-Dihydroxypropan-2-yl)-6-(hydroxymethyl)pyrrolo[3, 4-f] isoindole-1, 3, 5, 7(2H,6H)- tetraone
Figure imgf000046_0001
A stirred mixture of 2-(l,3-dihydroxypropyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.22 g, 0.74 mmol) and paraformaldehyde (44.5 mg, 1.48 mmol) in DMF (5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture, and was then left to stand at room temperature for 18 h. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a colourless solid (0.11 g, 0.34 mmol, 46%). 1H NMR (400 MHz d6-DMSO) δ 3.65-3.71 (2H, m), 3.80-3.86 (2H, m), 4.25-4.32 (1H, m), 4.91 (2H, dd, J = 6.9 and 5.2 Hz), 5.02 (2H, d, J = 7.1 Hz), 6.53 (1H, t, J = 7.1 Hz), 8.24 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 57.3 (CH), 58.1 (CH2), 60.7 (CH2), 117.4 (CH), 136.8 (C), 137.1 (C), 165.8 (C), 166.6 (C).
2-(l,3-Dihydroxypropyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000046_0002
A stirred mixture of pyromellitic imide anhydride (1.00 g, 4.61 mmol) and 2-aminopropane- 1,3-diol (0.39 mL, 5.07 mmol) in DMF (15 mL) was heated at 100 °C for 18 h. The reaction mixture was stirred for a further 3 h at 150 °C. After cooling to room temperature, water (100 mL) was added to the reaction mixture, and was then left to stand at room temperature for 18 h. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a colourless solid (1.00 g, 3.45 mmol, 75%). 1H NMR (400 MHz d6-DMSO) δ 3.64-3.70 (2H, m), 3.79-3.85 (2H, m), 4.24-4.32 (1H, m), 4.90 (2H, t, J = 6.0 Hz), 8.14 (2H, s), 11.83 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 57.2 (CH), 58.1 (CH2), 117.0 (CH), 136.8 (C), 137.9 (C), 166.7 (C), 167.6 (C); ESI-MS: m/z calcd for C13H10N2O6: 290.0539; found [M+Na]+: 313.0426.
Methyl 3-hydroxy-2-(6-(hydroxymethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- f]isoindol-2(1H)-yl)propanoate
Figure imgf000047_0001
A stirred mixture of methyl 3-hydroxy-2-(l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- /|isoindol-2(1H)-yl)propanoate (0.25 g, 0.79 mmol) and paraformaldehyde (47.2 mg, 1.57 mmol) in DMF (5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with water (3 x 20 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded the title compound as a colourless solid (0.14 g, 0.41 mmol, 53%). 1H NMR (400 MHz, d6-DMSO) δ 4.00-4.04 (2H, m), 5.03 (2H, d, J = 7.1 Hz), 5.07 (1H, dd, J = 8.5 and 6.5Hz), 5.15 (1H, t, J = 6.5 Hz), 6.56 (1H, t, J = 7.1 Hz), 8.33 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 52.5 (CH3), 54.4 (CH), 57.8 (CH2), 60.7 (CH2), 118.1 (CH), 136.5 (C), 137.3 (C), 165.6 (C), 165.7 (C), 167.6 (C).
Methyl 3-hydroxy-2-(l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)propanoate
Figure imgf000048_0001
A mixture of serine methyl ester hydrochloride (0.86 g, 5.53 mmol) and triethylamine (0.77 mL, 5.53 mmol) in DMF (10 mL) was stirred at room temperature for 30 min, before pyromellitic imide anhydride (1.0 g, 4.61 mmol) was added. The reaction mixture was then stirred at 100 °C for 18 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with water (2 x 20 mL). The organic layer was concentrated in vacuo to afford the title compound as a brown foam (0.30 g, 0.94 mmol, 20%). 1H NMR (400 MHz, d6-DMSO) δ 3.66 (3H, s), 4.00-4.04 (2H, m), 5.06 (1H, dd, J = 8.5 and 6.4 Hz), 5.14 (1H, t, 7 = 6.1 Hz), 8.22 (2H, s), 11.87 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 52.5 (CH3), 54.3 (CH), 57.8 (CH2), 117.7 (CH), 136.2 (C), 138.4 (C), 165.6 (C), 167.5 (C), 167.6 (C).
2-(2-Methoxyethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000048_0002
To a stirred mixture of pyromellitic diimide (2.00 g, 9.25 mmol) in DMF (200 mL) was added sodium hydride (0.22 g, 60% dispersion in mineral oil, 9.25 mmol). The reaction mixture was stirred at room temperature for 30 min before ethyl chloroformate (0.88 mL, 9.25 mmol) was added dropwise, and the reaction stirred for a further 1 h. 2-Methoxyethan-l -amine (0.80 mL, 9.25 mmol) was then slowly added, and the reaction mixture heated at 50 °C for 18 h. After cooling to room temperature, the reaction mixture was quenched with water (200 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were then washed with brine (3 x 200 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded the title compound as a colourless solid (0.30 g, 1.11 mmol, 24%). 1H NMR (400 MHz, d6-DMSO) δ 3.24 (3H, s), 3.57 (2H, t, J = 5.7 Hz), 3.80 (2H, t, J = 5.7 Hz), 8.15 (2H, s), 11.83 (1H, brs); 13C NMR (100 MHz, d6-DMSO) δ 37.4 (CH2), 57.8 (CH3), 68.4 (CH2), 117.3 (CH), 136.7 (C), 138.0 (C), 166.2 (C), 167.5 (C). 2-(Hydroxymethyl)-6-(2-(methylthio)ethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)- tetraone
Figure imgf000049_0001
A stirred mixture of 2-(2-(methylthio)ethyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.45 g, 1.55 mmol) and paraformaldehyde (93.1 mg, 3.10 mmol) in DMF (10 mL) was heated at 80 °C for 18 h. After cooling to room temperature, the reaction mixture was dried in vacuo to afford the title compound as a pale-yellow oil (0.40 g, 1.25 mmol, 81%). 1H NMR (400 MHz d6-DMSO) δ 2.11 (3H, s), 2.78 (2H, t, J = 6.8 Hz), 3.84 (2H, t, J = 6.8 Hz), 5.02 (2H, d, J = 7.0 Hz), 6.53 (1H, t, J = 7.0 Hz), 8.27 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 14.3 (CH3), 31.0 (CH2), 36.7 (CH2), 60.7 (CH2), 117.7 (CH), 136.9 (C), 137.0 (C), 165.8 (C), 166.0 (C).
2-(2-(Methylthio)ethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000049_0002
A stirred mixture of pyromellitic imide anhydride (1.00 g, 4.61 mmol) and 2- (methylthio)ethylamine (0.51 mL, 5.53 mmol) in DMF (10 mL) was heated at 100 °C for 18 h. The reaction mixture was stirred for a further 1 h at 150 °C. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with water (2 x 20 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 7:3) afforded the title compound as a pale-yellow solid (0.54 g, 1.87 mmol, 41%). 1H NMR (400 MHz, d6-DMSO) δ 2.10 (3H, s), 2.78 (2H, t, J = 6.8 Hz), 3.84 (2H, t, J = 6.8 Hz), 8.17 (2H, s), 11.83 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 14.2 (CH3), 31.0 (CH2), 36.6 (CH2), 117.3 (CH), 136.7 (C), 138.1 (C), 166.1 (C), 167.5 (C). 2-(Hydroxymethyl)-6-(2-(methylsulfonyl)ethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)- tetraone
Figure imgf000050_0001
A stirred mixture of 2-(2-(methylsulfonyl)ethyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)- tetraone (0.90 g, 2.79 mmol) and paraformaldehyde (168 mg, 5.59 mmol) in DMF (15 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a beige solid (0.70 g, 1.99 mmol, 71%). 1H NMR (500 MHz d6-DMSO) δ 3.08 (3H, s), 3.52 (2H, t, J = 6.9 Hz), 4.07 (2H, t, J = 6.9 Hz), 5.01 (2H, brs), 6.54 (1H, brs), 8.29 (2H, s); 13C NMR (125 MHz, d6-DMSO) δ 31.7 (CH2), 40.4 (CH3), 50.6 (CH2), 60.7 (CH2), 117.8 (CH), 137.0 (C), 137.1 (C), 165.8 (C).
2-(2-(Methylsulfonyl)ethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000050_0002
A mixture of 2-aminoethylmethylsulfone hydrochloride (0.88 g, 5.53 mmol) and triethylamine (0.77 mL, 5.53 mmol) in DMF (10 mL) was stirred at room temperature for 30 min, before pyromellitic imide anhydride (1.0 g, 4.61 mmol) was added. The reaction mixture was then stirred at 100 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a grey solid (1.20 g, 3.72 mmol, 81%). 1H NMR (500 MHz, d6-DMSO) δ 3.08 (3H, s), 3.52 (2H, t, J = 6.7 Hz), 4.07 (2H, t, J = 6.7 Hz), 8.19 (2H, s), 11.85 (1H, s); 13C NMR (125 MHz, d6-DMSO) δ 32.0 (CH2), 40.7 (CH3), 51.0 (CH2), 117.8 (CH), 137.2 (C), 138.5 (C), 166.3 (C), 167.9 (C).
2-(2-(Dimethylamino)ethyl)-6-((2-(trimethylsilyl)ethoxy)methyl)pyrrolo[3,4-f]isoindole-
1 ,3,5,7 (2H,6H ) -tetraone
Figure imgf000051_0001
To a stirred solution of ethyl l,3,5,7-tetraoxo-6-((2-(trimethylsilyl)ethoxy)methyl)-3,5,6,7- tetrahydropyrrolo[3,4-f|isoindole-2(1H)-carboxylate (0.30 g, 0.72 mmol) in THF (15 mL) was added N,N-dimcthylcthylcncdiaminc (0.078 mL, 0.71 mmol), and the reaction mixture stirred at 40 °C for 6 h. The reaction mixture was then concentrated in vacuo. Purification by flash column chromatography (CH2Cl2/MeOH, 9:1) afforded the title compound as a yellow oil (0.27 g, 0.65 mmol, 90%). 1H NMR (400 MHz, d6-DMSO) δ -0.03 (9H, s), 0.86-0.90 (2H, m), 2.17 (6H, s), 2.50-2.54 (2H, m), 3.59-3.63 (2H, m), 3.73 (2H, t, J = 6.4 Hz), 5.01 (2H, s), 8.27 (2H, s); 13C NMR (400 MHz, d6-DMSO) δ -1.4 (CH3), 17.3 (CH2), 36.1 (CH2), 56.2 (CH2), 66.3 (CH2), 66.9 (CH2), 117.7 (CH), 136.8 (C), 137.1 (C), 166.1 (C), 166.2 (C).
2-Benzyl-6-(hydroxymethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000051_0002
A stirred mixture of 2-benzylpyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (2.00 g, 6.53 mmol) and paraformaldehyde (0.29 g, 9.66 mmol) in DMF (30 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (60 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (30 mL), then dried in vacuo to afford the title compound as a beige solid (1.68 g, 5.00 mmol, 77%). 1H NMR (400 MHz, d6-DMSO) δ 4.83 (2H, s), 5.02 (2H, d, J = 7.1 Hz), 6.53 (1H, t, J = 7.1 Hz), 7.27-7.37 (5H, m), 8.27 (2H, s); 13C NMR (400 MHz, d6-DMSO) δ 41.4 (CH2), 60.7 (CH2), 177.8 (CH), 127.5 (CH), 127.6 (CH), 128.6 (CH), 136.1 (C), 136.8 (C), 137.2 (C), 165.8 (C), 166.1 (C).
2-Benzylpyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000052_0001
To a stirred solution of pyromellitic imide anhydride (2.00 g, 9.21 mmol) in DMF (25 mL) at 0 °C was added benzylamine (1.10 mL, 10.1 mmol) dropwise, then the reaction mixture was heated at 100 °C for 18 h. After cooling to room temperature, the insoluble by-products were filtered off, and water (60 mL) was added to the filtrate. The precipitated solids were collected by filtration, washed with water (30 mL), then dried in vacuo to afford the title compound as a beige solid (2.20 g, 7.18 mmol, 78%). 1H NMR (400 MHz, d6-DMSO) δ 4.82 (2H, s), 7.26- 7.36 (5H, m), 8.17 (2H, s), 11.84 (1H, brs); 13C NMR (400 MHz, d6-DMSO) δ 41.3 (CH2), 117.4 (CH), 127.5 (CH), 128.6 (CH), 136.2 (C), 136.8 (C), 138.1 (C), 166.2 (C), 167.7 (C).
2-(Hydroxymethyl)-6-(pyridin-2-ylmethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)- tetraone
Figure imgf000052_0002
A stirred mixture of 2-(pyridin-2-ylmethyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.37 g, 1.19 mmol) and paraformaldehyde (71.3 mg, 2.38 mmol) in DMF (7.5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a colourless solid (0.23 g, 0.67 mmol, 56%). 1H NMR (400 MHz, d6-DMSO) δ 4.97 (2H, s), 5.03 (2H, d, J = 7.1 Hz), 6.55 (1H, t, J = 7.1 Hz), 7.29 (1H, ddd, 7 = 7.5, 4.9 and 1.0 Hz), 7.47 (1H, d, J = 7.9 Hz), 7.78 (1H, td, J = 11.5 and 1.8 Hz), 8.44 (2H, s), 8.44 (1H, ddd, J = 4.8, 1.7 and 0.9 Hz); 13C NMR (100 MHz, d6-DMSO) δ 42.6 (CH2), 60.7 (CH2), 117.9 (CH), 121.5 (CH), 122.7 (CH), 136.9 (C), 137.0 (C), 137.2 (CH), 149.1 (CH), 154.6 (C), 165.8 (C), 166.1 (C).
2-(Pyridin-2-ylmethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000053_0001
A stirred mixture of pyromellitic imide anhydride (1.10 g, 5.07 mmol) and 2- (aminomethyl)pyridine (0.60 g, 5.57 mmol) in DMF (10 mL) was heated at 100 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL) and dried in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded the title compound as a colourless solid (0.39 g, 1.27 mmol, 25%). 1H NMR (400 MHz, d6-DMSO) δ 4.97 (2H, s), 7.28 (1H, ddd, J = 7.5, 4.9 and 1.1 Hz), 7.46 (1H, d, J = 7.9 Hz), 7.78 (1H, td, J = 11.6 and 1.8 Hz), 8.19 (2H, s), 8.43 (1H, ddd, 7 = 4.9, 1.8 and 1.0 Hz), 11.85 (1H, brs); 13C NMR (100 MHz, d6-DMSO) δ 42.5 (CH2), 117.4 (CH), 121.5 (CH), 122.6 (CH), 136.8 (C), 136.9 (CH), 138.3 (C), 149.1 (CH), 154.6 (C), 166.2 (C), 167.8 (C).
2-(6-(Hydroxymethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)acetic add
Figure imgf000053_0002
A stirred solution of 2-(l, 3,5, 7-tetraoxo-3, 5,6,7 -tetrahydropyrrolo[3,4-f|isoindol-2(1H)- yl)acetic acid (0.30 g, 1.09 mmol) and paraformaldehyde (50.0 mg, 1.67 mmol) in DMF (10 mL) was heated at 80 °C for 18 h. After cooling to room temperature, the reaction mixture was dried in vacuo to afford the title compound as a beige solid (0.30 g, 0.99 mmol, 91%). 1H NMR (400 MHz, d6-DMSO) δ 4.39 (2H, s), 5.02 (2H, d, J = 7.0 Hz), 6.55 (1H, t, J = 7.1 Hz), 8.33 (2H, s), 13.36 (1H, brs); 13C NMR (400 MHz, d6-DMSO) δ 39.5 (CH2), 60.7 (CH2), 118.1 (CH), 136.8 (C), 137.2 (C), 165.5 (C), 165.7 (C), 168.5 (C).
2-(l,3,5,7-Tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(1H)-yl)aceticacid
Figure imgf000054_0001
To a stirred solution of pyromellitic imide anhydride (0.75 g, 3.45 mmol) in DMF (10 mL) at 0 °C was added glycine (0.29 g, 3.86 mmol), then the reaction mixture was heated at 100 °C for 18 h. After cooling to room temperature, water (10 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (10 mL), then dried in vacuo to afford the title compound as a colourless solid (0.46 g, 1.68 mmol, 49%). 1H NMR (400 MHz, d6-DMSO) δ 4.39 (2H, s), 8.22 (2H, s), 11.86 (1H, s), 13.33 (1H, brs); 13C NMR (400 MHz, d6-DMSO) δ 39.5 (CH2), 117.7 (CH), 136.5 (C), 138.4 (C), 165.6 (C), 167.5 (C), 168.5 (C).
Methyl 2-(6-( hydroxy methyl)- 1,3.5, 7-tetraoxo-3,5, 6,7-tetrahydropyrrolo|3,4-f]isoindol-
2(lH)-yl)acetate
Figure imgf000054_0002
A stirred mixture of methyl 2-(l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f|isoindol- 2(1H)-yl)acetate (0.25 g, 0.87 mmol) and paraformaldehyde (52.1 mg, 1.73 mmol) in DMF (7.5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a yellow solid (0.12 g, 0.38 mmol, 43%). 1H NMR (400 MHz d6-DMSO) δ 3.71 (3H, s), 4.52 (2H, s), 5.02 (2H, d, J = 7.1 Hz), 6.55 (1H, t, J = 7.1 Hz), 8.33 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 39.1 (CH2), 52.6 (CH3), 60.7 (CH2), 118.2 (CH), 136.7 (C), 137.3 (C), 165.4 (C), 165.7 (C), 167.7 (C).
Methyl 2-(l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)-yl)acetate
Figure imgf000054_0003
A mixture of glycine methyl ester hydrochloride (0.64 g, 5.07 mmol) and triethylamine (0.71 mL, 5.07 mmol) in DMF (10 mL) was stirred at room temperature for 30 min, before pyromellitic imide anhydride (1.00 g, 4.61 mmol) was added. The reaction mixture was then stirred at 50 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL) and dried in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 1:1) afforded the title compound as a pale-yellow solid (0.27 g, 0.94 mmol, 20%). 1H NMR (400 MHz, d6-DMSO) δ 3.71 (3H, s), 4.52 (2H, s), 8.23 (2H, s), 11.87 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 38.9 (CH2), 52.6 (CH3), 117.8 (CH), 136.4 (C), 138.4 (C), 165.5 (C), 167.4 (C), 167.7 (C).
2-(6-(Hydroxymethyl)-l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4-f]isoindol-2(lH)- yl)-N,N -dimethylacetamide
Figure imgf000055_0001
A stirred mixture of N,N-dimethyl-2-(l,3,5,7-tetraoxo-3,5,6,7-tetrahydropyrrolo[3,4- |isoindol-2(177)-yl)acetamide (0.30 g, 1.00 mmol) and paraformaldehyde (59.8 mg, 1.99 mmol) in DMF (5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a colourless solid (0.21 g, 0.63 mmol, 64%). 1H NMR (400 MHz d6-DMSO) δ 2.85 (3H, s), 3.09 (3H, s), 4.58 (2H, s), 5.02 (2H, d, J = 7.1 Hz), 6.54 (1H, t, J = 7.1 Hz), 8.31 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 35.2 (CH3), 35.8 (CH3), 39.5 (CH2), 60.7 (CH2), 117.9 (CH), 137.0 (C), 137.2 (C), 164.9 (C), 165.8 (C), 165.9 (C). N,N -I)imethyl-2-( L3.5,7-tetraoxo-3.5,6,7-tetrahydropyrrolo|3.4-f]isoindol-2(1H)- yl)acetamide
Figure imgf000055_0002
A stirred mixture of pyromellitic imide anhydride (1.00 g, 4.61 mmol) and 2-amino- N,N- dimethylacetamide (0.56 g, 5.53 mmol) in DMF (20 mL) was heated at 100 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a colourless solid (0.50 g, 1.66 mmol, 36%). 1H NMR (400 MHz, d6-DMSO) δ 2.84 (3H, s), 3.09 (3H, s), 4.57 (2H, s), 8.20 (2H, s), 11.85 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 35.2 (CH3), 35.8 (CH3), 39.5 (CH2), 117.5 (CH), 136.7 (C), 138.3 (C), 164.9 (C), 166.0 (C), 167.5 (C).
2-(2,5,8,ll,14-Pentaoxahexadecan-16-yl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000056_0001
A stirred mixture of 2-(2,5,8,l l,14-pentaoxahexadecan-16-yl)pyrrolo[3,4-f|isoindole- l,3,5,7(2H,6H)-tetraone (0.45 g, 1.00 mmol) and paraformaldehyde (60.0 mg, 2.00 mmol) in DMF (10 mL) was heated at 80 °C for 18 h. After cooling to room temperature, the reaction mixture was dried in vacuo to afford the title compound as a colourless oil (0.44 g, 0.92 mmol, 92%). 1H NMR (400 MHz d6-DMSO) δ 3.25 (3H, s), 3.42-3.55 (16H, m), 3.68 (2H, t, J = 6.0 Hz), 3.82 (2H, t, J = 5.8 Hz), 5.04 (2H, brs), 6.55 (1H, brs), 8.28 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 37.7 (CH2), 58.0 (CH3), 60.6 (CH2), 66.8 (CH2), 69.5 (CH2), 69.6 (CH2), 69.7 (CH2), 71.2 (CH2), 117.6 (CH), 136.9 (C), 137.1 (C), 165.8 (C), 166.1 (C).
2-(2,5,8,ll,14-Pentaoxahexadecan-16-yl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000056_0002
A stirred mixture of pyromellitic imide anhydride (0.50 g, 2.30 mmol) and 2,5,8,11,14- pentaoxahexadecan-16-amine (0.58 g, 2.30 mmol) in DMF (5 mL) was heated at 80 °C for 18 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (30 mL) and washed with water (30 mL). The organic layer was washed with brine (2 x 30 mL), dried over anhydrous MgSO4, filtered and then concentrated in vacuo to afford the title compound as a colourless solid (0.47 g, 1.05 mmol, 46%). 1H NMR (400 MHz, d6-DMSO) δ 3.22 (3H, s), 3.40-3.53 (16H, m), 3.65 (2H, t, J = 5.9 Hz), 3.80 (2H, t, J = 6.0 Hz), 8.15 (2H, s), 11.83 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 37.7 (CH2), 58.0 (CH3), 66.8 (CH2), 69.4 (CH2), 69.5 (CH2), 69.6 (CH2), 69.7 (CH2), 71.2 (CH2), 117.3 (CH), 136.8 (C), 138.0 (C), 166.1 (C), 167.5 (C); ESI-MS: m/z calcd for C2IH26N2O9: 450.1638; found [M+Na]+: 473.1520.
2-(2-Hydroxyethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000057_0001
To a stirred solution of pyromellitic imide anhydride (2.00 g, 9.21 mmol) in DMF (25 mL) at 0 °C was added ethanolamine (0.56 mL, 9.28 mmol) dropwise, then the reaction mixture was heated at 100 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (40 mL), then dried in vacuo to afford the tile compound as a colourless solid (1.71 g, 6.57 mmol, 71%). 1H NMR (400 MHz, d6-DMSO) δ 3.59-3.64 (2H, m), 3.68-3.71 (2H, m), 4.85-4.88 (1H, m), 8.14 (2H, s), 11.82 (1H, brs); 13C NMR (400 MHz, d6-DMSO) δ 40.9 (CH2), 57.8 (CH2), 117.1 (CH), 137.0 (C), 137.9 (C), 166.4 (C), 167.6 (C).
2-(Hydroxymethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000057_0002
A stirred mixture of pyromellitic diimide (1 g, 4.63 mmol) and paraformaldehyde (111 mg, 3.70 mmol) in DMF (100 mL) was heated at 80 °C for 18 h. After cooling to room temperature, the reaction mixture was concentrated in vacuo. Purification by flash column chromatography (petroleum ether/EtOAc, 19:1) afforded the title compound as a colourless solid (170 mg, 0.69 mmol, 15%). 1H NMR (400 MHz, d6-DMSO) δ 5.01 (2H, d, J = 7.1 Hz), 6.52 (1H, t, J = 7.1 Hz), 8.20 (2H, s), 11.9 (1H, s); 13C NMR (100 MHz, d6-DMSO) δ 60.6 (CH2), 117.6 (CH), 116.7 (C), 138.2 (C), 165.8 (C), 167.5 (C).
Pyromellitic imide anhydride
Figure imgf000058_0001
To a solution of pyromellitic dianhydride (20 g) in tetrahydrofuran (200 mL) at room temperature was added water (11.4 mL), and the reaction stirred at room temperature for a further 15 min. After cooling to 0 °C, anhydrous magnesium sulfate (68.8 g) was added and the reaction stirred at 0 °C for a further 15 minutes, before allowing to warm to room temperature. After a further 30 minutes at room temperature the reaction mixture was filtered, and the collected solid washed with tetrahydrofuran (2 x 100 mL). The filtrate was then cooled to 0 °C and saturated with anhydrous ammonia gas - rapidly forming a precipitate (at which point 200 mL of acetone was added to increase the efficiency of the stirring - and the mixture then re-saturated with further ammonia gas). After stirring at 0 °C for a further 20 minutes the insoluble material was then collected by filtration, washed with acetone and dried in vacuo to afford 2,4,5-tricarboxybenzamide as a white solid (24.6 g), which was used without further purification.
A solution of 2,4,5-tricarboxybenzamide (24.6 g) in thionyl chloride (200 mL) was heated at reflux for 2 hours. Upon cooling, hexane (200 mL) was added and the mixture stirred at room temperature for a further 30 minutes. The insoluble material was then collected by filtration, washed thoroughly with hexane, followed by ice-cold DMF:toluene (200 mL, 1:4 v/v) to remove the impurities (pyromellitic dianhydride and pyromellitic diimide). The resulting solid was then dissolved in acetone (400 mL) and any insoluble salts were filtered off. The collected filtrate was then reduced in vacuo to afford the title compound as a light beige solid (16.4 g).
2-(2-Hydroxyethyl)-6-(hydroxymethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)-tetraone
Figure imgf000058_0002
A stirred mixture of 2-(2-hydroxyethyl)pyrrolo[3,4-f|isoindole-l,3,5,7(2H,6H)-tetraone (0.50 g, 1.92 mmol) and paraformaldehyde (87.0 mg, 2.90 mmol) in DMF (10 mL) was heated at 80 °C for 18 h. After cooling to room temperature, water (50 mL) was added to the reaction mixture. The precipitated solids were collected by filtration, washed with water (50 mL), then dried in vacuo to afford the title compound as a colourless solid (0.33 g, 1.14 mmol, 59%). 1H NMR (400 MHz d6-DMSO) δ 3.59-5.64 (2H, m), 3.69-3.72 (2H, m), 4.86 (1H, t, J = 6.0 Hz), 5.02 (2H, d, J = 7.0 Hz), 6.52 (1H, t, J = 7.1 Hz), 8.25 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 40.9 (CH2), 57.8 (CH3), 60.7 (CH2), 117.5 (CH), 136.8 (C), 137.3 (C), 165.8 (C), 166.3 (C).
2-(2,3-Dihydroxypropyl)-6-(2-hydroxyethyl)pyrrolo[3,4-f]isoindole-l,3,5,7(2H,6H)- tetraone
Figure imgf000059_0001
To a solution of pyromellitic dianhydride (1.00 g, 4.58 mmol) in DMF (50 mL) was added slowly ethanolamine (280 mg, 4.58 mmol) in DMF (5 mL) and 3 -aminopropane- 1,2-diol (418 mg, 4.58 mmol) in DMF (5 mL). The reaction mixture then was heated at 100 °C for 18 h, before being diluted with water (100 mL). The precipitated solids were then collected by filtration, washed with water (3 x 50 mL), then dried in vacuo. Purification by flash column chromatography (CH2CI2/McOH, 9:1) afforded the title compound as a colourless solid (100 mg, 0.30 mmol, 7%). 1H NMR (400 MHz, d6-DMSO) δ 3.34-3.46 (2H, m), 3.60-3.65 (4H, m), 3.70 (2H, t, J = 5.3 Hz), 3.80-3.88 (1H, m), 4.68 (1H, t, J = 5.6 Hz), 4.87 (1H, t, J = 6.1 Hz), 4.96 (1H, d, J = 5.1 Hz), 8.20 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 40.9 (CH2), 42.3 (CH2), 57.8 (CH2), 64.1 (CH2), 68.3 (CH), 117.0 (CH), 137.0 (2 x C), 166.39 (C), 166.44 (C).
2-(2,3-Dihydroxypropyl)-6-(l-hydroxypropan-2-yl)pyrrolo[3,4-f]isoindole-
1 ,3,5,7 (2H,6H ) -tetraone
Figure imgf000059_0002
To a solution of pyromellitic dianhydride (1.00 g, 4.58 mmol) in DMF (50 mL) was added slowly 2-amino-l -propanol (344 mg, 4.58 mmol) in DMF (5 mL) and 3 -aminopropane- 1,2- diol (418 mg, 4.58 mmol) in DMF (5 mL). The reaction mixture was then heated at 100 °C for 18 h, before being diluted with water (100 mL). The precipitated solids were then collected by filtration, washed with water (3 x 50 mL), then dried in vacuo. Purification by flash column chromatography (EtOAc, neat) afforded the title compound as a colourless solid (45 mg, 0.129 mmol, 3%). 1H NMR (400 MHz, d6-DMSO) δ 1.35 (3H, d, J = 7.1 Hz), 3.34-3.45 (2H, m), 3.53-3.58 (1H, m), 3.64 (2H, d, J = 6.5 Hz), 3.82-3.88 (2H, m), 4.26-4.35 (1H, m), 4.70 (1H, t, J = 5.7 Hz), 4.97 (1H, dd, J = 6.8 and 5.4 Hz), 4.98 (1H, d, J = 5.2 Hz), 8.18 (2H, s); 13C NMR (100 MHz, d6-DMSO) δ 14.3 (CH3), 42.3 (CH), 49.8 (CH2), 61.7 (CH2), 64.1 (CH2), 68.3 (CH), 116.9 (CH), 136.8 (C), 137.0 (C), 166.49 (C), 166.52 (C).
In vitro studies
The compounds of the present invention were initially screened for activity using an in vitro fully automated incubation system for the measurement of total gas production and gas composition (methane and hydrogen). The system used is described in detail in the paper by Muetzel et al. (2014; Animal Feed Science and Technology 196, 1-11). The system in this specification is referred to as the rumen in vitro system or RIV. The RIV system provides a relatively simple tool for determining the rate and production of methane and also whether the rate and production of methane is inhibited or reduced by a particular compound or feed.
Rumen in vitro assay method.
Rumen in vitro assays use rumen fluid from donor animals (typically either sheep or cattle) which is typically combined with a buffer and then incubated in sealed fermentation vessels at 39 °C for either 24 or 48 h. These assays systems have been well-characterised and used by the scientific community over the last two decades (Rymer et al. 2005). The system that has been used here to characterise the effects of inhibitors on methane production and gas production, and the formation of H2, was described by Muetzel et al. (2014). Rumen in vitro assay systems reflect what can occur in the rumen in vivo, but only short-term, due to their short incubation times (typically not exceeding 48 h) which is limited by its buffering capacity and the fact that it is a closed system. Stock solutions of inhibitors were re-suspended in dimethylformamide (DMF) at concentrations 1000-fold higher than the highest used for the assays. Any further dilutions were also prepared using DMF. The total amount of inhibitor solution added to the rumen fluidbuffer mixture (60 mL) was 60 pl. Incubations used 60 ml of medium containing 12 ml of filtered rumen fluid and 48 ml of buffer in serum bottles for 24-48 h, essentially as described by Muetzel et al. (2014). Two fistulated cattle were used as donor animals for rumen fluid and treatments were incubated in duplicate bottles. Sets of duplicate incubation vials that contained rye grass (with rumen fluid -buffer mixture), and rye grass with 30 pM bromoethanesulfonate (BES) were also incubated as negative and positive controls, respectively. One experiment consists of 2 replicates of an inhibitor at any one concentration run at the same time, and a run can contain up to 32 bottle, including positive (30 pM BES) and negative (no inhibitor added) controls.
Compound 12, for example, was tested using the 60 mL rumen in vitro assay system at 100 pM and 2 pM final concentrations and compared to a positive control with 30 pM BES. At 30 pM and 2 pM, inhibition levels were approximately 86.4 % and 16.6 % at 18 h, respectively.
Other compounds of Formula I were tested in the RIV using the same method. All the compounds were either sourced commercially from chemical supply companies or synthesised by chemical synthesis companies. It can be seen from the results of the RIV experimentation tabulated below, and the subsequent results of the in vivo testing that is described below, that the RIV results for the compounds of Formula I are somewhat predictive of in vivo activity.
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
In vivo studies
The animal experiments described in this specification were all reviewed and approved by the Grasslands Animal Ethics Committee (Palmerston North, NZ) and all animals were cared for according the Code of Ethical Conduct (Animal Welfare Act, 1999) and its amendments.
Example 1
Toxicity testing in mice
The potential toxicology for Compound 12 was tested according to OECD 423. Mice testing used two groups with three mice (female Swiss mice, non-pregnant), all at approximately 8-9 weeks of age. On the day of testing, food was withdrawn 1-2 hours prior to dosing. Following fasting, the animals were weighed and the test substances administered. Access to food was resumed 1-2 hours after administration of the compounds. The compounds were administered in a single dose by oral gavage in corn oil (maximum of 0.3 ml per dose) at the selected dose and the time recorded. The mice were observed individually after dosing at least once during the first 30 minutes, periodically over the first 24 hours, with special attention given to the first 4 hours, and then daily thereafter for a total of 14 days. The mice were weighed every seven days (0, 7 and 14 days). All observations were systematically recorded for each individual animal. These observations included (if present): tremors, convulsions, salivation, diarrhoea, lethargy, sleep, coma, and changes in fur, skin, eyes, mucus membranes, respiration, motor activity and behaviour. No adverse effects were noted for Compound 12 at 300 mg/kg.
In vivo trial methods
The trial for testing the effects of Compound 12 on methane emissions from sheep over a 48- hour period in methane measurement chambers was approved by the Grasslands Animal Ethics Committee, and the use of the compounds in animals by the New Zealand Food Safety Authority, as required under ACVM legislation. Three sheep were used for the Compound 12 treatment group. The animals received a general -purpose diet (GP) at 1.5 x maintenance energy requirements throughout the trials. Each 1000 g of GP diet consisted of the following proportions of 500 g hay, 100 g soybean meal, 290 g barley, 100 g molasses and 10 g mineral mix. During the time in the respiration chambers the animals had free access to water, with the feed offered twice a day at approximately 9:00 and 16:00 h in equal amounts. Feed refusals were weighed.
The animals were adapted to GP diet for 14 days prior to entering the methane measurement chambers.
After the 14-day acclimatisation period, methane emissions were measured in open circuit respiration chambers as described in Chapter 1 of the technical manual on respiration chamber design (http://www.globalresearchalliance.org/wp-content/uploads/2012/03/GRA-MAN- Facility-BestPract-2012-ch 13.pdf) . Dry matter intake (DMI, kg/d) was recorded during methane measurements from the weight difference of feed offered and refused. The first day in the chambers (24-hour period; Period 1 (Pl)) served as a control for each animal (to determine background methane emissions). For the next two days (Period 2 (P2)) the sheep received Compound 12 at 68 mg/day (evenly split between morning and afternoon feeds).
Results Methane gas outputs for the 72-hour period (first 24 hours for background measurements, followed by two days of dosing once each morning) in open circuit respiration chambers were determined and are summarised in Table 1.
Table 1: Data for 2-day dosing with Compound 12. P2 values are the average of the first and second days of dosing.
Figure imgf000067_0001
It can be seen from the results that Compound 12 at 68 mg/day caused a 21.6% inhibition (e.g., average of three animals) reductions in methane emissions (CH4 g/day) and a 21.4% inhibition (e.g., average of three animals) reductions methane yield (g CH4/kg feed) in Period 2, respectively. There was no drop in feed intake and H2 was observed in the Compound 12- treated group compared to the background measurements, indicative of a direct and specific effect on methane-forming microorganisms.
The present invention and its embodiments have been described in detail. However, the scope of the present invention is not intended to be limited to the particular embodiments of the invention described in the specification. Various modifications, substitutions, and variations can be made to the disclosed material without departing from the spirit and/or essential characteristics of the present invention. Accordingly, one of ordinary skill in the art will readily appreciate from the disclosure that later modifications, substitutions, and/or variations performing substantially the same function or achieving substantially the same result as embodiments described herein may be utilized according to such related embodiments of the present invention. Thus, the following claims are intended to encompass within their scope modifications, substitutions, and variations to the embodiments of the invention disclosed herein.

Claims

Claims
1 A compound of Formula I
Figure imgf000068_0001
Formula I wherein R1 is selected from -H, -alkyl, alkyl-ONO2, or -ONO2; wherein X is different to R1 and is selected from -H, alkyl-ONO2, -ONO2 or a substituent of Formula II, Formula II
Figure imgf000068_0002
wherein each n is independently selected from 0, 1, 2, 3, 4, 5 or 6 and when n is 0, 1, 2, 3, 4, 5, 6 any adjacent carbons are optionally unsaturated to form a double or triple bond, wherein R2 is selected from -H, -alkyl, - alkyl-ONO2, -aryl, -heteroaryl or -heterocyclyl or - CO2R3; wherein -R1 and -R2 together form an optionally substituted aliphatic ring which is optionally saturated; wherein -Z is selected from -H, -alkyl, -aryl, -heteroaryl, -heterocyclyl, -CO2R3, -C(=O)R3, - alkyl-OH, -(alkyl-OH)-alkyl-OH, -alkyl-(alkyl-OH)2, -alkyl-(alkyl-OH)3,
-(alkyl-O-alkyl)k, -(alkyl-O)k-L; -alkyl-(C=O)O-(alkyl-O)k-L; -alkyl-SH, -alkyl-S-alkyl, - alkyl-(S=O)-alkyl, -alkyl-(SO2)-alkyl; -alkyl-(SO2)-OH; -alkyl-(SO2)-NH2; -alkyl-(SO2)- NHalkyl, -alkyl-(SO2)-N(alkyl)2; -alkyl-(SO2)-NH(alkyl-O)k-L; -alkyl-(C=O)-NH2; -alkyl- (C=O)-NH(alkyl); -alkyl-(C=O)-N(alkyl)2; -alkyl-(C=O)-NH(alkyl-O)k-L; -alkyl heterocyclyl, wherein k is 1, 2, 3, 4, 5 or 6; and wherein L is -H, -alkyl; wherein R3 is selected from -H, -alkyl, -NHalkyl, -N(alkyl)2 or aryl with the proviso that R3 is only selected from -H, -alkyl, or -aryl for -CO2R3; or
Z is a substituent of Formula III
Figure imgf000069_0001
Formula III wherein m is 0, 1, 2, 3 or 4, and when m is 0, 1, 2, 3 or 4, any adjacent carbons are optionally unsaturated to form a double or triple bond; Y is selected from -CH2-, -O-, -C(=O)- -S-, -S(=O)- , -SO2-, -NH- or -N(alkyl)- p is selected from 0, or 1 ;
R4 when present is selected from -H, -alkyl, or -alkyl- ONO2;
R5 when present is selected from -H, -OH, -O-alkyl, -NH2, -alkyl, -NH(alkyl), -N(alkyl)2, - ONO2, -alkyl-ONO2, -aryl, -heteroaryl, -heterocyclyl, -C(= O)R3 or - CO2R3; or or a salt thereof.
2 The compound of Formula I as claimed in claim 1, which includes at least one -ONO2 substituent.
3 The compound of Formula I, as claimed in claim 1 or claim 2, wherein Ri is -H, X is - ONO2 and Z is a compound of Formula III.
4 The compound of Formula I as claimed in claim 1 , wherein Z is Formula III, R4 is selected from -H or -alkyl-ONO2 and R5 is selected from -H, -O-alkyl, -ONO2, -CO2-alkyl or aryl.
5 The compound of Formula I as claimed in claim 1 or claim 2, wherein Z is selected from -H, -alkyl, -aryl, -heteroaryl, -heterocyclyl, -CO2R3, -C(=O)R3 ; -(alkyl-O)k-L wherein R3 is selected from -H, -alkyl or -aryl; wherein k is 1, 2, 3, 4, 5 or 6; and wherein L is -H, -alkyl; or
Z is a substituent of Formula III
Figure imgf000070_0001
Formula III wherein m is 0, 1, 2, 3 or 4, and when m is 0, 1, 2, 3 or 4, any adjacent carbons are optionally unsaturated to form a double or triple bond; wherein Y is selected from -CH2-, -O-, -C(=O) -S-, -S(=O)-, -SO2-, -N(alkyl)-; wherein p is selected from 0, or 1 ; wherein R4 is selected from -H, or -alkyl-ONO2; wherein R5 is selected from -H, -OH, -NH2, -alkyl, -NH(alkyl), -N(alkyl)2, -ONO2, -alkyl- ONO2, -aryl, -heteroaryl, -heterocyclyl, C(=O)R3 or - CO2R3.
6 The compound of Formula I as claimed in claim 5, wherein Z is selected from -H, -alkyl, -aryl, -heteroaryl, -heterocyclyl, -CO2R3, -C(=O)R3 -(CH2CH2O)k-L, wherein R3 is selected from H, alkyl or aryl and wherein k isl, 2, 3, 4, 5 or 6; and wherein L is -H or -alkyl.
7 The compound of Formula I as claimed in claim 1 , wherein X is a compound of Formula II, n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2 and p is 0.
8 The compound of Formula I as claimed in claim 1, wherein R1 is -H, X is -ONO2; Z is -H, -alkyl or a substituent of Formula III.
9 The compound of Formula I as claimed in claim 1, wherein n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2.
10 The compound of Formula I as claimed in claim 1, wherein R1 is -H, X is Formula II, n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2.
11 The compound of Formula I as claimed in claim 1, wherein R1 is -H, X is Formula II, R2 is -CH2ONO2, n is 0, 1 or 2 and Z is a substituent of Formula III and m is 0, 1 or 2. 12 The compound of Formula I or a salt thereof as claimed in any one of claims 1 to 11 being selected from one or more of the following:
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0005
13 The compound of Formula I as claimed in claim 12 being
Figure imgf000074_0001
14 The compound of Formula I as claimed in claim 12 being
Figure imgf000074_0002
15 The compound of Formula I as claimed in claim 12 being or a salt thereof.
Figure imgf000074_0003
16 The compound of Formula I as claimed in claim 12 being
Figure imgf000074_0004
17 The compound of Formula I as claimed in claim 12 being or a salt thereof.
Figure imgf000075_0001
18 The compound of Formula I as claimed in claim 12 being or a salt thereof.
Figure imgf000075_0002
19 The compound of Formula I as claimed in claim 12 being or a salt thereof.
Figure imgf000075_0003
20 The compound of Formula I as claimed in claim 12 being or a salt thereof.
Figure imgf000075_0004
21 The compound of Formula I as claimed in claim 12 being
Figure imgf000075_0005
22 The use of a compound of Formula I as claimed in any one of claims 1 to 21 for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.
23 The use of a compound of Formula I as claimed in any one of claims 1 to 21 for reducing the formation of methane from the digestive actions of ruminants.
24 The use of a compound of Formula I as claimed in any one of claims 1 to 21 for reducing the formation of methane from the digestive actions of ruminants by at least 10%.
25 The use of a compound of Formula I as claimed in any one of claims 1 to 21 for reducing the formation of methane from the digestive actions of ruminants by at least 15%.
26 The use of a compound of Formula I as claimed in any one of claims 1 to 21 for reducing the formation of methane from the digestive actions of ruminants by at least 20%.
27 A method for reducing the production of methane emanating from a ruminant and/or for improving ruminant animal performance, comprising administering orally to the ruminant an effective amount of at least one compound of Formula I or a salt thereof as claimed in any one of claims 1 to 21 to the ruminant.
28 The method as claimed in claim 27, wherein the effective amount of at least one compound of Formula I or a salt thereof is administered at least once-daily to the ruminant.
29 The method as claimed in claim 28, wherein the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 10% per day.
30 The method as claimed in claim 29, wherein the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 15% per day.
31 The method as claimed in claim 30, wherein the effective amount of at least one compound of Formula I or a salt thereof reduces the production of methane emanating from the ruminant by at least 20% per day.
32 A composition for oral administration comprising at least one compound of Formula I or a salt thereof as claimed in any one of claims 1 to 21 for reducing the production of methane emanating from a ruminant, and the composition further including at least one agriculturally and orally acceptable excipient.
33 The composition as claimed in claim 32 being adapted for use as a feed additive.
34 The composition as claimed in claim 32 or claim 33 being adapted for use as a water additive.
35 The composition as claimed in claim 32 or claim 33 being adapted for use as a ruminant lick.
36 The composition as claimed in claim 32 being adapted for use as an oral drench.
37 The composition as claimed in claim 32 being adapted for use as a rumen bolus or capsule.
38 The composition as claimed in any one of claims 32 to 37 being adapted to reduce the production of methane emanating from the ruminant by at least 10% per day.
39 The composition as claimed in claim 38 being adapted to reduce the production of methane emanating from the ruminant by at least 15% per day.
40 The composition as claimed in claim 39 being adapted to reduce the production of methane emanating from the ruminant by at least 20% per day.
41 The composition as claimed in any one of claims 32 to 40 wherein the excipient includes one or more minerals and/or one or more vitamins.
42 The composition as claimed in claim 41 wherein the excipient includes one or more vitamins selected from vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3, vitamin B 12, biotin and choline, vitamin B l, vitamin B2, vitamin B6, niacin, folic acid or the like.
43 The composition as claimed in claim 42 wherein the excipient includes one or more minerals selected from calcium, phosphorus, sodium, manganese, zinc, iron, copper, chlorine, sulphur, magnesium, iodine, selenium, and cobalt or the like.
44 The composition as claimed in any one of claims 32 to 43 further including sunflower oil, electrolytes such as ammonium chloride, calcium carbonates, starch, proteins or the like.
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