WO2010130977A1 - Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants - Google Patents

Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants Download PDF

Info

Publication number
WO2010130977A1
WO2010130977A1 PCT/GB2010/000927 GB2010000927W WO2010130977A1 WO 2010130977 A1 WO2010130977 A1 WO 2010130977A1 GB 2010000927 W GB2010000927 W GB 2010000927W WO 2010130977 A1 WO2010130977 A1 WO 2010130977A1
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
halogenated
aryl
compound
alkenyl
Prior art date
Application number
PCT/GB2010/000927
Other languages
French (fr)
Inventor
Christopher Paul Larch
John Mervyn David Storey
Craig Williamson
Colin Marshall
Steven John Kemp
Original Assignee
Wista Laboratories Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wista Laboratories Ltd. filed Critical Wista Laboratories Ltd.
Priority to DK10718268.5T priority Critical patent/DK2430007T3/en
Priority to ES10718268.5T priority patent/ES2457227T3/en
Priority to PL10718268T priority patent/PL2430007T3/en
Priority to SI201030572T priority patent/SI2430007T1/en
Priority to EP10718268.5A priority patent/EP2430007B1/en
Priority to US13/318,907 priority patent/US8691979B2/en
Publication of WO2010130977A1 publication Critical patent/WO2010130977A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
    • C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
    • C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
    • C07D279/18—[b, e]-condensed with two six-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C381/00—Compounds containing carbon and sulfur and having functional groups not covered by groups C07C301/00 - C07C337/00
    • C07C381/02—Thiosulfates

Definitions

  • This invention pertains generally to the field of chemical synthesis and purification, and more specifically to methods of synthesizing and purifying certain 3,7-diamino- phenothiazin-5-ium compounds (referred to herein as "diaminophenothiazinium compounds”) including Methylthioninium Chloride (MTC) (also known as Methylene Blue).
  • MTC Methylthioninium Chloride
  • the present invention also pertains to the resulting compounds, and compositions comprising them (e.g., tablets, capsules).
  • Such compounds and compositions find use in methods of inactivating pathogens, and methods of medical treatment and diagnosis, etc., for example, for tauopathies, Alzheimer's disease (AD), skin cancer, melanoma, viral diseases, bacterial diseases and protozoal diseases.
  • Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about,” it will be understood that the particular value forms another embodiment.
  • Methylthioninium Chloride (also known as Methylene Blue)
  • Methylthioninium Chloride (also known as Methylene blue (MB); methylthionine chloride; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathionium chloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet; and Urolene Blue® is a low molecular weight (319.86), water soluble, tricyclic organic compound of the following formula:
  • Methylthioninium Chloride perhaps the most well known phenothiazine dye and redox indicator, has also been used as an optical probe of biophysical systems, as an intercalator in nanoporous materials, as a redox mediator, and in photoelectrochomic imaging.
  • MTC was first described in a German Patent in 1877 (Badische Anilin- und Soda- Fabrik, 1877). In that patent, MTC was synthesized by nitrosylation of dimethylaniline, subsequent reduction to form N,N-dimethyl-1 ,4-diaminobenzene, and subsequent oxidative coupling in the presence of hydrogen sulphide (H 2 S) and iron(lll) chloride (FeCI 3 ).
  • H 2 S hydrogen sulphide
  • FeCI 3 iron(lll) chloride
  • Bernthsen described subsequent studies of MTC and methods for its synthesis (see Bernthsen, 1885a, 1885b, 1889).
  • nitrosodimethylaniline is prepared from dimethylaniline by treatment with nitrite (NaNO 2 ) in aqueous acid (HCI) solution.
  • the nitroso compound is reduced to form p-aminodimethylaniline in aqueous acid (HCI) solution using zinc dust.
  • steps (c), (d), and (e) the p-aminodimethylaniline is oxidized in aqueous acid solution with another molecule of dimethylaniline, and simultaneously a thiosulfonic acid group is introduced; the ring is then closed using manganese dioxide or copper sulfate.
  • a clear neutral solution of p-aminodimethylaniline is acidified (H 2 SO 4 ), and a non-reducing zinc chloride solution is added (ZnCI 2 with Na 2 Cr 2 O 7 ).
  • Aluminium thiosulfate (AI 2 (S 2 O 3 ) 3 ) and sodium thiosulfate (Na 2 S 2 O 3 ) are added.
  • Sodium dichromate (Na 2 Cr 2 O 7 ) is added. The mixture is heated and aerated. Dimethylaniline is added.
  • Sodium dichromate (Na 2 Cr 2 O 7 ) is added. The mixture is heated, and becomes dark greenish-blue in colour due to the formation of the thiosulfonic acid of Bindschedler green.
  • Manganese dioxide or copper sulfate is added, and the mixture heated, and the dye precipitates from the concentrated zinc chloride solution.
  • MTC is synthesized commercially by oxidation of N,N-dimethyl-p-phenylene diamine with Na 2 Cr 2 O 7 in the presence of Na 2 S 2 O 3 , followed by further oxidation in the presence of N,N-dimethylamine.
  • Fierz-David et al., 1949 describes the synthesis of the zinc chloride double salt of MTC and the removal .of zinc by chelation with sodium carbonate followed by filtration to generate zinc free methylene blue.
  • WO 2006/032879 describes the synthesis of MTC, and other diaminophenothiazinium compounds, by a number of methods, including an adaptation of the Fierz-David and Blangley procedure described above. The methods described provide diaminophenothiazinium products having reduced levels of metals and contaminating organics.
  • WO 2006/032879 addresses the issue of residual Cr levels in a diaminophenothiazinium synthesis by reducing residual Cr(VI) to Cr(III), which is a much less toxic form, so that pharmaceutical standards can more easily be satisfied. This reduction was also found to greatly increase the yield of the final diaminophenothiazinium compound. Experimentally, it was also established that chromium can more easily be removed from a product when in the form of Cr(III) than when in the form of Cr(VI).
  • MTC and derivatives thereof have been found to be useful in the treatment of tauopathies (such as, for example, Alzheimer's disease) (see, for example, Wischik, CM., et al., 1996, 2002).
  • MTC MTC
  • Urolene Blue® Oral and parenteral formulations of MTC are commercially available in the United States, usually under the name Urolene Blue®. However, these formulations contain substantial amounts of metal impurities. These impurities are highly undesirable, and many (e.g., including Al 1 Cr, Fe, Cu) exceed the safety limits set by European health agencies.
  • diaminophenothiazinium compounds including MTC.
  • One aspect of the present invention pertains to a method of synthesis of diaminophenothiazinium compounds, including high purity diaminophenothiazinium compounds.
  • Another aspect of the present invention pertains to a method of purification of diaminophenothiazinium compounds.
  • Further aspects of the invention pertain to methods for the synthesis of intermediate compounds which are suitable for use in the synthesis of diaminophenothiazinium compounds.
  • Another aspect of the present invention pertains to diaminophenothiazinium compounds obtained or obtainable by the methods of the invention for use in therapy.
  • diaminophenothiazinium compounds of the following formula, collectively referred to herein as "diaminophenothiazinium compounds":
  • each of -R 1 and -R 9 is independently -H or -R A ; and each -R A is independently selected from C 1-4 alkyl; C 2 - 4 alkenyl; halogenated Ci -4 alkyl; C 5 .i 0 aryl; halogenated C 5 . 10 aryl; C 1-4 alkylene-C 5-1 oaryl and halogenated C 1-4 alkylene-C 5-10 aryl; each of -R 3NA and -R 3NB is independently selected from: C 1-4 alkyl; C ⁇ ⁇ alkenyl; halogenated Ci -4 alkyl; C 5-10 aryl; halogenated C 5 .
  • X " is an anionic counter ion.
  • diaminophenothiazinium compounds are themselves salts, they may also be provided in the form of a mixed salt (i.e., the diaminophenothiazinium compound in combination with another salt).
  • a mixed salt i.e., the diaminophenothiazinium compound in combination with another salt.
  • Such mixed salts are intended to be encompassed by the term "and pharmaceutically acceptable salts thereof.
  • a reference to a particular compound also includes salts thereof.
  • the diaminophenothiazinium compound is also referred to as compound Vl, and in certain embodiments, where X " is Cl " , compound VII.
  • the present invention pertains to methods for the preparation of certain 3,7-diamino-phenothiazin-5-ium compounds of the following formula:
  • the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "activated thio ether compounds” or “diaminoaryl sulphides”:
  • G-Il wherein A is an activating group and each of -R 10NA and -R 10NB is independently selected from: -H; C 1-4 alkyl; C 2-4 alkenyl; halogenated C 1-4 alkyl; C 5-10 aryl; halogenated C 5 . 10 aryl; C 1-4 alkylene-C 5- ioaryl and halogenated C 1-4 alkylene-C 5 . 10 aryl.
  • the groups -R 9 , -R 7NA and -R 7NB are as defined for the diaminophenothiazinium compounds.
  • the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "thiosulfonic acid compounds":
  • the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "activated imino compounds":
  • -R 1 , -R 9 , -R 3NA and -R 3NB , -R 7NA and -R 7NB are as defined for the diaminophenothiazinium compounds, and -A is as defined for the activated thio ether compounds (diaminoaryl sulphides).
  • the compound G-IV is shown as a cation, which may be used as a salt with a suitable counter ion, X " .
  • Compound G-IV may also be used in a zwitterionic form where a negative charge is associated with the group -A.
  • the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "imino compounds":
  • the C 1-4 alkyl groups are selected from: linear C 1-4 alkyl groups, such as -Me, -Et, -nPr, -iPr, and -nBu; branched C 3-4 alkyl groups, such as -iPr, -iBu, -sBu, and -tBu; and cyclic C 3-4 alkyl groups, such as -cPr and -cBu.
  • the halogenated C 1-4 alkyl groups are selected from: -CF 3 , -CH 2 CF 3 , and -CF 2 CF 3 .
  • the C 5- i 0 aryl groups are selected from C 6- iocarboaryl groups, such as phenyl or napthyl, and C 5 . 10 heteroaryl groups, such as thienyl, imidazolyl, pyrazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl or quinolinyl.
  • C 6- iocarboaryl groups such as phenyl or napthyl
  • C 5 . 10 heteroaryl groups such as thienyl, imidazolyl, pyrazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl or quinolinyl.
  • the halogenated C 5-10 aryl groups are selected from halogenated C 6-10 carboaryl groups, such as -4-fluoro-phenyl, -3-fluoro-phenyl, and -2-fluoro-phenyl, and halogenated C 5 .i 0 heteroaryl groups.
  • the group is benzyl.
  • the halogenated C 1 . 4 alkylene-C 5 . 1 oaryl groups are selected from halogenated C 1-4 alkylene-C 6 -iocarboaryl groups, such as -4-fluoro-benzyl, -3-fluoro- benzyl, and -2-fluoro-benzyl, and halogenated C 1-4 alkylene-C 5 .ioheteroaryl groups.
  • each of -R 1 and -R 9 is independently -H or -R A .
  • each -R 1 is independently -H.
  • each -R 9 is independently -H.
  • At least one -R 1 is independently -R A .
  • At least one -R 9 is independently -R A .
  • -R 1 and -R 9 are the same. In one embodiment, -R 1 and -R 9 , where present, are different.
  • only one -R 9 is other than -H. In one embodiment, only one -R 1 is other than -H.
  • each -R A is independently selected from C 1-4 alkyl; C 2 . 4 alkenyl; halogenated C 1-4 alkyl; C 5- i 0 aryl; halogenated C 5- ioaryl; Ci. 4 alkylene-C 5 .i 0 aryl and halogenated Ci. 4 alkylene-C 5- i 0 aryl;
  • each -R A is independently selected from C 1-4 alkyl
  • each -R A is independently -Me, -Et, or -CF 3 .
  • each -R A is independently C 1 . 4 alkyl. In one embodiment, each -R A , where present, is independently -Me. In one embodiment, each -R A , where present, is independently -Et.
  • each -R A is independently C 2-4 alkenyl.
  • each -R A is independently halogenated C 1-4 alkyl. In one embodiment, each -R A , where present, is independently -CF 3 .
  • each -R A where present, is independently C 5 .i 0 aryl.
  • each -R A is independently C 6- iocarboaryl.
  • each -R A where present, is independently phenyl.
  • each -R A is independently Ci -4 alkylene-C 5 .i 0 aryl. In one embodiment, each -R A , where present, is independently benzyl.
  • each -R A is independently halogenated C 5 .i 0 aryl.
  • each -R A where present, is independently halogenated
  • each -R A is independently -4-fluoro-phenyl
  • each of -R 3NA and -R 3NB is independently selected from: C 1-4 alkyl; C 2-4 alkenyl; halogenated Ci -4 alkyl; C 5-10 aryl; halogenated C 5 -i 0 aryl; Ci -4 alkylene-C 5 . 10 aryl and halogenated d ⁇ alkylene-Cs. ⁇ aryl.
  • each of -R 3NA and -R 3NB is independently selected from: C 1-4 alkyl; C 2 - 4 alkenyl; and halogenated Ci. 4 alkyl.
  • each of -R 3NA and -R 3NB is independently -Me, -Et, -nPr, -nBu,
  • each of -R 3NA and -R 3NB is independently C 1-4 alkyl.
  • each of -R 3NA and -R 3NB is independently -Me or -Et.
  • each of -R 3NA and -R 3NB is independently -Me.
  • each of -R 3NA and -R 3NB is independently -Et.
  • -R 3NA and -R 3NB are the same. In one embodiment, -R 3NA and -R 3NB are different.
  • each of -R 7NA and -R 7NB is independently selected from d -4 alkyl; C 2 . 4 alkenyl; halogenated C 1-4 alkyl; C 5- ioaryl; halogenated C 5-10 aryl; and halogenated C 1-4 alkylene-C 5-10 aryl.
  • each of -R 7NA and -R 7NB is independently selected from C 1-4 alkyl; C 2-4 alkenyl; and halogenated C 1-4 alkyl.
  • each of -R 7NA and -R 7NB is independently -Me, -Et, -nPr, -nBu,
  • each of -R 7NA and -R 7NB is independently C 1-4 alkyl.
  • each of -R 7NA and -R 7NB is independently -Me or -Et.
  • each of -R 7NA and -R 7NB is independently -Me.
  • each of -R 7NA and -R 7NB is independently -Et. In one embodiment, -R 7NA and -R 7NB are the same. In one embodiment, -R 7NA and -R 7NB are different.
  • -R 3NA and -R 7NA are the same. In one embodiment, -R 3NB and -R 7NB , where present, are the same.
  • the groups -N(R 3NA )(R 3NB ) and -N(R 7NA )(R 7NB ), where present, are the same.
  • the groups -N(R 3NA )(R 3NB ) and -N(R 7NA )(R 7NB ), where present, are the same, and are selected from: -NMe 2 and -NEt 2 .
  • the groups -N(R 3NA )(R 3NB ) and -N(R 7NA )(R 7NB ), where present, are each -NMe 2 .
  • the groups -N(R 3NA )(R 3NB ) and -N(R 7NA )(R 7NB ), where present, are each -NEt 2 .
  • the groups -N(R 3NA )(R 3NB ) and -N(R 7NA )(R 7NB ), where present, are other than -NMe 2 .
  • each of -R 10NA and -R 10NB is independently selected from: -H; C 1-4 alkyt; C 2 . 4 alkenyl; halogenated C 1-4 alkyl; C 5- i 0 aryl; halogenated C 5-10 aryl;
  • each of -R 3NA and -R 3NB is independently selected from: -H; -C 1-4 alkyl; C 2-4 alkenyl; and halogenated C 1-4 alkyl.
  • each of -R 10NA and -R 10NB is independently -H.
  • each of -R 10NA and -R 10NB is independently selected from:
  • each of -R 10NA and -R 10NB is independently -Me, -Et, -nPr, -nBu,
  • each of -R 10NA and -R 10NB is independently C 1-4 alkyl. In one embodiment, each of -R 10NA and -R 10NB is independently -Me or -Et. In one embodiment, each of -R 10NA and -R 10NB is independently -Me. In one embodiment, each of -R 10NA and -R 10NB is independently -Et.
  • -R 10NA and -R 10NB are the same. In one embodiment, -R 10NA and -R 10NB are different.
  • -A is an activating group.
  • the activating group -A activates the sulfur atom of the thio ether (diaminoaryl sulphide) for ring closure, such as a ring closure reaction described herein.
  • each -NR A2 R A3 is independently azetidino, pyrrolidino, imidazolidino, pyrazolidino, piperidino, piperazino, morpholino, azepino, or diazepino.
  • the group -A includes the ionic forms of the groups above, where appropriate.
  • the activated S-ether is a thiosulfonic acid (-SSO 3 H).
  • one or more of the carbon atoms is 11 C or 13 C. In one embodiment, one or more of the carbon atoms is 11 C. In one embodiment, one or more of the carbon atoms is 13 C. In one embodiment, one or more of the nitrogen atoms is 15 N.
  • one or more or all of the carbon atoms of one or more or all of the groups R 3NA , R 3NB , R 7NA and R 7NB is 13 C.
  • each of the groups -N(R 3NA )(R 3NB ) and -N(R 7NA )(R 7NB ), where present, is -N( 13 CH 3 ) 2 .
  • each of R 1 and R 9 where present, is -H
  • each of R 1 and R 9 where present, is -H; each of the groups -N(R 3NA )(R 3NB ) and -N(R 7NA )(R 7NB ), where present, is -N( 13 CH 3 ) 2 ; and X " is Cl ⁇
  • X ' is a counter anion to achieve electrical neutrality. In one embodiment, X " is one or more counter anions to achieve electrical neutrality. In one embodiment, X ' is a counter anion shared with other cations to achieve electrical neutrality.
  • X " is independently a halogen anion (i.e., halide).
  • X ' is independently F “ , Cl “ , Br, or I " .
  • X " is independently Cl “ , Br “ , or I “ .
  • X ' is independently Cl “ .
  • X " is independently NO 3 " (nitrate).
  • X " is independently CIO 3 " (perchlorate).
  • X " is independently selected from formate, propionate, benzoate and 4-hydroxybenzenesulfonate.
  • X " is independently S 2 O 8 " .
  • X " is S 2 O 3 "2 .
  • X " is SO 4 "2 (sulfate).
  • X " is succinate
  • the counter ion achieves electrical neutrality by combination with two of the recited cations.
  • X " is citrate.
  • the counter ion achieves electrical neutrality by combination with three of the recited cations.
  • the compound is in the form of a mixed salt, for example, a ZnCI 2 mixed salt.
  • Example diaminophenothiazinium compounds include the following:
  • Example thiosulfonic acid compounds include the following:
  • Example imino compounds include the following:
  • the present method avoids the use of a dichromate salt in the oxidative coupling step of a diaminophenothiazinium synthesis, such as those steps described by Fierz-David and Blangley and WO 2006/032879, amongst others.
  • the present method avoids the use of a dichromate salt in the thiosulfonic acid forming step of a diaminophenothiazinium synthesis, such as those steps described by Fierz-David and Blangley and WO 2006/032879, amongst others.
  • the present invention provides a method of preparing MTC that minimises the amount of dichromate salt required in the overall synthesis. In one embodiment, the present invention provides a method of preparing MTC that does not require a dichromate salt.
  • Sodium dichromate the preferred dichromate salt in the Fierz-David and Blangley and WO 2006/032879 syntheses, is registered under the International Maritime Dangerous Goods (IMDG) Code under Class 6.1 : Toxic substances.
  • IMDG International Maritime Dangerous Goods
  • Sodium dichromate is expensive to purchase and ship, and its disposal is likewise costly. Owing to the toxicity and carcinogenicity of sodium dichromate, specialist safety precautions are required to be put in place for its large scale use. The chromium-containing by-products from a chromate oxidation are likewise highly hazardous and must be dealt with appropriately.
  • the present inventors have established that an alternative range of oxidants may be used in a diaminophenothiazinium synthesis.
  • a persulfate salt may be used as an oxidant in a diaminophenothiazinium synthesis.
  • WO 2006/032879 addresses the issue of residual Cr levels in a diaminophenothiazinium synthesis.
  • the solution is to reduce residual Cr(VI) to Cr(III), which can more easily be removed from a product compared to Cr(VI).
  • Cr(VI) residual Cr(VI)
  • Cr(III) residual Cr(III)
  • chromium should not be used in a diaminophenothiazinium synthesis.
  • a reference herein to a TSAF step is also a reference to an activated thio ether (diaminoaryl sulphide) formation step, as described herein.
  • a reference herein to a Cr-TSAF step is also a reference to a Cr- mediated activated thio ether (diaminoaryl sulphide) formation step, as described herein.
  • the method of synthesis comprises an oxidative coupling (OC).
  • the method of synthesis comprises a thiosulfonic acid formation (TSAF).
  • TSAF thiosulfonic acid formation
  • the method of synthesis comprises, in order, an oxidative coupling (OC) and a ring closure (RC). In one embodiment, the method of synthesis comprises, in order: a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • TSAF thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • NR nitrosyl reduction
  • TSAF thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • NOS nitrosylation
  • NR nitrosyl reduction
  • TSAF thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: an ⁇ /,/V-disubstitution (NNDS); a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • NDS ⁇ /,/V-disubstitution
  • NOS nitrosylation
  • NR nitrosyl reduction
  • TSAF thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • Cr-TSAF Cr-mediated thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a nitrosyl reduction (NR); a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • NR nitrosyl reduction
  • Cr-TSAF Cr-mediated thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a nitrosylation (NOS); a nitrosyl reduction (NR); a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • NOS nitrosylation
  • NR nitrosyl reduction
  • Cr-TSAF Cr-mediated thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: an ⁇ /, ⁇ /-disubstitution (NNDS); a nitrosylation (NOS); a nitrosyl reduction (NR); a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
  • NDS ⁇ /, ⁇ /-disubstitution
  • NOS nitrosylation
  • NR nitrosyl reduction
  • Cr-TSAF Cr-mediated thiosulfonic acid formation
  • OC oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
  • TSAF thiosulfonic acid formation
  • Cr-OC Cr-mediated oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
  • NR nitrosyl reduction
  • TSAF thiosulfonic acid formation
  • Cr-OC Cr-mediated oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
  • NOS nitrosylation
  • NR nitrosyl reduction
  • TSAF thiosulfonic acid formation
  • Cr-OC Cr-mediated oxidative coupling
  • RC ring closure
  • the method of synthesis comprises, in order: an ⁇ /, ⁇ /-disubstitution (NNDS); a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
  • NDS ⁇ /, ⁇ /-disubstitution
  • NOS nitrosylation
  • NR nitrosyl reduction
  • TSAF thiosulfonic acid formation
  • Cr-OC Cr-mediated oxidative coupling
  • RC ring closure
  • the method of synthesis further includes the step of chloride salt formation (CSF) after a ring closure (RC) step.
  • CSF chloride salt formation
  • RC ring closure
  • the methods of the invention generally include at least one activated thio ether (diaminoaryl sulphide) activation step, which may be a thiosulfonic acid formation (TSAF) step, or at least one oxidative coupling (OC) step, as described below.
  • the method comprises both steps.
  • an ⁇ /, ⁇ /-disubstituted-diamino-substituted benzene, I is oxidized in the presence of an oxidizing agent that is or comprises persulfate to give an activated S-( ⁇ /, ⁇ /-disubstituted-diamino-phenyl) ether (a S-activated diaminoaryl sulphide), G-Il, as illustrated in the following scheme:
  • This reaction may be referred to as an activated thio ether (S-activated diaminoaryl sulphide) formation step.
  • the activating group reagent is or comprises [S-A]" ' or [S-A], where n is 1 or 2.
  • the activating group reagent is or may comprise the anion [S-SO 3 ] 2" (thiosulfate).
  • n is 1. In one embodiment, n is 2.
  • the activating group reagent additionally comprises a counter cation.
  • the counter cation is a sodium, potassium, or ammonium cation.
  • the counter cation is a sodium cation.
  • the activating group reagent is or comprises thiosulfate.
  • an ⁇ /, ⁇ /-disubstituted-diamino-substituted benzene, I is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises persulfate to give an amino-( ⁇ /, ⁇ /-disubstituted-amino)phenylthiosulfonic acid (a thiosulfonic acid S-( ⁇ /, ⁇ /-disubstituted-diamino-phenyl) ester) ), II, as illustrated in the following scheme:
  • This activated thio ether (S-activated diaminoaryl sulphide) formation reaction may be referred to as a thiosulfonic acid formation (TSAF) step.
  • TSAF thiosulfonic acid formation
  • the phenyl ring in compound I is substituted with an amino group, -NR 10NA R 10NB , at any one of the 2-, 3-, 4-, 5-, or 6-positions, and is substituted with three substituents -R 9 , at any three of the four remaining positions.
  • the final position is substituted with -H.
  • the phenyl ring in compound Il is substituted with an amino group, -NR 10NA R 10NB , at any of the 2-, 3-, A-, 5-, or 6-positions, and is substituted with three substituents -R 9 , at any three of the four remaining positions.
  • the final position is substituted with thiosulfonic acid, -S-SO 3 H.
  • compounds I and Il are compounds of formula Ia and Ha respectively as shown below:
  • compounds I and Il are compounds of formula Ib and Hb respectively as shown below:
  • an ⁇ /, ⁇ /-disubstituted-1 ,4-diamino-5-optionally substituted benzene, A is oxidized in the presence of a thiosulfate to give a thiosulfonic acid S- ⁇ 2- (amino)-3-(optionally substituted)-5-( ⁇ /, ⁇ /-disubstituted-amino)-phenyl ⁇ ester (an f amino-( ⁇ /, ⁇ /-disubstituted)aminophenylthiosulfonic acid), B, as illustrated in the following scheme:
  • an N,N-dimethyl-1 ,4-diamino-benzene, 1 is oxidized in the presence of a thiosulfate to give a thiosulfonic acid S- ⁇ 2-(amino)-5-(dimethylamino)- phenyl ⁇ ester (an amino-( ⁇ /, ⁇ /-disubstituted-amino)phenylthiosulfonic acid), 2, as illustrated in the following scheme:
  • Compound 1 may be obtained from commercial sources, or may be prepared directly or indirectly from the corresponding nitrosyl compound, as described below in the NOS and DNNDS steps.
  • the thiosulfate is or comprises S 2 O -2
  • the thiosulfate is or comprises Na 2 S 2 O 3 .
  • the thiosulfate is or comprises Na 2 S 2 O 3 or a hydrate thereof.
  • Na 2 S 2 O 3 may be obtained commercially, for example, as the anhydrous salt or as the pentahydrate.
  • the molar ratio of thiosulfate to diamine, I is 0.8 to 1.5. In one embodiment, the molar ratio is 0.8 to 1.3. In one embodiment, the molar ratio is about 1.0
  • the oxidizing agent is or comprises persulfate.
  • the persulfate is or comprises S 2 O 8 2" .
  • the persulfate is or comprises sodium persulfate, potassium persulfate, or ammonium persulfate.
  • the persulfate is or comprises sodium persulfate.
  • the persulfate is sodium persulfate, or a hydrate thereof.
  • the molar ratio of persulfate to diamine, I is 0.5 to 1.5. In one embodiment, the range is 0.8 to 1.3. In one embodiment, the range is about 1.0.
  • the amine, I 1 is added first, before the activating group reagent is added.
  • the activating group reagent is added before the persulfate is added.
  • the amine, I is added first, before the thiosulfate is added.
  • the thiosulfate is added before the persulfate is added.
  • the reaction is performed in an aqueous medium.
  • the pH of the aqueous medium is adjusted after the amine, I, is added.
  • the pH of the aqueous medium is adjusted after the amine, I, is added, and before the thiosulfate is added.
  • the pH of the aqueous medium is adjusted after the amine, I, is added, and before the thiosulfate and the persulfate are added.
  • the pH of the aqueous medium is adjusted to a pH of 2 to 7. In one embodiment, the pH of the aqueous medium is adjusted to a pH of 3 to 5. In one embodiment, the pH of the aqueous medium is adjusted to a pH of about 4.
  • the acidic conditions are adjusted using a strong acid. In one embodiment, the acidic conditions are adjusted using HCI.
  • the acidic conditions are adjusted using a strong base. In one embodiment, the acidic conditions are adjusted using a hydroxide base. In one embodiment, the hydroxide base is, or comprises, sodium hydroxide, potassium hydroxide, or lithium hydroxide. In one embodiment, the reaction is performed in an aqueous medium comprising an organic solvent.
  • the organic solvent is present at about 1 to 50 % v/v.
  • the organic solvent is present at about 5 to 40 % v/v.
  • the organic solvent is present at about 20 % v/v.
  • the organic solvent is miscible with water.
  • the organic solvent is THF, DMF, acetonitrile or saturated aliphatic C 1-6 alkyl alcohol.
  • the organic solvent is THF, acetonitrile or saturated aliphatic
  • the saturated aliphatic C 1-6 alkyl alcohol is methanol.
  • the reaction is performed in an aqueous medium comprising a surfactant.
  • the surfactant is an anionic surfactant.
  • the anionic surfactant is a sulfonic acid surfactant.
  • the anionic surfactant is a dodecylbenzenesulfonic acid salt.
  • the anionic surfactant is an alkyl sulfate salt.
  • the anionic surfactant is selected from alkyl benzene sulfonate, sodium laureth sulfate, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA or PFO).
  • the surfactant is a cationic surfactant.
  • the cationic surfactant is selected from benzethonium chloride (BZT), benzalkonium chloride (BAC), polyethoxylated tallow amine (POEA), cetylpyridinium chloride (CPC) and cetyl trimethylammonium bromide.
  • the surfactant is a nonionic surfactant.
  • the nonionic surfactant is a polysorbate.
  • the nonionic surfactant is a fatty alcohol.
  • the nonionic surfactant is selected from dodecyl dimethylamine oxide, Tween 20, Tween 80, cetyl alcohol, oleyl alcohol, decyl maltoside, octyl glucoside, cocamide MEA, cocamide DEA, alkylphenol poly(ethylene oxide) and alkyl poly(ethylene oxide).
  • the surfactant is a zwitterionic surfactant.
  • the zwitterionic surfactant is selected from coco ampho glycinate, dodecyl betaine and cocamidopropyl betaine.
  • the reaction is performed under acidic conditions. In one embodiment, the reaction is performed at a pH of less than 7. In one embodiment, the reaction is performed at a pH of 3 to 5. In one embodiment, the reaction is performed at a pH of about 4.
  • the thiosulfate is added in one portion. In one embodiment, the persulfate is added in one portion. In one embodiment, the persulfate is added portionwise.
  • the reaction temperature is 2 to 25°C. In one embodiment, the reaction temperature is 2 to 15°C. In one embodiment, the reaction temperature is 2 to 10 0 C. In one embodiment, the reaction temperature is about 5 0 C.
  • the reaction time is 10 to 240 minutes. In one embodiment, the reaction time is 30 to 120 minutes. In one embodiment, the reaction time is about 60 minutes.
  • reaction mixture is stirred during the reaction step.
  • isolation and purification is by filtration.
  • the S-activated diaminoaryl sulphide G-Il is isolated and purified.
  • the isolation and purification is by filtration followed by washing. In one embodiment, the isolation and purification is by filtration followed by drying.
  • the drying is air-drying. In one embodiment, the drying is air-drying for 10 min to 6 hours. In one embodiment, the drying is air-drying for 10 min to 2 hours. In one embodiment, the drying is air-drying for about 30 min. In one embodiment, the drying is air-drying on the filter.
  • the drying is oven-drying.
  • the drying is oven-drying for 2 to 48 hours.
  • the drying is oven-drying for 2 to 24 hours.
  • the drying is oven-drying for about 12 hours.
  • the drying is oven-drying at 30 to 6O 0 C for 2 to 24 hours.
  • the oven-drying is under vacuum.
  • the drying is air-drying followed by oven-drying. In one embodiment, after reaction, the reaction mixture is filtered and the filtrate collected.
  • isolation of the S-activated diaminoaryl sulphide is by filtration.
  • the purification comprises re-dissolving in an organic solvent and filtering.
  • the purification comprises re-dissolving in an organic solvent, heating at reflux, cooling and filtering.
  • the purification comprises re-dissolving in ethyl acetate, heating at reflux, cooling and then filtering.
  • the purification comprises re-dissolving in an organic solvent, heating at reflux, filtering and washing with an organic solvent.
  • heating at reflux is for a period of between 30 minutes and 120 minutes, preferably between 45 and 90 minutes.
  • a S-activated diaminoaryl sulphide an activated S-(amino-substituted-( ⁇ /, ⁇ /-disubstituted-amino)-phenyl) thio ether
  • G-IIa 1 is oxidatively coupled to an ⁇ /, ⁇ /-disubstituted-substituted-aniline, III, using an oxidizing agent that is or comprises persulfate, to give a S-activated ⁇ /-substituted ⁇ /'-substituted imino aminoaryl sulphide (activated ⁇ (thio ether)-(disubstituted amino)-(substituted)-phenyl-imino ⁇ -substituted cyclohexa-2,5-dienylidene- ⁇ /, ⁇ /-disubstituted ammonium), G-IV, as illustrated in the following scheme:
  • the compound G-IV is shown as a cation, which may be prepared as a salt with a suitable counter ion, X " .
  • Compound G-IV may also be prepared as a zwitterionic form where a negative charge is associated with the group -A.
  • a thiosulfonic acid S-(amino-substituted-( ⁇ /, ⁇ /-disubstituted- amino)-phenyl) ester (an amino-( ⁇ /, ⁇ /-disubstituted-amino)phenylthiosulfonic acid), Ha
  • the phenyl ring in compound Il is substituted with an amino group, -NH 2 , at any one of the 2-, 3-, 4-, 5-, or 6-positions, and is substituted with three substituents -R 9 , at any three of the four remaining positions.
  • the final position is substituted with thiosulfonic acid, -S-SO 3 H.
  • the phenyl ring in compound III is substituted with -H at the 4-position, and is substituted with four substituents -R 1 , at the 2-, 3-, 5-, and 6-positions.
  • the final position is substituted with thiosulfonic acid, -S-SO 3 H.
  • Ha and IV are compounds Hb and IVa respectively:
  • Ma, III and IV are compounds Mb, MIa and V respectively:
  • a thiosulfonic acid S- ⁇ 2-(amino)-5-(dimethylamino)-phenyl ⁇ ester (an amino-(disubstituted-amino)phenylthiosulfonic acid), 2 is oxidatively coupled to an ⁇ /, ⁇ /-dimethyl-aniline, 3, using an oxidizing agent that is or comprises persulfate, to give a [4- ⁇ 2-(thiosulfate)-4-(dimethylamino)-phenyl-imino ⁇ -cyclohexa-2,5-dienylidene]- ⁇ /, ⁇ /-dimethyl ammonium, 4, as illustrated in the following scheme:
  • the S-activated diaminoaryl sulphide, G-Ma is added first, before the aniline, III, is added.
  • the amino-( ⁇ /, ⁇ /-disubstituted-amino)phenylthiosulfonic acid , Ha is added first, before the aniline, III, is added.
  • the reaction is performed in an aqueous medium.
  • the aniline, III is added before the persulfate is added. In one embodiment, the aniline, III, is added in a mixture with aqueous acid. In one embodiment, the aqueous acid is an aqueous mineral acid. In one embodiment, the aqueous acid is aqueous H 2 SO 4 . In one embodiment, the aqueous acid is aqueous HCI.
  • the oxidizing agent is or comprises a persulfate.
  • the oxidizing agent is or comprises a persulfate salt.
  • the oxidizing agent is or comprises sodium persulfate, potassium persulfate, or ammonium persulfate.
  • the oxidizing agent is or comprises sodium persulfate.
  • the oxidizing agent is sodium persulfate, or a hydrate thereof.
  • the molar ratio of S-activated diaminoaryl sulphide, G-IIa, to aniline, III is 0.5 to 1.5.
  • the molar ratio of amino-( ⁇ /, ⁇ /-disubstituted- amino)phenylthiosulfonic acid, Ha, to aniline, III is 0.5 to 1.5.
  • the range is 0.8 to 1.2.
  • the range is about 1.0.
  • the molar ratio of aniline, III, to persulfate is 0.5 to 2.0. In one embodiment, the range is 0.8 to 1.5. In one embodiment, the range is 0.8 to 1.2. In one embodiment, the range is about 1.0.
  • the reaction is performed under basic conditions.
  • the basic conditions are obtained after the aniline has been added.
  • the reaction is performed at a pH of 7 or more. In one embodiment, the reaction is performed at a pH of more than 7. In one embodiment, the reaction is performed at a pH of 7.1 to 10.0. In one embodiment, the reaction is performed at a pH of 7.1 to 8.0. In one embodiment, the reaction is performed at a pH of about 7.5.
  • the basic conditions are obtained using a strong base.
  • the basic conditions are obtained using LiOH, NaOH 1 KOH, or
  • the basic conditions are obtained using NaOH.
  • the reaction temperature is 2 to 20 0 C. In one embodiment, the reaction temperature is 2 to 15°C. In one embodiment, the reaction temperature is about 5°C.
  • the reaction time is 30 minutes to 12 hours. In one embodiment, the reaction time is 60 minutes to 5 hours. In one embodiment, the reaction time is 60 minutes to 4 hours. In one embodiment, the reaction time is about 3 hours.
  • reaction mixture is stirred during the reaction step.
  • aniline, III is the same as aniline, IX.
  • the zwitterionic intermediate G-IV is isolated and purified.
  • the isolation and purification is by filtration.
  • the isolation and purification is by filtration followed by washing.
  • the washing is washing with H 2 O.
  • the washing is washing with H 2 O and tetrahydrofuran (THF).
  • the isolation and purification is by filtration followed by washing and drying.
  • the isolation and purification is by filtration followed by drying.
  • the drying is air-drying. In one embodiment, the drying is air-drying for 2 to 72 hours. In one embodiment, the drying is air-drying for 2 to 48 hours. In one embodiment, the drying is air-drying for 2 to 24 hours. In one embodiment, the drying is air-drying on the filter.
  • the drying is oven-drying.
  • the drying is oven-drying for 2 to 72 hours.
  • the drying is oven-drying for 2 to 48 hours.
  • the drying is oven-drying for 2 to 24 hours.
  • the drying is oven-drying at 30 to 6O 0 C for 2 to 48 hours.
  • the oven-drying is under vacuum.
  • the drying is air-drying followed by oven-drying.
  • the reaction mixture is filtered, and the residue (e.g., ⁇ 100 mmol crude product) is washed with H 2 O (e.g., 4 x 250 cm 3 ) and THF (e.g., 100 cm 3 ), and then air-dried overnight.
  • H 2 O e.g., 4 x 250 cm 3
  • THF e.g., 100 cm 3
  • the reaction mixture is filtered (e.g., through a Buchner filter under vacuum), the solid removed, added to another vessel with fresh water, the mixture stirred vigorously, and filtered again.
  • the "filter-recover-resuspend" process may be repeated a number of times.
  • the finally obtained solid may be used in subsequent steps.
  • a N, N-disubstituted N'-substituted aminoiminoaryl sulphide (a ⁇ (thio ether)-( ⁇ /, ⁇ /-disubstituted amino)-(substituted)-phenyl-imino ⁇ -substituted cyclohexa- 2, 5-dienylidene-N, N-disubstituted ammonium), G-V, is subjected to ring closure to give a bis( ⁇ /, ⁇ /-disubstituted-amino)-(substituted)-phenothiazin-5-ium salt, Vl, as illustrated in the following scheme:
  • the compound G-V is shown as a cation, which may be used as a salt with a suitable counter ion, X ' .
  • Compound G-V may also be used in a zwitterionic form where a negative charge is associated with the group -A.
  • a ⁇ (thiosulfate)-( ⁇ /, ⁇ /-disubstituted amino)-(substituted)-phenyl- iminoj-substituted cyclohexa-2,5-dienylidene- ⁇ /, ⁇ /-disubstituted ammonium, V is subjected to ring closure to give a bis( ⁇ /, ⁇ /-disubstituted-amino)-(substituted)- phenothiazin-5-ium salt, Vl 1 as illustrated in the following scheme:
  • the 5-position is substituted with -H 1 as shown.
  • the left-hand ring in compound Vl, as drawn, is linked to the right-hand ring through a sulphur atom at the 1 -position and the N atom at the 2-position.
  • the left-hand ring is substituted with an amino group, -NR 7NA R 7NB , at any one of the 3-, A-, 5-, or 6-positions, and is substituted with three substituents -R 9 , at the three remaining positions.
  • the right-hand ring in compound Vl, as drawn, is linked to the left-hand ring through the sulphur atom at the 1 -position and the N atom at the 2-position.
  • the right- hand ring is substituted with an amino group, -NR 3NA R 3NB , at the 5-position, and is substituted with three substituents -R 1 , at the three remaining positions.
  • compounds V and Vl are compounds of formula Va and Via respectively:
  • a [4- ⁇ 2-(thiosulfate)-4-( ⁇ /, ⁇ /-disubstituted amino)-6-(optionally substituted)-phenyl-imino ⁇ -3-(optionally substituted)-cyclohexa-2,5-dienylidene]-N,N- disubstituted ammonium, D is subjected to ring closure to give a 3,7-bis( ⁇ /,/V- disubstituted-amino)-1 ,9-(optionally substituted)-phenothiazin-5-ium salt, E, as illustrated in the following scheme:
  • a [ ⁇ 2-(thiosulfate)-4-(dimethylamino)-phenyl-imino ⁇ -cyclohexa-2,5- dienylidene]- ⁇ /, ⁇ /-dimethyl ammonium, 4, is subjected to ring closure to give a 3,7-bis(dimethylamino)-phenothiazin-5-ium salt, 5, as illustrated in the following scheme:
  • ring closure is achieved by treatment with an oxidizing agent.
  • the reaction is performed at reflux.
  • the reaction temperature is 30 to 95 0 C. In one embodiment, the reaction temperature is 50 to 90 0 C. In one embodiment, the reaction temperature is 60 to 9O 0 C. In one embodiment, the reaction temperature is about 85°C.
  • the reaction time is 10 to 120 minutes. In one embodiment, the reaction time is 20 to 90 minutes. In one embodiment, the reaction time is about 60 minutes.
  • the reaction is performed in an aqueous medium.
  • the reaction is performed until the reaction mixture changes colour, e.g., becomes a deep blue colour.
  • reaction mixture is stirred during the reaction step.
  • the reaction mixture is filtered and the filtrate collected.
  • the reaction mixture is first cooled, and the filtration is performed at about room temperature, to give a "cool" filtrate.
  • the filtration is performed at a temperature near to the reaction temperature, to give a "hot” filtrate.
  • a salt is added to the reaction mixture.
  • the salt is an alkali metal salt.
  • the salt comprises the counter ion X ' .
  • the product of the reaction is a salt, wherein X ' is S 2 O 8 ' .
  • the product of the reaction is a salt, wherein X ' is 0.5 S 2 O 3 "2 .
  • the product of the reaction is a salt, wherein X ' does not comprise a halogen ion.
  • the product of the reaction is a salt, wherein X ' does not comprise a chloride ion.
  • the product of the reaction is reacted with a chloride salt as a source of chloride to form a product where X ' is a chloride ion, as described below in the Chloride Salt Formation (CSF) step.
  • a chloride salt as a source of chloride to form a product where X ' is a chloride ion, as described below in the Chloride Salt Formation (CSF) step.
  • the oxidizing agent is or comprises Cu(II).
  • the oxidizing agent is or comprises Cu(II) sulfate.
  • the oxidizing agent is Cu(II) sulfate or a hydrate thereof.
  • the oxidizing agent is or comprises Cu(II).
  • ring closure is performed under acidic conditions. In one embodiment, ring closure is performed at a pH of 1 to 5. In one embodiment, ring closure is performed at a pH of 2 to 5. In one embodiment, ring closure is performed at a pH of 3 to 4.5. In one embodiment, ring closure is performed at a pH of 3.5 to 4.1. In one embodiment, ring closure is performed at a pH of about 2.0.
  • the desired pH is obtained by the addition of strong acid. In one embodiment, the desired pH is obtained by the addition of HCI.
  • the molar ratio of Cu(II) to ammonium, G-V is 0.02 to 0.2. In one embodiment, the molar ratio of Cu(II) to ammonium, V, is 0.02 to 0.2. In one embodiment, the range is 0.05 to 0.15 In one embodiment, the range is about 0.1.
  • the oxidizing agent is or comprises activated manganese dioxide
  • the oxidizing agent is activated manganese dioxide (MnO 2 ).
  • MnO 2 manganese dioxide
  • the molar ratio of MnO 2 to ammonium, G-V is 1.0 to 3.0.
  • the molar ratio of MnO 2 to ammonium, V is 1.0 to 3.0.
  • the molar ratio is 1.5 to 2.5. In one embodiment, the molar ratio is about 2.0.
  • strong acid e.g., concentrated H 2 SO 4 .
  • the strong acid dissolves the manganese salts (and other salts, if present).
  • a bis( ⁇ /, ⁇ /-disubstituted-amino)-(substituted)-phenothiazin-5-ium salt, Vl is reacted with chloride, to give a bis( ⁇ /, ⁇ /-disubstituted-amino)-(substituted)- phenothiazin-5-ium chloride salt, VII, as illustrated in the following scheme:
  • a 3,7-bis( ⁇ /, ⁇ /-disubstituted-amino)-1 ,9-(optionally substituted)- phenothiazin-5-ium salt, E is reacted with chloride, to give a 3,7-bis( ⁇ /, ⁇ /- disubstituted-amino)-1 ,9-(optionally substituted)-phenothiazin-5-ium chloride salt, F, as illustrated in the following scheme:
  • a 3,7-bis(dimethylamino)-phenothiazin-5-ium salt is reacted with chloride, to give a 3,7-bis(dimethylamino)-phenothiazin-5-ium chloride salt, 5 (i.e., MTC), as illustrated in the following scheme:
  • Vl is reacted with a chloride salt as a source of chloride.
  • the chloride is chloride salt.
  • the chloride is an alkali metal chloride.
  • the chloride is sodium chloride.
  • the molar ratio of chloride to salt, Vl is 5 to 100.
  • the molar ratio is 5 to 80.
  • the molar ratio is 5 to 50.
  • the molar ratio is about 8.
  • the reaction is performed in an aqueous medium.
  • the reaction temperature is 0 to 95°C.
  • the reaction temperature is 0 to 50 c C.
  • the reaction temperature is 0 to 30 0 C.
  • the reaction temperature is 2 to 30 0 C.
  • the reaction temperature is 2 to 20°C.
  • the reaction temperature is about 5 0 C.
  • the reaction time is 10 to 120 minutes. In one embodiment, the reaction time is about 60 minutes.
  • the reaction is performed until the reaction mixture (initially, e.g., a deep blue colour) becomes light blue to colourless.
  • reaction mixture is stirred during the reaction step.
  • the reaction mixture is allowed to cool following addition of the chloride to salt Vl at the reaction temperature, to yield the product as a precipitate. In one embodiment, the reaction mixture is allowed to cool to 0 to 30 0 C. In one embodiment, the reaction mixture is allowed to cool to 0 to 20 0 C. In one embodiment, the reaction mixture is allowed to cool to about 5°C.
  • reaction mixture is filtered hot following addition of the chloride to salt Vl at the reaction temperature, and the collected filtrate allowed to cool, to yield the product as a precipitate.
  • Vl is reacted with hydrochloric acid as a source of chloride.
  • the Chloride salt formation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein.
  • the sections relating to Chloride salt formation (CAF) at pages 32-33 and Chloride salt formation (CSF-2) at pages 58-59 are incorporated herein.
  • one or more additional steps may be performed, as discussed at pages 33 and 34, including the sulphide treatment described at pages 34-36, the dimethyldithiocarbamate treatment (DT) at pages 36- 37, the carbonate treatment (CT) at pages 37-38, the ethylenediaminetetraacetic acid treatment (EDTAT) at pages 39-40, and the organic extraction (OE) at pages 40-41 , which are incorporated herein.
  • an ⁇ /, ⁇ /-disubstituted-diamino-substituted benzene, Ia is oxidized in the presence of an oxidizing agent that is or comprises Cr(VI) to give an S-(amino- substituted-( ⁇ /, ⁇ /-disubstituted-amino)-phenyl) ether, (S-activated ⁇ /, ⁇ /-disubstituted diaminoaryl sulphide) G-IIa, as illustrated in the following scheme:
  • This reaction may be referred to as a Cr-mediated activated thio ether formation step.
  • Compound Ia is oxidised in the presence of an activating group reagent.
  • the activating group reagent and the group -A are as defined for the activated thio ether (S-activated diaminoaryl sulphide) formation step described above.
  • the activating group reagent is thiosulfate.
  • an ⁇ /, ⁇ /-disubstituted-diamino-substituted benzene, Ia is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises Cr(VI) to give a thiosulfonic acid S(-amino-substituted-( ⁇ /, ⁇ /-disubstituted-amino)-phenyl) ester (an amino-( ⁇ /, ⁇ /-disubstituted-amino)phenylthiosulfonic acid), Ha, as illustrated in the following scheme:
  • compounds I and Il are compounds of formula Ib and lib respectively as shown below:
  • an ⁇ /, ⁇ /-disubstituted-1 ,4-diarnino-5-optionally substituted benzene, A is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises Cr(VI) to give a thiosulfonic acid S- ⁇ 2-(amino)-3-(optionally substituted)- 5-( ⁇ /, ⁇ /-disubstituted-amino)-phenyl ⁇ ester (an amino-( ⁇ /, ⁇ /-disubstituted- amino)phenylthiosulfonic acid), B, as illustrated in the following scheme:
  • an ⁇ /, ⁇ /-dimethyl-1 ,4-diamino-benzene, 1 is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises Cr(VI) to give a thiosulfonic acid S- ⁇ 2-(amino)-5-(dimethylamino)-phenyl ⁇ ester, (a 2-amino-5-dimethyl- aminophenylthiosulfonic acid), 2, as illustrated in the following scheme:
  • the Cr-mediated thiosulfonic acid formation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein.
  • TSAF Thiosulfonic acid formation
  • TSAF-3 Thiosulfonic acid formation
  • TSAF-3 Thiosulfonic acid formation
  • Examples 1-8 and 17 relating to the synthesis of thiosulfonic acid S- ⁇ 2- (amino)-5-(dimethylamino)-phenyl ⁇ ester, (2-amino-5-(dimethylaminophenylthiosulfonic acid) and Example 20, relating to the synthesis of thiosulfonic acid S- ⁇ 2-(amino)-3- ethyl-5-(dimethylamino)-phenyl ⁇ ester (2-amino-3-methyl-5- dimethylaminophenylthiosulfonic acid).
  • the thiosulfate is or comprises S 2 O 3 "2 .
  • the thiosulfate is or comprises Na 2 S 2 O 3 .
  • the thiosulfate is Na 2 S 2 O 3 or a hydrate thereof.
  • the oxidation is by reaction with an oxidizing agent.
  • the oxidizing agent is or comprises Cr(VI).
  • the oxidizing agent is or comprises Cr 2 O 7 '2 .
  • the oxidizing agent is or comprises Na 2 Cr 2 O 7 .
  • the oxidizing agent is Na 2 Cr 2 O 7 or a hydrate thereof.
  • the molar ratio of Cr(VI) to diamine, I is 0.2 to 2.0.
  • the reaction is performed in an aqueous medium.
  • the reaction temperature is 2 to 25°C.
  • the oxidizing agent additionally comprises AI(III).
  • the molar ratio of AI(III) to diamine, I is 0.2 to 2.0.
  • the oxidizing agent further comprises a strong acid.
  • the molar ratio of acid protons to diamine, I is 1.0 to 4.0.
  • an activated S-(amino-substituted-( ⁇ /, ⁇ /-disubstituted-amino)-phenyl) thio ether, G-IIa is oxidatively coupled to an ⁇ /, ⁇ /-disubstituted-substituted-aniline, III, using an oxidizing agent that is or comprises Cr(VI), to give a S-activated N 1 N- disubstituted ⁇ /'-substituted aminoiminoaryl sulphide (an activated ⁇ (thio ether)-( N 1 N- disubstituted amino)-(substituted)-phenyl-imino ⁇ -substituted cyclohexa-2,5- dienylidene- ⁇ /, ⁇ /-disubstituted ammonium), G-IV, as illustrated in the following scheme:
  • a thiosulfonic acid S-(amino-substituted-( ⁇ /, ⁇ /-disubstituted- amino)-phenyl) ester (an amino-( ⁇ /, ⁇ /-disubstituted-amino)phenylthiosulfonic acid), Ua, is oxidatively coupled to an ⁇ /, ⁇ /-disubstituted-substituted-aniline, III, using an oxidizing agent that is or comprises Cr(VI), to give a ⁇ (thiosulfate)-( ⁇ /, ⁇ /-disubstituted amino)-(substituted)-phenyl-imino ⁇ -substituted cyclohexa-2,5-dienylidene- ⁇ /, ⁇ /- disubstituted ammonium, IV 1 as illustrated in the following scheme:
  • Ma and IV are compounds Mb and IVa respectively: lib IVa
  • Ha, III and IV are compounds lib, Ilia and V respectively:
  • a thiosulfonic acid S- ⁇ 2-(amino)-5-(dimethylamino)-phenyl ⁇ ester (a 2-amino-5-dimethylaminophenylthiosulfonic acid), 2, is oxidatively coupled to an ⁇ /, ⁇ /-dimethyl-aniline, 3, using an oxidizing agent that is or comprises Cr(VI), to give a [4- ⁇ 2-(thiosulfate)-4-(dimethylamino)-phenyl-imino ⁇ -cyclohexa-2,5-dienylidene]- ⁇ /, ⁇ /- dimethyl ammonium, 4, as illustrated in the following scheme:
  • the Cr-mediated oxidative coupling is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein.
  • OC Oxidative coupling
  • OC-3 Oxidative coupling
  • OC-3 Oxidative coupling
  • the oxidative coupling may be used in combination with any one of the Cr(VI) reduction (CR) and/or isolation and purification of zwitterinoic intermediate (IAPOZI) steps described in WO 2006/032879. See, in particular, pages 25-30, which are incorporated by reference herein.
  • the oxidizing agent is or comprises Cr 2 O 7 '2 . In one embodiment, the oxidizing agent is or comprises Na 2 Cr 2 O 7 . In one embodiment, the oxidizing agent is Na 2 Cr 2 O 7 .
  • the molar ratio of ester, G-IIa, to aniline, III is 0.5 to 1.5. In one embodiment, the molar ratio of ester, Ha, to aniline, III, is 0.5 to 1.5.
  • the molar ratio of Cr(VI) to aniline, III is 1.0 to 4.0.
  • the reaction is performed under acidic conditions.
  • the reaction is performed in an aqueous medium.
  • the reaction temperature is 2 to 20 0 C.
  • an ⁇ /, ⁇ /-disubstituted-substituted-nitrosyl aniline, VIII is reduced to form an ⁇ /, ⁇ /-disubstituted-diamino-substituted benzene, Ia, as illustrated in the following scheme:
  • an ⁇ /, ⁇ /-disubstituted-3-optionally substituted-4-nitrosyl aniline, G is reduced to form a ⁇ /, ⁇ /-disubstituted-1 ,4-diamino-5-optionally substituted benzene, A, as illustrated in the following scheme:
  • an N,N-dimethyl-4-nitrosyl aniline, 6, is reduced to form a N,N-dimethyl-1 ,4-diamino-benzene, 1, as illustrated in the following scheme:
  • nitrosylation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein.
  • the sections relating to Nitrosylation (NOS) at pages 19-20 and Nitrosylation (NOS-2) at pages 46-48 are incorporated herein, along with Examples 1-8 and 17 relating to the synthesis of N,N-dimethyl-1 ,4-diamino-benzene, and Example 19 relating to the synthesis of N,N-dimethyl-1 ,4-diamino-3-ethyl-benzene.
  • nitrosyl analine VIII is reduced with a reducing agent such as metallic iron under acidic conditions.
  • an N,N-disubstituted-substituted aniline, IX is nitrosylated to give an N,N-disubstituted-substituted-nitrosyl aniline, VIII, as illustrated in the following scheme:
  • an ⁇ /, ⁇ /-disubstituted-3-optionally substituted aniline H is 4-nitrosylated to give an ⁇ /, ⁇ /-disubstituted-3-optionally substituted-4-nitrosyl aniline, G, as illustrated in the following scheme:
  • an ⁇ /, ⁇ /-di methyl aniline, 3 is 4-nitrosylated to give an ⁇ /, ⁇ /-dimethyl-4-nitrosyl aniline, 6, as illustrated in the following scheme:
  • an ⁇ /, ⁇ /-dimethyl-3-ethyl-aniline, 8 is 4-nitrosylated to form the corresponding ⁇ /, ⁇ /-dimethyl-3-ethyl-4-nitrosyl aniline, 7, as illustrated in the following scheme:
  • nitrosylation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein.
  • the sections relating to Nitrosylation (NOS-1 ) at pages 19-20 and Nitrosylation (NOS-2) at pages 46-48 are incorporated herein, along with Examples 1-8, and 17 relating to the preparation of ⁇ /, ⁇ /-dimethyl-4-nitrosyl aniline, and Example 19 relating to the preparation of ⁇ /, ⁇ /-dimethyl-3-ethyl-4-nitrosyl aniline.
  • the aniline IX is treated with a nitrite such as an alkali metal nitrite under acidic conditions to give the nitrosyl aniline VIII.
  • a nitrite such as an alkali metal nitrite under acidic conditions
  • a substituted-aniline, X is ⁇ /, ⁇ /-disubstituted using an alkyl halide, an alkenyl halide, a haloalkyl halide, a halide or a halogenated halide, to give a ⁇ /, ⁇ /-disubstituted-substituted aniline, IXa, as illustrated in the following scheme:
  • each of -R 7NC and -R 7ND is independently selected from C 1-4 alkyl; C 2-4 alkenyl; halogenated C 1-4 alkyl; Ci. 4 alkylene-C 5 .ioaryl and halogenated C 1-4 alkylene-C 5 .ioaryl; and -R 9 is as defined previously.
  • a 3-optionally substituted-aniline, J is ⁇ /, ⁇ /-disubstituted using a alkyl halide, an alkenyl halide, a haloalkyl halide, a Ci- 4 alkylene-C 5 .i 0 aryl halide or a halogenated C 1-4 alkylene-C 5 .i 0 aryl halide, to give a ⁇ /, ⁇ /-disubstituted-3-optionally substituted-aniline, H, as illustrated in the following scheme:
  • each of -R 7NC and -R 7ND is independently selected from: Ci -4 alkyl;
  • each of -R 7NC and -R 7ND is independently -Me, -Et, -nPr, -nBu,
  • each of -R 7NC and -R 7ND is independently C 1-4 alkyl.
  • each of -R 7NC and -R 7ND is independently -Me or -Et.
  • each of -R 7NC and -R 7ND is independently -Me.
  • each of -R 7NC and -R 7ND is independently -Et.
  • -R 7NC and -R 7ND are the same. In one embodiment, -R 7NC and -R 7ND are different.
  • a 3-ethyl-aniline, 8, is ⁇ /, ⁇ /-dimethylated using a methyl halide, to give a ⁇ /, ⁇ /-dimethyl-3-ethyl-aniline, 9, as illustrated in the following scheme:
  • the disubstitution is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein.
  • the section relating to ⁇ /. ⁇ /-Disubstitution (NNDS-2) at pages 45-46 is incorporated herein, along with Example 19 relating to the preparation of ⁇ /, ⁇ /-dimethyl-m-ethyl-aniline.
  • aniline X is treated with an alkyl halide, an alkenyl halide, or a haloalkyl halide under basic conditions to give substituted aniline IXa.
  • a ⁇ /, ⁇ /-disubstituted-diamino-substituted benzene, Ia is ⁇ /, ⁇ /-disubstituted using an alkyl halide, an alkenyl halide, a haloalkyl halide, a Ci. 4 alkylene-C 5 .ioaryl halide or a halogenated halide, to give a di- ⁇ /, ⁇ /-disubstituted- substituted aniline, Ic, as illustrated in the following scheme:
  • each of -R 10NC and -R 10ND is independently selected from C 1-4 alkyl; C 2-4 alkenyl; halogenated Ci -4 alkyl; Ci. 4 alkylene-C 5 . 10 aryl and halogenated Ci -4 alkylene-C 5 .i 0 aryl; and -R 7NA , -R 7NB , and -R 9 are as previously defined.
  • the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.
  • the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al +3 .
  • Suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
  • suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH 3 J 4 + .
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic, and valeric.
  • a reference to a particular compound also includes salt forms thereof.
  • a reference to a salt also includes mixed salt forms thereof.
  • solvate is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
  • a reference to a particular compound also includes solvate and hydrate forms thereof.
  • the diaminophenothiazinium compounds may be provided in a composition or formulation for administration to a subject, for example a subject having AD. While it is possible for the compound to be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation.
  • a method of preparing a composition or formulation comprising a diaminophenothiazinium compound includes the synthesis of a diaminophenothiazinium compound comprising one or more of the steps described herein.
  • the method further comprises the step of admixing at least one diaminophenothiazinium compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
  • the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a diaminophenothiazinium compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • a pharmaceutical composition e.g., formulation, preparation, medicament
  • a pharmaceutically acceptable carrier e.g., diluent, or excipient.
  • the composition is a pharmaceutical composition comprising at least one diaminophenothiazinium compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
  • pharmaceutically acceptable carriers diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
  • the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.
  • Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA); Remington: The Science and Practice of Pharmacy, 20th Edition (ed. Gennaro et al.), 2000, Lippincott, Williams & Wilkins, Baltimore; and Handbook of Pharmaceutical Excipients, 2nd Edition (eds A. Wade and P.J. Weller), 1994, American Pharmaceutical Association, Washington and The Pharmaceutical Press, London.
  • pharmaceutically acceptable pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
  • carriers e.g., liquid carriers, finely divided solid carrier, etc.
  • the formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate).
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 ⁇ g/ml, for example from about 10 ng/ml to about 1 ⁇ g/ml.
  • the formulations may be presented in unit-dose or multi- dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • the present invention provides diaminophenothiazinium compounds and formulations comprising diaminophenothiazinium compounds as described herein for use in therapy.
  • the invention pertains to compounds obtained or obtainable by the methods described herein for use in therapy.
  • the compound is a compound obtained by the methods described herein.
  • the compounds and compositions described herein may reverse or inhibit the aggregation of tau protein (e.g. in the form of paired helical filaments (PHFs), optionally in neurofibrillary tangles (NFTs)) in the brain of a mammal.
  • tau protein e.g. in the form of paired helical filaments (PHFs), optionally in neurofibrillary tangles (NFTs)
  • PHFs paired helical filaments
  • NFTs neurofibrillary tangles
  • One aspect of the invention is the use of a diaminophenothiazinium compound (for example, obtained by the methods described herein) to reverse or inhibit the aggregation of tau protein.
  • This aggregation may be in vitro, or in vivo, and may be associated with a tauopathy disease state as discussed herein.
  • methods of reversing or inhibiting the aggregation of tau protein comprising contacting the aggregate or protein with a compound as described herein.
  • tauopathy disorders that have been recognized which feature prominent tau pathology in neurons and/or glia and this term has been used in the art for several years.
  • the similarities between these pathological inclusions and the characteristic tau inclusions in diseases such as AD indicate that the structural features are shared and that it is the topographic distribution of the pathology that is responsible for the different clinical phenotypes observed.
  • those skilled in the art can identify tauopathies by combinations of cognitive or behavioural symptoms, plus additionally through the use of appropriate ligands for aggregated tau as visualised using PET or MRI, such as those described in WO02/075318.
  • One aspect of the present invention pertains to a method of treatment or prophylaxis of a tauopathy condition in a patient, comprising administering to said patient a therapeutically-effective amount of a compound of formula (I), as described herein.
  • the compound of formula (I) is obtained by the methods described herein.
  • aspects of the present invention relate to "tauopathies".
  • AD Alzheimer's disease
  • PSP Progressive Supranuclear Palsy
  • FTDP fronto- temporal dementia
  • FTDP- 17 FTD with parkinsonism linked to chromosome 17
  • DDPAC disinhibition-dementia-parkinsonism-amyotrophy complex
  • PPND pallido- ponto-nigral degeneration
  • ALS Guam-amyotrophic lateral sclerosis
  • PNLD pallido-nigro-luysian degeneration
  • CBD cortico-basal degeneration
  • Dementia with Argyrophilic grains AgD
  • DP Dementia pugilistica
  • Senile dementia of Lewy body type and Alzheimer type are biochemically distinct in terms of paired helical filaments and hyperphosphorylated tau protein. Dementia 5, 215-228).
  • Tau-positive NFTs are also found in Postencephalitic parkinsonism (PEP) (Hof P. R., Charpiot, A., Delacourte A., Buee, L., Purohit, D., Perl D. P. and Bouras, C. (1992) Distribution of neurofibrillary tangles and senile plaques in the cerebral cortex in postencephalitic parkinsonism. Neurosci. Lett. 139, 10-14).
  • PEP Postencephalitic parkinsonism
  • Glial tau tangles are observed in Subacute sclerosing panencephalitis (SSPE) (Ikeda K., Akiyama H., Kondo H., Arai T., Arai N. and Yagishita S. (1995) Numerous glial fibrillary tangles in oligodendroglia in cases of subacute sclerosing panencephalitis with neurofibrillary tangles. Neurosci. Lett., 194, 133-135).
  • SSPE Subacute sclerosing panencephalitis
  • tau pathology may also contribute more generally to cognitive deficits and decline, including in mild cognitive impairment (MCI) (see e.g. Braak, H., Del Tredici, K, Braak, E. (2003) Spectrum of pathology. In Mild cognitive impairment: Aging to Alzheimer's disease edited by Petersen, R.C.; pp. 149-189).
  • MCI mild cognitive impairment
  • the tauopathy is selected from the list consisting of the indications above, i.e., AD, Pick's disease, PSP, FTD 1 FTDP-17, DDPAC, PPND, Guam-ALS syndrome, PNLD, and CBD and AgD, DS, SSPE, DP, PEP, DLB and MCI.
  • the tauopathy is Alzheimer's disease (AD).
  • One aspect of the present invention pertains to a compound of formula (I), as described herein, for use in a method of treatment or prophylaxis (e.g., of a tauopathy condition) of the human or animal body by therapy.
  • the compound of formula (I) is obtained by the methods described herein.
  • One aspect of the present invention pertains to use of a compound of formula (I), as described herein, in the manufacture of a medicament for use in the treatment or prophylaxis of a tauopathy condition.
  • the compound of formula (I) is obtained by the methods described herein.
  • a further embodiment is a method of treatment or prophylaxis of a disease of tau protein aggregation as described herein, which method comprises administering to a subject a diaminophenothiazinium compounds, or therapeutic composition comprising the same, such as to inhibit the aggregation of the tau protein associated with said disease state.
  • reaction mixture was stirred for 3 hours at 5 0 C and then warmed to 22 0 C over a period of 1 hour.
  • the dark solid which precipitated was collected via filtration.
  • the black solid was washed with water (50 ml) then dried at 40 0 C for 16 hours, 7.88 g (43 %).
  • ⁇ /', ⁇ /'-dimethyl-p-phenylene diamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and tetrahydrofuran (20 ml) and the solution was then cooled to 5 0 C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period.
  • the reaction mixture was stirred for 3 hours at 5 0 C and then warmed to 22 0 C over a period of 1 hour.
  • the black solid which precipitated was collected via filtration.
  • the black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0 C for 16 hours.
  • ⁇ /', ⁇ /-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added.
  • the slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0 C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration.
  • the purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0 C for 16 hours, 3.7 g (40 %).
  • ⁇ /'. ⁇ /'-dimethyl-p-phenylene diamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and acetonitrile (20 ml) and the solution was then cooled to 5 0 C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period.
  • the reaction mixture was stirred for 3 hours at 5 0 C and then warmed to 22 0 C over a period of 1 hour.
  • the black solid which precipitated was collected via filtration.
  • the black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 °C for 16 hours.
  • ⁇ f, ⁇ /'-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added.
  • the slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0 C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration.
  • the purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0 C for 16 hours, 4.3 g (47 %).
  • ⁇ /', ⁇ /'-dimethyl-p-phenylene diamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and N, ⁇ /-dimethylformamide (20 ml) and the solution was then cooled to 5 0 C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period.
  • the reaction mixture was stirred for 3 hours at 5 0 C and then warmed to 22 0 C over a period of 1 hour.
  • the black solid which precipitated was collected via filtration.
  • the black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0 C for 16 hours.
  • ⁇ /', ⁇ /-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added.
  • the slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0 C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration.
  • the purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0 C for 16 hours, 4.8 g (52 %).
  • ⁇ /', ⁇ /'-dimethyl-p-phenylene diamine 5 g, 0.036 mols
  • dodecylbenzenesulfonic acid, sodium salt (0.13 g, 0.00036 mols) were added to a round bottom flask.
  • De-ionised water 100 ml was added and the solution was then cooled to 5 0 C.
  • Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot.
  • Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added dropwise over a 15 minute period.
  • the reaction mixture was stirred for 3 hours at 5 0 C and then warmed to 22 0 C over a period of 1 hour.
  • the black solid which precipitated was collected via filtration.
  • the black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0 C for 16 hours.
  • ⁇ T, ⁇ /-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added.
  • the slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0 C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration.
  • the purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0 C for 16 hours, 4.6 g (50 %).
  • ⁇ /', ⁇ /'-dimethyl-p-phenylenediamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (100 ml) and the solution was then cooled to 5 0 C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0 C and then warmed to 22 0 C over a period of 1 hour.
  • the black solid which precipitated was collected via filtration.
  • the black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0 C for 16 hours.
  • ⁇ /', ⁇ /'-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added.
  • the slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0 C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration.
  • 2-amino-5-dimethylaminophenylthiosulfonic acid (15 g, 0.0605 mols) was added to a round bottom flask and de-ionised water (300 ml) was added. The solution was cooled to ⁇ 5 0 C and a pre-cooled mixture of ⁇ /, ⁇ /-dimethylaniline (7.32 g, 0.0605 mols), sulphuric acid (5.93 g, 0.0605 mols) and de-ionised water (7.32 ml) was added in one aliquot. The pH of the mixture was adjusted to pH -7.5 using 6 M sodium hydroxide ( ⁇ 30 ml).
  • ⁇ /', ⁇ /'-diethyl-p-phenylenediamine (5 g, 0.030 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and methanol (20 ml) and the solution was then cooled to 5 0 C. Sodium thiosulfate (8.31 g, 0.033 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (7.25 g, 0.030 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0 C and then warmed to 22°C over a period of 1 hour. The dark brown solid which precipitated was collected via filtration. The dark brown solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0 C for 16 hours, 4.43 g (52 %).
  • 2-amino-5-diethylaminophenyl thiosulfonic acid was added to a round bottom flask and ethyl acetate (240 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 C C. Once the solution had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (50 ml) and the solid was dried under vacuum (1000 mbar) at 40 0 C for 16 hours, 4.2 g.
  • the mixture was then allowed to increase in temperature to 22 0 C before being filtered.
  • the grey solid was washed with water (3 x 20 ml) and ethyl acetate (3 x 25 ml) to give the crude product (1.29 g, 37 %, MW 262.35 g/mol) as a light grey solid.
  • the pH of the mixture was then adjusted to pH ⁇ 7.5 with sodium hydroxide (6 M, ⁇ 1 ml) to give a black slurry.
  • Sodium persulfate (1.36g, 5.72 mmol, MW 238.10 g/mol, dissolved in 2 ml of water) was added drop-wise to the cooled near neutral slurry and the reaction was allowed to react for two hours.
  • the mixture was then allowed to increase in temperature to 24 0 C before being filtered.
  • the black solid was washed with water (3 x 10 ml) and air dried for two hours to give the crude product (0.42 g, 56 %, MW 392.52 g/mol) as a black solid.
  • the pH of the mixture was adjusted to pH -7.5 using 6 M sodium hydroxide ( ⁇ 400 ul).
  • a solution of sodium persulfate (0.395 g, 1.66 mmol) in water (2 ml) was added drop-wise over a period of 15 min.
  • the reaction mixture was stirred for 1 hour at 5 0 C and then warmed to room temperature (22 ° C) over a 1 hour period.
  • the solid was collected by filtration and washed with water (4 x 30 ml).
  • To the filtrate was added copper (II) sulphate (50 mg, 0.3 mmol) and the solution was heated at 85 ° C for 20 min.
  • reaction mixture was left to cool to room temperature (22 0 C) and sodium chloride (10 g, 171 mmol) was added and the mixture was stirred for 1 h.
  • the reaction mixture was then extracted with DCM (4 x 30 ml) and the combined organics were dried (Na 2 SO 4 ) and the solvent was removed under reduced pressure to give a blue oil that was purified by column chromatography (10 % MeOH in DCM) to give 13 as a dark blue solid.
  • ⁇ / 4 , ⁇ / 4 -Dimethyl-2-methyl-1 ,4-phenylene diamine monohydrochloride (6.0 g, 27.9 mmol, 1.0 eq.) was dissolved in water (80 cm 3 ), and methanol (20 cm 3 ) added. The solution was stirred by overhead stirrer and cooled to an internal temperature of ⁇ 2 0 C. Sodium thiosulfate (7.6 g, 30.1 mmol, 1.1 eq.) was dissolved in H 2 O (10 cm 3 ) and added to the reaction in one portion.
  • the pH of the medium was increased from 1.75 to 7.00 by addition of a saturated sodium carbonate solution, and volatiles were removed in vacuo.
  • the solution was extracted with ethyl acetate (3 x 100 cm 3 ). Upon the development of a characteristic deep blue colour in the aqueous residue, the solution was extracted with dichloromethane (100 cm 3 ), the extract dried with magnesium sulfate and solvent removed in-vacuo to yield an intensely blue oil.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

This invention pertains generally to the field of chemical synthesis and purification, and more specifically to methods of synthesizing and purifying certain 3,7-diamino- phenothiazin-5-ium compounds (referred to herein as "diaminophenothiazinium compounds") including Methylthioninium Chloride (MTC) (also known as Methylene Blue). In one embodiment, the method comprises the steps of, in order: a thiosulfonic acid formation (TSAF) as illustrated below, wherein the reaction is carried out in the presence of a thiosulfate and an oxidizing agent that is or comprises persulfate an oxidative coupling (OC); and a ring closure (RC). The resulting compounds and compositions comprising them (e.g., tablets, capsules) are useful in methods of medical treatment and diagnosis, etc., for example, tauopathies, or Alzheimer's disease (AD).

Description

METHODS OF CHEMICAL SYNTHESIS OF DIAMINOPHENOTHIAZINIUM COMPOUNDS INVOLVING THE USE OF PERSULFATE OXIDANTS
Technical Field
This invention pertains generally to the field of chemical synthesis and purification, and more specifically to methods of synthesizing and purifying certain 3,7-diamino- phenothiazin-5-ium compounds (referred to herein as "diaminophenothiazinium compounds") including Methylthioninium Chloride (MTC) (also known as Methylene Blue). The present invention also pertains to the resulting compounds, and compositions comprising them (e.g., tablets, capsules). Such compounds and compositions find use in methods of inactivating pathogens, and methods of medical treatment and diagnosis, etc., for example, for tauopathies, Alzheimer's disease (AD), skin cancer, melanoma, viral diseases, bacterial diseases and protozoal diseases.
Background
Throughout this specification, including any claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and any appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment.
Methylthioninium Chloride (MTC) (also known as Methylene Blue)
Methylthioninium Chloride (MTC) (also known as Methylene blue (MB); methylthionine chloride; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9; tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathionium chloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet; and Urolene Blue® is a low molecular weight (319.86), water soluble, tricyclic organic compound of the following formula:
Figure imgf000003_0001
Methylthioninium Chloride (MTC), perhaps the most well known phenothiazine dye and redox indicator, has also been used as an optical probe of biophysical systems, as an intercalator in nanoporous materials, as a redox mediator, and in photoelectrochomic imaging.
See, for example, Colour Index (Vol. 4, 3rd edition, 1971) and Lillie et al., 1979, and references cited therein.
MTC was first described in a German Patent in 1877 (Badische Anilin- und Soda- Fabrik, 1877). In that patent, MTC was synthesized by nitrosylation of dimethylaniline, subsequent reduction to form N,N-dimethyl-1 ,4-diaminobenzene, and subsequent oxidative coupling in the presence of hydrogen sulphide (H2S) and iron(lll) chloride (FeCI3).
Bernthsen described subsequent studies of MTC and methods for its synthesis (see Bernthsen, 1885a, 1885b, 1889).
Fierz-David and Blangley, 1949, also describes methods for the synthesis of MTC from dimethylaniline, as illustrated in the following scheme: Scheme 1.
Figure imgf000004_0001
dimethylaniline p-nitroso-dimethylaniline p-amino-dimethylaniline
Figure imgf000004_0002
Thiosulfonic acid of Thiosulfonic acid of p-amino-dimethylaniline Bindschedler green
Figure imgf000004_0003
MTC
In step (a), nitrosodimethylaniline is prepared from dimethylaniline by treatment with nitrite (NaNO2) in aqueous acid (HCI) solution. In step (b), the nitroso compound is reduced to form p-aminodimethylaniline in aqueous acid (HCI) solution using zinc dust. In steps (c), (d), and (e), the p-aminodimethylaniline is oxidized in aqueous acid solution with another molecule of dimethylaniline, and simultaneously a thiosulfonic acid group is introduced; the ring is then closed using manganese dioxide or copper sulfate. More specifically, a clear neutral solution of p-aminodimethylaniline is acidified (H2SO4), and a non-reducing zinc chloride solution is added (ZnCI2 with Na2Cr2O7). Aluminium thiosulfate (AI2(S2O3)3) and sodium thiosulfate (Na2S2O3) are added. Sodium dichromate (Na2Cr2O7) is added. The mixture is heated and aerated. Dimethylaniline is added. Sodium dichromate (Na2Cr2O7) is added. The mixture is heated, and becomes dark greenish-blue in colour due to the formation of the thiosulfonic acid of Bindschedler green. Manganese dioxide or copper sulfate is added, and the mixture heated, and the dye precipitates from the concentrated zinc chloride solution.
Very similar synthesis methods are described in the Colour Index (Vol. 4, 3rd edition, 1971), p. 4470.
Masuya et al., 1992, describe certain phenothiazine derivatives, and methods for their preparation and use in photodynamic therapy of cancer and in immunoassays utilizing chemiluminescence. The compounds are prepared by routes similar to those discussed above.
Leventis et al., 1997, describe methods for the synthesis of certain MTC analogs, which employ phenothiazine as a starting material and which add the desired 3,7- substituents by halogenation followed by amination. The authors assert that MTC is synthesized commercially by oxidation of N,N-dimethyl-p-phenylene diamine with Na2Cr2O7 in the presence of Na2S2O3, followed by further oxidation in the presence of N,N-dimethylamine.
Marshall and Lewis, 1975a, describes the purification of commercial MTC and Azure B by solvent extraction and crystallisation. They assert that aqueous MTC/Azure B mixtures at a buffered pH of 9.5 can be separated by extraction with carbon tetrachloride. The carbon tetrachloride removes the Azure B while leaving the MTC in the aqueous layer. They further assert that low temperature crystallisation of MTC at a concentration of 0.25 N with hydrochloric acid removes metal contaminants. However, the organic purity analysis reported therein is based on thin-layer chromatography, which is not suitable for quantification. Also, the microanalysis for sulphated ash does not indicate a metal free sample. (The preferred technique in 1975 was atomic absorption.)
Marshall and Lewis, 1975b, describes the analysis of metal contaminants in commercial thiazine dyes by atomic absorption spectrophotometry. They report 38 samples with metal concentrations that vary widely between 0.02% and 25.35% of individual samples; the metals examined were iron, potassium, sodium and zinc. They also report that other metals may be present which were not analysed. Aluminium, chromium, manganese, and copper, are all involved in synthetic procedures for MTC and are almost certain to be present. Importantly, they report large variations in the metal content of commercial samples of MTC.
Lohr et al., 1975, describes the purification of Azure B by column chromatography, specifically by separation to isolate the desired product followed by ion exchange back to the chloride. They assert that other cationic dyes such as MTC can be purified by this method. However, column chromatography is not a suitable method for the purification of MTC on a large scale.
Fierz-David et al., 1949, describes the synthesis of the zinc chloride double salt of MTC and the removal .of zinc by chelation with sodium carbonate followed by filtration to generate zinc free methylene blue. However, the authors acknowledge that this technique cannot be used on a large scale, because the yields are poor. WO 2006/032879 describes the synthesis of MTC, and other diaminophenothiazinium compounds, by a number of methods, including an adaptation of the Fierz-David and Blangley procedure described above. The methods described provide diaminophenothiazinium products having reduced levels of metals and contaminating organics.
In particular, WO 2006/032879 addresses the issue of residual Cr levels in a diaminophenothiazinium synthesis by reducing residual Cr(VI) to Cr(III), which is a much less toxic form, so that pharmaceutical standards can more easily be satisfied. This reduction was also found to greatly increase the yield of the final diaminophenothiazinium compound. Experimentally, it was also established that chromium can more easily be removed from a product when in the form of Cr(III) than when in the form of Cr(VI).
MTC and derivatives thereof (e.g., "diaminophenothiazinium compounds") have been found to be useful in the treatment of tauopathies (such as, for example, Alzheimer's disease) (see, for example, Wischik, CM., et al., 1996, 2002).
Oral and parenteral formulations of MTC are commercially available in the United States, usually under the name Urolene Blue®. However, these formulations contain substantial amounts of metal impurities. These impurities are highly undesirable, and many (e.g., including Al1 Cr, Fe, Cu) exceed the safety limits set by European health agencies.
Consequently, there is a great need for higher purity (e.g., pharmaceutical grade purity, e.g., a purity safe for human consumption, e.g., with low or reduced metal content) diaminophenothiazinium compounds, including MTC.
Summary of the Invention
One aspect of the present invention pertains to a method of synthesis of diaminophenothiazinium compounds, including high purity diaminophenothiazinium compounds.
Another aspect of the present invention pertains to a method of purification of diaminophenothiazinium compounds.
Further aspects of the invention pertain to methods for the synthesis of intermediate compounds which are suitable for use in the synthesis of diaminophenothiazinium compounds. Another aspect of the present invention pertains to diaminophenothiazinium compounds obtained or obtainable by the methods of the invention for use in therapy.
As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspects of the invention.
Detailed Description
Compounds
In general, the present invention pertains to methods for the preparation of certain diamino-phenothiazin-5-ium compounds of the following formula, collectively referred to herein as "diaminophenothiazinium compounds":
Figure imgf000007_0001
wherein: each of -R1 and -R9 is independently -H or -RA; and each -RA is independently selected from C1-4alkyl; C2-4alkenyl; halogenated Ci-4alkyl; C5.i0aryl; halogenated C5.10aryl; C1-4alkylene-C5-1oaryl and halogenated C1-4alkylene-C5-10aryl; each of -R3NA and -R3NB is independently selected from: C1-4alkyl; C∑^alkenyl; halogenated Ci-4alkyl; C5-10aryl; halogenated C5.10aryl; C1-4alkylene-C5.10aryl and halogenated Ci.4alkylene-C5-10aryl; each of -R7NA and -R7NB is independently selected from: C1-4alkyl; C2-4alkenyl; halogenated d-4alkyl; C5-10aryl; halogenated C5-i0aryl; C1-4alkylene-C5.10aryl and halogenated
Figure imgf000007_0002
and
X" is an anionic counter ion.
Although the diaminophenothiazinium compounds are themselves salts, they may also be provided in the form of a mixed salt (i.e., the diaminophenothiazinium compound in combination with another salt). Such mixed salts are intended to be encompassed by the term "and pharmaceutically acceptable salts thereof. Unless otherwise specified, a reference to a particular compound also includes salts thereof.
The diaminophenothiazinium compound is also referred to as compound Vl, and in certain embodiments, where X" is Cl", compound VII. In one embodiment, the present invention pertains to methods for the preparation of certain 3,7-diamino-phenothiazin-5-ium compounds of the following formula:
Figure imgf000008_0001
The above structure is only one of many equivalent resonance structures, some of which are shown below, and all of which are intended to be encompassed by the above structure:
Figure imgf000008_0002
In one aspect, the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "activated thio ether compounds" or "diaminoaryl sulphides":
Figure imgf000009_0001
G-Il wherein A is an activating group and each of -R10NA and -R10NB is independently selected from: -H; C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5-10aryl; halogenated C5.10aryl; C1-4alkylene-C5-ioaryl and halogenated C1-4alkylene-C5.10aryl. The groups -R9, -R7NA and -R7NB are as defined for the diaminophenothiazinium compounds.
In one embodiment, the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "thiosulfonic acid compounds":
Figure imgf000009_0002
In one aspect, the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "activated imino compounds":
Figure imgf000009_0003
G-IV wherein -R1, -R9, -R3NA and -R3NB, -R7NA and -R7NB are as defined for the diaminophenothiazinium compounds, and -A is as defined for the activated thio ether compounds (diaminoaryl sulphides). The compound G-IV is shown as a cation, which may be used as a salt with a suitable counter ion, X". Compound G-IV may also be used in a zwitterionic form where a negative charge is associated with the group -A. In one embodiment, the present invention pertains to methods for the preparation of compounds of the following formula, collectively referred to herein as "imino compounds":
Figure imgf000010_0001
Embodiments
The following embodiments may apply to each of the compounds described herein, as appropriate.
Alkyl and Alkenyl Groups
In one embodiment, the C1-4alkyl groups are selected from: linear C1-4alkyl groups, such as -Me, -Et, -nPr, -iPr, and -nBu; branched C3-4alkyl groups, such as -iPr, -iBu, -sBu, and -tBu; and cyclic C3-4alkyl groups, such as -cPr and -cBu.
In one embodiment, the C2.4alkenyl groups are selected from linear Ci-4alkenyl groups, such as -CH=CH2 (vinyl) and -CH2-CH=CH2 (allyl).
In one embodiment, the halogenated C1-4alkyl groups are selected from: -CF3, -CH2CF3, and -CF2CF3.
Aryl and Alkylenearyl Groups
In one embodiment, the C5-i0aryl groups are selected from C6-iocarboaryl groups, such as phenyl or napthyl, and C5.10heteroaryl groups, such as thienyl, imidazolyl, pyrazolyl, triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl or quinolinyl.
In one embodiment, the halogenated C5-10aryl groups are selected from halogenated C6-10carboaryl groups, such as -4-fluoro-phenyl, -3-fluoro-phenyl, and -2-fluoro-phenyl, and halogenated C5.i0heteroaryl groups.
In one embodiment, the
Figure imgf000010_0002
group is benzyl. In one embodiment, the halogenated C1.4alkylene-C5.1oaryl groups are selected from halogenated C1-4alkylene-C6-iocarboaryl groups, such as -4-fluoro-benzyl, -3-fluoro- benzyl, and -2-fluoro-benzyl, and halogenated C1-4alkylene-C5.ioheteroaryl groups.
-R1 and -R9
In one embodiment, each of -R1 and -R9 is independently -H or -RA.
In one embodiment, each -R1 is independently -H.
In one embodiment, each -R9 is independently -H.
In one embodiment, at least one -R1 is independently -RA.
In one embodiment, at least one -R9 is independently -RA.
In one embodiment, -R1 and -R9, where present, are the same. In one embodiment, -R1 and -R9, where present, are different.
In one embodiment, only one -R9 is other than -H. In one embodiment, only one -R1 is other than -H.
-RA
In one embodiment, each -RA, where present, is independently selected from C1-4alkyl; C2.4alkenyl; halogenated C1-4alkyl; C5-i0aryl; halogenated C5-ioaryl; Ci.4alkylene-C5.i0aryl and halogenated Ci.4alkylene-C5-i0aryl;
In one embodiment, each -RA, where present, is independently selected from C1-4alkyl;
C2-4alkenyl; and halogenated C1-4alkyl.
In one embodiment, each -RA, where present, is independently -Me, -Et, or -CF3.
In one embodiment, each -RA, where present, is independently C1.4alkyl. In one embodiment, each -RA, where present, is independently -Me. In one embodiment, each -RA, where present, is independently -Et.
In one embodiment, each -RA, where present, is independently C2-4alkenyl.
In one embodiment, each -RA, where present, is independently halogenated C1-4alkyl. In one embodiment, each -RA, where present, is independently -CF3.
In one embodiment, each -RA, where present, is independently C5.i0aryl.
In one embodiment, each -RA, where present, is independently C6-iocarboaryl.
In one embodiment, each -RA, where present, is independently phenyl.
In one embodiment, each -RA, where present, is independently Ci-4alkylene-C5.i0aryl. In one embodiment, each -RA, where present, is independently benzyl.
In one embodiment, each -RA, where present, is independently halogenated C5.i0aryl.
In one embodiment, each -RA, where present, is independently halogenated
C6-iocarboaryl.
In one embodiment, each -RA, where present, is independently -4-fluoro-phenyl,
-3-fluoro-phenyl or -2-fluoro-phenyl.
-R3NA, -R3NB, -R7NA, and -R7NB
In one embodiment, each of -R3NA and -R3NB is independently selected from: C1-4alkyl; C2-4alkenyl; halogenated Ci-4alkyl; C5-10aryl; halogenated C5-i0aryl; Ci-4alkylene-C5.10aryl and halogenated d^alkylene-Cs.^aryl.
In one embodiment, each of -R3NA and -R3NB is independently selected from: C1-4alkyl; C2-4alkenyl; and halogenated Ci.4alkyl.
In one embodiment, each of -R3NA and -R3NB is independently -Me, -Et, -nPr, -nBu,
-CH2-CH=CH2, or -CF3.
In one embodiment, each of -R3NA and -R3NB is independently C1-4alkyl.
In one embodiment, each of -R3NA and -R3NB is independently -Me or -Et.
In one embodiment, each of -R3NA and -R3NB is independently -Me.
In one embodiment, each of -R3NA and -R3NB is independently -Et.
In one embodiment, -R3NA and -R3NB are the same. In one embodiment, -R3NA and -R3NB are different.
In one embodiment, each of -R7NA and -R7NB is independently selected from d-4alkyl; C2.4alkenyl; halogenated C1-4alkyl; C5-ioaryl; halogenated C5-10aryl;
Figure imgf000012_0001
and halogenated C1-4alkylene-C5-10aryl.
In one embodiment, each of -R7NA and -R7NB is independently selected from C1-4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
In one embodiment, each of -R7NA and -R7NB is independently -Me, -Et, -nPr, -nBu,
-CH2-CH=CH2, or -CF3.
In one embodiment, each of -R7NA and -R7NB is independently C1-4alkyl.
In one embodiment, each of -R7NA and -R7NB is independently -Me or -Et.
In one embodiment, each of -R7NA and -R7NB is independently -Me.
In one embodiment, each of -R7NA and -R7NB is independently -Et. In one embodiment, -R7NA and -R7NB are the same. In one embodiment, -R7NA and -R7NB are different.
In one embodiment, -R3NA and -R7NA, where present, are the same. In one embodiment, -R3NB and -R7NB, where present, are the same.
In one embodiment, the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, are the same.
In one embodiment, the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, are the same, and are selected from: -NMe2, -NEt2, -N(nPr)2, -N(Bu)2, -NMeEt, -NMe(nPr), and -N(CH2CH=CHz)2.
In one embodiment, the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, are the same, and are selected from: -NMe2 and -NEt2.
In one embodiment, the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, are each -NMe2.
In one embodiment, the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, are each -NEt2.
In one embodiment, the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, are other than -NMe2.
-R10NA and -R10NB
In one embodiment, each of -R10NA and -R10NB is independently selected from: -H; C1-4alkyt; C2.4alkenyl; halogenated C1-4alkyl; C5-i0aryl; halogenated C5-10aryl;
Figure imgf000013_0001
In one embodiment, each of -R3NA and -R3NB is independently selected from: -H; -C1-4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
In one embodiment, each of -R10NA and -R10NB is independently -H.
In one embodiment, each of -R10NA and -R10NB is independently selected from:
-CT^alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
In one embodiment, each of -R10NA and -R10NB is independently -Me, -Et, -nPr, -nBu,
-CH2-CH=CH2, Or -CF3.
In one embodiment, each of -R10NA and -R10NB is independently C1-4alkyl. In one embodiment, each of -R10NA and -R10NB is independently -Me or -Et. In one embodiment, each of -R10NA and -R10NB is independently -Me. In one embodiment, each of -R10NA and -R10NB is independently -Et.
In one embodiment, -R10NA and -R10NB are the same. In one embodiment, -R10NA and -R10NB are different.
-A
In one embodiment, -A is an activating group. In one embodiment, the activating group -A activates the sulfur atom of the thio ether (diaminoaryl sulphide) for ring closure, such as a ring closure reaction described herein.
In one embodiment, -A is independently selected from: -S(=O)OH, -S(=O)2OH, -S(=O)ORA, -S(=O)2ORA, -S(=O)2RA,
-S(=O)2NH2, -S(=O)2NHRA, -S(=O)2NRA 2, -S(=O)2NRA2RA3,
-Se(=O)OH, -Se(=O)2OH,
-Se(=O)ORA, -Se(=O)2ORA,
-Se(=O)2RA,
-Se(=O)2NH2, -Se(=O)2NHRA, -Se(=O)2NRA 2,
-Se(=O)2NRA2RA3,
-P(=O)(OH)2l
-P(=O)(OH)2, -P(=O)(OH)(ORA), -P(=O)(ORA)2,
-Si(RA)3, and
-B(RA)2.
where -RA is as defined previously, and each -NRA2RA3, if present, is independently azetidino, pyrrolidino, imidazolidino, pyrazolidino, piperidino, piperazino, morpholino, azepino, or diazepino.
In one embodiment, the group -A includes the ionic forms of the groups above, where appropriate. For example, -S(=O)2OH encompasses the anionic form -S(=O)2O\
In one embodiment, -A is independently selected from: -S(=O)OH, -S(=O)2OH, -S(=O)ORA, -S(=O)2ORA, -S(=O)2RA,
-S(=O)2NH2, -S(=O)2NHRA, -S(=O)2NRA 2,
-S(=O)2NRA2RA3,
-Se(=O)OH, -Se(=O)2OH,
-Se(=O)ORA, -Se(=O)2ORA,
-Se(=O)2RA,
-Se(=O)2NH2, -Se(=O)2NHRA, -Se(=O)2NRA 2,
-Se(=O)2NRA2RA3.
In one embodiment, -A is independently selected from: -S(=O)OH, -S(=O)2OH, -S(=O)ORA, -S(=O)2ORA, -S(=O)2RA,
-Se(=O)OH, -Se(=O)2OH, -Se(=O)ORA, -Se(=O)2ORA, -Se(=O)2RA
In one embodiment, -A is independently selected from: -S(=O)OH, -S(=O)2OH, -S(=O)ORA, -S(=O)2ORA, -S(=O)2RA.
In one embodiment, -A is independently selected from -S(=O)2OH, -S(=O)2ORA, and -S(=O)2RA
In one embodiment, -A is independently -S(=O)2OH (also written as -SO3H or sulfonic acid). In this embodiment the activated S-ether is a thiosulfonic acid (-SSO3H).
Isotopes
In one embodiment, one or more of the carbon atoms is 11C or 13C. In one embodiment, one or more of the carbon atoms is 11C. In one embodiment, one or more of the carbon atoms is 13C. In one embodiment, one or more of the nitrogen atoms is 15N.
In one embodiment, one or more or all of the carbon atoms of one or more or all of the groups R3NA, R3NB, R7NA and R7NB is 13C. In one embodiment, each of the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, is -N(13CH3)2.
In one embodiment, each of R1 and R9, where present, is -H, and each of the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, is -N(13CH3)2.
In one embodiment, each of R1 and R9, where present, is -H; each of the groups -N(R3NA)(R3NB) and -N(R7NA)(R7NB), where present, is -N(13CH3)2; and X" is Cl\
X
In one embodiment, X' is a counter anion to achieve electrical neutrality. In one embodiment, X" is one or more counter anions to achieve electrical neutrality. In one embodiment, X' is a counter anion shared with other cations to achieve electrical neutrality.
In one embodiment, X" is independently a halogen anion (i.e., halide). In one embodiment, X' is independently F", Cl", Br, or I". In one embodiment, X" is independently Cl", Br", or I". In one embodiment, X' is independently Cl".
In one embodiment, X" is independently NO3 " (nitrate).
In one embodiment, X" is independently CIO3 " (perchlorate).
In one embodiment, X" is independently selected from formate, propionate, benzoate and 4-hydroxybenzenesulfonate.
In one embodiment, X" is independently S2O8 ".
In one embodiment, X" is S2O3 "2.
In one embodiment, X" is SO4 "2 (sulfate).
In one embodiment, X" is succinate.
In this embodiment, the counter ion achieves electrical neutrality by combination with two of the recited cations.
In one embodiment, X" is citrate. In this embodiment, the counter ion achieves electrical neutrality by combination with three of the recited cations.
In one embodiment, the compound is in the form of a mixed salt, for example, a ZnCI2 mixed salt. Combinations
Each and every compatible combination of the embodiments described above, and below, is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.
Example Compounds
Example diaminophenothiazinium compounds include the following:
Figure imgf000017_0001
Figure imgf000018_0001
Example thiosulfonic acid compounds include the following:
Figure imgf000019_0001
Example imino compounds include the following:
Figure imgf000019_0002
Synthesis
One important difference between known methods and the presently claimed methods is the minimisation or avoidance of a dichromate salt as an oxidising agent in the synthesis. In one aspect, the present method avoids the use of a dichromate salt in the oxidative coupling step of a diaminophenothiazinium synthesis, such as those steps described by Fierz-David and Blangley and WO 2006/032879, amongst others.
In another aspect, the present method avoids the use of a dichromate salt in the thiosulfonic acid forming step of a diaminophenothiazinium synthesis, such as those steps described by Fierz-David and Blangley and WO 2006/032879, amongst others.
In one embodiment, the present invention provides a method of preparing MTC that minimises the amount of dichromate salt required in the overall synthesis. In one embodiment, the present invention provides a method of preparing MTC that does not require a dichromate salt.
Sodium dichromate, the preferred dichromate salt in the Fierz-David and Blangley and WO 2006/032879 syntheses, is registered under the International Maritime Dangerous Goods (IMDG) Code under Class 6.1 : Toxic substances. Sodium dichromate is expensive to purchase and ship, and its disposal is likewise costly. Owing to the toxicity and carcinogenicity of sodium dichromate, specialist safety precautions are required to be put in place for its large scale use. The chromium-containing by-products from a chromate oxidation are likewise highly hazardous and must be dealt with appropriately.
Accordingly, the use of sodium dichromate on an industrial scale is impractical, and the diaminophenothiazinium syntheses described previously in the art would therefore not be amenable to large scale production.
The present inventors have established that an alternative range of oxidants may be used in a diaminophenothiazinium synthesis. In particular, the inventors have established that a persulfate salt may be used as an oxidant in a diaminophenothiazinium synthesis.
Previously, dichromate (in the form Cr(VI)) has been described for use (at least) in the oxidative coupling step of the diaminophenothiazinium synthesis. Residual Cr(VI) presents several serious problems. First, high levels of highly toxic contaminants such as residual Cr(VI) are unacceptable in products destined for use in pharmacy. Second, residual Cr(VI) destabilizes the zwitterionic intermediate and impedes the subsequent ring closure (RC) step, and thus reduces the yield of the final diaminophenothiazinium compound.
As indicated above, WO 2006/032879 addresses the issue of residual Cr levels in a diaminophenothiazinium synthesis. However, the solution is to reduce residual Cr(VI) to Cr(III), which can more easily be removed from a product compared to Cr(VI). There is no suggestion or indication that chromium should not be used in a diaminophenothiazinium synthesis.
In one embodiment, a reference herein to a TSAF step is also a reference to an activated thio ether (diaminoaryl sulphide) formation step, as described herein. In one embodiment, a reference herein to a Cr-TSAF step is also a reference to a Cr- mediated activated thio ether (diaminoaryl sulphide) formation step, as described herein.
In one embodiment, the method of synthesis comprises an oxidative coupling (OC).
In one embodiment, the method of synthesis comprises a thiosulfonic acid formation (TSAF).
In one embodiment, the method of synthesis comprises, in order, an oxidative coupling (OC) and a ring closure (RC). In one embodiment, the method of synthesis comprises, in order: a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: an Λ/,/V-disubstitution (NNDS); a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a nitrosyl reduction (NR); a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a nitrosylation (NOS); a nitrosyl reduction (NR); a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: an Λ/,Λ/-disubstitution (NNDS); a nitrosylation (NOS); a nitrosyl reduction (NR); a Cr-mediated thiosulfonic acid formation (Cr-TSAF); an oxidative coupling (OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
In one embodiment, the method of synthesis comprises, in order: an Λ/,Λ/-disubstitution (NNDS); a nitrosylation (NOS); a nitrosyl reduction (NR); a thiosulfonic acid formation (TSAF); a Cr-mediated oxidative coupling (Cr-OC); and a ring closure (RC).
In one embodiment, the method of synthesis further includes the step of chloride salt formation (CSF) after a ring closure (RC) step. Such a step may not be required where the product of the RC step is a chloride salt. General Methods
The methods of the invention generally include at least one activated thio ether (diaminoaryl sulphide) activation step, which may be a thiosulfonic acid formation (TSAF) step, or at least one oxidative coupling (OC) step, as described below. In one embodiment, the method comprises both steps.
Activated Thio Ether (Diaminoaryl Sulphide) Formation and Thiosulfonic Acid Formation (TSAF)
In this step, an Λ/,Λ/-disubstituted-diamino-substituted benzene, I, is oxidized in the presence of an oxidizing agent that is or comprises persulfate to give an activated S-(Λ/,Λ/-disubstituted-diamino-phenyl) ether (a S-activated diaminoaryl sulphide), G-Il, as illustrated in the following scheme:
Figure imgf000023_0001
G-Il
This reaction may be referred to as an activated thio ether (S-activated diaminoaryl sulphide) formation step.
Compound I is oxidised in the presence of an activating group reagent. In one embodiment, the activating group reagent is or comprises [S-A]"' or [S-A], where n is 1 or 2. For example, the activating group reagent is or may comprise the anion [S-SO3]2" (thiosulfate).
In one embodiment, n is 1. In one embodiment, n is 2.
In one embodiment, the activating group reagent additionally comprises a counter cation.
In one embodiment, the counter cation is a sodium, potassium, or ammonium cation.
In one embodiment, the counter cation is a sodium cation.
In one embodiment, the activating group reagent is or comprises thiosulfate. In one embodiment, an Λ/,Λ/-disubstituted-diamino-substituted benzene, I, is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises persulfate to give an amino-(Λ/,Λ/-disubstituted-amino)phenylthiosulfonic acid (a thiosulfonic acid S-(Λ/,Λ/-disubstituted-diamino-phenyl) ester) ), II, as illustrated in the following scheme:
Figure imgf000024_0001
This activated thio ether (S-activated diaminoaryl sulphide) formation reaction may be referred to as a thiosulfonic acid formation (TSAF) step.
The phenyl ring in compound I is substituted with an amino group, -NR10NAR10NB, at any one of the 2-, 3-, 4-, 5-, or 6-positions, and is substituted with three substituents -R9, at any three of the four remaining positions. The final position is substituted with -H.
The phenyl ring in compound Il is substituted with an amino group, -NR10NAR10NB, at any of the 2-, 3-, A-, 5-, or 6-positions, and is substituted with three substituents -R9, at any three of the four remaining positions. The final position is substituted with thiosulfonic acid, -S-SO3H.
In one embodiment, compounds I and Il are compounds of formula Ia and Ha respectively as shown below:
Figure imgf000024_0002
Ia Ma
In one embodiment, compounds I and Il are compounds of formula Ib and Hb respectively as shown below:
Figure imgf000025_0001
Ib lib
In one embodiment, an Λ/,Λ/-disubstituted-1 ,4-diamino-5-optionally substituted benzene, A, is oxidized in the presence of a thiosulfate to give a thiosulfonic acid S-{2- (amino)-3-(optionally substituted)-5-(Λ/,Λ/-disubstituted-amino)-phenyl} ester (an famino-(Λ/,Λ/-disubstituted)aminophenylthiosulfonic acid), B, as illustrated in the following scheme:
Figure imgf000025_0002
B
In one embodiment, an N,N-dimethyl-1 ,4-diamino-benzene, 1, is oxidized in the presence of a thiosulfate to give a thiosulfonic acid S-{2-(amino)-5-(dimethylamino)- phenyl} ester (an amino-(Λ/,Λ/-disubstituted-amino)phenylthiosulfonic acid), 2, as illustrated in the following scheme:
Figure imgf000025_0003
Compound 1 may be obtained from commercial sources, or may be prepared directly or indirectly from the corresponding nitrosyl compound, as described below in the NOS and DNNDS steps.
The thiosulfate is or comprises S2O -2
In one embodiment, the thiosulfate is or comprises Na2S2O3.
In one embodiment, the thiosulfate is or comprises Na2S2O3 or a hydrate thereof.
Na2S2O3 may be obtained commercially, for example, as the anhydrous salt or as the pentahydrate. In one embodiment, the molar ratio of thiosulfate to diamine, I, is 0.8 to 1.5. In one embodiment, the molar ratio is 0.8 to 1.3. In one embodiment, the molar ratio is about 1.0
In one embodiment, the oxidizing agent is or comprises persulfate.
In one embodiment, the persulfate is or comprises S2O8 2".
In one embodiment, the persulfate is or comprises sodium persulfate, potassium persulfate, or ammonium persulfate.
In one embodiment, the persulfate is or comprises sodium persulfate.
In one embodiment, the persulfate is sodium persulfate, or a hydrate thereof.
In one embodiment, the molar ratio of persulfate to diamine, I, is 0.5 to 1.5. In one embodiment, the range is 0.8 to 1.3. In one embodiment, the range is about 1.0.
In one embodiment, the amine, I1 is added first, before the activating group reagent is added.
In one embodiment, the activating group reagent is added before the persulfate is added.
In one embodiment, the amine, I, is added first, before the thiosulfate is added.
In one embodiment, the thiosulfate is added before the persulfate is added.
In one embodiment, the reaction is performed in an aqueous medium.
In one embodiment, the pH of the aqueous medium is adjusted after the amine, I, is added.
In one embodiment, the pH of the aqueous medium is adjusted after the amine, I, is added, and before the thiosulfate is added.
In one embodiment, the pH of the aqueous medium is adjusted after the amine, I, is added, and before the thiosulfate and the persulfate are added.
In one embodiment, the pH of the aqueous medium is adjusted to a pH of 2 to 7. In one embodiment, the pH of the aqueous medium is adjusted to a pH of 3 to 5. In one embodiment, the pH of the aqueous medium is adjusted to a pH of about 4.
In one embodiment, the acidic conditions are adjusted using a strong acid. In one embodiment, the acidic conditions are adjusted using HCI.
In one embodiment, the acidic conditions are adjusted using a strong base. In one embodiment, the acidic conditions are adjusted using a hydroxide base. In one embodiment, the hydroxide base is, or comprises, sodium hydroxide, potassium hydroxide, or lithium hydroxide. In one embodiment, the reaction is performed in an aqueous medium comprising an organic solvent.
In one embodiment, the organic solvent is present at about 1 to 50 % v/v.
In one embodiment, the organic solvent is present at about 5 to 40 % v/v.
In one embodiment, the organic solvent is present at about 20 % v/v.
In one embodiment, the organic solvent is miscible with water.
In one embodiment, the organic solvent is THF, DMF, acetonitrile or saturated aliphatic C1-6alkyl alcohol.
In one embodiment, the organic solvent is THF, acetonitrile or saturated aliphatic
C1-6alkyl alcohol.
In one embodiment, the saturated aliphatic C1-6alkyl alcohol is methanol.
In one embodiment, the reaction is performed in an aqueous medium comprising a surfactant.
In one embodiment, the surfactant is an anionic surfactant.
In one embodiment, the anionic surfactant is a sulfonic acid surfactant.
In one embodiment, the anionic surfactant is a dodecylbenzenesulfonic acid salt.
In one embodiment, the anionic surfactant is an alkyl sulfate salt.
In one embodiment, the anionic surfactant is selected from alkyl benzene sulfonate, sodium laureth sulfate, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA or PFO).
In one embodiment, the surfactant is a cationic surfactant. In one embodiment, the cationic surfactant is selected from benzethonium chloride (BZT), benzalkonium chloride (BAC), polyethoxylated tallow amine (POEA), cetylpyridinium chloride (CPC) and cetyl trimethylammonium bromide.
In one embodiment, the surfactant is a nonionic surfactant.
In one embodiment, the nonionic surfactant is a polysorbate.
In one embodiment, the nonionic surfactant is a fatty alcohol.
In one embodiment, the nonionic surfactant is selected from dodecyl dimethylamine oxide, Tween 20, Tween 80, cetyl alcohol, oleyl alcohol, decyl maltoside, octyl glucoside, cocamide MEA, cocamide DEA, alkylphenol poly(ethylene oxide) and alkyl poly(ethylene oxide).
In one embodiment, the surfactant is a zwitterionic surfactant.
In one embodiment, the zwitterionic surfactant is selected from coco ampho glycinate, dodecyl betaine and cocamidopropyl betaine. In one embodiment, the reaction is performed under acidic conditions. In one embodiment, the reaction is performed at a pH of less than 7. In one embodiment, the reaction is performed at a pH of 3 to 5. In one embodiment, the reaction is performed at a pH of about 4.
In one embodiment, the thiosulfate is added in one portion. In one embodiment, the persulfate is added in one portion. In one embodiment, the persulfate is added portionwise.
In one embodiment, the reaction temperature is 2 to 25°C. In one embodiment, the reaction temperature is 2 to 15°C. In one embodiment, the reaction temperature is 2 to 100C. In one embodiment, the reaction temperature is about 50C.
In one embodiment, the reaction time is 10 to 240 minutes. In one embodiment, the reaction time is 30 to 120 minutes. In one embodiment, the reaction time is about 60 minutes.
In one embodiment, the reaction mixture is stirred during the reaction step. In one embodiment, the isolation and purification is by filtration.
In one embodiment, the S-activated diaminoaryl sulphide G-Il is isolated and purified.
In one embodiment, the isolation and purification is by filtration followed by washing. In one embodiment, the isolation and purification is by filtration followed by drying.
In one embodiment, the drying is air-drying. In one embodiment, the drying is air-drying for 10 min to 6 hours. In one embodiment, the drying is air-drying for 10 min to 2 hours. In one embodiment, the drying is air-drying for about 30 min. In one embodiment, the drying is air-drying on the filter.
In one embodiment, the drying is oven-drying.
In one embodiment, the drying is oven-drying for 2 to 48 hours.
In one embodiment, the drying is oven-drying for 2 to 24 hours.
In one embodiment, the drying is oven-drying for about 12 hours.
In one embodiment, the drying is oven-drying at 30 to 6O0C for 2 to 24 hours.
In one embodiment, the oven-drying is under vacuum.
In one embodiment, the drying is air-drying followed by oven-drying. In one embodiment, after reaction, the reaction mixture is filtered and the filtrate collected.
In one embodiment, isolation of the S-activated diaminoaryl sulphide is by filtration.
In one embodiment, the purification comprises re-dissolving in an organic solvent and filtering.
In one embodiment, the purification comprises re-dissolving in an organic solvent, heating at reflux, cooling and filtering.
In one embodiment, the purification comprises re-dissolving in ethyl acetate, heating at reflux, cooling and then filtering.
In one embodiment the purification comprises re-dissolving in an organic solvent, heating at reflux, filtering and washing with an organic solvent.
In one embodiment heating at reflux is for a period of between 30 minutes and 120 minutes, preferably between 45 and 90 minutes.
In one embodiment heating at reflux for a period of about 1 hour.
Oxidative Coupling (OC)
In this step, a S-activated diaminoaryl sulphide (an activated S-(amino-substituted-( Λ/,Λ/-disubstituted-amino)-phenyl) thio ether), G-IIa1 is oxidatively coupled to an Λ/,Λ/-disubstituted-substituted-aniline, III, using an oxidizing agent that is or comprises persulfate, to give a S-activated Λ/-substituted Λ/'-substituted imino aminoaryl sulphide (activated {(thio ether)-(disubstituted amino)-(substituted)-phenyl-imino}-substituted cyclohexa-2,5-dienylidene- Λ/,Λ/-disubstituted ammonium), G-IV, as illustrated in the following scheme:
Figure imgf000029_0001
G-IIa III G-IV
The compound G-IV is shown as a cation, which may be prepared as a salt with a suitable counter ion, X". Compound G-IV may also be prepared as a zwitterionic form where a negative charge is associated with the group -A. In one embodiment, a thiosulfonic acid S-(amino-substituted-( Λ/,Λ/-disubstituted- amino)-phenyl) ester (an amino-( Λ/,Λ/-disubstituted-amino)phenylthiosulfonic acid), Ha, is oxidatively coupled to an Λ/,/V-disubstituted-substituted-aniline, III, using an oxidizing agent that is or comprises persulfate, to give a {(thiosulfate)-(disubstituted amino)-(substituted)-phenyl-imino}-substituted cyclohexa-2,5-dienylidene-N,N- disubstituted ammonium, IV, as illustrated in the following scheme:
Figure imgf000030_0001
The phenyl ring in compound Il is substituted with an amino group, -NH2, at any one of the 2-, 3-, 4-, 5-, or 6-positions, and is substituted with three substituents -R9, at any three of the four remaining positions. The final position is substituted with thiosulfonic acid, -S-SO3H.
The phenyl ring in compound III is substituted with -H at the 4-position, and is substituted with four substituents -R1, at the 2-, 3-, 5-, and 6-positions.
The left-hand phenyl ring in compound IV, as drawn, is substituted with an imino group, -N=, at any one of the 2-, 3-, 4-, 5-, or 6-positions, and is substituted with three substituents -R9, at any three of the four remaining positions. The final position is substituted with thiosulfonic acid, -S-SO3H. The right-hand cyclohexa-2,5-dienylidene ring in compound IV, as drawn, is substituted with an imino group, =N-, at the 4-position, and is substituted with four substituents -R1, at the 2-, 3-, 5-, and 6-positions.
In one embodiment, Ha and IV are compounds Hb and IVa respectively:
Figure imgf000030_0002
lib IVa In one embodiment, Ma, III and IV are compounds Mb, MIa and V respectively:
Figure imgf000031_0001
Hb Ilia
In one embodiment, a thiosulfonic acid S-{2-(amino)-3-(optionally substituted)-5-( N1N- disubstituted amino)-phenyl} ester (amino-( Λ/,Λ/-disubstituted- amino)phenylthiosulfonic acid), B, is oxidatively coupled to an Λ/,Λ/-disubstituted-3- optionally substituted-aniline, C, using an oxidizing agent that is or comprises persulfate, to give a [4-{2-(thiosulfate)-4-( Λ/,Λ/-disubstituted amino)-6-(optionally substituted)-phenyl-imino}-3-(optionally substituted)-cyclohexa-2,5-dienylidene]- N1N- disubstituted ammonium, D, as illustrated in the following scheme:
Figure imgf000031_0002
B
In one embodiment, a thiosulfonic acid S-{2-(amino)-5-(dimethylamino)-phenyl} ester (an amino-(disubstituted-amino)phenylthiosulfonic acid), 2, is oxidatively coupled to an Λ/,Λ/-dimethyl-aniline, 3, using an oxidizing agent that is or comprises persulfate, to give a [4-{2-(thiosulfate)-4-(dimethylamino)-phenyl-imino}-cyclohexa-2,5-dienylidene]- Λ/,Λ/-dimethyl ammonium, 4, as illustrated in the following scheme:
Figure imgf000031_0003
4
In one embodiment, the S-activated diaminoaryl sulphide, G-Ma, is added first, before the aniline, III, is added.
In one embodiment, the amino-( Λ/,Λ/-disubstituted-amino)phenylthiosulfonic acid , Ha, is added first, before the aniline, III, is added. In one embodiment, the reaction is performed in an aqueous medium.
In one embodiment, the aniline, III, is added before the persulfate is added. In one embodiment, the aniline, III, is added in a mixture with aqueous acid. In one embodiment, the aqueous acid is an aqueous mineral acid. In one embodiment, the aqueous acid is aqueous H2SO4. In one embodiment, the aqueous acid is aqueous HCI.
In one embodiment, the oxidizing agent is or comprises a persulfate.
In one embodiment, the oxidizing agent is or comprises a persulfate salt.
In one embodiment, the oxidizing agent is or comprises sodium persulfate, potassium persulfate, or ammonium persulfate.
In one embodiment, the oxidizing agent is or comprises sodium persulfate.
In one embodiment, the oxidizing agent is sodium persulfate, or a hydrate thereof.
In one embodiment, the molar ratio of S-activated diaminoaryl sulphide, G-IIa, to aniline, III, is 0.5 to 1.5.
In one embodiment, the molar ratio of amino-( Λ/,Λ/-disubstituted- amino)phenylthiosulfonic acid, Ha, to aniline, III, is 0.5 to 1.5.
In one embodiment, the range is 0.8 to 1.2.
In one embodiment, the range is about 1.0.
In one embodiment, the molar ratio of aniline, III, to persulfate, is 0.5 to 2.0. In one embodiment, the range is 0.8 to 1.5. In one embodiment, the range is 0.8 to 1.2. In one embodiment, the range is about 1.0.
In one embodiment, the reaction is performed under basic conditions.
In one embodiment, the basic conditions are obtained after the aniline has been added.
In one embodiment, the reaction is performed at a pH of 7 or more. In one embodiment, the reaction is performed at a pH of more than 7. In one embodiment, the reaction is performed at a pH of 7.1 to 10.0. In one embodiment, the reaction is performed at a pH of 7.1 to 8.0. In one embodiment, the reaction is performed at a pH of about 7.5.
In one embodiment, the basic conditions are obtained using a strong base.
In one embodiment, the basic conditions are obtained using LiOH, NaOH1 KOH, or
CsOH.
In one embodiment, the basic conditions are obtained using NaOH. In one embodiment, the reaction temperature is 2 to 200C. In one embodiment, the reaction temperature is 2 to 15°C. In one embodiment, the reaction temperature is about 5°C.
In one embodiment, the reaction time is 30 minutes to 12 hours. In one embodiment, the reaction time is 60 minutes to 5 hours. In one embodiment, the reaction time is 60 minutes to 4 hours. In one embodiment, the reaction time is about 3 hours.
In one embodiment, the reaction mixture is stirred during the reaction step.
In one embodiment, aniline, III, is the same as aniline, IX.
In one embodiment, the zwitterionic intermediate G-IV is isolated and purified.
In one embodiment, the isolation and purification is by filtration.
In one embodiment, the isolation and purification is by filtration followed by washing.
In one embodiment, the washing is washing with H2O.
In one embodiment, the washing is washing with H2O and tetrahydrofuran (THF).
In one embodiment, the isolation and purification is by filtration followed by washing and drying.
In one embodiment, the isolation and purification is by filtration followed by drying.
In one embodiment, the drying is air-drying. In one embodiment, the drying is air-drying for 2 to 72 hours. In one embodiment, the drying is air-drying for 2 to 48 hours. In one embodiment, the drying is air-drying for 2 to 24 hours. In one embodiment, the drying is air-drying on the filter.
In one embodiment, the drying is oven-drying.
In one embodiment, the drying is oven-drying for 2 to 72 hours.
In one embodiment, the drying is oven-drying for 2 to 48 hours.
In one embodiment, the drying is oven-drying for 2 to 24 hours.
In one embodiment, the drying is oven-drying at 30 to 6O0C for 2 to 48 hours.
In one embodiment, the oven-drying is under vacuum.
In one embodiment, the drying is air-drying followed by oven-drying. For example, in one embodiment, the reaction mixture is filtered, and the residue (e.g., ~100 mmol crude product) is washed with H2O (e.g., 4 x 250 cm3) and THF (e.g., 100 cm3), and then air-dried overnight.
For example, in one embodiment, the reaction mixture is filtered (e.g., through a Buchner filter under vacuum), the solid removed, added to another vessel with fresh water, the mixture stirred vigorously, and filtered again. The "filter-recover-resuspend" process may be repeated a number of times. The finally obtained solid may be used in subsequent steps.
Ring Closure (RC)
In this step, a N, N-disubstituted N'-substituted aminoiminoaryl sulphide (a {(thio ether)-( Λ/,Λ/-disubstituted amino)-(substituted)-phenyl-imino}-substituted cyclohexa- 2, 5-dienylidene-N, N-disubstituted ammonium), G-V, is subjected to ring closure to give a bis(Λ/,Λ/-disubstituted-amino)-(substituted)-phenothiazin-5-ium salt, Vl, as illustrated in the following scheme:
Figure imgf000034_0001
G-V Vl
The compound G-V is shown as a cation, which may be used as a salt with a suitable counter ion, X'. Compound G-V may also be used in a zwitterionic form where a negative charge is associated with the group -A.
In one embodiment, a {(thiosulfate)-( Λ/,Λ/-disubstituted amino)-(substituted)-phenyl- iminoj-substituted cyclohexa-2,5-dienylidene- Λ/,Λ/-disubstituted ammonium, V, is subjected to ring closure to give a bis(Λ/,Λ/-disubstituted-amino)-(substituted)- phenothiazin-5-ium salt, Vl1 as illustrated in the following scheme:
Figure imgf000034_0002
Vl The left-hand phenyl ring in compound V, as drawn, is substituted with thiosulfonic acid, -S-SO3 ", at the 1 -position, is substituted with an imino group, =N-, at the 2-position, is substituted with an amino group, -NR7NAR7NB, at any one of the 3-, 4-, 5-, or 6-positions, and is substituted with three substituents -R9, at the three remaining positions. The right-hand cyclohexa-2,5-dienylidene ring in compound V, as drawn, is substituted with an imino group, =N-, at the 4-position, and is substituted with three substituents -R1, at the 2-, 3-, and 6-positions. The 5-position is substituted with -H1 as shown.
The left-hand ring in compound Vl, as drawn, is linked to the right-hand ring through a sulphur atom at the 1 -position and the N atom at the 2-position. The left-hand ring is substituted with an amino group, -NR7NAR7NB, at any one of the 3-, A-, 5-, or 6-positions, and is substituted with three substituents -R9, at the three remaining positions. The right-hand ring in compound Vl, as drawn, is linked to the left-hand ring through the sulphur atom at the 1 -position and the N atom at the 2-position. The right- hand ring is substituted with an amino group, -NR3NAR3NB, at the 5-position, and is substituted with three substituents -R1, at the three remaining positions.
In one embodiment , compounds V and Vl are compounds of formula Va and Via respectively:
Figure imgf000035_0001
Va Via
In one embodiment, a [4-{2-(thiosulfate)-4-( Λ/,Λ/-disubstituted amino)-6-(optionally substituted)-phenyl-imino}-3-(optionally substituted)-cyclohexa-2,5-dienylidene]-N,N- disubstituted ammonium, D, is subjected to ring closure to give a 3,7-bis(Λ/,/V- disubstituted-amino)-1 ,9-(optionally substituted)-phenothiazin-5-ium salt, E, as illustrated in the following scheme:
Figure imgf000035_0002
In one embodiment, a [{2-(thiosulfate)-4-(dimethylamino)-phenyl-imino}-cyclohexa-2,5- dienylidene]- Λ/,Λ/-dimethyl ammonium, 4, is subjected to ring closure to give a 3,7-bis(dimethylamino)-phenothiazin-5-ium salt, 5, as illustrated in the following scheme:
Figure imgf000036_0001
In one embodiment, ring closure is achieved by treatment with an oxidizing agent.
In one embodiment, the reaction is performed at reflux.
In one embodiment, the reaction temperature is 30 to 950C. In one embodiment, the reaction temperature is 50 to 900C. In one embodiment, the reaction temperature is 60 to 9O0C. In one embodiment, the reaction temperature is about 85°C.
In one embodiment, the reaction time is 10 to 120 minutes. In one embodiment, the reaction time is 20 to 90 minutes. In one embodiment, the reaction time is about 60 minutes.
In one embodiment, the reaction is performed in an aqueous medium.
In one embodiment, the reaction is performed until the reaction mixture changes colour, e.g., becomes a deep blue colour.
In one embodiment, the reaction mixture is stirred during the reaction step.
In one embodiment, after reaction, the reaction mixture is filtered and the filtrate collected.
In one embodiment, the reaction mixture is first cooled, and the filtration is performed at about room temperature, to give a "cool" filtrate.
In one embodiment, the filtration is performed at a temperature near to the reaction temperature, to give a "hot" filtrate. In one embodiment, after reaction, a salt is added to the reaction mixture.
In one embodiment, the salt is an alkali metal salt.
In one embodiment, the salt comprises the counter ion X'.
In one embodiment, the product of the reaction is a salt, wherein X' is S2O8 '.
In one embodiment, the product of the reaction is a salt, wherein X' is 0.5 S2O3 "2.
In one embodiment, the product of the reaction is a salt, wherein X' does not comprise a halogen ion.
In one embodiment, the product of the reaction is a salt, wherein X' does not comprise a chloride ion.
In one embodiment, the product of the reaction is reacted with a chloride salt as a source of chloride to form a product where X' is a chloride ion, as described below in the Chloride Salt Formation (CSF) step.
Cu(II) Oxidising Agent
In one embodiment, the oxidizing agent is or comprises Cu(II).
In one embodiment, the oxidizing agent is or comprises Cu(II) sulfate.
In one embodiment, the oxidizing agent is Cu(II) sulfate or a hydrate thereof.
In one embodiment, the oxidizing agent is or comprises Cu(II).
In one embodiment, ring closure is performed under acidic conditions. In one embodiment, ring closure is performed at a pH of 1 to 5. In one embodiment, ring closure is performed at a pH of 2 to 5. In one embodiment, ring closure is performed at a pH of 3 to 4.5. In one embodiment, ring closure is performed at a pH of 3.5 to 4.1. In one embodiment, ring closure is performed at a pH of about 2.0.
In one embodiment, the desired pH is obtained by the addition of strong acid. In one embodiment, the desired pH is obtained by the addition of HCI.
In one embodiment, the molar ratio of Cu(II) to ammonium, G-V, is 0.02 to 0.2. In one embodiment, the molar ratio of Cu(II) to ammonium, V, is 0.02 to 0.2. In one embodiment, the range is 0.05 to 0.15 In one embodiment, the range is about 0.1.
MnO2 Oxidising Agent
In one embodiment, the oxidizing agent is or comprises activated manganese dioxide
(MnO2).
In one embodiment, the oxidizing agent is activated manganese dioxide (MnO2). In one embodiment, the molar ratio of MnO2 to ammonium, G-V, is 1.0 to 3.0. In one embodiment, the molar ratio of MnO2 to ammonium, V, is 1.0 to 3.0. In one embodiment, the molar ratio is 1.5 to 2.5. In one embodiment, the molar ratio is about 2.0.
In one embodiment, after completion of the reaction (a blue solution with precipitate is observed), strong acid (e.g., concentrated H2SO4) is added.
Without wishing to be bound by any particular theory, it is believed that the strong acid dissolves the manganese salts (and other salts, if present).
Chloride Salt Formation (CSF)
In this step, a bis(Λ/,Λ/-disubstituted-amino)-(substituted)-phenothiazin-5-ium salt, Vl, is reacted with chloride, to give a bis(Λ/,Λ/-disubstituted-amino)-(substituted)- phenothiazin-5-ium chloride salt, VII, as illustrated in the following scheme:
Figure imgf000038_0001
Vl VII
In one embodiment, a 3,7-bis(Λ/,Λ/-disubstituted-amino)-1 ,9-(optionally substituted)- phenothiazin-5-ium salt, E, is reacted with chloride, to give a 3,7-bis(Λ/,Λ/- disubstituted-amino)-1 ,9-(optionally substituted)-phenothiazin-5-ium chloride salt, F, as illustrated in the following scheme:
Figure imgf000038_0002
In one embodiment, a 3,7-bis(dimethylamino)-phenothiazin-5-ium salt is reacted with chloride, to give a 3,7-bis(dimethylamino)-phenothiazin-5-ium chloride salt, 5 (i.e., MTC), as illustrated in the following scheme:
Figure imgf000039_0001
in one embodiment, Vl is reacted with a chloride salt as a source of chloride.
In one embodiment, the chloride is chloride salt.
In one embodiment, the chloride is an alkali metal chloride.
In one embodiment, the chloride is sodium chloride.
In one embodiment, there is a large molar excess of (sodium) chloride.
In one embodiment, the molar ratio of chloride to salt, Vl, is 5 to 100.
In one embodiment, the molar ratio is 5 to 80.
In one embodiment, the molar ratio is 5 to 50.
In one embodiment, the molar ratio is about 8.
In one embodiment, the reaction is performed in an aqueous medium.
In one embodiment, the reaction temperature is 0 to 95°C.
In one embodiment, the reaction temperature is 0 to 50cC.
In one embodiment, the reaction temperature is 0 to 300C.
In one embodiment, the reaction temperature is 2 to 300C.
In one embodiment, the reaction temperature is 2 to 20°C.
In one embodiment, the reaction temperature is about 50C.
In one embodiment, the reaction time is 10 to 120 minutes. In one embodiment, the reaction time is about 60 minutes.
In one embodiment, the reaction is performed until the reaction mixture (initially, e.g., a deep blue colour) becomes light blue to colourless.
In one embodiment, the reaction mixture is stirred during the reaction step.
In one embodiment, the reaction mixture is allowed to cool following addition of the chloride to salt Vl at the reaction temperature, to yield the product as a precipitate. In one embodiment, the reaction mixture is allowed to cool to 0 to 300C. In one embodiment, the reaction mixture is allowed to cool to 0 to 200C. In one embodiment, the reaction mixture is allowed to cool to about 5°C.
In one embodiment, the reaction mixture is filtered hot following addition of the chloride to salt Vl at the reaction temperature, and the collected filtrate allowed to cool, to yield the product as a precipitate.
In other embodiments, Vl is reacted with hydrochloric acid as a source of chloride.
In other embodiments, the Chloride salt formation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein. In particular the sections relating to Chloride salt formation (CAF) at pages 32-33 and Chloride salt formation (CSF-2) at pages 58-59 are incorporated herein.
Following the chloride salt formation steps, one or more additional steps may be performed, as discussed at pages 33 and 34, including the sulphide treatment described at pages 34-36, the dimethyldithiocarbamate treatment (DT) at pages 36- 37, the carbonate treatment (CT) at pages 37-38, the ethylenediaminetetraacetic acid treatment (EDTAT) at pages 39-40, and the organic extraction (OE) at pages 40-41 , which are incorporated herein.
Cr-Mediated Activated Thio Ether (diaminoaryl sulphide) Formation and Thiosulfonic Acid Formation (Cr-TSAF)
In this step, an Λ/,Λ/-disubstituted-diamino-substituted benzene, Ia, is oxidized in the presence of an oxidizing agent that is or comprises Cr(VI) to give an S-(amino- substituted-(Λ/,Λ/-disubstituted-amino)-phenyl) ether, (S-activated Λ/,Λ/-disubstituted diaminoaryl sulphide) G-IIa, as illustrated in the following scheme:
Figure imgf000040_0001
Ia G-IIa
This reaction may be referred to as a Cr-mediated activated thio ether formation step. Compound Ia is oxidised in the presence of an activating group reagent. The activating group reagent and the group -A are as defined for the activated thio ether (S-activated diaminoaryl sulphide) formation step described above. In one embodiment, the activating group reagent is thiosulfate. In this embodiment, the group A is -S(=O)2OH.
In one embodiment, an Λ/,Λ/-disubstituted-diamino-substituted benzene, Ia, is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises Cr(VI) to give a thiosulfonic acid S(-amino-substituted-(Λ/,Λ/-disubstituted-amino)-phenyl) ester (an amino-(Λ/,Λ/-disubstituted-amino)phenylthiosulfonic acid), Ha, as illustrated in the following scheme:
Figure imgf000041_0001
Ia Ma
In one embodiment, compounds I and Il are compounds of formula Ib and lib respectively as shown below:
Figure imgf000041_0002
Ib Mb
In one embodiment, an Λ/,Λ/-disubstituted-1 ,4-diarnino-5-optionally substituted benzene, A, is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises Cr(VI) to give a thiosulfonic acid S-{2-(amino)-3-(optionally substituted)- 5-( Λ/,Λ/-disubstituted-amino)-phenyl} ester (an amino-( Λ/,Λ/-disubstituted- amino)phenylthiosulfonic acid), B, as illustrated in the following scheme:
Figure imgf000041_0003
B
In one embodiment, an Λ/,Λ/-dimethyl-1 ,4-diamino-benzene, 1, is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises Cr(VI) to give a thiosulfonic acid S-{2-(amino)-5-(dimethylamino)-phenyl} ester, (a 2-amino-5-dimethyl- aminophenylthiosulfonic acid), 2, as illustrated in the following scheme:
Figure imgf000042_0001
The Cr-mediated thiosulfonic acid formation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein. In particular the sections relating to Thiosulfonic acid formation (TSAF) at pages 22-23, and Thiosulfonic acid formation (TSAF-3) at pages 54-55 are incorporated herein, along with Examples 1-8 and 17 relating to the synthesis of thiosulfonic acid S-{2- (amino)-5-(dimethylamino)-phenyl} ester, (2-amino-5-(dimethylaminophenylthiosulfonic acid) and Example 20, relating to the synthesis of thiosulfonic acid S-{2-(amino)-3- ethyl-5-(dimethylamino)-phenyl} ester (2-amino-3-methyl-5- dimethylaminophenylthiosulfonic acid).
The thiosulfate is or comprises S2O3 "2.
In one embodiment, the thiosulfate is or comprises Na2S2O3.
In one embodiment, the thiosulfate is Na2S2O3 or a hydrate thereof.
In one embodiment, the oxidation is by reaction with an oxidizing agent.
In one embodiment, the oxidizing agent is or comprises Cr(VI).
In one embodiment, the oxidizing agent is or comprises Cr2O7 '2.
In one embodiment, the oxidizing agent is or comprises Na2Cr2O7.
In one embodiment, the oxidizing agent is Na2Cr2O7 or a hydrate thereof.
In one embodiment, the molar ratio of Cr(VI) to diamine, I, is 0.2 to 2.0.
In one embodiment, the reaction is performed in an aqueous medium.
In one embodiment, the reaction temperature is 2 to 25°C.
In one embodiment, the oxidizing agent additionally comprises AI(III). In one embodiment, the molar ratio of AI(III) to diamine, I, is 0.2 to 2.0.
In one embodiment, the oxidizing agent further comprises a strong acid.
In one embodiment, the molar ratio of acid protons to diamine, I, is 1.0 to 4.0. Cr-Mediated Oxidative Coupling (Cr-OC)
In this step, an activated S-(amino-substituted-( Λ/,Λ/-disubstituted-amino)-phenyl) thio ether, G-IIa, is oxidatively coupled to an Λ/,Λ/-disubstituted-substituted-aniline, III, using an oxidizing agent that is or comprises Cr(VI), to give a S-activated N1N- disubstituted Λ/'-substituted aminoiminoaryl sulphide (an activated {(thio ether)-( N1N- disubstituted amino)-(substituted)-phenyl-imino}-substituted cyclohexa-2,5- dienylidene-Λ/,Λ/-disubstituted ammonium), G-IV, as illustrated in the following scheme:
Figure imgf000043_0001
G-IIa G-IV
In one embodiment, a thiosulfonic acid S-(amino-substituted-( Λ/,Λ/-disubstituted- amino)-phenyl) ester (an amino-( Λ/,Λ/-disubstituted-amino)phenylthiosulfonic acid), Ua, is oxidatively coupled to an Λ/,Λ/-disubstituted-substituted-aniline, III, using an oxidizing agent that is or comprises Cr(VI), to give a {(thiosulfate)-( Λ/,Λ/-disubstituted amino)-(substituted)-phenyl-imino}-substituted cyclohexa-2,5-dienylidene-Λ/,Λ/- disubstituted ammonium, IV1 as illustrated in the following scheme:
Figure imgf000043_0002
In one embodiment, Ma and IV are compounds Mb and IVa respectively:
Figure imgf000044_0001
lib IVa
In one embodiment, Ha, III and IV are compounds lib, Ilia and V respectively:
Figure imgf000044_0002
Mb IMa
In one embodiment, a thiosulfonic acid S-{2-(amino)-3-(optionally substituted)-5-( N1N- disubstituted amino)-phenyl} ester (an amino-( Λ/,Λ/-disubstituted- amino)phenylthiosulfonic acid), B, is oxidatively coupled to an Λ/,Λ/-disubstituted-3- optionally substituted-aniline, C, using an oxidizing agent that is or comprises Cr(VI), to give a [4-{2-(thiosulfate)-4-( Λ/,Λ/-disubstituted amino)-6-(optionally substituted)- phenyl-imino}-3-(optionally substituted)-cyclohexa-2,5-dienylidene]-Λ/,Λ/-disubstituted ammonium, D, as illustrated in the following scheme:
Figure imgf000044_0003
B
In one embodiment, a thiosulfonic acid S-{2-(amino)-5-(dimethylamino)-phenyl} ester (a 2-amino-5-dimethylaminophenylthiosulfonic acid), 2, is oxidatively coupled to an Λ/,Λ/-dimethyl-aniline, 3, using an oxidizing agent that is or comprises Cr(VI), to give a [4-{2-(thiosulfate)-4-(dimethylamino)-phenyl-imino}-cyclohexa-2,5-dienylidene]-Λ/,Λ/- dimethyl ammonium, 4, as illustrated in the following scheme:
Figure imgf000044_0004
The Cr-mediated oxidative coupling is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein. In particular the sections relating to Oxidative coupling (OC) at pages 23-25 and Oxidative coupling (OC-3) at pages 55-56 are incorporated herein. In addition the use of a Cr oxidising agent as described in Examples 1 to 8, 17 and 20, with or without isolation of the intermediate, is also incorporated herein.
The oxidative coupling may be used in combination with any one of the Cr(VI) reduction (CR) and/or isolation and purification of zwitterinoic intermediate (IAPOZI) steps described in WO 2006/032879. See, in particular, pages 25-30, which are incorporated by reference herein.
In one embodiment, the oxidizing agent is or comprises Cr2O7 '2. In one embodiment, the oxidizing agent is or comprises Na2Cr2O7. In one embodiment, the oxidizing agent is Na2Cr2O7.
In one embodiment, the molar ratio of ester, G-IIa, to aniline, III, is 0.5 to 1.5. In one embodiment, the molar ratio of ester, Ha, to aniline, III, is 0.5 to 1.5.
In one embodiment, the molar ratio of Cr(VI) to aniline, III, is 1.0 to 4.0.
In one embodiment, the reaction is performed under acidic conditions.
In one embodiment, the reaction is performed in an aqueous medium.
In one embodiment, the reaction temperature is 2 to 200C.
Nitrosyl Reduction (NR)
In this step, an Λ/,Λ/-disubstituted-substituted-nitrosyl aniline, VIII, is reduced to form an Λ/,Λ/-disubstituted-diamino-substituted benzene, Ia, as illustrated in the following scheme:
Figure imgf000045_0001
In one embodiment, an Λ/,Λ/-disubstituted-3-optionally substituted-4-nitrosyl aniline, G, is reduced to form a Λ/,Λ/-disubstituted-1 ,4-diamino-5-optionally substituted benzene, A, as illustrated in the following scheme:
Figure imgf000046_0001
In one embodiment, an N,N-dimethyl-4-nitrosyl aniline, 6, is reduced to form a N,N-dimethyl-1 ,4-diamino-benzene, 1, as illustrated in the following scheme:
Figure imgf000046_0002
The nitrosylation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein. In particular the sections relating to Nitrosylation (NOS) at pages 19-20 and Nitrosylation (NOS-2) at pages 46-48 are incorporated herein, along with Examples 1-8 and 17 relating to the synthesis of N,N-dimethyl-1 ,4-diamino-benzene, and Example 19 relating to the synthesis of N,N-dimethyl-1 ,4-diamino-3-ethyl-benzene.
Typically the nitrosyl analine VIII is reduced with a reducing agent such as metallic iron under acidic conditions.
Nitrosylation (NOS)
In this step, an N,N-disubstituted-substituted aniline, IX, is nitrosylated to give an N,N-disubstituted-substituted-nitrosyl aniline, VIII, as illustrated in the following scheme:
Figure imgf000046_0003
IX VIII In one embodiment, an Λ/,Λ/-disubstituted-3-optionally substituted aniline H, is 4-nitrosylated to give an Λ/,Λ/-disubstituted-3-optionally substituted-4-nitrosyl aniline, G, as illustrated in the following scheme:
Figure imgf000047_0001
H
In one embodiment, an Λ/,Λ/-di methyl aniline, 3, is 4-nitrosylated to give an Λ/,Λ/-dimethyl-4-nitrosyl aniline, 6, as illustrated in the following scheme:
Figure imgf000047_0002
In one embodiment, an Λ/,Λ/-dimethyl-3-ethyl-aniline, 8, is 4-nitrosylated to form the corresponding Λ/,Λ/-dimethyl-3-ethyl-4-nitrosyl aniline, 7, as illustrated in the following scheme:
Figure imgf000047_0003
The nitrosylation is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein. In particular, the sections relating to Nitrosylation (NOS-1 ) at pages 19-20 and Nitrosylation (NOS-2) at pages 46-48 are incorporated herein, along with Examples 1-8, and 17 relating to the preparation of Λ/,Λ/-dimethyl-4-nitrosyl aniline, and Example 19 relating to the preparation of Λ/,Λ/-dimethyl-3-ethyl-4-nitrosyl aniline.
Typically, the aniline IX is treated with a nitrite such as an alkali metal nitrite under acidic conditions to give the nitrosyl aniline VIII. N,N-Disubstitution (NNDS)
In this step, a substituted-aniline, X, is Λ/,Λ/-disubstituted using an alkyl halide, an alkenyl halide, a haloalkyl halide, a
Figure imgf000048_0001
halide or a halogenated
Figure imgf000048_0002
halide, to give a Λ/,Λ/-disubstituted-substituted aniline, IXa, as illustrated in the following scheme:
Figure imgf000048_0003
Λ IXa
wherein each of -R7NC and -R7ND is independently selected from C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; Ci.4alkylene-C5.ioaryl and halogenated C1-4alkylene-C5.ioaryl; and -R9 is as defined previously.
In one embodiment, a 3-optionally substituted-aniline, J, is Λ/,Λ/-disubstituted using a alkyl halide, an alkenyl halide, a haloalkyl halide, a Ci-4alkylene-C5.i0aryl halide or a halogenated C1-4alkylene-C5.i0aryl halide, to give a Λ/,Λ/-disubstituted-3-optionally substituted-aniline, H, as illustrated in the following scheme:
Figure imgf000048_0004
In one embodiment, each of -R7NC and -R7ND is independently selected from: Ci-4alkyl;
C2-4alkenyl; and halogenated C1-4alkyl.
In one embodiment, each of -R7NC and -R7ND is independently -Me, -Et, -nPr, -nBu,
-CH2-CH=CH2, or -CF3.
In one embodiment, each of -R7NC and -R7ND is independently C1-4alkyl.
In one embodiment, each of -R7NC and -R7ND is independently -Me or -Et.
In one embodiment, each of -R7NC and -R7ND is independently -Me.
In one embodiment, each of -R7NC and -R7ND is independently -Et.
In one embodiment, -R7NC and -R7ND are the same. In one embodiment, -R7NC and -R7ND are different. In one embodiment, a 3-ethyl-aniline, 8, is Λ/,Λ/-dimethylated using a methyl halide, to give a Λ/,Λ/-dimethyl-3-ethyl-aniline, 9, as illustrated in the following scheme:
Figure imgf000049_0001
The disubstitution is performed according to the methods described in WO 2006/032879, which is incorporated by reference herein. In particular, the section relating to Λ/.Λ/-Disubstitution (NNDS-2) at pages 45-46 is incorporated herein, along with Example 19 relating to the preparation of Λ/,Λ/-dimethyl-m-ethyl-aniline.
Typically aniline X is treated with an alkyl halide, an alkenyl halide, or a haloalkyl halide under basic conditions to give substituted aniline IXa.
Di-N, N-Disubstitution (DNNDS)
In this step, a Λ/,Λ/-disubstituted-diamino-substituted benzene, Ia, is Λ/,Λ/-disubstituted using an alkyl halide, an alkenyl halide, a haloalkyl halide, a Ci.4alkylene-C5.ioaryl halide or a halogenated
Figure imgf000049_0002
halide, to give a di-Λ/,Λ/-disubstituted- substituted aniline, Ic, as illustrated in the following scheme:
Figure imgf000049_0003
Ia Ic
wherein each of -R10NC and -R10ND is independently selected from C1-4alkyl; C2-4alkenyl; halogenated Ci-4alkyl; Ci.4alkylene-C5.10aryl and halogenated Ci-4alkylene-C5.i0aryl; and -R7NA, -R7NB, and -R9 are as previously defined.
Substantially Purified Forms
One aspect of the present invention pertains to compounds, as described herein, in substantially purified form and/or in a form substantially free from contaminants. In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.
In one embodiment, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.
Salts
It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of a compound described herein. For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO ), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations such as Al+3. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 +) and substituted ammonium ions (e.g., NH3R+, NH2R2 +, NHR3 +, NR4 +). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3J4 +.
If the compound is cationic, or has a functional group which may be cationic (e.g., -NH2 may be -NH3 +), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
Unless otherwise specified, a reference to a particular compound also includes salt forms thereof. In one embodiment, a reference to a salt also includes mixed salt forms thereof.
Solvates and Hydrates
It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
Unless otherwise specified, a reference to a particular compound also includes solvate and hydrate forms thereof.
Formulations
The diaminophenothiazinium compounds may be provided in a composition or formulation for administration to a subject, for example a subject having AD. While it is possible for the compound to be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation.
In one aspect of the invention there is provided a method of preparing a composition or formulation comprising a diaminophenothiazinium compound. The method includes the synthesis of a diaminophenothiazinium compound comprising one or more of the steps described herein. In one embodiment, the method further comprises the step of admixing at least one diaminophenothiazinium compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
In one embodiment, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a diaminophenothiazinium compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
In one embodiment, the composition is a pharmaceutical composition comprising at least one diaminophenothiazinium compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
In one embodiment, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.
Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA); Remington: The Science and Practice of Pharmacy, 20th Edition (ed. Gennaro et al.), 2000, Lippincott, Williams & Wilkins, Baltimore; and Handbook of Pharmaceutical Excipients, 2nd Edition (eds A. Wade and P.J. Weller), 1994, American Pharmaceutical Association, Washington and The Pharmaceutical Press, London.
The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.
The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi- dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
Uses
In one aspect the present invention provides diaminophenothiazinium compounds and formulations comprising diaminophenothiazinium compounds as described herein for use in therapy.
In other aspects, the invention pertains to compounds obtained or obtainable by the methods described herein for use in therapy. In one embodiment, the compound is a compound obtained by the methods described herein.
The compounds and compositions described herein may reverse or inhibit the aggregation of tau protein (e.g. in the form of paired helical filaments (PHFs), optionally in neurofibrillary tangles (NFTs)) in the brain of a mammal.
One aspect of the invention is the use of a diaminophenothiazinium compound (for example, obtained by the methods described herein) to reverse or inhibit the aggregation of tau protein. This aggregation may be in vitro, or in vivo, and may be associated with a tauopathy disease state as discussed herein. Also provided are methods of reversing or inhibiting the aggregation of tau protein comprising contacting the aggregate or protein with a compound as described herein.
As discussed below, various tauopathy disorders that have been recognized which feature prominent tau pathology in neurons and/or glia and this term has been used in the art for several years. The similarities between these pathological inclusions and the characteristic tau inclusions in diseases such as AD indicate that the structural features are shared and that it is the topographic distribution of the pathology that is responsible for the different clinical phenotypes observed. In addition to specific diseases discussed below, those skilled in the art can identify tauopathies by combinations of cognitive or behavioural symptoms, plus additionally through the use of appropriate ligands for aggregated tau as visualised using PET or MRI, such as those described in WO02/075318. One aspect of the present invention pertains to a method of treatment or prophylaxis of a tauopathy condition in a patient, comprising administering to said patient a therapeutically-effective amount of a compound of formula (I), as described herein. In one embodiment, the compound of formula (I) is obtained by the methods described herein.
Aspects of the present invention relate to "tauopathies". As well as Alzheimer's disease (AD), the pathogenesis of neurodegenerative disorders such as Pick's disease and Progressive Supranuclear Palsy (PSP) appears to correlate with an accumulation of pathological truncated tau aggregates in the dentate gyrus and stellate pyramidal cells of the neocortex, respectively. Other dementias include fronto- temporal dementia (FTD); FTD with parkinsonism linked to chromosome 17 (FTDP- 17); disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC); pallido- ponto-nigral degeneration (PPND); Guam-amyotrophic lateral sclerosis (ALS) syndrome; pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration (CBD); Dementia with Argyrophilic grains (AgD); Dementia pugilistica (DP) wherein despite different topography, NFTs are similar to those observed in AD (Hof P. R., Bouras C1 Buee L., Delacourte A., Perl D. P. and Morrison J. H. (1992) Differential distribution of neurofibrillary tangles in the cerebral cortex of dementia pugilistica and Alzheimer's disease cases. Acta Neuropathol. 85, 23-30). Others ar,e discussed in Wischik et al. 2000, loc. cit, for detailed discussion - especially Table 5.1).
Abnormal tau in NFTs is found also in Down's Syndrome (DS) (Flament S., Delacourte A. and Mann D.M.A. (1990) Phosphorylation of tau proteins: a major event during the process of neurofibrillary degeneration. A comparative study between AD and Down's syndrome. Brain Res., 516, 15-19). Also Dementia with Lewy bodies (DLB) (Harrington, C.R., Perry, R.H., Perry, E.K., Hurt, J., McKeith, I.G., Roth, M. & Wischik, CM. (1994) Senile dementia of Lewy body type and Alzheimer type are biochemically distinct in terms of paired helical filaments and hyperphosphorylated tau protein. Dementia 5, 215-228). Tau-positive NFTs are also found in Postencephalitic parkinsonism (PEP) (Hof P. R., Charpiot, A., Delacourte A., Buee, L., Purohit, D., Perl D. P. and Bouras, C. (1992) Distribution of neurofibrillary tangles and senile plaques in the cerebral cortex in postencephalitic parkinsonism. Neurosci. Lett. 139, 10-14). Glial tau tangles are observed in Subacute sclerosing panencephalitis (SSPE) (Ikeda K., Akiyama H., Kondo H., Arai T., Arai N. and Yagishita S. (1995) Numerous glial fibrillary tangles in oligodendroglia in cases of subacute sclerosing panencephalitis with neurofibrillary tangles. Neurosci. Lett., 194, 133-135).
Additionally there is a growing consensus in the literature that a tau pathology may also contribute more generally to cognitive deficits and decline, including in mild cognitive impairment (MCI) (see e.g. Braak, H., Del Tredici, K, Braak, E. (2003) Spectrum of pathology. In Mild cognitive impairment: Aging to Alzheimer's disease edited by Petersen, R.C.; pp. 149-189).
In this and all other aspects of the invention relating to tauopathies, preferably the tauopathy is selected from the list consisting of the indications above, i.e., AD, Pick's disease, PSP, FTD1 FTDP-17, DDPAC, PPND, Guam-ALS syndrome, PNLD, and CBD and AgD, DS, SSPE, DP, PEP, DLB and MCI.
In one preferred embodiment the tauopathy is Alzheimer's disease (AD).
One aspect of the present invention pertains to a compound of formula (I), as described herein, for use in a method of treatment or prophylaxis (e.g., of a tauopathy condition) of the human or animal body by therapy. In one embodiment, the compound of formula (I) is obtained by the methods described herein.
One aspect of the present invention pertains to use of a compound of formula (I), as described herein, in the manufacture of a medicament for use in the treatment or prophylaxis of a tauopathy condition. In one embodiment, the compound of formula (I) is obtained by the methods described herein.
A further embodiment is a method of treatment or prophylaxis of a disease of tau protein aggregation as described herein, which method comprises administering to a subject a diaminophenothiazinium compounds, or therapeutic composition comprising the same, such as to inhibit the aggregation of the tau protein associated with said disease state.
Examples
The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.
Preparation of 2-amino-5-dimethylaminophenyl thiosulfonic acid (2)
'
Figure imgf000055_0001
(i): Na2S2O3 SH2O, Na2S2O8, H2O, MeOH, 5 0C Example 1
Synthesis of 2-amino-5-dimethylaminophenylthiosulfonic acid Λ/',Λ/'-dimethyl-p-phenylene diamine (10 g, 0.0735 mols) was added to a round bottom flask. To this was added de-ionised water (160 ml) and methanol (40 ml) and the solution was then cooled to 5 0C. Sodium thiosulfate (20 g, 0.0808 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (17.5 g, 0.0735 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0C and then warmed to 22 0C over a period of 1 hour. The dark solid which precipitated was collected via filtration. The black solid was washed with water (50 ml) then dried at 40 0C for 16 hours, 7.88 g (43 %).
Purification of 2-amino-5-dimethylaminophenylthiosulfonic acid 2-amino-5-dimethylaminophenylthiosulfonic acid (2.16 g, 0.0087 mols) was added to a round bottom flask and ethyl acetate (108 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (100 ml) and the solid was dried under vacuum (1000 mbar) at 40 0C for 16 hours, 1.82 g (84 %).
Mp: 195-197 0C vmax (KBr)/cm 1: 3454 (N-H), 3350 (N-H), 3033 (C-H), 1621 (C=C) 1463 (C-C or
O=S=O), 1170 (O=S=O), 1016 (O=S=O). δH(600 MHz, DMSO-d6) 2.98 (6 H, s, N-CH3), 7.03-7.08 (m, 2H, Ar-H), 7.19 (s, 1 H, Ar-
H).
Mass Spec {m/z) ESI high resolution: calculated: 249.0368, found: 249.0361 [M + H]+;
169.0800 [(M + H)-SO3J+.
Example 2
Λ/',Λ/'-dimethyl-p-phenylene diamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and tetrahydrofuran (20 ml) and the solution was then cooled to 5 0C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0C and then warmed to 22 0C over a period of 1 hour. The black solid which precipitated was collected via filtration. The black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0C for 16 hours. Λ/',Λ/-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0C for 16 hours, 3.7 g (40 %).
Mp: 197-198 0C v' mβx (KBr)/crrϊ1: 3452 (N-H), 3344 (N-H), 3032 (C-H), 1621 (C=C), 1463 (C-C or
O=S=O), 1165 (O=S=O), 1015 (O=S=O). δH(400 MHz, DMSO-d6) 2.98 (6 H, s, N-CH3), 6.99-7.01 (m, 1 H, Ar-H), 7.06-7.08
(d, 1 H, Ar-H- /6 = 8.4 Hz), 7.17 (s, 1 H, Ar-H).
Mass Spec (m/z) ESI high resolution: calculated: 249.0368, found: 249.0359 [M + H]+;
169.0794 [(M + H)-SO3J+.
Example 3
Λ/'.Λ/'-dimethyl-p-phenylene diamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and acetonitrile (20 ml) and the solution was then cooled to 5 0C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0C and then warmed to 22 0C over a period of 1 hour. The black solid which precipitated was collected via filtration. The black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 °C for 16 hours. Λf,Λ/'-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0C for 16 hours, 4.3 g (47 %).
Mp: 199 0C vmax (KBr)/cm'1: 3453 (N-H), 3347 (N-H), 3032 (C-H), 1621 (C=C), 1463 (C-C or
O=S=O), 1169 (O=S=O), 1010 (O=S=O). δH(400 MHz, DMSO-d6) 2.98 (6 H, s, N-CH3), 7.00-7.02 (m, 1H, Ar-H), 7.06-7.09
(d, 1H, Ar-H J*B= 9.2 Hz), 7.18 (s, 1H, Ar-H). Mass Spec (m/z) ESI high resolution: calculated: 249.0368, found: 249.0363 [M + H]+; 169.0794 [(M + H)-SO3J+.
Example 4
Λ/',Λ/'-dimethyl-p-phenylene diamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and N, Λ/-dimethylformamide (20 ml) and the solution was then cooled to 5 0C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0C and then warmed to 22 0C over a period of 1 hour. The black solid which precipitated was collected via filtration. The black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0C for 16 hours. Λ/',Λ/-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0C for 16 hours, 4.8 g (52 %).
Mp: 198-199 0C
Vmax (KBr)/cm"1: 3457 (N-H), 3346 (N-H), 3032 (C-H), 1621 (C=C), 1463 (C-C or O=S=O), 1 166 (O=S=O)1 101 1 (O=S=O). δH(400 MHz, DMSO-de) 2.98 (6 H, s, N-CIH3), 7.02 (m, 1 H, Ar-H), 7.06-7.09 (d, 1 H, Aril J*B = 8.4 Hz)1 7.18 (S1 1 H1 Ar-H).
Mass Spec (m/z) ESI high resolution: calculated: 249.0368, found: 249.0369 [M + H]+; 169.0802 [(M + H)-SO3]+.
Example 5
Λ/',Λ/'-dimethyl-p-phenylene diamine (5 g, 0.036 mols) and dodecylbenzenesulfonic acid, sodium salt (0.13 g, 0.00036 mols) were added to a round bottom flask. De-ionised water (100 ml) was added and the solution was then cooled to 5 0C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added dropwise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0C and then warmed to 22 0C over a period of 1 hour. The black solid which precipitated was collected via filtration. The black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0C for 16 hours. ΛT,Λ/-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0C for 16 hours, 4.6 g (50 %).
Mp: 198-199 0C vmax (KBr)/cm 1: 3450 (N-H), 3342 (N-H)1 3032 (C-H), 1620 (C=C), 1463 (C-C or
O=S=O), 1174 (O=S=O), 1010 (O=S=O). δH(400 MHz, DMSO-d6) 2.98 (6 H, s, N-CH3), 7.00-7.02 (d, /^ = 7.6 Hz, 1 H, Ar-H),
7.06-7.08 (d, /B = 8.8 Hz, 1 H, Ar-H), 7.18 (s, 1 H, Ar-H).
Mass Spec (m/z) ESI high resolution: calculated: 249.0368, found: 249.0363 [M + H]+;
169.0794 [(M + H)-SO3I+.
Example 6
Λ/',Λ/'-dimethyl-p-phenylenediamine (5 g, 0.036 mols) was added to a round bottom flask. To this was added de-ionised water (100 ml) and the solution was then cooled to 5 0C. Sodium thiosulfate (10.03 g, 0.040 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (8.75 g, 0.036 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0C and then warmed to 22 0C over a period of 1 hour. The black solid which precipitated was collected via filtration. The black solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0C for 16 hours. Λ/',Λ/'-dimethyl-p-phenylene diamine-5-thiosulfonic acid was added to a round bottom flask and ethyl acetate (100 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 0C. Once the slurry had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (50 ml) and dried under vacuum (1000 mbar) at 40 0C for 16 hours, 5.1 g (56 %). Actual value = 49 % by NMR with internal standard (DSS)
Mp: 201-202 0C vmax (KBr)/cπV1: 3455 (N-H), 3346 (N-H), 3033 (C-H), 1621 (C=C), 1463 (C-C or
O=S=O), 1170 (O=S=O), 1013 (O=S=O). δH(400 MHz, DMSO-CJ6) 2.96 (6 H, s, N-CH3), 7.00-7.03 (m, 2H, Ar-H), 7.15 (s, 1 H, Aril).
Mass Spec (m/z) ESI high resolution: calculated: 249.0368, found: 249.0376 [M + H]+; 169.0790 [(M + H)-SO3J+.
Synthesis 2 Preparation of thiosulfonic acid of Bindshedlers green (3)
Figure imgf000060_0001
(ii): H2O, H2SO4, (CHa)2NC6H5, Na2S2O8, H2O, NaOH(aq), 50C
2-amino-5-dimethylaminophenylthiosulfonic acid (15 g, 0.0605 mols) was added to a round bottom flask and de-ionised water (300 ml) was added. The solution was cooled to ~5 0C and a pre-cooled mixture of Λ/,Λ/-dimethylaniline (7.32 g, 0.0605 mols), sulphuric acid (5.93 g, 0.0605 mols) and de-ionised water (7.32 ml) was added in one aliquot. The pH of the mixture was adjusted to pH -7.5 using 6 M sodium hydroxide (~30 ml). Sodium persulfate (43.18 g, 0.181 mols) in water (62 ml) was added drop- wise over a period of ~20 minutes. The reaction mixture was stirred for 2 hours and then warmed to 22 0C over a 1 hour period. The black solid was collected via filtration and air dried for 16 hours.
vmax (KBr)/cm"1:1587 (C=C), 1360 (O=S=O), 1013 (O=S=O)
Mass Spec (m/z) ESI high resolution: calculated: 388.766, found: 388.0774 [M + Na]+. Synthesis 3 3, 7-Bis(dimethylamino)phenothiazin-5-ium chloride (methylthioninium chloride) (5)
Figure imgf000061_0001
(iii): CuSO4.5H2O, H2O, HCI, 850C, NaCI
De-ionised water (455 ml) was adjusted to pH 2 with hydrochloric acid and copper sulfate (2.06 g 0.0825 mols) was added. 3 was added to a round bottom flask and the copper sulfate solution was added. The reaction mixture was heated to ~85 0C and stirred for 1 hour. The deep blue solution was allowed to cool to 22 0C and then filtered away from insolubles. The filtrate was added to a round bottom flask and sodium chloride (100 g, 1.73mols) was added. The solution was stirred until precipitate was seen (~1 hour). The solid which precipitated was collected via filtration and dissolved in water (20 ml) and the solution was acidified to pH 1 using hydrochloric acid. The solution was stirred at room temperature for ~30 minutes and the resulting solid was collected via filtration and dried under vacuum (1000 mbar) at 40 0C for 16 hours. 3.44 g (16 %, calculated as MTC.2H2O from 2)
Mp: 190 0C (decomposed) vmax (KBr)/crrf1: 3418 (O-H, St (H2O as a solvate)), 1594 (C=C (Aromatic), St), 1489
(C=C (Aromatic), St)1 1393 (C-H (Aromatic), St), 1333 (C-N, St), 1175 (C-H
(Aromatic), St), 1 144 (C-H (Aromatic), St) δH(400 MHz, D2O): 3.09 (s, 12H, N-CH3), 6.84 (s, 2H, Ar-H), 7.03, 7.05 (d, /B = 8.8
Hz, 2H, A-H1), 7.36, 7.39 (d, J*8 = 9.2 Hz, 2H, Ar-H1). δc(150 MHz, D2O): 40.3 (N-CH3), 105.7 (Ar-C), 118.0 (Ar-C), 133.2 (Ar-C), 133.5 (Ar-
C), 135.9 (Ar-C), 152.7 (Ar-C).
Mass Spec (m/z) ESI high resolution: calculated: 284.1216, found: 284.1218 [M-Cl]+.
HPLC: retention time = 17.962 mins, peak area = 85.17 %
UV, λmax : 663 nm
Moisture analysis: 9.6% volatiles at 105 0C.
MT+ assay: MT+ content = 49 %; MTC.2H2O = 69 %; a significant amount of azure B was detected. Synthesis 4 2-amino-5-diethylaminophenyl thiosulfonic acid (6)
Figure imgf000062_0001
(i): Na2S2O3 SH2O, Na2S2O8, H2O, MeOH, 5 0C
Λ/',Λ/'-diethyl-p-phenylenediamine (5 g, 0.030 mols) was added to a round bottom flask. To this was added de-ionised water (80 ml) and methanol (20 ml) and the solution was then cooled to 5 0C. Sodium thiosulfate (8.31 g, 0.033 mols) in water (20 ml) was added to the solution in one aliquot. Sodium persulfate (7.25 g, 0.030 mols) in water (40 ml) was added drop-wise over a 15 minute period. The reaction mixture was stirred for 3 hours at 5 0C and then warmed to 22°C over a period of 1 hour. The dark brown solid which precipitated was collected via filtration. The dark brown solid was washed with water (50 ml) then dried under vacuum (1000 mbar) at 40 0C for 16 hours, 4.43 g (52 %).
2-amino-5-diethylaminophenyl thiosulfonic acid was added to a round bottom flask and ethyl acetate (240 ml) was added. The slurry was heated to reflux for one hour and then cooled to a temperature between 18-22 CC. Once the solution had cooled to the desired temperature the purple solid was collected via filtration. The purple solid was washed with ethyl acetate (50 ml) and the solid was dried under vacuum (1000 mbar) at 40 0C for 16 hours, 4.2 g.
Final yield = 49 % Actual value = 45 % by NMR with internal standard (DSS)
Mp: 203-204 0C vmax (KBr)/crτϊ1: 3434 (N-H, St)13336 (N-H1 St), 2985 (C-H, St), 1624 (C=C1 St), 1470
(C-C OrO=S=O), 1182 (O=S=O), 1019 (O=S=O). δH(400 MHz1 DMSO-de) 1.021 (6 H1 1, N-CH2CH3), 3.44 (m, 4H1 N-CH2CH3 ), 6.86-6.88
(d, 1 H1 Ar-H- J*8 = 8.0 Hz)1 7.16-7.18 (d, 1 H1 Ar-H J*B = 8.4 Hz), 7.40 (s, 1 H, Ar-H).
Mass Spec (m/z) ESI high resolution: calculated: 299.0495, found: 299.0503 [M +
Na]+; 197.1 1 12 [(M + H)-SO3J+. Synthesis 5
Preparation of 2-amino-5-ethylmethylaminophenyl thiosulfonic acid (8)
Figure imgf000063_0001
(i): Na2S2O3 SH2O, Na2S2O8, H2O, MeOH, 5 0C
To a 250 ml round bottom flask was added Λ/-ethyl-Λ/-methyl-p-phenylene diamine (2 g, 13.31 mmol, MW 150.23 g/mol), water (32 ml), methanol (8 ml) and sodium thiosulfate pentahydrate (3.63 g, 14.64 mmol, MW 248.18 g/mol, dissolved in 5 ml of water). The mixture was cooled over ice water to between 2-5 0C. Sodium persulfate (3.17 g, 13.31 mmol, MW 238.10 g/mol, dissolved in 8 ml of water) was added drop- wise to the cooled mixture and the reaction was allowed to react for three hours. The mixture was then allowed to increase in temperature to 22 0C before being filtered. The grey solid was washed with water (3 x 20 ml) and ethyl acetate (3 x 25 ml) to give the crude product (1.29 g, 37 %, MW 262.35 g/mol) as a light grey solid.
Mp: 197-198 0C (H2O) vmax (KBr)/crrr1; 3435 (NH), 3338 (WH)1 3004 [C-H), 2941 (=CH), 2706 (SO3H), 1626
(C=C)1 1467 (C-C or O=S=O), 1180 (O=S=O), 1017 (O=S=O) δH(400 MHz; D2O); 1.04 (3H, t, J = 7 Hz, CH3), 3.03 (3H,s, NCH3), 3.43 (4H, q, J = 7
Hz, NCH2) 6.98 (1 H, d, J = 6.8 Hz, ArH), 7.14 (1 H, brd s, ArH), 7.34 (1 H, brd s, ArH)
Mass Spec (m/z) ESI high resolution: calculated: 263.0524; found, 263.0513 [M + H]+;
183.0948 [(M + H)-SO3J+. Preparation of ethylmethylthiosulfonic acid of Bindschedlers green (9)
Figure imgf000064_0001
(ii): Λ/-ethyl-/V-methylaniline, Na2S2O8, H2O, H2SO4, NaOH(aq)
To a 50 ml round bottom flask was added 2-amino-5-ethylmethylaminophenyl thiosulfonic acid (0.5 g, 1.91 mmol, MW 262.35 g/mol) and water (10 ml).The mixture was cooled over ice water to between 2-5 0C. A homogeneous solution of Λ/-ethyl-Λ/- methylaniline (0.26 g, 1.91 mmol, 135.21 g/mol), water (0.5 ml) and sulphuric acid (0.19 g, 1.91 mmol, MW 98.08 g/mol) was added to the cooled mixture. The pH of the mixture was then adjusted to pH ~7.5 with sodium hydroxide (6 M, ~1 ml) to give a black slurry. Sodium persulfate (1.36g, 5.72 mmol, MW 238.10 g/mol, dissolved in 2 ml of water) was added drop-wise to the cooled near neutral slurry and the reaction was allowed to react for two hours. The mixture was then allowed to increase in temperature to 24 0C before being filtered. The black solid was washed with water (3 x 10 ml) and air dried for two hours to give the crude product (0.42 g, 56 %, MW 392.52 g/mol) as a black solid.
vmax (KBr)/crtV1; 2979 (C-H), 1594 (C=C), 1386 (O=S=O), 1021 (O=S=O)
Mass Spec (m/z) ESI high resolution: calculated: 396.1416, found: 396.1422 [M +
3H]+).
Preparation of ethylmethylthioninium chloride (10)
Figure imgf000064_0002
(iii): CuSO4.5H2O, H2O, HCI To a 50 ml round bottom flask was added Λ/-ethyl-Λ/-methylthiosulfonic acid of Bindschedlers green (0.4 g, 1.02 mmol, MW 392.52 g/mol), aqueous hydrochloric acid (5 ml, pH 2) and copper sulphate pentahydrate (0.04 g, 0.14 mmol, MW 249.69 g/mol). The mixture was heated to 85 0C and stirred at this temperature for 1 hour. The mixture was then allowed to cool to 24 0C before being filtered. The black solid was washed with water (3 x 0.5 ml). Sodium chloride was added to the deep blue filtrate to precipitate the crude product, which was collected by filtration (0.038 g, 11 %, MW 347.91 g/mol) as a purple solid. The crude solid was purified by column chromatography using silica 40-63μ 6θA with 10 % MeOH, 90% DCM as the eluent.
Mp: 178-18O 0C (MeOH) vmax (KBr)/crτV1; 2925 {=CH), 1594 (C=C), 1486 (C-C) δH(250 MHz; D2O); 1.20 (6H, t, J = 6.5 Hz, CH3), 3.10 (6H1S1 NCH3), 3.50 (4H, brd q,
NCH2) 6.87 (2H, s, ArH), 7.11 (2H, brd, ArH), 7.34 (2H, d, J = 9.5 Hz, ArH) δc(150 MHz; D2O); 11.5, (CH3), 38.3 (NCH3), 48.8 (NCH2), 106.0 (ArC), 1 18.4 (ArC),
133.9 (ArC), 134.2 (ArC), 136.5 (ArC), 152.6 (ArC)
Mass Spec (m/z) ESI high resolution: calculated: 312.1534, found: 312.1545 [M - Cl]+.
Synthesis 6
Preparation of N,N-di(prop-2-en-1-yl)aniline
Figure imgf000065_0001
To a stirred solution of aniline (5.62 g, 60.36 mmol) in EtOH (200 ml) and water (50 ml) was added allyl bromide (17.33 g, 143.3 mmol) in one portion followed by solid Na2CO3 (6.69 g, 62.5 mmol). The yellow mixture was then heated under reflux for 17 h, left to cool to room temperature (19 °C) and the EtOH was removed under reduced pressure. The aqueous residue was extracted with Et2O (5 x 200 ml) and the combined organics were washed with brine (3 x 200 ml) and dried (Na2SO4). The solvent was removed under reduced pressure to give a brown oil that was distilled over KOH pellets to give the title compound (7.81 g, 75%) as a pale yellow oil. vmax (KBr)/cnr1 3061 (C-H), 2979 (C-H)1 291 1 (C-H), 1642 (C=C), 1599 (Ar C=C), 1505
(Ar C=C) δH(400 MHz, CDCI3): 3.95 (ddd, J = 1.5, 1.5, 4.4 Hz, 4H, NCH2), 5.18 (ddt, J = 1.2, 1.6,
10.8 Hz, 2H, =CH2), 5.21 (ddt, J = 1.6, 1.9, 17.2 Hz, 2H, =CH2), 5.89 (ddt, J = 4.4,
10.8, 17.2 Hz, 2H, =CH), 6.69-6.74 (m, 3H, Ar-H), 7.20-7.24 (m, 2H, Ar-H) δc(100 MHz, CDCI3): 52.75 (NCH2), 112.34, 1 15.99, 116.30, 129.08, 134.04, 148.71
Prepared according to Ling et al (2007).
Preparation of 4-Nitro-N,N-di(prop-2-en-1-yl)aniline
Figure imgf000066_0001
A mixture of 4-fluoronitrobenzene (5.00 g, 35.44 mmol), diallylamine (3.48 g, 4.4 ml, 35.44 mmol) and K2CO3 (5.39 g, 38.98 mmol) in dry DMSO (40 ml) was heated at 100 °C under an atmosphere of argon for 3 h. The reaction mixture was then left to cool to room temperature (23 "C) and added to water (300 ml) and extracted with EtOAc (4 x 75 ml). The combined organics were washed with brine (100 ml) and dried (Na2SO4). The solvent was removed under reduced pressure to give a red oil that was purified by chromatography (2:1 Hexane/DCM) to give the title compound (4.60 g, 59%) as an orange oil.
vmax (KBr)/cm 1 3085 (C-H), 301 1 (C-H), 2983 (C-H), 2912 (C-H)1 1643 (alkene C=C),
1596 (NO2), 1516 (Ar C=C), 1488 (Ar C=C), 1315 (NO2) δH(400 MHz, CDCI3): 4.01-4.03 (ddd, J = 1.6, 1.6, 4.8 Hz, 4H, NCH2), 5.16 (ddt, J =
1.6, 1.9, 17.2 Hz, 2H, CH2=), 5.23 (dq, J = 1.6, 1.9, 10.4 Hz, 2H, CH2=), 5.84 (ddt, J =
4.4, 10.4, 17.2 Hz, 2H, =CH), 6.62 (d, J = 9.6 Hz, 2H, Ar-H), 8.09 (d, J = 9.6 Hz, 2H,
Ar-H) δc(100 MHz, CDCI3): 52.95, 1 10.73, 1 16.94, 119.27, 126.12, 131.75, 153.26
Mass Spec (m/z) ESI high resolution: calculated: 219.1134; found, 219.1131 [M + H]+.
Data consistent Yi-Chun Hsu et al (2005). Preparation of N,N-di(prop-2-en-1-yl)benzene-1 ,4-diamine
Figure imgf000067_0001
A mixture of 4-nitro-/V,Λ/-di(prop-2-en-1-yl)aniline (12.22 g, 55.98 mmol) and tin (II) chloride dihydrate (64.45 g, 279.9 mmol) in EtOH (280 ml) was stirred and heated under reflux for 21 h. The cooled reaction mixture was concentrated under reduced pressure to a volume of 100 ml, and neutralised by the addition of 5 M NaOH. The precipitate was removed by filtration and the filtrate was extracted with EtOAc (4 x 200 ml). The combined organics were washed with brine (2 x 200 ml) and dried (Na2SO4). The solvent was removed under reduced pressure to give a brown oil that was purified by chromatography (1 :1 Hexane/EtOAc) to give the title compound (7.79 g, 74%) as a brown oil.
vmax (KBr)/cnr1 3413 (NH), 3341 (NH), 3216 (C-H), 3075 (C-H), 3034 (C-H), 3004 (C-
H), 2977 (C-H), 1640 (alkene C=C), 1612 (Ar C=C), 1514 (Ar C=C) δH(400 MHz1 CDCI3): 2.98-3.68 (br s, 2H, NH2), 3.81 (d, J = 4.8 Hz, 4H, NCH2), 5.13
(ddt, J = 1.5, 2.0, 10.6 Hz, 2H, CH2=), 5.17 (ddt, J = 1.5, 2.0, 17.2 Hz, 2H, CH2=), 5.85
(ddt, J = 5.1 , 10.1 , 17.2 Hz, 2H, =CH), 6.63 (app br s, 4H, Ar-H)) δc(100 MHz, CDCI3): 53.87, 1 15.46, 1 16.22, 116.64, 134.71 , 137.46, 142.35
Mass Spec (m/z) ESI high resolution: calculated: 189.1392, found: 189.1383 [M + H]+.
Preparation of 2-amino-5-N,N-di(prop-2-en-1-yl)aminophenylthiosulfonic acid (12)
Example 1
A modification of Bernthsten's protocol for the preparation of 2-amino-5-Λ/,Λ/-di(prop-2- en-1-yl)aminophenylthiosulfonic acid has been executed. This preparation uses zinc chloride and aluminium sulfate.
Figure imgf000068_0001
(i): Na2S2O3 SH2O1 Na2S2O8, AI2(SO4J3.16H2O, ZnCI2, THF, H2O, 5 0C
Λ/,Λ/-di(prop-2-en-1-yl)benzene-1 ,4-diamine (1.00 g, 5.31 mmol) was added to a stirring solution of aluminium sulfate hexadeca hydrate (3.73 g, 5.68 mmol) in distilled water (10 mL). To the reaction mixture was added sequentially a solution of sodium thiosulfate (3.03 g, 12.21 mmol) in distilled water (5 ml) and a solution of zinc chloride (0.77 g, 5.57 mmol) in distilled water (1 ml). THF (4 ml) was added and the reaction mixture was cooled to 5 °C in an ice/water bath, and a solution of sodium persulfate (1.28 g, 5.31 mmol) in distilled water (3 ml) was added drop-wise over 15 min. The reaction mixture was stirred for 2 h at 5 °C then left to rise to room temperature (22 "C). After 39 h, the aqueous phase was decanted and the sticky green solid was collected by filtration and washed with water (2 x 80 ml), Et2O (5 x 50 ml) and acetone (5 x 100 ml). The solid obtained was dried at 74 °C for 2 h to give the title compound (0.30 g, 18%) as a pale green solid.
Mp 162-164 °C (decomposition) vmax (KBr)/crτV1 3433 (NH), 3347 (NH), 3228 (C-H), 2987 (C-H), 2922 (C-H), 1639
(alkene C=C), 1509 (Ar C=C), 1183 (O=S=O), 1019 (O=S=O). δH(400 MHz, DMSO-d6): 3.93 (d, J = 3.9 Hz, 4H, NCH2) 5.12-5.16 (m, 4H, =CH2), 5.82
(ddt, J = 5.1 , 9.8, 17.6 Hz, 2H, =CH), 6.76 (d, J = 7.8 Hz, 1 H1 Ar-H), 6.95 (br s, 1 H1 Ar-
H)1 7.18 (d, J = 9.0 Hz1 1 H, Ar-H)1 8.80-10.18 (br s, 3H, NH3) δc(100 MHz, DMSO-d6): 53.18 (NCH2), 114.16, 1 17.09, 120.75, 124.28, 127.58,
133.71 , 147.82
Mass Spec (m/z) ESI high resolution: calculated: 301.0681 , found: 301.0676 [M + H]+;
221.1104 [(M+H)-SO3]+ Example 2
Figure imgf000069_0001
(i): Na2S2O3 SH2O, Na2S2O8, MeOH, H2O1 5 0C
A stirred solution of Λ/,Λ/-di(prop-2-en-1-yl)benzene-1 ,4-diamine (1.00 g, 5.31 mmol) in MeOH (14 ml) and water (16 ml) was cooled to 5 °C in an ice/water bath, and a solution of sodium persulfate (1.28 g, 5.31 mmol) was added dropwise over 15 min. The reaction mixture was stirred for 2 h at 5 °C then left to rise to room temperature (22 'C) over 1 h. The reaction mixture was then extracted with DCM (3 x 40 ml) and the aqueous phase was neutralized with sat NaHCO3. After 48 h at room temperature (21-23 "C) the solid that had precipitated was collected by filtration and washed with water (50 ml) and acetone (4 x 50 ml). The solid obtained was dried at 74 °C for 5 h to give the title compound (0.31 g, 19%) as a green solid.
Mp 158-160 °C (decomp.) vmax (KBr)/crτr1 3433 (NH), 3347 (NH), 3228 (C-H), 3070 (C-H), 2987 (C-H), 2923 (C-H), 1639 (alkene C=C), 1508 (Ar C=C), 1240 (S=O), 1185 (C-N), 1019 (S=O) δH(400 MHz, DMSO-d6): 3.92 (d, J = 5.1 Hz, 4H, NCH2) 5.12-5.17 (m, 4H, =CH2), 5.81 (ddt, J = 5.1 , 9.8, 17.6 Hz, 2H, =CH), 6.75 (dd, J = 9.0, 2.7 Hz, 1H1 Ar-H), 6.93 (d, J = 2.4 Hz, 1 H1 Ar-H), 7.13 (d, J = 8.6 Hz, 1 H1 Ar-H)1 8.80-9.70 (br s, 3H, NH3) δc(100 MHz, DMSO-d6): 53.23 (NCH2), 1 14.38, 1 17.18, 120.86, 123.93, 127.29, 133.75, 147.55
Preparation of allylthioninium chloride (13)
Figure imgf000069_0002
(M):, Na2S2O8, H2SO4, H2O, 6M NaOH, 50C, (iii): CuSO4, HCI, H2O, 85 0C, NaCI To a stirred suspension of 2-amino-5-Λ/,Λ/-di(prop-2-en-1-yl)aminophenylthiosulfonic acid (0.500 g, 1.66 mmol) in distilled water (10 ml) at 5 °C was added a pre-cooled mixture of Λ/,Λ/-di(prop-2-en-1-yl)aniline (0.288 g, 1.66 mmol) and concentrated sulphuric acid (0.163 g, 1.66 mmol) in distilled water (1 ml). The pH of the mixture was adjusted to pH -7.5 using 6 M sodium hydroxide (~400 ul). A solution of sodium persulfate (0.395 g, 1.66 mmol) in water (2 ml) was added drop-wise over a period of 15 min. The reaction mixture was stirred for 1 hour at 5 0C and then warmed to room temperature (22 °C) over a 1 hour period. The solid was collected by filtration and washed with water (4 x 30 ml). To the filtrate was added copper (II) sulphate (50 mg, 0.3 mmol) and the solution was heated at 85 °C for 20 min. The reaction mixture was left to cool to room temperature (22 0C) and sodium chloride (10 g, 171 mmol) was added and the mixture was stirred for 1 h. The reaction mixture was then extracted with DCM (4 x 30 ml) and the combined organics were dried (Na2SO4) and the solvent was removed under reduced pressure to give a blue oil that was purified by column chromatography (10 % MeOH in DCM) to give 13 as a dark blue solid.
vmax (KBr)/cm 1 3424 (water, 0-H)1 3083 (C-H), 3013 (C-H), 2921 (C-H), 2854 (C-H),
1638 (alkene C=C), 1593 (Ar C=C) δH(400 MHz1 CD3OD): 4.36 (ddd, J = 1.6, 1.6, 4.7 Hz, 8H, NCH2), 5.26 (ddt, J = 1.2, x,
17.2 Hz, 2H1 =CH2), 5.32 (ddt, J = 1.2, x, 10.8 Hz, 2H, =CH2), 5.99 (ddt, J = 5.1 , 10.2,
17.2 Hz, 4H, =CH), 7.38 (d, J = 2.8 Hz, 2H), 7.49 (dd, J = 2.8, 9.4 Hz, 2H), 8.02 (d, J =
9.7 Hz, 2H) δc(100 MHz, CD3OD): 53.52 (NCH2), 106.3 (Ar-C), 116.9 (=CH2), 119.4 (Ar-C), 130.82
(=CH), 135.1 (Ar-C), 136.1 (Ar-C), 138.52 (Ar-C), 154.2 (Ar-C)
Mass Spec (m/z) ESI high resolution: calculated: 388.1842, found: 388.1481 [M-Cl]+;
348.1530 KM-CI)-CH2CHCH2I+, 268.1747 [(M-CI)-3(CH2CHCH2)]+.
Synthesis 7 Bis-diethylamino- 1, 9-dimethyl-phenothiazin-5-ylium (15)
Figure imgf000071_0001
(i): Na2S2O3-SH2O, Na2S2O8, H2O, MeOH, Na2CO3, EtOAc, DCM
Λ/4,Λ/4-Dimethyl-2-methyl-1 ,4-phenylene diamine monohydrochloride (6.0 g, 27.9 mmol, 1.0 eq.) was dissolved in water (80 cm3), and methanol (20 cm3) added. The solution was stirred by overhead stirrer and cooled to an internal temperature of ~2 0C. Sodium thiosulfate (7.6 g, 30.1 mmol, 1.1 eq.) was dissolved in H2O (10 cm3) and added to the reaction in one portion. Sodium persulfate (6.65 g, 27.9 mmol, 1.0 eq.) was dissolved in water (20 cm3) and added to the reaction drop-wise over 18 minutes. During the addition the internal temperature was held between 1.7 and 2.7 0C The solution was allowed to warm to 21 0C over ~1 hour, and stirred at this temperature for a further 3 hours.
The pH of the medium was increased from 1.75 to 7.00 by addition of a saturated sodium carbonate solution, and volatiles were removed in vacuo. The solution was extracted with ethyl acetate (3 x 100 cm3). Upon the development of a characteristic deep blue colour in the aqueous residue, the solution was extracted with dichloromethane (100 cm3), the extract dried with magnesium sulfate and solvent removed in-vacuo to yield an intensely blue oil.
HPLC analysis on an SBCN column using method "relsub.m" showed only one significant peak at RT = 25.056 minutes, λmax of peak = 657 nm. δH(400 MHz, CD3OD) 7.29 (2H, s, Ar), 7.16 (2H, s, Ar), 3.69 (8H, q, J 4.6, CH2), 2.71
(6H, s, CH3), 1.33 (12H, t, J 4.6, CH3).
Mass Spec (rn/z) ESI low resolution: calculated: 368.6, found: 369 [M + Hl]+. References:
The following references are hereby incorporated by reference in their entirety:
WO 2006/032879
Badische Anilin- und Soda-Fabrik, 1877, "Verfahren Zur Darstellung Blauer Farbstoffe Aus Dimethyl-Anilin Und Anderen Tertiaren Aromatischen Monaminen," German Patent No. 1886, published 15 December 1877.
Bernthsen, August, 1885a, "Studien in der Methylenblaugruppe," Justus Liebig's Annalen der Chemie, Band 230, pp. 73-136.
Bernthsen, August, 1885b, "Studien in der Methylenblaugruppe," Justus Liebig's Annalen der Chemie, Band 230, pp. 137-211.
Bernthsen, August, 1889, "Studien in der Methylenblaugruppe," Justus Liebig's Annalen der Chemie, Band 251 , pp. 1-96.
Colour Index, Vol. 4 (3rd Edition, 1971), p. 4470, Entry Number 52015.
Fierz-David and Blangley, 1949, "F. Oxazine and Thiazine Dyes," in: Fundamental Processes of Dye Chemistry, published by lnterscience (London, UK)1 pp. 308-314.
Leventis.N., et al., 1997, "Synthesis of Substituted Phenothiazines Analogous to Methylene Blue by Electrophilic and Nucleophilic Aromatic Substitutions in Tandem. A Mechanistic Perspective," Tetrahedron, Vol. 53, No. 29, pp. 10083-10092.
Lillie, R.D., et al., 1979, "Zinc Chloride Methylene Blue, I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples," Stain Technology. Vol. 54, No. 1 , pp. 33-39.
Ling Li and William D Jones, 2007, "Mechanistic investigation of the cobalt-catalysed selective conversion of diallylanilines to quinolines involving C-N and C-H activations", J Am Chem Soc, 129, pp. 10707-10713.
Lohr, W., Grubhoffer, N., Sohmer, I., Wittekind.D., 1975, "The azure dyes: their purification and physiochemical properties. Purification of Azure B," Stain Technology, Vol. 50 (3), pp. 149-156.
Marshall, P. N., Lewis, S. M., 1975a, "The purification of Methylene Blue and Azure B by solvent extraction and crystallisation," Stain Technology, Vol. 50 (6) pp. 375-381.
Marshall, P. N., Lewis, S. M., 1975b, "Metal contaminants in commercial dyes," Stain Technology, Vol. 50 (3), pp. 143-147.
Masuya, Hirotomo, 1992, "Phenothiazine Derivatives, Their Production and Use," European Patent Publication No 0 510 668 A2, published 28 October 1992.
Wischik, CM., et al., 1996, "Inhibition of Tau-Tau-Association," international (PCT) patent application publication number WO 96/30766 published 03 October 1996.
Wischik, CM., et al., 2002, "Materials and Methods Relating to Protein Aggregation in Neurodegenerative Disease," published international (PCT) patent application publication number WO 02/055720 published 18 July 2002.
Yi-Chun Hsu, Kim-Hong Gan and Shyh-Chyun Yang, 2005, "Palladium-catalysed allylation of acidic and less nucleophilic anilines using allylic alcohols directly", Chem Pharm Bull, VoI, 53 (10), pp. 1266-1269.

Claims

Claims:
1. A method for the preparation of a diaminophenothiazinium compound, the method comprising one or both of the steps of (i) Thiosulfonic Acid Formation (TSAF); and (ii) Oxidative Coupling (OC);
wherein the diaminophenothiazinium compound has the following formula:
Figure imgf000073_0001
and: each of -R1 and -R9 is independently -H or -RA; and each -RA is independently selected from
Figure imgf000073_0002
C2-4alkenyl; halogenated C1-4alkyl; Cs-ioaryl; halogenated C5-i0aryl; Ci.4alkylene-C5-1oaryl and halogenated C1-4alkylene-C5.1oaryl; each of -R3NA and -R3NB is independently selected from: C1-4alkyl; C2-4alkenyl; halogenated Ci-4alkyl; C5-10aryl; halogenated C5-ioaryl; C1-4alkylene-C5.1oaryl and halogenated C1-4alkylene-C5-ioaryl; each of -R7NA and -R7NB is independently selected from: C1.4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5-i0aryl; halogenated C5.10aryl; C1-4alkylene-C5.10aryl and halogenated C1-4alkylene-C5.i0aryl; and
X' is an anionic counter ion;
wherein step (i) Thiosulfonic Acid Formation (TSAF) comprises a step in which a compound of formula I is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises persulfate to give a compound of formula II:
Figure imgf000073_0003
I >ι wherein -R9, R7NA and -R7NB are as previously defined; and each of -R10NA and -R10NB is independently selected from: -H; C1-4alkyl; C2-4alkenyl; halogenated Ci-4alkyl; C5.10aryl; halogenated C5.10aryl; C1-4alkylene-C5-10aryl and halogenated C1-4alkylene-C5.10aryl; and wherein step (ii) Oxidative Coupling (OC) comprises a step in which a compound of formula Ua is oxidatively coupled to a compound of formula III, using an oxidizing agent that is or comprises persulfate, to give a compound of formula IV:
Figure imgf000074_0001
wherein: each of -R1 and -R9 is independently -H or -RA;
-RA _RJNA _R3NBi _R/NA and _R,NB are as previously defined.
2. A method of synthesis comprising the step of Thiosulfonic Acid Formation (TSAF)1 as defined in claim 1 , in which a compound of formula I is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises persulfate to give a compound of formula II:
Figure imgf000074_0002
wherein: each -R9 is independently -H or -RA; and each -RA is independently selected from C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5.10aryl; halogenated C5.iOaryl; C1.4alkylene-C5.1oaryl and halogenated Ci.4alkylene-C5.i0aryl; each of -R7NA and -R7NB is independently selected from: C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5-i0aryl; halogenated C5.10aryl; Ci.4alkylene-C5.i0aryl and halogenated
Figure imgf000074_0003
and each of -R10NA and -R10NB is independently selected from: -H; C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5-10aryl; halogenated C5.10aryl; Ci-4alkylene-C5.i0aryl and halogenated C1-4alkylene-C5.1oaryl.
3. The method according to claim 1 or claim 2, wherein each of -R7NA and -R7NB is independently selected from: C1-4alkyl; C2-4alkenyl; and halogenated Ci-4alkyl.
4. The method according to claim 3, wherein each of -R7NA and -R7NB is independently C1-4alkyl.
5. The method according to any one of the preceding claims, wherein each -R9 is independently selected from: -H; C1-4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
6. The method according to claim 5, wherein each -R9 is -H.
7. The method according to claim 5, wherein at least one -R9 is independently selected from: d.4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
8. The method according to any one of the preceding claims, wherein the thiosulfate is or comprises Na2S2O3 Or a hydrate thereof.
9. The method according to any one of the preceding claims, wherein the persulfate is or comprises sodium persulfate, potassium persulfate, or ammonium persulfate.
10. The method according to any one of the preceding claims, wherein the amine, I, is added first, before the thiosulfate is added.
11. The method according to any one of the preceding claims, wherein the thiosulfate is added before the persulfate is added.
12. The method according to any one of the preceding claims, wherein the reaction is performed in an aqueous medium comprising an organic solvent.
13. The method according to any one of the preceding claims, wherein the reaction is performed in an aqueous medium comprising a surfactant.
14. The method according to any one of the preceding claims, wherein the reaction is performed in an aqueous medium and the pH of the aqueous medium prior to the addition of the thiosulfate and persulfate is in the range of pH 2 to 7.
15. The method according to any one of the preceding claims, wherein each of -R10NA and -R10NB is independently selected from: -H; C1-4alkyl; C2.4alkenyl; and halogenated C1-4alkyl.
16. The method according to any one of claims 1 to 15, wherein each of -R10NA and -R10NB is -H.
17. The method according to claim 16, wherein I and Il are compounds of formula Ib and lib respectively:
Figure imgf000076_0001
Ib lib
18. The method according to claim 17, wherein Ib and lib are A and B respectively:
Figure imgf000076_0002
B
19. A method of synthesis comprising the step of Oxidative Coupling (OC) as defined in claim 1 , in which a compound of formula Ha is oxidatively coupled to a compound of formula III, using an oxidizing agent that is or comprises persulfate, to give a compound of formula IV:
Figure imgf000076_0003
wherein: each of -R1 and -R9 is independently -H or -RA; and each -RA is independently selected from C1-4alkyl; C2.4alkenyl; halogenated C5.10aryl; halogenated C5-10aryl; Ci-4alkylene-C5.i0aryl and halogenated
Figure imgf000076_0004
each of -R3NA and -R3NB is independently selected from: C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5-10aryl; halogenated C5-10aryl; Ci.4alkylene-C5.10aryl and halogenated C1-4alkylene-C5.ioaryl; and each of -R7NA and -R7NB is independently selected from: C1-4alkyl; C2.4alkenyl; halogenated C1-4alkyl; C5-10aryl; halogenated C5.i0aryl;
Figure imgf000077_0001
and halogenated C1-4alkylene-C5-1oaryl.
20. The method according to claim 19, wherein each of -R7NA and -R7NB is independently selected from: C^alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
21. The method according to claim 20, wherein each of -R7NA and -R7NB is independently C1-4alkyl.
22. The method according to any one claims 19 to 21 , wherein each of -R1 and -R9 is independently selected from: -H; C1-4alkyl; C2.4alkenyl; and halogenated C1-4alkyl.
23. The method according to claim 22, wherein each of -R1 and -R9 is -H.
24. The method according to claim 23, wherein at least one of each of -R1 and -R9 is independently selected from: Ci-4alkyl; C2-4alkenyl; and halogenated Ci.4alkyl.
25. The method according to any one of claims 19 to 24, wherein each of -R3NA and -R3NB is independently selected from: Ci-4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
26. The method according to claim 25, wherein each of -R3NA and -R3NB is independently C1-4alkyl.
27. The method according to any one of claims 19 to 26, wherein Il and IV are compounds of formula lib and IVa respectively:
Figure imgf000077_0002
lib IVa
28. The method according to any one of claims 19 to 26, wherein Hb, III and IVa are Hb, IHa and V respectively:
Figure imgf000078_0001
lib MIa
29. The method according to any one of claims 19 to 28, wherein the persulfate is or comprises sodium persulfate, potassium persulfate, or ammonium persulfate.
30. The method according to any one of claims 19 to 29, wherein the aniline, III, is added before the persulfate is added.
31. The method according to any one of claims 19 to 30, wherein the aniline, III, is added in a mixture with aqueous acid.
32 The method according to any one of claims 19 to 31 , wherein the reaction is performed under basic conditions.
33. The method according to any one of claims 19 to 32, wherein the zwitterionic intermediate IV is isolated and purified.
34. The method according to claim 33, wherein the isolation and purification is by filtration.
35. The method according to any one of claims 19 to 34, further comprising the preceding step of Thiosulfonic Acid Formation (TSAF) according to any one of claims 15 to 17.
36. The method of claim 1 , wherein step (i) Thiosulfonic Acid Formation (TSAF) comprises a method according to any one of claims 16 to 18; and step (ii) Oxidative Coupling (OC) comprises a method according to any one of claims 19 to 34.
37. The method according to claim 36, wherein each of -R7NA and -R7NB is independently selected from: C1-4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
38. The method according to claim 36, wherein each of -R7NA and -R7NB is independently C1-4alkyl.
39. The method according to any one claims 36 to 38, wherein each of -R1 and -R9 is independently selected from: -H; C1-4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
40. The method according to claim 39, wherein each of -R1 and -R9 is -H.
41. The method according to claim 40, wherein at least one of each of -R1 and -R9 is independently selected from: C1-4alkyl; C2-4alkenyl; and halogenated d.4alkyl.
42. The method according to any one of claims 36 to 41 , wherein each of -R3NA and -R3NB is independently selected from: C1-4alkyl; C2-4alkenyl; and halogenated C1-4alkyl.
43 The method according to claim 42, wherein each of -R3NA and -R3NB is independently C1-4alkyl.
44. The method according to any one of claims 36 to 43, further comprising the step of Ring Closure (RC), in which a compound of formula V is subjected to ring closure to give a compound of formula Vl:
Figure imgf000079_0001
Vl
wherein -R1 and -R9, -R3NA, -R3NB, -R7NA, -R7NB and X" are as defined according to the diaminophenothiazinium compound of any one of claims 36 to 43.
45. The method according to claim 44, wherein compounds V and Vl are D and E respectively:
Figure imgf000079_0002
46. The method according to claim 44 or claim 45, wherein ring closure is achieved by treatment with an oxidizing agent.
47. The method according to claim 46, wherein the oxidizing agent is or comprises Cu(II), or the oxidizing agent is or comprises activated manganese dioxide (MnO2).
48. The method according to any one of claims 35 to 47, further comprising the step of Chloride Salt Formation (CSF) in which a compound of formula Vl is reacted with chloride, to give a compound of formula VII:
Figure imgf000080_0001
Vl VII
wherein -R1 and -R9, -R3NA, -R3NB, -R7NA, -R7NB and X' are as defined according to the diaminophenothiazinium compound of any one of claims 34 to 43.
49. The method according to claim 48, wherein Vl is reacted with a chloride salt or hydrochloric acid as a source of chloride.
50. The method according to any one of claims 36 to 49, wherein the method comprises the step of (i) Thiosulfonic Acid Formation (TSAF) as defined in any one of claims 16 to 18; and a step of Cr-Mediated Oxidative Coupling (Cr-OC), in which a compound of formula Ha, is oxidatively coupled a compound of formula III, using an oxidizing agent that is or comprises Cr(VI), to give a compound of formula IV, wherein compounds Ha1 III and IV, are as defined according to any one of claims 19 to 28.
51. The method according to any one of claims 36 to 49, wherein the method comprises the step of (ii) Oxidative Coupling (OC) as defined in any one of claims 19 to 34; and a preceding step of Cr-Mediated Thiosulfonic Acid Formation (Cr-TSAF), in which a compound of formula I is oxidized in the presence of a thiosulfate and an oxidizing agent that is or comprises Cr(VI) to give a compound of formula II, wherein compounds I and Il are as defined according to any one of claims 15 to 17.
52. The method according to any one of the preceding claims further comprising the step of Nitrosyl Reduction (NR), in which a compound of formula VIII is reduced to form a compound of formula Ia:
Figure imgf000081_0001
VIII Ia wherein: each -R9 is independently -H or -RA; and each -RA is independently selected from d^alkyl; C2-4alkenyl; halogenated Ci-4alkyl; C5.10aryl; halogenated C5-10aryl; C1-4alkylene-C5.10aryl and halogenated Ci-4alkylene-C5.10aryl; and each of -R7NA and -R7NB is independently selected from: C1-4alkyl; C2-4alkenyl; halogenated d.4alkyl; C5-i0aryl; halogenated C5-i0aryl; Ci-4alkylene-C5-ioaryl and halogenated C1-4alkylene-C5.10aryl.
53. The method according to claim 52, wherein VIII and Ia are G and A respectively:
Figure imgf000081_0002
54. The method according to any one of the preceding claims further comprising the step of Nitrosylation (NOS), wherein a compound of formula IX is nitrosylated to give a compound of formula VIII:
Figure imgf000081_0003
IX VIII wherein: each -R9 is independently -H or -RA; and each -RA is independently selected from C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5-iOaryl; halogenated C5-i0aryl; Ci.4alkylene-C5.10aryl and halogenated C1-4alkylene-C5-ioaryl; and each of -R7NA and -R7NB is independently selected from: C1-4alkyl; C2-4alkenyl; halogenated C1-4alkyl; C5.10aryl; halogenated C5-i0aryl;
Figure imgf000082_0001
and halogenated C^alkylene-Cs-maryl.
55. The method according to claim 54, wherein IX and VIII are H and G respectively:
Figure imgf000082_0002
56. A process for the preparation of a pharmaceutical composition, the process comprising a method according to any one of the preceding claims and further comprising the step of admixing a diaminophenothiazinium compound, as defined in any one of claims 1 and 36 to 43, together with one or more other pharmaceutically acceptable ingredients.
PCT/GB2010/000927 2009-05-12 2010-05-11 Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants WO2010130977A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
DK10718268.5T DK2430007T3 (en) 2009-05-12 2010-05-11 Process for Chemical Synthesis of Diaminophenothiazinium Compounds Using Persulfate Oxidants
ES10718268.5T ES2457227T3 (en) 2009-05-12 2010-05-11 Chemical synthesis procedures of diaminophenothiazinium compounds that involve the use of persulfate oxidants
PL10718268T PL2430007T3 (en) 2009-05-12 2010-05-11 Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants
SI201030572T SI2430007T1 (en) 2009-05-12 2010-05-11 Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants
EP10718268.5A EP2430007B1 (en) 2009-05-12 2010-05-11 Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants
US13/318,907 US8691979B2 (en) 2009-05-12 2010-05-11 Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17744609P 2009-05-12 2009-05-12
US61/177,446 2009-05-12

Publications (1)

Publication Number Publication Date
WO2010130977A1 true WO2010130977A1 (en) 2010-11-18

Family

ID=42238536

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2010/000927 WO2010130977A1 (en) 2009-05-12 2010-05-11 Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants

Country Status (8)

Country Link
US (1) US8691979B2 (en)
EP (1) EP2430007B1 (en)
DK (1) DK2430007T3 (en)
ES (1) ES2457227T3 (en)
PL (1) PL2430007T3 (en)
PT (1) PT2430007E (en)
SI (1) SI2430007T1 (en)
WO (1) WO2010130977A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102634015A (en) * 2012-05-04 2012-08-15 中南大学 Synthesis method of poly(m-phenylenediamine) nanoparticles
WO2015052496A1 (en) * 2013-10-07 2015-04-16 Wista Laboratories Ltd. Methods of chemical synthesis of diaminophenothiazinium compounds including methylthioninium chloride (mtc)
EP3375777A1 (en) 2017-03-15 2018-09-19 MIKROCHEM spol. s.r.o. Method for preparation of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or bromide
US10968174B2 (en) 2016-12-21 2021-04-06 Wista Laboratories Ltd. Synthesis of a thiosulfonic acid by a step of periodate mediated oxidative coupling of a thiosulfonic acid with an aniline

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1886C (en) 1877-12-15 1877-12-15 Basf Ag Process for the preparation of blue dyes from dimethylaniline and other tertiary aromatic monamines
EP0510668A2 (en) 1991-04-26 1992-10-28 Takeda Chemical Industries, Ltd. Phenothiazine derivatives, their production and use
WO1996030766A1 (en) 1995-03-27 1996-10-03 F. Hoffmann-La Roche Ag Inhibition of tau-tau-association
WO2002055720A2 (en) 2001-01-15 2002-07-18 Univ Aberdeen Materials and methods relating to protein aggregation in neurodegenerative disease
WO2002075318A2 (en) 2001-03-20 2002-09-26 The University Court Of The University Of Aberdeen Neurofibrillary labels
WO2006032879A2 (en) 2004-09-23 2006-03-30 Wista Laboratories Ltd. Methods of chemical synthesis and purification of diaminophenothiazinium compounds including methylthioninium chloride (mtc)

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1886C (en) 1877-12-15 1877-12-15 Basf Ag Process for the preparation of blue dyes from dimethylaniline and other tertiary aromatic monamines
EP0510668A2 (en) 1991-04-26 1992-10-28 Takeda Chemical Industries, Ltd. Phenothiazine derivatives, their production and use
WO1996030766A1 (en) 1995-03-27 1996-10-03 F. Hoffmann-La Roche Ag Inhibition of tau-tau-association
WO2002055720A2 (en) 2001-01-15 2002-07-18 Univ Aberdeen Materials and methods relating to protein aggregation in neurodegenerative disease
WO2002075318A2 (en) 2001-03-20 2002-09-26 The University Court Of The University Of Aberdeen Neurofibrillary labels
WO2006032879A2 (en) 2004-09-23 2006-03-30 Wista Laboratories Ltd. Methods of chemical synthesis and purification of diaminophenothiazinium compounds including methylthioninium chloride (mtc)

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
BERNTHSEN: "Studien in der Methylenblaugruppe", JUSTUS LIEBIG'S ANNALEN DER CHEMIE, vol. 230, 18 August 1985 (1985-08-18), pages 137 - 211
BERNTHSEN: "Studien in der Methylenblaugruppe", JUSTUS LIEBIG'S ANNALEN DER CHEMIE, vol. 230, 18 August 1985 (1985-08-18), pages 73 - 136
BERNTHSEN: "Studien in der Methylenblaugruppe", JUSTUS LIEBIG'S ANNALEN DER CHEMIE, vol. 251, 18 August 1989 (1989-08-18), pages 1 - 96
FLAMENT S.; DELACOURTE A.; MANN D.M.A.: "Phosphorylation of tau proteins: a major event during the process of neurofibrillary degeneration. A comparative study between AD and Down's syndrome", BRAIN RES., vol. 516, 1990, pages 15 - 19, XP024267520, DOI: doi:10.1016/0006-8993(90)90891-E
HARRINGTON, C.R.; PERRY, R.H.; PERRY, E.K.; HURT, J.; MCKEITH, I.G.; ROTH, M.; WISCHIK, C.M.: "Senile dementia of Lewy body type and Alzheimer type are biochemically distinct in terms of paired helical filaments and hyperphosphorylated tau protein", DEMENTIA, vol. 5, 1994, pages 215 - 228
HOF P.R.; BOURAS C.; BUEE L.; DELACOURTE A.; PERL D.P.; MORRISON J.H.: "Differential distribution of neurofibrillary tangles in the cerebral cortex of dementia pugilistica and Alzheimer's disease cases", ACTA NEUROPATHOL., vol. 85, 1992, pages 23 - 30
HOF P.R.; CHARPIOT, A.; DELACOURTE A.; BUEE, L.; PUROHIT, D.; PERL D.P.; BOURAS, C.: "Distribution of neurofibrillary tangles and senile plaques in the cerebral cortex in postencephalitic parkinsonism", NEUROSCI. LETT., vol. 139, 1992, pages 10 - 14, XP024363562, DOI: doi:10.1016/0304-3940(92)90846-Y
IKEDA K.; AKIYAMA H.; KONDO H.; ARAI T.; ARAI N.; YAGISHITA S.: "Numerous glial fibrillary tangles in oligodendroglia in cases of subacute sclerosing panencephalitis with neurofibrillary tangles", NEUROSCI. LETT., vol. 194, 1995, pages 133 - 135
LEVENTIS,N. ET AL.: "Synthesis of Substituted Phenothiazines Analogous to Methylene Blue by Electrophilic and Nucleophilic Aromatic Substitutions in Tandem. A Mechanistic Perspective", TETRAHEDRON, vol. 53, no. 29, 1997, pages 10083 - 10092, XP004106125, DOI: doi:10.1016/S0040-4020(97)00349-9
LILLIE, R.D. ET AL.: "Zinc Chloride Methylene Blue, . Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples", STAIN TECHNOLOQY, vol. 54, no. 1, 1979, pages 33 - 39
LING LI; WILLIAM D JONES: "Mechanistic investigation of the cobalt-catalysed selective conversion of diallylanilines to quinolines involving C-N and C-H activations", J AM CHEM SOC, vol. 129, 2007, pages 10707 - 10713, XP055015929, DOI: doi:10.1021/ja070551q
LOHR, W.; GRUBHOFFER, N.; SOHMER, I.; WITTEKIND,D.: "The azure dyes: their purification and physiochemical properties. Purification of Azure B", STAIN TECHNOLOQY, vol. 50, no. 3, 1975, pages 149 - 156, XP009175680, DOI: doi:10.3109/10520297509117051
MARSHALL, P.N.; LEWIS, S.M.: "Metal contaminants in commercial dyes", STAIN TECHNOLOGY, vol. 50, no. 3, 1975, pages 143 - 147
MARSHALL, P.N.; LEWIS, S.M.: "The purification of Methylene Blue and Azure B by solvent extraction and crystallisation", STAIN TECHNOLOQV, vol. 50, no. 6, 1975, pages 375 - 381, XP009176225, DOI: doi:10.3109/10520297509117095
YI-CHUN HSU; KIM-HONG GAN; SHYH-CHYUN YANG: "Palladium-catalysed allylation of acidic and less nucleophilic anilines using allylic alcohols directly", CHEM PHARM BULL, vol. 53, no. 10, 2005, pages 1266 - 1269

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102634015A (en) * 2012-05-04 2012-08-15 中南大学 Synthesis method of poly(m-phenylenediamine) nanoparticles
CN102634015B (en) * 2012-05-04 2013-06-19 中南大学 Synthesis method of poly(m-phenylenediamine) nanoparticles
US10047062B2 (en) 2013-10-07 2018-08-14 Wista Laboratories Ltd. Methods of chemical synthesis of Diaminophenothiazinium compounds including methylthioninium chloride (MTC)
CN105683171A (en) * 2013-10-07 2016-06-15 维斯塔实验室有限公司 Methods of chemical synthesis of diaminophenothiazinium compounds including methylthioninium chloride (MTC)
JP2016531897A (en) * 2013-10-07 2016-10-13 ウィスタ ラボラトリーズ リミテッド Method for chemical synthesis of diaminophenothiazinium compounds containing methylthioninium chloride (MTC)
US9765042B2 (en) 2013-10-07 2017-09-19 Wista Laboratories Ltd. Methods of chemical synthesis of diaminophenothiazinium compounds including methylthioninium chloride (MTC)
WO2015052496A1 (en) * 2013-10-07 2015-04-16 Wista Laboratories Ltd. Methods of chemical synthesis of diaminophenothiazinium compounds including methylthioninium chloride (mtc)
CN110092764A (en) * 2013-10-07 2019-08-06 维斯塔实验室有限公司 Diaminophenothiazine compound includes the chemical synthesis process of methyl sulfonium chloride (MTC)
US10421733B2 (en) 2013-10-07 2019-09-24 Wista Laboratories Ltd. Methods of chemical synthesis of diaminophenothiazinium compounds including methylthioninium chloride (MTC)
CN110092764B (en) * 2013-10-07 2023-09-15 维斯塔实验室有限公司 Chemical synthesis method of diaminophenothiazinium compound including methyl sulfonium chloride (MTC)
US10968174B2 (en) 2016-12-21 2021-04-06 Wista Laboratories Ltd. Synthesis of a thiosulfonic acid by a step of periodate mediated oxidative coupling of a thiosulfonic acid with an aniline
EP3375777A1 (en) 2017-03-15 2018-09-19 MIKROCHEM spol. s.r.o. Method for preparation of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or bromide
WO2018167185A1 (en) 2017-03-15 2018-09-20 Mikrochem Spol.S R.O. Method for preparation of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or bromide

Also Published As

Publication number Publication date
PL2430007T3 (en) 2014-07-31
DK2430007T3 (en) 2014-05-12
EP2430007A1 (en) 2012-03-21
PT2430007E (en) 2014-04-14
SI2430007T1 (en) 2014-05-30
ES2457227T3 (en) 2014-04-25
US20120058995A1 (en) 2012-03-08
US8691979B2 (en) 2014-04-08
EP2430007B1 (en) 2014-02-12

Similar Documents

Publication Publication Date Title
AU2005286235C1 (en) Methods of chemical synthesis and purification of diaminophenothiazinium compounds including Methylthioninium Chloride (MTC)
DK2457905T3 (en) Methods for the synthesis and / or purification of diaminophenothiaziniumforbindelser
AU2009326173B2 (en) 3,6-disubstituted xanthylium salts as medicaments
AU2019203490B2 (en) Methods of Chemical Synthesis of Diaminophenothiazinium Compounds including Methylthioninium Chloride (MTC)
EP2430007B1 (en) Methods of chemical synthesis of diaminophenothiazinium compounds involving the use of persulfate oxidants
WO2020250186A1 (en) Novel improved method for synthesizing diaminophenothiazine compounds
EP3795565A1 (en) Improved process for the purification of methylene blue
AU2014280990A1 (en) 3,6-disubstituted xanthylium salts as medicaments

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10718268

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 13318907

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2010718268

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE