DAMAGE

This invention relates to compounds containing a catechol core. The invention further relates to said compounds for use as a medicament, for use in the prevention of DNA damage, and/or for use in the treatment or prevention of a disease associated with iron induced oxidative stress. The invention also relates to formulations comprising said compounds. Further, the invention relates to uses of said formulation, as well as methods of preventing DNA damage in the subject and/or methods of treating or preventing a disease associated with iron induced oxidative stress. Finally, the invention relates to the use of said compounds in the manufacture of a medicament for preventing DNA damage in the subject, and/or treating or preventing a disease associated with iron induced oxidative stress.

BACKGROUND

Prolonged exposure to ultraviolet (UV), such as from the sun, can increase a person’s risk of developing skin cancers, and accelerate the appearance of signs of skin aging, such as loss of skin elasticity, wrinkling and hyperpigmentation.

Ultraviolet radiation can be divided into three bands depending on wavelength: UVA, UVB, and UVC. UVA radiation is present in the sunlight reaching the earth's surface and has a wavelength of 320 to 400 nm. UVB radiation is present in the sunlight reaching the earth's surface and has a wavelength of 290 to 320 nm.

The main reason why UV, especially UVA, has such an undesirable effect on skin is because it results in the formation of reactive oxygen species (ROS) in the cells. ROS are a group of short-lived, highly reactive, oxygen-containing molecules that when present in excess, lead to oxidative stress, and ultimately cause damage to various cellular components, including DNA, mitochondrial DNA (mtDNA), RNA, proteins and lipids.

Exposure to UVA immediately causes free iron to act as a catalyst in the production of ROS. As free iron concentrations are especially high inside the mitochondrial matrix, the mitochondria are highly susceptible to oxidative stress caused by UVA exposure, which can result in mtDNA damage.

Tiron (4, 5-dihydroxy- 1 ,3-benzenedisulfonate) is a compound that acts as an iron chelator and is capable of penetrating the mitochondria where it can bind the free iron, preventing it from reacting upon exposure to UVA rays. The present invention aims to provide compounds that prevent DNA damage and/or mtDNA damage, e.g. compounds that are more active than Tiron in preventing mtDNA. These compounds may be iron chelators. These compounds may be useful in the context of a sunscreen. However, they may also be useful in preventing DNA damage in general, as well as treating and preventing diseases associated with iron induced oxidative stress. Such diseases include skin cancer and pre-cancer, as well as diseases where there is an iron overload, which may lead to increased iron induced oxidative stress and oxidative damage.

SUMMARY OF THE INVENTION

The present disclosure is based on the inventors’ development of compounds that may be free iron chelators, and may be particularly effective mitochondrial free iron chelators.

Free iron can be highly undesirable due to its ability to catalyse the formation of ROS which can cause oxidative damage in the form of DNA damage. Mitochondrial free iron can catalyse the formation of ROS in the mitochondria which can cause oxidative damage in the form of mtDNA damage. DNA and/or mtDNA damage can lead to cell death or contribute to the development of diseases such as skin cancer. UVA, the component of sunlight that initiates oxidation, is one of the main environmental factors the exposure to which results in oxidative damage due to the Fenton reaction. In this reaction, free iron acts as a catalyst to convert hydrogen peroxide, a product of mitochondrial oxidative respiration, into a highly toxic hydroxyl free radical.

Iron chelators are compounds that bind to the free iron and prevent it from acting as a catalyst in the formation of ROS. Mitochondrial iron chelators are compounds that may specifically bind to free iron in the mitochondria and prevent the formation of ROS inside these organelles.

Exposure to UVA is not the only cause for iron-induced oxidative damage. There are a number of diseases, where iron levels are simply too high, which can also lead to such oxidative damage. Such diseases may be referred to as diseases associated with iron induced oxidative stress. Diseases where mitochondrial iron levels are too high may be referred to as diseases associated with mitochondrial iron induced oxidative stress. An example of a disease associated with mitochondrial iron induced oxidative stress is Friedreich’s ataxia (FA). FA is caused by deficient expression of frataxin that leads to deleterious alterations in iron metabolism. These alterations lead to the accumulation of inorganic iron aggregates in the mitochondria that appear to play a key role in oxidative damage and subsequent degenerative features of this disease. Accordingly, targeted removal of cytosolic and/or mitochondrial free iron may be desirable for a number of reasons. Not only may this be an effective approach to protect cells against the harmful effects of UV, it may also be an effective approach to treating and preventing diseases associated with iron induced oxidative stress, such as diseases associated with mitochondrial iron induced oxidative stress .

The inventors have found that the compounds of the invention may prevent mtDNA and/or DNA damage. Certain compounds of the invention are more active than Tiron and can, for example, prevent mtDNA damage at a much lower concentration than Tiron. For example, one of the compounds of the invention can prevent mtDNA damage at a 60-fold lower concentration than Tiron. This ability to achieve a similar or improved effect at a lower concentration may be beneficial for a number of reasons. For example, it may result in reduced cell toxicity, greater freedom in formulation development, and reduction in manufacturing costs.

Accordingly, in a first aspect, is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof,

R ¹ and R ² are each independently selected from H, Ci-Cvalkyl and a prodrug moiety;

R ³ is independently at each occurrence selected from halo, OR ⁶ , NR ⁶ R ⁷ , C(0)0R ⁶ , C(0)NR ⁶ R ⁶ , S(0) ₂ 0R ⁶ , S(0) ₂ NR ⁶ R ⁶ , Ci-C ₄ -alkyl, C2-Cvalkenyl, C^Cvalkynyl, cyano and nitro;

R ⁶ is independently at each occurrence selected from H and Ci-Cvalkyl;

R ⁷ is independently at each occurrence selected from H, Ci-Cvalkyl, C(0)-Ci-C ₄ -alkyl and S(O)2-Ci-Cvalkyl;

-L ¹ - is absent or is a linker group;

-R ^A - is selected from an amino acid, a targeting group, a UV absorbent group, an antioxidant group, a fluorescent group; wherein R ^A is optionally substituted with groups that improve formulation properties and/or passage through the outer layer of dermis.

Where present, the prodrug moiety may be selected from an ester (e.g. a C(0)-C1-C ₄ -alkyl group) or amino acid (e.g. L-dopa). The linker group may comprise heteroatom linker groups (e.g. ether, amine, amide, ester, thioether) and/or alkyl chains (e.g. Ci-C8-alkylchains). The linker group may comprise an amino acid. The linker group may comprise a photocleavable group.

Where present, the targeting group will typically be a group that can target mitochondria, e.g. a peptide. An illustrative example of a peptide that targets mitochondria is elamipretide.

Where present, the UV absorbent group may be UVA absorbent or it may be UVB absorbent or it may be both. An illustrative example of a UV absorbent group is a cinnamic acid, ester or amide.

An illustrative example of suitable antioxidants include retinol, idebenone, Trolox and KH-176.

An illustrative example of a group that can improve formulation properties and/or passage through the outer layer of dermis is a polyethylene glycol (PEG) group. This might, for example attach to the carboxylic acid group of cinnamic acid.

The compound of formula (I) may be a compound of formula (II):

-X ¹ - is independently selected from -0-, -S- and -NR ⁵ -;

R ¹ and R ² are each independently selected from H, Ci-Cvalkyl and a prodrug moiety;

R ³ is independently at each occurrence selected from halo, OR ⁶ , NR ⁶ R ⁷ , C(0)0R ⁶ , C(0)NR ⁶ R ⁶ , S(0) ₂ 0R ⁶ , S(0) ₂ NR ⁶ R ⁶ , Ci-C ₄ -alkyl, CrC^alkenyl, CrC^alkynyl, cyano and nitro;

-L ² - is independently selected from -CHR ⁶ -, -L ³ -[C(R ⁹ )=C(R ⁹ )] _X - and -C^Cvalkylene-X ² -;

-L ³ - is independently selected from -CR ¹⁰ R ¹⁰ - and

-X ² - is independently selected from -O- and -NR ⁵ -; R ⁴ is independently selected from C(0)R ¹¹ , C3-C8-cycloalkyl, C6-C8-cycloalkenyl, phenyl and napthyl;

R ^s and R ⁶ are each independently at each occurrence selected from H and Ci-Cvalkyl;

R ⁷ is independently at each occurrence selected from H, Ci-Cvalkyl, C(0)-Ci-C4-alkyl and S(0)2-Ci-C ₄ -alkyl;

R ⁸ is independently selected from H, Ci-C8-alkyl and Ci-C3-alkylene-R ¹² ; or R ⁸ and R ⁵ together with the nitrogen to which they are attached form a 3- to 6-membered heterocycloalkyl group; R ⁹ and R ¹⁰ are each independently at each occurrence selected from H and Ci-Cvalkyl;

R ¹¹ is independently selected from OR ⁸ , NR ⁸ R ⁸ and 0-(C2-C4-alkylene-0)z-H

R ¹² is independently at each occurrence selected from phenyl, imidazole, indole, SR ⁸ , OR ⁸ ,

CO2R ⁸ , C0 ₂ NR ⁸ R ⁸ , NR ⁸ R ⁷ and NH(=NH)NH ₂ ; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0, 1, 2, 3 and 4; x is an integer selected from 0, 1, 2, 3, 4, 5 and 6; z is an integer from 1 to 500; wherein any of the aforementioned alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, phenyl, naphthyl and heteroaryl groups is optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from the group consisting of oxo, halo, nitro, cyano, NR ^a R ^a , NR ^a S(0) ₂ R ^a , NR ^a C(0)R ^a , NR ^a CONR ^a R ^a , NR ^a C0 ₂ R ^a , OR ⁸ , SR ^a , SOR ^a , S0 ₃ R ^a , S0 ₂ R ^a , S0 ₂ NR ^a R ^a , C0 ₂ R ^a , C(0)R ^a , CONR ^a R ^a , Ci-Cvalkyl, C2Cvalkenyl, C^Cvalkynyl; wherein R ^a is independently at each occurrence selected from H, Ci-Cvalkyl.

The compound may be a compound of formula (lla), or a pharmaceutically acceptable salt thereof:

-X ¹ - is independently selected from -0-, -S-, -NR ⁵ - and -NR ⁵ -(CHR ⁸ -C(0)NR ⁵ )q-;

R ¹ and R ² are each independently selected from H and a prodrug moiety;

R ³ is independently at each occurrence selected from halo, OR ⁸ , NR ⁸ R ⁷ , C(0)OR ⁸ , C(0)NR ⁸ R ⁸ , S(0) ₂ 0R ⁸ , S(0) ₂ NR ⁸ R ⁸ , Ci-C ₄ -alkyl, C2-C ₄ -alkenyl, C2-C ₄ -alkynyl, cyano and nitro;

-L ² - is independently selected from -CHR ⁸ -, -L ³ -[C(R ⁹ )=C(R ⁹ )] _x -and -CrC^alkylene-X ² -;

-L ³ - is independently selected from -CR ¹⁰ R ¹⁰ - and

-X ² - is independently selected from -O- and -NR ⁵ -;

R ⁴ is independently selected from C(0)R ¹¹ , C3-C8-cycloalkyl, C8-C8-cycloal kenyl , phenyl and napthyl;

R ^s and R ⁶ are each independently at each occurrence selected from H and Ci-Cvalkyl;

R ⁷ is independently at each occurrence selected from H, Ci-Cvalkyl, C(0)-Ci-C ₄ -alkyl and S(0) ₂ -Ci-C ₄ -alkyl;

R ⁸ is independently at each occurrence selected from H, Ci-C6-alkyl, Ci-C ₂ 4-alkylene-R ¹² ; or R ⁸ and R ⁵ together with the nitrogen to which they are attached form a 3- to 6-membered heterocycloalkyl group;

R ⁹ and R ¹⁰ are each independently at each occurrence selected from H and Ci-C4-alkyl;

R ¹¹ is independently selected from OR ⁸ , NR ⁸ R ⁸ and 0-(C ₂ -C ₄ -alkylene-0)z-H

R ¹² is independently at each occurrence selected from phenyl, imidazole, indole, SR ⁸ , OR ⁸ ,

C0 ₂ R ⁸ , C0 ₂ NR ⁸ R ⁸ , NR ⁸ R ⁷ , [NR ⁸ R ⁸ R ⁸ ] ⁺ , [P(R ¹⁵ )3] ⁺ , C(=NH)NH ₂ , and NH(=NH)NH ₂ ;

R ¹⁵ may be independently selected at each occurrence from Ci-C8-alkyl, Cs-C8-cycloalkyl, 3- to 8-membered heterocycloalkyl, phenyl, or 5- or 6-membered heteroaryl; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0, 1, 2, 3 and 4; x is an integer selected from 0, 1, 2, 3, 4, 5 and 6; z is an integer from 1 to 500; q is an integer selected from 1, 2, 3, 4 and 5; wherein any of the aforementioned alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, phenyl, naphthyl and heteroaryl groups is optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from the group consisting of oxo, halo, nitro, cyano, NR ^a R ^a , NR ^a S(0) ₂ R ^a , NR ^a C(0)R ^a , NR ^a CONR ^a R ^a , NR ^a C0 ₂ R ^a , OR ⁸ , SR ^a , SOR ^a , S0 ₃ R ^a , S0 ₂ R ^a , S0 ₂ NR ^a R ^a , C0 ₂ R ^a , C(0)R ^a , CONR ^a R ^a , Ci-Cvalkyl, C^Cvalkenyl, C^Cvalkynyl; wherein R ^a is independently at each occurrence selected from H, Ci-Cvalkyl.

In certain embodiments, the compound of formula (I) is a compound of formula (III): wherein R ³ , n, X ¹ , L ² and R ⁴ are as defined above for formula (II) or formula (I la).

In certain embodiments, the compound of formula (I) is a compound of formula (IV): wherein R ¹ , R ² , L ² and R ⁴ are as defined above for formula (II) or formula (I la).

In certain embodiments, the compound of formula (I) is a compound of formula (V): wherein L ² and R ⁴ are as defined above for formula (II) or formula (I la).

In certain embodiments, the compound of formula (I) is a compound of formula (VI): wherein R ¹ , R ² , R ³ , n, R ⁵ , m, R ⁹ , and R ¹¹ are as defined above for formula (II) or formula (I la).

In certain embodiments, the compound of formula (I) is a compound of formula (VII): wherein R ¹ , R ² , R ⁹ and R ¹¹ are as defined above for formula (II) or formula (lla).

In certain embodiments, the compound of formula (I) is a compound of formula (VIII): wherein R ⁹ and R ¹¹ are as defined above for formula (II) or formula (lla).

In certain embodiments, the compound of formula (I) is a compound of formula (IX): wherein R ¹ , R ² and R ¹¹ are as defined above for formula (II) or formula (lla).

In certain embodiments, the compound of formula (I) is a compound of formula (X): wherein R ¹¹ is as defined above for formula (II) or formula (I la). In certain embodiments, the compound of formula (I) is a compound of formula (XI): wherein R ¹ , R ² , R ³ , n and R ¹¹ are as defined above for formula (II) or formula (lla).

In certain embodiments, the compound of formula (I) is a compound of formula (XII): wherein R ³ , n and R ¹¹ are as defined above for formula (II) or formula (lla).

This disclosure also includes compound of formula (XIII): pharmaceutically acceptable salt thereof; wherein R ¹ , R ² , R ³ and n are as described above for compounds of formula (II) or formula (lla).

The following statements apply to compounds of any of formulae (I) to (XIII). These statements are independent and interchangeable. In other words, any of the features described in any one of the following statements may (where chemically allowable) be combined with the features described in one or more other statements below. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the statements below which describe a feature of that compound, expressed at any level of generality, may be combined so as to represent subject matter which is contemplated as forming part of the disclosure of this invention in this specification.

X ¹ may be O. X ¹ may be NR ⁵ , e.g. NH. X ¹ may be NR ⁵ (CHR ⁸ -C(0)NR ⁵ ) _q , e.g. NH(CHR ^e -C(0)NH) _q . In these embodiments, each R ⁸ of X ¹ may be the side chain of a naturally occurring proteinogenic amino add. Alternatively, R ⁸ may be selected from: H, Ci-C6-alkyl, Ci-^-alkylene-R ¹² and benzyl. R ⁸ may be benzyl. R ⁸ may be Ci-ie-alkylene-R ¹² . R ⁸ may be Ci-io-alkylene-R ¹² . R ⁸ may be C2-C8- alkylene-R ¹² . R ⁸ may be CrCe- alkylene-R ¹² . R ⁸ may be C^alkylene-R ¹² . R ⁸ may be C3-alkylene-R ¹² . R ⁸ may be Cvalkylene-R ¹² . In these embodiments, R ¹² is typically selected from NR ⁸ R ⁷ , [NR ⁸ R ⁸ R ⁸ ]*, [P(R ¹⁵ ) ₃ ] ⁺ , C(=NH)NH _2, and NH(=NH)NH ₂ .

R ¹⁵ may be independently selected at each occurrence from Ci-C8-alkyl, C3-C8-cydoalkyl, 3- to 8-membered heterocydoalkyl, phenyl, or 5- or 6-membered heteroaryl. R ¹⁵ may be phenyl, or 5- or 6-membered heteroaryl. Preferably, R ¹⁵ is phenyl.

R ¹ may be H. R ¹ may be Ci-Cvalkyl, e.g. Me. R ² may be H. R ² may be Ci-Cvalkyl, e.g. Me. R ¹ and R ² may both be Ci-Cvalkyl, e.g. Me. Preferably, R ¹ and R ² are both H. Compounds in which R ¹ and R ² are both H are effective iron-chelators and typically provide improved activity relative to compounds in which R ¹ and R ² are alkyl. n may be 0. n may be 1. If present on the phenyl ring to which OR ¹ and OR ² is attached, R ³ may be selected from halo and Ci-Cvalkyl.

-L ² - may be -CHR ⁸ -. R ⁸ may be the side chain of a naturally occurring proteinogenic amino add. R ⁸ may be selected from: H, Ci-C8-alkyl and benzyl. R ⁸ may be Ci-Ce-alkyl. R ⁸ may be C3-Cvalkyl. R ⁸ may be selected from H, methyl, isopropyl, isobutyl and benzyl. R ⁸ may be selected from methyl, isopropyl and isobutyl. In these embodiments, R ⁴ is typically C(0)R ¹¹ . R ¹¹ may be OR ⁸ , e.g. O-Ci-Cvalkyl.

-L ² -R ⁴ may be:

The stereochemical orientation at the carbon to which R ⁸ is attached may be that of the L- amino add. Alternatively, the stereochemical orientation at the carbon to which R ⁸ is attached may be that of the D-amino add. In a further alternative, there may be a mixture of L- and R- present.

-L ² - may be -L ³ -[C(R ⁹ )=C(R ⁹ )] ₃ r. -L ³ - may -L ³ - may be . m may be 0. m may be 1. If present on -L ³ -, R ³ may be selected from halo and Ci-Cvalkyl. In these embodiments, x may be 1. It may be that R ⁹ is at each occurrence H. In these embodiments, In these embodiments, R ⁴ is typically C(0)R ¹¹ . R ¹¹ may be OR ⁶ , e.g. O-Ci-Cvalkyl.

R ¹¹ may be OR ⁶ , e.g. O-Ci-Cvalkyl. R ¹¹ may be NR ⁶ R ⁶ , e.g. NHCi-Cvalkyl. R ¹¹ may be O- (C2-C4-alkylene-0)z-H. z may be an integer from 50-300.

-L ² - may be -CR ¹⁰ R ¹⁰ -. It may be that R ¹⁰ is independently at each occurrence H. In these embodiments, it may be that x is an integer selected from 2, 3, 4 and 5. It may be that x is 4. R ⁹ may be independently selected from H and Me. In these embodiments, R ⁴ may be C8-C8- cycloalkenyl. R ⁴ may be cyclohexenyl which may be optionally substituted with from 1 to 4 methyl groups. It may be that -L ² - and R ⁴ are selected such that HO-L ² -R ⁴ is retinol.

, wherein y is an integer selected from 1, 2, 3 and 4 and; R ¹³ is independently at each occurrence Ci-Cvalkyl. y may be 3. -L ² - may be -C^Cvalkylene-X ² -. -L ² -may be -CH2CH2-X ² -. -X ² - may be -0-. -X ² - may be - NR ⁵ -, e.g. -NH-. In these embodiments, R ⁴ may be selected from naphthyl or phenyl. R ⁴ may be naphthyl, e.g. 2-naphthyl. wherein z is an integer selected from 1, 2 and 3; and R ¹⁴ is independently at each occurrence selected from: halo, nitro, cyano, NR ^a R ^a , NR ^a S(0)2R ^a , NR ^a C(0)R ^a , NR ^a CONR ^a R ^a , NR ^a C0 ₂ R ^a , OR ^a , SR ^a , SOR ^a , S0 ₃ R ^a , S0 ₂ R ^a , S0 ₂ NR ^a R ^a , C0 ₂ R ^a , C(0)R ^a , CONR ^a R ^a , C1-Cvalkyl, C2-Cvalkenyl, C2-Cvalkynyl. R ¹⁴ may be at each occurrence SO3H. q may be an integer selected from 1, 2, and 3. q may be an integer selected from 1 and 2. Typically, q is 1.

The compounds of the invention may be iron chelators. In some examples, the compounds of the invention may be mitochondrial iron chelators. Such compounds may be selected from the group consisting of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, and compound 7.

In a further aspect, the present invention provides a compound for use as a medicament, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof.

In a further aspect, the present invention provides a compound for use in the prevention of DNA damage, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof. In certain embodiments, the DNA is mtDNA. This embodiment gives rise to a further aspect of the invention. Specifically, the present invention provides a compound of the invention for use in the prevention of mtDNA damage.

In a further aspect, the present invention provides a compound for use in the treatment or prevention of a disease associated with iron induced oxidative stress, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof.

In certain embodiments, the iron is mitochondrial iron. This embodiment gives rise to a further aspect of the invention. Specifically, the present invention provides a compound for use in the treatment or prevention of a disease associated with mitochondrial iron induced oxidative stress, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof.

In certain embodiments, the treatment or prevention of a disease associated with iron induced oxidative stress and/or a disease associated with mitochondrial iron induced oxidative stress is by preventing DNA damage. In certain embodiments, the DNA is mtDNA.

In certain embodiments, the disease associated with iron induced oxidative stress is a disease associated with mitochondrial iron induced oxidative stress selected from the group consisting of a cancer, a pre-cancer or a mitochondrial iron overload disease.

In certain embodiments, the disease associated with mitochondrial iron induced oxidative stress is selected from the group consisting of a cancer, a pre-cancer or a mitochondrial iron overload disease.

In certain embodiments, the cancer or pre-cancer is a skin cancer or skin pre-cancer, respectively.

In certain embodiments, the skin cancer is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and melanoma.

In certain embodiments, the mitochondrial iron overload disease is selected from the group consisting of Friedreich's ataxia, sideroblastic anaemia, erythropoietic protoporphyria, X- linked protoporphyria, cardiomyopathy (TTP), pulmonary hypertension (NFU1), Alzheimer's disease (AD), chronic obstructive pulmonary disease (CORD), Parkinson's disease (PD), Huntington's disease (HD), type 2 Wolfram syndrome, hereditary myopathy with lactic acidosis and adolescent onset autosomal recessive mitochondrial myopathy.

In a further aspect, the present invention provides a formulation comprising a compound of formula (I).

In certain embodiments, the formulation is for topical application.

In certain embodiments, the formulation is a cream, lotion, oil, gel, balm, liquid, aerosolizable liquid, or a foam.

In certain embodiments the formulation further comprises a photoprotective compound and/or an antioxidant compound.

In a further aspect, the present invention provides use of a formulation of the invention for protecting a subject from UV, and/or improving or restoring a subject’s appearance.

In certain embodiments, the UV is UVA, and optionally UVB.

In certain embodiments, protecting is by preventing DMA damage. In certain embodiments, DMA is mtDNA.

In certain embodiments, the subject’s appearance is improved or restored by reducing or preventing skin hyperpigmentation, reducing or preventing skin wrinkles, and/or increasing skin elasticity or preventing loss of skin elasticity.

In a further aspect, the present invention provides a method of preventing DMA damage in a subject, the method comprising providing the subject with a therapeutically effective amount of a compound, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof .

In a further aspect, the present invention provides a method of preventing or treating a disease associated with iron induced oxidative stress in a subject, the method comprising providing the subject with a therapeutically effective amount of a compound, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof. In a further aspect, the invention provides the use of a compound in the manufacture of a medicament for preventing DNA damage in the subject, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof.

In a further aspect, the invention provides the use of a compound in the manufacture of a medicament for treating or preventing a disease associated with iron induced oxidative stress, said compound being selected from a compound of formula (I), a compound of formula (XIII) or a pharmaceutically acceptable salt thereof.

It will be recognised that, except where the context requires otherwise, embodiments described in respect of one aspect of the invention will also be applicable to all of the aspects of the invention. Thus, for example, embodiments mentioned in the context of the compounds for use as described herein are also applicable to the methods described herein.

Various aspects and embodiments of the invention are described in further detail below.

The present application and invention further includes the subject-matter of the following numbered paragraphs:

1. A compound of formula (II), or a pharmaceutically acceptable salt thereof:

-X ¹ - is independently selected from -0-, -S- and -NR ⁵ -;

R ¹ and R ² are each independently selected from H, Ci-Cvalkyl and a prodrug moiety;

R ³ is independently at each occurrence selected from halo, OR ⁶ , NR ^S R ⁷ , C(0)0R ⁶ , C(0)NR ⁶ R ⁶ , S(0) ₂ 0R ⁶ , S(0) ₂ NR ⁶ R ⁶ , Ci-C ₄ -alkyl, C2-Cvalkenyl, C^Cvalkynyl, cyano and nitro;

-L ² - is independently selected from -CHR ⁶ -, -L ³ -[C(R ⁹ )=C(R ⁹ )] _X - and -C^Cvalkylene-X ² -;

-L ³ - is independently selected from -CR ¹⁰ R ¹⁰ - and

-X ² - is independently selected from -O- and -NR ⁵ -;

R ⁴ is independently selected from C(0)R ¹¹ , C3-C8-cycloalkyl, C6-C8-cycloalkenyl, phenyl and napthyl;

R ⁵ and R ⁶ are each independently at each occurrence selected from H and Ci-Cvalkyl;

R ⁷ is independently at each occurrence selected from H, Ci-Cvalkyl, C(0)-Ci-C ₄ -alkyl and S(0) ₂ -Ci-C ₄ -alkyl;

R ⁸ is independently selected from H, Ci-C8-alkyl and Ci-C3-alkylene-R ¹² ; or R ⁸ and R ⁵ together with the nitrogen to which they are attached form a 3- to 6-membered heterocycloalkyl group; R ⁹ and R ¹⁰ are each independently at each occurrence selected from H and Ci-C4-alkyl;

R ¹¹ is independently selected from OR ⁸ , NR ⁸ R ⁸ and 0-(C ₂ -C ₄ -alkylene-0)z-H

R ¹² is independently at each occurrence selected from phenyl, imidazole, indole, SR ⁸ , OR ⁸ ,

C0 ₂ R ⁸ , C0 ₂ NR ⁸ R ⁸ , NR ⁸ R ⁷ and NH(=NH)NH ₂ ; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0, 1, 2, 3 and 4; x is an integer selected from 0, 1, 2, 3, 4, 5 and 6; z is an integer from 1 to 500; wherein any of the aforementioned alkyl, alkylene, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, phenyl, naphthyl and heteroaryl groups is optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from the group consisting of oxo, halo, nitro, cyano, NR ^a R ^a , NR ^a S(0) ₂ R ^a , NR ^a C(0)R ^a , NR ^a CONR ^a R ^a , NR ^a C0 ₂ R ^a , OR ⁸ , SR ^a , SOR ^a , S0 ₃ R ^a , S0 ₂ R ^a , S0 ₂ NR ^a R ^a , C0 ₂ R ^a , C(0)R ^a , CONR ^a R ^a , Ci-Cvalkyl, C^Cvalkenyl, C^Cvalkynyl; wherein R ^a is independently at each occurrence selected from H, Ci-Cvalkyl.

2. A compound of paragraph 1, wherein X ¹ is O.

3. A compound of paragraph 1, wherein X ¹ is NH.

4. A compound of any one of paragraphs 1 to 3, wherein R ¹ and R ² are both H.

5. A compound of any one of paragraphs 1 to 3, wherein R ¹ and R ² are both Ci-C4-alkyl. 6. A compound of any one of paragraphs 1 to 3, wherein n is 0.

7. A compound of any one of paragraphs 1 to 6, wherein -L ¹ -R ⁴ is:

8. A compound of any one of paragraphs 1 to 6, wherein -L ¹ -R ⁴ is:

9. A compound of paragraph 8, wherein -L ¹ -R ⁴ is:

10. A compound of paragraph 8 or paragraph 9, wherein m is 0.

11. A compound of any one of paragraphs 8 to 10, wherein R ⁹ is at each occurence H.

12. A compound of any one of paragraphs 8 to 11 , wherein R ¹¹ is O-Ci-Cvalkyl.

13. A compound of any one of paragraphs 1 to 6, wherein -L ¹ -R ⁴ is wherein y is an integer selected from 1, 2, 3 and 4 and; R ¹³ is independently at each occurrence Ci-Cvalkyl.

13. A compound of any one of paragraphs 1 to 6, wherein -L ¹ -R ⁴ is wherein z is an integer selected from 1, 2 and 3; and R ¹⁴ is independently at each occurrence elected from: halo, nitro, cyano, NR ^a R ^a , NR ^a S(0) ₂ R ^a , NR ^a C(0)R ^a , NR ^a CONR ^a R ^a , NR ^a C0 ₂ R ^a , OR ^a , SR ^a , SOR ^a , S0 ₃ R ^a , S0 ₂ R ^a , S0 ₂ NR ^a R ^a , C0 ₂ R ^a , C(0)R ^a , CONR ^a R ^a , Ci-Cvalkyl, C^Cvalkenyl, C2-C4-alkynyl. R ¹⁴ may be at each occurrence SO3H.

14. A compound of paragraph 1 , wherein the compound of formula (I) is selected from:

H ₃ CO.

HgCO and

HO.

HO ^'

15. A compound of paragraph 1 , wherein the compound of formula (I) is selected from:

16. A compound for use as a medicament, said compound being selected from a compound of any one of paragraphs 1 to 15, a compound of formula (XIII) and a pharmaceutically acceptable salt thereof.

17. A compound for use in the prevention of DNA damage, said compound being selected from a compound of any one of paragraphs 1 to 15, a compound of formula (XIII) and a pharmaceutically acceptable salt thereof.

18. The compound of paragraph 17 wherein the DNA is mitochondrial DNA.

19. A compound for use in the treatment or prevention of a disease associated with iron induced oxidative stress, said compound being selected from a compound of any one of paragraphs 1 to 15, a compound of formula (XIII) and a pharmaceutically acceptable salt thereof.

20. A method of preventing or treating a disease associated with iron induced oxidative stress in a subject, the method comprising providing the subject with a therapeutically effective amount of a compound, said compound being selected from a compound of any one of paragraphs 1 to 15, a compound of formula (XIII) and a pharmaceutically acceptable salt thereof.

21. The compound for use according to paragraph 19 or the method according to paragraph 20, wherein the disease associated with iron induced oxidative stress is a disease associated with mitochondrial iron induced oxidative stress. 22. The compound for use or the method according to paragraph 21, wherein the treatment or prevention of the disease associated with mitochondrial iron induced oxidative stress is by preventing mitochondrial DNA damage.

23. The compound for use or the method according to paragraph 21 or 22, wherein the disease associated with mitochondrial iron induced oxidative stress is selected from the group consisting of a cancer, a pre-cancer or a mitochondrial iron overload disease.

24. The compound for use or the method according to paragraph 23, wherein the cancer or pre-cancer is a skin cancer or skin pre-cancer.

25. The compound for use or the method according to paragraph 24, wherein the skin cancer is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and melanoma.

26. The compound for use or the method according to paragraph 23, wherein the mitochondrial iron overload disease is selected from the group consisting of Friedreich's ataxia, sideroblastic anaemia, erythropoietic protoporphyria, X-linked protoporphyria, cardiomyopathy (TTP), pulmonary hypertension (NFU1), Alzheimer's disease (AD), chronic obstructive pulmonary disease (CORD), Parkinson's disease (PD), Huntington's disease (HD), type 2 Wolfram syndrome, hereditary myopathy with lactic acidosis and adolescent onset autosomal recessive mitochondrial myopathy.

27. A formulation comprising a compound of any one of paragraphs 1 to 15.

28. The formulation of paragraph 27, wherein the formulation is for topical application.

29. The formulation of paragraph 28, wherein the formulation is a cream, lotion, oil, gel, balm, liquid, aerosolizable liquid, or a foam.

30. The formulation of any one of paragraphs 27 to 29, further comprising a photoprotective compound and/or an antioxidant compound.

31. Use of a formulation according to any one of paragraphs 27 to 30 for protecting a subject from UV, and/or improving or restoring a subject’s appearance.

32. The use according to paragraph 31 , wherein the UV is UVA, and optionally UVB. 33. The use according to any one of paragraphs 31 or 32, wherein protecting is by preventing DNA damage.

34. The use according to paragraph 33, wherein the DNA is mtDNA.

35. The use according to any one of paragraphs 31 to 34, wherein the subject’s appearance is improved or restored by reducing or preventing skin hyperpigmentation, reducing or preventing skin wrinkles, and/or increasing skin elasticity or preventing loss of skin elasticity.

35. A method of preventing DNA damage in a subject, the method comprising providing the subject with a therapeutically effective amount of a compound, said compound being selected from a compound of any one of paragraphs 1 to 15, a compound of formula (XIII) and a pharmaceutically acceptable salt thereof.

36. Use of a compound in the manufacture of a medicament for preventing DNA damage in the subject, said compound being selected from a compound of any one of paragraphs 1 to 15, a compound of formula (XIII) and a pharmaceutically acceptable salt thereof.

37. The method of paragraph 35 or the use of a compound according to paragraph 36, wherein the DNA is mtDNA.

38. Use of a compound in the manufacture of a medicament for treating or preventing a disease associated with iron induced oxidative stress, said compound being selected from a compound of any one of paragraphs 1 to 15, a compound of formula (XIII) and a pharmaceutically acceptable salt thereof.

39. The use of a compound according to paragraph 38, wherein a disease associated with iron induced oxidative stress is a disease associated with mitochondrial iron induced oxidative stress.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows results of a mtDNA damage assay. Compound 5 is able to prevent mtDNA much more effectively than Tiron, even at significantly lower concentrations. In fact, 5 provided the same level of protection as foil. Compounds 3, 2 and 4 are also effective at preventing mtDNA damage at significantly lower concentrations than Tiron.

Figure 2 shows results of a mtDNA damage assay in which two different batches of the same compound 5 are compared.

DETAILED DESCRIPTION

Compounds of the invention

Throughout this specification, the term “compound of the invention” is generally intended to refer to a compound of any one of formulae (I) to (XII) or a pharmaceutically acceptable salt thereof. In the context of the uses, methods and/or formulations of the invention, the term compound of the invention is intended to refer to a compound of any one of formulae (I) to (XIII) or a pharmaceutically acceptable salt thereof.

Certain compounds of the invention are free iron chelators.

Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of the invention, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are add addition or base salts wherein the counter ion is optically active, for example, d-lactate or l-lysine, or racemic, for example, di-tartrate or dl-arginine.

Compounds of the invention containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound of the invention contains a double bond such as a C=C or C=N group, geometric ds/trans (or Z/E) isomers are possible. Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.

Where structurally isomeric forms of a compound are interconvertible via a low energy barrier, tautomeric isomerism (‘tautomerism’) can occur. This can take the form of proton tautomerism in compounds of the invention containing, for example, an imino, keto, or oxime group, or so- called valence tautomerism in compounds which contain an aromatic moiety. Conventional techniques for the preparation/isolation of individual enantiomers when necessary include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).

Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of the invention contains an addic or basic moiety, a base or add such as 1-phenylethylamine or tartaric add. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted into the corresponding pure enantiomer(s) by means well known to a skilled person.

Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.

When any racemate crystallises, crystals of two different types are possible. The first type is the racemic compound (true racemate) referred to above wherein one homogeneous form of crystal is produced containing both enantiomers in equimolar amounts. The second type is the racemic mixture or conglomerate wherein two forms of crystal are produced in equimolar amounts each comprising a single enantiomer.

While both of the crystal forms present in a racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures may be separated by conventional techniques known to those skilled in the art - see, for example, “Stereochemistry of Organic Compounds” by E. L. Eliel and S. H. Wilen (Wley, 1994).

It follows that a single compound may exhibit more than one type of isomerism.

The term Cm-C _n refers to a group with m to n carbon atoms.

The term “alkyl” refers to a monovalent linear or branched hydrocarbon chain. For example, Ci-Ce-alkyl may refer to methyl, ethyl, n-propyl, iso- propyl, n-butyl, sec-butyl, terf-butyl, n- pentyl and n-hexyl. An alkyl group may be unsubstituted or substituted by one or more substituents. Specific substituents for each alkyl group independently may be fluorine, OR ^® or NHRa.

The term “alkylene” refers to a bivalent linear hydrocarbon chain. For example, -C1-C3. alkyl may refer to methylene, ethylene or propylene. An alkylene group may be unsubstituted or substituted by one or more substituents. Specific substituents for each alkyl group independently may be methyl or ethyl.

The term “alkenyl” refers to a branched or linear hydrocarbon chain containing at least one double bond. The double bond(s) may be present as the £ or Z isomer. The double bond may be at any possible position of the hydrocarbon chain. For example, “C^Ce-alkenyl” may refer to ethenyl, propenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl. An alkenyl group may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each alkenyl group independently may be fluorine, ORa or NHRa.

The term “alkynyl” refers to a branched or linear hydrocarbon chain containing at least one triple bond. The triple bond may be at any possible position of the hydrocarbon chain. For example, “C2-C6-alkynyl” may refer to ethynyl, propynyl, butynyl, pentynyl and hexynyl. An alkynyl group may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each alkynyl group independently may be fluorine, OR ⁸ or NHR ^a .

The term “cycloalkyl” refers to a saturated hydrocarbon ring system containing 3, 4, 5 or 6 carbon atoms. For example, “C3-C6-cycloalkyl” may refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. A cycloalkyl group may be unsubstituted or substituted by one or more substituents. Specific substituents for each cycloalkyl group independently may be fluorine, ORa or NHRa.

The term ^u _y . _z -membered heterocycloalkyl” may refer to a monocyclic or bicyclic saturated or partially saturated group having from y to z atoms in the ring system and comprising 1 or 2 heteroatoms independently selected from O, S and N in the ring system (in other words 1 or 2 of the atoms forming the ring system are selected from O, S and N). By partially saturated it is meant that the ring may comprise one or two double bonds. This applies particularly to monocyclic rings with from 5 to 8 members. The double bond will typically be between two carbon atoms but may be between a carbon atom and a nitrogen atom. Examples of heterocycloalkyl groups include; piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, dihydrofuran, tetrahydropyran, dihydropyran, dioxane, azepine. Bicyclic systems may be spiro-fused, i.e. where the rings are linked to each other through a single carbon atom; vicinally fused, i.e. where the rings are linked to each other through two adjacent carbon or nitrogen atoms; or they may be share a bridgehead, i.e. the rings are linked to each other two non-adjacent carbon or nitrogen atoms. A heterocycloalkyl groups may be unsubstituted or substituted by one or more substituents. Specific substituents for any saturated carbon atom in each heterocycloalkyl group may independently be fluorine, OR ^a or NHR ^a .

The term “heteroaryl” may refer to any aromatic (i.e. a ring system containing 2(2n + 1)π electrons) 5-, 6-, 9- or 10- membered ring system comprising from 1 to 4 heteroatoms independently selected from O, S and N (in other words from 1 to 4 of the atoms forming the ring system are selected from O, S and N). Thus, any heteroaryl groups may be independently selected from: 5 membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-4 heteroatoms independently selected from O, S and N; and 6- membered heteroaryl groups in which the heteroaromatic ring is substituted with 1-3 (e.g.1- 2) nitrogen atoms; 9-membered bicyclic heteroaryl groups in which the heteroaromatic system is substituted with 1-4 heteroatoms independently selected from O, S and N; 10- membered bicyclic heteroaryl groups in which the heteroaromatic system is substituted with 1-4 nitrogen atoms. Specifically, heteroaryl groups may be independently selected from: pyrrole, fviran, thiophene, pyrazole, imidazole, oxazole, isoxazole, triazole, oxadiazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazine, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, indazole, benzimidazole, benzoxazole, benzothiazole, benzisoxazole, purine, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, pteridine, phthalazine, naphthyridine.

The present invention also includes the synthesis of all pharmaceutically acceptable isotopically-labelled compounds of formulae (I) to (XII) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as ² H and ³ H, carbon, such as ¹¹ C, ¹³ C and ¹⁴ C, chlorine, such as “Cl, fluorine, such as ¹⁸ F, iodine, such as ¹²³ l and ¹²⁵ l, nitrogen, such as ¹³ N and ¹⁵ N, oxygen, such as ¹⁵ 0, ¹⁷ 0 and ¹⁸ 0, phosphorus, such as “P, and sulphur, such as “S. Certain isotopically-labelled compounds, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ² H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some drcumstances.

Substitution with positron emitting isotopes, such as ¹¹ C, ¹⁸ F, ¹⁵ 0 and ¹³ N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

The compounds of the invention may be obtained, stored and/or administered in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic adds such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic adds, or salts of pharmaceutically acceptable organic adds such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, dtric, lactic, mudc, gluconic, benzoic, sucdnic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric adds. Suitable base salts are formed from bases which form non-toxic salts. Examples indude the aluminium, arginine, benzathine, caldum, choline, diethylamine, diolamine, glydne, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of adds and bases may also be formed, for example, hemisulfate and hemicaldum salts. Also induded are add addition or base salts wherein the counter ion is optically active, for example, d-lactate or l-lysine, or racemic, for example, dl-tartrate or dl- arginine.

In some instances, where the compounds of the invention are pharmaceutically acceptable salts (as described above), the R ¹² group may comprise the cationic and anionic groups of the salt. For the avoidance of doubt, R ¹² may be a covalent, inherently neutral moiety or may comprise a salt thereof having a charged (e.g. cationic) group assodated with an appropriate counterion (e.g. counteranion), thereby providing a salt as described above. For example, R ¹² may be -C(=NH)NH2 (i.e. covalent, neutral moiety) or may be -[C(=NH)NH3] ⁺ CI (i.e. a hydrochloric salt thereof).

Alternatively, R ¹² may be an ionic (e.g. cationic) entity associated with a counterion (e.g. counteranion), as described above. For example, R ¹² may be [NR ⁶ R ⁶ R ⁶ ] ⁺ or [P(R ¹⁵ )3] ⁺ associated with a counteranion.

Compounds of the invention may exist in a single crystal form or in a mixture of crystal forms or they may be amorphous. Thus, compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, or spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.

Uses and methods of the Invention

In one aspect, the present invention provides a compound of the invention for use as a medicament.

The term “medicament" as used herein refers to the compound’s ability to have any physiological, therapeutic, and/or prophylactic effect on a subject. The effect may be for example prevention of accumulation or reduction of free iron levels which may have further downstream effects. By way of example, such downstream effects may be selected from the group consisting of prevention or reduction of iron induced oxidative stress, prevention or reduction of the formation of ROS, prevention of DNA damage, and/or prevention or treatment of any disease, such as a disease associated with iron induced oxidative stress.

In one example, the effect may be prevention of accumulation or reduction of free mitochondrial iron levels. Merely by way of example, downstream effects of preventing or reducing free mitochondrial iron levels may be selected form the group prevention or reduction of mitochondrial iron induced oxidative stress, reduction or prevention of the formation of ROS catalysed by free mitochondrial iron, prevention of mtDNA damage, and/or prevention or treatment of any disease, such as a disease associated with mitochondrial iron induced oxidative stress.

As used herein, the term "oxidative stress" refers to pathophysiological effects of ROS on normal cellular structure and/or function. ROS is a term that collectively describes molecules that have a reactive oxygen moiety. Examples of ROS include hydroxyl radicals and/or superoxide. Oxidative stress may cause damage to DNA, RNA, proteins, lipids, or any other cellular components, such as mitochondria. Oxidative stress that causes damage to mitochondria and mitochondrial components, such as mtDNA, may be referred to as “mitochondrial oxidative stress”.

The term “iron induced oxidative stress” refers to oxidative stress caused by ROS, the production of which is catalysed by free iron in the cell, for example in the cytosol. Iron induced oxidative stress may be due to, for example, environmental factors (such as UV), and/or iron overloading. By the same token, in the context of the present description, “mitochondrial iron induced oxidative stress” refers to oxidative stress caused by ROS, the production of which is catalysed by free iron in the mitochondria. Mitochondrial iron induced oxidative stress may be due to, for example, environmental factors (such as UV) and/or mitochondrial iron overloading.

In further aspects, the invention provides a compound of the invention for use in the prevention of DNA damage. Suitably, the DNA is mtDNA.

The invention also provides a method of preventing DNA damage in the subject, the method comprising providing the subject with a therapeutically effective amount of a compound of the invention. Suitably, the DNA is mtDNA.

The term “DNA damage” as used herein refers to any change in DNA sequence or structure as compared to a wild-type and/or healthy DNA. The change may be caused by oxidative stress. The change in the sequence or structure may be any type of lesion (such as a base alteration, an apurinic site, a strand break, an adduct formation, deletion, insertion and/or duplication) in DNA. By the same token, the term "mtDNA damage" refers to any change in mtDNA sequence or structure as compared to a wild-type and/or healthy mtDNA. The change may be caused by oxidative stress. Suitably the oxidative stress may be mitochondrial iron induced oxidative stress. The change in the sequence or structure may be any type of lesion (such as a base alteration, an apurinic site, a strand break, an adduct formation, deletion, insertion and/or duplication) in mtDNA.

Methods for determining DNA damage are known in the art. Merely byway of example, mtDNA damage may be assessed directly (for example by PGR) or indirectly (for example by analysing downstream effects of mtDNA damage). Downstream effects that may be analysed to determine mtDNA damage include, but are not limited to, mitochondrial protein production, changes (such as a decrease) in mitochondrial oxidative phosphorylation or changes (such as a decrease) in mitochondrial ATP production. An exemplary protocol of measuring mtDNA damage is provided in the Examples section of the present specification.

The phrase “prevention of DNA damage” as used herein refers to delaying or inhibiting the development of changes in DNA in the subject’s cells caused by oxidative stress as compared to an appropriate control. Thus, by the same token, the phrase “prevention of mtDNA damage” as used herein refers to delaying or inhibiting the development of changes in mtDNA in the subject’s cells caused by oxidative stress (such as mitochondrial iron induced oxidative stress) as compared to an appropriate control.

The skilled person will be well aware of what constitutes an appropriate control. However, merely by way of example, in the context of DMA damage, a suitable control may be the DMA sequence of a healthy cell. A healthy cell may be one that has normal oxidative stress levels, normal free iron levels (for example normal free mitochondrial iron levels), and/or is from a subject that does not suffer a disease associated with iron induced oxidative stress.

In the context of mtDNA damage, a suitable control may be the mtDNA sequence of a healthy mitochondrion, a mitochondrial protein production reference value, and/or mitochondrial ATP production reference value derived from healthy mitochondria. Healthy mitochondria may be, for example, mitochondria from a healthy cell and/or a cell that has not been exposed to UV (for example UVA or UVB). Merely by way of example, the cell may be selected from the group consisting of a skin cell (such as a fibroblast, a keratinocyte, a melanocyte, a Merkel cell, or Langerhans cell), a muscle cell (such as a cardiac, skeletal or smooth muscle cell), a blood cell (such as a white blood cell, for example a neutrophil, an eosinophil, a basophile, a lymphocyte, ora monocyte), a nerve cell (such as a sensory cell, for example a retinal ganglion cell; or a motor neuron cell), a kidney cell (such as a glomerulus parietal cell, a glomerulus podocyte, a proximal tubule brush border cell, a loop of Henle thin segment cell, a thick ascending limb cell, a distal tubule cell, a collecting duct principal cell or a collecting duct intercalated cell) and a liver cell (such as a hepatocyte or a Kupffer cell).

The compounds and formulations of the invention may prevent DMA damage (for example mtDNA damage) by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% as compared to a suitable control. In some examples, the compounds and formulations of the invention may substantially completely prevent DNA damage (for example mtDNA damage). DNA damage has been linked to many diseases. It will be appreciated that DNA damage may be a downstream and/or upstream effect of a disease. DNA damage is a downstream effect of a disease when the disease causes the DNA damage. Such a disease is for example an iron overload disease. An iron overload disease may be caused by a mutation which results in excess free iron in the cell, for example in the cytoplasm and/or mitochondria. DNA damage is an upstream effect when the DNA damage causes or contributes to the disease, such as skin pre-cancer (also known as actinic keratosis) or skin cancer. It will be appreciated that in some conditions DNA damage may result in further oxidative stress, which in turn further exacerbates the disease. In such conditions, DNA damage is both an upstream and downstream effect of a disease.

Similarly, mtDNA damage has been linked to many diseases. It will be appreciated that mtDNA damage may be a downstream and/or upstream effect of a disease. mtDNA damage is a downstream effect of a disease when the disease causes the mtDNA damage. Such a disease is for example a mitochondrial iron overload disease. A mitochondrial iron overload disease may be caused by a mutation which results in excess free iron in the mitochondria. MtDNA damage is an upstream effect when the mtDNA damage causes or contributes to the disease, such as skin pre-cancer (also known as actinic keratosis) or skin cancer. It will be appreciated that in some conditions mtDNA damage may result in further oxidative stress, which in turn further exacerbates the disease. In such conditions, mtDNA damage is both an upstream and downstream effect of a disease.

In further aspects, the invention provides a compound of the invention for use in the treatment or prevention of a disease associated with iron induced oxidative stress. In one example, the disease associated with iron induced oxidative stress may be a disease associated with mitochondrial iron induced oxidative stress.

The invention also provides a method of preventing or treating a disease associated with iron induced oxidative stress in a subject, the method comprising providing the subject with a therapeutically effective amount of a compound of the invention. In one example, the disease associated with iron induced oxidative stress may be a disease associated with mitochondrial iron induced oxidative stress.

The term “disease associated with iron induced oxidative stress” as used herein is a disease which is caused by and/or which results in iron induced oxidative stress. Such iron induced oxidative stress may be caused by or result in increased free iron levels (for example in the cytosol) and/or DNA damage. Accordingly, a disease associated with iron induced oxidative stress may be prevented or treated by reducing free iron levels, preventing an increase in free iron levels, and/or preventing DNA damage. Examples of diseases associated with iron induced oxidative stress include pre-cancer (actinic keratosis), cancer (such as skin cancer, for example selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and melanoma), iron overload diseases (such as beta thalassemia). Exposure to certain compounds may also result in a disease associated with iron induced oxidative stress. An example of such a compound is Doxorubicin, a chemotherapeutic drug which may increase ROS (for example in cardiomyocytes).

Similarly, a “disease associated with mitochondrial iron induced oxidative stress” as used herein, herein is a disease which is caused by and/or which results in mitochondrial iron induced oxidative stress. Such mitochondrial iron induced oxidative stress may be caused by or result in increased free mitochondrial iron levels and/or mtDNA damage. Accordingly, a disease associated with mitochondrial iron induced oxidative stress may be prevented or treated by reducing free mitochondrial iron levels, preventing an increase in free mitochondrial iron levels, and/or preventing mtDNA damage. Examples of diseases associated with iron induced oxidative stress include pre-cancer (actinic keratosis), cancer (such as skin cancer, for example selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and melanoma), or mitochondrial iron overload diseases.

“Mitochondrial iron overload disease” as used herein refers to a disease caused by an increased amount of free iron in the mitochondrial matrix as compared to a suitable control. This increased amount of free iron may be caused, for example by dietary iron overload or a genetic mutation. Such a genetic mutation may result in abnormal retention and/or abnormal import of free iron into the mitochondrial matrix leading to increased mitochondrial ROS production and mitochondrial oxidative damage. Merely by way of example, the mutation may be in a gene selected from the group consisting of FXN, NFU1, SLC25A39, SLC22A4, and TMEM14C.

By way of example, a mitochondrial iron overload disease may be selected from the group consisting of Friedreich's ataxia, sideroblastic anaemia, erythropoietic protoporphyria, X- linked protoporphyria, cardiomyopathy, pulmonary hypertension, Alzheimer's disease, chronic obstructive pulmonary disease, Parkinson's disease, Huntington's disease, type 2 Wolfram syndrome, hereditary myopathy with lactic acidosis and adolescent onset autosomal recessive mitochondrial myopathy. The term “treatment" as used herein refers to an intervention which slows the progression, or reduces partially or completely, the clinical symptoms connected to a disease and/or the underlying cause of the clinical symptoms, in a subject It will be appreciated that the symptoms connected to a disease (for example mitochondrial iron induced oxidative stress) will depend on the specific disease, and generally will be well known to those skilled in the art. Merely by way of example, in the context of skin cancer or skin pre-cancer, the symptom may be a skin lesion. Thus, treatment of skin cancer or skin pre-cancer may include slowing down, preventing or reversing the progression of the size or severity of the lesion.

The term “treatment" encompasses not only the therapeutic use of the compounds of the invention in a subject with symptoms, but also use of the compounds in the treatment of a subject who does not exhibit the symptoms of the disease. Such uses may be of particular relevance to an asymptomatic subject, for example, known to carry a mutation which increases the subject’s likelihood of developing a disease associated with mitochondrial iron induced oxidative stress such as a mitochondrial iron overload disease.

The term “prevention” as used herein refers to inhibiting the development of a disease associated with iron induced oxidative stress (for example mitochondrial iron induced oxidative stress) and/or the development of clinical symptoms connected said disease.

The term “subject" as used herein refers to a human or any non-human animal that may benefit from a reduction of free iron levels (for example free mitochondrial iron levels), and downstream effects of reducing such free iron levels as described elsewhere in the present specification. By way of example, the non-human animal may be a farm animal (e.g., a horse, pig, cow, goat, or a sheep) or a pet (e.g., a dog or cat). In one example, the subject may be at risk of easily getting sunburned when exposed to sunlight. A subject that may be particularly at risk of easily getting sunburned when exposed to sunlight may have a fair skin complexion, blond or ruddy hair, and/or blue eyes. In another example, the subject may be particularly at risk of developing skin cancer. Such a subject may for example have pre-cancerous skin lesions, may spend extended amounts of time outdoors and/or may live in a part of the world where the UV levels are known to be high (for example Australia). In yet a further example, the subject may be suffering from an iron overload disease (for example mitochondrial iron overload disease).

The term “providing” as used herein refers to administering the compound of the invention (or the formulation of the invention) to the subject The amount, frequency and duration that the subject is provided the compound of the invention may differ depending on the formulation, clinical indication, age, and route of administration. Exemplary routes of administration are discussed elsewhere in the present specification.

The term “therapeutically effective amount" as used herein, refers to an amount of the compound or formulation of the invention, that when provided to the subject, is sufficient to reduce the amount of free iron in the cell (for example in the cytosol and/or in the mitochondria). Such a reduction of free iron may result in lower ROS levels (for example in mitochondria), less oxidative stress, and/or less oxidative damage (such as mtDNA damage).

Formulations of the Invention

In a further aspect, the invention provides a formulation comprising a compound of the invention.

The formulation may comprise a compound of the invention at a concentration of, for example, from about 0.01 mM to about 3 mM, from about 0.02 mM to about 2mM, from about 0.03 mM to about 1mM, or from about 0.04 mM to about 0.5 mM.

The formulation may comprise a compound of the invention at a concentration of about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, or about 0.1 mM. The formulation may comprise the compound of the invention at a concentration of about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM. The formulation may comprise the compound of the invention at a concentration of about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2 mM. The formulation may comprise the compound of the invention at a concentration of about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, or about 3 mM.

It will be appreciated that the concertation may depend on the specific compound of the invention in the formulation. For example, in the context of compound 5 or 4, the concentration may be, for example, from about 0.01 mM to about 0.1 mM, from about 0.025 mM to about 0.075 mM. Accordingly, the concertation of compound 5 or 4 may be for example about 0.01 mM, about 0.015 mM, about 0.02 mM, about 0.025 mM, about 0.03 mM, about 0.035 mM, about 0.04 mM, about 0.045 mM, about 0.05 mM, about 0.055 mM, about 0.06 mM, about 0.065 mM, about 0.07 mM, about 0.075 mM, about 0.08 mM, about 0.085 mM, about 0.09 mM, or about 1m mM. Suitably, the concertation of compound 5 or 4 may be 0.05mM.

In the context of compound 3 or 2, the concentration may be, for example, from about 0.1 mM to about 1 mM, from about 0.25 mM to about 0.75mM. Accordingly, the concertation of compound 3 or 2 may be for example about 0.1 mM, about 0.15 mM, about 0.2 mM, about 0.25 mM, about 0.3 mM, about 0.35 mM, about 0.4 mM, about 0.45 mM, about 0.5 mM, about 0.55 mM, about 0.6 mM, about 0.65 mM, about 0.7 mM, about 0.75 mM, about 0.8 mM, about 0.85 mM, about 0.9 mM, about 0.95 mM, or about 1mM. Suitably, the concertation of compound 3 or 2 may be 0.4 mM.

The formulation of the present invention may comprise one or more of the compounds of the invention. Thus, the formulation may comprise two, three, four or more compounds of the present invention. Merely as an example, a formulation that comprises two compounds of the invention may comprise compound 5 and 2, or compound 5 and 3, or compound 3 and 2. In a formulation comprising two or more compounds of the invention, each of the compounds may be at a concentration as mentioned hereinabove. Alternatively, the combined concentration of the two or more compounds of the invention may correspond to the concentrations mentioned hereinabove.

The formulation (or the compound of the invention itself) may be for topical administration meaning that it is for application to the skin. Suitably, the formulation for topical administration may be a cream, lotion, oil, gel, balm, liquid, aerosolizable liquid, or a foam. It will be appreciated that whilst a formulation for topical administration may be suitable for use as a therapeutic, such a formulation may be especially useful in the context of non-therapeutic (for example cosmetic) uses, for example as a sunscreen.

Alternatively, the formulation (or the compound of the invention itself) may be for systemic administration. Exemplary routes for systemic administration may be selected from the group consisting of oral, inhalation, subcutaneous, intramuscular, intravenous or rectal.

The formulation may further comprise a pharmaceutically or cosmetically acceptable diluent, carrier, excipient, salt, buffering agent, and/or another therapeutic or non-therapeutic agent.

The phrase "pharmaceutically or cosmetically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Diluents are diluting agents. Pharmaceutically or cosmetically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients (such as the compounds of any one of formulae (I) to (XIII)) of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically or cosmetically acceptable carriers are well known in the art A suitable carrier is therefore easily identifiable by one of ordinary skill in the art

Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. the compounds of the invention), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically and cosmetically acceptable excipients are well known in the art A suitable excipient is therefore easily identifiable by one of ordinary skill in the art By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

In an example, the formulation of the invention may comprise a photoprotective compound and/or an antioxidant compound.

A photoprotective compound is a compound that has the ability to filter UV. The photoprotective compound may be an organic chemical compound which absorbs UV, organic particulate which contain multiple chromophores, and inorganic chemical compound which reflects UV (a UV reflector). As UVC (radiation in the 100-290 nm) range is totally blocked by the ozone layer in the upper atmosphere of the Earth, the formulation may preferably comprise a photoprotective compound filters UVA and/or UVA light.

A photoprotective compound able to filter UVA may be selected from the group consisting of disodium 4,5-dihydroxy-1,3-benzenedisulfonate also known as Tiron, avobenzone or butyl methoxydibenzoylmethane, also known as Parsol 1789, Parsol A, Eusolex 9020, Escalol 517, BMBM, BMDBM, and related compounds, such as for example oxybenzone; Tinosorb S also known as bis-ethyl-hexyloxyphenol methoxyphenyl triazine, BEMT, bemotrizinol, anisotriazine, Escalol S, Tinosorb S Aqua; Tinosorb M also known as methylene bis- benzotriazolyl tetramethylbutyl-phenol, MBBT, Bisoctrizole; Mexoryl SX also known as terephthalylidene dicamphor sulfonic acid, TDSA, Ecamsule; Mexoryl XL also known as Drometrizole trisiloxane, Ecamsule; a combination of avobenzone and oxybenzone also known as Helioplex; Octocrylene also known as Uvinul N539T, OCR, Eusolex OCR; Oxybenzone also known as Benzophenone-3, BPS, Uvinul M40, Eusolex 4360, Escalol 567; Ensulizole also known as phenylbenzimiazole sulfonic acid, PBSA, Eusolex 232, Parsol HS; dioxybenzone also known as benzophenone-8; Meradimate also known as menthyl anthranilate; Sulisobenzone also known as benzophenone-4, BP4, uvinul MS40, Escalol 577; Disodium phenyl dibenzimidazole tetrasulfonate also known as Bisimidazylate, DPDP; Uvinal A Plus also known as diethylaminohydroxybenzoyl hexyl benzoate, DHHB; Uvasorb HEB also known as diethylhexyl butamidotriazone, DBT, Iscotrizinol;

A photoprotective compound able to filter UVB light may be selected from the group consisting of benzyl salicylate and salicylate derivatives, such as for example homosalate; benzyl cinnamate and cinnamate derivatives; Octinoxate also known as Octyl methoxy-cinnamate, OMC, Ethylhexyl methoxycinnamate, EHMC, Escalol 557, Parsol MCX, Eusolex 2292, Tinosorb OMC, Uvinul MC80; Octocrylene also known as Uvinul N539T, OCR, Eusolex OCR; a combination of avobenzone and oxybenzone also known as Helioplex; Tinosorb M also known as methylene bis-benzotriazolyl tetramethylbutyl-phenol, MBBT, Bisoctrizole; Tinosorb S also known as bis-ethyl-hexyloxyphenol methoxyphenyl triazine, BEMT, bemotrizinol, anisotriazine, Escalol S, Tinosorb S Aqua; Oxybenzone also known as Benzophenone-3, BPS, Uvinul M40, Eusolex 4360, Escalol 567; octisalate also known as octyl salicylate, ethylhexyl salicylate, EHS, Escalol 587; homosalate also known as homomethyl salicylate, HMS; uvinul T150 also known as octyltriazone, ethylhexyl triazone, EHT; cinoxate also known as 2- ethoxyethyl p-methoxycinnamate, Phiasol, Give Tan, Sundare; aminobenzoic acid, PABA; Padimate O also known as OD-PABA, octyldimethyl PABA, ethylhexyl dimethyl PABA, EH DP, Escalol 507; Ensulizole also known as phenylbenzimiazole sulfonic acid, PBSA, Eusolex 232, Parsol HS; dioxybenzone also known as benzophenone-8; Sulisobenzone also known as benzophenone-4, BP4, uvinul MS40, Escalol 577; trolamine salicylate also known as trienanolamine salicylate; 4-methylbenzylidene camphor; Uvasorb HEB also known as diethylhexyl butamidotriazone, DBT, Iscotrizinol; Parsol SLX also known as dimethico- diethylbenzal-malonate, Polysilicone-15, PS15; Amiloxate also known as Isoamyl p-Methoxy- cinnamate, IMG, Neo Heliopan E1000. For the avoidance of doubt, many photoprotective compounds are capable of filtering both UVA and UVB radiation.

A UV reflector compound is an inorganic particulate that reflects, scatters and absorbs UV length. A UV reflector may be selected from the group consisting of titanium dioxide, zinc oxide or a combination of both.

An antioxidant compound is a compound that reduces the biochemical side effects cause by ROS. The biochemical side effects caused by ROS may be for example by scavenging ROS, or by inhibiting molecule that catalyse ROS formation. Suitable antioxidants may be selected from the group consisting of water-soluble antioxidants, oil-soluble antioxidants, natural extracts, vitamins and vitamin derivates.

Water-soluble antioxidants are for example sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N- acetyl-cysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin, and ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbyl polypeptide).

Oil-soluble antioxidants are for example butylated hydroxytoluene, retinoids (e.g., retinol, tretinoin, and retinyl palmitate), tocopherols (e.g., tocopherol acetate), tocotrienols, alkylresorcinols, curcumin and its derivatives and ubiquinone. Natural extracts are for example extracts containing flavonoids and isoflavonoids and their derivatives e.g., genistein and diadzein, and extracts containing resveratrol.

Vitamins and vitamin derivatives include, for example, vitamin A, vitamin A propionate, vitamin A palmitate, vitamin A acetate, retinol, vitamin B, thiamine chloride hydrochloride (vitamin B.sub.1), riboflavin (vitamin B.sub.2), nicotinamide, vitamin C and derivatives (for example ascorbyl palmitate, magnesium ascorbyl phosphate, ascorbyl acetate), vitamin D, ergocalciferol (vitamin D.sub.2), vitamin E, DL-.alpha. -tocopherol, tocopherol E acetate, tocopherol hydrogensuccinate, vitamin K.sub.l, esculin (vitamin P active ingredient), thiamine (vitamin Bi), nicotinic acid (niacin), pyridoxine, pyridoxal, pyridoxamine, (vitamin B6), pantothenic acid, biotin, folic acid and cobalamine (vitamin Bi2) Preferred vitamins are, for example, vitamin A palmitate, vitamin C and derivatives thereof, DL-α-tocopherol, tocopherol E acetate, nicotinic acid, pantothenic acid and biotin. Vitamin E, which is often added to cosmetic and personal care products is also preferably stabilized by the compounds according to the invention. The formulation of the present invention may comprise a humectant The presence of a humectant may be especially desirable in the context of a formulation for topical administration. Exemplary humectants include, but are not limited to, glycerol, sorbitol, maltitol, polydextrose, triacetin, propylene glycol, and polyethylene glycols (PEG) such as PEG-4, PEG-6, PEG-8, PEG- 12, PEG-32, PEG-75 and PEG- 150, glycerol, propylene glycol, a polyethylene glycol, or a combination or mixture thereof.

Uses of the Invention

In a further aspect, the invention provides use of a formulation comprising a compound of the invention, or a compound of the invention itself, for protecting a subject from UV, and/or improving or restoring a subject’s appearance.

Accordingly, the invention also provides a method of protecting a subject from UV, and/or improving or restoring a subject’s appearance, the method comprising providing the subject with of a formulation comprising the compounds of the invention, or a compound of the invention itself.

The term “protecting” as used herein refers to preventing or reducing the effects of exposure to UV. The effects of exposure to UV may be invisible and/or visible to naked human eye. Examples of invisible effects include, increased ROS levels in cells exposed to UV and optionally surrounding cells, and/or increased DNA damage (for example mtDNA damage) to cells exposed to UV and optionally surrounding cells. Examples of visible effects include sunburn, hyperpigmentation, skin wrinkles and/or loss of skin elasticity. Preventing or reducing the effects of exposure to UV may also prevent skin pre-cancer or cancer.

In the context of the present application, the phrase “improving or restoring a subject’s appearance” refers to a cosmetic use with the intention to address a non-pathological condition in a subject, such as the signs of aging on the subject’s skin. The subject’s appearance may be restored or improved for example by reducing or preventing skin wrinkles, reducing or preventing skin hyperpigmentation and/or increasing skin elasticity or preventing loss of skin elasticity. It will be appreciated that these effects may be achieved by reducing the amount of free iron in the subject’s cells (for example in the mitochondria), and thereby reducing oxidative stress (such as mitochondrial oxidative stress) and oxidative damage (such as mitochondrial oxidative damage). Reduction of oxidative damage may be determined by assessing DNA damage. Reduction of mitochondrial oxidative damage may be determined by assessing mtDNA damage.

In the context of cosmetic uses, the subject may be any individual wishing to restore or improve their appearance.

In a further aspect, the invention provides the use of a compound of the invention in the manufacture of a medicament for preventing DNA damage in the subject. In one example, the DNA is mitochondrial DNA.

In a further aspect, the invention provides the use of a compound of the invention in the manufacture of a medicament for treating or preventing a disease associated with iron induced oxidative stress. In one example, the iron induced oxidative stress may be mitochondrial iron induced oxidative stress.

It will be recognised that, except where the context requires otherwise, embodiments described in respect of one aspect of the invention will also be applicable to all of the aspects of the invention. Thus, for example, embodiments mentioned in the context of the uses described herein are also disclosed in the context of the methods described herein.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Synthesis The skilled man will appreciate that adaptation of methods known in the art could be applied in the manufacture of the compounds of the present invention.

For example, the skilled person will be immediately familiar with standard textbooks such as "Comprehensive Organic Transformations - A Guide to Functional Group Transformations", RC Larock, Wiley-VCH (1999 or later editions), "March's Advanced Organic Chemistry - Reactions, Mechanisms and Structure”, MB Smith, J. March, Wiley, (5th edition or later) “Advanced Organic Chemistry, Part B, Reactions and Synthesis”, FA Carey, RJ Sundberg, Kluwer Academic/Plenum Publications, (2001 or later editions), Organic Synthesis - The Disconnection Approach", S Warren (Wiley), (1982 or later editions), "Designing Organic Syntheses" S Warren (Wiley) (1983 or later editions), "Guidebook To Organic Synthesis" RK Mackie and DM Smith (Longman) (1982 or later editions), etc., and the references therein as a guide.

The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound and will employ protecting groups as necessary. This will depend inter alia on factors such as the nature of other functional groups present in a particular substrate. Clearly, the type of chemistry involved will influence the choice of reagent that is used in the said synthetic steps, the need, and type, of protecting groups that are employed, and the sequence for accomplishing the protection / deprotection steps. These and other reaction parameters will be evident to the skilled person by reference to standard textbooks and to the examples provided herein.

Sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional methods, for example as described in “Protective Groups in Organic Synthesis” by TW Greene and PGM Wilts, John Wiley & Sons Inc (1999), and references therein.

The compounds of the invention can be synthesised according to or analogously to Examples 1 to 7 provided below. The compounds of the invention can be synthesised according to or analogously to General Scheme A. w

General Scheme A

Where R ¹ and R ² are Ci-Cvalkyl, compounds of the invention can be made by coupling a sulfonyl chloride A with an amine (formula B where X ¹ =NR ^S ) or an alcohol (formula B where X ¹ =OH) in the presence of a base (e.g. NEt3) to form a compound of formula C (a subset of compounds of the invention). Where it is desired that R ¹ and R ² are H, the coupling reaction can be performed using a compound of formula A in which R ¹ and R ² are methyl and the resulting compound of formula C can be subjected to demethylation, e.g. with BBra to provide a compound of formula D (a subset of compounds of the invention). Compounds in which R ¹ and R ² are esters can be formed via a subsequent esterification reaction, e.g. using an acid chloride of the desired acyl group.

EXAMPLES

1. Materials and methods

1.1 Compound synthesis

3,4-Dimethoxybenzenesulfonic acid (Gaspari, Gazette Chimica Italians 1896, 2611, 231)

3,4-Dimethoxybenzenesulfonyl chloride (0.596 g) was stirred with distilled water (3 mL) for 48 hrs at room temperature and then extracted with ethyl acetate (3 x 25 mL). Ethyl acetate combined, washed with distilled water (2 x 25 mL), saturated brine (25 mL), dried over anhydrous magnesium sulfate, decanted and solvent removed in vacuo to give a pale tan solid.

Yield: 0.596 g; 43 % pale tan solid; ¹ H NMR (300 MHz, CDCI): δ _Η 7.71 (dd, 1H, Ar H), 7.45 (d, 1H, Ar H), 7.02 (d, 1H, Ar H), 4.00 (s, 3H, OC H3), 3.98 (s, 3H, OC H ₃ ).

3,4-Dihydroxybenzenesulfonic acid (Cousin, Comptes Rendus Hebomadaires des Seances de I’Academie des Sciences 1893, 117, 113; Saito, Helv. Chim. Acta. 2006, 89, 1395)

To a solution of 1 ,2-dihydroxybenzene (5.50 g) in dimethylcartx)nate (14 mL) was added dropwise chlorosulfonic acid (3.37 mL) and the mixture was stirred for 2 hrs at 0°C and then allowed to warm to room temperature and stirred for 12 hrs. The mixture was then concentrated in vacuo to give a grey crystalline solid. The solid was dissolved in methanol (50 mL), dried over anhydrous magnesium sulfate, decanted, solvent removed in vacuo, followed by trituration with ethyl acetate then 40/60 petrol to give the desired product as a pale blue solid.

Yield: 8.754 g; 92 % pale blue solid;. ¹ H NMR (300 MHz, CD ₃ OD): δ _Η 7.30 (d, 1H, Ar H), 7.22 (dd, 1H, Ar H), 6.84 (d, 1H, Ar H). ¹³ C NMR (75 MHz, CD ₃ OD): δ _c 147.50, 144.60, 135.41, 117.78, 114.23, 113.05.

Methyl ((3,4-dimethoxyphenyl)sulfonyl)-L-leucinate 1

L-Leucine methyl ester hydrochloride (3.00 g) was added to a stirred solution of 3,4- dimethoxybenzene sulfonyl chloride (4.299g) and triethylamine (23 mL) in ethyl acetate (40 mL) and then stirred at room temperature for 18 hrs. Distilled water (50 mL) was added and the layers partitioned. The ethyl acetate layer was then washed with hydrochloric acid (2 x 50 mL, 1M), saturated sodium bicarbonate solution (50 mL), distilled water (50 mL), dried over anhydrous magnesium sulfate, decanted, solvent removed in vacuo to give the desired product as a pale tan solid. Yield: 3.521 g; 56 % pale tan solid; ¹ H NMR (300 MHz, CDCI): δ _Η 7.48 (dd, 1H, ArH), 7.30 (d, 1H, ArH), 6.94 (d, 1H, ArH), 5.16 (br d, N H), 3.94 (s, 3H, OCH ₃ ), 3.93 (s, 3H, OCH ₃ ), 3.48 (s, 3H, OC Hz), 3.28 (s, 1H, HNC H), 1.84 (m, 1H, HNCHCH ₂ ), 1.52 (m, 2H, CHCH ₂ CH), 1.16 (m, 1H, CH ₂ CH(CH ₃ ) ₂ ), 0.92 (m, 6H, CH ₂ CH(CH ₃ ) ₂ ). ¹³ C NMR (75 MHz, CDCI ₃ ): 5 _C 172.88, 152.65, 149.03, 131.28, 121.31, 110.38, 109.74, 56.23, 54.40, 52.34, 42.35, 24.32, 27.73, 21.44.

((3,4-Dihydroxyphenyl)sulfonyl)-L-leucine 2

BBr ₃ (23 mL, 1M in dichloromethane) was added dropwise to methyl ((3,4- dimethoxyphenyl)sulfonyl)-L-leucinate 1 (3.60 g) at 0°C over 30 mins then allowed to warm to room temperature over 2 hrs. The reaction was quenched with distilled water (50 mL) and then the dichloromethane was removed in vacuo. The resulting mixture was extracted with ethyl acetate (3 x 30 mL) and the combined extractions were washed with distilled water (50 mL), saturated brine (50 mL), dried over anhydrous magnesium sulfate, decanted, solvent removed in vacuo to give the desired product as a pale orange gum. Trituration with hexane gave the product as a pale orange solid.

Yield: 3.084 g; 97 % pale orange solid. ¹ H NMR (300 MHz, CDCI3): δ _Η 7.75 (br s, 1H, N H), 7.14 (d, 1H, ArH), 7.08 (dd, 1H, ArH), 6.83 (d, 1H, ArH), 3.58 (s, 1H, HNC H), 1.63 (m, 1H, HNCHCH ₂ ), 1.63 (m, 1H, CH ₂ CH(CH ₃ ) ₂ ), 1.37 (m, 1H, CH ₂ CH(CH ₃ ) ₂ ), 0.81 (m, 6H, CH ₂ CH(CH ₃ ) ₂ ). ¹³ C NMR (75 MHz, CDCI ₃ ): δ _c 172.88, 152.65, 149.03, 131.28, 121.31, 110.38, 109.74, 56.23, 54.40, 52.34, 42.35, 24.32, 27.73, 21.44.

Methyl ((3,4-dihydroxyphenyl)sulfonyl)-L-leucinate 3

Thionyl chloride was added dropwise to ((3,4-dihydroxyphenyl)sulfonyl)-L-leucine (1.587 g) in methanol (15 mL) at room temperature and then stirred for 18 hrs and then the solvent was removed in vacuo. Ethyl acetate (30 mL) was added and was washed with saturated sodium bicarbonate solution (2 x 30 mL), distilled water (30 mL), saturated brine (30 mL), dried over anhydrous magnesium sulfate, decanted, solvent removed in vacuo to give the desired product as a pale brown gum. Chromatography on silica (3:1 40/60 petrol: ethyl acetate) followed by tituration with hexane gave the desired product as a pale tan solid.

Yield: 1.352 g; 81 % pale tan solid; ¹ H NMR (300 MHz, CDCI): δ _Η 7.43 (d, 1H, Ar H), 7.32 (dd, 1H, Ar H), 6.95 (d, 1H, Ar H), 6.84 (br s, 1H, OH), 5.66 (d, 1H, N H), 3.99 (s, 1H, HNC H), 3.52 (s, 3H, OC Hz), 1.82 (m, 1H, CH ₂ CH(CH ₃ ) ₂ ), 1.52 (m 2H, CHCH ₂ CH), 0.90 (m, 6H, CH ₂ CH(CH ₃ ) ₂ ). ¹³ C NMR (75 MHz, (CDCI ₃ ): δ _C 173.71, 149.01, 143.94, 130.11, 121.18, 115.13, 114.11, 54.43, 52.71, 41.97, 24.33, 22.66, 21.31.

Ethyl (E)-3-(4-((3,4-((3,4-dimethoxyphenyl)sulfonamido)phenyl)acry late 4

Ethyl 4-aminocinammate (3.00 g) was added to a stirred solution of 3,4-dimethoxybenzene sulfonyl chloride (3.30 g) and triethylamine (17 mL) in ethyl acetate (40 mL) and then stirred at room temperature for 18 hrs. Distilled water (50 mL) was added and the layers partitioned. The ethyl acetate layer was then washed with hydrochloric acid (2 x 50 mL, 1M), saturated sodium bicarbonate solution (50 mL), distilled water (50 mL), dried over anhydrous magnesium sulfate, decanted, solvent removed in vacuo to give a orange gum. Chromatography on silica (3:1 40/60 petrol: ethyl acetate) followed by trituration with hexane gave the desired product as a pale cream solid.

Yield: 3.210 g; 59 % pale cream solid; ¹ H NMR (300 MHz, CDCI ₃ ): δ _Η 7.68 (br s, 1H N H), 7.60 (d, 1H, ArCHCHCO), 7.47 (dd, 1H, Ar H), 7.40 (d, 2H, Ar H), 7.29 (d, 1H, Ar H), 7.15 (d, 2H, Ar H), 6.86 (d, 1H, Ar H), 6.36 (d, 1H, ArCHCHCO), 4.28 (q, 2H, CH ₂ CH ₃ ), 3.87 (s, 3H, OCH ₃ ), 3.79 (s, 3H, OCH ₃ ), 1.34 (t, 3H, CH ₂ CH ₃ ). ¹³ C NMR (75 MHz, CDCI ₃ ): δ _c 167.07, 163.01, 149.15, 143.54, 138.05, 130.32, 129.19, 121.40, 120.71, 117.70, 110.48, 109.49, 60.60, 56.16, 56.14, 14.31.

Ethyl (E)-3-(4-((3,4-dihydroxyphenyl)sulfonamido)phenyl)acrylate 5

BBr ₃ (10 mL, 1M in dichloromethane) was added dropwise to ethyl (£)- 3-(4-((3,4-((3,4- dimethoxyphenyl)sulfonamido)phenyl)acrylate 4 (1.07 g) at 0°C over 30 mins. The reaction was quenched with distilled water (20 mL) and then the dichloromethane was removed in vacuo. The resulting mixture was extracted with ethyl acetate (2 x 30 mL) and the combined extractions were washed with distilled water (50 mL), saturated brine (50 mL), dried over anhydrous magnesium sulfate, decanted, solvent removed in vacuo to give a pale orange gum. Chromatography on silica (3:1 40/60 petrol: ethyl acetate) followed by trituration with hexane gave the desired product as a pale cream solid.

Yield: 0.582 g; 59 % pale cream solid; ¹ H NMR (300 MHz, (CD ₃ )2SO)): δ _Η 7.55 (d, 2H, Ar H), 7.49 (d, 1H, ArCHCHCO), 7.41 (dd, 1H, Ar H), 7.06 (d, 2H, Ar H), 6.78 (d, 1H, Ar H), 6.44 (d, 1H, ArCHCHCO), 4.14 (q, 2H, CH ₂ CH ₃ ), 3.87 1.20 (t, 3H, CH ₂ CH ₃ ). ¹³ C NMR (75 MHz, (CDs^O)): 5c 166.76, 150.50, 145.89, 144.22, 140.74, 129.94, 129.79, 129.40, 119.79, 119.14, 117.01, 115.79, 114.23, 60.37, 14.67.

8-((2-((3,4-dimethoxyphenyl)sulfonamido)ethyl)amino)napht halene-1 -sulfonic acid 6

8-(2-aminoethylamino)-1-naphthalenesulfonic acid (0.78 g) was added to a stirred solution of 3,4-dimethoxybenzene sulfonyl chloride (0.735 g) and triethylamine (3 mL) in ethyl acetate (20 mL) and /V,/V-dimethylfbrmamide (10 mL) then stirred at room temperature for 18 hrs and then the ethyl acetate was removed in vacuo. Distilled water (50 mL) was added and the pH adjusted to pH 1 with hydrochloric acid. Ethyl acetate was added and the mixture and stirred for 30 minutes at which point a colourless precipitate formed which was then filtered and washed with distilled water (50 mL), ethyl acetate (50 mL), hexane (50 mL) and then dried in vacuo to give a white solid.

Yield: 1.126 g; 84 % white solid; ¹ H NMR (300 MHz, (CD3)3SO)): δ _Η 7.68 (br s, 1H N H), 7.60 (d, 1H, ArCHCHCO), 7.47 (dd, 1H, Ar H), 7.40 (d, 2H, Ar H), 7.29 (d, 1H, Ar H), 7.15 (d, 2H, Ar H), 6.86 (d, 1H, Ar H), 6.36 (d, 1H, ArCHCHCO), 4.28 (q, 2H, CH ₂ CH ₃ ), 3.87 (s, 3H, OCH ₃ ), 3.79 (s, 3H, OCH ₃ ), 1.34 (t, 3H, CH ₂ CH ₃ ). ¹³ C NMR (75 MHz, CDCI ₃ ): δ ₀ 167.07, 163.01, 149.15, 143.54, 138.05, 130.32, 129.19, 121.40, 120.71, 117.70, 110.48, 109.49, 60.60, 56.16, 56.14, 14.31

8-((2-((3,4-dihydroxyphenyl)sulfonamido)ethyl)amino)napht halene-sulfonic acid 7 ΒΒΓ3 (3.1 mL, 1Μ in dichloromethane) was added dropwise to 8-((2-((3,4- dimethoxyphenyl)sulfonamido)ethyl)amino)naphthalene-1 -sulfonic acid 6 (0.651 g) at 0°C over 30 mins and then allowed to warm to room temperature over 2 hrs. The reaction was quenched with distilled water (10 mL) and then the dichloromethane was removed in vacuo. To the resulting mixture was added ethyl acetate (30 mL) and stirred for 30 mins resulting in white precipitate to form which was filtered and washed with distilled water (20 mL), ethyl acetate (20 mL), hexane (20 mL) and then dried in vacuo to give a white solid.

Yield: 0.500 g; 81 % white solid; ¹ H NMR (300 MHz, (CD ₃ )2SO)): δ _Η 8.32 (dd, 1H, Ar H), 8.15 (d, 1H, Ar H), 8.08 (d, 1H, Ar H), 7.71 (m, 3H, Ar H), 7.22 (d, 1H, Ar H), 7.18 (dd, 1H, Ar H), 6.91 (d, 1H, Ar H), 3.55 (t, 2H, NHCH ₂ CH ₂ NHAr), 3.17 (t, 2H, NHCH ₂ CH ₂ NHAr). ¹³ C NMR (75 MHz, (CD3)2SO): δc 150.11, 145.33, 140.28, 136.34, 133.10, 132.93, 130.06, 129.85, 128.87, 126.56, 125.87, 123.72, 121.94, 119.64, 115.87, 114.41, 52.40, 40.01.

1.2 MtDNA damage assay

Fibroblast mtDNA damage was determined following incubation with the test compounds for 24 hours, followed by UV irradiation, DNA extraction, and real-time quantitative Polymerase Chain Reaction (qPCR).

The lamps used to irradiate the cells were six iSOLde Cleo Performance 100 W-R bulbs (iSOLde, Germany) in a 6 foot sun bed, at 2 standard erythemal doses (SED). The Cleo Performance bulbs emit both UVA and UVB with approximate wavelengths of 310 nm to 400 nm.

Initially, cells were seeded at 1.5 x 10 ⁵ cells in 3 ml Dulbecco's Modified Eagle Medium (DMEM) containing Foetal Bovine Serum (FBS) and Penicillin-Streptomycin (PS) in 35 mm dishes, and incubated for approximately 16 hours at 37°C. Test compounds were made up to their chosen concentrations in either media or DMSO, and added to the cells for 24 hours of incubation. Following incubation, the test compounds were removed, and the cells were washed with PBS, and 3 ml serum-free DMEM (plus PS) was added per dish.

Dishes were irradiated at 2 SED, with non-irradiated cell dishes wrapped in foil. Following UV irradiation, media was removed and 1.5 ml Trypsin-EDTA (TE) was added to the cells, followed by incubation at 37°C for 5 minutes. TE was neutralised with complete DMEM, and the cell pellets were obtained. DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, UK) according to the manufacturer’s instructions, and total DNA concentration was determined using a NanoDrop ND2000 (ThermoFisher, UK). The level of UV-induced damage within the mtDNA was determined via real-time qPCR in a 1 kb section of the mtDNA, using a StepOnePlus Real- Time PGR System (Applied Biosystems, UK). MtDNA damage is expressed as a Ct value (where a 1 Ct difference is equivalent to a 2-fold difference in damage). An 83 bp qPCR assay was used to determine the relative amount of mtDNA present.

2. Results

Upon treating cells with the compounds described herein and exposing them to UV, the inventors measured mtDNA damage in the cells and found that compound 5 (Figure 1) provided complete protection to mtDNA damage. Other compound, such as 3 and 2 provided similar protection to Tiron, but advantageously at a much lower concentration than Tiron. Figure 2 confirms that the advantageous properties of compound 5 are not batch specific.