WO2012076842A1

WO2012076842A1 - Synthetic retinoids for control of cell differentiation

Info

Publication number: WO2012076842A1
Application number: PCT/GB2011/001691
Authority: WO
Inventors: Stefan Przyborski; Andrew Whiting; Todd Marder
Original assignee: University Of Durham
Priority date: 2010-12-09
Filing date: 2011-12-06
Publication date: 2012-06-14

Abstract

The present invention relates to synthetic retinoid compounds of formula (I) and their use in the control of cell differentiation or apoptosis.

Description

SYNTHETIC RETINOIDS FOR CONTROL OF CELL DIFFERENTIATION

Field of the Invention

The present invention relates to synthetic retinoid compounds and their use in the control of cell differentiation. There is also provided a method of medical treatment.

Background to the Invention

There are over 4000 retinoids, both natural and synthetic, structurally and often biochemically related to vitamin A,(l) All-fraws-retinoic acid 1 (ATRA), the major metabolite of vitamin A, and its two isomers, 9-ci.s-retinoic acid 2 (9CRA) and 13-ds-retinoic acid 3 (13CRA), have essential roles in many biological processes during both chordate embryogenesis and adult homeostasis. (2, 3) These include cellular differentiation, proliferation and apoptosis,(4) embryonic development^) and vision.(6) Due to the ability of retinoids to control differentiation and apoptosis in both normal and tumour cells, they have the potential to act as chemopreventative and chemotherapeutic agents, although toxicity has prevented widespread use.(7, 8) ATRA and other commercially available retinoids are, however, used to treat dermatological conditions.(9)

The use of retinoic acids as inducers of cell differentiation has some limitations, exemplified by ATRA which readily isomerises resulting in mixtures of ATRA, 9CRA, 13CRA and other species.(lO) This instability derives from the five conjugated double bonds which absorb light in the 300-400 nm region which can occur under laboratory lighting conditions.( 11-14) Murayama et a/.(l l) reported that isomers of ATRA differentially affect the ability of mammalian stem cells to differentiate along alternative lineages. The result, when applied to pluripotent stem cell differentiation can be increased culture heterogeneity due to differential initiation of key molecular pathways. (15, 16) In order to increase reproducibility in the differentiation process, stable retinoid derivatives are desirable; an approach adopted by us.(10) These types of stable compounds can maintain a constant concentration in the culture system, and are readily stored and handled. This is preferable to the alternative approach of attempting to reduce the sensitivity of ATRA to isomerisation by the use of additives such as bovine serum albumin (BSA), fibrogen, lysozyme, phosphatidylcholine N-ethylmaleimide and vitamin C.(17, 18) None of these additives completely suppress isomerisation, and indeed, they may affect cellular processes on their own.

Some known retinoid compounds have toxicity issues which have prevented their wide spread use. Retinoid compounds such as ATRA are commonly used to induce cell differentiation. However, ATRA exhibits poor stability, in particular upon exposure to light. ATRA compounds isomerise and degrade upon exposure to light. To overcome this, efforts are made to store and work with ATRA in the dark. Such precautions increase the cost associated with working with ATRA, and don't entirely mitigate the problem. As ATRA is liable to photoisomerisation and degradation upon storage, it is difficult to accurately predict the amount of active compound administered in a single dose.

Efforts have been made to overcome the problems associated with ATRA by synthesising stable retinoid compounds. It is generally believed that ATRA is susceptible to photoisomerisation due to its conjugated linker group. PCT/GB2007/003237 proposed new retinoid compounds which exhibited good stability and induced cell differentiation, of which EC23 4 is an example. This document explicitly taught away from the use of polyene linker groups, as it taught that the incorporation of such linker groups affected the stability of the retinoid compounds to exposure to light. As well as exhibiting good stability upon storage, the compounds of PCT/GB2007/003237 were also found to not be susceptible to metabolic degradation, and thus may have a relatively long associated half-life in the human or animal body.

Summary of the Invention

According to the present invention there is provided retinoid compounds comprising a conjugated linker group which exhibit good stability and can be used to control cell differentiation.

According to the present invention there is provided the compound as shown below and its use in the control of cell differentiation or apoptosis:

— Hydrogen Bond Donor

The aromatic group of the compound as shown above may be substituted or unsubstituted. The conjugated linker group comprises one or more carbon-carbon double bond, and/or one or more carbon-carbon triple bond. Preferably the conjugated linker group is substituted with one or more small alkyl group, in particular one or more methyl group.

Surprisingly, regardless of the conjugated linker group, the compound of the present invention exhibits good stability, and undergoes photoisomeriation far less easily than ATRA, whilst controlling cell differentiation and apoptosis to an extent commensurate with or greater than ATRA.

According to the present invention there is provided retinoid compounds useful in the control and inducement of cell differentiation. Surprisingly, the compounds of the present invention exhibit good stability upon storage even upon exposure to light. The compounds of the present invention do not degrade through photoisomerisation to any appreciable extent. In addition, the compounds exhibit very promising biological activity, generally commensurate to or greater than that exhibited by known retinoid compounds such as ATRA. The compounds of the present invention generally exhibit biological activity at concentrations far lower than those at which ATRA stops exhibiting biological activity. Despite not being susceptible to oxidative degradation, the compounds of the present invention are susceptible to metabolic degradation meaning that they are particularly well suited to therapeutic applications in the human and animal body. Metabolic degradation may lead to isomerisation of the compounds but it has generally been found that both isomers exhibit good biological activity. The metabolic degradation of the compounds means that they can be expelled easily from cells, thus reducing the associated half-life of the compounds of the present invention in the human or animal body. Description of Various Embodiments

Conjugated

As used herein the term "conjugated" is used to refer to a cyclic or acylic moiety comprising two or more alternating single and multiple bonds, in particular two or more alternating carbon-carbon single bonds and carbon-carbon multiple bonds.

Multiple Bond

A multiple bond is generally used to refer to a double or triple bond. Polyenic

The terms "polyene" and "polyenic" as used herein include reference to moieties comprising two or more conjugated carbon-carbon double bonds.

Hydrocarbyl

The term "hydrocarbyl" as used herein includes reference to moieties consisting exclusively of hydrogen and carbon atoms; such a moiety may comprise an aliphatic and/or an aromatic moiety. The moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of hydrocarbyl groups include Cl-6 alkyl (e.g. CI, C2, C3 or C4 alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl); Cl-6 alkyl substituted by aryl (e.g. benzyl) or by cycloalkyl (e.g cyclopropylmethyl); cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl); alkenyl (e.g. 2-butenyl); alkynyl (e.g. 2-butynyl); aryl (e.g. phenyl, naphthyl or fluorenyl) and the like.

Alkyl The terms "alkyl" and "CI -6 alkyl" as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. This term includes reference to groups such as methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl or tert- butyl), pentyl, hexyl and the like. In particular, alkyl may have 1, 2, 3 or 4 carbon atoms.

Small Alkyl Group

The term "small alkyl group" is used to refer to an alkyl group having 1 to 4 carbon atoms, generally 1 to 3 carbon atoms. Alkenyl

The terms "alkenyl" and "C2-6 alkenyl" as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one double bond, of either E or Z stereochemistry where applicable. This term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the like.

Alkynyl

The terms "alkynyl" and "C2-6 alkynyl" as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one triple bond. This term includes reference to groups such as ethynyl, 1-propynyl, 2- propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2- hexynyl and 3-hexynyl and the like.

Alkoxy

The terms "alkoxy" and "CI -6 alkoxy" as used herein include reference to -O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms. This term includes reference to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like.

Cvcloalkyl

The term "cycloalkyl" as used herein includes reference to an alicyclic moiety having 3, 4, 5, 6, 7 or 8 carbon atoms. The group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl and the like.

Aryi

The term "aryl" as used herein includes reference to an aromatic ring system comprising 6, 7, 8. 9, 10, 1 1 , 12, 13, 14, 15 or 16 ring carbon atoms. Aryl is often phenyl but may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.

Carbocyclyl

The term "carbocyclyl" as used herein includes reference to a saturated (e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or 16 carbon ring atoms. In particular, carbocyclyl includes a 3- to 10-membered ring or ring system and, in particular, a 5- or 6-membered ring, which may be saturated or unsaturated. A carbocyclic moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like. Heterocyclyl

The term "heterocyclyl" as used herein includes reference to a saturated (e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulphur. In particular, heterocyclyl includes a 3- to 10-membered ring or ring system and more particularly a 5- or 6-membered ring, which may be saturated or unsaturated.

A heterocyclic moiety is, for example, selected from oxiranyl, azirinyl, 1 ,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyI, pyrrolyi, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyraziny], pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, especially thiomorpholino, indolizinyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, cumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, [beta]-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl and the like.

Heterocvcloalkyl

The term "heterocycloalkyl" as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1 , 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulphur. The group may be a polycyclic ring system but more often is monocyclic. This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl and the like.

Heteroaryl

The term "heteroaryl" as used herein includes reference to an aromatic heterocyclic ring system having 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen and sulphur. The group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but is more often monocyclic. This term includes reference to groups such as pyrimidinyl, furanyl, benzo[b]thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridinyl, benzo[b]furanyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and the like.

Halogen

The term "halogen" as used herein includes reference to F, CI, Br or I. In a particular, halogen may be F or CI, of which F is more common.

Substituted

The term "substituted" as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described B2011/001691

8 substituents. The term "optionally substituted" as used herein means substituted or unsubstituted.

It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible. For example, amino or hydroxy groups with free hydrogen may be unstable if bound to carbon atoms with unsaturated (e.g. olefinic) bonds. Additionally, it will of course be understood that the substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restriction to appropriate substitutions as recognised by the skilled man.

Independently

Where two or more moieties are described as being "each independently" selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.

Compounds of the Present Invention

According to a first aspect of the present invention there is provided the compound of Formula I and its use in the control of cell differentiation and cell apoptosis

Formula I Wherein Rl to Rl l independently represent hydrogen, optionally substituted alkyl group, optionally substituted aryl group or Ry,

wherein each Ry is independently represents halogen, trifluoromethyl, cyano, nitro, oxo, NRz, -ORz, -C(0)Rz, -C(0)ORz, -OC(0)Rz, -S(0)IRz, -N(Rz)Ra, - C(0)N(Rz)Ra, - S(0)I (Rz)Ra and Rb;

Rz and Ra are each independently hydrogen or Rb; 1

9

Rb is selected from hydrocarbyl and -(CH2)k-heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, CI -6 alkyl and CI -6 alkoxy;

k is 0, 1, 2, 3, 4, 5 or 6; and

I is 0, 1 or 2;

or one or more Rl to Rl 1 taken together with the atoms to which they are attached form a carbocycle or a heterocycle, optionally substituted with one or more Ry;

wherein the backbone of the conjugated group has at least four carbon atoms; generally 4 - 10; typically 4 to 8; preferably 6 to 8; more preferably 6 carbon atoms; said conjugated group may be unsubstituted or may be substituted with one or more optionally substituted alkyl group, optionally substituted aryl group or Ry;

wherein X represents a hydrogen bond donor group, typically -C(0)Z, wherein Z may be selected from -OH, - C(0)OH, 0(Cl-6 alkyl), -NH2 and NHOH. Preferably Z is selected from -OH, OCH3 and NHOH.C02H, C(O).

According to one embodiment, the conjugated group comprises at least one carbon-carbon triple bond and at least one carbon-carbon double bond. Typically the conjugated group includes one carbon-carbon triple bond and two or three carbon-carbon double bonds. The conjugated group may comprise a carbon-carbon triple bond towards, or at the carbon atom closest to the carbocyclyl group. Alternatively, the carbon-carbon triple bond may towards, or at the carbon atom closest to the hydrogen bond donor X group.

Alternatively, all of the multiple carbon-carbon bonds of the conjugated group may be carbon-carbon triple bonds, or all may be carbon-carbon double bonds.

Typically the compound of Formula 1 has the following structure:

Generally Rl, R2 and R3 represent hydrogen.

Typically R4, R5, RIO and Rl l independently represent short alkyl group, generally comprising 1 to 6 carbon atoms; typically 1 to 3 carbon atoms. The alkyl group may be substituted or unsubstituted. Advantageously, the alkyl group is a methyl group.

According to one embodiment, R6, R7, R8 and R9 represent hydrogen.

Generally the conjugated group comprises at least three carbon - carbon multiple bonds; typically three or four carbon-carbon multiple bonds. Preferably, the conjugated group has three carbon-carbon multiple bonds. According to one embodiment, the conjugated group has one carbon-carbon triple bond and two carbon-carbon double bonds. The carbon-carbon triple bond may be located at the carbon atom closest to the carbocyclyl group. According to one embodiment, the conjugated group is substituted with one or more short alkyl groups, generally comprising 1 to 6 carbon atoms; typically 1 to 3 carbon atoms. The alkyl group may be substituted or unsubstituted. Advantageously, the alkyl group is a methyl group. Typically the conjugated group is substituted with one or two methyl groups, suitably one methyl group.

Suitably the conjugated group is substituted with an alkyl group at the double bond closest to the X group.

According to one embodiment the conjugated group is substituted with an alkyl group cis to the X group. Advantageously, the alkyl group is a methyl group.

According to one embodiment the conjugated group is substituted with an alkyl group trans to the X group. Advantageously, the alkyl group is a methyl group.

The backbone of the conjugated group may comprise one or more atoms other than carbon. Generally the conjugated group is directly attached to the carbocyclyl group of Formula 1. However, the conjugated group may be attached via a linker group.

X preferably represents CQ₂H.

According to one embodiment, there is provided a compound according to either one of Formula II and Formula III and its use in the control of cell differentiation and cell apoptosis:

Formula II

wherein Rl to Rl 1 and X are as defined above;

B represents a substituted or unsubstituted alkyl group generally comprising 1 to 6 carbon atoms;

the multiple bond within the square brackets of Formulae II and III may represent a carbon- carbon double bond or a carbon-carbon triple bond, 11 001691

12 and A represents 2, 3 or 4.

Generally Rl, R2, R3, R6, R7, R8 and R9 all represent hydrogen. Advantageously, R4, R5, RIO and Rl 1 independently represent a small alkyl group, typically methyl.

Typically B represents a substituted or unsubstiruted alkyl group comprising 1 to 3 carbon atoms. Advantageously, B represents a methyl group.

The carbon chain contained within the brackets of Formulae II and III represents the conjugated linker group. According to one embodiment the conjugated linker group includes at least one carbon-carbon triple bond and at least one carbon-carbon double bond. Typically a carbon-carbon triple bond is located at the carbon atom closest to the carbocyclyl group.

Generally A represents 2 or 3, preferably 2.

Where A represents 2, the conjugated linker group generally comprises one carbon-carbon triple bond (typically at the carbon atom closest to the carbocyclyl group), and one carbon- carbon double bond.

Where A represents 3, the conjugated linker group generally comprises one carbon-carbon triple bond (typically at the carbon atom closest to the carbocyclyl group), and two carbon- carbon double bonds.

According to one embodiment, the compound of formula 1 has one of the following structures:

Compound 31

Compound 42

Alternatively, the compound of formula 1 may have the structure as shown for compound 42 but with an E, E, Z configuration. Such compound may be named compound 43. Preferably the compound has the structure of compound 32. Compound 32 exhibits biological activity at relatively low concentrations. For example biological activity is exhibited at levels as low as 0.01 μΜ.

As noted above, retinoid compounds such as ATRA are unstable upon storage. In particular, such compounds are susceptible to photoisomerisation and degradation upon exposure to light in the 300 to 400 nm region. This instability derives from the five conjugated double bonds. It was long believed that the stability of synthetic retinoids having the cell differentiation properties of ATRA could be increased by removing the conjugated linker group, or minimising the length of the conjugated linker group. Surprisingly, the compounds of Formula I, II and III of the present invention are stable upon exposure to light and undergo far less photoisomerisation and degradation than ATRA. Typically the compounds of formula I, II and III do not undergo photoisomerisation to any appreciable extent.

Generally the compounds of formula 1 have far better stability than retinoids such as ATRA, in particular the compounds of formula 1 are far less susceptible to photoisomerisation.

Generally, following 3 days exposure to light having a wavelength of 300 to 400 nm, the compounds of the present invention undergo far less isomerisation and degradation than ATRA- Typically at least 60% by weight of the compounds of the present invention remain compared to less than 40% by weight ATRA.

Typically, where the compounds of the present invention are according to Formula II or III and A represents 2, at least 60% by weight of the compounds remain following 3 days exposure to light having a wavelength of 300 to 400 nm.

Typically, where the compounds of the present invention are according to Formula II or III and A represents 3, at least 50% by weight of the compounds remain following 3 days exposure to light having a wavelength of 300 to 400 nm. In addition, the amount of degradation of the compounds is far less than for ATRA.

As noted above, the compounds of the present invention are far more stable upon exposure to light than known retinoid compounds comprising a conjugated linker group. The compounds of the present invention do not tend to degrade greatly through photoisomerisation. However, the compounds of the present invention tend to be susceptible to metabolic degradation, for instance metabolically induced isomerisation. The compounds of the present invention may be degraded biologically, aiding the successful expulsion of the compounds from human or animal cells and thus reducing the half life associated with the compounds of the present invention in the human or animal body. The compounds of the present invention are thus extremely well suited to therapeutic uses in the human or animal body.

The compounds of formulae I, II and III control cell differentiation though arresting cell proliferation and inducing differentiation. Comparing the number of cells in a stem cell sample before and after exposure to a compound compared to a control, and compared to ATRA provides a good indication of the effectiveness of the compound in controlling cell differentiation.

Generally, following exposure of a stem cell sample to a compound of Formulae I, II or III for a 7 day period, the number of stem cells present has reduced due to the proliferation of the cells being arrested and their differentiation being induced. The number of stem cells following exposure to the compounds of the present invention is generally 70% or less of the number of stem cells following exposure to a control, typically 50% or less. Surprisingly, the compounds of the present invention exhibit enhanced biological activity compared to known retinoid compounds such as ATRA.

The number of stem cells following exposure to the compounds of the present invention is generally no more than 3 times greater than that following exposure of the sample to ATRA, typically no more than 2 times greater, suitably around 1.5 to 2 times greater. According to one embodiment, the number of stem cells following exposure of the compounds of the present invention is around the same as the number of stem cells following exposure to ATRA. Typically, where the compounds of the present invention are according to Formula II or III and A represents 2, the number of stem cells following exposure to the compounds of the present invention is generally around 50% of the number following exposure to the sample to a control. Typically, where the compounds of the present invention are according to Formula II or III and A represents 2, the number of stem cells following exposure to the compounds of the present invention is 1.5 to 2 times greater than that following exposure of the sample to ATRA. Compounds 16, 17 and 36 (as shown below) have a similar structure to the compounds of the present invention. However, following exposure of a stem cell sample to such compounds, the number of stem cells is greater than for the control composition. This illustrates how minor changes in the structure of a compound can result in large changes in the properties exhibited.

Assessing the strength of the retinoic acid response element (RARE) following exposure of a sample to a compound also provides a good indication of the effectiveness in activating the retinoid signalling pathways. This may be assessed by assessing whether the retinoid signalling pathway is activated by the compounds through activation of one or multiple RAR receptors. This is indicated through the extent of staining following exposure to beta- galactosidase.

Generally samples exposed to the compounds of Fomulae I, II or III exhibit substantially more beta-galactosidase staining than those exposed to the control composition.

Typically, samples exposed to the compounds according to Formula II or III where A represents 2, exhibit a level of staining commensurate with those exposed to ATRA.

As noted above, the compounds of the present invention induce differentiation of cells. This can be assessed by considering the expression of genes such as Nanog and Oct-4 which act as "gatekeepers" of pluripotency, or the expression of motor and/or neural phenotypes such as Pax-6. Alternatively, the expression of neuronal specific proteins can be investigated by assessing antibodies raised against neuronal specific markers such as β-ΙΙΙ-tubulin marker Tuj 1.

Exposure of cell samples to the compounds of the present invention affect the expression of such genes as much as or more than for samples exposed to compounds such as ATRA. Generally the expression of genes known as gatekeepers of pluripotency is suppressed. Typically the expression of such genes is suppressed at least 75% as much as samples exposed to ATRA; typically at least 80% as much; advantageously around 100% as much as samples exposed to ATRA preferably around 120% as much as samples exposed to ATRA, more preferably around 150% as much as samples exposed to ATRA. Generally the expression of neuronal specific proteins was increased following exposure of cell samples to the compounds of the present invention, typically commensurate with or greater than a sample exposed to ATRA. Typically the expression of such genes is increased at least 75% as much as samples exposed to ATRA; typically at least 80% as much; advantageously around 100% as much as samples exposed to ATRA, preferably around 120% as much as samples exposed to ATRA, more preferably around 150% as much as samples exposed to ATRA. A farther advantage of the compounds of the present invention is that they remain active as relatively low concentration. The compounds of the present invention generally exhibit biological activity at concentrations far lower than those at which ATRA stops exhibiting biological activity. According to one embodiment, the cell samples were exposed to the retinoids of interest for 3 to 14 days. Generally the cell samples were exposed to media supplemented with the retinoids of interest at a concentration of 0.001 μΜ to 10 μΜ; suitably 1 μΜ to 10 μΜ.

The expression of the proteins and genes of interest may be assessed using flow cytometry or RT-PCR techniques respectively.

Typically, the compounds of the present invention induce the differentiation of stem cells, such as human neural stem cells into neural sub-types. Generally the compounds of the present invention induce differentiation of cells to an extent commensurate to or greater than known retinoids such as ATRA.

Following exposure of a sample comprising stem cells, for instance a cell derived from the ventral mesencephalon of human foetal brain tissue, to media supplemented with the compounds of the present invention the number of differentiated cells expressing neuronal markers is substantially increased. Typically the sample is exposed to such media for around 7 days.

Following exposure of a stem cell sample to media supplemented with the compounds of the present invention the number of differentiated cells is generally at least 100% greater than a 1

18 sample exposed to a control media, typically at least 150% greater, suitably at least 200% greater.

Typically, following exposure of a stem cell sample to media supplemented with the compounds of the present invention the number of differentiated cells is at least 75% of the number of differentiated cells present for a sample exposed to media supplemented with ATRA; generally at least 85%; suitably around 100%.

The compounds of Formulae I, II and III provide control of cell differentiation to an extent commensurate with or greater than that provided by ATRA. In addition the compounds of the present invention generally exhibit biological activity at far lower concentrations than ATRA.

However, the compounds of Formulae I, II and III are far more stable than ATRA following exposure to light. The compounds of Formulae I, II and III are far less susceptible to photoisomerisation and degradation than known retinoid compounds such as ATRA. This enables the compounds of the present invention to be used and stored in light conditions. In addition, the amount of compound administered can be precisely and accurately determined even following storage of the composition.

A further advantage of the compounds of the present invention over retinoid compounds such as those disclosed in PCT/GB2007/003237 is that they are more susceptible to metabolic degradation, the compounds can be expelled from the human or animal body, meaning that their half life in the body is relatively short. The compounds of the present invention are extremely well suited to therapeutic applications in the human and animal body. Composition

According to a further aspect of the present invention there is provided a composition comprising one or more of the compounds of the present invention in combination with one or more pharmaceutically acceptable excipients. The composition of the present invention also includes one or more pharmaceutically acceptable carriers, excipients, adjuvants or diluents. The phrase "pharmaceutically 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, as the case may be, an animal without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

When the composition of the invention is prepared for oral administration, the compounds described above are generally combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. For oral administration, the composition may be in the form of a powder, a granular formation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The composition may also be presented as a bolus, electuary or paste. Orally administered compositions of the invention can also be formulated for sustained release, e.g., the compounds described above can be coated, microencapsulated, or otherwise placed within a sustained delivery device. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation. Thus, one or more suitable unit dosage forms comprising the compounds of the invention can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary, mucosal, intraocular and intranasal (respiratory) routes. The composition may also be formulated in a lipid formulation or for sustained release (for example, using microencapsulation, see WO 94/07529, and US Patent No. 4,962,091 incorporated herein by reference). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well-known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

Pharmaceutical formulations comprising the compounds of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the compound can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatine, and polyvinylpyrrolidone. Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds can also be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.

For example, tablets or caplets containing the compounds of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Suitable buffering agents may also include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxyl propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatine capsules containing at least one compound of the invention can contain inactive ingredients such as gelatine, microcrystalline cellulose, sodium lauryl sulphate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets containing one or more compounds of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.

The therapeutic compounds of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. The pharmaceutical formulations of the therapeutic compounds of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve. B2011/001691

21

Thus, the therapeutic compounds may be formulated for parenteral administration (e.g. by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The active compound(s) and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active compound(s) and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water before use.

It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colourings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and [alphaj-tocopherol and its derivates can be added.

These formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well-known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, acetic acid, ethanol, isopropyl alcohol, dimethyl sulphoxide, glycol ethers such as the products sold under the name "Dowanol", polyglycols and polyethylene glycols, CI- C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol", isopropyl mytrisate, animal, mineral and vegetable oils and polysiloxanes.

Preferably, the composition is in the form of a solvent or diluent comprising one or more of the compounds as described above. Solvents or diluents may include acid solutions, dimethylsulphone, N-(2- mercaptopropionyl) glycine, 2-n-nonyl-],3-dioxolane and ethyl alcohol. Preferably the solvent/diluent is an acidic solvent, for example, acetic acid, citric acid, boric acid, lactic acid, propionic acid, phosphoric acid, benzoic acid, butyric acid, malic acid, malonic acid, oxalic acid, succinic acid or tartaric acid,

The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non- limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0.

More preferably, the solvent is an acetic acid solution. The solvent, for example acetic acid solution, may be present in the composition at a concentration of less than 1%, 0.5%, 0.25%, 0.1%, 0.05% or 0.01 % acid, for example acetic acid .

The composition of the present invention may comprise one or more additional therapeutic agents. For instance, where the composition of the presen invention is useful in the treatment or prevention of cancer, one or more additional chemotherapeutic and or chemopreventative agents may be included. Where the composition is useful in skincare one or more additional skincare agent may be used such as one or more moisturising or antibacterial agent.

Additionally, the compounds of the present invention are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active compound, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g. stents, catheters, peritoneal dialysis tubing, draining devices and the like.

For topical administration, the active agents may be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, powders, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g. sprays or foams), soaps, detergents, lotions or cakes of soap. Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the therapeutic compounds of the invention can be delivered via patches or bandages for dermal administration. Alternatively, the therapeutic compounds can be formulated to be part of an 11 001691

23 adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.

Pharmaceutical formulations for topical administration may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.001 mg/ml and about 100 mg/ml, for example between 0.1 mg/ml and 10 mg/ml, of one or more of the compounds of the present invention specific for the indication or disease to be treated.

Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active compounds can also be delivered via iontophoresis, e.g., as disclosed in US Patent Nos. 4,140,122; 4,383,529; or 4,051,842 which are incorporated herein by reference. The percentage by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-85% by weight.

Drops, such as eye drops or nose drops, may be formulated with one or more of the therapeutic compounds in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays can be pumped, or are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, via a plastic bottle adapted to deliver liquid contents drop-wise, or via a specially shaped closure.

The therapeutic compound may further be formulated for topical administration in the mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatine and glycerine or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier. The compounds of the invention can also be administered to the respiratory tract. Thus, the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention. In general, such dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of a specific infection, indication or disease. Any statistically significant attenuation of one or more symptoms of an infection, indication or disease that has been treated pursuant to the method of the present invention is considered to be a treatment of such infection, indication or disease within the scope of the invention. Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g. gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered- dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newinan, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp. 197-224, Butterworths, London, England, 1984).

Therapeutic compounds of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form. Thus, other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.001 mg/ml and about 100 mg/ml of one or more of the compounds of the present invention specific for the indication or disease to be treated. Dry aerosol in the form of finely divided solid particles of the compounds described above that are not dissolved or suspended in a liquid are also useful in the practice of the present invention. Compounds of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5μπι, alternatively between 2 and 3 μη . Finely divided particles may be prepared by pulverization and screen filtration using techniques well-known in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic compounds of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those described in U.S. Patent Nos, 4,624,251; 3,703,173; 3,561,444; and 4,635,627 which are incorporated herein by reference. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co. (Valencia, CA). For intra-nasal administration, the therapeutic agent may also be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).

Furthermore, the active ingredients may also be used in combination with other therapeutic agents, for example one or more of pain relievers, anti-inflammatory agents, antihistamines, bronchodilators, chemoprotective agents, chemotherapeutic agents, antibacterial agents and the like.

Use

In a preferred use according to the invention there is provided the use of a compound or composition as defined herein in the differentiation of a stem cell into at least one differentiated cell type.

The stem cell may typically be a human or animal totipotent stem cell, in particular a non- human totipotent stem cell for example a totipotent cell of a mammal, for example a mouse, a rat or a rabbit. Alternatively, the stem cell may be a pluripotent stem cell of a human or animal, preferably a human pluripotent stem cell.

In an alternative preferred embodiment of the invention said stem cell is a multipotent stem cell of a human or animal.

In a preferred embodiment of the invention said multipotent stem cell is selected from the group consisting of: haemopoietic stem cell, neural stem cell, bone stem cell, muscle stem cell, mesenchymal stem cell, epithelial stem cell (derived from organs such as the skin, gastrointestinal mucosa, kidney, bladder, mammary glands, uterus, prostate and endocrine glands such as the pituitary), ectodermal stem cell, mesodermal stem cell or endodermal stem cell (for example derived from organs such as the liver, pancreas, lung and blood vessels).

According to a further aspect of the invention there is provided a method to induce the differentiation of a stem cell comprising the steps of:

i) forming a preparation of stem cells in a cell culture medium suitable for maintaining said stem cells wherein said culture medium comprises a compound according to Formula I; and ii) cultivating said stem cells in conditions that allow their differentiation into at least one differentiated cell type. In a preferred method of the invention said stem cell is a multipotent or pluripotent stem cell. According to one embodiment the stem cell is not a totipotent stem cell. Preferably said stem cell is human.

In a preferred method of the invention said differentiated cell is selected from the group consisting of a keratinocyte, a fibroblast (e.g. dermal, corneal, intestinal mucosa, oral mucosa, bladder, urethral, prostate, liver), an epithelial cell (e.g. dermal, corneal, intestinal mucosa, oral mucosa, bladder, urethral, prostate, liver), a neuronal glial cell or neural cell, a hepatocyte, a mesenchyma cell, a muscle cell (cardiomyocyte or myotube cell), a kidney cell, a blood cell (e.g.CD4+ lymphocyte, CD8+ lymphocyte), a pancreatic cell, or an endothelial cell.

Generally the medium has a concentration of 5 to 20 μΜ compound of the present invention; typically around 10 μΜ.

In a preferred method of the invention the method takes place in the presence of visible and/or UV light, temperatures not exceeding 50 degress Celsius (for example -80 degrees Celsius up to 50 degrees Celsius, typically -20 degrees Celsius up to about 44 degrees Celsius) and/or oxidative reagents for example air or DMSO. The method of the invention may take place ex vivo, in vivo or in vitro.

According to a further aspect of the present invention there is provided the use of a compound according to Formula I in the treatment or prevention of a disease or condition that would benefit from retinoid therapy.

The disease or condition typically benefits from the control of cell differentiation or apoptosis.

Diseases or conditions that may benefit from retinoid therapy include cancer (e.g. neural neoplasms), skin disorders such as acne, skin wounds e.g. burns, UV damage, aging skin.

The compounds of the present invention may act as chemotherapeutic, chemopreventative agents due to their ability to control differentiation and apoptosis in normal and tumour cells. In particular the compounds of the present invention may be particularly well suited to the treatment or prevention of precancerous or cancerous conditions including those of the skin, oral cavity, larynx, lung, bladder, vulva, breast, digestive tract. The compounds of the present invention may be used in the treatment or prevention of basal cell carcinomas, squamous cell carcinomas, including those of the head and neck, bladder tumours. Cancers particularly suited for treatment or prevention through use of the compounds of the present invention include leukaemia, such as myelogenous leukaemia, in particular acute promyelocyte leukaemia.

It is believed that the compounds of the present invention suppress transformation of cells in vitro and inhibit carcinogenesis. It is believed that the compounds of the present invention thus exhibit suppressive effects on tumor promotion, and/or tumor initiation. When used as chemotherapeutic agents, the compounds of the present invention generally arrest or reverse carcinogenic steps, reducing or avoiding the clinical consequences of overt malignancies. It is believed that the compounds of the present invention exhibit chemotherapeutic and/or chemopreventative properties due to their ability to modulate the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells in vitro and in vivo.

According to a further aspect of the present invention there is provided the use of the compounds of the present invention in the promotion of cell proliferation, for example skin or neural cell proliferation.

According to a further aspect of the present invention there is provided the use of the compounds of the present invention in promoting tissue health and development, in particular in promoting the health and development of the skin, bone, nerves teeth, hair and/or mucous membranes of the human or animal body. The compounds of the present invention may be used in the prevention or treatment of the signs of ageing (in particular, wrinkles and age spots), skin conditions such as acne (especially severe and/or recalcitrant acne), psoriasis, stretch marks, keratosis pilaris, emphysema, baldness.

According to a further aspect of the present invention, the compounds of the present invention may be used in the treatment or prevention of diseases or conditions of the eye, or may be used to maintain or maximise vision. According to a further aspect of the present invention, the compounds of the present invention may be used as antioxidants, in particular for use in or on the human or animal body. 1

29

The dosage of the compound of the present invention to be administered to the human or animal body is dependent on the intended use. For instance, formulations suitable for topical application generally comprise 0.025 to 1 wt % compound of the present invention, in particular, 0.025 to 0.1 wt %. For chemotherapeutic uses, a dosage of 20 to 80 mg/m2/day is usual, suitably 40 to 50 mg/m2/day, more suitably around 45 mg/m2/day.

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.

The present invention will now be described by way of example only with reference to the accompanying figures. Figure 1 provides a generic retinoid structure;

Figure 2A shows the NMR spectra of Compound 16 before exposure to fluorescent light and Figure 2B shows the NMR spectra of Compound 16 after exposure to fluorescent light with the percentage of Compound 16 remaining;

Figure 3 A shows the NMR spectra of Compound 17 before exposure to fluorescent light and Figure 3B shows the NMR spectra of Compound 17 after exposure to fluorescent light with the percentage of Compound 17 remaining;

Figure 4 A shows the NMR spectra of Compound 36 before exposure to fluorescent light and Figure 4B shows the NMR spectra of Compound 36 after exposure to fluorescent light with the percentage of Compound 3 remaining; Figure 5 A shows the NMR spectra of Compound 31 before exposure to fluorescent light and Figure 5B shows the NMR spectra of Compound 31 after exposure to fluorescent light with the percentage of Compound 31 remaining;

Figure 6A shows the NMR spectra of Compound 32 before exposure to fluorescent light and Figure 6B shows the NMR spectra of Compound 32 after exposure to fluorescent light with the percentage of Compound 32 remaining;

Figure 7A shows the NMR spectra of Compound 42 before exposure to fluorescent light and Figure 7B shows the NMR spectra of Compound 42 after exposure to fluorescent light with the percentage of Compound 42 remaining;

Figure 8 provides phase micrographs showing lacZ gene expression visualised through X-gal staining for ?-ga!actosidase for cell cultures exposed to A: no compounds (control), B: DMSO, C: 10 μΜ ATRA, D: 10 μΜ compound 31 , E: 10 μΜ compound 32, F: 10 μΜ compound 17, G: 10 μΜ compound 16, H: 10 μΜ compound 36, 1: 10 μΜ compound 42/43; Figure 9 shows an MTS assay indicating the number of viable cells in cultures exposed to various retinoids after 3 and 7 days;

Figure 10A shows the number of viable cells as a percentage of the total cell population of cell cultures exposed to various retinoids after 3 and 7 days;

Figure 10B shows the number of apoptopic cells as a percentage of the total cell population of cell cultures exposed to various retinoids after 3 and 7 days;

Figure 11 shows flow cytometric analysis of the induction of TERA2.cl.SP12 cell differentiation in response to ATRA, compound 31 and compound 32 using stem cell marker SSEA-3 (11 A), stem cell marker TRA-1-60 (1 IB) and neural differentiation marker A2B5 (1 1C);

Figure 12 shows flow cytometric analysis of the induction of TERA2.cl.SP12 cell differentiation in response to ATRA, compound 31 and compound 32 at different concentrations using stem cell marker SSEA-3 (12A), stem cell marker TRA-1-60 (12B) and neural differentiation marker A2B5 (12C);

Figure 13 shows RT-PCR analysis of the induction of TERA2.cl.SP12 cell differentiation in response to ATRA, compound 31 and compound 32 using stem pluropotency phenotype Nanog (13A), Oct-4 (13B) and motor and ventral neural phenotype Pax 6 (13C);

Figure 14 shows immunofluorescent micrographs showing the expression of ?-III-tubulin and mature neuronal marker neurofilament-200 in cultures comprising TERA2.cl.SP12 cells exposed to 10 μΜ ATRA, compound 31 and compound 32 for 21 days; Figure 15 shows phase and immunofluorescent micrographs showing the expression οίβ-ΙΙΙ- tubulin and mature neuronal marker neurofilament-200 in cultures comprising ReNcell 1 7VM cells exposed to 1 μΜ ATRA, compound 31 and compound 32 for 7 days;

Figure 16 shows quantification of the immunological data, showing the expression of J-III- tubulin and mature neuronal marker neurofilament-200 in cultures comprising ReNcell 197VM cells exposed to 1 μΜ ATRA, compound 31 and compound 32 for 7 days as performed by counting positively labelled cells;

Figure 17 shows immunofluorescent micrographs showing the expression of /Hll-tubulin and mature neuronal marker neurofilament-200 in cultures comprising ReNcell 197VM cells exposed to ATRA, compound 31 and compound 32 for 7 days at differing concentrations; Figure 18 shows quantification of the immunological data showing the expression of β-ΙΙΙ- tubulin and mature neuronal marker neurofilament-200 in cultures comprising ReNcell 197VM cells exposed to ATRA, compound 31 and compound 32 for 7 days at differing concentrations as performed by counting positively labelled cells.

Figure 19 (a) shows immunofluorescent micrographs showing that AH61 (compound 32) is a potent inducer of neurogenesis over a range of concentrations whereas ATRA is significantly less effective at lower levels; (b) shows an immunofluorescent micrograph and data showing the length of neurites developed from TERA2.cl.SP12 stem cells differentiated by different retinoids. Aggregates were differentiated by Ι μΜ of the compound for 21 days prior to removal of retinoid and induction of outgrowth by laminin. The maximum length of neurites from each treatment group was measured. An example image of AH61 treated cells is shown. A total of 15 neurites were measured for each treatment. Data demonstrates that the maximum length of neurites formed from aggregates differentiated by AH61 is significantly higher than ATRA.

Figure 20 (a) shows a histogram demonstrating that a single topical application of AH61 (compound 32) to the surface of murine skin results in significantly enhanced thickening of the epidermis. No significant effect to the dimensions of the skin was detected by ATRA at these concentrations when using a single dose; (b) Involucrin staining shows increased expression in skin samples treated with AH61 above control levels indicating enhanced biological activity; (c) shows a histogram demonstrating that a single topical application of AH61 and ATRA to the surface of murine skin results in significantly enhanced cell division as indicated by the cell proliferation marker Ki67. Higher levels of induced cell proliferation was achieved with AH61 compared to ATRA; (d) this image shows that Keratin 14, a marker of immature keratinocytes, was enhanced in skin samples exposed to AH61 compared to control and mice treated with ATRA.

Synthetic Retinoid Synthesis

Example 1 - Synthesis of Comparative Compounds 16 and 17

It was envisaged that the series of compounds 9 would be readily available as outlined in Scheme 1 , through a series of cross-coupling reactions, including Heck-Mizoroki and Sonogashira. Compounds of this type were expected to be suitable for probing the effect polyene double bond stereochemistry and chain length upon the ability of such compounds to effect cellular development processes, particularly in stem cells or stem cell models.

Scheme 1. Proposed synthetic sequence for the synthesis of isomeric retinoid analogues 9.

Alkyne 7 was prepared as a generic synthon, starting with iodination of 1 ,1 ,4,4-tetramethyl- 1,2,3,4-tetrahydronaphthalene to give 5, followed by cross-coupling to TMSA under Sonogashira conditions and finally deprotection (Scheme 2) according to optimised procedures.(lO)

Scheme 2. Synthesis of acetylene derivative 7 via iodide 5.

The synthesis of the polyene chains of different lengths began with the treatment of methyl propiolate 11 with sodium iodide in acetic acid(23, 24) to provide Z-iodoacrylate 12 in 58% yield and 100 % stereoselectivity. Generation of the £-isomer was achieved through isomerisation methyl 3-Z-iodoacrylate with hydroiodic acid in benzene(24, 25) to give methyl 3-£-iodoacrylate 13 in an 85% yield (Scheme 3). Both iodides were used in separate reactions to: 1) direct couple to acetylene 7 allowing for the formation of two 'short' retinoid like analogues; and 2) Heck-Mizoroki couple to a vinylboronate ester, followed by iododeboronation, leading to a dienyliodide for further elaboration to longer derivatives (vide infra). Hence, Sonogashira coupling of the two iodides 12 and 13 to the acetylene 7 under standard conditions(26, 27) provided each of the isomeric esters 14 and 15 in 91 and 71% yields, respectively. Saponification(28, 29) of the methyl esters 14 and 15 produced the corresponding carboxylic acids 16 and 17 in 84 and 85% yields, respectively (Scheme 3).

Scheme 3. Synthesis of synthetic retinoids 16 and 17 starting from methyl propiolate 11.

Example 2 - Synthesis of Compounds 31 and 32 of the Present Invention

In order to access longer chain analogues of 16 and 17, i.e. similar to that of natural ATRA, the addition of a further alkene unit was required The preparation of the Z,Z- and E,E- dienyliodides 20 and 21 commenced therefore with the coupling of the two iodides 12 and 13 to 4,4,6-trimethyl-2-vinyl-l,3,2-dioxaborane using modified Heck-Mizoroki conditions developed in our group.(24, 30, 31) This led to the isolation of the two boronate species 18 and 19 in 58 and 90% yields, respectively, which after stereoselective iododeboronation(24, P T/GB2011/001691

35

32-37) provided either the Z- or ^-dienyliodides 20 and 21 in 59 and 78% yields, respectively (Scheme 4).(38)

AgOAc, Pd(OAc¾

tri(i -toIyI)phosphine,

MeCN

1) Id, QCM, -78 °C 1) NaOMe, MeOH,

2) NaOMe, eOH, -78 °C

-78 ^CC - r.t. 2) ICI, DCM, -78 <>C

Scheme 4. Synthesis of dienyliodides 20 and 21.

Having isolated dienyliodides 20 and 21, they were subjected to Sonogashira coupling as employed for iodides 12 and 13, however, the resulting products from both dienes was a mixture of two alkene stereoisomers visualised through Ή NMR (Spectra not shown). Separation proved unsuccessful and exposure of the mixtures of stereoisomers to light caused further deterioration, making it clear that these types of retinoids were too unstable to be purified or further studied. It was therefore necessary to try and stabilise the polyenes so that single stereoisomers could be obtained; the addition of an extra methyl group, indeed with natural retinoid, was envisaged to be suitable for this purpose. The synthesis of methyl- substituted analogues was approached identically to that outlined in Scheme 4, however, starting with methyl tetrolate 22 rather than methyl propiolate 11. T/GB2011/001691

36

Methyl tetrolate 22 was treated with sodium iodide and acetic acid to yield Z-3-iodoalkene 23 as a single stereoisomer in 89% yield.(39, 40) Subsequent treatment with iodic acid in benzene for 20 hours resulted in equilibration to give a readily separable 1 :1 mixture of stereoisomers 23 and 24. Attempts to increase the yield of the trans stereoisomer 24 through increased reaction times provided a yield of 66% after 48 hours, with no further improvement in yield seen for longer reaction periods. Each stereoisomer 23 and 24 were then coupled with the vinylboronate ester (Scheme 5) to give the (2Z,4£)- and (2£',4£)-dienylboronates 25 and 26 in 78 and 74% yields, respectively. Subsequent iododeboronation provided corresponding Z,E- and i^-dienyliodides 27 and 28 in 98 and 86% yields, respectively.

27 28

Scheme 5. Synthesis of dienyliodides 27 and 28.

The dienyliodides 27 and 28 both showed improved stability towards light and in storage relative to the unmethylated derivatives 20 and 21, as readily determined by Ή NMR (data not shown). It was also possible to carry out Sonogashira couplings with acetylene 7 to provide each of the stereoisomer^ esters 29 and 30 in 88 and 76% yields, respectively (Scheme 6).

Scheme 6. Synthesis of ATRA-like synthetic retinoid esters 29 and 30.

Subsequent modified saponification(28, 29) of esters 29 and 30 produced the corresponding carboxylic acids 31 and 32 in 84 and 85% yields, respectively (Scheme 7).

Scheme 7. Synthesis of ATRA-like synthetic retinoids 31 and 32.

Example 3 - Synthesis of Comparative Compound 36

In order to study the effects of chain length and polyene geometry, different chain-length analogues were required with the inclusion of suitable stabilising methyl -substituents to stabilse the polyene sections of the synthetic retinoids. Shorter chain analogues were readily T B2011/001691

38 prepared in an corresponding manner to those in the previous series (Schemes 6 and 7), hence, coupling both the E- and Z-iodoalkene esters 23 and 24 to the acetylene 7 gave the corresponding methyl esters 33 and 34 in 63 and 99%, respectively. Saponification as previously described of the £-enyne methyl ester 34 gave the corresponding acid 36 in 31% yield, however the Z-enyne methyl ester 30 did not provide the expected product (Scheme 8).

Scheme 8. Synthesis of short retinoid analogue 33. Example 4 - Synthesis of Compounds 42 and 43 of the Present Invention

Saponification of ester 33 followed by an acidic work-up and silica gel chromatography yielded a bright yellow solid in 97% yield, which proved to be hemi-acetal lactone 35. Interestingly, this type of cyclisation reaction was not observed for the corresponding un- methylated retinoid analogue 16. The mechanism has not been fully elucidated, however, it can be proposed that the carboxylic function catalyses addition of water across the acetylene, as outlined in Scheme 9, followed by keto-enol tautomerisation and acyl-hemiacetal formation to give 35.

Scheme 9. Proposed mechanism for formation of acyl-hemiacetal 35.

To complete the series of synthetic retinoids, a longer polyene derivative was prepared using a similar approach, i.e. as outlined in Scheme 10. Hence, dienyliodide 28 was coupled with 4,4,6-trimethyl-2-vinyl-l,3,2-dioxaborane to generate the trienylboronate 39 in a 67% yield and iododeboronation gave trienyliodide 40 in a 65% yield. Coupling of 40 to acetylene 7 yielded the trienyne methyl ester 41 in 64% yield as a stable mixture of isomers. Subsequent saponification of the ester 41 gave the free acid 42 in an 87% yield. However, it was isolated as a mixture of the two stereoisomers confirmed by Ή NMR in a ratio of 1 :0.4 (Scheme 10). Due to the highly sensitive nature of the compound it was assumed that a single isomer was probably unattainable as isomerisation occurs under general laboratory light. Consequently, isomerisation occurs at all stages of the synthesis including in the reaction mixtures and work up procedures. Therefore, the mixture of the two inseparable stereoisomers 42 and 43 was examined for biological activity without further attempts at separation.

Scheme 10. Synthesis of the extended retinoid analogue 42. Example 5 - Stability of synthetic retinoids versus ATRA

The photostability of synthetic retinoids 16, 17, 31, 32, 36 and the inseparable mixture of 42 and 43 was tested and compared with that of ATRA. It was previously shown that ATRA was susceptible to photoisomerisation and degradation when exposed to laboratory (fumehood) fluorescent light in the visible to near-UV range using Ή NMR spectroscopy. After 3 days exposure, substantial isomerisation/degradation occurred with approximately 37% of the ATRA remaining. When the synthetic retinoids 16, 17, 31, 32, 36 and 42/43 were exposed to the same wavelength of fluorescent light and their Ή NMR spectra compared with control (unirradiated) samples, the severity of the isomerisation depended greatly upon the length of the conjugated linker. The shorter analogues, both methylated and un-methylated systems 16 (Fig. 2), 17 (Fig. 3) and 36 (Fig. 4) all remained completely unaltered. The ATRA-like analogues 31 and 32 showed varied degrees of isomerisation between the two isomers. Analogue 31 isomerised into two other isomers, (Fig. 5) identified as the, (E,E) ,or compound 32 in 29% and (Z,Z) in 1 1% respectively. Analogue 32 only isomerised to one other isomer (Fig. 6) identified as the (E,Z), or compound 31 in 17% respectively. The most susceptible bond to isomerisation can therefore be assumed to be the terminal alkene, as both analogues isomerise to mixtures of each other under UV light. However, there were no signs of degradation and >60% of compound 31 and >83% of compound 32 remained intact.

In contrast, the extended analogue, a mixture of analogues 42 and 43, showed greater isomerisation, not dissimilar to that seen for ATRA, but with reduced degradation (Fig. 7). Two other isomers were seen in a ratio of 1 :0.7:0.25, with approximately 50% of analogue 42 remaining after 72 hrs.

The inherent instability of ATRA lies within the conjugated linker region, as previously established and replacement of sections of the polyene chain with an acetylene moiety and phenyl ring provides increased stability.(lO) The stability of the systems reported herein varies as a function of both polyene-chian length and methyl substitution, however, there is sufficient stability to allow biological characterisation on the systems 1

41

Example 6 - Effects of the Synthetic Retinoids on F9 murine Embryonal Carcinoma Cells

The initial model chosen was a mouse F9 murine EC cell line containing a stable lacZ reporter line for the retinoic acid response element (RARE). The F9 cell line is characterised by the inability to differentiate spontaneously, thus it is termed a nullipotent cell line and was isolated in 1973 as a subline of the teratocarcinoma OTT6050.(41) It has the ability to differentiate into endodermal-like derivatives, depending on both reagents and culture conditions. Thus formulating its wide use in many laboratories as a model for the molecular mechanisms of differentiation.(42) To identify compounds which activate retinoid signalling pathways we have used a F9 cell line, chosen as it expresses endogenous α-, β-, and γ- retinoic acid receptors,(43) containing a reporter assay that makes use of a RARE to drive a lacZ reporter gene.(44) This RARE is located within the m-acting regulatory sequences of the human ?-retinoic acid receptor gene,(45) consists of 64 nucleotides, and responds to the α-, β-, and ^-retinoic acid receptor (RAR) subtypes. (46) The RARE had been transfected directly upstream of the E. coli lacZ gene, which confers retinoid responsivity to these genes. Thus the lacZ gene is consequently used for histochemical detection of retinoid-responsive cells, measured by assaying ?-galactosidase activity. To analyse whether the synthetic retinoids synthesised activate the classical retinoid signalling pathway, through activation of one or multiple RAR receptors (a, β, or γ), cultures of F9 murine embryonal carcinoma cells containing a lacZ reporter line were incubated with 10 μΜ concentrations of each compound tested. Similar controls were also assessed consisting of the vehicle, DMSO, and untreated cells. Those exposed to no compounds showed no positive results upon ^-galactosidase staining as expected, along with those treated with the vehicle, DMSO (Fig. 8 (A) and (B) respectively). This is in contrast to those cells treated with ATRA, which, as expected, stained highly in response to β-gal with a high percentage of blue cells visible (Fig. 8 (C)). Similarly, both analogues 31 and 32 showed very similar profiles to that of ATRA, again with a very high percentage of the cells staining positive (Fig. 8 (D) and (E)). All other synthetic retinoid analogues showed a very low percentage of cells staining positive, giving an early indication that they are not binding to the RAR receptors (Fig. 8 (F), (G), (H) and (I)). Example 7 - Effects of the Synthetic Retinoids on TERA2.cl.SP12 EC Cells.

EC cells are the stem cells of teratocarcinomas, and therefore retain their ability to differentiate into one or more of the embryonic layers. One of the earliest and most frequently used human EC cell lines is the TERA2 cell line, originally isolated from a lung metastasis originating from a testicular germ cell tumour.(47) A subsequent sub-clone culture, termed TERA2.cl.SP12, was developed in 2001 by immunomagnetic isolation followed by single cell selection and culture.(48) This cell line has been chosen for this study, as it has been previously shown to differentiate in response to ATRA. After 21 days of co-culture with ATRA a heterogeneous cell population is visualised containing multiple mature functioning neurons.(49) Consequently this makes the TERA2.cl.SP12 cell line an effective model system for the in vitro study of neurogenesis. To compare the effect of the synthetic retinoids versus both known natural retinoid responses and other synthetic retinoids previously synthesised within the group, cultures of TERA2.cl.SP12 EC cells were incubated with 10 μΜ concentrations of each compound synthesised. Simultaneously control cultures were assessed consisting of untreated, therefore undifferentiated, cells along with cultures exposed to the vehicle, DMSO.

Cell Viability

Retinoid treated cultures were maintained for a 7 day period, with cell viability analysed at 3 and 7 days. Analysis was undertaken using an MTS assay and on a Guave EasyCyte™ Plus System using the Guave ViaCount^® reagent. Untreated cultures, and those treated with the vehicle, continued to proliferate rapidly, to the point that, after 7 days the cultures were becoming highly confluent (Fig. 9). ATRA treatment resulted in a reduced cell number compared with both the untreated control and the vehicle, DMSO (Fig. 9). This is indicative of cells that are no longer proliferating but exiting the cell cycle and committing to differentiation. Decreased cell number was also observed in cultures treated with 31 and 32 comparable to that of ATRA (Fig. 9)..There was no difference between cell numbers in the control and those treated with the remaining synthetic retinoids, indicating the inability of these compounds to arrest cell proliferation and induce differentiation (Fig. 9).

Simultaneously, the number of apoptopic cells and viable cells were analysed. All compounds assessed showed similar levels of apoptopic cells and viable cells indicating at 01691

43 this concentration the compounds appear to not be toxic (Fig. 10). Thus further analysis can be carried out using concentrations equal or below 10 μΜ.

Differential Regulation of Key Developmental Mar rs

Induction of cellular differentiation in response to the synthetic retinoids was further evaluated by analysing the expression profile of known markers for both stem cell and differentiated phenotypes. Flow cytometry was performed on samples of cells at either 3, 7 or 14 days, treated with 10 μΜ ATRA, 31 and 32, tested for the expression of the stem cell antigens, SSEA-3 (globoseries stage specific embryonic antigen-3) and TRA-1-60 (keratin- sulfate-associated glycoprotein stem surface marker). A decrease in the expression level of SSEA-3 and TRA-1-60 indicates cells committing to differentiation, previously demonstrated using ATRA. As hypothesised similar expression profiles were visualised for 31 and 32 as was seen for ATRA over the 14 day period (Fig. 11). In cell cultures treated with the extended analogues 42/43 and short analogues 16, 17 and 36 expression levels of SSEA-3 and TRA-1-60 remained relatively high. Consequently cultures treated with 16, 17, 36 and 42/43 were cultured for only 7 days after which they were discarded due to overgrowth (data not shown).

As cell lines of the TERA2 lineage are well characterised in their ability to form neurons in response to ATRA,(50, 51) flow cytometry was according used to assess the expression of A2B5 (ganglioseries antigen marking early-stage neural cells), a marker which is expressed during the early stages of neuronal development. Consistent with previous data discussed, ATRA, 31 and 32 induced the expression of A2B5 comparatively (Fig. 11). All other retinoids tested showed no increase in expression levels, consistent with them having an inability to induce neurogenesis, and subsequently cultures were discarded after 7 days due to overgrowth (data not shown).

As all data pointed to the inability of the short and extended analogues to induce differentiation, all further discussions will centre on analogues 31 and 32. In an extension to this study, we applied both 31 and 32 and ATRA at a range of concentrations to investigate the dose response for each compound. As the concentration of ATRA is reduced by a factor of ten from 1 μΜ to 0.001 μΜ a clear dose response becomes apparent (Fig. 12). At 1 μΜ the expression levels are similar to those at 10 μΜ indicating a differentiated state. At 0.1 μΜ the expression levels of all three markers are significantly increase/reduced accordingly, so that at 0.01 pM and 0.001 μΜ expression levels are similar to undifferentiated control cultures (Fig. 12). The profile seen for analogue 31 is similar except that expression levels still indicate a differentiated state at 0.1 μΜ. More interesting is the expression profile exhibited by analogue 32, which retains activity down to a concentration of 0.01 M (Fig.12). The greater apparent potency of both 31 and 32 over ATRA may be a result of their increased stability under standard laboratory conditions. Then again, both 31 and 32 may be resistant to cellular metabolism, as sites known to be metabolically liable in ATRA have been blocked in both 31 and 32.(20-22) Either scenario would contribute to higher active levels of these retinoids being present in media/cells over a longer time period, and could therefore explain, at least in part, the enhanced levels of differentiation observed in the lower micromolar range. An explanation for the improved potency of 32 over 31 is that the structure of the molecule confers greater affinity with and/or activation of cognate cellular receptors.

Regulation of Key Transcripts During Development

Further assessment of cellular differentiation in response to the synthetic retinoids was evaluated through the expression profiles of know genes. Samples from the cells analysed for flow cytometry at 3, 7 and 14 days treated with retinoids at 10 pM concentration were simultaneously analysed through RT-PCR. Three genes of interest were chosen, Nanog and Oct-4, as gatekeepers of pluripotency, and Pax-6, as a motor and ventral neural phenotype. The expression of Nanog and Oct-4 was suppressed in cultures treated with both ATRA and analogues 31 and 32 all in a similar manner (Fig. 13). Consistent, Pax-6 expression increased to up to and just after day 7, after which it decreased slightly to day 14. Again all three retinoids, both natural and synthetic, showed a similar profile (Fig. 13). These data further indicate that both 31 and 32 synthetic retinoids can play a key role in inducing differentiation ofTERA2.cl.SP12.

Enhanced Expression of Neural Proteins

Conformation of neural development of TERA2.cI.SP12 cells in response to analogues 31 and 32 was demonstrated by immunocytochemical staining of cultures treated with each of the retinoid compounds at 10 μΜ over 21 days (Fig. 14). The expression of neuronal specific proteins was investigated using antibodies raised against the neuronal specific J-HI-tubulin marker Tuj l (Fig. 14). Low levels of expression are seen in TERA2.cl.SP12 cells, which was significantly up-regulated through cultures treated with either ATRA or analogues 31 and 32. The expression of the 200 kDa neurofilament protein (NF-200) was simultaneously examined as a cytoskeletal marker of mature neurons (Fig. 14). We previously know TERA2.cl.SP12 cells do not express NF200, though large numbers of NF-200 positive neurons were identified in cultures treated with either ATRA or the synthetic analogues. This data collectively demonstrates that both ATRA and the synthetic retinoids behave in a similar manner and induce the differentiation of pluripotent stem cells to form mature neural derivatives.

Example 9 - Differentiation of a commercial human neural progenitor cell line

Here we investigate the effects of incorporating ATRA, 31 or 32 into a commercial neural cell differentiation protocol. We chose to use the commercial neural progenitor line ReNcell 197VM, since effective differentiation protocols have already been devised for this cell line. The immortalised (v-myc insertion) cell line is derived from the ventral mesencephalon of human foetal brain, and is a useful system for the study of neuronal cells in vitro. Two methods have been published regarding the differentiation of these cells into neural subtypes^) along with a method involving retinoid incorporation into the standard differentiation protocol(53). It has been previously shown that addition ATRA, significantly increased the number of differentiated cells expressing neuronal markers and that ATRA not only accelerates neurogenesis in the ReNcell 197VM line, but that the maturity of neuronal derivatives is enhanced after 7 days in retinoid-supplemented media. The standard documented method is simply to remove the growth factors, fibroblast growth factor (FGF) and epidermal growth factor (EGF), from the proliferation media, and to allow the cells to differentiate for up to two weeks. (52) For the purposes of this study, retinoids were added at the point of growth factor withdrawal, which signifies the initiation of differentiation in the commercial protocol.

ReNcell 197VM cells were cultured as previously described by Donato el a/.(52) Undifferentiated cultures and control differentiated cultures were prepared alongside those treated with retinoids. The control differentiation method used followed the standard differentiation protocol, which involves the withdrawal of growth factors from the culture media. For cultures in which retinoids were incorporated as part of the differential protocol, either 1 μΜ ATRA, 1 μΜ 31 or 1 μΜ 32 were administered precisely at the same time that growth factors were withdrawn. All cultures were subsequently maintained in their respective media formulations for seven days. Media was replaced for each culture at the halfway point (3.5 days) of the experiment. Phase contrast micrographs of cells grown under the different experimental conditions indicate a number of characteristics (Fig.15). Removal of growth factors according to the standard ReNcell 197VM differentiation protocol resulted in the transition from a homogeneous population of proliferative cells (characterised by large uniform morphology) to a heterogeneous culture characterised by zones of cells which appear similar to those in the undifferentiated cultures, surrounded by more sparsely populated regions of neuron-like cells. Inclusion of retinoids in the differentiation medium resulted in greater homogeneity of cells, predominantly neuron-like, than in the standard differentiation counterparts. Therefore, phase contrast microscopy indicated that retinoid inclusion had resulted in accelerated differentiation of the neural progenitors.

Subsequent, immunocytochemical staining was performed to analyse the expression of the neuronal marker Mil tubulin and mature neuronal protein marker NF-200. Fluorescence micrographs for tubulin and NF-200 expression, alongside the corresponding nuclear stains (Hoechst 33342), from cells grown under control and retinoid-supplemented conditions display the significant advantages to retinoid supplement (Fig. 15). It has been previously published that the standard differentiation protocol results in approximately a 2.75-fold increase in the number of cells which stain positive for 5-III-tubulin compared to undifferentiation samples and there is only a minor increase in the visible expression of the more mature neuronal marker NF-200. Incorporation of either ATRA, 31 or 32 at ] μΜ concentration, resulted in a dramatic and highly significant increase in both Tuj 1 and NF-200 expression (Fig. 16). The induced levels of neurogenesis were comparable for ATRA and 31 while 32 displayed significantly greater properties over both based on these expression criteria. These results show that retinoid supplementation using 31 and 32 increases the number of neuronal cells derived from ReNcell 197VM cultures and significantly increases neuronal maturity, as evidenced by NF-200 expression, comparable to that of ATRA for 31 and significantly better when treated with 32.

To investigate the dose response effects of incorporated retinoids in differentiating ReNcell 197VM cultures, and to compare further the activities of ATRA, 31 and 32 we added retinoids at a range of concentrations (1, 0.1 , 0.01 and 0.001 μΜ). Optimal concentrations for the study of retinoid responses were obtained from studies previously undertaken on retinoid incorporation. As described previously, the numbers of cells expressing /Mil tubulin and NF- 200 were measured, alongside standard (control differentiation) conditions (Fig. 17). A dose response is observed when dilutions of ATRA are used in the 1 -0.001 μΜ retinoid range. Successively fewer cells stained positive for both neuronal markers as the retinoid concentration is decreased (Fig. 18). A similar dose response is seen between 1 and 0.001 μΜ 31 but although there was an observable dose response for 32, a drop in concentration from 1 μΜ to 0.01 μΜ 32 produced no significant drop in expression of β-lll tubulin. Example 10 - Effect of compound AH6 (compound 32) on neurite outgrowth:

Compound AH61 was evaluated for its ability to induce neural differentiation from human pluripotent stem cells with particular attention toward neurite outgrowth. TERA2.cl.SP12 embryonal carcinoma stem cells provide a robust model to assess neurogenesis. Cells were prepared and experiments performed as described in the methods section below. Results clearly demonstrate that AH61 is potent inducer of neural development by these stem cells and the figures below show enhanced neurite outgrowth.

Stem cell maintenance. Human pluripotent TERA2.cl.SP12 embryonal carcinoma stem cells were maintained in DMEM supplemented with 10% FCS, 2mM L-glutamine and 100 active units each of penicillin and streptomycin (DMEMFGP) at 37°C in 5% C0₂.

Induction of cell differentiation. Confluent human pluripotent TERA2.cl.SP12 embryonal carcinoma stem cells were treated with 0.25% trypsin EDTA for 2-3min to produce a single cell suspension for counting. Aggregate formation was induced by seeding 1.5 X 10⁶ cells in 20ml DMEMFGP onto 90mm bacteriological grade petri dishes. After 24 hours cells were induced to differentiate by addition of ec23, AH61 or ATRA to a final concentration of 0.1- 10μΜ, medium was changed every 3-4 days for 21 days.

Induction of neurite outgrowth. Differentiated aggregates were back -washed through a ΙΟΟμπι strainer into fresh media without retinoid. Aggregates were then seeded onto 48 well tissue culture plastic coated overnight with laminin (^g/ml) and Poly-D-Lysine (Ι Ομξ/^χηϊ). Aggregates were grown in the presence of mitotic inhibitors (Ι μΜ cytosine arabinosine, 10 μΜ 5-fluoro-2-deoxyuridine and 10 μΜ uridine) and cultured at 37°C in 5% C0₂. Neurite outgrowth was visible after 1-2 days culture, cultures were maintained for 7-10 days. Example 11 - Effect of compound AH61 (compound 32) on mouse epidermis:

Compound AH61 was evaluated for its ability to induce epidermal thickening in mammalian skin. An appropriate mouse model was selected, retinoid samples were applied topically to the surface of the skin, and biological evaluation of the effects of such compounds was subsequently assessed. Experimental procedures were performed as described in the methods section below. The results clearly demonstrate that AH61 is a potent inducer of epidermal thickening and promotes cell proliferation in the basal layers of the murine epidermis.

Experimental procedures were performed under a UK government Home Office licence, Animals of the same treatment group were housed together throughout the study, with access to food and water ad libitum. The study used sex-matched C57 BL6 mice, which were 8 weeks of age in order to ensure that all mice were in the telogen phase of the hair cycle. Retinoid solutions were prepared in acetone and kept at -80°C until needed. The back of each mouse (three mice per treatment group) was shaved and ΙΟΟμΙ of the retinoid solution was applied topically. The control mice received ΙΟΟμΙ of acetone only. In addition, each mouse was weighed immediately before treatment.

As retinoic acid treatment is known to cause adverse effects, mice were inspected daily for signs of irritation, such as redness and dryness. Changes in posture were also examined as this is a common indicator for ill health in mice. Four days after the single treatment, all mice were sacrificed by cervical dislocation. Mice were weighed and the skin at the site of treatment was harvested.

Skin samples were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Wax blocks were cut into 5μιη sections, which were stained with haematoxylin and eosin. Images of H&E-stained sections were taken at room temperature using a Leica microscope. Epidermal thickness (the distance from the dermal-epidermal junction to the upper layer of viable cells) was calculated from the mean of 75 measurements per section, with three repeats performed per sample. Statistical analysis of the measurements was carried out using repeated measures ANOVA and Dunnett's post-hoc test, relative to control. Asterisks indicate a P value <0.005. Error bars are S.E.M. Paraffin-embedded sections were used for involucrin antibody staining. Slides were deparaffinised in Histoclear and hydrated through a graded series of ethanols, before permeabilisation, blocking and incubation with the primary, then secondary antibody.

To determine the number of Ki67-positive keratinocytes, Ki67 antibody staining was carried out using the same method detailed above. The number of Ki67-positive keratinocytes in the epidermis was counted per ΙΟΟμιη length of interfollicular epidermis, with 20 lengths per section, with three repeats per sample. Statistical analysis of the measurements was carried out using repeated measures ANOVA and Dunnett's post-hoc test, relative to control. Asterisks indicate a P value <0.005. Error bars are S.E.M. Paraffin-embedded sections were used for keratin 14 antibody staining. Slides were deparaffinised in Histoclear and hydrated through a graded series of ethanols, before permeabilisation, blocking and incubation with the primary, then secondary antibody.

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Claims

1. A compound according to either one of Formula II and Formula III:

(HI) wherein Rl to Rl 1 independently represent hydrogen, optionally substituted alkyl group, optionally substituted aryl group or Ry,

wherein each Ry is independently represents halogen, trifluoromethyl, cyano, nitro, oxo, NRz, -ORz, -C(0)Rz, -C(0)ORz, -OC(0)Rz, -S(0)IRz, -N(Rz)Ra, - C(0)N(Rz)Ra, -S(0)I (Rz)Ra and Rb;

Rz and Ra are each independently hydrogen or Rb; Rb is selected from hydrocarbyl and -(CH2)k-heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, CI -6 alkyl and CI -6 alkoxy;

k is 0, 1, 2, 3, 4, 5 or 6; and

I is 0, 1 or 2;

wherein the conjugated group may be unsubstituted or may be substituted with one or more optionally substituted alkyl group, optionally substituted aryl group or Ry; wherein X represents -C(0)Z, wherein Z is selected from -OH, OCH3 and NHOH.C02H, C(O);

The multiple bond within the square brackets of Formulae II and III represents a carbon-carbon double bond or a carbon-carbon triple bond and

A represents 2, 3 or 4.

2. The compound as claimed in claim 1 wherein the conjugated linker group contained within the brackets of Formulae II and III includes at least one carbon-carbon triple bond and at least one carbon-carbon double bond.

3. The compound as claimed in claim 2 wherein a carbon-carbon triple bond is located at the carbon atom closest to the carbocyclyl group.

4. The compound as claimed in any preceding claim wherein A represents 2, and the conjugated linker group comprises one carbon-carbon triple bond, typically at the carbon atom closest to the carbocyclyl group, and one carbon-carbon double bond.

5. The compound as claimed in any one of claims 1 to 3 wherein A represents 3, and the conjugated linker group comprises one carbon-carbon triple bond, typically at the carbon atom closest to the carbocyclyl group, and two carbon-carbon double bonds.

6. The compound as claimed in any one of claims 1 to 3 wherein A represents 2 or 3 and B represents methyl.

7. The compound as claimed in any preceding claim wherein R4, R5, RIO and Rl l independently represent CI to 6 alkyl group; preferably methyl group.

8. The compound as claimed in any preceding claim wherein Rl, R2, R3, R6, R7, R8 and R9 represent hydrogen.

9. The compound as claimed in any preceding claim having one of the following structures:

Compound 31

Compound 32

Compound 42 the structure of compound 42 having an E,E, Z configuration.

10. The compound as claimed in any preceding claim wherein at least 60% by weight of the compound remains following 3 days exposure to light having a wavelength of 300 to 400 run.

1 1. The compound as claimed in claim 1 wherein A represents 2 and wherein at least 90% by weight of the compound remains following 3 days exposure to light having a wavelength of 300 to 400 nm.

12. The use of a compound as claimed in any preceding claim in the control of cell differentiation and cell apoptosis.

13. The use as claimed in claim 12 wherein the number of stem cells following exposure to the compounds of the present invention is 50% or less of the number of stem cells following exposure to a control.

14. The use as claimed in either one of claims 12 and 13 wherein the number of stem cells following exposure to the compounds of any one of claims 1 to 11 is 150 to 200% of the number of stem cells following exposure to ATRA.

15. The use as claimed in any one of claims 12 to 14 wherein the stem cell is a multipotent or a pluripotent stem cell.

16. The use as claimed in claim 15 wherein the stem cell is a multipotent stem cell is selected from the group consisting of: haemopoietic stem cell, neural stem cell, bone stem cell, muscle stem cell, mesenchymal stem cell, epithelial stem cell, ectodermal stem cell, mesodermal stem cell or endodermal stem cell.

17. A method to induce the differentiation of a stem cell comprising the steps of: i) forming a preparation of stem cells in a cell culture medium suitable for maintaining said stem cells wherein said culture medium comprises a compound according to any one of claims 1 to 11 ; and

ii) cultivating said stem cells in conditions that allow their differentiation into at least one differentiated cell type.

18. The method of claim 17 wherein said stem cell is a multipotent or pluripotent stem cell.

19. The method of either one of claims 17 and 18 wherein said differentiated cell is selected from the group consisting of a keratinocyte, a fibroblast (e.g. dermal, corneal, intestinal mucosa, oral mucosa, bladder, urethral, prostate, liver), an epithelial cell (e.g. dermal, corneal, intestinal mucosa, oral mucosa, bladder, urethral, prostate, liver), a neuronal glial cell or neural cell, a hepatocyte, a mesenchyma cell, a muscle cell (cardiomyocyte or myotube cell), a kidney cell, a blood cell (e.g.CD4+ lymphocyte, CD8+ lymphocyte), a pancreatic cell, or an endothelial cell.

20. The method of any one of claims 17 to 19 wherein the medium has a concentration of 0.001 to 10 μΜ compound of any one of claims 1 to 11.

21. The method as claimed in any one of claims 17 to 20, wherein the method takes place ex vivo, in vivo or in vitro.

22. The use of a compound as claimed in any one of claims 1 to 11 in the treatment or prevention of a disease or condition that would benefit from the control of cell differentiation or apoptosis.

23. The use as claimed in claim 22 wherein the disease or condition is cancer, in the treatment or prevention of precancerous or cancerous conditions including those of the skin, oral cavity, larynx, lung, bladder, vulva, breast and digestive tract.

24. The use as claimed in claim 23 wherein the cancer is leukaemia, in particular acute promyelocyte leukaemia.

25. The use as claimed in claim 22 in the promotion of the health and development, of the skin, bone, teeth, hair and/or mucous membranes of the human or animal body.

26. The use as claimed in claim 25 in the prevention or treatment of wrinkles, age spots Acne, psoriasis, stretch marks, keratosis pilaris, emphysema and/or baldness.