The present invention relates to compounds and their uses. In particular, though not exclusively, it concerns luminogenic compounds and their application in in vivo imaging techniques.

Catalysis of a substrate (e.g. luciferin) by a bioluminescence (BLI) enzyme (e.g. luciferase) results in the release of light. Thus, small animal bioluminescence imaging utilises emission of light by the transgenic expression, particularly of firefly luciferase (Flue), in selected tissue(s). This rapid, cheap and highly sensitive modality has revolutionised the use of small animal models, allowing facile longitudinal and quantitative tracking of labelled cells noninvasively. Furthermore, functionalised substrates and enzymes allow the imaging of biological events within the small animal. However, it has proven difficult to extend this modality to allow true tomographic and multi- parametric imaging, primarily because luciferases emit light (yellow-green colour) that is largely absorbed by haemoglobin. This causes scatter and differential attenuation depending on tissue density and light source depth. Shifting the emission of luciferase into the near infrared (nIR) would therefore overcome these barriers, since even complex mammalian tissues are quite transparent at these wavelengths (in the Optical window' between ca. 620-800 nm).

Despite much interest, no genetically encoded nIR BLI label currently exists. Furthermore, because BLI is currently restricted to relatively small animals, nIR emission with a respectable quantum yield would allow BLI in larger animals than is currently possible.

Firefly luciferase (Flue) has been heavily engineered, increasing its expression, stability and shifting its emission with natural firefly luciferin from green-yellow to red. However, it is apparent that current systems are near the limit of red-shifting (ca. 620nm) achievable by mutagenesis of Photinus pyralis Flue.

Firefly luciferin (often denoted LH ₂ ) is known to produce at least two colours of bioluminescence with different mutants of firefly luciferase, the natural yellow-green colour (peak wavelength - max 557nm) and a red colour ( max 620nm). Red mutants are useful for in vivo imaging since the attenuation of red light is less intense and variable. In addition, due to the dependence of photon scatter on wavelength (scatter is proportional to 1 /wavelength ⁴ ), red emission is expected to allow higher resolution and deeper tissue bioluminescence imaging. Due to the differential attenuation of the two colours and variable attenuation of the yellow green emission (depending on depth, haemoglobin concentration and tissue density) multiparametric bioluminescence in vivo imaging has thus far been impossible. For this, the information space above 600nm (the optical window above haemoglobin absorbance) must be utilised. Therefore, emission should ideally be shifted ca. 100 nm compared to current systems, into the far red or near infrared regions. It would also be beneficial for the maximum emission to be at different wavelengths with different mutants, and to have a narrow band of emission to allow for multiparametric imaging.

The chemical mechanism for the different colours observed with different mutant Flue and native LH ₂ is unknown and many studies with mutants and substrate analogues have tried to determine it without success (Navizet et al., J Am Chem Soc, 2010, 132, 706-12). One hypothesis is that the emitting product of the Flue reaction, oxyluciferin (OL) can exist in two tautomeric forms, i.e. red emitting 4-keto and yellow-green emitting 4-enolate (White et al., Bioorg Chem, 1971, 1, 92-122.); however, this theory is less probable since preadenylated 5- substituted dimethyl analogues (which cannot form an enolate) can produce green and red colours with wild-type and red mutant Flues, respectively (Branchini et al., J Am Chem Soc Comm, 2002, 124, 2112-3).

A number of other theories exist, including one solution that the entire molecule can exist in two different resonance forms dictated by electrostatic and/or H-bond interactions in the active site (Branchini et al., Biochemistry, 2004, 43, 7255-62). Although strictly unproven, this theory is supported by quantum mechanical calculations (Nakatani et al., J Am Chem Soc, 2007, 129, 8756-65). Furthermore, the postulated mechanism that leads to emission with LH ₂ (chemically induced electron-exchange luminescence - CIEEL) supports the notion of electron movement from 4- to 6'- groups (Koo et al, PNAS, 1978, 75, 30-3). However, it seems probable that the limit of red-shift achievable by enzyme engineering alone has been reached (Wood, Biolum Chemilum, 1990, 5, 107-14). The theoretical chemical mechanisms for resonance of luciferin are as follows:

1. Keto-enolate tautomeric forms of oxyluciferin (OL)

OL 4-keto anion OL 4-enolate anion

RED GREEN

2. Charge-based resonance delocalisation of oxyluciferin (OL)

OL 4-enolate form OL 4-keto form

RED GREEN

As the limit of red-shift available by enzyme engineering is thought to have been reached, modification of the chemical structure of luciferin (LH ₂ ) is an alternative option. A number of analogues of firefly LH ₂ have been previously synthesised to test theories of emission, to attempt to improve light yield or to shift emission colours. However, all have shown reduced light yields compared to native LH ₂ , and none has shifted emission sufficiently into the far red or near infrared regions. Thus, there is still a need for compounds which do not reduce the light yield too significantly, and which shift the light emission into a region that can be potentially useful outside of the window of haemoglobin absorbance.

Aminoluciferins, including those with the structures illustrated below, have been reported to have a higher affinity for Flue than LH ₂ (flash kinetics), lower light emission than LH ₂ and lower cell permeability in in vitro studies (Maki, J Electrochem Soc Japan, 2006, 9; Iwano et al., Tetrahedron 2013, http://dx.doi.Org/10.1016/j .tet.2013.03.050; Ugarova et al, Luminescence 2002, 17, 321-30; Japanese patent publication JP2009184932).

However, no data on the effect of pH on the emission wavelength maxima, or the use of mutated Flue to change the emitted wavelength have been reported. In addition, no in vivo studies have been reported.

Therefore, it is an object of the present invention to provide novel substrate analogues of luciferin that result in red-shifting of emission, particularly to the nIR range. Furthermore, it is desirable for such compounds to possess similar biocompatibility (e.g. good biodistribution and low toxicity) to natural luciferin, and to exhibit different emission colours (have distinct emitting forms) with different Flue mutants.

According to the invention, there is provided a compound according to the formula (I):

wherein

ring A is an optionally substituted 5- or 6-membered heterocyclyl, or heteroaryl ring;

ring B is an optionally substituted 5- or 6-membered heterocyclyl, heteroaryl, or hydrocarbon ring;

R ¹ is hydrogen or an optionally substituted alkyl group;

each R ² , when present, is a group independently selected from optionally substituted hydroxyl, optionally substituted amino, optionally substituted thiol, optionally substituted alkyl, and optionally substituted aryl;

R ³ is an optionally substituted acyl group; M is a group selected from O, S, and R ⁴ , wherein R ⁴ is a group selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

X is an optionally substituted unsaturated hydrocarbon group or an optionally substituted alkyl ene carbonyl; and

n is 0, 1, 2, or 3.

The compounds of formula (I) have been found to represent bioluminescent substrate analogues of luciferin which exhibit a red-shift in emission to the nIR range. Thus, they are the first examples of nIR BLI labels having true bioluminescence peaking in the nIR (i.e. not the product of resonance energy transfer or filtering). Structural features, such as the unsaturated linker group X, further allow different emission colours (have distinct emitting forms) with different mutant enzymes and/or under varying pH conditions. Such analogues have the potential to enable a new era in multispectral BLI, facilitating higher resolution multi-parametric imaging and enhanced depth penetration, and including the imaging of larger animals, such as dogs, cats, primates and human subjects.

The compounds of formula (I) have been found to be active in vivo, for example when injected into mice containing systemic Raji cell-derived lymphomas. The light generated could be imaged for analytical purposes. The compounds therefore provide the potential to increase depth penetration and resolution of BLI by reducing haemoglobin (Hb) attenuation and scatter (a higher percentage of photons are not absorbed by Hb and also travel for longer in a straight path), as well as high sensitivity multispectral BLI when used alone, or in combination with natural luciferin or further analogues.

The term 'C _x - _y alkyl' as used herein refers to a linear or branched saturated hydrocarbon group containing from x to y carbon atoms. For example, Ci-6 alkyl refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. Examples of Ci-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, hexyl, and isohexyl. The term 'C _x - _y alkylene' as used herein refers to a divalent hydrocarbon group obtained by removing one hydrogen atom from 'C _x - _y alkyl' above. Examples of Ci-6 alkylene groups include methylene, ethylene, propylene, butylene, pentylene, and hexylene. The term 'C _x - _y alkenyl' as used herein refers to a linear or branched hydrocarbon group containing one or more carbon-carbon double bonds and having from x to y carbon atoms. Examples of C2-6 alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, and 5-hexenyl.

The term 'C _x - _y alkenylene' as used herein refers to a divalent hydrocarbon group obtained by removing one hydrogen atom from 'C _x-y alkenyl' above. Examples of C2-6 alkenylene groups include ethenylene, propenylene, butenylene, 1,3-butadienylene, pentenylene, hexenylene, and 1,3,5 -hexatri enyl ene .

The term 'C _x-y alkynyl' as used herein refers to a divalent hydrocarbon group containing one or more carbon-carbon triple bonds and having from x to y carbon atoms. Examples of C2-6 alkynyl groups include ethynyl, propynyl, butynyl and pentynyl. The term 'C _x-y alkynylene' as used herein refers to a divalent hydrocarbon group obtained by removing one hydrogen atom from 'C _x-y alkynyl' above. Examples of C2-6 alkynylene groups include ethynylene, propynylene, butynylene, 1,3-butadiynylene, pentynylene, hexynylene, and 1,3,5 -hexatriy nyl ene . The term 'C _x-y alkoxy' as used herein refers to an -0-C _x-y alkyl group wherein C _x-y alkyl is as defined herein. Examples of Ci-6 alkoxy groups include methoxy, ethoxy, propoxy, iso- propoxy, butoxy, tert-butoxy, pentoxy and hexoxy.

The term 'x- to y-membered hydrocarbon ring' as used herein refers to a cycloalkyl, cycloalkenyl, or aryl ring as defined below in relation to formula (I).

The term 'x- to y-membered cycloalkyl' as used herein refers to a saturated monocyclic hydrocarbon ring of x to y carbon atoms. For example, 3- to 6-membered cycloalkyl refers to a saturated monocyclic hydrocarbon ring of 3 to 6 carbon atoms. Examples of 3- to 6-membered cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term 'x- to y-membered cycloalkenyl' as used herein refers to a monocyclic hydrocarbon ring containing one or more carbon-carbon double bonds of x to y carbon atoms. For example, C3-6 cycloalkenyl refers to an unsaturated monocyclic hydrocarbon ring of 3- to 6-carbon atoms. Examples of C3-6 cycloalkenyl groups include 2-cyclopenten-l-yl, 3-cyclopenten-l-yl, 2-cyclohexen-l-yl, and 3-cyclohexen-l-yl. The term 'aryl' as used herein refers to a 5- to 6-membered monocyclic hydrocarbon ring containing x to y carbon atoms, wherein the ring is aromatic. An example of such an aryl group is phenyl. Alternatively, the term 'C _x - _y aryl' as used herein refers to a monocyclic or bicyclic ring containing from x to y carbon atoms, wherein at least one ring is aromatic. Examples of C _6-14 aryl groups include phenyl, naphthyl, tetrahydronaphthalenyl, anthryl, phenanthryl, acenaphthylenyl, biphenylyl, anthracenyl, phenanthrenyl, and phenalenyl.

The term 'arylene' as used herein refers to a divalent hydrocarbon group obtained by removing one hydrogen atom from 'C _x - _y aryl' above. An example of such an arylene group is phenylene. Alternatively, the term 'C _x - _y arylene' as used herein refers to a monocyclic or bicyclic ring containing from x to y carbon atoms, wherein at least one ring is aromatic. Examples of C _6-14 arylene groups include phenylene, naphthylene, tetrahydronaphthalenylene, anthrylene, phenanthrylene, acenaphthylenylene, biphenylylene, anthracenylene, phenanthrenylene, and phenalenylene. The term 'heteroaryl' as used herein refers to a 5- or 6-membered monocyclic aromatic ring in which the monocyclic ring contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulphur. Examples of such monocyclic aromatic rings include thienyl, furyl, furazanyl, pyrrolyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, triazinyl, and tetrazinyl.

The term 'heterocyclyl' refers to a 5- or 6-membered monocyclic ring which may be saturated or partially unsaturated, in which the monocyclic ring contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulphur. Examples of such monocyclic rings include aziridinyl, oxiranyl, pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, dioxolanyl, dioxanyl, oxathiolanyl, oxathianyl, dithianyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, diazepanyl and azepanyl.

The term 'amino' as used herein refers to an organonitrogen compound with the connectivity - N(R')(R"), where R' and R" are each independently a hydrogen or an optional substituent as defined below in relation to formula (I).

The term 'acyl' as used herein refers to a group selected from:

(1) formyl;

(2) optionally substituted Ci-6 alkyl carbonyloxy;

(3) optionally substituted C2-6 alkenyl carbonyl;

(4) optionally substituted C2-6 alkynyl carbonyl;

(5) optionally substituted C ₆ -io aryl carbonyl;

(6) carboxyl;

(7) optionally substituted Ci-6 alkyl carbamoyl; and

(8) carbamoyl.

The term 'C _x - _y alkyl carbonyloxy' as used herein refers to an alkyl group wherein C _x-y alkyl is as defined herein and at least one methylene group (i.e. -CH2-) is replaced with an ester group (e.g. -CO2-). Examples of Ci-6 alkyl carbonyloxy groups include ethanoate, propanoate, butanoate, pentanoate, and hexanoate. The term 'carbonyloxy' as used herein refers to a single carbonyloxy group of the formula: -CO2-.

The term 'C _x - _y alkenyl carbonyl' as used herein refers to an alkenyl group wherein C _x-y alkenyl is as defined herein and at least one methylene group (i.e. -CH2-) is replaced with a carbonyl group (i.e. >C=0). Examples of C2-6 alkenyl carbonyl groups include ethenyl carbonyl, propenylcarbonyl, butenyl carbonyl, pentenylcarbonyl, and hexenyl carbonyl. The term 'C _x - _y alkynyl carbonyl' as used herein refers to an alkynyl group wherein C _x-y alkynyl is as defined herein and at least one methylene group (i.e. -CH ₂ -) is replaced with a carbonyl group (i.e. >C=0). Examples of C2-6 alkynyl carbonyl groups include ethynyl carbonyl, propynylcarbonyl, butynylcarbonyl, pentynylcarbonyl, and hexynyl carbonyl.

The term 'C _x - _y aryl carbonyl' as used herein refers to an aryl group wherein C _x-y aryl is as defined herein covalently linked to at least one carbonyl group (i.e. >C=0). Examples of C ₆ -io aryl carbonyl groups benzoyl, 1-naphthoyl, and 2-naphthoyl.

The term 'C _x-y alkyl carbamoyl' as used herein refers to an alkyl group wherein C _x-y alkyl is as defined herein and at least one methylene group (i.e. -CH ₂ -) is replaced with an amide group (e.g. -C(0) R-, where R is a hydrogen atom, a 5- or 6-membered heterocyclyl group, a 5- or 6- membered heteroaryl group, a 3- to 6-membered cycloalkyl group, a Ci-6 alkyl group, or a C ₆ -i4 aryl group, preferably a hydrogen atom). Examples of Ci-6 alkyl carbamoyl groups include ethyl carbamoyl, propyl carbamoyl, butyl carbamoyl, pentyl carbamoyl, and hexyl carbamoyl. The term 'carbamoyl' as used herein refers to a single carbamoyl group of the formula: - C(0) R-, where R is as defined above.

The term 'C _x-y alkoxy carbonyl' as used herein refers to an alkyl group wherein C _x-y alkyl is as defined herein and at least one methylene group (i.e. -CH ₂ -) is replaced with an ester group (e.g. -OC(O)-). Examples of Ci-6 alkyl carbonyl groups include ethyl oxycarbonyl, propyl oxycarbonyl, butyl oxycarbonyl, pentyl oxycarbonyl, and hexyl oxycarbonyl. The term 'oxycarbonyl' as used herein refers to a single oxycarbonyl group of the formula: -OC(O)-.

The term 'C _x-y alkylene carbonyl' as used herein refers to a divalent group as defined above for the group C _x-y alkylene and wherein at least one methylene group (i.e. -CH ₂ -) is replaced with at least one carbonyl group (i.e. >C=0). Examples of Ci-6 alkylene carbonyl groups include - CH ₂ CO-, -COCH ₂ -, -CH ₂ CH ₂ CO-, -COCH ₂ CH ₂ -, and CH ₂ COCH ₂ -.

The term 'C _x-y alkyl carbonyl' as used herein refers to an alkyl group wherein C _x-y alkyl is as defined herein and at least one methylene group (i.e. -CH ₂ -) is replaced with a carbonyl group (i.e. >C=0). Examples of Ci-6 alkyl carbonyl groups include ethanoyl, propanoyl, butanoyl, pentanoyl, and hexanoyl. The term 'carbonyl' as used herein refers to a single carbonyl group of the formula: >C=0.

The term 'unsaturated hydrocarbon group' as used herein refers to a hydrocarbon group containing at least one unsaturated carbon-carbon bond, i.e. at least one carbon-carbon double or triple bond. Suitable examples of unsaturated hydrocarbon groups include C2-6 alkenylene, C2-6 alkynylene, and C _6-14 arylene groups, and combinations thereof. For example, the unsaturated hydrocarbon group may be a C2-6 alkenylene group adjoined to a C _6-14 arylene group of the formula -(C2H2) _a -(C6H4)b-, where a and b are 1 or 2, or two C2-6 alkenylene groups flanking a C _6-14 arylene group of the formula -(C2H2) _a -(C6H4)b-(C2H2) _a -, where a and b are 1 or 2.

The term 'hydroxyl' as used herein refers to a group of the formula -OH. If such a group is substituted, the resulting group is an ether of the formula -0-.

The term 'thiol' as used herein refers to a group of the formula -SH. If such a group is substituted, the resulting group is a thioether of the formula -S-.

Each symbol in formula (I) is described in detail in the following.

Ring A is an optionally substituted 5- or 6-membered heterocyclyl, or heteroaryl ring. In a preferable embodiment, ring A is an optionally substituted 5-membered heterocyclyl, or heteroaryl ring, more preferably an optionally substituted 5-membered heteroaryl ring. For example, thiophenyl, furyl, pyrrolyl, imidazolyl, oxazolyl, and thiazolyl rings are preferred, especially thiazolyl.

Ring B is an optionally substituted 5- or 6-membered heterocyclyl, heteroaryl, or hydrocarbon ring. In a preferable embodiment, ring B is an optionally substituted 5-membered heterocyclyl, or heteroaryl ring, more preferably an optionally substituted 5-membered heteroaryl ring. For example, thiophenyl, furyl, pyrrolyl, imidazolyl, oxazolyl, and thiazolyl rings are preferred, especially thiazolyl. R ¹ is hydrogen or an optionally substituted alkyl group. In a preferable embodiment, R ¹ is hydrogen or an optionally substituted C _1-3 alkyl group. In particular, for reasons of improved resonance, and thus stability, R ¹ is preferably hydrogen. Each R ² , when present, is a group independently selected from optionally substituted hydroxyl, optionally substituted amino, optionally substituted thiol, optionally substituted alkyl, and optionally substituted aryl. In a preferable embodiment, R ² , when present, is a group independently selected from optionally substituted hydroxyl, optionally substituted amino, and optionally substituted alkyl. In particular, for reasons of improved resonance, R ² is preferably a group independently selected from hydroxyl and optionally substituted amino.

R ³ is an optionally substituted acyl group. In a preferable embodiment, R ³ is an acyl group group selected from:

(1) formyl;

(2) optionally substituted Ci-6 alkyl carbonyloxy;

(3) optionally substituted C2-6 alkenyl carbonyl;

(4) optionally substituted C2-6 alkynyl carbonyl;

(5) optionally substituted C ₆ -io aryl carbonyl;

(6) carboxyl;

(7) optionally substituted Ci-6 alkyl carbamoyl; and

(8) carbamoyl.

In a particularly preferred embodiment, R ³ is an acyl group group selected from optionally substituted Ci-6 alkyl carbonyloxy, optionally substituted C ₆ -io aryl carbonyl, carboxyl, optionally substituted Ci-6 alkyl carbamoyl, and carbamoyl, even more preferably R ³ is optionally substituted Ci-6 alkyl carbonyloxy or carboxyl. In particular, when R ³ is a C1-3 alkyl carbonyloxy group, a more stable compound is obtained, e.g. racemisation at the a-position to the carbonyl is reduced, and without any significant adverse effects in terms of bioluminescence. Indeed, common esterases cleave such groups in vivo to produce functional luciferin analogues having carboxyl functionality. Therefore, although R ³ is preferably hydrogen for reasons of improved resonance stability, R ³ is preferably a C1-3 alkyl carbonyloxy group for stability and handling ex vivo with the ester group being cleaved in vivo. M is a group selected from O, S, and R ⁴ , wherein R ⁴ is a group selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl. In a preferable embodiment, M is selected from O and S. In particular, M is preferably O. Furthermore, it has been found that compounds wherein M is O and R ¹ is hydrogen are particularly stable.

X is an optionally substituted unsaturated hydrocarbon group or an optionally substituted alkylene carbonyl. In a preferable embodiment, X is a group selected from optionally substituted alkenylene, optionally substituted alkynylene, and optionally substituted arylene groups, and combinations thereof, or an optionally substituted alkylene carbonyl group. In another preferred embodiment, X is a group selected from optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted aralkylene, and optionally substituted alkylene carbonyl. In particular, a C2-6 alkylene carbonyl group, or a group selected from C2-6 alkenylene, C2-6 alkynylene, C _6-14 arylene, and combinations thereof, is preferred. Most preferably, X is an optionally substituted alkenylene group. Preferably, X provides conjugation between the two ring systems, such that resonance stabilisation is provided. Furthermore, additional conjugation provided by X, i.e. by multiple unsaturated bonds such as -C2H2-C2H2-, has been found to further red-shift the emission wavelength into the nIR. The number of possible substituents, R ² , on the phenyl ring of the compound of formula (I) is denoted n, where n is 0, 1, 2, or 3. In a preferable embodiment, n is 0, 1, or 2, even more preferably n is 0 or 1.

Each of the optionally substituted heterocyclyl, optionally substituted heteroaryl, and optionally substituted hydrocarbon rings, and the optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted alkoxy, optionally substituted hydroxyl, optionally substituted thiol, optionally substituted unsaturated hydrocarbon, optionally substituted alkylene carbonyl, optionally substituted alkoxy carbonyl, optionally substituted alkyl carbonyloxy, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene, and optionally substituted amino groups, may be substituted by: (1) one or two groups selected from -J-aryl, -J-heteroaryl, -J-heterocyclyl and -J-C3-6 cycloalkyl, wherein J represents a bond or Ci-6 alkylene, and said aryl is selected from phenyl, said heteroaryl is selected from triazolyl, thiazolyl, thienyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, and pyridyl, said heterocyclyl is selected from pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and thiazolidinyl, and said C3-6 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and/or

(2) one to three substituents selected from

(a) Ci-6 alkyl (preferably methyl, ethyl or isopropyl),

(b) Ci-6 alkenyl (preferably propenyl),

(c) Ci-6 alkynyl (preferably ethynyl or propynyl),

(d) halogen (preferably CI or Br),

(e) haloCi-6 alkyl (preferably trifluoromethyl),

(f) cyano,

(g) amino, optionally mono-or di- substituted with Ci-6 alkyl, tert-butoxycarbonyl or benzyl,

(h) Ci-6 alkoxy (preferably methoxy),

(i) Ci-6 alkyl carbonyl, including ketones and derivatives thereof such as ketals and hemiketals, and aldehydes (e.g. formyl) and derivatives thereof such as acetals and hemiacetals (preferably acetyl),

(j) Ci-6 alkoxy carbonyl,

(k) Ci-6 alkyl carbonyloxy, including carboxyl,

(1) Ci-6 alkyl carbamoyl, including carbamoyl,

(m) Ci-6 alkyl thioether, or

(n) nitro.

In a preferred embodiment of the invention, there is provided a compound having the formula (II):

wherein

V, W, and Y are each independently selected from CR ⁵ , C(R ⁶ ) ₂ , N, R ⁷ , S, and O; and

Z is selected from C(R ⁶ ) ₂ , NR ⁷ , S, and O, wherein R ⁵ is a group selected from hydrogen, halogen, optionally substituted hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, and optionally substituted amino;

R ⁶ is a group selected from hydrogen, halogen, optionally substituted hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, and optionally substituted amino; R ⁷ is a group selected from hydrogen, halogen, optionally substituted hydroxyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl; and

the remaining groups are as defined above in relation to formula (I).

In this embodiment, R ⁵ is preferably a group selected from hydrogen, optionally substituted hydroxyl, optionally substituted alkyl, and optionally substituted amino. R ⁶ is preferably a group selected from hydrogen, optionally substituted hydroxyl, optionally substituted alkyl, and optionally substituted amino. R ⁷ is preferably a group selected from hydrogen, optionally substituted alkyl, and optionally substituted aryl.

Furthermore, V and Y are preferably each independently selected from CR ⁵ and N. W and Z are preferably each independently selected from R ⁷ , S, and O.

Preferably, there is provided a compound of formula (II):

wherein

R ¹ is hydrogen or optionally substituted alkyl (preferably hydrogen);

R ² , when present, is a group independently selected from optionally substituted hydroxyl, optionally substituted amino, and optionally substituted alkyl;

R ³ is optionally substituted Ci-6 alkyl carbonyloxy or carboxyl;

M is selected from O, S, and NR ⁴ , wherein R ⁴ is a group selected from hydrogen, optionally substituted alkyl, and optionally substituted aryl (preferably O); V, W, and Y are each independently selected from CR ⁵ and N;

Z is selected from R ⁷ , S, and O,

wherein R ⁵ is a group selected from hydrogen, optionally substituted hydroxyl, optionally substituted alkyl, and optionally substituted amino, and

R ⁷ is a group selected from hydrogen, optionally substituted alkyl, and optionally substituted aryl; and

n is 0 or 1.

Such compounds have been shown to possess an emission wavelength in the nIR, and therefore show improved spectral properties through haemoglobin, i.e. there is less interference and/or scatter when imaging is conducted in vivo. In addition, the compounds exhibit an excellent quantum yield, at least comparable to those known in the art, yet also provide an additional red-shift with Flue colour mutants. In another preferred embodiment of the invention, there is provided a compound having the formula (III):

wherein

V and Y are each independently selected from CR ⁵ and N;

W and Z are each independently selected from NR ⁷ , S, and O; and

the remaining groups are as defined above in relation to formula (I).

In this embodiment, it is preferable that V and Y are each N. It is also preferable that W and Z are each S.

Preferably, there is provided a compound of formula (III):

wherein R ¹ is hydrogen or optionally substituted alkyl (preferably hydrogen);

R ² is a group independently selected from optionally substituted hydroxyl, optionally substituted amino, and optionally substituted alkyl;

R ³ is optionally substituted Ci-6 alkyl carbonyloxy or carboxyl;

M is selected from O and S (preferably O);

V, W, and Y are each independently selected from CR ⁵ and N; and

Z is selected from R ⁷ , S, and O,

wherein R ⁵ is a group selected from hydrogen, optionally substituted hydroxyl, optionally substituted alkyl, and optionally substituted amino, and

R ⁷ is a group selected from hydrogen, optionally substituted alkyl, and optionally substituted aryl. Such compounds have been shown to possess an emission wavelength in the nIR, and therefore show improved spectral properties through haemoglobin, i.e. there is less interference and/or scatter when imaging is conducted in vivo. In addition, the compounds exhibit an excellent quantum yield, at least comparable to those known in the art, yet also provide an additional red-shift with Flue colour mutants. Furthermore, the compounds show good bioluminescence intensity in vivo, without requiring pre-adenylation.

Preferably, each of the optionally substituted heterocyclyl, optionally substituted heteroaryl, and optionally substituted hydrocarbon rings, and the optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted alkoxy, optionally substituted hydroxyl, optionally substituted thiol, optionally substituted unsaturated hydrocarbon, optionally substituted alkylene carbonyl, optionally substituted alkoxy carbonyl, optionally substituted alkyl carbonyloxy, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene, and optionally substituted amino groups, may be substituted by:

(1) a group selected from -J-aryl, -J-heteroaryl, -J-heterocyclyl and -J-C3-6 cycloalkyl, wherein J represents a bond or C _1-3 alkylene, and said aryl is selected from phenyl, said heteroaryl is selected from triazolyl, thiazolyl, thienyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, and pyridyl, said heterocyclyl is selected from pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, and thiazolidinyl, and said C3-6 cycloalkyl is selected from cyclopropyl, cyclopentyl and cyclohexyl; or

(2) one to three substituents selected from (a) Ci-6 alkyl (preferably methyl, ethyl or isopropyl),

(b) Ci-3 alkenyl (preferably propenyl),

(c) halogen (preferably CI or Br),

(e) haloCi-6 alkyl (preferably trifluoromethyl),

(d) cyano,

(e) amino, optionally mono-or di- substituted with C1-3 alkyl, tert-butoxycarbonyl or benzyl,

(f) Ci-3 alkoxy (preferably methoxy),

(g) Ci-6 alkyl carbonyl (preferably acetyl),

(h) Ci-3 alkoxy carbonyl,

(i) Ci-3 alkyl carbonyloxy, including carboxyl,

(j) Ci-3 alkyl carbamoyl, including carbamoyl.

The compounds of the present invention may be in any stereoisomeric form. It is preferable, nevertheless, that the compounds possess the same stereoisomeric form as natural luciferin, i.e. ^-stereochemistry at the a-position to the carbonyl functionality (on the basis of a dihydrothiazole ring).

In particular, the compound according to the invention may be selected from:

and optionally substituted Ci-6 alkyl esters thereof, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl (preferably methyl and ethyl). A compound according to the invention may be for use in in vivo imaging, preferably in vivo bioluminescence imaging. Furthermore, any of the preferred structural variants mentioned above in relation to formulae (I)-(III) also represent preferred aspects of this embodiment as in vivo imaging agents.

In particular, cells or animals bearing firefly luciferase may be detected using a number of common imaging devices. The most common form of such devices detects emitted photons using a lens focused onto a cooled charge-coupled device (CCD) with or without pre- intensification via a photomultiplier tube (e.g. IVIS imaging systems, by Perkin Elmer, MA, USA; and Photon Imager system, by Biospace Laboratories, Paris, France).

In addition, chemiluminescence reactions involving compounds according to the invention, or bioluminescence elicited using compounds according to the invention, and firefly luciferase in in vitro or diagnostic assays may be detected with common laboratory single tube or microplate luminometers, which employ either photomultiplier tube or CCD photon detectors (e.g. those sold by Promega, WI, USA).

In another aspect of the invention, there is provided a composition comprising a compound according to the invention, and one or more additional ingredients. Furthermore, any of the preferred structural variants mentioned above in relation to formulae (I)-(III) also represent preferred aspects of this composition.

The additional ingredients in the composition may include pharmaceutically acceptable diluents, excipients and carriers. Pharmaceutically acceptable diluents, excipients and carriers that may be used in the compositions include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, coenzyme A, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. The composition of the invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Preferably, the compositions are administered parenterally, more specifically by injection. The composition may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. The term parenteral as used herein includes intraperitoneal, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraocular, intraorbital, intralesional and intracranial injection or infusion techniques. Preferably, the route of administration of the composition is intraperitoneal, intravenous or intramuscular administration (most preferably intraperitoneal).

The composition may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.

In another aspect of the invention, there is provided a use of the compound according to the invention as a bioluminescent marker for in vitro measurement of luciferase expression. Furthermore, any of the preferred structural variants mentioned above in relation to formulae (I)-(III) also represent preferred aspects of this use.

Given that the compound of the invention has specific utility as an in vivo imaging agent, there is also provided a method of in vivo imaging, comprising administering to a subject a compound according to the invention, and a luciferase enzyme or a polynucleotide encoding a luciferase enzyme, and measuring the emission resulting from the action of the luciferase enzyme on the compound. Furthermore, any of the preferred structural variants mentioned above in relation to formulae (I)-(III) also represent preferred aspects of this method. The method may further comprising generating an image based on the measurements obtained.

In an additional aspect of the invention, there is provided a kit comprising a compound according to the invention, and a luciferase enzyme or a polynucleotide encoding a luciferase enzyme. Furthermore, any of the preferred structural variants mentioned above in relation to formulae (I)-(III) also represent preferred aspects of this kit.

The invention will now be described in more detail by way of example only, and with reference to the following figures. Description of the Figures

Figure 1

Fluorescence spectra of compound 9 (often denoted 'infraluciferin' or 'iLH ₂ ') in different pH conditions. The fluorescence spectra of compound 9 shows that it has redshifted absorbance, excitation and emission fluorescence spectra compared to native LH ₂ . In addition, higher pH further red-shifted the fluorescence excitation and emission, potentially indicating deprotonation and molecular conjugation in line with the resonance charge based mechanism. Importantly, compound 9 appeared to have emission maxima at different wavelengths dependent on the conditions.

Figure 2

Bioluminescence spectra of native LH ₂ and compound 9 ('analogue') with Flue colour mutants (x5 thermostable and x5 thermostable red). With a mutant that produces red emission with native LH ₂ ( max 620 nm), compound 9 produced a colour with max 700 nm, which is significantly more red-shifted than those in the art. Spectra experimental details: 50 μΙ_, of 5 μΜ Flue or red mutant in 100 mM tris-acetate, 10 mM magnesium sulfate and 2mM ethylenediaminetetraacetic acid (EDTA) (pH 7.8) was added to 20 μΙ_, 500 μΜ compound 9 with 70 μΙ_, PBS. Spectra were acquired in a Luminoskan Ascent machine (Thermo scientific, Waltham, MA, USA). Conversely, luciferin analogues known in the art do not show such a red-shift with Flue colour mutants.

Figure 3

Light emission kinetics of compound 8 added to Raji cells expressing thermostable Flue. The methyl (Me) ester (compound 8) was seen to be enantio-stable, and was found to be active in mammalian and bacterial cells. The kinetics of saponification in mammalian cells showed a slow rise (ca. 2min) to maximal activity. Experimental details: 20 μΐ 1 mg/ml compound 8 in citrate buffer (pH 5) was added to 10 μΐ containing 1 x 10 ⁶ Raji cells expressing thermsotable Flue and light emission was recorded over 2 min in a Turner Designs TD20/20 instrument (Turner Designs, CA, USA).

Figure 4

Relative light yield of compounds 8 and 12 in vitro compared to D-LH ₂ ethyl ester measured in an intensified CCD-based photon imager with good sensitivity to red light. The difference in light yield from compound 8 was seen to be only ca. 150-times less than from LH ₂ ethyl ester. Experimental details: 10 μΕ of ImM of the compounds was added to 50 μg pig liver esterase mixed with 5 mM ATP and 0.25 μΜ Flue enzymes. Light was captured in a Photon Imager machine (Biospace Lab, Paris, France).

Figure 5

In vitro bioluminescence spectra of luciferase mutants x5 (left series) and x5 red (right series), with compound 9. In vitro bioluminescence spectra were acquired by imaging the samples through different filter sets and confirmed the two colours of compound 9 with x5 and x5 red. It may be possible to image both separately by applying 550-600nm bandpass and 830 longpass filters, respectively. Experimental details: 10 μg purified porcine liver esterase in TEM buffer was added to 1 mL of compound 8 dissolved in TEM buffer and incubated at 37° C for 15min before assay as per details in Figure 1. Light emission was captured in a Photon Imager device for 30s using different bandpass filters (Biospace Labs, Paris, France).

Figure 6

Relative transmission of LH ₂ ethyl ester and compound 12 through blood with natural and red coloured Flue mutants. When Raji cells expressing x5 Flue injected into mice containing systemic Raji cell lymphomas, the produced light that was imaged was ca. 8-times less in intensity than native LH ₂ in the region of interest shown.

Figure 7

Relative light yield of compound 12 (middle image) in vivo compared to D-LH ₂ (right image) measured in an intensified CCD-based photon imager with good sensitivity to red light. The left image is a control with no filter and no substrate. The enhanced in vivo light yield compared to in vitro may be in part explained by the improved transmission of the emission of iLH ₂ through blood compared to LH ₂ .

Figure 8

Comparison of LH ₂ ethyl ester (termed 'luciferin' in the figure) and compound 8 (termed 'infraluciferin' in the figure) light emission in a subcutaneous colon carcinoma model. In a xenograft model of colon carcinoma in nude mice (MFI NuNu), light from sub cutaneously engrafted LS274T cells WT Flue expressing mice imaged with iLH ₂ ethyl ester was approximately 4 times less light than with compound 8. When subsequently imaged using an 830LP filter (infrared filter), the light yield from compound 8 was approximately 5-fold higher than from LH ₂ ethyl ester. Experimental details: mice were inoculated with 3 x 10 ⁶ LS174T cells expressing WT Flue and 5 days later were injected with 5mg of LH ₂ ethyl ester or compound 8 and imaged for a total of 30s in a Photon imager instrument (Biospace Labs, Paris, France). The infrared filter used was an 830 nm long pass (830 LP).

Figure 9

Quantification of LH ₂ ethyl ester and compound 8 light emission in a subcutaneous colon carcinoma model - no filter. Experimental details: M3 vision software (Biospace Labs, Paris, France) was used to process the light emission.

Figure 10

Quantification of LH ₂ ethyl ester and compound 8 light emission in a subcutaneous colon carcinoma model - 830LP filter. Experimental details: M3 vision software (Biospace Labs, Paris, France) was used to process the light emission. Figure 11

In vivo spectrum of light emission from LH ₂ ethyl ester (left series) and compound 8 (right series). A larger proportion of light emitted with compound 8 in vivo was in the nIR portion of the electromagnetic spectrum than LH ₂ ethyl ester. Experimental details as in Figure 8, but multiple acquisitions were acquired for 2 min each through a series of filters and corrected for light decay.

Figure 12

Light emission of compound 17 (denoted 'ssLH ₂ '). This compound was found to also generate light emission with purified x5 Flue, though this was less intense than emission with compound 8. Experimental details as in Figure 4.

Figure 13

iCCD spectrum of x5 Flue with compound 17. The in vitro emission peaked between 630-680 nm. Experimental details as in figure 5.

Figure 14

Fluorescence spectra of compound 8 against compound 17. The fluorescence spectra showed that compound 17 was further redshifted than compound 8 in its fluorescence excitation and emission properties, and also had access to two colour forms (pH dependent). Experimental details as in Figure 1.

Examples 6-Hydroxy-2-(2-(4S-carboxy-4, 5-dihydrothiazol-2-yl)ethenyl)benzothiazole (9)

Amino acid, D-TrtCysOMe was synthesised using an analogous method to that developed for L-cysteine by Rudolph et al., J. Med. Chem. 2001, 44, 619 and protected as the methyl ester using thionyl chloride (Mays et al, Tetrahedron Lett., 2007, 48, 4579).

6-Benzoxy-2-bromo-benzothiazole (2)

A solution of 1 (640 mg, 2.78 mmol) and BnBr (0.38 mL, 3.33 mmol) in acetone (50 mL) was treated with K2CO3 (1.09 g, 7.77 mmol) and stirred at rt for 16 h. After this time the reaction was filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (5 % EtO Ac/Pet. Ether) to give 2 (759 mg, 85 %) as a white solid, m.p. 58-60 °C; R _f = 0.61 (20 % EtOAc/Pet. Ether); IR v _m ax 2937 (V _C H), 2873 (V _C H), 1598, 1557, 1483, 1452, 1380, 1282, 1246, 1222, 1210, 1016, 946 cm ^"1 ; ¾ MR (CDCI3) δ 5.13 (2H, s, OG%Ar), 7.14 (1H, dd, J= 9.0, 2.6, ArH), 7.33 (1H, d, J= 2.5, ArH), 7.35-7.46 (6H, m, ArH), 7.87 (1H, d, J = 8.9, ArH); ¹³ C NMR (CDCI3) δ 70.8 (CH ₂ ), 105.0 (CH), 116.5 (CH), 123.4 (CH), 127.5 (CH), 128.3 (CH), 128.8 (CH), 135.7 (C), 136.4 (C), 138.6 (C), 147.1 (C), 157.2 (C); m/z (ES+) 322 (100 %), 320 (100 %, M ⁺ +H), 321 (18 %), 272 (7 %), 272 (7 %); HRMS Ci ₄ HnBrNOS calcd. 319.9745, found 319.9730; Anal. Calcd. for Ci ₄ HioBrNOS: C, 52.51; H, 3.15; N, 4.37. Found C, 52.19; H, 3.03; N, 4.33 %.

6-Benzoxy-2-formyl-benzothiazole (3)

A solution of 2 (120 mg, 0.374 mmol) in THF (4 mL) was cooled to -78 °C, treated with ⁿ BuLi (1.93 M, 0.22 mL, 0.412 mmol) (colour change from colourless to pale yellow) and stirred at - 78 °C for 15 min. After this time DMF (0.12 mL, 1.55 mmol) was added dropwise and the resultant solution stirred at -78 °C for 1 h. The reaction was quenched with NaHCC (aq) (2 mL), back extracted with EtO Ac (2 χ 10 mL), separated and concentrated in vacuo. Purification was achieved by flash column chromatography (5-10 % EtO Ac/Pet. Ether) to give 3 (98 mg, 96 %) as a white solid, m.p. 1 12-114 °C; R _f = 0.51 (20 % EtO Ac/Pet. Ether); IR Vmax 3035 (VCH), 2854 (V _C H), 1677 (vco), 1598, 1547, 1489, 1455, 1380, 1269, 1192, 1120, 999 cm ^"1 ; ¾ NMR (CDCI3) δ 5.19 (2H, s, OC%Ar), 7.30 (1H, dd, J = 9.1, 2.5, ArH), 7.38-7.48 (7H, m, ArH), 8.12 (1H, d, J = 9.1, ArH), 10.1 (1H, s, C(O)H); ¹³ C NMR (CDCI3) δ 70.8 (CH ₂ ), 105.0 (CH), 118.8 (CH), 126.7 (CH), 127.6 (CH), 128.5 (CH), 128.9 (CH), 136.0 (C), 138.6 (C), 148.5 (C), 159.5 (C), 163.2 (C), 185.2 (CH); m/z (ES+) 270 (100 %, M ⁺ +H), 210 (6 %), 132 (14 %, M ⁺ +H-C(0)H, OBn); HRMS Ci ₅ Hi ₂ N0 ₂ S calcd. 270.0589, found 270.0585; Anal. Calcd. for Ci ₅ HnN0 ₂ S: C, 66.89; H, 4.12; N, 5.20. Found C, 66.62; H, 4.04; N, 5.10 %. (E)-3-(6-benzoxy-benzothiazol-2-yl)-acrylic acid ethyl ester (4)

A solution of 3 (568 mg, 2.10 mmol) in PhMe (10 mL) was treated with (carbethoxymethylene)triphenylphosphorane (2.16 g, 6.31 mmol) and heated to reflux for 3 h. After this time the reaction was cooled to rt and concentrated in vacuo. Purification was achieved by flash column chromatography (10 % EtO Ac/Pet. Ether) to give 4 (660 mg, 92 %) as a pale yellow solid, m.p 79-80 °C; R _f = 0.38 (20 % EtOAc/Pet. Ether); IR v _m ax 3063 (VCH), 3038 (VCH), 2979 (VCH), 1703 (vco) 1595, 1551, 1488, 1445, 1386, 1368, 1254, 1106, 1018, 958 cm ^"1 ; 1.36 (3H, t, J = 7.1, CH ₂ C%), 4.31 (2H, q, J = 7.1, C%CH ₃ ), 5.16 (2H, s, OC%Ar), 6.71 (1H, d, J = 15.9, CHC(O)OEt), 7.20 (1H, dd, J = 9.0, 2.5, ArH), 7.35-7.48 (6H, m, ArH), 7.85 (1H, d, J = 15.9, CHC(N)S), 7.96 (1H, d, J = 9.0, ArH); ¹³ C NMR (CDC1 ₃ ) δ 14.3 (C¾), 61.2 (CH ₂ ), 70.7 (CH ₂ ), 105.2 (CH), 117.3 (CH), 124.7 (CH), 125.1 (CH), 127.6 (CH), 128.3 (CH), 128.8 (CH), 136.4 (C), 136.8 (C), 137.0 (CH), 148.7 (C), 158.0 (C), 161.2 (C), 165.8 (C); m/z (ES+) 340 (100%, M ⁺ +H), 339 (7%, M ⁺ ), 294 (7%, M ⁺ +H-OEt), 91 (7%, PhCH ₂ ); HRMS Ci ₉ Hi ₈ N0 ₃ S calcd. 340.1007, found 340.1003; Anal. Calcd. for Ci ₉ Hi ₇ N0 ₃ S: C, 67.24; H, 5.05; N, 4.13. Found C, 67.04; H, 5.02; N, 4.09 %.

(E)-3-(6-benzoxy-benzothiazol-2-yl)-acrylic acid (5)

5 A suspension of 4 (500 mg, 1.47 mmol) in ΨΐΟΗ (18.3 mL) was treated with NaOH (4.70 mL, 1.0 M) and stirred at rt overnight. An additional 10 mL of H ₂ 0 was added so that all precipitate had dissolved and the resultant solution acidified to pH 1 with HC1 (1 M). The yellow precipitate was isolated by filtration and washed with H ₂ 0 (30 mL) to give 5 (458 mg, quant.) as a fine yellow powder, m.p 174-176 °C; Rf = 0.40 (10 % MeOH/DCM); IR Vmax 3031 (VCH), 2866 (VCH), 2543 (VOH), 1694 (vco), 1599, 1558, 1497, 1451, 1312, 1259, 1205, 1051, 1013, 966 cm ^"1 ; ¾ MR (DMSO) δ 15.20 (2H, s, OC%Ar), 6.72 (1H, d, J = 15.9, CHC(O)OEt), 7.23 (1H, dd, J = 9.0, 2.5, ArH), 7.32-7.50 (5H, m, ArH), 7.69 (1H, d, J = 15.9, CHC(N)S), 7.83 (1H, d, J = 2.4, ArH), 7.97 (1Η, d, J = 9.0, ArH); ¹³ C NMR (DMSO) δ 69.9 (CH ₂ ), 105.8 (CH), 117.2 (CH), 124.2 (CH), 126.0 (CH), 127.9 (CH), 128.0 (CH), 128.5 (CH), 135.9 (CH), 136.6 (C), 136.6 (C), 147.9 (C), 157.4 (C), 160.8 (C), 166.4 (C); m/z (CI) 402 (26%), 312 (100%, M ⁺ +H), 294 (11%, M ⁺ +H-OH), 91 (5%, PhCH ₂ ); HRMS C17H14NO3S calcd. 312.0694, found 312.0691.

D-(Methyl 2-((E)-3-(6-benzoxybenzothiazol-2-yl)aciylamido)-3-(tritylth io)proponoate (6)

6 A solution of 5 (134 mg, 0.424 mmol) in DMF (4.2 mL) was treated with Et ₃ N (70 μί, 1.02 mmol) and cooled to 0 °C. The solution was treated with a solution of aminoacid (192 mg, 0.508 mmol) in DCM (2 mL) followed by a solution of BOP (231 mg, 0.508 mmol) in DCM (2.2 mL) and the resultant solution stirred at 0 °C for 2 h. After this time the reaction mixture was quenched with saturated NaHC0 ₃ (aq) (2 mL) and taken up in EtOAc (10 mL), the aqueous layer was back extracted using EtOAc (2 x 5 mL), organics dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (20 % EtOAc/Pet. Ether) to give 6 (220 mg, 80 %) as a yellow solid, m.p. 83-86 °C; R _f = 0.28 (30 % EtO Ac/Pet. Ether); [a] _D +62.6 (c 0.95, CHC1 ₃ ); CSP HPLC analysis (Chiracel OD-H (150 4.6 mm), eluent: hexane: ΨΓΟΗ, 70:30, flow 0.5 mL/min, 14 bar) determined > 98 % ee [t _R (minor) = 43.07 min, t _R (major) = 26.59 min]; IR Vmax 1741 (vco), 1662, 1627, 1596, 1488, 1446, 1346, 1319, 1260, 1196, 1051, 1001, 966 cm ^"1 ; ¾ NMR (CDC1 ₃ ) δ 2.73 (1H, dd, J = 12.7, 4.6, CH ₂ STrt), 2.82 (1Η, dd, J = 12.7, 5.6, CH ₂ STrt), 3.76 (3Η, s, OCH5), 4.76 (1Η, dt, J = 7.8, 5.2, CHCH ₂ ), 5.17 (2H, s, OCH ₂ Ph), 6.09 (1Η, d, J = 7.8, NH), 6.65 (1Η, d, J = 15.5, CHC(O)N), 7.20-7.49 (22Η, m, ArH), 7.73 (1Η, d, J = 15.5, CHC(N)S), 7.96 (1Η, d, J = 9.0, ArH); ¹³ C NMR (CDC1 ₃ ) δ 33.8 (CH ₂ ), 51.4 (CH), 52.9 (CH ₃ ), 67.2 (C), 70.7 (CH ₂ ), 105.2 (CH), 117.2 (CH), 124.6 (CH), 126.8 (CH), 127.1 (CH), 127.6 (CH), 128.1 (CH), 128.3 (CH), 128.8 (CH), 129.6 (CH), 134.2 (CH), 136.4 (C), 144.3 (C), 154.2 (C), 157.9 (C), 161.4 (C), 163.8 (C), 170.7 (C); m/z (ES+) 693 (96%, M ⁺ +Na), 595 (9%), 244 (18%), 243 (100%, M ⁺ - STrt,C0 ₂ Me, Bn, H), 228 (8%), 165 (22%); HRMS C ₄ oH34N20 ₄ NaS2 calcd. 693.1858, found 693.1855.

6-Benzoxy-2-(2-(4 ₎ 5'-methoxycarbonyl-4,5-dihydrothiazol-2-yl)ethenyl)ben zothiazole (7)

7

A solution of Ph ₃ PO (386 mg, 1.40 mmol) in DCM (10 mL) was cooled to 0 °C in an ice bath and treated with Tf ₂ 0 (0.160 mL, 0.874 mmol) added dropwise over 5 min. The resultant solution was stirred at 0 °C for 30 min and a solution of 6 (220 mg, 0.329 mmol) in DCM (4 mL) was then added dropwise over 5 min. The reaction was stirred at 0 °C for 10 min and then quenched with phosphate buffer (4 mL). Extracted into DCM (3 x 10 mL) and separated. The organic layer was dried over MgS04 , filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (20-40 % EtOAc/Pet. Ether) to give 7 (88 mg, 65 %) as a pale yellow solid, m.p. 98-100 °C; R _f = 0.32 (40 % EtOAc/Pet. Ether); IR v _m ax 3032, 2947 (VCH), 1726 (vco), 1599, 1569, 1557, 1480, 1454, 1431, 1380, 1319, 1261, 1242, 1225, 1199, 1176, 1043, 1018, 946 cm ^"1 ; ¾ MR (CDC1 ₃ ) δ 3.63 (1H, dd, J= 10.9, 9.3, CH ₂ S), 3.71 (1H, dd, J = 11.0, 9.3, CH ₂ S), 3.86 (3H, s, OC%), 5.15 (2H, s, OC%Ph), 5.26 (1H, t, J = 9.3, CHCH ₂ ), 7.18 (1H, dd, J= 9.0, 2.5, ArH), 7.31-7.48 (8H, m, ArH, 2 CHC(N)S), 7.93 (1H, d, J = 9.0, ArH); ¹³ C MR (CDC1 ₃ ) δ 35.1 (CH ₂ ), 53.1 (CH ₃ ), 70.7 (CH ₂ ), 78.2 (CH), 105.2 (CH), 117.2 (CH), 124.6 (CH), 127.6 (CH), 128.4 (CH), 128.8 (CH), 129.0 (CH), 135.2 (CH), 136.4 (C), 136.7 (C), 148.7 (C), 158.0 (C), 162.0 (C), 169.4 (C), 170.9 (C); m/z (ES+) 474 (32%), 411 (100%, M ⁺ +H), 320 (12%); HRMS C ₂ iHi ₉ N ₂ 0 ₃ S ₂ calcd. 411.0837, found 411.0831; Anal. Calcd. for C ₂ iHi ₈ N ₂ 0 ₃ S ₂ : C, 61.44; H, 4.42; N, 6.82. Found C, 61.38; H, 4.34; N, 6.74 %.

CSP HPLC analysis determined 45-95 % ee dependant on scale of reaction and purity of Tf ₂ 0. Optical purity could be achieved by dissolving 7 in MeCN and concentrating in vacuo until a precipitate appeared. Filtration and concentration of the filtrate gave 7. [α]ϋ -28.4 (c 0.91 , CHCb); CSP HPLC analysis (Chiracel OD-H, eluent: hexane: Ί>ΓΟΗ, 70:30, flow 0.5 mL/min, 14 bar) > 98 % ee [t _R (minor) = 21.8 min, t _R (major) = 30.7 min]. 6-Hydroxy-2-(2-(4 ₎ 5'-methoxycarbonyl-4,5-dihydrothiazol-2-yl)ethenyl)ben zothiazole (8)

8

A solution of 7 (55 mg, 0.133 mmol) in DCM (2 mL) was cooled to -78 °C in an acetone/C02( _S ) bath and treated with pentamethylbenzene (110 mg, 0.580 mmol) followed by BCI3 (0.39 mL, 0.399 mmol, 1 M in DCM) added dropwise over 5 min. The reaction was stirred at -78 °C for 20 min and then quenched with phosphate buffer (2 mL). Extracted into DCM (3 x 5 mL) and separated. The organic layer was dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (40-100 % EtOAc/Pet. Ether) to give 8 (34 mg, 79 %, 98 % b.r.s.m) as a pale yellow solid, m.p. 163-165 °C; R _f = 0.13 (40 % EtO Ac/Pet. Ether); IR v _m ax 3210 (V ₀ H), 3043 (V _C H), 2954, 1731 (vco), 1632, 1595, 1558, 1480, 1435, 1314, 1199, 1116, 1022, 949 cm ^"1 ; ¾ NMR (MeOD) δ 3.70 (1H, dd, J = 20.1, 11.2, C¾S), 3.72 (1H, dd, J = 20.8, 11.2, C¾S), 3.81 (3H, s, OC%), 5.33 (1H, t, J= 9.0, CHC0 ₂ Me), 7.03 (1H, dd, J= 8.9, 2.5, ArH), 7.29 (1H, d, J = 16.1, CHC(N)S), 7.31 (1H, d, J = 2.3, ArH), 7.41 (1H, d, J = 16.1, CHC(N)S), 7.82 (1H, d, J = 8.9, ArH); ¹³ C NMR (MeOD) δ 35.7 (CH ₂ ), 53.2 (CH ₃ ), 79.0 (CH), 107.4 (CH), 118.1 (CH), 125.1 (CH), 128.9 (CH), 136.0 (CH), 138.1 (C), 148.6 (C), 158.5 (C), 162.2 (C), 171.6 (C), 172.2 (C).

CSP HPLC analysis determined 65-90 % ee dependant on scale of reaction and purity of BC1 ₃ . Optical purity could be achieved by dissolving 8 in MeCN and concentrating in vacuo until a precipitate appeared. Filtration and concentration of the filtrate gave 8. CSP HPLC analysis (Chiracel AD, eluent: hexane: Ί>ΓΟΗ, 70:30, flow 0.5 mL/min, 23 bar) > 98 % ee [t _R (major) = 6.35 min, t _R (minor) = 9.90 min]. 6-Hydroxy-2-(2-(4 ₎ 5'-carboxy-4,5-dihydrothiazol-2-yl)ethenyl)benzothiazo le (9)

A solution of 8 in phosphate buffer (1 mg/mL) was treated with PLE (5 μg per 1 mg of 8) and incubated at 37 °C for 24 h. After this time the saponified ester 9 was used directly without isolation.

6-Hydroxy-2-(2-(4S-ethoxycarbonyl-4,5-dihydrothiazol-2-yl )ethenyl)

D-(Ethyl 2-((E)-3-(6-benzoxybenzothiazol-2-yl)acrylamido)- 3-(tritylthio)proponoate (10)

10

A solution of 5 (239 mg, 0.755 mmol) in DMF (7.6 mL) was treated with Et ₃ N (0.124 mL, 1.82 mmol) and cooled to 0 °C. The solution was treated with a solution of aminoacid (354 mg, 0.908 mmol) in DCM (3 mL) followed by a solution of BOP (414 mg, 0.908 mmol) in DCM (4.6 mL) and the resultant solution stirred at 0 °C for 2 h. After this time the reaction mixture was quenched with saturated NaHC0 ₃ ( _aq ) (5 mL) and taken up in EtOAc (20 mL), the aqueous layer was back extracted using EtOAc (2 x 10 mL), organics dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (20 % EtO Ac/Pet. Ether) to give 10 (422 mg, 82 %) as an orange solid, m.p. 75-78 °C; R _f = 0.23 (30 % EtO Ac/Pet. Ether); [a] _D +82.0 (c 1.05, CHC1 ₃ ); CSP HPLC analysis (Chiracel AD (300 4.6 mm), eluent: hexane: 'PrOH, 60:40, flow 1 mL/min, 26 bar) determined > 99 % ee [tR (minor) = 16.52 min, t _R (major) = 9.67 min]; IR v _ma x 1735 (vco), 1667, 1628, 1596, 1488, 1446, 1371, 1344, 1260, 1189, 1050, 1019, 966 cm ^"1 ; ¾ MR (CDC1 ₃ ) δ 1.27 (3H, t, J = 7.2, CH ₂ C%), 2.70 (1H, dd, J = 12.6, 4.5, CH ₂ STrt), 2.80 (1H, dd, J = 12.6, 5.5, CH ₂ STrt), 4.22 (2H, m, C%CH ₃ ), 4.75 (1H, dt, J = 7.7, 4.8, CHCH ₂ ), 5.17 (2H, s, OC%Ph), 6.14 (1H, d, J = 7.8, NH), 6.68 (1H, d, J = 15.5, CHC(O)N), 7.20-7.49 (22H, m, ArH), 7.74 (1H, d, J = 15.5, CHC(N)S), 7.96 (1H, d, J = 9.0, ArH); ¹³ C NMR (CDC1 ₃ ) δ 14.3 (CH ₃ ), 34.0 (CH ₂ ), 51.5 (CH), 62.2 (CH ₂ ), 67.1 (C), 70.7 (CH ₂ ), 105.2 (CH), 117.2 (CH), 124.6 (CH), 126.9 (CH), 127.1 (CH), 127.6 (CH), 128.2 (CH), 128.4 (CH), 128.9 (CH), 129.6 (CH), 134.2 (CH), 136.4 (C), 136.8 (C), 144.4 (C), 148.8 (C), 157.9 (C), 161.5 (C), 163.8 (C), 170.2 (C); m/z (ES+) 707 (100%, M ⁺ +Na), 685 (5%, M ⁺ +H), 338 (27%, M ⁺ +H-STrt, C0 ₂ Et), 243 (100%), 228 (23%), 165 (59%); HRMS C ₄ iH ₃₆ NaN ₂ 0 ₄ S ₂ calcd. 707.2014, found 707.2039; Anal. Calcd. for C ₄ iH ₃₆ N ₂ 0 ₄ S ₂ : C, 71.90; H, 5.30; N, 4.09. Found C, 71.43; H, 5.30; N, 3.98 %. 6-Benzoxy-2-(2-(4 ₎ 5'-ethoxycarbonyl-4,5-dihydrothiazol-2-yl)ethenyl)benz othiazole (11)

11

A solution of Ph ₃ PO (955 mg, 3.45 mmol) in DCM (26 mL) was cooled to 0 °C in an ice bath and treated with Tf ₂ 0 (0.39 mL, 2.16 mmol) added dropwise over 5 min. The resultant solution was stirred at 0 °C for 30 min and a solution of 10 (544 mg, 0.797 mmol) in DCM (8 mL) was then added dropwise over 5 min. The reaction was stirred at 0 °C for 10 min and then quenched with phosphate buffer (10 mL). Extracted into DCM (3 x 15 mL) and separated. The organic layer was dried over MgS0 ₄ , filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (20-40 % EtOAc/Pet. Ether) to give 11 (250 mg, 74 %) as a pale yellow solid, m.p. 114-117 °C; R _f = 0.39 (40 % EtOAc/Pet. Ether); IR v _m ax 2977 (V _C H), 1729 (vco), 1597, 1555, 1479, 1453, 1379, 1316, 1261, 1222, 1199, 1049, 1014, 947 cm ^"1 ; ¾ NMR (CDCls) δ 1.36 (3H, t, J = 6.6, CH ₂ C%), 3.63 (1H, dd, J = 1 1.0, 9.4, C¾S), 3.68 (1H, dd, J = 11.0, 9.4, C¾S), 4.27-4.38 (2H, m, CH2CH3), 5.16 (2H, s, OC%Ar), 5.24 (1H, t, J = 9.3, CHC0 ₂ Et), 7.19 (1H, dd, J = 9.0, 2.5, ArH), 7.30-7.49 (8H, m, ArH), 7.93 (1H, d, J = 9.0, ArH); ¹³ C NMR (CDCI3) δ 14.3 (CH ₃ ), 35.1 (CH ₂ ), 62.2 (CH ₂ ), 70.7 (CH ₂ ), 78.5 (CH), 105.2 (CH), 117.2 (CH), 124.6 (CH), 127.6 (CH), 128.4 (CH), 128.8 (CH), 129.2 (CH), 135.0 (CH), 136.4 (C), 136.6 (C), 148.7 (C), 158.0 (C), 162.1 (C), 169.2 (C), 170.5 (C); m/z (ES+) 425 (100%, M ⁺ +H), 334 (25%, M ⁺ -Bn), 293 (11%); HRMS C ₂₂ H ₂ iN ₂ 0 ₃ S ₂ calcd. 425.0994, found 425.0998.

CSP HPLC analysis determined 82-92 % ee dependant on scale of reaction and purity of Tf ₂ 0. Optical purity could be achieved by dissolving 11 in MeCN and concentrating in vacuo until a precipitate appeared. Filtration and concentration of the filtrate gave 11. CSP HPLC analysis (Chiracel AD, eluent: hexane: TrOH, 60:40, flow 1 mL/min, 26 bar) determined > 98 % ee [tR (minor) = 23.60 min, tR (major) = 11.19 min].

6-Hydroxy-2-(2-(4 ₎ 5'-ethoxycarbonyl-4,5-dihydrothiazol-2-yl)ethenyl)benz othiazole (12)

12

A solution of 11 (67 mg, 0.158 mmol) in DCM (2.7 mL) was cooled to -78 °C in an acetone/C0 ₂ ( _S ) bath and treated with pentamethylbenzne (130 mg, 0.788 mmol) followed by BCI3 (0.46 mL, 0.472 mmol, 1 M in DCM) added dropwise over 5 min. The reaction was stirred at -78 °C for 20 min and then quenched with phosphate buffer (2 mL). Extracted into DCM (3 x 5 mL) and separated. The organic layer was dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (40-100 % EtOAc/Pet. Ether) to give 12 (38 mg, 72 %, 96 % b.r.s.m) as a pale yellow solid, m.p. 155-157 °C; R _f = 0.11 (40 % EtOAc/Pet. Ether); IR vmax 3377 (V ₀ H), 2983 (V _C H), 1732 (vco) 1592, 1568, 1476, 1449, 1379, 1369, 1321, 1281, 1254, 1192, 1116, 1062, 1027, 944 cm ^"1 ; ¾ NMR (CDCI3) δ 1.36 (3H, t, J= 7.1, CH ₂ C%), 3.64 (1H, dd, J= 20.5, 10.8, C¾S), 3.68 (1H, dd, J = 20.3, 10.8, C¾S), 4.30-4.36 (2H, m, CH2CH3), 5.25 (1H, t, J = 9.2, CHC0 ₂ Et), 5.76 (1H, s, OH), 7.03 (1Η, dd, J = 8.8, 2.4, ArH), 7.28 (1Η, d, J = 16.1, CHC(N)S) 7.30 (1Η, d, J = 2.4, ArH), 7.38 (1Η, d, J = 16.1, CHC(N)S), 7.89 (1Η, d, J = 8.8, ArH); ¹³ C MR (CDCb) δ 14.3 (CH ₃ ), 35.1 (CH ₂ ), 62.3 (CH ₂ ), 78.4 (CH), 107.0 (CH), 116.7 (CH), 124.7 (CH), 129.1 (CH), 135.0 (CH), 136.7 (C), 148.6 (C), 155.0 (C), 162.0 (C), 169.5 (C), 170.6 (C); m/z (ES+) 335 (100%, M ⁺ +H), 307 (12%, M ⁺ +H-Et), 261 (48%, M ⁺ +H-C0 ₂ Et), 203 (47 %, M ⁺ +H- SCH ₂ CHC0 ₂ Et); HRMS Ci ₅ Hi ₅ N ₂ 0 ₃ S ₂ calcd. 335.0524, found 335.0526.

CSP HPLC analysis determined 80-92 % ee dependant on scale of reaction and purity of BC1 ₃ . Optical purity could be achieved by dissolving 12 in MeCN and concentrating in vacuo until a precipitate appeared. Filtration and concentration of the filtrate gave 12. CSP HPLC analysis (Chiracel AD, eluent: hexane: ¾>ΓΟΗ, 70:30, flow 1.0 mL/min, 23 bar) > 98 % ee [t _R (major) = 6.85 min, tR (minor) = 9.30 min].

Synthesis of 6-Hydroxy-2-(4-lE, 3E-(4S-ethoxycarbonyl-4, 5-dihydrothiazol-2-yl) buta-2, 4- dienyljbenzothiazole (17)

(E)-3-(6-Benzoxy-benzothiazol-2-yl)-prop-2-enal (13)

13 A solution of 3 (100 mg, 0.370 mmol) in DCM (2 mL) was treated with (triphenylphosphorarylidene)acetaldehyde (124 mg, 0.407 mmol) and stirred at rt for 2 h. The reaction was quenched with saturated NaHC0 ₃ ( _aq ) (2 mL), separated and back extracted using DCM (2 x 5 mL). The organic layers were dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (5 % EtO Ac/Pet. Ether) to give 13 (76 mg, 69 %) as a yellow solid.

m.p. 124-126 °C; R _f = 0.27 (20 % EtOAc/Pet. Ether); IR v _m ax 1674 (vco), 1589, 1555, 1481, 1464, 1389, 1264, 1245, 1224, 1201, 1112, 1095, 1052, 1003, 992, 966 cm ^"1 ; ¾ MR (CDC1 ₃ ) δ 5.17 (2H, s, OC%Ar), 6.89 (1H, dd, J = 16.0, 7.6, CHC(O)H), 7.23 (1H, dd, J = 9.0, 2.5, ArH), 7.34-7.50 (6H, m, ArH), 7.72 (1H, d, J = 16.0, CHC(N)S), 8.01 (1H, d, J = 9.0, ArH), 9.79 (1H, d, J = 7.2, C(O)H); ¹³ C NMR (CDC1 ₃ ) δ 70.8 (CH ₂ ), 105.0 (CH), 1 17.8 (CH), 125.2 (CH), 127.6 (CH), 128.4 (CH), 128.9 (CH), 133.5 (CH), 136.2 (C), 137.4 (C), 143.8 (CH), 148.9 (C), 158.5 (C), 160.8 (C), 192.6 (CH); m/z (CI) 296 (100%, M ⁺ +H), 220 (21%, M ⁺ +2H- Ph), 217 (12%, M ⁺ -H, Ph); HRMS C17H14NO2S calcd. 296.0745, found 296.0743. (E)-5-(6-Benzoxy-benzothiazol-2-yl)-2,4-pentadienoic acid (14)

14

A solution of diethylphosphonoacetic acid (80 mg, 0.41 1 mmol) in THF (4 mL) was cooled to 0 °C and treated with NaH (8 mg, 0.333 mmol, 95 %), gas evolution. The solution was stirred at 0 °C for 15 min and then treated with a solution of 13 (122 mg, 0.411 mmol) in THF (1 mL), added dropwise. The resultant red solution was stirred at 0 °C for 30 min and then allowed to warm to rt over 1 h. After this time the solution was concentrated, taken up in H ₂ 0, acidified (1 M HC1) and the resultant brown precipitate filtered to give give 14 (119 mg, 86 %) as a brown solid, m.p. 226-228 °C; R _f = 0.18 (5 % MeOH/DCM); IR Vmax 1678 (vco), 1610, 1594, 1553, 1482, 1449, 1362, 1321, 1269, 1241, 1208, 1181, 1149, 1058, 1021, 1002, 935 cm ^"1 ; ¾ NMR (DMSO) δ 5.19 (2H, s, OC%Ar), 6.28 (1H, d, J = 14.9, CHC(O)OH), 7.20 (1H, dd, J = 8.9, 2.5, ArH), 7.32-7.50 (8H, m, 3 xCHCH, 5><ArH), 7.79 (1H, d, J = 2.5, ArH), 8.01 (1H, d, J = 8.9, ArH); ¹³ C NMR (DMSO) δ 69.9 (CH ₂ ), 105.9 (CH), 116.8 (CH), 123.7 (CH), 126.3 (CH), 128.0 (CH), 128.1 (CH), 128.5 (CH), 131.9 (CH), 133.9 (CH), 136.1 (C), 136.7 (C), 142.3 (CH), 148.1 (C), 157.0 (C), 162.8 (C), 167.3 (C); m/z (ES ⁺ ) 338 (100%, M ⁺ +H), 320 (15%, M ⁺ -OH), 247 (19%, M ⁺ -H, Ph); HRMS C19H16NO3S calcd. 338.0851, found 338.0845.

Methyl 2-((2E,4E-5-(6-Benzoxy-benzothiazol-2-yl)penta-2,4-dienoylam ido)-3-(tritylthio) propononate (15)

15

A solution of 14 (118 mg, 0.350 mmol) in DMF (3.5 mL) was treated with Et ₃ N (58 μί, 0.841 mmol) and cooled to 0 °C. The solution was treated with a solution of aminoacid (158 mg, 0.419 mmol) in DCM (1.5 mL) followed by a solution of BOP (191 mg, 0.419 mmol) in DCM (2.0 mL) and the resultant solution warmed to rt and stirred for 16 h. After this time the reaction mixture was quenched with saturated NaHC0 ₃ (aq) (5 mL), separated and the aqueous layer was back extracted using DCM (2 x 10 mL), organics dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (20 % EtOAc/Pet. Ether) to give 15 (128 mg, 53 %) as an orange oil. R _f = 0.40 (40 % EtOAc/Pet. Ether); [a] _D +56.3 (c 1.00, CHC1 ₃ ); IR Vmax 3057 (V _C H), 1734 (vco), 1659, 1597, 1555, 1485, 1444, 1317, 1244, 1259, 1203, 1177, 1051, 996 cm ^"1 ; ¾ NMR (CDC1 ₃ ) δ 2.71 (1H, dd, J = 12.6, 4.7, C¾S), 2.76 (1H, dd, J= 12.6, 5.5, C¾S), 3.74 (3H, s, OC%), 4.68 (1H, dt, J= 7.6, 5.2, CHC0 ₂ Me), 5.16 (2H, s, OC%Ar), 6.05 (1H, d, J= 7.7, C(O)NH), 6.09 (1H, d, J= 14.9, CHC(O)NH), 7.10-7.50 (25H, m, 3 xCHCH, 22xArH), 7.92 (1H, d, J = 8.9, ArH); ¹³ C NMR (CDC1 ₃ ) δ 33.9 (CH ₂ ), 51.3 (CH ₃ ), 52.8 (CH), 67.1 (C), 70.7 (CH ₂ ), 105.3 (CH), 116.8 (CH), 124.1 (CH), 126.9 (CH), 127.0 (CH), 127.6 (CH), 128.1 (CH), 128.3 (CH), 128.8 (CH), 129.6 (CH), 131.9 (CHO, 133.2 (CH), 136.3 (C), 136.5 (C), 139.9 (CH), 144.3 (C), 148.7 (C), 157.6 (C), 163.1 (C), 164.7 (C), 170.9 (C); m/z (ES ⁺ ) 719 (100%, M ⁺ +Na), 697 (12%, M ⁺ +H), 643 (10%), 244 (22%, M ⁺ -Bn, C(C0 ₂ Me)(CH ₂ STrt))), 243 (78%, M ⁺ -H, Bn, C(C0 ₂ Me)(CH ₂ STrt))), 228 (12%, Trt), 165 (32%); HRMS C ₄₂ H ₃ 6N ₂ 0 ₄ NaS ₂ calcd. 719.2014, found 719.1990.

6-Benzoxy-2-(4-lE,3E-(4 ₎ 5'-methoxycarbonyl-4,5-dihydrothiazol-2-yl)buta-2,4-di enyl) benzothiazole (16)

16

A solution of Ph ₃ PO (217 mg, 0.784 mmol) in DCM (6.0 mL) was cooled to 0 °C in an ice bath and treated with Tf ₂ 0 (89 μΐ _^ , 0.491 mmol) added dropwise over 5 min. The resultant solution was stirred at 0 °C for 30 min and a solution of 15 (127 mg, 0.182 mmol) in DCM (2.5 mL) was then added dropwise over 5 min. The reaction was stirred at 0 °C for 20 min and then quenched with phosphate buffer (10 mL). Extracted into DCM (3 x 10 mL) and separated. The organic layer was dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (20-40 % EtO Ac/Pet. Ether) to give 16 (41 mg, 52 %) as a yellow solid, m.p. 104-107 °C; R _f = 0.36 (40 % EtO Ac/Pet. Ether); IR Vmax 3032 (V _C H), 2949 (VCH), 1739 (vco), 1596, 1560, 1484, 1448, 1260, 1232, 1204, 1173, 1053, 983cm ^"1 ; ¾ MR (CDC1 ₃ ) δ 3.58 (1H, dd, J = 11.0, 9.3, C¾S), 3.65 (1H, dd, J = 11.0, 9.3, C¾S), 3.84 (3H, s, OC%), 5.14, (2H, s, OC%Ar), 5.21 (1H, t, J = 9.3, 5.2, CHC0 ₂ Me), 6.78 (1H, d, J = 15.4, CHC(N)S), 6.97 (1H, dd, J = 15.4, 10.7, C CHCHC(N)S), 7.04 (1H, d, J = 15.5, CHC(N)S), 7.13-7.49 (8H, m, l xCHCH, 7><ArH), 7.90 (1H, d, J = 8.9, ArH); ¹³ C NMR (CDC1 ₃ ) δ 34.9 (CH ₂ ), 53.1 (CH ₃ ), 70.7 (CH), 78.1 (CH ₂ ), 105.3 (CH), 116.9 (CH), 124.1 (CH), 126.9 (CH), 127.6 (CH), 128.8 (CH), 129.9 (CH), 134.0 (CH), 136.3 (C), 136.5 (C), 140.3 (CH), 148.7 (C), 157.6 (C), 163.1 (C), 169.6 (C), 171.1 (C); m/z (ES ⁺ ) 459 (25%, M ⁺ +Na), 437 (100%, M ⁺ +H), 342 (42%, M ⁺ -Bn, 4H ), 279 (93%,), 243 (39%), 196 (22%), 165 (25%); HRMS C ₂₃ H ₂ iN ₂ 0 ₃ S ₂ calcd. 437.0994, found 437.0989. 6-Hydroxy-2-(4-lE E-(4 ₎ 5'-methoxycarbonyl-4,5-dihydrothiazol-2-yl)buta-2,4-di eny benzothiazole (17)

A solution of 16 (40 mg, 0.0916 mmol) in DCM (1.5 mL) was cooled to -78 °C in an acetone/C02( _S ) bath and treated with pentamethylbenzne (75 mg, 0.456 mmol) followed by BCb (0.27 mL, 0.275 mmol, 1 M in DCM) added dropwise over 5 min. The reaction was stirred at -78 °C for 20 min and then quenched with phosphate buffer (2 mL). Extracted into DCM (3 x 5 mL) and separated. The organic layer was dried over MgS04, filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (30-60 % EtOAc/Pet. Ether) to give 17 (17 mg, 54 %, 79 % b.r.s.m) as a pale yellow solid, m.p. 156-158 °C; R _f = 0.20 (40 % EtO Ac/Pet. Ether); IR Vmax 3210 (V ₀ H), 3046 (V _C H), 2920, 2853, 1731 (vco), 1631, 1595, 1557, 1480, 1435, 1314, 1199, 1116, 1022, 949 cm ^"1 ; ¾ NMR (MeOD) δ 3.64 (1H, dd, J = 19.9, 11.2, C¾S), 3.66 (1H, dd, J = 22.5, 11.2, C¾S), 3.81 (3H, s, OC%), 5.27 (1H, t, J = 9.0, CHC0 ₂ Me), 6.78 (1H, d, J = 15.4, CHC(N)S), 6.99 (1H, dd, J = 15.4, 2.4, CHCHC(N)S), 7.08-7.18 (2H, m, CHC(N)S, ArH), 7.25-7.31 (1H, m, CHC(N)S), 7.29 (1H, d, J = 2.4, ArH), 7.76 (1H, d, J = 8.9, ArH); ¹³ C NMR (MeOD) δ 35.3 (CH ₂ ), 53.1 (CH ₃ ), 78.6 (CH), 107.4 (CH), 117.7 (CH), 124.5 (CH), 129.3 (CH), 131.9 (CH), 135.5 (CH), 142.2 (CH), 148.4 (C), 158.1 (C), 164.2 (C), 172.3 (C), 172.6 (C); m/z (ES+) 347 (100%, M ⁺ +H), 279 (39%), 229 (15%), 202 (28%), 196 (58%); HRMS Ci6Hi5N ₂ 0 ₃ S ₂ calcd. 347.0524, found 347.0536. Synthesis of 2-(4-(6-hydroxyhen∑othiazoi-2-yi)aryi)-4,5-dihydro-thiazo acid (20)

20

The synthesis may begin with commercially available 2-amino-6-methoxybenzothiazole. The amine may then be converted into the bridged thiol (18) using KOH following the method described by Fink et al, Bioorg &Med Chem Lett, 2006 , 16, 1532.

4-(6-hydroxybenzothiazol-2-yl)benzonitrile (18)

19

A solution of 18 (104 mg, 0.371 mmol) and 4-cyanobenzaldehyde (99 mg, 0.757 mmol) in anhydrous DMSO (3 mL) was treated with sodium metabisulfate and heated to 140 °C for 3 h. After this time the reaction was cooled to rt and H ₂ 0 added (10 mL). The resultant brown precipitate was filtered giving 19 (55 mg, 29 %) as a brown solid. Rf = 0.51 (40 % EtO Ac/Pet. Ether); ¾ NMR (MeOD) δ 7.04 (IH, dd, J= 10.9, 2.3, ArH), 7.36 (IH, d, J = 2.3, ArH), 7.88 (2H, d, J = 8.4, ArH), 7.88 (IH, d, ArH), 8.19 (2H, d, J = 8.4, ArH); ¹³ C NMR (MeOD) δ 107.5 (CH), 1 14.7 (C), 117.9 (CH), 119.3 (C), 125.1 (CH), 128.6 (CH), 134.1 (CH), 138.2 (C), 138.9 (C), 148.9 (C), 158.1 (C), 163.6 (C). Synthesis of 6-hydroxy-2-(l-keto-2-(4 ₎ 5'-methoxycarbonyl-4,5-dihydrothiazol-2-yl)ethyl) benzothiazole (24) MeCN, nBuLi, THF

24 23

The synthesis may begin with commercially available 6-methoxy-2-methylbenzothiazole. The methyl group may then be oxidised using Se0 ₂ following the method described in US patent US4826833.

2-Hydroxy-3-(6-methoxy-benzothiazol-2-yl)-propionitrile (22)

22

THF (5 mL) was cooled to -78 °C using an acetone/C0 ₂ (s) bath and nBuLi (0.39 mL, 0.621 mmol, 1.6 M in hexanes) was added dropwise. The resultant solution was stirred for 5 min and then treated with MeCN (34 μΐ _^ , 0.650 mmol), stirred 15 min and a solution of 21 (100 mg, 0.518 mmol) in THF (2 mL) was added dropwise. The reaction mixture was removed from the /C0 ₂ (s) bath and stirred for 2 h. After this time the reaction was quenched using saturated NaHC0 ₃ (aq) (2 mL), extracted with EtOAc (10 mL), washed H ₂ 0 (5 mL), dried (MgS0 ₄ ), filtered and concentrated in vacuo. Purification was achieved by flash column chromatography (50 % EtO Ac/Pet. Ether) to give 22 (51 mg, 42 %) as an orange solid, m.p. 78-80 °C; R _f = 0.32 (50 % EtOAc/Pet. Ether); IR Vmax 3209 (VOH), 2013 (VCN), 1605 (vco), 1559, 1519, 1469, 1436, 1262, 1227, 1091, 1056, 1025 cm ^"1 ; ¾ NMR (CDC1 ₃ ) δ 3.30 (1H, dd, J = 16.8, 7.3, C¾CN), 3.20 (1H, dd, J = 16.8, 4.4, CH ₂ CN), 3.70 (1H, d, J= 5.7, OH), 3.90 (3H, s, OCH ₃ ), 5.38 (1H, app dd, J = 5.8, 10.7, CHOH), 7.12 (1H, dd, J = 8.9, 2.5, ArH), 7.36 (1H, J = 2.4, ArH), 7.88 (1H, J= 9.0, ArH); ¹³ C MR (CDC1 ₃ ) δ 26.8 (CH ₂ ), 56.0 (CH), 68.2 (CH), 104.2 (CH), 1 16.2 (CH), 116.7 (C), 123.9 (CH), 136.6 (C), 147.2 (C), 158.1 (C), 168.3 (C); m/z (EI ⁺ ) 234 (27%, M ⁺ ), 194 (100%, M ⁺ -CH ₂ CN), 166 (10%, M ⁺ -CH(OH)CH ₂ CN), 151 (29 %), 123 (14 %, M ⁺ - )Me, CCH(OH)CH ₂ CN), 95 (7%); HRMS CnHi ₀ N ₂ O ₂ S calcd. 234.0458, found 234.0459.

Oxidation and deprotection of 22 may then be achieved to provide 23, and 23 may be converted to 24 by manipulation of the nitrile group and condensation with cysteine.

Synthesis of 6-hydroxy-2-(2-(4S-methoxycarbonyl-4,5-dihydrothiazol-2-yl)e thynyl) benzothiazole (26)

Sonagashira coupling of 2 and ethynyltrimethylsilane, and subsequent (hydolysis and reaction with phenyl cyanate may provide nitrile 25. Ths may then be converted to 26 by manipulation of the nitrile andcondensation with cysteine.

Bioluminescence characterisation of compounds

Polynucleotides encoding mutant luciferases x5 luciferase:

ATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCACCACGCTATCCTCTAGAGGATGGA ACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCAGGTTCCTGGAACAATT GCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCC GTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTA TGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTT GCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATT TCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAA AAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGA TTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGAT TTTAAACCAGAAAGCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGA TCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCG

GTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGA TTT

CGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGAT TAC

AAAATTCAAAGTGCGTTGCTAGTACCAACCCTATTTTCATTCTTCGCCAAAAGCACT CTG ATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGGGGCGCACCTCTTTCG

AAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCCATCTTCCAGGGATACGACAAGGA TAT GGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGC GCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAA ACGCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACCTATGATTATGTCCGGT TATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCT GGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTTGAAGTCT TTAATTAAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTACAA CACCCCAACATCCGCGACGCCGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTT CCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGAT TACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGAC GAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATA AAGGCCAAGAAGGGCGGAAAGTAA

x5 red:

ATGGAGGACGCCAAGAACATCAAGAAGGGACCAGCCCCCAGATACCCCCTGGAGGACGGC ACAGCCGGCGAGCAGCTGCACAAGGCCATGAAGCGGTACGCCCAGGTGCCAGGCACCATC GCCTTCACCGACGCCCACATCGAGGTGAACATCACCTACGCCGAGTACTTCGAGATGAGC GTGCGGCTGGCCGAGGCCATGAAGCGGTACGGCCTGAACACCAACCACCGGATCGTGGTG TGCAGCGAGAACAGCCTGCAGTTCTTCATGCCCGTGCTGGGAGCCCTGTTCATCGGCGTG GCCGTGGCCCCAGCCAACGACATCTACAACGAGCGGGAGCTGCTGAACAGCATGAACATC AGCCAGCCCACCGTGGTGTTCGTGAGCAAGAAGGGCCTGCAGAAGATCCTGAATGTGCAG AAGAAGCTGCCCATCATCCAGAAGATCATCATCATGGACAGCAAGACCGATTACCAGGGC TTCCAGAGCATGTACACCTTCGTGACCAGCCACCTGCCCCCAGGCTTCAACGAGTACGAC TTCAAGCCCGAGAGCTTCGACCGGGACAAGACCATCGCCCTGATCATGAACAGCAGCGGC AGCACCGGCCTGCCCAAGGGCGTGGCCCTGCCCCACCGGACCGCCTGCGTGCGGTTCAGC CACGCCAGAGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCCATCCTGAGCGTG GTGCCCTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATCTGCGGCTTC CGGGTGGTGCTGATGTACAGGTTCGAGGAGGAGCTGTTCCTGCGGAGCCTGCAGGACTAC AAGATCCAGACCGCCCTGCTGGTGCCCACCCTGTTCAGCTTCTTCGCCAAGAGCACCCTG ATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCCTCTGGCGGAGCCCCACTGAGC AAGGAGGTGGGCGAGGCCGTGGCCAAGCGGTTCCACCTGCCAGGCATCCGGCAGGGCTAC GGCCTGACCGAGACCACCAGCGCCATCCTGATCACCCCCGAGGGCGACGACAAGCCCGGA GCCGTGGGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAG ACCCTGGGCGTGAACCAGAGAGGCGAGCTGTGCGTGAGAGGCCCCATGATCATGAGCGGC TACGTGAACAACCCCGAGGCCACCAACGCCCTGATCGACAAGGACGGCTGGCTGCACAGC GGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGC CTGATCAAGTACAAGGGCTACCAGGTGGCCCCAGCCGAGCTGGAGAGCATCCTGCTGCAG CACCCCAACATCTTCGACGCCGGAGTGGCCGGACTGCCCGACGACGACGCCGGAGAGCTG CCAGCCGCCGTGGTGGTGCTGGAGCACGGCAAGACCATGACCGAGAAGGAGATCGTGGAC TACGTGGCCAGCCAGGTGACCACCGCCAAGAAGCTGAGAGGCGGCGTGGTGTTCGTGGAC GAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCAGAAAGATCCGGGAGATCCTGATC AAGGCCAAGAAGGGCGGCAAGTAA

Bioluminescence

Table of bioluminescence emission wavelength maximum max) with native LH ₂ and compound 9. Name of mutant max with luciferin max with

(nm) compound 9(nm)

WT Flue 557 662

x5 Flue 557 638 x5 red Flue 620 700

Bioluminescence max values for LH ₂ and compound 9 were tested with purified enzymes. Experimental details as in Figure 2. These results show that compound 9 represents a near infrared (nIR) multiparametric luciferin analogue.

Saponification kinetics

Apparent kinetic parameters for LH ₂ ethyl ester and compound 12.

Michaelis-Menten kinetics: concentrations of each ester between 1.9mM and 0.38mM were incubated with 100μg of pig liver esterase at 37°C for 15min and allowed to come to room temperature before the initiation of light by addition of lOpmol Flue enzyme and 5.3mM ATP in 55μ1 reactions. Light was captured using the Photon Imager instrument (Biospace Labs, Paris, France) and the Hanes-Woolf plot was used to analyse kinetic parameters. PMT temperature varied between -9-12°C.

The kinetics of saponification in mammalian cells showed a slow rise (ca. 2min) to maximal activity. The apparent Km of compound 12 was seen to be the same as LH ₂ indicating similar affinity for the substrate, although the Kcat was lower, possibly due to emitter quenching.