Photo-switchable sulfonylureas and their uses

The present invention relates to sulfonylurea analogous compounds comprising a sulfonylurea moiety and a photoresponsive moiety. The present invention further relates to methods for generating the sulfonylurea analogous compounds of the invention and to pharmaceutical compositions comprising at least one sulfonylurea analogous compound of the invention. The present invention relates to the sulfonylurea analogous compounds or the pharmaceutical composition of the invention for use in medicine, in particular for use as anti-diabetic agent and/or in the prevention and/or treatment of diabetes conditions or a disease state requiring modulation of ATP-sensitive potassium (K ⁺ ) (KATP) channels. The present invention also relates to the use of the sulfonylurea analogous compounds for regulating ATP-sensitive potassium ( ⁺ ) (KATP) channels and/or for assessing the functional role and/or activation of KATP channels.

BACKGROUND OF THE INVENTION

Type 2 diabetes mellitus (T2DM) is a global healthcare epidemic associated with life- changing sequelae ranging from blindness to cancer (Currie et al., 2009; Stitt, 2010). This endocrine disease, which currently affects 1 in 12 of the adult population worldwide, involves a disturbance of normal glucose homeostasis due to failure of the pancreatic beta cell mass to adequately compensate for increased peripheral insulin resistance (Prentki & Nolan, 2006). As such, the rescue of insulin release through the coaxing of beta cell activity remains a therapeutically desirable approach for the long-term restoration of normal glucose levels.

Sulfonylureas, which target ATP-sensitive potassium (K ⁺ ) (KATP) channels, are a mainstay of diabetes therapy (see e.g. Fineman et al, 2003). KATP channels are hetero-octameric structures composed of four regulatory sulfonylurea receptor subunits (SUR1) and four Kir6.2 subunits, the latter forming a central ion pore that permits K ⁺ efflux (see e.g. Miki et al., 1999). By binding to SUR1, sulfonylureas block the Kir6.2 inward rectifier, leading to cell depolarization and opening of voltage-dependent Ca ²⁺ channels (VDCC) (see e.g. Seino & Miki, 2003). The ensuing Ca ²⁺ influx (Rorsman et al., 2012) along with K _A TP channel- independent signals (Henquin, 2009), drives various downstream processes which ultimately converge on the exocytosis of insulin. Elevated circulating insulin can then act on target tissues to improve glucose uptake, hepatic glycogenesis and fatty acid synthesis.

While sulfonylureas are widely prescribed because of their effectiveness and relative inexpensiveness, they have a range of off-target effects which limits their therapeutic use. For example, sulfonylureas can provoke prolonged episodes of low blood glucose due to hyperinsulinemia (Jennings et al., 1989), elevate cardiovascular disease risk (Evans et al., 2006), and induce weight gain (Nathan et al, 2006). Conversely, there is a lack of tools for the precise functional dissection of KATP channels located not only in the pancreas, but also in the brain (Lam et al., 2010; Hernandez- Sanchez et al., 2001 ), heart (Engler & Yellon, 1996) and vascular smooth muscle (Quayle et al., 1997).

There is a need in the art for improved sulfonylurea compounds.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by a sulfonylurea analogous compound comprising a sulfonylurea moiety and a photoresponsive moiety,

preferably having the general formula I

Ph - SU

(I)

wherein

Ph is the photoresponsive moiety, and

SU is the sulfonylurea moiety.

According to the present invention this object is solved by a method for synthesizing/generating the sulfonylurea analogous compounds of the present invention, comprising at least one of the following

the use of sulfanilamide and iV,iV-diethylaniline as the starting compounds,

the use of sulfanilamide and phenol as the starting compounds,

the use of 5-amino-l ,3,4-thiadiazole-2-sulfonamide and N,7V-diethylaniline as the starting compounds,

the use of nitroso compounds with anilines, the use of hydrazines and coupling partners using Pd-chemistry,

via sulfonamide azobenzenes,

the further derivatization to a sulfonylurea by the use of aliphatic isocyanates (e.g. cyclohexyl, o-butyl, /ra¾y-4-methyl-cyclohexyl)

and/or

the use of any sulfonylchlorides thereof together with ureas.

According to the present invention this object is solved by a pharmaceutical composition, comprising

(i) at least one sulfonylurea analogous compound according to the present invention,

(ii) optionally, pharmaceutically acceptable excipient(s) and/or carrier(s),

(iii) optionally, further compound(s).

According to the present invention this object is solved by the sulfonylurea analogous compounds of the present invention or the pharmaceutical compositions of the present invention for use in medicine.

According to the present invention this object is solved by the sulfonylurea analogous compounds of the present invention for use as anti-diabetic agent.

According to the present invention this object is solved by the sulfonylurea analogous compounds of the present invention or the pharmaceutical compositions of the present invention for use in the prevention and/or treatment of diabetes conditions or a disease state requiring modulation of ATP-sensitive potassium (K ⁺ ) (K _A TP) channels.

According to the present invention this object is solved by using the sulfonylurea analogous compounds of the present invention for regulating ATP-sensitive potassium (K ⁺ ) (KATP) channels and/or for assessing the functional role and/or activation of ATP-sensitive potassium ( ⁺ ) (KATP) channels.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.

Photo-switchable sulfonylurea compounds

As discussed above, the present invention provides sulfonylurea analogous compounds comprising a sulfonylurea moiety and a photoresponsive moiety.

The sulfonylurea analogous compound of the present invention preferably has the general formula I

Ph - SU

(I) wherein

Ph is the photoresponsive moiety, and

SU is the sulfonylurea moiety.

The term "sulfonylurea analogous compound" or "photo-switchable sulfonylurea compound" when used herein refers to a compound that has the same or similar biologic activity or action mechanism as sulfonylureas known in the art as anti-diabetic drugs (i.e. the sulfonylurea analogous compound/ photo-switchable sulfonylurea compound, as well as sulfonylureas bind to an ATP-dependent K ⁺ ( ATP) channel on the cell membrane of pancreatic beta and alpha cells and thus act by increasing insulin release and decreasing glucagon release). The sulfonylurea analogous compounds or photo-switchable sulfonylurea compounds of the present invention comprise a further moiety which allows the compound to be activated or inactivated by irradiation with light, or by illumination and dark relaxation.

The term "photoresponsive moiety" when used herein refers a moiety, preferably an azobenzene or a derivative thereof, that is a chemical unit that is responsive to light. The wavelength of the light response is determined by the electronic structure of the photoresponsive moiety, preferably the azobenzene or a derivative thereof, which can easily be modified/tuned.

The term "sulfonylurea moiety" when used herein refers to the pharmacological unit having the structure R-S0 ₂ -NH-C(=O)-NH-R' or R-S0 ₂ -NH-C(=0)-NH-R ₂ , which is common to the sulfonylurea drug family.

For example, the following (wherein R comprises an aromatic moiety):

Preferably, the photoresponsive moiety undergoes trans-cis isomerization by excitation with light, or by illumination and dark relaxation.

In particulai", the photoresponsive moiety undergoes cw-isomerization by excitation with light and /rarcs-isomerization by illumination and/or dark relaxation.

This intrinsic property can be used to induce a conformational change in the molecule and change binding affinity/efficacy towards a biological target accordingly.

In a preferred embodiment, the photoresponsive moiety is an azobenzene or a derivative thereof.

Examples for azobenzene derivatives are:

- heterocyclic azobenzene structure(s),

- substituted azobenzene structure(s) (e.g. tetra-ortho),

- bridged azobenzenes (e.g. by an 0,0 '-ethylene chain).

Preferably,

- the cis state or isomer is active / is in the binding conformation to close ATP- sensitive potassium (K ⁺ ) (KATP) channels,

and

- the trans state or isomer is inactive. In an embodiment, the reverse is also applicable,

- the cis state or isomer is inactive ,

and

- the trans state or isomer is active / is in the binding conformation to close ATP- sensitive potassium (K ⁺ ) (KATP) channels,.

KATP channels are ubiquitously-expressed ion channels and can be found in the pancreas, brain, muscle and heart, where they generally serve to link metabolism to cell activity (Engler & Yellon, 1996; Quayle et al.; 1997; Mild et al., 1999; Hernandez-Sanchez et al., 2001; Pocai et al, 2005; Lam et al, 2010). Their function is extensively detailed in pancreatic beta cells. Under resting conditions, KATP channels permit K ⁺ efflux, hypopolarizing the cell membrane and suppressing action potential firing and cell activity. Following a rise in blood glucose concentrations, such as that seen after a meal, the sugar is metabolized, leading to an increase in the cytosolic free ATP:ADP ratio, closure of KATP channels and membrane depolarization. Voltage-dependent Ca ²⁺ -channels (VDCC) then open, and the ensuing Ca ²⁺ -influx drives Ca -dependent exocytosis of insulin-containing granules via interactions with the exocytotic machinery. Insulin then binds its cognate receptor to increase glucose uptake and utilization (see e.g. Rorsman et al., 2012). As well as reduced insulin sensitivity, a hallmark of type 2 diabetes mellitus (T2DM) is defective beta cell function, resulting in impaired insulin secretion. KATP channel activity has therefore remained a popular target for the restoration of insulin secretion, by allowing defects in metabolism, exocytosis and cell number to be bypassed.

KATP channels are hetero-octameric structures assembled from four inwardly-rectifying subunits (e.g. Kir6.1 and Kir6.2 and four sulfonylurea receptor (SUR) subunits (e.g. SUR1 and SUR2A/B in the heart) (see Table below for subunit distribution and function) (see e.g. Miki et al., 1999). Kir6.x possesses two transmembrane regions and comprises the ion pore for K ⁺ efflux. In the pancreatic beta cell, Kir6.2 primarily transduces alterations in ATP:ADP to channel state via interactions between the β- and γ-phosphates of the nucleotide and N- and C- termini of the subunit. By contrast, SURs possess three transmembrane regions and, in the beta cell, SUR1 contains nucleotide binding pockets on the cytoplasmic portion which modulate ATP-sensitivity of the KATP channel complex. Importantly, the SUR also confers sensitivity to sulfonylurea drugs and sulfonylurea binding rapidly induces channel closure (Inagaki et al., 1996; Ashfield et al., 1999). Sublimits Distribution Role ir6.2/SUR1 o Pancreatic beta cell • Regulation of insulin secretion

» Hypothalamus • Central glucose homeostasis • Substantia nigra • Neuronal firing and behavior

Kir6.2/SUR2A © Cardiac muscle • Cardiac stress (contractility)

• Skeletal muscle • Exercise capacity

Kir6.2/SUR2B • Smooth muscle • Tissue perfusion

Kir6.1/SUR2B • Vasculature • Sepsis

In a preferred embodiment, the sulfonylurea moiety is derived from second and/or third generation sulfonylureas.

In a preferred embodiment, the sulfonylurea moiety is derived from glibenclamide and/or glimepiride, and/or the sulfonylurea moiety contains substitutent(s) on the terminal sulfonylurea nitrogen.

Such substitutent(s) can be aliphatic chains and/or rings, which can contain heteroatoms.

Preferably, the sulfonylurea moiety and the photoresponsive moiety are covalently attached to each other.

In one embodiment, the aromatic part of a known sulfonylurea is converted to an azobenzene. In order to be similar to or resemble the structure of the known sulfonylurea glimepiride, furthermore a cyclohexyl group was attached to the azobenzene.

In a preferred embodiment, the sulfonylurea analogous compound of the present invention has the general formula II

II wherein

R is selected from hydrogen, alkyl, alkylamino, hydroxy or substituted hydroxy groups, morpholines and/or piperazines,

and

R' is selected from cyclohexyl and substituted versions thereof, aliphatic linear chains (such as alkyl) and/or structures containing heteroatoms,

In a preferred embodiment, the sulfonylurea analogous compound of the present invention has the general formula III

III

wherein

Ar is selected from any (substituted) aromatic ring, such as (substituted) aryl (e.g. benzyl), and

R' is selected from cyclohexyl and substituted versions thereof, aliphatic linear chains and/or structures containing heteroatoms, and

X, Y and Z are, each independently, selected from C, N, S, O and/or multiples thereof.

The substituents of Ax are preferably as defined for R in general formula II, such as hydrogen, alkyl, alkylamino, hydroxy or substituted hydroxy groups, morpholines and/or piperazines

In a preferred embodiment, the sulfonylurea analogous compound of the present invention is (E)-N-(cyclohexylcarbamoyl)-4-((4-(diethylamino)phenyl)diaze nyl)berizenesulfonamide

(JB253), which is - a sulfonylurea analogous compound of the present invention having general formula II, wherein R is N,N-diemylamino- and wherein R' is cyclohexyl, and

- a sulfonylurea analogous compound of the present invention having general formula III, wherein Ar is Et ₂ N-C ₆ H4-, X is CHCH, Y and Z are CH and wherein R' is cyclohexyl.

In a preferred embodiment, the sulfonylurea analogous compound of the present invention is (i¾-N-(Cyclohexylcarbamoyl)-5-((4-(diethylamino)phenyl)-dia zenyl)-l ,3,4-thiadiazole-2- sulfonamide (JB558), which is a sulfonylurea analogous compound of the present invention having general formula III, wherein Ar is Et ₂ N-C6H ₄ -, X is S, Y and Z are N, and R' is cyclohexyl.

In a preferred embodiment, the sulfonylurea analogous compound of the present invention is selected from

&artS-JB5S8

Preferably, the activation wavelength is in the ultraviolet, visible or infrared ranges of the electromagnetic spectrum.

For example, for JB253, the activation wavelength is in the visible light range, such as in the range from about 400 to about 500 mn, while JB030 is activated with UV light (about 350 nm) and JB558 with green-yellow light (about 520 nm). N

k»r

n. o bioe light

a ^' s-J8253 frans-JB253

For example, for JB558, the activation wavelength is in the range of green-yellow light (around 520-560 nm).

s-JS558 tram- J B558 O

Preferably, the sulfonylurea analogous compound is active under irradiation with light, and inactive under either illumination or in the dark.

A sulfonylurea analogous compound of the invention has preferably at least one of the following characteristics:

(1) it is active under irradiation with light, and inactive under either illumination or in the dark,

(2) rapid conversion into active cz ^' s-state /conformation,

(3) rapid dark relaxation into irara-state,

(4) has a similar EC50 value to the sulfonylurea it is similar to/ its analogue it is (in case of JB253: similar EC50 value to glimepiride)

(5) allows regulation of ATP-sensitive potassium (K ⁺ ) (Κ _Α χρ) channel activity

preferably repeatedly and reversibly (such as by turning on and off of a light source)

(6) it is not cytotoxic

(7) it allows spatially and temporally restricted activation by illumination (8) can be modified / tailored

such as regarding

the activation wave length, and/or

pharmacological properties (i.e. substitution on the sulfonylurea).

Preferred or advantageous characteristics of the compound JB253 are:

(1) JB253 can be applied/administered exogenously and then washed out.

(2) JB253 allows specific photo switching of KATP channels.

(3) JB253 is very light sensitive and can be activated with e.g. a LED flashlight (which is about 100 times less intensity than required to activate optogenes).

Preferred or advantageous characteristics of the compound JB558 are:

(1) JB558 can be applied/administered exogenously and then washed out.

(2) JB558 allows photo switching of KATP channels.

(3) JB558 is very light sensitive and can be activated with a green-yellow light.

(4) JB558 allows photoswitching of Epac2.

(5) JB558 allows the optical control of intracellular calcium concentrations.

In an embodiment, the sulfonylurea analogous compound of the present invention can comprise further moiety/moieties or component(s),

such as

- label (e.g. radioisotopes, MRI responsive groups, IR dyes, fluorophores)

- tag(s) (e.g. antibodies, peptides),

- anchoring group(s),

- other pharmacological units (e.g. biguanides, incretins)

and/or

- cell penetrating peptides (e.g. TAT).

As discussed above, the present invention provides pharmaceutical compositions, comprising

(i) at least one sulfonylurea analogous compound according to the present invention,

(ii) optionally, pharmaceutically acceptable excipient(s) and/or carrier(s). In an embodiment, the phamiaceutical composition of the present invention can comprise further compound(s) or drug(s).

Said further compound(s) or drug(s) can be another anti-diabetic drug(s).

Said further compound(s) or drug(s) can be/include:

- allosteric glucagon-like peptide 1 receptor (GLP-1 R) modulators,

- GLP1-R agonists,

- dipeptidyl peptidase-4 (DPP4) inhibitors,

- biguanides,

- peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists,

- sodium-glucose co-transporter 2 (SGLT2) inhibitors,

- oxyntomodullin, and/or

- GLP-1 /glucagon co-infusions.

Method of generating the photo-switchable sulfonylurea compounds

As discussed above, the present invention provides methods for synthesizing/generating the sulfonylurea analogous compounds of the present invention.

Said method comprises at least one of the following

© the use of sulfanilamide and N,N-diethylaniline as the starting compounds,

• the use of sulfanilamide and phenol as the starting compounds,

• the use of 5-amino-l,3,4-thiadiazole-2-sulfonamide and N,N-diethylaniline as the starting compounds,

• the use of nitroso compounds with anilines,

• the use of hydrazines and coupling partners using Pd-chemistry,

• via sulfonamide azobenzenes,

» the further derivatization to a sulfonylurea by the use of aliphatic isocyanates (e.g. cyclohexyl, /7-butyl, fr<37«-4-methyl-cyclohexyl)

and/or

© the use of any sulfonylchlorides thereof together with ureas. embodiment, the method is a three-step synthesis: Commencing/starting with sulfanilamide that is diazotized and the resulting diazonium salt trapped with N,N-diethylaniline yielding a sulfonamide, which is further derivatized by using cyclohexyl isocyanate.

Medical uses of the photo-switchable sulfonylurea compounds

As discussed above, the present invention provides the sulfonylurea analogous compounds of the present invention or the pharmaceutical compositions of the present invention for use in medicine.

As discussed above, the present invention provides the sulfonylurea analogous compounds of the present invention for use as anti-diabetic agent.

As discussed above, the present invention provides the sulfonylurea analogous compounds of the present invention or the pharmaceutical compositions of the present invention for use in the prevention and/or treatment of diabetes conditions or a disease state requiring modulation of ATP-sensitive potassium (K ⁺ ) (KAT _P ) channels.

Preferably, diabetes or "diabetes conditions" is/refers to/comprises

- type 1 diabetes,

- type 2 diabetes,

- maturity onset diabetes of the young (MODY),

- permanent neonatal diabetes mellitus (PNDM),

- latent onset autoimmune diabetes of adults (LAD A),

- any disease state requiring restoration of normal glucose homeostasis,

- any disease state characterized by defective glucose tolerance, glucose counterregulation and/or insulin release.

A "disease state requiring modulation of ATP-sensitive potassium (K ⁺ ) (KATP) channels" refers to diseases that are either based on intrinsic malfunctioning KATP channels (e.g. due to mutations) and/or to disease states where signaling to or from KATP channels is disturbed.

In one embodiment, the use for the prevention and/or treatment according to the invention comprises at least one of ( 1 ) targeting of activity,

(2) spatially and temporally restricted activation by illumination,

(3) optical control of electrical activity,

(4) optical control over calcium fluxes,

(5) optical control over insulin release/secretion,

(6) optical control over glucagon release/secretion,

(7) optical control over somatostatin release/secretion,

(8) optical control over pancreatic peptide release/secretion,

(9) optical control over ghrelin release/secretion,

(10) repeated modulation of any cell/tissue expressing _ATP channels,

(1 1) pulsatile or oscillatory insulin release/secretion,

(12) pulsatile or oscillatory activation of any cell/tissue expressing KATP channels,

(13) tailoring of insulin release/secretion,

(14) optical control of exchange protein activated by cAMP (Epac).

In one embodiment, the use for the prevention and/or treatment of diabetes or diabetes conditions comprises at least one of

0) targeting of activity,

(2) spatially and temporally restricted activation by illumination,

(3) optical control of electrical activity,

(4) optical control over calcium fluxes,

(5) optical control over insulin release/secretion,

(6) optical control over glucagon release/secretion,

(7) optical control over somatostatin release/secretion,

(8) optical control over pancreatic peptide release/secretion.

(9) optical control over ghrelin release/secretion,

(Π) pulsatile or oscillatory insulin release/secretion,

(13) tailoring of insulin release/secretion,

(14) optical control of exchange protein activated by cAMP (Epac)

In one embodiment, the use for the prevention and/or treatment of a disease state requiring modulating of ATP-sensitive potassium (K ⁺ ) (KATP) channels comprises at least one of

(1) targeting of activity,

(2) spatially and temporally restricted activation by illumination. (10) repeated modulation of any cell/tissue expressing KATP channels,

(12) pulsatile or oscillatory activation of any cell/tissue expressing KATP channels.

In one embodiment, the use for the prevention and/or treatment according to the invention comprises photodynamic therapy or photopharmacology.

"Photodynamic therapy" (PDT) as used herein refers to a form of phototherapy using nontoxic light-sensitive compounds (i.e. the sulfonylurea analogous compounds of the present invention) that are exposed selectively to light, whereupon they become active.

PDT is used clinically to treat a wide range of medical conditions, including wet age-related macular degeneration and malignant cancers and has proven ability to kill microbial cells, including bacteria, fungi and viruses, and is recognized as a treatment strategy which is both minimally invasive and minimally toxic.

"Photoparmacology" as used herein refers to the use of light to finely regulate drug activity and modify biological processes accordingly.

Photopharmacology is used to reversibly target and regulate drug activity with the aim of more precisely controlling cell/tissue function, reducing drug side effects/off target activity, and preventing residual drug activity in the environment.

Further uses

As discussed above, the present invention provides the use of the sulfonylurea analogous compounds of the present invention for regulating ATP-sensitive potassium (K ⁺ ) (KATP) channels and/or for assessing the functional role and/or activation of ATP-sensitive potassium (K ⁺ ) (KATP) channels.

Methods of treatment

The present invention provides a method for the prevention and/or treatment of diabetes conditions or a disease state requiring modulation of ATP-sensitive potassium (K ⁺ ) (K _A TP) channels. Said method comprises the administration of a therapeutically effective amount of at least one sulfonylurea analogous compound of the present invention to a patient or subject in need thereof.

Preferably, diabetes or "diabetes conditions" is/refers to/comprises

- type 1 diabetes,

- type 2 diabetes,

- maturity onset diabetes of the young (MODY),

- permanent neonatal diabetes mellitus (PNDM),

- latent onset autoimmune diabetes of adults (LAD A),

- any disease state requiring restoration of normal glucose homeostasis,

- any disease state characterized by defective glucose tolerance, glucose counterregulation and/or insulin release.

Further description of preferred embodiments

Sulfonylureas are widely prescribed for the treatment of type 2 diabetes mellitus (T2DM). Through their actions on ATP-sensitive potassium (KATP) channels, sulfonylureas boost insulin release from the pancreatic beta cell mass to restore glucose homeostasis. A limitation of these compounds is the elevated risk of developing hypoglycemia and cardiovascular disease, both potentially fatal complications.

The inventors combined the glucose-lowering attributes of sulfonylureas with the exquisite spatiotemporal control conferred by possession of photoresponsive elements (Fortin et al., 2008; Fehrentz et al, 201 1 .

In this patent application, we describe the design and development of photoswitchable sulfonylureas, such as the compounds JB253 and JB558 - a "fourth-generation" sulfonylurea based on glimepiride that bears an azobenzene photoswitch, endowing KATP channels with remarkable photocontrollable properties (see Figure la and Figure 14b).

We demonstrate herein that the photoswitchable sulfonylureas according to the invention (such as compounds JB253 and JB558) offer sensitive, reversible and repeated manipulation of KATP channel state and beta cell activity with visible light. Using in situ imaging and hormone assays, we further show that photoswitchable sulfonylureas, in particular compounds JB253 and JB558, bestow light-sensitivity upon rodent and human pancreatic beta cell function. Thus, the photoswitchable sulfonylureas of the present invention, such as compounds JB253 and JB558, enable the optical control of insulin release and offer a valuable research tool for the interrogation of _A TP channel function in health and T2DM. The photoswitchable sulfonylureas according to the invention, such as compounds JB253 and JB558, allow the selective targeting of AT _P channels in the pancreas and elsewhere.

Discussion

In the present application, we describe the development and testing of photoswitchable sulfonylureas, such as the compound JB253, a chemical chimera of glimepiride and an azobenzene, as well as the compound JB558, which allow light-induced closure of KATP channels. In the primary tissue employed here, viz islets of Langerhans, this translates to activated Ca ²⁺ flux and insulin release.

The principles of photopharmacology, i.e. the control of biological function with small molecule photoswitches, are now well-established (Fehrentz et al., 201 1 ; Gorostiza & Isacoff, 2008; Velema et al, 2014. In particular, azobenzene photoswitches have been employed as photochromic neurotransmitters and neuromodulators (Stein et al, 2012; Tochitsky et al., 2012), ion channel blockers (Fortin et al., 2008; Mourot et al., 2012), covalently-bound ion channel gates (Lemoine et al., 2013) and enzyme inhibitors (Velema et al, 2013; Broichhagen et al., 2014). With respect to ion channels, however, they have mostly been used to optically control excitable cells in the mammalian nervous system, and none have directly targeted KATP channels. These are ubiquitously-expressed channels that contribute to membrane potential in a number of cell types including hypothalamic and hippocampal neurons, cardiac myocytes, vascular smooth muscle and neuroendocrine cells (Seino & Miki, 2003; Lam 2010; Hernandez-Sanchez et al., 2001 ; Engler & Yellon, 1996; Quayle et al., 1997; Proks et al., 2002). Importantly, KATP channels translate metabolic state to transmembrane potential and, in pancreatic beta cells, are central to glucose-stimulated insulin secretion (see e.g. Seino & Miki, 2003). Since electrical status is generally correlated to biological output in excitable tissues, JB253 (as well as JB558) provides a suitable tool for investigating KATP channel function under a range of normal and pathological states. The prevailing view of sulfonylurea action is one of SUR1 binding, ATP channel closure and alterations to beta cell membrane potential (see e.g. Mild et al, 1999). However, recent studies have also invoked a K _A TP-independent signaling pathway whereby sulfonylurea may alter insulin release via Epac2A interactions (see e.g. Zhang et al., 2009; Herbst et al., 2011). Using radioactive displacement assays in combination with FRET experiments, JB253 was found to interact with both SUR1 and Epac2A. While SUR1 affinity for JB253 was much lower than glimepiride, we were unable to properly assess the active cw-state due to rapid thermal back-relaxation. As such, a role for illumination in strengthening any interaction cannot be excluded e.g. by altering binding conformation due to isomerization. Nonetheless, JB253 and glimepiride possess similar EC ₅₀ values for intracellular Ca rises and, when applied at the same concentration, both compounds stimulated almost identical levels of insulin secretion. Thus, JB253 possesses a similar activity profile to glimepiride, most likely due to signaling via pathways generally acknowledged to underlie sulfonylurea action.

In addition to pliotopharmacology, optogenetic and artificial light-sensitive K ⁺ channels are equally applicable to the remote control of electrically-responsive cells, including beta cells (Reinbothe et al., 2014). However, therapeutic potential in humans is limited by the requirement for genetic manipulation, high activation irradiances and the hyperpolarizing effects of recombinantly-expressed Kir6.2. Alternatively, an implantable synthetic optogenetic transcription device has recently been shown to improve blood-glucose homeostasis in a mouse model of T2DM via the expression and secretion of incretin (Ye at al., 201 1). However, debate still exists as to whether incretin-based therapies are associated with increased risk of pancreatitis and pancreatic adenocarcinoma (Nauck & Friedrich, 2013). Nonetheless, similar concepts have recently been extended to designer fusion molecules and may in the future be adopted for insulin release (Rossger et al., 2013). By contrast, due to its favorable profile as an exogenously-applied sulfonylurea which is sensitive to light, JB253 (as well as JB558) has advantages both as a research tool and as an anti-diabetic agent.

In the context of photodynamic therapy, light penetration in human tissues has been studied in detail and is now well-understood (Grossweiner et al., 2005). Although, the current activation wavelength of JB253 (400-500 nni) limits deep tissue penetration, e.g. through the skin, valiants of the photo switchable sulfonylureas according to the invention can be used which can be switched at longer wavelengths, such as compound JB558. In addition, stimulated by the brisk development of optogenetics (Deisseroth 201 1), devices that can deliver light to target tissues with minimal invasiveness and high spatial precision have emerged (Baretto et al, 201 1 ; Kim et al., 2013).

Therefore, the photoswitchable sulfonylureas according to the invention, such as compounds JB253 and JB558, or related photoswitchable molecules, which regulate KATP channels, will have an impact on human medicine and research. A long-standing challenge in endocrinology has been the inability to properly recreate the dynamics that underlie pulsatile hormone release, a prerequisite for proper downstream organ function (Seino et al., 201 1). Furthermore, disparate biological systems can use similar or identical molecular components. For example, KATP channels are also expressed in the heart and brain, including in neuronal populations tasked with the central regulation of glucose homeostasis and counterregulatory responses (Lam 2010; Pocai et al., 2005). Photopharmacology has the ability to target drug activity to the primary site of dysfunction with high spatial and temporal resolution (Velema et al, 2014). JB253 holds particular promise in this regard. It is non-cytotoxic and can be used to repeatedly modulate rodent and human beta cell activity, the basis for recreating the oscillatory activity known to orchestrate hormone pulse, Its light-dependency means that JB253 activity is spatially-restricted by illumination, potentially reducing extra-pancreatic effects. Finally, the ability to 'turn on' or 'turn off JB253 action allows insulin secretion to be tailored to peak demand. Therefore, the photoswitchable sulfonylureas according to the invention, such as compound JB253, open up new avenues for the treatment of T2DM.

In summary, we have designed and synthesized light-sensitive sulfonylureas, in particular compounds JB253 and JB558, which have a broad spectrum of application due to conferment of photoswitching on K _A TP activity.

Furthermore. JB558 represents a blueprint for red-shifted light-sensitive sulfonylureas or AzoSulfonylureas based upon heterocyclic azobenzenes. JB558 is highly suitable as a research tool, such as for rapid KATP channel manipulation, Epac2 signalling and intracellular calcium influx.

The following examples and drawings illustrate the present invention without, however, limiting the same thereto. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Photopharmacology of KATP channels: design, synthesis and characteristics of JB253.

a) The logic of a photoswitchable sulfonylurea: upon photoisomerization to the cw-state, JB253 becomes more active, closing the K _A TP channel. Thermal relaxation makes the compound less active or leads to dissociation, restoring the open form of the channel. Closure of KATP channels leads to depolarization, promoting calcium influx and ultimately insulin release.

b) Chemical structure of tolbutamide and glimepiride, which served as templates for JB253. c) Synthesis, structure and switching characteristics of JB253. Sulfanilamide undergoes diazotization and is trapped with N,iV-diethylaniline to yield an azobenzene-sulfonamide, which is converted to JB253 by cyclohexyl isocyanate. trans-TB253 can be reversibly switched to C/5-JB253 with blue light and relaxes thermally.

d) UV/Vis spectra of JB253 in the dark (black) and during constant illumination with 460 nm (blue).

e) Crystal structure of trans-TB253 (CCDC: 1014606) and glimepiride (CSD: TOHBUN01) showing the structural similarity of both sulfonylureas.

Figure 2: JB253 binding and concentration-response studies.

a) Binding affinity of glimepiride (Glim) (red, IC ₅ 0 = 8.3 nM), trans-J 253 (black, IC50 = 17.6 μΜ) and CZJ- JB253 (blue, IC ₅₀ = 14.8 μΜ) were indirectly determined in SUR1- expressing HEK293t cells using displacement of [3H]-glibenclamide (datapoints fitted using the Hill equation) (n = 3 independent repeats). Note that, due to the potential for thermal dark- relaxation during the harvesting and washing steps, effects of photoswitching on JB253 binding affinity could not be excluded. Values represent the mean ± SD.

b) cis-JB253 and glimepiride (Glim) possess similar concentration-response curves for the stimulation of [Ca ²⁺ ]j in mouse islets, while trans-JB253 is largely ineffective. The concentration-response for glimepiride is right-shifted in the presence of a saturating concentration (100 μΜ) of trans-JB253 (datapoints fitted using non-linear regression) in = 3- 5 recordings).

c) HEK293t cells expressing a full length Epac2-camps probe respond to glimepiride with decreases in Forster resonance energy transfer (FRET) (represented here as an increase in R/Ro) (n = 28 cells from 4 recordings). d) As for c) but c/s-JB253. Values represent the mean ± SEM for b)-d). Figure 3: JB253 confers photoswitching on KATP channels:

a) Photocurrents recorded from HEK293t cells transfected with plasmids encoding Kir6.2 and SUR1 using the whole cell patch clamp configuration (holding potential -60 mV). JB2S3- treated cells respond to wavelengths between 400-500 nm with a reduction in the magnitude of the K ⁺ inward rectifier current.

b) JB253 allows reversible and repeated closure of KATP channels in response to light-dark cycles using λ = 400 nm to induce m-isomerization (purple) and relaxation in the dark (black).

c) Kinetics of light-triggered block and thermal restoration of K _A TP channel currents mediated by JB253.

In all cases, traces represent n = 4 cells.

Figure 4: JB253 allows optical manipulation of pancreatic beta cell activity.

a) Beta cells residing within JB253-treated islets display large increases in cytosolic Ca ²⁺ following exposure to 405 nm (purple) (scale bar, 1 10 μιτι) (linear LUT).

b) Illumination with 405 nm induces large and synchronous rises in Ca ²⁺ as indicated by Fluo- 2 (representative traces from n = 10 recordings).

c) JB253 can be used to impose complex dynamics including Ca ^" oscillations (representative traces from n = 5 recordings).

d) High dose tolbutamide (Tb) augments the effects of JB253 on K _A TP channel blockade (representative traces from n = 6 recordings).

e) Diazoxide (Dz) inhibits JB253 effects by opening the KATP channel ion pore (representative traces from n = 4 recordings).

Figure 5: Specific photoswitching of beta cell function in the violet-blue spectrum.

a) JB253-treated cells loaded with the red-shifted Ca indicator X-Rhodl (λ excitation— 561 nm) similarly respond to 405 nm with Ca rises (representative traces from n = 3 recordings). b) As for a) but imposition of oscillations (representative traces from n— 3 recordings).

2

c) JB253-treated beta cells display large increases in cytosolic Ca following exposure to 440 nm (representative traces from n = 6 recordings).

d) As for c), but following illumination with 491 nm (Tb, tolbutamide; positive control) (representative traces from n = 5 recordings). e) A single islet can be photo switched using a targeting laser while leaving its neighbor quiescent (~ 200 μηι center-center) (representative traces from n— 3 recordings). A global laser pulse evokes activity in both islets (grey, raw; red, smoothed).

f) Incubation of islets with JB253 does not alter cell viability as assessed by calcein AM and propidium iodide incoiporation (NS, non-significant versus DMSO-alone, Student's t-test) {n = 28 islets from four animals). Values represent mean ± SEM.

Figure 6: Manipulation of human islet activity using JB253.

a) JB253-treated beta cells residing within intact human islets respond to 440 nm with rapid rises in cytosolic Ca ²⁺ levels (representative traces from n = 8 recordings).

b) Almost 62% of X-Rhodl -loaded cells respond to JB253 with Ca ²⁺ rises.

c) As for a) but imposition of oscillations to demonstrate reversibility of JB253 effects in human tissue (Tb, tolbutamide; positive control) (n = representative traces from 4 recordings); d) Reversal of JB253 action using diazoxide to open the Κ _Α χρ chamiel ion pore (Dz, diazoxide; negative control) (n = representative traces from 5 recordings). In all cases, islets were derived from three donors.

Figure 7: JB253 yields optical control of insulin secretion.

Application of JB253 under dark conditions is unable to influence insulin release versus control (5 mM glucose-alone) during static incubation of isolated murine islets. By contrast, illumination with 405 nm and 485 nm significantly increases insulin secretion, and this is similar in magnitude to that achieved using glimepiride and tolbutamide (n = 9 mice) (*P<0.05 versus JB253 485 nm; **P<0.01 and ***P<0.001 versus G5; one-way ANOVA). Values represent mean ± SEM.

Figure 8

a) 'H NMR of JB253.

b) ^I3 C NMR of JB253.

c) 2D NMR of JB253 (HSQC)

d) 2D NMR of JB253 (HMBC).

Figure 9: pK _a measurement of JB253.

Ionization constant of trans-l 253 was determined with an absorbance-based platereader assay according to Martinez & DardonviUe (2013) in a DMSO/buffer mixture (1/1). All datapoints were acquired in triplicate (n = 3 assays) at the following pH- values: 3.33, 3.66, 4.00, 4.33, 4.66, 5.00, 5.33, 5.66, 6.00 and 6.33. Ionic strength was held constant (I = 0.1 mM) by the addition of KC1 to each buffer. Data was background subtracted and the spectral differences plotted against the pH. Sigmoidal fitting (IgorPro v6.22a) obtained a pK _a = 4.76. Values represent mean ± SD.

Figure 10: KATP channel block characteristics of JB253 in the dark.

a) To account for cell-cell variation in current densities, magnitude block with JB253 (dark) was calculated as a percentage versus that achieved with 500 μΜ tolbutamide in the same experiment (% ± SEM; n = 3 recordings).

b) Bar graph displaying the amplitude of the inward current (ΔΙ [pA]) at -60 mV elicited by application of either tolbutamide or JB253 in the dark (current + SEM, n = 3 recordings). c) Representative current-voltage (IV) relationships showing a minor decrease in membrane conductance upon application of JB253 in the dark (before drug = black; after drug = orange). This inhibition is reversibly enhanced by exposure to blue light (blue = ON; pink = OFF). d) Mean reversal potential (E _rCv ) before and after illumination of JB253 measured in recordings as depicted in c). (NS, non-significant, lrans-JB253 versus cw-JB253; Student's paired t-test) (mV ± SEM, n = 5 recordings).

Figure 11: JB253 reversibly blocks K _AT p currents in MIN6 beta cells.

a) Representative current- voltage (IV) relationships recorded straight after establishing the whole-cell configuration (ΡΓΘ-ΚΑΤΡ) and after development of the KATP current due to washout of ATP from the cell (Peak KATP current).

b) IVs from the same cell as a) in the presence of either trans-TB253 (light off) or czs- JB253 (light on).

c) Bar graph displaying mean data from experiments as shown in a) and b). Slope conductance was normalized to peak KATP current. Note that at this concentration (10 mM) JB253 only blocks the KATP current during illumination and this is readily reversed when the light source is shut off (*P<0.05 versus JB253 dark; one-way ANOVA) (n = 4 recordings).

Figure 12: Extended JB253 action spectrum.

JB253 is unable to photoswitch KATP currents in HEK293t cells transfected with Kir6.2 and SUR1 at wavelengths > 560 nm (holding potential -60 mV) (trace representative of n = 3 recordings). Figure 13: Extinction coefficient measurement of JB253.

Absorbance at two wavelengths (405 and 485 nm) was measured of a dilution series of JB253 (concentrations in μΜ: 0.01, 0.10, 1.00, 10.0, 25.0 50.0) in low K ⁺ buffer (containing in mM: 3 KC1, 1 18 NaCl, 25 NaHC0 ₃ , 2 CaCl ₂ , 1 MgCl ₂ , 10 HEPES, NaOH to pH 7.4) (« - 4). Data was background subtracted and the absorbance values plotted against the concentration to obtain the extinction coefficients via linear fitting. Values represent mean ± SD.

Figure 14: Design and synthesis of a red-shifted AzoSulfonylurea.

a) Structures of glimepiride (Glim) and the blue light-responsive AzoSulfonylurea JB253 for comparison.

b) The logic of a red-shifted AzoSulfonylurea. Following illumination with green-yellow light (1), the AzoSulfonylurea binds Epac2A, closing KATP channels (2) and opening voltage- dependent Ca ²⁺ channels (Ca _v ) (3). This allows optical control of Ca ²⁺ influx (4) and insulin secretion (5).

c) Synthesis of the AzoSulfonylurea JB558 that can be switched from the trans- to the cis- isomer using green/yellow light.

Figure 15: JB558 has a red-shifted absorption spectra in DMSO and can repeatedly be photoconverted to its cis-state with green-yellow light.

a) UV-Vis spectra of JB558 in DMSO following illumination with λ = 520 nm (green) or under dark-adapted conditions (black).

b) Robust photoswitching between cis- and trtms- JB558 induced with λ = 520 nm and dark, respectively.

Figure 16:

a) trans- JB558 and glimepiride (Glim) displace [3H]-glibenclamide from SUR1 (n - 3 repeats).

b) Glimepiride decreases FRET (shown here as an increase in R/R _m j„) in HEK293T cells expressing full length Epac2-camps (n = 32 cells).

c) As for (b) but c/s-JB558 (λ = 561 nm) (n = 41 cells).

d) As for (c) but irons- JB558 (dark) (n = 37 cells).

Values represent mean ± s.e.m. Figure 17: JB558 allows optical manipulation of pancreatic beta cell activity. a) JB558 increases intracellular Ca ²⁺ concentrations in 59% of beta cells residing within rodent islets of Langerhans following illumination with λ = 561 nm (scale bar = 75 μιτι) (n - 8 islets).

b) Photo switching is rapid following exposure to λ = 561 nm. (c) As for (b), but showing the absence of photoswitching with λ = 405 nm.

d) A high concentration (100 μΜ) of glimepiride (Glim) augments JB558-stimulated Ca ²⁺ rises.

e) Diazoxide (Dz) reverses cw-JB558-induced Ca ²⁺ fluxes.

f) Reversible manipulation of Ca ²⁺ transients can be achieved in the same islet following thermal back relaxation of JB558 in the dark (5 min between stimulation 1 and 2).

Traces represent n— 6-10 recordings from 3 animals. Islets were maintained in 5 raM D- glucose throughout.

Figure 18:

a) JB558 increases intracellular Ca ²⁺ concentrations in human beta cells in response to illumination with 561 nm to induce cz ^' s-formation (scale bar = 50 μηα).

b) Photoswitching is rapid following exposure to 561 nm and can be potentiated with glimepiride (Glim).

e) CW-JB588 activates 54% of beta cells (n = 4 islets).

d ) Reversible manipulation of Ca ²⁺ rises following thermal back relaxation of JB558 in the dark (5 min between stimulation 1 and 2).

Traces represent n = 3-9 recordings from a single donor. Islets were maintained in 5 mM D- glucose throughout.

Figure 19: JB5 ^' 58 yields optical control of insulin secretion.

a) JB558-treated islets respond to illumination with λ - 560 nm by increasing insulin secretion (**P<0.01 and NS, non-significant versus Con; one-way ANOVA).

b) Incubation with JB558 for 1 hr did not adversely alter cell viability versus dimethyl sulfoxide (DMSO), as assessed by the ratio of calcein (live):propidium iodide (dead) fluorescence (positive control; Triton X-100) (NS, non-significant versus Con; one-way ANOVA).

c) Representative images of islets stained with calcein and propidium iodide.

In all cases, n - 36 islets per treatment group from 6 animals. Values represent mean ± s.e.m. Figure 20:

AzoReductase from E. coli (BL21 DE3) is able to cleave methylred (MR) but not JB558. Figure 21 :

a) Representative whole cell voltage clamp recording in a HE 293T cell showing that 20 μΜ JB558 causes partial block (~ 280 pA) of K _A TP channel activity in its dark- adapted state (holding potential = -60 mV).

b) As for (a) but following application of 100 μΜ glimepiride (Glim), which produces complete KATP channel block (~ 850 pA).

Figure 22: JB558-treated islets respond to excitation with λ = 561 nm. but not λ = 491 nm, with alterations to Ca ²⁺ -spiking frequency and amplitude (representative trace from n = 3 recordings).

Figure 23: Voltage ramp experiments (from -120 to +20 mV over 1 s) in HEK293T cells show no light-dependent block of K _A TP channel activity in the presence of dark-adapted (black) or illuminated (green) 20 μΜ JB558.

EXAMPLES

Example 1 Methods 1.1 Chemical synthesis

(£)-4-((4-(diethylamino)phenyI)dia/.enyl)benzenesulfonamide

Sulfanilamide (2.00 g, 1 1.61 mmol, 1.0 eq.) was dissolved in 2.4 M HC1 and cooled to 0 °C. Under vigorous stirring, a solution of NaN0 ₂ (0.96 g, 13.91 mmol, 1.2 eq.) in 6 mL water was added drop wise until the solution turned pale yellow. The formed diazonium salt was stirred under ice-cooling for an additional 10 minutes before it was transferred into a solution of N,N- diethylaniline (1.73 g, 1 1.61 mmol, 1.84 mL, 1.0 eq.) in a 1/1 mixture of 1 M NaOAc/MeOH. The solution turned to dark red and was allowed to warm to r.t. under stirring. The crude product was extracted with EtOAc (3x), and the combined organic layers were washed with brine and dried over MgS0 ₄ . Flash column chromatography (25% EtOAc/z-hexanes) yielded 1.45 g (4.37 mmol) of the desired product as a red powder in 38% yield.

1H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 7.95 (d, J= 8.6 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.81 (d, J - 9.2 Hz, 2H), 7.45 (s, 2H), 6.82 (d, J - 9.3 Hz, 2H), 3.47 (q, J = 7.0 Hz, 4H), 1.15 (t, J = 7.0 Hz, 6H). ^I3 C NMR (101 MHz, DMSO-d ₆ ): δ [ppm] = 154.2, 150.8, 143.8, 142.2, 126.9, 125.8, 121.9, 1 11.1 , 44.2, 12.5. HRMS (ESI): calc. for C ₁₆ H ₂ iN ₄ 0 ₂ S ⁺ (M+H) ⁺ : 333.1380, found: 333.1377. R _t (LCMS; MeCN/H ₂ 0/formic acid = 10/90/0.1 -» 90/10/0.1 over 7 min) = 4.364 min. UV/Vis (LCMS): λ _η13Χ = 460 ran.

(£)-A ^L (cycIohexylcarbamoyl)-4-((4-(dieihylamino)phenyl)diaze nyl)benzenesulfonamide (JB253)

A mixture of (£)-4-((4-(diethylamino)phenyl)diazenyl)benzenesulfonamide (332 mg, 1.0 mmol, 1.0 eq.) and Cs ₂ C0 ₃ (1.30 g, 4.0 mmol, 4.0 eq.) in acetone (20 mL) was refluxed for 1 h before addition of cyclohexyl isocyanate (125 mg, 1.0 mmol, 1 19 μί, 1.0 eq.) diluted in acetone (20 mL). The reaction mixture was refluxed for an additional 3 h, before cooling to -40 °C. The crude solid was filtered and washed with small amounts of acetone before it was carefully dissolved in MeOH to yield 450 mg (0.98 mmol) of JB253 product in 98% yield.

Ή NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 7.83 (d, J= 8.5 Hz, 2H), 7.78 (d, J= 9.1 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 9.3 Hz, 2H), 5.62 (br s, 2H), 3.46 (q, J = 7.0 Hz, 4H), 3.20 (br s, 1 H), 1.86-1.38 (m, 5H), 1.33-0.92 (m, 1 1H). ¹³ C NMR (101 MHz, DMSO- d ₆ ): δ [ppm] = 172.7 (HMBC, see Figure 8d), 152.6, 150.2, 148.5, 142.2, 127.4, 125.3, 120.8, 1 1 1.0, 47.8, 44.1, 33.5, 25.5, 24.9, 12.5. HRMS (ESI): calc. for C ₂₃ H3 ₂ N ₅ 0 ₃ S ⁺ (M+H) ⁺ : 458.2220, found: 458.2219. R _t (LCMS; MeCN/H ₂ 0/formic acid = 10/90/0.1 -» 90/10/0.1 over 7 min) = 5.285 min. UV/Vis (100 μΜ in DMSO): X _max = 472 ran; (LCMS): _max = 468 mn. S405 nm ⁼ 18,501 mol ^"1 cm ^"1 ; s ₄ 8 _{5 n} m ⁼ 38,670 mol ^"1 cm ^"1 . IR (ATR): wavenumber/cm ^"1 = 3331, 2928, 2851 , 1652, 1626, 1602, 1576, 1537, 1514, 1390, 1349, 1174, 1130, 1086, 1042, 843, 820, 676. m.p. = 190 °C.

1.2 General Chemistry

Flash column chromatography was carried out on silica gel 60 (0.040-0.063 mm) purchased from Merck. RP flash column chromatography was earned out on Waters C18 silica gel (0.055-0105 mm, 125 A). Reactions and chromatography fractions were monitored by thin layer chromatography (TLC) on Merck silica gel 60 F254 glass plates. The spots were visualized either under UV light at 254 nm or with appropriate staining method (iodine, p- anisaldehyde, KM11O ₄ ) followed by heating.

NMR spectra were recorded in deuterated solvents on Varian Mercury 200, Bruker AXR 300, Varian VXR 400 S, Bruker AMX 600 and Bruker Avance III HD 400 (equipped with a CryoProbe™) instruments and calibrated to residual solvent peaks ( ¹ H/ ^lj C in ppm): DMSO-d ₆ (2.50/39.52). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, br— broad, m = multiple! Spectra are reported based on appearance, not on theoretical multiplicities derived from structural information.

A Varian MAT CH7A mass spectrometer was used to obtain low- and high-resolution electron impact (EI) mass spectra. Low- and high-resolution electrospray (ESI) mass spectra were obtained on a Varian MAT 711 MS instrument operating in either positive or negative ionization modes.

Solvents for column chromatography and reactions were purchased in HPLC grade or distilled over an appropriate drying reagent prior to use. If necessary, solvents were degassed either by freeze-pump-thaw or by bubbling N ₂ through the vigorously stirred solution for several minutes. Unless otherwise stated, all other reagents were used without further purification from commercial sources.

UV/Vis spectra were recorded on a Varian Gary 50 Bio UV-Visible Spectrophotometer using Helma Suprasil precision cuvettes (10 mm light path).

LC-MS was performed on an Agilent 1260 Infinity HPLC System, MS- Agilent 1 100 Series, Type: 1946D, Model: SL, equipped with a Agilent Zorbax Eclipse Plus C18 (100 x 4.6 mm, particle size 3.5 micron) RP column.

Infrared spectra were recorded on a Perkin Elmer Spectrum BX-59343 instrument as neat materials. For detection a Smiths Detection DuraSam-plIR II Diamond ATR sensor was used. The measured wave numbers are reported in cm ^"1 .

Melting points were measured on an EZ-Melt apparatus (Stanford Research Systems) and are uncorrected.

Extinction coefficients were measured on a BMG Labtech Omega Series FLUOstar microplate reader with clear flat-bottom white 96 wellplates by full spectra acquirement in low K ⁺ external bath buffer (containing in mM: 3 KC1, 1 18 NaCl, 25 NaHC0 ₃ , 2 CaCl ₂ , 1 MgCl ₂ , 10 HEPES; NaOH to pH 7.4). All JB253-containing solutions (dilution series, n = 4: 10 nM; 100 nM; 1 μΜ; 10 μΜ; 25 μΜ; 50 μΜ) were background substracted and fitted with a linear slope. Volumes were 100 pL each, which resulted in a path length of / = 2.94 mm. pK _a Measurements and data processing were performed using the same instrument and protocol according to Martinez & Dardonville (2013). Full absorbance spectra (280-800 nm) were acquired and background subtracted before spectral differences were calculated. The total change of maximal positive and maximal negative difference was calculated and plotted against pH. Sigmoidal fit of the obtained plot gave access to the pK _a .

1.3 Crystallography

X-Ray data collection was performed on a Bruker D8Venture at 173 K using MoKa -radiation (λ = 0.71073 A). The APEX2 (v2012.4-3, Bruker AXS Inc.) software and embedded programs were applied for the integration, scaling and multi-scan absorption correction of the data. The structures were solved by direct methods with SIR97 (Altomare et al., 1999) and refined by least-squares methods against F2 with SHELXL-97 (Sheldrick et al., 2008). All non-hydrogen atoms were refined anisotropically. The C-bound hydrogen atoms were placed in ideal geometry riding on their parent atoms, N-bound hydrogen atoms were refined freely. The crystallographic data for JB253 is available free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif (accession ref. CCDC 1014606).

1.4 Electrophysiology

HEK293t cells (obtained from Leibniz-Institut DSMZ: #305) were incubated in Dulbecco's Modified Eagle Medium (DMEM) + 10% FBS and used for electrophysiological recordings 24-48 hrs following Lipofectamine transfection with plasmids encoding ir6.2 (Genbank D50581), rat SUR1 (Genbank L40624) and GFP. Whole cell patch clamp experiments were performed using a standard electrophysiology setup equipped with a HEKA Patch Clamp EPC10 USB amplifier and PatchMaster software (HEKA Electronik). Micropipettes were generated from "Science Products GB200-F-8P with filament" pipettes using a vertical puller. Resistance varied between 5-7 ΜΩ. Bath solution contained in mM: 3 KCf 1 18 NaCl, 25 NaHC0 ₃ , 2 CaCl ₂ , 1 MgCl ₂ , 10 HEPES (NaOH to pH 7.4). Pipette solution contained in mM: 90 K-gluconate, 10 NaCl, 10 KC1. 1 MgCl ₂ , 10 EGTA, 60 HEPES (KOH to pH 7.3), and the holding potential was -60 mV. Illumination during electrophysiology and UV/Vis experiments was provided by a TILL Photonics Polychrome 5000 monochromator. JB253 was applied at 50 μΜ, as this concentration was found to be maximal for stimulating Ca ^~ rises.

ΜΓΝ6 cells (obtained from Dr Jun-ichi Miyazaki, Osaka University, see Miyazaki et al., 1990)) were cultured in DMEM supplemented with 15% FBS, 1% glutamine, 2% HEPES buffer, 0.0005% β-mercaptoethanol and 1% penicillin/streptomycin. Cells were seeded onto glass coverslips 24 hrs before whole cell patch clamp experiments using micropipettes generated from thin-walled borosilicate capillaries (3-6 ΜΩ). Bath solution contained in mM: 3 KC1, 118 NaCl, 25 HEPES, 3 MgCl ₂ and 2 CaCl ₂ . Pipette solution contained in mM: 150 KC1, 3 MgCl ₂ , 5 EGTA, 10 HEPES, 0.3 K?ATP (pH 7.2), and the holding potential was -60 mV. Illumination was provided using a X-Cite 120 mercury arc lamp (Lumen Dynamics) with a bandpass filter (470 ± 20 nm). Voltage ramps from -20 mV to -120 mV (500 ms duration) were applied every 5 s to produce current-voltage relationships in the presence and absence of JB253. All cells lines were regularly mycoplasma tested.

1.5 Mouse islet isolation

Male and female CD1 and C57BL6 mice (8-20 weeks) were maintained in a specific pathogen-free facility under a 12h light-dark cycle with ad libitum access to water and food. Animals were euthanized using a schedule- 1 method and pancreatic islets isolated by collagenase digestion. All procedures were regulated by the Home Office according to the Animals (Scientific Procedures) Act 1986 of the United Kingdom (PPL 70/7349), and study approval granted by the Animal Welfare and Ethical Review Body (A WERB) of Imperial College. No randomization was used for animal experimentation, since mice were only used as tissue donors.

1.6 Human islet isolation

Human islets were isolated from deceased heart-beating donors (n = 3) at transplantation facilities in Pisa and Edmonton with the relevant national and local ethical permissions, including consent from next of kin where required, and cultured in RPMI supplemented with 5.5 mM D-glucose, 10% foetal calf serum (FCS), 100 U penicillin, 100 μg streptomycin and 0.25/μ1 fungizone (37 °C, 5% C0 ₂ ). All studies involving human tissue were approved by the National Research Ethics Committee (NRES) London (Fulham), REC # 07/H071 1/1 14.

1.7 Calcium imaging

Islets were loaded for 30-45 min in fluo2-AM (10 μΜ) or for 2-5 min with X-RJiod 1 (5 μΜ) diluted with a mixture of DMSO (0.01%, wt/vol) and pluronic acid (0.001%, wt/vol; all Invitrogen) in a bicarbonate buffer containing in mM: 120 NaCl, 4.8 KC1, 1 .25 NaH ₂ P0 ₄ , 24 NaHC0 ₃ , 2.5 CaCl?, 1.2 MgCl ₂ and 5 D-glucose. fMCI was perfonned using a Zeiss Axiovert M200 fitted with a Nipkow spinning-disk head (Yokogawa CSU-10) and a 10x/0.3NA objective adjusted for chromatic aberration (EC Plan-Neofluar, Zeiss). Pulsed excitation (frequency = 0.5 Hz; exposure = 263 ms) was delivered at 491 nm and emitted signals recorded at 500-550 nm with a back-illuminated 16-bit EM-CCD camera (ImageEM 9100-13; Hamamatsu). During recording, islets were maintained at 35-36 °C in the presence of 50 μΜ JB253 using a custom-manufactured perfusion and heating system (Digital Pixel). Drugs were introduced through the perfusion system at the indicated time points and concentrations. Violet light was delivered by a 405 ± 5 nm laser coupled to the side port of the microscope and configured to fill the back of the objective with light using an Optospot (Caim Research). Blue light was delivered using 440 ± 5 nm and 491 ± 5 nm diode lasers controlled by a laser merge module (Spectral Applied Research) to allow simultaneous exposure and acquisition. For single islet targeting, a 473 ± 5 nm laser was coupled to a custom-manufactured dichroic array (Caim Research), allowing user-directed steering of a collimated laser spot across the field of view. Signals were normalized using F/Fmin where F is fluorescence at a given timepoint and Fmin is minimum fluorescence.

1.8 Cytotoxicity assay

Islets were incubated with either DMSO or JB253 for 1 hr before staining with 3 μΜ of calcein-AM (live) and 2.5 μΜ of propidium iodide (dead). Absorbance/emission was detected at 491 /525 nm and 561/620 nm for calcein and PI, respectively. The area of dead:live cells was calculated as a unitary ratio and the observer blinded to treatment identity.

1.9 EPAC2 imaging

For EPAC2 imaging, HE 293t were transfected with the full length construct for Epac2- camps containing the cAMP and sulfonylurea binding domains (obtained from Prof. Jin Zhang, Johns Hopkins University, see Herbst et al., 201 1) before imaging (Hodson et al., 2014) using a HEPES-bicarbonate buffer containing in mM: 120 NaCl, 4.8 C1, 24 NaHC0 ₃ , 0.5 Na ₂ HP0 ₄ , 5 HEPES, 2.5 CaCl ₂ , 1 .2 MgCl ₂ and 5 D-glucose. Excitation was delivered at 440 nm and emitted signals captured using cerulean (530 nm) and citrine (470 nm) filters. FRET was calculated as the ratio of Cerulean (CFP):Venus (YFP) fluorescence. Signals were normalized using R/Ro where R is the ratio at a given timepoint and R ₀ is the minimum ratio.

1.10 Measurements of insulin secretion fr m isolated islets

Insulin secretion was measured from 6 islets per well, incubated at 37 °C for 30 min in 0.5 ml of Krebs-HEPES-bicarbonate (KHB) solution (containing in mM: 130 NaCl, 3.6 KC1, 1.5 CaCl ₂ , 0.5 MgSC-4, 0.5 NaH ₂ P0 ₄ , 2 NaHC0 ₃ , 10 HEPES, and 0.1 % (wt/vol) BSA, pH 7.4) containing the indicated glucose concentration and tolbutamide (100 μΜ), glimeperide (50 μΜ), diazoxide (250 μΜ) and JB253 (50 μΜ). Illumination (λ = 405 ± 20 nm and 485 ± 6 nm) was performed using a Fluostar Optima microplate reader (BMG Labtech) set to deliver 30 s of light every 2 min to the designated wells. Insulin concentrations were determined in duplicate using specific radioimmunoassay (EMD Millipore).

1.11 |311]-GlibencIamide radioassay

SUR1 -expressing HEK293t cells were harvested and washed twice in assaying buffer containing in mM: 1 19 NaCl, 4.7 KC1, mM CaCl ₂ , 1.2 KH ₂ P0 ₄ , 1.2 MgS0 ₄ , 5 NaHC0 ₃ , and 20 HEPES, pH 7.4. In a 96-well plate, -200,000 cells/well were incubated for 50 min with [3H]-glibenclamide (PerkinElmer) and different concentrations of glimepiride (Sigma- Aldrich) or JB253. Incubation was terminated by rapid filtration through Whatman GF/C filters by means of a Brandel MWXR-96 TI harvester and filters were washed 3 times with ice-cold assay buffer. Radioactivity was counted 6 h after cell and filter lysis in 200 μΐ _^ Rotiszint EcoPlus (Roth) using a Packard microbeta scintillation counter (PerkinElmer).

1.12 Statistical analysis

Data distribution was determined using the D'Agostino omnibus test. Non-multifactorial pairwise comparisons were made using either the Student's t-test. Interactions between multiple treatments were assessed using one-way ANOVA followed by pairwise comparisons using Bonferroni's post-hoc test. Effect sizes in islets/cells are usually sufficiently large that multiple animals/independent replication is a more important determinant of power in studies requiring statistical comparison. Non-linear regression was used to calculate the EC50 of normalized and log-transformed concentration-response curves. For [3H]-glibenclamide displacement assays, data points were fitted to the Hill equation before calculation of the halfmax value. In all cases, analysis was performed using Graphpad Prism (Graphpad Software) and lgorPro, and experimental numbers reported as independent biological replicates. No animals or data were excluded from the analysis and results were considered significant at P<0.05.

Example 2 Results

2.1 Design and synthesis of JB253

Distinct substitution patterns are found within different classes of arylsulfonylurea drugs: while there may be a variety of moieties on the aryl-ring, ranging from a simple methyl group in tolbutamide to more complex structures like a linked pyrrolidinone in glimepiride (Figure lb), the terminal nitrogen in sulfonylureas is usually substituted with an aliphatic group. We reasoned that, to generate a photoswitchable analogue, the aromatic core of the sulfonylurea drugs could be extended to an azobenzene. Furthermore, we aimed for a cyclohexyl substituent on the urea moiety that mimics the corresponding substituent on glimepiride. Using a simple three-step procedure commencing with sulfanilamide, N,iV-diethylaniline and cyclohexyl isocyanate, JB253 could be synthesized rapidly and inexpensively in large quantities via the sulfonamide-azobenzene (£)-4-((4-(diethylamino)phenyl)- diazenyl)benzenesulfonamide (Figure lc) (Figure 8a-d). Initial photochromic characteristics of JB253 were measured using a UV/Vis spectrophotometer equipped with a monochromator, affording a single broad band as expected for a push-pull-azobenzene-system k _max = 472 nm) (Figure Id).

Azobenzenes are known to be photoconverted between their cis- and trans-state by excitation with different wavelengths of light, or alternatively by illumination and dark relaxation. Indeed, JB253 was readily converted to its cz ^' s-state by applying wavelengths ranging from λ = 400-500 nm (peak λ = 472 nm), while thermal relaxation to its trans-state occurred rapidly in the dark. X-ray diffractometry revealed a high degree of structural similarity between trans-JB253 and glimepiride crystals (Figure le) (see Table 1). Whereas glimepiride in solution rotates freely around its ethylene carbon chain to adopt various possible binding confonnations, JB253 is rigid unless illuminated and so can only adopt two conformations depending on isomeric state (i.e. trans- or cis-). Table 1: Crystallographic data for JB253

Attaching lipophilic azobenzene units normally renders molecules poorly soluble in water and aqueous buffers, an obvious drawback for their use in biological systems. JB253, however, demonstrates excellent water solubility (> 0.1 mM) when diluted from a 50 mM stock solution in DMSO, presumably due to its acidity (pK _a (trans-JB253) = 4.76; see Figure 9). These features were a promising entry point for our subsequent studies using mammalian tissue.

2.2 JB253 binding studies

To determine the binding affinity of JB253 to SUR1 relative to a known sulfonylurea (i.e. glimepiride), [3H]-glibenclamide displacement assays were performed. JB253 bound SUR1 with a 1000-fold lower affinity compared to glimepiride, and this was unaffected by illumination (IC50 = 8.3 nM versus 17.6 μΜ versus 14.8 μΜ for glimepiride versus trans- JB253 versus cz ^' s- JB253, respectively) (Figure 2a). However, due to the potential for rapid thermal dark-relaxation during the wash cycles (see below), we were unable to exclude a role for trans- to cis- isomerization in strengthening JB253 binding affinity. Therefore, to compare the activity profiles of trans- 253 , cz ^' s- JB253 and glimepiride using a functionally-relevant readout, concentration-response experiments were conducted in mouse islets. The EC 50 of cis- JB253 for cytosolic Ca ²⁺ rises was found to be 675 nM, similar to that obtained for glimepiride in the same system (EC50 glimepiride = 399 nM) (Figure 2b). The concentration- response curve for glimepiride was right-shifted in the presence of a saturating concentration of tra?7s-JB253, demonstrating the presence of competitive agonism even under dark conditions (Figure 2b).

Since most sulfonlyureas have been reported to bind and activate Exchange Protein directly Activated by cAMP 2A (Epac2A), an important mediator of insulin secretion (see e.g. Herbst et al., 2011), the presence of interactions with JB253 was assessed using a Foster resonance energy transfer (FRET)-based approach. To enable this, a full length Epac2 -camps biosensor containing the sulfonylurea binding site was encoded in HEK293t cells (Herbst et al., 201 1). Confirming the existence of sufonylurea-Epac2A interactions, application of either glimepiride (Figure 2c) or cz ^' s- JB253 (Figure 2d) decreased FRET to a similar extent (AR/R ₀ = 0.052 versus 0.064 AU, glimepiride versus JB253, respectively; NS, non-significant, Student's t-test).

2.3 JB253 allows photoswitehing of Κ _Α χρ channels

We sought first to investigate whether JB253 could yield optical control over K _A TP channel activity using a system free from confounding effects of glucose metabolism. To enable this, KATP channels were heterologously expressed in HEK293t cells by transfection with plasmids encoding the Kir6.2 and SUR1 subunits along with GFP. Tolbutamide and diazoxide-sensitive inward-rectifying currents could be recorded in transfected cells, confirming the functional assembly of KAT _P channels, in the dark state, JB253 partly reduced K ⁺ current amplitude within a few seconds. This was however a fraction of that observed during 500 μΜ tolbutamide application (Figure 10a and b). Subsequent illumination of JB253 with wavelengths between 400-500 nm further closed the channel (Figure 3a), with -45-72% block being achieved relative to that recorded using 500 μΜ tolbutamide (Table 2). The reversal potential was close to the expected equilibrium potential for K ⁺ , and this was unaffected by molecule orientation (-90.0 ± 1.8 versus -87.8 ± 1.6 mV, dark versus illuminated; nonsignificant) (Figure 10c and d). As such, JB253 possesses the advantageous property of becoming a high affinity K _A TP channel blocker upon illumination.

Using a wavelength of 400 nm, heterologously-expressed KATP channels could be repeatedly opened and closed in JB253-treated preparations without obvious desensitization (difference in Δ1 [pA] between first and last switch = 14.9 ± 7.6 %) (Figure 3b and c) (Tables 3 and 4). Upon exposure to 400 nm, rapid block was observed (τ _οη = 0.4 s) (Fig. 3c). When the light source was shut off, thermal relaxation was fast, returning the KAT _P channel to baseline levels within a couple of seconds {τ ₀ α = 1 .5 s) (Figure 3c) (see Tables 2 and 3). While maximal photoblock of KATP channels was observed at 460 nm, significant effects were also obtained with violet light {i.e. 405 nm), which was more compatible with fluorescence imaging of pancreatic beta cell function (see below). Confirming that JB253 could photoswitch endogenous KATP channels, hyperpolarizing currents were reversibly blocked in ΜΓΝ6 beta cells following illumination (Figure 1 1 ).

Table 2: Wavelength-dependent kinetics and current change in JB253-lreated cells.

On/off kinetics for various wavelengths are shown in ms ± SD (n = 3 recordings). Current change is expressed as percentage (% ± SD)-block versus that obtained with tolbutamide in the same experiment (n = 3 recordings). Current change (ΔΙ [pA]) for a single representative experiment is displayed. In all cases, cells were exposed to 500 μΜ tolbutamide before washout and application of 50 μΜ JB253.

λ [nm] τ _οη [ms] T _OFF [ms] % block ΔΙ [pA]

400 1176+385 2758+745 44.0+29.6 216

420 1309+438 2209±482 69.4+51.1 492

440 1246+421 2295+397 70.0±44.3 440

460 1181+447 1940+322 72.8±48.1 481

480 1 163+434 2183+422 72.4+49.0 487

500 1244±462 2002+354 70.2+49.5 482 Table 3: Kinetics and current change during a repetitive illumination cycle.

On/off kinetics are shown as ms ± SEM and current change as pA ± SEM calculated following four dark/400 nm cycles. Values are from a single cell.

Table 4: Current change for first and last switch for all experiments.

The difference in current change (ΔΙ [pA]) between the first and last switch (400:

represented as a percentage (%).

2.4 Functional interrogation of beta cells within mouse islets

Stimulus-secretion coupling in beta cells relies on the closure of K _ATP channels, Ca ²⁺ influx through VDCC and release of insulin granules. We therefore attempted to manipulate beta cell activity by optically controlling K _A TP channels with JB253.

Using functional multicellular Ca ²⁺ imaging (fMCI) to monitor cell activity directly in situ within intact islets (Hodson et al., 2013; Rutter & Hodson, 2013), increases in cytosolic free Ca ²⁺ , assumed largely to emanate from beta cells under the conditions used here (Quesada et al., 1999), could be evoked following global illumination using a 405 nm laser (Fig. 4a) (n = 10 recordings). Just over half (54 %) of the fluo-2-loaded population responded to illumination with synchronous Ca ²⁺ rises (Figure 4a and b). Demonstrating the utility of JB253 for the fine control of beta cell function, discrete Ca oscillations, thought to underlie generation of insulin pulses, could be imposed using repeat exposure to 405 nm (Figure 4c). As anticipated, high doses of tolbutamide and diazoxide were able to augment and suppress, respectively, the effects of JB253 (Figure 4d and e) (n = 4-6 recordings).

The wavelength required to excite Fluo-2 (λ = 491 nm), a commonly used Ca ²⁺ indicator, could potentially lead to K _A TP channel closure in JB253-treated islets due to cw-isomer formation. We therefore decided to repeat the above experiments using the red (λ = 561 nm)- excited Ca ²⁺ indicator X-Rhod-1. Identical results were obtained to studies with Fluo-2 (Figure 5a and b) (63% responsive X-Rhod-1 -loaded cells) (n = 3 recordings), confirming that the photostationary state of JB253 during brief (263 ms) pulses of 491 nm light was alone insufficient to close K _A TP channels in the islet preparation. Likewise, global Ca ²⁺ oscillations could be induced in JB253-treated islets by more prolonged illumination with 440 nm and 491 nm laser lines and, as expected from the electrophysiological recordings, both wavelengths appeared to activate a slightly larger cell population (Figure 5c and d). These observations were unlikely due to K _A T _P channel closure at 561 nm, since JB253 was unable to photoswitch K ⁺ currents at wavelengths > 560 nm (Figure 12).

Demonstrating the spatial precision of JB253, a single islet from a doublet could be activated using a targeting laser without significantly stimulating its neighbor (~ 200 μπι from center to center) (Figure 5e), in part aided by the high molecule extinction coefficient (38,670 mof ¹ cm ^"1 at 485 nm; see Figure 13). Lastly, JB253 did not appear to be cytotoxic to islets, as necrosis indices showed no significant differences in cell death versus DMSO-alone (Figure 5f).

2.5 Manipulation of human islet function using JB253

To underline the translational potential of JB253 for use in man, Ca 2+ -imaging experiments were repeated using isolated human islets of Langerhans. As observed for their mouse counterparts, beta cells within JB253-treated human islets responded to 440 nm and 491 nm with large intracellular Ca ²⁺ rises, and oscillations could be coaxed simply by turning the laser on and off (Figure 6a-c). JB253 effects appeared to be due to KATP blockade, as they could be mimicked and reversed using tolbutamide and diazoxide, respectively (Figure 6c and d).

2.6 Optical stimulation of insulin release using JB253

To cement the link between photocontrol of KATP channels, [Ca ]j and insulin secretion, islets (from n = 10 mice) were incubated in the presence of JB253 while exposing to either dark, 405 nm or 485 nm. Insulin release was similar in control experiments (5 mM glucose; shown to sensitize beta cells to sulfonylurea, Jonkers et al., 2001) and JB253-treated islets in the dark, suggesting that any KATP channel block and VDCC activity detected under these conditions was subthreshold for triggering Ca ²⁺ -activated exocytosis (Figure 7). By contrast, JB253-treated islets secreted almost 4-8-fold more insulin following illumination, and this could be partially reversed using diazoxide (Figure 7). When exposed to 485 nm light, JB253 was equipotent to glimepiride at stimulating insulin secretion (Figure. 7).

Example 3 A red-shifted photochromic sulfonylurea

3.1 Chemical synthesis

5-Amino-l, 3, 4-thiadia/oIe-2-sulfonamide (2)

iV-(5-Sulfamoyl-l,3,4-thiadiazol-2-yl)acetamide (acetazolamide) (3.00 g, 13.5 mmol, 1.0 eq.) was dissolved in 3 M HQ (23.4 mL) and refluxed at 1 10 °C for 3 h. The mixture was neutralized with 4 M NaOH (17.6 mL) and extracted with EtOAc (3 x 200 mL). The combined organic layers were washed with brine (1 x 200 mL) and concentrated in vacuo. 2.18 g (12.1 mmol) of the desired product were obtained in 90% yield as a white fluffy powder.

1H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 8.08 (br s, 2H), 7.90 (s, 2H).

^B C NMR (101 MHz, DMSO-d ₆ ): δ [ppm] = 171.7, 157.9.

FIRMS (ESI): calc. for C ₂ H ₅ N ₄ 0 ₂ S2 ⁺ [M+H] ⁺ : 180.9848, found: 180.9849.

R, (LCMS; MeCN/H ₂ 0/formic acid - 10/90/0.1 - 90/10/0.1 over 7 min) = 0.780 min.

UV/Vis (LCMS): _max = 278 nm.

IR (ATR): wave number/cm ^"1 = 3396, 3310, 3169, 1684, 1600, 1563, 1492, 1443, 1346, 1176, 1138, 1080, 974, 917 801.

m.p. = 202 °C ((4-(Diethylamino)phenyl)diazenyl)-l, 3, 4-thiadiazoIe-2-sulfonamide (3)

5-Amino-l, 3, 4-thiadiazole-2-sulfonamide (2) (800 mg, 4.44 mmol, 1.0 eq.) was dissolved in H ₂ 0 (32.0 mL) and cHCl (8.00 mL) at 0 °C. A 1 .2 M aqueous solution of sodium nitrite (738 mg, 10.7 mmol, 8.91 mL, 2.4 eq.) was added dropwise and the resulting reaction mixture was stirred for 10 min at 0 °C. The prepared yellow solution was added dropwise over 30 min to a solution of N, N-diethylaniline (748 μL·, 4.88 mmol, 1.1 eq.) in MeOH/1 M NaOAc (40.0 mL/10.0 mL) at 0 °C and stirred for 15 min at 0 °C. The reaction was allowed to warm to r.t. and stirred overnight. The reaction mixture was then acidified with 1 M HC1 and extracted with EtOAc (3 x 250 mL), washed with brine (1 x 250 mL) and the crude product was filtered through a pore III frit with EtOAc containing a plug of silica (50 mL). 908 mg (2.67 mmol) of 3 were obtained in 60% yield as a deep purple solid.

^J H NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 8.47 (br s, 2H), 7.86 (d, J = 9.2 Hz, 2H), 6.96 (d, J= 9.2, 2H), 3.59 (q, J= 6.8 Hz, 4H), 1.20 (t, J= 7.2 Hz, 6H).

¹³ C NMR (101 MHz, DMSO-d ₆ ): δ [ppm] = 183.3, 167.3, 153.8, 142.0, 1 12.6, 79.2, 44.9, 12.6.

HRMS (ESI): calc. for C ₁₂ Hi ₇ N ₆ 0 ₂ S ₂ ⁺ [M+H] ⁺ : 341.0849, found: 341.0846.

UV/Vis (LCMS): X _max = 540 ran.

R _t (LCMS; MeCN/H ₂ 0/formic acid - 10/90/0.1 90/10/0.1 over 7 min) = 3.845 min.

IR (ATR): wave number/cm ^"1 = 3303, 3060, 1713, 1 602, 1543, 1416, 1357, 1298, 1267, 1215,

1174, 1 142, 1068, 909, 829, 788.

m.p. = >275 °C

(/:)-A ^L (CycI<)hexylcarbam yl)-5-((4-(diethylamino)phcnyl)-dia/enyJ)-l , 3, 4-tfaiadiazole- 2-sulfonamide (JB558)

(£)-5-((4-(Diethylamino)phenyl)diazenyl)-l , 3, 4-thiadiazole-2-sulfonamide(3) (5.1 mg, 15.0 μmol, 1.0 eq.) was dissolved in dry MeCN (4.0 mL) under a N ₂ -atmosphere. NEt ₃ (2.08μί, 15.0 μιηοΐ, 1.0 eq.) and cyclohexyl isocyanate (48.0 μί, 376 μιηοΐ, 25.0 eq.) were added and the mixture was refluxed for 4 h at 90 °C. The solution was concentrated to 1 mL, filtered through a PTFE filter (CHROMAFIL® Xtra PTFE 0.45) and subjected to RP-HPLC (MeCN/HzO/formic acid = 5/95/0.1 80/20/0.1 over 40 min). 2.6 mg (5.6 μηιοΐ) of the desired product was obtained in 37% yield. Ή NMR (400 MHz, DMSO-d ₆ ): δ [ppm] = 7.86 (d, J= 9.2 Hz, 2H), 6.97 (d, J= 9.6 Hz, 2H), 6.68 (m, 1H), 3.60 (q, J- 7.2 Hz, 4H), 3.54-3.47 (m, 1H), 1.75-1.60 (m, 4H), 1 .55-1.45 (m, 2H), 1.25-1.14 (m, 10H).

¹³ C NMR (101 MHz, DMSO-d ₆ ): δ [ppm] = 184.2, 164.4 (formic acid), 163.1 , 158.2, 154.0, 150.6, 142.1, 112.8, 48.6, 45.0, 32.2, 24.3, 12.6.

HRMS (ESI): calc. for C ₁₉ H ₂₈ N ₇ 0 ₃ S2 ⁺ [M+H] ⁺ : 466.1690, found: 466.1690.

UV/Vis (LCMS): _max = 545 nm.

R _t (LCMS; MeCN/H ₂ 0/formic acid = 10/90/0.1 - 90/10/0.1 over 7 min) - 4.691 min.

IR (ATR): wave number/cm ^"1 = 2933, 1682, 1603, 1530, 1450, 1323, 1305, 1265, 1208, 1 139, 1072, 1009, 907, 826, 788, 696, 655.

m.p. = 171 °C

3.2 Chemistry

Solvents for chromatography and reactions were purchased in HPLC grade or distilled over an appropriate drying reagent prior to use. If necessary, solvents were degassed either by freeze- pump-thaw or by bubbling N ₂ through the vigorously stirred solution for several min. Unless otherwise stated, all other reagents were used without further purification from commercial sources.

Flash column chromatography was carried out on silica gel 60 (0.040-0.063 mm) purchased from Merck. Reactions and chromatography fractions were monitored by thin layer chromatography (TLC) on Merck silica gel 60 F254 glass plates. The spots were visualized under UV light at λ = 254 nm.

NMR spectra were recorded in deuterated solvents on a BRUKER Avance III HD 400 (equipped with a CryoProbe™) instrument and calibrated to residual solvent peaks ( H/ C in ppm): DMSO-d ₆ (2.50/39.52). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, br = broad, m = multiplet. Spectra are reported based on appearance, not on theoretical multiplicities derived from structural information.

Low- and high-resolution electrospray (ESI) mass spectra were obtained on a Varian MAT 711 MS instrument operating in either positive or negative ionization modes. UV/Vis spectra and time-resolved light-dependent JB558 activity were recorded on a Varian Gary 50 Bio UV- Visible Spectrophotometer using Helma SUPRASIL precision cuvettes (10 mm light path) equipped with an UHP-LED-520 (Prizmatic) connected to an AC/DC switching adapter (Mean Well Enterprises).

LC-MS was performed on an Agilent 1260 Infinity HPLC System, MS- Agilent 1100 Series, Type: 1946D, Model: SL, equipped with a Agilent Zorbax Eclipse Plus C18 (100 x 4.6 mm, particle size 3.5 micron) RP column with a constant flow-rate of 2 mL/min.

Melting points were measured on the apparatus BUCHI Melting Point B-540 from BOCHI Laborlechnik AG or on an EZ-Melt apparatus from Stanford Research Systems and are uncorrected.

Infrared spectra (IR) were recorded on a PERKIN ELMER Spectrum BX-59343 instrument as neat materials. For detection a SMITHS DETECTION DuraSam-plIR II Diamond ATR sensor was used. The measured wave numbers are reported in cm ^"1 .

3.3 Whole-cell electrophysiology in HE 293T cells

HEK293T cells were incubated at 37 °C (10% C0 ₂ ) in Dulbecco's Modified Eagle Medium (DMEM) + 10% FBS and were split at 80-90% confluency. Detached cells were diluted in growth medium and plated on acid-etched coverslips coated with poly-i-lysine in a 24 well plate. 50,000 cells were added to each well in 500 μΕ standard growth medium along with the DNA (per coverslip: 250 ng Kir6.2, 350 ng SURl, 50 ng YFP) and JetPRIME transfection reagents according to the manufacturer's instructions (per coverslip: 50 pL JetPRIME buffer, 0.5 μιΕ JetPRIME transfection reagent). The transfection medium was exchanged for normal growth media 4 hr after transfection and electrophysiological experiments were carried out 20-40 hr later. Whole cell patch clamp experiments were performed using a standard electrophysiology setup equipped with a ITEKA Patch Clamp EPC10 USB amplifier and PatchMaster software (HEKA Electronik). Micropipettes were generated from "Science Products GB200-F-8P with filament" pipettes using a Narishige PC- 10 vertical puller. The patch pipette resistance varied between 3-9 ΜΩ. The bath solution contained in mM: 3 KG, 118 NaCl, 25 NaHC0 ₃ , 2 CaCl ₂ , 1 MgCl ₂ , 10 HEPES (NaOH to pH 7.4). The pipette solution contained in mM: 90 K-gluconate, 10 NaCl, 10 KC1, 1 MgCl ₂ , 10 EGTA, 60 HEPES (KOH to pH 7.3). Compounds were applied by direct pipette of 1 pL DMSO stock (43.4 mM or 4.34 mM) directly into the recording bath. Voltage clamp measurements were carried out at room temperature with a holding potential of -60 raV. Compound switching was evaluated at λ = 520 nm using a UH-LED-520 light source and fibre optic placed ~l cm from the sample.

3.4 Isolation of rodent islets

C57BL6 mice were maintained in a specific pathogen-free facility under a 12 h light-dark cycle with ad libitum access to water and food. Euthanasia was carried out using a designated schedule-1 method and collagenase solution (1 mg/mL) injected into the bile duct to inflate the pancreas, before isolation of islets using Histopaque gradient separation. Islets were cultured overnight in solution containing Roswell Park Memorial Institute (RPMI) medium supplemented with 10% foetal calf serum and 100 U/mL penicillin & streptomycin 100 μg/mL. All procedures were regulated by the Home Office according to the Animals (Scientific Procedures) Act 1986 of the United Kingdom (PPL 70/7349).

3.5 Isolation of human islets

Islets were isolated with necessary local and national ethical permissions, including consent from the next of kin, and cultured as above except for further supplementation with 0.25 mg/mL fungizone. All studies involving human tissue were approved by the National Research Ethics Committee London (Fulham), REC #07/H0711/114.

3.6 Calcium and Epac2 imaging

Islets were loaded with Fluo2-AM (10 μΜ) for 45 min before performing functional multicellular Ca ²⁺ imaging (fMCI), as previously described (Hodson et al., 2013; Hodson et al., 2014). Briefly, pulsed λ = 491 nm light was delivered using a solid state laser (Cobalt) coupled to a Nipkow spinning disk head (Yokogawa CSU-10) and a Zeiss Axiovert M200 equipped with a 1 Ox/0.3 NA objective (EC Plan-Neofluar, Zeiss). Emitted signals were captured from A = 500-550 nm using a Hammamatsu ImageEM 9100-13 EM-CCD camera. Illumination to induce photos witching was performed using a λ = 561 ± 5 laser coupled to the microscope with a multimodal fibre optic.

Likewise, Epac2 imaging was performed as described (Hodson et al., 2014; Broichhagen et al., 2014-b) using HEK293T cells transiently transfected with full length Epac2-camps (as described in Herbst et al., 201 1) and emission filters centred on Cerulean (λ = 470 nm) and Venus (λ = 530 nm) (λ excitation — 440 nm). FRET was calculated as the ratio of Cerulean:Venus fluorescence and signals normalized using R/R _m in, where R _m j _n is the minimum ratio. Finally, Fura-2 (10 μM)-loaded islets were imaged using a monochromator set to alternately excite at 340 nm and 380 ran, with detection at = 500-550 nm using an Andor Zyla 5.5 sCMOS. Ca ²⁺ signals were presented as F340/F380, where F340 and F380 represent fluorescence intensity at 340 nm and 380 nm, respectively. In all cases, HEPES- bicarbonate buffer was used, containing in mM: 120 NaCl, 4.8 KC1, 24 NaHC0 ₃ , 0.5 Na ₂ HP0 ₄ , 5 HEPES, 2.5 CaCl ₂ , 1.2 MgCl ₂ and 5 D-glucose. Drugs were applied via the perfusion system, as for electrophysiological recordings.

3.7 Cytotoxicity assays

DMSO- or JB558-treated islets were incubated with 3 μΜ of calcein-AM (live) and 2.5 μΜ of propidium iodide (dead), and absorbance/emission detected at λ = 491/525 nm (calcein) and λ — 561/620 nm (propidium iodide). The area of dead:live cells was calculated as a unitary ratio.

3.8 Insulin secretion assays

Batches of six islets were incubated for 1 hr in 0.5 mL of Krebs-HEPES-bicarbonate solution containing in mM: 130 NaCl, 3.6 KC1, 1.5 CaCl ₂ , 0.5 MgS0 ₄ , 0.5 NaIi ₂ P0 ₄ , 2 NaHC0 ₃ , 10 HEPES, 5 D-glucose and 0.1% (wt/vol) bovine serum albumin, pH 7.4. Treatments were applied as indicated and λ - 560 nm delivered as pulses using a BMG Fluostar Optima platereader. Insulin concentrations in the supernatant were measured using a proprietary Cisbio HTRF assay according to the manufacturer's instructions.

3.9 |3H|-Glibenclamide radioassay

HEK293T cells expressing SUR1 were washed twice in assaying buffer, before incubation for 50 min with [3H]-glibenclamide and a concentration series of either glimepiride or JB558. Incubation was terminated by rapid filtration through Whatman GF/C filters subsequent to washing. Radioactivity was counted 6 hr after cell and filter lysis in 200 mL Rotiszint EcoPlus using a scintillation counter.

3.10 Azo Reductase assays

AzoReductase (EC 1.7.1.6) catalyses the reductive cleavage of an azobenzene diazene bond into the corresponding anilines (Mueller and Miller, 1949). Escherichia coli (BL21 DE3) cells served as a model system as they are known to carry azoreductase (Figure 20) (Raabe 1993). Escherichia coli (BL21 DE3) cells were transformed with pcDNA3.1 (Amp resistance) and grown in LB media with ampicillin to an OD ₆ oo = 1.0. Methyl red (MR) or JB558 was added from a 50 mM stock solution in DMSO to a final concentration of 50 μΜ with or without bacteria. DMSO alone served as control. Solutions were inoculated at 37 °C and 180 rpm and aliquots (100 μΐ,) were taken every 3 min and transferred into Eppendorf tubes. Eppendorf tubes were centrifuged for 1 min (4 °C and 12,000 rpm) and then stored immediately on ice for the duration of the experiment. The supernatant of each Eppendorf tube was transferred into a well of a 96-well plate and absorbance was measured at the respective wavelength with a plate reader. After 27 min, MR was not detectable anymore, assuming azoreductase performed complete diazene cleavage.

3.11 Statistics

Multifactorial comparisons were made using one-way ANOVA followed by Bonferonni's post-hoc test. Dose-response curves were fitted using a sigmoid equation (SD attributable to the unexplained variance = 52 cpm). All analyses were conducted using Graphpad Prism (Graphpad Software) and results deemed significant at P<0.05.

Example 4 A red-shifted photochromic sulfonylurea - Results

4.1 Design and synthesis of JB558

Spurred on by recent studies of ortho- or ara-substituted azobenzenes (see e.g. Kienzler et al., 2013; Rullo et al., 2014), we devised a novel approach for the synthesis of wavelength- tuned photopharmaceuticals with red-shifted photochromism. An AzoSulfonylurea based on glimepiride was achieved by installing a heterocyclic aromatic unit, rather than sterically bulky electron-donating halogen or amine moieties (Figure 14).

As can be seen in Figure 14: Starting with the deacetylation of acetazolamide (1) in refluxing HQ, heterocycle 2 was obtained that could be further diazotized with in situ generated ENO2. Trapping the resulting diazonium salt with N, N-diefhylaniline generated sulfonamide azobenzene 3. Finally, reaction with cyclohexyl isocyanate yielded JB558 via acylation of the sulfonamide, giving unprecedented access to a sulfonylurea containing a heterocyclic azobenzene. While yields were reduced compared to JB253 (37% versus 97%), this was most likely due to the presence of a less reactive sulfonamide intermediate, as predicted by the lower pK _a value for 3 (7.36) and JB558 (2.35). JB558 possessed a red-shifted absorption spectra _max - 526 nm) in DM SO (Figure 15a), and could be repeatedly photoconverted to its cw- state with green-yellow light (λ = 520 nm) (Figure 15b). Theraial back relaxation occurred rapidly in the dark and switching kinetics were within the millisecond range (x _C j _S = 64.9 ± 1.5 ms; Tt _rans = 410.8 ± 12.6 ms), without obvious decomposition (Figure 15b). JB558 was stable in the presence of Escherichia coli azoreductase, an enzyme expected to limit oral bioavailability through diazene cleavage in the intestine (Figure 20).

4.2 JB558 binding studies

To determine the binding affinity of JB558 to the KAT _P channel subunit SUR1, as well as Epac2A, [3H]-glibenclamide displacement and FRET assays were performed. While trans- JB558 bound SUR1 with -10,000-fold less affinity than glimepiride (IC ₅₀ (irara-JB558) = 37.3 μΜ; IC ₅ o (Glim) = 1.8 nM) (Figure 16a), it was able to strongly and light-dependently activate an Epac2A-camps biosensor containing the sulfonylurea binding domain (Herbstet al., 201 1) (Figure 16b-d).

Electrophysiological recordings of K ⁺ currents in HEK293T-SUR1-Kir6.2 cells revealed partial KATP channel blockade by /nmv-JB558, presumably due to the momentary stationary state favouring some continued czs-isomerisation (Figure 21).

4.3 Photoswitching properties of JB558

We next assessed the photoswitching properties of JB558 in native beta cells where sulfonylurea-mediated KATP channel-Epac2A signalling is intimately linked to voltage- dependent Ca ²⁺ channel (VDCC) activity and insulin exocytosis (see e.g. Zhang et al, 2009; Rutter & Hodson, 2013).

As expected, JB558 was able to evoke large increases in intracellular Ca ²⁺ concentrations in -60% of beta cells following exposure to yellow (λ = 561 ± 5 nm)- (Figure 17a and b), but not violet (λ = 405 ± 5 nm)-light (Figure 17c). These effects were potentiated using a high concentration of glimepiride (Figure 17d), and abrogated using diazoxide (Figure 17e) to force open the K _A TP channel pore. Repeated switching of cytosolic Ca ²⁺ concentrations could be achieved in the same islet following a brief period of dark exposure to induce trans-]B55S accumulation (Figure ΙΊΐ). Similar to the results observed in rodent tissue, cis-JB588 was able to confer light-sensitivity on Ca ²⁺ -spiking activity in human pancreatic islets (Figure 18a-c), and this effect could be reversed following 5 min relaxation in the dark (Figure 18d).

To link photocontrol of Ca ²⁺ levels with insulin secretion, batches of rodent islets were incubated with JB558 and exposed to either dark (no illumination) or light (λ = 560 ± 10 nm). JB558-treated islets kept under dark conditions were no different to controls (5 mM glucose- alone) (Figure 19a), suggesting that the observed stationary state KATP channel block was insufficient to elicit exocytosis. By contrast, irradiation dramatically stimulated insulin release (Figure 19a). Finally, cytotoxicity assays demonstrated that JB558 did not adversely affect cell viability, as assessed using the vital stain calcein and the necrosis indicator propidium iodide (Figure 19b and c).

The data presented here outline a synthetic route for the production of Azo Sulfonylureas with red-shifted photochromism. Consistent with its sulfonylurea backbone, JB558 was able to bind SUR1 and activate Epac2A. Formation of cz ^' s- JB558 occurred with green-yellow light (λ = 520-561 nm), and thermal back relaxation in the dark yielded lrans-JB558. While photoconversion between cis- and trans- forms was rapid in solution, it was slower in the tissue setting, taking minutes for reisomerisation. An effect of Fluo-2 excitation on the isomer equilibrium cannot be excluded, although illumination at λ = 491 nm per se was unable to evoke Ca ^' rises in either Fluo-2- or Fura2 (λ = 340 nm/380 nm)-loaded islets (Figure 22).

A more plausible explanation is the mactivation of beta cell Epac2A-signalling, which may lag behind that of JB558 due to persistent mobilisation of intracellular Ca ²⁺ . Such tissue effects may be desirable for the development of photopharmaceuticals, since pulsed illumination would reduce phototoxicity, while sustaining compound activity to match long- lasting (dozens of minutes) insulin peaks 8Seino et al., 201 1). Indeed, JB558 displayed almost 3-fold more potency than its blue-light activated predecessor JB253, most likely due to slower back-relaxation during the light pulses used in the secretion assays.

Neither were we able to detect photoswitching of K ⁺ currents in HEK293T cells overexpressing KATP channels, free from orthogonal wavelengths (Figure 23). This was likely because HEK293T cells do not express sufficient Epac2A to allow JB558 to properly toggle Κ _Λ τρ activity (Zhang et al., 2009; Tsalkova et al., 2012), and/or the inability to deliver sufficient illumination using the non-coherent source on our patch-clamp setup (ε _{5 0} nm (JB558) = 1.14 x 10 ⁵ mof ¹ cm ^"1 ).

We clearly show that JB558 light-dependently binds Epac2A, allowing optical control of cell function and insulin secretion with λ = 560 nm in the most physiologically-relevant testbed, viz the islets of Langerhans.

Thus, JB558 represents a blueprint for red-shifted Azo Sulfonylureas based upon heterocyclic azobenzenes. Furthermore, JB558 is highly suitable as a research tool, such as for rapid K _A TP channel manipulation.

The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

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