VISIBLE LIGHT SENSITIVE PHOTOREMOVABLE PROTECTING GROUPS, PREPARATION PROCESS THEREOF, PHOTOACTIVATABLE CONJUGATES COMPRISING THEM AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to visible light sensitive photoremovable protecting groups and their parent compounds X including a xanthene, xanthenium or related cores, which compounds can be attached to any desired chemically or biologically active agent either via a covalent bond, an oxycarbonyl linker or a self-immolative linker. In the resulting conjugates the active agent is rendered inactive (or less active). Irradiation of such photosensitive conjugates with visible light leads to the release of the agent with restored biological activity.

BACKGROUND OF THE INVENTION

Photolabile or photoremovable protecting groups (PPGs) or photocages (PCs) are light-sensitive chemical moieties that are able to mask the chemical or biological function of substrates (active agents) through covalent linkages rendering them inert or less active ^1-3 . Upon irradiation with a suitable wavelength, the photoremovable protecting group is removed via bond cleavage (photolysis) and the activity of the active agent reinstates ^4-6 . Examples for the use of photocaged compounds include chemical synthesis ⁷ , caged nucleotides ⁸ , neurotransmitters ⁹ , proteins ⁵ , pharmaceuticals ¹⁰ , ions ¹¹ , many other small molecules ¹² and biomolecules ¹³ . Among other external stimuli, the spatial and temporal resolution of light is unsurpassed. Therefore, using photocaged compounds including transiently disabled biologically active agents, the manipulation of chemical/biological systems becomes possible with external light control ¹⁴ . The most popular photoremovable protecting groups used in biological studies are the o-nitrobenzyl, phenacyl, acridinyl, benzoinyl, coumarinyl groups ¹⁵ . Unfortunately, a significant limitation of these photocages is that they absorb mostly in the ultraviolet (UV) range, where the limited penetration of UV light into tissues largely restricts their use ¹⁶ . Furthermore, prolonged exposure of cells or tissues to intense UV light can lead to cellular damage or death. Advantages of visible light irradiation include diminished phototoxicity and deeper optical penetration ¹⁴ . Recent work featuring redshifted photoremovable protecting groups resulted in K-extended coumarin ¹⁷ - ¹⁸ , BODIPY ^19-21 and cyanine frames ^22-24 as well as metal complexes ²⁵ - ²⁶ . In the red region, modification of the BODIPY scaffold led to highly efficient visible to NIR light activatable cores ²¹ - ²⁷ , however, at the cost of poor water solubility and increased molecular weight. On the other hand, cyanines require the presence of molecular oxygen limiting their use in oxygen-defficient environments, such as solid tumors ²³ - ²⁸ . Following the success of photodynamic therapy (PDT), where light is combined with exogenously delivered sensitizers to trigger spatiotemporally controlled generation of reactive oxygen species, a novel concept, termed 'photoactivated chemotherapy' (PACT) has emerged ²⁹ . Compared to clinically approved PDT, PACT is based on the use of photoremovable protecting groups attached transiently to a cytotoxic chemotherapeutic agent or an inhibitor ³⁰ . Upon light irradiation, the activity of the pharmaceutically active agent reinstates due to the liberation of the free agent. Since PACT does not rely on molecular oxygen, it would overcome the current limitations of PDT. Especially in malignant solid tumors, where the low oxygen concentration precludes the application of oxygen-dependent photosensitizers ²⁹ . Most PACT conjugates, however, are based on UV-absorbing photoremovable protecting groups ¹⁰ that inherently limit their applications in deeper tissues ¹⁶ . Therefore, photoremovable protecting groups absorbing in the optical window of tissues (600-900 nm) that are also water soluble and release their cargo units oxygen-independently are urgently needed, especially for phototherapeutic applications such as PACT ¹⁴ .

An ideal photoremovable protecting group should meet several criteria ¹⁵ . Accordingly, the PPG and its target conjugate: 1) should possess acceptable water-solubility within a given concentration range; 2) the target conjugate should be stable in the dark in aqueous media to prevent premature and uncontrolled release of the active agent; 3) to be suitable for clinical use, target conjugates with the photoremovable protecting groups should feature strong one-photon absorption above 550 nm with high molar absorption coefficient and efficient uncaging cross-sections (i.e., the photolysis rate should be fast to have sufficient photochemical quantum yields); 4) the target conjugate should be cell permeable; 5) the photolysis byproducts should be biologically inert and 6) synthesis of the target conjugates should allow late stage attachment of the cargo moiety, that is often a sensitive or hardly accessible drug, therefore it is of utmost importance to minimize any loss of it.

Development of novel PPGs is often inspired by the vast knowledge gained on the field of fluorescent dyes. Accordingly, the collection of visible-light sensitive photocages includes a wide variety of coumarins, BODIPYs, porphyrines or cyanines. The currently available palette, however, lacks one of the most popular scaffolds used exhaustively in fluorescent imaging schemes, i.e., xanthenium dyes such as rhodamines. The state of the art neither offers synthetic access to such compounds nor discloses the key structural details necessitated to their stability.

US 9,957,498 discloses a set of photoremovable protecting groups based on a BODIPY chromophore developed by Winter et al., ²¹ for the release of organic molecules, however, these compounds have limited photolysis rates and water solubility.

US 2002/0016472 discloses protecting groups derived either from a halogenated coumarin or a xanthene group. The protecting groups can be removed by irradiating the group with light, by using e.g., flash photholysis with ultraviolet radiation or pulsed infrared radiation. We note that although xanthene-based protecting groups are envisaged in this document, the only compound synthesized is 9- chloromethylxanthene. The concept of photoremovable protecting groups was not demonstrated with this class of compound since no examples were given beside the sole chloromethyl derivative and the chloride ion can not be considered as leaving group in this respect. Importantly, the described synthetic route to access the scaffold proved to be unsuitable for 1) attachment of a cargo moiety other than a simple chloride 2) rhodol- and rhodamine-based derivatives.

The disclosed PPG-s are not suitable for one-photon absorption above 550 nm (with efficient uncaging cross sections), the longer wavelength (pulsed infrared) activation was based on two-photon absorption that is certainly a useful methodology for uncaging small quantities but unsuitable for phototherapeutic applications due to the ultra-small size of the irradiated focal point (i.e., picoliters in volume).

WO 2021/071876 discloses photolytic compounds and triplet-triplet annihilation mediated photolysis. In this document, the disadvantages of using short wavelength light for photolysis are detailed, and it is mentioned that attempts to use far red light and near infrared light have failed. As a response to these drawbacks, the above document discloses a method of conducting photolysis mediated by triplet-triplet annihilation, which can be used with a pair of a PPG and a photosensitizer, a concept significantly different from the present invention (i.e., multiple components, nanoparticle requirements, etc.). Among the PPGs, anthracene derivatives are mentioned. Said anthracene derivatives are not suitable for phototherapeutic applications by themselves (as PPG moieties in target conjugates) due to their UV- absorbtion (no absorption in the visible range) and poor water solubility. As the inventors in said document presented, their anthracene (and other) derivatives are in fact not uncaging by themselves using visible light irradiation (see Table 12 for an example) and in the biology experiments only the nanoparticles were utilized.

Klan et al. ² mentions, among others, protecting groups based on xanthene and pyronine moieties. It is stated that (6-hydroxy-3-oxo-3H-xanthen-9-yl)methyl derivatives release diethyl phosphate or carboxylic acid upon irradiation with visible light, however, in their original report, ³¹ they could only isolate a fluorescein derivative in the form of a DDQ (2,3-dichloro-5,6-dicyano-l,4-benzoquinone) complex. Although pyronine analogues are suggested to be used in the field of photoremovable protecting groups, in their later article, ³² they admitted that attempts to install a saturated oxymethyl linker onto the C9 position, as well as many alternative synthetic procedures failed, so they decided to employ the said BODIPY compounds as alternative chromophores. Importantly, although Klan et al. mentioned the possibility of xanthenium PPGs, their lack of success clearly indicates that accessing such compounds is not trivial and further modifications are required. Kitamura et al. ³³ describe a rodamine based nitric oxide donor. The disclosed molecules contain a rhodamine moiety, but they cannot be regarded as protecting groups (the document does not even mention photoremovable protecting groups) since the released molecule (nitric oxide) cannot be replaced with any desired active agent.

To conclude, there are some examples in state of the art suggesting the use of xanthene-based groups, however, none of them offers any feasible photoremovable protecting group with xanthete-derived frames, and rather reports unsuccessful or cumbersome synthesis to access the envisioned structures.

Therefore, our aim was to provide photoremovable protecting groups with xanthenium scaffolds, which meet the above-mentioned general criteria for PPGs.

SUMMARY OF THE INVENTION

We have found that compounds X (represented by formulas (Xa) or (Xd)) can be readily synthesized and isolated, and are suitable precursor compounds of photoremovable protecting groups. The photoactivatable conjugates that can be prepared from compound X have significant advantages over conjugates containing known photoremovable protecting groups. The advantages of the photoactivatable conjugates are attributed to the novel photoremovable protecting groups derived from compound X. These advantages are (1) high solubility in aqueous media (2) one-photon absorbance between 550-700 nm (3) high photolysis rates and consequently high one-photon uncaging cross sections (4) cell permeability (5) simple synthesis enabling late stage attachment of the active agent. As will become evident to those skilled in the art, utilizing the derivatives of compounds X as photoremovable protecting groups attached to various active agents are optimal tools for manipulating biological systems with light as well as treating localized diseases.

Therefore, the present invention relates to compounds represented by formula (Xa), wherein

A is either C ⁺ or CH, when A is C ⁺ , the compound comprises a counter anion, which is preferably a pharmaceutically acceptable anion; Ri and R2 are each selected independently from hydrogen, Ci-Cg alkyl, Ca-Cg cycloalkyl, C2-C6 alkenyl, Cg- C10 aryl; or

Ri and R2, together with the nitrogen atom, to which they are attached, form a 3-10-membered heterocycloalkyl ring containing said N atom, and optionally containing a further heteroatom selected from N, O and S;

Y is selected from NRgRg or OR10, wherein

Ra and Rg are each independently selected from hydrogen, Ci-Cg alkyl, Ca-Cg cycloalkyl, C2-Cg alkenyl and Cg-Cio aryl; or

Ra and Rg, together with the nitrogen atom, to which they are attached, form a 3-10-membered heterocycloalkyl ring containing said N atom, and optionally containing a further heteroatom selected from N, O and S;

Rio is selected from hydrogen and OH-protecting group;

Ra, R4, Rs, Rg are each selected independently from H, halogen, Ci-Cg alkyl, Ca-Cg cycloalkyl or Ca-Cg cycloalkenyl, or

Ra and R2, R4 and Ri, R ₅ and Rg, and/or Rg and Rg together with the intervening atoms, may form a 5-7- membered non aromatic, saturated or unsaturated heterocycle containing a N atom, and optionally containing a further heteroatom selected from N, O and S,

R ₇ is Ci-Cg alkyl, Ca-Cg alkenyl, Ca-Cg cycloalkyl, Cg-Cio aryl or (Cg-Cio aryl)(Ci-Cg alkyl);

Q. is selected from NH, NRn, O, C(RH)2, Si(Rn)2, P=O(Ph) or P=O(OPh) or P=O(O ), wherein each R11 is independently selected from Ci-Cg alkyl, Ca-Cg cycloalkyl, C2-Cg alkenyl, Cg-Cio aryl and (Cg-Cio aryl)(Ci-C ₆ alkyl);

W is either

-OH,

- halogen, or a

- linker precursor moiety, wherein in the meaning of Ri to Rn each instance of alkyl and alkenyl is optionally substituted with one or more substituent(s) independently selected from the group of halogen, hydroxyl and CFa, in the meaning of Ri to Rn each instance of cycloalkyl is optionally substituted with one or more substituent(s) independently selected from the group of C1-C4 alkyl, halogen, hydroxyl and CFa, the 3-10-membered heterocycloalkyl formed by NRiRa or NRaRg is optionally substituted with 1-3 substituent(s) independently selected from the group of C1-C4 alkyl, halogen, hydroxyl and CFa, the 5-6-membered non aromatic, saturated or unsaturated heterocycle formed by Ra and R2, R4 and Ri, R ₅ and Rg, and/or Rg and Rg together with the intervening atoms is optionally substituted with one or more substituent(s) independently selected from the group of C1-C4 alkyl, halogen, hydroxyl and CF3, in the meaning of Ri, R2, R7-R9 and Rn each instance of aryl is optionally substituted with one or more substituent(s) independently selected from the group of C1-C4 alkyl, halogen and CF3, in any protonation states thereof, or salts thereof.

More specifically, in case A represents C ⁺ and Y represents OH, the deprotonated forms of the compounds are also included herein. Accordingly, the invention also relates to a compound represented by formula

(Xd) wherein Q, W and Ri to R ₇ are as defined above.

The invention also relates to a process for the preparation of a compound of formula (Xa), including: a) reacting a compound of formula (I): with a Grignard reagent of formula Ry-CHzMgHal, wherein Hal is halogen, or with a compound of formula R CHzLi, and converting the resulting adduct to an olefin derivative using first a proton source and then subsequent deprotonation using a strong base, to obtain a compound of formula subjecting the compound of formula (II) to hydroboration-oxidation reaction for the anti-Markovnikov addition of a hydroxyl group and a hydrogen to the olefin bond (e.g. using a borane reagent then hydrogen peroxide) to obtain a compound of formula (Xa) wherein A = CH and W = OH: or b) reacting a compound of formula (I): with a deprotonated vinyl ether derivative, e.g. which is performed by treatment of a vinyl ether of formula RuCH-OAIk with a strong base, wherein R14 represents e.g. CH2= or CH3-(CH2)n-CH= wherein n is an integer from 0 to 5, wherein said R14 can be considered as a precursor group of R ₇ and Aik refers to an alkyl group, and hydrolysing the resulting vinyl ether appended compound to obtain a compound of formula (III): reducing the compound of formula (III), to obtain a compound of formula (Xa) wherein A = CH and

W = OH: or c) reacting a compound of formula (I): with a Grignard reagent of formula FG-CHzMgHal, wherein Hal is halogen, or with a compound of formula R7CH2IJ, and converting the resulting adduct to an olefin derivative using first a proton source and then subsequent deprotonation using a strong base, to obtain a compound of formula reacting the compound of formula (II) with /V-halogenosuccinimide in the presence of water for the addition of HO-Hal to the olefin bond, then eliminating a hydroxide ion, to obtain a compound of formula (Xa) wherein A = C ⁺ and the compound comprises a counter anion, designated as "Anion" ", and W = halogen: and optionally d) converting the compounds of formula (Xa) wherein W = OH to compounds of formula (Xa) wherein W = oxycarbonyl linker precursor moiety using carbonic acid-derived activation reagents (such as DSC or 4-nitrophenyl chloroformate) and optionally e) converting the compounds of formula (Xa) wherein W = oxycarbonyl linker precursor moiety to compounds of formula (Xa) wherein W = self-immolative linker precursor moiety by reaction with a self-immolative linker precursor compound (e.g. with either /V-Boc-/V,/V'-dimethylethylenediamine in the presence of a base, or 4-hydroxymethylaniline in the presence of a base and 1-hydroxybenzotriazole), and optionally f) oxidizing the compounds of formula (Xa) wherein A = CH using chloranil and subsequent anion replacement to compounds of formula (Xa) wherein A = C ⁺ .

The invention also relates to the use of the compound represented by formula (Xa) or (Xd) as a precursor for a photoremovable protecting group.

Furthermore, the invention relates to photoactivatable conjugates comprising a photoremovable protecting group derived from a compound represented by formula (Xa) or (Xd), or salt thereof.

The photoremovable protecting group derived from a compound represented by formula (Xa) or (Xd) is represented by formulas (Xa ¹ ) or (Xd ¹ ): wherein A is C ⁺ and the compound comprises a counter anion,

Y, Q, and Ri to R ₇ are as defined above, and the wavy line represents the attachment point to the rest of the molecule.

The invention also relates to a pharmaceutical composition comprising the photoactivatable conjugate or a pharmaceutically acceptable salt thereof according to the invention, together with one or more pharmaceutically acceptable excipient.

The invention also relates to the photoactivatable conjugate or a pharmaceutically acceptable salt thereof for use in photoactivated therapy.

The invention furthermore relates to a method for photoreleasing an active agent Z from a photoactivatable conjugate of a formula X'-L-Z', comprising irradiating a biological sample selected from cell cultures or xenograft tumor models containing X'-L-Z' with a wavelength >550 nm so as to release the active agent Z, wherein X' is a photoremovable protecting group derived from a compound represented by formula (Xa) or (Xd), L is a covalent bond or a linker, and Z' is a moiety derived from an active agent.

More particular embodiments of the invention are set out in the dependent claims and in the detailed description. DEFINITIONS USED IN THE DESCRIPTION

In the description, the following definitions are used: chemical moiety: a part of a molecule with a distinct functionality, such as a chromophore, a protecting group, a self-immolative linker precursor group or a photoremovable protecting group. A chemical moiety is usually derived from a distinct chemical entity or a precursor molecule. chemical derivatization: when a specific functional group in a given molecule is converted to alter the function of the given molecule, it can be considered as chemical derivatization. For example, when two distinct molecules (A and B) are connected via a chemical linkage, the resulting conjugate (A-B) can be considered as a distinct molecule with two chemical moieties, a moiety (A') that is the result of chemical derivatization of A by B and a moiety (B') that is the result of chemical derivatization of B by A. active agent (Z): a chemically or biologically active small molecule or biomolecule that can be rendered inert (or less active) by disabling its key functional group by covalent modification. Such molecule could be, but not restricted to for example, an oligopeptide, protein, enzyme, nucleotide, nucleic acid, carbohydrate, lipid, neurotransmitter, catalyst, fragrance, pharmaceutically active ingredient or drug, anticancer agent, chemotherapeutic agent, small molecule inhibitor, fluorescent dye or fluorogenic dye. When the active agent is attached to an other molecule, such as compound X (via chemical derivatization), it is considered as the cargo unit (Z') of the resulting conjugate (also known in the literature as payload). In other words, the chemical moiety derived from the active agent is the cargo unit. compound X: these molecules are the necessary precursors for attachment of the photoremovable protecting group on a desired active agent. They are represented by formulas (Xa) and (Xd). Compound X therefore can be attached or coupled to any desired active agent resulting in the target conjugates. When compound X is attached to the active agent, it is no longer a distinct molecule but a chemical moiety that is considered as the photoremovable protecting group or photocage (X'). Therefore, in the description, X is also referred to as the parent compound of the X ¹ photoremovable protecting group. The X ¹ photoremovable protecting groups are represented by formulas (Xa ¹ ) and (Xd ¹ ). target conjugate or photoactivatable conjugate: attachment of compound X to an active agent results in the target conjugate, which is a distinct molecule. These molecules contain three chemical moieties that are the derivatives of the active agent Z (Z') and compound X (X') and a linker (L) between the chemical moieties. The target conjugates therefore consist of a photoremovable protecting group X', the cargo unit Z' and a linker. These are the actual photoactivatable conjugates. connecting chemical moiety or linker (L): the chemical moiety or covalent bond between the cargo unit and the photoremovable protecting group. oxycarbonyl linker: refers to an -O-(C=O)- or -O-(C=S)- moiety that is often required for the attachment of the cargo unit to a photoremovable protecting group. oxycarbonyl linker precursor moiety: a carbonic acid-derived group that readily reacts with -OH and -NH2 groups on the active agent, and forms an oxycarbonyl linker between the resulting photoremovable protecting group and the cargo unit. self-immolative linker: refers to a covalent chemical assembly, commonly used for therapeutic or bioanalytical aims, wherein a primary reaction initiates internal molecular rearrangements leading to the release of a substrate (e.g., a drug or a probe, in this case, the active agent). ³⁴ self-immolative linker precursor moiety: a chemical group that can be converted to a self- immolative linker by the attachment of the cargo unit.

ABBREVIATIONS

Bn benzyl

Boc tert-butoxycarbonyl

DCC /V,/V'-dicyclohexylcarbodiimide

DCM dichloromethane

DMAP 4-dimethylaminopyridine

DMF /V,/V-dimethylformamide

DMSO dimethyl sulfoxide

DSC /V,/V'-disuccinimidyl carbonate

EC50 half maximal effective concentration equiv. equivalent

Et ethyl-

EtOH ethanol

EtOAc ethyl-acetate

FBS Fetal Bovine Serum

GSH glutathione

HEPES /V-(2-hydroxyethyl)piperazine-/V'-(2-ethanesulfonic acid)

HOBt 1-hydroxybenzotriazole

HPLC high-performance liquid chromatography HRMS high-resolution mass spectrometry

KOtBu potassium tert-butoxide

IC50 half maximal inhibitory concentration

LC liquid chromatography

LED light-emitting diode

M mol/dm ³

Me methyl-

MeCN acetonitrile

MeOH methanol

MOM methoxymethyl- (protecting group)

MS mass spectrometry

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NBS /V-bromosuccinimide

NMR nuclear magnetic resonance on overnight

PBS phosphate-buffered saline

Ph phenyl-

Py pyridine

RT room temperature

TEA triethylamine

THF tetrahydrofuran

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a scheme of the concept of photoremovable protecting groups. First, compound X, the precursor of the photoremovable protecting group is attached to the active agent (i.e., a chemotherapeutic drug) via the connecting chemical moiety, resulting in the photoactivatable target conjugate after the appropriate chemical steps. The active agent is disabled in the conjugate form due to the chemical modification of a key functional group necessary for its biological activity. Upon irradiation with visible light, the active agent is released and its activity reinstates.

Figure 2a shows the general structure and examples of compounds X described herein (*) with additional analogous exemplary structures wherein A = CH and W = OH

Figure 2b shows examples of compounds X described herein (*) with additional exemplary structures wherein A = CH and W = OH or halogen precursor moiety Figure 2c shows examples of compounds X described herein (*) with additional exemplary structures wherein W = oxycarbonyl linker precursor moiety or self-immolative linker precursor moiety

Figures 3a, 3b and 3c show examples of target conjugates that represent the variability of the photoremovable protecting groups, connecting chemical moieties and cargo units that can be prepared from compounds X.

Figure 4 shows the structure of two photoactivatable conjugates X1'-SN38 and X2'-SN38 consisting of a photoremovable protecting group, a self-immolative linker and a cargo unit derived from a pharmaceutical agent, namely the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin, also known as SN38. Also shown is the mechanism of release and the byproducts of photolysis. The photoactivatable conjugate has low potency towards the target of the drug, topoisomerase I. Upon irradiation with red light using an LED light source, the active agent is released via light-induced bond cleavage and subsequent self-immolation reaction. The high potency of the active agent SN38 and its activity reinstates resulting in cell death.

Figure 5 shows the structure and absorption spectra of selected target conjugates.

Figure 6a shows uncaging experiments of compound 39 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using an orange LED light source (~605 nm). The release of phenylacetic acid (C) was monitored at 195 nm, the disappearance of the starting material (A) was monitored at 570 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions.

Figure 6b shows uncaging experiments of compound 41 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using a green LED light source (~550 nm). The release of phenylacetic acid (C) was monitored at 195 nm, the disappearance of the starting material (A) was monitored at 530 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions.

Figure 6c shows uncaging experiments of compound 43 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using a red LED light source ("'658 nm). The release of phenylacetic acid (C) was monitored at 195 nm, the disappearance of the starting material (A) was monitored at 640 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions.

Figure 6d shows uncaging experiments of compound 45 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using a red LED light source ("'658 nm). The release of phenylacetic acid (C) was monitored at 195 nm, the disappearance of the starting material (A) was monitored at 660 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions.

Figure 6e shows uncaging experiments of compound 49 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using a red LED light source ("'658 nm). The release of /V-phenylpiperazine (C) was monitored at 254 nm, the disappearance of the starting material (A) was monitored at 640 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions.

Figure 6f shows uncaging experiments of compound 51 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using a red LED light source ("'658 nm, 50% power). The release of phenol (C) was monitored at 195 nm, the disappearance of the starting material (A) was monitored at 640 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions. Note that in this case, the dark stability of the target conjugate is reduced.

Figure 6g shows uncaging experiments of compound X1'-SN38 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using an orange LED light source ("'605 nm). The release of the SN38-adduct (C) and SN38 (D) was monitored at 369 nm, the disappearance of the starting material (A) was monitored at 570 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions.

Figure 6h shows uncaging experiments of compound X2'-SN38 as followed by an HPLC-UV-MS instrument. Top: structure of the target conjugate and the scheme of the uncaging process. Left: partial chromatograms of the irradiated samples. A sample of 1 ml of the target conjugate with a concentration of 0.1 mM in water-MeCN 9:1 was irradiated for various time periods using a red LED light source ("'658 nm). The release of the SN38-adduct (C) and SN38 (D) was monitored at 369 nm, the disappearance of the starting material (A) was monitored at 640 nm. The photoproduct of the photoremovable protecting group (B) is also apparent. Right: partial chromatograms of the non-irradiated dark samples under similar experimental conditions.

Figure 7a shows the structure of the target conjugate X2'-SN38 (top) and confocal microscopy images showing the localization of the particular target conjugate in SK-OV-3 cells. The cells were incubated with 1 pM compound together with 50 nM MitoTracker Red or LysoTracker Red. The separate channels are shown as well as a trace that provides information on colocalization. From these images, it is evident that compound X2'-SN38 localizes in the lysosomes.

Figure 7b shows structure of the target conjugate X2'-SN38 (top) and confocal microscopy images showing the high targeting ability of light. The area marked with a square was irradiated using a 638 nm laser light source. The decrease of fluorescent signal solely at the area of irradiation demonstrates the high spatiotemporal control achievable by light. In each case, SK-OV-3 cells were treated with 1 pM of the compound.

Figure 8a shows the structure of the target conjugate X2'-SN38 (top) and cell viabilities from MTT assay. Cells were treated with different concentrations of the particular target conjugate and illuminated for various time periods. The viabilities are relative to vehicle (DMSO) treatment.

Figure 8b shows the structure of the target conjugate X1'-SN38 (top) and concentration-dependent viabilities for the EC50 value determination of the particular target conjugate. Cells were irradiated for 5 min by orange light (605 nm). The viabilities are relative to vehicle (DMSO) treatment.

Figure 8c shows the structure of the target conjugate X2'-SN38 (top) and concentration-dependent viabilities for the EC50 value determination of the particular target conjugate. Cells were irradiated for 60 s by red light (658 nm). The viabilities are relative to vehicle (DMSO) treatment. DETAILED DESCRIPTION OF THE INVENTION

1. Structure of the compounds X

The present invention relates to xanthene or xanthenium-derived compounds (abbreviated as compounds X) that can be attached to any desired active agent resulting in target conjugates or photoactivatable conjugates. Upon attachment of the active agent, the chemical moiety derived from compound X is called a photoremovable protecting group (abbreviated as moiety X') and the chemical moiety derived from the active agent becomes the cargo unit (abbreviated as moiety Z'). In other words, the target conjugates are composed of the photoremovable protecting group (X') the cargo unit (Z') and a connecting chemical moiety or linker (L) necessary to form the specific chemical connection between compound X and the active agent, i.e., the target conjugates can be described as X'-L-Z'. Importantly, when the active agent is in its photoactivatable (or photocaged) form, its chemical and biological activity is disabled or reduced. These target conjugates absorb in the upper visible range (>550 nm) and release the active agent upon irradiation.

1.1 General structure

The compound X is a substituted xanthenium or xanthene-based (or related) compound, represented by the following formula:

In formula (Xa), A is either C ⁺ or CH, and when A is C ⁺ , the compound comprises a counter anion.

Importantly, the structures wherein A = C ⁺ and CH can be interconverted via oxidation or reduction using standard methods known for those skilled in the art:

In those embodiments, wherein A = C ⁺ , the formulas can also be represented as other resonance structures that are equivalents of the original formula (i.e. the positive charge can be located on the N atom or on Y):

Resonance, also called mesomerism, is a way of describing bonding in certain molecules or polyatomic ions by the combination of several contributing formulas also known as resonance structures. Since in some embodiments of compound X (and its derivatives), the structure cannot be described as a simple Lewis structure due to the delocalization of the electrons, the resonance structures should also be considered. Although in formula (Xa) a single resonance structure is depicted, it is understood that several resonance structures are possible, as shown above, and in fact, the electron system is a combination of these resonance structures.

The distinction of embodiments wherein A = CH (xanthene-derivatives) and A = C ⁺ (xanthenium- derivatives) is mainly due to the synthetic access to the photoactivatable conjugates. The synthesis of compounds X requires an intermediate step wherein A = CH, however, such xanthene compounds do not absorb visible light due to the lack of the chromophore moiety. Compounds X wherein A = CH can be either oxidized to compounds X wherein A = C ⁺ (xanthenium), forming the visible light absorbing choromophore before attachment to the active agent, or, preferably, can be attached as such to the active agent (or self-immolative linker precursors) forming xanthene-derivatives. In this case the conjugates themselves can be converted to the visible light absorbing xanthenium derivatives, i.e. to the photoactivatable form, via oxidation. In some embodiments of compounds X, wherein A = C ⁺ and W = OH it was observed that the stability was insufficient for the formation of the photoactivatable conjugates, therefore compounds X wherein A = CH and W = OH were used for the attachment of the active agent, and the resulting conjugate was oxidized thereafter to the photoactivatable form.

Furthermore, the invention encompasses all protonation states of the compounds X, for example, their deprotonated forms.

More specifically, in those embodiments, wherein A = C ⁺ and Y = OH, the deprotonated forms of the original formula, represented by formula (Xd), should be also considered as embodiments of this invention:

The protonation-deprotonation naturally occurs in aqueous media, and leads to an equilibrium (depending on, among other things, the pH of the environment) between the protonated and deprotonated forms. In case of deprotonation, the counter ion (originally neutralizing the positive charge in X) can be regarded as the counter ion of the leaving proton (i.e. oxonium ion in aqueous media), it is no longer part of compound represented by formula (Xa). The compounds can be isolated from a solution, e.g. from a reaction mixture in either the salt form or in deprotonated form, depending on various factors, especially the pH.

As formulas (Xa) and (Xd) are closely related, in the description the compunds represented by them are commonly referred to as compounds X.

In connection with the above formula, the term "alkyl" means a straight or branched-chain alkyl group containing from 1 to 6, preferably 1 to 4 carbon atom(s), such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl and pentyl, etc. The alkyl group may be substituted by one or more, preferably by 1 to 3 substituent(s) independently selected from the group of halogen, hydroxyl and CF3.

The term "alkenyl " means a straight or branched-chain hydrocarbon group containing one or more double bond, and from 2 to 6, preferably 2 to 4 carbon atom(s), such as, vinyl, allyl, 1- butenyl, 2-butenyl, 3-butenyl, 1-pentenyl etc. The alkenyl group may be substituted by one or more, preferably by 1 to 3 substituent(s) independently selected from the group of halogen, hydroxyl and CF3. Preferably, the alkenyl group is attached via a saturated carbon atom to the rest of the molecule, i.e. it may be for example a 2-allyl, 2-butenyl, 3-butenyl, 4-pentenyl etc.

The term "cycloalkyl" means a monocyclic, saturated hydrocarbon group. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The cycloalkyl group may be substituted by one or more, preferably by 1 to 3 substituent(s) independently selected from the group of C1-C4 alkyl, halogen, hydroxyl and CF3. The term "heterocycloalkyl" means a saturated monocyclic group having at least one heteroatom selected from O, N or S, preferably from O and N. Examples of heterocycloalkyl groups include (but are not limited to): azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, acezanyl group. The heterocycloalkyl groups comprise 3 to 10, preferably 4 to 6 ring members, and may be substituted by one or more, preferably by 1 to 3 substituent(s) independently selected from the group of C1-C4 alkyl, halogen, hydroxyl and CF3.

The term "non aromatic, saturated or unsaturated heterocycle" encompasses the above mentioned heterocycloalkyl groups, as well as unsaturated but non aromaric rings, for example a dihydropyridine ring. The non aromatic, saturated or unsaturated heterocycle comprises a N atom, and optionally contains a further heteroatom selected from N, O and S, and preferably comprises 5 to 7 ring members. In a preferred emodiment, the heterocycle comprises only one N as heteroatom. The heterocycle may be substituted by one or more, preferably by 1 to 5 substituent(s) independently selected from the group of C1-C4 alkyl, halogen, hydroxyl and CF3.

When the heterocycle is formed by any one of the substituent pairs R3 and R2, R4 and Ri, R ₅ and Rs, and/or Re and Rg, said heterocycle is condensed with the xanthene (or xanthenium) skeleton, and in case two adjacent rings are formed, with each other.

The term "aryl" refers to six to ten-membered aromatic monocyclic or bicyclic hydrocarbon rings, which may be attached via one of the ring carbon atoms. The aryl group may be substituted by one or more, preferably by 1 to 3 substituent(s) independently selected from the group of C1-C4 alkyl, halogen and CF3. Specific examples include, but are not limited to, phenyl, tolyl, xylyl, trimethylphenyl, and naphthyl.

In connection with the above definitions, a preferred halogen substituent is fluorine.

Reference to a compound X herein is understood to include reference to salts thereof, unless otherwise indicated. The term "salt(s)", as employed herein, denotes salts formed with inorganic and/or organic acids. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Said pharmaceutically acceptable salts may be prepared from inorganic and/or organic acids. For example, inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids include acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like. A preferred salt is an iodide or trifluoroacetate salt.

The term "counter anion" refers to an inorganic or organic anion, which balances the charge of the cationic compound. Pharmaceutically acceptable anions are preferred. Examples of such anions include the following: Cl-, Br, r, AcO-, CFsCOO-, BFT, PFg-, sulfate, phosphate, methanesulfonate, formate, citrate, fumarate, malate, tartate, succinate, and salicylate. A preferred anion is T.

As used herein, the term "OH-protecting group" or "hydroxyl protecting group" refers to readily cleavable groups bonded to hydroxyl groups. The hydoxyl protecting group can be an acid-labile protecting group, a base-labile protecting group, or a protecting group that is removable under neutral conditions. The nature of the hydroxyl protecting groups is not critical so long as the derivatized hydroxyl group is stable. Examples of OH-protecting groups include Ci-Cg alkoxymethyl, Ci-Cg alkyl, Ca-Cg cycloalkyl, C ₇ -Cg alkenyl, aryl, arylmethyl, acetyl, benzoyl, tetrahydropyranyl, tetrahydrofuranyl and tetrahydrothiofuranyl. Further examples of a hydroxyl protecting group are silyl groups, which can be substituted with alkyl (trialkylsilyl), with an aryl (triarylsilyl) or a combination thereof (e.g., dialkylphenylsilyl). Specific examples include, but are not limited to, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), t- butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS).

Other suitable protecting groups for OH are well known to those of skill in the art (see Wuts, PGM and Greene, TW (2006) "Greene's Protective Groups in Organic Synthesis", 4th Edition, John Wiley & Sons, Inc., Hoboken, NJ, USA).

Preferred hydroxyl protecting groups are Ci-Cg alkoxymethyl, Ci-Cg alkyl, Ca-Cg cycloalkyl, C ₇ -Cg alkenyl, aryl, arylmethyl, acetyl, benzoyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiofuranyl, and Ci-Cg alkyl or aryl-substituted silyl groups. More preferred hydroxyl protecting groups are Ci-Cg alkoxymethyl, Ci-Cg alkyl, and Ci-Cg alkyl- or aryl-substituted silyl groups.

In formula (Xa) or (Xd), the substituents are as defined above under the "Summary of the invention".

In formula (Xa) (and, if applicable, in formula (Xd)), the preferred substituents are as follows.

Q. is preferably selected from O and C(Rn)2, wherein Rn is selected from Ci-Cg alkyl groups. More preferably Q. is C(Rn)2 wherein Rn is Ci-Cg alkyl, for example methyl or ethyl.

R ₇ is preferably hydrogen, Ci-Cg alkyl, C2-Cg alkenyl, Ca-Cg cycloalkyl or (Cg-Cio aryl)(Ci-Cg alkyl).

In one embodiment, R ₇ is Ci-Cg alkyl, C ₇ -Cg alkenyl or Ca-Cg cycloalkyl.

In another embodiment, R ₇ is Ci-Cg alkyl, Ca-Cg cycloalkyl or (Cg-Cio aryl)(Ci-Cg alkyl).

In a further embodiment, R ₇ is Ci-Cg alkyl or Ca-Cg cycloalkyl.

In yet a further embodiment, R ₇ is Ci-Cg alkyl or C ₇ -Cg alkenyl.

In more preferred embodiments, R ₇ is Ci-Cg alkyl.

Preferably, Ri and R2 are each selected independently from hydrogen and Ci-Cg alkyl; or

Ri and R2, together with the nitrogen atom, to which they are attached, form a 4-6-membered heterocycloalkyl ring containing said N atom, and optionally containing a further heteroatom selected from N and O. In one embodiment, Y is NRgRg, wherein preferably Rg and Rg are each independently selected from hydrogen and Ci-Cg alkyl; or

Rg and Rg, together with the nitrogen atom, to which they are attached, form a 4-6-membered heterocycloalkyl ring containing said N atom, and optionally containing a further heteroatom selected from N and O.

In another embodiment, Y is ORio, wherein Rio is selected from hydrogen and OH-protecting group.

Preferably, R ₃ , R4, Rs, Rg are each selected independently from H and Ci-Cg alkyl.

The substituents Ra and R2, R4 and Ri, and in case Y is NRgRg, Rs and Rg, and/or Rg and Rg together with the intervening atoms, may form a 5 to 7, preferably 6-membered non aromatic, saturated or unsaturated heterocycle containing a N atom and optionally containing a further heteroatom preferably selected from N and O, said heterocycle being optionally substituted with 1-6 C1-C4 alkyl. Most preferred heterocycles are piperidine and dihydropyridine rings that are optionally substituted.

Preferably, Rio is selected from hydrogen, Ci-Cg alkoxymethyl, Ci-Cg alkyl, C ₃ -Cg cycloalkyl, C ₃ -Cg alkenyl, aryl, arylmethyl, acetyl, benzoyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiofuranyl, and Ci-Cg alkyl or aryl-substituted silyl groups. More preferably, Rio is selected from hydrogen, Ci-Cg alkoxymethyl, Ci-Cg alkyl, and Ci-Cg alkyl- or aryl-substituted silyl groups.

In one embodiment, W is selected from

-OH, and

- halogen, preferably bromine.

In another embodiment, W is selected from

- an oxycarbonyl linker precursor moiety, preferably selected from:

- or a self-immolative linker precursor moiety, preferably selected from: wherein R12 and R13 are independently selected from hydrogen, Ci-Cg alkyl, C ₃ -Cg cycloalkyl, C2-Cg alkenyl; or R _i2 or R _i3 , together with the nitrogen atom to which they are attached and with one or both of the carbon atoms of the ethylene moiety, form a 4-6 membered heterocycloalkyl containing said N atom, and optionally containing a further heteroatom selected from N, O and S, and the wavy line

) represents the attachment point to the rest of the molecule. More preferably, R _i2 and R _i3 are independently selected from hydrogen, Ci-Cg alkyl groups; or R12 or R13, together with the nitrogen atom to which they are attached and with one or both of the carbon atoms of the ethylene moiety, form a 5- membered heterocycloalkyl containing said N atom.

We note that compounds represented by formula (Xa) or (Xd) wherein W represents a linker precursor group can be conveniently prepared from compounds represented by formula (Xa) or (Xd) wherein W represents an OH group or halogen.

Preferably, when A is C ⁺ , the counter anion is selected from Cl', Br, T, HCOO-, AcO-, CFaCOO-, BFT, PFg", methanesulfonate, sulfate, phosphate or citrate.

In the following, some preferred sub-structures of the compounds X are described. These are also subject matters of the invention.

1.2 Rhodamine-like structures (Y = NR ₈ R ₉ ) wherein A, Q, W, and Ri to Rg are as defined above.

1.3 Rhodol-like structures (Y = ORio) wherein A, Q, W, and Ri to R ₇ and Rio are as defined above.

Preferred meanings of Rio include hydrogen, Ci-Cg alkoxyalkyl or Ci-Cg alkyl, more preferably alkoxymethyl. In those embodiments, wherein A=C ⁺ and Rio= hydrogen, the deprotonated forms of the original formula are also included herein and should be also considered as embodiments of this invention.

1.4 O-rhodamine-like structures (Y = NR ₈ Rg, Q = 0) wherein A, W, and Ri to Rg are as defined above.

1.5 0-rhodol-like structures (Y = ORio, Q = 0) wherein A, W and Ri to R? and Rio are as defined above.

Preferred meanings of Rio include hydrogen, Ci-Cg alkoxyalkyl or Ci-Cg alkyl, more preferably alkoxymethyl.

In those embodiments, wherein A=C ⁺ and Rio= hydrogen, the deprotonated forms of the original formula are also included herein and should be also considered as embodiments of this invention.

1.6 Carborhodamine-like structures (Y = NR ₈ R ₉ , Q = CR2) wherein A, W and Ri to Rg and Rn are as defined above.

Preferred meanings of Rn include Ci-Cg alkyl, more preferably methyl. 1.7 Specific structures for compounds represented by formula (Xa) (A = CH, W = OH) 1.8 Specific structures for compounds represented by formula (Xa) (A = C+, W = OH)

1.9 Specific structures for compounds represented by formula (Xa) (A = CH, W = linker precursor moiety) 1.10 Specific examples for target conjugates: X'-SN38

2. Connecting chemical moieties between the photoremovable protecting groups and cargo units (moiety L from X'-L-Z')

The connecting chemical moiety or linker (L) that connects the cargo unit and the photoremovable protecting group is largely dependent on the selected functional group on the active agent that is to be blocked via the attachment of compound X. Usually, the functional group (i.e., NH2, COOH, OH, etc.) on the active agent that is selected to be caged by photoremovable protecting group X' is an essential element that interacts with the target of the agent (i.e., an H-donor group in the binding pocket of the target protein of a drug). In the present invention, the variability of functional groups that can be blocked by the attachment of compound X is demonstrated by exemplary target conjugates featuring different linkers and cargo units (in other words, caged functional groups). The formation of the target conjugates from compound X is also disclosed herein.

In the target conjugates, the photoremovable protecting group X' derived from compound X has the one of the following formulas: wherein the wavy line ( represents the attachment point to L,

A = C ⁺

Q, Y and Ri to R ₇ are as defined above.

We note that in the target conjugates, the photoremovable protecting group is also a chromophore (having absorption bands in the visible range), therefore in the above formula (Xa ¹ ) A = C ⁺ , meaning that (Xa') derived from compound represented by formula (Xa) is in its xanthenium form or oxidized state. As formula (Xd) is the deprotonated form of the oxidized form of compounds represented by formula (Xa) wherein Y=OH, the group derived therefrom, represented by formula (Xd ¹ ), is also in the oxidized state, i.e. in the photoactivatable form.

2.1 Covalent bond

In one embodiment, the connecting chemical moiety (L) is a covalent bond that connects the photoremovable protecting group (X') and the cargo unit (Z') by the replacement of moiety W in compound X with the cargo unit. Such target conjugates can be prepared via direct or indirect chemical methods of connecting a compound X wherein W is OH or halogen, with the active agent, followed by an oxidation step in case of A = CH.

Upon absorbing visible light, this connecting covalent bond is cleaved. Upon cleavage, in this embodiment the active agent is released directly.

In this embodiment, the functional groups on the active agent that may be connected to compound X via a covalent bond include, but are not restricted to hydroxyl groups (ethers in the target conjugates), primary, secondary and tertiary amino groups (secondary, tertiary and quaternary amines or ammoniums in the target conjugates), thiol groups (thioethers in the target conjugates), carboxyl or carboxylate groups (esters in the target conjugates), pyridyl groups (pyridiniums in the target conjugates), phosphate groups (phosphate esters in the target conjugates).

The replacement of moiety W in the general formula of compound X with the cargo unit and its subsequent photolysis can be better understood from the following scheme, wherein the wavy line

) represents the attachment point of the cargo unit to the photoremovable protecting group:

TARGET CONJUGATE active agents cargo unit with the defined functional group: W is replaced with: released active agent: or their deportonated forms

The following examples illustrate the formation of target conjugates (using model compounds with different functional groups) and their subsequent photolysis wherein the connecting chemical moiety is a chemical bond:

2.2 Oxycarbonyl linker

In another embodiment, the connecting chemical moiety between the photoremovable protecting group (X') and the cargo unit (Z') in the target conjugates can be an oxycarbonyl linker, such as -O-(C=O)- or -O- (C=S)-, wherein the oxygen is connected to X' and the carbonyl/thiocarbonyl carbon atom is connected to either an oxygen or a nitrogen atom of Z'. If the connecting atom on Z' = oxygen (originating from a hydroxyl functional group of Z), the resulting chemical moiety is called a carbonate with the formula -O- (C=O)-O-, or thiocarbonate with the formula -O-(C=S)-O-. If the connecting atom on Z' = nitrogen (originating from an amino functional group of Z), the resulting chemical moiety is called a carbamate with the formula -O-(C=O)-N-, or thiocarbamate with the formula -O-(C=S)-N-.

These compounds can be prepared by general chemical methods capable of connecting compound X (wherein W = OH) with the active agent through such moieties. These methods include the following steps.

First, the activation of compound X wherein W = OH with phosgene, thiophosgene, triphosgene, N,N'- carbonyldiimidazole, /V,/V'-dissucinimidyl carbonate, 4-nitrophenyl chloroformate, etc. This results in compounds wherein moiety W is an oxycarbonyl linker precursor.

The second step is the reaction of the resulting activated species with the active agent or its protected form. This is followed by an oxidation step in case of A = CH in the first step.

Alternatively, the active agent can also be activated with phosgene, thiophosgene, triphosgene, N,N'- carbonyldiimidazole, /V,/V'-dissucinimidyl carbonate, 4-nitrophenyl chloroformate, etc. The resulting activated compound (a derivative of the active agent) reacts with compound X wherein W = OH forming the target conjugate containing an oxycarbonyl linker. This is followed by an oxidation step in case of A = CH in compound X.

In either case, the resulting target conjugate releases an unstable derivative of the active agent in its anionic form (i.e., -(C=O)-O ^_ adducts of the cargo units) upon absorbing visible light, that rapidly releases a molecule of CO2 and the free active agent with restored activity.

The formation of target conjugates wherein the connecting chemical moiety is an oxycarbonyl linker and their subsequent photolysis can be better understood from the following scheme wherein the wavy line ) represents the attachment point of the linker to the photoremovable protecting group (X ¹ ) and

★ the asterisk ( ' ) represents the attachment point of the cargo unit to the linker:

1 ) reaction with compound X linker+cargo unit release of the released active agent: _{wherein w =} W is replaced with: unstable intermediate: active agent: The following examples illustrate the connection formation between compounds X and active agents (using model compounds with different functional groups) and the subsequent photolysis of the resulting target conjugates wherein the connecting chemical moiety is an oxycarbonyl linker: wherein W = OH

2.3 Self-immolative linker

In a further embodiment, the connecting chemical moiety (L) between the photoremovable protecting group (X') and the cargo unit (Z') in the target conjugates can be a self-immolative linker, which, may have, for example one of the following formulas: wherein the wavy line ) represents the attachment point to the photoremovable protecting group X'; the asterisk ( ' ★ ) represents the attachment point to the cargo unit Z';

Ri ₂ and R _i3 are as defined above.

These target conjugate compounds can be prepared via several steps.

First, a compound X, wherein W is OH or halogen, is converted to a compound X, wherein W is a self- immolative linker precursor moiety. Exemplary compounds that are useful for formation of a self- immolative linker (or its precursor moiety) are ethylenediamine derivatives, for example N,N'- dimethylethylenediamine, /V,/V'-diethylethylenediamine, /V-Boc-/V,/V'-dimethylethylenediamine, hydroxymethylphenol derivatives or hydroxymethylaniline derivatives, for example 4- hydroxymethylphenol, 2-hydroxymethylphenol, 4-hydroxymethylaniline, 2-hydroxymethylaniline, 4- hydroxymethyl-/V-methylaniline. These compounds can be attached to X (wherein X = OH) via direct covalent bond formation (as described in section 2.1) or via an oxycarbonyl linker (as described in section 2.2).

An exemplary structure of self-immolative linker precursor moieties that are attached through direct covalent bond to X ¹ is:

Exemplary structures of self-immolative linker precursor moieties that are attached through an oxycarbonyl linker to X ¹ are: Next, the obtained compound X wherein W is a self-immolative linker precursor moiety can be connected to the cargo unit through either direct covalent bond formation (as described in section 2.1) or via an oxycarbonyl linker formation (similarly as described in section 2.2, wherein either the free end of the linker precursor moiety or the selected functional group of the active agent can be activated for the oxycarbonyl linker formation).

Exemplary structure of self-immolative linkers that are attached through direct covalent bond to the cargo unit Z' are:

Exemplary structure of self-immolative linkers that are attached through an oxycarbonyl linker to the cargo unit Z' are:

Alternatively, the linker precursor moiety can be attached to the active agent in the first step, and thereafter the resulting structure can be attached to X (wherein X = OH). Direct covalent bond formation or oxycarbonyl linker formation can be applied in each step, similarly as described above.

Finally, an oxidation step is carried out in case of A = CH in compound X.

The resulting target conjugate, upon absorbing visible light, releases a derivative of the active agent that also contains a moiety derived from the self-immolative linker as an unstable chemical moiety. This drives a reaction cascade that transforms that moiety into an organic byproduct on the one hand and releases the free active agent on the other. In these target conjugates, the advantages of using a self-immolative linker are the larger distance between the photoremovable protecting group and the cargo unit and that it allows the transformation of the connecting chemical moiety to a physiologically more stable element, e.g., a carbonate to two carbamate groups.

The following examples demonstrate some possible embodiments of the connection between the linker- appended compounds X and cargo units and their subsequent photolysis wherein the connecting chemical moiety is a self-immolative linker:

released active agent

3. Synthesis of compounds X

We note that our initial attempts included the conversion of substituted xanthones to their 9- hydroxymethyl derivatives, however, it became evident that the oxidized 9-hydroxymethyl scaffolds are unstable. Following attachment of the cargo moiety, we could only isolate a mixture of products, which impeded the utilization of the target compound. Notably, even after subsequent purification steps, the target compound degraded significantly indicating that the 9-hydroxymethyl derivatives are not suitable as precursors of PPGs.

Strikingly, we have found that this problem can be solved by introducing an additional substituent onto the carbon atom adjacent to the the 9-position of the xanthene scaffold (i.e., the carbon atom bearing the leaving group or cargo moiety). Compounds X of the present invention, bearing an R ₇ substituent (other than a hydrogen atom) on the carbon atom adjacent to the the 9-position of the xanthene scaffold, can be readily synthesized, isolated and attached to cargo moieties. Some possible synthetic routes are set forth in the following sections. 3.1 Synthesis of compounds X from xanthone precursors wherein A = CH and W = OH

As mentioned above, the compounds X can be readily prepared from xanthone precursors, known extensively in the literature. These starting compound(s) can be prepared from readily accessible materials using methods described in the relevant literature. ^35-38 The compound X wherein A = CH and W = OH can be prepared either by a Grignard reaction followed by hydroboration-oxidation or using an umpolung strategy. The general scheme of the first strategy is depicted in the following scheme:

In detail, the starting xanthone compound is first reacted with a Grignard reagent such as ethylmagnesium bromide. Next, the resulting adduct is converted to an olefin derivative using first ammonium chloride and then deprotonation using a strong base, such as NaH. The resulting compound features an exo double bond that can be further functionalized using hydroborationoxidation (using a borane reagent then hydrogen peroxide) that enables the anti-Markovnikov addition of a hydroxyl group and a hydrogen resulting in compound X wherein A = CH and W = OH.

The second method that is disclosed uses an acyl anion umpolung strategy and is depicted in the following scheme:

In detail, the starting xanthone compound is reacted with a deprotonated vinyl ether derivative which is preformed by treatment of a vinyl ether of formula R^CH-OAIk with a strong base (using e.g. tert-butyl lithium), wherein R14 represents e.g. CH ₇ = or CH ₃ -(CH2)n-CH= wherein n is an integer from 0 to 5, wherein said R14 can be considered as a precursor group of R ₇ and Aik refers to an alkyl group. This results in the formation of a vinyl-ether appended xanthenium derivative that can be hydrolyzed to the acyl intermediate (during vinyl ether hydrolysis, R ₇ -C(=O)- is formed from Ri4=CH- O- due to enol-oxo tautomerism). Reduction of the carbonyl and the chromophore frame results in compounds wherein A = CH and W = OH. Note that the resonance structures of the acyl intermediate are also considered herein.

As it can be seen in the examples, this method gave quantitative yields in case of several structures, without the need for any subsequent purification by chromatography. Although multiple synthetic pathways can be envisaged by those skilled in the art for the formation of compounds X, the present inventors realized that the particularly important requirement for late-stage cargo attachment to the PPG is strongly dependent on the PPG precursors (compounds X). Therefore, the synthetic strategy towards the target conjugates were selected to be as modular as possible. Such modular access includes the primary formation of compounds X then its derivative with the connecting chemical moiety and subsequent cargo attachment in the last stage.

3.2 Synthesis of compounds X wherein A = C ⁺ and W = OH

The xanthene-derived compounds X wherein A = CH can be converted to their respective xanthenium derivatives (wherein A = C ⁺ ) as depicted in the following schemes:

In detail, regardless of moiety W, the reduced xanthene form can be oxidized with, for example, chloranil. Formal removal of a hydride ion from the xanthene ring results in the formation of the xanthenium chromophore. An ion exchange to iodide using, for example, hydrogen iodide, is usually beneficial for the stability of the xanthenium derivatives. Note that compounds X wherein A = C ⁺ and W = OH are usually unstable for longer time periods, therefore their reduced form (A = CH) are more suitable for chemical derivatization in the subsequent steps. In addition, the handling of the xantheniums (A = C ⁺ ) is more complicated due to the required darkness in the laboratory.

3.3 Synthesis compounds X wherein A = C ⁺ and W = halogen

The compounds X wherein W = halogen can be synthesized from xanthone precursors as depicted in the following scheme:

In detail, the starting xanthone compound is first reacted with a Grignard reagent such as ethylmagnesium bromide. Next, the resulting adduct is converted to an olefin derivative using first ammonium chloride and then deprotonation using a strong base, such as NaH or KOtBu. Next, the olefin is reacted with /V-bromosuccinimide in the presence of water for the addition of HOBr to the olefin bond. After the elimination of a hydroxide ion, the bromoethyl compound X is formed. Note that /V-bromosuccinimide can be replaced with /V-chlorosuccinimide or /V-iodosuccinimide as well to produce compounds X wherein W = Cl or I.

3.4 Synthesis compounds X wherein W = oxycarbonyl linker precursor moiety The compounds X wherein W = OH can be converted to compounds X wherein W = oxycarbonyl linker precursor moiety using carbonic acid-derived activation reagents such as DSC or 4-nitrophenyl chloroformate as depicted in the following schemes:

In detail, the OH group in compound X (W = OH) reacts with the carbonic-acid derived DSC or chloroformic acid-derived 4-nitrophenyl chloroformate in the presence of a base such as triethylamine to obtain 'mixed carbonate' compounds. These compounds are reactive towards nucleophiles such as amine or alcohol functional groups on the active agents in the presence of a base. Note that these embodiments are just examples of compounds X and as such they do not restrict the scope of the compounds. 3.5 Synthesis of compounds X wherein W = self-immolative linker precursor moiety

The compounds X wherein W = self-immolative linker precursor moiety can be prepared for example, from compounds X wherein W = oxycarbonyl linker precursor moiety as depicted in the following schemes:

In detail, the carbonate-derivatized compounds X are reacted with either N-Boc-N,N'- dimethylethylenediamine in the presence of a base or 4-hydroxymethylaniline in the presence of a base and 1-hydroxybenzotriazole to form compounds X wherein W = self-immolative linker precursor moiety and A = CH. These compounds can be oxidized to form compounds X wherein W = self-immolative linker precursor moiety and A = C ⁺ using chloranil and subsequent anion replacement.

4. Application of compound X as photoremovable protecting group for manipulating biological systems with light

The compounds of the present invention and the target conjugates can be used to study and manipulate biological systems and how those systems react when the active agent is photoreleased from the target conjugates prepared from compound X.

Upon irradiation with visible light with a wavelength >550 nm, the target conjugates can photorelease the active agents. In other words, attachment of compound X to the active agent via the chemical connecting moiety renders it inactive in their bound form (as cargo units). The active agents therefore become photoactivatable in the target conjugates. The various active agents used in a biological systems include but are not limited to any chemically/biologically active agent, including an oligopeptide, protein, enzyme, nucleoitide, nucleic acid, carbohydrate, lipid, neurotransmitter, catalyst, fragrance, pharmaceutically active ingredient or drug, anticancer agent, antibiotics, chemotherapeutic agent, small molecule inhibitor, fluorescent dye or fluorogenic dye. Preferably, the active agents are selected from cytotoxic chemotherapeutic agents or inhibitors used for the destruction of cancer cells including but not limited to nucleoside analogs such as cytarabine or fluorouracil, antifolates such as methotrexate, topoisomerase inhibitors such as campthothecin, irinotecan or SN38, anthracyclines such as doxorubicin, daunomycin or mitoxantrone, taxanes such as paclitaxel or docetaxel, vinca alcaloids such as vincristine and vinblastine, alkylating agents such as chlorambucil, melphalan or cyclophosphamide, platinum compounds such as carboplatin or cisplatin, or targeted antineoplastic agents such as tyrosine kinase inhibitors ibrutinib or dasatinib, histone deacetylase inhibitor vorinostat, or other agents such as methylprednisolone, retinoids, thalidomide.

The target conjugate, prepared from the attachment of compound X to an active agent can be used in a biological sample with external irradiation that enables the photorelease of the active agent from the target conjugates, using visible light with a wavelength >550 nm in a spatiotemporally highly controlled fashion. For example, an LED or laser light source can be used as a controlled external stimulus to trigger the photorelease of the active agent from the target conjugate. Upon the photorelease of the active agent, its original activity reinstates in a spatiotemporally highly controlled fashion.

The term "biological sample" includes but not restricted to in vitro cell cultures, e.g. dedicated or selected cells in a microscope, live cells, fixed cells, 3D cell cultures such as spheroids or organoids, tissue samples and preparations, xenograft tumor models or live animals such as mice.

More specifically, the target conjugate can be used in cells with irradiation that enables the photorelease of the active agent, using visible light with a wavelength >550 nm in a spatiotemporally highly controlled fashion, e.g., in dedicated or selected cells in a microscope. In such embodiments, the photoreleased active agents include but not restricted to a fluorescent dye, small molecular effector or a pharmaceutical agent capable of triggering a biological response or signaling event in a spatiotemporally highly controlled fashion. Upon photorelease of the active agent, its original activity reinstates in a spatiotemporally highly controlled fashion.

In another embodiment, the active agent consists of a pharmaceutical agent, chemotherapeutic agent or small molecule inhibitor and can be photoreleased from the target conjugate using visible light with a wavelength >550 nm in a spatiotemporally highly controlled fashion in live cells. Upon photorelease of the active agent, its original activity reinstates in a spatiotemporally highly controlled fashion. For example, the target conjugate can include but not restricted to compounds resulting from the attachment of compound X to a derivative of SN38 via a self-immolative linker as depicted here: The above mentioned target conjugate X1'-SN38 or X2'-SN38 for example, has a cytotoxicity towards cells in a cell culture up to 10-fold or 200-fold or 1000-fold or 10000-fold lower than the active agent SN38 (depending on the cell line used) enabling cell killing with the target conjugates and subsequent visible light irradiation with a wavelength >550 nm.

In another embodiment, the active agent consists of a pharmaceutical agent, a chemotherapeutic agent or small molecule inhibitor and can be photoreleased from the target conjugate using visible light with a wavelength >550 nm in a spatiotemporally highly controlled fashion in a subject in order to treat a highly localized disease, such as cancer using light delivered to the area of the disease via an optical probe/optical fiber setup to illuminate the given location within the said subject. Upon photorelease of the pharmaceutical agent, chemotherapeutic agent or small molecule inhibitor, highly localized effect restricted to the disease site can be achieved. Highly localized increase in the concentration of a said agent can increase the pharmaceutical effect while reducing off-site toxicities and further side-effects.

For example the target conjugate can be the above mentioned SN38 derivative for the treatment of solid tumors. Administering the compound to the subject and subsequent irradiation of the solid tumor can increase the therapeutic index of SN38 via the localized photorelease of the active agent.

In another embodiment, the invention provides a method for treating cancer using the target conjugate, wherein the active agent consists of a pharmaceutical agent, chemotherapeutic agent or small molecule inhibitor that can be photoreleased from the target conjugate using visible light with a wavelength >550 nm in a spatiotemporally highly controlled fashion in a subject with a solid tumor. For example, the target conjugate can be one of the above mentioned SN38 derivatives for the treatment of solid tumors.

5. Compositions and Methods of Administration

In one embodiment, the photoactivatable conjugate of the present invention comprises a photocaged active pharmaceutical agent.

Therefore, one aspect of the invention relates to pharmaceutical compositions including an effective amount of a photoactivatable conjugate of the invention and a pharmaceutically acceptable excipient. The compositions are suitable for veterinary or human administration.

Any suitable route of administration may be employed for providing a patient with an effective dosage of the photoactivatable compounds described herein. For example, oral or parenteral administration, and the like may be employed. Dosage forms include tablets, dispersions, suspensions, solutions, capsules, and the like. The compounds are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as "pharmacologically acceptable excipients") suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

After administration of the photocaged active agent to the subject, the locus of the disorder is irradiated with a wavelength >550 nm so as to release the active agent. An optical probe/optical fiber setup may be used to illuminate a location within the said subject.

Preferably the disorder is highly localizable in the body of the subject.

In one embodiment the photoactivatable conjugate comprises cytotoxic/chemotherapeutic agent or inhibitor used for the destruction of cancer cells, and the disorder is cancer and in particularly, cancer manifesting in solid tumors.

EXPERIMENTAL

General Methods

All starting materials were obtained from commercial suppliers and used without further purification. Analytical thin-layer chromatography (TLC) was performed on silica gel 60 F254 precoated aluminum TLC plates from Merck. Flash column chromatography was performed on Teledyne Isco CombiFlash® Rf+ automated flash chromatographer with silica gel (25-40 pm) from Zeochem. NMR spectra were recorded on a Varian Inova 500 MHz spectrometer. Chemical shifts (6) are given in parts per million (ppm) using solvent signals or TMS as the reference. Coupling constants (J) are reported in Hertz (Hz). Analytical RP- HPLC-UV/Vis-MS measurements were employed using a Shimadzu LCMS-2020 instrument applying a Gemini C18 column (100 x 2.00 mm I.D.) in which the stationary phase is 5 pm silica with a pore size of 110 A. The chromatograms were detected by a UV-Vis diode array (190-800 nm) and an ESI-MS detector. The following linear gradient elution profile was applied, 0 min 0% B; 6.5 min 100% B; 7 min 0% B; 8 min. 0% B) with eluent A (2% HCOOH, 5% MeCN and 93% water) and B (2% HCOOH, 80% MeCN and 18% water) at a flow rate of 1.0 mL min ¹ at 30°C. The samples were dissolved in MeCN - water mixtures. Spectroscopic measurements were performed on a Jasco FP 8300 spectrofluorometer. Quartz cuvettes with path length of 1 cm were used.

Uncaging experiments

The uncaging experiments were performed using green (4W input power, A _max = 549 nm half-width: 16 nm, output power: 72 mW), orange (4W input power, _max = 605 nm half-width: 10 nm, output power: 140 mW) and red (4W input power, A _max = 658 nm half-width: 10 nm, output power: 210 mW) LEDs as light source. Sample solutions were prepared, each containing 1 ml of solvent (90% v/v water-MeCN or 90% PBS-MeCN or 100% methanol for the photochemical quantum yield determination) and 0.1 mM or 0.05 mM concentration of the photoactivatable conjugates. The samples were irradiated for a given time using continuous water cooling for the light source. Then, the samples were transferred to the HPLC-UV- MS system and the chromatograms were recorded. The traces at 195 nm, 254 nm or 369 nm (dependent on the cargo moieties) and at the absorption maxima of the photoremovable protecting groups (530 nm, 570 nm or 640 nm) were compared and the corresponding peaks were integrated and compared to reference calibration sets. The possible photoproducts were identified by their corresponding m/z value and also from the UV/VIS spectra from the DAD-equipped instrument. For the dark stability experiments, the HPLC sample solutions were kept in the dark and chromatograms were recorded multiple times.

Cell sample preparation for fluorescence imaging experiments

SKOV3 (ATCC HTB-77) cells were maintained in McCoy's 5A (Modified) Medium, HEPES (Gibco 22330021) media supplemented with 10% FBS (Gibco 10500-064), 1% penicillin-streptomycin (Gibco 15140-122). The cells were cultured at 37°C in a 5% CO2 atmosphere and passaged - using trypsin (Gibco 25300-054) - every 3-4 days up to 20 passages.

Live cell fluorescence imaging

SKOV3 (4,5000 cell/well) cells were transferred into p-Slide 8 well plates (Ibidi 80827) and were incubated for 40 h at 37°C in a 5% CO2 atmosphere. After 30 min treatment with compounds X1'-SN38 and X2'- SN38 in the concentration of 1 pM and in some cases in combination with 1 nM MitoTracker Deep Red FM (Invitrogen M22426) or 1 nM LysoTracker Deep red (Invitrogen L12492) or 50 nM MitoTracker Red FM (Invitrogen M22425) or 50 nM LysoTracker red DND-99 (Invitrogen L7528). The samples were subjected to microscopy analyses.

Confocal images were acquired on a Leica TCS SP8 STED 3X microscope using 552 nm and 638 nm lasers for excitation. The images were taken using a Leica HC PL APO 100x/1.40 oil immersion objective along with Leica PMT and HyD detectors. All images were taken using dual detection (channel 1: PMT detector, channel 2: HyD detector). The channels were selected for minimal bleed-through between the green/yellow and red channels. The following wavelengths were selected in case of X2'-SN38: green/yellow channel (for the emission of the MitoTracker Red and LysoTracker Red) 564-600 nm using 552 nm excitation; red channel (for the emission of the photoremovable moieties) 715-800 nm using 638 nm excitation. For the trackers, a concentration of 50 nM was used, for compound X2'-SN38, concentrations of 100 nM and 0.1 uM were used. For compound X1'-SN38, MitoTracker Deep Red and LysoTracker Deep Red were used. In this case, the following wavelengths were used: green/yellow channel (for compound 11): 565-624 nm using 552 nm excitation; red channel (for the trackers): 700-800 nm using 638 nm excitation. The images were processed using ImageJ software. Cytotoxicity determination

A viability test was carried out to assess the toxicity of SN38 as well as the photoremovable protecting group-SN38 conjugates X1'-SN38 and X2'-SN38 after light irradiation and in the dark on SKOV3 cells. Cells were transferred into a 48-well plate (Thermo Fisher Scientific, 130187) (4,500 cells/well) and incubated for 20- 24 h at 37 °C in a 5% CO ₂ atmosphere in McCoy's 5A (Modified) Medium, HEPES (Gibco 22330021) supplemented with 10% FBS (Gibco 10500-064), 1% penicillin-streptomycin (Gibco 15140-122). Cells were treated with compounds in the concentration range of 10 ^_11 -10 ^-4 M for 90 min followed by light irradiation or kept in the dark at 37 °C in 5% CO ₂ atmosphere. After treatment and irradiation, cells were kept at 37 °C in 5% CO ₂ atmosphere for 72 hours. After the incubation period, supernatants were replaced with 0.5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma M5655-1G) solution (in complete DMEM: Dulbecco's modified Eagle's medium (DMEM, Gibco 41965-039), supplemented with 10% FBS (Gibco 10500-064), 1% penicillin-streptomycin (Gibco 15140-122), 1% Glutamax (Gibco 35050-061) and 1% sodium pyruvate (Life Technologies, Gibco 11360-070)) and incubated for 120 min at 37 °C in the dark. The insoluble formazan crystals were dissolved in 250 pl DMSO. Absorbance was detected at 540 nm using a Biotek Synergy 2 Cytation 3 imaging plate reader with Gen5 software version 3.08 (Biotek Winooski, VT, USA). Viability was expressed as percentage (n=3) of the readings of untreated control cells.

EXAMPLES

1. Synthesis of compounds X

1.1 Synthesis of compounds X wherein W = OH and A = CH

Synthesis of key intermediates from xanthones - Umpolung strategy

1.1.1 Synthesis of l-(3,6-di(pyrrolidin-l-yl)-9H-xanthen-9-yl)ethan-l-ol (3):

In a previously dried round-bottom flask anhydrous THF and ethyl-vinyl ether (846 uL, 8.97 mmol, 15 equiv.) was cooled to -78 °C under N ₂ atmosphere and tert-butyllithium (1.7 M in pentane, 2.46 mL, 4.186 mmol, 7.5 equiv.) was added dropwise. The resulting yellow solution was stirred for 20 min at -78 °C, then warmed up to 0 °C in an ice-water bath and kept at this temperature for 5 min. Next, the reaction mixture was cooled back down to -78 °C, then the suspension of 3,6-di(pyrrolidin-l-yl)-9H-xanthen-9-one (1) (200 mg, 0.598 mmol, 1 equiv.) in dry THF was added dropwise. The orange solution was stirred for 20 min at -78 °C, then the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature and stirred for one more hour. After completion, the reaction was quenched with saturated NH4CI (5 mL), then poured into 50 mL methanol. With the dropwise addition of cc. HCI (2.5 mL) the orange solution turned dark pink and was stirred for 16 hours at room temperature (hydrolysis step). The reaction was monitored using LC-MS and after the completion of the hydrolysis, water was added and the mixture was extracted with CH2CI2 5 times (5 x 50 mL), the combined organic layers were washed with brine and dried over Mg2SO4. After the evaporation of the volatiles, the dark pink solid (compound 2) was used without further purification. Yield: 236 mg, 0.595 mmol, 99%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ; d-TFA) 6 7.47 (d, J = 9.3 Hz, 2H), 6.95 (dd, J = 9.3, 2.3 Hz, 2H), 6.67 (d, J = 2.3 Hz, 2H), 3.61 - 3.58 (m, 8H), 2.71 (s, 3H), 2.11 - 2.08 (m, 8H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ; d-TFA) 6 203.0, 158.5, 158.2, 156.0, 130.5, 116.9, 114.7, 110.0, 98.2, 50.2, 25.8.

HRMS: [M] ⁺ : calcd for [C2 ₃ H25N2O ₂ ] ⁺ : 361.1910, found: 361.1906.

The crude acetyl-pyronine compound (2) (236 mg, 0.595 mmol, 1 equiv.) was dissolved in ethanol (30 mL) and NaBH4 (225 mg, 5.95 mmol, 10 equiv.) was added to the solution. The reaction was stirred for one hour at room temperature until the LC-MS indicated complete conversion. 20 mL water was added to the mixture at 0 °C and it was subsequently extracted with CH2CI2 3 times (3 x 50 mL). The combined organic layers were washed with water, brine and dried over Mg2SC>4. After filtration and evaporation, the pink solid (compound 3) was used without any further purification. Yield: 200 mg, 0.549 mmol, 92%. ^X H NMR (500 MHz, Chloroform-d) 6 7.15 - 7.02 (m, 2H), 6.36 - 6.28 (m, 4H), 3.84 - 3.73 (m, 2H), 3.27 - 3.21 (m, 8H), 2.01 - 1.95 (m, 8H), 0.97 (d, J = 5.9 Hz, 3H).

¹³ C NMR (126 MHz, Chloroform-d) 6 154.1, 153.8, 148.3, 148.2, 129.9, 129.7, 110.1, 109.2, 107.2, 107.1,

99.4, 99.2, 73.8, 47.9, 45.5, 25.6, 18.8.

HRMS: [M+H] ⁺ : calcd for [C ₂₃ H29N ₂ O2] ⁺ : 365.2223, found: 365.2217.

1.1.2 Synthesis of l-(10,10-dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethan-l-ol (6):

In a previously dried round-bottom flask anhydrous THF and ethyl-vinyl ether (785 uL, 8.32 mmol, 15 equiv.) was cooled to -78 °C under N2 atmosphere and tert-butyllithium (1.7 M in pentane, 2.45 mL, 4.16 mmol, 7.5 equiv.) was added dropwise. The resulting yellow solution was stirred for 20 min at -78 °C, then warmed up to 0 °C in an ice-water bath and kept at this temperature for 5 min. Next, the reaction mixture was cooled back down to -78 °C, the suspension of 10,10-dimethyl-3,6-di(pyrrolidin-l- yl)anthracen-9(10H)-one (4) (200 mg, 0.55 mmol, 1 equiv.) in dry THF was added dropwise. The orange solution was stirred for 20 min at -78 °C, then the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature and stirred for one more hour. After completion, the reaction was quenched with saturated NH4CI (5 mL), then poured into 50 mL methanol. With the dropwise addition of cc. HCI (2.5 mL) the orange solution turned dark blue and was stirred for 16 hours at room temperature (hydrolysis step). The reaction was monitored using LC-MS and after the completion of the hydrolysis, water was added and the mixture was extracted with CH2CI2 5 times (5 x 100 mL), the combined organic layers were washed with brine and dried over Mg ₂ SO4. After the evaporation of the volatiles, the dark blue solid (5) was used without further purification. Yield: 228 mg, 0.539 mmol, 98%. ^X H NMR (500 MHz, Acetonitrile-d ₃ ; d-TFA) 6 7.38 (d, J = 9.2 Hz, 2H), 7.03 (d, J = 2.4 Hz, 2H), 6.78 (dd, J =

9.2, 2.4 Hz, 2H), 3.68 - 3.63 (m, 8H), 2.67 (s, 3H), 2.11 - 2.07 (m, 8H), 1.70 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ; d-TFA) 6 206.3, 161.6, 158.7, 158.4, 158.1, 155.0, 135.7, 115.5, 113.9,

50.2, 42.7, 33.6, 25.8.

HRMS: [M] ⁺ : calcd for [C26H ₃ IN ₂ O] ⁺ : 387.2430, found: 387.2421.

The crude acetyl-pyronine compound (5) (228 mg, 0.539 mmol, 1 equiv.) was dissolved in ethanol (10 mL) and NaBH4 (204 mg, 5.539 mmol, 10 equiv.) was added to the solution. The reaction was stirred for one hour at room temperature until the LC-MS indicated complete conversion. 10 mL water was added to the mixture at 0 °C and it was subsequently extracted with CH2CI2 5 times (5 x 50 mL). The combined organic layers were washed with water, brine and dried over Mg ₂ SC>4. After filtration and evaporation, the blue solid (compound 6) was used without any further purification.

Yield: 211 mg, 0.541 mmol, 98%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 7.23 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H), 6.77 (dd, J = 5.4, 2.5 Hz, 2H), 6.55 - 6.50 (m, 2H), 3.85 - 3.77 (m, 2H), 3.35 - 3.32 (m, 8H), 2.19 (s, 1H), 2.06 - 2.02 (m, 8H), 1.71 (d, J = 7.2 Hz, 6H), 0.84 (d, J = 6.1 Hz, 3H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ) 6 148.19, 148.18, 146.5, 146.3, 131.0, 130.8, 123.1, 122.8, 111.03,

110.99, 110.5, 110.2, 75.2, 50.7, 48.6, 48.5, 39.7, 35.4, 33.8, 26.1, 20.3.

HRMS: [M+H] ⁺ : calcd for [C ₂₆ H ₃₅ N ₂ O] ⁺ : 391.2743, found: 391.2744.

1.1.3 Synthesis of the extended carbopyronin frame (9) In a previously dried round-bottom flask anhydrous THF (5 mL) and ethyl-vinyl ether (320 pL, 3.39 mmol, 15 equiv.) was cooled to -78 °C under N2 atmosphere and tert-butyllithium (1.7 M in pentane, 997 pL, 1.69 mmol, 7.5 equiv.) was added dropwise. The resulting yellow solution was stirred for 20 min at -78 °C, then warmed up to 0 °C in an ice-water bath and kept at this temperature for 5 min. Next, the reaction mixture was cooled back down to -78 °C, the suspension of the xanthone 7 (100 mg, 0.226 mmol, 1 equiv.) in dry THF (5 mL) was added dropwise. The orange solution was stirred for 20 min at -78 °C, then the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature and stirred for one more hour. After completion, the reaction was quenched with saturated NH4CI (20 mL), then poured into 15 mL methanol. With the dropwise addition of cc. HCI (2 mL) the orange solution turned dark blue and was stirred for 24 hours at room temperature (hydrolysis step). The reaction was monitored using LC-MS and after the completion of the hydrolysis, water was added and the mixture was extracted with CH2CI2 5 times (5 x 100 mL), the combined organic layers were washed with brine and dried over Mg2SO4. After the evaporation of the volatiles, the dark blue solid was used without further purification.

Yield: 113 mg, 0.224 mmol, 99%.

^X H NMR (500 MHz, Chloroform-d) 6 6.93 (s, 1H), 6.87 (s, 1H), 6.84 (s, 1H), 3.77 - 3.55 (m, 6H), 3.26 - 3.09 (m, 2H), 2.87 - 2.73 (m, 3H), 2.68 (s, 3H), 2.14 - 1.99 (m, 2H), 1.88 - 1.77 (m, 10H), 1.48 (s, 3H), 1.38 - 1.34 (m, 6H), 1.32 (d, J = 6.5 Hz, 3H).

MS: [M] ⁺ : calcd for [C ₃ 2H4iN ₂ O] ⁺ : 469, found: 469.

The acetyl-pyronine compound 8 (113 mg, 0.224 mmol, 1 equiv.) was dissolved in ethanol (30 mL) and NaBH4 (85 mg, 2.24 mmol, 10 equiv.) was added to the solution. The reaction was stirred for one hour at room temperature until the LC-MS indicated complete conversion. 20 mL water was added to the mixture at 0 °C and it was subsequently extracted with CH2CI2 5 times (5 x 100 mL). The combined organic layers were washed with water, brine and dried over Mg2SC>4. After filtration and evaporation, the blue solid (compound 9) was used without any further purification.

Yield: 104 mg, 0.220 mmol, 97%.

^X H NMR (500 MHz, Chloroform-d) 6 6.99 - 6.91 (m, 1H), 6.75 - 6.69 (m, 1H), 6.65 - 6.60 (m, 1H), 3.73 - 3.63 (m, 2H), 3.52 - 3.45 (m, 1H), 3.21 - 3.13 (m, 4H), 3.06 - 2.85 (m, 4H), 2.79 - 2.68 (m, 2H), 1.98 - 1.92 (m, 4H), 1.86 - 1.82 (m, 3H), 1.78 - 1.74 (m, 3H), 1.74 - 1.67 (m, 1H), 1.64 - 1.54 (m, 1H), 1.39 - 1.20 (m, 12H), 1.00 - 0.92 (m, 3H).

MS: [M] ⁺ : calcd for [C ₃ 2H45N ₂ O] ⁺ : 473, found: 473.

1.1.4 Synthesis of l-(3,6-Bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthrac en-9-yl)ethan-l-ol (12)

In a previously dried round-bottom flask anhydrous THF (30 mL) and ethyl-vinyl ether (4.59 mL, 48.63 mmol, 15 equiv.) was cooled to -78 °C under N2 atmosphere and tert-butyllithium (1.7 M in pentane, 14.3 mL, 24.32 mmol, 7.5 equiv.) was added dropwise. The resulting yellow solution was stirred for 20 min at -78 °C, then warmed up to 0 °C in an ice-water bath and kept at this temperature for 5 min. Next, the reaction mixture was cooled back down to -78 °C, the suspension of 3,6-bis(dimethylamino)-10,10- dimethylanthracen-9(10H)-one (10) (1.00 g, 3.24 mmol, 1 equiv.) in dry THF (15 mL) was added dropwise. The orange solution was stirred for 20 min at -78 °C, then the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature and stirred for one more hour. After completion, the reaction was quenched with saturated NH4CI (20 mL), then poured into 100 mL methanol. With the dropwise addition of cc. HCI (15 mL) the orange solution turned dark blue and was stirred for 1 hours at room temperature (hydrolysis step). The reaction was monitored using LC-MS and after the completion of the hydrolysis, water was added and the mixture was extracted with CH2CI2 5 times (5 x 100 mL), the combined organic layers were washed with brine and dried over Mg2SC>4. After the evaporation of the volatiles, the dark blue solid (compound 11) was used without further purification.

Yield: 1.133 g, 3.06 mmol, 94%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 7.40 (d, J = 9.4 Hz, 2H), 7.15 (d, J = 2.6 Hz, 2H), 6.91 (dd, J = 9.3, 2.6 Hz, 2H), 3.32 (s, 12H), 2.67 (s, 3H), 1.71 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-c/3) 6 206.1, 162.0, 158.3, 157.7, 135.8, 116.0, 114.7, 113.1, 42.9, 41.6,

33.6, 30.7.

MS: [M] ⁺ : calcd for [C ₂ 2H27N ₂ O] ⁺ : 335, found: 335.

The crude acetyl-pyronine compound 11 (1.133 g, 3.06 mmol, 1 equiv.) was dissolved in ethanol (100 mL) and NaBF (1.156 g, 30.55 mmol, 10 equiv.) was added to the solution. The reaction was stirred for one hour at room temperature until the LC-MS indicated complete conversion. 50 mL water was added to the mixture at 0 °C and it was subsequently extracted with CH2CI2 5 times (5 x 100 mL). The combined organic layers were washed with water, brine and dried over Mg2SO4. After filtration and evaporation, the blue solid (compound 12) was used without any further purification.

Yield: 1.003 g, 2.96 mmol, 97%.

^X H NMR (500 MHz, Chloroform-d) 6 7.20 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 2.6 Hz, 1H),

6.92 (d, J = 2.7 Hz, 1H), 6.69 (m, 2H), 3.83 (s, 1H), 3.76 (mlH), 2.98 (s, 12H), 1.74 (s, 3H), 1.67 (s, 3H), 1.03 (d, 7 = 6.2 Hz, 3H).

¹³ C NMR (126 MHz, Chloroform-d) 6 149.9, 149.8, 145.5, 145.5, 129.8, 129.6, 123.5, 123.4, 111.4, 111.3,

111.3, 110.9, 74.3, 50.7, 41.1, 39.4, 35.8, 32.9, 20.4.

MS: [M+H] ⁺ : calcd for [C ₂₂ H _3i N ₂ O] ⁺ : 339, found: 339.

Synthesis of key intermediates from xanthones - hydroboration-oxidation strategy

1.1.5 Synthesis of l-(3-(methoxymethoxy)-6-(pyrrolidin-l-yl)-9H-xanthen-9-yl)et han-l-ol (15):

Grignard reaction (step 1): in a dried round-bottom flask 3-(methoxymethoxy)-6-(pyrrolidin-l-yl)-9H- xanthen-9-one (13) (150 mg, 0.46 mmol, 1 equiv.) in anhydrous THF (5 mL) was cooled to -78 °C under N2 atmosphere and EtMgBr (1 M in THF, 4.6 mL, 4.6 mmol, 10 equiv.) was added dropwise. After stirring for 30 min, the cooling bath was removed, and the reaction was stirred for one more hour at room temperature. Then, saturated NH4CI (5 mL) was added dropwise and the mixture was extracted with EtOAc 3 times (3 x 50 mL). The collected organic phases were washed with brine and dried over Mg ₂ SO ₄ . After the evaporation, the dark orange solid (compound 14) was used immediately without purification. Crude NMR, mixture of the E/Z isomers of the exo-form (double bond located outside the ring):

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 7.60 (d, J = 8.7 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 6.87 (dd, J = 8.6, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 6.47 (dd, J = 8.7, 2.5 Hz, 1H), 6.34 (d, J = 2.5 Hz, 1H), 5.92 (q, J = 7.4 Hz, 1H), 5.29 (s, 2H), 3.56 (s, 3H), 3.38 - 3.30 (m, 4H), 2.16 (d, J = 7.5 Hz, 3H), 2.10 - 2.04 (m, 4H).

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 7.71 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 6.92 (dd, J = 8.6, 2.6 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.44 (dd, J = 8.7, 2.5 Hz, 1H), 6.28 (d, J = 2.5 Hz, 1H), 5.92 (q, J = 7.4 Hz, 1H), 5.32 (s, 2H), 3.57 (s, 3H), 3.38 - 3.30 (m, 4H), 2.16 (d, J = 7.5 Hz, 3H), 2.10 - 2.04 (m, 4H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ) 6 158.14, 158.13, 154.6, 154.5, 152.8, 152.7, 149.41, 149.37, 129.8, 129.7, 127.1, 127.0, 126.4, 125.2, 124.9, 120.7, 114.4, 113.7, 113.1, 111.8, 110.9, 109.6, 108.2, 104.8, 104.6, 99.3, 99.0, 95.4, 95.3, 56.5, 56.4, 48.4, 48.4, 30.7, 26.09, 26.07, 16.2, 16.1.

HRMS: [M] ⁺ : calcd for [C _2i H ₂₄ NO ₃ ] ⁺ : 338.1750, found: 338.1753.

Hydroboration-oxidation (steps 2 & 3): the resulting ethyl-pyronine (compound 14) was dissolved in anhydrous THF (5 mL) and NaH (74 mg, 1.84 mmol, 4 equiv.) was added to the solution under N ₂ atmosphere. After 15 min, the yellow solution was filtered through a syringe filter into a deoxygenated round-bottom flask. The solution was cooled to 0 °C and borane-dimethyl sulfide comlex (426 uL, 4.6 mmol, 10 equiv.) was added dropwise. The reaction mixture was stirred overnight at room temperature. Then, the mixture was poured into methanol (15 mL) and 3 M NaOH (3 mL) and 30% H ₂ O ₂ (3 mL) were added dropwise. After stirring for 30 min, the mixture was extracted with EtOAc 3 times (3 x 50 mL) and the combined organic phases were washed with Na ₂ S ₂ O3 and brine, dried over Mg ₂ SO4 and the solvents were evaporated. The crude product was purified by RP flash chromatography (eluent: water: MeCN 5% to 100%) to afford 15 as a colorless solid.

Yield for the 3 steps: 26 mg, 0.073 mmol, 16%.

Crude NMR, mixture of two diastereomers:

^X H NMR (500 MHz, Acetonitrile-c/3) 6 7.25 - 7.14 (m, 1H), 7.13 - 7.03 (m, 1H), 6.78 - 6.69 (m, 2H), 6.37 - 6.32 (m, 1H), 6.23 (d, J = 2.4 Hz, 1H), 5.17 (t, J = 2.4 Hz, 2H), 3.85 - 3.73 (m, 2H), 3.45 - 3.40 (m, 3H), 3.27 - 3.21 (m, 4H), 2.01 - 1.96 (m, 4H), 0.84 - 0.77 (m, 3H).

¹³ C NMR (126 MHz, Acetonitrile-c/3) 6 157.9, 157.8, 154.6, 154.4, 154.4, 154.1, 149.3, 149.3, 131.8, 131.4, 131.3, 130.9, 117.9, 117.2, 112.0, 111.9, 110.1, 109.7, 108.4, 104.8, 104.5, 99.5, 99.2, 95.5, 74.1, 74.1, 56.4, 48.5, 45.9, 45.7, 26.1, 18.9, 18.7.

HRMS: [M+H] ⁺ : calcd for [C ₂ IH ₂₆ NO ₄ ] ⁺ : 356.1856, found: 356.1850.

1.1.6 Synthesis of l-(3,6-di(pyrrolidin-l-yl)-9H-xanthen-9-yl)ethan-l-ol (3, alternative route):

Grignard reaction (step 1): in a dried round-bottom flask 3,6-di(pyrrolidin-l-yl)-9H-xanthen-9-one (1) (300 mg, 0.897 mmol, 1 equiv.) in anhydrous THF (5 mL) was cooled to -78 °C under N ₂ atmosphere and EtMgBr (1 M in THF, 8.97 mL, 8.97 mmol, 10 equiv.) was added dropwise. After stirring for 30 min, the cooling bath was removed, and the reaction was stirred for one more hour at room temperature. Then, saturated NH4CI (5 mL) was added dropwise and the mixture was extracted with EtOAc 3 times (3 x 50 mL). The collected organic phases were washed with brine and dried over Mg ₂ SO4. After the evaporation, the dark pink solid (compound 16) was used immediately without purification.

Crude NMR; dye-form (double bond located in the ring):

^X H NMR (500 MHz, Acetonitrile-c/ ₃ ; d-TFA) 6 7.90 (d, J = 9.4 Hz, 2H), 6.88 (dd, J = 9.4, 2.4 Hz, 2H), 6.44 (d, J = 2.3 Hz, 2H), 3.53 - 3.49 (m, 8H), 3.25 (q, J = 7.7 Hz, 2H), 2.10 - 2.06 (m, 8H), 1.32 (t, J = 7.7 Hz, 3H).

¹³ C NMR (126 MHz, Acetonitrile-c/ ₃ ; d-TFA) 6 163.6, 158.2, 155.5, 130.2, 115.9, 113.4, 97.3, 49.8, 25.9, 22.2, 16.1.

HRMS: [M] ⁺ : calcd for [C ₂₃ H ₂₇ N ₂ O] ⁺ : 347.2117, found: 347.2112. Hydroboration-oxidation (steps 2 & 3): the resulting ethyl-pyronine (compound 16) was dissolved in anhydrous THF (5 mL) and NaH (86 mg, 3.58 mmol, 4 equiv.) was added to the solution under N ₂ atmosphere. After 15 min, the yellow solution was filtered through a syringe filter into a deoxygenated round-bottom flask. The solution was cooled to 0 °C and borane-dimethyl sulfide comlex (850 uL, 8.97 mmol, 10 equiv.) was added dropwise. The reaction mixture was stirred overnight at room temperature. Then, the mixture was poured into methanol (20 mL) and 3 M NaOH (5 mL) and 30% H ₂ O ₂ (5 mL) were added dropwise. After stirring for 30 min, the mixture was extracted with EtOAc 3 times (3 x 50 mL) and the combined organic phases were washed with Na ₂ S ₂ O ₃ and brine, dried over Mg ₂ SO4 and the solvents were evaporated to afford 3 as a colorless solid.

Yield for the 3 steps: 87 mg, 0.239 mmol, 27%.

^X H NMR (500 MHz, Chloroform-d) 6 7.15 - 7.02 (m, 2H), 6.36 - 6.28 (m, 4H), 3.84 - 3.73 (m, 2H), 3.27 - 3.21 (m, 8H), 2.01 - 1.95 (m, 8H), 0.97 (d, J = 5.9 Hz, 3H).

¹³ C NMR (126 MHz, Chloroform-d) 6 154.1, 153.8, 148.3, 148.2, 129.9, 129.7, 110.1, 109.2, 107.2, 107.1,

99.4, 99.2, 73.8, 47.9, 45.5, 25.6, 18.8.

HRMS: [M+H] ⁺ : calcd for [C ₂₃ H ₂₉ N ₂ O ₂ ] ⁺ : 365.2223, found: 365.2217.

1.1.7 Synthesis of l-(10,10-dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethan-l-ol (6, alternative route):

Grignard reaction (step 1): in a dried round-bottom flask 10,10-dimethyl-3,6-di(pyrrolidin-l-yl)anthracen- 9(10H)-one (4) (50 mg, 0.139 mmol, 1 equiv.) in anhydrous THF (5 mL) was cooled to -78 °C under N ₂ atmosphere and EtMgBr (1 M in THF, 1.39 mL, 1.390 mmol, 10 equiv.) was added dropwise. After stirring for 30 min, the cooling bath was removed, and the reaction was stirred for one more hour at room temperature. Then, saturated NH4CI (5 mL) was added dropwise and the mixture was extracted with EtOAc 3 times (3 x 50 mL). The collected organic phases were washed with brine and dried over Mg ₂ SO4. After the evaporation, the dark blue solid (compound 17) was used immediately without purification.

Hydroboration-oxidation (steps 2 & 3): the resulting ethyl-pyronine (compound 17) was dissolved in anhydrous THF (5 mL) and NaH (16 mg, 0.417 mmol, 4 equiv.) was added to the solution under N ₂ atmosphere. After 15 min, the yellow solution was filtered through a syringe filter into a deoxygenated round-bottom flask. The solution was cooled to 0 °C and borane-dimethyl sulfide comlex (132 uL, 1.39 mmol, 10 equiv.) was added dropwise. The reaction mixture was stirred overnight at room temperature. Then, the mixture was poured into methanol (15 mL) and 3 M NaOH (3 mL) and 30% H ₂ O ₂ (3 mL) were added dropwise. After stirring for 30 min, the mixture was extracted with EtOAc 3 times (3 x 50 mL) and the combined organic phases were washed with Na ₂ S ₂ O ₃ and brine, dried over Mg ₂ SO4 and the solvents were evaporated. The crude product was purified by flash chromatography (eluent: hexane: EtOAc: 0% to 30%) to afford 6 as a colorless solid.

Yield for the 3 steps: 10 mg, 0.026 mmol, 18%.

Crude NMR, two form present in 1:1 ratio (dye- and exo-form):

Dye-form:

^X H NMR (500 MHz, Acetonitrile-d _3; d-TFA) 6 8.06 (d, J = 9.4 Hz, 2H), 6.95 (d, J = 2.5 Hz, 2H), 6.80 (dd, J =

9.4, 2.5 Hz, 2H), 3.64 - 3.60 (m, 8H), 3.30 (q, J = 7.7 Hz, 2H), 2.10 - 2.05 (m, 8H), 1.58 (s, 6H), 1.35 (t, J = 7.7 Hz, 3H).

Exo-form:

^X H NMR (500 MHz, Acetonitrile-d ₃ ; d-TFA) 6 7.79 (s, 1H), 7.76 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.51 (s, 1H), 7.50 (s, 1H), 6.37 (q, J = 7.3 Hz, 1H), 3.77 - 3.71 (m, 8H), 2.29 - 2.22 (m, 8H), 2.10 (d, J = 7.4 Hz, 3H), 1.66 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ; d-TFA) 6 170.2, 158.0, 154.7, 135.4, 130.4, 126.8, 126.4, 120.1, 114.8,

112.4, 68.4, 59.2, 49.8, 42.2, 34.6, 30.7, 28.1, 26.3, 25.9, 24.8, 24.7, 23.7, 17.1, 16.2.

HRMS: [M] ⁺ : calcd for [C ₂₆ H ₃₃ N ₂ ] ⁺ : 373.2638, found: 373.2621.

1.1.8 Synthesis of l-(3,6-bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthrac en-9-yl)butan-l-ol (19, alternative route):Grignard reaction (step 1): in a dried round-bottom flask 3, 6-bis(dimethylamino)-10, 10- dimethylanthracen-9(10H)-one (10) (154 mg, 0.50 mmol, 1 equiv.) in anhydrous THF (5 mL) was cooled to -78 °C under N ₂ atmosphere and butyl lithium (2.5 M in Hexane, 1.20 mL, 3.0 mmol, 6 equiv.) was added dropwise. After stirring for 30 min, the cooling bath was removed, and the reaction was stirred for one more hour at room temperature. Then, saturated NH4CI (5 mL) was added dropwise and the mixture was extracted with EtOAc 3 times (3 x 50 mL). The collected organic phases were washed with brine and dried over Mg ₂ SO4. After the evaporation, the dark blue solid (compound 18) was used immediately without purification.

Hydroboration-oxidation (steps 2): the resulting butyl-pyronine (compound 18) was dissolved in anhydrous THF (5 mL). The solution was cooled to 0 °C and borane-dimethyl sulfide comlex (300 uL, 3.0 mmol, 6 equiv.) was added dropwise. The reaction mixture was stirred overnight at room temperature. Then, the mixture was poured into methanol (15 mL) and 3 M NaOH (3 mL) and 30% H2O2 (3 mL) were added dropwise. After stirring for 30 min, the mixture was extracted with EtOAc 3 times (3 x 50 mL) and the combined organic phases were washed with Na2$2O3 and brine, dried over Mg2SO ₄ and the solvents were evaporated. The crude product was purified by flash chromatography (eluent: hexane: EtOAc: 0% to 30%) to afford 19 as a colorless solid.

Yield for the 2 steps: 32 mg, 0.094 mmol, 17 %.

^X H NMR (500 MHz, Chloroform-d) 6 7.19 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 11.1, 2.6 Hz, 2H), 6.69 (dt, J = 8.4, 2.7 Hz, 2H), 3.89 (d, J = 5.5 Hz, 1H), 3.60 (m, 1H), 2.98 (s, 12H), 2.05 (s, 1H), 1.71 (d, J = 21.7 Hz, 6H), 1.47 - 1.39 (m, 2H), 1.23 - 1.16 (m, 2H), 0.83 (t, J = 7.1 Hz, 3H).

MS: [M+H] ⁺ : calcd for [C2iH2 ₉ N2O ₂ ] ⁺ : 341, found: 341.

1.2 Synthesis of compounds X wherein W = OH and A = C

Oxidation of compounds X wherein A = CH

1.2.1 Synthesis of l-(9-(l-hydroxyethyl)-6-(methoxymethoxy)-3H-xanthen-3-yliden e)pyrrolidin-l-ium iodide (20) l-(3-(methoxymethoxy)-6-(pyrrolidin-l-yl)-9H-xanthen-9-yl)et han-l-ol (15) (15 mg, 0.042 mmol, 1 equiv.) was dissolved in CH2CI2 - MeOH 1 : 1 (2 mL) and p-chloranil (22 mg, 0.084 mmol, 2 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion. The reaction was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (Eluent: CH ₂ CI ₂ - MeOH 0% to 20%).

The product was redissolved in CH2CI2, one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica) was carried out, obtaining the final compound with L counterion, (eluent: CH2CI2- MeOH 0% to 20%).

Yield: 6 mg, 0.012 mmol, 30%.

MS: [M] ⁺ : calcd for [C ₂ IH ₂₄ NO ₄ ] ⁺ : 354, found: 354.

1.2.2 Synthesis of l-(9-(l-hydroxyethyl)-6-(pyrrolidin-l-yl)-3H-xanthen-3-ylide ne)pyrrolidin-l-ium iodide

(21) l-(3,6-Di(pyrrolidin-l-yl)-9H-xanthen-9-yl)ethan-l-ol (3) (15 mg, 0.041 mmol, 1 equiv.) was dissolved in CH2CI2- MeOH 1 : 1 (2 mL) and p-chloranil (21 mg, 0.082 mmol, 2 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion. The reaction was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (Eluent: CH2CI2- MeOH 0% to 20%).

The product was redissolved in CH2CI2, one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica) was carried out, obtaining the final compound with Lcounterion (eluent: CH ₂ CI ₂ - MeOH 0% to 20%).

Yield: 12 mg, 0.025 mmol, 59%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.46 (d, J = 9.6 Hz, 2H), 6.87 (dd, J = 9.6, 2.5 Hz, 2H), 6.49 (d, J = 2.4 Hz, 2H), 5.94 (q, J = 6.9 Hz, 1H), 2.20 (s, 16H), 1.65 (d, J = 6.9 Hz, 3H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ) 6 163.3, 157.5, 154.3, 130.5, 129.5, 114.4, 111.6, 96.4, 65.2, 48.7, 24.3.

MS: [M] ⁺ : calcd for [C ₂₃ H27N ₂ O2] ⁺ : 363, found: 363.

1.2.3 Synthesis of l-(10-(l-hydroxyethyl)-9,9-dimethyl-7-(pyrrolidin-l-yl)anthr acen-2(9H)- ylidene)pyrrolidin-l-ium iodide (22) l-(10,10-Dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethan-l-ol (6) (20 mg, 0.051 mmol, 1 equiv.) was dissolved in CH2CI2- MeOH 1 : 1 (2 mL) and p-chloranil (26 mg, 0.102 mmol, 2 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion. The reaction was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (Eluent: CH ₂ CI ₂ - MeOH 0% to 20%). The product was redissolved in CH2CI2, one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica) was carried out, obtaining the final compound with I counterion (eluent: CH ₂ CI ₂ - MeOH 0% to 20%).

Yield: 12 mg, 0.023 mmol, 45%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.16 (d, J = 2.3 Hz, 2H), 7.65 (dd, J = 8.4, 2.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 3.66 (d, J = 3.9 Hz, 1H), 2.35 - 2.29 (m, 8H), 2.11 (s, 3H), 1.82 (s, 3H), 1.53 (s, 3H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ) 6 157.4, 145.9, 139.9, 133.5, 131.0, 120.4, 119.68, 58.7, 58.3, 39.8, 33.8, 1 , 23.7.

MS: [M] ⁺ : calcd for [C ₂₆ H ₃₃ N ₂ O] ⁺ : 389, found: 389.

1.2.4 Synthesis of N-(7-(Dimethylamino)-10-(l-hydroxyethyl)-9,9-dimethylanthrac en-2(9H)-ylidene)-N- methylmethanaminium iodide (23) l-(3,6-Bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthrac en-9-yl)ethan-l-ol (12) (20 mg, 0.059 mmol, 1 equiv.) was dissolved in CH2CI2 - MeOH 1 : 1 (2 mL) and p-chloranil (30,1 mg, 0.118 mmol, 2 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion. The reaction was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (Eluent: CH2CI2- MeOH 0% to 20%).

The product was redissolved in CH2CI2, one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica, eluent: CH2CI2 - MeOH 0% to 20%) was carried out, obtaining the final compound with I counterion.

Yield: 12 mg, 0.026 mmol, 44%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.49 (d, J = 9.6 Hz, 2H), 7.09 (d, J = 2.8 Hz, 2H), 6.90 (dd, J = 9.7, 2.7 Hz, 2H), 5.89 (q, J = 6.9 Hz, 1H), 3.30 (s, 12H), 1.72 (d, J = 6.8 Hz, 3H), 1.67 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ) 6 158.5, 157.0, 152.9, 152.0, 136.5, 119.5, 113.4, 111.7, 67.5, 41.2,

34.3, 25.7.

MS: [M] ⁺ : calcd for [C ₂₂ H ₂₉ N ₂ O] ⁺ : 337, found: 337. 1.3 Synthesis of compounds X wherein W = Br and A = C ⁺

1.3.1 Synthesis of N-(10-(l-bromoethyl)-7-(dimethylamino)-9,9-dimethylanthracen -2(9H)-ylidene)-N- methylmethanaminium bromide (24)

In a dried round-bottom flask of 3,6-bis(dimethylamino)-10,10-dimethylanthracen-9(10H)-one (10) (93 mg, 0.30 mmol, 1 equiv.) in anhydrous THF (3 mL) was cooled to -78 °C under N2 atmosphere and EtMgBr (1 M in THF, 2.4 mL, 2.4 mmol, 8 equiv.) was added dropwise. After stirring for 30 min, the cooling bath was removed, and the reaction was stirred for one more hour at room temperature. Then, saturated NH4CI (5 mL) was added dropwise, and the mixture was extracted with EtOAc 3 times (3 x 50 mL). The collected organic phases were washed with brine and dried over MgzSC After the evaporation, the dark blue solid was dissolved in anhydrous THF (3 mL) and KC Bu (1 M in THF, 0.06 mL, 0.06 mmol, 0.2 equiv.) was added to the solution. The colour changed to light blue, then distilled water (0.5 mL) was added, and the reaction was cooled to 0 °C in ice-water bath. Then, /V-bromosuccinimide (59 mg, 0.33 mmol, 1.1 equiv.) was added in the dark, for which the reaction turned dark purple. After 15 min stirring at room temperature, 5 drops of cc HBr was added, and the mixture was extracted with CH2CI2 and water. The organic phase was dried over Mg2SC>4, and the solvent was evaporated. The crude product was purified by flash chromatography (eluent: CH2CI2- MeOH 0% to 20%) to obtain 24 as dark blue crystals.

Yield: 43 mg, 0.090 mmol, 30%.

^X H NMR (500 MHz, Chloroform-d) 6 8.28 (d, J = 9.4 Hz, 2H), 7.03 (d, J = 2.6 Hz, 2H), 6.98 - 6.88 (m, 2H), 6.03 (q, J = 7.1 Hz, 1H), 3.39 (s, 12H), 2.17 (d, J = 7.1 Hz, 3H), 1.67 (s, 6H).

MS: [M] ⁺ : calcd for [C ₂ 2H28BrN ₂ ] ⁺ : 399, found: 399.

1.3.2. Synthesis of l-(9-(l-bromoethyl)-6-(pyrrolidin-l-yl)-3H-xanthen-3-ylidene )pyrrolidin-l-ium bromide

(25) In a dried round-bottom flask 3,6-di(pyrrolidin-l-yl)-9H-xanthen-9-one (1) (100 mg, 0.30 mmol, 1 equiv.) in anhydrous THF (5 mL) was cooled to -78 °C under N2 atmosphere and EtMgBr (1 M in THF, 3.0 mL, 3.0 mmol, 10 equiv.) was added dropwise. After stirring for 30 min, the cooling bath was removed, and the reaction was stirred for one more hour at room temperature. Then, saturated NH4CI (5 mL) was added dropwise and the mixture was extracted with EtOAc 3 times (3 x 50 mL). The collected organic phases were washed with brine and dried over MgzSC After the evaporation, the dark pink solid was dissolved in anhydrous THF (3 mL) and KC Bu (1 M in THF, 0.06 mL, 0.06 mmol, 0.2 equiv.) was added to the solution. The colour changed to light pink, then distilled water (1 mL) was added, and the reaction was cooled to 0 °C in ice-water bath. Then N-bromosuccinimide (59 mg, 0.33 mmol, 1.1 equiv.) was added in dark, for which the reaction turned dark purple. After 15 min stirring at room temperature, 5 drops of cc HBr was added and extracted with CH2CI2 and water. The organic phase was dried over Mg2SC>4, and the solvent was evaporated. The crude product was purified by flash chromatography (eluent: CH2CI2- MeOH 0% to 20%) to obtain 25 as dark purple crystals.

Yield: 70 mg, 0.138 mmol, 46%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.02 (d, J = 9.5 Hz, 1H), 7.04 (dd, J = 9.6, 2.5 Hz, 1H), 6.68 (d, J = 2.4

Hz, 1H), 3.97 - 3.91 (m, 8H), 2.18 - 2.10 (m, 11H).

MS: [M] ⁺ : calcd for [C23H ₂₆ BrN2O] ⁺ : 425, found: 425.

1.3.3. Synthesis of 9-(l-bromoethyl)-6-(dimethylamino)-3H-xanthen-3-one (27)

In a dried round-bottom flask 3-(dimethylamino)-6-(methoxymethoxy)-9H-xanthen-9-one (26) (50 mg, 0.167 mmol, 1 equiv.) in anhydrous THF (5 mL) was cooled to -78 °C under N2 atmosphere and EtMgBr (1 M in THF, 1.67 mL, 1.67 mmol, 10 equiv.) was added dropwise. After stirring for 30 min, the cooling bath was removed, and the reaction was stirred for one more hour at room temperature. Then, saturated NH4CI (5 mL) was added dropwise, and the mixture was extracted with EtOAc 3 times (3 x 50 mL). The collected organic phases were washed with brine and dried over Mg2SO4. After the evaporation, the dark pink solid was dissolved in anhydrous THF (3 mL) and KC Bu (1 M in THF, 35 pL, 0.033 mmol, 0.2 equiv.) was added to the solution. The colour changed to light pink, then distilled water (1 mL) was added, and the reaction was cooled to 0 °C in ice-water bath. Then N-bromosuccinimide (33 mg, 0.184 mmol, 1.1 equiv.) was added in dark, for which the reaction turned dark purple. After 15 min stirring at room temperature, the mixture was extracted with CH2CI2 and water. The organic phase was dried over Mg2SC>4, and the solvent was evaporated.

The crude product was redissolved in CHzCL fl ml) and TFA (200 pL) was added dropwise. After 30 min stirring, the solvents were evaporated. The crude product was purified by flash chromatography (eluent: CH2CI2- MeOH 0% to 20%) to obtain 27 as dark red crystals.

Yield for 3 steps: 18 mg, 0.0517 mmol, 31%.

^X H NMR (500 MHz, Acetonitrile-d ₃ , d-TFA) 6 8.43 (d, J = 80.4 Hz, 2H), 7.36 (dd, J = 10.0, 2.6 Hz, 1H), 7.18 (dd, J = 9.3, 2.5 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H), 6.89 (d, J = 2.6 Hz, 1H), 6.24 (q, J = 7.1 Hz, 1H), 3.44 - 3.29 (m, 8H), 2.24 (d, J = 7.1 Hz, 3H).

MS: [M+H] ⁺ : calcd for [Ci7Hi ₇ BrNO ₂ ] ⁺ : 346, found: 346.

1.4 Synthesis of compounds X wherein W = oxycarbonyl linker precursor moiety

Activation of the hydroxyethyl derivatives with DSC

1.4.1 Synthesis of l-(10,10-Dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethyl (2,5- dioxopyrrolidin-l-yl) carbonate (28): l-(10,10-Dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethan-l-ol (6) (150 mg, 0.385 mmol, 1 equiv.) was dissolved in acetonitrile (7 mL), DSC (493 mg, 1.92 mmol, 5 equiv.), TEA (536 pL, 3.85 mmol, 10 equiv.) and DMAP (cat.) were added to the solution and stirred for overnight at room temperature. After the LC-MS indicated complete conversion, the reaction was extracted 5 times with saturated NaHCO ₃ , the organic phase was dried over MgzSC , and the solvent was evaporated. The crude product was used immediately without further purification.

Yield: 192 mg, 0.362 mmol, 94%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 7.18 (dd, J = 12.7, 8.5 Hz, 2H), 6.77 (dd, J = 9.7, 2.5 Hz, 2H), 6.56 (ddd, J = 8.5, 3.8, 2.5 Hz, 2H), 5.06 - 4.96 (m, 1H), 4.26 (d, J = 4.7 Hz, 1H), 3.34 (m, J = 6.0, 3.9, 3.1 Hz, 8H), 3.16 - 3.12 (m, 4H), 2.05 (m, J = 3.4 Hz, 8H), 1.72 (d, J = 8.1 Hz, 6H), 0.98 (d, J = 6.4 Hz, 3H).

MS: [M+H] ⁺ : calcd for [C ₃ IH38N3O ₅ ] ⁺ : 532, found: 532.

29 (95%)

1.4.2 Synthesis of l-(3,6-Bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthrac en-9-yl)ethyl (2,5- dioxopyrrolidin-l-yl) carbonate (29) l-(3,6-Bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthrac en-9-yl)ethan-l-ol (12) (250 mg, 0.729 mmol, 1 equiv.) was dissolved in acetonitrile (10 mL), DSC (946 mg, 3.69 mmol, 5 equiv.), TEA (1.03 mL, 7.39 mmol, 10 equiv.) and DMAP (cat.) were added to the solution and stirred for overnight at room temperature. After the LC-MS indicated complete conversion, the reaction was extracted 5 times with saturated NaHCOs, the organic phase was dried over MgjSC , and the solvent was evaporated. The crude product was used immediately without further purification.

Yield: 352 mg, 0.734 mmol, 99%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.08 (d, J = 6.4 Hz, 2H), 7.22 - 7.10 (m, 2H), 6.90 (t, J = 2.6 Hz, 2H), 6.68 - 6.64 (m, 2H), 2.93 (s, 16H), 1.66 (d, J = 4.0 Hz, 9H).

MS: [M+H] ⁺ : calcd for [C ₂ 7H3 ₄ N3O ₅ ] ⁺ : 480, found: 480. Activation of the hydroxyethyl derivatives with 4-nitrophenyl chloroformate

1.4.3 Synthesis of l-(10,10-dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethyl (4- nitrophenyl) carbonate (30) l-(10,10-Dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethan-l-ol (6) (20 mg, 0.051 mmol, 1 equiv.) was dissolved in dry THF and 4-nitrophenyl chloroformate (16 mg, 0.077 mmol, 1.5 equiv.) and pyridine (6.2 pL, 0.077 mmol, 1.5 equiv.) were added to the reaction. After completion, the reaction was extracted with CH2CI2 and water, the organic phase was dried over Mg2SC>4, and the solvent was evaporated. The crude product was used immediately without further purification.

Yield: 25 mg, 0.045, mmol, 88%.

MS: [M+H] ⁺ : calcd for [CaaHasNaOsf: 556, found: 556.

1.4.4 Synthesis of l-(3,6-bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthrac en-9-yl)ethyl (4- nitrophenyl) carbonate (31) l-(3,6-Bis(dimethylamino)-10,10-dimethyl-9,10-dihydroanthrac en-9-yl)ethan-l-ol (12) (25 mg, 0.079 mmol, 1 equiv.) was dissolved in dry THF and 4-nitrophenyl chloroformate (16 mg, 0.079 mmol, 1 equiv.) and pyridine (6.3 pL, 0.079 mmol, 1 equiv.) were added to the reaction. After completion, the reaction was extracted with CH2CI2 and water, the organic phase was dried over Mg2SC>4, and the solvent was evaporated. The crude product was used immediately without further purification.

Yield: 36 mg, 0.072, mmol, 91%.

^X H NMR (500 MHz, Acetonitrile-d3) 6 8.28 (d, J = 9.1 Hz, 2H), 7.35 (d, J = 9.2 Hz, 2H), 6.97 (dd, J = 6.0, 3.3

Hz, 4H), 6.72 (m, J = 7.0, 2.7, 1.4 Hz, 2H), 5.00 (m, J = 6.3 Hz, 1H), 4.20 (d, J = 5.7 Hz, 1H), 2.97 (s, 12H),

1.70 (s, 6H), 1.11 (d, J = 6.4 Hz, 3H).

MS: [M+H] ⁺ : calcd for [Cz^NaOsf: 504, found: 504. 1.5 Synthesis of compounds X wherein W = self-immolative linker

1.5.1 Compound 32 (carbamate formation)

Compound 28 (100 mg, 0,256 mmol, 1 equiv.) was dissolved in CH2CI2 (3 mL) then tert-butyl methyl(2- (methylamino)ethyl)carbamate (145 mg, 0.768 mmol, 3 equiv.), triethylamine (53 uL, 0.384 mmol, 1.5 equiv.) were added and the reaction was stirred for 1 hour at room temperature. After completion, the reaction was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane- EtOAc 0% to 10%) to afford 32 as a colourless oil. Yield: 120 mg, 0.199 mmol, 78%.

^X H NMR (500 MHz, Chloroform-d) 6 7.32 (t, J = 9.4 Hz, 1H), 7.18 - 7.09 (m, 1H), 6.67 (dd, J = 22.7, 2.5 Hz, 2H), 6.56 - 6.45 (m, 2H), 5.06 (s, 1H), 4.34 - 4.23 (m, 1H), 3.32 (dd, J = 8.4, 5.1 Hz, 8H), 3.06 - 2.96 (m, 4H), 2.96 - 2.81 (m, 6H), 2.02 (m, 8H), 1.70 (d, J = 11.2 Hz, 6H), 1.46 (s, 9H), 0.72 (d, J = 6.4 Hz, 3H).

¹³ C NMR (126 MHz, Chloroform-d) 6 146.8, 146.7, 144.7, 129.78, 119.6, 110.6, 109.8, 109.2, 60.4, 47.7,

45.8, 38.5, 34.5, 28.4, 28.4, 25.5, 21.0, 15.5, 14.2.

HRMS: [M+H] ⁺ : calcd for [C ₃ 6H5 ₃ N ₄ O ₄ ] ⁺ : 605.4061, found: 605.4052.

1.5.2 Compound 33 (oxidation and deprotection)

Step 1 (oxidation): Boc-protected compound 32 (120 mg, 0.198 mmol, 1 equiv.) was dissolved in CH2CI2- MeOH -1 : 1 (3 mL), and chloranil (152 mg, 0.596 mmol, 3 equiv.) was added to the solution. After 30 min, the solvent was evaporated, the dark blue solid was applied directly on silica gel column and purified by flash chromatography (Eluent: CH2CI2- MeOH 0% to 20%).

Step 2 (anion exchange): the purified product was redissolved in CH2CI2 (2 mL), one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica) was carried out (eluent: CH2CI2- MeOH 0% to 20%).

Step 3 (deprotection): Boc-protected iodide salt was dissolved in CHzCL mL), then trifluoroacetic acid (25% for the mixture) was added and stirred at room temperature for 1 hour. Then volatiles were evaporated and the solid was thoroughly dried. The blue product was used without further purification in the next step.

Yield for the 3 steps: 32% ^X H NMR (500 MHz, Acetonitrile-d ₃ , d-TFA) 6 8.24 (d, J = 9.5 Hz, 2H), 6.98 (d, J = 2.4 Hz, 2H), 6.80 (m, 2H), 6.58 (m, 1H), 3.66 (m, 8H), 2.99 (s, 2H), 2.61 (s, 2H), 2.11 - 2.07 (m, 8H), 1.88 (d, J = 7.0 Hz, 3H), 1.65 (s, 6H), 1.27 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ , d-TFA) 6 163.2, 158.1, 154.2, 135.8, 119.58, 119.57, 115.0, 112.99, 112.97, 72.0, 55.3, 42.8, 34.4, 34.3, 25.9, 22.9.

HRMS: [M] ⁺ : calcd for [C ₃ IH ₄₃ N ₄ O ₂ ] ⁺ : 503.3380, found: 503.3377.

2. Synthesis of target conjugates (attachment of compounds X to the active agents)

2.1 Synthesis of model target conjugates wherein the connecting chemical moiety between the photoremovable protecting group and the cargo unit is a covalent bond

Synthesis of model target conjugates from key intermediates - ethers

2.1.1 Synthesis of l,l '-(10-(l-(benzyloxy)ethyl)-9,9-dimethyl-9,10-dihydroanthrace ne-2,7- diyljdipyrrolidine (34)

Compound 6 (50 mg, 0.128 mmol, 1 equiv.) was dissolved in dryTHF (2 mL), then potassium tert-butoxide (22 mg, 0.192 mmol, 1.5 equiv.) and benzyl bromide (26 mg, 0.154 mmol 1.2 equiv.) were added. The reaction was stirred for 10 min at room temperature, then distilled water was added and the mixture was extracted with CH2CI23 times (3 x 50 mL) and dried over Mg ₂ SO ₄ . The solution was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc 0% to 10%) to afford 34 as a colourless oil. Yield: 10 mg, 0.021 mmol, 16%.

^X H NMR (500 MHz, Chloroform-d) 6 7.35 - 7.31 (m, 6H), 7.10 (d, J = 8.4 Hz, 1H), 6.69 (t, J = 2.8 Hz, 2H), 6.51 (ddd, J = 8.4, 3.5, 2.5 Hz, 2H), 4.59 (d, J = 17.4 Hz, 2H), 4.21 (d, J = 4.3 Hz, 1H), 3.69 (td, J = 6.3, 4.4 Hz, 1H), 3.33 (dt, J = 6.5, 3.3 Hz, 8H), 2.02 (q, J = 3.3 Hz, 8H), 1.70 (d, J = 20.6 Hz, 6H), 0.77 (d, J = 6.2 Hz, 3H).

¹³ C NMR (126 MHz, cdcl ₃ ) 6 146.7, 146.6, 145.3, 145.1, 139.3, 132.4, 130.4, 129.8, 128.4, 128.2, 127.8, 127.6, 127.2, 122.6, 121.0, 110.3, 110.1, 109.3, 109.1, 82.5, 70.7, 47.8, 45.9, 38.6, 34.9, 34.0, 25.4, 16.2. MS: [M+H] ⁺ : calcd for [C ₃ 3H ₄ IN ₂ O] ⁺ : 481, found: 481. 2.1.2 Synthesis of l-(10-(l-(benzyloxy)ethyl)-9,9-dimethyl-7-(pyrrolidin-l-yl)a nthracen-2(9H)- ylidene)pyrrolidin-l-ium iodide (35)

Compound 34 (10 mg, 0.021 mmol, 1 equiv.) was dissolved in 1 mL CH ₂ CI ₂ - MeOH 1 : 1 in the dark and p-chloranil (21 mg, 0.083 mmol, 4 equiv.) was added to the solution. The mixture was stirred until the LC- MS indicated full conversion (30 min). The reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH ₂ CI ₂ - MeOH 0% to 20%).

The product was redissolved in CH ₂ CI ₂ , one drop of HI (57% in water) was added, the mixture was stirred for 5 min and another flash chromatography (on silica, eluent: CH ₂ CI ₂ - MeOH 0% to 20%) was carried out, obtaining the final compound as blue crystals with L counterion. Yield: 5 mg, 0.008 mmol, 41%. HRMS: [M] ⁺ : calcd for [C ₃₃ H39N ₂ O] ⁺ : 479.3057, found: 479.3064.

2.1.3 10-(l-(Benzyloxy)ethyl)-N2,N2,N7,N7,9,9-hexamethyl-9,10-dihy droanthracene-2,7-diamine (36) Compound 12 (30 mg, 0.088 mmol, 1 equiv.) was dissolved in dry THF (2 mL), then potassium tert- butoxide (20 mg, 0.176 mmol, 2 equiv.) and benzyl bromide (16 pL, 0.132 mmol 1.5 equiv.) were added. The reaction was stirred for 10 min at room temperature, then distilled water was added and the mixture was extracted with CH ₂ CI ₂ 3 times (3 x 50 mL) and dried over Mg ₂ SC>4. The solution was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc 0% to 10%) to afford 36 as a colourless oil.

Yield: 22 mg, 0.052 mmol, 59%

^X H NMR (500 MHz, Chloroform-d) 6 7.29 - 7.24 (m, 5H), 7.23 - 7.19 (m, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.83 (dd, J = 4.5, 2.7 Hz, 2H), 6.63 (ddd, J = 8.4, 4.4, 2.7 Hz, 2H), 4.57 - 4.47 (m, 2H), 4.15 (d, J = 4.4 Hz, 1H), 3.63 (qd, J = 6.3, 4.5 Hz, 1H), 2.91 (s, 12H), 1.66 (s, 3H), 1.61 (s, 3H), 0.73 (d, J = 6.3 Hz, 3H).

¹³ C NMR (126 MHz, Chloroform-d) 6 149.5, 149.4, 145.2, 145.0, 139.1, 130.2, 129.7, 128.3, 127.7, 127.2, 124.0, 122.5, 111.5, 111.4, 110.9, 110.7, 82.2, 70.7, 46.0, 41.1, 41.0, 38.7, 34.9, 33.9, 16.2.

MS: [M+H] ⁺ : calcd for [C ₂ gH ₃₇ N ₂ O] ⁺ : 429, found: 429.

2.1.4 N-(10-(l-(Benzyloxy)ethyl)-7-(dimethylamino)-9,9-dimethylant hracen-2(9H)-ylidene)-N- methylmethanaminium iodide (37)

Compound 36 (30 mg, 0.088 mmol, 1 equiv.) was dissolved in 1 mL CH ₂ CI ₂ - MeOH 1 : 1 in the dark and p-chloranil (43 mg, 0.176 mmol, 2 equiv.) was added to the solution. The mixture was stirred until the LC- MS indicated full conversion (30 min). The reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH2CI2- MeOH 0% to 20%).

The product was redissolved in CH2CI2, one drop of HI (57% in water) was added, the mixture was stirred for 5 min and another flash chromatography (on silica, eluent: CH2CI2 - MeOH 0% to 20%) was carried out, obtaining the final compound as blue crystals with r counterion.

Yield: 20 mg, 0.036 mmol, 69%

^X H NMR (500 MHz, Acetonitrile-c/3) 6 8.47 (d, J = 9.6 Hz, 2H), 7.34 - 7.21 (m, 5H), 7.11 (d, J = 2.7 Hz, 2H), 6.90 (dd, J = 9.7, 2.7 Hz, 2H), 5.66 (q, J = 6.8 Hz, 1H), 4.54 - 4.39 (m, 2H), 3.32 (s, 12H), 1.77 (d, J = 6.9 Hz, 3H), 1.69 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-c/3) 6 165.7, 157.3, 156.1, 137.9, 134.9, 128.4, 128.0, 127.8, 118.9, 112.7, 110.9, 74.5, 71.6, 41.9, 40.3, 33.4, 23.0.

HRMS: [M] ⁺ : calcd for ^gHss^Of: 427.2744, found: 427.2747.

Synthesis of photocages from key intermediates - esters

2.1.5 Synthesis of l-(3-(methoxymethoxy)-6-(pyrrolidin-l-yl)-9H-xanthen-9-yl)et hyl 2-phenylacetate (38)

Hydroxyethyl derivative 15 (26 mg, 0.073 mmol, 1 equiv.) was dissolved in 1 ml CH2CI2 then phenylacetic acid (15 mg, 0.11 mmol, 1.5 equiv.), DCC (18 mg, 0.087 mmol, 1.2 equiv.) and DMAP (catalytic amount) were added to the solution and the resulting mixture was stirred for 30 min. After completion, the reaction mixture was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc, 0% to 10%) affording 38 as a colourless oil. Yield: 12 mg, 0.027 mmol, 38%.

Mixture of two diastereomers (double integrals):

^X H NMR (500 MHz, Chloroform-d) 6 7.37 - 7.33 (m, 4H), 7.31 - 7.28 (m, 6H), 7.16 (d, J = 8.5 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.78 - 6.75 (m, 2H), 6.75 - 6.70 (m, 3H), 6.63 - 6.59 (m, 1H), 6.31 (dd, J = 8.4, 2.4 Hz, 1H), 6.27 - 6.20 (m, 3H), 5.18 - 5.15 (m, 4H), 5.07 - 4.99 (m, 2H), 4.05 (d, J = 3.9 Hz, 2H), 3.66 - 3.62 (m, 4H), 3.50 (s, 3H), 3.49 (s, 3H), 3.31 - 3.26 (m, 8H), 2.03 - 1.98 (m, 8H), 0.90 - 0.86 (m, 6H).

¹³ C NMR (126 MHz, Chloroform-d) 6 171.0, 157.0, 153.7, 153.5, 153.5, 153.4, 153.0, 152.2, 148.2, 137.1, 134.1, 134.1, 130.6, 130.3, 129.9, 129.7, 129.4, 128.6, 128.6, 127.1, 127.0, 115.7, 114.1, 111.2, 111.0, 108.0, 107.5, 107.4, 107.3, 106.7, 104.1, 103.8, 98.8, 98.6, 94.6, 76.3, 56.0, 47.7, 47.7, 42.0, 41.9, 41.9, 41.8, 36.6, 25.5, 25.5, 24.7, 23.3, 14.7, 14.4.

HRMS: [M+H] ⁺ : calcd for [C29H ₃ 2NO ₅ ] ⁺ : 474.2274, found: 474.2267. 2.1.6 Synthesis of l-(3-oxo-6-(pyrrolidin-l-yl)-3H-xanthen-9-yl)ethyl 2-phenylacetate (39)

Deprotection (step 1): compound 38 (35 mg, 0.074 mmol, 1 equiv.) was dissolved in CH2CI2 (2 mL), and TFA (500 uL) was added and stirred at room temperature for 1 hour. After the completion of the deprotection, the volatiles were evaporated and the solid was thoroughly dried.

Oxidation (step 2): the resulting solid was dissolved in 1 mL CH2CI2- MeOH 1 : 1 and p-chloranil (36 mg, 0.148 mmol, 2 equiv.) was added to the solution in the dark. The mixture was stirred until the LC-MS indicated full conversion (30 min). Then, the reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH2CI2 - MeOH 0% to 20%) affording 39 as a red crystalline solid. Yield for the two steps: 20 mg, 0.048 mmol, 64%.

^X H NMR (500 MHz, Acetonitrile-cfe d-TFA) 6 8.23 (d, J = 9.9 Hz, 1H), 8.18 (d, J = 9.9 Hz, 1H), 7.30 - 7.25 (m, 3H), 7.20 - 7.16 (m, 2H), 7.07 - 6.98 (m, 3H), 6.73 - 6.67 (m, 2H), 3.78 - 3.72 (m, 2H), 3.71 (s, 2H), 3.67 - 3.60 (m, 2H), 2.15 - 2.10 (m, 4H), 1.80 (d, J = 7.0 Hz, 3H).

¹³ C NMR (126 MHz, Acetonitrile-cfe d-TFA) 6 171.6, 166.8, 159.6, 159.2, 157.9, 157.2, 134.6, 131.1, 130.9, 130.4, 129.6, 128.2, 119.4, 117.6, 115.8, 113.5, 103.7, 97.9, 69.5, 51.0, 50.8, 41.5, 25.9, 25.6, 21.4.

HRMS: [M+H] ⁺ : calcd for [C27H ₂₆ NO ₄ ] ⁺ : 427.1784, found: 427.2747.

2.1.7 Synthesis of l-(3,6-di(pyrrolidin-l-yl)-9H-xanthen-9-yl)ethyl 2-phenylacetate (40)

Hydroxyethyl derivative 3 (50 mg, 0.137 mmol, 1 equiv.) was dissolved in 2 mL CH2CI2 then phenylacetic acid (28 mg, 0.205 mmol, 1.5 equiv.), DCC (34 mg, 0.165 mmol, 1.2 equiv.) and DMAP (catalytic amount) were added to the solution and the resulting mixture was stirred for 30 min. After completion, the reaction mixture was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc, 0% to 10%) affording 40 as a colourless oil.

Yield: 46 mg, 0.095 mmol, 70%.

Mixture of two diastereomers:

^X H NMR (500 MHz, Chloroform-d) 6 7.38 - 7.28 (m, 5H), 7.12 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 6.30 (dd, J = 8.4, 2.4 Hz, 1H), 6.27 (d, J = 2.3 Hz, 2H), 6.23 (dd, J = 8.4, 2.5 Hz, 1H), 5.08 - 5.02 (m, 1H), 4.04 (d, J = 3.9 Hz, 1H), 3.66 (d, J = 3.0 Hz, 2H), 3.32 - 3.27 (m, 8H), 2.05 - 1.97 (m, 8H), 0.89 (d, J = 6.4 Hz, 3H). ¹³ C NMR (126 MHz, Chloroform-d) 6 171.1, 153.9, 153.5, 148.2, 134.4, 130.5, 129.8, 129.6, 129.3, 128.7, 128.7, 127.1, 109.0, 107.5, 107.3, 107.1, 98.9, 98.8, 76.8, 47.8, 42.1, 41.7, 25.6, 14.6.

HRMS: [M+H] ⁺ : calcd for [C3iH35N ₂ O ₃ ] ⁺ : 483.2642, found: 483.2624. 2.1.8 Synthesis of l-(9-(l-(2-phenylacetoxy)ethyl)-6-(pyrrolidin-l-yl)-3H-xanth en-3-ylidene)pyrrolidin-l- ium iodide (41)

Compound 40 (83 mg, 0.172 mmol, 1 equiv.) was dissolved in 2 mL CH2CI2- MeOH 1 : 1 and p-chloranil (170 mg, 0.688 mmol, 4 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion (30 min). Then, the reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH2CI2- MeOH 0% to 20%). The product was redissolved in C^CL. one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica) was carried out (eluent: CH2CI2- MeOH 0% to 20%) affording 41 as purple crystals. Yield: 62 mg, 0.102 mmol, 59%.

¹ H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.08 (d, J = 9.6 Hz, 2H), 7.31 - 7.23 (m, 3H), 7.21 - 7.17 (m, 2H), 6.85 (dd, J = 9.5, 2.4 Hz, 2H), 6.64 (q, J = 7.0 Hz, 1H), 6.59 (d, J = 2.4 Hz, 2H), 3.70 (s, 2H), 3.65 - 3.50 (m, 8H), 2.11 - 2.07 (m, 8H), 1.78 (d, J = 7.0 Hz, 3H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ) 6 170.5, 157.6, 156.4, 154.4, 133.7, 129.4, 129.2, 128.6, 127.1, 115.2, 111.0, 96.6, 68.4, 48.9, 40.6, 24.9, 20.5.

HRMS: [M] ⁺ : calcd for [C ₃ IH33N2O ₃ ] ⁺ : 481.2486, found: 481.2493.

2.1.9 Synthesis of l-(10,10-dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethyl 2- phenylacetate (42)

Hydroxyethyl derivative 3 (162 mg, 0.416 mmol, 1 equiv.) was dissolved in 5 mL CH2CI2 then phenylacetic acid (85 mg, 0.624 mmol, 1.5 equiv.), DCC (103 mg, 0.499 mmol, 1.2 equiv.) and DMAP (catalytic amount) were added to the solution and the resulting mixture was stirred for 30 min. After completion, the reaction mixture was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc, 0% to 10%) affording 42 as a colourless oil.

Yield: 60 mg, 0.118 mmol, 28%.

^X H NMR (500 MHz, Chloroform-d) 6 7.38 - 7.27 (m, 5H), 7.23 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 2.5 Hz, 1H), 6.65 (d, J = 2.5 Hz, 1H), 6.48 (dd, J = 8.4, 2.4 Hz, 1H), 6.41 (dd, J = 8.4, 2.5 Hz, 1H), 5.17 - 5.08 (m, 1H), 4.12 (d, J = 4.7 Hz, 1H), 3.67 (d, J = 4.3 Hz, 2H), 3.38 - 3.32 (m, 8H), 2.06 - 1.98 (m, 8H), 1.69 (d, J = 3.4 Hz, 7H), 0.80 (d, J = 6.4 Hz, 3H). ¹³ C NMR (126 MHz, Chloroform-d) 6 171.2, 147.0, 147.0, 145.9, 145.1, 134.5, 130.3, 130.0, 129.6, 129.4, 128.7, 128.7, 127.1, 121.5, 119.9, 110.6, 110.2, 110.1, 109.4, 109.4, 77.9, 47.9, 46.4, 42.2, 38.8, 35.1, 34.0, 25.6, 15.9.

HRMS: [M+H] ⁺ : calcd for [C ₃ 4H ₄ IN ₂ O2] ⁺ : 509.3162, found: 509.3154.

2.1.10 Synthesis of l-(9,9-dimethyl-10-(l-(2-phenylacetoxy)ethyl)-7-(pyrrolidin- l-yl)anthracen-2(9H)- ylidene)pyrrolidin-l-ium iodide (43)

Compound 42 (26 mg, 0.043 mmol, 1 equiv.) was dissolved in 1 ml CH2CI2- MeOH 1 : 1 and p-chloranil (21 mg, 0.086 mmol, 2 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion (30 min). Then, the reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH2CI2- MeOH 0% to 20%). The product was redissolved in C^CL. one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica, eluent: CH2CI2- MeOH 0% to 20%) was carried out, obtaining the final compound as blue crystals with I’ counterion. Yield: 24 mg, 0.038 mmol, 88%.

^X H NMR (500 MHz, Acetonitrile-c/3) 6 8.07 (d, J = 9.5 Hz, 2H), 7.26 - 7.20 (m, 3H), 7.13 (dd, J = 7.4, 2.1 Hz, 2H), 6.93 (d, J = 2.5 Hz, 2H), 6.63 (dd, J = 9.5, 2.5 Hz, 2H), 6.58 (q, J = 7.1 Hz, 1H), 3.67 - 3.61 (m, 8H), 2.11 - 2.07 (m, 8H), 1.82 (d, J = 7.1 Hz, 3H), 1.61 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-c/3) 6 170.4, 162.1, 156.9, 153.2, 134.44, 134.43, 133.7, 129.2, 128.5, 127.0, 118.0, 113.5, 111.5, 69.9, 48.9, 41.7, 40.8, 24.8, 21.2.

HRMS: [M] ⁺ : calcd for [C34H ₃₉ N2O2] ⁺ : 507.3006, found: 507.3006.

Hydroxyethyl derivative 9 (104 mg, 0.220 mmol, 1 equiv.) was dissolved in 5 mL CH2CI2 then phenylacetic acid (45 mg, 0.330 mmol, 1.5 equiv.), DCC (55 mg, 0.264 mmol, 1.2 equiv.) and DMAP (catalytic amount) were added to the solution and the resulting mixture was stirred for 30 min. After completion, the reaction mixture was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc, 0% to 10%) affording 44 as a colourless oil.

Yield: 45 mg, 0.076 mmol, 35%. Mixture of four diastereomers:

^X H NMR (500 MHz, Chloroform-d) 6 7.38 - 7.25 (m, 5H), 7.12 - 6.53 (m, 3H), 5.17 - 4.99 (m, 1H), 4.19 - 4.12 (m, 2H), 3.74 - 3.44 (m, 5H), 3.30 - 3.12 (m, 4H), 3.05 - 2.83 (m, 2H), 2.79 - 2.62 (m, 1H), 2.01 - 1.89 (m, 4H), 1.88 - 1.84 (m, 3H), 1.82 - 1.78 (m, 2H), 1.78 - 1.73 (m, 3H), 1.42 - 1.16 (m, 12H), 0.78 - 0.71 (m, 3H).

MS: [M+H] ⁺ : calcd for [C ₄ OH ₅ IN ₂ 02] ⁺ : 591, found: 591.

2.1.12 Synthesis of target conjugate 45

Compound 44 (22 mg, 0.037 mmol, 1 equiv.) was dissolved in 1 ml CH2CI2- MeOH 1 : 1 and p-chloranil (28 mg, 0.112 mmol, 3 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion (30 min). Then, the reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH2CI2- MeOH 0% to 20%). The product was redissolved in C^CL. one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica, eluent: CH2CI2- MeOH 0% to 20%) was carried out, obtaining the final compound as blue crystals with I’ counterion.

Yield: 13 mg, 0.018 mmol, 49%.

¹ H NMR (500 MHz, Acetonitrile-d ₃ - TFA-d) 6 7.96 - 7.86 (m, 1H), 7.80 - 7.75 (m, 1H), 7.26 - 7.18 (m, 3H), 7.13 - 7.05 (m, 2H), 6.90 - 6.82 (m, 1H), 6.71 - 6.61 (m, 1H), 3.77 - 3.50 (m, 8H), 3.11 - 3.06 (m, 2H), 2.94 - 2.68 (m, 3H), 2.02 - 1.96 (m, 2H), 1.87 - 1.81 (m, 3H), 1.77 - 1.71 (m, 6H), 1.48 - 1.44 (m, 3H), 1.38 - 1.26 (m, 12H).

MS: [M] ⁺ : calcd for [C ₄₀ H ₄₉ N2O2] ⁺ : 589, found: 589.

2.1.13 Synthesis of l-(3,6-bis(dimethylamino)-9H-xanthen-9-yl)butyl benzoate (46)

Hydroxyethyl derivative (19) (16 mg, 0.044 mmol, 1 equiv.) was dissolved in 2 mL CH2CI2 then benzoic acid (11 mg, 0.088 mmol, 2 equiv.), DCC (15 mg, 0.075 mmol, 1.7 equiv.) and DMAP (catalytic amount) were added to the solution and the resulting mixture was stirred for 30 min. After completion, the reaction mixture was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc, 0% to 10%) affording compound 46 as a colourless oil. Yield: 10 mg, 0.0225 mmol, 51%.

Mixture of two diastereomers:

^X H NMR (500 MHz, Chloroform-d) 6 8.19 - 8.13 (m, 2H), 7.63 - 7.56 (m, 1H), 7.50 (t, J = 7.7 Hz, 2H), 7.43 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 6.90 (d, J = 2.7 Hz, 1H), 6.86 (d, J = 2.6 Hz, 1H), 6.68 (td, J = 9.0, 2.6 Hz, 2H), 5.35 (dt, J = 8.5, 4.3 Hz, 1H), 4.38 (d, J = 4.2 Hz, 1H), 2.99 (d, J = 13.3 Hz, 12H), 1.73 (d, J = 25.7 Hz, 6H), 1.31 - 1.19 (m, 2H), 1.18 - 1.13 (m, 2H), 0.68 (t, J = 7.2 Hz, 3H).

MS: [M+H] ⁺ : calcd for [C ₂₈ H33N ₂ O ₃ ] ⁺ : 445, found: 445.

2.1.14 Synthesis of N-(9-(l-(benzoyloxy)butyl)-6-(dimethylamino)-3H-xanthen-3-yl idene)-N- methylmethanaminium (47)

Compound 46 (10 mg, 0.021mmol, 1 equiv.) was dissolved in 1 ml CH ₂ CI ₂ - MeOH 1 : 1 and p-chloranil (12 mg, 0.050 mmol, 2.5 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion (30 min). Then, the reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH ₂ CI ₂ - MeOH 0% to 20%), obtaining the final compound 47 as blue crystals.

Yield: 6 mg, 0.0135 mmol, 64%.

^X H NMR (500 MHz, Chloroform-d) 6 8.06 - 8.01 (m, 2H), 7.65 - 7.58 (m, 1H), 7.48 (t, J = 7.8 Hz, 2H), 7.06 (d, J = 2.6 Hz, 2H), 6.88 (dd, J = 9.7, 2.6 Hz, 2H), 6.77 (dd, J = 9.4, 5.2 Hz, 1H), 3.37 (s, 12H), 2.48 (m, 1H), 2.10 (m, 1H), 1.76 - 1.70 (m, 1H), 1.68 (s, 6H), 1.52 (m, 1H), 1.04 (t, J = 7.4 Hz, 3H).

MS: [M] ⁺ : calcd for [C ₂ 8H3iN ₂ O3] ⁺ : 444, found: 444.

2.2 Synthesis of model target conjugates wherein the connecting chemical moiety is an oxycarbonyl linker

Synthesis of model target conjugates from key intermediates - carbamates

2.2.1 Synthesis of l-(10,10-dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethyl 4- phenylpiperazine-1 -carboxylate (48) Compound 28 (177 mg, 0,128 mmol, 1 equiv.) was dissolved in 5 mL CH2CI2 (stabilized with amylene) then /V-phenylpiperazine (30 pL, 0,192 mmol, 1.5 equiv.), TEA (53 pL, 0.384 mmol, 3 equiv.) and DMAP (15 mg, 0.128 mmol, 1 equiv.) were added and the was stirred for 1 hour at room temperature. After completion, the mixture was concentrated onto celite in vacuo and purified by flash chromatography on silica (eluent: hexane-EtOAc 0% to 10%) to afford 48 as a colourless oil.

Yield: 31 mg, 0.054 mmol, 42%.

Mixture of two diastereomers:

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 7.27 (dd, J = 8.8, 7.2 Hz, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 8.3

Hz, 1H), 7.00 - 6.95 (m, 2H), 6.89 - 6.83 (m, 1H), 6.73 (d, J = 2.5 Hz, 1H), 6.70 (d, J = 2.5 Hz, 1H), 6.52 -

6.46 (m, 2H), 4.93 - 4.83 (m, 1H), 4.09 (d, J = 5.6 Hz, 1H), 3.70 - 3.45 (m, 4H), 3.31 - 3.26 (m, 8H), 3.18 -

3.07 (m, 4H), 2.03 - 1.97 (m, 8H), 1.66 (d, J = 1.6 Hz, 6H), 0.79 (d, J = 6.5 Hz, 3H).

¹³ C NMR (126 MHz, Chloroform-d) 6 155.2, 151.5, 147.0, 147.0, 146.9, 146.0, 145.0, 130.4, 129.9, 129.4,

121.8. 120.5, 116.9, 116.9, 110.7, 110.2, 109.9, 109.5, 109.4, 78.4, 61.6, 49.7, 49.6, 47.9, 46.2, 38.8, 34.8,

34.5, 25.6, 25.6, 15.9, 14.8.

HRMS: [M+H] ⁺ : calcd for [C37H ₄₇ N ₄ O2] ⁺ : 579.3693, found: 579.3686.

2.2.2 Synthesis of l-(9,9-dimethyl-10-(l-((4-phenylpiperazine-l-carbonyl)oxy)et hyl)-7-(pyrrolidin-l- yl)anthracen-2(9H)-ylidene)pyrrolidin-l-ium (49)

Compound 48 (10 mg, 0.017 mmol, 1 equiv.) was dissolved in 1 mL CH2CI2- MeOH 1 : 1 in the dark and p-chloranil (17 mg, 0.068 mmol, 4 equiv.) was added to the solution. The mixture was stirred until the LC- MS indicated full conversion (30 min). The reaction mixture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (Eluent: CH2CI2- MeOH 0% to 20%).

The product was redissolved in CH2CI2 (1 mL), one drop of HI (57% in water) was added, the mixture was stirred for 5 min and another flash chromatography (on silica, eluent: CH2CI2 - MeOH 0% to 20%) was carried out, obtaining the final compound as blue crystals with L counterion. Yield: 7 mg, 0.010 mmol, 57%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.28 (d, J = 9.5 Hz, 2H), 7.27 - 7723 (m, 2H), 6.97 (d, J = 2.5 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 6.86 (t, J = 7.3 Hz, 1H), 6.80 (dd, J = 9.6, 2.5 Hz, 2H), 6.56 (q, J = 7.1 Hz, 1H), 3.80 - 2.85 (m, 8 H and 4H), 2.10 - 2.07 (m, 8H), 1.87 (d, J = 7.1 Hz, 3H), 1.65 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-d ₃ ) 6 158.1, 154.8, 154.3, 135.7, 135.6, 130.6, 119.2, 114.7, 112.6, 71.5,

49.9, 42.8, 34.1, 30.4, 25.8, 22.5.

HRMS: [M] ⁺ : calcd for [C ₃₇ H ₄₅ N ₄ O ₂ ] ⁺ : 577.3537, found: 577.3538. Synthesis of model target conjugates from key intermediates - carbonates

Synthesis of l-(10,10-dimethyl-3,6-di(pyrrolidin-l-yl)-9,10-dihydroanthra cen-9-yl)ethyl phenyl carbonate

(50)

Compound 51 (50 mg, 0.128 mmol, 1 equiv.) was dissolved in CH2CI2 (3 mL) then phenol (18 mg, 0.192 mmol, 1.5 equiv.), triethylamine (53 pL, 0.384 mmol, 3 equiv.) and DMAP (16 mg, 0.128 mmol, 1 equiv.) was added and the reaction was stirred for 1 hour at room temperature. After completion, the reaction was concentrated onto celite and purified by flash chromatography on silica (eluent: hexane-EtOAc 0% to 10%) to afford 50 as a colourless oil.

Yield: 32 mg, 0.063 mmol, 49%.

Mixture of two diastereomers:

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 7.39 (dd, J = 8.5, 7.4 Hz, 2H), 7.28 - 7.24 (m, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.11 - 7.07 (m, 2H), 6.73 (dd, J = 9.0, 2.5 Hz, 2H), 6.55 - 6.47 (m, 2H), 4.90 (q, J = 6.3 Hz, 1H), 4.11 (d, J = 5.7 Hz, 1H), 3.32 - 3.27 (m, 8H), 2.03 - 1.98 (m, 8H), 1.68 (s, 6H), 1.03 (d, J = 6.4 Hz, 3H).

¹³ C NMR (126 MHz, Acetonitrile-c/3) 6 154.0, 152.3, 148.5, 148.4, 147.0, 146.4, 131.1, 130.8, 130.6, 130.4, 127.0, 122.4, 122.3, 121.6, 120.7, 111.2, 111.1, 110.4, 110.3, 82.5, 48.5, 48.5, 48.0, 39.7, 35.2, 33.5, 26.2, 25.7, 17.4.

HRMS: [M+H] ⁺ : calcd for 511.2955, found: 511.2951.

Synthesis of l-(9,9-dimethyl-10-(l-((phenoxycarbonyl)oxy)ethyl)-7-(pyrrol idin-l-yl)anthracen-2(9H)- ylidene)pyrrolidin-l-ium iodide (51)

Compound 50 (32 mg, 0.063 mmol, 1 equiv.) was dissolved in 1 mL CH2CI2- MeOH 1 : 1 in the dark and p-chloranil (64 mg, 0.251 mmol, 4 equiv.) was added to the solution. The mixture was stirred until the LC- MS indicated full conversion (30 min). The reaction miture was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (eluent: CH2CI2- MeOH 0% to 20%).

The product was redissolved in CH2CI2, one drop of HI (57% in water) was added, the mixture was stirred for 5 min and another flash chromatography (on silica, eluent: CH2CI2 - MeOH 0% to 20%) was carried out, obtaining the final compound as blue crystals with I counterion. Yield: 34 mg, 0.0534 mmol, 89%. ^X H NMR (500 MHz, Acetonitrile-c/3) 6 8.21 (d, J = 9.5 Hz, 2H), 7.38 (t, J = 7.9 Hz, 2H), 7.27 (t, J = 7.4 Hz, 1H), 7.12 - 7.05 (m, 2H), 7.00 (d, J = 2.5 Hz, 2H), 6.82 (dd, J = 9.5, 2.5 Hz, 2H), 6.64 (q, J = 7.0 Hz, 1H), 3.72 - 3.62 (m, 8H), 2.11 - 2.06 (m, 8H), 1.95 (d, J = 1.8 Hz, 3H), 1.66 (s, 6H).

¹³ C NMR (126 MHz, Acetonitrile-c/3) 6 160.3, 157.1, 153.4, 152.6, 151.0, 134.5, 129.7, 126.4, 121.2, 120.9, 118.1, 113.9, 111.9, 73.7, 49.0, 41.9, 24.8, 21.4.

HRMS: [M] ⁺ : calcd for [C33H37N ₂ O ₃ ] ⁺ : 509.2799, found: 509.2802.

2.3 Synthesis of target conjugates wherein the connecting chemical moiety between the photoremovable protecting group and the cargo unit is a self-immolative linker

Synthesis of photocage-SN38 conjugates

2.3.1 Synthesis of compound 53

SN-38 (177 mg, 0.45 mmol, 1 equiv.) was dissolved in dry DMF, then compound 52 (230 mg, 0.54 mmol, 1.2 equiv.) ³⁹ , triethylamine (136 pL, 1.35 mmol, 3 equiv.) and DMAP (2 mg, 0.035 mmol, 0.03 equiv.) was added under N2 atmosphere. The reaction was stirred at 80 °C for 2 hours. The solvent was evaporated, concentrated onto silica gel and purified by flash chromatography on silica (eluent: CH2CI2- MeOH 0% to 20%).

Yield: 400 mg, 0.356 mmol, 79%.

^X H NMR (500 MHz, DMSO-dg) 6 8.21 - 8.17 (m, 1H), 7.96 (s, 1H), 7.66 - 7.59 (m, 1H), 7.34 (s, 1H), 6.50 (s, 1H), 5.44 (s, 2H), 5.35 (s, 2H), 3.63 (t, J = 5.1 Hz, 1H), 3.52 - 3.42 (m, 3H), 3.23 - 3.13 (m, 4H), 2.90 (s, 3H), 2.74 (s, 3H), 1.93 - 1.82 (m, 2H), 1.45 - 1.36 (m, 9H), 1.34 - 1.29 (m, 3H), 0.89 (t, J = 7.3 Hz, 3H).

2.3.2 Synthesis of compound X1 -SN38

Compound 54: l-(3,6-di(pyrrolidin-l-yl)-9H-xanthen-9-yl)ethan-l-ol (3) (100 mg, 0.274 mmol, 1 equiv.) was dissolved in acetonitrile (5 mL), DSC (352 mg, 1.37 mmol, 5 equiv.), TEA (382 pL, 2.74 mmol, 10 equiv.) and DMAP (cat.) were added to the solution and stirred for overnight at room temperature. After the LC- MS indicated complete conversion, the reaction was extracted 5 times with saturated NaHCOs, the organic phase was dried over MgzSCU, and the solvent was evaporated. The crude product was used immediately without further purification. Yield: 136 mg, 0.270 mmol, 99%.

MS: [M+H] ⁺ : calcd for [CzsHszNsOeF: 506, found: 506.

Compound 56: SN38-Boc-protected self-immolative linker (53) (166 mg, 0.274 mmol, 1 equiv.) was dissolved in CH2CI2 and TFA (25% for the mixture) was added and stirred at room temperature for 1 hour. After the completion of the deprotection, the volatiles were evaporated and the solid was thoroughly dried.

The succinimidyl carbonate (54) (0.274 mmol, 1 equiv.) was dissolved in CH2CI2, the SN-38-self-immolative linker and triethylamine (114 pL, 0.822 mmol, 3 equiv.) was added, and stirred for 1 hour at room temperature. The reaction was concentrated onto silica gel and purified by flash chromatography on silica (eluent: CH2CI2- MeOH 0% to 20%). The compound was dissolved in acetonitrile and purified once more by reverse-phase chromatography (eluent: water-Acetonitrile 5% to 100%).

Yield: 127 mg, 0.142 mmol, 52%.

MS: [M+H] ⁺ : calcd for [C ₅ iH ₅ 7N ₆ 0g] ⁺ : 897, found: 897.

Compound X1/-SN38: the reduced photocage (56) (30 mg, 0.034 mmol, 1 equiv.) was dissolved in CH2CI2 - MeOH 1-1 mL) and p-chloranil (17 mg, 0.067 mmol, 2 equiv.) was added to the solution. The mixture was stirred until the LC-MS indicated full conversion (30 min). The reaction was evaporated and applied directly to a silica gel column and purified by flash chromatography on silica (Eluent: CH2CI2- MeOH 0% to 20%).

The product was redissolved in CH2CI2, one drop of HI (57% in water) was added, stirred for 5 min and another flash chromatography (on silica) was carried out, obtaining the final compound with r counterion (eluent: CH ₂ CI ₂ - MeOH 0% to 20%). Yield: 18 mg, 0.018 mmol, 52%.

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.43 - 8.02 (m, 5H), 7.57 - 7.49 (m, 1H), 7.01 - 6.90 (m, 2H), 6.72 - 6.51 (m, 1H), 6.13 (s, 1H), 5.72 - 5.64 (m, 1H), 5.51 - 5.30 (m, 3H), 3.65 - 3.46 (m, 11H), 3.33 - 3.14 (m, 4H), 3.08 (d, J = 2.7 Hz, 2H), 3.02 (s, 2H), 2.96 - 2.89 (m, 2H), 2.17 - 2.07 (m, 11H), 1.90 - 1.81 (m, 4H), 1.42 - 1.29 (m, 4H), 1.24 - 1.17 (m, 2H), 1.09 - 1.00 (m, 4H).

HRMS: [M] ⁺ : calcd for [C ₅ iH ₅ 5N ₆ 0g] ⁺ : 895.4025, found: 895.4028.

2.3.3 Synthesis of compound X2'-SN38

Compound 57 (112 mg, 0.198 mmol, 1 equiv.) was dissolved in dry DMF, then crude compound 33 (120 mg, 0.198 mmol, 1 equiv.), and DIPEA (69 pL, 0.396 mmol, 2 equiv.) were added and the reaction mixture was stirred for 30 min at room temperature. After the LC-MS indicated full conversion, the solvent was evaporated, the reaction was concentrated onto silica gel and purified by flash chromatography on silica gel (eluent: CH2CI2- MeOH 0% to 20%) to obtain compound X2'-SN38 as a blue solid. Yield: 107 mg, 0,116 mmol, 58%

^X H NMR (500 MHz, Acetonitrile-d ₃ ) 6 8.35 - 7.93 (m, 3H), 7.88 - 7.47 (m, 2H), 6.95 - 6.76 (m, 2H), 6.78 - 6.50 (m, 3H), 5.61 (d, J = 16.6 Hz, 1H), 5.38 (d, J = 16.6 Hz, 1H), 5.37 - 5.29 (m, 2H), 3.64 - 3.48 (m, 11H), 3.30 - 3.06 (m, 5H), 3.03 (s, 1H), 2.97 - 2.76 (m, 3H), 2.10 - 2.01 (m, 9H), 1.88 - 1.77 (m, 3H), 1.66 - 1.51 (m, 4H), 1.46 (d, J = 5.3 Hz, 2H), 1.38 - 1.21 (m, 5H), 1.02 - 0.93 (m, 3H).

HRMS: [M] ⁺ : calcd for [Cs^iNgC^: 921.4545, found: 921.4551.

2.4 Comparative examples

In these examples R7 = H, in which case the expected compounds could not be isolated in their dye form, only in the form of a chloranil complex. In this form, only the exo-form is present, which is demonstrated by the LC-MS chromatograms, where the mass is equal to the mass of the photocage and the chloranil together (with the characteristic isotope distribution of four Cl atoms of the chloranil). NMR characterisation also showed that the double bond is located outside of the ring system (singlet peak in the region of 7-9 ppm), also confirming that R7 = H compounds are only stable in the form of a complex that in fact does not absorb in the visible range, thus cannot be used as visible light photocages.

This observation is in accordance with the results of Klan et al. ³¹

2.4.1 Synthesis of (3,6-di(piperidin-l-yl)-9H-xanthen-9-ylidene)methyl benzoate chloranil complex (59): Compound 58 (18 mg, 0.037 mmol, 1 equiv.) was dissolved in 1 mL CH2CI2- MeOH 1 : 1 in the dark and p-chloranil (14 mg, 0.055 mmol, 1.5 equiv.) was added to the solution. The mixture was stirred for 1 h at room temperature. The LC-MS showed full conversion to the chloranil complex of the photocage. The solvent was evaporated and purified by reverse-phase column chromatography (Eluent: Water-MeCN- 0.1%TFA).

The expected product (dye-form of the photocage) could not be isolated, only in the form of the chloranil complex in exo-form.

^X H NMR (500 MHz, Chloroform-d) 6 8.49 (d, J = 9.7 Hz, 2H), 8.10 (s, 1H), 8.02 - 7.92 (m, 2H), 7.64 - 7.57 (m, 1H), 7.44 (t, J = 7.8 Hz, 2H), 7.21 (dd, J = 9.8, 2.5 Hz, 2H), 6.98 (d, J = 2.6 Hz, 2H), 3.77 (d, J = 5.2 Hz, 8H), 1.80 (d, J = 3.2 Hz, 8H).

MS for the chloranil complex: [M+H] ⁺ : calcd for [C37H33Cl4N2O ₅ ] ⁺ : 725, found: 725.

MS for the exo-form: [M+H] ⁺ : calcd for [C3iH33N2O3] ⁺ : 481, found: 481.

2.4.2 Synthesis of (3,6-bis(dimethylamino)-10,10-dimethylanthracen-9(10H)-ylide ne)methyl benzoate chloranil complex (61)

Compound 60 (20 mg, 0.0462 mmol, 1 equiv.) was dissolved in 1 mL CH2CI2- MeOH 1 : 1 in the dark and p-chloranil (28 mg, 0.115 mmol, 2.5 equiv.) was added to the solution. The mixture was stirred for 1 h at room temperature. The LC-MS showed full conversion to the chloranil complex of the photocage. The solvent was evaporated and purified by reverse-phase column chromatography (Eluent: Water-MeCN- 0.1%TFA).

The expected product (dye-form of the photocage) could not be isolated, only in the form of the chloranil complex in exo-form.

^X H NMR (500 MHz, Chloroform-d) 6 8.59 (d, J = 9.6 Hz, 2H), 8.19 (s, 1H), 8.05 - 7.98 (m, 2H), 7.64 - 7.59 (m, 1H), 7.46 (t, J = 7.7 Hz, 2H), 7.14 (d, J = 2.6 Hz, 2H), 6.95 (dd, J = 9.7, 2.6 Hz, 2H), 3.41 (s, 12H), 1.74 (s, 6H).

MS for the chloranil complex: [M+H] ⁺ : calcd for [Ca-iHaiCLNzC r: 671, found: 671.

MS for the exo-form: [M+H] ⁺ : calcd for [CjgHaiNzChr: 427, found: 427.

3. Spectroscopic properties of the target conjugates

The spectroscopic properties of the target conjugates were determined in PBS. All derivatives have one- photon absorption in the visible range and no signs of aggregation was observed (absorbance measured up to 25 pM) that demonstrates good water solubility for all compounds. Compounds with Q. = O have absorption bands in the green-yellow region while Q. = CMe2 derivatives absorb in the red (up to 700 nm). All of the compounds have moderate to high absorption coefficients and significant fluorescence. The spectroscopic details can be found in Table 1. In the table, A _max means absorption maximum; Z, _em means fluorescence emission maximum; s means molar absorption coefficient (at the absorption maximum). Furthermore, figure 5 shows the structure and absorption spectra of selected target conjugates.

Table 1

Optical properties of selected compounds 4. Uncaging studies of the target conjugates

The photouncaging was evaluated by HPLC-MS using green (4W input power, A _max = 549 nm half-width: 16 nm, output power: 72 mW), orange (4W input power, A _max = 605 nm half-width: 10 nm, output power: 140 mW) and red (4W input power, A _max = 658 nm half-width: 10 nm, for Q. = CMe2 output power: 210 mW) LED irradiation. For all compounds the experiments were performed in 90% water/MeCN mixtures as well as in 90% PBS/MeCN (0.1 mM concentration). In all cases, the irradiation led to the release of the model active agents with significant differences based on the photoremovable protecting group, cargo unit and the connecting chemical moiety as well as on the water content of the uncaging medium. Comparing the green light absorbing target conjugates, irradiation of 39 led to the release of 54% phenylacetic acid in 60 s (Figure 6b), while 180 s (for 50%) was required for the same result in case of 41. Approximately 65% of phenylacetic acid was released after 180s of irradiation with orange light from model target conjugate 41 (Figure 6a). The current state of the art BODIPY derivative (for the structure, see the next section), ²⁰ - ²¹ released only 12% phenylacetic acid after irradiation for 1800 s. Contrary to the general expectations that increasing the wavelength diminishes photolysis rates, the uncaging of the redshifted Q. = CMe2 derivative 43 was also very effective, reaching >70% release of phenylacetic acid in 120 s after red light irradiation (Figure 6c). The high efficiency of 43 as a red light-activatable photoremovable protecting group was also compared to a ^-extended BODIPY derivative (substrate 7 in REF ²¹ , using methanol as the solvent in a concentration of 0.1 mM), the current state of the art in this region of visible light. It was found that with a similar cargo unit, an approximately 5-fold increase in the photolysis rate could be observed. Also, the BODIPY reference was insoluble in water rendering it unsuitable for biological applications.

Importantly, except in the case of compounds wherein the connecting chemical moiety was a carbonate moiety (i.e., compound 51, Figure 6f), the target conjugates were stable in water as well as in PBS for 24 hours in the dark. The stability and uncaging rates of several leaving groups was also assessed. Comparing the most important functional groups (carboxylic acid, amine, phenol, alcohol) in potential active agents connected via the typical linkages in the target conjugates, the carbonate bond was the most labile (compound 51, >70% decomposition in 30 s with red light at 50% power) although the long-term dark stability was poor. None of the other model active agents were released in significant quantities, however, after 24-28 hours in the dark (for dark stability data see Figures 6a-6h, right side).

Using HPLC-MS, not only the appearance of the released model active agents could be monitored but also the products of photolysis were detectable. In all cases, the main product was the hydroxyethyl derivative also known as compound X wherein A = C ⁺ and W = OH, as can be seen on Figures 6a-6h. In case of photoactivatable conjugate X2'-SN38, irradiation with red light led to the release of the linker- appended SN38 conjugate after 5-10 mins in PBS containing 10% MeCN (0.1 mM) with subsequent self- immolation, liberating free SN38 (Figure 6h). Uncaging guantum yield (QY) determination

The relative photochemical quantum yields of the uncaging reactions together with the degradation quantum yields were determined from the results of the quantitative HPLC-UV experiments. In all cases, green light irradiation was used since all target conjugates absorbed in this range as well as the reference BODIPY. The photochemical quantum yields were determined using the following equation: where is the photochemical quantum yield of the sample

^> _{u re} f is the photochemical quantum yield of the reference k is the obtained reaction rate constant of the photoreaction of the sample k _re f is the obtained reaction rate constant of the photoreaction of the reference c is the concentration of the sample c _re f is the concentration of the reference

V is the volume of the sample

V _re f is the volume of the reference a is the absorption correction factor of the sample a _re f is the absorption correction factor of the reference

The absorption correction factors (a) were determined from the molar absorption coefficients and the green LED emission data obtained by the fluorimeter. We have calculated the transmittance (T) at each wavelength and calculated the efficiency of the illumination of each sample that resulted in the effectiveness of the irradiation. In other words, the transmittance at each wavelength together with the emission data of the light source can be used to calculate the percentage of absorbed photons according to the following equation: where

T(A) is the transmittance at each wavelength (determined from the molar absorption coefficients)

F(A) is the LED emission at each wavelength is the LED emission range. The rate constants of the photoreactions were determined by considering the reactions monoexponential with an initial linear phase (up to 10-20% degradation). First, the concentration vs. HPLC-UV peak were determined for each cargo moieties. Then, in each case, a linear fitting of the time vs. released cargo concentration traces was performed that resulted in the rate constant k as determined from the fitted linear equation. The rate constants were determined for both the reference (with known u) and the sample. For reference, a commonly known BODIPY derivative was used with a similar phenylacetic acid-derived cargo unit (substrate 7 from REF ⁴⁰ ), _u = 0.15%:

The calculated photochemical quantum yields together with uncaging cross-sections or quantum efficiencies (product of the molar absorption coefficient at a given wavelength, shown in parentheses and the photochemical quantum yield) are shown in Table 2. In the table, _u is the photochemical quantum yield of the release; deg is the photochemical quantum yield of degradation; s x <J _U is the uncaging cross-section or quantum efficiency of the release at a given wavelength in parentheses.

Table 2

Uncaging quantum yields of the photocages ^a Uncaging quantum yield, calculated from active agent release ^b Degradation quantum yield, calculated from the disappearance of the target conjugate ^c Uncaging quantum efficiencies with the wavelength in parentheses ^d Exact release rate was not determined due to the complexity of the mixture In general, the commonly accepted value of uncaging cross-section required for biological applications is above "'100 M^cm ¹ . ² As it is evident from Table 2, our PPGs exceed this value by an order of magnitude which is especially surprising considering their red-shifted absorption. It is generally expected that increasing the wavelength diminishes the photochemical quantum yield. Surprisingly, even the model target conjugates with the most redshifted absorption the uncaging cross-section reached 1400 M’ m ¹ , an unexpectedly high value.

Without wishing to be bound by any theory, it is believed that the surprisingly high efficiency of our PPGs is attributed to the low energy barrier of the heterolytic dissociation upon photoexcitation. The ~10% degradation quantum yield of the rhodol derivative 39 might be result of an energy barrier-free dissociation in the excited state facilitated by the lack of a positive charge on the chromophore scaffold.

5. Fluorescence microscopy experiments with the target conjugates X'-SN38

Due to their high fluorescence, the cell permeability of compounds X1'-SN38 and X2'-SN38 were tested in live SK-OV-3 (human ovarian carcinoma) cells using confocal microscopy. The colocalization was tested with commercially availabla MitoTrackers and LysoTrackers. In both cases, the compounds were cell permeable, an important feature of this invention. Interestingly, differing from most rhodamine derivatives that commonly localize in the mitochondria, fluorescence of compound X2'-SN38 (Q. = CMez) was observed only in the lysosomes (in concentrations 100 nM and 1 uM, no wash conditions, Figure 7a). In case of compound X1'-SN38 (Q. = O), clear localization in the mitochondria was observed, concominant with results observable with rhodamine B. The spatial targeting of light irradiation was also tested using the built-in red laser of the instrument. Only a small fraction of the cells or simply one cell was irradiated with high intensity 638 nm laser light (for about 5-10 frames). The decrease of fluorescence (related to the photolysis reaction) was only observed in the irradiated area. See Figure 7b for the images.

Overall, these experiments serve as evidence for the cell permeability, biocompatibility and excellent ability of this invention to determine the localization of the photoactivatable conjugates via fluorescence microscopy.

6. Cytotoxicity studies of the target conjugates X'-SN38

To determine phototoxic properties of compounds X1'-SN38 and X2'-SN38, standard MTT assays were performed on SK-OV-3 cells. To irradiate multiple specimens, an LED panel was custom-made for a 48- well plate consisting of 24 LEDs using green (4W input power, _max = 549 nm half-width: 16 nm, output power: 72 mW), orange (4W input power, _max = 605 nm half-width: 10 nm, output power: 140 mW) and red (4W input power, A _max = 658 nm half-width: 10 nm, for Q. = CMe2 output power: 210 mW) LED irradiation. The board was specifically designed for the plates and offer water cooling to minimize heat shock to cells. Every second well can be irradiated with an individual LED. First, the cytotoxicity of the free active agent, SN38 was determined to be EC50 ~ 6 nM. Treatment of cells with the photoactivatable SN38 derivatives for 72 hours in the absence of light (without washing) produced moderate cytotoxic effects, sigificantly lowering the effectiveness of the active agent. This confirms that SN38 is a suitable candidate for light-activated prodrug applications. Brief irradiation of the cells with red light (60 s) after treatment with compound 10 for 90 min resulted in almost complete restoration of the activity of SN38 (EC50 for X2'-SN38 after irradiation: ~24 nM), providing an excellent photoindex of 92, almost an order of magnitude higher than current state of the art ruthenium-based PACT agents ³⁰ . Reducing the irradiation time down to 10 s still produced significant phototoxicity in higher concentrations (see Figure 8a for compound X2'-SN38) even assuming incomplete uncaging as a result of the substantial dark stability of compound X2'-SN38. This effect is particularly important for further in vivo applications where lower irradiance can create significantly decreased uncaging yields.

For the complete uncaging of compound X1'-SN38, a higher irradiation time was required with orange LEDs, however, this compound still produced a significant phototoxic effect with a photoindex of 64.

Table 3

Concentration and light irradiation dependent cell viabilities of the target conjugate X2'-SN38 Table 4

Cytotoxicity of the photoactivatable SN38 derivatives

For compound X2'-SN38, red light irradiation was applied (60 s), for compound X1'-SN38, green light irradiation was applied (15 min) as well as orange light (5 min).

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