A tandem-locked chemiluminescent probe for imaging of macrophage polarization in cancer immunotherapy

Abstract

Disclosed herein is a compound of formula (I): (I) where: A represents a tandem-lock biomarker responsive group; B represents a self-immolative linker; and C represents a chemiluminescent group, or a pharmaceutically acceptable salt or solvate thereof.

Description

[0001] A TANDEM-LOCKED CHEMILUMINESCENT PROBE FOR IMAGING OF MACROPHAGE POLARIZATION IN CANCER IMMUNOTHERAPY

[0002] Field of Invention

[0003] The present invention generally relates to probes for imaging in cancer immunotherapy, and more particularly relates to tandem-locked chemiluminescent probes that have improved specificity and precision in the diagnosis field, and are particularly suited for real-time imaging of immune response in cancer immunotherapy.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Cancer immunotherapy, which stimulates the patients' own immune system to fight cancer, provides long-lasting protection against cancer. The infiltration and activation of leukocytes in the tumor microenvironment (TME) are closely associated with cancer progression and patient responses to immunotherapy. In particular, tumor-associated macrophages (TAMs) represent one of the main tumor-infiltrating immune cell types and play both anti- and pro-tumor functions based on different phenotypes. In contrast to M2 TAMs that promote tumor cell proliferation and invasion, M1 TAMs have anti-tumor effects and trigger tumor cell death by either directly releasing cytotoxic reactive oxygen species and nitric oxide (NO), or antibody-dependent cell- mediated cytotoxicity. Therefore, targeted M1 TAM polarization from naive MO macrophages and repolarization of M2 to M1 TAMs are effective tumor treatment options. To enable prognosis and improved patient outcomes, detection of M1 TAM activation during immunotherapy is essential. However, current clinical diagnostic approaches include histological, flow cytometry, or mass cytometry analyses of tumor specimens from biopsy, are invasive and suffer from risks of excessive bleeding and tumor cell seeding.

[0007] Molecular imaging that sensitively detects biomarker-of-interest offers a noninvasive approach to visualize biological processes at the cellular or subcellular levels in both fundamental studies and clinical practice. To detect M1 macrophage activation and repolarization, fluorescence imaging agents have been reported to actively target biomarkers such as GLUT 1 glucose transporter, and SLC18B1 transporter, which however show “always on” signal and thus background signal from nonspecific accumulation of probes. Moreover, activatable fluorescent probes that specifically turn on their fluorescence in the presence of biomarkers of M1 macrophages (such as NO, low pH and hypochlorite) have been developed. However, the activatable fluorescent probes fail to distinguish M1 TAMs over that in normal tissues or peripheral blood. Besides, they share the general drawback of fluorescence imaging that is tissue autofluorescence, resulting in poor signal-to-background ratio (SBR).

[0008] Without the requirement of real-time light excitation, chemiluminescence (CL) imaging have minimized background noise and thus higher SBR than fluorescence imaging. Recently, biomarker-activatable CL probes have been reported for a variety of biomarkers, including enzymes, reactive oxygen and nitrogen species, and metabolites. However, these CL probes typically emit visible light, and only nine of them emit near-infrared region (NIR) light. Besides, these CL probes suffer from drawbacks such as poor CL quantum yields and short CL halflives, which significantly hinder in vivo imaging applications. Despite the potential of CL imaging in in vivo detection of macrophage polarization, the challenges lie in the synthesis of highly efficient NIR CL probes with high specificity towards macrophages in the tumor tissues over normal tissues.

[0009] Therefore, to overcome at least one of the aforementioned problems, there exists a need for new chemiluminescent probes for real-time imaging of immune response in cancer immunotherapy.

[0010] Summary of Invention

[0011] Aspects and embodiments of the invention are provided in the following numbered clauses.

[0012] 1. A compound of formula (I):

[0013] A - B - C (I) where:

[0014] A represents a tandem-lock biomarker responsive group;

[0015] B represents a self-immolative linker; and

[0016] C represents a chemiluminescent group, or a pharmaceutically acceptable salt or solvate thereof.

[0017] 2. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Clause 1 , wherein B is selected from the group consisting of: where:

[0018] A represents NH, S, or O;

[0019] Ri represents H, F, or NO2;

[0020] R2represents H or F; the wiggly line represents the point of attachment to C; and the double-crossed line represents the point of attachment to A.

[0021] 3. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Clause 2, wherein B is selected from the group consisting of: where the wiggly line represents the point of attachment to C; and the double-crossed line represents the point of attachment to A, optionally wherein B is selected from the group consisting of: 4. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding clauses, where A is selected from: represents the point of attachment to the rest of the molecule, and R3 represents:

[0022]

[0023] , where the double crossed line represents the point of attachment to the rest of the molecule.

[0024] 5. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding clauses, wherein C is selected from:

[0025] (ai)

[0026] where:

[0027] X represents O, S, Se, or SO2; to provide a polymeric component having a number average molecular weight of from 500 to 10,000 Daltons, such as 500 Daltons, 1 ,000 Daltons, 2,000 Daltons, or 5,000 Daltons;

[0028] FU represents OH, OMe, NEt2, or NH2;

[0029] Rs represents H, Cl, or Br;

[0030] Re represents H, Cl, or Br; the double-crossed line represents the point of attachment to B; the wiggly line represents the point of attachment to the rest of the molecule; and EWG for (ai) to (aiv) is selected from:

[0031] R7 represents H, Cl, or Br; alternatively, the EWG for (aiii) and (aiv) is selected from the following:

[0032] , , where: the double-crossed line represents the point of attachment to the rest of the molecule,

[0033] Rs represents NH2, NMe2, OH, or OMe,

[0034] R9 represents H or CN, and

[0035] R10 represents H or halogen.

[0036] 6. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Clause 1 , wherein there is provided a tandem-locked biomarker-activatable chemiluminescent probe comprising:

[0037] (a) an adamantylidene-1 ,2-dioxetane-based chemiluminophore;

[0038] (b) wherein the adamantylidene-1 ,2-dioxetane-based chemiluminophore is caged at its hydroxyl group by a self-immolative linker (e.g. p-aminobenzyl alcohol), which is in turn connected to a GGT-cleavable y-glutamate, and the carboxyl group of the y-glutamate is further caged by a NO-cleavable o-phenylenediamine.

[0039] 7. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Clause 6, wherein:

[0040] (ai) the adamantylidene-1 , 2-dioxetane-based chemiluminophore further comprises an azide group; and

[0041] (aii) the adamantylidene-1 , 2-dioxetane-based chemiluminophore further comprises a DBCO-mPEG2k group conjugated to the rest of the molecule by a triazole group.

[0042] 8. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding clauses, wherein C is selected from:

[0043] 9. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding clauses, wherein the compound is

[0044] 10. An in vitro method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of: (a) contacting a sample obtained from a subject undergoing immunotherapy for cancer with a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Clauses 1 to 9 for a period of time; and

[0045] (b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not.

[0046] 11. A method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:

[0047] (a) administering to a subject undergoing immunotherapy for cancer a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Clauses 1 to 9 for a period of time; and

[0048] (b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not.

[0049] 12. Use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Clauses 1 to 9 in the manufacture of a medicament for determining whether an immunotherapy is effective in a subject undergoing immunotherapy for cancer.

[0050] 13. A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Clauses 1 to 9 for use in determining whether an immunotherapy is effective in a subject undergoing immunotherapy for cancer.

[0051] 14. A method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:

[0052] (a) administering to a subject undergoing a treatment for a disease involving an immune response, other than a cancer, a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Clauses 1 to 9 for a period of time; and

[0053] (b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not, optionally wherein the disease involving an immune response is an autoimmune disease.

[0054] 15. Use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Clauses 1 to 9 in the manufacture of a medicament for determining whether an immunotherapy is effective in a subject undergoing a treatment for a disease involving an immune response, other than a cancer, optionally wherein the disease involving an immune response is selected from an autoimmune disease.

[0055] 16. A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Clauses 1 to 9 for use in determining whether a drug-treatment is effective in a subject undergoing a treatment for a disease involving an immune response, other than a cancer, optionally wherein the disease involving an immune response is an autoimmune disease.

[0056] Drawings

[0057] FIG. 1 depicts (a) synthetic routes for chemiluminophores (DPDx, X = O or S): (i) compound 1x (X = O or S), 1 ,3-indandione, AC2O, 145 °C, 2 h, 83% product yield for compound 2o and 68% product yield for compound 2s; (ii) compound 2x (X = O or S), piperidine, hydroxybenzaldehyde-adamantan-2-ylidene (chemiluminescence intermediate hydroxybenzaldehyde-adamantan-2-ylidene (HBAY)), acetonitrile (ACN), N2, reflux, 2 h, 93% yield for compound 3o and 87% yield for compound 3s; (iii) compound 3x (X = O or S), methylene blue, white light, air, 0 °C, 2 h, 96% yield for compound DPDo and 93% yield for compound DPDs. (b) Synthetic routes for other chemiluminophores (BPDx, X = O or S): (iv) compound 1 x (X = O or S), benzo[b]thiophen-3(2H)-one-1 ,1 -dioxide, AC2O, 145 °C, 2 h, 57% yield for compound 4o and 53% yield for compound 4s; (v) compound 4x (X = O or S), piperidine, HBAY, ACN, N2, reflux, 2 h, 83% yield for compound 5o and 75% yield for compound 5s; (vi) compound 5x (X = O or S), methylene blue, white light, air, 0 °C, 2 h, 94% yield for compound BPDo and 95% yield for compound BPDs. (c) Synthetic routes for activated chemiluminophores (ADPDx and ABPDx, X = O or S): (vii) DPDx (X = O or S), triethylamine, dimethyl sulfoxide (DMSO) / H2O (9 / 1 , V / V), room temperature (r.t.), 4 h, 87% yield for ADPDo and 93% yield for ADPDs. (vii) BPDx (X = O or S), triethylamine (TEA), DMSO / H2O (9 / 1 , V / V), r.t., 4 h, 97% yield for ABPD0and 91 % yield for ABPDs.

[0058] FIG. 2 depicts photophysical properties of four NIR chemiluminophores. (a) Molecular design of NIR chemiluminophores based on Schaap's dioxetane scaffold with different substituent groups. UV / Vis absorption spectra (b), chemiluminescence (CL) spectra (c), and half-lives (d) of DPDo, DPDs, BPDo, and BPDs (50 pM) in PBS (10 mM, pH 7.4, 40% DMSO), respectively (n = 3 independent experiments, mean ± s.d.). (e) CL quantum yields and maximum CL emissions of reported NIR chemiluminophores and the present disclosure highlighted with circle. FIG. 3 depicts photophysical properties of activated chemiluminophores. UV / Vis absorption (a), and fluorescence spectra (b) of ADPDo, ABPDo, ADPDs, and ABPDg (50 pM) in PBS (10 mM, pH 7.4, 40 % DMSO), respectively.

[0059] FIG. 4 depicts (a) synthetic routes for modified acceptor compound 8: (i) 1 -azido-3- iodopropane, compound 6, K2CO3, dimethylformamide (DMF), 60 °C, overnight, 92% product yield; (ii) compound 7, 1 H-indene-1 ,3(2H)-dione, AC2O, 145 °C, 2 h, 67% product yield, (b) Synthetic routes for hydrophilic activatable DPDGN: (iii) o-Phenylenediamine, N- (Allyloxycarbonyloxy)succinimide, TEA, tetrahydrofuran (THF), N2, r.t., 6 h, - 90% yield; (iv) compound 9, Pd(Pphs)4, 1 ,3-Dimethylbarbituric acid (DMBA), MeOH, N2, r.t., 4 h, 97% yield; (v) compound 10, / V-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ), allyl-(2- aminophenyl)carbamate, THF, N2, r.t., 6 h, 86% yield; (vi) compound 11 , piperidine, ACN, 30 min, N2, r.t., 98% product yield; (vii) compound 12, N-(Allyloxycarbonyloxy)succinimide, TEA, N2, r.t., 76% product yield; (viii) compound 13, PBrs, THF, 0 °C, 4 h, without further purification; (ix) compound 14, HBAY, K2CO3, CS2CO3, ACN, N2, 4 h, r.t., 62% yield; (x) compounds 15 and 8, piperidine, ACN, reflux, 2 h; Pd(Pph3)4, DMBA, THF, r.t., 4 h, 37% product yield; (xi) compound 16, methylene blue, white light, air, 0 °C, 2 h, 92% product yield; (xii) compound 17, DBCO-mPEG2k, DCM, 0 °C, 2 h, -100% product yield.

[0060] FIG. 5 depicts design and synthesis of hydrophilic tandem-locked NIR chemiluminescent probe DPDGN. (a) Schematic illustration of the sequential CL signal activation from tandem- locked DPDGN in the presence of both NO from M1 macrophages and GGT from tumor cells, (b) Synthetic route of DPDGN. Reagents and conditions: (ix) compound 14, HBAY, K2CO3, CS2CO3, acetonitrile, N2, 4 h, room temperature (r.t.), 62% yield; (x) compounds 15 and 8, piperidine, Acetonitrile, reflux, 2 h; Pd(Pph3)4, 1 ,3-dimethylbarbituric acid, tetrahydrofuran, r.t., 4 h, 37% product yield; (xi) compound 17, methylene blue, white light, air, 0 °C, 2 h, 92% product yield; (xii) DBCO-mPEG2k, dichloromethane, 0 °C, 2 h, -100% product yield.

[0061] FIG. 6 depicts in vitro characterization of the detection capabilities of DPDGN. CL spectra (a) and and fluorescence spectra (b) of DPDGN (30 pM) in the presence of individual biomarkers or combined biomarkers in PBS (10 mM, pH 7.4) at 37 °C. For the individual biomarker, NONOate (NO donor, 60 pM) or GGT enzyme (-10 U / L) was added to a solution of DPDGN for 30 min incubation. For combined biomarkers, GGT was added and incubated for another 30min following NONOate incubation for 30 min. (c) CL signal changes of DPDGN (30 pM) after incubation with different enzymes (ALP, CatB, DPPiv, furin, p-gal, NTR, uPA, GGT, or NONOate for 30 min incubation at 37 °C in PBS buffer. Different groups on X axis: 1 : PBS, 2: ALP, 3: CatB, 4: DPPiv, 5: furin, 6: p-gal, 7: NTR, 8: uPA, 9: GGT, 10: NONOate, 1 1 : NONOate + GGT, 12: NONOate + GGT + GGsTop. Group NONOate + GGT + GGsTop: GGT inhibitor (GGsTop, 0.2 mM) was treated 6 h earlier than the combined biomarker incubation, (d) Schematic illustration for CL imaging of macrophage polarization in cancer immunotherapy. BMDM means bone marrow-derived macrophages, (e) CL intensity of BMDM+4T1 group and BMDM group treated with R837 (10 pg ml-1) at different treatment times (0, 36 and 72 h), and 4T1 group without R837 treatment, (f) Flow cytometry analysis of M1 (iNOS+CD206 ) and M2 macrophages (iNOS CD206+) polarization from BMDMs at post-treatment with R837 (10 pg ml-1), (g) Correlation studies between percentages of tumor-infiltrating M1 -type macrophages (iNOS+CD206 ) and CL intensity of turn-on DPDGN.

[0062] FIG. 7 depicts (a) UV absorbance of DPDGN (30 pM) in the presence of individual biomarkers or combined biomarkers in PBS (10 mM, pH 7.4) at 37 °C. For the individual biomarker, NONOate (60 pM) or GGT enzyme (~10 U / L) was added to a solution of DPDGN for 30 min incubation. For combined biomarkers, GGT was added and incubated for another 30min following NONOate incubation for 30 min. (b) Fluorescence changes of DPDGN (30 pM) treated with different enzymes (ALP, CatB, DPPiv, furin, p-gal, NTR, uPA, GGT, or NONOate for at 37°C in PBS buffer. Different groups on X axis: 1 : PBS, 2: ALP, 3: CatB, 4: DPPiv, 5: furin, 6: p-gal, 7: NTR, 8: uPA, 9: GGT, 10: NONOate, 1 1 : NONOate + GGT, 12: NONOate + GGT + GGsTop. Group NONOate + GGT + GGsTop: GGT inhibitor (GGsTop, 0.2 mM) was treated 6 h earlier than the combined biomarker incubation, (c) CL lifetimes of DPDGN (30 pM) in the presence of individual biomarkers or combined biomarkers in PBS (10 mM, pH 7.4) at 37 °C. (d) Limit of detection (LOD) of DPDGN (30 pM) after incubation with GGT at different concentrations (0, 0.125, 0.25, 0.5,1 and 2 U / L) after incubation with NONOate (60 pM) for 30 min.

[0063] FIG. 8 depicts (a) HPLC analysis of DPDGN (30 pM) in the presence of individual biomarkers or combined biomarkers in PBS (10 mM, pH 7.4) at 37 °C. For NO donor, NONOate (60 pM) was added to a solution of DPDGN for 30 min incubation. For combined biomarkers, GGT was added and incubated for 2 h following NONOate incubation for 30 min. (b) Liquid chromatography-mass spectrometry (LC-MS) analysis of the activated compound after treatment combined biomarkers in (a).

[0064] FIG. 9 depicts the cell viability of 4T1 cells and Raw264.7 after 24 h incubation with DPDGN at different concentrations.

[0065] FIG. 10 depicts in vivo real-time CL imaging of M1 macrophage in cancer immunotherapy, (a) Schematic illustration for the timeline of immune therapy and real-time imaging of DPDGN- 4T 1 cells were intravenously injected to living mice at day 0 and then R837 was treated at day 14, W and 18 through intraperitoneal injection. DPDGN (2 mM, 10 pl per mouse) was intratumorally administered to living mice at day 20. IF represents immunofluorescence, (b) Real-time CL imaging in orthotopic lung tumor after Intratracheal injection of DPDGN. The inhibitor (GGsTop) (20 mg kg1) was intraperitoneally injected 12 h before CL imaging, (c) Quantification of CL signal from the lung area of 4T1 -bearing mice in (b) (n = 3 independent mice, mean ± s.d.). (d) Flow cytometry analysis of M1 (iNQS+CD206 ) and M2 macrophages (iNQS CD206+) polarization in lung tumor tissues from each group, (e) H&E analysis of lung tissues in each group.

[0066] FIG. 11 depicts (a) real-time fluorescence imaging in metastasis tumor after intratracheal injection of DPDGN (2 mM, 10 pl). The inhibitor (GGsTop) (20 mg Kg1) was intraperitoneally injected 12 h before real-time imaging (excitation: 465 nm, emission: 720-740 nm, exposing time: 1 s). (b) Quantification of fluorescence signal from the lung area of 4T1 -bearing mice in (a).

[0067] FIG. 12 depicts the quantification of M1 macrophage population in lung tumor tissues from each group. The data are the mean ± s.d. (n = 3 independent mice). Two-tailed Student’s T- test: ***p < 0.001 .

[0068] FIG. 13 depicts H&E analysis of the slides from main organs after different treatments.

[0069] FIG. 14 depicts gating strategies for analysis of TAMs from tumor and in vitro samples.

[0070] Description

[0071] It has been surprisingly found the tandem-locked chemiluminescent probe for imaging of macrophage polarization in cancer immunotherapy. Thus, in a first aspect of the invention there is provided a compound of formula I:

[0072] A - B - C (I) where:

[0073] A represents a tandem-lock biomarker responsive group;

[0074] B represents a self-immolative linker; and

[0075] C represents a chemiluminescent group, or a pharmaceutically acceptable salt or solvate thereof. When used herein, the term “tandem-lock biomarker responsive group” refers to groups which can be activated only by two or more two or more distinct biomarkers in sequence.

[0076] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0077] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0078] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, and the like.

[0079] References herein (in any aspect or embodiment of the invention) to compounds of formula I include references to such compounds pe se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.

[0080] Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin. Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0081] Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic) , lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1 - hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

[0082] Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0083] As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography. The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

[0084] For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn eta!., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0085] Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula I”.

[0086] Compounds of formula I may contain double bonds and may thus exist as E (entgegen) and Z (zusammeri) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

[0087] Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

[0088] Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and / or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

[0089] Any suitable self-immolative linkers may be used in the compound of formula I. In some embodiments of the first aspect of the invention that may be mentioned herein, B may be selected from the group consisting of: where:

[0090] A represents NH, S, or O;

[0091] Ri represents H, F, or NO2;

[0092] R2represents H or F; the wiggly line represents the point of attachment to C; and the double-crossed line represents the point of attachment to A.

[0093] In some embodiments of the first aspect of the invention that may be mentioned herein, B may be selected from the group consisting of: where the wiggly line represents the point of attachment to C; and the double-crossed line represents the point of attachment to A, optionally wherein B is selected from the group consisting of:

[0094] Any suitable tandem-lock biomarker responsive groups may be used in the compound of formula I. In some embodiments of the first aspect of the invention that may be mentioned herein, A may be selected from:

[0095] represents the point of attachment to the rest of the molecule, and R3 represents:

[0096]

[0097] Any suitable chemiluminescent groups may be used in the compound of formula I. In some embodiments of the first aspect of the invention that may be mentioned herein, C may be selected from:

[0098] (ai)

[0099] where:

[0100] X represents O, S, Se, or SO2; to provide a polymeric component having a number average molecular weight of from 500 to 10,000 Daltons, such as 500 Daltons, 1 ,000 Daltons, 2,000 Daltons, or 5,000 Daltons;

[0101] FU represents OH, OMe, NEt2, or NH2;

[0102] Rs represents H, Cl, or Br;

[0103] Re represents H, Cl, or Br; the double-crossed line represents the point of attachment to B; the wiggly line represents the point of attachment to the rest of the molecule; and EWG for (ai) to (aiv) is selected from:

[0104] R7 represents H, Cl, or Br; alternatively, the EWG for (aiii) and (aiv) is selected from the following:

[0105] , , where: the double-crossed line represents the point of attachment to the rest of the molecule,

[0106] Rs represents NH2, NMe2, OH, or OMe,

[0107] R9 represents H or CN, and

[0108] R10 represents H or halogen.

[0109] When used herein, the term “EWG” refers to electron-withdrawing groups.

[0110] In some embodiments of the first aspect of the invention that may be mentioned herein, there may be provided a tandem-locked biomarker-activatable chemiluminescent probe comprising:

[0111] (a) an adamantylidene-1 ,2-dioxetane-based chemiluminophore;

[0112] (b) wherein the adamantylidene-1 ,2-dioxetane-based chemiluminophore is caged at its hydroxyl group by a self-immolative linker (e.g. p-aminobenzyl alcohol), which is in turn connected to a GGT-cleavable y-glutamate, and the carboxyl group of the y-glutamate is further caged by a NO-cleavable o-phenylenediamine. In such embodiments, (ai) the adamantylidene-1 , 2-dioxetane-based chemiluminophore may further comprise an azide group, and (aii) the adamantylidene-1 ,2-dioxetane-based chemiluminophore may further comprise a DBCO-mPEG2k group conjugated to the rest of the molecule by a triazole group.

[0113] Without wishing to be bound by theory, the azide group introduced onto adamantylidene-1 ,2- dioxetane-based chemiluminophore may improve water solubility of the chemiluminophore and this is advantageous for in vivo applications.

[0114] In some embodiments of the first aspect of the invention that may be mentioned herein, C may be selected from:

[0115] In particular embodiments of the first aspect of the invention that may be mentioned herein, the compound may be

[0116] As will be appreciated, the compounds, or pharmaceutically acceptable salts or solvates thereof, disclosed herein have high specificity toward macrophages in tumor tissues over normal tissues and are useful for in vivo detection of macrophage repolarization in cancer immunotherapy. In a second aspect of the invention, there is provided an in vitro method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:

[0117] (a) contacting a sample obtained from a subject undergoing immunotherapy for cancer with a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in the first aspect of the invention for a period of time; and

[0118] (b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not.

[0119] In a third aspect of the invention, there is provided a method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:

[0120] (a) administering to a subject undergoing immunotherapy for cancer a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in the first aspect of the invention for a period of time; and

[0121] (b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not.

[0122] In a fourth aspect of the invention, there is provided use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in the first aspect of the invention in the manufacture of a medicament for determining whether an immunotherapy is effective in a subject undergoing immunotherapy for cancer.

[0123] In a fifth aspect of the invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in the first aspect of the invention for use in determining whether an immunotherapy is effective in a subject undergoing immunotherapy for cancer.

[0124] In a sixth aspect of the invention, there is provided a method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:

[0125] (a) administering to a subject undergoing a treatment for a disease involving an immune response, other than a cancer, a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in the first aspect of the invention for a period of time; and

[0126] (b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not, optionally wherein the disease involving an immune response is an autoimmune disease. In a seventh aspect of the invention, there is provided use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in the first aspect of the invention in the manufacture of a medicament for determining whether an immunotherapy is effective in a subject undergoing a treatment for a disease involving an immune response, other than a cancer, optionally wherein the disease involving an immune response is an autoimmune disease.

[0127] In an eighth aspect of the invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 for use in determining whether a drug-treatment is effective in a subject undergoing a treatment for a disease involving an immune response, other than a cancer, optionally wherein the disease involving an immune response is an autoimmune disease.

[0128] Details of the chemiluminescent signal detection technique are provided in the examples section below.

[0129] Examples of autoimmune diseases include, but are not limited to rheumatoid arthritis, psoriasis, and multiple sclerosis.

[0130] For the avoidance of doubt, in the context of the present invention, the term “treatment’ includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and / or diagnosis of patients which are susceptible to the relevant disease states.

[0131] The terms “patient’ and “patients’’ include references to mammalian (e.g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

[0132] The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect). Further embodiments of the invention that may be mentioned include those in which the compound of formula I is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula I is not isotopically labelled.

[0133] The term "isotopically labelled", when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I. Thus, the term "isotopically labelled" includes references to compounds of formula I that are isotopically enriched at one or more positions in the compound.

[0134] The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and / or iodine. Particular isotopes that may be mentioned in this respect include2H,3H,11C,13C,14C,13N,15N,15O,17O,180,35S,18F,37CI,77Br,82Br and125l).

[0135] When the compound of formula I is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or nonradioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope.

[0136] Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

[0137] Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

[0138] Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0139] The amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is / are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.

[0140] For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w / w) active ingredient; from 0 to 99% (w / w) diluent or filler; from 0 to 20% (w / w) of a disintegrant; from 0 to 5% (w / w) of a lubricant; from 0 to 5% (w / w) of a flow aid; from 0 to 50% (w / w) of a granulating agent or binder; from 0 to 5% (w / w) of an antioxidant; and from 0 to 5% (w / w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w / w) of a release-controlling polymer.

[0141] A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w / w) active ingredient; and from 50% (w / w) to 99% (w / w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w / w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

[0142] Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof. However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0143] Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.

[0144] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0145] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0146] Examples

[0147] Materials

[0148] All the mentioned chemicals containing anhydrous and deuterium solvents were purchased from Sigma-Aldrich and TCI companies without further purification. DBCO-mPEG2k was purchased from BroadPharm company. All enzymes, such as y-Glutamyl Transferase (GGT), Cathepsin B (CatB), Dipeptidyl Peptidase IV (DPPiv), furin, |3-galactosidase (|3-gal), Nitroreductase (NTR), and Urokinase plasminogen activator (uPA), were purchased from Sigma-Aldrich Co., Ltd and R&D systems, inc.. The GGT inhibitor (GGsTop) and NO donor (NONOate) were ordered from MCE Co., Ltd. The cell RPMI 1640 Medium, Dulbecco's Modified Eagle Medium (DMEM), Gibco fetal bovine serum (FBS), streptomycin, and penicillin were purchased from Thermo Fisher Scientific Co., Ltd. MTS assay was supplied by Cell Signaling Technology Company. Recombinant mouse granulocyte-macrophage CSF (catalogue number 576304) and Antibodies against CD1 1b (M1 / 70, 1 :200), F4 / 80 (BM8, 1 :20), iNOS (W16030C, 1 :160) and CD206 (C068c2, 1 :100) were used for macrophage staining.

[0149] 1H and13C nuclear magnetic resonance (NMR) spectra were recorded by using a Bruker BBFO 400 MHz NMR. Liquid chromatography-mass spectrometry (LC-MS) analyses were tested with Triple Quadrupole LC / MS (Agilent 1260-6460). High-performance liquid chromatography (HPLC) analyses were carried out on an Agilent 1260 system using Methanol / water (1% TFA) or Acetonitrile / water (1% TFA) as the eluent. The ultraviolet-visible (UV-vis) spectra were measured on a Shimadzu UV-2450 spectrophotometer. The fluorescence spectra were tested on a Fluorolog 3-TCSPC spectrofluorometer or IVIS spectrum imaging system. The cytotoxicity was measured by using a microplate reader (SpectraMax M, Switzerland). Chemiluminescence spectra and half-lives were measured by an IVIS spectrum imaging system, and the region of interest was analyzed via the Living Image 4.3 software. Tissue slices were cut by Leica, Germany slicer and imaged by a Nikon ECLIPSE 80i microscope. The images of tissue sections and cells were recorded with Zeiss LSM800 confocal laser scanning microscope. The white light was provided by 150W LED High Bay Thermo Light. Flow cytometry assay was measured on a Fortessa X20 (BD Biosciences).

[0150] Example 1. Synthetic of ADPDx and ABPDx (FIG. 1)

[0151] Hvdroxybenzaldehvde-adamantan-2-ylidene (HBAY) and compound 1 x (X = O and S)

[0152] The phenol-adamantylidene derivative HBAY and compound 1 x (X = O and S) were successfully synthesized according to previous literature (Huang, J. et al., Angew. Chem. Int. Ed. 2021 , 60, 3999-4003).

[0153] General procedure: the mixture of compound 1x (X = O or S, 5 mmol) and 1 ,3-indandione (1461 mg, 10 mmol) was dissolved in acetic anhydride (40 ml) and then stirred for 2 h at 145 °C. After completion of the reaction, the mixture was concentrated under a vacuum to remove acetic anhydride after cooling down to room temperature. The residue was extracted with saturated NaHCOs (3 x 150 ml) and ethyl acetate (3 150 ml). The organic layer was collected and dried with anhydrous Na2SO4 to obtain the crude product which was purified with silica gel chromatography using hexane / ethyl acetate to give the corresponding compound.

[0154] Compound 2o (1196 mg, yield 83%) was obtained as an orange solid.1H NMR (400 MHz, CDCI3): 58.69 (dd, J= 8.3, 1.2 Hz, 1 H), 8.48 (s, 1 H), 7.84 - 7.81 (m, 2H), 7.72 - 7.65 (m, 3H), 7.46 - 7.41 (m, 2H), 2.51 (s, 3H).13C NMR (101 MHz, CDCI3): 5 163.5, 155.4, 151.8, 140.5, 134.3, 133.8, 133.7, 132.5, 124.3, 122.1 , 121.8, 119.2, 117.6, 1 13.9, 108.4, 21.1. MS (ESI+): m / z = 289.1 [M+H]+.

[0155] Compound 2s (1033 mg, yield 68%) was obtained as a pink solid. The crude product was purified by HPLC using ACN / Water as an eluent to give pure product.1H NMR (400 MHz, CDCh): 5 8.69 (dd, J = 8.3, 1.2 Hz, 1 H), 8.48 (s, 1 H), 7.83 (dt, J = 6.7, 3.4 Hz, 2H), 7.71 - 7.65 (m, 3H), 7.46 - 7.41 (m, 2H), 2.51 (s, 3H).13C NMR (101 MHz, CDCI3): 6 163.5, 155.4, 151.8, 140.5, 134.3, 133.8, 133.7, 132.5, 124.3, 122.1 , 121.81 , 1 19.2, 1 17.6, 113.9, 108.4, 21 .07. MS (ESI+): m / z = 304.1 [M+H]+.

[0156] Compound 3x

[0157] General procedure: to a solution of mixture of acceptor compound 2x (0.5 mmol) and phenol- adamantylidene derivative HBAY (164 mg, 0.55 mmol) in anhydrous acetonitrile (ACN, 4 ml) was added piperidine (~ 20 ul, 0.2 mmol) under N2 atmosphere. The mixture was refluxed for 2 h under. After completing the reaction, the mixture was extracted with brine (2 x 50 ml) and dichloromethane (DCM, 3 x50 ml), and then the organic solution was combined, and dried over anhydrous NaaSCV The crude product was obtained by removing organic solvent. The crude product was purified by HPLC using ACN and water as an eluent to afford the corresponding compound 3x.

[0158] Compound 3o was obtained as an orange solid (264 mg, 93% yield).1H NMR (400 MHz, DMSO-de): 6 8.57 (dd, J = 8.3, 7.0 Hz, 2H), 8.39 (t, J = 16.5 Hz, 1 H), 8.01 (t, J = 8.3 Hz, 1 H), 7.91 - 7.87 (m, 1 H), 7.78 (s, 4H), 7.53 - 7.44 (m, 3H), 7.24 (dd, J = 15.9, 2.6 Hz, 1 H), 6.97 (dd, J= 8.6, 2.5 Hz, 1 H), 3.12 (d, J= 4.7 Hz, 3H), 3.08 (s, 1 H), 2.27 (d, J = 12.3 Hz, 1 H), 2.08 - 1.46 (m, 12H).13C NMR (101 MHz, DMSO-d8): 6 160.2, 159.5, 153.9, 150.2, 140.0, 138.0, 136.6, 134.9, 134.0, 130.3, 128.4, 125.3, 118.5, 117.2, 1 17.1 , 116.9, 1 16.3, 1 13.3, 109.4, 56.4, 36.5, 32.2, 29.2, 27.7. MS (ESI ): m / z 567.4 [M-H]\

[0159] Compound 3s was obtained as a purple solid (254 mg, 87% yield).1H NMR (400 MHz, DMSO- d6): 6 9.12 (s, 1 H), 8.11 (d, J = 7.5 Hz, 1 H), 7.93 (dd, J = 1 1 .6, 8.4 Hz, 2H), 7.78 - 7.71 (m, 5H), 7.58 - 7.50 (m, 2H), 7.42 (d, J = 16.2 Hz, 1 H), 6.87 (dd, J= 8.6, 2.5 Hz, 1 H), 6.67 (d, J = 2.5 Hz, 1 H), 3.30 (s, 1 H), 3.22 (s, 3H), 2.02 (s, 1 H), 1 .93 - 1 .63 (m, 12H).13C NMR (101 MHz, DMSO-de): 6 159.1 , 152.6, 149.5, 140.2, 139.9, 137.4, 135.3, 134.8, 134.5, 134.1 , 131.9,

[0160] 130.2, 128.1 , 126.6, 126.5, 125.4, 125.4, 123.9, 123.8, 121.5, 117.7, 1 17.2, 1 16.3, 56.3, 36.5,

[0161] 32.2, 29.1 , 27.7. MS (ESI ): m / z 583.5 [M-H]’. Compound DPDx

[0162] The mixture of compound 3x (0.1 mmol) and a catalytic amount of methylene blue (4-6 mg) was dissolved in THF / DCM (5 ml / 20 ml). Air was bubbled through the solution while irradiating with white light (LED 150 W) at 0 °C for 3 h. The organic solvent was then removed by a rotary evaporator under a vacuum below 30 °C and the crude product was purified by HPLC using ACN / water or MeOH / water as an eluent to give the chemiluminophores.

[0163] DPDo was obtained as an orange solid (57.6 mg, 96% yield).1H NMR (400 MHz, DMSO-da): 6 8.57 (dd, J = 8.3, 7.0 Hz, 2H), 8.39 (t, J = 16.5 Hz, 1 H), 8.01 (t, J = 8.3 Hz, 1 H), 7.91 - 7.87 (m, 1 H), 7.78 (s, 4H), 7.53 - 7.44 (m, 3H), 7.24 (dd, J = 15.9, 2.6 Hz, 1 H), 6.97 (dd, J = 8.6, 2.5 Hz, 1 H), 3.12 (d, J = 4.7 Hz, 3H), 3.08 (s, 1 H), 2.27 (d, J = 12.3 Hz, 1 H), 2.08 - 1.46 (m, 12H).13C NMR (101 MHz, DMSO-d6): 5 166.9, 159.8, 159.0, 153.9, 150.2, 150.1 , 139.8, 135.1 , 134.5, 134.1 , 132.0, 131.9, 131.5, 129.7, 129.5, 126.2, 124.3, 124.2, 121.5, 1 18.5, 113.7, 11 1 .9, 109.7, 95.0, 49.6, 46.4, 38.5, 35.9, 35.57, 31 .6, 31 .5, 31 .3, 26.8, 25.5, 25.3. MS (ESI ): m / z 599.2 [M-Hp.

[0164] DPDswas obtained as a purple solid (57.3 mg, 93% yield).1H NMR (400 MHz, DMSO-de): 6 9.15 (d, J = 8.8 Hz, 1 H), 8.12 (d, J= 8.5 Hz, 1 H), 8.05 (d, J = 17.4 Hz, 1 H), 7.96 (t, J= 8.8 Hz, 2H), 7.82 - 7.72 (m, 5H), 7.53 (t, J = 7.7 Hz, 1 H), 7.42 (dd, J = 9.0, 6.9 Hz, 2H), 3.15 (s, 3H), 3.02 (s, 1 H), 2.07 - 1 .48 (m, 11 H), 1 .30-1 .21 (t, J= 1 1 .0 Hz, 2H).13C NMR (101 MHz, DMSO- ds): 6216.4, 166.8, 159.3, 158.7, 152.6, 149.0, 148.9, 139.9, 135.6, 135.3, 134.6, 134.2, 133.8, 132.0, 130.7, 129.4, 126.7, 126.6, 126.2, 126.0, 125.3, 124.6, 121.6, 1 19.9, 1 19.5, 118.5, 118.1 , 1 17.4, 1 16.9, 1 12.0, 95.0, 52.5, 49.5, 46.3, 38.5, 35.8, 35.6, 34.1 , 32.9, 32.2, 31 .6, 31 .4, 31.2, 26.8, 25.5, 25.3. MS (ESI ): m / z 614.8 [M-H]

[0165] The reaction procedure is similar to synthesis of compound 2x in the same scale.

[0166] Compound 4o was obtained as an orange solid (924 mg, 57% yield).1H NMR (400 MHz, CDCI3) 6 9.20 (d, J = 8.3 Hz, 1 H), 8.79 (s, 1 H), 8.02 (d, J = 7.4 Hz, 1 H), 7.96 (d, J = 7.5 Hz, 1 H), 7.81 (t, J = 7.0 Hz, 1 H), 7.75 (dd, J = 1 1 .7, 7.3 Hz, 2H), 7.56 (t, J = 7.6 Hz, 1 H), 7.49 (d, J= 8.3 Hz, 1 H), 2.51 (s, 3H).13C NMR (101 MHz, CDCI3): 6 163.4, 148.9, 142.0, 134.9, 134.4, 133.7, 130.6, 125.8, 123.9, 120.5, 1 18.3, 1 17.9, 105.1 , 21.0. MS (ESI-): m / z 614.8 [M+H]+.

[0167] Compound 4s was obtained as a red solid (901 mg, 53% yield). The crude solid was washed by hexane and cold diethyl ether to obtain the purple solid without further purification. The product yield was determined by HPLC analysis.

[0168] The reaction procedure is similar to the synthesis of compound 3x in the same scale.

[0169] Compound 5o was obtained as an orange solid (251 mg, 83% yield).1H NMR (400 MHz, CDCI3) 5 9.12 (d, J = 8.3 Hz, 1 H), 8.06 - 8.04 (m, 2H), 7.98 - 7.95 (m, 2H), 7.83 - 7.73 (m, 3H), 7.69 (d, J = 8.6 Hz, 1 H), 7.55 - 7.48 (m, 2H), 6.95 (dd, J = 8.6, 2.4 Hz, 1 H), 6.82 (dd, J = 12.1 , 9.3 Hz, 2H), 3.39 (s, 1 H), 3.31 (s, 3H), 2.13 (s, 1 H), 2.09 - 1 .70 (m, 12H).13C NMR (101 MHz, CDCI3): 6 160.5, 158.2, 154.4, 149.2, 141.8, 139.8, 139.0, 137.6, 134.8, 134.7, 133.8,

[0170] 133.8, 132.8, 130.8, 127.6, 127.2, 125.6, 124.0, 120.5, 1 18.5, 1 18.0, 1 18.0, 1 17.6, 116.3,

[0171] 115.7, 107.3, 57.1 , 39.2, 4.13, 32.8, 30.1 , 28.4. MS (ESI ): m / z 603.4 [M-Hp.

[0172] Compound 5s was obtained as a purplish red solid (232 mg, 75% yield).1H NMR (400 MHz, CDCh): 5 8.75 (s, 1 H), 8.62 (d, J = 7.0 Hz, 1 H), 8.03 (d, J = 7.3 Hz, 1 H), 7.96 (d, J = 7.5 Hz, 1 H), 7.74 (m, 6H), 7.60 - 7.55 (m, 1 H), 7.11 (d, J = 16.2 Hz, 1 H), 6.90 (dd, J = 8.5, 2.4 Hz, 1 H), 6.78 (d, J = 2.5 Hz, 1 H), 3.36 (s, 1 H), 3.29 (s, 3H), 2.1 1 (s, 1 H), 2.01 -1 .68 (m, 12H).13C NMR (101 MHz, CDCI3): 5 157.7, 151.8, 151.1 , 142.1 , 140.0, 138.5, 136.1 , 136.0, 134.8, 134.0,

[0173] 133.8, 133.3, 132.8, 131.8, 127.5, 127.3, 126.9, 125.9, 124.4, 124.1 , 121.9, 120.5, 118.0, 116.3, 57.1 , 39.2, 37.2, 32.8, 30.0, 28.4. MS (ESI ): m / z 619.3 [M-H]’.

[0174] The reaction procedure is similar to synthesis of compound DPDx in the same scale.

[0175] Compound BPDo was obtained as an orange solid (60 mg, 94% yield).1H NMR (400 MHz, DMSO-de); 59.03 (d, J= 7.9 Hz, 1 H), 8.91 (d, J= 14.0 Hz, 1 H), 8.43 (d, J = 15.7 Hz, 1 H), 8.10 (d, J = 7.0 Hz, 1 H), 8.03 - 7.91 (m, 5H), 7.65-7.63 (m, 2H), 7.44 (s, 1 H), 7.26 (dd, J = 28.5, 8.9 Hz, 1 H), 6.96 (dd, J= 8.5, 2.1 Hz, 1 H), 3.12 (s, 3H), 3.01 (s, 1 H), 1.96 (d, J= 17.7 Hz, 2H), 1.73 - 1.45 (m, 9H), 1 .33 - 1.17 (m, 2H).13C NMR (101 MHz, DMSO-ds): 5 179.2, 166.9,

[0176] 159.9, 159.7, 153.6, 141.1 , 135.8, 135.3, 134.5, 134.4, 132.4, 129.7, 129.4, 125.6, 123.5, 120.3, 119.7, 1 18.5, 1 17.4, 1 17.2, 1 1 1 .9, 106.3, 106.2, 95.0, 52.5, 49.5, 46.3, 43.7, 38.4, 35.9, 35.6, 33.9, 33.0, 32.2, 31.6, 31.4, 31.3, 26.8 25.5, 25.2, 22.2, 21.6. MS (ESI ): m / z 635.1 [M- HJ-.

[0177] Compound BPDs was obtained as a purplish red solid (62 mg, 95% yield).1H NMR (400 MHz, DMSO-d6): 5 8.09 (m, 2H), 8.03-7.91 (m, 6H), 7.79 (t, J = 7.6 Hz, 1 H), 7.74 - 7.55 (m, 2H), 7.49-7.42 (m, 2H), 6.95 (dd, J= 8.6, 2.5 Hz, 1 H), 3.15 (s, 3H), 3.03 (s, 1 H) , 2.05-1 .86 (m, 4H), 1 .73-1 .45 (m, 9H).13C NMR (101 MHz, DMSO-d6): 5 166.8, 159.4, 158.8, 141.6, 135.86, 134.9, 134.4, 133.9, 132.3, 132.2, 130.8, 129.6, 128.7, 127.3, 126.5, 125.8, 124.3, 123.8, 120.4, 119.8, 118.5, 117.4, 116.9, 112.0, 95.1, 52.5, 49.5, 46.3, 38.5, 38.1, 35.8, 35.6, 34.1, 32.9, 32.2, 31.6, 31.2, 29.8, 28.4, 26.8, 25.5, 25.2, 23.2, 22.4. MS (ESI ): m / z 651.1 [M-H]-.

[0178] Compound ABPDx

[0179] General procedure: to a solution of DPDxor BPDX(0.05 mmol) in solution of DMSO / H2O (3 ml, 9 / 1, V / V) was added triethylamine (70 pl, 0.5 mmol). The mixture was stirred at room temperature for 4 h and monitored by HPLC. The corresponding activated compound was purified by HPLC analysis.

[0180] ADPDo was obtained as an orange solid (19.6 mg, 87% yield).1H NMR (400 MHz, DMSO-ds): 68.59 - 8.56 (m, 2H), 8.37 (d, J = 15.5 Hz, 1 H), 8.00 (d, J = 8.3 Hz, 1 H), 7.87 - 7.69 (m, 7H), 7.49 (s, 1 H), 7.30 - 7.23 (m, 2H), 7.07 (d, J = 8.2 Hz, 1 H), 3.93 (s, 3H).13C NMR (101 MHz, DMSO-ds): 5166.9, 159.8, 159.0, 154.0, 150.2, 135.8, 134.9, 134.1, 131.9, 131.5, 129.5,

[0181] 126.2, 124.3, 119.8, 117.6, 116.8, 113.7, 109.7, 52.5. MS (ESI ): m / z 449.2 [M-H]'.

[0182] ADPDswas obtained as a purple solid (21.7 mg, 93% yield).1H NMR (400 MHz, DMSO-ds): 69.17 (s, 1H), 8.15 (dd, J = 11.7, 9.4 Hz, 2H), 7.99 (dd, J = 8.3, 5.7 Hz, 2H), 7.79-7.75 (m, 5H), 7.57-7.52 (m, 1H), 7.44 (d, J= 16.1 Hz, 1H), 7.32 (d, J= 2.6 Hz, 1H), 7.06 (dd, J= 8.6,

[0183] 2.6 Hz, 1H), 3.90 (s, 3H).13C NMR (101 MHz, DMSO-d6): 5166.8, 158.7, 152.6, 149.1, 139.9,

[0184] 135.6, 134.6, 134.2, 132.0, 130.7, 129.4, 126.7, 126.3, 125.3, 124.6, 121.7, 119.9, 118.1, 116.9, 52.5. MS (ESI ): m / z 465.4 [M-H]'.

[0185] ABPDo was obtained as an orange solid (23.6 mg, 97% yield).1H NMR (400 MHz, DMSO-ds): 69.03 (d, J = 8.4 Hz, 1H), 8.92 (s, 1H), 8.38 (d, J= 15.6 Hz, 1H), 8.10 (d, J= 7.6 Hz, 1H), 7.99 (dd, J= 15.2, 7.6 Hz, 3H), 7.92 (t, J= 7.2 Hz, 2H), 7.75 (d, J= 8.4 Hz, 1H), 7.63 (t, J =

[0186] 7.6 Hz, 1 H), 7.30 (d, J = 2.4 Hz, 1 H), 7.21 (d, J = 16 Hz, 1 H), 7.06 (dd, J = 8.8, 2.8 Hz, 1 H), 3.93 (S,3H).13CNMR(101 MHz, DMSO-ds): 6179.2, 166.9, 159.8, 159.1, 153.7, 147.4, 141.1,

[0187] 136.3, 135.8, 135.1, 134.4, 131.6, 129.6, 129.3, 126.0, 125.6, 123.5, 120.3, 119.7, 119.5,

[0188] 118.7, 116.8, 106.3, 52.5. MS (ESI ): m / z 485.2 [M-H]'.

[0189] ABPDs was obtained as a purple solid (22.8 mg, 91% yield).1H NMR (400 MHz, DMSO-ds): 610.57 (s, 1H), 8.50 (s, 1H), 8.17 (d, J= 16 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 8.06-7.97 (m, 5H), 7.94 (dd, J = 7.6, 1.2 Hz, 1 H), 7.82 - 7.78 (m, 1 H), 7.63 (t, J = 7.6 Hz, 1 H), 7.46 (d, J = 16.0 Hz, 1H), 7.32 (d, J= 2.8 Hz, 1H), 7.07 (dd, J= 8.4, 2.4 Hz, 1H), 3.90 (s, 3H).13C NMR (101 MHz, DMSO-ds): 5166.8, 158.9, 157.9, 157.6, 149.7, 141.6, 135.8, 135.0, 134.5, 132.2, 130.9, 129.6, 127.3, 126.4, 125.8, 123.9, 120.4, 1 19.9, 1 16.9, 52.5. MS (ESI ): m / z 501.2 [M- H]-.

[0190] Results and discussion

[0191] The near-infrared region (NIR) chemiluminophores (DPDo, DPDg, BPDo, and BPDg) were synthesized via a convergent approach based on Schaap's dioxetane scaffold (FIG. 1 ). Briefly, 2-methyl-4H-chromen-4-one (compound 1 o) and 2-methyl-4H-thiochromen-4-one (compound 1s) respectively reacted with 1 ,3-indanedione and benzo[b]thiophen-3(2H)one-1 ,1 -dioxide to yield the corresponding acceptors (compound 2o or 2s) via Knoevenagel condensation. The chemiluminophore precursors were then obtained by Knoevenagel reaction between the respective acceptors and HBAY; and subsequently, the chemiluminophores were synthesized by photosensitized oxidation of their precursors. The structures of all chemiluminophores were confirmed by1H,13C NMR, and LC-MS analyses.

[0192] Example 2. Photophysical Properties of DPDo, DPDg, BPDo, and BPDs

[0193] The photophysical properties of four chemiluminophores were studied by measuring their UV- vis absorption, fluorescence, and chemiluminescence (CL) spectra.

[0194] Kinetic studies of chemiluminophores

[0195] Chemiluminescence intensities of DPDx and BPDx (X= O or S, 50 pM) in 0.01 M PBS (pH 7.4, 40% DMSO) were acquired by using IVIS bioluminescence system with an open filter and 20s of acquisition time. The chemiluminescent intensities were plotted as a function of time.

[0196] Chemiluminescence quantum yield (<Pci)

[0197] Chemiluminescence quantum yields were determined by following the protocol provided in Huang, J. et al., Angew. Chem. Int. Ed. 2023, 62, e202303982. Briefly, chemiluminophore DBPD was used as the standard with a known 0ci of 0.08 einsteins / mol in PBS buffer. Chemiluminescence intensities of DBPD and unknown chemiluminophores (50 pM) in 0.01 M PBS (pH 7.4, 40% DMSO) were recorded by using the IVIS bioluminescence system under the same condition. The chemiluminescent intensities were plotted as a function of time. Then, according to the following equation, the presently disclosed probe’s chemiluminescence quantum yields can be calculated:

[0198] Where Qsampie represents total photons from the test sample, which was obtained by integrating chemiluminescent signal with time. Qreference presents the total photons from the standard chemiluminophore DBPD. Results and discussion

[0199] As shown in FIGS. 2a-b, all four chemiluminophores showed similar absorption spectra with maxima around 500 nm. The CL emission of DPDo, DPDs, BPDo, and BPDs had the maxima at -750, 800, 760 and 820 nm, respectively (FIG. 2c). In particular, sulfur-substituted DPDs and BPDs had record-long CL emission (> 800 nm) among reported chemiluminophores (Green, O. etal., J. Am. Chem. Soc. 2017, 139, 13243-13248; Huang, J. etal., Angew. Chem. int. Ed. 2023, 62, e202303982; and Huang, J. et al., Angew. Chem. Int. Ed. 2021 , 60, 3999- 4003) (Table 1 and FIG. 2e). This red-shifted emission could be attributed to the increased atomic radius and change in electronegativity. Besides, the chemiluminophores that contained strong EWGs (sulfuryl group) exhibited slightly red-shifted CL emission (DPDs: 760 nm; BPDs: 820 nm) than their corresponding ketone-substituted chemiluminophores (DPDo: 750 nm; BPDo: 800 nm). The CL spectra of all four chemiluminophores coincided well with their fluorescence spectra of activated forms (FIG. 3b). All chemiluminophores exhibited long CL half-lives due to intermolecular H-bonding: DPDo (~7.7 h), DPDs (5.7 h), BPDo (3.2 h), and BPDs (0.8 h). In addition, the CL quantum yield ( ci) of DPDo (2.7%) was the highest among all chemiluminophores (DPDs / 1 .0%, BPDo / 0.6%, and BPDs / 0.1 %), and also much higher than most of literature reported NIR chemiluminophores (Table 1 and FIG. 2e). Thus, combined molecular engineering approach endows NIR chemiluminophores with redshift CL emission and high After considering the physical properties, DPDo with the outstanding CL quantum yield and desired long half-life was selected for subsequent in vivo imaging.

[0200] Table 1. The summary of photophysical properties of NIR adamantylidene-1 ,2-dioxetane- based chemiluminescent substrates (emission > 700 nm).

[0201]

[0202] Example 3. Synthesis of DPDGN (FIG. 4)

[0203] Compound 7 The mixture of 1 -azido-3-iodopropane (844 mg, 4 mmol), K2CO3 (552.8 mg, 4 mmol) and commercial compound 6 (352.1 mg, 2 mmol) was added in anhydrous DMF (10 ml) and then stirred at 60 °C overnight. After completing the reaction, the mixture was extracted with EtOAc (3 x 80 ml) and brine (3 x 50 ml). The organic layer was collected and concentrated in vacuo to get the crude product. Then, the crude product was purified by silica gel chromatography using hexane / EtOAc to give compound 7 as a yellow solid (476.7 mg, 92%).1H NMR (400 MHz, CDCI3): 6 7.53 (d, J = 3.2 Hz, 1 H), 7.35 (d, J = 9.2 Hz, 1 H), 7.22 (dd, J = 8.8, 2.8 Hz, 1 H), 6.14 (s, 1 H), 4.13 (t, J = 5.6 Hz, 2H), 3.51 (t, J = 6.8 Hz, 2H), 2.37 (s, 3H), 2.07 (m, 2H).13C NMR (101 MHz, CDCI3): 6 178.1 , 166.0, 155.9, 151.5, 124.3, 123.6, 119.4, 110.0, 105.9, 65.4, 48.3, 28.8, 20.6. MS (ESI+): m / z = 175.1 [M+H]+.

[0204] The reaction procedure is similar to synthesis of compound 2x. Compound 8 as yellow solid (519 mg, 67% product yield).1H NMR (400 MHz, CDCI3): 6 8.53 (s, 1 H), 8.37 (d, J = 2.8 Hz, 1 H), 7.83 (dd, J = 5.2, 3.2 Hz, 2H), 7.65 (dd, J = 5.2, 2.8 Hz, 2H), 7.39 (d, J = 9.2 Hz, 1 H), 7.31 (dd, J = 9.2, 2.8 Hz, 1 H), 4.22 (t, J = 5.6 Hz, 2H), 3.56 (t, J = 6.4 Hz, 2H), 2.52 (s, 3H), 2.15 (p, J = 6.0 Hz, 2H).13C NMR (101 MHz, CDCI3): 6 163.4, 154.9, 152.1 , 150.6, 140.4, 133.6, 124.8, 121.9, 120.3, 1 18.7, 1 13.3, 1 13.3, 108.5, 65.6, 48.5, 28.8, 21.0. MS (ESI+): m / z = 388.2 [M+H]+.

[0205] Compound allyl-(2-aminophenyl)carbamate

[0206] N-(Allyloxycarbonyloxy)succinimide (1992 mg, 10 mmol) was added into a mixture of 0- phenylenediamine (1080 mg, 10 mmol) and triethylamine (~1.4 ml, 10 mmol) in anhydrous THE (20 ml) and stirred for 6 h under N2 atmospheres. By monitoring by thin layer chromatography (TLC), the mixture was extracted by brine (3 x 100 ml) and EtOAc (3 100 ml) and the organic phase was combined and dried with anhydrous Na2SC>4. The organic layer was concentrated in vacuo to get the crude product which was purified by silica gel chromatography using hexane / EtOAc to give compound as a white solid (1729 mg, 90%).1H NMR (400 MHz, CDC ): 6 7.29 (d, J = 6.8 Hz, 1 H), 7.03 (t, J = 7.6 Hz, 1 H), 6.79 (dd, J = 13.2, 7.6 Hz, 2H), 6.02-5.92 (m, 1 H), 5.38-5.24 (m, 2H), 4.67 (d, J = 6.0 Hz, 2H).13C NMR (101 MHz, CDCI3): 6 132.6, 126.8, 120.1 , 119.8, 118.3, 117.9, 117.8, 66.2. MS (ESI+): m / z = 193.2 [M+H]+.

[0207] Pd(Pphs)4 as a catalyst was added dropwise into a mixture of compound 9 (1 .028 g, 2 mmol) and 1 ,3-dimethylbarbituric acid (468 mg, 3 mmol) in anhydrous MeOH (20 ml) and stirred for 4 h at room temperature under N2 atmospheres. After monitoring by TLC, the mixture was concentrated in vacuo to afford the crude product and washed with cold diethyl ether to give pure product (yellow solid, 97% yield) which was confirmed by LC-MS. MS (ESI+): m / z = 475.4 [M+H]+. A mixture of compound 10 (711 mg, 1.5 mmol), allyl-(2-aminophenyl)carbamate (576 mg, 3 mmol) and N-Ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ) (742 mg, 3 mmol) was dissolved in anhydrous THF (15 ml) and stirred for 6 h at room temperature under N2atmospheres. After completing the reaction, the mixture was concentrated in vacuo to afford the crude product and washed with cold diethyl ether to give pure compound 11 (white solid, 836 mg, 86% yield).1H NMR (400 MHz, DMSO-d6): 6 7.89 (d, J = 7.2 Hz, 2H), 7.80 (d, J= 7.2 Hz, 1 H), 7.74 (d, J= 7.2 Hz, 2H), 7.55 - 7.52 (m, 3H), 7.41 (t, J = 7.2 Hz, 2H), 7.34 - 7.30 (m, 2H), 7.23 (d, J= 9.6 Hz, 2H), 7.20 - 7.11 (m, 2H), 5.92-5.82 (m, 1 H), 5.26 (d, J= 17.2 Hz, 1 H), 5.10 (dd, J = 10.4, 1.6 Hz, 1 H), 4.53 (d, J = 5.2 Hz, 2H), 4.43 (s, 2H), 4.53 - 4.22 (m, 4H), 2.48 - 2.44 m, 2H), 2.17 - 2.1 1 (m, 1 H), 2.07 - 1.93 (m, 1 H).13C NMR (101 MHz, DMSO-ds): 5 170.9, 170.3, 156.2, 153.7, 143.8, 143.7, 140.7, 137.8, 137.1 , 133.0, 127.6, 127.0, 126.8, 125.4, 125.3, 124.3, 120.1 , 1 18.8, 1 17.4, 65.8, 65.0, 62.6, 54.9, 46.6, 32.6, 27.1. MS (ESI+): m / z = 649.3 [M+H]+.

[0208] Compound 1 1 (778 mg, 1.2 mmol) was added into ACN (10 ml) containing 2 ml of piperidine and stirred for 30 min at room temperature under N2atmospheres. After monitoring by HPLC, the mixture was concentrated in vacuo to afford the crude product and washed with cold diethyl ether for three times to give pure product (pale white solid, 98% yield) which was confirmed by LC-MS. MS (ESI+): m / z = 427.4 [M+H]+.

[0209] Compound 13

[0210] N-(Allyloxycarbonyloxy)succinimide (398 mg, 2 mmol) was added into a mixture of compound 12 (426 mg, 1.0 mmol) and triethylamine (279 pl, 2 mmol) in anhydrous THF (10 ml) and stirred for 6 h under N2atmospheres. By monitoring by TLC, organic solvent was concentrated in vacuo to get the crude product which was purified by HPLC using MeOH / water as eluent to give compound 13 as a white solid (388 mg, 76% product yield).1H NMR (400 MHz, DMSO- ds) 6 7.53 (d, J = 8.0 Hz, 4H), 7.22 (d, J = 8.4 Hz, 2H), 7.21 - 7.12 (m, 2H), 6.00 - 5.88 (m, 2H), 5.37 - 5.30 (dd, J = 17.2, 9.6 Hz, 2H), 5.21 - 5.19 (m, 2H), 4.60 (d, J = 5.2 Hz, 2H), 4.51 - 4.50 (m, 2H), 4.42 (d, J = 5.6 Hz, 2H), 4.20 (dd, J = 13.2, 8.0 Hz, 1 H), 2.47 - 2.42 (m, 2H), 2.16 - 2.08 (m, 1 H), 1.98 - 1.89 (m, 1 H).13C NMR (101 MHz, DMSO-d6): 6 170.9, 170.3, 156.0, 153.8, 137.9, 137.1 , 133.5, 133.1 , 126.9, 125.4, 125.1 , 124.4, 1 18.8, 117.5, 1 17.2, 65.1 , 64.7, 62.6, 54.9, 32.6, 27.1 . MS (ESI+): m / z = 51 1 .3 [M+H]+.

[0211] Compound 14

[0212] PBr3(235 pl, 2.5 mmol) was added dropwise into a solution of compound 13 (255 mg, 0.5 mmol) was in anhydrous THF and stirred at 0 °C under N2atmospheres for 4 h. After monitoring the reaction by TLC, the mixture was extracted with EtOAc (3 x 50 ml) and saturated NaHCO3(3 x 60 ml). The organic solvent was combined and dried with anhydrous Na2SO4 to obtain the compound 14 which didn’t need further purification for the following reaction.

[0213] Compound 15

[0214] A mixture of dry compound 14, HBAY (75 mg, 0.25 mmol), K2CO3(35 mg, 0.25 mmol) and CS2CO3(81 mg, 0.25 mmol) in anhydrous ACN (5 ml) was stirred at room temperature under N2atmospheres for 4 h. The crude was obtained after removing solvent and purified by HPLC using MeOH / water as eluent to afford a white solid compound 15 (123 mg, 62%).1H NMR (400 MHz, DMSO-d6): 6 7.84 (d, J = 8.4 Hz, 1 H), 7.67 (d, J = 7.2 Hz, 1 H), 7.62 - 7.52 (m, 4H), 7.40-7.34 (m, 2H), 7.18 - 7.13 (m, 2H), 6.91 (d, J = 2.4 Hz, 1 H), 6.00-5.87(m, 2H), 5.32 (dd, J = 17.2, 8.8 Hz, 2H), 5.20 (s, 2H), 5.17 (s, 2H), 4.59 (d, J = 5.6 Hz, 2H), 4.50 (d, J = 4.0 Hz, 2H), 4.20 (dd, J = 13.6, 7.6 Hz, 1 H), 3.23 (s, 3H), 2.46 (s, 2H), 2.17 - 2.10 (m, 1 H), 2.06-1 .96 (m, 1 H), 1.92 - 1.62 (m, 13H).13C NMR (101 MHz, DMSO-d6): 5 190.2, 170.9, 170.5, 162.6, 156.0, 153.8, 140.8, 139.2, 138.5, 133.4, 133.1 , 132.5, 130.9, 130.5, 129.0, 128.6, 128.4, 127.9, 125.4, 125.1 , 124.4, 119.0, 1 17.5, 117.2, 1 16.7, 1 15.4, 69.6, 65.1 , 64.7, 56.8, 54.9, 38.2, 36.4, 32.7, 32.1 , 29.3, 28.6, 27.5, 27.1 . MS (ESI+): m / z = 791 .3 [M+H]+.

[0215] Compound 16

[0216] Piperidine (10 pl, 0.1 mmol) was addted into a mixture of compounds 15 (79 mg, 0.1 mmol) and 8 (46 mg, 0.12 mmol) in anhydrous ACN (5 ml) and stirred at reflux under N2atmospheres for 2 h. The organic solvent was removed to obtain the crude intermediate. Pd(Pph3)4 (23 mg, 0.02 mmol) as a catalyst was added dropwise into a mixture of intermediate and 1 ,3- dimethylbarbituric acid (23 mg, 0.15 mmol) in anhydrous THF (8 ml) and stirred for 4 h at room temperature under N2atmospheres. After monitoring by TLC, the mixture was concentrated in vacuo to afford the crude compound 16 (37 mg, 37% product yield) which was purified by flash column chromatography. The product yield was determined by HPLC analysis and confirmed by MS analysis ((ESI+): m / z = 992.5 [M+H]+.

[0217] Compound 17

[0218] The reaction procedure was similar to compound DPDx. Briefly, compound 16 (~10 mg, 0.01 mmol) and a catalytic amount of methylene blue (2 mg) was dissolved in THF / DCM (5 ml / 20 ml). Air was bubbled through the solution while irradiating with white light (LED 150 W) at 0 °C for 3 h. The organic solvent was then removed by a rotary evaporator under a vacuum below 30 °C and the crude product was purified by HPLC using MeOH / water as an eluent to give an orange solid compound 17 (9.4 mg, 92% product yield).1H NMR (400 MHz, CDCI3): 5 8.64 (s, 1 H), 8.31 (d, J= 2.4 Hz, 1 H), 7.94 (d, J= 16.0 Hz, 1 H), 7.84-7.82 (m, 2H), 7.72 - 7.69 (m, 2H), 7.64 (dd, J = 5.2, 2.8 Hz, 2H), 7.54 - 7.51 (m, 3H), 7.39 - 7.30 (m, 5H), 7.00 - 6.99 (m, 2H), 6.90 (d, J = 1 .2 Hz, 1 H), 6.85 (d, J = 15.6 Hz, 1 H), 5.04 (s, 2H), 4.26-4.17 (m, 2H), 4.12 (d, J = 7.2 Hz, 1 H), 3.56 (t, J = 6.4 Hz, 2H), 3.39 (s, 1 H), 3.29 (s, 3H), 2.66 - 2.55 (m, 2H), 2.36- 2.32 (m, 2H), 2.25 (d, J = 7.2 Hz, 2H), 1.86 - 1.62 (m, 13H). MS (ESI+): m / z = 1024.3 [M+H]+.

[0219] Compound DPDGN

[0220] The compound 17 (5.1 mg, 0.005 mmol) and DBCO-mPEG2k (1 1.8 mg, 0.005 mmol) in anhydrous DCM and THF (2 ml / 2 ml) was stirred at 0 °C for 2 h. After monitoring by TLC, the orange solid compound DPDGN (-16.8 mg, 100% yield) was obtained by removing the organic solvents.1H NMR (400 MHz, CDCI3): 5 8.72 - 8.64 (m, 1 H), 8.46 (dd, J = 16.0, 6.0 Hz, 1 H), 8.36 - 8.32 (m, 1 H), 7.94 (d, J = 15.6 Hz, 1 H), 7.83 - 7.81 (m, 2H), 7.74 - 7.64 (m, 5H), 7.58 - 7.44 (m, 7H), 7.39 - 7.32 (m, 5H), 7.17 - 7.10 (m, 1 H), 7.04 - 6.98 (m, 1 H), 6.91 -6.87 (m, 1 H), 6.83 - 6.77 (m, 1 H), 5.37 - 5.31 (m, 2H), 5.08 - 5.00 (m, 2H), 4.23 - 4.16 (m, 3H), 3.98 (s, 2H), 3.73 - 3.55 (m, 184H), 3.48 - 3.45 (m, 2H), 3.37 (s, 4H), 3.29 (s, 3H), 2.81 - 2.75 (m, 1 H), 2.65 - 2.58 (m, 3H), 2.52 - 2.46 (m, 4H), 1 .70 - 1 .59 (m, 13H).

[0221] Results and discussion

[0222] To achieve specific detection of macrophage polarization during cancer immunotherapy, a hydrophilic dual-locked NIR CL probe (DPDGN) was developed (FIG. 5a). DPDGN was designed by caging the hydroxyl group of DPDo with a self-immolative linker (p-aminobenzyl alcohol) connected to a GGT-cleavable y-glutamate, and the carboxyl group of y-glutamate was further locked by a NO-cleavable o-phenylenediamine. To improve its water solubility for subsequent in vivo applications, an azide group was introduced onto modified chemiluminophore (compound 17), which was then conjugated with DBCO-mPEG2k to afford DPDGN. AS shown in FIGS. 4 and 5b, the brominated tandem-locked responsive group with alloc protection was conjugated to the green chemiluminophore precursor HYCL to afford compound 15, which was then coupled to azide-functionalized compound 8 via Knoevenagel condensation reaction to yield compound 16. Followed by alloc group deprotection and photooxidation, water-soluble moiety PEG2k was introduced onto the dioxetane intermediate compound 17 via copper-free click reaction to afford probe DPDGN. The structures of all compounds were confirmed by1H,13C NMR, and LC-MS analyses.

[0223] Example 4. In Vitro Detection of DPDGN

[0224] Kinetic studies of tandem-locked chemiluminescence probe DPDGN Chemiluminescent probe DPDGN (30 pM) was incubated in the presence of individual biomarkers (NONOate) or combined biomarkers (NONOate and GGT) in PBS (10 mM, pH

[0225] 7.4) at 37 °C. For the individual biomarker, NONOate (NO donor, 60 pM) was added to a solution of DPDGN (30 pM) in PBS (10 mM, pH 7.4) at 37 °C for 30 min incubation. For combined biomarkers, GGT (~10 U / L) was added and incubated for another 30 min after 30 min incubation with NONOate (60 pM). The corresponding chemiluminescence intensities were acquired by using IVIS bioluminescence system with the open filter and 20 s of acquisition time. The chemiluminescent intensities were plotted as a function of time.

[0226] Selectivity of DPDGN towards combined biomarkers

[0227] Chemiluminescence and fluorescence signal changes of DPDGN (30 pM) in the presence of the individual biomarkers (NONOate or GGT), combined biomarkers (NONOate and GGT), or other different enzymes (ALP, CatB, DPPiv, p-gal , NTR, and uPA) (~ 10 U / L) were tested after 30 min incubation in 1 x PBS buffer (pH 7.4) for 30 min incubation at 37 °C. In the inhibitor group, GGT inhibitor (GGsTop, 0.2 mM) was treated 6 h earlier than the combined biomarker incubation.

[0228] LOD of DPDGN towards GGT enzyme

[0229] First, DPDGN (30 pM) was pretreated with NONOate (60 pM) for 30 min in 1 x PBS buffer (pH

[0230] 7.4). Then, different concentrations of GGT (0, 0.125, 0.25, 0.5, 1 , and 2 U / L) were added and incubated for another 30 min at 37 °C. Last, the chemiluminescence signal was acquired by using IVIS bioluminescence system with the open filter and 20s of acquisition time. The LOD was calculated by chemiluminescence intensities based on the equation: LOD = 3o / k, where o is the standard deviation of emission intensity of blank, and k is the slope of the plot of emission intensities.

[0231] Cytotoxicity assay. Cell viability of DPDGN.

[0232] 4T 1 and RAW 264.7 cells were seeded in 96-well plates which have 1 x 104cells per well and incubated with RPMI 1640 Medium and DMEM for 24 h, respectively. Then, DPDGN at different concentrations (0, 2.5, 5, 10, 25, 50, 100, and 150 pM) was added into 4T1 and RAW 264.7 cells, respectively. After 24 h incubation, MTS assay was added to the cell medium for another 4 h incubation. After 4 h incubation, the absorbance of MTS at 450 nm was recorded using a microplate reader. The assays were performed in five sets for each concentration.

[0233] Flow cytometry assay (He, S. et al., Nat. Biomed. Eng. 2023, 7, 281 -297)

[0234] Bone marrow-derived macrophages (BMDMs) were isolated from the bone marrow of BALB / c mouse and cultured in DMEM culture medium with macrophage CSF (20 ng ml"1), and BMDMs were treated with Imiquimod (R837) (10 pg / ml) for 36 and 72 h. For flow cytometry, M1 or M2 macrophages were co-stained with antibodies against CD11 b (M1 / 70, 1 :200), F4 / 80 (BM8, 1 :20), iNOS (W16030C, 1 : 160) and CD206 (C068c2, 1 : 100) . Then, the fluorescence of cells was measured using flow cytometry.

[0235] In vitro CL imaging

[0236] BMDMs were seeded in a Transwell 24-well plate with 1 x 105cells per well and incubated with DMEM for 24 h incubation, followed by incubation with R837 (10 pg ml1) for 0, 36 and 72 h. After incubation, 4T1 cells were seeded in the upper of the Transwell 24-well plate with 1 x 105cells per well. Then, DPDGN (30 pM) was added to the upper of Transwell 24-well plate for 30 min incubation. As control groups, DPDGN (30 pM) were solely incubated with 4T1 cells or BMDMs treated with R837 (10 pg ml1) for 0, 36 and 72 h. The chemiluminescence signals were relatively recorded by using the IVIS bioluminescence system with an open filter and 20s of acquisition time. The CL intensity was quantified by Living Image® Software.

[0237] Results and discussion

[0238] The sensing capability of DPDGN was tested by measuring CL spectra in the presence or absence of the biomarkers-of-interest. As shown in FIG. 6a, DPDGN intrinsically exhibited minimal CL signals, but showed a 26.6-fold CL enhancement at 725 nm in the presence of both biomarkers (NO and GGT). In comparison, its fluorescence only increased by -1.7-fold in the presence of both biomarkers because of the low fluorescence increase after cleavage (FIGS. 6b and 7b). This should be caused by the limited changes in intramolecular charge transfer process that methyl benzoate of activated form (ADPDo) could reduce the donation ability of oxygen of phenol. Meanwhile, the absorption maximum shifted from 476 to 465 nm, which was the characteristic peak of ADPDo (FIGS. 3a and 7a). In contrast, no CL or fluorescence enhancement was observed for DPDGN when only one biomarker or other interfering enzymes was present (FIG. 6c). The specificity was further confirmed by minimal CL enhancement after adding GGsTop (a potent GGT inhibitor) into the mixture of DPDGN, NO, and GGT. Moreover, the CL signal of DPDGN drastically enhanced and reached plateau within 20 min, followed by a slow decay with a half-life of 198 min (FIG. 7c). As validated by HPLC and LCMS analyses (FIG. 8a), a new HPLC peak appeared at 15.2 min corresponding to activated compound 17 (molecular weight: 549.2) (FIG. 8b), proving the sequential biomarker activation mechanism of DPDGN precursor (compound 17) by NO and GGT. Moreover, the limit of detection (LOD) for DPDGN to detect GGT was determined to be 0.12 U / L, which was more sensitive than other existing probes (FIG. 7d). The sensing capability of DPDGN towards macrophage polarization was then tested in living cells after confirming its low cytotoxicity (FIG. 9). To stimulate macrophage polarization in the TME, murine bone marrow-derived macrophages (BMDMs) and mammary carcinoma cells (4T1 ) were seeded in the lower and upper compartments of a transwell plate, respectively (FIG. 6d). The BMDMs in the lower compartment were firstly treated with imiquimod (R837, an immune response modifier and a selective toll like receptor 7 agonist) for 0 (R8370h), 36 (R8373Sh) or 72 h (R83772h) (Ryu, K. A. et al., J. Am. Chem. Soc. 2014, 136, 10823-10825); before the 4T1 -seeded upper compartment was introduced. The cell co-culture was then incubated with DPDGN (30 pM, 20 min) before CL imaging. As shown in FIG. 6e, CL signals from cell culture in R83772hgroup was 1 .7, 4.3-fold higher than R83736hand R8370h, respectively. No obvious CL signals from DPDGN solely incubated with BMDM or 4T1 cells was observed, demonstrating tandem-locked DPDGN showed a specific activation in the cell level. Flow cytometry analyses was conducted, showing the percentages of M1 macrophages (iNOS+CD206 ) from R83772hgroup was -2.0, 4.3-fold higher than R83735hand R8370hgroups, respectively (FIG. 6f). Moreover, population of M2 macrophages was continually decreased after prolonging R837 treatment. This trend was similar to the CL signals as it showed a very high correlation between CL intensity and population of M1 macrophage, confirming that the CL signals from activated DPDGN was specific to M1 macrophages (FIG. 6g). Thus, DPDGN specifically detected the polarization of macrophage to M1 macrophages.

[0239] Example 5. In Vivo CL Imaging of M1-type Tumor-associated Macrophages

[0240] Real-time CL imaging during immunotherapy

[0241] All the animal experiments (metastasis immunotherapy and imaging) were conducted and followed according to Care and Use of Laboratory Animals of the Nanyang Technological University-Institutional Animal Care.

[0242] 4T 1 cells (5 x 105cells) were intravenously injected into healthy BALB / c mice (female, 5 weeks old) to establish the metastasis tumor model. Each group has four mice. After 14-day implantation, one mouse in each group chosen for sacrifice is selected randomly to collect the lung tissue for analysis of metastasis tumor establishment. Then, R837 (50 pl, 0.5 mg ml'1) (~ 1.25 mg Kg1) was intraperitoneally injected for three dosages with an interval of 48 h. The control group was only treated with PBS for three dosages with an interval of 48 h. For the negative control group, tumor-bearing mice were pre-treated with GGsTop (20 mg kg ') 24 h earlier than probe DPDGN injection. DPDGN (2 mM, 10 pl) was intratumorally administered to living mice at day 20. The chemiluminescence and fluorescence signals in each group were recorded by using the I VIS bioluminescence and fluorescence system, respectively. The CL and FL intensities were quantified by Living Image® Software.

[0243] Histological and immunofluorescence analysis

[0244] Histological Analysis. After completing the immunotherapy and CL metastasis imaging, the mice in each group were sacrificed and the corresponding organs were collected. The collected main tissues (heart, liver, spleen, lung, kidney) were fixed with 4% formaldehyde solution for 48 h and dehydrated with 30% sucrose solution for 24 h. Then, dehydrated main tissues were embedded in an optimal frozen cutting temperature medium and cut into selection (10 pm) with Leica, CM 1950. For H&E staining, the sections were washed with ethanol, and immersed in hematoxylin and solution for 3 min and 1 min, respectively. The H&E-stained slices were washed with PBS and then observed by a Nikon ECLIPSE 80i microscope.

[0245] For flow cytometry, tumor-associated macrophages were co-stained with antibodies against CD11 b (M1 / 70, 1 :200), F4 / 80 (BM8, 1 :20), iNOS (W16030C, 1 :160), and CD206 (C068c2, 1 :100). Then, the fluorescence of gated cells was measured using flow cytometry.

[0246] Results and discussion

[0247] The ability of DPDGN for real-time imaging of macrophage polarization in cancer immunotherapy was tested in lung metastasis model (FIG. 10a). BALB / c mice received intravenous injection of 4T 1 cells and received immunotherapeutic agents 14 days post-tumor implantation.

[0248] Tumor-bearing mice received R837 (1.25 mg Kg1) for three dosages with an interval of 48 h (Xu, C. eta!., Nat. Biomed. Eng. 2023, 7, 298-312). For the control group, tumor-bearing mice were pre-treated with GGsTop or treated with PBS. At day 20, DPDGN was intratracheally injected into living mice, and longitudinal CL imaging were conducted for 4 h post-injection (FIG. 10b). The CL signal at lung metastatic region was quantitatively studied, a gradual CL signal increase was observed with a signal maximum at 30 min post injection, followed by gradual signal decrease due to body clearance (FIG. 10c). At 30 min post injection, the CL signal from mice receiving R837 treatment was 4.0, 15.7-fold higher than PBS or GGsTop- treated groups. In contrast, the fluorescence signals from DPDGN activation in mice receiving R837 treatment showed no statistically significant enhancement as compared to the control group (FIG. 11 ), which was similar to the in vitro data. To examine the close association of CL signals from activated DPDGN with the macrophage polarization, flow cytometry studies were conducted on the lung tissues from different treatment groups (FIG. 10d). In mice receiving R837-treatment, increased tumor infiltration of tumor infiltration of iNOS+CD206_M1 macrophages was 2.1 -fold higher than that for PBS-treated groups and decreased M2 TAMs was ~1 .7-fold lower than PBS-treated group due to R837-induced polarization from M2 to M1 . (FIG. 12). Histological studies showed significantly suppressed tumor size in R837-treated group as compared to PBS-treated group, demonstrating the anti-tumor effects of M1 macrophages (FIG. 10e). Moreover, no discernible indications of toxicity within major organs (heart, liver, spleen, and kidneys) were observed, validating the good biosafety of DPDGN (FIG. 13).

[0249] Conclusion

[0250] To screen out highly efficient NIR chemiluminophores, a combined molecular engineering approach involving both atomic alternation and introduction of electron-withdrawing group (EWG) was introduced into the adamantyliene-1 ,2-dioxetane scaffolds. Four NIR chemiluminophores (DPDo, DPDg, BPDo, and BPDs) were developed by the combined molecular engineering approach into the adamantyliene-1 ,2-dioxetane scaffold. Note that both CL half-lives and emission of 7.7 h and 820 nm are record-longer than existing chemiluminophores (Table 1). To realize specific detection of M1 TAMs during cancer immunotherapies, well-performed DPDo with the highest CL quantum yield (2.7% einsteins / mol) and the longest CL half-life (~ 7.7h) was screened out to be constructed into a tandem-locked NIR CL probe (DPDGN). In the present disclosure, we report a hydrophilic tandem-locked biomarker-activatable CL probe (DPDGN) for in vivo detection of macrophage repolarization in cancer immunotherapy. To ensure high specificity toward macrophages in TME, NO and gamma-glutamyl transferase (GGT) were chosen as the biomarkers for macrophages and cancer cells, respectively. In this way, the CL signal of DPDGN is only turned-on by M1 TAM. DPDGN is turned-on with 26.6-fold CL emission at 725 nm only in the presence of both NO and GGT, which are biomarkers associated with M1 macrophage and 4T 1 cancer cells, respectively. The detection capability of DPDGN was then tested in both living cells and tumor-bearing mice, and the CL signals from activated DPDGN highly correlated to population of TAM activation towards M1 -phenotype, which was validated by flow cytometry analyses.

[0251] Therefore, the present disclosure not only provides a combined alternation approach (EWG and atomic alteration) to develop NIR chemiluminophores, but also reports the first tandem- locked CL probes with a bright emission for specific detection of M1 TAM activation in cancer immunotherapy.

Claims

Claims1. A compound of formula (I):A - B - C (I) where:A represents a tandem-lock biomarker responsive group;B represents a self-immolative linker; andC represents a chemiluminescent group, or a pharmaceutically acceptable salt or solvate thereof.

2. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Claim 1 , wherein B is selected from the group consisting of:where:A represents NH, S, or O;Ri represents H, F, or NO2;R2represents H or F; the wiggly line represents the point of attachment to C; and the double-crossed line represents the point of attachment to A.

3. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Claim 2, wherein B is selected from the group consisting of:where the wiggly line represents the point of attachment to C; andthe double-crossed line represents the point of attachment to A, optionally wherein B is selected from the group consisting of:

4. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding claims, where A is selected from:the wiggly line represents the point of attachment to the rest of the molecule, and R3 represents:, where the double crossed line represents the point of attachment to the rest of the molecule.

5. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding claims, wherein C is selected from:X represents O, S, Se, or SO2;to provide a polymeric component having a number average molecular weight of from 500 to 10,000 Daltons, such as 500 Daltons, 1 ,000 Daltons, 2,000 Daltons, or 5,000 Daltons;FU represents OH, OMe, NEtz, or NHz;Rs represents H, Cl, or Br;R6represents H, Cl, or Br; the double-crossed line represents the point of attachment to B; the wiggly line represents the point of attachment to the rest of the molecule; and EWG for (ai) to (aiv) is selected from:R? represents H, Cl, or Br; alternatively, the EWG for (aiii) and (aiv) is selected from the following:, , where: the double-crossed line represents the point of attachment to the rest of the molecule,Rs represents NH2, NMe2, OH, or OMe,Rg represents H or CN, andR10 represents H or halogen.

6. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Claim 1 , wherein there is provided a tandem-locked biomarker-activatable chemiluminescent probe comprising:(a) an adamantylidene-1 ,2-dioxetane-based chemiluminophore;(b) wherein the adamantylidene-1 ,2-dioxetane-based chemiluminophore is caged at its hydroxyl group by a self-immolative linker (e.g. p-aminobenzyl alcohol), which is in turn connected to a GGT -cleavable y-glutamate, and the carboxyl group of the y-glutamate is further caged by a NO-cleavable o-phenylenediamine.

7. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to Claim 6, wherein:(al) the adamantylidene-1 , 2-dioxetane-based chemiluminophore further comprises an azide group; and(aii) the adamantylidene-1 , 2-dioxetane-based chemiluminophore further comprises a DBCO-mPEG2k group conjugated to the rest of the molecule by a triazole group.

8. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding claims, wherein C is selected from:

9. The compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, according to any one of the preceding claims, wherein the compound is10. An in vitro method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:(a) contacting a sample obtained from a subject undergoing immunotherapy for cancer with a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 for a period of time; and(b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not.

11. A method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:(a) administering to a subject undergoing immunotherapy for cancer a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 for a period of time; and(b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not.

12. Use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 in the manufacture of a medicament for determining whether an immunotherapy is effective in a subject undergoing immunotherapy for cancer.

13. A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 for use in determining whether an immunotherapy is effective in a subject undergoing immunotherapy for cancer.

14. A method of determining the efficacy of an immunotherapy treatment, the method comprising the steps of:(a) administering to a subject undergoing a treatment for a disease involving an immune response, other than a cancer, a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 for a period of time; and(b) after the period of time detecting a chemiluminescent signal to determine whether the immunotherapy is effective or not, optionally wherein the disease involving an immune response is an autoimmune disease.

15. Use of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 in the manufacture of a medicament for determining whether an immunotherapy is effective in a subject undergoing a treatment for a disease involving an immune response, other than a cancer, optionally wherein the disease involving an immune response is an autoimmune disease.

16. A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Claims 1 to 9 for use in determining whether a drug-treatment is effective in a subject undergoing a treatment for a disease involving an immune response, other than a cancer, optionally wherein the disease involving an immune response is an autoimmune disease.

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