Porphyrin-based photosensitizer for the treatment of cancer

Abstract

NOxPorfin is an on-demand porphyrin-based photosensitizer, described herein, designed to treat cancer via photodynamic therapy (PDT). A distinct advantage over existing FDA-approved photosensitizers is that NOxPorfin exhibits a suppressed singlet oxygen quantum yield (φΔ = 6%) prior to activation within cancer cells. This property translates to a severely compromised ability to harm healthy tissue. However, in the presence of nitric oxide, a common feature of all tumors, photosensitization increases by 650% (φΔ=45%). In addition, this is accompanied by a 70-fold fluorescence signal enhancement, which we designed to confirm NOxPorfin activation via clinical fluorescence imaging. The efficacy of NOxPorfin was successfully demonstrated in two murine models of cancer. First, we were able to selectively ablate aggressive breast cancer cells without causing collateral damage. Second, we were able to prevent the growth of nascent, non-small cell lung tumors. The latter study has implications in treating metastatic lesions.

Description

[0001] PORPHYRIN-BASED PHOTO SENSITIZER FOR THE TREATMENT OF CANCER

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) (or is related to) to U.S. Provisional Patent Application No. 63 / 593,094, filed October 25, 2023, which is incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] The tumor microenvironment (TME) is characterized by the complex interplay between residing cancer cells, the extracellular matrix, and the surrounding stroma. In addition, there is mounting evidence that infiltrating immune cells (e.g., macrophages) play a critical role in mediating tumor progression and metastasis by establishing a low-grade, local inflammatory response. At the center of this phenomenon is nitric oxide (NO), a gasotransmitter synthesized from L-arginine by inducible nitric oxide synthase (iNOS). Although its precise role is still being investigated, recent studies have demonstrated that inhibitors of iNOS may serve as potential anticancer agents. An alternative approach to leverage elevated NO for cancer treatment is the envisioned development of activatable chemotherapeutics. However, masking a drug payload with a NO-responsive warhead does not necessarily overcome resistance mechanisms that will inevitably arise. Moreover, owing to its high reactivity, fleeting nature, and low steady-state concentration (nM range), NO associated with cancer is difficult to detect, let alone be exploited for prodrug activation.

[0006] Conventional anticancer drugs are non-specific and therefore will lead to the indiscriminate destruction of healthy tissue once administered to a patient. Moreover, drug resistance will inevitably arise owing to incomplete eradication of cancer cells that result from sub-optimal dosing. Activitybased sensing (ABS) is an emerging strategy (Accounts of Chemical Research, American Chemical Society, January 21, 2020, p l) that is poised to overcome this long-standing challenge by capitalizing on the unique chemical reactivity of a given biomarker, such as NO. Accordingly, there is a need for such alternate therapeutic strategies that can effectively combat cancer.

[0007] SUMMARY

[0008] Photodynamic therapy (PDT) is an attractive treatment paradigm because it is less prone to resistance due to the rapid and typically complete destruction of cancer cells. Moreover, PDT agents are in theory safer because they can be induced to generate toxic species derived from molecular oxygen in a specific region of the body through controlled laser light irradiation. Nearly all FDA- approved PDT agents are based on the porphyrin scaffold and many notable studies have shown their efficacy in treating a range of cancer types. However, there is an important limitation associated with current PDT agents that we aim to address. Specifically, patients receiving PDT will suffer from painful lesions that result from severe ‘burning’ of the general area that arise because it is impossible to focus light only onto cancer cells. We solved this by inventing a porphyrin-based PDT agent named NOxPorfm that becomes activated only when it encounters and reacts with nitric oxide, an upregulated cancer biomarker. Once this chemical transformation takes place, the ability of NOxPorfm to harvest light to generate toxic, cancer killing reactive oxygen species increases by 650%. Moreover, we equipped NOxPorfm with companion diagnostic capabilities, where we will be able to use fluorescence monitoring of NO activation to determine which tumors, and ultimately which patients, will benefit the most from this form of treatment.

[0009] Accordingly, this disclosure provides a porphyrin compound comprising a phenylene group represented by Formula I: wherein,

[0010] Q is N(Rb)2 or ORC; and

[0011] Ra, Rb, and Rcare each independently H or -(Ci-Ce)alkyl; or

[0012] Q and NHRaform a fused heterocycle with the carbon atoms of the phenylene moiety to which they are attached.

[0013] In preferred embodiments, the porphyrin compound is represented by Formula II: wherein,

[0014] R1is the phenylene group represented by Formula I; and R2, R3, and R4are each independently unsubstituted phenyl, R1, or H.

[0015] This disclosure also provides a composition comprising a porphyrin compound described above and a liposomal nanoparticle, wherein the porphyrin compound is encapsulated by the liposomal nanoparticle.

[0016] Additionally, this disclosure provides a method for treating a subject having a cancer, comprising: a) administering a porphyrin compound or composition described above to a subject having a cancer; b) waiting for a sufficient period of time for the porphyrin compound to be activated by nitric oxide (NO) present in the cancer to form an activated compound within the cancer; c) optionally detecting fluorescence emitted from the activated compound within the cancer; and d) irradiating the activated compound within the cancer, wherein the activated compound is elevated to a higher energy state and then transfers its energy to ground state molecular oxygen present within the cancer to form singlet oxygen; wherein the singlet oxygen ablates the cancer by releasing its energy, and the subject is thereby treated.

[0017] The invention provides novel compounds of Formulas I-III, intermediates for the synthesis of compounds of Formulas I-III, as well as methods of preparing compounds of Formulas I-III. The invention also provides compounds of Formulas I-III that are useful as intermediates for the synthesis of other useful compounds. The invention provides for the use of compounds of Formulas I-III for the manufacture of medicaments useful for the treatment of cancer in a mammal, such as a human.

[0018] The invention provides for the use of the compositions described herein for use in medical therapy. The medical therapy can be treating cancer, for example, breast cancer, lung cancer, pancreatic cancer, prostate cancer, or colon cancer. The invention also provides for the use of a composition as described herein for the manufacture of a medicament to treat a disease in a mammal, for example, cancer in a human. The medicament can include a pharmaceutically acceptable diluent, excipient, or carrier.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0021] Figure 1. (a) Schematic demonstrating TME-mediated PDT agent activation through the release of PeT quenching. Product exhibits an increase in the singlet oxygen quantum yield and fluorescence signal, (b) Cartoon illustrating selective PDT agent activation within tumor cells via reaction with nitric oxide (NO). Subsequent irradiation and reaction with molecular oxygen result in localized cell death. Healthy cells, cancer cells, and ablated cells are annotated.

[0022] Figure 2A-F. (a) Fluorescence emission profiles of NOxPorfin-1 and tNOxPorfin-1 from 600 to 800 nm. Excitation = 418 nm. (b) Plot of the measurement of singlet oxygen quantum yield of NOxPorfin-1, tNOxPorfin-1, and tetraphenyl porphyrin. Y-axis represents change in the absorbance of DPBF (102 scavenger), (c) pH profile of NOxPorfin-1 (5 pM) in 20 mM Britton-Robinson buffer (1:1 v / v EtOH:H2O). pH values = 5.7, 6.3, 6.9, 7.5, 8.0, 8.3, 8.8, 9.3, 9.7, 10.2, 10.8. (d) Fluorescence spectra of NOxPorfin-1 (5 pM) treated with DEA NONOate (250 pM). Readings were collected over a period of 1 hour, (e) Fluorescence intensity at 418 nm with respect to the NO concentration (0-250 pM). (f) Fluorescence spectra of NOxPorfin-1 (5 pM) treated with a panel of biologically relevant analytes present at 20 equivalents excess relative to NOxPorfin-1. All data is reported as the mean ± standard deviation (n = 3). Error bars = SD.

[0023] Figure 3A-F. (a) Representative fluorescence images of A549 cells stained with NOxPorfin- 1 (5 pM) treated with different concentrations of DEA NONOate 30 minutes prior to imaging, (b) Quantitative analysis of results from panel a. (c) Representative fluorescence images of A549 cells pre-treated with a vehicle, LPS (1 mM) for 16 hours, or L-NMMA (1 mM) for two hours prior to staining with NOxPorfin-1 (5 pM). (d) Quantitative analysis of results from panel d. (e) Cell viability assay using MTT for varying concentrations of NOxPorfin-1 (0-20 pM) (f) Dose-dependent phototoxicity of NOxPorfin-1 (5 pM) in A549 cells upon irradiation for 2.5 minutes with 505 nm LED light, incubated with 20 pM and 100 pM DEA NONOate. Scale bar = 100 pm. All data is reported as the mean ± standard deviation (n = 10). Error bars = SD. Statistical analysis was performed using a two-tailed / -test (a = 0.05,* P < 0.001***, P < 0.0001****).

[0024] Figure 4A-D. (a) Representative fluorescence images of mice pretreated with vehicle control (saline, 50 pL) or L-NMMA (35 mM, 50 pL) prior to administration of NOxPorfin-1 -NP. (b) Quantitative analysis of normalized data from panel a. (c) Representative FL images of mice pretreated with vehicle control (saline, 50 pL) or L-NMMA (35 mM, 50 pL) prior to administration of Ctrl-NOxPorfm-l-NP. (d) Quantitative analysis of normalized data from panel c. Dotted white circles represent representative ROIs used for quantification. All data is reported as the mean ± standard deviation (n = 3). Statistical analysis was performed using a two-tailed paired / -test (a = 0.05,* P < 0.01, ns = not significant).

[0025] Figure 5. (a) Tumor volume monitored after NOxPorfin-1 -NP administration and treatment with light or without light, (b) Photographs of 4T1 tumors excised from BALB / c mice after PDT treatment with NOxPorfin-1 -NP. (c) Hematoxylin-and-eosin (H&E)-stained tumor tissues excised after 24 hours of treatment. Scale bar = 1 cm. All data is reported as the mean ± standard deviation (n = 3).

[0026] Figure 6A-C. (a) Timeline of NOxPorfin-1 -NP PDT treatment on A549 nascent-tumorbearing nude mice, (b) Photographs to show the effect of NOxPorfin-1 before and after PDT. Arrows indicate sites of tumor inoculation, (c) Tumor volume monitored after NOxPorfin-1 -NP administration and treatment with light or without light. All data is reported as the mean ± standard deviation (n = 4). Figure 7. (a) Representative fluorescence images of 4T1 cells pre-treated with a vehicle, LPS (1 pM) for 16 hours, or L-NMMA (1 mM) for 2 hours prior to staining with NOxPorfm-1 (5 pM). (b) Quantitative analysis of results from panel a. Scale bar = 100 pm. All data is reported as the mean ± standard deviation (n = 10). Error bars = SD. Statistical analysis was performed using a two- tailed Z-test (a = 0.05, P < 0.0001****).

[0027] Figure 8. (a) Representative fluorescence images of A549 cells pre-treated with a vehicle, LPS (1 pM) for 16 hours, or L-NMMA (1 mM) for 2 hours prior to staining with Ctrl -NOxPorfm-1 (5 pM). (b) Quantitative analysis of results from panel a. Scale bar = 100 pm. All data is reported as the mean ± standard deviation (n = 5). Error bars = SD. Statistical analysis was performed using a two-tailed Z-test (a = 0.05,* ns = not significant).

[0028] Figure 9. (a) Representative fluorescence images of 4T1 cells pre-treated with a vehicle, LPS (1 pM) for 16 hours, or L-NMMA (1 mM) for 2 hours prior to staining with Ctrl -NOxPorfm-1 (5 pM). (b) Quantitative analysis of results from panel a. Scale bar = 100 pm. All data is reported as the mean ± standard deviation (n = 5). Error bars = SD. Statistical analysis was performed using a two-tailed Z-test (a = 0.05, ns = not significant).

[0029] Figure 10. Phototoxicity of NOxPorfm-1 (2.5 pM) in A549 cells upon irradiation for 5 minutes at 505 nm, incubated with 100 pM DEA NONOate (data normalized to vehicle control). All data is reported as the mean ± standard deviation (n = 5). Error bars = SD.

[0030] Figure 11. Phototoxicity evaluation of Ctrl-NOxPorfm-1 (2.5 pM) in 4T1 cells upon irradiation for 5 minutes at 505 nm, incubated with 100 pM DEA NONOate. All data is reported as the mean ± standard deviation (n = 5). Error bars = SD.

[0031] Figure 12. Comparison of the normalized fluorescence intensities of the saline-treated tumor of group 1 mice (administered retro-orbitally with NOxPorfm-1 -NP) and group 2 mice (administered retro-orbitally with Ctrl -NOxPorfm-1 -NP). All data is reported as the mean ± standard deviation (n = 3). Error bars = SD. (Statistical analysis was performed using a two-tailed t-test (a = 0.05,* ns = not significant).

[0032] Figure 13A-C. Representative fluorescence imaging of a mouse with 4T1 orthotopic breast cancer before (left) and 24 hours after injection of Img / kg NOxPorfm-l-NP in PBS (right), (a) Representative fluorescence images, (b) Quantitative analysis of panel a. (c) Quantitative analysis at time points 3, 5, 8, and 24 hours. Data presented as mean ± standard deviation, n = 3. Error bars = SD. Statistical analysis was performed using a two-tailed Z-test (a = 0.05,* P < 0.01, ** P < 0.001***, ns = not significant).

[0033] Figure 14. Tumor volume monitored after PBS administration and treatment with light or without light.

[0034] Figure 15. Photographs of NU / J mice with their left flank nascent tumor inoculation site irradiated and the right kept in the dark. The treatment site is seen to be healing. DETAILED DESCRIPTION

[0035] Nitric oxide (NO) generated within the tumor microenvironment (TME) is an established driver of cancer progression and metastasis. Recent efforts have focused on leveraging this feature to target cancer through the development of diagnostic imaging agents and activatable chemotherapeutics. In this context, porphyrins represent an extraordinarily promising class of molecules owing to their demonstrated use within both modalities. However, the remodeling of a standard porphyrin to afford a responsive chemical that can distinguish elevated NO from physiological levels has remained a significant research challenge. In this study, we have employed a photoinduced electron transfer strategy to develop a panel of NO-activatable porphyrin photosensitizers (NOxPorfins) augmented with real-time fluorescence monitoring capabilities. The lead compound, NOxPorfin-1, features an o-phenylene diamine trigger that can effectively capture NO (via N2O3) to yield a triazole product that exhibits 7.5-fold enhancement and 70-fold tum-on response in the singlet oxygen quantum yield and fluorescence signal, respectively.

[0036] Beyond demonstrating excellent in vitro responsiveness and selectivity toward NO, we showcase the potent photodynamic therapy (PDT) effect of NOxPorfin-1 in murine breast cancer and human non-small cellular lung cancer cells. Further, to highlight in vivo efficacy, two key studies were executed. First, we utilized NOxPorfin-1 to ablate murine breast tumors in a site-selective manner without causing substantial collateral damage to healthy tissue. Second, we established a nascent human lung cancer model to demonstrate the unprecedented ability of NOxPorfin-1 to halt tumor growth and progression completely. Results from the latter study have tremendous implications for applying PDT to target metastatic lesions.

[0037] Additional information and data supporting the invention can be found in the following publication by the inventors: ACS Appl. Mater. Interfaces 2024, 16, 9680-9689 and its Supporting Information, which are incorporated herein by reference in its entirety.

[0038] Definitions.

[0039] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0040] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0041] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0042] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

[0043] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0044] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0045] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub- ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0046] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number ”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, ... , 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above.

[0047] The recitation of a), b), c), ... or i), ii), iii), or the like in a list of components or steps do not confer any particular order unless explicitly stated.

[0048] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0049] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0050] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

[0051] Alternatively, the terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment).

[0052] The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and / or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and / or prophylactic administration, as appropriate. As used herein, "subject" or “patient” means an individual having symptoms of, or at risk for, a disease or other malignancy. A patient may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, the patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods provided herein, the mammal is a human.

[0053] As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of a compound of the disclosure into a subject by a method or route that results in at least partial localization of the compound to a desired site. The compound can be administered by any appropriate route that results in delivery to a desired location in the subject.

[0054] The compound and compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.

[0055] The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.

[0056] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0057] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0058] This disclosure provides methods of making the compounds and compositions of the invention. The compounds and compositions can be prepared by any of the applicable techniques described herein, optionally in combination with standard techniques of organic synthesis. Many techniques such as etherification and esterification are well known in the art. However, many of these techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6; as well as standard organic reference texts such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed., by M. B. Smith and J. March (John Wiley & Sons, New York, 2001); Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modem Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing); Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983); for heterocyclic synthesis see Hermanson, Greg T., Bioconjugate Techniques, Third Edition, Academic Press, 2013.

[0059] The formulas and compounds described herein can be modified using protecting groups. Suitable amino and carboxy protecting groups are known to those skilled in the art (see for example, Protecting Groups in Organic Synthesis, Second Edition, Greene, T. W., and Wutz, P. G. M., John Wiley & Sons, New York, and references cited therein; Philip J. Kocienski; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), and references cited therein); and Comprehensive Organic Transformations, Larock, R. C., Second Edition, John Wiley & Sons, New York (1999), and referenced cited therein.

[0060] The term "halo" or "halide" refers to fluoro, chloro, bromo, or iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0061] The term "alkyl" refers to a branched or unbranched hydrocarbon having, for example, from 1-20 carbon atoms, and often 1-12, 1-10, 1-8, 1-6, or 1-4 carbon atoms; or for example, a range between 1-20 carbon atoms, such as 2-6, 3-6, 2-8, or 3-8 carbon atoms. As used herein, the term “alkyl” also encompasses a “cycloalkyl”, defined below. Examples include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl (Ao-propyl), 1 -butyl, 2-methyl-l -propyl (isobutyl), 2-butyl (secbutyl), 2-methyl-2-propyl (Lbutyl), 1 -pentyl, 2-pentyl, 3 -pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,

[0062] 3 -methyl- 1 -butyl, 2-methyl-l -butyl, 1 -hexyl, 2-hexyl, 3 -hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,

[0063] 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3 -pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The alkyl can be unsubstituted or substituted, for example, with a substituent described below or otherwise described herein. The alkyl can also be optionally partially or fully unsaturated. As such, the recitation of an alkyl group can include an alkenyl group or an alkynyl group. The alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., an alkylene).

[0064] An alkylene is an alkyl group having two free valences at a carbon atom or two different carbon atoms of a carbon chain. Similarly, alkenylene and alkynylene are respectively an alkene and an alkyne having two free valences at two different carbon atoms, or an alkenylene can have the two free valences on the same carbon.

[0065] The term "cycloalkyl" refers to cyclic alkyl groups of, for example, from 3 to 10 carbon atoms having a single cyclic ring or multiple condensed rings. Cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like. The cycloalkyl can be unsubstituted or substituted. The cycloalkyl group can be monovalent or divalent and can be optionally substituted as described for alkyl groups. The cycloalkyl group can optionally include one or more cites of unsaturation, for example, the cycloalkyl group can include one or more carbon-carbon double bonds, such as, for example, 1 -cyclopent- 1-enyl, 1 -cyclopent-2-enyl, 1 -cyclopent-3 -enyl, cyclohexyl, 1- cyclohex-l-enyl, 1 -cyclohex-2-enyl, 1 -cyclohex-3 -enyl, and the like.

[0066] The term “heteroatom” refers to any atom in the periodic table that is not carbon or hydrogen. Typically, a heteroatom is O, S, N, P. The heteroatom may also be a halogen, metal or metalloid.

[0067] The term "heterocycloalkyl" or “heterocyclyl” refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3- to 10-membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morpholino, 1,3 -diazapane, 1 ,4-diazapane, 1 ,4-oxazepane, and 1 ,4-oxathiapane. The group may be a terminal group or a bridging group.

[0068] The term "aryl" refers to an aromatic hydrocarbon group derived from the removal of at least one hydrogen atom from a single carbon atom of a parent aromatic ring system. The radical attachment site can be at a saturated or unsaturated carbon atom of the parent ring system. The aryl group can have from 6 to 30 carbon atoms, for example, about 6-10 carbon atoms. The aryl group can have a single ring (e.g., phenyl or phenylene) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. The aryl can be unsubstituted or optionally substituted with a substituent described below. For example, a phenyl moiety or group may be substituted with one or more substituents Rxwhere Rxis at the ortho-, meta-, or / % / ra-position, and X is an integer variable of 1 to 5. A phenyl moiety or group having two substituents other than hydrogen is referred to as phenylene. The phenylene group described herein is covalently bonded to a macrocycle (e.g., porphyrin) via a carbon-carbon bond and has two substituents (e.g., Q and NHRaas defined herein).

[0069] The term "heteroaryl" or "heterocycle" refers to a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. The heteroaryl can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, as described in the definition of "substituted". Typical heteroaryl groups contain 2-20 carbon atoms in the ring skeleton in addition to the one or more heteroatoms, wherein the ring skeleton comprises a 5-membered ring, a 6-membered ring, two 5 -membered rings, two 6-membered rings, or a 5 -membered ring fused to a 6-membered ring. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H- quinolizinyl, acridinyl, benzo [b]thienyl, benzothiazolyl, 0-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl, and xanthenyl. In one embodiment the term "heteroaryl" denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, aryl, or (Ci-C6)alkylaryl. In some embodiments, heteroaryl or heterocycle denotes an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

[0070] As used herein, the term "substituted" or “substituent” is intended to indicate that one or more (for example, in various embodiments, 1-10; in other embodiments, 1-6; in some embodiments 1, 2, 3, 4, or 5; in certain embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group indicated in the expression using “substituted” (or “substituent”) is replaced with a selection from the indicated group(s), or with a suitable group known to those of skill in the art, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a stable compound. Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, carboxyalkyl, alkylthio, alkylsulfinyl, and alkylsulfonyl. Substituents of the indicated groups can be those recited in a specific list of substituents described herein, or as one of skill in the art would recognize, can be one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, and cyano. Suitable substituents of indicated groups can be bonded to a substituted carbon atom include F, Cl, Br, I, OR', 0C(0)N(R')2, CN, CF3, OCF3, R', O, S, C(O), S(O), methylenedioxy, ethylenedioxy, N(R')2, SR', SOR', SO2R', SO2N(R')2, SO3R', C(O)R', C(O)C(O)R', C(O)CH2C(O)R', C(S)R', C(O)OR', OC(O)R', C(0)N(R')2, 0C(0)N(R')2, C(S)N(R')2, (CH2)O-2NHC(0)R', N(R')N(R')C(O)R', N(R')N(R')C(O)OR', N(R')N(R')C0N(R')2, N(R')SO2R', N(R')SO2N(R')2, N(R')C(O)OR', N(R')C(O)R', N(R')C(S)R', N(R')C(0)N(R')2, N(R')C(S)N(R')2, N(COR')COR', N(OR')R', C(=NH)N(R')2, C(O)N(OR')R', or C(=NOR')R' wherein R’ can be hydrogen or a carbon-based moiety (e.g., (Ci-Ce)alkyl), and wherein the carbon-based moiety can itself be further substituted. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond. When a substituent is divalent, such as O, it is bonded to the atom it is substituting by a double bond; for example, a carbon atom substituted with O forms a carbonyl group, C=O.

[0071] The term “IC50” is generally defined as the concentration required to inhibit a specific biological or biochemical function by half, or to kill 50% of the cells in a designated time period, typically 24 hours.

[0072] Embodiments of the Technology.

[0073] This disclosure provides a porphyrin compound comprising a phenylene group represented by Formula I: wherein,

[0074] Q is N(Rb)2 or ORC; and Ra, Rb, and Rcare each independently H or -(Ci-Ce)alkyl; or Q and NHRaform a fused heterocycle with the carbon atoms of the phenylene moiety to which they are attached.

[0075] In various embodiments, Q and NHRaare positioned ortho relative to each other on the phenylene moiety of Formula I. In some embodiments, the phenylene group is: In some embodiments, the phenylene group is:

[0076] In other embodiments, the phenylene group is:

[0077] In some embodiments, the porphyrin compound is represented by Formula II: wherein,

[0078] R1is the phenylene group represented by Formula I; and R2, R3, and R4are each independently unsubstituted phenyl, unsubstituted or substituted pyridyl, R1, or H. In some embodiments, phenyl is substituted. In some embodiments, phenyl is substituted with one or more or more of the substituents described in the definitions above.

[0079] In some embodiments, R2, R3, and R4are each independently phenyl or phenyl substituted with halo, OH, CO2H, CONH2, or SO3H.

[0080] In some embodiments, the porphyrin compound is represented by Formula III: In preferred embodiments, the porphyrin compound is:

[0081] This disclosure also provides a composition comprising a porphyrin compound described herein and a liposomal nanoparticle, wherein the porphyrin compound is encapsulated by the liposomal nanoparticle.

[0082] In various embodiments, the liposomal nanoparticle comprises l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-mPEG). In various embodiments, a %w / w of the porphyrin compound relative to DSPE-mPEG is about 0.05% to about 2.00%. In various embodiments, the liposomal nanoparticle has an average diameter of about 25 nm to about

[0083] 250 nm.

[0084] Additionally, this disclosure provides a method for treating a subject having a cancer, comprising: a) administering an effective amount of a porphyrin compound described herein or composition thereof to a subject having a cancer; b) waiting for a sufficient period of time for the porphyrin compound to be activated by nitric oxide (NO) present in the cancer to form an activated compound within the cancer; c) optionally detecting fluorescence emitted from the activated compound within the cancer; and d) irradiating the activated compound within the cancer, wherein the activated compound is elevated to a higher energy state and then transfers its energy to ground state molecular oxygen present within the cancer to form singlet oxygen; wherein the singlet oxygen ablates the cancer by releasing its energy, and the subject is thereby treated.

[0085] In various embodiments, the subject is administered about 0.1 mg / kg to about 10 mg / kg of the porphyrin compound, optionally in combination with a pharmaceutically acceptable carrier, diluent, and / or excipient. In various embodiments, the activated compound is irradiated with light having a wavelength of about 500 nm to about 600 nm. In various embodiments, the activated compound is irradiated for an effective amount of time, such as about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, or about 1 minute to about 10 minutes.

[0086] In various embodiments, the porphyrin compound administered is NOxPorfin-1, or NOxPorfin-1 encapsulated in a liposomal nanoparticle. In various embodiments, the activated compound is tNOxPorfin-1.

[0087] In some embodiments, the method further comprises: e) pausing irradiation and allowing the cancer to reoxygenate; and f) repeating steps c) and d) for a total of two or three cycles.

[0088] In some embodiments, steps a) to d) or steps a) to f) are repeated daily, every other day, or every six days for a total of two to five cycles.

[0089] In some embodiments, the cancer is a nascent cancer or a metastatic lesion. In some other embodiments, the cancer is breast cancer or non-small cellular lung cancer.

[0090] An activity-based nitric oxide-responsive porphyrin for theranostic monitoring of site-selective and nascent cancer ablation.

[0091] Utilizing a general approach involving activity -based sensing (ABS), our group (J Am Chem Soc 2021, 143 (18), 7196-7202) and others (Org Lett 2010, 12 (10), 2318-2321) have developed powerful imaging probes exhibiting the requisite reaction kinetics, sensitivity, and selectivity profile for the in vivo detection of NO in cancer, a previously unmet challenge. With the aim of expanding upon our previous work, we employed the principles of ABS to develop NO-responsive photodynamic therapy (PDT) agents augmented with a theranostic handle for real-time visualization of NO via fluorescence (Figure la). PDT is an attractive treatment paradigm because it is less prone to resistance due to the rapid destruction of cancer cells. Moreover, contrary to the systemic administration of a conventional anticancer drug, PDT agents are in theory safer because they can be induced to generate toxic reactive oxygen species derived from molecular oxygen in a specific region of the body through controlled laser light irradiation. PDT agents that are designed to be inefficient at photosensitization prior to interaction with NO (PS OFF-state), can further limit collateral damage to neighboring healthy cells. Once activated within NO-rich cells (PS ON-state), subsequent exposure to light and reaction with molecular oxygen can dramatically enhance cancer targeting (Figure lb).

[0092] Because nearly all FDA-approved PDT agents are based on porphyrins and numerous examples have been employed for fluorescence imaging, (Renal-Clearable PEGylated Porphyrin Nanoparticles for Image-Guided Photodynamic Cancer Therapy, Adv. Funct. Mater., 2017, 27 (34)) our PDT discovery campaign began with remodeling this scaffold. Our efforts resulted in the development of the NOxPorfins (Chart 1), a panel of NO-activatable porphyrin photosensitizers with an integrated fluorescent readout. In addition to demonstrating efficacy in multiple cancer cell lines, we employed NOxPorfin-1 to selectively target triple-negative murine breast tumors in live mice. Compared to non-treated (no photosensitizer) and dark (no light) controls, we show that complete destruction of the tumor is possible without causing substantial off-target toxicity. Lastly, we generated a nascent human lung cancer model to examine the ability of NOxPorfin-1 to target small tumors (a mimic of metastatic lesions). Even though nascent tumors are not known to be highly inflamed, NO-mediated activation of NOxPorfin-1 was still possible and enabled the successful inhibition of tumor growth and progression.

[0093] Chart 1. Chemical structures ofNOxPorfin PDT agents featuring different NO-responsive triggers shown in red. Ctrl -NOxPorfin-1 represents the non-responsive matching control of NOxPorfin-1.

[0094] NOxPorfm-4 Ctrl -NOxPorfin-1

[0095] Results and Discussion.

[0096] Design and Synthesis ofNOxPorfin Photosensitizers. When designing the NOxPorfin series, we deemed it would be critical to prioritize the following criteria. First, an ABS trigger must exhibit optimized reactivity to rapidly and irreversibly capture NO. Second, the trigger must be appended to the porphyrin scaffold in a manner where the interaction with NO can be relayed to activate photosensitization and fluorescence emission. With this in mind, we developed a panel of four NOxPorfin molecules featuring different NO-responsive triggers. Each of the nucleophilic triggers was strategically selected owing to their established NO reactivity (at least when installed onto other dye systems) and their potential to control the efficiency of intersystem crossing, which is necessary for photosensitization, as well as the fluorescence signature of the pendant porphyrin. We hypothesize the inclusion of at least one electron-rich amino functional group could serve as a PeT (photoinduced electron transfer) quencher to compete with the intersystem crossing and the radiative decay pathways.

[0097] To this end, we began by introducing an o-phenylene diamine trigger onto the mesosubstituted phenyl group of the porphyrin scaffold, to afford NOxPorfin-1. We posit that the triazole resulting from the reaction with NO (via N2O3) will be more electron-deficient than the corresponding diamine and no longer be an effective PeT quencher. In addition, NOxPorfm-2 and its regioisomer, NOxPorfm-3, are each equipped with an aminophenol group. Reaction with N2O3 is anticipated to completely remove the PeT quencher via a diazotization-deamination sequence. Lastly, we considered N-nitrosylation chemistry in designing NOxPorfm-4, which features a monoalkylated amino trigger, ortho to a methoxy group (Chart 1). Reaction with NO will result in the formation of an electron-deficient N-nitroso functional group which becomes less efficient at PeT quenching. In the context of rapid NO capture, we found that it was critical to enhance the nucleophilicity of each trigger by including an adjacent electron-rich functional group (e.g., -NH2, -OH, -OMe). We verified the importance of this design feature by preparing a non-responsive control (Ctrl-NoxPorfm-1), where the second amino group was shifted to the meta position (vide infra).

[0098] To prepare each of the NOxPorfms, we employed a Lewis acid-mediated macrocyclization protocol. Specifically, we found that it was essential to precisely control the stoichiometric ratio between pyrrole, benzaldehyde, and aldehyde precursors containing the NO-responsive trigger to obtain a product distribution that gives the desired product in the greatest conversion (Scheme 1). For instance, the synthesis of NOxPorfin-1 began with nitration of 4-acetamido benzaldehyde to afford the ort / zo-nitrated intermediate 1 in 95% yield. To the reagents, benzaldehyde, N-(4-formyl-2- nitrophenyl)acetamide and pyrrole, taken in a ratio of 3 : 1 :4, boron trifluoride etherate was added to initiate macrocycle formation. After silica gel column chromatography, 2 was obtained in 18% yield. This key intermediate was then subjected to treatment with TFA and cone. HC1 to promote the removal of the acetyl-protecting group. Finally, we employed reduction conditions using SnCb 2H2O to convert the aryl nitro group to the corresponding aniline, which gave NOxPorfin-1 in 95% yield. Additionally, the authentic tumed-over triazole product (tNOxPorfm-1) was synthesized quantitatively by adding DEA NONOate (an NO donor) to a solution of NOxPorfin-1. Preparation of the other NOxPorfms is detailed in the Examples, along with relevant characterization data. Scheme 1. Synthesis of NOxPorfin-1 and tNOxPorfin-1. In Vitro Characterization and Determination of NO Responsiveness. With the NOxPorfin series in hand, we began our analysis by comparing the singlet oxygen quantum yield and fluorescence properties before and after treatment with DEA NONOate, which we employed to generate the corresponding reaction products in situ (Table 1). With respect to the impact of NO on fluorescence, NOxPorfin- 1 gave a ~70-fold tum-on response and thereby dramatically outperformed each of the other congeners (Figure 2a). Likewise, the fold-change in the singlet oxygen quantum yield was also the largest for NOxPorfin- 1 (7.5 -fold), and this effect can be attributed to efficient PeT-mediated suppression of intersystem crossing prior to activation ( I> \ = 0.06). Importantly, achieving a low level of background photosensitization (< 0.10) is critical to limiting off-target effects during treatment. On the contrary, the <h \ after reaction with NO increases substantially (<h \ = 0.45) to reach levels comparable to tetraphenyl porphyrin, a potent reference photosensitizer (Figure 2b).

[0099] Table 1. Summary of fluorescent and singlet oxygen quantum yield properties of the NOxPorfin series.

[0100] Having identified NOxPorfm-1 as our lead compound, we focused our subsequent efforts on evaluating key properties pertinent to its in vivo use. First, because the OFF-ON design of NOxPorfm-1 relies on the PeT quenching ability of the amino functional group, a change in its protonation state while in circulation can impair this feature. Within a biologically relevant pH range of 5.5 to 11.0, we found the fluorescence emission to be invariant (Figure 2c). Next, we monitored the time-dependent response of NOxPorfin- 1 to 50 equivalents of the donor over a period of one hour (Figure 2d). Immediately upon initiating the reaction (even before the first scan), a colorimetric change was observed. Furthermore, the tum-on response had reached 6-fold by the one-minute mark and became saturated within 20 minutes.

[0101] To further assess NOxPorfm-1 reactivity, incubation with various concentrations of DEA NONOate were examined. We found the response to exhibit linear dependency and a significant signal change was noted when the donor was decreased to as low as two equivalents (10 pM) (Figure 2e). Of note, the ability to detect NO released from DEA NONOate at these levels is critical because it is commonly used to mimic basal levels of NO found in cancer cells. Finally, we incubated NOxPorfin-1 with various biologically relevant analytes to determine whether the signal would be affected to give false positive results (Figure 2f). For instance, transition metal ions such as Fe (II) and Cu (II) can form coordination complexes with the o-phenylenediamine moiety which can counteract the PeT quenching effect of the trigger. Likewise, reactive carbonyl species like formaldehyde can react with the trigger to generate an unquenched benzimidazole product. Lastly, various reactive oxygen species and reactive nitrogen species can alter the trigger and / or the porphyrin core. Despite using an excess of each analyte, no apparent change was observed for any of the conditions tested relative to NO treatment.

[0102] NO-mediated Activation of NOxPorfin-1 in Live Cells. After rigorous in vitro characterization, we turned our attention to assessing the responsiveness of NOxPorfin-1 to NO within living cells where there are numerous competing pathways (e.g., thiol 5-nitrosation of cysteine residues) that can decrease the effective concentration of NO. To this end, A549 lung cancer cells were co-incubated with NOxPorfin-1 (5 pM) and DEA NONOate at donor concentrations of 20 pM and 100 pM. In comparison to control cells, we found the cells treated with DEA NONOate were 1.21- and 1.80-fold more fluorescent, respectively (Figure 3a and 3b). Next, we incubated cells with L-NMMA, a non-selective inhibitor of nitric oxide synthase, to determine whether NOxPorfin-1 was sufficiently sensitive to detect basal NO. We hypothesized because NOS inhibition would decrease the production of NO, the fluorescence intensity will be lower due to attenuated NOxPorfin-1 activation. Conversely, we also stimulated the biosynthesis of NO via treatment with lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, to promote the expression of iNOS. Relative to untreated control cells, the normalized fluorescence signals were measured to be 0.77 ± 0.05 and 2.16 ± 0.34 for the L-NMMA and LPS conditions, respectively (Figure 3c and 3d). We attribute these results to different extents of NO-mediated NOxPorfin-1 activation, which we further confirmed by employing Ctrl-NOxPorfin-1. In this set of control experiments, the normalized intensity was not significantly different relative to the vehicle for both L-NMMA (1.02) and LPS (LOO). Lastly, we obtained similar results when each of these experiments was performed on 4T1 breast cancer cells. The microscopy images and quantified data are shown in Figure 7-9.

[0103] In Cellulo Photosensitization Using Activated NOxPorfin-1. To test the ability of NO to increase the extent of photosensitization, it was necessary to first determine dark toxicity of NOxPorfin-1. To our delight, when NOxPorfin-1 was cultured with A549 cells in the dark, we did not observe a reduction in viability even at NOxPorfin-1 concentrations up to 20 pM (16-hours incubation period) (Figure 3e). On the contrary, a significant loss of -13% and -40% viability was measured when cells were exposed to NOxPorfin-1 at 2.5 and 5 pM, respectively, and irradiated with light (505 nm) (Figure 10). This finding is remarkable because it indicates that basal levels of NO found in non-stimulated cancer cells are sufficient to activate NOxPorfin-1 for PDT applications (Figure 3f). To demonstrate a dose-dependent phototoxicity response, we co-treated cells with NOxPorfin-1 and DEA NONOate at two concentrations. Upon analysis, we found the MTT results to be consistent with the fluorescence imaging data in which 100 pM (8% viability) of the donor resulted in more cell death relative to 20 pM (30% viability) (Figure 3f). The corresponding experiments conducted with the control reagent support these findings (Figure 11).

[0104] In Vivo Evaluation of NOxPorfin-1 Performance. To facilitate uptake and accumulation in tumors, we leveraged the enhanced permeability and retention (EPR) effect by encapsulating NOxPorfin-1 or Ctrl -NOxPorfin-1 in DSPE-mPEG-based liposomes (Table 2 and 3). The corresponding nanoparticles are herein named NOxPorfin-1 -NP and Ctrl-NOxPorfm-l-NP, respectively. In order to retain the exceptional selectivity profile of NOxPorfin-1 and its responsiveness to NO in its encapsulated form, while simultaneously achieving an appropriate dynamic range for fluorescence enhancement and ensuring long-term stability in solution, it was necessary to systematically tune the porphyrin to DSPE-mPEG ratio. Specifically, the NPs must be formulated in a manner to ensure NOxPorfin-1 does not leak out while in circulation. Failing to meet this first objective will decrease the effective concentration of prodrug loading. At the same time, the porosity must enable NO to readily diffuse into the NP and react with NOxPorfin-1 to generate the activated light-sensitive drug (t-NOxPorfin-1). Compromising this second objective will attenuate how much ROS will eventually be generated upon irradiation.

[0105] Table 2. NOxPorfin-1 to DSPE-mPEG ratio optimization for the synthesis of NOxPorfin-1 -NP with maximum NO response.

[0106] Table 3. Stability analysis of NOxPorfin-1 -NP with various % w / w of NOxPorfin-1 :DSPE-mPEG over the period of seven days.

[0107] Once the liposomal formulation was optimized, we performed a set of in vivo experiments to determine biodistribution and response to endogenous NO synthesized within a tumor site. Specifically, we implanted 4T1 cells into both flanks of BALB / c mice to generate tumors. Tumors on the left flank received a direct injection of saline (vehicle), while the tumors on the right flank were treated with L-NMMA. The animals were then randomly divided into two groups, administered NOxPorfm-l-NP or Ctrl-NOxPorfm-l-NP via systemic injection, and monitored using an in vivo fluorescent imaging system. Representative images are shown in Figure 4a and Figure 4c, respectively. For both sets of animals, the fluorescence of the saline-treated tumors increased as a function of time. Although this result demonstrates the NPs are localizing to the tumor, the average intensity between the two conditions was not statistically significant (Figure 12). Thus, this result alone could not be used to determine the extent of NO-mediated activation of the encapsulated NOxPorfm-1. To distinguish this, we analyzed the intensity of the inhibitor-treated tumor on the right flank. While the fluorescence of the tumors in animals that received Ctrl-NOxPorfm-l-NP was similar (Figure 4d), a significant attenuation of 29.9% was observed in the inhibited tumors for the cohort treated with NOxPorfm-l-NP (Figure 4b). This compelling finding unequivocally shows selective activation of NOxPorfm-1 was indeed occurring.

[0108] In Vivo Photodynamic Activity ofNOxPorfin-1. The preceding in vivo imaging results prompted us to next evaluate NOxPorfm-1 -NP in the context of ablating breast tumors via PDT. A new cohort of BALB / c mice were inoculated with 4T1 cells to establish subcutaneous tumors. Once the tumors had reached a volume of -100 mm3, NOxPorfm-l-NP was administered systemically at a dose of 1 mg / kg body weight. After 24 hours, the time point at which accumulation and activation were determined to be maximal (Figure 13), tumors were subjected to a light exposure sequence comprised of three cycles of irradiation (7.5 minutes) and dark recovery (10 minutes). The latter was incorporated to allow for reoxygenation of the tumor. After this single treatment, we monitored the tumor volumes daily over a period of one week. By day six, the tumors were so small they became practically immeasurable via the caliper method (Figure 5a, with light). On the other hand, lesions not exposed to light increased in tumor volume by 5.2-fold (Figure 5a, without light). In addition, we treated a third and fourth group of tumor-bearing mice with a PBS vehicle (no NOxPorfm-1 -NP) and subjected them to the light exposure sequence or kept them in the dark away from light. Without NOxPorfm-l-NP, the tumors grew substantially with a relative increase of size by 5.52-fold and 6.24- fold, respectively (Figure 14). To confirm the measurements reported above, tumors were excised at the end of the study. NOxPorfm-1 had a dramatic impact on suppressing tumor growth and progression (Figure 5b) by inducing apoptosis as revealed by H&E staining (Figure 5c).

[0109] NOxPorfin-1 Suppresses Tumor Growth of Nascent Tumors in a Metastatic Model. Finally, we established a nascent tumor model by inoculating both flanks of Nu / J mice with A549 lung cancer cells. Typically, when 5 X 105cells are used, tumors are slow-growing and often require at least 6 weeks to develop before they become palpable. By the 10-day mark, the tumors are yet to establish. Although at this time point the average measurement was -3.5 mm3, it is critical to note that this mass was not a tumor but rather, due to the Matrigel matrix used during inoculation. With this model in hand, we examined whether NOxPorfm-l-NP could be utilized to treat small lesions. This application represents a major challenge (for any treatment modality) due to the general undetectable nature of such tumors. Moreover, in the context of NOxPorfm-l-NP activation, early-stage malignancies lack prominent inflammatory features and therefore are anticipated to generate NO at substantially lower levels compared to larger tumors. To further challenge the performance of NOxPorfm-l-NP, we reduced the dose of the NP formulation from 1 mg / kg to 0.5 mg / kg and exposed the tumors on one side of the animal to only a single dose of light for a duration of five minutes (repeated twice over a span of six days). Interestingly, no signs of cancer were observed throughout the entirety of the experiment. However, the flank that was protected from light grew rapidly and uncontrollably (Figure 15). By the day 14, the tumor volume had increased by ~33-fold (Figure 6c).

[0110] Conclusion. Porphyrins are time-tested molecules that have been a mainstay in the biomedical field for decades, owing at least in part to their clinical application as versatile PDT agents to treat cancer. In contrast to standard chemotherapeutic treatments, photosensitization is presumed to be a more precise option because incident light can be focused on a given region of the body to limit off-target effects. However, this does not account for the non-specific uptake of a photosensitizer by adjacent healthy tissue. Subsequent light exposure will result in severe “PDT bums”, which may require a patient to remain in the dark for up to several weeks after treatment to avoid further damage. Thus, a paradigm shift away from canonical photosensitizers that are always in an ON-state toward chemically engineered PDT agents that can be selectively activated within the TME represents the focus of our work. In this regard, various classes of activatable photosensitizers exist (Chem Rev 2010, 110 (5), 2839-2857); however, we achieved a significant breakthrough by developing a precision tuned activatable porphyrin-based PDT agent (NOxPorfin-1) that is capable of responding to exceedingly low levels of a disease biomarker (i.e., NO). Although NO is a prominent feature of nearly all solid tumors, its steady-state concentration is only in the sub-nM range, thus requiring the use of ABS tuning and a PeT design approach to achieve the requisite reactivity and selectivity profiles and tum-on response, respectively.

[0111] Importantly, the control of photosensitization via PeT is not trivial and thus, has only yielded several acid-responsive, tum-on porphyrins. However, it is notable that these molecules do not operate under biologically relevant conditions. In contrast, we have demonstrated via rigorous in vitro and in cellulo characterization studies that NOxPorfin-1 is optimized for use within living systems. For instance, when evaluating NOxPorfin-1 in vivo, we intentionally chose to irradiate breast tumors using an unfocused LED light source. Due to the OFF-ON feature of our activatable PDT agent, the usual burning effect of the surrounding tissue was minimized, and impacted regions healed rapidly. On the other hand, activation of NOxPorfin-1 by NO in cancer cells led to a dramatic reduction in the tumor volume after only one treatment, to an extent where accurate measurements of size became impossible. These results prompted us to test NOxPorfin-1 in the context of nascent tumors.

[0112] It is well established that the treatment of such small lesions with NO-responsive molecules is a significant challenge because inflammatory biomarkers like NO are present at even lower levels compared to developed tumors. Despite reducing the concentration of NOxPorfin-1 from a dose of 1 mg / kg to 0.5 mg / kg, along with lowering the overall light exposure duration by more than 50%, inoculation sites exposed to light were completely devoid of signs of cancer. These results are exciting because early metastatic tumors are notoriously difficult to target. We envision that by incorporating NOxPorfin-1 as part of standard adjuvant therapy protocols; it will be possible to limit instances of cancer relapse by simply irradiating known metastatic sites (e.g., the liver or lung) with light without concern of off-target effects. Another important consideration is that aberrant NO production is a feature of most tumors, regardless of the cancer type. As such, NOxPorfin-1 may emerge as a one-size-fits-all treatment option. Lastly, beyond theranostic applications, the enhancement of fluorescence upon NOxPorfin-1 activation can be paired with emerging medical procedures such as fluorescence-guided surgery to further improve treatment outcomes.

[0113] Pharmaceutical Formulations.

[0114] The compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier. The compounds may be added to a carrier in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and -glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

[0115] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

[0116] The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes. The compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1% of active compound. The percentage of the compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.

[0117] The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0118] The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.

[0119] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and / or gelatin.

[0120] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.

[0121] For topical administration, compounds may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer the active agent to the skin as a composition or formulation, for example, in combination with a dermatologically acceptable carrier, which may be a solid, a liquid, a gel, or the like.

[0122] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol / glycol blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0123] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0124] Examples of dermatological compositions for delivering active agents to the skin are known to the art; for example, see U.S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition.

[0125] Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.). The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.

[0126] In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg / kg, e.g., from about 10 to about 75 mg / kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg / kg / day, most preferably in the range of 15 to 60 mg / kg / day.

[0127] The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

[0128] The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg / m2, conveniently 10 to 750 mg / m2, most conveniently, 50 to 500 mg / m2of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0129] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0130] The compounds described herein can be effective anti-tumor agents and have higher potency and / or reduced toxicity as compared to other agents used for photodynamic therapy.

[0131] The invention provides therapeutic methods of treating cancer in a mammal, which involve administering to a mammal having cancer an effective amount of a compound or composition described herein. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like. Cancer refers to any various type of malignant neoplasm, for example, colon cancer, breast cancer, melanoma and leukemia, and in general is characterized by an undesirable cellular proliferation, e.g., unregulated growth, lack of differentiation, local tissue invasion, and metastasis.

[0132] The ability of a compound of the invention to treat cancer may be determined by using assays well known to the art. For example, the design of treatment protocols, toxicity evaluation, data analysis, quantification of tumor cell kill, and the biological significance of the use of transplantable tumor screens are known. In addition, ability of a compound to treat cancer may be determined using the Tests as described below.

[0133] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0134] EXAMPLES

[0135] Example 1. General Methods.

[0136] Materials: Materials were purchased from commercial vendors and used without further purification. Thin layer chromatography (TLC) was performed on glass-backed TLC plates precoated with silica gel containing an UV254 fluorescent indicator (Macherey-Nagel). TLCs were visualized with a 254 / 365 nm UV hand-held lamp (UVP). Flash silica gel chromatography was performed using 0.04 - 0.063 mm 60 M silica (Macherey-Nagel). All deuterated solvents were purchased from Cambridge Isotope Laboratories. Diethylammonium (Z)- l-(A / N-diethylamino)diazen- 1 -ium- 1 ,2- diolate (DEA NONOate) and L-N('-monomethyl Arginine acetate (L-NMMA) were purchased from the Cayman Chemical Company. Acetone, acetonitrile, cupric sulfate, dichloromethane, dimethylformamide, ethyl acetate, ferric chloride, glacial acetic acid, phosphate saline buffer (Coming), fuming nitric acid and tetrahydrofuran were purchased from Thermo Fisher Scientific. mDSPE-PEGMW 5,000 was purchased from Layson Bio. Centrifugal filters (50K) and Mohr’s salt were purchased from MilliporeSigma. Potassium carbonate, sodium bicarbonate, sodium borohydride, sodium sulfate (anhydrous), tin chloride and -chloranil were purchased from Oakwood Chemicals. Anhydrous methanol and hydrogen peroxide (30% v / v) were purchased from Macron Fine Chemicals. 2,4-Dinitrobenzaldehyde and 4-hydroxy 3 -nitrobenzaldehyde were purchased from Ambeed. 3 -Hydroxy 4-nitrobenzaldehyde and 4-acetamidobenzaldehyde were purchased from AK Scientific. Concentrated HC1 was purchased from VWR. Boron trifluoride etherate, 1,3- diphenylisobenzofuran, lipopolysaccharides from Escherichia coli Ol l i :B4 (purified by phenol extraction), and pyrrole were purchased from Sigma- Aldrich. Triethylamine was purchased from Alfa Aesar. Ethanol 200 proof was purchased from Decon Chemicals Inc. Matrigel Basement Membrane Matrix was purchased from Coming.

[0137] Instruments and Software: 'H and13C NMR spectra were acquired on a Varian 500 or Carver B500 spectrometer. The following abbreviations were used to describe coupling constants: singlet (s), doublet (d), triplet (t), quartet (q) or multiplet (m). Spectra were visualized and analyzed using MestReNova (version 14.2) and referenced to trace non-deuterated solvent. High-resolution mass spectra were acquired on a Waters Q-TOF Ultima ESI mass spectrometer or a Waters Synapt G2-Si ESI / LC-MS spectrometer. Absorption spectra was acquired on a JASCO V-770 UV-Vis-NIR spectrophotometer for wavelengths ranging from 190-1200 nm. Fluorescence spectra were acquired on a QuantaMaster-400 scanning spectrofluorometer with microfluorescence quartz cuvettes (Science Outlet). Dynamic light scattering measurements were acquired with a Malvern Zetasizer Nano ZS. MTT assays were analyzed using a SpectraMax M2 plate reader. A Countess® II FL Automated Cell Counter (Invitrogen, Thermo Fisher Scientific) was used for determining cell concentrations before plating, as well as tumor implantation. 505 nm LED (520 mW power output, 5.94 pW / mm2maximum irradiance) and 595 nm LED (1217 mW power output, 13.5 pW / mm2maximum irradiance) were purchased from Thorlabs, Inc. Cellular imaging was performed using EVOS FL epifluorescence microscope. Fluorescence imaging of mice was performed using the CRi Maestro in vivo fluorescence imaging system. All the images were analyzed using the ImageJ software and reported as the mean pixel intensity in ROIs of equal area. Data were analyzed using Origin® 2018, and Microsoft Excel. Schematic illustrations were prepared using Adobe Photoshop. Final figures were assembled and prepared in PowerPoint (version Microsoft 365).

[0138] Fluorescence Quantum Yield Measurements: The relative quantum yields (Q) of each NOxPorfin were determined using Perkin Elmer’s protocol. Tetraphenylporphyrin was used as the standard (fluorescence quantum yield = 0.11 in toluene).

[0139] Singlet Oxygen Quantum Yield Measurements: The singlet oxygen quantum yield of NOxPorfins, tNOxPorfins and Ctrl -NOxPorfin- 1 were determined using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger and tetraphenylporphyrin (Q(1O2) = 0.59) as the reference. Irradiation was carried out with a xenon arc lamp at 418 nm.

[0140] Example 2. Synthetic Procedures.

[0141] 5-(4-Acetamido-3-nitrophenyl)-10, 15, 20-triphenyl porphyrin (2). A solution of benzaldehyde (359 mg, 3.39 mmol,), A-(4-formyl-2-nitrophenyl) acetamide (241 mg, 1.16 mmol), and pyrrole (309 mg, 4.62 mmol) in CH2CI2 (150 mL) was purged with nitrogen for 15 minutes. A solution of 2.5 M BFs.OEt2 (45 pL) was added. The resulting mixture was stirred at room temperature for two hours, treated with 72-chloranil (2.3 g, 9.35 mmol), and stirred for an additional 12 hours. The volatiles were then removed under reduced pressure and the crude residue was purified using silica gel column chromatography (eluent = 4:1 v / v CH2CI2: Hexanes). The titled compound was obtained as dark purple crystals in 18% yield. 'H NMR (500 MHz, CDCh) 8 10.70 (s, 1H), 9.22 (d, J= 8.5 Hz, 1H), 9.08 (d, J= 2.1 Hz, 1H), 8.92 (d, J= 4.7 Hz, 2H), 8.88 (s, 4H), 8.80 (d, J= 4.7 Hz, 2H), 8.54 (dd, J = 8.5, 2.1 Hz, 1H), 8.24 (d, J= 6.6 Hz, 6H), 7.80 - 7.76 (m, 9H), 2.49 (s, 3H), -2.77 (s, 2H).13C NMR (126 MHz, CDCh) 8 169.38, 141.99, 141.95, 140.98, 137.66, 135.06, 134.59, 134.51, 130.30, 127.89, 126.79, 126.76, 120.94, 120.64, 120.50, 115.96, 25.86. 5-(4-Amino-3-nitrophenyl)-10,15,20-triphenyl porphyrin (3). A solution of compound 2 (50 mg, 0.069 mmol) in TFA (3 mL) and concentrated HC1 (4 mL) was stirred 65°C for two hours under a nitrogen atmosphere. Upon completion, as determined by consumption of 2 via TLC, the reaction was cooled to room temperature, neutralized with a 10% aq. NaOH (50 mL), and extracted with CHCh (50 mL). The organic was then washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified via passage through a neutral alumina plug using 4: 1 v / v CH2CT2: Hexanes as the eluent to afford the titled compound in 95% yield.XH NMR (500 MHz, CDCh) 8 8.97 (d, J= 1.9 Hz, 1H), 8.93 - 8.82 (m, 8H), 8.23 (d, J = 6.4 Hz, 6H), 8.20 (d, J= 2.0 Hz, 1H), 7.78 (p, J= 8.3 Hz, 9H), 7.12 (d, J= 8.4 Hz, 1H), 6.43 - 6.33 (m, 2H), -2.76 (s, 2H).13C NMR (126 MHz, CDCh) 5 144.17, 142.07, 141.06, 134.58, 131.29, 131.20, 130.81, 128.83, 127.81, 126.73, 120.50, 120.39, 117.29, 117.08.

[0142] NOxPorfin-1. To a solution of compound 3 (50 mg, 0.074 mmol) in cone. HC1 (10 mL), SnCh 2H2O (100 mg, 0.44 mmol) was added. The reaction was stirred for five minutes under a nitrogen atmosphere. The temperature was raised to 65°C and stirred for ~2 hours. Upon completion, as determined by consumption of 3 via TLC, the reaction was cool to room temperature, neutralized with 10% aq. NaOH, and extracted with CHCh (50 mL). The organic fraction was washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified via column chromatography on activated neutral alumina using CH2CI2 as the eluent. The titled compound was obtained as a purple crystalline solid in 95% yield. 'H NMR (500 MHz, CDCh) 8 8.99 (d, J= 4.7 Hz, 2H), 8.84 (d, J= 7.1 Hz, 6H), 8.23 (d, J= 6.2 Hz, 6H), 7.79 - 7.73 (m, 9H), 7.59 (d, J= 1.8 Hz, 1H), 7.57 (dd, J= 7.7, 1.9 Hz, 1H), 7.05 (d, J= 7.6 Hz, 1H), 3.67 (d, 4H), -2.73 (s, 2H).13C NMR (126 MHz, CDCh) 8 142.32, 142.28, 134.59, 134.52, 134.15, 132.82, 127.68, 126.68, 123.85, 120.94, 119.97, 119.78, 114.98). HR-MS calculated for C44H33N6 [M+H]+645.2770, found 645.2767.

[0143] Scheme 2. Synthesis of NoxPorfin-2.

[0144] NOxPorfin-2

[0145] 5-(3-Hydroxy-4-nitrophenyl)- 10, 15, 20-triphenyl porphyrin (4). A solution of benzaldehyde (0.359 g, 3.39 mmol), 3-hydroxy-4-nitrobenzaldehyde (0.193 g, 1.16 mmol), and pyrrole (0.309 g, 4.62 mmol) in CH2CI2 (150 mL) was purged with nitrogen for 15 minutes. A solution of 2.5 M BFs.OEt2(45 pL) was then added. The reaction mixture was stirred at room temperature for two hours, treated with (-chloranil (0.23 g, 0.935 mmol), and stirred for an additional 12 hours. The volatiles were then removed under reduced pressure. The residue was then purified using silica gel column chromatography (eluent = 1:1 v / v CH2CT2: Hexanes). The titled compound was obtained as dark purple crystals in 12% yield (0.094 g, 0.139 mmol).JH NMR (500 MHz, CDCh) 8 10.99 (s, 1H), 8.97 (d, J= 2.1 Hz, 1H), 8.94 - 8.86 (m, 8H), 8.81 (d, J= 4.6 Hz, 3H), 8.46 (d, J= 8.5 Hz, 1H), 8.24 (d, J= 6.5 Hz, 9H), 7.86 - 7.71 (m, 13H), 7.56 (d, J= 8.4 Hz, 2H).13C NMR (126 MHz, CDCh) 8 154.92, 143.10, 142.55, 142.00, 141.96, 134.78, 134.59, 132.48, 129.30, 127.88, 126.77, 120.87, 120.61, 118.75, 118.32, 116.08. HR-MS calculated for C44H30N5O3 [M+H]+676.2349, found 676.2351.

[0146] NOxPorfin-2. To a solution of compound 4 (50 mg, 0.074 mmol) in cone. HCI (10 mL), SnCh 2H2O (100 mg, 0.44 mmol) was added. After stirring for five minutes under an inert nitrogen atmosphere, the temperature was increased to 65 °C and stirred for an additional 2 hours. Upon completion, as determined by TLC analysis, the mixture was cooled to room temperature, neutralized with 10% aq. NaOH, and extracted with CHCh (50 mL). The organic fraction was then washed with water, filtered, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure without elevating the temperature of the water bath past 45 °C. The residue was then purified via column chromatography (activated neutral alumina) using CH2CI2 as the eluent. The product was obtained as purple crystalline solid in 99% yield (0.047 g, 0.073 mmol). 'H NMR (500 MHz, CDCh) 8 8.85 (t, J= 18.0 Hz, 8H), 8.22 (d, J= 28.0 Hz, 6H), 7.77 - 7.61 (m, 9H), 7.49 (s, 1H), 7.15 (s, 1H), 6.83 (s, 1H), 3.75 (br, 2H), -2.69 (br, 2H).13C NMR (126 MHz, CDCh) 8 142.18, 141.81, 134.61, 134.56, 132.85, 128.12, 127.69, 127.66, 126.70, 126.66, 121.86, 120.56, 120.10, 119.94, 114.53. HR-MS calculated for C44H32N5O [M+H]+646.2607, found 646.2606.

[0147] Scheme 3. Synthesis of NoxPorfin-3.

[0148] 5-(4-Hydroxy-3-nilrophenylj- 10.15, 20-triphenyl porphyrin (5). A solution of benzaldehyde (0.359 g, 3.39 mmol), 4-hydroxy-3 -nitrobenzaldehyde (0.193 g, 1.16 mmol), and pyrrole (0.309 g, 4.62 mmol) in CH2CI2 (150 mL) was purged with nitrogen for 15 minutes. A solution of 2.5 M BFs.OEt2(45 pL) was then added. The reaction mixture was stirred at room temperature for two hours, treated with (-chloranil (0.23 g, 0.935 mmol), and stirred for an additional 12 hours. The volatiles were then removed under reduced pressure. The residue was then purified using silica gel column chromatography (eluent = 1:1 v / v CH2CT2: Hexanes). The titled compound was obtained as dark purple crystals in 16% yield (0.12 g, 0.18 mmol). 'H NMR (500 MHz, CDCI3) 8 10.98 (s, 1H), 8.91 (d, J= 4.6 Hz, 2H), 8.90 - 8.82 (m, 6H), 8.48 (d, J= 8.5 Hz, 1H), 8.23 (d, J= 6.5 Hz, 6H), 8.06 (d, J= 1.6 Hz, 1H), 7.88 (dd, J= 8.5, 1.7 Hz, 1H), 7.82 - 7.75 (m, 9H), -2.76 (s, 2H).13C NMR (126 MHz, CDCI3) 8 153.51, 152.45, 141.94, 141.91, 134.56, 133.35, 127.91, 127.09, 126.80, 126.76, 125.94, 122.99, 121.22, 120.73, 116.12. HR-MS calculated for C44H30N5O3 [M+H]+676.2349, found 676.2354.

[0149] NOxPorfin-3. To a solution of compound 5 (50 mg, 0.074 mmol) in cone. HC1 (10 mL), SnCh 2H2O (100 mg, 0.44 mmol) was added. After stirring for five minutes under an inert nitrogen atmosphere, the temperature was increased to 65 °C and stirred for an additional 2 hours. Upon completion, as determined by TLC analysis, the mixture was cooled to room temperature, neutralized with 10% aq. NaOH, and extracted with CHCh (50 mL). The organic fraction was then washed with water, fdtered, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure without elevating the temperature of the water bath past 45 °C. The residue was then purified via column chromatography (activated neutral alumina) using CH2CI2 as the eluent. The product was obtained as purple crystalline solid in 99% yield (0.047 g, 0.073 mmol).JH NMR (500 MHz, DMSO) 8 9.58 (s, 1H), 9.00 (s, 1H), 8.80 (s, 6H), 8.28 (d, J= 33.2 Hz, 1H), 8.21 (d, J= 5.7 Hz, 6H), 7.82 (d, J= 4.9 Hz, 9H), 7.50 (s, 1H), 7.25 (d, J= 7.8 Hz, 1H), 7.08 (d, J= 7.7 Hz, 1H), 4.91 (s, 2H), -2.89 (s, 2H).13C NMR (126 MHz, DMSO) 8 144.67, 141.80, 135.43, 134.70, 132.86, 128.51, 127.47, 124.52, 122.27, 122.15, 120.22, 119.98, 113.30. HR-MS calculated for C44H32N5O

[0150] [M+H]+646.2607, found 646.2595.

[0151] Scheme 4. Synthesis of NOxPorfin-4. NOxPorfin-4

[0152] 5-(4-tert-Butoxycarbonylamino-3-hydroxyphenyl)-10, 15, 20-triphenyl porphyrin (6). To a solution of NOxPorfin-2 (0.250 g, 0.3871 mmol) in CH2CI2 (20 mL), di- / e / 7-butyl dicarbonate (0.845, 3.871 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours. The volatiles were then removed under reduced pressure. The residue was then purified using silica gel column chromatography (eluent = 5:1 v / v CTbCkHexanes). The titled compound was obtained as dark purple crystals in 73% yield (0.210 g, 0.283 mmol).JH NMR (400 MHz, CDCh) 8 8.90 - 8.81 (m, 8H), 8.22 (s, 6H), 7.76 (q, J= 6.6 Hz, 9H), 7.64 (d, J = 8.1 Hz, 1H), 7.61 (s, 1H), 7.42 (d, J= 8.0 Hz, 1H), 6.69 (s, 1H), 1.63 (s, 9H), -2.78 (s, 2H).13C NMR (126 MHz, CDCh) 8 146.78, 145.34, 142.18, 134.57, 127.72, 127.57, 126.69, 125.46, 120.15, 119.15, 85.22, 82.27, 28.39, 27.44. HR-MS calculated for C49H40N5O3 [M+H]+746.3131, found 746.3127.

[0153] 5-(3-Methoxy-4-tert-butoxycarbonylaminophenyl)-10, 15, 20-triphenyl porphyrin (7). To a solution of compound 6 (0.06 g, 0.081 mmol) and potassium carbonate (0.02 g, 4.62 mmol) in DMF (15 mL), iodomethane (0.342 g, 2.41 mmol) was added at room temperature. The temperature of the reaction mixture was elevated to 80 °C, under refluxing conditions and stirred for 12 hours. After that, the reaction was cooled to room temperature, and the reaction mixture was concentrated under reduced pressure. The crude residue was purified via column chromatography using silica gel (eluent = 1:2 v / v CH2CI2: Hexanes). The titled compound was obtained as dark purple crystals in 43% yield (0.026 g, 0.035 mmol).XH NMR (500 MHz, CDCh) 8 8.96 (d, J= 4.6 Hz, 2H), 8.87 (s, 6H), 8.51 (d, J= 7.2 Hz, 1H), 8.24 (d, J= 6.5 Hz, 6H), 7.87 (dd, J= 8.1, 1.7 Hz, 1H), 7.81 - 7.74 (m, 10H), 7.46 (s, 1H), 3.99 (s, 3H), 1.67 (s, 9H), -2.73 (s, 2H).13C NMR (126 MHz, CDCh) 8 153.09, 145.90, 142.23, 136.24, 134.58, 127.96, 127.92, 127.74, 126.72, 120.16, 120.12, 120.05, 117.05, 116.12, 80.69, 56.02, 28.52. HR-MS calculated for C50H42N5O3 [M+H]+760.3288, found 760.3284.

[0154] 5-(3-Methoxy-4-aminophenyl)-10, 15, 20-triphenyl porphyrin (8). A solution of compound 7 (50 mg, 0.067 mmol) in CH2CI2 (4 mL) was placed on an ice-bath. TFA (1 mL) was added dropwise to the reaction and was stirred for two hours at room temperature. Upon completion, as determined by consumption of 7 via TLC, the reaction mixture neutralized with a 10% aq. NaOH (20 mL) and extracted with CHCh (50 mL). The organic fraction was then washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified via passage through silica gel column chromatography using CH2CI2 as the eluent to afford the titled compound in 100% yield (0.044g, 0.067 mmol).JH NMR (500 MHz, CDCh) 8 9.00 (d, J= 4.7 Hz, 2H), 8.86 (s, 6H), 8.24 (dd, J= 7.6, 1.5 Hz, 6H), 7.80 - 7.74 (m, 9H), 7.71 (d, J= 1.8 Hz, 1H), 7.66 (dd, J= 7.7, 1.8 Hz, 1H), 7.08 (d, J= 7.7 Hz, 1H), 4.15 (s, 2H), 3.98 (s, 3H), -2.71 (s, 2H).13C NMR (126 MHz, CDCh) 8 145.64, 142.31, 142.26, 135.79, 134.57, 132.46, 128.18, 127.70, 126.70, 121.02, 120.03, 119.84, 117.67, 113.23, 55.79.

[0155] NOxPorfin-4. To a solution of compound 8 (0.035 g, 0.053 mmol) in 4: 1 v / v MeOHCTLCh, formaldehyde (250 pL of 37% in H2O) was added and the reaction was stirred for 15 hours. After that, the temperature was reduced to 0 °C, and NaBFL (0.0401 g, 1.06 mmol) was slowly added in portions over the course of next 15 minutes. The reaction was allowed to stir for two hours. After two hours, the mixture was quenched with sat aq. NH4CI and then basified with sat. aq NaHCOs, followed by extraction with CHCh. The organic fraction was then washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified via passage through silica gel column chromatography using 1:6 v / v EtOAc:Hexanes as the eluent to afford NOxPorfin-4 in 14% yield (5 mg, 0.007 mmol). 'H NMR (500 MHz, CDCh) 8 8.99 (d, J= 4.7 Hz, 2H), 8.83 (s, 6H), 8.22 (d, J= 7.7 Hz, 6H), 7.78 - 7.73 (m, 10H), 7.68 (d, J= 1.8 Hz, 1H), 6.97 (d, J= 7.9 Hz, 1H), 4.65 (s, 1H), 3.96 (s, 3H), 3.15 (s, 3H), -2.72 (s, 2H).13C NMR (126 MHz, CDCh) 8 145.35, 142.47, 134.69, 130.35, 128.80, 127.79, 126.80, 120.09, 119.80, 116.85, 107.76, 55.89, 30.72. HR-MS calculated for C46H36N5O [M+H]+674.2915, found 674.2920.

[0156] Scheme 5. Synthesis of Ctrl-NOxPorfin-1. Ctrl-NOxPorfin-1

[0157] 5-(2,4-Dinitrophenyl)-10, 15, 20-triphenyl porphyrin (9). A solution of benzaldehyde (0.359 g, 3.39 mmol), 2,4-dinitrobenzaldehyde (0.227 g, 1.16 mmol), and pyrrole (0.309 g, 4.62 mmol) in CH2CI2 (150 mL) was purged with nitrogen for 15 minutes. A solution of 2.5 M BFs.OEt2 (45 pL) was then added. The reaction mixture was stirred at room temperature for two hours, treated with p- chloranil (0.23 g, 0.935 mmol), and stirred for an additional 12 hours. The volatiles were then removed under reduced pressure. The residue was then purified using silica gel column chromatography (eluent = 1:1 v / v CH2CI2: Hexanes). The titled compound was obtained as a form with dark purple crystals in 15% yield (0.112 g, 0.174 mmol). *HNMR (500 MHz, CDCh) 8 9.28 (d, J= 2.3 Hz, 1H), 8.90 (d, J= 4.7 Hz, 2H), 8.88 - 8.85 (m, 4H), 8.76 - 8.73 (m, 1H), 8.58 (d, J= 4.7 Hz, 2H), 8.48 (s, 1H), 8.25 - 8.16 (m, 5H), 7.75-7.80 (m, 10H), -2.69 (s, 2H).13C NMR (126 MHz, CDCh) 8 151.81, 148.01, 143.11, 141.86, 141.67, 138.03, 134.59, 134.52, 128.68, 128.06, 127.97, 126.88, 126.76, 126.57, 124.71, 121.81, 121.08, 119.34, 110.60. HR-MS calculated for C44H29N6O4 [M+H]+705.2250, found 705.2247.

[0158] Ctrl-NOxPorfin-1. To a solution of compound 9 (50 mg, 0.074 mmol) in cone. HC1 (10 mL), SnCh 2H2O (100 mg, 0.44 mmol) was added. After stirring for five minutes under an inert nitrogen atmosphere, the temperature was increased to 65 °C and stirred for an additional 2 hours. Upon completion, as determined by TLC analysis, the mixture was cooled to room temperature, neutralized with 10% aq. NaOH, and extracted with CHCh (50 mL). The organic fraction was then washed with water, filtered, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was then purified via column chromatography (activated neutral alumina) using CH2CI2 as the eluent. The product was obtained as purple crystalline solid in 95% yield (0.045 g, 0.07 mmol).XH NMR (500 MHz, CDCh) 8 9.01 (d, J= 4.7 Hz, 2H), 8.87 (d, J= 6.9 Hz, 6H), 8.24 (d, J = 5.6 Hz, 6H), 7.81 - 7.74 (m, 9H), 7.66 (d, J= 7.9 Hz, 1H), 6.51 (d, J= 7.9 Hz, 1H), 6.36 (s, 1H), 3.66 (s, 4H), -2.70 (s, 2H).13C NMR (126 MHz, CDCh) 8 233.89, 147.74, 147.63, 142.24, 142.13, 135.81, 134.57, 127.74, 126.72, 120.34, 119.98, 118.47, 116.09, 105.31, 101.41. HR-MS calculated for C44H33N6 [M+H]+645.2767, found 645.2759.

[0159] General Procedure to Synthesize Turned-over NOxPorfin Products (tNOxPorfin-1 to -4). To synthesize turned-over product (tNOxPorfin), a 5 mM stock solution of the starting compound (NOxPorfin- 1 through NOxPorfin-4) was prepared in DMSO. From this stock, 20 pL was measured in a 0.2 mL PCR tube and was kept sealed. Concurrently, a 25 mM stock solution of DEA NONOate was prepared in PBS and maintained at 0°C. Immediately, 30 pL of the DEA NONOate stock solution was added to the NOxPorfin solution. The tube was sealed and vortexed vigorously for two minutes. This reaction induced an instantaneous color change from reddish-brown to a vibrant magenta-pink, indicating successful conversion. The resultant products were subsequently confirmed through HRMS analyses.

[0160] Example 3. Preparation of Liposome-based Nanoparticles Encapsulating NOxPorfin- 1 or Ctrl- NOxPorfin-1. a) NOxPorfin- 1-NP. NOxPorfin-1 (0.19 mg), and DSPE-mPEG (10 mg) in THF (1 mL) were rapidly injected into Milli-Q water (2 mL) under sonication. After sonication for an additional five minutes, THF was removed under rotary evaporation at room temperature. The NP solution was purified by ultrafiltration (50 K, 12 g, 10 minutes) followed by three washes with Milli-Q water (50 K, 17 g, 10 minutes). b) NOxPorfin- 1-NP: NOxPorfin-1 (0.0644 mg), and DSPE-mPEG (75 mg, 0.09% w / w) in THF (2.5 mL) were rapidly injected into Milli-Q water (5 mL) under sonication. After sonication for an additional five minutes, THF was removed under rotary evaporation at room temperature. The NP solution was purified by ultrafiltration (50 K, 12 g, 10 minutes) followed by three washes with Milli- Q water (50 K, 17 g, 10 minutes). c) NOxPorfin- 1-NP: NOxPorfin-1 (0.1288 mg), and DSPE-mPEG (75 mg, 0.17% w / w) in THF (2.5 mL) were rapidly injected into Milli-Q water (5 mL) under sonication. After sonication for an additional five minutes, THF was removed under rotary evaporation at room temperature. The NP solution was purified by ultrafiltration (50 K, 12 g, 10 minutes) followed by three washes with Milli- Q water (50 K, 17 g, 10 minutes). d) NOxPorfin- 1-NP: NOxPorfin-1 (0.0644 mg), and DSPE-mPEG (10 mg, 0.64% w / w) in THF (1 mL) were rapidly injected into Milli-Q water (2 mL) under sonication. After sonication for an additional five minutes, THF was removed under rotary evaporation at room temperature. The NP solution was purified by ultrafiltration (50 K, 12 g, 10 minutes) followed by three washes with Milli- Q water (50 K, 17 g, 10 minutes). e) NOxPorfin- 1-NP: NOxPorfin-1 (0.1932 mg), and DSPE-mPEG (10 mg, 1.93% w / w) in THF (1 mL) were rapidly injected into Milli-Q water (2 mL) under sonication. After sonication for an additional five minutes, THF was removed under rotary evaporation at room temperature. The NP solution was purified by ultrafiltration (50 K, 12 g, 10 minutes) followed by three washes with Milli- Q water (50 K, 17 g, 10 minutes).

[0161] Ctrl-NOxPorfin-l-NP. Ctrl-NOxPorfin-1 (0.19 mg), and DSPE-mPEG (10 mg) in THF (1 mL) were rapidly injected into Milli-Q water (2 mL) under sonication. After sonication for another 5 minutes, THF was removed under rotary evaporation at room temperature. The NP solution was purified by ultrafiltration (50 K, 12 g, 10 minutes) followed by three washes with Milli-Q water (50 K, 17 g, 10 minutes).

[0162] Example 4. In-vitro and in-vivo experimental details.

[0163] Fluorescence Imaging of Endogenous NO in Live Cells. Four- well chamber slides were coated with poly-L-lysine, seeded with A549 (500 pL of 2.0 x 105cells / mL) or 4T1 (500 pL of 1.0 x 105cells / mL) cells, and incubated at 37 °C with 5% CO2 for 24 hours. After reaching ~70% confluency, cells were subjected to the following conditions. LPS: Media was aspirated and replaced with fresh media containing LPS (1 pg / mL). After incubation for 16 hours, cells were treated with NOxPorfin-1. L-NMMA: Media was aspirated and replaced with fresh media containing L-NMMA (1 mM). After incubation for two hours, cells were treated with NOxPorfin-1. Control: Media was aspirated and replaced with PBS. The cells were immediately treated with NOxPorfin-1. The final concentration of NOxPorfin-1 was 5 pM for all conditions. Upon addition of NOxPorfin-1, cells were incubated for six hours prior to imaging. Fluorescent images were obtained using an EVOS epi- fluorescent microscope with a 20 x objective and a Cy5 filter cube.

[0164] Photodynamic Therapy Treatment of Cancer Cells. Transparent 96-well plates were seeded with A549 (100 pL of 2.0 x 105cells / mL) or 4T1 (100 pL of 1.0 x 105cells / mL) cells. After incubation at 37 °C with 5% CO2 for 24 hours, the culture media was aspirated and replaced with fresh media containing NOxPorfin-1 (5 pM for A549 and 2.5 pM 4T1), Ctrl-NOxPorfin-1 (at matching concentrations), or PBS. After incubation for 12 hours, the media was exchanged with fresh media containing DEA NONOate (20 pM or 100 pM) or a vehicle. After incubation for 30 minutes, the media was replaced with fresh serum-free media and subjected to light irradiation (505 nm LED). A549 cells were irradiated for 2.5 minutes and 4T1 cells were irradiated for 1.5 minutes at a distance of 2 cm (150 pW / mm2, current = 0.7 A, full power). After exposure to light, cells were incubated for 18 hours and subjected to standard MTT assay conditions. The absorbance of each well was measured at 570 nm using a SpectraMax M2 plate reader. The percentage of cell viability was normalized to the treatment with the vehicle control (n = 5).

[0165] Evaluation of NO Activation of NOxPorfin-1 -NP In Vivo. BALB / c mice (female and male, 5- 8 weeks old) were subcutaneously inoculated on both flanks with 4T1 cells (50 pL of 2.5 x 105cells / mL in 1:1 v / v serum-free RPMI 1640 media: Matrigel). Mice were divided into two groups (n = 3 for each). Upon reaching a tumor volume of 100-150 mm3, mice received either NOxPorfm-l-NP (1 mg / kg) or Ctrl-NOxPorfm-l-NP (1 mg / kg) via retroorbital administration. Immediately after, a sterile solution of saline (50 pL) was intratumorally injected in the left tumor. A solution of L- NMMA (35 mM) in sterile saline (50 pL) was intratumorally injected in the right tumor. After nine hours, fluorescence images were obtained using the Maestro in vivo imaging system using a filter set capable of collecting light beyond 630 nm. Spectral unmixing was performed, and quantification of the signal intensity was done by drawing an ROI around each tumor using the ImageJ software. Body weights and tumor volumes were recorded throughout the study. The caliper method was used to estimate tumor volumes. (U= LW2H, where V= volume, L = length of the tumor, and W= width of the tumor).

[0166] PDT Treatment of 4T1 Breast Tumors. BALB / c mice (female and male, 5-8 weeks old) were subcutaneously inoculated on both flanks with 4T1 cells (50 pL of 5.0 x 105cells / mL in 1: 1 v / v serum-free RPMI 1640 media: Matrigel). When the tumor volumes reached ~100 mm3, the mice were divided into two groups (n = 3 each). The first group of mice received NOxPorfm-l-NP (1 mg / kg) and the second group of mice received PBS via retroorbital administration. Only the left tumors were exposed to light (595 nm LED). Light exposure sequence = three cycles of irradiation (7.5 minutes) and dark recovery (10 minutes). The right tumor was shielded from light throughout the experiment. Tumor volume was monitored daily until the tumors became almost immeasurable. At the conclusion of the experiment, the tumors were excised and preserved in buffered formalin for fixation.

[0167] PDT Treatment of Nascent A549 Lung Tumors. Nu / J mice (female and male, 4-6 weeks old) were subcutaneously inoculated on both flanks with A549 cells (50 pL of 5.0 x 105cells / mL in 1:1 v / v serum-free RPMI 1640 media: Matrigel). When the tumor volumes reached ~100 mm3, the mice were divided into two groups (n = 4 each). After 10 days, mice received NOxPorfm-l-NP (0.5 mg / kg) via retroorbital administration. After 24 hours, the tumors located on the left flank were exposed to light (595 nm LED) for five minutes. The treatment was repeated after six days. The body weights and tumor volumes were recorded every two days over a two-week period.

[0168] Analyte Sensing Assay: The response of NOxPorfin-1 (5 pM) toward a panel of biologically relevant analytes was monitored as follows. Unless otherwise mentioned, NOxPorfin-1 was preincubated at 37 °C in 1: 1 v / v DMSO:PBS at 7.4 pH for at least 15 min. The initial fluorescence was measured followed by the addition of 100 pM of the analyte being tested. The solution was sealed and incubated at 37 °C for one hour. Pinal measurements were recorded, and the relative turnon was determined. The following notes pertain to the preparation of various analytes. Formaldehyde was prepared by depolymerizing saturated aqueous solution of paraformaldehyde at 100 °C before use. Perchlorate, nitrite, and nitrate solutions were prepared for their corresponding sodium salts in water. Superoxide anion was added as a solution of potassium superoxide in DMSO. Nitroxyl was generated in situ from a solution of Angeli’s salt in a degassed 10 mM potassium hydroxide solution. Peroxynitrite was prepared according using an established protocol.3NO was generated in situ from a solution of DEA NONOate in degassed PBS buffer. Cu2+was prepared from its chloride salt in water, while Fe(II) was prepared from FeSO4 (H2O)? in 0.1 M HC1. Hydroxy radical was generated in situ using the Fenton reaction between Mohr’s Salt and H2O2. All other analytes were prepared by dilution or dissolution from commercially available sources.

[0169] Cell Culture: A549 lung cancer cells and 4T1 breast cancer cells were acquired from ATCC. A549 cells were cultured in Ham’s F-12K Medium containing 10% FBS and 1% penicillin streptomycin solution, and 4T1 cells were cultured in RPMI-1640 Medium containing 10% FBS and 1% penicillin streptomycin solution. Cells were incubated at 37 °C with 5% CO2. Experiments were performed in 8-well plates (Nuc Lab-Tek Chambered Coverglass, Thermo Scientific) or 96-well plates (Nuclon Delta Surface Flat Bottom, Thermo Scientific).

[0170] MTT Cytotoxicity Assay: A transparent 96-well plate was seeded with 10,000 cells per well and incubated at 37 °C with 5% CO2 for 24 hours (-70% confluent). The media was removed and replaced with fresh medium (100 pL) containing vehicle control (1% DMSO in PBS) or NOxPorfins (1, 2, 5, 10, or 20 pM). After 16 hours of incubation, the media was removed and replaced with (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, 0.4 mg / mL in PBS). The cells were incubated with the MTT reagent for three hours under the same conditions, and then the media was removed, replaced with DMSO (100 pL), and the absorbance of each well was recorded at 570 nm on SpectraMax M2 plate reader. Percent viability was calculated relative to the vehicle control (n = 5).

[0171] Example 5. Pharmaceutical Dosage Forms.

[0172] The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a composition of a formula described herein, a composition specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Composition X'):

[0173] (i) Tablet 1 mg / tablet

[0174] 'Composition X' 100.0

[0175] Lactose 77.5

[0176] Povidone 15.0

[0177] Croscarmellose sodium 12.0

[0178] Microcrystalline cellulose 92.5

[0179] Magnesium stearate 3,0

[0180] 300.0

[0181] (ii) Tablet 2 mg / tablet

[0182] 'Composition X' 20.0

[0183] Microcrystalline cellulose 410.0

[0184] Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5,0

[0185] 500.0

[0186] (iii) Capsule mg / capsule

[0187] 'Composition X' 10.0

[0188] Colloidal silicon dioxide 1.5 Lactose 465.5

[0189] Pregelatinized starch 120.0 Magnesium stearate 3,0

[0190] 600.0

[0191] (iv) Injection 1 (1 mg / mL) mg / mL

[0192] 'Composition X' (free acid form) 1.0 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5

[0193] 1.0 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL

[0194] (v) Injection 2 (10 mg / mL) mg / mL

[0195] 'Composition X' (free acid form) 10.0 Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.0

[0196] 0.1 N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL

[0197] (vi) Aerosol mg / can

[0198] 'Composition X' 20

[0199] Oleic acid 10

[0200] T ri chloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000 Dichlorotetrafluoroethane 5,000

[0201] (vii) Topical Gel 1 wt.%

[0202] 'Composition X' 5% Carbomer 934 1.25%

[0203] Triethanolamine q.s.

[0204] (pH adjustment to 5-7) Methyl paraben 0.2%

[0205] Purified water q.s. to 100g

[0206] (viii) Topical Gel 2 wt.%

[0207] 'Composition X' 5% Methylcellulose 2% Methyl paraben 0.2% Propyl paraben 0.02% Purified water q.s. to 100g (ix) Topical Ointment

[0208] 'Composition X'

[0209] Propylene glycol

[0210] Anhydrous ointment base

[0211] Polysorbate 80

[0212] Methyl paraben

[0213] Purified water

[0214] (x) Topical Cream 1 wt.%

[0215] 'Composition X' 5% White bees wax 10% Liquid paraffin 30% Benzyl alcohol 5% Purified water q.s. to 100g

[0216] (xi ) Topical Cream 2 wt.%

[0217] 'Composition X' 5%

[0218] Stearic acid 10%

[0219] Glyceryl monostearate 3%

[0220] Polyoxyethylene stearyl ether 3%

[0221] Sorbitol 5%

[0222] Isopropyl palmitate 2 %

[0223] Methyl Paraben 0.2%

[0224] Purified water q.s. to 100g

[0225] These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Composition X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.

[0226] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

[0227] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. A porphyrin compound comprising a phenylene group represented by Formula I:wherein,Q is N(Rb)2 or ORC; andRa, Rb, and Rcare each independently H or -(Ci-Ce)alkyl; orQ and NHRaform a fused heterocycle with the carbon atoms of the phenylene moiety to which they are attached.

2. The porphyrin compound of claim 1 wherein Q and NHRaare positioned ortho relative to each other on the phenylene moiety of Formula I.

3. The porphyrin compound of claim 1 wherein the phenylene group is:The porphyrin compound of claim 1 wherein the phenylene group is:The porphyrin compound of claim 1 wherein the phenylene group is:

6. The porphyrin compound of claim 1 wherein the porphyrin compound is represented byFormula II:wherein,R1is the phenylene group represented by Formula I; andR2, R3, and R4are each independently unsubstituted phenyl, unsubstituted or substituted pyridyl, R1, or H.

7. The porphyrin compound of claim 1 wherein the porphyrin compound is represented byFormula III:The porphyrin compound of claim 1 wherein the porphyrin compound is:

9. A composition comprising the porphyrin compound of any one of claims 1 to 8 and a liposomal nanoparticle, wherein the porphyrin compound is encapsulated by the liposomal nanoparticle.

10. The composition of claim 9 wherein the liposomal nanoparticle comprises 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-mPEG).

11. The composition of claim 10 wherein a %w / w of the porphyrin compound relative to DSPE- mPEG is about 0.05% to about 2.00%.

12. The composition of claim 9 wherein the liposomal nanoparticle has an average diameter of about 25 nm to about 250 nm.

13. A method for treating a subject having a cancer, comprising: a) administering a porphyrin composition thereof of claim 9 to a subject having a cancer; b) waiting for a sufficient period of time for the porphyrin compound to be activated by nitric oxide (NO) present in the cancer to form an activated compound within the cancer; c) optionally detecting fluorescence emitted from the activated compound within the cancer; and d) irradiating the activated compound within the cancer, wherein the activated compound is elevated to a higher energy state and then transfers its energy to ground state molecular oxygen present within the cancer to form singlet oxygen; wherein the singlet oxygen ablates the cancer by releasing its energy, and the subject is thereby treated.

14. The method of claim 13 wherein the subject is administered about 0.1 mg / kg to about 10 mg / kg of the porphyrin compound.

15. The method of claim 13 wherein the activated compound is irradiated with light having a wavelength of about 500 nm to about 600 nm.

16. The method of claim 13 wherein the activated compound is irradiated for about 1 minute to about 15 minutes.

17. The method of claim 13 wherein the porphyrin compound administered is NOxPorfin-1, or NOxPorfin-1 encapsulated in a liposomal nanoparticle.

18. The method of claim 13 wherein the activated compound is tNOxPorfm-1.

19. The method of claim 13 further comprising: e) pausing irradiation and allowing the cancer to reoxygenate; and f) repeating steps c) and d) for a total of two or three cycles.

20. The method of claim 13 wherein steps a) to d) or steps a) to f) are repeated daily, every other day, or every six days for a total of two to five cycles.

21. The method of claim 13 wherein the cancer is a nascent cancer or a metastatic lesion.

22. The method of claim 13 wherein the cancer is breast cancer or non-small cellular lung cancer.

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