Patent Application
20250170098
This disclosure relates to the fields of biology, biochemistry, chemistry, pharmacology and medicine. In particular, new methods, compounds, and methods of treatment related to vibronic-driven action are disclosed.
While UV and visible light have only hundreds of microns to 1 mm of light penetration through human tissue (skin, muscle, fat), the near-infrared (NIR) window of 650 nm to 900 nm, also known as the optical therapeutic window, is ideally suited for in vivo applications because of minimal light absorption by hemoglobin and water with significant penetration through human tissue reaching ˜10 cm (Weissleder, 2001). Two-photon NIR activation of Feringa-type motors has been previously exploited for inducing rapid cellular necrosis, but the technique requires enormous laser-generated fluxes of photons and hence the depth of penetration is shallow, ˜0.5 mm, and the area of coverage is restricted to smaller-sized domains (Liu et al., 2019). Single photo NIR light is insufficient in energy to activate Feringa-type motors, which require excitation in the UV (˜360 nm) or blue visible light region (˜405 nm).
In a typical molecular absorbance of photons, an individual bond or small part of the molecule starts vibrating (
Additionally, US Patent Application No. 2020/0289676 relates to the use of near-infrared dye with conjugates for treating tumors. WO 2020/020905 relates to the use of near-infrared containg N-triazole chromophores that may be used in treatments such as photodynamtic therapy. WO 1997/040829 relates to the use of compounds for neuroendocrine resetting therapy or photodynamic therapy. U.S. Pat. No. 7,229,447 relates to the use of methylene blue in photodynamic disruption of cells. Finally, Yan et al. relates to the use of water-soluble cyanine dye for photodynamic therapy of cancer cells. Furthermore, WO 2022023496 relates to the preparation of isonitrile containing compound including fluorophores. US 2008/0233050 relates to cyanine and indocyanine dye conjugates that may be used to visualize and detect a tumor. WO 2011152046 relate to compositions of indocyanine dye and liposomes. Finally, WO 2005/082423 relates to methods of imaging the lympthatic or circulatory system using near IR dyes.
Therefore, there remains a need to find new and unique ways to achieve rapid cellular death that are distinct from photothermal therapy and ROS-based photodynamic therapies.
As provided herein, the present disclosure relates to methods of disrupting cell membranes using vibronic-driven actions.
In one aspect, the present disclosure provides methods of disrupting a membrane comprising:
In one aspect, the present disclosure provides compounds for use in disrupting a membrane comprising a compound is capable of generating a vibronic-driven action and optionally further comprising a targeting moiety; wherein the vibronic-driven action is sufficient to disrupt the membrane.
In one aspect, the present disclosure provides uses of a compound of disrupting a membrane comprising:
In another aspect, the present disclosure provides methods of disrupting a membrane comprising:
In another aspect, the present disclosure provides compound for use in disrupting a membrane comprising a compound, wherein the compound optionally further comprises a targeting moiety; and wherein the compound generates motion sufficient to disrupt the membrane and the energy source has an intensity of less than 250 mW/cm2.
In another aspect, the present disclosure provides uses of a compound for disrupting a membrane comprising:
In some embodiments, the membrane is the outer membrane of a cell. In other embodiments, the membrane is the inner membrane of a cell. In some embodiments, the inner membrane is the membrane of an organelle such as a mitochondria, a nucleus, an endoplasmic reticulum, or a golgi apparatus. In some embodiments, the membrane is the membrane of prokaryotic cell. In other embodiments, the membrane is the membrane of eukaryotic cell. In further embodiments, the membrane is the membrane of a human cell. In some embodiments, the human cell is a cancer cell. In other embodiments, the human cell is a healthy cell. In some embodiments, the human cell is an adipose cell.
In some embodiments, the membrane is a bacterial membrane, a viral membrane, a fungal membrane, or a protozoal membrane. In some embodiments, the membrane is a bacterial membrane. In other embodiments, the membrane is a viral membrane. In other embodiments, the membrane is a fungal membrane. In still other embodiments, the membrane is a protozoal membrane. In some embodiments, the membrane is a membrane of a parasite.
In some embodiments, the disruption creates a pore in the membrane. In some embodiments, the methods result in necrosis of the cell. In other embodiments, the methods result in death through the disruption of an organelle of the cell. In other embodiments, the methods result in death through the disruption of an nucleus of the cell.
In some embodiments, the compound comprises:
In some embodiments, the compound is an organomettalic compound. In further embodiments, the organometallic compound is not a nanoparticle. In some embodiments, the organometallic compound is an organic ligand bound individually to one or more metal atoms. In some embodiments, the organic ligand is bound to one metal atom. In other embodiments, the organic ligand is bound to two or more metal atoms. In some embodiments, the metal atom is bound to the organic ligand via a covalent bond. In other embodiments, the metal atom is bound to the organic ligand via an ionic bond. In some embodiments, the compound is an organic molecule. In further embodiments, the organic molecule exhibits either a longitudinal molecular plasmon or a transverse molecular plasmon. In still further embodiments, the organic molecule exhibits both a longitudinal molecular plasmon and a transverse molecular plasmon. In some embodiments, the compound is an organic dye.
In some embodiments, the present disclosure provides methods wherein the compound is further defined by the formula:
In some embodiments, the compound is further defined as:
In some embodiments, X1 and X2 are identical. In some embodiments, X1 is N. In some embodiments, X2 is N. In some embodiments, X1 and X2 are N.
In some embodiments, R1 or R2 are symmetric with R3 or R4. In some embodiments, R1 is taken together with R5 to form one, two, three, four, or five rings. In further embodiments, R1 is taken together with R5 to form two, three, or four rings. In still further embodiments, R1 is taken together with R5 to form three rings. In yet further embodiments, R1 is taken together with R5 to form three rings, wherein one ring is aliphatic and two rings are aromatic.
In some embodiments, R2 is alkyl(C≤18) or substituted alkyl(C≤18). In further embodiments, R2 is alkyl(C≤18). In further embodiments, R2 is alkyl(C≤8), such as methyl.
In some embodiments, R3 is taken together with R7 to form one, two, three, four, or five rings. In further embodiments, R3 is taken together with R7 to form two, three, or four rings. In still further embodiments, R3 is taken together with R7 to form three rings. In even further embodiments, R3 is taken together with R7 to form three rings, wherein one ring is aliphatic and two rings are aromatic.
In some embodiments, R4 is alkyl(C≤8) or substituted alkyl(C≤8). In some embodiments, R4 is alkyl(C≤18). In further embodiments, R4 is alkyl(C≤8), such as methyl.
In some embodiments, R6 is hydrogen.
In some embodiments, R5 and R7 are taken together and form one, two, or three rings. In some embodiments, R5 and R7 are taken together and form a single ring, such as a five, six, or seven membered ring.
In some embodiments, n is an integer from 1 to 10. In further embodiments, n is an integer selected from 2, 3, or 4. In some embodiments, n is 3.
In some embodiments, R4 is a cell targeting moiety with a linker. In some embodiments the linker is an alkyl chain, an alkenyl chain, an aryl chain, a peptide chain, a polyethylene glycol chain, or a polypropylene chain. In some embodiments, the linker further comprises one or more joining functional group selected from ether, amide, disulfide, ester, amine, or thioether. In further embodiments, the linker is two alkyl chains with an amide joining functional group.
In some embodiments, the the cell targeting moiety is a functional group that associates with the membrane, a carbohydrate or polysaccharide that binds to one or more markers on the membrane, a lipid that binds to one or more markers on the cell membrane, a small molecule that binds to one or more markers on the cell membrane, an aptamer that binds to one or more markers on the membrane, or a peptide or an antibody that binds to one or more markers on the membrane. In further embodiments, the cell targeting moiety is a functional group that associates with the cell membrane. In some embodiments, the functional group is an amine. In some embodiments, the amine is protonated. In other embodiments, the functional group is a natural product. In other embodiments, the functional group is a non-natural product small molecule.
In some embodiments, methods of the present disclosure are provided wherein the compound is further defined as:
In some embodiments, the compound is further defined as:
-A-NRaRa′Ra″
In some embodiments, the compound is at least one compound shown below:
In other embodiments, methods of the present disclosure are provided wherein the compound is further defined as:
In some embodiments, the compound is further defined as:
In some embodiments, X3 is —S—. In some embodiments, X4 is —N═. In some embodiments, R13 is dialkylamino(C≤8) or substituted dialkylamino(C≤8). In further embodiments, R13 is dialkylamino(c s), such as dimethylamino. In some embodiments, R9 is hydrogen. In some embodiments, R10 is hydrogen. In some embodiments, R11 is hydrogen. In some embodiments, R12 is hydrogen. In some embodiments, R14 is hydrogen. In some embodiments, X5 is +NR′R″. In further embodiments, R′ is alkyl(C≤8) or substituted alkyl(C≤8). In some embodiments, R′ is alkyl(C≤8), such as methyl. In some embodiments, R″ is alkyl(C≤8) or substituted alkyl(C≤8). In further embodiments, R″ is alkyl(C≤8), such as methyl.
In some embodiments, R″ is a cell targeting moiety. In some embodiments, the cell targeting moiety further comprises a linker.
In some embodiments, the compound is further defined as:
In some embodiments, the energy source is gamma rays, X-rays, ultraviolet (UV) light, visible (Vis) light, near-infrared (NIR) light, infrared light (IR), microwaves, radio waves, electric fields, ionizing radiation, magnetic fields, mechanical forces, ultrasound, or combinations thereof. In some embodiments, wherein the energy source is light. In further embodiments, the energy source is light with a wavelength from about 250 nm to about 2,000 nm. In some embodiments, the wavelength is from about 350 nm to about 1,000 nm. In further embodiments, the wavelength is from about 450 nm to about 900 nm. In some embodiments, the intensity of the energy source is less than 200 mW/cm2. In further embodiments, the intensity of the energy is less than 100 mW/cm2. In still further embodiments, the intensity of the energy is less than 25 mW/cm2.
In another aspect, the present disclosure provides methods of treating a disease or disorder in a patient comprising:
In another aspect, the present disclosure provides compounds for use in the preparation of a medicament for treating a disease or disorder in a patient comprising a compound, wherein the compound capable of generating a vibronic-driven action and optionally further comprising a cell targeting moiety; wherein the vibronic-driven action is sufficient to disrupt the cell membrane of at least one cell of said patient.
In another aspect, the present disclosure provides uses of a compound for treating a disease or disorder in a patient comprising:
In some embodiments, the methods further comprise administering the compound with a therapeutic agent. In some embodiments, the methods comprise administering the compound in combination with the therapeutic agent. In some embodiments, the contacting of step (A) comprises administering the compound. In some embodiments, the compound disrupts the cell membrane allowing the therapeutic agent to enter a cell. In some embodiments, the therapeutic agent is sufficient to treat or prevent the disease or disorder. In some embodiments, the compound is further defined as a compound disclosed in the present disclosure. In some embodiments, the patient is a mammal, such as a human.
In another aspect, the present disclosure provides methods of opening a cell membrane comprising:
In another aspect, the present disclosure provides compounds for use in opening a cell membrane comprising the compound capable of generating a vibronic-driven action and optionally further comprising a cell targeting moiety; and wherein the vibronic-driven action is sufficient to open the cell membrane.
In another aspect, the present disclosure provides use of a compound for opening a cell membrane comprising:
In some embodiments, the method comprises treating a disease or disorder. In some embodiments, the method comprises killing one or more cells. In some embodiments, the cell is killed by necrosis. In some embodiments, the cell is a parasitic cell. In further embodiments, the parasitic cell is a bacterial cell, a protozoan cell, a virus, or a fungal cell. In other embodiments, the cell is an abnormal human cell, such as a cancer cell. In some embodiments, the compound is further defined as a compound disclosed in the present disclosure.
In yet another aspect, the present disclosure provides methods of reducing the amount of adipose tissue in a patient comprising contracting the adipose tissue with a compound, wherein the compound capable of generating a vibronic-driven action and optionally further comprising a cell targeting moiety; and exposing the compound to an energy source sufficient to generate a vibronic-driven action, wherein the vibronic-driven action is sufficient to reduce the adipose tissue.
In yet another aspect, the present disclosure provides compounds for use in reducing the amount of adipose tissue in a patient comprising a compound, wherein the compound capable of generating a vibronic-driven action and optionally further comprising a cell targeting moiety; and wherein the vibronic-driven action is sufficient to reduce the adipose tissue.
In yet another aspect, the present disclosure provides uses of a compound for reducing the amount of adipose tissue in a patient comprising contracting the adipose tissue with a compound, wherein the compound capable of generating a vibronic-driven action and optionally further comprising a cell targeting moiety; and exposing the compound to an energy source sufficient to generate a vibronic-driven action, wherein the vibronic-driven action is sufficient to reduce the adipose tissue.
In some embodiments, the adipose tissue is an adipocyte cell. In some embodiments, the adipose tissue is a lipocyte cell. In some embodiments, the adipose tissue is a fat cell. In some embodiments, the method is sufficient to reduce the weight of the patient. In some embodiments, the method is sufficient to reduce the circumference of a part of the body of the patient. In some embodiments, the method further comprises a second exposure to the energy source. In some embodiments, the method further comprises applying the compound a second time, a third time, or more than three times. In further embodiments, the weight of the patient or the circumference of a part of the body of the patient is further reduced.
In still another aspect, the present disclosure provides methods of disrupting a cellular component comprising:
In still another aspect, the present disclosure provides compounds for use in disrupting a cellular component comprising a compound, wherein the compound is capable of generating a vibronic-driven action and optionally further comprising a targeting moiety; and wherein the vibronic-driven action is sufficient to disrupt the cellular component.
In still another aspect, the present disclosure provides use of a compound for disrupting a cellular component comprising:
In some embodiments, the cellular component is a carbohydrate or carbohydrate complex. In other embodiments, the cellular component is a protein or protein complex. In still other embodiments, the cellular component is a nucleic acid or nucleic acid complex. In some embodiments, the cellular component is a combination of a nucleic acid, a protein, a carbohydrate, a nucleic acid complex, a protein complex, or a carbohydrate complex. In some embodiments, the cellular component is a cellular component of a prokaryotic cell. In other embodiments, the cellular component is a cellular component of a eukaryotic cell. In some embodiments, the cellular component is a cellular component of a parasitic cell. In further embodiments, the parasitic cell is a bacterial cell, a protozoan cell, a virus, or a fungal cell. In other embodiments, the cellular component is a cellular component of a human cell. In further embodiments, the human cell is an abnormal human cell, such as a cancer cell.
In still yet another aspect, the present disclosure provides intermediate compounds are further defined by the formula:
wherein:
In some embodiments, X1 is N. In some embodiments, X2 is alkyl(C≤18) or substituted alkyl(C≤18). In other embodiments, X2 is amino, alkylamino(C≤12), or substituted alkylamino(C≤12). In other embodiments, X2 is carboxy. In some embodiments, R1 is taken together with R2 to form one, two, three, four, or five rings. In some embodiments, R1 is taken together with R2 to form two, three, or four rings. In some embodiments, R1 is taken together with R2 to form three rings. In some embodiments, R1 is taken together with R2 to form three rings, wherein one ring is aliphatic and two rings are aromatic. In some embodiments, n is an integer from 1 to 10. In some embodiments, n is an integer selected from 5, 6, or 7. In some embodiments, n is 6.
In some embodiments, the intermediate compounds are further defined as:
In still yet another aspect, the present disclosure provides compounds of the formula:
wherein:
-A-NRaRa′Ra″
In some embodiments, the compounds are further defined as:
wherein:
-A-NRaRa′Ra″
In some embodiments, the compounds are further defined as:
wherein:
-A-NRaRa′Ra″
In some embodiments, the compounds are further defined as:
wherein:
-A-NRaRa′Ra″
In some embodiments, the compounds are further defined as:
wherein:
-A-NRaRa′Ra″
In some embodiments, m is 1. In some embodiments, n is 1. In some embodiments, Ra is alkyl(C≤8). In other embodiments, Ra is hydrogen. In some embodiments, Ra′ is alkyl(C≤8). In other embodiments, Ra′ is hydrogen.
In some embodiments, the compounds are further defined as:
In still yet another aspect, the present disclosure provides methods of disrupting a cell membrane comprising contacting the cell membrane with a compound of formula:
exposing the membrane to an energy source capable of generating vibronic-driven action.
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. For example, a compound synthesized by one method may be used in the preparation of a final compound according to a different method.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Provided herein are methods and compounds that have been demonstrated to disrupt membranes. These methods and compounds may be useful to disrupt human cell membranes, bacterial cell membranes, a virus, fungal cell membranes, protozoal cell membranes, cell membranes of parasites, or adipose (also known as adipocyte, lipocyte and fat) cell membranes. These compounds may be used to treat one or more diseases or disorders for which disruption of a cell membrane may be useful. In some embodiments, these diseases or disorders include cancers, bacterial diseases, viral diseases, fungal diseases, protozoan diseases, or diseases carried by parasites. These methods may use vibronic driven action or a similar vibrational energy to achieve these therapeutic effects. Furthermore, the present methods include using very low intensity energy to complete the destruction of the membrane, biomolecule, or cellular component.
These methods may be used to target adipocytes or fat cells. These methods comprise contacting the fat cells with the compound and exposing the cells to an energy source. The energy source and the compounds may be applied once or two or more times over the course of several weeks to reduce the fat deposits. Such light assisted sculpting methods may be used to reduce the size of fat deposits in a patient. After exposure to the energy source, the resultant fat cells may be slowly absorbed over the course of the days or weeks after the energy exposure.
These methods or compounds may additionally be, in some embodiments, useful in selective regulation of the active site in enzymes, modulation of protein channels, or regulation of the structure or function of supramolecular biological assemblies. The compounds described in this application may be used to disrupt protein or protein complexes, nucleic acids or nucleic acid complexes, or carbohydrates or carbohydrate complexes. In these cases, the methods may be used to disrupt or damage these biomolecules and treat or prevent a disease or disorder. Furthermore, these compounds may represent an improvement over those known in the art as the compounds may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties. These and more details will be discussed in more detail below.
In some embodiments, the present disclosure provides methods of using vibronic-driven action to disrupt a cell membrane. In some embodiments, the methods use vibronic-driven action. In some embodiments, the method of disrupting a membrane may comprise contacting the membrane with a compound, wherein the compound comprises a moiety that generates a vibronic-driven action and optionally a cell targeting moiety and exposing the compound to an energy source sufficient to generate a vibronic-driven action. In some embodiments, the method of disrupting a membrane may comprise contacting the membrane with a compound, wherein the compound comprises a moiety that absorbs energy of less than 250 mW/cm2 and optionally a cell targeting moiety and exposing the compound to an energy source sufficient to destroy the membrane.
Vibronic coupling, also termed “vibronic mode”, refers to an alignment of vibrational and electronic modes, which may also be known as plasmonic modes and phonon modes, respectively. In a molecule, the vibronic mode may also be described as a “molecular plasmon” coupled to a “molecular phonon”. Upon absorbance of energy, vibrational modes of the atoms of a molecule may hybridize with the electronic transitions of the molecule to induce a vibronic mode. In some embodiments, the energy used to induce the vibronic mode is electromagnetic radiation, such as gamma rays, X-rays, ultraviolet (UV) light, visible (Vis) light, near-infrared (NIR) light, infrared light (IR), microwaves, or radio waves. In some embodiments, the energy source used to induce the vibronic mode may be light with a wavelength from about 250 nm to about 2,000 nm. In some embodiments, the wavelength is from about 350 nm to about 1,000 nm or about 450 nm to about 900 nm. In some embodiments, the wavelength of the light may be about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nm, or any range derivable therein. In some embodiments, the wavelength of light is about 730 nm. In some embodiments, other types of stimuli including electric fields, ionizing radiation, magnetic fields, mechanical forces, or ultrasound may also be used to induce vibronic coupling.
The present methods contemplate using different intensities or duration of light. The intensity of the light may be proportional to the effectiveness of the vibronic mode coupling at a particular wavelength. These intensities can range from 10 nW/cm2 to 10 W/cm2, from 100 nW/cm2 to 8 W/cm2, or from about 10 μW/cm2 to about 5 W/cm2. The intensity can be from about 10 nW/cm2, 50 nW/cm2, 100 nW/cm2, 250 nW/cm2, 500 nW/cm2, 750 nW/cm2, 1 W/cm2, 10 μW/cm2, 25 μW/cm2, 50 μW/cm2, 100 μW/cm2, 200 μW/cm2, 300 μW/cm2, 400 μW/cm2, 500 μW/cm2, 600 μW/cm2, 700 μW/cm2, 800 μW/cm2, 900 μW/cm2, 1 mW/cm2, 5 mW/cm2, 10 mW/cm2, 25 mW/cm2, 50 mW/cm2, 75 mW/cm2, 100 mW/cm2, 150 mW/cm2, 200 mW/cm2, 300 mW/cm2, 400 mW/cm2, 500 mW/cm2, 600 mW/cm2, 700 mW/cm2, 800 mW/cm2, 900 mW/cm2, 1 W/cm2, 2 W/cm2, 4 W/cm2, 5 W/cm2, 6 W/cm2, 8 W/cm2, to about 10 W/cm2, or any range derivable therein. The intensity of the light may be less than 250 mW/cm2, 200 mW/cm2, 175 mW/cm2, 150 mW/cm2, 125 mW/cm2, or 100 mW/cm2.
When considering depth of NIR light penetration in a patient, as a general rule, there is a loss of one order of magnitude (10×) of NIR photons per centimeter of light penetration through muscle and skin, and loss of two orders of magnitude (100×) of NIR photons per centimeter of light penetration through fat, such as in breast tissue. Fat contains higher water content, and water absorbs the NIR light. Hence, the starting intensity of the light can vary depending on the requisite penetration depth required for the treatment, and this fact accounts, in part, for the large intensity range. The other account depends on the efficiency of activation within the specific molecule.
The present methods may contemplate the use of an energy source with a specific intensity for a given amount of time. The amount of time may be from about 1 second to about 1 hour, from about 3 seconds to about 30 minutes, from about 5 seconds to about 10 minutes, or from about 10 seconds to about 5 minutes. The amount of time may be from about 1 second, 2 seconds, 3 seconds, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, to about 3 hours, or any range derivable therein. In some embodiments, these times may be the amount of time that the energy source is exposed to the compound in order to achieve necrosis.
In some embodiments, the compound may be an organic molecule. In particular, the organic compound may exhibit either or both of a longitudinal or a transverse molecular plasmon. In certain embodiments, the moiety that generates a vibronic-driven action has a net dipole, has a high degree of symmetry across the longitudinal and/or transverse axis, and has a resonance structure through a pi-bonded system. The net dipole of the moiety, in some embodiments, may be due to a charge, such as a cation, anion, radical cation, or radical anion. In some embodiments, the net dipole is due to a radical. In some embodiments, the moiety that generates a vibronic-driven action may be an organic dye. In particular, the moiety that generates a vibronic-driven action may be a cyanine dye. In other embodiments, the moiety that generates a vibronic-driven action may be a thiazine dye such as methylene blue, a boron containing dye such as 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), a xanthene dye such as fluorescein or rose bengal, a triarylmethylene such as phenol red, or a dye such as nile red. In some embodiments, the dye is Cy7.5 or derivatives thereof such as a Cy7.5-amine having the structure:
In other embodiments, the present disclosure relates to methods that may use a compound of the formula:
wherein:
-A-NRaRa′Ra″
In other embodiments, the methods comprise using a dye that is not Cy7.5-amine. In some embodiments, the methods comprise using methylene blue or a derivative thereof, such as a methylene blue having a formula:
In other aspects, the methods comprising a dye that is not methylene blue. In some aspects, the methods are applicable for at least one compound of Table 2. In some aspects, the methods are applicable for at least one compound denoted as BL-204, GL-308-2, BL-141-2, BL-142 of Table 2.
In other embodiments, the compounds may be an organometallic compound such as an organic ligand bound to one or more metal atoms. The ligands may be bound to one or more metal atoms of the same metal or a different metal. In some aspects, the organometallic compound does not comprise a nanoparticle. In some aspects, the organometallic compound comprises one metal atom. In other aspects, the methods comprise using an organometallic compound with two or more metal atoms. The organometallic compound may comprise two, three, four, or five metal atoms. In particular each of these metal atoms are individually bound to the organic ligand rather than another metal atom. In some embodiments, the metal atoms are not bound together to form some form of metal-metal bond. In some embodiments, the metal atom forms an ionic bond with the organic ligand. In other embodiments, the metal atom forms a covalent bond with the organic ligand.
The compounds may be used in an amount from about 100 nM to about 10 mM, from about 250 nM to about 5 mM, or from about 500 nM to about 2 mM. The amount of the compound used may be from about 50 nM, 100 nM, 200 nM, 250 nM, 500 nM, 750 nM, 1 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 750 μM, 800 μM, 900 μM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM, to about 10 mM, or any range derivable therein.
The compounds of the present disclosure are shown, for example, above, in the summary of the invention section, the Examples section, and in the claims below. They may be made using the synthetic methods outlined in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, (2013), which is incorporated by reference herein. In addition, the synthetic methods may be further modified and optimized for preparative, pilot- or large-scale production, either batch or continuous, using the principles and techniques of process chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in Anderson, Practical Process Research & Development—A Guide for Organic Chemists (2012), which is incorporated by reference herein.
All the cell membrane disrupting compounds of the present disclosure may in some embodiments be used for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. In some embodiments, one or more of the compounds characterized or exemplified herein as an intermediate, a metabolite, and/or prodrug, may nevertheless also be useful for the prevention and treatment of one or more diseases or disorders. As such unless explicitly stated to the contrary, all the cell membrane disrupting compounds of the present disclosure are deemed “active compounds” and “therapeutic compounds” that are contemplated for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting the public health by assuring the safety, effectiveness, quality, and security of human and veterinary drugs, vaccines and other biological products, and medical devices.
In some embodiments, the cell membrane disrupting compounds of the present disclosure have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more metabolically stable than, more lipophilic than, more hydrophilic than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the art, whether for use in the indications stated herein or otherwise.
The cell membrane disrupting compounds of the present disclosure may contain one or more asymmetrically-substituted carbon or nitrogen atom and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the cell membrane disrupting compounds of the present disclosure can have the S or the R configuration. In some embodiments, the present compounds may contain two or more atoms which have a defined stereochemical orientation.
Chemical formulas used to represent the cell membrane disrupting compounds of the present disclosure will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given compound, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.
In addition, atoms making up the cell membrane disrupting compounds of the present disclosure are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13C and 14C.
In some embodiments, the cell membrane disrupting compounds of the present disclosure function as prodrugs or can be derivatized to function as prodrugs. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the disclosure contemplates prodrugs of the cell membrane disrupting compounds of the present disclosure as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the disclosure may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a patient, cleaves to form a hydroxy, amino, or carboxylic acid, respectively.
In some embodiments, the cell membrane disrupting compounds of the present disclosure exist in salt or non-salt form. With regard to the salt form(s), in some embodiments the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.
It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates.” Where the solvent is water, the complex is known as a “hydrate.” It will also be appreciated that many organic compounds can exist in more than one solid form, including crystalline and amorphous forms. It is contemplated that the present methods include all different polymorphos of the compounds used herein. All solid forms of the cell membrane disrupting compounds provided herein, including any solvates thereof are within the scope of the present invention.
In another aspect, for administration to a patient in need of such treatment, pharmaceutical formulations (also referred to as a pharmaceutical preparations, pharmaceutical compositions, pharmaceutical products, medicinal products, medicines, medications, or medicaments) comprise a therapeutically effective amount of a cell membrane disrupting compound disclosed herein formulated with one or more excipients and/or drug carriers appropriate to the indicated route of administration. In some embodiments, the cell membrane disrupting compounds disclosed herein are formulated in a manner amenable for the treatment of human and/or veterinary patients. In some embodiments, formulation comprises admixing or combining one or more of the cell membrane-disrupting compounds disclosed herein with one or more of the following excipients: lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol. In some embodiments, e.g., for oral administration, the pharmaceutical formulation may be tableted or encapsulated. In some embodiments, the cell membrane disrupting compounds may be dissolved or slurried in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. In some embodiments, the pharmaceutical formulations may be subjected to pharmaceutical operations, such as sterilization, and/or may contain drug carriers and/or excipients such as preservatives, stabilizers, wetting agents, emulsifiers, encapsulating agents such as lipids, dendrimers, polymers, proteins such as albumin, nucleic acids, and buffers.
Pharmaceutical formulations may be administered by a variety of methods, e.g., orally or by injection (e.g. subcutaneous, intravenous, and intraperitoneal). Depending on the route of administration, the cell membrane disrupting compounds disclosed herein may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. To administer the active compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. In some embodiments, the active compound may be administered to a patient in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.
The cell membrane disrupting compounds disclosed herein may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
The cell membrane disrupting compounds disclosed herein can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compounds and other ingredients may also be enclosed in a hard or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the cell membrane disrupting compounds disclosed herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such pharmaceutical formulations is such that a suitable dosage will be obtained.
The therapeutic compound may also be administered topically to the skin, eye, ear, or mucosal membranes. Administration of the therapeutic compound topically may include formulations of the compounds as a topical solution, lotion, cream, ointment, gel, foam, transdermal patch, or tincture. When the therapeutic compound is formulated for topical administration, the compound may be combined with one or more agents that increase the permeability of the compound through the tissue to which it is administered. In other embodiments, it is contemplated that the topical administration is administered to the eye. Such administration may be applied to the surface of the cornea, conjunctiva, or sclera. Without wishing to be bound by any theory, it is believed that administration to the surface of the eye allows the therapeutic compound to reach the posterior portion of the eye. Ophthalmic topical administration can be formulated as a solution, suspension, ointment, gel, or emulsion. Finally, topical administration may also include administration to the mucosa membranes such as the inside of the mouth. Such administration can be directly to a particular location within the mucosal membrane such as a tooth, a sore, or an ulcer. Alternatively, if local delivery to the lungs is desired the therapeutic compound may be administered by inhalation in a dry-powder or aerosol formulation.
In some embodiments, it may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a patient. In some embodiments, active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal.
Single or multiple doses of the agents are contemplated. Desired time intervals for delivery of multiple doses can be determined by one of ordinary skill in the art employing no more than routine experimentation. As an example, patients may be administered two doses daily at approximately 12-hour intervals. In some embodiments, the agent is administered once a day.
The agent(s) may be administered on a routine schedule. As used herein a routine schedule refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical, or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between. Alternatively, the predetermined routine schedule may involve administration on a twice daily basis for the first week, followed by a daily basis for several months, etc. In other embodiments, the invention provides that the agent(s) may be taken orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the agent can be taken every morning and/or every evening, regardless of when the patient has eaten or will eat.
While hyperproliferative diseases can be associated with any disease which causes a cell to begin to reproduce uncontrollably, the prototypical example is cancer. One of the key elements of cancer is that the cell's normal apoptotic cycle is interrupted and thus agents that interrupt the growth of the cells are important as therapeutic agents for treating these diseases. In some embodiments, the cell membrane that may be disrupted is a human cell, such as a cancer cell. In some embodiments, the compounds of the disclosure may disrupt a human cell, such as an adipose cell. The methods described in the present disclosure contemplate the disruption of either or both a healthy cell or a cancerous cell. In this disclosure, the cell membrane disrupting compounds described herein may be used to lead to decreased cell counts and as such can potentially be used to treat a variety of types of cancer lines. In some embodiments, the cell membrane disrupting compounds described herein are contemplated to open the cell membrane. In further embodiments, the cell membrane disrupting compounds described herein thus allow at least a second therapeutic agent to enter the cell. In some aspects, it is anticipated that the cell membrane disrupting compounds described herein may be used to treat virtually any malignancy.
Cancer cells that may be treated with the compounds of the present disclosure include but are not limited to cells from the skin, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In certain aspects, the tumor may comprise an osteosarcoma, angiosarcoma, rhabdosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, neuroblastoma, or leukemia.
In some embodiments, the cell targeting moiety may target a bacterial cell, a protozoan cell, aa fungal cell, or another type of parasitic cell. In some aspects, the cell targeting moiety may target a virus. In this disclosure, the cell membrane disrupting compounds described herein may be used to lead to decreased cell counts and as such can potentially be used to treat a variety of diseases or conditions associated with or caused by bacteria, protozoa, viruses, a fungi, or other types of parasitic cells. In some embodiments, the cell membrane disrupting compounds described herein are contemplated to open the cell membrane to allow at least a second therapeutic agent to enter a bacterial cell, a protozoan cell, a virus, a fungal cell, or another type of parasitic cell. In some aspects, it is anticipated that the cell membrane disrupting compounds described herein may be used to treat virtually any malignancy associated with or caused by bacteria, protozoa, viruses, a fungi, or other types of parasitic cells.
i. Bacterial Pathogens
There are hundreds of bacterial pathogens in both the Gram-positive and Gram-negative families that cause significant illness and mortality around the word, despite decades of effort developing antibiotic agents. Indeed, antibiotic resistance is a growing problem in bacterial disease.
One of the bacterial diseases with highest disease burden is tuberculosis, caused by the bacterium Mycobacterium tuberculosis, which kills about 2 million people a year, mostly in sub-Saharan Africa. Some non-limiting examples of Mycobacterium tuberculosis antigens include recombinant Ag85A, Ag85B, ESAT6, TB10.4, or fragments thereof including those taught by Ottenhoff and Kaufmann, 2012, which is incorporated herein by reference. Pathogenic bacteria contribute to other globally important diseases, such as pneumonia, which can be caused by bacteria such as Streptococcus and Pseudomonas, and foodborne illnesses, which can be caused by bacteria such as Shigella, Campylobacter, and Salmonella. Pathogenic bacteria also cause infections such as tetanus, typhoid fever, diphtheria, syphilis, and leprosy.
Conditionally pathogenic bacteria are only pathogenic under certain conditions, such as a wound facilitates entry of bacteria into the blood, or a decrease in immune function. For example, Staphylococcus or Streptococcus are also part of the normal human flora and usually exist on the skin or in the nose without causing disease, but can potentially cause skin infections, pneumonia, meningitis, and even overwhelming sepsis, a systemic inflammatory response producing shock, massive vasodilation and death. Some species of bacteria, such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Mycobacterium avium, are opportunistic pathogens and cause disease mainly in people suffering from immunosuppression or cystic fibrosis.
Other bacterial invariably cause disease in humans, such as obligate intracellular parasites (e.g., Chlamydophila, Ehrlichia, Rickettsia) that are capable of growing and reproducing only within the cells of other organisms. Still, infections with intracellular bacteria may be asymptomatic, such as during the incubation period. An example of intracellular bacteria is Rickettsia. One species of Rickettsia causes typhus, while another causes Rocky Mountain spotted fever. Chlamydia, another phylum of obligate intracellular parasites, contains species that can cause pneumonia or urinary tract infection and may be involved in coronary heart disease. Mycobacterium, Brucella, Francisella, Legionella, and Listeria can exist intracellularly, though they are facultative (not obligate) intracellular parasites. Cell membranes for these bacteria may be disrupted using the methods described herein.
ii. Viral Pathogens
Viral pathogens are important health concerns. These pathogens include respiratory viruses such as Adenoviruses, Avian influenza, Influenza virus type A, Influenza virus type B, Measles, Parainfluenza virus, Respiratory syncytial virus (RSV), Rhinoviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, gastro-enteric viruses such as Coxsackie viruses, enteroviruses such as Poliovirus and Rotavirus, hepatitis viruses such as Hepatitis B virus, Hepatitis C virus, Bovine viral diarrhea virus (surrogate), herpesviruses such as Herpes simplex 1, Herpes simplex 2, Human cytomegalovirus, and Varicella zoster virus, retroviruses such as Human immunodeficiency virus 1 (HIV-1), and Human immunodeficiency virus 2 (HIV-2), as well as Dengue virus, Hantavirus, Hemorrhagic fever viruses, Lymphocytic choromeningitis virus, Smallpox virus, Ebola virus, Rabies virus, West Nile virus and Yellow fever virus. Some non-limiting viral antigens include hepatitis B virus HBV surface and core antigens, influenza virus haemagglutinin and neuroaminidase antigens, West Nile virus envelop protein (E) and premembrane protein (prM), Dengue virus 80E subunit protein, Ebola virus glycoprotein, HIV envelope protein gp41 and gp120, or fragments thereof. Other HIV antigens can be found in de Taeye, et al., 2016, which is incorporated herein by reference. The cell membranes for any of these viral pathogens may be disrupted using the methods described herein.
iii. Fungal Pathogens
Pathogenic fungi are fungi that cause disease in humans or other organisms. The following are but a few examples.
Candida species are important human pathogens that are best known for causing opportunist infections in immunocompromised hosts (e.g., transplant patients, AIDS sufferers, and cancer patients). Infections are difficult to treat and can be very serious. Aspergillus can and does cause disease in three major ways: through the production of mycotoxins; through induction of allergenic responses; and through localized or systemic infections. With the latter two categories, the immune status of the host is pivotal. The most common pathogenic species are Aspergillus fumigatus and Aspergillus flavus. Cryptococcus neoformans can cause a severe form of meningitis and meningo-encephalitis in patients with HIV infection and AIDS. The majority of Cryptococcus species lives in the soil and do not cause disease in humans. Cryptococcus laurentii and Cryptococcus albidus have been known to occasionally cause moderate-to-severe disease in human patients with compromised immunity. Cryptococcus gattii is endemic to tropical parts of the continent of Africa and Australia and can cause disease in non-immunocompromised people. Histoplasma capsulatum can cause histoplasmosis in humans, dogs and cats. Pneumocystis jirovecii (or Pneumocystis carinii) can cause a form of pneumonia in people with weakened immune systems, such as premature children, the elderly, transplant patients and AIDS patients. Stachybotrys chartarum or “black mold” can cause respiratory damage and severe headaches. It frequently occurs in houses in regions that are chronically damp. Cell membranes from these fungi may be disrupted using the methods described herein. Furthermore, the cell membrane disrupting compounds of the present disclosure may be used to treat onychomycosis.
iv. Parasites
Parasite presents a major health issue, particularly in under-developed countries around the world. Significant pathogenic parasites include Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Ascaris lumbricoides, Trichinella spiralis, Toxoplasma gondii, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, and Pneumocystis jiroveci. Cell membranes from these parasites may be disrupted using the methods described herein.
In some aspects, the present disclosure provides compounds conjugated directly or through linkers to a cell targeting moiety. In some embodiments, the conjugation of the compound to a cell targeting moiety increases the efficacy of the compound in treating a disease or disorder. Cell targeting moieties according to the embodiments may be, for example, an antibody, a lipid, a carbohydrate, a polysaccharide, a growth factor, a hormone, a peptide, an aptamer, a small molecule such as a hormone, an imaging agent, acofactor, an amino acid, a natural product, a small organic molecule other than a natural product, or a cytokine. In some embodiments, the cell targeting moiety is a functional group that associates with the cell membrane, a carbohydrate or polysaccharide that binds to one or more markers on the cell membrane, a lipid that binds to one or more markers on the cell membrane, a small molecule that binds to one or more markers on the cell membrane, an aptamer that binds to one or more markers on the cell membrane, or a peptide or an antibody that binds to one or more markers on the cell membrane. In some embodiments, the cell targeting moiety may target a human cell, such as a cancer cell. For instance, a cell targeting moiety according to the embodiments may bind to a liver cancer cell such as a Hep3B cell. It has been demonstrated that the gp240 antigen is expressed in a variety of melanomas but not in normal tissues. Thus, in some embodiments, the compounds of the present disclosure may be used in conjugates with an antibody for a specific antigen that is expressed by a cancer cell but not in normal tissues.
In certain embodiments, the cell targeting group is a functional group such as a positively charged group like an amine. The positively charged group may be used to associate with the negatively charged groups at the surface of the cell membrane. It is contemplated that this group might be used to associate with other negatively charged groups such as negatively charged proteins or nucleic acids.
In certain additional embodiments, it is envisioned that cancer cell targeting moieties bind to multiple types of cancer cells. For example, the 8H9 monoclonal antibody and the single chain antibodies derived therefrom bind to a glycoprotein that is expressed on breast cancers, sarcomas and neuroblastomas (Onda, et al., 2004). Another example is the cell targeting agents described in U.S. Patent Publication No. 2004/005647 and in Winthrop, et al. (2003) that bind to MUC-1, an antigen that is expressed on a variety cancer types. Thus, it will be understood that in certain embodiments, cell targeting constructs according the embodiments may be targeted against a plurality of cancer or tumor types.
Additionally, certain cell surface molecules are highly expressed in tumor cells, including hormone receptors such as human chorionic gonadotropin receptor and gonadotropin releasing hormone receptor (Nechushtan et al., 1997). Therefore, the corresponding hormones may be used as the cell-specific targeting moieties in cancer therapy. Additionally, the cell targeting moiety that may be used include a cofactor, a sugar, a drug molecule, an imaging agent, or a fluorescent dye. Many cancerous cells are known to over express folate receptors and thus folic acid or other folate derivatives may be used as conjugates to trigger cell-specific interaction between the conjugates of the present disclosure and a cell (Campbell, et al., 1991; Weitman, et al., 1992).
Since a large number of cell surface receptors have been identified in hematopoietic cells of various lineages, ligands or antibodies specific for these receptors may be used as cell-specific targeting moieties. IL-2 may also be used as a cell-specific targeting moiety in a chimeric protein to target IL-2R+ cells. Alternatively, other molecules such as B7-1, B7-2 and CD40 may be used to specifically target activated T cells (The Leucocyte Antigen Facts Book, 1993, Barclay, et al. (eds.), Academic Press). Furthermore, B cells express CD19, CD40 and IL-4 receptor and may be targeted by moieties that bind these receptors, such as CD40 ligand, IL-4, IL-5, IL-6 and CD28. The elimination of immune cells such as T cells and B cells is particularly useful in the treatment of lymphoid tumors.
Other cytokines that may be used to target specific cell subsets include the interleukins (IL-1 through IL-15), granulocyte-colony stimulating factor, macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor, leukemia inhibitory factor, tumor necrosis factor, transforming growth factor, epidermal growth factor, insulin-like growth factors, and/or fibroblast growth factor (Thompson (ed.), 1994, The Cytokine Handbook, Academic Press, San Diego). In some aspects, the targeting polypeptide is a cytokine that binds to the Fn14 receptor, such as TWEAK (see, e.g., Winkles, 2008; Zhou, et al., 2011 and Burkly, et al., 2007, incorporated herein by reference).
A skilled artisan recognizes that there are a variety of known cytokines, including hematopoietins (four-helix bundles) [such as EPO (erythropoietin), IL-2 (T-cell growth factor), IL-3 (multicolony CSF), IL-4 (BCGF-1, BSF-1), IL-5 (BCGF-2), IL-6 IL-4 (IFN-b2, BSF-2, BCDF), IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T-cell growth factor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM (OM, oncostatin M), and LIF (leukemia inhibitory factor)]; interferons [such as IFN-g, IFN-a, and IFN-b); immunoglobin superfamily (such as B7.1 (CD80), and B7.2 (B70, CD86)]; TNF family [such as TNF-α (cachectin), TNF-b (lymphotoxin, LT, LT-a), LT-b, CD40 ligand (CD40L), Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), and 4-1BBL)]; and those unassigned to a particular family [such as TGF-b, IL 1a, IL-1b, IL-1 RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NK cell stimulatory factor), MIF, IL-16, IL-17 (mCTLA-8), and/or IL-18 (IGIF, interferon-g inducing factor)]. Furthermore, the Fc portion of the heavy chain of an antibody may be used to target Fc receptor-expressing cells such as the use of the Fc portion of an IgE antibody to target mast cells and basophils.
Furthermore, in some aspects, the cell-targeting moiety may be a peptide sequence or a cyclic peptide. Examples, cell- and tissue-targeting peptides that may be used according to the embodiments are provided, for instance, in U.S. Pat. Nos. 6,232,287; 6,528,481; 7,452,964; 7,671,010; 7,781,565; 8,507,445; and 8,450,278, each of which is incorporated herein by reference.
Thus, in some embodiments, cell targeting moieties are antibodies or avimers. Antibodies and avimers can be generated against virtually any cell surface marker thus, providing a method for targeted to delivery of GrB to virtually any cell population of interest. Methods for generating antibodies that may be used as cell targeting moieties are detailed below. Methods for generating avimers that bind to a given cell surface marker are detailed in U.S. Patent Publications Nos. 2006/0234299 and 2006/0223114, each incorporated herein by reference.
Additionally, it is contemplated that the compounds described herein may be conjugated to a nanoparticle or other nanomaterial. Some non-limiting examples of nanoparticles include metal nanoparticles such as gold or silver nanoparticles or polymeric nanoparticles such as poly-L-lactic acid or poly(ethylene) glycol polymers. Nanoparticles and nanomaterials which may be conjugated to the instant compounds include those described in U.S. Patent Publications Nos. 2006/0034925, 2006/0115537, 2007/0148095, 2012/0141550, 2013/0138032, and 2014/0024610 and PCT Publication No. 2008/121949, 2011/053435, and 2014/087413, each incorporated herein by reference.
In particular, the compositions that may be used in treating a disease or disorder in a subject (e.g., a human subject) are disclosed herein. The compositions described above are preferably administered to a mammal (e.g., rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., slowing, stopping, reducing or eliminating one or more symptoms or underlying causes of disease). Toxicity and therapeutic efficacy of the compositions utilized in methods of the disclosure can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, body weight, age, the particular composition to be administered, time and route of administration, general health, the clinical symptoms and other drugs being administered concurrently. In some embodiments, amount of the cell membrane disrupting compounds used is calculated to be from about 0.01 mg to about 10,000 mg/day. In some embodiments, the amount is from about 1 mg to about 1,000 mg/day. In some embodiments, these dosings may be reduced or increased based upon the biological factors of a particular patient such as increased or decreased metabolic breakdown of the drug or decreased uptake by the digestive tract if administered orally. Additionally, the cell membrane disrupting compounds may be more efficacious and thus a smaller dose is required to achieve a similar effect. Such a dose is typically administered once a day for a few weeks or until sufficient achieve clinical benefit.
Also provided herein are uses of a composition in the treatment of a disease or disorder. These compositions may also be used in the preparation of a medicament for the treatment of a disease or disorder. Finally, the present disclosure also contemplates the use of a compound as described herein for the preparation of a medicament.
The therapeutic methods of the disclosure (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like).
It is envisioned that the cell membrane disrupting compounds described herein may be used in combination therapies with one or more additional therapies or a compound which mitigates one or more of the side effects experienced by the patient. It is common in the field of medicine to combine therapeutic modalities. The following is a general discussion of therapies that may be used in conjunction with the therapies of the present disclosure.
To treat diseases or disorders using the methods and compositions of the present disclosure, one would generally contact a cell or a subject with a cell membrane disrupting compound and at least one other therapy. These therapies would be provided in a combined amount effective to achieve a reduction in one or more disease parameter. This process may involve contacting the cells/subjects with both agents/therapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the cell/subject with two distinct compositions or formulations, at the same time, wherein one composition includes the compound and the other includes the other agent.
Alternatively, the compounds described herein may precede or follow the other treatment by intervals ranging from minutes to weeks. One would generally ensure that a significant period of time did not expire between the times of each delivery, such that the therapies would still be able to exert an advantageously combined effect on the cell/subject. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 1-2 hours. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
It also is conceivable that more than one administration of either the compound or the other therapy will be desired. Various combinations may be employed, where a compound of the present disclosure is “A,” and the other therapy is “B,” as exemplified below:
In some aspects, cell membrane disrupting compounds of this disclosure can be synthesized using the methods of organic chemistry as described in this application. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated by reference herein.
The synthetic methods described herein can be further modified and optimized for preparative, pilot- or large-scale production, either batch of continuous, using the principles and techniques of process chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in Practical Process Research & Development (2000), which is incorporated by reference herein. The synthetic method described herein may be used to produce preparative scale amounts of the compounds described herein.
When used in the context of a chemical group: “hydrogen” means —H; “hydroxy” means —OH; “oxo” means ═O; “carbonyl” means —C(═O)—; “carboxy” means —C(═O)OH (also written as —COOH or —CO2H); “halo” means independently —F, —Cl, —Br or —I; “amino” means —NH2; “hydroxyamino” means —NHOH; “nitro” means —NO2; imino means ═NH; “cyano” means —CN; “isocyanyl” means —N═C═O; “azido” means —N3; in a monovalent context “phosphate” means —OP(O)(OH)2 or a deprotonated form thereof; in a divalent context “phosphate” means —OP(O)(OH)O— or a deprotonated form thereof; “mercapto” means —SH; and “thio” means ═S; “thiocarbonyl” means —C(═S)—; “sulfonyl” means —S(O)2—; and “sulfinyl” means —S(O)—.
In the context of chemical formulas, the symbol “—” means a single bond, “═” means a double bond, and “≡” means triple bond. The symbol “----” represents an optional bond, which if present is either single or double. The symbol “” represents a single bond or a double bond. Thus, the formula
covers, for example,
And it is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol “—”, when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures thereof. The symbol “”, when drawn perpendicularly across a bond (e.g.,
for methyl) indicates a point of attachment of the group. It is noted that the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in unambiguously identifying a point of attachment. The symbol “” means a single bond where the group attached to the thick end of the wedge is “out of the page.” The symbol “
” means a single bond where the group attached to the thick end of the wedge is “into the page”. The symbol means a single bond where the geometry around a double bond (e.g., either E or Z) is “
” means a single bond where the geometry around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.
When a variable is depicted as a “floating group” on a ring system, for example, the group “R” in the formula:
then the variable may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When a variable is depicted as a “floating group” on a fused ring system, as for example the group “R” in the formula:
then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals —CH—), so long as a stable structure is formed. In the example depicted, R may reside on either the 5-membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter “y” immediately following the R enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.
For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” or “C=n” defines the exact number (n) of carbon atoms in the group/class. “C n” defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group/class in question. For example, it is understood that the minimum number of carbon atoms in the groups “alkyl(C≤8)”, “alkanediyl(C≤8)”, “heteroaryl(C≤8)”, and “acyl(C≤8)” is one, the minimum number of carbon atoms in the groups “alkenyl(C≤8)”, “alkynyl(C≤8)”, and “heterocycloalkyl(C≤8)” is two, the minimum number of carbon atoms in the group “cycloalkyl(C≤8)” is three, and the minimum number of carbon atoms in the groups “aryl(C≤8)” and “arenediyl(C≤8)” is six. “Cn-n′” defines both the minimum (n) and maximum number (n′) of carbon atoms in the group. Thus, “alkyl(C2-10)” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “C1-4-alkyl”, “C1-4-alkyl”, “alkyl(C1-4)”, and “alkyl(C≤4)” are all synonymous. Except as noted below, every carbon atom is counted to determine whether the group or compound falls with the specified number of carbon atoms. For example, the group dihexylamino is an example of a dialkylamino(C12) group; however, it is not an example of a dialkylamino(C6) group. Likewise, phenylethyl is an example of an aralkyl(C≤8) group. When any of the chemical groups or compound classes defined herein is modified by the term “substituted”, any carbon atom in the moiety replacing the hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted alkyl(C1-6). Unless specified otherwise, any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve.
The term “saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.
The term “aliphatic” signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl).
The term “aromatic” signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4n+2 electrons in a fully conjugated cyclic R system. An aromatic compound or chemical group may be depicted as a single resonance structure; however, depiction of one resonance structure is taken to also refer to any other resonance structure. For example:
Aromatic compounds may also be depicted using a circle to represent the delocalized nature of the electrons in the fully conjugated cyclic π system, two non-limiting examples of which are shown below:
The term “alkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups —CH3 (Me), —CH2CH3 (Et), —CH2CH2CH3 (n-Pr or propyl), —CH(CH3)2 (i-Pr, iPr or isopropyl), —CH2CH2CH2CH3 (n-Bu), —CH(CH3)CH2CH3 (sec-butyl), —CH2CH(CH3)2 (isobutyl), —C(CH3)3 (tert-butyl, t-butyl, t-Bu or tBu), and —CH2C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups —CH2— (methylene), —CH2CH2—, —CH2C(CH3)2CH2—, and —CH2CH2CH2— are non-limiting examples of alkanediyl groups. The term “alkylidene” refers to the divalent group ═CRR′ in which R and R′ are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH2, =CH(CH2CH3), and ═C(CH3)2. An “alkane” refers to the class of compounds having the formula H—R, wherein R is alkyl as this term is defined above.
The term “cycloalkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: —CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to a carbon atom of the non-aromatic ring structure. The term “cycloalkanediyl” refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group
is a non-limiting example of cycloalkanediyl group. A “cycloalkane” refers to the class of compounds having the formula H—R, wherein R is cycloalkyl as this term is defined above.
The term “alkenyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: —CH═CH2 (vinyl), —CH═CHCH3, —CH═CHCH2CH3, —CH2CH═CH2 (allyl), —CH2CH═CHCH3, and —CH═CHCH═CH2. The term “alkenediyl” refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups —CH═CH—, —CH═C(CH3)CH2—, —CH═CHCH2—, and —CH2CH═CHCH2— are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H—R, wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “α-olefin” are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.
The term “alkynyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups —C≡CH, —C≡CCH3, and —CH2C≡CCH3 are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H—R, wherein R is alkynyl.
The term “aryl” refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, —C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g., 4-phenylphenyl). The term “arenediyl” refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:
An “arene” refers to the class of compounds having the formula H—R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.
The term “aralkyl” refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
The term “heteroaryl” refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term “N-heteroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. A “heteroarene” refers to the class of compounds having the formula H—R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.
The term “heteroaralkyl” refers to the monovalent group -alkanediyl-heteroaryl, in which the terms alkanediyl and heteroaryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: pyridinylmethyl and 2-quinolinyl-ethyl.
The term “heterocycloalkyl” refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings are fused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term “N-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group.
The term “heterocycloalkalkyl” refers to the monovalent group -alkanediyl-heterocycloalkyl, in which the terms alkanediyl and heterocycloalkyl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: morpholinylmethyl and piperidinylethyl.
The term “acyl” refers to the group —C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above. The groups, —CHO, —C(O)CH3 (acetyl, Ac), —C(O)CH2CH3, —C(O)CH(CH3)2, —C(O)CH(CH2)2, —C(O)C6H5, and —C(O)C6H4CH3 are non-limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group —C(O)R has been replaced with a sulfur atom, —C(S)R. The term “aldehyde” corresponds to an alkyl group, as defined above, attached to a —CHO group.
The term “alkoxy” refers to the group —OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: —OCH3 (methoxy), —OCH2CH3 (ethoxy), —OCH2CH2CH3, —OCH(CH3)2 (isopropoxy), or —OC(CH3)3 (tert-butoxy). The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”, “heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as —OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term “alkylthio” and “acylthio” refers to the group —SR, in which R is an alkyl and acyl, respectively. The term “alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term “ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.
The term “alkylamino” refers to the group —NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: —NHCH3 and —NHCH2CH3. The term “dialkylamino” refers to the group —NRR′, in which R and R′ can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: —N(CH3)2 and —N(CH3)(CH2CH3). The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group —NHR, in which R is acyl, as that term is defined above. A non-limiting example of an amido group is —NHC(O)CH3.
When a chemical group is used with the “substituted” modifier, one or more hydrogen atom has been replaced, independently at each instance, by —OH, —F, —Cl, —Br, —I, —NH2, —NO2, —CO2H, —CO2CH3, —CO2CH2CH3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2, —C(O)NH2, —C(O)NHCH3, —C(O)N(CH3)2, —OC(O)CH3, —NHC(O)CH3, —S(O)2OH, or —S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: —CH2OH, —CH2Cl, —CF3, —CH2CN, —CH2C(O)OH, —CH2C(O)OCH3, —CH2C(O)NH2, —CH2C(O)CH3, —CH2OCH3, —CH2OC(O)CH3, —CH2NH2, —CH2N(CH3)2, and —CH2CH2C1. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. —F, —Cl, —Br, or —I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, —CH2C1 is a non-limiting example of a haloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups —CH2F, —CF3, and —CH2CF3 are non-limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl. The groups, —C(O)CH2CF3, —CO2H (carboxyl), —CO2CH3 (methylcarboxyl), —CO2CH2CH3, —C(O)NH2 (carbamoyl), and —CON(CH3)2, are non-limiting examples of substituted acyl groups. The groups —NHC(O)OCH3 and —NHC(O)NHCH3 are non-limiting examples of substituted amido groups.
The use of the word “a” or “an,” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or patients. Unless otherwise noted, the term “about” is used to indicate a value of 10% of the reported value, preferably a value of ±5% of the reported value. It is to be understood that, whenever the term “about” is used, a specific reference to the exact numerical value indicated is also included.”
An “active ingredient” (AI) or active pharmaceutical ingredient (API) (also referred to as an active compound, active substance, active agent, pharmaceutical agent, agent, biologically active molecule, or a therapeutic compound) is the ingredient in a pharmaceutical drug that is biologically active.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to the patient or subject, is sufficient to effect such treatment or prevention of the disease as those terms are defined below.
An “excipient” is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk up the composition (thus often referred to as “bulking agents,” “fillers,” or “diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.
The term “hydrate” when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid forms of the compound.
As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. The term “EC50” refers to an amount that is an effective concentration to results in a half-maximal response.
An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.
As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
“Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
A “pharmaceutically acceptable carrier,” “drug carrier,” or simply “carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and/or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.
A “pharmaceutical drug” (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined above) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).
“Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
“Prodrug” means a compound that is convertible in vivo metabolically into an active pharmaceutical ingredient of the present invention. The prodrug itself may or may not have activity with in its prodrug form. For example, a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound. Non-limiting examples of suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-p-hydroxynaphthoate, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates, and esters of amino acids. Similarly, a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.
A “stereoisomer” or “optical isomer” is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and/or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that that for any stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase “substantially free from other stereoisomers” means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of another stereoisomer(s).
“Treatment” or “treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.
The term “unit dose” refers to a formulation of the compound or composition such that the formulation is prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that may be used include but are not limited to a single tablet, capsule, or other oral formulations, or a single vial with a syringeable liquid or other injectable formulations.
The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.
The following is a list of the embodiments of the present disclosure.
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and exposing the membrane to an energy source capable of generating vibronic-driven action.
The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
The origin of the sub-bands in cyanines have been thoroughly studied, which without being bound by theory concluded that the presence of the shoulder next to the large absorption band is “primarily determined by a dominant vibration associated with its polymethine chain rather than a collection of singly excited vibrations” (Mustroph and Towns, 2018). The vibronic behavior, through the coupling of electronic and vibrational states, is a feature of the conjugated-backbone-near-symmetrical cyanines such as in Cy7-amine and C7.5-amine. In the case of conjugated-backbone-unsymmetrical cyanines, the absorption curves do not exhibit a vibrational fine structure, analogous to most spectra of merocyanines (Mustroph, 2021). Without being bound by theory, the vibronic mode in symmetrical cyanine structures is thought to result from the coupling of a dominant collective oscillation of electronic excitation (molecular plasmon) to a dominant collective vibrational excitation (phonon). The shoulders at ˜730 nm and ˜690 nm in the absorption spectrum of Cy7.5-amine and Cy7-amine, respectively, correspond to this collective vibrational mode.
Here the vibronic mode was selectively excited in a cell-membrane-bound Cy7.5-amine using a NIR light-emitting diode (LED) at 730 nm (
To rule out a photothermal effect, the temperature of the media was measured during the NIR light exposure when the treatment was done at room temperature versus when done on ice bath (
To confirm that a photodynamic ROS generation was not responsible for the necrosis, the permeabilization of A375 melanoma cells was repeated in the presence of ROS scavengers (
To confirm that the permeabilized cells to DAPI were indeed dead, the cells were cultured after treatment with 2 μM Cy7.5-amine, 30 min incubation, and subsequent illumination for 10 min with 730 nm light at 80 mW/cm2 and cell death was quantified by a crystal violet assay. In this test, the viable cells attach and grow in the culture dish while the dead cells detach or are easily detached in the washing step with phosphate buffered saline (PBS). As shown in
Finally, to confirm that the Cy7.5-amine binding to the cells was likely mediated by the charge on its pendant amine moiety, which without being bound by theory is probably due to interaction with the negatively charged phospholipids, acetic acid was added into the medium (0 mM-10 mM) to protonate the phosphates of the phospholipids. The permeabilization of the cells and flow cytometry analysis was conducted as before. The results, as shown by flow cytometry (
Another cyanine was also tried that has a vibronic absorption shoulder at 730 nm, indocyanine green (ICG), bearing sulfonate containing addends, as shown in
Molecular jackhammers (MJH) are chemical structures that support plasmon resonances in small organic molecules upon optical excitation. Here, four major plasmon resonances have been identified and proposed in cyanine-based molecular plasmons (
B. The Molecular Plasmon Index Correlates with the Activity to Open Cell Membranes.
The activity of plasmon-driven MJHs to open cell membranes by VDA upon NIR light-activation was evaluated in human melanoma A375 cells. When the cell membranes were open by VDA, DAPI enters immediately into the cells and stain the nucleus, and flow cytometry analysis was used to quantify the percentage of DAPI positive cells at variable concentrations of MJH. The flow cytometry analysis is conducted immediately after the cells were treated with 730 nm LED light at 80 mW/cm2 for 10 min. Typically, it took ˜30 s to start cell counting and observe that DAPI already had entered into the cell membrane-compromised cells but not in the controls without VDA action. This is indicative of a rapid necrotic cell death and cell membrane permeabilization to DAPI by VDA action. The effective concentration needed to permeabilize cells by 50% (VDA IC50) was estimated for each molecule. The library of MJHs compounds was ordered from most active (VDA IC50=0.12 μM) to least active (VDA IC50>>8 μM) in
The VDA activity, defined as VDA IC50, correlates with the plasmonicity index, defined here by first time as experimental plasmonicity index (EPI) in
The specific localization of MJHs in the cell was studied by confocal microscopy since cyanines are standard photostable and high yield fluorescent probes broadly use for live cell, small animal and even human imaging (
The plasmon resonance in Cy5.5-amine was activated upon laser excitation (λex=640 nm) under the confocal microscope while imaging real-time opening of the outer cellular membrane, DAPI entering into the cell throughout the holes and staining of nuclear DNA, and cytoskeleton was simultaneously disassembling (
The clonogenic assay was conducted to confirm that cancer cells treated by plasmon-driven MJH are indeed death upon NIR-light activation (
The octanol-water partition coefficient (log P value) was calculated on the MJH structures using online interactive log P calculator. This parameter informs of the lipophilicity or affinity of the MJH to the lipid bilayers.(29, 30) The higher the value the more likely the MJH will bind to the lipid bilayers. The protonation state of the MJH strongly modifies the polarity of the molecules and hence influences the log P values. The log P values were calculated considering the charged state of the arm (
The lack of full correlation between log P values and VDA activity does not mean that the affinity and loading into the lipid bilayers is not playing a role. Indeed, in a selected group of molecules (ICG, GL-328-2, GL-286, and GL-291-2) the log P values correlates well with the VDA activity. In this case, the correlation exists because the molecules have the same plasmonic core structure and the same length of the alkyl chain, the major variation is coming from a primary amine, secondary amine, quaternary amine and sulfonate. Therefore, the plasmon properties are expected to be similar among these molecules but the affinity to membranes defines a correlation with the VDA activity.
There is another group of molecules in which a correlation between the log P value and VDA activity could be expected because they have similar plasmonic core structure but different side arms. However, this is not the case. The plasmonicity index playing a stronger role specially on BL-204 and GL-308-2. In these two molecules, the protonated secondary amine, is too close to the core structure permitting the amine to act as an electron withdrawing group that enhances the plasmonicity. And second, comparing molecules such as BL-141-1, BL-141-2 and BL-142, suggest that docking by specific and selective interactions into the lipid bilayers could be playing a role which influences strongly the outcome of the VDA activity.
General information: All glassware was oven-dried overnight prior to use. All reactions were carried out under an N2 atmosphere unless otherwise noted. All other chemicals were purchased from commercial suppliers and used without further purification.
[a]The reactions were carried out with conditions: 1) 1 (0.25 mmol), Pd(PPh3)4 (10 mol %), Base (4 equiv) in solvent at 100° C. for 24 h;
[b]Yields were determined by HPLC on a C18 reverse-phase column;
[c]Isolated yield.
General procedure: To a screw-capped vial was added 1 (0.25 mmol), Pd(PPh3)4 (0.025 mmol, 10 mol %), Base (1 mmol, 4 equiv). The vial was sealed with a PTFE septum and then evacuated and backfilled with N2 for three times, followed by addition of solvent via syringe and vigorous stirring. The sealed reaction was heated to 100° C. in oil bath heating for 24 h, then the reaction was cooled to room temperature and concentrated under reduced pressure, followed by 6M HCl at 0° C. and stirred for 10 mins at room temperature. The precipitate was filtered, washed with H2O, Et2O and acetone, dried in vacuo to provide compound 2 as a dark red solid.
N-((E)-(3-((E)-(phenylimino)methyl)cyclohex-2-en-1-ylidene)methyl)aniline (2): 1H-NMR (600 MHz, MD3OD) δ 8.14 (s, 2H), 7.79 (s, 1H), 7.49-7.39 (m, 8H), 7.28-7.23 (m, 2H), 2.58 (t, J=6.2 Hz, 4H), 1.99-1.93 (m, 2H). 13C NMR (151 MHz, MD3OD) δ 163.58, 153.18, 140.74, 130.98, 127.16, 119.97, 119.18, 22.96, 21.58. HRMS (ESI) for calculated for [M+H, C20H21N2]+: 289.1626, found: 289.1707.
General procedure: To a screwed-capped vial charged with compound 3 (1 equiv) and alkyl halides (1.5 equiv) were heated to reflux in CH3CN until compound 3 consumed all. Subsequently, the mixture was cooled to room temperature, then diethyl ether was added to precipitate the product. That product was collected by filtration and washed with diethyl ether to obtained compound 4.
General Synthetic Procedure: All glassware was oven-dried overnight prior to use. All reactions were carried out under an N2 atmosphere unless otherwise noted. All other chemicals were purchased from commercial suppliers and used without further purification.
General procedure: To a screwed-capped vial charged with compound 3 (1 equiv) and alkyl halides (1.5 equiv) were heated to reflux in CH3CN until compound 3 consumed all. Subsequently, the mixture was cooled to room temperature, then diethyl ether was added to precipitate the product. That product was collected by filtration and washed with diethyl ether to obtained compound 4.
Procedure b: To a screw-capped vial charged with compound 4 (1 equiv), 2 (1 equiv), 4 (1 equiv) and NaOAc (3 equiv) were dissolved in absolute ethanol. The mixture was heated at 80° C. for overnight under N2 atmosphere. The final product was purified by silica column chromatography to obtain compound 5.
1,1,3-trimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-141-1): Prepared according to the general procedure from 4a (176 mg, 0.5 mmol, 1 equiv) and 2 (81 mg, 0.25 mmol, 1 equiv). Yield: 88% yield (149 mg). 1H-NMR (600 MHz, MD3OD) δ 8.24-8.21 (m, 2H), 8.00 (d, J=8.8 Hz, 2H), 7.97 (d, J=8.6 Hz, 2H), 7.87 (d, J=14.2 Hz, 2H), 7.64-7.60 (m, 2H), 7.57 (d, J=8.8 Hz, 2H), 7.53 (s, 1H), 7.48-7.44 (m, 2H), 6.19 (d, J=14.1 Hz, 2H), 3.72 (s, 6H), 2.61 (t, J=6.1 Hz, 4H), 2.03-1.95 (m, 14H). HRMS (ESI) calculated for [M, C40H41N2]+: 549.3264, found: 549.3275.
3-(6-(dimethylamino)hexyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-141-2): Prepared according to the general procedure from 4a (176 mg, 0.5 mmol, 1 equiv), 2 (162 mg, 0.5 mmol, 1 equiv) and 4e (248 mg, 0.5 mmol, 1 equiv). Yield: 14% yield (45 mg). 1H-NMR (600 MHz, MD3OD) δ 8.27-8.21 (m, 2H), 8.05-7.96 (m, 4H), 7.93-7.89 (m, 1H), 7.85 (d, J=14.0 Hz, 1H), 7.67-7.61 (m, 2H), 7.60 (d, J=8.8 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H), 7.52 (s, 1H), 7.50-7.45 (m, 2H), 6.21 (dd, J=23.7, 14.1 Hz, 2H), 4.24 (t, J=7.4 Hz, 2H), 3.75 (s, 3H), 3.04 (t, J=9.0 Hz, 2H), 2.80 (s, 6H), 2.63-2.60 (m, 4H), 2.06-1.90 (m, 14H), 1.75-1.70 (m, 2H), 1.59-1.54 (m, 2H), 1.51-1.47 (m, 2H). HRMS (ESI) calculated for [M, C47H56N3]+: 662.4469, found: 662.4476.
3-(6-(dimethylamino)hexyl)-2-((E)-2-((E)-3-((E)-2-(3-(6-(dimethylamino)hexyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-142): Prepared according to the general procedure from 2 (81 mg, 0.25 mmol, 1 equiv) and 4e (248 mg, 0.5 mmol, 2 equiv). Yield: 20% yield (40 mg). 1H-NMR (600 MHz, MD3OD) δ 8.25-8.22 (m, 2H), 8.03-7.96 (m, 4H), 7.89 (d, J=14.1 Hz, 2H), 7.66-7.62 (m, 2H), 7.60 (d, J=8.8 Hz, 2H), 7.56 (s, 1H), 7.50-7.45 (m, 2H), 6.24 (d, J=14.1 Hz, 2H), 4.27 (t, J=7.4 Hz, 4H), 4.27 (t, J=7.4 Hz, 4H), 2.82 (s, 12H), 2.63 (t, J=6.2 Hz, 4H), 2.05-1.96 (m, 12H), 1.95-1.90 (m, 6H), 1.76-1.72 (m, 4H), 1.60-1.55 (m, 4H), 1.52-1.47 (m, 4H). HRMS (ESI) calculated for [M, C54H71N4]+: 775.5673, found: 775.5662.
3-(2-(dimethylamino)ethyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-204): Prepared according to the general procedure from 4a (70 mg, 0.2 mmol, 1 equiv), 2 (65 mg, 0.2 mmol, 1 equiv) and 4b (107 mg, 0.2 mmol, 1 equiv). Yield: 18% yield (26 mg). 1H-NMR (600 MHz, MD3OD) δ 8.25 (d, J=8.6 Hz, 1H), 8.22 (d, J=8.7 Hz, 1H), 8.05-7.90 (m, 5H), 7.82 (d, J=13.9 Hz, 1H), 7.67-7.59 (m, 3H), 7.56-7.44 (m, 4H), 6.28 (d, J=14.2 Hz, 1H), 6.21 (d, J=14.1 Hz, 1H), 4.30 (t, J=7.4 Hz, 2H), 3.77 (s, 3H), 2.77 (t, J=7.4 Hz, 2H), 2.66-2.59 (m, 4H), 2.43 (s, 6H), 2.06-1.95 (m, 14H). HRMS (ESI) calculated for [M, C43H48N3]+: 606.3843, found: 606.3847.
1,3,3-trimethyl-2-((E)-2-((E)-3-((E)-2-(1,3,3-trimethyl-1,3-dihydro-2H-benzo[g]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-benzo[g]indol-1-ium (BL-205): Prepared according to the general procedure from 4j (70 mg, 0.2 mmol, 2 equiv) and 2 (33 mg, 0.1 mmol, 1 equiv). Yield: 74% yield (50 mg). 1H-NMR (600 MHz, MD3OD) δ 8.59 (d, J=8.7 Hz, 2H), 7.99 (d, J=8.2 Hz, 2H), 7.82 (d, J=8.9 Hz, 4H), 7.67-7.59 (m, 4H), 7.58-7.52 (m, 2H), 7.50 (s, 1H), 6.33 (d, J=14.0 Hz, 2H), 4.18 (s, 6H), 2.63 (t, J=6.2 Hz, 4H), 2.02-1.96 (m, 2H), 1.77 (s, 12H). HRMS (ESI) calculated for [M, C40H41N2]+: 549.3264, found: 549.3247.
3-(5-carboxypentyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-242): Prepared according to the general procedure from 4a (117 mg, 0.33 mmol, 1 equiv), 2 (107 mg, 0.33 mmol, 1 equiv) and 4g (134 mg, 0.33 mmol, 1 equiv). Yield: 18% yield (130 mg). 1H-NMR (600 MHz, MD3OD) δ 8.12 (d, J=8.5 Hz, 2H), 7.91-7.85 (m, 4H), 7.84-7.69 (m, 2H), 7.55-7.51 (m, 2H), 7.50-7.32 (m, 5H), 6.21-5.95 (m, 2H), 4.20-4.06 (m, 2H), 3.63 (s, 3H), 2.68-2.30 (m, 4H), 2.23 (t, J=7.3 Hz, 2H), 1.99-1.84 (m, 14H), 1.83-1.77 (m, 2H), 1.65-1.60 (m, 2H), 1.48-1.41 (m, 2H); 13C-NMR (151 MHz, MD3OD) δ 177.49, 174.88, 173.98, 163.31, 163.08, 162.84, 149.06, 148.67, 141.83, 141.22, 134.55, 133.35, 133.29, 132.30, 131.66, 131.61, 131.10, 129.68, 129.53, 129.46, 128.66, 125.84, 124.39, 123.33, 113.51, 111.95, 111.83, 100.54, 100.28, 54.81, 52.05, 44.83, 34.81, 31.83, 28.22, 27.60, 27.50, 27.41, 25.78, 25.02, 22.78, 22.07. HRMS (ESI) calculated for [M, C45H49N2O2]+: 649.3789, found: 649.3793.
3-(5-carboxypentyl)-2-((E)-2-((E)-3-((E)-2-(3-(5-carboxypentyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-243): Prepared according to the general procedure from 2 (33 mg, 0.1 mmol, 1 equiv) and 4 μg (81 mg, 0.2 mmol, 2 equiv). Yield: 45% yield (39 mg). 1H-NMR (600 MHz, MD3OD) δ 8.23 (d, J=8.5 Hz, 2H), 8.00 (d, J=8.9 Hz, 2H), 7.98 (d, J=8.0 Hz, 2H), 7.86 (d, J=14.0 Hz, 2H), 7.65-7.61 (m, 2H), 7.57 (d, J=8.8 Hz, 2H), 7.50-7.44 (m, 3H), 6.22 (d, J=14.1 Hz, 2H), 4.23 (t, J=7.5 Hz, 4H), 2.61 (t, J=6.2 Hz, 4H), 2.25 (t, J=7.4 Hz, 4H), 2.03-1.96 (m, 12H), 1.93-1.87 (m, 4H), 1.75-1.70 (m, 4H), 1.56-1.50 (m, 4H). HRMS (ESI) calculated for [M, C50H57N2O4]+: 749.4313, found: 749.4312.
3-(6-methoxy-6-oxohexyl)-2-((E)-2-((E)-3-((E)-2-(3-(6-methoxy-6-oxohexyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-246-1): Prepared according to the general procedure from 2 (65 mg, 0.2 mmol, 1 equiv) and 4h (167 mg, 0.4 mmol, 2 equiv). Yield: 18% yield (30 mg). 1H-NMR (600 MHz, MD3OD) δ 8.23 (d, J=8.5 Hz, 2H), 8.01 (d, J=8.9 Hz, 2H), 7.99 (d, J=8.1 Hz, 2H), 7.87 (d, J=14.1 Hz, 2H), 7.65-7.62 (m, 2H), 7.57 (d, J=8.8 Hz, 2H), 7.51-7.46 (m, 3H), 6.23 (d, J=14.1 Hz, 2H), 4.27-4.19 (m, 4H), 3.69 (s, 3H), 3.62 (s, 3H), 2.62 (t, J=6.2 Hz, 4H), 2.36 (dt, J=11.5, 7.3 Hz, 4H), 2.06-1.96 (m, 12H), 1.93-1.88 (m, 4H), 1.76-1.70 (m, 4H), 1.56-1.50 (m, 4H). HRMS (ESI) calculated for [M, C52H61N2O4]+: 777.4626, found: 777.4626.
2-((E)-2-((E)-3-((E)-2-(3-(5-carboxypentyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3-(6-methoxy-6-oxohexyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-246-2): Prepared according to the general procedure from 2 (65 mg, 0.2 mmol, 1 equiv) and 4h (167 mg, 0.4 mmol, 2 equiv). Yield: 17% yield (28 mg). 1H-NMR (600 MHz, MD3OD) δ 8.23 (d, J=8.5 Hz, 2H), 8.21-7.96 (m, 4H), 7.87 (dd, J=14.1, 9.3 Hz, 2H), 7.65-7.61 (m, 2H), 7.59-7.54 (m, 2H), 7.51-7.45 (m, 3H), 6.22 (t, J=14.0 Hz, 2H), 4.27-4.18 (m, 4H), 3.70-3.61 (m, 3H), 2.61 (t, J=6.4 Hz, 4H), 2.36 (dt, J=11.5, 7.2 Hz, 2H), 2.29 (t, J=7.3 Hz, 2H), 2.03-2.00 (m, 8H), 1.97 (s, 6H), 1.94-1.87 (m, 4H), 1.76-1.68 (m, 4H), 1.57-1.50 (m, 4H); HRMS (ESI) calculated for [M, C51H59N2O4]+: 763.4469, found: 763.4455.
2-((E)-2-((E)-3-(2-(3-(3-(dimethylamino)-3-oxopropyl)-1,1-dimethyl-2,3-dihydro-1H-benzo[e]indol-2-yl)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium (BL-248): Prepared according to the general procedure from 4a (70 mg, 0.2 mmol, 1 equiv), 2 (65 mg, 0.2 mmol, 1 equiv) and 4c (75 mg, 0.2 mmol, 1 equiv). Yield: 20% yield (28 mg). 1H-NMR (600 MHz, MD3OD) δ 8.16-8.11 (m, 2H), 7.94-7.88 (m, 2H), 7.86-7.81 (m, 3H), 7.69 (d, J=13.8 Hz, 1H), 7.56-7.49 (m, 3H), 7.43-7.38 (m, 2H), 7.35-7.32 (m, 1H), 7.30 (d, J=8.8 Hz, 1H), 6.17 (d, J=14.3 Hz, 1H), 5.88 (d, J=13.7 Hz, 1H), 5.08 (s, 2H), 3.67 (s, 3H), 3.22 (s, 3H), 2.94 (s, 3H), 2.50 (t, J=6.2 Hz, 2H), 2.46 (t, J=6.2 Hz, 2H), 1.96 (s, 6H), 1.91 (s, 6H), 1.88-1.82 (m, 2H). 13C-NMR (151 MHz, MD3OD) δ 174.77, 172.68, 165.98, 154.83, 149.06, 146.27, 140.60, 140.25, 133.71, 132.79, 132.18, 132.13, 131.72, 130.32, 129.93, 129.74, 129.66, 128.19, 127.96, 127.38, 127.08, 124.76, 124.06, 122.02, 121.85, 110.61, 110.18, 100.14, 98.32, 51.01, 50.46, 44.88, 35.61, 34.87, 30.69, 26.28, 26.00, 23.59, 21.37. HRMS (ESI) calculated for [M, C43H46N3O]+: 620.3635, found: 620.3634.
2,2′-((1E,1′E)-1,3-phenylenebis(ethene-2,1-diyl))bis(1,1,3-trimethyl-1H-benzo[e]indol-3-ium) (BL-250): Prepared according to the general procedure from m-Phthalaldehyde (70 mg, 0.52 mmol, 1 equiv) and 4a (365 mg, 1.04 mmol, 2 equiv). Yield: 50% yield (208 mg). 1H-NMR (600 MHz, MD3OD) δ 8.64 (d, J=16.5 Hz, 2H), 8.48-8.44 (m, 2H), 8.34-8.30 (m, 2H), 8.27 (d, J=8.9 Hz, 2H), 8.19 (d, J=8.2 Hz, 2H), 8.04 (d, J=8.9 Hz, 2H), 7.96 (d, J=16.5 Hz, 2H), 7.87-7.83 (m, 2H), 7.81 (t, J=7.7 Hz, 2H), 7.76-7.73 (m, 2H), 4.45 (s, 6H), 2.18 (s, 12H). HRMS (ESI) calculated for [M, C40H38N2]+: 546.3024, found: 546.3015.
2,2′-((1E,1′E)-1,3-phenylenebis(ethene-2,1-diyl))bis(1,1,3-trimethyl-1H-benzo[e]indol-3-ium) (BL-262): Prepared according to the general procedure from 4-Hydroxyisophthalaldehyde (50 mg, 0.33 mmol, 1 equiv) and 4a (232 mg, 0.66 mmol, 2 equiv). Yield: 80% yield (182 mg). 1H-NMR (600 MHz, MD3OD) δ 8.82 (s, 1H), 8.22 (d, J=16.0 Hz, 1H), 8.17 (d, J=8.5 Hz, 1H), 8.09 (d, J=8.8 Hz, 1H), 8.04 (d, J=8.1 Hz, 1H), 7.92 (d, J=8.6 Hz, 1H), 7.85 (d, J=16.0 Hz, 1H), 7.81 (dd, J=8.5, 1.3 Hz, 1H), 7.76 (d, J=8.5 Hz, 1H), 7.74-7.69 (m, 2H), 7.66-7.57 (m, 2H), 7.50 (d, J=10.3 Hz, 1H), 7.39-7.43 (m, 1H), 7.26-7.22 (m, 1H), 6.98 (d, J=8.6 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H), 5.89 (d, J=10.3 Hz, 1H), 4.56 (s, 3H), 2.84 (s, 3H), 2.06 (s, 3H), 2.06 (s, 3H), 1.62 (s, 3H), 1.35 (s, 3H). HRMS (ESI) calculated for [M, C40H37N2O]+: 561.2900, found: 561.2906.
2-((E)-2-((E)-3-(2-(3-(3-(dimethylamino)propyl)-1,1-dimethyl-2,3-dihydro-1H-benzo[e]indol-2-yl)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium (BL-260): Prepared according to the general procedure from 4a (70 mg, 0.2 mmol, 1 equiv), 2 (65 mg, 0.2 mmol, 1 equiv) and 4d (91 mg, 0.2 mmol, 1 equiv). Yield: 20% yield (28 mg). 1H-NMR (600 MHz, MD3OD) δ 8.24 (d, J=9.1 Hz, 1H), 8.23 (d, J=9.1 Hz, 1H), 8.04-7.95 (m, 4H), 7.91 (d, J=14.2 Hz, 1H), 7.85 (d, J=13.9 Hz, 1H), 7.67-7.56 (m, 4H), 7.54-7.43 (m, 3H), 6.30-6.21 (m, 2H), 4.27 (t, J=7.2 Hz, 2H), 3.76 (s, 3H), 2.65-2.60 (m, 4H), 2.58-2.51 (m, 2H), 2.55 (s, 6H), 2.09-1.96 (m, 14H). HRMS (ESI) calculated for [M, C44H50N3]+: 620.3999, found: 620.4000.
1,3,3-trimethyl-2-((E)-2-((E)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium (BL-264): Prepared according to the general procedure from 4i (60 mg, 0.2 mmol, 2 equiv) and 2 (33 mg, 0.1 mmol, 1 equiv). Yield: 87% yield (50 mg). 1H-NMR (600 MHz, MD3OD) δ 7.76 (d, J=14.0 Hz, 2H), 7.49-7.44 (m, 3H), 7.39 (td, J=7.7, 1.2 Hz, 2H), 7.27-7.21 (m, 4H), 6.15 (d, J=14.0 Hz, 2H), 3.60 (s, 6H), 2.57 (t, J=6.2 Hz, 4H), 1.98-1.92 (m, 2H), 1.71 (s, 12H). 13C-NMR (151 MHz, MD3OD) δ 173.41, 149.81, 144.46, 142.27, 133.79, 129.68, 125.85, 123.24, 111.46, 100.77, 50.15, 31.40, 27.91, 24.96, 22.73. HRMS (ESI) calculated for [M, C32H37N2]+: 449.2951, found: 449.2946.
3-(6-aminohexyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-272): Prepared according to the general procedure from 4a (70 mg, 0.2 mmol, 1 equiv), 2 (65 mg, 0.2 mmol, 1 equiv) and 4f (94 mg, 0.2 mmol, 1 equiv). Yield: 10% yield (14 mg). 1H-NMR (600 MHz, MD3OD) δ 8.24 (d, J=4.8 Hz, 1H), 8.22 (d, J=4.9 Hz, 1H), 8.03-7.95 (m, 4H), 7.90 (d, J=14.1 Hz, 1H), 7.85 (d, J=14.0 Hz, 1H), 7.65-7.61 (m, 2H), 7.58 (t, J=9.2 Hz, 2H), 7.54 (s, 1H), 7.49-7.44 (m, 2H), 6.25-6.17 (m, 2H), 4.25 (t, J=7.4 Hz, 2H), 3.74 (s, 3H), 2.95-2.90 (m, 2H), 2.64-2.58 (m, 4H), 2.04-1.96 (m, 14H), 1.94-1.88 (m, 2H), 1.72-1.66 (m, 2H), 1.59-1.48 (m, 4H). 13C-NMR (151 MHz, MD3OD) δ 175.21, 173.65, 156.10, 149.40, 148.36, 141.78, 141.29, 134.67, 134.44, 133.83, 133.51, 133.40, 133.22, 131.64, 131.11, 131.09, 129.99, 129.54, 129.42, 128.69, 128.68, 125.93, 125.85, 125.81, 123.35, 123.31, 118.00, 114.13, 111.97, 111.88, 100.80, 100.07, 52.13, 51.97, 44.80, 40.64, 31.90, 28.52, 28.40, 27.62, 27.56, 27.47, 27.29, 25.04, 22.80. HRMS (ESI) calculated for [M, C45H52N3]+: 634.4156, found: 634.4153.
3-(2-(dimethylamino)-2-oxoethyl)-2-((E)-2-((E)-3-((E)-2-(3-(2-(dimethylamino)-2-oxoethyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-273): Prepared according to the general procedure from 2 (33 mg, 0.1 mmol, 1 equiv) and 4c (75 mg, 0.2 mmol, 2 equiv). Yield: 26% yield (20 mg). 1H-NMR (600 MHz, MD3OD) δ 8.24 (d, J=8.5 Hz, 2H), 7.98-7.94 (m, 4H), 7.87 (d, J=14.2 Hz, 2H), 7.65-7.61 (m, 2H), 7.51 (s, 1H), 7.47 (t, J=7.6 Hz, 2H), 7.44 (d, J=8.8 Hz, 2H), 6.06 (d, J=14.0 Hz, 2H), 5.24 (s, 4H), 3.32 (s, 6H), 3.04 (s, 6H), 2.55 (t, J=6.2 Hz, 4H), 2.06 (s, 12H), 1.96-1.90 (m, 2H). HRMS (ESI) calculated for [M, C46H51N4O2]+: 691.4007, found: 691.4002.
General procedure: The corresponding pyridinium salt B (1 equiv.) and 4-bromoaniline (1 equiv.) were dissolved in methanol (4 mL) in an 8 mL of vial, and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt A (1 equiv.), C (1 equiv.) and sodium acetate (3 equiv.) were added. The reaction mixture was stirred for additional overnight at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol).
The corresponding pyridinium salt 175 (206 mg, 0.48 mmol) and 4-bromoaniline (103 mg, 0.3 mmol) were dissolved in methanol (4 mL, 7 mL/mmol), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt 144 (176 mg, 0.5 mmol), 302 (286 mg, 0.65 mmol) and sodium acetate (246 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1, then 10:1). Affording 58 mg of green solid GL308-2. Yield: 19%. 1H NMR (600 MHz, Methanol-d4) δ 8.27 (d, J=8.17 Hz, 1H), 8.23 (d, J=8.17 Hz, 1H), 8.11 (t, 1H), 8.06-7.98 (m, 5H), 7.69-7.63 (m, 4H), 7.55 (d, J=8.80 Hz, 1H), 7.52 (t, 1H), 7.48 (t, 1H), 6.67-6.58 (m, 2H), 6.42 (d, J=13.82 Hz, 1H), 6.27 (d, J=13.82 Hz, 1H), 4.29 (t, 2H), 3.79 (s, 3H), 2.77 (t, 2H), 2.43 (s, 6H), 2.02 (d, J=4.66 Hz, 12H). 13C NMR (150 MHz, Methanol-d4) δ 174.95, 171.89, 171.50, 149.47, 140.24, 139.83, 133.86, 132.70, 132.20, 131.76, 130.37, 130.24, 129.74, 129.69, 128.20, 127.95, 127.41, 127.24, 125.96, 125.61, 124.85, 124.32, 122.01, 121.84, 110.66, 110.25, 104.22, 102.16, 55.27, 51.09, 50.45, 48.16, 44.52, 41.59, 30.71, 26.27, 25.97. HRMS (ESI) for C40H44N3+ [M-Br]+: 566.3530. Found: 566.3534.
The corresponding pyridinium salt GL175 (209 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.65 mmol) were dissolved in pyridine (4 mL, 7 mL/mmol), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt GL220 (143 mg, 0.5 mmol), GL302 (286 mg, 0.65 mmol) and sodium acetate (246 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1, 10:1). 80 mg of green solid was obtained. Yield: 27.5%. 1H NMR (600 MHz, Methanol-d4) δ 8.48 (d, J=9.20 Hz, 1H), 0.8.23 (s, J=9.15 Hz, 2H), 8.13-8.00 (m, 4H), 7.87 (d, J=8.52 Hz, 2H), 7.75 (t, 1H), 7.62 (t, 1H), 7.52 (t, 1H), 7.33 (m, 3H), 6.96 (d, J=13.83 Hz, 1H), 6.69 (t, 1H), 6.42 (t, 1H), 5.84 (d, J=12.51 Hz, 1H), 4.31 (s, 3H), 4.08 (t, 2H), 2.70 (t, 2H), 2.45 (s, 6H), 1.95 (s, 6H), 13C NMR (150 MHz, Methanol-d4) δ 164.55, 155.60, 150.45, 141.82, 140.81, 140.41, 139.80, 133.82, 130.69, 129.88, 129.72, 129.58, 129.55, 128.69, 127.33, 126.82, 126.70, 126.28, 124.68, 122.86, 121.44, 119.68, 117.50, 114.24, 109.31, 98.05, 54.70, 48.70, 48.05, 47.88, 44.45, 40.41, 37.20, 26.46. HRMS (ESI) for C35H38N3+ [M-Br]+: 500.3060. Found: 500.3087.
The corresponding pyridinium salt GL175 (237 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL/mmol), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt GL176-1 (150 mg, 0.5 mmol), GL302 (286 mg, 0.65 mmol) and sodium acetate (246 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1, 10:1), affording 54 mg of GL-356-2 as green solid in 18.1% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.24 (d, J=8.43 Hz, 1H), 8.04-7.99 (m, 4H), 7.66-7.63 (m, 2H), 7.58 (d, J=8.95 Hz, 1H), 7.51 (m, 2H), 7.43 (m, 1H), 7.32-7.27 (m, 2H), 6.61 (t, 2H), 6.33 (m, 2H), 4.33 (t, 2H), 3.64 (s, 3H), 2.80 (t, 2H), 2.45 (s, 6H), 1.99 (s, 6H), 1.73 (s, 6H). 13C NMR (150 MHz, Methanol-d4) δ 142.95, 141.05, 139.67, 133.13, 131.97, 131.31, 130.31, 129.70, 128.94, 128.67, 128.36, 128.13, 127.31, 125.79, 124.79, 124.64, 124.53, 121.89, 121.26, 110.48, 110.40, 107.96, 103.83, 102.86, 70.74, 55.28, 50.70, 48.98, 44.48, 41.71, 30.20, 26.51, 26.25. HRMS (ESI) for C36H42N3+ [M-Br]+: 516.3373. Found: 516.3380.
The corresponding pyridinium salt GL175 (103 mg, 0.24 mmol) and 4-bromoaniline (49 mg, 0.29 mmol) were dissolved in methanol (4 mL), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt GL219 (105 mg, 0.3 mmol), GL344 (132 mg, 0.3 mmol) and sodium acetate (116 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1, then 10:1), affording GL365-2 as green solid in 22% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.62 (d, J=8.83 Hz, 1H), 8.23 (d, J=8.83 Hz, 1H), 8.08-7.98 (m, 4H), 7.88-7.82 (m, 2H), 7.71-7.63 (m, 3H), 7.58 (m, 2H), 7.48 (t, 1H), 6.69-6.60 (m, 2H), 6.51 (d, J=13.55 Hz, 1H), 6.31 (d, J=13.55 Hz, 1H), 4.34 (t, 2H), 4.22 (s, 3H), 2.84 (t, 2H), 2.49 (s, 6H), 2.01 (s, 6H), 1.78 (s, 6H). 13C NMR (150 MHz, Methanol-d4) δ 174.57, 139.71, 139.02, 137.44, 135.06, 131.85, 130.30, 129.70, 129.40, 128.17, 127.29, 126.73, 126.57, 125.71, 124.44, 121.87, 121.64, 121.30, 118.98, 110.30, 55.13, 53.40, 50.58, 49.00, 44.39, 43.06, 41.49, 36.15, 28.93, 26.38, 26.24, 19.84. HRMS (ESI) for C35H38N3+ [M-Br]+: 500.3060. Found: 500.3087.
The corresponding pyridinium salt 175 (206 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL/mmol), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt 144 (176 mg, 0.5 mmol), 288 (252 mg, 0.65 mmol) and sodium acetate (246 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1, 10:1, 8:1). affording GL291-2 as green solid in 19% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.13 (m, 1H), 7.91 (m, 3H), 7.54-7.48 (m, 2H), 7.37 (m, 1H), 6.52 (m, 1H), 6.24 (m, 1H), 3.38 (m, 2H), 3.02 (m, 2H), 2.79 (m, 12H), 1.89 (m, 6H), 1.81 (m, 2H), 1.64 (m, 2H), 1.42 (m, 2H), 1.33 (m, 2H). 13C NMR (150 MHz, Methanol-d4) δ 140.36, 139.80, 136.85, 133.37, 133.30, 131.899, 131.86, 130.91, 130.30, 130.27, 129.71, 129.68, 128.28, 128.13, 128.02, 127.31, 127.21, 124.58, 124.51, 122.99, 121.95, 121.92, 110.68, 110.52, 65.50, 57.55, 53.42, 42.17, 30.60, 27.83, 26.28, 26.12, 24.24, 20.65, 14.05. HRMS (ESI) for C45H52N3+ [M-Br]+: 622.4156. Found: 622.4162.
Synthesis affording GL291-3 as green solid in 10% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.26 (d, J=8.54 Hz, 2H), 8.08 (t, 2H), 8.03-8.00 (m, 3H), 7.97-7.94 (m, 2H), 7.78-7.77 (m, 2H), 7.66 (m, 2H), 7.60 (d, J=8.64 Hz, 2H), 7.51 (t, 2H), 6.64 (t, 2H), 6.37 (d, J=13.71 Hz, 2H), 4.25 (t, 4H), 3.03 (m, 4H), 2.80 (s, 12H), 2.03 (s, 12H), 1.93 (m, 4H), 1.73 (m, 4H), 1.59 (m, 4H), 1.50 (m, 4H). HRMS (ESI) for C51H67N3+ [M-Br]+: 735.5360. Found: 735.5368.
The corresponding pyridinium salt 183 (237 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL/mmol), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt 144 (176 mg, 0.5 mmol), 288 (322 mg, 0.65 mmol) and sodium acetate (246 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 30:1, 20:1, 10:1). affording GL297-2 as green solid in 22% yield. The product was found in LCMS at M:694.4 and [M2+]/2: 347.8. 1H NMR (600 MHz, Methanol-d4) δ 8.28 (m, 2H), 8.08-8.01 (m, 4H), 7.86-7.76 (m, 2H), 7.70-7.62 (m, 4H), 7.55-7.51 (m, 2H), 6.81-6.72 (m, 1H), 6.61-6.57 (m, 1H), 6.41-6.34 (m, 1H), 4.34-4.28 (m, 2H), 4.19 (s, 3H), 3.83 (d, J=6.96 Hz, 3H), 2.90 (m, 2H), 2.68 (m, 6H), 2.21 (d, J=12.22 Hz, 3H), 1.97 (s, 12H), 1.95 (m, 2H), 1.70 (m, 2H), 1.61 (m, 2H), 1.50 (m, 2H).
GL368-2 was obtained by use the similar procedure of GL297-2.
144 (105 mg, 0.3 mmol), 175 (103 mg, 0.24 mmol), 328 (154 mg, 0.3 mmol) and 4-bromoaniline (49 mg), NaOAc (116 mg) and methanol (4 mL) were mixed. The mixture was stirred at room temperature for overnight. After that, the methanol was removed by rotary evaporator. Then, the crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1 to 10:1), affording GL328-2 as green solid in 21% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.26-8.24 (m, 2H), 8.11-7.99 (m, 6H), 7.72-7.63 (m, 3H), 7.61 (t, 2H), 7.49 (m, 2H), 6.65 (m, 2H), 6.36 (d, J=13.67 Hz, 2H), 4.25 (t, 2H), 3.76 (s, 3H), 3.41-3.38 (m, 2H), 3.15 (s, 9H), 2.01 (d, J=1.89 Hz, 12H), 1.96-1.91 (m, 2H), 1.87-1.82 (m, 2H), 1.66-1.61 (m, 2H), 1.54-1.48 (m, 2H). 13C NMR (150 MHz, Methanol-d4) δ 140.35, 139.83, 133.45, 133.24, 132.05, 131.85, 130.29, 129.92, 129.72, 129.69, 128.15, 128.02, 127.99, 127.33, 127.32, 124.63, 124.51, 123.99, 121.95, 121.90, 110.61, 110.52, 66.33, 62.91, 52.23, 52.20, 52.18, 50.84, 50.67, 43.46, 30.55, 29.32, 27.21, 26.27, 26.09, 26.05, 25.79, 22.48. LCMS (ESI) for C47H56N32+ [M-Br]2+: 318.7. Found: 318.9.
The corresponding pyridinium salt 175 (206 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL/mmol), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt 144 (176 mg, 0.5 mmol), 162 (252 mg, 0.65 mmol) and sodium acetate (246 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. Afterwards, Et2O (21 mL/mmol) was added, and the mixture was placed to the freezer (−16° C.). The resulting precipitate was filtered, washed with water (2×10 mL/mmol), Et2O (2×10 mL/mmol) and dried on air. Then, the crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1 to 10:1), affording GL286 as green solid in 18% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.15-8.12 (m, 2H), 7.99-7.87 (m, 6H), 7.56-7.44 (m, 5H), 7.40-7.36 (m, 2H), 6.51 (t, 2H), 6.24-6.20 (m, 2H), 4.11 (t, 2H), 3.64 (s, 3H), 2.82 (t, 2H), 2.26 (s, 2H), 1.90 (s, 12H), 1.81 (m, 2H), 1.58 (m, 2H), 1.43 (m, 2H). HRMS (ESI) for C42H48N3+ [M-Br—HBr]+: 594.3843. Found: 594.3846.
GL144 (210 mg, 0.6 mmol), GL175 (208 mg, 0.5 mmol), GL148 (265 mg, 0.6 mmol) and 4-bromoaniline (100 mg) and NaOAc (232 mg) were dissolved in methanol (4 mL), and the mixture was stirred at room temperature for overnight (16 h). The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1 to 10:1), affording compound GL261 (87 mg, yield: 16%). 1H NMR (600 MHz, Methanol-d4) δ 8.24 (d, J=8.86 Hz, 2H), 8.09-7.99 (m, 6H), 7.66 (t, 2H), 7.59 (t, 2H), 7.49 (t, 2H), 6.62 (t, 2H), 6.33 (m, 2H), 4.23 (t, 2H), 3.74 (s, 3H), 2.34 (t, 2H), 2.01 (s, 12H), 1.93 (m, 2H), 1.74 (m, 2H), 1.56 (m, 2H). HRMS (ESI) for C42H45N2O2+ [M-Br]+: 609.3476. Found: 609.3481.
Compound GL261 (87 mg, 0.13 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl, 29 mg, 0.15 mmol) and N-hydroxysuccinimide (NHS, 20 mg, 0.16 mmol) were dissolved in 5 mL DMF and the resulting mixture was stirred at room temperature under nitrogen atmosphere for 24 h in the dark. Then NH2(CH2)6NHBoc (41 mg, 0.19 mmol) and N, N-diisopropylethylamine (DIPEA, 49 mg, 0.38 mmol) were added to the reaction solution and the mixture was stirred for an additional 6 h. The crude product was purified by silica gel column chromatography with gradient elution (dichloromethane/methane of 25:1 to 15:1) to afford GL264-2 as purplish red solid (80 mg, 69%). 1H NMR (600 MHz, Methanol-d4) δ 8.13 (d, J=7.92 Hz, 2H), 7.98-7.88 (m, 6H), 7.54 (t, 2H), 7.49-7.45 (m, 3H), 7.38 (t, 2H), 6.50 (t, 2H), 6.24 (m, 2H), 4.11 (t, 2H), 3.63 (s, 3H), 2.98 (t, 2H), 2.90 (t, 2H), 2.10 (t, 2H), 1.90 (s, 12H), 1.78 (m, 2H), 1.61 (m, 2H), 1.38 (m, 2H), 1.30 (m, 14H), 1.17 (m, 6H).
In an 8 ml of vial, 80 mg of Boc compound was dissolved in 10 mL DCM, and 1 ml of TFA added dropwise. The solution was stirred for 30 min, then, the excess of TFA was removed by vacuum, affording GL258-2 as green solid. 1H NMR (600 MHz, Methanol-d4) δ 8.13 (d, J=8.16 Hz, 2H), 7.98-7.85 (m, 6H), 7.57-7.45 (m, 5H), 7.38 (m, 2H), 6.50 (t, 2H), 6.23 (t, 2H), 4.10 (t, 2H), 3.63 (s, 3H), 3.01 (t, 2H), 2.78 (t, 2H), 2.02 (t, 2H), 1.89 (s, 12H), 1.77 (m, 2H), 1.60 (m, 2H), 1.52 (m, 2H), 1.42-1.32 (m, 4H), 1.29-1.19 (m, 4H).
Compound GL293-2 was obtained as green solid in 15% yield by use the similar procedure of GL261-1. 1H NMR (600 MHz, Methanol-d4) δ 7.84-7.71 (m, 11H), 7.41 (m, 2H), 7.31 (t, 1H), 6.59 (t, 2H), 6.32 (m, 2H), 4.38 (m, 2H), 2.28 (m, 2H), 1.90 (m, 5H), 1.79 (m, 2H), 1.65 (m, 2H). LCMS for C32H33N2O2+ [M-Br]+: 477.25. Found: 477.2.
Compound GL295-2 was afforded as green solid in 71% yield by use the similar procedure of GL264-2. LCMS for C43H55N403+ [M-Br]+: 675.42. Found: 675.4.
The corresponding pyridinium salt GL175 (103 mg, 0.24 mmol) and 4-bromoaniline (49 mg, 0.29 mmol) were dissolved in methanol (4 mL), and the mixture was stirred at room temperature for 30 min. Next, a heterocyclic salt GL144 (105 mg, 0.3 mmol), GL361 (129 mg, 0.3 mmol) and sodium acetate (116 mg) were added, and the reaction mixture was stirred for additional 16 h at room temperature. The crude product was purified by flash column chromatography (silica gel, dichlormethane/methanol, 20:1, 10:1), affording GL362-2 as green solid in 23% yield. 1H NMR (600 MHz, Methanol-d4) δ 8.24 (d, J=8.51 Hz, 2H), 8.09-7.99 (m, 6H), 7.70-7.57 (m, 5H), 7.49 (t, 2H), 6.62 (t, 2H), 6.34 (d, J=14.40 Hz, 2H), 4.22 (t, 2H), 3.74 (s, 3H), 3.18 (t, 2H), 2.01 (s, 12H), 1.93 (s, 3H), 1.88 (m, 2H), 1.55 (m, 4H), 1.46 (m, 2H). 13C NMR (150 MHz, Methanol-d4) δ 171.84, 140.40, 139.78, 133.39, 133.28, 131.98, 131.92, 130.32, 130.26, 129.71, 128.11, 128.05, 127.31, 125.57, 124.56, 121.92, 110.61, 110.47, 53.40, 50.76, 50.73, 48.47, 48.22, 43.60, 38.88, 30.44, 28.82, 27.28, 26.26, 26.23, 26.11, 26.08, 21.16. HRMS (ESI) for C44H50N3O+ [M-Br]+: 636.3948. Found: 636.3954.
GL342 (830 mg, 2 mmol), Pd(PPh3)4 (250 mg, 0.2 mmol), and K2CO3 (1.1 mg, 6 mmol) were mixed in 15 mL of isopropanol, and then stirred and heated at 90° C. for overnight. The dark solution was changed to yellow. After cooled down to room temperature, the solvents were removed by rotary evaporate. The crude product was purified by flash silica gel column (Hexane and ethyl acdetate 10:1), affording orange solid GL343. LCMS (ESI) for C24H28N2[M+H]+: 345.23. Found: 345.3.
GL144 (176 mg, 0.5 mmol), GL343 (172 mg, 0.5 mmol), compound GL345 (286 mg, 0.65 mmol) and sodium acetate (246 mg, 3 mmol) and Acetic anhydride (4 ml) were stirred and heated at 80° C. for overnight. The dark solution was cooled down and poured into ether. The green solid was filtered and collected. The crude product was purified by silica gel column (DCM and Methanol 10:1). Obtaining GL349-2 as green solid (yield: 10%). 1H NMR (600 MHz, Methanol-d4) δ 8.27 (d, J=8.82 Hz, 1H), 8.23 (d, J=8.82 Hz, 1H), 8.07-7.98 (m, 5H), 7.85 (d, J=13.24 Hz, 1H), 7.69-7.59 (m, 3H), 7.56-7.46 (m, 4H), 6.32 (d, J=14.42 Hz, 1H), 6.23 (d, J=14.22 Hz, 1H), 4.36 (t, 2H), 3.82 (s, 3H), 2.92 (m, 2H), 2.84 (t, 2H), 2.50 (s, 6H), 2.05 (m, 12H), 2.01 (s, 4H), 1.15 (s, 9H). 13C NMR (150 MHz, Methanol-d4) δ 174.35, 149.04, 140.27, 139.76, 132.16, 131.73, 130.34, 130.25, 129.75, 128.24, 127.94, 127.39, 127.22, 124.79, 124.22, 122.02, 121.84, 110.62, 110.07, 100.05, 98.09, 54.94, 51.06, 50.32, 44.40, 43.67, 41.11, 32.06, 30.72, 26.55, 26.29, 26.14, 26.06, 25.92. 19.64. HRMS (ESI) for C47H56N3+ [M-Br]+: 662.4469. Found: 662.4474.
GL144 (176 mg, 0.5 mmol), GL148 (202.2 mg, 0.5 mmol), (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde (86.3 mg, 0.5 mmol) and sodium acetate (86 mg, 1.05 mmol) and Acetic anhydride (5 ml) were stirred and heated at 70° C. for overnight. The dark solution was cooled down and poured into ether. The green solid was filtered and collected. The crude product was purified by silica gel column (DCM and Methanol 10:1). Obtaining GL149-2 as green solid (yield: 26.5%). 1H NMR (600 MHz, Methanol-d4) δ 8.58 (t, 1H), 8.30 (d, J=7.61 Hz, 1H), 8.07-8.02 (m, 2H), 7.69-7.63 (m, 2H), 7.53 (t, 1H), 6.35 (d, J=12.17 Hz, 1H), 4.33 (t, 1H), 3.83 (s, 3H), 2.80 (s, 2H), 2.34 (t, 1H), 2.12 (s, 6H), 2.04 (m, 2H), 1.96 (m, 2H), 1.75 (m, 2H), 1.57 (m, 2H). 13C NMR (150 MHz, Methanol-d4) δ 176.40, 174.90, 174.24, 173.96, 149.19, 149.11, 143.33, 143.18, 143.05, 142.97, 140.32, 139.71, 133.83, 133.81, 132.23, 132.15, 130.49, 130.44, 129.76, 129.34, 128.05, 127.95, 127.44, 127.43, 126.61, 126.57, 126.49, 126.43, 124.92, 124.86, 124.83, 122.04, 110.75, 110.64, 100.80, 100.35, 51.06, 48.16, 43.83, 33.65, 33.07, 30.72, 26.99, 26.48, 26.38, 26.02, 25.96, 25.92, 24.46, 24.24, 20.79. HRMS (ESI) for C45H48ClN2O2+ [M-Br]+: 683.3390. Found: 683.3312.
GL144 (176 mg, 0.5 mmol), GL162 (223.5 mg, 0.5 mmol), (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde (86.3 mg, 0.5 mmol) and sodium acetate (86 mg, 1.05 mmol) and Acetic anhydride (3 ml) were stirred and heated at 70° C. for overnight. The dark solution was cooled down and poured into ether. The green solid was filtered and collected. The crude product was purified by silica gel column (DCM and Methanol 10:1). Obtaining GL161-2 as green solid (yield: 26.5%). 1H NMR (600 MHz, Methanol-d4) 8.62-8.56 (m, 2H), 8.30 (m, 2H), 8.08-8.02 (m, 4H), 7.70-7.63 (m, 4H), 7.53 (m, 2H), 6.35 (m, 2H), 4.32 (t, 2H), 3.83 (s, 3H), 3.18 (t, 2H), 2.80 (m, 4H), 2.06 (d, 12H), 2.03 (m, 2H), 1.95 (m, 2H), 1.55 (m, 4H), 1.48 (m, 2H).
In a typical molecular absorbance of photons, an individual bond or small portion of the molecule starts vibrating (
Here, it is proposed that the feature of the higher-energy shoulder next to the large absorption band in symmetrical cyanine structures results because there is coupling of a molecular plasmon (a dominant collective oscillation of electronic excitation) to a dominant collective vibrational excitation, in agreement with the suggestion in the literature that the absorption sub-bands in cyanines are primarily determined by a dominant symmetric vibration rather than a collection of single vibrations. (Mustroph & Towns, 2018) This vibronic behavior, through the coupling of electronic and vibrational states, is a feature of the conjugated-backbone-near-symmetrical cyanines such as in Cy7-amine and Cy7.5-amine. The shoulder (λ˜730 nm) in the absorption spectrum of Cy7.5-amine corresponds to this collective vibrational mode (
Here the vibronic mode in a cell-membrane-bound Cy7.5-amine was selectively excite using a NIR light-emitting diode (LED) at 730 nm (
Consistent with the VDA proposed here, excitation of the 680 nm vibronic shoulder in Cy7-amine improves the MJH effect for opening cell membranes in A375 cells (
To rule out a photothermal effect, the temperature of the media was measured during the NIR light exposure when the treatment was done at room temperature versus when done in an ice bath (
To confirm that a photodynamic ROS generation is not responsible for the necrosis, the permeabilization of A375 melanoma cells was repeated in the presence of ROS scavengers (
To confirm that the permeabilized cells to DAPI are indeed dead, the cells were cultured after the treatment (with 2 μM Cy7.5-amine and illumination for 10 min with 730 nm light at 80 mWcm−2) and quantified cell death by crystal violet and clonogenic assays.
The FDA-approved indocynine green (ICG) bearing alkylsulfonate addends was also tried; it has a vibronic absorption shoulder at 730 nm, as shown in
The MJH Cy7.5-amine was applied to treat murine (B16-F10) and human (A375) melanoma tumors in mice (
The flow cytometry data was analyzed using FlowJo software version 10.5.3. The results were plot using the forward scattering area (FSC-A) vs side scattering area (SSC-A) as shown in
Cyanine molecules. Cy7.5-amine, Cy7-amine, Cy5.5-amine and Cy5-amine were purchased from Lumiprobe Corp. (Maryland, USA). DiD and DiR cyanines were purchased from Biotium (Fremont, CA).
LED illumination systems. The 730 nm LED (model UHP-F-730) and 630 nm LED (model UHP-F-630) were purchased from Prizmatix, Israel. The 680 nm LED and 740 nm LED were custom made and purchased from Keber Applied Research Inc. (Ontario, Canada).
Culture of human melanoma A375 cells. A375 cells were obtained from ATCC (CRL-1619). Cells were cultured in 10 cm polystyrene tissue culture treated dish (Corning) containing DMEM with L-glutamine, 4.5 g/L glucose, and sodium pyruvate (Corning Inc. 10013CV) and supplemented with 10% FBS (Corning, 35010CV), 2×MEM vitamin solution (Gibco, 11120052), 1×MEM non-essential amino acid solution (Gibco, 11140050) and penicillin/streptomycin. Typically, 0.5-1 million cells were inoculated per dish and cultured for 3-4 days in incubator at 37° C. and 5% CO2, then transferred to a new dish when confluency reached nearly 95-100%. For the passage step, cells were detached with 0.05% trypsin-EDTA (Gibco, 25-300-054).
Culture of mouse melanoma B16-F10 cells. Mouse melanoma B16-F10 cells were obtained from the ATCC (CRL-6475) and cultured in 10 cm polystyrene tissue culture treated dish (Corning) containing DMEM with 4.5 g/L glucose (Gibco, 11960-044) and supplemented with 10% FBS (SAFC Industries-Sigma-Aldrich, 12303C), 2×(10 mL) MEM vitamin solution (Corning, 25-020-C1), 1×(5 mL) non-essential amino acid (NEAA) mixture (Lonza, 13-114E), 1×(5 mL) of L-glutamine (Lonza, 17-605E), and 1×(5 mL) of penicillin/streptomycin (Hyclone, SV30010). Typically, 0.5-1 million cells were inoculated per dish, and cultured for 2-3 days in incubator at 37° C. and 5% CO2, then transferred to a new dish when confluency reached nearly 95-100%. For the passage to a new culture dish, cells are detached with 0.05% trypsin-EDTA (Gibco, 25-300-054).
Cell Permeabilization and flow cytometry analysis. A375 cells were cultured as described before. One day before the treatment, cells were inoculated at 5 million cells per dish (10 cm polystyrene tissue culture dish). The cells were harvested using 0.05% trypsin-EDTA (Gibco, 25-300-054), then the cells were counted and were adjusted to a cell density of 2×105 cells/mL in DMEM media with L-glutamine, 4.5 g/L glucose, and sodium pyruvate (Corning Inc. 10013CV) and supplemented with 10% FBS (Corning, 35010CV), 2×MEM vitamin solution (Gibco, 11120052), 1×MEM non-essential amino acid solution (Gibco, 11140050) and penicillin/streptomycin. 1 mL of this cell suspension containing 2×105 cells was used in each treatment. In a 1.5 mL Eppendorf tube, 1 mL of stock solution containing 2 mM Cy7.5-amine (or other cyanine molecule or other concentration) in DMSO (Fisher, 99.7%) was placed in the bottom of the tube, then 1 mL of the cell suspension was added into the tube to get final concentration of 2 μM of Cy7.5-amine containing 0.1% DMSO and 2×105 cells. The mixture was then incubated at 37° C. and 5% CO2 for 30 min. Then, 1 mM DAPI was added into the cell suspension. Then, the cells suspension was transferred to a 35 mL polystyrene tissue culture dish and immediately the cells were treated under the light beam of NIR light of 730 nm at 80 mW/cm2 (or adjusted powers down to 20 mW/cm2) for 10 min (or adjusted illumination times down to 30 s) using LED light source (PRIZMATIX, UHP-F-730, Israel) which covered the entire dish. While the cells were treated, the dish was placed on top of an aluminum block painted black, so that the excess NR light and that was not reflected back into the cell suspension while the aluminum block actED as a heat-sink, maintaining a constant temperature in the dish during the irradiation. The instrument for flow cytometry analysis (SONY, MA900 Multi-Application Cell Sorter) was already set up and calibrated by the time the light treatment was finished. Therefore, as soon as the 10-min light treatment was completed, the cell suspension was rapidly transferred from the 35 mm dish to a flow cytometry tube and the cells were analyzed for DAPI permeabilization and Cy7.5-amine binding. It took ˜30 s to load the sample and to start the analysis. Therefore, the permeabilization of cells was measured as DAPI positive cells and occur immediately due to the membrane permeabilization caused by Cy7.5-amine excitation with the 730 nm NIR light. The light intensity was measured using an Optical Power Meter from Thorlabs, sensor model S302C and console model PM100D.
Temperature measurements. The permeabilization of the cells and flow cytometry analysis was conducted as described above. The temperature of the cell suspension was measured using the temperature probe (Model SC-TT-K-30-36-PP; Omega Engineering, Inc.) immersed in the media. The same was repeated having the cell suspension on top of an ice bath, and the temperature of the cell suspension recorded in the same way during the NIR light illumination. The temperature of the media stayed constant at room temperature of −20° C. upon illumination of the media with the 730 nm LED light at 80 mW/cm2 for 10 min. There was only a minor temperature increase of 0.4° C. which was attributed to the light illumination absorption by the media components. Similarly, on ice the temperature of the media only increased 0.6° C. due to the illumination by the 730 nm LED on the media.
ROS scavenger experiments. The permeabilization of the cells and flow cytometry analysis was conducted as described before. But in this case, ROS scavengers were added into the cells suspension and incubated for 1.5-2 h at 37° C. and 5% CO2 before any treatment to allow the antioxidants interact first and protect the cells. Then the experiments were conducted exactly as described before with and without ROS scavengers present, and results were compared.
Crystal violet cell viability assay. The crystal violet assay was used to measures the cell viability. The principle of this method is that the viable cells adhere to the surface of the cell culture dish and keep growing and remain attached through the standard cell culture conditions during a period of 1-2 days and through the staining conditions in the assay. In contrast dead cells do not adhere to the surface of the cell culture dish, do not grow, and detach easily during the manipulation steps during the assay which includes removal of media and exchange with fresh media and washing steps with PBS buffer. For specific details, A375 cells were harvested and counted, and then 20,000 A375 cells per well were added in 24 cell culture well plate (Corning) and cultured for 1 day at standard incubation conditions of 37° C. and 5% CO2. The cells were treated in four experimental groups (4 samples per group): group 1) 0.1% DMSO, group 2) 0.1% DMSO+NIR light treatment, group 3) 2 μM Cy7.5-amine, and group 4) 2 μM Cy7.5-amine+NIR light. The treatments with 0.1% DMSO or 2 μM Cy7.5-amine were done by adding those respective concentrations to the cells in the media and then incubated for 60 min. Immediately after the incubation, the cells in the groups with “+NIR light”, were treated with 730 nm light at 80 mW/cm2 for 10 min. After the treatment with NIR light, the media in all the groups was removed and fresh media was added. Then, the cells were incubated for 2 days at 37° C. and 5% CO2. At the end of the incubation, the media was removed and the cells washed with 500 μL of PBS once. Then, the cells were stained with 500 μL of 0.05% w/v crystal violet solution in methanol for 5 min. Then, the crystal violet was removed and the excess of crystal violet was washed with water. The cells contained in the 24 well plate were dried at room temperature. Then, the crystal violet in each well was solubilized in 500 μL of 3.3% v/v acetic acid in water and the total crystal violet recover in this acidic solution. Then the crystal violet was quantified by its absorbance at 570 nm. The cell viability was calculated from the absorbance relative to the absorbance in the cells without any treatment. Cells without treatment were normalized to 100% cell viability.
Clonogenic assay. A375 cells were seeded in 35 mm cell culture dishes at predetermined densities to allow for an approximately equal number of resultant colonies. The next day, cells were treated with Cy7.5-amine at variable concentration and with or without 730 nm light at 80 mWcm−2 for 10 min. The cells were incubated with Cy7.5-amine for 50 min before the illumination, the media was replaced with fresh media after the illumination and cells were cultured for 6 days to allow for colony formation. Cells were then washed once with PBS and fixed-stained in a 0.5% (w/v) crystal violet in methanol/water solution (1:1) during 10 min. The excess of crystal violet was washed off with water, the plates were dried at room temperature and then the colonies were counted using ImageJ software version 1.52a, and the survival fraction was determined. All treatments were in triplicate.
ROS measurements using H2DCF-DA. H2DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate) is a cell permeant reagent. It is deacetylated by cellular esterases to form 2′,7′-dichlorodihydrofluorescein (H2DCF), a non-fluorescent compound, which is rapidly oxidized in the presence of ROS into 2′,7′-dichlorofluorescein (DCF). DCF is highly fluorescent and is detected with excitation/emission at 488 nm/535 nm. A375 cells in suspension containing 2×105 cells mL−1 were first prepared in DMEM media without phenol red. Then the cells were incubated for 30 min at 37° C. with Cy7.5-amine (or the other cyanines) typically at 2 μM concentration in the media. Then, H2DCF-DA (Sigma-Aldrich) was added to cells suspension in media to the final concentration of 5 μM (the stock of H2DCF-DA was at 5 mM in DMSO stored at ˜20° C.). Then transfer the cells to a 96 well plate, 100 μL to each well. Typically, it took 15 min to transfer all the samples and all the controls into the 96 well plate, 6 repetitions per each treatment condition: cyanine+light, cyanine only, DMSO+light, DMSO only, cells only, media only. Then, immediately after the cells were treated with NIR 730 nm light at 80 mWcm−2 for 10 min. From the time the H2DCF-DA was added into the cell suspension to the time the cells were treated there was in total a 20 min incubation. After the light treatment, the DCF fluorescence intensity was measured immediately after the light treatment using a 96 well plate reader at λex=488/9 nm and λem=535/20 nm. The measurements were normalized with respect to the fluorescence intensity in the media only.
Singlet oxygen measurements. For the measurement of singlet oxygen, the molecular probe DPBF (1,3-diphenylisobenzofuran) was used which decomposes in the presence of singlet oxygen and this was detected by the change in the absorbance of DPBF at 410 nm. DPBF was freshly prepared for every experiment by dissolving DPBF in methanol at 1 mM stock solution. Then dilute the DPBF in methanol and adjust the dilution volume to get an absorbance of ˜1.0 at 410 nm. For this purpose, typically 170 μL of the 1 mM DPBF stock in methanol was diluted in 2830 μL of methanol. Then to this mixture was added 1 μL of cyanine stock solution (8 mM cyanine solution in DMSO stored at ˜20° C.) to get a final concentration of 2.6 μM of cyanine. Then immediately after preparing the mixture, the solution was transferred to a clean spectrophotometer quartz cuvette, and the solution was irradiated with 730 nm LED light at a power intensity of 80 mWcm−2. The power intensity was calibrated to the distance at the top of the liquid on the quartz cuvette. The sample was irradiated every 30 s, and then absorption spectrum was recorded in between every irradiation interval until a total of 10 min of irradiation was accumulated.
In vivo studies. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas MD Anderson Cancer Center (Houston, TX). These studies used 7-8 weeks old female C57BL/6J mice (Jackson Laboratories, strain #000664) or 7-8 weeks old athymic female nude (nu/nu) mice from Envigo/Harlan labs.
Injection of B16-F10 cells subcutaneously in C57BL/6J mice to generate the melanoma tumors. The B16-F10 cells were cultured as described before. Cells were harvested from sub-confluent plates, ˜90%, and fresh media was added to the cells the day before harvesting. The cells were harvested using 0.05% trypsin-EDTA (Gibco, 25-300-054). The harvested cells were re-dispersed in DMEM media without supplements at 1×106 cells mL−1. The cell suspension was kept in ice. Then 100 μL of cells were injected per mouse (this was 100,000 cells per mouse) subcutaneously in the right flank of a 7-8-week-old female mouse (C57BL/6J), in which the hair in the right flank was previously depilated using a shaver. The tumors were allowed to grow for 12 days counting from the day of cell injection. And at day 12 the hair of the mouse was removed using hair remover cream (Nair Hair Remover Lotion). For this purpose, a drop of the cream was placed on the skin, on top of the area where the tumor was injected. The mice were anesthetized using isoflurane while the hair remover cream was applied. Starting at day 12 the tumors were measured using a caliper. The tumors can be observed as a black spot (due to the melanin present in the B16-F10 cells) under the skin after the cream depilation. The typical volume of the tumors at ˜15 days was ˜25 mm3. The volume of the tumor was calculated as: (½)×length×width×height. When the height was not possible to measure in the case of the tumors which were too small (usually <100 mm3), then the tumor volume was calculated as: (½)×length×width2.
Preparation of fresh solution of 200 μM Cy7.5-amine and 2.5% DMSO for in vivo studies. In the day of treatment, fresh solution of 200 μM Cy7.5-amine was prepared by diluting in PBS buffer the 8 mM Cy7.5-amine stock in DMSO, the final dilution contained 2.5% DMSO. As control 2.5% DMSO in PBS buffer was used.
Treatment of B16-F10 tumors with Cy7.5-amine and NIR 730 nm light. The tumors were treated at day 13 counting from the day of cell injection. Mice were divided in 3 groups: 1) Cy7.5-amine only (mice per group, n=4), 2) 2.5% DMSO+Light (n=5) and 3) Cy7.5-amine+light (n=5). The day of treatment, fresh solutions (200 μM of Cy7.5-amine in PBS and controls 2.5% DMSO in PBS) were prepared as described before. The mice were anesthetized with isoflurane using a vaporizer. Then, each mouse was injected with 50 μL of 200 μM Cy7.5-amine solution in PBS or 2.5% DMSO, intratumorally. Then mice were kept for 30 min in the cages to let the Cy7.5-amine solution or DMSO solution interact with the tumors. Then, after the 30 min of incubation, the mice were treated (under anesthesia, using isoflurane) with 730 nm LED light source from Prizmatix applying a power intensity of 150 mWcm−2 for 5 min. The light intensity was measured using an Optical Power Meter from Thorlabs, sensor model S302C and console model PM100D. While under light treatment the temperature at the tumor area was measured using an IR thermal camera (Model: Compact Seek Thermal for Android. Seek Thermal, Inc. Santa Barbara, CA). When the treatment was finished the mice were put back into the cages and housed in the animal facility. The treatment was repeated once daily for 4 days. The tumor sizes were measured every day starting the day of hair removal with cream. The tumors were measured using a caliper. Then after the 4 treatments, the tumors were measured every other day.
Injection of A375 cells subcutaneously in athymic nude mice to generate the human melanoma tumor model. The A375 cells were culture as described before. Cells were harvested from sub-confluent plates, ˜90%, and fresh media was added to the cells the day before harvesting. The cells were harvested using 0.05% trypsin-EDTA (Gibco, 25-300-054). The harvested cells were re-dispersed in DMEM media without supplements at 50×106 cells mL−1. The cell suspension was kept in ice. Then 100 μL of cells were injected per mouse (this was 5 million of cells per mouse) subcutaneously in the right flank of 7-8 weeks old female athymic nude mouse. Starting at day 2 the tumors were measured using a caliper. The typical volume of the tumors at day 2 was ˜33 mm3. The volume of the tumor was calculated as: (½)×length×width×height. When the height was not possible to measure in the case of the tumors that were too small (usually <100 mm3), then the tumor volume was calculated as: (½)×length×width2.
Treatment of A375 tumors with Cy7.5-amine and NIR 730 nm light. The tumors were treated at day 3 (since 5 million initial cells were injected per tumor site) when the average tumors size was 35 mm3. Forty mice were divided in 4 groups (number of mice per group, n=10): 1) Cy7.5-amine only 2) 2.5% DMSO only, 3) 2.5% DMSO+Light and 3) Cy7.5-amine+light. The day of treatment, fresh solutions (200 μM of Cy7.5-amine in PBS and controls 2.5% DMSO in PBS) were prepared as described before. The mice were anesthetized with isoflurane using a vaporizer. Then, each mouse was injected with 50 μL of 200 μM Cy7.5-amine solution in PBS or 2.5% DMSO, intratumorally. Then mice were kept for 25 min in the cages to let the Cy7.5-amine solution or DMSO solution interact with the tumors. Then, after the 25 min of incubation, the mice were treated (under anesthesia, using isoflurane) with 730 nm LED light source from Prizmatix applying a power intensity of 150 mWcm−2 for 5 min (other power intensities were 210 mWcm−2 for 5 min and 300 mWcm−2 for 5 min as described in the treatment schedule in
Time-Dependent Density Functional Theory Analyses (TDDFT). Starting from a ground-state DFT calculation to obtain the energies of all the occupied levels, the absorption spectra were calculated by TDDFT using the Liouville-Lanczos approach as coded in the program Quantum Espresso. (Malcioglu et al., 2011; Giannozzi et al., 2009) The charge density responses were visualized using the software VESTA. (Momma & Izumi, 2011)
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:
This application claims the benefit of priority to U.S. Provisional Application No. 63/314,094, filed on Feb. 25, 2022, the entire contents of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/063288 | 2/24/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63314094 | Feb 2022 | US |
