N-1 triazole substituted imidazoquinolines, conjugates thereof, and methods

Abstract

Disclosed are 1H-imidazo[4,5-c]quinolin-4-amine compounds having a triazole ring substituent at the 1-position, salts thereof, conjugates thereof, pharmaceutical compositions containing the compounds and conjugates, and methods for making the compounds and conjugates. Methods of using the compounds and conjugates as immune response modifiers to induce cytokine biosynthesis in humans and animals and in the treatment of diseases, including viral and neoplastic diseases, are also described.

Description

[Background technology]

[0001] Some drug compounds act by stimulating certain key aspects of the immune system as well as by suppressing certain other aspects. These compounds are sometimes referred to as immune response modifiers (IRMs). IRM compounds can act through a fundamental immune system mechanism known as Toll-like receptors (TLRs) to induce selected cytokine biosynthesis, induction of costimulatory molecules, and increased antigen-presenting capacity. Some IRM compounds have been shown to be effective in treating viral diseases, neoplastic diseases, and T. H Some IRM compounds are useful as vaccine adjuvants.

[0002] IRM compounds based on the following bicyclic and tricyclic ring systems have been reported: 1H-imidazo[4,5-c]quinolin-4-amines; 1H-imidazo[4,5-c]pyridin-4-amines; 1H-imidazo[4,5-c][1,5]naphthyridin-4-amines; thiazolo[4,5-c]quinolone-4-amines and oxazolo[4,5-c]quinolone-4-amines; 6,7,8,9-1H-tetrahydro-1H-imidazo[4,5-c]quinolin-4-amines; 2H-pyrazolo[3,4-c]quinolone-4-amines; and N-1 and 2-substituted 1H-imidazo[4,5-c]quinolin-4-amines. Conjugation of IRM compounds with polymeric materials or other active compounds is known. There is a need for new IRM compounds and new conjugates with IRM compounds that can effectively modulate the immune system. Summary of the Invention

[0003] Disclosed are novel compounds that may be useful for inducing cytokine biosynthesis in humans and animals, as well as conjugates thereof, methods of making the compounds and conjugates, and methods of using the compounds and conjugates. The disclosure provides IRM compounds of formula (I-III) (or salts thereof), IRM-containing conjugates of formula (IV) (or salts thereof), and enantiomers of formula (I-IV) (or salts thereof).

[0004] In certain embodiments, the present disclosure provides a compound of formula (I) (or a salt thereof):

[0005] [ka] wherein n is an integer of 0 or 1, R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl, R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl, and R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0006] In certain embodiments, the present disclosure provides a compound of formula (II) (or a salt thereof):

[0007] [ka] wherein n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; R3 is selected from the group consisting of alkyl, aryl, and aralkyl; the alkyl portion of the alkyl or aralkyl optionally contains one or more catenary non-peroxidized -O-atoms; the alkyl portion of the alkyl or aralkyl is optionally substituted with a functional group selected from the group consisting of amine (-NH2), carboxyl (-C(O)OH), hydroxyl (-OH), and thiol (-SH); and the aryl portion of the aryl or aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or a combination thereof.

[0008] In certain embodiments, the present disclosure provides a compound of formula (III) (or a salt thereof):

[0009] [ka] wherein n is an integer of 0 or 1, R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl, R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl, R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms, and R4 is

[0010] [ka] is a triazole ring selected from the group consisting of During the ceremony, `` * " indicates a covalent bond attached to R2, and " **" denotes a covalent bond attached to L1, L1 is an alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms, amine groups (-NH-), ester groups, amide groups (-NH-C(O)-), disulfide groups (-SS-), carbonyl groups (-C(O)-), carbonate groups (-OC(O)-O-), carbamate groups (-OC(O)-NH-), or combinations thereof, m is an integer of 0 or 1, and Q is a functional group for binding to a polymer moiety or a second active moiety.

[0011] The compounds and salts of these compounds (e.g., pharma- ceutically acceptable salts) can be used as immune response modifiers due to their ability to induce cytokine biosynthesis (e.g., induce the synthesis of at least one cytokine) and to modulate other immune responses when administered to humans or animals. Thus, these compounds can be used in the treatment of a variety of conditions, such as viral diseases and tumors, that respond to such alterations in the immune response.

[0012] The compounds may also be used in conjugates with polymeric materials or second active agents. Such conjugates include IRM-containing conjugates of formula (IV) (or a salt thereof):

[0013] [ka] wherein n is an integer of 0 or 1, R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl, R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl, R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms, and R4 is

[0014] [ka] is a triazole ring selected from the group consisting of During the ceremony, `` * " indicates a covalent bond attached to R2, and "** " denotes a covalent bond attached to L2, L2 being a bridging group, m being an integer of 0 or 1, Z being a polymer moiety or a second active moiety, and the -(L2) of the conjugate. m The -Z portion, with or without L2, optionally contains a labile bond.

[0015] Disclosed are pharmaceutical compositions (i.e., formulations) comprising an effective amount of a compound of formula (I), (II) or (III) (or a salt thereof, including a pharma- ceutically acceptable salt thereof), or an IRM-containing conjugate of formula (IV) (or a salt thereof, including a pharma- ceutically acceptable salt thereof), or a combination thereof. Also disclosed are methods of inducing cytokine biosynthesis in a human or animal, methods of treating viral diseases in a human or animal, and methods of treating neoplastic diseases in a human or animal by administering such formulations to the human or animal.

[0016] The term "alkyl" refers to a monovalent group that is a radical of an alkane, and includes straight-chain, branched, cyclic, and bicyclic alkyl groups, and combinations thereof. Unless otherwise specified, alkyl groups typically contain 1 to 30 carbon atoms. In some embodiments, alkyl groups contain 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of "alkyl" groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, t-butyl, isopropyl, n-octyl, n-heptyl, ethylhexyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, and the like.

[0017] The term "alkylene" refers to a divalent group that is a radical of an alkane, and includes groups that are straight chain, branched chain, cyclic, bicyclic, or combinations thereof. Unless otherwise specified, alkylene groups typically have 1 to 100 carbon atoms. In some embodiments, alkylene groups have 1 to 60 carbon atoms, 1 to 50 carbon atoms, 1 to 40 carbon atoms, 1 to 30 carbon atoms, 1 to 20 carbon atoms, 1 to 18 carbon atoms, 1 to 16 carbon atoms, 1 to 14 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some embodiments, an alkylene group has 2 to 60 carbon atoms, 2 to 50 carbon atoms, 2 to 40 carbon atoms, 2 to 30 carbon atoms, 2 to 20 carbon atoms, 2 to 18 carbon atoms, 2 to 16 carbon atoms, 2 to 14 carbon atoms, 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples of "alkylene" groups include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH2CH3)CH2CH2-, and -CH2CH2CH(CH(CH3)2)CH2CH2-.

[0018] The term "alkenylene" refers to a divalent hydrocarbon group having at least one carbon-carbon double bond, and includes groups that are linear, branched, cyclic, bicyclic, or combinations thereof. Unless otherwise specified, alkenylene groups typically have 2 to 60 carbon atoms. In some embodiments, alkenylene groups have 2 to 50 carbon atoms, 2 to 40 carbon atoms, 2 to 30 carbon atoms, 2 to 20 carbon atoms, 2 to 18 carbon atoms, 2 to 14 carbon atoms, 2 to 12 carbon atoms, 2 to 10, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Examples of "alkenylene" groups include -CH2CH2CH=CHCH2-, -CH2CH2CH2CHC=CHCH2CH2-, -CH2CH2CH2CH2CH=CHCH2CH2-, -CH2CH(CH3)CH2CH=CHCH2CH2-, -CH2CH(CH3)CH2CH=CHCH2CH(CH2CH2CH3)CH2CH2-, and -CH2CH2CH2CH=CHCH2CH2CH2CH=CHCH2CH2-.

[0019] For example, an alkyl, alkylene, or alkenylene group having a carbon atom optionally containing one or more catenary non-peroxidized -O- atoms means that the group has a carbon atom on either side of the -O-. Examples include -CH2CH2-O-CH2CH3, -CH2-CH2-O-CH2-CH2-O-CH2CH3, -CH2CH2-O-CH2CH2-, -CH2-CH2-O-CH2-CH2-O-CH2CH2-, -CH2CH2-O-CH2=CH2-, -CH2-CH2CH2-O-CH2CH=CHCH2CH2-O-CH2CH2-, and the like.

[0020] The term "alkoxy" refers to a monovalent group having an oxy group directly attached to an alkyl group.

[0021] The term "aryl" refers to a monovalent group that is aromatic and, optionally, carbocyclic. An aryl has at least one aromatic ring. Any additional rings may be unsaturated, partially saturated, saturated, or aromatic. Optionally, the aromatic ring may have one or more additional carbocyclic rings fused to the aromatic ring. Unless otherwise specified, an aryl group typically contains 6 to 30 carbon atoms. In some embodiments, an aryl group contains 6 to 20, 6 to 18, 6 to 16, 6 to 12, or 6 to 10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, biphenyl, phenanthryl, and anthracyl.

[0022] The term "aralkyl" refers to a monovalent group that is an alkyl group substituted with an aryl group (e.g., as in a benzyl group). Unless otherwise specified, for both groups, the alkyl portion often has 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms, and the aryl portion often has 6 to 20 carbon atoms, 6 to 18 carbon atoms, 6 to 16 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms.

[0023] The term "ar" refers to a divalent group that is a radical of an arene, such as phenylene (-C6H4-).

[0024] The terms "(Cx-Cy)alkyl", "(Cx-Cy)alkoxy", and "(Cx-Cy)alkylene" include straight chain, branched chain, cyclic groups, and combinations thereof, having X to Y carbon atoms. For example, "(C1-C5)alkyl" includes alkyl groups of 1 carbon, 2 carbon, 3 carbon, 4 carbon, and 5 carbon. Some examples of "(C1-C5)alkyl" include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, the isomeric pentyls, cyclopropyl, cyclopentyl, and -CH2-cyclopropyl.

[0025] "IRM-containing conjugate" and variations thereof refer to any conjugate (i.e., complex) that includes at least one immune response modifier (IRM) moiety derived from an IRM compound of formula (I) and at least one polymer moiety (e.g., a PEG moiety) or second active moiety.

[0026] "Moiety" and variations thereof refer to a portion of a chemical compound or polymer that exhibits a particular characteristic, such as, for example, a particular biological or chemical function (e.g., immunomodulatory and / or target specificity), or a physical property (e.g., size, hydrophilicity, or hydrophobicity).

[0027] A "crosslinking group" is derived from a heterobifunctional crosslinking compound that reacts with a functional group of an IRM compound to form a first bond and with a reactive functional group (e.g., including but not limited to, a hydroxyl (-OH), amino (-NH2), carboxylic acid, carboxylate ester, activated carboxylate ester, amide (-NHC(O)), aldehyde (-CH(O)), or thiol (-SH) group) of a polymer or a second active compound to form a second bond. Heterobifunctional crosslinking compounds (i.e., heterobifunctional crosslinkers) contain two different reactive groups and organic crosslinks of various lengths and compositions. More specifically, the "crosslinking group" is derived from a heterobifunctional crosslinking compound that reacts with an azide group of an IRM compound to form a triazole and reacts with a reactive group (e.g., including but not limited to, a hydroxyl (-OH), amino (-NH2), carboxylic acid, carboxylic acid ester, activated carboxylic acid ester, amide (-NHC(O)), aldehyde (-CH(O)), or thiol (-SH) group) of a polymer or a second active compound to form a second bond.

[0028] A "labile bond" refers to a bond that is readily cleaved in vivo such that the link between the IRM moiety and the polymer moiety or second active moiety is disrupted, thereby releasing a free, active IRM compound of Formula (II) or Formula (III) that is capable of contacting immune cells and inducing an immune response.

[0029] As used herein, the term "ester group" used to describe "L1" includes orientations in which the carbonyl portion of the ester group (-C(O)-) is bonded closer to the triazole ring than the oxygen portion of the ester group (-O-), as well as orientations in which the oxygen portion of the ester group (-O-) is bonded closer to the triazole ring than the carbonyl portion of the ester group (-C(O)-).

[0030] As used herein, the term "amide group (-NH-C(O)-)" used to describe "L1" includes an orientation in which the nitrogen portion of the amide group (-NH-) is bonded closer to the triazole ring than the carbonyl portion of the amide group (-(C(O)-), as well as an orientation in which the carbonyl portion of the amide group (-C(O)-) is bonded closer to the triazole ring than the nitrogen portion of the amide group (-NH-).

[0031] As used herein, the term "carbamate group (-OC(O)-NH-)" used to describe "L1" includes an orientation in which the oxygen portion of the carbamate group (-O-) is bonded closer to the triazole ring than the nitrogen portion of the carbamate group (-NH-), as well as an orientation in which the nitrogen portion of the carbamate group (-NH-) is bonded closer to the triazole ring than the oxygen portion of the carbamate group (-O-).

[0032] The term "antigen" refers to any substance that can be bound by an antibody in a manner that is immunospecific to a degree for a humoral immune response. As used herein, "antigen" also refers to any substance that can be bound by an antigen-presenting cell for a cell-mediated immune response. The antigens described herein can elicit antigenic activities, including, for example, any one or more of the following: production of antibodies specific to the antigen by B cells, maturation of immune cells, cytokine production by immune cells, and generation of antigen-presenting cells that present the antigen. Antigens useful for practicing the present disclosure include those that have very weak activity and / or no therapeutic benefit in the absence of an adjuvant (such as, for example, a compound or conjugate of the present disclosure). Exemplary antigens include peptides, polypeptides, proteins, glycoproteins, lipids, glycolipids, polysaccharides, carbohydrates, polynucleotides, prions, oligonucleotides (e.g., CpG), DNA, RNA, viruses, bacteria, fungi, parasites, toxins, or toxoids.

[0033] "Salts" of a compound or conjugate include pharma- ceutical acceptable salts such as those described in Berge, Stephen M., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 1977, 66, pages 1-19. For example, salts can be prepared by reacting a free base compound (i.e., not in salt form) with an inorganic or organic acid, such as, for example, hydrochloric acid, sulfuric acid, hydrobromic acid, methanesulfonic acid, ethanesulfonic acid, malic acid, maleic acid, acetic acid, trifluoroacetic acid, para-toluenesulfonic acid, salicylic acid, succinic acid, tartaric acid, citric acid, pamoic acid, xinafoic acid, oxalic acid, and the like.

[0034] As used herein, a "pharmaceutically acceptable carrier" includes a carrier capable of delivering a therapeutically or prophylactically effective amount of one or more of the disclosed compounds, salts, or conjugates to a subject by a selected route of administration, and which is generally tolerated by the subject and has an acceptable toxicity profile (preferably, minimal to no toxicity at the administered dose). Some suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences, 18 th Edition (1990), Mack Publishing Co. and can be readily selected by one of ordinary skill in the art. Exemplary pharma- ceutically acceptable salts include hydrochloride and dihydrochloride salts.

[0035] An "effective amount" (including "therapeutically effective amount" and "prophylactically effective amount") is defined as the amount of a compound, salt, or conjugate sufficient to induce a therapeutic or prophylactic effect, such as cytokine induction, immunomodulation, anti-tumor activity, and / or anti-viral activity. Depending on the disease or condition, the desired cytokine profile, and / or the tolerable level of side effects, the effective amount may vary. For example, a small amount of a highly active compound or salt, or a large amount of a less active compound or salt, may be used to avoid undesirable side effects.

[0036] "Treate" and "treatment" and variations thereof refer to any degree of reducing, limiting the progression of, ameliorating, preventing, or eliminating the symptoms or signs associated with a condition. "Ameliorate" and "ameliorating" refer to any decrease in the degree, severity, frequency, and / or likelihood of a symptom or clinical feature of a particular disease or condition.

[0037] As used herein, the term "comprises" and variations thereof do not have a limiting meaning when these terms appear in the specification and claims. It will be understood that such terms mean to include the recited step or element or group of steps or elements, but not to exclude any other step or element or group of steps or elements. "Consisting of" means to include and be limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and other elements may not be present. "Consisting essentially of" means to include any elements recited after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or action of the recited elements as specified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are optional and may be present or absent depending on whether they materially affect the activity or action of the recited elements. Any element or combination of elements recited herein with open-ended language (e.g., "comprising" and its derivatives) shall be deemed to be further recited with closed-ended language (e.g., "consisting of" and its derivatives) as well as partially closed-ended language (e.g., "consisting essentially of" and its derivatives).

[0038] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may provide certain benefits, under particular circumstances. However, other claims may also be preferred, under the same or other circumstances. Moreover, the recitation of one or more preferred claims does not imply that the other claims are not useful, nor is it intended to exclude the other claims from the scope of the present disclosure.

[0039] In this application, terms such as "a," "an," and "the" are not intended to refer to a singular entity only, but include a general class of which a particular example may be used for illustration. The terms "a," "an," and "the" are used interchangeably with the term "at least one." The phrases "at least one of" and "including at least one of," followed by a list, refer to any one of the items in the list, and any combination of two or more items in the list.

[0040] As used herein, the term "or" is generally used in its ordinary sense, including "and / or," unless the content clearly dictates otherwise.

[0041] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0042] Also, all numbers herein are intended to be modified by the term "about," and in certain embodiments, preferably by the term "exactly." When used herein in connection with a measured quantity, the term "about" refers to the variation in the measured quantity that would be expected by a person of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device used. As used herein, a number "up to" (e.g., up to 50) is inclusive of that number (e.g., 50).

[0043] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range as well as the endpoints (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0044] As used herein, the term "ambient temperature" or "room temperature" refers to a temperature between 20°C and 25°C or between 22°C and 25°C.

[0045] The terms "in the range" or "within a range" (and similar descriptions) include the endpoints of the stated range.

[0046] Grouping of alternative elements or embodiments disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found therein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed herein to include the group as modified and thus to satisfy the written description of all Markush groups used in the appended claims.

[0047] When a group is present more than once in the formula described herein, each group is "independently" selected, whether or not it is specifically described.For example, when there are multiple R groups in the formula, each R group is independently selected.For example, when multiple y's are used in the formula to specify the integers in the formula, each y is independently selected from the integer range described.

[0048] Throughout this specification, references to "one embodiment," "an embodiment," "particular embodiments," or "some embodiments" or the like mean that the particular features, configurations, compositions, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0049] The above summary of the disclosure is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly illustrates exemplary embodiments. In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. Thus, the scope of the disclosure should not be limited to the specific exemplary structures described herein, but rather extends to at least the structures described by the language of the claims, and equivalents of those structures. Any of the elements expressly recited herein as alternatives may be expressly included in or excluded from the claims, in any combination as appropriate. Various theories and possible mechanisms have been described herein, but in no event should such discussion serve to limit the patentable subject matter. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] IRM compounds The present disclosure provides a compound of formula (I) (or a salt thereof):

[0051] [ka]

[0052] In certain embodiments of Formula (I), n is an integer of 0 or 1. In certain embodiments of Formula (I), n is 0.

[0053] In certain embodiments of formula (I), R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl. In certain embodiments of formula (I), R is selected from the group consisting of halogen, hydroxyl, -(C1-C7)alkyl, -(C1-C7)alkoxy, and -C(O)-O-(C1-C5)alkyl. In certain embodiments of formula (I), R is selected from the group consisting of halogen, hydroxyl, alkyl, and alkoxy. In certain embodiments of formula (I), R is selected from the group consisting of hydroxyl, F, and Cl. In certain embodiments of formula (I), R is selected from the group consisting of F and Cl.

[0054] In certain embodiments of formula (I), R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl. In certain embodiments of formula (I), R1 is -CH2OCH3 or -CH2OCH2CH3. In certain embodiments of formula (I), R1 is -CH2OCH2CH3.

[0055] In certain embodiments of formula (I), R2 is a -(C2-C18) alkylene group, optionally including one or more catenary non-peroxidized -O- atoms. In certain embodiments of formula (I), R2 is a -(C2-C12) alkylene group, optionally including one or more catenary non-peroxidized -O- atoms. In certain embodiments of formula (I), R2 is a -(C2-C10) alkylene group, optionally including one or more catenary non-peroxidized -O- atoms. In certain embodiments of formula (I), R2 is a -(C2-C8) alkylene group, optionally including one or more catenary non-peroxidized -O- atoms. In certain embodiments of formula (I), R2 is a -(C2-C6) alkylene group, optionally including one or more catenary non-peroxidized -O- atoms. In certain embodiments of formula (I), R2 is a -(C2-C3) alkylene group, optionally containing one catenary -O- atom. In certain embodiments of formula (I), R2 is -CH2CH2-, -CH2CH2-O-CH2-, or -(CH2CH2-O) x-CH2-, where x is an integer from 1 to 8. In certain embodiments of Formula (I), R2 is -CH2CH2-.

[0056] In certain embodiments of Formula (I) (or a salt thereof), the compound is the following enantiomer of Formula (IA) (or a salt thereof):

[0057] [ka] wherein n, R, R1, and R2 are as described for formula (I).

[0058] The present disclosure provides a compound of formula (II) (or a salt thereof):

[0059] [ka]

[0060] In disclosing formula (II), n, R, R1, and R2 are as described herein for formula (I).

[0061] In certain embodiments of formula (II), n is an integer of 0 or 1. R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl, R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl, R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms, and R3 is selected from the group consisting of alkyl, aryl, and aralkyl, the alkyl portion of the alkyl or aralkyl optionally containing one or more catenary non-peroxidized -O-atoms, the alkyl portion of the alkyl or aralkyl optionally being substituted with a functional group selected from the group consisting of amine (-NH2), carboxyl (-C(O)OH), hydroxyl (-OH), and thiol (-SH), and the aryl portion of the aryl or aralkyl optionally being substituted with halogen, hydroxyl, alkyl, alkoxy, or a combination thereof.

[0062] In certain embodiments of formula (II), R3 is selected from the group consisting of -(C1-C10)alkyl, -(C6-C20)aryl, and -(C6-C20)ar-(C1-C10)alkyl, where the alkyl portion of the alkyl or aralkyl optionally contains one or more catenary non-peroxidized -O-atoms, the alkyl portion of the alkyl or aralkyl is optionally substituted with a functional group selected from the group consisting of amine, carboxyl, hydroxyl, and thiol, and the aryl portion of the aryl or aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or combinations thereof.

[0063] In certain embodiments of formula (II), R3 is selected from the group consisting of -(C1-C8)alkyl, -(C6-C18)aryl, and -(C6-C18)ar-(C1-C8)alkyl, where the alkyl portion of the alkyl or aralkyl optionally contains one or more catenary non-peroxidized -O-atoms, the alkyl portion of the alkyl or aralkyl is optionally substituted with a functional group selected from the group consisting of amine, carboxyl, hydroxyl, and thiol, and the aryl portion of the aryl or aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or combinations thereof.

[0064] In certain embodiments of formula (II), R3 is selected from the group consisting of -(C1-C6)alkyl, -(C6-C16)aryl, and -(C6-C16)ar-(C1-C6)alkyl, where the alkyl portion of the alkyl or aralkyl optionally contains one or more catenary non-peroxidized -O-atoms, the alkyl portion of the alkyl or aralkyl is optionally substituted with a functional group selected from the group consisting of amine, carboxyl, hydroxyl, and thiol, and the aryl portion of the aryl or aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or combinations thereof.

[0065] In certain embodiments of formula (II), R3 is selected from the group consisting of -(C1-C3)alkyl, -(C6-C12)aryl, and -(C6-C12)ar-(C1-C3)alkyl, where the alkyl portion of the alkyl or aralkyl optionally contains one or more catenary non-peroxidized -O-atoms. The alkyl portion of the alkyl or aralkyl is optionally substituted with a functional group selected from the group consisting of amine, carboxyl, hydroxyl, and thiol, and the aryl portion of the aryl or aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or combinations thereof.

[0066] In certain embodiments of Formula (II), R3 is

[0067] [ka] and wherein the alkyl portion optionally contains one or more catenary non-peroxidized -O- atoms, the alkyl portion is optionally substituted with a functional group selected from the group consisting of amine, carboxyl, hydroxyl, and thiol, and the aryl portion is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or combinations thereof.

[0068] In certain embodiments of Formula (II), R3 is a phenyl group or a benzyl group.

[0069] In certain embodiments of Formula (II) (or a salt thereof), the compound is an enantiomer of Formula (II-A) (or a salt thereof):

[0070] [ka] wherein n, R, R1, R2, and R3 are as described for formula (II).

[0071] The present disclosure provides a compound of formula (III) (or a salt thereof):

[0072] [ka]

[0073] In the disclosure of formula (III), n, R, R1, and R2 are as described herein for formula (I), and R4 is

[0074] [ka] is a triazole ring selected from the group consisting of During the ceremony, `` * " indicates a covalent bond attached to R2, and " ** " indicates a covalent bond attached to L1.

[0075] In certain embodiments of formula (III), L1 is an alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms, amine groups (-NH-), ester groups, amide groups (-NH-C(O)-), disulfide groups (-SS-), carbonyl groups (-C(O)-), carbonate groups (-OC(O)-O-), carbamate groups (-OC(O)-NH-), or combinations thereof.

[0076] In certain embodiments of formula (III), m is an integer of 0 or 1.

[0077] In certain embodiments of Formula (III), m is 1.

[0078] In certain embodiments of formula (III), L1 is a -(C1-C100) alkylene group, optionally including one or more catenary non-peroxidized -O- atoms, amine groups, ester groups, amide groups, disulfide groups, carbonyl groups, carbonate groups, carbamate groups, or combinations thereof. In certain embodiments of formula (III), L1 is a -(C1-C100) alkylene group including a -CH2-CH2-O- moiety, a -C(O)O- moiety, a -C(O)- moiety, a -OC(O)O- moiety, a -NH-C(O)- moiety, a -SS- moiety, or combinations thereof.

[0079] In a particular embodiment of formula (III), L1 is -(CH2) y -; -(CH2CH2O) y -CH2-; -(CH2CH2O) y -CH2CH2-; -CH2CH2-S-S-CH2CH2-; -(CH2CH2O) y -CH2CH2-NH-C(O)-CH2CH2-; -(CH2CH2O) y -CH2(CH2) y -NH-C(O)-CH2CH2-; -(CH2CH2O) y -CH2(CH2) y -NH-C(O)-CH2(CH2) y -; -(CH2) y -(CH2CH2O) y -CH2CH2-NH-C(O)-CH2CH2-; -(CH2) y -(CH2CH2O) y -CH2(CH2) y -NH-C(O)-CH2CH2-; -(CH2) y -(CH2CH2O) y -CH2(CH2) y -NH-C(O)-CH2(CH2) y -; -(CH2) y -O-(CH2) y -; -(CH2) y -(OCH2CH2) y -O(CH2) y -; -(CH2) y -(OCH2CH2) y -; -CH2(CH2) y -S-S-CH2(CH2) y -; -CH2OCH2(CH2) y -S-S-CH2(CH2) y -; -(CH2CH2O) y -CH2CH2-S-S-CH2(CH2) y-;-(CH2CH2O) y -CH2CH2-SS-CH2(CH2) y -; -(CH2CH2O) y -CH2(CH2) y -SS-CH2(CH2) y -;-(CH2) y -(CH2CH2O) y -CH2CH2-SS-CH2(CH2) y -; -(CH2) y -(CH2CH2O) y -CH2(CH2) y -SS-CH2(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0080] In certain embodiments of formula (III), L1 is -(CH2) y -;-(CH2CH2O) y -CH2-;-(CH2CH2O) y -CH2CH2-;-CH2CH2-SS-CH2CH2-; and -(CH2CH2O) y -CH2CH2-NH-C(O)-CH2CH2-, where each y is independently selected as an integer from 1 to 36.

[0081] In certain embodiments of formula (III), L1 is -CH2OCH2-; -CH2OCH2CH2-; -CH2CH2OCH2CH2-; -(CH2) y -;-CH2(OCH2CH2) y -OCH2-; -CH2(OCH2CH2) y and -CH2OCH2CH2-SS-CH2CH2-, wherein each y is independently selected as an integer from 1 to 36.

[0082] In certain embodiments of formula (III), L1 is -(CH2) y-O-(CH2) y -;-(CH2) y -;-(CH2) y -(OCH2CH2) y -O(CH2) y -;-(CH2) y -(OCH2CH2) y -; -CH2(CH2) y -SS-CH2(CH2) y -; and -CH2OCH2(CH2) y -SS-CH2(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0083] In certain embodiments of formula (III), L1 is -C(O)CH2CH2-; -C(O)CH2CH2C(O)NHCH2CH2(OCH2CH2). y -;-C(O)CH2CH2NHC(O)CH2CH2-; and -C(O)CH2CH2O(CH2CH2O) y CH2CH2NHC(O)CH2CH2-, where each y is independently selected as an integer from 1 to 36.

[0084] In certain embodiments of formula (III), L1 is -C(O)(CH2) y -;-C(O)CH2(CH2) y -;-C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y -; -C(O)CH2(CH2) y NHC(O)CH2(CH2) y - and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y NHC(O)CH2(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0085] In certain embodiments of formula (III), L1 is -C(O)CH2CH2(OCH2CH2) y -; and -C(O)NHCH2CH2CH2CH2-, where y is an integer from 1 to 36.

[0086] In certain embodiments of formula (III), L1 is -C(O)CH2(CH2) y (OCH2CH2) y -;-C(O)CH2(CH2) y (OCH2CH2) y O(CH2) y -;-C(O)NHCH2(CH2) y -; -C(O)NHCH2(CH2) y (OCH2CH2) y -; and -C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0087] In certain embodiments of Formula (III), each y is independently selected as an integer from 1 to 20 or from 2 to 20. In certain embodiments of Formula (III), each y is independently selected as an integer from 1 to 15 or from 2 to 15. In certain embodiments of Formula (III), each y is independently selected as an integer from 1 to 12 or from 2 to 12. In certain embodiments of Formula (III), each y is independently selected as an integer from 1 to 10 or from 2 to 10. In certain embodiments of Formula (III), each y is independently selected as an integer from 1 to 8 or from 2 to 8.

[0088] In certain embodiments of formula (III), Q is a functional group for binding to a polymeric portion or a second active moiety. In certain embodiments of formula (III), Q is an activated carboxylate or carbonate, an amine (-NH), an aminooxy (-O-NH), a carboxylic acid (-C(O)OH), an acyl hydrazide (-C(O)-NHNH), a hydroxyl (-OH), an aldehyde (-C(O)H), or a maleimide.

[0089] [ka] It is.

[0090] In this context, "activated" means that the carboxylic acid or carbonic acid has been modified to make it particularly susceptible to nucleophilic attack. With respect to activated carboxylic acid esters, the organic moiety on the alcohol component of the carboxylic acid ester is often electron-withdrawing, such that the carbonyl carbon of the ester has enhanced electrophilic properties. In one embodiment, an activated carboxylic acid ester is a carboxylic acid ester that is susceptible to reacting with a nucleophilic amine compound to form a carboxylic acid amide.

[0091] In certain embodiments of formula (III), Q is an amine (-NH), an aminooxy (-O-NH), a carboxylic acid (-C(O)OH), an acyl hydrazide (-C(O)-NHNH), a hydroxyl (-OH), an aldehyde (-C(O)H), N-Hydroxysuccinimide ester

[0092] [ka] N-Hydroxysuccinimide Carbonate

[0093] [ka] Maleimides

[0094] [ka] and pentafluorophenyl ester

[0095] [ka] is selected from the group consisting of:

[0096] In certain embodiments of formula (III), -triazole ring-(L1) of formula (III) m The -Q moiety is derived from a cycloaddition reaction that occurs by reacting a compound of formula (I) or (IA) with a heterobifunctional bridging compound that includes an alkynyl moiety, organic bridges of various lengths and compositions covalently bonded to the alkynyl moiety, and a functional group Q covalently bonded to the organic bridge (i.e., a heterobifunctional bridging compound having the general formula alkynyl moiety-organic bridge-Q). The azide moiety of formula (I) or (IA) reacts with the alkynyl portion of the heterobifunctional bridging compound to form a triazole ring. The alkynyl moiety may be directly bonded to the organic bridge portion or may be part of a ring or ring system that is bonded to the organic bridge. In certain embodiments, the functional group Q in the heterobifunctional cross-linking compound is an activated carboxylate or carbonate ester, an amine (-NH2), an aminooxy (-O-NH2), a carboxylic acid (-C(O)OH), an acylhydrazide (-C(O)-NHNH2), a hydroxyl (-OH), an aldehyde (-C(O)H), or a maleimide. In certain embodiments, the functional group Q in the heterobifunctional cross-linking compound is an N-hydroxysuccinimide ester, a pentafluorophenyl ester, a carboxylic acid, an amine, an aminooxy, a hydrazide, a maleimide, a hydroxyl, or an aldehyde.

[0097] In certain embodiments, the compound or salt of formula (III) is a compound of formula (I) or (IA) and a heterobifunctional bridge compound selected from the group consisting of:

[0098] [ka] 2-O-Propargyl-2-hydroxyacetic acid NHS (N-hydroxysuccinimide) ester;

[0099] [ka] 3-O-Propargyl-3-hydroxypropionic acid-NHS ester;

[0100] [ka] Propargyl-PEG y -2-hydroxyacetic acid-NHS ester;

[0101] [ka] Propargyl-PEG y -3-hydroxypropionic acid-NHS ester;

[0102] [ka] 2-O-Propargyl-2-hydroxyacetic acid pentafluorophenyl (PFP) ester;

[0103] [ka] Propargyl-PEG y -2-Hydroxyacetic acid pentafluorophenyl ester;

[0104] [ka] 2-O-Propargyl-2-hydroxyacetic acid;

[0105] [ka] Propargyl-PEG y -2-hydroxyacetic acid;

[0106] [ka] 3-O-Propargyl-3-hydroxypropionic acid;

[0107] [ka] Propargyl-PEG y -3-hydroxypropionic acid;

[0108] [ka] Propargyl-PEG y -amines;

[0109] [ka] Propargyl-PEG y -oxyamine;

[0110] [ka] Propargyl-PEG y -3-Hydroxypropionic acid hydrazide;

[0111] [ka] Propargyl-PEG y -3-hydroxyethylmaleimide;

[0112] [ka] Propargyl-PEG y -alcohol;

[0113] [ka] Propargyl-PEG y -2-Hydroxyacetaldehyde;

[0114] [ka] Propargyl-OCH2CH2-SS-propionic acid-NHS ester;

[0115] [ka] Propargyl-OCH2CH2-SS-propionic acid pentafluorophenyl ester;

[0116] [ka] Propargyl-OCH2CH2-SS-propionic acid;

[0117] [ka] Propargyl-OCH2CH2-SS-ethanol;

[0118] [ka] Propargyl-OCH2CH2-SS-ethylamine;

[0119] [ka] Alkynoic acid;

[0120] [ka] Propargyl-PEG y -3-Hydroxypropionic acid hydrazide;

[0121] [ka] Propargyl-PEG y -2-Hydroxyacetaldehyde;

[0122] [ka] endo-BCN-p-nitrophenyl carbonate;

[0123] [ka] Endo-BCN-PEG y -3-hydroxypropionic acid-NHS ester;

[0124] [ka] Endo-BCN-PEG y -3-Hydroxypropionic acid pentafluorophenyl ester;

[0125] [ka] endo-BCN-pentanoic acid;

[0126] [ka] Endo-BCN-PEG y -3-hydroxypropionic acid;

[0127] [ka] Endo-BCN-PEG y -alcohol;

[0128] [ka] Endo-BCN-PEG y -amines;

[0129] [ka] DBCO-succinic acid;

[0130] [ka] DBCO-succinic acid-NHS ester;

[0131] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -3-hydroxypropionic acid-NHS ester;

[0132] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -3-Hydroxypropionic acid pentafluorophenyl ester;

[0133] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -3-hydroxypropionic acid;

[0134] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -amines;

[0135] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -alcohol;

[0136] [ka] DBCO-amine;

[0137] [ka] DBCO-NHCOCH2CH2-PEG y -3-hydroxypropionic acid-NHS ester;

[0138] [ka] DBCO-NHCOCH2CH2-PEG y -3-hydroxypropionic acid;

[0139] [ka] DBCO-NHCOCH2CH2-PEG y -amines;

[0140] [ka] DBCO-NHCOCH2CH2-PEG y -alcohol;

[0141] [ka] DBCO-maleimide;

[0142] [ka] DBCO-PEG y -maleimide; and

[0143] [ka] DBCO-NHCOCH2CH2-PEG y - can be prepared by reaction with an alcohol, In the formula, y is an integer from 1 to 36.

[0144] In certain embodiments, a compound or salt of formula (III) can be prepared from the reaction of a compound of formula (I) or (IA) with a heterobifunctional bridging compound selected from the group consisting of:

[0145] [ka] Propargyl-PEG y -2-hydroxyacetic acid-NHS ester (y=3 or 4);

[0146] [ka] Propargyl-PEG y -3-hydroxypropionic acid-NHS ester (y=2, 3, 4, 5, 6, or 7);

[0147] [ka] Propargyl-PEG y -2-hydroxyacetic acid (y=3 or 4);

[0148] [ka] Propargyl-PEG y -3-hydroxypropionic acid (y=1, 2, 3, 4, 5, 6, 7, 9, 12, or 13);

[0149] [ka] Propargyl-PEG y -amine (y=2, 3, 4, 5, 6, 8, 9, 10, 12, or 14);

[0150] [ka] Propargyl-PEG y -oxyamines (y=1, 3, or 4);

[0151] [ka] Propargyl-PEG y -3-hydroxyethylmaleimide (y=3);

[0152] [ka] Propargyl-PEG y- alcohol (y=1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, or 17);

[0153] [ka] Alkynoic acids (y=10, 12, or 14);

[0154] [ka] Endo-BCN-PEG y -3-hydroxypropionic acid-NHS ester (y=1, 2, 3, 7, or 11);

[0155] [ka] Endo-BCN-PEG y -3-hydroxypropionic acid pentafluorophenyl ester (y=1 or 3);

[0156] [ka] Endo-BCN-PEG y -3-hydroxypropionic acid (y=1, 2, 3, 7, or 11);

[0157] [ka] Endo-BCN-PEG y -alcohol(y=2);

[0158] [ka] Endo-BCN-PEG y -Amin(y=4);

[0159] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y-3-hydroxypropionic acid-NHS ester (y=0, 3, 4, or 11);

[0160] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -3-hydroxypropionic acid pentafluorophenyl ester (y=1, 3, or 7);

[0161] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -3-hydroxypropionic acid (y=0, 1, 3, 4, 7, or 11);

[0162] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -amine (y=1, 2, 4, 6, 9, or 23);

[0163] [ka] DBCO-Succinic acid-NHCH2CH2-PEG y -alcohol (y=0, 3, or 7);

[0164] [ka] DBCO-NHCOCH2CH2-PEG y -3-hydroxypropionic acid-NHS ester (y=3, 4, or 12);

[0165] [ka] DBCO-NHCOCH2CH2-PEG y -3-hydroxypropionic acid (y=3, 4, or 12);

[0166] [ka] DBCO-NHCOCH2CH2-PEG y -amine (y=4); and

[0167] [ka] DBCO-PEG y -maleimide (y=3).

[0168] In certain embodiments of Formula (III) (or a salt thereof), the compound is an enantiomer of Formula (III-A) (or a salt thereof):

[0169] [ka] wherein n, m, R, R1, R2, R4, L1, and Q are as described for formula (III).

[0170] The present disclosure provides compounds (or salts thereof) of formulae (III-B) and (III-C):

[0171] [ka]

[0172] In the disclosure of formula (III-B) and formula (III-C), n, m, R, R1, R2, L1, and Q are as described herein for formula (III). Formula (III-C) is an enantiomer of formula (III-B) (or a salt thereof).

[0173] In certain embodiments of Formula (III-B) and Formula (III-C), L1 is -CH2OCH2-; -CH2OCH2CH2-; -CH2CH2OCH2CH2-; -(CH2) y -; -CH2(OCH2CH2) y-OCH2-;-CH2(OCH2CH2) y -OCH2CH2-;-CH2CH2-SS-CH2CH2-; and -CH2OCH2CH2-SS-CH2CH2-, where each y is independently selected as an integer from 1 to 36.

[0174] In certain embodiments of Formula (III-B) and Formula (III-C), L1 is -(CH2) y -O-(CH2) y -;-(CH2) y -;-(CH2) y -(OCH2CH2) y -O(CH2) y -; -(CH2) y (OCH2CH2) y -;-CH2(CH2) y -SS-CH2(CH2) y -; and -CH2OCH2(CH2) y -SS-CH2(CH2) y - selected from the group consisting of wherein each y is independently selected as an integer from 1 to 36.

[0175] The present disclosure provides compounds (or salts thereof) of formulae (III-D) and (III-E):

[0176] [ka]

[0177] In the disclosure of formula (III-D) and formula (III-E), n, m, R, R1, R2, L1 and Q are as described herein for formula (III). Formula (III-E) is an enantiomer of formula (III-D) (or a salt thereof).

[0178] In certain embodiments of formula (III-D) and formula (III-E), L1 is -C(O)CH2CH2-; -C(O)CH2CH2C(O)NHCH2CH2(OCH2CH2)y -; -C(O)CH2CH2NHC(O)CH2CH2-; and -C(O)CH2CH2O(CH2CH2O) y CH2CH2NHC(O)CH2CH2-; wherein each y is independently selected as an integer from 1 to 36.

[0179] In certain embodiments of Formula (III-D) and Formula (III-E), L1 is -C(O)(CH2) y -;-C(O)CH2(CH2) y -; -C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y -;-C(O)CH2(CH2) y NHC(O)CH2(CH2) y - and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y NHC(O)CH2(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0180] The present disclosure provides compounds (or salts thereof) of formulae (III-F) and (III-G):

[0181] [ka]

[0182] In the disclosure of formula (III-F) and formula (III-G), n, m, R, R1, R2, L1 and Q are as described herein for formula (III). Formula (III-G) is an enantiomer of formula III-F (or a salt thereof).

[0183] In certain embodiments of Formula (III-F) and Formula (III-G), L1 is -C(O)CH2CH2(OCH2CH2) y-; and -C(O)NHCH2CH2CH2CH2-, where y is an integer from 1 to 36.

[0184] In certain embodiments of Formula (III-F) and Formula (III-G), L1 is -C(O)CH2(CH2). y (OCH2CH2) y -; -C(O)CH2(CH2) y (OCH2CH2) y O(CH2) y -;-C(O)NHCH2(CH2) y -;-C(O)NHCH2(CH2) y (OCH2CH2) y -; and -C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0185] Preparation of IRM compounds The compounds of the present disclosure can be synthesized by synthetic routes that include processes analogous to those well known in the chemical arts, especially in light of the description contained herein.

[0186] The compounds of the present disclosure can be prepared, for example, according to Reaction Schemes I, II, III, IV, V, and VI, where R, R1, R2, R3, L1, Q, m, and n are as described above. In step (1) of Reaction Scheme I, (S)-2-(tert-butoxycarbonylamino)-3-(4-tert-butoxyphenyl)propanoic acid (a diprotected version of tyrosine) of formula (V) can be reacted with isobutyl chloroformate and N-methylmorpholine, followed by reaction with sodium borohydride in step (2) to obtain an alcohol of formula (VI). Alkylation of the alcohol of formula (VI) with an alkylating agent, such as a dialkyl sulfate or an alkyl halide, in step (3) can obtain an alkyl ether of formula (VII). In step (4) of Reaction Scheme I, the protecting group can be removed from the compound of formula (VII) using concentrated hydrochloric acid in ethanol with heating to obtain a compound of formula (VIII). In formulas (VII) and (VIII), "C 1-3 "Alkyl" includes -CH3, -CH2CH3, -CH2CH2CH3, and -CH(CH3)2.

[0187] Reaction Scheme I

[0188] [ka] In Reaction Scheme II, 4-chloro-3-nitroquinoline of formula (IX) is reacted with a compound of formula (VIII) in step (5) to give 3-nitroquinolin-4-amine of formula (X). The reaction can be carried out by adding the amine of formula (VIII) to a solution of formula (IX) in a suitable solvent, such as dichloromethane, in the presence of a tertiary amine, such as triethylamine. The 4-chloro-3-nitroquinoline compound of formula (IX) and substituted analogs are known compounds (see, for example, U.S. Pat. Nos. 3,700,674 (Diehl et al.), 5,389,640 (Gerster et al.), 6,110,929 (Gerster et al.), 7,923,560 (Wightman et al.), and references cited therein). In many cases, substituted analogs of formula (IX) (e.g., where n=1 and R is a halogen, alkoxy, or benzyloxy group) can be prepared starting from commercially available substituted anilines.

[0189] In step (6) of Reaction Scheme II, the nitro group of formula (X) can be reduced to an amino group. The reduction can be carried out in a pressure bottle using hydrogen, catalytic amounts of palladium or platinum on carbon, and a solvent such as methanol, acetonitrile, toluene, or combinations thereof. The reaction can be carried out using a Parr apparatus. Alternatively, the desired reduction can be accomplished using sodium dithionite and catalytic dioctyl viologen in a biphasic dichloromethane-water solvent system.

[0190] In step (7) of Reaction Scheme II, a 3,4-diamine of formula (XI) can be reacted with triethyl orthoformate to provide a 1H-imidazo[4,5-c]quinoline of formula (XII). The reaction can be carried out in an inert solvent such as propyl acetate or toluene. Optionally, a catalyst such as pyridine hydrochloride can be included.

[0191] In step (8) of Reaction Scheme II, the phenoxy group of the imidazoquinoline of formula (XII) can be converted to an ether of formula (XIII) using conventional synthetic methods. For example, the compound of formula (XII) can be reacted with a suitable alkylating agent of formula LG-R2-O-PG and a base (e.g., cesium carbonate) in an inert solvent (e.g., N,N-dimethylformamide), where LG is a leaving group, PG is an alcohol protecting group, and R2 is as defined above. Suitable leaving groups include, but are not limited to, bromide, iodide, methanesulfonyloxy, and p-toluenesulfonyloxy. For example, the compound of formula (XII) can be alkylated with (2-bromoethoxy)(tert-butyl)dimethylsilane to provide a compound of formula (XIII), where R2 is -CH2-CH2- and PG is tert-butyldimethylsilyl.

[0192] In step (9) of Reaction Scheme II, the protecting group (PG) on a compound of formula (XIII) can be removed using conventional methods to provide a compound of formula (XIV). For example, when PG is tert-butyldimethylsilyl, reaction with a fluoride source, such as tetrabutylammonium fluoride, in a solvent, such as tetrahydrofuran, provides the deprotected alcohol of formula (XIV).

[0193] In step (10) of Reaction Scheme II, an alcohol of formula (XIV) can be converted to a sulfonate ester by treatment with a sulfonyl chloride to provide a compound of formula (XV), where Ra is alkyl, aryl, or aralkyl. Suitable sulfonylating agents include alkyl or aryl sulfonyl chlorides, such as benzenesulfonyl chloride, methanesulfonyl chloride, or para-toluenesulfonyl chloride. The reaction can be carried out in the presence of a tertiary amine, such as triethylamine, in a suitable solvent, such as dichloromethane.

[0194] In step (11) of Reaction Scheme II, the sulfonate ester on a compound of Formula (XV) can be displaced with an azide to provide a compound of Formula (XVI). The reaction can be carried out using an azide salt, such as lithium azide or sodium azide, in a solvent such as N,N-dimethylformamide.

[0195] Alternatively, the phenoxy group of the imidazoquinoline of formula (XII) can be directly converted to an ether of formula (XVI) by reaction with a suitable alkylating agent of formula LG-R2-N3 and a base (e.g., cesium carbonate) in an inert solvent (e.g., N,N-dimethylformamide). LG is a leaving group and R2 is as defined above. Suitable leaving groups include, but are not limited to, bromide, iodide, methanesulfonyloxy, and p-toluenesulfonyloxy. For example, the compound of formula (XII) can be alkylated with 2-(2-azidoethoxy)ethyl methanesulfonate to give the compound of formula (XVI), where R2 is -CHCHOCHCH-; the compound of formula (XII) can be alkylated with 1-azido-2-(2-(2-bromoethoxy)ethoxy)ethane to give the compound of formula (XVI), where R2 is -CHCHOCHCHOCHCH-; and the compound of formula (XII) can be alkylated with 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate to give the compound of formula (XVI), where R2 is CHCHOCHCHOCHCHOCHCH-. Alkylating agents of formula LG-R2-N3 are commercially available (BroadPharm, San Diego, Calif.).

[0196] In step (12) of Reaction Scheme II, the 1H-imidazo[4,5-c]quinoline of Formula (XVI) can be oxidized to provide the 1H-imidazo[4,5-c]quinoline-5N-oxide using a conventional oxidizing agent capable of forming an N-oxide. Preferably, a solution of a compound of Formula (XVI) in a suitable solvent, such as chloroform or dichloromethane, is reacted with 3-chloroperbenzoic acid at ambient temperature.

[0197] In step (13) of Reaction Scheme II, the N-oxide compound can be aminated to provide 1H-imidazo[4,5-c]quinolin-4-amines of formula (XVII). Step (13) involves reacting the N-oxide compound with a sulfonylating agent and an aminating agent in an inert solvent such as dichloromethane or chloroform. Suitable sulfonylating agents include alkyl or aryl sulfonyl chlorides, such as benzenesulfonyl chloride, methanesulfonyl chloride, or para-toluenesulfonyl chloride. Ammonium hydroxide is a suitable aminating agent. Formula (XVII) is an embodiment of formula (I).

[0198] Reaction Scheme II

[0199] [ka] In reaction scheme III, an azide group of formula (XVII) can be reacted with an alkyne of formula (XVIII) to form a triazole of formula (XIX). The reaction can be catalyzed by a copper(I) salt such as cuprous bromide or by a copper(II) salt such as copper sulfate in the presence of a reducing agent such as sodium ascorbate. The reaction can be carried out in aqueous solution or with a co-solvent such as dimethylsulfoxide or N,N-dimethylformamide to enhance the homogeneity of the reaction mixture. For example, a compound of formula (XVII) can be reacted with phenylacetylene to obtain a compound of formula (XIX), where R3 is -Ph. Other suitable alkynes are commercially available or can be prepared using conventional synthetic methods. Formula (XIX) is an embodiment of formula (II).

[0200] Reaction Scheme III

[0201] [ka] In Reaction Scheme IV, an azide group of formula (XVII) can be reacted with an alkyne of formula (XX) to form a triazole ring of formula (XXI). The reaction can be catalyzed by a copper(I) salt such as cuprous bromide or by a copper(II) salt such as copper sulfate in the presence of a reducing agent such as sodium ascorbate. The reaction can be carried out in aqueous solution or with a co-solvent such as dimethylsulfoxide or N,N-dimethylformamide to enhance the homogeneity of the reaction mixture. As an example, a compound of formula (XVII) can be reacted with 3-O-propargyl 3-hydroxypropionic acid N-succinimidyl ester to give a compound of formula (XXI), where L1 is -CHOCHCH- and Q is -C(O)O-succinimidyl, to give a compound of formula (XXI), where L1 is -CHOCHCH- and Q is -C(O)O-succinimidyl, to give a compound of formula (XXI), where L1 is -CHOCHCH- and Q is -C(O)O-succinimidyl, to give a compound of formula (XXI). to give a compound of formula (XXI), where L1 is -CH2OCH2CH2OCH2CH2OCH2CH2- and Q is -NH2, and the compound of formula (XVII) can be reacted with O-(2-(prop-2-yn-1-yloxy)ethyl)hydroxylamine to give a compound of formula (XXI), where L1 is -CH2OCH2CH2- and Q is -O-NH2. Formula (XXI) is an embodiment of formula (III-B).

[0202] Reaction Scheme IV

[0203] [ka] In Reaction Scheme V, the azide group of formula (XVII) can be reacted with dibenzocyclooctyne (DBCO) of formula (XXII) to form a triazole ring of formula (XXIII). The reaction can be carried out in aqueous solution or using a co-solvent such as dimethylsulfoxide or N,N-dimethylformamide to enhance the homogeneity of the reaction mixture. As an example, the compound of formula (XVII) can be reacted with DBCO-NHS (BroadPharm, San Diego, CA) to form a triazole ring of formula (XXIII). The compound of formula (XVII) can be reacted with DBCO-PEG1-NHS ester (BroadPharm, Catalog No. BP-24018) to give a compound of formula (XXIII) where L1 is -C(O)CH2CH2C(O)NHCH2CH2OCH2CH2- and Q is -C(O)O-succinimidyl; the compound of formula (XVII) can be reacted with DBCO-amine (BroadPharm, Catalog No. BP-22066) to give a compound of formula (XXIII) where L1 is -C(O)CH2CH2- and Q is -NH2. Formula (XXIII) is an embodiment of formula (III-D).

[0204] Reaction Scheme V

[0205] [ka] In Reaction Scheme VI, the azide group of formula (XVII) can be reacted with bicyclo[6.1.0]nonyne (BCN) of formula (XXIV) to form a triazole ring of formula (XXV). The reaction can be carried out in aqueous solution or using a co-solvent such as dimethylsulfoxide or N,N-dimethylformamide to enhance the homogeneity of the reaction mixture. As an example, the compound of formula (XVII) can be reacted with endo-BCN-PEG2-NHS ester (BroadPharm, San Diego, CA) to form a triazole ring of formula (XXV). Diego, CA, catalog number BP-24081) to provide a compound of formula (XXV) where L1 is -OC(O)NHCH2CH2OCH2CH2OCH2CH2- and Q is -C(O)O-succinimidyl, and the compound of formula (XVII) can be reacted with endo-BCN-PEG4-amine (BroadPharm, catalog number BP-24459) to provide a compound of formula (XXV) where L1 is -OC(O)NHCH2C Compounds of formula (XVII) can be reacted with endo-BCN-PEG-PFP ester (BroadPharm, catalog number BP-21541, PFP=pentafluorophenyl) to give compounds of formula (XXV), where L is -OC(O)NHCHCHOCHCHOCHCHCH- and Q is -C(O)O-pentafluorophenyl. Formula (XXV) is an embodiment of formula (III-F).

[0206] Reaction Scheme VI

[0207] [ka] In some embodiments, the compounds of formula (XXI), (XXIII), and (XXV) are precursors of the IRM-containing conjugates of formula (IV), specifically, the IRM-containing conjugates of formula (IV-A), (IV-C), (IV-E), and (IV-G). The functional group "Q" in the compound can react with a complementary reactive functional group on a polymer or a second active compound to form a covalent linking bond in the resulting IRM-containing conjugate of formula (IV). The covalent linking bond in formula (IV) (i.e., the bond between L2 and Z in formula (IV)) can be an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0208] The compounds of the present disclosure can be prepared according to Reaction Schemes I, II, III, IV, V, and VI, where the starting compound of formula (V) is replaced with similarly di-protected versions of (S)-3-amino-4-(4-hydroxyphenyl)butanoic acid and (S)-4-amino-5-(4-hydroxyphenyl)pentanoic acid. The compounds of the present disclosure can also be prepared starting from the racemic versions of the compounds of formula (V), 3-amino-4-(4-hydroxyphenyl)butanoic acid and 4-amino-5-(4-hydroxyphenyl)pentanoic acid.

[0209] It will be appreciated by those skilled in the art that in the preparation of compounds of the present disclosure, it may be necessary to protect certain functional groups while reacting other functional groups of intermediate compounds. The need for such protection will vary depending on the nature of the particular functional group and the conditions of the particular reaction step. Reviews of reactions for protecting and deprotecting functional groups can be found in PGM Wets, Greene's Protective Groups in Organic Synthesis, John Wiley & Sons, 20 New York, USA, 2014.

[0210] Conventional methods and techniques of separation and purification can be used to isolate the IRM compounds used in the compositions of the present disclosure, which may include, for example, all types of chromatography (high performance liquid chromatography (HPLC), column chromatography using common adsorbents such as silica gel, and thin layer chromatography), recrystallization, and differential (i.e., liquid-liquid) extraction techniques.

[0211] The enantiomeric excess of the compounds of the present disclosure or salts thereof can be determined using standard analytical assays such as gas chromatography or HPLC using a column with a chiral stationary phase (CSP). Suitable columns with CSP are available from Chiral Technologies, Inc. (Westchester, PA).

[0212] The enantiomeric excess (ee%) is calculated according to Equation 1.

[0213]

number

[0214] The enantiomeric excess (ee%) can be calculated from the chiral HPLC chromatogram by comparing the peak areas of the major and minor enantiomer signals according to Equation 2.

[0215]

number

[0216] IRM-containing conjugates The present disclosure provides an IRM-containing conjugate (or a salt thereof) of formula (IV) (or a salt thereof):

[0217] [ka]

[0218] In disclosing formula (IV), n, m, R, R1, R2, and R4 are as described herein for formula (III).

[0219] In certain embodiments of Formula (IV), L2 is a bridging group.

[0220] In certain embodiments of Formula (IV), Z is a polymeric moiety or a second active moiety.

[0221] In certain embodiments of formula (IV), the second active moiety is an antigen or a monoclonal antibody.

[0222] In certain embodiments of formula (IV), with or without L2, the -(L2) m The -Z moiety optionally includes a labile bond.

[0223] In certain embodiments of Formula (IV) (or a salt thereof), the compound is an enantiomer of Formula (IV-A) (or a salt thereof):

[0224] [ka] wherein n, m, R, R1, R2, R4, L2, and Z are as described for formula (IV).

[0225] In certain embodiments, the IRM-containing conjugate is of formula (IV) or formula (IV-A) (or a salt thereof), where n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; L2 is a bridging group; m is an integer of 0 or 1; Z is a polymer moiety or a second active moiety; and -(L2) of the conjugate is a bridging group. m The -Z portion, with or without L2, optionally contains a labile bond.

[0226] In certain embodiments, the IRM-containing conjugate is of formula (IV) or formula (IV-A) (or a salt thereof), where n is an integer of 0 or 1, R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl, R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl, R2 is a -(C2-C18)alkenylene group, and optionally one or more catenary non-perfluoroalkyl groups. oxidized -O-atoms, L2 is a bridging group, the bridging group comprises an alkylene group, optionally comprising one or more catenary non-peroxidized -O-atoms, ester groups, amide groups, amine groups, disulfide groups, carbonyl groups (-C(O)-), carbonate groups, carbamate groups, or combinations thereof, m is an integer of 0 or 1, Z is a polymer moiety or a second active moiety, and the -(L2) of the conjugate m The -Z portion, with or without L2, optionally contains a labile bond.

[0227] In certain embodiments of formula (IV) and formula (IV-A), the bridging group comprises an alkylene group, optionally comprising one or more catenary non-peroxidized -O- atoms. In certain embodiments of formula (IV) and formula (IV-A), the bridging group comprises an alkylene group, optionally comprising one or more catenary non-peroxidized -O- atoms, disulfide groups, or combinations thereof. In certain embodiments of formula (IV) and formula (IV-A), the bridging group comprises an alkylene group, optionally comprising one or more catenary non-peroxidized -O- atoms, amine groups, or combinations thereof. In certain embodiments of formula (IV) and formula (IV-A), the bridging group comprises an alkylene group, optionally comprising one or more catenary non-peroxidized -O- atoms, carbonyl groups (-C(O)-), or combinations thereof. In certain embodiments of Formula (IV) and Formula (IV-A), the bridging group comprises an alkylene group, and optionally comprises one or more catenary non-peroxidized -O- atoms, amide groups, or combinations thereof.

[0228] The present disclosure provides IRM-containing conjugates (or salts thereof) of formula (IV-B) and (IV-C) below:

[0229] [ka]

[0230] In the disclosure of formula (IV-B) and formula (IV-C), n, m, R, R1, R2, L2, and Z are as described herein for formula (IV). Formula (IV-C) is an enantiomer of formula (IV-B) (or a salt thereof).

[0231] In certain embodiments of Formula (IV-B) and Formula (IV-C), Z is a polymer moiety or a second active moiety and L2 is a bridging group.

[0232] In certain embodiments of Formula (IV-B) and Formula (IV-C), the bridging group is -CH2OCH2C(O)-; -CH2OCH2CH2C(O)-; -CH2CH2OCH2CH2C(O)-;-(CH2) yC(O)-;-CH2(OCH2CH2) y -OCH2C(O)-;-CH2(OCH2CH2) y -OCH2CH2C(O)-;-CH2CH2-SS-CH2CH2C(O)-;-CH2OCH2CH2-SS-CH2CH2C(O)-; -CH2CH2-SS-CH2CH2NH-;-CH2OCH2CH2-SS-CH2CH2NH-;-CH2CH2-SS-CH2CH2O-; -CH2OCH2CH2-SS-CH2CH2O-;-CH2(OCH2CH2) y O-;-CH2(OCH2CH2) y NH-;-(CH2) y O-; and -(CH2) y NH-, where each y is independently selected as an integer from 1 to 36.

[0233] In certain embodiments of Formula (IV-B) and Formula (IV-C), the bridging group is -(CH) y -O-(CH2) y C(O)-;-(CH2) y C(O)-; -(CH2) y -(OCH2CH2) y -O(CH2) y C(O)-;-(CH2) y -(OCH2CH2) y C(O)-; -CH2(CH2) y -SS-CH2(CH2) y C(O)-;-CH2OCH2(CH2) y -SS-CH2(CH2) y C(O)-; -CH2(CH2) y -SS-CH2(CH2) y O-;-CH2OCH2(CH2) y -SS-CH2(CH2) y O-; -CH2(CH2) y -SS-CH2(CH2) yNH-;-CH2OCH2(CH2) y -SS-CH2(CH2) y NH-; -(CH2) y -(OCH2CH2) y O-; and -(CH2) y -(OCH2CH2) y NH-, where each y is independently selected as an integer from 1 to 36.

[0234] The present disclosure provides IRM-containing conjugates (or salts thereof) of formula (IV-D) and (IV-E) below:

[0235] [ka]

[0236] In the disclosure of formula (IV-D) and formula (IV-E), n, m, R, R1, R2, L2, and Z are as described herein for formula (IV). Formula (IV-E) is an enantiomer of formula (IV-D) (or a salt thereof).

[0237] In certain embodiments of Formula (IV-D) and Formula (IV-E), Z is a polymer moiety or a second active moiety and L2 is a bridging group.

[0238] In certain embodiments of Formula (IV-D) and Formula (IV-E), the bridging group is C(O)CH2CH2C(O)-; -C(O)CH2CH2C(O)NHCH2CH2(OCH2CH2) y C(O)-;-C(O)CH2CH2NHC(O)CH2CH2C(O)-; -C(O)CH2CH2NHC(O)CH2CH2(OCH2CH2) y C(O)-; -C(O)CH2CH2O(CH2CH2O) y CH2CH2NHC(O)CH2CH2C(O)-; -C(O)CH2CH2C(O)NHCH2CH2(OCH2CH2) yO-; -C(O)CH2CH2C(O)NHCH2CH2(OCH2CH2) y NH-; -C(O)CH2CH2NHC(O)CH2CH2(OCH2CH2) y O-; and -C(O)CH2CH2NHC(O)CH2CH2(OCH2CH2) y NH-, where each y is independently selected as an integer from 1 to 36.

[0239] In certain embodiments of Formula (IV-D) and Formula (IV-E), the bridging group is -C(O)(CH) y C(O)-;C(O)CH2(CH2) y C(O)-; -C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y C(O)-;-C(O)CH2(CH2) y NHC(O)CH2(CH2) y C(O)-; and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y NHC(O)CH2(CH2) y C(O)—, where each y is independently selected as an integer from 1 to 36.

[0240] In certain embodiments of Formula (IV-D) and Formula (IV-E), the bridging group is -C(O)(CH) y O-;-C(O)CH2(CH2) y O-; -C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y O-; -C(O)CH2(CH2) y NHC(O)CH2CH2(OCH2CH2) y O-;-C(O)CH2(CH2) y NHC(O)CH2(CH2) y O-; and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y NHC(O)CH2(CH2) y O-, where each y is independently selected as an integer from 1 to 36.

[0241] In certain embodiments of Formula (IV-D) and Formula (IV-E), the bridging group is -C(O)(CH) y NH-;-C(O)CH2(CH2) y NH-; -C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y NH-; -C(O)CH2(CH2) y NHC(O)CH2CH2(OCH2CH2) y NH-;-C(O)CH2(CH2) y NHC(O)CH2(CH2) y NH-; and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y NHC(O)CH2(CH2) y NH-, where each y is independently selected as an integer from 1 to 36.

[0242] The present disclosure provides IRM-containing conjugates (or salts thereof) of formula (IV-F) and (IV-G) below:

[0243] [ka]

[0244] In the disclosure of formula (IV-F) and formula (IV-G), n, m, R, R1, R2, L2, and Z are as described herein for formula (IV). Formula (IV-G) is an enantiomer of formula (IV-F) (or a salt thereof).

[0245] In certain embodiments of Formula (IV-F) and Formula (IV-G), Z is a polymer moiety or a second active moiety and L2 is a bridging group.

[0246] In certain embodiments of Formula (IV-F) and Formula (IV-G), the bridging group is -C(O)CH2CH2(OCH2CH2) y C(O)-; -C(O)CH2CH2(OCH2CH2) y O-;-C(O)CH2CH2(OCH2CH2) y NH-; -C(O)NHCH2CH2(OCH2CH2) y C(O)-;-C(O)NHCH2CH2(OCH2CH2) y O-; -C(O)NHCH2CH2(OCH2CH2) y NH-;-C(O)CH2CH2CH2CH2C(O)-; and -C(O)NHCH2CH2CH2CH2C(O)-, where each y is independently selected as an integer from 1 to 36.

[0247] In certain embodiments of Formula (IV-F) and Formula (IV-G), the bridging group is -C(O)CH2(CH2) y C(O)-;-C(O)CH2(CH2) y O-; -C(O)CH2(CH2) y NH-;-C(O)CH2(CH2) y (OCH2CH2) y C(O)-;-C(O)CH2(CH2) y (OCH2CH2) y O-; -C(O)CH2(CH2) y (OCH2CH2) y NH-;-C(O)CH2(CH2) y (OCH2CH2) y O(CH2) y C(O)-; -C(O)CH2(CH2) y (OCH2CH2) y O(CH2) yO-;-C(O)CH2(CH2) y (OCH2CH2) y O(CH2) y NH-; -C(O)NHCH2(CH2) y C(O)-;-C(O)NHCH2(CH2) y O-;-C(O)NHCH2(CH2) y NH-; -C(O)NHCH2(CH2) y (OCH2CH2) y C(O)-;-C(O)NHCH2(CH2) y (OCH2CH2) y O-; -C(O)NHCH2(CH2) y (OCH2CH2) y NH-;-C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y C(O)-; -C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y O-; and -C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y is selected from the group consisting of NH-, wherein each y is independently selected as an integer from 1 to 36.

[0248] In certain embodiments of formulas (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), each y is independently selected as an integer from 1 to 20 or from 2 to 20. In certain embodiments of formulas (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), each y is independently selected as an integer from 1 to 15 or from 2 to 15. In certain embodiments of formulas (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), each y is independently selected as an integer from 1 to 12 or from 2 to 12. In certain embodiments of Formulas (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), each y is independently selected as an integer from 1 to 10 or from 2 to 10. In certain embodiments of Formulas (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), each y is independently selected as an integer from 1 to 8 or from 2 to 8.

[0249] Crosslinking groups for IRM-containing conjugates "L2" of the IRM-containing conjugates present when m=1 is a bridging group derived from a heterobifunctional bridging compound. Heterobifunctional bridging compounds (i.e., heterobifunctional crosslinkers) contain two different reactive groups at either end and organic bridges of various lengths and compositions.

[0250] More specifically, the "crosslinking group" is derived from a heterobifunctional crosslinking compound that reacts with an azide group (-N3) of the IRM compound to form a triazole and reacts with a reactive functional group (e.g., a hydroxyl (-OH), amino (-NH2), amide (-NHC(O)), carboxylic acid, carboxylate ester (-OC(O)), aldehyde (-CH(O)), or thiol (-SH) group) of a polymer or a second active compound to form a second bond.

[0251] In some embodiments, the heterobifunctional crosslinking compound has an alkyne group (preferably a terminal alkyne group) and a second functional group. The alkyne is reacted with the azide group of formula (I) to give the structure of formula (III). The second functional group ("Q") is selected so that it can react with a reactive functional group on the polymer or the second active compound to give the structure of formula (IV). Useful functional groups that are often found on the components that form the Z group of the IRM-containing conjugate of formula (IV) include, but are not limited to, (-NH2); hydroxyl (-OH), thiol (-SH); aldehyde (-CHO), carboxylic acid, and carboxylic acid ester, which can be derivatized with crosslinkers containing amine reactive groups; hydroxyl reactive groups, thiol reactive groups; aldehyde reactive groups, carboxylic acid reactive groups, and carboxylic acid ester reactive groups, respectively. Examples of carboxylic acid and carboxylic acid ester reactive groups include amines and hydroxyls. Examples of amine reactive groups include aldehydes, carboxylic acids, and carboxylic acid esters. Examples of thiol reactive groups include maleimides. Examples of aldehyde reactive groups include amines, aminooxy groups, and acylhydrazides. Examples of useful alkyne-containing heterobifunctional crosslinkers include materials available from BroadPharm Company (San Diego, CA). In some embodiments, the second active compound can be an antigen, an antibody (e.g., a monoclonal antibody), or a targeting compound (or a compound derived from an antigen, an antibody (e.g., a monoclonal antibody), or a targeting compound).

[0252] In some embodiments of the IRM-containing conjugates of formula (IV), (IV-A), (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), L2 and Z (the polymer portion or the second active moiety) are covalently linked by an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0253] In some embodiments, compounds of formula (III) (including compounds of formulae (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), and (III-G)) are precursors to IRM-containing conjugates of formula (IV) (including conjugates of formulae (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), and (IV-G)). The functional group "Q" in the compound of formula (III) can react with a complementary reactive functional group on a polymer or a second active compound to form a covalent linking bond (i.e., a bond connecting L2 to Z in the IRM-containing conjugate) in the resulting IRM-containing conjugate.

[0254] In some embodiments, the IRM-containing conjugate of formula (IV) (including conjugates of formulae (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), and (IV-G)) is prepared by reacting a compound of formula (III) (including compounds of formulae (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), and (III-G)) with a polymer or a second active compound. The functional group "Q" in the compound of formula (III) can react with a functional group on the polymer or the second active compound to form a covalent linkage in the resulting IRM-containing conjugate (i.e., a linkage connecting L2 to Z in the IRM-containing conjugate). The covalent linkage in formula (IV) can be an ether linkage, a thioether linkage, an ester linkage, an amide linkage, an imine linkage, an oxime linkage, a hydrazone linkage, or an N-acylhydrazone linkage.

[0255] The present disclosure provides a method of making an IRM-containing conjugate of formula (IV) (including a conjugate of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, the method comprising reacting a compound of formula (III) (including a compound of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof with a polymer or a second active compound to form a covalent bond, wherein a functional group Q in the compound of formula (III) reacts with a functional group on the polymer or the second active compound to form a covalent bond.

[0256] The present disclosure provides a method of making an IRM-containing conjugate of formula (IV) (including a conjugate of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, the method comprising reacting a compound of formula (III) (including a compound of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof having a functional group Q with a polymer or a second active compound having a functional group, thereby forming a covalent bond by reacting the functional group Q with a functional group of the polymer or the second active compound.

[0257] The present disclosure provides a method of making an IRM-containing conjugate of formula (IV) (including a conjugate of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, the method comprising reacting a compound of formula (III) (including a compound of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof having a functional group Q with a second active compound having a functional group to form a covalent bond by reacting the functional group Q with a functional group of the second active compound.

[0258] In some embodiments of the method of making an IRM-containing conjugate of formula (IV), the covalent bond is an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0259] In some embodiments of the method of making an IRM-containing conjugate of formula (IV), the second active compound is an antigen or an antibody.

[0260] The present disclosure provides an IRM-containing conjugate of formula (IV) (including a conjugate of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, which is prepared by reacting a compound of formula (III) (including a compound of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof with a polymer or a second active compound to form a covalent bond, and reacting a functional group Q in the compound of formula (III) with a functional group on the polymer or the second active compound to form a covalent bond.

[0261] The present disclosure provides an IRM-containing conjugate of formula (IV) (including a conjugate of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, which is prepared by reacting a compound of formula (III) (including a compound of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof with a polymer or a second active compound, and reacting a functional group Q in the compound of formula (III) with a functional group on the polymer or the second active compound to form a covalent bond, wherein the covalent bond is an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0262] The present disclosure provides an IRM-containing conjugate of formula (IV) (including conjugates of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, which is prepared by reacting a compound of formula (III) (including compounds of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof having a first functional group Q with a polymer or a second active compound having a second functional group, and reacting the first functional group Q with a second functional group of the polymer or the second active compound to form a covalent bond.

[0263] The present disclosure provides an IRM-containing conjugate of formula (IV) (including a conjugate of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, which is prepared by reacting a compound of formula (III) (including a compound of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof having a first functional group Q with a polymer or a second active compound having a second functional group, and forming a covalent bond by reacting the first functional group Q with a second functional group of the polymer or the second active compound, wherein the covalent bond is an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0264] The present disclosure provides an IRM-containing conjugate of formula (IV) (including conjugates of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, which is prepared by reacting a compound of formula (III) (including compounds of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof having a first functional group Q with a second active compound having a second functional group, thereby forming a covalent bond by reacting the first functional group Q with the second functional group of the second active compound.

[0265] The present disclosure provides an IRM-containing conjugate of formula (IV) (including conjugates of formula (IV-A), (IV-B), (IV-C), (IV-D), (IE-E), (IV-F), or (IV-G)) or a salt thereof, which is prepared by reacting a compound of formula (III) (including compounds of formula (III-A), (III-B), (III-C), (III-D), (III-E), (III-F), or (III-G)) or a salt thereof having a first functional group Q with a second active compound having a second functional group to form a covalent bond, and reacting the first functional group Q with a second functional group of the second active compound to form a covalent bond, wherein the covalent bond is an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0266] Optional labile linkages in IRM-containing conjugates In certain embodiments of the IRM-containing conjugates of formula (IV), (IV-A), (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), the -(L2) m The -Z moiety, with or without L2, may optionally include a labile bond. A "labile bond" refers to a bond that is readily cleaved in vivo such that the link between the IRM moiety and the polymer moiety or the second active moiety is broken, thereby releasing the free, active IRM compound of Formula (II) or Formula (III) that can contact an immune cell to induce an immune response, and, in certain embodiments, the second activity.

[0267] A labile bond can be any covalent bond that links a second active moiety or polymer moiety to an IRM moiety at a location on the IRM moiety that is readily cleaved in vivo, causing a substantial reduction in the immunomodulatory activity of the IRM moiety. When the labile bond is intact (i.e., when the IRM moiety is linked to a second active moiety or polymer moiety), the IRM moiety can have substantially reduced immunomodulatory activity. However, when the labile bond is cleaved, a free, active IRM moiety is released and is capable of inducing an immune response.

[0268] In some cases, the reduction in immunomodulatory activity may be due primarily to the identity and nature (e.g., size and / or steric properties) of the substitution. In these cases, the substitution may reduce the immunomodulatory activity of the IRM moiety, for example, by masking a portion of the IRM moiety that binds to a receptor and initiates a cell signaling cascade that leads to an immune response.

[0269] Examples of suitable labile bonds include, but are not limited to, amide bonds, carbamate bonds, amidine bonds, ester bonds, disulfide bonds, or amide bonds of peptide units used with or without a self-immolative spacer such as those described in the literature (Toki, BE et al., J. Org. Chem., 2002, 67, 1866-1872; Jeffrey, SC et al., J. Med. Chem., 2005, 48, 1344-1358; Sun, MMC et al., Bioconjugate Chem. 2005, 16, 1282-1290), Tsuchikama, K. and An, Z. Protein Cell 2018, 9, 33-46, as well as WO 2005 / 082023 (Genentech, Inc.).

[0270] In some embodiments, the labile bond is selected from the group consisting of an amide bond, a carbamate bond, an amidine bond, an ester bond, and a disulfide bond. In other embodiments, the labile bond is selected from the group consisting of an amide bond, a carbamate bond, and an amidine bond. In some embodiments, the labile bond is an amide bond.

[0271] Labile bonds are readily cleaved in vivo, and cleavage can occur by a variety of mechanisms, including chemical (e.g., hydrolysis at physiological pH or in the lower pH environment found in certain tumors) or enzymatic (e.g., reaction with esterases) biotransformation.

[0272] For example, a conjugate designed for use in treating a tumor may include a tumor-specific targeting moiety and a labile bond selected to be more likely, faster, or more efficiently cleaved in the tumor environment than in the systemic environment. The tumor microenvironment is often characterized by having low oxygen tension, low extracellular pH, and low glucose concentration. A labile bond that can take advantage of one or more of these microenvironmental conditions (e.g., low pH) may form a labile bond that is particularly well suited for use in a conjugate designed to treat a tumor.

[0273] Polymer portion of IRM-containing conjugate In certain embodiments of the IRM-containing conjugates of formula (IV), (IV-A), (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), Z is a polymer moiety (i.e., the IRM-containing conjugate is an IRM-polymer conjugate). The polymer moiety can be derived from a wide variety of polymers.

[0274] Suitable polymers may be based on biopolymers or naturally occurring monomers and combinations thereof. Natural biopolymers may include single or double stranded RNA or DNA composed of nucleotides (e.g., adenosine, thymidine). Natural biopolymers may be peptides composed of amino acids. An example of this is poly(lysine). Biopolymers may be polysaccharides, which may include, but are not limited to, glycogen, cellulose, and dextran. Further examples include naturally occurring polysaccharides, including alginate and chitosan. Suitable polymers may also be composed of naturally occurring small molecules (e.g., lactic acid or glycolic acid) or copolymers of the two (i.e., PLGA). Suitable preformed particles may also be based on formulations (e.g., stabilized emulsions, liposomes, and polymersomes) or inorganic salts (which may include aluminum-based salts) that form particles suitable for conjugation or ion exchange at the surface of the particle.

[0275] In certain embodiments, the polymer is selected from polyethylene glycol (PEG), glycogen, cellulose, dextran, alginate, chitosan, polylactide, and combinations thereof, hi certain embodiments, the polymer is PEG.

[0276] The following description of PEG applies to other polymers that form the polymeric portion of an IRM-containing conjugate.

[0277] The PEG moiety may be or may be derived from any suitable PEG polymer. In some cases, the resulting IRM-PEG conjugate has a molecular weight of at least 16 kilodaltons (kDa). In some embodiments, the resulting IRM-PEG conjugate may have a molecular weight of at least 20 kDa. In other embodiments, the IRM-PEG conjugate has a molecular weight of at least 30 kDa.

[0278] In many embodiments, the IRM-PEG conjugate has a molecular weight of 500 kilodaltons (kDa) or less. In some embodiments, the IRM-PEG conjugate has a molecular weight of 200 kDa or less. In certain embodiments, the IRM-PEG conjugate has a molecular weight of 100 kDa or less, and often 50 kDa or less.

[0279] Various possible PEG polymers and methods for attaching PEG polymers to IRM compounds are described, for example, in International Patent Publication No. WO 2005 / 110013 (3M).

[0280] Some PEG polymers may contain multiple sites to which an IRM moiety may be attached. Thus, an IRM-PEG conjugate may contain multiple IRM moieties. In such cases, the multiple IRM moieties may be homogeneous (i.e., derived from the same IRM compound) or heterogeneous (i.e., derived from different IRM compounds).

[0281] IRM-PEG conjugates can provide active or potentially active IRM compounds to local tissue regions and / or tissue types while reducing the overall systemic activity of the IRM. In some cases, the IRM-PEG conjugates may be of a size and chemical nature that allows for preferential deposition in tissues (e.g., specific tissue types or local tissue regions), such as solid tumors. This can occur, for example, as a result of increased vascular permeability of the tissue to the IRM-PEG conjugate and decreased lymphatic drainage of tumor tissue.

[0282] One or more IRM moieties can be attached to the PEG moiety via either covalent or non-covalent bonds. Non-covalent attachment of an IRM moiety to a macromolecular moiety includes, for example, affinity binding (e.g., avidin-biotin).

[0283] Exemplary methods for covalently linking an IRM moiety to a PEG moiety include chemical crosslinkers, such as heterobifunctional crosslinking compounds, that react to form bonds between reactive groups in the immune response modifier (such as hydroxyl, amino, amide, or thiol groups) and other reactive groups (of similar nature) in the PEG. The bonds can be, for example, peptide bonds, disulfide bonds, thioester bonds, amide bonds, thioether bonds, and the like. IRM compounds can also be covalently linked to PEG by directly reacting an IRM containing a reactive group with a polymer containing a reactive group. Methods for linking an IRM moiety to a PEG moiety are described in detail, for example, in International Patent Publication No. WO 2005 / 110013 (3M).

[0284] Regardless of the particular method used to couple the IRM and PEG moieties, the bond can be cleaved, for example, by hydrolysis or enzymatic activity, to yield the free IRM compound.

[0285] In embodiments where the IRM-PEG conjugate provides an IRM prodrug, cleavage of the link between the IRM moiety and the PEG moiety can be controlled to some extent. For example, the bond can be designed to be hydrolyzed in a particular biological microenvironment. The extracellular environment of a tumor is known to be more acidic than that of normal tissues. Thus, the IRM-PEG conjugate can be designed as a prodrug in which the bond between the IRM moiety and the PEG moiety remains intact at the extracellular pH of normal tissues (7.4-7.5) but is hydrolyzed at the extracellular pH of solid tumors (below 7.2). Thus, a pharmaceutical composition comprising an IRM-PEG conjugate and an anti-tumor antigen can be administered in the vicinity of a solid tumor. The IRM-PEG conjugate and antigen can infiltrate the tumor environment (e.g., by diffusion from a thermoresponsive gel carrier), where the IRM-PEG conjugate is cleaved to produce the free IRM. This results in co-localization of the anti-tumor antigen and the free IRM, which can be co-delivered to immune cells in the vicinity of the tumor, thereby generating an antigen-specific, and therefore tumor-specific, immune response.

[0286] In other embodiments, the linkage between the IRM moiety and the PEG moiety can be designed such that the linkage is not cleaved unless and until the conjugate reaches the endosome of an immune cell (e.g., an antigen-presenting cell such as a dendritic cell).

[0287] The size and structure of the PEG moiety can affect the kinetics of the cleavage of the bond between the IRM moiety and the PEG moiety. For example, the PEG moiety can include a polyarm PEG. The number and size of the PEG arms can affect the kinetics of enzymatic cleavage of the IRM-PEG bond, thereby releasing the free IRM. As another example, the nature of the bond between the IRM moiety and the PEG moiety can affect the rate at which the bond is cleaved by hydrolysis. Amide bonds tend to be more easily hydrolyzed than carbamate bonds.

[0288] Second Active Moiety of the IRM-Containing Conjugate In certain embodiments of formulas (IV), (IV-A), (IV-B), (IV-C), (IV-D), (IV-E), (IV-F), and (IV-G), Z is a second active moiety (SAM) (i.e., the IRM-containing conjugate is an IRM-SAM conjugate). The second active moiety can be derived from a wide variety of second active compounds.

[0289] The second active moiety can be any moiety other than the second IRM moiety that has biological activity. For example, the second active moiety can include an antigen or a targeting moiety. The second active moiety can be a vaccine antigen. The second active moiety can be an infectious disease antigen or a tumor antigen.

[0290] The second active moiety can be a monoclonal antibody. The second active moiety can be an immunotherapeutic antibody. For example, the second active moiety can be an anti-tumor antibody. The immunotherapeutic antibody can block an immune checkpoint protein. The immune checkpoint protein can be a CTLA-4, PD-1, or PD-L1 protein.

[0291] The second active moiety can be an immune checkpoint protein inhibitor. The second active moiety can be an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody. The second active moiety can be an anti-HER2 antibody.

[0292] Conjugates comprising an antigen and an active IRM moiety have been described, for example, in U.S. Patent Application Publication No. 2004 / 091491 (Kedl et al.). These conjugates can enhance the immune response to the antigen by facilitating simultaneous delivery of the IRM compound and the antigen to antigen-presenting cells.

[0293] Some embodiments of the present disclosure include conjugates comprising an antigen and an IRM moiety, where the IRM moiety is inactive until the labile bond is cleaved to release the active IRM moiety. Such conjugates may be useful to allow the administered conjugate to reach the target tissue before inducing an immune response. This may provide a therapeutic benefit by inducing a more localized antigen-specific immune response. The IRM moiety may be kept inactive until the conjugate reaches the target tissue where antigen-specific immunotherapy is required, thereby reducing or even preventing a systemic immune response against the antigen that may be induced by the active IRM moiety before the conjugate can reach its target tissue.

[0294] In certain embodiments, the second active moiety may be a targeting moiety (i.e., a moiety that acts to target delivery of the conjugate to a particular tissue or cell population or to cause selective retention). The specific nature of the targeting moiety may be determined, in part, by the identity and nature of the intended target. For example, a suitable targeting moiety may actively provide directed binding to the target, such as in an antibody directed against an antigenic portion of a tumor, target cell, target tissue, or target organ. Active targeting may also be achieved by exploiting receptor-ligand affinity. In other cases, the targeting moiety may provide passive retention of the conjugate in the target. Passive retention may be achieved by exploiting differences in hydrophobicity / hydrophilicity, vascular porosity, etc., of the target versus non-target environment.

[0295] The targeting moiety may be any material capable of providing targeted delivery of the conjugate. In some embodiments, the targeting moiety may be a portion of an immunoglobulin (i.e., an antibody) sufficient to provide immunospecific targeting, i.e., promote immunospecific binding of the composition to a target antigen. However, aspects of the present disclosure may also be practiced using non-immunoglobulin targeting materials, such as, for example, receptor ligands for hormones (natural or synthetic), lipids, etc.

[0296] In some cases, the targeting moiety may be or may be derived from an antibody (i.e., at least a sufficient immunospecific portion of an antibody, e.g., sufficient light chain to provide some degree of immunospecificity). In other cases, however, the targeting moiety may be or may be derived from an agent that recognizes at least a portion of a tumor-specific marker, such as, for example, a ligand that binds to a receptor that is expressed with some degree of specificity by the target cell population. In such cases, the receptor may be considered a tumor-specific marker.

[0297] Conjugates designed for use in treating tumors may include a tumor-specific targeting moiety and a labile bond selected to be more likely, faster, or more efficiently cleaved in the tumor environment than in the systemic environment. The tumor microenvironment is often characterized by having low oxygen tension, low extracellular pH, and low glucose concentration. Labile bonds that can take advantage of one or more of these microenvironmental conditions (e.g., low pH) may form labile bonds that are particularly well suited for use in conjugates designed to treat tumors.

[0298] Leuteinizing hormone releasing hormone (LHRH) receptors are significantly elevated on breast cancer, prostate cancer, endometrial cancer, ovarian cancer, and melanoma cells. Thus, ligands of the LHRH receptor can be used as targeting moieties in conjugates to provide tumor-specific targeted delivery of IRM moieties to tumor sites. In animal models of human cancers as described above, LHRH-directed therapeutic agents selectively reach diseased tissues. Coupling an IRM to a ligand of the LHRH receptor (e.g., LHRH or a synthetic analog) can provide targeted delivery of the IRM to tumor cells of these cancers, thereby concentrating the IRM at the tumor site and increasing the therapeutic index beyond that observed with the IRM compound alone. Suitable LHRH receptor ligands can include LHRH decapeptides, analogs with agonistic or antagonistic activity, or small molecule receptor ligands.

[0299] The LHRH receptor is known to be overexpressed on many tumor cells (e.g., breast, prostate, melanoma) compared to normal organ tissues. Thus, a single IRM-LHRH receptor ligand conjugate may be useful for treating multiple types of cancer.

[0300] Folate receptor ligands can also be useful as targeting moieties to provide tumor-specific targeted delivery of IRM moieties. Folate receptor expression is increased on the surface of many tumor cells. Again, binding of folate receptor ligands to IRM moieties can result in selective accumulation of IRM at tumor sites, reducing the systemic availability of the IRM moiety and increasing the therapeutic index of the IRM moiety. Suitable folate receptor ligands include folic acid, analogs with agonist or antagonist activity, or small molecule receptor ligands.

[0301] In some alternative embodiments, the IRM moiety may be conjugated to a dendritic cell targeting moiety, which may be an antibody (e.g., an anti-DC antibody) or a non-antibody ligand that recognizes a DC-specific marker.

[0302] Suitable DC-specific markers can include, for example, costimulatory markers, such as any member of the TNFR superfamily (e.g., CD40), CD70, CD80, CD86, B7-CD, B7.1, B7.2, etc. Conjugates containing a targeting moiety that recognizes a costimulatory marker can be used to deliver two DC activation stimuli (i.e., an IRM moiety and a costimulatory moiety) with a single chemical entity.

[0303] As used herein, anti-DC antibody refers to an antibody that recognizes a dendritic cell antigen. A suitable dendritic cell targeting moiety can bind to any antigen that is qualitatively or quantitatively differentially expressed by dendritic cells. A suitable dendritic cell targeting moiety can bind to an antigen such as, for example, any one of DEC205, BDCA-1, BDCA-2, BDCA-3, BDCA-4, DC-SIGN, L-SIGN, HLR-DR, CD11c, CD13, CD14, CD21, CD33, CD35, CD123, C-type lectins, integrins (e.g., α4, α6, α1β1), and / or Toll-like receptors (TLRs).

[0304] Regardless of whether the targeting moiety recognizes a DC-specific marker or antigen, conjugating the IRM moiety to the targeting moiety can limit the systemic availability of the IRM moiety even when administered via a systemic delivery route. Furthermore, the conjugate, and therefore the IRM moiety, can be concentrated in the vicinity of dendritic cells, thereby more effectively maturing and activating the dendritic cells. Activated dendritic cells at the site of the tumor (or even inside the tumor mass) may be able to utilize tumor antigens present on the surface of tumor cells to mount an immune response against the tumor. This method can provide a generalized antitumor therapy without the need for tumor-specific antibodies.

[0305] In other alternative embodiments, the IRM moiety can be conjugated to an anti-macrophage targeting moiety. Macrophages are often localized in the vicinity of tumor cells. Thus, again, the systemic availability of the IRM moiety can be limited, and the IRM moiety can be concentrated in the vicinity of the target cells (i.e., macrophages), thereby activating the macrophages more efficiently. Activated macrophages are known to have anti-tumor activity. Thus, this method can provide generalized tumor therapy without the need for tumor-specific antibodies.

[0306] In other alternative embodiments, the IRM portion is, for example, CD8 + The IRM moiety may be conjugated to a target-specific moiety that recognizes a surface antigen on a cell type that can directly kill tumor cells, such as cytotoxic T cells, NK cells, or NKT cells. Again, even if the conjugate is administered systemically, the IRM moiety may be concentrated in the vicinity of the tumor-killing cells, thereby (a) activating the tumor-killing cells more effectively, and / or (b) limiting the systemic availability of the IRM moiety. The tumor-killing cells activated at the site of the tumor (or even inside the tumor mass) may be able to take advantage of tumor antigens present on the surface of the tumor cells to mount an immune response against the tumor. This method may provide a generalized tumor therapy without the need for tumor-specific antibodies.

[0307] In other alternative embodiments, the IRM moiety may be conjugated to a targeting moiety that recognizes, for example, an endothelial target. There are significant differences in the endothelial environment of tumor masses compared to normal capillary beds. For example, there are differences in the identity and degree to which certain endothelial surface proteins, adhesion molecules (e.g., integrins), extracellular matrix proteins, growth factor receptors, etc. are expressed. These differences can be exploited to target the delivery of the IRM moiety to tumor-associated endothelium. Several reagents that specifically target such differences have been demonstrated to be useful as antiangiogenic therapies. Conjugating such agents to the IRM moiety as a targeting moiety can combine two effective antitumor therapies: immunotherapy and antiangiogenic therapy.

[0308] Suitable anti-angiogenic reagents include, for example, anti-CD105 antibodies (CD105 is overexpressed on tumor endothelium), anti-ED-B antibodies (ED-B is a fibronectin isoform found in tumor masses), peptides recognized by tumor-associated endothelial integrins, and growth factors whose receptors are upregulated on tumor endothelium (e.g., vascular endothelial growth factor).

[0309] The use of antiangiogenic reagents in this manner may offer the possibility of combining antiangiogenesis with immunotherapy. Furthermore, targeted delivery of IRMs to tumor endothelium, as opposed to the tumor itself, may provide more effective long-term treatment, since endothelium is generally a less mutagenic tissue than the tumor mass. Thus, treatment directed to endothelium may be much less likely to cause drug resistance. Also, treatment directed to endothelium may be effective against virtually any vascularized tumor (e.g., breast, prostate, lung cancer) without the need for tumor-specific reagents.

[0310] In some embodiments, the targeting moiety may comprise an immunoglobulin or at least a functional portion of an immunoglobulin. Because immunoglobulins are proteins, it is understood that modifications can be made to a particular immunoglobulin without rendering the modified immunoglobulin unsuitable for use as a targeting moiety. For example, one or more portions of the amino acid sequence of an immunoglobulin may be deleted or substituted, or additional amino acids may be added to the immunoglobulin, and the immunoglobulin may still retain sufficient immunospecific characteristics to be suitable for use as a targeting moiety. Examples of suitable antibodies are described, for example, in US Patent Application Publication No. 2006 / 0142202 (Alkan et al.).

[0311] Pharmaceutical Compositions and Biological Activity Pharmaceutical compositions of the present disclosure are also contemplated. The pharmaceutical compositions of the present disclosure contain a therapeutically effective amount of a compound or salt or conjugate (i.e., conjugate) of the present disclosure (as described herein) in combination with a pharma- ceutically acceptable carrier.

[0312] The compound of formula (I), (IA), (II), (II-A), (III), or (III-A), or the IRM-containing conjugate of formula (IV) or (IV-A), its salt, or combinations thereof, may be provided in any pharmaceutical composition suitable for administration to a subject (human or animal) and may be present in the pharmaceutical composition in any suitable form (e.g., solution, suspension, emulsion, or any form of mixture). The pharmaceutical composition may be formulated with any pharma- ceutically acceptable carrier (e.g., excipient or vehicle). In some embodiments, the pharma- ceutically acceptable carrier comprises water (e.g., phosphate buffered saline or citrate buffered saline). In some embodiments, the pharmaceutical carrier comprises an oil (e.g., corn oil, sesame oil, cottonseed oil, soybean oil, or safflower oil). The pharmaceutical composition may further comprise one or more additives, including suspending agents, surfactants, dispersing agents, and preservatives (e.g., antioxidants).

[0313] In some embodiments of the pharmaceutical composition, the compound of formula (I), (IA), (II), (II-A), (III), or (III-A), or the IRM-containing conjugate of formula (IV) or (IV-A), its salt, or a combination thereof, can be incorporated into a homogeneously dispersed formulation. In some embodiments of the pharmaceutical composition, the compound of formula (I), (IA), (II), (II-A), (III), or (III-A), or the IRM-containing conjugate of formula (IV) or (IV-A), its salt, or a combination thereof, can be incorporated into an emulsified formulation. In some embodiments of the pharmaceutical composition, the compound of formula (I), (IA), (II), (II-A), (III), or (III-A), or the IRM-containing conjugate of formula (IV) or (IV-A), its salt, or a combination thereof, can be incorporated into an oil-in-water formulation. Oil-in-water formulations can include an oil component, an aqueous component, and one or more surfactants (e.g., formulations including soybean oil, TWEEN® 80, SPAN 85, and phosphate buffered saline). In some embodiments of the pharmaceutical composition, the compound of Formula (I), (IA), (II), (II-A), (III), or (III-A), or an IRM-containing conjugate of Formula (IV) or (IV-A), a salt thereof, or a combination thereof, can be incorporated into a liposomal formulation.

[0314] In some embodiments, the pharmaceutical composition may further comprise an antigen in an amount effective to generate an immune response to the antigen, hi some embodiments, the antigen is a vaccine.

[0315] The pharmaceutical compositions can be administered in any suitable manner (parenterally or non-parenterally). In some embodiments, the pharmaceutical compositions can be administered by intradermal, subcutaneous, intramuscular, or intravenous injection.

[0316] The exact amount of compound, salt, or IRM-containing conjugate to be used in the pharmaceutical compositions of the present disclosure will vary depending on factors known to those skilled in the art, such as the physical and chemical properties of the compound, salt, or IRM-containing conjugate, the properties of the carrier, and the intended dosage regimen.

[0317] In some embodiments, the concentration of the compound of Formula (I), (IA), (II), (II-A), (III), or (III-A), or the IRM-containing conjugate of Formula (IV) or (IV-A), a salt thereof, or a combination thereof in the pharmaceutical composition may be at least 0.0005 mg / mL, at least 0.001 mg / mL, or at least 0.05 mg / mL. In some embodiments, the concentration of the compound of Formula (I), (IA), (II), (II-A), (III), or (III-A), or the IRM-containing conjugate of Formula (IV) or (IV-A), a salt thereof, or a combination thereof in the pharmaceutical composition may be up to 2.4 mg / mL, up to 0.06 mg / mL, up to 0.01 mg / mL, or up to 0.005 mg / mL.

[0318] In some embodiments, the compositions of the present disclosure will contain sufficient active ingredient or prodrug to provide a subject with a dose of at least 100 nanograms per kilogram (ng / kg) or at least 10 micrograms per kilogram (μg / kg) of the compound, salt, or conjugate. In some embodiments, the compositions of the present disclosure will contain sufficient active ingredient or prodrug to provide a subject with a dose of up to 50 milligrams per kilogram (mg / kg) or up to 5 mg / kg of the compound, salt, or conjugate.

[0319] In some embodiments, the compositions of the present disclosure are administered to a subject over their body surface area (m 2 ) is the target weight:m 2 = (weight kg 0.425 ×Height (cm) 0.725 ) × 0.007184, for example, 0.01 mg / m 2 ~5.0mg / m 2 In some embodiments, the method contains sufficient active ingredient or prodrug to provide a dose of 0.1 mg / m, although in some embodiments, the method may be practiced by administering a dose of the compound, salt, or conjugate outside of this range. In some of these embodiments, the method includes administering a dose of 0.1 mg / m 2~2.0mg / m 2 A dose of, for example, 0.4 mg / m 2 ~1.2mg / m 2 The method includes administering to the subject a sufficient amount of the compound, salt, or conjugate to provide a dose of

[0320] In any of the embodiments of the pharmaceutical compositions comprising a compound of Formula (IA), (II-A), (III-A), (III-C), (III-E), or (III-G), the compound of Formula (IA), (II-A), (III-A), (III-C), (III-E), or (III-G), respectively, is present in the composition in at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, at least 96% enantiomeric excess, at least 96% enantiomeric excess, at least 97% enantiomeric excess, at least 98% enantiomeric excess, at least 99% enantiomeric excess, at least 99.5% enantiomeric excess, or at least 99.8% enantiomeric excess.

[0321] In any of the embodiments of the pharmaceutical compositions comprising a compound of Formula (IA), (II-A), (III-A), (III-C), (III-E), or (III-G), the opposite enantiomer of the compound is present in the composition at less than 10%, less than 5%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.25%, or less than 0.1%.

[0322] The compound, salt or conjugate of the present disclosure can be administered to humans or animals using various dosage forms.The dosage forms that can be used include, for example, tablets, lozenges, capsules, parenteral formulations, creams, ointments, topical gels, aerosol formulations, liquid formulations (e.g., aqueous formulations), transdermal patches, etc.These dosage forms can be prepared using conventional pharmaceutically acceptable carriers and additives using conventional methods, which generally include the step of associating active ingredient with carriers.Preferred dosage forms have one or more of the compound, salt or conjugate of the present disclosure dissolved in aqueous formulations.

[0323] The compounds, salts, or conjugates disclosed herein induce the production of certain cytokines in experiments performed as described in the Examples. These results indicate that the compounds, salts, or conjugates are useful for enhancing immune responses in several different ways, making them useful in the treatment of a variety of disorders.

[0324] The compounds, salts, or conjugates described herein may be administered as the sole therapeutic agent in a treatment regimen, or the compounds, salts, or conjugates described herein may be administered in combination with other active agents, including antivirals, antibiotics, proteins, peptides, oligonucleotides, antibodies, and the like.

[0325] The compounds, salts, or conjugates described herein induce the production of cytokines (e.g., IFN-α, IFN-γ, TNF-α, IP-10). These results indicate that the compounds, salts, or conjugates of the present disclosure are useful for activating immune responses in several different ways, making them useful in the treatment of a variety of disorders. Thus, the compounds, salts, or conjugates of the present disclosure are agonists of cytokine biosynthesis and production, in particular agonists of IFN-α, IFN-γ, TNF-α, and IP-10 cytokine biosynthesis and production.

[0326] One way in which the disclosed compounds, salts, or conjugates induce cytokine production is believed to be through activation of Toll-like receptors (TLRs) in the immune system, particularly TLR-7 and / or TLR-8, although other mechanisms may also be involved. In the immune system pathway (i.e., mechanism) for cytokine induction, the disclosed compounds, salts, or conjugates are believed to act primarily as agonists of TLR-7 and / or TLR-8, although other pathways or activities may also be involved.

[0327] Administration of the compounds, salts, or conjugates described herein can induce the production of interferon-alpha (IFN-α), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and IP-10 in cells. Cytokines whose biosynthesis can be induced by the compounds, salts, or conjugates of the present disclosure include IFN-α, IFN-γ, TNF-α, IP-10, and a variety of other cytokines. Among other effects, these cytokines can inhibit virus production and tumor cell proliferation, making the compounds or salts useful for treating viral and neoplastic diseases. Thus, the present disclosure provides a method of inducing cytokine biosynthesis in a human or animal by administering to the human or animal an effective amount of a compound, salt, or conjugate of the present disclosure. The human or animal to which the compound, salt, or conjugate is administered for induction of cytokine production may have one or more of the diseases, disorders, or conditions described below (e.g., viral or neoplastic diseases), and administration of the compound, salt, or conjugate may provide a therapeutic treatment. Alternatively, the compound, salt, or conjugate may be administered to the human or animal before the human or animal is afflicted with a disease, such that administration of the compound, salt, or conjugate may provide a prophylactic treatment. Animals that may benefit from administration of the compounds, salts, or conjugates of the present disclosure include, but are not limited to, non-human primates, rodents, dogs, cats, horses, pigs, sheep, goats, poultry, and cattle.

[0328] In addition to the ability to induce cytokine production, the compounds, salts, or conjugates described herein can affect other aspects of the innate immune response. For example, the activity of natural killer cells can be stimulated (an effect that can be attributed to cytokine induction). The compounds, salts, or conjugates can also activate macrophages, which in turn stimulate the secretion of nitric oxide and the production of additional cytokines. Furthermore, the compounds, salts, or conjugates can cause the proliferation and differentiation of B lymphocytes.

[0329] Conditions for which the compounds, salts, conjugates, or compositions identified herein may be used to treat include, but are not limited to, the following: Viral diseases, such as adenovirus, herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), poxvirus (e.g., orthopoxvirus such as smallpox or vaccinia, or molluscum contagiosum), picornavirus (e.g., rhinovirus or enterovirus), orthomyxovirus (e.g., influenza virus, avian influenza), paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus), diseases resulting from infection with a virus, such as respiratory syncytial virus (RSV), a coronavirus (e.g., SARS and SARS-CoV-2), a papovavirus (e.g., a papillomavirus, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., hepatitis C virus or dengue virus), or a retrovirus (e.g., a lentivirus such as HIV), an Ebola virus; Neoplastic diseases, such as bladder cancer, cervical dysplasia, cervical cancer, actinic keratosis, basal cell carcinoma, cutaneous T-cell lymphoma, mycosis fungoides, Sézary syndrome, HPV-associated head and neck cancer (e.g., HPV-positive oropharyngeal squamous cell carcinoma), Kaposi's sarcoma, melanoma, squamous cell carcinoma, renal cell carcinoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, hairy cell leukemia, esophageal cancer, and other cancers; Atopic dermatitis or eczema H 2-mediated atopic diseases, eosinophilia, asthma, allergies, allergic rhinitis, and Omenn's syndrome.

[0330] Diseases associated with wound repair, such as inhibition of keloid formation and other types of scarring (e.g., enhancing wound healing, including chronic wounds); and parasitic diseases, including, but not limited to, malaria, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosomal infections.

[0331] The IRM-containing conjugates described herein can be used as vaccines against diseases such as viral diseases, neoplastic diseases, bacterial diseases, fungal diseases, and parasitic diseases. Pharmaceutical compositions containing the IRM-containing conjugates described herein can be used as vaccines against diseases such as viral diseases, neoplastic diseases, bacterial diseases, fungal diseases, and parasitic diseases.

[0332] The compounds, salts, conjugates, or pharmaceutical compositions described herein may be used as vaccine adjuvants for use in conjunction with any agent that increases either the humoral and / or cell-mediated immune response, such as, for example, tumor antigens (e.g., MAGE-3, NY-ESO-1); live viral, bacterial, or parasitic immunogens; inactivated viral, protozoan, fungal, or bacterial immunogens; toxoids; toxins; polysaccharides; proteins; glycoproteins; peptides; cellular vaccines; DNA vaccines; mRNA vaccines; autologous vaccines; recombinant proteins, etc.

[0333] In one embodiment, the compounds, salts, conjugates, or pharmaceutical compositions described herein may be used as a vaccine adjuvant for use in conjunction with a DNA vaccine or an mRNA vaccine.

[0334] Examples of vaccines that may benefit from the use of a compound, salt, conjugate, or composition identified herein as a vaccine adjuvant include BCG vaccine, cholera vaccine, plague vaccine, typhoid vaccine, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, influenza A vaccine, influenza B vaccine, malaria vaccine, parainfluenza vaccine, polio vaccine, rabies virus vaccine, measles virus vaccine, mumps vaccine, rubella vaccine, yellow fever vaccine, tetanus vaccine, diphtheria vaccine, These include Haemophilus influenza b vaccine, tuberculosis vaccine, meningococcal and pneumococcal vaccines, adenovirus vaccines, coronavirus vaccines (e.g., SARS and SARS-CoV-2 vaccines), HIV vaccine, varicella vaccine, cytomegalovirus vaccine, dengue vaccine, feline leukemia vaccine, fowl plague vaccine, HSV-1 and HSV-2 vaccines, hog cholera vaccine, Japanese encephalitis vaccine, respiratory syncytial virus vaccine, rotavirus vaccine, papilloma virus vaccine, yellow fever vaccine, and Ebola virus vaccine.

[0335] The compounds, salts, conjugates, or pharmaceutical compositions identified herein may be particularly useful as vaccine adjuvants when used in conjunction with tumor antigens associated with colon cancer, head and neck cancer, breast cancer, lung cancer, and melanoma.

[0336] The compounds, salts, conjugates, or pharmaceutical compositions identified herein may be particularly useful in individuals with compromised immune function, for example, the compounds, salts, conjugates, or compositions may be used to treat opportunistic infections and tumors that arise after suppression of cell-mediated immunity in, for example, transplant patients, cancer patients, and HIV patients.

[0337] One or more of the above diseases or types of diseases, e.g., viral diseases or neoplastic diseases, can be treated in a human or animal in need thereof (having the disease) by administering a therapeutically effective amount of the compound, salt, conjugate, or composition to the human or animal.

[0338] In one embodiment, the IRM-containing conjugates described herein may be used as a coronavirus vaccine, a respiratory syncytial virus vaccine, a papillomavirus vaccine, an influenza A vaccine, an influenza B vaccine, or a pneumococcal vaccine. In one embodiment, the IRM-containing conjugates described herein may be used as a colon cancer vaccine, a head and neck cancer vaccine, a breast cancer vaccine, a lung cancer vaccine, and a melanoma vaccine.

[0339] Humans or animals may also be vaccinated by administering an effective amount of a compound, salt, conjugate, or composition described herein as a vaccine adjuvant. In one embodiment, a method of vaccinating a human or animal comprises administering an effective amount of a compound, salt, conjugate, or composition described herein to the human or animal as a vaccine adjuvant. The vaccine adjuvant may be co-administered with one or more humoral and cellular immune response enhancing materials by including each in the same composition. Alternatively, the vaccine adjuvant and materials that increase either the humoral and / or cellular immune response may be present in separate compositions.

[0340] The compounds, salts, conjugates, or compositions identified herein may be as prophylactic or therapeutic vaccine adjuvants in veterinary applications, for example, the compounds, salts, conjugates, or compositions identified herein may be administered to pigs, horses, cows, sheep, dogs, cats, poultry (e.g., chickens or turkeys), etc.

[0341] The compounds, salts, conjugates, or compositions identified herein may be particularly useful when administered in effective amounts to the human or animal body to treat bladder cancer, cervical dysplasia, actinic keratosis, basal cell carcinoma, genital warts, herpes virus infection, or cutaneous T-cell lymphoma. In these conditions, administration of the compounds, salts, or compositions of the disclosure is preferably topical (i.e., applied directly to the surface, such as a tumor, lesion, wart, or infected tissue).

[0342] In one embodiment, an effective amount of a compound, salt, conjugate, or composition described herein (e.g., an aqueous composition) is administered by intravesical instillation (e.g., administration using a catheter) to the bladder of a human or animal having at least one tumor in the bladder.

[0343] An effective amount of a compound, salt, or conjugate to induce cytokine biosynthesis typically causes one or more cell types (e.g., monocytes, macrophages, dendritic cells, and B cells) to produce increased (induced) amounts above background levels of one or more cytokines (e.g., IFN-α, IFN-γ, TNF-α, and IP-10). The exact dose will vary according to factors known in the art, but will typically be a dose of 100 ng / kg to 50 mg / kg, or 10 μg / kg to 5 mg / kg. In other embodiments, the amount is, for example, 0.01 mg / m 2 ~5.0mg / m 2 (calculated according to the Dubois method, supra), although in other embodiments, induction of cytokine biosynthesis may be achieved by administering a dose of the compound or salt outside of this range. In some of these embodiments, the method includes administering a dose of 0.1 mg / m 2 ~2.0mg / m 2 A dose of, for example, 0.4 mg / m 2 ~1.2mg / m 2 The method includes administering to the subject a sufficient amount of the compound, salt, conjugate, or composition to provide the subject with a dose of

[0344] Methods for treating viral infections in humans or animals, and methods for treating neoplastic diseases in humans or animals, may comprise administering to the human or animal an effective amount of a compound, salt, or conjugate described herein.

[0345] An effective amount for treating or inhibiting a viral infection may be an amount that will cause a decrease in one or more of the symptoms of a viral infection (e.g., viral lesions, viral load, viral production rate, and mortality rate) compared to an untreated human or animal. The exact amount that is effective for such treatment will vary according to factors known in the art, but is typically a dose of 100 ng / kg to 50 mg / kg or 10 μg / kg to 5 mg / kg.

[0346] An amount of a compound, salt, or conjugate effective to treat a neoplastic condition can be an amount that causes a reduction in tumor size or the number of tumor foci. The exact amount will vary according to factors known in the art, but is typically between 100 ng / kg and 50 mg / kg, or between 10 μg / kg and 5 mg / kg. In other embodiments, the amount is typically between 0.01 mg / m 2 ~5.0mg / m 2 (calculated according to the Dubois method, supra), although in some embodiments induction of cytokine biosynthesis may be achieved by administering a dose of the compound, salt, or conjugate outside of this range. In some of these embodiments, the method includes administering a dose of 0.1 mg / m 2 ~2.0mg / m 2 A dose of, for example, 0.4 mg / m 2 ~1.2mg / m 2 The method includes administering to the subject a sufficient amount of the compound, salt, conjugate, or composition to provide the subject with a dose of

[0347] Exemplary embodiments Embodiment 1 is a compound of formula (I) or a salt thereof:

[0348] [ka] wherein n is an integer of 0 or 1, R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl, R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl, and R2 is a -(C2-C18)alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0349] Embodiment 2 is a compound of formula (IA) or a salt thereof:

[0350] [ka] wherein n is an integer of 0 or 1, R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl, R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl, and R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0351] Embodiment 3 is a compound of formula (II) or a salt thereof:

[0352] [ka] wherein n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; R3 is selected from the group consisting of alkyl, aryl, and aralkyl; the alkyl portion of the alkyl or aralkyl optionally contains one or more catenary non-peroxidized -O-atoms; the alkyl portion of the alkyl or aralkyl is optionally substituted with a functional group selected from the group consisting of amine (-NH2), carboxyl (-C(O)OH), hydroxyl (-OH), and thiol (-SH); and the aryl portion of the aryl or aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or a combination thereof.

[0353] Embodiment 4 is a compound of formula (II-A) or a salt thereof:

[0354] [ka] wherein n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; R3 is selected from the group consisting of alkyl, aryl, and aralkyl; the alkyl portion of the alkyl or aralkyl optionally contains one or more catenary non-peroxidized -O-atoms; the alkyl portion of the alkyl or aralkyl is optionally substituted with a functional group selected from the group consisting of amine (-NH2), carboxyl (-C(O)OH), hydroxyl (-OH), and thiol (-SH); and the aryl portion of the aryl or aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or a combination thereof.

[0355] Embodiment 5 is a compound or a salt thereof selected from the group consisting of formula (III-B), formula (III-D), and formula (III-F):

[0356] [ka] wherein n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; L1 is an alkylene group, optionally containing one or more catenary non-peroxidized -O-atoms, amine groups, ester groups, amide groups, disulfide groups, carbonyl groups, carbonate groups, carbamate groups, or combinations thereof; m is an integer of 0 or 1; and Q is a functional group for binding to a polymer moiety or a second active moiety.

[0357] Embodiment 6 is a compound or a salt thereof selected from the group consisting of formula (III-C), formula (III-F), and formula (III-G):

[0358] [ka] wherein n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; L1 is an alkylene group, optionally containing one or more catenary non-peroxidized -O-atoms, amine groups, ester groups, amide groups, disulfide groups, carbonyl groups, carbonate groups, carbamate groups, or combinations thereof; m is an integer of 0 or 1; and Q is a functional group for binding to a polymer moiety or a second active moiety.

[0359] Embodiment 7 is a compound or salt according to any one of embodiments 5-6, wherein m is 0.

[0360] Embodiment 8 is a compound or salt according to any one of embodiments 5-6, wherein m is 1.

[0361] Embodiment 9 is a compound or salt according to any one of embodiments 5-8, wherein Q is an activated carboxylate, an activated carbonate, an amine, an aminooxy group, a carboxylic acid, a hydroxyl, an aldehyde, or a maleimide.

[0362] Embodiment 10 is a compound or salt of any one of embodiments 5-8, wherein Q is an amine, an aminooxy group, a carboxylic acid, an acyl hydrazide, a hydroxyl, an aldehyde, an N-hydroxysuccinimide ester, an N-hydroxysuccinimide carbonate, a maleimide, or a pentafluorophenyl ester.

[0363] Embodiment 11 is a compound or salt according to any one of embodiments 5 to 8, wherein Q is -NH2, -ONH2, -C(O)OH, -C(O)NHNH2, -OH, or -C(O)H.

[0364] In embodiment 12, L1 is -(CH2) y -O-(CH2) y -;-(CH2) y -; -(CH2) y (OCH2CH2) y -O(CH2) y -;-(CH2) y -(OCH2CH2) y -;-CH2(CH2) y -SS-CH2(CH2) y - and -CH2OCH2(CH2) y -SS-CH2(CH2) y-, wherein each y is independently selected as an integer from 1 to 36.

[0365] In embodiment 13, L1 is -C(O)(CH2) y -;-C(O)CH2(CH2) y -; -C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y -;-C(O)CH2(CH2) y NHC(O)CH2(CH2) y - and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y NHC(O)CH2(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0366] In embodiment 14, L1 is -C(O)CH2(CH2) y (OCH2CH2) y -; -C(O)CH2(CH2) y (OCH2CH2) y O(CH2) y -;-C(O)NHCH2(CH2) y -;-C(O)NHCH2(CH2) y (OCH2CH2) y -; and -C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y -, wherein each y is independently selected as an integer from 1 to 36.

[0367] Embodiment 15 is a compound or salt according to any one of embodiments 12-14, wherein each y is independently selected as an integer from 1 to 20, 1 to 12, or 1 to 8.

[0368] Embodiment 16 is a compound or salt according to any one of embodiments 12-14, wherein each y is independently selected as an integer from 2 to 20, from 2 to 12, or from 2 to 8.

[0369] Embodiment 17 is an IRM-containing conjugate selected from the group consisting of formula (IV-B), formula (IV-D), and formula (IV-F), or a salt thereof;

[0370] [ka] wherein n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; L2 is a bridging group; m is an integer of 0 or 1; Z is a polymer moiety or a second active moiety; and -(L2) of the conjugate is m The -Z portion, with or without L2, optionally contains a labile bond.

[0371] Embodiment 18 is an IRM-containing conjugate selected from the group consisting of formula (IV-C), formula (IV-E), and formula (IV-G), or a salt thereof;

[0372] [ka] wherein n is an integer of 0 or 1; R is selected from the group consisting of halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is -(C1-C3)alkylene-O-(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O-atoms; L2 is a bridging group; m is an integer of 0 or 1; Z is a polymer moiety or a second active moiety; and -(L2) of the conjugate is m The -Z portion, with or without L2, optionally contains a labile bond.

[0373] Embodiment 19 is a conjugate or salt according to any one of embodiments 17-18, wherein the bridging group and Z are covalently linked by an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0374] Embodiment 20 is a conjugate or salt according to any one of embodiments 17 to 19, wherein m is 0.

[0375] Embodiment 21 is a compound or salt according to any one of embodiments 17-19, wherein m is 1.

[0376] Embodiment 22 is a conjugate or salt according to any one of embodiments 17-21, wherein Z is a second active moiety.

[0377] Embodiment 23 is a conjugate or salt according to embodiment 22, wherein the second active moiety is a monoclonal antibody.

[0378] Embodiment 24 is a conjugate or salt of embodiment 23, wherein the second active moiety is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0379] Embodiment 25 is a conjugate or salt according to embodiment 23, wherein the second active moiety is an anti-HER2 antibody.

[0380] Embodiment 26 is a conjugate or salt according to embodiment 23, wherein the second active moiety is an immunotherapeutic antibody.

[0381] Embodiment 27 is a conjugate or salt according to embodiment 22, wherein the second active moiety is an antigen.

[0382] Embodiment 28 is a conjugate or salt according to embodiment 27, wherein the second active moiety is a tumor antigen.

[0383] Embodiment 29 is a conjugate or salt according to embodiment 27, wherein the second active moiety is a tumor antigen associated with colon cancer, head and neck cancer, breast cancer, lung cancer, or melanoma.

[0384] Embodiment 30 is an embodiment in which the bridging group is -(CH) y -O-(CH2) y C(O)-; -(CH2) y C(O)-;-(CH2) y -(OCH2CH2) y -O(CH2) y C(O)-;-(CH2) y -(OCH2CH2) y C(O)-; -CH2(CH2) y -SS-CH2(CH2) y C(O)-;-CH2OCH2(CH2) y -SS-CH2(CH2) y C(O)-; -CH2(CH2) y -SS-CH2(CH2) y O-;-CH2OCH2(CH2) y -SS-CH2(CH2) y O-; -CH2(CH2) y -SS-CH2(CH2) y NH-;-CH2OCH2(CH2) y -SS-CH2(CH2) y NH-;-(CH2) y-(OCH2CH2) y O-; and -(CH2) y -(OCH2CH2) y NH-, wherein each y is independently selected as an integer from 1 to 36.

[0385] Embodiment 31 is an embodiment in which the bridging group is -C(O)CH2CH2(OCH2CH2) y C(O)-; -C(O)CH2CH2(OCH2CH2) y O-;-C(O)CH2CH2(OCH2CH2) y NH-; -C(O)NHCH2CH2(OCH2CH2) y C(O)-;-C(O)NHCH2CH2(OCH2CH2) y O-; -C(O)NHCH2CH2(OCH2CH2) y NH-;-C(O)CH2CH2CH2CH2C(O)-; and 30. The conjugate or salt of any one of embodiments 17-29, wherein each y is independently selected as an integer from 1 to 36.

[0386] Embodiment 32 is an embodiment in which the bridging group is -C(O)CH2(CH2) y C(O)-; -C(O)CH2(CH2) y O-;-C(O)CH2(CH2) y NH-;-C(O)CH2(CH2) y (OCH2CH2) y C(O)-; -C(O)CH2(CH2) y (OCH2CH2) y O-;-C(O)CH2(CH2) y (OCH2CH2) y NH-; -C(O)CH2(CH2) y (OCH2CH2) yO(CH2) y C(O)-;-C(O)CH2(CH2) y (OCH2CH2) y O(CH2) y O-; -C(O)CH2(CH2) y (OCH2CH2) y O(CH2) y NH-;-C(O)NHCH2(CH2) y C(O)-;-C(O)NHCH2(CH2) y O-; -C(O)NHCH2(CH2) y NH-;-C(O)NHCH2(CH2) y (OCH2CH2) y C(O)-; -C(O)NHCH2(CH2) y (OCH2CH2) y O-;-C(O)NHCH2(CH2) y (OCH2CH2) y NH-; -C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y C(O)-;-C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y O-; and -C(O)NHCH2(CH2) y (OCH2CH2) y O(CH2) y NH-, wherein each y is independently selected as an integer from 1 to 36.

[0387] Embodiment 33 is an embodiment in which the bridging group is -C(O)(CH2) y O-; -C(O)CH2(CH2) y O-;-C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y O-; -C(O)CH2(CH2)y NHC(O)CH2CH2(OCH2CH2) y O-;-C(O)CH2(CH2) y NHC(O)CH2(CH2) y O-; and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y NHC(O)CH2(CH2) y 30. The conjugate or salt of any one of embodiments 17-29, wherein each y is independently selected as an integer from 1 to 36.

[0388] Embodiment 34 is an embodiment in which the bridging group is -C(O)(CH2) y NH-; -C(O)CH2(CH2) y NH-;-C(O)CH2(CH2) y C(O)NHCH2CH2(OCH2CH2) y NH-; -C(O)CH2(CH2) y NHC(O)CH2CH2(OCH2CH2) y NH-;-C(O)CH2(CH2) y NHC(O)CH2(CH2) y NH-; and -C(O)CH2CH2O(CH2CH2O) y CH2(CH2) y The conjugate or salt according to any one of embodiments 17 to 29, wherein the conjugate or salt is selected from the group consisting of NHC(O)CH2(CH2)yNH-, where y is an integer from 1 to 36.

[0389] Embodiment 35 is a conjugate or salt according to any one of embodiments 30-34, wherein each y is independently selected as an integer from 1 to 20, 1 to 12, 1 to 8, 2 to 20, 2 to 12, or 2 to 8.

[0390] Embodiment 36 is a compound, conjugate, or salt according to any one of embodiments 1 to 35, wherein R is selected from the group consisting of halogen, hydroxyl, -(C1-C7)alkyl, -(C1-C7)alkoxy, and -C(O)-O-(C1-C5)alkyl.

[0391] Embodiment 37 is a compound, conjugate, or salt according to any one of embodiments 1 to 36, wherein R is selected from the group consisting of hydroxyl, F, and Cl.

[0392] Embodiment 38 is a compound, conjugate, or salt according to any one of embodiments 1 to 37, wherein n is 0.

[0393] Embodiment 39 is a compound, conjugate, or salt according to any one of embodiments 1 to 38, wherein R1 is -CH2OCH3 or -CH2OCH2CH3.

[0394] Embodiment 40 is a compound, conjugate, or salt according to any one of embodiments 1 to 39, wherein R2 is a -(C2-C12)alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0395] Embodiment 41 is a compound, conjugate, or salt according to any one of embodiments 1 to 40, wherein R2 is a -(C2-C10) alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0396] Embodiment 42 is a compound, conjugate, or salt according to any one of embodiments 1 to 41, wherein R2 is a -(C2-C8)alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0397] Embodiment 43 is a compound, conjugate, or salt according to any one of embodiments 1 to 42, wherein R2 is a -(C2-C6)alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0398] Embodiment 44 is a compound, conjugate, or salt according to any one of embodiments 1 to 43, wherein R2 is a -(C2-C3) alkylene group, optionally containing one or more catenary non-peroxidized -O- atoms.

[0399] Embodiment 45 is an embodiment in which R2 is -CH2CH2-, -CH2CH2-O-CH2-, or -(CH2CH2-O) x The compound, conjugate, or salt according to any one of embodiments 1 to 39, wherein:

[0400] Embodiment 46 is a method of making an IRM-containing conjugate or salt of any one of embodiments 17-45, comprising reacting a compound or salt of any one of embodiments 5-16 with a polymer or a second active compound having a functional group to form a covalent bond by reacting a functional group Q of the compound with a functional group of the polymer or the second active compound.

[0401] Embodiment 47 is the method of embodiment 46, wherein the covalent bond is an ether bond, a thioether bond, an ester bond, an amide bond, an imine bond, an oxime bond, a hydrazone bond, or an N-acylhydrazone bond.

[0402] Embodiment 48 is an IRM-containing conjugate prepared according to the method of embodiment 46 or embodiment 47.

[0403] Embodiment 49 is a pharmaceutical composition comprising a compound or salt according to any one of embodiments 1-16 or 36-45 and a pharma- ceutically acceptable carrier.

[0404] Embodiment 50 is a pharmaceutical composition comprising an IRM-containing conjugate or salt according to any one of embodiments 17-45 and embodiment 48, and a pharma- ceutically acceptable carrier.

[0405] Embodiment 51 is the pharmaceutical composition of embodiment 49 or 50, wherein the pharmaceutical composition is a vaccine.

[0406] Embodiment 52 is a method of inducing cytokine biosynthesis in a human or an animal, comprising administering to the human or animal an effective amount of a compound, conjugate, or salt described in any one of embodiments 1 to 45 and embodiment 48.

[0407] Embodiment 53 is a method of inducing biosynthesis of IFN-alpha in a human or animal, comprising administering to the human or animal an effective amount of a compound, conjugate, or salt described in any one of embodiments 1 to 45 and embodiment 48.

[0408] Embodiment 54 is a method of inducing biosynthesis of IFN-gamma in a human or animal, comprising administering to the human or animal an effective amount of a compound, conjugate, or salt described in any one of embodiments 1 to 45 and embodiment 48.

[0409] Embodiment 55 is a method of inducing biosynthesis of TNF-alpha in a human or animal, comprising administering to the human or animal an effective amount of a compound, conjugate, or salt described in any one of embodiments 1 to 45 and embodiment 48.

[0410] Embodiment 56 is a method for inducing cytokine biosynthesis in a human or animal, comprising administering to the human or animal an effective amount of a pharmaceutical composition described in any one of embodiments 49 to 51.

[0411] Embodiment 57 is a method for inducing biosynthesis of IFN-alpha in a human or animal, comprising administering to the human or animal an effective amount of a pharmaceutical composition described in any one of embodiments 49 to 51.

[0412] Embodiment 58 is a method for inducing biosynthesis of IFN-gamma in a human or animal, comprising administering to the human or animal an effective amount of a pharmaceutical composition described in any one of embodiments 49 to 51.

[0413] Embodiment 59 is a method for inducing biosynthesis of TNF-alpha in a human or animal, comprising administering to the human or animal an effective amount of a pharmaceutical composition described in any one of embodiments 49 to 51.

[0414] Embodiment 60 is a method of treating a viral or neoplastic disease in a human or animal by administering to the human or animal an effective amount of a compound, conjugate, or salt described in any one of embodiments 1 to 45 and embodiment 48.

[0415] Embodiment 61 is a method for treating a viral disease or a tumor disease in a human or an animal by administering to the human or an animal an effective amount of the pharmaceutical composition according to any one of embodiments 49 to 51.

[0416] Embodiment 62 is a compound, conjugate, or salt according to any one of embodiments 1 to 45 and embodiment 48 for use in treating a viral or neoplastic disease in a human or animal by administration of the compound, conjugate, or salt to the human or animal.

[0417] Embodiment 63 is a pharmaceutical composition according to any one of embodiments 49 to 51, for use in treating a viral disease or a tumor disease in a human or an animal by administering the pharmaceutical composition to the human or an animal. EXAMPLES

[0418] The objects and advantages of the present disclosure are further illustrated by the examples provided herein. The specific materials and amounts thereof, as well as other conditions and details recited in these examples, are merely illustrative and are not intended to be limiting. Those skilled in the art will be able to use materials and conditions in addition to those specifically described in the examples after carefully studying the entirety of this disclosure.

[0419] Column chromatographic purification of the compounds was performed using an ISOLARA HPFC system (an automated high performance flash chromatography purification device available from Biotage, Inc., Charlottesville, VA). The eluents used for each purification are described in the Examples.

[0420] Proton nuclear magnetic resonance ( 1 1 H NMR analyses were performed using a BRUKER A500 NMR spectrometer (Bruker Corporation, Billerica, Mass.).

[0421] Cesium carbonate (Cs2CO3), sodium azide, 11-bromo-1-undecanol, and benzenesulfonyl chloride were obtained from Sigma-Aldrich Company, St. Louis, MO.

[0422] Methanesulfonyl chloride, tetrabutylammonium fluoride (1.0 M in THF), 2,6-lutidine (2,6-dimethylpyridine), pentamethyldiethylenetriamine (PMDTA), copper sulfate pentahydrate, maleic anhydride, potassium tert-butoxide, triethylene glycol, propargyl bromide, 4-(dimethylamine)pyridine (DMAP), triphenylphosphine, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), lysozyme, and sodium ascorbate were obtained from Alfa Aesar Company (Haverhill, MA).

[0423] (2-Bromoethoxy)(tert-butyl)dimethylsilane, phenylacetylene, tert-butyldimethylsilyl trifluoromethanesulfonate, copper(I) bromide, triethylamine, boron trifluoride etherate, ethyl diazoacetate (83% solution in CHCl), N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), and 3-chloroperbenzoic acid (approximately 70% MCPBA, as determined by iodometric titration according to Braun, G. Org. Synth., Collective Volume 1932, 1, 431) were obtained from Oakwood Products Incorporated (Estill, SC).

[0424] Example 1 (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine

[0425] [ka]

[0426] Part A To a stirred solution of (S)-4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenol (preparation described in US Patent Application Publication No. 2020 / 0385379) (1.20 g, 3.46 mmol) dissolved in 20 mL of anhydrous N,N-dimethylformamide (DMF) was added Cs2CO3 (1.69 g, 5.19 mmol) followed by (2-bromoethoxy)(tert-butyl)dimethylsilane (0.90 mL, 4.15 mmol). The reaction mixture was heated to 65 °C under a nitrogen atmosphere. After 4 h, the reaction mixture was diluted with 50 mL of ethyl acetate and 25 mL of water. The layers were separated and the organic portion was washed with water (3 x 25 mL) and brine, dried over Na2SO4, filtered and concentrated. Purification by column chromatography (SiO, 1% to 7.5% methanol / chloroform) gave 1.62 g of (S)-1-(1-(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline as an orange syrup.

[0427] Part B To a stirred solution of (S)-1-(1-(4-(2-((tert-butyldimethylsilyl)oxy)ethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline (1.62 g, 3.20 mmol) dissolved in 30 mL of tetrahydrofuran (THF) was added 0.32 mL of tetrabutylammonium fluoride solution (1 M in THF). After 2 h, the reaction mixture was diluted with 50 mL of ethyl acetate and 25 mL of water. The layers were separated and the organic portion was washed successively with water and brine (2×25 mL). The organic layer was dried over Na2SO4, filtered and concentrated. Purification by column chromatography (SiO2, 5% to 7.5% MeOH / chloroform) afforded 1.08 g of (S)-2-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)ethan-1-ol as an orange foam.

[0428] Part C To a stirred solution of (S)-2-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)ethan-1-ol (1.07 g, 2.74 mmol) dissolved in 10 mL of dichloromethane was added triethylamine (572 microliters, 4.11 mmol) and methanesulfonyl chloride (255 microliters, 3.28 mmol). After 2 hours, the reaction was quenched by the addition of saturated aqueous NaHCO3. The layers were separated and the organic portion was washed successively with water and brine, dried over Na2SO4, filtered, and concentrated to give 1.12 g of (S)-2-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)ethyl methanesulfonate as an amber syrup.

[0429] Part D To a solution of (S)-2-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)ethyl methanesulfonate (1.12 g, 2.39 mmol) dissolved in 6 mL of N,N-dimethylformamide (DMF) was added sodium azide (315 mg, 4.85 mmol) and the stirred mixture was heated at 60° C. overnight. The reaction mixture was then concentrated under reduced pressure and the resulting syrup was partitioned between 50 mL of ethyl acetate and 25 mL of water. The layers were separated and the organic portion was washed with water (2×25 mL) and brine, dried over Na2SO4, filtered and concentrated to give 0.95 g of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline as an amber syrup.

[0430] Part E A solution of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline (0.95 g, 2.28 mmol) dissolved in 25 mL of dichloromethane was combined with 617 mg of 3-chloroperbenzoic acid (MCPBA, 70%) and stirred for 90 min. The reaction mixture was cooled to -10°C, followed by the addition of 6 mL of concentrated NH4OH solution. The mixture was stirred rapidly and benzenesulfonyl chloride (0.35 mL, 2.74 mmol) was added. The reaction mixture was then allowed to warm to ambient temperature over 45 min. The reaction was quenched by the addition of 20 mL of water and the mixture was stirred for 15 min. The layers were then separated and the organic portion was washed successively with water, 5% aqueous Na2CO3 solution, water and brine. The organic portion was dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (SiO2, 4% methanol / chloroform saturated with NH4OH) gave 0.73 g of brown syrup. This syrup was dissolved in 15 mL of ethanol containing 0.5 mL of concentrated hydrochloric acid. The mixture was concentrated under reduced pressure, then concentrated from ethanol, and finally concentrated from acetonitrile to give a light brown solid. The solid was dissolved in 5 mL of hot acetonitrile and cooled to form a light brown solid. The solid was isolated by filtration, rinsed with cold acetonitrile, and dried under vacuum overnight to give 325 mg of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride as light brown crystals. 1 H NMR (500MHz, methanol-d4)δppm 8.59-8.69(m,1H)8.33(d,J=8.3Hz,1H)7.69-7.79(m,2H)7.57(t,J=7.4Hz,1H)7.04(d,J=8.6Hz,2H)6.69(d,J=8.7Hz,2H)5.66(br dd,J=9.0,5.0Hz,1H)4.07(d,J=5.0Hz,2H)3.96-4.03(m,2H)3.57(quind,J=7.1,7. 1,7.1,7.1,2.4Hz,2H)3.45-3.49(m,2H)3.43(d,J=5.4Hz,1H)1.13(t,J=7.0Hz,3H).

[0431] Example 2 (S)-1-(1-ethoxy-3-(4-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethoxy)phenyl)propan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine

[0432] [ka]

[0433] The filtrate from Example 1 Part E was concentrated and the residue was vigorously stirred in a mixture of 20 mL of dichloromethane and 10 mL of 5% aqueous Na2CO3. The organic layer was separated, washed successively with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (SiO2, 3-10% 2-propanol / chloroform) afforded 250 mg of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine free base as a light brown powder.

[0434] To a stirred solution of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine (250 mg, 0.58 mmol) and phenylacetylene (76 microliters, 0.70 mmol) in 3 mL of dimethylsulfoxide (DMSO) under a nitrogen atmosphere was added a solution of copper sulfate pentahydrate (29 mg) and sodium ascorbate (46 mg) dissolved in 3 mL of deionized water. An additional 3 mL of DMSO was added and the mixture was heated to 60° C. overnight. The reaction mixture was cooled to ambient temperature and diluted with 20 mL of water and 40 mL of dichloromethane. The layers were separated and the aqueous portion was extracted with an additional 20 mL of dichloromethane. The combined organic portion was washed successively with water (2×) and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (SiO2, 5% methanol / chloroform saturated with NH4OH) gave an amber solid. The solid was dissolved in 10 mL of ethanol containing 0.5 mL of concentrated hydrochloric acid. The mixture was concentrated under reduced pressure, then from ethanol (2x), and finally from acetonitrile to give a light brown solid. The solid was crystallized from acetonitrile, isolated by filtration, rinsed with cold acetonitrile, and dried under vacuum overnight to give 75 mg of (S)-1-(1-ethoxy-3-(4-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethoxy)phenyl)propan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride as off-white crystals. 1H NMR (500MHz, methanol-d4)δppm 8.57-8.64(m,1H)8.36(s,1H)8.28(d,J=8.3Hz,1H)7.76-7.83(m,2H)7.65-7.71(m,1H)7.61(t,J=7.6Hz,1 H)7.49(t,J=7.6Hz,1H)7.41-7.47(m,2H)7.32-7.39(m,1H)7.02(d,J=8.6Hz,2H)6.67-6.72(m,2H)5.61(br dd,J=8.7,5.1Hz,1H)4.77(t,J=5.1Hz,2H)4.24-4.36(m,2H)4.00(d,J=5.1Hz, 2H)3.48-3.58(m,2H)3.38-3.44(m,1H)3.25-3.32(m,1H)1.11(t,J=7.0Hz,3H).

[0435] Example 3 (S)-1-(1-(4-((11-azidoundecyl)oxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine

[0436] [ka]

[0437] Part A A solution of 11-bromoundecanol (2.51 g, 10.0 mmol) in 50 mL of dichloromethane was cooled to 0° C. under a nitrogen atmosphere. 2,6-lutidine (2.32 mL, 20.0 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (1.81 mL, 10.0 mmol) were added and the mixture was stirred overnight. The reaction mixture was quenched by the addition of saturated aqueous NaHCO3 and the layers were separated. The organic portion was washed sequentially with 1N hydrochloric acid solution, water (2×) and brine. The organic portion was dried over Na2SO4, filtered and concentrated to give a pale orange oil. The oil was passed through a small plug of silica gel eluting with 5% ethyl acetate / hexanes and the eluent was concentrated to give ((11-bromoundecyl)oxy)(tert-butyl)dimethylsilane as a colorless oil.

[0438] Part B To a stirred solution of (S)-4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenol (802 mg, 2.31 mmol) dissolved in 20 mL of anhydrous DMF was added Cs2CO3 (1.13 g, 3.46 mmol) followed by ((11-bromoundecyl)oxy)(tert-butyl)dimethylsilane (1.01 g, 2.77 mmol). The reaction mixture was heated to 65° C. under a nitrogen atmosphere. After 20 h, the reaction mixture was concentrated under reduced pressure and the resulting residue was partitioned between 50 mL of ethyl acetate and 25 mL of water. The layers were separated and the organic portion was washed successively with water (2×25 mL) and brine, dried over Na2SO4, filtered and concentrated. Purification by column chromatography (SiO, ethyl acetate to 10% methanol / ethyl acetate) gave 1.20 g of (S)-1-(1-(4-((11-((tert-butyldimethylsilyl)oxy)undecyl)oxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline as an orange syrup.

[0439] Part C To a stirred solution of (S)-1-(1-(4-((11-((tert-butyldimethylsilyl)oxy)undecyl)oxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline (1.20 g, 1.90 mmol) dissolved in 30 mL of THF was added 0.28 mL of tetrabutylammonium fluoride solution (1 M in THF). After stirring overnight, the reaction mixture was concentrated under reduced pressure. The resulting residue was partitioned between 50 mL of ethyl acetate and 25 mL of water. The layers were separated and the organic portion was washed sequentially with water (2×25 mL) and then with brine (2×25 mL). The organic layer was dried over Na2SO4, filtered and concentrated. Purification by column chromatography (SiO2, 10% methanol / ethyl acetate) gave 882 mg of (S)-11-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)undecane-1-ol as an amber syrup.

[0440] Part D To a stirred solution of (S)-11-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)undecan-1-ol (882 mg, 1.70 mmol) dissolved in 10 mL of dichloromethane was added triethylamine (355 microliters, 2.55 mmol) and methanesulfonyl chloride (159 microliters, 2.04 mmol). After 2 hours, the reaction was quenched by the addition of saturated aqueous NaHCO3 solution. The layers were separated and the organic portion was washed successively with water and brine, dried over Na2SO4, filtered and concentrated to give 981 mg of (S)-11-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)undecyl methanesulfonate as an amber syrup.

[0441] Part E To a solution of (S)-11-(4-(3-ethoxy-2-(1H-imidazo[4,5-c]quinolin-1-yl)propyl)phenoxy)undecyl methanesulfonate (981 mg, 1.65 mmol) dissolved in 5 mL of DMF was added sodium azide (214 mg, 3.29 mmol) and the stirred mixture was heated at 60° C. overnight. The reaction mixture was then concentrated under reduced pressure and the resulting syrup was partitioned between 50 mL of ethyl acetate and 25 mL of water. The layers were separated and the organic portion was washed successively with water (2×25 mL) and brine, dried over Na2SO4, filtered and concentrated to give 836 mg of (S)-1-(1-(4-((11-azidoundecyl)oxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline as a golden syrup.

[0442] Part F A solution of (S)-1-(1-(4-((11-azidoundecyl)oxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinoline (836 mg, 1.54 mmol) dissolved in 15 mL of dichloromethane was combined with 416 mg of MCPBA (70%) and stirred for 90 min. The reaction mixture was cooled to -10°C, followed by the addition of 6 mL of concentrated NH4OH solution. The mixture was stirred rapidly and benzenesulfonyl chloride (236 microliters, 1.85 mmol) was added. The reaction mixture was then allowed to warm to ambient temperature over 45 min. The reaction was quenched by the addition of 20 mL of water and the mixture was stirred for 15 min. The layers were then separated and the organic portion was washed successively with water, 5% aqueous Na2CO3 solution, water and brine. The organic portion was dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (SiO2, 1-7.5% methanol / chloroform) gave a brown syrup. The syrup was again purified by column chromatography (SiO2, 3.3% methanol / chloroform saturated with NH4OH) to give 525 mg of amber syrup. The amber syrup was dissolved in 10 mL of ethanol containing 0.2 mL of concentrated hydrochloric acid. The mixture was concentrated under reduced pressure, then concentrated from ethanol, and finally from acetonitrile to give a light brown solid. The solid was dissolved in 5 mL of hot acetonitrile and upon cooling a light brown solid formed. The solid was isolated by filtration, rinsed with cold acetonitrile, and dried under vacuum overnight to give 269 mg of (S)-1-(1-(4-((11-azidoundecyl)oxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride as light brown crystals. 1H NMR (500MHz, methanol-d4)δppm 8.62(m,1H)8.36(d,J=8.3Hz,1H)7.69-7.79(m,2H)7.56(t,J=7.6Hz,1H)7.03 (d,J=8.4Hz,2H)6.66(d,J=8.6Hz,2H)5.65(m,1H)4.03(d,J=5.0Hz,2H)3.80( t,J=6.4Hz,2H)3.55(m,2H)3.42(dd,J=5.8,14.3Hz,1H)3.31(m,1H)3.28(t,J =6.9Hz,2H)1.67(m,2H)1.59(m,2H)1.23-1.45(m,14H)1.13(t,J=7.0Hz,3H).

[0443] Example 4 (S)-1-(1-ethoxy-3-(4-((11-(4-phenyl-1H-1,2,3-triazol-1-yl)undecyl)oxy)phenyl)propan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine

[0444] [ka]

[0445] A stirred solution of (S)-1-(1-(4-((11-azidoundecyl)oxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride (80 mg, 0.135 mmol) and phenylacetylene (22 microliters, 0.20 mmol) dissolved in 3 mL of DMF was degassed with a stream of nitrogen. Copper(I) bromide (5 mg) and PMDTA (50 microliters) were added and the reaction was stirred overnight under a nitrogen atmosphere. The reaction mixture was then concentrated and the residue partitioned between 20 mL of dichloromethane and 10 mL of 0.1 N EDTA (ethylenediaminetetraacetic acid) solution. The layers were separated and the dichloromethane layer was washed successively with water (2x) and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography (SiO2, 4% methanol / chloroform saturated with NH4OH) gave an amber solid. The solid was dissolved in 5 mL of ethanol containing 0.1 mL of concentrated hydrochloric acid. The mixture was concentrated under reduced pressure, then from ethanol (2x), and finally from acetonitrile to give a light brown solid. The solid was crystallized from acetonitrile, isolated by filtration, rinsed with cold acetonitrile, and dried under vacuum overnight to give 24 mg of (S)-1-(1-ethoxy-3-(4-((11-(4-phenyl-1H-1,2,3-triazol-1-yl)undecyl)oxy)phenyl)propan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride as an off-white solid. 1H NMR (500MHz, methanol-d4)δppm 8.62(s,1H)8.35(d,J=8.3Hz,1H)8.34(s,1H)7.77-7.83(m,2H)7.67-7.76(m,2H)7.56(m,1H)7. 39-7.46(m,2H)7.32(m,1H)7.02(d,J=8.6Hz,2H)6.65(d,J=8.7Hz,2H)5.65(m,1H)4.46(t,J=7. 0Hz,2H)4.03(d,J=5.0Hz,2H)3.78(t,J=6.4Hz,2H)3.50-3.61(m,2H)3.42(dd,J=5.7,14.3Hz,1 H)3.32(dd,J=9.2,14.3Hz,1H)1.97(m,2H)1.63(m,2H)1.24-1.43(m,14H)1.13(t,J=7.0Hz,3H).

[0446] Cytokine induction in human cells Whole blood was obtained from healthy human donors by venipuncture and collected into vacutainer tubes or syringes containing EDTA. Human peripheral blood mononuclear cells (PBMCs) were purified from whole blood by density gradient centrifugation. Histopaque 1077 (15 mL, Sigma-Aldrich) was transferred to 6 × 50 mL sterile polypropylene conical tubes. The Histopaque was overlaid with 15–25 mL of blood diluted 1:2 with Hank's Balanced Salts Solution (HBSS) (Gibco, Life Technologies, Grand Island, NY). The tubes were then centrifuged at 1370 rpm for 30 min at 20 °C without brake (400 × g, GH 3.8A rotor).

[0447] The interface (buffy coat) containing PBMCs was collected and placed into a new sterile 50 mL conical polypropylene centrifuge tube. The PBMCs were mixed with an equal volume of HBSS (approximately 20 mL from the interface and approximately 20 mL of HBSS) and then centrifuged at 1090 rpm for 10 min at 20 °C with brake (270 × g, GH 3.8A rotor). After centrifugation was completed, the cells were resuspended in 2-3 mL of ACK red blood cell lysis buffer (ammonium chloride potassium solution, Gibco, Life Technologies) and incubated at 20 °C for 2-5 min. HBSS (40 mL) was then added to the cells and the sample was centrifuged at 270 × g for 10 min at 20 °C. The supernatant was decanted and the cell pellet was resuspended in 5 mL of AIM V medium (Gibco, Life Technologies). Cell aggregates and debris were removed by filtering the cell solution through a BD Falcon 70 micron nylon cell strainer (BD Biosciences, San Jose, Calif.).

[0448] The number of viable cells was determined by counting with a Moxi Z instrument (ORFLO Technologies, Ketchum, ID) or by using a hemocytometer. To determine cell viability with a hemocytometer, cells were diluted 1 / 10 in 0.4% trypan blue and HBSS (specifically, 50 microliters of trypan blue + 40 microliters of HBSS + 10 microliters of cell solution were added to a microcentrifuge tube and mixed). Ten microliters of diluted cells were then applied to the hemocytometer and the number of viable PBMCs was determined by microscopy.

[0449] PBMC samples were then plated in 96-well plates at 8 × 10 5The cells were resuspended at a concentration of 100 μM per well. Each compound was solubilized in DMSO to make a 3 mM stock solution. The stock solutions were then further diluted in AIM-V medium to prepare serial dilutions. The diluted compounds (100 microliters) were then transferred to PBMCs to make a test set with final compound concentrations of 30, 10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.01 micromolar. The plate also had both positive and negative controls. The negative control wells contained only AIM-V medium without the example compounds. The positive control wells contained a control set of imiquimod serially diluted to concentrations of 30, 10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.01 micromolar. The concentrations used in the control set were chosen to match those used in the test set. The plates were then incubated at 37°C / 5% CO2 for 21-24 hours. Cell-free supernatants were collected by centrifuging the 96-well plates at 2100 rpm for 10 minutes at 23° C. Approximately 160 microliters of supernatant was then stored in a NUNC 96-well plate, covered with a compression cap, and stored at −80° C. until cytokine analysis.

[0450] Cytokine levels (human IFN-α multisubtype, human IFN-γ, and human TNF-α) were measured in picograms / mL by Ella Simple Plex ELISA (ProteinSimple, San Jose, Calif.) according to the manufacturer's instructions.

[0451] The data was analyzed to determine the minimum effective concentration (MEC) of each compound at which induction of a particular cytokine was observed in the assay. Specifically, the minimum effective concentration (micromolar) of each compound was determined as the lowest concentration of compound that induced a cytokine response measured at a level (picograms / mL) at least two-fold greater than that observed in the negative control wells. The results are shown in Table 1. The symbol "≦0.01" indicates that cytokine induction was observed at the lowest concentration of compound evaluated in the assay.

[0452] [Table 1]

[0453] TLR activation and specificity HEK-BLUE-hTLR7 or hTLR8 reporter cells were obtained from InvivoGen (San Diego, CA). These reporter cells were prepared by co-transfection of HEK293 cells with an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene and either the human TLR7 or TLR8 gene, according to the manufacturer's instructions. The SEAP reporter gene was placed under the control of an IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. In the presence of TLR ligands, activation of NF-κB and AP-1 occurs, leading to a corresponding increase in SEAP levels.

[0454] Parental HEK293 cells expressing an inducible SEAP reporter but not TLR7 or TLR8 (null) were obtained from InvivoGen and used as a negative control in the assay.

[0455] In the assay, HEK cells were grown and maintained using standard cell culture techniques in growth medium containing Dulbecco's Modified Eagle Medium (ThermoFisher Scientific Incorporated, Waltham, MA) supplemented with 1% penicillin / streptomycin and 10% heat-inactivated Gibco fetal bovine serum (ThermoFisher Scientific). Each compound was solubilized in DMSO to make a 3 mM stock solution. The stock solution was then further diluted in growth medium to prepare serial dilutions. Each test compound was administered at 5×10 per well at concentrations of 30, 10, 3.3, 1.1, 0.37, 0.12, 0.04, and 0.01 micromolar. 4A 96-well format with cells and 200 microliters of growth medium was used for the study.

[0456] For each compound, hTLR7, hTLR8, and their respective null control HEK cells were screened. DMSO serially diluted in growth medium served as vehicle control. Cell culture supernatants containing the SEAP reporter were collected after a 16-20 h incubation period in a cell culture incubator (37 °C and 5% CO2) and either analyzed immediately or stored at -80 °C. SEAP levels were measured using a colorimetric enzymatic assay (QUANTI-BLUE (InvivoGen) according to the manufacturer's instructions.

[0457] The data was analyzed to determine the minimum effective concentration (MEC) of each compound at which activation was observed in the assay. Specifically, the minimum effective concentration (micromolar) of each compound was determined as the lowest concentration of compound that produced a SEAP expression response at least 2-fold greater than that observed in vehicle control wells. The results are shown in Table 2. The symbol "≦0.01" indicates that TLR activation was observed at the lowest concentration of compound evaluated in the assay.

[0458] [Table 2]

[0459] Example 5 (S)-1-(1-(4-(2-(4-((2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine

[0460] [ka]

[0461] Part A A 500 mL round bottom flask was charged with potassium tert-butoxide (3.29 g, 28.4 mmol) and 100 mL of anhydrous tetrahydrofuran (THF). The flask was placed under a nitrogen atmosphere and cooled to 0 °C in an ice bath. Triethylene glycol (8.45 g, 56.3 mmol) was added via syringe and the mixture was stirred for 30 minutes. Propargyl bromide (3.12 mL, 28.0 mmol) was then added and the mixture was stirred overnight. The reaction mixture was then filtered and the filtrate was concentrated to give an amber syrup. Column chromatography (silica gel, 50-70% ethyl acetate / hexanes) afforded 4.09 g of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-ol as a pale yellow oil.

[0462] Part B A 250 mL round bottom flask was charged with 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-ol (2.00 g, 10.6 mmol) and 25 mL of CHCl. ​​The flask was placed under a nitrogen atmosphere and cooled to 0° C. in an ice bath. Triethylamine (3.55 mL, 25.5 mmol) and DMAP (64 mg, 0.53 mmol) were added to the stirred reaction mixture, followed by the dropwise addition of methanesulfonyl chloride (1.00 mL, 12.9 mmol). The reaction mixture was allowed to warm to ambient temperature overnight. The reaction mixture was then quenched by the addition of saturated NaHCO solution and diluted with 50 mL of CHCl. ​​The mixture was transferred to a separatory funnel and the layers were separated. The organic portion was washed successively with 5% NaHPO solution and brine. The organic portion was then dried over Na2SO4, filtered, and concentrated to give 2.15 g of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl methanesulfonate as a light brown oil.

[0463] Part C A 250 mL round bottom flask was charged with 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl methanesulfonate (2.15 g, 8.08 mmol) and dissolved in 5 mL of anhydrous DMF. Sodium azide (630 mg, 9.70 mmol) was added and the stirred mixture was heated to 50° C. under a nitrogen atmosphere. After stirring overnight, the mixture was concentrated under reduced pressure. The resulting syrup was partitioned between 75 mL of ethyl acetate and 25 mL of water. The mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted with an additional 10 mL of ethyl acetate. The combined organic portions were washed with 10 mL of water, dried over Na2SO4, filtered and concentrated to give a golden syrup. Column chromatography (silica gel, 33% ethyl acetate / hexane) gave 1.32 g of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)prop-1-yne as a colorless oil.

[0464] Part D A mixture of 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy))prop-1-yne (1.32 g, 6.20 mmol) and triphenylphosphine dissolved in 20 mL of THF was stirred for 15 min. Water (0.3 mL) was then added and the reaction mixture was heated to 50° C. overnight. The reaction mixture was then concentrated under reduced pressure. Column chromatography (silica gel, 10% MeOH / CHCl3 saturated with concentrated NH4OH) afforded 1.08 g of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-amine as a colorless oil.

[0465] Part E A 100 mL round bottom flask was charged with 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-amine (114 mg, 0.609 mmol) and 3 mL of DMF. The flask was purged with nitrogen followed by the addition of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride (100 mg, 0.214 mmol) and copper(I) bromide (10 mg, 0.07 mmol). PMDTA (75 microliters, 0.360 mmol) was then added and the reaction mixture was stirred overnight. The reaction mixture was then concentrated under reduced pressure and the resulting blue syrup was partitioned between 20 mL of CHCl and 10 mL of water. The layers were separated and the aqueous layer was extracted with an additional 10 mL of CH2Cl2. The combined organic layers were concentrated under reduced pressure to give a light blue syrup. Column chromatography (silica gel, 10% MeOH / CHCl3 saturated with concentrated NH4OH) gave a colorless syrup which was then dissolved in 10 mL of CH2Cl2 and washed with three portions of water. The organic portion was dried over Na2SO4, filtered and concentrated to give 78 mg of (S)-1-(1-(4-(2-(4-((2-(2-(2-aminoethoxy)ethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine as a light brown syrup. 1H NMR(500MHz,chloroform-d)δppm 8.05(s,1H)8.02(dJ=8.2Hz,1H)7.83(dJ=8.3Hz,1H)7.75(s,1H)7.52(tJ=7. 6Hz,1H)7.31(tJ=7.6Hz,1H)7.04(dJ=8.3Hz,2H)6.76(dJ=8.3Hz,2H)5.52(br s,2H)5.32(m,1H)4.72(tJ=5.0Hz,2H)4.70(s,2H)4.30(tJ=5.0Hz,2H)3.8 2(dJ=3.5Hz,2H)3.58-3.74(m,8H)3.48-3.56(m,4H)3.46(ddJ=8.0,14.0Hz 1H)3.28(ddJ=5.9,14.0Hz,1H)2.87(br s,2H)1.22(tJ=7.0Hz,3H).

[0466] Example 6 (S)-1-2-(2-(2-((1-(2-(4-(2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-3-ethoxypropyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione

[0467] [ka]

[0468] Part A A mixture of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-amine (532 mg, 2.84 mmol) and maleic anhydride (279 mg, 2.84 mmol) was dissolved in 1 mL of anhydrous DMF and stirred overnight under nitrogen atmosphere. Sodium acetate (50 mg, 0.28 mmol) and acetic anhydride (0.47 mL, 4.97 mmol) were added and the mixture was heated to 100° C. for 60 min. The reaction was then concentrated under reduced pressure to give a dark oil. The dark oil was partitioned between 25 mL of CHCl and 20 mL of saturated NaHCO solution. The layers were separated and the aqueous portion was extracted twice more with 25 mL portions of CHCl. ​​The combined organic portions were washed with 10 mL of water followed by 10 mL of brine. The organic portion was dried over Na2SO4, filtered, and concentrated under reduced pressure to give an oil. Purification by column chromatography silica gel (3.3% MeOH / CHCl3) afforded 530 mg of 1-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione as a colorless syrup.

[0469] Part B A 100 mL round bottom flask was charged with 1-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione (72 mg, 0.207 mmol) and 3 mL of DMF. The flask was purged with nitrogen followed by the addition of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride (100 mg, 0.214 mmol) and copper(I) bromide (10 mg, 0.07 mmol). PMDTA (75 microliters, 0.360 mmol) was then added and the reaction mixture was stirred overnight. The reaction mixture was then concentrated under reduced pressure and the resulting blue syrup was partitioned between 30 mL of CHCl and 10 mL of water. The layers were separated and the aqueous layer was extracted with an additional 10 mL of CH2Cl2. The combined organic portions were washed with 10 mL of water followed by 10 mL of brine. The organic portions were dried over Na2SO4, filtered and concentrated under reduced pressure to give an oil. Purification by column chromatography (silica gel, 5% MeOH / CHCl3) afforded 115 mg of (S)-1-(2-(2-(2-((1-(2-(4-(2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-3-ethoxypropyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione as an amber syrup. 1H NMR (500MHz, chloroform-dδppm 8.06(s,1H)8.02(dJ=8.2Hz,1H)7.84(dJ=8.3Hz,1H)7.76(s,1H)7.52(tJ=7.6Hz,1 H)7.33(tJ=7.5Hz,1H)7.04(dJ=8.3Hz,2H)6.77(dJ=8.4Hz,2H)6.67(s,2H)5.68(br s,2H)5.31(m,1H)4.72(tJ=5.0Hz,2H)4.69(s,2H)4.30(tJ=5.0Hz,2H)3.82(dJ=3.3 Hz,2H)3.65-3.73(m,4H)3.56-3.65(m,8H)3.48-3.55(m,2H)3.45(ddJ=8.1,14.0Hz 1H)3.28(ddJ=5.8,13.9Hz,1H)1.22(tJ=7.0Hz,3H).

[0470] Example 7 (S)-1-(1-(2-(4-(2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-3-ethoxypropyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11-tetraoxatridecan-13-oic acid

[0471] [ka]

[0472] Part A A solution of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-ol (1.09 g, 5.79 mmol) in 20 mL of CHCl was cooled to 0° C. and stirred under a nitrogen atmosphere. Ethyl diazoacetate (83% solution in CHCl, 0.80 g, 5.79 mmol) was added, followed by boron trifluoride etherate (28 microliters, 0.23 mmol). The reaction mixture was allowed to reach ambient temperature over 90 min. The reaction was then quenched by the addition of water and diluted with an additional 20 mL of CHCl. ​​The layers were separated and the organic portion was washed with brine, dried over NaSO, filtered, and concentrated to give a yellow oil. Purification by column chromatography (silica gel, 25-67% ethyl acetate / hexanes) gave 470 mg of ethyl 3,6,9,12-tetraoxapentadec-14-ynoate as a colorless oil.

[0473] Part B To a solution of ethyl 3,6,9,12-tetraoxapentadec-14-ynoate (470 mg, 1.72 mmol) dissolved in 5 mL of THF was added 5.0 mL of 1N NaOH solution. After stirring for 60 min, 5.0 mL of 1.0N HCl solution was added and the mixture was concentrated under reduced pressure and then concentrated to dryness from toluene. The resulting residue was stirred with 10 mL of CHCl and filtered. The filtrate was concentrated to give 371 mg of 3,6,9,12-tetraoxapentadec-14-ynoic acid as a colorless oil.

[0474] Part C A 100 mL round bottom flask was charged with 3,6,9,12-tetraoxapentadeca-14-ynoic acid (371 mg, 1.51 mmol) and 10 mL of DMF. The flask was purged with nitrogen, followed by the addition of (S)-1-(1-(4-(2-azidoethoxy)phenyl)-3-ethoxypropan-2-yl)-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride (361 mg, 0.84 mmol) and copper(I) bromide (20 mg, 0.14 mmol). PMDTA (0.35 mL, 1.68 mmol) was then added and the reaction mixture was stirred for 2 h. The reaction mixture was then concentrated under reduced pressure and the resulting brown oil was partitioned between 50 mL of CHCl and 15 mL of 0.1 M EDTA solution. The layers were separated and the organic layer was discarded. The aqueous layer was acidified with 2 mL of 1N HCl solution and extracted with 50 mL of CHCl. ​​The organic layer was washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a syrup. Purification by column chromatography (silica gel, 10-50% MeOH / CHCl) gave 284 mg of (S)-1-(1-(2-(4-(2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-3-ethoxypropyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11-tetraoxatridecan-13-oic acid as an amber foam. 1 H NMR(500MHz,chloroform-d)δppm 8.20(m,1H)7.88(d,J=8.4Hz,1H)7.86(d,J=8.2Hz,1H)7.80(s,1H)7.46(t, J=7.7Hz,1H)7.28(m,1H)6.86(d,J=8.1Hz,2H)6.63(d,J=8.5Hz,2H)5.29(br s,1H)4.70(s,2H)4.66(m,2H)4.20(t,J=5.1Hz,2H)4.07(s,2H)3.92(d,J=3.9Hz,2H)3.62-3.79(m,12H )3.55(q,J=7.0Hz,2H)3.37(dd,J=14.2,6.0Hz,1H)3.24(dd,J=14.1,8.2Hz,1H)1.21(t,J=7.0Hz,3H).

[0475] Example 8. Preparation of IRM-containing conjugates A 0.1 M phosphate buffered saline (PBS) solution containing 2 mM EDTA was prepared and the pH was adjusted to 7.4 by the addition of 1 N NaOH. A 10 mg / mL lysozyme solution was prepared by dissolving 50 mg of lysozyme (obtained from Alfa Aesar Company) in 3 mL of PBS solution followed by the addition of 2 mL of DMSO. The solution was mixed gently for 30 minutes. A 1.00 mL sample of the lysozyme solution was added to a 1.5 mL Eppendorf tube. (S)-1-(2-(2-(2-((1-(2-(4-(2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-3-ethoxypropyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dione (20 mg, prepared in Example 6) was dissolved in 200 microliters of DMSO and a 100 microliter aliquot of the solution was added to the Eppendorf tube containing the lysozyme solution. The mixture was mixed gently overnight using a rotary mixer. The mixture was then purified by dialysis using a Thermo Scientific Slide-A-Lyzer dialysis apparatus (50 mL, obtained from ThermoFisher Scientific) and mixing with PBS buffer solution (45 mL) on a rotary mixer for 5 hours. The PBS buffer solution was removed and replaced with 45 mL of fresh buffer solution. The dialysis device was placed back on the rotary mixer and mixing was continued overnight. The solution was removed from the dialysis device and analyzed using MALDI-TOF MS and high resolution LC-MS. The results showed the presence of at least one IRM conjugated to approximately a 50% fraction of the total lysozyme protein.

[0476] Example 9. Preparation of IRM-containing conjugates In one drum vial, (S)-1-(1-(2-(4-(2-(4-amino-1H-imidazo[4,5-c]quinolin-1-yl)-3-ethoxypropyl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11-tetraoxatridecan-13-oic acid (38 mg, prepared in Example 7) and N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) (15 mg) were charged. The mixture was dissolved in 500 microliters of anhydrous DMSO and stirred for 5 minutes. 100 microliters of the sample was added to a 1.5 mL Eppendorf tube containing 1.00 mL of lysozyme solution (prepared as described in Example 8). The mixture was mixed gently overnight using a rotary mixer. The mixture was then purified by dialysis according to the procedure described in Example 8. The solution was removed from the dialyzer and analyzed by both MALDI-TOF MS and high resolution LC-MS. The results indicated the presence of at least one IRM conjugated to approximately a 50% fraction of the total lysozyme protein.

[0477] Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) Method for Analysis of IRM-Containing Conjugates A calibration mix of IRM-containing conjugate samples (diluted 1:20 in 10% methanol) and Bruker protein standards I and II (Bruker Company, Billerica, MA) were spotted onto a stainless steel MALDI target. Dried spots were analyzed using a Bruker Ultraflextreme MALDI-TOF / TOF mass spectrometer (Bruker Company) in positive ion mode. Linear detection was utilized with method settings of 5-50 kDa, collection speeds of 0.63 and 1.25 GS / s, and pulsed ion extraction of 490 ns. Laser power and number of laser shots were adjusted depending on the ionization efficiency of the sample. Data were smoothed from 40 to 60 m / z. The instrument was calibrated with Bruker protein standards immediately prior to analysis.

[0478] Liquid Chromatography-Mass Spectrometry (LC-MS) Method for Analyzing IRM-Containing Conjugates High-resolution LC-MS data was collected using a ThermoFisher ULTIMATE 3000 RSLCnano HPLC instrument and a ThermoFisher Scientific Orbitrap FUSION LUMOS TRIBRID mass spectrometer (ThermoFisher Scientific). Liquid chromatography (LC) was performed using an analytical ternary pump operated at 0.35 mL / min. Buffer A was 0.1% formic acid in water and Buffer B was 0.1% formic acid in acetonitrile. The gradient protocol for elution was as follows, sequentially: 10% Buffer B for 2 min hold, 85% Buffer B for 10 min gradient, 100% Buffer B for 2 min hold, 100% Buffer B for 2 min equilibration, 10% Buffer B for 4 min. The LC column was an AdvanceBio RP-mAb SB-C8, 2.1 x 100 mm, 3.5 micrometer (Agilent Technologies, Santa Clara, CA) maintained at 40°C. The mass spectrometer was tuned to intact protein mode prior to data collection. Data were collected in intact protein mode using 10 default charge states and RunStart Easy-IC mass calibration.

[0479] The complete disclosures of the patents, patent documents, and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated. Various modifications and alterations to the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the present invention. It is to be understood that the present invention is not intended to be unduly limited by the exemplary embodiments and examples described herein, and that such examples and embodiments are presented by way of example only, and that the scope of the present invention is intended to be limited only by the claims as set forth herein below.

Claims

1. A compound of formula (II) or a salt thereof, 【Chemistry 1】 During the ceremony, n is an integer of 0 or 1; R is halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is —(C1-C3)alkylene-O—(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O- atoms; R3 is selected from the group consisting of alkyl, aryl, and aralkyl; the alkyl or alkyl portion of the aralkyl optionally contains one or more catenary non-peroxidized —O— atoms; The alkyl or alkyl portion of the aralkyl may optionally be an amine (-NH ２ ), carboxyl (—C(O)OH), hydroxyl (—OH), and thiol (—SH); The compound of formula (II) or a salt thereof, wherein the aryl or the aryl portion of the aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or a combination thereof.

2. The compound of claim 1, which is of formula (II-A): 【Chemistry 2】

3. 2. The compound or salt of claim 1, wherein n is 0.

4. R1 is —CH ２ OCH ３ or -CH ２ OCH ２ CH ３ 2. The compound or salt of claim 1, wherein:

5. R3 is selected from the group consisting of -(C1-C10)alkyl, -(C6-C20)aryl, and -(C6-C20)ar-(C1-C10)alkyl, the alkyl or alkyl portion of the aralkyl optionally contains one or more catenary non-peroxidized —O— atoms; the alkyl or alkyl portion of the aralkyl is optionally substituted with a functional group selected from the group consisting of amine, carboxyl, hydroxyl, and thiol; 2. The compound or salt of claim 1, wherein the aryl or the aryl portion of the aralkyl is optionally substituted with halogen, hydroxyl, alkyl, alkoxy, or combinations thereof.

6. A compound of formula (III) or a salt thereof, 【Transformation 3】 During the ceremony, n is an integer of 0 or 1; R is halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is —(C1-C3)alkylene-O—(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O- atoms; R4 is, 【Chemistry 4】 is a triazole ring selected from the group consisting of During the ceremony, `` ＊ " indicates a covalent bond to R2, and " ＊＊ " indicates a covalent bond to L1, L1 is an alkylene group, optionally containing one or more catenary non-peroxide —O— atoms, amine groups, ester groups, amide groups, disulfide groups, carbonyl groups, carbonate groups, carbamate groups, or combinations thereof; m is an integer of 0 or 1; A compound of formula (III) or a salt thereof, wherein Q is a functional group for attaching to a polymer moiety or a second active moiety.

7. The compound of claim 6, which is of formula (III-A): 【Transformation 5】

8. 7. The compound or salt of claim 6, wherein n is 0 and m is 1.

9. R1 is —CH ２ OCH ３ or -CH ２ OCH ２ CH ３ 7. The compound or salt of claim 6, wherein:

10. R2 is —CH ２ CH ２ -, -CH ２ CH ２ -O-CH ２ - or -(CH ２ CH ２ -O) ｘ -CH ２ -, wherein x is an integer from 1 to 8.

11. Q is an amine (-NH ２ ), aminooxy (—O—NH ２ ), carboxylic acid (—C(O)OH), acyl hydrazide (—C(O)—NHNH ２ ), hydroxyl (—OH), aldehyde (—C(O)H), N-hydroxysuccinimide ester 【Transformation 6】 Maleimide 【Transformation 7】 and pentafluorophenyl ester 【Transformation 8】 7. The compound or salt of claim 6, selected from the group consisting of:

12. An IRM-containing conjugate of formula (IV) or a salt thereof, 【Chemistry 9】 During the ceremony, n is an integer of 0 or 1; R is halogen, hydroxyl, alkyl, alkoxy, and -C(O)-O-alkyl; R1 is —(C1-C3)alkylene-O—(C1-C3)alkyl; R2 is a -(C2-C18)alkenylene group, optionally containing one or more catenary non-peroxidized -O- atoms; R4 is, 【Chemistry 10】 is a triazole ring selected from the group consisting of During the ceremony, `` ＊ " indicates a covalent bond to R2, and " ＊＊ " indicates a covalent bond to L2, L2 is a bridging group; m is an integer of 0 or 1; Z is a polymer moiety or a second active moiety; -(L2) of the conjugate ｍ An IRM-containing conjugate of formula (IV) or a salt thereof, wherein the -Z moiety, with or without L2, optionally includes a labile bond.

13. The IRM-containing conjugate of claim 12 having formula (IV-A): 【Chemistry 11】

14. 13. The IRM-containing conjugate of claim 12, wherein n is 0 and m is 1.

15. R1 is —CH ２ OCH ３ or -CH ２ OCH ２ CH ３ 13. The IRM-containing conjugate of claim 12, wherein:

16. 13. The IRM-containing conjugate of claim 12, wherein Z is a second active moiety.

17. 13. The IRM-containing conjugate of claim 12, wherein the second active moiety is an antigen or an antibody.

18. 13. The IRM-containing conjugate of claim 12, wherein the second active moiety is an immune checkpoint inhibitor.

19. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising the IRM-containing conjugate or salt thereof of any one of claims 12 to 18 and a pharmaceutically acceptable carrier.

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