WO2021202921A1 - Composés du type imidazoquinoline et leurs utilisations - Google Patents

Composés du type imidazoquinoline et leurs utilisations Download PDF

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Publication number
WO2021202921A1
WO2021202921A1 PCT/US2021/025446 US2021025446W WO2021202921A1 WO 2021202921 A1 WO2021202921 A1 WO 2021202921A1 US 2021025446 W US2021025446 W US 2021025446W WO 2021202921 A1 WO2021202921 A1 WO 2021202921A1
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Prior art keywords
imidazoquinoline
antigen
tlr7
compound
moiety
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PCT/US2021/025446
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English (en)
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Bertrand Victor Gilbert GEORGES
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Altimmune Uk Limited
ANDERSON, Koren
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Publication of WO2021202921A1 publication Critical patent/WO2021202921A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • This application pertains generally to imidazoquinoline-type compounds as well as methods for preparing and using the same.
  • TLR7 and/or toll-like receptor 8 (TLR8) agonists are potent immune response modifiers (IRMs) that have received considerable attention for the treatment of various conditions such as cancer, viral infections and immune disorders.
  • IRMs immune response modifiers
  • the development of these IRMs have been hampered due to elevated systemic adverse effects as a result of strong systemic cytokine induction (cytokine storm) presumably resulting from rapid systemic diffusion.
  • the TLR7 agonist 852A resquimod
  • administered intravenously in patients with melanoma induced severe adverse events in almost 40% of patients (4 out of 13) that completed the first treatment cycle (Dummer et al. Clin Cancer Res.
  • TLR7 agonist imiquimod
  • imiquimod has been approved by regulatory agencies for the topical treatment of genital warts, superficial basal cell carcinoma and actinic keratosis.
  • Imiquimod is formulated as a 5% cream applied on the skin, thus limiting systemic diffusion of the small molecule TLR7 agonist and associated side effects, but also limiting its use because of the topical mode of application.
  • Both resiquimod and imiquimod have chemical structures based on the imidazoquinoline backbone (i.e., imidazoquinoline-type compounds).
  • imidazoquinoline-type compounds such as those that can be administered intratumorally (I.T.) or peritumorally (P.T.) and remain localized without inducing a systemic non-specific pro-inflammatory immune response, while also inducing a cell mediated immune response against tumor antigens.
  • I.T. intratumorally
  • P.T. peritumorally
  • Such imidazoquinoline-type compounds, as well as methods for preparing and using the same, are disclosed herein.
  • This disclosure relates to imidazoquinoline-type compounds that in some embodiments are useful as toll-like receptor (TLR) agonists, pharmaceutical compositions thereof and methods for preparing and using the same.
  • TLR toll-like receptor
  • the imidazoquinoline-type compounds are represented by the following Formulas (Ik), (II), and
  • R is -[A1J-NH-, wherein A1 is:
  • A2 is selected from:
  • A3 is -CO- or -NH-
  • A4 is nothing or p-aminobenzyloxy carbonyl (PABC)
  • A5 and A6 are independently -CO- or -NH-, one or more natural or non-natural amino- acids, or nothing; or, b) R is -NH-R 5 , wherein R 5 is selected from the group consisting of hydrogen, acetyl, -CO- tert-Bu (-Boc), CO-CFbPh (-Cbz), -CO-(CH 2 ) x -R 6 , C 1 -C 16 alkyl, -CO-4-(phenylboronic acid) a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, a protein antigen moiety, a polysaccharide antigen moiety, and a TLR 2 agonist moiety; R 6 is selected from the group consisting of hydrogen, alkyne, azido, carboxylic acid, and -C0NH-(CH 2 ) x -0- (CH 2 ) X -0-(CH 2 ) X -0-(CH
  • R 2 is selected from the group consisting of:
  • B is -O- or -NH-; m is any integer from 1-11; and, x is any integer from 1 to 12, preferably x is any integer from 2 to 12.
  • the disclosure provides pharmaceutical compositions comprising one or more imidazoquinoline-type compound(s) of any of Formula (Ik), Formula (II), or Formula (Im) and a pharmaceutical acceptable carrier or diluent, optionally in a preferred embodiment wherein the imidazoquinoline-type compound is soluble in an aqueous solution having a pH range of 3 to 9 or an ion concentration ranging from 0 mM to 600 mM.
  • DM-L-IM compounds undergo physical changes and exhibit improved properties under physiological conditions (e.g., after administration).
  • the methods comprise locally administering a liquid form of the pharmaceutical composition into the subject.
  • the methods induce an anti-tumor immune response when administered intratumorally or peri turn orally.
  • the methods induce a cell mediated immune response in the mucosa and/or bronchial tissue when administered nasally.
  • Other embodiments are also contemplated as will be apparent to those of ordinary skill in the art from this disclosure.
  • Figure 2 illustrates Scheme 2 for producing exemplary IM compounds.
  • Figure 3 illustrates Scheme 3 for producing exemplary IM compounds.
  • Figure 4 illustrates Scheme 4 for producing exemplary IM compounds.
  • Figure 5 illustrates Scheme 5 for producing exemplary IM compounds.
  • Figure 6 illustrates Scheme 6 for producing exemplary IM compounds.
  • Figure 7 illustrates Scheme 7 for producing exemplary IM compounds
  • Figure 8 illustrates the induction of systemic cytokines in mice.
  • Figure 9 illustrates the induction of systemic cytokines in mice.
  • Figure 11 illustrates tumor volume
  • Figure 12 illustrates the in vitro agonists activity of different compounds for the human TLR7 and TLR8 receptors.
  • Figure 13 illustrates the in vitro agonists activity of different compounds for the murine TLR7 receptor.
  • This disclosure relates to imidazoquinoline-type compounds that in some embodiments are useful as toll-like receptor (TLR) agonists (e.g., TLR7 and/or TLR8), pharmaceutical compositions thereof and methods for preparing and using the same.
  • TLR toll-like receptor
  • the imidazoquinoline-type compounds of this disclosure are represented by the following Formulas (Ik), (II), and (Im), and/or derivatives and/or analogues thereof:
  • R is -[A1J-NH-, wherein A1 is:
  • A2 is selected from: -A5-(CH 2 ) X -A6-,
  • A3 is -CO- or -NH-
  • A4 is nothing or p-aminobenzyloxy carbonyl (PABC)
  • A5 and A6 are independently selected from -CO- or -NH-, one or more natural or non-natural amino-acids, or nothing; or, b) R is -NH-R 5 , wherein R 5 is selected from the group consisting of hydrogen, acetyl, - CO-tert-Bu (-Boc), CO-CH 2 Ph (-Cbz), -CO-(CH 2 ) x -R 6 , Ci-Cie alkyl, -CO-4- (phenylboronic acid), -C(S)-NH-(CH 2 ) x -NH-(CH 2 ) x -NH-(CH 2 ) x -NH 2 , a reporter moiety, a tissue-specific moiety, a peptide antigen moiety, a protein antigen moiety, a polysaccharide antigen moiety, and a TLR2 agonist moiety; R 6 is selected from the group consisting of hydrogen, alkyne, azido
  • R 2 is selected from the group consisting of:
  • B is -O- or -NH-; m is any integer from 1-11; and, x is any integer from 1 to 12, preferably x is any integer from 2 to 12.
  • derivatives of the imidazoquinoline-type compounds contain or are modified by an amino or a carboxyl group to be used for conjugation to another compound (e.g., as defined by R in Formulas (Ik), (II) and (Im), and/or derivatives and/or analogues thereof).
  • the imidazoquinoline-type compounds of Formula (Ik), (II), or (Im) can be conjugated to or used in conjunction with a nanoparticle (e.g., gold nanoparticles (GNPs) immobilized with a-mannose as carrier (Shinchi, et al. Bioconj .
  • a nanoparticle e.g., gold nanoparticles (GNPs) immobilized with a-mannose as carrier
  • CDNPs b-cyclodextrin nanoparticles
  • alum e.g., Hu, et al. J. Transl. Med. 18(1): 112 (2020)
  • Other embodiments are also contemplated herein as would be understood by those of ordinary skill in the art.
  • derivatives of the imidazoquinoline-type compounds of this disclosure can include compounds having the formula DM-L-IM, wherein DM is the solubility component (and also referred to herein as a delivery/depot moiety), L is a linker, and IM is an imidazoquinoline-type compound of this disclosure (the immunostimulatory component; preferably a compound of Formula (Ik), (II), or (Im)).
  • the DM comprises a peptide from about 17 to about 45 amino acids in length comprising amino acid residues possessing helix forming properties, wherein the DM is configured to form an amphipathic a-helix structure, and wherein the peptide sequence is not derived from an antigen or immunogen and is a non-natural sequence.
  • the peptide does not comprise a T cell epitope and/or a B cell epitope (e.g., against an infective agent or tumor antigen, and/or that induces an immune response against the peptide).
  • the peptide (DM) does not comprise a T cell epitope or a B cell epitope (e.g., against an infective agent or tumor antigen, and/or that induces an immune response against the peptide; e.g., “immunogens”, as described in sources such as the immune epitope database at IEDB (https://www.iedb.org/home v3 php)).
  • a T cell epitope or a B cell epitope e.g., against an infective agent or tumor antigen, and/or that induces an immune response against the peptide; e.g., “immunogens”, as described in sources such as the immune epitope database at IEDB (https://www.iedb.org/home v3 php)).
  • the linker (L) can be a cleavable linker (e.g., a cathepsin B cleavable linker).
  • a cleavable linker When attached to a peptide conjugate (e.g., DM), the IM moiety has a lower ability to activate signaling through the receptor, compared to the free molecule, likely due to steric hindrance associated with the presence of a non-cleavable the linker.
  • a cleavable linker allows the TLR agonist to interact freely with the receptor and deliver its full potency.
  • the Examples section herein describes the benefits of a cleavable linker using the exemplary HL-6X2 and HL-6X3 conjugates.
  • Exemplary peptides comprise an amino acid sequence of RRLL(5)A(7)LAL(11)A(13)LLRRL (SEQ ID NO: 1) wherein amino acid positions (5), (7), (11) and (13) are each selected from A, L, or H; in some embodiments, an additional amino acid lysine (K) or Glutamic acid (E can be included on the N or C-terminus).
  • Coupling the DM comprising a peptide to the IM may, for example, reduce the pyrogenicity of the IM compared to the ‘free’ IM, and/or reduce the levels of in vivo inflammatory indicators, such as IL-1, TNF-a, IL-6 and/or IL-8 in the circulation.
  • the peptide can have the amino acid sequence RRLLHALLALLAHLLRRLE (SEQ ID NO. 2).
  • An exemplary cleavable linker conjugate having the formula DM-L-IM in which L is cleavable include HL-6X2 (Ac-RRLLHALLALLAHLLRRLE(PEG 6 -Val-Cit-PABC-IMDQ) where Ac is Acetyl; Val is valine; Cit is citrulline; PEG 6 is -NH-(CH 2 ) 2 -(0-CH 2 -CH 2 ) 6 -C0- PABC (p-aminobenzyloxy carbonyl); PAB is p-aminobenzyloxy; and IMDQ is the imidazoquinoline-based molecule as shown below:
  • a preferred exemplary conjugate having the formula DM-L-IM in which L is cleavable is HL-6X3 shown below:
  • the linker (L) can comprise or be: PEG6-Val- Cit-PABC or E-PEG6-Val-Cit-PABC.
  • the presence of the cathepsin B cleavable linker (L) (cathepsin B being present in the endosome of antigen presenting cells) in conjugates such as HL-6X2 or HL-6X3 provides for intracellular release of the TLR7/8 agonist (i.e., preferably IM3, Formula (Ik); see the Examples herein).
  • the TLR7/8 agonist i.e., preferably IM3, Formula (Ik); see the Examples herein).
  • the imidazoquinoline-type compounds of this disclosure can be used to induce an innate immune response, reduce one or more immune suppressive mechanisms, and/or stimulate a cell-mediated immune response (e.g., T cells, natural killer (NK) cells, and other like cells) at the site of administration, while limiting, reducing or avoiding a systemic proinflammatory response to the compositions.
  • a cell-mediated immune response e.g., T cells, natural killer (NK) cells, and other like cells
  • the imidazoquinoline-type compounds of this disclosure are ligands that can activate (i.e., are agonists for) toll-like receptor 7 (TLR7) and/or TLR8 (i.e., TLR7 or dual TLR7/TLR8 agonists) to induce an immunological response.
  • TLR7 toll-like receptor 7
  • TLR8 i.e., TLR7 or dual TLR7/TLR8 agonists
  • the imidazoquinoline-type compounds of this disclosure are agonists for TLR7 and/or TLR8.
  • the imidazoquinoline-type compounds of this disclosure are chosen from compounds of Formulas (Ik), (II), and (Im), as well as derivatives and/or analogues thereof, and pharmaceutically acceptable salts thereof, where the imidazoquinoline-type compound is capable of activating TLR7 and/or TLR8 (i.e., is a TLR7 and/or TLR8 agonist).
  • the imidazoquinoline-type compounds of this disclosure can be used to treat any condition associated with the activity of TLR7 and/or TLR8 such as, but not limited to, pathogen-associated molecular pattern (PAMP)-associated conditions, autoimmune disorders (e.g., rheumatic disorders), such as systemic lupus erythematosus (SLE), the regulation of antiviral immunity (e.g., against influenza viruses, Sendai virus, Cox sack ic B virus, hepatitis virus (e.g., hepatitis C (HCV)), human immunodeficiency virus (HIV)), cancer (e.g., anti-tumor immunotherapy), warts, actinic keratoses, and/or infections such as pulmonary tuberculosis, to induce the expression of cytokines such as interleukin- 12 (IL-12) (e.g., anti-tumor immunotherapy), or as a vaccine adjuvant,.
  • IL-12 interleuk
  • the imidazoquinoline-type compounds of this disclosure can be used to induce and/or enhance an immune response against an immunogen (e.g., the aforementioned peptide antigen moiety or a protein antigen moiety) derived from (or inducing an immune response against the immunogen and/or an organism or cell expressing the same, e.g., as an agonist of TLR7 and/or TLR8) of one or more viruses (e.g., viral target antigen(s)) including, for example, a double- stranded (dsDNA) virus (e.g.
  • an immunogen e.g., the aforementioned peptide antigen moiety or a protein antigen moiety
  • viruses e.g., viral target antigen(s)
  • dsDNA double- stranded
  • adenovirus adenovirus
  • herpesvirus Epstein-Barr virus
  • herpes simplex type 1 herpes simplex type 2
  • human herpes virus simplex type 8 human cytomegalovirus, varicella-zoster virus, poxvirus
  • single-stranded DNA (ssDNA) virus e.g., parvovirus, papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6 ⁇ In Papillomavirus and Human Cancer, edited by H. Pfister (CRC Press, Inc. 1990); Lancaster et ah, Cancer Metast. Rev. pp.
  • papillomavirus e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV1, BPV2, BPV3, BPV4, BPV5 and BPV6
  • double-stranded RNA (dsRNA) virus e.g., reovirus
  • single stranded RNA e.g., (+)ssRNA viruses
  • picomavirus coxsackie virus
  • coxsackie virus hepatitis A virus
  • poliovirus togavirus
  • rubella virus poliovirus
  • flavivirus hepatitis C virus
  • yellow fever virus dengue virus
  • west Nile virus coronavirus (e.g., severe acute respiratory syndrome eoronavirus 2 (SARS-CoV-2)
  • SARS-CoV-2 severe acute respiratory syndrome eoronavirus 2
  • ssRNA viruses e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rhabdovirus, rabies virus
  • ssRNA-RT viruses e.
  • bacterial species e.g., bacterial target antigen(s)
  • Bacillus spp. e.g., Bacillus anthracis
  • Bordetella spp. e.g., Bordetella pertussis
  • Borrelia spp. e.g., Borrelia burgdorferi
  • Brucella spp. e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis
  • Chlamydia spp. e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis
  • Clostridium spp. e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani
  • Corynebacterium spp. e.g., Cory neb acterium diptheriae
  • Enterococcus spp. e.g., Enterococcus faecalis, Enterococcus faecum
  • spp. Escherichia coli
  • Francisella spp. e.g., Francisella tularensis
  • Haemophilus spp. e.g., Haemophilus influenza
  • Helicobacter spp. e.g., Helicobacter pylori
  • Legionella spp. e.g., Legionella pneumophila
  • Leptospira spp. e.g., Leptospira interrogans
  • Listeria spp. e.g., Listeria monocytogenes
  • Mycobacterium spp. e.g., Mycobacterium leprae, Mycobacterium tuberculosis
  • Neisseria spp. e.g., Neisseria gonorrhea, Neisseria meningitidis
  • Pseudomonas spp. e.g., Pseudomonas aeruginosa
  • Rickettsia spp. e.g., Rickettsia rickettsii
  • Salmonella spp. e.g., Salmonella typhi, Salmonella typhinurium
  • Shigella spp. e.g., Shigella sonnei
  • Streptococcus spp. e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes
  • Treponema spp. e.g., Treponema pallidum
  • Vibrio spp. e.g., Vibrio cholerae
  • Yersinia pestis one or more parasitic organisms (spp.) (e.g., parasite target antigen(s)) including, for example, Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoelium dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp., Echinococcus spp. (e.g., E. granulosus, E.
  • parasitic organisms e.g., parasite target antigen(s)
  • Ancylostoma spp. e.g., A. duodenale
  • Anisakis spp. Ascaris lumbricoides
  • Fasciola spp. e.g., F. hepatica, F. magna, F. gigantica, F. jacksoni
  • Fasciolopsis buski Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp. (M. conjunctus, M. albidus), Necator americanus, Oestroidea spp.
  • Onchocercidae spp. Opisthorchis spp. (e.g., O. viverrini, O. felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S. japonicum, S. mekongi, S.
  • T. saginata T. solium
  • Toxocara spp. e.g., T. canis, T. cati
  • Toxoplasma spp. e.g., T.
  • tumor antigens such as cancer-testis (CT) antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens (i.e., Melan A/MART-1, tyrosinase, gplOO); mutational antigens (i.e., MUM-1, p53, CDK-4); overexpressed ‘self antigens (i.e., HER-2/neu, p53); and, viral antigens (i.e., HPV, EBV).
  • CT cancer-testis
  • melanocyte differentiation antigens i.e., Melan A/MART-1, tyrosinase, gplOO
  • mutational antigens i.e., MUM-1, p53, CDK-4
  • overexpressed ‘self antigens i.e., HER-2/neu, p53
  • viral antigens i.e., HPV, EBV
  • Suitable TAs include, for example, gplOO (Cox, et al., Science , 264: 716-719 (1994)), MART-l/Melan A (Kawakami, et al., ./. Exp. Med., 180: 347- 352 (1994)), gp75 (TRP-1) (Wang, et al., J Exp. Med., 186:1131-1140 (1996)), tyrosinase (Wolfel, et al., Eur. J.
  • pl5 Robots, et al., J. Immunol. 154: 5944-5950 (1995)
  • B-catenin Robots, et al., J. Exp. Med., 183: 1185-1192 (1996)
  • MUM-1 Muslie, et al., Proc. Natl. Acad. Sci. USA, 92: 7976-7980 (1995)
  • CDK4 cyclin dependent kinase-4 (CDK4)
  • p21 -ras Feossum, et al., Int. J.
  • BCR-a ⁇ Z Bocchia, et al., Blood, 85: 2680-2684 (1995)
  • p53 Theobald, et al., Proc. Natl. Acad. Sci. USA, 92: 11993-11997 (1995)
  • p 185 HER2/neu erb-Bl; Fisk, et al., J. Exp. Med., 181: 2109-2117 (1995)
  • EGFR epidermal growth factor receptor
  • CEA carcinoembryonic antigens
  • This disclosure also provides methods for inducing a cell-mediated immune response using the imidazoquinoline-type compounds of the disclosure.
  • the methods comprise locally administering a liquid form of the pharmaceutical composition to the subject.
  • the methods induce an immune response (e.g., anti tumor immune response) when administered intratum orally or peri turn orally.
  • the methods induce a cell mediated immune response in the mucosa and/or bronchial tissue when administered nasally.
  • this disclosure provides imidazoquinoline-type compounds for use in a method of treating the human or animal; a pharmaceutical composition comprising an imidazoquinoline-type compound of the disclosure, and a pharmaceutically acceptable carrier or diluent; a pharmaceutical composition of this disclosure for use in a method of treating the human or animal; a pharmaceutical composition of this disclosure for use in stimulating a cell-mediated immune response of an animal or human to a host antigen (e.g. tumor antigen); and the imidazoquinoline-type compound of this disclosure for use in the manufacture of a medicament for stimulating a cell mediated immune response to a host antigen.
  • a host antigen e.g. tumor antigen
  • the methods depend on the activity of TLR7 and/or TLR8 which are induced by the imidazoquinoline-type compounds of this disclosure.
  • Methods for making the imidazoquinoline-type compounds of this disclosure, and compositions comprising the same, are also provided herein. Other embodiments are also contemplated herein as would be understood by those of ordinary skill in the art.
  • the term "about” is used to refer to an amount that is approximately, nearly, almost, or in the vicinity of being equal to or is equal to a stated amount, e.g., the state amount plus/minus about 5%, about 4%, about 3%, about 2% or about 1%.
  • administering is meant introducing a compound of the present disclosure into a subject; it may also refer to the act of providing a composition of the present disclosure to a subject (e.g., by prescribing).
  • therapeutically effective amount refers to that amount of the compound being administered which will induce an immune response, in preferred embodiments a cell-mediated immune response (e.g., a T cell response).
  • a cell-mediated immune response e.g., a T cell response
  • the term also refers to an amount of the present compounds that will relieve or prevent to some extent one or more of the symptoms of the condition to be treated.
  • a therapeutically effective amount refers to that amount which has the effect of preventing the condition/disease from occurring in an animal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the condition/disease (prophylactic treatment), alleviation of symptoms of the condition/disease, diminishment of extent of the condition/disease, stabilization (e.g., not worsening) of the condition/disease, preventing the spread of condition/disease, delaying or slowing of the condition/disease progression, amelioration or palliation of the condition/disease state, and combinations thereof.
  • an “effective amount” refers to that amount of the compound being administered which will produce a reaction that is distinct from a reaction that would occur in the absence of the compound.
  • an “effective amount” is that amount which increases the immunological response in the recipient over the response that would be expected without administration of the compound.
  • animal refers to mammalian subjects, including humans, horses, dogs, cats, pigs, livestock, and any other mammal, along with birds. As referred to herein the term “animal” also includes an individual animal in all stages of development, including newborn, embryonic and fetal stages. In embodiments, a present subject is a human.
  • the term “host” or “organism” as used herein includes humans, mammals (e.g., cats, dogs, horses, etc.), insects, living cells, and other living organisms.
  • a living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal.
  • Typical hosts to which embodiments of the present disclosure relate are mammals, particularly primates, especially humans.
  • livestock such as cattle, sheep, goats, cows, swine, and the like
  • poultry such as chickens, ducks, geese, turkeys, and the like
  • domesticated animals particularly pets such as dogs and cats.
  • a wide variety of mammals are suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.
  • rodents e.g., mice, rats, hamsters
  • rabbits primates
  • swine such as inbred pigs and the like.
  • body fluids and cell samples of the above subjects are suitable for use, such as mammalian (particularly primate such as human) blood, urine, or tissue samples, or blood, urine, or tissue samples of the animals mentioned for veterinary applications.
  • Hosts that are “predisposed to” condition(s) can be defined as hosts that do not exhibit overt symptoms of one or more of these conditions but that are genetically, physiologically, or otherwise at risk of developing one or more of these conditions.
  • the term “moiety” means a chemical group on a compound or capable of being coupled to a compound that includes a functional group/subunit.
  • a “moiety” may include a compound with a specific function that is a part of a larger compound or capable of being coupled to a different compound to form a larger compound.
  • protein typically refers to the end product of transcription, translation, and post-translation modifications in a cell.
  • a “polypeptide” can refer to a “protein” or a “peptide”.
  • the peptide of the DM does not comprise a known T or B cell epitope and was not designed to be bound by an antibody.
  • peptide or “polypeptide” as used herein refers to proteins and fragments thereof. Peptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus.
  • amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
  • the peptides of the imidazoquinoline-type compounds having the formula DM-L-IM are not derived from nature, but the sequences are designed de novo.
  • the DM portion of the imidazoquinoline-type compound does not comprise peptides which are derivable from the naturally occurring sequences of a protein.
  • a peptide is said to be “derivable from a naturally occurring amino acid sequence” if it can be obtained by fragmenting a naturally occurring sequence, or if it can be synthesized based upon knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) that encodes this sequence.
  • compositions, formulations and methods of the present invention may comprise, consist essentially of, or consist of the components and ingredients of the present invention, as well as other ingredients described herein.
  • Consisting essentially of means that the compositions, formulations and methods may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed compositions, formulations and methods.
  • the term “configured” describes a system, apparatus, or other structure that is constructed or configured to perform a particular task or adopt a particular configuration.
  • the term “configured” can be used interchangeably with other similar phrases such as arranged and configured, constructed and arranged, adapted and configured, adapted, constructed, manufactured and arranged, and the like.
  • “pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.
  • inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.
  • a “pharmaceutical composition” refers to a mixture of one or more of the compounds described herein, derivatives thereof, or pharmaceutically acceptable salts thereof, with other chemical components, such as pharmaceutically acceptable carriers and excipients.
  • One purpose of a pharmaceutical composition is to facilitate administration of a compound to the organism.
  • a “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • TLR7 and/or TLR8 refer to a single stranded RNA (ssRNA) or synthetic small molecule that binds to or activates the TLR.
  • ssRNA single stranded RNA
  • TLR7 and TLR8 are innate immune receptors present in the endosomal compartment that are activated by ssRNA molecules of viral, as well as non-viral origin, inducing the production of inflammatory cytokines necessary for the development of adaptive immunity.
  • the main target cells of TLR7 agonists are plasmacytoid dendritic cells, producing IFN-a and thus acting on other immune cells.
  • dendritic cells acquire enhanced costimulatory and antigen-presenting capacity, priming an adaptive immune response.
  • antigen-specific T cells are the main terminal effectors of TLR7 agonists in tumor therapy.
  • Molecules that induce TLR7/8 represent potential cancer vaccine targets that can activate a host immune system against cancer antigens present in tumors.
  • Synthetic small molecule agonists of TLR7 and/or TLR8 that could in some embodiments be used with the imidazoquinoline-type compounds of this disclosure can, for instance, include the imidazoquinoline class of compounds such as gardiquimod [l-(4-amino- 2-((ethylamino)methyl)-lH-imidazo[4,5-c]quinolin-l-yl)-2-methylpropan-2-ol], imiquimod (l-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine), resiquimod/R848 (l-(4-amino-2- (ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl)-2-methylpropan-2-ol), 4-amino-2-(ethoxy- methyl)-lH-Imidazo[4,5-c]quinoline-l-butanamine, l-[[4-(aminomethyl)phenyl]-methyl
  • TLR7 and/or TLR8 agonist indicates a compound that induces a receptor molecule, for instance, a ligand that binds with and activates a TLR7 and/or TLR8 receptor molecule.
  • TLR7 and/or TLR8 agonist refers to a synthetic molecule that acts as a ligand for TLR7 and/or TLR8, including the imidazoquinoline-type compounds of this disclosure and derivatives thereof, such as nucleoside analogs (e.g., nucleoside bases, e.g., adenosine).
  • the imidazoquinoline- type compound compounds of this disclosure are ligands that can activate TLR7 and/or TLR8 (i.e., TLR7 or dual TLR7/TLR8 agonists), and thereby induce the same to generate an immunological response.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (e.g., not worsening) of disease, delaying or slowing of disease progression, substantially preventing spread of disease, amelioration or palliation of the disease state, and remission (partial or total) whether detectable or undetectable.
  • stabilization e.g., not worsening
  • substantially preventing spread of disease amelioration or palliation of the disease state
  • remission partial or total
  • “treat”, “treating”, and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • prophylactically treat or “prophylactically treating” refers completely, substantially, or partially preventing a disease/condition or one or more symptoms thereof in a host.
  • “delaying the onset of a condition” can also be included in “prophylactically treating” and refers to the act of increasing the time before the actual onset of a condition in a patient that is predisposed to the condition.
  • a “vaccine” can include an antigen or vector, along with other components of a vaccine formulation, including for example adjuvants, slow release compounds, solvents, etc.
  • vaccines are traditionally used to prevent or treat infectious diseases, vaccines are also able to modify the function of metabolites by binding signaling peptides or proteins or their receptors and by blocking antigens unique to certain abnormal cell types, such as for example, tumors. Accordingly, it is an embodiment of the invention to provide vaccines that improve immune response to any antigen regardless of the antigen source or its function, including antigens to alter physiological functions that are desirable to improve health, such as immunizing against cancer.
  • the imidazoquinoline-type compounds of this disclosure are formulated as a cancer vaccine wherein the imidazoquinoline-type compounds induce a cell mediated immune response against antigens present within tumors.
  • the imidazoquinoline-type compounds of this disclosure can be synthesized by or using any of Schemes 1-6 as illustrated in Figs. 1-6, and/or as described in Examples (e.g., using one or more of Methods A through F).
  • Method A can be used for the imidazoquinoline-type compound of Formula (Ik)
  • Methods B, C, D or E in conjunction with Method F can be employed to synthesize the imidazoquinoline-type compound of Formulas (II) and (Im).
  • Other methods may also be suitable as would be understood by those of ordinary skill in the art.
  • a solution of 2,4-dichloro-3-nitroquinoline (1, 1.0 eq) in anhydrous dichloromethane (DCM), triethylamine (EtaN) and the mono-protected diamine (2) can be used to produce purified compound 3; after which catalytic amounts of 10% platinum on carbon (10% Pt/C) and sodium sulfate (Na2S04) can be used to obtain crude compound 4 from compound 3; after which Et3N and acid chloride (5) can be used to produce crude compound 6 from compound 4; after which methanol (MeOH), ammonia (NH3), pressure and heat can be used to produce compound 7 from compound 6.
  • Variants of this method may also be suitable as may be determined by those of ordinary skill in the art using routine techniques.
  • aminomalononitrile p-toluenesulfonate (8) can be treated with Et3N, followed by the addition of orthoformate (10), and heated, after which additional orthoformate (10) can be added followed by additional heating. After cooling, Et3N and then l-amino-2-methylpropan-2-ol (9) (produced, e.g., as shown in Fig.
  • Catalyst e.g., palladium acetate (Pd(OAc) 2
  • Pd(OAc) 2 palladium acetate
  • Catalyst can then be introduced into a suspension with compounds 12, 13 and Na 2 CC> 3 , followed by processing (e.g., heating, cooling, diluting, extracting, washing, drying, filtering, concentrating and purifying) to produce compound 14.
  • Compound 14 can then be mixed with HC1 in dioxane, heated, cooled, and concentrated, followed by taking the residue up in MeOH (e.g., 10%) in EtOAc.
  • MeOH e.g. 10%
  • a number of substituted 2-aminophenylboronic acids (13) in which R 3 is H, Me, Et, iPr, tBu, cyclopropyl, CF 3 , F, Cl, Br, N0 2 , OPGi, OMe, OCF3, NHPG2, NMePG 2 , NEtPG 2 , NHCOMe, CN, CO2PG3, C0 2 Me, C0 2 Et, C0 2 iPr, CONHMe, CONHEt, or S0 2 Me; and R 4 is H, Me, CF3, F, Cl, NO2, OPGi, OMe, OCF3, CN, CO2PG3; where PGi is H, tBu, CH 2 Ph, TBDMS, COMe; PG2 H, Alloc, Boc, Cbz, Fmoc; and PG3 is H, tBu, or CH2PI1, can be used and are available commercially.
  • Ortho esters (10) can be used where, for example, R2 is CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, CH2OCH2CH3, CH2CH2OCH3, and can be made relatively easily using the Pinner reaction (McElvain, S.M.; Nelson, W. J. Am. Chem. Soc. 1942, 64, 1825-1827; Roger, R.; Neilson, D.G. Chem. Rev. 1961, 61, 179-211; Noe, M.; Perosa, A.; Selva, M. Green Chem.
  • Aryl boronic acids can also be prepared from organometallic reagents (i.e. Grignard reagents, organolithium compounds) and trialkyl borates (“Synthesis of Organoboronic Acids, Organoboronates, and Related Compounds” Chapter 6, in Practical Functional Group Synthesis, Stockland, R.
  • imidazoquinolines with different Nl- substituents can also be made through the process outlined in Method B through the use of different amino alcohols or diamines (Lason, P.; Kucaba, T.A.; Xiong, Z.; Olin, M.; Griffith, T.S.; Ferguson, D.M. ACS Med. Chem. Lett. 2017, 8, 1148-1152), such as 2. Variants of such methods may also be suitable as may be determined by those of ordinary skill in the art using routine techniques.
  • Compound 21 can then be reacted with Et3N and the acid chloride (5) (preferably one of acid chlorides 5a, 5b, 5c, 5e, 5f, 5g, 5h, 5i and 5j (Fig. 16)) to produce compound 22; which is then mixed with ME-MeOH and processed (e.g., using pressure) to produce compound 15 (e.g., imidazoquinoline 15a, 15b, 15c, 15e, 15f, 15g, 15h, 15i and 15j using acid chlorides 5a, 5b, 5c, 5e, 5f, 5g, 5h, 5i and 5j respectively (Fig. 3)).
  • the acid chloride (5) preferably one of acid chlorides 5a, 5b, 5c, 5e, 5f, 5g, 5h, 5i and 5j (Fig. 16)
  • compound 15 e.g., imidazoquinoline 15a, 15b, 15c, 15e, 15f, 15g, 15h, 15i and 15j using acid chlorides
  • acid chloride 5k provides imidazoquinoline 15k possessing an N-Cbz protecting group, which can be removed employing the same procedure described in Step E-12 (Example 1) to yield 15d.
  • Variants of these methods may also be suitable as may be determined by those of ordinary skill in the art using routine techniques.
  • synthesis of 15 begins with the nitration of 4-hydroxy quinoline (23) using the method of Step C-l (Example 1); the resulting 3-nitro-4-hydroxy quinoline (24) is then subjected to a sequence involving chlorination (Step C-2 (Example 1)) to provide compound 25; followed by reduction of the nitro group with Raney nickel in ethanol (e.g., Step C-4 (Example 1)) to give compound 26, and finally acylation (Step C-5 (Example 1) to yield compound 27; high temperature and concentrated reaction conditions are then used to produce an acid-induced, dehydrative ring closure of compound 27 experienced essentially simultaneously with displacement of the chloride by the amine of l-amino-2-methylpropan-2-ol (9) to produce compound 28; to which is then added meta-chloroperoxybenzoic acid and further processed to produce 29; which is then treated with concentrated ammonium hydroxide (NEEOH
  • This process can be employed with acid chlorides 5a, 5b, 5c, 5e, 5f, 5g, 5h, 5i and 5j to give the imidazoquinolines 15a, 15b, 15c, 15e, 15f, 15g, 15h, 15i and 15j, respectively (Fig. 4). Similar to Method C (Example 1), the procedure of Step E-12 (Example 1) can be employed to convert 15k (made from 5k) into 15d. Variants of these methods may also be suitable as may be determined by those of ordinary skill in the art using routine techniques.
  • 2-nitroacetaldehyde oxime is prepared, acidified with HC1 (cone.), added to anthranilic acid (30), and processed to produce product 31; which is then processed using acetic anhydride then potassium acetate to obtain 3-nitroquinolin-4-ol (24); compound 24 is then processed using phosphorous oxychloride, heated, evaporated, filtered, washed, dried and trituration of the residue with diethyl ether to gives 4-chloro-3-nitroquinoline (25); compound 25 is then treated with l-amino-2-methylpropan-2-ol (9) and N,N-diisopropylethylamine (DIPEA) in toluene and isopropanol (iPrOH) with heat to produce a precipitate which is cooled, filtered and washed sequentially with toluenedPrOH (7:3), diethyl ether
  • 35 is dissolved in anhydrous DCM and benzoyl isocyanate added, and then the mixture is heated, followed by concentration in vacuo and dissolution in anhydrous MeOH; excess NaOMe is the added, the reaction is refluxed for 2-3 h, and following evaporation, the crude residue is purified using flash chromatography to obtain 15.
  • acids 34a, 34b, 34c, 34e, 34f, 34g, 34h, 34i and 34j can be employed with acids 34a, 34b, 34c, 34e, 34f, 34g, 34h, 34i and 34j to give the imidazoquinolines 15a, 15b, 15c, 15e, 15f, 15g, 15h, 15i and 15j, respectively.
  • Imidazoquinolines 15k and 151 accessed from acids 34k and 341, respectively, are deprotected as described below using the procedures in Steps E-ll (Example 1) and E-12 (Example 1), respectively, to yield 15d. Variants of these methods may also be suitable as may be determined by those of ordinary skill in the art using routine techniques.
  • activated moieties can also be used here, such as pentafluorophenyl (OPfp) or succinimide (OSu).
  • the protecting group (PG) on 41 is preferably orthogonal to the Boc (i.e. Cbz, Fmoc, Alloc), but could also even be Boc, since at this stage, the 4-amino group does not require protection as its free state is not expected to interfere with subsequent transformations, so its removal should not be detrimental.
  • this disclosure provides pharmaceutical compositions comprising the imidazoquinoline-type compounds of the disclosure and at least one pharmaceutical acceptable carrier and/or diluent.
  • the composition comprises imidazoquinoline-type compounds of the formula of Formula (Ik), (II), or (Im) and at least one pharmaceutical acceptable carrier and/or diluent.
  • the disclosure provides pharmaceutical compositions comprising an imidazoquinoline-type compound having the structure of Formula (I) (DM-L-IM) and a pharmaceutical acceptable carrier or diluent, wherein the imidazoquinoline-type compound is soluble in an aqueous solution having a pH range of 3 to 9 or an ion concentration ranging from 0 mM to 600 mM, independent of the pH of the solution.
  • the diluent may comprise a stabilizer or bulking agent necessary for efficient lyophilization. Examples include sorbitol, mannitol, polyvinylpyrrolidone, trehalose, lactose, sucrose, glucose, polyethylene glycol and mixtures thereof, preferably mannitol.
  • Other excipients that may be present include preservatives such as antioxidants, lubricants, cryopreservatives and binders well known in the art.
  • the pharmaceutical compositions of the invention can be prepared in any standard manner known in the art.
  • the components of the pharmaceutical composition may be solubilized to disperse the components and form a clear, homogeneous solution.
  • This solution may be sterilized, such as by filtration, and then dried.
  • the term “solubilization” is used herein to mean the dispersion of the compound, and optionally other components of the composition, in a solvent to form a visually clear solution that does not lose material upon sterile filtration.
  • dispenser is meant dissolution of the compound, and optionally other components of the composition, in order to disrupt particulates and achieve solubility.
  • the input components for the pharmaceutical composition may be blended homogenously together to the desired ratios with any aggregates dispersed, rendered sterile and presented in a suitable format for administration.
  • Such examples could include the introduction of a vortexing and/or sonication post-blending or post-dilution stage to facilitate solubilization.
  • Other permutations of the manufacturing process flow could include sterile filtration being performed at an earlier stage of the process or the omission of lyophilization to permit a liquid final presentation.
  • solvents that may be used in some embodiments to disperse the compound in the blend include phosphate buffered saline (PBS), propan-2-ol, tert-butanol, acetone, acetic acid and other organic solvents.
  • each solvent used is typically able to solubilize the component it is being used to solubilize at relatively high concentrations (for example, up to 10 millimolar, such as up to 2 millimolar); water-miscible to facilitate dilution with water prior to lyophilization; compatible with lyophilization stabilizers, such as mannitol, that may be used in the manufacturing process; has a safety profile acceptable to the pharmaceutical regulatory authorities, for example, complies with the requirements of ICH Q3C (Note for Guidance on Impurities: Residual Solvents) and the requirements of Class III solvents, as defined by USP Residual Solvents ⁇ 467> (residual solvent limit of 50 mg/day in finished product or less than 5000 ppm or 0.5%); amenable to lyophilization, that is, sufficiently volatile to be removed to safe levels upon lyophilization; able to disperse the component molecules efficiently in a reproducible and uniform manner such that
  • the solution of the compound and optionally other components may be diluted.
  • the blend may be diluted in water.
  • Variations to the process flow are permitted, as known to one skilled in the art, to achieve the same resulting product characteristics; namely, that the input components are blended homogenously together to the desired ratios with any aggregates dispersed, rendered sterile and presented in a suitable format for administration. Such examples could include the introduction of a vortexing and/or sonication solubilization or post-dilution stage to facilitate solubilization.
  • Other permutations of the manufacturing process flow could include sterile filtration being performed at an earlier stage of the process or the omission of lyophilization to permit a liquid final presentation.
  • the solution containing the compound is preferably sterilized. Sterilization is particularly preferred where the formulation is intended for in vivo use. Any suitable means of sterilization may be used, such as heat sterilization, UV sterilization irradiation or filter sterilization. Preferably, filter sterilization is used. Sterile filtration may include a 0.45 pm filter followed by a 0.22 pm sterilizing grade filter train. Sterilization may be carried out before or after addition of any excipients and/or carriers.
  • the composition may be stored in a container, such as a sterile vial or syringe, prior to use.
  • the pharmaceutical composition may be in dried, such as lyophilized, form.
  • the composition of the invention may be an aqueous solution, for example an aqueous solution formed by dissolving a lyophilizate or other dried formulation in an aqueous medium.
  • the aqueous solution is typically pH neutral.
  • drying the formulation can facilitate long-term storage. Any suitable drying method may be used. Lyophilization is preferred but other suitable drying methods may be used, such as vacuum drying, spray drying, spray freeze-drying or fluid bed drying. The drying procedure can result in the formation of an amorphous cake within which the compound of the invention is incorporated.
  • the sterile composition may be lyophilized. Lyophilization can be achieved by freeze-drying.
  • Freeze-drying typically includes freezing and then drying.
  • Pharmaceutically acceptable compositions of the invention may be solid compositions.
  • the composition may be obtained in a dry powder form.
  • a cake resulting from lyophilization can be milled into powder form.
  • a solid composition according to the invention thus may take the form of free-flowing particles.
  • the solid composition typically is provided as a powder in a sealed vial, ampoule or syringe. If for inhalation, the powder can be provided in a dry powder inhaler.
  • the solid matrix can alternatively be provided as a patch.
  • a powder may be compressed into tablet form.
  • the dried, for example, lyophilized, composition may be reconstituted prior to administration.
  • substitution is understood to mean dissolution of the dried pharmaceutic composition product prior to use.
  • the compound preferably is reconstituted to form an isotonic, pH neutral, homogeneous suspension.
  • the formulation is typically reconstituted in the aqueous phase, for example by adding Water for Injection, histidine buffer solution (such as 28 mM L-histidine buffer), sodium bicarbonate, Tris-HCl or phosphate buffered saline (PBS) in the presence or absence of additional ions.
  • the reconstituted formulation is typically dispensed into sterile containers, such as vials, syringes or any other suitable format for storage or administration.
  • the composition may be stored in a container, such as a sterile vial or syringe, prior to use.
  • the imidazoquinoline-type compounds of this disclosure and pharmaceutical compositions thereof are suitable for administration to a subject.
  • the imidazoquinoline-type compounds can be used to induce and/or enhance an immunological response in the subject to a host antigen.
  • the imidazoquinoline-type compounds act as TLR7 and/or TLR8 agonists that induce immunological responses and/or other immunological responses such as, but not limited to, stimulating interferons (IFN-g).
  • IFN-g stimulating interferons
  • an “immunological response” refers to a response by the host's immune system to a stimulus.
  • the administration of the imidazoquinoline-type compounds of this disclosure activates the host immune system.
  • the methods are for inducing a cell mediated immune response in a subject.
  • the immunological response or immune responses resulting from administration of an effective amount or therapeutically effective amount referred to herein can be determined using any available methods and preferably those used in the Examples herein including, for example, an automated ELISpot reader system (CTL) (e.g., to determine spot forming cells (SFC) from splenocytes), cytokine measurements using a cytokine multiplex kit (e.g., Luminex analyzer (Luminex Corp., Austin, TX)) and/or using standard curve data (e.g., MasterPlexTM QT Analysis version 2 (MiraiBio, Alameda, CA)), in vitro agonist assays (e.g., using HEK-293 cell lines that functionally overexpress human or murine TLR proteins as well as a reporter gene which is a secreted alkaline phosphatase (SEAP); TLR7, TLR8 assays using,
  • the compounds disclosed herein can reduce or abolish the producing of cytokines such as CCL5, IL6, IFN-a, INF-b, CCL2, IL12 and compared to the same dose of a reference (e.g., control) compound (e.g., HL-6X2 compared to IMDQ (IM2)).
  • a reference e.g., control
  • the compounds of this disclosure can induce a delay in the CXCL10 response and CXCL9 compared to a reference (e.g., control) compound as determined using the assays mentioned above or that may be otherwise available to those of ordinary skill in the art.
  • the compounds of this disclosure can provide approximately five to approximately 10-fold higher activity as compared to a reference compound (see, e.g., Figs. 12-13).
  • the compounds of this disclosure induce a protective immune response against pathogens.
  • the compounds of this disclosure induce a therapeutic anti-tumor response.
  • Other methods for determining whether the compounds of this disclosure are also available as would be understood by those of ordinary skill in the art.
  • the imidazoquinoline-type compounds of this disclosure can be used to treat any condition associated with the activity of TLR7 and/or TLR8 such as, but not limited to, a pathogen-associated molecular pattern (PAMP)-associated conditions, autoimmune disorders (e.g., rheumatic disorders) such as systemic lupus erythematosus (SLE), the regulation of antiviral immunity (e.g., against influenza viruses, Sendai virus, Coxsackie B virus, hepatitis virus (e.g., hepatitis C (HCV)), human immunodeficiency virus (HIV)), cancer (e.g., anti-tumor immunotherapy), to induce the expression of cytokines such as interleukin- 12 (IL-12) (e.g., anti-tumor immunotherapy), warts, actinic keratoses, and/or infections such as pulmonary tuberculosis, among other conditions.
  • PAMP pathogen-associated molecular pattern
  • the methods can comprise locally administering a liquid form of the present pharmaceutical composition (comprising an imidazoquinoline-type compound of this disclosure) into the subject.
  • the imidazoquinoline-type compounds of this disclosure can be administered via intratumoral (I.T.) and induce an immune response against the tumor antigens present in the tumor.
  • the imidazoquinoline-type compounds of this disclosure may stimulate and/or “enhance” an immunological response to the tumor by reducing local tumor immune suppression, wherein suppressive and/or protumoral myeloid cells (such as MDSC, TAM and/or M2 cells) are converted into anti tumor myeloid cells (Ml).
  • the pharmaceutical compositions are used as a vaccine to induce an anti-tumor immune response.
  • such compositions include an imidazoquinoline-type compound of the present disclosure in an amount effective to treat the tumors.
  • the pharmaceutical compositions are used as a vaccine for cancers with solid tumors.
  • compositions include an imidazoquinoline- type compound of this disclosure in an amount effective to treat the cancers.
  • the cancers are selected from head and neck cancer, ovarian cancer, breast cancer, colon cancer, lung cancer, melanoma, gastric cancer, gallbladder cancer, bladder cancer, osteosarcoma, oral cancer, pancreatic cancer, gastric cancer, Merkel cell carcinoma, liver cancer, cervical cancer, kidney cancer, and/or lymphoma.
  • Intratumoral or peritumoral injection of the imidazoquinoline-type compounds of this disclosure can be achieved using needle systems including multipronged array needle, micro- needle or micro-needle array. For deeper lesions, 22-gauge needles may be used and for superficial lesions, needles as small as 30 gauge can be used. Intratumoral injection may be guided using imaging systems including ultrasonography, endoscopic ultrasonography, computed tomography, and/or magnetic resonance imaging (MRI). In some embodiments, the administration to local mucosal body sites may also be addressed through pulmonary administration, nasal administration, buccal administration, or intravesical administration.
  • needle systems including multipronged array needle, micro- needle or micro-needle array. For deeper lesions, 22-gauge needles may be used and for superficial lesions, needles as small as 30 gauge can be used.
  • Intratumoral injection may be guided using imaging systems including ultrasonography, endoscopic ultrasonography, computed tomography, and/or magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • TLR7 and/or TLR8 agonists may also be useful for the treatment of an airway disease.
  • the imidazoquinoline-type compounds of this disclosure i.e., TLR7 and/or TLR8 agonists
  • the imidazoquinoline-type compounds of this disclosure can be delivered through spray, aerosol and/or nebulization to form a depot in the mucosal environment preventing its systemic release, which would be highly beneficial for the treatment of these airway diseases.
  • the imidazoquinoline-type compounds of this disclosure i.e., TLR7 and/or TLR8 agonists
  • this disclosure provides methods for treating and/or preventing tumor growth by administration (i.e., systemic administration and/or intratumoral administration (by, e.g., injecting active agent(s) directly into a tumor) of one or more of the imidazoquinoline-type compound disclosed herein alone and/or with one or more additional anti-tumor agents.
  • administration i.e., systemic administration and/or intratumoral administration (by, e.g., injecting active agent(s) directly into a tumor) of one or more of the imidazoquinoline-type compound disclosed herein alone and/or with one or more additional anti-tumor agents.
  • the pharmaceutical compositions of this disclosure may benefit from concurrent combination with other systemic immunotherapies such as checkpoint inhibitors, adoptive T cell transfer including TILs (tumor-infiltrating lymphocytes) or CAR-T cells, monoclonal antibodies targeting tumor cells, CD3-bi-specific antibodies or T cell receptors, or virotherapy such as oncolytic viruses, vaccine cytokines.
  • the present pharmaceutical compositions may also benefit from combination treatment with other immune stimulants administered intratumorally or systemically such as TLR2, TLR3, TLR5, TLR9, STING, cGAS or NOD agonists, either administered temporally at the same time (co-formulated or not) or at different times.
  • growth factors such as Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, can be used with an imidazoquinoline-type compound of this disclosure (Nuhn, et al. Adv. Matter , 30(45): el803397 (2016)).
  • the one or more systemic immune checkpoint inhibitors can be directed at, without limitation, as PD-1, PD-L1, PD-L2, CD28, CD80, CD86, CTLA-4, B7RP1, ICOS, B7RPI, B7-H3, B7-H4, BTLA, HVEM, KIR, TCR, LAG3, CD 137, CD137L, 0X40, OX40L, CD27, CD70, CD40, CD40L, TIM3, GAIN, ADORA, CD276, VTCN1, IDOl, KIR3DL1, HAVCR2, VISTA or CD244.
  • the immune checkpoint inhibitor is preferably anti-PD-1 and/or anti-CTLA-4, and even more preferably anti-CTLA-4, particularly for extra-tumoral administration (i.e., non-injected tumors, systemic administration of the active agents).
  • a myeloid- derived suppressor cell (MDSC) inhibitor targets PGE-2, COX2, NOS2, ARGl, PI3K, CSF- 1R, caspase-8, CCL2, RON, ROSS100A8/A9 or liver-X nuclear receptor such as, for instance and without limitation, the MDSC inhibitor is PF-5480090, INCB7839, nitro-aspirine, SC58236, Celecoxib, IPI-549, PLX3397, BLZ945, GW2580, RG7155, IMC-CS4, AMG-820, ARRY-382, sildenafil, tadalafil, vardenafil, N-hydroxy-nor-L-Arg, imatinib, z-IETD-FMK, trabectedin, emricasan, anti-CCL2 antibody (carlumab, ABN912), tasquinimod, ASLAN002, IMC-RON8 , or
  • such methods induce tumor regression in tumors (e.g., injected tumors) above that observed in a control group (e.g., to whom or which the imidazoquinoline-type compound(s) were not administered).
  • a control group e.g., to whom or which the imidazoquinoline-type compound(s) were not administered.
  • the imidazoquinoline-type compounds of this disclosure and one or more additional active agents may be administered separately, and in some embodiments together (e.g., physically together and/or simultaneously administered to different anatomical sites).
  • the methods include intratumoral administration of one or more imidazoquinoline-type compounds of this disclosure combined with one or more systemic immune checkpoint inhibitors, either anti-PD-1 or anti-CTLA-4, preferably anti- CTLA-4, to induce tumor stabilization and/or regression in non-injected tumors (e.g., distal tumors such as metastases, a phenomenon generally referred to abscopal effect).
  • the methods result in a surprising synergistic effect of one or more of the imidazoquinoline-type compounds of this disclosure in combination with anti-CTLA-4 is observed).
  • this disclosure also provides methods of using one or more of the imidazoquinoline-type compounds of this disclosure for inducing stabilization of tumor volume over time (an antitumoral activity) with less or without side effects (e.g., in preferred embodiments reduced body weight and/or limiting the off-target side effects leading to the induction of a deleterious systemic burst of pro-inflammatory cytokines; e.g., an improved safety profile) observed following administration of other active agents (e.g., such as 3M-052 and/or R848).
  • other active agents e.g., such as 3M-052 and/or R848
  • this disclosure provides imidazoquinoline-type compounds of Formula (Ik), (II), or (Im) as shown herein wherein: R is -[A1]-NH-, wherein A1 is: A2-A3-(CH 2 ) X -CO-, A2-A3-CH 2 -0-CH 2 -C0-, A2-A3-(CH 2 ) X -0-(CH 2 ) X -0-(CH 2 ) X -CO-, A2-Valine-Alanine-A4-, A2-Valine-Citrulline-A4-, A2-Glutamate-Valine- Citrulline-A4-, or A2-Phenylalanine-Lysine-A4-; A2 is selected from A5-(CH 2 )x-A6-, A5- (CH 2 ) X -0-(CH 2 ) X -0-(CH 2 )
  • R is a linker covalently linking the compound of Formula (Ik), (II), or (Im) to a delivery/depot moiety (DM), wherein: DM comprises a peptide from 17 to 45 amino acids in length comprising amino acid residues possessing helix forming properties and configured to form an amphipathic a-helix structure, and wherein the peptide sequence is not derived from an antigen or immunogen and is a non-natural sequence.
  • this disclosure provides a peptide conjugate comprising or derived from such imidazoquinoline- type compound.
  • the peptide conjugate is Ac-
  • RRLLHALLALLAHLLRRLE PEG 6 - Val-Cit-PAB C-IM3 )-NH 2 where Ac is Acetyl; Peptide is RRLLHALLALLAHLLRRLE (SEQ ID NO. 2; HL-6X3); Val is valine; Cit is citrulline; PEG 6 is -NH-(CH 2 ) 2 -(0-CH 2 -CH 2 ) 6 -C0-; PABC is p-amino-benzyloxycarbonyl; PAB is p- aminobenzyloxy; and, IM3 is Formula (Ik) where R is -NH-, as shown below:
  • the imidazoquinoline-type compounds of this disclosure are capable of activating toll-like receptor 7 (TLR7) and/or toll-like receptor 8 (TLR8).
  • the imidazoquinoline-type compounds include Rs that comprises a reporter moiety comprising a reporter molecule chosen from a fluorescent molecule, an MRI agent, or a combination thereof.
  • the imidazoquinoline-type compounds include R5 that comprises an antigen moiety chosen from a peptide antigen moiety, a protein antigen moiety, or a polysaccharide antigen moiety, and wherein the imidazoquinoline-type compound is capable of activating TLR7 and/or TLR8.
  • the imidazoquinoline-type compounds of this disclosure can be conjugated to or used in conjunction with a nanoparticle, gold nanoparticles (GNPs) immobilized with a-mannose as carrier, hollow silica nanosheil, pH-responsive poly(lactide-co-glycolide) (PLGA) nanoparticle, PLGA hollow microneedle array, adamantane, b-cyclodextrin nanoparticle, and/or adjuvant that optionally comprises aluminum.
  • this disclosure provides vaccine adjuvant(s) comprising an imidazoquinoline-type compound of this disclosure.
  • this disclosure provides self-adjuvanting vaccines comprising an imidazoquinoline-type compound of this disclosure coupled to or associated with an antigen (e.g., a peptide antigen, a protein antigen, a polysaccharide antigen, or other suitable antigen).
  • an antigen e.g., a peptide antigen, a protein antigen, a polysaccharide antigen, or other suitable antigen.
  • this disclosure provides pharmaceutical compositions comprising the compounds and derivatives thereof disclosed herein and at least one pharmaceutical excipient.
  • this disclosure provides methods of immunizing a host comprising: administering to the host an antigen, and administering to the host an adjuvant comprising an imidazoquinoline-type compound of this disclosure, and/or derivative thereof, capable of activation of TLR7 or dual activation of TLR7 and TLR8, or a composition thereof, wherein the imidazoquinoline-type compound is administered with the antigen or is coupled to the antigen.
  • this disclosure provides methods of stimulating an anti -tumor immune response in a subject, comprising: locally administering intratumorally or peritumorally a liquid form of the pharmaceutical composition of this disclosure (preferably HL-6X3) into the subject, wherein in vivo physiological conditions reduce solubility of the DM component of the immunotherapy compound wherein the immunotherapy compounds form insoluble self-assemblies or aggregates in vivo; whereby the insoluble self-assemblies or aggregates induce an anti-tumor cell mediated immune response at the local site of administration and/or at a distant site from the site of administration of the pharmaceutical composition.
  • a liquid form of the pharmaceutical composition of this disclosure preferably HL-6X3
  • this disclosure provides the compounds and compositions disclosed herein for use as a medicament and/or for use in the treatment of a condition and/or disease (e.g., any of those mentioned herein or others as would be understood by those of ordinary skill in the art).
  • this disclosure provides imidazoquinoline-type compounds, compositions, or vaccines for stimulating a systemic anti -turn or immune response in a subject.
  • such methods can further comprise administering a systemic checkpoint inhibitor (e.g., anti -PD- 1 and/or anti- CTLA-4 antibody).
  • this disclosure provides methods of stimulating a systemic anti -tumor immune response in a subject, comprising locally administering intratumorally or peritumorally a liquid form of the pharmaceutical composition of this disclosure (preferably HL-6X3) into the subject, wherein the anti -tumor immune response is effective at a distant site from the site of administration of the pharmaceutical composition.
  • such methods can further comprise administering a systemic checkpoint inhibitor (e.g., an anti-PD-1 and/or anti-CTLA-4 antibody).
  • this disclosure provides probes comprising: a TLR7 ligand or a dual TLR7/TLR8 ligand comprising an imidazoquinoline-type compound of this disclosure, and/or a derivative thereof, and a reporter molecule capable of producing a detectable signal, coupled to the ligand.
  • this disclosure provides methods of imaging activation of TLR7, TLR 8, or both TLR7 and TLR8 comprising: contacting a sample comprising TLR7, TLR8 or both TLR7 and TLR8 with such a probe; and imaging the detectable signal produced by the reporter molecule.
  • Other embodiments are also contemplated and disclosed herein, as will be understood by those of ordinary skill in the art.
  • ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range.
  • the term “about” can include traditional rounding according to significant figures of the numerical value.
  • Scheme 1 presents the preparation of certain members of the imidazoquinoline class of TLR7/8 agonists such as represented by Formula (Ik) (Shukla, N.M.; Mutz, C.A.; Ukani, R.; Warshakoon, H.J.; Moore, D.S.; David, S.A. Bioorg. Med. Chem. Lett. 2010, 20, 6384- 6386). See Figure 1.
  • Step A-l Starting from a solution of 2,4-dichloro-3-nitroquinoline (1, 1.0 eq) in anhydrous dichloromethane (DCM), triethylamine (EtaN, 1.3 eq) and the mono-protected diamine (2, 1.1 eq) are each added sequentially and the resulting mixture is refluxed at 45°C. After 0.5 h, the reaction is allowed to cool to room temperature (r.t.), then evaporated in vacuo. The resulting crude product can be isolated using flash chromatography to produce purified compound 3.
  • DCM dichloromethane
  • Step A-2 To a solution of intermediate 3 in ethyl acetate (EtOAc) is added catalytic amounts of 10% platinum on carbon (10% Pt/C) and sodium sulfate (NaiSCri). The heterogeneous mixture is placed under hydrogen pressure (50-60 psi) for 4-6 h. The reaction is filtered through Celite, the filtered material is then washed with EtOAc (2x), and the combined filtrates evaporated in vacuo to obtain crude 4, typically of sufficient purity to be used for the next transformation.
  • EtOAc ethyl acetate
  • Step A-3 Triethylamine (1.5 eq) and acid chloride (5, 1.2 eq) are added to a solution of 4 (1.0 eq) in anhydrous tetrahydrofuran (THF), then the reaction mixture is stirred at r.t. for 4-8 h. The solvent is then removed in vacuo , and the crude residue taken up in EtOAc and washed with water (2x) and saturated aqueous sodium bicarbonate (NaHCOs). The organic layer is then dried over anhydrous Na2S04 and evaporated in vacuo to obtain crude 6, which can be purified by flash chromatography, but may be of sufficient quality to proceed directly to the final step.
  • THF tetrahydrofuran
  • Step A-4 Compound 6 is dissolved in a minimum amount of methanol (MeOH), treated with an excess of 2M ammonia (NEE) in MeOH, then transferred into a pressure vessel (e.g. Parr). The sealed vessel is heated to 145-150°C overnight (18-24 h). The solvent is then removed in vacuo and the residue purified by flash chromatography or crystallization to yield the desired structure 7.
  • a pressure vessel e.g. Parr
  • the sealed vessel is heated to 145-150°C overnight (18-24 h).
  • the solvent is then removed in vacuo and the residue purified by flash chromatography or crystallization to yield the desired structure 7.
  • the indicated imidazoquinoline-type compound of Formula I(k) can be synthesized from the specific diamine 4-(Boc-aminomethyl)-benzylamine (2), and the acid chloride 3-methoxypropanoyl chloride (5).
  • Scheme 2 outlines the synthesis of selected members of the imidazoquinoline class of TLR7/8 agonists (Shi, C.; Xiong, Z.; Chittepu, P.; Aldrich, C.C.; Ohlfest, J.R.; Ferguson, DM. ACS Med. Chem. Lett. 2012, 3, 501-504; Schiaffo, C.E.; Shi, C.; Xiong, Z.; Olin, M.; Ohlfest, J.R.; Aldrich, C.C.; Ferguson, D.M. J. Med. Chem. 2014, 57, 339-347). See Figure 2
  • Step B-l A suspension of aminomalononitrile p-toluenesulfonate (8, 1.0 eq) in THF is treated with Et3N (1.2 eq) at r.t. After stirring for 0.5 h, the solution became homogeneous and the orthoformate (10, 1.2 eq) added. The mixture is then heated to reflux for 3 h. If the reaction is not complete at that time (TLC), the reaction is removed from heat, additional orthoformate (0.6 eq) introduced and the solution heated at reflux for an additional 2 h. When completed, the mixture is allowed to cool to r.t. to provide a solution containing the intermediate imidate.
  • Step B-2 A solution of 11 (1.0 eq) in diiodomethane is heated to 80°C, then isoamylni trite (4.0 eq) in chloroform (CHCh) is added over a period of 0.25-0.5 h. After the addition is complete, heating is maintained for 0.5 h, then the reaction is allowed to cool to r.t. and the solvent concentrated in vacuo. The crude product is purified by flash chromatography to yield 12.
  • Step B-3 The catalyst is prepared from palladium acetate (Pd(OAc)2, 0.05 eq) and triphenylphosphine (PPI1 3 , 0.1 eq), which are placed together in a dry flask and purged with argon for 0.25 h, then 1,2-dimethyoxy ethane (DME) is added. The resulting suspension is stirred at r.t. for 5-10 min, then 12 (1.0 eq), 13 (1.5 eq) and 1.5 MNaiCCb (aq) (3.0 eq) added sequentially. The reaction is heated at 100°C for 3 h, then cooled to r.t. and the mixture is diluted with EtOAc and ThO.
  • Step B-4 A solution of 4 N HC1 in dioxane (16 eq) is added to 14 (1.0 eq), then heated at reflux for 5 h. The reaction is cooled to r.t. and concentrated in vacuo. The residue is taken up in 10% MeOH in EtOAc and washed with saturated NaHC0 3 (aq). The aqueous layer is extracted with 10% MeOH in EtOAc (3x). The combined organic layers are washed with saturated NaCl (aq), dried over anhydrous MgS0 4 , filtered, and the filtrate is evaporated in vacuo. The crude residue is then purified by flash chromatography to afford the desired imidazoquinoline 15.
  • Step B-5 Synthesis of l-amino-2-methylpropan-2-ol (9)
  • the title compound is prepared using a variation on the literature method (Close, W.J. J Am. Chem. Soc. 1951, 73, 95-98).
  • Isobutylene oxide (2,2-dimethyloxirane, 16, 1.0 eq) is combined with ammonium hydroxide (NH 4 OH, 2 mL/mmol) and MeOH (1 mL/mmol).
  • the mixture is stirred at r.t. for 12 h, and then slowly heated to 60°C and stirred at that temperature for 2-3 h.
  • the solvent is removed in vacuo , and the residue is distilled under atmospheric pressure to provide the title product 9 in low yield.
  • Analogous compounds can be made with different R2, R3 and R4 groups using Method B as well.
  • Scheme 3 outlines the synthesis of selected members of the imidazoquinoline class of TLR7/8 agonists. See Figure 3.
  • Step C-l A mixture of 2,4-dihydroxyquinoline (17) in concentrated nitric acid (HNO 3 , 0.25 mL/mmol) and glacial acetic acid (HO Ac, 1 mL/mmol) is stirred at 105°C for 1 h, then cooled to r.t. The reaction is quenched by the addition of H 2 O, upon which a yellow precipitate is formed. The solid is filtered, washed with cold H 2 O, and then dried to provide
  • HNO 3 concentrated nitric acid
  • HO Ac glacial acetic acid
  • Step C-2 To a solution of 18 (1.0 eq) in phosphorous oxychloride (POCI3, 1.65 mL/mmol) is added EtsN (1.0 eq), then the reaction mixture is heated to 120°C and stirred for 3 h. After cooling, the solvent is removed in vacuo. The residue is poured into ice-water and extracted with DCM (2-3x). The combined organic phase is washed sequentially with saturated NaHCCb (aq) and brine, dried over anhydrous NaiSCri, filtered and the filtrate is concentrated in vacuo to give the desired product 19, which could be used directly in the next step.
  • phosphorous oxychloride POCI3
  • EtsN 1.0 eq
  • Step C-3 To a solution of 19 (1.2 eq) and Et3N (1.5 eq) in DCM is added l-amino-2- methylpropan-2-ol (9, 1.0 eq, from Step B-5) dropwise. The mixture is stirred at reflux for 12 h. The solution is cooled to r.t., then washed with brine, dried over anhydrous NaiSCri, filtered, and the filtrate concentrated in vacuo to obtain crude product 20, which is purified by flash chromatography.
  • Step C-4 A mixture of 20 (1.0 eq) and 10% Pt/C (40 mg/mmol) in EtOAc is placed under a hydrogen atmosphere (50-55 psi) at r.t. for 4 h. The mixture is then filtered through Celite, the solid washed with EtOAc, and the combined filtrate concentrated in vacuo. The resulting crude 21 is purified by flash chromatography or used as is in the next step.
  • Step C-5 To a solution of 21 (1.0 eq) and Et3N (2.0 eq) in DCM is added the acid chloride (5, 1.2 eq (e.g., 5a though 5k)). The reaction is stirred for 3 h, then washed with saturated brine. The organic layer is concentrated in vacuo and the residue purified by flash chromatography to provide the desired product 22.
  • Step C-6 A mixture of compound 22 (1.0 eq) in excess NEE-MeOEl is placed in a pressure vessel, sealed and stirred at 160°C for 8 h. The solvent is then removed in vacuo , and the resulting crude residue is purified by flash chromatography (requires Et3N in the elution solvent) to yield 15.
  • Method D Alternative synthesis route for preparation of pre-cursor 15 a, 15b, 15c, 15d, 15e, 15f 15g, 15h, 15i and 15j compounds
  • Step C-2 The resulting 3-nitro-4-hydroxy quinoline (24) is then subjected to a sequence involving chlorination (Step C-2) to provide 25, reduction of the nitro group with hydrogen gas and Raney nickel in ethanol (e.g., Step C- 4) to give 26, and finally acylation (Step C-5) to yield 27.
  • Step C-5 acylation
  • Step C-5 high temperature, concentrated reaction conditions will lead to an acid-induced, dehydrative ring closure of 27 experienced essentially simultaneously with displacement of the chloride by the amine of l-amino-2-methylpropan-2-ol (9) to produce 28. See Figure 4.
  • Step D-l To a solution of 28 (1.0 eq) in DCM is added meta- chloroperoxybenzoic acid (70% active oxygen, 1.2 eq) at 0°C. The reaction is maintained at 0°C for 0.5 h, warmed to r.t. and stirred for 2 h. The mixture is then concentrated in vacuo. The resulting solid residue is dissolved in ThO and made slightly basic with dilute NaOH (aq), which leads to precipitation of the N-oxide. The solid is collected by vacuum filtration, washed with ThO, and air-dried to give still impure product. The collected solid is suspended in toluene, heated to reflux with stirring in order to azeotropically remove the ThO from the product. Once no further ThO is obtained, the solid is again collected by vacuum filtration, washed with toluene, and is dried to provide the slightly colored product 29.
  • meta- chloroperoxybenzoic acid 70% active oxygen, 1.2 eq
  • Step D-2 A solution of 29 (1.0 eq) in DCM is first treated with concentrated ammonium hydroxide (NThOH, NTT (aq)), followed by the dropwise addition of p-toluene- sulfonyl chloride (Tos-Cl, 1.0 eq) in DCM with vigorous agitation at 0°C over 0.25 h. A clear exothermic reaction is observed, and a solid precipitate formed during addition. Upon completion of the addition, the mixture is maintained at 0°C for 0.5 h, then warmed to r.t. and stirred for 2 h. The precipitate is collected by vacuum filtration, washed with DCM and ThO, then is pressed partially dry. The still moist solid is slurried with MeOH, collected by vacuum filtration, and dried. The solid is treated again with MeOH and refluxed for 5 min, then again collected and dried as before. Crystallization may be required to obtain pure product 15.
  • NThOH, NTT (aq) concentrated
  • Method E Alternative synthesis route for preparation of pre-cursor 15a, 15b, 15c, 15d, 15e, 15f 15g, 15h, 15i and 15j compounds
  • Step E-l Using a route adapted from the literature method for Nl- unsubstituted adenine derivatives (J. Med. Chem. 2006, 49, 3354-3361), 2-nitroacetaldehyde oxime is prepared in situ by adding nitromethane (1.1 eq) dropwise to a solution of NaOH (3.0 eq) in water at 0°C. The mixture is then warmed to 40°C and nitromethane (1.1 eq) is again introduced slowly at that temperature, which is maintained until the solution becomes clear. The reaction mixture is then heated to 50-55°C for 2-5 min, cooled to near r.t., and poured onto ice.
  • nitromethane 1.1 eq
  • This solution is acidified with HC1 (cone.), then immediately added to a filtered solution of anthranilic acid (30, 1.0 eq) in 0.5N HC1 in water.
  • the reaction mixture, from which a precipitate forms, is maintained at r.t. for 12 h.
  • the solid is collected by filtration, washed with water, and dried at 100-110°C to yield 31 as a yellow powder.
  • Step E-2 A heterogeneous mixture of 31 (1.0 eq) in acetic anhydride is heated to 100-105°C until a clear solution is obtained. Heating is removed and potassium acetate (1.03 eq) added. The reaction is then heated to reflux for 0.25 h with vigorous stirring, until a solid begins to form. The mixture is allowed to slowly cool to r.t., then the precipitate collected by filtration and washed with glacial acetic acid until no further color is seen in the wash. The solid is suspended in water, filtered, washed with water and dried at 100-110°C to obtain 3-nitroquinolin-4-ol (24).
  • Step E-3 Similar to Step C-2, 24 (1.0 eq) is carefully added to phosphorous oxychloride (13-15 eq) with stirring. The reaction mixture is heated to reflux for 0.5 h. The volatiles are then evaporated in vacuo and the liquid residue poured into crushed ice with stirring. After 1 h, the solid that is formed is collected by filtration and washed with cold H2O. This is then dissolved in DCM containing a minimum amount of MeOH, washed with ice cold 1 N NaOH (aq), and the organic layer dried over NaiSCri (anhydrous) and activated charcoal. The solution is filtered through Celite, washed with DCM and the combined filtrate evaporated in vacuo. Trituration of the residue with diethyl ether gives 4-chloro-3- nitroquinoline (25) after drying under vacuum.
  • Step E-4 To a solution of 25 (1.0 eq) and N,N-diisopropylethylamine (DIPEA) (2.5 eq) in toluene and isopropanol (iPrOH) (4:1) is added 9 (2.0 eq) and the mixture heated to 70°C for 0.5 h at which time a precipitate forms. The reaction is cooled and the solid collected by filtration, which is then washed sequentially with toluene :iPrOIT (7:3), diethyl ether and cold EhO. The residue is dried at 80°C to obtain 32 of sufficient purity to be used in the next reaction.
  • DIPEA N,N-diisopropylethylamine
  • iPrOH isopropanol
  • Step E-5 32 (1.0 eq) is dissolved in MeOH and hydrogenated using 10% Pd/C as catalyst under a Eh pressure of 50-60 psi for 4 h. The mixture is then filtered using Celite to remove the catalyst and the filtrate evaporated in vacuo to leave 33, which is purified by crystallization or flash chromatography if necessary.
  • DMF dimethylformamide
  • Step E-7 To a solution of 35 (1.0 eq) in DCM:CHCh (1:1) and MeOH (10% by volume) is added meta-chloroperoxybenzoic acid (2.5 eq) and the reaction heated to reflux for 30 min. The mixture is concentrated in vacuo and the residue purified using flash chromatography to give the N-oxide 36.
  • Step E-8 35 (1.0 eq) is dissolved in anhydrous DCM and benzoyl isocyanate (1.5 eq) added, then the mixture heated to reflux for 30 min. The reaction is concentrated in vacuo and the resulting residue dissolved in anhydrous MeOH. Excess NaOMe is added and the reaction again refluxed for 2-3 h. After evaporation of solvent in vacuo , the crude residue is purified using flash chromatography to obtain 15.
  • Step E-9 Synthesis of N-ethyl glycine methyl ester .
  • a solution of methyl glycinate 37 (3.0 eq) in anhydrous methanol is treated with acetaldehyde (1.0 eq), sodium cyanoborohydride (NaCNBEE, 1.0 eq) and 5-6 drops of acetic acid.
  • the resulting reaction is stirred for 12 h.
  • HC1 cone.
  • the solvent is then removed in vacuo and the crude residue purified using flash chromatography to afford 38.
  • Step E-10 Synthesis of Cbz-protected N-ethyl glycine To a solution of 38 (1.0 eq) in THF is added 2N NaOH (3.0 eq) and benzyl chloroformate (1.6 eq). After stirring 1 h at rt, the pH is adjusted to 1-2 and the layers separated. The aqueous phase is extracted with EtOAc (2x). The combined organic layers are dried (anhydrous MgSCE), filtered, and concentrated in vacuo to leave a crude residue, which is then purified by crystallization or flash chromatography to provide 34k.
  • Step E-l 1 Synthesis of Boc-protected N-ethyl glycine .
  • MeOH di-tert-butyl dicarbonate
  • Et3N 1.2 eq
  • the solvent is evaporated in vacuo , then the residue dissolved in THF:MeOH (3:1).
  • An aqueous solution of lithium hydroxide (LiOH, 4.0 eq) is added to the mixture, which is then stirred at r.t. for 12 h. The volatiles are removed in vacuo and H2O added to the residue.
  • Step E-12 Deprotection of Cbz protecting group.
  • a solution of 15k (1.0 eq) in 95% EtOH (0.1 M) with 10% Pd/C (0.1 eq) catalyst is stirred under an atmosphere of Eh for 72 h, after which time the mixture was filtered through a Celite pad. The pad was washed with EtOAc and the combined filtrate and washings are concentrated in vacuo to provide 15d, which is purified by crystallization or flash chromatography.
  • Step E-13 Deprotection of Boc protecting group. 151 (1.0 eq) is dissolved in 3 mL of TFA and stirred at r.t. for 0.5 h. The solvent is removed in vacuo to yield 15d as its trifluoroacetate salt, which can be neutralized and purified by flash chromatography to obtain the pure free base.
  • a standard method for its installation (Boc 2 0, Et 3 N, as in Step E-ll) is employed.
  • the alcohol in the resulting Boc-protected product 39 is then activated as its p-nitrophenyl carbonate by treatment with p-nitrophenyl chloroformate in the presence of base, with two alternative reactions for this transformation shown.
  • Other activated moieties can also be used here, such as pentafluorophenyl (OPfp) or succinimide (OSu).
  • the protecting group (PG) on 41 is preferably orthogonal to the Boc (i.e.
  • Example 2 Evaluation of in vivo Immunological Activity of Imidazoquinoline-type Compounds in Mice
  • Splenocytes are washed, counted and resuspended in complete media (RPMI GlutaMAXTM, Invitrogen), 10% fetal calf serum, 10 pg/mL gentamicin, sodium pyruvate and b-mercaptoethanol prior to incorporation in the IFNy ELIspot assay immunological assays.
  • PVDF plates (Millipore) were coated overnight at 4°C with rat anti-mouse IFNy antibody (BD Biosciences) and blocked for 1 h with complete Media.
  • Splenocytes are applied to plates at 5 x 10 5 cell/well and re-stimulated with ovalbumin at 10 pg/ml, complete media alone as a negative control and 0.25 pg/mL concanavalin A as positive control. After 18 h of culture in 5% CO2 incubator at 37°C, plates are washed and incubated with biotinylated rat anti-mouse IFNy followed by Streptavidin- HRP (BD Biosciences). Spots are visualized with AEC substrate and quantified using an automated ELISpot reader system (CTL). Results expressing spot forming cells (SFC) per million splenocytes are determined.
  • CTL automated ELISpot reader system
  • HL-6X2 was found to induce systemic cytokines in mice, while the same was not found for free immune stimulant IMDQ (EVb ((l-(4-(Aminomethyl)benzyl)- 2-butyl-lH-imidazo[4,5-c]quinolin-4-amine).
  • IMDQ (EVb ((l-(4-(Aminomethyl)benzyl)- 2-butyl-lH-imidazo[4,5-c]quinolin-4-amine).
  • IMDQ EMDQ
  • HL-6X2 is shown below: where Ac is Acetyl; Val is valine; Cit is citrulline; PEG 6 is -NH-(CH 2 ) 2 -(0-CH 2 -CH 2 ) 6 -C0- PABC (p-aminobenzyloxy carbonyl); PAB is p-aminobenzyloxy; and, IMDQ (IM2).
  • mice Fifty (50) female BALB/c mice were randomized according to the body weight into ten (10) groups of mice five (5) mice per group. Mice received on the right flank one (1) subcutaneous injection of either a dose-ranging of HL-6X2 or IMDQ (IM2) or a dose of 28mM L-Histidine/Mannitol dilution buffer as a vehicle control, according to the schedule table 14.1.1. The clinical examination (skin reaction and swelling persistence) at the injection sites are recorded post injection and before each blood sample. Blood tests for serum collection and later cytokine quantitative analysis were performed 2H and 24H or 6H and 48H post-test articles and vehicle administration according to the schedule Table 1. Body weight was assessed daily for the duration of the study.
  • IM2 IMDQ
  • the quantified cytokines in this example include TNF-a, IFN-a, IFN-b, IFN-g, IL12p40, IL6, MCP1 (CCL2), RANTES (CCL5), IP-10 (CXCL10) and CXCL9.
  • HL-6X2 at llnmol dramatically reduces or abolishes the producing of cytokines such as CCL5, IL6, IFN-a, INF-b, CCL2, IL12 and compared to the same dose of IMDQ (IM2).
  • HL-6X2 at llnmol induces a delay in the CXCL10 response and CXCL9 compared to IMDQ (IM2). Similar effects were observed at lower dose of HL-6X2 and IMDQ (IM2). TNF-a and IFN-g responses were very low and are not presented.
  • HL-6X2 due to its depot forming properties, promote a reduction or delay of the innate immune response compared to the free immune stimulant IMDQ (IM2).
  • C57BL/6J mice (Charles River) are used to assess the ability of different treatment groups to stimulate systemic proinflammatory cytokines responses as a measure of in vivo toxicity.
  • Another group of eight C57BL/6 is also topically treated with Aldara 5% (a commercial product containing imiquimod, a TLR-7 agonist) on the skin of shaved animals.
  • Negative control group C57BL/6 receive no treatment. Serum is collected 2 h after administration. Serum cytokine measurement is performed using a mouse cytokine multiplex kit. Sera is transferred to appropriate microtiter wells containing diluted antibody-coated bead complexes and incubation buffer. 50 pi of each homogenate sample is transferred to appropriate wells containing diluted antibody-coated bead complexes and incubation buffer. Samples are incubated for 2 hours. After washing with assay wash buffer (200 m ⁇ /well), 100 m ⁇ diluted biotinylated secondary antibody is added to the appropriate wells and incubated for 1 h.
  • Luminex analyzer Luminex Corp., Austin, TX
  • Minimums of 500 events (beads) are collected for each cytokine/sample and median fluorescence intensities obtained.
  • Cytokine concentrations are calculated based on standard curve data using MasterPlexTM QT Analysis version 2 (MiraiBio, Alameda, CA). Results expressing picograms of cytokines per ml of serum two hours after administration are determined.
  • the imidazoquinoline-type compounds of this disclosure are found not demonstrate induction of a systemic proinflammatory response, but to advantageously induce a cell-mediated immune response.
  • the imidazoquinoline-type compound of Formula (Ik), (II) or (Im) (“test articles”), designed for intracellular release of TLR7/8 agonist into a tumor (intratumoral administration), is/are administered to immunocompetent BALB/c mice comprising syngeneic tumors from CT-26 (colon carcinoma) tumor cells.
  • CT-26 colon carcinoma
  • One-hundred (100) female BALB/c mice (Charles River) are injected subcutaneously with 2xl0 5 CT26 cells on the right flank.
  • mice are randomized into five (5) groups of ten (10) mice per group and dosed with the test article(s) or the vehicle control.
  • mice receive one (1) intratumoral injection of either a dose-ranging of test article or DMSO/Mannitol buffer 1:10 solution as a vehicle control.
  • Tumor size is assessed daily, once tumors are palpable (e.g., day 3 tumor cell post-dosing). Tumor measurements begin daily up to the randomization day, and then tumor size and bodyweight are assessed three times per week for the duration of the study. Animals are removed from study when tumor volumes reach a 2000 mm 3 volume or according to animal care guidelines. The study will show that one or more of the test article(s) is capable of inhibiting and/or slowing the growth of cancer in vivo, as exemplified by the murine CT-26 model used herein. [00133] As described below, the activity of HL-6X2 administered intratumorally in the
  • CT26 tumor model was studied.
  • One-hundred female B ALB/c mice were injected subcutaneously with 2xl0 5 CT26 cells on the right flank.
  • mice were randomized into five groups of ten mice per group and dosed with the test articles or the vehicle control.
  • Mice received one intratumoral injection of either a dose-ranging of HL-6X2 or a single dose of HL-12 or 28mM L-Histidine/Mannitol dilution buffer as a vehicle control, according to the schedule table 1.
  • Tumor size were daily, once noticed palpable (day 3 post tumor cell dosing) up to randomization day. After randomization, tumor size and bodyweight were assessed three times a week for the duration of the study. Animals were removed from study when tumor volumes reach at least 2000mm3 volume or in the event of any deaths or animals found to be moribund.
  • HL-6X2 and HL-12 are shown below:
  • HL-6X2 Ac-RRLLHALLALLAHLLRRLE(PEG 6 -Val-Cit-PABC-IMDQ)-NH 2
  • HL-6X2 at 1.2nmol, 3.7nmol and especially 1 lnmol demonstrates a greater level of tumor growth control compared to HL- 12 tested at lOOnmol and the control group.
  • 3 complete regression were observed with each of the three doses of HL-6X2 compared to none with HL-12 tested at lOOnmol and the control group.
  • mice The synergy between the intratumoral treatment with and immune checkpoint inhibitors is examined in the CT26 colon carcinoma tumor in BALB/c mice.
  • Six to eight week old female BALB/c mice (Charles River) receive 2xl0 5 CT26 cells subcutaneously on both the right and the left flank.
  • mice are randomized into six groups of eight mice per group, approximately 10 days post tumor cell grafting, referred as day 0. After randomization, tumor size and bodyweight are assessed three times a week for the duration of the study.
  • mice are sacrificed according to the following humane endpoints: combined tumor volumes > 3000 mm 3 , presence of necrotic or ulcerated tumor, impaired mobility including transient prostration or hunched posture, interference with a vital physiological function.
  • On day 1 only largest tumors are intratumorally treated with 50 ul of injectable solution of a imidazoquinoline-type compound of Formulas I(k), 1(1), I(m), for groups 1, 2 and 3 or 28 mM L-Histidine/Mannitol dilution buffer as vehicle control for groups 4, 5 and 6. All mice receive three intratumoral injections once every two days, on day 1; day 3 and day 5.
  • results will show that intratumoral administration of a imidazoquinoline-type compound of Formulas I(k), 1(1), I(m) alone (group 3) induces tumor regression in the injected tumors in a larger proportion of animals compared with the control group 6, and further enhanced when intratumoral administration of kHL-12 is combined with systemic immune checkpoint inhibitors, either anti-PD-1 or anti-CTLA-4, administrated intraperitoneally, (group 1 and 2 respectively) compared to anti-PD-1 and anti- CTLA-4 alone groups 4 and 5 respectively.
  • the combination of an imidazoquinoline-type compound of Formulas I(k), 1(1), I(m) with anti-CTLA-4 shows a dramatic impact on tumor growth compared to all other groups in the non-injected tumors. These effects are observed at tumor level as reflected in terms of overall survival where a synergistic effect of an imidazoquinoline-type compound of Formulas I(k), 1(1), I(m) in combination with anti-CTLA-4 is observed.
  • An exemplary immunotherapy compound having the formula DM-L-IM is: Ac-RRLLHALLALLAHLLRRLE(PEG 6 -Val-Cit-PABC-IM3)-NH 2
  • HL-6X3 where Ac is Acetyl; Peptide is RRLLHALLALLAHLLRRLE (SEQ ID NO. 2); Val is valine; Cit is citrulline; PEG 6 is -NH-(CH 2 ) 2 -(0-CH 2 -CH 2 ) 6 -C0-; PABC is p-aminobenzyloxy carbonyl; PAB is p-aminobenzyloxy; and, IM3 is Formula (Ik) where R is -NH-.
  • HL-6X3 designed for intracellular release of TLR7/8 agonist through the presence of a cathepsin B cleavable linker (cathepsin B being present in the endosome of antigen presenting cells) is injected into a tumor (intratumoral administration), in immunocompetent B ALB/c mice comprising syngeneic (e.g. allograft) tumors from CT-26 (colon carcinoma) tumor cells.
  • CT-26 colon carcinoma
  • mice are randomized into five (5) groups of ten (10) mice per group and dosed with the test article (HL-6X3) or the vehicle control.
  • Mice receive one (1) intratumoral injection of either a dose-ranging of HL-6X3 (e.g., 11 nmol, 33 nmol, 100 nmol) in 28 mM L-Histidine/Mannitol solution or a dose of control kHL-12 (K(Ac)- RRLLHALLALLAHLLRRLKfADJ 12)-NH 2 , wherein peptide is
  • Tumor size is assessed daily, once tumors are palpable (e.g., day 3 tumor cell post-dosing). Tumor measurements begin daily up to the randomization day, and then tumor size and bodyweight are assessed three times per week for the duration of the study. Animals are removed from study when tumor volumes reach a 2000 mm 3 volume or according to animal care guidelines.
  • HL-6X3 is capable of inhibiting and/or slowing the growth of cancer in vivo , as exemplified by the murine CT-26 model used herein.
  • Example 7 Safety Profile of Oncology Therapy Compounds (Peptide Conjugates) Compared to Unmodified IM (TLR7 and/or TLR8 Agonist)
  • mice Female BALB/c mice (Charles River) are randomized according to the body weight into sixteen (16) groups of five (5) mice per group. Mice will receive on the right flank one (1) subcutaneous injection of either a dose-ranging of HL-6X3 or free TLR7/8 agonist (Formula (Ik) where R is -NFh (“Free IM”)) or a dose of kHL-12 or 28 mM L-Histidine/Mannitol dilution buffer as a vehicle control, according to the schedule in Table 4.
  • the clinical examination skin reaction and swelling persistence
  • Blood tests for serum collection and later pro-inflammatory cytokine quantitative analysis is performed 2 h and 24 h or 6 h and 48 h post-test article and vehicle administration according to the schedule in Table 4. Body weight is assessed daily for the duration of the study.
  • HL-6X3 is capable of reducing the induction of systemic serum cytokines compared to the free immunostimulant used herein.
  • Example 8 In vitro agonist activity of different compounds for the human TLR7 receptor, human TLR8 receptor and murine TLR7 receptor
  • HEK-293 cell lines functionally overexpress human or murine TLR proteins as well as a reporter gene which is a secreted alkaline phosphatase (SEAP). The production of this reporter gene is driven by an NFkB inducible promoter.
  • the TLR reporter cell line activation results are given as optical density values (OD).
  • HEK-Blue hTLR7, HEK-Blue hTLR8, HEK-Blue mTLR7 reporter cell lines from Invivogen were used.
  • Compound Cl-amine, C2-amine, C3-amine, C4-amine, C5-amine described in the table below were tested at different concentrations against compound 3M-052, IM2 and R848 used as internal controls.
  • Results are presented in Figure 12 (human TLR7 and Human TLR8) and Figure 13 (murine TLR7).
  • compound Cl -amine achieved an EC50 of 53nM that represent an activity approximately 10-fold higher compared to the reference compound R848 and 7-fold higher compared to 3M-052.
  • compound C2-amine achieved an EC50 of 412nM that represent an activity approximately 5.5-fold higher compared to the reference compound R848 and 7-fold higher compared to 3M-052.
  • compound Cl -amine showed an activity broadly equivalent to R848 but at least 5-fold higher compared to 3M-052.

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Abstract

La présente divulgation concerne des composés du type imidazoquinoline, leurs procédés de préparation, des compositions pharmaceutiques associées et leur utilisation, les composés du type imidazoquinoline, après une administration locale, formant des dépôts induisant une réponse immunitaire à médiation cellulaire tout en atténuant une réponse immunitaire pro-inflammatoire systémique.
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Publication number Priority date Publication date Assignee Title
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2
US11547761B1 (en) 2016-07-07 2023-01-10 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates

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US20060188913A1 (en) * 2001-10-12 2006-08-24 University Of Iowa Research Foundation Methods and products for enhancing immune responses using imidazoquinoline compounds
WO2020168017A1 (fr) * 2019-02-12 2020-08-20 Ambrx, Inc. Contenant de compositions, procédés et utilisations de conjugués anticorps-agonistes tlr

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US6451810B1 (en) * 1999-06-10 2002-09-17 3M Innovative Properties Company Amide substituted imidazoquinolines
US20060188913A1 (en) * 2001-10-12 2006-08-24 University Of Iowa Research Foundation Methods and products for enhancing immune responses using imidazoquinoline compounds
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11547761B1 (en) 2016-07-07 2023-01-10 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2

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