WO2023039662A1 - Dérivés de phénanthridinium utilisés en tant qu'inhibiteurs de ppm1a et leurs utilisations - Google Patents

Dérivés de phénanthridinium utilisés en tant qu'inhibiteurs de ppm1a et leurs utilisations Download PDF

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WO2023039662A1
WO2023039662A1 PCT/CA2022/051362 CA2022051362W WO2023039662A1 WO 2023039662 A1 WO2023039662 A1 WO 2023039662A1 CA 2022051362 W CA2022051362 W CA 2022051362W WO 2023039662 A1 WO2023039662 A1 WO 2023039662A1
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optionally substituted
group
compound according
independently
mtb
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PCT/CA2022/051362
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English (en)
Inventor
Jim Jian SUN
Weibo Yang
Zhongliang Xu
Lu Chen
Yi Chu LIANG
Andréanne LUPIEN
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University Of Ottawa
Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences
The Royal Institution For The Advancement Of Learning / Mcgill University
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Publication of WO2023039662A1 publication Critical patent/WO2023039662A1/fr

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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/133—Amines having hydroxy groups, e.g. sphingosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/10—Aza-phenanthrenes
    • C07D221/12—Phenanthridines
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/056—Ortho-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
    • C07D491/153—Ortho-condensed systems the condensed system containing two rings with oxygen as ring hetero atom and one ring with nitrogen as ring hetero atom

Definitions

  • the present disclosure relates to phenanthridinium-based compounds, methods of their production, and uses thereof.
  • Phenanthridine is a tricyclic aromatic heterocycle related to phenanthrene where a nitrogen atom is substituted for a CH group in the central ring. Alkylating the nitrogen atom results in phenanthridinium.
  • PPM1A Protein Phosphatase Mg 2+ /Mn 2+ -dependent 1A
  • PPM1A Protein Phosphatase Mg 2+ /Mn 2+ -dependent 1A
  • PPM1A is a member of the metal-dependent PP2C Ser/Thr phosphatase family, and is a key regulator of innate immune cell function. This has implications for multiple diseases, including infectious diseases by bacterial or viral pathogens, cancer, atherosclerosis, and neurodegeneration. As such, novel chemical compounds that can modulate the phosphatase activity of PPM1A may have therapeutic applications. While therapeutic intervention using kinase inhibitors is a well- established strategy, drug discovery efforts to develop targeted phosphatase inhibitors has been much more challenging.
  • Tuberculosis is an infectious lung disease caused by the bacterium Mycobacterium tuberculosis (Mtb).
  • Mtb Mycobacterium tuberculosis
  • Treatment for TB is complicated, typically involving a lengthy 6-month regimen of 2-4 antibiotics. This problem is compounded by the global emergence of multidrug-resistant TB (490,000 cases worldwide in 2016) and the lack of novel clinically approved anti-TB drugs, rendering treatment increasingly difficult.
  • the present disclosure provides compounds that inhibit PPM1A, which is a protein that is overproduced in Mtb- infected cells. Inhibiting PPM1A may boost the ability of the cell to kill Mtb.
  • Specific compounds according to the present disclosure may be identified using internal references (Groups I to IV; or “SMIP”) and be denoted by number, such as HI-22 or SMI P-76. The same compound may be referred to using different internal reference numbers. For example, a compound according to the formula may be referred to as either SMI P-6 or compound 1-9.
  • the present disclosure provides a compound according to
  • Ri to Rg independently denote: H; D; a halogen; an optionally substituted straight chain or branched alkyl group; an optionally substituted alkenyl group; an optionally substituted alkynyl group; an optionally substituted aryl group; an optionally substituted heteroaryl group; or an optionally substituted carbocyclic group.
  • Ri and R2 are optionally linked; R2 and R3 are optionally linked; R4 and Rs are optionally linked; Rs and Rs are optionally linked; Rs and R7 are optionally linked; R7 and Rs are optionally linked; Rs and R9 are optionally linked; and/or R9 and Ri are optionally linked.
  • X' is a pharmaceutically- or synthetically-acceptable anion.
  • Ri to R9 are not all H.
  • Ri to Rn independently denote: H; D; a halogen; an optionally substituted straight chain or branched alkyl group; an optionally substituted alkenyl group; an optionally substituted alkynyl group; an optionally substituted aryl group; an optionally substituted heteroaryl group; or an optionally substituted carbocyclic group.
  • Ri and R2 may be linked; R2 and R3 may be linked; R3 and R4 may be linked; R4 and Rs may be linked; Re and R7 may be linked; R7 and Rs may be linked; Rs and R9 may be linked; R9 and R w may be linked; R10 and R11 may be linked; and/or Rn and Ri may be linked.
  • R2 and R3 do not together form -OCH2O-
  • Re and R7 do not together form - OCH2O-.
  • X' is a pharmaceutically- or synthetically-acceptable anion.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a compound as disclosed herein, or a pharmaceutically acceptable solvate thereof, and (b) a pharmaceutically acceptable diluent, carrier, or excipient.
  • the present disclosure provides a method of inhibiting the enzymatic activity of a metal-dependent PP2C Ser/Thr phosphatase.
  • the method includes exposing the phosphatase to a compound or composition as disclosed herein.
  • the present disclosure provides a method of treating or preventing a metal-dependent PP2C Ser/Thr phosphatase-related disease or disorder in a patient.
  • the method includes administering to the patient a sufficient amount of a compound or composition as disclosed here to treat or prevent the disease or disorder.
  • the disease or disorder may be an infectious disease, such as a bacterial infection.
  • the disease or disorder is a Mycobacterium tuberculosis (Mtb) infection or a non-tuberculosis mycobacteria (NTM) infection.
  • the Mtb may be a mono-resistant or multidrug resistant (MDR) strain of Mtb.
  • the expression “mono-resistant” refers to a bacteria that is resistant to treatment with one antibiotic
  • the expression “multidrug resistant” refers to a bacteria that is resistant to treatment with more than one antibiotic.
  • the expression “MDR-TB” refers to a multidrug resistant strain of Mycobacterium tuberculosis.
  • An example of a multidrug resistant bacteria is Mtb resistant to treatment with both isoniazid and rifampicin.
  • the NTM infection may be an Mycobacterium abscessus (Mabs) or a Mycobacterium avium (Mav) infection.
  • an NTM infection may be an infection with a single species or with a combination of species.
  • a combination of subspecies may be referred to as a “complex”, or the combination of subspecies may be referred to only by the parent species.
  • the NTM infection may be, for example, an infection with Mycobacterium abscessus (Mabs) or Mycobacterium avium (Mav).
  • Mabs Mycobacterium abscessus
  • Mabs Mycobacterium abscessus
  • a Mabs complex may include, for example: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii.
  • the present disclosure provides a combination therapy for the treatment of a Mycobacterium tuberculosis (Mtb) infection in a patient.
  • the combination therapy includes: (a) a compound or composition as disclosed herein; and (b) at least one anti-tuberculosis medication.
  • the Mtb infection may be an infection by a monoresistant or a multidrug resistant (MDR) strain of Mtb.
  • MDR multidrug resistant
  • the present disclosure provides a process for forming a compound according to Formula I.
  • the process includes reacting reagents according to Scheme I (a), l(b), or l(c): aryl amination with
  • Fig. 1 is a blot associated with the experiments discussed below.
  • Fig. 2 shows graphs associated with the experiments discussed below.
  • Fig. 3 is a blot associated with the experiments discussed below.
  • Fig. 4 is a graph associated with the experiments discussed below.
  • Fig. 5 illustrates various compounds according to the present disclosure.
  • Fig. 6 is a graph associated with the experiments discussed below.
  • Fig. 7 is a graph associated with the experiments discussed below.
  • Fig. 8 is a graph associated with the experiments discussed below.
  • Fig. 9 is a blot associated with the experiments discussed below.
  • Fig. 10 is a graph associated with the experiments discussed below.
  • Fig. 11 is a graph associated with the experiments discussed below.
  • Fig. 12 is a graph associated with the experiments discussed below.
  • Fig. 13 is a graph associated with the experiments discussed below.
  • Fig. 14 is a graph associated with the experiments discussed below.
  • Fig. 15 is a graph associated with the experiments discussed below.
  • Fig. 16 is a graph associated with the experiments discussed below.
  • Fig. 17 is a graph associated with the experiments discussed below.
  • Fig. 18 is a graph associated with the experiments discussed below.
  • Fig. 19 is a graph associated with the experiments discussed below.
  • Fig. 20 shows a table summarizing predicted properties associated with a compound according to the present disclosure.
  • Fig. 21 shows a table summarizing predicted properties associated with another compound according to the present disclosure.
  • Fig. 22 show blots associated with the experiments discussed below.
  • Fig. 23 shows a graph associated with the experiments discussed below.
  • Fig. 24 shows graphs associated with the experiments discussed below.
  • Fig. 25 show blots associated with the experiments discussed below.
  • Fig. 26 show blots associated with the experiments discussed below.
  • Fig. 27 show blots associated with the experiments discussed below.
  • Fig. 28 shows a graph associated with the experiments discussed below.
  • Fig. 29 shows graphs associated with the experiments discussed below.
  • Fig. 30 show blots associated with the experiments discussed below.
  • Fig. 31 show blots associated with the experiments discussed below.
  • Fig. 32 show blots associated with the experiments discussed below.
  • Fig. 33 show blots associated with the experiments discussed below.
  • Fig. 34 show blots associated with the experiments discussed below.
  • Fig. 35 show blots associated with the experiments discussed below.
  • Fig. 36 show blots associated with the experiments discussed below.
  • Fig. 37 show blots associated with the experiments discussed below.
  • Fig. 38 show blots associated with the experiments discussed below.
  • Fig. 39 show blots associated with the experiments discussed below.
  • Fig. 40 show blots associated with the experiments discussed below.
  • Fig. 41 shows a graph associated with the experiments discussed below.
  • Fig. 42 shows a graph associated with the experiments discussed below.
  • Fig. 43 shows a graph associated with the experiments discussed below.
  • Fig. 44 show blots associated with the experiments discussed below.
  • Fig. 45 shows a graph associated with the experiments discussed below.
  • Fig. 46 shows a graph associated with the experiments discussed below.
  • Fig. 47 shows a graph associated with the experiments discussed below.
  • Fig. 48 shows a graph associated with the experiments discussed below.
  • Fig. 49 shows graphs associated with the experiments discussed below.
  • Fig. 50 shows a graph associated with the experiments discussed below.
  • Fig. 51 shows a graph associated with the experiments discussed below.
  • Fig. 52 shows a graph associated with the experiments discussed below.
  • Fig. 53 shows a graph associated with the experiments discussed below.
  • Fig. 54 shows graphs associated with the experiments discussed below.
  • Fig. 55 shows a graph associated with the experiments discussed below.
  • Fig. 56 shows graphs associated with the experiments discussed below.
  • Fig. 57 shows graphs associated with the experiments discussed below.
  • Fig. 58 shows graphs associated with the experiments discussed below.
  • Fig. 59 shows graphs associated with the experiments discussed below.
  • Fig. 60 shows graphs associated with the experiments discussed below.
  • the present disclosure provides a compound according to
  • Ri to R9 independently denote: H; D; a halogen; an optionally substituted straight chain or branched alkyl group; an optionally substituted alkenyl group; an optionally substituted alkynyl group; an optionally substituted aryl group; an optionally substituted heteroaryl group; or an optionally substituted carbocyclic group.
  • R1 and R2 may be linked; R2 and R3 may be linked; R 4 and Rs may be linked; Rs and Rs may be linked; Rs and R7 may be linked; R7 and Rs may be linked; Rs and R9 may be linked; and/or R9 and Ri may be linked.
  • X' is a pharmaceutically- or synthetically-acceptable anion.
  • Ri to R9 are not all H.
  • the present disclosure provides a compound A compound according to Formula II:
  • Ri to Rn independently denote: H; D; a halogen; an optionally substituted straight chain or branched alkyl group; an optionally substituted alkenyl group; an optionally substituted alkynyl group; an optionally substituted aryl group; an optionally substituted heteroaryl group; or an optionally substituted carbocyclic group.
  • Ri and R2 may be linked; R2 and R 3 may be linked; R3 and R4 may be linked; R4 and Rs may be linked; Re and R7 may be linked; R7 and Rs may be linked; Rs and R9 may be linked; R9 and R w may be linked; R10 and R11 may be linked; and/or Rn and Ri may be linked.
  • R2 and R 3 do not together form -OCH2O-
  • Re and R7 do not together form - OCH2O-.
  • X' is a pharmaceutically- or synthetically-acceptable anion.
  • An optional substituent according to the present disclosure may be a substituent independently selected from the group consisting of: D, halogen, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • an optional substituent may be independently selected from the group consisting of: -F, -CH3, - CH2CH3, -CH(CH 3 ) 2 , -CH 2 C(O)N(R) 2 , -CH2CH2OH, -CH2CH2CH2OH, -OH, -OCH3, -CH2CH2F, -CF 3 , -OCF3, -CH 2 CH 2 N(R) 2 , -C(O)N(R) 2 , -C(O)OR, -C(O)NH-R, -C(O)-R, -NHC(O)OR, - N(R) 2 , -CH 2 C(O)NHOR, -C(O)NHOR, -CH 2 C(O)NHCH 2 CH 2 OR, -NHSO2R, -SO2NR2, -
  • each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • An optionally substituted alkyl group according to the present disclosure may independently have: from 1 to 20 carbon atoms, wherein up to 10 CH or CH2 groups may independently be replaced by a group selected from -O-, -NR-, -NR2, -S-, -S(O)-, -SO2-, - S(O)(NR)-, -S(O 2 )(NR)-, -N(SO)R-, -C(O)-, -CO2-, -OC(O)-, -C(O)NR-, -NRC(O)-, -C(NOR)-, and R; wherein each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • each optionally substituted alkyl group may be independently selected from the group consisting of: -CH2CH2OH, -CH2CH2CH2OH, -OCH3, -CH2CH2F, -CF 3 , -OCF3, -CH2CH2NH2, -C(O)NH 2 , -C(O)OCH 2 CH 3 , - COOMe, -C(O)NH-R, -C(O)-R, -NHC(O)OtBu, -NH 2 , -CH 2 C(O)NHOH, -C(O)NHOH, - CH 2 C(O)NHCH 2 CH 2 OH, -NHSO2CH3, -SO2NH2, -C(NOH)CH 3 , and -C(NOCH 3 )CH 3 .
  • An optionally substituted alkenyl according to the present disclosure may independently have at least one carbon-carbon double bond, and may independently have 2 to 20 carbon atoms, where up to 10 CH or CH2 groups may be independently replaced by a group selected from -O-, -NR-, -NR 2 , -S-, -S(O)-, -SO2-, -S(O)(NR)-, -S(O 2 )(NR)-, -N(SO)R-, -C(O)-, -CO2-, -OC(O)-, -C(O)NR-, -NRC(O)-, -C(NOR)-, and R; where each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted
  • An optionally substituted alkynyl according to the present disclosure may independently have at least one carbon-carbon or carbon-nitrogen triple bond, and may independently have 2 to 20 carbon atoms, where up to 10 CH or CH2 groups may be independently replaced by a group selected from -O-, -NR-, -NR2, -S-, -S(O)-, -SO2-, - S(O)(NR)-, -S(O 2 )(NR)-, -N(SO)R-, -C(O)-, -CO2-, -OC(O)-, -C(O)NR-, -NRC(O)-, -C(NOR)-, and R; wherein each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or
  • An optionally substituted aryl group according to the present disclosure may be independently: a monocyclic aromatic ring, a multicyclic aromatic ring system, or a fused multicyclic aromatic ring system having 3- to 18-members.
  • Each monocyclic aromatic ring of the optionally substituted aryl group may be independently -CeHs or -CH2C6H5.
  • Each multicyclic aromatic ring of the optionally substituted aryl group may be independently - C6H4C6H5 or -CH2C6H4C6H5.
  • Each fused multicyclic aromatic ring of the optionally substituted aryl group may be independently -C10H7 or -CH2C10H7.
  • each optionally substituted aryl group may be independently selected from the group consisting of: [0085]
  • An optionally substituted heteroaryl group according to the present disclosure may be independently: a monocyclic aromatic ring, a multicyclic aromatic ring, or a fused multicyclic aromatic ring system having 3- to 18-members and containing 1 to 6 heteroatoms selected from N, O and S.
  • An optionally substituted carbocyclic group according to the present disclosure may be a saturated or an unsaturated carbocyclic ring having from 3 to 20 carbon atoms, where up to 10 CH or CH2 groups may independently be replaced by a group selected from - O-, N, -NR-, -S-, -S(O)-, -SO2-, -S(O)(NR)-, -N(SO)R-, -C(O)-, -CO2-, -OC(O)-, -C(O)NR-, and -NRC(O)-, where each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • Each optionally substituted carbocyclic group may be independently selected from the group consist
  • R1 and R2, R2 and R3, R4 and Rs, Rs and Rs, Rs and R7, R7 and Rs, Rs and R9, and/or R9 and R1 may be linked to form a five-, six-, or seven-membered saturated or unsaturated carbocyclic ring.
  • the five-membered saturated or unsaturated carbocyclic ring may be selected from the group consisting of: , and each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • R2, R4 and/or Rs are hydrogen;
  • R1, R2, and/or R9 is a halogen, preferably fluorine, or an optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, or carbocyclic group;
  • R4, Rs, Re and R7 are each independently H or an optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, or carbocyclic group, preferably -OR or -N(R)2 ; where each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group;
  • Rs and Rs are linked to form
  • Rs and Re may independently be H, -OR or -N(R)2 where each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group; or Rs and Re may be linked to form a five- or six-membered saturated or unsaturated carbocyclic ring.
  • R3 may be H, or an optionally substituted alkyl or alkenyl group, such as: -CH2CF3, -CH 2 OH, -CH2CH2OH, -CH 2 F, -CH 2 NH 3 + , -C(O)NH 2 , -
  • Ri is a halogen, or an optionally substituted alkyl, aryl or heteroaryl group.
  • the compound may have a formula according to Formula IV:
  • Ri may be: a halogen, an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group.
  • Ri may be: -F, -CF 3 , -C(O)OR, -C(O)NH-R, -CH 2 C(O)N(R) 2 , -C(O)-R, -N(R) 2 , -
  • H independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • Rs, Re and R? may independently be H, -OR or -N(R) 2 where each R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • R independently denotes: H, D, an optionally substituted straight chain or branched alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, or an optionally substituted carbocyclic group.
  • Rs and Re may be linked to form a five- or six-membered saturated or unsaturated carbocyclic ring.
  • R7 is H, -OCH3 or -OCH 2 CH3 and the others are H; or R7 is H, -OCH3 or -OCH 2 CH3, and Rs and Re are together -OCH 2 O- and form a five-membered ring.
  • R3 is H, or an optionally substituted alkyl or alkenyl group, such as: -CH 2 CF 3 , -CH 2 OH, -CH 2 CH 2 OH, -CH 2 F, -CH 2 NH 3 + , -C(O)NH 2 , -
  • Ri, R4, R9, R and/or Rn are hydrogen;
  • R2, R3, Re, R7, and/or Rs is a halogen, or an optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, or carbocyclic group;
  • R2 and R3 are linked to form a five-, six-, or seven-membered saturated or unsaturated carbocyclic ring;
  • Re and R7 are linked to form a five-, six-, or seven-membered saturated or unsaturated carbocyclic ring;
  • R7 and Rs are linked to form a five-, six-, or sevenmembered saturated or unsaturated carbocyclic ring; or (vi) any combination thereof.
  • Particular examples of a compound according to Formula II are:
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a compound according to the present disclosure, or a pharmaceutically acceptable solvate thereof, and (b) a pharmaceutically acceptable diluent, carrier, or excipient.
  • a compound or composition as disclosed herein may be used to inhibit the enzymatic activity of a metal-dependent PP2C Ser/Thr phosphatase by exposing the metaldependent PP2C Ser/Thr phosphatase to the compound or composition.
  • a compound or composition as disclosed herein may be used to treat or prevent a metal-dependent PP2C Ser/Thr phosphatase-related disease or disorder in a patient by administering to the patient a sufficient amount of the compound or the composition to treat or prevent the disease or disorder.
  • the metal-dependent PP2C Ser/Thr phosphatase may be Protein Phosphatase Mg 2+ /Mn 2+ -dependent 1A (“PPM1A”).
  • the metal-dependent PP2C Ser/Thr phosphatase-related disease or disorder may be an infectious disease, such as a bacterial infection.
  • the bacterial infection may be a Mycobacterium tuberculosis (Mtb) infection or a non-tuberculosis mycobacteria (NTM) infection.
  • the Mtb may be a monoresistant or a multidrug resistant (MDR) strain of Mtb.
  • the NTM infection may be an Mycobacterium abscessus (Mabs) or a Mycobacterium avium (Mav) infection.
  • Host Protein Phosphatase, Mg 2+ /Mn 2+ Dependent 1A is a factor for persistent Mtb infections in macrophages.
  • Mtb infection triggers the upregulation of PPM1A, which blocks both cytokine production and apoptosis of Mtb-infected macrophages, thereby favoring the intracellular survival of Mtb.
  • PPM1A dephosphorylates and antagonizes antiviral signaling proteins such as STING, TBK1 and IKKE.
  • the cGAS-STING-TBK1 pathway has been implicated in sensing the cytosolic DNA released by Mtb during infection, producing type-l IFN and triggering the autophagic response. Genetic ablation of PPM1A in human macrophages results in improved killing of intracellular Mtb.
  • Methods of treating or preventing an Mtb invention, such as a mono-resistant or an MDR Mtb infection, in a patient may additionally include administering at least one antituberculosis medication.
  • treating or preventing a mono-resistant or an MDR Mtb infection may include administering an anti-tuberculosis medication that includes a compound according to SMI P-6 or SMI P-9.
  • a compound or composition as disclosed herein may be in the form of a combination therapy for the treatment of a Mycobacterium tuberculosis (Mtb) infection in a patient.
  • the combination therapy would include at least one anti-tuberculosis medication.
  • a “combination therapy” would be understood to mean a combination of two or more therapeutics, administered together or separately, administered using the same or different dosage forms, at substantially the same time or at different times, to treat the Mtb infection.
  • the anti-tuberculosis medication may be a host-directed therapeutic, or (ii) may be a bacteria-directed therapeutic, for example inhibiting bacterial metabolism, cell wall, or protein synthesis.
  • the anti-tuberculosis medication may be a fluoroquinolone, capreomycin, kanamycin, amikacin, rifampicin, isoniazid, pyrazinamide, ethambutol, bedaquiline, delamanid, linezolid, or pretomanid.
  • a “host- directed therapeutic” would be understood to be a compound that acts via a host-mediated response to the pathogen.
  • a host-directed therapeutic may act to change the local environment (for example by modulating inflammation, or immune cell activation or recruitment) in which the pathogen exists to make it less favorable for the pathogen to live and/or grow.
  • a “bacteria-directed therapeutic” would be understood to be a compound that acts directly on the pathogen.
  • a bacteria-directed therapeutic may inhibit or negatively affect a bacterial secretion system, DNA or RNA synthesis, cell division, outer membrane protein localization or function, or biofilm formation.
  • a bacteria- directed therapeutic may activate, induce, or inhibit genes or proteins to transform bacteria from a latent to a non-latent state, or to prevent or inhibit bacteria from transforming from a non-latent to a latent state.
  • a compound or composition as disclosed herein may be formulated for intranasal, pulmonary, intravenous, intramuscular, oral, intrathecal, or intraperitoneal administration.
  • a compound or composition as disclosed herein may be formulated for administration one time, two times, three times, or four times every day; once every other day; once every two or three days; once a week; or three times every two weeks, for a period of time, such as until the Mtb or NTM infection is treated.
  • a compound or composition as disclosed herein may be formulated as a tablet, a solution, a powder such as a lyophilized powder, a suspension, a granule, or a capsule.
  • the compound “SMI P-30” is effective in reducing Mtb survival in vitro, in infected macrophages, and in vivo, synergizing with rifampicin to reduce the Mtb burden in mouse lungs.
  • SMI P-30 improves clearance of Mtb in vitro by boosting the autophagy pathway in a PPM1 A-dependent manner.
  • the underlying mechanism appears to rely on selective dephosphorylation of a novel PPM1A substrate, the autophagy receptor p62/SQSTM-1 on serine 403, a key residue for the recognition of ubiquitinated cytosolic bacilli and their delivery to lysosomal degradation via xenophagy.
  • the compounds “SMIP-6”, “SMIP-9”, “SMIP-19”, “SMIP-29”, and “SMIP-32” showed significantly improved activity compared to SG and inhibited the growth of Mtb by 85%.
  • Compounds SMIP-6 and SMIP-9 also showed activity against non-replicating Mtb; exhibited reduced cytotoxicity; showed efficacy against intracellular Mtb; showed efficacy against virulent and clinical MDR Mtb; and showed efficacy against non-tuberculosis mycobacteria (NTM) strains.
  • the present disclosure also provides a process for forming a compound according to Formula I.
  • the process includes reacting reagents according to Scheme l(a), Scheme I (b) , or Scheme l(c): Formula l(a) Formula I
  • the compounds of Formulas I (a), l(b), and l(c) may be produced using well-known organic chemistry techniques, for example using a Pd-catalyzed Suzuki coupling reaction to form the aryl-aryl bond.
  • the present disclosure also provides a process for forming a compound according to Formula II. The process includes reacting reagents according to Scheme I l(a), Scheme I l(b), or Scheme I l(c): Scheme I l(a)
  • the compounds of Formulas 11(a), 1(b), and 1(c) may be produced using well-known organic chemistry techniques, for example using a Pd-catalyzed reaction to form the aryl-aryl bond.
  • the Pd-catalyzed reaction may be a “Suzuki coupling”, for example using Pd(PPh3)4, or Pd(OAc)2/PPh3 as the catalyst.
  • THP-1 monocytes ATCC TIB-202
  • all the THP-1 derivative cell lines and PBMC were maintained in RPMI-1640 medium.
  • HEK 293T/17 ATCC CRL- 11268
  • HEK GP2-293 Clontech-Takara
  • RAW 264.7 ATCC TIB-71
  • BMDM BMDM-derived BMDM-derived BMDM-derived BMDM-derived derived derived derived from PBMC.
  • DMEM medium DMEM medium.
  • Medium was supplemented with 2 mM L-glutamine, penicillinstreptomycin (100 I.U./mL penicillin, 100 pg/ml streptomycin) and 10% heat-inactivated fetal bovine serum (FBS, Gibco).
  • RPMI-1640 was also supplemented with 10 mM HEPES.
  • THP-1 overexpressing PPM1A THP-1 PPM1A+ cells were generated previously (26). THP-1 cells were differentiated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA, Alfa Aesar) for 24- 72 h. For Tet-inducible PPM1A overexpressing THP-1, the cells were induced with doxycycline (Alfa Aesar) for 72 h and differentiated in PMA during the last 24 h.
  • PBMC peripheral blood mononuclear cells
  • PBMC Human peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Positive selection of monocytes was performed using anti-CD14 coated magnetic particles from StemCell Technologies according to manufacturer’s protocol.
  • Monocytes were differentiated with 5 ng/ml GM-CSF (Gibco) for 6 days to obtain human monocyte derived macrophages (hMDMs).
  • mBMDM Mouse bone marrow derived macrophages
  • Bacterial strains The Mycobacterium tuberculosis H37Rv and the derived auxotroph strain mc 2 6206 ( PanCD LeuCD) were grown in Middlebrook 7H9 medium (BD Biosciences) supplemented with 0.2% glycerol (Fisher Chemical), 0.05% Tween-80 (Acros Organics) and 10% OADC (BD Biosciences) or on Middlebrook 7H10 plates supplemented with 0.5% glycerol and 10% OADC. Growth medium of the auxotrophic M.
  • tuberculosis strain was supplemented with 24 pg/ml pantothenate (Thermo Fisher Scientific) and 50 pg/ml L- leucine (Alfa Aesar).
  • M. tuberculosis mc 2 6206 expressing GFP was generated previously (26).
  • Mtb mc 2 6206 expressing firefly luciferase was generated by transformation of the pSMT3-luciferase plasmid (generous gift from Dr. Zakaria Hmama). Both Mtb GFP and Luciferase were maintained in selection with 50 pg/ml Hygromycin B (Calbiochem). Liquid Mtb cultures were maintained at 37°C with slow shaking (50 rpm).
  • Escherichia coli strain DH5a and NEB Stable were used for plasmid propagation. Rosetta E. coli cells (kindly provided by Dr. Jean-Frangois Couture) were used for recombinant proteins expression. Cells were grown in Luria-Bertani broth at 37°C. The antibiotics ampicillin (Fisher BioReagents) or chloramphenicol (ACROS OrganicsTM) where used when required.
  • TH P-1 monocytes (ATCC TIB-202) were maintained at 37°C in a humidified atmosphere of 5% CO2 in RPMI 1640 media (Gibco, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES, penicillin (100 I.U./ml), streptomycin (100 pg/ml), and 2 mM L-glutamine purchased from Gibco.
  • FBS heat-inactivated fetal bovine serum
  • penicillin 100 I.U./ml
  • streptomycin 100 pg/ml
  • 2 mM L-glutamine purchased from Gibco.
  • TH P-1 macrophages For differentiation into TH P-1 macrophages, cells were resuspended in complete RPMI 1640 media (without antibiotics) and incubated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Alfa Aesar, Haverhill, MA). Cells were seeded at 50,000 cells/well into 96-well plates and incubated at 37°C for 3 days.
  • PMA phorbol 12-myristate 13-acetate
  • virulent strains Mtb Erdman, H37Rv, HN878, clinical MDR strains (isolates #50, 105, 116, 151 , 217), Mycobacterium kansasii (ATCC 12478), and Mycobacterium bovis BCG (BCG Pasteur) were cultured in Middlebrook 7H9 media (BD Biosciences, Franklin Lakes, NJ) supplemented with 0.05% Tween 80 (Acros Organics, Fair Lawn, NJ), 0.2% glycerol (Fisher Chemical, Waltham, MA), and 10% OADC (BD Biosciences).
  • Mtb mc 2 6206 strain D-pantothenic acid (24 pg/ml, Alfa Aesar) and L-leucine (50 pg/ml, Alfa Aesar) were also added.
  • M. kansasii 5 pg/ml streptomycin was also added (Fisher Scientific, Waltham, MA).
  • Mycobacterium smegmatis (ATCC 700084) was maintained in 7H9 media supplemented with 10% ADS enrichment: 50 g bovine serum albumin (VWR, Radnor, PA), 20 g dextrose (Fisher Scientific), 0.85% (w/v) sodium chloride (Fisher Chemical), 0.2% glycerol, and 0.05% tyloxapol (Acros Organics). Clinical MDR Mtb isolates were obtained from the McGill International TB Centre (Montreal, Canada).
  • Mtb mc 2 6206 expressing tdTomato was generated previously.
  • Escherichia coli strain NEB Stable New England Biolabs, Ipswich, MA
  • Pseudomonas aeruginosa strains PA01 and PA14 generous gift from Dr. Thien-Fah Mah, University of Ottawa
  • Salmonella enterica serovar Typhimurium strain SL1344 and Listeria monocytogenes strain 10403s both generous gifts from Dr. Subash Sad, University of Ottawa
  • Lysogeny Broth LB, Fisher Scientific. Bacteria were inoculated into LB from frozen glycerol stocks and grown at 37°C with shaking (200 rpm).
  • Resazurin microtiter assay (REMA). Bacteria were diluted to an initial OD of 0.02 at mid-log stage and incubated with sanguinarine (Tocris Bioscience, Toronto, Canada), RIF (Fisher Scientific), INH (Acros Organics), EMB (Alfa Aesar), MFX (Alfa Aesar), or SMIP compounds at 37°C. The incubation time was 5 days for Mtb and BCG, 20 h for M. smegmatis and 3 days for M. kansasii. Then, resazurin (Sigma-Aldrich, St.
  • the CRISPR/Cas9 knock-out plasmids targeting human PPM1A were generated by inserting single guide RNA (sgRNA) sequences into LentiCRISPRv2 vector, as previously reported (43).
  • sgRNA single guide RNA
  • Four sgRNAs targeting PPM1A and two non-targeting sgRNA sequences were designed (https://zlab.bio/guide-design-resources) to generate the plasmids pSL15-20.
  • the lentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid #52961 ; http://n2t.net/addgene:52961 ; RRID:Addgene_52961) (77).
  • the sequence encompassing the multi-cloning site (MCS) and EGFP were first amplified from mEGFP-N1 (gift from Michael Davidson, Addgene plasmid #54767; http://n2t.net/addgene:54767;RRID:Addgene_54767), and cloned into the lentiviral vector pLenti-CMV-MCS-GFP-SV-puro (gift from Paul Odgren (Addgene plasmid #73582; http://n2t.net/addgene: 73582; RRID:Addgene_73582) (78).
  • PPM1A was amplified from THP- 1 cDNA and cloned into the intermediate plasmid, resulting in the final pSL113 (PPM1A- GFP) plasmid.
  • PPM1A- GFP final pSL113
  • the MSG and EGFP sequence from mEFGP-C1 vector gift from Michael Davidson, Addgene plasmid #54759; http://n2t.net/addgene:54759;RRID:Addgene_54759 was first inserted into pLenti-CMV- MCS-GFP-SV-puro.
  • PPM1A cDNA was then cloned into the intermediate plasmid, resulting in pSL114.
  • Tet-inducible PPM1A-overexpressing THP-1 cell were generated cloning the human PPM1A cDNA into the pRetro-X Tet-One system (Clontech-Takara), following manufacturer’s instructions. All the plasmids were validated by Sanger sequencing. A list of plasmids and oligonucleotides used in the study is available in Tables 1 and 2.
  • HEK 293T/17 cells were seeded at 50% confluency and co-transfected with the newly generated lentiviral plasmids (pSL15-20, pSL113, pSL114, see Table 1), the pVSV-G envelope plasmid and the psPAX2 packaging plasmid using FuGENE (Promega).
  • psPAX2 was a gift from Didier Trono (Addgene plasmid #12260; http://n2t.net/addgene: 12260; RRID:Addgene_12260).
  • the retroviral plasmid (pRetro-X Tet-One PPM1A) was cotransfected with the pVSV-G envelope plasmid into HEK GP-293 cells using FuGENE. Culture supernatants were harvested after 48 h, aliquoted, and stored at -80 °C. Viral supernatants supplemented with 10 pg/ml DEAE-Dextran (Sigma-Aldrich) were used to transduce THP-1 cells. At 72 h post-transduction, the cells were selected using puromycin. The stable knock-out cell lines were analyzed by western blot. The expression of GFP- tagged PPM1A was evaluated via western blot, flow cytometry and immunofluorescence.
  • Lysis was performed by addition of 100 pg/mL of lysozyme (Alfa Aesar), 1 mM PMSF (OmniPurTM), 1 mM DTT (Fisher BioReagents), and 0.1 pL/mL PierceTM Universal Nuclease for Cell Lysis (Thermo Fisher Scientific) and by sonication at 1s/1s on/off for 5 minutes at 12W (Model 120 Sonic Dismembrator, Fisher Scientific).
  • Lysates were centrifuged and the soluble fraction was taken for protein purification using metal-ion affinity chromatography (Poly-Prep Chromatography Column, Bio-Rad) packed with 1 mL of HisPurTM Cobalt Resin (Thermo Fisher Scientific), according to manufacturer instructions. Buffer exchange was performed to remove imidazole from recombinant proteins PPM1A and PPM1B, using the 10,000 MWCO Vivaspin® 6 Centrifugal Concentrator (Sartorius). Purified protein was stored at -80°C in buffer supplemented with 10% glycerol and 1 mM DTT (protein buffer).
  • Mtb-luciferase infected cells were lysed using Gio-Lysis Buffer (Promega), according to manufacturer’s instructions. Lysates were transferred to white 96-well plates and the luciferase assay was performed using the Bright-GloTM Luciferase Assay System (Promega), according to manufacturer’s instructions. Luminescence was read with the SynergyTM H1 Hybrid Multi-Mode Reader (BioTek) and the relative light units (RLU) were used as measure of Mtb burden, as previously described (43).
  • CFU analysis in agar plates (7H10) was instead used to assess bacterial survival in Mtb-infected macrophages. Briefly, infected macrophages were washed several times in PBS and lysed in 0.025% SDS - PBS for 5 minutes at room temperature. Serial dilutions were then seeded in 7H10 agar plates. [00129] Protein cell lysates.
  • adherent cells were washed twice with PBS and lysed in RIPA buffer (150 mM NaCI, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing protease and phosphatase inhibitors (HaltTM Protease and Phosphatase Inhibitor Cocktail, ThermoFisher Scientific).
  • RIPA buffer 150 mM NaCI, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0
  • protease and phosphatase inhibitors HaltTM Protease and Phosphatase Inhibitor Cocktail, ThermoFisher Scientific.
  • macrophages were lysed in HNTG buffer (150 mM NaCI, 20 mM HEPES, 10% Glycerol, 0.1% Triton X-100) containing protease and phosphatase inhibitor
  • the cytoplasmic fraction was moved to a new tube and clarified by centrifugation at 12,000 x g for 10 minutes at 4°C, whereas the pellet containing the nuclei was washed twice in Buffer B (50 mM NaCI, 10 mM HEPES pH 8, 25% Glycerol, 0.1 mM EDTA) and spun down at 3000 rpm for 2 minutes at 4°C after each wash. After the washes, nuclei were lysed in Buffer C (350 mM NaCI, 10 mM HEPES pH 8, 25% Glycerol, 0.1 mM EDTA) at 4°C for 30 minutes in constant shaking, then clarified by centrifugation at 12,000 x g for 10 minutes at 4°C.
  • Buffer B 50 mM NaCI, 10 mM HEPES pH 8, 25% Glycerol, 0.1 mM EDTA
  • THP-APPM1A macrophages were infected with Mtb-WT (MOI 10) in presence of bafilomycin (100 nM) for 24 hours.
  • the adherent cells were harvested by centrifugation and lysed in cold Phosphatase Assay Reaction Buffer (100 mM NaCI + 25 mM HEPES, pH 7.2) with 0.1% Triton X-100.
  • Cells were passed 20x through a syringe (27G needle). No phosphatase/protease inhibitors or MnCh were added to the lysis buffer at this time. After 20 minutes in ice, lysates were spun down to remove the debris and supernatants were collected.
  • Protein concentration of the lysates was determined by DC Protein Assay (BioRad), according to the manufacturer’s recommendations.
  • Immunoprecipitation and western blot analysis Immunoprecipitation was performed according to standard protocols. Briefly, protein lysates (250 g per sample) were incubated with primary anti-GFP antibody (A-11122, at 1:500 dilution factor) for 3 hours at 4°C, in gentle rotation, followed by an incubation of 2 hours at 4°C with anti-protein A/G magnetic beads (ChlP-grade Protein A/G Magnetic Beads, Pierce). The following steps were performed according to manufacturer’s instructions.
  • Membranes were blocked in 5% skim milk, incubated with primary antibodies overnight at 4°C and with HRP-conjugated secondary antibodies for 1 h at room temperature (BioRad) for ECL reaction (ClarityTM Western ECL Substrate, BioRad). Imaging and densitometric analyses were performed with Image Quant LAS4000 and software (GE Healthcare, Cytiva). TBS with 0.1% tween-20 (TBS-T) was used for all washes, blocking buffer and antibody dilutions.
  • the primary antibodies used for western blot are: from Cell Signaling Technology LC3B (D11), PP2C-a (D18C10), pS403-SQSTM1/p62 (D8D6T), Vinculin (E1E9V), Beclin-1 (D40C5), Atg16L1 (D6D5), Atg7 (D12B11), Atg5 (D5F5U), Atg3, MDA-5 (D74E4), Rig-I (D14G6), MAVS, TBK1/NAK (D1B4), pS172- TBK1/NAK (D52C2), from Santa Cruz Biotechnology Fibrillarin (G-8), SQSTM1 (D-3), from Invitrogen GAPDH (GA1R), PPM1A (7F12), -Tubulin (BT7R), GFP (A-11122).
  • LC3B Cell Signaling Technology LC3B
  • PP2C-a D18C10
  • TH P-1 macrophages were fixed in 4% methanol-free formaldehyde (Pierce), permeabilized with 0.2% TritonX-100 in PBS for 10 minutes and blocked 2 hours in 1% BSA-PBS.
  • Cells were incubated with primary antibodies overnight at 4°C in a humid chamber and with the secondary AlexaFluor (AF)-conjugated antibodies for 1 hour at room temperature in the dark.
  • Primary and secondary antibodies were diluted in 1% BSA-PBS and washes were performed in 1X PBS. Nuclei were stained with Hoechst 33342, according to manufacturer’s recommendations (NucBlueTM Live ReadyProbesTM Reagent, Invitrogen).
  • Phosphatase assays were performed in reaction buffer (100 mM NaCI + 25 mM HEPES, pH 7.2) using either 10 pg of total protein lysates (previously described) or 40 mM of a synthetic peptide corresponding to residues surrounding Ser403 of human SQSTM1/p62 protein (ESLSQML[S]MGFSDEG, Thermo Fisher Scientific). Recombinant His-PPM1 A was added at the indicated concentrations. In samples treated with SMIP compounds and sanguinarine (“SG”), the recombinant His- PPM1A was pre-incubated 30 minutes with the inhibitors.
  • reaction buffer 100 mM NaCI + 25 mM HEPES, pH 7.2
  • MnCh or MgCh were added to the reaction.
  • the reaction was performed for 30 minutes at 37°C with constant shaking.
  • protein lysates were used, the reaction was stopped by boiling the samples for 10 minutes at 96°C in 4X Laemmli buffer with p-Mercapto-Ethanol (BioRad).
  • the results were evaluated by western blot, probing for phosphorylated S403-p62 and total p62 protein.
  • the synthetic peptide was used, the results were evaluated by Malachite Green Assay (Sigma-Aldrich), according to manufacturer’s instructions, measuring the amount of free phosphate.
  • pNPP assay 12.5-20 pg/mL of recombinant protein was combined with SMIP compounds or sanguinarine at the desired concentration in 96-well plates and incubated at room temperature for 15 minutes in reaction buffer with 4 mM MnCh.
  • pNPP Thermo Fisher Scientific
  • pNPP was added to a final concentration of 50 mM and the plates were incubated at 37°C for 20 minutes.
  • the reaction was stopped by adding one volume of 0.5 M EDTA (pH 10). Absorbance was measured at 405 nm using the SynergyTM H1 Reader (BioTek).
  • mice experiments BALB/c female mice of 6-8 weeks were purchased from Charles River Laboratories and allowed to acclimate for 2 weeks before any experiment.
  • SMI P-30 toxicity tests the animals were randomized and treated with different doses of SMIP-30 up to 100 mg/kg via intra-peritoneal injection.
  • reaction buffer 100 mM NaCI + 25 mM HEPES
  • the treatments were administered every second day.
  • Mtb infection experiments mice were infected via tail vein injection with 10 7 CFU of Mtb mc 2 6206, previously resuspended in PBS with 0.05% Tween-80.
  • mice were randomized in 4 different cohorts and treated with vehicle, SMIP-30 at 25 mg/kg, rifampicin at 20 mg/L or with the combination of the two drugs. All mice received an intra-peritoneal injection either with vehicle or SMIP-30 every two days, whereas rifampicin was administered in drinking water ad libitum and refreshed every second day. To avoid antibiotic carry-over in CFU analyses, rifampicin treatment was suspended 24 h before end point.
  • mice were sacrificed when a loss of body weight higher than 15% was shown or at the end of the course of treatment (14 or 21 days).
  • Mouse blood was collected via cardiac puncture, allowed to coagulate for 30 minutes at room temperature and centrifuged for 20 minutes at 1000 x g to separate the serum. Serum was immediately frozen at -80°C in aliquots. For CFU analysis, lungs and spleens were homogenized in PBS + 0.05% Tween-80.
  • RNA_01 Lungs were homogenized with GentleMACS Dissociator (Milteny) using the RNA_01 protocol, whereas spleens were manually homogenized with sterile pestles. Different serial dilutions were seeded in 7H10 agar plates and incubated for 3 weeks at 37 °C.
  • Cytokine and chemokine expression levels were measured in mouse serum and in culture supernatants harvested from Mtb-infected THP-1 macrophages at 24 h after infection.
  • the LEGENDplexTM Mouse Inflammation Panel (13-plex) and the Human Essential Immune Response Panel (13-plex) bead-based multiplex assays (BioLegend) were used for mouse serum and THP-1 supernatants, respectively, according to manufacturer’s protocols. Results were measured by flow cytometry (CytoFLEX, Beckman Coulter) and analyzed with LEGENDplexTM Data Analysis Software (BioLegend).
  • Mtb viability assays Mtb viability assays. Luminescence’. The direct killing ability of the compounds were assessed by the luminescence signal readout of Mtb-lux. Mid-log Mtb-lux was diluted to an OD of 0.03 and added into 96-well white plates at 100 pl in each well. 100 pl of serial diluted SMIP-6 and SMIP-9 (200-25 pM), RIF (1.2 pM, Fisher Scientific) and INH (3.6 pM, Acros Organics) were added into the wells. A readout of luminescence signal was performed immediately after plate setup (time 0). The plates were incubated in 37°C without shaking for 20 hours, followed by a final readout. Luminescence was measured with the Synergy H1 Microplate Reader with an optimized integration time of 10 seconds.
  • Colony forming unit (CFU) platina Middlebrook 7H10 agar plates (BD Biosciences) supplemented with 0.5% glycerol, 10% OADC, 24 pg/ml D-pantothenic acid, and 50 pg/ml L-leucine were prepared.
  • Midlog Mtb was washed and treated with serial diluted compounds (200-50 pM), RIF (250 ng/ml, 500 ng/ml) and untreated control in 96-well plates. The plates were incubated at 37°C for 20 hours. The bacteria in each well were serial diluted by 10-fold.
  • TH P-1 cells were differentiated into macrophages as described above. The amount of Mtb-lux was counted for a multiplicity of infection (MOI) of 10 using the conversion of 3x10 8 bacteria/ml for OD 1.0. Log-phase bacteria were resuspended in RPMI 1640 infection media: supplemented with 10% human serum (Millipore Sigma, Burlington, MA), 10 mM HEPES, 2 mM glutamine, D-pantothenic acid (24 pg/ml) and L-leucine (50 pg/ml).
  • MOI multiplicity of infection
  • THP-1 derived macrophages were washed with PBS and recovered in infection media for 1 hour, and then infected with Mtb-lux for 4 hours. Extracellular Mtb-lux was removed by three PBS washes, followed by addition of compounds. Intracellular survival of Mtb-lux was quantified 24 h postinfection by luminescence readouts with the Synergy H1 Microplate Reader with an integration time of 10 seconds.
  • Non-replicating Mtb (NR-Mtb) was generated using an stablished low-pH model. (Early, J. V.; Mullen, S.; Parish, T. A Rapid, Low PH, Nutrient Stress, Assay to Determine the Bactericidal Activity of Compounds against Non-Replicating Mycobacterium Tuberculosis.
  • NMR spectra were recorded at room temperature on the following spectrometers: Bruker Avance III 400 Spectrometer (400 MHz) and Bruker Avance III 500 (Cryo) Spectrometer (500 MHz). Chemical shifts are given in ppm and coupling constants in Hz. 1 H spectra were calibrated in relation to the reference measurement of TMS (0.00 ppm). 13 C spectra were calibrated in relation to deuterated solvents. The following abbreviations were used for 1 H NMR spectra to indicate the signal multiplicity: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet) as well as combinations of them.
  • Step a To a mixture of A1-1 (9.8 g, 80 mmol) and succinic anhydride (10 g, 100 mmol) in nitrobenzene (30 mL) was added AlCh (20 g, 160 mmol) in ice bath, and the reaction mixture was stirred at room temperature and monitored by TLC. After completion of the reaction, the reaction was quenched by ice water, and washed with water and ethyl acetate (EA). The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The residue was recrystallized to afford yellow solid (16 g, 90%). 1 H NMR (400
  • Step b and c A mixture of A2-1 (6 g, 25 mmol) and EtaSiH (7.25 g, 62.5 mmol) in TFA (18 mL) was stirred at 100°C for 7 h. The reaction mixture was cooled to room temperature and then diluted with water (100 mL). The product was extracted with EA (50 mLx3), then the organic extract was dried over NaaSCL and concentrated under reduced pressure. Polyphosphoric acid (12 mL) was added to the crude product dropwise at room temperature, the reaction mass was heated at 80°C and monitored by TLC. After the completion of reaction, the mixture was diluted with EA (20 mL) in 0°C, and quenched by ice water.
  • Step d A mixture of A4-1 in 48% HBr (70 mL) was refluxed at 125°C for 6 h. The reaction was cooled to room temperature and the brown crystal was filtered and dried to afford product (3.0 g, 73%).
  • Step e and f To a mixture of A4-2 in chloroform (10 mL), a solution of Br2 in chloroform (10 mL) was added dropwise at room temperature and the reaction was monitored by TLC. After the completion of reaction, the mixture was quenched by aqueous Na2SOs, washed with water and extracted with EA (30 mLx3). The organic phase was dried over Na2SC>4 and concentrated under reduced pressure. The crude product was dissolved in MeCN (15 mL), and DBU (3.74 g, 24.6 mmol) was added at 50°C, the mixture was stirred at 50°C for 10 min.
  • Step g To a solution of A6-2 (265 mg, 1.0 mmol) in DMF (5 mL) was added NaOH (80 mg, 2.0 mmol), after stirred at room temperature for 30 min, 2-bromo-2- methylpropanamide (332 mg, 2.0 mmol) was added, and stirred for 2 h at ambient temperature. The mixture was diluted with EA (30 mL), washed with water and dried over Na 2 SC>4, then purified by gel chromatography to give light yellow solid (172 mg, 49%).
  • Step h and i To a solution of A7-2 in DMF (12 mL) and DMTP (3 mL) was added NaH (60% dispersion in mineral oil, 268 mg, 6.7 mmol) at room temperature, the mixture was heated at 100°C for 4 h. The reaction was cooled to room temperature and quenched by aqueous NH4CI. The product was extracted with EA (30 mLx3), dried over Na2SC>4, concentrated under reduced pressure, then purified by gel chromatography to give light yellow solid (341 mg, 43%). ESI-MS m/z 266.1 [M+H + ],
  • Step j and k To a solution of A9-2 (424 mg, 1.6 mmol) in THF (5mL) was added formyl acetate (557 mg, 4.8 mmol), the mixture was stirred for 30 min at room temperature. The product was washed with water and EA (30 ml_x3), the organic phase was dried over Na2SC>4, concentrated under reduced pressure to afford A10-2 as white solid (400 mg, 85%). A mixture of A10-2 (400 mg, 1.36 mmol) and NaH (60% dispersion in mineral oil , 109 mg, 2.72 mmol) in DMF (5 mL) was stirred for 10 min at room temperature.
  • Step I and m To a solution of A11-2 (100 mg, 0.3 mmol) in 1 ,4-dioxane (2 mL) and water (2 mL), 2-(benzo[d][1 ,3]dioxol-5-yl)-4,4,5,5-tetramethyl-1 ,3,2-dioxaborolane (112 mg, 0.45 mmol), Pd(PPh3)4 (17 mg, 0.015 mmol) and K3PO4 (95 mg, 0.45 mmol) were added under nitrogen atmosphere, the suspension was heated at 90°C for 3 h.
  • reaction was cooled to room temperature, washed with water and EA (20 ml_x3), then concentrated under reduced pressure to give yellow oil.
  • POCh was added (0.5 mL) to the crude product, the mixture was heated at 60°C and monitored by TLC. After the completion of reaction, the mixture was diluted with DCM (10 mL), then yellow solid appeared and filtered to afford the product (64 mg, 43%).
  • I-4 was prepared according to the procedure for 1-1 synthesis with N,N- dimethyl-3-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)aniline as the reagent of Suzuki reaction, yellow solid, yield: 46%.
  • I-7 was prepared according to the procedure for 1-1 synthesis with 6- methoxytetralone as the starting material and 2-(4-methoxyphenyl)-4,4,5,5-tetramethyl-1 ,3,2- dioxaborolanea as the reagent of Suzuki reaction, yellow solid, yield: 57%.
  • I-8 was prepared according to the procedure for 1-1 synthesis with 6- methoxytetralone as the starting material and 2-(3-chlorophenyl)-4,4,5,5-tetramethyl-1 ,3,2- dioxaborolane as the reagent of Suzuki reaction, yellow solid, yield: 37%.
  • I-9 was prepared according to the procedure for 1-1 synthesis with 6- methoxytetralone as the starting material and N,N-dimethyl-3-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)aniline as the reagent of Suzuki reaction, yellow solid, yield: 52%.
  • 11-1 was prepared according to the procedure for 1-1 synthesis with 2-bromo-6- fluoroaniline as the starting material and 2-(4-methoxyphenyl)-4,4,5,5- tetramethyl-1 ,3,2- dioxaborolanea as the reagent of Suzuki reaction, yellow solid, yield: 36%.
  • 11-2 was prepared according to the procedure for 1-1 synthesis with 2-bromo-4- fluoroaniline as the starting material and N,N-dimethyl-3-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)aniline as the reagent of Suzuki reaction, yellow solid, yield: 42%.
  • 11-3 was prepared according to the procedure for 1-1 synthesis with 2-bromo-4- fluoroaniline as the starting material and 2-(4-methoxyphenyl)-4,4,5,5- tetramethyl-1 ,3,2- dioxaborolanea as the reagent of Suzuki reaction, yellow solid, yield: 50%.
  • 11-4 was prepared according to the procedure for 1-1 synthesis with 2-bromo-4- fluoroaniline as the starting material and 2-(benzo[d][1 ,3]dioxol-5-yl)-4,4,5,5-tetramethyl-1 ,3,2- dioxaborolane as the reagent of Suzuki reaction, yellow solid, yield: 30%.
  • 11-5 was prepared according to the procedure for 1-1 synthesis with methyl 3- amino-4-bromobenzoate as the starting material and 2-(benzo[d][1 ,3]dioxol-5-yl)-4, 4,5,5- tetramethyl-1 ,3,2-dioxaborolane as the reagent of Suzuki reaction, yellow solid, yield: 28%.
  • 11-6 was prepared according to the procedure for 1-1 synthesis with 2-bromo-5-
  • Step a To a solution of 2-bromo-5-fluoroaniline (190 mg, 1 mmol) in MeOH (3 mL), benzaldehyde (160 mg, 1.5 mmol) and AcOH (60 mg, 1 mmol) were added, the mixture was stirred at room temperature for 30 min. Then NaCNBHs (46 mg, 0.9 mmol) was added and the reaction was monitored by TLC. After the completion of reaction, the mixture was quenched with aqueous NH4CI, extracted with DCM, dried over Na2SO4 and concentrated under reduced pressure to give the crude product. ESI-MS m/z 280.1 [M+H + ],
  • Step b The crude product of step a was dissolved in DMF (4 mL), NaOH (80 mg, 2 mmol) was added and the mixture was stirred at room temperature for 10 min. Then Mel (426 mg, 3 mmol) was added and the reaction stirred for further 30 min. After the completion of reaction, the product was diluted with water (50 mL), extracted with DCM (30 mLx3), dried over Na2SC>4 and concentrated under reduced pressure to give the crude product.
  • Step c and d The crude product of step b was dissolved in toluene (5 mL), 1 ,10- phenanthroline (180 mg, 1 mmol) and fBuOK (670 mg, 6 mmol) were added in nitrogen atmosphere. The mixture was heated at 100°C for 3 h. The reaction was cooled to room temperature, washed with water, extracted with PE, dried over Na2SO4 and concentrated under reduced pressure to give brown oil. Next, the crude product was dissolved in toluene (30 mL), aqueous NaOH (10%, 3 mL) and DDQ (567 mg, 2.5 mmol) were added with vigorous stirring.
  • Step e B3-2 was prepared according to the procedure for 11-12 synthesis with methyl 3-amino-4-bromobenzoate as the starting material.
  • MeOH 2-aqueous LiOH
  • aqueous LiOH 120 mg, 2 mL
  • EA 30 ml_x3
  • Step f To a solution of B3-3 in DCM (3 mL), morpholine (49 mg, 0.56 mmol), EDCI (161 mg, 0.84 mmol) and HOBt (113 mg, 0.84 mmol) were added, the mixture was stirred at room temperature for 1 h. The product was extracted with DCM (20 mLx3), washed with water, dried over Na2SO 4 and concentrated under reduced pressure to afford B3-4. ESI-MS m/z 419.3 [M+H + ],
  • HI-2 was prepared according to the procedure for 111-1 synthesis with 2- aminopyridine as the reagent of condensation reaction, yellow solid, yield: 12%.
  • Step a To a solution of 5-bromo-2-iodoaniline (4.47 g, 15 mmol) in THF (20 mL), formyl acetate (3.96 g, 45 mmol) was added dropwise, the mixture was stirred at room temperature for 1 h. After the completion of reaction, the reaction was concentrated under reduced pressure to afford C2 as white solid (4.8 g, 98%).
  • ESI-MS m/z 325.9 [M+H + ],
  • Step b To a solution of C2 (1.96 g, 6.0 mmol) and 2-formylbenzeneboronic acid (1.08 g, 7.2 mmol) in DMF (30 mL), Pd(OAc)2 (67 mg, 0.3 mmol), PPhs (393 mg, 1.5 mmol) and CS2CO3 (3.9 g, 12 mmol) were added in nitrogen atmosphere, then the mixture was heated at 90°C for 3 h. The reaction was cooled to room temperature, washed with water, extracted with EA (30 ml_x3), dried over Na2SC>4 and concentrated under reduced pressure, then purified by gel chromatography to give C3 as yellow solid (660 mg, 43%).
  • Step c To a solution of C3 (102 mg, 0.4 mmol) and 1-Methyl-4-(4, 4,5,5- tetramethyl- 1 ,3,2-dioxaborolan-2-yl)-1 H-pyrazole (125 mg, 0.6 mmol) in 1 ,4-dioxane (3 mL) and waer (3 mL), Pd(PPh3)4 (23 mg, 0.02 mmol) and K3PO4 (127 mg, 0.6 mmol) were added in nitrogen atmosphere, the mixture was heated at 90°C for 1 h.
  • Step d-1 The crude product C4-1 was dissolved in acetone (3 mL), and Mel (1.13 g, 8 mmol) was added, the mixture was refluxed for 30 min. The resulting suspension was filtered to give HI-3 as yellow solid (20 mg, 12%).
  • HI-4 was prepared according to the procedure for HI-3 synthesis with 3,5- Dimethyl-4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl) isoxazole as the reagent of Suzuki reaction, yellow solid, yield: 20%.
  • Step d-2 To a solution of C3 (51 mg, 0.2 mmol) and 1 H-pyrrolo[3,2-b]pyridine (29 mg, 0.24 mmol) in toluene (3 mL), Cui (3.8 mg, 0.02 mmol), trans-N,N'-Dimethyl- 1 ,2- cyclohexanediamine (57 mg, 0.4 mmol) and K3PO4 (85 mg, 0.4 mmol) were added in nitrogen atmosphere, the mixture was heated at 110°C overnight. The reaction was cooled to room temperature, washed with water, extracted with DOM (30 ml_x3), dried over Na 2 SO4 and concentrated under reduced pressure to give C4-2.
  • HI-6 was prepared according to the procedure for HI-3 synthesis with 3-(N,N- dimethylamino)phenylboronic acid as the reagent of Suzuki reaction, yellow solid, yield: 20%.
  • Step d-3 To a solution of C3 (100 mg, 0.4 mmol) and morpholine(42 mg, 0.48 mmol) in toluene (3 mL), Pd 2 (dba)3 (9.2 mg, 0.01 mmol), fBuONa (54 mg, 5.6 mmol) and 2,2'- bis(diphenylphosphino)-1 ,1'-binaphthyl (18.7 mg, 0.03 mmol) were added in nitrogen atmosphere, the mixture was heated at 80°C for 8 h.
  • Step a To a solution of 2-bromo-5-methoxybenzaldehyde (1.08 g, 5 mmol) and 4-hydroxyphenylboronic acid (1.04 g, 7.5 mmol) in 1 ,4-dioxane (7 mL) and water (7 mL), Pd(PPh3)4 (289 mg, 0.25 mmol) and K3PO4 (1.59 g, 7.5 mmol) were added in nitrogen atmosphere, the mixture was heated at 90°C for 2 h.
  • Step b To a solution of D2 (244 mg, 1 mmol) and O-(4- cyanobenzoyl)hydroxylamine (243 mg, 1.5 mmol) in DMF (10 mL), fac-lr(ppy)3 (13 mg, 0.02 mmol) and 4-chlorobenzenesulfonic acid (19 mg, 0.1 mmol) were added in nitrogen atmosphere, the mixture was irradiated by white LED strips at room temperature for 36 h. After the completion of reaction, the product was extracted with DCM (30 mLx3), washed with water, dried over Na 2 SC>4 and concentrated under reduced pressure to give D3-1 as yellow solid (113 mg, 50%).
  • Step c To a solution of D3-1 (63 mg, 0.28 mmol) in THF (3 mL), 2-(morpholin- 4-yl)ethanol (73 mg, 0.56 mmol), DIAD (170 mg, 0.84 mmol) and PPha (220 mg, 0.84 mmol) were added, the mixture was stirred at room temperature for 2 h. After the completion of reaction, the product was extracted with DCM (30 ml_x3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure to afford D3-1-1. ESI-MS m/z 338.2 [M+H + ],
  • Step d The crude D4-1 was dissolved in acetone (3 mL), Mel (798 mg, 5.6 mmol) was added and the mixture was fluxed for 30 min. The resulting suspension was filtered to give HI-9 as yellow solid (25 mg, 19%).
  • 111- 14 was prepared according to the procedure for HI-9 synthesis with 1-[4-(2- hydroxyethyl)piperazin-1-yl] ethanone, hydrochloride as the reagent of Mitsunobu reaction, yellow solid, yield: 32%.
  • Step c D3-2 was prepared according to the procedure for HI-9 synthesis with methyl 4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)benzoate as the starting material, yellow solid, yield: 38%.
  • ESI-MS m/z 268.1 [M+H + ] To a solution of D3-2 (1.18 g, 5 mmol )in MeOH (15 mL) and THF (6 mL), aqueous LiOH (480 mg, 3 mL) was added, the mixture was stirred at room temperature for 1 h.
  • Step d To a solution of D3-3 (82 mg, 0.37 mmol) in DMF (3 mL), 4-amino-1- Boc-piperidine (148 mg, 0.74 mmol), EDCI (142 mg, 0.74 mmol) and HOBt (100 mg, 0.74 mmol) were added, the mixture was stirred at room temperature for 1 h. After the completion of reaction, the product was extracted with DCM (20 mLx3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure to give crude product. ESI-MS m/z 436.5 [M+H + ],
  • Step e The crude product of step d was dissolved in acetone (3 mL), Mel (1 .07 g, 7.4 mmol) was added and the mixture was refluxed for 30 min. The resulting suspension was filtered to afford yellow solid, yield: 49%.
  • Step d To a solution of D3-3 (82 mg, 0.37 mmol) in DMF (3 mL), NH 4 CI (119 mg, 1.1 mmol), HATLI (211 mg, 0.55 mmol) and DIPEA (191 mg, 1.48 mmol) were added, the mixture was stirred at room temperature for 2 h. After the completion of reaction, the product was extracted with DCM (20 ml_x3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure to give the crude product. ESI-MS m/z 253.2 [M+H + ],
  • Step e The crude product of step d was dissolved in DMSO (1 mL), Mel (1.07 g, 7.4 mmol) was added, the mixture was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (115 mg, 85%).
  • Step d To a solution of D3-3 (87 mg, 0.3 mmol) in DMF (3 mL), methoxyammonium chloride (30 mg, 0.36 mmol), EDCI (69 mg, 0.36 mmol) and 4- methylmorpholine (36 mg, 0.36 mmol) were added, the mixture was stirred at room temperature for 7 h. After the completion of reaction, the product was extracted with DCM (20 ml_x3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure to give the crude product. ESI-MS m/z 283.1 [M+H + ],
  • Step e The crude product of step d was dissolved in DMSO (1 mL), Mel (870 mg, 6.0 mmol) was added, the mixture was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (12 mg, 10%).
  • Step d To a mixture of D3-3 (17 mg, 0.06 mmol) and NaOH (24 mg, 0.6 mmol) in DCM (0.5 mL) and MeOH (1.0 mL) was added hydroxylamine (0.12 mL, 50 wt.% in water), the mixture was stirred at room temperature overnight. After the completion of reaction, HCI (1 mL, 4 M in 1 ,4-dioxane) was added, and the reaction stirred for further 10 min. The solvent was removed under reduced pressure to give the crude product. ESI-MS m/z 269.1 [M+H + ],
  • Step e The crude product of step d was dissolved in DMSO (0.5 mL), Mel (170 mg, 1.2 mmol) was added, the mixture was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (28 mg, 23%).
  • Step d and e To a solution of D3-3 (28 mg, 0.1 mmol) in EtOH (1 mL), hydrazine hydrate (0.03 mL, 85% in water) was added, the mixture was refluxed overnight. After the completion of reaction, the solvent was removed under reduced pressure, and the crude product was dissolved in DMSO (0.5 mL), Mel (284 mg, 2 mmol) was added, the mixture was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (40 mg, 89%).
  • Step c and d To a solution of D3-2 (40 mg, 0.15 mmol) in /PrOH (0.2 mL), K3PO4 (19 mg, 0.045 mmol) and ethanolamine (9 mg, 0.15 mmol) were added, the mixture was heated at 60°C overnight. The mixture was concentrated under reduced pressure and purified by flash chromatography to afford D3-3-6. The product was diluted with DMSO (0.5 mL), Mel (426 mg, 3 mmol) was added, the mixture was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (57 mg, 86%).
  • Step c and d D3-4 was prepared according to the procedure for III-9 synthesis with methyl 2-(4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl)acetate as the starting material, yellow solid, yield: 75%.
  • HI-23 was prepared according to the procedure for 111-19 synthesis with D3-4 as the intermediate, yellow solid, yield: 20%.
  • 1 H NMR (400 MHz, DMSO-cfe): 6 10.85 (s, 1 H), 10.16 (s, 1 H), 9.09 - 8.96 (m, 3H), 8.39 (s, 1 H), 7.99 (dd, J 25.4, 8.7 Hz, 3H), 4.64 (s, 3H), 4.04 (s, 3H), 3.73 (s, 2H).
  • 111-24 was prepared according to the procedure for 111-20 synthesis with D3-4 as the intermediate, yellow solid, yield: 83%.
  • 1 H NMR (400 MHz, DMSO-cfe): 6 10.17 (s, 1 H), 9.06 (d, J 9.2 Hz, 2H), 8.41 (s, 1 H), 8.08 - 7.96 (m, 3H), 6.09 (s, 2H), 4.65 (s, 3H), 4.04 (s, 3H).
  • HI-25 was prepared according to the procedure for 111-21 synthesis with D3-4 as the intermediate, yellow solid, yield: 20%.
  • Step c D3-9 was prepared according to the procedure for 111-9 synthesis with 4-cyanophenylboronic acid as the starting material, yellow solid, yield: 69%.
  • Step c D3-5 was prepared according to the procedure for 111-9 synthesis with 4-acetylphenylboronic acid as the starting material, yellow solid, yield: 62%.
  • Step c and d To a solution of D3-5 in EtOH/H 2 O (4:1 , 2 mL), KOAc (106 mg, 0.5 mmol) and hydroxylamine hydrochloride (49 mg, 0.7 mmol) were added, the mixture was refluxed for 2.5 h. After cooled to room temperature, the product was extracted with DCM (20 mLx3), washed with water, dried over Na 2 SC>4 and concentrated under reduced pressure. Then the mixture of crude product and Mel (568 mg, 4 mmol) in DMSO (1.5 mL) was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (72 mg, 88%).
  • HI-29 was prepared according to the procedure for HI-28 synthesis with methoxyammonium chloride as the starting material, yellow solid, yield: 70%.
  • HI-30 was prepared according to the procedure for III-9 synthesis with 4-
  • Step d A mixture of 111-31 (26 mg, 0.06 mmol) and TFA (2 mL) in DCM (1 mL) was stirred at room temperature for 3 h. The mixture was concentrated under reduced pressure and diluted with DCM (5 mL), the resulting suspension was filtered to afford III-32 as red solid (23 mg, 99%).
  • Step c D3-7 was prepared according to the procedure for HI-9 synthesis with 4-Boc-aminophenylboronic acid as the starting material, yellow solid, yield: 30%.
  • ESI-MS m/z 325.1 [M+H + ] To a solution of D3-7 (380 mg, 1.16 mmol) in DCM (2 mL) was added TFA (4 mL), the mixture was stirred at room temperature for 10 min. The mixture was concentrated under reduced pressure, extracted with DCM (20 mLx3), and washed with aqueous Na 2 CO3, dried over Na 2 SC>4 and concentrated under reduced pressure to give orange solid (255mg, 98%).
  • Step d A mixture of D3-8 (32 mg, 0.14 mmol) and Mel (398 mg, 2.8 mmol) in DMSO (0.5 mL) was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (60 mg, 95%).
  • Step d and e To a solution of D3-8 (32 mg, 0.14 mmol) and Et 3 N (56 mg, 0.56 mmol )in DMF (3 mL), lauroyl chloride (61 mg, 0.28 mmol) was added, the mixture was stirred at room temperature for 10 min. After the completion of reaction, the reaction was quenched with aqueous NH4CI, extracted with DCM (20 mLx3), dried over Na 2 SO4 and concentrated under reduced pressure. The crude product was dissolved in DMSO (0.5 mL), Mel (398 mg, 2.8 mmol) was added, the mixture was heated at 60°C for 30 min.
  • Step d and e To a solution of D3-8 (70 mg, 0.31 mmol) in DMF (1 mL), picolinic acid (42 mg, 0.34 mmol), EDCI (65 mg, 0.34 mmol) and HOBt (46 mg, 0.34 mmol) were added, the mixture was stirred at room temperature for 3 h. After the completion of reaction, the product was extracted with DCM (20 ml_x3), washed with water, dried over Na2SO4 and concentrated under reduced pressure. The crude product was dissolved in DMSO (0.5 mL), Mel (880 mg, 6.2 mmol) was added, the mixture was heated at 60°C for 30 min.
  • Step d and e A mixture of D3-8 and GDI (61 mg, 0.375 mmol) in DMF (1 mL) was stirred at room temperature for 10 min, and diethylamine (22 mg, 0.3 mmol) was added, the mixture was heated at 70°C for 6 h. After cooled to room temperature, the mixture was diluted with DCM (30 mL), washed with 1 M HCI (30 mL), dried over Na2SC>4 and concentrated under reduced pressure. The crude product was dissolved in DMSO (0.5 mL), Mel (710 mg, 5.0 mmol) was added, the mixture was heated at 60°C for 30 min.
  • HI-37 was prepared according to the procedure for HI-36 synthesis with morpholine as the starting material, yellow solid, yield: 38%.
  • Step d and e To a solution of D3-8 (45 mg, 0.2 mmol) and Et 3 N (81 mg, 0.8 mmol) in DMF (1 mL) was added methanesulfonic anhydride (70 mg, 0.4 mmol), the reaction was stirred at room temperature and monitored by TLC. The reaction was quenched by aqueous NH4CI, extracted with DCM (20 ml_x3), dried over Na2SC>4 and concentrated under reduced pressure. The crude product was dissolved in DMSO (0.5 mL), Mel (568 mg, 4.0 mmol) was added, the mixture was heated at 60°C for 30 min.
  • DMSO 0.5 mL
  • Mel 568 mg, 4.0 mmol
  • HI-39 was prepared according to the procedure for HI-38 synthesis with cyclopropanesulfonyl chloride as the starting material, red solid, yield: 43%.
  • HRMS calcd. for Ci8Hi9N 2 O 3 S[M + ]:
  • HI-40 was prepared according to the procedure for HI-38 synthesis with chlorosulfonamide as the starting material, yellow solid, yield: 44%.
  • Step c To a solution of D3-1 (610 mg, 2.5 mmol) and pyridine (400 mg, 5 mmol) in DCM (10 mL) was added triflic anhydride (846 mg, 3.0 mmol) at 0°C, the mixture was stirred at ambient temperature for 5 min. The product was extracted with DCM (30 mLx3), washed with water, dried over Na 2 SO4 and concentrated under reduced pressure, then purified by flash chromatography to afford D3-1-2 as yellow solid (300 mg, 34%).
  • Step d To a solution of D3-1-2 (383 mg, 1.02 mmol) and B2pin2 (518 mg, 2.04 mmol) in 1 ,4-dioxane (5 mL), Pd(dppf)Ch (90 mg, 0.12 mmol) and KOAc (300 mg, 3.06 mmol) were added in nitrogen atmosphere, the mixture was heated at 90°C for 3 h. After cooled to room temperature, the product was extracted with PE/EA (1 :1 , 40 ml_x2), washed with water, dried over Na2SC>4 and concentrated under reduced pressure to give D3-1-3. ESI-MS m/z 336.2 [M+H + ],
  • Step e To a solution of 3,3’-dithiodipropionic acid (3.15 g, 15 mmol) in DCM (30 mL), DMF (0.5 mL) and oxalyl chloride (5.7 g, 45 mmol) were added at 0°C, the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and dissolved in DCM (20 mL), then added to a solution of fBuNH 2 (15 mL) in DCM (10 mL) at 0°C.
  • Step f To a solution of H2 (4.0 g, 12.5 mmol) in DCE (63 mL) was added sulfuryl dichloride (5.06 g, 37.5 mmol) at 0°C, the mixture was stirred at room temperature for 3 h. The reaction was quenched with water, extracted with DCM (50 mLx3), dried over Na2SO4 and concentrated under reduced pressure.
  • Step g To a solution of H3 (1.24 g, 6.5 mmol) in DCM (33 mL) was added m-
  • Step h To a solution of D3-1-3 (392 mg, 1.0 mmol) and H4 (268 mg, 1.2 mmol) in 1 ,4-dioxane, Pd(dppf)Cl2*CH2Cl2 (139 mg, 0.17 mmol) and K2CO3 (690 mg, 5.0 mmol) were added in nitrogen atmosphere, the mixture was heated at 80°C overnight. After cooled to room temperature, the product was extracted with DCM (30 ml_x3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure. The residue was purified by flash chromatography to afford D3-11 as yellow solid (120 mg, 27%).
  • Step i To a solution of D3-11 (40 mg, 0.1 mmol) was added Mel (290 mg, 2.0 mmol), the mixture was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (50 mg, 92%).
  • Step j To a solution of D3-12 (15 mg, 0.03 mmol) in DMSO (0.5 mL) was added Mel (568 mg, 4.0 mmol), the mixture was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (13 mg, 87%).
  • Step c and d To a solution of D3-9 (126 mg, 0.5 mmol) in DMF/MeOH (1 :1 , 0.5 mL), CU2O (2.0 mg, 0.015 mmol) and TMSN3 (144 mg, 1.25 mmol) were added, the mixture was stirred at room temperature for 10 min, then heated at 80°C overnight. After cooled to room temperature, the mixture was filtered to afford D3-10 as yellow solid (64 mg, 43%). A mixture of D3-10 (20 mg, 0.07 mmol) and Mel (199 mg, 1.4 mmol) in DMSO (0.5 mL) was heated at 60°C for 30 min. The reaction was diluted with EA (20 mL), and the resulting suspension was filtered to afford yellow solid (25 mg, 83%). 1 H NMR (400 MHz, DMSO-cfe): 6
  • Step d and e To a solution of D3-10 (15 mg, 0.05 mmol) in DMF (2 mL), K2CO3 (14 mg, 0.1 mmol) and allyl bromide (6 mg, 0.05 mmol) were added, the mixture was stirred at room temperature for 3 h. After the completion of reaction, the product was extracted with DCM (10 ml_x3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure. The crude product was dissolved in DMSO (0.5 mL), Mel (142 mg, 1.0 mmol) was added, the mixture was heated at 60°C for 30 min.
  • HI-46 was prepared according to the procedure for HI-45 synthesis with 2- bromoacetamide as the starting material, yellow solid, yield: 98%.
  • Step d and e A11-3 was prepared according to the procedure for 1-1 synthesis with 2-bromo-4-fluoroaniline as the starting material and 4-hydroxyphenylboronic acid as the reagent of Suzuki reaction (yellow solid, 80%).
  • benzyl bromide 137 mg, 0.8 mmol
  • the product was extracted with EA (20 ml_x3), washed with water, dried over Na2SO4 and concentrated under reduced pressure.
  • HI-48 was prepared according to the procedure for HI-47 synthesis with 3- bromopropyne as the starting material, yellow solid, yield: 30%.
  • Step d and e To a solution of A11-3 (100 mg, 0.4 mmol) in THF, epolamine (67 mg, 0.8 mmol), DIAD (242 mg, 1.2 mmol) and PPha (314 mg, 1.2 mmol) were added, the mixture was stirred at room temperature for 1 h. After the completion of reaction, the product was extracted with EA (20 ml_x3), washed with water, dried over Na2SO4 and concentrated under reduced pressure. Next, added POCh (0.5 mL) to the product, the mixture was heated at 60°C and monitored by TLC.
  • Step c and d D3-13 was prepared according to the procedure for III-9 synthesis with 4-methoxyphenylboronic acid and 2-bromo-5-(hydroxy)benzaldehyde as the starting material, yellow solid, yield: 56%.
  • 111-51 was prepared according to the procedure for HI-50 synthesis with 1-(2- hydroxyethyl)-4-methylpiperazine as the starting material, yellow solid, yield: 90%.
  • 111-52 was prepared according to the procedure for 111-50 synthesis with 1-[4-(2- hydroxyethyl)piperazin-1-yl] ethanone, hydrochloride as the starting material, yellow solid, yield: 91%.
  • HI-53 was prepared according to the procedure for HI-50 synthesis with tetrahydro-4-pyranol as the starting material, yellow solid, yield: 79%.
  • 111-54 was prepared according to the procedure for 111-50 synthesis with (1- methylpiperidin-4-yl)methanol as the starting material, yellow solid, yield: 51%.
  • Step c To a solution of D3-13 (244 mg, 1.0 mmol) and pyridine (158 mg, 2.0 mmol) in DCM (10 mL) was added triflic anhydride (338 mg, 1.2 mmol) at O°C, the mixture was stirred at ambient temperature for 5 min. The product was extracted with DCM (20 ml_x3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure, then purified by flash chromatography to afford D3-13-2 as yellow solid (160 mg, 45%). ESI-MS m/z 358.3
  • Step d and e To a solution of D3-13-2 (188 mg, 0.5 mmol) and 1-methyl-4- (4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)-1 H-pyrazole (208 mg, 1.0 mmol) in DME/H2O (1 :1 , 3 mL), Pd(PPh3)4 (57.8 mg, 0.05 mmol) and K2CO3 (138 mg, 1.0 mmol) were added in nitrogen atmosphere, the mixture was heated at 80°C for 3.5 h. After cooled to room temperature, the product was extracted with DCM (20 ml_x3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure.
  • DCM 20 ml_x3
  • Step a To a solution of 2-bromo-4-fluoroaniline (2.37 g, 10 mmol) and B 2 pin 2 (5.08 g, 20 mmol) in 1 ,4-dioxane (30 mL), Pd(dppf)Ch (1.05 g, 1.2 mmol) and KOAc (2.94 g, 30 mmol) were added in nitrogen atmosphere, the mixture was heated at 90°C for 2.5 h. After cooled to room temperature, the product was extracted with PE (50 mLx2), washed with water, dried over Na2SO4 and concentrated under reduced pressure to give the product without further purification. ESI-MS m/z 238.1 [M+H + ],
  • Step b The product of step a was dissolved in DMA (30 mL), and 2-bromo-5- methoxybenzaldehyde (2.15 g, 10 mmol), Pd(OAc)2 (112 mg, 0.5 mmol), PPh 8 (656 mg, 2.5 mmol) and CS2CO3 (4.88 g, 15 mmol) were added in nitrogen atmosphere. The mixture was heated at 90°C for 3 h. After cooled to room temperature, the product was extracted with DCM (50 mLx3), washed with water, dried over Na2SC>4 and concentrated under reduced pressure, then purified by flash chromatography to afford E3 as red solid (600 mg, 27%).
  • Step c A mixture of E3 (43 mg, 0.2 mmol) and 1 -bromopropane (27 mg, 0.22 mmol) in MeCN (0.5 mL) was refluxed overnight. The mixture was concentrated under reduced pressure and purified by flash chromatography to afford I V-1 as yellow solid (5 mg, 7%).
  • IV-2 was prepared according to the procedure for IV-1 synthesis with 2- bromopropane as the starting material, yellow solid, yield: 8%.
  • IV-3 was prepared according to the procedure for IV-1 synthesis with 1-lodo- 3,3,3-trifluoropropane as the starting material, brown solid, yield: 10%.
  • Step a To a solution of 2-bromo-5-methoxybenzaldehyde (2.15 g, 10 mmol) and B2pin2 (5.08 g, 20 mmol) in 1 ,4-dioxane (30 mL), Pd(dppf)Ch (878 mg, 1.2 mmol) and KOAc (2.94 g, 30 mmol) were added in nitrogen atmosphere, the mixture was heated at 80°C for 4 h. After cooled to room temperature, the product was extracted with PE (50 ml_x2), washed with water, dried over Na2SO4 and concentrated under reduced pressure, then purified by flash chromatography to afford F2 as light oil (2.3 g, 88%).
  • Step b To a solution of F2 (2.62 g, 10 mmol) and 1-bromo-4-fluoro-2- iodobenzene (3.76 g, 12.5 mmol) in 1 ,4-dioxane/H2O (1 :1 , 30 mL), Pd(PPh3)2Cl2 (702 mg, 1 mmol) and K3PO4 (3.18 g, 15 mmol) were added in nitrogen atmosphere. The mixture was heated at 100°C for 2 h.
  • Step c To a solution of F3 (2.2 g, 6.7 mmol) in ethylene glycol (67 mL), CuCh (57 mg, 0.335 mmol) and ethanolamine (490 mg, 8.0 mmol) were added, the mixture was heated at 100°C for 24 h. After the completion of reaction, the mixture was concentrated under reduced pressure and purified by flash chromatography to afford IV-4 as yellow solid (1.17 g, 33%).
  • IV-5 was prepared according to the procedure for I V-1 synthesis with 1-fluoro- 2-iodoethane as the starting material, brown solid, yield: 15%.
  • I V-6 was prepared according to the procedure for I V- 1 synthesis with 3-bromo-
  • IV-7 was prepared according to the procedure for IV-1 synthesis with 2- bromoethylamine hydrobromide as the starting material, brown solid, yield: 6%.
  • IV-8 was prepared according to the procedure for IV-1 synthesis with 2- bromoacetamide as the starting material, brown solid, yield: 65%.
  • IV-9 was prepared according to the procedure for IV-1 synthesis with ethyl bromoacetate as the starting material, yellow solid, yield: 55%.
  • IV-10 was prepared according to the procedure for I -1 synthesis with tert-Butyl bromoacetate as the starting material, yellow solid, yield: 41%.
  • IV-12 was prepared according to the procedure for I V-1 synthesis with allyl bromide as the starting material, yellow solid, yield: 49%.
  • IV-13 was prepared according to the procedure for I V-1 synthesis with 2-bromo-
  • IV-15 was prepared according to the procedure for IV-1 synthesis with 4-(2- bromoethyl)morpholine as the starting material, yellow solid, yield: 24%.
  • HRMS calcd. for C 20 H 22 FN 2 O 2 [M + ]: 341.1660, found: 341.166
  • IV-16 was prepared according to the procedure for IV-1 synthesis with 2- (chloromethyl)-1H-benzo[d]imidazole as the starting material, yellow solid, yield: 25%.
  • IV-17 was prepared according to the procedure for IV-1 synthesis with N-(4- bromobutyl)phthalimide as the starting material, yellow solid, yield: 11%.
  • Step c A mixture of D3-5 (25.1 mg, 0.1 mmol) and 2-bromoethanol (125 mg, 1.0 mmol) in DMF (0.5 mL) was heated at 110°C overnight. The reaction was concentrated under reduced pressure and purified by flash chromatography to afford IV-18 as yellow solid (10 mg, 26%).
  • IV-19 was prepared according to the procedure for IV- 18 synthesis with D3-5-1 as the intermediate, yellow solid, yield: 8%.
  • IV-20 was prepared according to the procedure for IV- 18 synthesis with D3-2 as the intermediate, yellow solid, yield: 15%.
  • IV-22 was prepared according to the procedure for IV- 18 synthesis with D3-4-1 as the intermediate, yellow solid, yield: 12%.
  • IV-23 was prepared according to the procedure for IV- 18 synthesis with 4-
  • IV-24 was prepared according to the procedure for I V-18 synthesis with 4-Boc- aminophenylboronic acid as the starting material, yellow solid, yield: 11 %.
  • Fig. 1 is a representative western blot that shows the expression of PPM1A in controls and CRISPR KO cell populations after puromycin selection. Tubulin was used as loading control.
  • THP-1 wild type (WT) and PPM1A knock out (APPM1A) macrophages were infected with the virulent Mtb H37Rv or the auxotroph Mtb mc 2 6206 (41, 42) strain and bacteria burden was analysed at different time points (Fig. 2, panels A and B).
  • the auxotroph Mtb strain mc 2 6206 has recently become an attractive tool due to similar in vitro and intramacrophage replication rates, similar responses to anti-TB agents and whole genome sequence conservation when compared to its parent H37Rv strain, with the advantage of being approved for use in biosafety level (BSL) 2 facilities (41).
  • Mtb mc 2 6206 reporter strain expressing firefly luciferase to perform luminescence-based infection assays to determine the intracellular viability of Mtb in infected macrophages (43).
  • Extensive literature data and quality control experiments support the knowledge that luciferase expression systems in mycobacteria are strongly correlative with CFU data and that RLU maintains a linear relationship with CFU over several orders of magnitude (44-47), including within infected macrophages (43).
  • Mtb mc 2 6206 triggers the production of these three proinflammatory cytokines, which is consistent with the cytokine profile induced by virulent Mtb strains as previously reported (41).
  • Fig. 2 panel A THP-1 WT and APPM1A macrophages were infected with Mtb H37Rv and CFU analysis was conducted at the indicated time points.
  • Fig. 2 panel B THP-1 WT and APPM1A macrophages were infected with Mtb mc 2 6206 expressing luciferase (MOI 5). The luciferase signal was analysed at the indicated time points. Results are shown as mean ⁇ SD of three replicates. ***p ⁇ 0.001. RLU: relative light units.
  • Fig. 2 panels C-E expression profile of I L-1 Fig. 2 (panel C), TNF-a Fig. 2 (panel D), free active TGF-pi Fig.
  • SMIP-30 and SMI P-43 exhibited the strongest inhibitory activities, comparable to sanguinarine, while other prominent hits include SMIP-84, SMIP-107, and SMIP-112 (Fig. 4). All active SMIP compounds were titrated against PPM1A to determine their dose-dependent inhibitory activity. In line with the preliminary screening results (Fig. 4), SMIP-30 and SMIP-43 showed excellent inhibitory activity against PPM1A, reaching an IC50 of 1 pM and 2.5 pM, respectively (Fig.
  • SMIP-30 and SMIP-43 therefore possess greater than 40-fold and 15-fold selectivity, respectively for PPM1A than its closest homolog, PPM1 B.
  • Figs. 3 and 9 show His-tagged PPM1A and PPM1B genes were cloned into E. coli for expression and protein generation. Bacterial lysates were purified using metal-affinity chromatography. SDS-PAGE gel analysis of sonication and purification fractions was performed to verify the presence of PPM1A ( ⁇ 43 KDa, Fig. 3) and PPM1B protein ( ⁇ 53 KDa, Fig. 9).
  • Fig. 4 is an pNPP phosphatase assay performed with recombinant PPM1A treated with the indicated inhibitors (10 pM). Results are reported as relative activity of PPM1A compared to an untreated control and shown as the mean ⁇ SD of 3 replicates. **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG. 5 shows the molecular structures of the SMIP compounds used in Fig 4.
  • Figs. 6 and 10 pNPP phosphatase assays performed with recombinant PPM1A (Fig. 6), and PPM1 B (Fig 9) treated with sanguinarine, SMIP-30 and SMIP-43 at the indicated concentrations. Results are reported as relative activity of PPM1A (Fig. 6) and PPM1B (Fig. 10) compared to an untreated control and shown as the mean ⁇ SD of 3 replicates.
  • Figs. 7 and 8 are graphs illustrating dose-dependent activity of other active SMIP compounds. Titration of other active SMIP compounds was performed. Data is shown for SMIP-2, 11, 13, 15 and 16 in Fig. 7, and SMIP-84, 107, 112 in Fig. 8. Sanguinarine was used as the positive control in Fig. 7. SMIP-30 was used as control in Fig. 8. Data is shown as the mean ( ⁇ SD) of 3 replicates of the relative activity of PPM1A compared to an untreated control.
  • SM IP-30 is non cytotoxic in myeloid cells. Having confirmed that selected
  • SMIP compounds potently inhibit the enzymatic activity of PPM1A
  • the authors of the present disclosure next examined whether the newly synthesized compounds exhibited any cytotoxic effects in macrophages.
  • TH P-1 macrophages were treated with SMI P-30, -43, -84, -107, and -112 and cell viability was evaluated after 24 h using the resazurin cell viability assay (Fig.
  • the xCELLigence system allows for label-free and dynamic monitoring of cellular phenotypic changes in real time, using impedance as readout (50). Changes in impedance between electrodes at the bottom of E-well plates are then translated into a Cell Index (Cl) measurement, providing quantitative information about the biological status of the cells (50).
  • Cl Cell Index
  • An increase in the Cl generally reflects an increase in macrophage adherence (differentiation), whereas its decrease reflects the loss of macrophage viability as they detach from the bottom of the well (27, 50).
  • the results obtained using the RTCA technology confirmed the lack of SMI P-30 cytotoxicity in macrophages even at the highest tested concentration of 30 pM for prolonged time of treatment.
  • SMI P-30 the most active compound against PPM1A (Fig. 4) was used to treat THP-1 monocytes, primary human monocyte-derived macrophages (hMDM), RAW 264.7 macrophages and mouse bone marrow-derived macrophages (mBMDM) (Fig. 14).
  • hMDM primary human monocyte-derived macrophages
  • mBMDM mouse bone marrow-derived macrophages
  • the viability of human monocytes/macrophages was not affected by SMI P-30 treatment even at the highest tested dose (30 pM).
  • mouse cell cultures were more sensitive to the treatment, with reduced viability at concentrations higher than 10 pM.
  • doses of SMI P-30 up to 15 pM are well tolerated even in a multi-dose treatment scheme over a period of 6 days (Fig. 15).
  • FIG. 11 THP-1 macrophages were treated with SMIP compounds and sanguinarine at the indicated concentrations for 24 h. Cell viability was evaluated with the resazurin assay. The graph shows the relative cell viability compared to an untreated control, reported as mean ⁇ SD of 3 replicates.
  • THP-1 macrophages were treated with SMIP compounds at 15 pM for 6 days, changing medium every second day. Cell viability was evaluated with the resazurin assay. The graph shows the relative cell viability compared to an untreated control, reported as mean ⁇ SD of 3 replicates.
  • RTCA Real-Time Cell Analysis
  • SMIP-30 reduces M. tuberculosis burden in human macrophages.
  • the authors of the present disclosure infected THP-1 macrophages with Mtb and treated with subtoxic concentrations of SMIP-30 (15 pM) and sanguinarine (2 pM).
  • the sanguinarine treatment did not lead to a reduction in bacterial survival
  • treatment with SMIP-30 reduced the bacterial burden by ⁇ 75% compared to untreated cells (Fig. 16), and also displayed a dosedependent effect (Fig. 17).
  • SMIP-43 despite having similar potency as SMIP-30 in inhibiting PPM1A phosphatase activity (Fig. 4), did not enhance bacterial clearance in infected macrophages (Fig. 19), possibly due to the less favorable drug-likeness properties of SMIP- 43 compared to SMIP-30 (Figs. 20 and 21). Without wishing to be bound by theory, the authors of the present disclosure believe that SMIP-30 is effective at enhancing host-cell mediated bacterial killing in Mtb-infected macrophages at concentrations that do not affect the host cell viability.
  • THP-1 macrophages were treated with sub-toxic concentrations of sanguinarine and SMIP-30 and infected with Mtb mc 2 6206 expressing luciferase (MOI 5). Inhibitors were added 24 h before infection and maintained for the indicated time postinfection. Mtb survival was evaluated by luciferase assay (mean ⁇ SD of 3 replicates).
  • THP-1 macrophages were infected with Mtb mc 2 6206 expressing luciferase (MOI 5) and treated with SMIP-30 at the indicated concentrations.
  • the dosedependent bacterial killing was evaluated after 6 days with luciferase assays (mean ⁇ SD of 3 replicates). *p ⁇ 0.05, ****p ⁇ 0.0001.
  • Fig. 19 the graph illustrates Mtb survival upon treatment of infected THP- 1 macrophages with sub-toxic concentrations of sanguinarine and SMIP-43.
  • Cells were differentiated for 72 hours with PMA (100 ng/mL), infected with Mtb-luciferase (MOI 5) and survival was assessed with luciferase assays, as indicated. Data shown as the mean ( ⁇ SD) of 3 replicates showing the luminescence of treated and untreated cells. RLU: relative light units.
  • Figs. 20 and 21 illustrate in silica prediction of physicochemical properties and drug-likeness of SMIP-30 and SMIP-43, respectively, via Swiss-ADME (Swiss Institute of Bioinformatics).
  • THP-1 macrophages including WT, APPM1A, and PPM1A+ (overexpression of PPM1A (26)) were infected with Mtb in the presence of the autophagy flux inhibitor bafilomycin and treated with increasing doses of SMIP-30.
  • the authors of the present disclosure infected THP-1 WT, APPM1 A and PPM1A+ macrophages with Mtb-GFP in the presence or absence of SMIP-30 and analysed the cells 5 days post-infection, in line with the intracellular Mtb survival analysis in Fig. 16.
  • WT cells were treated with SMIP-30, the authors of the present disclosure observed a significant increase in Mtb positive for LC3+ puncta structures (Fig. 23).
  • the treatment did not show a significant effect on the number of LC3+Mtb in the genetically modified PPM1A cell lines when compared with their respective untreated controls (Fig. 24).
  • APPM1A macrophages do not show any modulation in S403-p62 after SMIP-30 treatment, it is important to observe that the S403-p62/p62 ratio is consistently much higher than in WT cells (Fig. 25). On average, APPM1A macrophages have 3.6-fold more active S403-p62 relative to untreated WT cells. Similar relative levels of S403-p62 are achieved in WT cells, only after treatment with high doses of SMIP-30 (at 20 pM of SMIP-30, S403-p62 levels are 3.7 times higher compared to the respective untreated control), indicating that the absence of PPM1A by itself associates with higher relative amount of phosphorylated p62.
  • TBK1 is one of the key kinases responsible for the phosphorylation of p62 on S403. This activity is dependent on phosphorylation of S172 residue of TBK1 (8, 24). Recently TBK1 has been described as substrate for PPM1A (31, 32). The authors of the present disclosure then evaluated if the PPM1A inhibitor SMIP-30 had any downstream effect on the phosphorylation of TBK1 , with the expectation that one would observe increased phosphorylation on S172 of TBK1. Surprisingly, the authors of the present disclosure did not observe any significant change in the levels of S172-TBK1 , which instead slightly decreased after SMIP-30 treatment (Fig. 25). This suggests that SMIP-30’s effect on S403-p62 is unlikely due to a modulation in the activity of TBK1 and may instead be a direct link to PPM1A.
  • THP-1 WT left
  • APPM1A mouse
  • PPM1A+ right macrophages were infected for 24 h with Mtb (MOI 5) in presence of bafilomycin (10 nM), and treated with SMIP-30, as indicated.
  • the representative western blot analysis shows the expression of LC3B-II.
  • the normalized quantification of LC3B-II is reported below the respective panel. GAPDH was used as loading control.
  • Fig. 24 is a graph illustrating the quantification of LC3 + Mtb in THP-1 APPM1A and PPM1A+ cells treated and analysed as in Fig. 23. ns: not significant.
  • Fig. 25 shows a representative western blot analysis of THP-1 WT and APPM1A macrophages infected for 24 h with Mtb-WT (MOI 5) in presence of bafilomycin (10 nM) and treated with SMIP-30, as indicated. Quantification for the representative western blot is reported below respective panels and shows the normalized levels of phosphorylated S403-p62 (over the total p62) and phosphorylated S172-TBK1 (over the total TBK1).
  • Fig. 26 shows a representative western blot of THP-1 WT and PPM1A+ macrophages treated as in A and D.
  • the expression level of phosphorylated S403-p62 over the total p62 is reported below panel, shown as fold over the respective untreated control.
  • PPM1A negatively regulates xenophagy during M. tuberculosis infection.
  • Fig. 27 is a representative western blot analysis of THP-1 WT, APPM1A and PPM1A+ macrophages infected with Mtb-WT (MOI 10) and treated with chloroquine (20 pM) as indicated, for 24 h.
  • Mtb-WT Mtb-WT
  • chloroquine (20 pM) as indicated, for 24 h.
  • LC3B-II over GAPDH
  • GAPDH was used as loading control.
  • Fig. 28 is graph illustrating an immunofluorescence analysis of THP-1 CTR, APPM1A and PPM1A+ macrophages infected with Mtb-GFP (MOI 10) for 2 hours in presence of 10% human serum. Bar: 10 pm. Arrows indicate LC3 + Mtb. On the right, the quantification of mycobacteria positive for LC3 puncta structure (LC3 + Mtb) is reported as percentage over the total number of intracellular Mtb per field. At least 10 random fields were used for the analyses. Results are shown as mean ⁇ SEM. ****p ⁇ 0.0001. Fig.
  • FIG. 29 are graphs that report the percentage of Mtb-infected cells that show p62 + Mtb (left), LAMP1 + Mtb (middle) and Mtb positive for both p62 and l_AMP1 (right). Data are expressed as mean ⁇ SEM, 10 fields for cell line. *p ⁇ 0.05 measured with Mann-Whitney test. Fig. 29 demonstrates that APPM1A show a higher number of mycobacteria positive for p62.
  • FIG. 30 is a representative western blot analysis of THP-1 WT, APPM1A and PPM1A+ macrophages infected with Mtb (MOI 10) and treated with bafilomycin (100 nM) as indicated, for 24 h.
  • the normalized quantification of phosphorylated S403-p62 (over the total p62) is shown below the panel and reported as fold increase over bafilomycin-only treated relative control sample. GAPDH was used as loading control.
  • Rapamycin an mTOR inhibitor widely used to induce autophagy and shown to increase Mtb clearance (53), does not further increase the phosphorylation on S403-p62, suggesting that the main contributor to this post-translational modification is the infection per se.
  • Figs. 27, 28, 30 and 31 demonstrate that PPM1A negatively regulates xenophagy during Mtb infection.
  • Fig. 31 is a representative western blot analysis of phosphorylated S403-p62 in tet-inducible PPM1A-overexpressing THP-1 macrophages.
  • Cells were induced with doxycycline (250 nM) for 48 h, differentiated with PMA (100 ng/ml) for 24 h and then infected with Mt-WT (MOI 10). Where indicated, rapamycin (125 nM) was added. Below the panels, the normalized expression of S403-p62 and PPM1A is shown, as indicated. A.U., Arbitrary Units. Fig.
  • Fig. 32 is a western blot that shows the expression of phospho S403-p62 and LC3B-II in THP-1 control macrophages Mtb-infected and treated as indicated in the table, for 24 hours.
  • Fig. 33 is a western blot that shows the expression of different autophagy markers in TH P-1 control macrophages Mtb-infected and treated as indicated in the table for 24 hours.
  • Fig. 34 is a protein expression analysis that shows the MOI-dependent phosphorylation levels of phospho S403-p62 in THP-1 control, APPM1A and PPM1A+ macrophages.
  • Fig. 34 is a protein expression analysis that shows the MOI-dependent phosphorylation levels of phospho S403-p62 in THP-1 control, APPM1A and PPM1A+ macrophages.
  • FIG. 35 is a western blot that shows the expression of phospho S403-62 at late time point (5 days) after infection with Mtb-WT at MOI 5 in control THP-1 macrophages.
  • GAPDH, vinculin, and tubulin were used as loading controls.
  • Figs. 36 and 37 are western blot analyses that show the expression of several markers of autophagy (Figs. 36 and 37) and RIG-l-like receptor (RLR) signaling pathway (Fig. 37) in different cell populations of control THP-1, APPM1A and PPM1A+ macrophages, treated as indicated for 24 h. Tubulin and GAPDH were used as loading controls. The genetic manipulation of PPM1A does not influence the expression of several autophagy and innate immunity markers.
  • Fig. 38 is a protein analysis performed using three different cell populations for APPM1A (expressing three different sgRNAs) and three different cell populations for PPM1A overexpressing cells (PPM1A+), infected 4 h with Mtb-WT and compared to their respective not-infected control.
  • THP-1 APPM1A show a higher expression of p62, phosphorylated S403-p62 and LC3 after infection with Mtb
  • THP-1 PPM1A+ show a reduction of these markers at the increase of PPM1 A expression.
  • GAPDH was used as loading control.
  • the differences in LC3 and p62 expression in Mtb-infected APPM1A and PPM1A+ macrophages are similarly maintained across different cell populations.
  • Fig. 39 is a western blot that shows the protein expression of phosphorylated S403-p62 and S172-TBK1 in cytoplasmic and nuclear fractions of THP-1 control, APPM1A and PPM1A+ macrophages.
  • the cells were infected and treated as indicated in the table above the panels, for 24 hours.
  • Tubulin and fibrillarin were used to evaluate the purity and loading of the cytoplasmic and nuclear fractions, respectively.
  • Fig. 40 is a representative phosphatase assay performed on protein lysates of THP-1 APPM1A macrophages, infected with Mtb-WT in presence of bafilomycin, and evaluated by western blot, probing for the endogenous phosphorylated S403-p62 and T172- TBK1.
  • the in vitro reaction was performed with increasing concentrations of recombinant PPM1A, as indicated, with or without MnCh (4 mM).
  • Fig. 41 is a graph that shows the relative levels of phosphorylated S403-p62 (over the total p62) in phosphatase assays performed as described in A. A.U., Arbitrary Units.
  • the present data show that PPM1 A expression and activity strongly influence p62 phosphorylation, supporting a key function for PPM1A-p62 signaling during Mtb infection.
  • the authors of the present disclosure designed a phospho-synthetic peptide containing the amino acid sequence surrounding S403 and performed an in vitro phosphatase assay by titrating increasing concentrations of recombinant PPM1A (Fig. 42). The reaction was performed in the presence of MnCh, MgCh or without any metal cofactors.
  • Fig. 42 is a graph that reports the phosphatase assay performed using a synthetic phospho-peptide corresponding to the residues surrounding S403 of human p62/SQSTM1 protein. The results were evaluated by malachite green assay, measuring the amount of free phosphate released during the phosphatase reaction. Recombinant PPM1A protein was added in increasing concentrations, in presence of MnCh (5 mM), MgCh (5 mM) or without metals as negative control. Sanguinarine or SMIP-30 were added at 30 pM in control samples, as indicated.
  • Fig. 43 shows the results of a PPM1A phosphatase assay performed using p62/SQSTM1 phospho-peptide and measured using the malachite green method. SMI P-30 and sanguinarine were titrated at the indicated concentrations with recombinant PPM1A. Results are shown as the relative activity compared to an untreated control, as mean ⁇ SD. ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • PPM1A binds p62 in M. tuberculosis-infected macrophages.
  • the authors of the present disclosure performed immunoprecipitation assays using macrophages expressing GFP-tagged PPM1A.
  • the authors of the present disclosure decided to clone PPM1A in both the N- or C-terminal of GFP and select cell populations expressing the two GFP-tagged forms of PPM1A (THP-1 GFP-PPM1A or PPM1A-GFP, where GFP is localized respectively at the N-terminus or C- terminus of PPM1A).
  • the N-terminus domain of PPM1A is crucial for its functions, substrate binding and subcellular localization (34, 55) and, if manipulated by adding GFP, it might result in altered structure and/or functions.
  • the authors of the present disclosure performed immunoprecipitation with lysates from all the cell lines.
  • PPM1A-GFP lysates show the highest ability of pulling-down p62, supporting a direct interaction between PPM1A and p62 (Fig. 44). The GFP control appears to pull down a small amount of p62 as well.
  • GFP-control THP- 1 cell lines indeed show a very high expression of GFP (lysates in Fig. 44, right panel) compared to the fusion proteins, levels that could induce degradation of the GFP protein to maintain the cell viability.
  • the efficient pull down of p62 occurs only when PPM1A conformation is not affected by the GFP-tag protein at PPM1A N-terminus.
  • Fig. 44 shows the immuno-precipitation with anti-GFP in THP-1 macrophages expressing GFP empty vector, GFP-PPM1A and PPM1A-GFP.
  • THP-1 APPM1A cells were used as negative control.
  • the protein lysates were collected at 24 h after Mtb infection (MOI 10) and bafilomycin treatment (100 nM). On the left, the proteins immunoprecipitated with GFP antibody are shown, probed with p62 and GFP antibodies. On the right, the lysates are shown, as reference for molecular weight and expression levels, probed for p62 and GFP.
  • IgG HC IgG Heavy Chain of the antibody anti-GFP.
  • p62 is a novel PPM1 A substrate and that PPM1A can directly bind and dephosphorylate p62 in human macrophages. Moreover, by regulating the phosphorylation on S403 of p62, PPM1A controls the activation of autophagy during Mtb infection.
  • SMIP-30 synergizes with rifampicin to promote clearance of M. tuberculosis in infected mice. Following elucidation of the molecular mechanism by which SMIP-30 improves macrophage control of Mtb, the authors of the present disclosure sought to test the important question of whether SMIP-30 would be effective for treatment of TB in a mouse model. While SMIP-30 remained relatively non-cytotoxic in human immune cells, the authors of the present disclosure did note increased sensitivity to the compound in murine macrophages (Fig. 14). As such, it was important to first assess the in vivo toxicity of SMIP- 30 to establish an appropriate and well-tolerated dose for the feasibility of in vivo treatment schemes.
  • mice Groups of 4 mice were treated with increasing doses of SMIP-30 (10, 25, 50, 100 mg/kg), administering the compound intra-peritoneum (i.p.) every other day for a total of 20 days.
  • doses of 25 mg/kg and lower were well tolerated for the whole duration of the treatment, whereas a dose of 50 mg/kg was compatible only with a shorter period of treatment (Fig. 45).
  • the authors of the present disclosure repeated the treatment for a shorter time course (12 days), more in line with the experimental scheme designed for the following in vivo infection experiments. Again, the results pointed to 25 mg/kg as an ideal dose in terms of tolerability (Fig. 46).
  • SMIP- 30 does not produce a hyperinflammatory response, even at the highest doses and after the longest treatment time point (Figs. 47 and 48), suggesting that in healthy mice, the treatment itself does not alter the immunomodulatory signals to harmful levels.
  • the graphs report the deposited Mtb CFU in lungs (left) and spleen (right) at the indicated time of control mice infected with 3x10 7 CFU of the auxotroph Mtb strain mc 2 6206 via tail vein injection. Each dot represents a mouse. *p ⁇ 0.05, **** p ⁇ 0.0001, measured with unpaired T-test.
  • mice were randomized in 4 cohorts and treated with vehicle, SMIP-30 (25 mg/kg, i.p.), low-dose rifampicin (20 mg/L, drinking water) or a combination of the two drugs.
  • Rifampicin a key first-line anti-tuberculosis drug, is associated with high levels of antibiotic resistance if used alone. As such, multiple studies have sought to combine it with other compounds in efforts lower its effective dose to prevent toxicity and antibiotic resistance (57, 58).
  • the authors of the present disclosure administered a low-dose rifampicin regimen in the present experiments to simulate a scenario where the dosage is below the limit required to cause a significant reduction in the Mtb burden in the infected mice.
  • mice were treated every other day for a total of 14 days after infection. The body weight showed that there were no signs of morbidity in the Mtb infected and treated animals (Fig. 50).
  • Fig. 50 BALB/c mice were infected with 10 7 CFU of Mtb mc 2 6206 via i.v. injection and treated with SMIP-30 and/or rifampicin. Treatments were administered every second day, starting at day 1 post-infection (DPI), and body weight was recorded just before administering the treatments.
  • SMIP-30 was administered via i.p. injection, at 25 mg/kg.
  • Figs. 51 and 52 The necropsy revealed a general reduction in the spleen weight of treated mice compared to the control cohort, even though the CFU analysis in the spleen did not show any significant difference among the cohorts.
  • Fig. 51 the graph shows the normalized weight of spleen in mice infected with Mtb and treated as indicated for 14 days. Each dot represents a mouse.
  • Fig. 52 Mtb CFU obtained from homogenized spleens in mice infected with Mtb and treated as indicated for 14 days. Each dot represents a mouse. Results are shown as mean ⁇ SEM.
  • cytokine profile of mouse serum showed that cytokines as IFN-y and MCP-1 have a significant reduction over the 2 weeks of infection, in line with the impaired proliferation of Mtb mc 2 6206 in vivo (Fig. 54, panels C and D). Importantly, no significant differences among the treatments at 2 weeks post-infection were found (Fig. 54 panels C to H), consistent with the cytokine production in THP-1 macrophages (Fig. 2, panels C to E). Given that high levels of systemic inflammation are known to lead to tissue damage and worsen the outcome of pharmacologic treatment, the absence of a hyper- inflammatory response in vivo after SMIP-30 treatment reasonably support its safety and compliance for HDT.
  • SMIP-30 can potentiate the effect of rifampicin to improve Mtb clearance in vivo.
  • the modulation of inflammatory cytokines does not appear to be responsible for the effect of the combination treatment, suggesting that other factors are more crucial in the mechanism of action of SMIP-30, or at least cooperate with it.
  • the mechanistic data in human macrophages clearly support a key role for PPM1A in controlling Mtb infection through induction of xenophagy.
  • Fig. 54 panels C to D, illustrates expression levels of IFN-y and MCP-1 in mouse serum collected at 1 DPI (in control cohort) and at 14 DPI (in cohorts as described in Figs 50 and 53).
  • Fig. 54 panels E to H, illustrates expression levels of IL-6 (E), IL-10 (F), IL-27 (G), I L-1 p (H) in mouse serum collected at the end of the experiment described in Fig. 50. Each dot represents one mouse.
  • Figs. 50, 53 and 54 demonstrate that SMI P-30 synergizes with rifampicin to accelerate clearance of Mtb in infected mice.
  • SMIP-30 is effective in reducing Mtb survival in vitro, in infected macrophages, and in vivo, synergizing with rifampicin to reduce the Mtb burden. This is due, at least partially, to the activation of xenophagy, as demonstrated in vitro by pharmacologic inhibition of PPM1A and its genetic ablation. Mechanistically, the authors of the present disclosure demonstrated that the selective autophagy receptor p62/SQSTM1 is a novel substrate of PPM1A and it is dephosphorylated by PPM1A on the Ser 403 residue.
  • the selective autophagy receptor p62 represents a key molecular determinant in anti-tuberculosis signaling and many studies support its role as the cytoplasmic bridge for ubiquitinated pathogens and autolysosomes (12, 60, 61).
  • Ubiquitin-decorated mycobacteria are indeed recognized via the Ubiquitin Associated Domain (UBA) localized at the C-terminus of p62, whereas the presence of an upstream LC3-lnteracting Region (LIR motif) allows the delivery of packaged cargos into newly formed autophagosomes (12).
  • the Ser 403 residue is localized in the UBA domain of p62 and, when phosphorylated, increases the binding activity of p62 for ubiquitinated targets (52).
  • Mycobacteria become ubiquitinated on surface proteins when they escape from the phagosome into the cytoplasm or when they are exposed in damaged phagosomes (17, 62, 63). The timing of this event is still not fully elucidated, but might be more relevant at later stages of infection (few days post infection), when evaluated in vitro (62).
  • TANK-binding kinase TANK-binding kinase
  • this Mtb strain retains all virulence associated genes unlike BCG or Mtb H37Ra, it is highly suitable for in vitro macrophage experiments (41).
  • the bacteria do not replicate in mice, which limited the evaluation of bacterial burden to the rate of clearance and not actual bacterial replication (56).
  • the i.p. injection route may not represent the best administration route for SMIP-30 to reach a therapeutic dosage at the site of infection, and more pharmacokinetic and pharmacodynamic tests must be performed.
  • Our decision to use i.p. administration was mainly based on the available information in terms of solubility, toxicity, and in vitro activity of SMIP-30 at the time of the experiments. Another important consideration is the effect of the combination treatment with rifampicin.
  • Rifampicin is one of the most important first line drugs in TB chemotherapy, but despite its efficacy, the increase in rifampicin-resistant TB has become a major health threat.
  • Several studies show that combining rifampicin with in vivo drugs that activate autophagy results in increased rifampicin activity or localized concentrations, and reduces Mtb burden in lungs (74, 75).
  • the mechanisms are not completely understood and may be different depending on the different combined drugs. Since rifampicin is an antibiotic, it requires direct contact with the bacteria to achieve a killing effect.
  • phenotypic activity screening against actively replicating Mtb identified 5 hits within the compounds disclosed above that possessed significantly improved anti-Mtb activity compared to sanguinarine (“SG”), which surprisingly only showed modest activity against Mtb.
  • SMI P-6 and SMIP-9 were characterized in detail in a series of experiments. SMIP-6 and SMIP-9 were shown to possess low-micromolar inhibitory activity against multiple mycobacterial species, but were inactive against other Gram-negative and Gram-positive bacteria, showing unique specificity against mycobacteria. Both compounds exhibit reduced cytotoxicity, a known undesirable property of sanguinarine, and showed efficacy against Mtb within infected human macrophages.
  • SMIP-6 and SMIP-9 was effective in inhibiting NR-Mtb.
  • both compounds exhibited activity against multiple multidrug resistant (MDR) Mtb clinical isolates, which demonstrates a unique mechanism of action compared to existing frontline TB antibiotics.
  • MDR multidrug resistant
  • SMIP-6 and SMIP-9 significantly inhibit the growth of mycobacteria.
  • the REMA growth inhibition assay was used to determine the dose-dependent inhibition activity of SMIP-6, -9, -29, -32 against Mtb mc 2 6206 (Fig. 56 panel A). This assay showed that SMIP-6 and SMIP-9 possessed the most potent anti-Mtb activity, reaching an MIC90 of 10 pM and 6 pM, respectively (Table 3). The activity of SMIP-6 and SMIP-9 thus show an 8-fold and 14-fold improvement over SG, respectively, confirming that these compounds possess significantly increased anti-Mtb activity.
  • SMIP-6 and -9 retained potent inhibitory effect against Mycobacterium bovis BCG, Mycobacterium kansasii, and Mycobacterium smegmatis, while showing no effect against other Gram-positive and Gram-negative bacteria (Table 3).
  • the specificity of an antibiotic to a particular pathogenic genus or species may have major advantages for treatment potential since it is less likely to adversely affect the host microbiome.
  • the specificity of SMIP-6 and -9 suggest a possible mechanism or target of inhibition that exists specifically in mycobacteria.
  • MIC90 of SMIP-6, SMIP-9 and Sanguinarine against various bacteria species n.a, no activity up to 100 pM; n.d., not determined.
  • EC Escherichia coir, LM, Listeria monocytogenes; PA, Pseudomonas aeruginosa; SE, Salmonella enterica Typhimurium.
  • Mtb-lux auto-luminescent Mtb mc 2 6206 strain
  • This recombinant Mtb-lux strain expresses a bacterial luciferase operon that allows the bacteria to generate a constitutive luminescence signal.
  • Luminescence signal depends not only on the proteins of the lux operon, but also requires ATP and NADPH .
  • the relative luminescence unit acts as a surrogate reporter for the viability of metabolically active Mtb.
  • panel D Mtb mc 2 6206 were mock-treated or treated with SMIP-6, SMIP-9 and RIF for 20 h.
  • Bacteria were serially diluted and select dilutions were inoculating on 7H10 agar plates for enumeration of colony forming units (CFU). Data represent the mean ⁇ SEM of 3 independent replicates.
  • SMIP-6 and SMIP-9 are active against non-replicating Mtb. Despite the lack of bactericidal activity, the ability of SMIP-6 and SMIP-9 to inhibit the metabolic activity of Mtb is comparable to or better than RIF. Bacteriostatic drugs targeting Mtb are valuable if they can also inhibit NR-Mtb, which have increased tolerance to most antibiotics. To determine the efficacy of SMIP-6 and SMIP-9 against Mtb with low metabolic activity, the established low pH model was used to generate NR-Mtb. (Early, J. V.; Mullen, S.; Parish, T.
  • Mtb has the ability to maintain a dormant state within macrophages and reactivate upon perturbations to the immune system.
  • This in vitro model of NR-Mtb simulates this dormancy state, verified by the low level of luminescence ( Figure 57, panel A).
  • This luminescence controlled by the lux operon, exploits fatty aldehydes as substrates to produce fatty acids and light, meaning luminescent signal is dependent on an aerobic and metabolically active environment.
  • the authors of the present disclosure observed resuscitation of untreated NR-Mtb upon incubation in complete growth media, due to restoration of metabolic activity and fatty acid biosynthesis.
  • SMIP-6 and SMIP-9 exhibit reduced cytotoxicity and are effective against intracellular Mtb.
  • the therapeutic potential of SG is limited given its cytotoxic properties due to non-specific targeting of essential eukaryotic proteins such as the Na+/K+-ATPase.
  • the results illustrated in Figure 58, panel A show that human THP-1 macrophages treated with SG show significant cytotoxicity, with an IC50 of 10 pM.
  • the IC50 of SG is 4-fold lower than the MIC50 of SG against Mtb, rendering it useless for targeting intracellular Mtb, which is critical given that Mtb is an intracellular pathogen.
  • SMIP-6 and SMIP-9 showed a cytotoxicity IC50 of 40 pM and 15 pM in macrophages, respectively ( Figure 58, panel A).
  • SMIP-9 and SMIP-6 have a cytotoxic IC50 that is ⁇ 5 to 6-fold higher than their respective MIC50 against Mtb.
  • the in vitro therapeutic ratio defined as IC501 MIC50 for the respective compounds, illustrates the potential and improvement of SMIP-6 and -9 compared to SG (Table 4).
  • SMIP-9 showed increased cytotoxicity compared to SMIP-6, suggesting that a unique structural feature of SMIP-9 contributes to its increased cytotoxicity.
  • Combination treatment of SMIP-6 and SMIP-9 compounds with rifampicin improves anti-Mtb activity in axenic conditions and in infected macrophages.
  • the combinatorial effectiveness of different antibiotics and drugs is one of the most emphasized aspects in drug development.
  • the combination checkerboard assay was adopted.
  • Combination effects are desirable since it can lower the effective concentrations of each drug to minimize cytotoxicity.
  • SMIP-6 and SMIP-9 accumulate within the bacterial cell. To gain insight into how these SMIP compounds may exert their antibacterial functions, the dynamics of the compounds’ interaction with bacteria were investigated. SMIP-6 and SMIP-9 have fluorescence properties (peak excitation/emission wavelengths of 420/485 nm) that can be exploited for visualization and quantification. To examine the uptake and localization of the compounds inside the bacteria, Mtb-RFP42 treated with sub-MIC concentrations of SMIP-6 and SMIP-9 were visualized by epifluorescence microscopy.
  • Fluorescence of SMIP-6 and SMIP-9 co-localized with RFP signal and was evenly distributed throughout the bacteria, indicating a possible accumulation of the compounds in the cytosol of Mtb cells. However, a portion of the Mtb did not appear to accumulate the compounds, suggesting incomplete uptake at this specific concentration.
  • SMIP-6 and SMIP-9 are effective against virulent and clinical MDR Mtb strains. Given the increasing prevalence of MDR-TB, anti-Mtb compounds that retain their efficacy against clinical and MDR strains of Mtb are highly desirable. Both SMIP-6 and SMIP- 9 retain their efficacy in the low micromolar MIC range against both virulent laboratory strains (Mtb H37Rv and Mtb Erdman) and the hypervirulent Mtb HN878 strain (Table 5).
  • SMIP6 and SMIP-9 are effective against multiple strains of virulent and MDR Mtb and that their mechanism of action is distinct from that of the frontline TB drugs, including rifampicin, isoniazid, pyrazinamide, streptomycin, and ethambutol.
  • SMIP-6 and SMIP-9 are effective against non-tuberculosis mycobacteria (NTM) strains relevant to human health.
  • NTM non-tuberculosis mycobacteria
  • Mabs Mycobacterium abscessus
  • Mav Mycobacterium avium
  • Both SMIP-6 and SMIP-9 were shown to be active against Mabs and Mav with an MICso in the range of 8-20 pM (Table 6).
  • Amikacin (AMK) was also tested as a positive control.
  • the cytosolic sensor cGAS detects Mycobacterium tuberculosis DNA to induce type I interferons and activate autophagy. Cell Host Microbe. 17, 811-819 (2015).

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Abstract

La présente divulgation concerne des composés selon les formules I et II : dans lesquelles R1 à R11 représentent indépendamment : H ; D ; un halogène ; un groupe alkyle linéaire ou ramifié éventuellement substitué ; un groupe alcényle éventuellement substitué ; un groupe alcynyle éventuellement substitué ; un groupe aryle éventuellement substitué ; un groupe hétéroaryle éventuellement substitué ; ou un groupe carbocyclique éventuellement substitué. X' est un anion acceptable sur le plan pharmaceutique ou synthétique. Ces composés sont des inhibiteurs PP2C Ser/Thr phosphatase dépendant du métal et peuvent être utilisés pour traiter des maladies associées, telles que des maladies infectieuses.
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