WO2015073804A2 - Method of blocking transmission of malarial parasite - Google Patents

Method of blocking transmission of malarial parasite Download PDF

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WO2015073804A2
WO2015073804A2 PCT/US2014/065671 US2014065671W WO2015073804A2 WO 2015073804 A2 WO2015073804 A2 WO 2015073804A2 US 2014065671 W US2014065671 W US 2014065671W WO 2015073804 A2 WO2015073804 A2 WO 2015073804A2
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alkyl
aryl
nitrogen
group
sulfur
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PCT/US2014/065671
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French (fr)
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WO2015073804A3 (en
Inventor
John C. Mckew
Wei Zheng
Kim C. WILLIAMSON
Wenwei Huang
Wei Sun
Takeshi Tanaka
Seameen Jean DEHDASHTI
Noel Terrence Southall
Crystal Tobin MAGLE
Xiuli HUANG
Paresma Rasiklal PATEL
Myunghoon Kim
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Loyola University Of Chicago
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Priority to US15/036,355 priority Critical patent/US20160264570A1/en
Publication of WO2015073804A2 publication Critical patent/WO2015073804A2/en
Publication of WO2015073804A3 publication Critical patent/WO2015073804A3/en

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    • 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
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • 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
    • A61K31/435—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
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • gametocytes are taken up in a blood meal by a mosquito, fertilization is stimulated and the resulting zygote differentiates into a motile ookinete that migrates across the midgut epithelium of the mosquito and forms an oocyst. Over the course of the next 2 weeks, tens of thousands of infectious sporozoites are generated and sequestered in the mosquito salivary glands until released into a vertebrate host for transmission during the next blood meal.
  • the invention provides a method of blocking transmission of a Plasmodium parasite comprising administering to a mammal in need of such treatment, a therapeutically effective amount of a first compound of formula I):
  • A is CR 12 or N
  • R 1 is an optionally substituted group selected from the group consisting of C 6 -io aryl
  • Ci-12 alkyl Ci -12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-10-membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C 6 alkyl, halo, CN, Ci-C 6 alkoxy, SO2NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
  • R 2 , R 10 , and R 1 1 are independently hydrogen, halogen, -NR 6 R 7 , -OR 8 , -SR 9 , or an optionally substituted group selected from the group consisting of Ci-i 2 acyl; C 6 .
  • R 3 and R 4 are independently selected from hydrogen, hydroxyl, OR 5 , halogen, optionally substituted C 6 -io aryl, and optionally substituted Ci -6 alkyl, R is Cj.i 2 alkyl, and
  • R 6 , R 7 , R 8 , and R 9 are independently hydrogen, an optionally substituted group selected from the group consisting of d.i 2 acyl; C -io aryl; C 6- io aryl Ci-i 2 alkyl; C4.7
  • Ci_i 2 alkyl Ci_i 2 alkyl; Ci_i 2 alkyl; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-7- membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and CM 2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
  • R and R are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1 -2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
  • R 12 is hydrogen, Ci -]2 alkyl, C6-io aryl, halogen, hydroxyl, or OR 5 ,
  • R is hydrogen, CM 2 alkyl or C 6- io aryl
  • the invention also provides a method of treating malaria by killing or arresting the growth of Plasmodium organisms in a mammal, wherein the Plasmodium organisms are in a gametocyte stage, the method comprising administering to a mammal a therapeutically effective amount of a first compound of formula I):
  • A is CR or N
  • R 1 is an optionally substituted group selected from the group consisting of C 6- 2 alkyl; Ci.i 2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-12-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoro methyl, Cj-C 6 alkyl, halo, CN, Ci-C 6 alkoxy, S0 2 NH 2 , piperizinyl, and 4-alkylcarbonylpiperazinyl,
  • R 2 , R 10 , and R 11 are independently hydrogen, halogen, -NR 6 R 7 , -OR 8 , -SR 9 , or an optionally substituted group selected from the group consisting of Ci_i 2 acyl; C 6 -io aryl; C 7-15 arylalkyl; C 6- i 5 heteroarylalkyl; C M 2 alkyl; C 1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C 6 alkylamino, di(Ci-C 6 alkyl)amino, CrC 6 alkyl
  • R 3 and R 4 are independently selected from hydrogen, hydroxyl, OR 5 , halogen, optionally substituted C 6- io aryl, and optionally substituted C 1-6 alkyl,
  • R 5 is C 1-12 alkyl
  • R 6 , R 7 , R 8 , and R 9 are independently hydrogen, an optionally substituted group selected from the group consisting of Cj.i2 acyl; C 6 -io aryl; C 6- io aryl C .n alkyl; C 4- 7 heteroaryl C . 2 alkyl; Ci -12 alkyl; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-7- membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and C 1.12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
  • R 6 and R 7 are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
  • R is hydrogen, C ⁇ . ⁇ 2 alkyl, C 6-10 aryl, halogen, hydroxyl, or OR ,
  • R is hydrogen, Ci- 12 alkyl or C6-10 aryl
  • Figure 1 depicts the structures of compounds in accordance with an embodiment of the invention.
  • Figure 2 A illustrates a protocol for a mouse model of gametocyte transmission in accordance with an embodiment of the invention.
  • Figure 2B illustrates the result of a malaria mouse model for Torin 2 using a 2 dose dosing regime.
  • Figure 2C illustrates the result of a malaria mouse model for Torin 2 using a 1 dose dosing regime.
  • Figures 3A-3D illustrate dose-concentration curves of panobinostat, CUDC-101 , primaquine, and Torin 2, respectively, against the drug sensitive 3D7 strain and against two asexual drug resistant strains HB3 and Dd2.
  • Figure 4 illustrates the results of a gametocyte viability assay for Torin 2 and Torin 1.
  • Figure 5 depicts the structures of Torin 2, Torin 1 , and WWH030.
  • Figure 6 A illustrates a protocol for a mouse model of gametocyte transmission in accordance with an embodiment of the invention.
  • NVP-AUY922 NVP-AUY922, and alvespimycin-treated mice are depicted in Fig. 6B.
  • the invention provides a method of blocking transmission of a Plasmodium parasite comprising administering to a mammal in need of such treatment, a therapeutically effective amount of a first compound of formula (I):
  • A is CR 12 or N
  • R 1 is an optionally substituted group selected from the group consisting of C 6- io aryl; Ci-i 2 alkyl; C M 2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-12-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoro methyl, Ci-C 6 alkyl, halo, CN, Ci-C 6 alkoxy, SO2NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
  • R 2 , R 10 , and R 1 1 are independently hydrogen, halogen, -NR 6 R 7 , -OR 8 , -SR 9 , or an optionally substituted group selected from the group consisting of Ci-i 2 acyl; 0 6 - ⁇ aryl; C7..15 arylalkyl; C 6 -i5 heteroarylalkyl; Ci -12 alkyl; Ci-i 2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Cj-C 6 alkylamino, di(Ci-C 6 alkyl)amino
  • R 3 and R 4 are independently selected from hydrogen, hydroxyl, OR 5 , halogen, optionally substituted C 6 -io aryl, and optionally substituted Ci -6 alkyl,
  • R 5 is Ci-12 alkyl
  • R 6 , R 7 , R 8 , and R 9 are independently hydrogen, an optionally substituted group selected from the group consisting of acyl; C 6 . 10 aryl; C 6-1 o aryl Ci-n alkyl; C 4- 7 heteroaryl C 1.12 alkyl; Ci-12 alkyl; 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-7- membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and C 1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
  • R 6 and R 7 are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
  • R is hydrogen, C 1-12 alkyl, C 6- io aryl, halogen, hydroxyl, or OR ,
  • R is hydrogen, CM2 alkyl or C 6- io aryl
  • A is CH or N
  • R 1 is Ci-C 6 alkyl, C 6 -Cio aryl, or heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C 6 alkyl, halo, CN, Ci-C 6 alkoxy, S0 2 NH 2 , piperizinyl, and 4-alkylcarbonylpiperazinyl,
  • R 2 is C 6 -Cio aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C 6 alkylamino, di(Ci- C 6 alkyl)amino, C
  • R 10 and R 11 are both hydrogen
  • R 3 and R 4 are individually selected from hydrogen, halo, optionally substituted d-C 6 alkyl, and OR 5 .
  • A is CH
  • R 2 is not 2-amino-5-pyridyl or
  • alkyl means a straight-chain or branched alkyl substituent containing from, for example, 1 to about 6 carbon atoms, preferably from 1 to about 4 carbon atoms, more preferably from 1 to 2 carbon atoms.
  • substituents include methyl, ethyl, propyl, isopropyl, /7-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, and the like.
  • aryl refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, and the term “C 6 -Cio aryl” includes phenyl and naphthyl. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 ⁇ electrons, according to Hiickel's Rule.
  • heteroaryl refers to a monocyclic or bicyclic 5 to 10-membered ring system as described herein, wherein the heteroaryl group is unsaturated and satisfies Hiickel's rule, and wherein the heteroaryl contains 1 -4 heteroatoms independently selected from nitrogen, oxygen and sulfur.
  • Non-limiting examples of suitable heteroaryl groups include furanyl, thiopheneyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, l,3,4-oxadiazol-2-yl, 1 ,2,4-oxadiazol-2-yl, 5- methyl-l,3,4-oxadiazole, 3 -methyl- 1 ,2,4-oxadiazole, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, benzothiopheneyl, indolyl, indazolyl, imidazolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolinyl, benzothiazolinyl, and quinazolin
  • heteroaryl groups can be attached at any open position on the heteroaryl groups.
  • heterocyclic or “heterocyclyl” refer to a 4 to 12-membered heterocyclic ring system as described herein, wherein the heterocycle contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the heterocycle is saturated or monounsaturated.
  • the heterocyclyl or heteroaryl group is optionally substituted with 1 , 2, 3, 4, or 5 substituents as recited herein such as with alkyl groups such as methyl groups, ethyl groups, and the like, or with aryl groups such as phenyl groups, naphthyl groups and the like, wherein the aryl groups can be further substituted with, for example halo, dihaloalkyl, trihaloalkyl, nitro, hydroxy, alkoxy, aryloxy, amino, substituted amino, alkylcarbonyl, alkoxycarbonyl, arylcarbonyl, aryloxycarbonyl, thio, alkylthio, arylthio, and the like, wherein the optional substituent can be present at any open position on the heterocyclyl or heteroaryl group.
  • alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms refers to a linear or branched alkyl group wherein one or more carbon atoms in the alkyl group is replace with the aforesaid atoms.
  • Non-limiting examples of alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms include, for example, methoxymethyl, methoxy ethyl, methylaminoethyl, and the like.
  • acyl refers to an alkylcarbonyl substituent.
  • alkylsulfonylamino refers to a group of the structure: alkyl-S0 2 -NH-.
  • aminosulfonyl refers to a group of the structure: H 2 NS0 2 -.
  • R 1 is Ci-C 6 alkyl, C 6 -Cio aryl, or heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C 6 alkyl, halo, CN, Ci-C 6 alkoxy, S0 2 NH 2 , piperizinyl, and
  • R 2 is C 6 -Cio aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C 6 alkylamino, di(Ci-C 6 alkyl)amino, and Ci-C 6 alkylcarbonylamino.
  • R 10 and R ! ! are both hydrogen.
  • R 3 and R 4 are both hydrogen.
  • A is CH.
  • R 1 is selected from 3-trifluoromethylphenyl, 4-piperazinylmethyl, ethyl, phenyl, 3-ethylphenyl, 3-chlorophenyl, 3-cyanophenyl, 3-methoxyphenyl, 3-(dimethylaminocarbonyl)phenyl, 3- sulfonamidophenyl, 3-phenoxyphenyl, 3-ethoxyphenyl, 4-(piperazin-4-yl)-3- trifluoromethylphenyl, 4-piperazinyl, l-acetylpiperidin-4-yl,cyclopropyl, 4- tetrahydropyranyl, cyclohexyl, and cyclopentyl.
  • R 2 is selected from 2-amino-pyridinyl, 4-pyridinyl, 2-amino-5-pyrimidinyl, 3-pyridyl, quinolin-3- yl, 5-pyrimidinyl, 2-amino-5-trifluoromethylpyrimidin-5-yl, 2-acetylamino-5-pyridyl, 2- amino-4-methylpyrimidin-5-yl, 1 -piperazinyl, indol-5-yl, lH-indazol-5-yl, 4-aminophenyl, l ,2,3,6-tetrahydropyridin-4-yl, 1 H-pyrazol-4-yl, lH-benzo[d]imidazol-5-yl, 4- sulfonylaminophenyl, 2-dimethylaminopyrimidin-5-yl, 3-trifluoromethylphenyl, bromo, 3- aminophenyl, vinyl, 4-amino
  • the compound has the formula:
  • the compound has the formula:
  • R 1 and R 2 are:
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoe)-2-aminoethyl
  • the compound is a compound of formula (I) and B is NR 12 .
  • A is CH.
  • R 10 and R 11 are both hydrogen.
  • R 13 is hydrogen or Ci-i 2 alkyl.
  • R 1 is 3-trifluoromethylphenyl.
  • R z is selected from the group consisting of 2-methyl-5-pyridyl, 4-aminophenyl, 2-acetylamino- 5-pyridyl, 4-hydroxyphenyl, 3-aminophenyl, 4-pyridyl, lH-benzo[d]imidazol-5-yl, 4- methlsulfonylphenyl, quinolin-3-yl, 2-aminopyrimidin-5-yl, 3-cyanophenyl, 3-pyridyl, and 4- aminocarbonylphenyl.
  • the second compound that is administered is selected from elesclomol, NSC 174938, NVP-AUY922, Maduramicin, Narasin, Alvespimycin, Omacetaxine, Thiram, Zinc pyrithione, Phanquinone, Bortezomib, Salinomycin sodium, Monensin sodium, Dipyrithione, Dicyclopentamethylene-thiuram disulfide, YM155,
  • the compound is NSC 174938, NVP-AUY922, Maduramicin, and Narasin.
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • composition comprising the compound and a pharmaceutically acceptable carrier.
  • compositions described herein for example, vehicles, adjuvants, excipients, or diluents, are well known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.
  • compositions of the present invention are merely exemplary and are in no way limiting.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,
  • microcrystalline cellulose acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
  • the compounds of the present invention can be made into aerosol formulations to be administered via inhalation.
  • aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l ,3-dioxolane- 4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexade
  • surfactant such as a soap or a detergent
  • suspending agent such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or
  • carboxymethylcellulose or emulsifying agents and other pharmaceutical adjuvants.
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (3) mixtures thereof.
  • the parenteral formulations will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • HLB hydrophile-lipophile balance
  • parenteral formulations can be presented in unit-dose or multi- dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.
  • sterile liquid carrier for example, water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the compounds of the present invention may be made into injectable
  • the compounds of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Suitable carriers and their formulations are further described in A.R. Gennaro, ed., Remington: The Science and Practice of Pharmacy (19th ed.), Mack Publishing Company, Easton, PA (1995).
  • the compound of the invention or a composition thereof can potentially be administered as a pharmaceutically acceptable acid-addition, base neutralized or addition salt, formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base, such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases, such as mono-, di-, trialkyl, and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids
  • the conversion to a salt is accomplished by treatment of the base compound with at least a stoichiometric amount of an appropriate acid.
  • the free base is dissolved in an inert organic solvent such as diethyl ether, ethyl acetate, chloroform, ethanol, methanol, and the like, and the acid is added in a similar solvent.
  • the mixture is maintained at a suitable temperature (e.g., between 0 °C and 50 °C).
  • the resulting salt precipitates spontaneously or can be brought out of solution with a less polar solvent.
  • the neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
  • solvates refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice.
  • the solvent incorporated in the solvate is water, the molecular complex is called a hydrate.
  • Pharmaceutically acceptable solvates include hydrates, alcoholates such as methanolates and ethanolates, acetonitrilates and the like. These compounds can also exist in polymorphic forms.
  • the Plasmodium parasite can be any suitable Plasmodium parasite.
  • suitable Plasmodium parasites include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi.
  • the Plasmodium parasite is Plasmodium falciparum.
  • the Plasmodium parasite is a Plasmodium gametocyte.
  • the Plasmodium gametocyte is a mature stage II-V gametocyte.
  • the Plasmodium gametocyte is a stage III-V gametocyte, e.g., a mature stage III-V gametocyte.
  • the Plasmodium gametocyte is a mature stage V gametocyte.
  • the compound effectively kills Plasmodium gametocytes.
  • the Plasmodium parasite is a drug-resistant strain.
  • drug-resistant strains of Plasmodium are described in Kun, J.F.J, et al., Antimicrob Agents
  • the Plasmodium parasite is in an asexual stage.
  • the Plasmodium parasite is in an asexual stage.
  • Plasmodium parasite can be a sporozoite, a liver stage parasite, a merozoite, an asexual erythrocyte-stage parasite, a zygote, an ookinete, or an oocyst.
  • the amount or dose of a compound of the invention or a salt thereof, or a composition thereof should be sufficient to affect a therapeutic or prophylactic response in the mammal.
  • the appropriate dose will depend upon several factors. For instance, the dose also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular compound or salt.
  • the attending physician will decide the dosage of the compound of the present invention with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound or salt to be administered, route of administration, and the severity of the condition being treated.
  • the dose of the compound(s) described herein can be about 0.1 mg to about 1 g daily, for example, about 5 mg to about 500 mg daily.
  • Further examples of doses include but are not limited to: 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.5 mg, 0.6 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 17 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 140 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg
  • the method further comprises administering to the mammal at least one additional antimalarial compound.
  • Any suitable antimalarial compound can be used, many of which are well known in the art.
  • suitable antimalarial compounds include primaquine, bulaquine, artemisinin and derivatives thereof, chloroquine, mefloquine, amodiaquine, piperaquine, pyronaridine, atovaquone, tafenoquine, methylene blue, trioxaquines, endoperoxides such as OZ 439 and OZ 277, decoquinate, 9-anilinoacridines, HIV-protease inhibitors, and natural products such as neem, epoxomicin, harmonine, and riboflavin.
  • the Malaria Box contained 400 drugs or tool compounds with the confirmed activities on blood-staged P. falciparum and assessed cytotoxicity against mammalian cells [39, 56].
  • the MIPE library was an internal collection of 550 kinase inhibitors, which contain approved drugs and drug candidates in preclinical and clinical stages [14]. Compounds from all libraries were obtained as powder samples and dissolved in DMSO as 10 mM stock solutions, except several hundreds from the approved drug library that were prepared as 4.47 mM stock solutions due to solubility limitations.
  • the eluted fractions were separated by SDS-PAGE and visualized by silver staining.
  • RBC infected with gametocytes (3D7 strain: Stage III-V) were washed 3 times with PBS and then lysed by 0.05% saponin treatment in PBS for 5 min at room temperature.
  • the prepared gametocytes were washed 3 times with PBS and frozen at -80° C.
  • the affinity precipitation experiment was processed as previously described [33, 57].
  • the frozen samples were lysed with homogenization buffer (60 mM glycerophosphate, 15 mM p-nitrophenyl phosphate, 25 mM MOPS (pH 7.2), 15 mM EGTA, 15 mM MgC12, ImM DTT, protease inliibitors (Roche Diagnostics, cat. no.
  • homogenization buffer 60 mM glycerophosphate, 15 mM p-nitrophenyl phosphate, 25 mM MOPS (pH 7.2), 15 mM EGTA, 15 mM MgC12, ImM DTT, protease inliibitors (Roche Diagnostics, cat. no.
  • DARTS drug affinity responsive target stability
  • the 3D7 gametocytes were lysed with M-PER supplemented with protease and phosphatase inhibitors as previously described [34]. After centrifugation at 16,000 x g for 20 min, protein concentration in the supernatant was quantified and 2 ⁇ g/ ⁇ l proteins were treated with 600 nM of Torin 2 or 600 nM of Torin 1 for 2 h at room temperature. The samples were treated with 46 ⁇ g/ml pronase (Sigma-Aldrich, cat. no. P691 1) for 30 min at room temperature. The digestion was stopped by adding the SDS-PAGE sample loading buffer and boiled at 70 °C for 10 min. The samples were separated on a 10% Bis-Tris gel and visualized by silver staining.
  • Plasmodium berghei ANKA (Pb) parasites were maintained by serial passage by intraperitoneal (i.p.) injection in outbred mice. Two days before feeding, female mice were infected i.p. with 200-400 ⁇ whole blood from a Pb- infected mouse with >10% parasitemia.
  • mice On the day of feeding, the mice were checked for exflagellation and injected intravenously (i.v.) with drug vehicle alone (10% N- methylpyrrolidnone, 40% PEG 400 in water), or (a) 2-4 mg/kg Torin 2 (one or two doses), (b) 8 mg/kg NVP-AUY922 (two doses), or (c) 8 mg/kg Alvespimycin (two doses).
  • drug vehicle alone 10% N- methylpyrrolidnone, 40% PEG 400 in water
  • Torin 2 one or two doses
  • mice 8 mg/kg NVP-AUY922 (two doses)
  • Alvespimycin two doses.
  • mice Two hours post treatment, mice were anesthetized and Anopheles stephensi mosquitoes were allowed to feed on infected mice for 15 minutes. Parasitemia, gametocytemia, and presence of exflagellation were examined as described previously [58].
  • Mosquitoes were maintained on 5% (w/v) glucose at 19 °C and 80%) relative humidity. At day 10 post feeding, mosquito midguts were dissected and transmission was measured by staining mosquito midguts with 0.2% mercurochrome and counting the numbers of oocysts per midgut.
  • Reverse phase column chromatography was performed on RediSep preparative C-18 column using the Teledyne ISCO combiflash Rf system. ⁇ spectra were recorded using an Inova 400 MHz spectrometer (Varian). Samples were analyzed on an Agilent 1200 series LC/MS. Method A used an Enomenex Kinetex 1.7 micron column and a flow rate of 1.1 mL/min. The mobile phase was a mixture of acetonitrile and H 2 0 each containing 0.05% trifluoroacetic acid. A gradient of 4% to 100% acetonitrile over 4 minutes was used during analytical analysis.
  • Method B used a ZorbaxTM Eclipse XDB-C18 reverse phase (5 micron, 4.6 x 150 mm) column and a flow rate of 1.1 mL/min.
  • the mobile phase was a mixture of acetonitrile and H 2 0 each containing 0.05% trifluoroacetic acid.
  • a gradient of 5% to 100% acetonitrile over 8 minutes was used during analytical analysis.
  • This example describes an assay for the identification of gametocytocidal compound in accordance with an embodiment of the invention.
  • P. falciparum strain 3D7 gametocytes were screened against 5,215 compounds at four concentrations ranging from 0.37 to 46 ⁇ using an alamarBlue viability assay [10, 1 1]. These compounds include 4,265 approved human or animal drugs [12], 400 from the Malaria Box library that are active against P. falciparum strain 3D7 asexual parasites in vitro [13], and 550 from an internal collection of kinase inhibitors [14]. A total of 27 novel active gametocytocidal compounds were identified and confirmed- with IC50 values ⁇ 1 ⁇ against gametocytes. Among these confirmed compounds, 21 had more than 10-fold selectivity against gametocytes over the mammalian cell line HepG2.
  • the gametocial activity is set forth in Table 1, and the cytotoxicity against the mammalian HepG2 cell line is set forth in Table 2.
  • NSC 174938, Torin 2, NVP-AUY922, maduramicin, and narasin were the most potent compounds against gametocytes with IC 50 values ranging from 3 to 50 nM (Table 1).
  • PQ (primaquine) and 7 other compounds with known gametocytocidal activity were present in the compound collection and were all identified in the screen (Table 1), validating the effectiveness of this screening method.
  • IC 5 o mean half-maximum inhibitory concentrations determined from at least 3 independent experiments against P. falciparum 3D7 gametocyte; * indicates compounds with previously reported activities against asexual parasites. ⁇ means compounds with previously reported activities against gametocytes.
  • PQ showed similar potencies against gametocytes from these three strains with IC50 values of 1.26, 0.68, and 1.08 ⁇ against 3D7, HB3, and Dd2, respectively.
  • concentration-response curves of strain selective compounds panobinostat and CUDC-101 in comparison with strain nonselective compounds primaquine and Torin 2 are depicted in FIG. 3.
  • Meflocmine hydrochloride 0.0 0.059 0.053
  • This example demonstrates activities of Torin 2 against gametocytes and asexual parasites in vitro in accordance with an embodiment of the invention.
  • This example demonstrates the efficacy of Torin 2 on gametocyte transmission from host to mosquitoes in a mouse model in accordance with an embodiment of the invention.
  • Torin 2 was tested in the same mouse model (Fig. 2C). A single dose of 2 mg/kg of Torin 2 significantly reduced oocyst production, while a single 4 mg/kg dose almost completely eliminated it. These results clearly demonstrate the ability of Torin 2 to completely block gametocyte transmission from infected mice to mosquitoes.
  • T2M was synthesized as an affinity resin for the pulldown experiment for identification of Torin 2 interacting proteins in P. falciparum gametocyte lysates.
  • the structure of T2M is shown as compound 10a in Example 7.
  • a negative control resin, TIM was similarly synthesized with a close analog of Torin 1, shown as compound 10b in Example 7.
  • the proteins precipitated from gametocyte lysate by T2M but not TIM were identified by mass spectrometric analysis [33].
  • the proteomics data revealed a total of 31 proteins selectively enriched by T2M. The results are set forth in Table 5.
  • a DARTS experiment [34] was also carried out to identify Torin 2 binding proteins by limited protease digestion of Torin 2-treated gametocyte lysates. Following treatment with either Torin 2 or the negative control Torin 1, gametocyte lysates were partially digested with pronase and size fractionated by SDS-PAGE.
  • Merozoite surface protein 1 (MSP1) PF3D7_093030C 196 kDa 2
  • Plasmodium exported protein (PHISTc), PF3D7_1 14870C 44 2 unknown function (GEXP12) kDa
  • RNA binding protein putative PF3D7J 454000 59 2 kDa
  • Plasmodium exported protein (PHISTb), PF3D7_040210C 68 1 unknown function kDa
  • 60S ribosomal protein L4 putative PF3D7_050710C 46 1 kDa ATP synthase subunit beta, PF3D7J2357O0 58 1 mitochondrial kDa
  • Aldehydes 1 were prepared using a reported procedure (J Med. Chem. 201 1 , 54(5): 1473-1480). A solution of 1 (300 ⁇ ) in 3 mL of THF were added 300 of Et 2 N Pr and R'CH 2 COCl (3000 ⁇ ). The mixture was heated in a microwave between 100 to 150 °C for 15 min. The crude product was purified by column chromatography on silica gel using dichloromethane in methanol (0-20%) as eluent to give 1'. A mixture of 1 ' (1.0 equiv), boronic acid or boronic acid pinacol ester (3.0 equiv),
  • reaction mixture was filtered through a plug of Celite and the filtrate was purified by reverse phase column chromatography using acetonitrile (containing 0.1% TFA)/water (containing 0.1% TFA) as an eluent to give 2.
  • Reagents and conditions (i) POCi 3 , DMF; (ii) triethyl phosphonoacetate, EtOH; K 2 C0 3 , r.t. (iii) R 2 NH 2 , 120-180 °C, 5-20 min; (iv) K 2 C0 3 , EtOH, MW, 150 °C, 15 min; (v) R B(OR') 2 , Pd(Ph 3 P) 4 , NaHC0 3 , DMF/water, MW, 120-150 °C, 15 min.
  • Polymer linked Torin 1 (16b) was prepared in a similar manner as polymer linked Torin 2.
  • This example demonstrates an In vitro drug activity on gametocytes.
  • Stage III-V gametocytes were enriched with treatment with 50 mM N- acetylglucosamine (NAG) and Percoll density gradient centrifugation as described
  • Asexual parasites of P. falciparum strain 3D7 were cultured as described previously (Trager, W. et al., J. Parasitol. 2005, 91(3): 484-486). Drug activity on asexual stage parasites was tested using a SYBR Green assay as described previously (Eastman, R.T. et al., Antimicrob. Agents Chemother. 2013, 57(1): 425-435; Smilkstein, M. et al,
  • This example demonstrates the efficacy of NVP-AUY922 and Alvespimycin on gametocyte transmission from host to mosquitoes in a mouse model in accordance with an embodiment of the invention.
  • Example 4 The experiment described in Example 4 was conducted using NVP-AUY922 and Alvespimycin in a two dose protocol at 8 mg/kg as test compounds. The protocol is depicted graphically in Fig. 6A. The oocyte number for vehicle, NVP-AUY922, and
  • filtrate was purified by reverse phase column chromatography using acetonitrile (containing 0.1% TFA)/water (containing 0.1% TFA) as an eluent to give 21.
  • This example demonstrates the gametocytocidal activity and activity against asexual parasites in accordance with an embodiment of the invention.
  • Baird JK Surjadjaja C (201 1) Consideration of ethics in primaquine therapy against malaria transmission. Trends in Parasitology 27: 1 1-16. 6. Song JP, Socheat D, Tan B, Dara P, Deng CS, et al. (2010) Rapid and effective malaria control in Cambodia through mass administration of artemisinin-piperaquine. Malaria Journal 9.
  • Tanaka TQ Williamson KC (201 1) A malaria gametocytocidal assay using
  • Tin F, Nyunt H (1984) Comparative drug trial of a sulfadoxine/pyrimethamine and a sulfalene/pyrimethamine combination against Plasmodium falciparum infections in semi- immune populations of Burma.
  • Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins. Proceedings of the National Academy of Sciences of the United States of America. -

Abstract

The invention provides a method of blocking transmission of a Plasmodium parasite and a method of treating or preventing malaria comprising administering to an animal an effective amount of a first compound of formula I: wherein A, B, R1, R2, R10, and R11 are described herein, either alone or in combination with a second compound selected from elesclomol, NSC 174938, NVP-AUY922, Maduramicin, Narasin, Alvespimycin, Omacetaxine, Thiram, Zinc pyrithione, Phanquinone, Bortezomib, Salinomycin sodium, Monensin sodium, Dipyrithione, Dicyclopentamethylene-thiuram disulfide, YM155, Withaferin A, Adriamycin, Romidepsin, AZD-1 152-HQPA, CAY10581, Plicamycin, CUDC-101, Auranofin, Trametinib, GSK-458, Afatinib, and Panobinostat.

Description

METHOD OF BLOCKING TRANSMISSION OF MALARIAL PARASITE
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 61/904,884, filed November 15, 2013, which is incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Malaria cases and deaths have dropped 50% in 29 countries since 2000 due to the combined effects of long-lasting insecticidal bed nets, indoor residual spraying, and artemisinin-based combination therapies (ACTs) [1], This success has raised hopes for malaria eradication and consequently stimulated interest in developing new reagents that block gametocyte transmission, such as novel and safe gametocytocidal drugs [2]. Previous drug development efforts have focused primarily on the asexual parasites that cause symptoms but not malaria transmission. To be transmitted from person to person via mosquitoes, the parasites must switch from asexual to sexual development and produce male and female gametocytes. Once gametocytes are taken up in a blood meal by a mosquito, fertilization is stimulated and the resulting zygote differentiates into a motile ookinete that migrates across the midgut epithelium of the mosquito and forms an oocyst. Over the course of the next 2 weeks, tens of thousands of infectious sporozoites are generated and sequestered in the mosquito salivary glands until released into a vertebrate host for transmission during the next blood meal.
[0003] Sexual stage P. falciparum gametocytes have a lifespan of over 3 weeks and are not cleared effectively by current antimalarial agents, except primaquine (PQ) [3,4] which is not widely used because it causes hemolytic anemia in patients with glucose-6-phosphate dehydrogenase deficiency [5]. Consequently, treatment with current antimalarial drugs often results in asymptomatic carriers who remain infectious for weeks after the clearance of asexual parasites. Despite the risks of PQ, its efficacy with artemisinin combination therapy (ACT) in reducing malaria transmission in the PQ-tolerant patients was recently
demonstrated in test regions. Other than PQ, the only other gametocytocidal candidate being tested is methylene blue.
[0004] Thus, a new generation of antimalarial agents with potent activities against both sexual and asexual parasites is urgently needed for better therapeutic effect and eradication of malarial infection globally. BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides a method of blocking transmission of a Plasmodium parasite comprising administering to a mammal in need of such treatment, a therapeutically effective amount of a first compound of formula I):
Figure imgf000004_0001
(I)
wherein A is CR12 or N,
B is CR3=CR4 or NR13,
R1 is an optionally substituted group selected from the group consisting of C6-io aryl;
Ci-12 alkyl; Ci-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-10-membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, SO2NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2, R10, and R1 1 are independently hydrogen, halogen, -NR6R7, -OR8, -SR9, or an optionally substituted group selected from the group consisting of Ci-i2 acyl; C6.10 aryl; 7-15 arylalkyl; C6-i 5 heteroarylalkyl; C M2 alkyl; C1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Cj-C6 alkylamino, di(Ci-C6alkyl)amino, Ci-C6 alkylcarbonylamino, C2-6 lkenyl, trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, alkylcarbonyl, alkylsulfonyl, hydroxyl, carboxy, C6-io aryl, heterocyclyl, and oxo,
R3 and R4 are independently selected from hydrogen, hydroxyl, OR5, halogen, optionally substituted C6-io aryl, and optionally substituted Ci-6 alkyl, R is Cj.i2 alkyl, and
R6, R7, R8, and R9 are independently hydrogen, an optionally substituted group selected from the group consisting of d.i2 acyl; C -io aryl; C6-io aryl Ci-i2 alkyl; C4.7
heteroaryl Ci_i2 alkyl; Ci_i2 alkyl; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-7- membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and CM2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
6 7
R and R are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1 -2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
R12 is hydrogen, Ci-]2 alkyl, C6-io aryl, halogen, hydroxyl, or OR5,
13
R is hydrogen, CM2 alkyl or C6-io aryl,
and/or a second compound selected from elesclomol, NSC174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamethylene- thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1 152-HQPA, CAY10581 , plicamycin, CUDC- 101 , auranofin, trametinib, GSK-458, afatinib, and panobinostat,
or a pharmaceutically acceptable salt thereof.
[0006] The invention also provides a method of treating malaria by killing or arresting the growth of Plasmodium organisms in a mammal, wherein the Plasmodium organisms are in a gametocyte stage, the method comprising administering to a mammal a therapeutically effective amount of a first compound of formula I):
Figure imgf000005_0001
(I)
wherein A is CR or N,
B is CR3=CR4 or NR13,
R1 is an optionally substituted group selected from the group consisting of C6- 2 alkyl; Ci.i2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-12-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoro methyl, Cj-C6 alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2, R10, and R11 are independently hydrogen, halogen, -NR6R7, -OR8, -SR9, or an optionally substituted group selected from the group consisting of Ci_i2 acyl; C6-io aryl; C7-15 arylalkyl; C6-i 5 heteroarylalkyl; C M2 alkyl; C1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C6 alkylamino, di(Ci-C6alkyl)amino, CrC6 alkylcarbonylamino, C2-6 alkenyl, trifluoromefhyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, alkylcarbonyl, alkylsulfonyl, hydroxyl, carboxy, C6-io aryl, heterocyclyl, and oxo,
R3 and R4 are independently selected from hydrogen, hydroxyl, OR5, halogen, optionally substituted C6-io aryl, and optionally substituted C1-6 alkyl,
R5 is C1-12 alkyl, and
R6, R7, R8, and R9 are independently hydrogen, an optionally substituted group selected from the group consisting of Cj.i2 acyl; C6-io aryl; C6-io aryl C .n alkyl; C4-7 heteroaryl C . 2 alkyl; Ci-12 alkyl; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-7- membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and C 1.12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
R6 and R7 are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
R is hydrogen, C\.\2 alkyl, C6-10 aryl, halogen, hydroxyl, or OR ,
R is hydrogen, Ci-12 alkyl or C6-10 aryl,
and/or a second compound selected from elesclomol, NSC 174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamethylene- thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1 152-HQPA, CAY10581 , plicamycin, CUDC-101, auranofin, trametinib, GSK-458, afatinib, and panobinostat,
or a pharmaceutically acceptable salt thereof.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0007] Figure 1 depicts the structures of compounds in accordance with an embodiment of the invention.
[0008] Figure 2 A illustrates a protocol for a mouse model of gametocyte transmission in accordance with an embodiment of the invention.
[0009] Figure 2B illustrates the result of a malaria mouse model for Torin 2 using a 2 dose dosing regime.
[0010] Figure 2C illustrates the result of a malaria mouse model for Torin 2 using a 1 dose dosing regime.
[0011] Figures 3A-3D illustrate dose-concentration curves of panobinostat, CUDC-101 , primaquine, and Torin 2, respectively, against the drug sensitive 3D7 strain and against two asexual drug resistant strains HB3 and Dd2.
[0012] Figure 4 illustrates the results of a gametocyte viability assay for Torin 2 and Torin 1.
[0013] Figure 5 depicts the structures of Torin 2, Torin 1 , and WWH030.
[0014] Figure 6 A illustrates a protocol for a mouse model of gametocyte transmission in accordance with an embodiment of the invention. The oocyte number for vehicle,
NVP-AUY922, and alvespimycin-treated mice are depicted in Fig. 6B. The structures of NVP-AUY922 and alvespimycin are depicted in Fig. 6C.
DETAILED DESCRIPTION OF THE INVENTION
[0015] In an embodiment, the invention provides a method of blocking transmission of a Plasmodium parasite comprising administering to a mammal in need of such treatment, a therapeutically effective amount of a first compound of formula (I):
Figure imgf000008_0001
wherein A is CR12 or N,
B is CR3=CR4 or NR13,
R1 is an optionally substituted group selected from the group consisting of C6-io aryl; Ci-i2 alkyl; C M2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-12-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoro methyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, SO2NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2, R10, and R1 1 are independently hydrogen, halogen, -NR6R7, -OR8, -SR9, or an optionally substituted group selected from the group consisting of Ci-i2 acyl; 06-ιο aryl; C7..15 arylalkyl; C6-i5 heteroarylalkyl; Ci-12 alkyl; Ci-i2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Cj-C6 alkylamino, di(Ci-C6alkyl)amino, Ci-C6 alkylcarbonylamino, C2-6 alkenyl, trifluoromethyl, Ci-C6 alkyl, halo, CN, C]-C6 alkoxy, alkylcarbonyl, alkylsulfonyl, hydroxyl, carboxy, C6-io ryl, heterocyclyl, and oxo,
R3 and R4 are independently selected from hydrogen, hydroxyl, OR5, halogen, optionally substituted C6-io aryl, and optionally substituted Ci-6 alkyl,
R5 is Ci-12 alkyl, and
R6, R7, R8, and R9 are independently hydrogen, an optionally substituted group selected from the group consisting of acyl; C6.10 aryl; C6-1o aryl Ci-n alkyl; C4-7 heteroaryl C 1.12 alkyl; Ci-12 alkyl; 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-7- membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and C1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
R6 and R7 are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
12
R is hydrogen, C1-12 alkyl, C6-io aryl, halogen, hydroxyl, or OR ,
13
R is hydrogen, CM2 alkyl or C6-io aryl,
and/or a second compound selected from elesclomol, NSC174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamefhylene- thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1 152-HQPA, CAY10581 , plicamycin, CUDC-101 , auranofin, trametinib, GSK-458, afatinib, and panobinostat,
or a pharmaceutically acceptable salt thereof.
[0016] In certain embodiments, the compound is of formula (I) and B is CR3=CR4.
[0017] In certain of these embodiments, A is CH or N,
[0018] R1 is Ci-C6 alkyl, C6-Cio aryl, or heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
[0019] R2 is C6-Cio aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C6 alkylamino, di(Ci- C6alkyl)amino, C|-C6 alkylcarbonylamino, sulfonyl, di(Ci-C6 alkyl)carbonylamino, trifluoromethyl, halo, C2-C6 alkenyl, cyano, Ci-C6 alkoxy, acyl, Ci-C6 alkyl, hydroxyl, heterocyclyl, oxo, aminosulfonyl, alkylsulfonylamino, Ci-C6 alkylaminomethyl, and di(Ci-C6 alkyl)aminomethyl,
[0020] R10 and R11 are both hydrogen, and
[0021] R3 and R4 are individually selected from hydrogen, halo, optionally substituted d-C6 alkyl, and OR5.
[0022] In certain embodiments, when A is CH, B is CR3=CR4, R3, R4, R10, and R1 1 are each hydrogen, and R1 is 3-trifluoromethylphenyl, R2 is not 2-amino-5-pyridyl or
3-quinolinyl. [0023] Referring now to terminology used generically herein, the term "alkyl" means a straight-chain or branched alkyl substituent containing from, for example, 1 to about 6 carbon atoms, preferably from 1 to about 4 carbon atoms, more preferably from 1 to 2 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, /7-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, and the like.
[0024] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, and the term "C6-Cio aryl" includes phenyl and naphthyl. It is understood that the term aryl applies to cyclic substituents that are planar and comprise 4n+2 π electrons, according to Hiickel's Rule.
[0025] The term "heteroaryl" refers to a monocyclic or bicyclic 5 to 10-membered ring system as described herein, wherein the heteroaryl group is unsaturated and satisfies Hiickel's rule, and wherein the heteroaryl contains 1 -4 heteroatoms independently selected from nitrogen, oxygen and sulfur. Non-limiting examples of suitable heteroaryl groups include furanyl, thiopheneyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, l,3,4-oxadiazol-2-yl, 1 ,2,4-oxadiazol-2-yl, 5- methyl-l,3,4-oxadiazole, 3 -methyl- 1 ,2,4-oxadiazole, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, benzothiopheneyl, indolyl, indazolyl, imidazolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolinyl, benzothiazolinyl, and quinazolinyl. The heteroaryl groups can be attached at any open position on the heteroaryl groups. The terms "heterocyclic" or "heterocyclyl" refer to a 4 to 12-membered heterocyclic ring system as described herein, wherein the heterocycle contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the heterocycle is saturated or monounsaturated. The heterocyclyl or heteroaryl group is optionally substituted with 1 , 2, 3, 4, or 5 substituents as recited herein such as with alkyl groups such as methyl groups, ethyl groups, and the like, or with aryl groups such as phenyl groups, naphthyl groups and the like, wherein the aryl groups can be further substituted with, for example halo, dihaloalkyl, trihaloalkyl, nitro, hydroxy, alkoxy, aryloxy, amino, substituted amino, alkylcarbonyl, alkoxycarbonyl, arylcarbonyl, aryloxycarbonyl, thio, alkylthio, arylthio, and the like, wherein the optional substituent can be present at any open position on the heterocyclyl or heteroaryl group.
[0026] The term " alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms" refers to a linear or branched alkyl group wherein one or more carbon atoms in the alkyl group is replace with the aforesaid atoms. Non-limiting examples of alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms include, for example, methoxymethyl, methoxy ethyl, methylaminoethyl, and the like.
[0027] The term "acyl" refers to an alkylcarbonyl substituent. The term
"alkylsulfonylamino" refers to a group of the structure: alkyl-S02-NH-. The term
"aminosulfonyl" refers to a group of the structure: H2NS02-.
[0028] In certain embodiments, the compound is a compound of formula (I) and B is CR3=CR4. In certain embodiments, R1 is Ci-C6 alkyl, C6-Cio aryl, or heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and
4-alkylcarbonylpiperazinyl. In certain embodiments, R2 is C6-Cio aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C6 alkylamino, di(Ci-C6alkyl)amino, and Ci-C6 alkylcarbonylamino. In certain embodiments, R10 and R! ! are both hydrogen. In certain embodiments, R3 and R4 are both hydrogen. In certain preferred embodiments, A is CH. In certain preferred embodiments, R1 is selected from 3-trifluoromethylphenyl, 4-piperazinylmethyl, ethyl, phenyl, 3-ethylphenyl, 3-chlorophenyl, 3-cyanophenyl, 3-methoxyphenyl, 3-(dimethylaminocarbonyl)phenyl, 3- sulfonamidophenyl, 3-phenoxyphenyl, 3-ethoxyphenyl, 4-(piperazin-4-yl)-3- trifluoromethylphenyl, 4-piperazinyl, l-acetylpiperidin-4-yl,cyclopropyl, 4- tetrahydropyranyl, cyclohexyl, and cyclopentyl. In certain preferred embodiments, R2 is selected from 2-amino-pyridinyl, 4-pyridinyl, 2-amino-5-pyrimidinyl, 3-pyridyl, quinolin-3- yl, 5-pyrimidinyl, 2-amino-5-trifluoromethylpyrimidin-5-yl, 2-acetylamino-5-pyridyl, 2- amino-4-methylpyrimidin-5-yl, 1 -piperazinyl, indol-5-yl, lH-indazol-5-yl, 4-aminophenyl, l ,2,3,6-tetrahydropyridin-4-yl, 1 H-pyrazol-4-yl, lH-benzo[d]imidazol-5-yl, 4- sulfonylaminophenyl, 2-dimethylaminopyrimidin-5-yl, 3-trifluoromethylphenyl, bromo, 3- aminophenyl, vinyl, 4-aminocarbonylphenyl, 3-cyanophenyl, 3-trifluoiOmethyl-5-pyridyl, tetrazolyl, 4-chlorophenyl, 4-methoxyphenyl, 3-aminocarbonylphenyl, 3-acetylphenyl, 2,3- dihydrobenzofuran-6-yl, 1 -methyl- lH-indol-5-yl, benzo[d][l,3]dioxo-5-yl, 4-fluorophenyl, 4- hydroxyphenyl, porpholin-l-yl, benzo[b]thiophen-l-yl, 4-methylsulfonylphenyl,
benzo[c][l ,2,5]oxadiazol-5-yl, 2-(piperidin-l -yl)-3-pyridinyl, 4-carboxyphenyl, 2-methyl-5- pyridyl, 4-methylsulfonylphenyl, 4-dimethylaminocarbonylphenyl, 4-phenylphenyl, 4- methylpenyl, 3-chloro-5-pyridyl, (3-pyrrolidin-l-yl)phenyl, 4-([piperizin-l- yl]carbonyl)phenyl, 4-([morpholin-l-yl]carbonyl)phenyl, 2-hydroxypyrimidin-5-yl, 3- aminosulfonylphenyl, 2-oxo- 1 ,2,3 ,4,tetrahydroisoquinolin-6-yl, 2-oxo- 1 ,2,3 ,4,- tetrahydroquinolin-6-yl, 4-(aminomethyl)phenyl, 4-(dimethylaminomethyl)ph( (methylaminocarbonyl)phenyl, l-oxoindolin-5-yl, and l-oxoisoindolin-5-yl.
[0029] In an embodiment, the compound has the formula:
Figure imgf000012_0001
[0030] In certain preferred embodiments, the compound has the formula:
O
R' .X
[0031] wherein R1 and R2 are:
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
-
17
Figure imgf000019_0003
[0032] In certain embodiments, the compound is
Figure imgf000019_0002
[0033] In certain other embodiments, the compound is a compound of formula (I) and B is NR12. In certain preferred embodiments, A is CH. In certain embodiments, R10 and R11 are both hydrogen. In certain embodiments, R 13 is hydrogen or Ci-i2 alkyl. In a certain preferred embodiment, R1 is 3-trifluoromethylphenyl. In certain preferred embodiments, Rz is selected from the group consisting of 2-methyl-5-pyridyl, 4-aminophenyl, 2-acetylamino- 5-pyridyl, 4-hydroxyphenyl, 3-aminophenyl, 4-pyridyl, lH-benzo[d]imidazol-5-yl, 4- methlsulfonylphenyl, quinolin-3-yl, 2-aminopyrimidin-5-yl, 3-cyanophenyl, 3-pyridyl, and 4- aminocarbonylphenyl.
Figure imgf000020_0001
Figure imgf000021_0001
[0035] In certain embodiments, the second compound that is administered is selected from elesclomol, NSC 174938, NVP-AUY922, Maduramicin, Narasin, Alvespimycin, Omacetaxine, Thiram, Zinc pyrithione, Phanquinone, Bortezomib, Salinomycin sodium, Monensin sodium, Dipyrithione, Dicyclopentamethylene-thiuram disulfide, YM155,
Withaferin A, Adriamycin, Romidepsin, AZD-1 152-HQPA, CAY10581, Plicamycin, CUDC- 101, Auranofin, Trametinib, GSK-458, Afatinib, and Panobinostat. In certain preferred embodiments, elesclomol, the compound is NSC 174938, NVP-AUY922, Maduramicin, and Narasin.
[0036] In accordance with an embodiment of the invention, the compound is
administered in the form of a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier.
[0037] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, or diluents, are well known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.
[0038] The choice of carrier will be determined in part by the particular active agent, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, interperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting. [0039] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,
microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
[0040] The compounds of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
[0041] Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l ,3-dioxolane- 4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a
pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.
[0042] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (3) mixtures thereof.
[0043] The parenteral formulations will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi- dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. -
22
[0044] The compounds of the present invention may be made into injectable
formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).
[0045] Additionally, the compounds of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
[0046] Suitable carriers and their formulations are further described in A.R. Gennaro, ed., Remington: The Science and Practice of Pharmacy (19th ed.), Mack Publishing Company, Easton, PA (1995).
[0047] The compound of the invention or a composition thereof can potentially be administered as a pharmaceutically acceptable acid-addition, base neutralized or addition salt, formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base, such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases, such as mono-, di-, trialkyl, and aryl amines and substituted ethanolamines. The conversion to a salt is accomplished by treatment of the base compound with at least a stoichiometric amount of an appropriate acid. Typically, the free base is dissolved in an inert organic solvent such as diethyl ether, ethyl acetate, chloroform, ethanol, methanol, and the like, and the acid is added in a similar solvent. The mixture is maintained at a suitable temperature (e.g., between 0 °C and 50 °C). The resulting salt precipitates spontaneously or can be brought out of solution with a less polar solvent.
[0048] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention. -
23
[0049] It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is
pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
[0050] It is further understood that the above compounds and salts may form solvates, or exist in a substantially uncomplexed form, such as the anhydrous form. As used herein, the term "solvate" refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is called a hydrate. Pharmaceutically acceptable solvates include hydrates, alcoholates such as methanolates and ethanolates, acetonitrilates and the like. These compounds can also exist in polymorphic forms.
[0051] The Plasmodium parasite can be any suitable Plasmodium parasite. Non-limiting examples of suitable Plasmodium parasites include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. In a preferred embodiment, the Plasmodium parasite is Plasmodium falciparum.
[0052] In an embodiment, the Plasmodium parasite is a Plasmodium gametocyte.
[0053] In embodiments, the Plasmodium gametocyte is a mature stage II-V gametocyte. In a preferred embodiment, the Plasmodium gametocyte is a stage III-V gametocyte, e.g., a mature stage III-V gametocyte. In another preferred embodiment, the Plasmodium gametocyte is a mature stage V gametocyte.
[0054] In certain preferred embodiments, the compound effectively kills Plasmodium gametocytes.
[0055] In embodiments, the Plasmodium parasite is a drug-resistant strain. Examples of drug-resistant strains of Plasmodium are described in Kun, J.F.J, et al., Antimicrob Agents
Chemother., 1999 September; 43(9): 2205-2208, and references cited therein.
[0056] In embodiments, the Plasmodium parasite is in an asexual stage. For example, the
Plasmodium parasite can be a sporozoite, a liver stage parasite, a merozoite, an asexual erythrocyte-stage parasite, a zygote, an ookinete, or an oocyst.
[0057] The amount or dose of a compound of the invention or a salt thereof, or a composition thereof should be sufficient to affect a therapeutic or prophylactic response in the mammal. The appropriate dose will depend upon several factors. For instance, the dose also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular compound or salt. Ultimately, the attending physician will decide the dosage of the compound of the present invention with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound or salt to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the compound(s) described herein can be about 0.1 mg to about 1 g daily, for example, about 5 mg to about 500 mg daily. Further examples of doses include but are not limited to: 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.5 mg, 0.6 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 17 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 140 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg/kg body weight per day.
[0058] In certain embodiments, the method further comprises administering to the mammal at least one additional antimalarial compound. Any suitable antimalarial compound can be used, many of which are well known in the art. Non-limiting examples of suitable antimalarial compounds include primaquine, bulaquine, artemisinin and derivatives thereof, chloroquine, mefloquine, amodiaquine, piperaquine, pyronaridine, atovaquone, tafenoquine, methylene blue, trioxaquines, endoperoxides such as OZ 439 and OZ 277, decoquinate, 9-anilinoacridines, HIV-protease inhibitors, and natural products such as neem, epoxomicin, harmonine, and riboflavin.
[0059] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
[0060] Materials and Methods
[0061] Cell culture. Asexual parasites of P. falciparum strain 3D7 were cultured as described previously [55]. Stage III-V gametocytes were selected and enriched with 3 -day treatment with 50 mM N-acetylglucosamine (NAG) and the following Percoll density gradient centrifugation after gametocyte production [10]. Gametocytes of HB3 and Dd2 strains were produced and then set up for assay in a similar process. HepG2 cells (ATCC, cat. no. 77400) were cultured in 175-cm2 tissue culture flasks with 30 ml growth medium at 37 °C in a 5% C02 humidified atmosphere. Growth medium was made with Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (FBS). Growth medium was replaced every other day and cells were passed at 75% confluence. [0062] Compound library and gametocyte assay screen. The approved drug library was collected with 4,265 compounds from traditional chemical suppliers, specialty collections, pharmacies and custom synthesis [12] that included 49% drugs approved for human or animal use by the US Food and Drug Administration (FDA), 23% approved in
Canada/UK/EU/Japan, and the remaining 28% either in clinical trials or research tool compounds. The Malaria Box contained 400 drugs or tool compounds with the confirmed activities on blood-staged P. falciparum and assessed cytotoxicity against mammalian cells [39, 56]. The MIPE library was an internal collection of 550 kinase inhibitors, which contain approved drugs and drug candidates in preclinical and clinical stages [14]. Compounds from all libraries were obtained as powder samples and dissolved in DMSO as 10 mM stock solutions, except several hundreds from the approved drug library that were prepared as 4.47 mM stock solutions due to solubility limitations.
[0063] Compound screening experiments were performed as previously described [1 1]. Briefly, 2.5 μΐ/well incomplete medium was dispensed into each well of 1,536-well plates using the Multidrop Combi followed by 23 nl compound transferring using the NX-TR Pintool (WAKO Scientific Solutions, San Diego, CA). Then, 2.5 μΐ/well of gametocytes was dispensed with a seeding density of 20,000 cells/well using the Multidrop Combi. The assay plates were incubated for 72 h at 37 °C with 5% C02. After addition of 5 μΐ/well of 2X AlamarBlue dye (Life Technologies, cat. no. DAL 1100), the plates were incubated for 24 h at 37 °C with 5% COz and then were read in a fluorescence detection mode (Ex = 525 nm, Em = 598 nm) on a ViewLux plate reader (PerkinElmer).
[0064] Small molecule pull-down. Affinity matrix: To make a bead-connected affinity probe of Torin 2, a tetraethylene glycol linker was attached to l-(piperazin-l-yl)propan-l-one of HWW030 and then coupled to Affi-Gel 10 resin (Bio-Rad Laboratories, cat. no. 153-6046) under mild basic conditions to afford Torin 2 matrix (T2M). See detailed version in Example 7. Torin 1 was similarly immobilized to resin and used as a negative control (TIM). The resultant affinities probes were incubated with gametocyte lysates, the bound proteins were eluted from resin by boiling in SDS-PAGE sample loading buffer. The eluted fractions were separated by SDS-PAGE and visualized by silver staining. RBC infected with gametocytes (3D7 strain: Stage III-V) were washed 3 times with PBS and then lysed by 0.05% saponin treatment in PBS for 5 min at room temperature. The prepared gametocytes were washed 3 times with PBS and frozen at -80° C. The affinity precipitation experiment was processed as previously described [33, 57]. The frozen samples were lysed with homogenization buffer (60 mM glycerophosphate, 15 mM p-nitrophenyl phosphate, 25 mM MOPS (pH 7.2), 15 mM EGTA, 15 mM MgC12, ImM DTT, protease inliibitors (Roche Diagnostics, cat. no.
1 1836170001), and 0.5% Nonidet P-40). Cell lysates were centrifuged at 16,000 x g for 20 min at 4°C, and the supernatant was collected. Protein concentration in the supernatant was determined by using a BCA protein assay kit (Pierce Chemical, cat. no. 23225). The lysate (0.5 mg) was then added to the packed affinity matrix, and bead buffer (50 mM Tris HC1 (pH 7.4), 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, protease inhibitors, and 0.1% Nonidet P-40) was added to a final volume of 1 ml. After rotating at 4°C for 2 h, the mixture was centrifuged at 16,000 x g for 2 min at 4°C, and the supernatant was removed. The affinity matrix was then washed (six times) with cold bead buffer and eluted by boiling with SDS-PAGE sample loading buffer at 95°C for 5 min. Supernatants were separated on a 10% Bis-Tris gel (Life Technologies, cat. no. NP0315BOX) and visualized by silver staining using a Pierce Silver Stain Kit for Mass Spectrometry (Pierce Chemical, cat. no. 24600).
[0065] DARTS (drug affinity responsive target stability). The 3D7 gametocytes were lysed with M-PER supplemented with protease and phosphatase inhibitors as previously described [34]. After centrifugation at 16,000 x g for 20 min, protein concentration in the supernatant was quantified and 2 μg/μl proteins were treated with 600 nM of Torin 2 or 600 nM of Torin 1 for 2 h at room temperature. The samples were treated with 46 μg/ml pronase (Sigma-Aldrich, cat. no. P691 1) for 30 min at room temperature. The digestion was stopped by adding the SDS-PAGE sample loading buffer and boiled at 70 °C for 10 min. The samples were separated on a 10% Bis-Tris gel and visualized by silver staining.
[0066] Malaria Mouse Model. Plasmodium berghei ANKA (Pb) parasites were maintained by serial passage by intraperitoneal (i.p.) injection in outbred mice. Two days before feeding, female mice were infected i.p. with 200-400 μΐ whole blood from a Pb- infected mouse with >10% parasitemia. On the day of feeding, the mice were checked for exflagellation and injected intravenously (i.v.) with drug vehicle alone (10% N- methylpyrrolidnone, 40% PEG 400 in water), or (a) 2-4 mg/kg Torin 2 (one or two doses), (b) 8 mg/kg NVP-AUY922 (two doses), or (c) 8 mg/kg Alvespimycin (two doses). Two hours post treatment, mice were anesthetized and Anopheles stephensi mosquitoes were allowed to feed on infected mice for 15 minutes. Parasitemia, gametocytemia, and presence of exflagellation were examined as described previously [58]. Mosquitoes were maintained on 5% (w/v) glucose at 19 °C and 80%) relative humidity. At day 10 post feeding, mosquito midguts were dissected and transmission was measured by staining mosquito midguts with 0.2% mercurochrome and counting the numbers of oocysts per midgut.
[0067] Data analysis. The primary screen data was analyzed using customized software developed internally [59]. IC5o values were calculated using the Prism software (Graphpad Software, Inc. San Diego, CA). Data were presented as means ± SEM with n = 3
independent experiments.
[0068] General materials and methods for chemical synthesis. All commercially available reagents, compounds, and solvents were purchased and used without further purification. 9-Bromo- 1 -(3 -(trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2( 1 H)-one was prepared according to the method described by Liu and coworkers (Liu, Q. et. al., J. Med. Chem. , 201 1, 1473-1480). Column chromatography on silica gel was performed on Biotage KPSil pre-packed cartridges using the Biotage SP-1 automated chromatography system. Reverse phase column chromatography was performed on RediSep preparative C-18 column using the Teledyne ISCO combiflash Rf system. Ή spectra were recorded using an Inova 400 MHz spectrometer (Varian). Samples were analyzed on an Agilent 1200 series LC/MS. Method A used an Enomenex Kinetex 1.7 micron column and a flow rate of 1.1 mL/min. The mobile phase was a mixture of acetonitrile and H20 each containing 0.05% trifluoroacetic acid. A gradient of 4% to 100% acetonitrile over 4 minutes was used during analytical analysis. Method B used a Zorbax™ Eclipse XDB-C18 reverse phase (5 micron, 4.6 x 150 mm) column and a flow rate of 1.1 mL/min. The mobile phase was a mixture of acetonitrile and H20 each containing 0.05% trifluoroacetic acid. A gradient of 5% to 100% acetonitrile over 8 minutes was used during analytical analysis.
EXAMPLE 1
[0069] This example describes an assay for the identification of gametocytocidal compound in accordance with an embodiment of the invention.
[0070] P. falciparum strain 3D7 gametocytes were screened against 5,215 compounds at four concentrations ranging from 0.37 to 46 μΜ using an alamarBlue viability assay [10, 1 1]. These compounds include 4,265 approved human or animal drugs [12], 400 from the Malaria Box library that are active against P. falciparum strain 3D7 asexual parasites in vitro [13], and 550 from an internal collection of kinase inhibitors [14]. A total of 27 novel active gametocytocidal compounds were identified and confirmed- with IC50 values < 1 μΜ against gametocytes. Among these confirmed compounds, 21 had more than 10-fold selectivity against gametocytes over the mammalian cell line HepG2. The gametocial activity is set forth in Table 1, and the cytotoxicity against the mammalian HepG2 cell line is set forth in Table 2. NSC 174938, Torin 2, NVP-AUY922, maduramicin, and narasin were the most potent compounds against gametocytes with IC50 values ranging from 3 to 50 nM (Table 1). Additionally, PQ (primaquine) and 7 other compounds with known gametocytocidal activity were present in the compound collection and were all identified in the screen (Table 1), validating the effectiveness of this screening method.
Table 1
Figure imgf000030_0001
-
29
Figure imgf000031_0001
Note: mean IC5o, mean half-maximum inhibitory concentrations determined from at least 3 independent experiments against P. falciparum 3D7 gametocyte; * indicates compounds with previously reported activities against asexual parasites.† means compounds with previously reported activities against gametocytes.
Table 2
Compound IC50 (μΜ) in HepG2 % Max response -
30
Figure imgf000032_0001
-
31
Primaquine 17.831 -65
Note: "Inactive": no significant activity at the highest tested compound concentration (46 μΜ).
EXAMPLE 2
[0071] This example demonstrates the profiles of gametocytocidal compounds against drug resistant strains in accordance with an embodiment of the invention.
[0072] Drug resistance is also a critical challenge for malaria treatment and eradication that has not been examined in gametocytes, though it has been extensively studied for the asexual parasites [25,26]. To evaluate whether existing antimalarial agents and newly identified gametocytocidal compounds are effective against well characterized drug resistant strains, the gametocytocidal activities of 52 selected compounds, including 27 newly identified compounds and 25 known antimalarial agents, was determined against gametocytes of P. falciparum strains Dd2 and HB3 in the alamarBlue viability assay. In contrast to 3D7, asexual Dd2 parasites are resistant to chloroquine, mefloquine and pyrimethamine while asexual HB3 parasites are resistant to pyrimethamine but not chloroquine or mefloquine [27]. Most of 52 compounds showed 5-fold or less differences in potency between these two parasite strains compared to the drug sensitive stain 3D7. The results are set forth in Table 3. Compared to the drug sensitive stain 3D7, chloroquine' s potency against Dd2 gametocytes was reduced 3.7-fold while it was 10-fold less potent against Dd2 asexual parasites [27]. Methylene blue was moderately more active against 3D7 gametocytes (IC50 = 0.307 μΜ) than those of HB3 (IC50 = 0.935 μΜ) and Dd2 (IC50 = 0.526 μΜ) (SI Table 6). PQ showed similar potencies against gametocytes from these three strains with IC50 values of 1.26, 0.68, and 1.08 μΜ against 3D7, HB3, and Dd2, respectively. The concentration-response curves of strain selective compounds panobinostat and CUDC-101 in comparison with strain nonselective compounds primaquine and Torin 2 are depicted in FIG. 3.
Table 3. Results of compound profiling against P. falciparum strains 3D7, HB3 and Dd2 gametocytes.
Figure imgf000033_0001
Dactinomvcin 0.0 0.019 0.033
Maduramicin ammonium 0.0 0.012 0.037
NVP-AUY922 0.0 0.042 0.047
Narasin 0.0 0.136 0.076
Artesunate 0.0 0.047 0.030
Omacetaxine mepesuccinate 0.0 0.017 0.037
Lumefantrine 0.0 0.033 0.013
Meflocmine hydrochloride 0.0 0.059 0.053
Artemether 0.0 0.047 0.053
Alvespimvcin 0.0 0.235 0.094
Artenimol 0.0 0.059 0.053
Thiram 0.0 0.148 0.148
Zinc Dvrithione 0.0 0.059 0.059
Tetraethvlthiuram disulfide 0.0 0.148 0.296
Disulfiram 0.0 0.108 0.096
Phanquinone 0.1 0.037 0.053
Salinomvcin sodium 0.1 0.372 0.296
Bortezomib 0.1 0.074 0.094
Diphenvleneiodonium 0.1 0.296 0.209
Artemisinin 0.1 0.074 0.061
Salinomvcin monosodium 0.1 0.469 0.296
Chloroauine diphosphate 0.2 0.935 0.935
Monensin sodium 0.2 0.264 0.372
Dipvrithione 0.2 0.743 0.590
Romidepsin 0.2 0.148 0.187
Dicvclopentamethylenethiuram 0.2 0.743 0.743
Methylene blue 0.3 0.935 0.526
Ouinine hemisulfate 0.3 0.235 0.083
Withaferin A 0.3 1.329 0.372
Figure imgf000034_0001
GSK-458 0.935 0.332 1.482
Puromycin 1.049 1.482 2.635
Primaquine diphosphate 1.262 0.679 1.077
Clotrimazole 1.866 1.482 1.482
Pvronaridine 1.866 2.635 6.619
Calcimvcin 2.635 5.899 1.663
CvclosDorin A 3.317 0.935 1.866
Torin- 1 6.619 3.722 5.258
Nizofenone 23.485 14.818 16.626
Note: Each compound was examined in 1 1 concentrations at a 1 :3 dilution for three times against 3D7, HB3 or Dd2 gametocytes. Compounds showing more than 5 -fold selectivity in two or three independent experiments against different strains were highlighted.
[0073] Interestingly, several of these newly identified gametocytocidal compounds exhibited similar or more favorable activities in these two asexual drug resistant strains compared to the drug sensitive 3D7 strain. For example, CUDC-101, a multi-target anticancer drug candidate [28] was 5.5-fold more potent against HB3 (IC5o = 0.152 μΜ) compared to 3D7 (IC50 = 0.833 μΜ) (Fig. 3). Additionally, panobinostat, a histone deacetylase inhibitor, was also 6.3 to 7.9 times more potent against Dd2 (IC50 = 0.148 μΜ) and HB3 (IC50 = 0.1 18 μΜ) compared to 3D7 (IC50 = 0.935 μΜ) (Fig. 3). These results suggest that these newly identified gametocytocidal compounds could be also be effective against a range of asexual drug resistant isolates.
EXAMPLE 3
[0074] This example demonstrates activities of Torin 2 against gametocytes and asexual parasites in vitro in accordance with an embodiment of the invention.
[0075] Torin 2, a known mTOR inhibitor [29, 30], was one of the most potent new gametocytocidal compounds (IC50 = 8 nM). In contrast, its structural analog, Torin 1, was 200-fold less potent (IC50 = 1.6 μΜ), regardless of their similar potencies on mTOR (IC50 values of 5.4 and 2.1 nM, respectively) [29, 31]. The difference in gametocytocidal activity between the two compounds was confirmed using the traditional gametocyte viability assay, optical microscopy of Giemsa stained smears as depicted in Fig. 4. The 200-fold difference in potencies against P. falciparum gametocytes suggests that Torin 2 and Torin 1 may act on -
34
a different target or targets rather than mTOR, consistent with the lack of mTOR homolog in P. falciparum [32].
[0076] Because an ideal new antimalarial agent should have similar activities against both sexual and asexual parasites, IC5o values of the two Torin compounds for asexual parasites in vitro were determined. In a viability assay using asexual parasites, Torin 2 exhibited an IC50 of 2.75 nM, while Torin 1 had an IC50 of 215 nM. Similarly, Torin 1 was 78 times less potent than Torin 2 against the asexual parasites and both compounds were slightly more potent against the asexual parasites compared to the gametocytes.
[0077] To assess the potential toxicity in mammalian cells, we examined both compounds in HepG2 cells. The results are set forth in Table 4. Torin 2 only exhibited partial cytotoxicity at the highest tested concentration (46 μΜ), indicative of greater than 1 , 000-fold selectivity against the parasites over the mammalian cells. Taken together, the results demonstrate the similar low nanomolar potencies of Torin 2 against both sexual and asexual stages of P. falciparum, as well as high selectivity against P. falciparum parasites over mammalian cells.
Table 4. Cytotoxicity of gametocytocidal compounds in mammalian HepG2 cell line.
Compound IC50 (μΜ) in HepG2 cells % Max response
NSC174938 Inactive 13
Torin 2 9.350 -46
Carfilzomib 1.177 -79
Dactinomycin Inactive 1
NVP-AUY922 0.148 -88
Maduramicin 37.221 -42
Narasin 27.041 -98
Artesunate Inactive 18
Arteme her Inactive 7
Alvespimycin 0.1 18 -94
Artenimol (DHA) Inactive 20
Omacetaxine Curve not complete -27
Thiram Inactive 23
Zinc pyrithione 2.148 -99
Phanquinone Inactive 23
Bortezomib 0.148 -82
Artemisinin Inactive 1 8
Salinomycin sodium 29.566 -69 -
35
Figure imgf000037_0001
Note: "Inactive": no significant activity at the highest tested compound concentration (46 μΜ).
EXAMPLE 4
[0078] This example demonstrates the efficacy of Torin 2 on gametocyte transmission from host to mosquitoes in a mouse model in accordance with an embodiment of the invention.
[0079] The transmission of Plasmodium berghei ANKA (Pb) from infected mice to Anopheles stephensi mosquitoes was examined to investigate the in vivo efficacy of Torin 2. After Pb infection, the mice were treated with either Torin 2 or a vehicle control (Fig. 2A), and mosquitoes were then allowed to feed on the infected mice. Oocyst production in these mosquitoes was used as an indication of malaria transmission. It was found that oocyst production in mosquitoes was completely blocked by the treatment of two 4 mg/kg doses of Torin 2 (Fig. 2B). To further evaluate the dose dependence, a single 2 or 4 mg/kg dose of , -
36
Torin 2 was tested in the same mouse model (Fig. 2C). A single dose of 2 mg/kg of Torin 2 significantly reduced oocyst production, while a single 4 mg/kg dose almost completely eliminated it. These results clearly demonstrate the ability of Torin 2 to completely block gametocyte transmission from infected mice to mosquitoes.
EXAMPLE 5
[0080] This example demonstrates the identification of potential molecular targets of Torin 2 in accordance with an embodiment of the invention.
[0081] The lack of an mTOR homologue in P. falciparum [32] and significant difference in the potencies of Torin 1 and Torin 2 against the parasites suggest the presence of distinct targets in the parasites. It was hypothesized that Torin 2 selectively interacts with an unknown P. falciparum protein (or proteins) that has a weaker binding affinity to Torin 1. To develop a probe for an affinity based pull-down experiment, a Torin derivative, WWH030, was synthesized, and the importance of the ortho-piperazine-amide on the (trifluoromethyl)- benzene of Torin 2 for its gametocytocidal activity was determined. The structures of Torin 2, Torin 1, and WWH030 are depicted in Fig. 5. The new derivative had an IC5o of 9 nM, similar to that of Torin 2 in the gametocyte assay. This result indicates that the ortho- piperazine-amide group on Torin 2 can be modified without a significant effect on its gametocytocidal activity. Therefore, T2M was synthesized as an affinity resin for the pulldown experiment for identification of Torin 2 interacting proteins in P. falciparum gametocyte lysates. The structure of T2M is shown as compound 10a in Example 7. A negative control resin, TIM, was similarly synthesized with a close analog of Torin 1, shown as compound 10b in Example 7.
[0082] The proteins precipitated from gametocyte lysate by T2M but not TIM were identified by mass spectrometric analysis [33]. The proteomics data revealed a total of 31 proteins selectively enriched by T2M. The results are set forth in Table 5. In parallel to the probe-protein precipitation experiment, a DARTS experiment [34] was also carried out to identify Torin 2 binding proteins by limited protease digestion of Torin 2-treated gametocyte lysates. Following treatment with either Torin 2 or the negative control Torin 1, gametocyte lysates were partially digested with pronase and size fractionated by SDS-PAGE. Four significant protein bands were enriched in the Torin 2-treated sample compared to the Torin 1 -treated sample and analyzed by mass spectrometry. After comparing with the results from the affinity precipitation experiment, it was found that phosphoribosylpyrophosphate -
37
synthetase (PF3D7_1325100, ribose-phosphate diphosphokinase), aspartate
carbamoyltransferase (PF3D7_1344800, ATCase), and a putative transporter
(PF3D7_0914700) were identified by both experiments. Thus, these three gametocyte proteins are potential drug targets for Torin 2 and they will need to be further confirmed by enzyme assays and binding assays using recombinant P. falciparum proteins.
Table 5. Predicted Torin 2 interacting proteins in gametocytes by mass spectrometry experiment.
Figure imgf000039_0001
-
38
Deoxyribodipyrimidine photolyase
(photoreactivating enzyme, DNA PF3D7_051360C 129 kDa 2 photolyase), putative
Multidrug resistance protein PF3D7_052300C 162 kDa 2 (MDR1)
Rifin PF3D7_0632700 42 2 (RIF) kDa
Sin3 associated polypeptide pi 8-like PF3D7_071 140C 88 2 protein kDa
Merozoite surface protein 1 (MSP1) PF3D7_093030C 196 kDa 2
Glycoprotease, putative PF3D7J 030600 70 2 kDa
Conserved Plasmodium protein, PF3D7J 14280C 35 2 unknown function kDa
Plasmodium exported protein (PHISTc), PF3D7_1 14870C 44 2 unknown function (GEXP12) kDa
Conserved Plasmodium protein, PF3D7J20890C 167 kDa 2 unknown function
DEAD/DEAH box ATP-dependent RNA PF3D7J25150C 83 2 helicase, putative kDa
Aspartate carbamoyltransferase PF3D7J 344800 43 2
(atcasE) kDa
Conserved Plasmodium protein, PF3D7J 349600 36 2 unknown function kDa
Alanyl-tRNA synthetase,Alanine~tRNA PF3D7 J 367700 165 kDa 2 ligase (AlaRS)
RNA binding protein, putative PF3D7J 454000 59 2 kDa
Conserved Plasmodium membrane protein, PF3D7J47460C 46 2 unknown function kDa
Plasmodium exported protein (PHISTb), PF3D7_040210C 68 1 unknown function kDa
60S ribosomal protein L4, putative PF3D7_050710C 46 1 kDa ATP synthase subunit beta, PF3D7J2357O0 58 1 mitochondrial kDa
[0083] Note: Protein bands in both positive (Torin 2 pull-down) and negative (Torin 1 pull-down) samples were destained, reduced, and digested for mass spectrum. The mass spectrum data were analyzed by SEQUEST using PlasmoDB genomic
database(http://www.plasmodb.org). Proteins with more than 1 unique peptide in positive samples and 0 unique peptide in negative samples were considered as Torin 2 selective interacting proteins.
EXAMPLE 6
[0084] This example demonstrates the synthesis of compounds in accordance with an embodiment of the invention.
0085] General procedure for the synthesis of compound 2:
Figure imgf000041_0001
[0086] Aldehydes 1 were prepared using a reported procedure (J Med. Chem. 201 1 , 54(5): 1473-1480). A solution of 1 (300 μιηοΐβ) in 3 mL of THF were added 300 of Et2N Pr and R'CH2COCl (3000 μηιοΐε). The mixture was heated in a microwave between 100 to 150 °C for 15 min. The crude product was purified by column chromatography on silica gel using dichloromethane in methanol (0-20%) as eluent to give 1'. A mixture of 1 ' (1.0 equiv), boronic acid or boronic acid pinacol ester (3.0 equiv),
tetrakis(triphenylphosphine)palladium (0.05 equiv), DMF (1.5 mL) and saturated NaHC03 aqueous solution (0.5 mL) was charged in a microwave vial. Nitrogen was bubbled through the mixture for 3 min. The vial was capped and heated in a microwave at 120-150 °C for 15 -
40
min. The reaction mixture was filtered through a plug of Celite and the filtrate was purified by reverse phase column chromatography using acetonitrile (containing 0.1% TFA)/water (containing 0.1% TFA) as an eluent to give 2.
[0087] 9-(2-Aminopyrimidin-5-yl)-l-(3- (trifluoromethyl)phenyl)benzo [h]
Figure imgf000042_0001
[0088] Ή NMR (400 MHz, DMSO-i¾) δ 9.15 (s, IH), 8.34 (d, J = 9.5Hz, IH), 8.08-8.10 (m, 2H), 8.04 - 7.81 (m, 6H), 6.91-6.99 (m, 4H); LC/MS (Method A): (electrospray +ve), mlz 434.1 (MH)+, tR =1.61 min, UV254 = 98%.
[0089] 9-(Pyrimidin-5-yl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin- 2(lH)-one
Figure imgf000042_0002
[0090] Ή NMR (400 MHz, DMSO-i/6) δ 9.24 (s, IH), 9.17 (s, IH), 8.55 (s, 2H), 8.37 (d, J = 9.5 Hz, IH), 8.24 - 8.10 (m, 3H), 8.05 - 7.98 (m, IH), 7.95 - 7.80 (m, 2H), 7.05 - 6.95 (m, 2H); LC/MS (Method A): (electrospray +ve), mlz 419.1 (MH)+, tR = 1.84 min, UV254 = 98 %.
[0091] 9-(Pyridin-3 -y 1)- 1 -(3 -(trifluoromethy l)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)- one
Figure imgf000042_0003
[0092] LC/MS (Method A): (electrospray +ve), mlz 418.1 (MH)+, tR = 1.51 min, UV254 = 98%. 9-(Pyridin-4-yl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin-2(lH)-
Figure imgf000043_0001
[0094] 1H NMR (400 MHz, DMSO- ¾) δ 9.22 (s, 1H), 8.59 (dd, J= 4.8, 1.6 Hz, 1 H), 8.43 - 8.33 (m, 2H), 8.23 - 7.80 (m, 6H), 7.57 - 7.41 (m, 2H), 7.07 - 6.93 (m, 2H); LC/MS (Method A): (electrospray +ve), mlz 418.1 (MH)+, tR = 1.65 min, UV254 = 100%.
[0095] 9-Phenyl- 1 -(3 -(trifluoromethyl)phenyl)benzo [h] [1 ,6] naphthyridin-2( 1 H)-one
Figure imgf000043_0002
[0096] Ή NMR (400 MHz, DMSO- 6) δ 9.19 (s, 1H), 8.35 (d, J= 9.4 Hz, 1H), 8.20 -
8.09 (m, 2H), 8.09 - 8.00 (m, 2H), 7.88 (dd, J= 8.4, 7.4 Hz, 1H), 7.84 - 7.76 (m, 1H), 7.42 -
7.32 (m, 3H), 7.15 - 7.05 (m, 3H), 6.96 (d, J= 9.4 Hz, 1H); LC/MS (Method A):
(electrospray +ve), mlz 417.1 (MH)+, tR = 2.39 min, UV25 = 95%.
[0097] 9-(4-Aminophenyl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin-
2(lH)-one
Figure imgf000043_0003
[0098] LC/MS (Method A): (electrospray +ve), mlz 432.1 (MH)+, tR
=95 %.
[0099] 9-(6- Amino-4-(trifluoromethyl)pyridin-3 -yl)-l-(3- (trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000044_0001
[0100] LC/MS (Method B): (electrospray +ve), m/z 501.1 (MH)+, tR
=95 %.
[0101] 9-(2-amino-4-methylpyrimidin-5-yl)- 1 -(3- (trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000044_0002
[0102] LC/MS (Method B): (electrospray +ve), m/z 448.1(MH)+, tR
=100 %.
[0103] N-(5-(2-Oxo-l-(3-(trifluoromethyl)phenyl)-l ,2- dihydrobenzo[h][l ,6]naphthyridin-9-yl)pyridin-2-yl)acetamide
Figure imgf000044_0003
[0104] LC/MS (Method B): (electrospray +ve), m/z 475.1 (MH)\ tR = 4.43 min, UV254 =98 %.
[0105] 9-(lH-Indazol-5-yl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin- 2(lH)-one
Figure imgf000044_0004
-
43
[0106] LC/MS (Method B): (electrospray +ve), miz 457.1 (MH)+, tR = 4.68 min, UV254 =95 %.
[0107] 9-(l H-Indol -5 -yl)- 1 -(3 - (trifluoromethy l)pheny l)benzo [h] [ 1 ,6] naphthyridin-2( 1 H)- one
Figure imgf000045_0001
[0108] LC/MS (Method B): (electrospray +ve), miz 456.1 (MH)+, tR
=95 %.
[0109] 9-(lH-Benzo[d]imidazol-6-yl)-l-(3- (trifluoromethyl)phenyl)benzo [h] [1 ,6] naphthyridin-2( 1 H)-one
Figure imgf000045_0002
[0110] LC/MS (Method B): (electrospray +ve), miz 457.1 (MH)+, tR = 3.75 min, UV254 =90 %.
[0111] 9-(l H-Pyrazol-4-yl)- 1 -(3-(trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin- 2(l H)-one
Figure imgf000045_0003
[0112] LC/MS (Method B): (electrospray +ve), miz 407.1 (MH)+, tR
=100 %.
[0113] 9-(6-(Piperidin- 1 -yl)pyridin-3-yl)- 1 -(3- (trifluoromethyl)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one -
Figure imgf000046_0001
[0114] H NMR (400 MHz, DMSO-c¾) δ 9.21 (s, 1H), 8.35 (d, J = 9.5 Hz, 1H), 8.15 - 7.99 (m, 4H), 7.99 - 7.81 (m, 3H), 7.17 (dd, J = 9.2, 2.5 Hz, 1H), 7.04 - 6.91 (m, 3H), 3.72 - 3.58 (m, 4H), 1.71 - 1.50 (m, 6H); LC/MS (Method B): (electrospray +ve), mlz 501.2(MH)+, tR = 4.31 min, UV254 =95 %.
[0115] 9-(6-(Dimethylamino)pyridin-3-yl)-l-(3- (trifluoromethyl)phenyl)benzo [h] [1 ,6] naphthyridin-2( 1 H)-one
Figure imgf000046_0002
[0116] LC/MS (Method B): (electrospray +ve), mlz 562.1 (MH)+, tR
=90 %.
[0117] N-(4-(2-Oxo-l-(3-(trifluoromethyl)phenyl)-l ,2- dihydrobenzo[h] [ 1 ,6]naphthyrid -9-yl)phenyl)methanesulfonamide
Figure imgf000046_0003
[0118] 1H NMR (400 MHz, DMSO-t/6) δ 9.90 (s, 1H), 9.20 (s, 1H), 8.35 (d, J = 9.5 Hz, 1H), 8.21 - 7.93 (m, 4H), 7.96 - 7.67 (m, 2H), 7.34 - 7.13 (m, 2H), 7.13 - 6.91 (m, 4H), 3.02 (s, 3H); LC/MS (Method B): (electrospray +ve), mlz 510.1(MH)+, tR = 4.49 min, UV254 =90
%.
[0119] 9-(2-(4-Acetylpiperazin- 1 -yl)pyrimidin-5-yl)- 1 -(3 - (trifluoromethyl)phenyl)benzo [h] [1 ,6] naphthyridin-2( 1 H)-one
Figure imgf000047_0001
[0120] 1H NMR (400 MHz, DMSO-t¾) δ 9.20 (s, 1H), 8.35 (d, J = 9.5 Hz, 1H), 8.15 - 7.99 (m, 4H), 7.96 - 7.80 (m, 2H), 7.18 (dd, J = 9.0, 2.6 Hz, 1H), 7.00 - 6.93 (m, 2H), 6.86 (d, J = 9.0 Hz, 1H), 3.52-3.64 (m, 8H), 2.06 (s, 3H); LC/MS (Method B): (electrospray +ve), mlz 545.2(MH)+, tR = 3.86 min, UV254 =98 %.
[0121] 9-(3 - Aminophenyl)- 1 -(3 -(trifluoromethyl)pheny l)benzo [h] [ 1 ,6]naphthyridin- 2(lH)-one
Figure imgf000047_0002
[0122] 1H NMR (400 MHz, DMSO-ifc) δ 9.12 (s, 1H), 8.31 (d, J = 9.4 Hz, 1H), 8.07 (d, J = 8.6 Hz, 2H), 8.04 (s, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.87 (s, 1H), 7.79 (s, 1H), 7.03 (d, J = 1.9 Hz, 1H), 6.94 (d, J= 7.8 Hz, 1H), 6.93 - 6.90 (m, 1H), 6.52 (dd, J = 7.8, 2.2 Hz, 1H), 6.44 (d, J = 2.1 Hz, 1 H), 6.04 - 5.99 (m, 1H), 5.04 (s, 2H); LC/MS (Method B): (electrospray +ve), m/z 432.1 (MH)+, tR = 4.045, UV254 = 100%
[0123] 9-(4-(Dimethylamino)phenyl)-l-(3- (trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin-2(lH)-one
Figure imgf000047_0003
[0124] LC/MS (Method B): (electrospray +ve), m/z 460.1 (MH)+, tR = 4.780, UV254 = 100%
[0125] 1 -(3 -(Trifluoromethyl)phenyl)-9-vinylbenzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one _ -
Figure imgf000048_0001
[0126] Ή NMR (400 MHz, DMSO-<¾) δ 9.12 (s, IH), 8.31 (d,J=9.4Hz, IH), 8.06 (s, IH), 8.04 (s, IH), 8.00 (d,J=8.6Hz, IH), 7.90 (t,J= 8.1 Hz, IH), 7.82 (dd,J=8.5, 1.8 Hz, 2H), 6.93 (d, J= 9.4 Hz, IH), 6.58 (d, J= 1.7 Hz, IH), 6.25 (dd, J= 17.6, 10.9 Hz, IH), 5.35 (d, J= 17.6 Hz, IH), 5.19 (d, J= 10.9 Hz, IH); LC/MS (Method B): (electrospray +ve), m/z 367.1 (MH)+, tR= 4.837, UV254 = 100%
[0127]
[0128] 4-(2-oxo-l-(3-(Trifluoromethyl)phenyl)-l,2-dihydrobenzo[h][l,6]naphthyridin-9- yl)benzamide
Figure imgf000048_0002
[0129] Ή NMR (400 MHz, DMSO-d6) δ 9.17 (s, IH), 8.34 (d, J= 9.5 Hz, IH), 8.15 (d, J = 2.0 Hz, IH), 8.13 (d,J=8.7Hz, IH), 8.06 (dd,J=8.7, 1.9 Hz, 2H), 8.02 (s, IH), 7.90- 7.79 (m, 4H), 7.40 (s, IH), 7.16 (d, J= 8.4 Hz, 2H), 7.11 (d,J= 1.9 Hz, IH), 6.94 (d,J=9.4 Hz, IH); LC/MS (Method B): (electrospray +ve), m/z 460.1 (MH)+, tR= 4.142, UV254 = 100%
[0130] l,9-Bis(3-(trifluoro 6]naphthyridin-2(lH)-one
Figure imgf000048_0003
[0131] Ή NMR (400 MHz, DMSO-^e) δ 9.20 (s, IH), 8.34 (d, J= 9.2 Hz, IH), 8.16 (d, J = 8.6 Hz, IH), 8.09 (s, IH), 8.06 (d, J= 8.4 Hz, IH), 7.96 (d, J= 7.3 Hz, IH), 7.87 (q, J = 8.8, 8.4 Hz, 2H), 7.72 (d, J= 7.8 Hz, IH), 7.61 (t, J= 7.8 Hz, IH), 7.48 (d, J= 8.0 Hz, IH), -
47
7.24 (s, 1H), 7.08 (s, 1H), 6.96 (s, 1H); LC/MS (Method B): (electrospray +ve), m/z 485.1 (MH)+, tR = 5.847, UV254 = 88%
[0132] 3-(2-Oxo-l-(3-(trifluoromethyl)phenyl)-l ,2-dihydrobenzo[h][l,6]naphthyridin-9- yl)benzonitrile
Figure imgf000049_0001
[0133] LC/MS (Method B): (electrospray +ve), m/z 442.1 (MH)+, tR = 5.148, UV254 = 100%
[0134] l -(3-(Trifluoromethyl)phenyl)-9-(6-(trifluoromethyl)pyridin-3- yl)benzo[h][l ,6]naphthyridin-2(l -one
Figure imgf000049_0002
[0135] LC/MS (Method B): (electrospray +ve), m/z 486.1 (MH)+, tR = 5.431 , UV254 = 100%
[0136] 9-(4-(lH-Tetrazol-5-yl)phenyl)-l -(3- (trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin-2(lH)-one
Figure imgf000049_0003
[0137] LC/MS (Method B): (electrospray +ve), m/z 485.1 (MH)+, tR = 4.470, UV254 = 100%
[0138] 9-(4-Chlorophenyl)- 1 -(3-(trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin- 2(lH)-one -
Figure imgf000050_0001
[0139] LC/MS (Method B): (electrospray +ve), m/z 451.0 (MH)+, tR = 5.762, UV254 = 100%
[0140] 9-(4-Methoxyphenyl)- 1 -(3-(trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin- 2(lH)-one
Figure imgf000050_0002
[0141] LC/MS (Method B): (electrospray +ve), m/z 447.1 (MH)+, tR = 5.306, UV254 = 100%
[0142] 3 -(2-Oxo- 1 -(3 -(trifluoromethyl)phenyl)- 1 ,2-dihydrobenzo [h] [ 1 ,6]naphthyridin-9- yl)benzamide
Figure imgf000050_0003
[0143] Ή NMR (400 MHz, DMSO- 6) δ 9.17 (s, 1H), 8.34 (d, J = 9.3 Hz, 1H), 8.14 (d, J
= 8.6 Hz, 1H), 8.10 (s, 1 H), 8.09 - 8.05 (m, 1H), 8.00 (d, J = 6.9 Hz, 2H), 7.87 (t, J = 7.9 Hz,
1H), 7.80 (d, J = 7.5 Hz, 3H), 7.41 (d, J = 2.9 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.05 (d, J =
1.9 Hz, 1H), 6.98 (d, J = 7.9 Hz, 1H), 6.94 (d, J = 9.4 Hz, 1H); LC/MS (Method B):
(electrospray +ve), m/z 459.7 (MH)+, tR = 4.196, UV254 = 100%
[0144] 9-(3-Acetylphenyl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l,6]naphthyridin-
2(lH)-one -
Figure imgf000051_0001
[0145] 1HNMR(400MHz,DMSO-i6)69.18(s, 1H), 8.34 (d, J= 9.5 Hz, 1H), 8.16- 8.12 (m, 2H), 8.09 (d, J= 2.0 Hz, 2H), 7.90 (dd, J= 17.4, 8.1 Hz, 3H), 7.81 (d, J= 8.0 Hz, 1H), 7.23 (d, J= 8.0 Hz, 2H), 7.13 (d, J= 1.9 Hz, 1H), 6.95 (d, J= 9.5 Hz, 1H), 2.59 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 458.7 (MH)+, tR= 5.044, UV254 = 100%
[0146] 9-(2,3-Dihydrobenzofuran-6-yl)-l-(3- (trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000051_0002
[0147] Ή NMR (400 MHz, DMSO-i¾) 59.11 (s, 1H), 8.32 (d, J= 9.3 Hz, 1H), 8.14 (s, 1H), 8.06 (dt, J= 8.2, 2.9 Hz, 3H), 7.94 (d, J= 8.9 Hz, 2H), 7.87 (t, J= 8.1 Hz, 1H), 7.78 (d, J= 8.0 Hz, 1H), 7.72 (d, J= 2.2 Hz, 1H), 6.99 (s, 1H), 6.91 (td, J= 6.0, 2.6 Hz, 3H), 6.83 (s, 1H), 6.74 - 6.67 (m, 2H); LC/MS (Method B): (electrospray +ve), m/z 458.8 (MH)+, tR = 5.155, UV254 = 100%
[0148] 9-(Benzo[d][l,3]dioxol-5-yl)-l-(3- (trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2(l H)-one
Figure imgf000051_0003
[0149] LC/MS (Method B): (electrospray +ve), m/z 461.1 (MH)+, tR= 5.147, UV254 100%
[0150] 9-(4-Fluorophenyl)- 1 -(3-(trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin- 2(lH)-one -
Figure imgf000052_0001
[0151] 1H NMR (400 MHz, DMSO- 6) δ 9.15 (s, 1H), 8.33 (d, J= 9.4 Hz, 1H), 8.13 (s, 1H), 8.10 (d, J= 8.2 Hz, 1H), 8.06 (d, J= 8.6 Hz, 1H), 7.98 (d, J= 8.1 Hz, 1H), 7.90 - 7.84 (m, 1H), 7.79 (d, J= 8.1 Hz, 1H), 7.21 - 7.15 (m, 2H), 7.11 (dd, J= 8.6, 5.5 Hz, 2H), 6.98 (s, 1H), 6.94 (d, J= 9.4 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 435.1 (MH)+, tR = 5.400, UV254 = 100%
[0152] 9-(4-Hydroxyphenyl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l,6]naphthyridin- 2(lH)-one
Figure imgf000052_0002
[0153] Ή NMR (400 MHz, DMSO-i¾) δ 9.63 (s, 1H), 9.11 (s, 1H), 8.31 (d,J=9.6Hz, 1H), 8.14 (s, 1H), 8.05 (d, J= 8.5 Hz, 2H), 7.93 (d, J= 8.8 Hz, 1H), 7.86 (t, J= 7.9 Hz, 1H), 7.75 (d, J= 8.1 Hz, 1H), 6.99 (s, 1H), 6.93 (d, J= 3.7 Hz, 2H), 6.90 (s, 1H), 6.71 (d, J= 8.0 Hz, 2H); LC/MS (Method B): (electrospray +ve), m/z 433.1 (MH)+, tR= 4.410, UV254 = 100%
[0154] 9-(4-Morpholinophenyl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l,6]naphthyridin- 2(lH)-one
Figure imgf000052_0003
[0155] LC/MS (Method B): (electrospray +ve), m/z 502.1 (MH)+, tR= 4.921, UV254 = 100%
[0156] 9-( 1 -Methyl- 1 H-indol-5-yl)- 1 -(3- (trifluoromethyl)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one -
Figure imgf000053_0001
[0157] Ή NMR (400 MHz, DMSO- 6) δ 9.15 (s, 1H), 8.34 (d, J= 9.4 Hz, 1H), 8.17 (s, 1H), 8.11 (d, J= 5.8 Hz, 1H), 8.10 (s, 1H), 8.07 (d, J= 9.0 Hz, 1H), 7.89 (t, J= 7.9 Hz, 1H), 7.81 (d, J= 8.0 Hz, 1H), 7.40 (d, J= 8.6 Hz, 1H), 7.38 (d, J= 3.2 Hz, 1H), 7.18 (d, J= 10.1 Hz, 2H), 6.99 (s, 1H), 6.95 (d, J= 9.4 Hz, 1H), 6.45 (d, J= 3.1 Hz, 1H), 3.81 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 470.1 (MH)+, tR= 5.239, UV25 = 100%
[0158] 9-(Benzo[b]thiophen-5-yl)-l-(3- (trifluoromethyl)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000053_0002
[0159] Ή NMR (400 MHz, DMSO-<fc) δ 9.20 (s, 1H), 8.35 (d, J= 9.6 Hz, 1H), 8.15 (d, J
= 8.5 Hz, 3H), 8.10 (d, J= 9.6 Hz, 2H), 7.98 (d, J= 8.4 Hz, 1H), 7.91 (t, J= 7.9 Hz, 1H),
7.83 (t, J= 7.1 Hz, 2H), 7.49 (s, 1H), 7.45 (d, J= 5.4 Hz, 1H), 7.17 (s, 1H), 7.14 (d, J= 8.5
Hz, 1H), 6.95 (d, J= 9.2 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 472.7 (MH)+, tR= 5.647, UV254 = 100%
[0160] 9-(4-(Methylsulfonyl)phenyl)-l-(3-
(trifluoromethyl)phenyl)benzo [h]
Figure imgf000053_0003
[0161] H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.41 (d, J= 9.5 Hz, 1H), 8.22 (d, J = 8.7 Hz, 1H), 8.20 - 8.15 (m, 2H), 8.14 (d, J- 9.3 Hz, 1H), 7.95 (d, J= 6.4 Hz, 3H), 7.89 (d, J= 7.9 Hz, 1H), 7.38 (d, J= 8.0 Hz, 2H), 7.17 (s, 1H), 7.02 (d, J= 9.2 Hz, 1H), 3.29 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 494.6 (MH)+, tR= 4.636, UV254 = 100% .
52
[0162] 9-(Benzo[c][l ,2,5]oxadiazol-5-yl)-l-(3- (trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin-2(lH)-one
Figure imgf000054_0001
[0163] LC/MS (Method B): (electrospray +ve), m/z 458.7 (MH)+, tR = 5.404, UV254 = 100%
[0164] 9-(6-(Piperidin-l -yl)pyridin-3-yl)-l-(3- (trifluoromethyl)phenyl)benzo [
Figure imgf000054_0002
[0165] LC/MS (Method B): (electrospray +ve), m/z 501.1 (MH)+, tR = 4.339, UV254 = 100%
[0166] 9-(2-(Dimethylamino)pyrimidin-5-yl)-l-(3- (trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000054_0003
[0167] Ή NMR (400 MHz, DMSO-i/6) δ 9.14 (s, 1H), 8.32 (d, J= 9.5 Hz, 1H), 8.08 (d, J = 8.0 Hz, 4H), 8.00 (d, J = 7.9 Hz, 1H), 7.96 (d, J= 8.7 Hz, 1H), 7.89 (t, J= 7.9 Hz, 1H), 7.80 (d, J= 8.0 Hz, 1H), 6.93 (d, J= 9.4 Hz, 1H), 6.89 (s, 1H), 3.14 (s, 6H); LC/MS (Method B): (electrospray +ve), m/z 462 A (MH)+, tR = 4.774, UV254 = 00%
[0168] 9-(6-Methylpyridin-3-yl)-l-(3- (trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2(lH)-one -
Figure imgf000055_0001
[0169] Ή NMR (400 MHz, DMSO-<¾) δ 9.20 (s, 1H), 8.34 (d, J- 9.4 Hz, 1H), 8.32 (s, 1H), 8.15 (d,J=8.7Hz, 1H), 8.11 (s, 1H), 8.06 (d,J=8.8Hz, 1H), 8.03 (d,J=7.5 Hz, 1H), 7.91 - 7.81 (m, 2H), 7.43 (d, J= 8.3 Hz, 1H), 7.37 (d, J= 8.3 Hz, 1H), 7.02 (s, 1H), 6.96 (d, J = 9.4 Hz, 1H), 2.53 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 432.1 (MH)+, tR = 3.741, UV254 = 100%
[0170] 4-(2-Oxo-l-(3-(trifluoromethyl)phenyl)-l,2-dihydrobenzo[h][l,6]naphthyridin-9- yl)benzoic acid
Figure imgf000055_0002
[0171] 1H NMR (400 MHz, DMSO-t¾) δ 13.06 (s, 1H), 9.18 (s, 1H), 8.34 (d, J= 9.3 Hz, 1H), 8.16 (s, 1H), 8.14(d,J=9.0Hz, 1H), 8.10 (d, J= 7.8 Hz, 1H), 8.06 (dd,J=8.8, 1.5 Hz, 1H), 7.90 (d, J= 8.0 Hz, 2H), 7.87 (d, J= 7.9 Hz, 1H), 7.80 (d, J= 8.0 Hz, 1H), 7.20 (d, J = 8.0 Hz, 2H), 7.12 (s, 1H), 6.95 (d, J= 9.4 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 461.1 (MH)+, tR= 4.491, UV254 = 100%
[0172] 4-(2-Oxo- 1 -(3-(trifluoromethyl)phenyl)- 1 ,2-dihydrobenzo [h] [ 1 ,6]naphthyridin-9- yl)benzenesulfonamide
Figure imgf000055_0003
[0173] 1H NMR (400 MHz, DMSO-c¾) δ 9.18 (s, 1H), 8.34 (d, J= 9.3 Hz, 1H), 8.15 (d, J = 9.0 Hz, 2H), 8.07 (dd,J=8.8, 1.8 Hz, 2H), 7.89 (t, J- 7.9 Hz, 1H),7.81 (d,J=7.1 Hz, 1H), 7.78 (d, J= 8.4 Hz, 2H), 7.42 (s, 2H), 7.26 (d, J= 8.1 Hz, 2H), 7.13 (d, J= 1.9 Hz, 1H), .
54
6.95 (d, J= 9.5 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 496.0 (MH)+, tR = 3.985, UV254 = 100%
[0174] N,N-Dimethyl-4-(2-oxo-l-(3-(trifluoromethyl)phenyl)-l,2- dihydrobenzo[h] [ 1 ,6]naphthyridi -9-yl)benzamide
Figure imgf000056_0001
[0175] Ή NMR (400 MHz, DMSO-i 6) δ 9.18 (s, 1H), 8.34 (d, J= 9.4 Hz, 1H), 8.16 -
8.1 1 (m, 2H), 8.09 - 8.03 (m, 2H), 7.89 (s, 1H), 7.81 (s, 1H), 7.37 (d, J= 7.9 Hz, 2H), 7.13
(d, J= 7.8 Hz, 2H), 7.08 (s, 1H), 6.95 (d, J= 9.3 Hz, 1H), 2.95 (d, J = 31.7 Hz, 6H); LC/MS
(Method B): (electrospray +ve), m/z 488.1 (MH)+, tR = 4.543, UV254 = 100%
[0176] 9-([l,r-Biphenyl]-4-yl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin-
2(lH)-one
Figure imgf000056_0002
[0177] 1H NMR (400 MHz, DMSO-i¾) δ 9.16 (s, 1H), 8.34 (d, J= 9.4 Hz, 1H), 8.16 (s, 1H), 8.14 - 8.10 (m, 2H), 8.07 (d, J= 8.7 Hz, 1H), 7.90 (t, J= 7.9 Hz, 1H), 7.81 (d, J= 7.8 Hz, 1H), 7.69 (d, J= 7.6 Hz, 2H), 7.65 (d, J= 8.0 Hz, 2H), 7.49 (t, J= 7.6 Hz, 2H), 7.39 (t, J = 7.4 Hz, 1H), 7.19 (d, J= 8.0 Hz, 2H), 7.13 (s, 1H), 6.94 (d, J= 9.4 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 493.1 (MH)+, tR = 6.137, UV254 = 100%
[0178] 9-(p-Tolyl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l ,6]naphthyridin-2(lH)-one
Figure imgf000056_0003
[0179] 1H NMR (400 MHz, DMSO-c¾) δ 9.14 (s, 1H), 8.32 (d, J= 9.5 Hz, 1H), 8.14 (s, 1H), 8.09 (d, J= 8.7 Hz, 1H), 8.05 (d, J= 7.9 Hz, 1H), 7.99 (d, J= 8.7 Hz, 1H), 7.86 (t, J= 7.9 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 7.07 (s, 1H), 6.99 (d, J = 7.8 Hz, 2H), 6.93 (d, J = 9.4 Hz, 1H), 2.31 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 431.1 (MH)+, tR = 5.546, UV2S4 = 100%
[0180] 9-(5-Chloropyridin-3-yl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l,6]naphthyridin- 2(lH)-one
Figure imgf000057_0001
[0181] LC/MS (Method B): (electrospray +ve), m/z 451.7 (MH)+, tR = 5.071 , UV254 = 100%
[0182] 9-(3-(Pyrrolidin- 1 -yl)phenyl)- 1 -(3- (trifluoromethyl)phenyl)benzo [h
Figure imgf000057_0002
[0183] LC/MS (Method B): (electrospray +ve), m/z 485.8 (MH)+, tR = 5.719, UV254 = 100%
[0184] 9-(4-(Piperazine-l-carbonyl)phenyl)-l -(3- (trifluoromethyl)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000057_0003
[0185] LC/MS (Method B): (electrospray +ve), m/z 528.7 (M), tR
[0186] 9-(4-(Morpholine-4-carbonyl)phenyl)- 1 -(3 - (trifluoromethyl)phenyl)benzo [h] [1 ,6] naphthyridin-2( 1 H)-one -
Figure imgf000058_0001
[0187] LC/MS (Method B): (electrospray +ve), m/z 529.7 (MH)+, tR = 4.496, UV254 = 100%
[0188] 9-(6-Hydroxypyridin-3-yl)-l-(3- (trifluoromethyl)phenyl)benzo[h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000058_0002
[0189] 1H NMR (400 MHz, DMSO-ifc) δ 11.94 (s, IH), 9.12 (s, IH), 8.32 (d, J= 9.4 Hz,
IH), 8.09 (s, 2H), 8.03 (s, IH), 7.91 (d, J= 8.2 Hz, 2H), 7.82 (d, J= 8.0 Hz, IH), 7.26 (s,
IH), 7.02 (d,J= 10.0 Hz, IH), 6.92 (s, IH), 6.80 (s, IH), 6.31 (d,J=9.5 Hz, IH); LC/MS
(Method B): (electrospray +ve), m/z 433.7 (MH)+, tR= 3.808, UV254 = 100%
[0190] 3-(2-Oxo-l-(3-(trifluoromethyl)phenyl)-l,2-dihydrobenzo[h][l,6]naphthyridin-9- yl)benzenesulfonamide
Figure imgf000058_0003
[0192] 1H NMR (400 MHz, DMSO-i¾) δ 9.18 (s, IH), 8.33 (d, J= 9.5 Hz, IH), 8.17 (d, J = 8.6 Hz, IH), 8.07 (s, IH), 8.02 (d, J= 7.6 Hz, IH), 7.98 (dd, J= 8.6, 2.0 Hz, IH), 7.88 (t, J = 7.7 Hz, IH), 7.83 (d,J=8.3 Hz, IH), 7.79 (d,J=7.4 Hz, 2H), 7.51 (t,J=8.0Hz, IH), 7.36 (s, 2H), 7.02 - 6.99 (m, 2H), 6.94 (d, J= 9.4 Hz, IH); LC/MS (Method B): (electrospray +ve), m/z 496.1 (MH)+, tR= 4.315, UV254 = 100%
[0193] 9-( 1 -Oxo- 1,2,3 ,4-tetrahydroisoquinolin-6-yl)- 1 -(3 - (trifluoromethyl)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one -
Figure imgf000059_0001
[0194] Ή NMR (400 MHz, DMSO- 6) 59.17 (s, 1H), 8.33 (d, J= 9.4 Hz, 1H), 8.14 - 8.11 (m, 2H), 8.09 (d, J= 7.8 Hz, 1H), 8.04 (dd, J= 8.7, 1.9 Hz, 1H), 7.95 (d, J= 2.8 Hz, 1H), 7.90 (t,J=7.9 Hz, 1H), 7.82 (d,J= 1.9 Hz, 1H), 7.79 (d,J=8.1 Hz, 1H), 7.11 (d,J= 1.9 Hz, 1H), 7.03 (dd,J = 8.0, 1.9 Hz, 1H), 6.97 (d,J= 1.8 Hz, 1H), 6.94 (d,J= 9.5 Hz, 1H), 3.39 (td, J= 6.7, 2.8 Hz, 4H); LC/MS (Method B): (electrospray +ve), m/z 486.1 (MH)+, tR = 4.359, UV254 = 100%
[0195] 9-(2-Oxo-l,2,3,4-tetrahydroquinolin-6-yl)-l-(3- (trifluoromethyl)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000059_0002
[0196] Ή NMR (400 MHz, DMSO-<¾) δ 10.13 (s, 1H), 9.13 (s, 1H), 8.31 (d,J=9.5Hz, 1H), 8.11 (d,J=2.1 Hz, 1H), 8.07 (d,J=6.0Hz, 1H), 8.05 (d,J=5.7Hz, 1H), 7.95 (dd,J = 8.7, 1.9 Hz, 1H), 7.89 (t,J= 7.9 Hz, 1H), 7.78 (dd,J=7.7, 1.8 Hz, 1H), 7.03 (d,J=1.9 Hz, 1H), 6.93-6.91 (m, 1H), 6.90 (d,J=6.1 Hz, 1H), 6.82 - 6.80 (m, 2H), 3.43 (s, 4H); LC/MS (Method B): (electrospray +ve), m/z 486.1 (MH)+, tR = 4.287, UV254 = 100%
[0197] 9-(4-(Aminomethyl)phenyl)-l-(3- (trifluoromethyl)phenyl)benzo[h] [1 ,6]naphthyridin-2(l H)-one
Figure imgf000059_0003
[0198] 1H NMR (400 MHz, DMSO-<¾) δ 9.16 (s, 1H), 8.33 (d, J = 9.4 Hz, 1H), 8.17 (d, J = 1.9 Hz, 1H), 8.12 (d, J = 8.7 Hz, 3H), 8.03 (dd, J = 8.7, 1.9 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.85 (t, J = 7.8 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.16 - 7.13 -
58
(m, 2H), 7.11 (d, J = 1.9 Hz, 1H), 6.94 (d, J = 9.4 Hz, 1H), 4.05 (d, J
(Method B): (electrospray +ve), m/z 446.1 (MH)+, tR = 3.630, UV254
[0199] 9-(4-((Dimethylamino)methyl)phenyl)-l-(3- (trifluoromethyl)phenyl)benzo[h][l,6]naphthyridin-2(lH)-one
Figure imgf000060_0001
[0200] Ή NMR (400 MHz, DMSO-cfe) δ 9.17 (s, 1H), 8.33 (d, J= 9.5 Hz, 1H), 8.17 (s, 1H), 8.13 (d, J= 8.6 Hz, 1H), 8.03 (dd, J= 8.7, 1.9 Hz, 1H), 7.98 (d, J= 7.8 Hz, 1H), 7.86 (t, J= 7.9 Hz, 1H), 7.79 (d, J= 7.9 Hz, 1H), 7.46 (d, J= 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 7.08 (d, J= 1.9 Hz, 1H), 6.94 (d, J= 9.4 Hz, 1H), 4.29 (d, J= 5.2 Hz, 2H), 2.72 (d, J= 4.6 Hz, 6H); LC/MS (Method B): (electrospray +ve), m/z 474.1 (MH)+, tR= 3.772, UV254 = 100%
[0201 ] N-Methyl-4-(2-oxo- 1 -(3 -(trifluoromethyl)phenyl)- 1,2- dihydrobenzo[h][l,6]naphthyrid -9-yl)benzamide
Figure imgf000060_0002
[0202] Ή NMR (400 MHz, DMSO-c6) δ 9.18 (s, 1H), 8.47 (d, J= 4.7 Hz, 1H), 8.33 (d, J = 9.4 Hz, 1H), 8.14 (s, 1H), 8.12 (d, J= 8.8 Hz, 1H), 8.07 - 8.03 (m, 2H), 7.86 (d, J= 7.8 Hz, 1H), 7.82 - 7.78 (m, 3H), 7.15 (d, J= 8.3 Hz, 2H), 7.10 (d, J= 1.9 Hz, 1H), 6.94 (d, J= 9.4 Hz, 1H), 2.78 (d, J - 4.4 Hz, 3H); LC/MS (Method B): (electrospray +ve), m/z 474.1 (MH)+, tR= 4.339, UV254 = 100%
[0203] 9-(2-Oxoindolin-5-yl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l,6]naphthyridin- 2(lH)-one
Figure imgf000060_0003
-
59
[0204] Ή NMR (400 MHz, DMSO-i¾) δ 10.47 (s, IH), 9.14 (s, IH), 8.31 (d, J = 9.5 Hz,
IH), 8.1 1 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 8.7 Hz, IH), 7.95 (dd, J = 8.7, 2.0 Hz, IH), 7.86 (d,
J = 7.9 Hz, IH), 7.78 (d, J = 7.9 Hz, IH), 6.99 (d, J = 1.9 Hz, IH), 6.97 - 6.94 (m, I H), 6.93
(d, J = 9.5 Hz, IH), 6.84 (s, IH), 6.76 (d, J = 8.1 Hz, IH), 3.48 (q, J = 22.3 Hz, 3H); LC/MS
(Method B): (electrospray +ve), m/z 472.0 (MH)+, tR = 4.214, UV254 = 100%
[0205] 9-(l-Oxoisoindolin-5-yl)-l-(3-(trifluoromethyl)phenyl)benzo[h][l,6]naphthyridin-
2(lH)-one
Figure imgf000061_0001
[0206] Ή NMR (400 MHz, DMSO- 6) δ 9.18 (s, IH), 8.61 (s, IH), 8.33 (d, J = 9.4 Hz, IH), 8.15 - 8.12 (m, 2H), 8.1 1 (d, J = 7.4 Hz, IH), 8.05 (dd, J = 8.7, 2.0 Hz, IH), 7.87 (t, J = 7.8 Hz, IH), 7.80 (d, J = 8.0 Hz, IH), 7.62 (d, J = 8.3 Hz, IH), 7.20 (s, IH), 7.19 (d, J = 3.8 Hz, IH), 7.09 (d, J = 1.9 Hz, IH), 6.94 (d, J = 9.5 Hz, IH), 4.44 - 4.31 (m, 2H); LC/MS (Method B): (electrospray +ve), m/z 472.1 (MH)+, tR = 4.204, UV254 = 100%
[0207] 2,4-Difluoro-N-(2-methoxy-5-(2-oxo-l-(3-(trifluoromethyl)phenyl)-l ,2- dihydrobenzo[h][l ,6]naphthyridin-9-yl)pyridin-3-yl)benzenesulfonamide
Figure imgf000061_0002
[0208] LC/MS (Method B): (electrospray +ve), m/z 638.6 (MH)+, tR = 5.26 min, UV254 = 95%.
[0209] 1 -( 1 - Acetylpiperidin-4-yl)-9-(6-aminopyridin-3-yl)benzo [h] [ 1 ,6]naphthyridin- 2(lH)-one ,
Figure imgf000062_0001
[0210] LC/MS (Method B): (electrospray +ve), mlz 414.2 (MH)+, tR = 2.90 min, UV254 98%.
[0211 ] 9-(6- Aminopyridin-3 -yl)-3 -methyl- 1 - (3 - (trifluoromethyl)phenyl)benzo [h]
Figure imgf000062_0002
[0212] LC/MS (Method B): (electrospray +ve), mlz 447.1 (MH)+, tR = 3.77 min, UV254 85%.
[0213] 9-(6-Aminopyridin-3-y -l -cyclopropylbenzo[h][l ,6]naphthyridin-2(lH)-one
Figure imgf000062_0003
[0214] LC/MS (Method B): (electrospray +ve), mlz 329.1 (MH)+, tR = 2.79 min, UV254 = 95%.
[0215] 9-(6-Aminopyridin-3-yl)-l-(tetrahydro-2H-pyran-4-yl)benzo[h][l ,6]naphthyridin- 2(lH)-one
Figure imgf000062_0004
[0216] LC/MS (Method B): (electrospray +ve), mlz 373.1 (MH)+, tR = 2.90 min, UV254 = 90%.
[0217] 9-(6- Aminopyridin-3 -y 1)- 1 -cyclohexylbenzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000063_0001
[0218] LC/MS (Method B): (electrospray +ve), mlz 371.2 (MH)+, tR = 3.45 min, UV254 80%.
[0219] 9-(6-Aminopyridin-3-y - 1 -cyclopentylbenzo[h] [ 1 ,6]naphthyridin-2(l H)-one
Figure imgf000063_0002
[0220] LC/MS (Method B): (electrospray +ve), mlz 357.2 (MH)+, tR
95%.
[0221] 9-(6-Aminopyridin-3-yl)-3-ethyl-l-(3- (trifluoromethyl)phenyl)benzo [
Figure imgf000063_0003
[0222] LC/MS (Method B): (electrospray +ve), mlz 460.8 (MH)+, tR
90%.
[0223] General procedure for the synthesis of compound 7:
-
62
Figure imgf000064_0001
Reagents and conditions: (i) POCi3, DMF; (ii) triethyl phosphonoacetate, EtOH; K2C03, r.t. (iii) R2NH2, 120-180 °C, 5-20 min; (iv) K2C03, EtOH, MW, 150 °C, 15 min; (v) R B(OR')2, Pd(Ph3P)4, NaHC03, DMF/water, MW, 120-150 °C, 15 min.
[0224] A two-necked flask equipped with a stirrer, condenser, and rubber septum was charged with dry DMF (300 mL) at 0 °C under a nitrogen atmosphere. Phosphoryl chloride (100 mL, 1002.4 mmol) was added dropwise to the flask at 0 °C. The mixture was allowed to warm up to room temperature and stirred at this temperature for 30 min. A solution of 3 (25.1 g, 1 17.3 mmol) in DMF (100 mL) was added dropwise and the mixture was heated at 60 °C for 4 h under nitrogen. The cooled mixture was added to crush ice and then neutralized with saturated NaHC03 solution. The solid was collected by filtration. The crude product was dissolved in dichloromethane and the solution was washed with water, dried over MgS04, filtered, and concentrated to give 4 (15.2 g, 48%) as a solid. 1H NMR (400 MHz,
Chloroform-; ) δ 10.69 (s, 1H), 9.26 (s, 1H), 8.54 (d, J=2.1 Hz, 1H), 8.04 (d, J=12.4Hz, 1H), 7.98 (dd, J=l 1.8, 2.1Hz, 1H); LC/MS (Method B): (electrospray +ve), mlz 269.9(MH)+, tR = 5.60 min, UV254 =98 %.
[00100] To a solution of 4 (5.04 g, 18.7 mmol) in 300 mL of ethanol were added triethyl phosphonoacetate (6.28 g, 28.0 mmol) and K2C03 (12.90 g, 93.0 mmol). The mixture was stirred at room temperature for overnight. The solid was filtered, washed with water and ethanol, and dried under vacuum to give 5 (4.41 g, 69% ) as a solid. LC/MS (Method B):
(electrospray +ve), mlz 340.0 (MH)+, tR = 6.52 min, UV254 =100 %.
[0225] A mixture of 5 (0.3 mmol) and amines (0.6 mmol) was heated at 120-180 °C for 5-20 min. After cooled down to room temperature, ethanol (5 mL) and K2C03 (1.8 mmol) were added. The mixture was heated at 80 °C for overnight. Ethyl acetate (50 mL) was added to the reaction and the mixture was washed with water (2x30 mL). The organic layer was -
63
dried over MgS04, filtered, and concentrated. The crude product was purified by column chromatography on silica gel using dichloromethane in methanol (0-20%) as eluent to give 6.
[0226] A mixture of 6 (0.2 mmol, 1.0 equiv), boronic acid or boronic acid pinacol ester (0.6 mmol, 3.0 equiv), tetrakis(triphenylphosphine)palladium (12 mg, 0.01 mmol, 0.05 equiv), DMF (2.5 mL) and saturated NaHC03 aqueous solution (0.5 mL) was charged in a microwave vial. Nitrogen was bubbled through the mixture for 3 min. The vial was capped and heated in a microwave at 120-150 °C for 15 min. The reaction mixture was filtered through a plug of Celite and the filtrate was purified by reverse phase column
chromatography using acetonitrile (containing 0.1% TFA)/water (containing 0.1% TFA) as an eluent to give 7.
[0227] 3-(9-(6-Aminopyridin-3-yl)-2-oxobenzo[h][l ,6]naphthyridin-l(2H)-yl)-N,N- dimethylbenzamide
Figure imgf000065_0001
[0228] 1H NMR (400 MHz, DMSO-ifc) δ 9.18 (s, 1H), 8.34 (d, J= 9.5 Hz, 1H), 8.16 - 8.04 (m, 1H), 8.03 - 7.89 (m, 2H), 7.78 - 7.53 (m, 5H), 7.26 - 6.91 (m, 4H), 2.97 (s, 3H), 2.83 (s, 3H); LC/MS (Method B): (electrospray +ve), mlz 436.2(MH)+, tR = 3.04 min, UV254 =90 %.
[0229] 9-(6- Aminopyridin-3 -yl)- 1 -(3 -isopropylphenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)- one
Figure imgf000065_0002
[0230] LC/MS (Method B): (electrospray +ve), mlz 407.1(MH)+, tR = 3.58 min, UV254 =95 %.
[0231 ] 3 -(9-(6- Aminopyridin-3 -y l)-2-oxobenzo [h] [ 1 ,6]naphthyridin- 1 (2H)- yl)benzenesulfonamide -
64
Figure imgf000066_0001
[0232] Ή NMR (400 MHz, DMSO-c/6) δ 9.20 (s, 1H), 8.36 (d, J= 9.5 Hz, 1H), 8.20 - 7.92 (m, 7H), 7.85 (t, J = 7.9 Hz, 1H), 7.73 (ddd, J= 7.9, 2.1 , 1.1 Hz, 1H), 7.65 (s, 2H), 7.55 - 7.31 (m, 1H), 6.98 (dd, J= 9.3, 3.6 Hz, 2H), 6.87 (d, J= 1.9 Hz, 1H); LC/MS (Method B): (electrospray +ve), mlz 444.1(MH)+, tR = 2.93 min, UV254 =90 %.
[0233] 9-(6-Aminopyridin-3- -l-phenylbenzo[h][l ,6]naphthyridin-2(lH)-one
Figure imgf000066_0002
[0234] LC/MS (Method B): (electrospray +ve), mlz 365.1 (MH)+, tR = 3.17 min, UV254 =98 %.
[0235] 9-(6-Aminopyridin-3-yl)-l-(piperidin-4-ylmethyl)benzo[h][l ,6]naphthyridin- 2(lH)-one
Figure imgf000066_0003
[0236] LC/MS (Method B): (electrospray +ve), mlz 386.1 (MH)+, tR = 2.64 min, UV254 =98 %.
[0237] 9-(6- Aminopyridin-3 -yl)- 1 -ethylbenzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000066_0004
[0238] LC/MS (Method B): (electrospray +ve), mlz 317.1 (MH)+, tR
=98 %. -
65
[0239] 9-(6-Aminopyridin-3-yl)-l-(2-methyl-5- (trifluoromethyl)phenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000067_0001
[0240] LC/MS (Method B): (electrospray +ve), mlz 447.1(MH)+, tR = 3.76 min, UV254 =98 %.
[0241 ] 9-(6- Aminopyridin-3 - - 1 -(m-tolyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000067_0002
[0242] LC/MS (Method A): (electrospray +ve), mlz 379.1(MH)+, tR = 3.17 min, UV254 =98 %.
[0243] 9-(6- Aminopyridin-3 -yl)- 1 -(3 -chlorophenyl)benzo [h] [1,6] naphthyridin-2( 1 H)-one
Figure imgf000067_0003
[0244] Ή NMR (400 MHz, DMSO-i/6) δ 9.19 (s, 1H), 8.34 (d, J = 9.5 Hz, 1H), 8.14 (d, J
= 8.7 Hz, 1H), 8.07-8.13 (m, 2H), 8.00 (dd, J = 8.7, 2.0 Hz, 1H), 7.89 - 7.65 (m, 4H), 7.55 -
7.47 (m, 2H), 7.04 - 6.98 (m, 2H), 6.96 (d, J = 9.4 Hz, 1H); LC/MS (Method B):
(electrospray +ve), mlz 399.0(MH)+, tR = 3.40 min, UV254 =98 %.
[0245] 3-(9-(6-Aminopyridin-3-yl)-2-oxobenzo[h] [1 ,6]naphthyridin- 1 (2H)- yl)benzonitrile .
66
Figure imgf000068_0001
[0246] Ή NMR (400 MHz, DMSO-<¾) δ 9.20 (s, 1H), 8.36 (d, J = 9.5 Hz, 1H), 8.04-8.23 (m, 5H), 8.00 (dd, J = 8.7, 2.0 Hz, 1H), 7.94 - 7.82 (m, 2H), 7.80 (dd, J = 2.5, 0.8 Hz, 1H), 7.50 (dd, J = 9.3, 2.3 Hz, 1H), 6.99 (dd, J = 9.6, 8.5 Hz, 2H), 6.92 (d, J= 1.9 Hz, 1H);
LC/MS (Method B): (electrospray +ve), mlz 390.1(MH)+, tR = 3.10 min, UV254 =98 %.
[0247] 9-(6- Aminopyridin-3 -y 1)- 1 -(3 -methoxyphenyl)benzo [h] [ 1 ,6] naphthyridin-2( 1 H)- one
Figure imgf000068_0002
[0248] LC/MS (Method B): (electrospray +ve), mlz 395.1 (MH)+, tR = 3.20 min, UV254 =100 %.
[0249] 9-(6-Aminopyridin-3-yl)- 1 -(3 -phenoxyphenyl)benzo [h] [ 1 ,6]naphthyridin-2( 1 H)- one
Figure imgf000068_0003
[0250] LC/MS (Method B): (electrospray +ve), mlz 457.1(MH)+, tR = 3.81 min, UV254 =98 %.
[0251] 9-(6-Aminopyridin-3-yl)- 1 -(2-ethoxyphenyl)benzo[h] [ 1 ,6]naphthyridin-2( 1 H)- one -
Figure imgf000069_0001
[0252] LC/MS (Method B): (electrospray +ve), mlz 409.1 (MH)+, tR = 3.33 min, UV254 =98 %.
[0253] 9-(6-Aminopyridin-3 -yl)- 1 -(4-(piperazin- 1 -yl)-3 - (trifluoromethyl)phenyl)benzo [h] [1,6] naphthyridin-2( 1 H)-one
Figure imgf000069_0002
[0254] LC/MS (Method B): (electrospray +ve), mlz 517.2 (MH)+, tR = 3.18 min, UV254 =95 %.
[0255] 1 -(4-((4-Acetylpiperazin- 1 -yl)sulfonyl)phenyl)-9-(6-aminopyridin-3- yl)benzo[h] [1 ,6]naphthyridin-2( 1 H)-one
Figure imgf000069_0003
[0256] LC/MS (Method B): (electrospray +ve), mlz 555.2 (MH)+, tR = 3.21 min, UV254 =95 %.
EXAMPLE 7
[0257] This example demonstrates the synthesis of compounds in accordance with an embodiment of the invention. [0258] Ethyl 6-bromo-4-((4-(4-(tert-butoxycarbonyl)piperazi
(trifluoromethyl)phenyl)amino)quinoline-3-carboxylate (8)
Figure imgf000070_0001
[0259] A mixture of tert-butyl 4-(4-amino-2-(trifluoromethyl)phenyl)piperazine-l - carboxylate (9, 0.691 g, 2.00 mmol) and ethyl 6-bromo-4-chloroquinoline-3-carboxylate (10, 0.629 g, 2.00 mmol) in 20 mL of THF was heated in a microwave for 15 min at 120 oc. The reaction mixture was poured into 50 mL of EtOAc. The solution was washed twice with NaOH solution (1 N, 2x30 mL), dried over MgS0 , filtered and concentrated. The crude product was purified by column chromatography on silica gel using 7-60% EtOAc in hexanes as eluent to give 8 (0.935 g, 75.0%) as a solid. 1H NMR (400 MHz,
CHLOROFORM-c/) δ ppm 10.50 (s, 1 H), 9.28 (s, 1 H),7.89 (d, J=9.00 Hz, 1 H), 7.72 (dd, J=9.00, 1.96 Hz, 1 H), 7.63 (d, J=2.35 Hz, 1 H), 7.36 (d, J=2.35 Hz, 1 H), 7.28 (d, J=9.00 Hz, 1 H), 7.14 (dd, J=8.61 , 2.35 Hz, 1 H), 4.46 (q, J=7.30 Hz, 2 H), 3.53 - 3.62 (m, 4 H), 2.83 -2.91 (m, 4 H), 1.49 (s, 9 H), 1.47 (t, J=7.30 Hz, 3 H); LC/MS: (electrospray +ve), m/z 623.1 (MH)+, tR = 5.90 min, UV254 = 00%.
[0260] ter/-Butyl 4-(4-((6-bromo-3-(hydroxymethyl)quinolin-4-yl)amino)-2- (trifluoromethyl)phenyl)piperazine-l-carboxylate (11)
[0261] To a solution of ethyl 6-bromo-4-((4-(4-(/ert-butoxycarbonyl)piperazin-l-yl)-3- (trifluoromethyl)phenyl)amino)quinoline-3-carboxylate (8, 1.24 g, 1.99 mmol) in 200 mL of ethanol was added NaBH4 (0.752 g, 19.9 mmol) at room temperature. After stirring for 4 h, the mixture was poured into 300 mL of EtOAc. The solution was washed with water (3x200 mL). The organic layer was dried over MgS04, filtered, and concentrated. The residue was purified by column chromatography through a CI 8 column using 5-100% ACN (containing 0.1% TF A)/ water (containing 0.1% TFA) as eluent. The combined pure fractions were neutralized using 1 N NaOH solution, extracted with ethyl acetate (3x50 mL). The organic layer was dried over MgS04, filtered, and concentrated to give 11 (342 mg,
0.59 mmol, 30%). 1H NMR (400 MHz, DMSO-<¾) δ 9.01 (s, 1H), 8.74 (s, 1H), 8.22 (d, J = 2.2 Hz, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.84 (dd, J = 8.9, 2.2 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 2.7 Hz, 1H), 6.80 (dd, J = 8.7, 2.7 Hz, 1H), 5.41 (t, J = 5.4 Hz, 1H), 4.43 (d, J = 5.5 Hz, 2H), 3.28-3.36 (m, 4H), 2.70-2.76 (m, 4H), 1.41 (s, 9H); LC/MS:
(electrospray +ve), mlz 581.1 (MH)+, tR = 5.23 min, UV254 >95%.
[0262] teri-Butyl 4-(4-((6-bromo-3 -formylquinolin-4-yl)amino)-2- (trifluoromethyl)phenyl)piperazine- 1 -carboxylate (12)
Figure imgf000071_0001
[0263] To a solution of tert-butyl 4-(4-((6-bromo-3-(hydroxymethyl)quinolin-4- yl)amino)-2- (trifluoromethyl)phenyl)piperazine-l -carboxylate (11, 335 mg, 0.576 mmol) in 30 mL of DCM was added Dess-Martin reagent (367 mg, 0.864 mmol). After stirring at room temperature for 2 h, the mixture was poured into 100 mL of ethyl acetate, washed with NaOH solution (1.0 N, 3x50 mL). The organic layer was dried over MgSo4, filtered, and concentrated. The crude product was purified on silica gel using 2-10% MeOH in DCM as eluent to give 12 (257 mg, 77%) as a solid. 1H NMR (400 MHz, DMSO-rfe) δ 10.38 (s, 1H), 10.01 (s, 1H), 8.91 (s, 1H), 8.16 (d, J = 2.1 Hz, 1H), 7.96- 7.83 (m, 2H), 7.57-7.46 (m, 2H), 7.40 (dd, J = 8.7, 2.6 Hz, 1H), 3.40-3.46 (m, 4H), 2.78- 2.82 (m, 4H), 1.42 (s, 9H); LC/MS: (electrospray +ve), m/z 579.1 (MH)+, tR = 5.54 min, Uv254 = 98%.
[0264] tert- uty\ 4-(4-(9-bromo-2-oxobenzo[h] [1 ,6]naphthyridin- 1 (2H)-yl)-2- (trifluoromethyl)phenyl)piperazi - 1 -carboxylate (13)
Figure imgf000071_0002
[0265] To a solution of tert-butyl 4-(4-((6-bromo-3-formylquinolin-4-yl)amino)-2- (trifluoromethyl)phenyl)piperazine-l -carboxylate (12, 251 mg, 0.434 mmol) and ethyl triethyl phosphonoacetate (146 mg, 0.650 mmol) in 3 mL of EtOH was added potassium carbonate (299 mg, 2.166 mmol). After heating at 150 °C for 15 min in a microwave, the reaction mixture was poured into 50 mL of EtOAc. The solution was washed with NaOH solution (1.0 N, 3x30 mL). The organic layer was dried over MgS04, filtered, and
concentrated. The crude product was purified by column chromatography on silica gel using 2-10% MeOH in DCM as eluent to give 13 (205 mg, 0.340 mmol, 78 %) as a solid. 1H NMR (400 MHz, DMSO-c ) δ 9.18 (s, 1H), 8.32 (d, J= 9.5 Hz, 1H), 8.00-7.84 (m, 3H), 7.76-7.83 (m, 2H), 6.96 (d, J = 9.4 Hz, 1H), 6.54 (d, J = 2.1 Hz, 1H), 3.48-3.53 (m, 4H), 2.96-3.32 (m, 4H), 1.44 (s, 9H); LC/MS: (electrospray +ve), m/z 603.1 (MH)+, tR = 6.46 min, UV254 = 98%.
[0266] teri-Butyl 4-(4-(9-(6-aminopyridin-3-yl)-2-oxobenzo[h][l ,6]naphthyridin-l(2H)- yl)-2- (trifluoromethyl)phenyl)piperazine- 1 -carboxylate (14a)
Figure imgf000072_0001
[0267] A mixture of 5-(4,4,5,5-tetramethyl-l ,3,2-dioxaborolan-2-yl)pyridin-2-amine (58.4 mg, 0.265 mmol), tert-butyl 4-(4-(9-bromo-2-oxobenzo[h][l ,6]naphthyridin-l(2H)-yl)- 2- (trifluoromethyl)phenyl)piperazine-l -carboxylate (13, 80.1 mg, 0.133 mmol) and
Pd(Ph3P)4 (15.3 mg, 0.013 mmol) in 3 mL of DMF and 0.6 mL of saturated Na2C03 aqueous solution was heated in a microwave for 10 min at 150 °C. The reaction mixture was filtered through a plug of celite and the filtrate was purified by column chromatography through CI 8 column using 5- 100% ACN (containing 0.1 %> TFA)/water (containing 0.1 % TFA) as eluent. The combined pure fractions were neutralized using 1 N NaOH solution, extracted with ethyl acetate (3x50 mL). The organic layer was dried over MgS04, filtered, and concentrated to give 14a (64.2 mg 78%). 'H NMR (400 MHz, DMSO-i 6) δ 9.09 (s, 1H), 8.30 (d, J = 9.5 Hz, 1H), 8.05 (d, J = 8.7 Hz, 1H), 8.00-7.96 (m, 2H), 7.81-7.67 (m, 3H), 7.10 (dd, J = 8.6, 2.6 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.91 (d, J = 9.4 Hz, 1H), 6.41 (dd, J = 8.6, 0.8 Hz, 1H), 6.18 (s, 2H), 3.47-3.53 (m, 4H), 2.86-2.94 (m, 4H), 1.45 (s, 9H). LC/MS: (electrospray +ve), m/z 617.2 (MH)+, tR = 4.63 min, UV254 = 95%.
[0268] ter t-Butyl 4-(4-((6-bromo-3 -formylquinolin-4-yl)amino)-2- (trifluoromethyl)phenyl)piperazine-l -carboxylate (14b)
Figure imgf000073_0001
[0269] A mixture of 3-quinolineboronic acid (46.2 mg, 0.267 mmol), tert-butyl 4-(4-(9- bromo-2- oxobenzo[h] [ 1 ,6]naphthyridin- 1 (2H)-yl)-2-(trifluoromethyl)phenyl)piperazine- 1 - carboxylate (13, 80.5 mg, 0.133 mmol) and Pd(Ph3P)4 (15.4 mg, 0.013 mmol) in 3 mL of DMF and 0.6 mL of saturated Na2C03 aqueous solution was heated in a microwave for
10 min at 150 °C. The reaction mixture was filtered through a plug of celite and the filtrate was purified by column chromatography through CI 8 column using 5-100% ACN (containing 0.1 % TFA)/water (containing 0.1% TFA) as eluent. The combined pure fractions were neutralized using 1 N NaOH solution, extracted with ethyl acetate (3x50 mL). The organic layer was dried over MgSC , filtered, and concentrated to give 14b (56.8 mg 65.3%). 1H NMR (400 MHz, DMSO-c 6) δ 9.25 (s, 1H), 8.61 -8.66 (m, 1H), 8.40 (d, J = 9.5 Hz, 1H), 8.34-8.14 (m, 3H), 8.10-7.99 (m, 3H), 7.90-7.69 (m, 4H), 7.13 (d, J = 1.8 Hz, 1H), 7.01 (d, J = 9.4 Hz, 1H), 3.33-3.38 (m, 2H), 3.21-3.26 (m, 2H), 2.67-2.56 (m, 2H), 2.46-2.52 (m, 2H), 1.47 (s, 9H); LC/MS: (electrospray+ve), m/z 652.2 (MH)+, tR = 5.96 min, UV254 = 95%.
[0270] ferr-Butyl 4-(4-((6-bromo-3-formylquinolin-4-yl)amino)-2- (trifluoromethyl)phenyl)piperazine-l -carboxylate (15c, WWH30)
Figure imgf000073_0002
[0271] To a solution of tert-butyl 4-(4-(9-(6-aminopyridin-3-yl)-2- oxobenzo [h] [ 1 ,6] naphthyridin- 1 (2H)- yl)-2-(trifluoromethyl)phenyl)piperazine- 1 -carboxylate (14a, 23.5 mg, 0.038 mmol) in 3 mL of dichloroethane was added 1 mL of TFA. The mixture was stirred at room temperature for 2 h. The solvent was removed and the residue was dissolved in 3 mL of MeOH. To this solution was added triethylamine (19.3 mg, 0.191 mmol) and propionyl chloride (7.1 mg, 0.076 mmol). The resulted mixture was stirred at room temperature for 4 h. The crude mixture was purified by column chromatography through C18 column using 5-100% ACN (containing 0.1% TFA)/water (containing 0.1% TFA) as eluent. The combined pure fractions were neutralized using 1 N aOH solution, extracted with ethyl acetate (3x50 mL). The organic layer was dried over MgS04, filtered, and concentrated to give 15c (16.7 mg, 77%). Ή NMR (400 MHz, DMSO- d6) 59.19 (s, 1H), 8.34 (d, J = 9.5 Hz, 1H), 8.13 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 2.2 Hz, 1H), 8.03-7.93 (m, 4H), 7.78-7.68 (m, 2H), 7.37 (dd, J= 9.3, 2.3 Hz, 1H), 7.00-6.84 (m, 3H), 3.63-3.80 (m, 2H), 3.47-3.57 (m, 2H), 2.87-3.00 (m, 2H), 2.83-2.67 (m, 2H), 2.39 (q, J = 7.4 Hz, 2H), 1.04 (t, J= 7.4 Hz, 3H). LC/MS: (electrospray +ve), mlz 573.2 (MH)+, tR = 3.85 min, UV254 = 100%.
[0272] tert-Butyl (15-(4-(4-(9-(6-aminopyridin-3-yl)-2-oxobenzo[h][l,6]naphthyridin- 1 (2H)-yl)-2- (trifluoromethyl)phenyl)piperazin- 1 -yl)- 15-oxo-3 ,6,9,12- tetraoxapentadecyl)carbamate (15a)
BocHN
Figure imgf000074_0001
[0273] To a solution of tert-butyl 4-(4-(9-(6-aminopyridin-3-yl)-2- oxobenzo[h] [ 1 ,6]naphthyridin- 1 (2H)- yl)-2-(trifluoromethyl)phenyl)piperazine- 1 -carboxylate (14a, 57.2 mg, 0.093 mmol) in 3 mL of dichloroethane was added 1 mL of TFA. The mixture was stirred at room temperature for 2 h. The solvent was removed under vacuum and the residue was dissolved in 5 mL of DMF. To this solution was added triethylamine (40 mg, 0.40 mmol), 2,2-dimethyl-4-oxo-3, 8,1 1 ,14,17- pentaoxa-5-azaicosan-20-oic acid (35.6 mg, 0.097 mmol), and HATU (51.7 mg, 0.1 1 1 mmol). The resulted mixture was stirred at room temperature for 6 h. The crude mixture was purified by column chromatography through C18 column using 5-100% ACN (containing 0.1% TFA)/water (containing 0.1% TFA) as eluent. The combined pure fractions were neutralized using 1 N NaOH solution, extracted with ethyl acetate (3x50 mL). The organic layer was dried over MgSC-4, filtered, and concentrated to give 15a (46.1 mg, 58%). 1H NMR (400 MHz, DMSO- d6) δ 9.09 (s, 1H), 8.30 (d, J= 9.4 Hz, 1H), 8.05 (d, J= 8.7 Hz, 1H), 7.99-7.87 (m, 2H), 7.79-7.69 (m, 3H), 7.14 (dd, J= 8.6, 2.6 Hz, 1H), 6.98-6.88 (m, 2H), 6.74 (t, J= 5.9 Hz, 1H),
6.42 (dd, J= 8.6, 0.8 Hz, 1H), 6.19 (s, 2H), 3.61 -3.70 (m, 5H), 3.45-3.55 (m, 10H), 3.23- 3.38 (m, 5H), 2.87-3.07 (m, 5H), 2.63-2.70 (m, 3H), 1.36 (s, 9H); LC/MS: (electrospray +ve), mlz 864.3 (MH)+, tR = 4.40 min, UV254 = 100%.
[0274] tert-Butyl (15-oxo-15-(4-(4-(2-oxo-9-(quinolin-3-yl)benzo[h][l,6]naphthyridin-
1 (2H)-yl)-2- (trifluoromethyl)phenyl)piperazin- 1 -yl)-3 ,6,9, 12-tetraoxapentadecyl)carbamate (15b)
[0275] To a solution of fert-butyl 4-(4-((6-bromo-3-formylquinolin-4-yl)amino)-2- (trifluoromethyl)phenyl)piperazine-l-carboxylate (14b, 50.6 mg, 0.078 mmol) in 3 mL of dichloroethane was added 1 mL of TFA. The mixture was stirred at room temperature for
2 h. The solvent was removed under vacuum and the residue was dissolved in 5 mL of DMF. To this solution was added triethylamine (19.3 mg, 0.191 mmol), 2,2-dimethyl-4-oxo-
3,8,1 1,14,17- pentaoxa-5-azaicosan-20-oic acid (29.8 mg, 0.082 mmol), and HATU (35.4 mg, 0.093 mmol). The resulted mixture was stirred at room temperature for 6 h. The crude mixture was purified by column chromatography through CI 8 column using 5-100% ACN (containing 0.1% TFA)/water (containing 0.1% TFA) as eluent. The combined pure fractions were neutralized using 1 N NaOH solution, extracted with ethyl acetate (3x50 mL). The organic layer was dried over MgS04, filtered, and concentrated to give 15a
(42.2 mg, 60%). 1H NMR (400 MHz, DMSO-c¾) δ 9.21 (s, 1H), 8.59 (d, J = 2.3 Hz, 1H), 8.36 (d, J = 9.5 Hz, 1H), 8.28 (dd, J = 2.3, 0.8 Hz, 1H), 8.25-8.13 (m, 2H), 8.10-7.96 (m, 3H), 7.63-7.90 (m, 4H), 7.13 (d, J = 1.7 Hz, 1H), 6.97 (d, J = 9.4 Hz, 1H), 6.73 (t, J = 5.5 Hz, 1H), 3.71-3.43 (m, 19H), 3.35 (t, J = 6.0 Hz, 1H), 3.04 (q, J = 6.0 Hz, 2H), 2.67-2.74 (m, 3H), 2.51 -2.62 (m, 3H), 1.36 (s, 9H); LC/MS: (electrospray +ve), mlz 899.3 (MH)+, tR =
5.43 min, UV254 >95%.
[0276] Polymer linked Torin 2 (16a)
Figure imgf000075_0001
-
74
[0277] To a mixture of Affi-Gel 10 (Bio-Rad Laboratories, cat. no. 153-6046), 3 mL gel, 45 μιτιοΐ) and 15a (12.9 mg, 15 μπιοΐε) in 10 mL of DMSO was added triethylamine (150 μπιοι). The mixture was shaken at room temperature for 6 h (15a disappeared from solution based on LC-MS analysis). Then ethanolamine (300 μηιοΐ) was added and the resulted mixture was shaken at room temperature for overnight. After washing with DMSO and PBS, the polymer linked Torin 2 (16a) was stored in PBS (containing 0.1% sodium azide) at 4 °C.
[0278] Polymer linked Torin 1 (16b) was prepared in a similar manner as polymer linked Torin 2.
Figure imgf000076_0001
EXAMPLE 8
[0279] This example demonstrates an In vitro drug activity on gametocytes.
[0280] Stage III-V gametocytes were enriched with treatment with 50 mM N- acetylglucosamine (NAG) and Percoll density gradient centrifugation as described
previously1. Briefly, 2.5 μΐ/well incomplete medium was dispensed into each well of 1,536- well plates using the Multidrop Combi followed by 23 nl compound transfer using the NX- TR Pintool (WAKO Scientific Solutions, San Diego, CA). Then, 2.5 μΐ/well of gametocytes was dispensed with a seeding density of 20,000 cells/well using the Multidrop Combi. The assay plates were incubated for 72 h at 37 °C with 5% C02. After addition of 5 μΐ/well of 2X AlamarBlue dye (Life Technologies, cat. no. DALl 100), the plates were incubated for 24 h at 37 °C with 5% C02 and then were read in a fluorescence detection mode (Ex = 525 nm, Em = 598 nm) on a ViewLux plate reader (PerkinElmer).
EXAMPLE 9
[0281] This example demonstrates In vitro drug activity on asexual parasites in accordance with an embodiment of the invention. -
75
[0282] Asexual parasites of P. falciparum strain 3D7 were cultured as described previously (Trager, W. et al., J. Parasitol. 2005, 91(3): 484-486). Drug activity on asexual stage parasites was tested using a SYBR Green assay as described previously (Eastman, R.T. et al., Antimicrob. Agents Chemother. 2013, 57(1): 425-435; Smilkstein, M. et al,
Antimicrob. Agents Chemother. 2004, 48(5): 1803-1806). Briefly, parasites were diluted to 0.5% parasitemia in complete culture medium with 2% hematocrit and drugs diluted in DMSO (<0.5%) and were loaded into a 96-well plate (200 μΐ/well). No drug and RBC alone wells were included as positive and background controls, respectively, and each testing condition was examined in duplicated. After 72 h incubation under the standard culture condition and a freeze-thaw lysis step at -80°C and room temperature, 100 μΐ/well of lysis buffer containing SYBR Green I was added to the parasite culture and incubated for 30 min at room temperature. The fluorescence of each well was measured at 520 nm following excitation at 490 nm using a FLUOstar Optima microplate reader (BMG Labtech).
EXAMPLE 10
[0283] This example demonstrates the efficacy of NVP-AUY922 and Alvespimycin on gametocyte transmission from host to mosquitoes in a mouse model in accordance with an embodiment of the invention.
[0284] The experiment described in Example 4 was conducted using NVP-AUY922 and Alvespimycin in a two dose protocol at 8 mg/kg as test compounds. The protocol is depicted graphically in Fig. 6A. The oocyte number for vehicle, NVP-AUY922, and
Alvespimycin-treated mice are depicted in Fig. 6B. The structures of NVP-AUY922 and Alvespimycin are depicted in Fig. 6C.
EXAMPLE 1 1
[0285] This example demonstrates the synthesis of compounds in accordance with an embodiment of the invention.
[0286] A reaction scheme for the synthesis of compounds 13 and 14 is as follows: - 6
Figure imgf000078_0001
18 19 20
Reagents and conditions, (i) 150 °C, 1 ,4-dioxanes; (ii) SnCI2, 65 °C, 3.5 h; (iii) CIC02CCI3, Et3N; (iv) Mel, NaH.THF
[0287] 6-Bromo-3-nitro-N-(3-(trifluoromethyl)phenyl)quinolin-4-amine
Figure imgf000078_0002
[0288] A solution of 6-bromo-4-chloro-3-nitroquinoline (1 g, 3.48 mmol) in 1,4-dioxane (1 1 mL) at room temperature was treated with 3-(trifluoromethyl)aniline (0.434 mL, 3.48 mmol). The mixture was allowed to heat at 150 °C for 2 hours and monitored via LC-MS for completion. The reaction mixture was treated with brine and extracted with ethyl acetate (3x). The organic layers were collected, dried, filtered, and concentrated. Purification by Si02 chromatography (0-50% Hex/EA) afforded the desired product as an off-white solid (1.32 g, 3.2 mmol, 92 %). LC/MS (Method A): (electrospray +ve), m/z 412.1 (MH)+, tR = 3.706, UV254 = 100%.
[0289] 6-Bromo-N4-(3 -(trifluoromethyl)phenyl)76quinoline-3 ,4-diamine
Figure imgf000079_0001
[0290] A solution of 6-bromo-N4-(3-(trifluoromethyl)phenyl)quinoline-3,4-diamine (1.3 0 g, 3.15 mmol) in ethyl acetate (12 mL) was treated with tin (II) chloride (3.56 g, 15.7 mmol) at room temperature. The mixture was heated at 65 °C for 3.5 hours and monitored via LC-MS for completion. The reaction mixture was cooled to room temperature and treated with 10N NaOH (20 mL). The mixture was filtered over Celite, and the filtrate was diluted with deionized water and extracted with ethyl acetate (3x). The organic layers were collected, dried, filtered and concentrated. Purification via Si02 chromatography (0-10% DCM-MeOH) afforded the desired product as a yellow solid (1.15 g, 3.01 mmol, 95 %). 1H NMR (400 MHz, DMSO-t¾) δ 8.63 (s, 1H), 8.21 (s, 1H), 7.83 (d, J = 2.2 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.46 (dd, J = 8.9, 2.1 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 6.82 (s, 1H), 6.66 (dd, J = 8.2, 2.2 Hz, 1H), 5.55 (s, 2H); LC/MS (Method A): (electrospray +ve), m/z 383.1 (MH)+, tR = 3.1 12, UV254 = 100%.
[0291] 8-Bromo-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5-c]quinolin-2(3H)-one
Figure imgf000079_0002
[0292] A solution of 6-bromo-N4-(3 -(trifluoromethyl)phenyl)quinoline-3 ,4-diamine (1.15 g, 3.01 mmol) and triethylamine (0.586 mL, 4.21 mmol) in dichloromethane (35 mL) was cooled to 0 °C. To the solution, trichloromethyl chloroformate (0.465 mL, 3.85 mmol) in a solution of dichloromethane (35 mL) was added. The mixture stirred for 30 minutes at 0 °C and monitored via LC-MS for completion. The reaction mixture was quenched with brine and extracted with dichloromethane (3x). The organic layers were combined, dried, filtered, and concentrated. Purification via Si02 chromatography (0-10% DCM-MeOH) afforded the desired product as a pale yellow solid (0.821 g, 2.012 mmol, 66.9 %). Ή NMR (400 MHz, DMSO-c/6) 1 1.89 (s, 1H), 8.80 (s, 1H), 8.15 - 8.09 (m, 1H), 8.04 (dt, J= 8.2, 1.5 Hz, 1H), 7.98 (dt, J = 8.0, 1.7 Hz, 1H), 7.94 (dd, J = 8.6, 7.1 Hz, 2H), 7.65 (dd, J = 9.0, 2.2 Hz, 1H), -
78
7.02 (d, J = 2.2 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 408.0 (MH)+, tR = 4.458, UV254 = 100%.
[0293] 8-Bromo-3-methyl-l -(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5-c]quinolin- 2(3H)-one
Figure imgf000080_0001
[0294] A room temperature solution of 8-bromo-l-(3-(trifluoromethyl)phenyl)-lH- imidazo[4,5-c]quinolin-2(3H)-one (0.40 g, 0.98 mmol) in tetrahydrofuran (7 mL) under N2 was treated with iodomethane (0.073 mL, 1.176 mmol) followed by sodium hydride (70.6 mg, 2.94 mmol). The reaction mixture was allowed to stir at room temperature overnight and monitored via LC-MS. The reaction was quenched with ammonium chloride and extracted with dichloromethane. The organic layers were combined, dried, filtered, and concentrated. Purification via Si02 chromatography (0-10% DCM-MeOH) afforded the desired product as an orange solid (0.315 g, 0.747 mmol, 76 %).Ή NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.1 1 (d, J = 2.2 Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.97 (dd, J = 8.7, 3.4 Hz, 2H), 7.92 (t, J = 7.9 Hz, 1H), 7.66 (dd, J = 9.0, 2.2 Hz, 1H), 7.02 (d, J = 2.2 Hz, 1H), 3.58 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 421.9 (MH)+, tR = 3.060, UV254 = 100%
[0295 General procedure fo the synthesis of 21:
Figure imgf000080_0002
19 or 20 21
[0296] A mixture of 19 or 20 (200 umole, 1.0 equiv), boronic acid or boronic acid pinacol ester (2.0 equiv), tetrakis(triphenylphosphine)palladium (0.05 equiv), DMF (1.5 mL) and saturated NaHC03 aqueous solution (0.5 mL) was charged in a microwave vial. Nitrogen was bubbled through the mixture for 3 min. The vial was capped and heated in a microwave at 120-150 °C for 40 min. The reaction mixture was filtered through a plug of Celite and the . _ _
79
filtrate was purified by reverse phase column chromatography using acetonitrile (containing 0.1% TFA)/water (containing 0.1% TFA) as an eluent to give 21.
[0297] 8-(4-Aminophenyl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5-c]quinolin- 2(3H)-one
Figure imgf000081_0001
[0298] Ή NMR (400 MHz, DMSO- 6) δ 12.20 (s, 1H), 8.91 (s, 1H), 8.25 (d, J = 2.0 Hz,
1H), 8.12 - 8.09 (m, 1H), 8.07 (d, J= 9.1 Hz, 1H), 8.06 - 8.02 (m, 1H), 7.99 - 7.94 (m, 2H),
7.04 - 7.01 (m, 2H), 7.00 (d, J= 1.9 Hz, 1H), 6.60 - 6.55 (m, 2H); LC/MS (Method B):
(electrospray +ve), m/z 421.1 (MH)+, tR = 3.575, UV254 = 100%
[0299] 8-(6-Methylpyridin-3-yl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5- c] quinolin-2(3 H)-one
Figure imgf000081_0002
[0300] Ή NMR (400 MHz, DMSO-c ) δ 12.1 1 (s, 1H), 8.92 (s, 1H), 8.45 (d, J= 2.4 Hz, 1H), 8.22 (d, J = 2.0 Hz, 1H), 8.17 (d, J= 8.9 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.04 (d, J= 1.9 Hz, 1H), 8.01 (dd, J = 8.9, 2.0 Hz, 1H), 7.96 (d, J= 7.9 Hz, 1H), 7.74 (dd, J = 8.1 , 2.5 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 2.51 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 421.1 (MH)+, tR = 3.296, UV254 = 100%
[0301] N-(5-(2-Oxo-l-(3-(trifluoromethyl)phenyl)-2,3-dihydro-lH-imidazo[4,5- c]quinolin-8-yl)pyridin-2-yl)acetamide 1
Figure imgf000081_0003
[0302] H NMR (400 MHz, DMSO-<¾) δ 10.64 (s, IH), 8.77 (s, IH), 8.20 (d, J= 2.2 Hz, IH), 8.18 (dd, J= 2.6, 0.8 Hz, IH), 8.09 (d, J= 3.8 Hz, IH), 8.07 (d, J= 3.8 Hz, IH), 8.07 - 8.00 (m, 3H), 7.94 (d, J= 7.9 Hz, IH), 7.90 (dd, J= 8.9, 2.0 Hz, IH), 7.78 (dd, J= 8.7, 2.6 Hz, IH), 7.1 1 (d, J= 2.0 Hz, IH), 2.08 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 464.1 (MH)+, tR = 3.710, UV254 = 100%
[0303] 8-(4-Hydroxyphenyl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5-c]quinolin- 2(3H)-one
Figure imgf000082_0001
[0304] H NMR (400 MHz, DMSO-i¾) δ 1 1.75 (s, IH), 9.61 (s, IH), 8.72 (s, IH), 8.20 (s, IH), 8.08 - 7.96 (m, 3H), 7.92 (t, J= 7.8 Hz, IH), 7.79 (dd, J= 8.9, 2.1 Hz, IH), 7.17 - 7.10 (m, 2H), 7.02 (d, J = 2.0 Hz, IH), 6.76 - 6.69 (m, 2H); LC/MS (Method B):
(electrospray +ve), m/z 422.1 (MH)+, tR = 3.910, UV254 = 100%
[0305] 8-(3-Aminophenyl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5-c]quinolin- 2(3H)-one
Figure imgf000082_0002
[0306] Ή NMR (400 MHz, DMSO-</6) δ 1 1.76 (s, IH), 8.73 (s, IH), 8.16 (s, IH), 8.03 (d, J= 8.8 Hz, IH), 8.02 - 7.98 (m, 2H), 7.93 (t, J= 7.8 Hz, IH), 7.72 (dd, J= 8.8, 2.1 Hz, IH), 7.09 (d, J= 2.0 Hz, IH), 6.95 (t, J= 7.8 Hz, IH), 6.62 (t, J= 2.0 Hz, IH), 6.51 (dd, J = 7.2, 2.3 Hz, IH), 6.35 - 6.32 (m, IH), 5.07 (s, 2H); LC/MS (Method B): (electrospray +ve), m/z 421.1 (MH)+, tR = 3.630, UV254 = 100%
[0307] 8-(6-Aminopyridin-3-yl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5- c]quinolin-2(3H)-one -
Figure imgf000083_0001
[0308] LC/MS (Method B): (electrospray +ve), m/z 421.8 (MH)+, tR = 3.09, UV254 = 100%
[0309] 8-(Pyridin-4-yl)- 1 -(3 -(trifluoromethyl)phenyl)- 1 H-imidazo [4,5 -c] quinolin-2(3 H)- one
Figure imgf000083_0002
[0310] 1H NMR (400 MHz, DMSO-c¼) δ 11.86 (s, IH), 8.81 (s, IH), 8.56 - 8.53 (m, 2H), 8.22 (s, IH), 8.13 (d, J = 8.9 Hz, IH), 8.05 (t, J= 9.6 Hz, 2H), 7.95 (d, J= 8.3 Hz, 2H), 7.31 - 7.28 (m, 2H), 7.25 (d, J = 2.1 Hz, IH); LC/MS (Method B): (electrospray +ve), m/z 407.1 (MH)+, tR = 3.1 16, UV254 = 100%
[0311] 8-(Pyridin-3-yl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5-c]quinolin-2(3H)- one
Figure imgf000083_0003
[0312] Ή NMR (400 MHz, DMSO-i¾) δ 1 1.93 (s, IH), 8.84 (s, IH), 8.53 (d, J = 4.3 Hz, IH), 8.51 (d, J = 2.6 Hz, IH), 8.20 (s, IH), 8.14 (d, J= 8.9 Hz, IH), 8.05 (s, IH), 8.02 (s, IH), 7.97 - 7.94 (m, IH), 7.94 - 7.90 (m, IH), 7.75 (dd, J= 7.9, 2.2 Hz, IH), 7.42 (dd, J = 7.9, 4.7 Hz, IH), 7.16 (d, J= 2.0 Hz, IH); LC/MS (Method B): (electrospray +ve), m/z 407.1 (MH)+, tR = 3.306, UV254 = 100%
[0313] 8-(lH-Benzo[d]irnidazol-5-yl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5- c]quinolin-2(3H)-one .
Figure imgf000084_0001
[0314] Ή NMR (400 MHz, DMSO-i¾) δ 12.52 (s, 1H), 1 1.78 (s, 1H), 8.74 (s, 1H), 8.21 (d, J= 13.4 Hz, 2H), 8.04 (dd, J= 15.0, 8.3 Hz, 3H), 7.91 (s, 2H), 7.65 - 7.43 (m, 2H), 7.17 (d, J= 2.0 Hz, 1H), 7.10 (dd, J= 21 A, 8.6 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 446.1 (MH)+, tR = 3.190, UV254 = 100%
[0315] 8-(4-(Methylsulfonyl)phenyl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5- c] quinolin-2(3 H)-one
Figure imgf000084_0002
[0316] 1H NMR (400 MHz, DMSO-ffc) 8 8.81 (s, 1H), 8.22 (d, J= 2.0 Hz, 1H), 8.13 (d, J = 8.8 Hz, 1H), 8.08 - 8.05 (m, 1H), 8.05 - 8.01 (m, 1H), 7.96 - 7.93 (m, 1H), 7.92 (d, J= 2.1 Hz, 1H), 7.92 - 7.89 (m, 2H), 7.59 - 7.55 (m, 2H), 7.22 (d, J= 2.0 Hz, 1H), 3.23 (s, 3H), 2.97 (d, J= 5.2 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 484.1 (MH)+, tR = 3.976, UV254 = 100%
[0317] 3 -(2-Oxo- 1 -(3 -(trifluoromethyl)phenyl)-2,3 -dihydro- 1 H-imidazo [4,5 -c] quinolin- 8-yl)benzonitrile
Figure imgf000084_0003
[0318] H NMR (400 MHz, DMSO-i^) δ 1 1.85 (s, 1H), 8.80 (s, 1H), 8.23 (s, 1H), 8.1 1 (d, J= 8.8 Hz, 1H), 8.03 (dd, J= 8.6, 1.5 Hz, 3H), 7.80 (dt, J= 7.6, 1.4 Hz, 1H), 7.69 (t, J= 1.7 Hz, 2H), 7.66 (dt, J= 8.0, 1.5 Hz, 1H), 7.59 (d, J= 7.7 Hz, 1H), 7.12 (d, J= 1.9 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 431.1 (MH)+, tR = 4.417, UV254 = 100% .
83
8 -(Quinolin-3 -yl)- 1 -(3 -(trifluoromethyl)phenyl)- 1 H-imidazo [4,5 -cjquinolin-
Figure imgf000085_0001
[0320] Ή NMR (400 MHz, DMSO-c/6) δ 1 1.85 (s, 1H), 8.82 (d, J = 2.4 Hz, 1H), 8.80 (s, 1H), 8.33 (d, J = 2.5 Hz, 1H), 8.22 (s, 1H), 8.16 (d, J = 8.9 Hz, 1H), 8.10 - 8.02 (m, 4H), 7.96 (d, J = 8.0 Hz, 1H), 7.95 - 7.91 (m, 1H), 7.77 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.66 (ddd, J = 8.1 , 6.9, 1.3 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 457.1 (MH)+, tR = 4.177, UV254 = 100%
[0321] 8-(2-Aminopyrimidin-5-yl)-l-(3-(trifluorornethyl)phenyl)-lH-irnidazo[4,5- c]quinolin-2(3H)-one
Figure imgf000085_0002
[0322] 1H NMR (400 MHz, DMSO-c¾) δ 1 1.78 (s, 1H), 8.73 (s, 1H), 8.20 (s, 2H), 8.16 (s, 1H), 8.03 (d, J = 8.9 Hz, 2H), 8.02 - 7.98 (m, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.83 (dd, J = 8.9, 2.1 Hz, 1H), 6.97 (d, J = 2.0 Hz, 1H), 6.87 (s, 2H).; LC/MS (Method B): (electrospray +ve), m/z 423.1 (MH)+, tR = 3.377, UV254 = 100%
[0323] 4-(2-Oxo-l-(3-(trifluoromethyl)phenyl)-2,3-dihydro-lH-imidazo[4,5-c]quinolin- 8-yl)benzamide
Figure imgf000085_0003
[0324] 'H NMR (400 MHz, DMSO-c¾) δ 1 1.86 (s, 1H), 8.80 (s, 1H), 8.22 (s, 1H), 8.10 (d, J = 8.9 Hz, 1H), 8.06 - 8.00 (m, 3H), 7.93 (d, J = 8.2 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), „
84
7.39 (s, 2H), 7.37 (s, 1H), 7.18 (d, J= 2:1 Hz, 1H); LC/MS (Method B): (electrospray +ve), m/z 449.1 (MH)+, tR = 3.540, UV254 = 100%
[0325] 8-(2-Aminopyrimidin-5-yl)-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5- c] quinolin-2(3 H)-one
Figure imgf000086_0001
[0326] Ή NMR (400 MHz, DMSO-cfe) δ 12.09 (s, 1H), 8.88 (s, 1H), 8.19 (s, 3H), 8.10 (d, J= 9.0 Hz, 1H), 8.07 (d, J= 7.0 Hz, 1H), 8.05 - 8.01 (m, 1H), 7.98 - 7.92 (m, 2H), 7.01 - 6.90 (m, 3H); LC/MS (Method B): (electrospray +ve), m/z 423.1 (MH)+, tR = 3.387, UV254 = 100%
[0327] 8-(4-Aminophenyl)-3-methyl-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5- c] quinolin-2(3 H)-one
Figure imgf000086_0002
[0328] 1H NMR (400 MHz, DMSO-i 6) δ 9.14 (s, 1H), 8.24 (s, 1H), 8.1 1 (d, J = 7.3 Hz, 1H), 8.08 (d, J = 9.1 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 2.0 Hz, 1H), 6.59 (d, J = 8.3 Hz, 2H), 3.61 (s, 4H), 2.51 (s, 1H); LC/MS (Method B): (electrospray +ve), m/z 435.1 (MH)+, tR = 3.774, UV254 = 100%
[0329]
[0330] 4-(3-Methyl-2-oxo-l-(3-(trifluoromethyl)phenyl)-2,3-dihydro-lH-imidazo[4,5- c] quinolin- 8 -yl)benzamide
Figure imgf000086_0003
. .
85
[0331] 1H NMR (400 MHz, DMSO-c¾) δ 9.12 (s, IH), 8.23 (s, IH), 8.16 (d, J = 8.9 Hz,
IH), 8.09 (d, J = 7.9 Hz, IH), 8.03 (t, J = 9.0 Hz, 3H), 7.96 (d, J = 7.9 Hz, IH), 7.86 (d, J =
8.3 Hz, 2H), 7.38 (d, J = 8.3 Hz, 3H), 7.19 (s, IH), 3.62 (s, 3H); LC/MS (Method B):
(electrospray +ve), m/z 463.1 (MH)+, tR = 3.741 , UV254 = 100%
[0332] N-(5-(3-Methyl-2-oxo-l -(3-(trifluoromethyl)phenyl)-2,3-dihydro-lH- imidazo[4,5-c]quinolin-8-yl)pyridin-2-yl)acetamide
[0333] 1H NMR (400 MHz, DMSO-<fc) δ 10.64 (s, lH), 9.10 (s, IH), 8.21 (s, IH), 8.18 (d, J = 2.5 Hz, IH), 8.15 (d, J - 8.9 Hz, I H), 8.08 (dd, J = 8.6, 2.4 Hz, 2H), 8.04 (d, J = 7.7 Hz, IH), 7.96 (d, J = 7.9 Hz, IH), 7.77 (dd, J - 8.7, 2.6 Hz, IH), 7.12 (d, J = 2.1 Hz, IH), 3.61 (s, 4H), 2.08 (s, 3H); LC/MS (Method B): (electrospray +ve), m/z 478.1 (MH)+, tR = 3.894, UV254 = 100%
[0334] 8-(3-Aminophenyl)-3-methyl-l-(3-(trifluoromethyl)phenyl)-lH-imidazo[4,5- c]quinolin-2(3H)-one
Figure imgf000087_0002
[0335] Ή NMR (400 MHz, DMSO-c¾) δ 9.15 (s, IH), 8.19 (s, IH), 8.14 (d, J = 8.9 Hz, IH), 8.07 (d, J = 7.7 Hz, IH), 8.04 (d, J = 8.1 Hz, IH), 7.98 (t, J = 7.8 Hz, IH), 7.88 (d, J = 8.8 Hz, IH), 7.12 (d, J = 2.0 Hz, IH), 7.04 (t, J = 7.8 Hz, IH), 6.73 (s, IH), 6.65 (d, J = 7.9 Hz, IH), 6.45 (d, J = 7.6 Hz, IH), 3.62 (s, 4H); LC/MS (Method B): (electrospray +ve), m/z 435.1 (MH)+, tR = 3.775, UV254 = 100%
[0336] 8-(2-Aminopyrimidin-5-yl)-3-methyl-l-(3-(trifluoromethyl)phenyl)-lH- imidazo [4,5 -c] quinolin-2(3 H)-one -
Figure imgf000088_0001
[0337] Ή NMR (400 MHz, DMSO-<¾) δ 9.13 (s, 1H), 8.20 (s, 2H), 8.19 (s, 1H), 8.12 (d, J = 8.9 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 8.04 (d, J = 8.2 Hz, 1H), 8.00 - 7.97 (m, 1H), 7.96 (d, J = 4.2 Hz, 1H), 7.04 - 6.90 (m, 3H), 3.61 (s, 4H); LC/MS (Method B): (electrospray +ve), m/z 437.1 (MH)+, tR = 3.531 , UV254 = 100%
[0338] 3-(3-Methyl-2-oxo-l-(3-(trifluoromethyl)phenyl)-2,3-dihydro-lH-imidazo[4,5- c]quinolin-8-yl)benzonitrile
Figure imgf000088_0002
[0339] Ή NMR (400 MHz, DMSO-ifc) 6 9.1 1 (s, 1H), 8.22 (s, 1H), 8.16 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 7.6 Hz, 2H), 8.00 (dd, J = 8.8, 2.0 Hz, 1H), 7.95 (t, J = 7.9 Hz, 1H), 7.81 (d, J = 7.4 Hz, 1H), 7.69 (d, J = 1.9 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 3.61 (s, 4H); LC/MS (Method B): (electrospray +ve), m/z 445.1 (MH)+, tR = 4.582, UV254 = 100%
EXAMPLE 12
[0340] This example demonstrates the gametocytocidal activity and activity against asexual parasites in accordance with an embodiment of the invention.
[0341] Compounds were screened against gametocytes and asexual parasites as described in Example 3. The results are set forth in Tables 6-8.
Table 6
Figure imgf000088_0003
. -
87
Figure imgf000089_0001
_
88
Figure imgf000090_0001
Figure imgf000091_0001
-
90
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
-
93
Figure imgf000095_0002
Table 7
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Table 8
Figure imgf000099_0001
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[0342] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0343] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly , .
103
contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0344] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 718775 HHlV/O 2015/073804· T-02 PCT/US2014/065671 104 CLAIM(S):
1. A method of blocking transmission of a Plasmodium parasite comprising administering to a mammal in need of such treatment, a therapeutically effective amount of a first compound of formula (I):
Figure imgf000106_0001
wherein A is CR12 or N,
B is CR3=CR4 or NR13,
R1 is an optionally substituted group selected from the group consisting of C6-io aryl; Cj.12 alkyl; d.12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoro methyl, Cj-C6 alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2, R10, and R11 are independently hydrogen, halogen, -NR6R7, -OR8, -SR9, or an optionally substituted group selected from the group consisting of C1-12 acyl; C6-io aryl; 07.15 arylalkyl; C6.15 heteroarylalkyl; CM2 alkyl; Ci_i2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, CpC6 alkylamino, di(CpC6alkyl)amino, CpC6 alkylcarbonylamino, C2.6 alkenyl, trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, alkylcarbonyl, alkylsulfonyl, hydroxyl, carboxy, C6.i0 aryl, heterocyclyl, and oxo,
R3 and R4 are independently selected from hydrogen, hydroxyl, OR5, halogen, optionally substituted C6-io aryl, and optionally substituted Ci-6 alkyl, Leydig 718775
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105
R5 is Ci_i2 alkyl, and
R6, R7, R8, and R9 are independently hydrogen, an optionally substituted group selected from the group consisting of Ci_i2 acyl; C6-io aryl; C6-io aryl Ci_i2 alkyl; C4-7 heteroaryl Cj-12 alkyl; Ci-12 alkyl; 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-12- membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and CM2 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
R6 and R7 are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
12
R is hydrogen, Ci-i2 alkyl, C6-io aryl, halogen, hydroxyl, or OR ,
R is hydrogen, Ci-i2 alkyl or C6-io aryl,
and/or a second compound selected from elesclomol, NSC174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamethylene- thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1 152-HQPA, CAY10581, plicamycin, CUDC-101 , auranofin, trametinib, GSK-458, afatinib, and panobinostat,
or a pharmaceutically acceptable salt thereof,
with the proviso that when A is CH, B is CR3=CR4, R3, R4, R10, and R1 1 are each hydrogen, and R 1 is 3-trifluoromethylphenyl, R 2 is not 2-amino-5-pyridyl or 3-quinolinyl.
2. The method of claim 1 , wherein the compound is of formula (I) and B is CR3=CR4.
3. The method of claim 2, wherein
A is CR12 or N,
R1 is C]-C6 alkyl, C6-Cio aryl, or heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2 is C6-Cio aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C6 alkylamino, di(Ci-C6alkyl)amino, Cj-C6 alkylcarbonylamino, sulfonyl, di(Ci-C6 alkyl)carbonylamino, trifluoromethyl, halo, C2-C6 alkenyl, cyano, C]-C6 alkoxy, acyl, Ci-C6 alkyl, hydroxyl, heterocyclyl, oxo, aminosulfonyl, alkylsulfonylamino, Ci-C6 alkylaminomethyl, and di(Ci-C6 alkyl)aminomethyl, Leydig 718775
Httwo 2015/073804 PCT/US2014/065671
106
R10 and R11 are both hydrogen, and
R3 and R4 are individually selected from hydrogen, halo, optionally substituted Cj-C6 alkyl, and OR5.
4. The method of claim 4, wherein A is CH.
5. The method of claim 3 or 4, wherein R3 and R4 are both hydrogen.
6. The method of claim 1 , wherein R1 is selected from 3-trifluoromethylphenyl, 4-piperazinylmethyl, ethyl, phenyl, 3-ethylphenyl, 3-chlorophenyl, 3 -cyanophenyl, 3- methoxyphenyl, 3-(dimethylaminocarbonyl)phenyl, 3-sulfonamidophenyl, 3-phenoxyphenyl,
3- ethoxyphenyl, 4-(piperazin-4-yl)-3-trifluoromethylphenyl, 4-piperazinyl, 1 -acetylpiperidin-
4- yl,cyclopropyl, 4-tetrahydropyranyl, cyclohexyl, and cyclopentyl.
7. The method of claim 5 or 6, wherein R is selected from 2-amino-pyridinyl, 4- pyridinyl, 2-amino-5-pyrimidinyl, 3-pyridyl, quinolin-3-yl, 5-pyrimidinyl, 2-amino-5- trifluoromethylpyrimidin-5-yl, 2-acetylamino-5-pyridyl, 2-amino-4-methylpyrimidin-5-yl, 1- piperazinyl, indol-5-yl, lH-indazol-5-yl, 4-aminophenyl, l,2,3,6-tetrahydropyridin-4-yl, 1H- pyrazol-4-yl, lH-benzo[d]imidazol-5-yl, 4-sulfonylaminophenyl, 2-dimethylaminopyrimidin-
5- yl, 3-trifluoromethylphenyl, bromo, 3-aminophenyl, vinyl, 4-aminocarbonylphenyl, 3- cyanophenyl, 3-trifluoromethyl-5-pyridyl, tetrazolyl, 4-chlorophenyl, 4-methoxyphenyl, 3- aminocarbonylphenyl, 3-acetylphenyl, 2,3-dihydrobenzofuran-6-yl, 1 -methyl- lH-indol-5-yl, benzo[d][l,3]dioxo-5-yl, 4-fluorophenyl, 4-hydroxyphenyl, porpholin-l-yl,
benzo[b]thiophen-l-yl, 4-methylsulfonylphenyl, benzo[c][l ,2,5]oxadiazol-5-yl, 2-(piperidin- l-yl)-3-pyridinyl, 4-carboxyphenyl, 2-methyl-5-pyridyl, 4-methylsulfonylphenyl, 4- dimethylaminocarbonylphenyl, 4-phenylphenyl, 4-methylpenyl, 3-chloro-5-pyridyl, (3- pyrrolidin-1 -yl)phenyl, 4-([piperizin-l -yl]carbonyl)phenyl, 4-([morpholin-l - yl]carbonyl)phenyl, 2-hydroxypyrimidin-5-yl, 3-aminosulfonylphenyl, 2-oxo- l,2,3,4,tetrahydroisoquinolin-6-yl, 2-oxo-l ,2,3,4,-tetrahydroquinolin-6-yl, 4- (aminomethyl)phenyl, 4-(dimethylaminomethyl)phenyl, 4-(methylaminocarbonyl)phenyl, 1- oxoindolin-5-yl, and l-oxoisoindolin-5-yl.
8. The method of claim 1 , wherein the compound is a compound of formula (I) and B is NR13.
9. The method of claim 8, wherein A is CH.
10. The method of claim 8 or 9, wherein R10 and R11 are both hydrogen.
1 1. The method of any one of claims 8-10, wherein R13 is hydrogen or Ci-i2 alkyl.
12. The method of claim 1 1, wherein R1 is 3-trifluoromethylphenyl. Leydis 718775
PCT/US2014/065671
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13. The method of claim 1 1 or 12, wherein R2 is selected from the group consisting of 2-methyl-5-pyridyl, 4-aminophenyl, 2-acetylamino-5-pyridyl, 4-hydroxyphenyl, 3-aminophenyl, 4-pyridyl, lH-benzo[d]imidazol-5-yl, 4-methylsulfonylphenyl, quinolin-3-yl, 2-aminopyrimidin-5-yl, 3-cyanophenyl, 3-pyridyl, and 4-aminocarbonylphenyl.
14. The method of claim 1 , wherein the second compound is selected from elesclomol, NSC 174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamethylene-thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1 152-HQPA, CAY10581 , plicamycin, CUDC-101 , auranofin, trametinib, GSK-458, afatinib, and panobinostat.
15. The method of claim 14, wherein the second compound is elesclomol, NSC 174938, NVP-AUY922, maduramicin, and narasin.
16. The method of any one of claims 1-15, wherein the first and/or second compound is administered in the form of a pharmaceutical composition.
17. The method of any one of claims 1-15, further comprising administering to the mammal at least one additional antimalarial compound.
18. The method of any one of claims 1-17, wherein the Plasmodium parasite is in a gametocyte stage.
19. The method of claim 18, wherein the Plasmodium gametocyte is a stage III-V gametocyte.
20. The method of claim 19, wherein the Plasmodium gametocyte is a mature stage III-V gametocyte.
21. The method of any one of claims 1-17, wherein the compound effectively kills Plasmodium parasites.
22. The method of any one of claims 1-17, wherein the Plasmodium parasite is a drug-resistant strain.
23. The method of any one of claims 1-17, wherein the Plasmodium parasite is in an asexual stage.
24. A method of treating malaria by killing or arresting the growth of Plasmodium organisms in a mammal, wherein the Plasmodium organisms are in a gametocyte stage, the method comprising administering to a mammal a therapeutically effective amount of a first compound of formula (I): Leydig 718775
ΗΗΛ ZT-02
WO 2015/073804 PCT/US2014/065671
108
Figure imgf000110_0001
(I)
wherein A is CR12 or N,
B is CR3=CR4 or NR13,
R is an optionally substituted group selected from the group consisting of C6-10 aryl; Ci-12 alkyl; Ci-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-12-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Q-Q alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2, R10, and R1 1 are independently hydrogen, halogen, -NR6R7, -OR8, -SR9, or an optionally substituted group selected from the group consisting of CM2 acyl; C6-io aryl; C7-i5 arylalkyl; C6-15 heteroarylalkyl; C1-12 alkyl; C1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1-2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Cj-C6 alkylamino, di(Ci-C6alkyl)amino, Ci-C6 alkylcarbonylamino, C2-6 alkenyl, trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, alkylcarbonyl, alkylsulfonyl, hydroxyl, carboxy, C6.10 aryl5 heterocyclyl, and oxo,
R3 and R4 are independently selected from hydrogen, hydroxyl, OR5, halogen, optionally substituted C6-io aryl, and optionally substituted Ci-6 alkyl,
R5 is Ci_i2 alkyl, and
R°, R', R°, and W are independently hydrogen, an optionally substituted group selected from the group consisting of C\.\z acyl; C6.10 aryl; C6-io aryl C .n alkyl; C4-7 heteroaryl .n alkyl; C].i2 alkyl; 5-10-membered heteroaryl having 1-4 heteroatoms Leydie 718775
L7CWO 2015/073804 T PCT/US2014/065671
109
independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-7- membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and C\.\i alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or
R6 and R7 are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1 -2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
R is hydrogen, Ci-12 alkyl, C6-io aryl, halogen, hydroxyl, or OR ,
R is hydrogen, d-12 alkyl or C6.10 aryl,
and/or a second compound selected from elesclomol, NSC 174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamethylene- thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1 152-HQPA, CAY10581 , plicamycin, CUDC-101 , auranofin, trametinib, GSK-458, afatinib, and panobinostat,
or a pharmaceutically acceptable salt thereof,
with the proviso that when A is CH, B is CR3=CR4, R3, R4, R10, and R1 1 are each hydrogen, and R1 is 3-trifluoromethylphenyl, R2 is not 2-amino-5-pyridyl or 3-quinolinyl.
25. The method of claim 24, wherein the compound is of formula (I) and B is CR3=CR4.
26. The method of claim 25, wherein
A is CR12 or N,
R1 is d-C6 alkyl, C6-Cio aryl, or heteroaryl, wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, d-C6 alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2 is C6-Cio aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, d -d alkylamino, di(Ci-C6alkyl)amino, d-d alkylcarbonylamino, sulfonyl, di(C]-C6 alkyl)carbonylamino, trifluoromethyl, halo, C2-d alkenyl, cyano, C] -C6 alkoxy, acyl, Ci-C6 alkyl, hydroxyl, heterocyclyl, oxo, aminosulfonyl, alkylsulfonylamino, Ci-C6 alkylaminomethyl, and di(d-C6 alkyl)aminomethyl,
R10 and R1 1 are both hydrogen, and
R3 and R4 are individually selected from hydrogen, halo, optionally substituted d -d alkyl, and OR5.
27. The method of claim 36, wherein A is CH. Leydig 718775
¾0 2015/073804 T'°2 PCT/US2014/065671
1 10
28. The method of claim 26 or 28, wherein R3 and R4 are both hydrogen.
29. The method of claim 28, wherein R1 is selected from 3-trifluoromethylphenyl, 4-piperazinylmethyl, ethyl, phenyl, 3-ethylphenyl, 3-chlorophenyl, 3-cyanophenyl, 3- methoxyphenyl, 3-(dimethylaminocarbonyl)phenyl, 3-sulfonamidophenyl, 3-phenoxyphenyl,
3- ethoxyphenyl, 4-(piperazin-4-yl)-3-trifluoromethylphenyl, 4-piperazinyl, 1 -acetylpiperidin-
4- yl,cyclopropyl, 4-tetrahydropyranyl, cyclohexyl, and cyclopentyl.
30. The method of claim 28 or 29, wherein R is selected from 2-amino-pyridinyl,
4- pyridinyl, 2-amino-5-pyrimidinyl, 3-pyridyl, quinolin-3-yl, 5-pyrimidinyl, 2-amino-5- trifluoromethylpyrimidin-5-yl, 2-acetylamino-5-pyridyl, 2-amino-4-methylpyrimidin-5-yl, 1- piperazinyl, indol-5-yl, l H-indazol-5-yl, 4-aminophenyl, l ,2,3,6-tetrahydropyridin-4-yl, 1H- pyrazol-4-yl, lH-benzo[d]imidazol-5-yl, 4-sulfonylaminophenyl, 2-dimethylaminopyrimidin-
5- yl, 3-trifluoromethylphenyl, bromo, 3-aminophenyl, vinyl, 4-aminocarbonylphenyl, 3- cyanophenyl, 3-trifluoromethyl-5-pyridyl, tetrazolyl, 4-chlorophenyl, 4-methoxyphenyl, 3- aminocarbonylphenyl, 3-acetylphenyl, 2,3-dihydrobenzofuran-6-yl, 1 -methyl- lH-indol-5-yl, benzo[d][l ,3]dioxo-5-yl, 4-fluorophenyl, 4-hydroxyphenyl, porpholin-l-yl,
benzo[b]thiophen-l-yl, 4-methylsulfonylphenyl, benzo[c][l,2,5]oxadiazol-5-yl, 2-(piperidin- l-yl)-3-pyridinyl, 4-carboxyphenyl, 2-methyl-5-pyridyl, 4-methylsulfonylphenyl, 4- dimethylaminocarbonylphenyl, 4-phenylphenyl, 4-methylpenyl, 3-chloro-5-pyridyl, (3- pyrrolidin- 1 -yl)phenyl, 4-([piperizin- 1 -yl]carbonyl)phenyl, 4-([morpholin- 1 - yl]carbonyl)phenyl, 2-hydroxypyrimidin-5-yl, 3-aminosulfonylphenyl, 2-oxo-
1 ,2,3,4,tetrahydroisoquinolin-6-yl, 2-oxo-l ,2,3,4,-tetrahydroquinolin-6-yl, 4- (aminomethyl)phenyl, 4-(dimethylaminomethyl)phenyl, 4-(methylaminocarbonyl)phenyl, 1- oxoindolin-5-yl, and l -oxoisoindolin-5-yl.
31. The method of claim 24, wherein the compound is a compound of formula (I) and B is NR13.
32. The method of claim 31 , wherein A is CH.
33. The method of claim 31 or 32, wherein R10 and R1 1 are both hydrogen.
34. The method of any one of claims 31-33, wherein R13 is hydrogen or Ci.12 alkyl.
The method of claim 34, wherein R1 is 3-trifluoromethylphenyl. The method of claim 34 or 35, wherein R2 is selected from the group consisting of 2-methyl-5-pyridyl, 4-aminophenyl, 2-acetylamino-5-pyridyl, 4-hydroxyphenyl,
Leydiv 71H775
WWcWO 2015/073804,T n7 PCT/US2014/065671
1 1 1
3-aminophenyl, 4-pyridyl, lH-benzo[d]imidazol-5-yl, 4-methlsulfonylphenyl, quinolin-3-yl, 2-aminopyrimidin-5-yl, 3-cyanophenyl, 3-pyridyl, and 4-aminocarbonylphenyl.
37. The method of claim 24, wherein the second compound is selected from elesclomol, NSC174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamethylene-thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1152-HQPA, CAY10581 , plicamycin, CUDC-101 , auranofin, trametinib, GSK-458, afatinib, and panobinostat.
38. The method of claim 37, wherein the second compound is elesclomol, NSC174938, NVP-AUY922, maduramicin, and narasin.
39. The method of any one of claims 24-38, wherein the first and/or second compound is administered in the form of a pharmaceutical composition.
40. The method of any one of claims 24-39, further comprising administering to the mammal at least one additional antimalarial compound.
41. The method of any one of claims 24-40, wherein the Plasmodium parasite is in a gametocyte stage.
42. The method of claim 41, wherein the Plasmodium gametocyte is a stage III-V gametocyte.
43. The method of claim 42, wherein the Plasmodium gametocyte is a mature stage III-V gametocyte.
44. The method of any one of claims 24-41 , wherein the compound effectively kills Plasmodium parasites.
45. The method of any one of claims 24-41, wherein the Plasmodium parasite is a drug-resistant strain.
46. The method of any one of claims 24-41 , wherein the Plasmodium parasite is in an asexual stage.
47. A first compound of formula (I):
Figure imgf000113_0001
Leydig 718775
PCT/US2014/065671
1 12
(I)
wherein A is CR12 or N,
B is CR3=CR4 or NR13,
R1 is an optionally substituted group selected from the group consisting of Ce-io aryl; C1 -12 alkyl; C1 -12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10-membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the alkyl, aryl, or heteroaryl is optionally substituted with one or more substituents selected from trifluoromethyl, Ci-C6 alkyl, halo, CN, Ci-C6 alkoxy, S02NH2, piperizinyl, and 4-alkylcarbonylpiperazinyl,
R2, R10, and Rn are independently hydrogen, halogen, -NR6R7, -OR8, -SR9, or an optionally substituted group selected from the group consisting of C1-12 acyl; C6-1o aryl; C7-15 arylalkyl; C6-i 5 heteroarylalkyl; C1-12 alkyl; C1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; 5-10- membered heteroaryl having 1 -4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and 4-7-membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; wherein the aryl or heteroaryl is optionally substituted with one or more groups selected from amino, Ci-C6 alkylamino, di(Ci-C6alkyl)amino, Ci-C6 alkylcarbonylamino, C2.6 alkenyl, trifluoromethyl, C\-C6 alkyl, halo, CN, Ci-C6 alkoxy, alkylcarbonyl, alkylsulfonyl, hydroxyl, carboxy, C6.10 aryl, heterocyclyl, and oxo,
R3 and R4 are independently selected from hydrogen, hydroxyl, OR5, halogen, optionally substituted C6-io aryl, and optionally substituted C1-6 alkyl,
R5 is Ci_i2 alkyl, and
R6, R7, R8, and R9 are independently hydrogen, an optionally substituted group selected from the group consisting of C1 -12 acyl; C6.10 aryl; C6-io aryl Ci_i2 alkyl; C4.J
heteroaryl Ci-12 alkyl; CM 2 alkyl; 5-10-membered heteroaryl having 1-4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; 4-12- membered heterocyclic having 1 -2 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and C1-12 alkyl wherein alkyl contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms; or Leyd'o- 71R77
,,,,,ννΌ 2015/073804
lino J ι - υ ι /u-r i_ J - PCT/US2014/065671
1 13
R6 and R7 are taken with the nitrogen atom to form a 4-7 membered heterocyclic ring having 1 -2 heteroatoms independently selected from nitrogen, oxygen, and sulfur,
R is hydrogen, d-12 alkyl, C6-io aryl, halogen, hydroxyl, or OR ,
R13 is hydrogen, d-12 alkyl or C6-io aryl,
and/or a second compound selected from elesclomol, NSC174938, NVP-AUY922, maduramicin, narasin, alvespimycin, omacetaxine, thiram, zinc pyrithione, phanquinone, bortezomib, salinomycin sodium, monensin sodium, dipyrithione, dicyclopentamethylene- thiuram disulfide, YM155, withaferin a, adriamycin, romidepsin, AZD-1 152-HQPA, CAY10581 , plicamycin, CUDC-101 , auranofin, trametinib, GSK-458, afatinib, and panobinostat,
or a pharmaceutically acceptable salt thereof,
with the proviso that when A is CH, B is CR3=CR4, R3, R4, R10, and R1 1 are each hydrogen, and R1 is 3-trifluoromethylphenyl, R2 is not 2-amino-5-pyridyl or 3-quinolinyl, for use in blocking transmission of a Plasmodium parasite comprising administering to a mammal or treating malaria by killing or arresting the growth of Plasmodium organisms in a mammal, wherein the Plasmodium organisms are in a gametocyte stage.
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WO2018071836A1 (en) * 2016-10-13 2018-04-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compounds and method for blocking transmission of malarial parasite
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