Background
Methods
Synthesis of ITT derivatives, ITT-001 to 006
1-(3,7-Bis(diethylamino)-10H-phenoxazin-10-yl)ethanone (ITT-001)
1-(3,7-Bis(diethylamino)-10H-phenoxazin-10-yl)-2,2-dimethylpropan-1-one (ITT-002)
3,7-Bis(diethylamino)-10H-phenoxazin-10-yl)(cyclopropyl)methanone (ITT-003)
(3,7-Bis(diethylamino)-10H-phenoxazin-10-yl)(phenyl)methanone (ITT-004)
1-(3,7-Bis(diethylamino)-10H-phenoxazin-10-yl)-2-(diethylamino)ethanone hydrochloride (ITT-005·HCl)
(3,7-Bis(diethylamino)-10H-phenoxazin-10-yl)(4-fluorophenyl)methanone (ITT-006)
Compounds
Parasite strains and culture
Characterization of anti-malarial activity in vitro
The automated haematology analyzer, XN-30
Microscopy
Measurement of anti-malarial efficacy using the XN-30 analyzer
Measurement of fluorescence spectrum
Fluorescence microscopy
Cytotoxic assay
In vivo anti-malarial activity
Statistical analysis
Results
Synthesis of ITT derivatives
Anti-malarial efficacy of ITT derivatives in vitro
3D7 | W2 | MRA-1239 | MRA-1240 | |
---|---|---|---|---|
IC50 (nM)a | IC50 (nM)a | IC50 (nM)a | IC50 (nM)a | |
basic blue 3 | 3.2 ± 0.19 | 4.8 ± 0.53 | 5.2 ± 1.1 | 5.9 ± 0.68 |
ITT-001 | 11.2 ± 1.3 | 11.1 ± 1.8 | 34.6 ± 9.1 | 39.0 ± 11.2 |
ITT-002 | 10.7 ± 1.5 | 10.4 ± 2.8 | 20.7 ± 1.6 | 24.4 ± 3.7 |
ITT-003 | 9.8 ± 1.0 | 9.6 ± 2.1 | 24.9 ± 3.1 | 31.3 ± 9.8 |
ITT-004 | 11.3 ± 2.4 | 12.3 ± 2.7 | 26.4 ± 2.9 | 30.5 ± 2.0 |
ITT-005 | 138.3 ± 9.0 | 145.6 ± 29.5 | 130.2 ± 9.3 | 183.7 ± 30.1 |
ITT-006 | 10.6 ± 1.6 | 16.0 ± 4.5 | 27.5 ± 3.4 | 29.4 ± 5.8 |
CQ | 8.6 ± 0.21 | 225.0 ± 23.2*** | 38.6 ± 5.2 | 58.0 ± 2.6* |
ART | ND | ND | 5.5 ± 0.018 | 13.2 ± 1.4** |
Localization of basic blue 3 in vitro
Cytotoxicity of ITT derivatives in vitro
HEK293T | HepG2 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
CC50 (μM)a | SI | CC50 (μM)a | SI | |||||||
3D7 | W2 | MRA-1239 | MRA-1240 | 3D7 | W2 | MRA-1239 | MRA-1240 | |||
basic blue 3 | 0.021 ± 0.0042 | 6.6 | 4.4 | 4.0 | 3.6 | 1.0 ± 0.064 | 312.5 | 208.3 | 192.3 | 169.5 |
ITT-001 | 0.32 ± 0.042 | 28.6 | 28.8 | 9.2 | 8.2 | 4.0 ± 0.85 | 357.1 | 357.1 | 90.1 | 12.6 |
ITT-002 | 0.20 ± 0.021 | 18.7 | 19.2 | 9.7 | 8.2 | > 50 | > 4672.9 | > 4807.7 | > 2415.5 | > 2049.2 |
ITT-003 | 9.2 ± 3.8 | 938.8 | 958.3 | 369.5 | 293.9 | > 50 | > 5102.0 | > 5208.3 | > 2008.0 | > 1597.4 |
ITT-004 | 10.5 ± 3.4 | 929.2 | 853.7 | 397.7 | 344.3 | > 50 | > 4424.8 | > 4065.0 | > 1893.9 | > 1639.3 |
ITT-005 | 0.73 ± 0.093 | 5.3 | 5.0 | 5.6 | 4.0 | 4.6 ± 0.26 | 33.2 | 31.6 | 35.3 | 25.0 |
ITT-006 | 13.6 ± 3.0 | 1283.0 | 850.0 | 494.5 | 462.6 | > 50 | > 3676.5 | > 3125.0 | > 1818.2 | > 1700.7 |
CQ | 2.9 ± 0.27 | 337.2 | 12.9 | 75.2 | 50.0 | 1.8 ± 0.14 | 209.3 | 8.0 | 46.6 | 31.0 |
Anti-malarial activity of ITT derivatives in vivo
% suppression on day 4 | MSD (day) | # of survived mice on day 30 | |
---|---|---|---|
basic blue 3a | > 99 | 20.3 | 1/4 |
ITT-001a | > 99 | 20.4 | 3/5 |
ITT-002a | > 99 | 19.2 | 2/5 |
ITT-003a | > 99 | 21.0 | 2/5 |
ITT-004a | > 99 | 29.4 | 4/5 |
ITT-005a | > 99 | 14.0 | 1/5 |
ITT-006a | 91.5 | 13.0 | 0/5 |
ITT-004 (50)b | > 99 | 11.8 | 0/5 |
Control | –c | 6.5 ± 0.9d | 0/5 (per group) |