Article
Reference
Optimization of the clofazimine structure leads to a highly water-soluble C3-aminopyridinyl riminophenazine endowed with
improved anti-Wnt and anti-cancer activity in vitro and in vivo
KOVAL, Alexey, et al.
Abstract
Triple-negative breast cancer (TNBC) is a cancer subtype critically dependent upon excessive activation of Wnt pathway. The anti-mycobacterial drug clofazimine is an efficient inhibitor of canonical Wnt signaling in TNBC, reducing tumor cell proliferation in vitro and in animal models. These properties make clofazimine a candidate to become first targeted therapy against TNBC. In this work, we optimized the clofazimine structure to enhance its water solubility and potency as a Wnt inhibitor. After extensive structure-activity relationships
investigations, the riminophenazine
5-(4-(chlorophenyl)-3-((2-(piperazin-1-yl)ethyl)imino)-N-(pyridin-3-yl)-3,5-dihydrophenazin-2-a mine (MU17) was identified as the new lead compound for the riminophenazine-based targeted therapy against TNBC and Wnt-dependent cancers. Compared to clofazimine, the water-soluble MU17 displayed a 7-fold improved potency against Wnt signaling in TNBC cells resulting in on-target suppression of tumor growth in a patientderived mouse model of TNBC.
Moreover, allowing the administration of reduced yet effective dosages, MU17 displayed no adverse effects, most [...]
KOVAL, Alexey, et al. Optimization of the clofazimine structure leads to a highly water-soluble C3-aminopyridinyl riminophenazine endowed with improved anti-Wnt and anti-cancer activity in vitro and in vivo. European Journal of Medicinal Chemistry, 2021, vol. 222, p. 113562
DOI : 10.1016/j.ejmech.2021.113562 PMID : 34116325
Available at:
http://archive-ouverte.unige.ch/unige:152351
Disclaimer: layout of this document may differ from the published version.
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1
SUPPORTING DATA
Optimization of the clofazimine structure leads to a highly water-soluble C3- aminopyridinyl riminofenazine endowed with improved anti-Wnt and anti-cancer
activity in vitro and in vivo
Alexey Koval*Ω, Ivan Bassanini*┴, Jiabin Xu*Ω£, Michele Tonelli§, Vito Boido§, Fabio Sparatore§, Frederic Amant†¥¶, Daniela Annibali†, Eleonora Leucci‡€, Anna Sparatore┴** and Vladimir L.
Katanaev Ω,£**
ΩDepartment of Cell Physiology and Metabolism, Translational Research Centre in Oncohaematology, Faculty of Medicine, University of Geneva, 1206 Geneva, Switzerland;
┴Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, 20133 Milano, Italy;
£Department of Biomedical Sciences, Faculty of Biology and Medicine, 1011 University of Lausanne, Lausanne, Switzerland;
§Dipartimento di Farmacia, Università di Genova, 16132 Genova, Italy;
†Gynecological Oncology Laboratory, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), 3000 Leuven, Belgium;
¥Department of Obstetrics and Gynecology, University Hospitals Leuven and Department of Oncology, 3000 Leuven, Belgium;
¶Centre for Gynecologic Oncology Amsterdam (CGOA), Antoni Van Leeuwenhoek-Netherlands Cancer Institute (AvL-NKI), University Medical Center (UMC), 1066 Amsterdam, The
Netherlands;
‡Laboratory for RNA Cancer Biology, Department of Oncology, KULeuven, 3000 Leuven, Belgium
€Trace, LKI Leuven Cancer Institute, KU Leuven, 3000 Leuven, Belgium
£School of Biomedicine, Far Eastern Federal University, 690922 Vladivostok, Russia
*equally contributing as first authors
**equally contributing as last authors
2
INDEX
1. Compounds’ solubility ... 3
2. Compounds’ activity in FopFlash against a panel of luciferase reporters ... 4
3. EB05: HR MS, 1H and 13C NRM, IR spectra ... 5
4. AR18: HR MS, 1H and 13C NRM, IR spectra ... 8
5. AR13: HR MS, 1H and 13C NRM, IR spectra ... 11
3 1. Compounds’ solubility
Supporting Table S1. Water solubility of clofazimine and of representative basic riminophenazines
Compound Xa Y Z n R Solubility (M)
Clofazimine CH Cl Cl 0 CH(CH3)2 < 3
SV21 CH CH3 CH3 1 a 182 ± 9
GM05 CH H H 4 b 5.5 ± 0.9
SV06 CH Cl Cl 1 c 128 ± 9
SV13 CH Cl Cl 2 c 126 ± 5
EB06 CH Cl Cl 3 d 114 ± 4
EB04 CH H Cl 3 d 209 ± 11
SV12 CH H H 2 e 93±2
GG13 N OCH3 H 4 b > 303000b
GG08 N H Cl 3 d > 414000b
MU05 N H Cl 3 e > 323000b
MU23 N H Cl 3 f > 323000b
MU17 N H Cl 2 g > 300000b
AR18 N H Cl 3 g > 75000b
AR13 N H Cl 4 g > 75000b
aWhen X=CH, compounds were in the form of di-hydrochloride, when X= N, compounds are tri-hydrochloride
bFor these compounds solubility exceeded the concentration of initial planned stock, hence this concentration is provided in the table for reference
4 2. Compounds’ activity in FopFlash against a panel of luciferase reporters
basal vehicle GG08(32) 7mM SV12(21) 7mM MU17(43) 1.7mM AR18(44) 4mM AR13(45) 3mM clofazimine 5mM
0.0 0.5 1.0 1.5 50 100 150 200
Response,foldoverbasal
TopFlash FopFlash
** **
*** ***
*** *
+Wnt3a
100
106 107 111 114 98 106
100
98 107 107 111 108 93
100
108 111 101 108 103 112
100
97 97 103
99 100 104
100
100 102 97 99 91 98
100
103 105 114 104 112 118
100
110 116 113 104 118 110
100
96 92 111
95 91 102
100
114 114 92 94 102 112
100
94 89 94 96 108
94 100
85 82 75 72 80 85
C/EBP NF-kB
HNF1a HNF3
CREB SOX2
STAT3
TA RXR RAR
Gli-1 vehicle
GG08(32) 7mM SV12(21)
7mM MU17(43)
1.5mM AR18(44)
4mM AR13(45)
3mM clofazimine
5mM
pathwayactivity,%ofbasal
Pathway reporter name
0 50 100
A B
Supporting Figure S1. (A) Derivatives or parental clofazimine do not show any inhibitory activity at selected concentrations in the cells transfected with FopFlash reporter plasmid. We have chosen individual concentrations of the compounds with maximal anti-Wnt efficacy before onset of unspecific cytotoxicity as assessed by Renilla luciferase levels (see Fig. 1 and Tables 1 and 2) (B) Clofazimine and its derivatives do not show unspecific inhibition in a panel of luciferase-based reporter assays. Despite a certain trend to decrease in Hedgehog reporter Gli-1, this decrease is not statistically significant. Statistical significance in each case was analyzed by two-way ANOVA with multiple comparisons, significance shown as on Figure 1.
Koval et al, Supplementary Figure 1
5 3. EB05: HR MS, 1H and 13C NRM, IR spectra
6
7
Sample Name Sample Form Operator Name Resolution
Scantime or Scans Sample Scans
Start Frequency Limit for File End Frequency Limit for File
EB-05 EB-05 Default 4 24 4000 400
Aperture Setting Beamsplitter Setting Detector Setting Source Setting Measurement Channel Acquisition Mode Phase Correction Mode Apodization Function
KBr
RT-DLATGS [Internal Pos.1]
MIR
Sample Compartment
Double Sided,Forward-Backward Power Spectrum
Blackman-Harris 3-Term
3341.96 2977.56 2881.17 1749.97 1696.65 1636.84 1610.67 1560.06 1521.42 1456.69 1376.79 1317.62 1226.86 1136.39 1097.87 1025.61 930.32 784.16 733.49 659.64 606.15 519.76 486.46 457.07 447.69
500 1000
1500 2000
2500 3000
3500
Wavenumber cm-1
-0.10-0.06-0.02
Absorbance Units
EB-05.0 2/18/2021
Default 3:46:13 PM
8 4. AR18: HR MS, 1H and 13C NRM, IR spectra
9
10
Sample Name Sample Form Operator Name Resolution
Scantime or Scans Sample Scans
Start Frequency Limit for File End Frequency Limit for File
AR-18 AR-18 Default 4 24 4000 400
Aperture Setting Beamsplitter Setting Detector Setting Source Setting Measurement Channel Acquisition Mode Phase Correction Mode Apodization Function
KBr
RT-DLATGS [Internal Pos.1]
MIR
Sample Compartment
Double Sided,Forward-Backward Power Spectrum
Blackman-Harris 3-Term
3425.56 2903.35 2811.90 2559.82 2364.17 2343.03 2149.35 2080.12 1991.03 1960.96 1896.08 1718.25 1615.99 1541.69 1457.79 1340.90 1291.24 1227.23 1138.25 1098.19 769.91 725.73 660.01 613.73 500.40 472.51 447.66
500 1000
1500 2000
2500 3000
3500
Wavenumber cm-1
-0.020-0.0050.010
Absorbance Units
AR-18.0 2/18/2021
Default 3:34:45 PM
11 5. AR13: HR MS, 1H and 13C NRM, IR spectra
12
13
Sample Name Sample Form Operator Name Resolution
Scantime or Scans Sample Scans
Start Frequency Limit for File End Frequency Limit for File
AR-13 AR-13 Default 4 24 4000 400
Aperture Setting Beamsplitter Setting Detector Setting Source Setting Measurement Channel Acquisition Mode Phase Correction Mode Apodization Function
KBr
RT-DLATGS [Internal Pos.1]
MIR
Sample Compartment
Double Sided,Forward-Backward Power Spectrum
Blackman-Harris 3-Term
3327.01 2971.81 2818.52 2174.55 1677.45 1610.32 1583.26 1522.46 1493.54 1381.54 1343.11 1315.27 1227.05 1136.04 1098.12 1047.29 1028.40 967.29 783.26 687.04 663.47 630.99 605.61 506.24 466.42 411.61
500 1000
1500 2000
2500 3000
3500
Wavenumber cm-1
020406080
Transmittance [%]
AR-13.4 2/18/2021
Default 5:28:04 PM