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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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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*, Ivan Bassanini*, Jiabin Xu*Ω£, Michele Tonelli§, Vito Boido§, Fabio Sparatore§, Frederic Amant†¥¶, Daniela Annibali, Eleonora Leucci‡€, Anna Sparatore** and Vladimir L.

Katanaev Ω,£**

Department 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

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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

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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

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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

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5 3. EB05: HR MS, 1H and 13C NRM, IR spectra

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6

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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

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8 4. AR18: HR MS, 1H and 13C NRM, IR spectra

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9

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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

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11 5. AR13: HR MS, 1H and 13C NRM, IR spectra

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12

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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

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