SUPPLEMENTARY INFORMATION

doi: 10.1038/nature07275 SUPPLEMENTARY INFORMATION Methods Reagents, cell lines and cell culture HhAntag was synthesized as described in Supplementar...
Author: Raymond Wiggins
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doi: 10.1038/nature07275

SUPPLEMENTARY INFORMATION Methods Reagents, cell lines and cell culture HhAntag was synthesized as described in Supplementary Fig.13. Cycoplamine was purchased from Toronto Research Chemicals, Inc.. The mouse anti-Shh monoclonal antibody, 5E129, was protein A-purified and confirmed for lack of aggregation and endotoxin, as well as the ability to block ligand-activated Hh signaling, following gel filtration. HEPM, human embryonic palatal mesenchyme cells30, were stably transfected with a luciferase reporter gene driven by 6 continuous repeats of the Gli DNA binding element. The human colon fibroblast line, CCD-18Co, was purchased from ATCC (CRL-1459). MEFs from CAGGCre-ER;SmoC/C and control mice were isolated according to published methods and treated with tamoxifen (1μM) in vitro for 5 days before testing for Hh responsiveness in vitro and/or mixing with HT29-luc cell for implantation into CD-1 nude mice. Cell viability was measured at 72h using the Celltiter-Glo Luminescent Cell Viability Assay kit (Promega), and the concentration of HhAntag resulting in 50% inhibition of cell viability was determined from a minimum of 2 experiments. Co-culture experiments were carried out by culturing tumor cells at a 4:1 ratio with C3H10T1/2 S12 fibroblast GLI-reporter cells11. Luciferase activity was measured using the SteadyLite HTS kit (Promega) after 24h. Assessment of Hh pathway target gene expression was performed by culturing cells in triplicate in 24-well plates for 24 hours with 300ng/ml rSHH and/or Hh antagonists in 0.5% serum containing medium. RNA was isolated using Qiagen RNeasy Mini Kit. Reporter gene assays Twenty-four hours after plating, cells were transfected for 18h with either Gli-luciferase or NFkB-luciferase reporter plasmids in combination with an HSV-TK plasmid utilizing the Fugene6 transfection reagent (Roche) then re-plated into 96 well culture plates. Six hours later, Hh antagonist, and/or 1µg/ml rSHH were added to cells in quadruplicate in 0.5% serum containing medium. Cultures were re-fed 24 hours later, and plates were assessed for firefly and renilla luciferase activity after an additional 24 hours utilizing the Promega Dual-Glo luciferase kit. Immunofluorescence

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To evaluate Hh pathway activation in vivo, Ptch1-lacZ; RAG2-/- mice were implanted with pancreatic tumor cells lines expressing differential levels of Hh ligands. Fourteen days following implantation, xenografts were excised and fixed in 4% paraformaldehyde prior to embedding in OCT. Sections were incubated with anti-βgal (1:10000, Cappel) and FITC conjugated anti-ESA (epithelial specific antigen) (1:100, Biomeda) overnight, followed by secondary Cy3-anti-rabbit (1:400, Jackson Immunoresearch) incubation for one hour. Nuclei were visualized with DAPI (blue) in Vectamount (Vector Laboratory). Xenograft models Primary tumor samples were provided by the National Disease Research Interchange (NDRI) and the Cooperative Human Tissue Network which is funded by the National Cancer Institute. Other investigators may have received samples from these same tissues. Primary human xenografts were established by direct implantation of surgical material into female CD1 nu/nu mice of 6-8 weeks of age (Charles River Laboratories, Inc., Wilmington, MA). All mice were housed and maintained according to the animal use guidelines of Genentech, Inc, conforming to California State legal and ethical practices. Tissue specimens were shipped in RPMI containing antibiotics on wet ice and implanted within 24 hours after rinsing in DMEM containing 0.11 mg/ml sodium pyruvate, 1.125 ug/ml Amphotericin B, and 1 mg/ml Kanamycin. Tissue was minced with scalpels to a size of < 1mm3, and approximately 100 mg of tissue was implanted in the subcutaneous space of the hind flank using blunt dissection and a 10G trocar. Tumor lines were serially passaged into larger cohorts of mice for efficacy testing. Mice were distributed into tumor volume-matched cohorts upon tumors reaching between 200 to 350 mm3. HT-29, HT55, and DLD-1 cell lines were purchased from ATCC and were established as xenografts by injection into the hindflank of 6-8 week old female CD1 nu/nu mice with 5 x 106 (HT-29 and HT55) or 10 x 106 cells (DLD-1) resuspended in Hanks’ balanced salt solution. Tumor-bearing mice were distributed into tumor volumematched cohorts when the tumors reached between 80 and 120 mm3. HhAntag, or a close derivative, was resuspended in 0.5% methyl-cellulose, 0.2% Tween-80 (MCT) and administered orally twice daily at either 100 or 75 mg/kg from a 10 mg/ml suspension as indicated. 5E1 or an isotypic control (IgG1) were dissolved in PBS and administered at 60 mg/kg on day 1 and then at 30 mg/kg weekly via intraperitoneal delivery. Tumor volume and animal weights were monitored twice weekly and tumor volume calculated as (L x W x W)/2. Expression studies www.nature.com/nature

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SUPPLEMENTARY INFORMATION

For the analysis of SHH and IHH mRNA expression in multiple human tissue specimens, data were obtained from Gene Logic, Inc. (Gaithersburg, MD). Microarray gene expression analysis of RNA extracted from primary xenograft tissue was carried out on two separate platforms, Human Genome U133 Plus 2.0 & Mouse Genome 430 2.0 arrays (Affymetrix). Preparation of complementary RNA, array hybridizations, and subsequent data analysis were carried out using the manufacturers’ protocols, with signal intensities being determined by the MAS5.0 algorithm. Hh pathway genes were quantitatively assessed by Taqman and transcript levels were normalized to the housekeeping genes β-glucuronidase (GUSB) or ribosomal protein L19 (RPL19). Results are expressed as normalized expression values (=2-ΔCt) or normalized expression relative to a cell line pool representative of multiple tissue types (=2-ΔΔCt), unless otherwise stated. For xenograft model profiling, gene expression in each compartment was normalized to the same species-specific housekeeping gene to ensure that observed differences weren’t due to differences in the degree of stromal infiltrate. Correlations in gene expression were evaluated by Spearman rank tests and p-values reported. Sequences of primer/probes are shown in Supplementary Table 3 and species-specificity of human/mouse Taqman assays is confirmed in Supplementary Table 2. FACS Cells were stained with either mouse anti-Hh antibody 5E1 or isotype control antibody and then followed by biotin-conjugated anti-mouse, streptavidin-PE, and PI. Stained live (PI-excluded) cells were analyzed on a FacsCalibur, and data were plotted using FlowJo software package. 30.

Yoneda, T., & Pratt, R.M. Mesenchymal cells from the human embryonic palate are highly responsive to epidermal growth factor. Science. 213, 563-565 (1981).

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Supplementary Table 1: Quantitative expression (qRT-PCR) of Hh pathway genes in pancreatic cell lines and relationship to in vitro HhAntagonist activity (IC50)

SHH

HhAntag

AsPC-1 BxPC-3 Capan-1 Capan-2 CFPAC HPAC HPAF-II Hs 766T HuP-T3 KP4 MIA PaCa-2 Pan C 02.03 Pan C 04.03 Pan C 05.04 Pan C 08.13 Panc 03.27 Panc 10.05 PANC-1 PA-TU-8902 PA-TU-8988T PL45 PSN1 SU.86.86 SW 1990

IC50 (μM)

≥30 5.4 20.0 nd 5.8 2.7 6.2 nd ≥30 10.3 ≥30 6.0 9.4 10.8 8.9 3.5 ≥30 ≥30 2.9 ≥30 ≥30 5.8 2.7 ≥30

SMO

Ct

2-ΔCt

Ct

2-ΔCt

23.3 26.9 23.1 26.5 26.1 28.8 25.7 26.2 33.4 27.0 25.9 32.0 32.2 27.9 26.5 25.5 32.0 23.9 27.0 32.9 30.4 23.6 28.8 26.1

0.9140 0.0823 0.9252 0.0647 0.0864 0.0246 0.1307 0.2199 0.0009 0.1012 0.0799 0.0036 0.0019 0.0569 0.1163 0.1677 0.0019 0.7840 0.1647 0.0012 0.0057 1.4144 0.0297 0.0767

31.4 38.7 28.3 35.2 27.8 >40 >40 29.9 27.1 24.0 24.1 31.7 34.3 25.3 38.8 26.8 36.0 28.4 39.2 24.6 34.6 26.1 25.3 27.2

0.0033 0.0000 0.0241 0.0002 0.0272

0.0172 0.0709 0.8241 0.2763 0.0044 0.0005 0.3479 0.0000 0.0693 0.0001 0.0337 0.0000 0.3932 0.0003 0.2502 0.3389 0.0378

PTCH1 2-ΔCt Ct 26.1 26.3 26.6 25.8 28.2 27.7 26.7 26.2 26.7 22.9 26.8 26.3 25.6 26.0 26.0 26.1 26.3 26.9 27.8 25.7 27.0 27.2 26.0 26.2

0.1236 0.1329 0.0768 0.1029 0.0213 0.0509 0.0665 0.2330 0.0908 1.7584 0.0421 0.1821 0.1763 0.2106 0.1656 0.1115 0.0987 0.0972 0.0907 0.1768 0.0605 0.1166 0.2023 0.0745

GLI1 Ct

2-ΔCt

26.3 35.6 32.2 31.2 34.8 33.3 30.9 29.1 33.5 23.2 30.6 33.2 32.7 33.0 32.2 33.3 33.8 27.7 33.9 31.7 34.6 31.1 32.6 28.6

0.1138 0.0002 0.0016 0.0025 0.0002 0.0010 0.0036 0.0297 0.0008 1.4079 0.0030 0.0015 0.0013 0.0017 0.0023 0.0008 0.0005 0.0570 0.0013 0.0027 0.0003 0.0081 0.0022 0.0143

nd: not done www.nature.com/nature

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doi: 10.1038/nature07275

Supplementary Table 2: Confirmation of the species-specificity of human and murine primer/probe sets for detection of Hh pathway genes using universal human or murine reference RNA sources. Cycling threshold (Ct) values are shown.

Cycling Threshold Primer/probe set

(100ng input RNA)

Universal Universal human mouse reference RNA reference RNA

Human_GUSB Human_GLI1 Human_PTCH1 Human_PTCH2 Human_SMO Murine_Gusb Murine_Gli1 Murine_Ptch1 Murin_Ptch2 Murine_Smo

22.6 26.9 24.0 28.4 21.8 nd nd nd nd nd

nd nd nd nd nd 22.1 27.6 22.6 30.9 22.9

no template control nd nd nd nd nd nd nd nd nd nd

nd, not detected (Ct above 40)

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Supplementary Table 3:

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Gene GUSB

species human

GLI1

human

PTCH1

human

PTCH2

human

SHH

human

IHH

human

SMO

human

Gusb

mouse

Gli1

mouse

Ptch1

mouse

Smo

mouse

qRT-PCR primer/probes Sequence

forward primer TGGTTGGAGAGCTCATTTGGA reverse primer GCACTCTCGTCGGTGACTGTT probe TTTGCCGATTTCATGACT forward primer GTTCACATGCGCAGACACACT reverse primer TTCGAGGCGTGAGTATGACTTC probe CACACAAGTGCACGTTT forward primer CGGCAGCCGCGATAAG reverse primer TTAATGATGCCATCTGCATCCA probe AGCCAGTTGACTAAACAG forward primer GCTTTGCCAGAGTGACTACCT reverse primer AGGGGCTGGATGGATGTA probe CATGACCGTGGCCATCCACC forward primer CGGCTTCGACTGGGTGTACT reverse primer GCAGCCTCCCGATTTGG probe CGAGTTCTCTGCTTTCA forward primer CTCACAAAGCATGGGACACT reverse primer GAGTCTCAGGGGCCAGAA probe CCGTGGCTGACCACCACCTG forward primer GTGCTGGCCCCAATCG reverse primer GCAGCATGGTCTCGTTGATCT probe CTCATCCGAGGAGTCAT forward primer CTGCCACGGCGATGGA reverse primer ACTGCATAATAATGGGCACTGTTG probe CCTCAACACCACTCTCATGTCGGTATCTTG forward primer GCAGTGGGTAACATGAGTGTCT reverse primer AGGCACTAGAGTTGAGGAATTGT probe CTCTCCAGGCAGAGACCCCAGC forward primer GCTACGACTATGTCTCTCACATCAACT reverse primer GGCGACACTTTGATGAACCA probe ATGGCGGCTGCCCTGTCTTCATT Applied Biosystems Cat. #: Mm01162702_g1

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1000

35.00

800

25.00

700 600

20.00

500 15.00

400 300

10.00

200 5.00 100 0

35.00

7000

C ur691IC 50(μM )

HhAntag IC50 (μM)

Cur691 IC50

HhAntag IC50 PTCH expression

PTCH 30.00

6000

25.00

5000

20.00

4000

15.00

3000

10.00

2000

5.00

1000

0.00

0

colo-rectal

pancreatic

NSCLC

PTCH1 expression

0.00

SMO expression

900

HhAntag IC50 SMO expression

SM Oexpression(unit)

SMO 30.00

P TC Hexpression(unit)

Cur691IC50(μM )

HhAntag IC50 (μM)

Cur691 IC50

SCLC

Supplementary Figure 1: No correlation of PTCH1 or SMO mRNA levels (Affymetrix -derived) with HhAntag activity in cell lines www.nature.com/nature

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35.0

1.0000 Cyclopamine IC50

30.0

GLI mRNA 0.1000

0.0100 20.0 0.0010 15.0 0.0001

GLI expression (2 -Δ Ct)

cyclopamine IC 50 (μ M)

25.0

10.0

0.0000

5.0

0.0

0.0000

Cell Lines Supplementary Figure 2: No correlation of GLI1 mRNA levels (qRT-PCR) with cyclopamine activity in cell lines www.nature.com/nature

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GLI-luciferase Activity

10

8

10T1/2

6

4

2

0

Control

SHH

GLI-luciferase Activity

10

8

CFPAC-1

6

4

2

0

Control

Supplementary Figure 3: rSHH does not induce GLI luciferase reporter activity in tumor cell lines. The mesenchymal cell line, 10T1/2, serves as a positive control www.nature.com/nature

GLI-luciferase Activity

10

8

SHH

BxPC-3

6

4

2

0

Control

SHH 9

SUPPLEMENTARY INFORMATION

doi: 10.1038/nature07275

CFPAC-1 1.2

1.2

1.0

GLI -luciferase Activity

GLI -luciferase Activity

BxPC-3 1.4

1.0 0 .8 0 .6 0 .4 0 .2

0.8 0.6 0.4 0.2 0.0

0 .0

Control

0.1µM

1µM

10µM

30µM

HSV-TK luciferase Activity

HSV-TK luciferase Activity

1.2 1.0 0.8 0.6 0.4 0.2 0.0

0.1µM

1µM

10µM

30µM

Control

0.1µM

1µM

10µM

30µM

Control

0.1µM

1µM

10µM

30µM

1.0 0.8 0.6 0.4 0.2 0.0

Control

0.1µM

1µM

10µM

30µM

1.4

1.2

1.2

NFκΒ -luciferase Activity

NFkB -luciferase Activity

Control 1.2

1.4

1.0 0.8 0.6 0.4 0.2

1.0

0.8

0.6

0.4

0.2

0.0

0.0

Control

0.1µM

1µM

10µM

30µM

Supplementary Figure 4: Normalized activity of transiently transfected pathway reporter constructs in cell lines treated with Hh antagonists (solid bars, cyclopamine; open bars, HhAntag). Note that Hh antagonists inhibit multiple pathway reporters at the concentrations required to inhibit growth in these cells www.nature.com/nature 10

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N417

400000 300000 200000 100000 0 0.01

0.1

1

10

100

900000 800000 700000 600000 500000 400000 300000 200000 100000 0 0.01

HhAntag cyclopamine forskolin 5E1

0.1

HhAntag cyclopamine forskolin 5E1

0.1

1

10

200000 100000

HhAntag cyclopamine forskolin 5E1

cell viability (RLU)

cell viability (RLU)

0.1

1

10

100

Drug concentration (μM or μg/mL)

DLD1

300000 250000 200000 150000 100000 50000 1

HhAntag cyclopamine forskolin 5E1

300000

0 0.01

100

HT55 350000

0.1

100

400000

Drug concentration (μM or μg/mL)

0 0.01

10

2Rv1 cell viability (RLU)

cell viability (RLU)

Panc05-04 275000 250000 225000 200000 175000 150000 125000 100000 75000 50000 25000 0 0.01

1

Drug concentration (μM or μg/mL)

Drug concentration (μM or μg/mL)

10

Drug concentration (μM or μg/mL)

100

1400000 1300000 1200000 1100000 1000000 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 0.01

HT29 HhAntag cyclopamine forskolin 5E1

0.1

1

10

Drug concentration (μM or μg/mL)

100

350000

cell viability (RLU)

cell viability (RLU)

HhAntag cyclopamine forskolin 5E1

500000

cell viability (RLU)

HUCCT 600000

HhAntag cyclopamine forskolin 5E1

300000 250000 200000 150000 100000 50000 0 0.01

0.1

1

10

100

Drug concentration (μM or μg/mL)

Supplementary Figure 5a: Hh pathway antagonist-mediated growth inhibition in select cell lines used in previous studies and/or colo-rectal cell lines used for in vivo studies in the accompanying manuscript www.nature.com/nature

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GLI1 mRNA expression (fold CTRL)

10 9 CTRL

8

SHH (1ug/ml)

7

HhAntag (10uM) SHH (1ug/ml) + HhAntag (10uM)

6

cyclopamine (10uM)

5

SHH (1ug/ml) + cyclopamine (10uM)

4

5E1 (10ug/ml)

3 2 1

nd

nd

0

DlD-1

HT55

HT29

Panc05.04

Panc-1

22RV1

N417

HUCCT

CCD18CO (fibroblast control)

Supplementary Figure 5b: Lack of Hh pathway activity in vitro in select cancer cell lines used in previous studies and/or colo-rectal cell lines used for in vivo studies in the accompanying manuscript; nd: not detected www.nature.com/nature

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100.000

c

SHH Expression (2-ΔΔCt)

a 10.000

d

b

e

1.000

0.100

0.010

0.001

IHH Expression (2-ΔΔCt)

1000.000 100.000 10.000

c

d

a b

e

1.000 0.100 0.010 0.001

pancreatic

colo-rectal

SCLC

NSCLC

Supplementary Figure 6. Quantitative expression of SHH and IHH mRNA in a panel of tumor cell lines. Expression values (2-ΔΔCt) are relative to gene expression determined from a pool of cell lines representative of multiple tissue types. The arrows and letters indicate the pancreatic cell lines HPAF-II (a) and PL45 (b), and the colon cell lines HT55 (c), HT-29 (d) and DLD-1 (e). www.nature.com/nature 13

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100

100

Panc 10.05

60

60

40

40

40

20

20

20

0 100

101 102 FL2 LOG: FL2 LOG

100

0

103

0 100

100

HPAF-II

101 102 FL2 LOG: FL2 LOG

103

100

60

40

40

20

20

20

100

101 102 FL2 LOG: FL2 LOG

100

103

0

0 100

101 102 FL2 LOG: FL2 LOG

100

DLD-1

100

103

80

80

60

60

60

40

40

40

20

20

20

101

102

103

104

0 100

101

102

103

101 102 FL2 LOG: FL2 LOG

100

HT-29

80

0 100

PANC-1

60

40

0

103

80 % of Max

% of Max

60

101 102 FL2 LOG: FL2 LOG

100

Panc 03.27

80

80

CFPAC-1

80 % of Max

% of Max

% of Max

80

60

% of Max

cell number (normalized)

80

100

BxPC3

104

0 100

103

HT-55

101

102

103

104

Fluorescence Intensity Supplementary Figure 7. Cell surface expression of Hh ligands in a panel of pancreatic and colon cancer cell lines determined by FACS analysis (mAb 5E1, black line). Grey shade represents isotype-matched control Ab staining. www.nature.com/nature

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HPAF-II

HPAF-II

PL45

PL45

Supplementary Figure 8: Stromal activation of the Hh pathway in HPAF-II, but not PL45, xenografts in vivo. Additional sections demonstrating β-galactosidase activity (red) in the stromal compartment following implantation of HPAF-II cells in Ptch1-lacZ;RAG2-/- mice with serial sections taken for hematoxylin and eosin stain (right panels). Epithelial-specific antigen staining is depicted in green. www.nature.com/nature

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A.

B.

C.

Supplementary Figure 9: Rapid infiltration of murine stromal cells in primary human xenograft models A. Primary human tumor hybridized with a mouse chromosome X painting probe fails to detect any positive cells within the tumor (arrow head) or stroma (arrow). B. Second passage xenograft tumors derived from this primary tumor reveal strong and ubiquitous hybridization within the stromal compartment (arrow) while the tumor cells (arrow head) fail to hybridize. C. Immunofluorescence of a separate section from the same xenograft was double labeled with BerEP4 (green) and vimentin (red) to highlight the tumor and stromal compartments, respectively. Hybridization was carried out as suggested by the manufacturer (Catalog 1189XMF-02, Cambio Ltd, 1 The Irwin Centre, Scotland Road, Dry Drayton, Cambridge, CB23 8AR). www.nature.com/nature

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hGLI1 / mGli1 Expression (2-ΔCt)

1.0000

0.1000

0.0100

0.0010

0.0001

0.0000

Human

Mouse

Supplementary Figure 10:Comparison of the GLI1 mRNA expression levels originating from the tumor epithelium and stromal microenvironment in human primary tumor implants. The red boxes reflect the human and mouse GLI signal respectively in a reference source representative of a pool of tissue types. www.nature.com/nature

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Supplementary Figure 11: Maximal inhibition of Gli1 in vivo is observed at concentrations required to inhibit tumor growth (ie. 100mg/kg) following HhAntag treatment www.nature.com/nature

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Smo relative expression

1.20

1.00

0.80

0.60

0.40

0.20

0.00

rSHH: tam:

-

+ Smoc/c MEFs

+

+ +

-

+ -

+

+ +

CAGGCre-ER;Smoc/c MEFs

Supplementary Figure 12: Reduction in Smo mRNA expression in tamoxifen treated MEFs derived from CAGGCreER;SmoC/C mice www.nature.com/nature

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Supplementary Figure 13a: Synthesis of HhAntag (N-[4-Chloro-3-(5dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide) N-[4-Chloro-3-(5-dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxybenzamide. N

Cl N N H

2HCl

O HN O O

1). 2-chloro-N-(5-(dimethylamino)-2-nitrophenyl)-5-nitrobenzamide. O

Cl

Cl

Cl N

O

NH2 NO2

NO2 ACN

N

NH

NO2

NO2

Dissolve N,N-dimethyl-4-nitrobenzene-1,3-diamine in ACN (50g/L) in a 3-neck flask equipped with overhead stirring and an addition funnel under nitrogen. Dissolve 2-chloro-5-nitrobenzoyl chloride in ACN (35g/100mL; 1.1equ.) and add drop-wise to the aniline while keeping the temperature under 25oC. After addition is complete, heat the reaction mixture to 75oC for 1 hour. (Note: LC-MS should indicate >95% product.) Cool reaction mixture to 0oC, filter, rinse with cold ACN and dry (Yields ~90%).

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Cont.

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Supplementary Figure 13b: Synthesis of HhAntag (N-[4-Chloro-3-(5dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide)

2). 2-(5-amino-2-chlorophenyl)-N,N-dimethyl-1H-benzimidazole. Cl O N

NH NO2

NO2

SnCl2 EtOH

N

Cl N N H

NH2

Dissolve 2-chloro-N-(5-(dimethylamino)-2-nitrophenyl)-5-nitrobenzamide in EtOH (50g/L) in a 3-neck flask equipped with overhead stirring and a reflux condenser and heat to 40oC. When temperature reaches 40oC, add 1st aliquot SnCl2/HCl (~7 equ. and 3 vol respectively, divided into 3 portions) and heat to 60oC. When the temperature reaches 60oC, add the 2nd aliquot of SnCl2/HCL and heat to 80oC. When the temperature reaches 80oC, add the 3rd aliquot SnCl2/HCl and heat to reflux (~87oC) for 2 hours). (Note: LC-MS should indicate >95% product.) Cool the reaction mixture to 0oC and begin NaOH addition (~30equ) while keeping the temperature under 10oC. When NaOH addition is complete, the pH is 12-13 and solution is white. Dilute the reaction mixture with EtOAc and 2.5M NaOH, stir overnight and extract. Back extract the aqueous layer with EtOAc, combine the organics and wash with NaOH followed by brine. Dry the organic layer with Na2SO4, concentrate and dry (Yields ~75% from the first crop).

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Cont.

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Supplementary Figure 13c: Synthesis of HhAntag (N-[4-Chloro-3-(5dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5-dimethoxy-benzamide) 3). (N-[4-Chloro-3-(5-dimethylamino-1H-benzoimidazol-2-yl)-phenyl]-3,5dimethoxy-benzamide. O O

Cl

N

N

Cl

O

N N H

THF

Cl N N H

2HCl

O HN

NH2

O O

Dissolve 2-(5-amino-2-chlorophenyl)-N,N-dimethyl-1H-benzimidazole in THF (25g/L) in a 3-neck flask equipped with overhead stirring and an addition funnel under nitrogen and cool to 0oC. Dissolve 3,5-dimethoxybenzoyl chloride in THF (50g/300mL; 1.2 equ.) and add dropwise while keeping the temperature under 10oC. After addition is complete, stir for an additional 30 minutes. Add additional THF to maintain consistency. (Note: LC-MS should indicate >95% product.) Dilute the reaction mixture with diethyl ether (1/2 volume of existing THF) and collect the solid by filtration. Dissolve the solid in EtOAc and extract with NaHCO3. Back extract the aqueous layer with EtOAc, combine the organics and wash with NaHCO3 followed by brine. Dry the organics with Na2SO4 and then concentrate to dryness (Yields ~60-70%; ~50% from the first crop). Dissolve the freebase in ACN (50g/1.5L) and heat to 60oC. When the solution is homogeneous, cool to 0oC and add 2.5N HCl dropwise. After HCl addition is complete, filter the product and dry in a vacuum oven (Yield from freebase ~95%).

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Characterization: 1H-NMR (400 MHz, CDCl3): δ 10.88 ppm (s, 1H), 8.51 ppm (d, 1H), 8.12 (m, 1H), 7.95 (bs, 1H), 7.88 (m, 1 H), 7.71 (m, 2H), 7.17 (d, 2H), 6.68 (t, 1H), 3.79 (s, 6H), 3.15 (s, 6H). MS (m/z): [M]+ calcd for C24H23ClN4O3, 450.15; found 450.2.

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