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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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
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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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doi: 10.1038/nature07275
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.
22