Synthesis of Small Molecule Inhibitors of N-WASP

7/30/2019 Synthesis of Small Molecule Inhibitors of N-WASP

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Synthesis of small moleculeinhibitors of N-WASP

Frances P. Rodrguez RiveraGiovanny Santana Green

Department of Chemistry

Mayra Pagn, Ph.D.

Claudia Ospina, Ph.D.


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Background

Group of proteins involved in

regulating the actin

cytoskeleton and cell migration

through induction ofmembrane protrusions at the

leading edge.

The human WASP family

currently has five members:WASP, N-WASP, WAVE1,

WAVE2, and WAVE3.

N-WASP is over expressed in

cancer cells.

Wiskott-Aldrich Syndrome Protein (WASP)1

Family

Figure 1. Crystal structure of N-WASP

in complex with skelectal actin


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Background

N-WASP stimulates actin polymerization by binding to and

activating the Arp2/3 complex

Inhibition of the activation of N-WASP is an ideal therapeutic

target for metastatic breast cancer.


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Background

Wiskostatin inhibits N-WASP activity with and IC50 = 10M.

This compound blocks N-WASP activity via stabilization of

the auto-inhibited conformation.

Wiskostatin Strucuture

Molecular Weight426.14562 [g/mol]

Molecular FormulaC17H18Br2N2O


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BackgroundMolecular modeling studies to predict binding modes of

N-WASP inhibitors**

** Results obtained by Dr. Hernndez, UPR-Medical Sciences Campus.


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Objectives

Synthesize new small molecule 1,4,5-trisubstituted-1,2,3-

triazole derivatives

Synthesize and characterize intermediate molecules

needed to obtain target molecule.

Perform the synthesis of triazoles via click chemistry

methodology.6

Optimize reaction conditions in order to synthesize similar

N-WASP inhibitors.

Investigate the binding and inhibitory potential of novel

compounds as N-WASP inhibitors.


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Design and Synthesis

IUPAC name1-[4-(7-chloroquinolin)]-4-(1-hydroxybutil)-5-[4-(metil-N-morpholine)benzene]-1,2,3-triazole

1

2Substructure

Substructure


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Experimental Scheme forSubstructure Synthesis

PBr3

0C

EtMgBr

THF

NaN3 /DMF

100C

Triton B

rt

1

2


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Experimental Scheme for TargetMolecule Synthesis

1

2


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Results

Synthesis and characterization of

4-azido-7-chloroquinoline

4-ethynylbenzyl bromide


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Synthesis and Characterization

Substrate 1: 4-azido-7-chloroquinoline

70 C

3 h 57.1%


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Chromatographic separation of 4-azide-chloroquinoline

Fractions: 61-95


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NMR Spectra for 4-azide-chloroquinoline in CDCl3

H1 NMR Spectrum (400 MHz)

C13 NMR Spectrum

(125 MHz)


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Infrared Spectrum of 4-azide-chloroquinoline


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Synthesis and Characterization

Substrate 2: 4-ethynylbenzyl bromide

76.45%

N2


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NMR Spectra for 4-ethynylbenzyl bromide in CDCl3

H1 NMR Spectrum (400 MHz)

C13 NMR Spectrum

(125 MHz)


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Infrared Spectrum of 4-ethynylbenzyl bromide


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Conclusions

Steps of the formation of the grignard reagent andthe synthesis of the triazole via click chemistry will be

the last steps to obtain target molecule.

Future work includes optimization of reaction

conditions in order to synthesize alternative N-WASPinhibitors

Substructures 1 and 2 were synthesized with a 57.1%

and 76.45% yield, respectively. Both compounds were

characterized by IR and NMR spectroscopy.

Separation by column chromatography and organic

synthesis under an inert atmosphere were useful

techniques acquired.


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Aknowledgements

Mayra Pagn, PhD

Claudia Ospina, PhD

Eliud Hernndez, PhD

Lab technicians

RISE (financial support)

III (financial support)


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References

1. (a) Worthylake, R.A., Lemoine, S., Watson, J.M., Burridge, K. RhoA is required for

monocyte tail retraction during transendothelial migration. J Cell Biol 2001, 154: 147160. (b) Takenawa, T., Miki, H. WASP and WAVE family proteins: key molecules for rapidrearrangement of cortical actin filaments and cell movement. J Cell Sci 2001, 114:18011809.

2. Rohatgi, R., Ma, L., Miki, H et al. The interaction between N-WASP and the Arp2/3complex links Cdc42-dependent signals to actin assembly. Cell 1999, 97, 221231.

3. (a) Khurana, S. Role of actin cytoskeleton in regulation of ion transport: examples fromepithelial cells. J Membr Biol 2000, 178, 7387. (b) Stamnes, M. Regulating the actincytoskeleton during vesicular transport. Curr Opin Cell Biol 2002, 14, 428433.

4. (a) Peterson, J.R., Bickford, L.C, Morgan, D., Kim, A.S, Ouerfelli, O., Kirschner, M.W,Rosen, M.K. Chemical inhibition of N-WASP by stabilization of a native autoinhibitedconformation. Nature Structural & Molecular Biology2004, 11, 747-755. (b) Chan A.Y.,Raft, S., Bailly, M., Wyckoff, J.B., Segall, J.E., Condeelis, J.S. EGF stimulates an increase

in actin nucleation and filament number at the leading edge of the lamellipod inmammary adenocarcinoma cells. Journal of Cell Science1998, 111, 199-211.

5. Guerriero, C. J., Weisz, O. A. N-WASP inhibitor wiskostatin nonselectively perturbsmembrane transport by decreasing cellular ATP levels. Am J Physiol Cell Physio 2007,292, C1562C1566.

6. Krasinski, A., Fokin, V. V., Sharpless, B. K. Direct synthesis of 1,5-disubstituted-4-magnesio-

1,2,3-triazoles, revisited. Organic Letters, 2004, 6 (8), 1237-1240.


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Synthesis of small moleculeinhibitors of N-WASP

Frances P. Rodrguez RiveraGiovanny Santana Green

Department of Chemistry

Mayra Pagn, Ph.D.

Claudia Ospina, Ph.D.


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Spectra of starting materials


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Infrared Spectrum of 4,7-dichloroquinoline


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Organic Synthesis: Overview

Intermediate Molecule Synthesis

Work up

Filtration

Extraction

Purification

Silica Gel Column Chromatography

Characterization

Infrared and UV Spectroscopy

Nuclear Magnetic Resonance (NMR)


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Background

N-WASP, Arp2/3 complex, activator, inhibitor interactions

Arp2/3

Arp2/3

Cdc42

Native conformation

Inhibited conformation


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Triazole formation

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Synthesis of Small Molecule Inhibitors of N-WASP