Mirza PhD defense on the Ugi reaction for anti-malarial screening

Khalid Baig Mirza Advisor : Prof. Jean-Claude Bradley Drexel University Prof Frank Ji (Chair) , Prof. Susan A. Varnum, Prof. Sally Solomon, Prof Peter Wade, Prof. Louis Scerbo, Prof. Jun Xi

Introduction
“ Open Notebook Science” is a term used to indicate that the primary record of a research in its entirety is made public over the internet as soon as it is conducted
Achieves and advocates complete transparency of research being performed
Serve as an open invitation to collaborate with like minded researchers / scientists who are capable and willing to participate in the project
The planned experimental procedure, the log, raw data, and a discussion of the analysis of the data, the assumptions and the conclusions drawn from the specific experiment and the project in general made public in real time.
The UsefulChem project – A collaborative effort to find a treatment/cure to malaria
Tools used – Wikis, blogs, YouTube, FriendFeed, Google Docs, JSpecView, ChemSpider, Mendeley, Online repositories, etc

Why Open Note Book Science?

Justification for the Open Notebook Science approach Oxidation of benzylic alcohols using sodium hydride
NaH is commonly used as a base, eg. enolate chemistry 1
References 1 Hudrlik,P., F.; Takacs, J., M.; J. Org. Chem; 43; 20; 3861-3865 (1978) 2 McConaghy Jr. J., S.; Bloomfield, J., J,; J. Org. Chem; 33; 9; 3425-3428 (1968) To a lesser extent it has also been used as a reducing agent 2

Sodium hydride as an oxidizing agent
In a JACS publication last year, Wang, et. al. reported that benzylic type
secondary alcohols under go oxidation when treated with NaH (2eq) in THF at
room temperature. Several examples were given 3 .
Well picked-up in the blogosphere community, facilitated by social networking
websites like Friendfeed and others.
References 3 Wang, X., Zhang, B., Wang, D., Z.; J Amer. Chem Soc ; DOI: 10.1021/ja904224y; (2009)

Wang’s ketones...

Experiments repeated by the scientific community from the blogosphere
Totally synthetic a :
UsefulChem b :
Experimental details were comprehensively documented with
photos and videos
a) Paul Docherty- totallysynthetic.com/blog/?p=1903; (12/05/2010) b) Khalid Mirza, Marshall Moritz, Jean-Claude Bradley; UsefulChem.wikispaces.com/Exp243. Reaction monitored before quenching; (12/05/2010)

Green: 1-phenylethanol Blue: acetophenone Red: product HNMR Overlay PPM

Green: acetophenone Blue:1-phenylethanol Red: product IR Overlay

After 19h no conversion to acetophenone was observed, paper mentioned 75% GC yield
However, totallysynthetic.com reported at 15% conversion to the ketone. Wang reported 85% isolated yield
A comment made by a reader on another blog Carbon based curiosities a mentions a JOC paper from 1968 which address the issue.
a http://www.coronene.com/blog/?p=842 Results

Lewis E., G.; J. Org. Chem ; 30; 7; 2433–2436 (1965) Scientific communication through social networks- Blog

Wang’s paper withdrawn from JACS
Social networking can/has played an important role in the development of science
Social networking + collaborative effort => unforeseen innovations (Chemistry, Medicine, Engineering, .. data curation as well.
Consequences

The UsefulChem Project

Malaria – Some Facts
Reported cases: 190-311m [ 2008, CDC]
Deaths: 708,000 - 1,003,000 [2008, WHO]
Half the worlds population (3.3 billion) susceptible to malaria.[CDC]
Second leading cause of deaths from infectious diseases in Africa after HIV/AIDS [CDC]
Approximately 1500 cases of malaria are reported in the US annually [CDC]
Drug resistance of Plasmodium falciparum to chloroquin, sufladoxine-pyrimethamine are well known. [WHO]
New anti-malarial agents needed.

Collaboration with Find-a-Drug
Obtained a small library of diketopiperazines designed to inhibit enoyl-reducatase, an essential enzyme for the fatty-acid metabolism of Plasmodium falciparium.
Target diketopiperazine Diketopiperazine library

Reterosynthesis of the target diketopiperazine
All starting materials except 3, 4-dihydrophenyl acetaldehyde (6) were commercially available.
Therefore a synthetic procedure had to be designed.

Proposed mechanism for the dehydration of adrenaline to DOPAL

Ugi reaction The reaction did not produce the desired Ugi product

Monitored the reaction with similar simpler reactants such as phenylacetaldehyde and boc-glycine
Mechanistic understanding of the reaction using NMR monitoring
Reactivity of the alpha protons on phenylacetaldehyde was one of the main reasons for the failure of reaction.
Monitoring the Ugi reaction

A Mechanistic Insight

Imine kinetics a- Alicia Holsey, b- James Giammarco, c- Sean Gardner 0.1043 c CD3OD 5-methylfurfurylamine 3,4,-dihydroxybenzaldehyde 0.008 b CD3OD t-butyl amine piperonal 0.1552 a CD3OD 5-methylfurfurylamine piperonal 0.07 a CDCl3 5-methylfurfurylamine piperonal 0.106 CD3OD 5-methylfurfurylamine veratraldehyde 0.01 CDCl3 5-methylfurfurylamine veratraldehyde Imine formation rate constant 1/(M*min) solvent amine aldehyde

Ugi reaction

Fufuryl Cleavage

Attempt at 2,5-diketopiperazine synthesis
The UDC strategy- Ugi deboc cyclization 4
4 Hulme, C.; Morrissette, M.; Volz, F., A.; Burns, C.; Tett. Lett. 39; 10; 1113-1116; (1998)

Furfuryl Cleavage The unusual elimination was seen in Ugi products containing the N-furfuryl group.

Reactivity of furan derivatives acidic media Butin, A.,V; Stroganova, T.; Lodina, I., V.; Krapivin, G., D.; Tet. Lett; 42; 10; 2031-2033; (2001) Reissert modification-indole synthesis

Furan ring opening with lewis acid Piancatelli, G.; Scettri, A.; David, G.; D’Auria, M.; Tetrahedron; 34; 18; 2775-; (1978)

1, 6-Hoffman elimination of quaternary amines Appropriately substituted quaternary ammonium hydroxides undergo loss of trimethyl amine across a furan or thiophene to unusual cyclic conjugated trienes, there dimmers and polymers Winberg, H., E.; Fawcett, F., S.; Mochel, W.,E.; Thebald, W., C.; J. Amer. Chem .Soc ; 82; 6; 1428-1435; (1959)

Furfuryl cleavage in Ugi products
Observed 5-methyl-2-furfuryl group on nitrogen of a tertiary amide, in the Ugi products.
Undergo a 1-6- elimination to yield a secondary amide when treated with trifluoroacetic acid in CDCl 3 or CD 3 OD

k = 0.9x10-3 min-1 k = 0.9x10 -3 min -1

k = 1.2x10 -3 min -1

k = 0.9x10 -3 min -1

Proposed mechanism for furfuryl cleavage

No methyl group- The cleavage is a thousand times slower than the methyl furfuryl analogs cleavage under the same conditions k= 4x10 -6 min -1 A different mechanism needed... Non-methylated furfuryl analog cleavage

Mechanism for the furfuryl cleavage for the non methylated analog

Optimization of the Ugi reaction

Automated optimization of a Ugi reaction*
The reaction is usually carried-out at room temperature while the product sometimes precipitates out from the reaction mixture.
When it precipitates out, the product is then just filtered and washed with the solvent.
The product precipitation although not consistent is very desirable when performing a scale-up, essentially eliminating an expensive chromatographic purification process.
In order to obtain high yield and purity of the product optimization studies were performed on a Ugi reaction, where the product was filtered directly and washed with the solvent.
Performed in collaboration with Prof. Kevin Owens, Drexel University

Automation performed using the 48-slot Mettler-Toledo MiniBlock quipped with filtration tubes.
Automation

Objective: To optimize the conditions to obtain highest yield
Concentration (0.4, 0.2, 0.07M)
Solvent (methanol, ethanol, acetonitrile and THF)
Excess of some reagents (1.2eq)
Parameters under consideration

Reactions performed in little tube with filters at the tip
Robot added the four components and solvent
Precipitated product then washed, weighed and NMR analyzed to confirm
Mettler-Toledo MiniBlock System

Results of the Optimization study Statistical analysis: Prof. Kevin Owens

Results of the Optimization study
Methanol and ethanol best solvents at 0.2M reagent concentration
Yields decreased from 0.2M to 0.07M in MeOH, EtOH & AcCN. Similar yields at 0.2M and 0.4M concentrations in methanol
In EtOH and MeOH higher yields resulted with imine or isonitrile excess. In AcCN amine, aldehyde or isonitrile excess gave better result. In THF imine excess resulted in higher yields. Over yields significantly lower for THF than other solvents
Significant interactions between the solvent choice, reagent concentration and identity of the reagent in excess found

Best yield was 66% at 0.4M in methanol with imine excess (1.2eq).
This was significantly higher than the initial reaction under equimolar conditions (49%)

Library Synthesis

Library synthesis
Most reactions were performed for the products which were predicted to possess some biological activity.
Docking and modeling studies were conducted by collaborators, Dr. Rajarshi Guha and Prof. Andrew Lang
Results of the ranked virtual library of Ugi products were provided in the form of smiles
Based on the ranking Ugi products were synthesized
The compounds were then sent for testing against facipain-2, an enzyme used by the malarial parasite to break down hemoglobin. This was done in collaboration with the Rosenthal group at UCSF

Ugi product precipitation
Very few Ugi reactions yielded solid products
Empirical modeling studies were performed by Prof. Andy Lang (ORU) to predict the likelihood of Ugi product precipitation.
Reactions were performed and the results were incorporated to further improve the model.
Ugi reactions performed – 511 Ugi reactions that precipitated a product – 104 (20%) Precipitate confirmed to be Ugi products – 65 (13%) Precipitate not a Ugi product – 8

Ugi reaction precipitation trends Correlation in terms of isocyanide Carboxylic acid – boc-glycine 35 % 0 0 107 tosylmethyl isocyanide 11 23 215 t-butyl isocyanide 4 5 120 n-butyl isocyanide 13 10 77 cyclohexyl isocyanide 7 3 43 benzyl isocyanide 0 0 13 2-morpholinoethyl isocyanide 0 0 4 2-chloro-6-methyl phenyl isocyanide 0 0 38 1-pentyl isocyanide 0 0 32 1,1,3,3-tetramethylbutyl isocyanide Percent precipitation number of product precipitations Number of reaction Isocyanide

Tosylmethyl isocyanide (TOSMIC)..
TOSMIC was used in 107 Ugi reactions due to its odor-free nature
No solid Ugi products were obtained.
Poor solubility of TOSMIC in most organic solvents
Reactions had to be performed at lower concentrations
Reactivity towards aldimine in basic conditions

Anti-malarial activity results

Anti-malarial activity
Ugi products show inhibition of falcipain-2, cystein protease inhibitor used by the malarial parasite, Plasmodium falciparum to degrade erythrocytic proteins especially hemoglobin.
Show inhibition of the Plasmodium falciparum.
UsefulChem top inhibitor IC 50 8.4uM

Solubility

Polyaromatic components in Ugi reactions
1-pyrenebutyric acid

Solubility issues 0.14 toluene 17 0.104 methanol 16 0.07 hexane 15 0.44 ethyl acetate 14 0.1 ethanol 13 0.1 diethyl ether 12 0 dichloromethane 11 0.03 cyclopentane 10 0.07 cyclohexane 9 0.04 chloroform 8 0.66 benzene 7 0.153 acetonitrile 6 1.29 THF 5 0.77 DMSO 4 1.25 DMF 3 0.07 2-propanol 2 0.02 1,1,2-trichlorotrifluoroethane 1 Ave. (M) Solvent Solubility of phenanthrene-9-carboxaldehyde 0 toluene 16 0.014 methanol 15 0 hexane 14 0.06 ethanol 13 0.02 diethyl ether 12 0.07 dichloromethane 11 0 cyclopentane 10 0 cyclohexane 9 0.03 chloroform 8 0 carbon tetrachloride 7 0 benzene 6 0 acetonitrile 5 0.55 THF 4 2.067 DMSO 3 1.88 DMF 2 0.06 2-propanol 1 Ave. (M) Solvent Solubility of 1-pyrenebutyric acid

Solubility..
Poor solubility of reactants resulted in decreased reagent concentration
Reduced likelihood of product precipitation
Need for a solubility model, to predict selective product precipitation
Need to measure solubility of several reactants and Ugi products in different organic solvents at room temperature
ONSChallenge a collaborative effort with Prof. Andew Lang (ORU) and Dr. Rajarshi Guha

Methods for solubility assessment
The shake flask method to saturate a solution
UV-VIS, HPLC, GC
Turbidimetry and nephlometry
Differential Scanning Calorimetry

Methods we used
Speed-Vac method

Hemiacetal of 4-nitrobenzaldehyde in methanol (3:1 aldehyde: hemiacetal ratio) Solute- Solvent reaction

2-chloro-5-nitrobenzaldehyde in methanol (2:3 aldehyde: hemiacetal ratio) Solute- Solvent reaction

NMR method and Semi Automated Measurement of Solubility (SAMS)
Web-service created by Prof. Bradley and Prof. Lang using Google spreadsheet.
Based on HNMR of a very small volume of the supernatant in deuterated solvent
JCAMP dx file of the spectrum is uploaded to the server
Density and molar mass of the solute and solvent is incorporated
Predicted densities from ChemSpider.com used for solids
A range in the solute to integrate with the number of corresponding H
A range in the solvent to integrate with the number of corresponding H

SAMS..
Spreadsheet calculates the molar ratio of solute to solvent
Molarity calculation is then done by assuming that the volumes of the two components are additive.

Problem..
Inconsistent integration of aromatic and non aromatic protons observed
Increasing the relaxation delay d1 from 0.3s to 50s resulted in uniform peak integrations per proton over the entire spectrum

Conclusions
An open, collaborative and more transparent approach to scientific research has been established
A mechanistic understanding in to the ‘furfuryl cleavage’ has been achieved
Conditions necessary for the Ugi reaction has been optimized
Library of Ugi reactions have been performed
Synthesis of potential anti-malarial agents accomplished
Solubility of the reagents and Ugi products determined to facilitate the successful construction of a model to predict the selective precipitation of the Ugi products from a reaction

Future work
Convenient web services for solubility measurement and prediction
Virtual library generator
Predictive Toxicology
Virtual docking
Integration of Multiple Web Services to expedite the process of drug discover.

Books published..

Acknowledgements
A very patient advisor – Prof. Jean-Claude Bradley
My Parents and families support
Committee members- Prof Frank Ji, Prof. Susan Jansen Varnum, Prof. Sally Solomon, Prof Peter Wade, Prof. Louis Scerbo and Prof. Jun Xi, Prof. Robert Hutchins (Late)
Collaborators – Prof. Kevin Owens, Dr. Rajarshi Guha, Prof. Andrew Lang, ONSChallenge Judges & Tom Osborne.
Prof. Lynn Penn, Prof. Anthony Wambsgans
Ed Doherty, Virginia Nesmith, Tina Lewinski, Ed Thorne, Tim Wade
Co-workers - Alicia Holsey, James Giammarco, Sean Gardner, Emily Messner, Shannon Oseback, Tim Bohinski, Cedric Tschakounte, Marshall Moritz.
Renata Szyszka, Neil Mukherjee, Sudipto Das, David Berke-Schlessel , Kerry Drake, Jonathan Soffer, Addy Kojtari, April Holcomb, Bill Erb, Jim Reiben, Molly O’connor, Tyson Reeves, Christopher Castillo, Nick Paparoidamis, Hung Le, Tom Measy, Andrew Haggarman, Joseph Depasquale, Natalie Dixon, Mukesh Kumar, Ismael Nieto, Marcela Garcia, KimChi Nguyen, Kyle Hess, William Hunt, Arben Kojtari, Michelle Livings, Chi Nguyen, Siobhan Toal and others I missed..

Questions ?
Thank You

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Mirza PhD defense on the Ugi reaction for anti-malarial screening

by Jean-Claude Bradley on Dec 13, 2010

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Mirza PhD defense on the Ugi reaction for anti-malarial screening — Presentation Transcript