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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 chemistry1
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)
O O-Na
+
NaH
To a lesser extent it has also been used as a reducing agent2
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 given3.
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 synthetica :
UsefulChemb:
Experimental details were comprehensively
documented with photos and videos
CH3
OH
CH3
O
2eq NaH
THFx
CH3
OH
Cl
CH3
O
Cl
2eq NaH
THF
15%
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)
CH3
OH
CH3
O
2eq NaH
THF, 19h
1-phenylethanol acetophenone
Green: 1-phenylethanolBlue: acetophenoneRed: product HNMR Overlay
PPM
Green: acetophenoneBlue:1-phenylethanolRed: product
CH3
OH
CH3
O
2eq NaH
THF, 19h
1-phenylethanol acetophenone
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 curiositiesa mentions a JOC
paper from 1968 which address the issue.
ahttp://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
OH
OH
O
O
CH3
NH2
N+
-C
O N CH3O
O
NH
S
CH3
NH
OO
CH3CH3
CH3
OH
OHmethanol
OO
CH3CH3
CH3
NH
O OH
S
CH3
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
aldehyde amine solvent
Imine formation rate constant 1/(M*min)
veratraldehyde 5-methylfurfurylamine CDCl3 0.01
veratraldehyde 5-methylfurfurylamine CD3OD 0.106
piperonal 5-methylfurfurylamine CDCl3 0.07 a
piperonal 5-methylfurfurylamine CD3OD 0.1552 a
piperonal t-butyl amine CD3OD 0.008 b
3,4,-dihydroxybenzaldehyde 5-methylfurfurylamine CD3OD 0.1043 c
a- Alicia Holsey, b- James Giammarco, c- Sean Gardner
Ugi reactionaldehyde amine carboxylic acid isocyanide Ugi product % Yield
H3CO
H3CO
CHO
O
NH2
CHO
CHO
CHO
O
NH
O
O
CH3
CH3
CH3N
O
O NH
CH3 CH3
CH3
ONH
O
O
CH3
CH3
CH3
N
O
ONH
CH3
CH3
CH3
OCH3H3CO
CH3CH3
CH3
NH O
N
(H2C)6CH3
O
CH3
CH3CH3
CH3
NH O
N
O
CH3
CH3
CH3 CH3
N+ C
-
O
O
CH3
CH3
CH3
NHO
OH
O
O
CH3
CH3
CH3
NHO
OH
CH3
CH3 CH3
N+ C
-
CH3
CH3 CH3
N+ C
-
CH3
CH3 CH3
N+ C
-
NH2
OH
O
CH3
CHO
O
O
CH3
CH3
CH3
NHO
OH
N+
C-
CH3(CH2)5
NH2
OH
O
CH3
O
NH2
CH3
N+
C- ONH
O
O
CH3
CH3
CH3
N
OCH3
ONH
OCH3
OCH3
O
NH2
H3CO
H3CO
CHO
O
O
CH3
CH3
CH3
NHO
OH 37
44
18
31
50
58.5
8
9
10
11
12
13
O
NH2
O
NH
O
O
CH3
CH3
CH3
N
O
O
NH
Fufuryl Cleavage
Attempt at 2,5-diketopiperazine synthesis
The UDC strategy- Ugi deboc cyclization4
R1
CHO
R4
NC-
Ugi reaction (4CR)
methanol, rt
Ugi product
10% TFA / CDCl 3 or CD3OD
10% TFA / CDCl 3 or CD3OD
xO
NH2
CH3 OCH3
N
O
NHO
R1
R4
NHBoc
R3
diketopiperazineCO2H
NHBoc
R3
NH
O
NHO
R1
R4
NH2
R3
OCH3
N O
O NH
R1
R3
4Hulme, C.; Morrissette, M.; Volz, F., A.; Burns, C.; Tett. Lett. 39; 10; 1113-1116; (1998)
CHO
H3CO
H3CO
CHO
O
O
CHO
ONH2
ONH2CH3 BocHN CO2H
NC-
CH3
NC-CH3
CH3
R1
CHO R2
NH2 R3
CO2H R4
NC-
OCH3
N
O
NHO
R1
R4
NHBoc
R3
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
O
N+(CH3)3
R
OCH2 CH2
R= H, CH3, C6H5
O
x
OO
furan analog of 2,2-[paracyclophane]
polymerization inhibitors20
21
22
23
Appropriately substituted quaternary ammonium hydroxides undergo loss of trimethyl amine across a furan or thiophene to unusual cyclic conjugated trienes, there dimmers and polymers
OCH2 CH2 N
+(CH3)3
HOH-
OCH2 CH2
N(CH3)3+ OH2+
21 2420
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 CDCl3 or CD3OD
N
R2
R1
OO
CH3
k = 0.9x10-3 min-1
k = 0.9x10-3 min-1
First Order Kinetics for 1,6-methyl furfuryl elimination
y = -0.0009x - 1.5593
-4.5
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
0 500 1000 1500 2000 2500 3000 3500
time (min)
ln[c
on
c]
(M)
H3CO
H3CO
N
O
NHBoc
NH
O
O
CH3
TFA-
H3CO
H3CO
NH
O
NH3+
NH
O10% TFA /CDCl3
11 13
k = 1.2x10-3 min-1
k = 0.9x10-3 min-1
Proposed mechanism for furfuryl cleavage
TFA-
10% TFA /CDCl 3
R1
N
O
NHBoc
NH
O
O
CH3
R2
R1
N+
O
NH3+
NH
O
O
CH2
R2
H
H
HR
1
NH
O
NHBoc
NH
O
R2
H
CH2
O
CH2
polymer
+10% TFA /CDCl 3
10% TFA /CDCl3
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 cleavagefor the non methylated analog
50% TFA /CDCl 3
R1
N
O
NHBoc
NH
O
O
R2
R1
N+
O
NH3+TFA
-
NH
O
O
R2
H
H50% TFA /CDCl 3
R1
NH
O
NH3+TFA
-
NH
O
R2
H
CH2
O+
Polymer
+
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 tipRobot added the four components and solventPrecipitated 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 synthesisMost 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 – 511Ugi reactions that precipitated a product – 104 (20%)Precipitate confirmed to be Ugi products – 65 (13%)
Precipitate not a Ugi product – 8
Ugi reaction precipitation trendsCorrelation in terms of isocyanide
Isocyanide
Number of
reaction
number of product
precipitations
Percentprecipitat
ion
1,1,3,3-tetramethylbutyl
isocyanide 32 0 0
1-pentyl isocyanide 38 0 0
2-chloro-6-methyl phenyl isocyanide 4 0 0
2-morpholinoethyl isocyanide 13 0 0
benzyl isocyanide 43 3 7
cyclohexyl isocyanide 77 10 13
n-butyl isocyanide 120 5 4
t-butyl isocyanide 215 23 11
tosylmethyl isocyanide 107 0 0Carboxylic acid – boc-glycine 35%
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 inhibitorIC50 8.4uM
Solubility
Polyaromatic components in Ugi reactions
1-pyrenebutyric acid
methanol; 1 week
R
NCH3
NH
O
CH3
CH3
CH3
O
O
NCH3 CH3
O
171F/6-4 171K/11-4
RCHO =
O
OH
CH3 NH2
CH3CH3
CH3N
+C
-
RCHO
CH3
O
173B/14
Solubility issues
Solubility of phenanthrene-9-carboxaldehyde
SolventAve. (M)
11,1,2-
trichlorotrifluoroethane 0.02
2 2-propanol 0.07
3 DMF 1.25
4 DMSO 0.77
5 THF 1.29
6 acetonitrile 0.153
7 benzene 0.66
8 chloroform 0.04
9 cyclohexane 0.07
10 cyclopentane 0.03
11 dichloromethane 0
12 diethyl ether 0.1
13 ethanol 0.1
14 ethyl acetate 0.44
15 hexane 0.07
16 methanol 0.104
17 toluene 0.14
Solubility of 1-pyrenebutyric acid
SolventAve. (M)
1 2-propanol 0.06
2 DMF 1.88
3 DMSO 2.067
4 THF 0.55
5 acetonitrile 0
6 benzene 0
7 carbon tetrachloride 0
8 chloroform 0.03
9 cyclohexane 0
10 cyclopentane 0
11 dichloromethane 0.07
12 diethyl ether 0.02
13 ethanol 0.06
14 hexane 0
15 methanol 0.014
16 toluene 0
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 generatorPredictive ToxicologyVirtual dockingIntegration 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