Center For Green Chemistry
and Green Engineering at Yale
Designing Future Products with Reduced Toxicity
Lecture #24
Date:
Course #
Center For Green Chemistry
and Green Engineering at Yale
Outline
• Approaches to hazard minimization through molecular design
• Case study: Codexis
• Alternatives to animal testing
• QSAR-Quantitative Structure Activity Relationship
• The nexus of chemistry and toxicology
• Sources of high throughput data
Center For Green Chemistry
and Green Engineering at Yale
Outline
• Approaches to hazard minimization through molecular design
• Case study: Codexis
• Alternatives to animal testing
• QSAR-Quantitative Structure Activity Relationship
• The nexus of chemistry and toxicology
• Sources of high throughput data
Center For Green Chemistry
and Green Engineering at Yale
By Changing the Molecular Charge: Cephalosporins
• Clinical nephrotoxicity was recognized early – damage to proximal tubular epithelia.
• Mechanism: the drug is rapidly transported across basolateral membranes via the organic anion
transporter, OAT1. Transport out of other side of cell into lumen is less well characterized (involves
other OAT, OAT4).
• Cephalosporin has a very low affinity for OAT4 due to its zwitterionic character, resulting in
considerable intracellular accumulation.
Cephaloridine
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Cephalothin
• In contrast, cephalothin, an anionic analog of the original zwitterionic drug,
rapidly moves across the epithelia from blood into urine.
• Is thus not a neurotoxin.
Cephalothin
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By Manipulating Sterics: Paraquat and Diquat
• Mechanism: After oral ingestion, a very small fraction is absorbed, but the blood levels remain
constant for many hours as paraquat is not metabolized by the liver. Instead, it accumulates in the
lungs and is retained there even after blood concentrations decrease.
• Paraquat was found to enter the lung via carrier-mediated transmembrane transport, specifically
the polyamine transport system.
Diquat Paraquat
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Through SAR Data: Nonylphenols and Glycidyl Ethers
Polyethoxylatednonylphenols
• Emulsifiers, surfactants
• When n = 14-19, intense myocardial necrosis at 40 mg/kg.day in dogs and guinea pigs
• When n<14>19, no myocardial effects
Glycidyl ethers
• When n = 7-9, mutagenic, cause testicular lesions
• When n = 11-13+, non-mutagenic
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By Manipulating Electrophilicity: Acrylates
• Acrylate is understood to be carcinogenic because it can readily undergo Michael addition
reactions.
• The addition of a methyl group to form methacrylate decreases the electrophilicity of the b-
carbon, and decreases tendency for Michael reactions.
• While this does not reduce the commercial efficacy, but whereas acrylate causes cancer in
experimental assays, methacrylate does not.
Acrylate Methacrylate
(carcinogenic) (non-carcinogenic)
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By Facilitating Detoxification: Benzene
• Benzene is known to cause hematoxicity and leukemia in humans.
• This is understood to be due to one of its metabolites, (E,E)-mucoanaldehyde.
• Toluene is comparatively much less toxic, as the methyl group is oxidized more easily than the benzene ring
to give benzoic acid.
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By Isosteric Substitution: Carbon with Silicon
• DDT is well known to be environmentally persistent.
• DDT analog with Silicon was synthesized.
• While less persistent, the analog turned out to be non-toxic to insects.
• This is due to the more readily oxidized (metabolized) Si-H bond compared to C-H
and also the larger size of the molecule.
DDT Si-DDT Analog
(Toxic to insects) (Non-toxic to insects)
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QSAR-Quantitative Structure Activity Relationship
• Assumes that similar chemicals behave similarly.
• Behavior of the chemical is derived from the structure.
• QSAR seeks for a relationship between chemical structure and activity.
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Why people use QSAR?
• Not sufficient data on the majority of chemicals.
• Not all chemicals will be tested.
• QSAR has been used for over 60 years.
• Better to predict than have nothing.
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QSAR Basics
• They are associated with a specific endpoint [like LC50 or oxidative stress].
• They ‘group’ chemicals based on toxicity.
• Predict endpoints directly from chemical structure.
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Examples of Popular Toxicity Prediction Tools
Current limitations of QSARS
• Lack of validation with up-to-date data.
• Application to a narrow range of
chemical structures.
• Qualitative output.
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New Approach:
Crossroads of Computational Chemistry and Toxicology
Computational chemistry Toxicology
Scientific Advances
Technological and
Organelles
Enzymes
Receptors Cells
Genes Tissues
Polypeptide Organs
DNA strands
Small molecules Whole organisms
System size
As toxicology has increased the understanding of underlying biological
processes leading to toxicity, computational chemistry has grown capable
of describing the chemical transformations involved in these processes.
Center For Green Chemistry
and Green Engineering at Yale Courtesy of Molecular Design Research Network and Dr. Adelina Voutchova
Understanding the Physicochemical Properties
Associated with Reduced Hazard
DESIGN
Chemical and Physical Properties
pKa mol. wt.
chain branching log P molecular refractivity
EHOMO ELUMO electronegativity hardness hydrolyzable groups
dipole log D7.4 # H-bond acceptor/donor
• Carcinogens
• Toxins
• Flammability • Pollution
• Radioactivity • Persistence
• Risk of Explosion • Depletion of resources
• Chemical Reactivity
Center For Green Chemistry
and Green Engineering at Yale Courtesy of Molecular Design Research Network and Dr. Adelina Voutchova
Property-Based Guidelines for Bioavailability
Acidic Chemical Basic
substance substance
pKa<7 pKa>7
Unionized at Un-ionized
Lipid
pH>pKa at pH>pKa
soluble
GI
Eyes
tract Absorbed across
intestine membrane
intoblood
Absorption
organs
Lungs Skin
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Courtesy of Molecular Design Research Network and Dr. Adelina Voutchova
Property-Based Guidelines for Bioavailability
molecular size < 500 Da
water solubility molecular size
log Pow 0 - 5
vapor pressure < 0.001 mmHg
Non-ionization at GI pH
GI
Eyes
tract
Absorption
organs
Particle size < 5um Lungs Skin molecular size < 400 Da
molecular size < 400 Da log Pow 0 - 6
vapor pressure < 0.001 presence of solvents
mmHg Ionization (polar, ionized)
Center For Green Chemistry Voutchkova, A.; Osimitz, T.; Anastas, T. Chem. Rev. 2010, 110, 5845
and Green Engineering at Yale
Adverse Outcome Pathways
Molecular Cellular Tissue Organ Organ System Individual Population Ecosystem
Macro- Cellular Organ Organism Population
Toxicant Molecular Responses Responses Responses Responses
Interactions
Gene Altered Lethality
Receptor/Ligand activation physiology Structure
Interaction Impaired
Chemical Protein Disrupted Development
DNA Binding production homeostasis Recruitment
Properties
Impaired
Protein Altered Altered tissue Reproduction Extinction
Oxidation signaling development/
function
• Mechanistic understanding is critical for evaluating toxicity pathways
• Design guidelines will be rationalized according to molecular initiation events
Center For Green Chemistry Ankley et al. 2010 ET&C; Source: Gary Ankley & Dan Villeneuve, EPA
and Green Engineering at Yale
Where is data coming from?
EPA ToxCast
Tox21
High throughput assays
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What is an assay?
Assays can test almost any biological
effect
In vitro assay in cell in vivo outcome in organism
For example: cytotoxicity, gene expression or interaction with protein
An ideal assay is:
• Reproducible: same result when repeated
• Reliable: same result with different people repeat it
• Sensitive: won’t miss an effect
• Specific: won’t say there is an effect if there is not
• Predictive of outcome of interest
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Data from ToxCast
Set Chemicals Assays Endpoints Completion Available
ToxCast Phase I 293 ~600 ~700 2011 Now
ToxCast Phase II 767 ~600 ~700 03/2013 10/2013
ToxCast Phase IIIa 1001 ~100 ~100 Just starting 2014
E1K (endocrine) 880 ~50 ~120 03/2013 10/2013
Tox21 8,193 ~25 ~50 Ongoing Ongoing
~600
Assays
0 Chemicals ~8,200
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Data from ToxCast
• ToxCast phase I:
primarily pesticides
• ToxCast phase II:
industrial chemicals
• ToxCast phase III:
diverse chemical space;
high production
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Data Endpoints
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If we can predict toxicity using computer models…
• Then the need for expensive toxicology studies is reduced.
• Then less time is needed to get a chemical into the market.
• We cab have more confidence in chemicals rather than facing unintended
consequences afterwards.
• We can design the chemical with specific functions before animal trials.
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Are we there yet?
• Sadly, no.
• Best prediction models offer only 65-70% accuracy for non-carcinogenic
compounds.
• Still cannot replace in vivo and in vitro studies.
• Once prediction power reaches 90-95%, the major shift in experimental strategy
can occur.
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