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A Hypothesis for the Physiological AntioxidantAction of the Salicylates.

I. Francis ChengDepartment of Chemistry

University of ArizonaTucson, Arizona 85721

Tel. (520) 621-6340ifcheng@u.arizona.edu

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Seminar Outline

A brief history of the salicylates

Accepted model for acetylsalicylic (aspirin) action.

Weakness of accepted model.

Hypothesis for salicylate action.

Experiments.

Discussion.

Proposed Studies.

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History of Aspirin

Plant Based Product

Folk remedy for centuries, known to relieve pains and fevers.

1828 - active ingredient isolated by Johann Buchner.

Found effective for fevers, inflammation, and pains but found to cause stomach irritation.

1898 - Felix Hofmann (Bayer) synthesizes and tests Acetylsalicylic Acid (Aspirin)

Just as effective but less irritating than salicylic acid.

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Accepted model for acetylsalicylic action.

Proposed in the 1970's - John Vane (1982 Nobel Prize)

Irreversible inactivation of Prostaglandin Synthase Action.

-Key enzyme in the arachidonic acid cascade

-Prostaglandins are local hormones that regulateinflammation

blood clotting

PG consists of two components, Aspirin works on cyclooxygenase.

-by acetylation of serine residue.

Inhibition of Cyclooxygenase results in reduction of inflammation.

Nature-New Biology 264 (1971) pp84-90.

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Weakness of the acetylation explanation.

Vane's Theory Describes The Action of Aspirin

But, How Does Salicylic Acid Exert Its Medicinal Action?

Lacks an Acetyl Group!

Pharmacological Literature Indicates That Salicylic Acid Exerts Anti-inflammatory Action Almost as Potent As Acetylsalicylic Acid.

Yet Salicylic Acid Lacks an Acetyl Group That Forms the Center Piece of

Vane's Theory for Acetylsalicylic Acid

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Other Weaknesses of the Acetylation Mechanism.

Does not explain other documented medicinal effects of aspirin.

Aspirin acts as a chemopreventative for...... Heart and circulatory diseases Parkinson’s and Alzheimer’s diseases Cancers Cataracts

All of the above may be due to oxidative damage by oxygen containing free radicals.

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Formation of Activated Oxygen

O2.- and H2O2 released as Respiration by-products,

[H2O2] = 10-7 [O2.-] = 10-11

Also, Inflammation response (pathogen defense) by white blood cells

Physiological oxidative damage linked to chronic inflammation

Physiological Reviews, 59 (1979) pp527-605.

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Goal of Respiration. (CH2O)n + O2 = nCO2 + nH2O

IncreasingReducingPow er

IncreasingOxidizingPower

O + 4H 2H O2+

2

O + 2H H O2 2 2+

O O2 2.-+ 1e

+ 2e

+ 4e

(CH O)2 n(sugars)(-)

(+)

G = nFE

E (pH7)o'

- 0 .45 volts

0.30 V

0.82 V

H2O2 & O2.- are known as “activated oxygen species”

RedoxPotential

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Dangers of Activated Oxygen Species

Hydrogen peroxide Fenton ReactionH2O2 + FeII(L)n = FeIII(L)n + HO- + HO.

HO. + e- = HO- Eo = 1.8 volts

Superoxide ion Disproportionation to H2O2

O2.- + O2

.- + 2H+ = H2O2 + O2

Reducing agent for Fenton rxn.

O2.- + FeIII(L)n = FeII(L)n + O2

Reduces Fe3+(insoluble) to Fe2+ (soluble)

physiological evidence indicates that O2.- is may be more toxic than H2O2.

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Hydroxyl Radical Damage to Biological Molecules Results in .....

Denaturation of lens proteins cataracts

DNA strand breakage damage to genes

aging

cancers mitochondrial dysfunction

Fatty acid cross linking circulatory diseases

Damage to nervous system Parkinson’s

Alzheimer’s diseases Summary

Hydroxyl radicals are the likely source of physiological oxidative damage

-Scientific American, December 1992, pp131-141.

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Iron complexes and activated oxygen are conspirators in the oxidative damage to physiological

components

FeII[complex] + H2O2 = FeIII[complex] + HO- + HO.

Fe and disease origins

Recently Discovered Statistical Implications in - Heart Diseases - Strokes - Cancers - Cataracts

- Alzheimer’s - Parkinson’s

Key Point Ailments due to active oxygen forms and iron are closely linked

Bioelectrochemistry and Bioenergetics, 18 (1987) pp105-116. Ibid, 18 (1987) pp3-11.

Biochemistry, 31 (1992) pp11255-11264. Circulation, 86 (1992) pp803-811.

New England Journal of Medicine, 320 (1989) 1012. Iron and Human Disease, CRC Press, Boca Raton, FL, 1992.

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Migration of Fe Under Conditions of Oxidative Stress

H2O2 + O2.-

Fe Containing Enzymes

Fe2+

OxidizedLigands

+ ATP, citrate

Fe(L)+ H2O2 + O2

.-

Fe(L) + HO.

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Hypothesized Antioxidant Properties of Salicylates.

Aspirin may play a role in the moderation of physiological oxidative damage.

Hypothesized because of aspirin’s ability to act as a

chemopreventative of many diseases associated with oxidative damage.

Free Radicals in Biology and Medicine 9, (1990) 299.

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Proposed Route of Antioxidant Action for Aspirin. (literature)

Proposed Route of Antioxidant Action for Aspirin. (literature)

Salicylates act as Hydroxyl Radicals Scavengers.

k 1010 M-1 s-1Xenobiotica, 18 (1988) pp459-470.

COOH

OH

COOH

OH

OH

COOH

OH

HO

++ HO.

A) B)

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Problems with Radical Scavenging Hypothesis.

Physiological concentration of aspirin (10-4 M) cannot compete with the oxidative damage to cellular components.

Most organics (physiological components) will react with HO. at the same rate as salicylates

k = 1010M-1 s-1 (diffusion limited kinetics).

Acetaminophen is a more effective hydroxyl radical scavenger.

k = 1.5 x 1010 M-1 s-1

lacks - chemopreventative effects- anti-inflammation

Summaryradical scavenging alone cannot explain the antioxidant characteristics of salicylates.

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Alternative Hypothesis forSalicylate Antioxidant Behavior.

Key Point Salicylates moderate iron activity rather than HO radical scavenging.

Salicylate may aid in one or more of the following antioxidant actions

I) Redox deactivation of Fe2+/3+ (observed in vitro)

II) Superoxide Dismutase Action.

III) Catalase Action.

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Proposed Hypothesis (Continued)

I) Storage and Transport of Fe. Redox Deactivation

Requires Fenton Inactive Forms

(shift Fe2+/3+ threshold to thermodynamically unfavorable potentials) Animals (Humans) - Ferritin, Transferrin Plants & Bacteria - Siderophores

II) Superoxide Dismutase (SOD) Action.

O2.- + 2H+ + e- = H2O2

III) Catalase Action.

2H2O2 = 2H2O + O2

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Salicylate as an inhibitor of Fenton processes.Redox Deactivation of Fe2+/3+

Salicylates as chelation agent of iron ions.

-may be plant siderophores - iron transport agents

Exact structure may vary with pH

Hand book of Chemical Equilibria in Analytical Chemistry, Chichester, U.K., Ellis Horwood Limited, 1985, p163.

log B3 = 35.5

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Outline of Experimental Section.

Electrochemistry - cyclic voltammetry experiments

Tells us something about thermodynamic ability to drive Fenton reaction.

DNA oxidations via Fenton reaction.

Examine the ability of salicylates to prevent the degradation of calf thymus DNA via Fenton

reaction.

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Redox Potential of Fe-Sal Indicates that it is a Fenton Inactive Complex.

Cyclic voltammogram of iron-salicylate (0.5 mM Ferric Nitrate with 2.0 mM Salicylate) at pH 7.2, 0.05 M phosphate buffer with a potential sweep rate of 5 mV/sec. The electrodes consisted of a 0.071 cm2 wax impregnated graphite disk with a Ag/AgCl, saturated KCl reference (0.197 volts vs. SHE).

0.4-0.4

Potential versus SHE

FeII[sal] FeIII[sal] + e-

FeII[sal] e- + FeIII[sal]

Eredox = 0.370 volts vs. SHE at pH 7.2

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Salicylate chelates iron into a Fenton inactive form

Thermodynamics of the Fenton Reaction

StrongerReducingAgents (-)

E0Fenton = 0.307 volts

EFe-sal = 0.370 volts

}EFe[EDTA]

EOxidases

EOxygenases

Fenton Active

x

Fenton Inactive

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Evidence for Fenton Reaction Inertness of Fe-salicylate from Cyclic Voltammetry experiments.

Electrochemical electrocatalytic wave for FeIII(EDTA) reduction in the presence of H2O2

Electrode: FeIII(EDTA) + e = FeII(EDTA) 0.090 volts SHE

Solution: FeII(EDTA) + H2O2 = FeIII(EDTA) + HO- + HO.

Results in enhanced electroreduction current for FeIII(EDTA) wave, no electro-oxidation wave for FeII(EDTA)

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Cyclic Voltammetry of FeII/III [EDTA] in the Absence and Presence of H2O2

0.4 A

1.0 A

Potential vs. Ag/AgCl

B

A

-0.7

CurrentA) 0.1 mM FeIII(EDTA)

B) +10 mM H2O2.

Potential sweep rate = 5 mV/secpH 7.2 0.05 M phosphate buffer with a potential sweep rate of 5 mV/sec

0.071 cm2 wax impregnated graphite disk

Ag/AgCl, saturated KCl reference (0.197 volts vs. SHE).

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Results of H2O2 electrocatalytic voltammetry.

Important Predictions. If Redox Deactivation Hypothesis Works Then….

Salicylate acts as an Antioxidant for Fe but not Cu.

EDTA acts as an Antioxidant for Cu but not Fe.

Potential H2O2 ReductionCuI(EDTA) 0.450 volts NoFeII(sal)3 0.370 No

H2O2 = HO- + HO. 0.307 ----

FeII(EDTA) 0.090 YesCuI(sal)2 0.050 Yes

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Important Predictions (continued).

If radical scavenging is the predominate mechanism for salicylate antioxidant action then…..

Salicylate (k =1010 M-1s-1) will act as a antioxidant for both Fe and Cu

EDTA (k = 109 M-1s-1) will act as a antioxidant for both Fe and

Cu.

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DNA as a Probe for Hydroxyl Radical Production.

+ .OHO

Base

OPO3

. H2O

OBase

OPO3

HO-PO4-H

OBase

O H

H

O3PO

OPO3

H

BaseO O3PO O3POO3PO

O

O

+ Base

DNA Strand is an efficient chelator of iron and copper ions.

Binding Constant 1012

Primarily through phosphate residues DNA-FeII ,- CuI complexes participates in Fenton type chemistries.

DNA degradation by .OH (or other oxidizing products) leads to attack on deoxyribose residues which releases bases from strands.

Adenine, Thymine, Guanine, Cytosine Products are easily quantifiable by HPLC.

UV detection at 254 nm

Key Point - DNA strand is a convenient probe for detection of hydroxyl radical.

JACS 1992, 114, pp2303-2312.

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DNA Incubation Studies.

Fe-DNA complex Eredox{FeII/III(DNA)} = -0.10 volts SHE

FeIII(DNA) + Ascorbate = FeII(DNA) + Deoxyascorbate

FeII(DNA) + H2O2 = FeIII(DNA) + HO- + HO.

Conditions 0.1 mM Fe(NO3)3, 1.0 mM ascorbate, and 7.8 mM H2O2 DNA (0.2 mM in base pairs), 120 minutes

Incubation of DNA with Fe-EDTA

FeIII(EDTA) + Ascorbate = FeII(EDTA) + Deoxyascorbate

FeII(EDTA) + H2O2 = FeIII(EDTA) + HO- + HO.

Conditions 0.1 mM Fe(NO3)3, 0.4 mM EDTA, 1.0 mM ascorbate, and

7.8 mM H2O2, DNA (0.2 mM in base pairs), 120 minutes

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HPLC chromatogram following incubation of calf thymus (CT) DNA

A) salicylate absent.

B) 0.4 mM salicylate present.

Salicylate retards oxidative

DNA damage due to Fenton

type processes

Retention times; Guanine, 1.09 mins.; Thymine, 1.44 mins.; Adenine 2.35 mins

Separation conditions: 50/1 water to methanol mobile phase, C18 reversed phase Zorbex cartridge column, absorbance detection at 254 nm.

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HPLC incubation results

DNA Incubation with…A) 0.1 mM Fe(NO3)3 B) 0.1 mM Fe[EDTA]

C) 0.1 mM Fe(NO3)3 and D) 0.1 mM Fe[EDTA] and 0.4 mM salicylate 0.4 mM salicylate

Salicylate decreases oxidative DNA damage due to

Both Fe-DNA and Fe(EDTA) complexes

0

20

40

60

80

100

HP

LC

Det

ecto

r R

esp

on

se(T

ho

us

and

s) Thymine

Adenine

A B C D

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Salicylates may compete for Fe chelation with oxidized EDTA

EDTA hydroxyl radical scavenging rate, k = 109 M-1 s-1

Under inflamed conditions Fe undergoes migration due to oxidative attack of low

molecular weight ligands

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Summary of DNA Incubation Experiments.

Incubation-10 Minutes Damage to CT-DNA

Control 0.5 mM Ascorbate NO5.0 mM H2O2

+ 0.1 mM Fe(EDTA) YES+ 0.1 mM Cu(EDTA) NO

+ 0.1 mM Fe(salicylate) NO+ 0.1 mM Cu(salicylate) YES

Confirms Redox deactivation hypothesis

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Summary of DNA Incubation Experiments

Excess Ligand (salicylate or EDTA)

Incubation 10 minutes Damage to CT-DNA

Control 0.5 mM Ascorbate NO5.0 mM H2O2

+ 0.1 mM Cu(salicylate) YES+ 10.0 mM salicylate

+ 0.1 mM Fe(EDTA) YES+ 50.0 mM EDTA

Indicates that radical scavenging is not an important mechanism.

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Incubation Results with Aspirin

Acetylsalicylic acid cannot chelate iron

– slowly hydrolyzes to salicylic acid (t1/2 = 20 min.)

– Radical scavenging rates; aspirin = salicylate

Incubation 10 minutes CT-Damage

Control 0.5 mM Ascorbate NO5.0 mM H2O2

+ 0.1 mM Fe(NO3)3 YES+ 0.4 mM aspirin

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Release of adenine with incubation time for controls, and presence of salicylate, and aspirin.

Adenine Release– Less than 10 minutes aspirin = control– Greater than 60 minutes aspirin = salicylic acid

Results consistent with acetylsalicylic acid to salicylic acid

Incubation Time (min)0 20 40 100

Control

Salicylic Acid

Acetylsalicylic Acid

HP

LC

Det

ecto

r R

espo

nse

(254

nm

)

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Outline of Discussion

Role of pH in the Fenton Reaction

• Implications in inflammation and cancer

pH and the FeII/III[salicylate] redox potential

• This is a key feature in salicylate’s antioxidant ability

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The role of H+ activity and physiological oxidative damage. Fenton Reaction is pH sensitive

H2O2 + e- = HO- + HO.

EFenton = 0.732 -(0.059 pH) where [H2O2] = [HO.] = 1

at pH 7.2

EFenton = 0.307 volts SHE

at pH 5.5

EFenton = 0.408 volts SHE

Fenton threshold becomes more facile with decreasing pH. Important consideration

Inflamed, damaged, or tumorous tissues may reach pH’s as low as

3.5

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FeII/III[salicylate] potential is pH dependent.

Measured by Cyclic Voltammetry

0

1

2

2 4 6 8 10 pH

Pot

enti

al (

volt

s vs

. SH

E)

EFe(sal) = 0.793 - (0.059 pH)

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pH dependence may be due to HO- complexation

FeIII(sal)n + HO- = FeIIIOH(sal)n

FeIIIOH(sal)n + e- = FeII(sal)n + HO-

E ERT

nF

Fe OH sal

Fe sal HO

IIIn

IIn

0 ln

[ ( )( ) ]

[ ( ) ][ ]

E = const - 0.059 pH

E = 0.793 - 0.059 pH

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Fenton threshold and the FeII/III(sal) redox potential

FeII/III(sal) redox potential closely parallels EoFenton

– Remains just slightly thermodynamically uphill

Why does salicylic acid not seek to maximize Fe deactivation?– By increasing FeII/III potential

Pot

enti

al (

volt

s S

HE

)

0

0.5

1

1.5

2

0 2 4 6 8 10 pH

E0Fenton = 0.732 - 0.059pH

E0Fe-Sal = 0.793 - 0.059pH

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Hypothesis Possible Significance of the close parallel of Fe II/III(sal)n and

Standard State Fenton threshold.

Superoxide Dismutation.

O2.- + 2H+ + e- = H2O2 Eo = 1.77 volts

E = 1.77 + 2(0.059)pH

Salicylic acid may seek to maximize

SOD activity with a minimum of

Fenton type reactivity.0

0.5

1

1.5

2

0 2 4 6 8 10

pH

Zone I

Zone II

Zone IIIFenton Threshold

Superoxide Dismutation

EFe-Sal

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Thermodynamic Suppression of HO. Production by Salicylate.

Reduction: H2O2 + e = HO- + HO.

Oxidation: FeII(sal)n = FeIII(sal)n + e

Ecell = Ered - Eox

Eox = 0.793 - 0.0591pH

Calculate equilibrium value for product/reactant ratio @ pH 7 (Ecell= 0)

Healthy Tissue Maintains [H2O2] = 10-9 - 10-7

(Physiological Reviews, 59 (1979) p564.)

Salicylic acid is a modest suppression agent of HO.

E pHH OHO

red 0 732 0 0591 0 05912 2

. . .[ ][ ].

log

[ ][ ]

..HO

H O2 20 0928

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Thermodynamic Analysis of Superoxide Dismutase Activity of Iron-Salicylate

Reduction: O2.- + 2H+ + e- = H2O2

Oxidation: FeII[sal] = FeIII[sal] + e-

[O ].

Ered pH 1 77 0 118 0 0591 2[H2O2]

. . . log

Eox = 0.793 - 0.0591 pH

@ pH 7 Ecell Spontaneous until

[ ]

[ ].

.O

H Ox2

2 2

102 94 10

Ecell = Ered - Eox

Salicylic acid may be an excellent suppression agent of O2.-

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Equilibrium SOD and Fenton Ratios vs. Iron Chelate Redox Potential

Equilibrium values (from Nernst equation) for SOD action and Fenton reaction moderation as a function of the redox potential of FeII/III transition of a chelate.

pH 7

-20

-15

-10

-5

0

5

10

-0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3

Redox Potential of Chelated Iron (SHE)

-20

-15

-10

-5

0

5

10 FeII/III[salicylate]

Fenton Rxn Moderation SOD Action

log[ ]

[ ]

.O

H O2

2 2

log[ ]

[ ]

.HO

H O2 2

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Conclusions

Antioxidant Action via Suppression of Fenton Reaction.

Redox inactivation, E = 0.793 - 0.059pH, rather than HO. radical scavenging

DNA Oxidation Studies with Fe2+/3+and Cu1+/2+ with salicylate and EDTA.

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Future Research Binding constant data, function of pH, potentiometric titrations Crystal structure of iron-salicylate complex Superoxide dismutase (SOD) action. Catalase action

H2O2 + 2H+ + 2e- = 2H2O

H2O2 = O2 + 2H+ + 2e-

2H2O2 = 2H2O + O2

-qualitatively observed during DNA oxidation studies. Prediction of Structure-Activity Relationships

-antioxidant characteristics of other NSAID, (ibuprofen)

-increase activity of salicylates

-quick screen for antioxidant characteristics of newly isolated natural products

Collaborative Research

-physiological Studies

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Quantitative Structure-Activity Relationships (QSAR) for Salicylates and Derivatives

(Anti-inflammatory action)

Rule 1. Substitution on either the carboxyl or the phenolic hydroxyl groups affect activity.

Rule 2. Placing the phenolic hydroxyl group meta or para to the carboxyl group abolishes activity.

Rule 3. Substitution of halogen atoms on the aromatic ring enhances potency.

Rule 4. Substitution of aromatic rings meta to the to the carboxyl and para to the phenolic hydroxyl groups increases anti-inflammatory activity.

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Rule 1. Substitution on either the carboxyl or the phenolic hydroxyl groups affect activity.

May Affect Chelation of Fe ions. Binding Constant to Fe Rate of hydrolysis to salicylate

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Rule 2. Placing the phenolic hydroxyl group meta or para to the carboxyl group abolishes

activity.

Meta and Para derivatives are not Fe chelators

COOH

OH

COOH

OH

COOH

HO

Salicylic Acid 3-hydroxyl benzoic acid 5-hydroxyl benzoic acid

Bidentate Chelation Site

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Rule 3. Substitution of halogen atoms on the aromatic ringenhances potency.

Rule 4. Substitution of aromatic rings meta to the to the carboxyl and para to the phenolic hydroxyl groupsincreases anti-inflammatory activity.

Increases electron withdrawing ability of salicylate raises FeII/III potential

e-FeII

O

COO

May improve Fenton deactivation

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???Anti-inflammatory action = Antioxidant action

???

If Fe chelation correlates to QSAR anti-inflammatory rules

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Other anti-inflammatory agents

All of the following NSAID’s are iron chelation agents. Iron chelation may play a role in their medicinal action.

N-ayrlanthranilic Acids

CONH2

OH

COOH

NH

R1

R2

R3

Mefenamic Acid, R1 = R2 = CH3, R3 = HMeclofenamic Acid, R1 = R3 = Cl, R3 = CH3Flufenamic Acid, R1 = R3 = H, R3 = CF3

SN

OH

OO

N

O

H

N

CH3

Piroxicam

N

H3CO

CH3

CH2COOH

O

Cl

Indomethacin

Salicylamide

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Acknowledgments

Seton Hall University Graduate Students (M.S.)Andris AmolinsChris Zhao

UndergraduatesMalgorzata GalazkaLeon Doneski

University of Arizona Dr. Quintus FernandoDr. Paul Oram

Equipment Ciba-GiegyUnion-CampFMC