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Biochemical and Biophysical Research Communications 262, 651–656 (1999)

Article ID bbrc.1999.1275, available online at http://www.idealibrary.com on

ovel Inhibitors of Advanced Glycation Endproducts

amuel Rahbar,*,1 Kiran Kumar Yernini,* Stephen Scott,* Noe Gonzales,* and Iraj Lalezari†Department of Diabetes, Endocrinology & Metabolism, City of Hope National Medical Center,uarte, California 91010-0269; and †Proscience Inc., New York

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Enhanced formation and accumulation of advancedlycation endproducts (AGE’s) have been proposed tolay a major role in the pathogenesis of diabetic com-lications, aging, atherosclerosis, and Alzheimer dis-ase leading to progressive and irreversible inter-olecular protein crosslinkings. This process is

ccelerated in diabetes and has been postulated toontribute to the development of a range of diabeticomplications including nephropathy, retinopathynd neuropathy. Several potential drug candidates asGE inhibitors have been reported recently. Amino-uanidine is the first drug extensively studied both initro and in vivo. We have developed a new class ofompounds as potent inhibitors of glycation and AGEormation. The novel inhibitors reported here are aryland heterocyclic) ureido, and aryl (and heterocyclic)arboxamido phenoxy isobutyric acids and relatedolecules, which were found by in vitro assay meth-

ds to be potent inhibitors of multiple stage of glyca-ion and AGE formation. © 1999 Academic Press

Nonenzymatic glycation is a complex series of reac-ions between reducing sugars and amino groups ofroteins, lipids and DNA, which lead to browning, flu-rescence, and crosslinking (1–3). The reaction is ini-iated with the reversible formation of Schiff ’s base,hich undergoes a rearrangement to form stable Ama-ori product. The Amadori product further undergoes aeries of reactions through dicarbonyl intermediates toorm advanced glycation endproducts (AGEs). Thishenomenon called “browning or Maillard” reactionas discovered early in this century by the food indus-

ry (4), however, the significance of a similar process iniology became evident only after the discovery of thelycosylated hemoglobins and their increased presencen diabetic patients (5–6). In human diabetic patientsnd in animal models of diabetes, these nonenzymaticeactions are accelerated and cause accumulation oflycation and AGE formation on long-lived structural

1 Corresponding author. Fax: (626) 301-8136. E-mail: srahbar@oh.org.

651

roteins such as collagen, fibronectin, tubulin, lensrystallin, myelin, laminin, actin, in addition of hemo-lobin, albumin, on LDL associated lipids and apopro-ein, and most recently reported to inactivate meta-olic enzymes (7).The structural and functional integrity of the af-

ected molecules which often have major roles in cellu-ar functions, become perturbed by these modifications,ith severe consequences on affected organs such asidney, eye, nerve, and micro-vascular functions (8–9).The Diabetic Control and Complications Trial

DCCT), has identified hyperglycemia as the main risk-actor for the development of diabetic complications10). Although, there is no consensus regarding theathogenic link between hyperglycemia and diabeticomplications, formation of advanced glycation end-roducts (AGEs) have been implicated as a majorathogenic process in the long-term complications ofiabetes, namely nephropathy, neuropathy and reti-opathy (11–15).Direct evidence indicating the contribution of AGE’s

n the progression of diabetic complications in differentesions of the kidneys, the rat lens, and in atheroscle-osis has been recently reported (16–20). Several linesf evidences indicate that; increase in reactive carbonylntermediates (methylglyoxal, glyoxal, 3-deoxygluco-one, malondialdehyde, and hydroxynonenal) as theonsequence of hyperglycemia in diabetes. Carbonyltress, leads to increased modification of proteins andipids, followed by oxidant stress and tissue damage21–23).

Methylglyoxal (MG) has recently received consider-ble attention as a common mediator to form AGE’s. Inatients with both insulin-dependent and non-insulinependent diabetes, the concentration of MG wasound to be increased 2–6-fold (24–25). Furthermore,

G has been found not only as the most reactive di-arbonyl AGE-intermediate in cross-linking of pro-eins, a recent report has found MG, to generate reac-ive oxygen species (ROS) (free radicals) in the coursef glycation reactions (26). The glyoxylase system (Ind II) and aldose reductase, catalyse the detoxifica-ion of MG to D-lactate. MG binds to and irreversibly

0006-291X/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 262, No. 3, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

odifies arginine and lysine residues in proteins. MGodified proteins have been shown to be ligands for theGE receptor (27), indicating that MG modified pro-

eins are analogous (28) to those found in AGEs. Mostecently, the effects of MG on LDL has been character-zed in vivo and in vitro (29).

Lipid peroxidation of polyunsaturated fatty acidsPUFA), such as arachidonate, also yield carbonyl com-ounds; some are identical to those formed from car-ohydrates (30), such as MG and GO, and others areharacteristic of lipid, such as malondealdehyde (MDA)nd 4-hydroxy-none-nal (HNE) (31). The latter car-onyl compounds produce lipoxidation products (31,2), A number of AGE compounds, fluorophores andonfluorescent are involved in cross-linking of proteinsave been characterized (21). Some reports indicatedhat “carbonyl stress” is the result of both increasedroduction and a deficit in detoxification of reactivearbonyl compounds (33).

Early pharmaceutical intervention against the long-erm consequences of hyperglycemia-induced-cross-inking, prevent the development of severe late com-lications of diabetes. The goals are to develop non-oxic and highly effective drugs that completely stoplucose-mediated crosslinking in the tissues and bodyuids. The prototype of the pharmaceutical compounds

nvestigated both in vitro and in vivo to intervene withhe formation of AGE’s on proteins is aminoguanidineAG) a small hydrazine-like compound (34–37). How-ver, AG is a well known inhibitor of nitric oxide (NO)37). A number of other compounds were found to haveuch an inhibitory effect on AGE formation. Examplesre D-lysine (38), desferrioxamine (39), D-penicill-mine (40), thiamine pyrophosphate and pyridoxamine41) which have no structural similarities to amino-uanidine. Also recently, the thiazolidine derivativePB-9195 compound was reported that inhibits bothGE formation and nephropathy in diabetic rats (42).In this communication, we report a new class of

otent inhibitors of glycation, AGE formation andGE-protein cross-linking. These compounds are 1 to 2

evels of magnitude more potent than AG, and moreffective than pyridoxine (41). The new compounds actt multiple steps of glycation and/or post glycationeactions, with potential therapeutic use of preventingiabetic complication and delaying aging process.

ATERIALS AND METHODS

Reagents. BSA (fraction V, essentially fatty acids free, low endo-oxin), glucose, d-gluconolactone (d-Glu), N-acetyl-glycyl-Lysineethyl ester (G.K.), peptide, ribose were from Sigma (St. Louis, MO).minoguanidine-hydrochloride from Aldrich (Milwaukee, WI). 0.2icron syringe filters from Gelman (Ann Arbor, MI), rat-tail-collagen

oatet 96 well plates were obtained from Biocoat (Beckton-ickenson). High titer anti-AGE-RNase was prepared in 2 femaleew Zealand white rabbits, following established protocols and used

n the immunochemical studies (specific ELISA assay).

652

atory are derivatives of aryl (and heterocyclic) uredio, and aryl (andeterocyclic) carboxamido phenoxy isobutyric acids. LR1, 4-[3-(6-chloro-2,-(1H, 3H) quinazolinedione)]phenoxyisobutyric acid; LR2, 4-(2-furoyl-arboxamido)phenoxyisobutyric acid; LR3, 4-(3,5-dichlorophenylureido)-henoxyisobutyric acid; LR4, 4-(4-ethylecarbamatophenylureido)-henoxyisobutyric acid; LR5, 4-(3, 4-dichlorophenylureido)phenoxyiso-utyric acid; LR6, 4-cyclohexylureidophenoxyisobutyric acid; LR7,-(2,3-dichlorophenylureido)phenoxyisobutyric acid; LR8, 4-(4-carbox-ldehydophenylureido)phenoxyisobutyric acid; LR9, 4-(2-naphthylcar-oxamido)phenoxyisobutyric acid; LR10, 4-(4-methoxyphenylureido)-henoxyisobutyric acid; LR11, 4-(3,4-dimethoxyphenylureido)phenoxy-

sobutyric acid; LR12, 4-(4-chloro-3-nitrophenylureido)phenoxyisobutyriccid; LR13, 4-(4-methylphenylureido)phenoxyisobutyric acid; LR14,-(3,4,5-trimethoxyphenylureido)phenoxyisobutyric acid; LR15, 4-(3-hlorophenylureido)phenoxyisobutyric acid; LR16, N-4-(nitrophthalimido)-henoxyisobutyric acid; LR17, 4-(2-thienylcarboxamido)phenoxyiso-utyric acid; LR18, 4-(4-pyridylureido)phenoxyisobutyric acid;R19, 4-(3,4,5-trichlorophenylureido)phenoxyisobutyric acid; LR20,-bis-[4-(4-chlorobenzamidophenoxisobutyryl)cystine; LR21, 4-(3,5ichlorophenylureido)phenoxyisobutyrylamidomethylcyclohexyl--carboxylic acid; LR22, DL-N-4-[(3,5-dichlorophenylureido)phen-xyisobutyryl]pipecolic acid; LR23, 4-(3,5-dichlorophenylureido)-henoxyisobutyryl-l–amidocyclohexane-1-carboxylic acid; LR24,-(4-iodophenylureido)phenoxyisobutyric acid; LR25, 4-(4-dimethylamino-henylureido)phenoxyisobutyric acid; LR26, 4-(2,4,6-trichlorophenylure-do)phenoxyisobutyric acid; LR27, 4-(2,4,6-trimethylphenylureido)-henoxyisobutyric acid; LR28, 4-(4-chlorophenoxacetamido)phenoxyisobu-yric acid; LR29, 4-(4-chloro-3-nitrobenzoylcarboxamido)phenoxyisobutyriccid; LR30, 4-chlorodiphenylurea-49-carboxylic acid; LR31, 4-(3,4-dichloro-henylacetamido)phenoxyisobutyric acid; LR32,diphenylurea-4-arboxylic acid; LR33, 4-(2-chloro-4-nitrophenylureido)phenoxy-sobutyric acid; LR34, 4-(nicotinylamido)phenoxyisobutyric acid;R35, 4-chlorophenoxyisobutyric acid; LR36, 4-(benzylsulfonamido)-henoxyisobutyric acid; LR37, 4-(2,5-dichlorobenzoylcarboxamido)-henoxyisobutyric acid; LR38, L-4-chlorobenzoylphenylalanine; LR39,-isopropyl-5-methylphenoxyisobutyric acid; LR40, 4-(3,4-dimethoxy-henylureido)phenoxyisobutyric acid; LR41, 4-(3-chloro-4-fluorophenyl-reido)phenoxyisobutyric acid; LR42, 4-(3,5-dichlorobenzamidoethyl)-henoxyisobutyric acid; LR43, 4-(phenylureido)phenoxyisobutyriccid; LR44, 4-(phenylureido-2-chloro)phenoxyisobutyric acid; LR45,-(2,6-dichloro-4-nitrobenzoylcarboxamido)phenoxyisobutyric acid; LR46,-(3,5-difluorophenylureido)phenoxyisobutyric acid; LR47, 4-(N-methyl--chlorobenzamido)phenoxyisobutyric acid; LR48, 4-(4-nitrophenyl-reido)phenoxyisobutyric acid; LR49, 4-(phenylureido)phenoxyaceticcid; LR50, 4-(4-chlorobenzoylcarboxamido)phenoxyisobutyric acid;R51, 4-(2-hydroxy-4-chlorobenzoylcarboxamido)phenoxyisobutyriccid; LR52, 4-(2-hydroxy-3,5-dichlorobenzoylcarboxamido)phenoxy-sobutyric acid; LR53, 4-(2-chloro-5-nitrophenylureido)phenoxyiso-utyric acid; LR54, 4-carboxyphenoxyisobutyric acid; LR55, 4-(4-arboxyphenylureido)phenoxyisobutyric acid; LR56, 4-ureidophenoxy-sobutyric acid; LR57, urea 1,3-bis-4-phenoxyisobutyric acid; LR58,-(4-morpholinosulfonylphenylureido)phenoxyisobutyric acid; LR59,-[(3,4-dichlorophenylmethyl)2-chlorophenylureido]phenoxyisobu-yric acid; LR60, 4-(3-pyridylureido)phenoxyisobutyric acid; LR61,-[(3,5-dichlorobenzoylamino)methyl]phenoxyisobutyric acid; LR62,-(2,4-dichlorophenacylamino)phenoxyisobutyric acid; and LR63,-(benzylureido)phenoxyisobutyric acid.Most of these compounds were easily soluble in 0.5 M phosphate

uffer pH 7.4 or phosphate buffered saline (PBS). A few compoundseeded to be briefly warmed in hot water.

Hemoglobin-d gluconolactone (d-Glu) assay. The d-Glu assays specific method for investigation of inhibitors of early stage oflycation.Evaluation of early glycation products (Amadori) formation on

emoglobin (HbA1C) is performed by incubating red blood cells withn oxidized form of glucose in the presence and the absence of thenhibitor compound followed by determination of (HbA1C) in the test

versus the control (43). d-Glu, an oxidized analogue of glucose, canritcabwotd3masffici

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eact rapidly with hemoglobin within the red cells and significantlyncreases (HbA1C) levels within hours after incubation. We have usedhis finding to devise an assay method to measure early stage gly-ation of hemoglobin (Amadori product) and an assay to evaluate thebility of an inhibitor to inhibit (HbA1C) formation. Briefly, freshlood was drawn in potassium-EDTA and prepared for incubationithin 30 minutes of collection by mixing 200 mL of blood with 40 mL

f either phosphate buffered-saline (PBS), pH 7.4. alone, PBS con-aining 50 millimoles/L d-Glu, or PBS containing 50 millimoles/L-Glu plus 1 millimoles/L inhibitor. After incubation for 16 hours at7°C, the percentage of glycated hemoglobin present was deter-ined. The percentage of glycated Hb (HbA1C) was determined usingdedicated ion-exchange HPLC system (BIORAD DIAMAT). Blood

amples were analyzed in triplicate. The % inhibition of HbA1C

ormation by the compound was calculated according to the followingormula: (B 2 C)/(B 2 A)) 3 100, in where A is HbA1C concentrationn the baseline control tube not treated with d-Glu, B is the HbA1C

ontent of the test tube treated with d-Glu and C is the HbA1c levelsn the sample treated with both d-Glu and the inhibitor compound.

BSA glucose assay. This test is used to evaluate the ability of thenhibitors to inhibit the glucose-mediated development of fluores-ence of BSA (44). BSA (fraction V) 50 mg/mL and 800 mmoles/Llycose (144 mg/mL) in 1.5 M phosphate buffer pH 7.4 containing.2 g/L NaN3 was incubated under aseptic conditions at 37°C for 7ays in the presence or absence of various concentrations of theompounds. After 7 days of incubation each sample was examined forhe development of specific fluorescence (excitation, 370 nm, emis-ion, 440 nm). The % inhibition of AGE formation in the test sampleersus control was calculated for each inhibitor compound. Amino-uanidine was used as a positive control.

N-Acetyl-glcyl-lysine methyl ester (G.K. peptide)-ribose assay.valuation of the late glycation products (AGE’s), and AGE-

nhibition by the new inhibitor compounds was tested by incubationf G.K. peptide in ribose in the presence or the absence of the agent,ollowed by determination of chromophores generated in the coursef glycation and AGE formation through determination of their spe-ific fluorescence (45). This test is used to evaluate the ability of theompounds of the present study to inhibit the crosslinking of-acetylglycyl-lysine methyl ester in the presence of ribose.Equal volumes (0.l mL) of 0.5 M sodium phosphate buffer pH 7.4

ontaining 0.2 g/L NaN3, GK peptide 80 mg/mL in 0.5 sodium phos-hate buffer pH 7.4 and Ribose 800 mmoles/L (120 mg/mL) in 0.5 Mhosphate buffer were mixed together, filtered through a 0.2 micron

FIG. 1. d-Gluconolaction-Hb assay. Effects of various inhibitorompounds on whole blood incubated for 18 hrs with d-Glu. Newompounds added at 1 mM final concentrations. Aminoguanidine at0 mM as control HbA1c levels present the results of three determi-ations. A, baseline control; B, d-Glu treated blood; C, d-Glu plus AG;, LR26; E, LR28; F, LR29; G, LR33; H, LR36; I, LR41; J, LR45;, LR49; L, LR62. Percent inhibition of Hb1c formation at 1 mmole

oncentration of various test compounds are as follows: AG (50 mM),2.8%; LR26, 73.8%; LR28, 50%; LR29, 52%; LR33, 40.4%; LR36,6.8%; LR41, 60.7%; LR45, 56.8%; LR49, 57.3%; LR62, 53.7%.

653

lter and incubated under aseptic conditions for 24 hours at 37°C.he inhibitor compounds were added to a final concentration of 1 mole/L. At the end of the incubation period, samples were analyzed

or their specific fluorescence (excitation, 340 nm; emission, 420 nm).he % inhibition by different concentrations of inhibitor was calcu-

ated as described above.

ELISA assay. We have used a special ELISA technique to eval-ate the ability of the compounds under this study to inhibit therosslinking of glycated-BSA (AGE-BSA) to a rat tail-tendon-ollagen coated 96 well plate (46). Cross-linking of AGE-BSA to a ratail-tendon-collagen coated plate was performed with and withouthe testing compound at the desired concentrations. The uncross-inked AGE-BSA was then removed by washing the wells. The AGE-SA cross-linked to the tail-tendon-collagen coated plate was thenuantified by a polyclonal antibody raised against AGE-RNase. Pos-tive results in this assay indicate that the inhibitor is capable ofeducing the amount of AGE-BSA which cross-links with collagen.minoguanidine was used as positive control.

ESULTS

The results of the d-Glu assay on representative 9ompounds from LR series and AG are demonstratedn Fig. 1.

HbA1c levels in each sample is represented by a barraph. Levels of HbA1c in “B”; the d-Glu treated bloods twice higher than the baseline control “A”. Variousnhibitors (C to L) show different levels of HbA1c, de-ending on their inhibitory potencies. Percent inhibi-ion of each compound is calculated at the bottom ofig. 1. AG, was used at 50 m moles/l, new compoundst 1 m mole/l concentration.Figure 2 demonstrates the data from a BSA-glucose

ssay in the form of a bar graph on the same 9 com-ounds and AG as positive control. Each bar representsercent inhibition of the control by the specific inhibi-or. This assay method is mostly for the inhibitors ofate glycation and AGE formation (post-Amadori). Theesults obtained by this assay show all 9 compoundsnvestigated here have strong inhibitory effects onost-Amadori glycation, AGE formation and AGE-rosslinking.Inhibition effects of the 9 compound assayed with the.K. peptide-ribose assay demonstrated in Fig. 3. The

FIG. 2. BSA-Glucose assay. Percent inhibition of AGE-formationy 1 mm/l of various new compounds, compared to 50 mm/l amino-uanidine. A, AG (50 mM); B, LR26; C, LR28; D, LR29; E, LR33;, LR36; G, LR41; H, LR45; I, LR49; J, LR62.

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Vol. 262, No. 3, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

esults of this assay also show that all 9 compoundsnvestigated here have strong inhibitory effects andlock specific fluorescence of proteins AGE in theseeparate determinations.Data from immunochemical studies on the inhibitory

ffects of the 2 representative (LR33, LR41) com-ounds using a specific ELISA are presented in Fig. 4.hese 2 compounds are among a number of strong

nhibitors of AGE-protein crosslinking. Percent inhibi-ions of the control were calculated to be 61% LR33 and7.4% for LR41 at 1 mmoles/L and 45.5% for AG at 50M concentrations.

ISCUSSION

The prototypes of the LR series were developed in984, as allosteric effectors of hemoglobin for loweringxygen affinity of human blood (47). It was also dem-nstrated that these compounds are capable of lower-ng blood cholesterol in rats. In the course of screeningifferent classes of organic compounds for investiga-ion of their possible inhibitory effects on advancedlycation endproudcts (AGE’s), we included some of theR series such as (LR3) and (LR5) and surprisingly

FIG. 3. G.K. peptide-ribose assay. Percent inhibition of AGE-ormation by 1 mm/l of various new compounds, compared to 50 mm/lminoguanidine. A, AG (50 mM); B, LR26; C, LR28; D, LR29;, LR33; F, LR36; G, LR41; H, LR45; I, LR49; J, LR62.

FIG. 4. Crosslinking of collagen-AG

654

ound these compounds to be strong inhibitors of earlynd post Amadori glycation. Since then, we have de-igned, synthesize other derivatives of these com-ounds and screened them using the above mentionedssay methods. Compounds comprising of the phenoxy-sobutyric acid alone were found effective (LR35). Com-ounds with additional aryl and (heterocylic) ureido orryl and (heterocyclic) carboxamido structure showigher inhibitory effects. Changes in the later part ofhe molecule greatly effects the activity of the com-ounds (see Table 1). There are a number of theseompounds which are approximately 40 times moreotent then aminoguanidine, and some are 2 to 3 timesore effective than pyridoxin (41) (data not shown).Despite the limitations of our current knowledge on

he mechanism of action the known inhibitors, trap-ing of reactive carbonyl compounds the chemical in-ermediates between hyperglycemia/hyperlipidemiand diabetic complications, may be a valuable strategyor inhibiting or delaying diabetic complications (15).onsidering the diversities of these carbonyl interme-iates, different drugs with diverse structures could beiscovered as inhibitors of carbonyl compounds.The mechanism of action of this class of compounds

s yet to be known. The present study indicates thathese compounds are powerful inhibitors that act atultiple steps of glycation and AGE formation, i.e.

arly stage of glycation as evidence by lowering HbA1c

evels in the d-Glu assay, which is a specific assay forhe early stage of glycation (type A inhibitor). Most ofhese compounds strongly inhibit the post-Amadorilycation as demonstrated by the G.K.-Ribose andSA-glucose assays (type B, D inhibitors) and a goodumber of them are inhibitors of AGE-proteinrosslinking as evidence by specific ELISA assay (type

inhibitors as described by Baynes et al. (32).The chemistry and synthesis of prototypes of LR

ompounds are reported elsewhere (48). Animal exper-mentation of a limited number of most efficient com-ounds in STZ induced diabetic rats will begin in theoming months.

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655

CKNOWLEDGMENT

The authors thank Almira Fontanilla for typing the manuscript.

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Summarized Data Obtained by the 3 Assay Methodson All 63 Compounds and AG as a Positive Control

Compound d-Glu assay G.K.-ribose assay BSA-glucose assay

AG 12.1 67.0 74.0LR1 28.8 11.2 24.8LR2 17.3 36.8 45.0LR3 25.0 40.0 46.2LR4 19.2 42.5 48.7LR5 36.5 33.6 66.2LR6 25.0 9.1 57.5LR7 19.2 27.5 39.6LR8 21.0 23.6 47.5LR9 17.7 0.0 31.2LR10 22.5 20.5 56.2LR11 25.8 18.6 50.0LR12 22.5 43.6 55.0LR13 21.0 27.5 48.1LR14 22.5 18.6 49.3LR15 22.5 30.0 58.1LR16 35.1 0.0 29.1LR17 46.4 26.4 12.5LR18 58.1 26.2 37.5LR19 41.0 40.0 28.4LR20 52.8 31.0 12.8LR21 50.1 15.0 7.1LR22 42.7 10.0 14.2LR23 45.0 70.0 42.6LR24 50.0 41.4 31.2LR25 52.0 30.5 41.5LR26 73.8 47.1 38.9LR27 50.0 52.5 52.0LR28 50.0 63.3 78.8LR29 52.0 53.1 44.2LR30 63.6 18.1 16.2LR31 54.5 9.6 13.3LR32 47.7 9.5 32.7LR33 70.4 25.1 41.1LR34 52.2 15.1 24.0LR35 47.7 5.7 42.4LR36 56.8 30.1 44.4LR37 40.9 41.2 47.7LR38 50.9 20.2 13.8LR39 56.8 18.6 21.2LR40 50.9 30.6 32.0LR41 60.7 35.4 37.4LR42 47.0 21.8 46.8LR43 58.8 21.5 44.0LR44 58.8 13.0 42.1LR45 56.8 31.7 49.5LR46 55.7 21.1 30.1LR47 54.0 30.5 34.7LR48 45.9 31.0 45.5LR49 57.3 22.9 41.3LR50 57.3 27.3 42.7LR51 52.0 0* 0*LR52 58.3 0* 0*LR53 54.1 13.6 20.4LR54 54.1 4.9 22.8LR55 56.2 11.0 36.8LR56 46.2 2.1* 39.1*LR57 48.1 0* 31.1*LR58 40.7 4.5* 49.0*LR59 48.1 8.0 39.4LR60 29.6 0* 47.8*LR61 46.2 13.1 62.0LR62 53.7 26.0 49.1LR63 40.7 10.9 60.2

* These compounds have an intrinsic fluorescence, which inter-eres with the assay.

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