Geranyl flavonoids from the leaves of Artocarpus altilis

Five geranyl dihydrochalcones, 1-(2,4-dihydroxyphenyl)-3-{4-hydroxy-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H-dibenzo[b,d]pyran-5-yl}-1-propanone (2), 1-(2,4-dihydroxyphenyl)-3-[3,4-dihydro-3,8-dihydroxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (4), 1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(3,4-epoxy-4-methyl-1-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (5), 1-(2,4-dihydroxyphenyl)-3-[8-hydroxy-2-methyl-2-(4-hydroxy-4-methyl-2-pentenyl)-2H-1-benzopyran-5-yl]-1-propanone (8), and 2-[6-hydroxy-3,7-dimethylocta-2(E),7-dienyl]-2',3,4,4'-tetrahydroxydihydrochalcone (9), along with four known geranyl flavonoids (1, 3, 6, 7), were isolated from the leaves of Artocarpus altilis. Their structures were established by spectroscopic means and by comparison with the literature values. Compounds 2, 4, and 9 exhibited moderate cytotoxicity against SPC-A-1, SW-480, and SMMC-7721 human cancer cells.     

Design, synthesis, stereochemistry and antioxidant properties of various 7-alkylated 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ones

We used the modified Mannich condensation to synthesize three closely-related series of 7-alkylated 3-ABNs 1-5 viz., 7-methylated 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ones (7-Me ABNs 1-5), 7-ethylated 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ones (7-Et ABNs 1-5) and 7-tert-pentylated 2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ones (7-tert-pentyl ABNs 1-5). All compounds yielded good as single isomers by the use of PPA·SiO(2) as a heterogeneous Bronsted acidic catalyst. The 1D, 2D NMR, and single-crystal XRD interpretations unambiguously characterized the stereochemistry of the synthesized compounds. In solution as well as solid-state, all compounds exist in the twin-chair conformation with equatorial orientations of all substitutions, despite their nature and positions. The chemical methods viz., DPPH, reducing power, and phospho-molybdenum methods identified some of the target curcumin analogs as active compounds. Among them, 7-Me ABN 4 (7-methyl-2,4-bis(3-methoxy-4-hydroxyphenyl)-3-azabicyclo[3.3.1]nonan-9)-one exerted the best antioxidant profile that comparable to standard l-ascorbic acid, α-tocopherol and curcumin. Hence, we evaluated further for its intracellular ROS inhibition potency on RAW 264.7 macrophage cells, and found to be effective as well as non-toxic at 100μM.     

Inhibitory effect on NO production of phenolic compounds from Myristica fragrans

Three new phenolics: ((7S)-8'-(benzo[3',4']dioxol-1'-yl)-7-hydroxypropyl)benzene-2,4-diol (1), ((7S)-8'-(4'-hydroxy-3'-methoxyphenyl)-7-hydroxypropyl)benzene-2,4-diol (2) and ((8R,8'S)-7-(4-hydroxy-3-methoxyphenyl)-8'-methylbutan-8-yl)-3'-methoxybenzene-4',5'-diol (3), along with four known compounds (4-7) were isolated from the seeds of Myristica fragrans. Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW264.7 cells.     

Synthesis and antiproliferative activity of 2,6-diamino-9-benzyl-9-deazapurine and related compounds

Treatment of 6-bromomethyl- or 6-dibromomethyl-5-nitropyrimidine-2,4-diamine with KCN gave the same product--(2,6-diamino-5-nitropyrimidinyl)acetonitrile. Benzylation of the nitrile took place on the alpha-carbon to the cyano group preferentially affording the corresponding mono- and dibenzyl derivative, whose reductive cyclization resulted in 7-benzyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine and 7,7-dibenzyl-7H-pyrrolo[3,2-d]pyrimidine-2,4,6-triamine, respectively. Suitability of the protection of N(2) and N(4) atoms with benzyl, acetyl, or benzoyl groups was also investigated. The in vitro evaluation of cell growth inhibition on CCRF-CEM, HL-60, HeLa S3, and L1210 cell lines showed significant activity in 8 new compounds. The most potent compounds were the above mentioned 6-dibromomethyl derivative (IC(50)=0.54, 1.7, 5.0, and 1.9 molL(-1)) and 7,N(2),N(4)-tribenzyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (IC(50)=1.9, 2.7, 7.3, and 1.0 molL(-1)).     

Eryvarins F and G,two 3-phenoxychromones from the roots of Erythrina variegata

Two 3-phenoxychromones, eryvarins F and G, were isolated from the roots of Erythrina variegata. Their structures were established to be 3-(2,4-dihydroxyphenoxy)-7-hydroxy-6,8-di(3,3-dimethylallyl)chromen-4-one and 3-(2,4-dihydroxyphenoxy)-8-(3,3-dimethylallyl)-2,2-dimethylpyrano[5,6:6,7]chromen-4-one on the basis of spectroscopic and chemical evidence. Eryvarins F and G are unusual 3-phenoxychromone derivatives with two isoprenoid groups.     

Synthesis of 3'-thioamido-modified 3'-deoxythymidine-5'-triphosphates and their use as chain terminators in Sanger-DNA sequencing

The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxo- propyl)amino]thymidine 1 and 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-((6-([(9H-(fluo-ren-9- ylmethoxy)carbonyl]-amino)-1-thioxohexyl)amino) thymidine 2 were synthesized by regioselective thionation of their corresponding amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lawesson's reagent). The thioamides were converted into the corresponding 5'-triphosphates 3 and 4. Compound 3 was chosen for DNA sequencing experiments and 4 was further labelled with fluorescein.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

Synthesis of new 2,4-Diaminopyrido[2,3-d]pyrimidine and 2,4-Diaminopyrrolo[2,3-d]pyrimidine inhibitors of Pneumocystis carinii,Toxoplasma gondii,and Mycobacterium avium dihydrofolate reductase

A concise new route allowing easy access to five previously unreported 2,4-diamino-6-(substituted benzyl)pyrido[2,3-d]pyrimidines (2a-e) was developed, involving condensation of 2,4-dipivaloylamino-5-bromopyrido[2,3-d]pyrimidine (6) with an organozinc halide in the presence of a catalytic amount of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II).CH(2)Cl(2), followed by removal of the pivaloyl groups with base. Also prepared via a scheme based on the Taylor ring expansion/ring annulation synthesis were three heretofore undescribed 2,4-diamino-5-(substituted benzyl)-7H-pyrrolo[2,3-d]pyrimidines (3b-c). Standard spectrophotometric assays were used to compare the ability of 2a-e and 3b-c to inhibit dihydrofolate reductase (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium, three examples of opportunistic pathogens to which AIDS patients are highly vulnerable because of their immunocompromised state. For comparison, 13 previously untested 2,4-diamino-6-(substituted benzyl)quinazolines (17a-m) were also evaluated as inhibitors of these enzymes, as well as the enzyme from rat liver. None of the quinazolines or pyridopyrimidines tested was more potent against the P. carinii enzyme than the structurally related reference compound piritrexim (1), and none showed selectivity for the P. carinii enzyme over the rat enzyme. One of the pyridopyrimidines (2c) showed 10-fold selectivity for T. gondii versus rat DHFR, and two of them (2b, 2c) showed selectivity for the M. avium enzyme. However, this gain in species selectivity was achieved at the cost of decreased in potency, as has been noted with many other lipophilic DHFR inhibitors.     

Photochemical reactions of transition metal organyl complexes with olefins Part 10. Light-induced formal [5+2] cycloadditions at manganese

Tricarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-manganese (1) forms upon UV irradiation in THF at 208 K solvent stabilized dicarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-tetrahydrofurane-manganese (2). With butynedioic acid dimethyl ester (3) and diphenylacetylene (5) complex 2 yields tricarbonyl-η⁵-1,2-dimethoxycarbonyl-4,6-dimethyl- cyclohepta-2,4-dien-1-yl-manganese (4) and tricarbonyl-η-4,6-dimethyl-1,2-diphenyl-cyclohepta-2,4-dien-1-yl- manganese (6) in a formal [5+2] cycloaddition. Addition of carbon monoxide and a 1,4-H shift completes the reaction. Propynoic acid methyl ester (7) forms the 2:1 adduct dicarbonyl-η^5:2-1,3-dimethyl-6-methoxycarbonyl-6- (E-2′-methoxycarbonylvinyl)-cyclohepta-2,4-dien-1-yl-manganese (8). The crystal and molecular structure of 8 was determined by X-ray structure analysis. The molecular structures of the complexes 4 and 6 were established by IR and NMR spectroscopy. Formation mechanisms of 4, 6 and 8 are discussed. Crystal data for 8: monoclinic space group P2₁/c, a=802.6(3), b=1136.6(1), c=8872.3(3) pm, β=93.14(2)°, V=1.705 nm³, Z=4.     

Callus induction and plantlet regeneration in damiana (Turnera diffusa,Willd.)

Experiments were performed to determine optimal conditions for callus induction and plantlet regeneration from damiana (Turnera diffusa, Willd.) leaf explants. MS media [7] and B5 media [4] were used; both of them were supplemented with different combinations of 2,4-dichlorophenoxyacetic, acid (2,4-D) and 6-benzyl-adenine (BA). In all cases the samples were incubated under continous fluorescent light, 100 mol.m^-2.sec^-1, at 25±2°C for 60 days. Highest callus percentage was obtained in MS media supplemented with 2,4-D 10^–5 M plus BA 10^-8M. Maximum percentage and multiple shoots and leaves were produced in MS media with BA 10^-5M and BA 10^-5M plus 2,4-D 10^-7M. These explants were transferred for rooting to MS medium without plant growth regulators. Later, rooted plantlets were successfully established in soil.     

Solid-phase synthesis of chiral stationary phases based on 2,4,5,6-tetrachloro-1,3-dicyanobenzene derivatives spaced from N-3,5-dinitrobenzoyl alpha-amino acids: comparative study of their resolution efficacy

Two new chiral stationary phases, 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-D-phenylglycinyl) aminoethyl]aminophen-1-yl] aminopropyl silica (CSP-1) and 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-L-leucinyl) aminoethyl] aminophen-1-yl] aminopropyl silica (CSP-2), were prepared by solid-phase synthesis. They comprise chiral unit, 3,5-dinitrobenzoyl derivative of the amino acid, D-PhGly or L-Leu, bound via spacer 1,2-diaminoethane to 2,4-positions of the persubstituted benzene ring, derived from compound 1, and possess pseudo-C2 symmetry. Preparation of model compounds 6 and 7 confirmed the structure of chiral selectors, which comprise pi-donor persubsituted aromatic ring and two strong pi-acceptor 3,5-dinitrobenzoyl amido units. CD spectra of model selectors 6 and 7, run in DMSO above 250 nm, exhibit negative exciton coupling (EC) between pi-acceptor and pi-donor chromophores, C(1) symmetric model compound 8 exhibited much weaker EC and 9, devoid of pi-donor unit, does not exhibit any significant CD. Combined pi-donor and pi-acceptor properties enable the new CSPs to separate a broad range of racemates. The columns with CSP-1 and CSP-2 were tested for the separation of 22 racemates by HPLC with two different mobile phase systems and the results are compared with those obtained by using a structurally related commercial column.     

Syntheses of 2″,3″-Dideoxy-L-Glycero-Pentofuranosyl C-Nucleosides

Abstract

2′,3′ -Dideoxy-L-C-nucleosides, 4-amino-8-(2,3-dideoxy-L-glyceropento-furanosyl)pyrazolo[1,5-a]-1,3,5-triazines (9 and 10), 4-amino-7-(2,3-dideoxy-L-glycero-pentofuranosyl)-3H,5H-pyrrolo[3,2-d]pyrimidines (17 and 18), 7-(2,3-dideoxy-L-glyceropentofuranosyl)-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidines (23 and 24) and 2,4-diamino-5-(2,3-dideoxy-L-glyceropentofuranosyl)pyrimidines (28 and 29) have been synthesized from L-gulonic γ-lactone 1.     

Synthesis of [18F]SU11248, a new potential PET tracer for imaging cancer tyrosine kinase

N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-[18F]fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide, a new potential positron emission tomography tracer for imaging cancer tyrosine kinase, has been prepared by the nucleophilic substitution of the nitro-precursor N-[2-(diethylamino)ethyl]-5-[(Z)-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide with K18F/Kryptofix 2.2.2 followed by a simple chromatography methodology combined solid-phase extraction with high-performance liquid chromatography purification procedures in 15-25% radiochemical yields.     

New strategies for the synthesis of A3 adenosine receptor antagonists

New A(3) adenosine receptor antagonists were synthesized and tested at human adenosine receptor subtypes. An advanced synthetic strategy permitted us to obtain a large amount of the key intermediate 5 that was then submitted to alkylation procedures in order to obtain the derivatives 6-8. These compounds were then functionalised into ureas at the 5-position (compounds 9-11, 18 and 19) to evaluate their affinity and selectivity versus hA(3) adenosine receptor subtype; in particular, compounds 18 and 19 displayed a value of affinity of 4.9 and 1.3 nM, respectively. Starting from 5, the synthetic methodologies employed permitted us to perform a rapid and a convenient divergent synthesis. A further improvement allowed the regioselective preparation of the N(8)-substituted compound 7. This method could be used as an helpful general procedure for the design of novel A(3) adenosine receptor antagonists without the difficulty of separating the N(8)-substituted pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines from the corresponding N(7)-isomers.     

Cytotoxic effects of 2-arylbenzofuran phytoestrogens on human cancer cells: modulation by adrenal and gonadal steroids

Although 2-arylbenzofuran phytoalexins are known for decades, their anticancer activity has not been studied systematically. We have previously reported on the isolation and the estrogen receptor (ER) modulation properties of three new 2-arylbenzofurans from Onobrychis ebenoides, ebenfuran I [2-(2,4-dihydroxyphenyl)-5-hydroxy-6-methoxy-benzofuran], ebenfuran II [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-benzofuran] and ebenfuran III [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-5-(3-methyl-buten-2-yl)-benzofuran]. We now show that, while I and II could stimulate the proliferation of MCF-7 cells, III was inhibitory in a proliferation-dependent manner. III inhibited the growth of all human cancer cells examined, regardless of ER or multidrug resistance status. Estradiol rendered MCF-7 cells more sensitive to III, and this coincided with the ability of the hormone at concentrations ≥0.1 nM to bind to the ER of the cells and stimulate their proliferation in the presence of III. Cell proliferation stimulating concentrations of I and II also enhanced the effect of III on MCF-7 cells. However, dehydroepiandrosterone and dihydrotestosterone were ineffective in this respect. III-treated MCF-7 cells exhibited G1 phase arrest followed by detachment-induced cell death and/or apoptosis in the adherent fraction, pronounced induction of Bax and suppression of estradiol induction of Bcl-2. Our data indicate that the largely unexplored pool of benzofuran phytoalexins includes entities potentially suitable for chemoprevention and treatment of human cancer.     

An alkaloid, two conjugate sesquiterpenes and a phenylpropanoid from Pachypodanthium confine Engl. and Diels

Two sesquiterpene-trimethoxystyrene conjugates (E)-1-[3'-(4',8'-dimethylnona-3',7'-dienyl)cyclohex-3'-enyl]-2,4,5-trimethoxybenzene (1) and (Z)-1-[3'-(4',8'-dimethylnona-3',7'-dienyl)cyclohex-3'-enyl]-2,4,5-trimethoxybenzene (2), a phenylpropanoid 1,2,4-trimethoxy-5-(1-methoxy-ethyl)-benzene (3), and an aporphine alkaloid N-acetylpachypodanthine (4), were isolated in addition to several known compounds from cyclohexane, dichloromethane and alkaloid extracts from bark of Pachypodanthium confine. The structures of these compounds were established based on the interpretation of their high resolution NMR (HSQC, HMBC, COSY and NOESY) spectral data.     

Synthesis and evaluation of in vitro antiviral activity of novel phenoxy acetic acid derivatives

Several substituted phenoxy acetic acid derived pyrazolines were synthesized by the reaction between 2-{4-[3-(2,4-dihydroxyphenyl)-3-oxo-1-propenyl]-2-methoxyphenoxy} acetic acid and substituted acid hydrazides and were tested for their in vitro cytotoxicity and antiviral activity. None of the compounds showed any specific antiviral activity [50% antivirally effective concentration (EC(50)) > or = 5-fold lower than minimum cytotoxic concentration]. The most cytotoxic of the series was 2-{4-[3-(2,4-dihydroxyphenyl)-1-(2-hydroxybenzoyl-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3(j)), with a minimum cytotoxic concentration of 0.16 microg/mL in human embryonic lung (HEL) cells.     

1,2,4]Triazole derivatives as 5-HT(1A) serotonin receptor ligands.

A series of new 4-amino-3-[3-[4-(2-methoxy or nitro phenyl)-1-piperazinyl] propyl]thio]-5-(substitutedphenyl)[1,2,4]triazoles 11a-t was synthesized in order to obtain compounds with high affinity and selectivity for 5-HT(1A) receptor over the alpha(1)-adrenoceptor. A series of isomeric 4-amino-2-[3-[4-(2-methoxy or nitro phenyl)-1-piperazinyl]propyl]-5-(substitutedphenyl)-2,4-dihydro-3H[1,2,4]triazole-3-thiones 12a-r was also isolated and characterized. New compounds were tested to evaluate their affinity for 5-HT(1A) receptor and alpha(1)-adrenoceptor in radioligand binding experiments. As a general trend, triazoles 11a-t showed a preferential affinity for the 5-HT(1A) receptor whereas isomeric 2,4-dihydro-3H[1,2,4]triazole-3-thiones 12a-r preferentially bind to the alpha(1)-adrenoceptor site. Several molecules showed affinities in the nanomolar range and 4-amino-3-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]thio]-5-(4-propyloxy-phenyl)[1,2,4]triazole (11o) was the most selective derivative for the 5-HT(1A) receptor (K(i) alpha(1)/K(i) 5-HT(1A)=55). The decrease in 5-HT(1A) receptor selectivity in 3-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]thio]-5-(substitutedphenyl)[1,2,4] triazole 14a-b, lacking in the amino group in 4-position of the triazole ring, in comparison with their analogues in the series 11a-t, suggest that the amino function represents a critical structural feature in determining 5-HT(1A) receptor selectivity in this class of compounds.     

Phenylpiperazine derivatives with strong affinity for 5HT1A, D2A and D3 receptors

Four 7-[3-(4-phenyl-1-piperazinyl)propoxy]coumarins were synthesized. The affinities of these compounds for DA (D_2A, D₃) and 5HT_1A receptors were evaluated for their ability to displace [³H]-raclopride and [³H]-8-OH-DPAT respectively from their specific binding sites. The affinities of the target compounds were all in the nanomolar range and followed the order 5-HT_1A > D₂ > D₃.     

Synthesis of C-6 Pyrimidine Acyclic Nucleoside Analogs as Potential Antiviral Agents

Abstract

A number of pyrimidine acyclic nucleosides in which the acyclic moiety is attached to the C-6 position rather than N-1 of the pyrimidine ring have been prepared. This was accomplished via treatment of lithiated 2,4-dimethoxy-5,6-dimethylpyrimidine, or, 2,4-dimethoxy-6-methylpyrirnidine with 1,3-bis-(benzyloxy)-2-propanone, benzyl chloromethyl ether or oxirane, respectively, to give the corresponding key intermediates 6-[3-benzyloxy-2-[(benzyloxy)methyl]-2-hydroxypropyl]-2,4-dimethoxy-5-methylpyrimidine (2a), 6-[3-Denzyloxy-2-[(benzyloxy)methyl]-2-hydroxypropyl]-2,4-dimethoxypyrimidine(2b), 6-(2-benzyloxyethyl)-2,4-dimethoxy-5-methylpyrimidine (3), and2,4-dunethoxy-6-(3-hydroxypropyl)-5-methylpyrimidine (4a). After acidic hydrolysis, followed by debenzylation with boron trichloride these key intermediates were converted to the target C-6 pyrimidine acyclic derivatives. Compounds 6–8b, 11–13, 15, 16, 20, 22, 26, and 29–32 were evaluated for activity against herpes viruses and human immunodeficiency virus. None of the compounds were active against the viruses nor were they cytotoxic at the highest concentration tested.