Novel synthesis of [1]-benzothiepino[5,4-b]pyridine-3-carbonitriles and their anti-inflammatory properties

Reaction of 4-arylmethylene-3,4-dihydro-[1]-benzothiepin-5(2H)-ones 1 with malononitrile in the appropriate alcohol in the presence of sodium afforded the 2-alkoxy-4-aryl-5,6-dihydro-[1]-benzothiepino[5,4-b]pyridine-3-carbonitriles 2 and not the isomeric forms [1]-benzothiepino[4,5-c]pyridine-1-carbonitriles 3 in high regioselective manner. The assumed structure of 2 was inferred through independent synthetic reaction of 3,4-dihydro-[1]-benzothiepin-5(2H)-one (4) with ylidenemalononitriles 5 under the same applied reaction conditions and confirmed by single crystal X-ray diffraction studies. However, reaction of 4 with arylidenecyanothioacetamides 6 in refluxing ethanol in the presence of basic catalyst (piperidine or morpholine) does not afford the expected 4-aryl-3-cyano-5,6-dihydro-[1]-benzothiepino[5,4-b]pyridine-2(1H)-thiones 7 and instead 4-aryl-3,5-dicyano-6-thioxo-2(1H)-pyridinethiolate monohydrates were isolated as piperidinium or morpholinium salts 8. On the other hand, reaction of 6 with cyanothioacetamide in the presence of a sufficient amount of basic catalyst yielded exclusively 2-amino-4-aryl-3,5-dicyano-2-pyridinethiolates as piperidinium or morpholinium salts 9. Meanwhile, 7 were prepared through the reaction of 1 with cyanothioacetamide in refluxing ethanol in the presence of a catalytic amount of piperidine. Anti-inflammatory activity screening of the prepared compounds using in vivo acute carrageenan-induced paw oedema in rats exhibited that all the tested compounds possess considerable activity. In addition, few synthesized derivatives reveal remarkable anti-inflammatory properties (2d, k, l) comparable with indomethacin which was used as a reference standard during the pharmacological activity screening studies.     

Synthesis of some new [1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and [1,2,4]triazolo[3,4-b][1,3,4] thiadiazoles starting from 5-nitro-2-furoic acid and evaluation of their antimicrobial activity

New series of fused 1,2,4-triazoles such as, 6-(aryl)-3-(5-nitrofuran-2-yl)-5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 4-8, 6-(alkyl/aryl amino)-3-(5-nitrofuran-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 9-13 and 6-(4-substituted phenyl)-3-(5-nitrofuran-2-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 14-18 have been synthesized via the reaction of 4-amino-5-(5-nitrofuran-2-yl)-4H-1,2,4-triazole-3-thiol 3 with various reagents such as hetero aromatic aldehydes, alkyl/aryl isothiocyanates and 4-substituted phenacyl bromides, respectively. The structures of the newly synthesized compounds have been confirmed on the basis of elemental analysis and spectral studies. The newly synthesized triazolo derivatives have been investigated for their in vitro antibacterial activity. Most of the tested compounds showed interesting antibacterial activity against Staphylococcus aureus. Furthermore, the most potent antibacterial compounds 11-13 were evaluated for their in vitro cytotoxic activity against human cancer cell lines. It was found that compounds 11 and 13 showed higher cytotoxicity against Hep-G2 cell line as compared to standard.     

Contribution of the anomeric effect to the solution and crystal structure of [1S,2S,6S,7s]-1,6-diaza-4,9-dioxa-2,7-dimethoxycarbonylbicyclo[4.4.1]undecane, a condensation product of L-serine methyl ester with formaldehyde

The reaction of L-serine methyl ester hydrochloride (1) with paraformaldehyde (2) in dichloromethane in the presence of triethylamine afforded a novel compound: [lS,2S,6S,7S]-1,6-diaza-4,9-dioxa-2,7-dimethoxycarbonylbicyclo[4.4.1]undecane (4) as a 2:3 adduct of 1 with 2. 1H and 13C NMR spectroscopy were unable to discriminate between two possible symmetrical structures. The latter was unambiguously proved by X-ray crystallography. The crystal structure established: (i) the existence of two identical seven-membered rings each containing a N-C-O grouping; (ii) the existence of a long C-O-C-N-C-N-C-O-C sequence in which each nitrogen belongs simultaneously to a N-C-O (oxazolidine) and to a N-C-N (aminal) motifs; (iii) the existence of a chair-like conformation for both seven-membered rings; (iv) the antiperiplanar geometry of pN-C-O and consequently the manifestation of a strong anomeric effect in both N-C-O groupings, whereas anomeric effect was virtually absent in the N-C-N sequence, as corroborated by bond distances and bond angles. Chemical shifts, coupling constants and NOE effects confirm that the conformational features of 4 are preserved in solution.     

New strapped calix[4]pyrrole based receptor for anions

Calix[4]pyrrole bearing catechol-derived diether strap linked via alkyl chains has been synthesized and characterized for the first time. The strap with 1,2-diether link is providing a relatively constrained geometry on its side of the calix[4]pyrrole moiety. As a result only one isomer (cis-type) of the receptor formed during reaction. The crystal structure reveals two molecules of methanol bound to the host. This calix[4]pyrrole also exhibits enhanced binding towards halide anions compared to simple calix[4]pyrrole apart from showing binding towards dihydrogenphosphate and acetate ions. The association constants are quite similar to that found for orcinol strapped calix[4]pyrrole towards halide anions in general, but having a higher preference for chloride than bromide ion in particular. Further it shows very strong preference towards fluoride ion.     

Synthesis and pharmacological evaluation of condensed heterocyclic 6-substituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole derivatives of naproxen

Some 6-substituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole derivatives (4a-f and 5a-d) have been synthesized by cyclisation of 4-amino-5-[1-(6-methoxy-2-naphthyl)ethyl]-3-mercapto-(4H)-1,2,4-triazole (3) with various substituted aromatic acids and aryl/alkyl isothiocyanates, through a single step reaction. The target compounds were pharmacologically evaluated for their anti-inflammatory and analgesic potentials by known experimental models. Several of these showed significant activity. Very low ulcerogenic index was observed for potent compounds.     

Substituent effects on the properties of fluorene-thieno[3,4-b]pyrazine derivatives for light-emitting applications

Five thieno[3,4-b]pyrazine-based model compounds were studied to explore the effects of the substituent groups (alkyl or aryl) on the structure, atomic charge, optical properties, ionization potential (IP), electron affinity (EA), and reorganization energy. Theoretical calculations were carried out by density functional theory (DFT) using the B3LYP hybrid function combined and CAM-B3LYP with the 6-31G(d) basis set. The lowest-lying absorption and emission spectra of 9,9'-diethylhexylfluorene-alt-5,7-dithien-2-yl-thieno[3,4-b] pyrazine (FDDTTP) with alkyl groups showed a blue-shift, while those of FDDTTP with aryl groups exhibited a red-shift. The results agree well with analytical data from reorganization energies. IPs are brought down by both alkyl and aryl groups. However, EAs are raised only by aryl units. The results indicate that aryl groups are more helpful in forming excitions for FDDTTP molecules. Consequently, FDDTTP with aryl groups are more efficient acceptor segments for designing donor-acceptor copolymers than those with alkyl groups.     

Radiosynthesis of [11C]BBAC and [11C]BBPC as potential PET tracers for orexin2 receptors

Radiosynthesis of [N-methyl-(11)C](S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide ([(11)C]BBAC or [(11)C]3) and [N-methyl-(11)C] (S)-N-([1,1'-biphenyl]-2-yl)-1-(3-(1-methyl-1H-benzo[d]imidazol-2-yl)propanoyl)pyrrolidine-2-carboxamide ([(11)C]BBPC or [(11)C]-4), two potential PET tracers for orexin2 receptors are described. Syntheses of non-radioactive standards 3, 4 and corresponding desmethyl precursors 1, 2 were achieved from common intermediate (S)-2-([1,1'-biphenyl]-2-yl)-1-(pyrrolidin-2-yl)ethanone. Methylation using [(11)C]CH(3)OTf in the presence of base in acetone afforded [(11)C]3 and [(11)C]4 in 30±5% yield (EOS) with >99 % radiochemical purities with a specific activity ranged from 2.5±0.5 Ci/μmol (EOB). The logP of [(11)C]3 and [(11)C]4 were determined as 3.4 and 2.8, respectively. The total synthesis time was 30 min from EOB. However, PET scans performed in a rhesus monkey did not show tracer retention or appropriate brain uptake. Hence [(11)C]3 and [(11)C]4 cannot be used as PET tracers for imaging orexin2 receptors.     

Synthesis of regiospecifically labeled [18O]glycolic acid and [18O]acyldihydroxyacetone phosphate

Methods are detailed for the preparation of [2-18O]glycolate from chloroacetic acid and for the direct conversion of these intermediates to regiospecifically labeled [2-18O]-2-O-acylglycolic acids containing approximately 90% 18O at the C-O-acyl bond. Methods are also detailed for optimization of reaction conditions and yields for each synthetic step in previously published methods for the preparation of 1-O-acyldihydroxyacetone-3-O-phosphate (DHAP) from acyloxyacetic acid (i.e., 2-O-acylglycolic acid), where acyl is tetradecanoyl, hexadecanoyl, or heptadecanoyl. The optimized reaction conditions generate 1-O-acyl DHAP in its acid form, both in high overall yield and in high purity, without requiring a final chromatographic purification of the product, 1-O-acyl DHAP. Combining these new methods, efficient and facile preparations of regiospecifically labeled [1-18O]-1-O-hexadecanoyl DHAP and [1-18O]-1-O-heptadecanoyl DHAP have now been demonstrated, in which approximately 90% 18O is specifically located only at the C-O-acyl position. Some mechanistic postulates are offered to account for the optimized yields, regioselectivities, and high 18O incorporation which are observed in the reactions we have employed to generate 1-O-acyl DHAP from glycolate intermediates.     

Synthesis and antiproliferative activity of [1,2,3,5]tetrazino[5,4-a]indoles, a new class of azolo-tetrazinones

Eight derivatives of the new ring system [1,2,3,5]tetrazino[5,4-a]indole-4-one 7, were synthesised in good yields by reaction of 2-diazoindoles with alkyl or aryl isocyanates. Compounds 7 were screened at National Cancer Institute (NCI) for their activity against a panel of approximately 60 human tumour cell lines. Some of them showed antiproliferative activity having generally GI50 in the micromolar range. The most sensitive cell lines were SF-295, SNB-75 and SF-539 of the CNS cancer sub-panel, SR of the leukaemia sub-panel, UACC-62 of the melanoma sub-panel and OVCAR-4 of the ovarian cancer sub-panel.     

Triazolbenzo[d]thiazoles: Efficient synthesis and biological evaluation as neuroprotective agents

A number of (1H-1,2,3-triazol-1-yl)benzo[d]thiazoles were synthesized utilizing a versatile Cu-catalyzed azide-alkyne click reaction (CuAAC) on tautomeric benzo[4,5]thiazolo[3,2-d]tetrazole (1) and 2-azidobenzo[d]thiazole (2) starting materials. Moreover, one of the resulting products of this investigation, triazolbenzo[d]thiazole 22, was found to possess significant neuroprotective activity in human neuroblastoma (SH-SY5Y) cells.     

Selective inhibitors of GABA uptake: synthesis and molecular pharmacology of 4-N-methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol analogues

A series of lipophilic diaromatic derivatives of the glia-selective GABA uptake inhibitor (R)-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol [(R)-exo-THPO, 4] were synthesized via reductive amination of 3-ethoxy-4,5,6,7-tetrahydrobenzo[d]isoxazol-4-one (9) or via N-alkylation of O-alkylatedracemic 4. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation or primary cultures of mouse cortical neurons and glia cells (astrocytes), as well as HEK cells transfected with cloned mouse GABA transporter subtypes (GAT1-4). The activity against isoniazid-induced convulsions in mice after subcutaneous administration of the compounds was determined. All of the compounds were potent inhibitors of synaptosomal uptake the most potent compound being (RS)-4-[N-(1,1-diphenylbut-1-en-4-yl)amino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (17a, IC50 = 0.14 microM). The majority of the compounds showed a weak preference for glial, as compared to neuronal, GABA uptake. The highest degree of selectivity was 10-fold corresponding to the glia selectivity of (R)-N-methyl-exo-THPO (5). All derivatives showed a preference for the GAT1 transporter, as compared with GAT2-4, with the exception of (RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (28d), which quite surprisingly turned out to be more potent than GABA at both GAT1 and GAT2 subtypes. The GAT1 activity was shown to reside in (R)-28d whereas (R)-28d and (S)-28d contributed equally to GAT2 activity. This makes (S)-28d a GAT2 selective compound, and (R)-28d equally effective in inhibition of GAT1 and GAT2 mediated GABA transport. All compounds tested were effective as anticonvulsant reflecting that these compounds have blood-brain barrier permeating ability.     

3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-substituted-3,6-diazabicyclo[3.1.1]heptanes as novel potent dopamine uptake inhibitors

A series of analogues 2a-i related to 3-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-8-(1H-indol-2-ylmethyl)-3,8-diazabicyclo[3.2.1]octane (1) in which the 3,8-diazabicyclo[3.2.1]octane core was replaced by 3,6-diazabicyclo[3.1.1]heptane ring system has been synthesized and evaluated for their ability to inhibit DA reuptake into striatal nerve endings (synaptosomes). Biological data showed that compound 2a, the closest analogue of lead 1, possessed an increased reuptake inhibition activity over 1 (2a, K(i)=5.5 nM). Replacement of the indole ring with bioisosteric aromatic rings--benzothiophene (2b), benzofurane (2c), or indene (2d)--resulted, with the exception of 2d, in a double digit nanomolar activity. Changing the indenyl moiety of 2d with simplified aryl groups led to compounds 2e-h which displayed a similar or slightly decreased activity with respect to the ground term. Naphthalene derivative (2i) demonstrated a weaker activity than aromatic analogues.     

1-[4-(1H-Benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea derivatives as small molecule heparanase inhibitors

A novel class of 1-[4-(1H-benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-ureas are described as potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea (7a) and 1,3-bis-[4-(5,6-dimethyl-1H-benzoimidazol-2-yl)-phenyl]-urea (7d), which displayed good heparanase inhibitory activity (IC(50) 0.075-0.27 microM). Compound 7a showed good efficacy in a B16 metastasis model.     

Synthesis and biological evaluation of indazolo[4,3-bc][1,5]naphthyridines (10-aza-pyrazolo[3,4,5-kl]acridines): a new class of antitumor agents

A series of potential DNA-binding antitumor agents, 2-[omega-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridines (2a-f), 10-aza derivatives of PZA, has been prepared by condensation of 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one (6) with the appropriate (omega-aminoalkyl)hydrazine in tetrahydrofuran/methanol. Compound 6 was obtained by heating at 100 degrees C in H(2)SO(4)5, yielded by the condensation of 2,6-dichloro-3-nitrobenzoic acid (4) and 6-methoxy-3-pyridinamine (3). The non-covalent DNA-binding properties of 2 have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives against human hormone-refractory prostate adenocarcinoma cell line (PC-3) are described and compared to that of parent drug PZA. We selected the most cytotoxic target derivatives 2c,d, the in vitro inactive 2f, and reference compound PZA to investigate whether in vitro treatment with these drugs was able to induce necrotic and/or apoptotic cell death. To this purpose, we evaluated the percentage of apoptotic cells in PC-3 treated with the target compounds 2c,d,f and reference compound PZA, by Annexin V staining and Propidium iodide (PI)/Annexin V, biparametric flow cytometric analysis and agarose gel electrophoresis.     

Design, synthesis, and pharmacological evaluation of new neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives with in vivo hypnotic and analgesic profile

The present study describes the synthesis and pharmacological profiles of four novel pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives 2-5, which were structurally designed by using the sedative and analgesic drug zolpidem 1 as lead compound. The heterotricyclic system present in the target compounds 2-5 was constructed in good yields, exploiting a regioselective hetero Diels-Alder reaction of the key azabutadiene derivative 7 and functionalized N-phenylmaleimides 9-12. Additionally, we identified that 1-methyl-7-(4-nitrophenyl)-3-phenyl-3,6,7,8-tetrahydropyrazolo[3,4-b]pyrrolo[3,4-d]pyridine-6,8-dione derivative (LASSBio-873, 5) presented not only the most potent ability to promote sedation, which was similar to that induced by the standard benzodiazepine drug midazolam, but also potent central antinociceptive effect.     

Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents

The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to give a series of 5-substituted 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7). Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8-13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.     

Reaction of DNA oligonucleotides with [Pt(dien)GSMe]2+ (GSMe =S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+: evidence of oligonucleotide platination via sulfur-coordinated platinum intermediates

To study the possibility of DNA platination via platinum-sulfur coordinated intermediates, the reactions of the complexes [Pt(dien)GSMe]2+ (GSMe=S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+ with the synthetic oligonucleotides d(ATATGCATAT), d(ATTACCGGTAAT), and d(ATCCTATTTTTTTTAGGAT) have been investigated. The reactions were studied using FPLC, NMR, and mass spectrometry. It was found that the sulfur atom of the platinum-thioether adduct is substituted by these oligonucleotides. For the reactions with [Pt(dien)GSMe]2+ at 310 K, half-lives were determined to be t 1/2 =147+/-7 h for d(ATATGCATAT), t 1/2 =84+/-4 h) for d(ATTACCGGTAAT), and t 1/2 = 21+/-1 h for d(ATCCTATTTTTTTTAGGAT. This study clearly shows that it is indeed possible for oligonucleotides to be platinated via Pt-thioether coordinated intermediates. The rates at which such substitutions occur, however, makes it improbable that such a mechanism contributes significantly to the antitumor activity of cisplatin.     

N-(sulfonamido)alkyl[tetrahydro-1H-benzo[e]indol-2-yl]amines: potent antagonists of human neuropeptide Y Y5 receptor

[3a,4,5,9b-Tetrahydro-1H-benzo[e]indol-2-yl]amines were prepared via reductive amination and concomitant cyclization of alpha-cyanomethyl-beta-aminotetralins. N-acylation with omega-sulfonamido-carboxylic acids and subsequent reduction afforded a series of N-(sulfonamido)alkyl[tetrahydro-1H-benzo[e]indol-2-yl]amines, which bound to the human neuropeptide Y Y5 receptor with nanomolar affinity.     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.     

Conjugation, immunoreactivity, and immunogenicity of calix[4]arenes; model study to potential calix[4]arene-based Ac3+ chelators.

For the development of calix[4]arene-based radiotherapeutic agents, the conjugation to biomolecules and immunogenicity in mice of potential 225Ac3+-chelating calix[4]arenes were studied. A calix[4]arene triethyl ester isothiocyanate and a bis(calix[4]arene) hexacarboxylic acid, containing a masked thiol functionality, were used in conjugation experiments to a mouse monoclonal antibody and serum albumins. All characterization techniques indicate that only the calix[4]arene carboxylic acid is successfully conjugated to the biomolecules. The immunoreactivity of the conjugates is not impaired when up to 6 equiv of calixarene are bound to the monoclonal antibody. Animal tests indicated that the immunogenicity toward the calix[4]arene is strongly influenced by the nature of the carrier, the dosage, and the injection method. No immune response occurred when a homologous carrier was used or when a heterologous carrier was applied at a dosage of 10 microg per immunization via intravenous injection. Under all other conditions, the presence of antibodies directed against the calix[4]arene was demonstrated. Thus, for the application in radioimmunotherapy, the conjugation of a calix[4]arene to a humanized antibody will probably not lead to an immune response, and the immunoreactivity will not be disturbed.