High affinity recognition of serotonin transporter antagonists defined by species-scanning mutagenesis. An aromatic residue in transmembrane domain I dictates species-selective recognition of citalopram and mazindol.

Human and Drosophila melanogaster serotonin (5-HT) transporters (SERTs) exhibit similar 5-HT transport kinetics and can be distinguished pharmacologically by many, but not all, biogenic amine transporter antagonists. By using human and Drosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid substitutions are made switching residues from one species to another, was employed on the eight amino acids that differ between human and Drosophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-like potencies. Strikingly, these potency changes were in opposite directions suggesting Tyr95 contributes both positive and negative determinants of antagonist potency. To gain insight into how the Y95F mutant might influence mazindol potency, we determined how structural variants of mazindol responded to the mutation. Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of mazindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of antagonist-binding sites for amine transporters.

Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited [3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-alpha]isoind ol-6-ol (23; IC50 1.0 nM; 8 x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-alpha ]isoindol-7-ol (28; IC50 0.26 nM; 32 x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.

Antitumor activity of the R- and S-enantiomers of RS-2-[[hydroxy[[2-[ (octadecyloxy)methyl]tetrahydrofuran-2-yl]methoxy]-phosphinyl]oxy]-N, N,N,-trimethylethylaminium hydroxide inner salt.

The R- and S-enantiomers of 2-[[hydroxyl[[2-[(octadecyloxy) methyl]tetrahydrofuran-2-yl]methoxy]-phosphinyl]oxy]-N,N,N,- trimethylethylaminium hydroxide salt (SRI 62-834) have been evaluated in several assays to determine potential antitumor activity. The S-enantiomer showed slightly greater cytotoxic activity than the R- or RS-forms against several murine tumor cell lines. In the mouse Meth A fibrosarcoma model, the S-enantiomer was ca. 4 times more effective than the R-isomer in controlling size of tumor growth and increasing the number of survivors.

Preclinical pharmacology and possible mechanism of action of the novel antitumor agent 5-(4'-piperidinomethylphenyl)-2,3-dihydroimidazo [2,1-a]isoquinoline.

SDZ 62-434 (CAS 115621-95-9, 5-(4'-piperidinomethylphenyl)-2,3-dihydroimidazo [2,1-a]isoquinoline dihydrochloride), a member of a novel class of antitumor agents, exhibited direct and macrophage-induced cytotoxicity against a variety of murine tumor cell lines. It is more effective than edelfosine in increasing survivors and reducing tumor volume in the oral mouse Meth A fibrosarcoma model. Preliminary studies suggest that an undefined cytotoxic effect, macrophage activation and possible effects on signal transduction may account for its antitumor mechanism of action. SDZ 62-434 is currently in Phase I clinical trials as a potential antitumor agent.

Antitumor activity of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinolines.

A series of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinolines previously reported to be platelet activating factor (PAF) receptor antagonists were evaluated for potential antitumor activity. Several compounds, such as the 5-(4'-tert-butylphenyl) (65), 5-[4'-(trimethylsilyl)phenyl] (69), and 5-(4'-cyclohexylphenyl) (71) analogs showed very good cytotoxicity against several tumor cell lines. 5-[4'-(Piperidinomethyl)phenyl]-2,3-dihydroimidazo[2,1- a]isoquinoline (SDZ 62-434, 53) was more effective on a milligram per kilogram basis than the clinical cytostatic agent edelfosine (1) in increasing survivors and decreasing tumor volume in the oral mouse Meth A fibrosarcoma assay. It was selected for further development and is currently in phase I clinical trials in cancer patients.

Antitumor activity of a piperidine phospholipid.

A piperidine phospholipid ((+/-)-2-[hydroxy] [1-octadecyloxycarbonylpiperidin-3-yl]methoxy-phosphinyl] oxy]-N,N,N, trimethylethaniminium hydroxide inner salt, SDZ 62-826) has been prepared that exhibited weak direct cytotoxicity and strong macrophage-induced cytotoxicity in vitro against a variety of murine and one human tumor cell lines. This compound was found to be as effective as ET-18-OCH3 and SRI 62-834, phospholipids with both strong direct and macrophage-induced cytotoxicity, in increasing survivors and reducing tumor volume when given either orally or intravenously in the mouse MethA fibrosarcoma model. These findings suggest that the macrophage-induced cytotoxicity exhibited by ET-18-OCH3 and other phospholipids may play an important role in this tumor model.

Structural modification of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinoline platelet activating factor receptor antagonists.

In an effort to determine the effect of modification of the imidazo[2,1-a]isoquinoline portion of the PAF-receptor antagonist SDZ 64-412 (1), several new analogs were prepared and evaluated in vitro and in vivo. One of these, 5-[4-[2-(3,4,5-trimethoxyphenyl)ethyl]phenyl]-2,3-dihydroimidazo [1,2-a]thieno[2,3-c]pyridine (6) was 4-5 times more potent than 1 in inhibiting PAF-induced bronchoconstriction and hemoconcentration when administered po to the guinea pig.

Lack of therapeutic effects of platelet activating factor antagonists in WEHI-3B leukemia, human xenotransplanted colorectal and lung cancer and Lewis-lung tumor in vivo.

Four new antagonists of platelet activating factor (PAF) from two different chemical classes (imidazoisoquinolines: SDZ 62-434, SDZ 63-135, SDZ 62-759; imidazopiperidines: SDZ 62-293) were tested for in vivo therapeutic activity in various tumor models including the murine myelomonocytic leukemia WEHl-3B, xenografts of human colon (HTB 38) and lung (HTB 119) cancer cell lines and the murine Lewis-lung tumor. After intraperitoneal (i.p.) injection of 1 x 10(3), 5 x 10(3) and 1 x 10(4) WEHl-3B cells into Balb/c mice, the drugs were given per os (p.o.) or i.p. over 6-14 days. Drug doses were pushed to exceed the lethal dose for 10% of the animals (LD10) and ranged from 1 to 100 mg/kg daily for p.o. treatment and from 1 to 75 mg/kg daily for i.p. treatment. In the xenotransplants and the Lewis-lung tumor experiments, PAF antagonists were given i.p. to nude Balb/c and C57 Black mice after intracutaneous (i.c.) tumor cell inoculation. None of the four compounds induced reproducible prolongation of life span, significant numbers of long term survivors, reduction of tumor size, or delay of tumor growth in any of the therapeutic models. Oral SDZ 62-759 had some activity in experiments in which there was slow WEHl-38 tumor growth in the controls. Toxicity of equivalent drugs doses was higher in the i.p. than in the p.o. schedules.

Some antagonists of platelet activating factor are cytotoxic for human malignant cell lines.

Nine new platelet activating factor (PAF) antagonists from 4 different chemical classes (thiopyrimidines: SDZ 59-015; thioimidazolines: SDZ 61-813; imidazoisoquinolines: SDZ 62-434, SDZ 62-759, SDZ 63-135, SDZ 63-596; and imidazopiperidines: SDZ 61-638, SDZ 62-293, SDZ 62-694) have been tested for cytostatic/antiproliferative ([3H]thymidine uptake) and cytotoxic (trypan blue dye exclusion) activity in neoplastic human cell lines of different histology in vitro. The antiproliferative activity of 3 of the 9 PAF antagonists (SDZ 61-638, SDZ 61-813, SDZ 62-694) was not stable after freezing and thawing. SDZ 59-015 showed only minor cytotoxic or antiproliferative effects in a dose range of 2-40 microns after 24, 48, and 72 h of incubation. SDZ 62-434 showed varying activity. There were no significant differences between the activities of the other 3 PAF antagonists from the imidazoisoquinoline class, which showed drug concentrations inhibiting 50% of the activity studied (IC50) and drug concentrations yielding a 50% decrease of trypan blue dye exclusion (LC50) of less than or equal to 20 microM at greater than or equal to 48 h, even in the K-562 cell line, which is known to be rather resistant for a variety of cytotoxic drugs related to PAF. SDZ 62-293 showed the best antineoplastic properties with IC50 and LC50 values less than or equal to 10 microM at greater than or equal to 48 h including K-562. SDZ 62-434, SDZ 62-759, SDZ 63-135, SDZ 63-596, and SDZ 62-293 have been further tested in a human tumor cloning assay in 5 cell lines. Colony formation was reproducibly suppressed to less than 30% of the controls only by SDZ 63-135 (less than or equal to 40 microM) and SDZ 62-293 (less than or equal to 20 microM) during continuous exposure. There was no correlation between the IC50 values for the antiproliferative activity of the test compounds and their IC50 values for PAF-induced human platelet aggregation. Furthermore, the antiproliferative activity of the most active compound, SDZ 62-293, could not be antagonized by preincubation with the specific PAF antagonists WEB 2170 or WEB 2086 or PAF itself in noncytotoxic doses.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and pharmacology of a novel class of long-lasting PAF receptor antagonists.

Several new charged PAF receptor antagonists were prepared, where the phosphate moiety has been replaced by a methylsulfonylcarbamoylpyridinium moiety, and evaluated for duration of inhibitory activity against PAF-induced bronchoconstriction and hemoconcentration in the guinea pig. One of these compounds (1d; SDZ 64-619) has shown potency and duration of inhibition in the range of CV-6209 (1c).

Cyclic oxygen analogues of alkyl-lysophospholipids. Synthesis and neoplastic cell growth inhibitory properties.

Ether phospholipids have demonstrated both in vitro and in vivo activity against a wide variety of tumor cell lines. The known cyclic ether phospholipid, SRI 62-834, was used as the model to prepare eight novel phospholipids containing a cyclic ether. All of the compounds were as effective as ET-18-OCH3 in their ability to activate macrophage-induced cytotoxicity against the Abelson-8.1 tumor cell line but varied in their direct cytotoxic effects. One of the new compounds, SDZ 62-406, was selected for in vivo studies and showed oral and i.v. activity in the mouse MethA fibrosarcoma model in the same range as ET-18-OCH3. No correlation was found between the direct or macrophage-activated cytotoxicity and the ability of the compounds to inhibit or promote platelet-activating factor (PAF)-induced aggregation of human platelets.

Biological effects of the orally active platelet activating factor receptor antagonist SDZ 64-412.

SDZ 64-412 is a trimethoxyphenylethylphenyl imidazo[2,1-a] isoquinoline molecule that displays marked in vitro inhibition of platelet activating factor (PAF)-induced human platelet aggregation (IC50 = 60 nM) but is without inhibition (at 100 microM) of epinephrine-, ADP- or collagen-induced aggregation. SDZ 64-412 antagonized receptor binding of radiolabeled PAF to human platelet membranes with an IC50 = 60 nM. In the rat, SDZ 64-412 inhibited 100 ng kg-1 PAF-induced hypotension when given i.v. (ED50 = 0.23 mg kg-1) or p.o. (ED50 = 13 mg kg-1). In the guinea pig, SDZ 64-412 inhibited 50 ng kg-1 PAF-induced bronchoconstriction (ED50 = 4.2 mg kg-1 p.o.) and hemoconcentration (ED50 = 5.0 mg kg-1 p.o.). SDZ 64-412 exhibited oral activity in the dog against 1.5 micrograms kg-1 PAF-induced hypotension (ED50 = 5.1 mg kg-1 p.o.) and hemoconcentration (ED50 = 4.9 mg kg-1) and 3.5 micrograms kg-1 PAF-induced hemoconcentration in the cebus primate (ED50 = 12.8 mg kg-1 p.o.). SDZ 64-412 protected in a dose-dependent manner against PAF-induced lethality (LD75 = 75 micrograms kg-1 i.v.) in mice, where 20 mg kg-1 p.o. improved survival from 25 +/- 4% to 77 +/- 8%. SDZ 64-412 afforded complete protection against endotoxin-induced lethality (LD90 = 7.5 mg kg-1 endotoxin i.v.) where the ED50 was 45 mg kg-1 twice predose.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor activity of SRI 62-834, a cyclic ether analog of ET-18-OCH3.

SRI 62-834, an analog of the antitumor agent ET-18-OCH3 in which the oxygen atom at carbon atom 2 has been incorporated into a five-membered heterocycle, has been prepared and evaluated as an antitumor agent. The compound exhibited good cytotoxicity in vitro against a variety of tumor cell lines and was as effective as ET-18-OCH3 given orally in the mouse Meth A sarcoma model. SRI 62-834 was shown to be an inhibitor of platelet-derived growth factor (PDGF), possibly at the receptor level, and platelet-activating factor (PAF) at the receptor level.

Lack of correlation between cytotoxicity of agonists and antagonists of platelet activating factor [paf-acether] in neoplastic cells and modulation of [3H]-paf-acether binding to platelets from humans in vitro.

The 3 ether-lipids ET-18-OCH3, SRI 63-154 and paf-acether, the TLP BM 41.440, the ester-linked 2-LPC and CV-3988, were tested for cytostatic/antiproliferative [3H]-thymidine uptake) and cytotoxic (trypan blue dye exclusion, HTCA) activity in 11 neoplastic human cell lines (U 698-M, Nall-1, Su-DHL-4, RPMI 8226, K 562-4, Li-A, HTB-47, HTB-38, CCL218, 85 HG-59, 85 HG-63) and 1 ALL in vitro. 2-LPC and paf-acether showed either no, or only minor, CV-3988 varying activity. There were no significant differences in the activity of ET-18-OCH3, SRI63-154 and BM 41.440, which showed IC50- and LC50-values of less than or equal to 10 micrograms/ml after incubation periods greater than or equal to 48 hours with or during continuous exposure to the cells. The latter three compounds were then tested for interaction with [3H]-paf-acether binding to intact human platelets: ET-18-OCH3 and SRI63-154 reduced [3H]-paf-acether binding in a time-dependent manner. BM 41.440 did not show this interaction. Thus, since the in vitro cytotoxicity of these lipids did not correlate with their modulation of [3H]-paf-acether binding to human platelets, it was concluded that cytotoxicity of ether-lipids is not mediated by specific paf-acether binding sites similar to those present on human platelets. This finding is important for the future design of antineoplastic lipids.

Platelet activating factor-induced ischemic bowel necrosis: the effect of PAF antagonists.

We have reported a model of ischemic bowel necrosis produced in the rat by injecting platelet activating factor (PAF) or PAF with bacterial endotoxin (LPS) into the mesenteric vasculature. In the present study, we examined the protecting effects of three PAF antagonists, i.e. (R,S)-3-[2-[(2-octadecylaminocarbonyloxymethyltetrahydro-2-fura nylmethoxy) -hydroxyphosphinyloxy]-ethyl]-thiazolium hydroxide inner salt 4-oxide (SRI 63-072), (+/-)-3-[4-[3-octadecylaminocarbonyloxy-2-methoxy)-propoxy]-butyl] -thiazolium bromide (SRI 63-119) and 1-O-hexadecyl-2RS-O-ethyl-3-O -(7-thiazolinoheptyl)-glycerol-methanesulfonate (ONO-6240), on PAF-induced bowel necrosis. The antagonists were injected into the vein 10 min before PAF. Two microgram of PAF or 20 micrograms LPS with 1 microgram of PAF were injected into the aorta above the renal arteries. The parameters assessed included blood pressure, hematocrit, white blood cell count, extent of bowel perfusion and microscopic changes of the bowel. We found that SRI 63-072 (3 mg/kg), SRI 63-119 (3 mg/kg) and ONO-6240 (2 mg/kg) significantly improved the initial hypotension, hemoconcentration and leukopenia caused by PAF. All three drugs also corrected the sustained hypotension and hypoperfusion and gross lesions of the bowel, although microscopic examination still revealed focal mild lesions. SRI 63-072 was also active at a much lower dose (0.3 mg/kg).

Inhibition of rhesus monkey airway and cutaneous responses to platelet-activating factor (PAF) (AGEPC) with the anti-PAF agent SRI 63-072.

Previous studies with synthetic platelet-activating factor (PAF) (AGEPC) showed that aerosol challenges in rhesus monkeys resulted in airway responses simulating acute antigen-induced responses and immediate-type skin reactions [1]. The current studies evaluated whether an antagonist of PAF (SRI 63-072) could inhibit the airway and cutaneous reactivity to PAF. Under the conditions of these experiments, the antagonist partially inhibited PAF activity in both experimental systems. Inhibition of endpoint cutaneous reactivity to PAF may be a suitable system for comparing potency of pharmacologic antagonists in primate skin.

Sleep-inducing N-alkyl-5-[m-(trifluoromethyl)phenyl]-5-hydroxy-2-pyrrolidinones and N-alkyl-3-(trifluoromethyl)cinnamamides.

A series of N-alkyl-3-[m-(trifluoromethyl)phenyl]-5-hydroxy-2-pyrrolidinones and N-alkyl-3-(trifluoromethyl)-cinnamamides were prepared and screened in a series of tests designed to detect potential sleep inducers. The more active members of the series were evaluated for their ability to induce sleep in Cebus monkeys. The most active compound, N-methyl-5-[m-(trifluoromethyl)phenyl]-5-hydroxy-2-pyrrolidinone, was equal to methaqualone.

Anti-inflammatory and other pharmacodynamic properties of five members of the 4-aryl-1-isopropyl-2(1H)-quinazolinone series.

A series of five 4-aryl-1-isopropyl-2(1H)-quinazolinone analogs were examined for their relative activities regarding analgesia, suppression of inflammation and pyresis, and associated phenomena. Two of these, proquazone (SaH 43-715, Biarsan, Biarison) and fluproquazone (SaH 46-790), are clinically effective anti-inflammatory and analgesic agents. Acetylsalicylic acid (ASA) and phenylbutazone were included for reference as first and second generation nonsteroidal anti-inflammatory drugs (NSAID), respectively. In general, substitutions in these five quinazolinone analogs produced noticeable changes in potency in several activities but changes of lesser degrees in others. Compared to ASA and phenylbutazone the quinazolinones exhibited better analgesic and related activities.