Febrifugine (1), a quinazoline alkaloid, isolated from Dichroa febrifuga roots, shows powerful antimalarial activity against Plasmodium falciparum. The use of 1 as an antimalarial drug has been precluded because of side effects, such as diarrhea, vomiting, and liver toxicity. However, the potent antimalarial activity of 1 has stimulated medicinal chemists to pursue compounds derived from 1, which may be valuable leads for novel drugs. In this study, we synthesized a new series of febrifugine derivatives formed by structural modifications at (i) the quinazoline ring, (ii) the linker, or (iii) the piperidine ring. Then, we evaluated their antimalarial activities. Thienopyrimidine analogue 15 exhibited a potent antimalarial activity and a high therapeutic selectivity both in vitro and in vivo, suggesting that 15 is a good antimalarial candidate.

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Gamete shedding can be induced in male and female surf clams (Spisula solidissima and sachalinensis) by injecting 5-hydroxytryptamine (5-HT) into the gonads. The administration of 0.4 ml of 2.0 mM 5-HT induced spawning in all the clams tested. Shedding of gametes occurred within 2 to 3 min after the treatment and continued for about 40 min. The majority of the shed oocytes retained their germinal vesicles and underwent maturation upon insemination. The fertilized oocytes developed normally to the trocophora stage. The motility of the shed sperm was normal. 5-HT added to the suspending medium to the final concentrations of 10 to 100 pM induced maturation of isolated Spisula oocytes in vitro. The oocytes underwent germinal vesicle breakdown, chromosomal condensation, extrusion of the first and second polar bodies, and pronuclei formation. The neurotransmitter-induced production of maturation-promoting factor. In conclusion, 5-HT induces gamete spawning and initiates oocyte maturation in the s u r f clam.Spawning in the molluscs can be induced by raising the temperature of the seawater (Galstoff, '38, '40; Kinoshita et al., '43; Yamamoto, '51). Although the mechanism of the temperature effect is not known, we hypothesize that a neurotransmitter is released and triggers spawning. The neurotransmitter may be 5-hydroxytryptamine (5-HT) since it induces spawning in scallops of both sexes (Matsutani and Nomura, '82).In the present report, evidence will be presented showing that 5-HT, injected into the gonads of the surf clams of both sexes, induces spawning and that this hormone in vitro induces oocyte maturation. A preliminary communication was presented at the scientific proceedings of the Marine Biological Laboratory, Woods Hole (Hirai et al., '84). MATERIALS AND METHODS 5-HT Spawning assayThe neurotransmitters were dissolved just before use in artificial seawater (ASW, MBL formula) at varying concentrations. A dose of 0.4 ml of the neurotransmitter solution was injected into the gonads and the time of onset and duration of spawning was recorded. The shed oocytes were examined under the microscope for intact germinal vesicles (GV) and sperm motility was evaluated. Oocyte maturation assayThe method of isolation of oocytes from surf clam is described in a previous publication (Sano et al., '79). The test substances were added to the suspending medium at final concentrations of 10 nM to 1.0 mM. After the addition of the neurotransmitters the oocytes were examined under the microscope for the occurrence of germinal vesicle

Quinazolinone type alkaloids, febrifugine (1) and isofebrifugine (2), isolated from Dichroa febrifuga roots, show powerful antimalarial activity against Plasmodium falciparum. Unfortunately, their emetic effect and other undesirable side effects have precluded their clinical use for malaria. Because of their antimalarial potency, analogues were searched for, with the goal of preserving the strong antimalarial activity, while dramatically reducing side effects. We expected that compounds useful in drug development would exist in metabolites derived from 1 and Df-1 (3), the condensation product of 1 with acetone, by mouse liver S9. Feb-A and -B (4 and 5) were isolated as the major metabolites of 1. In addition to 4 and 5, feb-C and -D (6 and 7) were also purified from the metabolic mixture of 3. Compounds 4 and 5 were compounds oxidized at C-6 and C-2 of the quinazolinone ring of 1, respectively. Compounds 6 and 7, derived from 3, also bear febrifugine type structures in which the 4' '- and 6' '-positions of the piperidine ring of 1 were oxidized. In vitro antimalarial and cytotoxic tests using synthetically obtained racemic 4-6 and enantiomerically pure 7 demonstrated that 4 and 6 had antimalarial activity against P. falciparum, of similar potency to that of 1, with high selectivity. The antimalarial activity of 5 and 7, however, was dramatically decreased in the test. The in vitro antimalarial activity of analogues 22 and 43, which are stereoisomers of 4 and 6, was also evaluated, showing that 22 is active. The results suggest that basicity of both the 1- and the 1' '-nitrogen atoms of 1 is crucial in conferring powerful antimalarial activity. Racemic 4 and 6 exhibited powerful in vivo antimalarial activity against mouse malaria P. berghei, and especially, no serious side effects were observed with 4. Thus, the metabolite 4 appears to be a promising lead compound for the development of new types of antimalarial drugs.