Novel N-methylated 8-oxoisoguanines from Pacific sponges with diverse neuroactivities

Abstract

Marine organisms have yielded a variety of metabolites with neuropharmacological applications. Here we describe the isolation and pharmacological characterization of four novel, neurologically active purines 1-4, isolated from Haplosclerida sponges collected in the Republic of Palau. The structures were determined by analyses of spectral and X-ray data. Compound 1 induced convulsions upon intracerebroventricular injection into mice, with a CD50 value of 2.4 nmol/mouse. Purines 2-4 were active in mouse bioassays at higher doses. The seizurogenic activity of 1 was correlated with inhibition of neuronal GABAergic transmission, with only a modest impact on excitatory signaling, in electrophysiological recordings from hippocampal neurons. Despite having a purine template structure, the inhibitory activity of 1 was not prevented by a nonselective adenosine receptor antagonist. Thus, 1 represents a novel substituted purine that elicits convulsions through its actions on inhibitory neurotransmission. These 8-oxoisoguanine analogs comprise a new family of compounds closely related in structure to endogenous neurosignaling molecules and commonly used CNS stimulants.

Figure 1

ORTEP diagram of 1 with atom-labeling scheme and thermal ellipsoids drawn at the 50% probability level. Broken lines indicate intermolecular hydrogen bonds.

Figure 2

Compound 1 alters neuronal signaling. (A) Representative whole-cell voltage clamp recording of mixed excitatory and inhibitory postsynaptic currents (PSCs) recorded from cultured hippocampal neurons before, during, and after application of 1 (10 µM, gray bar, applied for 5 min). The purine analog reliably altered the frequency, amplitude, and bursts of PSCs in qualitative assessments. Expanded time scales shown in the traces on the right were taken from the trace at the indicated time points. (B) Representative whole-cell current clamp recording of action potentials and subthreshold depolarizations recorded from cultured hippocampal neurons before, during, and after application of 1 (10 µM, gray bar, applied for 5 min). The dotted line and arrowhead indicate the resting membrane potential for this cell, from which a small tonic depolarization is noted in this example. Compound 1 had variable effects on action potential firing. Expanded time scales shown in the traces on the right were taken from the trace at the indicated time points.

Figure 3

Compound 1 reduces spontaneous inhibitory transmission in neuronal cultures. (A) Representative recordings of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from cultured hippocampal neurons before and in the presence of 1 (10 µM, applied for 5 min). Spontaneous EPSC burst frequency was not altered by the purine analog, as shown in the graph on the right. (B) Representative recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from cultured hippocampal neurons before and in the presence of 1 (10 µM, applied for 5 min). Spontaneous IPSC frequency was significantly reduced in the presence of 1 but returned to control levels following a 10 min wash-out period (p < 0.05, repeated measures ANOVA).

Figure 4

Compound 1 does not alter action-potential independent excitatory transmission in neuronal cultures. (A) Representative recordings of action potential-independent excitatory postsynaptic currents (miniature EPSCs, mEPSCs) recorded from cultured hippocampal neurons before and in the presence of 1 (10 µM, applied for 5 min). Gray circles indicate mEPSCs recorded in the presence of 1 µM TTX. (B) Representative cumulative probability histograms of the interevent interval and amplitude of mEPSCs recorded from a single neuron. Black lines are control, and red lines are data in the presence of 1. Neither parameter was altered to a significant degree in the presence of the compound (p > 0.05, repeated measures ANOVA).

Figure 5

Compound 1 reduces action-potential independent inhibitory transmission in neuronal cultures. (A) Representative recordings of action potential-independent inhibitory postsynaptic currents (miniature IPSCs, mIPSCs) recorded from cultured hippocampal neurons before in and in the presence of 1 (10 µM, applied for 5 min). Gray circles indicate mIPSCs recorded in the presence of 1 µM TTX. (B) Representative cumulative probability histograms of the interevent interval and amplitude of mIPSCs recorded from a single neuron. Black lines are control and red lines are data in the presence of 1. Interevent intervals were significantly increased in the presence of the compound, whereas mIPSCs amplitudes were unaffected. (C) Graph showing the mean mIPSC frequency in control (basal), compound 1, and after wash-out of 1 (**p <0.01, repeated measures ANOVA). (D) Graph showing the mean mIPSC frequency in control (basal), compound 1, and after wash-out of 1 in parallel experiments performed in the presence of the nonselective adenosine receptor antagonist CGS15943 (100 nM). The effect of 1 on mIPSC frequency was not occluded by the presence of the antagonist (**p < 0.01, repeated measures ANOVA).

Figure 6

Compound 1 reduces GABA_A receptor mediated inhibitory neurotransmission to CA1 pyramidal neurons. (A) EPSCs from CA1 pyramidal neurons were evoked by monopolar stimulation of Schaffer collateral axons in mouse hippocampal slices. Left: Representative traces of stimulus-evoked CA1 neuron EPSCs from juvenile mouse hippocampal slices in the absence (black traces) and presence (red traces) of 30 µM 1. Right: Average traces for all experiments showing the modest effect of 1 after a 5 min application (red bar) on CA1 EPSCs. The nonselective AMPA/KAR receptor antagonist CNQX (50 µM) eliminated the EPSCs, confirming their identity. (B) IPSCs from CA1 pyramidal neurons were evoked by monopolar stimulation in the stratum radiatum in mouse hippocampal slices. Left: Representative traces showing that 1 (red trace) significantly reduced CA1 IPSC amplitudes when compared to basal (predrug) conditions. Right: Average traces for all experiments showing the significant reduction of IPSCs by 1 after a 5 min application period (red bar). After a washout period the GABA_A antagonist bicuculline (10 µM) was added to confirm the IPSC recordings (n = 5; p < 0.05, paired Student’s t test). (C) Summary graph showing that compound 1 reduces IPSCs to a similar degree when coapplied with the nonselective α1 adrenoceptor antagonist prazosin (5 µM) or the selective GABA_B receptor antagonist SCH50911 (20 µM).

Figure 7

Compound 1 does not antagonize recombinant AMPA, kainate, or GABA_A receptors. (A) Representative traces showing currents evoked by glutamate from recombinant GluA4 receptors in the absence (black) and presence (red) of 1 (100 µM). Glutamate (10 mM) was fast-applied for 100 ms. (B) Analogous traces showing the lack of effect of 1 on glutamate-evoked currents from GluK2 kainate receptors. (C) Representative traces showing that the amplitude of currents gated by GABA_A receptors composed of α4β3γ2S subunits were unaffected by the presence of 1 (30 µM). GABA (1 mM) was applied for 2 s in these experiments. All recordings were in voltage-clamp mode at a command potential of −70 mV. (D) Summary graph showing the mean ± sem for current amplitudes in the presence of 1 expressed as a percentage of the control amplitudes in the absence of the purine.

Scheme 1

Scheme 2

HMBC and NOE Correlations for 2 and 3

Chart 1