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Unifiram (also known as DM-232), a piperazine derived ampakine-like drug, has potent nootropic effects in animal studies with significantly higher potency than piracetam. DM 232 and DM 235 are novel antiamnesic compounds structurally related to ampakines. The involvement of AMPA receptors in the mechanism of action of DM 232 and DM 235 was, therefore, investigated in vivo and in vitro. Both compounds (0.1 mg/kg(-1) i.p.) were able to reverse the amnesia induced by the AMPA receptor antagonist NBQX (30 mg/kg(-1) i.p.) in the mouse passive avoidance test. At the effective doses, the investigated compounds did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as revealed by the hole board test. DM 232 and DM 235 reversed the antagonism induced by kynurenic acid of the NMDA-mediated release of [(3)H]NA in the kynurenate test performed in rat hippocampal slices. This effect was abolished by NBQX. DM 232 increases, in a concentration dependent manner, excitatory synaptic transmission in the rat hippocampus in vitro. These results suggest that DM 232 and DM 235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system.
Physicochemical Properties
| Molecular Formula | C13H15FN2O3S | |
| Molecular Weight | 298.33 | |
| Exact Mass | 298.078 | |
| Elemental Analysis | C, 52.34; H, 5.07; F, 6.37; N, 9.39; O, 16.09; S, 10.75 | |
| CAS # | 272786-64-8 | |
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| PubChem CID | 9861054 | |
| Appearance | White to off-white solid powder | |
| Density | 1.5±0.1 g/cm3 | |
| Boiling Point | 479.5±55.0 °C at 760 mmHg | |
| Flash Point | 243.8±31.5 °C | |
| Vapour Pressure | 0.0±1.2 mmHg at 25°C | |
| Index of Refraction | 1.628 | |
| LogP | 0.66 | |
| Hydrogen Bond Donor Count | 0 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 20 | |
| Complexity | 482 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | S(C1C([H])=C([H])C(=C([H])C=1[H])F)(N1C([H])([H])C([H])([H])N2C(C([H])([H])C([H])([H])C2([H])C1([H])[H])=O)(=O)=O |
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| InChi Key | SNRTZFZAFBIBJP-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C13H15FN2O3S/c14-10-1-4-12(5-2-10)20(18,19)15-7-8-16-11(9-15)3-6-13(16)17/h1-2,4-5,11H,3,6-9H2 | |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | AMPA receptor |
| ln Vivo | DM 232 and DM 235 are novel antiamnesic compounds structurally related to ampakines. The involvement of AMPA receptors in the mechanism of action of DM 232 and DM 235 was, therefore, investigated in vivo and in vitro. Both compounds (0.1 mg/kg(-1) i.p.) were able to reverse the amnesia induced by the AMPA receptor antagonist NBQX (30 mg/kg(-1) i.p.) in the mouse passive avoidance test. At the effective doses, the investigated compounds did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as revealed by the hole board test. DM 232 and DM 235 reversed the antagonism induced by kynurenic acid of the NMDA-mediated release of [(3)H]NA in the kynurenate test performed in rat hippocampal slices. This effect was abolished by NBQX. DM 232 increases, in a concentration dependent manner, excitatory synaptic transmission in the rat hippocampus in vitro. These results suggest that DM 232 and DM 235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system [1]. |
| Cell Assay |
Extracellular recording [1] Test pulses (80 μs, 0.06 Hz) were delivered through a bipolar nichrome electrode positioned in the stratum radiatum. Evoked extracellular potentials were recorded with glass microelectrodes (2–10 MΩ) filled with 3 M NaCl, placed in the CA1 region of the stratum radiatum. Responses were amplified, digitised (sample rate, 33.33 kHz), and stored for later analysis using pCLAMP 6 software facilities. Stimulus-response curves were obtained by gradual increases in stimulus strength at the beginning of each experiment. The test stimulus pulse was then adjusted to produce a field excitatory postsynaptic potential (fEPSP) whose slope was 40–50% of the maximum and was kept constant throughout the experiment. In some experiments both the amplitude and the initial slope of fepsp were quantified, but since no appreciable differences were observed in the effect of drugs, only the measure of the amplitude was expressed in figure. |
| Animal Protocol | Drugs were dissolved in isotonic (NaCl 0.9%) saline solution immediately before use. Drug concentrations were prepared so that the necessary dose could be administered in a volume of 10 ml/kg−1 by i.p. injection for mice. For electrophysiological experiments DM 232 was dissolved in dimethylsulfoxide (DMSO) and stock solutions were made to obtain concentrations of DMSO of 0.05% and 0.01% in aCSF, respectively. Control experiments, carried out in parallel for an unrelated project, showed that this concentration of DMSO did not affect the amplitude of synaptic potential.[1] |
| References |
[1]. AMPA-receptor activation is involved in the antiamnesic effect of DM 232 (unifiram) and DM 235 (sunifiram). Naunyn Schmiedebergs Arch Pharmacol.2003 Dec;368(6):538-45. [2]. Design, synthesis and preliminary pharmacological evaluation of new analogues of DM232 (unifiram) and DM235 (sunifiram) as cognition modulators. Bioorg Med Chem. 2008 Dec 1;16(23):10034-42. 2003 Dec;368(6):538-45. |
| Additional Infomation |
A series of amides, structurally related to DM232 (unifiram) and DM235 (sunifiram), characterized by a 1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-6(2H)-one, 1,4-diamino-cyclohexane or 1,4-diaminobenzene ring, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, while some cyclohexane derivatives are endowed with amnesia inducing properties. [2] The present results evidence for the involvement of the AMPA receptors in the antiamnesic activity of DM 232 (unifiram) and DM 235 (sunifiram). Both compounds reversed the impairment of memory processes induced by the AMPA antagonist NBQX in “in vivo” studies. “In vitro” experiments, performed on hippocampal slices, also supported the hypothesis of a role of the AMPA receptors for DM 232 and DM 235. The amelioration of mouse memory processes induced by DM 232 and DM 235 is obtained without any induction of side effects. Both compounds, at the highest effective doses, did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as indicated by the hole board test. The lack of induction of hyper or hypo-excitability is also confirmed by the observation that, in the first session, the latency to enter the dark compartment of the light-dark box in the passive avoidance test was not modified by the administration of DM 232 and DM 235. It should be noted that deleterious behavioural effects were not present at doses 100-fold higher than the actives ones indicating that these compounds are endowed with an extremely low toxicity. DM 232 and DM 235 have been predicted to act as ampakine-like compounds and, as a direct consequence, they should be expected to ameliorate amnesic conditions through AMPA/kainate receptor-mediated mechanisms (Staubli et al. 1994a; Larson et al. 1995) as well as to reverse memory impairments chemically-induced by administering AMPA/kainate receptor antagonists (Kim et al. 1993; Quilfeldt et al. 1994; Filliat et al. 1998). As almost all the quinoxaline derivatives, the competitive AMPA antagonist NBQX fails to discriminate among AMPA/kainate receptor subtypes (Bleakman and Lodge 1998), but, differently from CNQX or DNQX, it displays low affinity for NMDA receptors (Sheardown et al. 1990). Here we show that DM 232 and DM 235 prevent amnesia caused by NBQX, suggesting a possible role of AMPA-mediated system in the ameliorative effects on memory processes exerted by the two investigated compounds. The administration of NBQX induced amnesia of intensity comparable to that produced by well-known amnesic compounds such as scopolamine, mecamylamine and baclofen. This effect is in agreement with previous results performed in the rat passive avoidance test (Burchuladze and Rose 1992) and more recently in the Morris Water Maze task (Filliat et al 1998). DM 232 and DM 235 did not show any procognitive activity in the passive avoidance test when given alone. However, an improvement in cognition of young animals, which have no memory impairment is difficult to demonstrate. As a matter of fact, not only the above-mentioned compounds, but also well-known nootropic drugs such as piracetam and aniracetam, do not show any memory facilitation in non-amnesic animals (Gouliaev and Senning 1994). The hypothesis that DM 232 and DM 235 exert their antiamnesic effect through the activation of AMPA receptors is also supported by “in vitro” results in which both compounds produced a NBQX-sensitive reversal of the kynurenate-induced antagonism in the “kynurenate test”. In 1995, a biochemical test for evaluation of cognition enhancers acting through glutamate receptors of the N-methyl-D-aspartate (NMDA) type was proposed (Pittaluga et al. 1995). In this test, called “the kynurenate test”, nootropics are evaluated for their ability to attenuate kynurenate antagonism of the NMDA-evoked NA release from rat hippocampal slices. The attention was focused on kynurenic acid since it is a broad spectrum endogenous antagonist at ionotropic glutamate receptors, showing high affinity for the glycinergic binding site of the NMDA receptor-complex (Stone 1993; Moroni 1999). Under physiological conditions, the levels of kynurenate in the human brain reach micromolar concentrations (Moroni et al. 1988; Turski et al. 1998), probably allowing the blockade of a very low percentage of NMDA receptors. Abnormally elevated concentrations of kynurenic acid, however, may occur in the CNS during ageing or psychopathologies such as AIDS associated dementia or Alzheimer’s disease (Gramsbergen et al. 1992; Baran et al. 1999; Bara et al. 2000). These conditions are typified by the development of cognitive deficits and have been proposed to be parallel by decrease of glutamate receptor functions. It was, therefore, postulated that learning and memory improvements obtained with some nootropics might be associated to a relief of the antagonism exerted by the endogenous compounds at glutamate receptors, especially the NMDA receptor complex subtypes. Several compounds were found to be active in this test: some of these drugs relieved the kynurenate antagonism probably by acting directly on the NMDA receptor (i.e. D-cycloserine, oxiracetam, CR2249; Pittaluga et al. 1995, 1997) while other compounds reverted the kynurenate antagonism through indirect mechanisms, involving receptor-receptor interaction (Pittaluga et al. 1999, 2001). Aniracetam and the ampakine 1-BCP were tested in this biochemical assay and were found to be potently active. While the effect of aniracetam was insensitive to the presence of NBQX, the reversal of the kynurenate antagonism mediated by 1-BCP was found to partly depend on AMPA receptor activation, since co-administration of the selective AMPA receptor antagonist NBQX abolished it (Pittaluga et al. 2001). One possible explanation to the DM-induced AMPA-mediated reversal of the “kynurenate test” is that AMPA receptors might influence NMDA receptors function, by directly modulating their activity. Actually, AMPA and NMDA receptors co-localised on noradrenergic terminals and they reciprocally influence their functions. Another possible explanation considers that, once slices are exposed to 100 μM NMDA, a release of endogenous glutamate occurs in the biophase that might induce AMPA receptor desensitisation (reviewed by Holmann and Heinemann 1994). The desensitisation is prevented, possibly, by the presence of ampakine-like compound, leading to a reinforcement of the AMPA-mediated effect and therefore to an apparent reversal of the kynurenate antagonism. Finally, it could be proposed that DM 232 and DM 235 might influence the AMPA-induced release of neurotransmitters others than noradrenaline, which might in turn facilitate NMDA receptor functions. Such an indirect mechanism underlies the reversal of the “kynurenate test” by the nootropic compound CGP36742. This drug acts as a very weak GABAB receptor antagonist, but it improves cognitive performances at low doses (0.01–1 μM) “in vivo”: its effect in the “kynurenate test” was found to be mediated by disinhibition of somatostatin release in hippocampal slice. The results obtained by electrophysiological recording in vitro demonstrate that DM 232 brought about a long lasting increase of neurotransmission in the CA1 region of rat hippocampal slices. This effect is concentration-dependent and not reversible upon interruption of drug application. Our data provide experimental evidences supporting the proposition that the long-lasting synaptic enhancements produced by DM 232 are similar to the hippocampal LTP, that represents a model for a cellular mechanism related to learning and memory (Staubli et al. 1994b). DM 232, through unknown mechanism(s) might enhance either the release of the putative neurotransmitter such as glutamate, as already demonstrated for FK960, a putative cognitive enhancer in the hippocampus (Hodgkiss and Kelly 2001) or the response to glutamate at post synaptic level probably on AMPA receptors, since NMDA receptors contribute little to the generation of fEPSP evoked by low frequency stimulation in the presence of physiological concentrations of Mg++ (Novak et al. 1984). Further experiments carried out in the presence of selective AMPA or NMDA antagonists will be devoted to clarify the exact mechanism. In conclusion, these results indicate that DM 232 and DM235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system. [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3520 mL | 16.7600 mL | 33.5199 mL | |
| 5 mM | 0.6704 mL | 3.3520 mL | 6.7040 mL | |
| 10 mM | 0.3352 mL | 1.6760 mL | 3.3520 mL |