The 3-brominated pyrroldione 1a served as starting material for the preparation of a series of substituted N-methyl 3-amino-pyrrol-2,5-diones, using a Cu catalysed substitution reaction. Initially the newly synthesised compounds were tested at a high dose for CNS depressant activity. When the activity was found significantly different from the control in the rota rod test and the wire mash grasping test at a high dose the active compounds were evaluated further at a low dose in anxiolytic assays. The pyrroldione 2p showed a good anxiolytic activity at low doses of 1/0.1 mg/kg in the light/dark box and the elevated plus-maze assay. The n-hexyl-amino pyrrol 2e served as lead structure for further structure optimisation. Based on 5-alkoxy 3,4-dichloro2(5H) furanones 3a and 3b, a second series of bis-substituted pyrroldiones was prepared under Cu catalysis and the pyrroldione 4f occurred the most potent anxiolytic activity.

Lattmann E, Dunn S, Schwalbe CH, Airarat W, Shortt AJ, et al. (2015) In vivo Evaluation of Substituted -1H-Pyrrole-2,5-Diones as AnxiolyticAgents. Ann Med Chem Res 1(2): 1007.

•    Novel 1H-pyrrole-2,5-diones
•    In vivo
•    X-maze
•    Anxiolytic-agents

Cyclic imides, such as succinimides and maleimides, have been found to be pharmaceutically useful [1] as antibacterial [2], anticonvulsant [3] and antitumor agents [4].

Chlorinated 1-arylamino-1H-pyrrole-2,5-diones occurred anti-fungicidal[5] activity and N-substituted imides showed both anti-micro bacterial [6], antidepressant [7], anxiolytic [8] and analgesic activity [9].

Bis-substituted succinimides (Figure 1) such as Ethosuximide represent useful agents in the treatment of epilepsy. A dichlorinated malimide showed analgesic properties against acetic acid-induced writhing in mice [10].

2-alkoxy-4-phenyl-pyrrol-2,5-diones [11] and structurally related 3-anilino-4-aryl-maleimides [12] have been prepared with the intent of treating and preventing clinical conditions such as inflammatory diseases and Alzheimer’s disease.

The dimethyl oxazolidindione and phenytoin (Figure 1) showed all sedating, CNS depressant [13] and anxiolytic properties and are mainly in use for the treatment of epilepsy.

These molecules all contain the substituted pyrroldione as privileged structure and served as rationale for the discovery of novel CNS drugs from this template.

The starting point behind this discovery programme was the desire to use an intermediate, which showed antibiotic properties [14], but was found too toxic in mice due to its high chemical reactivity. These halogenated reactive intermediates served here as ideal chemical starting point for the preparation of novel pyrroldiones by reacting them with simple commercially available amines.

New chemical entities, based on a particular molecular target, such as the 5HT7 - or CCK2 -antagonism did not reach the market, not even later clinical trials.

However, benzodiazepine anxiolytics were discovered by the classical approach using reliable animal models, and that justified evaluating these novel drug-like molecules in mice accordingly.

Chemistry

Maleimides, also known as pyrrole-2,5-diones, can be synthesised by a number of chemical approaches. The majority of these routes are based on reactions of the corresponding maleic anhydride with an amine [15]. Another alternative one-step method involves the action of ammonium acetate on the maleic anhydride in boiling acid [16].

Here, the N-substituents, such as the N-methyl group were introduced with the parent formamide, such as methylformamide. In terms of drug design, the substitution of the halogen attached to the double bond, enabled us the remove a chiral centre from the template, compared with succinimides, which are chiral. The expected desired reaction was according to an IPSO substitution, but proofed to deliver the desired molecules in low yields. The use of Cu catalysts finally gave the target molecules in good yields (Scheme 1).

The halogen of the brominated compound 1a and chlorinated entry 1b was readily displaced by a range of diverse primary and secondary amines. The brominated pyrrole 1a is the most preferred building block. A wide range of cyclic, acyclic and aromatic amines were reacted under mild conditions into the desired amino-pyrrols 2a-2s.

The metal catalysed coupling reaction provided the nonchiral unsaturated 1H-pyrroldione template in presence of triethyl amine and Cu (I) catalysis in high yields.

The building block was obtained from mucochloric acid [17], which is commercially available from furfural. Furfural itself is obtained by heating biomass with sulphuric acid. Any further chemical application of furfural represents another important example of using this renewable resource from biomass.

The unmethylated pyrrol template (not shown) also reacted into mono-substituted pyrrole-dione, according to the cupper catalysed reaction, but the building block was obtained in a much lower yield and is less lipophilic without the N-methyl group.

In the presence of water the yield is largely reduced. Acetonitril as a solvent could not be replaced by other solvents and a Ni catalyst (NiOAc) also increased the yield of the uncatalysed reaction from 30% range to a 50% range. Without Cu catalysis the aromatic amines 2f and 2g were not obtained at all. For the aninilino-pyrrols 2h and 2i the yield was approximately doubled by using a catalyst.

An overview of the synthesised structures using various chemically diverse amines is outlined in (Figure 2).

The crystal structure of the pyrroldione 2r is outlined in fig 3. Interestingly, the hydrogen of C10 formed a hydrogen bond with O7 and this resulted in a pseudo tricyclic structure for the piperazine 2r. Pyrroldione 2r, as well as the hydrazone 2q showed promising biological activity in further in vivo assays, but this could be due to the biologically active versatile piperazine template and not due the effects of the pyrrol scaffold, on which was focussed in this study (Figure 3).

Having realised the importance of a lipophilc substituent in general [18], in the n-hexyl derivative 2e and in particular for the N,N-substituted pyrrol 2p, further attemps were made to provide other lipophilic bis-substituted pyrrols. The 5-methyl- and the 5- isopropyloxy-3,4-dichloro-2(5H)-furanones 3a and 3b, which were described earlier as cytotoxic agents [19], were converted with primary amines into the desired bis-substituted pyrrols 4a4f. The building blocks 3a and 3b were obtained in high yields from mucochloric acid (Scheme 2).

The addition of triethylamine (TEA), facilitated the ringopening of the 2(5H)-furanone system and the chlorinated intermediate (Scheme 2) formed then, under Cu catalysis, with primary alkyl and cyclo-alkylamines the desired the 1,3-diaminopyrrol-2,5diones 4a to 4f in good yields. The 4-substitiuted amino-5-alkoxy-2(5H)-furanones [20], which were formed under these conditions only as a byproduct could be easily separated from the desired bis-substituted pyrroles by chromatography or extraction with petrol ether (PE). The 3-bromo-pyrrole template (not shown) was found of a similar reactivity and formed the same amino pyrroles in similar yields in ether at elevated RT. An overview of synthesised bispyrroldiones 4a-4f using primary amines is outlined in (Figure 4). The cyclohexyl-derivative 4e was recrystallised from ether and the crystal structure is outlined in (Figure 5).

In the crystal the substituents formed a nearly linear chain with a heterocyclic template in the centre. In terms of receptor interaction, the hexyl side chains are flexible and able to interact with a potential receptor via hydrophobic binding (van de Waals interactions), and the anxiolytic effects at low concentrations are likely to be a result of hydrogen binding of the pyrrol-dione (CO) and hydrophobic interactions.

SAR in vivo pharmacology

The first step of the pharmacological evaluation was to test for an impairment of co-ordination at a dose that is 100/1000 times higher, than the desired therapeutic dose, using the standard rota-rot and wire mesh grasping test.

The compounds 2a, 2b, 2d, 2j-2o, 2q and 2r were not found significantly different from the control at a 5% level.

If the compound was found initially inactive in these assays, NFI was concluded (no further interest). This is based on the assumption, that no CNS drug in general and no anxiolytic drug in particular is likely to exist, if at a 100/1000 times dose of the potentially active dose is not resulting in an impairment of co-ordination. If the compound was found active, it was tested further at two low doses (0.1 and 1 mg/kg) in 2 reliable anxiolytic assays, the light dark box and the elevated plus maze (Table 1).

The butyl derivative 2 c showed some initial activity in the light /dark box test and this was enhanced by increasing the size of the side chain, as observed for hexyl derivative pyrrol 2e.

The bis- heterocyclic compounds 2f and 2g showed some interesting analgesic effects, but only at the 1 mg/kg dose. The substituted anilines 2h and 2i displayed an anxiolytic effect in both assays, but were inactive at the 0.1 mg/kg dose. The cyclohexyl-ethyl derivative 2p was found active in both anxiolytic assays for both low doses of 1 and 0.1 mg/kg. Therefore, it was selected for a full dose evaluation.

The second compound of interest is the hexyl derivative 2e, which served as lead structure for further optimisation.

In order to assess the anxiolytic activity, the time spent in the open arms, as well as the number of entries, were observed in the elevated plus maze and these data are displayed in Figure 6 for the cyclohexyl-pyrrol derivative 2p (Figure 6). Diazepam served as positive control at 2 mg /kg. The low dose of the pyrrol 2p of 10µg/kg was found not different from the negative control (5% DMSO in water). A significant anxiolytic effect of 2p was observed from 100 µg/kg for the time spent in the open arm and the effect of 1 mg/kg 2p was increased dose dependently, similar to the 2 mg/ kg dose of diazepam standard. Interestingly the number of entries did not increase as seen for diazepam. Usually, for anxiolytics the time spent in the open arm is increased and the number of entries is increased dose dependently. Here, a mild muscle relaxant effect could be the cause for less spontaneous activity.

A series of disubstituted pyrroldiones 4a-4f was subsequently prepared, evaluated by the same concept and the results are outlined in Table 2. Out of 6 compounds 4 showed and a significant impairment of coordination and these 4 compounds were tested at the low doses for desired activity.4a, 4b and 4c dropped out at the low 0.1mg/kg dose and 4f is clearly an optimised, more lipophilic version of 4a.

For the bis-n-hexyl derivative 4f, the anxiolytic effects were determined for 0.01, 0.1, 1 mg/kg body weight in mice and the effects are displayed in (Figure 7). The pyrrole 4f was tested at a dose range of 0.01, 0.1 and 1 mg/kg compared with diazepam at a dose of 2 mg/kg as standard. The time, spend in the open arms and the number of entries increased from 0.01 mg/kg, via 0.10 mg/kg to 1 mg/kg in the elevated plus maze and the effects are similar to diazepam in mice. Compared to the previously reported 5-alkoxy 4-amino-furanones, these novel pyrroldiones-ones can be obtained as pure compounds by recrystallization, contain no chlorine in the 3-position, which could be considered a potential toxicophore and are achiral.

The versatile bioactivity of pyrroldiones justified to start our discovery programme and the use the metal catalyst, such as Cu I, finally enabled us to create these novel pyrroldiones in good yields, which otherwise would only be obtained as by products in low yields. 2 selected molecules of the 2 series were evaluated in vivo in mice using the elevated plus maze and in conclusions the novel chemical entities displayed promising anxiolytic activity.

Table 1: In vivo evaluation of selected pyrroldiones.

Cp	Rota-rod test 100 mg/kg	Wire mesh grasping test 100 mg/ kg	Light / dark Box 1 mg/kg / 0.1 mg/kg	Elevated plus maze 1mg/kg / 0.1mg/kg
2a	NS	NS	-	-
2b	NS	NS	-	-
2c	Yes	Yes	Yes/No	No / No
2d	NS	NS	-	-
2e	Yes	Yes	Yes /No	Yes /No
2f	Yes	Yes	No / No	No / No
2g	Yes	Yes	No / No	No / No
2h	Yes	Yes	Yes /No	Yes /No
2i	Yes	Yes	Yes /No	Yes /No
2j	NS	NS	-	-
2k	NS	NS	-	-
2l	NS	NS	-	-
2m	NS	NS	-	-
2n	NS	NS	-	-
2o	NS	NS	-	-
2p	Yes	Yes	Yes/Yes	Yes/Yes
2q	Refer	Refer	-	-
2r	Refer	Refer	-	-
Selection stage 1: (2 assays); high dose, 100mg/kg, impairment of coordination

Yes: Significant, P<0.05 active, No = NS: Not significant in assay at given 
dose.

Table 2: In vivo evaluation of selected pyrroldiones.

Cp	Rota-rod test 100 mg/kg	Wire mesh grasping test 100 mg/ kg	Light / dark Box 1 mg/kg /0.1mg/kg	Elevated plus maze 1mg/kg / 0.1mg/ kg
4a	Yes	Yes	Yes/ No	Yes/ No
4b	Yes	Yes	Yes/ No	Yes/ No
4c	Yes	Yes	Yes/ No	Yes/ No
4d	NS	NS	-	-
4e	NS	NS	-	-
4f	Yes	Yes	Yes / Yes	Yes / Yes

Yes: Significant, P<0.05 active, No = NS: Not significant in assay at given dose.

Materials and methods

Chemistry: The chemicals were purchased from Aldrich (Gillingham, UK) and Lancaster Synthesis (Lancaster, UK). Mass spectres were obtained by Atmospheric Pressure Chemical Ionisation (APCI), using a Hewlett-Packard 5989b quadrupole instrument (Vienna, Austria). Both proton and carbon NMR spectra were obtained on a Brucker AC 250 instrument (Follanden, Switzerland), calibrated with the solvent reference peak. Infra-red spectra were plotted from KBr discs on a Mattson 300 FTIR spectrophotometer (Coventry, UK). Melting points were recorded from a Stuart Scientific melting point apparatus (Essex, UK). Analytical Thin Layer Chromatography was obtained using aluminium sheets, silica gel60 F254, 250 µm and were visualized using ultraviolet light.

General methods for the preparation of substituted 1-methyl-pyrrole-2,5-diones

Method 1: 3-Bromo-1-methyl-pyrrole-2,5-dione (1a, 3.5x10-3 mol) was added to 3 ml acetonitril. 1.5 Equivalents of triethylamine and 1.5 equivalents of the appropriate amine (0.05 mol) with a catalytic amount of CuI (5%) were added and left to stir at 30-35 o C over night. The mixture was worked up using water and the desired compounds were extracted into diethyl ether. The resultant oily crystals were carefully re-crystallised from petrol ether.

Method 2: The same method as above was used, with the exception that (1b) 3-chloro-1-methyl-pyrrol-2,5-dione was used as reagent.

(2a): 1-Methyl-3-methylamino-pyrrole-2,5-dione

Yield = 88%; M.P: 141-144 o C; Rf (60% ether / 40% petrol ether) = 0.33

Molecular Weight: 140.1; Molecular Formula: C6 H8 N2 O2; MS (APCI (+)): 141 (M+1) m/z 1 H NMR (CDCl3 ) 250 MHz: δ = 5.42- 5.21 (bs, NH), 4.78 (s, CH), 2.92 (m, overlapping, 2xCH3 ) p.p.m.; 13C NMR (CDCl3 ) 250 MHz: δ = 168.6 (N-CO), 165.4 (CH-CO), 131.9 (CH), 131.3 (NH-C), 25.5 (NH-CH3 ), 24.6 (N-CH3 ) p.p.m. IR (KBr-disc) υ max: 3339, 3114, 2935, 2365, 1758, 1701, 1642, 1449, 1422, 985, 782 cm-1.

(2b): 3-Dimethylamino-1-methyl-pyrrole-2,5-dione

Yield = 82%; M.P: 103-105 o C; Rf (60% ether / 40% petrol ether) = 0.40; Molecular Weight: 154.1; Molecular Formula: C7 H10N2 O2; MS (APCI (+)): 155 (M+1) m/z 1 H NMR (CDCl3 ) 250 MHz: δ = 4.72 (s, CH), 2.81-3.58 (m, 3xCH3 ) p.p.m.; 13C NMR (CDCl3 ) 250 MHz: δ = 169.7 (N-CO), 161.6 (CH-CO), 144.3 (CH), 87.1 (C-NH), 44.0 (N-CH3 , 2xC), 23.3 (N-CH3 ) p.p.m.; IR (KBr-disc) υ max: 3449, 3118, 2924, 2359, 2337, 1706, 1627, 1442, 1375, 758 cm-1

(2c): 3-Butylamino-1-methyl-pyrrole-2,5-dione

Yield = 81%; M.P: N/A – Oily Solid; Rf (60% ether / 40% petrol ether) = 0.35; Molecular Weight: 182.2; Molecular Formula: C9 H14N2 O2; MS (APCI(+)): 183 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 5.35-5.45 (bs, NH), 4.82 (s, CH), 3.07-3.15 (q, NH-CH2 , J =5.1 Hz), 2.98 (s, N-CH3 ), 1.59-1.78 (m, NH-CH2 -CH2 ), 1.35-1.56 (m, CH3 -CH2 , ϑ = 4.2 Hz), 0.96-1.02 (t, CH3 -CH2 , J = 8.2 Hz) p.p.m.;13C NMR (CDCl3 ) 250 MHz: δ = 169.7 (N-CO), 160.4 (CH-CO), 148.0 (CH-N-C), 85.8 (CH-CO), 44.0 (NH-CH2 ), 30.5 (NH-CH2 -CH2 ), 23.7 (N-CH3 ), 22.8 (CH3 -CH2 ), 13.9 (CH2 -CH3 ) p.p.m.; IR (KBr-disc) υ max: 434, 3328, 2927, 2860, 2372, 2332, 1710, 1644, 1446, 1380, 1279, 1122, 1005, 775 cm-1

(2d): 3-Isobutylamino-1-methyl-pyrrole-2,5-dione

Yield = 81%; M.P: 174-177 o C; Rf (60% ether / 40% petrol ether) = 0.37; Molecular weight: 182.2;Molecular Formula: C9 H14N2 O2;MS (APCI(+)): 183 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 5.27-5.43 (bs, NH), 4.75 (s, CH), 2.91-3.17 (m, overlapping NH-CH2 & N-CH3 , 5H), 1.42-1.62 (m, CH2 -CH), 0.69- 1.03 (m, CH3 , 6H) p.p.m.;13C NMR (CDCl3 ) 250 MHz: δ = 169.3 (N-CO), 161.1 (CH-CO), 143.6 (CH-N-C), 88.1 (CH-CO), 49.4 (NHCH2 ), 31.9 (NH-CH2 -CH), 23.5 (N-CH3 ), 16.9 (CH2 -CH3 ) p.p.m.; IR (KBr-disc) υ max: 3448, 3305, 3105, 2957, 2914, 2864, 2367, 2335, 1699, 1639, 1435, 1282, 994, 758 cm-1

(2e): 3-Hexylamino-1-methyl-pyrrole-2,5-dione

Yield = 87%; M.P: 141-143 o C; Rf (60% ether / 40% petrol ether) = 0.37; Molecular Weight: 210.3; Molecular Formula: C11H18N2 O2; MS (APCI (+)): 211 (M+1) m/z;1 H NMR (CDCl3 ) 250 MHz: δ = 5.18 (s, CH), 4.71-4.95 (bs, NH-CH), 3.49-3.61 (t, J = 5.5 Hz, NH-CH2 ), 3.03 (s, N-CH3 ), 1.21-1.69 (m, overlapping, NH-CH2 - CH2 , NH- CH2 -CH2 -CH2 , NH-CH2 -CH2 -CH2 -CH2 , NH-CH2 -CH2 -CH2 - CH2 -CH2 , 8H), 0.72-0.99 (t, ϑ = 6.5 Hz, CH2 -CH3 ) p.p.m.; 13C NMR (CDCl3 ) 250 MHz: δ = 168.6 (N-CO), 161.3 (CH-CO), 143.4 (CHN-C), 87.4 (CH-CO), 44.0 (NH-CH2 ), 37.0 (NH-CH2 -CH2 ), 33.8 (NHCH2 -CH2 -CH2 ), 30.5 (NH-CH2 -CH2 -CH2 -CH2 ), 22.7 (N-CH3 ), 23.7 (NH-CH2 -CH2 -CH2 -CH2 -CH2 ), 14.2 (CH2 -CH3 ) p.p.m.; IR (KBr-disc) υ max: 3438, 3325, 2937, 2362, 2335, 1708, 1634, 1442, 1107, 784 cm-1

(2f): 1-Methyl-3-[1,2,4]triazol-1-yl-pyrrole-2,5-dione

Yield = 80%, M.P: 166-168 o C; Rf (60% ether / 40% petrol ether) = 0.28; Molecular Weight: 178.2; Molecular Formula: C7 H6 N4 O2; MS (APCI(+)): 179 (M+1) m/z 1 H NMR (CDCl3 ) 250 MHz: δ = 8.09-8.31 (d, C-N-CH), 8.18 (d, N-N-CH), 4.83 (s, CH), 3.05 (s, CH3 ) p.p.m.;13C NMR (CDCl3 ) 250 MHz: δ = 168.7 (N-CO), 161.2 (CH-CO), 153.3 (C-N-CH), 145.3 (N-N-CH), 133.3 (CH), 86.5 (C-NH), 24.2 (N-CH3 ) p.p.m. IR (KBr-disc) υ max: 3439, 3124, 2929, 2864, 2363, 2340, 1769, 1645, 1472, 1273, 1143, 980 cm-1

(2g): 1-Methyl-3-(3-methyl-pyrazol-1-yl)-pyrrole-2,5- dione

Yield = 73%; M.P: 169-171 o C; Rf (60% ether / 40% petrol ether) = 0.37; Molecular Weight: 191.2; Molecular Formula: C9 H9 N3 O2;MS (APCI(+)): 192 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 7.18-7.10 (d, N-CH), 6.69-6.60 (d, N-CH-CH), 6.49 (s, CH), 3.00 (s, N-CH3 ), 2.66-2.55 (m, C-CH3 ) p.p.m.; 13C NMR (CDCl3 ) 250 MHz: δ = 169.9 (N-CO), 161.3 (CH-CO), 150.6 (N-N-C), 144.3 (CH-N-C), 130.5 (N-CH), 111.2 (N-CH-CH), 88.8 (CH-CO), 24.1 (NCH3 ), 13.9 (C-CH3 ) p.p.m. IR (KBr-disc) υ max: 3437, 2929, 2860, 2365, 2338, 1784, 1719, 1701, 1436, 1376, 1289, 1074, 1042, 762 cm-1

(2h): 3-(3,4-Dimethyl-phenylamino)-1-methyl-pyrrole2,5-dione

Yield = 70%; M.P: 185-187 o C; Rf (60% ether / 40% petrol ether) = 0.26; Molecular Weight: 230.3; Molecular Formula: C13H14N2 O2; MS (APCI (+)): 231 (M+1) m/z 1 H NMR (CDCl3 ) 250 MHz: δ = 6.79-7.42 (m, Ar-H, 3H), 5.39 (s, CH), 4.52-4.71 (bs, NH), 3.09 (s, N-CH3 ), 2.55-2.68 (m, overlapping CH3 , 6H) p.p.m.; 13C NMR (CDCl3 ) 250 MHz: δ = 169.0 (N-CO), 161.6 (CH-CO), 142.4 (CH-N-C), 137.3 (ArC), 135.0 (ArC) 132.0 (ArC), 124.2, (ArC) 120.3 (ArC), 117.6 (Ar-C), 87.8 (N-CH-CH), 23.0 (N-CH3 ), 18.6 (ArC-CH3 , o-position) 17.2 (ArC-CH3 , p-position) p.p.m. IR (KBrdisc) υ max: 3444, 3299, 2924, 2856, 2368, 2336, 1707, 1640, 1450, 1386, 1273, 1124, 595 cm-1

(2i): 1-Methyl-3-(3-nitro-phenylamino)-pyrrole-2,5-dione

Yield = 71%; M.P: N/A – Oily Solid; Rf (60% ether / 40% petrol ether) = 0.22

Molecular Weight: 247.2; Molecular Formula: C11H19N3 O4; MS (APCI(+)): 248 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 7.79- 8.38 (m, 4H, Ar-H), 5.18-5.35 (bs, NH-CH), 4.78 (s, CH), 3.05 (s, N-CH3 ) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 169.2 (N-CO), 160.6 (CH-CO), 147.6 (CH-N-C), 137.8 (ArC), 125.7 (ArC), 121.2 (2xArC), 120.3 (ArC), 87.7 (CH-CO), 24.0 (N-CH3 ) p.p.m.; IR (KBrdisc) υ max: 3462, 3286, 3200, 3042, 2914, 2363, 2341, 1770, 1711, 1594, 1436, 1260, 1224, 1016, 749 cm-1

(2j): 3-Cyclopropylamino-1-methyl-pyrrole-2,5-dione

Yield = 86%; M.P: 125-127 o C; Rf (60% ether / 40% petrol ether) = 0.34; Molecular Weight: 166.2; Molecular Formula: C8 H10N2 O2; MS (APCI(+)):167 (M+1) m/z 1 H NMR (CDCl3 ) 250 MHz: δ = 5.41-5.61 (bs, NH), 5.02 (s, CH), 2.95 (s, N-CH3 ), 2.45- 2.62 (m, NH-CH), 0.58-1.10 (m, NH-CH-CH2 , 4xH) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 170.7 (N-CO), 161.3 (CH-CO), 143.3 (CHN-C), 89.8 (N-CH-CH), 23.7 (N-CH3 ), 23.0 (NH-CH), 6.8 (NH-CHCH2 ) p.p.m. IR (KBr-disc) υ max: 3445, 3333, 3105, 3011, 2957, 2921, 2366, 2335, 1708, 1632, 1448, 1390, 1256, 1018, 763 cm-1

(2k): 3-Cyclopentylamino-1-methyl-pyrrole-2,5-dione

Yield = 82%; M.P: N/A – Oily Solid; Rf (60% ether / 40% petrol ether) = 0.32

Molecular Weight: 194.2; Molecular Formula: C10H14N2 O2; MS (APCI(+)): 195 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 5.58- 5.38 (bs, NH), 4.83 (s, CH), 3.84-3.65 (m, N-CH2 ), 3.00 (s, N-CH3 ), 2.25-1.50 (m, overlapping, N-CH-CH2 , N-CH-CH2 -CH2 , 8H) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 172.7.0 (N-CO), 167.8 (CH-CO), 148.8 (CH-N-C), 84.4 (N-CH-CH), 55.7 (N-CH), 43.9 (N-CH-CH2 ), 32.5 (N-CH-CH2 , 2xC), 23.8 (N-CH-CH2 -CH2 , 2xC), 23.4 (N-CH3 ) p.p.m. IR (KBr-disc) υ max: 3531, 3331, 3099, 2931, 2364, 2338, 1706, 1635, 1509, 1445, 1393, 1122, 1029, 768 cm-1

(2l): 3-Cyclohexylamino-1-methyl-pyrrole-2,5-dione

Yield = 89%; M.P: N/A – Oily Solid; Rf (60% ether / 40% petrol ether) = 0.32

Molecular Weight: 208.3; Molecular Formula: C11H16N2 O2; MS (APCI(+)): 209 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 4.99 (s, CH), 4.01-4.22 (bs, NH-CH), 2.98 (s, N-CH3 ), 0.98-1.55 (m, overlapping CH-CH2 , CH-CH2 -CH2, CH-CH2 -CH2 -CH2 , 10H) p.p.m.13C NMR (CDCl3 ) 250 MHz: δ = 169.7 (N-CO), 161.3 (CH-CO), 145.2 (CH-N-C), 84.4 (CH-CO), 53.6 (NH-CH), 31.7 (NH-CH-CH2, 2xC), 25.4 (CH-CH2 -CH2 , 2xC), 24.4 (CH-CH2 -CH2 -CH2 ), 23.3 (NCH3 ) p.p.m. IR (KBr-disc) υ max: 3442, 3339, 3112, 3009, 2924, 2361, 2329, 1718, 1699, 1629, 1441, 1392, 1115, 1044, 1026, 994, 767 cm-1

(2m): 1’-Methyl-2,3,4,5-tetrahydro-[1,3’]bipyrrolyl-2’,5’- dione

Yield = 86%; M.P: N/A – Oily Solid; Rf (60% ether / 40% petrol ether) = 0.45

Molecular Weight: 180.2; Molecular Formula: C9 H12N2 O2; MS (APCI(+)): 181 (M+1) m/z;1 H NMR (CDCl3 ) 250 MHz: δ = 5.05 (s, CH), 2.81-3.51 (m, overlapping N-CH2 , N-CH3 , 7H), 1.69-2.40 (m, N-CH2 -CH2 , 4H) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 168.1 (NCO), 160.7 (CH-CO), 146.6 (C-N), 88.8 (CH-O), 48.4 (N-CH2 , 2xC), 37.5 (N-CH2 -CH2 , 2xC), 25.7 (N-CH3 ) p.p.m. IR (KBr-disc) υ max: 3433, 2926, 2848, 2361, 2325, 1694, 1616, 1443, 1369, 1340, 1253, 1167, 738 cm-1

(2n): 3-(2,6-Dimethyl-morpholin-4-yl)-1-methylpyrrole-2,5-dione.

Yield = 84%; M.P: 103-106 o C; Rf (60% ether / 40% petrol ether) = 0.32; Molecular Weight: 224.3; Molecular Formula: C11H16N2 O3; MS (APCI(+)): 225 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 4.82 (s, CH), 3.58-3.79 (m, O-CH, 2H), 2.90 (s, CH3 ), 2.55-2.69 (m, N-CH2 , 4H), 1.16-1.22 (d, overlapping O-CH3 , 6H) p.p.m.;13C NMR (CDCl3 ) 250 MHz: δ = 168.6 (N-CO), 161.2 (CHCO), 144.8 (CH-C-N), 89.1 (CH-CO), 64.0 (O-CH, 2xC), 54.9 (CH2 , 2xC), 20.4 (N-CH3 ), 19.4 (CH-CH3 , 2xC) p.p.m. IR (KBr-disc) υ max: 3439, 2975, 2924, 2855, 2367, 2335, 1709, 1607, 1444, 1379, 1133, 1082, 762 cm-1

(2o): 3-Benzylamino-1-methyl-pyrrole-2,5-dione.

Yield = 89%; M.P: 143-145 o C; Rf (60% ether / 40% petrol ether) = 0.27; Molecular Weight: 216.2; Molecular Formula: C12H12N2 O2; MS (APCI (+)): 217 (M+1) m/z 1 H NMR (CDCl3 ) 250 MHz: δ = 7.25-7.65 (m, Ar-H, 5H), 5.58-5.89 (bs, NH), 4.79 (s, CH), 4.32-4.48 (d, CH2 ), 2.98 (s, N-CH3 ) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 169.3 (N-CO), 161.1 (CH-CO), 143.1 (CH-N-C), 133.4 (ArC), 128.2 (2xArC), 126.6 (2xArC), 125.1 (Ar-C), 89.0 (N-CHCH), 44.0 (NH-CH2 ), 24.0 (N-CH3 ) p.p.m. IR (KBr-disc) υ max: 3322, 3023, 2965, 2363, 2336, 1694, 1626, 1450, 1120, 694 cm-1

(2p): 3-(Cyclohexyl-ethyl-amino)-1-methyl-pyrrole-2,5- dione.

Yield = 33%; M.P: N/A – Oily Solid; Rf (60% ether / 40% petrol ether) = 0.36; Molecular Weight: 236.3; Molecular Formula: C13H20N2 O2; MS (APCI(+)): 237 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 4.80 (s, CH), 2.89 (s, N-CH3 ), 2.49-2.73 (m, overlapping, N-CH, N-CH2 , 3H), 0.88-1.59 (m, overlapping, N-CH-CH2 , N-CHCH2 -CH2 , N-CH-CH2 -CH2 -CH2 , CH2 -CH3 ,13H) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 169.1 (N-CO), 160.6 (CH-CO), 143.6 (CHN-C), 89.1 (CO-CH), 60.9 (N-CH), 44.2 (N-CH2 ), 30.7 (N-CH-CH2 , 2xC), 24.1 (N-CH-CH2 -CH2 , 2xC), 23.1 (N-CH-CH2 -CH2 -CH2 ), 21.0 (N-CH3 ), 13.5 (CH2 -CH3 ) p.p.m. IR (KBr-disc) υ max: 3425, 2933, 2855, 2363, 2335, 1699, 1602, 1440, 1379, 762 cm-1

(2q): 1-Methyl-3-(N’-phenyl-hydrazino)-pyrrole-2,5- dione.

Yield = 72%; M.P: 179-181 o C; Rf (60% ether / 40% petrol ether) = 0.25; Molecular Weight: 217.2; Molecular Formula: C11H11N3 O2; MS (APCI(+)): 218 (M+1) m/z 1 H NMR (CDCl3 ) 250 MHz: δ = 7.24-7.51 (m, 3H, Ar-H), 6.797.06 (m, 2H, Ar-H), 6.25- 6.35 (bs, ArC-NH), 5.31 (s, CH), 4.49-5.33 (bs, C-NH), 3.03 (s, CH3 ) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 168.6 (N-CO), 160.9 (CHCO), 153.3 (C-NH), 138.0 (ArC), 127.9 (2xArC), 124.0 (2xArC), 121.9 (ArC), 89.3 (CH-CO), 24.0 (N-CH3 ) p.p.m. IR (KBr-disc) υ max: 3444, 3281, 3204, 2919, 2361, 2352, 1771, 1698, 1599, 1432, 1006, 762 cm-1

(2r): 1-Methyl-3-(4-phenyl-piperazin-1-yl)-pyrrole-2,5- dione.

Yield = 95%; M.P: 174-176 o C; Rf (60% ether / 40% petrol ether) = 0.29

Molecular Weight: 271.3; Molecular Formula: C15H17N3 O2; MS (APCI(+)): 272 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 7.31- 7.41 (m, 2H, Ar-H), 6.92-7.25 (m, 3H, Ar-H), 4.98 (s, CH), 3.80- 4.05 (m, ArC-N-CH2 , 4H) 3.28-3.41 (m, C-N-CH2 ,4H), 2.98 (s, CH3 ) p.p.m. 13C NMR (CDCl3 ) 250 MHz: δ = 169.5 (N-CO), 160.4 (CHCO), 151.3 (CH), 134.3 (ArC), 129.5 (2xArC), 128.0 (2xArC), 125.4 (ArC), 84.4 (C-NH), 51.5 (C-N-CH2 , 2xC), 46.8 (ArC-N-CH2 , 2xC), 24.7 (N-CH3 ) p.p.m. IR (KBr-disc) υ max: 3432, 2919, 2854, 2366, 1701, 1620, 1452, 1384, 1236, 945 758 cm-1

Crystal Data - (sample recrystallised from methanol):

Mr  = 271.32 T = 293(2) K Needle 0.80 x 0.20 x 0.15 mm Yellow, transparent Mo Kα radiation: λ = 0.71073 Å Monoclinic C2/c a = 20.6250(17) Å  b = 6.1567(9) Å  c = 21.840(2) Å  β = 91.424(8) o V = 2772.4(5) Å3 Z = 8 Dx  = 1.300 Mg/m-3 Dm not measured R [F2  > 2σ(F2 )] = 0.0516  wR(F2 ) = 0.1639 2699 reflections 182 parameters

Selected geometric parameters (Å, o )

N(5)-C(6)  O(7)-C(1)  O(8)-C(4)	1.444(3) 1.211(3) 1.216(3)	C(2)-C(3)  N(9)-C(2)  N(12)-C(15)	1.345(3) 1.344(3) 1.406(3)
C(1)-N(5)-C(4)  O(7)-C(1)-N(5)  O(8)-C(4)-N(5)	110.0(2) 125.0(2) 122.2(3)	N(9)-C(2)-C(3)  C(3)-C(2)-C(1)  C(10)-N(9)-C(14)	129.7(2) 106.5(2) 112.6(2)

General method for the preparation of bis-substituted amino-pyrrole-2,5-diones

0.5 g of the appropriate 5-alkoxy- furan-2-one (method 1/2) was dissolved in DMF (2 ml) and placed into 30 ml glass vials. Suitable amines (3 eq.) and 2 equivalents TEA with a catalytic amount of CuI (5%) were added carefully drop wise to each vial. The vials were placed in a heating block and heated to 45 o C for 48 hours. The compound was extracted with ether (10 ml) and washed twice with water (10 ml). The organic layer was dried using anhydrous magnesium sulphate and removed in vacuum to give a dark brown oil, which was purified using column chromatography (solvent system: 50/50 ether/petrol ether) to give the desired product.

Method 1: 3,4-Dichloro-5-methoxy-5H-furan-2-one 3a

Method 2: 3,4-Dichloro-5-isopropoxy-5H-furan-2-one 3b

(4a): 1-Butyl-3-butylamino-pyrrole-2,5-dione.

Yield = 63% (method 1); M.P: 135-137 o C; Rf (50% ether, 50% petrol ether) = 0.81

Molecular Weight: 224.3; Molecular Formula: C12H20N2 O2; MS (APCI(+)): 225 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: = 5.11- 5.30 (bs, NH), 4.69 (s, CO-CH), 3.40-3.52 (t, N-CH2 , J = 6.9 Hz), 3.05-3.18 (q, NH-CH2 , J = 4.9 Hz), 1.18-1.67 (m, overlapping CH2 , 8H), 0.82-1.03 (m, overlapping CH3 , 6H) p.p.m. 13C NMR (CDCl3 ) 250MHz: δ =170.4 (CN-C=O), 165.1 (CH-C=O), 150.4 (CO-CN), 84.9 (CH-CCl), 39.8 (NH-CH2 ), 39.4 (N-CH2 ), 31.3 (CH2 -CH2 -NH), 29.5 (CH2 -CH2 -N), 20.1 (NH-CH2 -CH2 -CH2 ), 19.7 (N-CH2 -CH2 -CH2 ), 13.7 (NH-CH2 -CH2 -CH2 -CH3 ), 13.6 (N-CH2 -CH2 -CH2 -CH3 ) p.p.m. IR (KBr-disc) υ max: 3431, 2966, 2925, 2867, 2361, 2338, 1701, 1638, 1458, 1399 1232, 1105, 1019, 893 cm-1

(4b): 1-Isobutyl-3-isobutylamino-pyrrole-2,5-dione.

Yield = 73% (method 1); M.P: 130-133 o C; Rf (50% ether, 50% petrol ether) = 0.77

Molecular Weight: 224.3;Molecular Formula: C12H20N2 O2;MS (APCI(+)): 181 (M+), 225 (M+1) m/z;1 H NMR (CDCl3 ) 250 MHz: δ = 5.33-5.58 (bs, NH), 4.83 (s, CO-CH), 3.26-3.34 (d, N-CH2 ), 2.97- 3.08 (t, NH-CH2, J = 6.0 Hz), 1.89-2.11 (m, overlapping CH-CH2 -N & CH-CH2 -NH, 2H), 0.87-1.09 (m, overlapping CH3 , 12H) p.p.m. 13C NMR (CDCl3 ) 250MHz: δ = 173.0 (CN-C=O), 167.8 (CH-C=O), 149.5 (CO-CN) 83.7 (CH-CCl), 52.0 (NH-CH2 ) 45.0 (N-CH2 ), 28.0 (CH-CH2 -NH), 27.9 (CH-CH2 -N), 20.2 (NH-CH2 -CH-CH3 , 2xC), 20.0 (N-CH2 -CH-CH3 , 2xC) p.p.m. IR (KBr-disc) υ max: 3339, 2961, 2941, 2882, 2365, 1705, 1635, 1519, 1449, 1416, 1297, 1131, 1035, 786 cm-1.

(4c): 1-sec-Butyl-3-sec-butylamino-pyrrole-2,5-dione

Yield = 69% (method 1); M.P: N/A – Oily Solid; Rf (50% ether, 50% petrol ether) = 0.80

Molecular Weight: 224.3; Molecular Formula: C12H20N2 O2; MS (APCI(+)): 169 (M+), 225 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 5.79 (s, CO-CH), 5.04-5.25 (bs, NH), 3.82-4.16 (m, overlapping N-CH& NH-CH, 2H), 1.46-1.73 (m, overlapping CH2 -CH-N & CH2 - CH-NH, 2H), 1.19-1.31 (m, overlapping CH-CH3 , 6H), 0.72-0.97 (m, overlapping CH2 -CH3 , 6H) p.p.m. 13C NMR (CDCl3 ) 250MHz: δ = 173.0 (CN-C=O), 168.3 (CH-C=O), 148.0 (CO-CN), 83.4 (CH-CCl), 51.9 (NH-CH), 48.3 (N-CH), 29.0 (CH2 -CH-NH), 27.1 (CH2 -CH-N), 19.4 (NH-CH-CH3 ), 18.4 (N-CH-CH3 ), 11.3 (NH-CH-CH2 -CH3 ), 10.3 (N-CH-CH2 -CH3 ) p.p.m. IR (KBr-disc) υ max: 3428, 2977, 2932, 2867, 2370, 2338, 1703, 1642, 1461, 1393 1229, 1106, 1019, 900 cm-1.

(4d): 1-Cyclopentyl-3-cyclopentylamino-pyrrole-2,5-dione.

Yield = 45% (method 1); M.P: 142-144 o C; Rf (50% ether, 50% petrol ether) = 0.78

Molecular Weight: 248.3; Molecular Formula: C14H20N2 O2; MS (APCI(+)): 249 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: = 5.26-5.41 (bs, NH), 4.82 (s, CH-O), 4.31-4.49 (m, N-CH, J = 8.8 Hz), 3.62-3.79 (m, NH-CH), 1.51-2.13 (m, overlapping CH2 , 16H) p.p.m. 13C NMR (CDCl3 ) 250MHz: = 168.6 (CN-C=O), 161.0 (CH-C=O), 148.3 (CHCCl), 84.3 (CH-CO), 55.6 (NH-CH), 50.5 (N-CH), 32.1 (NH-CH-CH2 , 2xC), 29.5 (NH-CH-CH2 , 2xC), 24.7 (NH-CH-CH2 -CH2 , 2xC), 23.8 (NH-CH-CH2 -CH2 , 2xC) p.p.m. IR (KBr-disc) υ max: 3441, 3252, 2924, 2855, 2376, 2338, 1698, 1651, 1623, 1399, 1125, 1075, 989, 892, cm-1.

(4e): 1-Cyclohexyl-3-cyclohexylamino-pyrrole-2,5-dione.

Yield = 57% (method 2); M.P: 146-148 o C; Rf (50% ether, 50% petrol ether) = 0.80

Molecular Weight: 276.4; Molecular Formula: C16H24N2 O2; MS (APCI(+)): 195 (M+), 277 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 5.19-5.40 (bs, NH), 4.66 (s, CH-O), 3.74-3.99 (m, N-CH), 2.99- 3.23 (m, NH-CH), 1.53-2.15 (m, overlapping CH2 , 12H), 1.07-1.49 (m, overlapping CH2 , 8H) p.p.m. 13C NMR (CDCl3 ) 250MHz: δ = 169.2 (CN-C=O), 162.0 (CH-C=O), 147.3 (CH-CCl), 83.4 (CH-CO), 53.1 (NH-CH), 50.0 (N-CH), 32.0 (NH-CH-CH2 , 2xC), 30.1 (NHCH-CH2 , 2xC), 26.0 (NH-CH-CH2 -CH2 , 2xC), 25.3 (NH-CH-CH2 -CH2 , 2xC), 24.7 (NH-CH-CH2 -CH2 -CH2 ), 24.4 (NH-CH-CH2 -CH2 -CH2 ) p.p.m. IR (KBr-disc) υ max: 3431, 2966, 2925, 2867, 2361, 2338, 1701, 1638, 1521, 1458, 1399, 1232, 1105, 1019, 893 cm-1.

(4f): 1-Hexyl-3-hexylamino-pyrrole-2,5-dione

Yield = 47% (method 2); M.P: 69-84 o C; Rf (50% ether, 50% petrol ether) = 0.84

Molecular Weight: 280.4; Molecular Formula: C16H28N2 O2; MS (APCI(+)): 281 (M+1) m/z; 1 H NMR (CDCl3 ) 250 MHz: δ = 5.22- 5.42 (bs, NH), 4.82 (s, CO-CH), 3.10-3.66 (m, overlapping N-CH2 & NH, 3H), 2.83-2.99 (m, NH-CH2 ), 1.18-1.67 (m, overlapping N-CH2 -CH2 & NH-CH2 -CH2 4H), 1.06-1.38 (m, overlapping CH2 , 12H), 0.64-0.98 (m, overlapping CH3 , 6H) p.p.m. 13C NMR (CDCl3 ) 250MHz: δ = 168.0 (CN-C=O), 163.0 (CH-C=O), 143.9 (CO-CN), 84.4 (CH-CCl), 40.0 (NH-CH2 ), 39.7 (N-CH2 ), 31.4 (CH2 -CH2 -NH), 31.3 (CH2 -CH2 -N), 29.7 (NH-CH2 -CH2 -CH2 ), 29.2 (N-CH2 -CH2 -CH2 ), 26.6 (NH-CH2 -CH2 -CH2 -CH2 ), 26.5 (N-CH2 -CH2 -CH2 -CH2 ), 22.6 (NH-CH2 -CH2 -CH2 -CH2 -CH2 ), 22.5 (N-CH2 -CH2 -CH2 -CH2 -CH2 ), 14.0 (NH-CH2 -CH2 -CH2 -CH2 -CH3 ), 13.96 (N-CH2 -CH2 -CH2 -CH2 -CH3 ) p.p.m. IR (KBr-disc) υ max: 3447, 2931, 2853, 2370, 2350, 1716, 1638, 1524, 1462, 1220, 1040, 972, 711 cm-1.

Animal studies

Experiments were conducted in male ICR mice obtained from the Animal House, Faculty of Medicine, Khon Kaen University. Each experimental group consisted of 6 animals and the treatment procedures were approved by the ethical committee, Faculty of Medicine, Khon Kaen University (HO 2434-76) accord with current UK legislation. Mice had free access to fresh water and food pellets. They were exposed to automated 12h light cycles.

Mice were intraperitoneal injected with either test compound dissolved in 5% DMSO at the volume not more than 0.2 ml/animal. At 30 min after treatment, animals were tested as described in the following sections.

Motor activity tests

The rota-rod test: Mouse was placed on the rotating drum with the acceleration speed (Acceler. Rota-rod, Jones & Roberts, for mice 7650, Ugo Basile, Italy). The time animal spent on the rod was recorded.

The wire mesh grasping test: Mouse was placed on a wire mesh (20x30 cm). After a few seconds, the mesh was turned 180o and the time the animal hold on the mesh was recorded.

Anxiolytic activity tests

The light/dark box: Mice were placed in the light part of the light/dark box. The box was a plexi glass cage, 25x50x20 cm, having one-third as a dark and two-third as a light compartment. A 40-W light bulb was used and positioned 10 cm above the centre of the light component. The animals could walk freely between dark and light parts through the opening. The time animals spent in light part during the 5 min interval was recorded. The mouse was considered to be in the light part when its 4 legs were in the light part.

The elevated plus-maze: The wooden elevated plus-maze consisted of two open arms (30x10 cm) without any walls, two enclosed arms of the same size with 5-cm high side walls and end wall, and the central arena (10x10 cm) interconnecting all the arms. The maze was elevated approximately 30 cm height from the floor. At the beginning of the experiment the mouse was placed in the central arena facing one of the enclosed arms. During a 5 min interval, the time animals spent in the open arms of plus-maze was recorded. The mouse was considered to be in the open part, when it had clearly crossed the line between the central arena and the open arm with its 4 legs.

Statistical methods

The data were expressed as mean ± SD and one-way analysis of variance (ANOVA) and supplementary Tukey test for pair wise comparison were tested to determine for any significant difference at p< 0.05.

1. Prado SR, Cechinel-Filho V, Campos-Buzzi F, Corrêa R, Cadena SM, De Oliveira MB. Biological evaluation of some selected cyclic imides: mitochondrial effects and in vitro cytotoxicity. Z Naturforsch C. 2004; 59: 663-672.

2. Zentz F, Hellio C, Valla A, De La Broise, D, Bremer G,Labia R. Antifouling activities of N-substituted imides : Antimicrobial activities and inhibition of Mytilus edulis Phenoloxidase. Mar Biotechnol (NY). 2002; 4: 431-440.

3. Clemson HC, Magarian EO, Reinhard JE. Synthesis and anticonvulsant properties of some derivatives of N-methyl-2-phenylsuccinimide II. J Pharm Sci.1970. 59:1137-1140.

4. Braña MF, Castellano JM, Morán M, Emling F, Kluge M, Schlick E, et al. Synthesis, structure and antitumor activity of new benz [d,e] isoquinoline-1,3-diones. Arzneimittel for schung. 1995; 45: 1311- 1318.

5. Yaroslavsky C, Bracha P. Glaser R. Preparation, proton and carbon-13 structure determination and fungicidal activity of chlorinated 1-arylamino-1H-pyrrol-2,5-diones. J. Hetero. Chem. 1989; 26: 1649- 1654.

6. Zentz F, Valla A, Le Guillou R, Labia R, Mathot AG, Sirot D. Synthesis and antimicrobial activities of N-substituted imides. Farmaco. 2002; 57: 421-426.

7. Pineyro, G. and Blier, P. 1996. Regulation of 5-hydroxytrytamine release from rat midbrain raphe nuclei by 5-hydroxytryptamine1D receptors: effects of tetrodotoxin, G-protein inactivation and long term anti-depressant administration. J Pharmacol Exp Ther. 1996; 276: 697-707.

8. Ishizumi, K, Kojima A, Antoku F. Synthesis and anxiolytic activity of N-substituted cyclic imides (1R*, 2S*, 3R*,4S*)-N- [4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-2,3-bicyclo[2.2.1] heptanedicarboximide (tandospirone) and related compounds. Chem. Pharm. Bull. 1991; 39: 2288-2300.

9. Filho VC, Vaz Z, Nunes RJ, Calixto JB, Yunes RA. Antinociceptive activity of phyllanthimide analogues : Structure-activity relationships. Pharm. Sci.1996; 2: 399.

10. Cechinel Filho V, Corrêa R, Vaz Z, Calixto JB, Nunes RJ, Pinheiro TR, et al. Further studies on analgesic activity of cyclic imides. Farmaco. 1998; 53: 55-57.

11. Boström J, Brickmann K, Holm P, Sandberg P, Swanson M, Westerlund C. Pyrrol-2,5-dione derivatives as liver X modulators. International Patent WO. 2005.

12. Smith DG, Buffet M, Fenwick AE, Haigh D, Ife RJ, Saunders M, et al. 3-Anilino-4-arylmaleimides: potent and selective inhibitors of glycogen synthase kinase-3 (GSK-3). Bioorg Med Chem Lett. 2001; 11: 635-639.

13. Tripathi M, Verma M, Palit G, Shanker K. Antipyrine congeners as antidepressant agents. Arzneimittelforschung. 1993; 43: 1045-1049.

14. Lattmann E, Dunn S, Niamsanit S, Sattayasai J, Sattayasai N. Antibacterial activity of 3-substituted pyrrole-2,5-diones against Pseudomonas aeruginosa, Lett Drug Design & Discovery. 2007; 4: 513-519.

15. Mehta NB, Phillips AP, Lui FF, Brooks RE. Maleamic and citraconamic acids, methyl esters and iminde. J. Org. Chem. 1960; 25: 1012-1015.

16. Earl RA, Clough FW, Townsend LB. Chemical investigations of citraconimide. J. Hetero. Chem.1978; 15: 1479-1483.

17. Duczek W, Jaehnisch K , Kunath A, Reck G, Winter G, Schulz B. Liebigs Ann. Chem.1992; 8: 781.

18. Lattmann E, Ayuko WO, Kinchinaton D, Langley CA, Singh H, Karimi L, et al. Synthesis and evaluation of 5-arylated 2(5H)-furanones and 2-arylated pyridazin-3(2H)-ones as anti-cancer agents. J Pharm Pharmacol. 2003; 55: 1259-1265.

19. Lattmann E, Kinchington D, Dunn S, Singh H, Ayuko WO, Tisdale MJ. Cytotoxicity of 3,4-dihalogenated 2(5H)-furanones. J Pharm Pharmacol. 2004; 56: 1163-1170.

20. Lattmann E, Sattayasai N, Schwalbe CS, Niamsanit S, Billington DC, Lattmann P, et al. Novel anti-bacterials against MRSA: synthesis of focussed combinatorial libraries of tri-substituted 2(5H)-furanones. Curr Drug Discov Technol. 2006; 3: 125-134.