Bioorg Med ChemBioorg. Med. ChemBioorganic & Medicinal Chemistry0968-08961464-3391Elsevier Science254874224346271S0968-0896(14)00776-710.1016/j.bmc.2014.11.002ArticleLigand-based virtual screening identifies a family of selective cannabinoid receptor 2 agonistsGianella-BorradoriMatteoa†ChristouIvyb†BatailleCarole J.R.aCrossRebecca L.aWynneGraham M.aGreavesDavid R.david.greaves@path.ox.ac.ukb⁎RussellAngela J.angela.russell@chem.ox.ac.ukac⁎Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UKSir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UKDepartment of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UKCorresponding authors. Tel.: +44 (0)1865 289150; fax: +44 (0)1865 275515 (D.R.G.); tel.: +44 (0)1865 275643; fax: +44 (0)1865 285002 (A.J.R.). david.greaves@path.ox.ac.ukangela.russell@chem.ox.ac.uk
The cannabinoid receptor 2 (CB2R) has been linked with the regulation of inflammation, and selective receptor activation has been proposed as a target for the treatment of a range of inflammatory diseases such as atherosclerosis and arthritis. In order to identify selective CB2R agonists with appropriate physicochemical and ADME properties for future evaluation in vivo, we first performed a ligand-based virtual screen. Subsequent medicinal chemistry optimisation studies led to the identification of a new class of selective CB2R agonists. Several examples showed high levels of activity (EC50 < 200 nM) and binding affinity (Ki < 200 nM) for the CB2R, and no detectable activity at the CB1R. The most promising example, DIAS2, also showed favourable in vitro metabolic stability and absorption properties along with a clean selectivity profile when evaluated against a panel of GPCRs and kinases.
The endocannabinoid system (ECS) is an endogenous signalling pathway that has a regulatory role in pain perception, the immune response, metabolism and mood. Among the key components of the ECS are the cannabinoid receptors 1 and 2 (CB1R and CB2R). While CB1R is predominantly expressed in the central nervous system, CB2R is mainly expressed in peripheral tissues, particularly immune cells,1,2 although studies over the last decade have shown that CB2R is also expressed in the CNS in microglia3 and brainstem neurons,4 and that it is upregulated in response to injury in dorsal root ganglia5 and sensory neurons.6
CB2R belongs to the G-Protein Coupled Receptor (GPCR) superfamily and mediates the downstream effects of endogenous secreted cannabinoid receptor ligands (endocannabinoids) such as anandamide and 2-arachidonoylglycerol (2-AG).7 Activation of the CB2R has been shown to lead to a number of downstream signalling events,8 the most widely studied of which involve inhibition of adenylyl cyclase and lowering of intracellular cyclic AMP levels,9 and mitogen-activated protein kinase (MAPK) activation10 via coupling through Gαi/o proteins.
CB2R has been proposed as a therapeutic target for the treatment of pain and inflammatory conditions.11 The main components of marijuana including the tetrahydrocannabinol Δ9-THC (1, Fig. 1) and other phytocannabinoids12 have long been known to exhibit analgesic activity, which has since been attributed to activation of the CB1R as well as the CB2R.8,13 However the clinical use of these agents is limited, in the most part due to their psychotropic side effects which are thought to arise from activation of the CB1R.14,15 Thus, there have been extensive studies in recent years to develop selective CB2R modulators, particularly for the management of pain.11,16 Indirect strategies to modulate the cannabinoid receptors through inhibition of endocannabinoid metabolism are also being explored.17
A variety of strategies have been explored to develop alternative CB2R agonists including structure activity relationship (SAR) analyses around the structure of Δ9-THC (1) leading to the discovery of compounds such as CP 55,940 (2, Fig. 1)18 and the selective CB2R agonist, HU-308 (5, Fig. 2).19 A homology model of the CB2R has been published using the crystal structure of bovine rhodopsin,20 and this and more recent refine models have been employed in virtual screening strategies to identify new classes of CB2R ligand.21,22 The most commonly employed strategies have been ligand-based virtual screening23 and high-throughput screening approaches,11 for example, in the recent discovery of 4 by Lilly (Fig. 1).24
Several CB2R selective agonists show efficacy in in vivo models of acute and chronic pain, and these effects are blocked upon co-treatment with CB2R antagonists such as SR14452825 and AM630,26 or in CB2R knockout animals, supporting a CB2R-mediated mechanism of action.11 Despite these promising preclinical data, only a few candidates have entered clinical trials to date for evaluation in the treatment of inflammatory pain, and none has progressed beyond Phase 2. For instance GW-842166X (3, Fig. 1)27 was recently evaluated in a Phase 2a trial for acute inflammatory pain following third molar extraction,28 but failed to demonstrate sufficient efficacy and has been discontinued. In this case the lack of efficacy was attributed to insufficient exposure and CB2R occupancy.28
Moreover, there is emerging in vivo data to support the role of CB2R as a therapeutic target in other disease indications including osteoporosis,29 multiple sclerosis30 or in other diseases with an inflammatory component31 such as allergic contact dermatitis,32 colitis33,34 and atherosclerosis.35 A non-psychoactive cannabinoid derivative, ajulemic acid, has also been reported to show anti-inflammatory effects.36 CB2R activation has been shown to induce monocyte chemotaxis,37 and reduce macrophage chemotaxis to a range of chemoattractants including MCP-1, MIP-1α, RANTES and fMLP.35,38,39 Support for CB2R being a physiologically relevant modulator of macrophage inflammation in vivo came from analysis of endocannabinoid levels in human atherosclerotic lesions in carotid arteries and reduced MMP-2 secretion by human inflammatory cells treated with the CB2R-selective agonist JWH-133.40 There is therefore still a real need for additional highly selective CB2R agonists with improved pharmacokinetic and pharmacodynamic profiles to evaluate their clinical efficacy as anti-inflammatory agents. To date most groups have focussed on CNS penetrant CB2R ligands, many of which have high lipophilicity.41
Despite the range of current CB2R agonists available in the published literature, there still exists a scientific need for new CB2R-selective tool compounds that are less lipophilic and peripherally restricted that can be used to specifically address the role of CB2R in modulating macrophage chemotaxis and activation in preclinical models of inflammation.34 We therefore sought to identify a new family of highly selective CB2R agonists with improved in vitro ADME properties to investigate their use as peripherally-restricted anti-inflammatory agents in a range of in vivo models. Herein we report our preliminary discovery efforts towards this overarching goal.
Results and discussion
We opted to employ a virtual ligand-based screening strategy in order to identify new chemical scaffolds which exhibited agonist activity at the CB2R to provide a starting point for initial SAR analysis and optimisation. A set of reported CB2R agonists and their respective affinities for CB1R and CB2R were assessed when making the final selection for the template ligand for the virtual screen (see Supplementary Table S1). HU-308 (5, Fig. 2A) was chosen as the query molecule for the virtual screen due to its high potency and degree of selectivity for CB2R over the CB1R (> 400 fold).
Ligand-based virtual screening
Using the Omega (OpenEye) package, 40 possible low energy conformers of HU-308 (5) were established based on the minimised energy conformations of the molecule (kcal/mol) (example in Fig. 2B).42 Up to 100 conformers were also generated of every molecule in our 25,000-member in-house library.43 Finally the 40 conformers of HU-308 (5) were screened against the virtual library using the programme rapid overlay of chemical structures (ROCS, OpenEye).44 The screen was performed under default parameters using full optimisation mode. The compounds were ranked in order according to their shape similarity compared to any of the 40 conformers of HU-308 (5). The top 100 compounds that presented most similarity in their overlay with HU-308 (5) were of interest, these overlay results were confirmed through the use of an independent alignment program, Forge (Cresset) (Fig. 2C).45 The overall score was based on a combination of two calculated parameters: the colour score46 and shape Tanimoto score47 combined, provided the combo score47 by which the compounds were ranked (see Supplementary Table S2). On the basis of this ranking the top 94 available compounds were selected for screening for CB2R activation.
Biochemical screening and hit identification
The 94 compounds were screened using a commercially available complementation immunoassay measuring levels of β-galactosidase (β-gal) labelled cAMP.48 The assay was performed using CB2R-expressing Chinese Hamster Ovary (CHO) cells stimulated with forskolin. In the absence of an agonist binding to the CB2R, intracellular cAMP competes with labelled cAMP for binding to the anti-cAMP antibody increasing levels of free β-gal labelled cAMP in the cell. The addition of a CB2R agonist leads to inhibition of adenylate cyclase activity by the Gαi complex formed upon receptor activation. This in turn leads to a lowering of the level of intracellular cAMP, increased sequestration of labelled cAMP by the anti-cAMP antibody and thus a decrease in the level of free β-gal labelled cAMP.
From the compounds tested several CB2R activators were identified as having modest activity. Six different cores were present in the top 16 hits. Interestingly the compound that ranked highest in the virtual screen did not demonstrate the highest activity in the in vitro assay. DIAS1 (6) was selected for further evaluation (Fig. 2D) on the basis of its activity, relatively low molecular weight (258 Da), and predicted lower lipophilicity compared to HU-308 (c log P HU-308, 6.9; c log P DIAS1, 4.2). This compound exhibited good activity and in a dose dependent manner at the CB2R, 3 μM (57.2%) and 1 μM (52.1%). The selected compound ranked 13th in the virtual screen based on its combo score (see Supplementary Table S2). An overlay of DIAS1 (6) with HU-308 (5) using Forge45 illustrates good alignment of the hydrophobic alkyl chains and the aromatic core in both molecules (Fig. 2C).
Following the identification of 6 as an active hit, our compound library was further mined using the 3-mercapto triazinoindole core of 6 to identify available molecules with the same substructure to allow screening of close analogues, and an evaluation of preliminary SARs. This search identified a small number of additional compounds with variations at C(3) and N(5) positions of the tricyclic core. The compounds were screened in the cAMP assay (Fig. S1) and one, DIAS2 (7) exhibited improved activity (EC50 = 120 nM), in comparison to DIAS1 (6) (EC50 = 296 nM) with a reduced c log P value (3.8). Interestingly DIAS2 was not amongst the top 16 ranked compounds in the virtual screen, nor did it appear in the top 400 compounds based on their combo score. This discrepancy may reflect the accuracy of the algorithms used but may also just reflect a limitation in this ligand-based screening method: for instance hits are ranked on the basis of similarity to a probe ligand (in this case HU-308) and not on the basis of predicted best fit with the CB2R.
Hit validation of DIAS1 (<bold>6</bold>) and DIAS2 (<bold>7</bold>)
Verification of the activity of DIAS1 (6) and DIAS2 (7) was carried out via the re-synthesis of an authentic sample. Compounds 6 and 7 were synthesised via a cyclisation of isatin 8 with thiosemicarbazide to yield the tricycle 9,49 followed by alkylation or benzylation of 9 to yield 6 and 7 in an overall yield of 76% and 79%, respectively (Scheme 1).50,51
The resynthesised samples of DIAS1 (6) and DIAS2 (7) gave EC50 values of 296 and 112 nM, respectively, in the cAMP assay. Having confirmed the functional activity of DIAS1 (6) and DIAS2 (7) as CB2R agonists, attention next turned to exploring preliminary structure–activity-relationships within this series in order to optimize activity.
DIAS2 optimisation: variation at C(3)
The first region of the tricyclic moiety to be explored was the C(3) benzyl thiol moiety. A synthetic route enabling a library to be efficiently enumerated was optimised from the previous synthesis of 7 via intermediate 9. This common intermediate was used to prepare a series of compounds representing S-alkyl, S-aryl and S-benzyl substituted derivatives. Intermediate 9 allowed efficient substitution of a range of alkyl and arylmethyl halides in the presence of Et3N to yield the S-substituted compounds 10–11 and 12–24, which were isolated in 50% to quantitative yield (Scheme 2).
The array of C(3)-substituted triazinoindoles were evaluated for activity at the CB2R using the CHO cell assay described above. The preliminary SAR assessment had demonstrated that the presence of linear thioalkyl groups C(3) larger than n-butyl led to a reduction in activity at the CB2R (Table S2), and it was therefore of interest to determine whether a small thiomethyl (10) substituent, or more bulky β-branched thiomethylcyclohexyl (11) would show activity at the CB2R. Compound 11 was also of particular interest as a fully saturated counterpart for comparison with the C(3)-thiobenzyl substituted analogues. Intermediate 9 was also tested and showed no detectable agonist activity at the CB2R, nor did the C(3)-thiomethyl- or C(3)-thiomethylcyclohexyl-substituted derivatives 10 and 11 (EC50 > 3000 nM, Table 1).
Having established a relatively restricted tolerance for variations in C(3)-thioalkyl substituents, attention turned to exploring systematic variations of C(3)-thiobenzyl substituents. To this end a selection of compounds with varying aryl substituents and regiochemistry were prepared and assayed (Table 1). Interestingly, removal of the cyano group (C(3)-thiobenzyl, 12) led to a reduction in activity at the CB2R (EC50 = 10,000 nM, Table 1). Regiochemistry was also found to influence activity, with 3-cyano substituted derivative 13 showing markedly reduced activity at the CB2R compared to its 2-cyano substituted counterpart DIAS2 7 (13: EC50 = 6450 nM; 7 EC50 = 112 nM, Table 1). Introduction of fluoro or chloro substituents at any site on the aryl ring showed no detectable activity (14–19; EC50 > 3000 nM, Table 1). In contrast, the 2-nitro substituted derivative 20 showed good levels of activity at the CB2R (EC50 = 33 nM, Table 1); regiochemistry was again found to influence activity, with the 4-nitro substituted counterpart (21) showing reduced activity at the CB2R (EC50 = 617 nM, Table 1).
These initial SAR observations suggested that there was a preference for electron withdrawing groups incorporating a hydrogen bond acceptor in the ortho site of the C(3)-thiobenzyl group. Previous studies have sought to rank and quantify hydrogen bond acceptor ability of various functional groups,52 and it was noted that examples incorporating a good hydrogen bond acceptor in the ortho site of the S-benzyl group such as nitro (20) and cyano (7) showed the highest levels of activity compared to poorer hydrogen bond acceptors such as halogens (14 and 17). Three further examples were therefore synthesized in order to further probe any relationship between electron withdrawing ability or hydrogen bond acceptor ability and activity: o-trifluoromethoxy (23), o-ethoxycarbonyl (24) and o-carboxylic acid (25, prepared through saponification of the corresponding ester 24). Trifluoromethoxy substituted derivative 23 showed no detectable activity at the CB2R (EC50 > 3000 nM, Table 1), further suggesting that a bulky group cannot be accommodated at this site. Interestingly, both ester 24 and carboxylic acid 25 displayed little or no detectable activity at the CB2R (24: EC50 > 3000 nM; 25 EC50 = 2185, Table 1) despite both groups being good hydrogen bond acceptors.
The effect of varying the linker between C(3) and the aryl group was also investigated. DIAS2 (7) was oxidised chemoselectively to the corresponding sulfoxide 26 in 95% yield using a mixture of trifluoroacetic acid and peroxytrifluoroacetic acid (Scheme 3). Compound 26 was also considered as a potential metabolite of DIAS2 so it was of further interest to determine the effect of S-oxidation on bioactivity. However, S-oxidation led to a significant reduction of activity at the CB2R (26: EC50 = 3550 nM, Scheme 3).
Representative compounds showing activity at the CB2R were also assessed for activity at the CB1R again using a complementation immunoassay measuring levels of β-galactosidase (β-gal) labelled cAMP, analogous to the assay used to measure CB2R activity.48 The assay was performed using CB1R-expressing CHO cells stimulated with forskolin and Δ9-THC, a non-specific CB1R agonist, was used as a positive control (data not shown). DIAS2 (7) and analogues 12, 13 and 20 were all evaluated for activity at the CB1R but none showed any detectable agonist activity up to the highest concentrations tested (EC50 > 30,000 nM, Table 1).
In addition to measuring the functional activity of these compounds at the CB1R and CB2R, the binding affinity of representative examples at both receptors was determined, in order to measure Ki values. Two representative compounds, 2-nitro- and 2-cyano-substituted S-benzyl derivatives (20 and 7) were thus assessed for CB1R and CB2R affinity using a radioligand displacement assay. Compound binding was measured by displacement of [3H]-CP 55,940 or [3H]-WIN55212-2 in CHO cells expressing the human CB1R or CB2R, respectively.53
These studies confirmed the binding of 20 and 7 to the CB2R (7: Ki = 355 nM; 20: Ki = 155 nM, Table 1). No binding was detected at the CB1R for either compound (Ki > 60,000 nM, Table 1) providing further support for the development of this series of compounds as selective CB2R agonists.
DIAS2 optimisation: variation at C(6)–C(9)
Having established preliminary structure–activity-relationships through variation at C(3), attention next turned to exploring the effect of introducing a substituent at C(6)–C(9) of the tricyclic core. In an initial effort to establish which position could tolerate further substitution, a bromo substituent was introduced at each of the four available sites. Following the same synthetic procedure developed for the preparation of DIAS2 (7), bromoisatins 27–30 were first treated with thiosemicarbazide to afford the corresponding tricyclic species 31–34 in 34–97% yield (Scheme 4). Intermediates 31–34 were subsequently treated with 2-cyanobenzyl bromide to give the bromo-substituted tricyclic derivatives 35–38 in 64–92% yield. 9-Bromo substituted tricycle 39 was also prepared in 66% yield by treating 35 with 2-nitrobenzyl bromide (Scheme 4).
The bromo-substituted tricycles 36–40 were assessed for CB2R activity using the CHO cell assay. Introducing a bromo substituent at C(6) (35), C(7) (36) or C(8) (37) was observed to lead to a loss of activity at the CB2R in all cases (EC50 > 3000 nM, Table 2). Conversely, 38 and 39 both of which incorporated a bromo substituent at C(9) maintained good levels of activity at the CB2R (EC50 = 39 and 37 nM, respectively, Table 2). The 9-bromo derivative 38 was selected for CB1 and CB2 receptor affinity studies for comparison with DIAS2 (7). Compound 38 showed high binding affinity at the CB2R and weak to moderate affinity at the CB1R (Ki = 22.5 and 3200 nM, respectively, Table 2). In accordance with the affinity studies, 38 also showed very weak agonist activity when evaluated in CB1R-expressing CHO cells (EC50 ≈ 20,000 nM, Table 2).
Having established that introducing a bromo substituent at C(9) was tolerated, we sought to further explore the effect of varying the C(9) group on activity and affinity at the CB2R. It was envisaged that a selection of groups could be introduced at this position via a Suzuki cross-coupling reaction using 4-bromoisatin 30 as starting material.54 4-(3′-Thienyl)isatin 41, 4-(2′-methylphenyl)isatin 42 and 4-(4′-methylphenyl)isatin 43 were all prepared in moderate to good yield via a Pd(PPh3)4Cl2-mediated cross-coupling using 4-bromoisatin and the corresponding potassium trifluoroborate salts.55 The intermediates 41–43 were next treated stepwise with thiosemicarbazide then 2-cyanobenzyl bromide to afford the C(9)-substituted derivatives 50–52 in 17–50% yield over the two steps (Scheme 5).
Treatment of 4-bromoisatin 30 with either cyclopropyl boronic acid or potassium cyclopropyl trifluoroborate under a range of conditions gave no conversion to the desired 4-cyclopropylisatin product. To circumvent this problem, isatin was first treated with 4-methoxybenzyl chloride and sodium hydride to give the corresponding N-PMB protected isatin 40. Treatment of 40 with potassium cyclopropyl trifluoroborate and Pd(dppf)2Cl2 afforded 44 in 96% yield. Cyclisation, S-benzylation and deprotection using trifluoroacetic acid gave 9-cyclopropyl substituted derivative 53 in 46% yield over the three steps (Scheme 5).
The array of C(9)-substituted compounds were evaluated for CB2R activity using the CHO cell assay. While 9-cyclopropyl-substituted derivative 53 showed good levels of activity at the CB2R (EC50 = 231 nM, Table 3), introducing a bulkier 2-thienyl substituent at C(9) led to a reduction in activity at the CB2R (52: EC50 = 1760 nM, Table 3). Of the two C(9)-aryl substituted derivatives tested (50 and 51) both showed markedly reduced activity at the CB2R (50: EC50 = 4300 nM; 51: EC50 > 3000 nM, Table 3).
In an effort to rationalise the steep SAR observed upon variation at C(9), lowest energy conformers were calculated for representative examples using the Maestro modelling package (Schrodinger): 51 (p-methylphenyl substituted), 52 (3-thienyl substituted) and 53 (cyclopropyl substituted) (Fig. 3). The results suggest that 51 and 52 both preferentially adopt a non-planar conformation about the exocyclic C(9)—C bond, consistent with the conformational preferences of ortho-substituted biaryls. A possible explanation for the reduced activity of 52 compared to 7 and 38, and the lack of detectable activity of 51, could be that this non-planar conformation cannot be accommodated upon binding to the CB2R.
Compound 53 is predicted to have a number of low energy conformers, one of which is depicted in Figure 3. The two carbons at the base of the cyclopropyl ring can either be oriented above or below the plane of the tricycle, and thus a possible explanation for the moderate CB2R agonist activity of 53 is that one of these conformers can be accommodated upon binding to the receptor.
DIAS2 optimisation: variation at N(5)
Having investigated the effects of varying substituents at C(3) and C(6)–C(9) attention next turned to exploring the effect of introducing a substituent at N(5). It was envisaged that a range of N-substituted derivatives could be readily prepared from the corresponding S-substituted tricycles. Several conditions were trialled, but the optimal route was found to be treatment of triazenoindoles 7, 14 and 20 with NaH (60% dispersion in mineral oil) in DMF followed by the requisite electrophile giving a range of N-alkyl, N-benzyl, N-sulfonyl, N-oxycarbonyl and N-acyl substituted analogues in 19–96% yield (Scheme 6). Three N-acyl substituted examples could not be accessed using this method, and were therefore prepared using alternative conditions by treating the triazenoindole with DMAP and the requisite acyl chloride: this afforded 73 from 20 in 73% yield, and 78–79 from 7 in 55% and 56% yield, respectively (Scheme 6).
The array of N(5)-substituted analogues were evaluated for activity at the CB2R in the CHO cell assay. A much broader range of substituents appeared to be tolerated, with most of the derivatives prepared and tested showing some agonist activity at the CB2R (Table 4). Of the N-alkyl substituted variants, N-methyl derivative 54 showed no detectable activity at the CB2R (EC50 > 3000 nM, Table 4), consistent with the preliminary SAR assessment (Table S2 and Fig. S1). In contrast larger linear N-alkyl groups, N-ethyl 55 and N-(n-butyl) 56, and β-branched N-alkyl groups, N-cyclopropylmethyl 57 and N-cyclohexylmethyl 58, were all tolerated and showed similar activities at the CB2R (55: EC50 = 674 nM; 56: EC50 = 106 nM; 57: EC50 = 144 nM; 58: EC50 = 270 nM, Table 4).
N-Benzyl substituted derivatives 59 and 61 also showed similar levels of activity at the CB2R compared to their unsubstituted counterpart DIAS2 7 (59: EC50 = 214 nM; 61: EC50 = 53 nM; Table 4). Interestingly, N-benzyl S-(2-fluorobenzyl) derivative 60 showed moderate activity at the CB2R in contrast to its N(5)-unsubstituted counterpart 14, which showed no activity up to the highest concentrations tested (60: EC50 = 850 nM, Table 4). In order to confirm the activity of the N-substituted derivatives and determine Ki values, N-benzyl substituted derivatives 59 and 60 and N-(2-nitro)benzyl 61 were selected as representative examples for CB2R and CB1R affinity studies. All were found to have moderate to high binding affinity for the CB2R (59: Ki = 11 nM; 60: Ki = 215 nM; 61: Ki = 290 nM; Table 4), and intriguingly both 59 and 60 also showed similar levels of binding affinity for the CB1R (59: Ki = 61 nM; 60: Ki = 275 nM; Table 4). These binding affinity measurements were supported by subsequent functional activity studies at the CB1R: 59 showed moderate agonist activity in CB1R-expressing CHO cells (EC50 = 1553 nM, Table 4). These results were in complete contrast to the analogues with no substituent at N(5) which showed no affinity or activity at the CB1R up to the highest concentrations tested. Furthermore N-(2-nitro)benzyl 61 showed no affinity or activity at the CB1R up to the highest concentrations tested (Ki, EC50 > 60,000 nM, Table 4), providing additional useful insights into the requirements for CB1R/CB2R selectivity in this series.
N-Sulfonyl and N-oxycarbonyl substituted derivatives were also investigated. N-Phenylsulfonyl derivative 62 showed comparable levels of activity at the CB2R to DIAS2 7 (EC50 = 76 nM, Table 4). However, introducing functional groups to the N-phenylsulfonyl substituent (exemplified by N-4-methylphenyl sulfonyl 63 and N-2-nitrophenyl sulfonyl 64) led to loss of detectable activity up to the highest concentration tested (EC50 > 3000 nM, Table 4). The reasons for this unexpectedly steep SAR are unclear at this time. N-Benzyloxycarbonyl derivative 65 showed no detectable activity at the CB2R (EC50 > 3000 nM; Table 4). Of more concern was the fact that the N-sulfonyl and N-oxycarbonyl derivatives were also found to show limiting solubility, even in the vehicle (DMSO), and therefore neither series were pursued further.
A series of aliphatic N-acyl substituted derivatives were next examined. While N-acetyl analogue 66 showed no detectable activity at the CB2R, all other aliphatic N-acyl substituted analogues tested showed good levels of activity: N-cyclopropylcarbonyl 67, N-cyclopentylcarbonyl 68 and N-cyclohexylcarbonyl 69 all showed comparable activity with DIAS2 7 (67: EC50 = 181 nM; 68: EC50 = 49 nM; 69: EC50 = 32 nM, Table 4). These observations were consistent with the trend in activity for the N-alkyl substituted series—small N-substituents show no or reduced activity at the CB2R. It was also of interest to determine whether polar groups could be accommodated within the N(5) substituent as it was envisaged these would aid with improving other important parameters such as solubility, lipophilicity and potentially metabolism. Thus, homologous esters 70 and 71 were prepared and evaluated for agonist activity at the CB2R: encouragingly both showed good levels of activity (70: EC50 = 48 nM; 71: EC50 = 42 nM, Table 4). N-Cyclopentyl 68, N-cyclohexyl 69 and ester 70 were also selected as representative examples for CB2R/CB1R binding affinity studies. All showed high affinity at the CB2R, consistent with the activity observed in CB2R-expressing CHO cells (68: Ki = 320 nM; 69: Ki = 30 nM; 70: Ki = 360 nM, Table 4), but no detectable activity up to the highest concentrations tested at the CB1R (Ki > 60,000 nM), Table 4).
The introduction of an N-benzoyl group 72 led to comparable levels of activity at the CB2R (EC50 = 113 nM, Table 4) to DIAS2 7 and its N-benzyl substituted counterpart 59. Similarly, N-benzoyl derivative 73 also showed comparable activity at the CB2R (EC50 = 75 nM, Table 4) to its N(5)-unsubstituted counterpart 20. Both N-benzoyl substituted derivatives 72 and 73 were selected for CB2R and CB1R affinity studies: both showed good affinity for the CB2R (72: Ki = 300 nM; 73: Ki = 235 nM; Table 4) and no detectable binding at the CB1R (72: Ki > 10,000 nM; 73: Ki > 60,000 nM, Table 4). Interestingly the lack of CB1R binding observed for N-benzoyl substituted species 72 was in contrast to the data obtained for its N-benzyl substituted counterpart 59 (Ki = 61 nM, Table 4). This suggests that the presence of the carbonyl functionality is not tolerated by the CB1R, possibly as it introduces a polar interaction or preferentially adopts a conformation that cannot be accommodated at the receptor.
Having established that the introduction of a benzoyl substituent at N(5) retained activity and affinity at the CB2R, and selectivity over the CB1R, a number of substituted analogues were next explored. All para- and meta-substituted analogues showed similar levels of activity at the CB2R compared to their N-benzoyl substituted counterpart 72 (74, 4-fluoro: EC50 = 57 nM; 75, m-trifluoromethyl: EC50 = 27 nM; 77, p-trifluoromethoxy: EC50 = 73 nM; 79, m-, p-dimethoxy: EC50 = 160 nM; Table 4), except for 4-dimethylamino substituted derivative 78 which showed no detectable activity at the CB2R (EC50 > 3000 nM, Table 4). It was noted that 78 was extremely poorly soluble under the assay conditions, and this may have led to the lack of observable activity. Introducing a trifluoromethyl group in the ortho site up to the highest concentrations tested (EC50 > 3000 nM, Table 4). Both of the examples investigated had comparatively bulky ortho substituents (A-values: CF3 = 2.1 kcal/mol; NO2 = 1.13 kcal/mol56) and it is therefore possible that the reduced conformational freedom and the preferred conformations cannot be accommodated at the CB2R.
A selection of heterocyclic substituents were next investigated as these were anticipated to confer more favourable physicochemical properties. N-(2-Furfuryl)carbonyl (80), N-(5-isoxazolyl)carbonyl (81) and N-(1-morpholino)carbonyl (82) derivatives were prepared and, as anticipated, all showed good activity at the CB2R (80: EC50 = 46 nM; 81: EC50 = 19 nM; 82: EC50 = 92 nM; Table 4). In subsequent affinity studies, representative examples 80 and 81 also showed good affinity for the CB2R (80: Ki = 490 nM; 81: Ki = 450 nM, Table 4), and no affinity at the CB1R (80: Ki > 10,000 nM; 81: Ki > 60,000 nM, Table 4).
Evaluation of physicochemical properties and metabolic stability of DIAS2 and analogues
An analysis of commonly employed synthetic and semi-synthetic cannabinoid receptor modulators revealed that the majority are highly lipophilic, with high c log P and log D values (HU-308: c log P = 8.0, Table S1). This is perhaps not surprising given the preference of cannabinoid receptors for lipophilic endogenous ligands. Moreover, the majority of cannabinoid receptor modulators to date have been developed and investigated for neuroinflammation and neuropathic pain indications and therefore require good distribution into the brain and CNS, a property often associated with higher than average log D values.57,58 To investigate the effects of CB2R agonists in peripheral pain and inflammation it was of interest to generate compounds with reduced lipophilicity in the hope that this would translate into reduced accumulation in the CNS and an improved pharmacokinetic profile. Given the relative expression patterns of CB2R (predominantly peripheral tissue)1,2 and CB1R (predominantly CNS)3,4 it was also antipated that aiming for a peripherally restricted molecule would lead to improved in vivo efficacy and reduced side-effects.
Values for c log P were therefore calculated for representative active compounds to allow comparisons to be made (Table 5). Relative values for lipophilic ligand efficiency (LLE) were also calculated based on the corresponding EC50 values for each compound. LLE has been shown to be a useful parameter in medicinal chemistry—optimal values for LLE have been shown in many studies to correlate with higher chances of success in the clinic.59 Moreover, a comparison of LLE within a series can give a good indication whether increasing activity may be directly related to increasing lipophilicity—that is, it is likely to be a function of the free energy of solvation and not an increase in the free energy of binding to the receptor.60 While the value for c log P for DIAS1 (6) was determined to be 4.2, c log P for DIAS2 (7) was determined to be 3.2, with a correspondingly lower value for LLE, and was therefore deemed to be a promising candidate for optimisation and development.59c,61 The c log P value for 20 was similar to DIAS2 7 (3.9), giving a proportionally higher value for LLE (3.7) due to its modestly increased activity. Unsurprisingly the c log P value for 9-bromo substituted derivative 38 was higher than DIAS2 (4.6), leading to a correspondingly lower value for LLE (2.8).
It was of particular interest to compare the values for c log P and LLE for a selection of the N-substituted variants. It was observed that a range of groups were tolerated at this site, and it was therefore anticipated that those examples incorporating polar functional groups would give more favourable values for c log P and thus proportionally higher values for LLE. As anticipated, N-alkyl- and N-benzyl substituted derivatives 56–59 all had higher c log P values (4.6–6.2), and correspondingly much lower values for LLE (0.3–2.3). Similarly, N-phenylsulfonyl (62), N-cyclopropylcarbonyl (67) and N-benzoyl (72) had similar or higher values for c log P to DIAS2 (62 = 5.1; 67 = 3.8; 72 = 4.6), and lower values for LLE (62 = 2.1; 67 = 3.0; 72 = 2.4). Conversely, all those derivatives incorporating a polar group within the N(5) substituent had lower c log P values and higher LLE values than DIAS2 (esters 70 and 71: c log P = 3.3 and 3.7, LLE = 4.0 and 3.7 respectively; 1-morpholinocarbonyl 82: c log P = 3.1, LLE = 3.9). Of particular interest was N-(5-isoxazolyl)carbonyl substituted derivative 81, which had a c log P value of 2.6 and, due to its high activity at the CB2R, a high value for LLE (5.2).
To further investigate any relationship between activity (in this case pEC50) and c log P within this hit series, a plot correlating these two parameters for each active compound was constructed (Fig. 4). Supporting the analyses in Table 5, the plot showed no correlation between c log P and activity (R2 < 0.2).
Besides c log P and LLE it was of real interest to optimise physicochemical properties within the series, particularly aqueous solubility. The majority of cannabinoid ligands described in the literature are relatively poorly soluble in aqueous solution, presumably as a consequence of their high lipophilicity, a property that limits their application as chemical tools. It was anticipated that given that polar groups could be accommodated within the N(5) substituent, aqueous solubility could also be tuned by varying this substituent. Thus, the kinetic solubility of a selection of active variants was measured at 37 °C, pH 7.0 (Fig. 5).62
DIAS1 (6) showed low aqueous kinetic solubility (2.0 μM), while DIAS2 (7) showed a slight improvement (3.75 μM, Table 5). A selection of derivatives incorporating N-acyl and/or polar groups within the N(5) substituent were next evaluated. While the introduction of an ester group within the N(5) substituent offered no improvement in aqueous kinetic solubility (70 and 71: 3.75 μM, Table 5), N-benzoyl substituted derivative 72 showed reduced solubility (2.0 μM) compared to its unsubstituted counterpart, DIAS2 (7). However, the heterocycle-containing analogues, N-(5-isoxazolyl)carbonyl 81 and N-(1-morpholino)carbonyl 82 both showed modestly improved solubility compared to DIAS2 (81: 6.5 μM; 82: 10.5 μM, Table 5) demonstrating that through varying the N(5)-substituent solubility could be improved within this series. Adequate solubility is a highly desirable parameter for a range of drug properties, and whilst some improvement was evident, further optimisation is still required.63
As the overall aim was to develop a selective CB2R agonist for evaluation in vivo, it was also of interest to investigate the metabolic stability of representative examples. To give an indication of in vivo metabolic stability, the compounds were incubated in vitro with mouse liver microsomes (MLM) and the concentration monitored by mass spectrometry over time allowing a value for half-life (t1/2) to be determined.64 Diazepam (t1/2 = 5–10 min) and diphenhydramine (t1/2 = 30–40 min) were used as controls. A representative selection of compounds were assessed using this in vitro stability assay: S-benzyl derivatives 7 and 20, C(9)-bromo substituted derivative 38, N-alkyl substituted derivatives 56–58, N-benzyl analogue 59, N-sulfonyl analogue 62, and N-acyl derivatives 67 and 72 (Table 5).
DIAS2 7 showed reasonable metabolic stability in the MLM assay (t1/2 = 46 min): interestingly the S-oxidation product 26 was not detected amongst the metabolites. Replacing the 2-cyanobenzyl with a 2-nitrobenzyl group 20 did not significantly alter the stability (20: t1/2 = 43 min), whereas incorporating a 9-bromo substituent (38) led to a ten-fold reduction in half-life (t1/2 = 5 min) compared to DIAS2 (7). The decreased stability of 38 may be due in part to its increased lipophilicity (c log P = 4.6): in many instances more lipophilic compounds have been shown to be better substrates for microsomal enzymes resulting in increased turnover.65 All examples tested incorporating an N(5) substituent showed a significant reduction in stability, regardless of the nature of the substituent: N-alkyl substituted derivatives 56–58, N-benzyl analogue 59, N-sulfonyl analogue 62, and N-acyl derivative 67 all gave values for t1/2 of less than 5 min. In the case of N-benzoyl derivative 72, no parent compound could be detected at the earliest time point (5 min) precluding the determination of a value for t1/2 and suggesting extremely rapid turnover. Importantly, the compounds were stable in the presence of microsomes in the absence of added cofactor (NADPH), suggesting a microsomal enzyme-mediated degradation and, moreover, in all of the studies carried out on the N(5)-substituted derivatives, DIAS2 (7) was observed as the principal metabolite. This observation suggested that the main origin of metabolic instability of the N(5)-substituted derivatives was the lability of the exocyclic N—C bond, independent of the nature of the linkage.
DIAS2 (<bold>7</bold>) further evaluation
On the basis of the activity, affinity, selectivity, solubility and in vitro metabolic stability analyses DIAS2 (7) and 20 were deemed to possess the most promising overall profiles. The nitrile functionality within 7 was favoured over the nitro group within 20 due to the well-documented possibility of toxicophores presented by the nitro group upon degradation,66 and thus DIAS2 (7) was selected for further evaluation.
It was of interest to investigate absorption properties of DIAS2 (7): ideally it was desired to generate an orally bioavailable compound that would not accumulate within the CNS. To give an indication of cellular permeability and efflux, and in particular an indication of whether significant CNS penetration was likely, Madin–Darby canine kidney cells transfected with multidrug resistance protein 1 (MDCK-MDR1) were used as an in vitro model. This cell line was selected as it can give an indication of p-glycoprotein-mediated efflux, a major pathway through which compounds are effluxed within the gut and at the blood brain barrier (BBB). DIAS2 (7) was determined to have a permeability of 3.21 × 10−6 cm s−1 predicting that the compound would be readily absorbed within the gut. The efflux ratio was determined to be <1 indicating DIAS2 (7) is not a p-glycoprotein substrate. This permeability data also predicts that the compound would be on the boundary of CNS penetration.67
It had already been demonstrated that DIAS2 (7) showed selectivity for the CB2R over the CB1R (Table 1), but it was also of interest to assess whether 7 also interacted with other targets, particularly related GPCRs and also kinases. Thus, 7 was screened for off-target activity against a selection of 24 GPCRs68 (Table S3) and 97 kinases69 (Table S4). Kinases were of interest to measure off target activity as they have been shown to bind polycyclic heteroaromatic structures, and as the cell-based activity screen measures intracellular cAMP levels it could in principle respond to a compound interacting with other cellular components. The GPCR screen used a radioligand displacement assay platform analogous to the affinity measurements at the CB1R and CB2R determined previously. Encouragingly at a concentration of 1 μM, no significant binding was observed for 7 at any of the GPCRs tested (Table S3), nor with any of the kinases when tested at a concentration of 10 μM (Table S4).69
Conclusions
In summary, a novel series of CB2R activating molecules has been identified following a ligand-based screening approach using a known CB2R agonist HU-308 as a template.
Systematical SAR analyses were conducted around the tricyclic core, varying substituents at C(3), C(6)–C(9) and N(5). At C(3), thiobenzyl substituents incorporating small electron withdrawing groups at the ortho site gave the highest levels of activity and affinity at the CB2R while maintaining selectivity over the CB1R. A small substituent was also found to be tolerated at C(9), however this was also found to give rise to modest affinity at the CB1R. A range of substituents were found to be tolerated at N(5), which allowed tuning of the selectivity (e.g., N-benzyl 59: CB1R Ki = 61 nM, CB2R Ki = 11 nM; N-benzoyl 72: CB1R Ki > 10,000 nM, CB2R Ki = 300 nM) and, importantly, other molecular properties. While the introduction of polar groups within the substituent at N(5) was found to improve solubility and reduce lipophilicity, all analogues examined were rapidly metabolised in in vitro mouse liver microsome stability studies: the major metabolite being DIAS2 (7) resulting from cleavage of the exocyclic N(5)—C bond. The use of these analogues as possible pro-drugs to liberate DIAS2 (7) in vivo is currently being explored.
DIAS2 (7) was deemed to possess the most promising overall profile in terms of CB2R activity, affinity, selectivity, lipophilicity and in vitro metabolic stability and was therefore advanced to further in vitro analyses. DIAS2 (7) was showed good in vitro absorption properties and a low efflux ratio. Moreover, no significant off-target activity was observed when DIAS2 (7) was screened against a panel of GPCRs and kinases. Overall we believe these properties make DIAS2 (7) a promising candidate for further investigation and application in vivo to assess the impact of CB2R activity in inflammation (and other pathologies). A detailed study of the formulation, in vivo pharmacokinetic properties, in vitro and in vivo efficacy of DIAS2 (7) in murine models of inflammation will be imminently forthcoming.
ExperimentalGeneral chemical methods
All reactions involving organometallic or moisture-sensitive reagents were carried out under an argon atmosphere using standard vacuum line techniques and glassware that was flame dried and cooled under argon before use. Solvents were dried according to the procedure outlined by Grubbs and co-workers.70 Water was purified by an Elix® UV-10 system. All other solvents and reagents were used as supplied (analytical or HPLC grade) without prior purification. Organic layers were dried over anhydrous MgSO4. Pet ether refers to the fraction of petroleum spirit boiling between 30 and 40 °C. Thin layer chromatography was performed on aluminium plates coated with 60 F254 silica. Plates were visualised using UV light (254 nM) or 1% aq KMnO4. Melting points were recorded either on a Gallenkamp Hot Stage apparatus (Gallenkamp) or a EZ-Melt Automated Melting Point Apparatus (EZ Melt) and are uncorrected. IR spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer with a diamond ATR module. Selected characteristic peaks are reported in cm−1. NMR spectra were recorded on Bruker Avance spectrometers in the deuterated solvent stated. The field was locked by external referencing to the relevant deuteron resonance. Chemical shifts (δ) are reported in ppm and coupling constants (J) are reported in Hz and are unaveraged. Low-resolution mass spectra were recorded on either a VG MassLab 20–250 or a Micromass Platform 1 spectrometer. Accurate mass measurements were run on either a Bruker MicroTOF internally calibrated with polyalanine, or a Micromass GCT instrument fitted with a Scientific Glass Instruments BPX5 column (15 m × 0.25 mm) using amyl acetate as a lock mass, by the mass spectrometry service of the Chemistry Research Laboratory, University of Oxford, UK. HPLC analysis was carried out on a Waters Xterra reverse phase C18 with gradients of water (0.1% TFA) and acetonitrile (0.1% TFA).
General synthesis proceduresGeneral procedure 1: S-substitution<xref rid="b0355" ref-type="bibr"><sup>71</sup></xref>
To a suspension of 5H-[1,2,4]triazino[5,6-b]indole-3-thiol (1 equiv) in methanol (5 mL) was added Et3N (1.5 equiv). To the resulting suspension was added the requisite electrophile (1 equiv) and the reaction mixture stirred at rt for 16 h. The resulting precipitate was filtered and washed with a solution of Et3N in water (1:25, 5% Et3N), which was dried in vacuo to yield the S-substituted product which was purified as specified in each example below.
General procedure 2: N-substitution<xref rid="b0355" ref-type="bibr"><sup>71</sup></xref>
A flask was flame dried and cooled under an atmosphere of argon. To the flask was added the S-substituted-5H-[1,2,4]triazino[5,6-b]indole-3-thiol (1 equiv) and anhydrous DMF or THF (5 mL) under argon. The resulting suspension was cooled to 0 °C using an ice bath. NaH (60% dispersion in mineral oil, 1.1–1.5 equiv) was added, resulting in a clear yellow solution, which was stirred for 15 min at 0 °C. The corresponding electrophile (1.5 equiv) was added gradually (dropwise or portionwise) at 0 °C. After addition was complete, the reaction mixture was allowed to warm to rt and stirred for 16 h under argon. The reaction was quenched with water and the product extracted as specified below.
Chemical synthesis5<italic>H</italic>-[1,2,4]Triazino[5,6-<italic>b</italic>]indole-3-thiol (<bold>9</bold>)<xref rid="b0250" ref-type="bibr"><sup>50</sup></xref>
To a stirred suspension of isatin 8 (5.00 g, 33.2 mmol) and K2CO3 (7.05 g, 51.0 mmol) in water (100 mL), was added thiosemicarbazide (3.09 g, 33.2 mmol). The mixture was heated under reflux for 16 h. Upon cooling the solution was acidified with glacial acetic acid to afford a precipitate, which was filtered and washed with a water/acetic acid (24:1, v/v) mixture. The resulting solid was triturated with hot DMF, filtered and dried to yield the product (9) as a yellow solid (6.25 g, 92%); mp (Gallenkamp) >300 °C [lit.50 mp >300 °C]; δH (400 MHz, DMSO-d6, 363 K) 7.34 (1H, app t, J = 7.6 Hz, C(8)H), 7.44 (1H, d, J = 8.1 Hz, C(6)H), 7.62 (1H, app t, J = 7.6 Hz, C(7)H), 8.00 (1H, d, J = 7.6 Hz, C(9)H); δC (100 Hz, DMSO-d6) 113.9, 118.6, 122.7, 123.9, 132.7, 136.5, 144.0, 150.0, 179.9; m/z (ESI−) 201 ([M−H]−, 100%).
Ethyl 2′-(((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzoate 24 (100 mg, 0.27 mmol), was added to a solution of aq NaOH (1 M, 270 μL, 0.27 mmol) in H2O (5 mL) and stirred at rt for 16 h. The solution was acidified with aq HCl (1 M) until a precipitate was formed, which was filtered and dried in vacuo to yield the final product (25) (92 mg, 99%); mp (Gallenkamp) >300 °C; νmax (solid) 3241 (v br), 1712, 1553, 1378, 1166, 1090; HPLC (Method 2) >99%, tR = 10.11 min; δH (500 MHz, DMSO-d6, 363 K) 4.99 (2H, s, S-CH2), 7.12–7.22 (3H, m, C(7)H, C(8)H and C(4′)H), 7.47–7.52 (3H, m, C(6)H, C(5′)H and C(6′)H), 7.68–7.72 (1H, m, C(3′)H), 8.15 (1H, d, J = 7.6 Hz, C(9)H); δC (125 MHz, DMSO-d6) 32.1, 114.9, 119.1, 119.7, 120.7, 126.0, 127.4, 129.2, 129.5, 129.6, 130.0, 137.2, 141.1, 142.5, 150.4, 166.4, 171.6; m/z (ESI−) 335 ([M−H]−, 100%); HRMS (ESI−) C17H11N4O2S− ([M−H]−) requires 335.0608; found 335.0612.
7-Bromoisatin 27 (1.00 g, 4.45 mmol), thiosemicarbazide (405 mg, 4.45 mmol) and K2CO3 (920 mg, 6.67 mmol) were suspended in water (50 mL). The reaction mixture was stirred at reflux for 16 h over which time the dark brown suspension became a clear light brown solution. The solution was carefully acidified by dropwise addition of acetic acid and the resulting precipitate was filtered. The precipitate was recrystallised from DMF to yield the title compound (31) as a red solid (416 mg, 34%); mp (Gallenkamp) >300 °C; νmax (solid) 2849 (br), 1629, 1603, 1581, 1386, 1151; δH (500 MHz, DMSO-d6, 363 K) 7.28 (1H, app t, J = 7.6 Hz, C(8)H), 7.84 (1H, d J = 7.6 Hz, C(7)H), 8.01 (1H, d, J = 7.6 Hz, C(9)H); δC (125 MHz, DMSO-d6) 116.1, 117.5, 123.8, 124.9, 126.3, 135.5, 144.6, 149.9, 176.6; m/z (ESI−) 279 ([79BrM−H]−, 100%), 281 ([81BrM−H]−, 100%); HRMS (ESI−) C9H5BrN4NaS ([79BrM−H]−) requires: 278.9335; found 278.9344, ([81BrM−H]−) requires 280.9314; found 280.9321.
6-Bromoisatin 28 (1.00 g, 4.45 mmol), thiosemicarbazide (405 mg, 4.45 mmol) and K2CO3 (920 mg, 6.67 mmol) were suspended in water (50 mL). The reaction mixture was stirred at reflux for 16 h over which time the dark brown suspension became a clear light brown solution. The solution was carefully acidified by dropwise addition of acetic acid and resulting precipitate was filtered. The precipitate was recrystallised from DMF to yield the title compound (32) as a red solid (1.12 g, 90%); mp (Gallenkamp) >300 °C; νmax (solid) 2919 (br), 1589, 1422, 1359, 1161; δH (500 MHz, DMSO-d6, 363 K) 7.50 (1H, d, J = 7.5 Hz, C(8)H), 7.60 (1H, s, C(6)H), 7.94 (1H, 1H, d, J = 7.5 Hz, C(9)H); δC (125 MHz, DMSO-d6) 105.5, 120.3, 121.3, 124.8, 134.6, 136.1, 142.4, 150.2, 179.8; m/z (ESI−) 279 ([79BrM−H]−, 100%), 281 ([81BrM−H]−, 100%); HRMS (ESI−) C9H5BrN4NaS ([79BrM−H]−) requires: 278.9335; found 278.9343, ([81BrM−H]−) requires 280.9314; found 280.9321.
5-Bromoisatin 29 (1.00 g, 4.45 mmol), thiosemicarbazide (405 mg, 4.45 mmol) and K2CO3 (920 mg, 6.67 mmol) were suspended in water (50 mL). The reaction mixture was stirred at reflux for 16 h over which time the dark brown suspension became a clear light brown solution. The solution was carefully acidified by dropwise addition of acetic acid and resulting precipitate was filtered. The precipitate was recrystallised from DMF to yield the title compound (33) as a red solid (1.23 g, 97%); mp (Gallenkamp) >300 °C; νmax (solid) 3097, 2858, 1603, 1589, 1446, 1311; δH (500 MHz, DMSO-d6, 363 K) 1.90 (1H, br s, NH), 7.38 (1H, d, J = 8.6 Hz, C(6)H), 7.74 (1H, dd, J = 8.6, 1.8 Hz, C(7)H), 8.15, (1H, d, J = 1.8 Hz, C(9)H); δC (125 MHz, DMSO-d6) 115.6, 115.9, 120.8, 125.0, 134.9, 135.8, 143.5, 150.4, 180.1; m/z (ESI−) 279 ([79BrM−H]−, 100%), 281 ([81BrM−H]−, 100%); HRMS (ESI−) C9H5BrN4NaS ([79BrM−H]−) 278.9335; found 278.9343, ([81BrM−H]−) requires 280.9314; found 280.9320.
4-Bromoisatin 30 (1.00 g, 4.45 mmol), thiosemicarbazide (405 mg, 4.45 mmol) and K2CO3 (920 mg, 6.67 mmol) were suspended in water (50 mL). The reaction mixture was stirred at reflux for 16 h over which time the dark brown suspension became a clear light brown solution. The solution was carefully acidified by dropwise addition of acetic acid and resulting precipitate was filtered. The precipitate was recrystallised from DMF to yield the title compound (34) as a red solid (1.18 g, 95%); mp (Gallenkamp) >300 °C; νmax (solid) 3389 (br), 3297, 1604, 1573, 1140; δH (500 MHz, DMSO-d6, 363 K) 7.43–7.52 (3H, m, C(6)H, C(7)H), and C(8)H), 14.68 (1H, br s, NH); δC (125 MHz, DMSO-d6) 113.0, 117.0, 118.3, 127.4, 133.5, 136.1, 145.3, 149.9, 180.0; m/z (ESI−) 279 ([79BrM−H]−, 100%), 281 ([81BrM−H]−, 100%); HRMS (ESI−) C9H5BrN4S ([79BrM−H]−) requires: 278.9335; found 278.9343, ([81BrM−H]−) requires 280.9314; found 280.9320.
4-Bromoisatin 30 (2.00 g, 8.85 mmol) was added to a suspension of NaH (60% dispersion in mineral oil, 550 mg, 8.85 mmol) in DMF (20 mL) at 0 °C. The resulting purple solution was stirred at 0 °C for 10 min before dropwise addition of p-methoxybenzyl chloride (1.12 mL, 8.23 mmol). The reaction mixture was stirred for 30 min at 0 °C, heated to 40 °C for 3 h, cooled down and quenched with water. The solution was extracted with EtOAc (3 × 30 mL), the combined organic phases were washed with brine, dried (Na2SO4), filtered and concentrated in vacuo to give the crude compound as an orange solid. Recrystallisation in EtOAc/petrol afforded the isatin 40 as an orange powder (2.10 g, 6.09); δH (CDCl3, 400 MHz) 3.80 (3H, s, O-CH3), 4.88 (2H,s, N-CH2), 6.76 (1H, d, J = 7.8 Hz, C(7)H), 6.88 (d, J = 8.3 Hz, 2H, phenyl ArH), 7.22 (d, J = 8.1 Hz, 1H, C(5)H), 7.26 (d, J = 8.3 Hz, 2H, phenyl ArH), 7.30 (t, J = 8.1 Hz, 1H, C(6)H); LRMS m/z (ESI+) 347 [M+H]+, 715 [2M+Na]+.
To a solution of 4-bromoisatin 30 (100 mg, 0.44 mmol) in degassed THF/water (3:1, 2.5 mL) was added potassium 2-methylphenyltrifluoroborate (114 mg, 0.62 mmol) and K3PO4 (338 mg, 1.59 mmol) followed by Pd(PPh3)2Cl2 (31 mg, 0.04 mmol). The reaction vessel was sealed and heated by microwave irradiation for 4 h at 130 °C. The reaction mixture was cooled to rt, diluted with EtOAc (5 mL) and filtered through Celite®. The organic solution was washed with brine (5 mL) and the resulting aqueous layer was further extracted with EtOAc (2×). The combined organic layers were dried (MgSO4), filtered and concentrated in vacuo to give the crude product. The product was purified via flash column chromatography (eluent 40–60 °C, petrol/EtOAc, 4:1) to afford the title compound (41) as an orange solid (40 mg, 42%); mp 161–162 °C; νmax (solid) 3249, 3017, 2926, 1727, 1603, 1480; δH (500 MHz, DMSO-d6) 2.15 (3H, s, CH3), 6.93 (2H, m, C(5)H and C(7)H), 7.14 (1H, d, J = 7.6 Hz, C(6′)H), 7.20–7.26 (1H, m, C(4′)H), 7.30 (1H, d, J = 7.3 Hz, C(3′)H), 7.32–7.37 (1H, m, C(5′)H), 7.56 (1H, app t, J = 7.9 Hz C(6)H), 8.84 (1H, br s, NH); δC (125 MHz, DMSO-d6) 19.7, 111.2, 115.7, 125.6, 126.3, 128.6, 128.8, 130.1, 135.7, 136.4, 137.9, 142.8, 149.3, 159.2, 182.0; m/z (ESI−) 236 ([M−H]−, 60%); HRMS (ESI−) C15H10NO2− ([M−H]−) requires 236.0717; found 236.0719.
To a solution of 4-bromoisatin 30 (100 mg, 0.44 mmol) in degassed THF/H2O (3:1, 3 mL) was added potassium 4-methyphenyltrifluoroborate (123 mg, 0.62 mmol) and K3PO4 (338 mg, 1.59 mmol) followed by Pd(PPh3)2Cl2 (31 mg, 0.04 mmol). The reaction vessel was sealed and heated by microwave irradiation for 4 h at 130 °C. The reaction mixture was cooled to rt, diluted with EtOAc (5 mL) and filtered through Celite®. The organic solution was washed with brine (5 mL) and the resulting aqueous layer was further extracted with EtOAc (2×). The combined organic layers were dried (MgSO4), filtered and concentrated in vacuo to give the crude product. The product was purified via flash column chromatography (eluent 30–40 °C petrol/acetone, 4:1) to afford the title compound (42) as an orange solid (72 mg, 68%); mp 178–179 °C; νmax (solid) 3274, 3051, 2906, 1741, 1609, 1486; δH (400 MHz, CDCl3) 2.43 (3H, s, CH3), 6.88 (1H, d, J = 8.0 Hz, C(7)H), 7.08 (1H, d, J = 8.0 Hz, C(5)H), 7.28 (2H, d, J = 8.5 Hz, C(3′)H and C(5′)H), 7.45 (2H, d, J = 8.5 Hz, C(6′)H and C(2′)H), 7.55 (1H, app t, J = 8.0 Hz, C(6)H), 8.70 (1H, br s, NH); δC (125 MHz, CDCl3) 21.4, 110.8, 114.4, 125.9, 128.8, 129.0, 133.1, 138.0, 139.4, 143.8, 154.2, 159.2, 178.3; m/z (ESI−) 236 ([M−H]−, 100%); HRMS (ESI−) C15H10NO2S− ([M−H]−) requires 236.0717; found 236.0716.
To a solution of 4-bromoisatin 30 (200 mg, 0.88 mmol) in THF/water (3:1, 3 mL) was added potassium 3-thienyltrifluoroborate (235 mg, 1.24 mmol) and K3PO4 (675 mg, 3.19 mmol) followed by Pd(PPh3)2Cl2 (62 mg, 0.09 mmol). The reaction vessel was sealed and heated by microwave irradiation for 4 h at 130 °C. The reaction mixture was cooled to rt, diluted with EtOAc (10 mL) and filtered through Celite®. The organic solution was washed with brine (10 mL) and the resulting aqueous layer was further extracted with EtOAc (2×). The combined organic layers were dried (MgSO4), filtered and concentrated in vacuo to give the crude product. The product was purified via flash column chromatography (eluent 30–40 °C petrol/acetone, 4:1) to afford the title compound (43) as an orange solid (120 mg, 59%); mp 213–216 °C; νmax (solid) 3098, 2927, 1727, 1584; δH (500 MHz, DMSO-d6) 6.85 (1H, dd, J = 7.9, 0.6 Hz, C(7)H), 7.20 (1H, dd, J = 7.9, 0.6 Hz, C(5)H), 7.50 (1H, dd, J = 4.9, 1.3 Hz, C(4′)H), 7.58 (1H, app t, J = 7.9 Hz, C(6)H), 7.62 (1H, dd, J = 4.9, 2.9 Hz, C(5′)H), 8.07 (1H, dd, J = 2.9, 1.3 Hz, C(2′)H), 11.13 (1H, br s, NH); δC (125 MHz, DMSO-d6) 110.8, 113.9, 123.8, 125.6, 126.3, 128.4, 135.8, 137.2, 138.0, 151.6, 158.9, 183.0; m/z (ESI−) 228 ([M−H]−, 100%); HRMS (ESI−) C12H6N4O2S− ([M−H]−) requires 228.0125; found 228.0127.
Following General procedure 2, 2′(((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was added to DMF (5 mL) and cooled to 0 °C. NaH (60% dispersion in mineral oil, 13.8 mg, 0.34 mmol) was added to the cooled solution and stirred at 0 °C for 10 min. Ethyl iodide (38 μL, 0.47 mmol) was added and reaction mixture was allowed to warm to rt and stirred for 16 h. The reaction mixture was quenched by dropwise addition of water until a precipitate was formed, this was collected by filtration and dried to yield the title compound (55) as a white powder (85 mg, 78%); mp (EZ Melt) 187–189 °C; νmax (solid) 2923, 2853, 2225, 1570, 1335, 1169; HPLC (Method 2) >97%, tR = 11.31 min; δH (500 MHz, DMSO-d6, 363 K) 1.34 (3H, t, J = 7.3 Hz, CH3), 4.45 (2H, q, J = 7.3 Hz, N-CH2), 4.77 (2H, s, S-CH2), 7.45–7.48 (1H, m, C(8)H), 7.49 (1H, app t, J = 7.6 Hz, C(5′)H), 7.67 (1H, app td, J = 7.6, 1.3 Hz, C(4′)H), 7.78 (1H, app t, J = 7.6 Hz, C(7)H), 7.82–7.88 (3H, m, C(6)H, C(3′)H and C(6′)H), 8.34 (1H, d, J = 7.6 Hz, C(9)H); δC (125 MHz, DMSO-d6) 13.4, 32.4, 36.0, 111.2, 112.0, 117.4, 117.6, 121.7, 122.9, 128.1, 130.2, 131.1, 133.0, 133.3, 140.5, 141.1, 141.6, 145.5, 165.9; m/z (ESI+) 368 ([M+Na]+, 100%); HRMS (ESI+) C19H15N5NaS ([M+Na]+) requires 368.0940; found 368.0936.
Following General procedure 2, 2′-(((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was added to DMF (5 mL), cooled to 0 °C followed by the addition of NaH (60% dispersion in mineral oil, 13.8 mg, 0.35 mmol) and stirred at 0 °C for 10 min. Bromomethylcyclopropane (31 μL, 0.32 mmol) was added and the resulting mixture stirred at rt for 16 h. The reaction mixture was quenched with water and resulting precipitate was filtered and washed with methanol to yield the title compound (57) as a pale yellow solid (62 mg, 52%); mp (Gallenkamp) 149–150 °C; νmax (solid) 3030, 2923, 2251, 1740; HPLC (Method 2) >99%, tR = 1.66 min; δH (500 MHz, DMSO-d6, 363 K) 0.37–0.50 (4H, m, C(2″)H2 and C(3″)H2), 1.19–1.33 (1H, m, C(1″)H), 4.32 (1H, d, J = 6.9 Hz, N-CH2), 4.73 (2H, s, S-CH2), 7.48 (2H, m, C(8)H and C(4′)H), 7.67 (1H, app t, J = 7.8 Hz, C(7)H), 7.77 (1H, app t, J = 7.6 Hz, C(5′)H), 7.8 (1H, d, J = 7.8 Hz, C(6)H), 7.81–7.99 (2H, m, C(3′)H and C(6′)H), 8.34 (1H, d, J = 7.8 Hz, C(9)H); δC (125 MHz, DMSO-d6) 3.8, 10.2, 32.4, 45.2, 111.6, 111.9, 117.3, 117.6, 121.6, 122.9, 128.1, 130.0, 131.0, 133.0, 133.4, 140.9, 141.0, 141.5, 145.8, 166.0; m/z (ESI+) 394 ([M+Na]+, 100%); HRMS (ESI+) C20H20N5NaS ([M+Na]+) requires 394.1094; found 394.1095.
Following General procedure 2, to 3-((2′-cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6-b]indole 7 (50 mg, 0.15 mmol) in THF at 0 °C, was added NaH (60% dispersion in mineral oil, 10 mg, 0.24 mmol) followed by benzyl bromide (25 μL, 0.24 mmol) and the resulting mixture stirred at rt for 16 h. The reaction mixture was quenched with water (30 mL) and CH2Cl2 (30 mL) added. The organic layer was separated and washed with brine (30 mL), dried (MgSO4), filtered and concentrated in vacuo to yield the title compound (59) as a light yellow solid (49 mg, 80%); mp (Gallenkamp) 197–200 °C; νmax (solid) 2215, 1571, 1520, 1468, 1328; HPLC (Method 2) >98%, tR = 11.96 min; δH (500 MHz, DMSO-d6, 363 K) 4.77 (2H, s, S-CH2), 5.68 (2H, s, N-CH2), 7.23–7.33 (5H, m, C(2″)H, C(3″)H, C(4″), C(5″)H and C(6″)H), 7.42–7.45 (1H, m, C(8)H), 7.47–7.51 (1H, m, C(4′)H), 7.53–7.56 (1H, m, C(7)H), 7.69–7.74 (2H, m, C(3′)H and C(6′)H), 7.75–7.81 (2H, m, C(6)H and C(5′)H), 8.37 (1H, d, J = 7.9 Hz, C(9)H); δC (125 MHz, DMSO-d6) 32.4, 44.2, 111.7, 111.9, 117.5, 117.6, 119.3, 121.7, 123.2, 127.3, 127.7, 128.1, 128.8, 130.2, 133.1, 133.2, 135.8, 140.7, 141.2, 141.5, 146.2, 166.2; m/z (ESI+) 430 ([M+Na]+, 100%); HRMS (ESI+) C24H17N5NaS ([M+H]+) requires 430.1097; found 430.1081.
Following General procedure 2, to 2′(((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (60 mg, 0.19 mmol) in THF (5 mL) was added NaH (60% dispersion in mineral oil, 11 mg, 0.28 mmol) followed by 2-nitro benzyl bromide (40 mg, 0.21 mmol) at 0 °C and the resulting mixture stirred for 16 h at rt. The reaction mixture was quenched with water until a precipitate formed. The precipitate was filtered and washed with methanol to give the title compound (61) as an off white solid (79 mg, 96%); mp (Gallenkamp) 225–226 °C (dec); νmax (solid) 3061, 2804, 2226, 1580, 1340, 1175; HPLC (Method 2) >96%, tR = 11.72 min; δH (500 MHz, pyr-d5, 363 K) 4.65 (2H, s, S-CH2), 6.02 (2H, s, N-CH2), 6.71 (1H, d, J = 7.1 Hz, C(6″)H), 7.42–7.72 (7H, m, 7× Ar-H), 7.81–7.89 (2H, m, C(3″ and C(6)H), 8.31 (1H, d, J = 7.8 Hz, C(3′)H), 8.40 (1H, d, J = 7.6 Hz, C(9)H); δC (125 MHz, pyr-d5) 35.0, 45.2, 112.9, 115.0, 119.3, 119.9, 120.7, 124.0, 126.7, 129.3, 129.8, 130.4, 132.2, 133.0, 134.8, 134.8, 135.4, 141.1, 142.7, 143.7, 143.9, 148.9, 155.1, 169.2; m/z (ESI+) 452 ([M+H]+, 10%), m/z (ESI−) 316 ([M−(2″−NO2Bn)H]−, 100%); HRMS (ESI+) C24H16N6NaO2S ([M−H]−) requires 475.0948; found 475.954.
Following General procedure 2, to 2′(((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) in DMF (5 mL) was added NaH (60% dispersion in mineral oil, 13.8 mg, 0.35 mmol) followed by cyclohexane carbonyl chloride (63 μL, 0.47 mmol) at 0 °C and the resulting mixture stirred for 16 h at rt. The reaction mixture was quenched with water until a precipitate formed. The precipitate was filtered and dried to give the title compound (69) as a pale yellow solid (98 mg, 71%); mp (Gallenkamp) 194–197 °C; νmax (solid) 2932, 2854, 2226, 1720, 1453, 1379; HPLC (Method 2) >95%, tR = 12.67 min; δH (500 MHz, DMSO-d6, 363 K) 1.18–1.38 (4H, m, C(3″)H2 and C(5″)H2), 1.45–1.55 (2H, m, C(4″)H2), 1.75–2.07 (4H, m, C(2″)H2 and C(6″)H2), 4.04–4.12 (1H, m, C(1″)H), 4.84 (2H, s, S-CH2), 7.53 (1H, app td, J = 7.6, 1.1 Hz, C(8)H), 7.60–7.65 (1H, m, C(4′)H or C(6′)H), 7.71 (1H, app td, J = 7.6, 1.1 Hz, C(7)H), 7.77–7.85 (2H, m, C(3′)H and C(4′)H or C(6′)H), 7.89–7.93 (1H, m, C(5′)H), 8.38 (1H, d, J = 6.9 Hz, C(6)H), 8.59 (1H, d, J = 8.5 Hz, C(9)H); δC (125 MHz, DMSO-d6) 25.1, 25.3, 28.5, 32.8, 44.7, 112.0, 112.8, 117.4, 117.5, 119.6, 121.1, 125.7, 128.5, 130.4, 132.1, 133.6, 139.6, 140.3, 142.6, 147.0, 166.5, 176.2; m/z (ESI+) 450 ([M+Na]+, 100%); HRMS (ESI+) C24H21N5NaOS ([M+Na]+) requires 450.1364; found 450.1362.
Following General procedure 2, 2′(((5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio)methyl)benzonitrile 7 (100 mg, 0.32 mmol) was added to DMF (5 mL), cooled to 0 °C followed by the addition of NaH (60% dispersion in mineral oil, 13.8 mg, 0.34 mmol) and stirred at 0 °C for 10 min. 1-Morpholine carbonyl chloride (47 μL, 0.32 mmol) was added, stirred for 16 h, cooled in an ice bath and quenched slowly with dropwise addition of water. The resulting precipitate was filtered and dried to yield the title compound (82) as a white solid (98 mg, 72%); mp (EZ Melt) 191–193 °C; νmax (solid) 2928, 2867, 2223, 1688, 1570, 1389; HPLC (Method 2) >99%, tR = 10.86 min; δH (500 MHz, DMSO-d6, 363 K) 3.40–3.79 (8H, m, 8× CH), 4.78 (2H, s, S-CH2), 7.50 (1H, app td, J = 7.8, 1.3 Hz, C(5′)H), 7.57 (1H, app t, J = 7.6 Hz, C(8)H), 7.68 (1H, app td, J = 7.8, 1.3 Hz, C(4′)H), 7.76 (1H, d, J = 7.8 Hz, C(3′)H), 7.79 (1H, app t, J = 7.6 Hz, C(7)H), 7.83 (1H, d, J = 7.6 Hz, C(6)H), 7.89 (1H, dd, J = 7.8, 1.3 Hz, C(6′)H), 8.37 (1H, d, J = 7.6 Hz, C(9)H); δC (125 MHz, DMSO-d6) 32.7, 44.7, 66.1, 112.0, 113.9, 117.5, 118.5, 121.6, 124.3, 128.3, 130.4, 131.5, 133.2, 133.3, 139.4, 140.9, 141.8, 145.7, 148.4, 166.3; m/z (ESI+) 453 ([M+Na]+, 100%); HRMS (ESI+) C22H18N6NaO2S ([M+Na]+) requires 453.1104; found 453.1087.
Pharmacology1hCB<sub>2</sub>R and hCB<sub>1</sub>R cAMP assay
The cAMP Hunter eXpress GPCR assay kit and cells used are from DiscoveRx Corporation (DiscoveRx Corporation, Birmingham, UK) and used according to the manufacturer’s protocol. The CB2R agonist 4-[4-(1,1-dimethylheptyl)-2,6-dimethoxyphenyl]-6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-methanol (HU-308 from Tocris Bioscience, UK) and the CB1R agonist N-(2-Chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA from Tocris Bioscience, UK) were used as positive controls in the hCB2R and hCB1R assays, respectively. Briefly, hCB2R or hCB1R CHO-K1 cells were plated in the provided medium (15,000 cells per well) in half-area 96-well plates (Corning, UK) and placed overnight in an incubator 37 °C 5% CO2. From this stage onwards, the assays for CB1R and CB2R CHO-K1 cells were identical. Next day, the medium was aspirated and replaced with a 1:3 solution of anti-cAMP antibody in provided buffer. In the presence of 20 μM forskolin, freshly reconstituted compounds or solvent alone (final concentration 0.2% DMSO) were added to the wells and incubated for 30 min at 37 °C, 5% CO2. After the incubation, a mixture of provided lysis solution and substrates was added to the wells and incubated in the dark for one hour at room temperature. The provided detection reagent EA was then added to the wells and incubated in the dark for 3 h at room temperature. The luminescence was then read using a standard luminometer (Perkin–Elmer, Seer Green, UK). The luminescence of the wells incubated only with forskolin and solvent (control wells) were normalised as 100% and the dose–response curves were expressed as a percentage of the control wells. The EC50 values shown are from pooling 3–4 independent experiments and were identified using GraphPad prism non-linear regression curve fit (4 PL parameters).
Radioligand binding assays: hCB<sub>1</sub>R and hCB<sub>2</sub>R
Radioligand binding assays were contracted to CEREP (Celle l’Evescault, France) where membranes from CHO-cells expressing either hCB1R or hCB2R were used to assess binding to the two cannabinoid receptors. Competition binding studies were performed by incubating membranes expressing hCB1R or hCB2R with unlabeled compounds and tritiated CP 55,940 (0.5 nM) or WIN-55212 (0.8 nM) respectively, for two hours at 37 °C. For CB1R and CB2R, non-specific binding was assessed using WIN-55212 at 10 and 5 μM, respectively. Two hours later, the binding of tritiated ligands was assessed by scintillation counting. The competition binding data for each compound are expressed as the percentage of the remaining bound tritiated ligand after the compound’s incubation with the membrane. Ki values (equilibrium dissociation constants for each compound) were calculated from CEREP using the Cheng–Prusoff equation and the Kd value (equilibrium dissociation constant for each radioligand).
In vitro molecular propertiesSolubility studies
The aqueous solubility assay was performed by Cyprotex using a high throughput turbidimetric assay. Initially, a stock DMSO solution was diluted in DMSO to produce a range of concentrations. These were then added to phosphate buffered saline at pH 7.4 (final test compound concentrations = 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, final DMSO concentration = 1%, 7 replicates per concentration) and incubated for 2 h at 37 °C. At the end of the incubation period, the absorbance at 620 nM was read for each concentration and each replicate. The assay provided a precipitation range, where the mid-point was used as the estimated precipitation concentration and recorded in μM.
Microsomal stability studies
The microsomal stability assay was performed by Cyprotex plc. The microsomes used were derived from mouse liver and were incubated with the test compound at 37 °C in the presence of added co-factor, NADPH. The reaction was terminated by the addition of methanol containing an internal standard. Following centrifugation, the supernatant was analysed by LC–MS/MS. The disappearance of test compound was monitored at 0, 5, 15 30, 45 min time points. Diazepam and diphenhydramine were used as the positive controls. Compound stability in the absence of added co-factor was measured at the 45 min time point.
Cellular permeability studies
Cell permeability studies were carried out by Cyprotex using MDR1-transfected (MDR1-MDCK) cells derived from Madin Darby canine kidney (MDCK) cells. The cells were plated on a Multiscreen plate (Millipore, MA, USA) and formed a confluent monolayer over a period of 4 days before the experiment. On day 4, the test compounds were applied to the apical side of the membrane and the transport of the compound across the monolayer was monitored over a 60 min time period. The efflux ratio of the desired compound was measured by determining transport of the compound from the basolateral area to the apical compartment.
Molecular modelling
Energy minimisations were performed for 51, 52 and 53 using Maestro molecular modelling software (version 9.0 Schrodinger). The calculations were performed ignoring solvent interactions, and the force field used was MM2. In each case an evaluation of the model was performed by a Ramachandran plot. The selection of most the plausible structural pose(s) was done by observation of the energy minimization score results.
References and notesGaliegueS.MaryS.MarchandJ.DussossoyD.CarriereD.CarayonP.BouaboulaM.ShireD.Le FurG.CasellasP.Eur. J. Biochem.2321995547556170MunroS.ThomasK.L.Abu-ShaarM.Nature3651993617689702NúñezE.BenitoC.PazosM.R.BarbachanoA.FajardoO.GonzálezS.TolónR.M.RomeroJ.Synapse53200420815266552Van SickleM.D.DuncanM.KingsleyP.J.MouihateA.UrbaniP.MackieK.StellaN.MakriyannisA.PiomelliD.DavisonJ.S.MarnettL.J.Di MarzoV.PittmanQ.J.PatelK.D.SharkeyK.A.Science310200532916224028AnandU.OttoW.R.Sanchez-HerreraD.FacerP.YiangouY.KorchevY.BirchR.BenhamC.BountraC.ChessellI.P.AnandP.Pain138200866718692962WotherspoonG.FoxA.McIntyreP.ColleyS.BevanS.WinterJ.Neuroscience135200523516084654WhitesideG.T.LeeG.P.ValenzanoK.J.Curr. Med. Chem.14200791717430144For a recent review see:BeltranoM.Mini Rev. Med. Chem.920091119149657SlipetzD.M.O’NeillG.P.FavreauL.DufresneC.GallantM.GareauY.GuayD.LabelleM.MettersK.M.Mol. Pharmacol.4819953527651369BouaboulaM.Poinot-ChazelC.MarchandJ.CanatX.BourriéB.Rinaldi-CarmonaM.CalandraB.Le FurG.CasellasP.Eur. J. Biochem.23719967048647116HanS.ThalteJ.JonesR.M.Ann. Rep. Med. Chem.442009227HanS.ThatteJ.BuzardD.J.JonesR.M.J. Med. Chem.562013822423865723GaoniY.MechoulamR.J. Am. Chem. Soc.8619641646for a recent review see:BursteinS.H.Bioorg. Med. Chem.222014283024731541Di MarzoV.De PetrocellisL.Annu. Rev. Med.57200655316409166BermanJ.S.SymondsC.BirchR.Pain112200429915561385WadeD.T.MakelaP.RobsonP.HouseH.BatemanC.Mult. Scler.10200443415327042For a recent reviews see: (a)WormK.DolleR.E.Curr. Pharm. Des.152009334519860684PatelK.D.DavisonJ.S.PittmanQ.J.SharkeyK.A.Curr. Med. Chem.1720101394TourteauA.Leleu-ChavainN.Body-MalapelM.AndrzejakV.BarczykA.DjouinaM.RigoB.DesreumauxP.ChavatteP.MilletR.Bioorg. Med. Chem. Lett.242014132224508127WeissmanA.MilneG.M.MelvinL.S.J. Pharmacol. Exp. Ther.22319825166290642HanušL.BreuerA.TchilibonS.ShiloahS.GoldenbergD.HorowitzM.PertweeR.G.RossR.A.MechoulamR.FrideE.Proc. Natl. Acad. Sci. U.S.A.9619991422810588688XieX.Q.ChenJ.Z.BillingsE.M.Proteins-Struct. Funct. Genet.53200330714517981SaloO.M.H.RaitioK.H.SavinainenJ.R.NevalainenT.Lahtela-KakkonenM.LaitinenJ.T.T.JärvinenT.PosoA.J. Med. Chem.482005716616279774RenaultN.LaurentX.FarceA.El BakaliJ.MansouriR.GervoisP.MilletR.DesreumauxP.FurmanC.ChavatteP.Chem. Biol. Drug Des.81201344223217060MarktP.FeldmannC.RollingerJ.M.RadunerS.SchusterD.KirchmairJ.DistintoS.SpitzerG.M.WolberG.LaggnerC.AltmannK.-H.LangerT.GertschJ.J. Med. Chem.52200936919143566HollinsheadS.P.TidwellM.W.PalmerJ.GuidettiR.SandersonA.JohnsonM.P.ChambersM.G.OskinsJ.StratfordR.AstlesP.C.J. Med. Chem.562013572223795771Rinaldi-CarmonaM.BarthF.MillanJ.DerocqJ.-M.CasellasP.CongyC.OustricD.SarranM.BouaboulaM.CalandraB.PortierM.ShireD.BrelièreJ.-C.FurG.L.J. Pharmacol. Exp. Ther.28419986449454810HosohataY.QuockR.M.HosohataK.MakriyannisA.ConsroeP.RoeskeW.R.YamamuraH.I.Eur. J. Pharmacol.3211997R19083796GiblinG.M.P.O’ShaughnessyC.T.NaylorA.MitchellW.L.EathertonA.J.SlingsbyB.P.RawlingsD.A.GoldsmithP.BrownA.J.HaslamC.P.ClaytonN.M.WilsonA.W.ChessellI.P.WittingtonA.R.GreenR.J. Med. Chem.502007259717477516OstenfeldT.PriceJ.AlbaneseM.BullmanJ.GuillardF.MeyerI.LeesonR.CostantinC.ZivianiL.NociniP.F.MilleriS.Clin. J. Pain27201166821540741OfekO.KarsakM.LeclercN.FogelM.FrenkelB.WrightK.TamJ.Attar-NamdarM.KramV.ShohamiE.MechoulamR.ZimmerA.BabI.Proc. Natl. Acad. Sci. U.S.A.103200669616407142PalazuelosJ.DavoustN.JulienB.HattererE.AguadoT.MechoulamR.BenitoC.RomeroJ.SilvaA.GuzmanM.NatafS.Galve-RoperhI.J. Biol. Chem.28320081332018334483For recent reviews on CB2R as a target for inflammatory and immune-related diseases see: (a)Leleu-ChavainN.Body-MalapelM.SpencerJ.ChavatteP.DesreumauxP.MilletR.Curr. Med. Chem.122012345722709008Leleu-ChavainN.DesreumauxP.ChavatteP.MilletR.Curr. Mol. Pharmacol.6201318324720538KarsakM.GaffalE.DateR.Wang-EckhardtL.RehneltJ.PetrosinoS.StarowiczK.SteuderR.SchlickerE.CravattB.MechoulamR.BuettnerR.WernerS.Di MarzoV.TutingT.ZimmerA.Science3162007149417556587KimballE.S.SchneiderC.R.WallaceN.H.HornbyP.J.Am. J. Physiol. Gastrointest. Liver Physiol.2912006G36416574988TourteauA.AndrzejakV.Body-MalapelM.LemaireL.LemoineA.MansouriR.DjouinaM.RenaultN.El BakaliJ.DesreumauxP.MuccioliG.G.LambertD.M.ChavatteP.RigoB.Leleu-ChavainaN.MilletR.Bioorg. Med. Chem.212013538323849204SteffensS.VeillardN.R.ArnaudC.PelliG.BurgerF.StaubC.KarsakM.ZimmerA.FrossardJ.L.MachF.Nature434200578215815632ZurierR.B.RossettiR.G.LaneJ.H.GoldbergJ.M.HunterS.A.BursteinS.H.Arthritis Rheum.4119981639433882ZurierR.B.RossettiR.G.BursteinS.H.BidingerB.Biochem. Pharmacol.65200364912566094TepperM.A.ZurierR.B.BursteinS.H.Bioorg. Med. Chem.222014324524856183MontecuccoF.BurgerF.MachF.SteffensS.Am. J. Physiol. Heart Circ. Physiol.2942008H114518178718RabornE.Marciano-CabralF.BuckleyN.MartinB.CabralG.J. Neuroimmune Pharmacol.3200811718247131SacerdoteP.MassiP.PaneraiA.E.ParolaroD.J. Neuroimmunol.109200015510996217MontecuccoF.Di MarzoV.da SilvaR.F.VuilleumierN.CapettiniL.LengletS.PaganoS.PiscitelliF.QuintaoS.BertolottoM.PelliP.GalanK.PiletL.KuzmanovicK.BurgerF.PaneB.SpinellaG.BraunersreutherV.Gayet-AgeronA.PendeA.VivianiG.L.PalomboD.DallegriF.Roux-LombardP.RobsonA.S.SantosR.A.S.StergiopulosN.SteffensS.MachF.Eur. Heart J.33201284622112961DiL.KernsE.H.Curr. Opin. Chem. Biol.7200340212826129(a) Openeye Scientific. OMEGA; OpenEye Scientific Software: Santa Fe, NM, http://www.eyesopen.com/omegaHawkinsP.C.SkillmanA.G.WarrenG.L.EllingsonB.A.StahlM.T.J. Chem. Inf. Model.50201057258420235588HawkinsP.C.NichollsA.J. Chem. Inf. Model.5220122919293623082786WestwoodI.M.KawamuraA.RussellA.J.SandyJ.DaviesS.G.SimE.Comb. Chem. High Throughput Screening142011117LaurieriN.CrawfordM.H.J.KawamuraA.WestwoodI.M.RobinsonJ.FletcherA.M.DaviesS.G.SimE.RussellA.J.J. Am. Chem. Soc.1322010323820170182LufinoM.M.P.SilvaA.M.NémethA.H.Alegre-AbarrateguiJ.RussellA.J.Wade-MartinsR.Hum. Mol. Genet.222013517323943791Openeye Scientific. ROCS; OpenEye Scientific Software: Santa Fe, NM, http://www.eyesopen.com/rocsCheeserightT.MackeyM.RoseS.VinterA.Expert Opin. Drug Discov.22007131144Forge v10; http://www.cresset-group.com/products/forge/23496041Grant, J. A.; Pickup, B. T. Comp Simulation of Biomolecular Systems. Springer Science and Business Media: Dordrecht, 1997; Vol 3.GrantJ.A.GallardoM.A.PickupB.T.J. Comput. Chem.1719961654http://www.discoverx.com/target-data-sheets/gpcr/cnr2.RamV.J.Arch. Pharm.3131980108El AshryE.S.H.RamadanE.S.HamidH.M.A.HagarM.Synlett2004723TomchinA.B.UryupovO.Y.ZhukovaT.I.KuznetsovaT.A.KostychevaM.V.SmirnovA.V.Pharm. Chem. J.311997125HunterC.A.Angew. Chem. Int. Ed.4320045310Rinaldi-CarmonaM.CalandraB.ShireD.BouaboulaM.OustricD.BarthF.CasellasP.FerraraP.Le FurG.J. Pharmacol. Exp. Ther.27819968718768742Adams, C. M.; Chamoin, S.; Hu, Q.-Y.; Zhang, C. WO 2010/130794.MolanderG.A.CanturkB.Angew. Chem. Int. Ed.12120099404SchneiderH.J.HoppenV.J. Org. Chem.4319783866HillA.P.YoungR.J.Drug Discovery Today15201064820570751LeesonP.SpringthorpeB.Nat. Rev. Drug Disc.62007881ShultzM.Bioorg. Med. Chem. Lett.232013598024018190TarcsayA.KeserűG.M.J. Med. Chem.562013178923356819WaringM.J.Expert Opin. Drug Discov.5201023522823020ArnottJ.A.PlaneyS.L.Expert Opin. Drug Discov.7201286322992175Abad-ZapateroC.BlasiD.Mol. Inform.302011122ShultzM.Bioorg. Med. Chem. Lett.232013599224054120LipinskiC.A.LombardoF.DominyB.W.FeeneyP.J.Adv. Drug Delivery Rev.2319973TeagueS.J.DavisA.M.LeesonP.D.OpreaT.Angew. Chem. Int. Ed.3819993743Cyprotex; Vol. http://www.cyprotex.com/admepk/physicochemical-properties/turbidimetric-solubility/.KernsE.H.DiL.CarterG.T.Curr. Drug Metab.9200887918991584Cyprotex; Vol. http://www.cyprotex.com/admepk/in-vitro-metabolism/microsomal-stability/.McLureJ.MinersJ.BirkettD.Br. J. Clin. Pharmacol.49200045310792203PurohitV.BasuA.K.Chem. Res. Toxicol.13200067310956054Cyprotex Table 1, Classification of brain uptake using Cyprotex’s MDR1-MDCK Permeability. http://www.cyprotex.com/admepk/in-vitro-permeability/mdr1-mdck-permeability/.http://www.cerep.fr/Cerep/Users/pages/productsservices/GPCRPlatform.asp.FabianM.A.BiggsW.H.IIITreiberD.K.AtteridgeC.E.AzimioaraM.D.BenedettiM.G.CarterT.A.CiceriP.EdeenP.T.FloydM.FordJ.M.GalvinM.GerlachJ.L.GrotzfeldR.M.HerrgardS.InskoD.E.InskoM.A.LaiA.G.LéliasJ.-M.MehtaS.A.MilanovZ.V.VelascoA.M.WodickaL.M.PatelH.K.ZarrinkarP.P.LockhartD.J.Nat. Biotechnol.23200532915711537KaramanM.W.HerrgardS.TreiberD.K.GallantP.AtteridgeC.E.CampbellB.T.ChanK.W.CiceriP.DavisM.I.EdeenP.T.FaraoniR.FloydM.HuntJ.P.LockhartD.J.MilanovZ.V.MorrisonM.J.PallaresG.PatelK.K.PritchardS.WodickaL.M.ZarrinkarP.P.Nat. Biotechnol.26200812718183025(c) http://www.discoverx.com/services/drug-discovery-development-services/kinase-profiling/kinomescan.PangbornA.B.GiardelloM.A.GrubbsR.H.RosenR.K.TimmersF.J.Organometallics1519961518GladychJ.M.Z.HornbyR.HuntJ.H.JackD.J. Med. Chem.1519722772814333824TrostB.M.MalhotraS.ChanW.H.J. Am. Chem. Soc.1332011732821520958VineK.L.LockeJ.M.RansonM.PyneS.G.BremnerJ.B.J. Med. Chem.502007510917887662Supplementary data
Supplementary Table S1
Structures and properties of selected reported CB2R agonists. a Determined using rat CBR. b Determined using human CBR. acalculated using the c Log P algorithm in ChemDraw v13.0.
Supplementary Table S2
Examples of results displayed by ROCS with the top 16 ranked compounds by their combo scores calculated by combining the shape tanimoto with the colour score. Molecule ranked at 13 was selected for further investigations. Activities are shown as a percentage residual activity of cAMP production vs control.
Supplementary data 3
Supplementary figures and tables.
Acknowledgments
We thank Dr. Raman Parkesh for technical advice in setting up the virtual screen. We also thank the British Heart Foundation for the provision of a studentship (I.C., FS/08/067) and a programme Grant (D.R.G., RG/10/15/28578), the British Heart Foundation Centre of Research Excellence at the University of Oxford for an award (A.J.R. and D.R.G.), Research Councils’ UK for a Fellowship (A.J.R.).
Structures of cannabinoid receptor agonists, Δ9-tetrahydrocannabinol (Δ9-THC, 1), CP 55,940 (2), GW-842166X (3) and Lilly’s recently disclosed CB2R agonist (4).
(A) Structure and properties of HU-308 (5); (B) 3-D representation of the lowest energy conformer of HU-308 (5) using Omega (OpenEye);42 (C) overlay of HU-308 (5) and DIAS1 (6) using Forge (Cresset);45 (D) structure and properties of DIAS1 (6) and DIAS2 (7). a Calculated using the c log P algorithm in ChemBioDraw 14.0.
3-D representations of calculated lowest energy conformers of 51, 52, and 53. Calculations were performed using Maestro (Schrodinger).
pEC50 and c log P correlation of triazinoindole series.
DIAS 2 (7) and its key pharmacological, physicochemical and in vitro ADME properties. a Calculated using ChemDraw v13.0. b Determined using MDCK-MDR1 cells. c <30% activity when tested at 10 μM against a panel of 12 GPCRs (see Supporting information). d <30% activity when tested at 10 μM against a panel of 96 kinases (see Tables S3 and S4).
Reagents and conditions: (i) thiosemicarbazide, K2CO3, H2O, reflux, 16 h; (ii) butyl iodide or 2-cyanobenzyl bromide, NEt3, MeOH, rt, 16 h.
Reagents and conditions: (i) alkyl halide, Et3N, MeOH, rt, 16 h; (ii) benzyl halide, Et3N, MeOH, rt, 16 h; (iv) NaOH (aq), rt, 16 h.
Reagents and conditions: (i) CF3CO3H, CF3CO2H, rt, 16 h.
Reagents and conditions: (i) thiosemicarbazide, K2CO3, H2O, rt, 16 h; (ii) benzyl halide, Et3N, MeOH, rt, 16 h.
Reagents and conditions: (i) NaH, 4-methoxybenzyl chloride, DMF, 0 °C, 30 min then 40 °C, 3 h; (ii) Potassium aryl trifluoroborate, K3PO4, Pd(PPh3)2Cl2, THF/H2O (3:1), 130 °C, MW, 4 h; (iii) Potassium cyclopropyl trifluoroborate, K3PO4, Pd(dppf)2Cl2, THF/H2O (3:1), 130 °C, MW, 4 h; (iv) thiosemicarbazide, K2CO3, H2O, reflux, 16 h; (v) 2-cyano benzyl bromide, Et3N, MeOH, rt, 16 h; (vi) CF3CO2H, 90 °C, 16 h.
Reagents and conditions: (i) electrophile, NaH (60% dispersion in mineral oil), DMF or THF, 0 °C–rt, 16 h; or DMAP, CH2Cl2, rt, 16 h.
Variation of sulfide substituent and effects on CBR activity and affinity. EC50 determined using CHO cells expressing hCB2R/hCB1R. Ki determined using radioligand displacement assay using either [3H]WIN-55212-2 (CB2R) or [3H]CP 55,940 (CB1R). All assays were performed in duplicate
Entry
ID
R1
R2
Yield from 9 (%)
EC50 (CB2R) (nM)
EC50 (CB1R) (nM)
Ki (CB2R) (nM)
Ki (CB1R) (nM)
1
10
Me
—
95
>3000
N.D.
N.D.
N.D.
2
6 (DIAS1)
n-Bu
—
80
296
N.D.
N.D.
N.D.
3
11
c-Hexylmethyl
—
67
>3000
N.D.
N.D.
N.D.
4
12
—
Ph
75
10,000
>30,000
N.D.
N.D.
5
7 (DIAS2)
—
2-CN–C6H4–
Quant.
112
>30,000
355
>60,000
6
13
—
3-CN–C6H4–
58
6450
>30,000
N.D.
N.D.
7
14
—
2-F–C6H4–
57
>3000
N.D.
N.D.
N.D.
8
15
—
4-F–C6H4–
82
>3000
N.D.
N.D.
N.D.
9
16
—
2,6-F2–C6H3–
54
>3000
N.D.
N.D.
N.D.
10
17
—
2-Cl–C6H4–
79
>3000
N.D.
N.D.
N.D.
11
18
—
3-Cl–C6H4–
73
>3000
N.D.
N.D.
N.D.
12
19
—
4-Cl–C6H4–
59
>3000
N.D.
N.D.
N.D.
13
20
—
2-NO2–C6H4–
98
33
>30,000
155
>60,000
14
21
—
4-NO2–C6H4–
62
617
N.D.
N.D.
N.D.
15
22
—
2-CF3–C6H4–
74
>3000
N.D.
N.D.
N.D.
16
23
—
2-OCF3–C6H4–
50
>3000
N.D.
N.D.
N.D.
17
24
2-CO2Et–C6H4–
83
>3000
N.D.
N.D.
N.D.
18
25
—
2-CO2H–C6H4–
99
2185
N.D.
N.D.
N.D.
N.D. = not determined.
Variation of the substitution pattern around the triazinoindole core and effects on CBR activity and affinity. EC50 determined using CHO cells expressing hCB2R/hCB1R. Ki determined using radioligand displacement assay using either [3H]WIN-55212-2 (CB2R) or [3H]CP 55,940 (CB1R). All assays were performed in duplicate
Entry
ID
EC50 (CB2R) (nM)
EC50 (CB1R) (nM)
Ki (CB2R) (nM)
Ki (CB1R) (nM)
1
7
112
>30,000
355
>60,000
2
35
>3000
N.D.
N.D.
N.D.
3
36
>3000
N.D.
N.D.
N.D.
4
37
>3000
N.D.
N.D.
N.D.
5
38
39
≈20,000
22.5
3200
6
39
37
N.D.
N.D.
N.D.
Variation of substituents at C9 of triazinoindole core and effects on CB2R activity and affinity. EC50 determined using CHO cells expressing hCB2R. All assays were performed in duplicate
Entry
ID
R
Yield (step (i) or (ii), %)
Yield (steps (iii)–(iv), %)
EC50 (CB2R) (nM)
1
7
H
—
—
112
2
50
o-MeC6H4–
42
55
4300
3
51
p-MeC6H4–
68
17
>3000
4
52
3-Thienyl
59
50
1760
5
53
Cyclopropyl
43
44
231
Variation of substituents at N5 of triazolyl-indole core and effects on CBR activity and affinity. EC50 determined using CHO cells expressing hCB2R/hCB1R. Ki determined using radioligand displacement assay using either [3H]WIN-55212-2 (CB2R) or [3H]CP 55,940 (CB1R). All assays were performed in duplicate
Entry
ID
R1
R3
Yield (%)
EC50 (CB2R) (nM)
EC50 (CB1R) (nM)
Ki (CB2R) (nM)
Ki (CB1R) (nM)
1
7
2-CN–C6H4CH2–
H
N/A
112
>30,000
355
>60,000
2
14
2-F–C6H4CH2–
H
N/A
>3000
N.D.
N.D.
N.D.
3
20
2-NO2–C6H4CH2–
H
N/A
33
>30,000
155
>60,000
4
54
2-CN–C6H4CH2–
Me
69
>3000
N.D.
N.D.
N.D.
5
55
2-CN–C6H4CH2–
Et
78
674
N.D.
N.D.
N.D.
6
56
2-CN–C6H4CH2–
n-Bu
68
106
N.D.
N.D.
N.D.
7
57
2-CN–C6H4CH2–
Cyclopropylmethyl
52
144
N.D.
N.D.
N.D.
8
58
2-CN–C6H4CH2–
Cyclohexylmethyl
19
270
N.D.
N.D.
N.D.
9
59
2-CN–C6H4CH2–
Benzyl
80
214
1553
11
61
10
60
2-F–C6H4CH2–
Benzyl
40
850
N.D.
215
275
11
61
2-CN–C6H4CH2–
2-NO2–C6H4–CH2–
96
53
>30,000
290
>60,000
12
62
2-CN–C6H4CH2–
PhSO2–
84
76
N.D.
N.D.
N.D.
13
63
2-CN–C6H4CH2–
4-Me–C6H4SO2–
92
>3000
N.D.
N.D.
N.D.
14
64
2-CN–C6H4CH2–
2-NO2–C6H4SO2–
83
>3000
N.D.
N.D.
N.D.
15
65
2-CN–C6H4CH2–
BnOCO–
96
>3000
N.D.
N.D.
N.D.
16
66
2-CN–C6H4CH2–
MeCO–
77
>3000
N.D.
N.D.
N.D.
17
67
2-CN–C6H4CH2–
Cyclopropylcarbonyl
76
181
N.D.
N.D.
N.D.
18
68
2-CN–C6H4CH2–
Cyclopentylcarbonyl
42
49
N.D.
320
>60,000
19
69
2-CN–C6H4CH2–
Cyclohexylcarbonyl
71
32
N.D.
30
>60,000
20
70
2-CN-C6H4CH2-
MeCO2CH2CH2CO–
81
48
N.D.
360
>60,000
21
71
2-CN–C6H4CH2–
MeCO2CH2CH2CH2CO–
81
42
N.D.
N.D.
N.D.
22
72
2-CN–C6H4CH2–
PhCO–
72
113
N.D.
300
>10,000
23
73
2-NO2–C6H4CH2–
PhCO–
73
75
N.D.
235
>60,000
24
74
2-CN–C6H4CH2–
4-F–C6H4CO–
67
57
N.D.
N.D.
N.D.
25
75
2-CN–C6H4CH2–
3-CF3–C6H4CO–
58
27
N.D.
N.D.
N.D.
26
76
2-CN–C6H4CH2–
2-CF3–4-F–C6H3CO–
42
>3000
N.D.
N.D.
N.D.
27
77
2-CN–C6H4CH2–
4-(CF3O)–C6H4CO–
67
73
N.D.
380
>60,000
28
78
2-CN–C6H4CH2–
4-NMe2–C6H4CO–
55
>3000
N.D.
N.D.
N.D.
29
79
2-CN–C6H4CH2–
3,4-(MeO)2–C6H3CO–
56
160
N.D.
N.D.
N.D.
30
80
2-CN–C6H4CH2–
2-Furfurylcarbonyl
78
46
N.D.
490
>10,000
31
81
2-CN–C6H4CH2–
5-Isoxazolylcarbonyl
85
19
N.D.
450
>60,000
32
82
2-CN–C6H4CH2–
1-Morpholinocarbonyl
72
92
N.D.
N.D.
N.D.
N.D. = not determined.
Variation of substituents at N(5) of triazinoindole core and effects on CB2R binding activity and affinity, solubility and mouse liver microsome metabolic stability
Entry
ID
R1
R2
R3
EC50 (CB2R) (nM)
c log Pa
LLE
Solubilityb (μM)
MLM stability t1/2 (min)
1
6
n-Bu
H
H
296
4.2
2.3
2.0
N.D.
2
7
2-CN–C2H4CH2–
H
H
112
3.8
3.2
3.75
46
3
20
2-NO2–C6H4CH2–
H
H
33
3.9
3.7
N.D.
43
4
38
2-CN–C2H4CH2–
Br
H
39
4.6
2.8
N.D.
5
5
56
2-CN–C2H4CH2–
H
n-Bu
106
5.2
1.5
N.D.
<5
6
57
2-CN–C2H4CH2–
H
Cyclopropylmethyl
144
4.6
2.3
N.D.
<5
7
58
2-CN–C2H4CH2–
H
Cyclohexylmethyl
270
6.2
0.3
N.D.
<5
8
59
2-CN–C2H4CH2–
H
Benzyl
214
5.1
1.6
N.D.
<5
9
62
2-CN–C2H4CH2–
H
PhSO2–
76
5.1
2.1
N.D.
<5
10
67
2-CN–C2H4CH2–
H
Cyclopropylcarbonyl
181
3.8
3.0
N.D.
<5
11
70
2-CN–C2H4CH2–
H
MeCO2CH2CH2CO–
48
3.3
4.0
3.75
N.D.
13
71
2-CN–C2H4CH2–
H
MeCO2CH2CH2CH2CO–
42
3.7
3.7
3.75
N.D.
14
72
2-CN–C2H4CH2–
H
PhCO–
113
4.6
2.4
2.0
—⁎
15
81
2-CN–C2H4CH2–
H
5-Isoxylcarbonyl
19
2.6
5.2
6.5
N.D.
16
82
2-CN–C2H4CH2–
H
1-Morpholinocarbonyl
92
3.1
3.9
10.5
N.D.
N.D. = not determined.
Calculated using the c log P algorithm in ChemBioDraw 14.0.
The value determined is the mid-point between the upper and lower solubility limits.
Not observable even at the earliest time point, suggesting stability is very low and t1/2 was not calculable.