Abstract
Dopamine-derived N6-substituents, compared to N6-(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A3 adenosine receptor (AR) affinity/selectivity, e.g. C2-phenylethynyl analogue 15 (MRS7591, Ki 10.9/17.8 nM, at human/mouse A3AR). 15 was a partial agonist in vitro (hA3AR, cAMP inhibition, 31% Emax; mA3AR, [35S]GTP-γ-S binding, 16% Emax) and in vivo, and also antagonized hA3AR in vitro. Distal H-bonding substitutions of the N6-(2-phenylethyl) moiety particularly enhanced mA3AR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mA3AR and hA3AR homology models. These hybrid models were based on an inactive antagonist-bound hA1AR structure for the upper part of TM2 and an agonist-bound hA2AAR structure for the remaining TM portions. These species-independent A3AR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the A3AR, a drug target of growing interest.
Keywords: adenosine receptor, G protein-coupled receptor, antagonist, homology modeling, structure activity relationship
Graphical Abstract
Introduction
The A3 adenosine receptor (AR) is a target for the treatment of inflammatory diseases, cerebral and cardiac ischemia, cancer and chronic neuropathic pain (agonists),1–5 as well as glaucoma and kidney disease (antagonists).6–8 Numerous selective A3AR antagonists, comprising both truncated nucleoside derivatives and diverse heterocyclic derivatives, have been reported.9 However, many of these antagonists suffer from a lack of general A3AR selectivity across species, and insufficient concern for the species differences can lead to misinterpretation of pharmacological data. For example, [1,2,4]triazolo[1,5-c]quinazoline derivative 1 (Chart 1) is selective for the human (h) A3AR, but not for the mouse (m) or rat (r) A3AR; in fact, 1 is ~10-fold selective for mA2AAR compared to mA3AR.10 For heterocycles such as 1 and its congeners, the ratio of A3AR affinity between primate and rodent species could reach or exceed 1000-fold.1,7,10 Previous studies have shown that nucleoside antagonists, such as the rigid spirolactam 2, tend to preserve A3AR selectivity across species. In addition, certain heterocyclic antagonists, among which are 1,3-diacyl-6-phenylpyridines (e.g. 3), 4-phenyl-5-pyridyl-1,3-thiazoles and a 2-amino-4,5-diaryl-1,3-oxazole derivative 4, were reported to be selective at the rA3AR in addition to hA3AR.10–12
Chart 1.
Various A3AR antagonists and their pharmacological properties in receptor binding and activation.7,11,15,20–22 Both non-nucleosides (1, 3, 4) and nucleosides (2, 5 – 8) are included. Species: h, human; r, rat; m, mouse; c, canine. % Emax (in inhibition of forskolin-stimulated cAMP production) in hA3AR-expressing CHO cells is reported relative to 37 as 100%; % Emax (in inhibition of forskolin-stimulated cAMP production) in mA3AR-expressing HEK293 cells is reported relative to 38 as 100%.14,15,17,22,23
Nucleoside derivatives are a general scaffold that often achieves drug-like physicochemical and pharmacokinetic properties.13 One means of reducing the functional efficacy of nucleoside derivatives as A3AR agonists is the truncation of the 5′ substituent.14–17 The loss or steric constraint of an otherwise flexible, polar 5′ substituent (typically, CH2OH or CONHCH3) impairs the ability of the nucleoside to stabilize its binding to an active A3AR conformation, and the efficacy can be sufficiently reduced such that the compound resembles pharmacologically an antagonist.14 The ribose moiety of nucleoside ligands engage transmembrane helical domains (TMs) 3, 6 and 7, which are involved in inducing AR activation,18,19 and truncation of the ribose functionality can affect these receptor conformational changes. Thus, 4′-truncated nucleosides 5 – 8 have all been reported as A3AR antagonists or low efficacy partial agonists that demonstrate this phenomenon.7,8,14–17
Variations of the ribose-like moiety upon which truncation has been shown to produce antagonists or partial agonists, include 4′-thioribose and (N)-methanocarba (bicyclo[3.1.0]hexyl), as well as the native-like ribose.1,10 In some cases, a residual low efficacy is detectable in functional assays, which also depends on the species, cell type, receptor density and the parameter measured in a signaling cascade.20,21 For example, truncated (N)-methanocarba derivative 6 lacked significant agonism in a functional assay of guanine nucleotide ([35S]GTPγS) binding in hA3AR-overexpressing mammalian (CHO) cells with only 1±3% maximal efficacy (Emax) of 5′-N-ethylcarboxamidoadenosine (37, NECA), but it reached 44% of the Emax of 37 in inhibition of cyclic AMP formation in the same cells.20 4′-Truncated nucleosides that behave as partial A3AR agonists in cell assays can act as A3AR antagonists in whole tissues or in vivo in rodents.7 An example of this generalization to living systems is in the reduction of intraocular pressure with topical application of nucleoside-related A3AR antagonist 2.24 We previously reported truncated (N)-methanocarba N6-2-phenylethyl-2-phenylethynyl derivative 8, with 14% Emax compared to 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine (38, Cl-IB-MECA) in mA3AR-mediated inhibition of forskolin-stimulated cyclic AMP production in HEK-293 cells.22 However, 8 achieved only moderate mA3AR affinity (480 nM). Here, we have elaborated on the observation that the extension of a N6-methyl to a N6-2-phenylethyl moiety, e.g. 7 vs 8, increases the mA3AR affinity. We expand the range of N6 and C2 substitutions, among which N6 substituents derived from dopamine increased affinity and selectivity at both m and hA3ARs.
Results and Discussion
Selection of target structures and chemical synthesis
We lacked a truncated methanocarba nucleoside that bound to mA3AR with both high affinity and selectivity. N6-2-Phenylethyl substitution in truncated nucleoside 8 was noted to enhance the mA3AR affinity of truncated (N)-methanocarba nucleosides compared to other N6 groups,22 and we probed its structure activity relationship (SAR) to further boost affinity in this species and at the hA3AR. Our initial aim was to examine whether functional group substitution of the N6-2-phenylethyl substituent of 8, which extends into the extracellular region of the receptor (see computational modeling described below), could further enhance its mA3AR affinity, without diminishing its hA3AR affinity. The AR affinity of ring-substituted N6-2-phenylethyl derivatives has been explored mainly in the ribose series.25,26 AR sequence analysis and receptor homology models indicated that the mA3AR extracellular region contained a large number of polar residues, suggesting that introducing polar interactions with the distal N6 substituent might be beneficial. Therefore, novel truncated (N)-methanocarba nucleosides containing various substitutions, particularly with polar N6-2-phenylethyl groups were synthesized (Table 1, 9 – 32). Also, substitutions of the C2-arylalkynyl group were included based on our previous study of full hA3AR agonists.27,28 The synthetic route (Scheme 1; Supporting Information) to these 4′-truncated (N)-methanocarba nucleosides closely followed previously reported synthesis.16,18,29 The N6 substituent was first installed by microwave-assisted nucleophilic displacement of 6-chloro of intermediate 41, followed by a Sonogshira reaction of an aryl acetylene with the 2-iodo group. Acidic deprotection of 55 – 70 provided the final nucleoside products in high yield.
Table 1.
Receptor binding affinity (or % inhibition at 10 μM) of truncated (N)-methanocarba nucleosides, including reference compounds 8and 22.
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Compound | R1 = | R2 = | A1AR Binding Ki, nM (species),a or % at 10 μM | hA2AAR Binding Ki, nM,a or % at 10 μM | A3AR Binding Ki, nM (species),a or % at 10 μM |
8b | Ph | Ph-(CH2)2 | 30±8% (h) | 22±5% | 20±6 (h), 480±90 (m) |
9 | Ph | 3-F-Ph-(CH2)2 | 2680±420 (h) | 27±6% | 35.9±3.3 (h), 264±32 (m) |
10 | Ph | 4-OH-Ph-(CH2)2 | 29±2% (h) | 32±11% | 45.6±24.6 (h), 76.4±3.9 (m) |
11 | Ph | 3-F-4-OH-Ph-(CH2)2 | 44±1% (h) | 17±8% | 9.03±1.60 (h), 61.6±4.2 (m) |
12 | Ph | 4-H2N-Ph-(CH2)2 | 30±3% (h) | 26±9% | 14.2±1.0 (h), 60.4±7.7 (m) |
13 | Ph | 3,4-di-OH-Ph-(CH2)2 | 28±6% (h), 1420±100 (m) | 27±7% | 48.8±22.2 (h), 39.8±5.4 (m) |
14 | Ph | 3-OH-4-OCH3-Ph-(CH2)2 | 49±8% (h) | 27±10% | 6.85±3.02 (h), 122±23 (m) |
15 | Ph | 3-OCH3-4-OH-Ph-(CH2)2 | 33±5% (h), 590±80 (m) | 39±11% | 10.9±4.4 (h), 17.8±2.3 (m) |
16 | Ph | 3,4-di-OCH3-Ph-(CH2)2 | 208±25 (h) | 28±13% | 16.3±2.8 (h), 331±54 (m) |
17 | 2-F-Ph | 3-OCH3-4-OH-Ph-(CH2)2 | 186±31 (h), 620±78 (m) | 5850±841 | 5.42±1.06 (h), 21.5±1.9 (m) |
18 | 3-F-Ph | 3-OCH3-4-OH-Ph-(CH2)2 | 38±9% (h) | 51±2% | 4.63±1.20 (h), 72.2±4.4 (m) |
19 | 4-F-Ph | 3-OCH3-4-OH-Ph-(CH2)2 | 39±5% (h) | 51±2% | 6.51±1.74 (h), 54.4±3.3 (m) |
20 | 3,4-F2-Ph | 3-OCH3-4-OH-Ph-(CH2)2 | 341±36 (h) | 33±3% | 12.8±1.1 (h), 75.0±7.4 (m) |
21 | 3-pyridyl | 3-OCH3-4-OH-Ph-(CH2)2 | 36±5% (h) | 39±3% | 6.70±2.12 (h), 155±10 (m) |
22b | 2-Cl-Ph | Ph-(CH2)2 | 37±7% (h) | 26±10% | 37.0±7.0 (h), 980±74 (m) |
23 | 5-Cl-thienyl | Ph-(CH2)2 | 10±3% (h) | 46±9% | 8.17±1.94 (h), 995±19 (m) |
24 | 5-Cl-thienyl | 3-F-Ph-(CH2)2 | 31±3% (h) | 55±3% | 42.2±12.4 (h), 488±20 (m) |
25 | 5-F-thienyl | 3-F-Ph-(CH2)2 | 2290±90 (h) | 48±40% | 55.8±15.7 (h), 404±55 (m) |
26 | 5-Cl-thienyl | 3-Cl-Ph-(CH2)2 | 39±3% (h) | 59±4% | 58.4±3.1 (h), 531±10 (m) |
27 | 5-Cl-thienyl | 3-CH3-Ph-(CH2)2 | 29±4% (h) | 50±0% | 106±10 (h), 675±34 (m) |
28 | 5-Cl-thienyl | 3,4-F2-Ph-(CH2)2 | 28±10% (h) | 57±1% | 47.1±10.6 (h), 2520±260 (m) |
29 | 5-Cl-thienyl | 3-OCH3-4-OH-Ph-(CH2)2 | 649±146 (h) | 38±0% | 9.52±0.50 (h), 74.7±9.8 (m) |
30 | 5-Br-thienyl | 3-OCH3-4-OH-Ph-(CH2)2 | 15±3% (h), 2840±170 (m) | 38±2% | 3.24±0.14 (h), 26.0±1.7 (m) |
31 | 5-Cl-thienyl | Ph-cPr (1S,2R) | 32±6% (h) | 44±0% | 80.5±1.8 (h), 1140±70 (m) |
32 | - | 3-OCH3-4-OH-Ph-(CH2)2 | 50±11% (h) | 49±3% | 9.1±1.5 (h), 92.5±18.3 (m) |
Binding in membranes of HEK293 cells stably expressing mA1, mA3, hA1, hA2A or hA3, unless noted.28 The binding affinity for hA1, hA2A and A3ARs was expressed as Ki values using agonists [3H]N6-R-phenylisopropyladenosine ([3H]R-PIA, 0.5 nM) 33, [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine ([3H]CGS21680, 5 nM) 34, or [125I]N6-(4-amino-3-iodobenzyl)-adenosine-5′-N-methyluronamide ([125I]I-AB-MECA, 0.1 nM) 35, respectively. A percent in italics refers to inhibition of specific radioligand binding at 10 μM. Nonspecific binding was determined using N-(2-aminoethyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide (XAC) 36 (10 μM). The binding affinity at mARs was determined as reported.23 Values are expressed as the mean ± SEM (n = 3, unless noted). Ki values were calculated as reported.31 The HEK293 cell lines were from American Type Culture Collection (ATCC, Manassas, VA), and the cDNA for the ARs was obtained from cdna.org.
ND, not determined.
Scheme 1.
Synthesis of truncated (N)-methanocarba nucleosides as A3AR partial agonists. Reagents and conditions: (i) R2NH2, DIPEA, 2-propanol, microwave, 65–84%; (ii) R1-alkynes, PdCl2(Ph3P)2, CuI, Et3N, DMF, 72–75%; (iii) 10% TFA, MeOH, 70 °C, 92% (32) or 54–87% for two steps (9 – 31).
Binding and functional data
Most compounds synthesized displayed high hA3AR affinity (Ki ~3 – 10 nM for 11, 14, 15, 17 – 19, 21, 23, 29, 30, and 32), but variable mA3AR affinity and generally low A1AR/A2AAR affinity. Affinity at mA3AR varied from weak with non-polar substitution (e.g. 9, 27 and 28) to strong with a 4-hydroxy (e.g. 10), combined with a 3-methoxy or 3-hydroxy group, or 4-amino (e.g. 12) group.
The effects on A3AR affinity of terminal aryl ring substitution of the N6 group were first probed. 3-Fluoro substitution of a phenyl ring in 9 did not improve the A3AR affinity, but 4-hydroxy substitution in 10 increased the mA3AR affinity 6-fold. Combination of 3-fluoro and 4-hydroxy substitution in 11 further increased the hA3AR affinity of 8 by 2-fold. 4-Amino substitution in 12 resulted in similar affinity to doubly substituted 11 (both Ki ~60 nM at mA3AR), while dopamine adduct 13 lost hA3AR affinity, but gained modestly at the mA3AR (Ki 40 nM). O-Methylation of the dopamine moiety of 13 had variable effects: 4-MeO (14) and 3,4-di-MeO (16) increased hA3AR and decreased mA3AR affinity, while 3-MeO in 15 achieved high affinity for both A3AR species homologues (Ki 10.9 nM at hA3AR, 17.8 nM at mA3AR). Reversal of the hydroxy and methoxy groups of 15, i.e. compound 14, reduced mA3AR affinity 7-fold. In addition to 15, the unmethylated dopamine adduct 13 displayed particularly high mA3AR affinity (Ki 40 nM). Thus, a H-bond donor in the para-position and a H-bond acceptor in the meta-position of the phenyl ring favored mA3AR binding.
Therefore, the N6-2-((4-hydroxy-3-methoxy)-phenyl)-ethyl group was retained in compounds 17 – 21, which contained fluorophenyl- or pyridyl-substituted C2-phenylalkynyl groups. The effects on mA3AR affinity of this arylalkynyl substitution was detrimental except for the 2-F analogue 17, which displayed a Ki value of 21.5 nM. The C2-arylalkynyl substituent was further modified in compounds 22 – 30, with generally reduced mA3AR affinity. The C2-(5-halo-thien-2-yl-ethynyl) group, which is particularly suitable for high affinity at both A3AR species when incorporated into methanocarba A3AR agonists,27 here reduced mA3AR affinity by 4-fold compared to C2-phenylethynyl (cf. 29 and 15). Curiously, the 5-chlorothien-2-yl group, shown in several species to enhance duration of action, pharmacokinetics and A3AR agonist affinity of (N)-methanocarba 5′-methylamide derivatives,27 had either no hA3AR affinity advantage (e.g. 9 compared to 24) or a small advantage (e.g. 8 compared to 23) in the truncated series. Nevertheless, N6-2-phenylethyl derivative 23 bound with high hA3AR affinity (Ki 8.2 nM). Combination of the N6-3-O-methyldopamine substitution with a C2-(5-bromothien-2-yl-ethynyl) group (30) provided mA3AR selectivity of >1000-fold. Applying a constraint of the N6-2-phenylethyl moiety using a cyclopropyl ring, which enhanced hA3AR agonist affinity,26 in the truncated series reduced the hA3AR affinity of 31 by ~10-fold with no significant effect on mA3AR affinity. A 2-iodo analogue 32 displayed high hA3AR and moderate mA3AR affinity, similar to the equivalent C2-(5-halo-thien-2-yl-ethynyl) analogue 29. In summary, the highest hA3AR affinity (Ki 3 – 11 nM) was observed for 2-phenylethynyl derivatives 11, 14, 15 and 17 – 19, 5-halo-thien-2-yl-ethynyl derivatives 23, 29 and 30, and 2-iodo derivative 32, and the highest mA3AR affinity (Ki 18 – 22 nM) with 15 and 17.
The hA1AR affinity was variable, with Ki values ranging from less than 400 nM (16, 17 and 20) to >10 μM. 2-F-phenylethynyl analogue 17 had the highest hA1AR affinity and was only 30-fold A3AR-selective. Other positions of the F atom in 18 and 19 did not have comparable effects on A1AR.
In functional assays, 15 was shown to be a low efficacy partial agonist (16% Emax of full agonist adenosine-5′-N-ethyluronamide 37) using a guanine nucleotide binding assay in HEK293 cells overexpressing the mA3AR (Figure 1A). In inhibition of forskolin-stimulated cAMP production in hA3AR-HEK293 cells, compounds 15, 23 and 30 (10 μM) were moderately potent partial agonists (Figure 1B; 31%, 80% and 50% Emax, respectively, relative to Cl-IB-MECA 38). Nevertheless, a fixed concentration of 15 greatly inhibited the activation of hA3AR by Cl-IB-MECA (Figure 1C); therefore, 15 has antagonist-like activity. The variability of residual efficacy in this series of truncated nucleosides is consistent with a dependence on factors such as N6 and C2 substitution, the species, functional activity measured and the receptor expression system. Note that 38 has been characterized as having either similar or lower maximal hA3AR efficacy than 37, depending on the functional parameter measured.29
Figure 1.
Assessment of selected compounds for activity in functional assays with h and mA3ARs, determined as reported.29,47 A. Stimulation of [35S]GTPγS binding by 15 (Emax 16%) and 37 (EC50 188 nM, Emax 100%) in assays with cell membranes prepared from HEK293 cells expressing the mA3AR (n=7). B. Inhibition of forskolin (10 μM)-stimulated cAMP formation in HEK293 cells expressing the hA3AR (EC50 (nM), maximal effect as percent of cyclic AMP content remaining; n=3): Cl-IB-MECA 38, 4.10, 31.7±3.2%; 15, 158, 75.5±1.4%; 30, 692, 61.0±4.3%; 23, 2410, 37.6±4.5%. C. In cAMP assays with the hA3AR, compound 15 (10 μM) shifted the concentration-response curve of Cl-IB-MECA 38 rightward. The EC50 value (nM) of 38 alone was 12.1 nM (n = 3).
Drug-likeness and off-target interactions of selected derivatives
The selective and high affinity hA3AR/mA3AR partial agonist 15 was predicted to have drug-like physicochemical properties. The molecule had 4 H-bond donors, a cLogP of 0.087, and total polar surface area (tPSA) of 122 Å2 (ChemDraw 19.0). The molecular weight was slightly below 500, and the ligand efficiency (LE) was calculated to be 0.30. The addition of a 2-F atom in the case of 17 did not change the tPSA and produced only a small change in cLogP (+0.143). Thus, the overall hydrophobicity in this series is in a drug-like range.
Off-target interactions of the truncated nucleosides were determined at 45 receptors, channels and transporters, as measured in radioligand binding assays.30 Compound 15 did not interact with other GPCRs (Table S1, Supporting Information). However, weak interactions were detected for 15 at (Ki, nM): translocator protein, TSPO (2220); σ1 (3570) and σ2 (1980) receptors, and many of the truncated nucleoside derivatives bound similarly to these proteins, with some of the σ1 and σ2 affinities reaching the 300–400 nM range. The most potent inhibition of TSPO was observed with N6-3-F and N6-3-Cl-phenylethyl analogues 24 and 26 (Ki, ~200 and ~350 nM, respectively), both of which contain a C2-(5-Cl-thien-2-yl-ethynyl) group. Other compounds in the series were found to bind weakly at 5HT2B serotonergic or α2 adrenergic receptors with Ki values >1 μM.
Compound 15 was studied in preclinic assessment of its absorption, distribution, metabolism and elimination (ADME)-toxicology (Supporting Information), using methods described.32 The plasma stability was high in three species (% remaining after 120 min): human (91.8%), rat (84.6%), mouse (94.6%). Similarly, in simulated gastric fluid (100%), simulated intestinal fluid (73.6%), compound 15 was relatively stable over 120 min. The IC50 values (μM) at CYPs were: 1A2, 2.89; 2C9, 14.4; 2C19, >30; 2D6, >30; 3A4, 2.23. However, the in vivo pharmacokinetics in rats (p.o.) was not favorable, with t1/2 values of 0.457 and 0.490 h (3 and 10 mg/kg, respectively, Table S2, Supporting Information). The oral bioavailability was only 1.5% and 3.5%, respectively. Therefore, the utility of 15 as a mA3AR-selective partial agonist is mainly limited to parenteral administration.
In vivo activity
hA3AR/mA3AR partial agonist 15 was tested in a mouse model of A3AR-induced hypothermia.33,34 We have shown this effect to be dependent on the mA3AR-induced degranulation of peripheral mast cells.33 Injection of a mA3AR agonist peripherally, but not intracerebroventricularly, reduced the core body temperature (Tb), and this effect was lost in A3AR KO mice. Conversely, low doses of centrally administered mA1AR agonist reduced Tb.32,34 Thus, this model is capable of distinguishing the AR subtype involved and the compartment in the body, either brain or periphery.
A high dose of 15 (10 mg/kg, i.p.) lowered core body temperature (Tb) in wild-type C57BL/6 (WT) mice only which was greatly attenuated in ADORA3−/− mice, indicating an in vivo A3AR agonist activity (Figure 2A). However, a smaller dose (3 mg/kg, i.p.) induced only weak hypothermia in WT mice (Figure 2B). Pretreatment of the mice with 3 mg/kg (Figure 2B) or 10 mg/kg (Figure S1A) 15 30 min prior to the administration of the full A3AR agonist (1S,2R,3S,4R,5S)-4-(6-((3-chlorobenzyl)amino)-2-((3,4-difluorophenyl)-ethynyl)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (39, MRS5698, 3 mg/kg)28,33 did not significantly attenuate its pronounced hypothermic effect. Thus, the antagonist property of 15 was not evident in this in vivo model.
Figure 2.
A,B. Effect of nucleoside derivative 15 on Tb in WT (C57BL/6J male) or A3AR-KO mice, in comparison to full agonist 39. Compound 15 at 10 mg/kg (A) or 3 mg/kg (B) and vehicle were dosed i.p. in wild-type mice (WT, A,B), or mice lacking A3AR (A). The i.p. dose (mg/kg) is indicated, with N=4 to 12/group. t-Test (2-tailed, paired or unpaired as appropriate).
The ambiguity of truncated nucleosides that may act as either antagonist or partial agonist, depending on the pharmacological context, has been addressed here. In transfected HEK293 cells, 15 activated m and hA3AR with low efficacy and also at a high concentration antagonized the effects of a more efficacious hA3AR agonist, consistent with pharmacological theory.35 At a high in vivo dose, 15 lowered mouse Tb, presumably by histamine release from peripheral mast cells. It is possible that the lack of intense hypothermia at the lower dose of 15, which is comparable to active doses used of other A3AR agonists,33 is related to its low efficacy. Partial GPCR agonists, for example A1AR and D2R partial agonists, often have advantages as therapeutic agents over full agonists or pure antagonists, with increased selectivity for a desired action in vivo.35,36 Partial A3AR agonists have not been extensively characterized in vivo, largely because of the low mA3AR affinity of most of the truncated nucleosides so far reported.22,23 Each intended application of A3AR ligands would have to be probed separately to see if these low efficacy partial agonists could achieve desired in vivo efficacy.
Computational studies
A molecular modeling study was carried out to provide a structural hypothesis of the binding mode of compounds 15 and 8 at hA3AR and mA3AR, aiming to explain their different binding affinities.
The hA3AR or mA3AR have no experimental structures, but we previously reported hybrid homology models of both homologues, featuring an outwardly displaced TM2 modeled on a β2 adrenergic receptor template in order to accommodate extended adenine C2 substituents.22 After recent reports of several hA1AR structures, it appeared that their antagonist-bound structures present a similar outward displacement of TM2 (PDB ID: 5UEN37, 5N2S38). Thus, we prepared a new hA3AR hybrid model, using the agonist-bound hA2AAR structure (PDB ID: 3QAK39) as a template for the main portion of the receptor, as in the previous study,22 and an inactive antagonist-bound hA1AR structure (PDB ID: 5UEN) for the upper part of TM2. Despite the availability of a hA1AR cryo-EM structure in the active state (PDB ID: 6D9H),40 an agonist-bound hA2AAR template in the intermediate-active state was considered more suitable to model truncated (N)-methanocarba nucleosides showing partial agonist behavior. Moreover, since in the A2AAR structure 3QAK, T4 lysozyme was fused within intracellular loop 3 (IL3), IL3 was modelled after the hA2AAR structure 4UHR41 (sequence alignment in Figure S2), where it was resolved. The newly obtained hA3AR homology model was then used as a template to build a homology model of mA3AR (~73% sequence identity, Figure S3). The hA3AR and mA3AR models were validated assessing their ability to bind adenosine-like ligands, beginning with docking of known agonist 39 (hA3AR 3.49 nM, mA3AR 3.08 nM). The predicted binding mode of compound 39 at hA3AR and mA3AR orthosteric sites resembled the conformation of adenosine and adenosine-like agonists in X-ray and cryo-EM structures of A2AAR and A1AR (Figure S4). The complexes were prepared for MD simulation, with the addition of a specific water molecule (Figure S5) identified using the AquaMMapS tool,42 and the geometry of the complexes appeared stable in the trajectories (Table S4), as reported in detail (Supporting Information).
Once the model and protocol were validated, 15 was docked at the hA3AR and mA3AR orthosteric sites, and, after equilibration, three different MD simulation runs of 30 ns each were performed to assess the docking pose stability. The main objective was a comparison with compound 8, whose conformation was built in place on the poses of compound 15 and then subjected to MD simulation, as well. In both hA3AR and mA3AR, 15 and 8 assumed a pose comparable to those of adenosine derivatives in A1AR or A2AAR experimental structures and to the predicted poses of 39 (Figure S6A-B), with N6.55 forming the typical bidentate H-bond with N7 and the exocyclic N6H, and H-bonding of S7.42 with 3’-OH and H7.43 with 2’-OH (using Ballesteros-Weinstein numbering43 for comparing hA3AR and mA3AR, with the corresponding residue numbers reported in SI). During the simulations the compounds maintained their positions with the (N)-methanocarba moiety pointing toward the bottom of the binding pocket, the nucleobase involved in a π-π stacking interaction with F168(h)/F169(m) of EL2, the C2-phenylethynyl substituent facing TM2, and the N6-2-phenylethyl substituent contacting the EL region. The contact frequency between ligand and each protein residue (within 4 Å, Figure S7) identified statistically relevant ligand-stabilizing amino acids. A pattern of eleven residues surrounded compounds 15 and 8 for >50% of all the replicates of both hA3AR and mA3AR: L3.32, L3.33, T3.36, F168(h)/F169(m) (EL2), M5.38, W6.48, L6.51, N6.55, L/M7.35, I7.39, H7.43 (S7.42 interacted with the ligand in all simulations except for 15-hA3AR), and among these, L3.32, T3.36, F168, M5.38, W6.48, L6.51, N6.55, I7.39, S7.42, H7.43 were proved to mediate hA3AR agonist binding and/or activation thanks to mutagenesis studies.44–46 L3.32, F168(h)/F169(m), M5.38, W6.48, L6.51, I7.39 may contribute to binding through a hydrophobic effect (not quantified here) and have been observed to interact with the compounds with negative average van der Waals contributions lower than −1 kcal/mol during MD (Figure S8–11D). The lowest van der Waals interaction is associated with F168/F169, involved in a π-π stacking interaction with the nucleobase, which is maintained during each simulation. Other intense contributions involve V169 (EL2) and R170 (EL2) in hA3AR and mA3AR, respectively, interacting with the 2-phenylethyl moiety.
In the hA3AR MD trajectories, compound 15 presented an average RMSD of 2.77 Å over the three replicates (Table S5) and stably maintained the H-bonding pattern with N6.55 and H7.43 (Figure S8), while S7.42 flipped during the simulations and its H-bond was not observed (Figure 3A and Video S1). Compound 8 (Figure 3B and Video S2) behaved similarly maintaining the full pattern of key interactions and an average RMSD of 3.04 Å over the replicates (Table S5 and Figure 3B). The presence of the 4-hydroxy and 3-methoxy substituents on the N6-(2-phenylethyl) moiety enabled compound 15 to form additional interactions with residues of the extracellular loops (ELs). In particular, transient H-bonds were observed: with E258 (EL3) in replicate 1 (Figure S8D) and with Q167 (EL2) and Q7.32 in replicates 2 and 3 (Figure S12). The 2-phenylethyl moiety of compound 8 deviated from its docking pose and inserted into a hydrophobic cleft defined by M172 (EL2), M5.35, M5.38 and V169 (EL2), showing intense negative van der Waals interactions (Figure S9D).
Figure 3.
A-B. Binding modes of compounds 15 (A) and 8 (B) at hA3AR binding site after 30 ns MD simulation. The receptor is depicted by light blue ribbon and key residues are highlighted by sticks. Compound 15 and 8 are depicted by orange and purple sticks respectively. C-D. Binding mode of compound 15 (C) and 8 (D) at mA3AR binding site after 30 ns MD simulation. The receptor is depicted by gray ribbon and key residues are highlighted by sticks. Compound 15 and 8 are depicted by orange and purple sticks, respectively. Correspondence of residue numbering28: T3.3694/95, N6.55250/251, S7.42271/272, H7.43272/273.
In MD of mA3AR, both 15 and 8 (Figure 3C-D and Video S3-S4) retained the conserved H-bonds with N6.55 and S7.42. H7.43 did not present a conformation compatible with the maintenance of a H-bond with 2’-OH but retained ligand contact during nearly all of each simulation. H-bonding of N6.55 persisted (~80%) during each replicate with compound 15, while it was more transient with compound 8. However, the interaction with S7.42 was more frequent for compound 8 than for compound 15 (Table S5 and Figure S10-S11). On average, compound 15 (1.97 Å average RMSD over the replicates) appeared more stably positioned than compound 8 (3.08 Å average RMSD). As observed during hA3AR simulation, the 2-phenylethyl moiety of 8 inserted into a cleft defined by EL2 and TM5 of mA3AR, surrounded by V173 (EL2), L5.35, M5.38 and R170 (EL2), showing negative van der Waals interactions (Figure S11D). Importantly, the 4-hydroxy and 3-methoxy substituents of 15 could interact through transient H-bonds with the backbone carbonyl of V258 (EL3) and NH of I260 (EL3) (Figure S10D).
In summary, the nucleoside scaffold of compounds 8 and 15 within the A3AR orthosteric binding site was predicted to be similar to agonist binding in A2A and A1 AR structures. The N6 hydroxyl and methoxyl groups present in 15 formed additional transient H-bonds with residues in EL2 and EL3 in either hA3AR or mA3AR, consistent with the higher affinity of 15 compared to 8. The enhancement of ~27-fold at mA3AR, compared to ~2-fold at hA3AR, aligned with the overall higher polarity of mA3AR ELs compared to hA3AR, suggesting that 15 might form additional dipolar interactions with the flexible ELs beyond those observed here. The two species homologues bear a high (73%) sequence identity, with greater EL variability (Figure S3). There are 34 polar amino acids in mA3AR ELs, including 12 charged residues, while 28 polar residues (6 charged) occur in hA3AR ELs. Furthermore, during MD simulations of the 15-mA3AR complex, the N6-2-((4-hydroxy-3-methoxy)-phenyl)-ethyl group approached polar residues for more than 33% of all replicates: H168 (EL2), R170 (EL2), S6.58, K259 (EL3), corresponding to the overall more hydrophobic Q167 (EL2), V169 (EL2), I6.58, E258 (EL3) in hA3AR (Figure S7).
Conclusions
In conclusion, we have synthesized novel 4′-truncated (N)-methanocarba nucleosides that have nearly nM affinity as antagonists/partial agonists at both h and mA3ARs, with effects of distal N6 substitution being particularly striking at the mA3AR. Dopamine-derived N6-substituents, compared to N6-(2-phenylethyl), enhanced mA3AR affinity by polar interactions with the ELs predicted using docking and MD simulation with newly constructed mA3AR and hA3AR homology models. These hybrid models were based on two templates, an inactive antagonist-bound hA1AR structure for the upper part of TM2 and an agonist-bound hA2AAR structure for the remaining TM portions. A N6−2-((4-hydroxy-3-methoxy)-phenyl)-ethyl group was conducive to high affinity, which with a C2-phenylethynyl group (15) achieved Ki values of 10.9 (hA3AR) and 17.8 nM (mA3AR). Highly potent and selective 15 displayed A3AR antagonist as well as partial agonist properties in cells (both h and m) and maintained in vivo agonism in the mouse over a >2 h period and can be considered a useful tool compound for in vivo use. This approach to species-independent A3AR-selective nucleosides as low efficacy partial agonists might provide novel, nuanced modulators of the A3AR, a drug target of growing interest.
Experimental section
Molecular Modeling
Homology Modeling
A homology model was built for hA3AR and mA3AR. hA3AR was obtained as a chimeric model using as templates the X-ray structures 3QAK39 of hA2AAR for the greater part of the receptor, 5UEN37 of hA1AR for the tip of TM2 and 4UHR41 of hA2AAR for IL3, with the alignment shown in Figure S2. EL2 and EL3 were removed from the templates being longer than in hA3AR. The Prime knowledge-based method was used to generate the model.48, 49 The histidine protonotation and tautomeric states were assigned by means of the Protein Preparation Wizard tool50 of the Schrödinger suite (Maestro 2019–1).51 Histidines H79, H95, H124 and H158 of hA3AR were protonated at Nε nitrogen (named HSE according to the CHARMM nomenclature), while H272 was protonated at Nδ (HSD). The model was minimized using the OPLS3 force field.52 The new hA3AR model was used as a template to build the mA3AR model using the knowledge-based Prime function. Histidines H80, H96, H108 of mA3AR were in the HSE tautomeric state, while H158 and H273 in the HSD form. The receptor was minimized with OPLS3.
Molecular Docking
Both known full agonist 39 and compound 15 were docked to the hA3AR homology model with Glide-XP53 scoring function, on a grid of 30 Å centered on the centroid of N250 and F168, setting a maximum number of poses to 10. Compounds 39 and 15 were docked to mA3AR structure with an induced fit procedure54 to accommodate protein flexibility, since residue R170 in EL2 hindered the entrance of the model’s orthosteric binding pocket. A grid of 30 Å centered on the centroid of N251 and F169 was employed, residues H168, R170, L175, S254 and M265 were trimmed, and residues within 5 Å from the ligand (except T95, F169, N251, S272 and H273) were optimized.
One pose for each compound and at each receptor was selected by visual inspection.
Compound 8 was built in place removing from the compound 15 poses the 4-hydroxy and 3-methoxy substituents.
Molecular Dynamics
30 ns apo MD simulations of both hA3AR and mA3AR were performed and analyzed with the AquaMMapS tool.42 A water hotspot geometrically close to the water molecule with RESID 2010 of X-ray structure 2YDO55 was identified. Structure 2YDO was superposed to models hA3AR and mA3AR, and the water molecule with RESID 2010 was minimized in the two models’ environment with OPLS3 force field and then used to build the initial system for all the MD simulations.
The HTMD56 module was employed to prepare the system for MD simulations. ACE and CT3 cappings were added to N-/C-termini respectively. Each protein-ligand complex was inserted into a 90 Å x 90 Å 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer generated with the VMD Membrane Plugin57 tool according to the orientation computed by the Positioning of Proteins in Membrane (PPM)58 web server. TIP3P59 water molecules and Na+/Cl- counter-ions were added respectively to solvate, neutralize the system and bring the salt concentration to the 0.154 M physiologic concentration.
The ACEMD60 molecular dynamics engine was used to perform the simulations, with CHARMM3661,62 force field for protein, lipids, water and ions, and CGenFF63,64 for the ligand. Missing ligand parameters were assigned by analogy using the ParamChem65 web service, with few modifications. The system was initially subjected to a 5000 conjugate-gradient steps of minimization and equilibrated through 40 ns MD simulation in the NPT ensemble, where positional harmonic restraints were applied to ligand and protein atoms (0.8 kcal mol−1 Å−2 for ligand atoms, 0.85 kcal mol−1 Å−2 for Cα carbon atoms, and 0.4 kcal mol−1 Å−2 for the other protein atoms) and linearly reduced in the last 20 ns. Three 30 ns MD simulations in the NVT ensemble were run for each equilibrated system. A Berendsen barostat (relaxation time 800 fs) was applied to keep the pressure at around 1 atm during equilibration and a Langevin thermostat was employed to maintain the temperature at 310 K in both equilibration and productive simulations (damping constant 1 ps−1 and 0.1 ps−1 for equilibration and production, respectively). In all simulations the timestep was set to 2 fs and the M-SHAKE66 algorithm was used to constrain bonds containing hydrogen atoms. A 9 Å cutoff was used for non-bonded interactions, with a switching distance of 7.5 Å, and the long-range electrostatic interactions beyond the non-bonded cutoff were computed with the Particle Mesh Ewald (PME)67 method, employing a grid spacing of 1Å. All the simulations were run on the NIH HPC Biowulf cluster (http://hpc.nih.gov, accessed Oct 15, 2019) using Tesla P100 GPUs.
Trajectory Analysis
Before analyzing the trajectories, the systems were aligned to the initial conformations by superposing protein Cα carbon atoms. An in-house Tcl script employing VMD 1.9.3 was used to perform the analysis.57 Ligand-protein electrostatic and van der Waals interactions were computed with NAMD.68 All the data were plotted using the Gnuplot (version 5.0) software.69
Chemistry
Chemical synthesis
Materials and instrumentation
All reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO). 1H NMR spectra were obtained with a Bruker 400 spectrometer using CDCl3, CD3OD and DMSO as solvents. Chemical shifts are expressed in δ values (ppm) with tetramethylsilane (δ 0.00) for CDCl3 and water (δ 3.30) for CD3OD. NMR spectra were collected with a Bruker AV spectrometer equipped with a z-gradient [1H, 13C, 15N]-cryoprobe. TLC analysis was carried out on glass sheets precoated with silica gel F254 (0.2 mm) from Aldrich. The purity of final nucleoside derivatives was checked using a Hewlett−Packard 1100 HPLC equipped with a Zorbax SB-Aq 5 μm analytical column (50 × 4.6 mm; Agilent Technologies Inc., Palo Alto, CA). Mobile phase: linear gradient solvent system, 5 mM TBAP (tetrabutylammonium dihydrogenphosphate) −CH3CN from 80:20 to 0:100 in 13 min; the flow rate was 0.5 mL/min. Peaks were detected by UV absorption with a diode array detector at 230, 254, and 280 nm. All derivatives tested for biological activity showed >95% purity by HPLC analysis (detection at 254 nm). Low-resolution mass spectrometry was performed with a JEOL SX102 spectrometer with 6-kV Xe atoms following desorption from a glycerol matrix or on an Agilent LC/MS 1100 MSD, with a Waters (Milford, MA) Atlantis C18 column. High resolution mass spectroscopic (HRMS) measurements were performed on a proteomics optimized Q-TOF-2 (Micromass-Waters) using external calibration with polyalanine, unless noted. Observed mass accuracies are those expected based on known performance of the instrument as well as trends in masses of standard compounds observed at intervals during the series of measurements. Reported masses are observed masses uncorrected for this time-dependent drift in mass accuracy. All of the monosubstituted alkyne intermediates were purchased from Sigma-Aldrich (St. Louis, MO), Small Molecules, Inc. (Hoboken, NJ), Anichem (North Brunswick, NJ), PharmaBlock, Inc. (Sunnyvale, CA), Frontier Scientific (Logan, UT) and Tractus (Perrineville, NJ).
(1R,2R,3S,4R,5S)-4-(6-((3-Fluorophenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (9)
A solution of compound 56 (32 mg, 0.06 mmol) in methanol (3 mL) and 10% trifluoromethane sulfonic acid (3 mL) was heated at 70 °C for 5 h. Solvent was evaporated under vacuum, and the residue was purified on flash silica gel column chromatography (CH2Cl2:MeOH = 40:1) to give the compound 9 (27 mg, 90%) as colorless syrup. 1H NMR (CD3OD, 400 MHz) δ 8.29 (s, 1H), 7.68–7.65 (m, 2H), 7.48–7.44 (m, 3H), 7.32–7.27 (m, 1H), 7.14–7.08 (m, 2H), 6.96–6.91 (m, 1H), 4.89 (s, 1H), 4.70 (t, J = 5.6 Hz, 1H), 3.92–3.91 (m, 3H), 3.05 (t, J = 7.2 Hz, 2H), 2.10–1.94 (m, 1H), 1.75–1.63 (m, 1H), 1.39–1.36 (m, 1H), 0.87–0.71 (m, 1H). HRMS calculated for C27H25N5O2F (M + H) +: 470.1992; found 470.2000.
(1R,2R,3S,4R,5S)-4-(6-((4-Hydroxyphenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (10)
Compound 10 (88%) was prepared from compound 57 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.19 (s, 1H), 7.65–7.63 (m, 2H), 7.44–7.41 (m, 3H), 7.13 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 4.86 (s, 1H), 4.68 (t, J = 5.6 Hz, 1H), 3.90 (d, J = 6.4 Hz, 1H), 3.83 (br s, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.00–1.97 (m, 1H), 1.73–1.69 (m, 1H), 1.38–1.35 (m, 1H), 0.80–0.74 (m, 1H). HRMS calculated for C27H26N5O3 (M + H) +: 468.2036; found 468.2038.
(1R,2R,3S,4R,5S)-4-(6-((3-Fluoro-4-hydroxyphenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (11)
Compound 11 (91%) was prepared from compound 58 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.02 (s, 1H), 7.66–7.64 (m, 2H), 7.45–7.43 (m, 3H), 7.14 (d, J = 8.4 Hz, 1H), 7.05–6.81 (m, 1H), 6.73 (d, J = 8.4 Hz, 1H), 4.69 (t, J = 5.2 Hz, 1H), 3.91–3.84 (m, 3H), 2.92 (t, J = 7.2 Hz, 2H), 2.01–1.98 (m, 1H), 1.73–1.70 (m, 1H), 1.39–1.36 (m, 1H), 0.81–0.75 (m, 1H). HRMS calculated for C27H25N5O3F (M + H) +: 486.1941; found 486.1946.
(1R,2R,3S,4R,5S)-4-(6-((4-Aminophenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (12)
Compound 12 (88%) was prepared from compound 59 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.66–7.64 (m, 2H), 7.45–7.44 (m, 3H), 7.08 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 4.80 (s, 1H), 4.70 (t, J = 5.6 Hz, 1H), 3.92 (d, J = 6.4 Hz, 1H), 3.82 (br s, 2H), 2.89 (d, J = 7.2 Hz, 2H), 2.03–1.98 (m, 1), 1.73–1.71 (m, 1H), 1.39–1.36 (m, 1H), 0.82–0.76 (m, 1H). HRMS calculated for C27H27N6O2 (M + H) +: 467.2195; found 467.2199.
(1R,2R,3S,4R,5S)-4-(6-((3,4-Dihydroxyphenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (13)
Compound 13 (90%) was prepared from compound 60 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.21 (s, 1H), 7.67–7.65 (m, 2H), 7.45–7.43 (m, 3H), 6.74–6.69 (m, 2H), 6.63–6.61 (m, 1H), 4.69 (t, J = 6.0 Hz, 1H), 3.91 (d, J = 6.4 Hz, 1H), 3.83 (br s, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.03–1.98 (m, 1H), 1.74–1.70 (m, 1H), 1.39–1.36 (m, 1H), 0.81–0.75 (m, 1H). HRMS calculated for C27H26N5O4 (M + H) +: 484.1985; found 484.1979.
(1R,2R,3S,4R,5S)-4-(6-((3-Hydroxy-4-methoxyphenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (14)
Compound 14 (93%) was prepared from compound 61 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.20 (s, 1H), 7.66–7.64 (m, 2H), 7.44–7.42 (m, 3H), 6.83–6.78 (m, 2H), 6.73–6.71 (m, 1H), 4.86 (m, 1H), 4.68 (t, J = 5.6 Hz, 1H), 3.90 (d, J = 6.4 Hz, 1H), 3.80–3.81 (m, 5H), 2.89 (t, J = 7.2 Hz, 2H), 2.02–1.97 (m, 1H), 1.73–1.69 (m, 1H), 1.38–1.35 (m, 1H), 0.80–0.75 (m, 1H). HRMS calculated for C28H28N5O4 (M + H) +: 498.2141; found 498.2148.
(1R,2R,3S,4R,5S)-4-(6-((4-Hydroxy-3-methoxyphenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (15)
Compound 15 (89%) was prepared from compound 62 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.19 (s, 1H), 7.65–7.62 (m, 2H), 7.44–7.42 (m, 3H), 6.88 (s, 1H), 6.71–6.72 (m, 2H), 4.85 (s, 1H), 4.67 (d, J = 6.8 Hz, 1H), 3.90–3.87 (m, 3H), 3.79 (s, 3H), 2.91 (t, J = 7.2 Hz, 2H), 2.03–1.96 (m, 1H), 1.73–1.69 (m, 1H), 1.38–1.35 (m, 1H), 0.79–0.74 (m, 1H). HRMS calculated for C28H28N5O4 (M + H) +: 498.2141; found 498.2145.
(1R,2R,3S,4R,5S)-4-(6-((3,4-Dimethoxyphenethyl)amino)-2-(phenylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (16)
Compound 16 (91%) was prepared from compound 63 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.26 (s, 1H), 7.66–7.64 (m, 2H), 7.46–7.45 (m, 3H), 6.92 (s, 1H), 6.83–6.84 (m, 2H), 4.81 (s, 1H), 4.69 (br s 1H), 3.91–3.90 (m, 3H), 3.83 (s, 6H), 2.96 (t, J = 6.8 Hz, 2H), 2.03–2.00 (m, 1H), 1.71–1.70 (m, 1H), 1.35–1.37 (m, 1H), 0.79–0.78 (m, 1H). HRMS calculated for C29H30N5O4 (M + H) +: 512.2298; found 512.2301.
(1R,2R,3S,4R,5S)-4-(2-((2-Fluorophenyl)ethynyl)-6-((4-hydroxy-3-methoxyphenethyl) amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (17)
Compound 17 (77%) was prepared from compound 32 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.23 (s, 1H), 7.69 (t, J = 6.8 Hz, 1H), 7.52–7.47 (m, 1H), 7.28–7.22 (m, 2H), 6.89 (s, 1H), 6.74–6.70 (m, 2H), 4.80 (s, 1H), 4.70 (t, J = 6.0 Hz, 1H), 3.91–3.88 (m, 4H), 3.82 (s, 3H), 2.93 (t, J = 7.2 Hz, 2H), 2.03–1.99 (m, 1H), 1.72–1.71 (m, 1H), 1.38–1.37 (m, 1H), 0.81–0.76 (m, 1H). HRMS calculated for C28H27N5O4F (M + H) +: 516.2047; found 516.2057.
(1R,2R,3S,4R,5S)-4-(2-((3-Fluorophenyl)ethynyl)-6-((4-hydroxy-3-methoxyphenethyl) amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (18)
Compound 18 (75%) was prepared from compound 32 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.23 (s, 1H), 7.47–7.44 (m, 2H), 7.40 (d, J = 8.8 Hz, 1Hz), 7.28–7.18 (m, 1H), 6.89 (s, 1H), 6.72 (s, 2H), 4.70 (t, J = 5.6 Hz, 1Hz), 3.92–3.87 (m, 3H), 3.82 (s, 3H), 2.93 (t, J = 7.2 Hz, 2Hz), 2.03–1.96 (m, 1H), 1.72–1.71 (m, 1H), 1.39–1.32 (m, 1H), 0.81–0.76 (m, 1H). HRMS calculated for C28H27N5O4F (M + H) +: 516.2047; found 516.2056.
(1R,2R,3S,4R,5S)-4-(2-((4-Fluorophenyl)ethynyl)-6-((4-hydroxy-3-methoxyphenethyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (19)
Compound 19 (73%) was prepared from compound 32 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.71–7.68 (m, 2H), 7.20 (t, J = 8.8 Hz, 2Hz), 6.89 (s, 1H), 6.74–6.70 (m, 2H), 4.81 (s, 1H), 4.69 (t, J = 5.6 Hz, 1Hz), 3.91–3.88 (m, 3H), 3.81 (s, 3H), 2.93 (t, J = 7.2 Hz, 2Hz), 2.04–1.98 (m, 1H), 1.73–1.71 (m, 1H), 1.39–1.36 (m, 1H), 0.81–0.76 (m, 1H). HRMS calculated for C28H27N5O4F (M + H) +: 516.2047; found 516.2054.
(1R,2R,3S,4R,5S)-4-(2-((3,4-Difluorophenyl)ethynyl)-6-((4-hydroxy-3-methoxyphenethyl) amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (20)
Compound 20 (72%) was prepared from compound 32 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.21 (s, 1H), 7.60–7.55 (m, 1H), 7.52–7.47 (m, 1H), 7.39–7.32 (m, 1H), 6.88 (s, 1H), 6.71–6.70 (m, 2H), 4.69 (t, J = 5.6 Hz, 1H), 3.91 (d, J = 6.8 Hz, 1H), 3.86 (br s, 2H), 3.81 (s, 3H), 2.92 (t, J = 7.2 Hz, 2H), 2.03–1.97 (m, 1H), 1.73–1.68 (m, 1H), 1.38–1.35 (m, 1H), 0.81–0.75 (m, 1H). HRMS calculated for C28H26N5O4F2 (M + H) +: 534.1953; found 534.1957.
(1R,2R,3S,4R,5S)-4-(6-((4-Hydroxy-3-methoxyphenethyl)amino)-2-(pyridin-3-ylethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (21)
Compound 21 (73%) was prepared from compound 32 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.82 (s, 1H), 8.61 (s, 1H), 8.23 (s, 1H), 8.11 (d, J = 8.8 Hz, 1H), 7.54–7.51 (m, 1H), 6.89 (s, 1H), 6.71 (s, 2H), 4.71 (t, J = 5.6 Hz, 1H), 3.92–3.83 (m, 3H), 2.93 (t, J = 6.8 Hz, 2H), 2.02–1.98 (m, 1H), 1.71–1.69 (m, 1H), 1.38–1.36 (m, 1H), 0.81–0.76 (m, 1H). HRMS calculated for C27H27N6O4 (M + H) +: 499.2094; found 499.2101.
(1R,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(phenethylamino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (23)
Compound 23 (87%) was prepared from compound 64 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.31–7.26 (m, 5H), 7.21–7.17 (m, 1H), 7.02 (d, J = 4.0 Hz, 1H), 4.85 (s, 1H), 4.68 (t, J = 5.6 Hz, 1H), 3.90–3.89 (m, 3H), 3.01 (t, J = 7.2 Hz, 2H), 2.03–1.97 (m, 1H), 1.73–1.69 (m, 1H), 1.38–1.35 (m, 1H), 0.80–0.75 (m, 1H). HRMS calculated for C25H23N5O2SCl (M + H) +: 492.1261; found 492.1255.
(1R,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-((3-fluorophenethyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (24)
Compound 24 (90%) was prepared from compound 65 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.28 (s, 1H), 7.31–7.26 (m, 2H), 7.12–7.01 (m, 3H), 6.92 (t, J = 8.4 Hz, 1H), 4.81 (s, 1H), 4.69 (s, 1H) 3.90–3.89 (m, 3H), 3.03 (t, J = 6.8 Hz, 2H), 2.02–1.98 (m, 1H), 1.75–1.68 (m, 1H), 1.37–1.36 (m, 1H), 0.91–0.75 (m, 2H). HRMS calculated for C25H22N5O2ClSF (M + H) +: 510.1167; found 510.1168.
(1R,2R,3S,4R,5S)-4-(6-((3-Fluorophenethyl)amino)-2-((5-fluorothiophen-2-yl)ethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (25)
Compound 25 (87%) was prepared from compound 66 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.31–7.26 (m, 1H), 7.18 (t, J = 4.0 Hz, 1H), 7.12–7.06 (m, 2H), 6.94–6.90 (m, 1H), 6.63–6.61 (m, 1H), 4.85 (s, 1H), 4.68 (t, J = 5.6 Hz, 1H), 3.90–3.88 (m, 3H), 3.02 (t, J = 7.2 Hz, 2H), 2.03–1.97 (m, 1H), 1.73–1.69 (m, 1H), 1.38–1.35 (m, 1H), 0.81–0.75 (m, 1H). HRMS calculated for C25H22N5O2SF2 (M + H) +: 494.1462; found 494.1459.
(1R,2R,3S,4R,5S)-4-(6-((3-Chlorophenethyl)amino)-2-((5-chlorothiophen-2-yl)ethynyl)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (26)
Compound 26 (89%) was prepared from compound 67 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.24 (s, 1H), 7.34–7.18 (m, 3H), 7.03–7.02 (m, 1H), 4.83 (s, 1H), 4.69 (d, J = 5.2 Hz, 1H), 3.91–3.89 (m, 3H), 3.01 (d, J = 7.2 Hz, 2H), 2.02–1.99 (m, 1H), 1.73–1.70 (m, 1H), 1.37–1.36 (m, 1H), 0.91–0.76 (m, 1H). HRMS calculated for C25H22N5O2SCl (M + H) +: 526.0871; found 526.0865.
(1R,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-((3-methylphenethyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (27)
Compound 27 (91%) was prepared from compound 68 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.20 (s, 1H), 7.29 (d, J = 3.6 Hz, 1H), 7.16–7.05 (m, 3H) 7.00 (d, J = 3.6 Hz, 2H), 4.83 (s, 1H), 4.67 (t, J = 4.8 Hz, 1H), 3.90–3.85 (m, 3H), 2.95 (t, J = 7.2 Hz, 2H), 2.02–1.99 (m, 1H), 1.72–1.68 (m, 1H), 1.37 (m, 1H), 0.80–0.74 (m, 1H). HRMS calculated for C26H25N5O2SCl (M + H) +: 506.1417; found 506.1418.
(1R,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-((3,4-difluorophenethyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (28)
Compound 28 (86%) was prepared from compound 69 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.28 (s, 1H), 7.34 (d, J = 2.8 Hz, 1H), 7.27–7.09 (m, 3H), 7.04 (d, J = 3.2 Hz, 1H), 4.81 (s, 1H), 4.69 (d, J = 5.6 Hz, 1H), 3.92–3.90 (m, 3H), 3.01 (d, J = 6.8 Hz, 1H), 2.02–1.99 (m, 1H), 1.78–1.71 (m, 1H), 1.37–1.36 (m, 1H), 0.91–0.76 (m, 2H). HRMS calculated for C25H21N5O2F2ClS (M + H) +: 528.1073; found 528.1066.
(1R,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-((4-hydroxy-3-methoxyphenethyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (29)
Compound 29 (76%) was prepared from compound 32 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.32 (d, J = 4.0 Hz, 1H), 7.03 (d, J = 4.0 Hz, 1H), 6.88 (s, 1H), 6.71 (s, 2H), 4.68 (t, J = 5.6 Hz, 1H), 3.90 (d, J = 6.4 Hz, 1H), 3.85 (br s, 2H), 3.82 (s, 3H), 2.92 (t, J = 7.2 Hz, 2H), 2.02–1.97 (m, 1H), 1.73–1.69 (m, 1H), 1.38–1.35 (m, 1H), 0.81–0.75 (m, 1H). HRMS calculated for C26H25N5O4SCl (M + H) +: 538.1316; found 538.1324.
(1R,2R,3S,4R,5S)-4-(2-((5-Bromothiophen-2-yl)ethynyl)-6-((4-hydroxy-3-methoxyphenethyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (30)
Compound 30 (74%) was prepared from compound 32 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.21 (s, 1H), 7.26 (d, J = 3.6 Hz, 1H), 7.13 (d, J = 3.6 Hz, 1H), 6.88 (s, 1H), 6.71 (s, 2H), 4.84 (s, 1H), 4.67 (t, J = 5.6 Hz, 1H), 3.88 (d, J = 6.8 Hz, 1H), 3.85 (s, 3H), 2.91 (t, J = 7.2 Hz, 2H), 2.02–1.97 (m, 1H), 1.73–1.68 (m, 1H), 1.38–1.36 (m, 1H), 0.80–0.75 (m, 1H). HRMS calculated for C26H25N5O4SBr (M + H) +: 582.0811; found 582.0801.
(1R,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(((1S,2R)-2-phenylcyclopropyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (31)
Compound 31 (89%) was prepared from compound 70 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.27 (s, 1H), 7.36–7.28 (m, 5H), 7.22–7.18 (m, 1H), 7.04 (d, J = 4.0 Hz, 1H), 4.81 (s, 1H), 4.69 (t, J = 5.6 Hz, 1H), 3.92 (d, J = 6.8 Hz, 1H), 3.18 (brs 1H), 2.22–2.14 (m, 1H), 2.03–1.93 (m, 1H), 1.73–1.67 (m, 1H), 1.45–1.30 (m, 3H), 0.85–0.74 (m, 1H). HRMS calculated for C26H23N5O2ClS (M + H) +: 504.1261; found 504.1260.
(1R,2R,3S,4R,5S)-4-(6-((4-Hydroxy-3-methoxyphenethyl)amino)-2-iodo-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol (32)
Compound 32 (92%) was prepared from compound 49 following the same method for compound 9. 1H NMR (CD3OD, 400 MHz) δ 8.06 (s, 1H), 6.84 (s, 1H), 6.72–6.67 (m, 2H), 4.79 (s, 1H), 4.71 (t, J = 5.6 Hz, 1H), 3.88 (d, J = 6.4 Hz, 1H), 3.82 (s, 3H), 3.76 (br s, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.00–1.94 (m, 1H), 1.66–1.61 (m, 1H), 1.32–1.29 (m, 1H), 0.78–0.73 (m, 1H). HRMS calculated for C20H23N5O4I (M + H) +: 524.0795; found 524.0800.
9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-N-(3-fluorophenethyl)-2-iodo-9H-purin-6-amine (43)
2-(3-Fluorophenyl)-ethan-1-amine (0.11 mL, 0.87 mmol) and DIPEA (0.29 mL, 1.7 mmol) was added to a solution of compound 41 (76 mg, 0.17 mmol) in isopropanol and heated at 75 °C for 1 hr. Solvent was evaporated under vacuum and residue was purified on flash silica gel column chromatography (hexane:ethyl acetate = 2:1) to give the compound 43 (63 mg, 67%) as colorless foamy solid. 1H NMR (CD3OD, 400 MHz) δ 8.03 (s, 1H), 7.32–7.27 (m, 1H), 7.11–7.03 (m, 2H), 6.95–6.91 (m, 1H), 5.36 (t, J = 6.4 Hz, 1H), 4.96 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.80 (br s, 2H), 2.99 (t, J = 7.2 Hz, 2H), 2.06–1.98 (m, 1H), 1.70–1.69 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 0.93–0.90 (m, 2H). HRMS calculated for C22H24N5O2FI (M + H) +: 536.0959; found 536.0961.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-yl)amino)ethyl)phenol (44)
Compound 44 (74%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.02 (s, 1H), 7.10 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 5.36 (t, J = 6.0 Hz, 1H), 4.95 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.72 (br s, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.07–2.02 (m, 1H), 1.72–1.67 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.93–0.87 (m, 2H). HRMS calculated for C22H25N5O3I (M + H) +: 534.1002; found 534.1006.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-yl)amino)ethyl)-2-fluorophenol (45)
Compound 45 (72%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.02 (s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.90–6.82 (m, 1H), 6.72 (d, J = 8.0 Hz, 1H), 5.36 (t, J = 6.4 Hz, 1H), 4.95 (s, 1H), 4.70 (d, J = 7.2 Hz, 1H), 3.73 (br s, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.07–2.01 (m, 1H), 1.72–1.67 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.93–0.87 (m, 2H). HRMS calculated for C22H24N5O3FI (M + H) +: 552.0908; found 552.0909.
N-(4-Aminophenethyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-amine (46)
Compound 46 (76%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.01 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 5.36 (t, J = 6.4 Hz, 1H), 4.95 (s, 1H), 4.70 (d, J = 7.2 Hz, 1H), 3.70 (br s, 2H), 2.83 (t, J = 7.6, Hz, 2H), 2.05–2.02 (m, 1H), 1.70–1.67 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.93–0.88 (m, 2H). HRMS calculated for C22H26N6O2I (M + H) +: 533.1162; found 533.1167.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-yl)amino)ethyl)benzene-1,2-diol (47)
Compound 47 (74%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.02 (s, 1H), 6.70–6.68 (m, 2H), 6.60 (d, J = 8.0 Hz, 1H), 5.36 (t, J = 6.4 Hz, 1H), 4.95 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.71 (br s, 2H), 2.81 (t, J = 7.2 Hz, 1H), 2.06–2.01 (m, 1H), 1.72–1.67 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 0.93–0.87 (m, 2H). HRMS calculated for C22H25N5O4I (M + H) +: 550.0951; found 550.0955.
5-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-yl)amino)ethyl)-2-methoxyphenol (48)
Compound 48 (73%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.01 (s, 1H), 6.84 (t, J = 8.4 Hz, 1H), 6.74–6.69 (m, 2H), 5.35 (t, J = 5.6 Hz, 1H), 4.95 (s, 1H), 4.70 (d, J = 7.2 Hz, 1H), 3.82 (s, 3H), 3.73 (br s, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.06–2.01 (m, 1H), 1.72–1.67 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.93–0.88 (m, 2H). HRMS calculated for C23H27N5O4I (M + H) +: 564.1108; found 564.1110.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-yl)amino)ethyl)-2-methoxyphenol (49)
Compound 49 (75%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.01 (s, 1H), 6.86 (m, 1H), 6.72–6.70 (m, 2H), 5.35 (t, J = 6.4 Hz, 1H), 4.94 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.84 (s, 3H), 3.75 (br s, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.05–2.02 (m, 1H), 1.71–1.66 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.94–0.87 (m, 2H). HRMS calculated for C23H27N5O4I (M + H) +: 564.1108; found 564.1115.
N-(3,4-Dimethoxyphenethyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-amine (50)
Compound 50 (74%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.01 (s, 1H), 6.89–6.84 (m, 2H), 6.81–6.79 (m, 1H), 5.35 (d, J = 6.4 Hz, 1H), 4.94 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.84–3.77 (m, 8H), 2.90 (t, J = 7.2 Hz, 2H), 2.05–2.00 (m, 1H), 1.71–1.66 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.93–0.87 (m, 2H). HRMS calculated for C24H29N5O4I (M + H) +: 578.1264; found 578.1266.
N-(3-Chlorophenethyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa [3,4] cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-amine (51)
Compound 51 (71%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.03 (s, 1H), 7.31–7.16 (m, 4H), 5.36 (t, J = 6.0 Hz, 1H), 4.95 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.79 (br s, 2H), 2.97 (t, J = 6.8 Hz, 2H), 2.07–2.02 (m, 1H), 1.72–1.67 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.93–0.87 (m, 2H). ). HRMS calculated for C22H24N5O2ClI (M + H) +: 552.0663; found 552.0660.
9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-N-(3-methylphenethyl)-9H-purin-6-amine (52)
Compound 52 (70%) was prepared from compound 41 following the same method for compound 43.
1H NMR (CD3OD, 400 MHz) δ 8.01 (s, 1H), 7.16–7.10 (m, 2H), 7.06–6.99 (m, 2H), 5.35 (t, J = 6.4 Hz, 1H), 4.94 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.76 (br s, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.31 (s, 3H), 2.05–2.02 (m, 1H), 1.71–1.66 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.92–0.88 (m, 2H). HRMS calculated for C23H27N5O2I (M + H) +: 532.1210; found 532.1202.
N-(3,4-Difluorophenethyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-9H-purin-6-amine (53)
Compound 53 (69%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.03 (s, 1H), 7.22–7.05 (m, 3H), 5.36 (t, J = 6.0 Hz, 1H), 4.95 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.78 (br s, 2H), 2.96 (t, J = 6.8 Hz, 2H), 2.03–2.02 (m, 1H), 1.72–1.68 (m, 1H), 1.51 (s, 3H), 1.25 (s, 3H), 0.93–0.89 (m, 2H). HRMS calculated for C22H22N5O2F2I (M + H) +: 554.0865; found 554.0874.
9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-iodo-N-((1S,2R)-2-phenylcyclopropyl)-9H-purin-6-amine (54)
Compound 54 (73%) was prepared from compound 41 following the same method for compound 43. 1H NMR (CD3OD, 400 MHz) δ 8.05 (s, 1H), 7.37–7.28 (m, 2H), 7.21–7.17 (m, 1H), 5.37 (t, J = 6.0 Hz, 1H), 4.97 (s, 1H), 4.72 (d, J = 7.2 Hz, 1H), 3.07 (br s, 1H), 2.18–2.14 (m, 1H), 2.08–2.02 (m, 1H), 1.72–1.68 (m, 1H), 1.52 (s, 3H), 1.40–1.28 (m, 2H), 1.26 (s, 3H), 0.94–0.88 (m, 2H). HRMS calculated for C23H25N5O2I (M + H) +: 530.1053; found 530.1063.
9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-N-(3-fluorophenethyl)-2-(phenylethynyl)-9H-purin-6-amine (56)
PdCl2(PPh3)2 (7.8 mg, 0.01 mmol), CuI (1.0 mg, 0.005 mmol), phenylacetylene (36 μL, 0.33 mmol) and triethylamine (76 μL, 0.54 mmol) were added to a solution of compound 43 (29.4 mg, 0.05 mmol) in anhydrous DMF (1.0 mL), and the mixture stirred at room temperature overnight. Solvent was evaporated under vacuum, and the residue was purified on flash silica gel column chromatography (ethyl acetate:MeOH = 100:1) to give the compound 56 (23 mg, 83%) as a syrup. 1H NMR (CD3OD, 400 MHz) δ 8.03 (s, 1H), 7.68–7.63 (m, 4H), 7.59–7.54 (m, 1H), 7.47–7.43 (m, 1H), 7.32–7.27 (m, 1H), 7.14–7.80 (m, 1H), 6.95–6.88 (m, 1H), 5.37 (t, J = 6.0 Hz, 1H), 5.09 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.91 (br s, 2H), 3.04 (t, J = 7.2 Hz, 2H), 2.12–2.07 (m, 1H), 1.81–1.76 (m, 1H), 1.53 (s, 3H), 1.26 (s, 3H), 1.01–0.91 (m, 2H). HRMS calculated for C30H29N5O2F (M + H) +: 510.2305; found 510.2296.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-(phenylethynyl)-9H-purin-6-yl)amino)ethyl)phenol (57)
Compound 57 (80%) was prepared from compound 44 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.67–7.65 (m, 2H), 7.44–7.43 (m, 3H), 7.13 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 5.35 (d, J = 6.4 Hz, 1H), 5.07 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.83 (br s, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.09–2.02 (m, 1H), 1.79–1.75 (m, 1H), 1.52 (s, 3H), 1.25 (s, 3H), 0.98–0.89 (m, 2H). HRMS calculated for C30H30N5O3 (M + H) +: 508.2349; found 508.2352.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-(phenylethynyl)-9H-purin-6-yl)amino)ethyl)-2-fluorophenol (58)
Compound 58 (82%) was prepared from compound 45 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.02 (s, 1H), 7.67–7.66 (m, 2H), 7.44–7.43 (m, 3H), 7.13 (d, J = 8.0 Hz, 1H), 7.01–6.90 (m, 1H), 6.72 (d, J = 8.0 Hz, 1H), 5.36 (t, J = 6.0 Hz, 1H), 5.80 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.84 (br s, 2H), 2.92 (t, J = 7.2 Hz, 1H), 2.10–2.02 (m, 1H), 1.78–1.76 (m, 1H), 1.52 (s, 3H), 1.25 (s, 3H), 1.00–0.90 (m, 2H). HRMS calculated for C30H29N5O3F (M + H) +: 526.2254; found 526.2260.
N-(4-Aminophenethyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-(phenylethynyl)-9H-purin-6-amine (59)
Compound 59 (80%) was prepared from compound 46 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.21 (s, 1H), 7.68–7.66 (m, 2H), 7.45–7.43 (m, 3H), 7.08 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 5.36 (t, J = 6.0 Hz, 1H), 5.08 (s, 1H), 4.70 (d, J = 7.2 Hz, 1H), 3.82 (br s, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.10–2.06 (m, 1H), 1.80–1.76 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 1.00–0.90 (m, 2H). HRMS calculated for C30H31N6O2 (M + H) +: 507.2508; found 507.2511.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-(phenylethynyl)-9H-purin-6-yl)amino)ethyl)benzene-1,2-diol (60)
Compound 60 (78%) was prepared from compound 47 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.02 (s, 1H), 7.59–7.55 (m, 2H), 7.45–7.44 (m, 3H), 6.74–6.69 (m, 2H), 6.63–6.61 (m, 1H), 5.36 (t, J = 6.0 Hz, 1H), 5.08 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.83 (br s, 2H), 2.87 (d, J = 7.2 Hz, 2H), 2.10–2.06 (m, 1H), 1.79–1.76 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 1.00–0.90 (m, 2H). HRMS calculated for C30H30N5O4 (M + H) +: 524.2298; found 524.2295.
5-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-(phenylethynyl)-9H-purin-6-yl)amino)ethyl)-2-methoxyphenol (61)
Compound 61 (83%) was prepared from compound 48 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.21 (s, 1H), 7.68–7.66 (m, 2H), 7.45–7.44 (m, 3H), 6.84–6.78 (m, 2H), 6.74–6.72 (m, 1H), 5.36 (t, J = 6.0 Hz, 1H), 5.08 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.84–3.82 (m, 5H), 2.89 (t, J = 7.2 Hz, 2H), 2.10–2.06 (m, 1H), 1.79–1.77 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 0.99–0.90 (m, 2H). HRMS calculated for C31H32N5O4 (M + H) +: 538.2454; found 538.2459.
4-(2-((9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-(phenylethynyl)-9H-purin-6-yl)amino)ethyl)-2-methoxyphenol (62)
Compound 62 (81%) was prepared from compound 49 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.67–7.66 (m, 2H), 7.44–7.45 (m, 3H), 6.90 (s, 1H), 6.70–6.71 (m, 2H), 5.36 (d, J = 6.0 Hz, 1H), 5.07 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.87–3.82 (m, 5H), 2.93 (t, J = 7.2 Hz, 2H), 2.10–2.07 (m, 1H), 1.77–1.76 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 0.99–0.91 (m, 2H). HRMS calculated for C31H32N5O4 (M + H) +: 538.2454; found 538.2453.
N-(3,4-Dimethoxyphenethyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa [3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-2-(phenylethynyl)-9H-purin-6-amine (63)
Compound 63 (78%) was prepared from compound 50 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.21 (s, 1H), 7.59–7.54 (m, 2H), 7.45–7.43 (m, 3H), 6.93 (s, 1H), 6.84–6.83 (m 2H), 5.36 (d, J = 5.6 Hz, 1H), 5.07 (s, 1H), 4.70 (d, J = 6.8 Hz, 1H), 3.88 (br s, 2H), 3.79 (s, 3H), 3.78 (s, 3H), 2.95 (t, J = 7.2 Hz, 2H), 2.10–2.06 (m, 1H), 1.79–1.76 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 1.00–0.90 (m, 2H). HRMS calculated for C32H34N5O4 (M + H) +: 552.2611; found 552.2612.
2-((5-Chlorothiophen-2-yl)ethynyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-N-phenethyl-9H-purin-6-amine (64)
Compound 64 (80%) was prepared from compound 42 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.22 (s, 1H), 7.34–7.27 (m, 5H), 7.22–7.17 (m, 1H), 7.03 (d, J = 4.0 Hz, 1H), 5.35 (t, J = 6.0 Hz, 1H), 5.06 (s, 1H), 4.69 (d, J = 7.2 Hz, 1H), 3.91 (br s, 2H), 3.01 (t, J = 7.2 Hz, 2H), 2.12–2.06 (m, 1H), 1.80–1.75 (m, 1H), 1.53 (s, 3H), 1.26 (s, 3H), 1.00–0.91 (m, 2H). HRMS calculated for C28H27N5O2SCl (M + H) +: 532.1574; found 532.1577.
2-((5-Chlorothiophen-2-yl)ethynyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydro cyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-N-(3-fluorophenethyl)-9H-purin-6-amine (65)
Compound 65 (82%) was prepared from compound 43 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.23 (s, 1H), 7.33–7.26 (m, 2H), 7.12–7.02 (m, 3H), 6.92 (t, J = 8.8 Hz, 1H), 5.35 (t, J = 5.6 Hz, 1H), 5.05 (s, 1H), 4.69 (d, J = 7.2 Hz, 1H), 3.88 (br s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.09–2.07 (m, 1H), 1.78–1.77 (m, 1H), 1.52 (s, 3H), 1.26 (s, 3H), 0.98–0.92 (m, 2H). HRMS calculated for C28H26N5O2ClSF (M + H) +: 550.1480; found 550.1478.
9-((3aR,3bR,4aS,5R,5aS)-2,2-Dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-N-(3-fluorophenethyl)-2-((5-fluorothiophen-2-yl)ethynyl)-9H-purin-6-amine (66)
Compound 66 (81%) was prepared from compound 43 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.23 (s, 1H), 7.32–7.19 (m, 2H), 7.13–7.06 (m, 2H), 6.95–6.90 (m, 1H), 6.64–6.62 (m, 1H), 5.35 (t, J = 6.0 Hz, 1H), 5.06 (s, 1H), 4.69 (d, J = 7.2 Hz, 1H), 3.88 (br s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.11–2.06 (m, 1H), 1.80–1.75 (m, 1H), 1.53 (s, 3H), 1.26 (s, 3H), 1.00–0.90 (m, 2H). HRMS calculated for C28H26N5O2SF2 (M + H) +: 534.1775; found 534.1781.
N-(3-Chlorophenethyl)-2-((5-chlorothiophen-2-yl)ethynyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-9H-purin-6-amine (67)
Compound 67 (78%) was prepared from compound 51 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.23 (s, 1H), 7.34–7.33 (m, 1H), 7.28–7.10 (m, 2H), 7.03–7.02 (m, 1H), 5.35 (t, J = 6.0 Hz, 1H), 5.05 (s, 1H), 4.69 (d, J = 6.8 Hz, 1H), 3.88 (br s, 2H), 3.01 (t, J = 7.2 Hz, 2H), 2.11–2.07 (m, 1H), 1.79–1.77 (m, 1H), 1.53 (s, 3H), 1.26 (s, 3H), 0.99–0.91 (m, 2H). HRMS calculated for C28H26N5O2SCl (M + H) +: 566.1184; found 566.1181.
2-((5-Chlorothiophen-2-yl)ethynyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydro cyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-N-(3-methylphenethyl)-9H-purin-6-amine (68)
Compound 68 (83%) was prepared from compound 52 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.20 (s, 1H), 7.31 (d, J = 3.2 Hz, 1H), 7.15–7.06 (m, 3H), 7.01 (d, J = 4.0 Hz, 2H), 5.34 (d, J = 5.6 Hz, 1H), 5.04 (s, 1H), 4.69 (d, J = 6.8 Hz, 1H), 3.85 (br s, 2H), 2.95 (d, J = 7.2 Hz, 2H), 2.30 (s, 3H), 2.09–2.06 (m, 1H), 1.77–1.76 (m, 1H), 1.52 (s, 3H), 1.25 (s, 3H), 0.98–0.90 (m, 2H). HRMS calculated for C29H29N5O2SCl (M + H) +: 546.1731; found 546.1733.
2-((5-Chlorothiophen-2-yl)ethynyl)-N-(3,4-difluorophenethyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-9H-purin-6-amine (69)
Compound 69 (82%) was prepared from compound 53 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 8.24 (s, 1H), 7.33 (d, J = 2.4 Hz, 1H), 7.27–7.09 (m, 3H), 7.03 (d, J = 2.0 Hz, 1H), 5.35 (t, J = 6.0 Hz, 1H), 5.06 (s, 1H), 4.69 (d, J = 7.2 Hz, 1H), 3.87 (br s, 2H), 3.00 (d, J = 7.2 Hz, 1H), 2.06–2.09 (m, 1H), 1.80–1.77 (m, 1H), 1.53 (s, 3H), 1.26 (s, 3H), 0.99–0.91 (m, 2H). HRMS calculated for C28H25N5O2F2ClS (M + H) +: 568.1386; found 568.1392.
2-((5-Chlorothiophen-2-yl)ethynyl)-9-((3aR,3bR,4aS,5R,5aS)-2,2-dimethylhexahydrocyclopropa[3,4]cyclopenta[1,2-d][1,3]dioxol-5-yl)-N-((1S,2R)-2-phenylcyclopropyl)-9H-purin-6-amine (70)
Compound 70 (81%) was prepared from compound 54 following the same method for compound 56. 1H NMR (CD3OD, 400 MHz) δ 7.95 (s, 1H), 7.52–7.28 (m, 4H), 7.25 (d, J = 4.0 Hz, 1H), 6.89 (d, J = 4.0 Hz, 1H), 5.36–5.31 (m, 1H), 5.13 (s, 1H), 4.62 (d, J = 6.8 Hz, 1H), 3.28 (br s, 1H), 2.25–2.20 (m, 1H), 2.14–2.11 (m, 1H), 1.72–1.69 (m, 1H), 1.57 (s, 3H), 1.49–1.44 (m, 1H), 1.34–1.26 (m, 4H), 1.04–0.92 (m, 2H). HRMS calculated for C29H27N5O2ClS (M + H) +: 544.1574; found 544.1567.
Supplementary Material
Acknowledgments:
We acknowledge support from the NIDDK Intramural Research Program (ZIA DK031117-28 and ZIA DK075063) and the National Institutes of Health (NHLBI R01 grant HL133589). We thank John Lloyd (NIDDK) for mass spectral determinations, Robert O’Connor (NIDDK) for NMR spectra, and Naili Liu for technical assistance with in vivo experiments. We thank Dr. Bryan L. Roth (Univ. North Carolina at Chapel Hill) and National Institute of Mental Health’s Psychoactive Drug Screening Program (Contract #HHSN-271-2008-00025-C) for screening data. This work utilized the computational resources of the NIH HPC Biowulf cluster. (http://hpc.nih.gov)
Abbreviations:
- AN
acetonitrile
- AR
adenosine receptor
- BM
binding mode
- CHO
Chinese hamster ovary
- Cl-IB-MECA
2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine
- DCM
dichloromethane
- DIPEA
diisopropylethylamine
- DMF
N,N-dimethylformamide
- DPTN
N-(4-(3,5-dimethylphenyl)-5-(pyridin-4-yl)thiazol-2-yl)nicotinamide
- EL
extracellular loop
- IL
intracellular loop
- GPCR
G protein-coupled receptor
- HEK
human embryonic kidney
- MD
molecular dynamics
- NECA
5′-N-ethylcarboxamidoadenosine
- PDSP
NIMH Psychoactive Drug Screening Program
- RMSD
root mean squared deviation
- TBAP
tetrabutylammonium dihydrogen phosphate
- THP
tetrahydropyran
- TM
transmembrane helical domain
- TSPO
translocator protein
Footnotes
Supporting information available:
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00235
NMR and mass spectra, HPLC analysis, pharmacological studies, off-target screening, and molecular modeling results (PDF)
Coordinates information for structure representation (PDB): docking pose of compound 39 (MRS5698) at hA3AR
Coordinates information for structure representation (PDB): docking pose of compound 39 (MRS5698) at mA3AR
Coordinates information for structure representation (PDB): last frame of the MD simulation of the 15-hA3AR complex (just protein and ligand coordinates)
Coordinates information for structure representation (PDB): last frame of the MD simulation of the 8-hA3AR complex (just protein and ligand coordinates)
Coordinates information for structure representation (PDB): last frame of the MD simulation of the 15-mA3AR complex (just protein and ligand coordinates)
Coordinates information for structure representation (PDB): last frame of the MD simulation of the 8-mA3AR complex (just protein and ligand coordinates)
MD video of 15 binding to the hA3AR (MP4)
MD video of 8 binding to the hA3AR (MP4)
MD video of 15 binding to the mA3AR (MP4)
MD video of 8 binding to the mA3AR (MP4)
Molecular formula strings and some data (CSV)
References
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