2-Oxabicyclo[2.2.2]octane as a new bioisostere of the phenyl ring

The phenyl ring is a basic structural element in chemistry. Here, we show the design, synthesis, and validation of its new saturated bioisostere with improved physicochemical properties − 2-oxabicyclo[2.2.2]octane. The design of the structure is based on the analysis of the advantages and disadvantages of the previously used bioisosteres: bicyclo[1.1.1]pentane, bicyclo[2.2.2]octane, and cubane. The key synthesis step is the iodocyclization of cyclohexane-containing alkenyl alcohols with molecular iodine in acetonitrile. 2-Oxabicyclo[2.2.2]octane core is incorporated into the structure of Imatinib and Vorinostat (SAHA) drugs instead of the phenyl ring. In Imatinib, such replacement leads to improvement of physicochemical properties: increased water solubility, enhanced metabolic stability, and reduced lipophilicity. In Vorinostat, such replacement results in a new bioactive analog of the drug. This study enhances the repertoire of available saturated bioisosteres of (hetero)aromatic rings for the use in drug discovery projects.

The phenyl ring is a basic structural element in chemistry.Here, we show the design, synthesis, and validation of its new saturated bioisostere with improved physicochemical properties − 2-oxabicyclo[2.2.2]octane.The design of the structure is based on the analysis of the advantages and disadvantages of the previously used bioisosteres: bicyclo[1.1.1]pentane,bicyclo[2.2.2] octane, and cubane.The key synthesis step is the iodocyclization of cyclohexane-containing alkenyl alcohols with molecular iodine in acetonitrile.2-Oxabicyclo[2.2.2]octane core is incorporated into the structure of Imatinib and Vorinostat (SAHA) drugs instead of the phenyl ring.In Imatinib, such replacement leads to improvement of physicochemical properties: increased water solubility, enhanced metabolic stability, and reduced lipophilicity.In Vorinostat, such replacement results in a new bioactive analog of the drug.This study enhances the repertoire of available saturated bioisosteres of (hetero)aromatic rings for the use in drug discovery projects.
The phenyl ring is a basic structural element in chemistry.It is one of the most common structural motifs in natural products 1 and bioactive compounds 2,3 .Moreover, more than five hundred drugs contain a fragment of para-substituted phenyl ring (Fig. 1a, b) 4 , including the well-known to everyone Paracetamol.However, organic compounds with more than two phenyl rings often suffer from poor solubility [5][6][7] .

Design
In the design of the improved phenyl bioisostere, we first needed to keep the advantages of the previously used cores: their conformational rigidity, metabolic stability, non-chirality, and collinearity of vectors (φ = 180°).At the same time, we needed to address their drawbacks: C-C distance, and lipophilicity.Considering the possible saturated structures (for the details of the design, please, see Supplementary Iinformation, page 5, Supplementary Fig. 1.), we decided to select the bicyclo[2.2.2]octane scaffold, because of its appropriate C-C distance, and decorate it with an oxygen atom.In particular, replacing one carbon atom with oxygen would give 2-oxabicyclo[2.2.2]octane with similar geometry and reduced lipophilicity (Fig. 1c).Also, this structure should be chemically stable as a simple derivative of tetrahydropyran.

Optimization
Synthesis of the 2-oxabicyclo[2.2.2]octane core has been previously reported.In 2014, Singh and Fukuda obtained compound 1 from diethyl malonate (2) in 15 steps using alkylation as a key reaction (Fig. 1d) 50 .In 2019, Harrison synthesized compound 3 from ester 4 in six steps employing an intramolecular Michael addition (Fig. 1d) 54 .The latter approach was limited only to aromatic substituents.We, however, needed a general modular method that would give 2-oxabicyclo[2.2.2]octanes with one or two functional groups that could be subsequently modified to obtain a wide variety of derivatives -bioisosteres of the monoand para-substituted phenyl rings.
Previously, we showed that smaller 2-oxabicyclo[2.1.1]hexanecould be assembled via the iodocyclization reaction of the corresponding cyclobutane alkenyl alcohols 56 .The reaction proceeded with I 2 /NaHCO 3 in the mixture of water and MeOtBu at room temperature.We hoped that similar cyclization would also take place with cyclohexane 5 (please, see its preparation below).However, under analogous conditions the expected product 6 was not formed (Table 1, entry 1).We repeated the reaction several times varying the time and the temperature, however, with the same negative outcome (Table 1, entries 2-4).The addition of the iodine molecule to the double C=C bond did take place, but the cyclization failed to occur.
Subsequently, we realized that in contrast to the already conformationally preorganized small cyclobutane, the larger and more flexible cyclohexane ring should adopt the highly energetic boat  1).The resulting entropic penalty seems to prevent the cyclization from occuring.We also tried other combinations of solvents with no success, however (Table 1, entries 5-8).Finally, we used solely dipolar aprotic solvents.Indeed, in dimethyl formamide, the formation of traces of the needed product was finally seen (Table 1, entry 9).A similar result was observed in dimethyl sulfoxide and N-methyl pyrrolidone (

Scalable synthesis
Having a working procedure in hand, we studied its scalability.The whole synthesis scheme commenced from the commercially available ketoester 7 (ca.3€/g, Fig. 2).Wittig reaction gave alkene 8 in 59% yield.
Treatment of the latter with LDA/methyl formate followed by the reduction of the intermediate aldehyde with NaBH 4 gave alcohol 5 in 86% combined yield.Finally, the key iodocyclization was attempted on a multigram scale.Pure iodide 6 was obtained as a white crystalline solid after column chromatography with a 36% yield.Despite a rather moderate yield, this protocol allowed us to obtain 135 g of the product in a single run.

Scope
Next, we studied the generality of the developed protocol.Treatment of alkene 8 with LDA/acetaldehyde gave the intermediate alcohol that was used in the subsequent iodocyclization under the developed conditions.The expected iodide 9 was isolated in 50% yield after column chromatography (Fig. 3a).Initially, we isolated the intermediate alcohol, but subsequently, we understood that performing the twostep procedure with a simple solvent swap ensured a better yield of the final product.
The reaction with aliphatic (10-12), aromatic (13-17), and heterocyclic (18-27) aldehydes gave the corresponding 2-oxabicyclo[2.2.2]octanes in good yields.Functional groups such as nitro, trifluoromethoxy, trifluoromethyl, nitrile, and halogen atoms tolerated the reaction conditions.The protocol was not without limitations, however.We could not obtain products 28, and 29 with thiazole and triazole heterocycles, due to the formation of complex mixtures (Fig. 3a).Ketones could also be used as electrophiles instead of aldehydes.As a representative example, the reaction of alkene 8 with LDA/ acetone followed by iodocyclization gave dimethyl-substituted product 30 in 81% yield.The structure of 30 was confirmed by X-ray crystallographic analysis (Fig. 3b, Supplementary Data 1).A reduction of 8 followed by iodocyclization gave iodide 31 in 58% yield.Interestingly, the cyclization was not efficient at room temperature, and required heating.Alkylation of 8 with MeI or BnOCH 2 Cl followed by reduction and iodocyclization gave the disubstituted products 32, 33 in 59-64% yield (Fig. 3b).
We also tried to assemble a 2-azabicyclo[2.2.2]octane skeleton using the developed strategy.An attempted iodocyclization of alkene 40 did not lead to the formation of the cyclic iodide 41 neither at room temperature nor under heating (Fig. 3d).However, the analogous reaction of the bridgehead-substituted alkene 42 at room temperature did give the needed product 43 in 31% yield.Under heating, the yield was improved to 41%.

Modifications
Several representative modifications of the obtained iodides were undertaken to obtain various monoand bifunctional 2-oxabicyclo[2.2.2]octanes ready for direct use in medicinal chemistry projects.Treatment of iodide 31 with potassium thioacetate followed by oxidation with NCS gave aliphatic sulfonyl chloride 44 in 85% yield.The reaction of 31 with potassium acetate and the subsequent alkali hydrolysis provided valuable alcohol 45.Oxidation of the latter afforded carboxylic acid 46 in 89% yield (Fig. 4).
Hydrogenative reduction of the C-I bond in iodide 6 followed by saponification of the ester group gave methyl acid 47.The Curtius reaction of the latter resulted in amine 48.The reaction of iodide 6 with LiAlH 4 gave alcohol 49 in 90% yield.O-Mesylation and the subsequent reaction with LiBr provided bromide 50.Swern oxidation of alcohol 49 gave aldehyde 51 in 63% yield.Isomeric methyl-substituted 2-oxabicyclo[2.2.2]octanes were obtained from iodide 32.Its reaction with sodium azide followed by the reduction formed amine 52.The reaction of iodide 32 with potassium acetate and hydrolysis gave alcohol 53 -isomer of alcohol 49.Oxidation of 53 formed carboxylic acid 54 -isomer of acid 47.Sulfonyl chloride 55 was also obtained from iodide 32 via a two-step procedure (Fig. 4).
From iodide 6 we also synthesized various bifunctional linkers for incorporation into bioactive compounds instead of the para-substituted phenyl ring.Saponification of ester 6 provided carboxylic acid 56 in 90% yield.The subsequent Curtius reaction afforded N-Boc iodide 57 in 87% yield.The structure of 57 was confirmed by X-ray crystallographic analysis (Supplementary Data 2).The reaction of the latter with potassium acetate, followed by ester hydrolysis (via 58) and N-Boc acidic deprotection gave amino alcohol 59.Oxidation of the alcohol group in 58 gave N-Boc protected amino acid 60a saturated analog of the para-aminobenzoic acid.The reaction of iodide 57 with NaN 3 (via azide 61) followed by reduction of the azide group formed diamine 62.The reaction of iodide 6 with NaN 3 (via azide 63), the subsequent reduction (via 64), N-Boc protection, and saponification gave another N-Boc protected amino acid 65.The Curtius reaction of the latter provided N-Boc diamine 66isomer of diamine 62.The reaction of iodide 6 with sodium azide followed by extensive reduction of the intermediate azide with LiAlH 4 gave amino alcohol 67.The structure of 67 was confirmed by X-ray crystallographic analysis (Supplementary Data 3).The reaction of iodide 6 with potassium acetate (via 68) followed by saponification of the ester group and oxidation gave linker 69.Its structure was also confirmed by X-ray crystallographic analysis (Supplementary Data 4).Worth noting that all the above-described syntheses depicted in Fig. 4 were realized on a multigram scale.

Chemical stability
We also examined the thermal and chemical stability of the synthesized 2-oxabicyclo[2.2.2]octanes.As representative examples, we selected three molecules: isomeric acids 47, 54, and amine 52.All 2-oxabicyclo[2.2.2]octanes were crystalline solids that were stable in air.We stored them in stock at room temperature in closed vials and observed no changes according to 1 H NMR after one year.Also, the compounds remained stable even under heating at 100 °C for five minutes.Treatment of the selected 2-oxabicyclo[2.2.2]octanes with aq. 1 M HCl, or aq. 1 M NaOH at room temperature for 1 h resulted in no decomposition either.

Crystallographic analysis
Next, we compared the geometric properties of 2-oxabicyclo[2.2.2] octanes with those of the para-substituted phenyl ring, and the previously used bioisosteres -bicyclo[2.2.2]octanes.For this purpose, we measured two C-C distances r and d to see the overall similarity of cores; and two angles φ 1 and φ 2 to estimate the collinearity of exit vectors (Fig. 5a).
In short summary, the replacement of the methylene group for an oxygen atom in the bicyclo[2.2.2]octane core did not affect its threedimensional geometry.Moreover, the formed 2-oxabicyclo[2.2.2] octane core resembled well the para-substituted phenyl ring, as the geometric parameters r, d, φ 1, and φ 2 remained very similar (please, see SI, page 277, Supplementary Fig. 8).

The acidity of functional groups
We also studied the influence of the replacement of the methylene group for an oxygen atom in the bicyclo[2.2.2]octane skeleton on the electronic properties.Towards this goal, we measured experimental pK a values of isomeric 2-oxabicyclo[2.2.2]octane carboxylic acids 47 and 54, bicyclo[2.2.2]octane carboxylic acid 84, and para-methyl benzoic acid (83) as a reference (Fig. 6).Replacement of the methylene group in 84 for the oxygen atom at the distal γ-position notably increased its acidity from pK a = 5.6 to 4.4 (47).However, analogous replacement at the β-position increased the acidity even more to pK a = 4.1 (54).
Important to mention that the acidity of aromatic carboxylic acid 83 and 2-oxabicyclo[2.2.2]octane 47 were almost identical (Fig. 6).The replacement of the phenyl ring in acid 83 with the bicyclo[2.2.2]octane core reduced the acidity: pK a = 4.5 (83) vs 5.6 (84).However, incorporation of the β-oxygen atom into the latter ideally restored it: pK a = 4.4 (47).Because the acidity/basicity of functional groups is often responsible for the potency, selectivity, and toxicity of bioactive compounds 65 , the fine-tuning of the pK a by replacing the phenyl ring with isomeric 2-oxabicyclo[2.2.2]octanes could become a preferred solution.

Incorporation into drugs
To demonstrate the practical utility of the 2-oxabicyclo[2.2.2]octane scaffold, we incorporated it into the structure of anticancer drugs Imatinib, and Vorinostat (SAHA) instead of the paraand mono-substituted phenyl rings, correspondingly (Figs. 7 and 8).
The reaction of iodide 56 with N-methyl piperazine, followed by acylation with the substituted aniline gave compound 85a saturated analog of Imatinib (Fig. 7).For comparison, we also synthesized compound 86 with the bicyclo[2.2.2]octane core (please, see SI, pages 52-54).The commercialized drug Imatinib is used in practice as a mesylate salt.However, to estimate the impact of the replacement of the phenyl ring with bioisosteres on the physicochemical properties, we prepared and studied all three compounds, -85, 86, Imatinib, -as free bases.
To study the replacement of the phenyl ring with saturated bioisosteres on lipophilicity, we used two characteristics: calculated (clogP) 66  In summary, the replacement of the para-substituted phenyl ring in Imatinib with common bicyclo[2.2.2]octane core (86) led to an undesired three-times decrease in water solubility.At the same time, analogous replacement with 2-oxabicyclo[2.2.2]octane (85) resulted in an improvement of all measured physicochemical parameters: increased solubility, enhanced metabolic stability, and reduced lipophilicity.

Biological activity
Finally, to answer a key question, -whether the 2-oxabicyclo[2.2.2] octane core could indeed mimic the phenyl ring in bioactive compounds, we measured the biological activity of Imatinib versus its analogs 85, 86; and Vorinostat versus its analogs 88, 89.
We studied the inhibitory effect of Imatinib, Staurosporine, and compounds 85, 86 on the catalytic activity of ABL1 kinase.While the expected activity of Imatinib and Staurosporine was confirmed; we did not observe any significant inhibitory effect of compounds 85, 86 on the ABL1 kinase (please, see SI, pages 294, 295; Supplementary Figs.13-15).The observed results correlate well with the previous study by Nicolaou, Vourloumis, and Stepan who demonstrated that the replacement of the para-substituted phenyl ring in Imatinib with various saturated cyclic cores, including bicyclo[1.1.1]pentaneand cubane, led to a dramatic loss of potency against the ABL1 kinase 67 .
To study the biological activity of Vorinostat and its analogs 88, 89, we evaluated their effect on human hepatocellular carcinoma cells HepG2 by fluorescent microscopy (please, see SI, pages 296-300; Supplementary Figs.16-19).The cells were incubated with the compounds for 48 hours.Staining with specific dyes revealed that all three compounds promoted caspase-dependent cell death, -apoptosis,that further precipitated in necrosis when the cellular membrane lost its integrity.Vorinostat treatment resulted in 7.2% and 12.2% of apoptotic cells upon incubation at concentrations 5 μΜ and 50 μΜ respectively (Fig. 9).Analogs 88 and 89 demonstrated similar efficacy only at 50 μΜ.
These primary biological results (Fig. 9) suggested that Vorinostat and both its analogs 88, 89 could have similar cytotoxic and cytostatic

Virtual libraries
To analyze how the replacement of the para-substitued phenyl ring with 2-oxabicyclo[2.2.2]octane affects 3D-shape of organic compounds, we generated two virtual libraries based on Cand N-terminus modifications of para-aminobenzoic acid and its 2-oxabicyclo[2.2.2] octane-containing analog.Each library contained 5000 molecules (Supplementary Data 6, Supplementary Data 7).According to principal moments of inertia (PMI) plots, both libraries occupied essentially the same region in 3D-chemical space.The same was true for FDAapproved drugs Aminopterin, Conivaptan, Deferasifox, Tetracaine, and their 2-oxabicyclo[2.2.2]octane-containing analogs (for details, please see SI, pages 301-304; Supplementary Table 8, Supplementary Figs.20 and 21).
In conclusion, we have designed, synthesized, and characterized a new saturated bioisostere of the phenyl ring -2-oxabicyclo[2.2.2] octane.In the design of the structure, we kept all advantages of the previously used cores (bicyclo[1.1.1]pentane,bicyclo[2.2.2]octane, cubane): conformational rigidity, metabolic stability, non-chirality, and collinearity of the exit vectors (Fig. 1c).In addition, we addressed their disadvantages: C-C distance and lipophilicity (Fig. 1c).Thus the 2oxabicyclo[2.2.2]octane scaffold designed here was synthesized from available starting materials on a multigram scale (Table 1) -up to 135 g in one run (Fig. 2).The key synthesis step was the iodocyclization of cyclohexane-containing alkenyl alcohols with molecular iodine in acetonitrile (Figs. 2 and 3).Crystallographic analysis revealed its high similarity with the para-substituted phenyl ring (Fig. 5).2-Oxabicyclo[2.2.2]octane core was incorporated into the structure of Imatinib and Vorinostat drugs instead of the para-substituted and the mono-substituted phenyl rings, correspondingly (Figs. 7 and 8).In the case of Imatinib, the formed saturated analog 85 possessed improved physicochemical properties over the drug: increased water solubility, enhanced metabolic stability, and reduced lipophilicity (Fig. 7).In the case of Vorinostat (SAHA), the formed saturated analog 88 exhibited a similar biological activity compared to that of the drug (Fig. 9).
This study enhances the repertoire of available saturated bioisosteres of (hetero)aromatic rings for use in drug discovery projects.

Table 1 |
Optimization of the synthesis of compound 6