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Article Synthesis of Cyclobutane Analogue 4: Preparation of and Carbocyclic Derivatives

Noha Hasaneen 1 , Abdelaziz Ebead 2, Murtaza Hassan 1, Hanan Afifi 3, Howard Hunter 1, Edward Lee-Ruff 1,*, Nadia S. El-Gohary 4, Azza R. Maarouf 4 and Ali A. El-Emam 4 1 Department of Chemistry, Faculty of Science, York University, Toronto, ON M3J 1P3, Canada 2 Chemistry Department, Faculty of Science, Arish University, Arish, Egypt 3 Basic Science Department, Faculty of Industrial Education, Beni-Suef University, Beni-Suef, Egypt 4 Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt * Correspondence: leeruff@yorku.ca; Tel.: 416-736-5443

 Academic Editors: Katherine L. Seley-Radtke and Theodore K. Dayie  Received: 2 August 2019; Accepted: 29 August 2019; Published: 5 September 2019 Abstract: The coupling of 2-bromo-3-benzoyloxycyclobutanone with purine under basic conditions produces two regioisomers consisting of the N-7 and N-9 alkylated products in equal amounts in their racemic forms. The distribution of the isomers is consistent with the charge delocalization between the N-7 and N-9 positions of the purinyl anion. The structural assignments and relative stereochemistry of each regioisomer were based on 1 and 2D NMR techniques. The relative stereochemistry of the C-2 and C-3 substituents in each regioisomer was the trans orientation consistent with steric factors in the coupling step. The N-9 regioisomer was reduced with sodium borohydride to give the all trans cyclobutanol as the major product in a stereoselective manner. The alcohol was debenzoylated with sodium methoxide in a transesterification step to give the nucleoside analogue. The regioisomeric pyrimidine were prepared by Vorbrüggen coupling of the 3-hydroxymethylcyclobutanone triflate with either thymine or uracil followed by stereoselective hydride addition. Regiospecificity of the coupling at the N-1 position was observed and stereoselective reduction to the trans-disubstituted cyclobutanol structure assignments was based on NMR data.

Keywords: nucleoside analogs; cyclobutanes; purine; uracil; thymine

1. Introduction Nucleosides constitute the components of nucleic acids. These are linked by phosphate units in the structures of DNA and RNA. Structural modifications of nucleosides (nucleoside analogs) result in changes to chemical and biological processes involving nucleic acids, resulting in their physiological properties as antiviral and anticancer agents [1,2]. Carbocyclic derivatives in which the ribose ring is replaced by a carbocycle enhances the metabolic stabilities of these potential drugs [3]. The cyclopentenyl derivatives Carbovir [4] and [5] developed in the 1990s are some of the early examples of effective antiviral agents. With the observation of potent antiviral activity of the natural occurring Oxetanocin A, [6] the development and commercialization of the synthetic carbocyclic analogues, Cyclobut-A and Cyclobut-G (Lobucavir), as well as related analogs were realized [7–13]. In view of rapid mutations of viruses becoming resistant to current drug therapies, the quest for new Reverse Transcriptase (RT) inhibitors with structural modifications is an ongoing pursuit. In the current study we report the stereospecific synthesis of cyclobutanols to which a is attached to the C-2 position of the carbocycle, and the C-3 position by a methylene pendant in a cis orientation to the alcohol function. Furthermore, the regiochemistry of both N-7 and N-9 isomers of purinonucleosides produced in the coupling reaction was unambiguously assigned.

Molecules 2019, 24, 3235; doi:10.3390/molecules24183235 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 18

attached to the C-2 position of the carbocycle, and the C-3 position by a methylene pendant in a cis orientation to the alcohol function. Furthermore, the regiochemistry of both N-7 and N-9 isomers of Moleculespurinonucleosides2019, 24, 3235 produced in the coupling reaction was unambiguously assigned. 2 of 16 We have already described the coupling of 6-chloropurine to the C-2 position and the C-3 positionWe haveby a methylene already described pendant the in coupling recent publications of 6-chloropurine [14,15]. to In the theC-2 currentposition study and we the reportC-3 position on the bycoupling a methylene reactions pendant of in recent the publications parent purine [14, 15]. nucleobase In the current to study the weC report-2 position on the coupling of 3- reactionshydroxylmethylcyclobutanol, of the parent purine nucleobase and the coupling to the C-2 reactionsposition of ofpyrimidine 3-hydroxylmethylcyclobutanol, bases to a methylene bridged and the couplingC-3 cyclobutanol. reactions These of pyrimidine compounds bases constitute to a methylene novel bridgednucleosideC-3 analogscyclobutanol. with potential These compounds biological constituteactivities. novel nucleoside analogs with potential biological activities.

2. Results and Discussion

2.1. Synthesis of Purine Carbocyclic Analogues

2.1.1. N N-Alkylation-Alkylation of P Purineurine to Bromocyclobutanone 1 DirectDirect alkylation of heterocyclic bases (purine and pyrimidine) with alkyl halides is well documented [1616,,17]. Such Such an an approach approach also also bears bears some similarity to the reported reported glycosylation glycosylation procedure ofof RobinsRobins [ 18[18],], where where the the sodium sodium salts salts of of a purinea purine base base react react with withα-halo-sugars α-halo-sugars to give to give the correspondingthe corresponding glyconucleosides. glyconucleosides. Based on these these observations, observations, we we prepared prepared cyclobutanone cyclobutanone derivatives derivatives possessing possessing purine purine as asan anα- αsubstituent-substituent using using the the following following method method [19]. [19 The]. Thepota potassiumssium salt of salt purine of purine (2), produced (2), produced in situ in using situ usingKOH KOHin the in presence the presence of tris(dioxa of tris(dioxa-3,6-heptyl)amine-3,6-heptyl)amine (TDA (TDA-1)-1) in acetonitrile, in acetonitrile, was was treated treated with with α- αbromocyclobutanone-bromocyclobutanone 1 1andand resulted resulted in in the the formation formation of of the the NN-9-9 coupledcoupled product product 33 inin an 11.7% yield as well as the N-7N-7 regioisomerregioisomer 44 inin anan 11.2%11.2% yieldyield (Scheme(Scheme1 1)).. TheThe assignmentassignment ofof thethe attachedattached sitesite ofof 1 13 the purine derivatives 33 and 4 was established usingusing 1D1D 1HH-- and 13CC-NMR-NMR as as well well as as 2D 2D COSY, COSY, HSQC HSQC,, and HMBC experiments.

Scheme 1.1. Base assisted coupling of purine with Bromoketone 1..

A number of NMR studies have been reported [20–22] forfor thethe N-7N-7 andand N-9N-9 purinepurine alkylatedalkylated derivatives and certain patternspatterns emergeemerge forfor thethe 11H-H- andand 1313CC-chemical-chemical shifts. The The signals signals of H H-8,-8, C C-1’,-1’, C-8,C-8, and more significantlysignificantly C-4C-4 are shifted upfieldupfield for the N-9N-9 isomerisomer relativerelative toto thethe correspondingcorresponding signals for the N-7N-7 isomer. By contrast,contrast, the C-5C-5 signal for the N-9N-9 derivatives is deshielded relative to that for the N-7N-7 isomerisomer duedue toto thethe largerlarger stericsteric congestioncongestion existingexisting inin thethe N-7N-7 isomer.isomer. The regiochemistryregiochemistry of of3 3and and4 was4 was supported supported by theby the larger larger chemical chemical shift shift values values of H-8 of ( δH8.41-8 (δ ppm), 8.41 C-1’ppm), (δ C69.8-1’ (δ ppm), 69.8 ppm), C-8 (δ C146.8-8 (δ 146.8 ppm), ppm) and, C-4and (Cδ-160.24 (δ 160.2 ppm) ppm) signals signals for for the theN-7 Nisomer-7 isomer4 (Figures4 (Figure1s and1 and2) as2) comparedas compared to theto the H-8 H ( -δ88.12 (δ 8.12 ppm), ppm), C-1’ C (δ-1’68.2 (δ 68.2 ppm), ppm), C-8 (Cδ-1448 (δ ppm), 144 ppm) and, C-4 and (δ C150.8-4 (δ ppm)150.8 signalsppm) signals for the forN-9 thealkylated N-9 alkylated derivatives derivatives3 (Figures 3 3(Figure and4).sThis 3 and regiochemistry 4). This regiochemistry was also supported was also bysupported the chemical by the shift chemical of the C-5shift signal of the (δ C133.6-5 signal ppm) (δ of 133.63, which ppm) was of shifted3, which downfield was shifted relative downfield to the C-5relative signal to (theδ 124.1 C-5 ppm)signal for (δ the124.1N-7 ppm)component for the N4 -(Figures7 component1 and2 4, Table(Figures1). The1 and High 2, Table Resolution 1). The Mass High Spectrometry data (HRMS) was consistent with the molecular composition of both 3 and 4. Molecules 2019, 24, x 3 of 18

Resolution Mass Spectrometry data (HRMS) was consistent with the molecular composition of both 3 and 4.

Molecules 2019, 24, x 3 of 18 Molecules 2019, 24, 3235 3 of 16 Resolution Mass Spectrometry data (HRMS) was consistent with the molecular composition of both 3 and 4.

Figure 1. 13C-NMR spectrum for ketone 4.

13 FigureFigure 1.1. 13CC-NMR-NMR spectrum spectrum for ketone for ketone 4. 4.

Molecules 2019, 24, x 1 4 of 18 FigureFigure 2. 2.1H-NMRH-NMR spectrum spectrum for ketone for 4. ketone 4.

Figure 2. 1H-NMR spectrum for ketone 4.

13 FigureFigure 3 3.. 13C-C-NMRNMR spectrum spectrum for ketone for 3 ketone. 3.

Figure 4. 1H-NMR spectrum for ketone 3.

Table 1. 1H- and 13C-NMR chemical shifts (ppm) for 3 and 4 ketones.

N-9-ketone 3 N-7-ketone 4 H-8 8.12 8.41 C-1’ 68.2 69.8 C-4 150.8 160.2 C-5 133.6 124.1 C-8 144 146.8

The 2D HMBC spectra for 3 and 4 also supported the regiochemical assignments. Correlations between H-1’ and C-8 and C-4 signals were observed with the absence of any correlation with C-5 for Molecules 2019, 24, x 4 of 18

Figure 3. 13C-NMR spectrum for ketone 3.

Molecules 2019, 24, 3235 4 of 16

1 FigureFigure 4. 1H 4.-NMRH-NMR spectrum spectrum for ketone for ketone3. 3. Table 1. 1H- and 13C-NMR chemical shifts (ppm) for 3 and 4 ketones. Table 1. 1H- and 13C-NMR chemical shifts (ppm) for 3 and 4 ketones.

N-9-ketoneN-9 3-ketone N-7-ketone 3 4 N-7-ketone 4 H-8H-8 8.12 8.128.41 8.41 C-1’ 68.2 69.8 C-1’C-4 150.8 68.2160.2 69.8 C-4C-5 133.6 150.8124.1 160.2 C-8 144 146.8 C-5 133.6 124.1 The 2D HMBC spectraC-8 for 3 and 4 also supported 144 the regiochemical assignments. 146.8 Correlations between H-1’ and C-8 and C-4 signals were observed with the absence of any correlation with C-5 for The 2D HMBC spectra for 3 and 4 also supported the regiochemical assignments. Correlations between H-1’ and C-8 and C-4 signals were observed with the absence of any correlation with C-5 for the N-9 isomer 3 (Figure5). Correlations between H-1’ and C-8 and C-5 signals were observed without any correlation with C-4 for the N-7 isomer 4 (Figure6). The relative stereochemistry of both the N-7 and N-9 alkylated derivatives 3 and 4 were assigned the trans-configurations based on 2D NOESY experiments. None of the cis-ketone was detected. The NOESY data for 3 (Figure7) showed correlations between the H-1’ and H-3’a signals, and between the H-2’ and H-3’b signals while no correlation between H-1’ and H-3’b signals, nor between H-2’ and H-3’a signals was observed, indicating that H-1’ and H-2’ are on opposite sides (trans-configuration) of the ring. The trans-configuration was also supported with the similarity of the 1H-NMR spectrum for 3 (Figure4) with that of trans-ketone 5 (Figure8) the stereochemistry of which was established by X-ray diffraction analysis [14]. The stereochemistry for 4 was determined by the NOESY spectrum (Figure9), which showed cross-peaks between H-1’ with one of the two H-5’ protons, which indicated their cis-configuration and was consistent with the trans-configuration for H-1’ and H-2’ protons. Molecules 2019, 24, x 5 of 18 Molecules 2019, 24, 3235 5 of 16 the N-9 isomer 3 (Figure 5). Correlations between H-1’ and C-8 and C-5 signals were observed without any correlation with C-4 for the N-7 isomer 4 (Figure 6).

Figure 5. 2D-HMBC spectrum for ketone 3. Molecules 2019, 24, x Figure 5. 2D-HMBC spectrum for ketone 3. 6 of 18

Figure 6. 2D-HMBC spectrum for ketone 4. Figure 6. 2D-HMBC spectrum for ketone 4.

The relative stereochemistry of both the N-7 and N-9 alkylated derivatives 3 and 4 were assigned the trans-configurations based on 2D NOESY experiments. None of the cis-ketone was detected. The NOESY data for 3 (Figure 7) showed correlations between the H-1’ and H-3’a signals, and between the H-2’ and H-3’b signals while no correlation between H-1’ and H-3’b signals, nor between H-2’ and H-3’a signals was observed, indicating that H-1’ and H-2’ are on opposite sides (trans- configuration) of the ring. The trans-configuration was also supported with the similarity of the 1H- NMR spectrum for 3 (Figure 4) with that of trans-ketone 5 (Figure 8) the stereochemistry of which was established by X-ray diffraction analysis [14]. Molecules 2019, 24, x 7 of 18 Molecules 2019, 24, 3235 6 of 16

Molecules 2019, 24, x 7 of 18

Figure 7. 2D-NOESY spectrum for ketone 3.

Figure 7. 2D-NOESY spectrum for ketone 3. Figure 7. 2D-NOESY spectrum for ketone 3.

FigureFigure 8. 8.1 1H-NMRH-NMR spectrumspectrum for ketoneketone 5 [14].]. Reproduced from thesis of of A.E. A.E. York York University 2011 2011 permission grantedpermission by supervisor granted E.L-R. by and supervisor York University. E.L-R. and York University. Figure 8. 1H-NMR spectrum for ketone 5 [14]. Reproduced from thesis of A.E. York University 2011 The stereochemistry for 4 was determined by the NOESY spectrum (Figure 9), which showed permission granted by supervisor E.L-R. and York University. cross-peaks between H-1’ with one of the two H-5’ protons, which indicated their cis-configuration andThe wa stereochemistrys consistent with for the 4 transwas -determinedconfiguration by for the H NOESY-1’ and Hspectrum-2’ protons. (Figure 9), which showed cross-peaks between H-1’ with one of the two H-5’ protons, which indicated their cis-configuration and was consistent with the trans-configuration for H-1’ and H-2’ protons. Molecules 2019, 24, 3235 7 of 16 Molecules 2019, 24, x 8 of 18

Figure 9. 2D-NOESY spectrum for ketone 4. 2.1.2. Reduction of Ketone DerivativesFigure 9. 2D-NOESY spectrum for ketone 4.

2.1.2.Although Reduction we of needed Ketone to Derivatives reduce the ketone group as well as deprotect the hydroxymethyl group in ketone 3, we decided to reduce the ketone group first and then carry out the deprotection step, since the Although we needed to reduce the ketone group as well as deprotect the hydroxymethyl group resulting diol diastereomers would be very polar and difficult to separate by chromatography. Therefore, in ketone 3, we decided to reduce the ketone group first and then carry out the deprotection step, a chemoselective reduction of the ketone group was affected without touching the benzoate group. since the resulting diol diastereomers would be very polar and difficult to separate by Reduction of 3 using NaBH4 in MeOH at room temperature for 14 h resulted in the formation chromatography. Therefore, a chemoselective reduction of the ketone group was affected without of a mixture of trans-1’,2’-trans-1’,4’-cyclobutanol 6 and trans-1’,2’-cis-1’,4’-alcohol 7a in a ratio 1:0.3, touching the benzoate group. respectively (Scheme2), with a total yield of 51.3%, which could not be separated by either flash Reduction of 3 using NaBH4 in MeOH at room temperature for 14 h resulted in the formation of chromatography (5–7% methanol in chloroform using gradient elution) or thin layer chromatography a mixture of trans-1’,2’-trans-1’,4’-cyclobutanol 6 and trans-1’,2’-cis-1’,4’-alcohol 7a in a ratio 1:0.3, (5% methanol in chloroform). No starting material 3 was observed. respectively (Scheme 2), with a total yield of 51.3%, which could not be separated by either flash The structures of 6 and 7 were supported by the appearance of new downfield proton signals at δ chromatography (5–7% methanol in chloroform using gradient elution) or thin layer chromatography = 4.79 and 5.13 ppm (ratio 1:0.3), respectively. Furthermore, aromatic proton signals for the heterocycle (5% methanol in chloroform). No starting material 3 was observed. in the δ 9–7.5 range showed the same intensity ratio reflecting the stereoisomeric distribution. The following 2D-NMR analysis was based on the more intense signals of the major alcohol 6. The absence of the cyclobutanone carbonyl peak in the IR spectrum pointed to complete reaction in the reduction step. HRMS analysis confirmed the molecular composition for 6 and 7. Molecules 2019, 24, 3235 8 of 16 Molecules 2019, 24, x 9 of 18

SchemeScheme 2. 2.Reduction Reduction of of Ketone Ketone3 .3.

TheThe stereochemistry structures of 6 and of the 7 were major supported alcohol 6bywas the assignedappearance based of new on 1D-downfield1H, 13C proton and 2D-COSY, signals at NOESY,δ = 4.79 and and HSQC 5.13 NMR ppm experiments. (ratio 1:0.3), Therespectively. 2D-NOESY Furthermore, spectrum (Figure aromatic 10) showed proton thatsignals the forH-3’a the signalheterocycle correlated in with the δthose 9–7.5 for range protons show H-2’ed and the H-4’, same but intensitynot with H-1’, ratio while reflecting the H-3’b the signal stereoisomeric showed adistribution. correlation with The thefollowing H-1’ signal 2D-NMR but notanalysis with eitherwas based H-2’ on or H-4’the more signals, intense which signals indicated of the the majorcis relationshipalcohol 6. The between absence H-2’ of andthe H-4’cyclobutanone and the trans carbonylrelationship peak in for the H-1’. IR spectrum This was consistentpointed to withcomplete the assignedreaction stereochemistry in the reduction of step.6 to beHRMS the trans analysis-1’,2’- confirmedtrans-1’,4’-substitution the molecular pattern. composition Since reduction for 6 and of 7. the ketoneMoleculesThe produces 2019stereochemistry, 24, onlyx two epimers, of the major structure alcohol7 was 6 was assigned assigned the other based epimer. on 1D-1H, 13C and 2D-COSY,10 of 18 NOESY, and HSQC NMR experiments. The 2D-NOESY spectrum (Figure 10) showed that the H-3’a signal correlated with those for protons H-2’ and H-4’, but not with H-1’, while the H-3’b signal showed a correlation with the H-1’ signal but not with either H-2’ or H-4’ signals, which indicated the cis relationship between H-2’ and H-4’ and the trans relationship for H-1’. This was consistent with the assigned stereochemistry of 6 to be the trans-1’,2’-trans-1’,4’-substitution pattern. Since reduction of the ketone produces only two epimers, structure 7 was assigned the other epimer.

Figure 10. 2D-NOESY spectrum for the major signals of alcohol 6. Figure 10. 2D-NOESY spectrum for the major signals of alcohol 6.

The formation of the trans, trans-alcohol 6 as the major products in the reduction of the N-9 ketone 3 was likely due to the directing effect of the proximal lone pair of N-3 of the purine ring, which coordinated with the boron of the borohydride, leading to the introduction of the hydride ion on the same side of the purine ring, resulting in the hydroxyl group being on the opposite side, i.e., trans-configuration.

2.1.3. Debenzoylation of Purine Alcohols Debenzoylation of the N-9 alcohols mixture 6 and 7 using saturated methanolic NaOMe at room temperature for 3 hours afforded a mixture of the purine carbocyclic nucleosides 8 and 9 in an excellent yield (96.3%) in the same 1:0.3 ratio as the original stereoisomeric benzoylated mixture, respectively (Scheme 3). This was indicated from the 1H-NMR spectrum (Figure 11). The structures of 8 and 9 were confirmed by the absence of the benzoyl signals in the 1H-NMR spectra (Figure 11). The HRMS data for the 8 and 9 mixture were consistent with their molecular formulae. Molecules 2019, 24, 3235 9 of 16

The formation of the trans, trans-alcohol 6 as the major products in the reduction of the N-9 ketone 3 was likely due to the directing effect of the proximal lone pair of N-3 of the purine ring, which coordinated with the boron of the borohydride, leading to the introduction of the hydride ion on the same side of the purine ring, resulting in the hydroxyl group being on the opposite side, i.e., trans-configuration.

2.1.3. Debenzoylation of Purine Alcohols Debenzoylation of the N-9 alcohols mixture 6 and 7 using saturated methanolic NaOMe at room temperature for 3 hours afforded a mixture of the purine carbocyclic nucleosides 8 and 9 in an excellent Molecules 2019, 24, x 11 of 18 yield (96.3%) in the same 1:0.3 ratio as the original stereoisomeric benzoylated mixture, respectively (Scheme3). This was indicated from the 1H-NMR spectrum (Figure 11). The structures of 8 and 9 were confirmed by the absence of the benzoyl signals in the 1H-NMR spectra (Figure 11). The HRMS data forMolecules the 8 2019and, 249, mixturex were consistent with their molecular formulae. 11 of 18

Scheme 3. Debenzoylation of esters 6 and 7. Scheme 3. Debenzoylation of esters 6 and 7.

Scheme 3. Debenzoylation of esters 6 and 7.

1 FigureFigure 11. 11. 1HH-NMR-NMR spectrum spectrum for for mixture mixture of of purine purine nucleosides nucleosides 88 andand 99 (1:0.3(1:0.3 ratio). ratio).

SeparationSeparation of of the the diol diol mixture mixture 88 andand 99 couldcould not not be be accomplished accomplished by by either either flash flash or or thin thin-layer-layer chromatographychromatography after after numerous numerous attempts. attempts. Possible Possible solutions solutions to to this this problem problem would would include include 1 derivatizationderivatizationFigure of of either either 11. Halcohols alcohols-NMR spectrum 66 andand 77, , foror or mixturediols diols 88 andofand purine 99.. nucleosides 8 and 9 (1:0.3 ratio).

2.2. SynthesisSeparation of Pyrimidine of the diol Carbocyclic mixture 8 Nucleoside and 9 could Analogues not be accomplished by either flash or thin-layer chromatography after numerous attempts. Possible solutions to this problem would include derivatizationWe reported of either recently alcohols on the 6 and 6-chloropurine 7, or diols 8 andN-alkylation 9. to 3-hydroxymethylcyclobutanone triflate 10 [15]. In order to complete the series of these related nucleoside analogs we undertook to couple2.2. Synthesis the pyrimidine of Pyrimidine Carbocyclic Nucleoside thymine andAnalogues uracil with ketone 10. However, unlike 6- chloropurine, the attempted coupling under basic conditions through the anions did not lead to any We reported recently on the 6-chloropurine N-alkylation to 3-hydroxymethylcyclobutanone meaningful yields for the coupling products. The lack of coupling of the as compared to triflate 10 [15]. In order to complete the series of these related nucleoside analogs we undertook to 6-chloropurine under basic conditions may be due to the lower pKa for the N–H group in the couple the pyrimidine nucleobases thymine and uracil with ketone 10. However, unlike 6- pyrimidine nucleobases. Thus, we resorted to the classical Silyl–Hilbert–Johnson (or Vorbruggen chloropurine, the attempted coupling under basic conditions through the anions did not lead to any coupling) reaction [23]. This protocol entails the use of silyl protected nucleobases in the coupling meaningful yields for the coupling products. The lack of coupling of the pyrimidines as compared to with a substrate possessing a labile leaving group under Lewis acid conditions. The specific silyl 6-chloropurine under basic conditions may be due to the lower pKa for the N–H group in the protected nucleobases 11 and 12 were prepared under standard conditions (Scheme 4) [24]. pyrimidine nucleobases. Thus, we resorted to the classical Silyl–Hilbert–Johnson (or Vorbruggen coupling) reaction [23]. This protocol entails the use of silyl protected nucleobases in the coupling with a substrate possessing a labile leaving group under Lewis acid conditions. The specific silyl protected nucleobases 11 and 12 were prepared under standard conditions (Scheme 4) [24]. Molecules 2019, 24, 3235 10 of 16

2.2. Synthesis of Pyrimidine Carbocyclic Nucleoside Analogues We reported recently on the 6-chloropurine N-alkylation to 3-hydroxymethylcyclobutanone triflate 10 [15]. In order to complete the series of these related nucleoside analogs we undertook to couple the pyrimidine nucleobases thymine and uracil with ketone 10. However, unlike 6-chloropurine, the attempted coupling under basic conditions through the anions did not lead to any meaningful yields for the coupling products. The lack of coupling of the pyrimidines as compared to 6-chloropurine under basic conditions may be due to the lower pKa for the N–H group in the pyrimidine nucleobases. Thus, we resorted to the classical Silyl–Hilbert–Johnson (or Vorbruggen coupling) reaction [23]. This protocol entails the use of silyl protected nucleobases in the coupling with a substrate possessing a labile leaving group under Lewis acid conditions. The specific silyl protected nucleobases 11 and 12 wereMolecules prepared 2019, 24, underx standard conditions (Scheme4)[24]. 12 of 18

Scheme 4.4. Coupling of ketone 10 with thymine and uracil.

The coupling of 11 and 12 to 13 and 14, respectively, was accomplished using 3-hydroxymethylcyclobutanone triflate 10 generated in situ prepared from 3-hydroxycyclobutanone and reacting with the silylated nucleobase 11 or 12 giving 13 and 14, in yields of 45 and 35%, respectively, as crystalline solids. The final reduction step was carried out with the bulky hydride donor, lithium aluminum t-butoxide. Unlike the corresponding 6-chloropurine analog, which gave a single stereoisomer, pyrimidine ketones gave stereoisomeric mixtures of 15 and 16 for 13, and 17 and 18 for 14 (Scheme5). The cis stereoisomers 15 and 17 still predominated by at least 10:1 in each case as expected from hydride delivery to the less hindered side of the cyclobutanone ring. The lower stereoselectivity in the reduction of 13 and 14 is attributed to the smaller steric bulk of pyrimidine as compared to 6-chloropurine. The regio- and stereochemistry for 15 and 17 were assigned by the following 2D NMR analyses as seen by their NMR spectra (see Supplementary Materials).

Scheme 5. Reduction of ketones 13 and 14.

The coupling of 11 and 12 to 13 and 14, respectively, was accomplished using 3- hydroxymethylcyclobutanone triflate 10 generated in situ prepared from 3-hydroxycyclobutanone and reacting with the silylated nucleobase 11 or 12 giving 13 and 14, in yields of 45 and 35%, respectively, as crystalline solids. The final reduction step was carried out with the bulky hydride donor, lithium aluminum t- butoxide. Unlike the corresponding 6-chloropurine analog, which gave a single stereoisomer, pyrimidine ketones gave stereoisomeric mixtures of 15 and 16 for 13, and 17 and 18 for 14 (Scheme 5). The cis stereoisomers 15 and 17 still predominated by at least 10:1 in each case as expected from hydride delivery to the less hindered side of the cyclobutanone ring. The lower stereoselectivity in the reduction of 13 and 14 is attributed to the smaller steric bulk of pyrimidine as compared to 6- chloropurine. The regio- and stereochemistry for 15 and 17 were assigned by the following 2D NMR analyses as seen by their NMR spectra (see supplementary material). Molecules 2019, 24, x 12 of 18

Molecules 2019, 24, 3235 11 of 16 Scheme 4. Coupling of ketone 10 with thymine and uracil.

SchemeScheme 5.5. Reduction of ketones 13 and 14.14.

ForThe 15 couplingthe vinyl of protons 11 and from 12the to uracil13 and ring 1 were4, respectively, readily identified was accomplished at 5.623 and 7.551 using ppm 3- forhydroxymethylcyclobutanone positions 5 and 6, respectively triflate (see 10 Scheme generated6 for positionin situ prepared numbering). from Proton3-hydroxycyclobutanone 5 showed 1H–13C multipleand reacting bond with correlations the silylated to carbons nucleobase 4 and 6 at11 167.25 or 12and giving 147.90 13 ppm,and respectively,14, in yields in of the 45 2D and1H– 35%,13C HMBCrespectively, spectrum. as crystalli Protonne 6 solids. showed 1H–13C multiple bond correlations to carbon-2 at 153.38 ppm as well asThe to final a methylene reduction carbon step was at 54.68 carried ppm. out This with carbon the bulky would hydride have correspondeddonor, lithium to aluminum C-7 and the t- correlationbutoxide. Unlike would only the have corresponding been possible 6-chloropurine if the alkyl substitution analog, which wasat gave nitrogen a single 1. Likewise, stereoisomer, the 2D 1pyrimidineH–13C HMBC ketones also showedgave stereoisomeric correlations mixtures from protons of 15 7and to carbons16 for 13 2,, and 6 (at 1 147.907 and 1 ppm),8 for 1 84 (at(Scheme 26.78 ppm),5). The and cis stereoisomers 9 (at 38.26 ppm) 15 andand provided17 still predominated convincing evidence by at least of alkyl10:1 in substitution each case as at expected the 1 position. from Usinghydride the delivery 2D 1H– 1toH the COSY less spectrum, hindered protons side of 8,the 9, cyclobutanone and 10 could be ring. identified. The lower Position stereoselectivity 9 had identical in Moleculesshiftsthe reduction with 2019, prochiral 24 , ofx 13 and protons. 14 is attributed Protons on to one the face smaller of the steric cyclobutane bulk of pyrimidine ring had a shiftas compared of 1.65013 ppm, toof 186- whilechloropurine. the opposite face had a shift of 2.357 ppm. The regio- and stereochemistry for 15 and 17 were assigned by the following 2D NMR analyses as seen by their NMR spectra (see supplementary material).

SchemSchemee 6. StructureStructure numbering numbering for for 2D 2D NMR analysis.

1 1 ForThe 1 2D5 theH– vinylH NOESY protons also from indicated the uracil that ring protons were 6 andreadily 7 were identified close together at 5.623 in and space. 7.551 In addition,ppm for positionsthere was 5 an and intense 6, respectively correlation (see between Scheme proton 6 for 7 and position proton numbering) 9 at 1.650. ppm, Proton suggesting 5 showed that 1H– this13C multipleinteraction bond was correlations between protons to carbons on the 4 and same 6 at side 167.25 of the and cyclobutane 147.90 ppm ring., respectively Conversely,, in thethe protons2D 1H– 13atC positions HMBC spectrum. 8 and 10 hadProton more 6 show intenseed correlations1H–13C multiple to proton bond 9correlations at 2.357 ppm to carbon than they-2 at did 153.38 to proton ppm as9 atwell 1.650. as to This a methylene evidence carbon supports at the54.68 structure ppm. This with carbon protons would 8 and have 10 correspond both on theed same to C side-7 and of the correlationcyclobutane would ring. only have been possible if the alkyl substitution was at nitrogen 1. Likewise, the 2D 1HFor–13C17 HMBCthe aromatic also show anded methyl correlations protons from from protons the thymine 7 to carbons ring were 2, readily6 (at 147.90 identified ppm), at 8 7.401 (at 26.78 and 1 13 ppm)1.855, ppm, and 9 respectively. (at 38.26 ppm) The and methyl provide protond convincing at 1.855 ppm evidence showed of H–alkylC substitution multiple bond at the correlations 1 position. to 1 13 Usingcarbons the 4, 2D 5, and1H–1 6H at COSY 167.36, spectrum, 111.45, and protons 143.79 8, ppm,9, and respectively, 10 could be inidentified. the 2D H– PositionC HMBC 9 hadspectrum. identical shifts with prochiral protons. Protons on one face of the cyclobutane ring had a shift of 1.650 ppm, while the opposite face had a shift of 2.357 ppm. The 2D 1H–1H NOESY also indicated that protons 6 and 7 were close together in space. In addition, there was an intense correlation between proton 7 and proton 9 at 1.650 ppm, suggesting that this interaction was between protons on the same side of the cyclobutane ring. Conversely, the protons at positions 8 and 10 had more intense correlations to proton 9 at 2.357 ppm than they did to proton 9 at 1.650. This evidence supports the structure with protons 8 and 10 both on the same side of the cyclobutane ring. For 17 the aromatic and methyl protons from the thymine ring were readily identified at 7.401 and 1.855 ppm, respectively. The methyl proton at 1.855 ppm showed 1H–13C multiple bond correlations to carbons 4, 5, and 6 at 167.36, 111.45, and 143.79 ppm, respectively, in the 2D 1H–13C HMBC spectrum. In addition, the proton at 7.401 ppm (6) showed 1H–13C multiple bond correlations to carbon 2 at 153.52 ppm. Proton 6 also showed 1H–13C multiple bond correlations to a methylene carbon at 54.4 ppm. This carbon would have corresponded to 8 and the correlation would have only been possible if the alkyl substitution was at nitrogen 1. Likewise, the 2D 1H–13C HMBC also showed correlations from proton 8 to carbons 2, 5, and 6 and provided convincing evidence of alkyl substitution at the 1 position. Using the 2D 1H–1H COSY spectrum, protons 9, 10, and 11 could be identified. Position 10 had identical shifts with prochiral protons. Protons on one face of the cyclobutane ring had a shift of 1.653 ppm, while the opposite face had a shift of 2.347 ppm. The 2D 1H–1H NOESY also indicated that protons 6 and 8 were close by in space. In addition, there was an intense correlation between protons 8 and 10 at 1.653 ppm, suggesting that this interaction was between protons on the same side of the cyclobutane ring. Conversely, the protons at positions 9 and 11 had more intense correlations to proton 10 at 2.347 ppm than they did to protons 10 at 1.653. This evidence supports the structure with protons 9 and 11 both on the same side of the cyclobutane ring.

Molecules 2019, 24, 3235 12 of 16

In addition, the proton at 7.401 ppm (6) showed 1H–13C multiple bond correlations to carbon 2 at 153.52 ppm. Proton 6 also showed 1H–13C multiple bond correlations to a methylene carbon at 54.4 ppm. This carbon would have corresponded to 8 and the correlation would have only been possible if the alkyl substitution was at nitrogen 1. Likewise, the 2D 1H–13C HMBC also showed correlations from proton 8 to carbons 2, 5, and 6 and provided convincing evidence of alkyl substitution at the 1 position. Using the 2D 1H–1H COSY spectrum, protons 9, 10, and 11 could be identified. Position 10 had identical shifts with prochiral protons. Protons on one face of the cyclobutane ring had a shift of 1.653 ppm, while the opposite face had a shift of 2.347 ppm. The 2D 1H–1H NOESY also indicated that protons 6 and 8 were close by in space. In addition, there was an intense correlation between protons 8 and 10 at 1.653 ppm, suggesting that this interaction was between protons on the same side of the cyclobutane ring. Conversely, the protons at positions 9 and 11 had more intense correlations to proton 10 at 2.347 ppm than they did to protons 10 at 1.653. This evidence supports the structure with protons 9 and 11 both on the same side of the cyclobutane ring.

3. Summary and Conclusions Two classes of cyclobutane nucleoside analogs were prepared. Parent purine was coupled with 2-bromo-3-hydroxymethylcyclobutanone to give both the N-7 and N-9 regioisomers by way of the purinyl anion. The anion has the negative charge delocalized at both N-7 and N-9 centers. The N-9 coupled ketone was structurally elaborated to the diol in two steps in order for the attempted separation of the stereoisomeric mixture in the initial reduction with NaBH4 in which the trans, trans-alcohol predominates in a 3:1 ratio. This mixture was debenzoylated with sodium methoxide to give the diol mixture in the same ratio. The other class of cyclobutane analogs, where the nucleobase is bonded to the cyclobutane ring via a methylene pendant at the C-3 position, was accomplished by treating 3-hydroxymethylcyclobutanone triflate and coupling with the silylated uracil and thymine under Vorbruggen conditions. Only the N-1 coupled products were observed for both uracil and thymine. The ketones were stereoselectively reduced to the cyclobutanols with the bulky hydride donor, sodium t-butoxide, to give the cis-alcohols in both cases. In view of the reported antiviral activities of the carbocyclic analogs of oxetanocin, these novel structurally related compounds may be good candidates to possess biological activity. It should be noted that the cyclobutane purine analogs were prepared in their racemic modifications from racemic 1, the starting precursor. Once biological activity is established the enantiomeric forms will be resolved in order to identify the specifically active component. The pyrimidine analogs possess a plane of symmetry and hence exist as single stereoisomers.

4. Experimental Melting points (mp) were obtained on a Fisher–Johns melting point apparatus (Thermo Fisher Scientific, Carlsbad, CA, USA) and were uncorrected. Fourier transform infrared spectra were recorded on a Genesis II Mattson 3000 FT-IR spectrometer (Mattson Technology, Fremont, CA, USA) as thin films and on a Nicolet 380 FT-IR spectrometer (Nicolet Instrument Inc., Madison, WI, USA)as a solid disk. UV spectra were determined using an Ultrospec 4300 pro UV spectrometer (GE, Boston, MA, USA). HRMS analysis was performed at Queen’s University (Kingston, ON, Canada). LC–MS analyses were obtained on a Waters 2695LC-Quatro Ultima MS instrument (Waters Corporation, Milford, MA, USA). Elemental analysis was performed at Guelph Chemical Laboratories Limited (Guelph, ON, Canada).The 1D and 2D-NMR analyses were recorded on Bruker AV 400 (400.13 MHz) and DRX 600 (599.86 MHz) NMR spectrometers (Bruker Biospin, Billerica, MA, USA). Data for 1H-NMR were referenced relative to the solvent residual proton signals, CDCl3 (δ 7.27), DMSO-d6 (δ 2.50), and CD3OD 13 (δ 3.31). Data for C-NMR were referenced relative to the solvent residual carbon signals, CDCl3 (δ 77.16), DMSO-d6 (δ 39.52), and CD3OD (δ 49.00). The 1D-NMR chemical shifts were quoted in delta (δ) values in parts per million (ppm) downfield from TMS (δ = 0). Tabulation of 1D-NMR data was Molecules 2019, 24, 3235 13 of 16 reported in the following order: chemical shifts (δ in ppm), multiplicity (s = singlet, d = doublet, t = triplet, dxd = doublet of doublets, m = multiplet), coupling constant (J in Hz), and integration (number of protons).

4.1. N-Alkylation of Purine Potassium hydroxide (0.61 g, 10.8 mmol) and TDA-1 (0.29 g, 0.9 mmol) were added to purine (1.08 g, 9 mmol) in 100 mL of acetonitrile and the mixture was stirred overnight at room temperature. A solution of α-bromocyclobutanone 1 (3.06 g, 10.8 mmol) in acetonitrile (15 mL) was added dropwise and the mixture was stirred overnight at room temperature. Insoluble material was filtered off and the solvent was evaporated under vacuum. The residue was purified by chromatography (2% methanol in chloroform) to afford the trans-N-9-alkylated derivative 3 (0.34 g (11.7%) as well as 0.33 g (11.2%) of the trans-N-7-alkylated derivative 4 as an oil. 1 Trans-(3-oxo-2-(9H-purin-9-yl)cyclobutyl)methyl benzoate (3). H-NMR (400 MHz, CDCl3), δ 9.06 (s, 1H), 8.83 (s, 1H), 8.12 (s, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 6.8 Hz, 1H), 7.32 (t, J = 7.6 Hz, 2H), 5.77 (d, J = 8.0 Hz, 1H), 4.70 (d, J = 5.6 Hz, 2H), 3.54 (m, 1H), 3.35 (m, 1H), 3.16 (m, 1H); 13C-NMR (400 MHz, CDCl3), δ 197.5, 166.2, 152.5, 150.8, 148.6, 144.0, 133.6, 133.4, 129.3, 129.0, 128.4, 68.2, 64.9, 45.0, + 32.8; HRMS (EI) m/z calculated for C17H14N4O3 (M ) 322.1066, found 322.1057. 1 Trans-(3-oxo-2-(7H-purin-7-yl)cyclobutyl)methyl benzoate (4). H-NMR (400 MHz, CDCl3), δ 9.02 (s,1H), 8.90 (s, 1H), 8.41 (s, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 7.8 Hz, 2H), 13 5.97 (d, J = 7.2 Hz,1H), 4.83 (m, 1H), 4.74 (m, 1H), 3.33 (m, 3H); C-NMR (400 MHz, CDCl3), δ 196.5, 166.3, 160.2, 153.3, 148.6, 140.5, 133.6, 129.4, 128.6, 128.5, 124.1, 69.8, 64.8, 44.8, 33.6; HRMS (EI) m/z + calculated for C17H14N4O3 (M ) 322.1066, found 322.1053. Benzoic acid 2-(6-chloro-purin-9-yl)-3-oxo-cyclobutylmethyl ester (5)[14]. 1H-NMR (400 MHz, CDCl3), δ 8.65 (s, 1H), 8.15 (s, 1H), 7.90 (d, J = 7.6 Hz, 2H), 7.57 (t, J = 6.8 Hz, 1H), 7.41 (t, J = 7.8 Hz, 2H), 5.76 (d, J = 8.0 Hz, 1H), 4.77 (d, J = 5.2Hz, 2H), 3.56 (m, 1H), 3.44 (m, 1H), 3.23 (dxd, J = 8.8, 18.0Hz, 1H); (Figure8).

4.2. Reduction of N-9-Purine Ketone 3 To a solution of trans-N9-ketone 3 (0.499 g, 1.55 mmol) in methanol (30 mL), 0.059 g (1.55 mmol) of sodium borohydride (NaBH4) was added and the mixture was stirred at r.t for 14 h. Saturated aqueous solution of NH4Cl (35 mL) was added and stirred for 10 min. The solvent was removed under vacuum and the organic mixture was extracted from the residue using 25% MeOH in CHCl3. The solvent was evaporated, and the residue was purified by column chromatography (5–7% methanol in chloroform using gradient elution) to give 0.258 g (51.3%) of a mixture of alcohols 6 and 7 as a gum with a ratio 1:0.3, respectively (based on 1H-NMR spectrum).

4.3. Major Alcohol

1 Trans, trans-3-hydroxy-2-(9H-purin-9-yl)cyclobutyl)methyl benzoate (6). H-NMR (400 MHz, CDCl3), δ 9.04 (s, 1H), 8.86 (s, 1H), 8.21 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 7.2 Hz, 1H), 7.40 (t, J = 7.8 Hz, 2H), 4.79 (dxd, J = 8.0, 15.6 Hz, 1H), 4.62 (dxd, J = 5.0, 11.8 Hz, 1H), 4.55 (t, J = 8.0 Hz, 1H), 4.49 (dxd, J = 6.8, 11.6 Hz, 1H); 2.94, (m, 1H), 2.63 (dxd, J = 8.2, 19.0 Hz, 1H), 1.94 (dxd, J = 10.4, 19.6 Hz, 1H); 13 C-NMR (400 MHz, CDCl3), δ 166.3, 151.7, 151.5, 148.0, 144.3, 133.9, 133.3, 129.3, 129.2, 128.4, 67.9, + 65.5, 61.7, 31.7, 28.2; HRMS (for alcohol mixture) (EI) m/z calculated for C17H16N4O3 (M ) 324.1222, found 324.1212.

4.4. Minor Alcohol

1 Cis, trans-3-hydroxy-2-(9H-purin-9-yl)cyclobutyl)methyl benzoate (7). H-NMR (400 MHz, CDCl3), δ 8.90 (s, 1H), 8.79 (s, 1H), 8.52 (s, 1H), 7.79 (d, J = 7.6 Hz, 2H), 7.52 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 8.0 Hz, 2H), 5.13 (dxd, J = 5.4, 9.0 Hz, 1H), 4.86 (m, 1H), 4.47 (m, 2H), 3.76 (m, 1H), 2.30 (dxd, J = 3.4, 9.4 Hz, Molecules 2019, 24, 3235 14 of 16

13 2H); C-NMR (400 MHz, CDCl3), δ 166.2, 151.5, 151.0, 166.7, 166.2, 133.9, 133.2, 129.2, 128.4, 128.3, 68.4, 65.4, 54.0, 39.5, 29.0.

4.5. Deprotection of Purine Alcohols 6 and 7 To 0.16 g (0.49 mmol) of N-9-alcohol mixture 6 and 7, 4 mL of saturated solution of NaOMe in methanol was added and the mixture was stirred at room temperature for 3 h. Saturated aqueous solution (12 mL) of NaHCO3 was added and the mixture was stirred for 15 min. The solvent was evaporated under vacuum and the organic mixture was extracted from the residue using 5% methanol in chloroform. The solvent was removed to obtain 0.105 g (96.3%) of a mixture of purine carbocyclic nucleosides 9 and 10 as a gum in a ratio 1:0.3, respectively. 1 Trans, trans-3-hydroxymethyl-2-purin-9-yl-cyclobutanol (8). H-NMR (400 MHz, CD3OD), δ 9.08 (s, 1H), 8.93 (s, 1H), 8.66 (s, 1H), 4.82 (dxd, J = 8.0, 15.6 Hz, 1H), 4.69 (m, 1H), 3.72 (m, 2H), 2.80 (m, 1H); 13 2.44, (m, 1H), 1.72 (dxd, J = 10.2, 19.0 Hz, 1H); C-NMR (400 MHz, CD3OD), δ 151.6, 151.5, 147.2, 146.0, 133.8, 67.7, 62.1, 60.4, 34.7, 27.5; HRMS (for 172 and 173 mixture) (EI) m/z calculated for C10H12N4O2 (M+) 220.0960, found 220.0969. 1 Cis, trans-3-hydroxymethyl-2-purin-9-yl-cyclobutanol (9). H-NMR (400 MHz, CD3OD), δ 9.06 (s, 1H), 8.91 (s, 1H), 8.76 (s, 1H), 5.16 (dxd, J = 5.4, 8.6 Hz, 1H), 4.62 (t, J = 5.4 Hz, 1H), 3.72 (m, 2H), 3.52 13 (m, 1H), 2.22 (m, 1H), 2.06 (m, 1H).; C-NMR (400 MHz, CD3OD), δ 151.6, 151.3, 146.8, 146.7, 133.2, 67.0, 62.4, 53.1, 41.4, 27.8.

4.6. Preparation of Ketone 13 In round bottom flask, 3-(hydroxymethyl)cyclobutan-1-one (0.998 mmol, 100 mg) in 10 mL dry methylene chloride was added and cooled to 78 C. Hünig’s base (3.1936 mmol, 0.556 mL) was added − ◦ and after 5 min this was followed by addition of trifluoromethane sulfonic anhydride (1.098 mmol, 0.185 mL). The reaction mixture was stirred at 78 C for 10 min then warmed to 0 C and continued for − ◦ ◦ 1 h. To this flask was added silylated uracil 11 (0.8316 mmol, 213.28 mg) in 10 mL dry acetonitrile and stirred overnight at room temperature. This was then followed by evaporation of the solvents under vacuum and purification by column chromatography packed with silica gel using 3%MeOH/CHCl3. We recovered 83 mg (43%) of a white solid, melting point 203–206 ◦C, Rf: 0.357, with 5%MeOH/CHCl3. The IR spectrum can be found in the Supplementary Materials.

4.7. Preparation of Ketone 14 In round bottom flask, 3-(hydroxymethyl)cyclobutan-1-one (1.997 mmol, 200 mg) in 20 mL dry methylene chloride was added and cooled to 78 C. To this solution was added (6.39 mmol, 1.112 − ◦ mL) Hünig’s base, and after 5 min trifluoromethane sulfonic anhydride (2.1967 mmol, 0.37 ml) was added. The mixture was stirred at 78 C for 10 min then warmed to 0 C and continued for 1 h. To − ◦ ◦ this flask was added silylated thymine 12 (1.664 mmol, 450 mg) in 20 mL dry acetonitrile, and stirring continued overnight at room temperature. The solvents were evaporated under vacuum and purified using column chromatography packed with silica gel using 20% hexane/ethyl acetate as eluent. After further purification using preparative TLC with 7% MeOH/DCM, 121.3 mg (35%) of a white solid was obtained, melting point 199–204 ◦C, Rf: 0.277, with 3% MeOH/DCM. The IR spectrum can be found in the Supplementary Materials.

4.8. Preparation of Alcohol 15 In a round bottom flask, ketone 13 (0.257 mmol, 50 mg) in 10 mL dry ether was placed and cooled to 78 C, followed by addition of LiAl(OtBu) H 1 M in dry THF (0.386 mL, 0.386 mmol) dropwise − ◦ 3 and stirred for 2.5 h at 78 C, then overnight at room temperature. The mixture was quenched with − ◦ saturated aqueous ammonium chloride solution and stirred for 20 minutes. Evaporation of the solvents under vacuum and extraction with 25% MeOH/DCM, followed by drying with anhydrous MgSO4, and evaporation under vacuum gave a mixture that was further purified using preparative TLC with Molecules 2019, 24, 3235 15 of 16

8% MeOH/DCM. A 43 mg quantity of a colorless viscous oil (43 mg) was obtained; yield 85%, Rf: 0.2 on TLC using 5% MeOH/DCM as eluent. HRMS: Calculated m/e 197.77052; Found 197.07747. Details of the NMR data can be found in the Supplementary Materials.

4.9. Preparation of Alcohol 17 In a round bottom flask, ketone 14 (0.24 mmol, 50 mg) in 10 mL dry ether was placed and cooled to 78 C, followed by addition of LiAl(OtBu) H 1 M in dry THF (0.36 mL, 0.36 mmol) drop wise − ◦ 3 and stirred for 2.5 h at 78 C then overnight at room temperature. The mixture was quenched with − ◦ saturated aqueous ammonium chloride solution and stirred for 20 minutes. Evaporation of the solvents under vacuum, followed by extraction of the product with 25% MeOH/DCM, drying with anhydrous MgSO4, and evaporation under vacuum gave a mixture that was further purified using preparative TLC with 7.5% MeOH/DCM. A 33 mg quantity of a white solid was obtained; yield 60.6%, Rf: 0.138 on TLC with 3% MeOH/DCM as eluent. HRMS: Calculated m/e 209.09317; Found 209.09289. Details of the NMR data can be found in the Supplementary Materials.

Supplementary Materials: The following are available online, the IR spectra of Ketones 13 and 14, the 1D and 2D NMR data for alcohols 15 and 17. Author Contributions: Conceptualization, E.L.-R.; Data curation, H.A. and H.H.; Formal analysis, N.H. and M.M.H.; Investigation, E.L.-R., N.H. and A.E.; Methodology, A.E. and M.M.H.; Supervision, E.L.-R., N.S.E.-G., A.A.E.-E. and A.R.M. Funding: We wish to thank York University for funding in the form of a PER grant. No external funding for this project was obtained. Acknowledgments: We would like to thank the Centre for Mass Spectrometry for providing the high resolution MS data. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 13 and 14 are available from the authors.

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