Synthesis of 2, 2, 6-Trisubstituted 5-Methylidene-Tetrahydropyran-4

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Article Synthesis of 2,2,6-Trisubstituted 5-Methylidene- † tetrahydropyran-4-ones with Anticancer Activity

Tomasz Bartosik 1, Jacek K˛edzia 1, Joanna Drogosz-Stachowicz 2, Anna Janecka 2, Urszula Krajewska 3, Marek Mirowski 3 and Tomasz Janecki 1,*

1 Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego˙ 116, 90-924 Łód´z,Poland; [email protected] (T.B.); [email protected] (J.K.) 2 Department of Biomolecular Chemistry, Medical University of Łód´z,Mazowiecka 6/8, 92-215 Łód´z,Poland; [email protected] (J.D.-S.); [email protected] (A.J.) 3 Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Faculty of Pharmacy, Medical University of Łód´z,Muszy´nskiego1, 90-151, Łód´z,Poland; [email protected] (U.K.); [email protected] (M.M.) * Correspondence: [email protected]; Tel.: +48-426313220 Dedicated to Professor Grzegorz Mlosto´nof University of Łód´zon the occasion of his 70th birthday. † Academic Editor: René Csuk Received: 14 January 2020; Accepted: 28 January 2020; Published: 30 January 2020

Abstract: In our continuous search for new, relatively simple 2-alkylidene-1-oxoheterocycles as promising anticancer drug candidates, herein we report an eﬃcient synthesis of 2,2,6-trisubstituted 5-methylidenetetrahydropyran-4-ones. These compounds were obtained in a four step reaction sequence, in which starting diethyl 2-oxopropylphosphonate was transformed into 2,2-disubstituted 5-diethoxyphosphoryldihydropyran-4-ones, followed by Michael addition of various Grignard reagents and Horner-Wadsworth-Emmons reaction of the obtained adducts with formaldehyde. Stereochemistry of the intermediate Michael adducts is also discussed. Final 5-methylidenetetrahydropyran-4-ones were tested for their possible antiproliferative eﬀect against three cancer cell lines and 6-isopropyl-2,2-dimethyl-5-methylidenetetrahydropyran-4-one (11c), which showed very high cytotoxic activity against HL-60 human leukemia cells and was three times more active than known anticancer drug carboplatin, was selected for further biological evaluation, in order to disclose its mechanism of action. The obtained results indicated that 11c induced apoptosis in HL-60 cells and caused the arrest of the cell cycle in the G2/M phase, which may suggest its cytotoxic and cytostatic activity.

Keywords: methylidenedihydropyran-4-ones; Michael addition; Horner-Wadsworth-Emmons oleﬁnation; cytotoxic activity; apoptosis; cell cycle

1. Introduction In the continuation of our search for relatively simple structural motifs with anticancer potential [1–3], we turned our attention to 3-alkylidenetetrahydropyran-4-ones 1. Compounds containing this skeleton are common in nature and display interesting biological activities. This structural motif is present in a number of biologically active and medicinally important natural products. For example, norperovskatone 2, recently isolated from Perovskia atriplicifolia, possesses remarkable anti-HBV (Hepatitis B Virus) activity [4] and (+)-okilactomycin 3 [5] exhibits in vitro cytotoxicity against a number of human cancer cell lines, including lymphoid leukemia L1210 or leukemia P388. This compound also shows in vivo activity against Ehrlich ascites carcinoma. Chrolactomycin 4 displays signiﬁcant cytotoxicity in vitro against several human cancer

Molecules 2020, 25, 611; doi:10.3390/molecules25030611 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 12 Molecules 2020, 25, 611 2 of 12 compound also shows in vivo activity against Ehrlich ascites carcinoma. Chrolactomycin 4 displays significant cytotoxicity in vitro against several human cancer cell lines: ACHN, A431, MCF-7, and cellT24 lines:[6]. The ACHN, structures A431, of MCF-7,natural compounds and T24 [6]. contain The structuresing 3-alkylidenetetrahydropyran of natural compounds-4 containing-one motif 3-alkylidenetetrahydropyran-4-oneare shown in Figure 1. motif are shown in Figure1.

O O O O O O O OMe O O R R O O O O O O OH OH 1 2 3 4 FigureFigure 1. Natural 1. Natural compounds compounds containing containing 3-alkylidenetetrahydropyran-4-one3-alkylidenetetrahydropyran-4-one skeleton. skeleton.

However,However, literature search search revealed revealed that that synthetic synthetic methodologies methodologies that that can can be beapplied applied to the to thepreparation preparation of 3- ofalkylidenetetrahydropyran 3-alkylidenetetrahydropyran-4-ones-4-ones 1 with1 diversewith diverse substitution substitution patterns patterns are limited. are limited.There are There several are methods several methods which enable which the enable introduction the introduction of arylidene of arylidene group grouponto the onto plain the plaintetrahydropyran tetrahydropyran-4-one-4-one ring ringusing using a reaction a reaction with with aromatic aromatic aldehydes aldehydes in in the the presence presence of catalytic amountamount ofof TMSNMe TMSNMe2 and2 and MgBr MgBr2 Et2 2EtO[2O7 ][ or7] pyrrolidineor pyrrolidine [8]. There[8]. There are also are twoalso literature two literature reports reports which describewhich describe preparation preparation of speciﬁc of 3-methylidenetetrahydropyran-4-ones, specific 3-methylidenetetrahydropyran and-4 thus-ones 8-methylidene-9-, and thus 8- oxo-6-oxaspiromethylidene-9- [oxo5,6]- decane6-oxaspir waso prepared[5,6] decane by carbocyclization was prepared of unsaturatedby carbocyclization thioesters underof unsaturated palladium catalysisthioesters [9 ]under and 2-butyl-6,6-dimethyl-3-methylidenetetrahydropyran-4-one palladium catalysis [9] and 2-butyl-6,6-dimethyl-3-methylidenetetrahydropyran was obtained in a three-step-4- reactionone was sequence obtained starting in froma three 6-ethoxy-2-methyl-5-diphenylphosphorylhex-3-yn-5-en-2-ol-step reaction sequence starting from 6-ethoxy-2-methyl [10]. -5- diphenylphosphorylhexIn this report, we- present3-yn-5-en a-2 novel,-ol [10] general,. and eﬃcient synthesis of 2,2,6-trisubstituted 5-methylidenetetrahydropyran-4-onesIn this report, we present a novel11, usinggeneral Horner-Wadsworth-Emmons, and efficient synthesis of 2 methodology,2,6-trisubstituted [11,12 5].- Themethylidenetetrahydropyran obtained compounds have-4 been-ones evaluated 11 using for Horner their possible-Wadsworth cytotoxic-Emmons activity methodology against three human[11,12]. cancerThe obtain cell lines:ed compounds NALM-6 andhave HL-60 been leukemia evaluated and for the their MCF-7 possible breast cytotoxic cancer cell activity line. against three human cancer cell lines: NALM-6 and HL-60 leukemia and the MCF-7 breast cancer cell line. 2. Results and Discussion

2.1.2. Results Chemistry and Discussion

2.1. ChemistryWe start our research from the synthesis of diethyl 4-hydroxy-2-oxoalkylphosphonates 7a–d using a procedure described by Wada and coworkers [13]. In this respect, diethyl 2-oxopropylphosphonate (5) wasWe reacted start our with research 1,1 equiv. from of the sodium synthesis hydride of diethyl at r.t. and4-hydroxy next with-2-oxoalkylphosphonates 1,1 equiv. of 2,5 M n-BuLi 7a–d solutionusing a inprocedure hexane at described40 C to obtain by Wada dianion and6, which, coworkers after cooling[13]. In to this70 respectC was,treated diethyl with 2- − ◦ − ◦ symmetricoxopropylphosphonate ketones, such (5 as) was acetone, reacted cyclohexanone, with 1,1 equiv benzophenone,. of sodium hydride and ﬂuoren-9-one at r.t. and next (Scheme with 1).,1 Afterequiv. standard of 2,5 M n work-up,-BuLi solution expected in hexane phosphonates at −40 °C 7a–dto obtainwere dianion obtained 6, which in moderate, after cooling to good to − yields70 °C (Tablewas treated1). Next, with7a –symmetricd were subjected ketones to, asuch reaction as acetone with dimethylformamide, cyclohexanone, benzophenone dimethyl acetal, and (DMF-DMA) fluoren-9- inone the (Scheme presence 1). ofAfter triﬂuoroborate standard work diethyl-up, ex etheratepected phosphonates BF Et O, applying 7a–d were toluene obtained as a solvent. in moderate Initial to 3· 2 results,good yields using (Table one equiv.1). Next, of 7a DMF-DMA–d were subjected and BF to Eta reactionO and performingwith dimethylformamide the reaction atdimethyl r.t. for 3· 2 24acetal h were (DMF unsatisfactory-DMA) in the presence with 15–20% of trifluoroborate conversion to diethyl the desired etherate pyran-4-ones BF3 Et2O, applying9. To our toluene delight, as a solvent. Initial results, using one equiv. of DMF-DMA and BF3 Et2O and performing the reaction the optimization of this reaction was fully successful and carrying out∙ the reaction with 3 equiv. at r.t. for 24 h were unsatisfactory with 15–20% conversion to the desired pyran-4-ones 9. To our of DMF-DMA and 1 equiv. of BF3 Et2O for 3 h at 100 ◦C (for∙ pyran-4-ones 9a–c) or at r.t. (for pyran-4-onedelight, the 9doptimization) gave desired of this 3-diethoxyphosphoryldihydropyran-4-ones reaction was fully successful and carrying9a–d outin good the reaction yields (Table with1 ).3 Apparently,equiv. of DMF this-DMA reaction and proceeds1 equiv. of via BF the3 Et Knoevenagel2O for 3 h at 100 type °C condensation, (for pyran-4-ones with 9a the–c)formation or at r.t. (for of dimethylaminovinylphosphonatespyran-4-one 9d) gave desired 3-diethoxyphosphoryldihydropyran8a–d as intermediates, followed-4- byones intramolecular 9a–d in good oxy-Michaelyields (Table cyclization.1). Apparently When, this progress reaction ofproceeds these reactions via the Knoevenagel was monitored type by condensation31P NMR technique,, with the signalsformation with of chemicaldimethylaminovinylphosphonates shift ~25 ppm initially appeared, 8a–d whichas intermediates could be attributed, followed to intermediateby intramolecular vinylphosphonates oxy-Michael 31 8cyclization.. Only when When BF Et progressO was addedof these to thereactions reaction was mixture monitored did these by signalsP NMR gradually technique disappear, signals withwith 3· 2 thechemical simultaneous shift ~25 appearance ppm ofinitially pyran-4-ones appeared9 signals., which could be attributed to intermediate vinylphosphonates 8. Only when BF3 Et2O was added to the reaction mixture did these signals gradually disappear with the simultaneous appearance of pyran-4-ones 9 signals. ∙

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1. NaH (1,1 equiv.), r. t., 0.5 h. 1. NaH (1,1 equiv.), r. t., 0.5 h. 1 1 2. n-BuLi (1,1 equiv.), R C(O)R (1,2 equiv.) O O O O R1C(O)R1 (1,2 equiv.) O O OH O O 2. n-BuLi (1,1 equiv.), o O O OH 1 o O O THF, 1.5 h, -78 C R1 (EtO) P -40 oC, 0.5 h., THF o (EtO) P R 2 -40 C, 0.5 h., THF (EtO)2P THF, 1.5 h, -78 C 2 1 (EtO)2P (EtO) P (EtO)2P R 2 R1

5 6 7a - d 5 6 7a - d

Me OMe Me OMe O O N O O O O O O N (EtO)2P Me OMe (EtO)2P (EtO) P Me OMe BF . Et O (EtO)2 P toluene BF3 . Et2O 2 toluene Me 3 2 Me N HO R1 R1 N HO 1 R1 O R1 R1 1 Me R O R1 Me R 8a-d 9a-d 8a-d 9a-d

Scheme 1. SynthesisSynthesis of diethyl of diethyl 4-hydroxy 4-hydroxy-2-oxoalkylphosphonates-2-oxoalkylphosphonates 7a–d7a –andd and3- Scheme 1. Synthesis of diethyl 4-hydroxy-2-oxoalkylphosphonates 7a–d and 3- diethoxyphosphoryldihydropyran3-diethoxyphosphoryldihydropyran-4-ones-4-ones9a– 9ad.–d. diethoxyphosphoryldihydropyran-4-ones 9a–d. Table 1. Yields of diethyl 4-hydroxy-2-oxoalkylphosphonates 7a–d and Table 1. Yields of diethyl 4-hydroxy-2-oxoalkylphosphonates 7a–d and 3- 3-diethoxyphosphoryldihydropyran-4-onesTable 1. Yields of diethyl 4-hydroxy9a–d. -2-oxoalkylphosphonates 7a–d and 3- diethoxyphosphoryldihydropyran-4-ones 9a–d. diethoxyphosphoryldihydropyran-4-ones 9a–d. Yield [%] a 1, 1 a Compound R R Yield [%] a Compound R1, R1 Yield [%]7 9 Compound R1, R1 7 9 7 9 a a MeMe,, Me 66 6684 84 a Me, Me 66 84 b b --(CH(CH22)5- 83 8390 90 b -(CH2)5- 83 90 c Ph, Ph 83 70 c c PhPh,, Ph 83 8370 70

d 77 66 d d 77 7766 66

a a Yield of pure, isolated products based on 5 or 7, respectively. Yielda ofYield pure of pure,, isolated isolated products productsbased based on on5 or 57 ,or respectively. 7, respectively. With 3-diethoxyphosphoryldihydropyran-4-ones 9a–d in hand, we used them as Michael With 3-diethoxyphosphoryldihydropyran-4-ones3-diethoxyphosphoryldihydropyran-4-ones9a –9ad in–d hand, in hand we used, we them used as them Michael as acceptorsMichael acceptors in the reaction with Grignard reagents, to introduce various substituents into position 2. inacceptors the reaction in the with reaction Grignard with reagents, Grignard to introducereagents, variousto introduce substituents various into substituents position 2. in Theto additionposition of2. The addition of ethyl-, n-butyl- and i-propylmagnesium chlorides proceeded smoothly when three ethyl-,The additionn-butyl- of and ethyli-propylmagnesium-, n-butyl- and i-propylmagne chlorides proceededsium chlorides smoothly proceeded when three smoothly equiv. ofwhen Grignard three equiv. of Grignard reagent were used, while the addition of phenylmagnesium chloride was effective reagentequiv. of were Grignard used, whilereagent the were addition used, of while phenylmagnesium the addition of chloridephenylmagnesium was eﬀective chloride only in was the presenceeffective only in the presence of 3 equiv. of copper iodide (I). Standard work-up of the reaction mixtures ofonly 3 equiv.in the ofpresence copper of iodide 3 equiv (I).. Standardof copper work-up iodide (I) of. Standard the reaction work mixtures-up of the followed reaction by mixtures column followed by column chromatography purification furnished 6-substituted 5- chromatographyfollowed by puriﬁcationcolumn furnishedchromatography 6-substituted purification 5-diethoxyphosphoryltetrahydropyran-4-ones furnished 6-substituted 5- diethoxyphosphoryltetrahydropyran-4-ones 10a–p in good yields (Scheme 2, Table 2). Interestingly, 10adiethoxyphosphoryl–p in good yieldstetra (Schemehydropyran2, Table2-4).-ones Interestingly, 10a–p in pyran-4-onesgood yields (Scheme10a–p were2, Table formed 2). Interestingly as mixtures, pyran-4-ones 10a–p were formed as mixtures of diasteroisomers trans-10a–p and cis-10a–p along with ofpyran diasteroisomers-4-ones 10a–ptrans were-10a formed–p and ascis mixtures-10a–p alongof diasteroisomers with the enol trans form,-10a enol-–p 10aand– cisp. -10a–p along with the enol form, enol-10a–p. the enol form, enol-10a–p.

2 R2MgCll ((3 equiiv..)) orr O O R2MgCl (3 equiv.) or O O O OH O O O O R2MgCl (3 equiv.) and O OH O O O R2MgCl (3 equiv.) and O O O OH O O (EtO) P R MgCl (3 equiv.) and(EtO)2P (EtO)2P (EtO)2P (EtO)2P CuI (3 equiv.) (EtO)2P (EtO)2P (EtO)2P (EtO) P CuI (3 equiv.) (EtO) P (EtO) P (EtO) P 2 CuI (3 equiv.) 2 2 2 0 1 1 1 1 THF, 0 0C, 2h R1 R1 R1 R THF, 0 C, 2h 2 R R R O 1 0 R2 O 1 R2 O 1 R2 O 1 O 1R THF, 0 C, 2h R O 1R R O 1R R O 11R O R1 R2 O R R2 O R R2 O R R1 R1 R1 R1 9a--d ttrrans--10a--p enoll--10a--p ciis--10a--p 9a-d trans-10a-p enol-10a-p cis-10a-p

SchemeScheme 2. Synthesis 2. Synthesis of of6-substituted 6-substituted 5 5-diethoxyphosphoryldihydropyran-4-ones-diethoxyphosphoryldihydropyran-4-ones10a– 10ap. –p. Scheme 2. Synthesis of 6-substituted 5-diethoxyphosphoryldihydropyran-4-ones 10a–p.

Table 2. Adducts 10a–p and 5-methylidenetetrahydropyran-4-ones 11a–p obtained. Table 2. Adducts 10a–p and 5-methylidenetetrahydropyran-4-ones 11a–p obtained. 10 11 Compound R1, R1 R2 10 11 Compound R1, R1 R2 Trans a Enol a Cis a Yield b [%] Yield b [%] Trans a Enol a Cis a Yield b [%] Yield b [%] a Et 72 16 12 70 97 a Me, Me Et 72 16 12 70 97 b Me, Me n-Bu 78 6 16 68 96 b n-Bu 78 6 16 68 96

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Table 2. Adducts 10a–p and 5-methylidenetetrahydropyran-4-ones 11a–p obtained.

10 11 1 1 Compound R ,R R2 Yield b Yield b Trans a Enol a Cis a [%] [%] a Et 72 16 12 70 97 b n-Bu 78 6 16 68 96 Me, Me Molecules 2019, 24, xc FOR PEER REVIEW i-Pr 71 9 20 80 51 4 of 12 d Ph 52 33 15 69 76 Molecules 2019c, 24, x FOR PEER REVIEWi- Pr 71 9 20 80 51 4 of 12 ed Ph Et52 7933 15 6 1569 7376 99 fe c Etn -Bui-Pr 79 71 716 9 1715 20 1273 80 719951 92 d -(CH2)5- Ph 52 33 15 69 76 gf n-Bui-Pr 71 6317 1012 2771 7592 55 e -(CH2)5- Et 79 6 15 73 99 hg i-PrPh 63 6610 2327 1175 7055 75 f n-Bu 71 17 12 71 92 h -(CH2)5-Ph 66 23 11 70 75 i g Eti-Pr 63 4010 3427 2675 7055 95 i Et 40 34 26 70 95 j h n-BuPh 66 3923 3611 2570 7575 91 j Ph, Ph n-Bu 39 36 25 75 91 k i Ph, Ph i-PrEt 40 3034 6726 370 6695 57 k j i-Prn -Bu 30 39 67 36 3 25 66 75 5791 l Ph, Ph Ph 23 72 5 55 85 l k Ph i-Pr 23 30 72 67 5 3 55 66 8557 m Et 68 17 15 82 82 m l Et Ph 68 23 17 72 15 5 82 55 8285 nn m n-Bun-Bu Et 70 68 7015 17 1515 15 1561 82 619982 99 oo n i-Prin-Pr -Bu 59 70 5930 15 3011 15 1140 61 405899 58 pp o Ph Phi-Pr 41 59 4151 30 518 11 875 40 759858 98 a 31 b Ratiosa Ratios of oftrans/enol transp/enol /cis/cis isomers isomers were were takenPh taken from 3141 fromP NMR spectraP51 NMR of 8 thespectra crude of products.75 the crudeb Yield products.98 of pure isolated Yieldproducts aof Ratios pure based of isolated trans/enol on 9 or 10products, respectively./cis isomers based were on 9 taken or 10 from, respectiv 31P NMRely. spectra of the crude products. b Yield of pure isolated products based on 9 or 10, respectively. 1 13 CarefulCareful analysis analysis of ofH 1andH and C 13 NMRC NMR spectra spectra allowed allowed us not us notonly only to propose to propose the therelative relative 1 13 configurationconﬁgurationCareful of both ofanalysis diastereoisomers both of diastereoisomers H and butC NMR also but their spectra also preferred allowed their preferredconformation. us not only conformation. Forto propose example Forthe, coupling relative example, constantsconfiguration 3JH5-H6, 3 JofH6 -bothP 3and diastereoisomers 3JC1′3-P for trans but3-10f also and their cis preferred-10f (Figure conformation. 2) unambiguously For example indicated, coupling coupling constants JH5-H6, JH6-P and JC10-P for trans-10f and cis-10f (Figure2) unambiguously constants 3JH5-H6, 3JH6-P and 3JC1′-P for trans-10f and cis-10f (Figure 2) unambiguously indicated diequatorialindicated and diequatorial axial/equatorial and axialposition/equatorial of n-Bu positionand phosphoryl of n-Bu groups and phosphorylin these diastereoisomers groups in these, diequatorial and axial/equatorial position of n-Bu and phosphoryl groups in these diastereoisomers, respectively.diastereoisomers, Because respectively. similar sets Because of coupling similar constants sets of werecoupling observed constants for were other observed pairs of for respectively. Because similar sets of coupling constants were observed for other pairs of diastereoisomersother pairs of diastereoisomerswe believe that we this believe assignment that this assignmentis valid for is validall for6-substituted all 6-substituted 5- diethoxyphosphoryldiastereoisomerstetra wehydropyran believe -4that-ones this10a –passignment. In turn enol is formvalid of forpyran all-4 -ones6-substituted 10a–p had 5 - 5-diethoxyphosphoryltetrahydropyran-4-onesdiethoxyphosphoryltetrahydropyran-4-ones 10a10a–p–. pIn. Inturn turn enol enol form form of ofpyran pyran-4-ones-4-ones 10a10a–p –hadp had characteristic doublets of hydroxyl group with chemical shift in the range of 11–12 ppm and coupling characteristiccharacteristic doublets doublets of of hydroxyl hydroxyl group groupwith with chemicalchemical shift in in the the range range of of 11 11–12–12 ppm ppm and and coupling coupling constant 4JH-P4 ~1 Hz. Presence of such coupling constant is yet another confirmation of the existence constantconstantJ H-P4JH-P~1 ~1 Hz.Hz. PresencePresence of such coupling coupling constant constant is is yet yet another another confirmation conﬁrmation of ofthe the existence existence of resonance assisted hydrogen bond (RAHB) in organophosphorus compounds [14]. Relative ratios of resonanceof resonance assisted assisted hydrogen hydrogen bond bond (RAHB) (RAHB) in in organophosphorus organophosphorus compounds compounds [[1414].]. RelativeRelative ratios of of trans-, cis- and enol-10a–p, taken from the corresponding 3131P NMR spectra, are given in Table 2. transof -,transcis--, and cis- enol-and enol10a-–10ap, taken–p, taken from from the the corresponding correspondingP 31P NMR NMR spectra, spectra, are are given given inin TableTable2 2..

6 6 O H 6 6 O H H6 O H H 6 O O O H H H P(O)(OEt) 5 5 O O P(O)(O2 Et)2 O O O O HH n-Bu n-Bu n-Bu RAHB n-Bu H5 5 n-Bu O O RAHB n-Bu H (EtO(E)2tOP)2P P(PO(O)(O)(OEtE)2t)2

trans-t1ra0nfs-10f enoel-n1o0lf-10f cisc-i1s-01f0f

3 4 4 3 J = 9.7 Hz JH-PJ =H- P1 .=2 1H.2z Hz 3 3 JH5-H6 H=5 -9H.67 Hz JHJ5H-H56-H =6 =3 .35. 5H Hz z 3 3J = 6.9 Hz 3 3 JH6-P =H 6-.P9 Hz JHJ6H-P6 -=P =3 83.87. 7H Hz z 3 3 3 J J =C 1~'-P 0 = H ~z 0 Hz 3J JC1' -=P =4 .47. 7H Hz z C1'-P C1'-P FigureFigureFigure 2. Preferred 2. Preferred 2. Preferred conformations conformations conformations ofof trans-,trans of transcis-, cis- and,- cis and- enol- and enol10f enol-10fand-10f and characteristic and characteristic characteristic coupling coupling coupling constants. constants.constants.

In the final step, the target 2,2,6-triisubstituted-5-methylidenetetrahydropyran-4-ones 11a–p In the final step, the target 2,2,6-triisubstituted-5-methylidenetetrahydropyran-4-ones 11a–p were obtained by olefination of formaldehyde using 5-diethoxyphosphoryltetrahydropyran-4-ones were obtained by olefination of formaldehyde using 5-diethoxyphosphoryltetrahydropyran-4-ones 10a–p as Horner-Wadsworth-Emmons reagents (Scheme 3). Very good yields were obtained when 10a–p as Horner-Wadsworth-Emmons reagents (Scheme 3). Very good yields were obtained when formalin and potassium carbonate were used and the reaction mixture was stirred at 0 °C for 2 h formalin and potassium carbonate were used and the reaction mixture was stirred at 0 °C for 2 h (Table 2). However, to get 2,2-disubstituted 6-isopropyl-5-methylidenetetrahydropyran-4-ones (Table11c 2),g. ,kHowever,o in reasonable, to get yields 2,2 -(60disubstituted–70%), stirring 6- isopropylof the reaction-5-methyliden mixture atetetrahydropyran r.t. for 3 h was necessary.-4-ones 11c,g,k,o in reasonable yields (60–70%), stirring of the reaction mixture at r.t. for 3 h was necessary.

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In the ﬁnal step, the target 2,2,6-triisubstituted-5-methylidenetetrahydropyran-4-ones 11a–p were obtained by oleﬁnation of formaldehyde using 5-diethoxyphosphoryltetrahydropyran-4-ones 10a–p as Horner-Wadsworth-Emmons reagents (Scheme3). Very good yields were obtained when formalin and potassium carbonate were used and the reaction mixture was stirred at 0 ◦C for 2 h (Table2). However, to get 2,2-disubstituted 6-isopropyl-5-methylidenetetrahydropyran-4-ones 11c,g,k,o in reasonable yieldsMolecules (60–70%), 2019, 24, x FOR stirring PEER of REVIEW the reaction mixture at r.t. for 3 h was necessary. 5 of 12

O HCHO (10 equiv.) K CO (2 equiv.) 10a-p 2 3 0 THF, 0 C, 2h R1 R2 O or rt, 3h R1 Molecules 2019, 24, x FOR PEER REVIEW 5 of 12 11a-p O SchemeScheme 3. Synthesis 3. Synthesis of of2,2 2,2,6-trisubstituted-5-methylidenetetrahydropyran-4-ones,6-trHCisubstitutedHO (10 equiv.) -5-methylidenetetrahydropyran-4-ones11a– 11ap. –p. K CO (2 equiv.) 10a-p 2 3 0 THF, 0 C, 2h R1 2.2. Biological Screening of Novel Pyranones R2 O 2.2. Biological Screening of Novel Pyranonesor rt, 3h R1 Novel 2,2,6-trisubstituted 5-methylidenetetrahydropyran-4-ones11a-p 11a–p were tested for their Novel 2,2,6-trisubstituted 5-methylidenetetrahydropyran-4-ones 11a–p were tested for their possibleScheme antiproliferative 3. Synthesis of 2, activity2,6-trisubstituted on two-5 human-methylidenetetrahydropyran leukemia cell lines,-4- acuteones 11a promyelocytic–p. leukemia possibleHL-60 and antiproliferative lymphoblastic activity leukemia on two NALM-6, human leukemia and on cell a solid lines, tumor-derivedacute promyelocytic human leukemia breast 2.2.HLadenocarcinoma Biological-60 and Screening lymphoblastic cell of Novel line MCF-7,Pyranones leukemia using NALM the tetrazolium-6, and on derivative a solid reductiontumor-derived assay (MTT)human (Table breast3). adenocarcinoma cell line MCF-7, using the tetrazolium derivative reduction assay (MTT) (Table 3). AnNovel anticancer 2,2,6-trisubstituted drug, carboplatin, 5-methylidenetetrahydropyran and a sesquiterpene-4-ones lactone,11a–p were parthenolide, tested for their containing the An anticancer drug, carboplatin, and a sesquiterpene lactone, parthenolide, containing the same α, possiblesame α antiproliferative, β-unsaturated activity carbonyl on two function human leukemia as analogs cell lines11a, acute–p, were promyelocytic used as leukemia reference compounds. HLβConcentration-response-unsaturated-60 and lymphoblastic carbonyl analysisleukemia function was NALM as performed -6analogs, and on to 11aa determine solid–p, tumorwere drug- derivedused concentrations humanas reference breast required compounds. to inhibit adenocarcinomaConcentration -cellresponse line MCF analysis-7, using was the tetrazoliumperformed derivative to determine reduction drug assay concentrations (MTT) (Table required 3). to inhibit the growth of cancer cells by 50% (IC50) after 48 h incubation. Anthe anticancer growth of drug cancer, carboplatin cells by, and 50% a sesquiterpene(IC50) after 48 lactone h incubation., parthenolide , containing the same α, β-unsaturated carbonyl function as analogs 11a–p, were used as reference compounds. Table 3. In vitro cytotoxic activity of 5-methylidenetetrahydropyran-4-ones 11a–p tested on three Concentration-response analysis was performed to determine drug concentrations required to inhibit Tablecancer cell3. In lines, vitro HL-60, cytotoxic NALM-6, activity and of MCF-7. 5-methylidenetetrahydropyran-4-ones 11a–p tested on the growth of cancer cells by 50% (IC50) after 48 h incubation. three cancer cell lines, HL-60, NALM-6, and MCF-7. a IC50 [µM] Table 3. In vitro cytotoxic activity1 1 of 5-methylidenetetrahydropyran2 -4-ones 11aa –p tested on 11 R ,R R IC50 [μM] three cancer cell lines,11 HL -60, NALMR1, -R61, and MCFR2 -7. HL-60 NALM-6 MCF-7 HL-60 NALM-6 MCF-7 a a EtEt 35.51IC 35.51±50 3.99[μM] a 3.991.57 ± 0.19 1.57 12.850.19 ± 0.35 12.85 0.35 11 R1, R1 R2 ± ± ± b nHL-Bu-60 NALM 4.98 -6 0.41MCF-7 0.35 0.07 14.50 0.14 b Me, Me n-Bu 4.98 ± 0.41± 0.35 ± 0.07 ±14.50 ± 0.14 ± c a Me, EtMe 35.51i-Pr ± 3.99 1.57 1.02 ± 0.190.03 12.85 ± 0.35 0.27 0.04 8.30 0.30 c i-Pr 1.02 ± 0.03± 0.27 ± 0.04 ±8.30 ± 0.30 ± d b n-Bu 4.98Ph ± 0.41 0.35 35.88 ± 0.07 2.6414.50 ± 0.14 5.45 0.24 46.90 0.20 d Me, Me Ph 35.88 ± 2.64 5.45 ± 0.24 46.90 ± 0.20 c i-Pr 1.02 ± 0.03 0.27 ± 0.04± 8.30 ± 0.30 ± ± e Et 45.70 2.75 5.18 0.68 18.30 0.70 d e Ph 35.88Et ± 2.6445.70 5.45 ± 2.75 ± 0.24± 5.1846.90 ± ±0.68 0.20 ±18.30 ± 0.70 ± f n-Bu 29.16 2.30 4.72 0.34 20.2 0.40 e f (CH2)5 Et 45.70n-Bu ± 2.7529.16 5.18 ± 2.30 ± 0.68± 4.7218.30 ± ±0.34 0.70 ±20.2 ± 0.40 ± g (CH2)5 i-Pr 6.76 0.48 0.74 0.06 11.00 1.20 f g n-Bu 29.16i-Pr ± 2.306.76 4.72 ± 0.48 ± 0.34± 0.7420.2 ± ± 0.06 0.40 ±11.00 ± 1.20 ± h (CH2)5 Ph 61.09 6.06 55.97 4.28 75.10 7.00 g h i-Pr 6.76Ph ± 0.4861.09 0.74 ± 6.06 ± 0.06± 55.9711.00 ± ± 4.281.20 ±75.10 ± 7.00 ± i h i Ph 61.09EtEt ± 6.0621.15 55.97 21.15± 0.64 ± 4.28 0.64 6.0575.10 ± ±0.49 7.00 6.05 56.350.49 ± 3.32 56.35 3.32 ± ± ± j i Et 21.15n-Bu ± 0.64 6.05 16.63 ± 0.49 1.8856.35 ± 3.32 5.63 0.14 6.90 0.21 j j Ph, Ph n-Bu 16.63n-Bu ± 1.8816.63 5.63 ± 1.88 ± 0.14± 5.636.90 ± ± 0.14 0.21 ±6.90 ± 0.21 ± k Ph, Ph Ph, Ph i-Pr 2.90 0.07 1.15 0.21 11.90 0.99 k k i-Pr 2.90i-Pr ± 0.072.90 1.15 ± 0.07 ± 0.21± 1.1511.90 ± ±0.21 0.99 ±11.90 ± 0.99 ± l Ph 39.47 2.97 6.31 0.41 81.10 4.90 l l Ph 39.47Ph ± 2.9739.47 6.31 ± 2.97 ± 0.41± 6.3181.10 ± ±0.41 4.90 ±81.10 ± 4.90 ± m m m Et 5.20EtEt ± 0.575.20 2.70 ± 5.200.57 ± 0.14 0.57 2.7018.10 ± ±0.14 0.28 2.70 18.100.14 ± 0.28 18.10 0.28 ± ± ± n n n-Bu 16.95n-Bu ± 0.78 7.45 16.95 ± 0.07 0.7830.30 ± 4.07 7.45 0.07 30.30 4.07 n n-Bu 16.95 ± 0.78± 7.45 ± 0.07 ±30.30 ± 4.07 ± o o i-Pr 1.10i-Pr ± 0.0 0.39 1.10 ± 0.01 0.04.50 ± 0.28 0.39 0.01 4.50 0.28 o i-Pr 1.10 ± 0.0 ± 0.39 ± 0.01 ±4.50 ± 0.28 ± p p Ph 47.28Ph ± 5.20 9.00 47.28 ± 0.75 5.2026.60 ± 2.25 9.00 0.75 26.60 2.25 p Ph 47.28 ± 5.20± 9.00 ± 0.75 ±26.60 ± 2.25 ± CarboplatinCarboplatin 2.90 ± 0.10 0.70 2.90 ± 0.300.10 3.80 ± 0.45 0.70 0.30 3.80 0.45 ± ± ± ParthenolideParthenolideCarboplatin 5.43± 0.852.90 2.82± ± 5.430.10 0.08 0.85 0.709.02± ± 0.300.18 2.82 3.800.08 ± 0.45 9.02 0.18 a Compound concentrationParthenolide required to inhibit metabolic5.43± 0.85activity± by2.82± 50%. 0.08 The ±cells9.02± were 0.18 ± a Compound concentration required to inhibit metabolic activity by 50%. The cells were incubated with the analogs incubateda with the analogs for 48 h. Values are expressed as mean ± SEM from the forCompound 48 h. Values areconcentration expressed as mean requiredSEM fromto inhibit the concentration-response metabolic activity curves by of 50%. at least The three cells experiments were ± concentrationincubatedusing a nonlinear - withresponse estimation the curves analogs (quasi-Newton of at forleast 48 three algorithm) h. experiments Values method. are using expressed a nonlinear as meanestimation ± SEM from the (quasi-Newton algorithm) method. concentration-response curves of at least three experiments using a nonlinear estimation All(Allquasi 16 16 analogs- analogsNewton were werealgorithm) found found to possess method. to possess antiproliferative antiproliferative activity in the activity tested incell the lines tested in the cell µM lines in the µM range.range. The The IC IC5050 valuesvalues below below 5 5µMµM were were obtained obtained for for 4 4 compounds compounds (11b (11b,c,k,,co,)k on,o) on HL- HL-60,60, 8 8 compounds compoundsAll 16 ( 11aanalogs,b,c,f,g ,werek,m,o )found on NALM to possess-6, and 1 antiproliferativeanalog (11o) on the activity MCF-7 cell in line.the testedCarboplatin cell lines in the µM inhibitedrange. Thethe growthIC50 values of all three below types 5 of µM cells werewith IC obtained50 values below for 45 µM compounds and the potency (11b ,orderc,k,o ) on HL-60, 8 NALMcompounds-6 > HL -(6011a > MCF,b,c,f-7.,g ,Parthenolidek,m,o) on NALMwas about-6, 2and-3-fold 1 analogless cytotoxic (11o )than on carboplatinthe MCF- 7on cell the seline. Carboplatin cell lines. The obtained data indicate that methylidenepyranones 11a–p were more cytotoxic against inhibited the growth of all three types of cells with IC50 values below 5 µM and the potency order leukemia cells than against breast cancer cells. Analog 11c was the most cytotoxic, with a three-fold lowerNALM IC-506 than > HL carboplatin-60 > MCF on-7. HL Parthenolide-60 and NALM was-6 cell abouts (1.02 2 and-3-fold 0.27 lessµM ascytotoxic compared than to 2.9 carboplatin and on these cell lines. The obtained data indicate that methylidenepyranones 11a–p were more cytotoxic against leukemia cells than against breast cancer cells. Analog 11c was the most cytotoxic, with a three-fold lower IC50 than carboplatin on HL-60 and NALM-6 cells (1.02 and 0.27 µM as compared to 2.9 and

Molecules 2020, 25, 611 6 of 12

(11a,b,c,f,g,k,m,o) on NALM-6, and 1 analog (11o) on the MCF-7 cell line. Carboplatin inhibited the growth of all three types of cells with IC50 values below 5 µM and the potency order NALM-6 > HL-60 > MCF-7. Parthenolide was about 2-3-fold less cytotoxic than carboplatin on these cell lines. The obtained data indicate that methylidenepyranones 11a–p were more cytotoxic against leukemia cells than against breast cancer cells. Analog 11c was the most cytotoxic, with a three-fold lower IC50 than carboplatin on HL-60 and NALM-6 cells (1.02 and 0.27 µM as compared to 2.9 and 0.7 µM for carboplatin, respectively). None of the analogs were more cytotoxic than carboplatin on MCF-7 cells. Analysis of the structure-activity relationship revealed that the cytotoxicity of 5-methylidenetetrahydropyran-4-ones 11a–p strongly depended on the nature of the substituent in position 6 (R2) and, to a much lesser extent, on the structure of geminal substituents in position 2 (R1,R1). This observation can be rationalized with the reasonable assumption that the conjugated methylidene group in pyranones 11 acts as an effective Michael acceptor towards various bionucleophiles through a reaction with mercapto groups (-SH) of cysteine residues in proteins and intracellular glutathione [15]. Such alkylation of cellular thiols disrupts key biological processes and affects multiple targets in cancer cells [16]. Therefore, R2 substituents that are much closer to the reaction site (methylidene group) than R1 substituents can influence the effectiveness of the alkylation to a much greater extent. Among position 2 substituents (R1,R1), two phenyl groups (11i-l) or a fluorenyl moiety (11m-p) were more advantageous for growth inhibition on all three tested cell lines than the non-aromatic substituents. Among analogs with various R2 alkyl groups introduced into position 6, those having isopropyl were the most active. The only exception was pyran-4-one 11k (R1 = Ph, R2 = i-Pr, 1 2 IC50 = 11.9 µM), which was less active than pyran-4-one 11j (R = Ph, R = n-Bu, IC50 = 6.9 µM) against the MCF-7 cell line. As opposed to position 2, phenyl moiety in position 6 (as R2) was the one contributing less to the compounds’ cytotoxicity. Evidently, the most cytotoxic compound of the series was pyran-4-one 11c (R1 = Me, R2 = i-Pr), which had the highest activity among all tested analogs on HL-60 and NALM-6 cell lines (IC50 = 1.02 µM and 0.27 µM, respectively). Therefore, analog 11c was selected for further evaluation of its anticancer activity. Since the mortality rates for acute myeloid leukemia are very high [17], necessitating the search for novel chemotherapeutic candidates, additional tests were performed on the HL-60 cell line. We wanted to determine whether the effect of 11c on cell viability was due to apoptosis. DNA damage and the subsequent induction of apoptosis is a primary cytotoxic mechanism of many anticancer agents. Cytotoxicity of 11c in HL-60 cells was once again measured after a shorter incubation time (24 h) using a broad range of analog concentrations (0–25 µM) and the obtained IC50 value was 2.2 µM (Figure3A). Phosphorylation of H2AX histone was used as a marker of DNA double-strand breaks. To assess the ability of 11c to phosphorylate H2AX, HL-60 cells were incubated with this compound for 24 h at 2.2 and 4.4 µM concentration (IC50 and 2 IC50, respectively). At 4.4 µM, 11c caused a significant H2AX phosphorylation, indicative of DNA damage (Figure3B). The induction of apoptosis was assessed by detection of the 89 kDa-cleaved fragment of PARP (poly (ADP-ribose) polymerase). The tested analog caused about 4-fold increase in the number of cleaved PARP positive (apoptotic) cells as compared to the control (Figure3C). Analysis of the cell cycle phases was then performed using flow cytometry. After 24 h incubation with 11c, HL-60 cells were treated with a fluorescent dye (4,6-diamidino-2-phenylindole, DAPI) that quantitatively stains DNA. The fluorescence intensity of the stained cells correlates with the amount of DNA they contain and identifies cell cycle position in the major phases (G0/G1 versus S versus G2/M phase). Representative profiles are shown in Figure4. Analog 11c at a concentration of 4.4 µM caused a significant increase of cells in the G2/M phase (from 18.9% in control to 31.4%), suggesting an antimitotic effect. Together, the obtained data may indicate that the novel pyranone 11c produced both cytotoxic and cytostatic effects. Molecules 2019, 24, x FOR PEER REVIEW 6 of 12

0.7 µM for carboplatin, respectively). None of the analogs were more cytotoxic than carboplatin on MCF-7 cells. Analysis of the structure-activity relationship revealed that the cytotoxicity of 5- methylidenetetrahydropyran-4-ones 11a–p strongly depended on the nature of the substituent in position 6 (R2) and, to a much lesser extent, on the structure of geminal substituents in position 2 (R1, R1). This observation can be rationalized with the reasonable assumption that the conjugated methylidene group in pyranones 11 acts as an effective Michael acceptor towards various bionucleophiles through a reaction with mercapto groups (-SH) of cysteine residues in proteins and intracellular glutathione [15]. Such alkylation of cellular thiols disrupts key biological processes and affects multiple targets in cancer cells [16]. Therefore, R2 substituents that are much closer to the reaction site (methylidene group) than R1 substituents can influence the effectiveness of the alkylation to a much greater extent. Among position 2 substituents (R1, R1), two phenyl groups (11i-l) or a fluorenyl moiety (11m-p) were more advantageous for growth inhibition on all three tested cell lines than the non-aromatic substituents. Among analogs with various R2 alkyl groups introduced into position 6, those having isopropyl were the most active. The only exception was pyran-4-one 11k (R1 = Ph, R2 = i-Pr, IC50 = 11.9 μM), which was less active than pyran-4-one 11j (R1 = Ph, R2 = n-Bu, IC50 = 6.9 μM) against the MCF- 7 cell line. As opposed to position 2, phenyl moiety in position 6 (as R2) was the one contributing less to the compounds’ cytotoxicity. Evidently, the most cytotoxic compound of the series was pyran-4-one 11c (R1 = Me, R2 = i-Pr), which had the highest activity among all tested analogs on HL-60 and NALM-6 cell lines (IC50 = 1.02 μM and 0.27 μM, respectively). Therefore, analog 11c was selected for further evaluation of its anticancer activity. Since the mortality rates for acute myeloid leukemia are very high [17], necessitating the search for novel chemotherapeutic candidates, additional tests were performed on the HL-60 cell line. We wanted to determine whether the effect of 11c on cell viability was due to apoptosis. DNA damage and the subsequent induction of apoptosis is a primary cytotoxic mechanism of many anticancer agents. Cytotoxicity of 11c in HL-60 cells was once again measured after a shorter incubation time (24 h) using a broad range of analog concentrations (0-25 µM) and the obtained IC50 value was 2.2 µM (Figure 3A). Phosphorylation of H2AX histone was used as a marker of DNA double-strand breaks. To assess the ability of 11c to phosphorylate H2AX, HL-60 cells were incubated with this compound for 24 h at 2.2 and 4.4 µM concentration (IC50 and 2 IC50, respectively). At 4.4 µM, 11c caused a significant H2AX phosphorylation, indicative of DNA damage (Figure 3B). The induction of apoptosis was assessed by detection of the 89 kDa-cleaved fragment of PARP (poly (ADP-ribose) Moleculespolymerase)2020, 25, 611. The tested analog caused about 4-fold increase in the number of cleaved PARP positive7 of 12 (apoptotic) cells as compared to the control (Figure 3C).

Molecules 2019, 24, x FOR PEER REVIEW 7 of 12

Analysis of the cell cycle phases was then performed using flow cytometry. After 24 h incubation with 11c, HL-60 cells were treated with a fluorescent dye (4,6-diamidino-2-phenylindole, DAPI) that quantitatively stains DNA. The fluorescence intensity of the stained cells correlates with the amount of DNA they contain and identifies cell cycle position in the major phases (G0/G1 versus S versus G2/M phase).FigureFigure 3.RepresentativeInﬂuence 3. Influence of 11c proofon 11c HL-60files on are HL cells shown-60 after cells 24 in after h Fig incubation 24ure h 4incubation. Analog (A): Metabolic 11c (A) :at Metabolic a activity, concentration (activityB): DNA, of( damageB )4.4: µM caused ameasured significantDNA by damage H2AXincrease phosphorylationmeasured of cells by inH2AX the changes phosphorylationG2/M and ph (Case): apoptosis (from changes induction18.9% and in(C measured) control: apoptosis to by 31.4%)PARPinduction cleavage., suggesting an measured by PARP cleavage. (B,C): Results are expressed as mean ± SEM of a triplicate antimitotic(B,C): Results effect. are expressed Together as, meanthe obtainedSEM of data a triplicate may indicate assay; *** thatp < 0.001, the novel ** p < pyranone0.01. 11c produced both cytotoxicassay; *** p and < 0.001 cytostatic, ** p < 0.01. effects ± .

FigureFigure 4. Eff ect4. Effect of 11c ofon 11c the cellon the cycle cell progression cycle progression in HL-60 cells. in HL (A-):60 Representative cells. (A): Representative flow cytometry flow resultscytometry of the cell results cycle changes. of the cell (B): Percentagecycle changes. quantification (B): Percentage of the total quantification cell populations of inthe each total of cell the cellpopulations cycle phases in expressedeach of the as meancell cycleSEM phases of a triplicate expressed assay; as ***meanp < 0.001. ± SEM of a triplicate assay; ± *** p < 0.001. 3. Materials and Methods

3.1.3. Chemistry Materials and Methods

3.1.1.3.1. General Chemistry Information NMR spectra were recorded on a Bruker DPX 250 (Billerica, MA, USA) or Bruker Avance II 3.1.1. General Information instrument (Billerica, MA, USA) at 250.13 MHz or 700 MHz for 1H, 62.9 MHz or 176 MHz for 13C, 31 and 101.3NMR MHz spectra for Pwere NMR recorded with tetramethylsilane on a Bruker DPX used 250 as (Billerica, an internal MA, and US) 85% or Bruker H3PO4 Avanceas an II externalinstrument standard. (Billerica,31P NMR MA, spectra US) at were250.13 recorded MHz or using 700 MHz broadband for 1H, proton62.9 MHz decoupling. or 176 MHz IR for spectra 13C, and were101.3 recorded MHz on for a 31 BrukerP NMR Alpha with ATRtetramethylsilane spectrophotometer. used as Melting an internal points and were 85% determined H3PO4 as inan open external capillariesstandard. and are31P uncorrected. NMR spectra Column were recorded chromatography using broadband was performed proton on silica decoupling. gel 60 (230–400 IR spectra mesh) were (Aldrich,recorded Saint on Louis, a Bruker MO, US). Alpha Thin-layer ATR spectr chromatographyophotometer. wasMelting performed points onwere the determined pre-coated TLCin open sheetscapillaries of silica and gel 60are F254 uncorrected. (Aldrich). Column The purity chromatography of the synthesized was performed compounds on was silica confirmed gel 60 (230 by–400 combustionmesh) (Aldrich elemental, Saint analyses Louis, (CH, MO, elemental US). Thin analyzer-layer chromatography EuroVector 3018, was Elementar performed Analysensysteme on the pre-coated GmbH,TLC Langenselbold, sheets of silica Germany.gel 60 F254 MS (Aldrich). spectra ofThe intermediates purity of the were synthesized recorded compounds on Waters 2695-Waters was confirmed ZQ 2000by co LCmbustion/MS apparatus. elemental EI mass analyses spectra of (CH the,final elemental compounds analyzer were recorded EuroVector on a GCMS-QP2010 3018, Elementar ULTRAnalysensysteme A instrument (Shimadzu, GmbH, Langenselbold, Kyoto, Japan). Germany Mass. spectra MS spectra were of obtained intermediates using were the following recorded on operatingWaters conditions: 2695-Waters electron ZQ 2000 energy LC/MS of 70 apparatus. eV and ion sourceEI mass temperature spectra of ofthe 200 final◦C. Samplescompounds were were introducedrecorded via on a directa GCMS insertion-QP2010 probe ULTR heated A instrumentfrom 30 ◦C to (Shimadzu 300 ◦C. All, K reagentsyoto, Japan). and starting Mass materialsspectra were wereobtained purchased using from commercialthe following vendors operating and used conditions: without furtherelectron purification. energy of Organic 70 eV solventsand ion were source driedtemperature and distilled of prior200 °C. to Samples use. Standard were introduced syringe techniques via a direct were insertion used for probe transferring heated from dry solvents. 30 °C to 300 °C.General All reagents procedures and starting and characterization materials were datapurchased for diethyl from commercial 4-hydroxy-2-oxoalkylphosphonates vendors and used without 7a–dfurther, 3-diethoxyphosphoryldihydropyran-4-ones purification. Organic solvents were dried9a– dandand distilled 2,2-dialkyl(diaryl)-6-alkyl(aryl)-5- prior to use. Standard syringe methylenetetrahydrotechniques were used -4H-pyran-4-ones for transferring10a dry–p aresolvents. given in Supplementary Materials. General procedures and characterization data for diethyl 4-hydroxy-2-oxoalkylphosphonates 7a–d, 3- diethoxyphosphoryldihydropyran-4-ones 9a–d and 2,2-dialkyl(diaryl)-6-alkyl(aryl)-5-methylenetetrahydro - 4H-pyran-4-ones 10a–p are given in Supplementary Materials.

Molecules 2020, 25, 611 8 of 12

3.1.2. General Procedure for the Synthesis of 2,2,6-Triisubstituted-5-Methylidenetetrahydropyran- 4-Ones 11a–p To a vigorously stirred solution of 5-diethoxyphosphoryltetrahydropyran-4-ones 10a–p (0.2 mmol) in dry THF (2 mL), formaldehyde (36–38% solution in water, 0.167 mL, ca. 2.00 mmol) was added at 0 ◦C, followed by addition of K2CO3 (55.3 mg, 0.40 mmol) in water (0.56 mL). The resulting mixture was stirred vigorously at 0 ◦C for 2 h (for pyran-4-ones 10a,b,d–f,h–j,l–n,p) or at r.t. for 3 h (for pyran-4-ones 10c,g,k,o). Next Et2O (10 mL) was added and the layers were separated. The water fraction was washed with Et2O (5 mL). Organic fractions were combined, washed with brine (10 mL), and dried over MgSO4. The solvents were evaporated under reduced pressure and the resulting crude product was puriﬁed by column chromatography (eluent DCM). 6-Ethyl-2,2-dimethyl-5-methylidenetetrahydro-4H-pyran-4-one (11a) (32.6 mg, 97%) Colorless oil. 1H NMR (700 MHz, Chloroform-d) δ 0.97 (t, J = 7.3 Hz, 3H), 1.28 (s, 3H), 1.29 (s, 3H), 1.60–1.69 (m, 1H), 1.79–1.89 (m, 1H), 2.39–2.51 (m, 2H), 4.47 (ddd, J = 5.6, 2.0, 2.0 Hz, 1H), 5.27 (dd, J = 2.0, 1.0 Hz, 1H), 6.14 (dd, J = 2.0, 1.0 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 9.3, 25.3, 28.0, 30.8, 51.1, 72.3, 72.9, 119.6, 144.9, + 197.9. EI-MS [M] = 168.0. Anal. Calcd. for C10H16O2: C, 71.39; H, 9.59. Found: C, 71.21; H, 9.61. 6-Butyl-2,2-dimethyl-5-methylidenetetrahydro-4H-pyran-4-one (11b) (37.7 mg, 96%) Colorless oil. 1H NMR (700 MHz, Chloroform-d) δ 0.91 (t, J = 7.2 Hz, 3H), 1.27 (s, 3H), 1.28 (s, 3H), 1.30–1.42 (m, 3H), 1.43–1.52 (m, 1H), 1.62 (dddd, J = 14.1, 10.2, 7.8, 4.7 Hz, 1H), 1.77 (dddd, J = 14.1, 10.6, 5.4, 3.7 Hz, 1H), 2.46 (d, J = 16.7 Hz, 2H), 4.50 (dddd, J = 7.8, 3.9, 2.0, 2.0 Hz, 1H), 5.27 (dd, J = 2.0, 1.0 Hz, 1H), 6.11 (dd, J = 2.1, 1.0 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 14.2, 22.8, 25.4, 27.3, 30.8, 34.7, 51.1, 71.8, 72.4, + 119.4, 145.3, 197.9. EI-MS [M] = 196.0. Anal. Calcd. for C12H20O2: C, 73.43; H, 10.27. Found: C, 73.60; H, 10.30. 6-Isopropyl-2,2-dimethyl-5-methylidenetetrahydro-4H-pyran-4-one (11c) (18.6 mg, 51%) Colorless oil. 1H NMR (700 MHz, Chloroform-d) δ 0.86 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 1.26 (s, 3H), 1.29 (s, 3H), 1.96 (heptd, J = 6.8, 3.2 Hz, 1H), 2.37–2.48 (m, 2H), 4.44 (ddd, J = 3.2, 2.0, 2.0 Hz, 1H), 5.27 (dd, J = 2.0, 1.1 Hz, 1H), 6.20 (dd, J = 2.0, 1.1 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 15.7, 19.4, 25.0, 30.8, + 33.7, 51.0, 71.8, 76.8, 120.6, 144.1, 198.1. EI-MS [M] = 182.0. Anal. Calcd. for C11H18O2: C, 72.49; H, 9.95. Found: C, 72.71; H, 9.92. 2,2-Dimethyl-5-methylidene-6-phenyltetrahydro-4H-pyran-4-one (11d) (32.9 mg, 76%) Colorless oil. 1H NMR (700 MHz, Chloroform-d) δ 1.40 (s, 3H), 1.42 (s, 3H), 2.65 (s, 2H), 4.82 (dd, J = 2.2, 1.2 Hz, 1H), 5.52 (dd, J = 2.2, 2.2 Hz, 1H), 6.15 (dd, J = 2.2, 1.2 Hz, 1H), 7.30 – 7.34 (m, 1H), 7.35 – 7.40 (m, 4H). 13C NMR (176 MHz, Chloroform-d) δ 25.5, 30.8, 51.5, 73.5, 76.0, 123.0, 128.0 (2 C), 128.4, 128.7 (2 C), + × × 140.5, 145.4, 197.1. EI-MS [M] = 216.0. Anal. Calcd. for C14H16O2: C, 77.75; H, 7.46. Found: C, 77.71; H, 7.47. 2-Ethyl-3-methylidene-1-oxaspiro [5.5]undecan-4-one (11e) (41.2 mg, 99%) Colorless oil. 1H NMR (700 MHz, Chloroform-d) δ 1.05 (t, J = 7.3 Hz, 3H), 1.25–1.92 (m, 12H), 2.38 (d, J = 16.7 Hz, 1H), 2.44 (d, J = 16.7 Hz, 1H), 4.37 (dddd, J = 7.8, 3.6, 2.1, 2.1 Hz, 1H), 5.25 (dd, J = 2.1, 1.0 Hz, 1H), 6.11 (dd, J = 2.1, 1.0 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 9.9, 21.8, 21.8, 25.5, 28.1, 33.2, 39.3, 50.9, 71.7, 73.1, 119.3, + 145.6, 198.2. EI-MS [M] = 208.0. Anal. Calcd. for C13H20O2: C, 74.96; H, 9.68. Found: C, 74.76; H, 9.70. 2-Butyl-3-methylidene-1-oxaspiro[5.5]undecan-4-one (11f) (43.5 mg, 92%) Colorless oil. 1H NMR (700 MHz, Chloroform-d) δ 0.92 (t, J = 7.2 Hz, 3H), 1.18–1.91 (m, 16H), 2.37 (d, J = 16.7 Hz, 1H), 2.43 (d, J = 16.7 Hz, 1H), 4.32–4.49 (m, 1H), 5.25 (dd, J = 2.1, 1.0 Hz, 1H), 6.08 (dd, J = 2.1, 1.0 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 14.2, 21.8, 21.8, 22.8, 25.5, 27.7, 33.2, 34.8, 39.3, 50.9, 70.6, 73.1, 119.1, 145.9, + 198.2. EI-MS [M] = 236.0. Anal. Calcd. for C15H24O2: C, 76.23; H, 10.24. Found: C, 76.18; H, 10.25. 2-Isopropyl-3-methylidene-1-oxaspiro[5.5]undecan-4-one (11g) (24.5 mg, 55%) Colorless oil. 1H NMR (700 MHz, Chloroform-d) δ 0.86 (d, J = 6.8 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 1.16–1.91 (m, 10H), 2.00 (hd, J = 6.8, 3.0 Hz, 1H), 2.32 (d, J = 17.1 Hz, 1H), 2.38 (d, J = 17.1 Hz, 1H), 4.37 (ddd, J = 3.0, 2.2, 2.2 Hz, 1H), Molecules 2020, 25, 611 9 of 12

5.24 (dd, J = 2.2, 1.1 Hz, 1H), 6.18 (dd, J = 2.2, 1.1 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 15.3, 19.7, 21.7, 21.9, 25.6, 32.9, 33.4, 39.2, 50.8, 72.4, 75.2, 120.1, 144.7, 198.3. EI-MS [M]+ = 222.0. Anal. Calcd. for C14H22O2: C, 75.63; H, 9.97. Found: C, 75.80; H, 9.96.

3-Methylidene-2-phenyl-1-oxaspiro[5.5]undecan-4-one (11h) (38.5 mg, 75%) White solid mp 128 – 130 ◦C. 1H NMR (700 MHz, Chloroform-d) δ 1.04–2.12 (m, 10H), 2.57 (d, J = 16.5 Hz, 1H), 2.64 (d, J = 16.5 Hz, 1H), 4.82 (dd, J = 2.2, 1.2 Hz, 1H), 5.48 (dd, J = 2.2, 2.2 Hz, 1H), 6.14 (dd, J = 2.2, 1.2 Hz, 1H), 7.31–7.35 (m, 1H), 7.36–7.40 (m, 4H). 13C NMR (176 MHz, Chloroform-d) δ 21.8, 21.9, 25.5, 33.7, 39.2, 50.6, 74.4, 74.9, 122.7, 128.0 (2 C), 128.2, 128.6 (2 C), 140.8, 145.8, 197.4. EI-MS [M]+ = 256.0. Anal. Calcd. for × × C17H20O2: C, 79.65; H, 7.86. Found: C, 79.87; H, 7.85. 6-Ethyl-5-methylidene-2,2-diphenyltetrahydro-4H-pyran-4-one (11i) (55.6 mg, 95%) Pale yellow oil. 1H NMR (700 MHz, Chloroform-d) δ 1.14 (t, J = 7.3 Hz, 3H), 1.79–1.87 (m, 1H), 1.89–1.98 (m, 1H), 2.90 (d, J = 17.1 Hz, 1H), 3.58 (d, J = 17.2 Hz, 1H), 4.21 (dddd, J = 7.8, 3.9, 2.2, 2.2 Hz, 1H), 5.19 (dd, J = 2.2, 1.0 Hz, 1H), 6.13 (dd, J = 2.2, 1.0 Hz, 1H), 7.26 (m, 10H). 13C NMR (176 MHz, Chloroform-d) δ 9.7, 28.0, 50.98, 73.5, 80.1, 120.2, 125.1 (2 C), 127.0, 127.9 (2 C), 127.9, 128.3 (2 C), 128.8 (2 C), 142.2, 144.4, 147.6, + × × × × 196.5. EI-MS [M] = 292.0. Anal. Calcd. for C20H20O2: C, 82.16; H, 6.90. Found: C, 82.37; H, 6.89. 6-Butyl-5-methylidene-2,2-diphenyltetrahydro-4H-pyran-4-one (11j) (55.8 mg, 91%) White solid 141 – 143 1 ◦C. H NMR (700 MHz, Chloroform-d) δ 0.88–0.98 (m, 1H), 1.02 (t, J = 7.3 Hz, 3H), 1.42–1.48 (m, 1H), 1.52–1.61 (m, 1H), 1.71–1.79 (m, 1H), 1.85–1.92 (m, 2H), 2.94 (d, J = 17.1 Hz, 1H), 3.63 (d, J = 17.1 Hz, 1H), 4.28 (dddd, J = 6.9, 4.4, 2.1, 2.1 Hz, 1H), 5.25 (dd, J = 2.1, 1.1 Hz, 1H), 6.16 (dd, J = 2.1, 1.1 Hz, 1H), 7.26 (m, 10H). 13C NMR (176 MHz, Chloroform-d) δ 14.2, 22.8, 27.4, 34.7, 51.0, 72.4, 80.1, 120.1, 125.1 (2 C), 127.0, 127.9 (2 C), 127.9, 128.3 (2 C), 128.8 (2 C), 142.2, 144.8, 147.6, 196.6. EI-MS [M]+ = × × × × 320.0. Anal. Calcd. for C22H24O2: C, 82.46; H, 7.55. Found: C, 82.66; H, 7.53. 6-Isopropyl-5-methylidene-2,2-diphenyltetrahydro-4H-pyran-4-one (11k) (34.9 mg, 57%) Pale yellow oil. 1H NMR (700 MHz, Chloroform-d) δ 1.09 (d, J = 6.8 Hz, 3H), 1.23 (d, J = 6.9 Hz, 3H), 2.10 (heptd, J = 6.8, 2.8 Hz, 1H), 2.90 (d, J = 17.5 Hz, 1H), 3.59 (d, J = 17.5 Hz, 1H), 4.16–4.29 (m, 1H), 5.17 (dd, J = 2.0, 1.0 Hz, 1H), 6.21 (dd, J = 2.0, 1.0 Hz, 1H), 7.18–7.51 (m, 10H). 13C NMR (176 MHz, Chloroform-d) δ 15.7, 19.9, 33.5, 50.8, 77.0, 79.6, 121.2, 125.0 (2 C), 127.0, 127.89 (3 C), 128.3 (2 C), 128.8 (2 C), 142.0, + × × × × 143.3, 147.7, 196.5. EI-MS [M] = 306.0. Anal. Calcd. for C21H22O2: C, 82.32; H, 7.24. Found: C, 82.07; H, 7.22. 5-Methylidene-2,2,6-triphenyltetrahydro-4H-pyran-4-one (11l) (57.9 mg, 85%) Pale yellow oil. 1H NMR (700 MHz, Chloroform-d) δ 3.12 (d, J = 17.0 Hz, 1H), 3.70 (d, J = 17.0 Hz, 1H), 4.76 (dd, J = 2.2, 1.1 Hz, 1H), 5.20 (dd, J = 2.2, 2.2 Hz, 1H), 6.13 (dd, J = 2.2, 1.1 Hz, 1H), 7.17–7.46 (m, 15H). 13C NMR (176 MHz, Chloroform-d) δ 51.5, 76.6, 81.3, 123.3, 125.2 (2 C), 127.2, 128.0 (2 C), 128.2 (3 C), 128.4 (2 C), × × × × 128.5, 128.7 (2 C), 129.0 (2 C), 140.3, 142.0, 145.2, 147.2, 196.0. EI-MS [M]+ = 340.0. Anal. Calcd. for × × C24H20O2: C, 84.68; H, 5.92. Found: C, 84.45; H, 5.90.

60-Ethyl-50-methylidene-50,60-dihydrospiro[ﬂuorene-9,20-pyran]-40(30H)-one (11m) (47.6 mg, 82%) Pale yellow oil. 1H NMR (700 MHz, Chloroform-d) δ 0.99 (t, J = 7.4 Hz, 3H), 1.78–1.88 (m, 1H), 1.96 (ddt, J = 14.6, 7.3, 3.6 Hz, 1H), 2.87 (d, J = 16.7 Hz, 1H), 3.12 (d, J = 16.7 Hz, 1H), 5.00 (dddd, J = 6.1, 4.0, 2.1, 2.1 Hz, 1H), 5.53 (dd, J = 2.1, 1.2 Hz, 1H), 6.49 (dd, J = 2.1, 1.2 Hz, 1H), 7.24 (dd, J = 8.4, 7.5 Hz, 1H), 7.33 (dd, J = 8.4, 7.3 Hz, 1H), 7.39 (dd, J = 8.4, 7.6 Hz, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.64 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.6 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 9.0, 27.8, 47.3, 75.5, 82.6, 120.1, 120.8, 121.0, 123.9, 124.7, 127.6, 128.5, 129.5, 129.5, 139.0, 140.3, 144.7, 145.3, 147.4, 196.3. + EI-MS [M] = 290.0. Anal. Calcd. for C20H18O2: C, 82.73; H, 6.25. Found: C, 83.01; H, 6.23.

60-Butyl-50-methylidene-50,60-dihydrospiro[ﬂuorene-9,20-pyran]-40(30H)-one (11n) (63.04 mg, 99%) Pale yellow oil. 1H NMR (700 MHz, Chloroform-d) δ 0.87 (t, J = 7.3 Hz, 3H), 1.26–1.49 (m, 4H), 1.79 (dddd, J = 14.0, 10.5, 7.1, 4.8 Hz, 1H), 1.90 (dddd, J = 14.3, 10.7, 5.7, 3.8 Hz, 1H), 2.88 (d, J = 16.6 Hz, 1H), 3.09 (d, J = 16.7 Hz, 1H), 5.03 (dddd, J = 7.2, 4.0, 2.1, 2.1 Hz, 1H), 5.53 (dd, J = 2.1, 1.2 Hz, 1H), 6.46 (dd, J = 2.1, Molecules 2020, 25, 611 10 of 12

1.2 Hz, 1H), 7.24 (dd, J = 8.6, 7.5 Hz, 1H), 7.33 (dd, J = 8.6, 7.4 Hz, 1H), 7.37–7.40 (m, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 14.1, 22.8, 26.9, 34.6, 47.3, 74.7, 82.7, 120.1, 120.8, 120.8, 124.0, 124.7, 127.7, 128.5, 129.5, + 129.5, 139.0, 140.3, 145.22, 145.4, 147.5, 196.4. EI-MS [M] = 318.0. Anal. Calcd. for C22H22O2: C, 82.99; H, 6.96. Found: C, 82.75; H, 6.97.

60-Isopropyl-50-methylidene-50,60-dihydrospiro[ﬂuorene-9,20-pyran]-40(30H)-one (11o) (35.3 mg, 58%) Pale yellow oil. 1H NMR (700 MHz, Chloroform-d) δ 0.99 (d, J = 6.9 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 2.11–2.17 (m, 1H), 2.71 (d, J = 17.1 Hz, 1H), 3.15 (d, J = 17.1 Hz, 1H), 4.86 (ddd, J = 3.2, 2.1 Hz, 1H), 5.55 (dd, J = 2.1, 1.2 Hz, 1H), 6.56 (dd, J = 2.2, 1.2 Hz, 1H), 7.21 (dd, J = 8.6, 7.5 Hz, 1H), 7.34 (dd, J = 8.5, 7.6 Hz, 1H), 7.36–7.43 (m, 3H), 7.47 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 15.7, 19.6, 33.9, 47.7, 79.3, 82.4, 120.0, 120.8, 122.0, 124.0, 124.9, 127.7, 128.5, 129.4, 129.5, 139.0, 140.4, 144.3, 145.2, 147.6, 196.3. EI-MS [M]+ = 304.0. Anal. Calcd. for C21H20O2: C, 82.86; H, 6.62. Found: C, 82.92; H, 6.61.

50-Methylidene-60-phenyl-50,60-dihydrospiro[ﬂuorene-9,20-pyran]-40(30H)-one (11p) (66.3 mg, 98%) Pale yellow oil. 1H NMR (700 MHz, Chloroform-d) δ 3.02 (d, J = 16.6 Hz, 1H), 3.30 (d, J = 16.5 Hz, 1H), 5.05 (dd, J = 2.1, 1.4 Hz, 1H), 5.99 (dd, J = 2.1, 2.1 Hz, 1H), 6.47 (dd, J = 2.1, 1.4 Hz, 1H), 7.28–7.44 (m, 9H), 7.51 (d, J = 7.6 Hz, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H). 13C NMR (176 MHz, Chloroform-d) δ 47.6, 78.6, 83.4, 120.2, 120.9, 124.1, 124.5, 124.8, 127.8, 128.2 (2 C), × 128.6 (2 C), 128.7 (2 C), 129.7, 129.7, 139.2, 140.0, 140.5, 144.8, 145.71, 146.9, 195.8. EI-MS [M]+ = × × 338.0. Anal. Calcd. for C24H18O2: C, 85.18; H, 5.36. Found: C, 85.01; H, 5.35.

3.2. Biology

3.2.1. Cell Culture The breast cancer MCF-7, leukemia HL-60, and NALM-6 cell lines were obtained from the European Collection of Cell Cultures (ECACC). The MCF-7 cells were cultured in Minimum Essential Medium Eagle (MEME, Sigma-Aldrich, St. Louis, MO, USA) with glutamine (2 mM), Men Non-essential amino acid solution, gentamycin (5 µg/mL) and 10% heat-inactivated fetal bovine serum (FBS). HL-60 and NALM-6 cells were maintained in RPMI 1640 + Glutamax medium (Gibco/Life Technologies, Carlsbad, CA, USA), supplemented with 10% FBS, streptomycin and penicillin. Cells were maintained in the logarithmic phase at 37 ◦C under a 5% carbon dioxide atmosphere.

3.2.2. Cytotoxicity Determination (MTT Assay) The MTT assay was performed according to a known procedure [18]. The cells were incubated with the analogs for 48 or 24 h. The absorbance of the blue formazan product was measured at 560 nm using FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, LLC, San Jose, CA, USA) and compared with the control (untreated cells). All experiments were performed in triplicate.

3.2.3. Analysis of DNA Damage and Apoptosis by Flow Cytometry DNA damage and apoptotic cell death were determined by ﬂow cytometry using ﬂuorescent antibodies for phosphorylated H2AX (Alexa Fluor 647 Mouse Anti-H2AX (pS139)) and cleaved PARP (PE Mouse Anti-Cleaved PARP (Asp214) Antibody) (BD Bioscience, San Jose, CA, USA), according to the manufacturer’s guidelines, as described in detail elsewhere [19].

3.2.4. Cell Cycle Analyses by Flow Cytometry The cell cycle kinetics were determined using DAPI staining solution (BD Bioscience), according to the manufacturer guidelines, as described in detail elsewhere [20]. Cells were analyzed by ﬂow cytometry using CytoFLEX (Beckman Coulter, Inc., Brea, CA, USA). Molecules 2020, 25, 611 11 of 12

3.2.5. Statistical Analysis Statistical analyses were performed using Prism 4.0 (GraphPad Software Inc., San Diego, CA, USA). The data were expressed as means SEM. Statistical comparisons were assessed by a one-way ± ANOVA, followed by a post-hoc multiple comparison Student-Newman-Keuls test or Student t test. A probability level of 0.05 or lower was considered statistically signiﬁcant; *** p < 0.001, ** p < 0.01, * p < 0.05.

4. Conclusions The reported results showed that biologically important 5-methylidenetetrahydropyran-4-ones 11 can be eﬀectively synthesized using the Horner-Wadsworth-Emmons reaction of easily accessible 3-diethoxyphosphoryldihydropyran-4-ones 10 with formaldehyde. Various substituents in position 2 can be introduced to the target compounds performing the reaction of dianion 6 with both, acyclic or cyclic symmetrical ketones. Furthermore, additional substituents can be placed in position 6 by the addition of selected Grignard reagents to 3-diethoxyphosphoryldihydropyran-4-ones 9. Therefore, the described methodology has a broad scope, as a variety of substituents can be introduced in these positions. Currently, the reaction of aldehydes and unsymmetrical ketones with dianion 6 is investigated to broaden the scope of the presented methodology. The biological evaluation of the obtained 5-methylidenetetrahydropyran-4-ones 11 conﬁrmed our assumption that these compounds possess a strong cytotoxic activity against several cancer cell lines. Investigation of structure-activity relationship revealed that the i-propyl substituent in position 6 is crucial for activity. 6-Isopropyl-2,2-dimethyl-5-methylidenetetrahydropyran-4-one 11c, which was recognized as the most active compound against the HL-60 cancer cell line, was further tested in order to disclose its mechanism of action. The preliminary tests indicated that 11c induced apoptosis in HL-60 cells and caused the arrest of the cell cycle in the G2/M phase, which may suggest its cytotoxic and cytostatic activity. Further biological experiments are necessary to fully determine the mode of action of this analog in leukemic cells.

Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/3/ 611/s1, General procedures and characterization data for diethyl 4-hydroxy-2-oxoalkylphosphonates 7a–d, 3-diethoxyphosphoryldihydropyran-4-ones 9a–d and 2,2-dialkyl(diaryl)-6-alkyl(aryl)-5-methylenetetrahydro -4H-pyran-4-ones 10a–p. Copies of 1H, 13C and 31P NMR spectra of all obtained compounds. Author Contributions: Conceptualization, T.J., A.J and M.M.; Data curation, T.B., J.D.-S. and U.K.; Formal analysis, T.B., J.K. and J.D.-S.; Funding acquisition, T.J., A.J. and M.M.; Investigation, T.B., J.K., J.D.-S. and U.K.; Methodology, T.J., J.K., J.D.-S. and A.J.; Project administration, T.J. and A.J.; Resources, T.B., J.K. and J.D.-S.; Supervision, T.J. and A.J.; Validation, J.K. and J.D.-S.; Visualization, T.B. and J.D.-S.; Writing—original draft T.J., A.J. and T.B.; Writing—review & editing, T.J. and A.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Young Scientists’ Fund at the Faculty of Chemistry, Lodz University of Technology, Grant W-3D/FMN/24G/2019 and by a grant from the Medical University of Łód´z No. 503/1-156-02/503-11-001-19-00. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Sample Availability: Samples of the compounds 7a–d, 9a–d and 10a–n,p are available from the authors.

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