Preparation and Chiral HPLC Separation of the Enantiomeric Forms of Natural Prostaglandins

Article Preparation and Chiral HPLC Separation of the Enantiomeric Forms of Natural Prostaglandins

Márton Enesei 1,2,László Takács 2, Enik˝oTormási 3, Adrienn Tóthné Rácz 3, Zita Róka 3, Mihály Kádár 3 and Zsuzsanna Kardos 2,*

1 Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, M˝uegyetemrakpart 3, 1111 Budapest, Hungary; [email protected] 2 CHINOIN, a member of Sanofi Group, PG Development, Tó utca 1-5, 1045 Budapest, Hungary; laszlo.takacs@sanofi.com 3 CHINOIN, a member of Sanofi Group, PG Analytics, Tó utca 1-5, 1045 Budapest, Hungary; robertne.tormasi@sanofi.com (E.T.); adrienn.tothneracz@sanofi.com (A.T.R.); zita.roka@sanofi.com (Z.R.); mihaly.kadar@sanofi.com (M.K.) * Correspondence: zsuzsanna.kardos@sanofi.com

Received: 15 June 2020; Accepted: 10 August 2020; Published: 18 August 2020

Abstract: Four enantiomeric forms of natural prostaglandins, ent-PGF α (( )-1), ent-PGE ((+)-2) 2 − 2 ent-PGF α (( )-3), and ent-PGE ((+)-4) have been synthetized in gram scale by Corey synthesis used 1 − 1 in the prostaglandin plants of CHINOIN, Budapest. Chiral HPLC methods have been developed to separate the enantiomeric pairs. Enantiomers of natural prostaglandins can be used as analytical standards to verify the enantiopurity of synthetic prostaglandins, or as biomarkers to study oxidation processes in vivo.

Keywords: preparation; ent-PGF2α; ent-PGF1α; ent-PGE2; ent-PGE1; chiral HPLC; enantiomeric separation; mirror images; enantiopurity

1. Introduction Interest in the field of prostaglandins has been steadily growing since the basic work of Bergström Samuelsson, and Vane [1–3]. Their discoveries revealed the structure, the enzyme-controlled biochemical synthesis from arachidonic acid, and the main physiological effects of prostaglandins as well as their related substances. The biological behavior of prostaglandins, namely the regulation of the functions of all key organs in mammals including humans, has opened promising prospects for their therapeutic use. The request for systematic studies of natural prostaglandins and their synthetic derivatives has forced researchers all over the world to develop economical and scalable syntheses to produce these substances that were previously available only from natural sources [4–6]. The first generally applicable prostaglandin synthesis was a linear one, developed by Corey [7]. The key intermediate in the synthesis is lactone ( )-5, commonly referred to as Corey lactone, from − which the omega and the alpha side chains of prostaglandins can be constructed [8–10]. The linear approach was followed by convergent syntheses, a more versatile one, a two-component coupling was first applied by Sih [11,12]. Noyori developed the idea of the shortest, highly convergent synthesis, the three-component coupling reaction. Noyori’s process provides the prostaglandins or derivatives in a one-pot reaction. The starting material is a chiral cyclopentenone; the omega side chain is introduced by an organo copper-mediated conjugate addition of the optically pure omega side chain. The enolate formed is trapped by the alfa side chain containing alkyl halide [13–15]. Recently, Aggarwal has developed a short, stereocontrolled organocatalytic synthesis starting with the double aldol reaction of succinaldehyde in the presence of a chiral auxiliary. The key intermediate is a methoxy acetal

Chemistry 2020, 2, 727–741; doi:10.3390/chemistry2030047 www.mdpi.com/journal/chemistry Chemistry 2020, 2 728

Chemistry 2020, 2, x 2 from which the omega side chain, which is constructed by a conjugate coupling reaction, then the is a methoxy acetal from which the omega side chain, which is constructed by a conjugate coupling alpha side chain is formed by a Wittig reaction [16–18]. For reviews of prostaglandin syntheses see reaction, then the alpha side chain is formed by a Wittig reaction [16–18]. For reviews of refs. [19–21]. Scheme1 shows the structures of the key intermediates of the prostaglandin synthesis prostaglandin syntheses see refs. [19–21]. Scheme 1 shows the structures of the key intermediates of and the structures of PGF2α and an isoprostane, IPF2α. the prostaglandin synthesis and the structures of PGF2α and an isoprostane, IPF2α.

Protected Corey Explanation Protected cyclopentenone Methoxy acetal lactone (−)-5

Name of key intermediates R = protecting group

Structure of alpha side chain PGF2α omega side chain

9 1 In isoprostanes (IP-s) the side chains are Structure of IPF2α attached to the cyclopentane ring in cis 8-iso-PGF2α 11 position

Scheme 1. Key intermediates of prostaglandin syntheses and structures of PGF2α and IPF2α. Scheme 1. Key intermediates of prostaglandin syntheses and structures of PGF2α and IPF2α.

VersatileVersatile chemical chemical syntheses syntheses from from widely widely available available starting starting materials materials have removed have removed barriers barriers from the usefrom ofnatural the use prostaglandins of natural prostaglandins and synthetic and derivatives synthetic in derivativeshuman [19] in and human veterinary [19] therapiesand veterinary [22,23 ]. Thetherapies main uses [22,23] in human. The therapy main uses are treatments in humanof therapy ocular hypertension are treatments and of glaucoma ocular hypertension [24,25], pulmonary and arterialglaucoma hypertension [24,25], pulmonary (PAH) [26 arterial,27], lumbar hypertension spinal stenosis (PAH) [[26,27]28], gastric, lumbar and spinal duodenal stenosis ulcer [28] [29, gastric], labor inductionand duodenal [30], congenital ulcer [29], heartlabor diseaseinduction in [30] infants, congenital [31,32], andheart chronic disease idiopathic in infants [31,32] constipation, and chronic [33]. idiopathicThe selection constipation of successful [33]. drug candidates required the preparation of thousands of prostaglandin The selection of successful drug candidates required the preparation of thousands of derivatives, among them the enantiomers and epimers of natural prostaglandins as well. The idea that prostaglandin derivatives, among them the enantiomers and epimers of natural prostaglandins as enantiomers of natural prostaglandins retain their biological activity but metabolize in vivo more well. The idea that enantiomers of natural prostaglandins retain their biological activity but slowly has led to contradicting results; therefore, no drugs have been developed from the prostaglandin metabolize in vivo more slowly has led to contradicting results; therefore, no drugs have been enantiomers and the interest in these derivatives has been pushed to the periphery [34–42]. The situation developed from the prostaglandin enantiomers and the interest in these derivatives has been pushed changed when Morrow and Roberts reported that prostaglandin-like compounds are generated in vivo to the periphery [34–42]. The situation changed when Morrow and Roberts reported that from arachidonic acid by the peroxidation of free radicals independently of the cyclooxygenase prostaglandin-like compounds are generated in vivo from arachidonic acid by the peroxidation of pathway [43–47]. Unbalanced free radicals, reactive oxygen or nitrogen species (ROS or RNS), can free radicals independently of the cyclooxygenase pathway [43–47]. Unbalanced free radicals, cause oxidative stress in the body, contributing to the development of cardiovascular, neurological, reactive oxygen or nitrogen species (ROS or RNS), can cause oxidative stress in the body, contributing respiratory,to the development and kidney of cardiovascular, disease, and even neurological, cancer [48 ].respiratory, and kidney disease, and even cancer [48]Once. discovered, isoprostanes are used as important biomarkers to study the oxidative processes in humans.Once Prostaglandin discovered, isoprostanes enantiomers, are which, used unlike as important natural prostaglandins, biomarkers to stareudy also the formed oxidative from arachidonicprocesses inacid humans. by free radicalProstaglandin reactions, enantiomers, are in the spotlight which, unlike again naturalas promising prostaglandins, biomarkers. are also formed from arachidonic acid by free radical reactions, are in the spotlight again as promising 2. Aim of the Work biomarkers. Evaluation of the literature data revealed that syntheses providing enantiomers of natural prostaglandins2. Aim of the inWork a larger quantity are still missing. The aim of our work is to prepare prostaglandin enantiomersEvaluation in a of practical the literature way using data revealed our processes that syntheses that yield providing numerous enantiomers prostaglandin of natural active pharmaceuticalprostaglandins ingredients in a larger quantity in our prostaglandin are still missing. plants. The aim of our work is to prepare prostaglandin enantiomersThe enantiomeric in a practical forms of way natural using prostaglandins our processes can that be used yield for numerous scientiﬁc prostag purposeslandin as reference active standardspharmaceutical for studying ingredients oxidative in our processes prostaglandin in the body.plants. Natural prostaglandins are synthesized from

ChemistryChemistry2020 20, 220, 2, x 3 729

The enantiomeric forms of natural prostaglandins can be used for scientific purposes as arachidonicreference acid standards by COX-1 for / studyingCOX-2 enzymes. oxidative In processes contrast, in enantiomers the body. Natural of the natural prostaglandins prostaglandins are cansynth be formedesized byfrom free arachidonic radical oxidation. acid by COX-1/COX-2 enzymes. In contrast, enantiomers of the natural prostaglandinsAnother important can be applicationformed by free of enantiomericradical oxidation. forms is in the analytical tests of the prostaglandin active ingredients.Another important The enantiomeric application forms of can enantiomeric be used as analytical forms is standards in the analytical to verify tests the optical of the purity prostaglandin active ingredients. The enantiomeric forms can be used as analytical standards to of the active ingredients to ensure the quality that meets the requirements of the pharmaceutical authorities. verify the optical purity of the active ingredients to ensure the quality that meets the requirements of theWe pharmaceutical plan to prepare authorities. the enantiomeric forms of all prostaglandins that are in our portfolio. This workWe has plan been to prepare started the by enantiomeric synthetizing forms the enantiomeric of all prostaglandins forms of that the are natural in our prostaglandins portfolio. This that are manufacturedwork has been started in our by plants, synthetizing because the theyenantiomeric can be used forms for of the two natural diﬀerent prostaglandins purposes. that Hereinafter, are enantiomericmanufactured forms in ofour the plants, modiﬁed because prostaglandin they can be derivatives used for two will different be prepared. purposes. The Hereinafter, latter can only be usedenantiomer for analyticalic forms of purposes, the modified as theyprostaglandin are not derivatives synthesized will in be the prepared. human The body. latter In can the only present study,be used the synthesis for analytical of ent purposes,-PGF1α ,asent they-PGF are2α not, ent synthesized-PGE2 and inent the-PGE human1, starting body. In from the presentent-Corey study lactone,, andthe methods synthesis for of the ent separation-PGF1α, ent-PGF of the2α, ent enantiomeric-PGE2 and ent pairs-PGE by1, sta chiralrting analyticalfrom ent-Corey HPLC lactone are presented., and See structuresmethods for of the the separation Corey lactone of the enantiomers enantiomeric (( pairs)-5 andby chiral (+)-5 analytical)) and the HPLC prepared are presented.ent-prostaglandins See structures of the Corey lactone enantiomers ((−)-5− and (+)-5)) and the prepared ent-prostaglandins in in Scheme2. Scheme 2.

Corey lactone (R = protecting group) ent-Corey lactone (R = protecting group) (−)-5 (+)-5 starting material of natural prostaglandins starting material of the mirror images, ent-PGs

6 5 1

ent-PGF2α ent-PGF1α (−)-1 (−)-3

ent-PGE2 ent-PGE1 (+)-2 (+)-4

Scheme 2. Structure of Corey lactone enantiomers and ent-PGF1α, ent-PGF2α, ent-PGE2 and ent-PGE1. Scheme 2. Structure of Corey lactone enantiomers and ent-PGF1α, ent-PGF2α, ent-PGE2 and ent-PGE1.

3. Material3. Material and and Methods Methods TheThe commercially commercially availableavailable reagents reagents and and analytical analytical grade gradesolvents solvents were purchased were purchased from Merck from Merck(Darmstadt, (Darmstadt, Germany). Germany). Merck DC Merck Fertigplatten DC Fertigplatten 60 silicagel 60 TLC silicagel plates with TLC fluorescence plates with indicator ﬂuorescence 254 nm was used for TLC chromatography, and Merck silica gel 60 (0.063–230 mm) for column indicator 254 nm was used for TLC chromatography, and Merck silica gel 60 (0.063–230 mm) for chromatography. column chromatography. Waters HPLC system equipped with a PDA detector and Empower V3 electronic data proceWatersssing HPLC system system was used equipped for the withHPLC a method PDA detector development. and Empower V3 electronic data processing system was used for the HPLC method development. 1H and 13C NMR spectra were recorded on Bruker AvanceIII (Billerica, MA, USA) instrument at

500.15 MHz and 125.8 MHz, respectively, in the DMSO-d6 solvents. Chemical shifts (δ) are given in parts per million (ppm) relative to TMS, coupling constants (J) in hertz (Hz). The solvent signals were used as references and the chemical shifts converted to the TMS scale (deuterated dimethyl sulfoxide Chemistry 2020, 2, x 4 Chemistry 2020, 2 730 1H and 13C NMR spectra were recorded on Bruker AvanceIII (Billerica, MA, USA) instrument at 500.15 MHz and 125.8 MHz, respectively, in the DMSO-d6 solvents. Chemical shifts (δ) are given in

(DMSO-partsd6 ):perδ Cmillion 39.52 (ppm) ppm relative (sep) in to DMSO-TMS, couplingd6, δH constants 2.50 ppm (J) (m),in hertz residual (Hz). The peak solvent in DMSO- signals dwere6)[49 ]. In theused report, as references the full assignment and the chemical of the shifts1H and converted13C NMR to the spectra TMS scale have (deuterated been achieved dimethyl by utilizingsulfoxide the attached(DMSO proton-d6): testδC 39.52 (APT) ppm and (sep) the in two-dimensional DMSO-d6, δH 2.50 HSQC,ppm (m), ed-HSQC, residual peak HMBC, in DMSO COSY,-d6) [49] NOESY. In the and report, the full assignment of the 1H and 13C NMR spectra have been achieved by utilizing the ROESY measurements. attached proton test (APT) and the two-dimensional HSQC, ed-HSQC, HMBC, COSY, NOESY and 4. ResultsROESY and measurements. Discussions

4.1. Synthesis4. Results of and Mirror Discussions Images of Natural Prostaglandins

In4.1. our Synthesis prostaglandin of Mirror Images plant, of weNatural use Prostaglandins modified version of Corey synthesis [48] to produce a variety ofIn natural our prostaglandin and structurally plant, we modified use modified prostaglandins version of Corey as active synthesis pharmaceutical [48] to produce ingredientsa variety (PG API-s)of natural [50– 69and]. structurally This versatile modified synthesis prostaglandins allows the as production active pharmaceutical of different ingredients types of prostaglandins (PG API-s) from a[50 common–69]. This intermediate, versatile synthesis the Corey allows lactone the production (( )-5). of The different same strategytypes of prostaglandins was used to design from a the − synthesiscommon of enantiomeric intermediate, forms the Corey of natural lactone prostaglandins. ((−)-5). The same Logically, strategy was in thisused case, to design the starting the synthesis material was theof enantiomeric mirror image forms of theof natural Corey prostaglandins. lactone, called Logically,ent-Corey in this lactone case, the ((+ starting)-5), from material which was each the of the fourmirror enantiomeric image of the prostaglandins Corey lactone, could called be ent prepared.-Corey lactone The ent ((+)-Corey-5), from lactone which can each be of synthetized the four by knownenantiomeric methods prostaglandins from (+)-bicyclic could lactone,be prepared. the The “wrong” ent-Corey enantiomer lactone can of be the synthetized resolution by step known of the prostaglandinmethods from synthesis (+)-bicyclic [48,70 lactone,,71]. Preparationthe “wrong” enantiomer and resolution of the resolution of rac-bicyclic step of lactone the prostaglandin is shown in synthesis [48,70,71]. Preparation and resolution of rac-bicyclic lactone is shown in Scheme 3. The ent- Scheme3. The ent-Corey lactone and Corey lactone are also commercially available [72]. Corey lactone and Corey lactone are also commercially available [72].

Reductive [2+2] cycloaddition dehalogenization Monomerization Zn, HCl 200 °C Dichloro acetylchloride

Dicyclopentadiene Cyclopentadiene Dichloro ketone Bícyclic ketone

Bayer-Williger oxidation Resolution

m-Chloroperbenzoic acid 1. NaOH-MeOH 2. (R)-(+)-alpha methylbenzylamine rac-Bicyclic lactone 3. HCl-CH Cl 2 2 (-)-Bicyclic lactone (-)-Bicyclic lactone 3,3a,6,6a-tetrahydrocyclopenta[b]furan-2-one

Corey lactone ent-Corey lactone

SchemeScheme 3. Preparation 3. Preparation and and resolution resolutionof ofrac rac-bicyclic-bicyclic lactone lactone..

The initialThe initial reaction reaction steps steps in thein the synthesis synthesis of ofent ent-prostaglandins,-prostaglandins, were were the the transformation transformation of the of the ent-Coreyent-Corey lactone lactone ((+)- ((+)5)- to5) to the the unprotected unprotected lactol ( (1111).). First, First, the theprimary primary hydroxyl hydroxyl-group-group in the ent in- the ent-CoreyCorey lactone lactone ((((+)+)--5)) was was oxidized oxidized by by Anelli Anelli oxidation oxidation [73]. [Dichloromethane73]. Dichloromethane solution solutionof the crude of the crudealdehyde aldehyde (6 ()6 )was was reacted reacted with with the the sodium sodium salt salt dimethyl dimethyl (2-oxo)heptylphosphonate (2-oxo)heptylphosphonate (7) to form (7) to the form the omegaomega side side chain chain under under the the conditions conditions of of Horner–Wadsworth–Emmons Horner–Wadsworth–Emmons (HWE) (HWE) reaction reaction [74– [7476]–.76 ]. Protected enone (8) was purified by crystallization. Then, the 15-oxo-group was stereoselectively Protected enone (8) was puriﬁed by crystallization. Then, the 15-oxo-group was stereoselectively reduced with in situ prepared catecholborane in the presence of (S)-2-Methyl-CBS catalyst [77]. After reduced with in situ prepared catecholborane in the presence of (S)-2-Methyl-CBS catalyst [77]. work-up, the lactone group of protected enol (9) was reduced to the lactol (10). The undesired After work-up,diastereomer, the lactone the derivatized group of protected side-product enol of (9 ) 15 was-oxo reduced-reduction, to the (S) lactol-10 was (10 ). removed The undesired by diastereomer,chromatography the derivatized and crystallization. side-product ofThe 15-oxo-reduction, methanolysis of (SPPB)-10-protectingwas removed group by chromatography provided the and crystallization.unprotected lactol The (11 methanolysis), which was purified of PPB-protecting by crystallization. group Reaction provided steps the are unprotected shown in Scheme lactol 4. (11 ), which was puriﬁed by crystallization. Reaction steps are shown in Scheme4. Chemistry 2020, 2 731

a. Oxidation (6) d. Lactone reduction (10) b. HWE reaction (8) e. Deprotection (11) c. Oxo reduction (9) 15 R

(+)-5 9 11

PPB- = p-phenylbenzoyl group dimethyl (2-oxoheptyl)phosphonate (7)

Scheme 4. Preparation of lactone (11) from ent-Corey lactone (+)-5.(a) NaOCl, KBr, NaHCO3, TEMPO

catalyst, dichloromethane, water, 0–10 ◦C; (b) 7, NaOH, dichloromethane, water, 0–10 ◦C, crystallization

from diisopropyl ether-hexane; (c) BH3-DMS, pyrocatechol, (S)-2-Me-CBS, THF, ( 15)–( 10) C; (d) − − ◦ DIBAL-H, THF, toluene, ( 75)–( 70) C, chromatography with toluene-EtOAc, crystallization from − − ◦ toluene-hexane; (e) K2CO3, MeOH, 35–40 ◦C, crystallization from EtOAc-diisopropyl ether.

The unprotected lactol (11), containing a masked aldehyde functionality, is a common building block of all four enantiomeric prostaglandins. The alpha side chain for ent-PGF α (( )-1) was prepared 2 − by Wittig reaction with the phosphorane liberated from 4-carboxybutyl triphenylphosphonium bromide t (CBP-Br) (12) by the strong base, KOBu . We have not crystallized yet the oily crude ent-PGF2α (1), instead it was converted to its stable, crystalline tromethamine salt. For the preparation of the remaining three derivatives ent-PGE ((+)-2), ent-PGF α (( )-3), 2 1 − ent-PGE1 ((+)-4), the lactol group of 11 was reoxidized to lactone (13) with iodine in an aqueous medium containing KI and KHCO3. This reoxidation is required for the selective formation of the 9-oxo group in the PGE derivatives. Free hydroxyl groups were protected with dihydropyran by an acid catalyzed reaction, supplying the bis-THP lactone (14). To form the alpha side chain of the prostaglandin structure, the lactone group was reduced to lactol to make the molecule suitable for the Wittig reaction. The alpha side chain was built again with the phosphorane prepared from CBF-Br t (12) with KOBu as the base in THF solution, giving the ent-THP2-PGF2α (16), that is the last common intermediate for the preparation of ent-PGE ((+)-2), ent-PGF α (( )-3) and ent-PGE ((+)-4). 2 1 − 1 Preparation of ent-PGE2 ((+)-2), happened by the oxidation of the free 9-hydroxyl group of acid (16) with pyridinium chlorochromate (PCC) in ethyl acetate. The pH of the reaction mixture was buffered by sodium acetate and acetic acid. The crude ent-THP2-PGE2 (17) was filtered through silica gel to remove the residue of the oxidant. In the last step of this series, the THP-protecting groups were removed in isopropanol. The reaction was catalyzed by 1 mol/L hydrochloric acid. Crude ent-PGE2 ((+)-2) was purified by chromatography using hexane-ethyl acetate eluent, and crystallization from ether-diisopropyl ether. To prepare ent-PGF α (( )-3) and ent-PGE ((+)-4) the cis double bound in the alpha side chain of 16 1 − 1 must be selectively reduced. This selective reduction is a key step because it allows derivatives containing either one or two double bonds to be prepared from a common intermediate. This transformation is performed by catalytic hydrogenation in diisopropyl ethylamine-dichloromethane solution using 10% Pd/C catalyst. The quantity of crude ent-THP2-PGF1α (18) was divided into two parts. One part was dissolved in isopropanol containing 1 mol/L hydrochloric acid to remove the THP-protecting groups, giving ent-PGF α. Crude ent-PGF α (( )-3) was purified by double crystallization from ethyl acetate 1 1 − followed by ethyl acetate-hexane. 1

Chemistry 2020, 2 732

The 9-hydroxyl group of the remaining quantity of 18 was oxidized with PCC to the 9-oxo derivative,Chemistryent-THP 2020, 2, 2x-PGE 1 (19). Crude 19 was puriﬁed by column chromatography. The evaporated6 main fraction was dissolved in isopropanol containing 1 mol/L hydrochloric acid to remove the main fraction was dissolved in isopropanol containing 1 mol/L hydrochloric acid to remove the protecting groups. Crude ent-PGE1 ((+)-4) was crystallized from diisopropyl ether-hexane. Reaction protecting groups. Crude ent-PGE1 ((+)-4) was crystallized from diisopropyl ether-hexane. Reaction steps for the preparation of ent-prostaglandins are shown in Scheme5. steps for the preparation of ent-prostaglandins are shown in Scheme 5.

t t SchemeScheme 5. Preparation 5. Preparation of ent of-prostaglandins ent-prostaglandins from from lactol lactol11 .(11a.) ( CBP-Br,a) CBP-Br, KOBu KOBu, THF,, THF,( (-10)-(10)-(- 5) ◦C;(b) 5) °C; (b) TAM, MeOH, acetone, hexane, 25 °C 0 °C; (c) I2, KI, KHCO3, water; (d) Dihydropyran,− − TAM, MeOH, acetone, hexane, 25 ◦C 0 ◦C; (c)I2, KI, KHCO3, water; (d) Dihydropyran, pTsOH.H2O, pTsOH.H2O, toluene, THF, 20–50 →°C; (e) DIBAL-H, toluene, THF (−75)–(−70) °C; (f) CBP-Br, KOBut, toluene, THF, 20–50 C; (e) DIBAL-H, toluene, THF ( 75)–( 70) C; (f) CBP-Br, KOBut, THF, toluene, THF, toluene, ◦ (−10)–(−5) °C; (g) PCC, NaOAc, AcOH,− EtOAc,− 3◦0–40 °C, chromatography with ( 10)–( 5) C; (g) PCC, NaOAc, AcOH, EtOAc, 30–40 C, chromatography with diisopropyl ether- − −diisopropyl◦ ether-acetone mixture as eluent; (h) 1 mol/L HCl,◦ H2O, i-PrOH, 20 °C, chromatography acetonewith mixture hexane as-EtOAc eluent; mixtures (h) 1 mol as eluent,/L HCl, crystallization H2O, i-PrOH, from 20ether◦C,-diisopropyl chromatography ether; (i) cat with H2, hexane-EtOAcPd/C, mixtures as eluent, crystallization from ether-diisopropyl ether; (i) cat H2, Pd/C, diisopropyl ethylamine, dichloromethane, rt; (j) 1 mol/L HCl, H2O, i-PrOH, 20 ◦C, crystallization from EtOAc then EtOAc-hexane; (k) PCC, NaOAc, EtOAc, AcOH, 30–40 ◦C, chromatography with diisopropyl ether-EtOAc mixture as eluent; (l) 1 mol/L HCl, H2O, i-PrOH, 20 ◦C, crystallization from diisopropyl ether-hexane. Chemistry 2020, 2 733

4.2. Separation of Natural Prostglandin Enantiomers by Chiral HPLC Having the optically pure mirror images of four natural prostaglandins in our hands (chemical purities are given in the Supplementary Material), the next goal was to develop chiral HPLC methods for the separation of the enantiomeric pairs. To date, only one scientific article has reported the chiral reversed phase HPLC separation of PGE2 enantiomers. According to the article, various chiral HPLC columns were tested, but the enantiomeric separation was only successful when the Phenomenex Lux Amylose2 column was used. Elution of the Lux Amylose2 column with methanol:water or 2-propanol:water did not resolve the enantiomers. When a low concentration of acetonitrile (25% in water with 0.1% formic acid) and a slow flow rate (50 µL/min) were used, the enantiomers separated, but the peaks were broadened. The best results were obtained when two columns, connected in series were used for the enantiomeric separation [78]. As the known procedure presented a rather sophisticated method and that method was only applied for the separation of PGE2 enantiomers, we have decided to work out a simplified procedure which is suitable for routine tests of the prostaglandins manufactured in our plant. Reverse phase methods are promising for the chromatographic analysis of acidic compounds, including prostaglandin acids, so we have been looking for a reverse phase chiral column. Based on literature data and our previous experience, we have chosen Chiracel OJ-RH as the chiral HPLC column. This type of column has been successfully applied for the enantiomeric separation of chemically distinct racemic organic acids [79]. In selecting the values of the test parameters, we partly took into account the column supplier’s recommendation [80], and partly our chromatographic experience. The reversed phase HPLC conditions are summarized in Table1.

Table 1. Reversed phase HPLC conditions.

Parameter Value Apparatus: Waters HPLC system equipped with a PDA detector and Empower V3 electronic data processing system Column: Chiracel OJ-RH, 150 4.6 mm, 5 µm × Column temperature: 25 or 40 ◦C (5–40 ◦C) * Flow rate: 0.5 mL/min (0.5–1.0 mL) * Injected volume: 5 µL Concentration of sample: 0.25 mg/mL Composition of eluent: see Table2 Composition of sample solvent: acetonitrile:methanol:water 30:10:60 Wavelength: 200, 210 nm Run time: 20–40 min * The optimal parameters recommended by the column supplier are shown in the brackets.

Based our preliminary experiments, sample solutions were prepared by dissolving equal amounts of the pure enantiomers in the sample solvent (acetonitrile:methanol:water = 30:10:60) and aliquots from the solution of the corresponding enantiomeric pairs were mixed prior to the injection. The eluent was a three-component mixture containing acetonitrile:methanol:water (pH = 4). It is noted here that pH of the water was adjusted to 4 with phosphoric acid (85 w/w% in H2O) in every case. This pH = 4 value provides an adequate separation and elution rate for the prostaglandin acids, without causing decomposition of their acid-sensitive structure. Phosphoric acid was also recommended for adjusting the pH of Chiracel OJ-RH columns at this pH value to avoid degradation of the column. Composition of the eluents were varied in a wide range to achieve a good resolution. The composition of the eluents is summarized in Table2. Chemistry 2020, 2, x 8

case. This pH = 4 value provides an adequate separation and elution rate for the prostaglandin acids, without causing decomposition of their acid-sensitive structure. Phosphoric acid was also recommended for adjusting the pH of Chiracel OJ-RH columns at this pH value to avoid degradation Chemistry 2020, 2 734 of the column. Composition of the eluents were varied in a wide range to achieve a good resolution. The composition of the eluents is summarized in Table 2. Table 2. Eluent composition. Table 2. Eluent composition. Composition Run Composition Run AcetonitrileAcetonitrile Methanol Methanol Water (pH = 4) Water (pH = 4) 11 3030 10 1060 60 22 2525 10 1065 65 33 2424 10 1066 66 44 2323 10 1067 67 55 2020 10 1070 70 6 20 15 65 6 20 15 65 7 15 20 65 7 15 20 65 8 15 15 70 8 15 15 70

TheThe results results for for individual individual runs runs were were evaluated evaluated by comparing comparing the the resolution resolution values values (R) calculated (R) calculated accordingaccording to United to United States States Pharmacopeia Pharmacopeia (USP (USP-NF,-NF, < <621621 > Chromatography).> Chromatography). To achieve To achieve at least R at = least R = 1.21.2 resolutionresolution the composition composition of of the the eluent eluent and and column column temperature temperature were were varied. varied. When When optimizing optimizing the method,the method, not only not only the resolution, the resolution, but also but thealso time the time and eluent and eluent consumption consumption were were taken taken into into account. account. The effect of the variable parameters on the resolution of enantiomers of PGE2 is shown in The eﬀect of the variable parameters on the resolution of enantiomers of PGE2 is shown in Figure1. Figure 1.

Departm ent: PG_DEV Additional_Comments: 20-114/0300 SampleName: PGE2+ent-PGE2 Batch_No: - Operation_ID: - HPLC Result Sam ple Type: Unknow n System Name: UJP_Q276_LC114 Sample No: - Sample Weight (mg): 1.000 Processed Channel Descr.: 2998 (191-300)nm Parameters Resolution Notebook: PG-1789/1 Dilution (ml): 1.00 Processing Method: PGE2_királis Instrument Method Id: 1940 WeighingBook: - Acq Method Set: PGE2_királis_30_10_60 Processing Method Id: 2049 Injection Volume (µl): 5.00 Project Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Eluent Column R Vial: 18 Date Acquired: Result05 Mar 2020 14:43:05 CET Channel Id: 1977 Date Processed: 06 Mar 2020 12:38:25 CET Result Id.: 2147 Informations: A: ACN B:MeOH C:H2O (pH=3.6)=30:10:60 Calibration Id: 2127 MeCN MeOH H2O T (°C ) Column_type_and_No: Chiralcel OJ-RH 150*4.6mm,5um (211) 0.10

0.08

PGE2 -PGE2 6.703 ent-PGE2 - 6.787 0.06 Departm ent: PG_DEV Additional_Comments: 20-114/0301 SampleName: PGE2+ent-PGE2 Batch_No: - Operation_ID: -

AU Sam ple Type: Unknow n HPLC0.04 Result System Name: UJP_Q276_LC114 30 10 60 25 - Sample No: - Sample Weight (mg): 1.000 Processed Channel Descr.: 2998 (191-300)nm Notebook: PG-1789/1 Dilution (ml): 1.00 Processing Method: PGE2_királis Instrument Method Id: 1979 WeighingBook: - 0.02 Acq Method Set: PGE2_királis_25_10_65 Processing Method Id: 2049 Injection Volume (µl): 5.00 Project Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Vial: 18 Date Acquired: 05 Mar 2020 15:20:45 CET Channel Id: 1987 Date Processed: 06 Mar 2020 12:38:38 CET 0.00 Result Id.: 2148 Informations: A: ACN B:MeOH C:H2O (pH=3.6)=25:10:65 Calibration Id: 2127 Column_type_and_No: Chiralcel OJ-RH 150*4.6mm,5um (211) 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Minutes 0.10 Solvent_Content Water_Content Unit_Correction_DIV Unit_Correction_MULT Keyed_Unit 1 1.00 100.00 - 0.08 Peak Results Name RT Area % Area Content 1 PGE2 6.703 1013811 35.60 0.06 -PGE2 10.757 2 ent-PGE2 6.787 1833772 64.40 Departm ent: PG_DEV Additional_Comments: 20-114/0302

SampleName: PGE2+ent-PGE2 Batch_No: - Operation_ID: - AU 25 10 65 25 - 0.04HPLC Result Sam ple Type: Unknow n System Name: UJP_Q276_LC114 Sample No: - Sample Weight (mg): 1.000 Processed Channel Descr.: 2998 (191-300)nm Notebook: PG-1789/1 Dilution (ml): 1.00 Processing Method: PGE2_királis 0.02Instrument Method Id: 2011 WeighingBook: -

Acq Method Set: PGE2_királis_20_10_70ent-PGE2 - 11.175 Processing Method Id: 2049 Injection Volume (µl): 5.00 Project Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Vial: 18 Date Acquired: 05 Mar 2020 15:57:20 CET 0.00Channel Id: 2018 Date Processed: 06 Mar 2020 12:38:52 CET Result Id.: 2149 Informations: A: ACN B:MeOH C:H2O (pH=3.6)=20:10:70 Calibration Id: 2127 Column_type_and_No: Chiralcel OJ-RH 150*4.6mm,5um (211) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Minutes Solvent_Content0.10 Water_Content Unit_Correction_DIV Unit_Correction_MULT Keyed_Unit 1 1.00 100.00 - 0.08 Peak Results Name RT Area % Area Content 1 PGE2 10.757 1243447 44.14 0.06

2 ent-PGE2 11.175 1573691 55.86 AU

20 10 70 25 - 0.04

PGE2 -PGE2 26.076 ent-PGE2 - 27.386

0.02

0.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes Solvent_Content Water_Content Unit_Correction_DIV Unit_Correction_MULT Keyed_Unit 1 1.00 100.00 -

Peak Results Figure 1. Cont. Name RT Area % Area Content 1 PGE2 26.076 1317173 47.53

2 ent-PGE2 27.386 1453928 52.47

Chemistry 2020, 2 735 Chemistry 2020, 2, x 9

Chemistry 2020, 2, x 9

Departm ent: PG_DEV Additional_Comments: 20-114/0308 SampleName: PGE2+ent-PGE2 Batch_No: - Operation_ID: - HPLC Result Sam ple Type: Unknow n System Name: UJP_Q276_LC114 20 10 70 40 1.1 Departm ent: PG_DEV Additional_Comments: 20-114/0308 Sample No: - Sample Weight (mg): 1.000 Processed Channel Descr.: 2998 (191-300)nm SampleName: PGE2+ent-PGE2 Batch_No: - Operation_ID: - Notebook: PG-1789/1 Dilution (ml): 1.00 Processing Method: PGE2_királis HPLCInstrument Result Method Id: 2100 Sam ple Type: Unknow n System Name: UJP_Q276_LC114 20 10 70 40 1.1 WeighingBook: - Acq Method Set: PGE2_királis_20_15_65_40C Sample No: - Sample Weight (mg): 1.000 Processing Method Id: 2049 Injection Volume (µl): 5.00 Processed Channel Descr.: 2998 (191-300)nm Notebook: PG-1789/1 Dilution (ml): 1.00 Project Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Vial: 18 Date Acquired: 06 Mar 2020 10:02:15 ProcessingCET Method: PGE2_királis Instrument Method Id: 2100 WeighingBook: - Channel Id: 2106 Date Processed: 06 Mar 2020 12:41:40Acq CET Method Set: PGE2_királis_20_15_65_40C Processing Method Id: 2049 Result Id.: 2154 Injection Volume (µl): 5.00 Project Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Vial: 18 Informations: A: ACN B:MeOH C:H2O (pH=3.6)=20:15:65,40°C Calibration Id: 2140 Date Acquired: 06 Mar 2020 10:02:15 CET Column_type_and_No: Chiralcel OJ-RH 150*4.6mm,5um (211) Channel Id: 2106 Date Processed: 06 Mar 2020 12:41:40 CET Result Id.: 2154 Informations: A: ACN B:MeOH C:H2O (pH=3.6)=20:15:65,40°C Calibration Id: 2140 0.10 Column_type_and_No: Chiralcel OJ-RH 150*4.6mm,5um (211) 0.10 0.08

0.08 -PGE2 13.371 Additional_Comments: 20-114/0304

Departm0.06 ent: PG_DEV ent-PGE2 - 14.290 PGE2 -PGE2 13.371 SampleName: PGE2+ent-PGE2 Additional_Comments:Batch_No: - 20-114/0304 Operation_ID: - Departm0.06 ent: PG_DEV ent-PGE2 - 14.290

SampleName:HPLC Result PGE2+ent-PGE2 Batch_No:Sam ple - Type: Unknow nOperation_ID:System - Name: UJP_Q276_LC114 AU 0.04

HPLCSample Result No: - SamSample ple Type: Weight Unknow(mg): 1.000 n SystemProcessed Name: Channel UJP_Q276_LC114 Descr.: 2998 (191-300)nm AU 20 15 65 40 1.2 0.04Notebook: PG-1789/1 Dilution (ml): 1.00 20 15 65 40 1.2 Sample No: - Sample Weight (mg): 1.000 ProcessedProcessing Channel Descr.: Method: 2998 PGE2_királis (191-300)nm Notebook:Instrument PG-1789/1 Method Id: 2020 Dilution WeighingBook: (ml): 1.00 - 0.02 ProcessingAcq Method: Method PGE2_királis Set: PGE2_királis_15_15_70_40C InstrumentProcessing Method Method Id: 2020 Id: 2049 WeighingBook:Injection Volume - (µl): 5.00 0.02 Acq MethodProject Set: PGE2_királis_15_15_70_40C Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Processing Method Id: 2049 Vial: 18 InjectionDate Volume Acquired: (µl): 5.00 05 Mar 2020 18:15:14 CETProject Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Vial:Channel 18 Id: 2031 Date Acquired: 05 Mar 2020 18:15:14 CET 0.00 Date Processed: 06 Mar 2020 12:39:30 CET Channel Id: 2031 0.00Result Id.: 2151 Date Processed:Informations: 06 Mar A: ACN 2020 B:MeOH12:39:30 CET C:H2O (pH=3.6)=15:15:70,40°C ResultCalibration Id.: 2151 Id: 2127 Informations: A: ACN B:MeOH C:H2O (pH=3.6)=15:15:70,40°C Calibration0.00 Id: 21272.00 4.00 6.00 8.00 10.00 Column_type_and_No:12.00 14.00 16.00 Chiralcel18.00 OJ-RH20.00 150*4.6mm,5um22.00 24.00 (211) 26.00 28.00 30.00 0.00 2.00 4.00 6.00 8.00 10.00 Column_type_and_No:12.00 14.00 16.00 ChiralcelMinutes18.00 OJ-RH20.00 150*4.6mm,5um22.00 24.00 (211) 26.00 28.00 30.00 Minutes Solvent_Content Water_Content Unit_Correction_DIV Unit_Correction_MULT Keyed_Unit Solvent_Content0.10 Water_Content Unit_Correction_DIV Unit_Correction_MULT Keyed_Unit 1 0.10 1.00 100.00 - 1 1.00 100.00 - PeakPeak Results Results 0.080.08NameName RT RT AreaArea% Area% AreaContentContent 1 1PGE2PGE2 13.37113.3716451142645114249.7949.79

2 2ent-PGE2ent-PGE214.29014.2906505903650590350.2150.21 0.060.06

DepartmDepartm ent: ent: PG_DEV PG_DEV Additional_Comments:Additional_Comments: 20-114/0305 20-114/0305 AU SampleName:SampleName:AU PGE2+ent-PGE2 PGE2+ent-PGE2 Batch_No:Batch_No: - - Operation_ID:Operation_ID: - - 0.040.04 15 15 70 40 1.5 HPLC Result Sam ple Type: Unknow n 15 15 70 40 1.5 HPLC Result Sam ple Type: Unknow nSystemSystem Name: Name:UJP_Q276_LC114 UJP_Q276_LC114 Sample No: - Sample Weight (mg): 1.000 Processed Channel Descr.: 2998 (191-300)nm Sample No: - Sample Weight (mg): 1.000 Processed Channel Descr.: 2998 (191-300)nm Notebook: PG-1789/1 Dilution (ml): 1.00

Processing Method: PGE2_királis -PGE2 40.672

Notebook:0.02 PG-1789/1 Dilution (ml): 1.00 ent-PGE2 - 44.448 Instrument Method Id: 2040 Processing Method: PGE2_királis -PGE2 40.672 0.02 WeighingBook: - ent-PGE2 - 44.448 Instrument Method Id: 2040 Acq Method Set: PGE2_királis_15_20_65_40C Processing Method Id: 2049 WeighingBook: - Acq Method Set: PGE2_királis_15_20_65_40C Injection Volume (µl): 5.00 Project Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Processing Method Id: 2049 Vial: 18 Date InjectionAcquired: Volume 06 Mar 2020(µl): 06:31:365.00 CET Project Name: PGAF\LC114\LC114_DEV\LC114_PGF2a_dev_01 Vial: 18 Channel0.00 Id: 2046 Date Processed:Date Acquired: 06 Mar 06 2020 Mar 12:40:30 2020 06:31:36 CET CET Channel Id: 2046 Result Id.: 0.002152 Informations:Date Processed: A: ACN B:MeOH 06 Mar C:H2O 2020 12:40:30 (pH=3.6)=15:20:65,40°C CET CalibrationResult Id.: Id: 2152 2127 Column_type_and_No:Informations: A: ChiralcelACN B:MeOH OJ-RH C:H2O 150*4.6mm,5um (pH=3.6)=15:20:65,40°C (211) Calibration0.00 Id: 2127 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Column_type_and_No: Chiralcel OJ-RH 150*4.6mm,5um (211) 0.00 5.00 10.00 15.00 20.00 25.00Minutes 30.00 35.00 40.00 45.00 50.00 55.00 60.00 0.10 Minutes Solvent_Content Water_Content Unit_Correction_DIV Unit_Correction_MULT Keyed_Unit 0.10 1 Solvent_Content Water_Content Unit_Correction_DIV1.00 Unit_Correction_MULT100.00 - Keyed_Unit 1 1.00 100.00 - 0.08 Peak Results 0.08 Name RTPeakArea Results% Area Content 0.061 PGE2Name 40.672RT 1435006Area 51.32% Area Content 2 ent-PGE2 44.448 1361019 48.68 0.061 PGE2 40.672 1435006 51.32

AU 2

15 20 65 40 1.6 0.04 ent-PGE2 44.448 1361019 48.68 AU

0.04 -PGE2 25.680 15 20 65 40 1.6 ent-PGE2 - 28.276

0.02

PGE2 -PGE2 25.680 ent-PGE2 - 28.276 0.02 0.00

0.000.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 Minutes Solvent_Content Water_Content Unit_Correction_DIV Unit_Correction_MULT Keyed_Unit 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 1 1.00 100.00 - Minutes Figure 1. Effect of the variable parameters on theSolvent_Content resolutionPeakWater_Content Results Unit_Correction_DIV of PGEUnit_Correction_MULT2 enantiomers.Keyed_Unit Figure 1. Eﬀect of the variable parameters on the1 Name resolutionRT Area % Area Content of1.00 PGE2 100.00enantiomers.- 1 PGE2 25.680 1431094 51.44 Figure 1. Effect of the variable parameters on the resolutionPeak Results of PGE2 enantiomers. 2 ent-PGE2 28.276 1350951 48.56 Name RT Area % Area Content By varying the indicated parameters, we performed the1 PGE2 chiral25.680 1431094 separation51.44 of all four enantiomeric By varying the indicated parameters, we performed the2 ent-PGE2 chiral28.276 1350951 48.56 separation of all four enantiomeric pairsBy varying with excellent the indicated resolution, parameters, R ≥ 1.5. The we best performed resolutions the are chiral shown separation in Figures of 2all–5 four. enantiomeric pairs with excellent resolution, R 1.5. The best resolutions are shown in Figures2–5. pairs with excellent resolution, R≥ ≥ 1.5. The best resolutions are shown in Figures 2–5. 0.40 0.35 0.40 0.30 0.35

0.25 PGF2aTAM - 5.910 0.30

ent-PGF2aTAM ent-PGF2aTAM - 5.267 0.20

AU 0.25 PGF2aTAM - 5.910

0.15 ent-PGF2aTAM - 5.267 0.20

AU 0.10 0.15 0.05

0.10 4.901

3.624

4.118

16.267

17.075

8.103

9.395

15.183

11.039

13.228 0.00

0.05 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 Minutes

4.901

3.624

4.118

16.267

17.075

8.103

9.395

15.183

11.039 13.228 0.00 Additional_Comments: 20-114/0310 SampleName: PGF2aTAM + ent-PGF2aTAM Acq Method Set: PGF2aTAM_királis_30_10_60 Result Id: 2281 0.00 2.00Figure4.00 2. Chiral6.00 8.00separation10.00 12.00of the14.00 optical16.00 isomers18.00 20.00of PGF22.002α (tested24.00 as26.00 TAM28.00 salt),30.00 R =32.00 1.5. Optimized34.00 36.00 38.00 40.00 Minutes parameters: Eluent: MeCN:MeOH:water (pH = 4) = 30:10:60, column temperature 25 °C, wavelength Additional_Comments: 20-114/0310 SampleName: PGF2aTAM + ent-PGF2aTAM Acq Method Set: PGF2aTAM_királis_30_10_60 Result Id: 2281 200 nm. FigureFigure 2. 2Chiral. Chiral separation separation of of the the optical optical isomers isomers of of PGF PGF2α2α (tested as TAM salt), R = 1.5. Optimized parameters:parameters: Eluent: Eluent: MeCN:MeOH:waterMeCN:MeOH:water (pH(pH == 4)4) = 30:10:60, column temperature temperature 25 25 °C,◦C, wavelength wavelength 200200 nm. nm.

Chemistry 2020, 2 736

ChemistryChemistry 20 202020, ,2, 2, ,x, x x 1010

0.1100.110 0.110 0.1000.100 0.100

0.0900.090 0.090

0.0800.080 0.080

0.070 - 13.467 ent-PGF1a 0.070 - 13.467 ent-PGF1a 0.070 - 13.467 ent-PGF1a

0.0600.060 0.060

PGF1a PGF1a 15.777 -

AU 0.0500.050 PGF1a 15.777 - 0.050 PGF1a 15.777 -

0.0400.040 0.040

0.0300.030 0.030

0.0200.020 0.020

0.0100.010 0.010

10.970

3.677

7.899

8.113

10.970

3.677

11.979

9.835

7.899

8.113

11.979

9.835

10.970

3.677

7.899

8.113

11.979 0.0000.000 9.835 0.000 0.000.00 2.002.00 4.004.00 6.006.00 8.008.00 10.0010.00 12.0012.00 14.0014.00 16.0016.00 18.0018.00 20.0020.00 22.0022.00 24.0024.00 26.0026.00 28.0028.00 30.0030.00 32.0032.00 34.0034.00 36.0036.00 38.0038.00 40.0040.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 MinutesMinutes Minutes Additional_Comments:Additional_Comments: 20-114/0309 20-114/0309 SampleName: SampleName: PGF1a PGF1a + +ent-PGF1a ent-PGF1a Acq Acq Method Method Set: Set: PGF1a_királis_23_10_67 PGF1a_királis_23_10_67 Result Result Id: Id: 2280 2280 Additional_Comments: 20-114/0309 SampleName: PGF1a + ent-PGF1a Acq Method Set: PGF1a_királis_23_10_67 Result Id: 2280 Figure 3. Chiral separation of the optical isomers of PGF1α, R = 1.7. Optimized parameters: Eluent: FigureFigure 3. 3.Chiral. Chiral separation separation of of the the optical optical isomers isomers of of PGF PGF1α1α1α,, RR == 1.7.1.7. Optimized parameters: parameters: Eluent: Eluent: MeCN:MeOH:water(pHMeCN:MeOH:water(pHMeCN:MeOH:water(pH = = 4) 4) 4) = == 23:10:67, 23:10:67, column column temperature temperature temperature 25 25 25 °C, °C,◦C, wavelength wavelength wavelength 200 200 200 nm. nm. nm.

0.0220.022 0.022 0.0200.020 0.020 0.0180.018 0.018 0.016

0.016 PGE2 - 28.191 0.016 PGE2 - 28.191

PGE2 - 28.191 0.0140.014 0.014 0.0120.012 0.012 ent-PGE2 - 31.048

ent-PGE2 ent-PGE2 - 31.048

0.010AU 0.010 0.010 0.0080.008 0.008 0.0060.006 0.006 0.0040.004 0.004 0.0020.002

37.304

3.989

0.002 37.304

3.989

37.304

3.989 0.0000.000 0.000 0.000.00 2.002.00 4.004.00 6.006.00 8.008.00 10.0010.00 12.0012.00 14.0014.00 16.0016.00 18.0018.00 20.0020.00 22.0022.00 24.0024.00 26.0026.00 28.0028.00 30.0030.00 32.0032.00 34.0034.00 36.0036.00 38.0038.00 40.0040.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 MinutesMinutes Minutes

Additional_Comments:Additional_Comments: 20-114/0312 20-114/0312 SampleName: SampleName: PGE2 PGE2 + +ent-PGE2 ent-PGE2 Acq Acq Method Method Set: Set: PGE2_királis_15_20_65_40C PGE2_királis_15_20_65_40C Result Result Id: Id: 2283 2283 Additional_Comments: 20-114/0312 SampleName: PGE2 + ent-PGE2 Acq Method Set: PGE2_királis_15_20_65_40C Result Id: 2283 FigureFigure 4.4. Chiral separation of the optical isomers of PGE 2,, RR == 1.5.1.5. Optimized Optimized parameters: parameters: Eluent: Eluent: Figure 4.. Chiral separation of the optical isomers of PGE222,, R = 1.5. Optimized parameters: Eluent: MeCN:MeOH:water(pHMeCN:MeOH:MeCN:MeOH:waterwater(pH(pH(pH = = 4) 4) 4) = == 15:20:65, 15:20:65, column column temperature temperature temperature 40 40 40 °C, °C,◦C, wavelength wavelength wavelength 210 210 210 nm. nm. nm.

0.200.20 0.20

0.180.18 0.18

0.160.16 0.16

PGE1 - 7.999

0.140.14 PGE1 - 7.999 0.14

0.120.12 0.12 0.10 AU 0.10

ent-PGE1 ent-PGE1 - 9.138

0.10 ent-PGE1 - 9.138

ent-PGE1 ent-PGE1 - 9.138 0.080.08 0.08

0.060.06 0.06

0.040.04 0.04

0.020.02 0.02

15.848

13.533

7.323

6.441

5.233

6.636

3.643

12.123

5.475

5.895

3.399

4.312

17.953

15.848

13.533

7.323

6.441

5.233

6.636

3.643

12.123

5.475

5.895

3.399

4.312

17.953

15.848

13.533

7.323

6.441

5.233

6.636

3.643

12.123

5.475

5.895

3.399

4.312 0.000.00 17.953 0.00 0.000.00 2.002.00 4.004.00 6.006.00 8.008.00 10.0010.00 12.0012.00 14.0014.00 16.0016.00 18.0018.00 20.0020.00 22.0022.00 24.0024.00 26.0026.00 28.0028.00 30.0030.00 32.0032.00 34.0034.00 36.0036.00 38.0038.00 40.0040.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 MinutesMinutes Minutes Additional_Comments:Additional_Comments: 20-114/0311 20-114/0311 SampleName: SampleName: PGE1 PGE1 + +ent-PGE1 ent-PGE1 Acq Acq Method Method Set: Set: PGE1_királis_30_10_60 PGE1_királis_30_10_60 Result Result Id: Id: 2282 2282 Additional_Comments: 20-114/0311 SampleName: PGE1 + ent-PGE1 Acq Method Set: PGE1_királis_30_10_60 Result Id: 2282 Figure 5. Chiral separation of the optical isomers of PGE1, R = 1.8. Optimized parameters: Eluent: FigureFigure 5. 5.Chiral Chiral separation separation ofof thethe optical optical isomersisomers ofof PGEPGE111,,, RR == 1.8.1.8. Optimized parameters: Eluent: Eluent: MeCN:MeOH:water(pHMeCN:MeOH:water(pHMeCN:MeOH:water(pH = = 4) 4) 4) = == 30:10:60, 30:10:60, column column temperature temperature temperature 25 25 25 °C, °C,◦C, wavelength wavelength wavelength 200 200 200 nm. nm. nm. Enantiopurity of the individual pure ent-prostaglandins has been also determined by the developed EnantiopurityEnantiopurity of of the the individual individual pure pure entent-prostaglandins--prostaglandins has has been been also also determined determined by by thethethe chiral methods. HPLC area percentages and enantiomeric excesses are summarized in Table3. developeddeveloped chiralchiral methods.methods. HPLCHPLC areaarea percentagespercentages andand enantiomericenantiomeric excessesexcesses are are summarizedsummarized inin TableTable 3. 3. Table 3. Table 3. Enantiopurity of the ent-prostaglandins.

ent-PGTableTable 3. 3. Enantiopurity Enantiopurity HPLC of of Area the the ent %ent-prostaglandins.--prostaglandins. Enantiomeric Excess ent-PGF .TAMent (ent)--PG-1PG.TAM HPLCHPLC 98.2 Area Area % % EnantiomericEnantiomeric 0.964 Excess Excess 2a ent− -PG HPLC Area % Enantiomeric Excess entent-PGF-PGE2a2.(TAM+)-2 (−)-1.TAM 99.998.2 0.964 0.998 ent--PGF2a2a..TAM (−)(−)--1.TAM.TAM 98.2 0.964 ent-PGF ( )-3 99.3 0.986 ent1α-PGE2 (+)-2 99.9 0.998 ent--PGE− 22 (+)(+)--2 99.9 0.998 ent-PGE1 (+)-4 99.9 0.998 ent-PGF1α (−)-3 99.3 0.986 ent--PGF1α1α (−)(−)--3 99.3 0.986 ent-PGE1 (+)-4 99.9 0.998 ent--PGE11 (+)(+)--4 99.9 0.998

Chemistry 2020, 2 737

5. Conclusions

Four mirror images of the natural prostaglandins, ent-PGF α (( )-1), ent-PGE ((+)-2), ent-PGF α 2 − 2 1 (( )-3), and ent-PGE ((+)-4) were synthesized by the modified Corey synthesis. The starting material − 1 was the ent-Corey lactone ((+)-5), which is a side-product of our prostaglandin production. Using ent-Corey lactone ((+)-5) the mirror images of natural prostaglandins and numerous modified derivatives can be synthesized. The mirror images of prostaglandins can be used as reference standards to justify the optical purity of the pharmaceutical active ingredients. In addition, enantiomeric forms of natural prostaglandins may be important biomarkers to study the oxidative stress of the human body to better understand the mechanism of development of many serious, or even fatal, diseases. Chiral HPLC methods have been also developed for the analytical separation of the enantiomeric pairs. Enantiopurity of the pure ent-prostaglandins has been determined. The HPLC method developed proved to be quite general. The Chiracel OJ-RH column was suitable for the separation of all four enantiomeric pairs. The eluent mixture contained acetonitrile:methanol:water (pH = 4) in each case, but the solvents were mixed in different proportions. The column temperature was 25 ◦C for the separation of the enantiomeric pairs of PGF2α, PGF1α and PGE1, but adequate resolution of the PGE2 enantiomers was achieved at a higher column temperature, at 40 ◦C. In contrast to the known method, the procedure developed is suitable for the routine analytical tests to check the enantiopurity of the prostaglandins produced in our plants. Our long-term goal is to synthesize the mirror images of all prostaglandins that are in our portfolio in order to test the enantiopurity of the active ingredients of the prostaglandin drugs.

Supplementary Materials: Preparation of ent-PGF2α (( )-1), ent-PGE2 ((+)-2) ent-PGF1α (( )-3), and ent-PGE1 ((+)-4) is available online at http://www.mdpi.com/2624-8549− /2/3/47/s1. − Author Contributions: Conceptualization, Z.K., L.T. and E.T.; methodology, L.T.; E.T.; Z.R.; M.E.; A.T.R.; formal analysis, M.K.; investigation, M.E.; A.T.R.; writing—original draft preparation, Z.K., E.T.; writing—review and editing, L.T., Z.R.; visualization, E.T.; supervision, Z.K.; project administration, Z.K.; All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors thank Zoltán Szeverényi and Klára Osapay˝ for reviewing the manuscript before submission. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Bergström, S. The prostaglandins: From the Laboratory to the Clinic (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 1983, 22, 858–866. [CrossRef] 2. Samuelsson,B.Fromstudiesofbiochemicalmechanismtonovelbiologicalmediators: Prostaglandinendoperoxides, thromboxanes, and leukotrienes (Nobel lecture). Angew. Chem. Int. Ed. Engl. 1983, 22, 805–815. [CrossRef] 3. Vane, J.R. Adventures and Excursions in bioassay: The steppingstones to prostacyclin (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 1983, 22, 741–752. [CrossRef] 4. Kurzok, R.C.; Lieb, C. Biochemical studies of human semen. II. The action of semen on the human uterus. Proc. Soc. Exp. Biol. Med. 1930, 28, 268–272. [CrossRef] 5. Goldblatt, M.W. Properties of human seminal plasma. J. Phisyol. 1935, 84, 208–218. [CrossRef][PubMed] 6. von Euler, U.S. On the speciﬁc vasodilating and plain muscle stimulating substances from accessory genital glands in man and certain animals (prostaglandin and vesiglandin). J. Phisyol. 1937, 88, 213–234. 7. Corey, E.J. The logic of chemical synthesis: Multistep synthesis of complex carbogenic molecules (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 1991, 30, 455–465. [CrossRef]

8. Corey, E.J.; Weinshenker, N.M.; Schaaf, T.K.; Huber, W. Stereo-controlled synthesis of prostaglandins F2a and E2 (dl). J. Am. Chem. Soc. 1969, 91, 5675–5677. [CrossRef] 9. Corey, E.J.; Schaaf, T.K.; Huber, W.; Koelliker, U.; Weinshenker, N.M. Total synthesis of prostaglandins F2a and E2 as the naturally occurring forms. J. Am. Chem. Soc. 1970, 92, 397–398. [CrossRef] Chemistry 2020, 2 738

10. Corey, E.J.; Noyori, R.; Schaaf, T.K. Total synthesis of prostaglandins F1a,E1,F2a, and E2 (Natural forms) from a common synthetic intermediate. J. Am. Chem. Soc. 1970, 92, 2586–2587. [CrossRef] 11. Sih, C.J.; Salomon, R.G.; Price, P.; Peruzzotti, G.; Sood, R.G. Total synthesis of ( )-15-deoxyprostaglandin E . ± 1 J. Chem. Soc. Chem. Commun. 1972, 4, 240–241. [CrossRef] 12. Sih, C.J.; Price, P.; Sood, R.; Salomon, R.G.; Peruzzotti, G.; Casey, M. Total synthesis of prostaglandins II.

Prostaglandin E1. J. Am. Chem. Soc. 1972, 94, 3643–3644. [CrossRef][PubMed] 13. Noyori, R.; Suzuki, M. Prostaglandin synthesis by tree-component coupling. Angew. Chem. Int. Ed. Engl. 1984, 23, 847–876. [CrossRef] 14. Suzuki, M.; Morita, Y.; Koyano, H.; Koga, M.; Noyori, R. Three-component coupling synthesis of prostaglandins. A simpliﬁed, general procedure. Tetrahedron 1990, 46, 4809–4822. [CrossRef] 15. Noyori, R. Asymmetric catalysis: Science and opportunities (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 2002, 41, 2008–2022. [CrossRef] 16. Coulthard, G.; Erb, W.; Aggarwal, V.K. Stereocontrolled organocatalytic synthesis of prostaglandin PGF2a in seven steps. Nature 2012, 489, 278–281. [CrossRef]

17. Baars, H.; Classen, M.J.; Aggarwal, V.K. Synthesis of Alfaprostol and PGF2a through 1,4-addition of an alkyne to an enal intermediate as the key step. Org. Lett. 2017, 19, 6008–6011. [CrossRef] 18. Pelss, A.; Gandhamsetty, N.; Smith, J.R.; Mailhol, D.; Silvi, M.; Watson, A.J.A.; Perez-Powell, I.; Prévost, S.; Schützenmeister, N.; Moore, P.R.; et al. Reoptimization of the organocatalyzed double aldol domino process 12 to a key enal intermediate and its application to the total synthesis of delta prostaglandin J3. Chem. Eur. J. 2018, 24, 9542–9545. [CrossRef] 19. Collins, P.W.; Djuric, S.W. Synthesis of therapeutically useful prostaglandin analogs. Chem. Rev. 1993, 93, 1533–1564. [CrossRef] 20. Das, S.; Chandrasekhar, S.; Yadav, J.S.; Grée, R. Recent developments in the synthesis of prostaglandin analogues. Chem. Rev. 2007, 107, 3286–3337. [CrossRef] 21. Peng, H.; Chen, F.-E. Recent advances in asymmetric total synthesis of prostaglandins. Org. Biomol. Chem. 2017, 15, 6281–6301. [CrossRef][PubMed] 22. Cooper, M.J. Prostaglandins in veterinary practice. In Pract. 1981, 3, 30–34. [CrossRef][PubMed] 23. Cooper, M.J.; Hammond, D.; Schultz, R.H. Veterinary uses of prostaglandins. In Practical Applications of Prostaglandins and Their Synthesis Inhibitors, 1st ed.; Kerim, S.M.M., Ed.; Springer: Berlin/Heidelberg, Germany; MPP Press Ltd.: Dordrecht, The Netherlands, 1979; pp. 190–209. 24. Lee, C.W.; Buckley, F.; Costello, S.; Kelly, S. A systematic review of the characteristics of randomised control trials featuring prostaglandins for the treatment of glaucoma. Curr. Med. Res. Opin. 2008, 24, 2265–2270. [CrossRef][PubMed] 25. Toris, C.; Gulati, V. The biology, pathology and therapeutic use of prostaglandins in the eye. Clin. Lipidol. 2011, 6, 577–591. [CrossRef] 26. Lee, S.H.; Rubin, L.J. Current treatment strategies for pulmonary arterial hypertension. J. Internal Med. 2005, 258, 199–215. [CrossRef] 27. Buckley, M.S.; Berry, A.J.; Kazem, N.H.; Patel, S.A.; Librodo, P.A. Clinical utility of treprostinil in the treatment of pulmonary hypertension: An evidence based review. Core Evid. 2014, 9, 71–80. [CrossRef] 28. Kim, H.J.; Kim, J.H.; Park, Y.S.; Suk, K.S.; Lee, J.H.; Park, M.S.; Moon, S.H. Comparative study of the efficacy of limaprost and pregabalin as single agents and in combination for the treatment of lumbar spinal stenosis: A prospective, double-blind, randomized controlled non-inferiority trial. Spine J. 2016, 16, 756–763. [CrossRef] 29. Satoh, H.; Takeuchi, K. Management of NSAID/Aspirin-Induced Small Intestinal Damage by GI-Sparing NSAIDs, Anti-Ulcer Drugs and Food Constituents. Curr. Med. Chem. 2012, 19, 82–89. [CrossRef] 30. Jozwiak, M.; Oude Rengerink, K.; TenEikelder, M.L.G.; van Pampus, M.G.; Dijksterhuis, M.G.K.; de Graaf, I.M.; van der Post, J.A.M.; van der Salm, P.;Scheepers, H.C.J.; Schuitemaker, N.; et al. Foley catheter or prostaglandin

E2 inserts for induction of labour at term: An open-label randomized controlled trial (PROBAAT-P trial) and systematic review of literature. Eur. J. Obst. Gynecol. Reprod. Biol. 2013, 170, 137–145. [CrossRef] 31. Distefano, G. The pharmacological manipulation of Botallo’s duct in the duct-dependent congenital cardiopathies and in the preterm infants with respiratory distress. A review and personal findings. Minerva Pediatrica 2005, 57, 21–34. Chemistry 2020, 2 739

32. Huang, F.K.; Lin, C.C.; Huang, T.C.; Weng, K.P.; Liu, P.Y.; Chen, Y.Y.; Wang, H.P.; Ger, L.P.; Hsieh, K.S. Reappraisal of the prostaglandin E1 dose for early newborns with patent ductus arteriosus-dependent pulmonary circulation. Pediatr. Neonat. 2013, 54, 102–106. [CrossRef][PubMed] 33. Baker, D.E. Lubiprostone: A new drug for the treatment of chronic idiopathic constipation. Rev. Gastroent. Disord. 2007, 7, 214–222. 34. Corey, E.J.; Andersen, N.H.; Carlson, R.M.; Paust, J.; Vedejs, E.; Vlattas, I.; Winter, R.E.K. Total synthesis

of prostaglandins. Synthesis of the pure dl-E1, -F1a,F1b, -A1 and -B1. J. Am. Chem. Soc. 1968, 90, 3247. [CrossRef][PubMed] 35. Corey, E.J.; Vlattas, I.; Harding, K. Total synthesis of natural (levo) and enantiomeric (dextro) forms of

prostaglandin E1. J. Am. Chem. Soc. 1969, 91, 535–536. [CrossRef] 36. Slaters, H.L.; Zelawski, Z.S.; Taub, D.; Wendler, N.L. A new stereoselective total synthesis of prostaglandin

E1 and its optical antipodes. J. Chem. Soc. Chem. Commun. 1972, 6, 304–305. [CrossRef] 37. Slaters, H.L.; Zelawski, Z.S.; Taub, D.; Wendler, N.F. A stereoselective total synthesis of nat( )-prostaglandin − E1 and its optical antipode. Tetrahedron 1974, 30, 819–830. [CrossRef] 38. Taub, D.; Hoﬀsommer, R.D.; Kuo, C.H.; Zelaeski, Z.S.; Wendler, N.L. A stereoselective total synthesis of

PGE1. Chem. Comm. 1970, 1258–1259. 39. Cooper, E.L.; Yankee, E.W. Enantiomeric prostaglandins. J. Am. Chem. Soc. 1974, 96, 5876–5894. [CrossRef] 40. Andersen, N.H.; Imamoto, S.; Picker, D.H. Synthesis of bis-unsaturated prostaglandins. Prostaglandins 1977, 14, 61–101. [CrossRef] 41. Ramwell, P.W.; Shaw, J.E.; Corey, E.J.; Andersen, N. Biological activity of synthetic prostaglandins. Nature 1969, 221, 1251–1253. [CrossRef] 42. Miller, W.L.; Sutton, M.J. Relative biological activity of certain prostaglandins and their enantiomers. Prostaglandins 1976, 11, 77–84. [CrossRef] 43. Morrow, J.D.; Harris, T.M.; Roberts, L.J., II. Non-cyclooxygenase oxidative formation of a series of novel prostaglandins: Analytical ramifications for measurement of eicosanoids. Anal. Biochem. 1990, 184, 1–10. [CrossRef] 44. Morrow, J.D.; Hill, K.E.; Burk, R.F.; Nammour, T.M.; Badr, K.F.; Roberts, L.J., II. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA 1990, 87, 9383–9387. [CrossRef][PubMed] 45. Milne, G.L.; Dai, Q.; Roberts, L.J., II. The isoprostanes 25 years later. Biochim. Biophis. Acta 2015, 1851, 433–445. [CrossRef][PubMed] 46. Oge, K.; Cuyamendous, C.; de la Torre, A.; Candy, M.; Guy, A.; Bultel-Poncé, V.; Durand, T.; Galano, J.M. History of chemical routes towards cyclic non-enzymatic oxygenated metabolites of polyunsaturated fatty acids. Synthesis 2018, 50, 3257–3280. 47. Antus, B.; Kardos, Z. Oxidative stress in COPD: Molecular background and clinical monitoring. Curr. Med. Chem. 2015, 22, 627–650. [CrossRef] 48. Pizzino, G.; Irrera, N.; Cucinotta, M.; Pallio, G.; Mannino, F.; Arcoraci, V.; Squadrito, F.; Altavilla, D.; Bitto, A. Oxidative stress: Harms and benefits for human health. Oxid. Med. Cell. Longev. 2017, 2017, 1–13. [CrossRef] 49. Gottlieb, H.E.; Kotlyar, V.; Nudelman, A. NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 1997, 62, 7512–7515. [CrossRef] 50. Tömösközi, I.; Gruber, L.; Kovács, G.; Székely, I.; Simonidesz, V. Regiospecific Prins reaction, a new way to prostanoids. Tetrahedron Lett. 1976, 50, 4639–4642. [CrossRef] 51. Szabó, T.; Dalmadi, G.; Remport, R.I.; Forró, L.; Hermecz, I.; Sárközi, P.; Nemes, J. Process for the Preparation of PGF2a Tromethamine Salt. Patent HU 56060 A2, 29 July 1991. 52. Ivanics, J.; Szabó, T.; Hermecz, I.; Dalmadi, G.; Kovacs, G.; Resul, B. Chemical Process: Preparation of 13,14-dihydro-15(R)-17-phenyl-18,19,20-trinor-PGF2a Esters. Patent WO 1993000329 A1, 7 January 1993. 53. Dalmadi, G.; Hajdu, F.; Hermecz, I.; Mozsolits, K.; Szabo, T.; Szeverényi, Z.; Vajda, E. Process for the Preparation of Prostaglandin E1. Patent WO 1997022881 A1, 26 June 1997. 54. Galambos, G.; Benno, N.; Povarny, B.M.; Dalmadi, G.; Ebinger, K.; Forró, L.; Hajdu, F.; Hermecz, I.; Kardos, Z.; Lukacs, G.; et al. Process for the Selective Reduction of α,β-Unsaturated Ketones as Prostaglandin Intermediates. Patent WO 1997022602 A2, 26 June 1997. 55. Szabó, T.; Bódi, J.; Dalmadi, G.; Kardos, Z.; Szeverényi, Z. Process for the Preparation of Beraprost and Its Salts. Patent WO 2003011849 A1, 13 February 2003. Chemistry 2020, 2 740

56. Kardos, Z.; Szeverényi, Z.; Dalmadi, G.; Szabó, T.; Bódi, J. Production of Beraprost Ester by Selective reduction. Patent HU 0103088 A2, 28 July 2001. 57. Havasi, G.; Kiss, T.; Hortobágyi, I.; Kardos, Z.; Lászlófi, I.; Bischof, Z.; Bódis, Á. Novel Processes for the Preparation of Prostaglandin Amides. Patent WO 2012164324 A1, 6 December 2012. 58. Kardos, Z.; Kiss, T.; Lászlófi, I.; Hortobágyi, I.; Bischof, Z.; Bódis, Á.; Havasi, G. Process for the Preparation of Travoprost. Patent WO 2013093528 A1, 27 June 2013. 59. Bischof, Z.; Bódis, Á.; K˝om˝uves,M.M. Alternative Process for the Preparation of Travoprost. Patent WO 2014083367 A1, 5 June 2014. 60. Vajda, E.; Hortobágyi, I.; Lászlófi, I.; Buzder-Lantos, P.; Havasi, G.; Takács, L.; Kardos, Z. New Process for the Preparation of High Purity Prostaglandins. Patent WO 2015136317 A1, 17 September 2015. 61. Juhász, I.; Hortobágyi, I.; Altsach, T.; Lászlófi, I.; Borkó, N.Á.; Rozsumberszki, I.; Havasi, G.; Kardos, Z.; Buzder-Lantos, P. Process for the Preparation of Treprostinil and Its Salt Forms. Patent WO 2016055819 A1, 14 April 2016. 62. Buzder-Lantos, P.; Kardos, Z.; Hortobágyi, I.; Lászlófi, I.; Juhász, I.; Fónagy, L.; Váradi, C.; Borkó, N.Á. Process for the Preparation of Carboprost and Its Tromethamine Salt. Patent WO 2017093770 A1, 8 June 2017. 63. Takács, L.; Fekete, I.; Buzder-Lantos, P.; Lászlófi, I.; Hortobábyi, I.; Havasi, G.; Kardos, Z. Preparation of Latanoprostene Bunod of Desired, Pre-Defined Quality by Gravity Chromatography. Patent WO 2017093771 A1, 8 June 2017. 64. Hortobágyi, I.; Lászlófi, I.; Kardos, Z.; Molnár, J.; Takács, L.; Bán, T. Process for the Preparation of Triple-Bond Containing Optically Active Carboxylic Acids, Carboxylate Salts and Carboxylic Acid Derivatives. Patent WO 2017152667 A1, 14 September 2017. 65. Hortobágyi, I.; Lászlófi, I.; Kardos, Z.; Molnár, J.; Takács, L.; Tormási, R. Preparation of Epoprostenol Sodium of Enhanced Stability. Patent WO 2017162668 A1, 28 September 2017. 66. Hortobágyi, I.; Lászlófi, I.; Kardos, Z.; Molnár, J.; Takács, L.; Bán, T. Process for the Optically Active Beraprost. Patent WO 2017174439 A1, 12 October 2017. 67. Hortobágyi, I.; Lászlófi, I.; Kardos, Z.; Molnár, J.; Takács, L.; Horváth, K. Process for the preparation and purification of misoprostol. Patent WO 2019011668 A1, 19 January 2019. 68. Hortobágyi, I.; Lászlófi, I.; Varga, Z.; Juhász, I.; Ritz, I.; Kardos, Z. Process for the preparation of polymorph form B of treprostinil diethanolamine salt. Patent WO 2019170093 A2, 12 September 2019. 69. Rozsumberszki, I.; Váradi, C.; Bán, T.; Kardos, Z.; Hortobágyi, I.; Szabó, T. Process for the preparation of iloprost. Patent WO 2019202345 A2, 24 October 2019. 70. Corey, E.J.; Arnold, Z.; Hutton, J. Total synthesis of prostaglandins E2 and F2a (dl) via a tricarbocylic intermediate. Tetrahedron Lett. 1970, 4, 307–310. [CrossRef] 71. T˝oke,L.; Szabó, G.; Fogassy, E.; Ács, M.; Nagy, L.; Árvai, L. Verfahren zur Trennung von racemischen Alkalimatellsalzen und vom Lacton der cis-2-Hydroxy-cyclopent-4-en-1-yl-essigsaure. Patent CH 647 496 A5, 9 January 1979. 72. Sigma-Aldrich Products, Chemical and Biochemical Product. Available online: https: //www.sigmaaldrich.com/catalog/search?term=Corey+lactone&interface=All&N=0&mode=match% 20partialmax&lang=fr®ion=FR&focus=product (accessed on 26 July 2020). 73. Anelli, P.L.; Biffi, C.; Montanari, F.; Quici, S. Fast and selective oxidation of primary alcohols to aldehydes or to carboxylic acids and of secondary alcohols to ketones mediated by oxoammonium salts under two-phase conditions. J. Org. Chem. 1987, 52, 2559–2562. [CrossRef] 74. Wadsworth, W.S.; Emmons, W.D. The utility of phosphonate carbanion in olefin synthesis. J. Am. Chem. Soc. 1961, 83, 1733–1738. [CrossRef] 75. Horner, L.; Hofmann, H.; Klink, W.; Ertel, H.; Toscano, V.G. PO aktivierte Verbindung als Olefinierungreagentin. Chem. Ber. 1962, 95, 581–601. [CrossRef] 76. Bruckner, R.; Harmata, M. (Eds.) Organic Mechanisms—Reactions, Stereochemistry and Synthesis; Springer: Berlin/Heidelberg, Germany, 2010; pp. 471–475. 77. Corey, E.J.; Bakshi, R.K.; Shibata, S. Highly enantioselective borane reduction of ketones catalyzed by oxazaborolidines. Mechanism and synthetic implications. Am. Chem. Soc. 1987, 109, 5551–5553. [CrossRef]

78. Brose, S.A.; Thuen, B.T.; Golokovo, M.Y. LC/MS/MS method for analysis of E2 series prostaglandins and isoprostanes. J. Lip. Res. 2011, 52, 850–859. [CrossRef] Chemistry 2020, 2 741

79. Overbeke, A.V.; Baeyens, W.; Oda, H.; Aboul-Enein, H.Y. Direct enantiomeric HPLC separation of several 2-aryl-propionic acids, barbituric acids and benzodiazepines on Chiracel OJ-R chiral stationary phase. Chromatographia 1996, 43, 599–606. [CrossRef] 80. Chiral Technologies, Daicel-Chromatography Market Leader. Available online: https://mz-at.de/downloads/ chiral_chiralcel_oj-rh_instruction_manual.pdf (accessed on 26 July 2020).

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