α-Halogenated oxaphosphinanes: Synthesis, unexpected reactions and evaluation as inhibitors of cancer cell proliferation

European Journal of Medicinal Chemistry 104 (2015) 33e41

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Research paper

a-Halogenated oxaphosphinanes: Synthesis, unexpected reactions and evaluation as inhibitors of cancer cell proliferation Rachida Babouri a, b, Marc Rolland e, Odile Sainte-Catherine c, Zahia Kabouche b, €l Volle a, **, David Virieux a, *, Marc Lecouvey c, ***, Norbert Bakalara d, Jean-Noe a Jean-Luc Pirat a

ICG MontpelliereUMR 5253, Equipe AM2N, ENSCM, 8, Rue de l'Ecole Normale, 34296 Montpellier CEDEX 5, France Universit e Mentouri-Constantine, D epartement de chimie, Laboratoire d’Obtention de Substances Th erapeutiques (LOST), Campus Chaabet Ersas, 25000 Constantine, Algeria c Universit e Paris 13, Sorbonne Paris Cit e, CSPBAT, CNRS UMR 7244, 74, Rue Marcel Cachin, F-93017 Bobigny, France d INSERM U-1051, Institut des Neurosciences de Montpellier, 80 Rue Augustin Fliche, 34091 Montpellier, France e Institut Europ een des Membranes, cc047 Universit e de Montpellier 2, 34095 Montpellier, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 June 2015 Received in revised form 19 September 2015 Accepted 21 September 2015 Available online 28 September 2015

This paper describes the preparation and the biological evaluation of a-halogenated oxaphosphinanes. These halogen derivatives were synthetized from a short and stereoselective synthetic sequence starting by previously described hydroxy-precursors 1 and 2 with respectively a glucose and mannose-like configuration. The in vitro biological tests of these unnatural halogenated phosphinosugars, on several cell lines, highlighted, for some of them, their antiproliferative and anti migration and invasion properties at nanomolar concentration. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: a-Halogenated oxaphosphinanes Phosphinosugars Phase-transfer catalysis Triflate halogen exchange Sigmatropic rearrangement Antiproliferative properties

1. Introduction Glioblastoma are the most frequent brain tumors in humans [1]. The traditional treatments of these cancers, i.e. resection, followed by radiotherapy and chemotherapy with Temozolomide, have a limited efficacy and the median survival rarely exceeds 15 months [2]. A family of phosphorus glycomimetics, has been described by our groups for antiproliferative, anti-migratory and anti-invasive effects on several glioblastoma cell lines [3,4]. The discovery of these original structures may afford a timely therapeutic response for patients. Indeed if the antiproliferative efficiency of these phosphinosugars are in the mM ranges on brain cancer cell lines and

* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (M. Lecouvey), jean-noel. [email protected] (J.-N. Volle), [email protected] (D. Virieux). http://dx.doi.org/10.1016/j.ejmech.2015.09.027 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

primary cultures, some of them exhibited antimigratory properties on C6 and SNB75 cell lines as well as on Gli 4 and Gli 7 brain cancer primary cultures in nanomolar range [4]. Thousands of natural halogenated organic molecules were already discovered, mainly are organochlorines [5e7] and/or bromines [6e8], and to a lesser extent natural compounds incorporating iodine [9], or fluorine atoms [10,11]. All of them exhibit interesting and noteworthy biological properties and cytotoxicity is commonly reported. Njardarson highlighted the preeminence of halogen elements featured in commercial drugs approved by the FDA [12]. In this context, the contribution of fluorine is typically well documented. This relatively small and highly electronegative element has generally a high impact on pharmacological parameters, modifying pKa, lipophilicity and even conformation [13,14]. Consequently, metabolic stability and increased binding affinity by direct interaction of fluorine atom with the protein or by modification of the polar surface of other groups are often observed. In order to complete the [1,2]-oxaphosphinane structure-activity relationship (SAR), we investigated the stereoselective

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preparation of halogenated analogues 3 and 4 starting from the enantiopure oxaphosphinane 1 and 2 respectively considered as having a glucose-like and a mannose-like configuration. Besides, the preparation of fluorine analogues to study the in vivo biodistribution of oxaphosphinanes 1 and 2 was very attractive, due to the possibility to introduce 18F isotope for imagery by positron emission tomography (PET) [14] Fig. 1. 2. Chemistry The synthesis of the 3-hydroxyoxaphosphinanes 1 and 2 was previously described using a one-step additionecyclization reaction of ethyl phenylphosphinate to 2,3,5-tri-O-benzyl-D-arabinofuranose under basic activation [3,15]. Stereoselective preparation of halogenated-derivatives 3aed and 4aed from glucose-like and mannose-like oxaphosphinanes 1 and 2, may be accomplished by a modified version of the Finkelstein reaction. The formation of triflate derivatives 5 and 6 which generally occurs with retention of configuration could be followed by halogen-triflate exchange. This reaction proceeds generally through a SN2 process with inversion of configuration Fig. 2. Introduction of triflate on a-hydroxy-phosphonate is already referred, and can be achieved in presence of triflic anhydride with pyridine [16,17], or 2,6-lutidine [18,19] as base. However, a rapid screening of literature did not allow to find any procedure where stereocontrol is required or where the leaving group is a substituent of a phosphorus heterocycle. We already published the preparation of a diastereomeric mixture of triflate derivatives 5 and 6 from a 60:40 ratio of phosphinosugars 1 and 2 using a large excess of triflic anhydride and pyridine [3]. Nevertheless, the purification of each diastereoisomer 5 and 6 by chromatography on silica revealed very intricate. Therefore all triflate derivatives were prepared preferentially from the enantiopure precursors 1 and 2. Starting from glucose-like oxaphosphinane 1, triflate derivative 5 was obtained quantitatively (98%). By contrast, the mannose-like diastereomer 2 gave, in the same conditions, moderate yield of the expected triflate 6 (44%). Surprisingly 2,6-lutidine has a dramatic effect on stereoselectivity. Indeed, inversion of configuration was observed affording exclusively triflate 5 (96% from 31P-NMR and 53% isolated yield). This unexpected stereoselectivity might be explained by the difference of ion pair interactions between pyridinium/triflate and lutidinium/triflate. Indeed, the sterically hindered lutidinium cation would prevent an intimate electrostatic interaction with its counter anion, allowing to the free triflate anion a nucleophilic displacement of the preformed derivative 6 favoring finally the thermodynamic triflate 5 (Scheme 1). Each diastereomeric triflates 5 and 6 were converted into the corresponding iodides 3a and 4a in good yields, respectively 62% and 70% by treatment with 4 equivalents of potassium iodide in acetonitrile at 80
C (Table 1, entries 1 and 2) [20]. Bromination of triflate 5 was accomplished in low yield from the reaction of potassium bromide and tetraethylammonium bromide (10 mol %) as phase-transfer agent [21]. Under these conditions only 21% of the bromide derivative 3b was isolated after 24 h at 80
C (entry 3). After optimization using stoichiometric amounts of tetraethylammonium bromide (entry 4), yield of 52% was obtained.

Fig. 1. Oxaphosphinane 1e2 and targeted halogenated analogues 3 and 4.

Fig. 2. Stereocontrolled approach for preparation of halogenated oxaphosphinane 3aed and 4aed.

Scheme 1. Preparation of triflates 5 and 6 from oxaphosphinanes 1 and 2.

Similarly, the diastereomer 4b was isolated in 67% yield from the reaction of epimer 6 (entry 5). Monocrystals of brominated 4b have been obtained by crystallization from a mixture of acetone and hexanes. X-ray data showed that bromide derivative 4b adopted a chair conformation with the bromine atom laying in equatorial position confirming that the reaction occurred through a clean SN2 pathway (Fig. 3). Chlorinated oxaphosphinanes 3c and 4c were obtained respectively in 62% and 35% yields by reaction of the triflates 5 and 6 with stoichiometric quantity of tetraethylammonium chloride in acetonitrile (entries 6 and 7). This methodology was consecutively applied for the synthesis of the fluoride derivative 3d from glucose-like oxaphosphinane 5 using potassium fluoride (entry 8). Instead of the expected triflatefluorine exchange, a rearrangement occurred giving two different compounds (Scheme 2). They were characterized by two doublets respectively at 46.2 ppm and 46.0 ppm and strong 31Pe19F coupling constants in 31P-NMR. With values of 1058 and 1048 Hz, coupling constants were unambiguously attributed to a 1JPF. These PeF intermediates were rapidly hydrolyzed by addition of water in the reaction mixture giving only one signal centered at 21.4 ppm in 31PNMR. Fluorine derivatives were identified as the diastereomeric mixture of 2-fluorophosphino tetrahydrofurans 7a and 7b. The action of water led to the corresponding furanosylphosphinic acid 8 as a single enantiomer consecutively to the loss of asymmetric phosphorus center. Mechanistically, the chemoselectivity observed during the process can be rationalized using the hard-soft-acidbase theory (HSAB). Fluoride anion considered as a “hard” nucleophile interacted preferentially with the “hardest” electrophilic site, i.e. phosphinolactone group. We excluded the nucleophilic substitution of fluoride to the phosphorus atom. If the reaction proceeded by this SN(P) reaction only one stereoisomer would be obtained. An alternate mechanism might be the formation of the pentacoordinated phosphorane (Scheme 2). Configurational

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Table 1 Formation of halogenated oxaphosphinanes 3, 4 and compounds 8 and 9.

Entry

Precursor

Reaction conditions

Product

Yield %

1 2 3 4 5 6 7 8 9 10

5 6 5 5 6 5 6 5 6 3a

KI (4 eq.), MeCN, 80
C, 2 h KI (4 eq.), MeCN, 80
C, 1.5 h KBr (4 eq.), Et4NBr (0.1 eq.), MeCN, 80
C, 24 h Et4NBr (1 eq.), MeCN, 80
C, 3 h Et4NBr (1 eq.), MeCN, 80
C, 2 h Et4NCl (1 eq.), MeCN, 80
C, 1 h Et4NCl (1 eq.), MeCN, 80
C, 3 h KF (4 eq.), MeCN, 80
C, 5 h KF (6 eq.), DMSO, 100e115
C, 4 h CsF (4 eq.), DMSO, 95
C, 1 h 15

3a 4a 3b 3b 4b 3c 4c 8 9 9

62b 70b 21b 52b 67b 62b 35.5b 84b (95a) 41a 80b (95a)

a b

Estimated by 31P NMR from the crude mixture with a sealed capillary tube containing DMSO-d6 as lock solvent. Isolated yield after purification on silica column.

Fig. 3. X-ray diffraction diagram of bromooxaphosphinane 4b.

flexibility in pentavalent compounds, through stereomutation mechanism is well established and could explain the epimerization of the phosphorus atom [22]. At this level, the resulting phosphoranes could collapse following two pathways: 1) by cleavage of oxygen-phosphorus bond and oxaphosphinane ring-opening. Then the resulting alkoxide attacked the electrophilic carbon through a SN2 reaction, leading to both diastereomers 7a and 7b, which upon hydrolysis furnished the enantiopure furanosylphosphinic acid 8

(Scheme 2, Pathway A), 2) or by sigmatropic rearrangement affording diastereomers 7a and 7b (Scheme 2, Pathway B). The other diastereomeric triflate 6 also gave an unexpected product on treatment with potassium fluoride in dimethylsulfoxide (entry 9) which was clearly identified as the oxaphosphinene 9. The formation of this structure can be explained by the basic character of fluoride, leading preferentially to an elimination process rather than a nucleophilic displacement of iodide atom. Suitable monocrystals for X-ray studies were obtained from slow crystallization in ethyl acetate and hexane confirming the presence of the enol ether functional group (Scheme 3). Formation of oxaphosphinene 9 was deeply improved in yield up to 80% by employing cesium fluoride as fluorine source and iodooxaphosphinane 3a rather than triflate 6 (entry 10). A common reagent used for transformation of alcohols into fluorides is diethyl amino sulfur trifluoride (DAST) [17,23]. Direct fluorination of glucose and mannose-like oxaphosphinanes 1 and 2 were tested using DAST in dichloromethane. Compound 1 failed to give the fluorine derivative 3d whatever the conditions. Nevertheless, the glucose-like fluorooxaphosphinane 4d was isolated by column chromatography on silica in 3% yield (Scheme 4).

Scheme 2. Attempt of nucleophilic fluorination of triflate 5.

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R. Babouri et al. / European Journal of Medicinal Chemistry 104 (2015) 33e41

Scheme 3. Synthesis and X-ray structure of oxaphosphinene 9.

3. Biological evaluation 3.1. a-Halogenated oxaphosphinanes effects on cell viability The a-halogenated oxaphosphinanes were screened on a panel of six cancer cell lines. The panel comprised human breast adenocarcinoma (pleural metastasis) cell line (MDAMB231), human melanoma cell lines (MDAMB435) and mouse melanoma cell lines (B16F10), human colorectal carcinoma cell line (CaCo2), human hepatocarcinoma cell line (HuH7), prostatic carcinoma cell line (DU145). To evaluate the antiproliferative activity of these a-halogenated oxaphosphinanes and related analogues, we have used a MTT test. Interestingly, among the six cancer lines tested only the human colorectal adenocarcinoma does not respond to the ahalogenated oxaphosphinanes (Table 2). Human (MDAMB435) and mouse (B16F10), melanoma cell lines proliferation inhibition reached 70% inhibition at the highest concentration tested. DU145 and HuH7 cell lines proliferations were only inhibited by 50%. MDAMB231 cell line proliferation was inhibited by 30%. It has to be noticed that for a given cell line the maximum inhibition of proliferation was almost the same for seven of the eight a-halogenated oxaphosphinanes but with different IC50 values. Only the fluorinated one 4d was not active against MDAMB231 and DU145 cell lines. The other a-halogenated oxaphosphinanes and the nonhalogenated one (compound 9) exhibited IC50's in the 15e500 nM range. 3.2. a-Halogenated oxaphosphinanes effects on cell migration and invasion To study the impact of compounds 3 aec and 4 aed on B16F10 and MDAMB435 cell lines migration according to the matrix context, we performed all comparisons of compoundematrix context and measured the serum motility response using a 2Dmigration assay over the course of 24 h in presence and absence of the glycoside mimetics (Table 3). As shown in Table 3, most of the synthetic a-halogenated oxaphosphinanes strongly inhibited the migration of B16F10 and

Scheme 4. Direct fluorination of glucose and mannose-like oxaphosphinanes 1 and 2 by DAST.

MDAMB435 cell lines on matrix made of fibronectin, vitronectin or laminin in the nM range. The compound 4c and 4a displayed moderate anti-migratory activity of B16F10 and MDAMB435 cells respectively on vitronectin but these compounds cause cell cytotoxicity. Best migration inhibition of B16F10 cells by 3b on laminin gave Ki value of 10 nM. In the presence of HGF in the lower part of the Boyden migration chamber, the B16F10 and MDAMB435 cells invaded the Matrigel and were visualized at lower surface of the membrane. a-halogenated oxaphosphinanes reduced the invasion of the cells at nanomolar range but the maximum of inhibition did not exceed 70% for B16F10 cells and 50% for MDAMB435 cells. 4. Conclusion In conclusion, we succeeded to obtain stereoselectively the desired halogen-based oxaphosphinanes from a two-step sequence. In contrast to the other halogens, introduction of fluorine failed when performed by this triflate/halogen exchange and the reaction was deeply affected by the stereochemistry of the triflate group: when triflate layed in equatorial position, an unusual rearrangement was observed leading to furanosylphosphinic acid 8 whereas the formation of the enol ether 9 was observed from the other epimer both in high yields. The glucose-like fluorinated oxaphosphinane 4d was obtained by direct fluorination using DAST. We consecutively demonstrated that these oxaphosphinanes were active against melanoma, epidermoid car, hepatocarcinoma, prostatic carcinoma and breast adenocarcinoma cell lines. Activities on these cancer cell lines did not depend on the nature of the halogen atom. By contrast with our previous results on the hydroxyl derivatives 1 and 2, no stereoselectivity was observed. Although a-halogenated oxaphosphinanes exhibited antiproliferative activity in the nM range for MDAMB231, MDAMB435, B16F10, HuH7, DU145 cell lines, inhibition did not reach 100% of cell viability at the highest concentration tested (100 mM). One hypothesis could be that a-halogenated oxaphosphinanes are more cytostatic than cytotoxic. Alternatively, this observation may reflect cell heterogeneity concerning either the expression of the a-halogenated oxaphosphinanes targets or the impact of the a-halogenated oxaphosphinanes on these targets. In a second screening step, we tested the selected phostines 3aec and 4aed for their migration inhibition activity against B16F10 and MDAMB435 cell lines. As the cytotoxicity, the position of halogen on the carbon 2 didn't change Ki values. We observed for the two epimers the same range for the migration inhibition. The nature of halogen is not a determining parameter because variations of Ki values between 16 and 50 nM on fibronectin, 10 and 42 nM on laminin are not significant. In contrast, the nature of halogen plays a more important role on vitronectin migration inhibition. Ki value (380 nM) decreased drastically for B16F10 migration inhibition if the halogen is a chlorine (compound 4c). This effect was also observed for MDAMB435 migration inhibition with the compound 4a bearing iodine on a position. These experimental data suggest that ahalogenated oxaphosphinanes affect proliferation, migration and

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Table 2 Antiproliferative Values (IC50, nM) of the oxaphosphinanes on MDAMB231, MDAMB435 (Cell Line Obtained from Breast Cancer Tissue), B16F1 (Cell Lines Obtained from Skin Cancer Tissues), Caco2 (Cell Line Obtained from Colon Cancer Tissue), HuH7 (Cell Line Obtained from Intestine Cancer Tissue), DU145 (Cell Line Obtained from Prostate Cancer Tissue). IC50 values (nM)

Compounds

3a 4a 3b 4b 6c 4c 9 4d

MDAMB231a

MDAMB435b

B16F10c

Caco2

HuH7d

DU145e

466 478 445 432 404 359 286 NA

0.55 0.3 0.2 0.35 1.1 1.6 0.52 1.45

7.7 4 4.6 3.8 8 7.7 4.4 1.7

20,500 28,600 24,718 29,500 26,200 24,800 28,400 NA

229 13 11.6 15.8 270 1170 37.5 12.1

215 (±3) 104 (±0.2) 102 (±0.2) 86.5 (±0.5) 15.5 (±0.2) 226 (±0.3) 116 (±0.2) NA

(±2) (±2) (±1) (±135) (±1) (±14) (±1)

(±0.08) (±0.01) (±0.01) (±0.01) (±0.02) (±0.01) (±0.001) (±0.01)

(±0.01) (±0.01) (±0.01) (±0.01) (±0.02) (±0.02) (±0.02) (±0.02)

(±1) (±0.2) (±0.1) (±0.1) (±1) (±4) (±0.1) (±0.1)

a The maximum inhibition of cell proliferation was 30% at 100 mM. The IC50 has been calculated from 0 to 35% of inhibition for compound concentrations ranging from 10 nM to 100 mM. b The maximum inhibition of cell proliferation was 60% at 100 mM. The IC50 has been calculated from 0 to 60% of inhibition for compound concentrations ranging from 10 nM to 100 mM. c The maximum inhibition of cell proliferation was 80% at 100 mM. The IC50 has been calculated from 0 to 80% of inhibition for compound concentrations ranging from 0.1 nM to 100 mM. d The maximum inhibition of cell proliferation was 55% at 100 mM. The IC50 has been calculated from 0 to 55% of inhibition for compound concentrations ranging from 10 nM to 100 mM. e The maximum inhibition of cell proliferation was 45% at 100 mM. The IC50 has been calculated from 0 to 55% of inhibition for compound concentrations ranging from 10 nM to 100 mM. The remaining cells appeared to be resistant.

Table 3 Antimigratory values (Ki) of a-halogenated oxaphosphinanes on B16F10 and MDAMB435 cell lines. Strain

Products

Kia (nM) support FN

B16F10

3a 4a 3b 4b 3c 4c 4d

Strain

MDAMB435

16 20 46 30 49 18 22 Products

3a 4a 3b 4b 3c 4c 4d

LN ± ± ± ± ± ± ±

1 1 1 1 1 1 1

17 16 10 20 19 13 16

± ± ± ± ± ± ±

1 1 1 1 1 1 1

VN

INVb

44 ± 1 35 ± 1 21 ± 1 50 ± 1 34 ± 1 380 ± 1 40 ± 1

29 37 17 32 35 37 33

± ± ± ± ± ± ±

1 1 1 1 1 1 1

Ki (nM) support FN

LN

VN

INVd

29 ± 1 28c ±1 39 ± 1 28c ±1 28c ±1 36 ± 1 28c ±1

25 ± 1 23c ±1 34 ± 1 42 ± 1 23c ±1 32 ± 1 34 ± 1

43 ± 1 101 ± 1 46 ± 1 78 ± 1 41c ±1 52 ± 1 66

28 35 13 30 36 35 31

± ± ± ± ± ± ±

1 1 1 1 1 1 1

FN: fibronectin; LN: laminin; VN: vitronectin; INV: invasivity. a Ki estimation was determined with serf/cell and maps software using the Hill equation. b Maximum inhibition does not exceed 70%. c Maximum inhibition does not exceed 90%. d Maximum inhibition does not exceed 50%.

invasion. Our study provides a novel type of small molecule therapeutic agents that aim to block tumor progression and the metastasis formation. 5. Experimental 5.1. Chemistry 5.1.1. General considerations Before use, all solvents were dried to help of MBRAUN solvent purification system or by drying according to current method, and stored under nitrogen. Reactions were monitored by 31P NMR using DMSOed6 as internal reference. Purifications by column chromatography were performed on silica gel (60 AC. 35e70 mm). Melting

point were measured on a Büchi B-540 apparatus. Spectrums 1H, 19 F, 31P and 13C were recorded on a Bruker DRX 400 MHz spectrometer operating respectively at frequency of 400 MHz, 188 MHz, 162 MHz and 101 MHz. All NMR experiments performed on phosphorus are indicated uncoupling of hydrogen. In order to facilitate NMR description of oxaphosphinane, proton and carbon of ring was numbered from right to left and started by adjacent position of phosphorus atom. The spectrometer used for high resolution mass spectra was SYNAPT G2-S of Water. Intensities reflections were measured with an Xcalibur, Sapphire 3, Gemini diffractometer [24] (graphite monochromated CuKa radiation (k ¼ 1.54184 Å), CCDdetector, u scanning). Data reduction was performed with CrysAlisPro [25], and an empirical absorption correction was applied. Equivalent reflections, other than Friedel pairs were merged. The structures were solved by direct method using the SHELXS and refined using SHELXL [25], with full-matrix least-squares refinement against F2 in anisotropic approximation for non-hydrogen atoms. H atoms were treated by a mixture of independent and constrained refinement. Refinement of the absolute structure Flack parameters [26], while the Hooft analysis [27], gave absolutes parameters which confidently confirms that the refined coordinates represent the true enantiomorph.

5.1.2. (2Sp,3S,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3-Otrifluoromethane sulfonyl-2-phenyl-2-oxo-2l5-[1,2]oxaphosphinane 5 Under N2 and at 70
C, trifluoromethanesulfonic anhydride (222 mL, 1.32 mmol) was added dropwise to a solution of 1 (0.60 g, 1.1 mmol) and pyridine (178 mL, 2.2 mmol) in dichloromethane (3.6 mL). After 1 h at 0
C, the organic layer was washed twice with water, dried over sodium sulfate, filtered and concentrated under vacuum to give 5 as a white solid (0.73 g, 98%), m.p. 135.8e136.4
C. 31 P NMR (CDCl3): d 28.07 (s). 19F NMR (CDCl3): d 75.03 (s). 1H NMR (CDCl3): d 3.78 (dd, 2JHH ¼ 11.2 Hz, 3JHH ¼ 2.0 Hz, 1H, OCH2), 3.98 (ddd, 2JHH ¼ 11.2 Hz, 4JHP ¼ 2.6 Hz, 3JHH ¼ 2.4 Hz, 1H, OCH2), 4.19 (dd, 3 JHH ¼ 9.8 Hz, 3JHH ¼ 9.5 Hz, 1H, 3CH), 4.45 (ddd, 3JHH ¼ 10.5 Hz, 3 JHH ¼ 9.5 Hz, 3JHP ¼ 3.1 Hz, 1H, 2CH), 4.53 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.62 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.64 (dddd, 3JHH ¼ 9.8 Hz, 3 JHP ¼ 4.2 Hz, 3JHH ¼ 2.4 Hz, 3JHH ¼ 2.0 Hz, 1H, 4CH), 4.68 (d, 2 JHH ¼ 10.7 Hz, 1H, OCH2), 4.89 (d, 2JHH ¼ 10.7 Hz, 1H, OCH2), 4.90 (d, 2 JHH ¼ 10.4 Hz, 1H, OCH2), 4.98 (d, 2JHH ¼ 10.4 Hz, 1H, OCH2), 5.08

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R. Babouri et al. / European Journal of Medicinal Chemistry 104 (2015) 33e41

(dd, 3JHH ¼ 10.5 Hz, 2JHP ¼ 1.2 Hz, 1H, 1CH), 7.15e7.17 (m, 2H, CHar), 7.29e7.37 (m, 13H, CHar), 7.56e7.61 (m, 2H, CHar), 7.70e7.74 (m, 1H, CHar), 7.87e7.92 (m, 2H, CHar). 13C NMR (CDCl3): d 67.88 (d, 3 JCP ¼ 9.4 Hz, BnOCH2), 73.50 (s, PhCH2), 75.51 (d, 2JCP ¼ 5.9 Hz, 4 CH), 75.64 (s, PhCH2), 76.39 (s, PhCH2), 78.48 (s, 3CH), 80.32 (d, 2 JCP ¼ 4.3 Hz, 2CH), 81.42 (d, 1JCP ¼ 96.5 Hz, 1CH), 117.96 (q, 1 JCF ¼ 319.6 Hz, CF3), 124.82 (d, 1JCP ¼ 145.1 Hz, Car), 127.58, 127.67, 127.92, 128.02, 128.37, 128.52, 129.03 (d, 3JCP ¼ 14.2 Hz, CHar), 132.42 (d, 2JCP ¼ 10.8 Hz, CHar), 134.49 (d, 4JCP ¼ 2.9 Hz, CHar), 137.01 (s, CarBn), 137.31 (s, CarBn), 137.54 (s, CarBn). HRMS (m/z) calcd for C33H33F3O8PS: 677.1586. Found: 677.1590. 5.1.3. (2Sp,3R,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3-Otrifluoromethane sulfonyl-2-phenyl-2-oxo-2l5-[1,2]oxaphosphinane 6 On the same manner than above and after chromatography column on silica gel using a mixture of n-heptane/ethyl acetate (60:40) as eluent, compound 6 was obtained as a colorless oil (0.33 g, 44%). 31P NMR (CDCl3): d 29.36 (s). 19F NMR (CDCl3): d 75.21 (s). 1H NMR (CDCl3): d 3.68 (dd, 2JHH ¼ 11.2 Hz, 3 JHH ¼ 2.0 Hz, 1H, OCH2), 3.83 (ddd, 2JHH ¼ 11.2 Hz, 3JHH ¼ 3.0 Hz, 4 JHP ¼ 2.9 Hz, 1H, OCH2), 4.13 (dd, 3JHH ¼ 9.9 Hz, 3JHH ¼ 9.8 Hz, 1H, 3 CH), 4.38 (dd, 3JHH ¼ 9.8 Hz, 3JHH ¼ 2.4 Hz, 1H, 2CH), 4.48 (d, 2 JHH ¼ 12.3 Hz, 1H, OCH2), 4.50 (d, 2JHH ¼ 11.00 Hz, 1H, OCH2), 4.51 (dddd, 3JHH ¼ 9.9 Hz, 3JHP ¼ 3.1 Hz, 3JHH ¼ 3.0 Hz, 3JHH ¼ 2.0 Hz, 1H, 4 CH), 4.53 (d, 2JHH ¼ 11.4 Hz, 1H, OCH2), 4.55 (d, 2JHH ¼ 12.3 Hz, 1H, OCH2), 4.80 (d, 2JHH ¼ 11.0 Hz, 1H, OCH2), 4.83 (d, 2JHH ¼ 11.4 Hz, 1H, OCH2), 5.37 (dd, 2JHP ¼ 4.7 Hz, 3JHH ¼ 2.4 Hz, 1H, 1CH), 7.04e7.06 (m, 2H, CHAr), 7.14e7.30 (m, 13H, CHAr), 7.41e7.46 (m, 2H, CHAr), 7.57e7.61 (m, 1H, CHAr), 7.79e7.84 (m, 2H, CHAr). 13C NMR (CDCl3): d 68.46 (d, 3JCP ¼ 9.0 Hz, BnOCH2), 73.12 (s, PhCH2), 73.21 (d, 2 JCP ¼ 1.6 Hz, 3CH), 73.37 (s, PhCH2), 75.86 (s, PhCH2), 76.55 (d, 3 JCP ¼ 6.2 Hz, 4CH), 79.05 (d, 2JCP ¼ 3.4 Hz, 2CH), 79.73 (d, 1 JCP ¼ 93.3 Hz, 1CH), 117.88 (q, 1JCF ¼ 319.6 Hz, CF3), 124.02 (d,1JCP ¼ 147.5 Hz, Car), 127.60, 127.83, 127.92, 128.07, 128.25, 128.42, 128.48, 128.52, 128.85 (d, 3JCP ¼ 14.2 Hz, CHar), 133.21 (d, 2 JCP ¼ 10.5 Hz, CHar), 134.51 (d, 4JCP ¼ 2.9 Hz, CHar), 136.65 (s, CarBn), 137.54 (s, CarBn), 137.87 (s, CarBn). HRMS (m/z) calcd for C33H33F3O8PS: 677.1586. Found: 677.1592. 5.1.4. (2Sp,3S,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3iodo-2-phenyl-2-oxo-2l5-[1,2]-oxaphosphinane 3a Under nitrogen, potassium iodine (0.068 g, 0.41 mmol) was added to a solution of 5 (0.070 g, 0.10 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 80
C for 2 h, and solvent was evaporated under vacuum, Ethyl acetate (9 mL) was added to residue and organic solution was washed with water (3 mL). The organic layer was dried over sodium sulfate, filtered and solvent was evaporated under vacuum. A normal phase chromatography on silica gel using a mixture of n-heptane/ethyl acetate (60:40) as eluent, afforded 3a as a colorless oil (0.042 g, 62%). 31P NMR (CDCl3): d 35.87 (s). 1H NMR (CDCl3): 3.80 (ddd, 3JHH ¼ 8.7 Hz, 3 JHH ¼ 3.7 Hz, 3JHP ¼ 2.3 Hz, 1H, 2CH), 3.82 (dd, 2JHH ¼ 11.4 Hz, 3 JHH ¼ 2.0 Hz, 1H, OCH2), 3.99 (ddd, 2JHH ¼ 11.4 Hz, 3JHH ¼ 3.3 Hz, 4 JHP ¼ 2.6 Hz, 1H, OCH2), 4.27 (dd, 3JHH ¼ 9.9 Hz, 3JHH ¼ 8.7 Hz, 1H, 3 CH), 4.56 (dd, 2JHP ¼ 8.6 Hz, 3JHH ¼ 3.7 Hz, 1H, 1H), 4.59 (d, 2 JHH ¼ 11.2 Hz, 1H, OCH2), 4.61 (d, 2JHH ¼ 10.8 Hz, 1H, OCH2), 4.62 (d, 2 JHH ¼ 12.2 Hz, 1H, OCH2), 4.68 (m, 3JHH ¼ 9.9 Hz, 3JHH ¼ 3.3 Hz, 3 JHH ¼ 2.0 Hz, 1H, 4CH), 4.73 (d, 2JHH ¼ 11.2 Hz, 1H, OCH2), 4.76 (d, 2 JHH ¼ 12.2 Hz, 1H, OCH2), 4.93 (d, 2JHH ¼ 10.8 Hz, 1H, OCH2), 7.21e7.23 (m, 2H, CHar), 7.28e7.44 (m, 13H, CHar), 7.50e7.54 (m, 2H, CHar), 7.61e7.66 (m, 1H, CHar), 7.92e7.97 (m, 2H, CHar). 13C NMR (CDCl3): d 22.60 (d, 1JCP ¼ 80.0 Hz, 1CH), 68.98 (d, 3JCP ¼ 9.1 Hz, BnOCH2), 71.34 (s, PhCH2), 73.36 (s, PhCH2), 75.46 (s, PhCH2), 75.99 (d, 2JCP ¼ 1.1 Hz, 3CH), 76.25 (d, 2JCP ¼ 5.7 Hz, 4CH), 78.28 (s, 2CH),

126.61 (1 transition, Car), 127.56, 127.76 (d, 3JCP ¼ 13.9 Hz, CHar), 128.01, 128.03, 128.06, 128.21, 128.40, 128.45, 128.52, 133.12 (d, 2 JCP ¼ 9.6 Hz, CHar), 133.46 (d, 4JCP ¼ 2.7 Hz, CHar), 137.22 (s, CarBn), 137.89 (s, CarBn), 138.18 (s, CarBn). HRMS (m/z) calcd for C32H33IO5P: 655.1110. Found: 655.1105.

5.1.5. (2Sp,3R,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3iodo-2-phenyl-2-oxo-2l5-[1,2]oxaphos phinane 4a Under nitrogen, potassium iodine (0.077 g, 0.46 mmol) was added to a solution of 6 (0.078 g, 0.12 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 80
C for 1.5 h, and solvent was evaporated under vacuum. A normal phase chromatography on silica gel using a mixture of n-heptane/ethyl acetate (60:40) as eluent, afforded compound 4a as a white solid (0.053 g, 70%), m.p. 127e128
C. 31P NMR (CDCl3): d 34.52 (s). 1H NMR (CDCl3): d 3.69 (dd, 2JHH ¼ 11.2 Hz, 3JHH ¼ 2.0 Hz, 1H, OCH2), 3.81 (dd, 3 JHH ¼ 11.5 Hz, 2JHP ¼ 4.2 Hz, 1H, 1CH), 3.91 (dt, 2JHH ¼ 11.2 Hz, 3 JHH ¼ 2.6 Hz, 4JHP ¼ 2.6 Hz, 1H, OCH2), 3.98 (dd, 3JHH ¼ 9.9 Hz, 3 JHH ¼ 9.2 Hz, 1H, 3CH), 4.20 (ddd, 3JHH ¼ 11.5 Hz, 3JHH ¼ 9.2 Hz, 3 JHP ¼ 2.3 Hz, 1H, 2CH), 4.46 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.55 (d, 2 JHH ¼ 12.0 Hz, 1H, OCH2), 4.56 (m, 3JHH ¼ 9.9 Hz, 3JHP ¼ 4.1 Hz, 3 JHH ¼ 2.6 Hz, 3JHH ¼ 2.0 Hz, 1H, 4CH), 4.62 (d, 2JHH ¼ 10.9 Hz, 1H, OCH2), 4.83 (d, 2JHH ¼ 10.9 Hz, 1H, OCH2), 4.85 (d, 2JHH ¼ 10.1 Hz, 1H, OCH2), 4.94 (d, 2JHH ¼ 10.1 Hz, 1H, OCH2), 7.13e7.15 (m, 2H, CHar), 7.19e7.33 (m, 13H, CHar), 7.42e7.47 (m, 2H, CHar), 7.56e7.59 (m, 1H, CHar), 7.81e7.86 (m, 2H, CHar). 13C NMR (CDCl3): d 20.63 (d, 1 JCP ¼ 81.2 Hz, 1CH), 68.40 (d, 3JCP ¼ 9.3 Hz, BnOCH2), 73.47 (s, PhCH2), 75.47 (s, PhCH2), 75.53 (d, 2JCP ¼ 5.5 Hz, 4CH), 76.28 (s, PhCH2), 79.30 (s, 3CH), 82.22 (d, 2JCP ¼ 1.2 Hz, 2CH), 126.61 (d, 1 JCP ¼ 146.9 Hz, Car), 127.60, 127.61, 127.79, 127.94, 128.04, 128.09, 128.45, 128.53, 128.73 (d, 3JCP ¼ 13.9 Hz, CHar), 132.36 (d, 2 JCP ¼ 10.1 Hz, CHar), 133.64 (d, 4JCP ¼ 2.9 Hz, CHar), 137.48 (s, CarBn), 137.64 (s, CarBn), 137.84 (s, CarBn). HRMS (m/z) calcd for C32H33IO5P: 655.1110. Found: 655.1116.

5.1.6. (2Sp,3S,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3bromo-2-phenyl-2-oxo-2l5-[1,2]-oxaphosphinane 3b Under nitrogen, tetraethylammonium bromide (0.032 g, 0.15 mmol) was added to a solution of 5 (0.104 g, 0.15 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 80
C for 3 h, and solvent was evaporated under vacuum. A normal phase chromatography on silica gel using a mixture of n-heptane/ethyl acetate (60:40) as eluent afforded compound 3b as a colorless oil (0.049 g, 52%). 31P NMR (CDCl3): d 34.46 (s). 1H NMR (CDCl3): d 3.72 (dd, 2 JHH ¼ 11.4 Hz, 3JHH ¼ 1.9 Hz, 1H, OCH2), 3.91 (dt, 2JHH ¼ 11.4 Hz, 3 JHH ¼ 3.0 Hz, 4JHP ¼ 3.0 Hz, 1H, OCH2), 4.25 (dd, 3JHH ¼ 9.9 Hz, 3 JHH ¼ 9.0 Hz, 1H, 3CH), 4.37 (ddd, 3JHH ¼ 9.0 Hz, 3JHH ¼ 3.3 Hz, 3 JHP ¼ 0.9 Hz, 1H, 2CH), 4.41 (dd, 2JHP ¼ 6.9 Hz, 3JHH ¼ 3.3 Hz, 1H, 1 CH), 4.52 (d, 2JHH ¼ 12.1 Hz, 1H, OCH2), 4.53 (d, 2JHH ¼ 10.7 Hz, 1H, OCH2), 4.56 (d, 2JHH ¼ 11.3 Hz, 1H, OCH2), 4.56 (dddd, 3JHH ¼ 9.9 Hz, 3 JHP ¼ 3.2 Hz, 3JHH ¼ 3.0 Hz, 3JHH ¼ 1.9 Hz, 1H, 4CH), 4.65 (d, 2 JHH ¼ 12.1 Hz, 1H, OCH2), 4.66 (d, 2JHH ¼ 11.3 Hz, 1H, OCH2), 4.85 (d, 2 JHH ¼ 10.7 Hz, 1H, OCH2), 7.11e7.14 (m, 2H, CHar), 7.21e7.33 (m, 13H, CHar), 7.41e7.45 (m, 2H, CHar), 7.53e7.58 (m, 1H, CHar), 7.84e7.90 (m, 2H, CHar). 13C NMR (CDCl3): d 43.40 (d, 1JCP ¼ 83.9 Hz, 1 CH), 68.88 (d, 3JCP ¼ 9.2 Hz, BnOCH2), 71.62 (s, PhCH2), 73.37 (s, PhCH2), 74.54 (d, 3JCP ¼ 1.7 Hz, 3CH), 75.63 (s, PhCH2), 76.23 (d, 2 JCP ¼ 5.8 Hz, 4CH), 78.71 (d, 2JCP ¼ 1.7 Hz, 2CH), 126.24 (d,1JCP ¼ 149.8 Hz, Car), 127.57, 127.71, 127.83, 127.97, 127.99, 128.03, 128.22 (d, 3JCP ¼ 14.0 Hz, CHar), 128.41, 128.45, 128.55, 133.20 (d, 2 JCP ¼ 9.7 Hz, CHar), 133.57 (d, 4JCP ¼ 2.9 Hz, CHar), 137.23 (s, CarBn), 137.91 (s, 1C, CarBn), 138.11 (s, CarBn). HRMS (m/z) calcd for C32H33BrO5P: 607.1249. Found: 607.1255.

R. Babouri et al. / European Journal of Medicinal Chemistry 104 (2015) 33e41

5.1.7. (2Sp,3R,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3bromo-2-phenyl-2-oxo-2l5-[1,2]-oxaphosphinane 4b Under nitrogen, tetraethylammonium bromide (0.031 g, 0.147 mmol) was added to a solution of 6 (0.100 g, 0.147 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 80
C for 2 h, and solvent was evaporated under vacuum. A normal phase chromatography on silica gel using a mixture of n-heptane/ethyl acetate (60:40) as eluent afforded compound 4b as a white solid (0.060 g, 67%), m.p. 138.5e140.6
C. 31P NMR (CDCl3): d 32.42 (s). 1H NMR (CDCl3): d 3.69 (dd, 2JHH ¼ 11.2 Hz, 3JHH ¼ 2.0 Hz, 1H, OCH2), 3.84 (dd, 3JHH ¼ 11.2 Hz, 2JHP ¼ 3.1 Hz, 1H, 1CH), 3.90 (dt, 2JHH ¼ 11.2 Hz, 3 JHH ¼ 2.6 Hz, 4JHP ¼ 2.6 Hz, 1H, OCH2), 3.97 (dd, 3JHH ¼ 10.0 Hz, 3 JHH ¼ 9.2 Hz, 1H, 3CH), 4.23 (ddd, 3JHH ¼ 11.2 Hz, 3JHH ¼ 9.2 Hz, 3 JHP ¼ 2.3 Hz, 1H, 2CH), 4.45 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.54 (d, 2 JHH ¼ 12.0 Hz, 1H, OCH2), 4.56 (m, 3JHH ¼ 10.0 Hz, 3JHH ¼ 2.6 Hz, 3 JHH ¼ 2.0 Hz, 1H, 4CH), 4.61 (d, 2JHH ¼ 10.9 Hz, 1H, OCH2), 4.83 (d, 2 JHH ¼ 10.2 Hz, 1H, OCH2), 4.83 (d, 2JHH ¼ 10.9 Hz, 1H, OCH2), 4.93 (d, 2 JHH ¼ 10.2 Hz, 1H, OCH2), 7.12e7.14 (m, 2H, CHar), 7.20e7.30 (m, 13H, CHar), 7.42e7.47 (m, 2H, CHar), 7.54e7.59 (m, 1H, CHar), 7.80e7.85 (m, 2H, CHar). 13C NMR (CDCl3): d 44.29 (d, 1JCP ¼ 85.4 Hz, 1 CH), 68.39 (d, 3JCP ¼ 9.3 Hz, BnOCH2), 73.50 (s, PhCH2) 75.34 (d, 2 JCP ¼ 5.4 Hz, 4CH), 75.60 (s, PhCH2), 76.79 (s, PhCH2), 79.32 (s, 3CH), 82.54 (d, 2JCP ¼ 2.1 Hz, 2CH), 126.36 (d, 1JCP ¼ 146.3 Hz, Car), 127.63, 127.67, 127.82, 127.94, 128.02, 128.07, 128.46, 128.47, 128.51, 128.78 (d, 3JCP ¼ 13.9 Hz, CHar), 132.22 (d, 2JCP ¼ 10.2 Hz, CHar), 133.73 (d, 4 JCP ¼ 2.9 Hz, CHar), 137.62 (s, CarBn), 137.67 (s, CarBn), 137.81 (s, CarBn). HRMS (m/z) calcd for C32H33BrO5P: 607.1249. Found: 607.1246. 5.1.8. (2Sp,3S,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3chloro-2-phenyl-2-oxo-2l5-[1,2]-oxaphosphinane 3c Under nitrogen, tetraethylammonium chloride (0.025 g, 0.15 mmol) was added to a solution of 5 (0.104 g, 0.15 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 80
C for 1 h, and solvent was evaporated under vacuum. A normal phase chromatography on silica gel using a mixture of n-heptane/ethyl acetate (60:40) as eluent afforded compound 3c as a colorless oil (0.054 g, 62%). 31P NMR (CDCl3): d 34.76 (s). 1H NMR (CDCl3): d 3.83 (dd, 2 JHH ¼ 11.3 Hz, 3JHH ¼ 2.0 Hz, 1H, OCH2), 4.01 (ddd, 2JHH ¼ 11.3 Hz, 3 JHH ¼ 3.3 Hz, 4JHP ¼ 2.7 Hz, 1H, OCH2), 4.37 (dd, 3JHH ¼ 10.0 Hz, 3 JHH ¼ 9.3 Hz, 1H, 3CH), 4.52 (dd, 2JHP ¼ 6.9 Hz, 3JHH ¼ 3.2 Hz, 1H, 1 CH), 4.62 (d, 2JHH ¼ 12.6 Hz, 1H, OCH2), 4.64 (d, 2JHH ¼ 10.7 Hz, 1H, OCH2), 4.64 (ddd, 3JHH ¼ 9.3 Hz, 3JHH ¼ 3.2 Hz, 3JHP ¼ 0.7 Hz, 1H, 2 CH), 4.65 (m, 1H, 4CH), 4.70 (d, 2JHH ¼ 11.4 Hz, 1H, OCH2), 4.75 (d, 2 JHH ¼ 12.6 Hz, 1H, OCH2), 4.78 (d, 2JHH ¼ 11.4 Hz, 1H, OCH2), 4.97 (d, 2 JHH ¼ 10.7 Hz, 1H, OCH2), 7.23e7.25 (m, 2H, CHar), 7.30e7.43 (m, 13H, CHar), 7.52e7.57 (m, 2H, CHar), 7.65e7.69 (m, 1H, CHar), 7.95e8.00 (m, 2H, CHar). 13C NMR (CDCl3): d 52.37 (d, 1JCP ¼ 87.2 Hz, 1 CH), 68.85 (d, 3JCP ¼ 9.1 Hz, BnOCH2), 71.87 (s, PhCH2), 73.39 (s, PhCH2), 73.74 (d, 3JCP ¼ 1.8 Hz, 3CH), 75.70 (s, PhCH2), 76.21 (d, 2 JCP ¼ 5.8 Hz, 4CH), 79.38 (d, 2JCP ¼ 2.0 Hz, 2CH), 125.66 (d, 1 JCP ¼ 148.4 Hz, Car), 127.59, 127.73, 127.84, 127.95, 127.98, 128.06, 128.32 (d, 3JCP ¼ 13.9 Hz, CHar), 128.42, 128.46, 128.57, 133.24 (d, 2 JCP ¼ 9.8 Hz, CHar), 133.65 (d, 4JCP ¼ 2.9 Hz, CHar), 137.27 (s, CarBn), 137.94 (s, CarBn), 137.09 (s, CarBn). HRMS (m/z) calcd for C32H33ClO5P: 563.1754. Found: 563.1755. 5.1.9. (2Sp,3R,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3chloro-2-phenyl-2-oxo-2l5-[1,2]-oxaphosphinane 4c Under nitrogen, tetraethylammonium chloride (0.025 g, 0.15 mmol) was added to a solution of 6 (0.088 g, 0.13 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 80
C for 3 h, and solvent was evaporated under vacuum. A normal phase chromatography on silica gel using a mixture of n-heptane/ethyl acetate (60:40) as eluent afforded compound 4c as a white solid (0.026 g,

39

35.5%), m.p. 127.1e129.3
C. 31P NMR (CDCl3): d 32.37 (s). 1H NMR (CDCl3): d 3.79 (dd, 2JHH ¼ 11.2 Hz, 3JHH ¼ 2.0 Hz, 1H, OCH2), 3.98e4.03 (m, 1H, OCH2), 4.00 (dd, 1H, 3JHH ¼ 10.9 Hz, 2JHP ¼ 3.1 Hz, 1 CH), 4.06 (dd, 3JHH ¼ 10.2 Hz, 3JHH ¼ 9.1 Hz, 1H, 3CH), 4.30 (ddd, 3 JHH ¼ 10.9 Hz, 3JHH ¼ 9.1 Hz, 3JHP ¼ 2.4 Hz, 1H, 2CH), 4.56 (d, 2 JHH ¼ 12.0 Hz, 1H, OCH2), 4.65 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.65 (m, 1H, 4CH), 4.70 (d, 2JHH ¼ 10.9 Hz, 1H, OCH2), 4.92 (d, 2 JHH ¼ 10.3 Hz, 1H, OCH2), 4.94 (d, 2JHH ¼ 10.9 Hz, 1H, OCH2), 5.02 (d, 2 JHH ¼ 10.3 Hz, 1H, OCH2), 7.22e7.24 (m, 2H, CHar), 7.30e7.39 (m, 13H, CHar), 7.54e7.58 (m, 2H, CHar), 7.66e7.70 (m, 1H, CHar), 7.90e7.95 (m, 2H, CHar). 13C NMR (CDCl3): d 55.19 (d, 1JCP ¼ 88.9 Hz, 1 CH), 68.36 (d, 3JCP ¼ 9.3 Hz, BnOCH2), 73.49 (s, PhCH2), 75.26 (d, 2 JCP ¼ 5.4 Hz, 4CH), 75.64 (s, PhCH2), 77.01 (s, PhCH2), 78.89 (s, 3CH), 83.05 (d, 2JCP ¼ 2.4 Hz, 2CH), 126.25 (d, 1JCP ¼ 144.9 Hz, Car), 127.65, 127.70, 127.83, 127.94, 128.01, 128.08, 128.46, 128.47, 128.51, 128.83 (d, 3JCP ¼ 13.9 Hz, CHar), 132.14 (d, 2JCP ¼ 10.2 Hz, CHar), 133.78 (d, 4 JCP ¼ 2.8 Hz, CHar), 137.69 (s, CarBn), 137.70 (s, CarBn), 137.79 (s, CarBn). HRMS (m/z) calcd for C32H33ClO5P: 563.1754. Found: 563.1755. 5.1.10. (2Sp,3S,4S,5S,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-3fluoro-2-phenyl-2-oxo-2l5-[1,2]-oxaphosphinane 4d Under nitrogen, compound 2 (0.82 g, 1.5 mmol) in DCM (6 mL), was added slowly at 78
C to a solution of DAST (1.0 mL, 7.5 mmol) in DCM (6 mL). The reaction mixture was stirred for 1 h at 78
C, and overnight at 40
C. At 0
C, a saturated aqueous solution of sodium hydrogenocarbonate was added in the reaction mixture. The aqueous phase was extracted with dichloromethane, and the organic phase was dried with MgSO4, and evaporated. After two successive column chromatography using a mixture of n-heptane/ ethyl acetate (60:40) as eluent, compound 4d was obtained as a white solid (0.022 g, 2.7%), m.p. 93.1e94.5
C. 31P NMR (CDCl3): d 29.46 (d, 2JPF ¼ 66.0 Hz). 19F NMR (CDCl3): d 212.54 (d, 2 JFP ¼ 66.0 Hz). 1H NMR (CDCl3): d 3.69 (dd, 2JHH ¼ 11.2 Hz, 3 JHH ¼ 2.1 Hz, 1H, OCH2), 3.89 (ddd, 2JHH ¼ 11.2 Hz, 4JHP ¼ 2.5 Hz, 3 JHH ¼ 2.4 Hz, 1H, OCH2), 3.94 (dd, 3JHH ¼ 9.9 Hz, 3JHH ¼ 9.7 Hz, 1H, 3 CH), 4.35 (dddd, 3JHF ¼ 11.8 Hz, 3JHH ¼ 9.7 Hz, 3JHH ¼ 9.7 Hz, 3 JHP ¼ 2.2 Hz, 1H, 2CH), 4.45 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.52 (dddd, 3JHH ¼ 9.9 Hz, 3JHP ¼ 4.4 Hz, 3JHH ¼ 2.4 Hz, 3JHH ¼ 2.1 Hz, 1H, 4 CH), 4.54 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.57 (d, 2JHH ¼ 10.8 Hz, 1H, OCH2), 4.75 (d, 2JHH ¼ 10.8 Hz, 1H, OCH2), 4.78 (ddd, 2JHF ¼ 49.4 Hz, 3 JHH ¼ 9.7 Hz, 2JHP ¼ 3.8 Hz, 1H, 1CH), 4.85 (d, 2JHH ¼ 10.8 Hz, 1H, OCH2), 4.85 (d, 2JHH ¼ 10.8 Hz, 1H, OCH2), 7.11e7.13 (m, 2H, CHar), 7.19e7.27 (m, 13H, CHar), 7.44e7.49 (m, 2H, CHar), 7.56e7.60 (m, 1H, CHar), 7.78e7.83 (m, 2H, CHar). 13C NMR (CDCl3): d 68.31 (d, 3 JCP ¼ 9.2 Hz, BnOCH2), 73.48 (s, PhCH2), 74.94 (d, 2JCP ¼ 5.3 Hz, 4 CH), 75.73 (s, PhCH2), 76.04 (d, 4JCP ¼ 2.5 Hz, PhCH2), 77.10 (d, 3 JCF ¼ 7.1 Hz, 3CH), 82.15 (dd, 2JCF ¼ 15.8 Hz, 2JCP ¼ 4.8 Hz, 2CH), 91.35 (dd, 1JCF ¼ 194.1 Hz, 1JCP ¼ 101.7 Hz, 1CH), 126.70 (d,1JCP ¼ 140.1 Hz, Car), 127.65, 127.78, 127.82, 127.88, 127.91, 128.11, 128.42, 128.44, 128.45, 128.91 (d, 3JCP ¼ 13.7 Hz, CHar), 131.88 (d, 2 JCP ¼ 10.5 Hz, CHar), 133.75 (d, 4JCP ¼ 2.8 Hz, CHar), 137.76 (s, CarBn), 137.77 (s, CarBn), 137.86 (s, CarBn). HRMS (m/z) calcd for C32H33FO5P: 547.2050. Found: 547.2039. 5.1.11. (3,4,5-tri-O-benzyl-a-D-arabinofuranosyl) (phenyl) phosphinic acid 8 Under nitrogen, potassium fluoride (0.035 g, 0.6 mmol) was added to a solution of 5 (0.104 g, 0.15 mmol) in acetonitrile (3 mL). The reaction mixture was stirred at 80
C for 5 h, and solvent was evaporated. An aqueous solution of potassium hydroxide (9 mL, 1.0 M) was added to the residue and the mixture was extracted with DCM (3  3 mL). The organic layer was dried over sodium sulfate, filtered and evaporated under vacuum to give compound 8 as a pale yellow solid (0.070 g, 84%), m.p. 74.5e77.0
C. 31P NMR (CDCl3):

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d 21.37 (s). 1H NMR (CDCl3): d 3.13 (dd, 2JHH ¼ 10.0 Hz, 3JHH ¼ 5.4 Hz,

1H, OCH2), 3.21 (dd, 2JHH ¼ 10.0 Hz, 3JHH ¼ 7.4 Hz, 1H, OCH2), 3.61 (dd, 3JHH ¼ 3.9 Hz, 3JHH ¼ 2.5 Hz, 1H, 3CH), 3.82 (d, 2JHH ¼ 11.5 Hz, 1H, OCH2), 3.87 (d, 2JHH ¼ 11.5 Hz, 1H, OCH2), 3.90 (dd, 3 JHH ¼ 5.6 Hz, 2JHP ¼ 1.7 Hz, 1H, 1CH), 4.02 (ddd, 3JHP ¼ 11.5 Hz, 3 JHH ¼ 5.6 Hz, 3JHH ¼ 2.5 Hz, 1H, 2CH), 4.10 (m, 1H, 4CH), 4.13 (d, 2 JHH ¼ 12.4 Hz, 1H, OCH2), 4.19 (d, 2JHH ¼ 12.5 Hz, 1H, OCH2), 4.22 (d, 2 JHH ¼ 12.5 Hz, 1H, OCH2), 4.24 (d, 2JHH ¼ 12.4 Hz, 1H, OCH2), 4.51 (s, 1H, OH), 6.78e6.80 (m, 2H, CHar), 6.99e7.16 (m, 16H, CHar), 7.75e7.79 (m, 2H, CHar). 13C NMR (CDCl3): d 68.52 (s, BnOCH2), 71.16 (s, PhCH2), 71.63 (s, PhCH2), 72.90 (s, PhCH2), 81.42 (d, 3JCP ¼ 5.0 Hz, 4 CH), 82.65 (d, 1JCP ¼ 110.3 Hz, 1CH), 85.06 (d, 3JCP ¼ 5.1 Hz, 3CH), 85.67 (d, 3JCP ¼ 5.0 Hz, 2CH), 127.29, 127.49, 127.60, 127.62, 127.77, 127.95, 128.06, 128.07, 128.30, 128.33, 130.19, 132.50 (d, JCP ¼ 8.5 Hz), 137.44 (d, 1JCP ¼ 122.5 Hz, Car), 137.63 (s, CarBn), 137.98 (s, CarBn), 138.13 (s, CarBn). HRMS (m/z) calcd for C32H34O6P: 545.2093. Found: 545.2093. 5.1.12. (2Rp,5R,6R)-4,5-bis-benzyloxy-6-benzyloxymethyl-2phenyl-2-oxo-2l5-[1,2]oxa phosphine-3-ene 9 Under nitrogen, cesium fluoride (0.245 g, 1.61 mmol) was added to a solution of 3a (0.264 g, 0.40 mmol) in DMSO (8 mL). The reaction mixture was stirred at 95
C for 75 min. The solvent was evaporated and dichloromethane was added. The organic layer was washed with water, dried over sodium sulfate and concentrated under vacuum. The residue was purified by column chromatography on silica gel with n-heptane/ethyl acetate (30/70) to give product 9 (0.170 g, 80%). m.p. 95.8e96.6
C. 31P NMR (CDCl3): d 27.96 (s). 1H NMR (CDCl3): d 3.72 (dd, 2JHH ¼ 11.2 Hz, 3JHH ¼ 2.1 Hz, 1H, OCH2), 3.87 (dt, 2JHH ¼ 11.2 Hz, 3JHH ¼ 2.5 Hz, 4JHP ¼ 2.5 Hz, 1H, OCH2), 4.41 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.47 (d, 2JHH ¼ 10.4 Hz, 1H, OCH2), 4.54 (d, 2JHH ¼ 12.0 Hz, 1H, OCH2), 4.58 (ddd, 3JHH ¼ 9.6 Hz, 4 JHP ¼ 2.0 Hz, 4JHH ¼ 1.2 Hz, 1H, 3CH), 4.66 (dddd, 3JHH ¼ 9.6 Hz, 3 JHP ¼ 4.9 Hz, 3JHH ¼ 2.5 Hz, 3JHH ¼ 2.1 Hz, 1H, 4CH), 4.74 (d, 2 JHH ¼ 11.3 Hz, 1H, OCH2), 4.79 (d, 2JHH ¼ 10.4 Hz, 1H, OCH2), 4.86 (d, 2 JHH ¼ 11.3 Hz, 1H, OCH2), 4.90 (dd, 2JHP ¼ 12.2 Hz, 4JHH ¼ 1.2 Hz, 1H, 1 CH), 7.05e7.09 (m, 2H, CHar), 7.14e7.30 (m, 13H, CHar), 7.35e7.39 (m, 2H, CHar), 7.45e7.49 (m, 1H, CHar), 7.72e7.77 (m, 2H, CHar). 13C NMR (CDCl3): d 68.70 (d, 3JCP ¼ 8.6 Hz, BnOCH2), 70.43 (d, 4 JCP ¼ 1.6 Hz, PhCH2), 72.25 (d, 3JCP ¼ 4.1 Hz, 3CH), 73.56 (s, PhCH2), 74.64 (d, 2JCP ¼ 6.4 Hz, 4CH), 75.44 (s, PhCH2), 87.58 (d, 1 JCP ¼ 133.5 Hz, 1CH), 127.74, 127.78, 127.93, 127.99, 128.33, 128.36, 128.40, 128.53 (d, 3JCP ¼ 6.5 Hz, CHar), 128.72, 131.84 (d, 2 JCP ¼ 10.7 Hz, CHar), 131.86 (d,1JCP ¼ 152.3 Hz, Car), 132.39 (d, 4 JCP ¼ 2.8 Hz, CHar), 134.95 (s, CarBn), 137.42 (s, CarBn), 137.97 (s, CarBn), 169.18 (d, 2JCP ¼ 11.8 Hz, 2C). HRMS (m/z) calcd for C32H32O5P: 527.1987. Found: 527.1986. 5.2. X-ray experiments Using Olex2 [28], the crystals structures were solved with the ShelXT [29] structure solution program using Direct Methods and refined with the ShelXL [26] refinement package using Least Squares minimization. Refinement of the absolute structure Flack parameters [27], while the Hooft analysis [28] gave absolutes parameters 0.001(17) for 4b and 0.020(18) for 9 which confidently confirms that the refined coordinates represent the true enantiomorph. Crystal data for 4b: Formula ¼ 0.5(C32H32BrO5P), C32H33BrO5P T ¼ 175.00(14) K, Mr ¼ 607.45 g mol1, crystal size ¼ 0.30  0.40  0.35 mm3, Monoclinic, space group P21, a ¼ 5.61523(10) Å, b ¼ 23.1126(3) Å, c ¼ 22.1330(3) Å, a ¼ 90
, b ¼ 91.4033(15)
, g ¼ 90
, V ¼ 2871.62(8) Å3, Z ¼ 4, rc ¼ 1.405 g/cm3, m(CuKa) ¼ 2.800 mm1, qmax ¼ 67.613, 24,229 reflections measured, 10,273 unique, 9002 with I > 2s (I), refined parameters ¼ 703, R1

(I > 2s (I)) ¼ 0.0468, wR2 (I > 2s (I)) ¼ 0.1449, R1 (all data) ¼ 0.0574, wR2 (all data) ¼ 0.1498, S (all data) ¼ 0.916, Dr(min/max) ¼ 0.45/ 0.48 eA3. Crystal data for 9: Formula ¼ C32H31PO5, T ¼ 175.00(10) K, Mr ¼ 526.54 g mol1, crystal size ¼ 0.40  0.40  0.40 mm3, Orthorhombic, space group P212121, a ¼ 10.4652(3) Å, b ¼ 15.0297(4) Å, c ¼ 18.0287(4) Å, a ¼ 90
, b ¼ 90
, g ¼ 90
, V ¼ 2835.72(12) Å3, Z ¼ 4, rc ¼ 1.233 g/cm3, m(CuKa) ¼ 1.170 mm1, qmax ¼ 61.953, 8167 reflections measured, 4362 unique, 4076 with I > 2s (I), refined parameters ¼ 343, R1 (I > 2s (I)) ¼ 0.0468, wR2 (I > 2s (I)) ¼ 0.1109, R1 (all data) ¼ 0.0498, wR2 (all data) ¼ 0.1134, S (all data) ¼ 1.027, Dr(min/max) ¼ 0.22/0.33 eA3. Data of 4b (CCDC 1051163) and 9 (CCDC 1061496) can be obtained free of charge from the Cambridge Crystallographic Data Centre at www. ccdc.cam.ac.uk/data_request/cif 5.3. Pharmacology 5.3.1. Cell lines MDA-MB-231, MDA-MB-435, B16F10, Caco2, HuH7 and DU145 cells were obtained from the American Type Culture Collection. These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U mL1 streptomycin. 5.3.2. Cell viability experiment. The compounds are solubilized in 100% DMSO and tested at 1% DMSO for the first concentration Cell viability was evaluated using the MTT microculture tetrazolium assay [30]. Cells were seeded at a density of 5  104 cells/ well in 96-well flat-bottom plates (Falcon, Strasbourg, France) and incubated in complete culture medium for 24 h. Then, medium was removed and replaced by 2% FCS (Fetal Calf Serum)-medium containing increasing concentrations of a-halogenated oxaphosphinanes. After 72 h incubation, cells were washed with phosphate buffered saline h72 h (PBS, Life Technologies) and incubated with 0.1 mL of MTT (2 mg/mL, SigmaeAldrich) for additional 4 h at 37
C. The insoluble product was then dissolved by addition of DMSO (SigmaeAldrich). Optical density was measured at 570 nm using a Labsystems Multiskan MS microplate reader. Experiments were performed in triplicate. 5.3.3. Measurements of a-halogenated oxaphosphinanes effects on cancer cell line cultures (B16, MDAMB435), migration and invasion For migration inserts of Boyden chambers were coated overnight with 20 mg/mL of either laminin), fibronectin or vitronectin (human plasma, BD Biosciences) washed in PBS and blocked with 0.5% BSA for 1 h. For invasion 100 mL of matrigel (BD Matrigel TM Matrix, mouse EHS tumor, LDEV; BD Biosciences) diluted 33 times in PBS was added in the insert. Cells (50  105) resuspended in DMEM supplemented with 0.1% BSA were plated into the upper compartment and incubated for 24 h with and without treatment. The upper side of the filter was wiped off to remove non-migrating cells, and cells attached to the lower side were fixed for 10 min with methanol, stained with crystal violet and fully counted using a Zeiss axiophot microscope (x200). The % of migration inhibition (% Inh) was calculated as follows: % Inh ¼ 100  (number of cell migrating without treatment/number of cell migrating under treatment) x 100. Experiments were performed in triplicate. 5.3.4. Statistical analyses The presented experiments were carried out at least in triplicate. Depending on the number of independent variables Student's or ANOVA statistical tests was applied, using serf software (bram.org/serf/CellsAndMaps.php). EC50 values and Ki were calculated using the Hill equation of the doseelog response curves.

R. Babouri et al. / European Journal of Medicinal Chemistry 104 (2015) 33e41

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