Design, synthesis, antiviral and cytostatic evaluation of novel isoxazolidine nucleotide analogues with a carbamoyl linker

DOI: 10.1016/j.bmc.2013.01.007

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Bioorganic and Medicinal Chemistry p. 1097 - 1108 (2013)

Update date:2022-08-05

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Authors:

Kokosza, Kamil

Balzarini, Jan

Piotrowska, Dorota G.

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Article abstract of DOI:10.1016/j.bmc.2013.01.007

5-Arylcarbamoyl-2-methylisoxazolidin-3-yl-3-phosphonates have been synthesised from N-methyl-C-diethoxyphosphorylnitrone and N-arylacrylamides in good yields. cis- and trans-isoxazolidine phosphonates obtained herein were evaluated for activity against a broad range of DNA and RNA viruses. None of the compounds were endowed with antiviral activity at subtoxic concentrations. Isoxazolidines having phenyl substituted with halogen (Ar = 2-F-C ₆H₄; 3-Br-C₆H₄; and 4-Br-C ₆H₄) have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 100-170 μM range.

Full text of DOI:10.1016/j.bmc.2013.01.007

Bioorganic & Medicinal Chemistry 21 (2013) 1097–1108

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Design, synthesis, antiviral and cytostatic evaluation of novel

isoxazolidine nucleotide analogues with a carbamoyl linker

Kamil Kokosza ^a, Jan Balzarini ^b, Dorota G. Piotrowska ^a,

⇑

Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of Łódz, Muszynskiego 1, 90-151 Łódz, Poland

^bRega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

a r t i c l e i n f o

a b s t r a c t

Article history:

5-Arylcarbamoyl-2-methylisoxazolidin-3-yl-3-phosphonates have been synthesised from N-methyl-C-

diethoxyphosphorylnitrone and N-arylacrylamides in good yields. cis- and trans-isoxazolidine phospho-

nates obtained herein were evaluated for activity against a broad range of DNA and RNA viruses. None of

the compounds were endowed with antiviral activity at subtoxic concentrations. Isoxazolidines having

phenyl substituted with halogen (Ar = 2-F-C₆H₄; 3-Br-C₆H₄; and 4-Br-C₆H₄) have been found to inhibit

Received 15 November 2012

Revised 27 December 2012

Accepted 2 January 2013

Available online 16 January 2013

proliferation of L1210, CEM as well as HeLa cells with IC₅₀in the 100–170 lM range.

Keywords:

1,3-Dipolar cycloaddition

Isoxazolidines

Phosphonates

Antiviral

Cytostatic

1. Introduction

logues with anticancer or antiviral activity (Fig. 2). Nucleoside ana-

logue 1 [(ꢀ)-AdFU] having a ﬂuorouracil residue attached to the

isoxazolidine ring induces apoptosis on lymphoid and monocytoid

cells and exhibits low level of cytotoxicity.¹⁹Phosphonylated isox-

azolidines 2 inhibit reverse transcriptase of HTLV-1 with activity

comparable to that of AZT and protect human peripheral blood

mononuclear cells against HTLV-1 transmission.²⁰Furthermore,

compounds of general formula 3 show high cytotoxic activity

against several cancer cell lines comparable to the known antican-

cer drugs, namely, Mitomycin C, Paclitaxel and 5-Fluorouracil, used

as positive controls.²¹Synthesis and promising antiproliferative

properties of isoxazolidines 4 have been reported by Bortolini

et al.²²

Nucleoside analogues are of great interest in medicinal chemis-

try due to their broad spectrum of biological activities. Extensive

search for modiﬁed nucleosides has led to the discovery of many

potent drugs for treatment of various viral infections¹and diverse

types of neoplasms (Fig. 1).²The adverse effects of the available

therapies, low selectivity and the observed drug resistance have

become a driving force in a search for new analogues with im-

proved pharmacokinetic and pharmacodynamic properties.

Numerous modiﬁcations of naturally occurring nucleosides have

provided analogues with altered sugar and/or nucleobase subunits.

Heterocycles containing one or more heteroatoms and carbocycles

of various sizes as well as straight or branched chains also with

heteroatoms have been applied as an alternative to the furanose

ring.^3,4So far less attention has been paid to the synthesis of ana-

logues having modiﬁed nucleobase residues in comparison to su-

gar-modiﬁed analogues due to ensuring base pairing, but

recently it has been proven that other aromatic rings are able to

base-pair as well.⁵A long list of nucleoside analogues continues

to expand by incorporation of several linkers such as a 1,2,3-tria-

zole group,^6–9a carbamoyl^10–17or an ureidyl function¹⁸among

others.

Recently, a series of 3,5-disubstituted isoxazolidine nucleosides

^23,24as well as their further modiﬁcations 6 with an 1,2,3-triazole

spacer²⁵have been obtained and their antiviral and cytotoxic prop-

erties were evaluated. Isoxazolidines 5 substituted with 1- and 2-

naphthyl at C5 were found cytotoxic against HeLa and K562 cell

lines (R = 1-naphthyl and 2-naphthyl; IC₅₀0.05 and 0.09 mM,

respectively),²³while cis-conﬁgured 5-ﬂuorouracil and 5-thymine

derivatives 5 completely inhibited the reverse transcriptase activ-

ity of Avian Moloney Virus (AMV) and Human Immunodeﬁciency

Virus (HIV).²⁴Although (1,2,3-triazolyl)isoxazolidinephosponates

6 did not show antiviral activity at subtoxic concentrations, deriv-

atives of 6 having the unsubstituted and ﬂuorosubstituted phenyl

at C4 in the 1,2,3-triazole ring proved slightly cytostatic.²⁵Encour-

aged by these results a new series of 5-substituted (3-diethoxy-

The isoxazolidine framework has successfully been applied as a

surrogate for a furanose ring in the synthesis of nucleoside ana-

⇑

Corresponding author. Tel.: +48 42 677 9235; fax: +48 42 678 8398.

E-mail address: dorota.piotrowska@umed.lodz.pl (D.G. Piotrowska).

phosphoryl)isoxazolidines

was designed (Scheme 1).

http://dx.doi.org/10.1016/j.bmc.2013.01.007

1098

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

NH₂

Cladribine

Citarabine

Gemcitabine

Fludarabine

NH₂

H₂N

Acyclovir

Didanosine

Stavudine

Lamivudine

Figure 1. Examples of clinically applied nucleoside analogues with anticancer or antiviral activity.

Me(O)C

R = Ph, Me, H

Ar = Ph, p-OMe-C₆H₄

p-NO₂-C₆H₄

(EtO)₂(O)P

B = Thy, Cyt,

Ade, 5-FU

p-Cl-C₆H₄

1 [(-)-AdFU]

_nR

(EtO)₂(O)P

R = substituted aryls,

R = Me, Ph, Bn, t-Bu

Ar = Ph, o,m,p-X-C6H4, furyl

B = Thy, Ura, Cyt, 5-F-Cyt,

Ade, Gua

R=nucleobase, aromatic,

heteroaromatic group

n=0,1

Thy, FUra

Figure 2. Examples of isoxazolidine nucleosides with cytotoxic and antiviral properties.

Compounds 7 could be regarded as nucleotide prodrugs due to

incorporation of a bioisosteric diethoxyphosphoryl function at C3

of the isoxazolidine ring which mimics nucleoside monophos-

phate.²⁶Moreover, it was anticipated that insertion of a carbamoyl

linker between the isoxazolidine moiety and careful selection of

mono-, di- or trisubstituted phenyl groups as nucleobase replacer

would improve their interaction within DNA/RNA strands by form-

ing stronger hydrogen bonds.

of acrylamides 9 was employed (Scheme 2). Most of substituted

acrylamides 9 used in this paper have already been described in

the literature. However, compounds 9ab, 9ad, 9an, 9az and 9ba

were prepared according to the standard procedure from commer-

cially available substituted anilines and acryloyl chloride in the

presence of triethylamine.²⁹Cycloadditions of nitrone 8 with

acrylamides 9aa–9ba were carried out in toluene at 70 °C and

afforded mixtures of diastereoisomeric (3-diethoxyphospho-

ryl)isoxazolidines trans-10aa–10ba and cis-11aa–11ba (Scheme 2,

Table 1). In all cases moderate to good trans/cis diastereoselectivi-

ties (de 50–80%) were observed. The crude mixtures of the respec-

tive cycloadducts were subjected to column chromatography and

in almost all cases (except for 10ae, 10ah and 10aq) pure major

trans-isomers 10 were separated in moderate to good yields (Ta-

ble 1). Isolation of pure minor cis-isomers 11, which are very cru-

2. Results and discussion

2.1. Chemistry

To synthesise the desired isoxazolidines 10 and 11 1,3-dipolar

cycloaddition of N-methyl C-phosphorylnitrone 8^27,28with a series

(EtO)₂(O)P

R = F, Br, Cl, Me, OMe, OEt, CN, NO₂, C(O)CH₃

Scheme 1. Retrosynthesis of isoxazolidynylphosphonates 7 with a carbamoyl linker.

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

1099

70 ºC, 24 h

toluene

(EtO)₂(O)P

Scheme 2. Synthesis of compounds 10 and 11.

Table 1

Isoxazolidines 10 and 11 obtained according to Scheme 2

Entry

Acrylamide 9

10:11 ratio

Yield (%)

2-F-C₆H₄

3-F-C₆H₄

4-F-C₆H₄

2,4-diF-C₆H₃

2-Br-C₆H₄

3-Br-C₆H₄

4-Br-C₆H₄

2-Cl-C₆H₄

3-Cl-C₆H₄

4-Cl-C₆H₄

2-NO₂-C₆H₄

3-NO₂-C₆H₄

4-NO₂-C₆H₄

3-CN-C₆H₄

4-CN-C₆H₄

2-CH₃C(O)-C₆H₄

3-CH₃C(O)-C₆H₄

4-CH₃C(O)-C₆H₄

3-CH₃-C₆H₄

88:12

78:22

80:20

85:15

90:10

77:23

78:22

86:14

75:25

86:14

83:17

80:20

78:22

80:20

81:19

83:17

78:22

77:23

76:24

79:21

84:16

80:20

81:19

80:20

10aa (43)^a+ 11aa (6)^a+ 10aa and 11aa (24)^b

10ab (55)^a+ 11ab (6)^a+ 10ab and 11ab (25)^b

10ac (48)^a+ 11ac (6)^a+ 10ac and 11ac (34)^b

10ad (56)^a+ 10ad and 11ad (38)^b

10ae and 11ae (94)^b

10af (37)^a+ 10af and 11af (51)^b

10ag (68)^a+ 10ag and 11ag (16)^b

10ah and 11ah (91)^b

10ai (48)^a+ 11ai (3)^a+ 10ai and 11ai (39)^b

10aj (60)^a+ 11aj (8)^a+ 10aj and 11aj (20)^b

10ak (18)^a+ 10ak and 11ak (69)^b

10al (26)^a+ 11al (8)^a+ 10al and 11al (57)^b

10am (60)^a+ 11am (14)^a

10an (17)^a+11an (6)^a+ 10an and 11an (67)^b

10ao (10)^a+ 10ao and 11ao (85)^b

10ap (22)^a+ 11ap (7)^a+ 10ap and 11ap (55)^b

10aq (53)^a+11aq (5)^a+ 10aq and 11aq (31)^b

10ar (51)^a+ 11ar (11)^a

10as (40)^a+ 11as (7)^a

4-CH₃-C₆H₄

10at (70)^a+ 11at (10)^a

3-CH₃O-C₆H₄

4-C₂H₅O-C₆H₄

3,4-diCH₃O-C₆H₃

3,5-diCH₃O-C₆H₃

3,4,5-triCH₃O-C₆H₂

4,5-diCH₃O-2-CN-C₆H₂

4,5-diCH₃O-2-CH₃C(O)-C₆H₂

10au (42)^a+ 10au and 11au (48)^b

10av (38)^a+ 11av (4)^a+ 10av and 11av (47)^b

10aw (17)^a+11aw (4)^a+ 10aw and 11aw (44)^b

10ax (25)^a+ 10ax and 11ax (70)^b

10ay (39)^a+11ay (9)^a+ 10ay and 11ay (40)^b

10az (26)^a+ 10az and 11az (40)^b

10ba (10)^a+ 10ba and 11ba (74)^b

Yield of pure isomer.

Yield of pure mixture of cis- and trans-isomers.

cial for biological evaluation, was not a trivial task. However, sev-

eral puriﬁcations of the enriched diastereoisomeric mixtures of the

respective isoxazolidines 10/11 on silica gel columns proved fruit-

ful for isoxazolidines 11aa, 11ab, 11ac, 11ai, 11aj, 11al, 11am,

11an, 11ap, 11aq, 11ar, 11as, 11at, 11av, 11aw and 11ba (Table 1)

making minute quantities of cis-11 available.

_H5= 5.0–5.7 Hz and J_H4b–H5= 8.5–9.2 Hz] extracted from the ¹H

and ¹³C NMR spectra of compounds 10al–10ao, 10aq, 10at and

10aw–10ba preferred ₃E conformation of the isoxazolidine ring

(Fig. 3) was established. In this conformation the diethoxyphos-

phoryl group resides in the equatorial position of the isoxazolidine

ring while carbamoyl substituents are located pseudoequatorially.

Furthermore, a similar spectral pattern was previously observed

for structurally related methyl trans-3-(diethoxyphosphoryl)-2-

methylisoxazolidin-5-yl-5-carboxylate.^27,28

To provide an additional piece of evidence for the already estab-

lished relative conﬁguration at C3 and C5 in isoxazolidines trans-

10 and cis-11 2D NOE experiments were performed for trans-

10ay and cis-11ay (Fig. 4). The occurrence of NOE signal between

HC5 and HC3 was noticed for cis-11ay, while the spectrum of

trans-10ay lacks such correlation.

Stereochemistry of the cycloaddition of N-substituted C-phos-

phorylated nitrones to various alkenes has already been described

and the relative conﬁgurations of trans- and cis-isoxazolidine cyc-

loadducts were established based on detailed conformational anal-

yses.^{23–25,27,28}Indeed, the assignment of relative conﬁgurations in

isoxazolidines has often been difﬁcult due to conformational ﬂex-

ibility of substituted ﬁve-membered ring, but in the case of isoxaz-

olidines containing the diethoxyphosphoryl group at C3

stereochemically valuable data are extended over PCCH^30,31and

PCCC^31–34vicinal couplings, which appeared to be extremely useful

in establishing the stereochemistries of phosphorus-labelled het-

erocycles.^35,36For the major isomers of all obtained isoxazolidines

10aa–10ba trans-conﬁguration was assigned taking advantage of

our previous observations regarding stereochemistry of cycloaddi-

tion of N-methyl-C-phosphorylated nitrone 8 with terminal al-

kenes.^27,28In this series a similar approach to conﬁgurational

assignment was applied. Thus, based on the values of vicinal cou-

H_β

αH

(EtO)₂(O)P

pling constants [J_CCCP= 8.6–9.5 Hz, J_H3–H4= 7.7–8.3 Hz, J_H3–

_H4b= 8.5–9.2 Hz, J_{H4 –P}= 8.0–9.8 Hz, J_H4b–P= 15.4–16.1 Hz, J_H4

Figure 3. The preferred conformations of trans-isoxazolidines 10.

a–

1100

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

OMe

MeO

P(O)(OEt)₂

H_β

MeO

P(O)(OEt)₂

H_β

H_α

MeO

H_α

trans-10ay

cis-11ay

OMe

Figure 4. Observed NOEs for trans-10ay and cis-11ay.

Table 2

2.2. Antiviral and cytostatic evaluation

Inhibitory effect of several 5-arylcarbamoyl-2-methylisoxazolidin-3-yl-3-phospho-

nates against the proliferation of murine leukemia (L1210), human T-lymphocyte

(CEM) and human cervix carcinoma cells (HeLa)

5-Arylcarbamoyl-2-methylisoxazolidin-3-yl-3-phosphonates

trans-10 and cis-11 were evaluated for inhibitory activity against a

wide variety of DNA and RNA viruses, using the following cell-

based assays: (a) human embryonic lung (HEL) cells: herpes sim-

plex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia virus,

vesicular stomatitis virus and herpes simplex virus-1 (TK^ꢀKOS

ACV^r); (b) HeLa cell cultures: vesicular stomatitis virus, Coxsackie

virus B4 and respiratory syncytial virus; (c) Vero cell cultures:

para-inﬂuenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus

B4, Punta Toro virus; (d) MDCK cell cultures: inﬂuenza A virus

(H1N1 and H3N2 subtypes) and inﬂuenza B virus; (e) CrFK cell cul-

tures: feline herpes virus (FHV) and feline corona virus (FIPV) and

(f) CEM cell cultures: human immunodeﬁciency virus type 1 (HIV-

1) and HIV-2. Ganciclovir, cidofovir, acyclovir, brivudin, (S)-9-(2,3-

dihydroxypropyl)adenine [(S)-DHPA], oseltamivir carboxylate,

amantadine, rimantadine, ribavirin, dextran sulfate (molecular

weight 5000, DS-5000), Hippeastrum hybrid agglutinin (HHA) and

Urtica dioica agglutinin (UDA) were used as the reference com-

pounds. The antiviral activity was expressed as the EC₅₀: the com-

pound concentration required to reduce virus plaque formation

(VZV) by 50% or to reduce virus-induced cytopathogenicity by

50% (other viruses). Unfortunately, no inhibitory activity against

Compound

IC₅₀

L1210

CEM

>250

HeLa

10aa

2-F-C₆H₄

>250

P250

11aa

10ab

2-F-C₆H₄

3-F-C₆H₄

130 24

>250

145 30

>250

177 42

>250

11ab

10af

11af/10af (77:23)

10ag

3-F-C₆H₄

228 12

>250

P250

140 16

227 32

102

>250

P250

3-Br-C₆H₄

4-Br-C₆H₄

4-Cl-C₆H₄

180 35

136 11

P250

136 11

P250

156

177 20

11ag/10ag (87:13)

116

10aj

11aj

10ak

11ak

10ar

11ar

>250

170 27

168 24

4-Cl-C₆H₄

>250

2-NO₂-C₆H₄

4-CH₃C(O)-C₆H₄

Not available

>250

150

>250

227 18

>250

211 55

50% Inhibitory concentration or compound concentration required to inhibit

tumor cell proliferation by 50%.

three tumor cell lines. 5-Arylcarbamoyl derivatives 10 and 11

substituted with CN, Me, MeO or EtO did not show any appreciable

cytostatic activity on the tested tumour cell lines. Among 5-arylcar-

bamoyl derivatives substituted with ﬂuorine atom, compound 11aa

(Ar = 2-F-C₆H₄) is the most cytostatic. According to Kool, a 2,4-ﬂuo-

rophenyl group could be regarded as an uracil non-polar isoster due

to the steric and electrostatic similarities.^37–40To our surprise intro-

duction of additional ﬂuorine atoms into the aromatic ring resulted

in loss of cytostatic activity (10ad/11ad; Ar = 2,4-diF-C₆H₃).

any virus was detected for the evaluated compounds at 250 lM.

The cytotoxicity of the tested compounds toward the uninfected

host cells was deﬁned as the minimum compound concentration

(MCC) that caused a microscopically detectable alteration of normal

cell morphology. The 50% cytostatic inhibitory concentration (IC₅₀),

causing a 50% decrease in cell proliferation was determined against

murine leukemia L1210, human lymphocyte CEM and human cer-

vix carcinoma HeLa cells. None of the tested compounds affected

cell morphology of HEL, HeLa, Vero, MDCK and CrFK cells at concen-

3. Conclusions

trations up to 100 lM. However, several compounds, having phenyl

residue substituted with F, Br, Cl, NO₂and CH₃C(O) groups, were

able to inhibit cell proliferation by 50% (CC₅₀) at concentrations

A new series of 5-arylcarbamoyl-2-methylisoxazolidin-3-yl-3-

phosphonates have been efﬁciently obtained from N-methyl-C-diet-

hoxyphoshporylnitrone and the respective N-arylacrylamides via

the 1,3-dipolar cycloaddition. All synthesised isoxazolidinepospho-

nates trans-10 and cis-11 were evaluated against a variety of DNA

ranging from 116 to 228

lM for L1210 cells, and from 102 to

227 M for CEM and HeLa cells (Table 2).

Structure–activity relationship studies for a series of 5-arylcar-

bamoyl-2-methylisoxazolidin-3-yl-3-phosphonates trans-10 and

cis-11 described in this paper revealed that, in general, cis-isomers

11 are more cytostatic toward tested tumour cell lines than the

respective trans-10 (11aa vs 10aa, 11ab vs 10ab, 11af vs 10af,

11ag vs 10ag, 11aj vs 10aj and 11ar vs 10ar). Isoxazolidines 11ab

(Ar = 3-F-C₆H₄), 10ag (Ar = 4-Br-C₆H₄), 11aj (Ar = 4-Cl-C₆H₄), 10ak

(Ar = 2-NO₂-C₆H₄) and 11ar (Ar = 4-CH₃C(O)-C₆H₄) slightly inhibit

cell proliferation of murine leukemia (L1210), while they are less

active or inactive toward human T-lymphocyte (CEM) and human

and RNA viruses but were not active at 250 lM.

Cytostatic activity of trans-10 and cis-11 compounds were per-

formed on three tumor cell lines (L1210, CEM and HeLa) and

showed that cis-conﬁgurated isoxazolidines containing phenyl

substituted with halogen [cis-11aa (Ar = 2-F-C₆H₄), cis-11af

(Ar = 3-Br-C₆H₄), and cis-11ag (Ar = 4-Br-C₆H₄)] are the most active

toward all tested cancerous cell lines (IC₅₀100–180 lM).

Further studies on isoxazolidinephosphonates of general for-

mula 7 containing natural or modiﬁed nucleobases instead of aryl

groups are in progress and will be published in due course.

cervix cells (HeLa) at 250 lM. Compounds containing phenyl

substituted with halogen, namely, cis-11aa (Ar = 2-F-C₆H₄), cis-

11af (Ar = 3-Br-C₆H₄), and cis-11ag (Ar = 4-Br-C₆H₄) appeared to

be the most active toward L1210, CEM as well as HeLa cells at

4. Experimental section

¹H NMR spectra were taken in CDCl₃or CD₃OD on the following

spectrometers: Varian Mercury-300 and Bruker Avance III

IC₅₀’s consistently ranging between 100 and 200 lM against all

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

1101

(600 MHz) with TMS as internal standard. ¹³C NMR spectra were

recorded for CDCl₃solution on the Varian Mercur-300 machine

at 75.5 MHz. ³¹P NMR spectra were performed in CDCl₃solution

on the Varian Mercury-300 at 121.5 MHz. IR spectra were mea-

sured on an Inﬁnity MI-60 FT-IR spectrometer. Melting points were

determined on a Boetius apparatus and are uncorrected. Elemental

analyses were performed by the Microanalytical Laboratory of this

Faculty on Perkin–Elmer PE 2400 CHNS analyzer. The following

adsorbents were used: column chromatography, Merck silica gel

60 (70–230 mesh); analytical TLC, Merck TLC plastic sheets silica

Calcd for C₁₀H₈N₂O: C, 69.76; H, 4.68; N, 16.27; found: C, 69.83;

H, 4.85; N, 16.31.

4.1.4. N-(2-Cyano-4,5-dimethoxyphenyl)acrylamide (9az)

Yield: 91%; yellow amorphous solid mp 158–162 °C; IR (KBr,

cm^ꢀ1

) m_max: 3247, 2225, 1663, 1610, 1514, 1450, 1356, 1283,

1226, 1109; ¹H NMR (600 MHz, CDCl₃) d: 8.22 (s, 1H), 7.65 (br s,

1H, NH), 6.99 (s, 1H), 6.51 (dd, 1H, J = 16.8, 0.9 Hz, CH@CH₂), 6.34

(dd, 1H, J = 16.8, 10.3 Hz, CH@CH₂), 5.90 (dd, 1H, J = 10.3, 0.9 Hz,

CH@CH₂), 3.99 (s, 3H, CH₃O), 3.91 (s, 3H, CH₃O); ¹³C NMR

(151.0 MHz, CDCl₃) d: 163.65 (s, C(O)), 153.71, 145.68, 136.33,

130.59 (s, CH@CH₂), 129.07 (s, CH@CH₂), 116.80 (s, CN), 112.91,

104.97, 92.54, 56.32 (s, CH₃O), 56.17 (s, CH₃O). Anal. Calcd for

C₁₂H₁₂N₂O₃: C, 62.06; H, 5.21; N, 12.06; found: C, 62.07; H, 5.28;

N, 11.97.

gel 60 F₂₅₄

Starting materials: All solvents were dried according to the liter-

ature methods. Nitrone 8 was previously reported.²⁷

4.1. General procedure for the preparation of acrylamides 9

To a solution of substituted aniline (1.00 mmol) in dichloro-

methane (2 mL) triethylamine (1.10 mmol) was added. The mix-

ture was cooled in an ice bath and acryloyl chloride (1.05 mmol)

was added dropwise. The reaction mixture was stirred for 24 h at

room temperature and extracted with water (3 ꢁ 3 mL). Subse-

quently, the inorganic layer was extracted with ethyl ether

(3 ꢁ 5 mL). The combined organic layers were dried over anhy-

drous MgSO₄and ﬁltered. After evaporation of solvents the residue

was puriﬁed on a silica column with chloroform:methanol mix-

tures (100:1, 50:1 v/v) as eluents to afford the respective acryla-

mides 9.

4.1.5. N-(2-Acethyl-4,5-dimethoxyphenyl)acrylamide (9ba)

Yield: 88%; yellow amorphous solid; mp 89–90 °C; IR (KBr,

cm^ꢀ1

) m_max: 3447, 3120, 2938, 1683, 1615, 1522, 1366, 1253,

1207, 1153; ¹H NMR (600 MHz, CDCl₃) d: 8.67 (s, 1H), 7.32 (s,

1H), 6.45 (dd, 1H, J = 16.9, 1.0 Hz, CH@CH₂), 6.34 (dd, 1H, J = 16.9,

10.2 Hz, CH@CH₂), 5.81 (dd, 1H, J = 10.2, 1.0 Hz, CH@CH₂), 4.02 (s,

3H, CH₃O), 3.94 (s, 3H, CH₃O), 2.65 (s, 3H, CH₃–C(O)); ¹³C NMR

(151.0 MHz, CDCl₃) d: 200.82 (s, C(O)), 164.63 (s, C(O)NH),

154.74, 143.76, 137.88, 132.53 (s, CH@CH₂), 127.22 (s, CH@CH₂),

114.50, 113.86, 103.65, 56.43 (s, CH₃O), 56.21 (s, CH₃O), 28.37 (s,

CH₃C(O)). Anal. Calcd for C₁₃H₁₅NO₄: C, 62.64; H, 6.07; N, 5.62;

found: C, 62.69; H, 6.16; N, 5.74.

4.1.1. N-(3-Fluorophenyl)acrylamide (9ab)

4.2. General procedure for preparation of isoxazolidines 10 and

Yield: 46%; white amorphous solid (crystallised from chloro-

form/hexane) mp 125–126 °C; IR (KBr, cm^ꢀ1

) m_max: 3277, 1666,

1611, 1549, 1491, 1443, 1223, 774, 677; ¹H NMR (300 MHz, CDCl₃)

d: 7.52–7.48 (m, 1H), 7.34 (br s, 1H, NH), 7.24–7.11 (m, 2H), 6.79–

6.73 (m, 1H), 6.38 (dd, 1H, J = 16.9, 1.2 Hz, CH@CH₂), 6.17 (dd, 1H,

J = 16.9, 10.1 Hz, CH@CH₂), 5.73 (dd, 1H, J = 10.1, 1.2 Hz, CH@CH₂);

¹³C NMR (75.5 MHz, CDCl₃) d: 164.68 (s, C(O)), 162.73 (d,

J = 243.3 Hz, C3), 139.62 (d, J = 10.9 Hz, C1), 130.89 (s, CH@CH₂),

129.89 (d, J = 9.2 Hz, C5), 127.85 (s, CH@CH₂), 115.52 (s, C6),

110.96 (d, J = 21.2 Hz, C4), 107.53 (d, J = 26.1 Hz, C2). Anal. Calcd

for C₉H₈FNO: C, 65.45; H, 4.88; N, 8.48; found: C, 65.24; H, 4.63;

N, 8.56.

A mixture of nitrone 8 (1.00 mmol), acrylamide 9 (1.00 mmol)

and toluene (2 mL) was stirred at 70 °C for 24 h or until disappear-

ance of the starting nitrone. After evaporation of the solvent under

reduced pressure the crude products were puriﬁed by silica gel

chromatography with chloroform/methanol mixtures as eluents.

4.2.1. Diethyl (3RS,5SR)-5-(2-ﬂuorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10aa)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3396, 2983, 1700, 1531,

1456, 1238, 1053, 1025, 757; ¹H NMR (300 MHz, CDCl₃) d: 8.56

(br s, 1H, NH), 8.34–8.29 (m, 1H), 7.18–7.08 (m, 3H), 4.65 (dd,

1H, J = 8.8, 4.9 Hz, HC5), 4.27–4.14 (m, 4H, 2 ꢁ CH₂OP), 3.15–2.93

(m, 2H, H_bC4 and HC3), 3.05 (s, 3H, CH₃N), 2.90–2.80 (m, 1H,

4.1.2. N-(2,4-Diﬂuorophenyl)acrylamide (9ad)

Yield: 56%; white plates (crystallised from chloroform/hexane)

mp 105–106 °C; IR (KBr, cm^ꢀ1

) m_max: 3277, 1668, 1549, 1503,

H C4), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.35 (t, 3H, J = 7.0 Hz,

1214, 1142, 1099, 847, 808, 699; ¹H NMR (300 MHz, CDCl₃) d:

8.41–8.33 (m, 1H), 7.36 (br s, 1H, NH), 6.94–6.85 (m, 2H), 6.47

(dd, 1H, J = 16.9, 1.2 Hz, CH=CH₂), 6.28 (dd, 1H, J = 16.9, 10.2 Hz,

CH@CH₂), 5.83 (dd, 1H, J = 10.2, 1.2 Hz, CH@CH₂); ¹³C NMR

(75.5 MHz, CDCl₃) d: 163.87 (s, C(O)), 158.84 (dd, J = 246.3,

11.7 Hz, C4), 153.02 (dd, J = 245.4, 10.6 Hz, C2), 130.66 (s,

CH@CH₂), 128.41 (s, CH@CH₂), 123.65 (d, J = 9.8 Hz, C6), 122.36

(dd, J = 10.7, 3.8 Hz, C5), 111.21 (dd, J = 21.8, 3.7 Hz, C1), 103.69

(dd, J = 26.6, 23.5 Hz, C3). Anal. Calcd for C₉H₇F₂NO: C, 59.02; H,

3.85; N, 7.65; found: C, 58.83; H, 3.94; N, 7.68.

CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 169.13 (s, C(O)),

152.61 (d, J = 243.6 Hz, C2⁰), 125.37 (d, J = 10.0 Hz, C1⁰), 125.04 (d,

J = 7.7 Hz, C4⁰), 124.67 (d, J = 3.7 Hz, C6⁰), 121.51 (s, C5⁰), 115.03

(d, J = 18.9 Hz, C3⁰), 76.51 (d, J = 9.4 Hz, C5), 63.59 (d, J = 6.6 Hz,

CH₂OP), 63.51 (d, J = 168.6 Hz, C3), 62.78 (d, J = 6.9 Hz, CH₂OP),

47.08 (s, CH₃N), 36.90 (s, C4), 16.76 (d, J = 5.4 Hz, CH₃CH₂OP),

16.69 (d, J = 5.2 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d:

21.20. Anal. Calcd for C₁₅H₂₂FN₂O₅P: C, 50.00; H, 6.15; N, 7.77;

found: C, 49.83; H, 6.04; N, 7.96.

4.2.2. Diethyl (3RS,5RS)-5-(2-ﬂuorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11aa)

4.1.3. N-(3-Cyanophenyl)acrylamide (9an)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3397, 2983, 1699, 1619,

Yield: 86%; yellow amorphous solid; mp 125–126 °C; IR (KBr,

cm^ꢀ1

) m_max: 3253, 3077, 2230, 1665, 1606, 1556, 1484, 1415,

1531, 1457, 1236, 1053, 1024, 970; ¹H NMR (300 MHz, CDCl₃) d:

8.95 (br s, 1H, NH), 8.37–8.28 (m, 1H), 7.15–7.06 (m, 3H), 4.63–

4.58 (m, 1H, HC5), 4.16–4.05 (m, 4H, 2 ꢁ CH₂OP), 3.02 (s, 3H,

CH₃N), 3.10–2.82 (m, 3H, H₂C4 and HC3), 1.28 (t, 3H, J = 7.1 Hz,

CH₃CH₂OP), 1.17 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR signals of

cis-11aa were extracted from the spectrum of a 65:35 mixture of

trans-10aa and cis-11aa, ¹³C NMR (75.5 MHz, CDCl₃) d: 170.50

1328, 1212; ¹H NMR (600 MHz, CDCl₃) d: 8.02 (s, 1H), 7.84–7.83

(m, 1H), 7.58 (br s, 1H, NH), 7.48–7.42 (m, 2H), 6.50 (dd, 1H,

J = 16.9, 0.8 Hz, CH@CH₂), 6.29 (dd, 1H, J = 16.9, 10.3 Hz, CH@CH₂),

5.87 (dd, 1H, J = 10.3, 0.8 Hz, CH@CH₂); ¹³C NMR (151.0 MHz,

CDCl₃) d: 164.40, 139.13, 130.77 (s, CH@CH₂), 129.87, 128.64 (s,

CH@CH₂), 127.68, 124.38, 123.26, 118.65 (s, CN), 112.66. Anal.

1102

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

(s, C(O)), 152.58 (d, J = 243.7 Hz, C2⁰), 125.89 (d, J = 10.1 Hz, C1⁰),

124.54 (d, J = 3.8 Hz, C6⁰), 124.46 (d, J = 8.3 Hz, C4⁰), 121.26 (s,

C2⁰), 114.89 (d, J = 19.1 Hz, C3⁰), 75.70 (d, J = 8.0 Hz, C5), 63.80 (d,

J = 169.3 Hz, C3), 63.58 (d, J = 6.0 Hz, CH₂OP), 62.75 (d, J = 6.8 Hz,

CH₂OP), 45.99 (s, CH₃N), 36.97 (s, C4), 16.56 (d, J = 6.1 Hz,

CH₃CH₂OP), 16.41 (d, J = 5.7 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 21.51. Anal. Calcd for C₁₅H₂₂FN₂O₅P: C,

50.00; H, 6.15; N, 7.77; found: C, 49.97; H, 6.01; N, 7.94.

C₁₅H₂₂FN₂O₅P: C, 50.00; H, 6.15; N, 7.77; found: C, 50.04; H,

5.94; N, 8.00.

4.2.6. Diethyl (3RS,5RS)-5-(4-ﬂuorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11ac)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3279, 2983, 1688, 1532,

1510, 1227, 1052, 1025, 971; ¹H NMR (300 MHz, CDCl₃) d: 8.81

(br s, 1H, NH), 7.58–7.54 (m, 2H), 7.05–6.98 (m, 2H), 4.64–4.60

(m, 1H, HC5), 4.21–4.06 (m, 4H, 2 ꢁ CH₂OP), 3.12–2.97 (m, 2H,

HC3 and H_bC4), 2.96 (s, 3H, CH₃N), 2.88–2.73 (m, 1H, H C4), 1.31

4.2.3. Diethyl (3RS,5SR)-5-(3-ﬂuorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ab)

White amorphous solid (crystallised from ether/hexane); mp

(t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.21 (t, 3H, J = 7.0 Hz, CH₃CH₂OP);

¹³C NMR signals of cis-11ac were extracted from the spectrum of

a 48:52 mixture of trans-10ac and cis-11ac, ¹³C NMR (75.5 MHz,

CDCl₃) d: 169.77 (s, C(O)), 159.28 (d, J = 242.9 Hz, C4⁰), 133.56 (d,

J = 2.8 Hz, C1⁰), 121.38 (d, J = 7.9 Hz, C2⁰and C6⁰), 115.82 (d,

J = 22.5 Hz, C3⁰and C5⁰), 75.99 (d, J = 6.8 Hz, C5), 63.78 (d,

J = 169.7 Hz, C3), 63.56 (d, J = 6.5 Hz, CH₂OP), 63.09 (d, J = 6.8 Hz,

CH₂OP), 46.28 (s, CH₃N), 36.69 (s, C4), 16.69 (d, J = 5.9 Hz,

CH₃CH₂OP), 16.57 (d, J = 5.7 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 22.14. Anal. Calcd for C₁₅H₂₂FN₂O₅P: C,

50.00; H, 6.15; N, 7.77; found: C, 49.98; H, 6.16; N, 7.82.

85–87 °C. IR (KBr, cm^ꢀ1

) m_max: 3267, 3084, 3051, 2995, 1698,

1623, 1562, 1445, 1215, 1021, 877, 583; ¹H NMR (300 MHz, CDCl₃)

d: 8.24 (br s, 1H, NH), 7.56–7.51 (m, 1H), 7.33–7.25 (m, 1H), 7.20–

7.17 (m, 1H), 6.88–6.82 (m, 1H), 4.62 (dd, 1H, J = 8.8, 5.2 Hz, HC5),

4.27–4.14 (m, 4H, 2 ꢁ CH₂OP), 3.10–2.90 (m, 2H, H_bC4 and HC3),

3.02 (s, 3H, CH₃N), 2.89–2.77 (m, 1H, H C4), 1.37 (t, 3H,

J = 7.0 Hz, CH₃CH₂OP), 1.35 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(75.5 MHz, CDCl₃) d: 168.88 (s, C(O)), 162.87 (d, J = 244.5 Hz, C3⁰),

138.39 (d, J = 10.9 Hz, C1⁰), 130.18 (d, J = 9.2 Hz, C5⁰), 115.14 (d,

J = 2.9 Hz, C6⁰), 111.53 (d, J = 21.5 Hz, C4⁰), 107.37 (d, J = 26.3 Hz,

C2⁰), 76.45 (d, J = 8.9 Hz, C5), 63.52 (d, J = 167.8 Hz, C3), 63.51 (d,

J = 6.3 Hz, CH₂OP), 62.78 (d, J = 6.9 Hz, CH₂OP), 46.83 (s, CH₃N),

36.51 (s, C4), 16.72 (d, J = 5.4 Hz, CH₃CH₂OP), 16.65 (d, J = 5.2 Hz,

CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.16. Anal. Calcd for

4.2.7. Diethyl (3RS,5SR)-5-(2,4-diﬂuorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ad)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3397, 2984, 1698, 1534,

1432, 1236, 1054, 1025, 964, 848; ¹H NMR (300 MHz, CDCl₃) d:

8.43 (s, 1H, NH), 8.31–8.23 (m, 1H), 6.93–6.87 (m, 2H), 4.63 (dd,

1H, J = 8.5, 4.6 Hz, HC5), 4.27–4.14 (m, 4H, 2 ꢁ CH₂OP), 3.20–2.95

(m, 2H, HC3 and H_bC4), 3.04 (s, 3H, CH₃N), 2.95–2.78 (m, 1H,

C₁₅H₂₂FN₂O₅P: C, 50.00; H, 6.15; N, 7.77; found: C, 49.99; H,

5.92; N, 7.98.

H C4), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.35 (t, 3H, J = 7.0 Hz,

4.2.4. Diethyl (3RS,5RS)-5-(3-ﬂuorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11ab)

CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 169.06 (s, C(O)),

158.82 (dd, J = 246.5, 11.5 Hz, C4⁰), 152.69 (dd, J = 246.6, 11.8 Hz,

C2⁰), 122.57 (dd, J = 9.1, 2.2 Hz, C6⁰), 121.70 (dd, J = 10.3, 3.7 Hz,

C5⁰), 111.34 (dd, J = 21.7, 3.8 Hz, C1⁰), 103.79 (dd, J = 26.6,

23.2 Hz, C3⁰), 76.36 (d, J = 9.4 Hz, C5), 63.54 (d, J = 6.6 Hz, CH₂OP),

63.48 (d, J = 167.8 Hz, C3), 62.72 (d, J = 6.9 Hz, CH₂OP), 46.98 (s,

CH₃N), 36.85 (s, C4), 16.73 (d, J = 5.2 Hz, CH₃CH₂OP), 16.66 (d,

J = 5.4 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.25. Anal.

Calcd for C₁₅H₂₁F₂N₂O₅P: C, 47.62; H, 5.60; N, 7.40; found: C, 47.38;

H, 5.66; N, 7.31.

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3276, 2984, 2931, 1690,

1613, 1536, 1445, 1230, 1052, 1026, 966; ¹H NMR (300 MHz,

CDCl₃) d: 8.92 (br s, 1H, NH), 7.59–7.55 (m, 1H), 7.29–7.20 (m,

2H), 6.84–6.78 (m, 1H), 4.61 (dd, 1H, J = 8.7, 5.7 Hz, HC5), 4.23–

4.02 (m, 4H, 2 ꢁ CH₂OP), 3.17–2.99 (m, 2H, HC3 and H_bC4), 3.02

(s, 3H, CH₃N), 2.90–2.74 (m, 1H, H C4), 1.31 (t, 3H, J = 7.0 Hz,

CH₃CH₂OP), 1.21 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR signals of

cis-11ab were extracted from the spectrum of a 68:32 mixture of

trans-10ab and cis-11ab, ¹³C NMR (75.5 MHz, CDCl₃) d: 170.09 (s,

C(O)), 162.96 (d, J = 245.0 Hz, C3⁰), 139.04 (d, J = 10.9 Hz, C1⁰),

130.09 (d, J = 9.5 Hz, C5⁰), 115.08 (d, J = 3.1 Hz, C6⁰), 111.08 (d,

J = 21.2 Hz, C4⁰), 107.18 (d, J = 26.3 Hz, C2⁰), 76.06 (d, J = 6.7 Hz,

C5), 63.78 (d, J = 169.8 Hz, C3), 63.31 (d, J = 6.8 Hz, CH₂OP), 63.14

(d, J = 6.8 Hz, CH₂OP), 46.21 (s, CH₃N), 36.48 (s, C4), 16.70 (d,

J = 5.9 Hz, CH₃CH₂OP), 16.57 (d, J = 5.8 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 22.08. Anal. Calcd for C₁₅H₂₂FN₂O₅P: C,

50.00; H, 6.15; N, 7.77; found: C, 50.03; H, 6.24; N, 7.82.

4.2.8. Diethyl (3RS,5SR)-5-(2-bromophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ae)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3354, 2980, 1698, 1524,

1439, 1245, 1052, 1025, 755; (signals of trans-10ae were extracted

from the spectra of a 86:14 mixture of trans-10ae and cis-11ae); ¹H

NMR (300 MHz, CDCl₃) d: 8.94 (br s, 1H, NH), 8.42–8.38 (m, 1H),

7.58–7.55 (m, 1H), 7.36–7.31 (m, 1H), 7.04–6.99 (m, 1H), 4.64

(dd, 1H, J = 8.8, 4.6 Hz, HC5), 4.27–4.14 (m, 4H, 2 ꢁ CH₂OP), 3.14–

2.90 (m, 2H, HC3 and H_bC4), 3.09 (s, 3H, CH₃N), 2.90–2.77 (m,

4.2.5. Diethyl (3RS,5SR)-5-(4-ﬂuorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ac)

White amorphous solid (crystallised from ether/hexane) mp

1H, H C4), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.35 (t, 3H,

J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 169.24 (s,

C(O)), 134.84, 132.36, 128.41, 125.61, 121.34, 113.59, 76.39 (d,

J = 9.7 Hz, C5), 63.52 (d, J = 6.6 Hz, CH₂OP), 63.44 (d, J = 168.6 Hz,

C3), 62.67 (d, J = 7.2 Hz, CH₂OP), 47.07 (s, CH₃N), 37.09 (s, C4),

16.71 (d, J = 5.2 Hz, CH₃CH₂OP), 16.64 (d, J = 5.4 Hz, CH₃CH₂OP);

³¹P NMR (121.5 MHz, CDCl₃) d: 21.15. Anal. Calcd for

59–62 °C. IR (KBr, cm^ꢀ1

) m_max: 3278, 3084, 2999, 1701, 1512,

1211, 1021, 834, 582; ¹H NMR (300 MHz, CDCl₃) d: 8.17 (br s,

1H, NH), 7.54–7.48 (m, 2H), 7.07–7.00 (m, 2H), 4.60 (dd, 1H,

J = 8.7, 5.4 Hz, HC5), 4.26–4.13 (m, 4H, 2 ꢁ CH₂OP), 3.15–2.90 (m,

2H, HC3 and H_bC4), 3.01 (s, 3H, CH₃N), 2.87–2.76 (m, 1H, H C4),

C₁₅H₂₂BrN₂O₅P: C, 42.77; H, 5.26; N, 6.65; found: C, 42.52; H,

1.36 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.34 (t, 3H, J = 7.0 Hz,

CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 168.65 (s, C(O)),

159.43 (d, J = 243.6 Hz, C4⁰), 132.92 (d, J = 2.8 Hz, C1⁰), 121.67 (d,

J = 7.7 Hz, C2⁰and C6⁰), 115.66 (d, J = 22.6 Hz, C3⁰and C5⁰), 76.40

(d, J = 9.2 Hz, C5), 63.45 (d, J = 168.9 Hz, C3), 63.42 (d, J = 6.3 Hz,

CH₂OP), 62.71 (d, J = 6.9 Hz, CH₂OP), 46.86 (s, CH₃N), 36.48 (s,

C4), 16.66 (d, J = 5.4 Hz, CH₃CH₂OP), 16.59 (d, J = 5.4 Hz,

CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.38. Anal. Calcd for

5.07; N, 6.71 (obtained on a 86:14 mixture of trans-10ae and cis-

11ae).

4.2.9. Diethyl (3RS,5SR)-5-(3-bromophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10af)

White amorphous solid (crystallised from ether/hexane); mp

111–112 °C. IR (KBr, cm^ꢀ1

)

_max: 3254, 3107, 3070, 2995, 1699,

1612, 1546, 1422, 1215, 1022, 582; ¹H NMR (300 MHz, CDCl₃) d:

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

1103

8.16 (br s, 1H, NH), 8.20–7.81 (m, 1H), 7.49–7.45 (m, 1H), 7.29–

4.2.13. Diethyl (3RS,5RS)-5-(3-chlorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11ai)

7.18 (m, 2H), 4.61 (dd, 1H, J = 8.5, 5.2 Hz, HC5), 4.27–4.13 (m, 4H,

2 ꢁ CH₂OP), 3.10–2.92 (m, 2H, HC3 and H_bC4), 3.02 (s, 3H, CH₃N),

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3256, 3071, 2908, 1698,

2.89–2.76 (m, 1H, H C4), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.35

1596, 1547, 1426, 1258, 1217, 1050, 1022, 973; ¹H NMR

(300 MHz, CDCl₃) d: 8.87 (br s, 1H, NH), 7.72–7.71 (m, 1H), 7.44–

7.40 (m, 1H), 7.26–7.21 (m, 1H), 7.09–7.06 (m, 1H), 4.61 (dd, 1H,

J = 9.0, 5.4 Hz, HC5), 4.23–4.01 (m, 4H, 2 ꢁ CH₂OP), 3.12–2.94 (m,

(t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d:

168.86 (s, C(O)), 138.20, 130.35, 127.71, 122.74, 122.63, 118.33,

76.45 (d, J = 8.9 Hz, C5), 63.51 (d, J = 167.5 Hz, C3), 63.47 (d,

J = 6.9 Hz, CH₂OP), 62.78 (d, J = 7.2 Hz, CH₂OP), 46.94 (s, CH₃N),

36.50 (s, C4), 16.73 (d, J = 5.4 Hz, CH₃CH₂OP), 16.66 (d, J = 5.4 Hz,

CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.27. Anal. Calcd for

2H, HC3 and H_bC4), 2.96 (s, 3H, CH₃N), 2.88–2.72 (m, 1H, H C4),

1.31 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.21 (t, 3H, J = 7.0 Hz,

CH₃CH₂OP); ¹³C NMR signals of cis-11ai were extracted from the

spectrum of a 70:30 mixture of trans-10ai and cis-11ai, ¹³C NMR

(75.5 MHz, CDCl₃) d: 170.05 (s, C(O)), 138.52, 134.44, 129.85,

124.25, 119.62, 117.63, 75.88 (d, J = 6.9 Hz, C5), 63.50 (d,

J = 167.2 Hz, C3), 63.17 (d, J = 6.9 Hz, CH₂OP), 63.01 (d, J = 6.9 Hz,

CH₂OP), 46.12 (s, CH₃N), 36.31 (s, C4), 16.62 (d, J = 5.7 Hz,

CH₃CH₂OP), 16.51 (d, J = 5.7 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 21.97. Anal. Calcd for C₁₅H₂₂ClN₂O₅P: C,

47.82; H, 5.89; N, 7.44; found: C, 47.84; H, 5.61; N, 7.56.

C₁₅H₂₂BrN₂O₅P: C, 42.77; H, 5.26; N, 6.65; found: C, 42.88; H,

4.99; N, 6.90.

4.2.10. Diethyl (3RS,5SR)-5-(4-bromophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ag)

White amorphous solid (crystallised from ether/hexane); mp

81–82 °C. IR (KBr, cm^ꢀ1

) m_max: 3260, 3057, 2988, 1705, 1547,

1489, 1219, 1025, 827, 577; ¹H NMR (300 MHz, CDCl3) d: 8.22

(br s, 1H, NH), 7.52–7.38 (m, 4H), 4.59 (dd, 1H, J = 8.8, 5.5 Hz,

HC5), 4.25–4.12 (m, 4H, 2 ꢁ CH₂OP), 3.15–2.90 (m, 2H, H-C3 and

4.2.14. Diethyl (3RS,5SR)-5-(4-chlorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10aj)

White amorphous solid (crystallised from ether/hexane); mp

H_b-C4), 3.00 (s, 3H, CH₃N), 2.89–2.75 (m, 1H, H -C4), 1.35 (t, 3H,

J = 7.0 Hz, CH₃CH₂OP), 1.34 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(75.5 MHz, CDCl₃) d: 168.77 (s, C(O)), 135.99, 132.06, 121.44,

117.47, 76.48 (d, J = 9.2 Hz, C5), 63.67 (d, J = 161.4 Hz, C3), 63.54

(d, J = 6.6 Hz, CH₂OP), 62.79 (d, J = 6.9 Hz, CH₂OP), 46.91 (s,

CH₃N), 36.57 (s, C4), 16.76 (d, J = 5.4 Hz, CH₃CH₂OP), 16.69 (d,

J = 5.2 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 20.63. Anal.

Calcd for C₁₅H₂₂BrN₂O₅P: C, 42.77; H, 5.26; N, 6.65; found: C,

42.95; H, 5.06, N, 6.86.

75–76 °C. IR (KBr, cm^ꢀ1

) m_max: 3259, 3059, 1705, 1549, 1493,

1302, 1219, 1027, 832, 577; ¹H NMR (300 MHz, CDCl₃) d: 8.18

(br s, 1H, NH), 7.55–7.49 (m, 2H), 7.33–7.25 (m, 2H), 4.60 (dd,

1H, J = 8.7, 5.1 Hz, HC5), 4.26–4.13 (m, 4H, 2 ꢁ CH₂OP), 3.15–2.89

(m, 2H, HC3 and H_bC4), 3.01 (s, 3H, CH₃N), 2.88–2.76 (m, 1H,

H C4), 1.36 (t, 3H, J = 7.2 Hz, CH₃CH₂OP), 1.35 (t, 3H, J = 7.0 Hz,

CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 168.50 (s, C(O)),

135.31, 129.39, 128.75, 120.88, 76.20 (d, J = 9.2 Hz, C5), 63.28 (d,

J = 167.2 Hz, C3), 63.18 (d, J = 6.6 Hz, CH₂OP), 62.48 (d, J = 6.9 Hz,

CH₂OP), 46.58 (s, CH₃N), 36.21 (s, C4), 16.45 (d, J = 5.1 Hz,

CH₃CH₂OP), 16.38 (d, J = 5.4 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 21.31. Anal. Calcd for C₁₅H₂₂ClN₂O₅P: C,

47.82; H, 5.89; N, 7.44; found: C, 47.93; H, 5.82; N, 7.58.

4.2.11. Diethyl (3RS,5SR)-5-(2-chlorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ah)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3477, 3368, 2982, 1699, 1593,

1528, 1442, 1242, 1055, 1027, 756; (signals of trans-10ah were ex-

tracted from the spectra of a 81:19 mixture of trans-10ah and cis-

11ah); ¹H NMR (300 MHz, CDCl₃) d: 8.95 (br s, 1H, NH), 8.43–8.40

(m, 1H), 7.41–7.38 (m, 1H), 7.32–7.27 (m, 1H), 7.11–7.05 (m, 1H),

4.65 (dd, 1H, J = 8.8, 4.6 Hz, HC5), 4.27–4.14 (m, 4H, 2 ꢁ CH₂OP),

3.07 (s, 3H, CH₃N), 3.15–2.78 (m, 3H, H₂C4 and HC3), 1.37 (t, 3H,

J = 6.9 Hz, CH₃CH₂OP), 1.35 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(75.5 MHz, CDCl₃) d: 169.27 (s, C(O)), 133.75, 129.18, 127.82,

125.19, 123.19, 121.15, 76.51 (d, J = 9.7 Hz, C5), 63.59 (d, J = 6.6 Hz,

CH₂OP), 63.52 (d, J = 165.8 Hz, C3), 62.76 (d, J = 6.9 Hz, CH₂OP),

47.13 (s, CH₃N), 37.07 (s, C4), 16.76 (d, J = 5.2 Hz, CH₃CH₂OP),

16.68 (d, J = 5.4 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d:

21.17. Anal. Calcd for C₁₅H₂₂ClN₂O₅P: C, 47.82; H, 5.89; N, 7.44;

found: C, 47.52; H, 5.60; N, 7.53 (obtained on a 81:19 mixture of

trans-10ah and cis-11ah).

4.2.15. Diethyl (3RS,5RS)-5-(4-chlorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11aj)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3286, 3264, 2983, 2924,

1691, 1537, 1494, 1234, 1051, 1025, 971; ¹H NMR (300 MHz,

CDCl₃) d: 8.86 (br s, 1H, NH), 7.58–7.53 (m, 2H), 7.31–7.26 (m,

2H), 4.61 (dd, 1H, J = 9.0, 4.8 Hz, HC5), 4.23–4.01 (m, 4H,

2 ꢁ CH₂OP), 3.12–2.99 (m, 2H, HC3 and H_bC4), 2.95 (s, 3H, CH₃N),

2.88–2.72 (m, 1H, H C4), 1.31 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.20

(t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR signals of cis-11aj were ex-

tracted from the spectrum of a 51:49 mixture of trans-10aj and cis-

11aj, ¹³C NMR (75.5 MHz, CDCl₃) d: 169.91 (s, C(O)), 136.12,

129.21, 128.9, 120.93, 76.04 (d, J = 6.9 Hz, C5), 63.56 (d,

J = 168.4 Hz, C3), 63.25 (d, J = 6.9 Hz, CH₂OP), 63.13 (d, J = 6.0 Hz,

CH₂OP), 46.89 (s, CH₃N), 36.40 (s, C4), 16.67 (d, J = 6.8 Hz,

CH₃CH₂OP), 16.58 (d, J = 7.1 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 22.16. Anal. Calcd for C₁₅H₂₂ClN₂O₅P: C,

47.82; H, 5.89; N, 7.44; found: C, 47.84; H, 5.81; N, 7.40.

4.2.12. Diethyl (3RS,5SR)-5-(3-chlorophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ai)

White amorphous solid (crystallised from ether/hexane); mp

106–109 °C. IR (KBr, cm^ꢀ1

) m_max: 3256, 3071, 2963, 1699, 1597,

1548, 1425, 1258, 1216, 1022, 583; ¹H NMR (300 MHz, CDCl₃) d:

8.24 (br s, 1H, NH), 7.70–7.68 (m, 1H), 7.42–7.40 (m, 1H), 7.27–

7.23(m, 1H), 7.14–7.11 (m, 1H), 4.63 (dd, 1H, J = 8.8, 4.6 Hz, HC5),

4.27–4.14 (m, 4H, 2 ꢁ CH₂OP), 2.95–3.20 (m, 2H, HC3 and H_bC4),

4.2.16. Diethyl (3RS,5SR)-5-(2-nitrophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ak)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3320, 2982, 1704, 1503,

3.03 (s, 3H, CH₃N), 2.90–2.75 (m, 1H, H C4), 1.37 (t, 3H,

1278, 1238, 1051, 1023, 969, 745; ¹H NMR (300 MHz, CDCl₃) d:

8.84–8.80 (m, 1H), 8.28–8.25 (m, 1H), 7.71–7.66 (m, 1H), 7.28–

7.22 (m, 1H), 4.66 (dd, 1H, J = 8.9, 5.6 Hz, HC5), 4.28–4.15 (m, 4H,

2 ꢁ CH₂OP), 3.14–2.98 (m, 2H, HC3 and H_bC4), 3.12 (s, 3H, CH₃N),

J = 7.0 Hz, CH₃CH₂OP), 1.35 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(75.5 MHz, CDCl₃) d: 168.87 (s, C(O)), 138.07, 134.62, 130.04,

124.75, 119.92, 117.84, 76.45 (d, J = 8.9 Hz, C5), 63.52 (d,

J = 167.8 Hz, C3), 63.45 (d, J = 6.6 Hz, CH₂OP), 62.56 (d, J = 6.9 Hz,

CH₂OP), 46.89 (s, CH₃N), 36.49 (s, C4), 16.71 (d, J = 5.2 Hz,

CH₃CH₂OP), 16.64 (d, J = 5.7 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 21.17. Anal. Calcd for C₁₅H₂₂ClN₂O₅P: C,

47.82; H, 5.89; N, 7.44; found: C, 47.92; H, 5.75; N, 7.63.

2.89–2.75 (m, 1H, H C4), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.36

(t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d:

170.43 (s, C(O)), 136.77, 135.86, 133.74, 125.85, 123.86, 122.00,

76.30 (d, J = 9.7 Hz, C5), 63.43 (d, J = 166.8 Hz, C3), 63.37 (d,

J = 6.4 Hz, CH₂OP), 62.63 (d, J = 6.9 Hz, CH₂OP), 46.57 (s, CH₃N),

1104

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

37.27 (s, C4), 16.49 (d, J = 6.1 Hz, CH₃CH₂OP), 16.41 (d, J = 5.7 Hz,

CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.06. Anal. Calcd for

3.20–3.00 (m, 2H, H_bC4 and HC3), 2.95 (s, 3H, CH₃N), 2.87–2.71

(m, 1H, H C4), 1.26 (t, 3H, J = 7.1 Hz, CH₃CH₂OP), 1.23 (t, 3H,

C₁₅H₂₂N₃O₇P: C, 46.51; H, 5.73; N, 10.85; found: C, 46.32; H,

J = 7.1 Hz, CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 170.35 (s,

C(O)), 142.85, 142.85, 125.14, 119.33, 76.58 (br s, C5), 63.69 (d,

J = 6.9 Hz, CH₂OP), 63.65 (d, J = 171.2 Hz, C3), 63.16 (d, J = 6.9 Hz,

CH₂OP), 46.11 (s, CH₃N), 36.14 (s, C4), 16.73 (d, J = 5.2 Hz,

CH₃CH₂OP), 16.66 (d, J = 5.4 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 22.20. Anal. Calcd for C₁₅H₂₂N₃O₇P: C,

46.51; H, 5.73; N, 10.85; found: C, 46.59; H, 5.73; N, 10.96.

5.82; N, 10.95.

4.2.17. Diethyl (3RS,5SR)-5-(3-nitrophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10al)

Yellowish amorphous solid (crystallised from ether/hexane);

mp 139–142 °C; IR (KBr, cm^ꢀ1

) m_max: 3271, 3103, 2982, 1704,

1608, 1530, 1353, 1227, 1050, 1040, 977, 738; ¹H NMR

(300 MHz, CDCl₃) d: 8.54 (s, 1H, NH), 8.47–8.45 (m, 1H), 8.03–

7.98 (m, 2H), 7.56–7.50 (m, 1H), 4.68 (dd, 1H, J = 8.5, 5.7 Hz,

HC5), 4.28–4.15 (m, 4H, 2 ꢁ CH₂OP), 3.10–3.08 (br m, 1H, HC3),

3.05 (s, 3H, CH₃N), 3.00 (dddd, 1H, J = 15.9, 13.0, 8.5, 8.5 Hz,

4.2.21. Diethyl (3RS,5SR)-5-(3-cyanophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10an)

White amorphous solid (crystallised from ether/hexane); mp

85–86 °C; IR (KBr, cm^ꢀ1

) m_max: 3268, 3081, 2984, 2231, 1695,

H_bC4), 2.86 (dddd, 1H, J = 13.0, 9.5, 8.3, 5.7 Hz, H C4), 1.38 (t, 3H,

1590, 1537, 1485, 1432, 1305, 1231, 1050, 1026, 971, 796; ¹H

NMR (600 MHz, CDCl₃) d: 8.36 (br s, 1H, NH), 8.02–8.01 (m, 1H),

7.79–7.77 (m, 1H), 7.48–7.43 (m, 2H), 4.64 (dd, 1H, J = 8.8,

5.6 Hz, H-C5), 4.26–4.18 (m, 4H, 2 ꢁ CH₂OP), 3.13–3.08 (m, 1H,

HC3), 3.04 (s, 3H, CH₃N), 3.02 (dddd, 1H, J = 16.0, 12.7, 8.8,

J = 7.0 Hz, CH₃CH₂OP), 1.36 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(75.5 MHz, CDCl₃) d: 169.29 (s, C(O)), 148.41, 138.36, 129.86,

125.60, 119.16, 114.71, 76.61 (d, J = 8.6 Hz, C5), 63.52 (d,

J = 168.6 Hz, C3), 63.43 (d, J = 6.6 Hz, CH₂OP), 62.94 (d, J = 6.9 Hz,

CH₂OP), 46.79 (s, CH₃N), 36.20 (s, C4), 16.70 (d, J = 5.4 Hz,

CH₃CH₂OP), 16.63 (d, J = 5.2 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 20.93. Anal. Calcd for C₁₅H₂₂N₃O₇P: C,

46.51; H, 5.73; N, 10.85; found: C, 46.51; H, 5.73; N, 10.87.

8.8 Hz, H_bC4), 2.84 (dddd, 1H, J = 12.7, 9.8, 8.3, 5.6 Hz, H C4),

1.39 (t, 3H, J = 7.1 Hz, CH₃CH₂OP), 1.37 (t, 3H, J = 7.1 Hz,

CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d: 169.19 (s, C(O)),

137.88, 129.94, 128.12, 123.89, 122.99, 118.26 (s, CN), 113.19,

76.35 (d, J = 8.8 Hz, C5), 63.55 (d, J = 165.5 Hz, C3), 63.33 (d,

J = 6.5 Hz, CH₂OP), 62.63 (d, J = 7.2 Hz, CH₂OP), 46.62 (s, CH₃N),

36.28 (s, C4), 16.54 (d, J = 2.0 Hz, CH₃CH₂OP), 16.52 (d, J = 4.8 Hz,

CH₃CH₂OP); ³¹P NMR (243.0 MHz, CDCl₃) d: 20.11. Anal. Calcd for

4.2.18. Diethyl (3RS,5RS)-5-(3-nitrophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11al)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3222, 3095, 2988, 1708,

1570, 1509, 1330, 1231, 1024, 974; ¹H NMR (600 MHz, CDCl₃) d:

9.28 (s, 1H, NH), 8.52–8.51 (m, 1H), 8.01–7.98 (m, 2H), 7.53–7.50

(m, 1H), 4.69 (dd, 1H, J = 9.2, 4.5 Hz, HC5), 4.27–4.08 (m, 4H,

2 ꢁ CH₂OP), 3.15–3.06 (m, 2H, H_bC4 and HC3), 2.98 (s, 3H, CH₃N),

C₁₆H₂₂N₃O₅P: C, 52.31; H, 6.04; N, 11.44; found: C, 52.52; H,

6.07; N, 11.44.

2.87–2.79 (m, 1H, H C4), 1.35 (t, 3H, J = 7.1 Hz, CH₃CH₂OP), 1.25

4.2.22. Diethyl (3RS,5RS)-5-(3-cyanophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11an)

(t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d:

170.39 (s, C(O)), 148.63, 138.85, 129.70, 125.39, 118.79, 114.49,

76.09 (d, J = 6.4 Hz, C5), 63.56 (d, J = 170.8 Hz, C3), 63.23 (d,

J = 7.1 Hz, CH₂OP), 62.92 (d, J = 7.1 Hz, CH₂OP), 45.94 (s, CH₃N),

36.02 (s, C4), 16.42 (d, J = 5.7 Hz, CH₃CH₂OP), 16.31 (d, J = 5.7 Hz,

CH₃CH₂OP); ³¹P NMR (243.0 MHz, CDCl₃) d: 21.14. Anal. Calcd for

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3212, 3079, 2980, 2228,

1703, 1594, 1432, 1211, 1048, 1015, 961; ¹H NMR (600 MHz,

CDCl₃) d: 9.20 (s, 1H, NH), 8.07–8.06 (m, 1H), 7.82–7.80 (m, 1H),

7.45–7.40 (m, 2H), 4.67 (dd, 1H, J = 9.4, 4.4 Hz, HC5), 4.26–4.08

(m, 4H, 2 ꢁ CH₂OP), 3.15–3.05 (m, 2H, H_bC4 and HC3), 2.96 (s,

C₁₅H₂₂N₃O₇P: C, 46.51; H, 5.73; N, 10.85; found: C, 46.58; H,

3H, CH₃N), 2.83–2.76 (m, 1H, H C4), 1.35 (t, 3H, J = 7.1 Hz,

CH₃CH₂OP), 1.25 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(151.0 MHz, CDCl₃) d: 170.23 (s, C(O)), 138.59, 129.78, 127.64,

123.79, 122.78, 118.49 (s, CN), 113.03, 76.14 (d, J = 5.8 Hz, C5),

63.58 (d, J = 171.3 Hz, C3), 63.29 (d, J = 7.0 Hz, CH₂OP), 62.90 (d,

J = 6.8 Hz, CH₂OP), 45.96 (s, CH₃N), 35.98 (s, C4), 16.43 (d,

J = 5.8 Hz, CH₃CH₂OP), 16.32 (d, J = 5.7 Hz, CH₃CH₂OP); ³¹P NMR

(243.0 MHz, CDCl₃) d: 21.22. Anal. Calcd for C₁₆H₂₂N₃O₅P: C,

52.31; H, 6.04; N, 11.44; found: C, 52.50; H, 6.18; N, 11.46.

5.66; N, 10.89.

4.2.19. Diethyl (3RS,5SR)-5-(4-nitrophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10am)

Yellowish amorphous solid (crystallised from ether/hexane);

mp 98–99 °C; IR (KBr, cm^ꢀ1

) m_max: 3221, 3095, 2990, 2910, 1710,

1600, 1570, 1510, 1332, 1300, 1270, 1230, 1050, 1025, 974; ¹H

NMR (300 MHz, CDCl₃) d: 8.56 (s, 1H, NH), 8.25–8.21 (m, 2H),

7.79–7.76 (m, 2H), 4.67 (dd, 1H, J = 8.8, 5.5 Hz, HC5), 4.28–4.15

(m, 4H, 2 ꢁ CH₂OP), 3.10–3.08 (m, 1H, HC3), 3.04 (s, 3H, CH₃N),

3.01 (dddd, 1H, J = 15.7, 12.7, 8.8, 8.8 Hz, H_bC4), 2.84 (dddd, 1H,

4.2.23. Diethyl (3RS,5SR)-5-(4-cyanophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ao)

J = 12.7, 8.8, 8.3, 5.5 Hz, H C4), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP),

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3261, 2985, 2224, 1701,

1.36 (t, 3H, J = 6.9 Hz, CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d:

169.30 (s, C(O)), 143.78, 142.85, 125.04, 119.41, 76.57 (d,

J = 8.6 Hz, C5), 63.52 (d, J = 166.3 Hz, C3), 63.49 (d, J = 6.3 Hz,

CH₂OP), 62.93 (d, J = 7.2 Hz, CH₂OP), 46.79 (s, CH₃N), 36.30 (s,

C4), 16.73 (d, J = 5.2 Hz, CH₃CH₂OP), 16.66 (d, J = 5.4 Hz,

CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 20.97. Anal. Calcd for

1600, 1521, 1410, 1311, 1233, 1025, 970, 842; ¹H NMR

(300 MHz, CDCl₃) d: 8.36 (s, 1H, NH), 7.73–7.71 (m, 2H), 7.67–

7.65 (m, 2H), 4.64 (dd, 1H, J = 8.8, 5.6 Hz, HC5), 4.27–4.18 (m, 4H,

2 ꢁ CH₂OP), 3.13–3.08 (m, 1H, HC3), 3.04 (s, 3H, CH₃N), 3.03 (dddd,

1H, J = 16.1, 13.0, 8.8, 8.8 Hz, H_bC4), 2.84 (dddd, 1H, J = 13.0, 9.8,

8.3, 5.6 Hz, H C4), 1.39 (t, 3H, J = 6.9 Hz, CH₃CH₂OP), 1.38 (t, 3H,

₁₅H₂₂N₃O₇P: C, 46.51; H, 5.73; N, 10.85; found: C, 46.60; H,

J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d: 169.28 (s,

C(O)), 141.11, 133.22, 119.60, 118.64 (s, CN), 107.62, 76.46 (d,

J = 8.6 Hz, C5), 63.42 (d, J = 167.6 Hz, C3), 63.29 (d, J = 6.5 Hz,

CH₂OP), 62.75 (d, J = 6.8 Hz, CH₂OP), 46.61 (s, CH₃N), 36.04 (s,

C4), 16.46 (d, J = 5.8 Hz, CH₃CH₂OP), 16.40 (d, J = 6.1 Hz,

CH₃CH₂OP); ³¹P NMR (243.0 MHz, CDCl₃) d: 20.00. Anal. Calcd for

5.66; N, 10.94.

4.2.20. Diethyl (3RS,5RS)-5-(4-nitrophenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11am)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3215, 3085, 2920, 1715,

1607, 1580, 1500, 1260, 1250, 1057, 1020; ¹H NMR (300 MHz,

CDCl₃) d: 9.49 (s, 1H, NH), 8.24–8.19 (m, 2H), 7.83–7.78 (m, 2H),

4.68 (dd, 1H, J = 9.2, 4.7 Hz, HC5), 4.28–4.03 (m, 4H, 2 ꢁ CH₂OP),

C₁₆H₂₂N₃O₅P: C, 52.31; H, 6.04; N, 11.44; found: C, 52.39; H,

6.11; N, 11.67.

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

1105

4.2.24. Diethyl (3RS,5SR)-5-(2-acetylphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ap)

spectrum of a 65:35 mixture of trans-10aq and cis-11aq, ¹³C

NMR (151.0 MHz, CDCl₃) d: 197.77 (s, C(O)), 170.34 (s, C(O)NH),

137.99, 137.87, 129.27, 124.23, 124.12, 119.28, 75.88 (d,

J = 7.4 Hz, C5), 63.49 (d, J = 169.1 Hz, C3), 63.16 (d, J = 5.9 Hz,

CH₂OP), 62.88 (d, J = 7.2 Hz, CH₂OP), 46.70 (s, CH₃N), 36.50 (s,

C4), 26.63 (s, CH₃C(O)), 16.39 (d, J = 5.6 Hz, CH₃CH₂OP), 16.29 (d,

J = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 22.01. Anal.

Calcd for C₁₇H₂₅N₂O₆P: C, 53.12; H, 6.56; N, 7.29; found: C, 52.90;

H, 6.54; N, 7.22.

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3472, 3217, 2981, 2912,

1691, 1660, 1580, 1518, 1451, 1251, 1050, 1023, 964; ¹H NMR

(300 MHz, CDCl₃) d: 8.78–8.74 (m, 1H), 7.93–7.90 (m, 1H), 7.59–

7.54 (m, 1H), 7.19–7.14 (m, 1H), 4.60 (dd, 1H, J = 9.1, 5.0 Hz,

HC5), 4.27–4.12 (m, 4H, 2 ꢁ CH₂OP), 3.16 (s, 3H, CH₃N), 3.12–

2.95 (m, 2H, H_bC4 and HC3), 2.80–2.68 (m, 1H, H C4), 2.67 (s,

3H, CH₃C(O)), 1.36 (t, 3H, J = 7.1 Hz, CH₃CH₂OP), 1.34 (t, 3H,

J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d: 201.92 (s,

C(O)), 170.60 (s, C(O)NH), 139.52, 134.75, 131.54, 122.98, 122.66,

120.76, 76.51 (d, J = 9.7 Hz, C5), 63.28 (d, J = 6.5 Hz, CH₂OP),

63.27 (d, J = 167.2 Hz, C3), 62.49 (d, J = 6.9 Hz, CH₂OP), 46.42 (s,

CH₃N), 37.26 (s, C4), 28.36 (s, CH₃C(O)), 16.40 (d, J = 5.7 Hz,

CH₃CH₂OP), 16.33 (d, J = 5.8 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 21.55. Anal. Calcd for C₁₇H₂₅N₂O₆P: C,

53.12; H, 6.56; N, 7.29; found: C, 52.86; H, 6.80; N, 7.35.

4.2.28. Diethyl (3RS,5SR)-5-(4-acetylphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ar)

White amorphous solid (crystallised from ether/hexane); mp

66–67 °C; IR (KBr, cm^ꢀ1

) m_max: 3258, 3188, 3103, 1707, 1678,

1600, 1642, 1269, 1226, 1050, 1017, 957; ¹H NMR (300 MHz,

CDCl₃) d: 8.39 (s, 1H, NH), 7.95–7.92 (m, 2H), 7.67–7.64 (m,

2H), 4.44 (dd, 1H, J = 8.5, 5.5 Hz, HC5), 4.24–4.11 (m, 4H,

2 ꢁ CH₂OP), 3.06–2.91 (m, 2H, H_bC4 and HC3), 3.01 (s, 3H,

4.2.25. Diethyl (3RS,5RS)-5-(2-acetylphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11ap)

CH₃N), 2.87–2.78 (m, 1H, H C4), 2.56 (s, 3H, CH₃C(O)), 1.34 (t,

3H, J = 7.0 Hz, CH₃CH₂OP), 1.33 (t, 3H, J = 6.9 Hz, CH₃CH₂OP);

¹³C NMR (75.5 MHz, CDCl₃) d: 196.77 (s, C(O)), 169.05 (s,

C(O)NH), 141.16, 133.33, 129.29, 119.64, 76.42 (d, J = 9.2 Hz,

C5), 63.40 (d, J = 167.5 Hz, C3), 63.52 (d, J = 6.3 Hz, CH₂OP),

62.81 (d, J = 6.9 Hz, CH₂OP), 47.00 (s, CH₃N), 36.54 (s, C4),

26.99 (s, CH₃C(O)), 16.80 (d, J = 5.4 Hz, CH₃CH₂OP), 16.73 (d,

J = 5.4 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.16.

Anal. Calcd for C₁₇H₂₅N₂O₆P: C, 53.12; H, 6.56; N, 7.29; found:

C, 53.37; H, 6.47; N, 7.10.

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3482, 2980, 2915, 1690,

1670, 1585, 1520, 1450, 1250, 1050, 1025; ¹H NMR (300 MHz,

CDCl₃) d: 8.82–8.79 (m, 1H), 7.91–7.87 (m, 1H), 7.56–7.51 (m,

1H), 7.15–7.10 (m, 1H), 4.54 (dd, 1H, J = 8.5, 5.2 Hz, HC5), 4.15–

3.92 (m, 4H, 2 ꢁ CH₂OP), 3.10 (s, 3H, CH₃N), 2.96–2.88 (m, 3H,

H₂C4 and HC3), 2.65 (s, 3H, CH₃C(O)), 1.27 (t, 3H, J = 7.0 Hz,

CH₃CH₂OP), 1.08 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(151.0 MHz, CDCl₃) d: 201.49 (s, C(O)), 172.25 (s, C(O)NH),

139.94, 134.68, 131.45, 122.83, 122.62, 120.57, 75.51 (d,

J = 8.5 Hz, C5), 63.83 (d, J = 166.3 Hz, C3), 63.44 (d, J = 6.0 Hz,

CH₂OP), 62.15 (d, J = 7.1 Hz, CH₂OP), 45.36 (s, CH₃N), 36.79 (s,

C4), 28.44 (s, CH₃C(O)), 16.33 (d, J = 6.2 Hz, CH₃CH₂OP), 16.11 (d,

J = 5.7 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.95. Anal.

Calcd for C₁₇H₂₅N₂O₆P: C, 53.12; H, 6.56; N, 7.29; found: C, 53.38;

H, 6.68; N, 7.37.

4.2.29. Diethyl (3RS,5RS)-5-(4-acetylphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11ar)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3258, 3188, 3060, 3001,

1710, 1679, 1599, 1542, 1270, 1230, 1051, 1025; ¹H NMR

(300 MHz, CDCl₃) d: 9.11 (s, 1H, NH), 7.96–7.92 (m, 2H), 7.72–

7.69 (m, 2H), 4.64 (dd, 1H, J = 8.4, 5.4 Hz, HC5), 4.23–4.05 (m, 4H,

2 ꢁ CH₂OP), 3.11–3.02 (m, 2H, H_bC4 and HC3), 2.97 (s, 3H, CH₃N),

4.2.26. Diethyl (3RS,5SR)-5-(3-acetylphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10aq)

Yellowish amorphous solid (crystallised from ether/hexane);

2.85–2.79 (m, 1H, H C4), 2.58 (s, 3H, CH₃C(O)), 1.31 (t, 3H,

J = 7.0 Hz, CH₃CH₂OP), 1.19 (t, 3H, J = 7.1 Hz, CH₃CH₂OP); ¹³C

NMR (151.0 MHz, CDCl₃) d: 196.87 (s, C(O)), 170.32 (s, C(O)NH),

141.88, 133.04, 129.68, 118.97, 76.07 (d, J = 6.6 Hz, C5), 63.64 (d,

J = 170.3 Hz, C3), 63.07 (d, J = 6.7 Hz, CH₂OP), 63.03 (d, J = 7.4 Hz,

CH₂OP), 45.99 (s, CH₃N), 36.24 (s, C4), 26.38 (s, CH₃C(O)), 16.41

(d, J = 5.6 Hz, CH₃CH₂OP), 16.30 (d, J = 5.6 Hz, CH₃CH₂OP); ³¹P

NMR (121.5 MHz, CDCl₃) d: 21.94. Anal. Calcd for C₁₇H₂₅N₂O₆P:

C, 53.12; H, 6.56; N, 7.29; found: C, 52.91; H, 6.43; N, 7.18.

mp 99–103 °C; IR (KBr, cm^ꢀ1

) m_max: 3244, 3084, 2974, 1701,

1596, 1433, 1212, 1047, 1016, 953; ¹H NMR (300 MHz, CDCl₃) d:

8.32 (s, 1H, NH), 8.05–8.04 (m, 1H), 7.94–7.91 (m, 1H), 7.75–7.72

(m, 1H), 7.49–7.44 (m, 1H), 4.62 (dd, 1H, J = 8.8, 5.3 Hz, HC5),

4.27–4.14 (m, 4H, 2 ꢁ CH₂OP), 3.13–3.08 (m, 1H, HC3), 3.04 (s,

3H, CH₃N), 3.04 (dddd, 1H, J = 16.0, 12.7, 8.8, 8.8 Hz, H_bC4), 2.86

(dddd, 1H, J = 12.7, 8.9, 8.3, 5.3 Hz, H C4), 2.62 (s, 3H, CH₃C(O)),

1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.35 (t, 3H, J = 7.1 Hz,

CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 197.64 (s, C(O)),

169.08 (s, C(O)NH), 137.85, 137.40, 129.53, 124.75, 124.44,

119.33, 76.42 (d, J = 9.2 Hz, C5), 63.42 (d, J = 167.6 Hz, C3), 63.58

(d, J = 6.6 Hz, CH₂OP), 62.81 (d, J = 6.9 Hz, CH₂OP), 47.00 (s,

CH₃N), 36.72 (s, C4), 26.99 (s, CH₃C(O)), 16.80 (d, J = 5.4 Hz,

CH₃CH₂OP), 16.73 (d, J = 5.4 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 21.30. Anal. Calcd for C₁₇H₂₅N₂O₆P: C,

53.12; H, 6.56; N, 7.29; found: C, 52.97; H, 6.52; N, 7.23.

4.2.30. Diethyl (3RS,5SR)-5-(m-tolylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10as)

White amorphous solid (crystallised from ether/hexane); mp

96–97 °C; IR (KBr, cm^ꢀ1

) m_max: 3273, 3104, 2899, 1698, 1621,

1600, 1566, 1293, 1262, 1214, 1055, 1020, 959; ¹H NMR

(300 MHz, CDCl₃) d: 8.09 (s, 1H, NH), 7.33–7.26 (m, 2H), 7.18–

7.13 (m, 1H), 6.91–6.88 (m, 1H), 4.55 (dd, 1H, J = 8.7, 5.4 Hz,

HC5), 4.20–4.06 (m, 4H, 2 ꢁ CH₂OP), 3.00–2.88 (m, 2H, H_bC4 and

HC3), 2.96 (s, 3H, CH₃N), 2.85–2.76 (m, 1H, H C4), 2.28 (s, 3H,

4.2.27. Diethyl (3RS,5RS)-5-(3-acetylphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11aq)

CH₃), 1.30 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.28 (t, 3H, J = 7.0 Hz,

CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 168.62 (s, C(O)),

138.89, 136.73, 128.80, 125.51, 120.38, 116.88, 76.45 (d,

J = 9.4 Hz, C5), 63.47 (d, J = 167.5 Hz, C3), 63.38 (d, J = 6.6 Hz,

CH₂OP), 62.61 (d, J = 6.9 Hz, CH₂OP), 46.84 (s, CH₃N), 36.58 (s,

C4), 21.56 (s, CH₃), 16.65 (d, J = 5.2 Hz, CH₃CH₂OP), 16.58 (d,

J = 5.4 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.22. Anal.

Calcd for C₁₆H₂₅N₂O₅P: C, 53.93; H, 7.07; N, 7.86; found: C, 54.16;

H, 7.30; N, 7.99.

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3274, 2981, 1700, 1680,

1617, 1564, 1237, 1046, 1016, 972; ¹H NMR (300 MHz, CDCl₃) d:

8.93 (s, 1H, NH), 8.11 (s, 1H), 7.93–7.90 (m, 1H), 7.72–7.69 (m,

1H), 7.46–7.41 (m, 1H), 4.63 (dd, 1H, J = 8.9, 4.9 Hz, H-C5), 4.20–

4.06 (m, 4H, 2 ꢁ CH₂OP), 2.98 (s, 3H, CH₃N), 3.15–2.92 (m, 2H,

H_bC4 and HC3), 2.90–2.76 (m, 1H, H C4), 2.62 (s, 3H, CH₃C(O)),

1.30 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.22 (t, 3H, J = 7.1 Hz,

CH₃CH₂OP); ¹³C NMR signals of cis-11aq were extracted from the

1106

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

4.2.31. Diethyl (3RS,5RS)-5-(m-tolylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11as)

CH₂OP), 55.11 (s, CH₃O), 46.57 (s, CH₃N), 36.17 (s, C4), 16.42 (d,

J = 5.3 Hz, CH₃CH₂OP), 16.37 (d, J = 5.2 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 21.17. Anal. Calcd for C₁₆H₂₅N₂O₆P: C,

51.61; H, 6.77; N, 7.52; found: C, 51.72; H, 6.88; N, 7.54.

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3216, 2971, 2900, 1700, 1600,

1565, 1453, 1290, 1210, 1050, 1020; ¹H NMR (600 MHz, CDCl₃) d:

8.66 (s, 1H, NH), 7.44 (s, 1H), 7.39–7.38 (m, 1H), 7.24–7.22 (m, 1H),

6.96–6.94 (m, 1H), 4.61 (dd, 1H, J = 8.6, 5.1 Hz, HC5), 4.22–4.06 (m,

4H, 2 ꢁ CH₂OP), 3.08–2.99 (m, 2H, H_bC4 and HC3), 3.00 (s, 3H,

4.2.35. Diethyl (3RS,5SR)-5-(4-ethoxyphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10av)

CH₃N), 2.90–2.81 (m, 1H, H C4), 2.37 (s, 3H, CH₃), 1.33 (t, 3H,

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3270, 2980, 1683, 1512,

J = 7.1 Hz, CH₃CH₂OP), 1.23 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(151.0 MHz, CDCl₃) d: 169.91 (s, C(O)), 138.84, 137.36, 128.77,

125.13, 120.29, 116.82, 75.83 (d, J = 7.6 Hz, C5), 63.86 (d,

J = 168.7 Hz, C3), 63.24 (d, J = 6.6 Hz, CH₂OP), 62.68 (d, J = 6.8 Hz,

CH₂OP), 46.09 (s, CH₃N), 36.56 (s, C4), 21.45 (s, CH₃), 16.40 (d,

J = 5.6 Hz, CH₃CH₂OP), 16.30 (d, J = 5.8 Hz, CH₃CH₂OP); ³¹P NMR

(243.0 MHz, CDCl₃) d: 21.82. Anal. Calcd for C₁₆H₂₅N₂O₅P: C,

53.93; H, 7.07; N, 7.86; found: C, 54.07; H, 7.20; N, 7.98.

1237, 1050, 1026, 969, 826; ¹H NMR (300 MHz, CDCl₃) d: 7.46–

7.43 (m, 2H), 8.09 (s, 1H, NH), 6.88–6.85 (m, 2H), 4.61 (dd, 1H,

J = 8.8, 5.2 Hz, HC5), 4.27–4.13 (m, 4H, 2 ꢁ CH₂OP), 4.02 (q, 2H,

J = 6.9 Hz, CH₃CH₂O), 3.15–2.92 (m, 2H, H_bC4 and HC3), 3.02 (s,

3H, CH₃N), 2.90–2.80 (m, 1H, H C4), 1.41 (t, 3H, J = 6.9 Hz,

CH₃CH₂O), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.35 (t, 3H,

J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (75.5 MHz, CDCl₃) d: 168.22 (s,

C(O)), 155.77, 129.59, 121.37, 114.53, 76.21 (d, J = 9.2 Hz, C5),

63.53 (s, CH₂CH₃), 63.29 (d, J = 168.6 Hz, C3), 63.25 (d, J = 6.6 Hz,

CH₂OP), 62.45 (d, J = 6.9 Hz, CH₂OP), 46.67 (s, CH₃N), 36.44 (s,

C4), 16.49 (d, J = 5.4 Hz, CH₃CH₂OP), 16.42 (d, J = 5.4 Hz,

CH₃CH₂OP), 14.78 (s, CH₃CH₂O); ³¹P NMR (121.5 MHz, CDCl₃) d:

21.37. Anal. Calcd for C₁₇H₂₇N₂O₆P: C, 52.84; H, 7.04; N, 7.25;

found: C, 52.61; H, 7.01; N, 7.26.

4.2.32. Diethyl (3RS,5SR)-5-(p-tolylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10at)

White amorphous solid (crystallised from ether/hexane); mp

62–64 °C; IR (KBr, cm^ꢀ1

) m_max: 3264, 3125, 2974, 1699, 1614,

1550, 1516, 1299, 1264, 1210, 1050, 1017, 952, 819; ¹H NMR

(300 MHz, CDCl₃) d: 8.16 (br s, 1H, NH), 7.46–7.43 (m, 2H), 7.17–

7.14 (m, 2H), 4.63 (dd, 1H, J = 9.0, 5.4 Hz, HC5), 4.27–4.14 (m, 4H,

2 ꢁ CH₂OP), 3.10–3.04 (m, 1H, HC3), 3.03 (s, 3H, CH₃N), 2.96 (dddd,

1H, J = 15.7, 13.0, 9.0, 9.0 Hz, H_bC4), 2.85 (dddd, 1H, J = 13.0, 8.3,

4.2.36. Diethyl (3RS,5RS)-5-(4-ethoxyphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11av)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3270, 2981, 1680, 1607, 1513,

7.8, 5.4 Hz, H C4), 2.33 (s, 3H, CH₃), 1.37 (t, 3H, J = 7.0 Hz,

1298, 1234, 1050, 1024, 970; ¹H NMR (300 MHz, CDCl₃) d: 8.61 (s,

1H, NH), 7.49–7.46 (m, 2H), 6.87–6.84 (m, 2H), 4.60 (dd, 1H,

J = 8.7, 5.5 Hz, HC5), 4.19–4.06 (m, 4H, 2 ꢁ CH₂OP), 4.01 (q, 2H,

J = 6.9 Hz, CH₃CH₂O), 3.07–2.99 (m, 2H, H_bC4 and HC3), 2.97 (s, 3H,

CH₃CH₂OP), 1.35 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(75.5 MHz, CDCl₃) d: 168.59 (s, C(O)), 134.55, 134.28, 129.60,

120.22, 76.45 (d, J = 9.5 Hz, C5), 63.50 (d, J = 167.5 Hz, C3), 63.58

(d, J = 6.3 Hz, CH₂OP), 62.76 (d, J = 6.9 Hz, CH₂OP), 46.73 (s,

CH₃N), 36.71 (s, C4), 20.91 (s, CH₃), 16.70 (d, J = 5.5 Hz, CH₃CH₂OP),

16.55 (d, J = 5.5 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d:

21.24. Anal. Calcd for C₁₆H₂₅N₂O₅P: C, 53.93; H, 7.07; N, 7.86;

found: C, 53.95; H, 7.09; N, 7.92.

CH₃N), 2.85–2.77 (m, 1H, H C4), 1.40 (t, 3H, J = 6.9 Hz, CH₃CH₂O),

1.30 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.21 (t, 3H, J = 7.0 Hz, CH₃CH₂OP);

¹³C NMR signals of cis-11av were extracted from the spectrum of a

55:45 mixture of trans-10av and cis-11av, ¹³C NMR (75.5 MHz,

CDCl₃) d: 169.57 (s, C(O)), 155.71, 130.49, 121.36, 114.75, 75.90 (d,

J = 7.2 Hz, C5), 63.84 (d, J = 168.7 Hz, C3), 63.79 (s, CH₂CH₃), 63.40

(d, J = 6.6 Hz, CH₂OP), 62.75 (d, J = 6.6 Hz, CH₂OP), 46.26 (s, CH₃N),

36.60 (s, C4), 16.62 (d, J = 6.2 Hz, CH₃CH₂OP), 16.54 (d, J = 6.4 Hz,

CH₃CH₂OP), 15.10 (s, CH₃CH₂O); ³¹P NMR (121.5 MHz, CDCl₃) d:

22.06. Anal. Calcd for C₁₇H₂₇N₂O₆P: C, 52.84; H, 7.04; N, 7.25; found:

C, 52.83; H, 7.07; N, 7.15.

4.2.33. Diethyl (3RS,5RS)-5-(p-tolylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11at)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3260, 2970, 2890, 1700,

1615, 1300, 1210, 1045, 1020; ¹H NMR (300 MHz, CDCl₃) d: 8.65

(br s, 1H, NH), 7.47–7.44 (m, 2H), 7.13–7.11 (m, 2H), 4.60 (dd,

1H, J = 8.6, 5.4 Hz, HC5), 4.21–4.03 (m, 4H, 2 ꢁ CH₂OP), 3.07–3.01

(m, 2H, H_bC4 and HC3), 2.97 (s, 3H, CH₃N), 2.89–2.79 (m, 1H,

4.2.37. Diethyl (3RS,5SR)-5-(3,4-dimethoxyphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10aw)

H C4), 2.31 (s, 3H, CH₃), 1.30 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.20

(t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d:

168.62 (s, C(O)), 134.48, 134.32, 129.56, 119.87, 76.39 (d,

J = 9.1 Hz, C5), 63.52 (d, J = 169.5 Hz, C3), 63.34 (d, J = 6.4 Hz,

CH₂OP), 62.53 (d, J = 7.1 Hz, CH₂OP), 46.74 (s, CH₃N), 36.53 (s,

C4), 20.85 (s, CH₃), 16.40 (d, J = 5.6 Hz, CH₃CH₂OP), 16.31 (d,

J = 5.6 Hz, CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 21.96. Anal.

Calcd for C₁₆H₂₅N₂O₅P: C, 53.93; H, 7.07; N, 7.86; found: C, 53.87;

H, 7.18; N, 7.87.

White amorphous solid (crystallised from ether/hexane); mp

83–84 °C; IR (KBr, cm^ꢀ1

) m_max: 3274, 2981, 2934, 2837, 1682,

1608, 1515, 1453, 1234, 1050, 969, 806, 765; ¹H NMR (600 MHz,

CDCl₃) d: 8.09 (br s, 1H, NH), 7.38–7.37 (m, 1H), 6.95–6.93 (m,

1H), 6.85–6.83 (m, 1H), 4.61 (dd, 1H, J = 9.0, 5.3 Hz, HC5), 4.25–

4.19 (m, 4H, 2 ꢁ CH₂OP), 3.91 (s, 3H, CH₃O), 3.88 (s, 3H, CH₃O),

3.12–3.07 (m, 1H, HC3), 3.04 (s, 3H, CH₃N), 3.01 (dddd, 1H,

J = 15.9, 12.9, 9.0, 9.0 Hz, H_bC4), 2.86 (dddd, 1H, J = 12.9, 8.9, 8.3,

5.3 Hz, H C4), 1.38 (t, 3H, J = 7.1 Hz, CH₃CH₂OP), 1.37 (t, 3H,

4.2.34. Diethyl (3RS,5SR)-5-(3-methoxyphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10au)

J = 7.1 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d: 168.54 (s,

C(O)), 149.25, 146.31, 130.52, 111.86, 111.50, 104.82, 76.37 (d,

J = 9.2 Hz, C5), 63.53 (d, J = 166.3 Hz, C3), 63.34 (d, J = 6.4 Hz,

CH₂OP), 62.54 (d, J = 7.0 Hz, CH₂OP), 56.16 (s, CH₃O), 55.98 (s,

CH₃O), 46.72 (s, CH₃N), 36.52 (s, C4), 16.49 (d, J = 5.7 Hz,

CH₃CH₂OP), 16.43 (d, J = 5.6 Hz, CH₃CH₂OP); ³¹P NMR

(243.0 MHz, CDCl₃) d: 20.33. Anal. Calcd for C₁₇H₂₇N₂O₇P: C,

50.74; H, 6.76; N, 6.96; found: C, 50.53; H, 6.93; N, 6.90.

White amorphous solid; mp 83–84 °C; IR (KBr, cm^ꢀ1

) m_max:

3265, 3212, 3088, 2896, 1700, 1600, 1559, 1453, 1213, 1048,

1018, 957; ¹H NMR (300 MHz, CDCl₃) d: 8.20 (s, 1H, NH), 7.40–

7.35 (m, 1H), 7.35–7.31 (m, 1H), 7.04–7.01 (m, 1H), 6.72–6.69

(m, 1H), 4.61 (dd, 1H, J = 9.1, 5.5 Hz, HC5), 4.27–4.14 (m, 4H,

2 ꢁ CH₂OP), 3.82 (s, 3H, CH₃O), 3.07–2.92 (m, 2H, H_bC4 and HC3),

3.03 (s, 3H, CH₃N), 2.89–2.82 (m, 1H, H C4), 1.38 (t, 3H,

J = 6.9 Hz, CH₃CH₂OP), 1.37 (t, 3H, J = 6.9 Hz, CH₃CH₂OP); ¹³C

NMR (75.5 MHz, CDCl₃) d: 168.56 (s, C(O)), 159.78, 138.01,

129.40, 111.86, 110.22, 105.44, 76.35 (d, J = 8.9 Hz, C5), 63.24 (d,

J = 167.9 Hz, C3), 63.13 (d, J = 6.5 Hz, CH₂OP), 62.49 (d, J = 6.9 Hz,

4.2.38. Diethyl (3RS,5RS)-5-(3,4-dimethoxyphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (cis-11aw)

Colourless oil; IR (ﬁlm, cm^ꢀ1

)

_max: 3263, 2965, 2928, 1690,

1618, 1513, 1447, 1232, 1212, 1055, 1021, 972; ¹H NMR

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

1107

(300 MHz, CDCl₃) d: 8.62 (br s, 1H, NH), 7.41–7.40 (m, 1H), 6.97–

6.90 (m, 1H), 6.82–6.79 (m, 1H), 4.60 (dd, 1H, J = 8.6, 5.1 Hz,

HC5), 4.22–4.06 (m, 4H, 2 ꢁ CH₂OP), 3.89 (s, 3H, CH₃O), 3.86 (s,

3H, CH₃O), 2.98 (s, 3H, CH₃N), 3.12–2.92 (m, 2H, H_bC4 and HC3),

J = 170.6 Hz, C3), 63.17 (d, J = 6.7 Hz, CH₂OP), 62.78 (d, J = 6.8 Hz,

CH₂OP), 60.92 (s, CH₃O), 56.16 (s, 2 ꢁ CH₃O), 46.77 (s, CH₃N),

36.50 (s, C4), 16.41 (d, J = 5.9 Hz, CH₃CH₂OP), 16.35 (d, J = 5.7 Hz,

CH₃CH₂OP); ³¹P NMR (121.5 MHz, CDCl₃) d: 22.04. Anal. Calcd for

2.90–2.76 (m, 1H, H C4), 1.31 (t, 3H, J = 7.0 Hz, CH₃CH₂OP), 1.22

C₁₈H₂₉N₂O₈P: C, 50.00; H, 6.76; N, 6.48; found: C, 50.23; H, 6.97;

(t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR signals of cis-11aw were ex-

tracted from the spectrum of a 79:21 mixture of trans-10aw and

cis-11aw, ¹³C NMR (151.0 MHz, CDCl₃) d: 169.64 (s, C(O)),

149.11, 145.91, 131.15, 111.70, 111.45, 104.62, 75.83 (d,

J = 7.2 Hz, C5), 63.52 (d, J = 168.2 Hz, C3), 63.21 (d, J = 6.9 Hz,

CH₂OP), 62.74 (d, J = 6.7 Hz, CH₂OP), 56.13 (s, CH₃O), 55.95 (s,

CH₃O), 46.73 (s, CH₃N), 36.53 (s, C4), 16.41 (d, J = 5.3 Hz,

CH₃CH₂OP), 16.35 (d, J = 5.6 Hz, CH₃CH₂OP); ³¹P NMR

(121.5 MHz, CDCl₃) d: 22.06. Anal. Calcd for C₁₇H₂₇N₂O₇P: C,

50.74; H, 6.76; N, 6.96; found: C, 50.83; H, 6.94; N, 6.99.

N, 6.48.

4.2.42. Diethyl (3RS,5SR)-5-(2-cyano-4,5-

dimethoxyphenylcarbamoyl)-2-methylisoxazolidin-3-yl-3-

phosphonate (trans-10az)

White amorphous solid (crystallised from ether/hexane); mp

79–80 °C; IR (KBr, cm^ꢀ1

) m_max: 3342, 2982, 2207, 1696, 1593,

1521, 1451, 1223, 1023, 965, 751; ¹H NMR (600 MHz, CDCl₃) d:

8.87 (br s, 1H, NH), 8.10 (s, 1H), 6.99 (s, 1H), 4.65 (dd, 1H, J = 9.1,

5.0 Hz, HC5), 4.27–4.19 (m, 4H, 2 ꢁ CH₂OP), 3.98 (s, 3H, CH₃O),

3.91 (s, 3H, CH₃O), 3.13 (s, 3H, CH₃N), 3.16–3.10 (m, 1H, HC3),

3.08 (dddd, 1H, J = 15.4, 12.7, 9.1, 9.1 Hz, H_bC4), 2.84 (dddd, 1H,

4.2.39. Diethyl (3RS,5SR)-5-(3,5-dimethoxyphenylcarbamoyl)-2-

methylisoxazolidin-3-yl-3-phosphonate (trans-10ax)

J = 12.7, 8.7, 7.7, 5.0 Hz, H C4), 1.39 (t, 3H, J = 7.1 Hz, CH₃CH₂OP),

White amorphous solid; mp 118–118.5 °C. IR (KBr, cm^ꢀ1

)

m_max

1.38 (t, 3H, J = 7.1 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d:

169.59 (s, C(O)), 153.67, 145.87, 135.42, 116.46 (s, CN), 112.94,

104.44, 93.12, 76.58 (d, J = 9.5 Hz, C5), 63.35 (d, J = 167.7 Hz, C3),

63.31 (d, J = 6.4 Hz, CH₂OP), 62.62 (d, J = 6.8 Hz, CH₂OP), 56.23 (s,

CH₃O), 56.19 (s, CH₃O), 46.89 (s, CH₃N), 37.07 (s, C4), 16.46 (d,

J = 5.6 Hz, CH₃CH₂OP), 16.41 (d, J = 5.9 Hz, CH₃CH₂OP); ³¹P NMR

(243.0 MHz, CDCl₃) d: 20.03. Anal. Calcd for C₁₇H₂₃N₃O₇P: C,

49.52; H, 5.62; N, 10.19; found: C, 49.39; H, 5.83; N, 9.98.

3272, 3213, 3113, 3002, 2892, 1704, 1615, 1563, 1480, 1452, 1264,

1250, 1212, 1160, 1050, 1017, 966, 581; ¹H NMR (600 MHz, CDCl₃)

d: 8.13 (br s, 1H, NH), 6.81–6.80 (m, 2H), 6.30–6.29 (m, 1H), 4.61

(dd, 1H, J = 8.9, 5.3 Hz, HC5), 4.27–4.18 (m, 4H, 2 ꢁ CH₂OP), 3.82

(s, 6H, 2 ꢁ CH₃O), 3.12–3.07 (m, 1H, HC3), 3.04 (s, 3H, CH₃N),

3.01 (dddd, 1H, J = 15.9, 13.0, 8.9, 8.9 Hz, H_bC4), 2.86 (dddd, 1H,

J = 13.0, 8.9, 8.2, 5.3 Hz, H C4), 1.39 (t, 3H, J = 7.1 Hz, CH₃CH₂OP),

1.38 (t, 3H, J = 7.1 Hz, CH₃CH₂OP); ¹³C NMR (151.0 MHz, CDCl₃) d:

168.81 (s, C(O)), 161.17, 138.58, 98.12, 97.21, 76.41 (d, J = 9.2 Hz,

C5), 63.48 (d, J = 170.9 Hz, C3), 63.47 (d, J = 6.5 Hz, CH₂OP), 62.55

(d, J = 7.1 Hz, CH₂OP), 55.40 (s, 2 ꢁ CH₃O), 46.72 (s, CH₃N), 36.46

(s, C4), 16.48 (d, J = 5.7 Hz, CH₃CH₂OP), 16.45 (d, J = 4.0 Hz,

CH₃CH₂OP); ³¹P NMR (243.0 MHz, CDCl₃) d: 20.27. Anal. Calcd for

4.2.43. Diethyl (3RS,5SR)-5-(2-acetyl-4,5-

dimethoxyphenylcarbamoyl)-2-methylisoxazolidin-3-yl-3-

phosphonate (trans-10ba)

White amorphous solid (crystallised from ether/hexane); mp

108–112 °C; IR (KBr, cm^ꢀ1

) m_max: 3535, 3148, 2973, 2916, 1649,

C₁₇H₂₇N₂O₇P: C, 50.74; H, 6.76; N, 6.96; found: C, 50.88; H, 6.97;

1586, 1529, 1272, 1066, 1028, 963; ¹H NMR (600 MHz, CDCl₃) d:

8.57 (s, 1H), 7.33 (s, 1H), 4.61 (dd, 1H, J = 9.2, 5.0 Hz, HC5), 4.28–

4.18 (m, 4H, 2 ꢁ CH₂OP), 4.01 (s, 3H, CH₃O), 3.94 (s, 3H, CH₃O),

3.17 (s, 3H, CH₃N), 3.16–3.12 (m, 1H, HC3), 3.05 (dddd, 1H,

J = 15.8, 13.0, 9.2, 9.2 Hz, H_bC4), 2.82 (dddd, 1H, J = 13.0, 8.0, 8.0,

N, 7.01.

4.2.40. Diethyl (3RS,5SR)-5-(3,4,5-trimethoxyphenylcarbamoyl)-

2-methylisoxazolidin-3-yl-3-phosphonate (trans-10ay)

White amorphous solid (crystallised from ether/hexane); mp

5.0 Hz, H C4), 2.64 (s, 3H, CH₃C(O)), 1.39 (t, 3H, J = 7.0 Hz,

123–124 °C; IR (KBr, cm^ꢀ1

)

m_max: 3277, 3143, 2982, 2938, 1688,

CH₃CH₂OP), 1.37 (t, 3H, J = 7.0 Hz, CH₃CH₂OP); ¹³C NMR

(151.0 MHz, CDCl₃) d: 199.98 (s, C(O)), 170.77 (s, C(O)NH),

154.37, 144.02, 136.31, 115.31, 113.87, 103.77, 76.58 (d,

J = 9.5 Hz, C5), 63.37 (d, J = 165.7 Hz, C3), 63.30 (d, J = 6.3 Hz,

CH₂OP), 62.44 (d, J = 7.0 Hz, CH₂OP), 56.43 (s, CH₃O), 56.16 (s,

CH₃O), 46.39 (s, CH₃N), 37.41 (s, C4), 28.28 (s, CH₃C(O)), 16.48 (d,

J = 5.7 Hz, CH₃CH₂OP), 16.42 (d, J = 5.7 Hz, CH₃CH₂OP); ³¹P NMR

(243.0 MHz, CDCl₃) d: 20.68. Anal. Calcd for C₁₉H₂₉N₂O₈P: C,

51.35; H, 6.58; N, 6.30; found: C, 51.23; H, 6.47; N, 6.22.

1606, 1540, 1505, 1453, 1415, 1231, 1127, 1050, 1023, 969; ¹H

NMR (600 MHz, CDCl₃) d: 8.09 (br s, 1H, NH), 6.87 (s, 2H), 4.61

(dd, 1H, J = 8.9, 5.4 Hz, HC5), 4.26–4.17 (m, 4H, 2 ꢁ CH₂OP), 3.88

(s, 6H, 2 ꢁ CH₃O), 3.83 (s, 3H, CH₃O), 3.14–3.07 (m, 1H, HC3),

3.04 (s, 3H, CH₃N), 3.00 (dddd, 1H, J = 16.0, 12.9, 8.9, 8.9 Hz,

H_bC4), 2.85 (dddd, 1H, J = 12.9, 9.1, 8.2, 5.4 Hz, H C4), 1.38 (t, 3H,

J = 7.1 Hz, CH₃CH₂OP), 1.37 (t, 3H, J = 7.1 Hz, CH₃CH₂OP); ¹³C

NMR (151.0 MHz, CDCl₃) d: 168.69 (s, C(O)), 153.46, 135.26,

132.91, 97.66, 76.37 (d, J = 9.1 Hz, C5), 63.49 (d, J = 169.1 Hz, C3),

63.36 (d, J = 6.5 Hz, CH₂OP), 62.58 (d, J = 6.8 Hz, CH₂OP), 60.94 (s,

CH₃O), 56.20 (s, 2 ꢁ CH₃O), 46.72 (s, CH₃N), 36.46 (s, C4), 16.50

(d, J = 3.0 Hz, CH₃CH₂OP), 16.47 (d, J = 3.8 Hz, CH₃CH₂OP); ³¹P

NMR (243.0 MHz, CDCl₃) d: 20.27. Anal. Calcd for C₁₈H₂₉N₂O₈P: C,

50.00; H, 6.76; N, 6.48; found: C, 50.06; H, 6.92; N, 6.45.

4.3. Antiviral activity assays

The compounds were evaluated against the following viruses:

herpes simplex virus type

1 (HSV-1) strain KOS, thymidine

kinase-deﬁcient (TK^ꢀ) HSV-1 KOS strain resistant to ACV (ACV^r),

herpes simplex virus type 2 (HSV-2) strains Lyons and G, vaccinia

virus Lederle strain, respiratory syncytial virus (RSV) strain Long,

vesicular stomatitis virus (VSV), Coxsackie B4, Parainﬂuenza 3,

Inﬂuenza virus A (subtypes H1N1, H3N2), inﬂuenza virus B, Reovi-

rus-1, Sindbis, Punta Toro, human immunodeﬁciency virus type 1

strain IIIB and human immunodeﬁciency virus type 2 strain ROD.

The antiviral, other than anti-HIV, assays were based on inhibition

of virus-induced cytopathicity or plaque formation in human

embryonic lung (HEL) ﬁbroblasts, African green monkey cells

(Vero), human epithelial cells (HeLa) or Madin-Darby canine kid-

ney cells (MDCK). Conﬂuent cell cultures in microtiter 96-well

plates were inoculated with 100 CCID₅₀of virus (1 CCID₅₀being

the virus dose to infect 50% of the cell cultures) in the presence

4.2.41. Diethyl (3RS,5RS)-5-(3,4,5-trimethoxyphenylcarbamoyl)-

2-methylisoxazolidin-3-yl-3-phosphonate (cis-11ay)

Colourless oil; IR (ﬁlm, cm^ꢀ1

) m_max: 3278, 2984, 1697, 1620,

1560, 1510, 1235, 1124, 1054, 1015, 971; ¹H NMR (300 MHz,

CDCl₃) d: 8.64 (br s, 1H, NH), 6.89 (s, 2H), 4.60 (dd, 1H, J = 8.6,

5.5 Hz, HC5), 4.20–4.07 (m, 4H, 2 ꢁ CH₂OP), 3.86 (s, 6H, 2 ꢁ CH₃O),

3.82 (s, 3H, CH₃O), 3.06–2.99 (m, 2H, H_bC4 and HC3), 2.98 (s, 3H,

CH₃N), 2.94–2.74 (m, 1H, H C4), 1.32 (t, 3H, J = 7.0 Hz, CH₃CH₂OP),

1.23 (t, 3H, J = 6.9 Hz, CH₃CH₂OP); ¹³C NMR signals of cis-11ay were

extracted from the spectrum of a 74:26 mixture of trans-10ay and

cis-11ay, ¹³C NMR (151.0 MHz, CDCl₃) d: 169.79 (s, C(O)), 153.32,

134.87, 133.54, 97.56, 75.86 (d, J = 7.5 Hz, C5), 63.50 (d,

1108

K. Kokosza et al. / Bioorg. Med. Chem. 21 (2013) 1097–1108

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2010, 20, 5831.

of varying concentrations of the test compounds. Viral cytopathic-

ity was recorded as soon as it reached completion in the control

virus-infected cell cultures that were not treated with the test

compounds. Antiviral activity was expressed as the EC₅₀or com-

pound concentration required to reduce virus-induced cytopathog-

enicity or viral plaque formation by 50%.

9. Lee, Y.-S.; Park, S. M.; Kim, H. M.; Park, S.-K.; Lee, K.; Lee, C. W.; Kim, B. H.

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4.4. Anti-HIV activity assays

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Inhibition of HIV-1(III_B)- and HIV-2(ROD)-induced cytopathicity

in CEM cell cultures was measured in microtiter 96-well plates

containing 3 ꢁ 10⁵CEM cells/mL infected with 100 CCID₅₀of HIV

per milliliter and containing appropriate dilutions of the test com-

pounds. After 4ꢀ5 days of incubation at 37 °C in a CO₂-controlled

humidiﬁed atmosphere, CEM giant (syncytium) cell formation

was examined microscopically. The EC₅₀(50% effective concentra-

tion) was deﬁned as the compound concentration required to

inhibit HIV-induced giant cell formation by 50%.

4.5. Cytostatic activity assays

All assays were performed in 96-well microtiter plates. To each

well were added (5ꢀ7.5) ꢁ 10⁴tumor cells and a given amount of

the test compound. The cells were allowed to proliferate for 48 h

(murine leukemia L1210 cells) or 72 h (human lymphocytic CEM

and human cervix carcinoma HeLa cells) at 37 °C in a humidiﬁed

CO₂-controlled atmosphere. At the end of the incubation period,

the cells were counted in a Coulter counter. The IC₅₀(50% inhibi-

tory concentration) was deﬁned as the concentration of the com-

pound that inhibited cell proliferation by 50%.

21. Singh, R.; Bhella, S. S.; Sexana, A. K.; Shanmugavel, M.; Faruka, A.; Ishar, M. P. S.

Tetrahedron 2007, 63, 2283.

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Acknowledgments

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1999, 47, 1006.

The authors wish to express their gratitude to Mrs. Jolanta

Płocka, Mrs. Leentje Persoons, Mrs. Frieda De Meyer, Mrs. Leen In-

gels and Mrs. Lizette van Berckelaer for excellent technical assis-

tance. The preliminary experiments in this project was

conducted by Miss Katarzyna Raj, M.Sc. The synthetic part of this

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work was supported by the Medical University of Łódz internal

funds (503/3-014-1/503-01 and 502-03-/3-014-01/502-34-020).

The biological part of this work was supported by the KU Leuven

(GOA 10/014).

References and notes

38. Somoza, A.; Chelliserrykattil, J.; Kool, E. T. Angew. Chem., Int. Ed. 2006, 45, 4994.

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