New isoxazolidine-conjugates of quinazolinones-synthesis, antiviral and cytostatic activity

Article abstract of DOI:10.3390/molecules21070959

A novel series of (3-diethoxyphosphoryl)isoxazolidines substituted at C5 with various quinazolinones have been synthesized by the 1,3-dipolar cycloaddition of N-methyl-C- (diethoxyphosphoryl)nitrone with N3-substitued 2-vinyl-3H-quinazolin-4-ones. All isoxazolidines were assessed for antiviral activity against a broad range of DNA and RNA viruses. Isoxazolidines trans-11f/cis-11f (90:10), trans-11h and trans-11i/cis-11i (97:3) showed weak activity (EC₅₀ = 6.84, 15.29 and 9.44 μM) toward VZV (TK⁺ strain) which was only one order of magnitude lower than that of acyclovir used as a reference drug. Phosphonates trans-11b/cis-11b (90:10), trans-11c, trans-11e/cis-11e (90:10) and trans-11g appeared slightly active toward cytomegalovirus (EC₅₀ = 27-45 μM). Compounds containing benzyl substituents at N3 in the quinazolinone skeleton exhibited slight antiproliferative activity towards the tested immortalized cells with IC₅₀ in the 21-102 M range.

Full text of DOI:10.3390/molecules21070959

molecules
Article
New Isoxazolidine-Conjugates of
Quinazolinones—Synthesis, Antiviral and
Cytostatic Activity
1
,
2
2
2
Dorota G. Piotrowska *, Graciela Andrei , Dominique Schols , Robert Snoeck and
Magdalena Grabkowska-Dru z˙ yc 1
1
Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of Lodz, Muszy n´ skiego 1,
9
0-151 Lodz, Poland; magdalena.grabkowska-druzyc@umed.lodz.pl
2
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium;
Graciela.Andrei@rega.kleuven.be (G.A.); Dominique.Schols@rega.kleuven.be (D.S.);
Robert.Snoeck@rega.kleuven.be (R.S.)
*
Correspondence: dorota.piotrowska@umed.lodz.pl; Tel.: +48-42-677-92-35; Fax: +48-42-678-83-98
Academic Editor: Derek J. McPhee
Received: 16 June 2016; Accepted: 19 July 2016; Published: 22 July 2016
Abstract: A novel series of (3-diethoxyphosphoryl)isoxazolidines substituted at C5 with
various quinazolinones have been synthesized by the 1,3-dipolar cycloaddition of N-methyl-C-
(
diethoxyphosphoryl)nitrone with N3-substitued 2-vinyl-3H-quinazolin-4-ones. All isoxazolidines
were assessed for antiviral activity against a broad range of DNA and RNA viruses. Isoxazolidines
trans-11f/cis-11f (90:10), trans-11h and trans-11i/cis-11i (97:3) showed weak activity (EC50 = 6.84
,
+
15.29 and 9.44
µM) toward VZV (TK strain) which was only one order of magnitude lower
than that of acyclovir used as a reference drug. Phosphonates trans-11b/cis-11b (90:10),
trans-11c, trans-11e/cis-11e (90:10) and trans-11g appeared slightly active toward cytomegalovirus
(
EC50 = 27–45 µM). Compounds containing benzyl substituents at N3 in the quinazolinone skeleton
exhibited slight antiproliferative activity towards the tested immortalized cells with IC50 in the
1–102 µM range.
2
Keywords: phosphonate; isoxazolidine; quinazolinones; antiviral; cytostatic
1
. Introduction
Nitrogen-containing heterocycles form the core of natural products (e.g., alkaloids) and they
are also present in many pharmacophores as well as in numerous marketed drugs. Among them,
quinazolines and quinazolinones have drawn special attention due to the broad spectrum of
biological activities of their derivatives, including sedative [
antibacterial [13 15], antifungal [15 16], anti-inflamatory [15 17
Several reviews focused on the synthetic strategies and biological activities of these compounds
have been published [22 29]. The significant impact of various functional groups installed into
1–
3
], anticancer [
4–
7], antiviral [
8–12],
–
,
,
–19] and antifibrotic [20
,21] activities.
–
quinazoline/quinazolinone frameworks on pharmacological properties have been proven.
In the last decades several compounds containing the quinazolin-4-one framework, which
exhibited promising anticancer as well as antiviral properties, have been obtained (Figure 1).
Furthermore, some biologically active substituted quinazolin-4(3H)-ones were isolated from various
fungi and bacteria species. For example, 2-(4-hydroxybenzyl)quinazolin-4(3H)-one (1) was
found in an entomopathogenic fungus Isaria farinosa and its strong inhibitory properties on the
replication of tobacco mosaic virus (TMV) [30] were recognised, whereas its 2-(4-hydroxybenzoyl)
analogue
Moreover, compound
2
present in fungus from Penicillium genus appeared only slightly active toward TMV [30].
exhibited significant cytotoxicity toward various cancer cell lines [31 32].
1
,
Molecules 2016, 21, 959; doi:10.3390/molecules21070959
www.mdpi.com/journal/molecules

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Quinazolinone
synthetic pyridine-containing analogue
and their slight activity against influenza A virus was revealed [34]. On the other hand, various
,3-disubstitued quinazolin-4(3H)-ones, including compounds 10, have been found to possess
antitumor activity [35].
3
isolated from Streptomyces sp. appeared cytotoxic against Vero cells [33]. Very recently
4
and its 3-substituted derivatives and have been obtained
5
6
2
7–
Figure 1. Examples of quinazolinones with antiviral and anticancer activity.
In continuation of our studies on antiviral and cytostatic activity of isoxazolidine analogues
of C-nucleoside analogues, we designed a new series of compounds of the general formula 11
containing a substituted quinazolinone moiety as a false nucleobase at C5 in the isoxazolidine
ring and the diethoxyphosphoryl function attached at C3. Our synthetic strategy to compounds
trans-11/cis-11 relies on the 1,3-dipolar cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone
1
2 [36] with 2-vinyl-3H-quinazolin-4-ones 13 substituted at N3 (Scheme 1).
Scheme 1. Retrosynthesis of (isoxazolidinyl) phosphonates trans-11/cis-11.
2
. Results and Discussion
2
.1. Chemistry
2
-Vinyl-3H-quinazolin-4-ones 13 modified at N3 with substituted benzyl groups were synthesized
from commercially available 2-aminobenzamide (14) by acylation with 3-chloro-propionyl chloride
followed by cyclization and dehydrohalogenation to prepare 2-vinyl-3H-quinazolin-4-one (13a) as a key
intermediate [37] and a subsequent reaction with substituted benzyl bromides 13b
–i [38] (Scheme 2).
Moreover, compounds 13j (R = Me) and 13k (R = Et) were also obtained with intention to determine the
influence of the benzyl substituent on biological activity of the designed isoxazolidines trans-11/cis-11
.
1
In the H-NMR spectra of compounds 13a
–
k
characteristic signals for vinyl protons were observed in
the 6.94–5.59 ppm (three doublets of doublets).

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Scheme 2. Synthesis of Compounds 13a–k. Reaction and conditions: a. 3-chloropropionyl chloride,
˝
˝
1
6
,4-dioxane, 0 C; b. 5% NaOH-EtOH (2:1); c. RBr, KOH, CH CN, 105 C; d. MeI or EtI, KOH, CH CN,
0 C.
3
3
˝
The 1,3-dipolar cycloaddition of a nitrone 12 with 2-vinylquinazolinones 13a
–
k
led to the
formation of diastereoisomeric mixtures of 5-substituted (3-diethoxyphosphoryl)isoxazolidines trans-11
and cis-11 with good (80%–88%) diastereoselectivities (Scheme 3, Table 1). Ratios of cis/trans
diastereoisomers were calculated from 31P-NMR spectra of crude reaction mixtures and confirmed
1
by the analysis of H-NMR spectral data. Crude mixtures of isoxazolidine cycloadducts were then
subjected to purification on silica gel columns. However, attempts to isolate pure diastereoisomers
were fruitful for trans-11a (R = H), trans-11c (R = 2-NO -C H -CH ), trans-11g (R = 3-F-C H -CH ),
2
6
4
2
6
4
2
trans-11h (R = 4-F-C H -CH ) and trans-11j (R = Me) only.
6
4
2
Scheme 3. Synthesis of Isoxazolidines cis-11a
–
k
and trans-11a–k. Reaction and conditions: a. toluene,
˝
7
0 C, 24 h.
Table 1. Isoxazolidines trans-11 and cis-11 obtained according to Scheme 3.
Quinazolinone 13
R
Ratio of
Trans-11:Cis-11
Entry
Yield (%)
trans-11a (23) a + trans-11a and cis-11a (70) b
trans-11b and cis-11b (84) b
a
b
c
d
e
f
g
h
i
H
92:8
90:10
90:10
92:8
90:10
90:10
90:10
90:10
92:8
C6H5-CH2
trans-11c (20) a + trans-11c and cis-11c (73)
b
2-NO2-C6H4-CH2
3-NO2-C6H4-CH2
4-NO2-C6H4-CH2
2-F-C6H4-CH2
3-F-C6H4-CH2
4-F-C6H4-CH2
2,4-diF-C6H3-CH2
Me
b
trans-11d and cis-11d (98)
trans-11e and cis-11e (91)
trans-11f and cis-11f (94)
b
b
trans-11g (7) a + trans-11g and cis-11g (89) b
a
b
trans-11h (22) + trans-11h and cis-11h (70)
trans-11i and cis-11i (94) b
trans-11j (19) a + trans-11j and cis-11j (78)
b
j
k
94:6
92:8
trans-11k and cis-11k (86) b
Et
a
yield of pure isomer; b yield of pure mixture of cis- and trans-isomers.
The relative configurations of isoxazolidines trans-11a and cis-11a were established based on our
previous studies on stereochemistry of cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone (12
)

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1
with various vinyl aryls [39,40] since similar H-NMR spectral patters for the respective series of trans-
and cis-isoxazolidines were observed. Since for compound trans-11a all necessary coupling constants
1
13
were successfully extracted from the H- and C-NMR spectra, detailed conformational analysis was
performed based on these data {J(H3-H4α) = 9.3 Hz [41], J(H3-H4β) = 8.3 Hz, J(H4α-P) = 9.9 Hz [42 43],
J(H4β-P) = 16.9 Hz, J(H4α-H5) = 6.2 Hz, J(H4β-H5) = 8.3 Hz, J(CCCP) = 8.5 Hz [44 45]} and revealed that
,
,
isoxazolidine ring in trans-11a adopts a E conformation in which the diethoxyphosphoryl group
3
resides in the equatorial position of the isoxazolidine ring while a quinazolinone substituent is located
pseudoequatorially (Figure 2). On the other hand, cis configuration of the minor isomer was established
from the corresponding couplings [J(H3-H4α) = 9.0 Hz, J(H3-H4β) = 6.5 Hz, J(H4α-P) = 11.2 Hz, J(H4β-P)
=
20.0 Hz, J(H4α-H5) = 9.1 Hz, J(H4β-H5) = 3.9 Hz, J(CCCP) = 7.3 Hz] indicating the E conformation of
2
the isoxazolidine ring (Figure 2). The additional arguments to support our assignments follow from
shielding of the CH CH OP protons observed for the cis isomer (∆δ ca. 0.1 ppm) when compared with
3
2
1
the trans-11a. Furthermore, it was found that on a H-NMR spectrum taken on the 83:17 mixture of
cis- and trans-11a, the H-N proton in the quinazolinone ring of cis-11a was significantly deshielded
(
∆δ = 0.7 ppm) when compared with the trans isomer, highly likely, as a result of the hydrogen
bond formation with the phosphoryl oxygen amide, a phenomenon spatially achievable in the cis
isomer only.
Since introduction of various substituents at N3 of quinazolinone moiety has no influence on the
stereochemical outcome of the cycloaddition therefore configuration of the all major isoxazolidines 11
were assigned as trans, thereby minor ones as cis.
Figure 2. The preferred conformations of trans-isoxazolidine trans-11a.
2
.2. Antiviral and Cytostatic Evaluation
2
.2.1. Antiviral Activity
All obtained phosphonates trans-11a, trans-11c, trans-11g, trans-11h and trans-11j as well as
mixtures of diastereoisomeric phosphonates trans-11b/cis-11b (90:10), trans-11d/cis-11d (90:10),
trans-11e/cis-11e (90:10), trans-11f/cis-11f (90:10), trans-11i/cis-11i (97:3) and trans-11k/cis-11k (97:3)
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 simplex virus-1 (KOS),
herpes simplex virus-2 (G), thymidine kinase deficient (acyclovir resistant) herpes simplex virus-1
´
r
(
TK KOS ACV ), vaccinia virus, adeno virus-2, vesicular stomatitis virus, cytomegalovirus (AD-169
+
´
strain -1 and Davis strain), varicella-zoster virus (TK VZV Oka strain and TK VZV 07-1 strain);
(
(
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, yellow fever virus; (e) CRFK cell cultures: feline corona virus (FIPV) and feline herpes virus
FHV) and (d) MDCK cell cultures: influenza A virus (H1N1 and H3N2 subtypes) and influenza B virus.
(
Ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, zanamivir, alovudine, amantadine, rimantadine,
ribavirin, dextran sulfate (molecular weight 10,000, DS-10000), mycophenolic acid, Hippeastrum
hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA) were used as the reference compounds.

Molecules 2016, 21, 959
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The antiviral activity was expressed as the EC : the compound concentration required to reduce virus
50
plaque formation (VZV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses).
+
´
Several isoxazolidines trans-11/cis-11 were able to weakly inhibit the replication of TK and TK
VZV strains with EC50 values in the range of 6.84–100 M (Table 2). Among them, phosphonates
trans-11f/cis-11f (90:10) (R = 2-F-C H -CH ) (EC = 6.84 M), trans-11h (R = 4-F-C H -CH )
µ
µ
6
4
2
50
6
4
2
(
EC50 = 15.29 µM), trans-11i/cis-11i (97:3) (R = 2,4-diF-C H -CH ) (EC = 9.44 µM) were the most
6 3 2 50
+
´
active toward TK VZV Oka strain, while exhibiting no activity toward TK VZV strain. The activity
of these isoxazolidines trans-11/cis-11 against TK VZV Oka strain was 8- to 22-folds lower than that
+
of the reference drug acyclovir.
Table 2. Cytotoxicity and antiviral activity against varicella-zoster virus (VZV) in HEL cell cultures.
Antiviral Activity EC50 (µM) a
Cytotoxicity (µM)
Compound
R
TK+ VZV Strain
TK VZV Strain
´
Cell Morphology MCC b
trans-11c
trans-11e/cis-11e (90:10)
trans-11f/cis-11f (90:10)
trans-11h
trans-11i/cis-11i (97:3)
trans-11k
2-NO2-C6H4-CH2
4-NO2-C6H4-CH2
2-F-C6H4-CH2
4-F-C6H4-CH2
2,4-diF-C6H3-CH2
CH3CH2
46.47
34.20
6.84
15.29
9.44
38.80
0.71
0.019
100
42.87
>20
>20
>20
41.57
39.69
25.59
>100
100
100
100
100
>100
>100
>100
Acyclovir
Brivudin
a
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque
b
forming units (PFU); Minimum cytotoxic concentration that causes a microscopically detectable alternation of
cell morphology.
´
On the other hand, the EC50 values for the TK VZV 07-1 strain (which is an acyclovir resistant
strain) of the phosphonates trans-11e/cis-11e (90:10) (R = 4-NO -C H -CH ) (EC50 = 42.87 µM
)
2
6
4
2
and trans-11k/cis-11k (97:3) (R = Et) (EC50 = 41.57
µM) were comparable to that of acyclovir
+
´
(
EC50 = 39.69 µM). These derivatives showed similar EC ’s for TK and TK VZV strains and
50
+
therefore their potency against TK VZV was approximately 50-fold lower compared to acyclovir.
Furthermore, compounds trans-11b/cis-11b (90:10) (R = C H -CH ), trans-11c (R = 2-NO -C H -
6
5
2
2
6
4
CH ), trans-11e/cis-11e (90:10) (R = 4-NO -C H -CH ) and trans-11g (R = 3-F-C H -CH ) showed
2
2
6
4
2
6
4
2
some activity against human cytomegalovirus (EC50 = 27–45 µM), although they were less active
than ganciclovir and cidofovir used as the reference compounds (Table 3). None of the phosphonate
derivatives here described showed activity against the other tested DNA and RNA viruses.
Table 3. Antiviral activity and cytotoxicity against human cytomegalovirus in HEL cell cultures.
Antiviral Activity EC50 (µM) a
Cytotoxicity (µM)
Compound
R
Cell Morphology MCC b
AD-169 Strain
Davis Strain
trans-11b/cis-11b (90:10)
trans-11c
trans-11e/cis-11e (90:10)
trans-11g
C6H5-CH2
44.72
>100
44.72
>100
10.52
1.49
>20
44.72
20
27.59
0.63
0.23
ě100
ě20
>100
100
>350
>300
2-NO2-C6H4-CH2
4-NO2-C6H4-CH2
3-F-C6H4-CH2
Ganciclovir
Cidofovir
a
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque
b
forming units (PFU); Minimum cytotoxic concentration that causes a microscopically detectable alternation of
cell morphology.
2
.2.2. Cytostatic Activity
The 50% cytostatic inhibitory concentration (IC ) causing a 50% decrease in cell proliferation was
50
determined against murine leukemia L1210, human lymphocyte CEM, human cervix carcinoma HeLa
and immortalized human dermal microvacsular endothelial cells (HMEC-1). Isoxazolidines trans-11a
(R = H) and trans-11j (R = Me) did not inhibit cell proliferation at the highest tested concentration

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(
i.e., 250
cell lines (IC50 = 85–101
substituents at N3 in the quinazolinone moiety showed lower IC50 values (IC50 = 21–102
µ
M), whereas trans-11k/cis-11k (97:3) (R = Et) appeared slightly cytostatic towards the tested
M). On the other hand (Table 4, entries to ), compounds having benzyl
M) thereby
µ
b
i
µ
indicating that installation of functionalized benzyl groups was profitable for inhibitory properties.
Table 4. Inhibitory effect of the tested compounds against the proliferation of murine leukemia (L1210),
human T-lymphocyte (CEM), human cervix carcinoma (HeLa) and immortalized human dermal
microvascular endothelial cells (HMEC-1).
IC50 a (µM)
Compound
R
L1210
CEM
HeLa
HMEC-1
trans-11a
trans-11b/cis-11b (90:10)
trans-11c
trans-11d/cis-11d (90:10)
trans-11e/cis-11e (90:10)
trans-11f/cis-11f (90:10)
trans-11g
trans-11h
trans-11i/cis-11i (97:3)
trans-11j
H
>250
49 ˘ 23
87 ˘ 22
28 ˘ 11
59 ˘ 32
33 ˘ 7
>250
28 ˘ 4
76 ˘ 9
21 ˘ 4
34 ˘ 12
29 ˘ 13
26 ˘ 6
30 ˘ 12
24 ˘ 8
>250
>250
82 ˘ 5
>250
83 ˘ 16
92 ˘ 1
58 ˘ 0
102 ˘ 3
77 ˘ 0
74 ˘ 3
78 ˘ 1
67 ˘ 4
>250
C H -CH
6
5
2
2-NO -C H -CH
3-NO -C H -CH
4-NO -C H -CH
97 ˘ 0
2
6
4
2
2
2
50 ˘ 4
2
6
4
76 ˘ 4
2
6
4
2-F-C H -CH
76 ˘ 11
62 ˘ 9
6
4
2
2
2
3-F-C H -CH
35 ˘ 12
26 ˘ 1
6
4
4-F-C H -CH
58 ˘ 5
6
4
2,4-diF-C H -CH
26 ˘ 2
55 ˘ 10
6
3
2
Me
Et
>250
>250
trans-11k/cis-11k (97:3)
101 ˘ 17
85 ˘ 13
97 ˘ 10
0.54 ˘ 0.12
86 ˘ 11
5
-Fluorouracil
0.33 ˘ 0.17
18 ˘ 5
n.d.
a
50% Inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%;
n.d.—not determined.
3
. Experimental Section
3
.1. General
1
H-NMR spectra were taken in CDCl on the following spectrometers: Gemini 2000BB (200 MHz
3
Varian, Palo Alto, CA, USA), and Avance III (600 MHz, Bruker Instruments, Karlsruhe, Germany)
1
3
with TMS as internal standard.
Bruker Avance III at 151.0 MHz.
C-NMR spectra were recorded for CDCl solution on the
3
3
1
P-NMR spectra were performed in CDCl solution on the
3
Varian Gemini 2000 BB at 81.0 MHz or on Bruker Avance III at 243.0 MHz. IR spectra were
measured on an Infinity MI-60 FT-IR spectrometer (ATI Instruments North America-Mattson, Madison,
WI, USA). Melting points were determined on a Boetius apparatus (VEB Kombinat NAGEMA,
Dresden, Germany) and are uncorrected. Elemental analyses were performed by the Microanalytical
Laboratory of this Faculty on Perkin-Elmer PE 2400 CHNS analyzer (Perkin-Elmer Corp., Norwalk,
CT, USA). The following adsorbents were used: column chromatography, Merck silica gel 60
(70–230 mesh); analytical TLC, Merck (Merck KGaA, Darmstadt, Germany) TLC plastic sheets silica gel
6
3
0 F254. N-methyl-C-(diethoxyphosphoryl)nitrone (12) [36], 2-vinyl-3H-quinazolin-4-one (13a) [37] and
-methyl-2-vinyl-3H-quinazolin-4-one (13j) [38] were obtained according to the literature procedures.
1
13
31
H-, C- and P-NMR spectra of all new synthesised compounds are provided in Supplementary
Materials (Figures S1–S54).
3
.2. General Procedure for the Synthesis of N3-Benzylated 2-Vinyl-3H-quinazolin-4-ones 13b–i
To the solution of 2-vinyl-3H-quinazolin-4-one (13a, 1.00 mmol) in acetonitrile (15 mL) potassium
carbonate (3.00 mmol) was added. After 15 min the respective benzyl bromide (1.10 mmol) was added
and the reaction mixture was stirred under reflux for 4 h. A solvent was removed and the residue
was extracted with water (3
crude product was purified on a silica gel column with a methylene chloride: hexane mixture (7:3, v/v)
followed by crystallisation (chloroform-petroleum ether) to give pure quinazolinones 13b and 13g–i.
ˆ
10 mL). An organic layer was dried (MgSO ), concentrated and the
4
–e

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˝
˝
C
3
-Benzyl-2-vinylquinazolin-4(3H)-one (13b).
White amorphous solid, m.p.
= 85 C–87
νmax: 3027, 2950, 2925, 1677, 1615, 1574, 1425,
= 8.20–8.19 (m, 1H), 7.92–7.88 (m,
´
1
(reference [46]—colorless viscous oil). IR (KBr, cm
)
1
1
1
312, 1271, 952, 845, 778, 755, 585. H-NMR (200 MHz, CDCl ):
δ
3
3
3
H), 7.83–7.78 (m, 1H), 7.58–7.49 (m, 3H), 7.46–7.36 (m, 3H), 6.94 (dd, J = 17.2 Hz, J = 10.1 Hz, 1H,
CH=CH ), 6.73 (dd, J = 17.2 Hz, J = 2.3 Hz, 1H, CH=CH ), 5.80 (dd, J = 10.1 Hz, J = 2.3 Hz, 1H,
3
2
3
2
2
2
13
CH=CH ), 5.69 (s, 2H, N-CH ). C-NMR (151 MHz, CDCl3):
δ
= 166.12 (s, C(O)), 159.97, 151.63, 137.17,
2
2
1
36.44, 133.57, 128.60, 128.28, 128.25, 127.67, 126.60, 123.81, 123.61, 115.59, 68.32 (s, N-CH ). Anal.
2
Calcd. for C H N O: C, 77.84; H, 5.38; N, 10.68. Found: C, 77.69; H, 5.21; N, 10.88.
17
14
2
˝ ˝
-(2-Nitrobenzyl)-2-vinylquinazolin-4(3H)-one (13c). Yellowish amorphous solid, m.p. = 88 C–91 C.
3
´
1
IR (KBr, cm
H- NMR (600 MHz, CDCl3):
)
νmax: 2952, 2851, 1615, 1574, 1525, 1493, 1396, 1312, 988, 1104, 988, 938, 780, 723, 664.
= 8.24–8.22 (m, 1H), 8.18–8.16 (m, 1H), 7.96–7.94 (m, 1H), 7.87–7.82
1
δ
3
3
(
(
1
1
m, 2H), 7.69–7.67 (m, 1H), 7.59–7.53 (m, 2H), 6.91 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H, CH=CH ), 6.64
2
3
2
3
2
dd, J = 17.2 Hz, J = 1.2 Hz, 1H, CH=CH ), 6.14 (s, 2H, N-CH ), 5.78 (dd, J = 10.4 Hz, J = 1.2 Hz,
2
2
H, CH=CH2). 13C-NMR (151 MHz, CDCl3):
δ
= 165.51 (s, C(O)), 159.83, 151.80, 147.86, 136.77, 133.78,
33.65, 132.77, 129.02, 128.67, 128.86, 127.86, 126.85, 124.97, 124.12, 123.56, 123.26, 115.26, 64.79 (s,
N-CH ). Anal. Calcd. for C H N O : C, 66.44; H, 4.26; N, 13.67. Found: C, 66.23; H, 4.11; N, 13.38.
2
17 13
3
3
˝ ˝
-(3-Nitrobenzyl)-2-vinylquinazolin-4(3H)-one (13d). Yellowish amorphous solid, m.p. = 90 C–93 C.
3
´
1
IR (KBr, cm
H-NMR (600 MHz, CDCl3):
)
νmax: 3093, 3020, 2958, 2941, 1615, 1574, 1562, 1523, 1345, 1044, 991, 965, 801, 764, 687.
= 8.46 (brs, 1H), 8.25–8.23 (m, 1H), 8.22–8.20 (m, 1H), 7.94–7.89 (m,
1
δ
3
3
2H), 7.86–7.83 (m, 1H), 7.63–7.60 (m, 1H), 7.57–7.55 (m, 1H), 6.94 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H,
3
2
3
2
CH=CH ), 6.71 (dd, J = 17.2 Hz, J = 1.6 Hz, 1H, CH=CH ), 5.82 (dd, J = 10.4 Hz, J = 1.6 Hz, 1H,
2
2
13
CH=CH ), 5.80 (s, 2H, N-CH ). C-NMR (151 MHz, CDCl3):
δ
= 165.61 (s, C(O)), 159.72, 151.74,
48.48, 138.58, 136.98, 133.89, 133.82, 129.61, 127.82, 126.88, 123.89, 123.33, 123.15, 122.98, 115.24, 66.83
s, N-CH ). Anal. Calcd. for C H N O : C, 66.44; H, 4.26; N, 13.67. Found: C, 66.12; H, 3.97; N, 13.40.
2
2
1
(
2
17 13
3
3
˝ ˝
-(4-Nitrobenzyl)-2-vinylquinazolin-4(3H)-one (13e). Yellowish amorphous solid, m.p. = 131 C–132 C.
3
´
1
1
IR (KBr, cm
)
νmax: 2944, 2849, 1608, 1570, 1514, 1488, 1338, 1162, 984, 854, 812, 704, 680. H-NMR
(
600 MHz, CDCl3): δ = 8.30–8.28 (m, 2H), 8.24–8.21 (m, 1H), 7.96–7.94 (m, 1H), 7.87–7.84 (m, 1H),
3
3
7
.73–7.72 (m, 2H), 7.58–7.55 (m, 1H), 6.93 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H, CH=CH ), 6.68 (dd,
2
3
2
3
2
J = 17.2 Hz, J = 1.8 Hz, 1H, CH=CH ), 5.82 (s, 2H, N-CH ), 5.80 (dd, J = 10.4 Hz, J = 1.6 Hz,
2
2
13
1
1
H, CH=CH2). C-NMR (151 MHz, CDCl3):
δ
= 165.59 (s, C(O)), 159.74, 151.78, 147.77, 143.81,
36.97, 133.87, 128.31, 127.89, 126.91 123.91, 123.85, 123.27, 115.25, 66.78 (s, N-CH ). Anal. Calcd. for
2
C H N O : C, 66.44; H, 4.26; N, 13.67. Found: C, 66.23; H, 4.03; N, 13.33.
17
13
3
3
´
3
1
-(2-Fluorobenzyl)-2-vinylquinazolin-4(3H)-one (13f). Colorless oil; IR (film, cm 1) νmax: 3061, 1676,
1
618, 1496, 1422, 1349, 1104, 988, 941, 759, 680, 661. H-NMR (200 MHz, CDCl ):
δ
= 8.17–8.13 (m,
3
3
1
H), 7.92–7.74 (m, 2H), 7.61–7.45 (m, 2H), 7.41–7.29 (m, 1H), 7.26–7.08 (m, 2H), 6.93 (dd, J = 17.2 Hz
J = 10.0 Hz, 1H, CH=CH ), 6.74 (dd, J = 17.2 Hz, J = 2.4 Hz, 1H, CH=CH ), 5.79 (dd, J = 10.0 Hz
,
3
3
2
3
,
2
2
2
13
J = 2.4 Hz, 1H, CH=CH ), 5.76 (s, 2H, N-CH ). C-NMR (151 MHz, CDCl ):
δ
= 165.95 (s, C(O)),
= 3.5 Hz, C4),
2
2
3
3
1
161.15 (d, J
= 249.1 Hz, C2), 159.93, 151.69, 137.12, 133.57, 130.54 (d, J
= 8.5 Hz, C6), 127.71, 126.61, 124.17 (d, J
(
CF)
(CCCF)
3
4
1
30.13 (d, J
= 3.4 Hz, C5), 123.85, 123.66 (d,
= 14.4 Hz, C3), 123.56, 115.54 (d, J(CCF) = 21.1 Hz, C1), 115.51, 62.16 (d, J
(
CCCF)
(CCCCF)
2J(
2
3
= 4.4 Hz,
(CCCF)
CCF)
N-CH ). Anal. Calcd. for C H FN O: C, 72.85; H, 4.67; N, 9.99. Found: C, 72.78; H, 4.41; N, 9.80.
2
17 13
2
˝ ˝
-(3-Fluorobenzyl)-2-vinylquinazolin-4(3H)-one (13g). White amorphous solid, m.p. = 58 C–59 C.
3
´
1
1
IR (KBr, cm
) νmax: 3095, 3057, 3032, 1618, 1578, 1498, 1422, 1349, 1106, 988, 945, 681, 520. H-NMR
(
200 MHz, CDCl3):
δ
= 8.20–8.15 (m, 1H), 7.93–7.88 (m, 1H), 7.84–7.75 (m, 1H), 7.55–7.47 (m, 1H),
.39–7.22 (m, 3H), 7.10–7.01 (m, 1H), 6.93 (dd, 3J = 17.2 Hz, J = 10.1 Hz, 1H, CH=CH ), 6.70 (dd,
3
7
2
3
2
3
2
J = 17.2 Hz, J = 2.3 Hz, 1H, CH=CH ), 5.79 (dd, J = 10.1 Hz, J = 2.3 Hz, 1H, CH=CH ), 5.67 (s, 2H,
2
2
N-CH2). 13C-NMR (151 MHz, CDCl ):
1
δ
= 165.88 (s, C(O)), 162.95 (d, J
1
= 246.6 Hz, C3), 159.88,
(CF)
= 8.8 Hz, C1), 127.75, 126.72,
(CCCF)
3
51.69, 138.97 (d, 3J(CCCF) = 7.8 Hz, C5), 137.09, 133.67, 130.15 (d, J
3

Molecules 2016, 21, 959
8 of 15
1
2J(
23.84, 123.53 (d, 4J(CCCCF) = 2.4 Hz, C6), 123.48, 115.45, 115.09 (d, 2J(CCF) = 21.0 Hz, C4), 114.93 (d,
= 22.0 Hz, C2), 67.39 (s, N-CH ). Anal. Calcd. for C H FN O: C, 72.85; H, 4.67; N, 9.99. Found:
2 17 13 2
CCF)
C, 72.66; H, 4.38; N, 9.95.
˝
˝
3
-(4-Fluorobenzyl)-2-vinylquinazolin-4(3H)-one (13h). White amorphous solid, m.p. = 125 C–127 C. IR
´
1
1
(
KBr, cm
)
νmax: 3058, 1677, 1618, 1493, 1425, 1349, 1102, 992, 942, 757, 683, 658. H-NMR (200 MHz,
CDCl3):
δ = 8.12–8.07 (m, 1H), 7.87–7.82 (m, 1H), 7.78–7.73 (m, 1H), 7.51–7.40 (m, 3H), 7.10–6.97
3
3
3
2
(m, 2H), 6.88 (dd, J = 17.2 Hz, J = 10.1 Hz, 1H, CH=CH ), 6.66 (dd, J = 17.2 Hz, J = 2.3 Hz, 1H,
2
3
2
13
CH=CH ), 5.74 (dd, J = 10.1 Hz, J = 2.3 Hz, 1H, CH=CH ), 5.59 (s, 2H, N-CH ). C-NMR (151 MHz,
2
2
2
1
CDCl ): δ = 165.99 (s, C(O)), 162.71 (d, J
= 249.1 Hz, C4), 159.90, 151.69, 137.19, 133.59, 132.26 (d,
(CF)
= 7.7 Hz, C2, C6), 127.74, 126.62, 123.70, 123.49, 115.55 (d,
3
4
2
3
J(CCCCF) = 3.2 Hz, C1), 130.22 (d, J
J(CCF) = 21.3 Hz, C3, C5), 67.58 (s, N-CH ). Anal. Calcd. for C H FN O: C, 72.85; H, 4.67; N, 9.99.
(CCCF)
2
17 13
2
Found: C, 72.67; H, 4.30; N, 9.70.
˝
˝
.
3
-(2,4-Difluorobenzyl)-2-vinylquinazolin-4(3H)-one (13i). Yellowish amorphous solid, m.p. = 67 C–68 C
´
1
IR (KBr, cm
)
νmax: 3076, 3015, 2929, 1681, 1606, 1574, 1505, 1351, 1279, 1100, 964, 851, 772, 666, 683.
= 8.10–8.05 (m, 1H), 7.86–7.82 (m, 1H), 7.78–7.68 (m, 1H), 7.57–7.40
m, 2H), 6.90–6.78 (m, 2H), 6.88 (dd, J = 17.2 Hz, J = 10.1 Hz, 1H, CH=CH ), 6.67 (dd, J = 17.2 Hz,
1
H-NMR (200 MHz, CDCl3):
δ
3
3
3
(
2
2
3
2
J = 2.3 Hz, 1H, CH=CH ), 5.74 (dd, J = 10.1 Hz, J = 2.3 Hz, 1H, CH=CH ), 5.65 (s, 2H, N-CH ).
2
2
2
13
1
3
C-NMR (151 MHz, CDCl ):
δ
= 165.84 (s, C(O)), 163.11 (dd, J
= 249.9 Hz, J = 12.1 Hz,
= 12.1 Hz, C4), 159.85, 151.69, 137.11, 133.64, 131.70 (dd,
= 5.5 Hz, C6), 127.75, 126.66, 123.81, 123.46, 119.69 (dd, J(CCF) = 14.6 Hz
3
(CF)
(CCCF)
1
3
C2) 161.40 (dd, J
= 251.2 Hz, J
(
CF)
(CCCF)
3
3
2
J(CCCF) = 9.8 Hz, J
J(CCCCF) = 3.6 Hz, C1), 115.44, 111.40 (dd, J
,
(CCCF)
4
2
4
= 21.0 Hz, J
= 3.4 Hz, C5), 104.07 (dd,
(CCCCF)
= 4.0 Hz, N-CH ). Anal. Calcd. for
(CCCF)
2
(CCF)
2J(
= 25.3 Hz, J
2
= 25.0 Hz, C3), 61.61 (d, J
3
CCF)
(CCF)
C H F N O: C, 68.45; H, 4.06; N, 9.39. Found: C, 68.19; H, 3.87; N, 9.45.
17
12
2
2
3
.3. General Procedure for the Synthesis of N3-Alkylated 2-Vinyl-3H-quinazolin-4-ones 13j and 13k
To the solution of 2-vinyl-3H-quinazolin-4-one (13a, 1.00 mmol) in acetonitrile (15 mL) potassium
carbonate (3.00 mmol) was added. After 15 min. iodomethane (2.00 mmol) or iodoethane (1.10 mmol)
˝
was added and the reaction mixture was stirred at 60 C for 5 h. The solvent was removed and a
residue was extracted with water (3 10 mL). Organic layer was dried (MgSO ), concentrated and the
crude product was purified on a silica gel column with methylene chloride:hexane mixture (7:3, v/v)
followed by crystallization (chloroform : petroleum ether) to give pure quinazolinones 13j 35] or 13k
ˆ
4
[
.
˝ ˝
-Methyl-2-vinylquinazolin-4(3H)-one (13j). Amorphous solid, m.p. = 122 C–124 C (reference [35]
m.p. = 123 C–125 C).
3
˝
˝
´
3
1
7
-Ethyl-2-vinylquinazolin-4(3H)-one (13k). Yellowish oil; IR (film, cm 1) νmax: 3066, 2979, 2927, 2855,
1
684, 1621, 1576, 1423, 1381, 1163, 962, 768, 681. H-NMR (200 MHz, CDCl ):
δ
= 8.16–8.11 (m, 1H),
3
3
3
.90–7.85 (m, 1H), 7.81–7.73 (m, 1H), 7.52–7.44 (m, 1H), 6.91 (dd, J = 17.2 Hz, J = 10.1 Hz, 1H,
3
2
3
2
CH=CH ), 6.69 (dd, J = 17.2 Hz, J = 2.3 Hz, 1H, CH=CH ), 5.76 (dd, J = 10.1 Hz, J = 2.3 Hz, 1H,
CH=CH ), 4.68 (q, J = 7.1 Hz, 2H, CH CH ), 1.52 (t, J = 7.1 Hz, 3H, CH CH ). C-NMR (151 MHz,
2
2
3
3
13
2
2
3
2
3
CDCl3):
δ
= 166.31 (s, C(O)), 160.09, 151.51, 137.29, 133.35, 127.62, 126.39, 123.55, 115.66, 62.70 (s,
0.25 H O: C, 70.40; H, 6.15; N, 13.68.
CH CH ), 14.34 (s, CH CH ). Anal. Calcd. for C H N O
ˆ
2
3
2
3
12 12
2
2
Found: C, 70.73; H, 5.92; N, 13.80.
3
.4. General Procedure for the Synthesis of Isoxazolidines Trans-11 and Cis-11
A solution of the nitrone 12 (1.0 mmol) and the respective vinyl quinazolinone (1.0 mmol) in
˝
toluene (2 mL) was stirred at 70 C until the disappearance (TLC) of the starting nitrone. All volatiles
were removed in vacuo and crude products were subjected to chromatography on silica gel columns
with a chloroform/methanol (100:1, 50:1, 20:1, v/v) mixtures as eluents.
Diethyl trans-(2-methyl-5-(4-oxo-3,4-dihydroquinazolin-2-yl)isoxazolidin-3-yl)phosphonate (trans-11a).
´
1
Yellowish oil; IR (film, cm
) νmax: 3085, 2980, 2929, 2782, 1687, 1610, 1469, 1331, 1132, 1098, 1052,

Molecules 2016, 21, 959
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9
68, 775. 1H-NMR (600 MHz, CDCl3):
δ
= 10.13 (s, 1H, NH), 8.31–8.29 (m, 1H), 7.80–7.78 (m,
3
3
1H), 7.71–7.69 (m, 1H), 7.53–7.50 (m, 1H), 5.04 (dd, J(
= 8.3 Hz, J
= 6.2 Hz, 1H,
= 16.9 Hz,
(H4β–P)
H5–H4β)
(H5–H4α)
3
HC5), 4.29–4.20 (m, 4H, 2
ˆ
CH OP), 3.28–3.24 (m, 1H, HC3), 3.13 (dddd, J
2
2
3
3
J(H4β–H4α) = 12.8 Hz, J
= 8.3 Hz, J
= 8.3 Hz, 1H, H C4), 3.05 (s, 3H, CH N), 2.96
(H4β–H3)
(H4β–H5)
β
3
3
dddd, 2J(H4α–H4β) = 12.8 Hz, J
3
= 9.9 Hz, J
3
= 9.3 Hz, J
= 6.2 Hz, 1H, H C4),
(
α
(H4α–P)
(H4α–H3)
(H4α–H5)
3
3
13
1
.40 (t, J = 7.1 Hz, 3H, CH CH OP), 1.38 (t, J = 7.2 Hz, 3H, CH CH OP). C-NMR (151 MHz, CDCl ):
3
2
3
2
3
3
δ
= 161.75 (s, C(O)), 153.76, 148.61, 134.82, 127.45, 127.15, 126.60, 121.61, 75.82 (d,
J
= 8.5 Hz
,
,
(CCCP)
C5), 63.76 (d, 1J(CP) = 170.0 Hz, C3), 63.36 (d, J
CH OP), 46.33 (s, CH N), 37.77 (d, J
2
= 6.5 Hz, CH OP), 62.59 (d, J(COP) = 7.3 Hz
2
(COP)
2
3
2
= 1.9 Hz, C4), 16.53 (d, J
= 5.7 Hz, CH CH OP),
(CCOP)
3 2
2
3
(CCP)
31
3
16.46 (d, J(
= 5.6 Hz, CH CH OP). P-NMR (243 MHz, CDCl3):
δ
= 20.64. Anal. Calcd. for
CCOP)
3
2
C H N O P ˆ 0.25 H O: C, 51.68; H, 6.10; N, 11.30. Found: C, 51.63; H, 6.09; N, 11.32.
16
22
3
5
2
Diethyl cis-(2-methyl-5-(4-oxo-3,4-dihydroquinazolin-2-yl)isoxazolidin-3-yl)phosphonate (cis-11a). Data noted
´
1
below correspond to a 17:83 mixture of trans-11a and cis-11a. Yellowish oil; IR (film, cm
) νmax: 3202,
3
086, 2980, 2926, 2791, 1688, 1610, 1567, 1469, 1392, 1330, 1231, 1163, 1100, 1024, 973, 776, 573. (signals of
1
cis-11a were extracted from the spectra of a 17:83 mixture of trans-11a and cis-11a); H-NMR (600 MHz,
CDCl3):
δ
= 10.48 (s, 1H, NH), 8.31–8.29 (m, 1H), 7.77–7.75 (m, 1H), 7.67–7.65 (m, 1H), 7.50–7.47 (m,
3
3
1
H), 5.10 (dd, J
= 9.1 Hz, J
= 3.9 Hz, 1H, HC5), 4.27–4.11 (m, 4H, 2
ˆ
CH OP), 3.21
2
(
H5–H4α)
(H5–H4β)
3
2
3
3
(dddd, J(
= 11.2 Hz, J
= 12.6 Hz, J
= 9.1 Hz, J
(H4α–H3)
= 9.0 Hz, 1H, H C4),
α
H4α–P)
(H4β–H4α)
(H4α–H5)
3
3
2
3
.10 (ddd, J
= 9.0 Hz, J
= 6.5 Hz, J
= 3.9 Hz, 1H, HC3), 3.01 (s, 3H, CH N), 2.85
(
H3–H4α)
(H3–H4β)
(H3-P)
3
3
2
3
3
(dddd, J
= 20.0 Hz, J
= 12.6 Hz, J
= 6.5 Hz, J
= 3.9 Hz, 1H, H C4),
(H4β–P)
(H4α–H4β)
(H4β–H3)
(H4β–H5)
13
β
3
3
1
.31 (t, J = 7.5 Hz, 3H, CH CH OP), 1.25 (t, J = 6.5 Hz, 3H, CH CH OP). C-NMR (151 MHz, CDCl ):
3
2
3
2
3
3
δ
= 161.52 (s, C(O)), 156.11, 148.53, 134.53, 127.14, 126.88, 126.67, 121.83, 75.51 (d, J(CCCP) = 7.3 Hz
,
1
2
2
C5), 63.66 (d, J
= 168.5 Hz, C3), 63.23 (d, J
= 6.6 Hz, CH OP), 63.03 (d, J
= 6.7 Hz,
(
CP)
(COP)
2
3
(COP)
3
CH OP), 45.62 (d, J
= 5.4 Hz, CH N), 37.96 (s, C4), 16.38 (d, J
= 5.5 Hz, CH CH OP),
(CCOP)
3 2
2
(CNCP)
3
3
31
16.35 (d, J(
= 5.5 Hz, CH CH OP). P-NMR (243 MHz, CDCl3):
δ
= 20.87. Anal. Calcd. for
CCOP)
3
2
C H N O P
ˆ
0.25 H O: C, 51.68; H, 6.10; N, 11.30. Found: C, 51.63; H, 6.05; N, 11.32 (obtained on a
2
16
22
3
5
9
0:10 mixture of trans-11a and cis-11a).
Diethyl trans-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl)phosphonate
´
1
(
1
trans-11b). Yellowish oil; IR (film, cm
)
νmax: 3065, 3033, 2981, 2915, 2779, 1687, 1620, 1574, 1498,
1
455, 1347, 1239, 1104, 1026, 966, 774, 683. H-NMR (600 MHz, CDCl ):
δ
= 8.22–8.20 (m, 1H), 7.97–7.95
3
(
m, 1H), 7.84–7.82 (m, 1H), 7.57–7.54 (m, 1H), 7.43–7.41 (m, 1H), 7.38–7.36 (m, 1H), 5.67 (s, 2H,
3
3
N-CH ), 5.28 (dd, J
3
3
= 7.4 Hz, J
= 6.5 Hz, 1H, HC5), 4.33–4.20 (m, 4H, 2 ˆ CH OP),
2
(H5–H4β)
(H5–H4α)
2
3
.45–3.42 (m, 1H, HC3), 3.06 (s, 3H, CH N), 3.07–2.97 (m, 2H, H
H, CH CH OP), 1.39 (t, J = 7.1 Hz, 3H, CH CH OP). 13C-NMR (151 MHz, CDCl3):
α
C4, H C4), 1.42 (t, J = 7.0 Hz,
3
β
3
δ
= 167.04 (s,
3
2
3
2
C(O)), 162.94, 151.27, 136.14, 133.71, 128.59, 128.30, 128.24, 127.86, 127.12, 123.50, 115.60, 80.30 (d,
3
1
2
J(CCCP) = 8.6 Hz, C5), 68.73 (s, CH N), 64.44 (d, J
= 168.2 Hz, C3), 63.25 (d, J
= 6.5 Hz,
= 5.6 Hz,
2
(CP)
(COP)
2
3
CH OP), 62.55 (d, J
CH CH OP), 16.51 (d, J
= 6.9 Hz, CH OP), 46.63 (s, CH N), 37.95 (s, C4), 16.56 (d, J
2
(COP)
3
2
3
(CCOP)
31
= 5.6 Hz, CH CH OP). P-NMR (243 MHz, CDCl3): δ = 22.19. Anal.
3 2
3
2
(CCOP)
Calcd. for C H N O P: C, 60.39; H, 6.17; N, 9.19. Found: C, 59.99; H, 6.21; N, 9.15.
23
28
3
5
Diethyl trans-(2-methyl-5-(3-(2-nitrobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)isoxazolidin-3-yl)-phosphonate
´
1
(
1
trans-11c). Yellowish oil; IR (film, cm
)
νmax: 3067, 2973, 2918, 1620, 1576, 1526, 1347, 1337,
1
264, 1050, 1018, 964, 790, 733, 584, 571.
H), 7.93–7.69 (m, 3H), 7.60–7.42 (m, 3H), 6.01 (s, 2H, N-CH ), 5.15 (dd, J
J(H5–H4α) = 6.1 Hz, 1H, HC5), 4.29–4.07 (m, 4H, 2
H-NMR (200 MHz, CDCl3):
δ
= 8.18–8.07 (m,
3
2
= 7.8 Hz,
= 10.6 Hz,
2
(H5–H4β)
3
3
ˆ
CH OP), 3.28 (ddd, J
2
(H3–H4α)
3J(
(
1
= 8.5 Hz
,
2
J(H3-P) = 1.2 Hz, 1H, HC3), 2.93 (s, 3H, CH N), 2.99–2.82 (m, 1H, H C4), 2.76
H3–H4β)
3
β
dddd, 3J(H4α–P) = 12.5 Hz, 2J(H4α–H4β) = 12.5 Hz, J
3
= 10.6 Hz, J
3
= 6.1 Hz, 1H, H C4),
(H4α–H3)
(H4α–H5)
13
α
3
3
.33 (t, J = 7.0 Hz, 3H, CH CH OP), 1.31 (t, J = 7.0 Hz, 3H, CH CH OP). C-NMR (151 MHz, CDCl3):
3
2
3
2
δ
= 166.51 (s, C(O)), 162.99, 151.40, 147.85, 133.98, 133.68, 132.46, 129.10, 128.82, 128.08, 127.44, 125.06,
3
1
1
23.19, 115.30, 80.23 (d, J
= 8.4 Hz, C5), 65.34 (s, N-CH ), 64.33 (d, J
= 168.4 Hz, C3), 63.19 (d,
(CP)
(
CCCP)
2

Molecules 2016, 21, 959
10 of 15
2J(COP) = 6.5 Hz, CH OP), 62.44 (d, J
2
= 7.1 Hz, CH OP), 46.58 (d, J
3
= 3.8 Hz, CH N), 37.98
2
(COP)
2
(CNCP)
3
s, C4), 16.55 (d, 3J
= 6.4 Hz, CH CH OP), 16.49 (d, J
= 22.85. Anal. Calcd. for C H N O P
3
= 5.7 Hz, CH CH OP). P-NMR
0.25 H O: C, 54.49; H, 5.47; N, 11.05.
2
31
(
(
(
δ
CCOP)
3
2
(CCOP)
3
2
81 MHz, CDCl3):
ˆ
23
27
4
7
Found: C, 54.42; H, 5.28; N, 10.89.
Diethyl trans-(2-methyl-5-(3-(3-nitrobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)isoxazolidin-3-yl)-phosphonate
(
trans-11d). Data noted below correspond to a 92:8 mixture of trans-11d and cis-11d. A yellowish oil;
´
1
IR (film, cm
) νmax: 3070, 2982, 2930, 2910, 1620, 1574, 1531, 1497, 1415, 1298, 1103, 1025, 774, 733,
6
82. (signals of trans-11d were extracted from the spectra of a 92:8 mixture of trans-11d and cis-11d);
1
H-NMR (600 MHz, CDCl3):
δ = 8.46–8.44 (m, 1H), 8.25–8.22 (m, 2H), 8.00–7.98 (m, 1H), 7.91–7.88 (m,
3
3
2
H), 7.63–7.60 (m, 2H), 5.78 (s, 2H, N-CH ), 5.28 (dd, J
= 6.8 Hz, J
= 6.8 Hz, 1H, HC5),
(H5–H4α)
2
(H5–H4β)
4
2
H
.33–4.20 (m, 4H, 2
ˆ
CH OP), 3.40–3.38 (m, 1H, HC3), 3.07–3.00 (m, 1H, H C4), 3.05 (s, 3H, CH N),
2
β
3
3
2
3
3
.98 (dddd, J
= 12.2 Hz, J
= 12.2 Hz, J
= 9.8 Hz, J = 6.8 Hz, 1H,
(H4α–H5)
(
H4α–P)
3
(H4α–H4β)
(H4α–H3)
C4), 1.42 (t, J = 7.1 Hz, 3H, CH CH OP), 1.40 (t, J = 6.7 Hz, 3H, CH CH OP). 13C-NMR (151 MHz,
3
α
3
2
3
2
CDCl3):
1
δ
= 166.54 (s, C(O)), 162.63, 151.38, 148.50, 138.29, 134.06, 133.97, 129.65, 128.02, 127.45, 123.25,
23.00, 115.30, 80.14 (d, 3J(CCCP) = 8.4 Hz, C5), 67.21 (s, N-CH ), 64.42 (d, J
1
= 168.5 Hz, C3), 63.24 (d,
(CP)
2
2
2
J(COP) = 6.5 Hz, CH OP), 62.44 (d, J
= 7.2 Hz, CH OP), 46.33 (s, CH N), 37.84 (s, C4), 16.56 (d,
2 3
2
(COP)
3
3J(
= 5.6 Hz, CH CH OP), 16.46 (d, J
= 4.2 Hz, CH CH OP). P-NMR (243 MHz, CDCl3):
(CCOP)
3 2
31
CCOP)
3
2
δ
= 22.07. Anal. Calcd. for C H N O P: C, 54.98; H, 5.42; N, 11.15. Found: C, 54.62; H, 5.24; N, 11.14
23 27 4 7
(obtained on a 92:8 mixture of trans-11d and cis-11d).
Diethyl trans-(2-methyl-5-(3-(4-nitrobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)isoxazolidin-3-yl)-phosphonate
(
trans-11e). Data noted below correspond to a 90:10 mixture of trans-11e and cis-11e. A yellowish
´
1
oil; IR (film, cm
) νmax: 3073, 2982, 2929, 2781, 1687, 1608, 1523, 1498, 1342, 1106, 968, 851, 739.
(
signals of trans-11e were extracted from the spectra of a 90:10 mixture of trans-11e and cis-11e);
1
H-NMR (600 MHz, CDCl3):
δ
= 8.29–8.27 (m, 2H), 8.23–8.21 (m, 1H), 8.00–7.98 (m, 1H), 7.89–7.86
3
(
m, 1H), 7.72–7.70 (m, 2H), 7.62–7.60 (m, 1H), 5.77 (s, 2H, N-CH ), 5.26 (dd, J
= 6.8 Hz,
(H5–H4β)
2
3J
= 6.8 Hz, 1H, HC5), 4.33–4.19 (m, 4H, 2
ˆ
CH OP), 3.38–3.35 (m, 1H, HC3), 3.05 (s, 3H,
(
H5–H4α)
2
3
3
CH N), 3.04–2.91 (m, 2H, H
CH CH OP). 3C-NMR (151 MHz, CDCl3):
1
6
4
α
C4, H C4), 1.41 (t, J = 7.2 Hz, 3H, CH CH OP), 1.39 (t, J = 7.2 Hz, 3H,
3
β
3
2
1
δ
= 166.51 (s, C(O)), 162.65, 151.35, 147.82, 143.45, 134.04,
3
2
3
28.90, 128.45, 128.07, 127.48, 123.84, 123.19, 115.29, 80.10 (d, J
= 8.8 Hz, C5), 67.18 (s, N-CH ),
(
CCCP)
2
1
2
2
4.42 (d, J(
= 168.6 Hz, C3), 63.25 (d, J
= 5.3 Hz, CH OP), 62.46 (d, J
= 6.9 Hz, CH OP),
CP)
(COP)
2
(COP)
2
3
3
6.58 (s, CH N), 37.84 (s, C4), 16.55 (d, J
= 5.6 Hz, CH CH OP), 16.50 (d, J
= 5.7 Hz,
(CCOP)
3
(CCOP)
3
2
31
CH CH OP). P-NMR (243 MHz, CDCl3):
5
δ
= 22.05. Anal. Calcd. for C H N O P
ˆ
0.5 H O: C,
2
3
2
23 27
4
7
4.01; H, 5.52; N, 10.95. Found: C, 53.91; H, 5.24; N, 10.95 (obtained on a 90:10 mixture of trans-11e and
cis-11e).
Diethyl trans-(5-(3-(2-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl)-phosphonate
(
trans-11f). Data noted below correspond to a 90:10 mixture of trans-11f and cis-11f. A colorless oil; IR
´
1
(film, cm
)
νmax: 3067, 2981, 2930, 2909, 1620, 1574, 1498, 1457, 1418, 1346, 1298, 1162, 1101, 1025, 965,
7
70, 682. (signals of trans-11f were extracted from the spectra of a 90:10 mixture of trans-11f and cis-11f);
1
H-NMR (600 MHz, CDCl3):
2
δ
= 8.17–8.15 (m, 1H), 7.95–7.93 (m, 1H), 7.83–7.80 (m, 1H), 7.58–7.52 (m,
2
H), 7.36–7.32 (m, 1H), 7.17–7.15 (m, 1H), 7.13–7.10 (m, 1H), 5.73 (AB, J = 12.6 Hz, 1H, N-CH ), 5.70
AB
2b
2
3
3
(
(
(
AB, J = 12.6 Hz, 1H, N-CH ), 5.26 (dd, J
m, 4H, 2
t, J = 7.0 Hz, 3H, CH CH OP), 1.38 (t, J = 7.1 Hz, 3H, CH CH OP). C-NMR (151.0 MHz, CDCl ):
= 7.3 Hz, J
= 6.7 Hz, 1H, HC5), 4.32–4.18
AB
2a
(H5–H4β)
(H5–H4α)
ˆ
CH OP), 3.43–3.40 (m, 1H, HC3), 3.04 (s, 3H, CH N), 3.03–2.93 (m, 2H, H C4, H C4), 1.40
α
2
3
β
3
3
13
3
2
3
2
3
1
3
δ = 166.86 (s, C(O)), 162.84, 161.08 (d, J
C4’), 130.26 (d, J
= 248.6 Hz, C2’), 151.19, 133.76, 130.60 (d, J
= 3.4 Hz,
(
CF)
(CCCF)
3
4
= 8.6 Hz, C6’), 127.85, 127.17, 124.17 (d, J(CCCCF) = 3.3 Hz, C5’), 123.45, 123.31
(
CCCF)
2
2
3
(d, J(
= 14.3 Hz, C3’), 115.54 (d,
J
= 21.4 Hz, C1’), 115.49, 80.27 (dd, J
= 8.7 Hz, C5),
(CCCP)
CCF)
1
(CCF)
2
3
6
4.41 (d, J
= 168.3 Hz, C3), 63.23 (d, J
= 6.3 Hz, CH OP), 62.60 (d, J = 4.3 Hz, CH N), 62.39
(
CP)
(COP)
2
2
2
3
(d, J(
= 6.8 Hz, CH OP), 46.61 (s, CH N), 37.94 (s, C4), 16.56 (d, J
= 5.6 Hz, CH CH OP),
(CCOP)
3 2
COP)
2
3
31
3
16.50 (d, J(
= 5.6 Hz, CH CH OP). P-NMR (243 MHz, CDCl3):
δ
= 22.16. Anal. Calcd. for
CCOP)
3
2

Molecules 2016, 21, 959
11 of 15
C H FN O P
ˆ
0.5 H O: C, 57.02; H, 5.83; N, 8.67. Found: C, 57.30; H, 5.58; N, 8.62 (obtained on a
23
27
3
5
2
9
0:10 mixture of trans-11f and cis-11f).
Diethyl trans-(5-(3-(3-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl)-phosphonate
´
1
(
1
trans-11g). Colorless oil; IR (film, cm
)
νmax: 3066, 2982, 2909, 1620, 1575, 1497, 1452, 1416, 1343, 1299,
= 8.22–8.20 (m, 1H), 7.98–7.96 (m, 1H), 7.86–7.84
1
163, 1104, 966, 775, 682. H-NMR (600 MHz, CDCl ):
δ
3
(
5
m, 1H), 7.59–7.57 (m, 1H), 7.40–7.37 (m, 1H), 7.31–7.28 (m, 2H), 7.08–7.06 (m, 1H), 5.66 (s, 2H, N-CH ),
2
3
3
.27 (dd, J(
= 7.4 Hz, J
= 6.7 Hz, 1H, HC5), 4.33–4.21 (m, 4H, 2 ˆ CH OP), 3.42–3.40
H5–H4β)
(H5–H4α)
2
3
(
m,1H, HC3), 3.05 (s, 3H, CH N), 3.04–2.94 (m, 2H, H
α
C4, H C4) 1.42 (t, J = 7.1 Hz, 3H, CH CH OP),
3
β
3
2
3
13
1
1J(
.39 (t, J = 7.0 Hz, 3H, CH CH OP). C-NMR (151 MHz, CDCl ):
δ
= 166.80 (s, C(O)), 162.92 (d,
3
2
3
3
= 246.6 Hz, C3’), 162.80, 151.25, 138.65 (d, J
= 7.6 Hz, C5’), 133.84, 130.17 (d, J = 8.7 Hz
,
CF)
(CCCF)
4
2
C1’), 127.94, 127.27, 123.60 (d, J
1
1J(
= 2.6 Hz, C6’), 123.39, 115.47, 115.18 (d, J(CCF) = 21.0 Hz, C4’),
(
CCCCF)
2
3
3
15.00 (d, J(
= 22.0 Hz, C2’), 80.22 (dd, J
= 7.9 Hz, C5), 67.80 (d, J = 1.6 Hz, CH N), 64.43 (d,
CCF)
(CCCP)
2
2
2
= 168.3 Hz, C3), 63.26 (d, J
= 6.7 Hz, CH OP), 62.41 (d, J
= 6.4 Hz, CH OP), 46.59 (s,
2
CP)
(COP)
2
(COP)
3
3
CH N), 37.92 (s, C4), 16.56 (d, J
= 5.6 Hz, CH CH OP), 16.50 (d, J
= 5.6 Hz, CH CH OP).
(CCOP)
3 2
3
(CCOP)
3
2
3
1
P-NMR (243 MHz, CDCl3):
δ
= 22.14. Anal. Calcd. for C H FN O P
ˆ
0.5 H O: C, 57.02; H, 5.83;
2
23
27
3
5
N, 8.67. Found: C, 57.30; H, 5.66; N, 8.74.
Diethyl trans-(5-(3-(4-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl)-phosphonate
´
1
(
1
7
=
trans-11h). Colorless oil; IR (film, cm
) νmax: 3069, 2982, 2930, 2910, 1620, 1574, 1512, 1498, 1226,
1
104, 966, 823, 774, 683. H-NMR (600 MHz, CDCl3):
.84–7.81 (m, 1H), 7.56–7.51 (m, 3H), 7.11–7.08 (m, 2H), 5.62 (s, 2H, N-CH ), 5.27 (dd, J
= 6.7 Hz, 1H, HC5), 4.32–4.20 (m, 4H, 2
.07–2.99 (m, 1H, H C4), 3.06 (s, 3H, CH N), 2.99 (dddd, J
J(H4α–H5) = 6.7 Hz, 1H, H
J = 6.4 Hz, 3H, CH CH OP). C-NMR (151 MHz, CDCl ):
1J( = 246.9 Hz, C4’), 151.21, 133.76, 131.93 (d, J
C2’, C6’), 127.89, 127.18, 123.41, 115.53 (d, J
= 168.1 Hz, C3), 63.25 (d, J
J(COP) = 6.9 Hz, CH OP), 46.62 (s, CH N), 37.96 (s, C4), 16.56 (d, J
δ
= 8.17–8.15 (m, 1H), 7.96–7.94 (m, 1H),
3
2
(H5–H4β)
3
6.7 Hz, J
ˆ
CH OP), 3.43–3.40 (m, 1H, HC3),
(
H5–H4α)
2
3
2
3
= 12.5 Hz, J
= 12.5 Hz,
(H4α–H4β)
β
3
(H4α–P)
3
3
3
J(H4α–H3) = 10.3 Hz
,
α
C4), 1.41 (t, J = 7.1 Hz, 3H, CH CH OP), 1.39 (t,
3
2
3
13
δ
= 166. 90 (s, C(O)), 162.84, 162.73 (d,
3
2
3
4
3
= 3.2 Hz, C1’), 130.28 (d, J
= 8.0 Hz,
CF)
(CCCCF)
(CCCF)
2
= 21.4 Hz, C3’, C5’), 115.52, 80.23 (d, 3J
= 8.1 Hz,
= 6.4 Hz, CH OP), 62.39 (d,
2
(CCF)
(CCP)
1
2
C5), 68.02 (s, N-CH ), 64.45 (d, J
2
(CP)
(COP)
3
2
= 5.6 Hz, CH CH OP),
3 2
2
3
(CCOP)
3
31
1
6.50 (d, J(
= 5.5 Hz, CH CH OP). P-NMR (243 MHz, CDCl3): δ = 22.13. Anal. Calcd. for
CCOP)
3 2
C H FN O P: C, 58.10; H, 5.72; N, 8.84. Found: C, 57.74; H, 5.70; N, 8.77.
23
27
3
5
Diethyl
trans-(5-(3-(2,4-difluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl)-
phosphonate (trans-11i). Data noted below correspond to a 92:8 mixture of trans-11i and cis-11i
.
´
1
A colorless oil; IR (film, cm
)
νmax: 3068, 2982, 2930, 2910, 1621, 1574, 1498, 1416, 1162, 1102, 1025,
9
61, 773, 682. (signals of trans-11i were extracted from the spectra of a 92:8 mixture of trans-11i
1
and cis-11i); H-NMR (600 MHz, CDCl3):
δ
= 8.18–8.16 (m, 1H), 7.98–7.96 (m, 1H), 7.86–7.84
2
(
m, 1H), 7.62–7.56 (m, 2H), 6.95–6.89 (m, 2H), 5.71 (AB, JAB = 12.4 Hz, 1H, N-CH ), 5.71 (AB,
2b
2J = 12.4 Hz, 1H, N-CH ), 5.29 (dd, J
3
= 6.6 Hz, J = 6.2 Hz, 1H, HC5), 4.34–4.22
3
AB
2a
(H5–H4β)
(H5–H4α)
(
(
H
m, 4H, 2
ˆ
CH OP), 3.44–3.41 (m, 1H, C3), 3.07 (s, 3H, CH -N), 3.04–3.02 (m, 1H, H C4), 3.01
2
3
β
dddd, 3J(
= 12.4 Hz
,
2
J(H4α–H4β) = 12.4 Hz, J
3
= 10.1 Hz, J
3
= 6.2 Hz, 1H,
H4α–P)
(H4α–H3)
(H4α–H5)
3
3
13
α
C4), 1.43 (t, J = 7.1 Hz, 3H, CH CH OP), 1.41 (t, J = 7.0 Hz, 3H, CH CH OP). C-NMR
3
2
3
2
1
3
(
151 MHz, CDCl ):
δ
= 165.94 (s, C(O)), 163.05 (dd, J
= 265.3 Hz, J
= 12.1 Hz, C2’), 162.71,
3
(CF)
(CCCF)
61.44 (dd, 1J(CF) = 266.1 Hz
,
3
J(CCCF) = 14.4 Hz, C4’), 151.19, 133.76, 131.87 (dd, J(CCCF) = 9.9 Hz
3
,
= 3.3 Hz,
= 25.3 Hz,
= 168.2 Hz,
= 3.2 Hz,
1
3J(
2
4
= 5.5 Hz, C6’), 127.85, 127.18, 123.33, 119.34 (dd, J
= 14.5 Hz, J
CCCF)
(CCF)
(CCCCF)
2
2
4
C1’), 115.38, 111.35 (dd, J(CCF) = 21.0 Hz, J
= 3.3 Hz, C5’), 104.02 (dd, J
(CCCCF)
(CCF)
2
3
3
1
J(CCF) = 25.3 Hz, C3’), 80.20 (dd, J
= 8.7 Hz, J
= 2.3 Hz, C5), 64.41 (d, J
(CCCP)
(CCCF)
(CP)
C3), 63.19 (d, 2J(
= 6.1 Hz, CH OP), 62.35 (d, J
2
= 7.3 Hz, CH OP), 62.04 (d, J
3
COP)
2
(COP)
2
2
(CCCF)
CH N), 46.45 (d, 3J
= 2.6 Hz, CH N), 37.89 (d, J
= 1.7 Hz, C4), 16.52 (d, J
3
= 5.5 Hz,
(CCOP)
2
(CNCP)
3
3
(CCP)
31
CH CH OP), 16.45 (d, J
= 5.5 Hz, CH CH OP). P-NMR (243 MHz, CDCl3): δ = 22.13. Anal.
3 2
3
2
(CCOP)
Calcd. for C H F N O P
ˆ
0.5 H O: C, 54.98; H, 5.42; N, 8.36. Found: C, 55.08; H, 5.14; N, 8.26
2
23
26
2
3
5
(obtained on a 92:8 mixture of trans-11i and cis-11i).

Molecules 2016, 21, 959
12 of 15
Diethyl
trans-(2-methyl-5-(3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)isoxazolidin-3-yl)phosphonate
´
1
(trans-11j). Colorless oil; IR (film, cm
)
νmax: 2983, 2955, 2924, 2854, 1679, 1601, 1474, 1435, 1340,
1
1
299, 1241, 1053, 1022, 945, 781, 695, 653, 600, 559. H-NMR (600 MHz, CDCl ):
δ
= 8.28–8.25 (m, 1H),
3
3
3
7
.75–7.72 (m, 1H), 7.68–7.66 (m, 1H), 7.50–7.47 (m, 1H), 5.18 (dd, J
= 7.7 Hz, J(H5–H4α) = 5.6 Hz
,
(
H5–H4β)
3
= 14.0 Hz, 2J
1
H, HC5), 4.30–4.22 (m, 4H, 2
ˆ
CH OP), 3.75 (dddd, J
= 12.7 Hz,
(H4α–H4β)
2
(H4α–P)
3
3
= 5.6 Hz, 1H, H C4), 3.74 (s, 3H, CH N), 3.37 (ddd, 3J(H3–H4α) = 9.1 Hz
J(H4α–H3) = 9.1 Hz, J
,
(H4α–H5)
β
3
3
3
3
2
= 2.7 Hz, 1H, HC3), 2.79 (dddd, 3
2
J(H3–H4β) = 7.7 Hz, J
J
= 15.4 Hz, J = 12.7 Hz,
(H3-P)
(H4β–P)
(H4β–H4α)
3
3
J(H4β–H3) = 7.7 Hz, J
= 7.7 Hz, 1H, H C4), 1.40 (t, J = 7.1 Hz, 3H, CH CH OP), 1.39 (t,
(H4β–H5)
β
3
2
13
J = 7.1 Hz, 3H, CH CH OP). C-NMR (151 MHz, CDCl ):
δ
= 162.50 (s, C(O)), 152.24, 146.52, 134.08,
3
2
3
3
1
1
27.62, 127.41, 126.79, 120.98, 76.37 (d, J
= 7.8 Hz, C5), 64.38 (d, J
= 170.6 Hz, C3), 63.02
(
CCCP)
(CP)
3
2
2
(d, J(
= 6.6 Hz, CH OP), 62.70 (d, J
= 6.9 Hz, CH OP), 47.21 (d, J
= 6.3 Hz, CH N),
COP)
2
(COP)
3
2
(CNCP)
3
3
34.41 (s, C4), 30.77 (s, CH N) 16.56 (d, J
= 5.0 Hz, CH CH OP), 16.53 (d, J
= 4.9 Hz,
(CCOP)
3
(CCOP)
3
2
31
CH CH OP). P-NMR (243 MHz, CDCl3):
δ
= 22.09. Anal. Calcd. for C H N O P
ˆ
H O: C, 51.13;
2
3
2
17 24
3
5
H, 6.56; N, 10.52. Found: C, 51.23; H, 6.17; N, 10.49.
Diethyl trans-(5-(3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl)phosphonate (trans-11k).
Data noted below correspond to a 94:6 mixture of trans-11k and cis-11k. Yellowish oil; IR (film, cm 1)
´
νmax: 3068, 2981, 2930, 2870, 1620, 1574, 1501, 1424, 1382, 1340, 1240, 1164, 1105, 1053, 1023, 967, 774,
6
84, 581. (signals of trans-11k were extracted from the spectra of a 94:6 mixture of trans-11k and cis-11k);
1
H-NMR (600 MHz, CDCl ):
δ = 8.19–8.17 (m, 1H), 7.96–7.94 (m, 1H), 7.84–7.82 (m, 1H), 7.58–7.55 (m,
3
3
3
3
1H), 5.26 (dd, J
= 7.4 Hz, J
= 6.4 Hz, 1H, HC5), 4.69 (q, J = 7.1 Hz, 2H, CH CH ),
(
H5–H4β)
(H5–H4α)
3
2
4
.34–4.21 (m, 4H, 2
ˆ
CH OP), 3.49–3.46 (m, 1H, HC3), 3.08 (s, 3H, CH N), 3.10–2.96 (m, 2H, H
α
C4,
2
3
3
3
3
H C4), 1.55 (t, J = 7.1 Hz, 3H, CH CH ), 1.42 (t, J = 7.0 Hz, 3H, CH CH OP), 1.39 (t, J = 7.1 Hz, 3H,
CH CH OP). C-NMR (151 MHz, CDCl ):
β
3
2
3
2
13
δ =167.25 (s, C(O)), 163.16, 151.03, 133.51, 127.77, 126.92,
3
2
3
3
1
2
1
23.44, 115.60, 80.36 (d, J
= 8.3 Hz, C5), 64.38 (d, J
= 168.2 Hz, C3), 63.23 (d, J
= 6.3 Hz, CH N), 34.41 (s, C4), 29.64 (s,
3
= 6.4 Hz,
(COP)
(
CCCP)
(CP)
2
3
CH OP), 62.36 (d, J
CH CH ) 16.53 (d, J
CH CH ). P-NMR (243 MHz, CDCl3):
= 6.7 Hz, CH OP), 47.21 (d, J
2
(COP)
3
2
(CNCP)
3
= 5.7 Hz, CH CH OP), 16.47 (d, J
= 5.8 Hz, CH CH OP), 14.29 (s,
(CCOP)
3 2
3
2
(CCOP)
3
2
31
δ = 22.23. Anal. Calcd. for C H N O P: C, 54.68; H, 6.63; N,
18 26 3 5
3
2
1
0.63. Found: C, 54.93; H, 6.51; N, 10.21 (obtained on a 94:6 mixture of trans-11k and cis-11k).
3
.5. Antiviral Activity Assays
The compounds were evaluated against different herpesviruses, including herpes simplex virus
´
type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK ) HSV-1 KOS strain resistant to ACV
(
r
´
ACV ), herpes simplex virus type 2 (HSV-2) strain G, varicella-zoster virus (VZV) strain Oka, TK
VZV strain 07-1, human cytomegalovirus (HCMV) strains AD-169 and Davis as well as feline herpes
virus (FHV), the poxvirus vaccinia virus (Lederle strain), para-influenza-3 virus, reovirus-1, Sindbis
virus, Coxsackie virus B4, Punta Toro virus, respiratory syncytial virus (RSV), feline coronovirus
(
(
FIPV) and influenza A virus subtypes H1N1 (A/PR/8), H3N2 (A/HK/7/87) and influenza B virus
B/HK/5/72) and human immune deficiency virus (5HVV-1 and HIV-2). The antiviral assays, other
than HIV, were based on inhibition of virus-induced cytopathicity or plaque formation in human
embryonic lung (HEL) fibroblasts, African green monkey kidney cells (Vero), human epithelial cervix
carcinoma cells (HeLa), Crandell-Rees feline kidney cells (CRFK), or Madin Darby canine kidney cells
(
(
MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus
1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU)
and the cell cultures were incubated in the presence of varying concentrations of the test compounds.
Viral cytopathicity or plaque formation (VZV) 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 EC50 or compound concentration required to reduce virus-induced cytopathicity
or viral plaque formation by 50%. Cytotoxicity of the test compounds was expressed as the minimum
cytotoxic concentration (MCC) or the compound concentration that caused a microscopically detectable
alteration of cell morphology.

Molecules 2016, 21, 959
13 of 15
3
.6. Cytostatic Activity against Immortalized Cell Lines
Murine leukemia (L1210), human T-lymphocyte (CEM), human cervix carcinoma (HeLa)
and immortalized human dermal microvascular endothelial cells (HMEC-1) were suspended at
00,000–500,000 cells/mL of culture medium, and 100 L of a cell suspension was added to 100
L of an appropriate dilution of the test compounds in 200 L-wells of 96-well microtiter plates. After
3
µ
µ
µ
˝
incubation at 37 C for two (L1210), three (CEM) or four (HeLa) days, the cell number was determined
using a Coulter counter. The IC50 was defined as the compound concentration required to inhibit cell
proliferation by 50%.
4
. Conclusions
A series of isoxazolidine-containing quinazolinones trans-11 and cis-11 have been
synthesised from N-methyl-C-diethoxyphosphorylnitrone (12) and the respective N3-substituted
-vinyl-quinazolin-ones 13 via the 1,3-dipolar cycloaddition. The obtained isoxazolidine phosphonates
2
trans-11 or the respective mixtures of trans-11/cis-11 were evaluated against a variety of DNA and
RNA viruses. Among all tested compounds, isoxazolidines trans-11f/cis-11f (90:10), trans-11h and
+
trans-11i/cis-11i were slightly active toward TK VZV strain (EC50 = 6.84, 15.29 and 9.44
µM) without
exhibiting cytotoxicity toward uninfected cells at concentration up to 100
µM. On the other hand,
phosphonates trans-11b/cis-11b (90:10), trans-11c, trans-11e/cis-11e (90:10) and trans-11g showed weak
antiviral properties against cytomegalovirus (EC50 = 27–45 µM).
Compounds equipped with benzyl substituents at N3 in the quinazolinone skeleton exhibited
some antiproliferative activity toward the tested immortalized cells (IC50 = 21–102
µM), while
analogues lacking substituent or having alkyl group at N3 were inactive. These results encourage
us to continue the search for more active compounds with special focus on modification the
quinazoline-4-one unit especially at N3.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
2
1/7/959/s1.
Acknowledgments: The authors wish to express their gratitude to Leentje Persoons, Frieda De Meyer,
Ellen De Waegenaere and Lizette van Berckelaer for excellent technical assistance. The synthetic part of
this work was supported by the Medical University of Lodz internal funds (503/3-014-01/503-31-001 and
5
02-03/3-014-01/502-34-067). The biological part of this work was supported by the KU Leuven (GOA 15/19 TBA).
Author Contributions: Research group from Medical University of Lodz (D.G.P. and M.G.-D.) conceived the
research project, participated in all steps of the research, interpreted the results, discussed the experimental data
and prepared the manuscript. Research group from KU Leuven (G.A., D.S. and R.S.) conducted the biological
assays and provided the experimental procedures and results. All authors read, commented and approved
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).