Isoxazolidine conjugates of N3-substituted 6-bromoquinazolinones—synthesis, anti-varizella-zoster virus, and Anti-cytomegalovirus activity

Article abstract of DOI:10.3390/molecules23081889

1,3-Dipolar cycloaddition of N-methyl C-(diethoxyphosphoryl) nitrone to N3-substituted 6-bromo-2-vinyl-3H-quinazolin-4-ones gave (3-diethoxyphosphoryl) isoxazolidines substituted at C5 with quinazolinones modified at N3. All isoxazolidine cycloadducts were screened for antiviral activity against a broad spectrum of DNA and RNA viruses. Several isoxazolidines inhibited the replication of both thymidine kinase wild-type and deficient (TK⁺ and TK^?) varicella-zoster virus strains at EC₅₀ in the 5.4–13.6 μM range, as well as human cytomegalovirus (EC₅₀ = 8.9–12.5 μM). Isoxazolidines trans-11b, trans-11c, trans-11e, trans-11f/cis-11f, trans-11g, trans-11h, and trans-11i/cis-11i exhibited moderate cytostatic activity towards the human lymphocyte cell line CEM (IC₅₀ = 9.6–17 μM).

Full text of DOI:10.3390/molecules23081889

molecules
Article
Isoxazolidine Conjugates of N3-Substituted
6-Bromoquinazolinones—Synthesis,
Anti-Varizella-Zoster Virus, and
Anti-Cytomegalovirus Activity
1
2
2
Magdalena Grabkowska-Druzyc , Graciela Andrei , Dominique Schols , Robert Snoeck 2 and
˙
Dorota G. Piotrowska 1,
*
1
Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of Lodz, Muszyn´skiego 1,
90-151 Lodz, Poland; magdalena.grabkowska-druzyc@umed.lodz.pl
Rega Institute for Medical Research, KU Leuven, Herestraat 49, 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.)
2
*
Correspondence: dorota.piotrowska@umed.lodz.pl; Tel.: +48-42-677-92-33; Fax: +48-42-678-83-98
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 June 2018; Accepted: 27 July 2018; Published: 28 July 2018
Abstract: 1,3-Dipolar cycloaddition of N-methyl C-(diethoxyphosphoryl) nitrone to N3-substituted
6-bromo-2-vinyl-3H-quinazolin-4-ones gave (3-diethoxyphosphoryl) isoxazolidines substituted at C5 with
quinazolinones modified at N3. All isoxazolidine cycloadducts were screened for antiviral activity against
a broad spectrum of DNA and RNA viruses. Several isoxazolidines inhibited the replication of both
thymidine kinase wild-type and deficient (TK+ and TK−) varicella-zoster virus strains at EC50 in the
5.4–13.6 µM range, as well as human cytomegalovirus (EC50 = 8.9–12.5 µM). Isoxazolidines trans-11b,
trans-11c, trans-11e, trans-11f/cis-11f, trans-11g, trans-11h, and trans-11i/cis-11i exhibited moderate
cytostatic activity towards the human lymphocyte cell line CEM (IC50 = 9.6–17 µM).
Keywords: phosphonate; isoxazolidine; quinazolinones; antiviral; cytostatic
1. Introduction
Herpesviruses are enveloped, icosahedral viruses containing double-stranded DNA.
What fundamentally distinguishes herpesviruses from others is the ability to establish latent infection
and reactivating when the host immune system is impaired. Human cytomegalovirus (HCMV) and
varicella zoster virus (VZV) belong to the family of herpesviruses which cause infections usually
acquired in childhood [1–3]. For the treatment of viral infections caused by herpesviruses, including
VZV and HCMV, several drugs, such as acyclovir (e.g., Zovirax), valacyclovir (e.g., Valtrex), famciclovir
(e.g., Famvir), brivudin (e.g., Zostex, Helpin), and ganciclovir (e.g., Cymevene), have been used [3–5].
Despite the wide range of available drugs, effective treatment of viral infections is still limited. This is
mainly due to the specificity of viral infections and the ability of viruses to mutate, which is associated with
the acquisition of drug resistance. Therefore, new compounds with potential biological activity are being
sought all the time. Quinazolinone and its derivatives have been extensively studied because of their wide
range of biological activities, including antiviral [
antitubercular [12 13], anti-inflammatory [11 14], anticonvulsant [15], hypolipidemic [16], analgesic [17],
or immunotropic properties [18].
6], anticancer [7,8], antifungal [9], antibacterial [10,11],
,
,
The antiviral activity of compounds containing the C6-substituted quinazolin-4-one
framework has been discovered in recent years (Figure 1). For example, 5-bromo-2-(6-bromo-
4-oxo-2-phenyl-4H-quinazolin-3-yl)-benzoic acid
1
was reported to possess distinct antiviral activity against
Molecules 2018, 23, 1889; doi:10.3390/molecules23081889
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Molecules 2018, 23, 1889
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both herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) and vaccinia virus (VACV) (EC50 = 12
µg/mL) [19],
whereas compound
1.92 g/mL) [20]. Another analogue, i.e., 2-methyl-3-(substituted-benzalamino)-4(3H)-quinazolinone
, was found to exhibit antiviral activity against tobacco mosaic virus (TMV) in vivo. Compound
showed curative effects of 54%, which was slightly higher than that of a reference drug,
Ningnanmycin [21]. Very recently quinazolin-4(3H)-ones were synthesized and evaluated for
inhibitory action on the replication of influenza A virus (H5N1), as well as to test toxicity on in vitro
cell lines. In general, quinazolin-4(3H)-ones containing a chalcone skeleton , thiosemicarbazone
showed moderate antiviral activity against H5N1 (inhibition rate: 38%, 47%, 25%,
) compared to a reference drug, Zanamivir [22]. In addition, compounds and
2 exhibited activity against vaccinia virus in E6SM cell cultures (MIC of
µ
3
3
4–6
4
5,
and hydrazide
respectively, for
6
4
,
5,
6
7
8
possessing a nitro group at C6 were reported as potent inhibitors of Venezuelan Equine Encephalitis
Virus (VEEV) (EC50 = 0.8 M, CC50 = 50 M) [23]. Quinazolinone (aS)- displayed high selectivity
for PI4KIII and appeared to be potent inhibitors of hepatitis C virus (HCV) replication in vitro
Moreover, compound (aS)- exhibited higher potency for PI4KIII than its atropisomer aR (pIC50 of
µ
µ
9
α
.
9
α
8.3 vs. 6.9) and an improved selectivity range (2.7–3.2 vs. 1.4–2.1) against the other lipid kinases [24].
Figure 1. Examples of C6-substituted quinazolinone derivatives with antiviral activity.
Recently, we have succeeded in synthesis of a series of quinazolinones substituted at C3 with
a (diethoxyphosphoryl)isoxazolidine moiety 10. Several of the synthesized analogues exhibited
moderate activity against varicella zoster virus (VZV) and human cytomegalovirus (HCMV) [25].
On the basis of these observations, and in continuation of our search for new biologically active
compounds [25], a novel series of quinazolinone derivatives of general formula 11 having an
isoxazolidine ring at C2, different substituents at N3, and an additional bromine atom at C6 was

Molecules 2018, 23, 1889
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synthesized. The synthetic strategy for our new isoxazolidine-conjugates of quinazolinones relies
on the 1,3-dipolar cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone 12
6-bromo-2-vinyl-3H-quinazolin-4-ones 13 (Scheme 1).
[26] with selected
Scheme 1. Retrosynthesis of isoxazolidine conjugates of 6-bromoquinazolinones.
2. Results and Discussion
2.1. Chemistry
The respective 3-substituted 6-bromo-2-vinyl-3H-quinazolin-4-ones 13a
from the commercially available isatoic acid anhydride, which was first reacted with bromine and then
transformed into 5-bromoanthranilic acid amide 14 according to the procedure in the literature [27 28].
–k were synthesized starting
,
Reaction 5-bromoanthranilic acid amide 14 with 3-chloropropionyl chloride followed by cyclization and
dehydrohalogenation led to the formation of 6-bromo-2-vinyl-3H-quinazolin-4-one 13a, which was then
used as a key intermediate in the preparation of N3-substituted derivatives 13b–13k via alkylation with
the respective benzyl bromides, as well as with methyl or ethyl iodide [29] (Scheme 2).
Scheme 2. Synthesis of 3-substituted 6-bromo-2-vinylquinazolinones 13a–13k. Reaction and conditions:
a. 3-chloropropionyl chloride, 1,4-dioxane, 0 ◦C; b. 5% NaOH–EtOH (2:1); c. RBr, KOH, 105 ◦C; d.
◦
MeI or EtI, KOH, CH3CN, 60 C.
The 1,3-dipolar cycloadditions of nitrone 12 with the respective 6-bromo-2-vinylquinazolinones
–
13k were carried out at 70 ◦C in toluene and afforded mixtures of diastereoisomeric isoxazolidines
13a
trans-11a
–11k and cis-11a–11k (Scheme 3, Table 1). Ratios of trans/cis diastereoisomers were determined on
the basis of the 31P-NMR spectra of crude products and confirmed by the analysis of 1H-NMR spectra data.
In all cases good trans/cis diastereoselectivities were observed (in the range of 76–88%) and trans-isomers
predominated. The mixtures of isoxazolidine cycloadducts were separated chromatographically on silica
gel. The isolation of pure isomers was successfully accomplished for the major isomers trans-11a (R = H),
trans-11b (R = C6H5CH2), trans-11c (R = 2-NO2-C6H4-CH2), trans-11d (R = 3-NO2-C6H4-CH2), trans-11e
(R = 4-NO2-C6H4-CH2), trans-11g (R = 3-F-C6H4-CH2), trans-11h (R = 4-F-C6H4-CH2), trans-11j (R = CH3),
and trans-11k (R = CH3CH2), as well as for a minor isomer cis-11a (R = H).

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Scheme 3. Synthesis of isoxazolidines trans-11a–k and cis-11a–k. Reaction and conditions: a. toluene,
◦
70 C, 24 h.
Table 1. Isoxazolidines trans-11 and cis-11 obtained according to Scheme 3.
Quinazolinone 13
Entry
Yield (%)
Ratio of trans-11:cis-11
R
trans-11a (23) a + cis-11a (5) a + trans-11a and cis-11a (62) b
trans-11b (8) a + trans-11b and cis-11b (83) b
trans-11c (30) a + trans-11c and cis-11c (62) b
trans-11d (14) a + trans-11d and cis-11d (80) b
trans-11e (18) a + trans-11e and cis-11e (72) b
trans-11f and cis-11f (96) b
a
b
c
d
e
f
g
h
i
H
92:8
92:8
94:6
92:8
90:10
90:10
92:8
92:8
93:7
94:6
88:12
C6H5-CH2
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
CH3
trans-11g (10) a + trans-11g and cis-11g (72) b
trans-11h (12) a + trans-11h and cis-11h (86) b
trans-11i and cis-11i (82) b
trans-11j (32) a + trans-11j and cis-11j (65) b
j
k
trans-11k (14) a + trans-11k and cis-11k (80) b
CH3CH2
a
yield of pure isomer, b yield of pure mixture of cis- and trans-isomers.
Stereochemistry of the cycloaddition of a nitrone 12 to N3-substituted derivatives of
2-vinyl-3H-quinazolin-4-ones has recently been described and relative configurations of trans-
and cis-isoxazolidine cycloadducts were established based on conformational analysis [25].
Since the introduction of the bromine atom at C6 of the quinazolin-4-one framework has no
influence on the stereochemical outcome of the reaction of nitrone 12 with N3-susbstituted
2-vinyl-3H-quinazolin-4-ones, the trans and cis configurations of the major trans-11a
cis-11a–k isomers were assigned taking advantage of our previous observations.
–k and minor
2.2. Antiviral and Cytostatic Evaluation
2.2.1. Antiviral Activity
Pure isomers trans-11a, trans-11b, trans-11c, trans-11d, trans-11e, trans-11g, trans-11h, trans-11j
,
trans-11k, and cis-11a, as well as diastereoisomeric mixtures of isoxazolidines trans-11f/cis-11f (95:5) and
trans-11i/cis-11i (92:8), were tested for inhibitory activity against a wide variety of DNA and RNA viruses.
The following cell-based assays were used: (a) human embryonic lung (HEL) cells (herpes simplex virus-1
(KOS strain), herpes simplex virus-2 (G strain), thymidine kinase deficient (acyclovir resistant) herpes
−
simplex virus-1 (TK KOS ACVr), vaccinia virus, adenovirus-2, vesicular stomatitis virus, cytomegalovirus
(AD-169 and Davis strains), varicella-zoster virus (TK+ VZV and TK VZV strains)); (b) HeLa cell cultures
−
(vesicular stomatitis virus, Coxsackie virus B4, and respiratory syncytial virus); (c) Vero cell cultures
(parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, yellow fever virus);
(d) CrFK cell cultures (feline corona virus (FIPV) and feline herpes virus (FHV)); and (e) 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 10000, DS-10000), mycophenolic acid, Hippeastrum hybrid agglutinin (HHA), and Urtica dioica
agglutinin (UDA) were used as the reference compounds. The antiviral activity was expressed as the
EC50: the compound concentration required to reduce virus plaque formation (VZV) by 50% or to reduce
virus-induced cytopathogenicity by 50% (other viruses).

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Almost all synthesized isoxazolidines inhibited the replication of both TK+ and TK− VZV
strains at EC50’s in the 5.4–13.6 M range (Table 2), at the same time they were inactive toward
µ
other tested viruses, except for HCMV. In general, the inhibitory activity of the tested compounds
toward TK− VZV 07-1 strain was better than that of the reference drugs acyclovir and brivudin
(EC50 = 39.2 µM and EC50 = 31.9 µM, respectively), which require activation by the viral enzyme.
On the other hand, the activity of these compounds against the TK+ VZV OKA strain was five to
nine-fold and 360 to 587-fold lower than that of the reference drugs acyclovir and brivudin, respectively.
While exhibiting significant activity toward both TK+ and TK− VZV stains, compounds trans-11b
,
trans-11d, trans-11g, and trans-11h exhibited the lowest cytotoxicity (CC50 = 33–42
mentioning that incorporation of the bromine atom at C6 in the quinazolinone skeleton resulted in a
significant increase in potency of isoxazolidine-conjugates 11b 11i against VZV (up to five-fold higher)
µM). It is worth
–
when compared with previously described analogous conjugates 10b–10i having an unsubstituted
quinazoline skeleton [25].
Table 2. Antiviral activity and cytotoxicity against varicella-zoster virus (VZV) in HEL cell cultures.
Antiviral Activity EC50 (µM) a
Cytotoxicity (µM)
Compound
R
TK+ VZV Strain (OKA)
TK VZV Strain (07-1)
Cell Growth CC50
−
c
Cell Morphology MCC b
cis-11a
trans-11a
trans-11b
trans-11c
trans-11d
H
H
>100
>100
13.5 ± 7.1
10.3 ± 1.1
8.3 ± 1.4
6.84
>100
66.87
13.6 ± 9.1
5.4 ± 1.0
5.8 ± 1.4
7.51
>100
>100
100
100
100
20
n.d.
n.d.
42.3 ± 0.3
9.4 ± 0.4
37.0 ± 4.3
n.d.
C6H5-CH2
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
CH3
trans-11e
trans-11f/cis-11f (95:5)
trans-11g
trans-11h
trans-11i/cis-11i (92:8)
trans-11j
7.76
9.56
20
n.d.
11.6 ± 5.3
12.6 ± 2.6
8.7 ± 3.2
>4
7.7 ± 6.2
7.5 ± 5.4
10.5 ± 0.3
>4
>20
>20
>20
20
33.0 ± 3.5
39.4 ± 5.2
15.6 ± 3.2
n.d.
trans-11k
Acyclovir
Brivudin
CH3CH2
>20
>20
100
>440
>300
n.d.
>350
>300
1.55 ± 1.0
39.2 ± 3.6
0.023 ± 0.008
31.9 ± 16.1
a Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units
(PFU), b Minimum cytotoxic concentration that causes a microscopically detectable alternation of cell morphology, c
Cytotoxic concentrations required to reduce cell growth by 50%, n.d.—not determined.
The synthesized isoxazolidine phosphonates also showed marked activity against human
cytomegalovirus (HCMV) (Table 3) and, among them, trans-11d, trans-11g, trans-11h, and trans-11i/cis-11i
(92:8) were the most potent ones. Activities of the tested compounds against both AD-169 and Davis strains
were comparable (EC50 = 8.9–12.5 µM) to that of Ganciclovir (EC50 = 16.9 and 7.7 µM) used as a reference
drug, but still lower than that of the other reference drug, Cidofovir (EC50 = 1.5–1.7 µM).
Table 3. Antiviral activity and cytotoxicity against human cytomegalovirus in HEL cell cultures.
Antiviral Activity EC50 (µM) a
Cytotoxicity (µM)
Compound
R
c
Cell Morphology MCC b
AD-169 Strain
Davis Strain
Cell Growth CC50
cis-11a
trans-11a
trans-11b
H
H
>100
>100
20
100
>100
20
20
100
20
n.d.
n.d.
n.d.
C6H5-CH2
trans-11c
trans-11d
trans-11e
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
CH3
>20
15.29
100
100
100
20
100
100
>20
20
n.d.
10.4 ± 0.8
11.6 ± 2.5
37.0 ± 4.3
>20
>20
n.d.
trans-11f/cis-11f (95:5)
trans-11g
trans-11h
trans-11i/cis-11i (92:8)
trans-11j
8.94
8.94
n.d.
10.5 ± 2.2
12.5 ± 3.9
9.4 ± 0.46
>20
8.94 ± 0
11.2 ± 3.1
9.7 ± 1.1
>100
33.0 ± 3.5
39.4 ± 5.2
15.6 ± 3.2
n.d.
trans-11k
Ganciclovir
Cidofovir
CH3CH2
>20
44.72
20
>350
>300
n.d.
>350
>300
16.9 ± 6.9
7.7 ± 0.9
1.5 ± 0.2
1.7 ± 0.4
a Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units
(PFU), b Minimum cytotoxic concentration that causes a microscopically detectable alternation of cell morphology,
c
Cytotoxic concentrations required to reduce cell growth by 50%, n.d.—not determined.

Molecules 2018, 23, 1889
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2.2.2. Cytostatic Activity
The 50% cytostatic inhibitory concentration (IC50) causing a 50% decrease in cell proliferation was
determined against murine leukemia L1210, human lymphocyte CEM, human cervix carcinoma
HeLa, and immortalized human dermal microvacsular endothelial cells (HMEC-1). Almost all
compounds 11a
–11k showed inhibitory activity against the proliferation of tumour cell lines (Table 4).
They appeared to be the most cytostatic toward the human T-lymphocyte (CEM) cell line with an
inhibitory effect in the 9.6–33
µ
M range. Thus, isoxazolidines trans-11b (IC50 = 16
M), trans-11e (IC50 = 17 12 M), trans-11f/cis-11f (95:5) (IC50 = 13
M), trans-11h (IC50 = 9.6 2.2
±
±
3
1
µ
M), trans-11c
M), trans-11g
(IC50 = 14
(IC50 = 10
±
4
0
µ
±
µ
µ
±
µ
±
µ
M), and trans-11i/cis-11i (92:8) (IC50 = 9.8
±
4.8 µM)
exhibited activity toward the CEM line higher than of 5-fluorouracil (IC50 = 18 ± 5 µM).
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).
a
IC50 (µM)
Compound
R
L1210
CEM
HeLa
HMEC-1
cis-11a
trans-11a
trans-11b
trans-11c
trans-11d
H
H
>250
126 ± 19
20 ± 2
19 ± 3
26 ± 1
242 ± 7
148 ± 2
16 ± 3
>250
196 ± 20
26 ± 7
140 ± 54
85 ± 13
30 ± 0
30 ± 0
30 ± 0
26 ± 6
30 ± 0
30 ± 0
29 ± 1
30 ± 0
>250
C6H5-CH2
2-NO2-C6H4-CH2
3-NO2-C6H4-CH2
14 ± 4
22 ± 11
24 ± 9
33 ± 25
17 ± 12
13 ± 1
trans-11e
4-NO2-C6H4-CH2 18 ± 76
24 ± 9
trans-11f/cis-11f (95:5)
trans-11g
trans-11h
trans-11i/cis-11i (92:8)
trans-11j
trans-11k/cis-11k (97:3)
5-Fluorouracil
2-F-C6H4-CH2
3-F-C6H4-CH2
4-F-C6H4-CH2
2,4-diF-C6H3-CH2
CH3
17 ± 2
16 ± 2
25 ± 7
10 ± 0
25 ± 7
16 ± 0
9.6 ± 2.2
9.8 ± 4.8
128 ± 21
44 ± 18
18 ± 5
25 ± 7
16 ± 1
22 ± 6
138 ± 19
102 ± 14
0.33 ± 0.17
175 ± 105
113 ± 44
0.54 ± 0.12
CH3CH2
121 ± 41
n.d.
a
50% Inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%,
n.d.—not determined.
Structure–activity relationship studies indicated that the isoxazolidine derivatives possessing
hydrogen (cis-11a, trans-11a), methyl (trans-11j), or ethyl (trans-11k) at N3 in the quinazolinone ring
were not active against tested viruses and they did not inhibit proliferation of the tested cell lines.
Compounds substituted at N3 with benzyl moieties were found effective against VZV, HCMV, and were
able to inhibit tumour cell line growth. Moreover, isoxazolidine phosphonates having 3-nitrobenzyl
(trans-11d) and 3-fluorobenzyl (trans-11g) substituents at N3 of the quinazolinone core showed the
highest antiviral activity with an EC50 values in the range of 5.8–11.6 µM.
On the other hand, compounds possessing 3-fluorobenzyl (trans-11g), 4-fluorobenzyl (trans-11h
)
and 2,4-difluorobenzyl (trans-11i/cis-11i (92:8)) substituents at the N3 position showed an inhibitory
effect against the proliferation of murine leukemia (L1210) and human T-lymphocyte (CEM) cell lines,
whereas isoxazolidines having 2-nitrobenzyl (trans-11c) and 2,4-difluorobenzyl (trans-11i/cis-11i (92:8))
substituents at the same position of a quinazolinone moiety appeared to be the most active toward the
human cervix carcinoma (HeLa) cell line, however, their inhibitory concentration was much lower
than that of the reference drugs.
3. Experimental Section
3.1. General
1H-NMR spectra were taken in CDCl3 on the following spectrometers: Gemini 2000BB (200 MHz
Varian, Palo Alto, CA, USA), and Bruker Avance III (600 MHz, Bruker Instrument, Karlsruhe, Germany)

Molecules 2018, 23, 1889
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with TMS as internal standard. 13C-NMR spectra were recorded for CDCl3 solution on the Bruker
Avance III at 151.0 MHz. 31P-NMR spectra were performed in CDCl3 solution on the 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, DDR—currently Germany) and are uncorrected.
Elemental analyses were performed by the Microanalytical Laboratory of this faculty on a 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 sheet silica gel 60 F254
.
N-methyl-C-(diethoxyphosphoryl)nitrone [26] and 5-bromoanthranilic acid amide [27,28] were
obtained according to the procedures in the literature.
3.2. Synthesis of 6-Bromo-2-vinyl-3H-quinazolin-4-one 13a
To a solution 5-bromoanthranilic amide (2.40 g, 11.2 mmol) in 1,4-dioxane (5 mL) 3-chloropropionyl
chloride (0.527 mL, 5.60 mmol) was added over 20 min. at 0 ◦C under an argon atmosphere. The mixture
was stirred at room temperature for 1 h and then diluted with water until a precipitate appeared, which was
collected and washed with water (3
×
10 mL). A mixture of 2-(3-chloropropionylamino)benzamide (2.03 g,
6.63 mmol) in 5% sodium hydroxide (16.4 mL) and ethanol (8.2 mL) was stirred under reflux for 5 min.,
the solution was allowed to cool for 15 min. and then acidified with acetic acid (1.6 mL). The precipitate
was collected and washed with water (3 × 10 mL). The crude 6-bromo-2-vinylquinazolin-4(3H)-one was
purified by crystallization from methanol.
Off-white amorphous solid, m.p. = 223–225 ◦C (re-crystallized from methanol). IR (KBr, cm−1
3165, 3088, 3019, 2943, 2738, 1673, 1638, 1591, 1462, 1337, 1292, 1245, 1207, 1114, 1065, 977, 829, 793, 687, 633.
)
ν
max
:
1H-NMR (200 MHz, DMSO): = 12.51 (s, 1H, NH), 8.17 (d, 4J = 2.4 Hz, 1H, HC5), 7.90 (dd, 3J = 8.7 Hz,
δ
4J = 2.4 Hz, 1H, HC7), 7.81 (d, 3J = 8.7 Hz, 1H, HC8), 6.63 (dd, 3J = 17.5 Hz, 2J = 3.7 Hz, 1H, CH=CH2), 6.53
(dd, 3J = 17.5 Hz, 3J = 8.1 Hz, 1H, CH=CH2), 5.86 (dd, 3J = 8.1 Hz, 2J = 3.7 Hz, 1H, CH=CH2). 13C-NMR
(151 MHz, DMSO): δ = 161.11 (C=O), 152.00, 148.23, 137.80, 131.00, 130.18, 128.45, 126.31, 123.40, 119.37.
Anal. Calcd. for C10H7BrN2O: C, 47.84; H, 2.81; N, 11.16. Found: C, 47.88; H, 2.49; N, 11.01.
3.3. General Procedure for the Synthesis of N3-Benzylated 6-bromo-2-vinyl-3H-quinazolin-4-ones 13b–13i
To a solution of 6-bromo-2-vinyl-3H-quinazolin-4-one (0.251 g, 1.00 mmol) in acetonitrile (15 mL)
potassium hydroxide (0.168 g, 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. The solvent was
removed and the residue was re-dissolved in methylene chloride (10 mL) and extracted with water
(
3 × 10 mL). An organic phase was dried (MgSO4), concentrated, and the crude product was purified
on a silica gel column with a methylene chloride:hexane (7:3, v/v) mixture followed by crystallization
(chloroform-petroleum ether or ethyl acetate) to give pure quinazolinones.
3-Benzyl-6-bromo-2-vinylquinazolin-4(3H)-one (13b). A white amorphous solid, m.p. = 71–72 ◦C
(re-crystallized from ethyl acetate). IR (KBr, cm−1
)
ν
max: 3097, 3036, 2926, 1608, 1571, 1553, 1470,
= 8.30–8.27 (m, 1H), 7.88–7.82
1
1290, 1153, 1112, 989, 841, 678, 581, 437. H-NMR (200 MHz, CDCl3):
δ
(m, 1H), 7.77–7.73 (m, 1H), 7.57–7.50 (m, 2H), 7.48–7.37 (m, 3H), 6.91 (dd, 3J = 17.2 Hz, 3J = 9.9 Hz,
1H, CH=CH2), 6.73 (dd, 3J = 17.2 Hz, 2J = 2.5 Hz, 1H, CH=CH2), 5.82 (dd, 3J = 9.9 Hz, 2J = 2.5 Hz, 1H,
CH=CH2), 5.67 (s, 2H, N-CH2). 13C-NMR (151 MHz, CDCl3):
δ = 165.16 (C=O), 160.28, 150.36, 137.00,
136.88, 136.02, 129.45, 128.67, 128.45, 126.09, 124.36, 120.03, 116.67, 68.69 (N-CH2). Anal. Calcd. for
C17H13BrN2O: C, 59.84; H, 3.84; N, 8.21. Found: C, 59.92; H, 3.68; N, 8.29.
6-Bromo-3-(2-nitrobenzyl)-2-vinylquinazolin-4(3H)-one (13c). A yellowish amorphous solid, m.p.
◦
117–119 C (re-crystallized from chloroform-petroleum ether). IR (KBr, cm−1
)
ν
max: 3078, 2980,
2925, 2854, 1689, 1613, 1573, 1526, 1489, 1390, 1354, 1287, 1242, 1116, 1053, 1024, 968, 836, 791, 730.

Molecules 2018, 23, 1889
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1H-NMR (600 MHz, CDCl3):
δ
= 8.33 (d, 4J = 2.2 Hz, 1H, HC5), 8.18–8.16 (m, 1H), 7.90 (dd, 3J = 8.9 Hz,
4J = 2.2 Hz, 1H, HC7), 7.81 (d, 3J = 8.9 Hz, 1H, HC8), 7.80–7.78 (m, 1H), 7.71–7.69 (m, 1H), 7.56–7.53
(m, 1H), 6.87 (dd, 3J = 17.2 Hz, 3J = 10.4 Hz, 1H, CH=CH2), 6.64 (dd, 3J = 17.2 Hz, 2J = 1.5 Hz, 1H,
CH=CH2), 6.10 (s, 2H, N-CH2), 5.80 (dd, 3J = 10.4 Hz, 2J = 1.5 Hz, 1H, CH=CH2). 13C-NMR (151 MHz,
CDCl3):
δ = 164.62 (C=O), 160.12, 150.32, 147.84, 137.31, 136.31, 133.78, 132.33, 129.51, 129.05, 128.87,
125.76, 125.07, 124.92, 120.38, 116.31, 65.14 (N-CH2). Anal. Calcd. for C17H12BrN3O3: C, 52.87; H, 3.13;
N, 10.88. Found: C, 52.50; H, 2.80; N, 10.80.
6-Bromo-3-(3-nitrobenzyl)-2-vinylquinazolin-4(3H)-one (13d). A yellowish amorphous solid, m.p.
◦
147–149 C (re-crystallized from chloroform-petroleum ether). IR (KBr, cm−1
)
ν
max: 3112, 3071,
= 8.44
1
2924, 2854, 1610, 1573, 1529, 1456, 1382, 1294, 1119, 836, 668. H-NMR (600 MHz, CDCl3):
δ
(brs, 1H), 8.32–8.30 (m, 1H), 8.26–8.24 (m, 1H), 7.91–7.88 (m, 2H), 7.80–7.78 (m, 1H), 7.64–7.61 (m, 1H),
6.91 (dd, 3J = 17.2 Hz, 3J = 10.4 Hz, 1H, CH=CH2), 6.73 (dd, 3J = 17.2 Hz, 2J = 1.4 Hz, 1H, CH=CH2),
5.84 (dd, 3J = 10.4 Hz, 2J = 1.4 Hz, 1H, CH=CH2), 5.78 (s, 2H, N-CH2). 13C-NMR (151 MHz, CDCl3):
δ
= 164.69 (C=O), 160.05, 150.49, 148.51, 138.14, 137.28, 136.68, 134.10, 129.73, 129.60, 125.84, 124.47,
123.37, 123.27, 120.35, 116.34, 67.22 (N-CH2). Anal. Calcd. for C17H12BrN3O3: C, 52.87; H, 3.13; N,
10.88. Found: C, 52.81; H, 2.98; N, 10.60.
◦
6-Bromo-3-(4-nitrobenzyl)-2-vinylquinazolin-4(3H)-one (13e). Off-white amorphous solid, m.p. 121–123 C
(re-crystallized from chloroform-petroleum ether). IR (KBr, cm−1
)
ν
max: 3418, 2924, 2853, 1739, 1608,
1
1573, 1524, 1343, 1292, 1117, 945, 833, 661. H-NMR (200 MHz, CDCl3):
δ
= 8.30–8.26 (m, 3H), 7.96–7.92
(m, 1H), 7.81–7.76 (m, 1H), 7.73–7.67 (m, 2H), 6.88 (dd, J = 17.2 Hz, J = 10.1 Hz, 1H, CH=CH2),
6.65 (dd, 3J = 17.2 Hz, 2J = 2.1 Hz, 1H, CH=CH2), 5.80 (dd, 3J = 10.1 Hz, 2J = 2.1 Hz, 1H, CH=CH2),
3
3
5.77 (s, 2H, N-CH2). 13C-NMR (151 MHz, CDCl3):
δ = 164.69 (C=O), 160.03, 150.41, 147.93, 143.27,
137.37, 136.59, 129.58, 128.50, 125.80, 124.58, 123.93, 120.44, 116.33, 67.14 (N-CH2). Anal. Calcd. for
C17H12BrN3O3: C, 52.87; H, 3.13; N, 10.88. Found: C, 52.81; H, 2.98; N, 10.60.
6-Bromo-3-(2-fluorobenzyl)-2-vinylquinazolin-4(3H)-one (13f). A white amorphous solid, m.p. 91–92 ◦C
(re-crystallized from ethyl acetate). IR (KBr, cm−1
1238, 1115, 938, 828, 760, 675. H-NMR (600 MHz, CDCl3):
)
ν
max: 3078, 2967, 1568, 1555, 1486, 1420, 1348, 1293,
1
δ
= 8.31 (d, 4J = 2.2 Hz, 1H, HC5), 7.89
(dd, 3J = 8.9 Hz, 4J = 2.2 Hz, 1H, HC7), 7.68 (d, 3J = 8.9 Hz, 1H, HC8), 7.61–7.58 (m, 1H), 7.42–7.38
(m, 1H), 7.23–7.20 (m, 1H), 7.18–7.15 (m, 1H), 6.92 (dd, 3J = 17.2 Hz, 3J = 10.4 Hz, 1H, CH=CH2), 6.78
(dd, 3J = 17.2 Hz, 2J = 1.7 Hz, 1H, CH=CH2), 5.84 (dd, 3J = 10.4 Hz, 2J = 1.7 Hz, 1H, CH=CH2), 5.78
(s, 2H, N-CH2). 13C-NMR (151 MHz, CDCl3):
δ
= 164.99 0(C=O), 161.20 (d, J(CF) = 248.8 Hz, C20),
1
160.24, 150.39, 137.03, 136.81, 130.70 (d, 3J(CCCF) = 4.0 Hz, C4 ), 130.39 (d, 3J(CCCF) = 7.9 Hz, C6 ), 129.47,
0
0
0
126.04, 124.44, 124.25 (d, 4J(CCCCF) = 3.4 Hz, C5 ), 123.24 (d, 2J(CCF) = 14.5 Hz, C3 ), 120.06, 116.59, 115.63
0
(d, 2J(CCF) = 21.5 Hz, C1 ), 62.51 (d, 3J(CCF) = 4.4 Hz, N-CH2). Anal. Calcd. for C17H12BrFN2O: C, 56.85;
H, 3.37; N, 7.80. Found: C, 56.62; H, 2.99; N, 7.79.
6-Bromo-3-(3-fluorobenzyl)-2-vinylquinazolin-4(3H)-one (13g). A yellowish amorphous solid, m.p.
78–80 ◦C (re-crystallized from ethyl acetate). IR (KBr, cm−1
1490, 1416, 1336, 1286, 1115, 1055, 967, 836, 790. H-NMR (600 MHz, CDCl3):
1H, HC5), 7.89 (dd, 3J = 8.9 Hz, 4J = 2.2 Hz, 1H, HC7), 7.80 (d, 3J = 8.9 Hz, 1H, HC8), 7.43–7.39 (m, 1H),
7.34–7.32 (m, 1H), 7.28–7.26 (m, 1H), 7.11–7.08 (m, 1H), 6.92 (dd, 3J = 17.1 Hz, 3J = 10.4 Hz, 1H, CH=CH2),
6.73 (dd, 3J = 17.1 Hz, 2J = 1.7 Hz, 1H, CH=CH2), 5.84 (dd, 3J = 10.4 Hz, 2J = 1.7 Hz, 1H, CH=CH2),
)
ν
max: 3070, 2980, 2925, 2853, 1614, 1568,
1
δ
= 8.32 (d, 4J = 2.2 Hz,
5.69 (s, 2H, N-CH2). 13C-NMR (151 MHz, CDCl3):
δ
= 164.95 (C=O), 162.95(d, 1J(CF) = 246.6 Hz, C30),
0
0
160.19, 150.43, 138.50 (d, 3J(CCCF) = 7.5 Hz, C5 ), 137.13, 136.79, 130.25 (d, J = 8.1 Hz, C1 ), 129.53, 125.98,
4
2
124.42, 123.74 (d, J(CCCCF) = 3.1 Hz, C60), 120.18, 116.54, 115.33 (d, J(CCF) = 21.0 Hz, C40), 115.14
(d, 2J(CCF) = 22.0 Hz, C20), 67.77 (d, 4JCCCCF = 1.5 Hz, N-CH2). Anal. Calcd. for C17H12BrFN2O: C,
56.85; H, 3.37; N, 7.80. Found: C, 56.99; H, 2.97; N, 7.84.
6-Bromo-3-(4-fluorobenzyl)-2-vinylquinazolin-4(3H)-one (13h). A yellowish amorphous solid, m.p.
◦
100–103 C (re-crystallized from chloroform-petroleum ether). IR (KBr, cm−1
) νmax: 3064, 2926,

Molecules 2018, 23, 1889
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1608, 1565, 1510, 1487, 1354, 1219, 1118, 989, 832, 697, 501. 1H-NMR (200 MHz, CDCl3):
(d, J = 2.1 Hz, 1H, HC5), 7.85 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H, HC7), 7.75 (d, J = 8.9 Hz, 1H,
HC8), 7.55–7.48 (m, 2H), 7.15–7.06 (m, 2H), 6.90 (dd, 3J = 17.2 Hz, 3J = 11.5 Hz, 1H, CH=CH2), 6.72
(dd, 3J = 17.2 Hz, 2J = 2.3 Hz, 1H, CH=CH2), 5.82 (dd, 3J = 11.5 Hz, 2J = 2.3 Hz, 1H, CH=CH2), 5.63
δ
= 8.26
4
3
4
3
0
δ
= 165.04 (C=O), 162.82 (d, 1J(CF) = 247.1 Hz, C4 ), 160.20,
(s, 2H, N-CH2). 13C-NMR (151 MHz, CDCl3):
150.40, 137.04, 136.88, 131.84 (d, 4J(CCCCF) = 3.1 Hz, C10), 130.39 (d, 3J(CCCF) = 8.6 Hz, C20, C60), 129.50,
125.99, 124.28, 120. 08, 116.60, 115.62 (d, 2J(CCF) = 21.9 Hz, C30, C50), 67.94 (N-CH2). Anal. Calcd. for
C17H12BrFN2O: C, 56.85; H, 3.37; N, 7.80. Found: C, 56.50; H, 3.01; N, 7.76.
6-Bromo-3-(2,4-difluorobenzyl)-2-vinylquinazolin-4(3H)-one (13i). A yellowish amorphous solid, m.p.
88–90 ◦C (re-crystallized from chloroform-petroleum ether). IR (KBr, cm−1
1614, 1565, 1508, 1488, 1416, 1349, 1279, 1099, 955, 834, 728, 538. H-NMR (600 MHz, CDCl3):
)
ν
max: 3079, 2923, 2852,
= 8.27
1
δ
4
3
4
3
(d, J = 2.1 Hz, 1H, HC5), 7.88 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H, HC7), 7.78 (d, J = 8.9 Hz, 1H,
HC8), 7.59–7.55 (m, 1H), 6.96–6.89 (m, 2H), 6.89 (dd, 3J = 17.2 Hz, 3J = 10.0 Hz, 1H, CH=CH2), 6.72
3
2
3
2
(dd, J = 17.2 Hz, J = 2.4 Hz, 1H, CH=CH2), 5.82 (dd, J = 10.0 Hz, J = 2.4 Hz, 1H, CH=CH2),
5.68 (s, 2H, N-CH2). 13C-NMR (151 MHz, CDCl3)
δ
: 164.86 (C=O), 163.23 (dd, J(CF) = 250.4 Hz,
1
3J(CCCF) = 12.0 Hz, C20), 161.45 (dd, 1J(CF) = 251.8 Hz, 3J(CCCF) = 12.1 Hz, C40), 160.15, 150.38, 137.06,
136.80, 131.83 (dd, J(CCCF) = 9.8 Hz, J(CCCF) = 5.4 Hz, C60), 129.49, 125.95, 124.39, 120.12, 119.29
(dd, 2J(CCF) = 15.0 Hz, 4J(CCCCF) = 4.0 Hz, C10), 116.48, 111.50 (dd, 2J(CCF) = 21.7 Hz, 4J(CCCCF) = 4.1 Hz,
C50), 104.16 (dd, 2J(CCF) = 25.3 Hz, 2J(CCF) = 25.3 Hz, C30), 61.93 (d, 3J(CCCF) = 3.4 Hz, N-CH2) Anal.
Calcd. for C17H11BrF2N2O: C, 54.13; H, 2.94; N, 7.43. Found: C, 54.42; H, 3.14; N, 7.04.
3
3
3.4. General Procedure for the Synthesis of N3-Alkylated 6-bromo-2-vinyl-3H-quinazolin-4-ones 13j and 13k
To the solution of 6-bromo-2-vinyl-3H-quinazolin-4-one (0.251 g, 1.00 mmol) in acetonitrile (15 mL)
potassium hydroxide (0.168 g, 3.00 mmol) was added. After 15 min iodomethane (0.124 mL, 2.00 mmol)
◦
or iodoethane (0.088 mL, 1.10 mmol) was added and the reaction mixture was stirred at 60 C for
5 h. The solvent was removed and the residue was re-dissolved in methylene chloride (10 mL) and
extracted with water (3
×
10 mL). The organic layer was dried (MgSO4), concentrated, and the crude
product was purified on a silica gel column with a methylene chloride:hexane mixture (7:3, v/v) mixture
followed by crystallization (chloroform:petroleum ether).
◦
6-Bromo-3-methyl-2-vinylquinazolin-4(3H)-one (13j). A white amorphous solid, m.p. = 147–148 C
(re-crystallized from chloroform-petroleum ether). IR (KBr, cm−1
)
ν
max: 2956, 2924, 1675, 1661, 1555,
= 8.21 (d, 4J = 2.1 Hz, 1H, HC5),
7.79 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H, HC7), 7.67 (d, J = 8.9 Hz, 1H, HC8), 6.83 (dd, J = 17.2 Hz,
3J = 9.9 Hz, 1H, CH=CH2), 6.66 (dd, 3J = 17.2 Hz, 2J = 2.5 Hz, 1H, CH=CH2), 5.74 (dd, 3J = 9.9 Hz,
1
1468, 1337, 1260, 1025, 981, 791, 645. H-NMR (200 MHz, CDCl3):
δ
3
4
3
3
2J = 2.5 Hz, 1H, CH=CH2), 4.14 (s, 3H, CH3). 13C-NMR (151 MHz, CDCl3):
δ = 161.06 (C=O), 152.56,
146.20, 137.33, 129.34, 129.29, 128.92, 127.19, 121.95, 120.12, 30.95. Anal. Calcd. for C11H9BrN2O: C,
49.84; H, 3.42; N, 10.57. Found: C, 50.11; H, 3.42; N, 10.56.
6-Bromo-3-ethyl-2-vinylquinazolin-4(3H)-one (13k). A white amorphous solid, m.p. = 62–64 ◦C
(re-crystallized from chloroform-petroleum ether). IR (KBr, cm−1
) νmax: 3097, 3035, 2956, 2926,
1
2853, 1610, 1571, 1554, 1488, 1428, 1347, 1291, 1153, 1018, 842, 807, 678. H-NMR (200 MHz, CDCl3):
4
4
3
δ
= 8.22 (d, J = 2.2 Hz, 1H, HC5), 7.79 (dd, 3J = 8.9 Hz, J = 2.2 Hz, 1H, HC7), 7.68 (d, J = 8.9 Hz,
1H, HC8), 6.82 (dd, 3J = 17.2 Hz, 3J = 10.0 Hz, 1H, CH=CH2), 6.63 (dd, 3J = 17.2 Hz, 2J = 2.4 Hz, 1H,
CH=CH2), 5.73 (dd, 3J = 10.0 Hz, 2J = 2.4 Hz, 1H, CH=CH2), 4.62 (q, 3J = 7.1 Hz, 2H, CH3CH2), 1.47
(t, 3J = 7.1 Hz, 3H, CH3CH2). 13C-NMR (151 MHz, CDCl3):
δ = 161.06 (C=O), 160.37, 150.12, 136.90,
136.78, 129.34, 126.10, 124.19, 119.83, 116.71, 63.10, 14.28. Anal. Calcd. for C12H11BrN2O: C, 51.63; H,
3.97; N, 10.04. Found: C, 51.94; H, 3.75; N, 9.94.

Molecules 2018, 23, 1889
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3.5. General Procedure for the Synthesis of Isoxazolidines trans-11 and cis-11
A solution of the nitrone (0.195 g, 1.00 mmol) and the respective 6-bromo-2-vinylquinazolin-4(3H)-one
(1.00 mmol) in toluene (2 mL) was stirred at 70 ◦C until the disappearance (TLC) of the starting nitrone.
Solvents were evaporated in vacuo and crude products were subjected to chromatography on silica gel
columns with chloroform:methanol (100:1, 50:1, 20:1, v/v) mixtures.
Diethyl trans-[5-(6-bromo-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl]phosphonate (trans-11a).
Colorless oil. IR (film, cm−1
) νmax: 3316, 3171, 3090, 2980, 2974, 2783, 1660, 1625, 1486, 1412, 1301, 1234,
0
δ
= 10.63 (s, 1H, NH), 8.44 (d, 4J = 2.3 Hz, 1H, HC5 ),
1054, 968, 834, 775, 575. 1H-NMR (600 MHz, CDCl3):
0
0
7.84 (dd, 3J = 8.6 Hz, 4J = 2.3 Hz, 1H, HC7 ), 7.54 (d, 3J = 8.6 Hz, 1H, HC8 ), 5.02 (dd, 3J(H5–H4β) = 8.4 Hz,
3J(H5–H4α) = 6.2 Hz, 1H, HC5), 4.30–4.21 (m, 4H, 2
×
CH2OP), 3.25–3.21 (m, 1H, HC3), 3.04 (s, 3H, CH3N),
3
2
3
3
3.12 (dddd, J(H4β–P) = 16.7 Hz, J(H4β–H4α) = 12.9 Hz, J(H4β–H5) = 8.6 Hz, J(H4β–H3) = 8.3 Hz, 1H,
HβC4), 2.94 (dddd, 3J(H4α–P) = 12.9 Hz, 2J(H4α–H4β) = 12.9 Hz, 3J(H4α–H3) = 10.0 Hz, 3J(H4α–H5) = 6.2 Hz,
3
3
1H, HαC4), 1.40 (t, J = 7.1 Hz, 3H, CH3CH2OP), 1.38 (t, J = 7.0 Hz, 3H, CH3CH2OP). 13C-NMR
(151 MHz, CDCl3):
δ = 160.30 (C=O), 156.50, 147.35, 137.66, 129.26, 128.96, 123.27, 120.46, 74.68
3
1
2
(d, J(CCCP) = 8.6 Hz, C5), 64.60 (d, J(CP) = 168.1 Hz, C3), 63.51 (d, J(COP) = 6.6 Hz, CH2OP), 62.61
2
3
(d, J(COP) = 6.9 Hz, CH2OP), 46.07 (CH3N), 40.92 (C4), 16.55 (d, J(CCOP) = 6.2 Hz, CH3CH2OP),
3
16.48 (d, J(CCOP) = 5.5 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
δ = 20.51. Anal. Calcd. for
C16H21BrN3O5P: C, 43.07; H, 4.74; N, 9.42. Found: C, 43.09; H, 4.52; N, 9.35.
Diethyl cis-[5-(6-bromo-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl]phosphonate (cis-11a).
Colorless oil. IR (film, cm−1
)
ν
max: 3090, 2959, 2925, 2865, 1661, 1626, 1601, 1461, 1336, 1234,
1054, 969, 834, 575. 1H-NMR (600 MHz, CDCl3):
δ
= 10.63 (s, 1H, NH), 8.44 (d, J = 2.3 Hz, 1H,
4
3
4
3
HC50), 7.84 (dd, J = 8.6 Hz, J = 2.3 Hz, 1H, HC70), 7.54 (d, J = 8.6 Hz, 1H, HC80), 5.07 (dd,
3
3J(H5–H4α) = 9.2 Hz, J(H5–H4β) = 4.3 Hz, 1H, HC5), 4.23–4.12 (m, 4H, 2
×
CH2OP), 3.21 (dddd,
3J(H4β–P) = 19.8 Hz, 2J(H4β–H4α) = 10.9 Hz, 3J(H4β–H3) = 6.8 Hz, 3J(H4β–H5) = 4.3 Hz, 1H, HβC4), 3.12
3
3
2
(ddd, J(H3–H4β) = 6.8 Hz, J(H3–H4α) = 9.6 Hz, J(H3–P) = 4.4 Hz 1H, HC3), 3.00 (s, 3H, CH3N), 2.83
(dddd, 3J(H4α–P) = 13.1 Hz, 2J(H4α–H4β) = 10.9 Hz, 3J(H4α–H3) = 9.6 Hz, 3J(H4α–H5) = 9.2 Hz, 1H, HαC4),
1.32 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.27 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
3
δ
= 160.33 (C=O), 156.50, 147.35, 137.65, 129.26, 128.96, 123.27, 120.46, 75.58 (d, J(CCCP) = 6.6 Hz,
1
2
2
C5), 63.58 (d, J(CP) = 169.1 Hz, C3), 63.18 (d, J(COP) = 6.5 Hz, CH2OP), 63.15 (d, J(COP) = 6.4 Hz,
CH2OP), 45.58 (d, J(CNCP) = 5.8 Hz, CH3N), 37.71 (C4), 16.38 (d, J(CCOP) = 5.3 Hz, CH3CH2OP),
16.37 (d, J(CCOP) = 5.4 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
3
3
3
δ = 20.92. Anal. Calcd. for
C16H21BrN3O5P: C, 43.07; H, 4.74; N, 9.42. Found: C, 43.12; H, 4.35; N, 9.33.
Diethyl
trans-[5-(3-benzyl-6-bromo-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylisoxazolidin-3-yl]phosphonate
(trans-11b). A yellowish oil. IR (film, cm−1
)
ν
max: 3040, 2980, 2926, 2853, 1613, 1568, 1490, 1418, 1353,
1
1239, 1056, 1025, 835, 822, 699. H-NMR (600 MHz, CDCl3):
δ
= 8.33 (d, 4J = 2.3 Hz, 1H, HC50), 7.90 (dd,
3J = 8.9 Hz, 4J = 2.3 Hz, 1H, HC70), 7.83 (d, 3J = 8.9 Hz, 1H, HC80), 7.55–7.53 (m, 2H), 7.45–7.42 (m, 2H),
7.40–7.37 (m, 1H), 5.66 (s, 2H, N-CH2), 5.25 (dd, 3J(H5–H4β) = 6.8 Hz, 3J(H5–H4α) = 6.4 Hz, 1H, HC5), 4.33–4.19
(m, 4H, 2
×
CH2OP), 3.42–3.39 (m, 1H, HC3), 3.07–2.94 (m, 2H, HαC4, HβC4), 3.05 (s, 3H, CH3N), 1.42
3
3
(t, J = 7.0 Hz, 3H, CH3CH2OP), 1.39 (t, J = 6.9 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
δ
= 166.08 (C=O), 163.35, 149.91, 137.20, 135.69, 129.67, 128.67, 128.65, 128.52, 128.45, 125.97, 120.69, 80.15
(d, 3J(CCCP) = 8.5 Hz, C5), 69.14 (N-CH2), 64.41 (d, 1J(CP) = 168.8 Hz, C3), 63.26 (d, 2J(COP) = 6.5 Hz, CH2OP),
62.42 (d, J(COP) = 7.2 Hz, CH2OP), 46.62 (CH3N), 37.90 (C4), 16.58 (d, 3J(CCOP) = 5.6 Hz, CH3CH2OP),
2
16.52 (d, J(CCOP) = 5.6 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
δ = 22.04. Anal. Calcd. for
3
C23H27BrN3O5P: C, 51.50; H, 5.07; N, 7.83. Found: C, 51.78; H, 5.11; N, 7.70.
Diethyl trans-{5-[6-bromo-3-(2-nitrobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11c). A yellowish oil. IR (film, cm−1
)
ν
max: 3444, 3077, 2979, 2925, 2853, 1690, 1614, 1575, 1527,
1
1489, 1413, 1355, 1337, 1055, 1024, 835, 791, 730, 574. H-NMR (600 MHz, CDCl3):
δ
= 8.36 (d, 4J = 2.0 Hz,
3
4
4
1H, HC50), 8.20–8.18 (m, 1H), 7.95 (dd, J = 8.9 Hz, J = 2.3 Hz, 1H, HC70), 7.87 (d, J = 8.9 Hz,

Molecules 2018, 23, 1889
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1H, HC80), 7.78–7.76 (m, 1H), 7.72–7.69 (m, 1H), 7.57–7.55 (m, 1H), 6.10 (AB, JAB = 14.6 Hz, 1H,
3
3
N-CH2b), 6.07 (AB, JAB = 14.6 Hz, 1H, N-CH2a), 5.21 (dd, J(H5–H4β) = 8.0 Hz, J(H5–H4α) = 6.1 Hz,
1H, HC5), 4.31–4.22 (m, 4H, 2 CH2OP), 3.36–3.33 (m, 1H, HC3), 3.00 (s, 3H, CH3N), 2.99
(dddd, 2J(H4β–P) = 16.6 Hz, 3J(H4β–H4α) = 12.6 Hz, 3J(H4β–H3) = 8.2 Hz, 3J(H4β–H5) = 8.0 Hz, 1H, HβC4),
2.38 (dddd, 2J(H4α–H4β) = 12.6 Hz, 3J(H4α–P) = 10.2 Hz, 3J(H4α–H3) = 8.8 Hz, 3J
= 6.1 Hz, 1H,
×
(H4α–H5)
HαC4), 1.41 (t, 3J = 7.1 Hz, 3H, CH3CH2OP), 1.39 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz,
CDCl3): = 165.56 (C=O), 163.42, 150.09, 147.81, 137.49, 133.80, 132.03, 129.85, 129.14, 129.00, 125.66,
125.15, 121.03, 116.40, 80.08 (d, 3J(CCCP) = 7.8 Hz, C5), 65.64 (N-CH2), 64.30 (d, 1J(CP) = 168.5 Hz, C3),
δ
2
2
3
63.21 (d, J(COP) = 6.3 Hz, CH2OP), 62.49 (d, J(COP) = 6.8 Hz, CH2OP), 46.61 (d, J(CNCP) = 4.2 Hz,
CH3N), 37.91 (C4), 16.54 (d, 3J(CCOP) = 5.6 Hz, CH3CH2OP), 16.49 (d, 3J(CCOP) = 5.5 Hz, CH3CH2OP).
31P-NMR (243 MHz, CDCl3):
Found: C, 47.22; H, 4.40; N, 9.35.
δ = 21.85. Anal. Calcd. for C23H26BrN4O7P: C, 47.52; H, 4.51; N, 9.64.
Diethyl trans-{5-[6-bromo-3-(3-nitrobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11d). A yellowish oil. IR (film, cm−1
)
ν
max: 3078, 2980, 2926, 2854, 1613, 1566, 1531, 1489,
1
1416, 1348, 1242, 1115, 1053, 1024, 966, 836, 805, 733, 671. H-NMR (600 MHz, CDCl3):
δ = 8.44–8.42
4
3
4
(m, 1H), 8.32 (d, J = 2.0 Hz, 1H, HC50), 8.24–8.22 (m, 1H), 7.92 (dd, J = 8.9 Hz, J = 2.0 Hz, 1H,
HC70), 7.90–7.88 (m, 1H), 7.84 (d, 3J = 8.9 Hz, 1H, HC80), 7.63–7.60 (m, 1H), 5.77 (s, 2H, N-CH2), 5.29
(dd, 3J(H5–H4β) = 8.0 Hz, 3J(H5–H4α) = 6.4 Hz, 1H, HC5), 4.34–4.23 (m, 4H, 2
×
CH2OP), 3.44–3.37 (m,
1H, HC3), 3.10–2.94 (m, 2H, HαC4, HβC4), 3.07 (s, 3H, CH3N), 1.43 (t, 3J = 7.1 Hz, 3H, CH3CH2OP),
3
1.41 (t, J = 7.0 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
δ = 165.59 (C=O), 163.05,
150.03, 148.49, 137.81, 137.49, 134.31, 129.78, 129.76, 125.74, 123.45, 123.29, 121.03, 116.42, 80.00 (d,
1
2
3J(CCCP) = 8.4 Hz, C5), 65.61 (N-CH2), 64.38 (d, J(CP) = 168.5 Hz, C3), 63.26 (d, J(COP) = 6.5 Hz,
2
3
CH2OP), 62.50 (d, J(COP) = 6.8 Hz, CH2OP), 46.57 (CH3N), 37.77 (C4), 16.56 (d, J(CCOP) = 5.6 Hz,
CH3CH2OP), 16.50 (d, 3J(CCOP) = 5.9 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
= 21.52. Anal.
Calcd. for C23H26BrN4O7P: C, 47.52; H, 4.51; N, 9.64. Found: C, 47.71; H, 4.51; N, 9.44.
δ
Diethyl trans-{5-[6-bromo-3-(4-nitrobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11e). A yellowish oil. IR (film, cm−1
)
ν
max: 3069, 2969, 2925, 2854, 1610, 1571, 1523, 1490,
1343, 1285, 1241, 1114, 1027, 968, 837. 1H-NMR (600 MHz, CDCl3):
δ
= 8.35 (d, J = 2.0 Hz, 1H,
4
HC50), 1H), 8.30–8.28 (m, 2H), 7.95 (dd, 3J = 8.9 Hz, 4J = 2.0 Hz, 1H, HC70), 7.86 (d, 3J = 8.9 Hz, 1H,
HC80), 7.72–7.71 (m, 2H), 5.76 (s, 1H, N-CH2), 5.24 (dd, 3J(H5–H4β) = 6.4 Hz, 3J(H5–H4α) = 6.0 Hz, 1H,
HC5), 4.32–4.22 (m, 4H, 2
×
CH2OP), 3.37–3.33 (m, 1H, HC3), 3.07–2.98 (m, 1H, HβC4), 3.03 (s, 3H,
CH3-N), 2.94 (dddd, 3J(H4α–P) = 12.4 Hz, 2J(H4α–H4β) = 12.4 Hz, 3J(H4α–H3) = 9.2 Hz, 3J(H4α–H5) = 6.0 Hz,
3
3
1H, HαC4), 1.41 (t, J = 7.0 Hz, 3H, CH3CH2OP), 1.39 (t, J = 7.0 Hz, 3H, CH3CH2OP). 13C-NMR
(151 MHz, CDCl3):
δ = 165.57 (C=O), 163.06, 150.04, 147.95, 142.93, 137.55, 129.83, 128.67, 125.69, 123.92,
121.09, 116.42, 79.97 (d, 3J(CCCP) = 8.6 Hz, C5), 67.55 (s, N-CH2), 64.40 (d, 1J(CP) = 168.3 Hz, C3), 63.28
(d, 2J(COP) = 6.5 Hz, CH2OP), 62.46 (d, 2J(COP) = 7.1 Hz, CH2OP), 46.55 (CH3N), 37.77 (C4), 16.56 (d,
3J(CCOP) = 5.7 Hz, CH3CH2OP), 16.50 (d, 3J(CCOP) = 5.8 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
δ
= 21.85. Anal. Calcd. for C23H26BrN4O7P: C, 47.52; H, 4.51; N, 9.64. Found: C, 47.75; H, 4.54; N, 9.39.
Diethyl trans-{5-[6-bromo-3-(2-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11f). Data presented below were extracted from spectra of a 88:12 mixture of trans-11f and
cis-11f. Yellowish oil. IR (film, cm−1
)
ν
max: 3069, 2981, 2929, 2909, 1614, 1567, 1490, 1456, 1353,
1
1285, 1116, 1025, 964, 869, 760, 691. H-NMR (600 MHz, CDCl3):
δ
= 8.31 (d, 4J = 2.1 Hz, 1H, HC50),
3
4
3
7.90 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H, HC70), 7.84 (d, J = 8.9 Hz, 1H, HC80), 7.58–7.55 (m, 1H),
7.40–7.36 (m, 1H), 7.21–7.18 (m, 1H), 7.16–7.13 (m, 1H), 5.73 (AB, JAB = 12.4 Hz, 1H, N-CH2b), 5.71
(AB, JAB = 12.4 Hz, 1H, N-CH2a), 5.25 (dd, 3J(H5–H4β) = 7.9 Hz, 3J(H5–H4α) = 6.2 Hz, 1H, HC5), 4.33–4.18
(m, 4H, 2
×
CH2OP), 3.42–3.39 (m, 1H, C3), 3.05 (s, 3H, CH3-N), 3.03–2.96 (m, 1H, HβC4), 2.96
(dddd, 3J(H4α–P) = 12.5 Hz, 2J(H4α–H4β) = 12.5 Hz, 3J(H4α–H3) = 8.9 Hz, 3J(H4α–H5) = 6.2 Hz, 1H, HαC4),
1.42 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.39 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
δ
= 165.94 (C=O), 163.31, 161.19 (d, 1J(CF) = 248.8 Hz, C2”), 149.96, 137.25, 130.81 (d, 3J(CCCF) = 3.5 Hz,

Molecules 2018, 23, 1889
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C4”), 130.52 (d, 3J(CCCF) = 8.0 Hz, C6”), 129.68, 125.94, 124.26 (d, 4J(CCCCF) = 3.4 Hz, C5”), 122.93 (d,
2J(CCF) = 14.5 Hz, C3”), 120.75, 116.65, 115.66 (d, 2J(CCF) = 21.0 Hz, C1”), 80.14 (dd, 3J(CCCP) = 8.1 Hz,
C5), 64.42 (d, 1J(CP) = 168.3 Hz, C3), 63.25 (d, 2J(COP) = 6.5 Hz, CH2OP), 63.03 (d, 3J = 4.3 Hz, N-CH2),
62.43 (d, 2J(COP) = 7.2 Hz, CH2OP), 46.59 (CH3N), 37.89 (C4), 16.55 (d, 3J(CCOP) = 5.7 Hz, CH3CH2OP),
3
16.50 (d, J(CCOP) = 5.8 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
δ = 22.01. Anal. Calcd.
for C23H26BrFN3O5P: C, 49.83; H, 4.73; N, 7.58. Found: C, 49.49; H, 4.53; N, 7.71 (obtained on a
88:12 mixture of trans-11f and cis-11f).
Diethyl trans-{5-[6-bromo-3-(3-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11g). Yellowish oil. IR (film, cm−1
1345, 1285, 1255, 1114, 1055, 1025, 966, 836, 789, 749. H-NMR (200 MHz, CDCl3):
)
ν
max: 3071, 2979, 2926, 2853, 1613, 1572, 1490, 1452, 1415,
1
δ = 8.35–8.33 (m, 1H),
7.91–7.89 (m, 1H), 7.84–7.82 (m, 1H), 7.41–7.37 (m, 1H), 7.30–7.28 (m, 1H), 7.26–7.24 (m, 1H), 7.08–7.05
(m, 1H), 5.64 (s, 1H, N-CH2), 5.24 (dd, 3J(H5–H4β) = 7.9 Hz, 3J(H5–H4α) = 6.5 Hz, 1H, HC5), 4.33–4.17 (m,
4H, 2
×
CH2OP), 3.39–3.36 (m, 1H, C3), 3.04 (s, 3H, CH3-N), 3.05–2.99 (m, 1H, HβC4), 2.95 (dddd,
3J(H4α–P) = 12.7 Hz, 2J(H4α–H4β) = 12.7 Hz, 3J(H4α–H3) = 9.7 Hz, 3J(H4α–H5) = 6.5 Hz, 1H, HαC4), 1.41
(t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.39 (t, 3J = 6.9 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
δ
= 165.84 (C=O), 163.22, 162.91, (d, 1J(CF) = 246.7 Hz, C30), 149.97, 138.18 (d,03J(CCCF) = 7.6 Hz, C50),
137.31, 130.26 (d, J = 7.9 Hz, C1 ), 129.72, 125.84, 123.80 (d, 4J(CCCCF) = 2.4 Hz, C6 ), 120.83, 116.59, 115.45
0
(d, J(CCF) = 21.0 Hz, C40), 115.21 (d, J(CCF) = 22.0 Hz, C20), 80.06 (dd, J(CCCP) = 8.0 Hz, C5), 68.19
(d, 3J = 1.7 Hz, N-CH2), 64.42 (d, 1J(CP) = 168.3 Hz, C3), 63.24 (d, 2J(COP) = 6.4 Hz, CH2OP), 62.44 (d,
2J(COP) = 7.3 Hz, CH2OP), 46.57 (d, 3J(CNCP) = 3.7 Hz, CH3N), 37.84 (s, C4), 16.55 (d, 3J(CCOP) = 5.6 Hz,
2
2
3
CH3CH2OP), 16.49 (d, 3J(CCOP) = 5.9 Hz, CH3CH2OP). 31P-NMR (121.5 MHz, CDCl3):
δ = 21.97. Anal.
Cald. for C23H26BrFN3O5P: C, 49.83; H, 4.73; N, 7.58. Found: C, 49.49; H, 4.53; N, 7.71.
Diethyl trans{5-[6-bromo-3-(4-fluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11h). A yellowish oil. IR (film, cm−1
max: 3072, 2981, 2926, 2853, 1611, 1568, 1512, 1490,
)
ν
1430, 1351, 1285, 1227, 1160, 1114, 1099,1056, 1026, 965, 835.1H-NMR (600 MHz, CDCl3):
δ = 8.31
4
3
4
3
(d, J = 2.2 Hz, 1H, HC50), 7.91 (dd, J = 8.9 Hz, J = 2.2 Hz, 1H, HC70), 7.84 (d, J = 8.9 Hz, 1H,
3
HC80), 7.55–7.53 (m, 2H), 7.14–7.11 (m, 2H), 5.63 (s, 1H, N-CH2), 5.26 (dd, J(H5–H4 = 6.4 Hz,
β
)
3J(H5–H4 = 6.4 Hz, 1H, HC5), 4.34–4.22 (m, 4H, 2
×
CH2OP), 3.41–3.39 (m, 1H, C3), 3.07–3.00 (m, 1H,
α
)
HβC4), 3.06 (s, 3H, CH3-N), 2.98 (dddd, 3J(H4 –P) = 13.4 Hz, 2J(H4
) = 13.4 Hz, 3J(H4 –H3) = 10.4 Hz,
α
3
α
α
–H4β
3
3J(H4
= 6.4 Hz, 1H, HαC4), 1.42 (t, J = 7.0 Hz, 3H, CH3CH2OP), 1.40 (t, J = 7.1 Hz, 3H,
α
–H5)
CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
δ = 165.94 (C=O), 163.26, 162.84 (d, J(CF) = 247.6 Hz,
1
C4”), 149.94, 137.25, 131.52 (d, 4J(CCCCF) = 3.1 Hz, C1”), 130.49 (d, 3J(CCCF) = 8.0 Hz, C2”, C6”), 129.69,
2
3
125.89, 120.75, 116.65, 115.62 (d, J(CCF) = 21.9 Hz, C3”, C5”), 80.09 (d, J(CCP) = 8.1 Hz, C5), 68.40
(N-CH2), 64.43 (d, 1J(CP) = 168.3 Hz, C3), 63.26 (d, 2J(COP) = 6.5 Hz, CH2OP), 62.45 (d, 2J(COP) = 6.8 Hz,
CH2OP), 46.59 (CH3N), 37.87 (C4), 16.56 (d, 3J(CCOP) = 5.8 Hz, CH3CH2OP), 16.46 (d, 3J(CCOP) = 5.3 Hz,
CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
4.73; N, 7.58. Found: C, 50.20; H, 4.62; N, 7.32.
δ = 22.00. Anal. Calcd. for C23H26BrFN3O5P: C, 49.83; H,
Diethyl trans-{5-[6-bromo-3-(2,4-difluorobenzyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}
phosphonate (trans-11i). Data presented below were extracted from spectra of a 92:8 mixture of trans-11i
and cis-11i. Yellowish oil. IR (film, cm−1
)
ν
max: 3079, 2959, 2924, 2853, 1738, 1689, 1613, 1565, 1509,
1
1490, 1416, 1351, 1280, 1141, 1100, 961, 836, 798. H-NMR (600 MHz, CDCl3):
δ
= 8.30 (d, 4J = 2.1 Hz,
3
4
3
1H, HC50), 7.92 (dd, J = 8.8 Hz, J = 2.1 Hz, 1H, HC70), 7.85 (d, J = 8.8 Hz, 1H, HC80), 7.59–7.55
(m, 1H), 6.95–6.89 (m, 2H), 5.68 (AB, JAB = 13.3 Hz, 1H, N-CH2b), 5.66 (AB, JAB = 13.3 Hz, 1H,
N-CH2a), 5.25 (dd, 3J(H5–H4β) = 7.7 Hz, 3J(H5–H4α) = 6.5 Hz, 1H, HC5), 4.33–4.18 (m, 4H, 2
×
CH2OP),
3.41–3.38 (m, 1H, HC3), 3.07–3.01 (m, 1H, HβC4), 3.05 (s, 3H, CH3-N), 2.98 (dddd, 3J(H4α–P) = 12.8 Hz,
3
3
3
2J(H4α–H4β) = 12.8 Hz, J(H4α–H3) = 9.7 Hz, J(H4α–H5) = 6.5 Hz, 1H, HαC4), 1.41 (t, J = 7.0 Hz,
3H, CH3CH2OP), 1.40 (t, J = 7.0 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
(C=O), 163.32 (dd, J(CF) = 250.9 Hz, J(CCCF) = 12.1 Hz, C2”), 163.20, 161.50 (dd, J(CF) = 251.4 Hz,
δ = 165.82
3
1
3
1
3J(CCCF) = 12.2 Hz, C4”), 149.87, 137.31, 132.09 (dd, 3J(CCCF) = 9.8 Hz, 3J(CCCF) = 4.9 Hz, C6”), 129.71,

Molecules 2018, 23, 1889
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125.87, 120.81, 118.98 (dd, 2J(CCF) = 14.4 Hz, 4J(CCCCF) = 3.4 Hz, C1”), 116.58, 111.51 (dd, 2J(CCF) = 21.1 Hz,
4J(CCCCF) = 3.5 Hz, C5”), 104.21 (dd, 2J(CCF) = 25.3 Hz, 2J(CCF) = 25.4 Hz, C3”), 80.09 (dd, 3J(CCCP) = 7.9 Hz,
1
2
3
C5), 64.43 (d, J(CP) = 168.3 Hz, C3), 63.26 (d, J(COP) = 6.5 Hz, CH2OP), 62.47 (d, J(CCCF) = 2.7 Hz,
N-CH2), 62.43 (d, 2J(COP) = 6.3 Hz, CH2OP), 46.57 (d, 3J(CNCP) = 4.0 Hz, CH3N), 37.89 (C4), 16.56 (d,
3J(CCOP) = 5.7 Hz, CH3CH2OP), 16.50 (d, 3J(CCOP) = 5.6 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
δ
= 21.98. Anal. Calcd. for C23H25BrF2N3O5P
×
0.75 H2O: C, 47.15; H, 4.56; N, 7.17. Found: C, 46.84;
H, 4.18; N, 6.96 (obtained on a 92:8 mixture of trans-11i and cis-11i).
Diethyl trans-{5-[6-bromo-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11j). A yellowish oil. IR (film, cm−1
)
ν
max: 3521, 3477, 2976, 2912, 2855, 1687, 1606, 1470,
1308, 1265, 1050, 1023, 972, 849, 574. 1H-NMR (600 MHz, CDCl3):
δ
= 8.42 (d, J = 2.3 Hz, 1H,
4
3
4
3
HC50), 7.82 (dd, J = 8.7 Hz, J = 2.3 Hz, 1H, HC70), 7.56 (d, J = 8.7 Hz, 1H, HC80), 5.18 (dd,
3J(H5–H4β) = 7.6 Hz, 3J(H5–H4α) = 5.7 Hz, 1H, HC5), 4.29–4.23 (m, 4H, 2
×
CH2OP), 3.75 (s, 3H, CH3), 3.71
(dddd, 3J(H4α–P) = 12.6 Hz, 2J(H4α–H4β) = 11.3 Hz, 3J(H4α–H3) = 9.1 Hz, 3J(H4α–H5) = 5.7 Hz, 1H, HαC4),
3.35 (ddd, 3J(H3–H4α) = 9.1 Hz, 3J(H3–H4β) = 7.6 Hz, 2J(H3-P) = 2.8 Hz, 1H, HC3), 2.87 (s, 3H, CH3N), 2.80
(dddd, 3J(H4β–P) = 15.3 Hz, 2J(H4β–H4α) = 11.3 Hz, 3J(H4β–H3) = 7.6 Hz, 3J(H4β–H5) = 7.6 Hz, 1H, HβC4),
1.41 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.40 (t, 3J = 7.1 Hz, 3H, CH3CH2OP). 13C-NMR (151 MHz, CDCl3):
δ
= 161.31 (C=O), 152.70, 145.34, 137.33, 129.44, 129.37, 122.31, 121.02, 76.29 (d, 3J(CCCP) = 7.8 Hz, C5),
64.35 (d, 1J(CP) = 170.1 Hz, C3), 62.89 (d, 2J(COP) = 6.6 Hz, CH2OP), 62.73 (d, 2J(COP) = 7.2 Hz, CH2OP),
47.15 (d, 3J(CNCP) = 6.4 Hz CH3N), 34.38 (C4), 30.97 (CH3), 16.56 (d, 3J(CCOP) = 4.9 Hz, CH3CH2OP),
16.53 (d, J(CCOP) = 5.1 Hz, CH3CH2OP). 31P-NMR (243 MHz, CDCl3):
δ = 21.93. Anal. Calcd. for
3
C17H23BrN3O5P: C, 44.36; H, 5.04; N, 9.13. Found: C, 44.15; H, 4.75; N, 9.03.
Diethyl trans-{5-[6-bromo-3-ethyl-4-oxo-3,4-dihydroquinazolin-2-yl]-2-methylisoxazolidin-3-yl}phosphonate
(trans-11k). Colorless oil. IR (film, cm−1
)
ν
max: 3055, 2981, 2929, 2854, 1687, 1613, 1569, 1493, 1430,
1383, 1285, 1241, 1117, 1056, 1024, 967, 836. 1H-NMR (600 MHz, CDCl3):
δ
= 8.32 (d, J = 2.2 Hz,
4
3
4
3
1H, HC50), 7.90 (dd, J = 8.8 Hz, J = 2.2.Hz, 1H, HC70), 7.82 (d, J = 8.8 Hz, 1H, HC80), 5.23 (dd,
3J(H5–H4β) = 8.0 Hz, 3J(H5–H4α) = 6.0 Hz, 1H, HC5), 4.68 (q, 3J = 7.2 Hz, 2H, CH3CH2), 4.33–4.21 (m,
4H, 2
×
CH2OP), 3.45–3.43 (m, 1H, HC3), 3.07 (s, 3H, CH3N), 3.06–3.01 (m, 1H, HβC4), 2.97 (dddd,
3J(H4α–P) = 12.6 Hz, 2J(H4α–H4β) = 12.6 Hz, 3J(H4α–H3) = 9.4 Hz, 3J(H4α–H5) = 6.0 Hz, 1H, HαC4), 1.55 (t,
3J = 7.2 Hz, 3H, CH3CH2), 1.42 (t, 3J = 7.0 Hz, 3H, CH3CH2OP), 1.39 (t, 3J = 6.8 Hz, 3H, CH3CH2OP).
13C-NMR (151 MHz, CDCl3):
δ =166.33 (C=O), 163.63, 149.78, 137.01, 129.63, 126.03, 120.47, 116.78,
80.22 (d, 3J(CCCP) = 7.9 Hz, C5), 64.38 (d, 1J(CP) = 168.0 Hz, C3), 63.28 (d, 2J(COP) = 6.4 Hz, CH2OP), 62.41
(d, 2J(COP) = 6.7 Hz, CH2OP), 47.21 (CH3N), 38.01 (C4), 29.69 (CH3CH2) 16.56 (d, 3J(CCOP) = 5.5 Hz,
CH3CH2OP), 16.50 (d, 3J(CCOP) = 6.2 Hz, CH3CH2OP), 14.29 (CH3CH2). 31P-NMR (243 MHz, CDCl3):
δ
= 22.08. Anal. Calcd. for C18H25BrN3O5P: C, 45.58; H, 5.31; N, 8.86. Found: C, 45.37; H, 5.31; N, 8.54.
3.6. 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
−
(ACVr), 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 vaccinia virus, adeno
virus-2, vesicular stomatitis virus, 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). The antiviral
assays were based on the 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.

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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. Alternatively, the cytostatic activity of the test compounds was measured
based on inhibition of cell growth. HEL cells were seeded at a rate of 5
microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations
of the test compounds was added. After three days of incubation at 37 C, the cell number was
×
103 cells/well into 96-well
◦
determined with a Coulter counter. The cytostatic concentration was calculated as the CC50, or the
compound concentration required to reduce cell proliferation by 50% relative to the number of cells in
the untreated controls.
3.7. 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
300,000–500,000 cells/mL of culture medium, and 100
µL of a cell suspension was added to 100
µL of an
appropriate dilution of the test compounds in 200
µ
L-wells of 96-well microtiter plates. After 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-conjugates of quinazolinones trans-11a
–
k
and cis-11a–k
were obtained from N-methyl C-(diethoxyphosphoryl)nitrone and selected N3-substituted
6-bromo-2-vinylquinazolinones. The trans-isoxazolidine cycloadducts (trans-11a, trans-11b, trans-11c
trans-11d, trans-11e, trans-11g, trans-11h, trans-11j, trans-11k) and isoxazolidine cis-11a, or the
respective mixtures of isoxazolidines (trans-11f/cis-11f (95:5) and trans-11i/cis-11i (92:8)) were
evaluated for their antiviral activity toward variety of DNA and RNA viruses. Almost all compounds
were active against VZV and among them trans-11b, trans-11d, trans-11g, and trans-11h were the most
,
potent (EC50 = 5.8–11.6
lines (CC50 = 33–42 M). Furthermore, (isoxazolidine)phosphonates trans-11d, trans-11g, trans-11h
and trans-11i/cis-11i (92:8) showed the highest activity against HCMV (EC50 = 8.9–12.5 M). On the
µM) and, at the same time, exhibited lower cytotoxicity toward uninfected cell
µ
,
µ
other hand, several compounds exhibited moderate cytostatic effect toward the CEM cell line
(IC50 = 9.6–17 µM), however, slightly higher than that of 5-fluorouracil used as the reference drug.
It was proved that installation of functionalized benzyl groups at N3 in the quinazolinone moiety
is essential for inhibitory properties toward both VZV and HCMV. At the same time, it was noticed that
incorporation of the bromine atom at C6 in a quinazolinone skeleton resulted in a significant increase
in potency of isoxazolidine-conjugates 11b
(up to three-fold higher) when compared with previously described analogous conjugates 10b
lacking a bromine substituent at C6 of the quinazolinone moiety. Moreover, inhibitory properties of
the newly synthesized compounds 11b 11k toward tested cell lines were also slightly higher than
–11i toward both VZV (up to five-fold higher) and HCMV
–
10i
–
those of previously described analogues 10b–10k.
Author Contributions: The research group from the 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. The 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 on,
and approved the manuscript.
Funding: This research was funded by the Medical University of Lodz (internal fund 503/3-014-01/503-31-001)
and by the KU Leuven (GOA 15/19 TBA).

Molecules 2018, 23, 1889
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Acknowledgments: The authors wish to express their gratitude to Leentje Persoons, Ellen De Waegenaere,
Bianca Stals, Kirsten Lepaige, Niels Willems, and Nathalie Van Winkel for their excellent technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
König, B.; Grätzei, M.A. Novel Immunosensor for Herpes Viruses. Anal. Chem. 1994, 66, 341–344. [CrossRef]
[PubMed]
Hollier, L.M.; Grissom, H. Human Herpes Viruses in Pregnancy: Cytomegalovirus, Epstein-Barr Virus,
and Varicella Zoster Virus. Clin. Perinatol. 2005, 32, 671–696. [CrossRef] [PubMed]
Field, H.J.; Vere Hodge, R.A. Recent developments in anti-herpesvirus drugs. Br. Med. Bull. 2013, 106,
213–249. [CrossRef] [PubMed]
4.
5.
Cohen, J.I. Herpes Zoster. N. Engl. J. Med. 2013, 369, 255–263. [CrossRef] [PubMed]
De Clercq, E. Antivirals for the treatment of herpes virus infections. J. Antimicrob. Chemother. 1993, 32
(Suppl. A), 121–132. [CrossRef] [PubMed]
6.
7.
Ma, J.; Li, P.; Li, X.; Shi, Q.; Wan, Z.; Hu, D.; Jin, L.; Song, B. Synthesis and Antiviral Bioactivity of Novel
3-((2-((1E,4E)-3-Oxo-5-arylpenta-1,4-dien-1-yl)phenoxy)methyl)-4(3H)-quinazolinone Derivatives. J. Agric.
Food Chem. 2014, 62, 8928–8934. [CrossRef] [PubMed]
Dohle, W.; Jourdan, F.L.; Menchon, G.; Prota, A.E.; Foster, P.A.; Mannion, P.; Hamel, E.; Thomas, M.P.;
Kasprzyk, P.G.; Ferrandis, E.; et al. Quinazolinone-Based Anticancer Agents: Synthesis, Antiproliferative
SAR, Antitubulin Activity, and Tubulin Co-crystal Structure. J. Med. Chem. 2018, 61, 1031–1044. [CrossRef]
[PubMed]
8.
9.
Al-Suwaidan, I.A.; Abdel-Aziz, A.A.-M.; Shawer, T.Z.; Ayyad, R.R.; Alanazi, A.M.; El-Morsy, A.M.;
Mohamed, M.A.; Abdel-Aziz, N.I.; El-Sayed, M.A.-A.; El-Azab, A.S. Synthesis, antitumor activity and
molecular docking study of some novel 3-benzyl-4(3H)-quinazolinone analogues. J. Enzyme Inhib. Med. Chem.
2016, 31, 78–89. [CrossRef] [PubMed]
Zhang, J.; Liu, J.; Ma, Y.; Re, D.; Cheng, P.; Zhao, J.; Zhang, F.; Yao, Y. One-pot synthesis and antifungal
activity against plant pathogens of quinazolinone derivatives containing an amide moiety. Bioorg. Med.
Chem. Lett. 2016, 26, 2273–2277. [CrossRef] [PubMed]
10. Raju, G.N.; Sai, K.B.; Resshma, V.; Sudarshini, N.; Sowmya, P.L.; Nalini, Y.; Nadendla, R.R.
Potential antimicrobial activities of quinazolinone derivatives. J. Chem. Pharm. Res. 2015, 7, 1279–1287.
11. Rakesh, K.P.; Ramesh, S.; Kumar, H.M.M.; Chandan, S.; Gowda, D.C. Quinazolinones linked amino acids
derivatives as a new class of promising antimicrobial, antioxidant and anti-inflammatory agents. Eur. J. Chem.
2015, 6, 254–260. [CrossRef]
12. Kuneš, J.; Bažant, J.; Pour, M.; Waisser, K.; Šlosárek, M.; Janota, J. Quinazoline derivatives with antitubercular
activity. Il Farmaco 2000, 55, 725–729. [CrossRef]
13. Pandit, U.; Dodiya, A. Synthesis and antitubercular activity of novel pyrazole–quinazolinone hybrid analogs.
Med. Chem. Res. 2013, 22, 3364–3371. [CrossRef]
14. Kumar, A.; Bansal, D.; Bajaj, K.; Sharma, S.; Srivastava, V.K. Synthesis of some newer derivatives of 2-amino
benzoic acid as potent anti-inflammatory and analgesic agents. Bioorg. Med. Chem. 2003, 11, 5281–5291.
[CrossRef]
15. Noureldin, N.A.; Kothayer, H.; Lashine, E.-S.M.; Baraka, M.M.; El-Eraky, W.; El Awdan, S.A.
Synthesis, Anticonvulsant Activity, and SAR Study of Novel 4-Quinazolinone Derivatives. Arch. Pharm.
Chem. Life Sci. 2017, 350, e1600332. [CrossRef] [PubMed]
16. Mokale, S.N.; Palkar, A.D.; Dube, P.N.; Sakle, N.S.; Miniyar, P.B. Design, synthesis and in vivo screening of
some novel quinazoline analogs as anti-hyperlipidemic and hypoglycemic agents. Bioorg. Med. Chem. Lett.
2015, 6, 272–276. [CrossRef] [PubMed]
17. Alafeefy, A.M.; Kadi, A.A.; Al-Deeb, O.A.; El-Tahir, K.E.H.; Al-Jaber, N.A. Synthesis, analgesic and
anti-inflammatory evaluation of some novel quinazoline derivatives. Eur. J. Med. Chem. 2010, 45, 4947–4952.
[CrossRef] [PubMed]
18. Nawrocka, W.; Zimecki, M. Syntheses of Novel 3-Amino-2(1H)-thioxo-4(3H)-quinazolinones and Evaluation
of Their Immunotropic Activity. Part III. Arch. Pharm. Pharm. Med. Chem. 1997, 330, 399–405. [CrossRef]

Molecules 2018, 23, 1889
16 of 16
19. Selvam, P.; Murugesh, N.; Chandramohan, M.; Pannecouque, C.; De Clercq, E. Synthesis, Antiviral and
Cytotoxic Activities of 2-(2-Phenyl carboxylic acid)-3-Phenylquinazolin-4(3H)-one Derivatives. Indian J.
Pharm. Sci. 2010, 72, 806–809. [CrossRef] [PubMed]
20. Dinakaran, M.; Selvam, P.; De Clercq, E.; Sridhar, S.K. Synthesis, Antiviral and Cytotoxic Activity of
6-Bromo-2,3-disubstituted-4(3H)-quinazolinones. Biol. Pharm. Bull. 2003, 26, 1278–1282. [CrossRef]
[PubMed]
21. Gao, X.; Cai, X.; Yan, K.; Song, B.; Gao, L.; Chen, Z. Synthesis and Antiviral Bioactivities of 2-Aryl- or
2-Methyl-3-(substituted-Benzalamino)-4(3H)-quinazolinone Derivatives. Molecules 2007, 12, 2621–2642.
[CrossRef] [PubMed]
22. Abbas, S.Y.; El-Bayouki, K.A.M.; Basyouni, W.M.; Mostafa, E.A. New series of 4(3H)-quinazolinone
derivatives: Syntheses and evaluation of antitumor and antiviral activities. Med. Chem. Res. 2018, 27,
571–582. [CrossRef]
23. Schroeder, C.E.; Yao, T.; Sotsky, J.; Smith, R.A.; Roy, S.; Chu, Y.-K.; Guo, H.; Tower, N.A.; Noah, J.W.;
McKellip, S.; et al. Development of (E)-2-((1,4-Dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide,
ML336: Novel 2-Amidinophenylbenzamides as Potent Inhibitors of Venezuelan Equine Encephalitis Virus.
J. Med. Chem. 2014, 57, 8608–8621. [CrossRef] [PubMed]
24. Leivers, A.L.; Tallant, M.; Shotwell, J.B.; Dickerson, S.; Leivers, M.R.; McDonald, O.B.; Gobel, J.; Creech, K.L.;
Strum, S.L.; Mathis, A.; et al. Discovery of Selective Small Molecule Type III Phosphatidylinositol 4-Kinase
Alpha (PI4KIII
[PubMed]
α) Inhibitors as Anti Hepatitis C (HCV) Agents. J. Med. Chem. 2014, 57, 2091–2106. [CrossRef]
˙
25. Piotrowska, D.G.; Andrei, G.; Schols, D.; Snoeck, R.; Grabkowska-Druzyc, M. New Isoxazolidine-Conjugates
of Quinazolinones—Synthesis, Antiviral and Cytostatic Activity. Molecules 2016, 21, 959. [CrossRef]
[PubMed]
26. Piotrowska, D.G. N-Substituted C-diethoxyphosphorylated nitrones as useful synthons for the synthesis of
α-aminophosphonates. Tetrahedron Lett. 2006, 47, 5363–5366. [CrossRef]
27. Erb, B.; Akue, R.; Rigo, B. Synthesis of 2-Aminoquinazoline-4(3H)-one Derivatives as Potential Potassium
Channel Openers. J. Heterocycl. Chem. 2000, 37, 253–260. [CrossRef]
28. Dolzhenko-Podchezertseva, A.V.; Korkodinova, L.M.; Vasilyuk, M.V.; Kotegov, V.P. Synthesis and
antiinflamatory activity of N-acyl-5-bromoanthranilic acid amides. Pharm. Chem. J. 2002, 36, 647–648.
[CrossRef]
29. Witt, A.; Bergman, J. Synthesis and reactions of some 2-vinyl-3H-quinazolin-4-ones. Tetrahedron 2000, 56,
7245–7253. [CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
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2018 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/).