Design, antiviral and cytostatic properties of isoxazolidine-containing amonafide analogues

Article abstract of DOI:10.1016/j.bmc.2015.04.079

Abstract A novel series of 5-arylcarbamoyl- and 5-arylmethyl-2-methylisoxazolidin-3-yl-3-phosphonates have been synthesized via cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone with N-substituted naphthalimide acrylamides and N-allylnaphthalimides. All cis- and trans-isoxazolidine phosphonates obtained herein were assessed for antiviral activity against a broad range of DNA and RNA viruses. Isoxazolidines trans-9d and trans-9f exhibited the highest activity (EC₅₀ = 8.9 μM) toward cytomegalovirus. Compounds cis- and trans-9d as well as cis- and trans-9f were found potent against HSV and Vaccinia viruses (EC₅₀ in the 45-58 μM range), whereas isoxazolidines 10a and 10d suppressed replication of Coxsackie B4 and Punta Toro viruses (EC₅₀ in the 45-73 μM range). Antiproliferative evaluation of all obtained isoxazolidines revealed the promising activity of cis-9b, cis-9d, trans-9d, cis-9e, trans-9e, cis-9f and trans-9f toward tested cancer cell lines with IC₅₀ in the 1.1-19 μM range.

Full text of DOI:10.1016/j.bmc.2015.04.079

Accepted Manuscript
Design, antiviral and cytostatic properties of isoxazolidine-containing amona-
fide analogues
Kamil Kokosza, Graciela Andrei, Dominique Schols, Robert Snoeck, Dorota G.
Piotrowska
PII:
S0968-0896(15)00395-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.079
BMC 12291
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 March 2015
28 April 2015
29 April 2015
Please cite this article as: Kokosza, K., Andrei, G., Schols, D., Snoeck, R., Piotrowska, D.G., Design, antiviral and
cytostatic properties of isoxazolidine-containing amonafide analogues, Bioorganic & Medicinal Chemistry (2015),
doi: http://dx.doi.org/10.1016/j.bmc.2015.04.079
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Design, antiviral and cytostatic properties of isoxazolidine-containing amonafide
analogues
a
Kamil Kokosza, Graciela Andrei, Dominique Schols, Robert Snoeck and Dorota G. Piotrowska ∗
b
b
b
a,
a
Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of Łódź, Muszyńskiego 1, 90-151 Łódź, Poland
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
b
ARTICLE INFO
ABSTRACT
Article history:
Received
A novel series of 5-arylcarbamoyl- and 5-arylmethyl-2-methylisoxazolidin-3-yl-3-phosphonates
have been synthesized via cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone with N-
substituted naphthalimide acrylamides and N-allylnaphthalimides. All cis- and trans-
isoxazolidine phosphonates obtained herein were assessed for antiviral activity against a broad
range of DNA and RNA viruses. Isoxazolidines trans-9d and trans-9f exhibited the highest
activity (EC50 = 8.9 µM) toward cytomegalovirus. Compounds cis- and trans-9d as well as cis-
and trans-9f were found potent against HSV and Vaccinia viruses (EC50 in the 45–58 µM
range), whereas isoxazolidines 10a and 10d suppressed replication of Coxsackie B4 and Punta
Toro viruses (EC50 in the 45–73 µM range). Antiproliferative evaluation of all obtained
isoxazolidines revealed the promising activity of cis-9b, cis-9d, trans-9d, cis-9e, trans-9e, cis-9f
and trans-9f toward tested cancer cell lines with IC50 in the 1.1–12 µM range.
Received in revised form
Accepted
Available online
Keywords:
Phosphonates
Isoxazolidines
Naphthalimides
Antiviral
Cytostatic
2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Despite significant achievements in cancer chemotherapy a
solid tumors, however most of them showed poor therapeutic
indices.
search for more selective compounds with capability to better
differentiate malignant tumors from normal cells and able to
minimize side effects of current drugs continues. Taking into
account their mechanisms of action, therapeutic agents belong to
different pharmacological classes. Among them, those which
interact with DNA, such as DNA alkylating agents, intercalators
On the other hand, various functional groups present in the
structure of intercalators may also contribute to the binding of a
drug to DNA. The amino group of a sugar residue in doxorubicin
3 forms an ionic bond with the phosphate function of the DNA
which results in additional and very efficient stabilization of
1
0
locking the drug into an active site. There are also known
groups of intercalators which activities are related to the specific
shape of molecule rather than their aromatic ring system. Studies
on intercalating properties of 2-[(arylmethyl)amino]-2-methyl-
1,3-propanodiols (AMAPs) 4 proved that the amino side chain is
responsible for DNA binding due to electrostatic interactions and
enhanced activity which was observed for derivatives with 2-
1–3
or groove binders, are of special interest.
Intercalators
constitute a well explored class of compounds because their
mode of action is the most predictable and relies on inhibition of
4
,5
replication process by reversible binding to DNA. The planar
aromatic fragments of intercalators insert into the DNA double
helix, thereby distorting the DNA backbone conformation and
6
11,12
poison the DNA topoisomerases I or II. Several factors are
amino-1,3-propanediol moiety (Fig. 1).
responsible for stabilization a drug–DNA complex, namely
stacking π-bond interactions, van der Waals forces and
eventually hydrogen bonding between the aromatic fragment of a
drug moiety and the purine/pyrimidine bases present in DNA
strains. Intercalators can be classified according to their
aromatic/heteroaromatic subunit or electrostatic potential they
2
,3
posses. Among them, naphthalimide derivatives have been
found promising as anticancer agents since they are able to
7
,8
intercalate into DNA and some of them, including amonafide 1
9
and mitonafide 2, have reached clinical trials for treatment of
———
Figure 1. Structures of known intercalators.
∗
Corresponding author. Tel.: +48-42-677-9235; fax: +48-42-678-8398; e-mail: dorota.piotrowska@umed.lodz.pl

substituted acrylamides 11 were synthesized starting from
commercially available naphthalic anhydride 13 via multistep
procedures including nitration followed by a standard reduction
Compounds
5
(Fig. 2) have been designed as new
27
isoxazolidine-containing intercalators equipped with planar
polycyclic aromatic frameworks at C3 and their cytotoxic and
apoptotic properties have already been described.
28
of the nitro group, transformation of the aminoanhydride 15
into naphthalimides 16a-f by treatment with alkyl amines or
1
3,14
Recently,
29
ethylenediamines and finally preparation of acrylamides 11a–f
from 16a–f and acryloyl chloride in the presence of triethylamine
we succeeded in the preparation of compounds 6 having 1- and 2-
naphthyl substituents at C5 of an isoxazolidine ring and they
proved cytotoxic against HeLa and K562 cell lines with IC50
(
the reaction of naphthalic anhydrides 13, 14 and 15 with
Scheme 2). The series of dipolarophiles 12a–c were obtained in
1
5
values 50 and 90 µM, respectively. Moreover, isoxazolidines 7
containing at C5 a carbamoyl linker which separates the
isoxazolidine ring and substituted phenyls suppressed divisions
of three cancer cell lines at concentrations ranging from 228 to
29
allylamine. Furthermore, naphthalimide 12c was converted into
its acetyl derivative 12d.
30
1
6
1
02 µM.
Figure 2. Biologically active isoxazolidines.
Herein,
arylmethylisoxazolidines
a
new series of 5-arylcarbamoyl- and 5-
substituted at C3 with
a
Scheme 2. Reaction conditions: a) H
R′NH , EtOH, see Table 1; d) acryloyl chloride, NEt
allylamine, EtOH; f) Ac O.
2
SO
4
, HNO
3
; b) SnCl
2
, HCl; c)
dialkoxyphosphoryl functionality 9 and 10 have been designed as
a continuation of an ongoing project directed towards the
construction of compounds with antineoplastic activity based on
application of the phosphoryl nitrone 8 (Scheme 1).
2
3
, see Table 1; e)
2
3
1
1
naphthalimides 11a–f were performed in toluene or a toluene–
,3-Dipolar cycloadditions of
a
nitrone
8
with
chloroform mixture at 70°C and led to the formation of
diastereoisomeric
diethoxyphosphoryl)isoxazolidines trans-9a–f and cis-9a–f
Scheme 3; Table 1). In all cases moderate trans/cis
mixtures
of
(3-
(
diastereoselectivities were observed with trans-isomers 9a–f
predominating (de 56–72%). Purification of the crude mixtures
of cycloadducts on silica gel columns resulted in separation of all
major diastereoisomers trans-9a-f as well as all minor isomers
cis-9a-f.
Scheme 1. Retrosynthesis of (isoxazolidinyl)phosphonates 9 and 10.
An idea behind designing compounds 9 and 10 is based on the
combination of two biologically active moieties/pharmacophores,
namely naphthalimide and (3-diethoxyphosphoryl)isoxazolidine
units, with intention to obtain their active hybrids or conjugated
1
7
drugs. Recently, similar approach was successfully applied by
several research groups and resulted in synthesizing various
1
8
series
naphthalimides conjugated with carbazole, benzodiazepine,
of
amonafide-containing chimeras,
including
20–22
19
2
3
24
benzoic acid, aliphatic diamine chain as well as peptide
2
5
nucleic acid. Our approach to the synthesis of phosphonylated
intercalators 9 and 10 relies on the assumption that the designed
molecules contain both aromatic systems able to intercalate into
DNA and the diethoxyphosphoryl function at C3 of the
isoxazolidine ring which could be further phosphorylated but also
Scheme 3. Reaction conditions: toluene or toluene–chloroform,
0°C.
7
26
Table 1. (appended at the end of the paper)
contains a non-hydrolysable C–P bond.
Reactions of a nitrone 8 with N-allylated naphthalimides 12a–
d were carried out in toluene or toluene–chloroform solutions at
2
2
. Results and discussion
.1. Chemistry
7
0°C and gave mixtures of isomeric isoxazolidines trans-10a–d
and cis-10a–d with good trans/cis diastereoselectivities (de 72–
2%). Crude mixtures of cycloadducts were subjected to
8
To the best of our knowledge all acrylamides 11 as well as
allylated naphthalimides 12, except for 12a and 12b have not
been described in the literature. For the purpose of this study, N-
purification on silica gel columns and subsequent crystallization.
However, attempts at separating diastereoisomers trans-10a-d

+
–
and cis-10a-d failed leading to mixtures enriched in isomers
trans-10 (Scheme 4; Table 2).
varicella-zoster virus (TK VZV strain and TK VZV 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; (d) MDCK cell cultures: influenza A virus
(
H1N1 and H3N2 subtypes) and influenza B virus and (e) CrFK
cell cultures: feline herpes virus (FHV) and feline corona virus
FIPV). Ganciclovir, cidofovir, acyclovir, brivudin, (S)-9-(2,3-
(
dihydroxypropyl)adenine [(S)-DHPA], oseltamivir carboxylate,
amantadine, rimantadine, ribavirin, dextran sulfate (molecular
weight 5000, DS-5000), Hippeastrum hybrid agglutinin (HHA)
and Urtica dioica agglutinin (UDA) were used as the reference
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).
Scheme 4. Reaction conditions: toluene or toluene–chloroform,
7
0°C.
Table 2. (appended at the end of the paper)
Some compounds of the series of isoxazolidines cis-9/trans-9
were found active against human cytomegalovirus with activities
below 60 µM (Table 3). Among them, trans-9d (EC50 = 8.9 µM)
and trans-9f (EC50 = 8.9–20 µM) exhibited the highest activity
with EC50 values comparable to those found for the reference
compound ganciclovir which is also an approved drug for
HMCV, and approximately an order of magnitude lower than that
assayed for cidofovir used as the second reference. However, the
new-generation drug letermovir showed EC50 = 0.0046±0.0019
µM against HCMV in AD169 strain and recently reached phase
The relative configurations of the isoxazolidines trans-9a–f
and cis-9a–f were determined taking advantage of our previous
studies on stereochemistry of cycloaddition of N-methyl-C-
1
6
diethoxyphosphorylnitrone 8 with (hetero)arylacrylamides since
1
similar H NMR spectral patterns for the respective series of
isoxazolidines trans-9a–f and cis-9a–f were observed as
compared to the previously described trans- and cis-5-
(
phosphonates.
hetero)arylcarbamoyl-2-methylisoxazolidin-3-yl-3-
16,32
3
Briefly, for the major trans-isomers 9a–f the E
conformation (Fig. 3) of an isoxazolidine ring was established
taking advantage of the diagnostic values of vicinal coupling
3
4
IIb of clinical trials.
constants [JCCCP = 7.7–8.9 Hz, JH3–H4α = 8.1–8.3 Hz, JH3–H4β
=
=
Among the series of isoxazolidines cis-9/trans-9, majority of
+
the derivatives was able to inhibit replication of TK and TK
–
8
5
.5–8.9 Hz, JH4α–P = 9.4–10.3 Hz, JH4β–P = 15.7–16.1 Hz, JH4α–H5
.5–5.8 Hz and JH4β–H5 = 8.7–8.9 Hz] extracted from the
VZV strains with EC50 values in the range of 20–45 µM (Table
4
1
13
+
). The potency against TK VZV strain of the most active trans-
respective H and C NMR spectra. In this conformation the
diethoxyphosphoryl group resides in the equatorial position of
the isoxazolidine ring while carbamoyl substituents are located
pseudoequatorially. Similarly, relative configurations of
isoxazolidines trans-10a–d and cis-10a–d were established based
on very close stereochemical outcomes found for the reaction
between a nitrone 8 and N-allylated naphthalimides 12a–d as
compared with already elaborated analogous reactions of a
9
d (EC50 = 14–15 µM) was found an order or three orders of
magnitude lower than that of the reference acyclovir (also the
approved drug) or brivudin, respectively. However, activities of
–
isoxazolidines studied (except trans-9c) against the TK VZV
strain compare favorably (EC50 = 20–45 µM) with that of the
reference acyclovir (EC50 = 33–44 µM) being an order of
magnitude less active than the second reference brivudin (EC50
1
=
3
3
nitrone 8 and N-allylated nucleobases.
.0–4.9 µM).
On the other hand, none of the compounds from the series
trans-10/cis-10 was found active either towards HCMV or
against VZV.
Table 3. (appended at the end of the paper)
Table 4. (appended at the end of the paper)
Figure 3. The
f.
3
E conformation of an isoxazolidine ring in trans-9a–
Although the isoxazolidine derivatives equipped with N,N-
dimethylaminoethyl (trans-9d and cis-9d) and 2-(pirolidyn-1-
yl)ethyl (trans-9f) functions were found slightly active against
three strains of herpes simplex virus with EC50 values ranging
from 45 to 100 µM (Table 5) their potency seems marginal when
compared with that of the four reference compounds (brivudin,
cidofovir, ganciclovir and the FDA-approved acyclovir).
However, activities of trans-9d, cis-9d, trans-9f and cis-9f
against vaccinia virus (EC50 = 45–100 µM) are of considerable
value since the reference acyclovir and ganciclovir were inactive
while the two other reference compounds brivudin and cidofovir
showed EC50 values of 17–22 and 22 µM (Table 5), respectively.
Moreover, these compounds were not cytotoxic toward HEL cells
(in which the viruses were replicated) at concentrations up to 100
µM or displayed a minimum cytotoxic concentration (MCC) of
100 µM.
2
.2. Antiviral and cytostatic evaluation
2
.2.1. Antiviral activity
Pure 5-arylcarbamoyl-2-methylisoxazolidin-3-yl-3-
phosphonates trans-9a–f and cis-9a–f and inseparable mixtures
of 5-arylmethyl-2-methylisoxazolidin-3-yl-3-phosphonates trans-
1
0a–d/cis-10a–d 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 (TK
r
KOS ACV ), vaccinia virus, vesicular stomatitis virus and,
adenovirus-2, cytomegalovirus (AD-169 strain and Davis strain),

cytostatic activity against all three tested cancer cell lines (IC50
from 20 to 180 µM). Isoxazolidines trans-10b/cis-10b substituted
with the nitro group at C5 of a naphthalimide unit emerged as the
most cytostatic with the highest potency toward HeLa cell line
Table 5. (appended at the end of the paper)
(
1
IC50 = 20 µM). None of the evaluated compounds trans-10/cis-
0 decreased viability of MDCK and CrFK cells at concentration
In the second series of isoxazolidine phosphonates 10, trans-
0a/cis-10a and trans-10d/cis-10d inhibited the replication of
up to 100 µM.
1
Coxsackie virus B4 at concentrations 45–73 µM which was
significantly better than ribavirin (Table 6). On the other hand,
only trans-10d/cis-10d showed a weak activity against Punta
Toro virus (EC50 = 50–100 µM) which again was higher than that
of ribavirin (Table 6). These compounds did not alter morphology
of Vero cells where the antiviral assays were performed. Since no
drugs for Coxackie and Punta Toro viruses have been approved
so far, the urgency of search for active compounds is justified.
Table 7. (appended at the end of the paper)
3
. Conclusions
Two new series of 5-arylcarbamoyl-2-methylisoxazolidin-3-
yl-3-phosphonates trans-9 and cis-9 and 5-arylmethyl-2-
methylisoxazolidin-3-yl-3-phosphonates trans-10 and cis-10
Table 6. (appended at the end of the paper)
have
diethoxyphosphoryl)nitrone 8 and the respective N-substituted
naphthalimide acrylamides or N-allylated naphthalimides via the
been
obtained
from
N-methyl-C-
(
None of the isoxazolidines cis-9/trans-9 and trans-10 and cis-
0 phosphonates showed activity against the other tested viruses,
1
1
,3-dipolar cycloaddition.
including adenovirus-2, vesicular stomatitis virus, respiratory
syncytial virus, para-influenza-3 virus, reovirus-1, Sindbis virus,
influenza A and B viruses, feline herpes virus, and feline corona
virus.
All synthesized isoxazolidine phosphonates trans-9 and cis-9
as well as the respective mixtures of trans-10/cis-10 were
evaluated against a variety of DNA and RNA viruses. Several of
these derivatives showed some activity against varicella-zoster
virus and cytomegalovirus. Among all tested compounds,
isoxazolidines trans-9d and trans-9f exhibited the highest
activity (EC50 = 8.9 µM) toward cytomegalovirus, comparable to
the activity of ganciclovir, the approved drug which was used as
the reference compound. The isoxazolidines 9 (except trans-9c)
2
.2.2. Cytostatic activity
The 50% cytostatic inhibitory concentration (IC50) causing a
0% decrease in cell proliferation was determined against murine
5
leukemia L1210, human lymphocyte CEM and human cervix
carcinoma HeLa cells. The synthesized compounds showed
differences in their antiproliferative activity with IC50 ranging
from 1.1 to 180 µM (Table 7). Among isoxazolidines having a
carbamoyl linker, structure–activity relationship studies indicated
that the presence of a tertiary nitrogen atom in a side chain
significantly increased cytostatic activity of the tested
compounds. Phosphonates trans-9a–c and cis-9a–c having
–
appeared active against the TK VZV strain and the potency of
compound cis-9d (EC50 = 20 µM) compares favorably with that
of the reference acyclovir (EC50 = 33–44 µM) which is also the
approved drug.
Some of the tested compounds were also endowed with the
antiviral activity against HSV and Vaccinia (cis- and trans-9d,
cis- and trans-9f, EC50 in the 45–58 µM range), Coxsackie B4
and Punta Toro (10a and 10d, EC50 in the 45–73 µM range)
viruses and although for the Coxsackie virus B4 compounds 10a
and 10d are even more active than ribavirin used as the reference
compound their potency is not large enough to deserve further
studies.
straight aliphatic chains were considerably less cytostatic (IC50
2
=
0–85 µM) than those containing tertiary amino functions,
namely trans-9d–f and cis-9d–f. Moreover, compounds trans-9a
and cis-9c decreased viability of the MDCK cell line at
concentrations of 46.2 and 51.8 µM (CC50), respectively.
Furthermore, both trans- and cis-isoxazolidines 9d, 9e and 9f
having aminoalkyl substituents appeared to be the most cytostatic
towards L1210, CEM and HeLa cell lines with IC50’s below 23
µM. Among them, trans-9d and cis-9d, which can be considered
as amonafide analogues, as well as trans-9f and trans-9f proved
highly cytostatic toward murine leukemia L1210 and human
cervix carcinoma HeLa cells (IC50 = 1.1–3.3 µM) and their
potency was only an order of magnitude lower than that of a
reference 5-fluorouracil. Besides their high cytostatic activity,
compounds trans-9d, cis-9d, trans-9f and cis-9f were found to
reduce viability of MDCK and CrFK cells at concentrations in
the range of 95.2–62.2 µM (CC50). In addition, phosphonates
trans-9a, trans-9b, cis-9b, cis-9c, trans-9e and cis-9e exhibited
high cytostatic activity against human lymphocyte cells (CEM),
comparable to that of 5-fluorouracil, while trans-9d and trans-9f
were even five-fold more active than 5-fluorouracil. Furthermore,
although no correlation between configuration of isoxazolidines
trans-9a–c/cis-9a–c and trans-9e/cis-9e and their biological
activity was observed, trans-configured isoxazolidines trans-9d
and trans-9f were found two-fold more cytostatic than the
corresponding cis-isomers cis-9d and cis-9f.
Cytostatic activity of trans-9, cis-9 and trans-10/cis-10 was
evaluated on L1210, CEM and HeLa cell lines and for the most
cystostatic compounds (cis-9b, cis-9d, trans-9d, cis-9e, trans-9e,
cis-9f and trans-9f), IC50 values were found in the 1.1–12 µM
range. Generally, compounds with a carbamoyl linker trans-
9/cis-9 were more active than trans-10/cis-10 having a methylene
bridge.
4
. Experimental section
1
H NMR spectra were taken in CDCl3 on the following
spectrometers: Varian Mercury-300 and Bruker Avance III (600
1
3
MHz) with TMS as internal standard. C NMR spectra were
recorded for CDCl solution on the Varian Mercur-300 machine
at 75.5 MHz, while for DMSO solution on Bruker Avance III at
3
3
1
151.0 MHz. P NMR spectra were performed in CDCl solution
3
on the Varian Mercury-300 at 121.5 MHz or on Bruker Avance
III at 243.0 MHz. IR spectra were measured on an Infinity MI-60
FT-IR spectrometer. Melting points were determined on Boetius
apparatus and are uncorrected. Elemental analyses were
The second series of 5-arylmethyl-2-methylisoxazolidin-3-yl-
-phosphonates trans-10/cis-10 also showed considerable
3

performed by the Microanalytical Laboratory of this Faculty on
Perkin-Elmer PE 2400 CHNS analyzer. The following adsorbents
were used: column chromatography, Merck silica gel 60 (70–230
mesh); analytical TLC, Merck TLC plastic sheets silica gel 60
4.1.4. N-(2-propenyl-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-5-yl)acetamide (12d)
N-(2-propenyl)-3-amino-1,8-naphthalimide 12c (1.00 mmol)
was treated with acetic anhydride (3.00 mL, 31.55 mmol) and
stirred for 5 h at room temperature. The reaction mixture was
cooled to room temperature and cold water (10 mL) was added.
Yellow precipitate was filtered off, washed several times with
water and dried to afford naphthalimide 12d. Yield: 86%; yellow
254
F .
4.1. General procedure for the preparation of 1,8-
naphthalimides 16a-f and 12a-c
-
1
amorphous solid mp 253–254°C; IR (KBr, cm ) νmax: 3291,
3
1
8
213, 1703, 1663, 1630, 1565, 1465, 1424, 1335, 1264, 1236,
A suspension of 1,8-naphthalic anhydride 13, 14 or 15 (1.00
mmol), appropriate aliphatic amine, substituted ethylenediamine
or allylamine (2.00 mmol) in ethanol (10 mL) was heated under
reflux for 3 h. After evaporation of a solvent under reduced
pressure the crude product was purified on a silica gel column
with chloroform:methanol mixtures (100:1, 50:1 v/v) to give the
corresponding 1,8-naphthalimides 16a-f and 12a-c.
1
180, 880, 783, 745; H NMR (300 MHz, CDCl
.50 (d, 1H, J = 7.1 Hz), 8.27 (d, 1H, J = 2.2 Hz), 8.18 (d, 1H, J
8.3 Hz), 7.73 (dd, 1H, J = 8.3, 7.1 Hz), 7.69 (br s, 1H, NH),
.98 (ddt, 1H, J = 17.1, 10.5, 5.5 Hz, CH CH=CH ), 5.31 (d, 1H
J = 17.1 Hz, CH CH=CH ), 5.21 (d, 1H, J = 10.5 Hz,
CH CH=CH ), 4.80 (d, 2H, J = 5.5 Hz, CH CH=CH ), 2.31 (s,
H, CH ); C NMR (151.0 MHz, DMSO) δ: 169.50 (s,
C(O)NH), 163.46 (s, C(O)), 163.24 (s, C(O)), 138.47, 134.11,
33.29, 132.49, 129.29, 127.86, 124.28, 124.15, 122.80, 122.03,
120.93, 116.83, 42.21 (s, CH CH=CH ), 24.56 (s, CH C(O)).
3
) δ: 8.95 (s, 1H),
=
5
2
2
,
2
2
2
2
13
2
2
3
3
4
.1.1. N-propyl-3-amino-1,8-naphthalimide (16a)
1
Yield: 74%; yellow amorphous solid (crystallized from
-
1
2
2
3
chloroform/hexane) mp 202–203°C; IR (KBr, cm ) νmax: 3473,
1
17 14 2 3
Anal. Calcd. for C H N O : C, 69.38; H, 4.79; N, 9.52; found:
C, 69.14; H, 4.88; N, 9.76.
3
(
368, 1688, 1646, 1580, 1341, 1308, 1220, 778, 744; H NMR
300 MHz, CDCl ) δ: 8.31 (dd, 1H, J = 7.3, 1.1 Hz), 8.02 (d, 1H,
J = 2.3 Hz), 7.92 (dd, 1H, J = 8.3, 1.1 Hz), 7.60 (dd, 1H, J = 8.3,
3
7
2
1
.3 Hz), 7.30 (d, 1H
H, J = 7.5 Hz, CH
.01 (d, 3H, J =7.5 Hz, CH
) δ: 164.51 (s, C(O)), 164.28 (s, C(O)), 145.33, 133.50,
31.71, 127.54, 127.30, 123.79, 122.67, 122.62, 122.11, 114.05,
2.18 (s, CH CH CH ), 21.71 (s, CH CH CH ), 11.85 (s,
CH CH ). Anal. Calcd. for C15 : C, 70.85; H, 5.55; N,
1.02; found: C, 70.61; H, 5.70; N, 10.77.
,
J = 2.3 Hz), 4.18 (br s, 2H, NH
2
), 4.13 (d,
CH CH ),
2 3
CH CH ); C NMR (75.5 MHz,
4
1
.2. General procedure for the preparation of acrylamides
1a-f
2
CH CH ), 1.81–1.69 (m, 2H, CH
2
3
2
2
3
13
2
CDCl
1
4
CH
1
3
To a solution of an appropriate 3-amino-1,8-naphthalimide
6a-f (1.00 mmol) in dichloromethane (2 mL) triethylamine
1.10 mmol) was added. The mixture was cooled in an ice bath
1
2
2
3
2
2
3
(
2
2
3
14 2 2
H N O
and acryloyl chloride (1.05 mmol) was added dropwise. The
reaction mixture was stirred for 24 h at room temperature and
extracted with water (3 × 3 mL). Subsequently, the inorganic
layer was extracted with ethyl ether (3 × 5 mL). The combined
4
.1.2. N-isobutyl-3-amino-1,8-naphthalimide
(
16c)
Yield: 77%; yellow amorphous solid (crystallized from
4
organic layers were dried over anhydrous MgSO and filtered.
-
1
After evaporation of solvents the residue was purified on a silica
column with chloroform:methanol mixtures (100:1, 50:1 v/v) to
afford the respective acrylamides 11a-f.
chloroform/hexane) mp 185–186°C; IR (KBr, cm ) νmax: 3452,
3
7
357, 1692, 1657, 1627, 1581, 1447, 1343, 1297, 1222, 1072,
1
83; H NMR (300 MHz, CDCl ) δ: 8.33 (dd, 1H, J = 7.3, 1.0
3
Hz), 8.03 (d, 1H, J = 2.4 Hz), 7.93 (dd, 1H, J = 8.3, 1.0 Hz), 7.60
dd, 1H, J = 8.3, 7.3 Hz), 7.31 (d, 1H J = 2.4 Hz), 4.15 (br s, 2H,
NH ), 4.03 (d, 2H, J = 7.3 Hz, CH CH(CH ), 2.28–2.18 (m, 1H,
CH CH(CH ), 0.98 (d, 6H, J = 6.7 Hz, CH
NMR (75.5 MHz, CDCl ) δ: 164.79 (s, C(O)), 164.57 (s, C(O)),
45.33, 133.50, 131.67, 127.61, 127.31, 123.75, 122.72, 122.59,
22.21, 114.05, 47.40 (s, CH CH(CH ), 27.69 (s,
CH(CH ), 20.62 (s, CH CH(CH ). Anal. Calcd. for
: C, 71.62; H, 6.01; N, 10.44; found: C, 71.42; H,
4.2.1. N-(2-propyl-1, 3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-5-yl)propenamide (11a)
(
,
2
2
3 2
)
Yield: 57%; slightly yellowish amorphous solid (crystallized
-1
1
3
C
2
3
)
2
2
CH(CH
3
)
2
);
from chloroform/hexane) mp 223–224°C; IR (KBr, cm ) νmax
:
3
1
3
(
308, 2960, 1703, 1663, 1623, 1564, 1337, 1225, 786; H NMR
300 MHz, CDCl ) δ: 9.05 (s, 1H), 8.49 (dd, 1H, J = 7.3, 1.0 Hz),
.30 (d, 1H, J = 2.2 Hz), 8.17 (dd, 1H, J = 8.3, 1.0 Hz), 7.87 (br
1
1
CH
3
2
3 2
)
8
2
3
)
2
2
3 2
)
s, 1H, NH), 7.72 (dd, 1H, J = 8.3, 7.3 Hz), 6.55 (dd, 1H, J = 16.8,
16 16 2 2
C H N O
1
5
.2 Hz, CH=CH
.89 (dd, 1H, J = 10.1, 1.2 Hz, CH=CH
CH CH ), 1.79–1.69 (m, 2H, CH CH
7.4 Hz, CH ); C NMR (151.0 MHz, DMSO) δ: 164.24
s, C(O)), 163.80 (s, C(O)), 163.58 (s, C(O)), 138.24, 134.12,
2
), 6.37 (dd, 1H
,
J = 16.8, 10.1 Hz, CH=CH
), 4.13 (t, 2H, J = 7.6 Hz,
CH ), 1.01 (t, 3H, J =
2
),
5
.84; N, 10.19.
2
4
.1.3. N-(2-propenyl)-3-amino-1,8-naphtalimide
CH
2
2
3
2
2
3
1
3
(
12c)
Yield: 92%; orange amorphous solid mp 221–222°C; IR
2 2 3
CH CH
(
-
1
1
1
1
32.50, 131.97, 129.47, 128.22, 128.00, 124.54, 124.30, 123.12,
22.27, 121.48, 41.73 (s, CH CH CH ), 21.31 (s, CH CH CH ),
8.82 (s, CH CH CH ). Anal. Calcd. for C18 : C, 70.12;
(
KBr, cm ) νmax: 3455, 3364, 1688, 1644, 1615, 1578, 1448,
1
1
379, 1330, 1237, 1180, 777, 744; H NMR (300 MHz, CDCl
δ: 8.32 (dd, 1H, J = 7.3, 1.0 Hz), 8.03 (d, 1H, J = 2.4 Hz), 7.93
dd, 1H, J = 8.1, 1.0 Hz), 7.60 (dd, 1H, J = 8.1, 7.3 Hz), 7.30 (d,
J = 2.4 Hz), 5.99 (ddt, 1H, J = 17.0, 10.3, 5.7 Hz,
CH=CH ), 5.31 (dq, 1H J = 17.0, 1.3 Hz, CH CH=CH ),
.20 (dq, 1H, J = 10.3, 1.3 Hz, CH CH=CH ), 4.79 (dt, 2H, J =
.7, 1.3 Hz, CH CH=CH ), 4.17 (br s, 2H, NH
3
)
2
2
3
2
2
3
2
2
3
16 2 3
H N O
H, 5.23; N, 9.09; found: C, 70.37; H, 5.13; N, 8.98.
(
1H
,
4.2.2. N-(2-butyl-1,3-dioxo-2, 3-dihydro-1H-
benzo[de]isoquinolin-5-yl)propenamide (11b)
CH
5
5
2
2
,
2
2
2
2
Yield: 70%; slightly yellowish amorphous solid (crystallized
-1
1
3
2
2
2
); C NMR
from chloroform/hexane) mp 235–236°C; IR (KBr, cm ) νmax
:
258, 2956, 1696, 1661, 1624, 1547, 1338, 1269, 1227, 1130,
(
151.0 MHz, DMSO) δ: 163.92 (s, C(O)), 163.75 (s, C(O)),
3
7
8
1
7
1
1
48.35, 134.05, 133.46, 132.03, 127.39, 125.96, 122.93, 122.26,
22.13, 121.11, 116.65, 112.35, 42.08 (s, CH CH=CH ). Anal.
: C, 71.42; H, 4.79; N, 11.10; found: C,
1.14; H, 4.88; N, 10.96.
1
86; H NMR (300 MHz, CDCl ) δ: 9.06 (d, 1H, J = 2.2 Hz),
3
2
2
.49 (dd, 1H, J = 7.3, 1.0 Hz), 8.30 (d, 1H, J = 2.2 Hz), 8.17 (dd,
H, J = 8.3, 1.0 Hz), 7.80 (br s, 1H, NH), 7.73 (dd, 1H, J = 8.3,
12 2 2
Calcd. for C15H N O
7
2 ,
.3 Hz), 6.55 (dd, 1H, J = 16.9, 1.2 Hz, CH=CH ), 6.36 (dd, 1H
J = 16.9, 10.1 Hz, CH=CH ), 5.89 (dd, 1H, J = 10.1, 1.2 Hz,
2

CH=CH
(
0
2
), 4.17 (t, 2H, J = 7.5 Hz, CH
m, 2H, CH CH CH CH ), 1.51–1.41 (m, 2H, CH
.97 (t, 3H, J = 7.1 Hz, CH
2
CH
2
CH
2
CH
CH
3
3
), 1.76–1.66
CH CH ),
); C NMR (151.0
132.29, 130.83, 129.99, 128.90, 127.51, 124.84, 124.37, 122.88,
122.78, 122.00, 50.07 (s, CH CH N(CH CH ), 47.66 (s,
), 38.36 (s, CH CH N(CH CH ), 12.36 (s,
). Anal. Calcd. for C21 : C, 69.02; H,
2
2
2
3
2
2
2
3
2
2
2
3 2
)
1
2
CH
2
CH
2
CH
3
CH
CH
2
CH
CH
2
N(CH
N(CH
2
CH
3
)
)
2
2
2
2
3 2
)
MHz, DMSO) δ: 164.24 (s, C(O)), 163.78 (s, C(O)), 163.55 (s,
C(O)), 138.24, 134.13, 132.50, 131.97, 129.47, 128.23, 128.01,
2
2
2
CH
3
2
23 3 3
H N O
6.34; N, 11.50; found: C, 68.88; H, 6.19; N, 11.34.
1
24.54, 124.31, 123.15, 122.30, 121.50, 40.43 (s,
NCH CH CH CH ), 30.11 (s, CH CH CH CH ), 20.28 (s,
CH CH CH CH ), 14.15 (s, CH CH CH CH ). Anal. Calcd. for
: C, 70.79; H, 5.63; N, 8.69; found: C, 70.44; H, 5.78;
4.2.6. N-(2-pirolidyn-1-yl)ethyl-1,3-dioxo-2,3-
dihydro-1H-benzo[de]isoquinolin-5-
yl)propenamide (11f)
2
2
2
3
2
2
2
3
2
2
2
3
2
2
2
3
19 18 2 3
C H N O
Yield: 43%; yellowish amorphous solid (crystallized from
-
N, 8.43.
1
chloroform/hexane) mp 188–189°C; IR (KBr, cm ) νmax: 3254,
3096, 2966, 2786, 1698, 1661, 1562, 1467, 1418, 1349, 1268,
4.2.3. N-(2-isobutyl-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-5-yl)propenamide (11c)
1
1232, 1131, 787; H NMR (300 MHz, CDCl ) δ: 8.77 (d, 1H, J =
3
Yield: 76%; slightly yellowish amorphous solid (crystallized
-
1.9 Hz), 8.37 (dd, 1H, J = 7.1, 1.0 Hz), 8.37 (br s, 1H, NH), 8.10
(d, 1H, J = 1.9 Hz), 7.99 (dd, 1H, J = 8.3, 1.0 Hz), 7.66 (dd, 1H,
1
from chloroform/hexane) mp 213–214°C; IR (KBr, cm ) νmax
:
316, 2959, 1702, 1661, 1621, 1562, 1467, 1418, 1371, 1337,
3
1
1
1
J = 8.3, 7.1 Hz), 6.53 (dd, 1H, J = 16.9, 1.6 Hz, CH=CH
(dd, 1H J = 16.9, 10.1 Hz, CH=CH ), 5.87 (dd, 1H, J = 10.1, 1.6
Hz, CH=CH ), 4.37 (t, 2H, J = 6.6 Hz, CH CH N(CH CH ),
2.94 (t, 2H, J = 6.6 Hz, CH CH N(CH CH ), 2.80‒2.71 (m, 4H,
CH CH N(CH CH ), 1.86–1.79 (m, 4H, CH CH N(CH CH );
C NMR (75.5 MHz, CDCl ) δ: 164.25 (s, C(O)), 164.20 (s,
2
), 6.36
1
264, 1227, 888, 787; H NMR (300 MHz, CDCl
3
) δ: 9.06 (d,
,
2
H, J = 1.8 Hz), 8.49 (dd, 1H, J = 7.2, 1.1 Hz), 8.31 (d, 1H, J =
.8 Hz), 8.17 (dd, 1H, J = 8.1, 1.1 Hz), 7.89 (br s, 1H, NH), 7.72
2
2
2
2
2 2
)
2
2
2
2 2
)
(
dd, 1H, J = 8.1, 7.2 Hz), 6.55 (dd, 1H, J = 16.9, 1.2 Hz,
CH=CH ), 6.38 (dd, 1H J = 16.9, 10.1 Hz, CH=CH ), 5.89 (dd,
H, J = 10.1, 1.2 Hz, CH=CH ), 4.04 (d, 2H, J = 7.3 Hz,
CH CH(CH ), 2.29–2.16 (m, 1H, CH CH(CH ), 0.97 (d, 6H, J
6.5 Hz, CH ); C NMR (75.5 MHz, CDCl ) δ:
2
2
2
2
)
2
2
2
2
2 2
)
1
3
2
,
2
3
1
2
C(O)), 163.60 (s, C(O)), 136.68, 133.83, 132.24, 130.87, 129.87,
128.84, 127.60, 124.70, 123.70, 122.95, 122.11, 121.95, 54.87 (s,
2
3
)
2
2
3 2
)
13
=
2
CH(CH
3
)
2
3
CH
2
CH
2
N(CH
2
CH
2
)
2
and CH
), 23.91 (s, CH
: C, 69.41; H, 5.82; N, 11.56; found: C,
69.53; H, 5.74; N, 11.67.
2
CH
2
N(CH
2
CH
2
)
2
), 39.38 (s,
1
1
1
64.93 (s, C(O)), 164.66 (s, C(O)), 164.47 (s, C(O)), 137.36,
34.02, 132.51, 130.84, 130.03, 128.37, 127.46, 124.94, 124.45,
CH
2
CH
2
N(CH
2
CH
2
)
2
2
CH N(CH
2
2
CH ). Anal.
2 2
)
21 3 3
Calcd. for C21H N O
22.79, 122.71, 122.07, 47.43 (s, CH
CH(CH ), 20.42 (s, CH CH(CH
: C, 70.79; H, 5.63; N, 8.69; found: C, 70.63; H, 5.84;
2
3 2
CH(CH ) ), 27.57 (s,
CH
2
3
)
2
2
3 2
) ). Anal. Calcd. for
4.3. General procedure for the preparation of isoxazolidines
trans-9a–f and cis-9a–f
19 18 2 3
C H N O
N, 8.44.
A mixture of the nitrone 8 (1.00 mmol), acrylamide 11a–f
1.00 mmol) and toluene or a toluene-chloroform mixture (2 mL,
:1, v/v) was stirred at 70°C for 24 h or until disappearance of the
4
.2.4. N-(2-dimethylamino)ethyl-1,3-dioxo-2,3-
(
1
dihydro-1H-benzo[de]isoquinolin-5-
yl)propenamide (11d)
starting nitrone. After evaporation of solvents under reduced
pressure the crude products were purified by silica gel
chromatography with chloroform:methanol mixtures.
Yield: 30%; yellowish amorphous solid (crystallized from
1
-
chloroform/hexane) mp 208–210°C; IR (KBr, cm ) νmax: 3275,
3
7
1
8
1
096, 2774, 1660, 1623, 1561, 1467, 1415, 1267, 1231, 1138,
1
86; H NMR (300 MHz, CDCl
3
) δ: 8.67 (br s, 1H, NH), 8.64 (s,
4.3.1. Diethyl cis-5-(N-propylnaphthalimide-3-
ylcarbamoyl)-2-methylisoxazolidin-3-yl-3-
phosphonate (cis-9a)
H), 8.33 (dd, 1H, J = 7.3, 1.0 Hz), 7.99 (s, 1H), 7.90 (d, 1H, J =
.2 Hz), 7.63 (dd, 1H, J = 8.2, 7.3 Hz), 6.52 (dd, 1H, J = 16.9,
-
1
.6 Hz, CH=CH
dd, 1H, J = 9.9, 1.6 Hz, CH=CH
CH CH N(CH ), 2.86 (t, 2H, J = 6.1 Hz, CH
.49 (s, 6H, CH
2
), 6.38 (dd, 1H
,
J = 16.9, 9.9 Hz, CH=CH
), 4.35 (t, 2H, J = 6.1 Hz,
CH N(CH ),
); C NMR (75.5 MHz, CDCl ) δ:
2
), 5.86
max
Yellow oil; IR (film, cm ) ν : 3317, 2968, 2934, 1696,
1
1661, 1542, 1430, 1339, 1234, 1052, 1025, 972; H NMR (300
(
2
2
2
3
)
2
2
2
3 2
)
MHz, CDCl ) δ: 9.31 (br s, 1H, NH), 8.94 (d, 1H, J = 2.1 Hz),
3
13
2
1
1
1
2
CH
2
3
N(CH )
2
3
8.50 (dd, 1H, J = 7.3, 1.0 Hz), 8.41 (d, 1H, J = 2.1 Hz), 8.18 (dd,
1H, J = 8.1, 1.0 Hz), 7.73 (dd, 1H, J = 8.1, 7.3 Hz), 4.70 (dd, 1H,
64.37 (s, C(O)), 163.98 (s, C(O)), 163.35 (s, C(O)), 136.76,
33.62, 131.85, 130.95, 129.50, 128.43, 127.28, 124.17, 123.55,
J = 8.7, 5.0 Hz, HC5), 4.24–4.05 (m, 6H, 2 × CH OP and
2
22.33, 121.67, 121.67, 57.85 (s, CH
CH N(CH ), 37.89 (s, CH CH N(CH
: C, 67.64; H, 5.68; N, 12.46; found: C, 67.60; H,
2
CH
2
N(CH
3
)
2
), 46.05 (s,
CH CH CH ), 3.20–2.98 (m, 2H, HC3 and H C4), 3.02 (s, 3H,
β
2
2
3
CH
2
2
3
)
2
2
2
3
)
2
). Anal. Calcd. for
CH N), 2.94–2.81 (m, 1H, H C4), 1.80–1.70 (m, 2H,
3
α
19 19 3 3
C H N O
CH
2
CH CH
2
3
3 2
), 1.30 (t, 3H, J = 7.0 Hz, CH CH OP), 1.21 (t, 3H, J
13
5
.43; N, 12.16.
3 2 2 2 3
= 7.0 Hz, CH CH OP), 1.02 (t, 3H, J = 7.4 Hz, CH CH CH ); C
NMR (151.0 MHz, CDCl
C(O)), 163.81 (s, C(O)), 136.32, 133.63, 132.55, 129.90, 127.49,
25.22, 124.20, 123.54, 122.55, 121.89, 76.02 (d, J = 6.8 Hz,
C5), 63.72 (d, J = 169.4 Hz, C3), 63.07 (d, J = 7.1 Hz, CH OP),
62.02 (d, J = 6.8 Hz, CH OP), 46.04 (d, J = 5.8 Hz, CH N), 41.99
(s, CH CH CH ), 36.41 (s, C4), 21.39 (s, CH CH CH ), 16.43 (d,
J = 6.0 Hz, CH CH OP), 16.36 (d, J = 5.6 Hz, CH CH OP),
1.49 (s, CH CH CH ) δ: 21.31.
Anal. Calcd. for C24 P: C, 57.25; H, 6.01; N, 8.35; found:
C, 57.32; H, 5.92; N, 8.48.
3
) δ: 170.81 (s, C(O)NH), 164.11 (s,
4.2.5. N-(2-diethylamino)ethyl-1,3-dioxo-2, 3-
dihydro-1H-benzo[de]isoquinolin-5-
yl)propenamide (11e)
1
2
Yield: 64%; yellowish amorphous solid (crystallized from
-
1
2
3
chloroform/hexane) mp 196–197°C; IR (KBr, cm ) νmax: 3278,
1
2
2
3
2
2
3
2
969, 1700, 1662, 1624, 1542, 1466, 1341, 1263, 1227, 784; H
NMR (300 MHz, CDCl ) δ: 8.96 (d, 1H, J = 2.1 Hz), 8.42 (dd,
H, J = 7.2, 1.1 Hz), 8.41 (br s, 1H, NH), 8.30 (d, 1H, J = 2.1
Hz), 8.08 (dd, 1H, J = 8.2, 1.1 Hz), 7.67 (dd, 1H, J = 8.2, 7.2
Hz), 6.54 (dd, 1H, J = 16.9, 1.4 Hz, CH=CH ), 6.40 (dd, 1H J =
6.9, 10.0 Hz, CH=CH ), 5.86 (dd, 1H, J = 10.0, 1.4 Hz,
CH=CH ), 4.27–4.22 (m, 2H, CH CH N(CH CH ), 2.78–2.73
m, 2H, CH CH N(CH CH ), 2.64 (q, 4H, J = 7.1 Hz,
), 1.05 (t, 6H, 7.1 Hz,
) δ: 164.49
3
2
3
2
3
31
1
2
2
3 3
); P NMR (121.5 MHz, CDCl
1
30 3 7
H N O
2
,
1
2
4.3.2. Diethyl trans-5-(N-propylnaphtalimide-3-
ylcarbamoyl)-2-methylisoxazolidin-3-yl-3-
phosphonate (trans-9a)
2
2
2
2
3 2
)
(
2
2
2
3 2
)
CH
CH
2
CH
CH
2
N(CH
N(CH
2
CH
3
)
)
2
J
=
Yellowish
amorphous
solid
(crystallized
-1
from
1
3
2
2
2
CH
3
2
); C NMR (75.5 MHz, CDCl
3
max
chloroform/hexane) mp 175–176°C; IR (KBr, cm ) ν : 3241,
1
3067, 2967, 1697, 1664, 1567, 1339, 1266, 1214, 1049, 1018; H
(
s, C(O)), 163.84 (s, C(O)), 163.77 (s, C(O)), 136.85, 133.81,

NMR (300 MHz, CDCl
3
) δ: 8.93 (d, 1H, J = 2.2 Hz), 8.57 (br s,
H, NH), 8.51 (dd, 1H, J = 7.2, 1.1 Hz), 8.33 (d, 1H, J = 2.2 Hz),
CH
CH
Hz, CH
CH CH
Calcd. for C25
58.13; H, 6.20; N, 7.92.
3
N), 40.37 (s, CH
CH CH CH ), 20.51 (s, CH
CH OP), 16.64 (d, J = 5.1 Hz, CH
CH
2
CH
2
CH
2
CH
3
), 36.56 (s, C4), 30.31 (s,
CH CH ), 16.74 (d, J = 5.1
CH OP), 14.02 (s,
); P NMR (121.5 MHz, CDCl ) δ: 20.55. Anal.
P: C, 58.02; H, 6.23; N, 8.12; found: C,
1
8
4
2
2
2
3
2
CH
2
2
3
.18 (dd, 1H, J = 8.3, 1.1 Hz), 7.74 (dd, 1H, J = 8.3, 7.2 Hz),
.69 (dd, 1H, J = 8.7, 5.8 Hz, HC5), 4.29–4.14 (m, 4H, 2 ×
3
2
3
2
3
1
2
2
2
CH
3
3
CH
2
OP), 4.15 (t, 2H, J = 7.5 Hz, CH
H, HC3), 3.08 (s, 3H, CH N), 3.07 (dddd, 1H, J = 16.1, 12.8,
.7, 8.7 Hz, H C4), 2.88 (dddd, 1H, J = 12.8, 9.9, 8.2, 5.8 Hz,
C4), 1.80‒1.72 (m, 2H, CH CH CH ), 1.39 (t, 3H, J = 7.0 Hz,
CH CH OP), 1.37 (t, 3H, J = 7.0 Hz, CH CH OP), 1.02 (t, 3H, J
7.4 Hz, CH ); C NMR (75.5 MHz, CDCl ) δ: 169.45
s, C(O)NH), 163.72 (s, C(O)), 163.37 (s, C(O)), 135.71, 133.47,
2
CH
2
CH
3
), 3.19–3.06 (m,
H N
32 3
O
7
1
8
3
β
4.3.5. Diethyl cis-5-(N-isobutylnaphthalimide-3-
ylcarbamoyl)-2-methylisoxazolidin-3-yl-3-
phosphonate (cis-9c)
H
α
2
2
3
3
2
3
2
1
3
=
2
CH
2
CH
3
3
-1
Yellow oil; IR (film, cm ) νmax: 2963, 1701, 1662, 1543,
(
1
1
9
7
3
337, 1234, 1054, 1027, 755; H NMR (300 MHz, CDCl ) δ:
.30 (br s, 1H, NH), 8.95 (d, 1H, J = 2.2 Hz), 8.50 (dd, 1H, J =
.3, 1.1 Hz), 8.40 (d, 1H, J = 2.2 Hz), 8.18 (dd, 1H, J = 8.3, 1.1
1
7
32.18, 129.89, 127.46, 125.00, 124.00, 123.25, 122.23, 122.12,
6.58 (d, J = 8.6 Hz, C5), 63.57 (d, J = 164.3 Hz, C3), 63.41 (d, J
=
6.3 Hz, CH
CH N), 42.03 (s, CH
CH CH CH ), 16.71 (d, J = 5.1 Hz, CH
.1 Hz, CH CH OP), 11.67 (s, CH CH
MHz, CDCl ) δ: 20.52. Anal. Calcd. for C24
H, 6.01; N, 8.35; found: C, 57.34; H, 5.92; N, 8.58.
2
OP), 62.84 (d, J = 6.9 Hz, CH
CH CH ), 36.52 (s, C4), 21.48 (s,
CH OP), 16.61 (d, J =
CH
2
OP), 46.84 (s,
Hz), 7.73 (dd, 1H, J = 8.3, 7.3 Hz), 4.69 (dd, 1H, J = 8.3, 4.6 Hz,
HC5), 4.24–4.02 (m, 4H, 2 × CH OP), 4.05 (d, 2H, J = 7.3 Hz,
CH CH(CH ), 3.16–3.01 (m, 2H, HC3 and H C4), 3.01 (d, 3H,
J = 1.0 Hz, CH N), 2.93–2.83 (m, 1H, H C4), 2.28‒2.19 (m, 1H,
CH CH(CH ), 1.30 (t, 3H, J = 7.0 Hz, CH CH OP), 1.21 (t, 3H,
J = 7.1 Hz, CH CH OP), 0.99 (d, 6H, J = 6.7 Hz, CH CH(CH );
C NMR (151.0 MHz, CDCl ) δ: 170.80 (s, C(O)NH), 164.37 (s,
3
2
2
3
2
2
2
3
3
2
3
1
2
3
)
2
β
5
3
2
2
2
3
); P NMR (121.5
30 3 7
H N O P: C, 57.25;
3
α
3
2
3
)
2
3
2
3
2
2
3 2
)
1
3
4.3.3. Diethyl cis-5-(N-butylnaphthalimide-3-
ylcarbamoyl)-2-methylisoxazolidin-3-yl-3-
phosphonate (cis-9b)
3
C(O)), 164.08 (s, C(O)), 136.33, 133.59, 132.52, 129.95, 127.48,
125.23, 124.29, 123.46, 122.48, 121.86, 76.01 (d, J = 6.8 Hz,
-1
Yellow oil; IR (film, cm ) νmax: 2962, 2932, 1697, 1662,
543, 1466, 1430, 1340, 1233, 1053, 1026, 753; H NMR (300
C5), 63.70 (d, J = 166.5 Hz, C3), 63.06 (d, J = 6.5 Hz, CH
63.02 (d, J = 6.8 Hz, CH OP), 47.21 (s, CH CH(CH ), 46.01 (d,
J = 5.7 Hz, CH N), 36.39 (s, C4), 27.39 (s, CH CH(CH ), 20.28
(s, CH CH(CH ), 16.42 (d, J = 5.6 Hz, CH CH OP), 16.34 (d, J
= 5.6 Hz, CH CH OP); P NMR (121.5 MHz, CDCl ) δ: 21.36.
Anal. Calcd. for C25 P: C, 58.02; H, 6.23; N, 8.12; found:
C, 58.15; H, 6.25; N, 7.87.
2
OP),
1
1
2
2
3 2
)
MHz, CDCl
3
) δ: 9.30 (br s, 1H, NH), 8.94 (d, 1H, J = 2.1 Hz),
.50 (dd, 1H, J = 7.3, 1.2 Hz), 8.40 (d, 1H, J = 2.1 Hz), 8.17 (dd,
H, J = 8.3, 1.2 Hz), 7.72 (dd, 1H, J = 8.3, 7.3 Hz), 4.71–4.67
OP), 4.16 (t, 2H, J =
), 3.14–3.05 (m, 2H, HC3 and H C4),
.01 (d, 3H, J = 0.6 Hz, CH N), 2.87–2.78 (m, 1H, H C4), 1.77–
.67 (m, 2H, CH CH CH CH ), 1.51–1.39 (m, 2H,
), 1.30 (t, 3H, J = 7.1 Hz, CH CH OP), 1.21 (t,
CH OP), 0.98 (t, 3H, J = 7.3 Hz,
); C NMR (151.0 MHz, CDCl ) δ: 170.80 (s,
3
2
3 2
)
8
1
2
3
)
2
3
2
3
1
3
2
3
(
m, 1H, HC5), 4.24–4.04 (m, 4H, 2 × CH
2
32 3 7
H N O
7
3
1
CH
3
.4 Hz, CH
2
CH
2
CH
2
CH
3
β
3
α
4
3
.3.6. Diethyl trans-5-(N-isobutylnaphthalimide-
-ylcarbamoyl)-2-methylisoxazolidin-3-yl-3-
phosphonate (trans-9c)
Yellowish amorphous
chloroform/hexane) mp 165–166°C; IR (KBr, cm ) νmax: 3273,
2
2
2
3
2
CH
2
CH
2
CH
3
3
2
H, J = 7.0 Hz, CH
3
2
1
3
solid
(crystallized
from
CH
2
CH
2
CH
2
CH
3
3
-
1
C(O)NH), 164.09 (s, C(O)), 163.78 (s, C(O)), 136.31, 133.62,
3
243, 2986, 2955, 1699, 1661, 1564, 1337, 1228, 1052, 1034,
1
1
7
32.53, 129.87, 127.48, 125.19, 124.18, 123.52, 122.54, 121.87,
6.01 (d, J = 6.8 Hz, C5), 63.70 (d, J = 169.6 Hz, C3), 63.07 (d, J
9
Hz), 8.57 (br s, 1H, NH), 8.51 (dd, 1H, J = 7.3, 1.0 Hz), 8.33 (d,
73, 783; H NMR (300 MHz, CDCl ) δ: 8.93 (d, 1H, J = 2.2
3
=
6.3 Hz, CH
.8 Hz, CH N), 40.28 (s, CH
s, CH CH CH CH ), 20.35 (s, CH
.8 Hz, CH CH OP), 16.35 (d, J = 5.9 Hz, CH
CH CH CH
Calcd. for C25
2
OP), 62.03 (d, J = 6.4 Hz, CH
CH N(CH ), 36.39 (s, C4), 30.21
CH CH CH ), 16.43 (d, J =
CH OP), 13.81 (s,
); P NMR (121.5 MHz, CDCl ) δ: 21.37. Anal.
P: C, 58.02; H, 6.23; N, 8.12; found: C,
8.30; H, 6.23; N, 8.00.
2
OP), 46.03 (d, J =
1
8
4
H, J = 2.2 Hz), 8.17 (dd, 1H, J = 8.3, 1.2 Hz), 7.74 (dd, 1H, J =
.3, 7.3 Hz), 4.69 (dd, 1H, J = 8.7, 5.7 Hz, HC5), 4.29–4.18 (m,
5
(
5
CH
3
2
2
3 2
)
2
2
2
3
2
2
2
3
H, 2 × CH
2
OP), 4.04 (d, 2H, J = 7.5 Hz, CH
N), 3.06 (dddd, 1H, J = 15.7, 12.8,
C4), 2.87 (dddd, 1H, J = 12.8, 10.3, 8.1, 5.7 Hz,
C4), 2.28–2.19 (m, 1H, CH CH(CH ), 1.38 (t, 3H, J = 7.1
Hz, CH CH OP), 1.37 (t, 3H, J = 7.1 Hz, CH CH OP), 0.98 (d,
6H, J = 6.5 Hz, CH CH(CH ) ); C NMR (75.5 MHz, CDCl ) δ:
2 3 2
CH(CH ) ), 3.16–
3
2
3
2
3
1
3.05 (m, 1H), 3.07 (s, 3H, CH
.7, 8.7 Hz, H
3
2
2
2
3
3
8
H
β
32 3 7
H N O
α
2
3 2
)
5
3
2
3
2
1
3
4.3.4. Diethyl trans-5-(N-butylnaphthalimide-3-
ylcarbamoyl)-2-methylisoxazolidin-3-yl-3-
phosphonate (trans-9b)
2
3
2
3
169.47 (s, C(O)NH), 164.21 (s, C(O)), 163.90 (s, C(O)), 135.69,
133.65, 132.43, 130.23, 127.68, 125.34, 124.13, 123.53, 122.46,
122.26, 76.53 (d, J = 8.9 Hz, C5), 63.68 (d, J = 166.9 Hz, C3),
Yellowish
amorphous
solid
(crystallized
1
from
-
chloroform/hexane) mp 157–158°C; IR (KBr, cm ) νmax: 3278,
63.54 (d, J = 6.6 Hz, CH
47.38 (s, CH CH(CH ), 46.93 (br s, CH
27.60 (s, CH CH(CH ), 20.52 (s, CH CH(CH
5.1 Hz, CH CH OP), 16.71 (d, J = 5.4 Hz, CH
NMR (121.5 MHz, CDCl δ: 20.53. Anal. Calcd. for
P: C, 58.02; H, 6.23; N, 8.12; found: C, 58.12; H,
5.99; N, 7.88.
2
OP), 62.89 (d, J = 6.9 Hz, CH
N), 36.72 (s, C4),
), 16.78 (d, J =
2
OP),
1
2
(
958, 1698, 1661, 1565, 1341, 1229, 1052, 1033, 970; H NMR
300 MHz, CDCl ) δ: 8.93 (d, 1H, J = 2.2 Hz), 8.57 (br s, 1H,
NH), 8.51 (dd, 1H, J = 7.3, 1.2 Hz), 8.33 (d, 1H, J = 2.2 Hz),
2
3
)
2
3
3
2
)
3 2
2
3 2
)
3
1
3
2
3
2
CH OP);
P
8
4
.18 (dd, 1H, J = 8.3, 1.2 Hz), 7.74 (dd, 1H, J = 8.3, 7.3 Hz),
.69 (dd, 1H, J = 8.5, 5.6 Hz, HC5), 4.29–4.16 (m, 4H, 2 ×
3
)
25 32 3 7
C H N O
CH
m, 1H, HC3), 3.07 (s, 3H, CH
2.7, 8.5, 8.5 Hz, H C4), 2.88 (dddd, 1H, J = 12.7, 10.1, 8.1, 5.6
Hz, H C4), 1.76‒1.66 (m, 2H, CH CH CH CH ), 1.51‒1.39 (m,
H, CH CH CH CH ), 1.38 (t, 3H, J = 7.1 Hz, CH CH OP), 1.37
t, 3H, J = 7.1 Hz, CH CH OP), 0.98 (t, 3H, J = 7.3 Hz,
); C NMR (75.5 MHz, CDCl ) δ: 169.47 (s,
C(O)NH), 163.79 (s, C(O)), 163.45 (s, C(O)), 135.70, 133.55,
2
OP), 4.18 (t, 2H, J = 7.4 Hz, CH
2 2 2 3
CH CH CH ), 3.18–3.05
(
3
N), 3.07 (dddd, 1H, J = 15.9,
4
.3.7. Diethyl cis-5-[N-(2-
1
β
dimethylamino)ethylnaphthalimide-3-
ylcarbamoyl]-2-methylisoxazolidin-3-yl-3-
phosphonate (cis-9d)
α
2
2
2
3
2
2
2
2
3
3
2
(
3
2
-1
13
Yellow amorphous solid; mp 119–120°C; IR (KBr, cm ) νmax
274, 2980, 2776, 1695, 1662, 1544, 1465, 1430, 1341, 1237,
026, 971, 751; H NMR (300 MHz, CDCl ) δ: 9.29 (br s, 1H,
:
CH
2
CH
2
CH
2
CH
3
3
3
1
1
3
1
7
32.26, 129.98, 127.54, 125.10, 124.01, 123.36, 122.33, 122.17,
6.59 (d, J = 8.9 Hz, C5), 63.59 (d, J = 167.2 Hz, C3), 63.47 (d, J
NH), 8.95 (d, 1H, J = 2.2 Hz), 8.50 (dd, 1H, J = 7.4, 1.2 Hz),
8
.41 (d, 1H, J = 2.2 Hz), 8.18 (d, 1H, J = 8.3 Hz), 7.72 (dd, 1H, J
=
2 2
6.6 Hz, CH OP), 62.87 (d, J = 6.9 Hz, CH OP), 46.86 (s,

=
=
3
2
2
8.3, 7.4 Hz), 4.69 (dd, 1H, J = 8.9, 4.8 Hz, HC5), 4.34 (t, 2H, J
6.9 Hz, CH CH N(CH ), 4.23–4.05 (m, 4H, 2 × CH OP),
.18–3.00 (m, 2H, HC3 and H C4), 3.01 (s, 3H, CH N), 2.87–
.78 (m, 1H, H C4), 2.69 (t, 2H, J = 6.9 Hz, CH CH N(CH ),
.38 (s, 6H, CH CH N(CH ), 1.30 (t, 3H, J = 7.0 Hz,
CH OP), 1.20 (t, 3H, J = 7.0 Hz, CH
151.0 MHz, CDCl ) δ: 170.83 (s, C(O)NH), 164.13 (s, C(O)),
CH
CH
3
CH
CH
2
OP), 16.36 (d, J = 6.0 Hz, CH
3
CH
); P NMR (121.5 MHz, CDCl
37 4 7
H N O P: C, 57.85; H, 6.65; N, 9.99; found:
2
OP), 12.06 (s,
31
2
2
3
)
2
2
2
2
N(CH
2
CH
3
)
2
3
) δ: 21.31.
β
3
Anal. Calcd. for C27
C, 57.67; H, 6.62; N, 9.85.
α
2
2
3 2
)
2
2
3 2
)
1
3
4.3.10. Diethyl trans-5-[N-(2-
diethylamino)ethylnaphthalimide-3-
ylcarbamoyl]-2-methylisoxazolidin-3-yl-3-
phosphonate (trans-9e)
-1
CH
(
3
2
3 2
CH OP); C NMR
3
1
1
63.83 (s, C(O)), 136.33, 133.73, 132.56, 129.98, 127.49,
25.26, 124.25, 123.45, 122.46, 121.93, 76.00 (d, J = 6.8 Hz,
Yellowish oil; IR (film, cm ) νmax: 3249, 2974, 2933, 1698,
662, 1563, 1466, 1430, 1341, 1235, 1054, 1027, 972, 755; H
C5), 63.71 (d, J = 169.5 Hz, C3), 63.07 (d, J = 6.7 Hz, CH
3.02 (d, J = 7.0 Hz, CH OP), 56.95 (s, CH CH N(CH ), 46.03
d, J = 5.9 Hz, CH N), 45.67 (s, CH CH N(CH ), 38.15 (s,
), 36.41 (s, C4), 16.43 (d, J = 5.6 Hz,
2
OP),
1
1
6
2
2
2
3 2
)
NMR (600 MHz, CDCl
H, NH), 8.51 (dd, 1H, J = 7.3, 0.8 Hz), 8.35 (d, 1H, J = 2.2 Hz),
8.18 (d, 1H, J = 8.0 Hz), 7.74 (dd, 1H, J = 8.0, 7.3 Hz), 4.71 (dd,
H, J = 8.8, 5.6 Hz, HC5), 4.30 (t, 2H, J = 7.5 Hz,
CH CH N(CH CH ), 4.28–4.19 (m, 4H, 2 × CH OP), 3.18–3.11
m, 1H, HC3), 3.09 (s, 3H, CH N), 3.08 (dddd, 1H, J = 16.0,
3
) δ: 8.93 (d, 1H, J = 2.2 Hz), 8.64 (br s,
(
3
2
2
3 2
)
1
CH
2
CH
2
N(CH
OP), 16.36 (d, J = 5.9 Hz, CH
) δ: 21.32. Anal. Calcd. for C25
H, 6.25; N, 10.52; found: C, 56.20; H, 6.49; N, 10.39.
3 2
)
3
1
CH
3
CH
2
3
CH
2
OP); P NMR (121.5
P: C, 56.39;
1
MHz, CDCl
3
33 4 7
H N O
2
2
2
3
)
2
2
(
3
4
.3.8. Diethyl trans-5-[N-(2-
dimethylamino)ethylnaphthalimide-3-
ylcarbamoyl]-2-methylisoxazolidin-3-yl-3-
phosphonate (trans-9d)
12.7, 8.8, 8.8 Hz, H C4), 2.89 (dddd, 1H, J = 12.7, 9.5, 8.3, 5.6
β
α
2
2
2
3 2
Hz, H C4), 2.80 (t, 2H, J = 7.5 Hz, CH CH N(CH CH ) ), 2.68
(q, 4H, J = 7.1 Hz, CH CH N(CH CH ) ), 1.40 (t, 3H, J = 7.0 Hz,
2
2
2
3 2
CH CH OP), 1.38 (t, 3H, J = 7.0 Hz, CH
= 7.1 Hz, CH
3
2
3
CH
2
OP), 1.10 (t, 6H, J
) δ:
-
1
13
); C NMR (75.5 MHz, CDCl
Yellowish oil; IR (film, cm ) νmax: 3248, 3092, 2943, 2776,
700, 1662, 1562, 1465, 1430, 1340, 1236, 1052, 1025, 970, 751;
2
CH
2
N(CH
2
CH
3
)
2
3
1
169.46 (s, C(O)NH), 163.81 (s, C(O)), 163.47 (s, C(O)), 135.71,
133.68, 132.34, 130.04, 127.59, 125.16, 124.01, 123.35, 122.32,
122.23, 76.57 (d, J = 7.7 Hz, C5), 63.64 (d, J = 167.8 Hz, C3),
1
H NMR (300 MHz, CDCl
s, 1H, NH), 8.50 (d, 1H, J = 7.1 Hz), 8.32 (d, 1H, J = 2.1 Hz),
3
) δ: 8.91 (d, 1H, J = 2.1 Hz), 8.61 (br
8
1
.16 (d, 1H, J = 8.5 Hz), 7.73 (dd, 1H, J = 8.5, 7.1 Hz), 4.69 (dd,
H, J = 8.7, 5.8 Hz, HC5), 4.33 (t, 2H, J = 6.8 Hz,
63.48 (d, J = 6.6 Hz, CH
2
OP), 62.86 (d, J = 6.9 Hz, CH
CH ), 47.72 (s, CH CH N(CH CH
N), 38.16 (s, CH CH N(CH CH ), 36.63 (s, C4),
CH OP), 16.65 (d, J = 5.4 Hz,
CH N(CH
) δ: 20.17. Anal. Calcd. for C27
H, 6.65; N, 9.99; found: C, 57.87; H, 6.71; N, 9.91.
2
OP),
49.87 (s, CH
46.96 (s, CH
2
CH
2
N(CH
2
3
)
2
2
2
2
3
)
2
),
CH
2
CH
H, HC3), 3.07 (s, 3H, CH
.7, 8.7 Hz, H C4), 2.88 (dddd, 1H, J = 12.8, 9.6, 8.2, 5.8 Hz,
C4), 2.66 (t, 2H, J = 6.8 Hz, CH CH N(CH ), 2.36 (s, 6H,
CH CH N(CH ), 1.38 (t, 3H, J = 7.1 Hz, CH CH OP), 1.37 (t,
H, J = 7.0 Hz, CH OP); C NMR (75.5 MHz, CDCl ) δ:
2
3
N(CH )
2
), 4.29–4.16 (m, 4H, 2 × CH
2
OP), 3.18–3.05 (m,
3
2
2
2
3 2
)
1
8
3
N), 3.06 (dddd, 1H, J = 16.0, 12.8,
16.76 (d, J = 4.9 Hz, CH
CH CH OP), 12.36 (s, CH
MHz, CDCl
3
2
31
β
3
2
2
2
2
CH
3
)
2
); P NMR (243.0
H
α
2
2
3
)
2
3
H N O P: C, 57.85;
37 4 7
2
2
3
)
2
3
2
13
3
1
1
1
6
5
3
CH
2
3
4
.3.11. Diethyl cis-5-[N-(2-pirolidyn-1-
yl)ethylnaphthalimide-3-ylcarbamoyl]-2-
methylisoxazolidin-3-yl-3-phosphonate (cis-9f)
69.42 (s, C(O)NH), 163.92 (s, C(O)), 163.53 (s, C(O)), 135.71,
33.69, 132.30, 130.08, 127.58, 125.13, 123.93, 123.31, 122.28,
22.09, 76.57 (d, J = 8.9 Hz, C5), 63.65 (d, J = 170.9 Hz, C3),
-
1
Yellowish oil; IR (film, cm ) νmax: 3276, 2971, 2791, 1691,
662, 1543, 1430, 1338, 1234, 1052, 971, 785, 750; H NMR
3.50 (d, J = 6.6 Hz, CH
7.21 (s, CH CH N(CH
CH N(CH ), 38.33 (s, CH
6.79 (d, J = 5.1 Hz, CH CH
OP); P NMR (121.5 MHz, CDCl
Calcd. for C25 P: C, 56.39; H, 6.25; N, 10.52; found: C,
6.18; H, 6.48; N, 10.45.
2
OP), 62.86 (d, J = 6.9 Hz, CH
), 46.95 (s, CH N), 45.90 (s,
CH N(CH ), 36.57 (s, C4),
OP), 16.68 (d, J = 5.2 Hz,
) δ: 20.56. Anal.
2
OP),
1
1
2
2
3
)
2
3
3
(600 MHz, CDCl ) δ: 9.30 (br s, 1H, NH), 8.96 (d, 1H, J = 2.0
Hz), 8.52 (d, 1H, J = 7.2 Hz), 8.43 (d, 1H, J = 2.0 Hz), 8.19 (d,
CH
1
CH
2
2
3
)
2
2
2
3 2
)
3
2
3
1
1H, J = 8.1 Hz), 7.74 (dd, 1H, J = 8.1, 7.2 Hz), 4.71 (dd, 1H, J =
3
CH
2
3
9
.2, 5.0 Hz, HC5), 4.40 (t, 2H, J = 7.3 Hz, CH
.24–4.08 (m, 4H, 2 × CH OP), 3.15–3.08 (m, 2H, HC3 and
C4), 3.04 (s, 3H, CH N), 2.93–2.88 (m, 1H, H C4), 2.88–2.83
(m, 2H, CH CH N(CH CH ) ), 2.71 (br s, 4H,
CH CH N(CH CH ) ), 1.83 (br s, 4H, CH CH N(CH CH ) , 1.32
2 2 2 2 2
CH N(CH CH ) ),
33 4 7
H N O
4
H
2
5
β
3
α
4
.3.9. Diethyl cis-5-[N-(2-
2
2
2
2 2
diethylamino)ethylnaphthalimide-3-
ylcarbamoyl]-2-methylisoxazolidin-3-yl-3-
phosphonate (cis-9e)
2
2
2
2
2
2
2
2
2 2
(
CH
t, 3H, J = 7.0 Hz, CH
3
CH
2
OP), 1.23 (t, 3H, J = 7.0 Hz,
) δ: 170.84 (s,
13
3
CH
2
OP); C NMR (151.0 MHz, CDCl
3
-
1
Yellowish oil; IR (film, cm ) νmax: 3301, 2973, 2932, 1696,
662, 1544, 1466, 1430, 1342, 1235, 1026, 972, 785; H NMR
C(O)NH), 164.07 (s, C(O)), 163.75 (s, C(O)), 136.32, 133.72,
132.58, 129.95, 127.50, 125.26, 124.21, 123.50, 122.51, 121.92,
76.00 (d, J = 7.3 Hz, C5), 63.72 (d, J = 169.1 Hz, C3), 63.08 (d, J
= 6.7 Hz, CH OP), 62.99 (d, J = 6.8 Hz, CH OP), 54.35 (s,
1
1
(
300 MHz, CDCl
Hz), 8.49 (dd, 1H, J = 7.3, 1.2 Hz), 8.40 (d, 1H, J = 2.2 Hz), 8.18
d, 1H, J = 8.3 Hz), 7.72 (dd, 1H, J = 8.3, 7.3 Hz), 4.69 (dd, 1H,
8.7, 4.7 Hz, HC5), 4.31 (t, 2H, 7.1 Hz,
CH N(CH CH ), 4.23–4.04 (m, 4H, 2 × CH OP), 3.20–2.98
m, 2H, HC3 and H C4), 3.01 (s, 3H, CH N), 2.98–2.80 (m, 1H,
C4), 2.83 (t, 2H, J = 7.1 Hz, CH CH N(CH CH ), 2.69 (q,
CH N(CH CH ), 1.29 (t, 3H, J = 7.0 Hz,
OP), 1.20 (t, 3H, J = 7.0 Hz, CH CH OP), 1.11 (t, 6H, J
); C NMR (151.0 MHz, CDCl
δ: 170.86 (s, C(O)NH), 164.08 (s, C(O)), 163.78 (s, C(O)),
3
) δ: 9.29 (br s, 1H, NH), 8.95 (d, 1H, J = 2.2
2
2
(
2 2 2 2 2 2 2 2 2 2
CH CH N(CH CH ) ), 53.63 (s, CH CH N(CH CH ) ), 46.04 (d,
J
=
J
=
3 2 2 2 3 2
J = 5.7 Hz, CH N), 39.21 (s, CH CH N(CH CH ) ), 36.44 (s,
CH
2
2
2
3
)
2
2
2 2 2 2 2
C4), 23.64 (s, CH CH N(CH CH ) , 16.43 (d, J = 5.7 Hz,
31
CH CH OP), 16.36 (d, J = 5.6 Hz, CH CH OP); P NMR (243
3 2 3 2
(
β
3
H
α
2
2
2
3
)
2
MHz, CDCl ) δ: 20.86. Anal. Calcd. for C H N O P: C, 58.06;
3
27 35
4
7
4
H, J = 7.0 Hz, CH
CH CH
7.0 Hz, CH
2
2
2
3
)
2
H, 6.32; N, 10.03; found: C, 57.84; H, 6.39; N, 9.75.
3
2
3
2
1
3
4.3.12. Diethyl trans-5-[N-(2-pirolidyn-1-
yl)ethylnaphthalimide-3-ylcarbamoyl]-2-
methylisoxazolidin-3-yl-3-phosphonate (trans-9f)
=
2
CH
2
N(CH
2
CH
3
)
2
3
)
1
1
36.34, 133.77, 132.60, 129.95, 127.53, 125.23, 124.20, 123.44,
22.45, 121.98, 76.01 (d, J = 6.8 Hz, C5), 63.71 (d, J = 169.4 Hz,
-
1
Yellowish oil; IR (film, cm ) νmax: 3271, 2970, 1698, 1661,
563, 1465, 1430, 1337, 1233, 1027, 970, 784, 749; H NMR
1
1
C3), 63.08 (d, J = 6.7 Hz, CH
CH OP), 49.69 (s, CH CH
CH ), 46.04 (d, J = 5.8 Hz, CH
CH ), 36.45 (s, C4), 16.43 (d, J = 5.6 Hz,
2
OP), 63.01 (d, J = 6.8 Hz,
N(CH CH ), 47.67 (s,
N), 37.85 (s,
(
600 MHz, CDCl
NH), 8.53 (dd, 1H, J = 7.3, 0.9 Hz), 8.36 (d, 1H, J = 2.1 Hz),
.19 (d, 1H, J = 8.0 Hz), 7.76 (dd, 1H, J = 8.0, 7.3 Hz), 4.71 (dd,
3
) δ: 8.94 (d, 1H, J = 2.1 Hz), 8.60 (br s, 1H,
2
2
2
2
3 2
)
2
CH
CH
2
N(CH
2
CH
CH
3
)
)
2
3
8
2
2
N(CH
2
3
2

1
1
H, J = 8.9, 5.5 Hz, HC5), 4.39 (t, 2H, J = 7.3 Hz,
CH CH N(CH CH ), 4.29–4.19 (m, 4H, 2 × CH OP), 3.17–3.09
m, 1H, HC3), 3.10 (s, 3H, CH N), 3.08 (dddd, 1H, J = 16.1,
C4), 2.89 (dddd, 1H, J = 12.9, 9.4, 8.3, 5.5
C4), 2.85 (t, 2H, J = 7.3 Hz, CH CH N(CH CH ), 2.70
br s, 4H, CH CH N(CH CH ), 1.82 (br s, 4H,
CH CH N(CH CH ), 1.41 (t, 3H, J = 7.1 Hz, CH CH OP), 1.39
t, 3H, J = 7.1 Hz, CH OP); C NMR (151.0 MHz, CDCl
δ: 169.48 (s, C(O)NH), 163.88 (s, C(O)), 163.55 (s, C(O)),
3
and cis-10b); H NMR (300 MHz, CDCl ) δ: 9.32 (d, 1H, J = 2.2
2
2
2
2
)
2
2
Hz), 9.14 (d, 1H, J = 2.2 Hz), 8.79 (dd, 1H, J = 7.3, 1.2 Hz), 8.44
(dd, 1H, J = 8.2, 1.2 Hz), 7.95 (dd, 1H, J = 8.2, 7.3 Hz), 4.56 (dd,
(
3
1
2.9, 8.9, 8.9 Hz, H
β
1H, J = 12.5, 7.7 Hz, CH
4.0 Hz, HC5), 4.30 (dd, 1H, J = 12.5, 4.0 Hz, CH
(m, 4H, 2 × CH OP), 3.09 (ddd, 1H, J = 9.6, 7.2, 2.4 Hz, HC3),
2.88 (d, 3H, J = 1.0 Hz, CH N), 2.69 (dddd, 1H, J = 18.1, 12.5,
7.2, 7.2 Hz, H C4), 2.41 (dddd, 1H, J = 12.5, 11.9, 9.6, 7.2 Hz,
C4), 1.34 (t, 3H, J = 7.1 Hz, CH CH OP), 1.33 (t, 3H, J = 7.0
OP); C NMR (75.5 MHz, CDCl ) δ: 163.07 (s,
2
N), 4.47 (dddd, 1H, J = 7.7, 7.2, 7.2,
Hz, H
(
α
2
2
2
2
)
2
2
N), 4.25–4.12
2
2
2
2
)
2
2
2
2
2
2
)
2
3
2
3
1
3
(
3
CH
2
3
)
α
H
β
3
2
1
3
1
1
35.68, 133.64, 132.40, 130.09, 127.59, 125.27, 123.97, 123.49,
22.43, 122.20, 76.38 (d, J = 8.9 Hz, C5), 63.67 (d, J = 170.4 Hz,
Hz, CH
3
CH
2
3
C(O)), 162.50 (s, C(O)), 146.43, 135.66, 134.61, 131.04, 130.24,
129.09, 129.02, 124.55, 124.44, 123.07, 74.53 (d, J = 7.6 Hz,
C3), 63.35 (d, J = 6.5 Hz, CH
CH OP), 54.34 (s, CH CH
CH 46.72
CH 36.56
CH
2
OP), 62.69 (d, J = 6.9 Hz,
N(CH CH ), 53.64 (s,
(s, CH N),
(s, C4),
2
2
2
2
2
)
2
C5), 63.94 (d, J = 169.3 Hz, C3), 63.13 (d, J = 6.5 Hz, CH
62.42 (d, J = 6.7 Hz, CH OP), 46.46 (s, CH N), 43.05 (s, CH
36.34 (s, C4), 16.51 (d, J = 6.3 Hz, CH CH OP), 16.47 (d, J = 6.1
OP); P NMR (121.5 MHz, CDCl ) δ: 22.49. Anal.
Calcd. for C21 P: C, 52.83; H, 5.07; N, 8.80; found: C,
2
OP),
2
CH
CH
CH
2
2
2
N(CH
N(CH
N(CH
2
2
2
CH
CH
CH
2
3
2
)
)
)
2
),
),
3
39.27
23.61
(s,
(s,
2
3
2
N),
2
2
3
2
3
1
2
2
, 16.52 (d, J = 5.9 Hz, CH
3
CH
OP); P NMR (243.0 MHz, CDCl
0.13. Anal. Calcd. for C27 P: C, 58.06; H, 6.32; N,
0.03; found: C, 58.22; H, 6.28; N, 9.83.
2
OP), 16.45
Hz, CH
3
CH
2
3
3
1
(
d, J = 5.7 Hz, CH
3
CH
2
3
) δ:
24 3 8
H N O
2
1
H
35
N
4
O
7
52.73; H, 4.82; N, 8.52 (obtained on a 94:6 mixture of trans-10b
and cis-10b).
4
.4.3. Diethyl trans-5-(5-amino-1,3-dioxo-2,3-
dihydro-1H-benzo[de]izochinolin-2-ylmethyl)-2-
methylisoxazolidin-3-yl-3-phosphonate (trans-
0c)
Yellow
chloroform/hexane) mp 126–127°C; IR (KBr, cm ) νmax: 3431,
353, 1692, 1651, 1619, 1236, 1056, 1021, 969, 778; (signals of
trans-10c were extracted from the spectra of a 93:7 mixture of
4.4. General procedure for the preparation of isoxazolidines
trans-10a–d and cis-10a–d
1
amorphous
solid
(crystallized
from
A
mixture of the nitrone
8
(1.00 mmol), N-(2-
-
1
propenyl)naphthalimide 12a–d (1.00 mmol) and toluene or a
toluene–chloroform mixture (2 mL, 1:1, v/v) was stirred at 70°C
for 24 h or until disappearance of the starting nitrone. After
evaporation of solvents under reduced pressure the crude
products were purified by silica gel chromatography with
chloroform:methanol mixtures.
3
1
trans-10c and cis-10c); H NMR (300 MHz, CDCl
1
8
Hz), 4.53–4.42 (m, 2H, HC5 and CH
CH N), 4.23–4.11 (m, 4H, 2 × CH
7
1
11.8, 9.6, 7.2 Hz, H
1.32 (t, 3H, J = 7.1 Hz, CH
CDCl
3
) δ: 8.30 (dd,
H, J = 7.3, 1.0 Hz), 8.00 (d, 1H, J = 2.3 Hz), 7.93 (dd, 1H, J =
.2, 1.0 Hz), 7.59 (dd, 1H, J = 8.2, 7.3 Hz), 7.29 (d, 1H, J = 2.3
N), 4.31–4.25 (m, 1H,
OP), 3.09 (ddd, 1H, J = 9.6,
.2, 2.2 Hz, HC3), 2.89 (d, 3H, J = 0.8 Hz, CH N), 2.64 (dddd,
H, J = 18.2, 12.5, 7.2, 7.2 Hz, H C4), 2.43 (dddd, 1H, J = 12.5,
C4), 1.33 (t, 3H, J = 7.1 Hz, CH CH OP),
OP); C NMR (75.5 MHz,
) δ: 164.41 (s, C(O)), 164.12 (s, C(O)), 145.70, 133.25,
2
2
2
4
.4.1. Diethyl trans-5-(1, 3-dioxo-2,3-dihydro-1H-
benzo[de]izochinolin-2-ylmethyl)-2-
methylisoxazolidin-3-yl-3-phosphonate (trans-
0a)
White amorphous solid (crystallized from chloroform/hexane)
3
α
β
3
2
13
1
3 2
CH
3
-1
1
7
31.78, 127.23, 126.91, 123.04, 122.85, 122.07, 121.87, 113.86,
5.17 (d, J = 7.4 Hz, C5), 63.92 (d, J = 170.9 Hz, C3), 63.26 (d, J
mp 175–176°C; IR (KBr, cm ) νmax: 2981, 1702, 1662, 1591,
328, 1234, 1056, 1035, 973; (signals of trans-10a were
extracted from the spectra of a 93:7 mixture of trans-10a and cis-
1
=
CH N), 42.64 (s, CH N), 36.36 (s, C4), 16.77 (d, J = 5.7 Hz,
2 2
6.5 Hz, CH OP), 62.74 (d, J = 6.9 Hz, CH OP), 46.76 (br s,
1
3
2
1
0a); H NMR (300 MHz, CDCl
Hz), 8.23 (dd, 2H, J = 8.1, 1.1 Hz), 7.76 (dd, 2H, J = 8.1, 7.2
Hz), 4.56–4.44 (m, 2H, HC5 and CH N), 4.33–4.28 (m, 1H,
CH N), 4.22–4.11 (m, 4H, 2 × CH OP), 3.09 (ddd, 1H, J = 9.5,
.2, 2.3 Hz, HC3), 2.89 (d, 3H, J = 0.8 Hz, CH N), 2.65 (dddd,
H, J = 18.0, 12.5, 7.2, 7.2 Hz, H C4), 2.44 (dddd, 1H, J = 12.5,
1.6, 9.5, 6.9 Hz, H C4), 1.33 (t, 3H, J = 7.0 Hz, CH CH OP),
.32 (t, 3H, J = 7.0 Hz, CH OP); C NMR (75.5 MHz,
) δ: 164.16 (s, 2 × C(O)), 134.14, 131.60, 131.47, 128.21,
26.98, 122.50, 74.96 (d, J = 7.6 Hz, C5), 64.06 (d, J = 168.6 Hz,
C3), 63.34 (d, J = 6.6 Hz, CH OP), 62.53 (d, J = 6.9 Hz,
CH OP), 46.71 (br s, CH N), 42.77 (s, CH N), 36.55 (s, C4),
6.81 (d, J = 6.0 Hz, CH CH OP), 16.73 (d, J = 6.0 Hz,
OP); P NMR (121.5 MHz, CDCl ) δ: 22.67. Anal.
Calcd. for C21 P: C, 58.33; H, 5.83; N, 6.48; found: C,
8.28; H, 5.59; N, 6.61 (obtained on a 93:7 mixture of trans-10a
and cis-10a).
3
) δ: 8.61 (dd, 2H, J = 7.2, 1.1
3
1
CH
3
CH
2
OP), 16.70 (d, J = 5.7 Hz, CH
3
CH
2
OP); P NMR (121.5
P: C, 56.37;
H, 5.86; N, 9.39; found: C, 56.42; H, 5.88; N, 9.36 (obtained on a
3
26 3 6
MHz, CDCl ) δ: 22.73. Anal. Calcd. for C21H N O
2
2
2
9
3:7 mixture of trans-10c and cis-10c).
7
1
1
1
3
β
4
.4.4. Diethyl trans-5-(5-acetamido-1,3-dioxo-2,3-
α
3
2
dihydro-1H-benzo[de]izochinolin-2-ylmethyl)-2-
methylisoxazolidin-3-yl-3-phosphonate (trans-
10d)
13
3
CH
2
CDCl
1
3
Yellow
amorphous
solid
(crystallized
from
-
1
2
chloroform/hexane) mp 166–168°C; IR (KBr, cm ) νmax: 3299,
2
3
2
2
1
1
982, 1628, 1660, 1553, 1255, 1044, 973, 784; (signals of trans-
0d were extracted from the spectra of a 93:7 mixture of trans-
1
CH
3
2
3
1
3
CH
2
3
1
0d and cis-10d); H NMR (300 MHz, CDCl
3
) δ: 8.81 (d, 1H, J
25 2 6
H N O
=
2.0 Hz), 8.41 (d, 1H, J = 7.1 Hz), 8.35 (br s, NH), 8.22 (d, 1H,
J = 2.0 Hz), 8.07 (d, 1H, J = 8.1 Hz), 7.67 (dd, 1H, J = 8.1, 7.1
Hz), 4.55–4.46 (m, 2H, HC5 and CH N), 4.29–4.13 (m, 5H,
CH N and 2 × CH OP), 3.17 (ddd, 1H, J = 9.5, 7.0, 2.3 Hz,
HC3), 2.91 (s, 3H, CH N), 2.72 (dddd, 1H, J = 18.0, 12.7, 7.0,
7.0 Hz, H C4), 2.45 (dddd, 1H, J = 12.7, 11.9, 9.5, 7.0 Hz,
C4), 2.30 (s, 3H, CH C(O)), 1.34 (t, 3H, J = 7.1 Hz,
CH CH OP), 1.33 (t, 3H, J = 7.1 Hz, CH
(75.5 MHz, CDCl ) δ: 169.74 (s, C(O)NH), 163.98 (s, C(O)),
5
2
4
.4.2. Diethyl trans-5-(5-nitro-1,3-dioxo-2,3-
dihydro-1H-benzo[de]izochinolin-2-ylmethyl)-2-
methylisoxazolidin-3-yl-3-phosphonate (trans-
0b)
Grey amorphous solid (crystallized from chloroform/hexane)
2
2
3
α
1
H
β
3
1
3
3
2
3 2
CH OP); C NMR
-1
3
mp 197–198°C; IR (KBr, cm ) νmax: 2985, 1715, 1672, 1600,
541, 1343, 1326, 1233, 1033, 974, 799; (signals of trans-10b
were extracted from the spectra of a 94:6 mixture of trans-10b
1
1
63.47 (s, C(O)), 137.34, 133.80, 132.20, 129.62, 127.16,
24.42, 124.24, 122.37, 121.80, 121.66, 75.25 (d, J = 6.2 Hz,
1

C5), 63.93 (d, J = 169.4 Hz, C3), 63.34 (d, J = 6.7 Hz, CH
3.03 (d, J = 7.1 Hz, CH OP), 46.87 (br s, CH N), 42.70 (s,
CH N), 36.27 (s, C4), 24.69 (s, CH C(O)), 16.78 (d, J = 5.5 Hz,
CH CH OP), 16.71 (d, J = 5.6 Hz, CH
MHz, CDCl ) δ: 22.70. Anal. Calcd. for C23
H, 5.77; N, 8.59; found: C, 56.53; H, 5.64; N, 8.67 (obtained on a
2
OP),
03-/3-014-01/502-34-020). The biological part of this work was
supported by the KU Leuven (GOA 10/014).
6
2
3
2
3
3
1
3
2
3
CH
2
OP); P NMR (121.5
References and notes
3
28 3 7
H N O
P: C, 56.44;
1. Hendry, L. B.; Mahesh, V. B.; Bransome Jr., E. D.; Ewing, D. E.
Mutat. Res. 2007, 623, 53–71.
9
3:7 mixture of trans-10d and cis-10d).
2
3
4
.
.
.
Martínez, R.; Chacón-García, L. Curr. Med. Chem. 2005, 12, 127–
151.
4
.5. Antiviral Activity Assays
Braña, M. F.; Cacho, M.; Gradillas, A.; de Pascual-Teresa, B.;
Ramos, A. Curr. Pharm. Design 2001, 7, 1745–1780.
Lerman, L. S. J. Mol. Biol. 1961, 3, 18–30.
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
5. Hogan, M., Dattagupta, N., Crothers, D. M. Biochemistry 1979,
18, 280–288.
r
resistant to ACV (ACV ), herpes simplex virus type 2 (HSV-2)
6
7
.
.
Pommier, Y. Chem. Rev. 2009, 109, 2894–2902.
Scheithauer, W.; Dittrich, C.; Kornek, G.; Haider, K.; Linkesch,
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strain G, varicella-zoster virus (VZV) strains Oka and YS, TK-
VZV strains 07-1 and YS-R, 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
8
9
.
.
Costanza, M. E.; Berry, D.; Henderson, I. C.; Ratain, M. J.; Wu,
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(
(
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
1
1. Bair, K. W.; Andrews, C. W.; Tuttle, R. L.; Knick, V. C.; Cory,
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J. Med. Chem. 1990, 33, 2385–2393.
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
1
(
HEL) fibroblasts, African green monkey kidney cells (Vero),
13. Rescifina, A.; Chiacchio, M. A.; Corsaro, A.; De Clercq, E.;
Iannazzo, D. Mastino, A. Piperno, A.; Romeo G.; Romeo, R.
Valveri V. J. Med. Chem. 2006, 49, 709–715.
human epithelial cervix carcinoma cells (HeLa), Crandell-Rees
feline kidney cells (CRFK), or Madin Darby canine kidney cells
(
1
4. Rescifina, A.; Chiacchio, U.; Piperno, A.; Sordino, S. New J.
Chem. 2006, 30, 554–561.
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
1
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concentration that caused a microscopically detectable alteration
of cell morphology. Selectivity indexes were calculated as the
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Acknowledgments
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The authors wish to express their gratitude to Mrs. Leentje
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Mrs. Lizette van Berckelaer for excellent technical assistance.
The synthetic part of this work was supported by the Medical
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13.

Table 1. Isoxazolidines trans-9 and cis-9 obtained according to Scheme 3.
Acrylamide 11
Ratio of
Entry
Yield (%)
R
trans-9:cis-9
a
trans-9a (63) + cis-9a (11) + trans-9a and cis-9a (11)
a
b
A
81:19
a
a
trans-9b (71) + cis-9b (14) + trans-9b and cis-9b (2)
b
B
C
D
84:16
79:21
79:21
a
trans-9c (67) + cis-9c (19) + trans-9c and cis-9c (5)
a
b
a
trans-9d (33) + cis-9d (8) + trans-9d and cis-9d (52)
a
b
N
a
a
trans-9e (32) + cis-9e (11) + trans-9e and cis-9e (54)
b
E
F
86:14
78:22
N
N
a
trans-9f (31) + cis-9f (8) + trans-9f and cis-9f (50)
a
b
a
yield of pure isomer,
b
yield of pure mixture of cis- and trans-isomers.
Table 2. Isoxazolidines trans-10 and cis-10 obtained according to Scheme 4.
Imide 9
Ratio of
trans-10:cis-10
91:9
a
Entry
Yield (%)
R
H
A
B
C
D
trans-10a and cis-10a (76%; trans-10a:cis-10a = 93:7)
trans-10b and cis-10b (67%; trans-10b:cis-10b = 94:6)
trans-10c and cis-10c (32%; trans-10c:cis-10c = 93:7)
trans-10d and cis-10d (73%; trans-10d:cis-10d = 93:7)
NO
NH
NHC(O)CH
2
91:9
2
86:14
3
88:12
a
yield of pure mixture of cis- and trans-isomers

Table 3. Cytotoxicity and antiviral activity against cytomegalovirus in HEL cell cultures.
a
Antiviral activity EC50 (µM)
AD-169 strain
Cytotoxicity (µM)
b
Cell morphology MCC
Compound
R
Davis strain
cis-9a
(
exp. 1)
exp. 2)
45
45
45
45
>100
>100
(
trans-9a
(
exp. 1)
exp. 2)
>20
49
55
45
>100
>100
(
cis-9b
(
exp. 1)
exp. 2)
>20
>20
>20
>20
100
100
(
trans-9b
(
exp. 1)
exp. 2)
>20
>20
>20
>20
100
100
(
cis-9c
(
(
exp. 1)
exp. 2)
45
45
45
45
>100
>100
trans-9c
(
(
exp. 1)
exp. 2)
>
100
100
>100
>100
>100
>100
>
cis-9d
(
exp. 1)
exp. 2)
45
37
41
37
>100
>100
(
trans-9d
(
exp. 1)
exp. 2)
8.9
8.9
8.9
8.9
100
100
(
cis-9e
(
exp. 1)
exp. 2)
100
100
100
>100
>100
>100
(
trans-9e
(
exp. 1)
exp. 2)
55
55
49
63
>100
>100
(
cis-9f
(
exp. 1)
exp. 2)
45
45
45
45
>100
>100
(
trans-9f
(
exp. 1)
exp. 2)
8.9
10.9
10.9
20
100
100
(
Cidofovir
(
exp. 1)
exp. 2)
0.92
0.92
1.02
1.14
>350
>350
(
Ganciclovir
(
exp. 1)
exp. 2)
7.1
6.3
7.1
7.9
>350
>350
(
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,

Table 4. Cytotoxicity and antiviral activity against varicella-zoster virus (VZV) in HEL cell cultures.
a
Antiviral activity EC50 (µM)
Cytotoxicity (µM)
b
Cell morphology MCC
Compound
R
+
TK VZV strain
–
TK VZV strain
cis-9a
(
exp. 1)
exp. 2)
20
32
38
45
>100
>100
(
trans-9a
(
exp. 1)
exp. 2)
32
28
40
41
>100
>100
(
cis-9b
(
exp. 1)
exp. 2)
20
32
38
45
>100
>100
(
trans-9b
(
exp. 1)
exp. 2)
32
28
40
41
>100
>100
(
cis-9c
(
exp. 1)
exp. 2)
45
45
45
45
>100
>100
(
trans-9c
(
exp. 1)
exp. 2)
>
>
100
100
>100
>100
>100
>100
(
cis-9d
(
exp. 1)
exp. 2)
>20
11
20
20
100
100
(
trans-9d
(
exp. 1)
exp. 2)
14
15
26
34
>100
>100
(
cis-9e
(
exp. 1)
exp. 2)
>20
41
36
44
>100
>100
(
trans-9e
(
exp. 1)
exp. 2)
30
>20
32
>20
100
100
(
cis-9f
(
exp. 1)
exp. 2)
>20
>20
>20
79
100
>100
(
trans-9f
(
exp. 1)
exp. 2)
>20
>20
>20
>20
100
100
(
Acyclovir
(
exp. 1)
exp. 2)
3.6
1.5
44
33
>440
>440
(
Brivudin
(
(
exp. 1)
exp. 2)
0.042
0.029
1.0
4.9
>300
>300
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,

Table 5. Cytotoxicity and antiviral activity in HEL cell cultures.
b
a
EC50 (µM)
MCC
Compound
R
HSV-1 (KOS)
HSV-1 (TK¯
KOS ACVr)
HSV-2 (G)
Vaccinia virus
(
µM)
trans-9d
(
(
exp. 1)
exp. 2)
≥100
≥100
45
45
45
45
45
>100
45
>100
N
cis-9d
(
(
exp. 1)
exp. 2)
>100
>100
50
58
50
58
50
58
45
>100
trans-9f
(
(
exp. 1)
exp. 2)
≥100
≥100
45
45
45
45
45
>100
45
>100
N
cis-9f
(
(
exp. 1)
exp. 2)
>100
>100
>100
>100
>100
>100
58
100
50
58
Brivudin
(
(
exp. 1)
exp. 2)
>250
>250
0.01
0.05
2.0
10
146
250
17
22
Cidofovir
(
(
exp. 1)
exp. 2)
>250
>250
1.0
2.0
1.2
2.0
1.0
1.5
22
22
Acyclovir
(
(
exp. 1)
exp. 2)
>250
>250
0.2
0.2
2.0
2.0
0.08
0.2
>250
>250
Ganciclovir
(
(
exp. 1)
exp. 2)
>100
>100
0.03
0.02
0.8
0.5
0.03
0.03
>100
>100
a
minimum cytotoxic concentration that causes a microscopically detectable alternation of cell morphology,
b
effective concentration that is required to reduce virus-induced cytopathogenicity by 50 %
Table 6. Cytotoxicity and antiviral activity in Vero cell cultures.
b
EC50 (µM)
Coxsackie virus B4
a
Compound
R
H
MCC (µM)
Punta Toro
virus
trans-10a/cis-10a (93:7)
(
exp. 1)
exp. 2)
>100
>100
45
73
>100
>100
(
trans-10d/cis-10d (93:7)
(
exp. 1)
exp. 2)
NHC(O)CH
3
>100
>100
45
58
50
100
(
DS-10.000 (µg/ml)
(
exp. 1)
>100
>100
>100
>100
45
100
(exp. 2)
Ribavirin
(
exp. 1)
exp. 2)
>250
>250
>250
>250
112
250
(
a
minimum cytotoxic concentration that causes a microscopically detectable alternation of cell morphology,
b
effective concentration that is required to reduce virus-induced cytopathogenicity by 50 %

Table 7. Inhibitory effect of the tested compounds against the proliferation of murine leukemia (L1210), human T-lymphocyte (CEM) and human cervix
carcinoma cells (HeLa).
Compound
a
b
IC50 (µM)
CC50 (µM)
R
L1210
76 ± 16
41 ± 7
CEM
47 ± 18
28 ± 4
19 ± 1
25 ± 7
26 ± 3
HeLa
70 ± 41
74 ± 11
25 ± 3
MDCK
CrFK
>100
>100
>100
>100
>100
cis-9a
trans-9a
cis-9b
>100
46.2
28 ± 7
85 ± 54
73 ± 27
>100
>100
51.8
trans-9b
cis-9c
78 ± 3
49 ± 21
trans-9c
cis-9d
72 ± 52
2.9 ± 0.2
1.5 ± 0.3
16 ± 4
48 ± 10
6.0 ± 1.1
3.4 ± 0.4
23 ± 0
56 ± 27
3.3 ± 0.3
1.8 ± 0.9
7.3 ± 2.4
>100
>100
75.2
>100
62.2
95.2
>100
N
trans-9d
cis-9e
>100
trans-9e
N
11 ± 0
21 ± 1
13 ± 5
>100
>100
cis-9f
4.4 ± 0.1
1.7 ± 0.4
12 ± 1
3.0 ± 0.6
1.1 ± 0.0
>100
87.3
89.1
trans-9f
N
3.9 ± 0.0
>100
trans-10a/cis-10a (93:7)
H
180 ± 47
34 ± 6
95 ± 42
111 ± 14
51 ± 6
99 ± 2
77 ± 20
20 ± 3
>100
>100
>100
>100
―
>100
>100
>100
>100
―
trans-10b/cis-10b (94:6)
trans-10c/cis-10c (93:7)
trans-10d/cis-10d (93:7)
NO
NH
2
2
101 ± 58
111 ± 3
NHC(O)CH
3
136 ± 14
0.33 ± 0.17
125 ± 4
18 ± 5
5
-Fluorouracil
0.54 ± 0.12
a
5
0% Inhibitory concentration or compound concentration required to inhibit tumor cell proliferation by 50%,
b
5
0% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay.