Synthesis, toxicity and chemo-sensitization of HeLa cells to etoposide, of some 2-methyl amino acid ester-substituted-1,3-benzoxazines

Article abstract of DOI:10.1007/s00044-015-1338-4

A number of new L- or LD-2-amino acid ester-(substituted)-benz[1,3]oxazines 12-17 were synthesized from the reaction of free L- or LD-amino acid ester 9a-d with 2-methylthio-1,3-benzoxazines 11a-g. The structures of the new products 12-17 were confirmed from their ¹H, ¹³CNMR and IR spectra and CHN microanalysis. Some of these compounds weakly inhibited DNA-PK and platelet aggregation. Studies of the toxicity for some of the new compounds showed mostly no inhibitory effects on HeLa cell growth at 1 and 10 μM and some up to 40 μM. The chemo-sensitization to etoposide by some of the compounds revealed that the most effective chemo-sensitizers at 10 μM were 14c (1.83 fold), 12e (1.42 fold), 15a (0.8 fold), 16d (0.76 fold) and 1c (0.74). However, at 1 μM in the presence of etoposide, some compounds were shown to be more effective. No direct link was observed between the type of the L-amino acid methyl ester as well as the 7-, 8-, or 7, 8-substitution on the aromatic ring on the effectiveness of the chemo-sensitizers; however, the 7-hydroxy group did lower the effective chemo-sensitizers values.

Full text of DOI:10.1007/s00044-015-1338-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1338-4
ORIGINAL RESEARCH
Synthesis, toxicity and chemo-sensitization of HeLa cells
to etoposide, of some 2-methyl amino acid ester-substituted-
1,3-benzoxazines
•
Saleh K. Ihmaid Cheree Fitzgibbon
•
Jasim M. A. Al-Rawi
Received: 5 September 2014 / Accepted: 2 February 2015
Ó Springer Science+Business Media New York 2015
Abstract A number of new L- or LD-2-amino acid ester-
(substituted)-benz[1,3]oxazines 12–17 were synthesized
from the reaction of free L- or LD-amino acid ester 9a–
d with 2-methylthio-1,3-benzoxazines 11a–g. The struc-
tures of the new products 12–17 were confirmed from their
1H, 13CNMR and IR spectra and CHN microanalysis. Some
of these compounds weakly inhibited DNA-PK and platelet
aggregation. Studies of the toxicity for some of the new
compounds showed mostly no inhibitory effects on HeLa
cell growth at 1 and 10 lM and some up to 40 lM. The
chemo-sensitization to etoposide by some of the com-
pounds revealed that the most effective chemo-sensitizers
at 10 lM were 14c (1.83 fold), 12e (1.42 fold), 15a (0.8
fold), 16d (0.76 fold) and 1c (0.74). However, at 1 lM in
the presence of etoposide, some compounds were shown to
be more effective. No direct link was observed between the
type of the L-amino acid methyl ester as well as the 7-, 8-,
or 7, 8-substitution on the aromatic ring on the effective-
ness of the chemo-sensitizers; however, the 7-hydroxy
group did lower the effective chemo-sensitizers values.
Introduction
The a-Amino acids are the fundamental building blocks in
nature. N-aryl-a-amino acids are found as core structural
components of antimicrobial, antiviral and pharmaco-
logically active molecules (Ma and Yao, 1996). 2-Amino-
1,3-benzoxazines can exhibit herbicidal, insecticidal,
fungicidal and analgesic properties (Grigat et al., 1964;
Tomita and Murakami, 1979; Schroth et al., 1989; Bereznak
and Marshall, 2000). In addition, 2-amino-1,3-benzoxazines
have been found to exhibit antiatherogenic activity giving
them usefulness in the treatment and prophylaxis of
atherosclerosis (Schroth et al., 1989). In particular, 2-mor-
pholino-1,3-benzoxazine analogues 1 and 2, 2-thiomor-
pholino-1,3-benzoxazine 3 and 2-(4-methylpiperazino)-1,3-
benzoxazine 4 (Fig. 1) were reported to reduce arterial
cholesterol accumulation in the SEA Japanese quail model
(Gammill et al., 1990). Additionally, the 7-acetoxy-8-
methyl analogue of 2 has been reported to inhibit cell pro-
liferation as well as ADP-induced platelet aggregation
(Gammill et al., 1991).
Keywords 2-Amino acid ester-substituted-benz[1,3]
oxazines ꢀ Toxicity ꢀ Chemo-sensitization to etoposide
To date general method for methyl 2-a-amino acid-
benz[1,3]oxazines synthesis has not been achieved. Previ-
ously 2-((8-methyl-4-oxo-4H-benz[e][1,3]oxazin-2-yl)a-
mino)acetic acid was prepared from the reaction of ethyl
2-cyanato-3-methylbenzoate and glycine in low yield
(B30 %; Grigat et al., 1964). The development of a general
synthetic method of 2-a-amino acid-benz[1,3]oxazines is
challenging as the amino acids themselves contain nonpo-
lar, polar neutral, acidic and basic side chains, which may
complicate or interfere with the reactions.
S. K. Ihmaid ꢀ C. Fitzgibbon ꢀ J. M. A. Al-Rawi (&)
School of Pharmacy and Applied Science, La Trobe Institute for
Molecular Science, La Trobe University, P.O. Box 199,
Bendigo, Australia
e-mail: j.al-rawi@latrobe.edu.au
However, it is of importance as these families of com-
pounds have been found to enhance the efficacy of anti-
cancer treatments through inhibition and activation of
different pathways. For example, DNA-PK is a key enzyme
S. K. Ihmaid
e-mail: saleh_ihmaid@yahoo.com.au
C. Fitzgibbon
e-mail: cafitzgibbon@students.latrobe.edu.au
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Med Chem Res
Results and discussion
Chemistry
Methyl esterification of a-amino acids
This step involves the protection of the carboxylic function
of the a-amino acid 7 to prevent the formation of a zwit-
terion, which is a simple and reversible protection method.
The a-amino acids 7 were converted to their corresponding
methyl ester derivatives 8 then the hydrochloric salt com-
pound 8 which was neutralized and gave product 9 using
the previously reported procedure (McKerrow et al., 2010;
Scheme 1).
Fig. 1 Some structures of the biologically important 2-amino-1,3-
benzoxazines 1–6
Synthesis of 2-amino acid ester-substituted-
benz[1,3]oxazines 12–17
involved in the non-homologous end joining (NHEJ)
pathway repair of double strand DNA breaks, caused by
some anticancer treatments (Matsuoka et al., 2007).
Recently, we found that some 2-morpholino-1,3-ben-
zoxazines compounds showed high-to-moderate DNA-PK
inhibition activity (ca. 0.28–6.80 lM IC50) from which the
The 2-thio-1,3-benzoxazines 10a–g were synthesized ac-
cording to the previously reported procedure (Scheme 1;
Pritchard et al., 2005). Furthermore, the 2-methylthio-1,3-
benzoxazines 11a–g were synthesized according to the
previously reported procedure (Scheme 1; Ihmaid et al.,
2012).
most active compound was product
IC50 = 0.28 lM. We also found that the 2-(N-substituted
(pyridin-3-ylmethyl)amino)-1,3-benzoxazines showed
5
with an
The reaction of compounds 11a–g according to general
procedure A with free amino acid ester 9a–e gave 2-amino
acid-benz[1,3]oxazines 12–17 (Scheme 1).
moderate-to-low (ca. 2.5–25.2 lM IC50) DNA-PK in-
hibitory activity, with the most potent being 6 with an
IC50 = 2.5 lM (Fig. 1; Ihmaid et al., 2012).
Radhamani et al. (2014) found compound 5 to be an
effective radio-sensitizing agent that inhibits DSB repair
and promotes apoptosis in both irradiated lung cancer and
colon cancer cells. We proposed that its mechanism of
action is through the inhibition of DNA-PK.
Structural conformation of 2-amino acid-1,3-benzoxazines
methyl esters
The structures of the new 2-amino acid-1,3-benzoxazines
methyl esters 12a–g, 13a–c, 14a–e, 15a–c, 16a–d and 17a–
d were confirmed using IR, 1H, 13CNMR spectroscopy and
microanalysis. The IR spectra of compounds 12–17
showed two carbonyl groups, one related to the amino acid
ester C-20 at mmax 1,752, and the other carbonyl related to
the benzoxazine C-4 at mmax * 1,655 (C=O). The 1HNMR
spectra showed the expected signals for the 2-amino acid
ester-1,3-benzoxazine with characteristic signals appearing
in the proton NMR of all the products 12–17, as multiple
signals d * 4.5 ppm for H10 and the signal for
OCH3 * 3.7 ppm as a singlet.
Furthermore, it was found (Fitzgibbon et al., 2013) that
compound 12b (LTUSI54), which was found to have no
DNA-PK or PI3K inhibitory activity ([100 lM), still
sensitized HeLa cells to the effects of etoposide. The
study hypothesized that product 12b promotes cell cycle
arrest through activation of p38a pathways, independent
of p53 mechanisms, results in a decrease of p53 stabi-
lization and hence, restricted apoptosis and arrest in cell
growth.
The aim of this study was to develop a simplified, re-
producible method of 2-a-amino acid-benz[1,3]oxazines
synthesis that can be used as a general method in future.
Furthermore, through use of this new method, we aimed to
develop a family of 2-a-amino acid-benz[1,3]oxazines and
assess them for their chemo-sensitizing abilities against the
ovarian cancer, HeLa cell line.
It is well known that the phenomenon of geminal ani-
sochrony (U2 protons) can be observed in chiral species
such as CXYZ–CU2V and CXYZC : CU2V (Stiles,
1977). As a result of chirality, some of 2-amino acid esters-
1,3-benzoxazines showed magnetic nonequivalent protons
and carbon-13 chemical shift for the chemically equivalent
groups (McKerrow et al., 2010).
123

Med Chem Res
Scheme 1 Esterification of
amino acids, and the synthesis
of substituted of 2-(amino acid)-
1,3-benzoxazine 12–17
Comp.
11a, 12a
11b, 12b
11c, 12c
11c*12c*
11d, 12d
11e, 12e
11e,*12e*
11f, 12f
11g, 12g
13a
R2
R1
R
Yield Comp.
R2
H
R1
Ph
H
R
Yield
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
75%
72%
67%
63%
65%
70%
60%
70%
60%
CH2CH(CH3)2 60%
CH2CH(CH3)2 55%
CH2CH(CH3)2 67%
14c
14d
14e
15a
15b
15c
16a
16b
16c
16d
17a
17b
17c
17d
H
CH3
Ph
Ph
H
OH
H
OH CH3
H
H
CH3
H
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2Ph
63%
60%
55%
55%
55%
50%
53%
60%
68%
60%
63%
OCH2CH3
OCH3
OCH3
OH
OH
H
OH
H
OH CH3
H
H
CH3
Ph
H
H
H
CH2Ph
CH3
CH3
H
OH
CH2Ph
CH(CH3)CH2CH3 55%
CH(CH3)CH2CH3 51%
CH(CH3)CH2CH3 60%
OH CH3
CH2Ph
OH
OH
H
H
H
L-Proline
L-Proline
L-Proline
L-Proline
13b
CH3
H
H
CH3
H
13c
CH2CH(CH3)2
65%
OH
14a
H
CH3
CH2CH(CH3)2
70%
OH CH3
14b
*DL amino acid is used to produce the product.
Hydrolysis of methyl 3-methyl-2-((8-methyl-4-oxo-4H-
L-valine derivatives 15a–c showed two different proton
and carbon-13 chemical shifts for the 40- and 50-methyl
groups. Similarly, the methyl groups 50 and 60 in leucine
derivatives 14a–e showed two different proton and carbon-
13 chemical shifts. However, the CH2 protons of C-40 in
isoleucine derivatives 13a–c showed two nonequivalent
protons. Similarly, the C-30 CH2 protons in phenylalanine
derivatives 16a–d were nonequivalent.
benz[e][1,3]oxazin-2-yl)amino)butanoate 15a
Product 15a was hydrolyzed to the corresponding acid 18
using dilute NaOH solution under mild condition to keep the
amino acid chirality unchanged in the final product (see
‘‘Experimental’’). Structureofproduct18 wasconfirmedfrom
the CHN microanalysis, IR, 1H and 13C NMR spectroscopy.
Biological activity
Chirality and anisochrony (magnetic nonequivalence)
Inhibition of platelet aggregation of some 2-methyl amino
It is worth noting t substitution of 2-methylthio group of
compound 11 with a-amino acid methyl esters occurs
through a 1,2-addition elimination reaction. Therefore, no
racemization would be expected to take place as the C-30 is
not involved in the reaction.
acid-substituted-1,3-benzoxazines 12b, 14b and 14c
Platelet aggregation was determined by the optical method
in a two-channel platelet aggregometry (Chrono-Log) us-
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Med Chem Res
Fig. 2 SRB results of HeLa cells treated with increasing concentrations of a 16b, b 16d or c 13c for 48 h. Graph indicates mean of four
replicates SEM. Statistical significance was determined using two-tailed t tests, asterisk indicates results statistically significant to the control
ing the previously reported protocol (Pritchard et al.,
2007). Compounds 12b, 14b and 14c showed no activity at
100 lM regardless of 8-substituent (Me or Ph) of the
2-methyl amino acid-1,3-benzoxazines (L-Alanine,
L-Phenylalanine and L-Leucine 12b, 14b, and 14c,
respectively).
DNA-PK IC50 for select 2-amino acid-substituted-1,3-
benzoxazines methyl ester 12b, 14c and 16b
The DNA-PK assay was performed on compounds 12b,
14c and 16b by Reaction Biology Corporation, One Great
Valley Parkway, Suite 2 Malvern, PA 19355 USA ac-
cording to the previously reported method (Morrison et al.,
2014). Compound 12b, 14b, and 14c showed low-to-
moderate DNA-PK activity with IC50 of 81.0, 25.7 and
10.0 lM, respectively.
Fig. 3 SRB results of HeLa cells treated with 25 lM etoposide both
in the presence and absence of test compounds at either 1 lM (open
bars) or 10 lM (shaded bars) for 48 h. Graph indicates mean
SEM of four replicates. Statistical significance was determined using
two-tailed t tests, asterisk indicates combination treatments sig-
nificantly different to the etoposide alone treatment group
Toxicity of select 2-amino acid-substituted-1,3-
benzoxazines methyl ester and the corresponding amino
acid derivative 18
When investigating the effects of the compounds 12d–g,
13c, 14a–c, 15a, c, 16b, d, 17b, d and 18 alone on
HeLa cervical cancer cells, no inhibitory effects were
observed at low concentrations (1, and 10 lM). How-
ever, 13c, 14b, 16b, 16d and 17d were found to sig-
nificantly (p value \0.05) inhibit HeLa cell growth
when used at the high concentration of 40 lM when
compared to the DMSO control (Fig. 2). Figure 2 shows
that 16b, 16d and 13c have a dose-dependent effect on
HeLa cell number, whereby there is no significant effect
on cell growth until high concentrations of 40 lM. All
other tested compounds were found to have no sig-
nificant effect (p [ 0.05) on cell number as indicated by
the lack of change in absorbance across all tested
concentrations.
Chemo-sensitization to etoposide by selected 2-amino acid-
substituted-1,3-benzoxazines methyl ester
and the corresponding amino acid derivative 18
We previously found (Fitzgibbon et al., 2013) that com-
pound 12b works synergistically with etoposide to inhibit
growth of HeLa cells with no corresponding increase in
cell death, as indicated by sub G1 analysis. This result is
believed to be due to the observed enhancement of cell
cycle arrest at both the S and G2 checkpoints.
In this study, we investigated the effect of select, newly
synthesized compounds, on enhancing the chemo-toxic
123

Med Chem Res
Table 1 SRB results of HeLa cells treated with 25 lM etoposide both in the presence and absence of test compounds 1 or 10 lM for 48 h
Etop ? 1 lM Etop ? 10 lM
1.56 1.37
Etop ? 1 lM Etop ? 10 lM
0.55 0.80
12d
15a
12e
1.25
1.42
15c
0.71
0.24
12g
0.58
0.68
16b
0.04
0.27
13c
0.31
0.74
16d
17b
0.53
0.76
14a
0.37
0.40
0.17
0.29
14b
0.49
0.44
17d
0.63
0.51
14c
1.31
1.83
18
0.99
1.35
Folds were calculated using etoposide alone at 25 lm as 12.5 % chemo-sensitizing effects on HeLa cells
effects of etoposide on the HeLa cervical cancer cell line. It
was observed that 25 lM etoposide treatment resulted in a
significant reduction in cell number from absorbance of
0.181–0.158 (p \ 0.05). Figure 3 shows the effect ob-
served by etoposide treatment as well as the chemo-sen-
sitization effects of a selection of compounds. Table 1
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Med Chem Res
summarizes chemo-sensitization effects observed for select
compound relative to the chemo-sensitization of etoposide.
As can be seen in Fig. 3, 25 lM etoposide treatment re-
sulted in a significant reduction in HeLa cell number from
absorbance of 0.181–0.158 (p \ 0.05). This observation was
further enhanced by 14c, which was found to be the most
effective chemo-sensitizer at 10 lM (1.83 fold) as indicated
by the lowest optical absorbance reading of 0.12. This was
found to be a significant reduction in HeLa cell numbers
when compared to the f etoposide-only treatment (1.83 fold,
p \ 0.0001). Similarly, when 14c was used at the lower
concentration (1 lM), there was a significant reduction ob-
served in HeLa cell number with an absorbance of 0.129
when compared to etoposide-only treatment (1.31 fold,
p \ 0.0001). Compound 12d was found to be the second
most effective chemo-sensitizer when used at 1 lM in con-
junction with etoposide resulting in an absorbance reading of
0.123, which is again a significant reduction in cell number
compared to the etoposide-only treated HeLa cells (1.56
folds, p \ 0.0001). However, when compound 12d was used
at a higher concentration of 10 lM in combination with
etoposide, there was still a significant sensitization by
comparison to the etoposide-only treated cells (1.37 fold,
p \ 0.0001) although this was not as effective as the lower
1 lM concentration. Similarly, compound 15c was found to
be more effective at enhancing the effects of etoposide on
HeLa cells when used at a lower concentration. When 1 lM
15c was used in the presence of etoposide, there was a sig-
nificant reduction (0.71 fold)in cell numbers when compared
to the etoposide alone treatment (absorbance 0.142,
p = 0.0102). However, when 10 lM 15c was used in com-
bination with etoposide, there was no significant change
(0.24 fold) in HeLa cell numbers observed. Conversely, 17b
was found to have no chemo-sensitizing effect on HeLa cells
when used in combination with etoposide at 1 lM (0.17 fold)
or 10 lM (0.29 fold) concentration with an absorbance of
0.154 and 0.152, respectively. The only other two com-
pounds to share this observation were 14a and 14b, and all
other compounds were found to have chemo-sensitizing ef-
fects on HeLa cells in combination with etoposide at one or
both the concentrations tested (Table 1).
correlation with the type of L-amino acid methyl ester
substitution at position 2 nor with the 7- or 8-substitution or
7, 8-bis-substitution on the aromatic ring. However, the
7-hydroxy group substitution appears to lower the efficacy
of chemo-sensitization (Table 1).
It is worth noting that compound 14c enhances the
chemo-sensitizing effects of etoposide on HeLa cells by
1.83-fold at 10 lM, a concentration at which it is found to
have no effect on either DNA-PK activity or cell growth.
Conclusion
A number of new L- or LD-2-amino acid ester-substituted-
benz[1,3]oxazines 12–17 and the corresponding acid ana-
logue 18 were synthesized for the first time using simple
synthetic methods. A selection of the novel compounds
produced were assessed for their biological activity as
chemo-sensitizers. Studies of the chemo-sensitization of
HeLa cells to etoposide using selected new compounds
revealed that some are more effective chemo-sensitizer at
10 lM, while others were more effective at 1 lM. Com-
pound 14c was the most effective chemo-sensitizer at
10 lM (1.83 fold), while compound 12d was found to be
the most effective chemo-sensitizer (1.56 folds) when used
at 1 lM. 2-Amino acid-8-methyl-1,3-benzoxazines acid 18
enhanced the chemo-sensitizing effects on HeLa cells in
combination with etoposide at both the concentrations
tested (1 and 10 lM) by 0.99- and 1.35-fold, respectively.
Alone they showed no negative effect on HeLa cell
growth at concentrations up to 10 lM.
Experimental
Chemistry
Infrared spectra were obtained using a Perkin Elmer FT-IR
1
1720 9 spectrometer. H and 13CNMR spectra were ob-
tained using a Bruker AC 200 NMR spectrometer at 200
1
and 50 MHz, respectively. All H and 13CNMR spectral
Additionally, hydrolysis of 2-amino acid-8-methyl-1,3-
benzoxazines methyl ester 15a to the corresponding acid
compound 18 enhanced the chemo-sensitizing effects on
HeLa cells in combination with etoposide at both the
concentrations tested (1 and 10 lM) by 0.99- and 1.35-
fold, respectively, compared with the methyl ester ana-
logue 15a (Table 1).
results are recorded as chemical shifts (d) relative to the
internal TMS for proton and 77.0 ppm in CDCl3 solvent
and 39.4 ppm in DMSO-d6 solvent 13CNMR. Micro-
analysis was performed by Chemical and Micro Analytical
Services (CMAS), Australia. Melting point determinations
were carried out using a Stuart Scientific (SMP3) melting
point apparatus, and all melting points are uncorrected.
Structure activity relationship
Starting material
It is worth noting that enhanced effects of etoposide on
HeLa cells (effective chemo-sensitizer) showed no direct
The starting reagents L, Dl a-amino-acids, sodium hydro-
gen carbonate, cesium carbonate, and methyl iodide and
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Med Chem Res
(C-8a), 134.3 (C-7), 124.4 (C-8), 123.9 (C-5), 123.6 (C-6),
116.8 (C-4a), 51.3 (OCH3), 49.1 (C-10), 16.1 (C-30), 13.4
(CH3); (Found C, 59.47; H, 5.40; N, 10.75; C13H14N2O4,
requires C, 59.54; H, 5.38; N, 10.68).
bromine were purchased from Aldrich Chemical Company
and were used as received.
Triphenylphosphine was purchased from Merck chemi-
cal company Germany.
L-Methyl 2-((4-oxo-8-phenyl-4H-benz[e][1,3]oxazin-2-yl)-
amino)propanoate 12c 8-Phenyl-2-(methylthio)-4H-benz
[e][1,3]oxazin-4-one (0.67 g, 2.5 mmol) 11c was allowed
to react with L-alanine methyl ester 9a (0.31 g, 3 mmol)
according to general procedure A. The crude solid was
collected and recrystallized from ethanol to give 12c
(0.54 g, 67 % yield), mp 204 °C. mmax (KBr)/cm-1 3,056,
2,911 (N–H), 1,751 (C=O), 1,655 (C=O), 1,603 (C=N);
1HNMR (200 MHz, d6-DMSO) d 8.60 (bs, 1H, N–H), 7.90
(dd, 1H, JH5,H7 = 1.7 Hz, JH5,H6 = 7.72 Hz, H-5), 7.63
(dd, 1H, JH7,H5 = 1.7 Hz, JH7,H6 = 7.7 Hz, H-7),
7.50–7.40 (m, 6H, H-6, H-10, H-11 & H-12), 4.40 (q, 1H,
J = 7.3 Hz, H-10), 3.62 (s, 3H, OCH3), 1.43 (d, 3H,
J = 7.3 Hz, H-30); 13CNMR (50 MHz, d6-DMSO) d 172.2
(C-20), 163.5 (C-4), 152.3 (C-2), 150.0 (C-8a), 134.5 (C-9),
134.3 (C-7), 128.1 (C-11), 127.7 (C-10), 127.2 (C-12),
125.8 (C-6), 124.9 (C-5), 109.5 (C-8), 117.6 (C-4a); 51.6
(C-10), 49.4 (OCH3), 16.1 (C-30); (Found C, 66.36; H, 5.09;
N, 8.56; C18H16N2O4, requires C, 66.66; H, 4.97; N, 8.64).
Synthesis of 2 L, or DL-a-amino acid-1,3-benzoxazines
General procedure A
The appropriate 2-methylthio-1,3-benzoxazine 11a–
g (2.5 mmol) was suspended in dry 1,4-dioxane (10 mL) in
a 50-mL round-bottomed flask. The appropriate a-amino
acid methyl ester-free amine 9 (3 mmol) was then added,
with stirring before the reaction mixture was heated to
reflux for 4 h. At completion, the reaction mixture was
evaporated to dryness under reduced pressure and triturated
with minimal diethyl ether. The resulting solid was col-
lected by vacuum filtration and recrystallized from an ap-
propriate solvent.
L-Methyl 2-((4-oxo-4H-benz[e][1,3]oxazin-2-yl)amino)-
propanoate 12a 2-(Methylthio)-4H-benz[e][1,3]oxazin-
4-one 11a (0.48 g, 2.5 mmol) was allowed to react with L-
alanine methyl ester 9a (0.31 g, 3 mmol) according to
general procedure A. The crude solid was collected and
recrystallized from toluene to give 12a (0.46 g, 75 %
yield), mp 211 °C. mmax (KBr)/cm-1 3,279, 2,923 (N–H),
1,771 (C=O), 1,665 (C=O), 1,624 (C=C), 1,489 (C=N);
1HNMR (200 MHz, d6-DMSO) d 8.61 (bs, 1H, N–H), 7.92
(d, 1H, J = 6.5 Hz, H-5), 7.71 (t, 1H, J = 7.3 Hz, H-7),
7.53 (t, 1H, J = 7.0 Hz, H-6), 7.32 (d, 1H, J = 7.0 Hz,
H-8), 4.5 (q, 1H, J = 7.3 Hz, H-10), 3.7 (s, 3H, OCH3), 1.4
(d, 3H, J = 7.3 Hz, H-30); 13CNMR (50 MHz, d6-DMSO)
d 172.7 (C-20), 166.1 (C-4), 154.4 (C-2), 151.6 (C-8a),
132.3 (C-7), 130.4 (C-5), 121.9 (C-6), 120.6 (C-8), 114.8
(C-4a), 51.3 (OCH3), 48.1 (C-10), 17.1 (C-30); (Found C,
58.47; H, 4.95; N, 11.75; C12H12N2O4, requires C, 58.06;
H, 4.87; N, 11.29).
D,L-methyl 2-((4-oxo-8-phenyl-4H-benz[e][1,3]oxazin-2-yl)-
amino)propanoate 12c* 8-Phenyl-2-(methylthio)-4H-benz
[e][1,3]oxazin-4-one 11c (0.67 g, 2.5 mmol) was allowed
to react with D, L-alanine methyl ester 9a*(0.31 g, 3 m-
mol) according to general procedure A. The crude solid
was collected and recrystallized from ethanol to give 12c*
(0.51 g, 63 % yield), mp 210–213 °C. mmax (KBr)/cm-1
3,056, 2,911 (N–H), 1,751 (C=O), 1,655 (C=O), 1,603
(C=N); 1HNMR (200 MHz, d6-DMSO) d 8.65 (bs, 1H, N–
H), 7.90 (dd, 1H, JH5,H7 = 1.7 Hz, JH5,H6 = 7.7 Hz, H-5),
7.62 (dd, 1H, JH7,H5 = 1.7 Hz, JH7,H6 = 7.7 Hz, H-7),
7.50–7.40 (m, 6H, H-6, H-10, H-11 & H-12), 4.43 (q, 1H,
J = 7.3 Hz, H-10), 3.62 (s, 3H, OCH3), 1.4 (d, 3H,
J = 7.3 Hz, H-30); 13CNMR (50 MHz, d6-DMSO) d 172.2
(C-20), 163.5 (C-4), 152.3 (C-2), 150.0 (C-8a), 134.5 (C-9),
134.3 (C-7), 128.1 (C-11), 127.7 (C-10), 127.2 (C-12),
125.8 (C-6), 124.9 (C-5), 109.5 (C-8), 117.6 (C-4a); 51.6
(C-10), 49.4 (OCH3), 16.1 (C-30); (Found C, 66.36; H, 5.09;
N, 8.56; C18H16N2O4, requires C, 66.66; H, 4.97; N, 8.64).
L-Methyl 2-((8-methyl-4-oxo-4H-benz[e][1,3]oxazin-2-yl)
yl)amino)propanoate 12b 8-Methyl-2-(Methylthio)-4H-
benz[e][1,3]oxazin-4-one (0.52 g, 2.5 mmol) 11b was al-
lowed to react with L-alanine methyl ester 9a (0.31 g,
3 mmol) according to general procedure A. The crude solid
was collected and recrystallized from toluene to give 12b
(0.47 g, 72 % yield), mp 217–220 °C. mmax (KBr)/cm-1
3,279, 2,923 (N–H), 1,751 (C=O), 1,663 (C=O), 1,624
(C=C), 1,489 (C=N); 1HNMR (200 MHz, d6-DMSO) d
8.63 (bs, 1H, N–H), 7.72 (d, 1H, J = 7.5 Hz, H-5), 7.53 (d,
1H, J = 7.5 Hz, H-7), 7.21 (t, 1H, J = 7.5 Hz, H-6), 4.49
(q, 1H, J = 7.3 Hz, H-10), 3.71 (s, 3H, OCH3), 2.42 (s, 3H,
8-CH3). 1.4 (d, 3H, J = 7.3 Hz, H-30); 13CNMR (50 MHz,
d6-DMSO) d 171.7 (C-20), 165.1 (C-4), 157.4 (C-2), 151.6
L-Methyl 2-((7-ethoxy-4-oxo-4H-benz[e][1,3]oxazin-2-yl)-
amino)propanoate
12d 7-Ethoxy-2-(methylthio)-4H-
benz[e][1,3]oxazin-4-one 11d (0.59 g, 2.5 mmol) was al-
lowed to react with L-alanine methyl ester 9a (0.31 g,
3 mmol) according to general procedure A. The crude solid
was collected and recrystallized from ethyl acetate to give
12d (0.48 g, 65 % yield), mp 203 °C. mmax (KBr)/cm-1
3,234, 2,838 (N–H), 1,765 (C=O), 1,674 (C=O), 1,558
1
(C=N); HNMR (200 MHz, d6-DMSO) d 8.91 (s, 1H, N–
123

Med Chem Res
H), 7.73 (d, 1H, J = 8.6 Hz, H-5), 6.92 (dd, 1H,
JH6,H8 = 2.4 Hz, JH6,H5 = 8.7 Hz, H-6), 6.81 (d, 1H,
J = 2.2 Hz, H-8), 4.43 (q, 1H, J = 7.3 Hz, H-10), 4.12 (q,
2H, J = 5.6, OCH2), 3.72 (s, 3H, OCH3), 1.40 (d, 3H,
J = 7.3 Hz, H-30), 1.30 (t, 3H, J = 5.6, CH3); 13CNMR
(50 MHz, d6-DMSO) d 171.5 (C-20), 169.1 (C-4), 162.7
(C-7), 157.0 (C-2), 154.4 (C-8a), 127.7 (C-5), 112.8 (C-6),
110.1 (C-4a), 99.8 (C-8), 63.7 (OCH2), 51.3 (C-10), 49.1
(OCH3), 16.1 (C-30), 13.6 (CH3); (Found C, 57.34; H, 5.56;
N, 9.72; C14H16N2O5, requires C, 57.53; H, 5.52; N, 9.58).
3 mmol) according to general procedure A. The crude solid
was collected and recrystallized from ethanol to give 12f
(0.46 g, 70 % yield), mp 199–201 °C. mmax (KBr)/cm-1
3,300–2,954 (O–H), 3,246, 2,867 (N–H), 1,743 (C=O),
1,655 (C=O), 1,573 (C=N); 1HNMR (200 MHz, d6-
DMSO) d 10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–H), 7.53
(d, 1H, J = 8.4 Hz, H-5), 6.82 (d, 1H, J = 8.4 Hz, H-6),
6.6 (s, 1H, H-8), 4.5 (q, 1H, J = 7.3 Hz, H-10), 3.7 (s, 3H,
OCH3),1.40 (d, 3H, J = 7.3 Hz, H-30); 13CNMR (50 MHz,
d6-DMSO) d 171.6 (C-20), 164.9 (C-4), 159.7 (C-7), 157.1
(C-2), 152.7 (C-8a), 124.3 (C-5), 112.2 (C-6), 109.5 (C-8),
108.9 (C-4a), 51.2 (C-10), 49.1 (OCH3), 16.2 (C-30); (Found
C, 54.66; H, 4.79; N, 10.79; C12H12N2O5, requires C,
54.55; H, 4.58; N, 10.60).
L-methyl 2-((7-methoxy-4-oxo-4H-benz[e][1,3]oxazin-2-yl)
amino)propanoate 12e 7-Methoxy-2-(methylthio)-4H-
benz[e][1,3]oxazin-4-one 11e (0.56 g, 2.5 mmol) was al-
lowed to react with L-alanine methyl ester 9a (0.31 g,
3 mmol) according to general procedure A. The crude solid
was collected and recrystallized from ethyl acetate to give
12e (0.49 g, 70 % yield), mp 176 °C. mmax (KBr)/cm-1
3,234, 2,838 (N–H), 1,765 (C=O), 1,674 (C=O), 1,558
(C=N); 1HNMR (200 MHz, d6-DMSO) d 8,62 (bs, 1H, N–
H), 7.83 (d, 1H, J = 8.6 Hz, H-5), 6.92 (dd, 1H,
JH6,H8 = 2.4 Hz, JH6,H5 = 8.7 Hz, H-6), 6.81 (d, 1H,
J = 2.2 Hz, H-8), 4.50 (q, 1H, J = 7.3 Hz, H-10), 3.91 (s,
3H, 7-OCH3), 3.72 (s, 3H, OCH3), 1.40 (d, 3H,
J = 7.3 Hz, H-30); 13CNMR (50 MHz, d6-DMSO) d 171.4
(C-20), 164.2 (C-4), 163.5 (C-7), 157.0 (C-2), 154.4 (C-8a),
127.7 (C-5), 112.4 (C-6), 110.2 (C-4a), 99.3 (C-8), 55.4 (7-
OCH3), 51.3 (C-10), 49.1 (OCH3), 16.1 (C-30); (Found C,
55.95; H, 5.06; N, 10.21; C13H14N2O5, requires C, 56.11;
H, 5.07; N, 10.07).
L-Methyl-2-((7-hydroxy-8-methyl-4-oxo-4H-benz[e][1,3]ox-
azin-2-yl)amino)propanoate 12g 7-hydroxy-8-methyl-2-
(methylthio)-4H-benz[e][1,3]oxazin-4-one 11g (0.56 g,
2.5 mmol)was allowed to react with L-alanine methyl ester
9a (0.31 g, 3 mmol) according to general procedure A. The
crude solid was collected and recrystallized from ethanol to
give 12g (0.45 g, 60 % yield), mp 204 °C. mmax (KBr)/
cm-1 3,200–2,954 (OH), 3,246, 2,867 (N–H), 1,746
(C=O), 1,650 (C=O), 1,569 (C=N); 1HNMR (200 MHz, d6-
DMSO) d 10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–H), 7.50
(d, 1H, J = 8.4 Hz, H-5), 6.83 (d, 1H, J = 8.4 Hz, H-6),
4.50 (q, 1H, J = 7.3 Hz, H-10), 3.71 (s, 3H, OCH3), 2.22
(s, 3H, 8-CH3). 1.40 (d, 3H, J = 7.3 Hz, H-30); 13CNMR
(50 MHz, d6-DMSO) d 171.6 (C-20), 164.9 (C-4), 159.7
(C-7), 157.1 (C-2), 152.7 (C-8a), 124.3 (C-5), 112.2 (C-6),
109.5 (C-8), 108.9 (C-4a), 51.2 (C-10), 49.1 (OCH3), 16.2
(C-30), 6.8 (8-CH3); (Found C, 56.06; H, 5.09; N, 10.06;
C13H14N2O5, requires C, 56.11; H, 5.07; N, 10.07).
D,L-Methyl 2-((7-methoxy-4-oxo-4H-benz[e][1,3]oxazin-
2-yl)amino)propanoate 12e* 7-Methoxy-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11e (0.56 g, 2.5 mmol) was
allowed to react with D,L-alanine methyl ester 9a* (0.31 g,
3 mmol) according to general procedure A. The crude solid
was collected and recrystallized from ethyl acetate to give
12e* (60 % yield), mp 176 °C. mmax (KBr)/cm-1 3,234,
2,838 (N–H), 1,765 (C=O), 1,674 (C=O), 1,558 (C=N);
1HNMR (200 MHz, d6-DMSO) d 8,62 (bs, 1H, N–H), 7.82
(d, 1H, J = 8.6 Hz, H-5), 6.91 (dd, 1H, JH6,H8 = 2.4 Hz,
JH6,H5 = 8.73 Hz, H-6), 6.82 (d, 1H, J = 2.2 Hz, H-8),
4.53 (q, 1H, J = 7.3 Hz, H-10), 3.91 (s, 3H, 7-OCH3), 3.72
(s, 3H, OCH3), 1.40 (d, 3H, J = 7.3 Hz, H-30); 13CNMR
(50 MHz, d6-DMSO) d 171.4 (C-20), 164.2 (C-4), 163.5
(C-7), 157.0 (C-2), 154.4 (C-8a), 127.7 (C-5), 112.4 (C-6),
110.2 (C-4a), 99.3 (C-8), 55.4 (7-OCH3), 51.3 (C-10), 49.1
(OCH3), 16.1 (C-30); (Found C, 55.95; H, 5.06; N, 10.21;
C13H14N2O5, requires C, 56.11; H, 5.07; N, 10.07).
L-Methyl 3-methyl-2-((8-methyl-4-oxo-4H-benz[e][1,3]ox-
azin-2-yl)amino)pentanoate 13a 8-Methyl-2-(methylth-
io)-4H-benz[e][1,3]oxazin-4-one 11b (0.52 g, 2.5 mmol)
was allowed to react with L-Isoleucine methyl ester ac-
cording 9b (0.44 g, 3 mmol)to general procedure A. The
crude solid was collected and recrystallized from toluene to
give 13a (0.42 g, 55 % yield), mp 175–177 °C. mmax (KBr)/
cm-1 3,280, 2,868 (N–H), 1,761 (C=O), 1,671 (C=O),
1,625 (C=C), 1,487 (C=N); 1HNMR (200 MHz, d6-DMSO)
d 8.61 (bs, 1H, N–H), 7.73 (d, 1H, J = 7.5 Hz, H-5), 7.51
(d, 1H, J = 7.5 Hz, H-7), 7.22 (t, 1H, J = 7.5 Hz, H-6),
4.51 (m, 1H, H-10), 3.70 (s, 3H, OCH3), 2.40 (s, 3H,
8-CH3), 1.72 (m, 3H, H-30/H-40), 0.97 (d, 3H,
J = 6.0 Hz,C60H3), 0.94 (t, 3H, J = 6.5 Hz, C50H3);
13CNMR (50 MHz, d6-DMSO) d 171.7 (C-20), 165.0 (C-4),
157.7 (C-2), 151.6 (C-8a), 134.3 (C-7), 124.4 (C-8), 123.9
(C-6), 123.6 (C-5), 116.7 (C-4a), 52.2 (C-10), 51.2 (OCH3),
40.5 (C-40), 23.9 (C-30), 23.0, 21.3 (C-50/C-60), 13.4 (CH3);
(Found C, 63.06; H, 6.67; N, 9.14; C16H20N2O4, requires
C, 63.14; H, 6.62; N, 9.20).
L-Methyl
2-((7-hydroxy-4-oxo-4H-benz[e][1,3]oxazin-2-
yl)amino)propanoate 12f 7-Hydroxy-2-(methylthio)-4H-
benz[e][1,3]oxazin-4-one 11f (0.52 g, 2.5 mmol)was al-
lowed to react with L-alanine methyl ester 9a (0.31 g,
123

Med Chem Res
L-Methyl-2-((7-hydroxy-4-oxo-4H-benz[e][1,3]oxazin-2-yl)a-
mino)-3-methylpentanoate 13b 7-Hydroxy-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11f (0.52 g, 2.5 mmol)was
allowed to react with L-Isoleucine methyl ester 9b (0.44 g,
3 mmol) according to general procedure A. The crude
material was collected and recrystallized from ethanol to
give 13b (0.39 g, 51 % yield), mp 225 °C. mmax (KBr)/
cm-1 3,300, 2,956 (O–H), 3,246, 2,867 (N–H), 1,752
(C=O), 1,655 (C=O), 1,583 (C=N); 1HNMR (200 MHz, d6-
DMSO) d 10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–H), 7.62
(d, 1H, J = 7.5 Hz, H-5), 6.80 (d, 1H, J = 7.5 Hz, H-6),
4.52 (t, 1H, J = 7.0 Hz, H-10), 3.70 (s, 3H, OCH3), 2.22 (s,
3H, 8-CH3), 1.70 (m, 3H, H-30/H-40), 0.98 (d, 3H,
J = 6.0 Hz, H-50/H-60), 0.94 (d, 3H, J = 6.0 Hz, H-60/H-
H-8), 4.51 (t, 1H, J = 7.0 Hz, H-10), 3.70 (s, 3H, OCH3),
1.70 (m, 3H, H-30/H-40), 0.99 (d, 3H, J = 6.0 Hz, H-50/H-
60), 0.94 (d, 3H, J = 6.0 Hz, H-60/H-50); 13CNMR
(50 MHz, d6-DMSO) d 171.3 (C-20), 164.7 (C-4), 157.9
(C-2), 153.2 (C-8a), 133.5 (C-7), 126.2 (C-5), 124.7 (C-6),
117.1 (C-4a), 115.0 (C-8), 52.2 (C-10), 51.2 (OCH3), 40.5
(C-40), 23.9 (C-30), 21.8, 20.8 (C-50/60). (Found C, 62.06;
H, 6.30; N, 9.50; C15H18N2O4, requires C, 62.06; H, 6.25;
N, 9.65).
L-Methyl 4-methyl-2-((8-methyl-4-oxo-4H-benz[e][1,3]ox-
azin-2-yl)amino)pentanoate 14b 8-Methyl-2-(methylth-
io)-4H-benz[e][1,3]oxazin-4-one 11b (0.52 g, 2.5 mmol)
was allowed to react with L-Leucine methyl ester 9c
(0.44 g, 3 mmol)according to general procedure I. The
crude solid was collected and recrystallized from toluene to
give 14b (0.53 g, 70 % yield), mp 177 °C. mmax (KBr)/
cm-1 3,280, 2,868 (N–H), 1,761 (C=O), 1,671 (C=O),
1,625 (C=C), 1,487 (C=N); 1HNMR (200 MHz, d6-DMSO)
d 8.70 (s, 1H, N–H), 7.70 (d, 1H, J = 7.5 Hz, H-5), 7.52
(d, 1H, J = 7.5 Hz, H-7), 7.2 (t, 1H, J = 7.5 Hz, H-6),
4.51 (t, 1H, J = 7.0 Hz, H-10), 3.70 (s, 3H, OCH3), 2.42 (s,
3H, 8-CH3), 1.71 (m, 3H, H-30/H-40), 0.99 (d, 3H,
J = 6.0 Hz, H-50/H-60), 0.94 (d, 3H, J = 6.0 Hz, H-60/H-
13
50); CNMR (50 MHz, d6-DMSO) d 171.9 (C-20), 167.3
(C-4), 160.3 (C-7), 152.7 (C-2), 151.8 (C-8a), 124.3 (C-5),
112.2 (C-6), 109.4 (C-8), 108.9 (C-4a), 52.2 (C-10), 51.2
(OCH3), 41.5 (C-40), 23.9 (C-30), 21.8, 20.8 (C-50/C-60), 6.8
(8-CH3); (Found C,58.75; H, 5.94; N, 8.80; C15H18N2O5,
requires C, 58.82; H, 5.92; N, 9.15).
L-Methyl-2-((7-hydroxy-8-methyl-4-oxo-4H-benz[e][1,3]oxaz-
in-2-yl)amino)-3-methylpentanoate 13c 7-Hydroxy-8-
methyl-2-(methylthio)-4H-benz[e][1,3]oxazin-4-one 11g
(0.56 g, 2.5 mmol) was allowed to react with L-Isoleucine
methyl ester 9b (0.44 g, 3 mmol)according to general
procedure A. The crude material was collected and re-
crystallized from ethanol to give 13c (0.48 g, 60 % yield),
mp 152 °C. mmax (KBr)/cm-1 3,300–2,956 (O–H), 3,246,
2,867 (N–H), 1,752 (C=O), 1,655 (C=O), 1,583 (C=N);
1HNMR (200 MHz, d6-DMSO) d 10.22 (bs, 1H, O–H),
8,61 (bs, 1H, N–H), 7.62 (d, 1H, J = 7.5 Hz, H-5), 6.81 (d,
1H, J = 7.5 Hz, H-6), 4.50 (t, 1H, J = 7.0 Hz, H-10), 3.70
(s, 3H, OCH3), 2.22 (s, 3H, 8-CH3), 1.70 (m, 3H, H-30/H-
40), 0.98 (d, 3H, J = 6.0 Hz, H-50/H-60), 0.94 (d, 3H,
J = 6.0 Hz, H-60/H-50); 13CNMR (50 MHz, d6-DMSO) d
171.9 (C-20), 167.3 (C-4), 160.3 (C-7), 152.7 (C-2), 151.8
(C-8a), 124.3 (C-5), 112.2 (C-6), 109.4 (C-8), 108.9 (C-4a),
52.2 (C-10), 51.2 (OCH3), 41.5 (C-40), 23.9 (C-30), 21.8,
20..8 (C-50, 60), 6.8 (8-CH3); (Found C, 59.75; H, 6.10; N,
8.87; C16H20N2O5, requires C, 59.99; H, 6.29; N, 8.74).
13
50); CNMR (50 MHz, d6-DMSO) d 172.6 (C-20), 166.0
(C-4), 158.4 (C-2), 151.9 (C-8a), 135.5 (C-7), 124.8 (C-8),
125.2 (C-6), 124.5 (C-5), 117.2 (C-4a), 53.1 (C-10), 52.4
(OCH3), 40.5 (C-40), 24.7 (C-30), 23.0, 21..3 (C-50, 60), 14.6
(CH3); (Found C, 63.06; H, 6.67; N, 9.14; C16H20N2O4,
requires C, 63.14; H, 6.62; N, 9.20).
L-Methyl 4-methyl-2-((4-oxo-8-phenyl-4H-benz[e][1,3]ox-
azin-2-yl)amino)pentanoate 14c 8-Phenyl-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11c (0.67 g, 2.5 mmol) was
allowed to react with L-Leucine methyl ester according 9c
(0.44 g, 3 mmol)to general procedure A. The crude solid
was collected and recrystallized from toluene to give 14c
(0.55 g, 60 % yield), mp 184 °C. mmax (KBr)/cm-1 3,243,
2,867 (N–H), 1,745 (C=O), 1,689 (C=O), 1,622 (C=C),
1
1
1,475 (C=N); H NMR (200 MHz, d6-DMSO) d HNMR
(200 MHz, d6-DMSO) d 8.51 (s, 1H, N–H), 7.92 (dd, 1H,
JH5,H7 = 1.5 Hz, JH5,H6 = 7.7 Hz, H-5), 7.70 (dd, 1H,
JH7,H5 = 1.5 Hz, JH7,H6 = 7.7 Hz, H-7), 7.60–7.42 (m,
4H, H-6/H-10/H-11/H-12), 4.42 (m, 1H, H-10), 3.62 (s, 3H,
OCH3), 1.70 (m, 3H, H-30/H-40), 0.93 (d, 3H, J = 6.0 Hz,
H-50/H-60), 0.87 (d, 3H, J = 6.0 Hz, H-60/H-50); 13CNMR
(50 MHz, d6-DMSO) d 171.6 (C-20), 163.5 (C-4), 152.3
(C-2), 150.0 (C-8a), 134.6 (C-7), 134.3 (C-9), 128.8 (C-8),
128.6 (C-11), 127.7 (C-10), 127.2 (C-12), 125.5 (C-6),
124.5 (C-5), 117.0 (C-4a), 52.1 (C-10), 51.2 (OCH3), 41.5
(C-40), 24.7 (C-30), 23.0, 21.3 (C-50/60); (Found C, 68.81;
H, 6.16; N, 7.40; C21H22N2O4, requires C,68.84; H, 6.05;
N, 7.65).
L-Methyl 4-methyl-2-((4-oxo-4H-benz[e][1,3]oxazin-2-yl)
amino)pentanoate 14a 2-(Methylthio)-4H-benz[e][1,3]
oxazin-4-one 11a (0.48 g, 2.5 mmol) was allowed to react
with L-Leucine methyl ester 9c (0.44 g, 3 mmol)according
to general procedure A. The crude solid was collected and
recrystallized from toluene to give 14a (0.47 g, 65 %
yield), mp 145–147 °C. mmax (KBr)/cm-1 3,240, 2,870 (N–
H), 1,755 (C=O), 1,682 (C=O), 1,619 (C=C), 1,471 (C=N);
1HNMR (200 MHz, d6-DMSO) d 8.62 (s, 1H, N–H), 7.91
(d, 1H, J = 6.5 Hz, H-5), 7.70 (t, 1H, J = 7.3 Hz, H-7),
7.53 (t, 1H, J = 7.0 Hz, H-6), 7.30 (d, 1H, J = 7.0 Hz,
123

Med Chem Res
L-Methyl-2-((7-hydroxy-4-oxo-4H-benz[e][1,3]oxazin-2-yl)a-
mino)-4-methylpentanoate 14d 7-Hydroxy-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11f (0.52 g, 2.5 mmol) was
allowed to react with L-Leucine methyl ester 9c (0.44 g,
3 mmol)according to general procedure A. The crude solid
was collected and recrystallized from ethanol to give 14d
(0.42 g, 55 % yield), mp 210–213 °C. mmax (KBr)/cm-1
3,300–2,956 (O–H), 3,258, 2,867 (N–H), 1,749 (C=O),
1,654 (C=O), 1,583 (C=N); 1HNMR (200 MHz, d6-
DMSO) d 10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–H), 7.61
(d, 1H, J = 7.5 Hz, H-5), 6.8 (d, 1H, J = 7.5 Hz, H-6),
4.50 (t, 1H, J = 7.0 Hz, H-10), 3.70 (s, 3H, OCH3), 2.22 (s,
3H, 8-CH3), 1.70 (m, 3H, H-30/H-40), 0.98 (d, 3H,
J = 6.0 Hz, H-50/H-60), 0.94 (d, 3H, J = 6.0 Hz, H-60/H-
1H, H-30), 1.0 (d, 6H, J = 7.3 Hz, H-40/H-50), 0.99 (d, 3H,
J = 7.3 Hz, H-40/H-50); 13CNMR (50 MHz, d6-DMSO) d
171.2 (C-20), 167.4 (C-4), 158.5 (C-8a), 154.5 (C-2), 141.5
(C-7), 128.8 (C-5), 123.4 (C-4a), 119.8 (C-6), 109.4 (C-8),
55.4 (C-10), 51.9 (OCH3), 29.3 (C-30), 18.1, 17.7 (C40,50);
(Found C, 62.56 %; H, 6.45 %; N, 9.64 %; C15H18N2O4,
requires C, 62.06 %; H, 6.25 %; N, 9.65 %).
L-Methyl 2-((7-hydroxy-4-oxo-4H-benz[e][1,3]oxazin-2-yl)
amino)-3-methylbutanoate 15b 7-Hydroxy-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11e (0.52 g, 2.5 mmol) was
allowed to react with L-valine methyl ester 9d (0.39 g,
3 mmol)according to general procedure A. The crude
material was collected and recrystallized from ethanol to
give 15b (0.44 g, 60 % yield), mp decomp 220 °C. mmax
3,249–2,963 (OH), 3,246, 2,867 (N–H), 1,745 (C=O),
13
50); CNMR (50 MHz, d6-DMSO) d 171.9 (C-20), 167.3
(C-4), 160.3 (C-7), 152.7 (C-2), 151.8 (C-8a), 124.3 (C-5),
112.2 (C-6), 109.4 (C-8), 108.9 (C-4a), 52.2 (C-10), 51.2
(OCH3), 41.5 (C-40), 23.9 (C-30), 21.8, 20.8 (C-50, 60), 6.8
(8-CH3); (Found C, 59.85; H, 6.20; N, 8.33; C16H20N2O5,
requires C, 59.99; H, 6.29; N, 8.74).
1,655 (C=O), 1,598 (C=N) cm-1
MHz,DMSO-d6) d 10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–
H), 7.70 (d, 1H, J = 8.4 Hz, H-5), 6.82 (dd, 1H, JH6,H8
;
1HNMR (200
=
2.4 Hz, JH6,H5 = 8.4 Hz, H-6), 6.62 (d, 1H, J = 2.2 Hz,
H-8), 4.32 (d, 1H, J = 7.2 Hz, H-10), 3.70 (s, 3H, OCH3),
2.22 (m, 1H, H-30), 1.00 (d, 3H, J = 7.3 Hz, H-40/H-50),
0.99 (d, 3H, J = 7.3 Hz, H-40/H-50). 13CNMR (50
MHz,DMSO-d6) d 170.5 (C-20), 164.4 (C-4), 162.2 (C-7),
157.5 (C-2), 154.5 (C-8a), 127.8 (C-5), 113.4 (C-6), 109.0
(C-4a), 100.4 (C-8), 59.4 (C-10), 51.0 (OCH3), 29.3 (C-30),
18.1, 17.7 (C40,50); (Found C, 55.56; H, 5.45; N, 9.04;
C14H16N2O5, 1/2 H2O requires C, 57.53; H, 5.52; N, 9.58).
L-methyl-2-((7-hydroxy-8-methyl-4-oxo-4H-benz[e][1,3]oxaz-
in-2-yl)amino)-4-methylpentanoate 14e 7-Hydroxy-8-
methyl-2-(methylthio)-4H-benz[e][1,3]oxazin-4-one 11g
(0.56 g, 2.5 mmol) was allowed to react with L-Leucine
methyl ester 9c (0.44 g, 3 mmol)according to general
procedure A. The crude material was collected and re-
crystallized from ethanol to give 14e (0.54 g, 67 % yield),
mp 195–197 °C. mmax 3,300–2,956 (OH), 3,246, 2,867 (N–
L-Methyl-2-((7-hydroxy-8-methyl-4-oxo-4H-benz[e][1,3]oxaz-
in-2-yl)amino)-3-methylbutanoate 15c 7-Hydroxy-8-methyl-
2-(methylthio)-4H-benz[e][1,3]oxazin-4-one 11g (0.56 g,
2.5 mmol)was allowed to react with L-valine methyl ester 9d
(0.39 g, 3 mmol) according to general procedure A. The
crude material was collected and recrystallized from ethanol
to give 15c (0.42 g, 55 % yield), mp 220 °C. mmax
3,200–2,954 (OH), 3,246, 2,867 (N–H), 1,746 (C=O), 1,653
(C=O), 1,589 (C=N) cm-1; 1HNMR (200 MHz, DMSO-d6)
d 10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–H), 7.51 (d, 1H,
J = 8.6 Hz, H-5), 6.8 (d, 1H, J = 8.6 Hz, H-6), 4.32 (d, 1H,
J = 7.2 Hz, H-10), 3.70 (s, 3H, OCH3), 2.32 (m, 1H, H-30),
2.22 (s, 3H, 8-CH3), 1.00 (d, 3H, J = 7.3 Hz, H-40/H-50),
0.99 (d, 3H, J = 7.3 Hz, H-40/H-50). 13CNMR (50 MHz,
DMSO-d6) d 170.6 (C-20), 165.1 (C-4), 159.7 (C-7), 157.1
(C-2), 152.5 (C-8a), 124.3 (C-5), 112.2 (C-6), 109.6 (C-8),
108.9 (C-4a), 59.4 (C-10), 50.9 (OCH3), 29.3 (C-30), 18.1,
17.8 (C40, 50), 6.9 (8-CH3); (Found C, 58.35; H, 5.83; N,
9.52; C15H18N2O5, requires C, 58.82; H, 5.92; N, 9.15).
H), 1,752 (C=O), 1,655 (C=O), 1,583 (C=N) cm-1
;
1HNMR (200 MHz, DMSO-d6) d 10.22 (bs, 1H, O–H),
8,61 (bs, 1H, N–H), 7.61 (d, 1H, J = 7.5 Hz, H-5), 6.83 (d,
1H, J = 7.5 Hz, H-6), 4.52 (t, 1H, J = 7.0 Hz, H-10), 3.70
(s, 3H, OCH3), 2.22 (s, 3H, 8-CH3), 1.70 (m, 3H, H-30/H-
40), 0.98 (d, 3H, J = 6.0 Hz, H-50/H-60), 0.94 (d, 3H,
J = 6.0 Hz, H-60/H-50). 13CNMR (50 MHz, DMSO-d6) d
171.9 (C-20), 167.3 (C-4), 160.3 (C-7), 152.7 (C-2), 151.8
(C-8a), 124.3 (C-5), 112.2 (C-6), 109.4 (C-8), 108.9 (C-4a),
52.2 (C-10), 51.2 (OCH3), 41.5 (C-40), 23.9 (C-30), 21.8,
20.8 (C-50, 60), 6.8 (8-CH3); (Found C, 59.85; H, 6.20; N,
8.33; C16H20N2O5, requires C, 59.99; H, 6.29; N, 8.74).
L-Methyl 3-methyl-2-((8-methyl-4-oxo-4H-benz[e][1,3]ox-
azin-2-yl)amino)butanoate 15a 8-Methyl-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11b (0.52 g, 2.5 mmol) was
allowed to react with L-valine methyl ester 9d (0.39 g,
3 mmol)according to general procedure A. The crude solid
was collected and recrystallized from ethyl acetate to give
15a (0.46 g, 63 % yield), mp 207–209 °C. mmax (KBr)/
cm-1 3,280, 2,868 (N–H), 1,761 (C=O), 1,671 (C=O),
1,625 (C=C), 1,487 (C=N); 1HNMR (200 MHz, d6-DMSO)
d 8.61 (bs, 1H, N–H), 7.70 (d, 1H, J = 7.5 Hz, H-5), 7.51
(d, 1H, J = 7.5 Hz, H-7), 7.22 (t, 1H, J = 7.5 Hz, H-6),
4.32 (d, 1H, J = 7.2 Hz, H-10), 3.71 (s, 3H, OCH3), 2.2 (m,
L-Methyl 2-((8-methyl-4-oxo-4H-benz[e][1,3]oxazin-2-yl)
amino)-3-phenylpropanoate 16a 8-Methyl-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11b (0.56 g, 2.5) mmol)was
allowed to react with L-Phenylalanine methyl ester 9e
(0.54 g, 3 mmol) according to general procedure A. The
123

Med Chem Res
crude solid was collected and recrystallized from
toluene/cyclohexane to give 16a (0.46 g, 55 % yield), mp
205 °C. mmax (KBr)/cm-1 3,189, 2,867 (N–H), 1,755
(C50/C-90), 125.9 (C-70), 124.3 (C-5), 112.2 (C-6), 109.5
(C-8), 108.9 (C-4a), 55.0 (C-10), 51.2 (OCH3), 36.2 (C-30);
(Found C, 63.80; H, 4.90; N, 8.59; C18H16N2O5, requires
C, 63.52; H, 4.74; N, 8.23).
1
(C=O), 1,675 (C=O), 1,625 (C=C), 1,476 (C=N); HNMR
(200 MHz, d6-DMSO) d 8.62 (s, 1H, N–H), 7.81 (d, 1H,
Hz, J = 7.5 Hz, H-5), 7.51 (d, 1H, J = 7.6 Hz, H-7), 7.42
(t, 1H, J = 7.5 Hz, H-6), 7.22 (m, 5H, ArH50/90), 4.61 (m,
1H, H-10), 3.62 (s, 3H, OCH3), 3.12 (AB part of ABX
system, 2H, 13.5 Hz, H-30); 13CNMR (50 MHz, d6-
DMSO) d 171.2 (C-20), 166.7 (C-4), 158.4 (C-2), 150.8 (C-
8a), 136.4 (C-40), 134.1 (C-7), 128.6, 128.3, 127.8, 127.5,
127.3, (CH, of C-50/C-60/C-70/C-80C-90), 124.8 (C-8), 125.4
(C-6), 124.5 (C-5), 117.4 (C-4a), 55.0 (C-10), 52.3 (OCH3),
35.9 (C-30); (Found C, 67.59; H, 5.54; N, 8.95;
C19H18N2O4, requires C, 67.44; H, 5.36; N, 8.28).
L-Methyl-2-((7-hydroxy-8-methyl-4-oxo-4H-benz[e][1,3]oxaz-
in-2-yl)amino)-3-phenylpropanoate
16d 7-Hydroxy-8-
methyl-2-(methylthio)-4H-benz[e][1,3]oxazin-4-one 11g
(0.5,6 g, 2.5 mmol) was allowed to react with L-Pheny-
lalanine methyl ester 9e (0.54 g, 3 mmol) according to
general procedure A. The crude solid was collected and
recrystallized from ethanol to give 16d (0.49 g, 53 %
yield), mp 195–197 °C. mmax (KBr)/cm-1 3,300–2,954 (O–
H), 3,246, 2,867 (N–H), 1,746 (C=O), 1,650 (C=O), 1,569
1
(C=N); HNMR (200 MHz, d6-DMSO) d 10.22 (bs, 1H,
O–H), 8,61 (bs, 1H, N–H), d 7.61 (d, 1H, J = 8.4 Hz,
H-5), 6.81 (d, 1H, J = 8.4 Hz, H-6), 4.52 (q, 1H,
J = 7.3 Hz, H-10), 3.70 (s, 3H, OCH3), 2.22 (s, 3H,
8-CH3). 1.43 (d, 3H, J = 7.3 Hz, H-30); 13CNMR
(50 MHz, d6-DMSO) d 171.6 (C-20), 164.9 (C-4), 159.7
(C-7), 157.1 (C-2), 152.7 (C-8a), 124.3 (C-5), 112.2 (C-6),
109.5 (C-8), 108.9 (C-4a), 51.2 (C-10), 49.1 (OCH3), 16.2
(C-30), 6.8 (8-CH3); (Found C, 64.65; H, 5.10; N, 8.05;
C19H18N2O5, requires C, 64.40; H, 5.12; N, 7.91 %).
L-Methyl 2-((4-oxo-8-phenyl-4H-benz[e][1,3]oxazin-2-yl)a-
mino)-3-phenylpropanoate 16b 8-Phenyl-2-(methylthio)-
4H-benz[e][1,3]oxazin-4-one 11c (0.67 g, 2.5 mmol) was
allowed to react with L-Phenylalanine methyl ester 9e
(0.54 g, 3 mmol) according to general procedure I. The
crude solid was collected and recrystallized from
toluene/cyclohexane to give 16b (0.55 g, 55 % yield), mp
183 °C. mmax (KBr)/cm-1 3,189, 2,867 (N–H), 1,745
1
(C=O), 1,681 (C=O), 1,645 (C=C), 1,476 (C=N); HNMR
L-Methyl 1-(4-oxo-4H-benz[e][1,3]oxazin-2-yl)pyrrolidine-2-
carboxylate 17a 2-(Methylthio)-4H-benz[e][1,3]oxazin-
4-one 11a (0.48 g, 2.5 mmol) was allowed to react with L-
Proline methyl ester 9f (0.39 g, 3 mmol) according to
general procedure A. The crude solid was collected and
recrystallized from toluene to give 17a (0.41 g, 60 %
yield), mp 139–141 °C. mmax (KBr)/cm-1 1,741 (C=O),
1,675 (C=O), 1,619 (C=C), 1,466 (C=N); 1HNMR
(200 MHz, d6-DMSO) d 8.61 (bs, 1H, N–H), 7.92 (d, 1H,
J = 6.5 Hz, H-5), 7.70 (t, 1H, J = 7.3 Hz, H-7), 7.51 (t,
1H, J = 7.0 Hz, H-6), 7.32 (d, 1H, J = 7.0 Hz, H-8), 4.70
(m, 1H, H-10), 3.70 (s, 3H, OCH3), 3.70 (m, 1H and m, 1H,
C50H2), 2.2 (m, 4H, C30H2 and C40H2); 13CNMR (50 MHz,
d6-DMSO) d 171.2 (C-20), 164.4 (C-4), 155.7 (C-2), 152.9
(C-8a), 133.4 (C-7), 126.2 (C-5), 124.9 (C-6), 116.8 (C-4a),
115.1 (C-8), 58.9 (C-10), 51.4 (OCH3), 46.5 (C-50), 29.0 (C-
30), 22.6 (C-40).
(200 MHz, d6-DMSO) d 8.61 (s, 1H, N–H), 7.90 (dd, 1H,
JH5,H7 = 1.6 Hz, JH5,H6 = 7.6 Hz, H-5), 7.70 (dd, 1H,
JH7,H5 = 1.6 Hz, JH7,H6 = 7.6 Hz, H-7), 7.60–7.40 (m,
4H, H-6/H-10/H-11/H-12), 7.2 (m, 5H, ArH50/90), 4.61 (m,
1H, H-10), 3.63 (s, 3H, OCH3), 3.12 (AB part of ABX
system, 2H, 13.5 Hz, H-30); 13CNMR (50 MHz, d6-
DMSO) d 170.2 (C-20), 164.7 (C-4), 157.7 (C-2), 149.8 (C-
8a), 136.4 (C-40), 134.4 (C-9), 134.1 (C-7), 128.6, 128.3,
127.8, 127.5, 127.3, 125.9 (CH, of C10, 11, 12 and C50, 60,
70, 80, 90), 125.4 (C-6), 124.5 (C-5), 117.4 (C-4a), 55.0 (C-
10), 51.3 (OCH3), 35.9 (C-30); (Found C, 72.09; H, 5.14; N,
6.95; C24H20N2O4, requires C, 71.99; H, 5.03; N, 7.00).
L-Methyl-2-((7-hydroxy-4-oxo-4H-benz[e][1,3]oxazin-2-yl)a-
mino)-3-phenylpropanoate 16c 7-Hydroxy-2-(methylth-
io)-4H-benz[e][1,3]oxazin-4-one 11f (0.5,2 g, 2.5 mmol)
was allowed to react with L-Phenylalanine methyl ester 9e
(0.54 g, 3 mmol) according to general procedure A. The
crude solid was collected and recrystallized from ethanol to
give 16c (0.43 g, 50 % yield), mp 195–197 °C. mmax (KBr)/
cm-1 3,300–2,954 (O–H), 3,246, 2,867 (N–H), 1,743
(C=O), 1,655 (C=O), 1,573 (C=N); 1HNMR (200 MHz, d6-
DMSO) d 10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–H), 7.51
(d, 1H, J = 8.5 Hz, H-5), 7.22 (m, 5H, ArH50/90), 6.83 (d,
1H, J = 8.5 Hz, H-6), 6.62 (s, 1H, H-8), 4.7 (m, 1H, H-10),
3.7 0 (s, 3H, OCH3), 3.12 (AB part of ABX system, 2H,
13.5 Hz, H-30). 2.12 (s, 3H, 8-CH3); 13CNMR (50 MHz,
d6-DMSO) d 170.8 (C-20), 165.1 (C-4), 159.8 (C-7), 157.5
(C-2), 152.4 (C-8a), 136.7 (C-40), 128,3 (C60/C-80), 127,6
L-Methyl 1-(8-methyl-4-oxo-4H-benz[e][1,3]oxazin-2-yl)pyrro-
lidine-2-carboxylate
17b 8-Methyl-2-(methylthio)-4H-
benz[e][1,3]oxazin-4-one 11b (0.56 g, 2.5) mmol)was al-
lowed to react with L-Proline methyl ester 9f (0.39 g,
3 mmol) according to general procedure I. The crude solid
was collected and recrystallized from toluene\cyclohexane
to give 17b (0.49 g, 68 % yield), mp 164 °C. mmax (KBr)/
cm-1 1,756 (C=O), 1,676 (C=O), 1,621 (C=C), 1,483
(C=N); 1HNMR (200 MHz, d6-DMSO) d 8.64 (bs, 1H, N–
H), 7.70 (d, 1H, J = 7.5 Hz, H-5), 7.51 (d, 1H,
J = 7.5 Hz, H-7), 7.32 (t, 1H, J = 7.5 Hz, H-6), 4.70 (m,
123

Med Chem Res
1H, H-10), 3.70 (s, 3H, OCH3), 3.73 (m, 1H and m, 1H,
C50H2), 2.32 (s, 3H, 8-CH3), 2.12 (m, 4H, C30H2 and
C40H2); CNMR (50 MHz, d6-DMSO) d 171.0 (C-20),
164.9 (C-4), 155.4 (C-2), 151.3 (C-8a), 134.2 (C-7), 124.4
(C-8), 124.1 (C-6), 123.6 (C-5), 116.6 (C-4a), 58.7 (C-10),
51.4 (OCH3), 46.5 (C-50), 29.1 (C-30), 22.6 (C-40), 13.2
(CH3); (Found C, 62.46; H, 5.38; N, 9.86; C15H16N2O4,
requires C, 62.49; H, 5.59; N, 9.72).
overnight at room temperature. At the completion, the re-
action mixture was neutralized with 10 % hydrochloric
acid and the solid was separated by vacuum filtration and
recrystallized from ethanol and gave white solid 18
(0.16 g, 58 %), mp decomp 195–198. mmax (KBr)/cm-1
3,286, (N–H), 1,706 (carboxylic C=O), 1,654 (C=O), 1,624
(C=C), 1,443 (C=N); 1HNMR (300 MHz, d6-DMSO) d
10.80 (bs, 1H, OH), 8.69 (d, 1H, J = 8.1 Hz N–H), 7.84
(d, 1H, J = 7.2 Hz, H-5), 7.34 (d, 1H, J = 7.2 Hz, H-7),
6.83 (t, 1H, J = 7.7 Hz, H-6),), 4.24 (d,d, 1H,
13
L-Methyl 1-(7-hydroxy-4-oxo-4H-benz[e][1,3]oxazin-2-yl)pyrro-
lidine-2-carboxylate
17c 7-Hydroxy-2-(methylthio)-4H-
JNH = 8.4 Hz, JH3 = 4.8, H-10), 2.22 (s, 3H, 8-CH3). 2.19
0
benz[e][1,3]oxazin-4-one 11f (0.52 g, 2.5) was allowed to
react with L-Proline methyl ester 9f (0.39 g, 3 mmol) ac-
cording to general procedure A. The crude solid was col-
lected and recrystallized from ethanol to give 17c (0.44 g,
60 % yield), mp 201 °C. mmax (KBr)/cm-1 3,058–2,851
(m, 1H, H-30) 0.94, 0.92 (2d, 6H 2 9 CH3, J = 6.9 and
6.6); CNMR (75 MHz, d6-DMSO) d 172.4 (C-20), 169.8
(C-4), 157.6 (C-2), 152.8 (C-8a), 136.0 (C-7), 127.2 (C-5),
126.8 (C-8), 119.1 (C-6), 115.9 (C-4a), 57.9 (C-10), 30.3
(C-30), 19.1 (8-CH3)17.7, 15.9 (2 9 CH3), (Found C,
60.96; H, 5.79; N, 10.23; C15H16N2O5, requires C, 60.86;
H, 5.84; N, 10.14).
13
1
(O–H), 1,736 (C=O), 1,660 (C=O), 1,539 (C=N); HNMR
(200 MHz, d6-DMSO) d 10.22 (bs, 1H, O–H), 8,61 (bs,
1H, N–H), 7.70 (1H, J = 8.6 Hz, H-5), 6.82 (d, 1H,
J = 8.6 Hz, H-6), 6.61 (s, 1H, H-8), 4.61 (m, 1H, H-10),
3.70 (s, 3H, OCH3), 3.73 (m, 1H and m, 1H, C50H2), 2.22
(m, 4H, C30H2 and C40H2); 13CNMR (50 MHz, d6-DMSO)
d 171.1 (C-20), 164.4 (C-4), 163.5 (C-7), 155.2 (C-2), 154.5
(C-8a), 127.5 (C-5), 114.1 (C-6), 108.0 (C-4a), 100.5 (C-8),
58.8 (C-10), 51.4 (OCH3), 46.5 (C-50), 29.0 (C-30), 22.6 (C-
40); (Found C, 58.05; H, 4.54; N, 9.49;C14H14N2O5, re-
quires C, 57.93; H, 4.86; N, 9.65).
HeLa cervical cancer cell line used was obtained from
the American Type Culture Collection and was cultured in
Dulbecco’s Modified Eagle Medium (DMEM), Invitrogen,
Grand Island) containing phenol red, supplemented with
10 % fetal bovine (FBS, Sigma-Aldrich, St Louis).
Preparation of stock solutions
L-Methyl-1-(7-hydroxy-8-methyl-4-oxo-4H-benz[e][1,3]oxazin-
2-yl)pyrrolidine-2-carboxylate 17d 7-Hydroxy-8-methyl-2-
(methylthio)-4H-benz[e][1,3]oxazin-4-one 11g (0.5,6 g,
2.5 mmol) was allowed to react with L-Proline methyl ester
9f (0.39 g, 3 mmol) according to general procedure A. The
crude solid was collected and recrystallized from ethanol to
give 17d 0.46 g, (60 % yield), mp decomp, 264 °C. mmax
(KBr)/cm-1 3,300–2,956 (O–H), 1,737 (C=O), 1,656
(C=O), 1,541 (C=N); 1HNMR (200 MHz, d6-DMSO) d
10.22 (bs, 1H, O–H), 8,61 (bs, 1H, N–H), 7.61 (d, 1H,
J = 7.5 Hz, H-5), 6.82 (d, 1H, J = 7.5 Hz, H-6), 4.70 (m,
1H, H-10), 3.70 (s, 3H, OCH3), 3.69 (m, 1H and m, 1H,
C50H2), 2.22 (m, 4H, C30H2 and C40H2), 2.12 (s, 3H,
All stock solutions were made up using DMSO (Sigma-
Aldrich, St. Louis), and Compounds and etoposide (Sigma-
Aldrich, St. Louis) were made up to a 10 mM stock. It
should be noted that in accordance with Lee et al. (2006),
the amount of DMSO added to both test and control media
never exceeded 0.5 % of the total volume, and control
cultures were treated with equivalent volumes of DMSO
that was used to prepare stock solutions of the drug to
eliminate adverse effects of DMSO.
Toxicity and chemo-sensitization (SRB assay)
In order to determine the toxicity of the compounds alone
as well as their sensitizing effects in combination with
etoposide, the sulphonamide blue assay (SRB assay) was
utilized. The method put forward by Freshney (1992) was
used to carry out the assay with slight variations. Cells
were seeded on 24-well plates at a concentration of
2 9 103 cells/well. After cells had adhered overnight,
culture media was removed and replaced by 1 ml of
treatment media. Treatment media was removed after 48 h
before cells were fixed, stained and absorbance was mea-
sured at 538 nm using a Flex Station 3 (Molecular Devices,
California). Absorbance at 538 nm is directly proportional
to overall cell number.
13
8-CH3); CNMR (50 MHz, d6-DMSO) d 171.6 (C-20),
164.9 (C-4), 159.6 (C-7), 155.7 (C-2), 152.7 (C-8a), 124.3
(C-5), 112.4 (C-6), 109.5 (C-8), 108.8 (C-4a), 58.7 (C-10),
51.4 (OCH3), 46.5 (C-50), 29.1 (C-30), 22.6 (C-40), 6.6 (8-
CH3); (Found C, 58.96; H, 5.49; N, 9.13; C15H16N2O5,
requires C, 59.21; H, 5.30; N, 9.21).
L-3-methyl-2-((8-methyl-4-oxo-4H-benz[e][1,3]oxazin-2-
yl)amino)butanoic acid 18 The L-Methyl 3-methyl-2-
((8-methyl-4-oxo-4H-benz[e][1,3]oxazin-2-yl)amino)bu-
tanoate 15a (0.29 g, 1 mmol) was dissolved in anhydrous
methanol (5 mL),to this solution 1 M NaOH (1.1 ml,
1.1 mmol) was added dropwise. The mixture was stirred
123

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DNA-PK inhibition assay
The DNA-PK assays were performed by Reaction Biology
Corporation, One Great Valley Parkway, Suite 2 Malvern,
PA 19355 USA.
Ihmaid SK, Al-Rawi JMA, Bradley CJ, Angove MJ, Robertson MN
(2012) Synthesis, DNA-PK inhibition, antiplatelet activity studies
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All Compounds were dissolved in DMSO and tested for
their ability to inhibit human DNA-PK. Compounds were
tested in a 10-dose IC50 profile with fourfold serial dilution
starting at 100 lM. The Control compound, LY294002,
was tested in a 10-dose IC50 profile with threefold serial
dilutions starting at 10 lM. Reactions were carried out
using 20 lM Peptide substrate [EPPLSQEAFADLWKK],
10 lg/ml DNA and 10 lM ATP using the HTRF assay
format.
Platelet aggregometry
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