NMR studies of new heterocycles tethered to purine moieties with anticancer activity

Full text of DOI:10.1002/mrc.4871

Camacho M Encarnacion (Orcid ID: 0000-0003-1214-5103)
Mikhova Bozhana (Orcid ID: 0000-0001-8851-971X)
NMR studies of new heterocycles tethered to purine moieties with anticancer activity
*
*
Nerea Fernández-Sáez, Joaquín M. Campos, M. Encarnación Camacho, M. Dora Carrión
Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de
Granada, 18071 Granada, Spain.
1
13
1
13
KEYWORDS: H and C 1D NMR; H/ C 2D NMR (HSQC, HMBC); 2D NOESY;
benzoxazine, quinoline, pyridoxazine.
*Corresponding author: M. Encarnación Camacho. Departamento de Química Farmacéutica y
Orgánica. Facultad de Farmacia. Universidad de Granada. c/ Campus de Cartuja, s/n. 18071
Granada, Spain. E-mail: ecamacho@ugr.es. Tfno. +34958243844.
*
Corresponding author: M. Dora Carrión. Departamento de Química Farmacéutica y Orgánica.
Facultad de Farmacia. Universidad de Granada. c/ Campus de Cartuja, s/n. 18071 Granada, Spain.
E-mail: dcarrion@ugr.es. Tfno. +34958240728.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/mrc.4871
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Introduction
Cancer is one of the greatest threats of our society because is one of the leading causes of
death. Many active compounds have been developed to face this problem in recent years.[1-3]
Some purine derivatives with an interesting anti-proliferative activity have been previously
synthesized by combining benzo-fused heterocycles linked to substituted purines.[4-10] In
order to improve the activity and have a deeper idea of the structure-activity relationship, new
compounds have been obtained. Such changes include some bioisosteric replacements as
elimination of oxygen atom of the heterocycle or elongation of the linking chain between the
six-membered heterocycles and the substituted purines.
In this way the synthesis and biological evaluation of these novel families of purine
derivatives linked to six-membered heterocyclic moieties 3a-d, 5a-d and 11a-b, which were
designed and evaluated as anticancer agents, have been described.[11]
1
13
The structures of these new compounds have been determined by H and C NMR and mass
spectrometry MS). Some of them have been studied more detailed in order to corroborate
their skeleton by using two-dimensional techniques, and an elemental analysis have been
performance to all the final compounds.
1
13
The present study reports the unambiguous assignment of each signal in the H and C NMR
spectra in benzoxazine derivatives (3a-d), including elongation of the side chain,
tetrahydroquinolines (5a-d) and pyridoxazine derivatives (11a-b) in which a pyridine ring is
merged to the oxazine heterocycle, using one- and two-dimensional resonance techniques.
The assignment of derivatives 1, 2, 4, 6-10, the precursors in their synthetic pathway, are also
included.
Experimental
NMR spectra
1
13
Nuclear magnetic resonance (NMR) spectra were made on a 400-MHz H and 101 MHz C
1
13
NMR Agilent Varian Direct Drive, and a 500-MHz H and 125-MHz C NMR Agilent
Varian Innova Unity spectrometers at 298 K. In DEPT experiments the employed parameters
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were the following: pulse width (135°), 9.0 ms; recycle time, 1 s; ½ J (CH) = 4 ms; 65 536
data points acquired and transformed from 1024 scans; spectral width, 15 KHz; and line
broadening, 1.3 Hz. Chemical shifts (δ) are quoted in parts per million (ppm) and are
1
13
referenced to the residual solvent peak: CDCl
3
, δ = 7.26 ppm ( H), δ = 77.4 ppm ( C) or
1
13
(
CD
3
)
2
SO, δ = 2.50 ppm ( H), δ = 39.52 ppm ( C). Spin multiplicities are given as s
(singlet), bs (broad singlet), d (doublet), dd (doublet of doublet), ddd (doublet of doubletof
doublet), td (triplet of doublet), t (triplet), tt (triplet of triplet), q (quadruplet), and m
multiplet). Coupling constant (J) are given in Hz.
(
The HSQC spectra were calculated with a pulse sequence gc2hsqcse (Standard sequence,
Agilent Vnmrj_3.2A software). The HMBC spectra were determined with a pulse sequence
gc2hmbc (standard sequence, Agilent Vnmrj_3.2A software) optimized for 8 Hz (inter-pulse
delay for the evolution of long-range couplings: 62.5 ms).
Nuclear Overhauser spectra were recorded on a Agilent Varian Direct Drive spectrometer,
operating at 500 MHz, with a spectral width of 8.01 KHz at 16K complex points (acquisition
time 2s). The mixing time in 1D-NOESY experiment was 0.5 s. Data processing with zero
filling at 64K and apodization with exponential function (LB = 0.5 Hz).
Results and Discussion
Schemes 1-3 depict the synthetic route carried out in the preparation of final compounds 3a-
d, 5a-d and 11a-b, previously reported[11] and Tables 1-4 show the spectroscopic data for
both the intermediate and final synthesized compounds.
Schemes 1-3 may be inserted here
Table 1-2 may be inserted here
The structure of these compounds has been elucidated by routine H and 13C NMR
techniques. Nevertheless, a definitive assignment of all signals was accomplished with the
help of NMR techniques. Such procedures include the following: i) DEPT experiments for
determining the presence of primary, secondary and tertiary carbon atoms; ii) HSQC spectra
to assign the 13C resonances of the primary, secondary and tertiary carbons; iii) HMBC
1
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sequences to corroborate the signals of quaternary carbons via two-bond and three-bond
interactions.
1
Tables 1 and 2 show the H NMR signals of each proton for molecules 1-11, whereas Tables
1
3
3
and 4 show the corresponding C NMR chemical shifts for the same compounds. The
NMR spectra of the intermediates were carried out in CDCl solutions. The NMR spectra of
the final compounds were performed in CDCl solutions, except for 5c, which was
accomplished in DMSO-d for solubility reasons. As a consequence, some significant
variations are observed in the chemical shifts depending of the solvent.
3
3
6
1
13
In relation to the benzoxazine family, chemical shifts of H and C NMR of the tosylated
intermediate 2 and the final derivatives 3a-d are similar for H-2, H-3a, H-3b, H-5, H-6, H-7
and H-8; nevertheless, in the untosylated intermediate 1, H-2 appears at a greater chemical
shift than H-3a. In the same way, C-2 of the intermediate 1 appears at a higher shift than C-2
in both compounds, the tosylated intermediate 2 and the tosylated purines 3a-d. Finally, the
presence of trifluoromethyl group in 3d is justified due to the following C-F couplings:
1
45.20 (q, JC-CF3  37.1, C-6’) and 120.69 (q, JCF  274.9, CF ).
3
1
In addition, in the dihydroquinoline series, the H NMR signals are similar in both, the
intermediate 4 and the final purines 5a-d, except for the CH
derivative 4 the CH linked to the OH group appears as a multiplet at δ = 3.53 ppm, while in
a-d the CH peak, which is linked to the purine moiety, appears as two doublet of doublets
2
signal of the side chain. In
2
5
2
between δ 4.12 - 4.21 ppm and δ 4.20 - 4.32 ppm respectively. Nevertheless, the methylene
group of 5c appears as multiplet centered at δ 4.26 ppm. Moreover, in this family, the main
1
3
difference in C is the CH
2
aliphatic signal, which appears at δ 64.34 ppm in the intermediate
4
and between δ 46.10 - 47.00 in purines 5a-d. Otherwise, the trifluoromethyl group presents
1
3
in 5d is corroborated by the two quadruplets in the CNMR spectrum which appear at δ
45.80 ppm (JC-CF3  37.7, C-6’) and δ 120.80 ppm (JCF  275.9, CF ), respectively.
1
3
In the last pyridoxazine family, the intermediate 6, structurally different from derivatives 7 –
0, does not follow the same coupling pattern of the latter, being similar the signals of the
1
remaining intermediates except for H-3. This proton appears between δ 3.74 – 3.76 ppm in
the untosylated derivatives (7, 8) and at δ 4.95 – 4.98 ppm in the tosylated ones (9, 10),
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respectively. In addition, target molecules 11a and 11b show similar chemical shifts with
respect to their tosylated intermediates, except for the CH X and CH CH X aliphatic signals,
2
2
2
due to the different nature of the heteroatom linked to the aliphatic chain (X = O in the
intermediate compounds; X = N in the final derivatives).
Table 3 may be inserted here
Table 4 may be inserted here
HSQC and HMBC experiments were performed on an intermediate and a final molecule of
each family. Table 5 shows the HSQC correlations for compounds 2, 4, 7, 3c, 5b and 11a,
whereas Figure 1 illustrates the more important connectivities found in the HMBC spectra
ofthe same molecules.
Table 5 may be inserted here
HSQC experiments performed on compounds 2, 4, and 7 allows the assignment of the
secondary carbon atoms chemical shifts C-3, CH
2
OR and CH
2
CH OR, and the assignment of
2
the chemical shifts for tertiary carbon atoms C-5, C-6, C-7 and C-8 in the intermediate
derivatives 1, 2, 4, 6-8 and 10. These atoms show signals in ranges of δ 48.47- 72.24 (C-2), δ
3
1
5.25 - 50.92 (C-3), δ 33.60 - 64.34 (CH
2
OR), δ 33.33 - 35.26 (CH
2
CH OR), δ 115.35 -
2
29.52 (C-5), δ 120.76-140.52 (C-6), δ 113.18 - 125.82 (C-7) and δ 116.60 - 124.46 (C-8).
1
3
Similar HSQC experiments performed on 3c, 5b and 11a indicate that the C NMR signals
for the secondary and tertiary carbon atoms in the purine derivatives 3a-d, 5a-d and 11a-b
are in similar ranges. Such chemical shifts are the following: δ 48.22 - 68.66 (C-2), δ 32.35 -
5
1
8
1.21 (C-3), δ 40.15- 47.00 (CH
2
N), δ 30.63 - 31.88 (CH
2
CH N), δ 30.30 - 31.09 (C-4), δ
2
17.15 - 129.79 (C-5), δ 121.31 - 140.94 (C-6), δ 120.31 - 126.44 (C-7), δ 123.47 -124.79 (C-
), δ 151.02-153.19 (C-2’) (except for 3a, 5a and 11a where C-2’ is a quaternary carbon
atom) and δ 145.03 - 147.92 (C-8’).
The quaternary carbon signals were confirmed by HMBC spectra on the intermediate (2, 4
and 7) and final (3c, 5b and 11a) compounds (Figure 1). In 2, correlation between H-6 (δ
6
.92) and the 13C at 123.51 ppm, allows the unequivocal assignment of C-4a; another
correlation between H-7 (δ 7.04) and the peak at 146.44 ppm allows the identification of C-
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1
3
8
a. In the same way, the H-2’ and H-6’ signals (δ 7.54) correlate with the C peak at 144.07
ppm, making possible to assign this peak to C-4’, and the H-3’ and H-5’ signals (δ 7.23)
correlate to peak at δ 135.41 ppm, being identified as C-1’ accordingly. In 4, correlations
1
3
between H-6 (δ 7.15) and H-5 (δ 7.04) with the C signal which appears at 137.01 ppm allow
to identify it as C-4a. Assignation of C-8a has been possible due to the interactions between
1
3
the signals at δ 7.06 ppm (H-7) and δ 7.68 ppm (H-8) and the C peak at 129.27 ppm.
1
3
Correlation between H-4 (δ 2.24 and δ 2,59 ppm) and the same C peak permits us to assign
this peak to C-8a. Furthermore, the quaternary carbons of the tosyl group C-1’ and C-4’ were
1
3
assigned by the observed correlation among δ 7.21 ppm (H-3’ and H-5’) and the C atom at
36.90 ppm (C-1’). Correlation between δ 7.51 ppm (H-2’ and H-6’) and δ 143.78 ppm
allows us to assign inequivocally this signal to C-4’. Finally, in 7, correlations of H-2a (δ
1
1
3
4
.14), H-2b (δ 3.82) and H-7 (δ 6.49) and the C at δ 139.08 ppm enables the assignment of
C-8a. In addition, the assignment of C-4a is possible due to the observed correlation between
H-6 (δ 7.57) and H-8 (δ 6.93) with the peak at δ 146.99 ppm.
The HMBC experiment performed on 3c indicates correlations between H-3 (δ 4.19) and H-6
(δ 6.93) with the signal δ 123.42 ppm (C-4a). Other correlation exits between H-7 (δ 7.06)
1
3
and the peak which appears at δ 145.88 ppm (C-8a). In the same way, the C at δ 150.39
ppm that correlates with H-2’ (δ 8.69), H-8’ (δ 7.98) and CH N (δ 4.44) can be identified as
2
1
3
C-4’. A correlation of H-2’ (δ 8.69) and the C peak at δ 143.07 ppm allows to identify C-6’
and finally, correlation of H-8’ (δ 7.98) and the signal at 135.20 ppm permits us to identify it
as C-5’. C-1’’ corresponds to a signal appearing at δ 134.11 ppm due to its correlation with
H-3’’ and H-5’’ (δ 7.06). C-4’’ is assigned to the 13C peak at δ 144.55 ppm because is
correlated with H-2’’ and H-6’’ (δ 7.36).
Figure 1 may be inserted here
In 5b, the assignment of the C-4a and C-8a signals of the quaternary carbons included in the
tetrahydroquinoline heterocycle has been deduced through the correlations between H-6 (δ
1
3
7
.20) and the C at 136.33 ppm (C-4a). In the same way, correlations between δ 7.08 (H-7),
δ 6.98 (H-5) and H-4 (δ 2.30 and 2.59) with δ 127.23, permits the latter to be assigned to C-
a. In addition, the quaternary carbons of the purine moiety and the tosyl group have been
8
assigned by similar correlations to those of their benzoxazine isosteres, being these signals C-
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4
4
’ (δ 151.84 ppm), C-5’ (δ 131.59 ppm), C-6’ (δ 151.33 ppm), C-1’’ (δ 136.21 ppm) and C-
’’ (δ 143.99 ppm).
The HMBC experiment performed on 11a, shows that H-6 (δ 8.02) and H-8 (δ 7.15) are
1
3
correlated with the C signal at δ 137.00 ppm, being assigned this to C-4a. H-7 (δ 6.96) and
H-2a (δ 4.18) are correlated with the peak at δ 140.34 ppm (C-8a). Furthermore, the signal
appearing at δ 153.13 ppm is assigned to C-4’ due to its correlation with H-8’ (δ 8.30) and
CH N (δ 4.44 and δ 4.52). In addition, H-8’ is correlated with a signal at δ 130.95, which can
2
be assigned to C-5’. Moreover, the quaternary carbon atoms identification of the tosyl moiety
has been deduced similary as in their two isosteric families, being C-1’’ the peak at δ 136.01
ppm and C-4’’ the δ 144.51 ppm signal. At last, the characterization of quaternary atoms in
the purine skeleton C-2’ and C-6’ has been deduced by analogy to the corresponding purine
chemical shifts already justify in the two previous isosteric families (see Figure 1).
Intermediate 6, precursor of the pyridoxazine family 11a-b, has been studied in detail to
prove its structure. In this way, to obtain only the desired 3-regioisomer we have started from
2
-aminopyridin-3-ol, followed by acylation of its amino group by treatment with acetic
anhydride and pyridine, to decrease the nucleophilicity of the amino group. Subsequently,
cyclization of this intermediate with 4-bromobut-2-enoate (ethyl crotonate) in ethanol and
K
2
CO
described by other authors who obtained a mixture of two pyridoxazine regioisomers (ethyl-
-acetyl-3,4-dihidro-2H-pyrido[3,2-b][1,4]oxazine-2-carboxylate and ethyl-4-acetyl-3,4-
dihidro-2H-pyrido[3,2-b][1,4]oxazine-3-carboxylate), by reaction between N-(3-
hydroxypyridin-2-yl)acetamide and ethyl 2,3-dibromopropionate in the presence of K CO
employing CH CN as solvent [12].
3
only gave 6, but not its 2-regioisomer 12 (Scheme 4). This is different to that
4
2
3
,
3
Scheme 4 may be inserted here
In order to unequivocally determine the structure of 6, 2D NMR (HSQC and HMBC)
experiments have been carried out. HSQC (Table 5) allows the unequivocal assignment of the
tertiary carbon in the pyrido[3,2-b][1,4]oxazine ring C-3, C-6, C-7 and C-8, and the methyl
group of both, acetyl and ethyl carboxylate moieties. These atoms show signals at δ 44.57,
1
39.56, 120.60, 124.30, 25.70 and 13.95 ppm, respectively. In addition, the secondary
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carbons C-2 (δ 67.17 ppm), the CH
2
linker carbon (δ 33.60 ppm) and the CH of the ethyl
2
carboxylate group (δ 60.69 ppm) have also been assigned.
The HMBC experiment to determine the quaternary carbons (Figure 1) shows that H-2a (δ
1
3
4
5
.47 ppm) and H-8 (δ 6.93 ppm) are correlated with the C at δ 140.67 ppm (C-8a). H-3 (δ
.41 ppm) and H-6 (δ 7.96 ppm) peaks are correlated with the signal at δ 138.46 (C-4a). In
addition, both ester and amide CO groups show very similar chemical shifts (δ 170.21 and δ
69.69 ppm). The signal at δ 4.12 ppm of the CH ethyl carboxylate group is correlated with
a peak at δ 170.21 ppm (CO ester group). Furthermore, a corrrelation between the CH amide
1
2
3
13
group (δ 2.59 ppm) and the C at δ 169.69 ppm indicates that this peak is the CO amide
moiety.
Scheme 4 shows two possible structures (6 and 12) that could be formed in the cyclization
1
13
reaction. In our case, we have only obtained 6, as demonstrated by the H/ C HMBC study.
1
3
This experiment corroborates that H-3 (δ 5.41 ppm) is correlated with the C chemical shift
of the amide group (δ 169.69). This correlation confirms the proximity between these groups
and proves that 6 but not 12 is the obtained regioisomer.
Due to 6 is the intermediate of the final purines 11a-b, it is demonstrated that the side chain
links throughthe 3-position of the heterocycle, unlike derivatives 3a-d, whose purine residue
binds through its 2-position.
Nuclear Overhauser Spectroscopy experiments performed on compound 3c (X = Br, Y = H)
shows the existence of a NOESY effect between H-2 and H-8’ and within both multiplet
hydrogen atoms CH
2
CH
2
N of the linear chain and H-8’, A correlation with CH N and the
2
same hydrogen of the purine skeleton H-8’ is observed, but no NOESY effects are noticed
among H-3a or H-3b and H-8’. These facts are compatible with a preferred conformational
arrangement in which the imidazole ring of the purine is located below that of the
benzoxazine moiety (Figure 2a).
Figure 2 may be inserted here
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Acknowledgements
o
This work was partially supported by the Junta de Andalucía (project n CS2016.1).
References
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1]
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1
Table 1. H NMR signal assignments of intermediate compounds 1, 2, 4, 6-10
Compound
H-2a
H-2b
H-3a
H-3b
H-4
H-5
H-6
H-7
H-8
CH
2
OR
2 2
CH CH OR
3
4
.36 (dd, 11.6,
3.17 (dd, 11.6,
7.7)
3.19 (dd, 14.3,
9.9)
6.60 (dd, 7.7,
1.5)
6.79 (dd, 8.1,
1.6)
6.66 (dd, 7.7,
1.5)
6.92 (ddd, 8.1,
7.3, 1.6)
1.84 (m), 1.94
(m)
1
2
4.30 (m)
-
-
2.99 (bs)
6.77 (m)
6.77 (m)
3.87 (m)
3.76 (m)
2
.4)
.32 (dd, 14.3,
.4)
7.04 (ddd, 8.2,
7.3, 1.6)
7.83 (dd, 8.2,
1.6)
3.58 (m)
-
1.77 (m)
2
2
.24 (dd, 16.5,
4
.12 (dd, 13.4,
3.42 (dd, 13.3,
9.4)
4
1.94 (m)
-
9.1), 2.59 (dd,
7,04 (m)
7,15 (m)
7,06 (m)
7.68 (d, 8.3)
3.53 (m)
-
-
4
.2)
.47 (dd, 11.4,
.2)
.15 (dd, 10.3,
.8)
.21 (dd, 10.7,
.9)
.37 (dd, 11.1,
.6)
.27 (dd, 11.2,
.5)
1
6.5, 6.0)
4
4
4
4
4
4.07 (dd, 11.4,
2.9)
3.82 (dd, 10.3,
1.3)
3.87 (dd, 10.6,
7.0)
4.02 (dd, 11.1,
2.5)
7.96 (dd, 4.6,
1.4)
7.57 (dd, 5.1,
1.4)
7.66 (dd, 5.0,
1.4)
7.94 (dd, 4.7,
1.6)
7.00 (dd, 8.1,
4.6)
6.49 (dd, 7.8,
5.1)
6.52 (dd, 7.7,
5.0)
6.90 (dd, 8.0,
4.7)
7.20 (dd, 8.1,
1.4)
6.93 (dd, 7.8,
1.4)
6.94 (dd, 7.7,
1.4)
7.12 (dd, 8.0,
1.6)
6
7
8
9
5.41 (m)
3.76 (m)
3.74 (m)
4.98 (m)
4.95 (m)
-
-
-
-
-
-
-
-
-
-
-
1
3.87 (m),
3.97(m)
5.12 (bs)
1.70 (m)
1.70 (m)
1.73 (q, 6.4)
1.79 (m)
2
5.32 (bs)
3.80 (m)
3.65 (m)
2
-
-
1
3.86 (dd, 11.2,
2.5)
7.97 (dd, 4.7,
1.6)
6.92 (dd, 8.1,
4.7)
7.14 (dd, 8.1,
1.6)
3.71 (m), 3.82
(m)
1
0
1
Solvent used CDCl
3
.
Chemical shifts (in CDCl
3
) are reported in δ (ppm) relative to CDCl
H signals for the tosyl substituent: 2, H-2’, H-6’: 7.54 (m), H-3’, H-5’: 7.23 (m), CH
H-6’: 7,93 (d, 8.3), H-3’, H-5’: 7.26 (d, 8.1), CH : 2.38 (s).
: 0.91 (s), (CH
: 4.12 (m), CH
s, singlet; bs, broad singlet; d, doublet; dd, doublet doublet; ddd, doublet doublet doublet; t, triplet; q, quadruplet; m, multiplet.
3
; multiplicities and coupling constants (Hz) are given in parentheses.
1
3
: 2.38 (s); 4, H-2’, H-6’: 7,51 (d, 8.0), H-3’, H-5’: 7,21 (d, 8.0), CH
3
: 2.38 (s); 9, H-2’, H-6’: 8,01 (d, 8.3), H-3’, H-5’: 7.28 (d, 5.9), CH
3
: 2.40 (s); 10, H-2’,
3
1
H signals for the sylil substituent: 8: (CH
3
)
3
3
3
)
2
: 0.07 (s); 9: (CH
)
3 3
3 2
: 0.90 (s), (CH ) : 0.05 (s).
Other signals of compound 6: COOCH CH
2
2
COOCH CH : 2.47 (m), COCH
2
3
3
: 2.59 (s), COOCH CH : 1.22 (t, 7.2).
2 3
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1
Table 2. H NMR signal assignments of final compounds 3a-d, 5a-d, 11a-b.
Compound
H-2a
H-2b
H-3a
H-3b
H-4
H-5
H-6
H-7
H-8
H-2’
-
H-8’
CH
2
N
2 2
CH CH N
4
.19 (dd,
4.2, 2.3)
.20 (dd,
4.1, 1.9)
.19 (dd,
4.2, 2.3)
4.20 (dd,
14.2, 2.4)
3.21 (dd,
14.2, 9.6)
3.21 (dd,
14.1, 9.7)
3.21 (dd,
14.2, 9.7)
3.23 (dd,
14.3, 9.6)
6.76 (dd, 8.2,
1.6)
6.78 (dd, 8.2,
1.5)
6.77 (dd, 8.2,
1.5)
6.74 (dd, 8.2,
1.5)
7.77 (dd, 8.3,
1.6)
7.79 (dd, 8.3,
1.5)
7.77 (dd, 8.3,
1.6)
7.76 (dd, 8.3,
1.6)
4.43 (dd, 7.7,
5.5)
2.02 (m),
2.23 (m)
2.27 (m),
2.02 (m)
2.04 (m),
2.28 (m)
2.08 (m),
2.28 (m)
3
a
3.35 (m)
3.31 (m)
-
-
-
-
-
-
-
6.94 (m)
6.93 (m)
7.08 (m)
7.05 (m)
7.06 (m)
7.05 (m)
7.96 (s)
7.96 (s)
7.98 (s)
8.13 (s)
1
1
1
4
3
b
8.75 (s)
8.69 (s)
9.08 (s)
4.44 (m)
4.44 (m)
4.52 (m)
4
6.93 (ddd,
8.8, 7.3, 1.5)
6.93 (ddd,
3
c
3.34 (m)
3
.44 (tt, 9.7,
3
d
-
2.8)
8.6, 7.3, 1.5)
2
.31 (dd,
4.12 (dd,
14.2, 6.7),
4.20 (dd,
14.2, 7.3)
4.15(dd,
3
.98 (dd,
3.42 (dd,
13.5, 8.7)
15.9, 9.0),
2.59 (dd,
6.98 (dd, 7.6,
1.5)
7.06 (td, 7.5,
1.2)
7.69 (dd, 8.3,
1.1)
5
a
2.38 (m)
-
7.16 (m)
.
8.07 (s)
-
13.5, 3.8)
1
2
5.9, 5.2)
.30 (dd,
4
.01 (dd,
3.45 (dd,
13.5, 8.9)
15.9, 9.1),
2.59 (dd,
6.98 (dd, 7.6,
1.5)
7.2 (td, 7.9,
1.5)
7.08 (td, 7.5,
1.2)
7.73 (dd, 8.3,
1.1)
14.2, 6.7),
4.25 (dd,
14.2, 7.2)
5
b
2.39 (m)
2.09 (m)
2.48 (m)
-
-
8.74 (s)
8.76 (s)
9.08 (s)
8.04 (s)
8.70 (s)
8.21 (s)
-
-
13.5, 4.1)
1
5.9, 5.3)
.30 (m),
2.55 (dd,
6.5, 5.3)
2
4.05 (dd,
5
ca
3.35 (m)
7.07 (d, 7.5)
6.99 (d, 7.6)
7.17 (m)
7.07 (d, 7.5)
7.07 (d, 7.3)
7.61 (d, 8.3)
7.72 (d, 8.3)
4,26 (m)
13.5, 4.0)
1
4.21 (dd,
2
.31 (d, 8.6),
2.61 (dd,
4.02 (dd,
3.48 (dd,
13.5, 8.5)
14.4, 6.4),
4.32 (dd,
5
d
-
-
7.20 (t, 7.8)
-
13.5, 3.7)
1
5.9, 5.1)
14.3, 6.6)
4
1
4
1
.18 (dd,
1.4, 1.4)
.18 (dd,
1.4, 1.4)
3.75 (dd,
11.4, 2.4)
3.77 (dd,
11.4, 2.3)
8.02 (dd, 4.6,
1.5)
8.02 (dd, 4.7,
1.5)
6.96 (dd, 8.1,
4.6)
6.96 (dd, 8.0,
4.6)
7.15 (dd, 8.1,
1.5)
7.15 (dd, 8.0,
1.5)
4.44 (m),
4.52 (m)
1
1a
1b
4.77 (d, 10.2)
4.78 (d, 9.8)
-
-
-
-
-
8.30 (s)
8.30 (s)
2.13 (m)
2.44 (m),
2.15 (m)
1
8.73 (s)
4.51 (m)
a
SSolvent used CDCl
3
. Solvent used DMSO-d
) are reported in δ (ppm) relative to CDCl
H signals for the tosyl substituent: 3a, H-2’’, H-6’’: 7.40 (d, 8.2), H-3’’, H-5’’: 7.08 (m), CH
H-6’’: 7.39 (m), H-3’’, H-5’’: 7.10 (d, 8.0), CH : 2.37 (s); 5a, H-2’’, H-6’’: 7.38 (d, 8.0), H-3’’, H-5’’: 7.16 (m), CH
m), CH : 2.30 (s); 5d, H-2’’, H-6’’: 7.40 (d, 7.9), H-3’’, H-5’’: 7.12 (m), CH
s, singlet; d, doublet; dd, doublet doublet; ddd, doublet doublet doublet; t, triplet; td, triplet doublet; tt, triplet triplet; m, multiplet.
6
.
Chemical shifts (in CDCl
3
3
; multiplicities and coupling constants (Hz) are given in parentheses.
: 2.39 (s); 3b, H-2’’, H-6’’: 7.35 (d, 8.1), H-3’’, H-5’’: 7.05 (m), CH
1
3
3
: 2.39 (s); 3c, H-2’’, H-6’’: 7.36 (m), H-3’’, H-5’’: 7.06 (m), CH
3
: 2.39 (s); 3d, H-2’’,
3
3
: 2.35 (s); 5b, H-2’’, H-6’’: 7.39 (d, 8.1), H-3’’, H-5’’: 7.12 (d, 8.1), CH
3
: 2.36 (s); 5c, H-2’’, H-6’’: 7.25 (d, 7.9), H-3’’, H-5’’: 7.16
(
3
: 2.35 (s); 11a, H-2’’, H-6’’: 7.82 (m), H-3’’, H-5’’: 7.25 (d, 10.3), CH : 2.38 (s); 11b, H-2’’, H-6’’: 7.84 (d, 8.1), H-3’’, H-5’’: 7.25 (d, 8.1), CH : 2.38 (s).
3 3 3
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Table 3. 13C NMR signal assignments of intermediate compounds 1, 2, 4, 6-10.
Compound
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
2
CH OR
2 2
CH CH OR
3
3
5.16
1
2
4
6
72.24
69.97
48.47
67.17
45.31
48.47
35.25
44.57
-
132.95
123.51
137.01
138.46
115.35
117.25
121.20
120.76
126.62
139.56
118.75
125.82
124.93
120.60
116.60
123.98
123.60
124.30
143.30
146.44
129.37
140.67
59.40
58.70
64.34
33.60
4.88
-
29.48
-
1
29.52
-
-
-
3
3.35
7
8
9
68.70
69.27
49.25
48.64
-
-
146.99
147.53
-
-
139.02
140.52
113.18
114.18
121.72
121.11
139.08
139.59
60.06
60.67
35.26
6
6
7.75
7.44
50.92
50.83
-
-
140.66
140.50
-
-
140.11
140.21
119.64
119.82
123.88
124.46
137.96
137.39
59.39
58.28
33.33
33.40
1
0
Solvent used CDCl
Chemical shifts (in CDCl
9.79, C-4’: 143.78,CH : 21.65; 9, C-1’: 137.98, C-2’, C-6’: 128.26, C-3’, C-5’: 128.95, C-4’: 143.98, CH
C signals for the sylil substituent: 8: C: 18.69, (CH : 26.38, (CH : 4.96; 9: C: 18.03, (CH : 25.76, (CH
2 3
Other signals of compound 6: COOCH CH : 170.21, COCH : 169.69, COOCH CH : 60.69, COCH : 25.70, COOCH CH : 13.95.
3
.
3
) are reported in δ (ppm) relative to CDCl
3
; 13C signals for the tosyl substituents: 2, C-1’: 135.41, C-2’, C-6’: 127.05, C-3’, C-5’: 129.70, C-4’: 144.07, CH
: 21.39; 10, C-1’: 136.66, C-2’, C-6’: 128.16, C-3’, C-5’: 129.17, C-4’: 143.98, CH
: 5.65.
3
: 21.39; 4, C-1’: 136.90, C-2’, C-6’: 127.07, C-3’, C-5’: 1
: 21.39.
2
3
3
3
13
3
)
3
3
)
2
)
3 3
3 2
)
2
3
3
2
3
3
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Table 4. 13C NMR signal assignments of final compounds 3a-d, 5a-d, 11a-b.
Compound
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
2
CH N
2 2
CH CH N
C-2’
C-4’
C-5’
C-6’
C-8’
3
a
68.66
68.33
68.32
48.31
48.18
48.08
-
-
-
123.68
123.52
123.42
117.40
117.25
117.17
121.60
121.42
121.31
126.44
126.29
126.18
124.35
124.29
124.13
145.98
145.98
145.88
40.61
40.33
40.29
31.88
31.75
31.67
153.19
151.97
151.80
153.17
151.72
150.39
130.86
131.62
135.20
151.91
151.09
146.07
145.24
145.03
3
b
3
c
143.07
1
1
45.20 (q,
3
d
68.62
48.07
-
123.44
117.15
121.36
126.17
124.00
145.80
40.15
31.77
152.40
153.65
129.92
147.29
37.1)
5
a
48.22
48.47
48.51
32.35
32.85
32.58
30.43
30.67
30.30
136.11
136.33
136.04
129.29
129.34
129.73
127.03
127.19
126.71
125.06
125.20
125.07
123.60
123.97
123.47
126.87
127.23
128.77
46.71
46.70
46.10
-
-
-
153.00
152.01
151.78
152.86
151.84
150.95
130.58
131.59
133.63
151.78
151.33
145.96
145.22
147.62
5
b
5
ca
141.96
45.80 (q,
5
d
48.90
33.24
31.09
136.73
129.79
127.64
125.62
124.27
127.51
47.00
-
152.40
154.25
130.36
147.92
37.7)
1
1a
1b
66.92
67.02
51.13
51.21
-
-
137.00
137.15
-
-
140.94
140.93
120.46
120.36
124.79
124.72
140.34
140.34
41.74
41.60
30.63
30.85
152.63
151.02
153.13
151.81
130.95
131.82
151.66
151.68
146.72
145.96
1
Solvent used CDCl
Chemical shifts (in CDCl
3
. aSolvent used DMSO-d
) are reported in δ (ppm) relative to CDCl
C signals for the tosyl substituents: 3a, C-1’’: 135.49, C-2’’, C-6’’: 127.08, C-3’’, C-5’’: 129.95, C-4’’: 144.83, CH
C-3’’, C-5’’: 129.64, C-4’’: 144.55, CH : 21.57; 3d, C-1’’: 135.30, C-2’’, C-6’’: 126.83, C-3’’, C-5’’: 129.69, C-4’’: 144.57, CH
’’: 126.83, C-3’’, C-5’’: 129.72, C-4’’: 143.99, CH : 21.51; 5c, C-1’’: 135.76, C-2’’, C-6’’: 126.63, C-3’’, C-5’’: 129.95, C-4’’: 144.43, CH
36.01, C-2’’, C-6’’: 128.18, C-3’’, C-5’’: 129.41, C-4’’: 144.51, CH : 21.49; 11b, C-1’’: 136.21, C-2’’, C-6’’: 128.23, C-3’’, C-5’’: 129.37, C-4’’: 144.43, CH
substituent: 3d: 120.69 (q, 274.9); 5d: 120.80 (q, 275.9).
6
.
3
3
; multiplicities and coupling constants (Hz) are given in parentheses.
: 21.77; 3b, C-1’’: 135.29, C-2’’, C-6’’: 126.87, C-3’’, C-5’’: 129.72, C-4’’: 144.65, CH
: 21.37. 5a, C-1’’: 135.94, C-2’’, C-6’’: 126.66, C-3’’, C-5’’: 129.63, C-4’’: 144.01, CH
: 21.13; 5d, C-1’’: 136.63, C-2’’, C-6’’: 127.25, C-3’’, C-5’’: 130.15, C-4’’: 144.45, CH
: 21.50.
13
3
3
: 21.61; 3c, C-1’’: 134.11, C-2’’, C-6’’: 126.78,
: 21.38; 5b, C-1’’: 136.21, C-2’’, C-
: 21.90; 11a, C-1’’:
3
3
3
6
1
3
3
3
3
3
13
C signals for the CF
3
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Table 5. HSQC correlations found for compounds 2, 4, 6, 7, 3c, 5b and 11a
1H/13
C
2
3.58
69.97
4.32, 3.19
48.47
-
-
6.79
115.35
6.92
120.76
7.04
125.82
7.83
123.98
4
6
4.47, 4.07
67.17
5.41
44.57
-
7
3c
3.34
68.32
4.19, 3.21
48.08
-
5b
4.01, 3.45
48.47
2,39
32.85
2.30
30.67
6.98
129.34
7.2
127.19
7.08
125.2
7.73
123.97
8,74
152.01
8.04
145.22
4.15,4.25
46.7
-
11a
4.18, 3.75
66.92
4.77
51.13
-
-
-
-
8.02
140.34
6.96
120.46
7.15
124.79
-
153.13
8.30
146.72
4.44, 4.52
41.74
2.13
30.63
-
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
H-7
C-7
H-8
C-8
H-2’
C-2'
H-8’
C-8'
4.12, 3.42
48.47
1.94
35.25
2.24
29.48
7.04
129.52
7.15
126.62
7.04
124.93
7.8
123.60
-
-
4.15, 3.82
68.7
3.76
49.25
5.12
-
-
-
7.57
139.02
6.49
113.18
6.93
121.72
-
-
-
-
-
-
6.77
117.17
6.93
121.31
7.06
126.18
7.77
124.13
8.69
151.8
7.98
145.03
4.44
40.29
2.04, 2.28
31.67
-
7.96
139.56
7.00
120.60
7.20
124.30
-
-
-
-
-
-
-
-
-
-
-
-
CH
CH
2
X
X
3.76
58.7
1.77
34.88
-
-
-
-
-
-
3.53
64.34
2.47
33.60
-
3.87, 3.97
60.06
1.70
33.35
2
CH
CH
2
CH
CH
2
X
X
-
-
-
-
-
-
-
-
2
2
-
-
-
-
-
-
-
-
COOCH
COOCH
COOCH
COOCH
COCH
COCH
2
2
2
2
CH
CH
CH
CH
3
3
3
3
4.12
60.69
1.22
13.95
2.59
25.70
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
3
-
-
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CH3
CH3
4
''
4
'
5''
6''
3''
2''
5
'
3'
2'
6'
1'
1''
O S O
O S O
H
5
8
5
8
4
5
8
N4
NH2
N4
N
(i)
ii)
(iii)
(iv)
3
6
7
3
6
7
6
7
3
8
'
9'
N
(
2
2
N7'
OH
O
2
OR
O
OR
O
1
1
1
4'
5'
1, R = H
2, R = H
3'N
X
6'
N
'
1
2
'
Y
3
3
3
a (X = Cl,Y = Cl)
b (X = Cl, Y = H)
c (X = Br, Y = H)
(
vii)
3
d (X = CF , Y = H)
3
Scheme 1. Synthesis of compounds 3a-d included in the benzoxazine family. Reagents
and conditions: (i) ethyl 4-bromobut-2-enoate, NaHCO , EtOH, 3 h, rt, then K CO , 30 min;
ii) LiAlH , Et O, 1 h, 0ºC to rt; (iii) TsCl, py, 12 h, 0ºC to rt; (iv) 2,6-dihalopurine or 6-
halopurine, DIAD, Ph
3
2
3
(
4
2
3
P, 36 h, -20ºC to rt; (v) MFSDA, CuI, HMAP, DMF, 12 h, 70ºC.
This article is protected by copyright. All rights reserved.

CH3
CH3
4
''
4
'
5
''
''
3''
2''
5
'
3'
2'
6
6
'
1
'
1''
O S O
N1
O S O
N1
H
8
5
8
5
7'
N 5
N
N
(
i), (ii)
(v)
2
7
6
2
8'
7
6
(vi)
'
X
(
iii), (iv)
OH
OR
N
3
6'
CN
3
9'
4
4
4'
'N
N
3
1'
4, R = H
2'
Y
5
5
5
a (X = Cl,Y = Cl)
b (X = Cl, Y = H)
c (X = Br, Y = H)
(vii)
5d (X = CF , Y = H)
3
Scheme 2. Synthesis of the tetrahydroquinoline family compounds (5a-d). Reagents and
conditions: (i) aq. NaOH, EtOH, 20 h, reflux, then HCl 1N; (ii) SOCl , EtOH, 4 h, reflux; (iii)
NaBH CN, THF, MeOH, HCl, Et O, 6 h, rt; (iv) LiAlH , THF, 1 h, 0ºC; (v) TsCl, py, 12 h,
ºC to rt; (vi) 2,6-dihalopurine or 6-halopurine, DIAD, Ph P, 36 h, -20ºC to rt; (vii) MFSDA,
2
3
2
4
0
3
CuI, HMAP, DMF, 12 h, 70ºC.
This article is protected by copyright. All rights reserved.

1
8
O1
2
8
1
8
O1
8
O
OH
O
2
3
7
6
2
3
(iii)
(iv)
7
6
2
3
(v)
vi)
7
6
(vii)
(
i)
ii)
7
6
8
'
3
9'
N
4'
COOEt
(
N7'
5'
(
N
5
N4
OR
N
5
N4
N
NH2
N
5
N4
N
5
N4
H
OR
O
S
1'
O
O
S
1''
O
O
N
X
7
, R = H
2''
3''
3'
6'
6'
2'
6''
5''
N
2' 1'
6
8, R = Si(CH3)2C(CH3)3
3
'
Y
5
'
4
'
4''
CH3
CH3
9
1
, R = Si(CH3)2C(CH3)3
0, R = H
11a (X = Cl, Y = Cl)
11b (X = Cl, Y = H)
Scheme 3. Synthesis of compounds belonging to the pyridoxazine family (11a-b).
Reagents and conditions: (i) Ac O/py, 5 min, reflux, then NaOH; (ii) ethyl 4-bromobut-2-
enoate, K CO , EtOH, 24 h; (iii) LiAlH , THF, 1 h, 0ºC to rt; (iv) TBDMSCl, Et N, DMAP,
DCM, 12 h, rt; (v) TsCl, Et N, DMAP, DCM, 12 h, rt; (vi) AcOH, H O, THF, 12 h, rt; (vii)
,6-dichloropurine or 6-halopurine, DIAD, Ph P, -20ºC to rt, 36 h.
2
2
3
4
3
3
2
2
3
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Scheme 4. Detailed route leading to the intermediate regioisomer 6 from 2-aminopyridin-3-
ol.
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CH3
CH3
H5'
H3'
H '
H3'
H '
5
H6'
H2'
H '
6
H8
N
2
H8
N
O S O
N
H O S O
8
H7
H6
O
N
H2a
H7
O
H2a
H2b
H6
H7
H7
H6
N
O
H3
O
OH
H6
N
H
OH
O
OH
H
O
H5 H4
2
4
6
7
CH3
CH3
H5''
H3''
H8
N
H5''
H6''
H3''
H2''
H7
H6
O
H2a
H8'
H6''
H2''
O S O
N
N
N
N
O S O
N
H
H6
H3
H8'
O S O
H7
H8'
N
X
H6''
H2''
N
N
H7
O
N
N
N
H6
X
H2
Y
H5''
H3''
H5 H4
H
N
N
N
X
CH3
N
Y
Y
3
c
5b
11a
1
13
Figure 1. Main connectivities found in the HMBC H/ C spectra of intermedia compounds
(2, 4, 6, 7) and final derivatives (3c, 5b and 11a).
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CH3
CH3
O S O
N
O S O
N
H3a
H3a
N
H8'
H3b
H3b
N
Hb
Hb
O
O
N
N
H2
Br
H2
N
Ha
N
Ha
H8'
N
Br
N
b)
a)
Figure 2. Selected NOESY correlations for compound 3c.
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Graphical table of Contents
CH3
CH3
X = Cl, Br, CF3
Y = H, Cl
Z = CH , O
2
n = 1, 2
O S O
N
N
O S O
N
X
N
N
N
X
N
N
n
Z
N
O
N
Y
N
Y
The NMR assignment of several heterocyclic families linked to purine moieties has been
unequivocally established by using the concerted application of one- and two-dimensional
NMR experiments (DEPT, HSQC and HMBC).
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