1H and 13C assignments of 6-, 8-, 9- substituted purines

Full text of DOI:10.1002/mrc.4743

1H and 13C Assignments of 6-, 8-, 9- Substituted Purines
Álvaro Lorente-Macías1,2, Manuel Benítez-Quesada1, Ignacio J. Molina2,3, Asier Unciti-
Broceta4, Juan José Díaz-Mochón*1,5, María José Pineda de las Infantas Villatoro*1.
(1) Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Campus de
Cartuja, Universidad de Granada, Granada Spain.
(2) Instituto de Biopatología y Medicina Regenerativa, Centro de Investigación Biomedica,
Parque Tecnológico de Ciencias de la Salud, Universidad de Granada. 18016 Armilla,
Granada, Spain.
(3) Instituto de Investigación Biosanitaria ibs.GRANADA. Granada University Hospitals and
University of Granada, Spain.
(4) Ediburgh Cancer Research UK Centre MBC Institute of Genetics and Molecular
Medicine. University of Edinburgh, Crewe Road South, Edinburgh EH4, 2XR UK.
(5) Centro Pfizer-Universidad de Granada-Junta de Andalucía de Genómica e Investigación
Oncológica (GENYO), Avenida de la Ilustración 114, Parque Tecnológico de Ciencias de la
Salud, 18016 Granada, Spain.
Corresponding Author:
* Email: juandiaz@ugr.es Phone: (+)34-958-249360. Fax: (+)34-958-243845.
* Email: mjpineda@ugr.es Phone: (+)34-958-249360. Fax: (+)34-958-243845.
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doi: 10.1002/mrc.4743
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Introduction
Purines are heterocyclic aromatic organic compounds consisting of two 5 and 6
membered fused rings containing nitrogen. They are present in numerous compounds,
including natural products, with potent biological activity. Purine analogs are used for the
treatment of acute leukemias. Thiopurine derivatives are effective antiviral (acyclovir,
ganciclovir) or antitumor agents such as vidarabine, among other clinical uses [1].
Over the years, the purine nucleus has become one important pharmacophoric
group. It is capable of interfering in the synthesis and function of enzymes and nucleic acids.
It is also frequently used in the development of protein kinase inhibitors [2].
Our research group has developed new routes to produce purine libraries. One of
[3]
such routes is the one-pot synthesis
to obtain 6-, 8-, and 9-substituted purines from 4-
alkylamino-5-amino-6-chloropirimidines, alcohols and N,N-dimethylamides. We also
presented a new approach [4], to obtain trisubstituted purines. We were able to prepare a
library of polysubstituted purines. Some compounds from this library were found to be
[5]
specific inhibitors of the Death-Associated Protein Kinase-1
(DAPK1). The leading
compounds of this library are potent inducers of apoptosis in tumor lymphocytes and also
reduce viability of trypanosomes [4, 6]
.
1
13
We herein report the unambiguous assignment of H and C of a subset of these
new purine derivatives. Assignments were carried out in compounds 3k and 3n which have,
as substituents, phenyl and benzyl at positions 6 and 8, respectively. We also characterize
compound 3o, which has benzyl, phenyl and phenethyl substituents at positions 6, 8 and 9,
respectively. We therefore provide better knowledge of the molecular structure of our
compounds, while obtaining further insight for future structure-activity studies and for
improving the structural determination of other purine derivatives.
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Experimental
Synthesis
Purines were prepared as described elsewhere [4], [5]
Scheme 1 shows the previously reported synthetic pathway to obtain the novel family
of purine derivatives 3a-o. Trisubstituted purines at positions 6, 8 and 9 were prepared
starting from 6-chloro-4,5-diaminopyrimidine, alcohols and N, N-dimethylamides in basic
conditions without metal catalysis. Depending on the size of the R1 substituent of the amides
(Scheme 1), two different synthetic routes may occur. Route A results in purine analogs
whose C8 substituents are derived from the amide, whereas Route B gives rise to purines
with C8 substituents coming from the alcohol. Route A proceeds via in situ generation of N-
alkylimidate species, which are created by reaction between amides and alkoxides leading to
purine derivatives with either H or methyl in C8 when using N,N-dimethylformamide or N,N-
dimethylacetamide, respectively. When steric hindrance of amides increases, such as in the
cases of N,N-dimethylbenzamide or N,N-dimethylpropionamide, Route A is then impeded
and a metal-free tandem alcohol oxidation/annulation reaction occurs giving rise to Route B
products. Therefore, competition between N-alkylimidate formation and metal-free oxidative
coupling of primary alkoxides and diaminopyrimidines with Schiff base formation and
subsequent annulation can be controlled. In both routes SNAr of the chloro atom at C6 by
alkoxides takes place and, along with the R3 group of exocyclic nitrogen located at C4 of the
starting pyrimidine, increases structural diversity of these one-pot synthesis.
Scheme 1 may be inserted about here.
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This synthetic platform, therefore, allows the creation of a diversity of purine
analogues in a parallel and straightforward fashion using a variety of amides and alcohols
with different pyrimidines under the same reaction conditions. Scheme 2 shows the set of 15
purines presented here, which were obtained through either Route A or B.
Scheme 2 may be inserted about here.
NMR Techniques
1H and 13C NMR data (chemical shifts multiplicity and coupling constants) for
compounds 3a-n are shown in Tables below. Unambiguous assignments for all NMR signals
1
13
were accomplished by combined analysis of H, C, 90º DEPT, HSQC and HMBC, COSY
and TOCSY NMR experiments.
¹H Nuclear magnetic resonance spectra were recorded on a Varian Inova Unity (300
MHz), Varian Direct Drive (400 MHz) and/or Varian Direct Drive (500 MHz). Chemical
shifts (δ) are referenced to the residual solvent peak: CDCl3, δ 7.26 (1H), δ 77.16 (13C). Spin
multiplicities are given as s (singlet), bs (broad singlet), d (doublet), dd (double doublet), ddd
(double double doublet), t (triplet), dt (double triplet), pt (pseudotriplet), q (quadruplet) and m
13
(multiplet). Coupling constants (J) are given in Hz. C NMR were recorded on a Varian
Direct Drive (400 MHz) and Varian Direct Drive (500 MHz). DEPT experiments were
carried out using the standard pulse sequence [7]. The HMBC, HSQC, H2BC, COSY,
TOCSY DEPT spectra were measured with a pulse sequence gc2hmbc, gc2hsqcse, gc2h2bc,
gCOSY, gTOCSY, gDEPT respectively (Standard sequence Agilent Vnmrj 4.2A software),
CRISIS type [8].
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Results and discussion
Analysis of 1H and 13C-NMR spectra
To facilitate the analysis of all purines, NMR data are presented in tables. Substituent
at 6, 8 and 9 positions of the purine ring are named as R1, R2 and R3 respectively. Table 1 and
2 show 1H and 13C-NMR chemical shifts (δ) for compounds (3a-o).
Table 3 shows the family of compounds derived from purine as well as the number
scheme of the compounds used.
Table 3 may be inserted here.
In particular, we focused the analysis in protons belonging to phenyl, benzyl and
phenethyl aromatic rings within a same molecule, as for compounds 3o, 3k and 3n. TOCSY
method was used to identify unequivocally these aromatic protons of our compounds. Purine
3o was firstly analyzed. Irradiation frequency of δ 7.55 presents an associated spin system
which corresponds with aromatic protons of the R1 benzyl substituent located at C6 of the
purine ring. In the case of irradiation frequency of δ 7.50, the associated spin system
corresponds to protons of the aromatic ring appearing at position 8 of the purine ring (R2), a
phenyl substituent. Finally, irradiation frequency of δ 7.19 present an associated spin system
which corresponds to protons of the R3 substituent located at C9 of the purine ring, a
phenethyl group.
Once chemical shifts of the protons of the three types of aromatic rings at purine substituents
were identified, coupling techniques to identify C-H and H-H interactions (HSQC, HMBC,
COSY, TOCSY and H2BC) were used to unequivocally assign protons of the three different
aromatic rings at 6, 8 and 9 positions of compound 3o as shown in Table 1. Therefore, by
means of TOCSY studies we unequivocally assigned the protons of the aromatic rings which
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appear in positions 6, 8 and 9 of the purine ring (Table 1). A similar procedure was carried
out to assign the aromatic protons of compounds 3k and 3n.
13
1
Table 2 and 3 shows the C-NMR data. Similar to our H-NMR analysis, unambiguous
assignment of 13C chemical shifts was performed for the three aromatic rings, phenyl, benzyl
and phenethyl substituents at positions 6, 8 and 9 of purine 3o, as well as for carbons of the
aromatic substituents, benzyl and phenyl, located at C6 and C8 of purines 3k and 3n.
13
For compound 3o, quaternary carbons were assigned using 90º DEPT and C-NMR
spectra. Chemical shifts at δ 160.32, 153.96, 153.34, 137.27, 136.40, 129.66 and 121.40
correspond to C4, C5, C6 and C8 of the purine ring of phenyl, benzyl and phenethyl rings,
located at C6, C8 and N9. These assignments were confirmed by analysis of HSQC spectra.
HMBC spectrum indicated that the methylene protons of the benzyl substituent located at C6
of the purine ring showed coupling with the quaternary carbon appearing at δ 160.32,
corresponding to C6. The same methylene protons were coupled to the quaternary carbon of
the aromatic ring substituent at C6. Carbon C1’ was at δ 136.40. The proton at C2 of the
purine ring (δ 8.58) is coupled long-distance with C6 and with C4 at δ 153.96. The methylene
protons of the phenethyl group (δ 5.71), substituent at C9 are coupled to C1’ (δ 137.27) and
with C4. The quaternary carbon at δ 121.40 was assigned to C5, since was not coupled with
any hydrogen. The quaternary carbon δ 153.34 wa assigned to C8, since in the HMBC
spectrum it showed coupling to the aromatic protons of the substituent phenyl ring. The
signal of the quaternary carbon at δ 129.66 was assigned to C1 of the phenyl group of the
substituent at C8 of the purine ring.
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Acknowledgments
ALM is the recipient of a FPU fellowship from the Ministry of Education, Culture and
Sports, Spain.
This work was supported by grant number CS2016.4 from Consejería de Salud, Regional
Government of Andalucia, Spain, and a philanthropic donation.
References:
[1] Z. O’Brien, M. Moghaddam, Expert Opin. Drug Metab. Toxicol. 2013, 9, 1597.
[2] L. Meijer, E. Raymond, Acc. Chem. Res. 2003, 36, 417.
[3] P.G. Baraldi, A. Unciti-Broceta, M.J. Pineda de las Infantas, J.J. Díaz-Mochón, A.
Espinosa and R. Romagnoli, Tetrahedron. 2002, 58, 7607.
[4] M.J. Pineda de las Infantas, J.D. Unciti-Broceta, R. Contreras, J.A. García, M.A. Gallo,
A. Unciti-Broceta and J.J. Díaz-Mochón, Sci. Rep. 2015, 5, 9139.
[5] M.J. Pineda de las Infantas, S. Torres-Rusillo, J.D. Unciti-Broceta, P. Fernández-Rubio,
M.A. Luque-González, M.A. Gallo, A. Unciti-Broceta, I.J. Molina and J.J. Díaz-Mochón,
Org. Biomol. Chem. 2015, 13, 5234.
[6] L. Harmse, R. van Zyl, N. Gray, P. Schultz, S. Leclerc, L. Meijer, C. Doerig, I. Havlik,
Biochem Pharmacol. 2001, 62, 341.
[7] D.M. Doddrell, D.T. Pegg, M.R. Bendall, J. Magn. Reson (1969). 1982, 48, 323.
[8] R. Boyer, R. Johnson, K. Krishnamurthy, J. Magn .Reson. 2003, 2, 165.
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1H-NMR chemical shifts () and coupling constants (J, Hz) of purine derivatives (3a-o)
Table 1
Compound
H-2
H-8
R1
R2
R3
3a
4.63 (q, 7),
1.47 (t, 7)
4.85 (m),
8.48 (s) 7.96 (s)
-
1.59 (d, 7)
3b
3c
4.62 (q, 7),
1.49 (t, 7)
4.74 (m),
8.42 (s)
-
2.64 (s)
1.68 (d, 6.5)
5.68 (s),
7.54 (H2´-6´, d, 7.5),
7.36 (H3´-5´, dd, 7.5, 1.5),
7.31 (H4´, dd, 7.5, 1.5)
4.89 (m),
8.54 (s) 7.99 (s)
-
1.63 (d, 7)
3d
5.64 (s),
7.53 (H2´-6´, d, 7.5),
7.34 (H3´-5´, dd, 7.5, 2),
7.30 (H4´, dd, 7.5, 2)
4.74 (m),
8.46 (s)
-
2.64 (s)
1.68 (d, 7)
3e
5.67 (m),
4.88 (m),
8.51 (s) 7.95 (s)
-
1.62 (d, 6.90)
1.47 (d, 6)
3f
5.40 (s),
4.66 (q, 7),
1.51 (t, 7)
8.55 (s)
7.88(s)
-
7.35- 7.30 (m),
7.27 (dd, 8)
5.40 (s),
3g
4.65 (q, 7),
1.51 (t, 7)
7.32 (H4´, dd, 6, 2),
7.28 (H3´-5´, dd, 6, 2),
7.14 (H2´-6´,dd, 6, 2)
8.50 (s)
-
2.51 (s)
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3h
5.69 (s),
5.41 (s),
8.58 (s) 7.89 (s)
7.54 (d, 7.5),
7.38-7.34 (m)
-
7.34-7.30 (m),
7.26 (dd, 8)
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Compound
3i
H-2
H-8
R1
R2
R3
4.65 (q, 7.2),
1.51 (t, 7.2)
8.50 (s) 7.98 (s)
-
1.81 (s)
3j
7.25-7.17 (m),
7.02 (dd, 8.41),
4.46 (t, 7.21),
3.16 (t, 7.21)
4.64 (q, 7.21),
1.49 (t, 7.21)
8.52 (s) 7.50 (s)
-
3k
5.69 (s),
7.66 (m),
4.76 (m),
8.54 (s)
-
7.57-7.50 (m)
1.73 (d, 6.90)
7.37-7.29 (m)
3l
4.89 (m),
8.53 (s) 7.98 (s)
4.18 (s)
-
1.62 (d, 6.5)
3m
4.52 (t, 7.20),
1.26 (m),
2.94 (q ,7.2),
1.43 (t, 7.2)
4.69 (m),
8.43 (s)
-
1.70 (d, 6.90)
1.05 (t, 7.50)
3n
5.65 (s),
7.46 (d, 7.5),
7.45-7.38 (m)
8.55 (s)
-
7.54 (d, 7.5),
7.35-7.27 (m)
1.66 (s)
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3o
7.25-7.17 (m),
6.91 (dd, 2.4),
4.56 (t, 4.8),
3.10 (t, 4.8)
5.71 (s),
7.48 (d, 4.4),
7.45-7.43 (m)
8.58 (s)
-
7.55 (d, 5.2),
7.37-7.31 (m)
Table 1.- Family of compounds derivative of 6,8,9 poly-substituted purine.
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13C-NMR chemical shifts () of purine derivatives (3a-o)
Table 2
Compound
3a
C2
C4
C5
C6
C8
R1
R2
R3
14.6 (CH3),
63.1 (CH2)
47.5 (CH),
22.7 (CH3)
151.8
152.0
122.0
160.9
139.7
-
3b
3c
14.7 (CH3),
62.8 (CH2)
48.5 (CH),
21.4 (CH3)
150.7
150.6
120.9
159.9
133.2
15.4
CH2 (68.5),
C-1´ph (136.4),
C-2´,6´ph (128.6),
C-3´,5´ph (128.5),
C-4´ph(128.2)
47,6 (CH),
22,8 (CH3)
151.8
152.1
122.0
160.7
139.9
-
3d
CH2 (68.23),
C-1´ph (136.59),
C-2´,6´ph (128.60),
C-3´,5´ph (128.52),
C-4´ph(128.15)
48.5 (CH),
21.4 (CH3)
150.5
150.9
120.9
159.6
140.0
15.4
3e
70.3 (CH),
22.8 (CH3)
47.5 (CH),
22.2 (CH3)
152.0
150.2
122.2
160.8
139.5
-
CH2 (47.6),
C-1´ph (135.5),
C-2´,6´ph (129.2),
C-3´,5´ph (127.9),
C-4´ph(128.6)
3f
14.7 (CH3),
63.3 (CH2)
152.5
151.7
152.3
151.6
121.5
120.5
161.1
160.0
142.0
153.7
-
CH2 (46.2),
3g
14.5 (CH3),
63.1 (CH2)
14.7
C-1´ph (135.7),
C-2´,6´ph (129.1),
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C-3´,5´ph (127.1),
C-4´ph(128.3)
3h
CH2 (68.6),
C-1´ph (136.3),
C-2´,6´ph (128.6),
C-3´,5´ph (128.5),
C-4´ph(128.3)
CH2 (47.6),
C-1´ph (135.4),
C-2´,6´ph (129.3),
C-3´,5´ph (127.9),
C-4´ph(128.7)
152.4
152.5
121.6
160.8
142.2
-
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Compound
3i
C2
C4
C5
C6
C8
R1
R2
R3
14.7 (CH3),
63.0 (CH2)
57.7 (C4º),
29.2 (CH3)
151.1
152.4
123.1
161.1
139.8
-
N-CH2(45.7)
CH2(36.3),
3j
14.6 (CH3),
63.2 (CH2)
C-1´ph (137.3),
C-2´,6´ph (128.9),
C-3´,5´ph (128.8),
C-4´ph (127.2)
152.2
152.0
121.6
160.9
142.1
-
3k
CH2 (68.3),
C-1´ph (136.6),
C-2´,6´ph (128.7),
C-3´,5´ph (128.5),
C-4´ph(128.2)
C-1´ph (135.5),
C-2´,6´ph (129.7),
C-3´,5´ph (128.9),
C-4´ph(130.3)
49.9 (CH),
21.4 (CH3)
150.9
153.7
122.0
160.4
153.0
3l
47.6 (CH),
22.8 (CH3)
151.9
150.6
148.8
150.2
122.0
133.9
161.2
160.2
139.9
155.2
54.3
-
3m
OCH2(68.6)
CH2 (22.4)
CH3(10.5)
12.4 (CH3),
22.1 (CH2)
48.5 (CH),
21.4 (CH3)
3n
CH2 (68.3),
C-1´ph (136.5),
C-2´,6´ph (128.8),
C-3´,5´ph (128.5),
C-4´ph(128.18)
C-1´ph (134.9),
C-2´,6´ph (130.0),
C-3´,5´ph (128.0),
C-4´ph(129.6)
60.9 (C4º),
31.0 (CH3)
150.3
151.7
154.4
154.0
121.7
121.4
160.5
160.3
153.3
153.3
3o
CH2 (68.5),
C-1´ph (129.7),
N-CH2(45.6)
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C-1´ph (136.4),
C-2´,6´ph (128.6),
C-3´,5´ph (128.5),
C-4´ph(128.2)
C-2´,6´ph (129.3),
C-3´,5´ph (129.8),
C-4´ph(130.3)
CH2(35.7),
C-1´ph (137.2),
C-2´,6´ph (128.8),
C-3´,5´ph (128.8),
C-4´ph (127.0)
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Summary of13C-NMR chemical shifts () of purine derivatives (3a-o)
Table 3
Compound
C2
C4
C5
C6
C8
3i-o
151.9 - 152.2
148.8 - 154.4
121.4 - 133.9
160.2 - 161.2
139.8 - 155.2
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Scheme 1.- Proposed mechanisms of action for routes A and B.
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Scheme 2.- Synthetic routes to generate purine types "A" or "B" and set of 15 purines
prepared through either Route A or B.
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1H and 13C Assignments of 6-, 8- 9- Substituted Purines
Álvaro Lorente-Macías, Manuel Benítez-Quesada, Ignacio J. Molina, Asier Unciti-Broceta,
Juan José Díaz-Mochón*, María José Pineda de las Infantas Villatoro*.
1
13
Using H and C-NMR, DEPT, HMBC, HSQC, DQF-COSY and 1D-TOCSY techniques,
proton and carbon atoms present in a purine rings substituted by benzyl and phenyl groups at
positions 6 and 8 respectively, as well as of benzyl, phenyl and phenethyl substituents at
positions 6, 8 and 9 respectively of the purine ring have been unequivocally assigned.
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