Complete and unambiguous assignments of 1H and 13C chemical shifts of new arylamino derivatives of ortho-naphthofuranquinones

Article abstract of DOI:10.1002/mrc.2250

Six new nor-β-lapachones have been synthesized fromreaction of 3-bromo-nor-β-lapachone with arylamines. These derivatives have potent anticancer properties against several cell lines. Here, we report complete unambiguous assignments of ¹H and <

Full text of DOI:10.1002/mrc.2250

Spectral Assignments and Reference Data
Received: 21 December 2007
Revised: 14 April 2008
Accepted: 15 April 2008
Published online in Wiley Interscience: 4 June 2008
(www.interscience.com) DOI 10.1002/mrc.2250
Complete and unambiguous assignments
of 1H and 13C chemical shifts of new arylamino
derivatives of ortho-naphthofuranquinones
a
a
b
ˆ
ˆ
Eufranio N. da Silva Ju´nior, Maria Cecília B. V. de Souza, Antonio V. Pinto,
Maria do Carmo F. R. Pinto,b Christiane M. Nogueira,a Vitor F. Ferreiraa and
Rodrigo B. V. Azeredoa∗
Six newnor-β-lapachoneshavebeensynthesizedfrom reactionof3-bromo-nor-β-lapachonewitharylamines. Thesederivatives
have potent anticancer properties against several cell lines. Here, we report complete unambiguous assignments of 1H and 13
C
chemical shifts of the new compounds. The assignments were made using a combination of one- and two-dimensional NMR
techniques (1H, 13C, 1H–1H COSY, 1H–13C HSQC, and 1H–13C HMBC). Copyright ꢀ 2008 John Wiley & Sons, Ltd.
c
Keywords: NMR; 1H NMR; 13C NMR; naphthofuranquinones; nor-β-lapachones
Introduction
models for the assignment of compounds belonging to the 2H-
naphtho[2,3-b]-furan-4-one ring system. The assignments were
made by means of a combination of one- and two-dimensional
Quinones are an important class of synthetic and natural
products with much benefits due to several related biological
activities.[1] Recently, new quinones have been found as potential
drug candidates for antitumor,[2] trypanocidal,[3] molluscicidal,[4]
leischmanicidal,[5] anti-inflammatory,[6] and antifungal[7]activities.
Among the quinones that deserves mention is lapachol.[8]The
quinones promote DNA scission through the redox cycling-based
generation of super-oxide anion radicals.[9] β-Lapachone, an
isomer of lapachol, is a natural product that can be found in
the heartwood of Tabebuia sp. of South and Central America. It
directly targets Topoisomerase I[10] and II,[11] and has been the
subject of investigation for clinical use in cancer therapy.[12]
Among the quinones, the furan derivatives, more specifically,
the naphtho[2,3-b]furan-4,9-dione derivatives, are widely present
in nature and they have several important biological activities,
such as anticancer, antibacterial, and anti-inflammatory.[13]
Recently, we described our efforts on the synthesis of new
arylamino derivatives of nor-β-lapachone and nor-α-lapachone.
The modified arylamino quinones showed anticancer activities on
human tumor cell lines indicating their potential as interesting
new lead compounds in anticancer drug development.[2]
The information derived from the spectral analysis of naphtho-
quinones can be used to differentiate between ortho- and para-
substituted isomers and establish the size of the heterocyclic ring
attached on the naphthoquinone moieties.[14–16] Although some
reports on NMR spectra of para and ortho-naphthofuranquinones
have appeared in the literature in the past,[17–19] some of them
have incorrect 1H or 13C assignments,[7,16,17] mainly due to the lack
of high-field spectrometers and adequate pulse sequences at the
time.
1
1
NMR techniques: H and 13C NMR, 1H–1H COSY, H–13C HSQC
and 1H–13C HMBC.
Experimental
Methods
NMR spectra were collected in CDCl3 solution at 25 ◦C on a Varian
UnityPlusspectrometeroperatingat299.9 MHzforproton.Allone-
dimensionaland1H–1HCOSYspectrawereobtainedusinga5 mm
switchable broadband probe, while 1H–13C HSQC and 1H–13
C
HMBC spectra were obtained using a 5 mm indirect detection
broadbandprobe. Thesampleconcentrationswereapproximately
60 mM for 13C analysis, and 20 mM for the other experiments.
Chemical shifts (δ) were reported in ppm and coupling constants
(J)inHz.Thechemical-shiftstandardwasinternaltetramethylsilane
(TMS) for both 1H and 13C.
1H spectra were acquired with a 4000 Hz spectral width, using
a 90◦ pulse of 16.1 µs and a 2 s relaxation delay. The 16-transient
free-induction decay (FID) was collected with 16 384 data points,
multiplied by a 0.15 Hz Lorentzian function, and zero-filled to
32 768 data points, prior to Fourier transformation (FT).
∗
Correspondence to: Rodrigo B. V. Azeredo, Universidade Federal Fluminense,
ˆ
Instituto de Química, Deptartamento de Química Organica, Campus do
´
Valonguinho, 24020-141, Niteroi, RJ, Brazil. E-mail: bagueira@vm.uff.br
a
Universidade Federal Fluminense, Instituto de Química, Departamento de
In this article, we report the complete and unambigu-
ous assignments of 1H and 13C chemical shifts of six ortho-
naphthofuranquinones 2–7. These results can be used as
ˆ
´
Química Organica, Campus do Valonguinho, 24020-141, Niteroi, RJ, Brazil
b
Universidade Federal do Rio de Janeiro, 21944-971, Rio de Janeiro, RJ, Brazil
c
Magn. Reson. Chem. 2008, 46, 1158–1162
Copyright ꢀ 2008 John Wiley & Sons, Ltd.

Spectral assignments and reference data
13C spectra were acquired with a 19502.7 Hz spectral width,
using a 30◦ pulse of 4.6 µs and a 0.4 s relaxation delay. The 1868
transient FID was collected with 71 680 data points, multiplied by
a 1.0 Hz Lorentzian function, and zero-filled to 143 360 data points,
prior to FT.
The1H–1HCOSYspectrawereobtainedasamatrixof256×1024
(t1 × t2) data points, with eight transients per increment, a
1 s relaxation delay, and a spectral width of 4000 Hz in both
dimensions. The t2 and t1 domains were multiplied by a squared
sine bell function, and zero-filled to 2048 data points, prior to
2D FT.
The multiplicity edited 1H–13C HSQC spectra were obtained as
matrices of 256 × 2048 (t1 × t2) data points, with 12 transients
per increment and a 1 s relaxation delay. The spectral width was
4000 HzinF2and10 778.8 HzinF1withthetransmitterforGlobally
optimizedAlternatingphaseRectangularPulse(GARP)decoupling
Scheme 1. Syntheses of naphthofuranquinones 2–7 from 3-bromo-nor-β-lapachone.
Table 1. 1H NMR chemical shifts (δ, ppm), multiplicities and coupling constants (J, Hz) for compounds 2–7
Naphthofuranquinones
H
2
3
4
5
6
7
3
4.85 (s)
4.72 (s)
4.78 (s)
4.78 (s)
4.84 (s)
4.78 (d, 6.9)
6a 8.14 (ddd, 7.7,1.3, 0.5) 8.10 (ddd, 7.7,1.2, 0.4) 8.12 (ddd, 7.7,1.3, 0.5) 8.10 (ddd, 7.7,1.2, 0.4) 8.01 (ddd, 7.7,1.2, 0.4) 8.10 (ddd, 7.7,1.3, 0.5)
7a 7.65 (ddd, 7.7, 7.6, 1.2) 7.64 (ddd, 7.7, 7.6, 1.1) 7.65 (ddd, 7.7, 7.6, 1.2) 7.65 (ddd, 7.7, 7.6, 1.2) 7.64 (ddd, 7.7, 7.7, 1.1) 7.64 (ddd, 7.7, 7.6, 1.2)
8a 7.71 (ddd, 7.6, 7.6, 1.3) 7.71 (ddd, 7.6, 7.6, 1.2) 7.70 (ddd, 7.6, 7.6, 1.3) 7.70 (ddd, 7.6, 7.6, 1.2) 7.71 (ddd, 7.7, 7.6, 1.2) 7.70 (ddd, 7.6, 7.6, 1.3)
9a 7.75 (ddd, 7.6, 1.2, 0.5) 7.75 (ddd, 7.6, 1.1, 0.4) 7.73 (ddd, 7.6, 1.2, 0.5) 7.73 (ddd, 7.6, 1.2, 0.4) 7.75 (ddd, 7.6, 1.1, 0.4) 7.73 (ddd, 7.6, 1.2, 0.5)
2ꢁ
3ꢁ
4ꢁ
5ꢁꢁ
6
1ꢁꢁ
2ꢁꢁ
R1
R4
–
6.52 (dd, 9.2, 4.5b)
6.56 (t, 2.2)
6.72 (t, 1.5)
7.38 (t, 1.9)
6.27 (dt, 11.3b, 2.3)
6.95 (d, 7.5)
6.53 (d, 7.5)
–
6.89 (t, 9.2c)
–
–
6.86 (dd, 7.9, 1.5)
7.03 (t, 7.9)
6.50 (dd, 7.9, 1.5)
1.58 (s)
–
7.49 (dd, 8.0, 1.9)
7.21 (t, 8.0)
6.85 (dd, 8.0, 1.9)
1.59 (s)
–
–
6.72 (ddd, 8.0, 2.2, 0.7)
6.43 (ddd, 8.6b, 8.2, 2.3)
6.89 (t, 9.2c)
7.09 (t, 8.0)
7.10 (ddd, 8.2, 8.2, 6.7b)
6.34 (s)
1.58 (s)
1.72(s)
6.52 (dd, 9.2, 4.5b)
6.46 (ddd, 8.0, 2.2, 0.7)
6.35 (dd, 8.2, 2.3)
1.58 (s)
1.58 (s)
1.58 (s)
1.66 (s)
1.68 (s)
1.68 (s)
1.73 (s)
1.68 (s)
2.08 (s)
2.30 (s)
–
–
–
–
–
–
–
–
–
–
a The chemical shifts and the coupling constants were extracted by simulation.
b JHF
.
c JHH = JHF
.
c
Magn. Reson. Chem. 2008, 46, 1158–1162
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

E. N. da Silva Ju´nior et al.
set to the center of F1. A one-bond coupling constant value of
140 Hz was used to select direct correlations. The t2 domain was
multiplied by a Gaussian function and zero-filled to 2048 data
points. In t1, forward linear prediction was used to predict 1024
data points, prior to multiplication by a Gaussian function, zero
filling to 4096 data points and 2D FT.
Table 2. 13C NMR chemical shifts (δ, ppm) multiplicities and coupling
constants (J, Hz) for compounds 2–7
Naphthofuranquinones
C
2
3
4
5
6
7
The 1H–13C HMBC spectra were obtained as matrices of
256 × 2048 (t1 × t2) data points each with 84 transients and
a 1 s relaxation delay. The spectral width was 4000 Hz in F2 and
15 083.0 Hz in F1. Long-range correlations were optimized for
a 10 Hz coupling, and a value of 140 Hz was used to suppress
direct correlations. The t2 domain was multiplied by a shifted
Gaussianfunctionandzero-filledto2048datapoints. Int1, forward
linear prediction was used to predict 1024 data points, prior to
multiplication by a sine bell function, zero filling to 4096 data
points and 2D FT.
2
3
96.8
61.8
96.7
62.4
96.7 96.7 96.6
61.4 61.3 61.1
114.7 114.7 114.3
175.3 175.3 175.2
180.8 180.8 180.7
131.1 131.1 130.9
129.5 129.5 129.3
132.6 132.5 132.6
134.6 134.6 134.5
125.1 125.1 125.1
127.2 127.2 127.0
169.7 169.8 170.2
96.6
61.6
3a 115.3
115.0
175.4
180.8
131.1
129.5
132.5
134.6
125.1
127.3
169.6
143.5
114.8
175.3
180.8
131.2
129.5
132.6
134.6
125.1
127.3
169.6
4
5
175.3
180.9
5a 131.2
6
7
8
9
129.5
132.5
134.6
125.1
All spectra were processed and analyzed using the MestReC
4.6 software (Mestrelab Research SL, Spain). The spin-systems
simulation was performed using SpinWorks 2.5 software (Dr Kirk
Marat, Canada).
9a 127.5
9b 169.4
1ꢁ 145.2
148.3 148.4 148.0 149.0 (d, 10.6)a
112.8 115.7 106.6
99.9 (d, 25.4)a
2ꢁ 119.5 114.1 (d, 7.7)a
3ꢁ 130.2 115.7 (d, 22.6)a 135.0 123.2 148.9 164.0 (d, 241.4)a
4ꢁ 118.4 155.0 (d, 234.8)a 118.0 120.9 112.2 104.6 (d, 21.3)a
5ꢁꢁ 136.5 115.7 (d, 22.6)a 130.3 130.6 129.5 130.4 (d,10.0)a
Materials
6ꢁꢁ 111.1 114.1 (d, 7.7)a
111.3 111.7 118.6
21.7 21.7 21.6
27.3 27.4 27.3
108.9 (d,1.7)a
The syntheses for compounds 2–7 have been published
elsewhere.[2] The naphthofuranquinones 2–7 were obtained, as
a racemic mixture, in good yields by nucleophilic substitution of
the bromide group of 3-bromo-nor-β-lapachone (1) by arylamines
and the compounds were purified by column chromatography in
silica gel, eluted with an increasing polarity gradient mixture of
hexane and ethyl acetate (9/1–7/3) (Scheme 1). The purity of the
compounds was observed by high-resolution mass spectra.
1
21.6
27.3
17.0
21.7
21.7
27.4
–
21.7
27.3
–
2ꢁꢁ
R1
R4
–
–
–
–
–
–
–
–
a JCF
.
betweenH-3andtheC-2ꢁꢁ (methylgroup). Thefullcharacterization
of compounds 2–7 is presented in Tables 1 and 2.
Results and Discussion
1H multiplets from the naphthoquinone rings exhibit inconve-
nient second-order effects at 7.05 T. In this case, a computer-aided
spectralanalysiswasperformedtomaketheextractionofchemical
shiftsandcouplingconstantseasier.Figure 1showsthepartialsim-
ulated spectra of an ABCD spin system formed by the hydrogens
of the naphthoquinone ring.
The 1H and 13C NMR spectra of the compounds 2–7 were
well resolved and chemical-shift assignments were based on the
analysis of the multiplicity patterns of proton resonances and also
ontheuseofhomonuclear1H–1HCOSYandheteronuclear1H–13
C
1
HSQC and H–13C HMBC spectra. The nuclear Overhauser effect
(NOE) experiment helped to establish the relative configuration
Figure 1. Partial simulated and experimental spectra showing the ABCD spin system formed by the hydrogen naphthoquinone ring from compound 5.
c
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2008, 46, 1158–1162

Spectral assignments and reference data
Although the NMR experiments and the assignments
δ = 148.9 ppm (2JCH), C-4ꢁ at δ = 112.2 ppm, and C-6ꢁ at
δ = 118.6 ppm(3JCH). TheH-4ꢁ signalatδ = 7.49 ppmiscorrelated
with the carbon resonances for C-3ꢁ at δ = 148.9 ppm (2JCH), C-2ꢁ
at δ = 106.6 ppm, and C-6ꢁ at δ = 118.6 ppm (3JCH). The H-5ꢁ
signal at δ = 7.21 ppm is correlated with the carbon resonances
for C-1ꢁ at δ = 148.0 ppm, and C-3ꢁ at δ = 148.9 ppm (3JCH).
The H-6ꢁ signal at δ = 6.85 ppm is correlated with the carbon
resonances for C-2ꢁ at δ = 106.6 ppm, and C-4ꢁ at δ = 112.2 ppm
(3JCH) (Fig. 3). On the use of homonuclear 1H–1H COSY spectrum
there were observed correlations between H-4ꢁ and H-5ꢁ, H-5ꢁ,
and H-6ꢁ. Additionally, for the compounds 3 and 7, there were
observed the heteronuclear coupling constants, JHF and JCF, for
the 1H and 13C signals of the arylamine ring.
studies were made for all compounds, the quinone
6
(2,2-dimethyl-3-(3-nitro-phenylamino)-2,3-dihydro-naphtho[1,2-
1
b]furan-4,5-dione) was taken as an example to illustrate the H
and 13C assignments of the quinones 2–7. We can identify, in
1H NMR spectrum, singlets at δ = 1.59, 1.73, and 4.84 ppm,
corresponding to the hydrogens of the two methyl groups and
H-3. To confirm the assignments based on the 1H–13C HSQC and
1H–1H COSY spectra, and to assess more information about the
structure of quinone 6, a 1H–13C HMBC spectrum was recorded.
From this spectrum we can conclude the following:
The H-3 furan ring hydrogen at δ = 4.84 ppm shows long-range
correlation with the carbon resonances for C-3a at δ = 114.3 ppm
and C-2 at δ = 96.6 ppm (2JCH), C-1ꢁ at δ = 148.0 ppm, C-2ꢁꢁ at
δ = 27.3 ppm and C-9b at δ = 170.2 ppm (3JCH). The H-1ꢁꢁ signal
at δ = 1.59 ppm is correlated with the carbon resonances for
C-2 at δ = 96.6 ppm (2JCH), for C-3 at δ = 61.1 ppm and C-2ꢁꢁ at
δ = 27.3 ppm (3JCH). The H-2ꢁꢁ signal at δ = 1.73 ppm is correlated
with the carbon resonances for C-2 at δ = 96.6 ppm (2JCH), C-3
at δ = 61.1 and C-1ꢁꢁ at δ = 21.6 ppm (3JCH) (Fig. 2). The relative
configuration between the C-2ꢁꢁ and the hydrogen H-3 of the
furan ring was assigned employing the NOE experiment: the H-2ꢁꢁ
signal (δ = 1.73 ppm) was enhanced when the H-3 hydrogen
(δ = 4.84 ppm) was irradiated.
TheH-6naphthoquinonering hydrogenatδ = 8.01 ppmshows
long-range correlation with the carbon resonances for C-5 at
δ = 180.7 ppm, C-8 at δ = 134.5 ppm, and C-9a at δ = 127.0 ppm
(3JCH). H-7 signal at δ = 7.64 ppm is correlated with the carbon
resonances for C-5a at δ = 130.9 ppm, and C-9 at δ = 125.1 ppm
(3JCH). The H-8 signal at δ = 7.71 ppm is correlated with the
carbon resonances for C-6 at δ = 129.3 ppm (3JCH). The H-9 signal
is correlated for C-7 at δ = 132.6, and C-9b at δ = 170.2 ppm
(3JCH) (Fig. 4). The conclusive correlations of the naphthoquinone
ring between H-6 and C-5, and H-9 and C-9b can be observed in
1
Fig. 5. On the use of homonuclear H–1H COSY spectrum there
The H-2ꢁ benzene ring hydrogen at δ = 7.38 ppm shows
long-range correlation with the carbon resonances for C-3ꢁ at
were observed correlations between the hydrogens H-6 and H-7,
H-7 and H-8, and H-8 and H-9.
Figure 4. Connectivities found in the compound 6 HMBC spectrum for the
naphthoquinone ring.
Figure 2. Connectivities found in the compound 6 HMBC spectrum for the
furan ring.
Figure 3. Connectivities found in the compound 6 HMBC spectrum for the
benzene ring.
Figure 5. HMBC spectrum of derivative 6.
c
Magn. Reson. Chem. 2008, 46, 1158–1162
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

E. N. da Silva Ju´nior et al.
In conclusion, we have shown in this article the complete and
unambiguous assignments of 1H and 13C chemical shifts of six new
ortho-naphthofuranquinones 2–7. We observed that the nature of
the substituents on the arylamine ring did not affect significantly
the1Hand13Cchemicalshiftsofthenaphthofuranquinonesystem.
Therefore, this work can be used as a model for 1H and 13C
assignments of other compounds possessing the 2H-naphtho[2,3-
b]-furan-4-one system in their structures.
[4] T. P. Barbosa, C. A. Camara, T. M. S. Silva, R. M. Martins, A. C. Pinto,
M. D. Vargas, Bioorg. Med. Chem. 2005, 13, 6464.
[5] N. M. F. Lima, C. S. Correia, L. L. Leon, G. M. C. Machado, M. F.
Madeira, A. E. G. Sant’Ana, M. O. F. Goulart, Mem. Inst. Oswaldo Cruz
2004, 99, 757.
[6] D.-O. Moon, Y. H. Choi, N.-D. Kim, Y.-M. Park, G.-Y. Kim, Int.
Immunopharmacol. 2007, 7, 506.
[7] S. Gafner,
J.-L. Wolfender,
M. Nianga,
H. Stoeckli-Evans,
K. Hostettmann, Phytochemistry 1996, 42, 1315.
[8] H. Hussain, K. Krohn, V. U. Ahmad, G. A. Miana, I. R. Green, Arkivoc
2007, (ii), 145.
[9] M. N. Silva, V. F. Ferreira, M. C. B. V. de Souza, Quim. Nova 2003, 26,
407.
Acknowledgements
[10] J. Chiang, L. Averboukh, A. B. Pardee, J.Biol.Chem. 1993, 268, 22463.
[11] B. Frydman, L. J. Marton, S. J. Sun, K. Neder, T. D. Witiak, A. A. Liu,
H. M. Wang, Y. Mao, H. Y. Wu, M. M. Sanders, L. F. Liu, Cancer Res.
1997, 57, 620.
[12] L. Huang, A. B. Pardee, Mol. Med. 1999, 5, 711.
[13] C. L. Zani, A. B. de Oliveira, S. M. Starling, Quim. Nova 1994, 17, 43.
[14] R. A. Tapia, C. Salas, A. Morello, J. D. Maya, A. T. Labbe`, Bioorg. Med.
Chem. 2004, 12, 2451.
We would like to thank Mr Raymond McInerney, University of
Limerick, Ireland, for reviewing the manuscript. This work was
supported by CNPq (National Council of Research of Brazil), CAPES,
FAPERJ, UFF, FINEP, and UFRJ.
References
[15] C. Salas, V. Armstrong, C. Lo´pez, R. Tapia, Magn. Reson. Chem. 2008,
45, 1.
[1] E. P. Sacau, A. E. Braun, A. G. Ravelo, D. G. Yapu, A. G. Turba, Chem.
Biodivers. 2005, 2, 264.
[2] E. N. Silva Ju´nior, M. C. B. V. de Souza, A. V. Pinto, M. C. F. R. Pinto,
M. O. F. Goulart, F. W. A. Barros, C. Pessoa, L. V. Costa-Lotufo,
R. C. Montenegro, M. O. de Moraes, V. F. Ferreira, Bioorg.Med.Chem.
2007, 15, 7035.
[3] V. F. Ferreira,A. Jorqueira,A. M. T. Souza,M. N. daSilva,M. C. B. V. de
Souza, R. M. Gouveˆa, C. R. Rodrigues, A. V. Pinto, H. C. Castro,
D. O. Santos, H. P. Arau´jo, S. C. Bourguignon, Bioorg. Med. Chem.
2006, 14, 5459.
[16] U. S. Kumar, A. K. Tiwari, S. V. Reddy, P. Aparna, R. J. Rao, A. Z. Ali,
J. M. Rao, J. Nat. Prod. 2005, 68, 1615.
[17] F. W. Ribeiro, M. C. F. R. Pinto, A. V. Pinto, C. G. T. de Oliveira,
V. F. Ferreira, J. Braz. Chem. Soc. 1990, 1, 55.
[18] C. E. Heltzel, A. A. L. Gunatilaka, T. E. Glass, D. G. I. Kingston, J. Nat.
Prod. 1993, 56, 9 1500.
[19] H. Itokawa, K. Matsumoto, H. Morita, K. Takeya, Phytochemistry
1992, 31, 3 1061.
c
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