Synthetic chalcones as efficient inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

Article abstract of DOI:10.1016/j.bmcl.2008.09.105

In the search for lead compounds for new drugs for tuberculosis, the activity of 38 synthetic chalcones were assayed for their potential inhibitory action towards a protein tyrosine phosphatase from Mycobacterium tuberculosis - PtpA. The compounds were ob

Full text of DOI:10.1016/j.bmcl.2008.09.105

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6227–6230
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic chalcones as efficient inhibitors of Mycobacterium tuberculosis
protein tyrosine phosphatase PtpA
Louise Domeneghini Chiaradia a, , Alessandra Mascarello a, , Marcela Purificação b, Javier Vernal b,
Marlon Norberto Sechini Cordeiro a, María Emilia Zenteno b, Andréa Villarino b, Ricardo José Nunes a,
Rosendo Augusto Yunes a, Hernán Terenzi b,
*
a Departamento de Química, Universidade Federal de Santa Catarina, Campus Trindade, 88040-900 Florianópolis, SC, Brazil
b Departamento de Bioquímica, Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Campus Trindade, CEP: 88040-900, Florianópolis, SC, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
In the search for lead compounds for new drugs for tuberculosis, the activity of 38 synthetic chalcones
were assayed for their potential inhibitory action towards a protein tyrosine phosphatase from Mycobac-
terium tuberculosis– PtpA. The compounds were obtained by aldolic condensation between aldehydes and
acetophenones, under basic conditions. Five compounds presented moderate or good activity. The struc-
ture–activity analysis reveals that the predominant factor for the activity is the molecule planarity/
hydrophobicity and the nature of the substituents.
Received 18 August 2008
Revised 25 September 2008
Accepted 29 September 2008
Available online 5 October 2008
Hernán Terenzi dedicates this paper to the
memory of his mentor, colleague and friend,
Pr. Mario Zakin – Institut Pasteur – Paris.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Chalcones
Tuberculosis
PtpA
Mycobacterium tuberculosis is responsible for tuberculosis infec-
tion, and the emergence in recent years of multi-resistant strains
has made it a serious challenge in terms of international public
health. Annually 10 million new cases of tuberculosis appear and
about two million people die each year as a consequence of the
disease.1
As generally described for intracellular pathogens, mycobacte-
rial infection promotes the secretion of proteins by the pathogen,
especially phosphatases, which contribute to the disease installa-
tion. For instance, M. tuberculosis secretes PI3P-phosphatase which
inhibits phagosomal maturation2; Yersinia pseudotuberculosis, once
in the host cell, releases phosphatase YopH, which inhibits bacte-
rial phagocytosis by dephosphorylation of Cas and other focal
adhesion proteins.3,4 Similarly, the phosphatase SptP of Salmonella
enterica serovar Typhimurium, is directly responsible for the rever-
sal of the actin cytoskeletal changes induced after bacteria inter-
nalization into non-phagocytic cells.5
a cellular context. Phosphotyrosine appears as a critical cell-signal-
ing element and PTP activity is essential to control appropriate re-
sponses.6 M. tuberculosis has two phosphotyrosine phosphatases
(PTPs), PtpA and PtpB,7 and has no tyrosine kinase genes annotated
in its genome sequence database,8 suggesting that the targets of
these two phosphatases may be present in the host cell, and in-
volved in host–pathogen interactions.
It was recently demonstrated that PtpA is secreted by M. tuber-
culosis in infected human macrophages,9 and it was also very ele-
gantly shown that VPS33B, a regulator of membrane fusion, is a
PtpA substrate. This target, VPS33B, when dephosphorylated by
PtpA inhibits phagosome–lysosome fusion, a process arrested in
mycobacterial infections. Considered together, these data, demon-
strate that PtpA is essential for mycobacterial intracellular persis-
tence, emerging as a promising target for therapeutic intervention.
Chalcones are essential intermediate compounds in flavonoid
biosynthesis in plants. Several reports have documented active bio-
logical properties of natural and synthetic chalcones.10,11 Many
studies have demonstrated antileukemic,12,13 antitumoral,14–20
antiinflammatory,21,22 anti-ulcerogenic,23 antioxidant,14,24,25 anti-
malarial,26 antileishmaniosis26,27 and hypotensive28 activities for
chalcones, besides other pharmacological effects. Chalcones are also
cited as being antibacterial,29 including M. tuberculosis, by Lin et
al.,30 however, no information on the target of action of the com-
Tyrosine phosphatases (PTPs) constitute a family of closely re-
lated key regulatory enzymes that, in conjunction with protein-
tyrosine kinases, control the state of tyrosine phosphorylation in
* Corresponding author. Tel.: +55 48 3721 6919; fax: +55 48 3721 9672.
E-mail address: hterenzi@ccb.ufsc.br (H. Terenzi).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.105

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L. D. Chiaradia et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6227–6230
pounds is available. Nayyar and Jain (2005) cited chalcones as agents
with chemotherapeutic potential against M. tuberculosis.31
cones of the series 3 are derived from 2,4,6-trimethoxyacetophe-
none; chalcones of the series are derived from 1-
4
The main objective of this study is the search for a specific
inhibitor of the protein tyrosine phosphatase PtpA. We synthesized
and characterized 38 synthetic chalcones, which were assayed
in vitro using a recombinant purified PtpA from M. tuberculosis.
The chalcones were prepared by aldolic condensation as shown
in Figure 1. Chalcones 1 and 2 are derived from xanthoxyline; chal-
naphthaldehyde; chalcones of the series 5 are derived from 2-
naphthaldehyde; and the chalcones of the series 6 are derived from
3,4-methylenedioxybenzaldehyde; the obtained yields were be-
tween 36% and 97% (Table 1).
The structures of all compounds, including those still not pub-
lished (4c, 4f, 4l, 5c, 5i, 6i and 6c), were confirmed by chemical
Figure 1. Synthesis of the chalcones. (a) Methanol, KOH 50%, rt, 24 h.

L. D. Chiaradia et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6227–6230
6229
Table 1
Table 2
Yields of the synthesis and activity of the Mycobacterium tuberculosis PtpA in the
IC50 of the compounds more actives against Mycobacterium tuberculosis PtpA, assayed
in concentrations of 5–60 M.
presence of 25
l
M of the chalcones.
l
Chalcone Yield Activity of the
Chalcone Yield Activity of the
Chalcone
Ring A
Ring B
IC50
(lM
error)
(%)
enzyme PtpA
(% error)
(%)
enzyme PtpA
(% error)
5i
4d
4ª
5j
5a
2-OCH3-4-OH
2-OH
2,5-OCH3
3,4-OCH3
2,5-OCH3
2-Naphtyl
1-Naphtyl
1-Naphtyl
2-Naphtyl
2-Naphtyl
53.7 1.3
50.2 2.1
39.5 1.1
23.1 1.6
8.4 0.9
1
2
51
36
91
43
89
65
58
54
57
63
57
65
70
59
51
58
83
90
71
92 15
87 21
5d
5e
5f
5g
5h
5i
66
88
73
95
97
39
96
88
74
77
40
62
76
85
66
88
47
72
95
74.6
89.2
89
7
6
1
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4l
5ª
5b
5c
98
100
5
4
88 33
99 12
109
6
54
82.4
88
7
7
6
61 23
31.5 18
104 13
5j
5l
the substituents were 2,5-dimethoxy in the A ring and 2-naphthyl
in the B ring, with an excellent IC50 of 8.4 M (compound 5a). Com-
pound 5j also with two methoxy groups in the A ring (3,4-dime-
thoxy) and a 2-naphthyl group in the B ring, had an IC50 value of
23.1 lM. These results permit us to observe that the substitution
of the phenyl group for 2-naphthyl group in the B ring, seems to
be a predominant factor to reduce the activity of PtpA, when asso-
ciated with the ideal position of the methoxyl groups in the A ring.
Compound 4a, which has two methoxyl groups in A ring, and the
phenyl group substituted by 1-naphtyl group as B ring, had rela-
tively moderate activity against the enzyme, with an IC50 value
l
24 15
75 17
6a
6b
6c
6d
6e
6f
6g
6h
6i
91.3
94
9
4
82
62.7
102
5
4
1
74 12
103
7
86 10
80.5 17
80 10
79.5
84.4
42.5
9
8
7
103
82.4
8
7
80 23
99 10
91.9 12
81.5 13
84
6j
6l
3
of 39.5
an IC50 value of 50.2
l
M. The moderate activity observed for compound 4d, with
M, may be due to the presence of the hydro-
l
identification data using 1H NMR, 13C NMR, IR and elementary
analysis.
xyl group in the A ring that may eventually mediate hydrogen
bonding with the enzyme.
On analyzing the relationships between structure and activity it
was observed that the position and nature of the substituents in A
ring, probably the hydrophobicity or planarity of the naphtyl
groups in B rings are predominant factors to inhibition of PtpA.
The presence of substituents that increase the electronic density
of the A ring appear to be necessary for the activity of the
chalcones.
In summary, from the 38 assayed chalcones, the synthetic com-
pound, 5a is highly effective against M. tuberculosis PtpA and 4a,
4d, 5i and 5j are moderately effective. These results encourage us
to elucidate the possible binding mode of these compounds to
PtpA.
The PtpA coding region was subcloned from M. tuberculosis geno-
mic DNA, and the expression vector pET28a-PtpA was used to pro-
duce recombinant His-tagged protein as previously described.32
Initial assays were performed with a single compound concen-
tration (25 lM) to assess the potential of the compounds as inhibi-
tors.33 The activity of PtpA in vitro in the presence of the
compounds was determined as previously described 34 and is shown
in Table 1.
It can be observed from Table 1 that compounds 4a,37 4d,36
5a,39 5i35 and 5j38 show important inhibitory activity towards
PtpA. The data given in Table 1 indicate that the change in the sub-
stituent of the phenyl A ring affects the activity of the enzyme.
When the B ring is substituted for 1 or 2-naphthyl groups, the com-
pounds generally show potential bioactivity, such as 5i, 4a, 5a, 5j
and 4d, decreasing the relative activity of the enzyme to 61.0%,
54.0%, 42.5%, 31.5% and 24.0%, respectively, at a chalcone final con-
Acknowledgments
This research was supported by grants and fellowships from
CNPq (Conselho Nacional de Desenvolvimento Científico e Tec-
nológico), MCT, FAPESC and CAPES (Coordenação de Pessoal de
Nível Superior). This work is part of the theses of L.D. Chiaradia
and A. Mascarello, who are doctoral and master’s students in
chemistry. We like to thank the Universidade Federal de Santa Cat-
arina, Department of Chemistry and Department of Biochemistry
for the equipment facilities.
centration of 25 lM. We can observe a relationship, between the
substituents that increase the electronic density of the A ring, such
as methoxyl, and a 1 or 2-naphthyl group in the B ring. To assess
the activity of these compounds, further assays were carried out
and their IC50 values in relation to mycobacterial PtpA (5–60
of compound) were established and are given in Table 2.
lM
Chalcones where the B ring is 3,4-methylenedioxy (all series 6)
and chalcones with electron acceptor substituents in the B ring (1,
2, 3a, 3b and 3c), or groups that decrease the electronic density of
the A ring, such as bromo, chloro and nitro (compounds 1, 2, 3a, 3b,
3c, 4c, 4e, 4f, 5c, 5e, 5f, 6c, 6e and 6f), present a low inhibition of
PtpA activity.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.105.
The compounds with the phenyl B-ring substituted for naphthyl
groups and with three methoxyl groups (4h, 5l and 5h), without
substituents (4g and 5g) or with only one methoxyl or hydroxyl
group in the A ring (4b, 4j, 5b and 5d), did not show satisfactory
reduction of the activity of PtpA, with the exception of 4d. Com-
pounds that exhibit similar changes in the molecule due to the
substituents in position 2 and 6 of the A ring (4l and 5l) also exhibit
low PtpA inhibitory activity.
In Table 2 the activity of chalcones when the B ring is a naphthyl
group and two electron-donors substituents are present in the A
ring is depicted. We now observed that the inhibitory effect is con-
siderably increased. Highest inhibitory activity was achieved when
References and notes
1. Maher, D.; Raviglione, M. Clin. Chest Med. 2005, 26, 167.
2. Hestvik, A. L.; Hmama, Z.; Av-Gay, Y. Infect. Immun. 2003, 71, 5514.
3. Ivanov, M. I.; Stuckey, J. A.; Schubert, H. L.; Saper, M. A.; Bliska, J. B. Mol.
Microbiol. 2005, 55, 1346.
4. Black, D. S.; Bliska, J. B. EMBO J. 1997, 16, 2730.
5. Fu, Y.; Galan, J. E. Nature 1999, 401, 293.
6. Bishop, A. C.; Zhang, X. Y.; Lone, A. M. Methods 2007, 42, 278.
7. Zhang, Z. Y. Curr. Opin. Chem. Biol. 2001, 5, 416.
8. Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher, C.; Harris, D.; Gordon, S.
V.; Eiglmeier, K.; Gas, S.; Barry, C. E., 3rd; Tekaia, F.; Badcock, K.; Basham,
D.; Brown, D.; Chillingworth, T.; Connor, R.; Davies, R.; Devlin, K.; Feltwell,
T.; Gentles, S.; Hamlin, N.; Holroyd, S.; Hornsby, T.; Jagels, K.; Krogh, A.;

6230
L. D. Chiaradia et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6227–6230
McLean, J.; Moule, S.; Murphy, L.; Oliver, K.; Osborne, J.; Quail, M. A.;
Rajandream, M. A.; Rogers, J.; Rutter, S.; Seeger, K.; Skelton, J.; Squares,
R.; Squares, S.; Sulston, J. E.; Taylor, K.; Whitehead, S.; Barrell, B. G. Nature 1998,
393, 537.
also the IC50 values were determined in triplicate in two independent
experiments.
35. Compound 4a – (2E)-1-(20,50-dimethoxyphenyl)-3-(1-naphthyl)-2-propen-1-one.
Yellow solid, mp 89–90 °C. 1H NMR (CDCl3) d 3.88 (s, 3H, OCH3), 3.83 (s, 3H,
OCH3), 6.96 (d, 1H, J = 8.2 Hz, H40), 7.06 (dd, J = 8.0 Hz, 1H, H4), 7.49–7.59 (m,
5H, H3, H5, H6, H7, H8), 7.85 (d, 1H, J = 7.2 Hz, H30), 7.89 (d, 1H, J = 16.0 Hz,
9. Bach, H.; Papavinasasundaram, K. G.; Wong, D.; Hmama, Z.; Av-Gay, Y. Cell Host
Microbes 2008, 3, 316.
10. Dimmock, J. R.; Elias, D. W.; Beazely, M. A.; Kandepu, N. M. Curr. Med. Chem.
1999, 6, 1125.
11. Ni, L.; Meng, Q. C.; Sikorski, J. A. Expert Opin. Ther. Patents 2005, 14, 1669.
12. Saydam, G.; Aydin, H. H.; Sahin, F.; Kucukoglu, O.; Erciyas, E.; Terzioglu, E.;
Buyukkececi, F.; Omay, S. B. Leuk. Res. 2003, 27, 57.
Ha
), 7.89 (s, 1H, H60), 8.24 (d, 1H, J = 8.0 Hz, H2), 8.50 (d, 1H, J = 16.0 Hz, Hb).
13C NMR (CDCl3) d 56.12 (OCH3), 56.72 (OCH3), 113.60 (C30), 114.72 (C60),
119.71 (C40), 123.85 (C ), 125.40 (C10), 125.72 (C10), 126.45 (C3), 127.06 (C9),
128.96 (C5), 129.69 (C4), 129.86 (C8), 130,72 (C6), 132.01 (C2), 132.85 (C7),
133.96 (C1), 140.20 (Cb), 152.97 (C50), 153.90 (C20), 192.45 (C@O). IR max/cmꢀ1
a
m
13. Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. Bioorg. Med. Chem. 2004, 12, 2679.
14. Go, M. L.; Wu, X.; Liu, X. L. Curr. Med. Chem. 2005, 12, 483.
15. Won, S. J.; Liu, C. T.; Tsao, L. T.; Weng, J. R.; Ko, H. H.; Wang, J. P.; Lin, C. N. Eur. J.
Med. Chem. 2005, 40, 103.
16. Takahashi, T.; Baba, M.; Nischino, H.; Okuyama, T. Cancer Lett. 2006, 231, 319.
17. LeBlanc, R.; Dickson, J.; Brown, T.; Stewart, M.; Pati, H. N.; VanDerveer, D.;
Arman, H.; Harris, J.; Pennington, W.; Holt, H. L.; Lee, M. Bioorg. Med. Chem.
2005, 13, 6025.
18. Cabrera, M.; Somoens, M.; Falchi, G.; Lavaggi, M. L.; Piro, O. E.; Castellano, E. E.;
Vidal, A.; Azqueta, A.; Monge, A.; Ceráin, A. L.; Sagrera, G.; Seoane, G.;
Cerecetto, H.; González, M. Bioorg. Med. Chem. 2007, 15, 3356.
19. Kachadourian, R.; Day, B. J. Free Radic. Biol. Med. 2006, 41, 65.
20. Hsu, Y. L.; Kuo, P. L.; Lin, C. C. Life Sci. 2005, 77, 279.
21. Liu, Y. C.; Hsieh, C. W.; Wu, C. C.; Wung, B. S. Life Sci. 2007, 80, 1420.
22. Ko, H. H.; Tsao, L. T.; Yu, K. L.; Liu, C. T.; Wang, J. P.; Lin, C. N. Bioorg. Med. Chem.
2003, 11, 105.
1657 (C@O), 1588 (C@C), 1223, 1043 (C–O), 2947, 1493, 977, 807, 787 (KBr)
Anal. Calcd for C21H18O3: C, 79.23; H, 5.70. Found: C, 79.24; H, 6.11. Yield: 65%.
36. Compound 4d
–
(2E)-1-(20-hydroxyphenyl)-3-(1-naphthyl)-2-propen-1-one:
1
Yellow solid, mp 106–108 °C. H NMR (CDCl3) d 6.97 (dd, 1H, J = 7.4 Hz, H50),
7.06 (d, 1H, J = 8.4 Hz, H30), 7.51–7.62 (m, 4H, H3, H6, H7, H40), 7.76 (d, 1H,
J = 15.2 Hz, H
8.79 (d, 1H, J = 15.2 Hz, Hb), 12.88 (s, 1H, OH). 13C NMR (CDCl3) d 118.93 (C30),
119.17 (C50), 122.99 (C ), 123.67 (C2), 125.59 (C8), 125.68 (C10), 126.67 (C3),
127.41 (C6), 129.07 (C4, C5), 130.00 (C9, C60), 131.48 (C10), 136.74 (C1, C40),
142.66 (Cb), 163.93 (C20), 194.12 (C@O). IR max/cmꢀ1 3451, 1635, 1351, 1576,
a
), 7.90–7.99 (m, 4H, H60, H4, H5, H8), 8.29 (d, 1H, J = 8.4 Hz, H2),
a
m
1203, 1015, 3047, 1435, 1162, 972, 760 (KBr). Anal. Calcd for C19H14O2: C,
83.19; H, 5.14. Found: C, 83.88; H, 5.18. Yield: 57%.
37. Compound 5a – (2E)-1-(20,50-dimethoxyphenyl)-3-(2-naphthyl)-2-propen-1-one.
Light yellow, mp 120–121 °C. 1H NMR (CDCl3) d 3.83 (s, 3H, o-OCH3), 3.89 (s,
3H, m-OCH3), 6.97 (d, 1H, J = 8.0 Hz, H40), 7.06 (d, 1H, J = 8.0 Hz, H30), 7.23 (s,
1H, H60), 7.51–7.53 (m, 2H, H6, H7), 7.53 (d, 1H, J = 16.0 Hz, H
a), 7.75 (d, 1H,
23. Yamamoto, K.; Kakegawa, H.; Ueda, H.; Matsumoto, H.; Sudo, T.; Miki, T.;
Satoh, T. Planta Med. 1992, 58, 389.
24. Anto, R. J.; Sukumran, K.; Kuttan, G.; Rao, M. N. A.; Subbaraju, V.; Kuttan, R.
J = 8.0 Hz, H3), 7.81 (d, 1H, J = 16.0 Hz, Hb), 7.83–7.88 (m, 3H, H4, H5, H8), 7.99
13
(s, 1H, H1). C NMR (CDCl3) d 55.89 (o-OCH3), 56.56 (m-OCH3), 109.77 (C60),
113.42 (C30), 114.40 (C40), 119.17 (C10), 123.76 (C
a), 126.68 (C3), 127.07–
Cancer Lett. 1995, 97, 33.
25. Zhan, C.; Yang, J. Pharmacol. Res. 2006, 53, 303.
127.23 (C6, C7), 127.78 (C5), 128.63 (C8), 129.73 (C1), 130.51 (C4), 132.66
(C10), 133.36 (C9), 134.27 (C2), 143.46 (Cb), 152.58–153.64 (C20, C50), 192.48
26. Liu, M.; Wilairat, P.; Croft, S. L.; Tan, A. L. C.; Go, M. L. Bioorg. Med. Chem. 2003,
11, 2729.
27. Boeck, P.; Falcão, C. A. B.; Leal, P. C.; Yunes, R. A.; Cechinel, V.; Torres-Santos, E.
C.; Rossi-Bergamann, B. Bioorg. Med. Chem. 2006, 14, 1538.
28. Ogawa, H.; Okada, Y.; Kamisako, T.; Baba, K. Clin. Exp. Pharmacol. Physiol. 2007,
34, 238.
29. Pappano, N. B.; Puig de Centorbi, O.; Debattista, N. B.; Calleri de Milan, C.;
Borkowski, E. J.; Ferretti, F. H. Rev. Argent. Microbiol. 1985, 17, 27.
30. Lin, Y. M.; Zhou, Y.; Flavin, M. T.; Zhou, L. M.; Nie, W.; Chen, F. C. Bioorg. Med.
Chem. 2002, 10, 2795.
31. Nayyar, A.; Jain, R. Curr. Med. Chem. 2005, 12, 1873.
32. Purificação, M.; Razzera, G.; Terenzi, H.; Villarino, A., unpublished work.
33. Manger, M.; Scheck, M.; Prinz, H.; von Kries, J. P.; Langer, T.; Saxena, K.;
Schwalbe, H.; Fuerstner, A.; Rademann, J.; Waldmann, H. Chem. Bio Chem. 2005,
6, 1749.
(C@O). IR m
max/cmꢀ1 1644 (C@O), 1570 (C@C), 1336, 1130 (C–O), 3012, 2946,
2837, 1508, 1227, 1005, 693 (Ar) (KBr). Anal. Calcd for C21H18O3: C, 79.23; H,
5.70. Found: C, 79.69; H, 6.00. Yield: 83%.
38. Compound 5i – (2E)-1-(30-methoxy-40-hydroxyphenyl)-3-(2-naphthyl)-2-propen-
1-one: Light yellow solid, mp: 166–168 °C. 1H NMR (CDCl3) d 4.01 (s, 3H, OCH3),
6.10 (s, 1H, OH), 7.02 (d, 1H, J = 8.0 Hz, H50), 7.29 (s, 1H, H20), 7.52–7.54 (m, 1H,
H60), 7.66–7.72 (m, 3H, H3, H6, H7), 7.83 (d, 1H, J = 15.6 Hz, H
a
), 7.80–7.87 (m,
3H, H4, H5, H8), 7.97 (d, 1H, J = 15.6 Hz, Hb), 8.05 (s, 1H, H1). 13C NMR (DMSO-
d6) d 56.37 (m-OCH3), 110.74 (C20), 114.05 (C50), 121.99 (C3), 123.96 (C
a),
126.97 (C60), 127.51 (C6), 128.03 (C7), 128.85 (C1), 128.91 (C8), 130.68 (C4,
C5), 131.33 (C10), 132.81 (C10), 133.63 (C9), 134.54 (C2), 144.30 (Cb), 147.15
(C40), 150.64 (C30), 188.74 (C@O). IR max/cmꢀ1 3265 (OH), 1643, 1202 (C@O),
m
1280, 1025 (C–O), 1563 (C@C), 2950, 2835, 1522, 1445, 970, 844, 816, 779 (Ar)
(KBr). Anal. Calcd for C20H16O3: C, 78.93; H, 5.30. Found: C, 78.86; H, 5.76.
Yield: 39%.
34. Measurement of phosphatase activity and inhibition: The phosphatase assays
39. Compound 5j – (2E)-1-(30,40-dimethoxyphenyl)-3-(2-naphthyl)-2-propen-1-one:
Light yellow solid, mp 168–170 °C. 1H NMR (CDCl3) d 3.97 (s, 3H, OCH3), 3.98
(s, 3H, OCH3), 6.94 (d, 1H, H60), 7.50–7.53 (m, 2H, H6, H7), 7.65 (s, 1H, H1), 7.67
were carried out in 96-well plates containing 5
ll of diluted compound or
solvent (100% DMSO) in each well. After dilution in PtpA buffer (25 mM bis
Tris–HCl [pH 8.0], 20 mM Imidazol [pH7.0], 40 mM DL-dithiothreitol, 40 mM p-
nitrophenyl phosphate [pNPP]), 2 ll of recombinant PtpA (1.0 lg/ll) diluted in
PtpA buffer was added in order to start the reaction. After 20 min at 37 °C, the
absorbance was measured (at 410 nm with readings every 2 min) on an ELISA
plate spectrophotometer (TECAN). Negative controls were performed in the
absence of enzyme, and positive controls were carried out in the presence of
enzyme with DMSO 100% in place of the compound. The percentage of activity
was calculated by the average of two experiments carried out in triplicate, and
(d, 1H, J = 15.6 Hz, H
a
), 7.73 (d, 1H, H50), 7.81–7.87 (m, 4H, H3, H4, H5, H8),
7.95 (d, 1H, J = 15.6 Hz, Hb), 8.02 (s, 1H, H20). 13C NMR (CDCl3) d 56.31 (m-e p-
OCH3), 110.23 (C50), 111.04 (C20), 121.99 (C ), 123.30 (C60), 123.93 (C3), 126.97
a
(C6), 127.52 (C5), 128.02 (C7), 128.90 (C4, C8), 130.70 (C1), 131.60 (C10),
132.78 (C10), 133.61 (C9), 134.52 (C2), 144.29 (Cb), 149.50 (C30), 153.52 (C40),
188.76 (C@O). IV m
max/cmꢀ1 1652 (C@O), 1583 (C@C), 1261, 1021 (C–O), 3008,
2935, 2841, 1510, 1448, 1415, 975, 920, 844, 810 (Ar) (KBr). Anal. Calcd for
C21H18O3: C, 79.23; H, 5.70. Found: C, 79.06; H, 6.14. Yield: 96%.