New 5-carba-pterocarpans: Synthesis and preliminary antiproliferative activity on a panel of human cancer cells

Article abstract of DOI:10.1016/j.bioorg.2020.104584

Natural pterocarpans and synthetic 5-carba-pterocarpans are isosteres in which the oxygen atom at position 5 in the pyran-ring of pterocarpans is replaced by a methylene group. These 5-carba-analogues were obtained in good yields through the palladium-catalyzed oxyarylation of alcoxy-1,2-dihydronaphthalens with o-iodophenols in PEG-400. They were evaluated on human cancer cell lineages derived respectively from prostate tumor (PC3, IC₅₀ = 11.84 μmol L^?1, SI > 12)) and acute myeloid leukemia (HL-60, IC₅₀ = 8.81 μmol L^?1, SI > 16), highly incident cancer types presenting resistance against traditional chemotherapeutics. Compound 6c (LQB-492) was the most potent (IC₅₀ = 3.85 μmol L^?1, SI > 37) in SF-295 cell lineage (glioblastoma). Such findings suggest that 5-carba-pterocarpan can potentially be new hit compounds for further development of novel antiproliferative agents.

Full text of DOI:10.1016/j.bioorg.2020.104584

Bioorganic Chemistry 107 (2021) 104584
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New 5-carba-pterocarpans: Synthesis and preliminary antiproliferative
activity on a panel of human cancer cells
,
,
,2
Francisco V. Gaspara 1, Soraya Marques Ribeirob 1, Júlio C.F. Barcellosa, Samuel Monteiroa
,
,
2
Jorge L.O. Domingosc, Maria Claudia dos Santos Lucianob, Carlos R.K. Paierb
,
b
a
´
Claudia Pessoa , Paulo R.R. Costa
a
´
ˆ
ˆ
Laboratorio de Química Bioorganica, Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saúde, Bloco H, Ilha da
´
Cidade Universitaria, 21941-590 Rio de Janeiro, RJ, Brazil
b
´
´
´
Laboratorio de Oncologia Experimental, Núcleo de Pesquisa e Desenvolvimento de Medicamentos, Universidade Federal do Ceara, Rodolfo Teofilo, 60430-275
Fortaleza, CE, Brasil
c Instituto de Química, Universidade do Estado do Rio de Janeiro, R.S. Francisco Xavier 524, Rio de Janeiro 20550-900, RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Natural pterocarpans and synthetic 5-carba-pterocarpans are isosteres in which the oxygen atom at position 5 in
the pyran-ring of pterocarpans is replaced by a methylene group. These 5-carba-analogues were obtained in good
yields through the palladium-catalyzed oxyarylation of alcoxy-1,2-dihydronaphthalens with o-iodophenols in
PEG-400. They were evaluated on human cancer cell lineages derived respectively from prostate tumor (PC3,
Palladium catalyzed oxyarylation
5-Carba-pterocarpans
Antiproliferative
Isosteres of pterocarpans
3-Hydroxy-4-iodo-benzaldehyde
IC50 = 11.84
μ
mol Lꢀ 1, SI > 12)) and acute myeloid leukemia (HL-60, IC50 = 8.81
μ
mol Lꢀ 1, SI > 16), highly
incident cancer types presenting resistance against traditional chemotherapeutics. Compound 6c (LQB-492) was
the most potent (IC50 = 3.85
μ
mol Lꢀ 1, SI > 37) in SF-295 cell lineage (glioblastoma). Such findings suggest that
5-carba-pterocarpan can potentially be new hit compounds for further development of novel antiproliferative
agents.
1. Introduction
Miller [14] and co-workers (Fig. 1a) and pterocarpen (3) and its carba-
analogue (4) presented similar affinity for α- and β-estrogenic human
Pterocarpans are the second-largest group of isoflavonoids and are
isolated from Fabaceaea, Leguminosae, Papilionaceae and Bituminaria
plant families [1,2], They act as phytoalexins, metabolites produced de
novo in stressful conditions like resource deprivation, pathogen invasion
and UV radiation-mediated injuries [3]. Pterocarpan-producing plants
are extensively used in traditional medicines [4], some of their reported
bioactivities are snake anti-venom [5], anti-HIV [6], antiprotozoal [7],
antifungal [1,7], antibiotic [1], antioxidant anti-inflammatory [8] and
antiproliferative [9–12]. Natural pterocarpans and synthetic 5-carba-
pterocarpans are isosteres in which the oxygen atom at position 5 in
the pyran-ring of pterocarpans is replaced by a methylene group. Despite
this structural similarity, bioactive 5-carba-pterocarpans were poorly
reported in the literature. We previously reported that the 11-aza-pter-
ocarpan (1) and its isostere 5-carba-analogue (2) showed anti-
proliferative properties on some cell lineages and 2 was more potent
(Fig. 1a) [12,13]. The isosteric concept was also successfully used by
receptors, higher than estradiol, the natural ligand [14] (see Fig. 1c).
In this note we disclose the synthesis of a series of 5-carba-pterocar-
pans (6–8) structurally related to the natural (+)-2,3,9-trimethox-
ypterocarpan (5) (Fig. 2). This compound was isolated from
Platymiscium floribundum in the northeast of Brazil [15–17] and its
promising antiproliferative activity against four different leukemic cell
lines was studied [15–17]. Pessoa and collaborators [16,17] reported
that this pterocarpan blocked DNA synthesis in HL-60 cells [16,17].
Cancer is still the third most common death cause in the world [18],
although new antineoplastic drugs have been developed in the last
years. Several new aggressive tumor with MDR (multidrug resistance)
phenotype have been identified, exihibiting a widespread resistance to
known drugs, enhancing the need for constant research and develop-
ment of new compounds and multidrug treatments [19].
In this work the comparison between the biological activities of
compounds 5 and its 5-carba-analogues 6–8 is our main goal (Fig. 1c).
1
Equal contribution to this work.
2
Undergraduate students.
https://doi.org/10.1016/j.bioorg.2020.104584
Received 13 August 2020; Received in revised form 18 December 2020; Accepted 21 December 2020
Available online 1 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

F.V. Gaspar et al.
Bioorganic Chemistry 107 (2021) 104584
Fig. 2. 5-carbapterocarpans 6, 7 and 8 synthesized in this work.
Fig. 1. Bioisosterism in pterocarpans and pterocarpenes.
Scheme 1. Synthesis of Alkoxy-dihydronaphthalens.
The pattern of substitution at the A-ring and its relevance was evaluated
through the comparison of the cytotoxic profile of compounds type 6,
dimethoxylated at the A-rind as the natural product, with compounds
type 7 and 8, respectively mono-methoxylated at position 2 or not
substituted at the A-ring. Regarding the pattern of substitution at the D-
ring, we have methoxy groups, as in the natural product. Alternatively,
the presence of a hydroxymethyl group, that could mimic at some degree
a phenol group [20,21] provides structural diversity at the D-ring. The
aldehyde group present in the structure of some compounds can be used
to prepare new derivatives or to connect the structure to carrier systems
[20].
good yields when oxyarylated with the same o-iodophenols. These
oxyarylations were accomplished under conditions previously described
by our laboratory [21]: 10 mol% Pd(OAc)2, 1.1 equiv. Ag2CO3 in PEG-
400, at 140 ◦C 10–40 min. Under these conditions the reactions are
faster and yields are better when compared to previous conditions using
refluxing acetone [10]. The stereospecificity of the reaction was the
same as observed in previous works, 5-carba-pterocarpans were ob-
tained exclusively in the cis configuration, confirmed by the character-
istically 1H NMR displacement and coupling constant of H11a (d, J ≅
8.2 Hz, H11a - H6b) [21]. No regioisomeric products were observed as
was recently identified by our group when oxyarylating electron-rich
2H-chromenes in similar conditions [24].
2. Results and discussion
The aldehydes 6b,c and 7b,c were reduced by NaBH4 to the corre-
sponding hydroxymethyl derivatives 6d,e and 7d,e in excellent yields
(Scheme 3). 8c was not subjected to any further transformations as itself
did not presented any interesting cytotoxicity (See Table 1).
Dihydronaphthalen (11c) is commercially available and the alkoxy-
dihydronaphthalens 11a,b were prepared from the corresponding
α
-tetralones 9a,b (Scheme 1) [21]. On the other hand o-iodophenols
12a-c were obtained according to standard literature procedures
[22,23] (Scheme 2).
3. Preliminary antiproliferative evaluation
Olefins 11a-c were oxyarylated with 12a-c leading to 5-carba-ptero-
carpans 6a-c, 7a-c and 8c in moderated to good yield (Scheme 3). It is
worth to mention that while phenols containing electron releasing
methoxy group, as in 12a and 12c reacts with 2H-chromenes leading to
adducts in low chemical yields[10], we were pleased to know that
dihydronaphthalens 11a,b lead to adducts 6a,c and 7a,c in moderate to
In Table 1 are shown the antiproliferative activity of the synthesized
5-carba-pterocarpans 6a–e, 7a–e, 8c and the pterocarpan (5) in its
racemic and enantiomeric pure forms. The cytotoxic activity of these
compounds was assessed through colorimetric MTT assay [25,26],
performed to estimate the half-maximum inhibitory concentrations
2

F.V. Gaspar et al.
Bioorganic Chemistry 107 (2021) 104584
Table 1
Cytotoxic activity of pterocapan 5 and 5-carbapterocarpans after a 72 h-expo-
sure expressed by IC50 (µmol Lꢀ 1).
Compound*
SF-295
PC3
HL-60
HCT-116
L929
(+-)5
27.67 ±
1.94
6.33 ±
0.15
1.78 ±
0.06
7.2 ± 0.72
80.5±
11.05
62.16 ±
15.3
(+)-5
8.3 ± 0.03
4.42 ±
0.01
0.42 ±
0.04
0.41 ±
0.06
(-)-5
80.52 ±
2.24
50.93 ±
1.99
57.07 ±
4.05
63.05 ±
0.33
99.54 ±
6.22
LQB-485 (6a)
99.56 ±
3.24
> 160
22.18 ±
0.75
98.4 ±
4.03
> 160
LQB-488
(6b)
>160
76.24 ±
1.94
15.9 ±
1.1
32.04 ±
1.21
> 160
>160
> 160
>160
LQB-492 (6c)
3.85 ±
0.52
42.26 ±
2.26
24.41 ±
1.34
42.3 ±
2.11
LBQ-487
(6d)
39.34 ±
2.62
76.77 ±
2.83
16.75 ±
1.12
28.01 ±
1.33
LBQ-507 (6e)
33.93 ±
1.82
11.84 ±
0.87
8.81 ±
0.68
20.24 ±
0.75
LQB-493 (7a)
LQB-496
> 160
15.31 ±
4.36
> 160
29.55 ±
0.08
> 160
19.05 ±
0.33
> 160
29.6 ±
1.34
>160
>160
(7b)
LQB-500 (7c)
107.27 ±
6.01
>160
14.24 ±
1.35
90.6 ±
0.93
>160
>160
>160
>160
LQB-509
(7d)
86.85 ±
5.2
106.22 ±
0.76
38.11 ±
0.81
64.7 ±
2.01
Scheme 2. Synthesis of 5-carba-pterocarpans 6a-c, 7a-c and 8c.
LQB-511 (7e)
136.19 ±
1.32
46.73 ±
3.30
45.36 ±
0.95
101.39 ±
5.27
LQB-499 (8c)
Doxorubicin†
111.98 ±
4.14
>160
59.89 ±
0.24
53.7 ±
0.42
0.26 ±
0.03
0.43 ±
0.02 ±
0.004
0.14 ±
0.02
0.84 ±
0.10
0.06
*Estimated through nonlinear regression from three independent experiments
with cancer and non-cancer cell lines.
Table 2
Selectivity index (SI) of 5-carbapterocarpans for selected tumour cell lineages.
Compound*
SF-295
PC3
HL-60
HCT116
(+/-)-5
2.91
12.72
14.03
1.94
45.22
147.62
1,73
11.18
151.22
1.57
(+)-5
7.47
(-)-5
1.23
LQB-488 (6b)
LQB-492 (6c)
LQB-487 (6d)
LQB-507 (6e)
LQB-500 (7c)
Doxorubicin (Positive control)
>1.01
>37.92
>4.07
>4.30
0.85
>2.11
>3.45
>2.08
>12.33
0.57
>10.13
>5.97
>9.55
>16.57
6.39
>5.02
>3.45
>5.71
>7.21
1.00
3.23
1.95
42.00
6.00
Scheme 3. Preparation of hydroxymethyl group 6d,e and 7d,e.
decreased.
Compounds 7a-e were in general far less potent than their 6 analogs,
corroborating our suspicion that the methoxy groups in C2 and C3 are
important for the biological activity. An exception was the compound
7b, which was more potent than 6b in almost all the cell lines, having
the second lowest IC50 against SF-295, PC3 and HCT-116
(IC50) of these compounds against tumor and non-tumor cell lines
(Table 1) (see Table 2).
We firstly focused our attention in comparing the activity of natural
product 5, previously studied by Pessoa’s group [15–17] with its isostere
6a. Surprisingly 6a is inactive on those cell lines where 5 is active,
suggesting the oxygen atom at the pyran ring in 5 may be involved in the
interaction with the biological target.
It is worth mentioning that (+)-5 is more potent than the racemate
and the bioselectivity (+/-)-5/(+)-5 ranged from 1.4 (PC3), 3.3 (SF-
295), 4.2 (HL-60) to 17 (HCT-116). Therefore, we can reasonably
speculate, based on the previous results, that enantiopure carba-
analogues could be more potent than the corresponding racemates.
The selectivity indexes (SIs) of compounds with better anti-
proliferative activities were calculated through the ratio between the
IC50 on the L929 non-tumor cell line and the IC50 of the tumor cell line
[26]. The natural product 5 presents high SI, specifically for HL-60 and
HCT-116, and as expected the enantiomer (+)-5 was more selective than
the racemate in all cases. Concerning the 5-carba-analogues, 6c is the
most active compound against SF-295 cells, even when compared to the
natural product, and presents a SI about five times higher than (+)-5 for
this cell line. The racemate 6e, the more active carba-analogue in PC3,
HL-60, and HCT-116, presented selectivity comparable to (+)-5 and
Interestingly, compound 6b was less potent than 5 and showed
higher potency than 6a, except for SF-295 cells, probably suggesting
involvement of the aldehyde group is in the molecular recognition by
the biological target”. Compound 6c, which presents a 2,3-dimethoxy
group at the A-ring like the natural product and a methoxy group in
C10 and an aldehyde at C8, was the most potent molecule (IC50 3.85
µmol Lꢀ 1) against SF-295 in comparison to any other compound in the
series, including the natural product itself. The presence of hydrox-
ymethyl group in 7d did not enhance or diminish its activity compared
with 7b. The hydroxymethyl group in the D ring of compound 6e (LQB-
507) substantially enhanced its biological activity against PC3, HL-60,
and HCT-116 cell lineages, however, the inhibitory activity on SF-295
3

F.V. Gaspar et al.
Bioorganic Chemistry 107 (2021) 104584
(+/-)-5 in PC3 linages.
5.1.1. 2,3,9-trimethoxy-5,6,6a,11a-tetrahydronaphtho[1,2-b]benzofuran
(6a)
Our group had previously studied the effect of natural pterocarpan 5
in a large panel of prostatic cancer cells [27], considering the relevance
of this cancer and its incidence around the world. For this reason, we
were inclined to pursue our effort in the investigation of LQB-507 (6e).
The mechanism of growth inhibition of 6e in PC3 cell lines was initially
investigated by flow cytometry.
White solid, (35.12 mg, 45% yield) mp 110–115 ◦C. 1H NMR (500
MHz, CDCl3): δ 7.11 (d, J = 8.1 Hz, 1H), 7.03 (s, 1H), 6.63 (s, 1H), 6.44
(d, J = 8.3 Hz, 1H), 6.39 (s, 1H), 5.62 (d, J = 8.2 Hz, 1H), 3.93 (s, 3H),
3.87 (s, 3H), 3.76 (s, 3H), 3.57 (dd, J = 13.5, 8.1 Hz, 1H), 2.72 – 2.50 (m,
2H), 2.03 (d, J = 12.8 Hz, 2H). 13C NMR (126 MHz, CDCl3): δ 160.5,
149.2, 147.8, 144.9, 132.9, 131.3, 124.0, 120.7, 112.8, 111.6, 111.0,
84.5, 55.8, 55.9, 56.0, 40.6, 27.7, 26.8. HRMS (ESI). Calculated for
The PC3 cells were treated with (+)-5, Taxol (Tx), and 6e in five
different concentrations (3, 7, 12, 18, and 24 µmol Lꢀ 1). The evaluation
of tumor cells by flow cytometry after a 24 h exposure to 6e indicated a
pattern of concentration-dependent cell cycle arrest at the G2/M phase,
starting with the 12 µmol Lꢀ 1 concentration (Table 3 and Supplementary
Fig. 1). This pattern was similar to that of (+)-5 and Taxol both in this
work and in previous assays evaluating the racemate of natural pter-
ocarpan 5 [17,27]. These results indicated a disruption of the mitotic
process in the PC3 cell line [27], the similar patterns found in the cell
cycle evaluations of (+)-5 and its derivative 6e could indicate a
resembling mechanism of action for these compounds.
C
38H40O8 (2 M+Na). Expected mass 2 M+Na 647, 1362; obtained mass
647.1358.
5.1.2. 2,3-dimethoxy-5,6,6a,11a-tetrahydronaphtho[1,2-b]benzofuran-9-
carbaldehyde (6b)
White solid, (53 mg, 68% yield) mp 140 ◦C. 1H NMR (400 MHz,
CDCl3): δ 9.88 (s, 1H), 7.41 (d, J = 7.4 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H),
7.23 (d, J = 5.1 Hz, 1H), 7.02 (s, 1H), 6.61 (s, 1H), 5.66 (d, J = 8.3 Hz,
1H), 3.91 (s, 1H), 3.84 (s, 1H), 3.67 (dd, J = 13.8, 8.4 Hz, 1H), 2.62 (dd,
J = 10.0, 5.1 Hz, 1H), 2.07 (dd, J = 12.8, 6.2 Hz, 1H), 1.88 – 1.76 (m,
1H). 13C NMR (101 MHz, CDCl3): δ 191.7, 159.9, 149.0, 147.8, 139.2,
137.8, 124.4, 124.3, 124.2, 112.4, 111.0, 110.1, 109.0, 82.4, 56.0, 55.9,
55.8, 41.0, 29.6, 27.4. HRMS (ESI): Calculated for C19H18O4 (M + Na).
Expected mass M + Na 333,1003; obtained mass: 333,1010.
4. Conclusions
5-Carba-pterocarpan is a new prototype for antiproliferative com-
pounds. Further studies are in progress to better understanding the
mechanism of action of the more promising compounds. 5-Carbaptero-
carpan 6c is more potent against glioblastoma-derived tumor cells (SF-
295) than the natural product, presenting a high SI (>37.92). On the
other hand, 6e showed cytotoxic and cytostatic activity against the
prostate cancer-derived cells (PC3) and leukemia (HL-60) with SI > 12
and SI > 16, respectively. The aldehyde group in 6c is probably
responsible for the activity against SF-295 and although this function
can be quickly metabolized in vivo, the conjugation of this group with
carrier systems could further be explored.
5.1.3. 2,3,10-trimethoxy-5,6,6a,11a-tetrahydronaphtho[1,2-b]
benzofuran-8-carbaldehyde (6c)
White solid, (52 mg, 54% yield) mp 135 ◦C. 1H NMR (500 MHz,
CDCl3): δ 1,93–1.89 (m, 1H), 2.11–2.05 (m, 1H), 2.63 – 2.55 (m, 2H),
3.77–3.62 (m, 1H), 3.79 (s, 3H), 3.87 (s, 3H), 3.92 (s, 3H), 5.84 (d, J =
8.6 Hz, 1H), 6.63 (s, 1H), 7.09 (s, 1H) 7.32 (s, 1H), 7.43 (s, 1H), 9.84 (s,
1H). 13C NMR (101 MHz, CDCl3): δ: 26.8, 27.7, 40.6, 55.8, 55.9, 56.0,
84.5, 111.0, 111.6, 112.8, 120.7, 124.0, 131.2, 131.3, 132.9, 144.9,
147.8, 149.2, 153.7, 190.6.). HRMS (ESI): Calculated for C20H20O5 (M
+ H) 341, 1354; obtained mass: 341.1384.
5. Experimental
5.1. General procedure for oxyarylation reactions.
5.1.4. 2-methoxy-5,6,6a,11a-tetrahydronaphtho[1,2-b]benzofuran-9-
carbaldehyde (7b)
In a sealed reaction tube were added 0.25 mmol (0.048 g) of 6,7-di
methoxy-1,2-dihydronaphthalene (11a), 0.5 mmol (0.125 g) of 4-hy-
droxy-3-iodo-5-methoxybenzaldehyde (12a), 0.275 mmol (0.076 g)
silver carbonate (1.1 equiv.), 10 mol% (0.0056 g) of palladium acetate
and 2 mL of PEG-400 as solvent. Then reaction tube was sealed with the
cap and stirred at 140 ◦C. The reaction was monitored by TLC till
completion. The reaction was extracted with 5 mL EtOAc, washed with
brine 5 × 10 mL, the organic phase was dried with anhydrous MgSO4,
filtered and evaporated under vacuum. The pure compound (45% yield)
was obtained after flash chromatography (EtOAc/ hexane; 10/90).
White solid, (43 mg, 62% yield) mp 130 ◦C. 1H NMR (500 MHz,
CDCl3): δ 1.77–1,82 (m, 1H), 2.18 – 2.04 (m, 1H), 2.70 – 2.59 (m, 2H),
3.66–3.60 (m, 1H), 3.90 (s, 3H), 5.90 (d, J = 8.8 Hz, 1H), 6.8 (d, J = 7.5
Hz, 1H 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.20(s, 1H), 7.32 (s, 1H), 7.49 (d, J
= 7.3 Hz 1H) 7.52 (d, J = 7.4 Hz, 1H) 9.89 (s, 1H). 13C NMR (101 MHz,
CDCl3): δ 26.8, 27.7, 40.6, 55.8, 84.5, 109.0, 114.2, 115.2, 124.2, 124.6,
129.4, 130.4, 133.5, 137.4, 139.1, 158.4, 160.0, 190.6 . HRMS.
Calculated for C18H16NaO3 (M + Na). Expected mass M + Na 303,0997;
obtained mass: M + Na) + 303.1002.
5.1.5. 2,10-dimethoxy-5,6,6a,11a-tetrahydronaphtho[1,2-b]benzofuran-
8-carbaldehyde (7c)
White solid, (55.6 mg, 72% yield) mp 142 ◦C. 1H NMR (500 MHz,
CDCl3): δ 1.90 – 1.72 (m, 1H), 2.18 – 2.04 (m, 1H), 2.70 – 2.59 (m, 2H),
3.90 (s, 3H), 3.84 (s, 3H), 5.90 (d, J = 8.8 Hz, 1H), 6.8 (dd, J = 7.4, 5.5
Hz, 1H), 7.14 (d, J = 7.4 Hz, 1H), 7.20 (d, J = 5.6 Hz, 1H), 7.32 (s, 1H),
7.52 (s,1H) 9.89 (s, 1H).13C NMR (101 MHz, CDCl3): δ (ppm): 27.1,
28.0, 40.7, 56.0, 84.5, 111.7, 120.8, 126.8, 128.4, 128.8, 130.6, 131.2,
132.1, 132.9, 138.9, 144.8, 153.9, 190.5. HRMS. Calculated for
Table 3
Flow cytometry profile of PC3 cells treated with (+)-5a, Taxol (Tx) and LQB-507
(6e) in 5 different concentrations (3, 7, 12, 18 and 24 µmol Lꢀ 1). SE, Standard
error of the mean (*p < 0.05).
Cell Cycle Phases
G0/G1
S
G2/M
Mean (%)
SE
Mean (%)
SE
Mean (%)
SE
C
19H18O4 (M + Na). Expected mass M + Na 333,1103; obtained mass:
333.1097.
Negative Control
(+)-5
55.4
0.8
2.3
2.3
1.7
2.0
2.3
2.0
2.1
22.3
6.9*
16.5
25.5
25.2
11.44*
31.6
26.0
1.5
1.4
1.6
1.9
2.5
2.2
2.6
3.8
22.3
1.6
2.5
1.8
1.1
1.5
3.3
3.7
2.2
16.26*
18.45*
60.6
76.84*
65.06*
14.0
Taxol
5.1.6. 10-methoxy-5,6,6a,11a-tetrahydronaphtho[1,2-b]benzofuran-8-
carbaldehyde (8c)
6e 3.0
6e 7.3
6e 12
6e 18
6e 24
μ
μ
mol Lꢀ 1
mol Lꢀ 1
61.1
13.7
White solid, (40 mg, 58% yield) mp 150 ◦C. 1H NMR (500 MHz,
CDCl3): δ 1.90–1.72 (m, 1H), 2.18 – 2.04 (m, 1H), 2.76 – 2.59 (m, 2H),
3.84 (m, 1H), 3.90 (s, 3H), 5.90 (d, J = 8.8 Hz, 1H), 7.30 – 7.25 (m, 1H),
7.14 (d, J = 6.8 Hz, 1H), 7.43 (s, 1H), 7.32 (s, 1H), 7.59 (d, J = 6.6 Hz,
1H), 9.83 (s, 1H). 13C NMR (101 MHz, CDCl3): δ (ppm): 27.1, 28.0, 40.7,
μ
μ
μ
mol Lꢀ 1
mol Lꢀ 1
mol Lꢀ 1
43.67*
14.44*
22.86*
44.89*
53.96*
51.11*
Table 3. Patterns of cell cycle arrest in PC3 cells treated with vehicle (a), and
natural pterocarpan (+)-5 (b), Taxol (c), 6e (d) at 18 µmol Lꢀ 1
.
4

F.V. Gaspar et al.
Bioorganic Chemistry 107 (2021) 104584
55.9, 84.4, 111.7, 120.8, 126.8, 128.3, 128.8, 130.6, 131.2, 132.1,
132.9, 138.9, 144.8, 153.9, 190.5. HRMS. Calculated for C18H16NaO3
(M + Na). Expected mass M + Na 303,0997; obtained mass: (M + Na) +
303.09916.
37 ◦C at a 5% CO2 atmosphere. For cytotoxicity assays, PC3 (prostate
cancer), SF295 (glioblastome), HL-60 (acute myeloid leukemia), HCT-
116 (colorectal cancer), and L929 (murine non-tumor fibroblasts) cell
lines were plated in 96-well plates respectively at 1.0 × 104, 1.0 × 104,
3.0 × 104, 7.0 × 104 and 1.0 × 104 cells/ml. These cells were provided
by the National Cancer Institute (USA), except for L929 obtained from
5.2. General procedure for reduction of aldehydes
´
BCRJ (Banco de Celulas do Rio de Janeiro).
To 34,1 mg (0.1 mmol) (of 5-carbapterocarpan (6c) in 1 mL of
methanol, 5.7 mg (0.15 mmol) of NaBH4 was added. After total con-
sumption of starting materials observed by TLC analysis (10–20 min), 5
mL of water was added, extractions with ethyl acetate (3x10 mL) were
performed. The organic layers were evaporated in vacuum giving the
respective alcohols. The pure compound was obtained after flash chro-
matography (EtOAc/ hexane;30/70).
5.3.2. MTT assay for cytotoxicity assessment
This method is based on the reduction of the yellow tetrazolium salt,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
in a formazan purple crystal by the mitochondrial dehydrogenases and
other NADPH-dependent cellular oxidoreductases [25]. The cells were
plated in 96-well plates and incubated for 72 h as previously described.
At the end of the treatment time the formazan precipitate was solubi-
lized in DMSO and the solution was read in a spectrophotometer at 595
nm.
5.2.1. (2,3-dimethoxy-5,6,6a,11a-tetrahydrobenzo[d]naphtho[1,2-b]
furan-9-yl)methanol(6d)
White solid, (41 mg, 98% yield) mp 112–115 ◦C. 1H NMR (400 MHz,
CDCl3): δ 7.20 (d, J = 7.5 Hz, 1H), 7.02 (s, 1H), 6.88 (d, J = 7.5 Hz, 1H),
6.79 (s, 1H), 6.61 (s, 1H), 5.61 (d, J = 8.3 Hz, 1H), 4.61 (s, 2H), 3.91 (s,
3H), 3.85 (s, 3H), 3.60 (dd, J = 14.4, 7.2 Hz, 1H), 2.68 – 2.53 (m, 2H),
5.3.3. Flow cytometry analysis
The PC3 cells at a density of 5 × 105 cells/mL were seeded in a 6-well
plate and let to adhere on to the surface overnight. Cells were exposed to
2.03 (dt, J = 11.5, 5.0 Hz, 1H), 1.79 (dtd, J = 12.8, 8.5, 4.3 Hz, 1H). 13
C
different concentrations (3, 7.3, 12, 18 and 24 μM) of pterocarpan de-
rivative LQB-507 for 24 h. The vehicle DMSO was used as negative
control, Taxol (Tx) and the natural dextrorotatory pterocarpan were
used as positive controls. Cells were washed thoroughly with PBS.
Processed cells were incubated at room temperature in propidium iodide
NMR (101 MHz, CDCl3): δ 159.5, 148.8, 147.7, 141.6, 131.1, 130.9,
125.0, 124.1, 119.2, 112.4, 111.0, 108.3, 82.1, 65.3, 55.9, 55.9, 55.8,
55.8, 40.7, 29.6, 27.6, 27.1.
(50
μ
g mLꢀ 1) for 30 min. The analysis was performed in the Guava flow
5.2.2. 2,3,10-trimethoxy-5,6,6a,11a-tetrahydrobenzo[d]naphtho[1,2-b]
furan-8-yl)methanol (6e)
cytometry system (Merk, Darmstadt, Germany).
White solid, (43 mg, 85% yield) mp ꢀ 138–140 ◦C. 1H NMR (400
MHz, CDCl3) δ 7.13 – 7.05 (m, 1H), 6.86 (s, 1H), 6.79 (d, J = 14.9 Hz,
1H), 6.60 (s, 1H), 5.67 (d, J = 8.3 Hz, 1H), 4.61 (d, J = 4.9 Hz, 2H), 3.88
(s, 3H), 3.83 (s, 6H), 3.69 – 3.57 (m, 1H), 2.72 – 2.49 (m, 2H), 2.02 (dt, J
= 12.1, 5.2 Hz, 1H), 1.80 (ddd, J = 17.6, 8.7, 4.5 Hz, 1H). 13C NMR
(101 MHz, CDCl3): δ 148.8, 147.6, 147.0, 144.2, 134.2, 132.6, 131.1,
124.7, 115.4, 112.7, 110.8, 110.4, 82.8, 65.6, 56.0, 55.8, 55.8, 41.4,
27.7, 27.2. HRMS (ESI): Calculated for C20H20O5 (M + Na) calculated
for 363,1202; found: 363,1259.
5.4. Data analysis
Cell viability was calculated as a percentage of cell growth in vehicle-
treated cell culture (negative control) and IC50 was estimated through
non-linear regression using the GraphPad Prism version 6.0.
The cell cycle data were exported and analyzed using the ModFit 4.1
trial version. All the experiments were accomplished for at least three
independent times. The mean of cell cycle experiments was used for
statistical analysis in GraphPad Prism 6.0. The results were presented by
the mean of ±SE (standard error of the mean). Entire statistic differences
were calculated after one-way anova followed by the Tukey’s test for
post-hoc analysis (*p < 0.05).
5.2.3. 2-methoxy-5,6,6a,11a-tetrahydrobenzo[d]naphtho [1,2-b]furan-9-
yl)metanol (7d)
White solid, (41 mg, 97% yield) mp 120 ◦C. 1H NMR (400 MHz,
CDCl3): δ 7.20 (d, J = 7.5 Hz, 1H), 7.06 (dd, J = 10.9, 5.5 Hz, 2H), 6.88
(d, J = 7.6 Hz, 1H), 6.83 – 6.76 (m, 2H), 5.63 (d, J = 8.5 Hz, 1H), 4.61 (s,
2H), 3.82 (s, 3H), 3.65 (dd, J = 13.7, 8.3 Hz, 1H), 2.69 – 2.50 (m, 2H),
2.11 – 1.95 (m, 1H), 1.77 – 1.42 (m, 1H). 13C NMR (101 MHz, CDCl3) δ
159.6, 158.2, 141.6, 134.1, 130.8, 130.7, 129.3, 124.2, 119.3, 114.9,
114.9, 114.2, 108.2, 108.2, 82.2, 82.2, 65.3, 55.4, 55.3, 40.8, 40.7, 29.6,
28.0, 26.6. HRMS (ESI): Calculated for C17H14NaO4:305.0789; found:
305.0788.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
˜
This study was financed in part by the Coordenacao de Aperfeiçoa-
5.2.4. 2,10-dimethoxy-5,6,6a,11a-tetrahydrobenzo[d]naphtho[1,2-b]
furan-8-yl)methanol (7e)
mento de Pessoal de Nível Superior-Brasil (CAPES). The authors are also
grateful to Conselho Nacional de Desenvolvimento Científico e Tec-
White solid, (43 mg, 94% yield) mp 140 ◦C. 1H NMR (400 MHz,
CDCl3): δ 7.15 (d, J = 2.5 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 6.87 (d, J =
7.2 Hz, 2H), 6.86 – 6.77 (m, 4H), 5.71 (d, J = 8.6 Hz, 1H), 4.63 (s, 2H),
3.87 (s, 3H), 3.83 (s, 3H), 3.68 (dd, J = 13.8, 8.2 Hz, 1H), 2.69 – 2.51 (m,
2H), 2.10 – 2.01 (m, 1H), 1.81 (ddd, J = 17.3, 8.5, 4.2 Hz, 1H). 13C NMR
(101 MHz, CDCl3): δ 159.6, 158.2, 141.6, 134.1, 130.8, 130.7, 129.3,
124.2, 119.3, 114.9, 114.9, 114.2, 108.2, 108.2, 82.2, 82.2, 65.3, 55.4,
55.3, 40.8, 40.7, 29.6, 28.0, 26.6.
´
nologico (CNPq) for the financial support of the project besides the
research sponsorships of C. Pessoa (PQ-1B, Process nr: 303102/2013-6).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104584.
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