One-step partial synthesis of (±)-asperteretone B and related hPTP1B1–400 inhibitors from butyrolactone I

Article abstract of DOI:10.1016/j.bmc.2020.115817

Protein tyrosine phosphatase 1B (PTP1B) is a validated target for developing antiobesity, antidiabetic and anticancer drugs. Over the past years, several inhibitors of PTP1B have been discovered; however, none has been approved by the drug regulatory agencies. Interestingly, the research programs focused on discovering PTP1B inhibitors typically use truncated structures of the protein (PTP1B_1-300, 1–300 amino acids), leading to the loss of valuable information about the inhibition and selectivity of ligands and repeatedly misleading the optimization of putative drug leads. Up to date, only six inhibitors of the full-length protein (hPTP1B_1-400), with affinity constants ranging from 1.3 × 10⁴ to 3.3 × 10⁶ M^?1, have been reported. Towards the discovery of new ligands of the full-length human PTP1B (hPTP1B_1-400) from natural sources, herein we describe the isolation of a γ-lactone (1, butyrolactone I) from the fungus Aspergillus terreus, as well as the semisynthesis, inhibitory properties (in vitro and in silico), and the structure-activity relationship of a set of butyrolactone derivatives (1 and 2, and 6–12) as hPTP1B_1-400 inhibitors, as well as the affinity constant (k_a = 2.2 × 10⁵ M^?1) of the 1-hPTP1B₁–₄₀₀ complex, which was determined by fluorescence quenching experiments, after the inner filter effect correction.

Full text of DOI:10.1016/j.bmc.2020.115817

Bioorg. Med. Chem. 28 (2020) 115817
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Bioorganic & Medicinal Chemistry
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One-step partial synthesis of (±)-asperteretone B and related hPTP1B1–400
inhibitors from butyrolactone I
a
,
*
a
b
´
´
´
Jose Rivera-Chavez , Diego Coporo-Blancas , Jesús Morales-Jimenez
a
´
´
´
Departamento de Productos Naturales, Instituto de Química, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria 04510, Ciudad de Mexico, Mexico
b
´
´
´
´
´
CONACYT-Consorcio de Investigacion, Innovacion y Desarrollo para las Zonas Aridas (CIIDZA), Instituto Potosino de Investigacion Científica y Tecnologica A. C.,
´
´
Camino a la Presa San Jose 2055, Lomas 4a seccion, 78216 San Luis Potosí, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
Protein tyrosine phosphatase 1B (PTP1B) is a validated target for developing antiobesity, antidiabetic and
anticancer drugs. Over the past years, several inhibitors of PTP1B have been discovered; however, none has been
approved by the drug regulatory agencies. Interestingly, the research programs focused on discovering PTP1B
inhibitors typically use truncated structures of the protein (PTP1B1-300, 1–300 amino acids), leading to the loss of
valuable information about the inhibition and selectivity of ligands and repeatedly misleading the optimization
of putative drug leads. Up to date, only six inhibitors of the full-length protein (hPTP1B1-400), with affinity
constants ranging from 1.3 × 104 to 3.3 × 106 Mꢀ 1, have been reported. Towards the discovery of new ligands of
the full-length human PTP1B (hPTP1B1-400) from natural sources, herein we describe the isolation of a γ-lactone
(1, butyrolactone I) from the fungus Aspergillus terreus, as well as the semisynthesis, inhibitory properties (in vitro
and in silico), and the structure-activity relationship of a set of butyrolactone derivatives (1 and 2, and 6–12) as
hPTP1B1-400 inhibitors, as well as the affinity constant (ka = 2.2 × 105 Mꢀ 1) of the 1-hPTP1B1–400 complex,
which was determined by fluorescence quenching experiments, after the inner filter effect correction.
Full-length PTP1B
Semisynthesis
Krapcho decarboxylation
Fungal natural products
Butyrolactone I
1. Introduction
was demonstrated that the use of truncated structures of this protein
(hPTP1B1-300) in drug discovery programs lead to the loss of valuable
Protein tyrosine phosphatase 1B (PTP1B) has recently emerged as a
target for developing antiobesity, antidiabetic and anticancer drugs. In
diabetes and obesity, it negatively regulates the insulin and leptin signal
transduction pathways, respectively, enhancing the insulin receptor’s
susceptibility and stimulating glucose transporters translocation and
glucose uptake in insulin-sensitive cells.1 Whereas in cancer it controls
several pathways involved in tumor growth, tumorigenesis, and
metastasis, for example, the Ras/Raf/MAPK and PI3K/AKT pathways.2,3
Since the validation of PTP1B as a target to treat chronic diseases,
numerous programs have focused on the synthesis or isolation (from
natural sources) of both competitive and non-competitive inhibitors.4
Such programs have resulted in the discovery of more than 500 PTP1B1-
300 inhibitors.5 Two of the most promising drugs developed to date are
ertiprotafib (a competitive inhibitor of the truncated model, PTP1B1-
300), and trodusquemine, a non-competitive inhibitor of the full-length
protein (1–405 residues). However, while both advanced to phase II
clinical trials, they were discontinued due to their low selectivity,
adverse effects, and/or pharmacokinetic properties.6 In a recent study, it
information about the inhibition and selectivity of the ligands, which
could be associated with the low rate of success of PTP1B inhibitors.7
The discovery of an allosteric site in PTP1B, and the expression of a
full-length protein (1–400 amino acids) have shifted the approaches to
discover inhibitors. The allosteric site in PTP1B is a hydrophobic pocket
constituted by helices α3, α6, and α7, situated on the C-terminus of the
protein and located 20 Å away from the catalytic domain.8 This pocket
regulates the catalytic domain’s flexibility, which results in a decrease of
the phosphatase activity.8–10 Whereas the C-terminal domain of this
protein has lower sequence conservation and higher specificity,9
significantly increasing the chances of discovering more specific mole-
cules with less toxic effects.
Thus, as part of our ongoing research program focused on discov-
ering non-competitive inhibitors of hPTP1B1–400 from natural sources,
particularly from fungi, the bioactive extract of an Aspergillus terreus (IQ-
046) was chemically investigated. This study led to the isolation of
butyrolactone I (1), butyrolactone IV (2), lovastatin (3), methyl 3,4,5-
trimethoxy-2-(2-(nicotinamido)benzamido)benzoate
(4),
and
* Corresponding author.
´
E-mail address: jrivera@iquimica.unam.mx (J. Rivera-Chavez).
https://doi.org/10.1016/j.bmc.2020.115817
Received 2 September 2020; Received in revised form 1 October 2020; Accepted 8 October 2020
Available online 15 October 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

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J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
chrysamide B (5), exhibiting the privileged metabolic potential of this
strain. Furthermore, the biological properties of compounds 1 and 2 as
hPTP1B1–400 inhibitors were screened in vitro, displaying IC50 values of
2.2. Partial synthesis of butyrolactone I (1) derivatives (6–12)
Thus, to explore the reactivity of butyrolactone I (1, the most
abundant compound in the extract of IQ-046), and to expand the
chemical space of this interesting bioactive metabolite, several one-step
reactions were performed (Scheme 1). Considering the features of
compound 1, it was initially speculated that the 4′, 4′′ phenolic groups
could be methylated using diazomethane or iodomethane. Unfortu-
nately, these methodologies did not result in the methylation of the
product. Instead, diazomethane generated an unstable methylated
product at the 4-OH position, while iodomethane was unreactive.
Challenged by these results, we hypothesized that 1 could be methylated
at the 4′, 4′′ phenolic groups using dimethyl carbonate (DMC), and tet-
rabutylammonium iodide (TBAI).17 Interestingly, under our reaction
settings, the dimethylated product was not obtained. Instead, two
35 and higher than 450 μM, respectively. Motivated by the differences in
biological activity of compounds 1 and 2, several semisynthetic de-
rivatives of 1 [asperteretone B (6), (±)-5′′-iodoasperteretone B (7),
(±)-asperteretone D (8), aspernolide A (9), 4′,4′′-butyrolactone I
′′ ′′
dipropionate (10), Δ7 ,8 -9′′-hydroxy-4′,4′′-butyrolactone I dipropionate
(11), and 3′,5′,5′′,8′′-tetrabromoaspernolide A (12)] were prepared via
one-step reactions. Principal component analysis of the chemical space
of butyrolactone I (1) derivatives allowed a structure-activity relation-
ship to be established, indicating that small differences in these ligands’
structure significantly affect their IC50 values. Molecular docking studies
of butyrolactone I (1) and its derivatives with a homologated model of
hPTP1B1–400 indicated that compounds (1, 2, and 6–12) bind to four
different sites within the protein. Three of them in the macromolecule’s
C-terminal domain, suggesting that some of them may behave as allo-
steric modulators. Finally, after inner filter effect correction, fluores-
cence quenching experiments determined the binding affinity of the
complex 1-hPTP1B1–400 and supported that butyrolactone I (1) may
behave as an allosteric inhibitor of the protein.
1
decarboxylated products of 1 were generated, as evidenced by the H
NMR, 13C NMR, and HR-DART-MS data (Figs. S29–S31, and S34–S36,
for compounds 6 and 7, respectively, Supporting Information). Careful
analysis of the 1D and 2D NMR (Figs. S32–S33, and S37–S38, respec-
tively, Supporting Information) and the CD data (Fig. S64, Supporting
Information) established the structure of the decarboxylated products as
(±)-asperteretone B (6), a secondary metabolite recently isolated from
Aspergillus terreus,18 and its 5′′-iodo derivative (7). Further insights about
the formation of compound 6 from 1 were obtained by dissolving the
later in THF, THF-DMC, and THF-TBAI, and heated up to 80 ◦C from six
to 60 h. Interestingly, compounds 6 or 7 were not obtained under these
conditions, indicating they are generated via a one-pot reaction
involving TBAI and DMC. Mechanistically, compound 6 could be pro-
duced from 1 via opening and methylation of the γ-lactone ring, medi-
ated by methoxide -generated in situ through a bimolecular base-
catalyzed alkyl cleavage mechanism (BAI2)-,19 and a re-arrangement of
the γ-lactone, followed by a Krapcho-like20 decarboxylation process
(Scheme 2).
2. Results and discussion
2.1. Isolation of natural products from Aspergillus terreus IQ-046
The extract of Aspergillus terreus (IQ-046, Fig. S1, Supporting Infor-
mation) was pursued for chemical investigation, as it inhibited the ac-
tivity of hPTP1B1–400 in an in vitro assay (95% inhibition at 50 ppm).
Fractionation of the extract led to the isolation of butyrolactone I and IV
(1 and 2),11,12 lovastatin (3, mevinolin),13 methyl 3,4,5-trimethoxy-2-
(2-(nicotinamido)benzamido)benzoate (4),14 and chrysamide B (5).
The latter being a nitro-compound previously isolated from Penicillium
chrysogenum SCSIO41001 (Fig. 1).15,16 All compounds were character-
ized by comparing their spectroscopic and spectrometric data with those
previously described in the literature (Figs. S2–S28, Supporting Infor-
mation). In vitro biological evaluation of compounds 1 and 2 as hPTP1B1-
(±)-5′′-Iodoasperteretone B (7) was isolated as a white amorphous
powder. Its molecular formula was established as C23H23O5I, based on
HR-DART-MS data ([M+H]+ m/z = 507.0681, calcd. for C23H24O5I, Δ =
+3.5 ppm), (Fig. S34, Supporting Information), indicating an index of
hydrogen deficiency of 12. The 1H and 13C NMR spectra (Figs. S35 and
S36, Supporting Information) were similar to those obtained for 6. The
main differences were observed in the aromatic region, particularly the
loss of the doublet attributed to the 5′′-H at δH = 6.75 ppm, the change in
400 inhibitors (IC50 = 35 and >450
μM, respectively) triggered our in-
terest in the structure-activity relationship (SAR) of these molecules.
Fig. 1. Structure of compounds isolated from A. terreus (IQ-046).
2

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J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
Scheme 1. Semisynthesis of compounds 6–12.
the coupling pattern from an ABB’ system in 6 to an AB system in 7
(Table S1, Supporting Information), and the shielding of the C-5′′ from
δC 116.3 in 6 to 86.4 in 7 (Table S2, Supporting Information).
Compound 8 was obtained by reacting 1 with DMC and TBAI in
double bound and an oxygenated sp3 carbon (C-9′′). Thoughtful in-
spection of the HMBC spectrum (Fig. S58, Supporting Information)
′′ ′′
established the structure of 11 as Δ7 ,8 -9′′-hydroxy-4′,4′′-butyrolactone
I dipropionate.
1
acidified (HCl) THF at 80 C. The H, 13C NMR, and HR-ESI-MS data
(Figs. S39–S41, Supporting Information) agreed with those previously
reported.18 Further analysis of the 2D NMR spectra (Figs. S42 and S43,
Supporting Information) confirmed the structure of 8 as (±)-asperter-
etone D.
Unsurprisingly, nature has demonstrated that the incorporation of
halogens (Cl, Br, and I) onto the scaffolds of organic compounds is a
typical process. Up to date, more than 5000 halogenated natural prod-
◦
ucts with numerous exciting activities have been reported in the liter-
24
ature.
In this regard, chlorination and bromination are routine
Asperteretones B and D (6 and 8) were recently isolated as racemic
mixtures from the extract of a coral-associated A. terreus. Both the 4S and
modifications in natural products’ structural diversification, since hal-
ogens play a crucial role in bioactivity by increasing the affinity and
selectivity of molecules for their targets.25–30 This phenomenon is
mainly associated with the ability of these atoms to interact with bio-
macromolecules by halogen bonding (interactions of the type
R–X⋅⋅⋅Y–R’, where X acts as a Lewis acid and Y is an electron donor
atom),29 as well as their capacity to increase the lipophilicity and steric
hindrance.30 In fact, the success of pairing halogens with bioactive
organic molecules has led to a growing trend in the number of iodinated
and brominated drugs reaching clinical phases.31 Conversely, some aryl
bromides have been associated with the occurrence of hepatotoxicity,32
whereas the presence of iodide in medicines or foods is related with
hypersensitivity and thyroids diseases,33 this stigma (high toxicity) have
seriously limited the research in this field, limiting our understanding of
the biological properties of halogen-containing natural products.
Considering the positive impact of halogenation on the bioactivity of
organic compounds, the 3′,5′,5′′,8′′-tetrabrominated analog of asperno-
lide A (12) was prepared reacting 1 with Br2 in a 1:1 MeOH-CH2Cl2
mixture at rt. Compound 12 was obtained as a white amorphous pow-
der. Its molecular formula was established as C24H20O7Br4 based on the
isotopic pattern and the HR-ESI-MS data ([M¡H]ꢀ = 734.7877 m/z,
calculated for C24H19O7Br4, Δ = 1.0 ppm) (Fig. S59, Supporting Infor-
mation), indicating an index of hydrogen deficiency of 13. The 1H
spectrum (Fig. S60, Supporting Information) displayed signals
4R enantiomers of 6 and 8 showed potent α-glucosidase inhibitory ac-
tivity (a molecular target for treating type II diabetes mellitus), with IC50
values ranging from 15.7 to 19.2
μ
M.18 Remarkably, Liu and coworkers
proposed the structures of 6 and 8 as corrections of the previously
described (±)-asperteretal D, and (4S)-4-decarboxylflavipesolide C,21
two specialized metabolites obtained from a different strain of A. terreus
(OUCMDZ-2739), grown under static conditions in liquid media sup-
plemented with 10 μM trichostatin A.
Compound 9 was obtained by reacting 1 with 1 M HCl at rt. The 1H,
13C NMR, and HR-ESI-MS data (Figs. S44–S46, Supporting Information)
were in agreement with those previously reported.22 Additional analysis
of the 2D NMR spectra (Figs. S47 and S48, Supporting Information)
confirmed the structure of 9 as aspernolide A.
To explore the reactivity of the phenolic groups at 4′ and 4′′ in
compound 1, it was reacted with propionic anhydride in pyridine
(Scheme 1). As expected,23 the 4′,4′′-butyrolactone I dipropionate (10)
was obtained, as evidenced by the 1H, 13C, HSQC, and HMBC NMR
spectra, and the HR-ESI-MS data (Figs. S49–S53, Supporting Informa-
tion). Interestingly, under the reaction conditions, a second product was
generated (11). The 1D, 2D NMR, and the HR-ESI-MS data
(Figs. S54–S56, Supporting Information) of this compound resembled
those of product 10. The main differences were the presence of an E-
3

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J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
Scheme 2. Proposed mechanism of reaction for the formation of (±)-asperteretone B (6), (±)-5′′-iodoasperteretone B (7), and (±)-asperteretone D (8).
attributable to an A2 spin system (δH 7.79), an aromatic AB system (δ
To gain further insights about the potential of compounds 1, 2, and
6–12 as hPTP1B1–400 inhibitors, these molecules were evaluated in vitro
to determine the effect of the introduced or eliminated fragments on the
hydrolase activity of the protein (Fig. 2 and Table 1). Compounds 2, 8,
and 9 showed moderate to weak activity with IC50 values ranging from
H
6.83 and 6.55), an oxymethylene (δH 3.51 and 3.34), an AMX spin sys-
tem (δ 4.18, 3.26 and 3.10), a methoxy (δ 3.83) and two methyl
H
groupsH(δH 1.55 and 1.40). The 13C NMR, together with the HSQC and
HMBC (Figs. S61–S63, Supporting Information) experiments, further
confirmed the structure of compound 12.
2.3. hPTP1B1–400 inhibition and principal components analysis (PCA)
PTP1B has gained significant interest since its validation as a target
for developing anti-obesity, antidiabetic, and anticancer drugs.1
Recently, we reported the expression and purification of a full-length
human PTP1B (hPTP1B1–400), as we detected a gap in the literature
regarding the use of truncated models of this macromolecule in drug
discovery programs (both in vitro or in silico). The use of these models
often leads to the loss of valuable information about the mechanisms of
inhibition and selectivity of ligands, while simultaneously misleading
the optimization of putative drug leads. In a recent report, four dimeric
phenalenones isolated from an ant-hill associated Talaromyces sp. were
reported as putative allosteric modulators of hPTP1B1–400, being only
the second class of inhibitors of the full-length enzyme reported in the
literature.34
Fig. 2. Concentration-response curves for enzymatic inhibitory activity of
compounds 1, 2, 6–12, and ursolic acid (positive control) against hPTP1B1-400
.
These data correspond to three independent experiments.
4

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J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
experimental IC50 values were considered. Principal components one
and two (PC1 and PC2) explained 73.6% of the covariance, while
PC1–PC3 described 96.1%. Remarkably, the number of rotatable bonds,
the accptHB, the PSA, and #NandO, had the most significant contribu-
tion to PC1. In contrast, all the descriptors except for the number of
rotatable bonds contributed to PC2. WPSA and the number of halogens
significantly contributed to PC3.
Table 1
Experimental IC50 values for compounds 1, 2, and
6–12 as hPTP1B1–400 inhibitors.
compound
IC50 (μM)
1
53.9 ± 1.1
>450
2
6
78.8 ± 3.6
34.6 ± 1.0
229.4 ± 9.8
186.1 ± 4.9
57.3 ± 1.3
96.3 ± 1.9
61.8 ± 1.4
33.1 ± 1.8
7
The 2D representation of the PCA (PC1 vs. PC2, and PC1 vs. PC3,
Fig. 3) revealed that active and poorly active analogs of 1 occupy
different chemical space regions. For example, compounds that bear an
isoprenyl group at C-3′′ (1, 6a, 6b, 7a, 7b, 10, and 11, the most active
products) grouped together, while separately clustered are the moder-
ately active compounds 2a, 2b, 8a, 8b, and 9, which lack this moiety.
8
9
10
11
12
Ursolic acid
186 to higher than 450
μ
M, while products 6, 7, and 10–12 displayed
2.4. Molecular docking
IC50 values from 35 to 97 μM, similar to that of ursolic acid, an allosteric
inhibitor of the full-length protein (IC50 = 33.1 μM, positive control),
To gaining further structural insights about the putative binding
mode of the butyrolactone I (1) analogs included in the PCA with
hPTP1B1–400, and explain the outcomes from bioactivity evaluation,
docking studies were performed using a homologated model of the
enzyme, as so far, there is no crystallographic structure reported for the
full-length protein (Fig. 4). Initially, the performance of AutoDock (AD)
was validated using trodusquemine, an aminosterol that binds to the C-
duclauxin, xenoclauxin, bacillisporin G, and talaromycesone B, which
display IC50 values ranging from 12.7 to 82.1
μ
M.7
Up to date, several natural and semisynthetic triterpenes (ursolic,
moronic, and oleanolic acids)35–37 have been reported as inhibitors of
the truncated model of PTP1B. These molecules inhibit the protein’s
phosphatase activity by binding to an extended allosteric site, or directly
interacting with the catalytic domain, as predicted by docking studies
and demonstrated by kinetics experiments.35–37 Additionally, numerous
PTP1B1-300 inhibitors with different scaffolds, including γ and δ-lac-
tones, have been isolated from fungi. Some examples include chrys-
opyrones (competitive inhibitors),38 deflectins,39 tylopilusin D (non-
competitive inhibitor),40 betulactone B,41 and verruculide B,42 high-
lighting the versatility of fungi to produce small molecules with PTP1B
inhibitory properties. Moreover, it does high point the affinity of these
structural moieties (γ and δ-lactones) for the macromolecule.
terminal domain of hPTP1B1–400 (Arg371, Arg373 and Val375 in α
9′ and
residues Leu299, His310, Ile311, Val334, and Ser393) with stoichiom-
etry 2:1, and ka values of 3.3 × 106 and 3.3 × 105 Mꢀ 1, respectively. The
results from molecular coupling showed that AD successfully predicts
the binding pocket of trodusquemine, which docked at the interface of
both the N (Pro89, Cys92, Leu119, Glu123, Met133, Ile134, Phe135, Glu136
,
and Asp137) and C-terminal (Thr368, Glu369, Val370, Arg371, and Ser372
)
domains of the protein.
The docking of hPTP1B1–400 with butyrolactone derivatives exhibi-
ted well-established bonds with amino acids in four different binding
sites within the protein (Figs. 4, S67–S80, Supporting Information);
three of them including residues of the C-terminal domain (residues
301–400). Interestingly, none of the molecules bonded to the active site
of the enzyme. In all cases, the forces that governed the interactions
were primarily hydrophobic (Van der Waals) and hydrogen bonding.
The putative binding site for compounds 1, 2a, 6a, 6b, 8a, 8b, 9, and
To explore the chemical space of natural (2) and semisynthetic de-
rivatives (6–12) of 1, detect the critical topologies that impact the in-
hibition of the phosphatase activity of hPTP1B1–400, and to establish a
SAR, principal component analysis (PCA) of 14 analogs (Fig. S65, Sup-
porting Information) was carried out. With this purpose, the number of
rotatable bonds, the polar component of the solvent-accessible surface
area (WPSA), the estimated number of hydrogen bonds that would be
accepted (accptHB), the polar surface area (PSA), the number of N and O
atoms (#NandO), the number of atoms in rings, and the number of ring
atoms not able to form conjugated aromatic systems, were predicted
using QikProp. Additionally, the number of halogens, as well as the
12a was formed by residues of the α
1′ helix (Glu4, Phe7, Glu8, Asp11), the
WPD loop (Ser187, Pro188, non-catalytic), the α3 helix (Ala189), α6 and α7
helices (Leu272, Ile275, Glu276, Lys279, Val287, Gln290, Trp291, Leu294), and
residues of the C-terminal domain (Cys324, Glu326, Phe327, Phe328, and
Fig. 3. Representation of the chemical space of natural butyrolactones (1 and 2), and semisynthetic analogs (6–12). The 2D plots were generated with the principal
component analysis of nine descriptors. Components 1–3 described 96.1% of the covariance.
5

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J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
Fig. 4. 3D representation of the interaction of the
complex 1, 2a, 2b, 6a–12b, and hPTP1B1-400. In
pale-cyan, the C-terminal domain of the protein
(residues 300–400). In pale-green, the
(residues 189–201). In pale-yellow, the
(residues 265–281). In pale-pink, the
α
3 helix
6 helix
7 helix
α
α
(residues 286–295). In black sticks, the residues
involved in the catalytic triad (Asp181, Cys215
,
and Arg221). In brown sticks trodusquemine. For
clarity, the full-size images of the 3D models of
the interaction of compounds 1, 2a, 2b, and
6a–12b in complex with hPTP1B1-400 are pro-
vided in the Supporting Information file
(Figs.
S67–S80,
Supporting
Information).
Graphics generated with PyMOL.
Val334). The interaction of these molecules with residues of the allosteric
incorporation of a hydroxy group at C-9′′ (compounds 2 and 11), and
decarboxylation and re-arrangement of the γ-lactone negatively affect
the phosphatase activity of hPTP1B1-400 (Fig. 5). However, docking
studies predicted two major binding pockets for this set of molecules
within PTP1B1-400, suggesting that the established structure-activity-
relationship must be taken cautiously. Further experiments such as
1H-15N NMR TROSY and comparison of the IC50 values of butyrolactone
derivatives as inhibitors of the truncated and full-length protein are
required to confirm the binding pockets and selectivity for these mole-
cules experimentally.
site (helices α3, α6, and α7) and the C-terminus of hPTP1B1–400, suggest
that this set of ligands may induce conformational changes onto the
protein and putatively behave as allosteric inhibitors. Conversely, the
binding pocket for 2b (4R, 8S, poorly active compound) was constructed
by Lys342, Cys344, Pro345, Asn355, Ala357, Pro358, and Tyr359, all of them
residues of the C-terminal domain. These results indicated that the
interaction of molecules with these amino acids, do not significantly
affect the hydrolase activity of hPTP1B1–400
The union site for molecules 10, 11, and 12b was similar to that
predicted for trodusquemine. It consisted of residues of the 2 helix
(Pro89, Asn90, Cys92, Gly93), β sheets 2–7 (Leu119, Ala122, Gln123, Gln127
.
α
,
2.5. Steady-state fluorescence quenching experiments of hPTP1B1-400
Glu132, Met133, Ile134, Phe135, Asp137), and one residue of the C-terminus
of the protein (Thr368), predicting a non-competitive type of inhibition.
Interestingly, the acylation of the phenolic groups at C-4′ and C-4′′ in 1
does not affect the bioactivity; however, these modifications direct
compounds 10 and 11 to a different binding pocket within the protein,
likely due to the conformational changes and steric hindrance imposed
by the propionate moieties, which also form hydrogen bonds with Gln123
stabilizing the interaction. In contrast, the binding site for compounds
7a and 7b, the most active compound of the series (iodinated), con-
with butyrolactone I (1)
To confirm the interaction of butyrolactone I (1, the most abundant
product in the extract of A. terreus IQ-046) with hPTP1B1–400, and sup-
port the outcomes from docking studies, steady-state fluorescence
quenching experiments were carried out. The high sensitivity of tryp-
tophan (Trp, a fluorescent amino acid) to its local environment results in
changes in its emission spectra. This phenomenon allows for monitoring
conformational transitions, subunit associations, and binding of a sub-
strate or ligand, providing valuable insight into a protein’s structure and
function.43,44 In this experiment, the Trp residues were used as intrinsic
fluorophores. The samples were excited at 280 nm, and the emission
spectra recorded from 300 to 500 nm. The marked reduction in the
fluorescence intensity of hPTP1B1–400 at increasing concentrations of
tained amino acids Leu71, Lys73, Gln78, Arg79, Ser80, Glu101, Gln102
,
Lys103, Ser203, Leu204, Ser205, Pro206, His208, Glu207, Gly209, Pro210, and
Val211, all of them residues from the N-terminal domain, and far away
from the active site of the enzyme. Remarkably, these residues have 87%
homology with TCPTP, which may compromise the in vivo selectivity of
these molecules. Complementary studies are needed to support this
statement fully.
compound 1 suggested that this molecule interacts with hPTP1B1–400
,
inducing conformational changes that directly affect its phosphatase
activity (allosteric inhibition, Fig. 6).
Briefly, close inspection of the structures, bioactivity, PCA, and
molecular docking data for the 14 analogs included in this study,
revealed that (i) the isoprenyl group at C-3′′ is a key feature for bioac-
tivity, (ii) incorporation of bulky moieties onto the structure of 1 direct
molecules to different binding sites within the protein, (iii) halogenation
of 1 and its derivatives positively contributes to the bioactivity, (iv)
Furthermore, the affinity of the complex butyrolactone I (1)-
hPTP1B1–400 was measured (Fig. 6). Unsurprisingly, the well-known
inner filter effect was observed by butyrolactone I (1), since it absorbs
light at the excitation and emission wavelengths (Fig. S66, Supporting
Information), which reduces the fluorescence emission of the
6

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J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
Fig. 5. Sites in 1 for bioactivity modulation.
Fig. 6. (A) Fluorescence spectra and titration curve (insets) of hPTP1B1–400 (0.5
μ
M) with increasing concentrations of butyrolactone I (1) (0–11
μ
M) before inner
M)
filter effect correction. (B) Fluorescence spectra and titration curve (inset) of hPTP1B1–400 (0.5
μ
M) with increasing concentrations of butyrolactone I (1) (0–11
μ
upon inner filter effect correction. Samples were excited at 280 nm, and the emission spectra recorded from 300 to 500 nm. The changes in maximal fluorescence
emission were corrected for light scattering and plotted against the total concentration of protein. The insets’ red line comes from fitting the data to the binding
model (Eq. (3)) to obtain the ka (1/kd). Experiments were carried out in HEPES 50 mM, NaCl 100 mM, and DTT 1.5 mM at pH 6.8.
complex.43,45,46 The emission spectra of the hPTP1B1–400, and 1-
hPTP1B1–400 complex, before (Fig. 6A) and upon the inner filter effect
correction (Fig. 6B) display a fluorescence quenching of 86.2% and
63.2%, respectively.43–45 These data evidenced the strong absorption of
butyrolactone I (1) at the excitation and emission wavelengths. Even
higher than 450 μM. It is worth noting that so far, it is the second report
of fungal natural products binding to the full-length protein (1–400
amino acids). Previous reports have used a truncated model of the
protein (1–300 or 1–323 amino acids), which often leads to the lack of
information about the mechanism of action of the molecules. Up today,
only five natural products have been reported as ligands of this
macromolecule, including trodusquemine and four dimeric phenale-
nones from a Talaromyces species, the later reported by our research
team, highlighting the lack of information regarding inhibitors of the
full-length protein, and further supporting the medicinal chemistry
performed in butyrolactone I (1). Furthermore, PCA of the chemical
space of butyrolactone I (1) derivatives indicated that small differences
in the structure of these ligands have a significant effect on their IC50
values. Molecular docking studies of butyrolactone I (1) and its de-
rivatives with a homologated model of hPTP1B1–400 suggested that some
of these molecules may behave as allosteric modulators. Finally, after
inner filter effect correction, fluorescence quenching experiments
determined the binding affinity of the complex 1-hPTP1B1–400 and
supported that butyrolactone I (1) may behave as an allosteric inhibitor
of the protein. Altogether, the results from this investigation highlight
the applicability of one-step semisynthetic approaches to expand the
chemical space of fungal natural products and detect the important
features that impact and improve their bioactivity, and the use of full-
length models of PTP1B in drug discovery programs.
though the concentration of 1 was low (11 μM), it created a significant
inner filter effect and contributed 23% of the quenching.
The association constant (ka) for the butyrolactone I (1)-hPTP1B1–400
complex was calculated before (2.6 × 105 Mꢀ 1) and after (2.2 × 105
Mꢀ 1) the inner filter effect correction.43–45 As expected, there are dif-
ferences in the values, stressing the importance of the correction.43,45,46
Butyrolactone I (1) showed higher affinity than ursolic acid (ka of 1.3 ×
104 Mꢀ 1),47 and is comparable with the ka reported for trodusquemine
(3.3 × 105 Mꢀ 1).34
3. Conclusion
In summary, chemical investigation of the bioactive extract of
Aspergillus terreus (IQ-046) as a hPTP1B1–400 inhibitor led to the isolation
of butyrolactone I (1), butyrolactone IV (2), lovastatin (3), methyl 3,4,5-
trimethoxy-2-(2-(nicotinamido)benzamido)benzoate (4), and chrys-
amide B (5). Additionally, the reactivity of 1 was explored using one-
step reactions, which allowed the semisynthesis of seven derivatives,
including the recently reported (±)-asperteretone B (6) and (±)-asper-
teretone D (8), the known compound aspernolide A (9), as well as the
four new derivatives (±)-5′′-iodoasperteretone B (7), 4′,4′′-butyr-
4. Experimental section
′′ ′′
olactone I dipropionate (10), Δ7 ,8 -9′′-hydroxy-4′,4′′-butyrolactone I
dipropionate (11), and 3′,5′,5′′,8′′-tetrabromoaspernolide A (12). The
biological properties of compounds 1, 2, and 6–12 as hPTP1B1–400 in-
hibitors were screened in vitro, displaying IC50 values ranging from 35 to
4.1. General experimental procedures
NMR experiments were conducted in CDCl3. NMR instrumentation
7

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J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
was a Bruker Advance III 500 (1H 500 MHZ, 13C 125 MHZ) or a Bruker
Ascend III 700 MHz NMR spectrometer equipped with a cryoprobe,
operating at 700 MHz for 1H and 175 MHz for 13C. The chemical shifts
are reported in ppm and are referenced to the residual solvent reso-
nances (7.26 ppm for δH, and 77.16 ppm for δC). HR-ESI-MS data were
obtained in a Thermo QExactive Plus Hybrid Quadrupole-Orbitrap mass
spectrometer (ThermoFisher, San Jose, CA, USA) coupled to a Waters
Acquity Ultraperformance Liquid Chromatography (UPLC) system
(Waters Corp.), or a Jeol, AccuTOF JMS-T100LC mass spectrometer
(HR-DART-MS). The UPLC separations were performed using an Acquity
BEH C18 column (50 mm × 2.1 mm, internal diameter, 1.7 µm) equili-
brated at 40 ◦C and a flow rate of 0.3 mL/min. The mobile phase con-
sisted of a linear CH3CN-H2O (acidified with 0.1% formic acid) gradient
starting at 15% CH3CN to 100% CH3CN over 8 min. The mobile phase
was held for 1.5 min at 100% CH3CN before returning to the starting
conditions. HPLC separations were performed using a Waters system
(2535 quaternary pump) connected to a 2707 autosampler and a 2998
PDA detector. Data acquisition and analysis were made with the
Empower 3 software (Waters). Analytical and semipreparative HPLC
were performed on Gemini-NX 5 µm particle size C18 columns (4.6 ×
250 mm, and 10.0 × 250 mm for analytical and semipreparative runs,
respectively; Phenomenex, Torrance, CA, USA).
The bottom layer was drawn off and evaporated to dryness. The solid
was resuspended in 300 mL of 1:1 MeOH-CH3CN mixture and parti-
tioned with 300 mL of hexane in a separatory funnel. The bottom layer
was evaporated to dryness.57 The extract (2.5 g) was adsorbed onto a
minimal amount of silica gel. This material was fractionated via open
column chromatography, using 200 g of silica gel 230–400 mesh
(Macherey-Nagel), and a gradient solvent system of hexane-EtOAc
100:0 → 0:100, to afford 28 fractions (F1-F28). Butyrolactone I (1)
(860.2 mg) was obtained as a yellow vitreous solid from fractions F14
-
F16. Fraction F20 (17 mg) was subjected to semi-preparative reversed-
phase chromatography on a GeminiNX C18 column using an isocratic
system of CH3CN-H2O (0.1% formic acid) 45:55 for 10 min at a flow rate
of 4.6 mL/min, to afford compound 2 (7.9 mg). Fraction F21 (~30 mg)
was subjected to semipreparative HPLC using a gradient system initiated
with 50:50 CH3CN-H2O (0.1% formic acid) to 70% CH3CN over 30 min
at a flow rate of 4.6 mL/min to generate compound 3 (8.5 mg). Fraction
F23 (~30 mg) was resolved by semipreparative HPLC using an isocratic
system of CH3CN-H2O (0.1% formic acid) 45:55 for 25 min at a flow rate
of 4.6 mL/min, to afford compound 4 (4.6 mg). Finally, fraction F22 (~8
mg) was resolved by semipreparative HPLC using a gradient system
initiated with 20:80 CH3CN-H2O (0.1% formic acid) to 80% CH3CN over
30 min at a flow rate of 4.6 mL/min to generate compound 5 (1.3 mg).
4.3.1. Butyrolactone I (1)
4.2. Fungal strain isolation and molecular identification
1H NMR (CDCl3, 500 MHz) δ: 7.63 (2H, d, J = 8.9 Hz, H-2′, H-6′),
6.91 (2H, d, J = 8.9 Hz, H-3′, H-5′), 6.60 (1H, dd, J = 8.2, 2.2 Hz, H-6′′),
6.54 (1H, d, J = 8.2 Hz, H-5′′), 6.52 (1H, d, J = 2.2 Hz, H-2′′), 5.99 (1H, s,
OH-4′), 5.47 (1H, s, OH-4′′), 5.10 (1H, dddd, J = 8.7, 5.7, 2.8 Hz, 1.4, H-
8′′), 3.78 (3H, s, 6-OMe), 3.56 (1H, d, J = 14.7 Hz, H-5a), 3.50 (1H, d, J
= 14.7 Hz, H-5b), 3.14 (2H, d, J = 7.2 Hz, H-7′′), 1.69 (3H, s, H-11′′),
1.65 (3H, s, H-10′′). 13C NMR (CDCl3, 125 MHz) δ: 169.9 (C-6), 169.5 (C-
1), 156.7 (C-4′), 153.3 (C-4′′), 137.4 (C-2), 134.6 (C-9′′), 132.0 (C-2′′),
129.7 (C-2′, C-6′), 129.4 (C-6′′), 128.3 (C-3), 126.7 (C-3′′), 124.8 (C-1′′),
122.4 (C-1′), 121.6 (C-8′′), 116.2 (C-3′, C-5′), 115.3 (C-5′′), 86.3 (C-4),
53.8 (6-OMe), 38.7 (C-5), 29.3 (C-7′′), 25.9 (C-11′′), and 17.9 (C-10′′);
HR-ESI-MS, m/z 425.1595 [M+H]+ (calcd for C24H25O7, 425.1595).
´
IQ-046 was isolated from wetland sediment (Tamiahua-El Idolo,
´
´
´
Veracruz, Mexico) by Jesús Morales-Jimenez and Jose Rivera-Chavez.
´
The strain was identified by Jesús Morales-Jimenez using the sequence
of internal transcribed spacer (ITS) regions of the rDNA. ITS regions
were amplified using the primers ITS1 (5′-GGAAGTAAAAGTCGTAA-
CAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) by PCR.48,49
Final concentrations for 25 µL PCR reactions were as follows: 10 ng of
fungal genomic DNA, 0.8 pM of each primer, 0.2 mM dNTPs, 2.5 mM
MgCl2, 1 U of Taq polymerase, and 1 × Taq polymerase buffer (Invi-
trogen Life Technologies, Sao Paulo, Brazil). The reaction conditions
were 94 ◦C for 10 min; 35 cycles of 45 s at 94 ◦C, 45 s at 58 ◦C, and 60 s at
◦
◦
72 C; and a final extension at 72 C for 10 min. PCR products were
purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA,
USA) and sequenced in an ABI PRISM 310 genetic analyzer (Applied
Biosystems, Foster City, CA, USA) using the ITS1 primer.
4.3.2. Butyrolactone IV (2)
1H NMR (CDCl3, 700 MHz) δ: 7.61 (2H, d, J = 7.2 Hz, H-2′, H-6′),
6.91 (2H, d, J = 7.2 Hz, H-3′, H-5′), 6.65 (1H, brs, H-2′′), 6.57 (1H, d, J =
8.2 Hz, H-6′′), 6.52 (1H, d, J = 8.2 Hz, H-5′′), 4.53 (dd, J = 9.4, 8.6 Hz, H-
8′′), 3.78 (3H, s, 6-OMe), 3.56 (1H, d, J = 14.9 Hz, H-5a), 3.49 (1H, d, J
= 14.9 Hz, H-5b), 3.02 (1H, dd, J = 15.6, 8.6 Hz, H-7a’’), 2.96 (1H, dd, J
= 15.6, 9.4 Hz, H-7b’’), 1.30 (3H, s, H-11′′), 1.15 (3H, s, H-10′′). 13C
NMR (CDCl3, 175 MHz) δ: 169.8 (C-6), 169 (C-1), 159 (C-4′′), 156.5 (C-
4′), 137.2 (C-2), 130.2 (C-6′′), 129.7 (C-2′, C-6′), 127.6 (C-3), 127.1(C-
2′′), 127.0 (C-3′′), 124.8 (C-1′′), 122.6 (C-1′), 116.2 (C-3′, C-5′), 108.6 (C-
5′′), 89.5 (C-8′′), 86.1 (C-4), 72.1 (C-9′′), 53.7 (6-OMe), 39.0 (C-5), 30.7
(C-7′′), 26.3 (C-11′′), and 24.0 (C-10′′); HR-ESI-MS, m/z 458.1807
[M+NH4]+ (calcd for C24H28NO8, 458.1809).
The sequence from the ITS region of the fungal isolate IQ-046 was
compared with the nonredundant GenBank library using a BLAST
search.50 A collection of taxonomically related sequences was obtained
from the National Center for Biotechnology Information Taxonomy
Browser. DNA sequences were aligned using CLUSTAL X,51 edited and
confirmed visually in BIOEDIT.52 Maximum likelihood analyses were
performed using PhyML.53 The GTR + G + I model (
α = 0.401 for the
gamma distribution; A = 0.18, C = 0.31, G = 0.28, T = 0.23; p-inv =
0.281) was selected for the tree search. The confidence at each node was
assessed by 1000 bootstrap replicates.54 Histoplasma capsulatum was
used as the outgroup. The sequence for the ITS region of fungal isolate
IQ-046 was deposited in the GenBank database under the accession
number MT254737.
4.4. Semisynthesis of products 6–12
4.4.1. (±)-Asperteretone B (6)
4.3. Fermentation, extraction and isolation
To a cold solution of butyrolactone I (1, 15 mg, 0.035 mmol) in THF
(1 mL) was added 27 mL of DMC (0.32 mmol), and 1.0 mL of TBAI 78.5
mM in THF. The reaction mixture was stirred and heated at 80 ◦C for 6 h.
The final solution was then adsorbed onto Celite and eluted with MeOH.
Product 6 (3.3 mg) was purified by reverse phase (C-18) semipreparative
HPLC, using an isocratic method of 50:50 CH3CN:H2O (0.1% formic
acid) for 30 min at 4.6 mL/min, (Rt 9.5–10.5 min). The spectroscopic
data were consistent with those reported in the literature. 1H NMR
(CDCl3, 700 MHz) δ: 7.41 (2H, d, J = 8.7 Hz, H-2′, H-6′), 6.89 (2H, d, J =
8.7 Hz, H-3′, H-5′), 6.87 (1H, d, J = 7.9 Hz, H-6′′), 6.86 (1H, d, brs, H-
2′′), 6.75 (1H, d, J = 7.9 Hz, H-5′′), 5.46 (1H, s, H-4), 5.28 (1H, dddd, J =
7.2, 5.9, 2.7, 1.4 Hz, H-8′′), 5.20 (1H, s, 4′-OH), 5.12 (1H, s, 4′′-OH), 3.98
A seed culture of the fungal strain IQ-046 was grown on potato-
dextrose-agar (PDA). Then, the mycelium transferred into potato-
dextrose-broth (PDB, 15 mL × 8) and incubated for seven days at rt
with agitation at 100 rpm. Each seed culture was then transferred into
an E-flask (250 mL) containing 10 g of autoclaved cereal (Cheerios ×
8).25 All flasks were incubated at rt for 28 days. To the large scale solid
fermentation of IQ-046, 500 mL of 1:1 CH2Cl2-MeOH mixture was added
and shaken for 24 h at 100 rpm.55,56 Next, the solution was filtered, and
750 mL of each H2O and CH2Cl2 were added to a final volume of 2 L. The
mixture was stirred for 30 min and partitioned in a separatory funnel.
8

´
J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
(1H, d, J = 15.2 Hz, H-5a), 3.60 (1H, d, J = 15.2 Hz, 5b), 3.55 (3H, s, 4-
OMe), 3.32 (2H, d, J = 7.2 Hz, H-7′′), 1.77 (3H, s, H-10′′), 1.77 (3H, s, H-
11′′). δ: 13C NMR (CDCl3, 175 MHz) 171.2 (C-1), 156.5 (C-4′), 156.2 (C-
3), 153.6 (C-4′′), 135.3 (C-9′′), 130.8 (C-2′, C-6′), 130.5 (C-2′′), 129.2 (C-
2), 128.3 (C-1′′), 127.9 (C-6′′), 127.7 (C-3′′), 121.7 (C-1′), 121.6 (C-8′′),
116.3 (C-5′′), 115.8 (C-3′, C-5′), 102.5 (C-4), 57.3 (4-OMe), 32.0 (C-5),
29.9 (C-7′′), 25.9 (C-10′), and 18.1 (C-11′′); HR-DART-MS, m/z 381.1693
[M+H]+ (calcd for C23H25O5, 381.1697).
8′′), 1.27 (3H, s, H-10′′), 1.27 (3H, s, H-11′′). δ: 13C NMR (CDCl3, 175
MHz) 169.9 (C-6), 169.1 (C-1), 156.5 (C-4′), 153.3 (C-4′′), 137.2 (C-2),
131.7 (C-2′′), 129.7 (C-2′, C-6′), 129.3 (C-6′′), 127.6 (C-3), 123.7 (C-1′′),
122.6 (C-1′), 120.5 (C-3′′), 116.8 (C-5′′), 116.1 (C-3′, C-5′), 86.2 (C-4),
74.3 (C-9′′), 53.7 (6-OMe), 38.8 (C-5), 32.8 (C-8′′), 27.0 (C-10′′), 26.9 (C-
11′′), and 22.4 (C-7′′); HR-ESI-MS, m/z 442.1860 [M+NH4]+ (calcd for
C24H28NO7, 442.1860).
4.4.5. 4′,4′′-Butyrolactone I dipropionate (10)
4.4.2. (±)-5′′-Iodoasperteretone B (7)
Compound 10 was obtained by reacting 10.0 mg (0.024 mmol) of 1,
To a cold solution of butyrolactone I (1, 15 mg, 0.035 mmol), in THF
(1 mL) was added 27 mL of DMC (0.32 mmol), and 1.0 mL of TBAI 78.5
mM in THF. The reaction mixture was stirred and heated at 80 ◦C for 6 h.
The final solution was then adsorbed onto Celite and eluted with MeOH.
Product 7 (1.3 mg) was purified by reverse phase (C-18) semipreparative
HPLC, using an isocratic method of 50:50 CH3CN:H2O (0.1% formic
acid) for 30 min at 4.6 mL/min, (Rt 13.0–14.0 min). Compound 7 was
isolated as a white solid powder. 1H NMR (CDCl3, 700 MHz) δ: 7.40 (2H,
d, J = 8.7 Hz, H-2′, H-6′), 7.29 (1H, d, J = 2.2 Hz, H-6′′), 6.91 (2H, d, J =
8.7 Hz, H-3′, H-5′), 6.86 (1H, d, J = 2.2 Hz, H-2′′), 5.46 (1H, s, 4′′-OH),
5.45 (1H, s, H-4), 5.25 (1H, ddq, J = 8.8, 5.9, 1.4 Hz, H-8′′), 5.02 (1H, s,
4′-OH), 3.94 (1H, d, J = 15.3 Hz, H-5a), 3.57 (1H, d, J = 15.3 Hz, H-5b),
3.57 (3H, s, 4-OMe), 3.34 (2H, dd, J = 5.9 Hz, 5.8, H-7′′), 1.75 (3H, d, J
= 1.3 Hz, H-11′′), 1.72 (3H, d, J = 1.3 Hz, H-10′′). δ: 13C NMR (CDCl3,
175 MHz) 170.9 (C-1), 156.5 (C-4′), 155.2 (C-3), 152.2 (C-4′′), 136.1 (C-
6′′), 134.8 (C-9′′), 130.9 (C-2′′), 130.8 (C-2′, C-6′), 130.1 (C-3′′), 129.6
(C-2), 128.7 (C-1′′), 121.6 (C-1′), 121.3 (C-8′′), 115.8 (C-3′, C-5′), 102.4
(C-4), 86.4 (C-5′′), 57.5 (4-OMe), 31.5 (C-5) 30.2 (C-7′′), 26.0 (C-11′′),
18.0 (C-10′′); HR-DART-MS, m/z 507.0681 [M+H]+ (calcd for
with propionic anhydride (0.40 mmol) in 500 μL of pyridine. Compound
10 (4.5 mg) was purified by reverse phase semipreparative HPLC using a
gradient system of 40:60–60:40 CH3CN:H2O (0.1% formic acid) over 30
min at a flow rate of 4.6 mL/min, (Rt 10.5–12.0 min). 1H NMR (CDCl3,
700 MHz) δ: 7.72 (2H, d, J = 8.8 Hz, H-2′, H-6′), 7.20 (2H, d, J = 8.8 Hz,
H-3′, H-5′), 6.78 (1H, d, J = 8.2 Hz, H-5′′), 6.71 (1H, dd, J =, 8.2, 2.2 Hz,
H-6′′), 6.66 (1H, d, J = 2.2 Hz, H-2′′), 5.00 (1H, tt, J = 7.2, 1.5 Hz, H-8′′),
3.79 (3H, s, 6-OMe), 3.63 (1H, d, J = 14.7 Hz, H-5a), 3.54 (1H, d, J =
14.7 Hz, H-5a), 3.04 (2H, d, J = 7.2 Hz, H-7′′), 1.67 (3H, d, J = 1.3 Hz, H-
11′′), 1.58 (3H, d, J = 1.3 Hz, H-10′′), 2.65 (2H, q, J = 7.6 Hz,
–
–
COOCH2CH3), 1.29 (2H, t, J = 7.6 Hz, COOCH2CH3), 2.57 (2H, q, J
–
–
= 7.6 Hz, COOCH2CH3), 1.24 (2H, t, J = 7.6 Hz, COOCH2CH3). δ:
13C NMR (CDCl3, 175 MHz) 169.6 (C-6), 168.6 (C-1), 151.3 (C-4′), 148.3
(C-4′′), 138.9 (C-2), 133.4 (C-3′′), 133.3 (C-9′′), 132.1 (C-2′′), 130.5 (C-
1′′), 129.1 (C-6′′), 129.0 (C-2′, C-6′), 127.2 (C-1′), 126.5 (C-3), 122.4 (C-
3′, C-5′), 121.9 (C-5′′), 121.4 (C-8′′), 85.8 (C-4), 53.8 (6-OMe), 38.9 (C-
′′
′′
′′
–
–
5), 28.5 (C-7 ), 25.8 (C-11 ), 17.9 (C-10 ), 173.0 ( COOCH2CH3), 27.7
–
–
–
(
(
COOCH2CH3), 9.3 ( COOCH2CH3), 172.7 ( COOCH2CH3), 27.9
–
COOCH2CH3), and 9.2 ( COOCH2CH3); HR-ESI-MS, m/z 554.2383
C
23H24IO5, 507.0663).
[M+NH4]+ (calcd for C30H36NO9, 554.2384).
′′ ′′
4.4.3. (±)-Asperteretone D (8)
4.4.6. Δ7 ,8 -9′′-hydroxy-4′,4′-butyrolactone I dipropionate (11)
To a cold solution of butyrolactone I (1, 15 mg, 0.035 mmol), in THF
(1 mL) was added 27 mL of DMC (0.32 mmol), and 1.0 mL of TBAI 78.5
mM in THF. The reaction mixture was stirred and heated at 80 ◦C for 6 h.
The final solution was then acidified with HCl 1 M, and then adsorbed
onto Celite and eluted with MeOH. Product 8 (1.3 mg) was purified by
Compound 11 was obtained by reacting 10.0 mg (0.024 mmol) of 1,
with propionic anhydride (0.40 mmol) in 500 μL of pyridine. Compound
11 (1.5 mg) was purified by reverse phase semipreparative HPLC using a
gradient system of 40:60–60:40 CH3CN:H2O (0.1% formic acid) over 30
min at a flow rate of 4.6 mL/min, (Rt 8.5–9.5 min). 1H NMR (CDCl3, 700
MHz) δ: 7.72 (2H, d, J = 8.8 Hz, H-2′, H-6′), 7.21 (2H, d, J = 8.8 Hz, H-3′,
H-5′), 6.87 (1H, s, H-2′′), 6.84 (1H, d, J = 1.3 Hz, H-5′′), 6.84 (1H, d, J =
1.3 Hz, H-6′′), 6.41 (2H, d, J = 16.4 Hz, H-7′′), 5.90 (1H, d, J = 16.4 Hz,
H-8′′), 3.82 (3H, s, 6-OMe), 3.64 (1H, d, J = 14.8 Hz, H-5a), 3.59 (1H, d,
J = 14.8 Hz, H-5b), 1.36 (3H, s, H-10′′), 1.36 (3H, s, H-11′′), 2.63 (2H, q,
reverse phase (C-18) semipreparative HPLC, using
a gradient
50:50–100:0 CH3CN:H2O (0.1% formic acid) over 30 min at 4.6 mL/
min, (Rt 12.5–13.5 min). Compound 8 was isolated as a white powder;
the spectroscopic data agreed with those previously reported in the
literature. 1H NMR (CDCl3, 700 MHz) δ: 7.43 (2H, d, J = 8.6 Hz, H-2′, H-
6′), 6.90 (2H, d, J = 8.6 Hz, H-3′, H-5′), 6.88 (1H, dd, J = 8.4 Hz, 2.3, H-
6′′), 6.81 (1H, d, J = 2.2 Hz, H-2′′), 6.72 (1H, d, J = 8.4 Hz, H-5′′), 5.50
(1H, s, H-4), 3.98 (1H, d, J = 15.2 Hz, H-5a), 3.58 (1H, d, J = 15.2 Hz, H-
5b), 3.58 (3H, s, 4-OMe), 2.73 (2H, t, J = 6.8 Hz, H-7′′), 1.79 (2H, t, J =
6.8 Hz, H-8′′), 1.33 (3H, s, H-10′′), 1.33 (3H, s, H-11′′). δ: 13C NMR
(CDCl3, 175 MHz) 171.1 (C-1), 156.4 (C-4′), 156.3 (C-3), 153.3 (C-4′′),
130.8 (C-2′, C-6′), 129.8 (C-2′′), 129.1 (C-2), 127.8 (C-6′′), 127.1 (C-1′′),
121.8 (C-1′), 121.6 (C-3′′), 117.8 (C-5′′), 115.7 (C-3′, C-5′), 102.4 (C-4),
74.5 (C-9′′), 57.3 (4-OMe), 32.8 (C-8′′), 31.9 (C-5), 27.0 (C-10′′), 27.0 (C-
11′′), and 22.6 (C-7′′); HR-ESI-MS, m/z 398.1959 [M+NH4]+ (calcd for
–
–
J = 7.6 Hz, COOCH2CH3), 1.29 (2H, t, J = 7.6 Hz, COOCH2CH3),
–
2.58 (2H, q, J = 7.6 Hz, COOCH2CH3), 1.26 (2H, t, J = 7.6 Hz,
COOCH2CH3). δ: 13C NMR (CDCl3, 175 MHz) 169.5 (C-6), 168.6 (C-1),
–
151.3 (C-4′), 147.5 (C-4′′), 138.9 (C-2), 136.2 (C-8′′), 130.7 (C-1′′), 130.6
(C-6′′), 129.0 (C-2′, C-6′), 128.6 (C-3), 128.6 (C-3′′), 127.3 (C-1′), 123.4
(C-7′′), 122.6 (C- 3′, C-5′), 122.4 (C-2′′), 122.4 (C-5′′), 85.8 (C-4), 82.3
(C-9′′), 53.9 (6-OMe), 38.7 (C-5), 24.5 (C-11′′), 24.4 (C-10′′), 173.1
–
–
– –
–
(
(
COOCH2CH3), 27.8 ( COOCH2CH3), 9.4 ( COOCH2CH3), 172.9
–
COOCH2CH3), 27.9 ( COOCH2CH3), and 9.1 ( COOCH2CH3); HR-
ESI-MS, m/z 570.2332 [M+NH4]+ (calcd for C30H36NO10, 570.2333).
C
23H28NO5, 398.1962).
4.4.7. 3′,5′,5′′,8′′-Tetrabromoaspernolide A (12)
4.4.4. Aspernolide A (9)
To a solution of butyrolactone I (1, 10 mg, 0.024 mmol) in CH2Cl2-
Compound 9 was obtained by reacting 10.1 mg of 1 (0.024 mmol) in
MeOH, 1:1, was added Br2 in excess (50 μL, 2.7 mmol in 500 μL of
MeOH (1 mL) with 50
μL of HCl 1 M. Aspernolide A (9, 4.6 mg) was
CH2Cl2), and stirred for 3 h at rt. 3′,5′,5′′,8′′-tetrabromoaspernolide A
(12, 1.8 mg) was purified by semipreparative reverse phase HPLC, using
an isocratic method of 40:30:30 CH3CN-MeOH-H2O (0.1% formic acid)
at a flow rate of 4.6 mL/min. 1H NMR (CDCl3, 700 MHz) δ: 7.79 (2H, s,
H-2′, H-6′), 6.83 (1H, d, J = 2.1 Hz, H-6′′), 6.55 (1H, d, J = 2.1 Hz, H-2′′),
4.18 (1H, dd, J = 9.2, 5.6 Hz, H-8′′), 3.83 (3H, s, 6-OMe), 3.51 (1H, d, J
= 15.0 Hz, H-5a), 3.34 (1H, d, J = 15.0 Hz, H-5b), 3.26 (1H, dd, J =
17.0, 6.0 Hz, H-7a’’), 3.10 (1H, dd, J = 17.0, 9.2 Hz, H-7b’’), 1.55 (3H, s,
H-11′′), 1.40, (3H, s, H-10′′). δ: 13C NMR (CDCl3, 175 MHz) 169.1 (C-6),
168.0 (C-1), 150.3 (C-4′), 149.1 (C-4′′), 138.9 (C-2), 133.4 (C-6′′), 131.4
purified by semipreparative reverse phase HPLC using a gradient system
of 40:60–60:40 CH3CN:H2O (0.1% formic acid) over 30 min at a flow
rate of 4.6 mL/min, (Rt 19.5–21.5 min). Compound 9 was isolated as a
white powder and its spectroscopic data agreed with those reported in
the literature. 1H NMR (CDCl3, 700 MHz) δ: 7.61 (2H, d, J = 8.8 Hz, H-2′,
H-6′), 6.92 (2H, d, J = 8.8 Hz, H-3′, H-5′), 6.56 (1H, s, H-2′′), 6.51 (1H, s,
H-5′′), 6.51 (1H, s, H-6′′), 3.78 (3H, s, 6-OMe), 3.54 (1H, d, J = 14.8 Hz,
H-5a), 3.44 (1H, d, J = 14.8 Hz, H-5b), 2.62 (1H, dt, J = 16.7, 6.8 Hz, H-
7a’’), 2.59 (1H, dt, J = 16.7, 6.8 Hz, H-7b’’), 1.72 (1H, t, J = 6.8 Hz, H-
9

´
J. Rivera-Chavez et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115817
(C-2′, C-6′), 130.2 (C-2′′), 125.6 (C-3), 124.5 (C-1′), 124.5 (C-1′′), 121.1
(C-3′′), 111.1 (C-5′′), 110.7 (C-3′, C-5′), 85.7 (C-4), 78.5 (C-9′′), 54.0 (6-
OMe), 51.7 (C-8′′), 38.5 (C-5), 34.3 (C-7′′), 27.1 (C-11′′), and 21.9 (C-
10′′); HR-ESI-MS, m/z 734.7877 [M ꢀ H]ꢀ (calcd for C24H19O7Br4,
734.7870).
DTT). The excitation wavelength was 280 nm, and the emission wave-
lengths from 300 to 500 nm were recorded. The inner filter effect due to
the absorption of butyrolactone I (1) at the excitation and emission
wavelengths was corrected by treating the fluorescence data with Eq.
(2), “where Fcor and Fobs are the corrected and measured fluorescence
intensities, respectively, and Aex and Aem are the differences in the
absorbance values of the sample upon the addition of the ligand at the
excitation (280 nm) and emission (300–400 nm) wavelengths,
respectively.”45
4.5. hPTP1B1–400, expression and purification
Human wild-type PTP1B (hPTP1B1–400, residues 1–400) containing
an N-terminal 6 × His tag was cloned into a pET28 vector. The plasmid
was transformed into an E. coli Rosetta (DE3) pLysS strain and grown
using LB media. Protein expression was induced using 1 mM IPTG at
+A
)
Fcor = Fobs10(A
(2)
ex
em
2
The saturation degree of hPTP1B1-400 with butyrolactone I (1), and
the ka for the 1-hPTP1B1-400 complex was calculated by non-linear
regression utilizing Eq. (3), where F is the measured fluorescence, and
F0 and Fc are the fluorescence in the absence of the ligand and the fully
saturated protein, respectively, [P]t and [L]t are the total protein and
total ligand concentration, and kd = (1/ka) is the dissociation constant.45
20 ◦C for 16 h. Purification of hPTP1B1–400 was carried out following the
7
´
procedure previously described by Jimenez-Arreola, et al. 2020.
Briefly, cells were resuspended in lysis buffer (50 mM HEPES pH 7.2,
150 mM NaCl, 10 mM imidazole, 2 mM TCEP) and sonicated in an ice
◦
bath (4 C). Lysates were then centrifuged and purified using affinity
chromatography, by 50 mM HEPES pH 7.4, 100 mM NaCl, 300 mM
imidazole, and 1.5 mM DTT, with subsequent buffer exchange using 50
mM HEPES pH 7.4, 100 mM NaCl, and 1.5 mM DTT. The protein was
used immediately or stored at ꢀ 20 ◦C in 50 mM HEPES pH 7.4, 100 mM
NaCl, 2 mM DTT, and 4% glycerol.34,58
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ꢀ
)
[P]t + [L]t + kd ꢀ
[P] + [L] + kd 2 ꢀ 4[P]t[L]t
F0 ꢀ F
t
t
=
(3)
F0 ꢀ Fc
2[P]t
Declaration of Competing Interest
4.6. hPTP1B1–400 inhibition assay
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.
Pure compounds and ursolic acid (positive control) were dissolved in
DMSO. Aliquots of 0–10
μL of testing materials (in triplicate) were
incubated for 10 min with 5 μL of enzyme stock solution (3 μM) in 50
Acknowledgments
mM HEPES pH 6.8, 100 mM NaCl, 1.5 mM DTT. After incubation, 10 μL
of the substrate (p-NPP 30 mM, Sigma-Aldrich) was added and incu-
◦
This work was supported by a grant from DGAPA, PAPIIT, UNAM
(IA200818/IA203220). This research received partial financial support
from CONACyT, through infrastructure project no. 295057. The authors
would like to thank Dr. Beatriz Quiroz-García, Dra. María del Carmen
bated for 20 min at 37 C, and the absorbance at 405 nm was deter-
mined. All assays were performed in a final volume of 100 μL. The IC50
values were calculated by regression analysis using Eq. (1), with
GraphPad Prism.
´
García-Gonzalez, and Dr. Celia Bustos-Brito (Instituto de Química,
A100
UNAM) for the collection of spectroscopic and spectrometric data, and
%Inh =
(1)
(
)
S
́
technical assistance. This study made use of UNAMs NMR Laboratory:
I
1 +
IC50
LURMN at IQ-UNAM, which is funded by CONACyT-Mexico (Project:
0224747) and UNAM, as well as the instrumentation acquired through
the BGSI-IQ node. J. R-C. would like to thank MSc. Tyler N. Graff and
Jacklyn M. Gallagher, and Dr. Huzefa Raja for their assistance in the
revision of the manuscript.
4.7. Computational details
4.7.1. Proteins and ligands
Polar hydrogens and charges (Kollman) were allocated to the ho-
mologated model for hPTP1B1-400 using AutoDockTools (ADT) 1.5.6,
and the files saved in .pdbqt format for their use in AutoGrid 4.0 and
AutoDock 4.0 interfaces. All docked compounds were built and energy-
optimized using Spartan 10 using a semi-empiric method (PM3). The
ligands were prepared in ADT 1.5.6 by adding the Gasteiger-Marsili
charges.
Appendix A. Supplementary material
1D NMR (1H and 13C), 2D NMR (COSY, HSQC, and HMBC), and
HRMS data for compounds 1-12. Summarized 1D NMR data for com-
pounds 1, 2, and 6-12. Molecular docking for compounds 1, 2, and 6-12.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115817.
4.7.2. Molecular docking
Simulations were performed using AutoDock 4.0. Previously, the
docking protocols were validated to ensure the integrity of the data. In
this regard, trodusquemine was used as a probe to validate the simula-
tions generated for hPTP1B1-400.7 After validation, a blind docking was
performed for each ligand at the interface as the first ligand binding
position in a grid box with dimensions of 126 × 126 × 126 Å. Docking
simulations were performed with Lamarckian Genetic Algorithm cal-
culations of 100 runs. The obtained poses were analyzed with ADT 1.5.6
using cluster analysis, and visualized with PyMOL.
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