Neoflavonoids as inhibitors of HIV-1 replication by targeting the Tat and NF-κB pathways

Article abstract of DOI:10.3390/molecules22020321

Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter gene. Inhibition of HIV transcription and Tat function were teste

Full text of DOI:10.3390/molecules22020321

molecules
Article
Neoflavonoids as Inhibitors of HIV-1 Replication by
Targeting the Tat and NF-κB Pathways
Dionisio A. Olmedo 1,*, José Luis López-Pérez 1, Esther del Olmo 1, Luis M. Bedoya 4,5
,
Rocío Sancho 2, José Alcamí 4, Eduardo Muñoz 2, Arturo San Feliciano 1 and Mahabir P. Gupta 3,
*
1
Pharmaceutical Chemistry Area, Department of Pharmaceutical Sciences, University of Salamanca,
Faculty of Pharmacy, CIETUS, IBSAL, Campus Miguel de Unamuno, 37007 Salamanca, Spain;
lopez@usal.es (J.L.L.-P.); olmo@usal.es (E.d.O.); artsf@usal.es (A.S.F.)
Department of Cellular Biology, Physiology and Immunology, University of Córdoba,
2
Faculty of Medicine Avda de Menendez Pidal s/n, 14004 Córdoba, Spain;
Rocio.Sancho@cancer.org.uk (R.S.); fi1muble@uco.es (E.M.)
CIFLORPAN, Center for Pharmacognostic Research on Panamanian Flora, College of Pharmacy,
3
University of Panama, P.O. Box 0824-00172 Panama, Panama
National Centre of Microbiology, Institute Carlos III, Crt. Majadahonda a Pozuelo, 28220 Majadahonda,
4
Madrid, Spain; lmbedoya@ucm.es (L.M.B.); ppalcami@isciii.es (J.A.)
Pharmacology Department, College of Pharmacy, Complutense University. Pz. Ramón Y Cajal s/n,
5
28040 Madrid, Spain
*
Correspondence: dolmedoagudo@hotmail.com (D.A.O.); mahabirpgupta@gmail.com (M.P.G.);
Tel.: +507-523-6311 (M.P.G.); Fax: +507-264-0789 (M.P.G.)
Academic Editor: Derek J. McPhee
Received: 12 January 2017; Accepted: 16 February 2017; Published: 19 February 2017
Abstract: Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1.
Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter
gene. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the
HIV-LTR and Tat protein. Seven 4-phenylchromen-2-one derivatives showed HIV transcriptional
inhibitory activity but only the phenylchrome-2-one 10 inhibited NF-
simultaneously. Compounds 10 14, and 25, inhibited HIV replication in both targets at concentrations
<25 M. The assays of these synthetic 4-phenylchromen-2-ones may aid in the investigation of some
κB and displayed anti-Tat activity
,
µ
aspects of the anti-HIV activity of such compounds and could serve as a scaffold for designing better
anti-HIV compounds, which may lead to a potential anti-HIV therapeutic drug.
Keywords: neoflavonoids; 4-phenyl-chromen-one; AIDS; Tat protein; NF-κB inhibition; anti-HIV activity
1. Introduction
UNAIDS report in 2016 indicated that tuberculosis remains the leading cause of death among
people living with HIV, accounting for around one in three AIDS-related deaths. In 2014, the percentage
of identified HIV-positive tuberculosis patients who started or continued on ART reached 77% [1].
This relevant fact should promote further studies to take advantage of this therapeutic ambivalence
and to evaluate the possibility of using 4-phenylchromene-2-ones for treating patients suffering from
both AIDS and tuberculosis.
Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome
(AIDS) which is one of the leading causes of 2.9% of mortality in the world [2]. Although modern
antiretroviral therapy (ART) using a combination of anti-HIV drugs has been highly effective in
suppressing HIV load and decreasing mortality in AIDS patients, the emergence of drug resistances
in HIV and the toxicity of the therapies currently in use have made the continued search for novel
anti-HIV drugs necessary [3,4]. On the other hand, failures in efforts to develop an effective vaccine
Molecules 2017, 22, 321; doi:10.3390/molecules22020321
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against HIV-1 infection [5] have emphasized the importance of ART in treating HIV-1-infected patients.
Therefore, medicinal chemists are interested in the development of novel anti-HIV agents that might
be particularly effective in controlling strains of HIV that are resistant to the current drugs [6].
The HIV viral cycle can be divided into early and late stages. Early stages comprise several steps,
from viral attachment on the cell surface to integration in the host genome. Late stages include the
processes of HIV mRNA synthesis, protein expression and morphogenesis. Once integrated, HIV can
remain in a latent state in resting lymphocytes or undergo active replication. Transition from latency
to HIV expression occurs mainly when cells are activated and requires the concerted action of cellular
transcription factors and regulatory HIV proteins [
LTR transactivation, the HIV proximal enhancer contains three binding sites for SP1 transcription
factor and two binding sites for NF- B. The NF- B/Rel family of transcription factors represents a
major inducible regulatory element involved in HIV transcription [ ]. Located downstream of the
basal promoter TAR sequence is the RNA target for the viral protein Tat, which acts in concert with
other cellular factors [10], to generate full-length RNA transcripts [11]. Furthermore, NF- B and Tat
7,8]. Among the transcription factors involved in
κ
κ
9
κ
cooperate in driving HIV replication from the state of latency. Therefore, inhibition of the activity of
these critical proteins should result in an effective blocking of viral replication [12–14].
The neoflavonoids with anti-HIV activity possessing 4-phenylcoumarin skeleton have been
obtained mainly from Calophyllaceae family that includes the genera: Calophyllum [15–21]; Mammea [21–26]
;
Mesua [27
–33]; Kielmeyera [34–38]; and Marila [39], from which several 4-phenyl chromen-2-one
derivatives have been isolated.
The discovery, structural modification and structure-activity relationships studies of natural
neoflavonoids with anti-HIV activity:(+)-Inophyllum B [40], (+)-Inophyllum C [19], and Inophyllum
P [40], and a number of their synthetic derivatives have been successfully obtained in this work.
These 4-phenylcoumarins have been proposed as suppressors of LTR-dependent transcription,
but the mechanism of action has not been fully characterized [41]. In addition, isomesuol and
mesuol inhibit TNF-
(NF- B) pathway. Mesuol inhibited the phosphorylation and the transcriptional activity of the NF-
p65 subunit in TNF
inhibitors, which inhibit both, NF-
α
-induced HIV-1-LTR transcriptional activity by targeting the nuclear factor-
κ
B
B
κ
κ
α
-stimulated cells [42]. Isodispar B and Disparinol A are HIV transcription
B and Tat targets, affecting the HIV replication by synergistic
κ
effect [43]. Synthetic 4-phenylchromen-2-ones have been also reported to show antimicrobial [30
anti-mycobacterial [46] and anti-inflammatory activities [47].
,44,45],
In a previous paper we reported the anti-HIV activity of natural 4-phenylcoumarins isolated
from Marila pluricostata. They were structurally related to Inophyllum coumarins series, but with one
prenyl and other cyclized group across the hydroxyl group at position C-7 [43]. Furthermore, these
compounds showed moderate anti-mycobacterial activity. This relevant fact induced us to prepare new
similar, but simpler, derivatives with the idea in mind to obtain compounds with activity against both
HIV and tuberculosis, and also to increase the structural diversity. With that, a better structure-activity
relationship could be established. In this paper we reported the preparation and the anti-HIV activity
of several neoflavone derivatives that showed anti-mycobacterial activity [48].
2. Results and Discussion
2.1. Chemistry
The 4-phenylchromen-2-ones or neoflavones
condensation between ethyl benzoylacetate and different phenol derivatives that is, resorcinol for
phloroglucinol for , hydroxyhydroquinone for , 3,4-methylenedioxyphenol for , 3-methoxycatechol
for , 2,3-dimethoxyphenol for and -naphthol for in presence of concentred H2SO4 as condensing
1–7 have been obtained by the Peckman
1,
2
3
4
5
6
β
7
agent (Scheme 1). Some differences in the yield of the different compounds can be appreciated
depending on the phenol derivative used (see the Experimental Section).

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Scheme 1. Preparation of 4-phenylcoumarins 1–7.
Phloroglucinol, as the starting material (Scheme 1), was treated with ethyl benzoylacetate by the
Pechmann-Duisberg reaction giving 5,7-dihydroxy-4-phenylcoumarin ( ). Friedel-Craft acylation
or benzoylation of compound , by refluxing the phenol derivative with the corresponding
2
2
acylchloride in a carbon disulfide/nitrobenzene mixture, and in the presence of aluminum trichloride,
followed by Fries rearrangement provided mixtures of the 6-, 8-monoacylated and benzoylated
neoflavones and 6,8-diacylated or dibenzoylated neoflavones [49
–52]. Workup of the crude reaction
products led to the isolation of neoflavones 21 whose spectroscopic properties were consistent
8
–
with the structures shown in the Scheme 2. On the basis of detailed analysis of the H-H COSY
(Correlation Spectroscopy), H-C HMQC (Heteronuclear Single Quantum Correlation), and H-C HMBC
(Heteronuclear Multiple-Bond Correlation) 2D-NMR spectra, all of the compounds described were
correctly characterized and their 13C-NMR data will be introduced in NAPROC-13 RMN spectroscopic
database [53], for posterior online identification of natural products and their analogs and derivatives.
Scheme 2. Preparation of neoflavanones 8–21.

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Neoflavones 22
–
28 were obtained by the condensation of substituted cinnamic acids with the
corresponding phenols, that is, phenol for 22, resorcinol for 23 and 24, 3,5-dichlorophenol for 25
,
3,4-methylenedioxyphenol for 26 and 27, and 3-methoxybenzene-1,2-diol for 28. The reaction occurred
in the presence of milder Friedel-Crafts catalyst BF3–Et2O and POCl3 in 54%–75% yields (Scheme 3).
Previous attempts towards condensation of substituted cinnamic acids with corresponding phenols
for the preparation of 4-phenylchromen-2-ones 22–28 in the presence of concentrated HCl and HCl
gas were unsuccessful. Additionally, some of these compounds were synthesized in low yields by
microwave irradiation.
Scheme 3. Preparation of neoflavones 22–28.
Some compounds prepared in this study have been described already in the literature (see the
Experimental Section) and their structures were verified from an iterative search by 13C-NMR chemical
shifts carried out within our NAPROC-13 RMN spectroscopic database [53], and later identified
1
unambiguously. The rest of the prepared 4-phenylcoumarins were established on the basis of H-
and 13C-NMR spectra. A combination of COSY, HMQC, and NOE experiments was utilized when
necessary for a correct assignment of 1H and 13C chemical shifts.
The synthesized neoflavonoids
in Table 1.
1
–8
were evaluated in vitro against HIV-1 with the results shown
2.2. Evaluation of Antiviral Activity
The neoflavonoids synthesized (
1
–28) belong to three groups: simple 4-phenylchromen-2-ones,
acyl-4-phenylchromen-2-ones and 3,4-dihydro-4-phenylchromen-2-ones, which have been evaluated
in the anti HIV bioassay. The analysis of the results of inhibitory activity of NF- B indicates that
4-phenylchromenones 10 13 14, and 15 show a fair inhibitory activity at 50 M. Regarding the
specific HeLa-Tat-Luc assay results, compounds 13, and 15 were nonspecific, whereas compounds
10 and 14 showed specificity. Furthermore compound 14 is only slightly toxic at 50 M, but not
toxic at 25 M, and their activity as NF- B and tat inhibitors is still strong (83.06% for NF- B and
41.87% for Tat). Compound 14 turned out to be identical to the natural neoflavonoid Isodispar B
previously isolated from Marila pluricostata [43]. Compound 10 NF- B activity is also strong (70.53 at
25 M) and it is nontoxic at 10 M. Interestingly, the dichlorinated 3,4-dihydroflavonoid 25 showed
κ
9,
,
,
µ
9
,
µ
µ
κ
κ
κ
µ
µ
specific anti-Tat activity, whereas all other 3,4-dihydroanalogs resulted inactive in this assay. It must
be noted that simple structural differences within this series of 4-phenylchromen-2-ones and acyl
4-phenylchromen-2-ones, determine substantial changes in activity and selectivity. As an example, we
could compare the specific NF-κB inhibitor 8-isovaleroyl-4-phenylchromen-2-one 14 and the specific

Molecules 2017, 22, 321
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Tat inhibitor 3,4-dihydro-5,7-dichloro-4-phenyl-chroman-2-one 25, also a 4-phenylchromen related.
The drastic difference in bioactivity for these compounds should be due either to the presence of an
isovaleroyl or heptanoyl group in C-6 or C-8, which potentiates the activity in both targets. However,
regarding the comparison of compounds
ring fused to the chromenone moiety, increases the anti-Tat activity, but the introduction 8-isovaleroyl
or 6,8-diacetyl groups enhances the NF- B inhibitory activity. Compound 10, when compared to
7 and 14, it is worth noting that the presence of a benzene
κ
Disparinol A, showed higher anti-HIV potency than this natural neoflavonoid. The acyl derivatives of
4-phenylchromen-2-ones are the most potent dual-target inhibitors.
The assays of these 4-phenyl-chromen-2-one derivatives may aid in the investigation of some
aspects of the anti-HIV activity of this kind of compound that inhibited the transcription and could
serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential HIV
therapeutic drug.
Table 1. Anti-HIV Activity of neoflavonoids.
Specificity
(HeLa-Tet-On-Luc)
Toxicity
MT2 (%)
Compound
NF-κB (5.1 LTR)
Hela-Tat-luc
25 µM
NT
NT
50 µM
−11.28
−4.10
34.27
21.66
23.83
19.12
51.71
9.25
68.74
68.19
37.81
20.40
67.29
86.60
79.41
−17.05
83.70
35.05
66.04
10.70
15.20
11.94
43.21
53.99
13.37
20.48
5.67
25 µM
50 µM
27.88
−7.43
6.59
34.64
44.60
16.25
94.69
21.74
80.84
83.32
26.63
5.81
66.72
69.32
80.37
20.87
44.93
30.20
12.99
22.30
6.06
−34.54
−18.03
72.27
−28.08
15.08
−65.28
71.30
50 µM
NT
NT
NT
S
S
NT
U
NT
U
S
S
S
U
S
U
S
U
S
S
S
50 µM
18.40
8.78
2.22
3.55
11.30
2.88
4.00
8.33
NT
<10
NT
NT
NT
17.02
NT
NT
NT
NT
1
2
3
4
5
6
7
8
9
NT
NT
NT
5.58
−2.63
NT
NT
NT
NT
NT
NT
NT
NT
41.87
NT
NT
NT
NT
NT
NT
NT
NT
NT
57.46
NT
NT
NT
NT
NT
7.30
22.98
NT
NT
NT
NT
70.53
NT
NT
NT
83.06
NT
NT
NT
NT
59.86
NT
NT
10
11
12
13
14
15
16
17
18
19
20
22
23
24
25
26
27
28
Mesuol
NT
NT
NT
NT
NT
S
NT
NT
NT
S
1.61
2.55
2.00
3.50
2.59
4.73
6.13
>4 µM
NT
36.95
35.20
NT
NT
NT
71.00
77.90
S = Specific activity; U = Unspecific mode of action; NT = Not tested.
3. Experimental Section
3.1. General Information
All of the reagents for synthesis were commercially available and either used without further
purification or purified by standard methods prior to use. Melting points were determined on a Büchi
510-K melting point apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and are uncorrected. IR
1
spectra were recorded (KBr 1%) in a Nicolet Impact 410 spectrophotometer. H-, 13C-NMR, COSY,

Molecules 2017, 22, 321
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HMQC, and HMBC were recorded on Brüker AC 200 (200 MHz) and Brüker DRX 400 (400 MHz)
instruments. Chemical shifts ( ) are expressed in parts per million (ppm) relative to the residual solvent
δ
peak, and coupling constants are reported in Hertz (Hz). All signals assigned to hydroxyl groups were
exchangeable with D2O. Reaction progress was monitored using analytical thin-layer chromatography
(TLC) on precoated Merck silica gel Kiesegel 60 F254 plates, and the spots were detected under UV
light (254 nm). The flash chromatography was conducted using silica gel 230–400 mesh. For EIMS
and HRFABMS analysis, a VG-TS250 mass spectrometer (70 eV) was used. Elementary analyses were
obtained with a LECO CHNS-932 and were within ±0.4% of the theoretical values.
3.2. General Procedures I for the Synthesis of Compounds 1–7
To a mixture of appropriate phenol (2 mmol) and ethyl benzoyl acetate (2 mmol), concentrated
H2SO4, (1 mL) was added and stirred at room temperature for four days; after which the mixture
was poured over crushed ice and extracted with AcOEt, (50 mL
×
5). Evaporation gave a brown
1:1), afforded the corresponding
solid which, after chromatography (silica gel, hexane/AcOEt 10:1
→
4-phenylcoumarin (1–7) as a white solid (yield, 25%–30%).
◦
7-Hydroxy-4-phenyl-2H-chromen-2-one (
1
). Yield 85%; A white solid; m.p. 232–234 C (MeOH).
The spectral data (1H-NMR) were quite comparable with the data reported in [54].
◦
5,7-Dihydroxy-4-phenyl-2H-chromen-2-one (2). Yield 70%; A white solid; m.p. 227–229 C (MeOH).
The spectral data (1H-NMR) were quite comparable with the data reported in [52].
6,7-Dihydroxy-4-phenyl-2H-chromen-2-one (3). Yield 68%; A white solid; m.p. 230–232 ◦C (CHCl3/MeOH)
;
IR (KBr):
ν δ 7.52 (m, 2H), 7.52 (m, 3H), 6.86 (s,
= 3437, 3414, 1686, 1617, 1562 cm−1; 1H-NMR (MeOD)
1H), 6.83 (s, 1H), 6.13 (s, 1H); 13C-NMR (MeOD)
δ 104.0, 111.3, 111.9, 112.3, 129.4, 129.4, 129.9, 129.9,
130.6, 137.2, 144.4, 150.5, 152.0, 158.4, 164.1. MS (EI) m/z: 254 (M+ C15H10O4, 8), 252 (52), 224 (100),
152 (80), 139 (13).
8-Phenyl-6H-[1,3]dioxolo[4,5-g]chromen-6-one (
described in the general procedure I. Yield 65%; A white solid; m.p. 190–192 ◦C (CDCl3/MeOH). IR
(KBr): 7.50 (m, 3H), 7.41 (m, 2H), 6.86 (s, 1H), 6.82
= 1712, 1627, 1563, 1503 cm−1; 1H-NMR (CDCl3)
4). Prepared from benzo[d][1,3]dioxol-5-ol (2 mol) as
ν
δ
(s, 1H), 6.22 (s, 1H), 6.04 (s, 2H); 13C-NMR (CDCl3)
δ 98.5, 102.3, 104.3, 112.1, 112.8, 128.2, 128.2, 128.8,
128.8, 129.6, 135.6, 144.8, 151.1, 151.2, 155.8, 161.1. MS (EI) m/z: 266 (M+ C16H10O4, 66), 265 (88), 238
(100), 152 (22).
8-Hydroxy-7-methoxy-4-phenyl-2H-chromen-2-one (
5
). Prepared as described in general procedure I from
◦
3-methoxybenzene-1,2-diol (2 mmol), yield 65%; A white solid; m.p. 220–222 C (CDCl3/MeOH);
IR (KBr):
ν
= 3342, 2937, 2838, 1697, 1562 cm–1; 1H-NMR (MeOD)
δ
7.47 (m, 2H), 7.47 (m, 3H), 6.95
56.7, 108.7,
(d, J = 9.1 Hz, 1H), 6.87 (d, J = 9.1 Hz, 1H), 6.15 (s, 1H), 3.94 (s, 3H); 13C-NMR (MeOD)
δ
112.1, 114.3, 118.4, 129.2, 129.2, 129.5, 129.5, 130.4, 136.5, 143.8, 151.7, 158.2, 162.7. MS (EI) m/z: 268
(M+ C16H12O4, 60), 265 (88), 225 (100), 152 (19), 141 (65).
7,8-Dimethoxy-4-phenyl-2H-chromen-2-one (
as described in the general procedure I, yield 70%; A white solid; m.p. 175–177 ◦C (CDCl3); IR (KBr):
= 2968, 2933, 2844, 1718, 1602, 1557 cm−1; 1H-NMR (CDCl3)
7.51 (m, 3H), 7.43 (m, 2H), 7.18 (d,
J = 9.2 Hz, 1H), 6.83 (d, J = 9.2 Hz, 1H), 6.22 (s, 1H), 4.02 (s, 3H), 3.95 (s, 3H); 13C-NMR (CDCl3)
56.4,
6). This was prepared from 2,3-dimethoxyphenol (2 mol)
ν
δ
δ
61.5, 108.1, 112.3, 113.8, 122.2, 128.4, 128.4, 128.8, 128.8, 129.6, 135.6, 136.5, 148.4, 155.5, 155.9, 160.6. MS
(EI) m/z: 282 (M+ C17H14O4, 100), 267 (8), 254 (19), 239 (42), 152 (24), 139 (53).
4-Phenyl-2H-benzo[g]chromen-2-one (7). Prepared as described in general procedure II from naphthalen-2-ol
(2 mmol), yield 60%; A white solid; m.p. 211–213 (CDCl3); IR (KBr):
1553 cm–1; 1H-NMR (CDCl3) 8.57 (m, 1H), 7.83 (m, 1H), 7.52–7,62 (m, 9H), 6.44 (s, 1H); 13C-NMR
(CDCl3) 114.1, 114.5, 122.3, 122.7, 123.3, 123.9, 127.1, 127.6, 128.5, 128.5, 128.9, 128.9, 128.9, 129.6, 134.8,
ν = 3054, 1722, 1633, 1593,
δ
δ

Molecules 2017, 22, 321
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135.6, 151.4, 156.5, 160.8. MS (EI) m/z: 272 (M+ C19H12O2, 52), 245 (18), 244 (100), 215 (64), 189 (10),
139 (7).
3.3. General Procedures II: Synthesis of Compounds 8–22
Anhydrous aluminum trichloride (0.4 mmol) was added to a stirred suspension of compound
2
(0.1 mmol) in carbon disulfide (6 mL). Nitrobenzene (2 mL) was then added over 40 min, forming
a homogeneous solution with evolution of HCl. The solution was heated under reflux for 30 min,
appropriate acyl chloride (0.1 mmol) in nitrobenzene (1 mL) was added over 40 min before allowing it
to cool with stirring. The mixture was poured onto ice/water and aqueous HCl and was extracted
with ethyl acetate (25 mL, twice). Workup of the crude product by chromatography on silica gel led to
the isolation of the different acyl derivatives products.
Following the general procedure II using allyl bromide and benzyl bromide the crude reaction
product was chromatographed and eluted with hexane/EtOAc, to yield 8 and 21.
◦
6-Allyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one (
(CHCl3/MeOH); IR (KBr):
= 3217, 1686, 1626, 1591 cm–1; 1H-NMR (CDCl3)
3H), 6.55 (s, 1H), 6.36 (s, 1H), 5.70 (m, 1H), 4.82 (t, J = 1.8 Hz, 1H) 4.76 (dd, J = 1.2 Hz, 1H), 3.16 (d,
8
). Yield 41%; A white solid; m.p. 196–198 C
ν
δ 7.24 (m, 2H), 7.22 (m,
J = 6.2 Hz, 2H); 13C-NMR (CDCl3)
δ 26.7, 95.8, 110.8, 110.9, 114.5, 114.6, 127.2, 128.9, 129.4, 135.9, 137.3,
154.3, 154.4, 155.3, 160.1, 161.8. (EI) m/z: 294 (M+ C18H14O4, 88), 281 (100), 268 (24), 253(46), 139 (15).
5,7-Dibenzyloxy-4-phenyl-2H-chromen-2-one (21). Yield 45%; A white solid; m.p. 175–177 ◦C (CH2Cl2).
IR (KBr):
1H-NMR (CDCl3)
6.41 (d, J = 2.4 Hz, 1H), 5.10 (s, 1 H), 4.72 (s, 1H). 13C-NMR (CDCl3)
ν
= 3089, 3059, 3031, 2927, 2865, 1720, 1611, 1597, 1432, 1337, 1159, 1111, 1064, 726 cm–1.
δ
7.38 (m, 3H), 7.20 (m, 5H), 7.18 (m, 10 H), 5.99 (s, 1H), 6.61 (d, J = 2.4 Hz, 1H),
70.5, 70.8, 94.37, 94.87, 103.8,
δ
113.10, 127.03, 127.11, 127.51, 127.62, 127.73, 127.99, 128.25, 128.40, 128.80, 135.08, 135.78, 139.71, 156.74,
157.21, 157.35, 160.74, 162.32. (EI) m/z: 434 (M+ C29H22O4, 17), 343 (12), 181 (14), 139 (6), 114 (6), 92 (52),
91 (100).
Following the general procedure II using acetyl chloride, the crude reaction product was
chromatographed and eluted with hexane/EtOAc, to yield 12 and 17.
◦
8-Acetyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one (12). Yield 25%; A white solid; m.p. 206–208 C
(Hex:AcOEt); IR (KBr):
ν
= 3224, 3083, 1689, 1627, 1592 cm–1; 1H-NMR (CDCl3)
δ
14.01 (s, 1H), 7.57
32.9,
(m, 3H), 7.42 (m, 2H), 6.25 (s, 1H), 6.02 (s, 1H), 5.85 (s, 1H), 2.93 (s, 3H); 13C-NMR (CDCl3)
δ
99.3, 101.8, 103.7, 110.9, 126.8, 127.2, 127.9, 139.0, 157.9, 158.0, 159.9, 162.0, 167.8, 202.7. (EI) m/z, 272
(M+ C19H12O2, 52), 245 (18), 244 (100), 215(64), 189 (10). (EI) m/z: 296 (M+ C17H12O5, 18), 295 (100),
277 (22), 221(16), 165 (20), 139 (43).
6,8-Diacetyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one or (1,1’-(5,7-dihydroxy-2-oxo-4-phenyl-2H-chromene-
6,8-diyl-diethanone) (17). Yield 52%; A white solid; m.p. 162–164 ◦C (Hex/AcOEt); IR (KBr):
ν
= 3467,
16.53 (s, 1H), 15.92 (s, 1H), 7.40 (m, 3H), 7.28 (m, 2H), 6.04
33.4, 33.5, 102.8, 106.3, 112.4, 126.9, 127.8, 128.5,
1733, 1717, 1593 cm–1; 1H-NMR (CDCl3)
(s, 1H), 2.95 (s, 3H), 2.75 (s, 3H); 13C-NMR (CDCl3)
δ
δ
138.9, 156.5, 158.0, 161.5, 170.2, 172.2, 203.9, 205.5. (EI) m/z: 338 (M+ C19H14O6, 100), 323 (94), 313 (48),
295(90), 277 (26), 139 (29).
Following the general procedure II using propionyl chloride, the crude reaction product was
chromatographed and eluted with hexane/EtOAc, to yield 13 and 18.
5,7-Dihydroxy-4-phenyl-8-propionyl-2H-chromen-2-one (13). Yield 28%; A white solid; m.p. 216–218 ◦C
(Hex:AcOEt); IR (KBr):
ν
= 3218, 3067, 1693, 1616, 1592 cm−1; 1H-NMR (MeOD)
δ
7.37 (m, 2H), 7.37 (m,
8.6,
3H), 6.18 (s, 1H), 5.99 (s, 1H), 3.36 (c, J = 7.3 Hz, 2H), 1.26 (t, J = 7.3 Hz, 3H); 13C-NMR (MeOD)
δ
38.5, 100.2, 111.5, 127.6, 127.9, 128.6, 140.1, 158.3, 160.9, 162.6, 168.7, 206.7. (EI) m/z: 310 (M+ C18H14O5,
36), 282 (18), 281 (100), 252 (8), 171 (7), 139 (8).

Molecules 2017, 22, 321
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◦
5,7-Dihydroxy-4-phenyl-6,8-dipropionyl-2H-chromen-2-one (18). Yield 49%; A white solid; m.p. 152–154 C
(Hex:AcOEt); IR (KBr):
15.97 (s, 1H), 7.40 (m, 3H), 7.28 (m, 2H), 6.03 (s, 1H), 3.41 (q, J = 7.3 Hz, 2H), 3.19 (q, J = 6.7 Hz, 2H),
ν δ 16.64 (s, 1H),
= 3468, 3437, 3067, 2978, 2937, 2876 cm–1; 1H-NMR (CDCl3)
1.28 (t, J = 7.3 Hz, 3H), 1.15 (t, J = 6.7 Hz, 3H); 13C-NMR (CDCl3)
δ 8.2, 8.5, 38.2, 38.2, 101.4, 102.4, 106.1,
112.2, 126.9, 127.8, 128.5, 139.0, 156.7, 161.2, 169.9, 170.0, 172.0, 207.3, 208.7. (EI) m/z: 366 (M+ C21H18O6,
36), 338 (44), 337 (100), 319 (27), 308 (28), 281 (40), 178 (9), 138 (14).
Following the general procedure II using 3-methylbutanoyl chloride, the crude reaction product
was chromatographed and eluted with hexane/EtOAc, to yield 9, 14, and 19.
5,7-Dihydroxy-6-(3-methylbutanoyl)-4-phenyl-2H-chromen-2-one (
206–208 ◦C (Hex:AcOEt); IR (KBr): = 3139, 3111, 2956, 2928, 2870, 1689, 1617, 1580 cm–1; 1H-NMR
(CDCl3) 15.79 (s, 1H), 11.39 (s, 1H), 7.52 (m, 3H), 7.39 (m, 2H), 6.63 (s, 1H), 5.98 (s, 1H), 2.90 (d,
J = 6.3 Hz, 2H), 2.23 (m, 1H), 0.92 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.3 Hz, 3H); 13C-NMR (CDCl3)
22.5,
9). Yield 12%; A white solid; m.p.
ν
δ
δ
22.5, 25.0, 53.0, 94.8, 101.4, 106.9, 111.3, 127.1, 127.4, 128.1, 139.0, 157.2, 159.6, 161.1, 163.7, 164.9, 207.3.
(EI) m/z: 338 (M+ C20H18O5, 10), 282 (18), 281(100), 253 (3), 225(1), 171 (6), 139 (4).
5,7-Dihydroxy-8-(3-methylbutanoyl)-4-phenyl-2H-chromen-2-one (14). Yield 13%; A white solid; m.p.
◦
210–212 C (Hex:AcOEt); IR (KBr):
1H-NMR (CDCl3)
14.14 (s, 1H), 7.55 (m, 3H), 7.43 (m, 2H), 6.25 (s, 1H), 6.01 (s, 1H), 6.00 (s, 1H), 3.17
(d, J = 6.3 Hz, 2H), 2.28 (m, 1H), 1.05 (d, J = 6.3 Hz, 3H), 1.05 (d, J = 6.3 Hz, 3H); 13C-NMR (CDCl3)
ν
= 3293, 2959, 2934, 2874, 2454, 1745, 1685, 1620, 1592 cm–1;
δ
δ
22.7, 22.7, 25.5, 53.6, 101.6, 101.8, 104.9, 112.0, 127.5, 129.6, 130.3, 136.5, 154.1, 155.7, 158.7, 159.7, 168.8,
205.9. (EI) m/z: 338 (M+ C20H18O5, 20), 323 (12), 281 (100), 254 (9), 171(6), 141 (6).
5,7-Dihydroxy-6,8-bis(3-methylbutanoyl)-4-phenyl-2H-chromen-2-one or 1,10-(5,7-dihydroxy-2-oxo-4-phenyl-
2H-chromene-6,8-diyl)bis(3-methylbutan-1-one) (19). Yield 50%; A white solid; m.p. 148–150 ◦C (Hex:AcOEt)
IR (KBr):
;
ν δ 16.77 (s, 1H), 16.09
= 3469, 3435, 2959, 2931, 2871, 1756, 1618, 1597 cm–1; 1H-NMR (CDCl3)
(s, 1H), 7.40 (m, 3H), 7.28 (m, 2H), 6.02 (s, 1H), 3.18 (d, J = 6.7 Hz, 2H), 3.02 (d, J = 6.6 Hz, 2H), 2.27
(m, 1H), 2.27 (m, 1H), 1.06 (d, J = 6.7 Hz, 3H), 1.06 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.95 (d,
J = 6.6 Hz, 3H); 13C-NMR (CDCl3)
δ 22.7, 22.7, 22.7, 22.7, 24.9, 25.8, 53.3, 53.3, 101.5, 102.6, 106.4, 112.2,
126.9, 127.8, 128.4, 139.1, 156.6, 158.0, 161.8, 170.3, 172.4, 206.6, 207.9. (EI) m/z: 422 (M+ C25H26O6, 34),
418 (25), 394 (36), 381 (43), 365 (100), 347 (36), 337(32), 281 (49), 171 (25), 139 (36).
Following the general procedure II using heptanoyl chloride, the crude reaction product was
chromatographed and eluted with hexane/EtOAc, to yield 10, 15, and 20.
6-Heptanoyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one (10). Yield 12%; A white solid; m.p. 210–212 ◦C
(Hex:AcOEt); IR (KBr):
(CDCl3) 14.03 (s, 1H), 10.11 (s, 1H), 7.44 (m, 3H), 7.35 (m, 2H), 6.99 (s, 1H), 5.99 (s, 1H), 3.10 (t,
J = 6.7 Hz, 2H), 1.64 (m, 4H), 1.24 (m, 4H), 0.87 (t, J = 6.7 Hz, 3H); 13C-NMR (CDCl3)
14.1, 22.6, 24.4,
ν
= 3596, 3254, 3058, 2954, 2928, 2856, 1774, 1688, 1717, 1593 cm–1; 1H-NMR
δ
δ
29.0, 31.7, 44.7, 95.9, 101.9, 107.1, 111.6, 127.4, 128.4, 128.9, 138.3, 157.7, 159.3, 161.8, 164.0, 164.8, 207.8.
(EI) m/z: 366 (M+ C22H22O5, 32), 351 (14), 310 (30), (309 (100), 281 (82), 253(11), 171 (18), 139 (20).
8-Heptanoyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one (15). Yield 27%; A white solid; m.p. 212–214 ◦C
1
(Hex:AcOEt); IR (KBr):
ν
= 3246, 2951, 2928, 2868, 2844 cm−1, 1685, 1637, 1591; H-NMR (CDCl3)
δ
14.11 (s, 1H), 7.54 (m, 3H), 7.43 (m, 2H), 6.25 (s, 1H), 6.11 (s, 1H), 6.00 (s, 1H), 3.30 (t, J = 6.7 Hz,
2H), 1.73 (m, 4H), 1.33 (m, 4H), 0.89 (t, J = 6.7 Hz, 3H); 13C-NMR (CDCl3)
δ
14.1, 22.6, 24.6, 29.0, 31.8,
44.9, 101.5, 105.0, 111.9, 127.5, 129.7, 130.2, 136.4, 154.0, 158.3, 158.6, 159.6, 168.7, 206.2. (EI) m/z: 366
(M+ C22H22O5, 44), 351 (31), 309 (91), 281 (100), 267 (3), 253 (18), 171 (23), 139 (39).
6,8-Diheptanoyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one or 1,1’-(5,7-dihydroxy-2-oxo-4-phenyl-2H-chromene-
◦
6,8-diyl)diheptan-1-one (20). Yield 50%; A white solid; m.p. 164–166 C (Hex:AcOEt); IR (KBr):
ν = 3458,
34240, 2958, 2930, 2855, 1744, 1620, 1582 cm–1; 1H-NMR (CDCl3)
3H), 7.28 (m, 2H), 6.02 (s, 1H), 3.33 (t, J = 7.3 Hz, 2H), 3.14 (t, J = 7.3 Hz, 2H), 1.76 (m, 4H), 1.64 (m, 4H),
1.32 (m, 4H), 1.32 (m, 4H), 0.90 (m, 3H), 0.90 (m, 3H); 13C-NMR (CDCl3)
14.0, 14.0, 22.6, 22.6, 24.3,
24.8, 29.0, 29.0, 31.6, 31.8, 44.8, 44.8, 101.4, 102.5, 106.2, 112.3, 127.0, 127.8, 128.5, 139.1, 156.6, 158.0,
δ 16.69 (s, 1H), 16.02 (s, 1H), 7.40 (m,
δ

Molecules 2017, 22, 321
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161.2, 170.1, 172.2, 207.0, 208.4. (EI) m/z: 478 (M+ C29H34O6, 24), 421 (34), 393 (100), 323 (38), 293(52),
171 (20), 139 (14).
Following the general procedure II using benzoyl chloride, the crude reaction product was
chromatographed and eluted with hexane/EtOAc, to 11 and 16.
◦
6-Benzoyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one (11). Yield 18%; A white solid; m.p. 245–247 C
(AcOEt). The spectral data (IR, 1H-NMR and 13C-NMR) were quite comparable with the data reported
in [46,48].
◦
8-Benzoyl-5,7-dihydroxy-4-phenyl-2H-chromen-2-one (16). Yield 34%; A white solid; m.p. 253–256 C
(AcOEt). The spectral data (IR, 1H-NMR and 13C-NMR) were quite comparable with the data reported
in [50,51].
3.4. General Procedures III: Synthesis of Compounds 22–28
To a mixture of POCl3 (10 mmol) and BF3–Et2O (20 mmol) at ◦0 ◦C, appropriated cinnamic acid
(5 mmol) was added and the reaction mixture stirred for 15 min at 0 C. Phenol (5 mmol) was added to
◦
the above reaction mixture in small portions and stirring continued at 25–28 C for 4–12 h. The reaction
mixture was poured on to ice-water; sodium acetate (1 g) was added and the mixture was warmed
on a water bath for 2 min. It was cooled, extracted with ethyl acetate (2
×
150 mL), washed with
water (150 mL), dried, and the solvent removed under reduced pressure to obtain the crude product,
which was purified by column chromatography using acetone–chloroform as eluent to afford pure
4-phenyldihydro-coumarins 22–28 in 60%–75% yields.
4-(4-Methoxyphenyl)chroman-2-one (22). This was prepared from p-methoxycinnamic acid and phenol
◦
using the general procedure III. Yield 62%; A white solid; m.p. 160–162 C (CH2Cl2/MeOH); IR (KBr):
1
ν
= 2943, 2927, 2907, 2833, 1832, 1717, 1701, 1608 cm–1; H-NMR (CDCl3)
δ
7.31 (br d, J = 8.1 Hz,
1H), 7.12 (d, J = 8.2 Hz, 2H), 7.07 (br d, J = 8.2 Hz, 1H), 6.97 (m, 2H), 6.87 (d, J = 8.2 Hz, 3H), 4.30 (t,
J = 7.3 Hz, 1H), 3.79 (s, 3H), 3.08 (dd, J = 7.3 Hz, 15.7 Hz, 1H), 2.96 (dd, J = 7.3, 15.7 Hz, 2H); 13C-NMR
(CDCl3)
δ 37.2, 40.0, 55.3, 114.5 (2C), 117.1, 124.7, 126.3, 128.3, 128.6 (3C), 132.2, 151.7, 159.0, 167.8. (EI)
m/z: 254 (M+ C16H14O3, 65), 226 (14), 212 (15), 211 (72), 197 (22), 182 (15), 181 (100), 168 (13), 139 (12).
4-(3,4-Dimethoxyphenyl)-7-hydroxychroman-2-one (23). This was prepared from 3,4-dimethoxycinnamic
◦
acid and resorcinol as described in the general procedure III. Yield 67%; A white solid; m.p. 167–169 C
(CHCl3/MeOH); IR (KBr):
ν δ 6.83 (d,
= 3434, 2960, 2936, 1762, 1624, 1595 cm–1; 1H-NMR (CDCl3)
J = 7.7 Hz, 1H), 6.68 (dd, J = 7.7, 2.4 Hz, 1H), 6.68 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 7.7 Hz, 1H), 6.66
(s, 1H), 6.59 (dd, J = 8.2, 2.4 Hz, 1H), 6.46 (br s, 1H), 4.21 (t, J =7.3 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 3H),
3.06 (dd, J = 7.3 Hz, 14.9 Hz, 1H), 2.95 (dd, J = 7.3, 14.9 Hz, 1H); 13C-NMR (CDCl3)
δ 37.5, 39.6, 55.9,
55.9, 104.2, 110.6, 111.6, 112.1, 117.5, 119.8, 129.1, 133.2, 148.3, 149.3, 152.1, 156.5, 168.7. (EI) m/z: 300
(M+ C17H16O5, 100), 269 (14), 257 (36), 243 (26), 227 (81), 190 (14), 139 (8).
4-(4-Hydroxyphenyl)-7-methoxychroman-2-one (24). This was prepared from p-coumaric acid and resorcinol
as described in the general procedure III. Yield 68%; A white solid; m.p. 169–171 ◦C (CHCl3/MeOH);
IR (KBr):
ν δ 6.98 (d, J = 8.3 Hz, 2H), 6.89
= 3436, 2938, 2904, 2840, 1762, 1615 cm–1; 1H-NMR (CDCl3)
(d, J = 8.3 Hz, 1H), 6.77 (d, J = 8.3 Hz, 2H), 6.66 (d, J = 2.4 Hz, 1H), 6.64 (dd, J = 8.3, 2.4 Hz, 1H), 5.54 (br
s, 1H), 4.22 (t, J = 6.8 Hz, 1H), 3.80 (s, 3H), 3.05 (dd, J = 6.8, 15.1Hz, 1H), 2.93 (dd, J = 6.8, 15.1 Hz, 1H);
13C-NMR (CDCl3)
δ 37.4, 39.3, 55.6, 102.4, 110.7, 115.6, 115.6, 118.0, 128.7, 128.7, 128.8, 132.6, 152.3,
155.0, 159.9, 168.1. (EI) m/z: 270 (M+ C16H14O4, 38), 242 (17), 228 (10), 227 (100), 211 (40), 184 (15),
128 (18).
5,7-Dichloro-4-phenylchroman-2-one (25). This was prepared from cinnamic acid and 3,5◦-dichorophenol
as described in the general procedure III. Yield 70%; A white solid; m.p. 230–232 C (CHCl3); IR
(KBr):
(d, J = 1.9 Hz, 1H), 7.06 (m, 2H), 4.64 (dd, J = 5.4, 3.4 Hz, 1H), 3.10 (dd, J = 3.4, 12.2 Hz, 1H), 3.04 (dd,
ν δ 7.27 (m, 3H), 7.22 (d, J = 1.9 Hz, 1H), 7.11
= 3077, 1713, 1599, 1567 cm–1; 1H-NMR (CDCl3)

Molecules 2017, 22, 321
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J = 5.4, 12.2 Hz, 1H); 13C-NMR (CDCl3)
δ
36.7, 38.5, 116.5, 122.2, 125.6, 126.7, 126.7, 127.9, 129.2, 129.2,
134.5, 138.9, 152.9, 165.7. (EI) m/z: 293 (M+ C15H10O2Cl2, 36), 291 (59), 276 (23), 274 (36), 256 (13), 251
(64), 249 (100), 215 (19), 152 (33).
8-(4-Hydroxy-3,5-dimethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]chromen-6-one (26). This was prepared
from 4-hydroxy-3,5-dimethoxycinnamic acid and sesamol as described in the general procedure III.
Yield 69%; A white solid; m.p. 162–164 ◦C (eter); IR (KBr):
ν
= 3458, 3023, 3007, 2956, 2930, 2913, 2836,
6.65 (s, 1H), 6.41 (s, 1H), 6.36 (s, 2H), 5.96 (s, 2H), 5.52 (br s,
1H), 4.13 (t, J =7.3 Hz, 1H), 3.84 (s, 3H), 3.84 (s, 3H), 3.09 (dd, J = 7.3, 15.8 Hz, 1H), 2.92 (dd, J = 7.3,
1742, 1627, 1609 cm–1; 1H-NMR (CDCl3)
δ
15.8 Hz, 1H); 13C-NMR (CDCl3)
δ 37.2, 40.8, 56.4, 56.4, 99.1, 101.7, 104.2, 104.2, 107.2, 118.2, 131.5, 134.2,
144.5, 146.1, 147.5, 147.5, 147.5, 167.8. (EI) m/z: 344 (M+ C18H16O7, 100), 326 (13), 295 (14), 271 (82), 256
(21), 167 (37), 133 (12).
8-(3,4,5-Trimethoxyphenyl)-7,8-dihydro-[1,3]dioxolo[4,5-g]chromen-6-one (27). This was prepared from
3,4,5-trimethoxycinnamic acid and sesamol as described in the general procedure III. Yield 61%; A
white solid; m.p. 162–164 ◦C (diethyl ether); IR (KBr):
ν
= 3023, 3007, 2956, 2930, 2913, 2836, 1742,
6.66 (s, 1H), 6.43 (s, 1H), 6.35 (s, 2H), 5.96 (s, 2H), 4.15 (t,
J = 6.8 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 6H), 3.05 (dd, J = 6.8, 16.1 Hz, 1H), 2.92 (dd, J = 6.8, 16.1 Hz, 1H);
1627, 1609, 1584 cm–1; 1H-NMR (CDCl3)
δ
13C-NMR (CDCl3)
δ 37.1, 41.0, 56.1, 56.1, 60.8, 99.2, 101.8, 104.4, 104.5, 107.2, 117.8, 136.1, 136.1, 137.5,
144.5, 146.1, 147.6, 153.7, 167.6. (EI) m/z: 358 (M+ C19H18O7, 100), 325 (29), 315 (21), 285 (83), 241 (27),
215 (35), 181 (81), 133 (38).
8-Hydroxy-7-methoxy-4-(3,4,5-trimethoxyphenyl)chroman-2-one (28).
3,4,5-trimethoxycinnamic acid and 3-methoxybenzene-1,2-diol as described in the general procedure
III. Yield 60%; A white solid; m.p. 164–166 ◦C (CHCl3/MeOH); IR (KBr):
= 3309, 3001, 2951, 2841,
1759, 1629, 1588 cm–1; 1H-NMR (CDCl3)
6.64 (d, J = 8.5 Hz, 1H), 6.48 (d, J = 8.5 Hz, 1H), 6.37 (s, 2H),
5.73 (s, 1H), 4.24 (t, J = 7.3 Hz, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.80 (s, 6H), 3.09 (dd, J = 7.3, 14.1 Hz, 1H),
This was prepared from
ν
δ
2.97 (dd, J = 7.3, 14.1 Hz, 1H); 13C-NMR (CDCl3)
δ 37.2, 40.8, 56.4, 56.4, 56.4, 60.8, 104.6, 104.6, 107.0,
117.9, 119.2, 133.8, 136.2, 137.4, 139.4, 147.2, 153.6, 153.6, 166.8. (EI) m/z: 360 (M+ C19H20O7, 100), 345
(22), 327 (35), 317 (19), 287 (99), 272 (24), 217 (17), 181 (31), 173 (22).
3.5. Antiviral Activity Assays
The anti-HIV activity of these neoflavonoids on Tat and NF-
To this aim, we have used two stably transfected cell lines. The previously described 5.1 cell line [55
is a Jurkat-derived clone stably transfected with a plasmid containing the luciferase gene under the
control of HIV-LTR. In this cell clone, activation with TNF induces NF- B activation and subsequent
κB functions has been evaluated.
]
α
κ
HIV-1 expression. We have also analysed the anti-HIV activity in HeLa-Tat-Luc cells, in which the
HIV-1 LTR is directly activated by the HIV-1 Tat protein. A compound was considered active in
one assay if it inhibited the target function by more than 50% (NF-κB) or 30% (Tat) at either 25
or 50 M concentration. The active compounds were submitted for further evaluation through a
µ
HeLa-Tet-ON assay, as previously described [56]. In the Hela-Tet-ON cells the luciferase expression is
under control of an artificial promoter that can be activated by tetracycline. Therefore, compounds
that inhibit tetracycline-induced luciferase activity were considered non-specific for luciferase-based
anti-HIV assays.
Cell viability was evaluated in non-infected treated cultures following the same protocol as in the
recombinant virus assay and measuring cell toxicity with a classical MTT assay. IC50 were calculated
using GraphPad Prism software (non-linear regression, log (inhibitor) vs. response).
4. Conclusions
A series of twenty-eight neoflavonoids have been synthesized and evaluated against HIV-1
in vitro. Ten 4-phenylchromen-2-one derivatives displayed HIV specific transcriptional inhibition and
five displayed nonspecific mechanisms of action. The heptanoylchromen-one 10 was the more potent

Molecules 2017, 22, 321
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Tat antagonist, while compound 14 showed high inhibition of the NF-
κ
B pathway. A preliminary SAR
analysis established that the presence of the acyl group is essential for the anti HIV in both targets.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/22/2/321/s1,
Figure S1: Spectroscopic data of neoflavonoid derivatives 3–10, 12–15, 17–28.
Acknowledgments: This research is part of the following projects funded by the Spanish Ministry of Economy
and Competitiveness and Instituto de Salud Carlos III (PI16/CIII/034); the Spanish AIDS Research Network
(RD16CIII/0002/0001) that is included in the Spanish I+D+I Plan and is co-financed by ISCIII-Subdirección
General de Evaluacion and European Funding for Regional Development (FEDER), Acknowledgements are
also due to the University of Panama, and to the National Secretariat of Science, Technology and Innovation
(SENACYT) of Panama for SNI distinguished scientist stimulus award to MPG.
Author Contributions: The authors of these research have participated as follows: “José Luis López-Pérez,
Esther Del Olmo and Arturo San Feliciano” conceived and designed structures and synthesis project;
“
Dionisio A. Olmedo” performed all the synthetic experiments; “José Luis López-Pérez and Dionisio A. Olmedo”
interpreted the results, discussed the experimental data and prepared the manuscript; “Rocío Sancho,
Eduardo Muñoz, Luis M. Bedoya and José Alcamí” conducted the biological assay and provided the experimental
procedure and results; “Dionisio A. Olmedo, José Luis López-Pérez, Arturo San Feliciano, Luis M. Bedoya and
Mahabir P. Gupta contributed in overall redaction and revision of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from José Luis López-Pérez, E-Mail: lopez@usal.es.
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2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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