Synthesis and bioevaluation of substituted chalcones, coumaranones and other flavonoids as anti-HIV agents

Article abstract of DOI:10.1016/j.bmc.2016.04.045

A series of chalcone, flavone, coumaranone and other flavonoid compounds were screened for their anti HIV-1 activity in two cell culture models using TZM-bl and PM1 cells. Within the systems evaluated, the most promising compounds contained either an α- or β-hydroxy-carbonyl motif within their structure (e.g., 8 and 9). Efficacious substituents were identified and used to design new HIV inhibitors with increased potency and lower cytotoxicity. Of the scaffolds evaluated, specific chalcones were found to provide the best balance between anti-HIV potency and low host cell toxicity. Chalcone 8l was shown to inhibit different clinical isolates of HIV in a dose-dependent manner (e.g., IC₅₀ typically ≤ 5 μM). Inhibition of HIV infection experiments using TZM-bl cells demonstrated that chalcone 8l and flavonol 9c had IC₅₀ values of 4.7 μM and 10.4 μM, respectively. These insights were used to design new chalcones 8o and 8p. Rewardingly, chalcones 8o and 8p (at 10 μM) each gave >92% inhibition of viral propagation without impacting PM1 host cell viability. Inhibition of viral propagation significantly increased (60-90%) when PM1 cells were pre-incubated with chalcone 8o, but not with the related flavonol 9c. These results suggested that chalcone 8o may be of value as both a HIV prophylactic and therapy. In summary, O-benzyl-substituted chalcones were identified as promising anti-HIV agents for future investigation.

Full text of DOI:10.1016/j.bmc.2016.04.045

Accepted Manuscript
Synthesis and Bioevaluation of Substituted Chalcones, Coumaranones and other
Flavonoids as anti-HIV agents
Amy L. Cole, Sandra Hossain, Alex M. Cole, Otto Phanstiel IV
PII:
S0968-0896(16)30288-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.045
BMC 12960
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 February 2016
15 April 2016
21 April 2016
Please cite this article as: Cole, A.L., Hossain, S., Cole, A.M., Phanstiel, O. IV, Synthesis and Bioevaluation of
Substituted Chalcones, Coumaranones and other Flavonoids as anti-HIV agents, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.045
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Bioevaluation of Substituted Chalcones, Coumaranones and other
Flavonoids as anti-HIV agents
Amy L. Cole†, Sandra Hossain‡, Alex M. Cole†, and Otto Phanstiel IV‡*
†Burnett School for Biomedical Sciences
‡Department of Medical Education
College of Medicine
University of Central Florida
12722 Research Parkway
Orlando FL 32826-3227
407-823-6545
otto.phanstiel@ucf.edu
Page 1

Abstract. A series of chalcone, flavone, coumaranone and other flavonoid compounds were
screened for their anti HIV-1 activity in two cell culture models using TZM-bl and PM1 cells.
Within the systems evaluated, the most promising compounds contained either an α- or β-
hydroxy-carbonyl motif within their structure (e.g., 8 and 9). Efficacious substituents were
identified and used to design new HIV inhibitors with increased potency and lower cytotoxicity.
Of the scaffolds evaluated, specific chalcones were found to provide the best balance between
anti-HIV potency and low host cell toxicity. Chalcone 8l was shown to inhibit different clinical
isolates of HIV in a dose-dependent manner (e.g., IC50 typically ≤ 5 µM). Inhibition of HIV
infection experiments using TZM-bl cells demonstrated that chalcone 8l and flavonol 9c had IC50
values of 4.7 µM and 10.4 µM, respectively. These insights were used to design new chalcones
8o and 8p. Rewardingly, chalcones 8o and 8p (at 10 µM) each gave >92% inhibition of viral
propagation without impacting PM1 host cell viability. Inhibition of viral propagation
significantly increased (60% to 90%) when PM1 cells were pre-incubated with chalcone 8o, but
not with the related flavonol 9c. These results suggested that chalcone 8o may be of value as
both a HIV prophylactic and therapy. In summary, O-benzyl-substituted chalcones were
identified as promising anti-HIV agents for future investigation.
Page 2

1.1 Introduction
Human immunodeficiency virus-1 (HIV-1) leads to acquired immunodeficiency
syndrome (AIDS) via destruction of T-cells after the virus successfully enters and replicates
within the human host. 1 The current treatment for HIV/AIDS includes the use of antiretroviral
therapy (ART) to control viral load and indirectly minimize the spread of the virus.1 This
treatment consists of a multi-drug cocktail that inhibits various processes required for HIV
propagation. Although relatively successful in slowing disease progression and enabling HIV
patients to live longer, long-term ART often leaves patients susceptible to unwanted side effects
and drug-resistant viral quasi-species.2 Furthermore, HIV-infected individuals have higher rates
of chronic disease, cancer, and immune defects compared to the general population3 and
HIV/AIDS remains an important epidemic of global concern. According to the 2014 UNAIDS
report, there are nearly 35 million infected people worldwide and roughly 2.1 million new cases
each year.3a Alarmingly, the HIV-1 acquisition rate continues to outpace morbidity from
HIV/AIDS, causing a steady global increase in the number of infected people each year.
Without a vaccine, attention must be given to identifying candidate drugs that are inexpensive to
produce in large scale and show promise for preventing transmission of HIV-1 between
serodiscordant sexual partners or for suppressing viral load with minimal daily pill burden and
side effects.
Flavonoids are a class of compounds that are typically found in nature in plants, and have
demonstrated a number of medicinal properties. They exhibit antitumor, anti-inflammatory, and
antiviralactivity, as well as the ability to act as an antioxidant.4 Chalcones are common synthetic
precursors to flavones and other flavonoid structures. The chalcones themselves have also
demonstrated inhibitory activity against a variety of enzymes and have been shown to be useful
Page 3

in treating inflammatory or infectious diseases.5 Their broad range of medicinal properties,
along with the simplicity of synthesizing these compounds make them a desirable compound
class to be further investigated.
Flavonoids have also been evaluated as antiviral agents both in our laboratory6 and
others.7 For example, baicalein 1 and quercetin 2 (Figure 1) are examples of naturally-occurring
flavonoids with activity against human cytomegalovirus (HCMV),8 as well as human
immunodeficiency virus (HIV).9 The chalcone butein 3 has been shown to have moderate
activity against HIV-1 protease10 and xanthohumol 4 has been shown to have antiviral activity
against several herpes viruses, including HCMV.11 Previous literature also indicates that 5-
bromo-2-hydroxy-3-[3-(2,3,6-trichlorophenyl)acryloyl benzoic acid 5 is effective against HIV-1
integrase and inhibits the replication of HIV-1 with an IC50 of 8.7 µM.2
Prior work in our group investigated the ability of both chalcone and flavonoid structures
to inhibit the US28 signaling of HCMV.6 Since there was literature precedent for the anti-HIV
activity of these types of compounds (Figure 1),4b, 12 we elected to test the efficacy of a small
library of chalcone and flavonoid compounds against HIV.6 Specifically, we examined the anti-
HIV-1 activities of the flavonoid library in two different cell culture assays. In addition, we
assessed each compound’s toxicity profile in two cell lines. These studies demonstrated that the
chalcone series (8l-8p) represents a drug scaffold capable of HIV-1 suppression in the low µM
range along with low toxicity to human cells.
In this report, we describe our findings with this flavonoid series (Figure 1) in the
inhibition of HIV as well as our efforts to improve their efficacy and to reduce their toxicity to
host cells.
Page 4

Figure 1. Structures of bioactive flavones (1, 2) and chalcone motifs 3-5 and synthetic target
structures
2.1 Materials and Methods.
General. The compounds 7a-d, 8a-m, 9a-j, and 11-13 were prepared previously and the new
chalcones 8n-8p were prepared using published methods. 6 The coumaranones 14a and 14b were
made using a modified hydrogen peroxide method for generating aurones.13 Coumaranone 14c
was made previously and was incorrectly assigned as flavonol 9f in a prior reference.6 All tested
compounds were ≥ 95% pure as evidenced by their elemental analyses. Elemental analyses for
8n, 8o, 8p, 14a and 14b are provided in the Supporting Information.
5-Bromo-2-(octyloxy)benzaldehyde (7e).14 5-Bromosalicylaldehyde (1.01 g, 5 mmol) and 1-
bromooctane (1.46 g, 7.5 mmol), K2CO3 (1.38 g: 10 mmol) were combined with DMF (5 mL)
and stirred at rt for 2 days. The DMF was removed under reduced pressure and CH2Cl2 added
and the solution washed with 1M HCl. The organic layer was separated, dried over anhydrous
Page 5

Na2SO4, filtered and concentrated to give 2.63 g of crude. Column chromatography (10%
CH2Cl2 in hexane to elute the 1-bromooctane impurity followed by 40% CH2Cl2 in hexane to
elute the product) gave 7e as a white solid, 1.26 g (80% yield). 1H NMR (CDCl3): δ 10.34 (s, 1H,
CHO), 7.84 (d, 1H), 7.53 (dd, 1H), 6.80 (d, 1H), 3.98 (t, 2H), 1.77 (m, 2H), 1.41 (m, 2H), 1.24
(m, 8H), 0.82 (t, 3H). 13C NMR (CDCl3): δ 188.5, 160.4, 138.2, 130.8, 126.2, 114.6, 113.2, 69.0,
31.8, 29.3, 29.2, 29.0, 26.0, 22.6, 14.1.
1-(2-Benzyloxy-6-hydroxy-phenyl)-3-(5-bromo-2-octyloxy-phenyl)-propenone
(8n).
Aldehyde 7e (0.155g: 0.5mmol) and 6-O-benzyl-2-hydroxy-acetophenone6 (0.12 g: 0.5 mmol)
were combined and dissolved in methanol (3 mL) and combined with 40% KOH in MeOH (2
mL) at rt. The orange solution was stirred for 2h at 80oC. Workup included evaporating the
solvent and adding water (20 mL) followed by addition of CH2Cl2. 1M HCl (20 mL) was added
until the pH of the water layer was pH 1. The organic layer was then separated, dried over
anhydrous Na2SO4, filtered, and concentrated to provide a crude orange oil. Column
chromatography was performed (100% CH2Cl2, then 50:50 Hexane/CH2Cl2) to obtain the pure
1
product (179 mg, 67%). 8n: m.p. 138-140oC, H NMR (CDCl3): δ 13.09 (s, 1H, OH), 8.04 (d,
1H, J= 15.9Hz), 7.80 (d, 1H, J= 15.9 Hz), 7.40 (m, 2H), 7.37 (m, 2H), 7.33 (d, 1H, J = 2.4 Hz),
7.28 (m, 3H), 6.73 (d, 1H, J=8.8 Hz), 6.65 (dd, 1H, J=8.3 Hz and 1 Hz), 6.53 (dd, 1H, J= 8.3 Hz
and 1 Hz), 5.14 (s, 2H), 3.91 (t, 2H, J=6.7 Hz), 1.78 (m, 2H), 1.43 (m, 2H), 1.29 (m, 8H), 0.87 (t,
13
3H, J=6.85 Hz). C NMR (CDCl3): δ 194.58, 164.76, 160.01, 156.99, 136.18, 135.81, 135.46,
133.75, 130.28, 128.84, 128.68, 128.58, 128.45, 127.46, 126.11, 113.69, 112.64, 112.22, 111.11,
102.58, 71.26, 68.83, 31.72, 29.26, 29.14, 28.96, 25.94, 22.60, 14.05. Anal. C30H33BrO4: C, H.
HRMS C30H33BrO4 (M + H): theory 537.1635; found 537.1636.
Page 6

1-(2-Benzyloxy-6-hydroxy-phenyl)-3-(5-bromo-2-ethoxy-phenyl)-propenone (8o). 5-Bromo-
2-ethoxy-benzaldehyde 7a (473 mg, 2.06 mmol), 1-(2-Benzyloxy-6-hydroxy-phenyl)-ethanone6
(500 mg, 2.06 mmol), and MeOH (9 mL) were combined and KOH in methanol (40%
weight/volume, 8.24 mL) added at room temperature. A water condenser was attached to the
flask, and the orange solution quickly turned dark red upon heating and was stirred at reflux in an
oil bath at 85 °C for 3h. Workup involved evaporation of the solvent and addition of 1N HCl
until the solution was at pH 1, followed by extraction with ethyl acetate (twice). The organic
layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated to give an orange
solid. Column chromatography was performed (10% EtOAc:hexane) to purify the product. A
crystallization step was then performed (ethyl acetate/hexane) to obtain the pure product (75%
1
yield). 8o: m.p. 107-109oC, H NMR (CDCl3): δ 13.08 (s, 0.76H), 8.07 (d, 1H), 8.03 (d, 0.53H),
7.81 (d, 0.58H), 7.78 (d, 0.47H), 7.41 (m, 2H), 7.37 (m, 2H), 7.33 (d, 1H), 7.29 (m, 3H), 6.73 (d,
2H, J = 8.8 Hz), 6.66 (d, 1H, J = 1.2 Hz), 6.64 (d, 1H, J = 1 Hz), 6.55 (d, 1H, J = 1 Hz), 6.53 (d,
13
1H, J = 0.7 Hz), 5.14 (s, 2H), 4.00 (1, 2H), 1.54 (s, 0.93H), 1.41 (t, 3H). C NMR (CDCl3): δ
165.05, 160.17, 157.1, 136.20, 133.87, 130.38, 128.76, 127.59, 126.20, 113.68, 112.76, 102.71,
71.36, 64.31, 14.68; Anal. C24H21BrO4: C, H; HRMS For C24H22BrO4 (M + H): theory 453.0701;
found 453.0691.
1-(2-Benzyloxy-6-hydroxy-phenyl)-3-(5-chloro-2-ethoxy-phenyl)-propenone (8p). 5-Chloro-
2-ethoxy-benzaldehyde 7c (500 mg, 2.7mmol), 1-(2-Benzyloxy-6-hydroxy-phenyl)-ethanone6
(655 mg, 2.7mmol), and MeOH (10 mL) were combined and KOH in methanol (40%
weight/volume, 10 mL) added at room temperature. A water condenser was attached to the flask,
and the yellow solution quickly turned red upon heating and was stirred at reflux in an oil bath at
85 °C for 4 hours. Workup involved evaporation of the solvent and addition of 1N HCl until the
Page 7

solution was at pH 1, followed by extraction with ethyl acetate (twice). The organic layer was
separated, dried over anhydrous Na2SO4, filtered, and concentrated to give an orange/yellow
solid (1.3 g). Column chromatography was performed (15% ethyl acetate:hexane) to obtain the
1
pure product (56% yield). 8p: m.p. 103-105oC, H NMR (CDCl3): δ 13.08 (s, 0.76H), 8.09 (d,
2H, J = 15.9 Hz), 7.82 (d, 2H, J = 15.7 Hz), 7.78 (d, 0.47H), 7.43-7.25 (m, 8H), 7.15 (d, 2 H, J =
2.7 Hz), 6.79 (d, 2H, J = 8.8), 6.66 (dd, 2H, J = 8.6), 6.55 (dd, 2H, J = 8.2 Hz), 5.14 (s, 2H), 4.00
(q, 2H), 1.54 (s, 0.93H), 1.41 (t, 3H). 13C NMR (CDCl3): δ 194.79, 164.98, 160.01, 156.37,
136.49, 135.92, 135.53, 130.93, 128.77, 128.60, 127.64, 127.35, 125.62, 125.46, 113.26, 112.25,
111.24, 102.61, 71.40, 64.38, 14.69. Anal. C24H21ClO4: C, H.; HRMS C24H22ClO4 (M + H):
theory 409.1207; found 409.1211.
4-Benzyloxy-2-(5-bromo-2-ethoxy-benzylidene)-benzofuran-3-one
(14a).
(E)-1-(2-
(benzyloxy)-6-hydroxyphenyl)-3-(5-bromo-2-ethoxyphenyl)prop-2-en-1-one, 8o (174 mg, 0.38
mmol) was dissolved in a 3 M KOH solution in 96% ethanol (2.7 mL). Then 35% H2O2 (0.8 mL)
was added dropwise to the mixture cooled in an ice bath (the mixture turned viscous) and stirred
for 2 h. TLC (10% ethyl acetate:hexane) showed the completion of the reaction via the
consumption of 8o. The mixture was the acidified to pH 1 with 1M HCl at 0°C. A precipitate
formed as the acid was added. A crude solid was collected by vacuum filtration and purified by
column chromatography (60% CH2Cl2:hexane) to obtain the bromo-coumaranone derivative 14a
1
(74 mg, 43%). 14a: m.p. 153-155oC, H NMR: δ 8.36 (d, 1H, J = 2.4 Hz), 7.53 (m), 7.41 (m),
7.34 (m), 7.33 (s), 7.31 (m), 6.92 (d, 1H, J = 0.5 Hz), 6.91 (d, 1H, J = 0.5 Hz), 6.79 (d, 2H, J =
8.8 Hz), 6.64 (d, 2H, J = 8.3Hz), 5.35 (s), 4.10 (q), 2.18 (s), 1.57 (s), 1.49 (t). 13C NMR (CDCl3):
δ 182.00, 166.90, 157.49, 157.01, 147.28, 138.16, 136.15, 133.95, 133.37, 128.63, 127.91,
Page 8

126.69, 123.57, 113.26, 112.92, 111.40, 107.22, 105.14, 104.44, 70.65, 64.51, 14.71. Anal.
C24H19BrO4: C, H.; HRMS For C24H19BrO4 (M + H): theory: 451.0539 ; found 451.0530.
4-Benzyloxy-2-(5-chloro-2-ethoxy-benzylidene)-benzofuran-3-one (14b). 1-(2-Benzyloxy-6-
hydroxy-phenyl)-3-(5-chloro-2-ethoxy-phenyl)-propenone 8p (300 mg, 0.73 mmol) was
dissolved in a 3 M KOH solution in 96% ethanol (5.3 mL). Then 35% H2O2 (1.49 mL) was
added dropwise to the mixture cooled in an ice bath (the mixture turned viscous) and stirred for
30 min. The ice bath was removed and the reaction stirred at room temperature for 3 h. TLC
(15% EtOAc:hexane) showed the completion of the reaction via the consumption of 8p. The
mixture was the acidified to pH 1 with 1M HCl at 0°C. A precipitate formed as the acid was
added. A crude solid was collected by vacuum filtration and purified by column chromatography
(85% CHCl3: hexane) to give the chloro-coumaranone derivative 14b (60% yield). 14b: m.p.
148-149oC; 1H NMR (CDCl3): δ 8.22 (d, 2H, J = 2.7H), 7.52 (m), 7.39 (t), 7.31 (m), 7.27 (d, 1 H,
J = 2.7 Hz), 7.26 (s), 6.91 (d, 2H, J = 8.1 Hz), 6.83 (d, 2H, J = 9 Hz), 6.64 (d, 2H, J = 8.3 Hz),
5.33 (s), 4.09 (q), 1.48 (t). 13C NMR (CDCl3): δ 181.87, 166.97, 156.70, 147.42, 138.15, 131.10,
130.45, 128.63, 127.94, 126.70, 125.62, 123.05, 112.83, 111.0, 107.25, 105.13, 104.59, 70.67,
64.59, 14.76, Anal. C24H19ClO4: C, H; HRMS For C24H20ClO4 (M + H): theory: 407.1050 ;
found : 407.1050.
Cell lines and viruses. TZM-bl cells (Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme,
Research Triangle Park, NC), and PM1 cells (Dr. Marvin Reitz) were acquired from the AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Both cell lines
express high levels of HIV-1 co-recepters CD4, CCR5, and CXCR4, and TZM-bl cells are also
endowed with an HIV-1 tat luciferase reporter construct that is useful for quantifying HIV
infection in microtiter assays. TZM-bl cells were maintained in Dulbecco’s modified Eagles’s
Page 9

medium (DMEM) (4.5 g/L glucose) containing penicillin, streptomycin, and 10% (v/v) fetal
bovine serum (FBS) (termed “D10” hereafter). PM1 cells are a pro-myelocytic cell line capable
of propagating HIV-1 virions following exposure to infectious virus. These were maintained in
RPMI 1640 supplemented with penicillin, streptomycin, 10mM HEPES, and 20% (v/v) FBS
(“R20”). The following HIV-1 isolates were acquired from the NIH AIDS Research and
Reference Reagent Program: BaL (R5), 92UG029 (X4, subtype A), 93UG070 (X4, subtype D),
93UG067 (R5X4). Stocks were prepared via propagation in PM1 cells for 5-11 days (isolate-
dependent), and cell supernatants containing fresh virions were clarified of cellular debris by
centrifugation followed by filtration through a 0.45 uM syringe filter, and stored at -80C in
aliquots to be used as viral stocks. Quantification of viral stocks were performed by HIV-1 p24
ELISA (Perkin Elmer, Waltham, MA), and viral infectious titer was determined by infecting
PM1 and TZM-bl cells with serial dilutions of viral stock and identification of the dilution that
resulted in infection of 50% of the culture wells (TCID50/mL).
CHO cells were grown as previously described.6 Briefly, cell growth was assayed in sterile 96-
well microtiter plates (Costar 3599, Corning, NY, USA) in the presence of each compound.
Experiments were conducted in triplicate. CHO cells were plated at 10,000 cells/mL. Drug
solutions were prepared in 100% DMSO and dosed so that the final DMSO concentration was
<1%. Drug additions occured after an initial overnight incubation of CHO cells in each well. For
example, 1 μL of flavonoid solution was added to the CHO cells plated in each well in 100 μL of
media. After flavonoid compound was added, the cells were incubated in 5% CO2 for 48 h at
37oC. The MTS reagent (Promega Cell Titer 96 AQueous non-radiactive cell proliferation
reagent) was added (20 μL) and the CHO cells were incubated for an additional 4h and then
absorbance at 490 nm was measured on a BioTek Synergy MX plate reader. Controls run using
Page 10

1% DMSO in the media and no compound showed no toxicity over the 48 h period compared to
CHO cells grown in media only. IC50 values were determined from the corresponding plot of
relative absorbance at 490 nm vs flavonoid concentration. The data is tabulated in Table 1.
Antiviral and cytotoxicity assays. TZM-bl cells were trypsinized and seeded to 6 × 104
cells/ml (6000 cells/well) in 96-well dishes, then incubated (37°C/5% CO2) overnight to allow
for cell attachment. The following day, when cells were 50-60% confluent, maintenance media
was aspirated and cells were treated in triplicate with 50 μL of D10 containing diluted compound
or the volume equivalent of DMSO vehicle. After 5 min (or for select experiments, 3 hr) HIV
was diluted in D10 and applied to pre-treated wells (50 uL/well) at a final concentration of 60
TCID50/0.1mL (MOI=0.01) and incubated at 37°C/5% CO2. After 24 h, cells were lysed and
processed for luciferase assay (Bright Glo luciferase system, Promega, Madison, WI), and
luciferase was quantified with a Spectramax luminometer (Molecular Devices Corp., Sunnyvale,
CA, USA), with an integration time of 5 s/well. Ability to prevent HIV-1 infection was measured
as a percentage reduction in luciferase (relative light units) compared to the positive viral control
(medium, DMSO vehicle, and virus), while the negative control (medium, DMSO, no virus)
represented the baseline. To test whether reduced luciferase activity could be due to cytotoxic
effects of certain compounds rather than reduced HIV infection, replicate dishes of treated TZM-
bl cells were assayed for dead cell and total cell numbers using CytoTox Glo reagent (Promega).
Percent differences in live cell luminescence were calculated for each compound versus the
matched DMSO vehicle control. Select compounds were assayed additionally for cytotoxic
effects using confluent TZM-bl cells and MTT reduction assay (R&D Systems, Minneapolis,
MN) to monitor cell metabolic activity. CHO IC50 values were determined using established
methods.6
Page 11

PM1 cells (1.5×105/0.1 mL) were treated with compound or matched DMSO volume-equivalent
diluted in R20 maintenance media, incubated for 10 min, then infected with HIV-1 BaL (150
TCID50/0.1 mL, MOI=0.001) for 2 h. Cells were then washed and resuspended in fresh R20
containing compounds or DMSO vehicle. Culture supernatants were collected on day 3, and
cells were re-suspended and incubated in treatment media until day 5, when supernatant was
collected and assayed for HIV p24 by ELISA. Cell viability was monitored by trypan blue dye
exclusion.
3.1 Results and Discussion
The new target structures were inspired by the bioactive structures 1-5 and the previously
designed compounds 6-13 (Figure 2).
Page 12

Page 13

Figure 2. Aryl Precursors and final Chalcone and Flavonoid compounds
3.2 Synthesis. The synthesis of compounds 8a-m, 9a-j and 10-13 from their precursors 6 and 7
has been previously described.6 During the course of our biological investigations of 8 and 9 as
antivirals, we discovered that certain ring substituents provided enhanced efficacy against HIV
and significantly reduced toxicity to host cells. This insight led to the design of new molecules
which combined these favorable substituents onto the same scaffold.
While many of the required benzaldehyde and acetophenone starting materials were
commercially available, several were made via O-alkylation of 2-(hydroxy)phenyl derivatives, 6.
As shown in Scheme 1, the desired alkyl groups were readily introduced in good yields.6
Scheme 1a
aReagents: a) MeI or EtBr or n-octylbromide, K2CO3, DMF, 2 days, rt
The newly-designed target chalcones were synthesized following Scheme 2 and the chalcones
8n, 8o, and 8p were generated in good yields. The chalcones were accessed using strong base at
85°C via the crossed aldol condensation of the respective carbonyl compound 7 and
acetophenone 10b to obtain the chalcone products, 8n-8p. We found that a reaction time of 3 h
produced higher yields, and the products were isolated using column chromatography.
Page 14

These compounds incorporated the most effective substituents, such as bromine or
chlorine at the R4 position, as well lengthening the alkyl chain at the R1 position. Compounds 8o
and 8p incorporated an O-ethyl substituent, whereas 8n incorporated an O-(n-octyl) substituent
to evaluate the effects of increasing lipophilicity at this position. This strategy was predicated
upon indications in our initial HIV screens that the O-ethyl substituent was desirable. The new
generation of compounds also contained an O-benzyl substituent at the R3 position (See Scheme
2) found to be so effective in prior antiviral studies. 6
The conversion of the chalcones 8o and 8p to the substituted benzyl-coumaranones 14a
and 14b was accomplished with 35% H2O2 in the presence of KOH (Scheme 2).15 Upon mixing
the reagents at 0oC, the solution was allowed to slowly warm to room temperature to facilitate
conversion. The final yields of these compounds, however, were still low due to the formation of
significant by-products. The presence of the R3 alkoxy group (Scheme 2) is known to direct ring
closure to form the five membered ring in lieu of the six membered ring.13 Indeed systems
lacking oxygen at this position generated six membered rings under these conditions.6 The
coumaranones 14a and 14b were isolated and purified via column chromatography.
Page 15

Scheme 2a
aReagents : a) 40%KOH/MeOH, 85oC ; b) 35% H2O2, KOH/EtOH, then 1M HCl
In summary, the expanded library of compounds was tested for their activity in inhibiting HIV
infection and cytotoxicity using infectious HIV-1 and the human target cell lines TZM-bl and
PM1.
3.3 Bioevaluation. To determine the efficacy of these flavonoid derivatives against HIV-1,
compounds were initially screened at 10 µM using the target cell line TZM-bl, which expresses
an HIV-1 tat luciferase reporter construct for quantifying HIV infection.16 To provide a strict
test of antiviral activity, cells were treated and infected while at 50-60% confluence, which
provided more surface area for viral fusion and entry than if the cells formed a confluent
monolayer. Luciferase production in response to HIV-1 tat expression was measured after 24 hr.
Page 16

Compounds were tested for cytotoxicity on replicate dishes of TZM-bl cells, as well as on CHO
cells for comparison. CHO cells were chosen in order to compare the relative toxicities of the
new flavonoid derivatives to previously investigated compounds.6 Table 1, column 2, displays
the mean % inhibition of HIV-1 infection for each compound. The third column in Table 1
shows the relative % decrease each compound had on uninfected TZM-bl cell viability. In order
to select potent non-toxic compounds for further investigation, we applied a screening criteria to
the datasets obtained. Compounds, which gave >50% HIV-1 inhibition at 10 µM and showed
limited toxicity to unifected TZM-bl cells (arbitrarily set as a ≤25% decrease in cell viability for
this early screen, see column 3), are highlighted in bold in Table 1, column 2. The fourth column
in Table 1 shows the cell proliferation IC50 value for each compound in CHO cells after 48 h.
Taken together, this efficacy and safety information allowed for an assessment of the
selectivity of each compound to target the virus and not the host cell. A relative ranking of the
compounds via their toxicity profiles was also performed. In each case, the chalcone system 8
was more toxic than the corresponding ring-closed 4H-chromen-4-one 9. With the exception of
11a (CHO IC50 = 17.5 µM), compounds 11-13 were found to be relatively non-toxic to CHO
cells (CHO IC50 values > 64 µM). In sum, these studies provided general toxicity profiles of each
compound as well as insights into the molecular design needed to lower cell toxicity and still
maintain potency against HIV-1.
Page 17

Table 1. Effect of selected compounds 8-9 and 11-14 on HIV-1 infection, viability of TZM-bl
cells treated with each compound at 10 µM, and the cell proliferation IC50 values (µM) in CHO
cells. a,b,c
Compound % Inhibition of HIV-1
infection by 10 µM
% decrease in TZM-bl cell
viability by 10 µM compound cell proliferation
CHO 48 h
compound (mean±SEM)
(mean±SEM)
IC50 value (µM)a
8a
8b
8c
8d
8e
8f
none
16.59.1
25.5  2.7
78.61.0
88.10.2
85.81.7
28.65.3
95.10.8
99.70.2
96.40.5
86.11.4
63.11.0
ND
62.67.0
51.96.6
72.34.4
1.99.6
90.44.5
99.61.2
99.53.6
86.43.9
46.93.5
ND
8.7  0.3
7.2  0.5
7.2  0.4
16.9  1.3
6.1  0.8
6.7  0.7
8.4  0.6
6.8  0.9
14.2  2.7
ND
8g
8h
8i
8j
8k
8l
86.55.4
53.33.5
21.33.6
22.41.6
51.71.5
25.90.8
43.60.7
61.71.2
60.71.2
61.41.5
13.00.9
23.44.9
19.95.0
none
8.1  1.0
15.6  1.0
>100
8m
9a
9b
9c
9d
9e
9g
9h
9i
none
88.5  10.1
13.5  1.3
>100
none
none
none
18.3  2.5
17.9  1.6
25.0  2.2
>100
15.33.5
23.55.3
21.95.9
4.85.8
9j
54.6  4.2
Page 18

ND
11a
11b
11c
11d
12a
12b
13b
14a
14b
14c
ND
17.5  1.9
96.6  5.6
64.8  8.1
>100
16.41.6
74.80.9
38.41.9
83.80.9
52.41.5
2.82.8
12.69.3
15.77.5
9.21.1
6.55.5
39.54.9
23.68.4
44.55.6
34.83.7
none
>100
75.2  8.7
67.9  9.3
ND
8.47.9
none
ND
0.28.6
17.3  1.9
a
CHO cells were incubated for 48 h at 37°C with the respective compound and relative cell
viability assessed. We noted that compounds 9a, 9j, 11b and 13b demonstrated low toxicity
b
across multiple cell types but were relatively ineffective against HIV-1. Compounds, which
gave >50% HIV-1 inhibition at 10 µM and showed limited toxicity to unifected TZM-bl cells
(arbitrarily set as a ≤25% decrease in TZM-bl cell viability, see column 3), are highlighted in
bold in column 2 and were selected for further study. cCompound 8k was not evaluated in
bioassays due to its previously observed high toxicity in HEK 293T cells (see ref 6).
As shown in Table 1, interpreting the efficacy of each compound against HIV-1 required
an understanding of the compound’s toxicity because dead or damaged cells would not be able to
synthesize luciferase protein following HIV-1 infection. For example, a highly toxic compound
like 8g which inhibits 99% of HIV-1 but also damaged 99% of the host cells at the same
concentration (10 µM) was undesirable. In contrast, a compound like 8l (10 µM) provided a
better balance between efficacy (86.5% inhibition of HIV-1 infection) and toxicity (23.4%
decrease in host cell viability). The flavonols 9c, 9g, 9h, and 9i each had >50% inhibition of
Page 19

HIV-1 and had modest toxicity (<23.5% decrease in host cell toxicity). Interestingly, the nature
of the 2-alkoxy substituent in the 5-bromo derivatives 9b (22.4% inhibition of HIV-1) and 9c
(51.7%) suggested that the O-ethyl substituent was more advantageous than OMe. However, this
was not the case with the related 5-chloro derivatives 9h (OMe) and 9i (OEt) which had similar
activities (~61% inhibition of HIV-1). The nature of the 5-halo group was interesting in the OMe
series (9b, 9g, and 9h) and suggested that the 5-chloro and 5-iodo substituents (both gave ~61%
HIV inhibition) may be more efficacious than the 5-bromo (9b, 22.4%). In the presence of the 2-
O-ethyl group, however, this difference is less pronounced where 9c (Br/OEt, 51.7% HIV
inhibition) and 9i (Cl/OEt, 61.7% HIV inhibition).
Next, we screened selected compounds from Table 1 for their dose-dependent
effectiveness in protecting TZM-bl cells from HIV-1 infection. These studies allowed for the
determination of IC50 values for this subset of compounds and are listed in Table 2.
Page 20

Table 2. Anti-HIV potency of each compound (anti-HIV IC50 value), the toxicity of each
compound when tested in uninfected TZM-bl cells at the anti-HIV IC50 concentration. Each
compound’s substituents are shown for comparisons.
Anti-HIV
HIV-1 BAL
IC50 (µM)
4.7
% Toxicity at its
respective
HIV-1 BAL IC50 dose
not toxic
Compound
Substituents
OMe/Br/OBn/benzene core
OMe/Br/H/naphthyl core
H/OMe
8l
8m
9a
20
30.4
90%
not toxic
43.7
10.4
not toxic
not toxic
Br/OMe
Br/OEt
9b
9c
30.9
12.6
not toxic
Br/ diOMe
Cl/OEt
9d
9i
20%
42.3
4.4
not toxic
OMe/Br/OBn
OMe
11b
12a
42%
As shown in Table 2, compound 12a was the most potent (anti-HIV IC50: 4.4 µM) but
also resulted in significant toxicity at 4.4 µM (42% toxicity). In this regard, compound 8l was
selected as the better scaffold to pursue as it also displayed good potency (4.7 µM) along with
derivatives 9c (10.4 µM) and 9i (12.6 µM). The other systems 11b and 12a were not pursued due
to either low efficacy or high toxicity properties. As shown in Figure 3, compound 8l was also
shown to inhibit diverse isolates of HIV-1 in a dose-dependent manner (Panel A, Fig 3). The
laboratory strain BaL is an R5 (M-tropic) strain representing the type of HIV-1 typcially spread
by sexual contact. Clinical isolates 92UG029, 93UG070, and 93UG067 were tested because
they represent both dual tropic (R5X4) and T-tropic (X4) strains isolated from patients with
advanced disease. Compound 8l was not toxic to the host cells (Panel B, Fig 3) and no changes
in relative cell metabolic activity were noted when 8l was dosed up to 10 µM (Panel C, Fig 3).
Note: Compound 8l gave a CHO 48h IC50 value of 8.1 µM in Table 1, but this toxicity was not
Page 21

observed in the TZM-bl cells at 4.7 µM (Table 2) nor in Figure 3 at 10 µM and is likely cell line
dependent.
Figure 3. Chalcone 8l dose-dependently inhibits diverse isolates of HIV-1. Panel A)
Compound 8l was tested in TZM-bl HIV reporter cells at 2.5-10 µM and compared with volume-
matched DMSO vehicle controls. Presented is the mean % inhibition of infection for assays
performed in triplicate. Panels B-C) Compound 8l (red lines) or matched vehicle (black lines)
was applied to confluent TZM-bl cells and assessed for cytotoxicity using a live/dead cell
detection assay (CytoTox Glo, Promega, panel B) or MTT reduction assay (C). Mean ± SEM,
n=3.
Page 22

We noted that the most active compounds of all those tested had similar O-alkyl and
halogen substituents. The most potent chalcone 8l also contained the unique 5’-O-benzyl
substituent suggesting that this modification was key to its performance as 8b, which does not
contain this group, was less effective and significantly more toxic to TZM-bl cells (Table 1). We
noted that 8f, which contains a smaller OMe group at the 5’-position, was also more toxic than
8l, which contained the 5’-O-benzyl group. Moreover, 8c (2-OEt) was less toxic and more
potent than 8b (2-OMe) suggesting that the longer O-alkyl group improved performance. This
trend was also seen in the 9 series in Table 2, where the 2-O-ethyl derivative 9c was significantly
more potent (anti HIV IC50: 10.4 µM) than its related 2-O-methyl analogue 9b (43.7 µM), again
suggesting that increased length of the alkoxy substituent may be beneficial. This relationship,
however, was not evident in 9h (OMe) and 9i (OEt) which had similar toxicities in TZM-bl cells
in Table 1 (~23 µM). Therefore, we designed a new series of chalcones, 8n-8p, which pursued
these insights in order to increase potency and lower toxicity. Compound 8n contained a long n-
octyl substituent to further improve lipophilicity at the 2-position, whereas 8o and 8p
investigated the role of the 5-halo substituent in the presence of the 5’-O-benzyl substituent.
Figure 4. New inhibitor designs, 8n-8p
We then synthesized the new compounds that contained the desirable 5’-O-benzyl group
and longer 2-O-alkyl substituents (8n-8p, described above in the Synthesis section).
Interestingly, the n-octyl derivative 8n was devoid of anti-HIV activity and was not pursued,
Page 23

while 8o and 8p exhibited anti-HIV-1 activity and were essentially non-toxic under the
experimental conditions (Table 3). To test whether exposure time impacted efficacy, TZM-bl
cells were pre-treated with each compound for either 5 min (standard assay) or 3 hours prior to
inoculation with HIV-1. The anti-HIV activity of compounds 8o and 8p was higher when tested
using longer pre-incubation times (Table 3). Both 8l (2-OMe/5’-OBn) and 8o (2-OEt/5’-OBn)
inhibited HIV infection by ~90% after a three hour pre-incubation period. In contrast, the
potency of the 9 series compounds (9c and 9i) did not improve with the extended pre-incubation
period.
Table 3: Bioevaluation of selected compounds (10 µM) in TZM-bl cells challenged with
HIV-1 a
Compound HIV % Inhibition HIV % Inhibition % Inhibition of Cell Viability
after 5 min
pre-treatment
(at 10 μM)
after 3 hr
pre-treatment
(at 10 μM)
(compd at 10 μM)
86.2±5.4
<10
90.5±3.5
27.9±10.6
89.9±2.2
79.0±12.9
54.9±6.2
54.1±6.8
23.4±4.9
ND
8l
8n
8o
8p
9c
9i
60.2±6.7
64.8±5.3
57.2±4.5
67.2±5.6
none
8.7±5.1
none
none
a ND = not determined
Having shown that these compounds can inhibit HIV-1 infection of TZM-bl cells, we assessed
their ability to inhibit viral propagation by PM1 cells, a pro-myelocytic cell line that is readily
infected by HIV. As shown in Table 4, compounds 9c and 9i had no effect on HIV-1 infection
of cultured PM1 cells compared to the DMSO control at 10 µM. In contrast, the chalcones 8l-8p
Page 24

showed >92% inhibition of HIV propagation at 10 µM and >65% inhibition at 5 µM. Regarding
host-cell viability, compound 8l was toxic to PM1cells at 10 µM (38% viability), whereas the
chalcones 8o and 8p were relatively non-toxic to PM1 cells at 10 µM (96.8 and 87.7 % viability,
respectively, Table 4).
Table 4. Select compounds effectively inhibit HIV-1 propagation by host PM1 cells without
impacting cell viability
% inhibition of HIV propagation % inhibition of Cell Viability
Compound
10 µM
99.9±0.1
92.3±2.7
95.8±1.7
1.3
5 µM
80.0±13.9
72.2±9.2
65.8±12.4
none
10 µM
61.7±5.8
none
12.3±7.2
23.1
5 µM
0.6±7.2
none
none
16.5
8l
8o
8p
9c
9i
none
ND
25.8±14.2
ND
We noted that the 5-bromo-substituent in 8b (64.2% decrease in cell viability) provided a
compound which was less toxic to TZM-bl cells than 8h, which contained the 5-chloro-
substituent (98.9% decrease in cell viability). In the HIV propagation assay, however, the bromo-
containing 8o and chloro-containing 8p had similar toxicity profiles and potencies in PM1 cells
(Table 4).
In summary, chalcone 8o was found to provide a good balance between potency and
toxicity and was effective in inhibiting both HIV-1 infection and the spread of infection while at
the same time remaining relatively non-toxic to the cell lines tested (TZM-bl and PM1). In
contrast, the other systems investigated (e.g., substituted flavones 11 and coumaranones 14) were
found to be ineffective by comparison. These new chalcones 8o and 8p not only demonstrated
Page 25

good anti-viral activity, but were significantly less toxic than the previously screened derivatives,
showing an improvement in the overall drug design.
A comparison of the related pairs 11d and 13b indicated that the presence of the α,β-
unsaturated system of 11d increased toxicity to TZM-bl host cells. Michael acceptors (α,β-
unsaturated carbonyls) like the chalcones explored here are often considered PAINS in medicinal
chemistry due to their ability to react with a variety of biological nucleophiles and generate
significant off-target effects.17 However, some PAINS constructs can act as privileged scaffolds,
which allow for the “quick identification of hits with sufficient target affinity whose selectivity
18
can be tuned in subsequent development steps.”
In the library of flavonoids evaluated here,
we noted that not all Michael acceptors within the same series performed the same. For example,
9b and 9c were both not toxic to TZM-bl host cells at 10 µM (Table 1), but 9b and 9c gave
significantly different potencies in terms of HIV inhibition (22.4% and 51.7% HIV inhibition,
respectively). A comparison across related Michael scaffolds showed that 81 and 11d had nearly
identical toxicity to TZM-bl cells (23% decrease in TZM-bl cell viability at 10 µM, Table 1) but
8l outperformed 11d in terms of HIV inhibition in Table 1 (86.5% vs 38.4%, respectively). Thus,
our results suggest that one can maintain HIV inhibition properties and reduce host cell toxicity
via substituent changes on a chalcone platform.
Lastly we also noted that one of the best HIV inhibitors 8l was also the immediate
synthetic precursor to the HCMV inhibitor 11b.6 We speculated that this arrangement of 5-
bromo, 2-alkoxy and 5’-benzyloxy substituents on a core platform may provide unique anti-
virals. This hypothesis was tested via the related coumaranone 14a and flavone 11b. Both were
shown to be ineffective even though they represent these same substituents annealed to a related
core platform. This suggested that the activity we observed in HIV may be due, in part, to the
Page 26

additional presence of the β-hydroxyketone of 8 and α-hydroxyketone of 9; an observation which
may also partially explain why β-hydroxyflavone 12b has anti-HIV-1 activity, whereas
flavanone 13b did not (Table 1). In addition, flavone 11d retains α,β-unsaturation, but lacks the
adjacent hydroxyl group and underperforms in terms of HIV inhibition in Table 1 (% Inhibition
of HIV-1 infection by 10 µM compound; 8l: 86.5%; 11d: 38.4%). Indeed, these hydroxyl-
carbonyl motifs and related bidentate ligands are well known to interact with HIV integrase12b,
12d, 12e
and is the central motif of the HIV integrase inhibitor, Raltegravir (Isentress).19 In
addition, Courter et al have described inhibitors of HIV-1 viral entry predicated upon related
oxalamide structures.20 Future work will explore these possible mechanisms of action.
4.1 Conclusion. In conclusion, 33 flavonoids including chalcones, flavones, flavanones, and
coumaranones were screened for their anti HIV activity. In general, compounds which contained
an α- or β-hydroxy carbonyl motif were typically more potent. Of the series tested, the chalcones
8o and 8p provided the best balance between high anti-viral potency and low cytotoxicity. These
chalcones significantly inhibited HIV propagation at low micromolar doses (≤ 10 µM) and were
relatively non-toxic. We note that the low micromolar potencies observed with chalcones 8l-o
are in the range of other known small molecule HIV inhibitors. For example, small molecule
allosteric HIV-1 integrase inhibitors in pre-clinical development have demonstrated antiviral
activity in the 0.1-5 µM range.21 In addition, small ubiquitin-like modifier (SUMO)-specific
protease inhibitors represent a novel class of anti-HIV-1 agents, and are active in the 1-100 µM
22
range and human antimicrobial peptides (1-5 kD) capable of inhibiting HIV-1 fusion exhibit
EC50 values of 0.5-5 µM.23 In this regard, chalcone 8o provides a good starting point for future
drug development.
Page 27

1
5.1 Supporting Information. H NMR and 13C NMR spectra and elemental analyses for the
new compounds 8n-8p, 14a and 14b. This material is available free of charge via the Internet.
Author Information.
Corresponding Author. *Email Otto.Phanstiel@ucf.edu; phone 407-823-6545.
Notes. The authors declare no financial interest.
Acknowledgements. The authors wish to thank the Office of Research at the UCF College of
Medicine for their support of this work (Grant #25060753) and Dr. Nivedita Jena for her help in
completing this manuscript.
Abbreviations Used. HIV-1, Human immunodeficiency virus-1; AIDS, acquired
immunodeficiency syndrome; ART, antiretroviral therapy; HCMV, human cytomegalovirus;
CHO, Chinese hamster ovary; TCID50, tissue culture infectious dose, i.e. the dose that resulted in
infection of 50% of the cells in culture; MOI, multiplicity of infection; DMSO,
dimethylsulfoxide
Keywords. Flavonoids, HIV, chalcones
Page 28

6.1 References
1.
Zhao, Y.; Jiang, F.; Liu, P.; Chen, W.; Yi, K., Catechins containing a galloyl moiety as
potential anti-HIV-1 compounds. Drug Discov Today 2012, 17 (11-12), 630-635.
2.
chalcones: a mini review. Eur J Med Chem 2014, 85, 758-777.
3. (a) Nations, U., UNAIDS report on the global AIDS epidemic 2014.
http://www.unaids.org/sites/default/files/media_asset/20140716_FactSheet_en.pdf 2014,
Singh, P.; Anand, A.; Kumar, V., Recent developments in biological activities of
unaids.org; (b) van den Heuvel, D.; Driessen, G. J.; Berkowska, M. A.; van der Burg, M.;
Langerak, A. W.; Zhao, D.; Charif, H.; Hartwig, N. G.; van Rossum, A. M.; Fraaij, P. L.; van
Dongen, J. J.; van Zelm, M. C., Persistent subclinical immune defects in HIV-1-infected children
treated with antiretroviral therapy. AIDS 2015, 29 (14), 1745-1756; (c) Buckhold, F. R., 3rd,
Primary Care of the Human Immunodeficiency Virus Patient. Med Clin North Am 2015, 99 (5),
1105-1122.
4.
(a) Olivero-Verbel, J.; Pacheco-Londono, L., Structure-activity relationships for the anti-
HIV activity of flavonoids. J Chem Inf Comput Sci 2002, 42 (5), 1241-1246; (b) Wu, J. H.;
Wang, X. H.; Yi, Y. H.; Lee, K. H., Anti-AIDS agents 54. A potent anti-HIV chalcone and
flavonoids from genus Desmos. Bioorg Med Chem Lett 2003, 13 (10), 1813-1815.
5.
Structural and molecular target perspectives. Eur J Med Chem 2015, 101, 496-524.
6. Kralj, A.; Nguyen, M. T.; Tschammer, N.; Ocampo, N.; Gesiotto, Q.; Heinrich, M. R.;
Mahapatra, D. K.; Bharti, S. K.; Asati, V., Chalcone scaffolds as anti-infective agents:
Phanstiel, O., IV, Development of flavonoid-based inverse agonists of the key signaling receptor
US28 of human cytomegalovirus. J Med Chem 2013, 56 (12), 5019-5032.
Page 29