Synthesis and skin gene analysis of 4′-acetoxy-resveratrol (4AR), therapeutic potential for dermal applications

Article abstract of DOI:10.1016/j.bmcl.2016.05.068

Resveratrol (RV) 1, a plant polyphenol, has proven effective in commercial products yet drawbacks include low bioavailability due to rapid metabolism. Structural modifications have led to a 4′-acetoxy analog 2 (4AR) now produced using a selective one-step esterification reaction. The one-step synthesis is shown together with expression of skin genes using human dermal models to establish 4AR 2 benefits to skin health. 4AR 2 at 1% in qPCR experiments using a human skin model significantly increased gene expression of the anti-aging factor, SIRT 1 by over 3.3-fold, extracellular matrix proteins collagen III, IV, elastin and tissue inhibitors of metalloproteinases (TIMP 1, 2), anti-oxidants CAT, LOX, superoxide dismutase (SOD 1, 2), metallothioneins (MT1H, MT1H), skin aging biomarkers fibrillin (FBN1), laminin (LAMB1), proliferating cell nuclear antigen (PCNA), skin growth factors (HBEGF, IGF1, NGF and TGF). 4AR 2 also decreased gene expression of inflammatory and skin-aging molecules (IL-1, IL-6, IL-8, COX-2, TNGRSF) and S100 calcium binding proteins A8, A9. These findings suggest that 4AR 2 has potential for topically treatment and prevention of skin aging.

Full text of DOI:10.1016/j.bmcl.2016.05.068

Accepted Manuscript
Synthesis and Skin Gene Analysis of 4’-Acetoxy-Resveratrol (4AR), Thera-
peutic Potential for Dermal Applications
Edwin D. Lephart, Mark J. Acerson, Merritt B. Andrus
PII:
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http://dx.doi.org/10.1016/j.bmcl.2016.05.068
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21 May 2016
Please cite this article as: Lephart, E.D., Acerson, M.J., Andrus, M.B., Synthesis and Skin Gene Analysis of 4’-
Acetoxy-Resveratrol (4AR), Therapeutic Potential for Dermal Applications, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.068
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Synthesis and Skin Gene Analysis of 4'-
Acetoxy-Resveratrol (4AR), Therapeutic
Potential for Dermal Applications
Edwin D. Lephart,a Mark J. Acerson,b Merritt B. Andrusb

Synthesis and Skin Gene Analysis of 4'-Acetoxy-Resveratrol (4AR),
Therapeutic Potential for Dermal Applications
Edwin D. Lephart,a Mark J. Acerson,b Merritt B. Andrusb,*
Department of Physiology and Developmental Biology/The Neuroscience Center, Brigham
Young University, Provo, Utah, USA, 84602a
Department of Chemistry, Brigham Young University, Provo, Utah, USA, 84602b
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract- Resveratrol (RV) 1, a plant polyphenol, has proven effective in commercial products yet drawbacks
include low bioavailability due to rapid metabolism. Structural modifications have led to a 4'-acetoxy analog 2
(4AR) now produced using a selective one-step esterification reaction. The one-step synthesis is shown together
with expression of skin genes using human dermal models to establish 4AR 2 benefits to skin health. 4AR 2 at 1%
in qPCR experiments using a human skin model significantly increased gene expression of the anti-aging factor,
SIRT 1 by over 3.3-fold, extracellular matrix proteins collagen III, IV, elastin and tissue inhibitors of
metalloproteinases (TIMP 1, 2), anti-oxidants CAT, LOX, superoxide dismutase (SOD 1, 2), metallothioneins
(MT1H , MT1H), skin aging biomarkers fibrillin (FBN1), laminin (LAMB1), proliferating cell nuclear antigen
(PCNA), skin growth factors (HBEGF, IGF1, NGF and TGF). 4AR 2 also decreased gene expression of
inflammatory and skin-aging molecules (IL-1, IL-6, IL-8, COX-2, TNGRSF) and S100 calcium binding proteins
A8, A9. These findings suggest that 4AR 2 has potential for topically treatment and prevention of skin aging.
______________________________________________________________________________
Article info
Keywords: Resveratrol, Polyphenol, Esterification, Extra Cellular Matrix, Gene Expression,
Human Skin
* Corresponding Author. Tel.: 801 422-8171; fax: 801 422-0153
E-mail address: mbandrus@chem.byu.edu
3

Resveratrol 1 (3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic compound, known for
its potent activation of sirtuins.1a,b The sirtuins are a class of NAD+-dependent protein
deacetylase enzymes that regulate a wide variety of cellular activities which promote cell
survival and delay or attenuate many age-related changes such as preventing early mortality in
obese animals.1a,c Thus, activators of sirtuins may benefit fundamental cellular processes that
protect cells from stress and potentially treat age-related conditions, and lengthen a healthy
life.1a–d In fact, the “French Paradox” suggests that abundant levels of resveratrol in red wines
may be responsible for the surprisingly normal lifespan of the French, in spite of heavy
consumption of fatty foods that cause heart disease.1a,b
Moreover, resveratrol 1, as a stilbene phytoalexin has received increased attention for its
role in mitigating numerous human pathological processes including inflammation,
atherosclerosis, and carcinogenesis.1a,b Resveratrol is also known for its activity as an
antioxidant, cyclooxygenase inhibitor, vasodilator, inhibitor of tumor initiation, neuroprotectant
and antiviral compound.1a,b,e, 2a,b
Polyphenolic compounds, due to their positive benefits, in recent years have been
incorporated into medicinal, OTC and cosmetic products.3a In fact, botanicals or plant
polyphenols in skin products function as: protectants, whitening agents, anti-wrinkle and anti-
aging ingredients and resveratrol’s use for skin disorders has been reviewed.3b However,
resveratrol 1 as a natural compound in commercial products has been problematic due it its rapid
metabolism, especially when taken orally. To address this challenge, we developed a novel one-
step synthesis of a resveratrol analog 2 (4AR, 4'-acetoxy-resveratrol) to obtain enhanced
physical/chemical properties such as improved oil/water partition coefficient, and clinical and
cosmetic utility to advance dermal health.3b
A convenient, one-step esterification of resveratrol 1 was developed by exploring various
bases, acylating agents, and solvents under both kinetic and thermodynamic conditions (Table
1).4 Using acetic anhydride (1.5 eq.), 4AR 2 was produced from RV 1 with 5:1 selectivity over
the 3-acetoxy-RV product using NaH as base in THF (65 °C). This selectivity can be attributed
to the stability of the extended phenolate intermediate formed by removal of the more acidic 4'-
OH proton. Use of K2CO3 in ethanol at 0 °C lead to a reversal in selectivity producing the 3-
acetoxy RV product (1.3:1) over 4AR 2. Acylation of RV 1 with acetic anhydride with InCl3 in
acetonitrile at room temperature also favored 3-acetoxy-RV over 4AR 2 (2.7:1).4 The higher
4

temperature 4'-selective process proved amenable to gram scale using triethylamine in DMSO at
65 °C giving an isolated 47% yield of 4AR 2.4
Table 1. Selective Acetylation of Resveratrol 1
Our previous data on RV 1 utilized human monolayer fibroblast cell cultures to test its
ability to stimulate collagen and microarray gene analysis of several skin-related biomarkers in
order to determine how 1 influenced dermal health in a positive manner.5 This pilot work lead to
the current investigation where the purpose was to investigate 4AR 2 on the expression of skin
genes using human dermal models to determine whether 4AR 2 may benefit skin health and if it
could potentially exceed the influence of RV 1 on several biomarkers.
In brief, 4AR 2 at 1% in qPCR experiments5,6 using a human skin model significantly
increased gene expression of: 1) the anti-aging factor, SIRT 1 by 3.35-fold, 2) the extracellular
matrix proteins and 3) anti-oxidants biomarkers. Other dermal components were significantly
increased, including TGF beta, IGF-1, nerve growth factor, fibrillin, laminin and proliferating
cell nuclear antigen while at the same time 4AR 2 significantly down-regulated gene expression
of inflammatory and skin-aging molecules, such as: IL-1, IL-6, IL-8, COX-2, TNFRSF and the
S100 calcium binding proteins A8 and A9. These findings suggest that 4AR has potential for
topical use for the treatment and prevention of skin aging by dramatically influencing important
dermal components.
Since 4AR is not soluble in any aqueous buffers, pilot studies revealed that 100% DMSO
could be utilized as a skin penetrating agent via MTT viability analysis as reported elsewhere.6 A
20 µL application of 4AR in 100% DMSO was established in preliminary studies (and confirmed
5

by MatTek personnel via personal communication that indicated cell viability was above 90
percent under these conditions). Therefore, EFT skin cultures (n = 6) were exposed for 24 h to 20
µL of: (1) 100% DMSO vehicle (control) or (2) 4AR at 1.0 % in the DMSO vehicle.
Additionally, while DMSO is usually not employed as a topical formulation as a skin
penetrating agent, several organic compounds like ethanol, propylene or butylene glycol as well
as others can be used in human topical formulations. Finally, DMSO was utilized in the present
study for ease of solubility and use along with validation of the DMSO applications in cell
viability in the MatTek EFT skin cultures in order to provide proof of principle. Finally, the log
P value of resveratrol is 3.1 compared to 4AR 2 which is 3.7 (calculated), suggested that 4AR 2
would be more permeable in human skin compared to resveratrol.
Control testing and validation medium components were established following reported
protocols.5,6 Notably, in preliminary studies, multiple experiments were performed to evaluate
the DMSO vehicle in short-term (24 hr) incubations as to the potential influence each medium
component may have on the measured parameters. The DMSO vehicle or the tissue medium
alone were assayed and there were no significant influences (either decreases or increases) in
gene expression (array/mRNA levels), except that DMSO alone significantly decreased collagen
levels and significantly increased MMPs levels. Thus, the displayed results took into account the
influence of DMSO on these parameters.
In brief, the EFT skin cultures obtained from MatTek enable investigators to test
fibroblast-keratinocyte cellular mechanisms, functions and responses. The stratum corneum is
present along with a well-developed basement membrane at the dermal/epidermal junction
representing normal human skin morphology. Keratinoctyes compose the epidermal layers while
in the dermis, fibroblasts are present that produce a variety of extra-cellular matrix compounds
including collagen, elastin, matrix metalloproteinases (MMPs), etc. Each single well EFT skin
culture was visually inspected for physical imperfections and each had a surface area of 1.0 cm2.
The 20 µL treatment sample was placed across each EFT skin culture well following the protocol
published elsewhere.5
EpiDerm full thickness (EFT) gene array/mRNA and histology analysis were then
performed. EFT skin cultures (MatTek, Ashland, MA, USA) were employed in that these
cultures represent human skin barrier equivalents. A 1.0 % concentration of 4AR 2 was used in
order to penetrate the multiple-cell layers of the epidermal/dermal components.6 Pilot studies
6

revealed that 100 % DMSO could be utilized as the skin penetrating agent via MTT viability
analysis (data not shown).6 Application of 4AR 2 (20 µL) in 100 % DMSO, established in
preliminary studies, was used in EFT skin cultures exposed to 100 % DMSO vehicle or 4AR 2 at
1.0 % in DMSO (plus untreated controls as reference standard for histology purposes only). At
the end of 24 hr incubations, total RNA was isolated using Qiagen RNEasy fibrous tissue
isolation where the RNA quality was confirmed with optical density (OD) values from 1.8-2.1
(i.e. OD260/OD280 ratios).
For qPCR analysis, RNA (1 µg) from each tissue EFT sample was converted to cDNA.
Custom Taqman Low Density Arrays (TLDAs) were then created using validated gene
expression assays that contained target genes and five common endogenous control genes for
amplification/fluorescence detection. Standard cycling conditions were employed and mRNA
levels were quantified as previously described.5 To validate the integrity of the EFT skin
cultures after application of the various treatments, the slides were prepared and stained
revealing cellular structures and epidermal/dermal borders. All treatment slides displayed intact
and healthy epidermal layers (stratum corneum and keratinocytes) and dermal (fibroblasts)
components (Figure 1).
Untreated Controls
DMSO Controls
4´ Acetoxy-Resveratrol 1 %
100 microns
10x
10x
10x
100 microns
20x
20x
20x
40x
fibroblasts
100 microns
epi
SC
40x
40x
Figure 1. Validation of Epidermal Full Thickness (EFT) skin culture integrity. Representative
EFT histological sections across the treatments for the gene array experiments are displayed at
7

10x, 20x and 40x magnification. Marker bars are shown in the middle panels. The skin sections
were stained with hematoxylin/eosin and all treatment slides displayed intact and healthy
epidermal layers (SC = stratum corneum and K = keratinocytes), dermal (fibroblasts)
components and epidermal/dermal borders.
The Limma unpaired t-test was used to detect significant changes in gene expression
between the treatment and control (DMSO) groups (p < 0.005 expressed as the mean + SEM).
Inhibition of gene expression was detected by significantly lower copy numbers and significant
stimulation of gene expression was detected by higher copy numbers compared to control
(DMSO) values for each biomarker.
Topical application of 4AR 2 significantly stimulated gene expression of SIRT 1 and
ECM proteins in vitro. In this gene array experiment, we examined the ability of 4AR 2 (1%) in
DMSO to penetrated the human skin barrier [EFT] equivalents and either stimulate or inhibit
several important skin-related genes and quantify directly by transcribed mRNA levels using
Taqman real time PCR. Thus, when the anti-aging gene SIRT 1 was examined, 1% 4AR 2
significantly stimulated SIRT 1 expression by 335 % over control (Figure 2). When extracellular
matrix components were studied, 4AR 2 significantly stimulated several important dermal genes
including procollagen type III by 220 %, type IV collagen alpha 1 by 170 %, type IV collagen
alpha 2 by 180 %, elastin by 280 % and tissue inhibitor matrix metalloproteinases (TIMP 1 and
TIMP 2) by 250 and 130 %, respectively (Figure 3).
Anti-Aging Skin Gene Biomarker
500
335
400
300
200
100
0
SIRT1
8

Figure 2. Topical application of 4AR 2 significantly stimulated gene expression of SIRT 1
compared to DMSO controls. SIRT = silent information regulator protein 1; shown as the mean
+ SEM, p < 0.005.
Skin Extracellular Matrix (ECM)
Component Gene Markers
500
280
400
300
200
100
0
250
220
180
170
130
ProCol III
Col IV
alpha 1
Col IV
alpha 2
Elastin
TIMP 1
TIMP 2
Figure 3. Topical application of 4AR 2 significantly stimulated gene expression of extracellular
matrix proteins compared to DMSO controls. Collagen = ProCol III, Col IV alpha 1, Col IV
alpha 2, Elastin (ELN), tissue inhibitor of matrix metalloproteinases (TIMP 1 and TIMP 2);
shown as the mean + SEM, p < 0.005.
Topical application of 4AR 2 significantly influenced the expression of skin antioxidant
and skin aging biomarkers in-vitro. As shown (Figure 4), the application of 1.0 % 4AR 2 in
DMSO significant increased the expression of the antioxidant genes catalase (CAT by 160%),
lysyl oxidase (LOX by 190 %), superoxide dismutase 1 (SOD 1 by 160 %), superoxide
dismutase 2 (SOD 2 by 170 %), metallotheonein 1H (MT1H by 6,400 %) and metallotheonein
2A (MT2A by 340 %). In addition, for these genes the negative dermal components were
significantly inhibited (i.e., S100 calcium-binding protein A8 by 270 % and S100 calcium-
binding protein A9 by 160 %) whereas, the positive aging biomarkers were significantly
stimulated such as, fibrillin (FBN 1 by 130 %), laminin (LAMB 1 by 140 %) and proliferating
cell nuclear antigen (PCNA by 540 %; Figure 5).
9

Skin Anti-oxidant Biomarkers
100000
10000
1000
100
6400
340
160
190
170
160
10
1
CAT
LOX
SOD1
SOD2
MT1H
MT2H
Figure 4. Topical application of 4AR 2 significantly stimulated skin anti-oxidant biomarkers
compared to DMSO controls. Catalase (CAT), lysyl oxidase (LOX), superoxide dismutases
(SOD 1 and SOD 2) and metallotheoneins (MT1H and MT2A); shown as the mean + SEM on a
log scale, p < 0.005.
Skin Aging Biomarkers
900
700
540
500
300
140
130
100
0
-100
-300
-500
-160
-270
FBN1
LAMB1
S100A8
S100A9
PCNA
Figure 5. Topical application of 4AR 2 significantly stimulated or inhibited skin aging
biomarkers compared to DMSO controls. Significant stimulation: fibrillin (FBN 1), laminin
(LAMB 1) and proliferating cell nuclear antigen (PCNA); significant inhibition: S100 calcium-
binding protein (S100A8 and S100A9); shown as the mean + SEM, p < 0.005.
10

Topical application of 4AR 2 also significantly stimulated the expression of skin growth
factor and inhibited pro-inflammatory genes. For example, the growth factors stimulated by
4AR 2 included heparin-binding epidermal growth factor (HBEGF by 190 %), insulin like
growth factor I (IGF I by 264 %), nerve growth factor (NGF by 672 %), and transforming
growth factor (TGF by 200 %; Figure 6). When skin inflammatory biomarkers were examined,
4AR 2 treatment significantly decreased the inflammatory genes: interleukin-1a (IL-1a by
1,010%), interleukin-1 receptor II (IL-1RII by 190 %), interleukin-6 (IL-6 by 3,520 %),
interleukin-8 (IL-8 by 380 %), cyclooxygenase 2 (COX 2 by 170 %) and tumor necrosis factor
receptor super family (TNFRSF by 140 %; Figure 7). These data along with the other significant
changes in the dermal biomarkers suggest a positive role of 4AR 2 in improving skin health.
Growth Factor Skin Biomarkers
900
800
672
700
600
500
400
264
300
200
190
200
100
0
HBEGF
IGFI
NGF
TGF
Figure 6. Topical application of 4AR 2 significantly stimulated gene expression of growth factor
skin biomarkers compared to DMSO controls. Heparin-binding epidermal growth factor
(HBEGF), insulin like growth factor I (IGF I), nerve growth factor (NGF) and transforming
growth factor (TGF); shown as the mean + SEM, p < 0.005.
11

Skin Inflammatory Biomarkers
0
-500
-140
-190
-170
-380
-1000
-1500
-2000
-2500
-3000
-3500
-4000
-4500
-1010
-3520
IL-6
IL-1a
IL-1RII
IL-8
COX-2
TNFRSF
Figure 7. Topical application of 4AR 2 significantly inhibited skin inflammatory markers
compared to DMSO controls. Interleukin-1a (IL-1a), interleukin-1 receptor II (IL-1RII),
interleukin-6 (IL-6), interleukin-8 (IL-8), cyclooxygenase 2 (COX 2) and tumor necrosis factor
receptor super family (TNFRSF); shown as the mean + SEM, p < 0.005.
While RV 1 percutaneous absorption7a and use for skin disorders has been reported,3c, 7b
this is the first study to reveal the topical and in vitro effects of a resveratrol analog to address
the important issues of increased stability and bio-availability. Specifically, we examined the
influence of 4AR 2 at a 1 % concentration on dermal components using gene analysis, conducted
on human equivalent skin barrier cultures. Since RV 1 results have been previously reported by
our laboratory,5 noted comparisons to the 4AR 2 results are described below for several
prominent skin biomarkers. For example, in general 4AR 2 significantly stimulated several
different types of collagen; whereas RV 1 yielded influence on fewer biomarkers in this
category.5 Additionally, 4AR 2 significantly stimulated elastin by 280 %, while RV 1 only
stimulated this biomarker to 180 % over controls.5
While 4AR 2 did not directly inhibit matrix metalloproteinases (MMPs), RV 1 has been
shown to significantly inhibit MMP1 and MMP9.5 Notably, 4AR 2 did significantly increase
SIRT 1 expression by over 3.3-fold which has been shown to inhibit MMPs,8a,b suggesting an
indirect pathway to protecting collagen and elastin.8c Notably, RV 1 only stimulated SIRT 1 to
1.8-fold as reported previously.5 To support this notion, 4AR 2 also significantly increased the
gene expression of tissue inhibitor of the matrix metalloproteinases TIMP 1 and TIMP 2 that
12

presumably resulted in significant increases in collagen and elastin levels, important for optimal
skin health especially with aging.8c Finally in this regard, SIRT 1 has been shown to be
expressed in human dermal fibroblasts that decreases cellular aging and provides protection
against UV-induced DNA fragmentation/apoptosis.8d,e
Furthermore, when the skin anti-oxidant biomarkers were examined, CAT, LOX and the
super oxidase dismutases (SOD 1 and SOD 2) along with the metallothioneins (MT1H and
MT2H) were significantly increased with 4AR 2. The present results were similar to that
reported5 by RV 1 suggesting enhanced skin benefits with protection from oxidant agents and
heavy metals due to their known metal-binding properties.9a,b
When the skin aging and growth factor biomarkers were quantified, 4AR 2 treatment
significantly enhanced gene expression of HBEGF, IGF 1, NGF, TGF, FBN 1, LAMB 1 and
PCNA, all which improve skin health and provide anti-aging influences.10a In this regard, RV 1
has been shown to induce TGF expression along with several other skin biomarkers examined in
this class similar to the present 4AR 2 results.5 Moreover, TGF signaling has been shown to
exhibit anti-inflammatory, anti-oxidant and anti-proliferative effects.10b This notion is supported
by the present results where 4AR 2 significantly inhibited the skin inflammatory biomarkers (IL-
1, IL-6, IL-8, COX-2 and TNFRSF), which has been reported to be a property of other plant
derived polyphenolics including RV 1.5,10c Additionally, while not investigated specifically in the
present study, RV analogs have been shown to be photo-protective of ultraviolet radiation,
lessening photo-aging.10d Collectively, the gene array results suggest potential for 4AR 2 in
topical applications to improve human skin health. Particularly when overall comparisons were
made, 4AR 2 is approximately 1.6-fold more potent compared to RV 1 for the various
biomarkers reported.5 This may not appear as a major increase in effectiveness for 4AR 2 over
RV 1. Yet, these findings gain considerable significance in that these skin fibroblasts with 4AR 2
show positive dermal biomarkers regulation within a wide range of health related genes.
Notably, the present study was not designed to address the issue of 4AR 2 stability and
bioavailability in human skin. However, based upon the known information about phase 2
metabolism (conjugation pathways) in human skin and the known comparison of gene array
results of human skin biomarkers between RV 1 and 4AR 2 this issue can be addressed.
First, it is known that phase 2 metabolism (conjugation) does occur for a variety of
compounds in human skin.11 However, the conjugation enzymatic activities in human skin are in
13

the pmol/mg skin/hr range. As noted by Manevski et al.11, “comparison of the skin and liver
organ clearances implies that the skin contributes only maginally to the total systemic phase II
enzyme-mediated metabolic elimination in humans.” Thus, it is reasonable to suggest that only a
minor portion of the 4AR 2 tested in the present study undergoes phase 2 metabolism in human
skin and that the 4AR 2 remains, mostly, intact and does not act as a prodrug of RV 1.
Second, when the previous gene array results5 of RV 1 are compared to the present
results, in general, 4AR 2 is approximately 1.6-fold more potent than RV 1 in the stimulation
and/or inhibition of human skin-related biomarkers. Finally, putting the two known concepts
together of the very low phase 2 metabolism that occurs in human skin and the comparison of the
gene array results for RV 1 vs. 4AR 2 suggests that 4AR 2 is not rapidly metabolized and has
good stability and bioavailability within human skin cells.
The convenient one-step synthesis of 4AR 2 provides a quick and cost-effective method
of generating RV analogs for various applications in cosmetics, OTC drugs, and
pharmaceuticals. These findings suggest that 4AR 2 has the potential to be used topically for the
treatment of skin aging by the enhancement of dermal components to optimize the expression of
gene and gene-product(s) to provide skin aging benefits. Specifically, 4AR 2 significantly
increased SIRT 1 and extracellular matrix proteins, along with the enhancement of anti-oxidants
and growth factors, while at the same time the expression of skin aging and inflammatory
biomarkers were significantly inhibited.
Acknowledgements
The authors express thanks to Dr. Anna Langerveld at Genemarkers LLC for assistance
with the gene analysis. This work was supported, in part, by an LS/TTO BYU 19-22160 funding
to EDL and a BYU Cancer Center Fellowship to MJA.
Supplementary Data
Experimental procedures, characterization, NMR spectra, and in vivo methods.
Supplementary data associated with this article can be found, in the online version, at
ScienceDirect.
14

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