(+)-Usnic acid enamines with remarkable cicatrizing properties

Article abstract of DOI:10.1016/j.bmc.2013.01.045

Wound healing is a significant concern in many pathologies (post-surgeries, burns, scars) and the search for new chemical entities is advisable. The lichen compound (+)-usnic acid (1) has found application in dermatological and cosmetic preparations, due to its bacteriostatic and antioxidant activities. The compound has also been shown to stimulate the wound closure of keratinocyte monolayers at subtoxic doses. Here we describe the design and synthesis of usnic acid enamines (compounds 2-11), obtained through nucleophilic attack of amino acids or decarboxyamino acids at the acyl carbonyl of the enolized 1,3 diketone. The wound repair properties of these derivatives were evaluated using in vitro and in vivo assays. Compounds 8 and 9 combine low cytotoxicity with high wound healing performance, suggesting their possible use in wound healing-promoting or antiage skin preparations.

Full text of DOI:10.1016/j.bmc.2013.01.045

Bioorganic & Medicinal Chemistry 21 (2013) 1834–1843
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
(+)-Usnic acid enamines with remarkable cicatrizing properties
Michela Bruno a,b, Beatrice Trucchi a,b, Bruno Burlando c, Elia Ranzato c, Simona Martinotti c,
Esra Küpeli Akkol d, Ipek Süntar d, Hikmet Keleßs e, Luisella Verotta a,b,
⇑
a Dipartimento di Chimica, Universita’ degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy
b Consorzio Interuniversitario Nazionale ‘‘Metodologie e Processi Innovativi di Sintesi’’, I-20133 Milano, Italy
c Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, Viale Teresa Michel 11, 15121 Alessandria, Italy
d Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, Etiler 06330, Ankara, Turkey
e Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Pathology, 03200 Afyonkarahisar, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Wound healing is a significant concern in many pathologies (post-surgeries, burns, scars) and the search
for new chemical entities is advisable. The lichen compound (+)-usnic acid (1) has found application in
dermatological and cosmetic preparations, due to its bacteriostatic and antioxidant activities. The com-
pound has also been shown to stimulate the wound closure of keratinocyte monolayers at subtoxic doses.
Here we describe the design and synthesis of usnic acid enamines (compounds 2–11), obtained through
nucleophilic attack of amino acids or decarboxyamino acids at the acyl carbonyl of the enolized 1,3 dike-
tone. The wound repair properties of these derivatives were evaluated using in vitro and in vivo assays.
Compounds 8 and 9 combine low cytotoxicity with high wound healing performance, suggesting their
possible use in wound healing-promoting or antiage skin preparations.
Received 24 November 2012
Revised 15 January 2013
Accepted 18 January 2013
Available online 4 February 2013
Keywords:
(+)-Usnic acid
Mannich bases
Scratch wound assay
In vivo wound healing assays
Wound healing therapy
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
We have recently described the potential of (+)-usnic acid as a
wound healing agent, due to its property to stimulate wound clo-
Impaired tissue repair is a significant concern in many condi-
tions (post-surgeries, burns, scars). Hence, of great interest is the
search for new chemical entities to be used as cicatrizing agents
(alone or in formulations with other drugs) to treat chronic
wounds, burns, topical otitis, hemorrhoids, vaginal lesions, and
mouth infections.
Wound healing is a complex process that can be divided into
three stages: inflammation, proliferation and remodeling. These
phases involve a variety of coordinated cellular activities, such as
migration to the wounded area, proliferation, and deposition and
remodelling of extracellular matrix, mainly the collagen lattice.1
Various materials for wound dressing, skin substitutes, and recom-
binant growth factors have been shown to enhance the healing
process, and some of them have been introduced into the clinical
setting with therapeutic efficacy.2 Many natural products are also
known to possess wound healing properties, based on both ane-
doctal and scientific evidence.3 Hence, there is a great interest to
find new chemical entities that could share antibiotic, anti-inflam-
matory and cell remodeling properties, and to characterize their
mechanism of action.
sure of HaCaT monolayers at subtoxic doses. The mechanism of ac-
tion was correlated to its ability to stimulate cell motility.4
Previous results indicated usnic acid sodium salt as a wound heal-
ing enhancer through the secretion of growth factor and accelerat-
ing the cell migration in a dose dependent manner.5 In vivo
experiments confirmed the property of usnic acid sodium salt to
accelerate skin wound healing, but the effect was not due to stim-
ulation of fibroblast proliferation.6 Recent publications report the
use of liposome-loaded usnic acid formulations for dermal burn
healing.7
The most investigated lichen metabolite, usnic acid, is univer-
sally known as an antibiotic (see Merck Index) a definition that
has found confirmation in the many biological data so far obtained.
(+)- and (ꢀ)-usnic acid are active against Gram positive bacteria on
susceptible and multidrug resistant strains.8 Some metal com-
plexes of usnic acid hydrazones have been found to be more active9
and all the results so far obtained confirm a bacteriostatic rather
than a bactericidal action.
Usnic acid is a potent antioxidant and has been largely used in
dermatological and cosmetic products as a preservative.10
(+)-Usnic acid is now commercially available, easily isolated
from Usnea or Ramalina species of lichens where it occurs up to
26%.11 As a pure compound it has been formulated into creams,
toothpaste, mouthwash, deodorants, antibiotic ointments and
⇑
Corresponding author.
E-mail address: luisella.verotta@unimi.it (L. Verotta).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.045

M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
1835
sunscreen products. Usnea barbata extracts, usnic acid, and its cop-
per salt have been formulated as antimicrobial preparations. In
most preparations, usnic acid is employed both as an active agent
and a preservative.
easy oxidation of the reagent and the most convenient conditions
result in a two stage process, by preparing the disulfur through
condensing cystamine (10) that was later reduced to the desired
thiol (8) (Fig. 2).
Data concerning usnic acid toxicity, mainly deal with the sys-
temic use of usnic acid containing preparations to promote weight
loss.12 Research on toxicity performed at the National Center for
Toxicological Research (NCTR), USA, attested that Usnea lichen
preparations containing equivalent concentrations of usnic acid
produced greater toxicity than pure usnic acid.
Contact dermatitis and allergenicity of usnic acid has been re-
ported as weak, mainly due to the (ꢀ)-enantiomer,13 probably cor-
related to its general toxicity.
The thiol (8) is very stable under usual preserving conditions, in
any case, the two compounds were both submitted to biological
testing. Enaminousnic (11) (Fig. 4) was also prepared as non acidic
derivative by refluxing usnic acid in ammonia solution.14c The
compounds so far obtained exhibits intrinsic wound healing prop-
erties with respect to the parent compound and, in most cases, an
overall increase in water solubility.
2.2. In vitro scratch wound healing
The wound healing ability of usnic acid derivatives has been
tested on an in vitro wound healing model consisting of monolay-
ers of HaCaT keratinocytes. These cells represent an in vitro model
of proliferating and migrating keratinocytes. The HaCaT cell line
mimics many properties of normal epidermal keratinocytes, is
not invasive and can differentiate under appropriate experimental
conditions.15,16 HaCaT have been previously used in wound healing
studies by our and other laboratories as an in vitro system of re-
epithelialization, a typical phase of the wound healing process.17
On the basis of IC50 values (Table 1), 4, 5 and 6 resulted signif-
icantly more toxic than 1, 3 had almost the same toxicity, and 2, 7,
8 and 9 were less toxic. These results indicate that most of the moi-
eties attached to the usnic acid molecule have significantly modi-
fied the chemical and biological properties of the original
compound. The derivatives found to be more toxic than 1 on HaCaT
cells, were also tested on A431 epidermoid carcinoma cells, used as
2. Results and discussion
2.1. Synthesis of usnic acid derivatives
(+)-Usnic acid (1) is a benzofurandione, phloroglucinol like, sec-
ondary metabolite from lichens, occurring in nature up to 26% in
species growing in China11 and now commercially available. The
most reactiveportion of the moleculeis thetriketone moietyof poly-
ketide origin, which is also mainly responsible for activity.14 But its
high lipophilicity due to the strong intramolecular hydrogen bon-
dings, probably also increases its toxicity, rendering the molecule
permeable to membranes. Thus (+)-usnic acid is a good candidate
to investigate factors involved in its biological behavior: it is easily
affordable, it has recognized and proved in vitro activities, but it
has limits in exploitation due to in vivo toxicity.12 Toxicity draw-
backs are generally overcome in cosmetic preparations through
the use of complexes with liposomes or other formulations.
a chemoresistant model cell line. The IC50 (lg/mL) of 4 and 5, 36
(32–41) and 29 (17–52), respectively, were higher or not different
from that of 1, 15 (11–21), whereas the IC50 of 6 was significantly
lower, 4.1 (2.4–7.1), thus confirming a stronger toxicity for this lat-
ter compound also on cancer cells and suggesting its possible use
as an anticancer drug.
Scratch wound analysis was performed on confluent monolay-
ers of HaCaT, by selecting 4, 7, 8 and 9, which resulted most active
in preliminary tests, and comparing them with 1. The analysis was
carried out as described in Ranzato et al.18 Compound 1 showed a
significant wound healing effect, confirming our previous find-
ings,4 while all the derivatives displayed stronger wound healing
properties (Fig. 3). The GABA–usnic acid derivative 9 induced the
highest wound closure rate, almost approaching the wounding po-
tential of the platelet lysate, a blood derivative used as reference
substance (see Section 4).
We have synthesized and tested some chemical derivatives ob-
tained by nucleophilic attack of a number of amines and amino
containing natural products at the acetyl carbonyl at 2-position
of the enolized 1,3 diketone, selected on the basis of their intrinsic
very low toxicity, commercially available at low cost. The choice
fell on amino acids or decarboxyamino acids, with a particular
emphasis to those that contain chemical groups involved in cell-
to-cell interaction (i.e., redox systems or adhesion mechanisms).
Usnic acid underwent addition of amino acids systems in neutral
or basic conditions (2–7, 9) (Fig. 1). Reaction with cysteamine oc-
curred at low yields and limited reproducibility perhaps due to
2.3. In vivo assessment of wound healing activity
For the evaluation of in vivo wound healing activity, the com-
pounds were mixed with a vehicle consisting of glycol stearate,
1,2 propylene glycol, liquid paraffin (3:6:1) in 1% concentration.
Linear incision by using tensiometer and circular excision wound
models were employed. Skin samples were also evaluated
histopathologically.
As shown in Table 2, topical application of the ointment pre-
pared with the compound 9 onto the incised wounds demon-
strated the best wound tensile strength by the highest value of
47.6% (p <0.01) on day 10. Moreover, 1, 7, and 8 were found gener-
ally highly effective (31.9%, 29.3% and 35.2%, respectively) in linear
incision wound model.
The contraction values of the progression healing of wounds on
circular excision wound model for vehicle, negative control, com-
pounds and reference drug treated groups were shown in Table 3.
Usnic acid (1) and compounds 9, 7 and 8, were found to have wound
healing potential, while the other groups showed no statistically
Figure 1. Reagents and conditions: (i) Amines or amino acid derivatives (1–
3 equiv), abs. EtOH or abs. EtOH/triethylamine or anhydrous CH2Cl2/MeOH or abs.
EtOH/pyridine, reflux, N2.

1836
M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
Figure 2. Reagents and conditions: (i) Cystamine dihydrochloride (0.5 equiv), abs. EtOH/pyridine, reflux, N2, 8 h, quantitative yield; (ii) triphenylphosphine (1.5 equiv),
acetone/H2O, rt, helium, 4 days, 92%.
Table 1
Table 2
In vitro toxicity of usnic acid and its derivatives to HaCaT keratinocytes, as assessed
by the NRU endpoint after 24-h exposures
Effect of the compounds on linear incision wound model
Material
Mean S.E.M.
Tensile strength (%)
Compound
IC50
(
l
g/mL)
IC05 (lg/mL)
Vehicle
Negative control
1
7
11.75 2.48
10.62 2.63
15.50 2.14
15.19 2.02
15.89 1.98
17.34 1.92
13.62 1.84
18.87 1.39
10.6
—
1
2
3
4
5
6
7
8
9
24 (18–32)
163 (108–244)
36 (22–57)
0.5 (0.2–1.3)
16 (8.0–30)
1.7 (0.5–6.5)
1.3 (0.5–3.5)
2.5 (1.6–4.0)
0.2 (0.04–1.1)
nd
31.9**
29.3*
35.2**
47.6**
15.91
60.6***
13 (9–18)
8
9
10
5.3 (4.7–6.3)
5.8 (3.5–9.5)
47 (39–58)
155 (116–207)
150 (140–161)
MadecassolÒ
43 (18–103)
67 (55–80)
S.E.M.: Standard error of mean. Percentage of tensile strength values: the vehicle
group was compared to negative control; compounds and the reference material
MadecassolÒ were compared to the vehicle group.
IC50
:
median effective concentration. IC05
:
toxicity threshold. 95% confidence
intervals are shown in parentheses.
Statistically-significant values in bold.
*
p <0.05.
**
p <0.01.
***
p <0.001.
e
300
d
Treated skin samples were also assessed for their hydroxypro-
line content, which gives an estimate of collagen concentration.
Collagen is the major component of extracellular tissue. It is com-
posed of the amino acid, hydroxyproline, which has been used as a
biochemical marker for tissue collagen.19 As shown in Table 4, high
hydroxyproline content was determined for the tissues treated
with the ointments of usnic acid, and compounds 7, 8, and 9.
Extracellular matrix (ECM) is composed of proteoglycans and
matrix proteins such as collagen and elastin. Collagen provides
supportive framework to the cell, elastin maintains the skin’s elas-
ticity and hyaluronic acid keeps the moisture. All three compo-
nents help wound healing.20 Therefore, inhibition of the enzymes
that break down these ECM components could improve wound
healing. In the present study, we compared the hyaluronidase
and collagenase enzyme inhibitory activities of usnic acid (1), 8,
9, 10, and 11 (Tables 5 and 6), which indicate a possible mecha-
nism of actions of the wound healing process.
c
c
c
b
1
200
100
0
a
ctrl
4
7
8
9
PL
Figure 3. Scratch wound closure rates of HaCaT confluent monolayers. Cells
cultured in 12-well plates were mechanically scratched and exposed to equimolar
doses (5
l
M) of usnic acid (1) (1.7
lg/mL), 4 (2.5 lg/mL), 7 (2.2 lg/mL), 8 (2.0 lg/
mL), and 9 (2.1
l
g/mL). One sample was exposed to 20% platelet lysate (PL) as
positive control. Wound closure measurements are expressed as the difference
between wound width at 0 and 24 h. Bars represent mean S.D. of two independent
experiments, each with n = 20. The mean of controls was set at 100%. Different
letters on bars indicate groups significantly different from each other according to
the Tukey’s test (p <0.01).
Considering the low toxic effect observed on keratinocytes and
the good wound healing activity, 8 and 9 seem to possess the most
interesting properties.21
We can now draw interesting information regarding the pecu-
liar molecular features that compounds should possess to show
wound healing properties. Compound 7 bears a free thiol group
which is a weak acid, 8 and 9 all bear an acid functionality, the
compounds lacking such portion (like enaminousnic 11) have no
activity. A limited action on matrix protein is here reported. Com-
pound 8 bears a free thiol group which is a light acid and also has
recognized antioxidant properties. It shows wound healing proper-
ties, while the disulfur 10 has no activity. Thus acidic groups seem
to be significant for the maintaining of activity.
Figure 4. Reagents and conditions: (i) Ammonium hydroxide, abs. EtOH, reflux, N2,
2 h, 77%.
We then synthesized the new derivatives 12 and 13 (Fig. 5) by
condensing two known wound healing agents (c-aminobutyric
significant wound healing activity. The wound contractions were
52.42% (p <0.01) and 82.95% (p <0.001) for 9, 30.36% (p <0.05) and
52.19% (p <0.01) for 1, 37.55% (p <0.05) and 40.83% (p <0.01) for 7
and 45.23% (p <0.01) and 64.08% (p <0.01) for 8 on day 8 and 10,
respectively. These values were compared to reference drug Made-
cassolÒ [72.24% (p <0.01)—100% (p <0.001)].
acid and N-acetylcysteine) to the most active compound in our ser-
ies (9). Acidic groups are still present and the labile thioester bond-
ing in 12 should be easily hydrolyzed on skin and release two
therapeutic agents.21 Future bioassays will confirm the hypothesis.
These data will be reported elsewhere.

M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
1837
Table 3
Effect of the compounds on circular excision wound model
Material
Wound area S.E.M. (contraction %)
0 day
2 days
4 days
6 days
8 days
10 days
Vehicle
19.63 2.13
18.02 2.61
—
16.02 2.68
—
13.32 1.90
(3.41)
8.07 1.11
(2.18)
3.87 1.04
(6.29)
Negative control
1
19.55 2.10
19.59 2.01
17.99 2.71
17.04 2.16
(5.44)
15.86 2.63
14.10 2.12
(11.99)
13.79 1.76
10.25 1.84
(23.05)
8.25 1.28
5.62 1.75
(30.36)⁄
4.13 1.43
1.85 0.63
(52.19)⁄⁄
7
19.33 2.15
19.46 2.08
19.70 2.25
19.25 2.40
19.49 2.11
17.74 2.03
(1.55)
16.41 1.99
(8.93)
16.03 1.87
(11.04)
17.95 2.36
(0.39)
14.52 2.01
(9.36)
13.74 1.67
(14.23)
12.81 1.36
(20.04)
15.92 1.89
(0.62)
11.42 1.38
(14.26)
10.25 1.54
(23.05)
5.04 1.61
2.29 0.58
(37.55)⁄
(40.83)⁄⁄
8
4.42 1.10
(45.23)⁄⁄
3.84 1.12
(52.42)⁄⁄
6.48 1.23
(19.70)
1.39 0.61
(64.08)⁄⁄
0.66 0.22
(82.95)⁄⁄⁄
2.96 0.74
(23.51)
9
9.01 1.51
(32.36)⁄
10
12.48 1.77
(7.51)
MadecassolÒ
15.21 1.74
(15.59)
10.45 1.17
6.47 1.29
2.24 0.52
0.00 0.00
(34.77)⁄
(51.43)⁄⁄
(72.24)⁄⁄
(100.00)⁄⁄⁄
S.E.M.: Standard error of the mean. Percentage of contraction values: the vehicle group was compared to negative control; compounds and reference material were compared
to the vehicle group.
Statistically-significant values in bold.
*
p <0.05.
p <0.01.
p <0.001.
**
***
Table 4
number of wound healing products are currently present on the
market, deriving from natural, chemical, and biomedical sources.
However, in most cases these products are complex mixtures that
are used on an empirical basis, while the mechanisms of action
and active principles have not been clearly identified. For instance,
most effective results in the healing of chronic or severe wounds
are currently obtained in clinical settings by using platelet deriva-
tives containing various growth factors and a number of low molec-
ular-weight compounds.22
In contrast, in this study we have been able to attribute signif-
icant wound healing properties to single usnic acid derivatives. The
results of in vitro and in vivo assays were quite consistent, showing
lowest cytotoxicity combined to highest healing performance for 8
and 9, which in most cases were preferable to their precursor usnic
acid. These data suggest the possible use of these compounds in
wound healing-promoting or antiage skin preparations.
Effect of topical ointments for 7 days on hydroxyproline content
Material
Hydroxyproline (mg/g) S.E.M.
Vehicle
Negative control
1
7
17.1 2.08
16.9 2.44
36.1 1.89*
30.8 1.92*
39.8 1.84*
43.0 1.71**
20.4 1.99
49.7 1.10***
8
9
10
MadecassolÒ
S.E.M.: Standard error of the mean.
Statistically-significant values in bold.
*
p <0.05.
p <0.01.
**
***
p <0.001 significant from the control.
Table 5
4. Experimental
4.1. Chemistry
Hyaluronidase enzyme inhibitory activity
Material
Concentration (
l
g/ml)
Inhibition (%) S.E.M.
1
50
100
50
100
50
100
50
100
50
14.45 1.33
33.14 0.99*
10.19 1.03
15.64 1.11
25.78 1.05
39.42 0.78*
10.03 1.21
18.34 1.18
35.32 0.47**
41.85 0.34**
76.31 0.95***
4.1.1. General
8
All reagents were purchased from Sigma–Aldrich and Fluka and
used without further purification. Yields are given after purifica-
tion, unless differently stated. When reactions were performed un-
der anhydrous conditions, the mixtures were maintained under
nitrogen. All melting points were taken on a Büchi apparatus and
are uncorrected. NMR spectra were recorded on a Bruker Avance
400 spectrometer (400 MHz for 1H NMR, 100 MHz for 13C). 2D-
NMR experiments (1H,1H COSY, 1H,13C HSQC, and 1H,13C HMBC
experiments) were recorded using sequences available in Bruker
software. Data for 1H NMR are reported as follows: chemical shift
(d ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet), integration and coupling constant (Hz), whereas
13C NMR analyses are reported in terms of chemical shift. Elemen-
tal analyses were performed with a Perkin–Elmer series II CHNS/O
Analyzer 2400. Mass spectra were recorded on a LCQ Advantage
Thermo Fisher Mass Spectrometer equipped with a ESI source
(spray voltage: 4.5 kV, capillary temperature: 275.90 °C, sheat gas
flow rate: 15 arbitrary units). LC–MS analysis was performed using
the following equipment: HPLC Agilent 1100 with quaternary
9
10
11
100
100
Tannic acid
*
p <0.05.
p <0.01.
p <0.001.
**
***
3. Conclusion
Active principles to be used on skin in dermatological or cosmetic
preparations for regenerative and antiaging purposes, should ideally
possess the lowest possible cytotoxicity on skin cells, the highest
antibacterial activity on infectious or opportunistic pathogens, and
the strongest skin regeneration or wound healing properties. A wide

1838
M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
Table 6
Collagenase and elastase enzyme inhibitory activities
Material
Concd (lg/ml)
Collagenase inhibition (%) S.E.M.
Elastase inhibition (%) S.E.M.
1
50
100
50
100
50
100
50
100
50
27.14 0.99
51.22 0.56***
19.46 1.27
27.32 1.10
21.42 1.13
49.24 0.68**
20.82 1.23
29.33 1.20
20.68 1.43
33.10 0.85*
41.16 0.81**
22.08 1.17
36.42 1.19
15.28 1.02
20.44 1.18
25.12 1.52
30.32 1.21
21.29 1.02
30.15 1.24
13.26 1.41
15.32 1.08
88.17 1.18***
8
9
10
11
EGCG
100
100
S.E.M.: Standard error of the mean; EGCG: epigallocatechin gallate.
*
p <0.05.
p <0.01.
p <0.001.
**
***
Figure 5. Reagents and conditions: (i) NMM (1.2 equiv), DMTMM (1 equiv), THF, rt, N2, 30 min, then N-acetyl-
L
-cysteine (1 equiv), THF, rt, N2, 3 days, 4%; (ii) NMM
(1.2 equiv), DMTMM (2 equiv), THF, rt, N2, 45 min, then 4-aminobutyric acid (1 equiv), THF, rt, N2, 3 h and 30 min, 25%.
pump, diode array detector, autosampler, thermostated column
holder, Bruker ion-trap Esquire 3000+ Mass Spectrometer with
ESI; method: column Supelco Ascentis-Express 50 ꢁ 4.6 mm,
CH3-18), 3.13 (m, 2H, CH2-50), 4.26 (m, 1H, CH2-20), 5.69 (s, 1H, CH-
4), 7.62 (br s, 3H), 8.89 (br s, 1H,) 12.43 (br s, 1H, OH-10), 13.28 (br
s, 1H, OH-8), 13.35 (br s, 1H, NH). 13C NMR 400 MHz (DMSO-d6) d
7.81 (C-16), 19.36 (C-15), 25.14 (C-40), 30.45 (C-30), 31.39 (C-18),
32.48 (C-13), 56.68 (C-12), 56.68 (C-50), 58.87 (C-20), 101.19
(C-7), 101.97 (C-2), 102.90 (C-4), 105.56 (C-11), 106.81 (C-9),
156.09 (C-6), 157.68 (C-60), 158.08 (C-10), 163.08 (C-8), 172.54
(C-5), 172.77 (C-10), 173.03 (C-14), 188.2 (C-3), 197.38 (C-1),
200.95 (C-17). HRMS (ESI), positive, m/z found: 501.1 [M+H]+,
Calcd for C24H28N4O8: 500.19. Anal. found: C 55.18%, H 5.72%,
N 10.84%, Calcd for C24H28N4O8: C 55.59%, H 5.83%, N 10.81%.
2.7 lm; phase A: Milli-Q water containing 0.05% (v/v) TFA; phase
B: acetonitrile (LC–MS grade) containing 0.05% TFA; gradient: from
40% B to 100% B in 6 min, washing at 100% B for 1 min, equilibra-
tion at 5% B in the next; flow: 1 mL/min, temperature: 40 °C; UV
detection at 220 and 254 nm with reference at 500 nm (40 nm
bandwith); ESI+ detection in the 50–2000 m/z range with alternat-
ing MS/MS. Chromatographic separations were performed on a
silica gel column by gravity chromatography (Kieselgel 40,
0.063–0.200 mm; Merck) or flash chromatography (Kieselgel 40,
0.040–0.063 mm; Merck). Reverse phase chromatography was per-
formed on a Biotage SP1 instrument (pressure limit: 7 bar, range of
flow rate: 1–100 mL/min) equipped with a UV detector (220 and
254 nm) and a Windows XP operating system control. The reac-
tions were monitored by analytical TLC using silica gel 60 F254
precoated glass plates (0.25 mm thickness) and C18 reverse phase
F254 precoated glass plates (0.25 mm thickness). Visualization was
accomplished by irradiation with a UV lamp and staining with
molibdic reagent ((NH4)6MoO4, Ce(SO4)2, H2SO4, H2O) or vanillin
solution. Optical rotations were recorded on a JASCO P-1030 Polar-
imeter, using a 1 dm cell at the sodium D line (k = 589 nm).
Concentrations are expressed in g/100 mL of solvent.
4.1.2.2. Triethylammonium (S)-2-(((E)-1-((R)-6-acetyl-7,9-dihy-
droxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo[b,d]furan-
2(3H)-ylidene)ethyl)amino)-3-(1H-imidazol-4-yl)propanoate
(3).
L-Histidine (205 mg, 0.85 mmol) was added to a suspen-
sion of (+)-usnic acid (296 mg, 0.85 mmol), triethylamine (237
ll,
1.7 mmol) in H2O (3 mL) and absolute EtOH (15 mL) and refluxed
under nitrogen for 4 h. The mixture was stirred at room tempera-
ture for further 15 h. The solid obtained by concentration in vac-
uum was crystallized from diisopropyl ether/EtOH 9:1 giving
322 mg (78%) of a yellow solid. Mp: 209–211 °C. ½a D20
ꢂ
+185 (c
d ppm 1.16 (t,
0.05, MeOH). 1H NMR 400 MHz (DMSO-d6)
J = 8.2 Hz, 9H, CH3-triethylamine), 1.61 (s, 3H, CH3-13), 1.94 (s,
3H, CH3-16), 2.50 (s, 3H, CH3-15), 2.61 (s, 3H, CH3-18), 2.95–3.06
(m, 7H, CH2-triethylamine, CH2-30), 3.18 (m, 1H, CH2-30), 4.60 (m,
1H, CH-20), 5.80 (s, 1H, CH-4), 6.83 (s, 1H, CH-50), 7.69 (d,
J = 0.8 Hz, 1H, CH-60), 12.39 (br s, 1H, OH), 13.25 (br s, 1H, OH)
13.38 (br s, 1H, NH). 13C NMR 100 MHz (DMSO-d6) d ppm 8.0 (C-
16), 8.9 (CH3-triethylamine), 18.9 (C-15), 31.3 (C-30), 31.4 (C-18)
32.1 (C-13), 45.6 (CH2-triethylamine), 56.5 (C-12), 58.8 (C-20),
101.2 (C-7), 102.0 (C-2), 102.9 (C-4), 105.6 (C-11), 106.6 (C-9),
118.0 (C-50), 132.8 (C-40), 135.3 (C-60), 156.2 (C-6), 158.2 (C-10),
162.9 (C-8), 171.8 (C-10), 173.0 (C-5), 173.9 (C-14), 188.9 (C-3),
197.9 (C-1), 201.19 (C-17). MS (ESI), positive, m/z found: 504.1
[M+Na]+, Calcd for: C24H23N3O8 481.15. For the bioassays the com-
pound was acidified and extracted with CH2Cl2. Anal. found: C
4.1.2. Synthesis
4.1.2.1. (S)-2-(((E)-1-((R)-6-Acetyl-7,9-dihydroxy-8,9b-dimethyl-
1,3-dioxo-1,9b-dihydrodibenzo[b,d]furan-2(3H)-yli-
dene)ethyl)amino)-5-guanidinopentanoic acid (2).
202 mg
(1.16 mmol) of -arginine were added to a suspension of (+)-usnic
L
acid (400 mg, 1.16 mmol) in absolute EtOH (15 ml) and refluxed
under nitrogen for 5 h, then stirred at room temperature for further
15 h. The solid which was obtained by concentration in vacuo was
crystallized from diisopropylether/EtOH 9:1 giving 0.516 g (89%) of
a pale yellow solid. Mp: 232–234 °C. ½a D20
ꢂ
+262 (c 0.05, MeOH). 1
H
NMR 400 MHz (DMSO-d6) d 1.58 (m, 5H, CH3-13, CH2-40), 1.81 (m,
2H, CH2-30), 1.92 (s, 3H, CH3-16), 2.57 (s, 3H, CH3-15), 2.57 (s, 3H,

M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
1839
58.73%, H 6.41%, N 8.80%, Calcd for: C24H23N3O8 ꢁ 2H2O C 58.24%,
(C-6), 158.2 (C-10), 162.9 (C-8), 169.6 (C-20), 172.9 (C-5), 173.6
(C-14), 188.4 (C-3), 197.6 (C-1), 201.3 (C-17). MS (ESI), negative,
m/z found: 400.10 [MꢀH]ꢀ, Calcd for C20H19NO8: 401.11. Anal.
H 6.84%, N 9.06%.
4.1.2.3. (S)-2-(((E)-1-((R)-6-Acetyl-7,9-dihydroxy-8,9b-dimethyl-
1,3-dioxo-1,9b-dihydrodibenzo[b,d]furan-2(3H)-ylidene)ethyl)
found:
C
57.75%,
H
6.75%,
N
5.41%, Calcd for
C
20H19NO8 ꢁ
Et3N ꢁ H2O: C 59.99%, H 6.97%, N 5.38%.
amino)-3-(4-hydroxyphenyl)propanoic acid (4).
L-Tyrosine
(157 mg, 0.87 mmol) was added to a suspension of (+)-usnic acid
(300 mg, 0.87 mmol) in absolute EtOH (10 mL) and H2O (3 mL)
and refluxed under nitrogen for 6 h, then stirred at room tempera-
ture for further 15 h. The solid which was obtained by concentra-
tion in vacuum was crystallized from diisopropyl ether/EtOH 9:1
4.1.2.6. (R,E)-2-((1-(6-Acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-
dioxo-1,9b-dihydrodibenzo[b,d]furan-2(3H)-yli-
dene)ethyl)amino)ethanesulfonic acid (7).
Procedure A:
Taurine (751 mg, 6 mmol) was dissolved in aqueous EtOH
(20 mL, 1:1). KOH was added to adjust pH ꢃ9.5, and the mixture
was refluxed for 1 h on water bath. Then a suspension of (+)-usnic
acid (689 mg, 2 mmol) in absolute EtOH (5 mL) was added in por-
tion over a period of 30 min, and the mixture was refluxed for 10 h.
Solvent was evaporated until dryness, the crude product was di-
luted with CH2Cl2 and washed with HCl 1 N until pH 4. The aque-
ous layers were collected, concentrated under reduced pressure
and washed with CH3OH/H2O 9:1 (20 mL) to remove the excess
of taurine. The solution was evaporated until dryness and crystal-
lized from diisopropylether/EtOH 9:1 to give 687 mg (1.52 mmol,
giving 406 mg of a yellow solid (92%). Mp: 183–185 °C. ½a D20
ꢂ
+95
(c 0.05, MeOH). 1H NMR 400 MHz (DMSO-d6) d ppm 1.63 (s, 3H,
CH3-13), 1.96 (s, 3H, CH3-16), 2.36 (s, 3H, CH3-15), 2.62 (s, 3H,
CH3-18), 3.01 (dd, J = 7.2 Hz, 14 Hz, 1H, CH2-30), 3.15 (dd,
J = 4.8 Hz, 14 Hz, 1H, CH2-30), 4.95 (m, 1H, CH-20), 5.85 (s, 1H, CH-
4), 6.65 (d, J = 8.4 Hz, 2H, CH-50, CH-90), 6.97 (d, J = 8.4 Hz, 2H,
CH-60, CH-80), 12.11 (s, 1H, OH), 13.28 (br s, 2H, OH, NH). 13C
NMR 100 MHz (DMSO-d6) d ppm 7.7 (C-16), 18.9 (C-15), 31.4 (C-
18), 32.0 (C-13), 37.9 (C-30), 57.1 (C-12), 58.4 (C-20), 101.2 (C-7),
102.4 (C-2), 103.0 (C-4), 105.6 (C-11), 107.2 (C-9), 116.0 (C-60, C-
80), 126.1 (C-40), 131.3 (C-50, C-90), 157.2 (C-70), 157.3(C-6),
158.60 (C-10), 163.0 (C-8), 171.5 (C-10), 173.4 (C-5), 174.9 (C-14),
189.2 (C-3), 198.0 (C-1), 201.4 (C-17). LC–MS (ESI), negative, m/z
found: 506.14 [MꢀH]ꢀ, Calcd for C27H25NO9: 507.15.
76%) of the desired product as brown solid. ½a D20
ꢂ
+180 (c 0.11,
MeOH). 1H NMR 400 MHz (DMSO-d6) d 1.65 (s, 3H, CH3-13), 1.98
(s, 3H, CH3-16), 2.60 (s, 3H, CH3-15), 2.65 (s, 3H, CH3-18), 2.78 (t,
2H, J = 6.4 Hz, CH2-20), 3.77–3.81 (m, 2H, CH2-10), 5.86 (s, 1H, H-
4), 7.72 (br s, 1H, SO3H), 12.43 (s, 1H, OH-10), 12.90-12.93 (m,
1H, NH), 13.42 (s, 1H, OH-8). 13C NMR 400 MHz (DMSO-d6) d
8.64 (C-16), 19.26 (C-15), 32.17 (C-18), 32.83 (C-13), 42.13
(CH2-10), 50.63 (CH2-20), 57.26 (C-12), 102.01 (C-7), 102.86 (C-2),
103.63 (C-4), 106.40 (C-11), 107.39 (C-9), 156.95 (C-6), 158.94
(C-10), 163.64 (C-8), 173.83 (C-5), 175.63 (C-14), 189.42 (C-3),
198.07 (C-1), 202.10 (C-17).
4.1.2.4. (R,E)-2-(1-((2-(1H-Indol-3-yl)ethyl)amino)ethylidene)-
6-acetyl-7,9-dihydroxy-8,9b-dimethyldibenzo[b,d]furan-
1,3(2H,9bH)-dione (5).
0.139 g (0.87 mmol) of tryptamine
were added to a suspension of (+)-usnic (0300 g, 0.87 mmol) in
absolute EtOH (10 mL) and refluxed under nitrogen for 5 h, then
stirred at room temperature for further 16 h. The solid which
was obtained by concentration in vacuo was crystallized from
diisopropylether/EtOH 9:1 giving 0.393 (93%) of a yellow solid.
Mp: 218–220 °C. 1H NMR 400 MHz (CDCl3) d ppm 1.69 (s, 3H,
CH3-13), 2.12 (s, 3H, CH3-16), 2.55 (s, 3H, CH3-15), 2.69 (s, 3H,
CH3-18), 3.22 (m, 2H, CH2-100), 3.82 (m, 2H, CH2-110), 5.78 (s, 1H,
CH-4), 7.17–7.24 (m, 3H, CH-50, CH-60, CH-20), 7.41 (d, J = 8.0 Hz,
1H, CH-70), 7.60 (d, J = 7.8 Hz, 1H, CH-40), 8.38 (s, 1H, NH-Ar),
11.98 (br s, 1H, OH), 13.36 (br s, 2H, NH, OH). 13C NMR 400 MHz
(CDCl3) d ppm 7.4 (C-16), 18.5 (C-15), 25.1 (C-100), 31.1 (C-18),
32.1 (C-13), 44.46 (C-110), 101.26 (C-7), 102.4 (C-4), 105.16 (C-2),
107.94 (C-11), 110.7 (C-9), 110.9 (C-30), 111.6 (C-70), 118.1 (C-60),
119.7 (C-40), 122.4 (C-50), 123.0 (C-20), 126.7 (C-30a), 136.4
(C-70a), 155.7 (C-6), 158.5 (C-10), 163.7 (C-8), 174.9 (C-5), 175.0
(C-14), 188.8 (C-3), 198.2 (C-1), 200.7 (C-17). HRMS (ESI), negative,
m/z found: 485.17 [MꢀH]ꢀ, Calcd for C28H26N2O6: 486.18.
Procedure B (triethylammonium salt): To a solution of usnic
acid (400 mg, 1.16 mmol) in 10 mL of EtOH and 6 mL of water, tau-
rin (145 mg, 1.16 mmol) and 2 mL of triethylamine were added at
room temperature. After stirring at 75 °C for 4 h and at room tem-
perature overnight, the reaction mixture was concentrated under
reduced pressure. The crude product was crystallized from diiso-
propylether/EtOH 9:1 to give 551 mg (1.02 mmol, 88%) of yellow
solid (88%).
1H NMR 400 MHz (DMSO-d6) d 1.17 (t, J = 7.3 Hz, 9H, CH3-tri-
ethylamine), 1.63 (s, 3H, CH3-13), 1.96 (s, 3H, CH3-16), 2.60 (s,
3H, CH3-15), 2.62 (s, 3H, CH3-18), 2.79 (t, J = 6.4 Hz, 2H, CH2-20),
3.08 (q, J = 7.3 Hz, 6H, CH2-triethylamine), 3.78 (m, 2H, CH2-10),
5.82 (s, 1H, CH-4), 12.37 (s, 1H, OH-10), 12.92 (t, J = 5.3 Hz, 1H,
NH), 13.38 (s, 1H, OH-8). 13C NMR 400 MHz (DMSO-d6) d 8.0
(C-16), 9.15, 18.5 (C-15), 31.5 (C-18), 32.1 (C-13), 40.7 (CH2),
46.3 (CH2), 50.0 (CH2), 56.6 (C-12), 101.3 (C-7), 102.2 (C-2), 102.9
(C-4), 105.7 (C-11), 106.7 (C-9), 156.4 (C-6), 158.3 (C-10), 162.3
(C-8), 173.3 (C-5), 175.1 (C-14), 188.7 (C-3), 197.3 (C-1), 201.4
(C-17). HRMS (ESI), negative, m/z found: 450.08568 [MꢀH]ꢀ, Calcd
for C20H19NO9S: 450.08643.
4.1.2.5. Triethylammonium (R,E)-2-((1-(6-acetyl-7,9-dihydroxy-
8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo[b,d]furan-2(3H)-
ylidene)ethyl)amino)acetate
1.16 mmol) was added to a suspension of (+)-usnic acid (400 mg,
1.16 mmol), triethylamine (324 l, 2.32 mmol) in H2O (3 mL) and
(6).
L
-Glycine
(145 mg,
l
4.1.2.7.
ethyl)amino)ethylidene)-8,9b-dimethyldibenzo[b,d]furan-
1,3(2H,9bH)-dione (8). Procedure A: To a suspension of (+)-
(R,E)-6-Acetyl-7,9-dihydroxy-2-(1-((2-mercapto-
absolute EtOH (15 mL) and refluxed under nitrogen for 4 h. The
mixture was stirred at room temperature for further 15 h. The solid
obtained by concentration in vacuum was crystallized from diiso-
propyl ether/EtOH 9:1 giving 375 mg (78%) of a yellow solid.23 Mp:
usnic acid (1 g, 2.90 mmol) in 6 mL of anhydrous CH2Cl2 and
30 mL of anhydrous MeOH, a solution of cysteamine (224 mg,
2.90 mmol) was added under nitrogen. The reaction mixture was
refluxed at 65 °C for 4 h, then at room temperature overnight
and again refluxed at 65 °C for 2 h. The solvent was removed until
dryness and the solid was purified by flash-chromatography (tolu-
ene/EtOAc 8:2) to obtain the desired product as yellow solid
(511 mg, 1.27 mmol, 44%). Mp: 142–143 °C. Procedure B: To a solu-
tion of 10 (2.680 g, 3.33 mmol) in 120 mL of acetone, triphenyl-
phosphine (1.310 g, 4.99 mmol) and 18 mL of H2O were added
210–212 °C. ½a 2D0
ꢂ
+207 (c 0.04, MeOH). 1H NMR 400 MHz (DMSO-
d6) d ppm 1.16 (t, J = 7.2 Hz, 9H, CH3-triethylamine), 1.62 (s, 3H,
CH3-13), 1.94 (s, 3H, CH3-16), 2.54 (s, 3H, CH3-15), 2.61 (s, 3H,
CH3-18), 3.01 (q, J = 7.2 Hz, 6H, CH2-triethylamine), 4.06 (m, 2H,
CH-10), 5.81 (s, 1H, CH-4), 12.43 (br s, 1H, OH), 12.94 (s, 1H, OH),
13.38 (br s, 1H, NH). 13C NMR 100 MHz (DMSO-d6) d ppm 7.9
(C-16), 8.9 (CH3-triethylamine), 19.7 (C-15), 31.4 (C-18), 32.23
(C-13), 45.49 (CH2-triethylamine), 47.9 (C-10), 56.5 (C-12), 101.2
(C-7), 102.1 (C-2), 103.0 (C-4), 105.7 (C-11), 106.7 (C-9), 156.3

1840
M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
under helium and the reaction mixture was stirred at room
temperature for 4 days. Solvent was evaporated until dryness,
the yellow solid was dissolved in CH2Cl2, the organic layer was
washed with brine, dried over Na2SO4, filtered and evaporated to
dryness. The crude product was purified by flash chromatography
(toluene/EtOAc 100:0 to 95:5) to give 1.233 g (3.05 mmol, 92%) of
201.30 (C-17). LC–MS (ESI), positive, m/z found: 805.2 [M+H]+,
Calcd for C40H40N2O12S2: 804.2.
4.1.2.10. (R,E)-6-Acetyl-2-(1-aminoethylidene)-7,9-dihydroxy-
8,9b-dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione
(11).
To a suspension of (+)-usnic acid (300 mg, 0.87 mmol) in
yellow solid. ½a D20
ꢂ
+318 (c 0.11, MeOH). 1H NMR 400 MHz (CDCl3) d
3 mL of absolute EtOH, 0.4 mL of concentrated ammonium hydrox-
ide was added under nitrogen and the reaction mixture was stirred
at 80 °C for 2 h. After cooling in an ice bath, the yellow solution was
concentrated to approximately one third the original volume, acid-
ified with 1 N hydrochloric acid and extracted twice with EtOAc.
The combined extracts were washed with H2O and brine solution,
dried over anhydrous Na2SO4 and evaporated under reduced pres-
sure. The residue was crystallized from CHCl3 to produce pale yel-
low plates of the desired product (230 mg, 0.67 mmol, 77%). Mp
ppm 1.63 (t, J = 8.6 Hz, 1H, SH), 1.71 (s, 3H, CH3-13), 2.10 (s, 3H,
CH3-16), 2.65 (s, 3H, CH3-15), 2.68 (s, 3H, CH3-18), 2.83-2.88 (m,
2H, CH2-20), 3.69–3.73 (m, 2H, CH2-10), 5.80 (s, 1H, CH-4), 11.91
(br s, 1H, OH-10), 13.36 (s, 1H, OH-8), 13.74 (br s, 1H, NH). Adding
D2O, the signal at 2.83–2.88 ppm became a triplet (J = 6.4 Hz) and
the triplet at 1.63 disappeared. 13C NMR 100 MHz (CDCl3) d ppm
8.16 (C-16), 19.13 (C-15), 24.46 (C-20), 31.95 (C-18), 32.63 (C-13),
47.38 (C-10), 57.95 (C-12), 102.04 (C-2 + C-7), 103.00 (C-4),
105.67 (C-11), 108.69 (C-9), 156.52 (C-6), 158.88 (C-10), 164.17
(C-8), 174.97 (C-5), 175.65 (C-14), 191.54 (C-3), 199.14 (C-1),
201.33 (C-17). LC–MS (ESI), positive, m/z found: 403.9 [M+H]+,
425.8 [M+Na]+, Calcd for C20H21NO6S: 403.1.
249 °C. ½a 2D0
ꢂ
+378 (c 0.08, CHCl3). 1H NMR 400 MHz (DMSO-d6) d
ppm 1.64 (s, 3H, CH3-13), 1.98 (s, 3H, CH3-16), 2.53 (s, 3H, CH3-
15), 2.65 (s, 3H, CH3-18), 5.87 (s,1H, CH-4), 9.83 (br s, 1H, NH,
D2O exchangeable), 11.54 (br s, 1H, NH, D2O exchangeable),
12.32 (s, 1H, OH-10, D2O exchangeable), 13.42 (s, 1H, OH-8, D2O
exchangeable). 13C NMR 100 MHz (DMSO-d6) d ppm 8.60 (C-16),
25.63 (C-15), 32.11 (C-18), 32.79 (C-13), 57.17 (C-12), 101.95 (C-
7), 102.36 (C-2), 103.64 (C-4), 106.21 (C-11), 107.40 (C-9), 156.77
(C-6), 158.77 (C-10), 163.62 (C-8), 174.07 (C-5), 176.94 (C-14),
189.59 (C-3), 198.69 (C-1), 201.94 (C-17). MS (EI), positive, m/z
found: 343 [M+ꢀ], Calcd for C18H17NO6: 343.
4.1.2.8. (R,E)-4-((1-(6-Acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-
dioxo-1,9b-dihydrodibenzo[b,d]furan-2(3H)-yli-
dene)ethyl)amino)butanoic acid (9).
To a solution of (+)-us-
nic acid (1 g, 2.9 mol) in absolute EtOH(30 mL) and 2 mL of
triethylamine, 4-aminobutyric acid (300 mg, 2.9 mol) was added
at room temperature. After stirring at 75 °C for 5 h, the mixture
was concentrated under reduced pressure, diluted with CH2Cl2
and washed with 0.1 N HCl. The organic layer was dried, evaporated
to dryness and crystallized with diisopropyl ether/EtOH 9:1 to give
the desired product (0.914 g, 73%) as a yellow solid. Mp: 202–
4.1.2.11. (R)-2-Acetamido-3-((4-(((E)-1-((R)-6-acetyl-7,9-dihy-
droxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo[b,d]furan-
2(3H)-ylidene)ethyl)amino)butanoyl)thio)propanoic
(12). In an oven dried round bottomed flask, 8 (200 mg,
0.47 mmol) was dissolved in 2.5 mL of anhydrous THF (previously de-
acid
205 °C. ½a 2D0
ꢂ
+292 (c 0.12, MeOH). 1H NMR 400 MHz (DMSO-d6) d
ppm 1.66 (s, 3H, CH3-13), 1.86 (m, 2H, CH2-20), 1.98 (s, 3H, CH3-
16), 2.36 (t, 2H, J = 7.3 Hz, CH2-30), 2.59 (s, 3H, CH3-15), 2.65 (s, 3H,
CH3-18), 3.59 (m, 2H, CH-10), 5.89 (s, 1H, CH-4), 12.26 (s, 1H, COOH),
gassed by sparging helium). 4-Methylmorpholine (62
ll, 0.56 mmol)
and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholine
(130 mg,
0.47 mmol) were added and the reaction mixture was stirred at rt for
30 min. N-Acetyl- -cysteine (77 mg, 0.47 mmol) was added and the
reaction mixture was stirred at rt for 7 h, then overnight. Starting mate-
rial was still visible, so 4-methylmorpholine (26 l, 0.235 mmol) and
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholine (65 mg, 0.235 mmol)
were added and, after 30 min also N-acetyl- -cysteine (38 mg,
12.34 (s, 1H, OH), 13.05 (t, 1H, J = 5.2 Hz, NH), 13.42 (s, 1H, OH). 13
C
L
NMR 100 MHz (DMSO-d6) d ppm 8.0 (C-16), 18.6 (C-15), 24.4 (C-20),
31.1 (C-30), 31.5 (C-18), 32.2 (C-13), 43.3 (C-10), 56.8 (C-12), 101.4 (C-
7), 102.1 (C-2), 102.8 (C-4), 105.6 (C-11), 106.7 (C-9), 156.2 (C-6),
158.2 (C-10), 162.9 (C-8), 173.5 (C-5), 174.2 (COOH), 175.5 (C-14),
189.4 (C-3), 197.7 (C-1), 201.4 (C-17). MS (ESI), negative, m/z found:
428.0 [MꢀH]ꢀ, Calcd for C22H23NO8: 429.1.
l
L
0.235 mmol) was added. The reaction mixture was stirred for 2 days.
The reaction mixture was diluted with CH2Cl2, then washed five times
with brine. The combined organic layers were dried over Na2SO4, fil-
tered and evaporated to dryness to give 234 mg of crude product as
yellow solid. The crude product was purified by column chromatogra-
phy (CH2Cl2/MeOH/EtOH 9:0.5:0.5 to 8:1:1, silica weight 50 g, volume
fraction: 30 mL, fractions collected from the 43th to the 49th) and
then by reverse phase using Biotage SP1 instrument (SNAP KP-
4.1.2.9. (S,E)-6-Acetyl-2-(1-((2-((2-(((E)-1-((R)-6-acetyl-7,9-dihy-
droxy-8,9b-dimethyl-1,3-dioxo-1,9b-dihydrodibenzo[b,d]furan-
2(3H)ylidene)ethyl)amino)ethyl)disulfanyl)ethyl)amino)ethyli-
dene)-7,9-dihydroxy-8,9b-dimethyldibenzo[b,d]furan-
1,3(2H,9bH)-dione (10).
24 ll of dry pyridine were added to a
solution of 65 mg (0.29 mmol) of cystamine dihydrochloride in
3 mL of absolute EtOH. After stirring 30 min at room temperature,
a suspension of 200 mg (0.58 mmol) of usnic acid in 3 mL of abso-
lute EtOH was added, and dry pyridine was added until usnic acid
dissolution. The reaction was refluxed under nitrogen atmosphere
for 8 h. The solvent was concentrated under reduced pressure, and
the crude material was acidified with 1 N HCl until pH 4, then ex-
tracted twice with EtOAc. The organic layers were collected and
concentrated under reduced pressure, giving 173.2 mg of pale yel-
low solid, quantitative yield. 1H NMR 400 MHz (CDCl3) d ppm 1.68
(s, 3H, CH3-13), 2.07 (s, 3H, CH3-16), 2.64 (s, 3H, CH3-15), 2.66 (s,
3H, CH3-18), 3.00 (t, J = 6.5 Hz, 2H, CH2-20), 3.85–3.89 (m, 2H,
CH2-10), 5.76 (s, 1H, CH-4), 11.81 (br s, 1H, OH-10), 13.33 (s, 1H,
OH-8), 13.72 (br s, 1H, NH). 13C NMR 100 MHz (CDCl3) d ppm
8.18 (C-16), 19.14 (C-15), 31.94 (C-18), 32.66 (C-13), 37.26 (C-20),
42.98 (C-10), 58.14 (C-12), 102.04 (C-2 + C-7), 102.92 (C-4),
105.58 (C-11), 108.73 (C-9), 156.48 (C-6), 158.80 (C-10), 164.22
(C-8), 174.97 (C-5), 175.69 (C-14), 191.63 (C-3), 199.20 (C-1),
C18-HS 12 g, 35–70 lm, flow rate 15 mL/min, ACN/H2O 20:80 to
80:20) to obtain 11 mg (0.019 mmol, 4%) of the desired product
as yellow solid. ½a D20
ꢂ
+120 (c 0.14, MeOH). 1H NMR 400 MHz
(DMSO-d6) d ppm 1.66 (s, 3H, CH3-13), 1.80 (s, 3H, CH3CONH),
1.89-1.93 (m, 2H, CH2-30), 1.98 (s, 3H, CH3-16), 2.58 (s, 3H, CH3-
15), 2.65 (s, 3H, CH3-18), 2.64–2.69 (m, 2H, CH2-20), 3.04–3.09
(dd, J = 12.6 Hz, J = 6.2 Hz, 1H, SCH2), 3.37–3.41 (dd, J = 12.6 Hz,
J = 4.6 Hz, 1H, SCH2), 3.55–3.58 (m, 2H, CH2-40), 4.02–4.07 (m, 1H,
NHCH), 5.89 (s, 1H, CH-4), 7.51 (d, J = 6.8 Hz, 1H, NHCH), 12.27
(br s, 1H), 13.04 (br s, 1H, NH), 13.41 (br s, 1H), 13.45 (s, 1H). 13C
NMR 100 MHz (DMSO-d6) d ppm 8.65 (C-16), 19.26 (C-15), 23.82
(CH3CONH), 25.57 (C-30), 32.17 (C-18), 32.82 (C-13), 32.92 (SCH2),
41.28 (C-20), 43.62 (C-40), 53.59 (NHCH), 57.46 (C-12), 102.01 (C-
7), 102.80 (C-2), 103.49 (C-4), 106.31 (C-11), 107.44 (C-9), 156.90
(C-6), 158.84 (C-10), 163.63 (C-8), 169.79 (CH3CONH), 172.42
(COOH), 174.14 (C-5), 176.18 (C-14), 190.09 (C-3), 198.38 (C-1),
199.14 (C-10), 202.06 (C-17). LC–MS (ESI), negative, m/z found:
572.9 [MꢀH]ꢀ, Calcd for C27H30N2O10S: 574.2.

M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
1841
where f is the fraction of the endpoint affected by dose D and m is
4.1.2.12. (R,E)-4-(4-((1-(6-Acetyl-7,9-dihydroxy-8,9b-dimethyl-
1,3-dioxo-1,9b-dihydrodibenzo[b,d]furan-2(3H)-ylidene)ethyl)
the slope shape or Hill’s coefficient.25
amino)butanamido)butanoic acid (13).
(50 mg, 0.12 mmol) in 1.5 mL of anhydrous THF under nitrogen, N-
methylmorpholine (15 l, 0.14 mmol) was added and the reaction
mixture was stirred at room temperature. After 30 min, 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
To a solution of 9
4.3. Scratch wound assay
l
Cells were grown in multiwell plates until confluence and
thereafter, cell layers were scratched by using a sterile 0.1–10 ll
pipette tip. After washing away suspended cells, cultures were re-
fed with medium and exposed to 1 or its derivatives, for 24 h. One
series of samples, used as positive controls, were exposed to a dose
of 20% (v/v) of a platelet lysate (PL). This preparation had been pre-
viously shown to promote scratch wound healing in HaCAT cells,
and was obtained from blood samples as previously described.17b
After exposures, cells were fixed in 3.7% formaldehyde in PBS for
30 min, and then stained with 0.1% toluidine blue at room temper-
ature for 30 min. The width of the wound space was measured at
wounding and at the end of treatments, using an inverted micro-
scope equipped with a digital camera (Leica Microsystems, Milan,
Italy), and the NIH ImageJ software. Wound closure was deter-
mined as the difference between wound width at 0 and 24 h.
(65 mg, 0.23 mmol)was added and the reaction mixture was stirred
at room temperature for 15 min. 4-Aminobutyric acid (12 mg,
0.12 mmol) was added and the reaction mixture was stirred at room
temperature for 3 h and 30 min. The solvent was removed under re-
duced pressure and the solid obtained was diluted with CH2Cl2 and
extracted twice with 0.1 N HCl. The combined organic layers were
washed with brine, dried over Na2SO4 and evaporated to dryness
to obtain 64 mg of orange solid. The crude product was then purified
by flash-chromatography using Biotage SP1 intrument (prepacked
21X55 mm SNAP silica gel cartridge, silica weight 10 g, flow rate
10 mL/min, CH3OH/CH2Cl2 0:100 to 10:90) to obtain 15 mg
(0.029 mmol, 25%) of orange solid (Rf 0.31, eluent: CH3OH/CH2Cl2
1:9). ½a 2D0
ꢂ
+231 (c 0.09, CHCl3). 1H NMR 400 MHz (DMSO-d6) d
ppm 1.58–1.62 (m, 2H, CH2-300), 1.66 (s, 3H, CH3-13), 1.82-1.89 (m,
2H, CH2-30), 1.98 (s, 3H, CH3-16), 2.18-2.22 (m, 4H, CH2-200 and
CH2-20), 2.59 (s, 3H, CH3-15), 2.64 (s, 3H, CH3-18), 3.03–3.08 (m,
2H, CH2-400), 3.53–3.57 (m, 2H, CH2-40), 5.88 (s, 1H, CH-4), 7.94 (br
s, 1H, NH-CO), 12.32 (s, 1H, OH-10), 13.04 (br s, 1H, NH), 13.41 (s,
1H, OH-8). 13C NMR 100 MHz (DMSO-d6) d ppm 8.65 (C-16), 19.25
(C-15), 25.72 (C-30), 25.80 (C-3), 32.17 (C-18), 32.51 (C-2), 32.84
(C-13), 33.22 (C-20), 39.14 (C-4), 44.19 (C-40), 57.45 (C-12), 102.02
(C-7), 102.76 (C-2), 103.49 (C-4), 106.31 (C-11), 107.46 (C-9),
156.92 (C-6), 158.86 (C-10), 163.66 (C-8), 172.12 (C-10), 174.16
(C-5), 175.72 (C-100), 176.11 (C-14), 190.08 (C-3), 198.36 (C-1),
202.07 (C-17). MS (ESI), negative, m/z found: 513.4 [MꢀH]ꢀ, Calcd
for C26H30N2O9: 514.2.
4.4. In vivo bioassays
4.4.1. Animals
Male, Sprague–Dawley rats (160–180 g), and Swiss albino mice
(20–25 g), were purchased from the animal breeding laboratory of
Saki Yenilli (Ankara, Turkey).
Animals were left for 3 days at room conditions for acclimatiza-
tion, and then maintained on standard pellet diet and water ad libi-
tum throughout the experiment. A minimum of six animals were
used in each group. The study was permitted by the Institutional Ani-
mal Ethics Committee and was performed according to the interna-
tional rules considering animal experiments and biodiversity right.
4.4.2. Preparation of test samples for bioassay
4.2. In vitro tests on keratinocytes
Incision and excision wound models were used to evaluate the
wound healing activity. For the in vivo wound models, test samples
were prepared at 1% concentration in an ointment base (vehicle)
consisting of glycol stearate, 1,2-propylene glycol, liquid paraffin
(3:6:1). Amounts of 0.5 g of each test ointment were applied topi-
cally on the wounded site immediately after wound was created by
a surgical blade. The animals of the vehicle group were treated
with the ointment base only, whereas the animals of the reference
drug group were treated with 0.5 g of MadecassolÒ (Bayer,
00001199), containing 1% extract of Centella asiatica.
All reagents were from Sigma Chemical Co., unless otherwise
indicated. Cells were maintained at 37 °C, 5% CO2, in Dulbecco-
modified Eagle’s medium (DMEM) supplemented with 10% foetal
bovine serum (FBS, Euroclone, Pero, Italy) and 1% antibiotic mix-
ture. Compounds 3, 6 and 7 were submitted to bioassays in the acid
form. The corresponding triethylammonium salts were acidified
and extracted with CH2Cl2.
4.2.1. Cell viability assay
Cell viability was assessed on HaCaT keratinocytes and on A431
human epidermoid carcinoma cells. For this analysis we used the
neutral red uptake (NRU) test by following the method reported
by Borenfreund et al.24 Such a cell viability assay is based on the
incorporation of neutral red dye into the lysosomes of viable cells
after incubation with the test agent. Briefly, cells were seeded on
96-well plates (20,000 cells/ well), grown for 24 h prior to experi-
ments, and then exposed for 24 h to various concentrations of us-
nic acid or of its derivatives. After removing the medium, a 0.05%
solution of neutral red was added to each well, followed by incuba-
tion for 3 h at 37 °C. Cells were then washed with PBS, followed by
the addition of a solution of 1% glacial acetic acid in 50% ethanol, in
order to fix the cells and extract the neutral red dye incorporated
into lysosomes. The absorption of the supernatants was measured
at 540 nm by using a microplate reader. Estimates of IC50, IC05 and
of their 95% confidence intervals where obtained by using a Micro-
soft ExcelÒ sheet developed by CSIRO, Australia, which makes use
of a downhill logistic curve:
4.4.3. Linear incision wound model
Rats, seven animals in each group, were anaesthetized with
0.15 ml KetalarÒ, the hairs on the dorsal part of the rats were
shaved and cleaned with 70% alcohol. Two 5 cm-length linear-par-
avertebral incisions were made with a sterile blade through the
shaved skin at the distance of 1.5 cm from the dorsal midline on
each side. Three surgical sutures were placed each 1 cm apart.
The ointments prepared with test samples, the reference drug
(MadecassolÒ), or ointment alone [glycol stearate:propylene gly-
col:liquid parafin (3:6:1)] were topically applied on the dorsal
wounds in each group of animals once daily throughout 9 days.
All the sutures were removed on the last day and tensile strength
of previously wounded and treated skin was measured by using a
tensiometer (Zwick/Roell Z0.5, Germany).26
4.4.4. Circular excision wound model
This model was used to monitor wound contraction and wound
closure time. Mice, seven animals in each group, were anaesthe-
tized by 0.01 cc KetalarÒ, back hairs were depilated by shaving,
and a circular wound was created on the dorsal interscapular
1
f ¼
ð1 þ expðꢀm ꢄ ðlogðDÞ ꢀ logðIC50ÞÞ

1842
M. Bruno et al. / Bioorg. Med. Chem. 21 (2013) 1834–1843
region by excising the skin with a 5 mm biopsy punch (Nopa
instruments, Germany); wounds were left open.27 Test samples,
the reference drug (MadecassolÒ), and the vehicle ointment were
applied topically once a day till the wound was completely healed.
The progressive changes in wound area were monitored by a cam-
era (Fuji, S20 Pro, Japan) every two days, and the wound area was
then evaluated by using AutoCAD software. Wound contraction
was calculated as percentage of the reduction in wounded area.
A sample of tissue was isolated from the healed skin of each group
of mice for histopathological examination.28
405 nm. HNE inhibition activities were calculated as reported
above for the ChC inhibition activity.
4.7. Statistical analysis of data
Data were analyzed using one-way analysis of variance (ANO-
VA). The values of p 60.05 were considered statistically significant.
Acknowledgment
4.5. Hydroxyproline estimation
Work supported by MIUR (PRIN 2008).
Tissues were dried in hot air oven at 60–70 °C until consistent
weight was achieved. Afterwards, samples were hydrolyzed with
6 N HCl for 3 h at 130 °C. The hydrolyzed samples were adjusted
to pH 7 and subjected to chloramin T oxidation. The colored adduct
formed with Ehrlich reagent at 60 °C was read at 557 nm. Standard
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.045.
hydroxyproline was also run, and values were reported as
lg/mg
dry weight of tissue.29
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The inhibition of hyaluronidase was assessed by the measure-
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hyaluronate.20,30 A volume of 50
l
l of bovine hyaluronidase (7900
units/ml) was dissolved in 0.1 M acetate buffer (pH 3.6). This solu-
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was added instead of the oils. After 20 min incubation at 37 °C,
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l
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Shiu, W. K. P.; Gibbons, S. Planta Med. 2007, 73, 176.
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incubated for 3 min in boiling waterbath. A volume of 1.5 ml of
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(0.4 M NaCl, 0.01 M CaCl2, pH 7.5) and preincubated at 25 °C for
5 min. Thereafter, 2 mM N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala
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25
l
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