Some thiocarbamoyl based novel anticathepsin agents

Article abstract of DOI:10.1016/j.bioorg.2020.104174

Cathepsins have emerged as important targets in various tissues degenerative disorders due to their involvement in degradation of extracellular matrices and endogenous protein turnover. Elevated cathepsins levels vis-à-vis decreased concentration of endogenous inhibitors has been reported at different diseased sites. The design and synthesis of specific potential anti-cathepsin agents is therefore of great significance. Most of potential anti-cathepsin agents developed have peptide based structures with an active warhead. Due to oral instability and immunogenic problems related to peptidyl inhibitors drift the synthesis and evaluation of non-peptide cathepsin inhibitors in last two decades. The present work provides a detailed structure activity relationship for developing potential non-peptide anticathepsin agents based on in-vitro inhibition studies of a library of synthesized thiocarbamoyl- non-peptide inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.104174

See related Commentaries on pages iv and vi
Ultraviolet B-Induced DNA Damage in Human Epidermis Is
Modified by the Antioxidants Ascorbic Acid and D-a-Tocopherol
Ã
Ã
Ã
Ã
Marianne Placzek, Sabine Gaube, Urs Kerkmann, Klaus-Peter Gilbertz,w Thomas Herzinger, Ekkehard
Ã
Haen,z and Bernhard Przybilla
Ã
Klinik und Poliklinik f u¨ r Dermatologie und Allergologie, Ludwig-Maximillians-Universit a¨ t, Frauenlobstr. 9-11, 80337 Munich, Germany; wInstitut f u¨ r Radiobiologie
der Bundeswehr, Neuherbergstr. 11, 80937 Munich, Germany; zKlinische Pharmakologie, Klinik und Poliklinik f u¨ r Psychiatrie der Universit a¨ t im Bezirksklinikum
Regensburg, Universit a¨ tsstraꢀe 84, 93042 Regensburg, Germany
DNA damage caused by ultraviolet (UV) irradiation is considered the main etiologic factor contributing to the
development of skin cancer. Systemic or topical application of antioxidants has been suggested as a protective
measure against UV-induced skin damage. We investigated the effect of long-term oral administration of a com-
bination of the antioxidants ascorbic acid (vitamin C) and D-a-tocopherol (vitamin E) in human volunteers on UVB-
induced epidermal damage. The intake of vitamins C and E for a period of 3 mo significantly reduced the sunburn
reaction to UVB irradiation. Detection of thymine dimers in the skin using a specific antibody revealed a significant
increase of this type of DNA damage following UVB exposure. After 3 mo of antioxidant administration, significantly
less thymine dimers were induced by the UVB challenge, suggesting that antioxidant treatment protected against
DNA damage.
Key words: human skin/ultraviolet radiation/antioxidants/DNA damage/thymine dimers
J Invest Dermatol 124:304 –307, 2005
Skin cancer is very frequent in Caucasians, and its inci-
dence is increasing steadily. This development is caused
inter alia by demographic changes (increased life expect-
ancy) and by the intake of photosensitizing drugs (Placzek
et al, 1999). These two effects together with an increased
ultraviolet (UV) exposure by changes in the recreational be-
havior are a major cause of skin cancer. The exposure to UV
radiation promotes the development of squamous cell car-
cinoma (SCC) and its precursor lesions, actinic keratoses.
Epidemiologic data also imply UV as a major factor in the
etiology of melanoma and basal cell carcinoma (BCC), al-
though the role of the cumulative UV dose is less clear for
these cutaneous malignancies than for SCC.
of vitamins C and E decreased the sunburn reaction
and protected epidermal cells against the induction of
thymine dimers.
Results
Subjects took 1 g of ascorbic acid and 500 IU of D-a-to-
copherol twice daily over a period of 3 mo. None of the
volunteers complained of any adverse events. The mean
vitamin C serum level at the beginning was 11.6 ꢂ 4.8 mg
per liter (normal range: 4–20 mg per liter). After 1 mo of
treatment vitamin C blood concentration was increased to
1
9.3 ꢂ 7.5 mg per liter (po0.001). This increase remained
stable, levels being 19.0 ꢂ 6.8 mg per liter after 2 mo and
8.2 ꢂ 6.8 mg per liter after 3 mo. Vitamin E serum levels
were 21.0 ꢂ 12.2 mg per liter at baseline (normal range:
Both DNA damage caused by direct absorbance of UV
radiation and indirect DNA damage contributed by reactive
oxygen species (ROS) may lead to mutations, which can
result in UV-induced skin cancer. The most prominent direct
DNA damages are the cyclobutane pyrimidine dimers, i.e.
1
5
–20 mg per liter) and increased to 36.0 ꢂ 14.4 mg per liter
after 1 mo (po0.001). After 2 or 3 mo, they were 39.6 ꢂ 17.9
or 38.8 ꢂ 17.8 mg per liter, respectively. These data dem-
onstrate that serum levels of vitamins E and C can be in-
creased significantly by exogenous oral intake.
thymine dimers, and the (6-4) photoproducts. Indirect DNA
1
damage is inferred by ROS such as singlet oxygen ( O ) and
2
ꢁ
ꢀ
free radicals such as the superoxide anion radical ( O ), the
2
ꢀ
ꢀ
perhydroxyl radical (HO ), or the hydroxyl radical ( OH).
2
To assess the potential protective effects of antioxidant
treatment against UVB-induced sunburn, we determined
the minimal dose of UVB required to elicit a clearly de-
markable erythema (reddening) of the skin on a previously
unexposed body site (minimal erythema dose (MED)) before
and after the 3 mo course of vitamins C plus E. After 90 d of
Administration of antioxidants like ascorbic acid and D-
a-tocopherol that scavenge ROS has been promoted as
a strategy to decrease UV-induced skin damage and
ultimately to also prevent skin cancer. In this study, we in-
vestigate the protective effect of long-term oral administra-
tion of antioxidant vitamins against UV-induced epidermal
damage in human volunteers. We find that a combination
antioxidant administration, the median MED rose from 80 to
2
1
13 mJ per cm (p ¼ 0.002, Wilcoxon’s test; Fig 1) demon-
strating that oral vitamins E plus C decreases the skin’s
susceptibility to sunburn.
Abbreviation: UV, ultraviolet
Copyright r 2004 by The Society for Investigative Dermatology, Inc.
3
04

1
24 : 2 FEBRUARY 2005
UVB-INDUCED DNA DAMAGE IN HUMAN EPIDERMIS 305
Discussion
This study shows that antioxidants modify skin reactivity to
UVB in vivo. The long-term combined oral intake of ascorbic
acid and D-a-tocopherol may lead to a significant physio-
logic increase of antioxidant capacity of human skin. This
was demonstrated by a reduction of sunburn reaction and
thymine dimers in human skin.
We used a combination of ascorbic acid and D-a-tocop-
herol, because there is evidence for a synergistic interaction
of vitamins C and E in antioxidant defense: UV-activated
molecules oxidize cellular components; particularly, they
induce a chain reaction of lipid peroxidation in membranes
rich in polyunsaturated fatty acids. D-a-tocopherol, by
acting as an antioxidant, is oxidized to the tocopheroxyl
radical in this process (Chow, 1991; Fryer, 1993), and it is
regenerated by ascorbic acid to D-a-tocopherol (Packer
et al, 1979). This synergism has been shown with un-
saturated lipids in micelles (Barclay et al, 1983) or in
phosphatidylcholine liposomes (Niki et al, 1985). Further-
more, when compared with other vitamin E compounds, a-
tocopherol has the best bioavailability (Burton and Traber,
Figure 1
Minimal erythema dose before and after antioxidant treatment.
1990) and reaches the highest concentration in epidermis
(Fuchs et al, 2003).
A protective effect of topical or systemic antioxidants
against UV-induced skin damage has been demonstrated in
several experimental systems (reviewed in Pinnell, 2003).
Several studies have documented photoprotective effects
when vitamin E was applied to animal skin. Topical a-to-
copherol protected rabbit or mouse skin against UV-in-
duced erythema (Roshchupkin et al, 1979; Record et al,
1
991; Trevithick et al, 1992). In mice, topical vitamin E de-
creased UV-induced effects such as lipid peroxidation
Lopez-Torres et al, 1998), cutaneous photoaging (Bissett
et al, 1990; Jurkiewicz et al, 1995), immunosuppression
Gensler and Magdaleno, 1991; Yuen and Halliday, 1997;
(
(
Steenvoorden and Beijersbergen v Henegouven, 1999), and
photocarcinogenesis (Bissett et al, 1990; Gensler and
Magdaleno, 1991; Yuen and Halliday, 1997; Burke et al,
2000). Topical application of vitamin E to human skin has
also been found to reduce UVB- and PUVA-induced skin
erythema (Potapenko et al, 1980, 1984).
Figure 2
Thymine-dimer-positive cells per mm epidermis before or after 3
mo antioxidant (AO) administration (NS, not significant).
Topical L-ascorbic acid protected porcine skin from UVB-
and UVA-phototoxic injury as measured by erythema and
sunburn cell formation (Darr et al, 1992), and also protected
against UV-induced immunosuppression and systemic tol-
erance to contact allergies in mice (Nakamura et al, 1997).
Topical application of combined vitamins C and E on
porcine skin provided significant protection against ery-
thema, sunburn cell formation, and thymine-dimer forma-
tion. Ascorbic acid or D-a-tocopherol alone were also
protective against erythema and sunburn cell formation
but the combination was superior. Only the combination of
vitamins C and E reduced thymine dimers (Lin et al, 2003).
In mice, systemic vitamin E reduced MED, lipid oxida-
tion, and incorporation of thymine dimers in DNA (Record
et al, 1991). Systemic application of vitamin C decreased
the incidence and delayed the onset of the malignant skin
lesions (Dunham et al, 1982).
Biopsy specimens were taken from uninvolved skin prior
to irradiation and from a sunburned (2-fold MED) test field
2
4 h after UVB exposure. Thymine dimers were visualized
by immunohistochemistry using a monoclonal antibody.
Prior to UVB irradiation, no thymine-dimer-positive cells
could be detected in the epidermis. Twenty-four hours after
irradiation the median number of thymine-dimer-positive
cells per mm epidermis was 82.0, demonstrating the in-
duction of DNA damage by a 2-fold MED of UVB. Following
3
mo of antioxidant treatment, the number of thymine-
dimer-positive cells after 2 MED UVB reduced to 48.2 per
mm (p ¼ 0.003, Wilcoxon’s test), showing that the protection
provided by antioxidants against clinical sunburn was re-
flected on the molecular level by a protective effect against
UV-inflicted DNA damage (Fig 2).
Few studies have assessed the photoprotective potential
of systemical antioxidant treatment in humans. Ingestion of

3
06 PLACZEK
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
400 IU D-a-tocopherol acetate per d or of a selenium–cop-
per–vitamin complex (containing 40 mg vitamin E per d) did
not significantly influence the MED (la Ruche and C e´ sarini,
1
991; Werninghaus et al, 1994). But we demonstrated in a
previous study that the combined oral administration of vi-
tamins C and E provided protection against UV-induced
erythema after only 1 wk (Eberlein-K o¨ nig et al, 1998). This
finding was confirmed by Fuchs (1998) and Fuchs and Kern
(
1998) who further showed that either vitamin alone was
ineffective.
In this study, we show that by extending the administra-
tion of ascorbic acid and D-a-tocopherol to a period of 3 mo
the protective effect against UVB-induced erythema could
be increased even further: whereas after 1 wk of oral vita-
mins C and E, the median MED was increased by 21%, the
increase was 41% after 3 mo. The decreased sensitivity to
sunburn was paralleled by an increase of blood levels of
vitamins C and E. A significant rise of vitamin serum con-
centration was found after only 1 mo of administration; be-
yond this point no further increase of levels was found.
Serum levels achieved by oral intake for 1 mo were on av-
erage higher than levels after only 1 wk in our previous
study. This suggests that saturation levels of vitamins E and
C in serum are not yet reached after 1 wk, and that the
increased protective effect after long-term administration of
vitamins E and C is caused by higher serum (and, possibly,
tissue) concentrations.
Proceeding from the protective effect of antioxidants on
the sunburn response to UVB, we assumed that adminis-
tration of vitamins C and E might also protect against UV-
induced DNA damage, as has been shown previously with
topical antioxidant treatment in pig skin (Lin et al, 2003). In
accordance with this, we found a reduction of thymine
dimers also in UVB-irradiated human skin after 3 mo of
systemic administration of antioxidants.
Figure 3
Wavelength spectrum of TL 20 W/12 fluorescent tubes.
UV irradiation was performed using fluorescent tubes with a
main emission between 275 and 365 nm and a maximum at about
315 nm (TL 20 W/12, Philips, Hamburg, Germany); the spectral
distribution is shown in Fig 3. Irradiance was 1.0 mW per cm for
2
2
UVB and 0.4 mW per cm for UVA at a distance of 40 cm as
measured by an integrating instrument (UV-Meter, Waldmann,
Villingen-Schwenningen, Germany). On the lower back, 10 circular
areas each measuring 1.8 cm were exposed to increasing UVB
2
2
doses (20, 28, 40, 57, 80, 113, 124, 160, 175, and 200 mJ per cm ).
The MED was the lowest dose causing a sharply defined, homo-
geneous erythema 24 ꢂ 2 h after exposure. The second test after
90 d of administration of antioxidants was carried out on the other
side of the lower back.
Biopsies were taken from unirradiated skin and the day after
from the UVB-irradiated site that had received a 2-fold MED before
and after 3 mo intake of antioxidants. Only 17 patients agreed to
the biopsies.
Evaluation of ascorbic acid and a-tocopherol levels Evaluation
of ascorbic acid and a-tocopherol levels in the serum/plasma
was carried out by high-pressure liquid chromatography. For
vitamins C and E analysis, we used commercial kits (ascorbic acid:
Immundiagnostik, Bensheim, Germany; a-tocopherol: Chromsys-
tems, Munich, Germany).
In summary, our findings suggest that twice daily oral
administration of ascorbic acid plus D-a-tocopherol repre-
sents a convenient and well-tolerated prophylactic treat-
ment against the hazardous effects of solar UV irradiation.
Further studies are required to confirm whether the photo-
protective effects of this treatment result indeed in protec-
tion against the development of skin cancer.
Immunohistochemistry For detection of thymine dimers, we used
the labeled streptavidin–biotin method (LSAB 2 System, Alkaline
Phosphtase; Dako, Carpinteria, California) applying a monoclonal
mouse anti-thymine-dimer antibody (Kamiya Biomedical Company,
Seattle, Washington). Paraffin-embedded sections were deparaffini-
zed and then incubated in a moist chamber with 0.1% trypsin
(Sigma, Steinheim, Germany) for 30 min at room temperature. After
washing with distilled water, unspecific binding was blocked by in-
cubation at room temperature for 20 min with 20% fetal calf serum
Patients and Methods
Study design Twelve males and six females (21–77 y old) were
included in the study, according to the Declaration of Helsinki
principles after informed written consent had been obtained.
Experiments were approved by the Ethikkonmission des Fachber-
eichs Medizin of the Ludwig-Maximillians-Universit a¨ t M u¨ nchen. Six-
teen participants had skin type II, and two had skin type III
(
PAA, Linz, Austria) in Tris-buffered saline (TBS). Then sections were
covered with anti-thymine-dimer antibody (1:80) at room tempera-
ture for 90 min. Sections were rinsed with TBS three times and
incubated with biotinylated secondary antibody (ready-to-use, en-
closed in the LSAB 2 System, Alkaline Phosphtase; Dako) in PBS
buffer for 7 min and, after washing, with streptavidin-conjugated al-
kaline phosphatase (ready-to-use, enclosed in the LSAB 2 System,
Alkaline Phosphatase; Dako) in PBS buffer for further 7 min. Sections
were rinsed again and staining was achieved by incubation for 10
min with Fuchsin chromogen. Counterstaining was performed with
hematoxylin blue, and samples were embedded in Kaiser’s glycerol
gelatine.
(
according to Fitzpatrick). Fourteen patients were recruited during
follow-up examinations at the outpatient department of oncology.
The patients had a history of melanoma, BCC, or SSC. Four patients
had no UV-induced skin cancer. All participants agreed to refrain
from sun exposure and from vitamin supplements for 2 wk before
and during the study. UV sensitivity by determining the MED was
assessed at the start of the trial, and again after 90 d of oral intake of
ascorbic acid 2 g per d and D-a-tocopherol 1000 IU per d (both
Kloesterl Apotheke, Munich, Germany). Blood vitamin concentra-
tions were measured at the beginning and every 30 d during the trial.
Statistical evaluation Statistical analysis was performed with the
Student’s t test (blood vitamin levels) and Wilcoxon’s test for paired
comparisons (MED, thymine dimers) with the assistance of the

1
24 : 2 FEBRUARY 2005
UVB-INDUCED DNA DAMAGE IN HUMAN EPIDERMIS 307
‘
‘Institut f u¨ r Medizinische Informationsverarbeitung, Biometrie und
Epidemiologie’’, Ludwig-Maximillians-Universit a¨ t, Munich. Values
of po0.05 were regarded as significant.
Gensler HL, Magdaleno M: Topical vitamin E inhibition of immunosuppression
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1991
Jurkiewicz BA, Bisset DL, Buettner GR: Effect of topically applied tocopherol on
ultraviolet radiation-mediated free radical damage in skin. J Invest De-
rmatol 104:484–488, 1995
This work was supported by Bayerischer Forschungsverbund Bay-
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DOI: 10.1111/j.0022-202X.2004.23560.x
Manuscript received May 5, 2004; revised July 20, 2004; accepted for
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