Design, synthesis and evaluation of novel levoglucosenone derivatives as promising anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2020.127247

A series of levoglucosenone-derived 1,2,3-triazoles and isoxazoles featuring a flexible spacer between the heteroaromatic and anhydropyranose cores have been designed and synthesized following an hetero Michael // 1,3-dipolar cycloaddition path. The use o

Full text of DOI:10.1016/j.bmcl.2020.127247

Journal Pre-proofs
Design, synthesis and evaluation of novel levoglucosenone derivatives as
promising anticancer agents
Yi-hsuan Tsai, Carla M. Borini Etichetti, Soledad Cicetti, Javier E. Girardini,
Rolando A. Spanevello, Alejandra G. Suárez, Ariel M. Sarotti
PII:
S0960-894X(20)30352-8
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127247
BMCL 127247
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
12 March 2020
25 April 2020
2 May 2020
Please cite this article as: Tsai, Y-h., Borini Etichetti, C.M., Cicetti, S., Girardini, J.E., Spanevello, R.A., Suárez,
A.G., Sarotti, A.M., Design, synthesis and evaluation of novel levoglucosenone derivatives as promising
anticancer agents, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127247
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Design, synthesis and evaluation of novel levoglucosenone derivatives as promising
anticancer agents
Yi-hsuan Tsai,a Carla M. Borini Etichetti,b,§ Soledad Cicetti,a,§ Javier E. Girardini,c Rolando A. Spanevello,a Alejandra G. Suárez,a
and Ariel M. Sarotti a*
a) Instituto de Química Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000,
Argentina. b) Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET). Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina. §Both authors contributed equally to this work. *Corresponding author: sarotti@iquir-
conicet.gov.ar (AMS)
A series of levoglucosenone-derived 1,2,3-triazoles and isoxazoles featuring a flexible spacer between the
heteroaromatic and anhydropyranose cores have been designed and synthesized following an hetero Michael //
1,3-dipolar cycloaddition path. The use of a design of experiments approach allowed the optimization of the oxa-
Michael reaction with propargyl alcohol as nucleophile, a key step for the synthesis of the target compounds. All
of the compounds were tested for their anticancer activity on MDA-MB-231 cells, featuring mutant p53. The
results highlighted the importance of the introduction of the flexible spacer as well as the higher activity of oxa-
Michael isoxazole-derivatives. The most prominent compounds also showed anti-proliferative activities against
lung and colon cancer cell lines. The compounds showed enhanced cytotoxic effects in the presence of mutant
p53, determined both by endogenous mutant p53 knock down (R280K) and by reintroducing p53 R280K in cells
lacking p53 expression.
Levoglucosenone (1), is the main product of the pyrolytic transformation of acid-pretreated cellulosic materials.1 Its structural rigidity and versatile
functionality were exploited in the synthesis of natural products,1 valuable intermediates,2 new tools of asymmetric synthesis,3 and development
of greener solvents.4 It also emerges as a prominent pharmacophore in medicinal chemistry.1 In particular, some of its derivatives have shown
anticancer activities related with the restoration of p53,5 a transcription factor acting as a tumor suppressor which can induce apoptosis or
senescence in cases of persistent oncogenic stress or DNA damage.6 Mutation of the p53 gene (TP53) is the most common genetic alteration in
human cancer (more than 50% in some cases).6c In most cases, missense mutations are found in the DNA binding domain. As a consequence,
human tumors often show abundant expression of point mutant p53 proteins which have lost tumor suppressor function. The possibility to restore
wt (wild type) function by a refolding process induced upon the interaction of mutant p53 proteins with small organic molecules was proposed. In
this way, a robust cytotoxic response may be unleashed in tumor cells.Several small molecules able to restore wt p53-associated responses were
identified .6d However, direct interaction with the mutant protein may not to be required in all cases. Even though the underlying mechanisms are
still under investigation, this leading strategy in anticancer drug development resulted in the discovery of PRIMA-1 and other compounds which
have reached clinical trials.6e
Recently we reported the synthesis of levoglucosenone-derivatives featuring triazol units attached to the C-4 position. Some derivatives exhibited
satisfactory in vitro anti-proliferative activity against MDA-MB-231 breast cancer cell line, and showed enhanced activity in the presence of mutant
p53, suggesting a possible path to be further exploited.5f
This exciting discovery motivated us to explore structural modifications for SAR studies. We foresaw evaluating the effect of introducing a flexible
spacer between the anydrosugar and triazole cores, which would significantly change the conformational freedom and topology of the molecule.
This modification could be done by exploiting the known reactivity of 1 as Michael acceptor (Scheme 1), allowing access to additional elements of
diversity, such as the nature of the heteroatom at C-4) and the possibility to synthesize other heteroaromatic cores.
O
XH
O
O
O
2 (X=O)
3 (X=NH)
R-N3
O
O
6
X
O
Cu(I)
O
N
X
O
7 (X=O)
8 (X=NH)
1
N
4 (X=O)
5 (X=NH)
N
R
Scheme 1. Synthesis of novel levoglucosenone derivatives.
Despite its great synthetic utility, the oxa-Michael reaction is one of the less studied variants of the Michael-type reactions. The major drawbacks
are related with the poor nucleophilicity of alcohols and the reversibility of the addition stage.7 Levoglucosenone is a reactive enone system
suitable for 1,4-conjugate additions with a variety of reagents,1 but the reaction using alcohols has been almost unexplored. Initially we carried out
the base-catalyzed addition of 2 to levoglucosenone using the procedure reported by Shafizadeh et al for methanol as nucleophile (0.6 eq. NEt3,
r.t., 3 h, 100%),8a but the results obtained were disappointing. The desired product 4 was obtained in low yield (<40%), with a significant
dimerization of levoglucosenone (vide infra). We also tested the conditions reported by the same group for the oxa-Michael reaction with benzyl
alcohol (BnOK/BnOH, r.t., 5 min),8b but the isolated yields of 4 were still very low. On the basis of the difficulties encountered to efficiently achieve

this transformation with known procedures, we undertook the optimization of the acid-promoted and base-promoted oxa-Michael reaction of 1
and 2 with the aid of a design of experiments approach (DOE), which allows the exploration of the multidimensional chemical space on a more
rational basis.9 Since the oxa-Michael reactions can be typically carried out in acid media (activation of the Michael acceptor) or basic media
(activation of the Michael donor),7 we decided to evaluate both conditions.
Combining a survey of inexpensive promoters, variable selection by factorial designs, and final optimization through central composite analysis
(detailed in the ESI), the desired 4 could be successfully obtained in high yields (Scheme 2). The by-products (ketal 9 or dimer 10) were kept as
traces at the optimized protocols. In the acid-catalyzed process, HCl was the most promising acid promoters. Interestingly, the central composite
analysis yielded a second-order polynomial model with a strong quadratic behavior with factor A (HCl normality). Using concentrated HCl solutions
decreased the yield of 4 through formation of the by-product 9, whereas this side reaction could be suppressed by the water content provided by
more diluted HCl. However, highly diluted HCl feedstocks somehow displaced the equilibrium towards 1. The optimal reaction conditions were
predicted around 1.5 eq. HCl 5N, 16 h, 25 ºC, affording a 90% of 4 (higher than those obtained in all previous experiments), demonstrating the
power of the conducted DOE methodology. The value was reproducible after several replications, and showing minor changes upon scaling. To the
best of our knowledge, this is the first report of the acid-catalyzed oxa-Michael reaction using levoglucosenone as acceptor.
In the case of base-catalyzed conditions, the central composite design uncovered a linear relationship with negative slopes between reaction time
and Nº eq. of NEt3 (the most promising base under study) with the yield of 4. This prompted us to evaluate the reactions at descending quantities
of NEt3 keeping the reaction time fixed at 6 min (shorter reaction times resulted operationally impractical). In perfect agreement with the DOE
predictions, the yields of 4 increased gradually to reach a maximum around 0.1-0.15 eq (see SI). Even lower amounts of NEt3 resulted in a dramatic
reduction in the catalytic activity. The optimal reaction conditions were then set at 0.1 eq. NEt3, 6 min, 25 °C, affording an overall yield of 87%.
These results were in clear contrast to the experimental reaction conditions found for Shafizadeh et al. using methanol as nucleophile (0.6 eq.
NEt3,
3
h),8a suggesting that the nature of the alcohol is closely related with the optimal reaction parameters.
By-products
O
O
O
O
OH
O
O
O
O
2
OR
OR
+
O
O
O
O
RO
HCl or NEt3
O
4
9
10
1
90% (HCl)
87% (NEt3)
O
O
obtained in
acid media
obtained in
basic media
Scheme 2. Design of experiments-optimized synthesis of 4. Optimal conditions: 1.5 eq. HCl 5N, 16 h, 25 ºC (acid conditions); or 0.1 Eq. NEt3, 6 min, 25 ºC (basic conditions).
The synthesis of 5 was more straightforward, obtaining the desired product in ca. 100% conversion. However, after purification by column
chromatography, compound 5 experienced significant retro-aza-Michael decomposition. Such behavior (particularly important with aliphatic
amines) was recently reported by Greatrex and co-workers.2b To overcome this limitation, we foresaw that acetylation of the secondary amine in 5
would reduce the tendency for the retro-aza-Michael decomposition. Upon treating the reaction crude of 5 with freshly distilled Ac2O, the
acetamide 11 could be isolated in 81% overall yield after column chromatography (Scheme 3). As in the case of 4, the 1,6-anhydro bridge allowed
excellent levels of diastereoselectivity, obtaining only the isomers resulting from the attack of the nucleophiles through the α face of the enone.
Ac2O
DCM/py
DMAP
O
O
NH2
O
O
O
O
3
AcN
O
HN
O
NEt3
DCM
81%
(2 steps)
O
11
5
1
Scheme 3. Synthesis ofacetamide 11
Once the two propargyl precursors were synthesized, the 1,3-dipolar cycloaddition with organic azides was carried out to obtain the desired 1,2,3-
triazoles. The aliphatic azides were prepared by standard SN2 reactions of NaN3 and the corresponding primary or secondary alkyl chloride or
bromide, whereas the aromatic azides were synthesized via the Sandmeyer reaction. The azides were chosen to provide diversity of alkyl, vinyl and
aryl groups with different substitution patterns and electronic characters. Using a modified protocol of Kim (0.5 mmol of 4/11, 1.5 eq. azide, 40
mol% sodium ascorbate, 10 mol% CuSO4.5H2O, CH2Cl2:H2O 1:1),10 the desired triazoles were obtained in good yields (Table 6).
The pharmacokinetic properties of new compounds represent an obstacle that must be overcome to reach the clinical trials. Hence, an early
estimation of the ADME properties is mandatory, allowing a preliminary drug-likeness evaluation in a simple and rapid fashion. Using the online
open access tool Molinspiration (www.molinspiration.com) we computed the following molecular descriptors: the partition coefficient (log P), the
molecular weight (MW), the topological polar surface area (TPSA), the number of hydrogen bond acceptors (nON), the number of hydrogen bond
donors (nOHNH), and the number of rotatable bonds (nrotb). All of the compounds in the library show no violation of the Lipinski´s rule of five. All
of them have a MW lower than 500 kDa, a logP lower than 5, nON less than 10 and nOHNH less than 5. Moreover, they all showed a polar surface
area lower than 140 Å2 and 10 or less rotable bonds, which indicate good cell permeability. Altogether, this in silico study supported the library
choice.
The in vitro antiproliferative activity was studied on the MDA-MB-231 cell line, originally derived from Triple Negative Breast cancer (TNBC). This
cell line lacks a wt allele at the TP53 locus but retained a mutated one, allowing exclusive expression of endogenous p53 R280K mutant protein.
Upon treatment with each individual compound for 48 h at different concentrations, living cells were quantified using the MTT viability assay and
normalized comparing with untreated cells. The half maximal inhibitory concentration (IC50) obtained for all compounds are also shown in Table 1.
2

Interestingly, while none of the nitrogenated compounds 12 exhibited interesting levels of cytotoxicity (IC50> 50 μM), most of the oxigenated
counterparts were active in the range 18.6-36.0 μM, comparable with the cytotoxicity of PRIMA-1 determined on the same line and similar
experimental conditions.11 The lack of activity of the alkyne precursors (IC50 > 100 μM) suggested that the incorporation of the triazole unit was
important in terms of cytotoxicity, whereas the larger amount of bioactive compounds (regarding the original series) validated our structural
hypothesis.
To analyze the influence of the nature of the heterocycle, we foresaw the synthesis and evaluation of isoxazole analogues, considered a prime
scaffold for drug discovery.12
Table 1. Synthesis and evaluation of triazoles 7 and 12 on survival of MDA-MB-231 breast cancer cells.a
O
O
O
O
R-N3
9
8
CuSO4.5H2O
Na ascorbate
DCM/H2O
X
O
X
O
R
N
4 (X=O)
11 (X=NAc)
7 (X=O)
12 (X=NAc)
N
N
Entry
1
2
3
4
5
6
7
8
X
O
O
O
O
NAc
NAc
NAc
NAc
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
R
-Bn
-CH2CO2Me
-CH2C(O)Ph
-Octyl
Product
7a
7b
7c
7d
12a
12b
12c
12d
7e
7f
7g
7h
7i
7j
Yield
IC50 (μM)
>50
21.3
25.9
>50
>50
>50
>50
>50
24.8
26.1
25.2
23.7
27.9
26.7
26.4
22.5
23.2
21.9
26.0
34.8
25.4
23.7
36.0
18.6
24.3
78%
58%
79%
85%
67%
77%
76%
91%
81%
77%
70%
78%
66%
74%
81%
79%
75%
96%
55%
~100%
~100%
98%
67%
87%
48%
-Bn
-CH2CO2Me
-CH2C(O)Ph
-nOctyl
-4-NO2Ph
-4-ClPh
-Ph
-4-FPh
-Butyl
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
-CH2C(Me)=CH2
-2-ClPh
7k
7l
-2-BrPh
-4-CO2HPh
-α-naphtyl
-2,3-dichloroPh
-CH2(4-OCH3)Ph
-CH2(4-NO2)Ph
-CH2(β-naphtyl)
-α-cyclohexanone
fluorenil
7m
7n
7o
7p
7q
7r
7s
7t
7u
-cyclohexyl
aGeneral procedure: 0.5 mmol of 4 or 11, 1.5 eq. azide, 40 mol% sodium ascorbate, 10 mol% CuSO4.5H2O, CH2Cl2:H2O 1:1). bIsolated yield obtained after column chromatography. IC50
:
c
concentration achieving 50 % reduction in viability determined by MTT assay.
Taking advantage of the alkyne precursor 4, the synthesis of the targets were carried out following the known 1,3-dipolar cycloaddition with nitrile
oxides.13 Starting from commercially available aldehydes, the imidoyl chlorides 13 were easily prepared in 2 steps (Scheme 4). Exposure of 13a
(R=Ph) with NaHCO3 afforded the corresponding nitrile oxide, which further reacted with alkyne 4 under Sharpless conditions (Cu(I), t-BuOH/H2O)
to give 14a in low yield (36%).13 Analysis of the reaction mixture by 1H NMR evidenced significant retro-oxa-Michael decomposition. To prevent this
side reaction, we next evaluated the CH2Cl2/H2O solvent system with the aim of minimizing the contact between 4 and the base in the bifasic
system. To our delight, this simple modification allowed the isolation of 14a as the only detectable isomer in very good yield. We used this
modified procedure for the synthesis of a small library of isoxazoles featuring diversity (Table 2). In all cases the corresponding 3,5-disubstituted
isoxazoles were obtained as single regioisomers, with high conversion (as determined by TLC analysis of the reaction mixtures) and isolated yields
varying from discrete to very good depending on the nature of the substituent at the isoxazole core. The modest yields observed in some cases
were mainly attributed to partial decomposition that took place during the chromatographic purification stage. In general, aromatic fragments
afforded cleaner reactions than aliphatic or vinylic moieties, which is consistent with the relative stability of the corresponding imidoyl chloride.13
Table 2. Synthesis and evaluation of isoxazoles 14 on survival of MDA-MB-231 breast cancer cells.a
3

OH
Cl
N
O
O
O
O
R
NaHCO3
CuSO4.5H2O
Na ascorbate
DCM/H2O
O
O
O
O
R
4
14
O
N
Entry
1
2
3
4
5
6
7
8
R
-Ph
Product
14a
14b
14c
14d
14e
14f
14g
14h
14i
Yield
76%
70%
52%
59%
88%
62%
82%
65%
35%
66%
IC50 (μM)
21.9
21.9
16.5
21.2
22.9
23.9
17.9
15.8
26.4
13.8
-4-OMe-Ph
-4-NO2-Ph
2-Naphtyl
-CH=CH-Ph
3,5-(CF3)2-Ph
-4-Cl-Ph
-cyclohexyl
-CH=CH-4-OMe-Ph
-5-Cl-2-furyl
9
10
14j
aGeneral procedure: 0.25 mmol of 4, 1 eq. of imidoyl chloride, 10 mol% of sodium ascorbate, 2 mol% of CuSO4.5H2O and 4.5 eq of KHCO3 in DCM:H2O 1:1bIsolated yield obtained after
column chromatography . cIC50: concentration achieving 50 % reduction in viability determined by MTT assay.
The biological assays performed with compounds 14a-j showed that all isoxazoles displayed cytotoxicity, with IC50 values ranging 13.8-26.4 μM.
These values were lower than those obtained with the 1,2,3-triazole derivatives 7, suggesting that the pendant isoxazole ring is a more prominent
structural motif. This can be evidenced by comparing the activities observed for compounds with the same R substituent at the aromatic fragment
(for example, 14g, 14h and 14c with 7f, 7u and 7e, respectively), being the isoxazoles ~43% more potent (on average) than the corresponding
triazole analogues. In fact, the most active compound synthesized in this work was 14j, featuring a 5-Cl-2-furyl substituent, with an IC50 value of
13.8 μM. In order to expand our observations, we selected compounds 14a, 14c, 14e, 14g, 14h and 14j, as well as compounds 7e-g, 7j, 7t, and 7u
for further studies. The selection was made to include the most prominent members of each library, and to cover different elements of structural
diversity.
First, we tested the anti-proliferative activity in other cancer cell lines, such as lung (H1299) and colon (HT29). As shown in Table 3, the selected
compounds exhibited interesting results, with IC50 values slightly lower than those observed with MDA-MB-231 cells. Isoxazoles were still more
active than triazoles, being compounds 14h and 14j the most prominent members of the library.
Table 3. Anti-proliferative activity of selected compounds against lung (H1299) and colon (HT29) cancer cell lines.
IC50 (μM)
Entry
1
2
3
4
5
6
7
8
Compound
MDA-MB-231
24.8
H1299
10.3
10.6
10.9
14.3
9.0
12.0
9.8
11.2
7.1
HT29
18.3
14.7
13.4
17.2
15.6
11.9
16.9
19.8
13.7
14.5
9.0
7e
7f
7g
7j
7t
26.1
25.2
26.7
18.6
24.3
22.0
23.0
15.9
7u
14a
14c
14e
14g
14h
14j
9
10
11
12
18.3
15.2
13.6
9.0
6.1
7.6
10.1
To further characterize the selected compounds, we tested their effect on the non-tumorigenic Vero cell line in similar conditions. We found that
in all cases the IC50 increased comparing with the three tumorigenic cell lines, indicating a selective antiproliferative effect towards malignant cells
(see ESI). Comparing with H1299 and HT29 cells, IC50 values changed more than three-fold for several compounds. Although the differences were
less marked when we compared Vero with MDA-MB-231 cells, compounds 7t and 7u showed a clear increase of more than two-fold. Interestingly,
these same compounds also showed the highest selectivity index values (SI: IC50 non tumor/IC50 tumor) comparing Vero with H1299 and HT29 cells (with
up to almost 5- and 6-fold IC50 increase in the cases of 7u and 7t, respectively. Thus, our results indicate that some of the synthesised compounds
have a selective antiproliferative effect against tumor cells, particularly 7t and 7u.
Finally, we wondered if the cytotoxic effect depends on the presence of mutant p53. To test this hypothesis, similar survival assays were
performed upon p53 R280K knock down. MDA-MB-231 cells were transduced with plasmids coding for a specific shRNA for p53 (shp53) or an
unrelated sequence as a control (shControl).14 The reduction in mutant p53 levels was confirmed by western blot.
We found that survival was significantly increased upon mutant p53 knock down when cells were treated with all compounds tested (Figure 1a),
supporting the idea that mutant p53 is necessary to achieve higher activity. To further explore the dependence of the cytotoxic effect on the
presence of mutant p53 we analysed the effect of reintroducing p53 R280K in cells lacking expression of any p53 form. To that end we used H1299
cells, which lost expression of any p53 form due to a spontaneous deletion of the gene. Cells were transduced with plasmids coding for p53 R280K
of GFP as a control and the expression of mutant p53 was confirmed by western blot. In line with our results, we found that cytotoxicity was
significantly increased in cells expressing mutant p53 in all cases (Figure 1b). Therefore, our results showed that the analysed compounds displayed
a selective effect on mutant p53 expressing cells. This is a relevant feature, since p53 mutants are absent in normal cells. Consequently, selective
action on mutant p53 expressing cells may enhance specificity of the treatment, reducing the risk of adverse effects.
4

80
60
40
20
0
100
80
60
40
20
0
*
**
**
*
*
**
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*
*
*
7e
7f
7g
7j
7t
7u
14a
14c
14e
14g
14h
14j
7e
7f
7g
7j
7t
7u
14a
14c
14e
14g
14h
14j
Figure 1. a)
Cytotoxicity of selected triazoles and isoxazoles is reduced in MDA-MB-231 cells upon mutant p53 knock down. Survival assays for the indicated compounds (25 μM) on control cells
(shControl, black bars) or cells with stable knock down of p53 R280K (shp53,white bars). b) Cytotoxicity of selected triazoles and isoxazoles is enhanced upon introduction of p53 R280K in
p53 null H1299cells. Survival assays for the indicated compounds (10 μM) on p53 null H1299 lung carcinoma cells (black bars) or H1299 cells expressing p53 R280K (white bars). In both
cases, survival was normalized to DMSO control treatment and expressed as mean value and standard error of the mean (sem). One tail T-test, n=3, * p<0,05, ** p<0,01.
In conclusion, novel levoglucosenone-derived compounds have been designed and synthesized through an hetero Michael addition and 1,3-dipolar
cycloaddition sequence. The oxa-Michael reaction with propargyl alcohol, key to obtain the most active compounds, was optimized with the aid of
a design of experiments approach. All compounds were further evaluated as anticancer agents against MDA-MB-231 cell line. The incorporation of
a flexible spacer proved to be relevant in terms of the biological activity, as well as the incorporation of an oxygen atom at the C-4 position of the
anhydro pyranose. On the other hand, the nature of the heteroaromatic ring at the pendant side chain was also evaluated, being isoxazole more
active than the 1,2,3-triazole analogues. The most active compounds were tested against two additional cancer cell lines showing very good
results. A higher antiproliferative activity against tumor cells was observed for several of the identified compounds. Among them, 7t and 7u are
particularly interesting since they showed the highest selectivity for tumor cells, suggesting that they could provide a useful therapeutic window. In
addition, the identified compounds showed a significant dependence on the presence of mutant p53, a specific marker of tumor cells, further
underlining the potential of these compounds as leading molecules for antitumoral strategies.
The mechanism of action of compounds like PRIMA-1 and PRIMA-1MET, originally identified with the aim to reactivate wt p53-like functions in cell
expressing mutant p53, is still a matter of debate. A direct interaction between derivatives of these compounds and mutant p53 was
demonstrated.15 However, mechanisms that do not require direct interaction with mutant p53 were also proposed.16 In particular, PRIMA-1 and
PRIMA-1MET are able to enhance oxidative stress or activation of the Unfolded Protein Response, even in cells lacking p53 or bearing the wt form.
In some cases, the presence of mutant p53 may promote cytotoxicity by sensitizing cells to the stress conditions imposed by the presence of drug
treatment. Collectively, the available evidence suggests that these compounds may activate several mechanisms, inducing a response that is dose
and context dependent. In spite of its complexity, the presence of pleiotropic effects may also represent an advantage, since they may allow to
expand the application to wt or null p53 tumors, or to engage in synergistic responses with other anti-cancer agents. In the case of the compounds
characterized in this work, we cannot exclude the possibility that they act through more than one mechanism, which may even not require direct
interaction with mutant p53. Moreover, different mechanisms may be activated, depending on the specific chemical properties of each compound.
Future studies aimed to understand the involvement of mutant p53-dependent mechanisms in the observed effects will be important to
understand the potential application of these compounds. Nevertheless, our results showed at least partial selectivity towards the presence of
mutant p53, which may represent a potential advantage to increase the selective action on tumor cells. In this regard, these compounds provide
useful information to further explore structural features able to enhance activity and selectivity.
Acknowledgement
This research was supported by UNR (BIO 316 and 504), CONICET (PIP 0660), Ancient (PICT-2016-0116 and PICT-2016-2307), and
PhosAgro/UNESCO/IUPAC (Green Chemistry for Life Program, project 121). Y.H.T and C.M.B.E thank CONICET for the award of a fellowship. We
thank Drs. Maitena Martinez-Amezaga, Mario O. Salazar and Ricardo L. E. Furlan for helpful assistance with HPLC analysis.
Supplementary Data. Experimental details, optimization of the oxa-Michael reaction between 1 and 2, analytical data, copies of NMR spectra of all
compounds and HPLC data of selected compounds.
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INSTITUTO DE QUIMICA ROSARIO (CONICET)
FACULTAD DE CIENCIAS BIOQUIMICAS Y FARMACEUTICAS
UNIVERSIDAD NACIONAL DE ROSARIO
Suipacha 531, Rosario (2000), Argentina
Dr. Ariel M. Sarotti
Phone/Fax: +54-341-4370477
E-mail: sarotti@iquir-conicet.gov.ar
March 12th 2020
Dear Editor,
We have no competing interests to declare.
Yours sincerely,
Dr. Ariel M. Sarotti
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