Benzofuro[3,2-d]pyrimidines inspired from cercosporamide CaPkc1 inhibitor: Synthesis and evaluation of fluconazole susceptibility restoration

Article abstract of DOI:10.1016/j.bmcl.2018.05.044

In a context of growing resistance to classical antifungal therapy, the design of new drugs targeting alternative pathways is highly expected. Benzofuro[3,2-d]pyrimidine derivatives, derived from (?)-cercosporamide, were synthesized and evaluated as potential Candida albicans PKC inhibitors in the aim of restoring susceptibility to azole treatment. Co-administration assay of benzofuropyrimidinedione 23 and fluconazole highlighted a synergistic effect on inhibition of cell growth of a Candida albicans resistant strain.

Full text of DOI:10.1016/j.bmcl.2018.05.044

Accepted Manuscript
Benzofuro[3,2-d]pyrimidines inspired from cercosporamide CaPkc1 inhibitor:
synthesis and evaluation of fluconazole susceptibility restoration
Viet Hung Dao, Isabelle Ourliac-Garnier, Marc-Antoine Bazin, Catherine
Jacquot, Blandine Baratte, Sandrine Ruchaud, Stéphane Bach, Olivier Grovel,
Patrice Le Pape, Pascal Marchand
PII:
S0960-894X(18)30456-6
https://doi.org/10.1016/j.bmcl.2018.05.044
BMCL 25861
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 March 2018
20 May 2018
23 May 2018
Please cite this article as: Dao, V.H., Ourliac-Garnier, I., Bazin, M-A., Jacquot, C., Baratte, B., Ruchaud, S., Bach,
S., Grovel, O., Pape, P.L., Marchand, P., Benzofuro[3,2-d]pyrimidines inspired from cercosporamide CaPkc1
inhibitor: synthesis and evaluation of fluconazole susceptibility restoration, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.044
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Bioorganic & Medicinal Chemistry Letters
Benzofuro[3,2-d]pyrimidines inspired from cercosporamide CaPkc1 inhibitor:
synthesis and evaluation of fluconazole susceptibility restoration
Viet Hung Dao a, Isabelle Ourliac-Garnier b, Marc-Antoine Bazin a, Catherine Jacquot b, Blandine Baratte c,
Sandrine Ruchaud c, Stéphane Bach c, Olivier Grovel d, Patrice Le Pape b, Pascal Marchand a,
a Université de Nantes, Nantes Atlantique Universités, Département de Chimie Thérapeutique, EA1155 – IICiMed, Institut de Recherche en Santé 2, F-44200
Nantes, France
b Université de Nantes, Nantes Atlantique Universités, Département de Parasitologie et Mycologie Médicale, EA1155 – IICiMed, Institut de Recherche en Santé
2, F-44200 Nantes, France
c Sorbonne Université, CNRS, Plateforme de criblage KISSf (Kinase Inhibitor Specialized Screening facility), USR Protein Phosphorylation and Human
Diseases, Station Biologique, Place Georges Teissier, F-29688 Roscoff, France
d Université de Nantes, Nantes Atlantique Universités, EA2160 – Mer Molécules Santé, Faculté de Pharmacie, F 44035 Nantes, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
In a context of growing resistance to classical antifungal therapy, the design of new drugs targeting
alternative pathways is highly expected. Benzofuro[3,2-d]pyrimidine derivatives, derived from (–)-
cercosporamide, were synthesized and evaluated as potential Candida albicans PKC inhibitors in the
aim of restoring susceptibility to azole treatment. Co-administration assay of
Accepted
Available online
benzofuropyrimidinedione 23 and fluconazole highlighted a synergistic effect on inhibition of cell
growth of a Candida albicans resistant strain.
Keywords:
Cercosporamide
Benzofuro[3,2-d]pyrimidine
Antifungal activity
Candida albicans Pkc1
Fluconazole susceptibility restoration
———
 Corresponding author. Tel.: +33-253-009-155; e-mail: pascal.marchand@univ-nantes.fr (P. Marchand)

Invasive fungal infections are a major cause of mortality
worldwide, especially among vulnerable patients.1 Among the
35876 invasive fungal disease cases identified in France on the
2001-2010 period, 43.4% are candidemia; this number increases
among patients with hematologic malignancies and those with
chronic renal failure.2 Candida albicans remains the most
prevalent species in invasive candidiasis in the United States3 and
Europe.4 It is noteworthy that the proportion of infections caused
by non-albicans Candida (NAC) species such as C. glabrata, C.
parapsilosis, C. tropicalis and C. krusei has increased over the
last two decades, species involvement depending on the infection
site and the geography.5 Candida pathogenicity is caused by a
large number of mechanisms,6 such as adherence to the host
tissues and medical devices (biofilm), host recognition through
binding to host cells and proteins and production of extracellular
hydrolytic enzymes.7 Its virulence depends on fungal but also on
host factors in opportunistic situations.
justifying the strategy of keeping dihydroxybenzofuran-
carboxamide part of the natural product model for the design of
new tricyclic compounds (Figure 1).20
OH
O
O
CH3
HO
CH3
OH
H2N
O
O
(-)-Cercosporamide
OH
OH
H
N
O
N
NH
HO
NH
HO
O
O
O
O
H2N
H2N
O
O
19
OH
N
23
N
HO
H2N
O
27
Benzofuro[3,2-d]pyrimidine derivatives
NH2
In models of experimental infection, strains deleted for
elements of MAPK-mediated signal transduction pathways
exhibit a reduction or loss of virulence8 and decrease in biofilm
formation.9 In Candida albicans, protein kinase C (called
CaPkc1), one of the key proteins involved in MAPK pathways, is
described as a regulator of cell wall integrity during growth,
morphogenesis and response to cell wall stress.10 In addition, La
Fayette et al.11 established a new role for PKC signaling in drug
tolerance mechanisms. Given the limited number of antifungals
used in clinic staments12 and the emergence of drug resistance,13
there is an urgent need to identify alternative targets in order to
speed up the development of new generation of antifungals either
more effective or able to restore susceptibility to classical
antifungal drugs.14 In this context, targeting PKC-mediated signal
transduction pathway represents an new attractive strategy for
antifungal therapy.15
O
Fig. 1. Design of target compounds
In addition, our expertise in the synthesis of pyrimidine-fused
heterocycles as biological agents targeting kinases was used for
the design of the new compounds, constituting the third ring of
the benzofuro[3,2-d]pyrimidine derivatives described in this
study.23
Benzofuran scaffold was first built from phloroglucinol 1 to
achieve the suitable substitutions on the benzene ring and at the
positions 2 and 3 for subsequent ring closure sequence (Scheme
1). 3,5-Dibenzyloxyphenol 4 was obtained in three steps by an
initial tribenzylation of triacetoxyphloroglucinol 2 to avoid
additive C-benzylation by direct O-benzylation of phloroglucinol
1.21,24 Mono-deprotection was carried out under transfer
hydrogenation conditions using Pd/C and cyclohexene, in a
mixture of ethyl acetate/ethanol at reflux, to afford compound 4
in a moderate yield (Scheme 1).25 2-Halo-3,5-
dibenzyloxyphenols 5 and 6 were then synthesized by a mono-
bromination or a mono-iodination procedure, respectively, very
quickly at low temperature.26 Afterwards, O-alkylation was
realized using NaH as a base and ethyl bromoacetate at room
temperature providing esters 7 and 8 in excellent yields.27 In the
next step, cyanation in the presence of copper cyanide in DMF
gave ester 9 in a good yield from iodinated precursor 8 but the
corresponding reaction, from the brominated counterpart 7,
remained less effective (19% of yield).23b Cyclization of the
ethoxycarbonylmethylether 9 was performed in the presence of
NaH to furnish benzofuran derivative 10 bearing amino group at
position 3 and ester function at position 2.28 Finally,
(–)-Cercosporamide is a natural product isolated from the
phytopathogen fungus Cercosporidium henningsii.16 It was
identified as a broad-spectrum antifungal agent displaying in
vitro mean MIC value of 89 µg/mL17 and of 10 µg/mL18 against
C. albicans. Interestingly, it appeared to act as a potent CaPkc1
ATP-competitive inhibitor with an IC50 of 44 nM.18 Furthermore,
cercosporamide inhibited human PKC(IC50 = 1 µM) and
PKC (IC50 = 0.3 µM)18 and was later shown to inhibit other
human kinases, including Mnk1/2, Jak3, GSK3, ALK4 and
Pim1, from nanomolar to low micromolar ranges.19,20
To the best of our knowledge, heterocyclic compounds
displaying antifungal activity associated with CaPkc1 inhibition
are not highlighted in the literature, except cercosporamide.18
Consequently, in continuation of our successful attempts in the
search of biologically active cercosporamide inspired
derivatives,21 we report here the synthesis and antifungal
evaluation of benzofuro[3,2-d]pyrimidines targeting CaPkc1.
benzofuro[3,2-d]pyrimidin-4-one derivative 11 was obtained
through a ring closure reaction from benzofuran precursor 10 via
a formamide intermediate reacting with ammonia.27
The strategy of mimicking natural product for the design of
antifungal agents to combat fungal resistance was very recently
validated by the discovery of xanthones derived from α-
Unfortunately, the carbamoyl group in position 6 could not be
incorporated directly by electrophilic aromatic substitution using
chlorosulfonyl isocyanate (CSI) followed by an acidic hydrolysis
step.21
mangostin.22 In addition, cercosporamide was found to recognize
ATP-binding site of Mnk2 kinase through hydrogen bond
network due to the 3-OH and the 4-CONH2 of the phenyl portion,

OBn
X
OBn
X
OH
OAc
OBn
OBn
i
iii
ii
iv
v
OEt
BnO
O
BnO
OH
HO
OH
AcO
OAc
BnO
OBn
BnO
OBn
OH
O
N
5: X=Br
6: X=I
7: X=Br
8: X=I
1
2
3
4
OBn
OBn
CN
OBn
OBn
NH
NHCHO
O
N
2
NH
vi
vii
viii
ix
X
NH
O
O
OEt
O
O
BnO
BnO
O
O
O
O
OEt
OEt
BnO
BnO
BnO
CONH
O
2
9
10
11
12
Scheme 1. (i) Ac2O, pyridine, 120 °C, 5 h, 87%; (ii) BnCl, NaH, DMF, H2O, 0 °C to rt, 10 h, 96%; (iii) C6H10, Pd-C 10%, AcOEt/EtOH (3/1), 110 °C, 2 h, 45%;
(iv) NBS (1.0 eq.), CH2Cl2, -78 °C, 2 min, 95% for 5 or NIS (1.0 eq.), CH2Cl2, -78 °C, 2 min, 76% for 6; (v) NaH, BrCH2CO2Et, DMF, rt, 12 h, 83% for 7 and
96% for 8; (vi) CuCN, DMF, 160 °C, 1 h, 19% from 7 and 92% from 8; (vii) NaH, DMF, 0 °C, 30 min, 66%; (viii) HC(OEt)3, MW, 200 °C, 15 min then
NH3/MeOH 7N, MW, 140 °C, 15 min, 31%; (ix) CSI, CH3CN, rt, 24h then HCl 1N, rt, 24 h, failure.
To circumvent this issue, we decided to introduce CONH2
group at the beginning of the synthesis (Scheme 2). A direct
aminocarbonylation of phenol derivative 4 in the presence of CSI
afforded benzamide 13 after hydrolysis of the corresponding
chlorosulfonyl intermediate. Afterwards, the same reaction
sequence, as previously described in scheme 1, was applied to
obtain benzofuro[3,2-d]pyrimidin-4-one derivative 12 bearing the
carbamoyl appendage at C-6 position of the azaheterocycle. The
only difference was the possibility to perform the mono-
iodination at 0 °C vs -78 °C and according to the yields, it was
better to carry out first the cyanation (compound 17) and then O-
alkylation (compound 16). In the last step, benzyl cleavage was
accomplished with concentrated sulfuric acid to provide the
target compound 19.
The synthetic route developed for the preparation of the 2,4-
dioxo-1,2,3,4-tetrahydro[1]benzofuro[3,2-d]pyrimidine-6
carboxamide 23 is outlined in Scheme 3. To this end, O-
alkylation of the benzamide derivative 17 was performed by the
formation of the sodium salt in the presence of sodium hydride as
base followed by its reaction with 2-iodoacetamide as described
for the corresponding ester 16 (Scheme 2) but warming the
medium at 60 °C.29 Cyclization using KOH/EtOH at 60 °C
furnished benzofuran-2,7-dicarboxamide 21 in acceptable yield.
Indeed, we observed that these basic conditions were more
efficient than NaH/DMF combination (50% vs 60% of yield).
OBn
OBn
OBn
OBn
OBn
NH
2
2
OBn
I
CN
I
O
i
ii
iii
iv
v
vi
O
OEt
BnO
OH
CONH
BnO
OH
BnO
OCH CO Et
BnO
BnO
OCH CO Et
2
2
2
2
BnO
OH
CONH
CONH
2
CONH
CONH
2
2
2
4
13
14
15
16
18
iv
iii
OBn
OH
OBn
N
N
NH
NH
O
CN
OH
vii
O
O
O
BnO
BnO
HO
CONH
CONH
CONH
2
2
2
12
19
17
Scheme 2. (i) CSI, CH3CN, 0 °C, 10 min then HCl 1N, rt, 10 h, 53%; (ii) NIS, CH2Cl2, 0 °C, 10 min, 80%; (iii) NaH, BrCH2CO2Et, DMF, rt, 24 h, 86% for 15
and 90% for 16 (iv) CuCN, DMF, 160 °C, 6 h, 20% for 16 and 73% for 17; (v) NaH, DMF, 0 °C, 10 min, 67%; (vi) HC(OEt)3, MW, 200 °C, 15 min then
NH3/MeOH 7N, MW, 140 °C, 15 min, 62%; (vii) H2SO4cc, rt, 15 min, 53 %.
Benzofuropyrimidinedione derivative 22 was then synthesized
from benzofuran precursor 21 via an urea intermediate.30 Finally,
the dihydroxy analogue 23 was formed by treatment with
concentrated sulfuric acid as procedure of debenzylation.
O
O
H
N
H
N
OBn
OBn
OBn
NH
OBn
OBn
OH
2
NHCONH
2
CN
CN
O
NH
O
NH
O
iv
i
ii
iii
O
NH
BnO
OH
BnO
2
O
O
BnO
OCH CONH
2
NH
2
O
O
2
BnO
BnO
HO
CONH
CONH
CONH
2
2
2
CONH
2
CONH
CONH
2
2
17
20
21
22
23
Scheme 3. (i) NaH, ICH2CONH2, DMF, 60 °C, 12 h, 69%; (ii) KOH, EtOH, 60 °C, 30 min, 60%; (iii) 1) KOCN, CH3COOH, 100 °C, 1 h - 2) KOH, EtOH, 90
°C, 1 h, 56%; (iv) H2SO4cc, rt, 5 min, 48%.
Synthesis of the 4-aminobenzofuro[3,2-d]pyrimidine-6-
carboxamide derivative 27 started by O-alkylation of 3-cyano-2-
hydroxybenzamide 17 in the presence of K2CO3 and
furnished readily 3-amino-2-cyanobenzofuran-7-carboxamide 25
with suitable substitutions for the production of the fused 4-
aminopyridine ring (compound 26). Previous debenzylation
conditions were then applied leading to the unprotected analogue
27.
bromoacetonitrile in DMF at 80 °C.22b Heteroannulation of the
corresponding cyanomethylether intermediate 24 using NaH

OBn
OBn
OBn
NH
2
OBn
OH
2
N
N
CN
OH
CN
N
N
iv
i
ii
iii
CN
O
BnO
BnO
NH
NH
2
BnO
OCH CN
2
O
O
2
BnO
HO
CONH
CONH
CONH
2
2
CONH
CONH
2
2
17
24
25
26
27
Scheme 4. (i) BrCH2CN, K2CO3, DMF, 80 °C, 24 h, 72%; (ii) NaH, DMF, 0 °C, 5 min, 64%; (iii) HC(OEt)3, MW, 200 °C, 15 min then NH3/MeOH 7N, MW,
140 °C, 15 min, 45%; (iv) H2SO4cc, rt, 5 min, 66%.
Inhibition of PKC activity (CaPkc1) was initially investigated
in order to check the involvement of this protein as the putative
target of benzofuro[3,2-d]pyrimidine derivatives. Pkc activity of
C. albicans (CAAL2) total protein extracts was measured using
ELISA-based PKC Kinase Activity Assay (Enzo Life Sciences,
Inc.) as previously described.31 PKC inhibition activity was
expressed as the ratio between the absorbance measured for
protein extract treated with compounds 19, 23 or 27 (100 µM)
and untreated protein extract. The reference natural (–)-
As depicted in Figure 2A, when used at 100 M
cercosporamide displayed a moderate Pkc inhibitory activity of
393% in our conditions. Interestingly, compounds 19, 23, and
27 showed statistically (p < 0.05) significant higher inhibition
with values of 882%, 715% and 813%, respectively, at 100
M. The same experiments performed with active human PKC
showed that all the compounds also displayed inhibition of the
human enzyme counterpart (Figure 2B). All together these data
supported a moderate activity on PKCs. Moreover the absence of
selectivity for fungal CaPkc1 suggested potential additional
fungal targets for these compounds as discussed below.
cercosporamide was provided by extraction and purification from
a Mycosphaerella henningsii culture using a modified procedure
of the literature (see Supplementary Material).16,32
Fig 2. Percentage of inhibition of Pkc activity on protein extract of CAAL2 (A) and percentage of inhibition human PKCactivity (B). (cerco: cercosporamide,
19: compound 19, 23: compound 23, 27: compound 27). Mean percentage of inhibition of PKC activity were obtained for each compound (100 µM) and
compared with mean results obtained for cercosporamide (100 µM) applying a one-way ANOVA followed by multiple comparisons with an uncorrected Fisher’s
LSD test. p values were expressed for uncorrected Fisher’s LSD test.
Afterwards, compounds 19, 23 and 27 were assessed for their in
vitro antifungal properties alone or in combination with
(33 µg/mL) on CAAL93, CAAL97 and CAAL2 strains in
agreement with previous findings.17,18 Unfortunately, even at 300
µM (100 µg/mL), i. e. the highest tested concentration based on
Lafayette et al. previous work,11 no activity was measured for
cercosporamide on resistant strains CAAL28, CAAL111 and
CAAL117 (Table 1). Against all 6 strains studied, MICs for
compounds 19, 23, and 27 were greater than 300 µM.
Consequently, two hypotheses may arise from the intrinsic
antifungal inactivity of the compounds: they might not reach the
fungal cytosol or C. albicans Pkc1 would not be a main protein
to target suggesting that pkc is not an essential gene.
fluconazole against six C. albicans clinical isolates. Among the
six strains chosen for this study, CAAL93 and CAAL97 were
susceptible to fluconazole and CAAL2, CAAL28, CAAL111 and
CAAL117 exhibited resistance through well characterized and
distinct mechanisms, as we previously described.33 Minimal
inhibitory concentrations (MICs) were determined according to a
slightly modified EUCAST reference method34,35 and expressed
as the compound concentration that produced 50% of growth
inhibition compared to the drug-free growth control. In the
experiments, cercosporamide exhibited MICs of about 100 µM
Table 1. Activity of compounds 19, 23 and 27, cercosporamide and fluconazole on 6 clinical isolates of C. albicans and HeLa
MIC values (µM)a
Cpd
Structure
HeLa
CAAL93
CAAL97
CAAL 2
CAAL28
CAAL111
CAAL117

OH
N
NH
O
227.53
±27.97
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 100
> 300
> 300
> 300
> 300
> 100
> 300
> 300
> 300
> 300
> 100
19
O
HO
CONH
2
O
H
N
OH
NH
O
> 300
> 300
< 3
23
O
HO
CONH
2
OH
N
N
27
NH
O
2
HO
CONH
2
OH
O
O
3
CH
HO
H N
103.2
±23.2
93.3
±18.5
90.1
±11.3
CERCOb
CH
3
O
2
OH
O
0.14
±0.07
0.19
±0.11
FLUb
> 100
a Values represent the mean ±SD of experiments performed in triplicate. MIC were determined as the compound concentration that produced 50% of growth
inhibition relative to that of the drug-free growth control.
b CERCO: cercosporamide; FLU: fluconazole.
Initial hypothesis was that benzofuro[3,2-d]pyrimidine
derivatives could restore susceptibility of fluconazole-resistant
strains. Therefore, a checkerboard assay was performed with the
same methodology as MIC determination (see Supplementary
Material).36 MICs of compounds 19, 23, and 27 alone and in
combination with fluconazole were determined to calculate a
fractional inhibitory concentration index (FICI) (Table 2).37 FICI
values were used to classify interactions between fluconazole and
compounds 19, 23, or 27 as recommended by Odds:38 “synergy”
(FICI ≤ 0.5), “antagonism” (FICI > 4.0) and “no interaction”
(FICI > 0.5–4.0). Combination of cercosporamide and
(FICI = 0.50). Compound 23 and fluconazole showed a
promising synergistic effect on inhibition of cell growth of
CAAL111 strain (FICI = 0.35) contrary to combination of
cercosporamide and fluconazole against that strain. Interestingly,
this synergistic effect demonstrated interaction of the
benzofuro[3,2-d]pyrimidine derivative 23 at cellular level.
Compound 27 and fluconazole had no effect on cell growth when
combined, whatever the strain assessed. Compound 19 combined
with fluconazole decreased cell growth of CAAL2 and
CAAL117 strains but calculation of FICI did not allow to
conclude to any interaction.
fluconazole exhibited a synergistic effect only against CAAL2
Table 2. MIC results of checkerboard assays testing the combination of compounds 19, 23 and 27 with fluconazole against 4
fluconazole-resistant C. albicans clinical isolates.
MIC (µM) and FICIa determination
FLU
19
FLU/19b
FICI19 23
FLU/23b
FICI23 27
FLU/27b FICI27 cerco FLU/cercob
FICIcerco
0.50
2.0
> 100
> 100
> 100
> 100
> 300 4/300
> 300 > 100/> 300 2.0
> 300 > 100/> 300 2.0
> 300 > 100/> 3002.0
> 300 > 100/> 3002.0
> 300 > 100/> 3002.0
> 300 > 100/> 3002.0
100
0.5/50
CAAL2
1.06
> 300 > 100/> 300 2.0
> 300 > 100/> 300 2.0
> 300 > 100/> 300
> 300 > 100/> 300
> 300 100/> 300
CAAL28
CAAL111
CAAL117
> 300 50/30
2.0
0.35
> 300 50/300
> 300 > 100/> 300 2.0
2.0
1.25
a FICI was the sum of the FICs of each of the drugs, which in turn is defined as the MIC of each drug when used in combination divided by the MIC of the drug
when used alone. Highest FICI value could not be greater than 2.0 due to highest concentrations tested for each compound.
b MIC in combination expressed as [FLU]/[compound]
As MAP kinases were shown to be involved in biofilm
formation,9 an assay was performed to explore the potential
antibiofilm effect of cercosporamide and compound 19.
CAAL121, CAAL124 and CAAL146 were selected for their
ability to form biofilm. After 24h of incubation, mature biofilm
was treated for 24h with cercosporamide or compound 19.
Minimal inhibitory concentrations (MICs) was determined as the
antifungal concentration that produced 50 % of biofilm growth
inhibition compared to the drug-free growth control. For
cercosporamide, MICs of about 120 µM were measured on
CAAL124 and CAAL146, whereas no effect was observed on
CAAL121, even at 300 µM. In addition, no effect on biofilm
growth was observed for compound 19 on any strain. Taking into
account the results obtained for cercosporamide, inhibiting Pkc1
could be of interest in targeting biofilm growth. However, more
experiments are necessary on all the benzofuro[3,2-d]pyrimidine
series to develop more in depth the first biological data.
To investigate in vitro cytotoxic activity of benzofuro[3,2-
d]pyrimidine derivatives, assay on Hela cells was performed.
Compounds 19, 23, and 27 proved to be, at least 100 times, less
cytotoxic than cercosporamide (Table 1).
Due to the common reports of tricyclic pyrimidine as kinase
inhibitors19,20 and the reported polypharmacology of the class,
compounds 19, 23, and 27 and cercosporamide were evaluated by
a kinase screen to investigate their selectivity profile.23 Kinases
available for this study were HsCDK2/CyclinA, HsCDK5/p25,
HsCDK9/CyclinT, HsPIM1, HsHaspin, MmCLK1, RnDYRK1A,
SscGSK3α/β, SscCK1δ/ε and HsAurora B. As described in the
literature,19 cercosporamide showed inhibitory activity at sub-
micromolar range against CDK2 (IC50= 0.77 µM) and CDK9
(IC50= 0.22 µM) (Table 3). In addition, lower inhibitory activity
was measured against Aurora B (IC50= 2.40 µM), CDK5 (IC50=
5.60 µM) and PIM1 (IC50= 8.10 µM), and no activity was
detected against the other kinases. Tricyclic pyrimidinone 19 and
its pyrimidinedione counterpart 23 remained inactive on the

panel of kinases suggesting that compound 23 restoring
susceptibility of fluconazole-resistant strains (CAAL111), could
be the result of PKC inhibition. Finally, 4-aminopyrimidine
derivative 27 exhibited significant kinase inhibition towards
PIM1 (IC50= 1.29 µM), CLK1 (IC50= 2.69 µM) and DYRK1A
(IC50= 8.23 µM) offering new potential targets for this
sequences with C. albicans genome, Fun31p, a serine/threonine
kinase of this pathogen exhibits 51 % similarity with the kinase
domain of human PIM1. This fungal kinase being implicated in
cell wall damage regulation in Candida albicans could be a
putative target for compound 27.39
compound. Indeed, after performing alignment of human kinase
Table 3. Inhibitory activity of benzofuro[3,2-d]pyrimidines 19, 23, 27 and cercosporamide against a panel of 10 protein kinases.
IC50 (M)a
Cpd
Structure
CDK2/
CyclinA
CDK5/
p25
CDK9/
CyclinT
Haspin
>10
PIM1
>10
CLK1
>10
DYRK1A
>10
GSK3α/β
CK1δ/ε
AurKB
>10
OH
N
NH
O
>10
>10
>10
0.77
>10
>10
>10
5.60
>10
>10
>10
0.22
>10
>10
19
O
HO
CONH
2
O
H
N
OH
NH
O
>10
1.29
8.10
>10
>10
>10
>10
2.69
>10
>10
8.23
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.40
23
O
HO
HO
CONH
2
OH
N
N
27
NH
O
2
CONH
2
OH
O
O
3
CH
HO
H N
CH
3
CERCOb
O
2
OH
O
a Values are the mean of at least two independent determinations and are within ± 15% SD.
b (–)-Cercosporamide is used as reference compound.
5.
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pharmacomodulations are still requested to improve the effects
observed in this study.
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Authors are thankful to T. Robiou du Pont for performing
fungal seedings and cultures, and M.-C. Boumard for helping in
separation and purification of cercosporamide. They also
acknowledge Dr. B. Misme-Aucouturier for helping in statistical
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(Infrastructures en Biologie Santé et Agronomie, France),
Biogenouest (Western France life science and environment core
facility network) for supporting kinase screening facility (KISSf,
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Graphical abstract
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Benzofuro[3,2-d]pyrimidines inspired from
cercosporamide CaPkc1 inhibitor: synthesis
and evaluation of fluconazole susceptibility
restoration
Leave this area blank for abstract info.
Viet Hung Dao, Isabelle Ourliac-Garnier, Marc-Antoine Bazin, Catherine Jacquot, Blandine Baratte, Sandrine
Ruchaud, Stéphane Bach, Olivier Grovel, Patrice Le Pape, Pascal Marchand
OH
OH
CH3
H
N
O
O
O
MIC values ( M) on C. albicans strain
HO
CH3
OH
NH
HO
FLU
23
FLU / 23*
O
H2N
O
> 100
> 300
50 / 30
O
H2N
O
O
*synergistic
combination
23
(-)-Cercosporamide

HIGHLIGHTS
Heterocyclic compounds inspired from
cercosporamide were synthesized
Synergistic effect with fluconazole on inhibition of
cell growth of a Candida albicans resistant strain is
reported.
Kinase inhibitory activity was detected