Synthesis and antiproliferative evaluation of glucosylated pyrazole analogs of K252c

Article abstract of DOI:10.1016/j.tet.2018.01.017

Pyrazole analogs of the staurosporine aglycon K252c were recently described as potent inhibitors of the three Pim protein kinase isoforms. To evaluate the impact of the introduction of a sugar moiety on the biological activities of this heterocyclic scaffold, four new glucosylated pyrazole analogs of K252c were synthesized. Their biological evaluation demonstrated that most active compounds 11 and 19 substituted by a β-D-glucosyl moiety at N12 or N13 positions exhibited antiproliferative activities toward HCT116 cells.

Full text of DOI:10.1016/j.tet.2018.01.017

Accepted Manuscript
Synthesis and antiproliferative evaluation of glucosylated pyrazole analogues of
K252c
Bachir Douara, Yannick J. Esvan, Elisabeth Pereira, Francis Giraud, Yulia L.
Volodina, Dmitry N. Kaluzhny, Alexander A. Shtil, Fabrice Anizon, Pascale Moreau
PII:
S0040-4020(18)30039-5
10.1016/j.tet.2018.01.017
TET 29232
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 December 2017
Revised Date: 8 January 2018
Accepted Date: 9 January 2018
Please cite this article as: Douara B, Esvan YJ, Pereira E, Giraud F, Volodina YL, Kaluzhny DN, Shtil
AA, Anizon F, Moreau P, Synthesis and antiproliferative evaluation of glucosylated pyrazole analogues
of K252c, Tetrahedron (2018), doi: 10.1016/j.tet.2018.01.017.
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ACCEPTED MANUSCRIPT
Graphical Abstract
N
N
HN
HN
N
N
H
N
H
N
O
OH
O
OH
HO
HO
HO
OH
OH
OH
11
IC50 HCT116 = 12 µM
19
IC50 HCT116 = 11 µM
1

ACCEPTED MANUSCRIPT
Synthesis and antiproliferative evaluation of glucosylated pyrazole analogues of K252c
Bachir Douara‡,§,┴, Yannick J. Esvan‡,┴, Elisabeth Pereira‡, Francis Giraud‡, Yulia L. Volodinaǁ,
Dmitry N. Kaluzhny#, Alexander A. Shtilǁ,†, Fabrice Anizon‡,*, Pascale Moreau‡,*
‡ Université Clermont Auvergne, CNRS, ICCF, F-63000 Clermont-Ferrand, France
§ Laboratoire de Phytochimie et de Pharmacologie, Département de Chimie, Faculté des Sciences
Exactes et Informatique, Université Mohamed Seddik Ben Yahia, 18000 Jijel, Algeria
ơ Blokhin National Medical Center of Oncology, 115478 Moscow, Russian Federation
# Engelhardt Institute of Molecular Biology, 119991 Moscow, Russian Federation
† ITMO University, 197101 Saint-Petersburg, Russian Federation
┴ Both authors (BD, YJE) equally contributed to this work.
*Author to whom correspondence should be addressed:
Phone number: +33 (0) 4 73 40 79 63 (PM)
Fax number: +33 (0) 4 73 40 77 17.
E-mail address: pascale.moreau@uca.fr
Phone number: +33 (0) 4 73 40 53 64 (FA)
Fax number: +33 (0) 4 73 40 77 17.
E-mail address: fabrice.anizon@uca.fr
2

ACCEPTED MANUSCRIPT
Abstract:
Pyrazole analogs of the staurosporine aglycon K252c were recently described as potent inhibitors
of the three Pim protein kinase isoforms. To evaluate the impact of the introduction of a sugar
moiety on the biological activities of this heterocyclic scaffold, four new glucosylated pyrazole
analogues of K252c were synthesized. Their biological evaluation demonstrated that most active
compounds 11 and 19 substituted by a β-D-glucosyl moiety at N12 or N13 positions exhibited
antiproliferative activities toward HCT116 cells.
Keywords: indolocarbazole, K252c analogs, glucosylated derivatives, antiproliferative activity.
3

ACCEPTED MANUSCRIPT
1.
Introduction
A wide range of natural or synthetic indolocarbazole derivatives demonstrated important
biological activities such as antibiotic and antitumor properties [1]. Staurosporine (Figure 1A) was
the first indolocarbazole to be isolated in 1977 from Streptomyces staurosporeus [2]. It was found
to be a highly potent, broad spectrum protein kinase inhibitor [3], and is now frequently used as a
positive control research tool. However, due to this large range of protein kinase inhibition,
staurosporine is far too toxic to be used as a drug. Therefore numerous structure-activity
relationship studies including the modification of the heteroaromatic part and/or the sugar moiety
were undertaken to improve the inhibitory/pharmacological profile of new staurosporine analogs.
This approach was successful for Midostaurin (PKC412, Figure 1A) which is a multi-targeting
protein kinase inhibitor that recently reached the market for treatment of acute myeloid leukemia
with FLT3 mutation in combination with other antitumor drugs. Midostaurin (RYDAPT®) was also
approved for treatment of aggressive systemic mastocytosis, systemic mastocytosis with associated
hematological neoplasm, or mast cell leukemia [4].
A)
H
N
H
N
H
N
O
H
O
O
N
N
N
N
N
N
O
O
H
H
CH3
H
O
CH3
K252c
OCH3
OCH3
NHCH3
NCH3
Staurosporine
Midostaurin, PKC412
B)
6
N
5
HN
N
N
7
HN
HN
4
8
3
9
2
10
N
N
H
N
N
N
N
13 12
1
11
H
H
H
O
OR
O
OR
Compound A
RO
RO
RO
OR
OR
OR
11
19
This work
R = Bn, H
Previous work
Figure 1. A) Chemical structures of staurosporine, midostaurin and K252c. B) Previous work and
this work.
4

ACCEPTED MANUSCRIPT
As part of our research program dedicated to the discovery of new heteroaromatic
derivatives with biological potential, we recently reported the synthesis and activities of
pyrazol(on)e analogs of the staurosporine aglycon K252c (Figure 1A) [5]. The most active
compound in that series was A (Figure 1B) that inhibited the three Pim (proviral integration site for
Moloney murine leukemia virus) protein kinase isoforms in the submicromolar range; IC50 values
were 0.15 µM, 0.59 µM and 22 nM for Pim-1, Pim-2 and Pim-3, respectively. Whereas protein
kinase C family enzymes (PKCs) are known targets of K252c, these results demonstrated that the
modification of the upper ring changed the kinase inhibition profile leading to a Pim inhibitor
without activity toward PKCs [6]. As Pim kinases are involved in cell cycle progression and
apoptosis inhibition [7] and are activated in human cancers such as acute myeloid leukemia and
colon carcinoma [8,9], the antiproliferative activity of compound A was evaluated toward leukemia
K562 and colon carcinoma HCT116 cell lines. Micromolar potencies were determined for both cell
lines. The comparison of K252c (K562 IC50 = 7.8 µM, HCT116 IC50 = 1.72 µM) and staurosporine
(K562 IC50 = 0.071 µM, HCT116 IC50 = 0.006 µM) demonstrated that the presence of a sugar
moiety enhanced the potency by > 100- to 250-fold [10–13] due to a better cellular penetration and
kinase inhibition. The nature of the sugar residue introduced on the heteroaromatic aglycon of
staurosporin analogues affects considerably the related biological activities [14]. Numerous studies
reported the preparation of new glycosylated analogues of indolocarbazoles either by chemical
synthesis (e.g. aza-analogs of staurosporin) [15] or by combinatorial biosynthesis [16]. To start this
study, in order to improve the cellular potencies of compound A, we prepared glucosylated analogs
bearing a β-D-glucosyl unit on the 12- or 13-position of the indolocarbazole moiety (Figure 1B).
Four new synthesized compounds, with sugar hydroxyl groups protected or not, were evaluated in
regard to their antiproliferative activity.
2.
Results and discussion
2.1 Chemistry
As reported for the synthesis of compound A (Scheme 1), a pyrazolone moiety was first
synthesized from racemic ketoester 2 in the presence of hydrazine and acetic acid, with concomitant
cleavage of the Boc protecting groups [5]. The pyrazole nucleus was then obtained in two steps
(triflation and reduction) before a photo-induced cyclization/oxidation in the presence of iodine to
give compound A.
5

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of compound A.
The ketoester 2 was prepared from carboxylic acid 3 and ester 4 (Scheme 2). In order to
prepare the two regioisomeric glucosylated analogs of compound A, we firstly foresaw that this
coupling reaction could also be performed from glucosylated analogs of 3 or 4 (compounds 7 and
13, respectively, Scheme 2). Therefore, for the first regioisomer, we prepared compound 7 in two
steps from benzylated β-D-glucopyranosyl-1H-indole 5, whose synthesis was also optimized based
on our previously described procedure [17]. Unfortunately, the activated carboxylic acid prepared
from 7 by reaction with oxalyl chloride or the corresponding methyl ester showed no reactivity with
the enolate prepared from ester 4.
6

ACCEPTED MANUSCRIPT
O
COOEt
COOEt
CO2H
R1= β-D-glucosyl, R2= Boc
+
or
N
R1
R1= Boc, R2= β-D-glucosyl
N
R2
R2= Boc
N
N
R1
R2
3
7
R1= Boc
4
2 R1=R2=Boc
R1= β-D-glucosyl 13 R2= β-D-glucosyl
CHO
N
1- D-glucose, EtOH, H2O
2- NaH, BnBr, THF
3- DDQ, AcOEt
N
POCl3, DMF
89%
OBn
O
OBn
O
N
H
58% (3 steps)
BnO
BnO
OBn
OBn
OBn
OBn
5
6
KH2PO4, NaClO2
2-methylbut-2-ene
H2O, tBuOH
63%
N
HN
CO2H
1- activation
2- LDA, 4
N
N
N
H
no reaction
OBn
O
OR
O
BnO
RO
OBn
R = H, Bn
OBn
OR
OR
7
Scheme 2. First synthetic pathway to prepare glucosyled derivatives.
Therefore another synthetic pathway was examined. Initially, for the synthesis of compound
A from 2 and during our attempts to carry out the pyrazolone formation with hydrazine, we applied
a method reported by Braña et al. using hydrazine hydrate in the presence of camphoric acid [18].
Under these conditions pyrazolone was not formed but the cleavage of a single Boc protecting
group was achieved leading to ketoester 8 (Scheme 3). The structure of 8 was confirmed by 2D
NMR experiments, showing that the deprotected indole was the one attached at the C3 position to
the ketone function. This mono-deprotection was an opportunity to perform a regioselective
glycosylation. Accordingly, the NH being more acidic due to the presence of the carbonyl group at
the C3 position, a Mitsunobu reaction could be performed between 8 and 2,3,4,6-tetra-O-benzyl-α-
D-glucopyranose to obtain a mixture of two diastereoisomers. Then, as for compound A, the
pyrazole derivative 9 was synthesized after pyrazolone formation in the presence of hydrazine and
acetic acid, then triflation and reduction to pyrazole in two steps. The indolocarbazole was then
obtained after photo-induced cyclization/oxidation in the presence of iodine, leading to compound
7

ACCEPTED MANUSCRIPT
10 in 60% yield. Finally, debenzylation using boron tribromide was achieved in 76% yield to
produce glucosylated indolo[2,3-a]pyrazolo[3,4-c]carbazole 11.
.
NH2-NH2 H2O
H
O
COOEt
O
COOEt
H
camphoric acid
EtOH
70%
N
N
N
N
Boc
Boc
H
Boc
2
8
1- PPh3, DIAD,
2,3,4,6-tetra-O-benzyl- -D-glucopyranose
.
2- NH2-NH2 H2O, EtOH/AcOH 1:1
3- PhN(Tf)2, NEt3, CH2Cl2
4- Pd(OAc)2, dppf, HCO2NH4, DMF
15% (4 steps)
N
N
HN
HN
h , I2
CH3CN
N
N
N
N
H
H
60%
OR
OBn
O
O
RO
BnO
9
OR
OR
OBn
OBn
10 R= Bn
BBr3, CH2Cl2
76%
11 R = H
Scheme 3. Synthesis of compounds 10 and 11. For compound 8, arrows indicate long range 1H-13C
coupling observed in 2D NMR HMBC experiment.
For the synthesis of the regioisomer 19 glucosylated at N12 position, we used a similar
strategy as in Scheme 2: glycosylated indole-3-acetate 13 was first prepared in two steps from
compound 5 (Scheme 4). First, the ethyl oxoacetate 12 was synthesized in 83% yield before
reduction with triethylsilane to give compound 13 in 43% yield. The lithium enolate of this ester
was prepared and reacted with activated carboxylic acid synthesized from 3 to give ketoester 14 as a
mixture of diastereoisomers. Pyrazole formation was then performed from keto-ester 14 as
presented above. However, with the conditions used for the pyrazolone/pyrazole formation, the Boc
protecting group was only partially cleaved. This led to a mixture of major Boc-protected and minor
Boc-deprotected products (compounds 15a and 15b) that were separated before the palladium-
catalyzed reduction of the triflate intermediates. The major product 15a led to pyrazole 16 after
reduction, and an additional Boc-deprotection step with K2CO3/MeOH/H2O yielded compound 17.
Compound 17 can also be prepared from the minor Boc-deprotected triflate intermediate 15b. To
summarize, pyrazole 17 was obtained in 42% yield from starting material 13. Finally, 17 was
cyclized and then deprotected as described above for 9, to give compound 19.
8

ACCEPTED MANUSCRIPT
O
CO2Et
CO2Et
Et3SiH, TFA
CH2Cl2
1- (COCl)2, Et2O
2- EtOH, NEt3
N
N
N
OBn
O
OBn
O
OBn
O
BnO
83%
43%
BnO
BnO
OBn
OBn
OBn
OBn
13
OBn
OBn
12
5
O
HO
(COCl)2
pyridine, DMF
LDA, THF
N
Boc
3
N
N
O
TfO
TfO
EtO2C
NH
NH
.
1- NH2-NH2 H2O
EtOH/AcOH 1:1
N
N
N
N
N
N
Boc
+
Boc
H
OBn
14
O
2- PhN(Tf)2, NEt3
CH2Cl2
OBn
15a major
OBn
OBn
15b minor
OBn
O
O
BnO
BnO
BnO
OBn
OBn
OBn
OBn
Pd(OAc)2, dppf
HCO2NH4, DMF
Pd(OAc)2, dppf
HCO2NH4, DMF
N
N
N
NH
NH
NH
h , I2
K2CO3
CH3CN
MeOH/H2O
N
N
H
N
N
N
N
Boc
65%
H
89%
OBn
O
OBn
16
O
OBn
O
BnO
BnO
BnO
18
17
OBn
OBn
OBn
OBn
OBn
OBn
BBr3, CH2Cl2
69%
39% from 13
8% from 13
N
NH
N
N
H
OH
O
HO
19
OH
OH
Scheme 4. Synthesis of compounds 18 and 19.
9

ACCEPTED MANUSCRIPT
2.2 Biological evaluations
Firstly, we evaluated the antiproliferative activities of compounds 10, 11, 18 and 19 toward
HCT116 human colon carcinoma cell line. As indicated in Table 1, only compounds 11 and 19
bearing a deprotected β-D-glucosyl moiety at N13 or N12 positions were found to be active with
similar IC50 values of 12 µM and 11 µM, respectively. Compounds 10 and 18 bearing a benzylated
sugar moiety were inactive with IC50 values > 50 µM, probably due to a too high lipophilicity and a
poor cellular penetration (cLogP > 5). These results demonstrated that in this pyrazole series, the
introduction of a β-D-glucosyl moiety on the heteroaromatic moiety did not enhance the
antiproliferative potency.
% residual activity (± SD)
HCT116 cell line
DNA Kb
104 M-1
Cpds
cLogP
(IC50, µM)
Pim-1
PKCα
108 ± 3
118 ± 3
70 ± 2
95 ± 9
3.58
9.06
1.68
9.06
1.68
4.2
> 50
12
2 ± 0
A
IC50 = 16 µM [5]
4.6 ± 0.4
118 ± 5
101 ± 2
95 ± 33
86 ± 5
10
11
18
19
1.92 ± 0.03
2.35 ± 0.05
2.12 ± 0.04
> 50
11
117 ± 32
Table 1. cLogP calculated with Osiris DataWarrior [19,20]. Antiproliferative activities of
compounds A, 10, 11, 18 and 19. Kinase inhibitory potencies are expressed as % of residual kinase
activity at 10 µM compound concentration. DNA binding constants (Kb) determined by fluorescent
titration.
Then we evaluated the activity of compounds 10, 11, 18 and 19 toward known cellular
targets of indolocarbazoles (Pim-1, PKCα, DNA binding). Compared to the non-glycosylated
analogue A (Figure 1) [5] we observed a significant decrease of activity toward Pim-1 indicating
that the glycosylation was detrimental to Pim-1 inhibition. None of the compounds tested exhibited
significant activities toward the targets evaluated. Therefore, the antiproliferative activity of
compounds 11 and 19 is associated with the modulation of other cellular targets that have not been
identified yet.
3.
Conclusion
10

ACCEPTED MANUSCRIPT
In our efforts to design original aza-heterocyclic compounds with antitumor properties, we
synthesized new glucosylated pyrazole analogues of K252c. Most active compounds 11 and 19
were substituted by a deprotected glucosyl moiety at N12 or N13 positions. Compared to non-
glycosylated analogue A, these compounds exhibited similar antiproliferative activities toward
HCT116 cells with IC50 values around 10 µM. Altogether, these results indicated that the
antiproliferative activities of these compounds are not correlated to the inhibition of Pim-1 and
PKCα kinases or DNA binding. The synthesis of new analogues and the identification of the
cellular targets involved in the antiproliferative activities of this series are currently under progress.
4. Experimental
4.1. Chemistry
4.1.1. General
Starting materials were obtained from commercial suppliers and used without further purification.
IR spectra were recorded on a Perkin-Elmer Spectrum 65 FT-IR spectrometer ( in cm-1). NMR
13
spectra, performed on a Bruker AVANCE 400 III HD (1H: 400 MHz, C: 101 MHz), or a Bruker
AVANCE III HD 500 (1H: 500 MHz, 13C: 126 MHz), are reported in ppm using the solvent residual
peak as an internal standard; the following abbreviations are used: singlet (s), doublet (d), triplet (t),
quadruplet (q), doublet of doublets (dd), doublet of doublet of doublets (ddd), multiplet (m), broad
signal (br s). High resolution mass spectra were determined on a high-resolution Waters Micro Q-
Tof or Thermo Scientific Q Exactive Q-Orbitrap apparatus (UCA START, Université Clermont
Auvergne, Clermont-Ferrand, France). Chromatographic purifications were performed by column
chromatography using 40–63 ꢀm silica gel. Reactions were monitored by TLC using fluorescent
silica gel plates (60 F254 from Merck). Melting points were measured on a Stuart SMP3 apparatus
and are uncorrected.
4.1.2. 1-(2,3,4,6-Tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-1H-indole (5)
Step 1. -Glucose (7.05 g, 39 mmol) was added to a solution of indoline (9.4 mL, 84 mmol) in
D
ethanol (200 mL) and water (10 mL). The mixture was refluxed for 24 h and the solvents were
evaporated to give a brown oil (16.85 g). TLC Rf = 0.33 (EtOAc/MeOH 9:1).
Step 2. A solution of intermediate from step 1 (5 g) in anhydrous THF (120 mL) was cooled to 0 °C
and NaH (60% dispersion in mineral oil, 5.52 g, 138 mmol) and then benzyl bromide (16.2 mL, 136
mmol) were added. The solution was refluxed for 17 h and the solvent was evaporated. EtOAc (250
11

ACCEPTED MANUSCRIPT
mL) was added and the mixture was washed with water and dried over MgSO4. TLC Rf = 0.26
(cyclohexane/EtOAc 9:1). The solution was directly used for step 3.
To characterize the indoline intermediate, a part of the crude from step 2 was also purified by
column chromatography (cyclohexane/EtOAc) to give 1-(2,3,4,6-tetra-O-benzyl-1-deoxy-β-D-
glucopyranos-1-yl)-2,3-dihydro-1H-indole as a beige solid. mp 119 °C; IR (ATR): 1606, 1491,
1
1454 cm-1; H NMR (400 MHz, DMSO-d6) δ 2.83–3.01 (2H, m), 3.44–3.65 (5H, m), 3.66–3.72
(1H, m), 3.74 (1H, t, J = 9 Hz), 3.82 (1H, t, J = 9 Hz), 4.40 (1H, d, J = 12 Hz), 4.47 (1H, d, J = 12
Hz), 4.56 (1H, d, J = 11 Hz), 4.73–4.89 (5H, m), 5.07 (1H, d, J = 9 Hz), 6.66 (1H, t, J = 7.5 Hz),
13
6.77 (1H, d, J = 8 Hz), 7.02 (1H, t, J = 7.5 Hz), 7.07 (1H, d, J = 7 Hz), 7.17 –7.34 (20H, m); C
NMR (101 MHz, DMSO-d6) δ 27.7, 46.0, 68.6, 72.1, 73.1, 74.0, 74.6 (CH2), 75.4, 78.0, 78.5, 84.4,
85.3 (CH), 108.1, 118.3, 124.5, 127.0, 127.339, 127.344, 127.41, 127.47, 127.49, 127.54, 127.6,
127.7, 128.08, 128.14, 128.20, 128.22 (CHarom), 129.8, 138.2 (2C), 138.61, 138.64, 150.4 (Carom);
HRMS (ESI+) calcd for C42H44NO5 (M+H)+ 642.3214, found 642.3217.
Step 3. DDQ (6 g, 26.4 mmol) was added by portions to the EtOAc solution from step 2. After
addition, the mixture was stirred at room temperature for 45 min, washed with a 10% aqueous
Na2SO3 solution and dried over MgSO4. After evaporation, the residue was purified by column
chromatography (cyclohexane/EtOAc 9:1) to give 5 as a brown gum (4.33 g, 6.77 mmol, 58%).
TLC Rf = 0.21 (cyclohexane/EtOAc 9:1). The 1H NMR spectra of the product corresponds to that of
1-(2,3,4,6-tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-1H-indole reported in the literature [14].
4.1.3. 1-(2,3,4,6-Tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-1H-indole-3-carbaldehyde (6)
POCl3 (0.43 mL, 4.6 mmol) was slowly added to anhydrous DMF (1.5 mL) at 0 °C under argon
atmosphere. The solution was stirred at 0 °C for 40 min and a solution of 5 (2.66 g, 4.16 mmol) in
DMF (1 mL) was added at 0 °C and stirred for 1 h and at 35 °C for an additional hour. Ice and an
aqueous NaOH solution (1.85 g in 5 mL H2O) were added. The mixture was heated at 100 °C for 30
min. After cooling and extraction with EtOAc, the combined organic phases were washed with
water, brine and dried over MgSO4. The residue was purified by column chromatography
(cyclohexane/EtOAc 8:2) to give 6 (2.47 g, 3.7 mmol, 89%) as a beige gum. TLC Rf = 0.47
1
(cyclohexane/EtOAc 7:3); IR (ATR): 1662, 1535, 1455 cm-1; H NMR (400 MHz, DMSO-d6) δ
3.67–3.74 (2H, m), 3.77 (1H, d, J = 11 Hz), 3.80 (1H, t, J = 9.5 Hz), 3.94–3.99 (1H, m), 3.95 (1H, t,
J = 9 Hz), 4.14 (1H, t, J = 9 Hz), 4.36 (1H, d, J = 11 Hz), 4.47 (1H, d, J = 12 Hz), 4.52 (1H, d, J =
12 Hz), 4.63 (1H, d, J = 11 Hz), 4.81 (1H, d, J = 11 Hz), 4.86 (2H, s), 5.93 (1H, d, J = 9 Hz), 6.67
(2H, d, J = 7.5 Hz), 7.04 (2H, t, J = 7.5 Hz), 7.12 (1H, t, J = 7.5 Hz), 7.21–7.36 (17H, m), 7.78–7.84
12

ACCEPTED MANUSCRIPT
13
(1H, m), 8.13–8.20 (1H, m), 8.56 (1H, s), 9.97 (1H, s); C NMR (101 MHz, DMSO-d6) δ 68.5,
72.3, 73.7, 74.2, 74.6 (CH2), 76.5, 77.5, 80.0, 84.0, 84.5 (CH), 112.1, 121.2, 123.1, 124.0, 127.47,
127.54, 127.56, 127.59, 127.62, 127.65, 127.67, 127.8, 127.9, 128.23, 128.26, 128.28, 138.7
(CHarom), 118.5, 124.6, 136.9, 137.1, 138.0, 138.1, 138.4 (Carom), 185.3 (C=O); HRMS (ESI+) calcd
for C43H42NO6 (M+H)+ 668.3007, found 668.3010.
4.1.4. 1-(2,3,4,6-Tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-1H-indole-3-carboxylic acid (7)
A solution of KH2PO4 (3.76 g, 27.6 mmol) and NaClO2 (80% unstabilized, 4.16 g, 36.8 mmol) in
H2O (15 mL) was added to a solution of 6 (2.46 g, 3.68 mmol) in t-butanol (20 mL) and 2-
methylbut-2-ene (8 mL). The mixture was vigorously stirred at room temperature for 24 h. The two
layers were separated and the aqueous phase was extracted with EtOAc. The combined organic
phases were dried over MgSO4 and evaporated. The residue was purified by column
chromatography (cyclohexane/EtOAc 8:2) to give 7 (1.58 g, 2.31 mmol, 63%) as a beige solid. TLC
1
Rf = 0.48 (cyclohexane/EtOAc 6:4); mp 70 °C; IR (ATR): 1662, 1537, 1455 cm-1; H NMR (400
MHz, DMSO-d6) δ 3.65 (1H, d, J = 10.5 Hz), 3.68–3.74 (2H, m), 3.80 (1H, t, J = 9.5 Hz), 3.89–
3.94 (1H, m), 3.91 (1H, t, J = 9 Hz), 4.16 (1H, d, J = 9 Hz), 4.30 (1H, d, J = 10.5 Hz), 4.47 (1H, d, J
= 12 Hz), 4.53 (1H, d, J = 12 Hz), 4.62 (1H, d, J = 11 Hz), 4.81 (1H, d, J = 11 Hz), 4.85 (2H, s),
5.88 (1H, d, J = 9 Hz), 6.68 (2H, d, J = 7.5 Hz), 7.06 (2H, t, J = 7.5 Hz), 7.10–7.15 (1H, m), 7.21–
7.36 (17H, m), 7.76–7.81 (1H, m), 8.09–8.14 (1H, m), 8.30 (1H, s), 12.24 (1H, br s); 13C NMR (101
MHz, DMSO-d6) δ 68.6, 72.3, 73.6, 74.2, 74.5 (CH2), 76.6, 77.4, 80.0, 84.1, 84.5 (CH), 112.0,
121.1, 122.0, 122.8, 127.46, 127.51, 127.56, 127.59, 127.64, 127.7, 127.8, 127.9, 128.23, 128.24,
128.3, 133.0 (CHarom), 108.4, 126.5, 136.3, 137.2, 138.09, 138.11, 138.5 (Carom), 165.5 (C=O),
between 127.4 and 128.3 ppm, one benzylic CHarom peak could not be observed; HRMS (ESI–)
calcd for C43H40NO7 (M-H)– 682.2799, found 682.2808.
4.1.5. tert-Butyl 3-(1-ethoxy-3-(1H-indol-3-yl)-1,3-dioxopropan-2-yl)-1H-indole-1-carboxylate (8)
To a solution of compound 2 (330 mg, 0.6 mmol) and camphoric acid (120 mg, 0.6 mmol) in EtOH
(6 mL) was added hydrazine monohydrate (30 µL, 0.6 mmol). After stirring for 2 h at room
temperature, hydrazine monohydrate (0.3 mL) was additionally added and the mixture was stirred at
reflux for 1 h. After concentration under reduced pressure, the residue was dissolved in EtOAc, the
solution was washed with a saturated aqueous NaHCO3 solution, brine, dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by flash chromatography
(cyclohexane/EtOAc 8:2) yielding compound 8 (188 mg, 0.42 mmol, 70%) as a yellowish foam.
13

ACCEPTED MANUSCRIPT
TLC Rf =0.3 (cyclohexane/EtOAc 7:3); mp 179 °C; IR (ATR): 3193, 1740, 1724, 1619, 1147 cm-1;
1H NMR (400 MHz, DMSO-d6) δ 1.17 (3H, t, J = 7.1 Hz), 1.62 (9H, s), 4.09–4.23 (2H, m), 6.11
(1H, s), 7.16–7.25 (3H, m), 7.30 (1H, ddd, J1 = 8.4 Hz, J2 = 7.3 Hz, J3 = 1.4 Hz), 7.44–7.50 (1H,
m), 7.81 (1H, s), 7.82 (1H, d, J = 7.8 Hz), 8.02 (1H, d, J = 8.2 Hz), 8.14–8.17 (1H, m), 8.63 (1H, s),
13
12.11 (1H, br s); C NMR (101 MHz, DMSO-d6) δ 14.0, 27.7 (CH3), 61.0 (CH2), 52.0 (CH), 84.0
(C), 112.3, 114.6, 120.8, 121.3, 122.2, 122.5, 123.3, 124.4, 125.3, 135.5 (CHarom), 114.9, 115.0,
125.6, 129.3, 134.6, 136.7 (Carom), 148.9, 168.8, 187.6 (C=O); HRMS (ESI+) calcd for C26H27N2O5
(M+H)+ 447.1915, found 447.1924.
4.1.6. 3-(4-(1H-Indol-3-yl)-1H-pyrazol-5-yl)-1-(2,3,4,6-tetra-O-benzyl-1-deoxy-β-
D-glucopyranos-
1-yl)-1H-indole (9)
Step 1. Diisopropyl azodicarboxylate (738 µL, 3.75 mmol) was added dropwise to a cooled solution
(–78 °C) of 8 (405 mg, 0.91 mmol), triphenylphosphine (1.0 g, 3.81 mmol) and 2,3,4,6-tetra-O-
benzyl-α-D-glucopyranose (2.02 g, 3.74 mmol) in THF (25 mL). The mixture was allowed to reach
room temperature and stirred for 18 h. The solvent was removed under reduced pressure and the
residue was chromatographed on silica gel (gradient from pure cyclohexane to EtOAc/cyclohexane
1:9) yielding β-glucosylated product diastereoisomers (530 mg, TLC Rf
=
0.55
(EtOAc/cyclohexane 2:8) as a colorless oil. The crude product was used in the next step without
further purifications.
Step 2. In a sealed reaction tube, a solution of the glucosylated intermediate from step 1 (574 mg)
and hydrazine monohydrate (1.34 mL, 27.6 mmol) in a 1:1 v/v EtOH/AcOH mixture (6 mL) was
heated at 110 °C (oil bath) for 24 h. After cooling, water was added and the mixture was extracted
with EtOAc, the assembled organic phases were washed with water, brine, and dried over MgSO4.
After evaporation, brown oil was obtained which was dissolved in a minimum of acetone before
precipitation by addition of cyclohexane. After evaporation under reduced pressure, the pyrazolone
intermediate (523 mg) was obtained as a beige gum used without further purifications.
Step 3. To a solution of the pyrazolone intermediate from step 2 (517 mg) and PhNTf2 (355 mg,
0.99 mmol) in CH2Cl2 (8 mL), at 0 °C under argon, was added NEt3 (0.9 mL, 6.5 mmol) and the
mixture was allowed to reach room temperature. After 1 hour of stirring, solvent was evaporated
under reduced pressure. EtOAc was added and the mixture was washed with water, brine and dried
over MgSO4. After evaporation, the residue was chromatographed on silica gel
(EtOAc/cyclohexane 5:95) yielding the triflate intermediate (380 mg, TLC Rf = 0.39
14

ACCEPTED MANUSCRIPT
EtOAc/cyclohexane 2:8, HRMS (ESI+) calcd for C54H48F3N4O8S (M+H)+ 969.3139, found
969.3139) as a pale yellow oil.
Step 4. In a sealed reaction tube, a mixture of pyrazolyl triflate from step 3 (380 mg), Pd(OAc)2 (22
mg, 0.098 mmol), dppf (54 mg, 0.098 mmol) and HCO2NH4 (249 mg, 3.95 mmol) in DMF (14 mL)
was heated at 90 °C for 2 h. After cooling, EtOAc was added and the mixture was filtered through a
Celite pad. The filtrate was washed with water, dried over MgSO4 and evaporated under reduced
pressure. The residue was purified by column chromatography (cyclohexane/EtOAc 7:3) yielding
compound 9 (122 mg, 0.148 mol, 15% from 8) as a colorless oil. TLC Rf = 0.3
(EtOAc/cyclohexane 4:6); IR (ATR): 3418, 1455, 1361, 1068 cm-1; 1H NMR (500 MHz, CD3CN) δ
3.47 (1H, d, J = 10.5 Hz), 3.62–3.80 (6H, m), 4.10 (1H, d, J = 10.5 Hz), 4.45 (1H, d, J = 12.0 Hz),
4.50 (1H, d, J = 12.0 Hz), 4.61 (1H, d, J = 10.9 Hz), 4.79–4.85 (3H, m), 5.48 (1H, d, J = 8.6 Hz),
6.62–6.65 (2H, m), 6.88 (1H, ddd, J1 = 8.0 Hz, J2 = 7.1 Hz, J3 = 1.0 Hz), 7.00–7.13 (6H, m), 7.19
(1H, ddd, J1 = 8.3 Hz, J2 = 7.1 Hz, J3 = 1.1 Hz), 7.22–7.34 (15H, m), 7.39 (1H, d, J = 8.2 Hz), 7.41
(1H, d, J = 8.2 Hz), 7.43 (1H, s), 7.57 (1H, d, J = 8.3 Hz), 7.74 (1H, d, J = 8.0 Hz), 7.80 (1H, br s),
9.19 (1H, br s), the pyrazole NH was not observed; 13C NMR (126 MHz, CD3CN) δ 69.8, 73.8,
75.0, 75.6, 76.0 (CH2), 78.2, 78.6, 82.0, 85.6, 85.9 (CH), 111.8, 112.4, 120.31, 120.34, 121.5,
122.3, 122.6, 123.4, 124.2, 125.6, 128.44, 128.46, 128.47, 128.6, 128.69, 128.74, 128.90, 128.92,
128.96, 129.2, 129.3, 134.6a (CHarom), 109.0, 110.2, 113.3b, 128.0, 128.1, 137.17, 137.22, 138.5,
139.38, 139.39, 139.8, 140.9b (Carom), between 128.4 and 129.3 ppm, one benzylic CHarom peak
a
1
could not be observed; chemical shift measured from a HSQC H–13C experiment, b chemical
1
shift measured from a HMBC H–13C experiment; HRMS (ESI+) calcd for C53H49N4O5 (M+H)+
821.3675, found 821.3701.
4.1.7. 13-(2,3,4,6-Tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-12,13-dihydro-5H-indolo[2,3-
a]pyrazolo[3,4-c]carbazole (10)
A solution of compound 9 (122 mg, 0.149 mmol) in freshly distilled CH3CN (130 mL) in a Pyrex
photochemical reactor was degassed with argon for 30 min, and iodine (84 mg, 0.33 mmol) was
added. The solution was additionally degassed for 15 min and was irradiated for 45 min with a 400
W medium-pressure mercury lamp. The solvent was removed under reduced pressure. The residue
was dissolved in EtOAc and the solution was washed with a 1% aqueous sodium metabisulfite
solution, water and dried over MgSO4. The residue was purified by column chromatography
(CH2Cl2/EtOAc 9:1) to give 10 (73 mg, 0.089 mmol, 60%) as a pale yellow solid. TLC Rf = 0.4
1
(EtOAc/Cyclohexane 4:6); mp 115°C; IR (ATR): 3350, 1455, 1329, 1067 cm-1; H NMR (500
15

ACCEPTED MANUSCRIPT
MHz, CD3CN) δ 2.78 (1H, d, J = 10.0 Hz), 3.65 (1H, d, J = 10.0 Hz), 3.84 (1H, dd, J1 = 10.4 Hz, J2
= 2.4 Hz), 3.97–4.02 (3H, m), 4.10–4.14 (1H, m), 4.32–4.39 (1H, m), 4.71 (1H, d, J = 12.6 Hz),
4.74 (1H, d, J = 12.6 Hz), 4.76 (1H, d, J = 10.8 Hz), 4.81 (1H, d, J = 11.4 Hz), 4.83 (1H, d, J = 11.4
Hz), 4.95 (1H, d, J = 10.8 Hz), 6.02–6.05 (2H, m), 6.23–6.28 (1H, m), 6.76–6.81 (2H, m), 6.91–
6.95 (1H, m), 7.19–7.29 (10H, m), 7.31–7.39 (7H, m), 7.48–7.55 (3H, m), 7.82 (1H, d, J = 8.2 Hz),
8.34–8.37 (1H, m), 8.49–8.52 (1H, m), 8.70 (1H, s), 10.64 (1H, br s), the pyrazole NH was not
observed; 13C NMR (126 MHz, CD3CN) δ 67.8, 74.3, 74.9, 75.8, 76.0 (CH2) 77.2, 77.9, 82.0, 85.2,
85.8 (CH) 112.0, 113.0, 120.8, 121.6, 121.7, 122.1, 125.5 (2C), 128.3, 128.4, 128.56, 128.57, 128.7,
128.9, 129.0, 129.14, 129.16, 129.3, 129.5, 129.6, 131.5 (CHarom), 108.0, 113.7, 113.8, 123.5,
124.2, 124.8, 127.3, 133.9, 137.6, 138.4, 139.3, 139.6, 140.1, 140.6 (Carom); HRMS (ESI+) calcd for
C53H47N4O5 (M+H)+ 819.3541, found 819.3541.
4.1.8. 13-(1-deoxy-β-D-glucopyranos-1-yl)-12,13-dihydro-5H-indolo[2,3-a]pyrazolo[3,4-
c]carbazole (11)
To a solution of 10 (73 mg, 0.089 mmol) in CH2Cl2 (30 mL) at –78 °C was added BBr3 (1 M in
CH2Cl2, 1.07 mL, 1.07 mmol). After stirring 15 min at –78 °C, water was added and the mixture
was allowed to reach room temperature. After extraction with EtOAc, the assembled organic phases
were dried over MgSO4 and the solvent was evaporated under reduced pressure. The residue was
purified by column chromatography (EtOAc to EtOAc/MeOH 9:1) to give 11 (31 mg, 0.068 mmol,
76%) as a greenish beige solid. TLC Rf = 0.53 (EtOAc/MeOH 9:1); mp 246 °C; IR (ATR): 3700–
1
3000, 1460, 1328 cm-1; H NMR (500 MHz, DMSO-d6) δ 3.55–3.61 (1H, m), 3.64–3.70 (1H, m),
3.80–3.85 (1H, m), 3.91–3.95 (1H, m), 4.02–4.13 (2H, m), 4.83 (1H, d, J = 5.5 Hz), 5.11 (1H, d, J =
5.4 Hz), 5.35 (1H, d, J = 5.2 Hz), 5.88 (1H, t, J = 4.1 Hz), 6.21 (1H, d, J = 8.9 Hz), 7.27 (1H, ddd,
J1 = 7.9 Hz, J2 = 7.0 Hz, J3 = 1.0 Hz), 7.34 (1H, ddd, J1 = 7.8 Hz, J2 = 7.1 Hz, J3 = 0.7 Hz), 7.39
(1H, ddd, J1 = 8.1 Hz, J2 = 7.0 Hz, J3 = 1.1 Hz), 7.44 (1H, ddd, J1 = 8.2 Hz, J2 = 7.1 Hz, J3 = 1.1
Hz), 7.64 (1H, d, J = 8.1 Hz), 7.91 (1H, d, J = 8.6 Hz), 8.39 (1H, d, J = 7.8 Hz), 8.64 (1H, d, J = 7.6
13
Hz), 8.79 (1H, s), 11.28 (1H, s), 13.66 (1H, br s); C NMR (101 MHz, DMSO-d6) δ 58.4 (CH2),
67.6, 73.0, 76.9, 78.5, 84.6 (CH), 111.52, 112.05, 119.2, 120.1, 120.60, 120.63, 123.7 (2C), 131.5a
a
(CHarom), 106.6b, 111.47, 111.99, 121.7, 123.0, 123.2, 125.7, 131.3b, 138.7, 139.8 (Carom),
1
b
chemical shift measured from a HSQC H–13C experiment, chemical shift measured from a
1
HMBC H–13C experiment; HRMS (ESI+) calcd for C25H23N4O5 (M+H)+ 459.1663, found
459.1671.
16

ACCEPTED MANUSCRIPT
4.1.9. Ethyl 2-(1-(2,3,4,6-tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-1H-indol-3-yl)-2-
oxoacetate (12)
Oxalyl chloride (0.84 mL, 9.6 mmol) was added to a solution of 5 (3.13 g, 4.89 mmol) in Et2O (75
mL) at 0 °C and the mixture was stirred at room temperature for 8 h. After evaporation, the residue
was dissolved in EtOH (40 mL) and NEt3 (0.7 mL, 5 mmol) was added. After stirring overnight, the
solvent was evaporated and the residue was purified by column chromatography
(cyclohexane/EtOAc 85:15) to give 12 (3 g, 4.05 mmol, 83%) as a light brown gum. TLC Rf = 0.49
1
(cyclohexane/EtOAc 8:2); IR (ATR): 1726, 1649, 1455 cm-1; H NMR (400 MHz, DMSO-d6) δ
1.32 (3H, t, J = 7 Hz), 3.69–3.76 (3H, m), 3.84 (1H, t, J = 9.5 Hz), 3.91–3.96 (1H, m), 3.92 (1H, t, J
= 9 Hz), 4.18 (1H, t, J = 9 Hz), 4.34–4.40 (3H, m), 4.48 (1H, d, J = 12 Hz), 4.53 (1H, d, J = 12 Hz),
4.63 (1H, d, J = 11 Hz), 4.81 (1H, d, J = 11 Hz), 4.86 (2H, s), 5.95 (1H, d, J = 9 Hz), 6.67 (2H, d, J
= 7.5 Hz), 7.03 (2H, t, J = 7.5 Hz), 7.11 (1H, t, J = 7.5 Hz), 7.21–7.40 (17H, m), 7.83–7.88 (1H, m),
13
8.20–8.26 (1H, m), 8.65 (1H, s); C NMR (101 MHz, DMSO-d6) δ 13.9 (CH3), 61.8, 68.4, 72.2,
73.8, 74.2, 74.6 (CH2), 76.7, 77.3, 79.7, 84.6 (2C) (CH), 112.63, 121.5, 123.7, 124.3, 127.46,
127.54, 127.59, 127.61, 127.68, 127.70, 127.86, 127.92, 128.2, 128.26, 128.28, 138.5 (CHarom),
112.65, 126.0, 136.2, 137.0, 138.0, 138.1, 138.4 (Carom), 163.1, 179.4 (C=O), between 127.4 and
128.3 ppm, one benzylic CHarom peak could not be observed; HRMS (ESI+) calcd for C46H46NO8
(M+H)+ 740.3218, found 740.3224.
4.1.10. Ethyl 2-(1-(2,3,4,6-tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-1H-indol-3-yl)acetate
(13)
Triethylsilane (0.69 mL, 4.3 mmol) and TFA (2.13 mL, 28 mmol) were added to a solution of 12
(540 mg, 0.73 mmol) in CH2Cl2 (8 mL). The mixture was stirred at room temperature for 36 h.
After addition of a saturated aqueous NaHCO3 solution, the mixture was extracted with EtOAc and
the combined organic phases were dried over MgSO4 and evaporated. The residue was purified by
column chromatography (cyclohexane/EtOAc 85:15) to give 13 (230 mg, 0.32 mmol, 43%) as a
light brown gum. TLC Rf = 0.43 (cyclohexane/EtOAc 8/2); IR (ATR): 1731, 1461, 1363, 1071 cm-
1
1; H NMR (400 MHz, Acetone-d6) δ 1.15 (3H, t, J = 7 Hz), 3.66 (1H, d, J = 10.5 Hz), 3.77–3.81
(1H, m), 3.80 (2H, s), 3.84–3.93 (4H, m), 4.01–4.08 (1H, m), 4.08 (2H, q, J = 7 Hz), 4.20 (1H, d, J
= 10.5 Hz), 4.53 (1H, d, J = 12 Hz), 4.61 (1H, d, J = 12 Hz), 4.74 (1H, d, J = 11 Hz), 4.91 (1H, d, J
= 11 Hz), 4.93 (1H, d, J = 11 Hz), 4.96 (1H, d, J = 11.5 Hz), 5.67 (1H, d, J = 9 Hz), 6.77 (2H, d, J =
7 Hz), 7.07 (2H, t, J = 7 Hz), 7.10–7.22 (3H, m), 7.23–7.40 (15H, m), 7.51 (1H, s), 7.65 (1H, d, J =
13
7.5 Hz), 7.69 (1H, d, J = 8 Hz); C NMR (101 MHz, Acetone-d6) δ 14.5 (CH3), 31.7, 60.9, 69.8,
17

ACCEPTED MANUSCRIPT
73.8, 74.7, 75.5, 75.9 (CH2), 78.3, 78.7, 81.9, 86.1, 86.2 (CH), 112.1, 120.0, 120.7, 122.8, 125.8,
128.18, 128.21, 128.3, 128.41, 128.44, 128.68, 128.69, 129.03, 129.06, 129.09 (CHarom); 110.1,
129.5, 137.3, 138.6, 139.5, 139.6, 139.9 (Carom), 171.9 (C=O), between 128.1 and 129.1 ppm, two
benzylic CHarom peaks could not be observed; HRMS (ESI+) calcd for C46H48NO7 (M+H)+
726.3425, found 726.3440.
4.1.11. tert-Butyl 3-(4-(1-(2,3,4,6-tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-1H-indol-3-yl)-
1H-pyrazol-5-yl)-1H-indole-1-carboxylate (16)
Preparation of the activated carboxylic acid. Oxalyl chloride (0.33 mL, 3.8 mmol) was added
dropwise to a solution of 3 (0.5 g, 1.9 mmol), pyridine (0.93 mL, 11.5 mmol) and DMF (7 ꢀL) in
CH2Cl2 (6 mL) under argon. The mixture was stirred at room temperature for 15 h and the solvent
was evaporated under reduced pressure. The residue was purified by column chromatography
(EtOAc/cyclohexane, 2:8) to give activated carboxylic acid as a white solid (436 mg).
Step 1. BuLi (2.5 M in hexanes, 0.62 mL, 1.55 mmol) was added to a solution of diisopropylamine
(0.26 mL, 1.84 mmol) in anhydrous THF (4 mL) at 0 °C. After 30 min, the mixture was cooled to –
78 °C and a solution of 13 (500 mg, 0.69 mmol) in THF (2 mL) was added. The mixture was stirred
at –78 °C for 1 h and a solution of activated carboxylic acid (210 mg) in THF (4 mL) was added
dropwise. The mixture was allowed to reach room temperature and stirred for 2 h. After
evaporation, EtOAc was added and the mixture was washed with an 8% aqueous NaHCO3 solution,
dried over MgSO4 and evaporated. The residue was chromatographed on silica gel
(cyclohexane/AcOEt 9:1) to give the ketoester intermediate 14 as a mixture of diastereoisomers
(465 mg) as a light brown solid which was used directly for the next step.
Step 2. In a sealed reaction tube, a solution of the ketoester intermediate from step 1 (400 mg) and
hydrazine monohydrate (0.93 mL, 19 mmol) in a 1:1 v/v EtOH/AcOH mixture (4 mL) was heated at
110 °C (oil bath) for 45 min. After cooling, water was added and the mixture was extracted with
EtOAc, the assembled organic phases were washed with a 1 M aqueous HCl solution, brine, and
dried over MgSO4. After evaporation, a brown oil was obtained which was dissolved in a minimum
of acetone before precipitation by addition of cyclohexane. After evaporation under reduced
pressure, the pyrazolone intermediate (360 mg) was obtained as a brown foam which was used
directly for the next step.
Step 3. To a solution of the pyrazolone intermediate from step 2 (360 mg) and PhNTf2 (247 mg,
0.69 mmol) in CH2Cl2 (5 mL), at 0 °C under argon, was added NEt3 (0.58 mL, 4.16 mmol) and the
mixture was allowed to reach room temperature. After 1 hour of stirring, solvent was evaporated
18

ACCEPTED MANUSCRIPT
under reduced pressure. EtOAc was added and the mixture was washed with water, brine and dried
over MgSO4. After evaporation, the residue was chromatographed on silica gel
(cyclohexane/EtOAc 7:3) to give the pyrazolyl triflate 15a as a major product (384 mg, TLC Rf 0.83
in cyclohexane/EtOAc 6:4, light brown oil, HRMS (ESI+) calcd for C59H56F3N4O10S (M+H)+
1069.3664, found 1069.3671) and its Boc deprotected analog 15b (67 mg, TLC Rf 0.53 in
cyclohexane/EtOAc 6:4, beige oil, HRMS (ESI+) calcd for C54H48F3N4O8S (M+H)+ 969.3140,
found 969.3139), which were used without further purification for the next step.
Step 4. In a sealed reaction tube, a mixture of pyrazolyl triflate 15a (210 mg), Pd(OAc)2 (4.8 mg,
0.022 mmol), dppf (11.95 mg, 0.022 mmol) and HCO2NH4 (71 mg, 1.12 mmol) in DMF (2.5 mL)
was heated at 90 °C for 2 hours. After cooling, EtOAc was added and the mixture was filtered
through a Celite pad. The filtrate was washed with water, dried over MgSO4 and evaporated under
reduced pressure. The residue was purified by column chromatography (cyclohexane/EtOAc 7:3) to
give 16 (116 mg, 0.126 mmol, 39% from 13) as a beige solid. TLC Rf = 0.61 (cyclohexane/EtOAc
1
6:4); mp 88 °C; IR (ATR): 3500–3000, 1732, 1453, 1367, 1153, 1054 cm-1; H NMR (400 MHz,
CD3CN) δ 1.38 (9H, s), 3.58 (1H, d, J = 10.5 Hz), 3.70–3.86 (5H, m), 3.96 (1H, t, J = 8.8 Hz), 4.16
(1H, d, J = 10.5 Hz), 4.48 (1H, d, J = 12.0 Hz), 4.53 (1H, d, J = 12.0 Hz), 4.65 (1H, d, J = 10.9 Hz),
4.82–4.89 (3H, m), 5.54 (1H, d, J = 8.9 Hz), 6.64–6.67 (2H, m), 6.90–6.95 (2H, m), 7.01–7.06 (1H,
m), 7.09 (1H, ddd, J1 = 8.0 Hz, J2 = 7.1 Hz, J3 = 0.9 Hz), 7.14 (1H, ddd, J1 = 8.1 Hz, J2 = 7.6 Hz, J3
= 1.0 Hz), 7.20–7.34 (17H, m), 7.39 (1H, s), 7.39–7.42 (1H, m), 7.48 (1H, s), 7.65 (1H, d, J = 8.3
13
Hz), 7.76 (1H, s), 8.01 (1H, d, J = 7.9 Hz), 8.05 (1H, d, J = 8.3 Hz), 10.60–11.90 (1H, br s); C
NMR (101 MHz, CD3CN) δ 28.1 (CH3), 69.9, 73.8, 75.0, 75.6, 76.1 (CH2), 78.2, 78.7, 81.8, 86.05,
86.14 (CH), 84.6 (C), 112.3, 115.8, 121.2, 121.4, 122.7, 123.4, 123.8, 125.46, 125.50, 125.6, 128.4,
128.5, 128.6, 128.72, 128.73, 128.8, 128.96, 128.97, 129.24, 129.25, 129.29, 132.9 (CH), 110.1,
113.0, 114.1, 129.4, 130.1, 136.1, 137.3, 138.6, 139.4, 139.5, 139.8, 143.3 (C), 150,2 (C=O),
between 128.4 and 129.3 ppm, one benzylic CHarom peak could not be observed; HRMS (ESI+)
calcd for C58H57N4O7 (M+H)+ 921.4222, found 921.4230.
4.1.12. 3-(5-(1H-Indol-3-yl)-1H-pyrazol-4-yl)-1-(2,3,4,6-tetra-O-benzyl-1-deoxy-β-
D-glucopyranos-
1-yl)-1H-indole (17)
To a solution of 16 (138 mg, 0.15 mmol) in 3:1 v/v MeOH/H2O (16 mL) was added K2CO3 (77 mg,
0.56 mmol) and the mixture was refluxed for 3 h. After cooling, a saturated aqueous NH4Cl solution
was added. After extraction with EtOAc, drying over MgSO4 and evaporation, the residue was
purified by column chromatography (cyclohexane/EtOAc 6:4) to give 17 (110 mg, 0.134 mmol,
19

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89%) as a beige solid. TLC Rf = 0.24 (cyclohexane/EtOAc 6:4); mp 82 °C; IR (ATR): 3416, 1455
cm-1; 1H NMR (500 MHz, CD3CN) δ 3.59 (1H, d, J = 10.5 Hz), 3.69–3.79 (4H, m), 3.82 (1H, t, J =
9 Hz), 3.90 (1H, t, J = 9 Hz), 4.18 (1H, d, J = 10.5 Hz), 4.47 (1H, d, J = 12 Hz), 4.52 (1H, d, J = 12
Hz), 4.63 (1H, d, J = 11 Hz), 4.82–4.85 (2H, m), 4.86 (1H, d, J = 11.5 Hz), 5.54 (1H, d, J = 9 Hz),
6.68 (2H, d, J = 7.5 Hz), 6.91 (1H, ddd, J1 = 8 Hz, J2 = 7 Hz, J3 = 1 Hz), 7.01 (1H, ddd, J1 = 8 Hz,
J2 = 7 Hz, J3 = 1 Hz), 7.02–7.06 (2H, m), 7.08 (1H, ddd, J1 = 8 Hz, J2 = 7 Hz, J3 = 1 Hz), 7.10–7.14
(2H, m), 7.18 (1H, ddd, J1 = 8.5 Hz, J2 = 7 Hz, J3 = 1 Hz), 7.23–7.37 (17H, m), 7.38 (1H, s), 7.59
(1H, d, J = 8.5 Hz), 7.71 (1H, d, J = 8 Hz), 7.75 (1H, s), 9.20 (1H, s), 9.6–12.6 (1H, br s); 13C NMR
(126 MHz, CD3CN) δ 69.9, 73.9, 75.0, 75.6, 76.1 (CH2), 78.2, 78.8, 82.0, 85.8, 86.1 (CH), 111.9,
112.42, 120.7, 121.1, 121.2, 121.4, 122.9, 123.2, 124.9, 125.1, 128.51, 128.53, 128.54, 128.7,
128.80, 128.83, 128.9, 128.977, 128.982, 129.28, 129.31, 129.32, 135.8a (CHarom), 108.3, 110.9,
a
112.45, 127.1, 129.01, 137.1, 137.4, 138.7, 139.43, 139.47, 139.8, 140.7b (Carom), chemical shift
1
b
1
measured from a HSQC H–13C experiment, chemical shift measured from a HMBC H–13C
experiment; HRMS (ESI+) calcd for C53H49N4O5 (M+H)+ 821.3698, found 821.3706.
Compound 17 can also be directly prepared from the minor Boc-deprotected pyrazolyl triflate
intermediate 15b. In a sealed reaction tube, a mixture of 15b (126 mg), Pd(OAc)2 (7.3 mg, 0.0325
mmol), dppf (18 mg, 0.032 mmol) and HCO2NH4 (82 mg, 1.3 mmol) in DMF (6 mL) was heated at
90 °C for 2 hours. After cooling, EtOAc was added and the mixture was filtered through a Celite
pad. The filtrate was washed with water, dried over MgSO4 and evaporated under reduced pressure.
The residue was purified by column chromatography (cyclohexane/EtOAc 7:3) to give 17 (70 mg,
0.085 mmol, 8% from 13) as a beige solid.
4.1.13. 12-(2,3,4,6-Tetra-O-benzyl-1-deoxy-β-D-glucopyranos-1-yl)-12,13-dihydro-5H-indolo[2,3-
a]pyrazolo[3,4-c]carbazole (18)
A solution of 17 (110 mg, 0.134 mmol) in freshly distilled CH3CN (120 mL) in a Pyrex
photochemical reactor was degassed with argon for 30 min, and iodine (76 mg, 0.30 mmol) was
added. The solution was additionally degassed for 15 min and was irradiated for 15 min with a 400
W medium-pressure mercury lamp. The solvent was removed under reduced pressure. The residue
was dissolved in EtOAc and the solution was washed with a 1% aqueous sodium metabisulfite
solution, water and dried over MgSO4. The residue was purified by column chromatography
(cyclohexane/EtOAc 6:4) to give 18 (71 mg, 0.087 mmol, 65%) as a beige solid. TLC Rf = 0.5
1
(cyclohexane/EtOAc 1:1); mp 215 °C; IR (ATR): 3337, 1454, 1240, 1070 cm-1; H NMR (400
MHz, CD3CN) δ 2.70 (1H, d, J = 9.9 Hz), 3.58 (1H, d, J = 9.9 Hz), 3.77 (1H, dd, J1 = 10.4 Hz, J2 =
20

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2.3 Hz), 3.88–3.97 (3H, m), 4.03–4.08 (1H, m), 4.27–4.35 (1H, m), 4.64 (1H, d, J = 12.6 Hz), 4.69
(1H, d, J = 12.6 Hz), 4.72 (1H, d, J = 10.9 Hz), 4.76 (1H, d, J = 11.4 Hz), 4.79 (1H, d, J = 11.4 Hz),
4.91 (1H, d, J = 10.9 Hz), 5.99–6.04 (2H, m), 6.16–6.23 (1H, m), 6.75–6.80 (2H, m), 6.89–6.95
(1H, m), 7.15–7.55 (20H, m), 7.78 (1H, d, J = 8.3 Hz), 8.39 (1H, d, J = 7.7 Hz), 8.43–8.48 (1H, m),
13
8.72 (1H, s), 10.84 (1H, s), the pyrazole NH was not observed; C NMR (101 MHz, CD3CN) δ
67.9, 74.4, 74.8, 75.8, 76.0 (CH2), 77.2, 77.9, 82.1, 85.4, 85.8 (CH), 112.1, 112.9, 121.3, 121.5,
121.7, 121.9, 125.4, 125.8, 128.34, 128.38, 128.55, 128.59, 128.7, 128.96, 129.04, 129.14, 129.16,
129.3, 129.5, 129.6, 132.4 (CHarom), 106.9, 112.6, 114.8, 123.55, 123.62, 124.8, 128.1, 134.4,
137.7, 138.4, 139.3, 139.57, 139.64, 141.1 (Carom); HRMS (ESI+) calcd for C53H47N4O5 (M+H)+
819.3541, found 819.3547.
4.1.14. 12-(1-Deoxy-β-D-glucopyranos-1-yl)-12,13-dihydro-5H-indolo[2,3-a]pyrazolo[3,4-
c]carbazole (19)
To a solution of 18 (54 mg, 0.066 mmol) in CH2Cl2 (22 mL) at –78 °C was added BBr3 (1 M in
CH2Cl2, 0.79 mL, 0.79 mmol). After stirring 15 min at –78 °C, water was added and the mixture
was allowed to reach room temperature. After extraction with EtOAc, the assembled organic phases
were dried over MgSO4 and the solvent was evaporated under reduced pressure. The residue was
purified by column chromatography (EtOAc to EtOAc/MeOH 9:1) to give 19 (21 mg, 0.046 mmol,
69%) as a greenish beige solid. TLC Rf = 0.45 (EtOAc/MeOH 9:1); Mp = 251 °C; IR (ATR): 3700–
3000, 1458, 1329, 1239 cm-1; 1H NMR (500 MHz, DMSO-d6) δ 3.55–3.63 (2H, m), 3.84 (1H, d, J =
10.5 Hz), 3.93 (1H, dd, J1 = 9.8 Hz, J2 = 1.9 Hz), 4.03 (1H, t, J = 9.1 Hz), 4.09 (1H, d, J = 10.8 Hz),
4.79 (1H, br s), 5.09 (1H, br s), 5.34 (1H, br s), 5.92 (1H, br s), 6.18 (1H, d, J = 8.4 Hz), 7.28–7.34
(2H, m), 7.41 (1H, ddd, J1 = 8.2 Hz, J2 = 7.1 Hz, J3 = 1.1 Hz), 7.43 (1H, ddd, J1 = 8.3 Hz, J2 = 7.1
Hz, J3 = 1.2 Hz), 7.67 (1H, d, J = 8.1 Hz), 7.89 (1H, d, J = 8.6 Hz), 8.39 (1H, d, J = 7.7 Hz), 8.64
(1H, d, J = 7.7 Hz), 8.80 (1H, s), 11.48 (1H, s), 13.69 (1H, br s); 13C NMR (126 MHz, DMSO-d6) δ
58.5 (CH2), 67.6, 73.1, 77.0, 78.4, 84.6 (CH), 111.7, 111.9, 119.71, 119.74, 120.6, 120.7, 123.7,
123.9, 131.8a (CHarom), 104.8b, 111.2, 113.4, 122.0, 122.1, 122.9, 126.8, 132.1b, 138.2, 140.3
(Carom); achemical shift measured from a HSQC 1H–13C experiment, bchemical shift measured from
1
a HMBC H–13C experiment; HRMS (ESI+) calcd for C25H23N4O5 (M+H)+ 459.1663, found
459.1665.
4.2. Biological studies
4.2.1. Cell culture and cytotoxicity assays
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The human colon carcinoma HCT116 cell line (American Type Culture Collection;
Manassas, VA) was propagated in Dulbecco modified Eagle’s medium supplemented with 5% fetal
calf serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 °C, 5%
CO2 in a humidified atmosphere. Cells in logarithmic phase of growth were used in the
experiments. Compounds 10, 11, 18 and 19 were dissolved in DMSO as 10 mM stock solution
followed by serial dilutions in water immediately before experiments. The cytotoxicity was
determined in a formazan conversion assay (MTT-test) [21]. Briefly, cells (5x103 in 190 µL of
culture medium) were plated into a 96-well plate (Becton Dickinson, Franklin Lakes, NJ) and
treated with 0.1% DMSO (vehicle control) or with 10, 11, 18 and 19 (0.1–50 µM; each
concentration in duplicate) for 72 h. After the completion of drug exposure, 50 µg of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were added into each well for an additional
2 h. Formazan was dissolved in DMSO, and the absorbance at 540 nm was measured. Cell viability
at a given drug concentration (% MTT conversion) was calculated as the percentage of absorbance
in wells with drug-treated cells to that of vehicle control cells (100%).
4.2.2. In vitro kinase inhibition assays
The procedures for in vitro protein kinase assays have been described previously [22]. All full
length protein kinases were of human origin. All proteins were either expressed as GST (glutathione
transferase) fusion proteins in Escherichia coli or as hexahistidine (His6)-tagged proteins in Sf21
(Spodoptera frugiperda 21) insect cells. GST fusion proteins were purified by affinity
chromatography on glutathione-Sepharose, and His6-tagged proteins on nickel/nitrilotriacetate-
agarose. The assays (25.5 ꢀL volume) were carried out robotically at room temperature (21 °C) and
were linear with respect to time and enzyme concentration under the conditions used. Assays were
performed for 30 min using Multidrop Micro reagent dispensers (Thermo Electron Corporation,
Waltham, MA, U.S.A.) in a 96-well format. The concentration of magnesium acetate in the assays
was 10 mM; [γ-33P]ATP (800 c.p.m./pmol) was used at 20 ꢀM for Pim-1 and PKCα, in order to be
at or below the Km for ATP for each enzyme. The assays were initiated with MgATP, stopped by
the addition of 5 ꢀL of 0.5 M orthophosphoric acid and spotted on P81 filter plates using a unifilter
harvester (PerkinElmer, Boston, MA). Kinase substrates were RSRHSSYPAGT (300 ꢀM) for Pim-
1 or histone H1 (0.1 mg/mL) for PKCα. Pim-1 was diluted in a buffer containing 50 mM Tris/HCl,
pH 7.5, 0.1 mM EGTA, 1 mg/mL BSA and 0.1% 2-mercaptoethanol and assayed in a buffer
comprising 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA and 0.1% 2-mercaptoethanol. PKCα was
diluted in 20 mM HEPES (pH 7.4)/0.03% Triton X-100 and assayed in the same buffer
22

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supplemented with 0.1 mg/mL phosphatidylserine, 10 ꢀg/mL diacylglycerol and 0.1 mM CaCl2.
The inhibitory profile of tested compounds was expressed as the percentage of residual kinase
activity for the inhibitor concentration of 10 ꢀM. The results are shown as the mean ± standard
deviation for duplicate determinations.
4.2.3. Interaction with double stranded DNA
The binding of compounds to DNA was determined in100 mM NaCl, 10 mM Na phosphate
buffer, pH 6.5 at 25°C. Fluorescence spectra were recorded with Cary Eclipse fluorescence
spectrophotometer (Varian Inc., USA); excitation was 360 nm for 10 and 18 and 305 nm for 11 and
19. The concentration of calf thymus DNA (ctDNA) in moles of base pairs was determined in a
sodium phosphate buffered solution at 20 °C using the molar extinction coefficient ε[ctDNA] =
13200 M (bp)-1 cm-1. Binding constants were determined by linear approximation of the ratio
[L]·[DNA]/(F0-F) where [L] and [DNA] are concentrations of the compound and ctDNA,
respectively; F0 and F are fluorescence intensities of the compound in the absence or presence of
DNA, respectively. Error value was calculated as standard deviation of binding constants
determined by fluorescence at three wavelengths (410, 435, 460 nm for 10 and 18; and 375, 395,
410 nm for 11 and 19) [23,24].
Acknowledgements
The French Ministry of Higher Education and Research is acknowledged for Y. J. E. PhD
fellowship, and the University Mohamed Seddik Ben Yahia (Jijel, Algeria) for B. D. doctoral
mobility fellowship.
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