Linker structure-activity relationships in fluorodeoxyglucose chlorambucil conjugates for tumor-targeted chemotherapy

Article abstract of DOI:10.1016/j.bmc.2017.08.043

Nitrogen mustards, such as chlorambucil (CLB), can cause adverse side-effects due to ubiquitous distribution in non-target organs. To minimize this toxicity, strategies of tumor-targeting drug delivery have been developed, where a cytotoxic warhead is linked to a tumor-cell-specific small ligand. Malignant cells exhibit marked glucose avidity and an accelerated metabolism by aerobic glycolysis, known as the Warburg effect, and recognized as a hallmark of cancer. A targeting approach exploiting the Warburg effect by conjugation of CLB to 2-fluoro-2-deoxyglucose (FDG) was previously reported and identified two peracetylated glucoconjugates 2 and 3 with promising antitumor activities in vivo. These results prompted us to investigate the importance of the spacer in this tumor-targeting glucose-based conjugates. Here we report the chemical synthesis and an in vitro cytotoxicity evaluation, using a 5-member panel of human tumor cell lines and human fibroblasts, of 16 new CLB glucoconjugates in which the alkylating drug is attached to the C-1 position of FDG via different linkages. We studied the structure-activity relationships in the linker, and evidenced the positive impact of an aromatic linker on in vitro cytotoxicity: compound 51 proved to be the most active FDG-CLB glucoside, characterized by a bis-aromatic spacer tethered to CLB through an amide function.

Full text of DOI:10.1016/j.bmc.2017.08.043

Accepted Manuscript
Linker structure-activity relationships in fluorodeoxyglucose chlorambucil con-
jugates for tumor-targeted chemotherapy
Mostafa El Hilali, Bastien Reux, Eric Debiton, Fernand Leal, Marie-Josephe
Galmier, Magali Vivier, Jean-Michel Chezal, Elisabeth Miot-Noirault, Pascal
Coudert, Valérie Weber
PII:
S0968-0896(17)31014-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.043
BMC 13947
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 May 2017
28 July 2017
25 August 2017
Please cite this article as: El Hilali, M., Reux, B., Debiton, E., Leal, F., Galmier, M-J., Vivier, M., Chezal, J-M.,
Miot-Noirault, E., Coudert, P., Weber, V., Linker structure-activity relationships in fluorodeoxyglucose
chlorambucil conjugates for tumor-targeted chemotherapy, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.08.043
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Linker structure-activity relationships in fluorodeoxyglucose chlorambucil
conjugates for tumor-targeted chemotherapy
Mostafa El Hilalia, Bastien Reuxa, Eric Debitona, Fernand Leala, Marie-Josephe
Galmiera, Magali Viviera, Jean-Michel Chezala, Elisabeth Miot-Noiraulta, Pascal
Couderta, Valérie Weber a*.
(a) Université Clermont Auvergne, INSERM, U1240 Imagerie Moléculaire et Stratégies
Théranostiques, F-63000 Clermont-Ferrand, France
*Corresponding author: Valérie Weber - Phone: 0033 4 73 15 08 21, Fax: 003 4 73 15 08 01,
e-mail: valerie.weber@uca.fr
Abstract
Nitrogen mustards, such as chlorambucil (CLB), can cause adverse side-effects due to
ubiquitous distribution in non-target organs. To minimize this toxicity, strategies of tumor-
targeting drug delivery have been developed, where a cytotoxic warhead is linked to a tumor-
cell-specific small ligand. Malignant cells exhibit marked glucose avidity and an accelerated
metabolism by aerobic glycolysis, known as the Warburg effect, and recognized as a hallmark
of cancer. A targeting approach exploiting the Warburg effect by conjugation of CLB to 2-
fluoro-2-deoxyglucose (FDG) was previously reported and identified two peracetylated
glucoconjugates 2 and 3 with promising antitumor activities in vivo. These results prompted
us to investigate the importance of the spacer in this tumor-targeting glucose-based
conjugates. Here we report the chemical synthesis and an in vitro cytotoxicity evaluation,
using a 5-member panel of human carcinoma cell lines and human fibroblasts, of 16 new CLB
glucoconjugates in which the alkylating drug is attached to the C-1 position of FDG via
different linkages. We studied the structure-activity relationships in the linker, and evidenced
the positive impact of an aromatic linker on in vitro cytotoxicity: compound 51 proved to be
the most active FDG-CLB glucoside, characterized by a bis-aromatic spacer tethered to CLB
through an amide function.
Keywords :
Chlorambucil, 2-fluoro-2-deoxy-D-glucose derivatives, tumor-targeting drug delivery, in vitro
cytotoxicity
1

1. Introduction
Targeted chemotherapy is one key issue for a successful outcome in cancer therapy. The main
limitations of conventional cancer chemotherapy arise from the suboptimal biodistribution of
cytotoxics in the body: lack of drug-specific affinity for tumor cells and systemic toxicity lead
to many negative, sometimes life-threatening side effects. If a drug can instead be selectively
delivered to the targeted tumor, with less drug reaching critical normal tissues, then cancer
therapy and patients’ quality of life can be substantially improved. Much effort has
accordingly been made to develop new drug delivery systems that mediate drug release
selectively at the tumor site.
Active drug delivery systems range widely, and use various kinds of targeting carriers, such
as antibodies or small-molecule ligands.1 Although immunoconjugates show substantial
clinical promise, as represented by several approved monoclonal antibody-drug conjugates for
treating cancer (e.g. lymphoma and breast cancer),2 they present major drawbacks, including
immunogenic responses and (or) suboptimal biodistribution, leading to unwanted side-effects.
Low-molecular-weight molecules targeting specific tumor biomarkers have now been well-
documented as an alternative to antibodies for targeted drug delivery,3 given the inherent
advantages of their small size, nonimmunogenic nature, and much more manageable
synthesis. To date, the most clinically advanced low-molecular-weight ligands are folate-
conjugated drugs,4,5 such as vintafolide and EC-0225. Other small ligands that have also
stirred much interest in recent years for tumor targeting are glucose derivatives.6–12
To fuel cell growth and division, cancer cells can reprogram their glucose metabolism with
increased glucose uptake and need for glycolysis, known as Warburg effect13 and recognized
as one of the hallmarks of cancer.14 This metabolic transformation is driven by increased
surface expression of the glucose transporters15,16 and enhanced activity of glycolytic enzymes
(hexokinase and phosphofructokinase).17,18 Glucose is transported into cells via two classes of
hexose transporters: the SGLT family (sodium-dependent glucose transporter, SLC5A family)
and GLUT family (SLC2A family). GLUT1 is the most common glucose transporter in
humans and it is aberrantly expressed in several tumors, including hepatic, pancreatic, breast,
esophageal, brain, lung, cutaneous, colorectal cancers. But other GLUT and SGLT isoforms
have also been reported for cancers with tissue specific expression differences: GLUT2
(gastric, breast, colon, and liver carcinomas), GLUT3 (B-cell non-Hodgkin’s lymphoma),
GLUT12 (prostate adenocarcinomas), and SGLT1–2 (colorectal and metastatic lung
carcinomas).16,19 The high demand for glucose in cancer cells is the basis for the detection and
2

staging of tumors using a radiolabeled analogue of glucose (2-18F-fluorodeoxyglucose, 2-
[18F]FDG)20 in PET imaging. Glucose transporters, especially GLUT1 and GLUT3, and
hexokinase seem to play a crucial role on the 2-FDG accumulation. Some tumors do not
accumulate 2-FDG, which is a substrate for GLUTs but not for SGLTs, increasing interest in
the expression of SGLTs in cancer. This Na+-dependent co-transporter was reported in the
literature to mediate the uptake of a glucose-isophosphoramide mustard conjugate
(glufosfamide), the first-in-class glucose conjugate reaching advanced clinical trial (Phase III
for treatment of metastatic pancreatic cancer)21 in a strategy using the high demand for
glucose into tumor cells to deliver chemotherapeutic agents.
Indeed, targeting the Warburg effect has been developed in the last decades in therapeutic
area using several glucose-based conjugates designed to more specifically deliver the attached
drug to cancer cells. The synthesis and biological evaluation of glycoconjugate anticancer
therapeutics was firstly reported in the literature in 1995 by Wiessler et al22 with
glufosfamide. Since the introduction of glufosfamide, there has been many examples of
glucose-conjugated drugs in preclinical or clinical evaluation6 (paclitaxel23,24, adriamycin25,
DNA alkylator including chlorambucil7,11,26,27, cyclophosphamide28, platinum29–31).
In the study reported here, FDG was used as active tumor-specific drug carrier. The choice of
FDG was motivated by the markedly increased uptake and retention of this fluorinated
glucose analogue in malignant cells, and by its potential to inhibit glycolysis. Also, 2-deoxy-
D-glucose (2-DG) has been described as a potential potent antitumor agent through depletion
of cellular energy, increased oxidative stress, interference with N-linked glycosylation, and
induction of autophagy.32 Neoplastic cells treated with 2-DG accelerate their own demise, and
2-DG also enhances the cytotoxic effects of anticancer agents when used in combination.33,34
We have previously reported preclinical results of this targeting approach,35–37 mainly with
chlorambucil (CLB). CLB served as basis for our FDG-glycolysis-mediated targeting proof-
of-concept, because its cell uptake occurs through passive diffusion, and like all alkylating
agents, its lack of tumor specificity causes serious side-effects, such as nausea, bone marrow
suppression and anemia. Three peracetylated glucoconjugates 1-3 demonstrated an increased
cytotoxicity in vitro compared with the free CLB (Figures 1 and 2) for all the cell lines
tested.35 In vivo, a single-dose-finding study to determine the maximum tolerated dose (MTD)
using the intraperitoneal route (IP) showed that the three peracetylated glucoconjugates 1-3
were less toxic than CLB itself. When given to tumor-bearing mice (melanoma and colon
carcinoma models), according to a q4d × 3 schedule (i.e. three doses at 4-day intervals),
compounds 2 and 3 demonstrated promising antitumor activities, with log cell kill (LCK)
3

values higher than 1.3 in both syngenic colon carcinoma and melanoma models (CT-26 and
B16F0).36 The difference in in vivo antitumor activity between conjugated forms 2-3 and
CLB, and their better tolerance, make them promising candidates for further work to develop
new highly active antineoplastic compounds.
Figure 1. Structures of FDG, chlorambucil (CLB) and CLB-peracetylated FDG conjugates 1,
2 and 3.
The success of a tumor-targeting drug delivery system depends on the nature of the cytotoxic
warhead and the tumor-seeking moiety, but also on the chemical scaffold linking these two
parts. We therefore undertook a more thorough pharmacomodulation investigation of this
linker. The composition of the linker can be designed to contain stable or cleavable functional
groups, and at the same time residues that may influence physical properties such as the
hydrophilic-lipophilic balance of the designed conjugate. The nature of the linker thus
influences how successful drug delivery is, and its outcome. Ideally, as the conjugate is meant
to reach the malignant site, the spacer arm has to be designed in such a way as to ensure its
stability in blood, while also allowing the action of the active cytotoxic warhead addressed to
tumor cells. Too-stable linkers can curb the activity of the associated drug, resulting in a low-
potency conjugate. Conversely, a too-labile linking moiety can lead to poor target specificity
4

and high systemic toxicity. Several chemical functions can form the linker. The most common
ones are amide, reverse amide, ester and carbamate subjected to enzymatic and non-
enzymatic hydrolysis, or labile linkers that rely on more specific modes: enzymatic, reductive
or pH-controlled release. In our study, the CLB provides a carboxyl group that can be
appropriately functionalized to bind to the linker or to the FDG residue by the formation of an
ester or an amide bond, largely used in targeted drug strategies.
In this report, we extended the pharmacomodulation of the previously selected compounds 1-
3, with the synthesis, characterization and in vitro cytotoxicities of new glycoconjugate
analogs to provide more knowledge on the structure-activity relationships between the nature
of the linker and the in vitro cytotoxicity of linked drugs.
2. Results and Discussion
2.1. Chemistry
As stated above, the purpose of this study was to pursue the pharmacomodulation of
the lead compounds 1-3, chosen on the basis of their in vitro and in vivo activity, and their
tolerance in tumor-bearing mice.35,36 We also designed analogues of these derivatives,
particularly of compound 3, which presented the most promising antitumor activity, and can
be retained as a highly active antitumor drug on the basis of the NCI criteria in two solid
tumor models. Compound 3 exhibited LCK values higher than 1.3 (1.52 in the colon
carcinoma xenograft model and 1.81 in the melanoma model). Schemes 1-5 describe the
syntheses of the targeted CLB-FDG conjugates analogues of compounds 1, 2 and 3 with the
following linker modifications: CLB was attached to a spacer arm through amide, ester,
peptide and reverse peptide bonds, and the FDG moiety was linked via its C-1 position to the
spacer arm through amide, ester, carbamate or glycosidic bonds. Published results suggest that
a β-conformation for glucose conjugates is preferred for GLUT-1 transport.6 We accordingly
synthesized all the compounds in the β-conformation.
For compounds 1 and 2, the spacer between FDG and CLB was made up of two amide
linkages and an aromatic or glycyl group. Also, for the analogues of these two derivatives, the
amide was replaced by an ester, a reverse amide or a carbamate bond in compounds 10, 11,
15, 18, 22 and 25. The syntheses are described in schemes 1 and 2.
5

Scheme 1a
Cl
OAc
O
OAc
O
N
(iv)
O
Cl
H
N
H
N
AcO
AcO
AcO
AcO
X
7
X
OR
O
F
F
O
O
(ii)
6
R = Bn : : X = C6H4
;
:X = CH2
OAc
OAc
O
10 : X = C6H4
11
: X = CH2
(iii)
R = H : 8 :X = C6H4 ; 9 : X = CH2
(i)
O
AcO
AcO
AcO
AcO
NH2
F
F
Cl
Br
5
4
(v)
OAc
O
N
OAc
Cl
H
N
H
N
(vii)
O
H
N
AcO
AcO
AcO
AcO
OR
F
F
(viii)
O
O
O
O
12
: R = Bn
: R = H
(vi)
15
13
H
N
R
OAc
OAc
O
(x)
O
AcO
AcO
AcO
O
AcO
O
O
Cl
N
F
F
O
O
16 : R = NO2
17 : R = NH2
18
Cl
(ix)
a
Reagents and conditions: (i) (1) NaN3, acetone/water, rt, 1 h, 84%; (2) H2, Pd/C,
THF/MeOH, rt, 1 h, 98%; (ii) DCC, HOBt, DMF, rt: 4 d from 4-benzyloxybenzoic acid, (6),
45%; 21 h from benzyloxyacetic acid, (7), 83%; (iii) H2, Pd/C, rt, 5 h, THF, 46% (8),
THF/MeOH, 99% (9); (iv) DCC, DMAP, DCM, rt, 20 h, 55% (10), 81% (11); (v) malonic
acid monobenzyl ester, DCC, HOBt, DMF, rt, 48 h, 57%; (vi) LiOH, EtOH/H2O, rt, 12 h,
89%; (vii) 4-(3-aminopropyl)-N,N-bis(2-chloroethyl)aniline 14, DCC, HOBt, DMF, rt, 24 h,
13%; (viii) 4-nitrobenzoic acid, NaOH, rt, 4 h 30, 37%; (ix) H2, Pd/C, THF/EtOH, rt, 10 h,
quantitative yield; (x) CLB, TEA, ClCO2Et, DCM, 30 h, 50%.
Scheme 2a
a
Reagents and conditions: (i) 4-nitrophenylchloroformate, TEA, THF, rt, 24 h, 77% yield for
α- and β-anomers; 35% yield for β-anomer; (ii) 4-nitroaniline, ClCO2Et, TEA, DCM, rt, 17 h,
24%; (iii) H2, Pd/C, THF/EtOH, rt, 3.5 h, 75%; (iv) 19, TEA, DMF, rt, 25 h, 42%; (v) 4-
6

nitrophenol, DCC, DMAP, DCM, 24 h, rt, 93%; (vi) H2, Pd/C, EtOH, 20 h, rt, 93%; (vii) 19,
TEA, DMF, rt, 40 h, 14%.
To access compounds 10, 11 and 15, with FDG anchored to the spacer through an
amide bond, the synthetic methods depicted in Scheme 1 were designed starting from β-
aminosugar 5, easily produced from the α-bromo compound 4 through the β-anomeric azid35.
The N-acylation of the β-aminosugar 5 with 4-benzyloxybenzoic acid, benzyloxyacetic acid
or benzyl malonate half ester, the latter prepared by reaction of Meldrum’s acid with benzyl
alcohol at 120 °C,38 led respectively to derivatives 6, 7 and 12 in moderate yields. Cleavage of
the benzyl protecting group by catalytic hydrogenation provided the corresponding hydroxyl
derivatives 8, 9 and 13. Intermediates 8 and 9 were then directly esterified using CLB
activated by DCC and DMAP to produce compounds 10 and 11. Derivative 15 was obtained
by coupling acid 13 with 4-(3-aminopropyl)-N,N-bis(2-chloroethyl)aniline 14,39 synthesized
via the Curtius rearrangement.
For compound 18, with FDG anchored to the spacer through an ester bond, a first synthetic
route starting from 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-α,β-D-glucose35 was considered.
Despite repeated attempts, compound 16 was always isolated as an inseparable mixture of the
two α/β anomers. Another reaction pathway was therefore tested starting from α-bromo
compound 4. Reaction with 4-nitrobenzoic acid successfully gave compound 16 exclusively
as the β-form, but with a moderate yield (37%). The β-stereochemistry of 16 was assigned
1
based on its H NMR signature, in particular on the multiplicity at and near the anomeric
center: the C1 anomeric proton signal appears at 6.05 ppm as a doublet of doublets (J1-2 = Jax-
= 8.0 Hz, J1-F = 3.0 Hz) and the C2 proton signal appears at 4.66 ppm as a doublet of
ax
doublets of doublets (J1,2 = Jax-ax = 8.0 Hz, J2-3 = Jax-ax = 9.1 Hz, J2-F = 50.8 Hz). For the α-
anomer, the J1-2 coupling constant appears smallest (4 Hz), characteristic of a Jax-eq coupling
constant. Finally, reduction to amine 17 followed by coupling using ethylchloroformate and
CLB gave compound 18 in 49% yield.
For compounds 22 and 25, with FDG anchored to the spacer through a carbamate bond
(scheme 2), the synthetic pathway designed required the production of carbonate 19, where
the anomeric hydroxyl group of the sugar was activated with 4-nitrophenylchloroformate,
followed by substitution with an amine group included in the linker unit. The sequence chosen
had to ensure a β-configuration of the carbamoyl linkage at the anomeric center. During the
activation
of
3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-α,β-D-glucose
with
4-
7

nitrophenylchloroformate, we faced the problem of stereospecificity. This reaction, performed
in THF at room temperature in the presence of TEA, gave an anomeric mixture of carbonate
19 (α/β = 20/80) as already described for glucuronate derivatives.40 Other carbamoylation
methods such as activation through N,N-carbonyldiimidazole41 or utilization of isocyanate
derivatives42,43 also lead to αβ mixtures. However, the difference in solubility of the two
anomers in THF and diisopropryl ether allowed us to isolate the pure β-anomer in 35% yield.
1
The β-stereochemistry of 19 was confirmed by coupling constants observed in the H NMR
spectra (J1-2 = 8.0 Hz, J2-3 = 9.2 Hz, typical of ax-ax coupling). The β-carbonate derivative 19
was then treated with two different synthetic aromatic amines 21 and 2435 in DMF in the
presence of TEA to afford compounds 22 and 25 in 14% and 42% yield respectively
(enantiomerically pure as shown by 1H NMR analyses).
In an attempt to improve the antitumor efficacy of our lead O-glycoside 3, we
modulated the spacer arm of this derivative. To evaluate the importance of the type of linkage
binding the spacer domain, the FDG moiety and the drug, and also the importance of the
nature of the spacer domain (lipophilicity, length), we synthesized compounds with FDG
anchored through an amide bond (30, scheme 3) or a carbamate bond (33, 38, 41) (scheme 4)
and other O-glycosides with several spacer arms (45, 48, 51 and 56) (scheme 5).
Scheme 3a
a
Reagents and conditions: (i) 4-nitrobenzoyl chloride, TEA, THF, rt, 4 h, 67%; (ii) H2, Pd/C,
THF/MeOH, rt, 3 h, 91%; (iii) N-Cbz-glycine, ClCO2Et, TEA, DCM, rt, 22 h, 50%; (iv) H2,
Pd/C, THF/EtOH, rt, 5 h, quant. yield; (v) CLB, DCC, HOBt, DMF, rt, 24 h, 58%.
8

Scheme 4a
H
N
OAc
O
O
H
N
AcO
AcO
O
O
Cl
N
N
F
H
O
38
Cl
(vii)
R
OAc
O
O
H
AcO
AcO
O
N
O
N
F
H
O
O
Cl
H2N
N
37 : R = NH2
36
24
(vi)
: R = NO2
Cl
(i)
(v)
R
O
O
O
H
N
O2
N
OAc
O
O
RHN
O
Cl
N
AcO
AcO
N
N
NHR
O
O
H
H
N
Cl
N
O
F
H
O
31 : R = Cbz
NO2
Cl
39
: R = NO2
34 : R = Cbz
(ii)
19
(iv)
32
: R = H
(ix)
(viii)
40 : R = NH2
35
: R = H, HBr
Cl
(iii)
(x)
OAc
O
O
OAc
O
O
H
N
H
AcO
AcO
AcO
O
N
O
Cl
O
AcO
O
N
N
F
H
N
F
H
O
O
N
H
33
O
Cl
Cl
N
41
Cl
a Reagents and conditions: (i) N-Cbz-glycine, DCC, HOBt, DMF, rt, 24 h, 90%; (ii) H2, Pd/C,
THF/EtOH, rt, 4 h, 99%; (iii) TEA, DMF, rt, 24 h, 59%; (iv) HBr/AcOH, AcOH, rt, 2 h 45,
87%; (v) (a) from 35, TEA, DMF, rt, 1.5 h, (b) TEA, DMF, rt, 5 h, 89%; (vi) H2, Pd/C,
THF/EtOH, 5 h, rt, 93%; (vii) CLB, ClCO2Et, TEA, DCM, rt, 4 h, 86%; (viii) (a) TEA, DCM,
rt, 2.5 h, (b) CLB, ClCO2Et, TEA, DCM, rt, 3 d, 92%; (ix) H2, Pd/C, THF/EtOH, rt, 24 h,
88%; (x) TEA, DMF, rt, 20 h, 37%.
9

Scheme 5a
a
Reagents and conditions: (i) CLB, DCC, DMAP, DCM, rt, 48 h, 85%; (ii) H2, Pd/C,
THF/EtOH, rt, 2 h, 97%; (iii) ClCOOEt, TEA, DCM, rt, 18 h, 48%; (iv) malonic acid
monobenzyl ester, DCC, HOBt, DMF, rt, 22 h, 61%; (v) H2, Pd/C, THF/MeOH, rt, 2 h, quant.
yield; (vi) 4-(3-aminopropyl)-N,N-bis(2-chloroethyl)aniline (14), DCC, HOBt, DMF, rt, 39 h,
56%; (vii) 4-nitrobenzoic chloride, TEA, THF, 0 °C, 30 mn, rt, 20 h, 40%; (viii) H2, Pd/C,
THF/MeOH, rt, 24 h, 88%; (ix) CLB, ClCO2Et, TEA, DCM, rt, 15 h, 43%; (x) N-Cbz-Gly,
DCC, HOBt, DMF, rt, 28 h, 89%; (xi) HCO2NH4, Pd/C, THF/EtOH, 70 °C, 50 mn, 98%; (xii)
CLB, ClCO2Et, TEA, DCM, rt, 24 h, 54%; (xiii) LiOH, EtOH/H2O, rt, 5 h, 99%; (xiv) DCC,
HOBt, DMF, rt, 24 h, 28%.
The synthetic method for the preparation of compound 30 with an amide bond instead
of the glucoside bond is depicted in Scheme 3. Starting from the β-aminosugar 5, N-acylation
with 4-nitrobenzoyl chloride preceded reduction of the nitro function to provide amino
derivative 27.35 N-Acylation with N-Cbz-glycine activated by ethylchloroformate and
10

triethylamine, followed by cleavage of the benzyloxycarbonyl (Cbz) protecting group by Pd/C
catalytic hydrogenation provided the corresponding amino derivative 29. Final coupling with
CLB afforded compound 30 in 18% overall yield.
For compounds 33, 38 and 41, with the FDG moiety grafted to the spacer through a
carbamate bond, syntheses were carried out starting from compound 19 and different amines
32, 35 and 40 (scheme 4). Compound 33 was obtained in three steps in 53% overall yield
from amine 24: N-acylation with N-Cbz-glycine activated by DCC/HOBt, cleavage of the
benzyloxycarbonyl protecting group by catalytic hydrogenation before final formation of the
carbamate linkage by reaction with carbonate derivative 19. The synthesis of derivatives 38
and 41 differed from the CLB-glycyl-aromatic sequence, but 2-amino-N-(4-
nitrophenyl)acetamide 35 was used as common key intermediate. This compound was
obtained by reaction of 4-nitroaniline with N-Cbz-glycine, activated by ethylchloroformate
followed by selective cleavage of the Cbz protecting group of the glycine residue by hydrogen
bromide in acetic acid. The synthesis of compound 38 was carried out according to the
following sequence: reaction of amine 35 with the carbonate derivative 19, then reduction of
the aromatic nitro function and coupling of CLB. Using amine 35, the same sequence of
reactions was used, but in reverse, to obtain derivative 41: coupling of CLB with amine 35,
reduction of the aromatic nitro function and finally reaction with the carbonate derivative 19.
The introduction of a spacer via a glycosidic bond needed the synthesis of compound
42 (Scheme 5), following the protocol of Reux et al.35 For the synthesis of compound 45, the
requisite acid 44 was readily accessible in 85% yield from benzyl glycolate44 through a two-
step sequence: reaction of CLB with benzyl glycolate, using DCC and DMAP as condensing
reagents and hydrolysis of the benzyl ester with hydrogen using Pd/C. Activation of 44 with
ethylchloroformate in the presence of triethylamine before reaction with the O-glucoside 42
provided derivative 45 in 48% yield. DCC/HOBt coupling of 42 with benzyl malonate half
ester38 gave compound 46. Hydrogenolytic removal of the benzyl carbamate led in
quantitative yield to acid 47, and finally a further DCC/HOBt coupling with 4-(3-
aminopropyl)-N,N-bis(2-chloroethyl)aniline 14 afforded expected derivative 48.
Compound 51 was obtained in three steps starting from the O-glucoside 42: N-acylation with
4-nitrobenzoyl chloride, reduction of the nitro function by catalytic hydrogen atmosphere and
finally coupling with CLB using ethylchloroformate instead of DCC/HOBt to avoid problems
typically faced during purification by silica gel column chromatography due to the presence
of dicyclohexylurea.
11

Finally in this set of compounds with FDG anchored to the spacer through a glucosidic bond,
we performed the synthesis of glycoside 56 for which two glycine moieties were condensed
next to the aromatic of O-glucoside 24 to extend the spacer domain.
The synthetic route designed for compound 56 used derivative 53, a key intermediate in the
production of the lead compound 3.35 In a first attempt, we designed a three-step reaction
pathway including (i) coupling of compound 53 to N-Cbz-glycine (65% or 53% yield
respectively under the standard DCC/HOBt or ethylchloroformate coupling conditions),
(ii) hydrogenolytic removal of the benzyl carbamate (quantitative yield), and finally (iii) N-
acylation to introduce CLB. Unfortunately, our attempts to introduce CLB under the standard
DCC/HOBt or ethylchloroformate coupling conditions all failed to produce derivative 56 in
the high chemical purity required for further biological evaluation. Consequently, a
convergent pathway was preferred: acid 55 was readily accessible from glycine methyl ester
through a two-step sequence: coupling of glycine methyl ester with CLB (54% yield) and
hydrolysis of the methyl ester with aqueous lithium hydroxide (quantitative yield). Finally,
compound 56 was obtained by DCC/HOBt condensation of acid 55 and amine 53 in 28%
yield.
2.2. In vitro cytotoxicity
The antiproliferative activity (i.e., IC50) of all the target fluoroglycosides was
evaluated relative to CLB, using a 5-member panel of human carcinoma cell lines from
prostate (PC3), breast (MCF-7), lung (A549), melanoma (M4Beu), ovarian (PA1) and a
primary culture of human fibroblasts (see Figure 2). On the basis of this study, whatever the
cell line considered, almost all the compounds were more potent than CLB, which is only
moderately cytotoxic in these conditions (with IC50 values higher than 40 µM except for PA1
cells, which showed high sensitivity in all cases. The cytotoxicity of FDG or peracetylated
FDG alone, or in combination with CLB at 50 µM was assessed on A549 cells and no
additive effect was observed for concentrations up to 200 µM (data not shown).
12

Figure 2. Cytotoxicity induced by the newly synthesized CLB-peracetylated FDG-conjugates
on human normal and tumor cells. (A) Summary of IC50 data from cytotoxicity assay of all
fluoroglucoconjugates (reciprocal values displayed). Comparisons were made against
previously identified compounds 1, 2 and 3, and CLB. (B) IC50 data and error values are
13

given. Human cell lines tested included PC3 (prostate), MCF-7 (breast), A549 (lung), M4Beu
(melanoma), PA1 (ovarian) and a primary culture of fibroblasts.
Only compounds 10, 11, 25 and 45 did not reveal any cytotoxic effect, with IC50
values above 50 µM, except in A549 and PA1 cells. These four compounds with CLB linked
via an ester bond showed a drop in activity compared with amide bond analogues 1, 2, 22 and
3. The negative impact of the ester bond was already observed with compound 33, showing
overall IC50 values above 30 µM, compared with compound 38, with an IC50 values ranging
from 6 to 18 µM, i.e. approximately half. As the ester bond opens a route for CLB release due
to its significant susceptibility to hydrolysis when exposed to aqueous media and cellular
medium in vitro, we surmise that the decreased potencies of these glucoconjugates may be
due to hydrolysis and early release of CLB. In the literature, we can find several examples
where drugs linked with the spacer via an ester bond are released due to hydrolysis or
enzymatic degradation.45–47 The rate of release generally decreases in the order ester > amide.
Such an early hydrolysis could not be excluded in vivo in the blood circulation, preventing
any active targeting.
However, an ester bond in another position, namely the anomeric position of FDG, given an
ester-type glycoside linkage did not induce a negative impact on cytotoxic activity, with IC50
values in the same range (0.3 to 6 µM) for glycosyl ester 18 and amide 1. Considering these
results, namely that the ester bond induced an unchanged or decreased activity, and
difficulties in synthesis of pure β-anomer of compound 18, no other ester compound was
synthesized. Amide or reverse amide bond led to barely any change in cytotoxicity, as shown
by compounds 2 and 15. Regarding compounds 1 and 22 differing only in one connection, an
amide or a carbamate function grafted to the FDG moiety, a difference in cytotoxicity patterns
was observed. Interestingly, carbamate 22 did not induce cytotoxicity on human fibroblasts
(IC50 > 50 µM), but was still active on tumor cell lines, especially prostatic adenocarcinoma
PC3, lung carcinoma A549 and ovary adenocarcinoma PA1 cell lines (IC50 < 4 µM). These
preliminary results suggest a different activity between tumor and these healthy cells.
Since compound 3 presented the most promising antitumor activity in two in vivo solid tumor
models, we further designed analogues in an attempt to understand the impact of each
fragment of the linker in term of cytotoxicity. For our lead compound 3, an extend structure-
activity relationship study was considered: the glycoside bond was replaced by an amide
function, affording compound 30 or replaced by a carbamate, leading to compounds 38 and
14

41. For amide 30, quite no change was obtained in term of activity on all cancer cell lines. For
derivatives 38 and 41, the carbamate function was chosen in order to obtain similar results as
thus obtained for carbamate 22, where a several fold higher IC50 value was observed in
fibroblast cells as compared to tumour cells. Unfortunately, no differencial activity was
observed with these derivatives 38 and 41, having a quite similar potency on every cell lines,
i.e. tumor cells or fibroblasts.
We also inverted methylene and phenyl groups in compound 38 and no difference in IC50
values was observed. Regarding compound 51, a spacer arm with two aromatic groups
seemed to be beneficial for the cytotoxic activity (IC50 values were similar to that of
compound 1, the most active compound in this in vitro study). Extension of the linker, with
two glycine fragments, afforded derivative 56, which clearly appears unpromising in terms of
cytotoxicity, with IC50 > 20 µM. Finally, inversion of the amide bond (compound 48) seemed
to favor a specific pattern, with no activity on normal human fibroblasts and lung carcinoma
A549, unlike the observation made above for compound 15, with IC50 values below 13 µM
for all cell lines tested.
3. Conclusions
The synthesis of this set of glycoconjugates focused on the use of FDG designed to enhance
sugar-mediated uptake, and led to overall improvement in antiproliferative effect compared
with CLB. The present study revealed that in vitro antitumoral efficiency was optimized using
an aromatic spacer, which seemed to favor the cytotoxicity on all human carcinoma cell lines
tested as previously described.35 With IC50 values similar to that of lead compound 1,
compound 51 appears as one of the most active FDG-CLB glycoside in this series in terms of
in vitro cytotoxicities. However, compounds with CLB tethered through an ester bond lose
their overall cytotoxicities, perhaps by early hydrolysis. Concerning the cell lines, no real
sensitivity or resistance was evident for this set of compounds, except for the two derivatives
22 and 48, which did not show any activities on human fibroblasts (IC50 > 50 µM), but
maintained their effect on tumor cell lines. To conclude, this work adds to our understanding
of the structure-activity relationships concerning the linker, and highlights the positive impact
of an aromatic linker on in vitro cytotoxicity, but raises questions about the precise
mechanisms of cytotoxicity. These will be addressed in further experiments, including:
−DNA alkylation assessment, (ii)
(i) fluoroglycoside
transport/receptor-mediated uptake
(GLUT or SGLT), (iii) biodistribution of [14C]-radiolabeled compound 51 in tumor-bearing
15

mice in order to study its tumor uptake, (iv) stability of the alkylating agent, and (v) in vivo
antitumor activity in tumor-bearing mice.
16

4. Materials and Methods
4.1. Chemistry
General
Solvents and reagents were purchased from Aldrich, Acros Organics, or Carlo Erba, and used
without further purification. Column chromatography was performed with silica gel A normal
phase (Chromagel, 35–70 µm, SDS) or neutral aluminum oxide 90 standardized (63−200 µm,
Fluka) using the indicated solvent mixture expressed as volume/volume ratios. Analytical
thin-layer chromatography (TLC) was conducted on precoated silica gel aluminum plates
(SDS, 60 F254, 0.2 mm thick) or neutral aluminum oxide aluminum plates (Fluka, 60F254, 0.2
mm thick). The plates were visualized with ultraviolet light (254 nm) and (or) by development
with vanillin in sulfuric acid. Melting points were determined on an electrothermal digital
apparatus (Reichert), are uncorrected, and only given for purified compounds. Infrared spectra
were recorded in KBr pellets or with NaCl plates on an FTIR-Nicolet Impact 410
spectrophotometer. Nuclear magnetic resonance spectra (1H NMR and 13C NMR) were
1
performed on a Bruker AM 200 spectrometer (200 MHz for H, 50 MHz for 13C) or a Bruker
1
DRX 500 spectrometer (500 MHz for H, 125 MHz for 13C) (Bruker Biospin SAS,
Wissembourg, France). Chemical shifts are reported in parts per million relative to the
internal tetramethylsilane standard for 1H NMR and the solvent for 13C NMR (acetone-d6, δ =
29.8 ppm; DMSO-d6, δ = 39.5 ppm; CDCl3, δ = 77.2 ppm). The abbreviations used for signal
patterns are: br, broad; s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet
of doublets; t, triplet; dt, doublet of triplets; q, quadruplet; qt, quintuplet; m, multiplet. 19F
NMR spectra (470 MHz) were recorded on a Bruker DRX 500 apparatus using
tetrafluorotoluene as internal reference (δ -63 ppm); δ values were expressed in ppm, coupling
constants (J values) are in hertz. Mass spectra were recorded on a Bruker Esquire-LC
spectrometer. Electrospray ionization mass spectrometry (ESI-MS) was used in positive
mode. Microanalyses were performed by the Central Analysis Service (CNRS, Vernaison,
France) for C, H, and N; the results were within 0.4% of the theoretical values.
Abbreviations for reactions, reagents and protecting groups: Ac, acetyl; Cbz,
benzyloxycarbonyl; DCC, dicyclohexylcarbodiimide; DMAP, N,N-4-dimethylaminopyridine;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; HOBt, 1-hydroxybenzotriazole;
TEA, triethylamine; THF, tetrahydrofuran.
17

General procedure for DCC/HOBt coupling
The amine was dissolved in dry DMF and the suitable acid (0.8-1.5 equiv), DCC (1.1 equiv),
and HOBt (1.1 equiv) were added. The solution was stirred for several hours at room
temperature, filtered, concentrated under vacuo, and purified by silica gel chromatography to
give the expected compound.
General procedure for DCC/DMAP coupling
The suitable acid (1.1 equiv), DCC (1.1 equiv) and a catalytic amount of DMAP (0.1-0.2
equiv.) were added to a solution of the alcohol in anhydrous DCM, and stirred at room
temperature for several hours. The solid was removed by filtration, and the filtrate was
washed successively with a 1N aqueous acetic acid solution and water, dried over MgSO4,
filtered, and concentrated under vacuum. The resulting residue was purified by silica gel
chromatography to yield the expected compound.
General procedure for ethylchloroformate coupling
A stirred solution of the acid in anhydrous DCM was treated with triethylamine. The mixture
was cooled to 0-10 °C, treated with ethyl chloroformate, and stirred at room temperature for 2
h. The amine was then added, and the mixture was stirred for several hours at room
temperature, followed by tlc monitoring. The quantities of reagents used are reported for each
reaction. The mixture was suspended in saturated aqueous Na2CO3 and extracted twice with
ethyl acetate. The organic extracts were combined, dried over MgSO4, filtered, and
concentrated under reduced pressure before purification by column chromatography (SiO2 or
Al2O3) to give the expected compound.
General reduction or N-Cbz/OBn deprotection procedure using hydrogen
To a suspension of 10% palladium on charcoal (25 wt %) in THF or THF/MeOH or
THF/EtOH was added the nitro or protected compound. The mixture was hydrogenated at
atmospheric pressure for several hours, filtered on celite® 545, and concentrated to yield the
reduced or deprotected product, which was used in the next step without further purification
unless otherwise specified.
General carbamoylation procedure
To a solution of carbonate 19 in DMF, were added the suitable amine (0.8-1.3 equiv.) and
TEA (1.4-1.5 equiv.) and stirring was maintained at room temperature for several hours. After
18

evaporation of the volatiles, the crude product was chromatographed on silica gel to give the
title β-anomer.
N-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl)-4-(benzyloxy)benzamide
(6).
Coupling of amino-sugar 5 (500 mg, 1.63 mmol) with 4-benzyloxybenzoic acid (410 mg, 1.79
mmol) was carried out according to the DCC/HOBt general procedure in DMF (44 mL) and 4
days stirring. After filtration and evaporation under vacuum, the crude residue was purified by
silica gel chromatography (petroleum ether/ethyl acetate, 5/5, v/v) to give compound 6 (382
1
mg, 45%): H NMR (200 MHz, CDCl3) δ 7.82 (d, 2H, J = 8.8 Hz, HAr), 7.42-7.36 (m, 5H,
HAr), 7.20 (d, 1H, J = 9.3 Hz, NH), 7.00 (d, 2H, J = 8.8 Hz, HAr), 5.61 (td, 1H, J1,NH = J1,2
=
8.8 Hz, J1,F = 1.4 Hz, H-1), 5.43 (td, 1H, J2,3 = J3,4 = 9.2 Hz, J3,F = 13.7 Hz, H-3), 5.11 (s, 2H,
CH2Ph), 5.06 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-4), 4.55 (td, 1H, J1,2, J2,3, J2,F = 49.9 Hz, H-2), 4.40
(dd, 1H, J6a,6b = 12.3 Hz, J5,6a = 4.0 Hz, H-6a), 4.04 (m, 1H, H-6b), 3.91 (ddd, 1H, J4,5, J5,6a
,
J5,6b = 1.8 Hz, H-5), 2.08, 2.06, 2.04 (each s, 3×3H, OAc); 13C NMR (50 MHz, CDCl3) δ
170.77, 170.16, 169.93, 167.14 (CO), 162.18 (CArO), 136.27 (CArCH2), 129.53, 128.81,
128.37, 127.60 (CHAr), 125.60 (CArCO), 114.88 (CHAr), 88.51 (C-2, J2,F = 190.5 Hz), 78.18
(C-1, J1,F = 22.4 Hz), 73.80-73.50 (C-3, C-5), 70.27 (CH2Ph), 68.11 (C-4, J4,F = 6.1 Hz),
19
−
61.63 (C-6), 20.73 (CH3); F NMR (CDCl3) δ 197.67.
N-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl)-2-benzyloxyacetamide
(7).
DCC/HOBt coupling of amino-sugar 5 (350 mg, 1.14 mmol) with 4-benzyloxyacetic acid
(284 mg, 1.71 mmol) was carried out with DCC (352 mg, 1.71 mmol) and HOBt (31 mg, 0.23
mmol) in DMF (12 mL) and 21 h stirring, to give after silica gel purification (petroleum
ether/ethyl acetate, 6/4 then 5/5, v/v), compound 7 (429 mg, 83%) as a pale green solid: mp
149 °C; IR (NaCl) ν 3355, 3066, 3032, 1751, 1700, 1524, 1368, 1230, 1097, 1070, 1036 cm-1;
1H NMR (200 MHz, CDCl3) δ 7.41-7.23 (m, 5H, HAr), 5.45-5.28 (m, 2H, H-1, H-3), 5.04 (t,
1H, J3,4 = J4,5 = 9.8 Hz, H-4), 4.58 (s, 2H, CH2Ph), 4.34 (td, 1H, J1,2 = J2,3 = 9.1 Hz, J2,F
=
50.5 Hz, H-2), 4.30 (dd, 1H, J5,6a = 4.4 Hz, J6a,6b = 12.5 Hz, H-6a), 4.11-4.03 (m, 3H, H-6b,
COCH2), 3.84 (ddd, 1H, J4,5, J5,6a, J5,6b = 2.1 Hz, H-5), 2.08, 2.05, 2.04 (each s, 3×3H, OAc);
13C NMR (50 MHz, CDCl3) δ 172.30, 169.70 (CO), 137.02 (CArCH2), 128.81, 128.15, 128.20
(CHAr), 88.70 (C-2, J2,F = 191.0 Hz), 76.81 (C-1, J1,F = 23.0 Hz), 74.05 (C-5), 73.51 (C-3, J3,F
19

= 19.1 Hz), 73.57 (CH2Ph), 69.80 (C-4, J4,F = 7.0 Hz), 66.92 (COCH2), 20.78 (CH3); 19F
NMR (CDCl3) δ −198.10.
N-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl)-4-hydroxybenzamide
(8).
Hydrogenation at atmospheric pressure for 5 h of a suspension of compound 6 (118 mg, 0.228
mmol) and 10% palladium on charcoal (30 mg) in THF (7 mL) yielded phenol 8 (45 mg,
46%) as a white solid after purification by silica gel chromatography (petroleum ether/ethyl
acetate, 5/5, v/v): mp 155 °C; IR (KBr) ν 3361, 1757, 1646, 1511, 1369, 1232, 1074, 1031
1
cm-1; H NMR (200 MHz, acetone-d6) δ 9.17 (br s, 1H, OH), 8.47 (d, 1H, J = 9.2 Hz, NH),
7.86 (d, 2H, J = 8.7 Hz, HAr), 6.92 (d, 2H, J = 8.7 Hz, HAr), 5.73 (td, 1H, J1,NH = J1,2 = 9.2 Hz,
J1,F = 2.5 Hz, H-1), 5.55 (td, 1H, J2,3 = J3,4 = 9.2 Hz, J3,F = 14.2 Hz, H-3), 5.01 (t, 1H, J3,4
=
J4,5 = 9.7 Hz, H-4), 4.65 (td, 1H, J1,2, J2,3, J2,F = 50.4 Hz, H-2), 4.27 (dd, 1H, J6a,6b = 12.3 Hz,
J5,6a = 4.6 Hz, H-6a), 4.15-4.02 (m, 2H, H-5, H-6b), 2.04, 2.02, 1.99 (each s, 3×3H, OAc); 13C
NMR (50 MHz, acetone-d6) δ 170.17, 167.35 (CO), 161.82 (CArO), 130.46 (CHAr), 125.65
(CArCO), 115.96 (CHAr), 89.47 (C-2, J2,F = 187.2 Hz), 78.77 (C-1, J1,F = 22.7 Hz), 74.28 (C-3,
J3,F = 19.7 Hz), 73.98 (C-5), 69.10 (C-4, J4,F = 7.4 Hz), 62.68 (C-6), 20.59 (CH3); 19F NMR
(acetone-d6) δ −198.11.
N-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyransyl)-2-hydroxyacetamide
(9).
Deprotection of compound 7 (400 mg, 0.879 mmol) using standard hydrogenation conditions
in THF/MeOH (15/15 mL) with 3.5 h stirring, yielded alcohol 9 (320 mg, 99%) as white solid
which was used in the next step without further purification: IR (NaCl) ν 3537, 3343, 1760,
1
1663, 1516, 1369, 1252, 1232, 1068, 1045 cm-1; H NMR (200 MHz, CDCl3) δ 7.42 (d, 1H,
J1,NH = 9.5 Hz, NH), 5.51-5.31 (m, 2H, H-1, H-3), 5.05 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 4.39
(td, 1H, J1,2 = J2,3 = 9.1 Hz, J2,F = 50.6 Hz, H-2), 4.34-4.06 (m, 4H, H-6, CH2OH), 3.87 (ddd,
1H, J6a,6b = 10.1 Hz, J5,6a = 4.3 Hz, J5,6b = 2.0 Hz, H-5), 2.11, 2.09, 2.05 (each s, 3×3H, OAc);
13C NMR (50 MHz, CDCl3) δ 172.31, 170.70, 169.90, 169.69 (CO), 88.40 (C-2, J2,F = 191.3
Hz), 76.98 (C-1, J1,F = 23.1 Hz), 73.91 (C-5), 73.36 (C-3, J3,F = 19.1 Hz), 67.84 (C-4, J4,F
=
6.9 Hz), 62.33, 61.61 (C-6, CH2OH), 20.74, 20.69, 20.60 (CH3); 19F NMR (CDCl3) δ −198.07.
4-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosylcarbamoyl)phenyl
4-{4-[bis(2-
chloroethyl)amino]phenyl}butanoate (10). A standard DCC/DMAP procedure starting from
20

compound 7 (262 mg, 0.612 mmol) and CLB (204 mg, 0.673 mmol) in DCM (27 mL) and 16
h stirring, followed by purification of the crude product by silica gel chromatography
(petroleum ether/ethyl acetate, 6/4, v/v) yielded 10 (242 mg, 55%) as a white solid: mp 103
°C; IR (NaCl) ν 3376, 1749, 1676, 1520, 1367, 1235, 1036 cm-1; 1H NMR (200 MHz, CDCl3)
δ 7.86 (d, 2H, J = 8.6 Hz, HAr), 7.18-7.07 (m, 5H, HAr, NH), 6.64 (d, 2H, J = 8.7 Hz, HAr),
5.59 (td, 1H, J1,2 = J1,NH = 9.2 Hz, J1,F = 1.7 Hz, H-1), 5.43 (td, 1H, J2,3 = J3,4 = 9.2 Hz, J3,F
=
13.6 Hz, H-3), 5.06 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 4.50 (td, 1H, J1,2, J2,3, J2,F = 50.5 Hz, H-
2), 4.38 (dd, 1H, J6a,6b = 12.5 Hz, J5,6a = 4.3 Hz, H-6a), 4.07 (dd, 1H, J6a,6b, J5,6b = 1.9 Hz, H-
6b), 3.91 (ddd, 1H, J4,5, J5,6a, J5,6b, H-5), 3.76-3.58 (m, 8H, N(CH2CH2Cl)2), 2.65, 2.59 (each t,
2×2H, J = 7.4 Hz, CH2CH2CH2), 2.08-1.96 (m, 11H, OAc, CH2CH2CH2); 13C NMR (50 MHz,
CDCl3) δ 171.77, 170.75, 170.23, 169.93, 166.85 (CO), 153.88 (CArO), 144.38 (CArN),
130.61 (CAr), 129.86, 129.10, 121.93, 112.57 (CHAr), 88.36 (C-2, J2,F = 190.7 Hz), 78.18 (C-
1, J1,F = 22.5 Hz), 73.77-73.41 (C-3, C-5), 68.08 (C-4, J4,F = 6.7 Hz), 61.59 (C-6), 53.78
(NCH2), 40.52 (CH2Cl), 34.00, 33.75 (CH2CH2CH2), 26.64 (CH2CH2CH2), 20.74 (CH3); 19F
NMR (CDCl3) δ –197.82; MS (ESI) m/z 713.49 [M+H]+ (Exact Mass: 713.20); Anal.
(C33H39Cl2FN2O10) C, H, N.
2-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosylamino)-2-oxoethyl
4-{4-[bis(2-
chloroethyl)amino]phenyl}butanoate (11). A standard DCC/DMAP procedure starting from
compound 9 (319 mg, 0.870 mmol) and CLB (345 mg, 1.14 mmol) in DCM/DMF (5/5 mL)
and 24 h stirring, followed by purification of the crude product by silica gel chromatography
(7/3 to 5/5, v/v, petroleum ether/ethyl acetate gradient), yielded 11 (461 mg, 81%) as a beige
solid: mp 73 °C; IR (NaCl) ν 3348, 1748, 1519, 1367, 1229, 1070, 1032 cm-1; 1H NMR (200
MHz, CDCl3) δ 7.09-7.02 (m, 3H, HAr, NH), 6.64 (d, 2H, J = 8.7 Hz, HAr), 5.48-5.31 (m, 2H,
H-1, H-3), 5.04 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 4.61 (s, 2H, COCH2O), 4.38 (td, 1H, J1,2
=
J2,3 = 9.1 Hz, J2,F = 50.5 Hz, H-2), 4.32 (dd, 1H, J5,6a = 4.5 Hz, J6a,6b = 12.7 Hz, H-6a), 4.05
(dd, 1H, J6a,6b, J5,6b = 2.2 Hz, H-6b), 3.87 (ddd, 1H, J4,5, J5,6a, J5,6b, H-5), 3.75-3.58 (m, 8H,
N(CH2CH2Cl)2), 2.59 and 2.43 (each t, 2H, J = 7.4 Hz, CH2CH2CH2), 2.08, 2.06, 2.04 (each s,
3×3H, OAc), 1.95 (qt, 2H, 3J = 7.4 Hz, CH2CH2CH2); 13C NMR (50 MHz, CDCl3) δ 171.94,
170.63, 169.88, 169.74, 167.78 (CO), 144.62 (CArN), 130.18 (CArCH2), 129.84, 112.42
(CHAr), 88.45 (C-2, J2,F = 191.6 Hz), 77.09 (C-1, J1,F = 23.0 Hz), 74.11 (C-5), 73.40 (C-3, J3,F
= 19.3 Hz), 67.86 (C-4, J4,F = 7.0 Hz), 62.84, 61.67 (C-6, COCH2O), 53.72 (NCH2), 40.63
21

(CH2Cl), 33.90, 33.20 (CH2CH2CH2), 26.46 (CH2CH2CH2), 20.82, 20.75, 20.67 (CH3); 19F
NMR (CDCl3) δ −198.10; MS (ESI) m/z 651.43 [M+H]+ (exact mass = 651.19). Anal.
(C28H37Cl2FN2O10) C, H, N.
Benzyl (3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosylamino]-3-oxopropanoic acid
(12). Coupling of amine 5 (500 mg, 1.63 mmol) with malonic acid monobenzyl ester (320 mg,
1.80 mmol) was carried out according to the DCC/HOBt general procedure in DMF (25 mL)
and 2 days stirring. Chromatography on silica gel (cyclohexane/ethyl acetate, 6/4, v/v) yielded
compound 12 (450 mg, 57%) as a pale oil: IR (KBr) ν 3368, 1751, 1560, 1382, 1230, 1036
cm-1; NMR 1H (200 MHz, CDCl3) δ 8.12 (d, 1H, J = 9.0 Hz, NH), 7.37 (s, 5H, Ph), 5.45-5.29
(m, 2H, H-1, H-3), 5.19 (s, 2H, OCH2Ph), 5.05 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-4), 4.35 (td, 1H,
J1,2 = J2,3 = 8.9 Hz, J2,F = 50.6 Hz, H-2), 4.29 (dd, 1H, J5,6a = 4.2 Hz, J6a,6b = 12.5 Hz, H-6a),
4.08 (dd, 1H, J6a,6b, J5,6b = 1.9 Hz, H-6b), 3.83 (ddd, 1H, J4,5, J5,6a, J5,6b, H-5), 3.44 (s, 2H,
COCH2CO), 2.09, 2.08, 2.04 (3s, 3×3H, OAc); NMR 13C (50 MHz, CDCl3) δ 170.71, 169.98,
169.72, 169.18 (COO), 165.60 (NHCO), 134.79 (CArCH2), 128.88, 128.63 (CHAr), 88.50 (C-
2, J2,F = 191.2 Hz), 77.29 (C-1, J1,F = 23.2 Hz), 73.87 (C-5), 73.46 (C-3, J3,F = 19.5 Hz), 67.92
(C-4, J4,F = 6.5 Hz), 61.70 (C-6), 40.70 (COCH2CO), 20.84, 20.78, 20.69 (CH3).
3-(3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosylamino)-3-oxopropanoic acid ( ).
13
Benzyl ester
(430 mg, 0.889 mmol) in ethanol/water (20/10 mL) was treated with lithium
12
hydroxide (80 mg, 3.34 mmol) for 12 h at room temperature. After addition of water (10 mL)
and DCM (30 mL) and separation of the layers, the aqueous one was acidified with HCl 1M
(2 mL) and extracted with DCM (3×25 mL). The organic extracts were combined, dried over
MgSO4, filtered, and concentrated under reduced pressure to yield the acid 13 (310 mg, 89%)
as an oil which was used in the next step without further purification: IR (KBr) ν 3350, 1740,
1708, 1655, 1568, 1381, 1238, 1044 cm-1; NMR 1H (200 MHz, CDCl3) δ 8.14 (d, 1H, J = 8.4
Hz, NH), 6.71 (br.s, 1H, COOH), 5.45-5.31 (m, 2H, H-1, H-3), 5.05 (t, 1H, J3,4 = J4,5 = 9.6
Hz, H-4), 4.55 (t, ½ H, J1,2 = J2,3 = 8.9 Hz, H-2), 4.29 (m, 1.5H, ½ H-2, H-6a), 4.09 (m, 1H,
H-6b), 3.46 (m, 1H, H-5), 3.46 (s, 2H, COCH2CO), 2.08, 2.05 (each s, 9H, OAc); NMR 13C
(50 MHz, DMSO-d6) δ 170.02, 169.55, 169.36, 168.70, 166.53 (CO), 88.38 (C-2, J2,F = 186.1
Hz), 76.47 (C-1, J1,F = 22.8 Hz), 72.68 (C-3, J3,F = 19.0 Hz), 71.95 (C-5), 67.74 (C-4, J4,F
=
7.3 Hz), 61.58 (C-6), 42.91 (COCH2CO), 20.54, 20.45, 20.38 (CH3), 19F NMR (CDCl3) δ
−197.45.
22

N-(3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyloxy)-N’-[3-{4-[bis(2-
chloroethyl)amino]phenyl}propyl]propanediamide (15). Coupling of 4-(3-aminopropyl)-N,N-
bis(2-chloroethyl)aniline 14 (100 mg, 0.363 mmol) with acid 13 (140 mg, 0.356 mmol) was
carried out according to the general DCC/HOBt procedure in DMF (5 mL). After 24 h at
room temperature, 10 mL of water were added and the reaction medium was extracted with
DCM (3×10 mL). The organic extracts were combined, washed with saturated aqueous
NaHCO3 (2×5 mL) and aqueous acetic acid 1N (2×5 mL) solution, dried over MgSO4, filtered
and concentrated under reduced pressure. Purification by silica gel chromatography
(cyclohexane/ethyl acetate, 3/7 to 5/5, v/v) provided compound 15 (30 mg, 13%) as a white
powder: mp 180 °C; IR (KBr) ν 3466, 1753, 1654, 1520, 1367, 1232, 1069, 1038 cm-1; NMR
1H (200 MHz, CDCl3) δ 8.50 (d, 1H, J = 8.9 Hz, C1NH), 7.05 (d, 2H, J = 8.4 Hz, HAr), 6.61
(d, 3H, J = 8.4 Hz, HAr, NHCH2), 5.44-5.28 (m, 2H, H-1, H-3), 5.04 (t, 1H, J3,4 = J4,5 = 9.7
Hz, H-4), 4.37 (td, 1H, J1,2 = J2,3 = 9.1 Hz, J2,F = 50.7 Hz, H-2), 4.27 (m, 1H, H-6a), 4.07 (m,
1H, H-6b), 3.83 (m, 1H, H-5), 3.72-3.57 (m, 8H, N(CH2CH2Cl)2), 3.28 (q, 2H, J = 6.6 Hz,
NHCH2), 3.21 (s, 2H, COCH2CO), 2.56 (t, 2H, J = 7.4 Hz, CH2Ph), 2.08, 2.07, 2.04 (each s,
3×3H, OAc), 1.80 (qt, 2H, J = 7.3 Hz, CH2CH2CH2); NMR 13C (50 MHz, CDCl3) δ 170.72,
170.03, 169.68 (COCH3), 168.06, 166.96 (NHCO), 144.53 (CArN), 130.28 (CArCH2), 129.67,
112.43 (CHAr), 88.43 (C-2, J2,F = 190.9 Hz), 77.63 (C-1), 73.84 (C-5), 73.47 (C-3, J3,F = 19.3
Hz), 67.93 (C-4, J4,F = 7.1 Hz), 61.70 (C-6), 53.71 (NCH2), 42.79 (COCH2CO), 40.62
(CH2Cl), 39.53 (CH2NH), 32.11 (CH2Ph), 31.03 (CH2CH2CH2), 20.84, 20.78, 20.69 (CH3);
19F NMR (CDCl3) δ −197.98; MS (ESI) m/z 650.24 [M+H]+ (exact mass = 650.20).
3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-1-(4-nitrobenzyl)-β-D-glucopyranose
(16).
4-
nitrobenzoic acid (580 mg, 3.47 mmol) was dissolved in MeOH (30 mL) and NaOH (140 mg,
3.5 mmol) was added. The mixture was stirred for 1h30 at room temperature. After
evaporation under vacuum, the residue was dissolved in DMF (15 mL) and bromo sugar 4
(700 mg, 1.88 mmol) was added. The mixture was kept at room temperature for 3h. DMF was
evaporated and the crude product was dissolved in water (60 mL) before extraction with
DCM (5 × 40 mL). The organic extracts were combined, washed with saturated aqueous
NaHCO3 solution (2 × 30 mL), dried over MgSO4, filtered and then evaporated to afford, after
purification by silica gel chromatography (cyclohexane/ethyl acetate, 5/5, v/v) and
precipitation in diisopropyl ether, the pure product 16 (320 mg, 37%) as a white powder: mp
109 °C ; IR (KBr) ν 1744, 1535, 1367, 1242, 1084, 1033 cm-1; NMR 1H (500 MHz, CDCl3) δ
23

8.33 (d, 2H, J = 8.8 Hz, HAr), 8.28 (d, 2H, J = 8.8 Hz, HAr), 6.05 (dd, 1H, J1,2 = 8.0 Hz, J1,F
3.0 Hz, H-1), 5.46 (td, 1H, J2,3 = J3,4 = 9.1 Hz, J3,F = 14.4 Hz, H-3), 5.15 (t, 1H, J3,4 = J4,5 = 9.7
Hz, H-4), 4.66 (ddd, 1H, J1,2, J2,3, J2,F = 50.8 Hz, H-2), 4.33 (dd, 1H, J6a,6b = 12.6 Hz, J6a,5
=
=
4.6 Hz, H-6a), 4.15 (dd, 1H, J6a,6b, J5,6b = 2.2 Hz, H-6b), 3.98 (ddd, 1H, J4,5, J5,6a, J5,6b, H-5),
2.12, 2.08, 2.07 (each s, 3×3H, OAc); RMN 13C (50 MHz, CDCl3) δ 170.58, 169.95, 169.63
(CO), 162.85 (COPh), 151.26 (CArNO2), 133.98 (CArCO), 131.49, 123.68 (CHAr), 92.49 (C-1,
J1,F = 24.2 Hz), 88.36 (C-2, J2,F = 191.4 Hz), 73.19 (C-5), 72.74 (C-3, J3,F = 19.6 Hz), 67.68
(C-4, C4,F = 7.1 Hz), 61.41 (C-6), 20.76, 20.68 (CH3).
3,4,6-Tri-O-acetyl-1-(4-aminobenzyl)-2-deoxy-2-fluoro-β-D-glucopyranose (17). Reduction
of nitro compound 16 (300 mg, 0.65 mmol) using standard conditions in THF/EtOH (10/5
mL) during 10 h, yielded aniline 17 (280 mg, quant.) as a white solid: IR (KBr) ν 3478, 3383,
1747, 1603, 1368, 1243, 1069, 1040 cm-1; NMR 1H (200 MHz, CDCl3) δ 7.90 (d, 2H, J = 8.7
Hz, HAr), 6.64 (d, 2H, J = 8.7 Hz, HAr), 6.01 (dd, 1H, J1,2 = 8.0 Hz, J1,F = 3.1 Hz, H-1), 5.44
(td, 1H, J2,3 = J3,4 = 9.1 Hz, J3,F = 14.3 Hz, H-3), 5.13 (t, 1H, J3,4 = J4,5 = 9.6 Hz, H-4), 4.61
(ddd, 1H, J1,2, J2,3, J2,F = 50.7 Hz, H-2), 4.33 (dd, 1H, J6a,6b = 12.5 Hz, J6a,5 = 4.4 Hz, H-6a),
4.18 (br.s, 2H, NH2), 4.11 (dd, 1H, J6a,6b, J6b,5 = 2.2 Hz, H-6b), 3.93 (ddd, 1H, J4,5, J5,6a, J5,6b
,
H-5), 2.10, 2.06, 2.05 (3s, 3×3H, OAc); NMR 13C (50 MHz, CDCl3) δ 170.71, 170.03, 169.71
(CO), 164.41 (COPh), 151.95 (CArNH2), 132.60 (CHAr), 117.69 (CArCO), 113.88 (CHAr),
91.57 (C-1, J1,F = 24.0 Hz), 88.53 (C-2, J2,F = 190.9 Hz), 73.08 (C-3, J3,F = 19.7), 72.79 (C-5),
67.91 (C-4, C4,F = 7.0 Hz), 61.60 (C-6), 20.80, 20.70 (CH3).
N-[4-(3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl-oxycarbonyl)phenyl]-4-{4-
[bis(2-chloroethyl)amino]phenyl}butanamide (18). Ethylchloroformate coupling, starting
from amine 17 (260 mg, 0.61 mmol), CLB (220 mg, 0.72 mmol), TEA (140 µL, 1.01 mmol)
and ethylchloroformate (84 µL, 0.88 mmol) in DCM (20 mL) and with a reaction time of 30
min, afforded compound 18 (290 mg, 50%) as a white powder after chromatography on silica
gel (ethyl acetate/DCM, 15/85, v/v): mp 87 °C; IR (KBr) ν 3366, 1746, 1597, 1519, 1367,
1243, 1070, 1043 cm-1; NMR 1H (200 MHz, CDCl3) δ 8.05 (d, 2H, J = 8.8 Hz, HAr), 7.61 (d,
2H, J = 8.8 Hz, HAr), 7.34 (s, 1H, NH), 7.08 (d, 2H, J = 8.6 Hz, HAr), 6.63 (d, 2H, J = 8.6 Hz,
HAr), 6.03 (dd, 1H, J1,2 = 8.0 Hz, J1,F = 3.0 Hz, H-1), 5.45 (td, 1H, J3,2 = J3,4 = 9.1 Hz, J3,F
=
14.3 Hz, H-3), 5.14 (t,1H, J3,4 = J4,5 = 9.8 Hz, H-4), 4.64 (td, 1H, J1,2 ≈ J2,3, J2,F = 50.7 Hz, H-
24

2), 4.33 (dd, 1H, J6a,6b = 12.4 Hz, J6a,5 = 4.3 Hz, H-6a), 4.12 (dd, 1H, J5,6a, J6b,H5 = 1.9 Hz, H-
3
6b), 3.95 (ddd, 1H, J4,5, J5,6a, J5,6b, H-5), 3.74-3.56 (m, 8H, N(CH2CH2Cl)2), 2.63 (t, 2H, J =
3
7.3 Hz, PhCH2), 2.38 (t, 2H, J = 7.4 Hz, COCH2), 2.11-2.10 (m, 11H, OAc, CH2CH2CH2);
NMR 13C (50 MHz, CDCl3) δ 171.44 (CONH), 170.69, 170.02, 169.68 (CO), 163.95 (COPh),
144.62, 143.20 (CArN, CArNH), 131.73 (CHAr), 130.35 (CArCH2), 129.86 (CHAr), 123.73
(CArCO), 118.80, 112.40 (CHAr), 91.88 (C-1, J1,F = 24.1 Hz), 88.46 (C-2, J2,F = 191.2 Hz),
72.96 (C-3, J3,F = 19.7), 72.93 (C-5), 67.83 (C-4, J4,F = 7.2 Hz), 61.53 (C-6), 53.72 (NCH2),
40.65 (CH2Cl), 37.03 (COCH2), 34.01 (CH2Ph), 26.91 (CH2CH2CH2), 20.81, 20.78, 20.69
(CH3); 19F NMR (CDCl3) δ −200.69; MS (ESI) m/z 713.29 [M+H]+ (Exact Mass: 713.20);
Anal. (C33H39Cl2FN2O10) C, H, N.
3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl 4-nitrophenylcarbonate (19). To a
solution of compound 3,4,6-tri-O-acetyl-2-desoxy-2-fluoro-α,β-D-glucose (4.4 g, 14.3 mmol)
in THF (70 mL), cooled to 0 °C were added 4-nitrophenylchloroformate (2.87 g, 14.2 mmol)
and TEA (2.4 mL, 17.3 mmol). The reaction mixture was stirred at room temperature for 24 h.
The formed white solid was filtered, dissolved in ethyl acetate (150 mL) and the resulting
solution was washed successively with water (4×40 mL) and brine (60 mL). After drying over
MgSO4, filtration and concentration under reduced pressure, a white solid corresponding to
the β-anomer of compound 19 (1.5 g, 3.17 mmol) was obtained. The former filtrate was
evaporated under reduced pressure and the residue was purified by silica gel chromatography
(cyclohexane/ethyl acetate, 6/4, v/v) to afford carbonate 19 (3.7 g, 7.82 mmol) as a mixture of
α- and β-anomers. From this mixture, a supplementary fraction of β-anomer (890 mg, 1.88
mmol) can be obtained by crystallization in diisopropryl ether: 77% yield for α- and β-
anomers; 35% yield for β-anomer; mp 191 °C (β-anomer); IR (KBr) ν 1779, 1740, 1526,
1
1346, 1241, 1079, 1040 cm-1; H NMR (200 MHz, CDCl3) δ 8.32 (d, 2H, J = 9.1 Hz, HAr),
7.44 (d, 2H, J = 9.1 Hz, HAr), 5.75 (dd, 1H, J1,F = 3.3 Hz, J1,2 = 8.0 Hz, H-1), 5.44 (td, 1H, J2,3
= J3,4 = 9.2 Hz, J3,F = 14.3 Hz, H-3), 5.13 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 4.57 (ddd, 1H, J1,2,
J2,3, J2,F = 50.6 Hz, H-2), 4.34 (dd, 1H, J6a,6b = 12.6 Hz, J5,6a = 4.4 Hz, H-6a), 4.17 (dd, 1H,
J6a,6b, J5,6b = 2.2 Hz, H-6b), 3.93 (ddd, 1H, J4,5, J5,6a, J5,6b, H-5), 2.12, 2.10, 2.06 (each s, 3×3H,
OAc); 13C NMR (50 MHz, CDCl3) δ 170.59, 169.93, 169.56 (COCH3), 154.99, 150.95
(OCOCArO), 145.96 (CArNO2), 125.59, 121.84 (CHAr), 95.58 (C-1, J1-F = 24.4 Hz), 88.09 (C-
2, J2-F = 191.9 Hz), 73.19 (C-5), 72.41 (C-3, J3-F = 19.6 Hz), 67.43 (C-4, J4-F = 7.3 Hz), 61.26
(C-6), 20.79, 20.74, 20.65 (CH3); 19F NMR (CDCl3) δ −200.81.
25

4-{4-[bis(2-chloroethyl)amino]phenyl}-N-(4-nitrophenyl)butanamide (20). Prepared from p-
nitroaniline (240 mg, 1.74 mmol) and CLB (500 mg, 1.64 mmol) in the presence of TEA (380
µL, 2.73 mmol) and ethylchloroformate (170 µL, 1.78 mmol) in DCM (16 mL) according to
the standard ethylchloroformate coupling. After stirring for 17 h, purification by
chromatography on alumina (cyclohexane/ethyl acetate, 7/3, v/v) yielded compound 20 (170
mg, 24%) as a yellow powder: mp 105 °C; IR (KBr) ν 3331, 1681, 1520, 1508, 1349 cm-1; 1H
NMR (200 MHz, CDCl3) δ 8.16 (d, 3H, J = 9.0 Hz, HAr, NH), 7.73 (d, 2H, J = 9.0 Hz, HAr),
7.05 (d, 2H, J = 8.5 Hz, HAr), 6.59 (d, 2H, J = 8.5 Hz, HAr), 3.72-3.55 (m, 8H,
N(CH2CH2Cl)2), 2.60 (t, 2H, J = 7.3 Hz, CH2Ph), 2.43 (t, 2H, J = 7.4 Hz, COCH2), 2.01 (qt,
2H, J = 7.4 Hz, CH2CH2CH2); 13C NMR (50 MHz, CDCl3) δ 171.63 (CO), 144.66, 143.87,
143.54 (CArN, CArNO2, CArNH), 130.18 (CArCH2), 129.85, 125.24, 119.05, 112.38 (CHAr),
53.69 (NCH2), 40.66 (CH2Cl), 36.99 (COCH2), 33.97 (CH2Ph), 26.86 (CH2CH2CH2).
N-(4-aminophenyl)-4-{4-[bis(2-chloroethyl)amino]phenyl}butanamide (21). Reduction of
nitro compound 20 (360 mg, 0.848 mmol) in THF/EtOH (10/10 mL) for 4 h using standard
conditions, yielded amine 21 (250 mg, 75%) as a light yellow solid after purification by silica
gel chromatography (cyclohexane/ethyl acetate, 4/6, v/v): mp 130 °C; IR (KBr) ν 3463, 3279,
1647, 1517 cm-1; 1H NMR (200 MHz, CDCl3) δ 7.24 (d, 2H, J = 8.4 Hz, HAr), 7.08 (d, 3H, J
= 8.7 Hz, HAr, NH), 6.62 (2d, 2×2H, J = 8.4 Hz, J = 8.7 Hz, HAr), 3.74-3.56 (m, 8H,
N(CH2CH2Cl)2), 2.60 (t, 2H, J = 7.3 Hz, CH2Ph), 2.30 (t, 2H, J = 7.4 Hz, COCH2), 1.99 (qt,
2H, J = 7.4 Hz, CH2CH2CH2); 13C NMR (50 MHz, CDCl3) δ 170.98 (CO), 144.47, 143.34
(CArN, CArNH2), 130.71 (CArCH2), 129.84 (CHAr), 129.39 (CArNH), 122.10, 115.48, 112.30
(CHAr), 53.70 (NCH2), 40.67 (CH2Cl), 36.75 (COCH2), 34.11 (CH2Ph), 27.34 (CH2CH2CH2).
(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl)
4-(4-{4-[bis(2-chloroethyl)
amino]phenyl}butanamido)phenylcarbamate (22) was prepared according to the standard
carbamoylation procedure, starting from amine 21 (100 mg, 0.254 mmol) and β-carbonate 19
(118 mg, 0.249 mmol) in DMF (10 mL) with a reaction time of 25 h. Chromatography on
silica gel (DCM/ethyl acetate, 9/1, v/v) afforded β anomer 22 (77 mg, 42%) as a white
1
powder: mp 119 °C; IR (KBr) ν 3422, 1752, 1519, 1222, 1071, 1037 cm-1; H NMR (200
MHz, CDCl3) δ 7.43 (d, 3H, J = 8.8 Hz, HAr, NH), 7.31 (d, 2H, J = 8.8 Hz, HAr), 7.24 (br s,
1H, NH), 7.07 (d, 2H, J = 8.5 Hz, HAr), 6.61 (d, 2H, J = 8.5 Hz, HAr), 5.83 (dd, 1H, J1,2 = 8.1
26

Hz, J1,F = 2.9 Hz, H-1), 5.41 (td, 1H, J2,3 = J3,4 = 9.2 Hz, J3,F = 14.3 Hz, H-3), 5.09 (t, 1H, J3,4
J4,5 = 9.7 Hz, H-4), 4.48 (td, 1H, J1,2 ≈ J2,3, J2,F = 50.8 Hz, H-2), 4.33 (dd, 1H, J6a,6b = 12.8 Hz,
J5,6a = 4.3 Hz, H-6a), 4.10 (dd, 1H, J6a,6b, J5,6b = 3.1 Hz, H-6b), 3.89 (ddd, 1H, J4,5, J5,6a, J5,6b
=
,
H-5), 3.74-3.56 (m, 8H, N(CH2CH2Cl)2), 2.60 (t, 2H, J = 7.3 Hz, CH2Ph), 2.33 (t, 2H, J = 7.4
Hz, COCH2), 2.10-1.96 (m, 11H, OAc, CH2CH2CH2); 13C NMR (50 MHz, CDCl3) δ 171.43
(NHCO), 170.71, 170.00, 169.70 (COCH3), 150.92 (OCONH), 144.51 (CArN), 134.40, 133.17
(CArNH), 130.52 (CArCH2), 129.79, 120.95, 119.90, 112.32 (CHAr), 92.49 (C-1, J1,F = 23.8
Hz), 88.27 (C-2, J2,F = 191.3 Hz), 72.83 (C-3, J3,F = 19.0 Hz), 72.65 (C-5), 67.74 (C-4, J4,F
=
7.4 Hz), 61.44 (C-6), 53.65 (NCH2), 40.67 (CH2Cl), 36.77 (COCH2), 34.08 (CH2Ph), 27.18
(CH2CH2CH2), 20.74, 20.63 (CH3); 19F NMR (CDCl3) δ −200.71; MS (ESI) m/z 728.25
[M+H]+ (Exact Mass: 728.20); Anal. (C33H40Cl2FN3O10) C, H, N.
3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl) 4-(4-{4-[bis(2-chloroethyl)amino]
phenyl}butanoyloxy)phenylcarbamate (25). Carbamoylation of amine 2435 (280 mg, 0.708
mmol) with β-anomer 19 (250 mg, 0.528 mmol) was carried out in DMF (15 mL) with 40 h
stirring, according to the standard procedure. The crude product was chromatographed on
silica gel (DCM/ethyl acetate, 19/1, v/v) to give compound 25 (54 mg, 14%) as a white
1
powder: mp 130 °C; IR (KBr) ν 3361, 1753, 1519, 1218, 1071, 1038 cm-1; H NMR (200
MHz, CDCl3) δ 7.38 (d, 2H, J = 8.8 Hz, HAr), 7.10 (d, 3H, J = 8.7 Hz, NH, HAr), 7.01 (d, 2H,
J = 8.8 Hz, HAr), 6.64 (d, 2H, J = 8.7 Hz, HAr), 5.84 (dd, 1H, J1,2 = 8.1 Hz, J1,F = 2.9 Hz, H-1),
5.42 (td, 1H, J2,3 = J3,4 = 9.2 Hz, J3,F = 14.2 Hz, H-3), 5.09 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-4),
4.49 (td, 1H, J1,2 ≈ J2,3, J2,F = 50.8 Hz, H-2), 4.34 (dd, 1H, J6a,6b = 12.7 Hz, J5,6a = 4.2 Hz, H-
6a), 4.10 (dd, 1H, J6a,6b, J5,6b = 2.0 Hz, H-6b), 3.90 (ddd, 1H, J4,5, J5,6a, J5,6b, H-5), 3.75-3.57
(m, 8H, N(CH2CH2Cl)2), 2.64 (t, 2H, J = 7.2 Hz, PhCH2), 2.56 (t, 2H, J = 7.4 Hz, COCH2),
2.10-1.95 (m, 11H, OAc, CH2CH2CH2); 13C NMR (50 MHz, CDCl3) δ 172.30 (COCH2),
170.69, 169.97, 169.71 (COCH3), 150.76 (OCONH), 147.03 (CArO), 144.53 (CArN), 134.51
(CArNH), 130.33 (CArCH2), 129.85, 122.27, 120.09, 112.27 (CHAr), 92.49 (C-1, J1-F = 23.9
Hz), 88.23 (C-2, J2-F = 191.3 Hz), 72.80 (C-3, J3-F = 19.4 Hz), 72.67 (C-5), 67.69 (C-4, J4-F
=
7.2 Hz), 61.41 (C-6), 53.65 (NCH2), 40.62 (CH2Cl), 34.01, 33.68 (COCH2CH2CH2Cl), 26.76
(CH2CH2CH2), 20.76, 20.74, 20.65 (CH3); 19F NMR (CDCl3) δ −200.73; MS (ESI) m/z
729.19 [M+H]+ (Exact Mass: 729.20); Anal. (C33H39Cl2FN2O11) C, H, N.
27

Benzyl
2-[4-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosylcarbamoyl)phenyl
amino]-2-oxoethylcarbamate (28) was prepared according to the standard ethylchloroformate
coupling, starting from N-Cbz-glycine (215 mg, 1.03 mmol), amine 2735 (400 mg, 0.938
mmol), TEA (141 µL, 1.01 mmol) and ethylchloroformate (100 µL, 1.05 mmol) in DCM (25
mL). After 20 h stirring, chromatography on alumina (ethyl acetate/ethanol, 9/1, v/v) yielded
compound 28 (290 mg, 50%) as a white powder: mp 225 °C; IR (KBr) ν 3374, 1745, 1677,
1
1656, 1525, 1367, 1242, 1067, 1036 cm-1; H NMR (200 MHz, DMSO-d6) δ 10.28 (s, 1H,
PhNHCO), 9.23 (d, 1H, J = 9.1 Hz, C1NH), 7.88 (d, 2H, J = 8.7 Hz, HAr), 7.71 (d, 2H, J = 8.7
Hz, HAr), 7.60 (br t, 1H, J = 6.3 Hz, CH2NH), 7.37 (m, 5H, Ph), 5.74-5.51 (m, 2H, J3,F = 14.3
Hz, H-1, H-3), 5.05 (s, 2H, CH2Ph), 4.90 (t, 1H, J3,4 = J4,5 = 9.1 Hz, H-4), 4.67 (td, 1H, J1,2
=
J2,3 = 8.9 Hz, J2,F = 50.2 Hz, H-2), 4.21-4.16 (m, 2H, 2H-6), 3.98 (m, 1H, H-5), 3.84 (d, 2H, J
= 6.1 Hz, CH2NH), 2.05, 2.00, 1.98 (each s, 3×3H, OAc); 13C NMR (50 MHz, DMSO-d6) δ
170.53, 170.08, 169.88, 169.08, 166.60 (CO), 157.14 (NHCOO), 142.91 (CArNH), 137.57
(CArCH2), 129.16, 128.90, 128.34, 128.26 (CHAr), 127.93 (CArCO), 118.83 (CHAr), 88.70 (C-
2, J2,F = 185.1 Hz), 77.71 (C-1, J1,F = 22.8 Hz), 73.41 (C-3, J3,F = 19.6 Hz), 72.49 (C-5), 68.36
(C-4, J4,F = 7.8 Hz), 66.04 (CH2Ph), 62.16 (C-6), 44.73 (COCH2NH), 21.06, 20.99, 20.92
(CH3) ; 19F NMR (DMSO-d6) δ −198.84.
N-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl)-4-(2-aminoacetamido)
benzamide (29). The deprotection of the N-Cbz protected compound 28 (390 mg, 0.631
mmol) was performed under standard hydrogenation conditions in THF/EtOH (40/10 mL) and
yielded compound 29 (305 mg, 100%) as a white solid, used directly for the next step: IR
1
(KBr) ν 3446, 1747, 1653, 1540, 1249, 1036 cm-1; H NMR (200 MHz, CDCl3) δ 9.77 (br s,
1H, NH), 7.80 (d, 2H, J = 7.7 Hz, NH, HAr), 7.61 (d, 2H, J = 7.7 Hz, HAr), 5.59-5.32 (m, 2H,
H-1, H-3), 5.07 (t, 1H, J3,4 = J4,5 = 9.6 Hz, H-4), 4.63 (td, 1H, J1,2 = J2,3 = 8.5 Hz, J2,F = 50.4
Hz, H-2), 4.36-3.87 (m, 3H, H-5, 2H-6), 3.56 (s, 2H, CH2NH2), 2.74 (br s, 2H, NH2), 2.07,
2.03 (each s, 9H, OAc); 13C NMR (50 MHz, DMSO-d6) δ 171.64, 170.01, 169.56, 169.38,
166.08 (CO), 142.16 (CArNH), 128.63 (CHAr), 127.47 (CArCO), 118.31 (CHAr), 88.24 (C-2,
J2,F = 185.9 Hz), 77.15 (C-1, J1,F = 22.8 Hz), 72.87 (C-3, J3,F = 19.3 Hz), 72.00 (C-5), 67.84
(C-4, J4,F = 7.6 Hz), 61.62 (C-6), 45.03 (CH2NH2), 20.53, 20.46, 20.39 (CH3); 19F NMR
(DMSO-d6) δ −198.88.
28

N-(3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-β-D-glucopyranosyl)-4-[2-(4-{4-[bis(2-chloro
ethyl)amino]phenyl}butanamido)acetamido]benzamide (30). Coupling of amine 29 (140 mg,
0.290 mmol) with CLB (97 mg, 0.319 mmol) was carried out in DMF (15 mL) according to
the DCC/HOBt general procedure to yield after 24 h stirring and purification on silica gel
(cyclohexane/ethyl acetate, 3/7, v/v) the title compound 30 (130 mg, 58%) as a white solid:
mp 190 °C; IR (KBr) ν 3385, 1748, 1654, 1522, 1507, 1244, 1039 cm-1; 1H NMR (200 MHz,
DMSO-d6) δ 10.28 (s, 1H, PhNHCO), 9.23 (d, 1H, J = 8.8 Hz, C1NH), 8.19 (br.t, 1H, J = 5.8
Hz, CH2NH), 7.88 (d, 2H, J = 8.9 Hz, HAr), 7.70 (d, 2H, J = 8.9 Hz, HAr), 7.04 (d, 2H, J = 8.6
Hz, HAr), 6.66 (d, 2H, J = 8.6 Hz, HAr), 5.73-5.50 (m, 2H, J2,3 = J3,4 = 9.3 Hz, J3,F = 14.1 Hz,
H-1, H-3), 4.89 (t, 1H, J3,4 = J4,5 = 9.3 Hz, H-4), 4.66 (td, 1H, J1,2 = J2,3 = 9.0 Hz, J2,F = 50.2
Hz, H-2), 4.20-3.93 (m, 3H, 2H-6, H-5), 3.89 (d, 2H, J = 5.6 Hz, CH2NH), 3.70 (s, 8H,
N(CH2CH2Cl)2), 2.47 (m, 2H, CH2Ph), 2.16 (t, 2H, J = 7.3 Hz, COCH2CH2), 2.05, 2.00, 1.98
3
(each s, 3×3H, OAc), 1.75 (qt, 2H, J = 7.3 Hz, CH2CH2CH2); 13C NMR (50 MHz, DMSO-
d6) δ 172.61, 169.96, 169.52, 169.34, 168.47, 166.04 (CO), 144.39 (CArN), 142.32 (CArNH),
129.97 (CArCH2), 129.33, 128.58 (CHAr), 127.41 (CArCO), 118.30, 111.90 (CHAr), 88.23 (C-2,
J2,F = 185.8 Hz), 77.09 (C-1, J1,F = 22.9 Hz), 72.83 (C-3, J3,F = 18.9 Hz), 71.96 (C-5), 67.79
(C-4, J4,F = 7.8 Hz), 61.58 (C-6), 52.23 (NCH2), 42.76 (CH2NH), 41.16 (CH2Cl), 34.58, 33.56
(CH2CH2CH2), 27.32 (CH2CH2CH2), 20.51, 20.44, 20.37 (CH3); 19F NMR (DMSO-d6) δ
−198.87; MS (ESI) m/z 769.35 [M+H]+ (Exact Mass: 769.23); Anal. (C35H43Cl2FN4O10) C, H,
N.
4-(2-(benzyloxycarbonylamino)acetamido)phenyl
4-(4-(bis(2-chloroethyl)amino)phenyl)
butanoate (31). Coupling of compound 24 (1.19 g, 3.01 mmol) with N-Cbz-glycine (640 mg,
3.06 mmol) was carried out according to the DCC/HOBt general procedure in DMF (70 mL)
and with stirring during 24 h, to yield after purification on silica gel (petroleum ether/ethyl
acetate, 5/5, v/v) the title compound 31 (1.60 g, 90%) as a white powder: IR (KBr) ν 3337,
1746, 1692, 1670 cm-1; 1H NMR (200 MHz, CDCl3) δ 8.37 (br.s, 1H, NHPh), 7.47 (d, 2H, Jo
= 8.8 Hz, HAr), 7.34 (m, 5H, HAr), 7.10 (d, 2H, J’o = 8.5 Hz, HAr), 6.97 (d, 2H, Jo, HAr), 6.63
3
(d, 2H, J’o, HAr), 5.70 (br t, 1H, NHCH2), 5.14 (s, 2H, PhCH2CO), 3.98 (d, 2H, J = 5.5 Hz,
NHCH2CO), 3.75-3.57 (m, 8H, N(CH2CH2Cl)2), 2.64, 2.55 (t, 2H, 3J = 7.3 Hz, CH2CH2CH2),
3
2.02 (qt, 2H, J = 7.3 Hz, CH2CH2CH2); 13C NMR (50 MHz, CDCl3) δ 172.42, 167.42,
157.04 (CO), 147.08 (CArO), 144.53 (CArN), 136.08, 135.20 (CArNH, CArCH2O), 130.33
(CArCH2CH2), 129.84, 128.69, 128.40, 128.10, 122.06, 121.05, 112.28 (CHAr), 67.45
29