Structure-activity relationship study of hypoxia-activated prodrugs for proteoglycan-targeted chemotherapy in chondrosarcoma

Article abstract of DOI:10.1016/j.ejmech.2018.08.060

Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-resistant. Surgical resection with wide margins remains the mainstay of treatment. To address the lack of therapy, our strategy aims to increase anticancer drugs targeting and delivery in the tumour, by leveraging specific chondrosarcoma hallmarks: an extensive cartilaginous extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia. A dual targeted therapy for chondrosarcoma was investigated by conjugation of a hypoxia-activated prodrug (HAP) to quaternary ammonium (QA) functions which exhibit a high affinity for polyanionic sites of proteoglycans (PGs), the major components of the chondrosarcoma extracellular matrix. Based on preclinical results, an imidazole prodrug, ICF05016, was identified and provided the basis for a lead optimization study. A series of 27 QA-phosphoramide mustard conjugates, differing by the type of QA function and the length of the alkyl linker, was yielded by a common multi-step sequence involving phosphorylation of a key 2-nitroimidazole alcohol. Then, a screening was realized by surface plasmon resonance technology to assess biomolecular interactions between QA derivatives and aggrecan, the most abundant PG in chondrosarcoma. Results revealed that affinity depends more on the type of QA function, than on the linker length. Moreover, the presence of a benzyl group enhanced affinity to aggrecan. Twelve compounds were shortlisted and evaluated for antiproliferative activity (i.e., growth inhibiting concentration 50), under normoxic and hypoxic conditions using the human extraskeletal myeloid chondrosarcoma cell line (HEMC-SS). For all prodrugs, hypoxic selectivity was maintained and even increased, compared with the lead. From this study, compound 31f emerged as the most effective PG-targeted HAPs with a dissociation constant of 2.10 μM in the SPR experiment, a hypoxia cytotoxicity ratio of 24 and an efficient reductive cleavage under chemical and enzymatic conditions.

Full text of DOI:10.1016/j.ejmech.2018.08.060

Accepted Manuscript
Structure-activity relationship study of hypoxia-activated prodrugs for proteoglycan-
targeted chemotherapy in chondrosarcoma
Donia Ghedira, Aurélien Voissière, Caroline Peyrode, Jamil Kraiem, Yvain Gerard,
Elise Maubert, Magali Vivier, Elisabeth Miot-Noirault, Jean-Michel Chezal, Farhat
Farhat, Valérie Weber
PII:
S0223-5234(18)30730-X
10.1016/j.ejmech.2018.08.060
EJMECH 10671
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 May 2018
Revised Date: 20 August 2018
Accepted Date: 22 August 2018
Please cite this article as: D. Ghedira, Auré. Voissière, C. Peyrode, J. Kraiem, Y. Gerard, E. Maubert,
M. Vivier, E. Miot-Noirault, J.-M. Chezal, F. Farhat, Valé. Weber, Structure-activity relationship study
of hypoxia-activated prodrugs for proteoglycan-targeted chemotherapy in chondrosarcoma, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.060.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Hypoxia-activated prodrug with a quaternary ammonium function as proteoglycan-targeting
ligand for a better management of chondrosarcoma: synthesis, Surface Plasmon Resonance
screening, in vitro hypoxic selectivity and reductive activation.

ACCEPTED MANUSCRIPT
Structure-activity relationship study of Hypoxia-Activated Prodrugs for
Proteoglycan-targeted chemotherapy in chondrosarcoma
a,b
a
a
b
a
Authors: Donia Ghedira , Aurélien Voissière , Caroline Peyrode , Jamil Kraiem , Yvain Gerard ,
a
a
a
a
b
Elise Maubert , Magali Vivier , Elisabeth Miot-Noirault , Jean-Michel Chezal , Farhat Farhat ,
a,
Valérie Weber *
Affiliations:
a
Université Clermont Auvergne, INSERM, U1240 Imagerie Moléculaire et Stratégies
Théranostiques, F-63000 Clermont-Ferrand, France
b
Laboratoire de Développement Chimique, Galénique et Pharmacologique des Médicaments,
Faculté de Pharmacie de Monastir, Université de Monastir, 5000 Monastir, Tunisia
*Corresponding author: Valérie Weber - Phone : 0033 4 73 15 08 21, Fax : 0033 4 73 15 08 01,
e-mail: valerie.weber@uca.fr
Keywords: Hypoxia-activated prodrug, Quaternary ammonium, Chondrosarcoma,
Proteoglycan,
1

ACCEPTED MANUSCRIPT
Abstract
Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic
extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-
resistant. Surgical resection with wide margins remains the mainstay of treatment. To address
the lack of therapy, our strategy aims to increase anticancer drugs targeting and delivery in the
tumour, by leveraging specific chondrosarcoma hallmarks: an extensive cartilaginous
extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia.
A dual targeted therapy for chondrosarcoma was investigated by conjugation of a hypoxia-
activated prodrug (HAP) to quaternary ammonium (QA) functions which exhibit a high affinity
for polyanionic sites of proteoglycans (PGs), the major components of the chondrosarcoma
extracellular matrix. Based on preclinical results, an imidazole prodrug, ICF05016, was
identified and provided the basis for a lead optimization study. A series of 27 QA-
phosphoramide mustard conjugates, differing by the type of QA function and the length of the
alkyl linker, was yielded by a common multi-step sequence involving phosphorylation of a key
2-nitroimidazole alcohol. Then, a screening was realized by surface plasmon resonance
technology to assess biomolecular interactions between QA derivatives and aggrecan, the
most abundant PG in chondrosarcoma. Results revealed that affinity depends more on the type
of QA function, than on the linker length. Moreover, the presence of a benzyle group enhanced
affinity to aggrecan. Twelve compounds were shortlisted and evaluated for antiproliferative
activity (i.e., growth inhibiting concentration 50), under normoxic and hypoxic conditions using
the human extraskeletal myeloid chondrosarcoma cell line (HEMC-SS). For all prodrugs, hypoxic
selectivity was maintained and even increased, compared with the lead. From this study,
compound 31f emerged as the most effective PG-targeted HAPs with a dissociation constant of
2.10 µM in the SPR experiment, a hypoxia cytotoxicity ratio of 24 and an efficient reductive
cleavage under chemical and enzymatic conditions.
2

ACCEPTED MANUSCRIPT
1. Introduction
Chondrosarcomas represent a heterogeneous group of rare and slowly-growing types of
bone sarcomas sharing common features such as their mesenchymal origin and the presence of
a cartilaginous extracellular matrix. Among all primary malignant bone sarcomas,
1
chondrosarcoma is the second most common after osteosarcoma, but the first in adults. To
2
,3
date, the therapeutic tools available for the management of the disease are very few. Surgical
resection with wide margins in surrounding non-tumour tissue is still considered the mainstay
of treatement and the only recommended therapy for non-metastatic chondrosarcoma, all
grades and subtypes included. Well-conducted surgery has been reported to correlate with
better control of the disease, i.e. lower frequency of local recurrence, and improved overall
survival. Nevertheless, wide margin excision is not always possible (in some anatomic locations)
2
,4–6
or could entail a significant morbidity for the patient.
Also, the current clinical challenge is
to prevent recurrences and to offer new treatment options for patients with inoperable
primary, recurrent disease, and/or metastases. As regards to radiotherapy and conventional
7
–9
chemotherapy, chondrosarcoma is known as a resistant lesion due to tumour’s specific
hallmarks. Like hyaline cartilage, tumour tissue is marked by a dense and extensive extracellular
matrix with poor blood and lymph vascularity, on which a low percentage of dividing cells is
embedded. The extracellular matrix creates a physical semi-permeable barrier, which prevents
cytotoxic agents reaching their target, i.e tumour chondrocytes, while reduced blood circulation
1
0
creates severe chronic hypoxia. The latter is also well known to be associated with radio- and
1
1–13
chemo-resistance in cancer.
Given the lack of significant improvement in terms of patient survival over the past
three decades, new therapeutic targets, such as isocitrate dehydrogenase, m-TOR and
Hedgehog pathway inhibitors have been investigated and are currently used in rational drug
1
4
design for the development of new targeted therapies . The challenges of translating these
1
5,16
drugs into clinical use unfortunately failed and no substance has been approved so far
.
In clinic, commonly used chemotherapies currently target either the highly proliferative
nature of cancer cells, or specific receptors that are upregulated in cancer but often result in
side effects leading to potential toxicity, and/or causing treatment failure in resistant
3

ACCEPTED MANUSCRIPT
populations. A new promising option for improving cancer therapies appeared, based on
1
7,18
targeting the tumour microenvironnement
. For chondrosarcoma, two physical and chemical
hallmarks of the tumour could also be considered as therapeutic opportunities rather than
barriers, namely hypoxia and the hyaline extracellular matrix, more precisely, its high
proteoglycan (PG) content.
For more than five decades, hypoxia is well recognized as a key feature in cancer
1
1
progression . Hypoxia leads to low oxygen concentration areas and then to the emergence of
tumour cell subpopulations with highly aggressive phenotypes, including increased drug-
resistance and amplified anti-apoptotic and metastatic potential. The differences between cells
in well-oxygenated normal tissue and hypoxic neoplastic cells were put to use in the
development of bioreductive prodrugs activated preferentially in hypoxic regions to specifically
1
9–23
release active cytotoxic species.
This approach emerged more than three decades ago, with
tirapazamine being the first hypoxia-activated prodrug (HAP) to undergo clinical trial. However,
while multiple different bioreductive prodrugs demonstrating antitumour cytotoxicity against
hypoxic cells, like AQ4N, apaziquone, SN30000, evofosfamide (TH-302), PR-104, etc. have been
described in literature, none are so far available in the clinical setting. HAPs have in common
chemical moities used as a “trigger” or “oxygen concentration sensor” ensuring activation of
the prodrug through a bioreduction process via one or two electrons cellular oxidoreductases.
Five different chemical groups, i.e. nitroaromatics, aromatic N-oxides, aliphatic N-oxides,
1
9–21
quinones and transition metals, are reported in literature as triggers
. After the reduction
step, the link between the trigger and the anti-cancer entity is cleaved and converts the non-
toxic prodrug into a toxic molecule achieving hypoxia-selective killing associated with a
bystander effect. By means of HAP, the therapeutic window could be extended and higher
doses could be administered, providing therefore greater therapeutic potential.
In chondrosarcoma, HAP activity could be hampered by inefficient physical distribution
to tumour chondrocytes due to the cartilaginous matrix barrier. Using the physical and chemical
properties of the extensive cartilaginous extracellular matrix of chondrosarcoma, namely the
high negative fixed charge density created by the presence of PGs, may be an attractive way of
adding to these HAP therapies targeted-drug delivery. In effect, PGs are glycoproteins highly
4

ACCEPTED MANUSCRIPT
2
4
expressed in the chondrosarcoma extracellular matrix , with numerous sulphate and
carboxylate groups on their glycosaminoglycan moieties. This extracellular PG-targeting
approach using a quaternary ammonium (QA) function was previously validated in
2
5,26
chondrosarcoma preclinical models, with radiotracers for scintigraphic imaging
or DNA-
More recently, this dual targeting therapy for
chondrosarcoma was investigated and led to the identification of the first PG-targeting HAP, 3-
{[bis(2-chloroethyl)amino][(1-methyl-2-nitro-1H-imidazol-5-yl)methoxy]phosphoryl}amino)-
2
7,28
alkylating agents for therapy.
(
2
9
N,N,N-trimethyl-propane-1-aminium iodide, named ICF05016 (Table 1). The latter is a
nitroheteroaryl-based compound designed as follows: a phosphoramidic mustard
functionalized with a QA function, and a 2-nitroimidazole group tethered to the phosphorous
atom to trigger fragmentation and then release the bis-alkylating mustard anion by
bioreduction in hypoxic tissues. Aggrecan binding of ICF05016 was confirmed from the
dissociation constant determined by surface plasmon resonance (SPR), as well as hypoxic-
cytotoxicity ratio (HCR) on the HEMC-SS cell line. In a HEMC-SS tumour-bearing mice model,
treatment with ICF05016 provides a significant inhibition of tumour growth with no associated
haematological side effects, a decrease in the mitotic index and increased DNA damage
predominantly found in pimonidazole-positive hypoxic areas.
Based on these results, we intend to begin a lead optimization study of ICF05016.
Herein, we report the synthesis and characterization of a series of QA-targeted phosphoramide
mustard conjugates 28-32 differing by the nature of the QA function and the length of the alkyl
linker binding the QA to the mustard (Table 1). Then, we screened by SPR their direct binding
ability to aggrecan. Short-listed compounds were evaluated in terms of HCR in the HEMC-SS cell
line to provide more knowledge on the structure-activity relationships existing between the
nature of the linker and the in vitro cytotoxicity of these QA-targeted HAPs. Finally, stability in
both phosphate buffer and plasma, activation and phosphoramide mustard release under
reductive chemical conditions, and nitroreductase-based activation were demonstrated for the
most potent derivative.
5

ACCEPTED MANUSCRIPT
2
. Results and discussion
.1. Chemistry
2
Target compounds 28-32 were obtained by quaternisation of the tertiary amine function
of compounds 23-27 via a two-step reaction sequence: phosphorylation of the key
hydroxymethyl 2-nitroimidazole intermediate 6 by phosphoramidic dichloride 8, followed by
the introduction of the QA alkyl chain by substitution of the remaining chloride by a series of
diamines 18-22 (Scheme 1). The latter were prepared according to a Gabriel synthesis protocol
starting from the appropriate alkyl halide. Briefly, N-bromoalkylphthalimides 9-12 with alkyl
chain ranging from two to five carbons were produced in moderate to excellent yields (43-98%)
by alkylation of commercial potassium phthalimide with the corresponding dibromoalcane
3
0
following a slightly modified procedure of the method described by Hu et al. . Nucleophilic
substitution of derivatives 9-12 in acetonitrile with dimethyl-, diethyl-, diisopropyl- or
3
1,32
benzylmethylamines afforded intermediates 13-17
. Hydrazinolysis of the latters under
reflux afforded directly N-benzyl-N-alkyldiamines 21a-d and 3-(piperidin-1-yl)propanamine 22
in good to excellent yields (76 to 98%), whereas short-path distillation was required for N,N-
dialkyldiamines 18-20 leading to moderate yields (22 to 61%). To note, elimination of the
Gabriel phthalhydrazide by-product was insured by a slightly modified work up protocol
3
1
compared to the litterature , namely a twice acidic precipitation at low temperature. Following
this general three-steps procedure, the twelve diamines 18a-b, 19a-b, 20a-c, 21a-d and 22 were
obtained from potassium phthalimide in overall yields ranging from 15 to 77 %.
The (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (6) is the key intermediate for
synthesis of our QA-targeted HAPs. The 1-methyl-2-nitroimidazole group was chosen as
common trigger for the designed molecules. Among bioreductive nitroaryl-triggers used in
HAPs, like the 4-nitrobenzyl group, nitro-imidazole-, nitrofuran- and nitrothiophene-based
groups, the 1-methyl-2-nitroimidazole group has been used the most widely, generally showing
the best tendency to undergo bioreduction and, consecutively selective cleavage at low oxygen
concentrations. Hence, a 1-methyl-2-nitroimidazole group was embedded in the well-
investigated evofosfamide, a HAP of bromo-isophosphoramide mustard reaching phase III
6

ACCEPTED MANUSCRIPT
3
3
34,35
clinical trials against soft tissue sarcoma and pancreatic cancer (MAESTRO study)
.
Evofosfamide appeared for the first time in a series of nitroaromatics including nitrothiophenes
3
6
and nitrofuranes, developed by Duan et al. in 2008 and emerged as the most potent HAP of
the series.
The synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (6) was built on the work
3
7
38
published by Cavalleri et al. and patented by Matteucci et al. , starting from N-methylglycine
3
9,40
(1) and improved later by O’Connor et al.
. Synthesis of 1-methyl-2-nitroimidazole
derivatives appears as a challenging and fastidious task, as recently reviewed. O’Connor et al.
4
0
published in 2016 a careful and detailed description of the experimental protocol with
emphasis on critical steps, precautions to be taken and the optimal reaction times for each
step. More precisely, it turned out that the synthesis of the intermediate amino ester 4
represents the most critical step, associated with the lowest yields (ranging from 10-25%
according to the Matteuci et al. patent to 50 % in the O’Connor protocol).
According to the procedure outlined in Scheme 1, N-methylglycine was first converted
to the corresponding ethyl ester hydrochloride salt, which in turn was N-formylated by ethyl
formate in the presence of potassium carbonate. Then, a one-pot three-steps procedure was
applied to yield the 2-aminoimidazole 4: (i) C-formylation of the amino ester 3 at the α-carbon
by treatment with sodium hydride and ethylformate, (ii) removal of the unwanted N-formyl
group under acidic conditions at reflux, and (iii) subsequent Marckwald cyclisation with
3
9
cyanamide under reflux at pH 3 as suggested by O’Connor et al. to minimise ester hydrolysis.
This sequence finally provided the desired 2-aminoimidazole 4 in good yields (53-61 %
3
9,40
compared to the previous 50 % yield in the O’Connor protocol
). Then, diazotization of the
amino group in the presence of excess sodium nitrite afforded nitroester 5 in a 75% yield.
Finally, reduction of the ester function with LiBH formed in situ via the metathesis reaction
4
between NaBH and LiBr, allowed to obtain compound 6 in 68% yield. LiBH was reported as a
4
4
selective reducing agent for esters, with reducing power greatly enhanced by utilizing ether-
4
1
4
type solvents containing methanol . In our procedure, a mixture of LiBH -THF-MeOH was used
with a strict control of the reaction medium temperature (below 10°C) in order to avoid the
reduction of the nitro function.
7

ACCEPTED MANUSCRIPT
Tertiary amine derivatives 23-27 were synthesized via a one-pot reaction according to a
4
2
modified procedure of Hernick et al. Synthesis began with the phosphorylation of the 2-
nitroimidazole key intermediate 6 at -78 °C by sequential treatment with lithium
bis(trimethylsilyl)amide and the electrophile phosphorylating agent 8 (prepared according to a
4
3
literature procedure , on a 10 g scale from commercially available N,N-bis(2-chloroethyl)amine
hydrochloride salt (7)). Then, addition of the appropriate diamine 18-22 ensured the formation
of the corresponding phosphoramide 23-27. For accurate management of this one-pot
3
1
procedure, the progress of the reaction was monitored using P NMR, initiated at addition of
the N,N-bis(2-chloroethyl)phosphoramidic dichloride (8). When nearly complete disappearance
of dichloride 8 (used in slight excess) concurrent with the formation of the phosphoramidic
monochloride intermediate was observed, the appropriate amine was added to obtain the
3
1
desired compounds 23-27. For example, P NMR monitoring for compound 24d is depicted in
Figure 1: (1) at T , two signals at 15.59 and 15.17 ppm appeared, relative to dichloride 8 and
0
monochloride intermediate formed respectively; (2) after 15 min, the signal at 15.59 ppm had
almost disappeared and 2-(N,N-diethylamino)ethylamine was added; (3) only 10 min later, the
intermediate was consumed giving rise to compound 24d with a signal at 17.24 ppm and an
impurity at 18.97 ppm. It should be noted that this phosphorylation step was unpredictable and
3
1
not always reliable, requiring a P NMR monitoring to control the time of consumption of
dichloride 8, ranging from 5 to 80 min in different experiments. We also observed that the best
results, in terms of kinetic and conversion rate, were obtained when the solution of
dichlorophosphoramidate 8 in THF was pre-cooled at -78 °C and added at once in order to avoid
the undesired bis-addition of alcohol 6. We finally noted that the solubility of 2-nitroimidazole 6
in THF at -78 °C was a critical factor, partial solubility correlating with longer times and lower-
yielding phosphorylation.
Quaternization of the series of tertiary amines 23-25, 27 was achieved using alkyl iodide
(methyl or ethyl iodide), leading to the corresponding ammonium derivatives 28-30, 32. For
compounds 31a-h, two synthesis pathways have been considered, i.e. direct methylation or
ethylation of benzyl compounds 26 or benzylation of dialkyl derivatives 23 and 24. In order to
establish the best route, QA derivative 31c was synthesized from 26c by treatment with methyl
8

ACCEPTED MANUSCRIPT
4
4
iodide or from 23c by alkylation with freshly prepared benzyl iodide . In the latter case, a
supplementary work-up of the crude product was necessary in order to remove excess benzyle
iodide, affording the desired product in a lower yield compared with the methyl iodide route
(30% from 23c versus 93% from 26c). Thus, alkyl iodides (methyl or ethyl) were used for the
preparation of all ammonium derivatives bearing a benzyl group within the series. It should be
emphasized that the reaction took place in different ways within the series, in terms of reaction
time, number of alkyl iodide equivalents required and purity status of the obtained crude
product. Most QA compounds were obtained in excellent yields (mostly > 80 %), with a
satisfactory purity, only after evaporation of the excess methyl or ethyl iodide. Analytical purity
1
13
was evaluated by the absence of impurities revealed in the H and C spectra combined with
3
1
P impurities estimated at less than 5 % in global integrals. However, for five compounds (i.e.
3
1
2
8g, 29g, 29h, 31g and 31h) belonging to the n = 2 or 3 series, P spectra analyses revealed the
presence of an impurity after ethylation emphasized by a signal at 24-25 ppm (in CD OD), and
3
ranging from 6 to 15% for 28g, 29g, 29h, 31g, and 87% for 31h. Purification of these QA
compounds by preparative high performance liquid chromatography (C18 column, gradient
acetonitrile in water) with subsequent lyophilisation was thus required to afford analyticaly
pure compounds 28g, 29g, 29h and 31g. To better understand this side reaction, the impurity
3
1
from the synthesis of compound 29h (signal at 24.35 ppm in P spectrum) was isolated for
structure elucidation. HRMS data showed molecular ion at m/z 451.1984, which could
correspond to the loss of a neutral HCl (36 amu). A comprehensive spectral analysis based on
1
13
31
HRMS and RMN ( H, C, COSY, HSQC, P) was in favour of the formation of the corresponding
2-oxide-diazaphospholidine obtained by intramolecular 1,5-N-alkylation of compound 29h. For
synthesis of compound 31h, total conversion of 21d required 5 days’ reaction in the presence
of a considerable excess of ethyl iodide (100 eq.) and led to the 2-oxide-diazaphospholidine
3
1
impurity as the major product (> 80 % highlighted by P spectrum) preventing obtention of 31h
in sufficient yield to allow biological assessment. This unwanted intramolecular 1,5-N-alkylation
seems to be correlated with the length of the alkyl chain and/or steric hindrance of the tertiary
amine substituents which hampered quaternization with ethyl iodide, requiring longer reaction
time and greater excess of ethyl iodide.
9

ACCEPTED MANUSCRIPT
2.2. Biology
The interaction between synthesized QA-targeted prodrugs 28-32 and aggrecan was evaluated
by Surface plasmon resonance (SPR) analyses. SPR technology has emerged as a powerful
technique to investigate the interaction between low-molecular-weight molecules and target
4
5–47
proteins and also as an effective tool for drug screening.
Aggrecan (≈ 250 kD), with
approximately 150 glycosaminoglycan side chains of chondroitin sulphate and keratan sulphate,
2
4,48
is the most abundant PG in chondrosarcoma.
A preliminary screening of the different
prodrugs was performed by binding level analysis (Figure 2A). Compound ICF05016 was used as
positive control and its non-QA equivalent (O-[(1-methyl-2-nitro-1H-imidazol-5-yl)methyl]-N,N-
2
9
bis(2-chloroethyl)-N’-propylphosphorodiamidate named ICF05017 ) as negative control for
aggrecan binding measurements. The hit selection by initial screening of all synthesized QA-HAP
was based on at least equivalent relative binding to aggrecan (Response Units) compared with
ICF05016.
Some structure-activity relationships appeared in terms of interaction with aggrecan:
affinity seemed to be related more to the type of the QA function, rather than the length of the
alkyl chain between the QA function and the nitrogen mustard, since the twelve short-listed
compounds were of various chain lengths (n = 2, 3, 4, 5). Concerning QA substituents, the first
emerging conclusion was that the presence of a benzyl group enhanced the affinity to
aggrecan. In fact, all synthesized compounds bearing a benzyl moiety showed a higher binding
affinity than the lead compound (almost two-fold difference in relative binding, except for
compounds 31f and 31c) with the best affinity obtained for analogue 31a, characterized by a
pentyl chain and a benzyl-dimethyl QA function. Improved binding affinity to aggrecan was also
observed with derivatives with ethyl substituents on the QA function. None isopropyl
derivatives were short-listed, suggesting that steric hindrance perhaps hampers the inter-
molecular recognition process between the isopropyl QA function and aggrecan.
10

ACCEPTED MANUSCRIPT
Based on this selection criterion, twelve hits with Response Units over 24 were further
evaluated to determine their dissociation constants (K
secondary experiment (Figure 2B). This analysis yielded K values ranging from 2.10 ± 0.13 to
.03 ± 0.21 mM (Table 1) for all selected prodrugs, which demonstrates fairly similar affinity for
aggrecan as ICF05016.
D
) in a concentration-dependent
D
5
The selected prodrugs were evaluated for antiproliferative activity (i.e., Growth
Inhibiting Concentration 50: IC ) respectively to ICF05016 and evofosfamide, under normoxic
5
0
and hypoxic conditions using the human extraskeletal myeloid chondrosarcoma cell line
HEMC-SS). The IC values for all tested compounds are given in Table 2 and confirmed their
(
5
0
relevance as hypoxia-activated prodrugs. Based on the inhibition of proliferation, almost all
compounds were more potent than ICF05016, with a higher activity observed in hypoxia
compared to normoxia. Hypoxic-cytotoxic ratio (HCR, IC50 under normoxia vs. IC50 under
hypoxia) was maintained and even increased, compared with the HCR of 7 obtained for
ICF05016. Substitution of methyl groups by ethyl on the QA function or modification of the alkyl
chain length did not lead to significant change in potency or in hypoxic selectivity of the
prodrugs, as compared with ICF05016. However, substitution of one methyl by a benzyl group
appears more effective in terms of hypoxic selectivity, as for all benzylated prodrugs (31a-g)
selectivity was maintained and even increased of 2-fold to 3-fold for three compounds, mainly
for compound 31f, emerging as the most effective (HCR = 24). Compound 31f exhibited
selectivity for hypoxia similar to evofosfamide with a HCR of 23 in HEMC-SS cells. However, the
potency of evofosfamide on HEMC-SS cells in normoxia was higher (IC50 = 2.9 ± 1.5 µM)
compared to compound 31f (IC50 = 40.7 ± 8.8 µM). This cytotoxicity could be attributed to the
superoxide species, generated under normoxia and is time-dependent with longer drug
4
9
exposures as previously reported . This may lead to toxicity in healthy cells, which could be
expected to occur to a lesser extent with compound 31f.
2.3. Chemical Stability in PBS and plasma
11

ACCEPTED MANUSCRIPT
A stability evaluation, carried out in phosphate buffer solution (10 mM, pH 7.4, 0.13% DMSO)
on compound 31f at 37 °C and monitored by reversed-phase HPLC, demonstrated promising
results. Compound 31f remains structurally intact under these conditions with no significant
changes over a 24 h period (< 5%). Stability in rabbit plasma at 37 °C was also demonstrated by
3
1
P NMR experiments, as no cleavage or degradation over a 24 h period was observed (Figure
B).
3
2.3. Reductive Chemical Activation
3
1
A P NMR experiment was carried out on compound 31f under model physiological conditions
(pH 7.4, 37 °C) to ascertain that initial reductive activation of the phosphoramidate prodrug 31f
leds to the rapid formation of the corresponding phosphoramidate anion, hypothesized as
being the active cytotoxic species. Prodrug 31f was proved to be stable in these conditions over
a period of 24 h. The phosphoramidate prodrug 31f was chemically reduced with 3 equivalents
of sodium dithionite (0.1 M cacodylate buffer, pH 7.4), a reductant system used to mimick
3
1
bioreduction in hypoxic tissue. Figure 3 is an example of a P NMR stack plot showing the
results from the dithionite reduction of 31f. As expected, the resonance corresponding to
prodrug 31f at 17.99 ppm rapidly disappears (half-life < 2 min) with the concurrent appearance
3
1
of the resonance at 12.39 ppm, which corresponds to the P signal of the substituted bis(2-
chloroethyl)phosphoramidate anion. The phosphoramidate anion is a highly reactive specie and
give raised to the formation of a variety of by-products arising both from subsequent solvolysis
5
0,51
reactions (bisulfite and water substitution) and cleavages of P-N bonds
. At least 90%
conversion of prodrug 31f occured in less than 8 min at 37 °C under chemical reductive
conditions, efficiently leading to the release of the bis-alkylating mustard anion under hypoxia
mimic conditions (Figure 3B)
.3. Bioreductive activation via nitroreductase
Prodrug 31f was evaluated as substrate of an oxygen-insensitive nitroreductase from E.
.
2
coli, to verify activation following an enzymatic reduction. This enzyme, which involves a two
electrons reduction process, provides an experimental setting not affected by the
environmental oxygen level. This offers the great advantage to bypass the oxygen-sensitive
12

ACCEPTED MANUSCRIPT
prodrug radical, which is reoxidised to the original prodrug in the presence of oxygen and thus
hampered the phosphoramidate anion release, as observed for nitroaromatic HAPs. The
activation was evaluated by incubating compound 31f in phosphate buffer solution (10 mM, pH
7.4) at 37 °C with nitroreductase in the presence of NADPH as the cofactor. This latter doesn’t
induce enzyme-free reaction, as demonstrated by HPLC analysis. Aliquots were withdrawn at
various time points and analyzed by reversed-phase HPLC. The half-lives were estimated on the
basis of the disappearance of the prodrug at two nitroreductase concentrations (15 and 25
µg/mL) (Figure 4). For compound 31f, the both estimated half-lives were below 1 min vs 5 and
15 min respectively for the lead ICF05016, used as a control in our experiment. Prodrugs 31f
was substrate for nitroreductase and was readily reduced with at least 90 % conversion after 3-
5
min, showing proof of principle for this HAP strategy.
3. Conclusions
The aim of this work was to synthesize a series of QA-HAP conjugates differing by the nature of
the PG-targeted QA function for effective chemotherapy of chondrosarcoma, a hypoxic and PG-
rich tumour. The present study revealed that all prodrugs led to overall improvement in hypoxic
selectivity compared with ICF05016 and highlights the positive impact of a benzyl QA function
on affinity to aggrecan, the most abundant PG in chondrosarcoma. Because of its attractive
hypoxic selectivity (HCR = 24) and cytotoxicity in hypoxia compared to that of lead ICF05016,
coupled with rapid in vitro reductive activation, compound 31f emerged as the most effective
HAP and will undergo further preclinical studies, including in vivo efficacy on the HEMC-SS
xenograft model on mice.
13

ACCEPTED MANUSCRIPT
Scheme 1 : Reagents and conditions : (a) EtOH, SOCl , -10 °C ,then 55 °C, overnight, 97%; (b) ethyl formate, K CO , EtOH, rt, overnight, 85%; (c) (1)
2
2
3
2 2 2
ethylformate, NaH, THF anh., 0 °C, rt, overnight, (2) HCl 37%, EtOH, 110 °C, 2 h, (3) NH CN, EtOH/H O, pH 3, reflux, 1.5 h, 61%; (d) NaNO , AcOH, -5 °C, then rt,
overnight, 75%; (e) NaBH , LiBr, THF/MeOH/H O, <10 °C, rt, 19 h, 68%; (f) POCl , 130-140 °C, 80 h, 57%; (g) HNR R , K CO , ACN or THF, reflux, 24 h, 35-93%;
2
4
2
3
1
2
3
3
1
(h) hydrazine hydrate (60% aqueous solution), EtOH, reflux, 12 h, 22-99%; (i) (1) LiN(TMS)
2
, THF anh., 8, -78 °C, 15-80 min (duration determined by P NMR
3
monitoring); (2) amines 18-22 or commercial amines, THF anh., -78 °C, 5-75 min (duration determined by P NMR monitoring), 22-78%; (j) alkyl iodide, K CO ,
1
2
3
ACN anh., rt, 4-96 h, 60-100%.
14

ACCEPTED MANUSCRIPT
A : T0
B : T 15 min
C : T 20 min
31
Figure 1. Example of NMR P monitoring obtained for the synthesis of compound 24d.
A. Promply after addition of dichlorophosphoramidate 8, two signals at 15.59 and 15.17 ppm
appeared, relative respectively to dichloride 8 and monochloride intermediate formed;
B. After 15 min, phosphorylation reached an optimal ratio ~0.1/1 between the remaining
dichlorophosphoramidate 8 and the formed intermediate.
C. After the addition of 2-(N,N-diethylamino)ethylamine, the two signals at 15.59 and 15.17
disappeared immediately, generating the corresponding tertiary amine prodrug 24d with a
signal at 17.24 ppm and an impurity at 18.97 ppm.
15

ACCEPTED MANUSCRIPT
A.
B.
Figure 2. SPR response of compounds 28-32.
A. The SPR responses of the all QA-compounds from the first round of screening. ICF05016 was
used as positive control and its non PG-targeted equivalent ICF05017 as negative control.
Twelve compounds were selected, based on a Response Unit greater than or equal to the
ICF05016 value (dotted line).
B. Representative sensorgram showing binding of compound 31a to immobilized aggrecan at
height concentrations (0.2, 0.4, 0.5, 0.6, 0.8, 1, 1.5 and 2 mM from bottom to top) is presented
2
(K
D
= 2.3 mM; Chi = 0.76).
16

ACCEPTED MANUSCRIPT
2
Compd
n
R1
R2
R3
Kd (mM)
Chi
ICF05016
3
5
4
2
5
4
3
2
5
4
3
2
5
4
3
2
5
4
3
2
5
4
3
2
5
4
3
2
3
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Me
Me
Me
Me
Et
Et
Et
Et
Me
Me
Me
Me
Et
Et
Et
Et
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Me
1.29 ± 0.92
4.22 ± 1.25
0.56 ± 0.41
0.39 ± 0.02
2
2
8a
8b
-
-
2
8c
-
3.56 ± 0.78
2.02 ± 0.49
-
-
0.36 ± 0.42
0.24 ± 0.18
-
2
2
8d
8e
28f
2
2
8g
9a
-
-
-
4.97 ± 1.41
-
0.40 ± 0.08
2
2
9b
9c
-
-
-
-
-
-
2
2
9d
9e
29f
Et
Et
Et
Et
Et
Et
-
3.49 ± 0.45
-
0.35 ± 0.32
2
9g
29h
30a
30b
-
-
-
-
-
-
-
-
i-Pr
i-Pr
i-Pr
i-Pr
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
i-Pr
i-Pr
i-Pr
i-Pr
Me
Me
Me
Me
Me
Me
Me
Me
3
0c
30d
31a
31b
-
-
2.55 ± 0.35
5.27 ± 0.21
2.99 ± 0.57
2.90 ± 0.28
3.47 ± 1.14
2.10 ± 0.13
ND
0.7 ± 0.09
1.50 ± 1.33
0.18 ± 0.06
1.30 ± 0.51
4.85 ± 2.92
0.62 ± 0.52
3
1c
3
3
1d
1e
3
1f
1g
1h
3
3
ND*
-
3
2
piperidinyl
-
Evofosfamide
TH-302)
2
.9 ± 1.5
(
2
Table 1. Binding to immobilized aggrecan by SPR. Dissociation constants (K ) and Chi were determined for each
D
derivative by “steady-state affinity analysis”. All experiments were carried out in duplicate. ND: Not Determined.
*
no evaluation due to low degree of purity
17

ACCEPTED MANUSCRIPT
IC (µM)
Hypoxia
50
Compd
n
R1
R2
R3
Normoxia
29.3 ± 8.0
25.8 ± 6.3
15.8 ± 7.7
10.2 ± 0.3
13.4 ± 4.3
9.7 ± 3.5
28.9 ± 2.6
18.0 ± 2.6
5.4 ± 0.9
HCR
7
ICF05016
3
5
5
4
4
3
5
4
3
2
5
4
3
Me
Me
Me
Me
Et
Me
Me
Me
Me
Et
Me
Me
Et
Et
Me
Et
Me
Me
Me
Me
Et
4.3 ± 1.2**
2.4 ± 0.4**
1.5 ± 0.8**
1.5 ± 0.1**
2.4 ± 0.7**
1.4 ± 1.0**
2.8 ± 1.2**
1.8 ± 0.9**
0.4 ± 0.1**
1.1 ± 0.4**
0.8 ± 0.3**
1.7 ± 0.9**
2.1 ± 1.6**
28a
11
11
7
6
7
10
10
14
6
16
24
10
28d
28e
29b
29g
31a
31b
Et
Et
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Me
Me
Me
Me
Me
Me
Me
3
1c
3
3
1d
1e
6.4 ± 2.1
12.9 ± 3.5
40.7 ± 8.8
20.8 ± 8.7
3
1f
1g
Evofosfamide
TH-302)
Et
Et
3
2.9 ± 1.5
0.13 ± 0.02**
23
(
Table 2. Cytotoxicity of HAP derivatives on HEMC-SS cells in normoxic (21% O
2 2 2
) and hypoxic (N , O <
0.3%) conditions. Cultures were exposed to drugs for 24 h followed by incubation for 48 h in normoxia. Notes:
** significant difference vs. normoxic condition, p<0.01. Abbreviations: IC : Growth Inhibiting Concentration 50;
HCR: Hypoxic-Cytotoxic Ratio = IC in normoxia/IC in hypoxia.
50
50
50
18

ACCEPTED MANUSCRIPT
A.
2
4 h
c
2
5’39
1
4’49
4
’49
1
’39
b
T0
a
B.
Figure 3. Drug release in reductive conditions (3 equiv. sodium dithionite, CH CN/0.1 M cacodylate
3
3
1
31
buffer, pH 7.4, 37 °C) and stability in plasma assessed by NMR P: A. Stacked NMR P spectra; a.
prodrug 31f, 17.99 ppm; b. phosphoramidate anion, 12.39 ppm (proven by MS-HPLC, data not shown);
c. inorganic phosphate, 0 ppm. Other signals are relatives to solvolysis, buffer and reductive adducts or
transient unknown by-products. Chemical shifts are reported relative to the Ph PO reference. B.
3
Representative time course of phosphoramide 31f cleavage (p), substituted bis(2-
chloroethyl)phosphoramidate anion formation (Ø) and inorganic phosphate formation (×) upon
incubation of the prodrug with sodium dithionite in cacodylate buffer; Stability of phosphoramide 31f in
31
cacodylate buffer (æ) and in rabbit plasma (□). Data points were measured from NMR P areas
(average of two runs).
1
9

ACCEPTED MANUSCRIPT
100
Prodrug 31f; 15 µg/mL of nitroreductase
ICF05016; 15 µg/mL of nitroreductase
Prodrug 31f; 25 µg/mL of nitroreductase
ICF05016; 25 µg/mL of nitroreductase
80
60
40
20
0
0
1000
2000
3000
4000
5000
Time (s)
Figure 4. Percent of prodrugs 31f and ICF05016 remaining after incubation in phosphate buffer (pH 7.4,
7 °C) with nitroreductase in the presence of NADPH. Reactions were followed by RP-HPLC at 322 nm.
The lines represent the best-fit values calculated from GraphPadPrism.
3
2
0

ACCEPTED MANUSCRIPT
4
. Experimental protocols
.1. Synthesis
4
All commercially available reagents and solvents were purchased at the following commercial
suppliers: Sigma Aldrich (Saint-Quentin Fallavier, France), Acros Organics (Geel, Belgium), Fisher
Scientific (Illkirch, France), Carlo Erba Reagents (Val de Reuil, France), VWR (Fontenay-sous-
Bois, France) and Alfa Aesar (Karlsruhe, Germany) and were used without further purification.
All solvents were dried using common techniques. Air and moisture sensitive reactions were
carried out under anhydrous argon atmosphere. Analytical thin-layer chromatography (TLC)
was performed on precoated silica gel aluminium plates (60 F , 0.2 mm thick, Macherey or
2
54
SDS) using the indicated solvent mixture expressed as volume/volume ratios. The plates were
visualized with ultraviolet light (254 nm) and (or) by development with ninhydrine ethanolic
solution (0.2%) or a 4-(4'-nitrobenzyl)pyridine (NBP)/potassium hydroxide dyeing reagent used
for the determination of alkylating agents (the chromatography plate was immersed in the NBP
solution (2.5% in acetone), heated for few min and then immersed in potassium hydroxide
solution (10% in methanol)). Column chromatography was performed on silica gel 60A normal
phase, 35-70 μm (Merck or SDS or Carlo Erba). Uncorrected melting points (mp) were measured
on an electrothermal capillary Digital Melting Point Apparatus (IA9100, Bibby Scientific, Roissy,
−1
France). Infrared spectra (IR) were recorded in the range 4000-600 cm on a IS10 with
attenuated total reflectance (ATR) accessory Nicolet (Fisher Scientific). Nuclear magnetic
resonance spectra (1H NMR and 13C NMR) were performed on a Bruker AM 200 spectrometer
(200 MHz for 1H, 50 MHz for 13C), a Broker Avance DPX300 spectrometer (300 MHz for 1H, 75
MHz for 13C) or a Bruker DRX 500 spectrometer (500 MHz for 1H, 125 MHz for 13C) (Bruker
Biospin SAS, Wissembourg, France). Chemical shift values (δ) are quoted in parts per million
1
13
31
(
ppm) and calibrated to the deuterated solvent reference peak for H and C spectra. P NMR
1
spectra (202 MHz) were recorded on a Bruker Avance 500 apparatus with broadband H
decoupling and chemical shifts were reported relative to a 1% phosphoric acid solution in
deuterium oxide as a coaxial reference (0 ppm). Coupling constants (J) are quoted in Hz. To
describe spin multiplicity, standard abbreviations such as s, d, dd, t, q, qt, hept, td, m, br.s
referring to singlet, doublet, doublet of doublet, triplet, quartet, quintet, heptuplet, doublet of
21

ACCEPTED MANUSCRIPT
triplet, multiplet, broad singlet respectively, are used. When necessary, chemical shifts
1
13
assignments in H and C spectra were supported by two dimensional NMR experiments (COSY
and HSQC). Compounds were analyzed by High-Resolution Mass Spectrometry in positive mode
(HRMS, Waters® Micromass® Q-Tof micro™ Mass Spectrometer, UCA-Partner, Clermont
Auvergne University, Clermont-Ferrand, France). Preparative high performance liquid
chromatography was performed on a CombiflashEZprep (Teledyne ISCO). The purification of
QA-derivatives was carried out on a C column using the following conditions: total experiment
1
8
time: 30 min, flow rate = 15 mL/min, eluent mixture: H O/MeCN (v/v), gradient: 95/5 for 2 min,
2
then 95/5 → 70/30 for 6 min, then 70/30 for 2 min, then 70/30 → 60/40 for 6 min, then 60/40
for 3 min, and 60/40 min → 10/90 for 11 min, λ = 254 and 323 nm.
Abbreviations: ACN, acetonitrile; DCM, dichloromethane; NBP, 4-(4'-nitrobenzyl)pyridine; TEA,
triethylamine; THF, tetrahydrofuran; rt, room temperature.
4
.1.1. Preparation of the common required 5-hydroxymethyl-1-N-methyl-2-nitro-1H-
imidazole (6)
.1.1.1. Ethyl N-methylglycinate hydrochloride salt (2)
4
Thionyle chloride (65 mL, 900 mmol) was added dropwise under stirring to a solution of
sarcosine (1) (20.0 g, 224 mmol) in EtOH (250 mL) cooled in an ice-water bath, while
maintaining temperature around -10 °C. Then the reaction mixture was gently heated at 55 °C
overnight until the mixture became clear. Solvent and traces of thionyl chloride were removed
by evaporation under reduced pressure and the solid residue was washed with Et O (3 × 50
2
mL). The remaining solid was well dried under vacuum to afford compound 2 (33.5 g, 218
mmol) as a white powder, which was used in the next step without further purification. Yield
5
2
-1
1
9
7%; mp 126 °C (Lit. mp 125-127 °C); IR (ATR)
ν
cm 2970-2440, 1742, 1229; H NMR (CDCl
3
,
+
2
3
J
3
J
400 MHz) δ 9.64 (br.s, 2H, NH
), 4.24 (q, 2H,
= 7.1 Hz, CH
2 3
CH ), 3.84 (t, 2H,
= 5.7 Hz,
+
3
+
2
3
J
13
NH
2
CH
2
), 2.80 (t, 3H,
J
= 5.2 Hz, NH
3
CH ), 1.26 (t, 3H,
2 3 3
= 7.1 Hz, CH CH ); C NMR (CDCl
,
+
+
1
01 MHz) δ 166.18 (CO ), 62.62 (CH CH ), 48.94 (NH CH ), 33.34 (NH CH ), 14.03 (CH CH ).
2
2
3
2
2
2
3
2
3
22

ACCEPTED MANUSCRIPT
4.1.1.2. Ethyl N-formyl-N-methylglycinate (3)
Ethyl N-methylglycinate hydrochloride salt (2) (30.0 g, 195 mmol) was suspended in a mixture
of EtOH (200 mL) and ethyl formate (126 mL). Potassium carbonate (40.6 g, 294 mmol) was
added under vigorous stirring and the suspension was stirred at rt overnight. The reaction
mixture was then filtered and the precipitate was washed with EtOH (150 mL). The filtrate was
concentrated and dissolved in a minimum amount of water (10 mL), followed by extraction
with EtOAc (4 × 200 mL). The combined organic layers were dried over MgSO , filtered and
4
concentrated under reduced pressure to afford compound 3 (24.3 g, 167 mmol) as a pale
1
yellow liquid. Yield 85%; H NMR (CDCl , 500 MHz) δ (two rotamers) 7.93 and 7.86 (s, 1H,
3
3
J
CHO), 4.04 and 4.02 (q, 2H,
2 3 2
= 7.2 Hz, CH CH ), 3.91 and 3.85 (s, 2H, NCH ), 2.87 and 2.74 (s,
3
13
3
1
3
H, NCH
68.76, 168.31 (CO
5.10, 30.76 (NCH ), 14.09 (OCH CH ).
3
), 1.12 and 1.10 (t, 3H,
J
= 7.1 Hz, CH
2
CH
3
); C NMR (CDCl
3
, 75 MHz) δ (two rotamers)
2
), 163.09, 162.85 (CHO), 61.62, 61.29 (OCH
2
CH ), 50.93, 45.72 (NCH ),
3
2
3
2
3
4.1.1.3. Ethyl 2-amino-1-N-methyl-1H-imidazole-5-carboxylate (4)
To a solution of N-formyl sarcosine ethyl ester (3) (29.6 g, 204 mmol) in an equal mixture of
ethyl formate and THF (190 mL) with cyclohexane (12 mL), was added slowly NaH (60% wt in
mineral oil, 12.5 g, 313 mmol) at room temperature. After the addition was completed and
hydrogen release stopped, the reaction mixture was allowed to stirred during 3.5 h. The
reaction mixture was concentrated under vacuum. The obtained solid was suspended in a
solution of EtOH (250 mL) containing concentrated aq. HCl 32 % (61 mL) and refluxed for 2 h.
The hot reaction mixture was filtered and the resulting colourless solid was washed with boiling
EtOH (2 × 150 mL). The filtrate was concentrated under vacuum and diluted with a mixture of
EtOH/water (500 mL, 70/30, v/v). The pH of the solution was adjusted to 3, using an aqueous
5
M solution of NaOH and cyanamide (17.5 g, 416 mmol) was added. The resulting mixture was
refluxed for 1.5 h, then cooled to rt and concentrated under reduced pressure to approximately
/8 of the initial volume. The pH of the remaining solution was adjusted to 9-10 with a
1
saturated aqueous solution of potassium carbonate, after cooling in an ice-water bath. The
precipitate formed was removed by filtration, washed with water (2 × 10 mL) and dried under
vacuum at 40 °C overnight to afford compound 4 (16.9 g, 99.9 mmol) as a pale yellow to orange
23

ACCEPTED MANUSCRIPT
solid. A supplementary fraction can be yielded after extraction of the remaining filtrate with
ethyl acetate (3 × 100 mL). The combined organic layers were dried over MgSO
evaporated under reduced pressure. The residue was purified by column chromatography on
silica gel using ethyl acetate/ethanol (95/5, v/v with 1% NH OH) as eluent to yield compound 4
4
, filtered and
4
4
0
(
3.78 g, 22.3 mmol). Yield 61%; Rf 0.33 (SiO , EtOAc/EtOH, 9/1, v/v); mp 131 °C (Lit. mp 130-
2
-
1
1
1
33 °C); IR (ATR) ν cm 3390, 3119, 1647, 1542, 1168, 750, 737; H NMR (CDCl , 400 MHz) δ
3
3
7
.39 (s, 1H, CH ), 4.66 (br.s, 2H, NH ), 4.25 (q, 2H,
J = 7.1 Hz, CH CH ), 3.65 (s, 3H, NCH ), 1.32
2 3 3
Ar
2
3
13
(
t, 3H, J = 7.1 Hz, CH CH ); C NMR (DMSO-d6, 126 MHz) δ 159.68 (CO), 154.27 (C NH ),
2 3 Ar 2
1
36.02 (CH ), 116.88 (C CO), 58.82 (OCH ), 30.17 (NCH ), 14.30 (CH CH ).
Ar Ar 2 3 2 3
4.1.1.4. Ethyl 1-N-methyl-2-nitro-1H-imidazole-5-carboxylate (5)
To a solution of sodium nitrite (18.3 g, 266 mmol) in water (55 mL) cooled around -5 °C in an
ice-salt bath, was added dropwise a solution of the amino ester 4 (6.42 g, 38.0 mmol) in acetic
acid (42 mL). The temperature was allowed to rise gradually to rt and the reaction mixture was
stirred overnight. The reaction mixture was extracted with dichloromethane (3 × 50 mL). The
4
combined organic layers were dried over MgSO , filtered and evaporated under reduced
pressure. The residue was purified by column chromatography on silica gel using
cyclohexane/ethyl acetate (7/3, v/v) as eluent. After concentration under vacuum, the residue
was washed with water (2 × 50 mL). The combined organic layers were dried over MgSO4,
filtered and evaporated under reduced pressure to yield nitro ester 5 as yellow crystals (5.67 g,
4
0
37
2
8.5 mmol). Yield 75%; mp 56-58 °C (Lit. 56-58 °C and 65-66 °C ); Rf 0.40 (SiO ,
2
-1
cyclohexane/ethyl acetate, 7/3, v/v); IR (ATR) ν cm 1723, 1552, 1486, 1519, 1364, 1233, 767;
1
3
H NMR (CDCl , 300 MHz) δ 7.70 (s, 1H, CH ), 4.39 (q, 2H,
J = 7.1 Hz, OCH CH ), 4.31 (s, 3H,
2 3
3
Ar
3
13
NCH ), 1.39 (t, 3H,
J = 7.1 Hz, OCH CH ); C NMR (DMSO-d6, 126 MHz) δ 158.71 (CO), 147.61
2 3
3
2 2 3 2 3
(CArNO ), 133.70 (CHAr), 126.07 (CArCO), 61.34 (OCH ), 35.14 (NCH ), 13.91 (CH CH ).
4
.1.1.5. (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (6)
The ester 5 (11.0 g, 55.2 mmol) was dissolved in a mixture of anhydrous THF/MeOH (200 mL,
/2, v/v) in a 1 L three-necked flask fitted with a mechanical stirrer, a 500 mL dropping funnel, a
bubbler and maintained in an ice-water bath. A suspension of NaBH (6.33 g, 167 mmol) in THF
8
4
24

ACCEPTED MANUSCRIPT
(50 mL) and a solution of LiBr (14.6 g, 168 mmol) in THF (50 mL) were cooled at 0 °C, introduced
in the same dropping funnel with water (50 m) and added dropwise to the nitroimidazole
solution, at such a rate that the internal temperature did not exceed 10 °C. The reaction
mixture was stirred at rt until total conversion of the starting nitroester 5, according to TLC
monitoring (19 h). Ammonium chloride (3 g) was then added at 0 °C and stirring was prolonged
for 30 min. The precipitate was filtrated and washed with THF (3 × 50 mL). After concentration
under vacuum, the yellow solid was taken up in a mixture of EtOAc/MeOH (98/2, v/v) and the
resulting solution was passed through a pad of silica gel surmounted by a pad of Celite®545 to
afford pure alcohol 6 as pale yellow to orange crystals (5.91 g, 37.7 mmol). Yield 68%; Rf 0.50
4
0
-1
(SiO
2
, EtOAc); mp 140 °C (lit. 141-143 °C); IR (ATR) ν cm 3231, 1491, 1358, 1186, 1040, 832;
1
3
J
3
J =
6
H NMR (DMSO-d , 300 MHz) δ 7.10 (s, 1H, CHAr), 5.40 (t, 1H,
= 5.3 Hz, OH), 4.55 (d, 2H,
1
3
5
.3 Hz, CH
2
OH), 3.93 (s, 3H, NCH
3
); C NMR (DMSO-d
6
, 126 MHz) δ 145.63 (CArNO ), 138.60
2
(
C CH ), 126.50 (CH ), 52.96 (CH ), 34.03 (NCH ).
Ar
2
Ar
2
3
4.1.2. N,N-bis(2-chloroethyl)phosphoramidic acid dichloride (8)
N,N-bis(2-chloroethyl)amine HCl (7) (10.0 g, 56.0 mmol) was refluxed at 130-140 °C in the
presence of an excess of POCl (21 mL, 224 mmol) during a period of 80 h, until a clear and
3
brown mixture was obtained. The excess of POCl was removed under reduced pressure and
3
the remaining brown residue purified by column chromatography on silica gel
(EtOAc/cyclohexane, 20/80, v/v). The obtained solid was thoroughly washed with cyclohexane
and dried under vacuum to afford pure compound 8 as white crystals (8.0 g, 31 mmol). Yield
4
3
-1
5
5%; Rf 0.39 (SiO
2
, cyclohexane/EtOAc, 8/2, v/v); mp 57 °C (Lit. 57-59 °C); IR (ATR) ν cm
1
1
272, 1264 (νP=O, νC-N), 1116, 1102 (νP-O, νP-N); H NMR (CDCl
3
, 200 MHz) δ 3.841-3.51 (m, 8H);
1
3
2
J
3
C NMR (CDCl
3
, 50 MHz) δ 49.61 (d, 2C,
2 2
C-P = 4.1 Hz, NCH CH Cl), 40.91 (d, 2C,
JC-P = 2.7 Hz,
3
1
NCH CH Cl); P NMR (CDCl , 202 MHz) δ 17.42.
2
2
3
4.1.3. General procedure for the synthesis of non-targeted prodrugs 23 to 27
25

ACCEPTED MANUSCRIPT
To a solution of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (6) (1 eq.) in freshly distilled
anhydrous THF (10 mL for 3 mmol of alcohol 6) was added dropwise lithium
bis(trimethylsilyl)amide (1M in THF, 1.1 eq.) at −78 °C under an inert atmosphere. The reacꢀon
mixture was stirred around 5 min at −78 °C, and a solution of bis(2-chloroethyl)phosphoramidic
dichloride (8) (1.1 eq.) in THF (3.3 mmol in 10 mL), previously cooled at −78 °C, was added all at
once at the same temperature (T ). The reaction mixture was stirred for 15 to 80 min, before
0
the addition of a solution of the appropriate amine 18-22 (2-2.2 eq.) in THF (3 mL for 5 mmol of
amine) and stirring was maintained at −78°C for 5 min to 75 min. These reacꢀon ꢀmes were
3
1
determined by P NMR monitoring for each compound. The reaction was stopped by addition
of water (20 mL for 3 mmol of alcohol 6), concentrated in vacuum and then extracted with
EtOAc (3 × 50 mL for 3 mmol of alcohol 6). The combined organic extracts were dried over
MgSO , filtered and concentrated under reduced pressure. The residue was purified by column
4
chromatography on silica gel using an eluent gradient (EtOAc/EtOH with TEA or NH OH) to
4
afford compounds 23-27 as yellow to orange oils.
(1-methyl-2-nitro-1H-imidazol-5-yl)methyl
N,N-bis(2-chloroethyl)-N’-[5-(dimethylamino)
pentyl]phosphorodiamidate (23a)
Starting from 500 mg (3.18 mmol) of alcohol 6, the coupling reaction with bis(2-
chloroethyl)phosphoramidic dichloride (8) was performed for 60 min before the addition of the
prepared amine 18a (826 mg, 6.34 mmol). The reaction was then stopped after 10 min of
stirring. The crude product was purified by silica gel chromatography (EtOAc/EtOH/NH
4
OH
ranging from 90/10/1 to 50/50/3, v/v/v) to afford 23a (457 mg, 0.965 mmol) as a yellow-orange
-
1
oil. Yield 30%; Rf 0.35 (SiO , EtOAc/EtOH/TEA, 70/30/3, v/v/v); IR (ATR) ν cm 3389, 3223,
2
1
1
1
9
4
2
537, 1490, 1351, 1216, 1191, 1007, 979, 960, 834, 744; H NMR (200 MHz, CDCl ) δ 7.19 (s,
3
2
3
2
3
H, CH ), 5.19 (dd, 1H, JH-H = 13.3 Hz, JH-P = 7.5 Hz, CH’O), 4.98 (dd, 1H, JH-H = 13.3 Hz, JH-P
=
Ar
3
2
.0 Hz, CH”O), 4.07 (s, 3H, NCH ), 3.95 (td, 1H, JH-H = 6.8 Hz, JH-P = 11.3 Hz, NHP), 3.64-3.51 (m,
3
H, N(CH
.79 (s, 6H, N(CH
), 133.43 (d,
2
CH
2
Cl)
2
), 3.53-3.25 (m, 4H, N(CH
2
CH
2
Cl)
2
), 3.04-2.89 (m, 4H, CH
2
NHP, CH
2 3 2
N(CH ) ),
1
3
3
)
2
), 1.90-1.53 (m, 6H, CH
2
CH CH
2
2
CH
2
CH
2
); C NMR (50 MHz, CDCl
3
) δ 146.41
3
J
2
J
(C
ArNO
2
C-P = 7.1 Hz, CArCH
2
), 129.11 (CHAr), 57.62 (CH
2 3
N(CH )
2
), 56.44 (d,
C-P = 4.4
26

ACCEPTED MANUSCRIPT
2
Hz, CH O), 49.33 (d, 2C, JC-P = 3.9 Hz, N(CH CH Cl) ), 43.18 (2C, N(CH ) ), 42.81 (2C,
2
2
2
2
3 2
3
J
N(CH
2
CH
2
2
Cl) ), 40.17 (CH
2 3
NHP), 34.78 (NCH ), 30.12 (d,
C-P = 6.4 Hz, CH
2
CH
2
NHP), 23.92, 23.34
3
1
+
(CH CH
2 2
2
CH N(CH
3 2
)
3
); P NMR (202 MHz, CD OD) δ 18.33; HRMS (ESI) m/z 473.1607 [M+H]
+
(
calculated for [C H Cl N O P] 473.1594).
1
6
32
2 6 4
4.1.4. General procedure for the synthesis of targeted prodrugs 28 to 32
To a solution of amine 23-27 (1 eq.) in anhydrous ACN (5 mL for 50-100 mg of amine) under an
inert atmosphere, was added potassium carbonate (2.5 eq) and appropriate alkyl iodide (5-24
eq.). Stirring was maintained at rt in a sealed flask for several hours until TLC monitoring
showed total conversion of the initial product (further addition of alkyl iodide was sometimes
necessary). The suspension was filtered and the precipitate was thoroughly washed with ACN.
After evaporation of the filtrate, the residue was dissolved in acetone (10 mL for 50 mg of
amine) and the mixture was filtered to remove the remaining potassium carbonate. After
evaporation to dryness under reduced pressure, the corresponding quaternary ammoniums 28-
32 were obtained analytically pure or purified by preparative high performance liquid
chromatography and lyophilised.
4.1.4.1.
5-({[bis(2-chloroethyl)amino][(1-methyl-2-nitro-1H-imidazol-5-
yl)methoxy]phosphoryl}amino)-N,N,N-trimethylpentane-1-aminium iodide (28a)
Alkylation of amine 23a (34 mg, 71.8 µmol) with methyl iodide (24 eq.) using the standard
procedure (reaction time: 4 h) afforded compound 28a analytically pure, as a yellow
-
1
hygroscopic solid (43 mg, 69.2 µmol). Yield 96%; IR (ATR) ν cm 3426, 3230, 1537, 1489, 1349,
1
1
219, 1190, 1007, 960, 835, 743; H NMR (500 MHz, CD OD) δ 7.25 (s, 1H, CH ), 5.16 (dd, 2H,
3
Ar
2
3
2
3
JH-H = 13.3 Hz, JH-P = 8.2 Hz, CH’O), 5.12 (dd, 2H, JH-H = 13.3 Hz, JH-H = 8.8 Hz, CH”O), 4.07 (s, 3H,
3
J
4
J
3
NCH ), 3.67 (td, 4H,
H-H = 7.0 Hz,
H-P = 2.3 Hz, N(CH
2
CH
2 2 2 2 2
Cl) ), 3.54-3.32 (m, 8H, N(CH CH Cl) ,
+
+
+
CH
2
N ), 3.19 (s, 9H, N (CH
3 3 2
) ), 2.96-2.84 (m, 2H, CH NHP), 1.84-1.77 (m, 2H, CH
2
CH
2
N ), 1.59 (qt,
3
J
3
J
13
2H,
H-H = 7.2 Hz, CH
2
CH
2
NHP), 1.41 (qt, 2H,
H-H = 7.5 Hz, CH
2
CH
2
CH
2
NHP); C NMR (126 MHz,
3
J
+
3 2
CD OD) δ 147.64 (CArNO ), 135.10 (d,
C-P = 6.8 Hz, CArCH
2
), 129.27 (CHAr), 67.81 (CH
2
N ), 57.73
2
+
2
(
d, JC-P = 4.7 Hz, OCH ), 53.75, 53.72, 53.68 (N (CH ) ), 50.30 (d, 2C, JC-P = 4.6 Hz, N(CH CH Cl) ),
2
3 3
2
2
2
27

ACCEPTED MANUSCRIPT
3
4
2
4
3.24 (2C, N(CH CH Cl) ), 41.37 (CH NHP), 35.23 (NCH ), 32.19 (d, JC-P = 5.5 Hz, CH CH NHP),
2
2
2
2
3
2
2
+
31
4.37 (CH
2
CH
2
CH
2
NHP), 23.58 (CH
2
CH
2
3
N ); P NMR (202 MHz, CD OD) δ 18.28; HRMS (ESI) m/z
+
+
87.1744 [M] (calculated for [C17
2
H34Cl N
6
O
4
P] 487.1751).
4.1.4.2.
4-({[bis(2-chloroethyl)amino][(1-methyl-2-nitro-1H-imidazol-5-
yl)methoxy]phosphoryl}amino)-N,N,N-trimethylbutane-1-aminium iodide (28b)
Alkylation of amine 23b (78 mg, 0.171 mmol) with methyl iodide (12 eq.) using the standard
procedure (reaction time: 4 h), afforded compound 28b analytically pure, as a yellow
-1
hygroscopic solid (95 mg, 0.158 mmol). Yield 92%; IR (ATR) ν cm 3404, 3223, 1537, 1488,
1
1
349, 1217, 1190, 1009, 960, 835, 743; H NMR (500 MHz, CD
3
OD) δ 7.25 (s, 1H, CHAr), 5.21-
CH Cl) ), 3.55-3.34 (m, 6H,
), 3.01-2.88 (m, 2H, CH NHP), 1.89-1.77 (m, 2H,
5.07 (m, 2H, OCH
2
), 4.07 (s, 3H, NCH
3
), 3.74-3.61 (m, 4H, N(CH
2
2
2
+
+
N(CH
2
CH
2
Cl)
2
, CH
2
N ), 3.15 (s, 9H, N (CH
)
3 3
2
+
13
CH CH N ), 1.63-1.50 (CH CH NHP); C NMR (126 MHz, CD OD) δ 147.64 (C NO ), 135.06 (d,
2
2
2
2
3
Ar
2
3
+
2
JC-P = 7.2 Hz, C CH ), 129.26 (CH ), 67.50 (CH N ), 57.78 (d, JC-P = 4.8 Hz, OCH ), 53.78, 53.76,
Ar
2
Ar
2
2
+
2
5
3.72 (N (CH ) ), 50.23 (d, 2C, JC-P = 4.7 Hz, N(CH CH Cl) ), 43.23 (2C, N(CH CH Cl) ), 41.05
3 3 2 2 2 2 2 2
3
+
31
(
CH NHP), 35.25 (NCH ), 29.41 (d, JC-P = 4.6 Hz, CH CH NHP), 21.36 (CH CH N ); P NMR (202
2 3 2 2 2 2
+
+
MHz, CD OD) δ 18.25; HRMS (ESI) m/z 473.1603 [M] (calculated for [C H Cl N O P]
3
16 32
2 6 4
473.1594).
4.1.4.3.
2-({[bis(2-chloroethyl)amino][(1-methyl-2-nitro-1H-imidazol-5-
yl)methoxy]phosphoryl}amino)-N,N,N-trimethylethane-1-aminium iodide (28c)
Alkylation of amine 23d (114 mg, 0.264 mmol) with methyl iodide (5 eq.) using the standard
procedure (reaction time: 24 h) and purification by preparative high performance liquid
chromatography (gradient ACN in water) afforded compound 28c as a hygroscopic lyophilisate
-
1
(
116 mg, 0.202 mmol). Yield 77%; IR (ATR) ν cm 3436, 3196, 1537, 1488, 1350, 1222, 1190,
1
2
1
005, 959, 835, 744; H NMR (500 MHz, CD OD) 7.27 (s, 1H, CH ), 5.20 (dd, 2H, JH-H = 13.4 Hz,
3
Ar
3
2
3
J
H-P = 8.4 Hz, CH’O), 5.17 (dd, 2H,
.66 (m, 4H, N(CH CH Cl)
C NMR (126 MHz, CD OD) δ 147.73 (CArNO
J
H-H = 13.4 Hz,
J
3
H-H = 8.3 Hz, CH”O), 4.08 (s, 3H, NCH ), 3.75-
+
+
3
2
2
2
), 3.55-3.35 (m, 8H, N(CH N ), 3.22 (s, 9H, N (CH
CH Cl)
2 2 2
, CH CH
2 2
3 3
) );
1
3
3
3
2
), 134.64 (d, JC-P = 7.3 Hz, CArCH ), 129.37 (CHAr),
2
28