Design, synthesis and evaluation of targeted hypoxia-activated prodrugs applied to chondrosarcoma chemotherapy

DOI: 10.1016/j.bioorg.2020.103747

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Bioorganic Chemistry (2020)

Update date:2022-08-16

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Authors:

Canitrot, Damien

Chezal, Jean-Michel

Galmier, Marie-Josephe

Gaumet, Vincent

Gerard, Yvain

Ghedira, Donia

Maubert, Elise

Miot-Noirault, Elisabeth

Peyrode, Caroline

Tarrit, Sebastien

Voissière, Aurélien

Weber, Valérie

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Article abstract of DOI:10.1016/j.bioorg.2020.103747

The tumor microenvironment in chondrosarcoma (CHS), a chemo- and radio-resistant cancer provides unique hallmarks for developing a chondrosarcoma targeted drug‐delivery system. Tumor targeting could be achieved using a quaternary ammonium function (QA) as a ligand for aggrecan, the main high negative charged proteoglycan of the extracellular matrix of CHS, and a 2-nitroimidazole as trigger that enables hypoxia‐responsive drug release. In a previous work, ICF05016 was identified as efficient proteoglycan-targeting hypoxia-activated prodrug in a human extraskeletal myxoid chondrosarcoma model in mice and a first study of the structure-activity relationship of the QA function and the alkyl linker length was conducted. Here, we report the second part of the study, namely the modification of the nitro-aromatic trigger and the position of the proteoglycan-targeting ligand at the aromatic ring as well as the nature of the alkylating mustard. Synthetic approaches have been established to functionalize the 2-nitroimidazole ring at the N-1 and C-4 positions with a terminal tertiary alkyl amine, and to perform the phosphorylation step namely through the use of an amine borane complex, leading to phosphoramide and isophosphoramide mustards and also to a phosphoramide mustard bearing four 2-chloroethyl chains. In a preliminary study using a reductive chemical activation, QA-conjugates, except the 4-nitrobenzyl one, were showed to undergo efficient cleavage with release of the corresponding mustard. However N,N,N-trimethylpropylaminium tethered to the N-1 or C-4 positions of the imidazole seemed to hamper the enzymatic reduction of the prodrugs and all tested compounds featured moderate selectivity toward hypoxic cells, likely not sufficient for application as hypoxia-activated prodrugs.

Full text of DOI:10.1016/j.bioorg.2020.103747

Bioorganic Chemistry 98 (2020) 103747

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Design, synthesis and evaluation of targeted hypoxia-activated prodrugs

applied to chondrosarcoma chemotherapy

Yvain Gerard, Aurélien Voissière, Caroline Peyrode, Marie-Josephe Galmier, Elise Maubert,

Donia Ghedira, Sebastien Tarrit, Vincent Gaumet, Damien Canitrot, Elisabeth Miot-Noirault,

⁎

Jean-Michel Chezal, Valérie Weber

Université Clermont Auvergne, INSERM, U1240 Imagerie Moléculaire et Stratégies Théranostiques, F-63000 Clermont-Ferrand, France

A R T I C L E I N F O

A B S T R A C T

Keywords:

The tumor microenvironment in chondrosarcoma (CHS), a chemo- and radio-resistant cancer provides unique

hallmarks for developing a chondrosarcoma targeted drug‐delivery system. Tumor targeting could be achieved

using a quaternary ammonium function (QA) as a ligand for aggrecan, the main high negative charged pro-

teoglycan of the extracellular matrix of CHS, and a 2-nitroimidazole as trigger that enables hypoxia‐responsive

drug release. In a previous work, ICF05016 was identiﬁed as eﬃcient proteoglycan-targeting hypoxia-activated

prodrug in a human extraskeletal myxoid chondrosarcoma model in mice and a ﬁrst study of the structure-

activity relationship of the QA function and the alkyl linker length was conducted. Here, we report the second

part of the study, namely the modiﬁcation of the nitro-aromatic trigger and the position of the proteoglycan-

targeting ligand at the aromatic ring as well as the nature of the alkylating mustard. Synthetic approaches have

been established to functionalize the 2-nitroimidazole ring at the N-1 and C-4 positions with a terminal tertiary

alkyl amine, and to perform the phosphorylation step namely through the use of an amine borane complex,

leading to phosphoramide and isophosphoramide mustards and also to a phosphoramide mustard bearing four 2-

chloroethyl chains. In a preliminary study using a reductive chemical activation, QA-conjugates, except the 4-

nitrobenzyl one, were showed to undergo eﬃcient cleavage with release of the corresponding mustard. However

N,N,N-trimethylpropylaminium tethered to the N-1 or C-4 positions of the imidazole seemed to hamper the

enzymatic reduction of the prodrugs and all tested compounds featured moderate selectivity toward hypoxic

cells, likely not suﬃcient for application as hypoxia-activated prodrugs.

Hypoxia-activated prodrug

Phosphoramide

Quaternary ammonium

Chondrosarcoma

Proteoglycan

. Introduction

Surgical resection with wide margins remains the mainstay for the

extracellular matrix is due to the numerous sulfate and carboxylate

anionic functions of aggrecan (≈ 250 MD), the most abundant PG in

chondrosarcoma with approximately 150 glycosaminoglycan side

chains of chondroitin sulfate and keratan sulfate [7].

management of chondrosarcoma, the malignant cartilage tumor [1].

For patients with inoperable primary disease, metastases, or relapse, a

lack of appropriate therapy persists due to chemo- and radio-resistance

related to the extensive cartilaginous extracellular matrix expressed by

tumor cells, a physical semi-permeable barrier, and to hypoxia [1–4].

To circumvent this problem, new therapeutic tools have been devel-

oped such as: isocitrate dehydrogenase, m-TOR, tyrosine kinase and

Hedgehog pathway inhibitors, but so far no drug have been approved

for clinical use [5,6]. Our strategy aims at providing a dual targeted

therapy of chondrosarcoma leveraging two speciﬁc hallmarks of this

tumor, namely hypoxia and hyaline extracellular matrix, more pre-

cisely, its high negative ﬁxed charge density due to the presence of

proteoglycans (PGs). This latter feature of the chondrosarcoma

Hypoxia is a common feature of various solid tumors inﬂuencing

many aspects of cancer biology, including increased resistance to

therapy and disease progression through angiogenesis, glycolytic

switch in metabolism, and ampliﬁed anti-apoptotic and metastatic po-

tentials [8]. Therefore, the development of cancer-targeted therapies

directed against hypoxia is of great importance, especially with hy-

poxia-activated prodrugs (HAP) [9], like AQ4N [10], apaziquone

[11,12], SN30000 [13,14], evofosfamide (TH-302) [15–20], PR-104

[21,22] or TH-4000 [23] for example (Fig. 1). HAPs are designed to be

bioactivated by one-electron oxygen-sensitive reductases, such as

NADPH cytochrome P450 oxidoreductase (POR), to speciﬁcally release

active cytotoxic species in hypoxic regions of the tumor

⁎

Corresponding author.

E-mail address: valerie.weber@uca.fr (V. Weber).

https://doi.org/10.1016/j.bioorg.2020.103747

Received 18 November 2019; Received in revised form 13 February 2020; Accepted 9 March 2020

Available online 13 March 2020

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

Fig. 1. Structures of some hypoxia-activated prodrugs reviewed in literature.

microenvironment, using a “trigger” or “oxygen concentration sensor” series), or the nature of the cytotoxic agent (Fig. 3, axis 2).

[

24–28]. Nitroaromatic-based HAP are well reported in the literature,

In one hand, we were interested in the reduction of the sensitive

nitroaromatic trigger. Although the nitroimidazole appears as the most

widely studied, diﬀerent other cycles have been used in HAP design.

Concerning HAP undergoing clinical trials, the 2-nitroimidazole trigger

is used in the structure of TH-302 and TH-4000, whereas in the case of

PR-104, the cytotoxin is released by bioreduction of a 4-nitrophenyl

group. Many other nitroaryl-based HAP were studied in preclinical

models, like camptothecin, paclitaxel, doxorubicine conjugates

[29,30,37] and tyrosine kinase inhibitors [31,32]. In addition to

widespread used 4-nitrophenyl and 2- or 5-nitroimidazole groups, ni-

trofuran and nitrothiophene groups have also been used as bioreduc-

tion-sensitive moieties. The most important considerations in the choice

of the bioreductive trigger may be the hypoxic threshold required for

activation and the propensity to undergo bioreduction, in part corre-

lated with the trigger’s electrochemical reduction potential. The nitro-

compounds are encompassed in class II HAP, which depends on severe

hypoxia for activation. The 1-methyl-2-nitroimidazole group, with an

estimated reduction potential of −390/−400 mV [38,39], should un-

dergo easier reduction than the 4-nitrobenzyl group (showing a lower

reduction potential, E = −490 mV), and a harder reduction than 2-

nitrofuran (E = −330 mV). To deﬁne the best trigger in our series of

PG-targeted HAP, the selected subset 2-nitroimidazole-, 2-nitrofuran-

and 4-nitrobenzyl-based conjugate were synthesized.

the 2-nitroimidazole moiety being by far the most widely documented

trigger [24–26,29–32].

This dual targeted therapy for chondrosarcoma was previously in-

vestigated, by conjugation of a hypoxia-activated prodrug to a qua-

ternary ammonium function (QA) validated as PG-targeting ligand

[

33,34] and led to the identiﬁcation of the ﬁrst PG-targeted HAP,

named ICF05016 [35], a phosphorodiamidic mustard functionalized

with a QA function and a nitroimidazole heterocycle, which may un-

derwent diﬀerent reductive processes, causing the release of the cyto-

toxic mustard (Fig. 2). Aggrecan binding of ICF05016 by surface

plasmon resonance (SPR), as well as hypoxic-cytotoxicity ratio (HCR;

IC50 under normoxia vs. IC50 under hypoxia) on the HEMC-SS (human

extraskeletal myxoid chondrosarcoma) cell line were conﬁrmed. In a

HEMC-SS tumor-bearing mice model, ICF05016 treatment provided a

signiﬁcant tumor growth inhibition, a decrease in the mitotic index and

an increase in DNA damages predominantly found in pimonidazole-

positive hypoxic areas, with no associated hematological side eﬀects.

Based on these results, a ﬁrst lead optimization study of ICF05016

was initiated and recently published. This work concerned the mod-

iﬁcation of the structure of the QA function and the length of the alkyl

linker (Fig. 3, axis 1) [36]. Thirty QA-phosphoramide mustard con-

jugates were synthesized and a preliminary screening by SPR revealed

that aﬃnity depends more on the type of QA function, than on the

linker length and highlighted the positive impact of a benzyl QA

function on the aﬃnity to aggrecan. For all tested prodrugs, hypoxic

selectivity was maintained and even increased, compared to the

lead ICF05016, mainly with compound ICF05089 that emerged as the

most eﬀective QA HAPs. Herein, we extend this study to a series of

prodrugs diﬀering both by the hypoxia-sensitive trigger, the position of

the N,N,N-trimethylpropylaminium targeting moiety (N-1 or C-4

In a second hand, we were interested in the position of the N,N,N-

trimethylpropylaminium targeting chain, so far tethered to the phos-

phorodiamidic mustard. To this end, we synthesized compounds

bearing this chain on the N-1 or C-4 position of the 2-nitroimidazole

ring. This strategy will allow the possibility to release under hypoxic

conditions only the alkylating agent free of the PG-targeting ligand.

Our ﬁrst attempts to validate the concept of PG-targeted HAP were

focused on phosphoramide mustard. Cyclophosphamide belongs with

Fig. 2. Proposed mechanism of ICF05016 activated via enzymatic metabolism under hypoxic condition.

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

Fig. 3. Pharmacomodulation of compound ICF05016.

ifosfamide and trofosfamide to the oxazaphophorines alkylating class,

commonly used in clinical practice for their wide spectrum of anti-

neoplasic activity. Cyclophosphamide and ifosfamide both require

metabolic activation from the cytochrome P450 to subsequently pro-

duce phosphoramide and isophosphoramide mustards (PM, IPM),

which induce DNA cross-linking. These active nitrogen mustards PM

and IPM show an isomeric structure with one or two 2-chloroethyl

groups linked to the same nitrogen. We also decided to synthesize

isomeric analogs of PM derivatives 15a and 26a (Schemes 2 and 3),

with IPM 15c and 26c, and also a phosphoramide mustard bearing four

without intermediate puriﬁcation: (i) C-formylation of 7 at the α-

carbon position using sodium hydride (added carefully in order to avoid

reaction thermal runaway and explosion) and ethyl formate in anhy-

drous THF, (ii) removal of the unwanted N-formyl group under reﬂux in

concentrated water-alcohol hydrochloric acid solution, and ﬁnally (iii)

Markwald cyclisation with cyanamide under reﬂux at pH 3. According

to this procedure, aminoimidazole 8 was obtained after silica gel

column chromatography with 21% yield. The nitro group was then

introduced by diazotization of the aromatic amine in the presence of an

excess of sodium nitrite with good yield (71%). Due to low stability of

product 9, reduction of the ester function with lithium borohydride

(formed in situ via the metathesis reaction between sodium borohydride

and lithium bromide) needs to be performed immediately after pur-

iﬁcation. Indeed, prolonged storage of compound 9 at 4 °C led to the

-chloroethyl chains (TPM) (15b and 26b).

A preliminary screening of all the ﬁnal QA-conjugates produced was

performed to evaluate their suitability as substrates for reduction-

mediated cleavage (chemical and enzymatic) leading to the release of

the phosphorodiamidic mustard, as well as their in vitro hypoxia cyto-

toxity ratio.

formation of

a tetrahydroimidazo[1,2-a]pyrimidine derivative 11

(supporting data), resulting from a nucleophilic aromatic substitution

of the nitro group by the tertiary amine of the ester 9, probably allowed

by the presence of the electron withdrawing ester in the 5-position,

followed by demethylation of the QA intermediate. Lee et al. [44] al-

. Results and discussion

2.1. Chemistry

ready described a similar S Ar reaction by tertiary amine with 2,4-di-

chloropyrimidines, further substituted with an electron-withdrawing

substituent, followed by in situ N-dealkylation of the intermediate. Even

if compound 9 was engaged quickly in the next step, the desired alcohol

10 was only obtained in 7% yield, while the major fraction isolated was

identiﬁed as the corresponding amine-borane complex (45% yield). The

The reagents required to tether the 4-nitrobenzyl- and 2-nitrofuran-

based groups to phosphoramidic mustard are readily available from

commercial sources, contrary to the 1-methyl-2-nitroimidazole pre-

cursor 5 previously described for the synthesis of ICF05016 and its

derivatives [35,36]. Also, 2-hydroxymethyl-5-nitrofuran 4 was ob-

structure of the borazane 10-BH was validated using mass spectro-

tained by reduction of 5-nitro-2-furaldehyde with NaBH

in 79% yield.

metry and X-Rays diﬀraction (supporting data); the HRMS spectrum

showed the protonated complex, m/z 243.1616, and the sodium adduct

at m/z 265.1434. After diﬀerent attempts of complex dissociation under

acidic [45,46] of basic [47] conditions, the best results were obtained

with boiling ethanol [48,49]. Both reactions, reduction with lithium

borohydride, followed after work-up by complex dissociation in boiling

ethanol aﬀorded after silica gel puriﬁcation the alcohol 10 from 9 in

49% yield.

As for ICF05016, compounds 3a-b were obtained after phosphorylation

of the appropriate hydroxymethyl nitroaromatic by the phosphoramidic

dichloride 1 followed by the introduction of the N,N-dimethylamino-

propyl chain by substitution of the remaining chloride by 3-(N,N-di-

methylamino)propylamine and ﬁnally quaternisation of the tertiary

amine function using excess of methyl iodide (Scheme 1).

Our ﬁrst attempts to validate the concept of PG-targeted HAP were

focused on phosphoramide mustard then followed by the synthesis of

isomeric analogs of PM derivatives 15a and 26a, with TPM and IPM

alkylating moieties. To that end, a similar sequence of phosphorylation

and quaternisation used for compounds 3a-b was considered to obtain

targeted compounds 15a and 26a, starting from the appropriate alcohol

Starting from 10, initial attempts to synthesize phosphor-

odiamidates 14a-b following published synthetic methods from

Matteucci et al. [43] and previously adapted for ICF05016 derivatives

[36], resulted respectively in ineﬀective reaction or rapid degradation

of the synthesized products, as observed by phosphorus NMR mon-

itoring. Assuming that these results may be due to the nucleophilicity of

the tertiary amine on the N-1 position, we decided to use the amine-

0 and 21, respectively (Scheme 2 and 3). We previously reported the

synthesis of alcohol precursor 5 [36] using O’Connor et al. optimized

method [40,41]. The same synthetic sequence was applied to obtain the

alcohol 10, common precursor of the N-1 functionalized derivatives

with a 3-(N,N-dimethylamino)propyl chain instead of a methyl group.

The ﬁrst step of the synthesis of derivative 10 was a nucleophilic sub-

stitution involving 3-(N,N-dimethylamino)propylamine and ethyl-

bromo acetate [42]. Then the secondary amine of 6 was protected by

formylation with ethyl formate under basic conditions [43] to give

compound 7 in good yield. The next three steps were performed

borane complex 10-BH , described as stable in the presence of strong

bases such as n-butyllithium [49], to perform the phosphorous coupling

reaction. The alcohol 10-BH

was deprotonated with LiHMDS at

−78 °C and the appropriate phosphorous compound 1 or 12 (synthe-

sized as described in the literature [50,51]) was then added. For com-

pound 14a-BH , the reaction was carried out at −78 °C in THF for 1 h

before bubbling of ammonia gas and was almost quantitative. To obtain

compound 14b-BH3, the reaction had to be warmed to rt after the

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

Scheme 1. Reagents and conditions: (i) (a) LiN(TMS)

, anh. THF, −78 °C, 30 min, (b) 3-(N,N-dimethylamino)propylamine, anh. THF, −78 °C, 40 min, 57%; (ii)

I, anh. THF, rt, 3 h, 99%; (iii) NaBH

, THF/H

O, 0 °C, rt, 1 h 30, 79%; (iv) (a) LiN(TMS) , bis(2-chloroethyl)phosphoramidic acid dichloride (1), anh. THF,

−78 °C, 30 min, (b) 3-(N,N-dimethylamino)propylamine, anh. THF, −78 °C, 2 h, 42%; (v) CH

phosphoramidic dichloride (1), anh. THF, −78 °C, 40 min, (b) 3-(N,N-dimethylamino)propylamine, anh. THF, −78 °C, 20 min, 43%; (vii) CH

9%.

I, anh. THF, rt, 12 h, 81%; (vi) (a) LiN(TMS)

, bis(2-chloroethyl)

I, anh. THF, rt, 3 h 30,

addition of compound 12. With these conditions, the kinetic follow-up

(due to the presence of excess of DABCO). Following this procedure,

quaternary ammonium 15a-c were obtained with 75%, 21% and 57%

yield, respectively including one-pot borazane dissociation and alky-

lation steps.

by phosphorus NMR showed a complete reaction after 20 h, leading to

compound 14b-BH in 70% yield. For the last isophosphorodiamidate

analog 14c-BH , a Mitsunobu reaction was considered as previously

described [20] to access to evofosfamide and needed the prior synthesis

of the isophosphorodiamidic acid (IPM). This kind of compound is

usually synthesized by hydrolysis of the corresponding phosphor-

odiamidyl chloride [20,40]. In all our attempts, this methodology ap-

peared not reliable and we decided to focus our attention to a two-steps

sequence involving synthesis of the corresponding benzyl ester followed

by hydrogenation as described in the literature [52] and leading to IPM

in 80% overall yield (supporting data). However, Mitsunobu conditions

For the 4-C-(N,N-dimethylamino)propylamino) derivatives 26a-c,

alcohol 5 was functionalized using a Stille coupling reaction. According

to the procedure outlined in Scheme 3, the imidazole cycle was ﬁrst

brominated in the 4-position with NBS [55], followed by protection of

the hydroxyl function using tri(isopropyl)silylchloride to aﬀord the si-

lylether 17. Then the Stille coupling reaction was performed with 66%

yield following a procedure described by Jiao et al. [55] using tetrakis

(triphenylphosphine)palladium as catalyst in anhydrous DMF and N,N-

dimethyl-N-[3-(tributylstannyl)-2-propenyl]amine (18). The latter was

synthesized by hydrostannylation of N,N-dimethylpropargylamine ac-

cording to the methodology developed for the N,N-diethyl analog [56].

Under the Stille conditions used, compound 19 was obtained only as the

(E) isomer, starting either from the pure (E) isomer or the isomers

mixture of 18. After silylether deprotection of compound 19 using

TBAF, reduction with diimide was favored to selectively reduced the

double bond of compound 20 without impacting on the nitro function

[57]. First we tried to use potassium azodicarboxylate (PADA) as de-

scribed in the literature [55] but no reduction reaction was evidenced

according to TLC monitoring. A method involving p-tosylhydrazine in

reﬂuxing pyridine to generate in situ diimide [58] successfully con-

verted the alkene 20 to its saturated analog 21 in 67% yield. These two

last steps could also be performed in reverse order, i.e. reduction before

silyl deprotection, but this sequence involving intermediate compound

22 led to a lower overall yield (i.e. 11% vs 60% for the two steps).

To access respectively to the cyclophosphamide mustards deriva-

tives 23 and 24a starting from the alcohols 20 and 21, we tried initially

our previous method used for the synthesis of ICF05016 from

dichloride 1. Under these conditions, phosphorodiamidate inter-

mediates 23 and 24a were obtained with 31% and 30% yields, re-

spectively. Each compound was further methylated with methyl iodide

under basic conditions to yield the corresponding quaternary ammo-

niums 25 and 26a. Nevertheless, the phosphorylation reaction with

both alcohols 20 and 21 was not reproducible. Therefore and based on

between IPM and 10-BH led to a poor yield of the desired product 14c-

(5%) while a large amount of starting material 10-BH (76%) has

been recovered. To circumvent this problem, we turned our attention

on a two-step one-pot reaction, previously described by Misiura et al.

[

53] for a 4-substituted benzyl alcohol, and involving the phosphor-

ylation of 10-BH using phosphorus oxychloride in the presence of TEA,

to yield the dichlorophosphate intermediate, which subsequently was

treated, without isolation, with two equivalents of 2-chloroethylamine

hydrochloride followed by another base addition. This sequence suc-

cessfully aﬀorded compound 14c-BH in moderate yield (48%). As

expected with the use of phosphorus oxychloride and previously pub-

lished with phosphorus trichloride [20], the side product 1-N-[3-(N,N-

dimethylamino)propyl]-2-nitro-5-chloromethylimidazole 13 was also

isolated (10% yield).

To dissociate the boron complex, we tried similar conditions as

described for the alcohol 10-BH , namely heating in ethanol but also

acidic conditions. Unfortunately, only several by-products and low or

no amount of desired product were observed, highlighting the in-

stability of phosphorodiamidate under heating or acidic conditions. To

remove the borane group, a transchelation reaction with 1,4-diazabi-

cyclo[2.2.2]-octane (DABCO; 5 eq.) was then assessed using the method

described by Tam et al. [54] slightly modiﬁed due to the instability of

the phosphorodiamidate intermediate. Indeed, the number of equiva-

lents of DABCO was increased (5 eq. vs 1) to decrease reaction time and

the free amine was directly alkylated using an excess of methyl iodide

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

Scheme 2. Reagents conditions: (i) BrCH

Et, K

, PhMe, 0 °C then rt, 18 h, 54%; (ii) HCO

CN, EtOH/H O (70/30) (v/v), pH = 3.0, reﬂux, 90 min, 21%; (iv) NaNO

, LiBr, THF/MeOH (80/20) (v/v), H O, 0 °C then rt, 17 h; (b) EtOH, reﬂux, 4 h, 49%; (vi) NaBH , LiBr, THF/MeOH (80/20) (v/v),

O, 0 °C then rt, 15 h, 7% of 10 and 45% of 10-BH ; (vii) (a) LiHMDS, anh. THF, −78 °C, 5 min; (b) N,N-bis(2-chloroethyl)phosphoramidic acid dichloride (1), anh.

, −78 °C, 2 min, 93%; (ix) (a) DABCO, anh. THF, 30–40 °C, 1 h; (b) CH I, 0 °C to rt, 2 h 30, 75%; (x) (a) LiHMDS, anh. THF, −78 °C,

min; (b) compound 12, anh. THF, −78 °C, 1 h then rt, 20 h, 70%; (xi) (a) DABCO, anh. THF, 40 °C, 1 h; (b) CH I, 0 °C to rt, 2 h, 21%; (xii) POCl , Et N, DCM,

78 °C, 1 h; (xiii) NH CH CH Cl.HCl, TEA, −78 °C to 5 °C, 18 h, 48%; (xiv) (a) DABCO, anh. THF, 40 °C, 1 h; (b) CH I, 0 °C to rt, 1 h 30, 57%.

Et, K

, EtOH, rt, 25 h, 90%; (iii) (a) HCO

Et, NaH (60%), THF,

cyclohexane, rt, 3 h; (b) HCl (37%), EtOH, reﬂux, 2 h; (c) NH

, AcOH (66% vol. in H

O),

°C then rt, 2 h, 71%; (v) (a) NaBH

THF, −78 °C, 1 h; (viii) NH

−

the previous results obtained in the N-1 series, we explored the use of

2.2. Biological assessments for HAP screening

2.2.1. Reductive chemical activation

amine borane complex to further perform phosphorylation. The com-

plex 21-BH was obtained in 70% yield from compound 21 and its

structure was conﬁrmed by X-rays diﬀraction (supporting data) (note

that borane complex of alkene 20 was not studied due to the presence

of the double bond). The phosphorylation from borazane complex 21-

The stability of all QA-compounds in aqueous media was tested ﬁrst

as this parameter is essential for the biological evaluation of HAP.

Under physiological conditions (cacodylate buﬀer pH 7.4, 37 °C) and in

the absence of a reductant system, no cleavage or degradation over a

aﬀorded compounds 24a-c-BH in yield ranging from 33% to 63%.

The boron complex of 24a-c was then dissociated using DABCO and the

tertiary amine directly converted onto the corresponding QA function

using methyl iodide to aﬀord ﬁnal products 26a-c.

24 h period was observed by P NMR experiments. To evaluate the

sensitivity to reduction of the nitroheteroaromatic rings, the three

phosphoramidate prodrugs, ICF05016, 3a and 3b, were evaluated in

All the seven PG-targeted prodrugs 15a-c, 25 and 26a-c were pur-

iﬁed by reverse phase (C18) preparative column and freeze-dried in

aliquots before further evaluation. Analytical purity was evaluated by

terms of reductive chemical activation by P NMR experiments. In the

presence of three equivalents of sodium dithionite (0.1 M cacodylate

buﬀer, pH 7.4), a reductant system used to mimick bioreduction in

hypoxic tissue, the two prodrugs ICF05016 and 3b were rapidly acti-

vated (Table 1 and Fig. 4A). The signals related to the prodrugs

ICF05016 and 3b disappeared with a half-time of about 3–4 min

leading to the rapid formation of a signal at 12 ppm corresponding to

the active cytotoxic specie, the substituted bis(2-chloroethyl)phos-

phorodiamidate anion [36] (as exempliﬁed on Fig. 4B for ICF05016).

In these conditions, only 15% of the prodrug 3a were reduced (Table 1

the absence of impurities revealed in the H and C spectra combined

with P impurities estimated at < 5% in global integrals, and by HPLC

analysis (supporting data).

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

Scheme 3. Reagents conditions: (i) NBS, DMF, 60 °C, 2 h 30, 82%; (ii) TIPSCl, imidazole, DMF, rt, 18 h, 97%; (iii) Bu

SnH, AIBN, PhMe, 85 °C, 3 h 30, reﬂux, 1 h,

5%; (iv) [Pd(PPh

)

], 105 °C, 1 h 30, 66%; (v) TBAF, anh. THF, 0 °C, 90 min, 89%; (vi) TsNHNH

, K

, pyridine, reﬂux, 3 h, 67%; (vii) TsNHNH

, K

CO ,

pyridine, reﬂux, 19 h, 24%; (viii) TBAF, anh. THF, 0 °C, 1 h, 47%; (ix) BH

·THF, anh. CHCl

, 0 °C, 1 h, 70%; (x) (a) LiHMDS, anh. THF, −78 °C, 5 min; (b) N,N-bis(2-

I, K CO , anh. ACN, rt, 1 h 45, 65%; (xii) (a) LiHMDS,

, −78 °C, 2 min, 30%; (xiii) CH I, anh. THF, rt,

h, 52%; (xiv) (a) LiHMDS, anh. THF, −78 °C, 5 min; (b) N,N-bis(2-chloroethyl)phosphoramidic dichloride (1), anh. THF, −78 °C, 1 h; (c) NH , −78 °C, 2 min,

3%; (xv) (a) DABCO, anh. THF, 40 °C, 1 h; (b) CH I, 0 °C, 2 h, 46%; (xvi) (a) LiHMDS, anh. THF, −78 °C, 5 min; (b) N,N,N’,N’-tetrakis(2-chloroethyl)phos-

: 33% or (a) POCl , TEA, anh. DCM, −78 °C, 1 h; (b) NH CH CH Cl.HCl, TEA, −78 °C to

: 41%; (xvii) (a) DABCO, anh. THF, 40 °C, 1 h; (b) CH I, 0 °C, 1–2 h, 26b: 35%; 26c: 56%.

chloroethyl)phosphoramidic dichloride (1), anh. THF, −78 °C, 45 min; (c) NH

, −78 °C, 2 min, 31%; (xi) CH

anh. THF, −78 °C, 5 min; (b) N,N-bis(2-chloroethyl)phosphoramidic dichloride (1), anh. THF, −78 °C, 1 h 15; (c) NH

phoramidic acid chloride (12), anh. THF, −78 °C to 5 °C, 24 h, 24b-BH

°C, 15 h, 24c-BH

and Fig. 4A). Nine equivalents of reducing agent were required for a

total activation. As expected with the estimated reduction potential for

the 1-methyl-2-nitroimidazole group, compound ICF05016 was more

easily reduced compared to the 4-nitrobenzyl analog 3a. In addition,

reductions of ICF05016 and 2-nitrofuran derivatives 3b were similar.

For compounds 15a-c, 25 and 26a-c, the imidazole ring is func-

tionalized on the C-4 or N-1 position with a N,N,N-trimethylpropyla-

minium or N,N,N-trimethylpropyl-2-enaminium that may hamper re-

ductive activation of the nitro group due to steric hindrance. To

ascertain the reductive activation of the compounds and release of the

Table 1

Chemical reduction, NTR activation and in vitro potency on HEMC-SS cells in normoxic (21% O

) and hypoxic (N

, O

< 0.3%) conditions of HAP

derivatives. [a] Chemical reduction and subsequent fragmentation of HAP derivatives (3 eq. sodium dithionite, CH

CN/0.1 M cacodylate buﬀer, pH 7.4, 37 °C)

assessed by P NMR as a function of time, Half-life times were determined using GraphpadPrism; [b] Enzymatic reduction and subsequent fragmentation of HAP

derivatives (17 µM) by incubation with NADPH (0.9 mM) and NTR (25 µg/mL) in sodium phosphate buﬀer (10 mM, pH 7.4, 37 °C), calculated from RP-HPLC

chromatogram peak areas at 322 nm as a function of time (experiments were performed in triplicate); [c] Cultures were exposed to drugs for 24 h followed by

incubation for 48 h in normoxia; [d] IC50 (Growth Inhibiting Concentration 50) values are an average of at least three independent experiments with standard error;

[

e] HCR: Hypoxic-Cytotoxic Ratio = IC50 in normoxia/IC50 in hypoxia. Notes: signiﬁcant diﬀerence vs. normoxic condition, * p < 0.05 and ** p < 0.01. NC: not

calculable.

Compound

Chemical reduction^[^a^]

Half-live

NTR activation^[^b^]

Cytotoxicity^[^c^]

IC50 (µM)^[^d^]

Normoxia

Amount of substrate

after 24 h [%]

Amount of substrate

after 15 min [%]

Amount of substrate

after 2 h [%]

HCR^[^e^]

[

min]

Hypoxia

ICF05016

4 ± 0.5

13 ±

6 ±

29.3 ± 8.0

56.9 ± 13.1

0.9 ± 0.3

6.7 ± 0.3

7.2 ± 2.4

12.9 ± 5.7

2.7 ± 0.3

4.4 ± 1.1

10.4 ± 1.8

6.9 ± 0.9

4.3 ± 1.2**

29.7 ± 3.8*

0.9 ± 0.3

6.8 [35]

1.9

2.5

< 1

86 ±

6 ±

49 ± 23

30 ±

< 2

2 ±

1.1

2 ± 0.5

1 ± 0.5

2 ± 0.5

5.2 ± 1.5

1.3

94 ±

100

60 ±

90 ±

82 ±

92 ±

75 ±

97 ±

41 ±

69 ±

50 ±

76 ±

7.3 ± 2.5

1.0

3.6 ± 1.5*

1.6 ± 0.2**

3.1 ± 0.2

3.6

17 ±

3 ±

–

1,7

1.4

–

4.4 ± 2.1**

3.6 ± 1.7*

2.4

–

1.9

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

B.1.

24h

ICF05016

4’

’

2’20

Fig. 4. Drug release in reductive conditions (3 eq. sodium dithionite, CH CN/0.1 M cacodylate buﬀer, pH 7.4, 37 °C) assessed by P NMR as a function of

time: A. Percent of phosphorodiamidate prodrugs remaining after addition of sodium dithionite in cacodylate buﬀer; B.1. Stacked P NMR spectra; a. prodrug

ICF05016, 18.22 ppm; b. phosphoramidate anion, 12.4 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.2. Representative time

course of ICF05016 cleavage (•), substituted bis(2-chloroethyl)phosphorodiamidate anion formation (□) and inorganic phosphate formation (♢) upon incubation of

these prodrugs with sodium dithionite in cacodylate buﬀer; stability of ICF05016 in cacodylate buﬀer (▾). Data points were measured from NMR P areas

(

experiments were performed in duplicate); C. P NMR spectrum of: a. pure synthesized IPM (11.2 ppm), b. 27c reduction medium at 5 min, c. 27c reduction

medium at 5 min spiked with pure synthesized IPM, d. 15c reduction medium at 5 min.

corresponding PM, ³P kinetic experiments were ﬁrstly carried out on

compounds 15a, 25, 26a. Cleavage studies were also performed with

compounds 15b and 15c, bearing respectively IPM and TPM, in order

to evaluate the impact of the phosphorodiamidic mustard on the re-

duction and conﬁrm the release of the three corresponding mustards.

The three prodrugs 15a-c functionalized on the N-1 position showed a

similar reduction proﬁle with a short half-life (< 1 min) (Table 1 and

Fig. 4A). While compound 26a bearing the N,N,N-trimethylpropyla-

minium chain on the C-4 position showed comparable kinetic (half-life

of about 5 min) as the lead compound ICF05016, the alkene analog 25

displayed a slower reduction proﬁle with a half live exceeding 1 h. For

medium of 27c at 5 min, leading to an increase of the signal level in the

spiked medium (Fig. 4C). Similar experiments have been carried out for

the other QA-phosphorodiamidates proving each time the release of the

corresponding mustard and conﬁrming the relevance of these com-

pounds as reduction-activated prodrugs (supporting data, Fig. S1).

.2.2. Bioreductive activation using nitroreductase enzyme

Nitroaromatic-based HAP are activated through nitroreductases

(

NTR) that catalyze the reduction of nitro compounds using NAD(P)H.

Reduction with NTRs to form nitroso, hydroxylamine and amine me-

tabolites and subsequent release of the mustard can occur via two dif-

ferent pathways. Type I NTRs are oxygen-insensitive enzymes and

catalyze two electron reduction of the nitro group, while type II NTRs

are oxygen-sensitive enzymes and performs single electron reduction.

For experimental setting not aﬀected by the environmental oxygen level

avoiding the futile-redox cycle, the prodrugs 3a-b, 15a-c, 25, 26a-c

were evaluated, compared to ICF05016, as substrate of an oxygen-in-

sensitive I NTR from E. coli to verify enzymatic reductive activation.

The prodrugs dissolved in PBS (pH 7.4) were incubated with this I NTR

and NADPH as cofactor at 37 °C, aliquots were withdrawn at various

time points and the biological reduction process was monitored by

HPLC (Fig. 5 and Table 1). The reduction was estimated on the basis of

the disappearance of the prodrugs. In phosphate buﬀer supplemented

with NADPH, no enzyme-free reaction was observed by HPLC analysis.

Compared to the lead compound ICF05016, 3b was readily reduced in

these conditions with less than 5% of the prodrug being detectable

all compounds, P prodrugs signals observed around 17–19 ppm dis-

appeared with the concurrent appearance of a resonance near 12 ppm

(

as exempliﬁed on Fig. 4B for ICF05016). The rapid kinetics of re-

duction of the imidazole prodrugs 15a-c and the high reactivity of the

corresponding mustards, gave rapidly rise to the disappearance of the

ﬁrst signals at 11–12 ppm and the formation of a variety of by-products.

In order to clearly assign the signal at 11–12 ppm to the corresponding

mustard anions, reduction experiments were performed with the lower

reduction-sensitivity 4-nitrobenzyl analogs 27a-c already described in

the literature [53,59,60] (synthesis in supporting data). Indeed for

compound 27c, a maximum of 40% of reduction was observed in the

presence of 3 equivalents of sodium dithionite compared with more

than 95% for compound 15c, both giving rise to the same signal at a

chemical shift of 11.2 ppm. The latter signal is clearly assigned to IPM

as demonstrated with pure synthesized IPM added in the reduction

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

with compound 3b and already reported in literature [30,31]. Even if,

the enzymatic cleavage experiment allowed obtaining a ﬁrst in tubo

result on the sensitivity of molecule to undergo reductive cleavage, this

experiment remains far away from the cytotoxicity test, given the

variety of diﬀerent mechanisms underlying the prodrug activity and

selectivity in the entire biological environment.

. Experimental protocols

3.1. Synthesis

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

Fig. 5. Percent of prodrugs remaining after incubation in phosphate buﬀer

(PBS 1x, 37 °C, pH = 7.4) with nitroreductase in the presence of NADPH;

(

Illkirch, France), Carlo Erba Reagents (Val de Reuil, France), VWR

Fontenay-sous-Bois, France) and Alfa Aesar (Karlsruhe, Germany) and

Data points were calculated from RP-HPLC chromatogram peak areas at 322 nm

and experiments were performed in triplicate. The lines represent the best-ﬁt

values calculated from GraphPadPrism.

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 chroma-

tography (TLC) was performed on precoated silica gel aluminium plates

already after 15 min. The bioreductive process via NTR was somewhat

milder with all other derivatives. As previously noted, 4-nitrophenyl

derivative 3a was the less sensitive to reduction. Moreover, in the N-1

series of prodrugs 15a-c nearly no bioreduction appended, with 75 to

(

60 F254, 0.2 mm thick, Macherey or 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 ninhy-

drine ethanolic solution (0.5%) or a 4-(4′-nitrobenzyl)pyridine (NBP)/

potassium hydroxide dyeing reagent used for the revelation of alky-

lating agents (the chromatography plate was immersed in the NBP so-

lution (2.5% in acetone), heated for few min and then immersed in

potassium hydroxide solution (10% in methanol)). Column chromato-

graphy was performed on silica gel 60A normal phase, 35–70 μm

5% of the prodrugs being unchanged after 2 h of reaction. Prodrugs 25

and 26a-c of the C-4 series, revealed to be better substrates of NTR with

0%, 70%, 50% and 80% respectively of the native molecule at 2 h.

These results highlighted that N,N,N-trimethylpropylamimium tethered

to the N-1 or C-4 position of the imidazole seemed to hamper the en-

zymatic reduction of the prodrug. Like ICF05016, compounds 3b ap-

peared as the best candidate for hypoxia-activated prodrugs according

to this bioreductive assay.

(

Merck or SDS or Carlo Erba). Uncorrected melting points (mp) were

measured on an electrothermal capillary Digital Melting Point

Apparatus (IA9100, Bibby Scientiﬁc, Roissy, France). Infrared spectra

−1

.2.3. Cytotoxicity evaluation in normoxic versus hypoxic conditions

(IR) were recorded in the range 4000–600 cm

on a IS10 spectro-

The nine prodrugs were evaluated for their in vitro cytotoxicity

photometer with attenuated total reﬂectance (ATR) accessory Nicolet

under normoxic and hypoxic (< 0.3% O

) conditions using human

(Fisher Scientiﬁc). Nuclear magnetic resonance spectra ( H NMR and

myxoïd extraskeletal chondrosarcoma cell line (HEMC-SS). The IC50

values (i.e., Growth Inhibiting Concentration 50: IC50) of the tested

compounds were determined and listed in Table 1, in comparison with

ICF05016 as a positive control. For all newly synthesized derivatives

except benzyl compound 3a, the IC50 values ranged between 0.9 and

C NMR) were performed on a Bruker AM 200 spectrometer (200 MHz

1 13

for H, 50 MHz for C), a Bruker Avance DPX300 spectrometer

(400 MHz for H) or a Bruker DRX 500 spectrometer (500 MHz for H,

125 MHz for

C) (Bruker Biospin SAS, Wissembourg, France).

Chemical shift values (δ) are quoted in parts per million (ppm) and

2.9 µM whatever the oxygenation conditions. The higher selectivity

calibrated to the deuterated solvent reference peak for H and

ratio between normoxia and hypoxia (HCR) was observed for com-

pound 15c (HCR = 3.6), respectively to a value of 6.8 for ICF05016.

All tested compounds featured moderate selectivity toward hypoxic

cells, likely not suﬃcient for application as hypoxia-activated prodrugs.

The limited HCR of these molecules compared to ICF05016 or evo-

fosfamide (HCR = 23) [36] might be attributed either to partial trigger

cleavage under the in vitro hypoxic assay conditions, leading to low

concentration of the cytotoxic mustard, or conversion to less cytotoxic

derivatives, which may cause decreased hypoxia cytotoxicity. Com-

pounds may have be substrates of other enzymes besides oxygen-sen-

sitive reductases even in normoxic conditions, which may cause in-

creased normoxia cytotoxicity. Although bioreductive assay highlighted

compound 3b as suitable for hypoxia-activated prodrugs, this was not

conﬁrmed by cytotoxicity values obtained which were equivalent in

normoxic and hypoxic conditions.

spectra. P NMR 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 triplet, multiplet, broad

singlet respectively, are used. When necessary, chemical shifts assign-

ments in H and C spectra were supported by two dimensional NMR

experiments ( H- H COSY and H- C HSQC). Compounds were ana-

lyzed by High-Resolution Mass Spectrometry in positive mode (HRMS,

Waters® Micromass® Q-Tof micro™ Mass Spectrometer, UCA-Partner,

Clermont Auvergne University, Clermont-Ferrand, France or MS,

Bruker® Esquire, Wissembourg, France). Preparative reverse phase high

performance liquid chromatography was performed on

In conclusion, a series of seven phosphoramide-based HAP were

prepared and in vitro assays demonstrated almost similar cytotoxicities

whatever the oxygen conditions used and no eﬃcient enzymatic clea-

vage in the presence of oxygen-insensitive nitroreductase, except for the

furane compound 3b. These results highlighted that N,N,N-trimethyl-

propylamimium tethered to the N-1 or C-4 positions of the imidazole

seemed to hamper eﬃcient reductive enzymatic activation, essential for

the drug release to hypoxic tumor cells. Moreover, in this series no

correlation between sensitivity to enzymatic reduction and hypoxic

selectivity against H-EMC-SS cells was obtained, as namely observed

CombiﬂashEZprep (Teledyne ISCO). The puriﬁcation of QA-derivatives

was carried out on a C18 column (Teledyne, Redisep Prep C18, 100 Å,

5 µm, 200 × 250 mm, ﬂow rate = 15 mL/min, eluent mixture: H

MeCN (v/v), λ = 254 and 323 nm).

Abbreviations: ACN, acetonitrile; DCM, dichloromethane; NBP, 4-

(4′-nitrobenzyl)pyridine; NBS, N-bromosuccinimide; TBAF, tetra-

butylammonium ﬂuoride; TEA, triethylamine; THF, tetrahydrofuran; rt,

room temperature.

4-nitrobenzyl

N,N-bis(2-chloroethyl)-N′-[3-(dimethylamino)propyl]

phosphorodiamidate (2a). To

solution of 4-nitrobenzylic acid

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

(

1 g, 6.53 mmol) in anhydrous THF (10 mL) was added dropwise li-

53.75 (CH

(CH

)

), 50.16 (d, 2C, JC-P = 4.7 Hz, N(CH

Cl) ),

thium bis(trimethylsilyl)amide (1 M in THF, 7.18 mL, 7.18 mmol) at

43.16 (d, 2C, JC-P = 1.7 Hz, N(CH

Cl)

), 38.79 (CH NHP), 26.35

−

78 °C under an inert atmosphere. The reaction mixture was stirred

(d, JC-P = 4.7 Hz, CH

); P NMR (202 MHz, CD

OD) δ 18.10;

around 5 min at −78 °C, and a solution of bis(2-chloroethyl)phos-

HRMS (ESI) m/z 455.24 [M] (calculated for [C17

H30Cl

O P]

phoramidic dichloride (1) (1.84 g, 7.12 mmol) in anhydrous THF

455.14).

(

20 mL) previously cooled at −78 °C, was added all at once at the same

3-({[bis(2(chloroethyl)amino][(5-nitrofuran-2-yl)methoxy]phos-

temperature (T

0 min, and a solution of 3-(N,N-dimethylamino)propylamine (2 eq.)

). The reaction mixture was stirred at −78 °C for

phoryl}amino)-N,N,N-trimethylpropane-1-aminium iodide (3b) was pre-

pared from amine 2b (115 mg, 0.267 mmol) as described for the pre-

paration of 3a (reaction time: 12 h) to aﬀord compound analytically

pure, as a yellow hygroscopic solid (124 mg, 0.216 mmol). Yield 81%;

was added. The reaction was completed after an additional 40 min.

These reaction times were determined by P NMR monitoring for each

compound. The reaction was stopped by the addition of water (40 mL)

and the reaction mixture was slowly warmed to rt. The aqueous layer

was extracted three times with ethyl acetate (3 × 40 mL). The organic

layers were combined, dried over magnesium sulfate, ﬁltrated and

evaporated under reduced pressure. The crude product was puriﬁed by

silica gel column chromatography (EtOAc/EtOH, 50/50, v/v + 2%

−1

IR (ATR) ν cm

3444, 1500, 1353, 1242, 1218, 1020; H NMR

(200 MHz, CD

OD) δ 7.46 (d, 2H, J = 3.7 Hz, CHAr), 6.85 (d, 2H,

J = 3.7 Hz, CHAr), 5.09 (d, 2H, JH-P = 8.9 Hz, OCH

), 3.65–3.72 (m,

4H, NCH

Cl), 3.35–3.53 (m, 6H, NCH

Cl, CH

), 3.18 (s, 9H,

(CH

)

), 3.04 (td, 2H,

H,H = 6.3 Hz,

H-P = 12.5 Hz,

NHP), 2.03–2.10 (m, 2H, CH

);

C NMR (50 MHz,

OH) to aﬀord compound 2a (1.64 g, 3.72 mmol) as a yellow-orange

OD) δ 154,70 (d, J = 7.2 Hz, CArCH

), 153.75 (CArNO

), 114.38,

oil. Yield 57%; Rf 0.35 (SiO

, EtOAc/EtOH, 50/50, v/v, + 12%

113.31 (CHAr), 65.85, 65.82, 65.80 (CH

(CH

)

), 60.14 (d,

C-

−

OH); IR (ATR) ν cm

1522, 1347, 1220, 1046; H NMR

= 4.6 Hz, OCH

), 53.85, 53.82, 53.78 (N (CH

) ), 50.11 (d,

(

500 MHz, CD

OD) δ 8.26 (d, 2H, J = 7.7 Hz, CHAr), 7.67 (d, 2H,

= 4.6 Hz, NCH

Cl), 43.07 (d, JC-P = 1.4 Hz, NCH

Cl), 38.82

J = 7.7 Hz, CHAr), 5.12 (d, 2H, JH-P = 7.5 Hz, OCH

), 3.63–3.70 (m,

(CH

NHP), 26.31 (d, J C-P = 4.5 Hz, CH

); P NMR (202 MHz,

H, NCH

Cl), 3.35–3.50 (m, 4H, NCH

Cl), 2.95 (td, 2H,

OD)

17,97; MS (ESI) m/z 445.21 [M]

(calculated for

H,H = 6.9 Hz, JH-P = 10.8 Hz, CH

NHP), 2.37–2,44 (m, 2H, CH

N),

[C15

28Cl

P] 445.12).

.26 (s, 6H, CH

), 1.71 (qt, 2H, J = 7.3 Hz, CH

); C NMR

Ethyl N-[3-(N′,N′-dimethylamino)propyl]glycinate (6). To a solution

at 0 °C of 3-(N,N-dimethylamino)propylamine (60 mL, 477 mmol) and

potassium carbonate (54.5 g, 394 mmol) in toluene (260 mL) was

added dropwise a solution of ethyl bromoacetate (26 mL, 234 mmol) in

toluene (260 mL) for 3 h 30. The resulting solution was allowed to

stirred at rt for 18 h. The reaction mixture was ﬁltrated and the solid

was washed with toluene (3 × 200 mL). The ﬁltrate was evaporated

under reduced pressure to aﬀord a slightly yellow liquid. The residue

was puriﬁed by silica gel column chromatography (eluent: EtOAc/

EtOH, 80/20, v/v + 5% TEA) to yield the secondary amine 6 (23.9 g,

(

50 MHz, DMSO‑d

) δ 145.63 (d,

C-P = 7.3 Hz, CArCH

), 129.21,

24.70 (4C, CHAr), 67.06 (d, JC-P = 4.4 Hz, OCH

), 57.39 (CH

NH ),

0.23 (d, JC-P = 4.6 Hz, NCH

Cl), 44.45 (2C, N(CH

) ), 43.10 (d,

C-P = 1.4 Hz, NCH

Cl), 39.50 (CH

OD) δ 18.30.

N,N-bis(2-chloroethyl)-N′-[3-dimethyla-

NHP), 29.10 (d, J = 5.2 Hz,

); P NMR (202 MHz, CD

(

5-nitrofuran-2-yl)methyl

mino)propyl]phosphorodiamidate (2b) was prepared from alcohol 5-

nitro-2-hydroxymethylfurane (4) (526 mg, 3.68 mmol) as described for

the preparation of 2a (after addition of 3-(N,N-dimethylamino)propy-

lamine, the reaction was stirred for an additional 2 h). The crude pro-

duct was puriﬁed by silica gel column chromatography (EtOAc/EtOH,

127 mmol). Yield 54%; Rf 0.71 (SiO

, EtOAc/EtOH, 80/20, v/v + 5%

−

TEA); IR (ATR) ν cm 3336, 1737, 1191; H NMR (200 MHz, CDCl

)

0/50, v/v with NH

OH ranging from 0 to 5%) to aﬀord compound 2b

δ 4.15 (q, 2H, J = 7.1 Hz, OCH

), 3.36 (s, 2H, CH

CO), 2.62 (t, 2H,

(

720 mg, 1.67 mmol) as a yellow-orange oil. Yield 48%; Rf 0.35 (SiO

J = 7.1 Hz, NHCH

), 2.30 (t, 2H, J = 7.2 Hz, (CH

)

NCH ), 2.19

−

EtOAc/EtOH, 50/50, v/v, + 5% NH

OH); IR (ATR) ν cm

1504,

(s, 6H, N(CH

CH CH CH

CDCl ) δ 172.55 (CO

(NHCH CO), 48.11 (NHCH

(CH CH CH ), 14.29 (CH CH

)

), 1.98 (s, 1H, NH), 1.64 (qt, J = 7.1 Hz, 2H,

352, 1241, 1034; H NMR (500 MHz, DMSO‑d

) δ 7.69 (d, 1H,

), 1.24 (t, 3H, J = 7.1 Hz, CH

), 60.74 (OCH

); C NMR (50 MHz,

J = 3.7 Hz, CHAr), 6.92 (d, 1H, J = 3.7 Hz, CHAr), 4.96 (d, 2H, JH-

), 57.90 (CH

N(CH

) ), 51.07

= 8.7 Hz, OCH

), 4.88 (td, 1H,

H,H = 6.7 Hz,

H-P = 11.7 Hz,

), 45.55 (2C, N(CH

) ), 28.06

NHP), 3.61–3.69 (m, 4H, NCH

Cl), 3.20–3.36 (m, 4H,

NCH

Cl), 2,75 (qd, 2H, JH-P = 11.0 Hz, JH,H = 6.7 Hz, CH

NHP),

N-formyl-N-[3-(N′,N′-dimethylamino)propyl]glycinate (7). To a stirred

solution of the diamino ester 6 (24.2 g, 129 mmol) in ethanol (86 mL)

were added potassium carbonate (17.7 g, 128 mmol) and ethyl formate

(77 mL, 957 mmol). The reaction mixture was stirred for 25 h at rt. The

resulting mixture was ﬁltrated and the solid was washed with ethanol

(3 × 50 mL). The ﬁltrate was evaporated under reduced pressure, be-

fore addition of water (100 mL) and extraction with ethyl acetate

(3 × 200 mL). The aqueous layer was saturated with sodium chloride

and extracted with ethyl acetate (3 × 200 mL). The organic layers were

combined, dried over magnesium sulfate, ﬁltrated and evaporated

under reduced pressure to yield the formylated product 7 (25.7 g,

119 mmol) analytically pure as a yellowish liquid. Yield 90%; Rf 0.85

.24 (t, 2H, J = 6.8 Hz, CH

N), 2,12 (s, 6H, N(CH

)

), 1,53 (qt, 2H,

) δ 154.26 (d,

J = 6.9 Hz, CH

); C NMR (50 MHz, DMSO‑d

C,P = 8.1 Hz, CArCH

), 151.58 (CArNO

), 113.69, 113,59 (CHAr),

8.14 (d,

C-P = 3.8 Hz, OCH

), 54.30 (CH

NH ), 48.34 (d,

C-

= 4.3 Hz, NCH

Cl), 42.35 (d, JC-P = 1.5 Hz, NCH

Cl), 41.99

(

2C, N(CH

)

), 37.37 (CH

NHP), 25,94 (d,

C-P

= 5,2 Hz,

); P NMR (202 MHz, CD

OD) δ 17.79; MS (ESI) m/z

31.18 [M+H] (calculated for [C14

25Cl

O P] 431,10).

-({(4-nitrobenzyloxy)[bis(2-chloroethyl)amino]phosphoryl}amino)-

N,N,N-trimethylpropane-1-aminium iodide (3a). To a solution of amine 2a

(

1.34 g, 3.03 mmol) in anhydrous THF (45 mL) under an inert atmo-

−

sphere, was added methyl iodide (1 mL, 16.1 mmol). Stirring was

maintained at rt in a sealed ﬂask for 3 h. After evaporation to dryness

under reduced pressure, compound 3a was obtained analytically pure,

as a yellow hygroscopic solid (1.76 g, 3.02 mmol). Yield 99%; IR (ATR)

(Al

, EtOAc/EtOH, 80/20, v/v + 0.1% NH

OH); IR (ATR) ν cm

) δ (two rotamers) 8.14

), 4.04 and 3.98 (s, 2H,

CH ), 2.29–2.16

), 1.27 and 1.26

) δ (two ro-

1745, 1668, 1196; H NMR (200 MHz, CDCl

and 8.03 (s, 1H, CHO), 4.27–4.10 (m, 2H, OCH

NCH

CO), 3.27–3.08 (t, 2H, J = 6.9 Hz, NCHOCH

−

ν cm

1520, 1348, 1221, 1046; H NMR (500 MHz, CD

OD) δ 8.27

(m, 8H, (CH

N(CH

)

), 1.77–1.56 (m, 2H, CH

(

d, 2H, J = 8.7 Hz, CHAr), 7.68 (d, 2H, J = 8.6 Hz, CHAr), 5.17 (d,

(t, 3H, J = 7.1 Hz, CH

); C NMR (50 MHz, CDCl

H, JH-P = 7.4 Hz, OCH

), 3.68–3.72 (m, 4H, NCH

Cl), 3.41–3.50

tamers) 168.99, 168.20 (CO

56.27, 55.33 (CH N(CH

(NCHOCH CH ), 44.90 (2C, N(CH

), 163.00 (CHO), 61.20, 60.87 (OCH

(

m, 6H, NCH

H,H = 6.4 Hz,

CH CH

d, JC,P = 7.7 Hz, CArCH

= 4.7 Hz, OCH

Cl, CH

), 3.17 (s, 9H, CH

(CH

)

), 3.05 (td, 2H,

)

), 48.79, 43.73 (NCH CO), 45.55, 41.70

H-P = 12.0 Hz, CH

NHP), 1.97–2.07 (m, 2H,

)

), 25.65, 24.81 (CH

CH ),

); C NMR (126 MHz, CD

OD) δ 149.14 (CArNO

), 145.55

13.73 (CH

); MS (ESI) m/z 217.14 [M+H] (calculated for

(

), 129.28, 124.71 (4C, CHAr), 67.16 (d, JC-

[C10H

N O

]

217.15)

), 65.83, 65.80, 65.78 (CH

(CH

)

), 53.81, 53.78,

Ethyl 2-amino-1-N-(3-(N′,N′-dimethylamino)propyl)-1H-imidazole-5-

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

carboxylate (8). To a solution of N-formyl compound 7 (19.2 g,

not exceed 10 °C. The temperature was allowed to rise gradually to rt

and the reaction mixture was stirred for 17 h. The reaction mixture was

cooled at 0 °C and a saturated aqueous solution of ammonium chloride

(40 mL) was added. After the evaporation of the THF under reduced

pressure, the pH of the resulting solution was adjusted to 9–10 by the

addition of a saturated aqueous solution of sodium carbonate (40 mL)

and saturated with brine. The solution was then extracted with ethyl

acetate (3 × 200 mL) maintaining the pH of the solution at 10. The

combined organic layers were dried over magnesium sulfate, ﬁltered

and evaporated under reduced pressure. The residue was diluted in

ethanol (180 mL) and the resulting solution was reﬂuxed for 4 h. After

cooling down to rt, ethanol was evaporated under reduced pressure and

the residue was puriﬁed by silica gel column chromatography (eluent:

DCM/methanol, 95/5, v/v + 5% TEA) to yield the alcohol 10 (1.49,

8.8 mmol) in an equal mixture of ethyl formate and THF (90 mL)

containing cyclohexane (5 mL), was added slowly NaH (60% wt in

mineral oil, 5.33 g, 133 mmol) at rt (the reaction was carefully acti-

vated by heat until hydrogen gas evolution and color change were

observed. At this point, the reaction mixture was cooled down with a

water/ice bath). After the addition was completed and hydrogen gas

release ceased, the reaction mixture was allowed to stirred at rt for 3 h.

The reaction mixture was concentrated under reduced pressure. The

obtained solid was suspended in a solution of EtOH (145 mL) con-

taining concentrated aq. HCl 37% (29 mL, 353 mmol) and reﬂuxed for

h. The hot reaction mixture was ﬁltered and the resulting white solid

was washed with boiling EtOH (3 × 50 mL). The ﬁltrate was con-

centrated under vacuum and diluted with a mixture of EtOH/water

(

170 mL, 70/30, v/v). The pH of the solution was adjusted to 3, using

6.53 mmol) as an orange oil. Yield 49%; Rf 0.18 (SiO

, DCM/MeOH,

−

an aqueous 5 M solution of NaOH and cyanamide (7.46 g, 177 mmol)

was added. The resulting mixture was reﬂuxed for 1 h 30, then cooled

to rt and concentrated under reduced pressure to approximately 1/8 of

the initial volume. After cooling at 0 °C, the pH of the remaining so-

lution was adjusted to 9–10 with a saturated aqueous solution of po-

tassium carbonate. The mixture was extracted with ethyl acetate

95/5, v/v + 5% TEA); IR (ATR) ν cm

3239, 1481, 1335, 1031;

NMR (200 MHz, CDCl

) δ 7.03 (s, 1H, HAr), 5.29 (br.s, 1H, CH

OH),

4.58 (s, 2H, CH

OH), 4.46 (t, 2H, J = 6.8 Hz, NArCH

), 2.32 (t, 2H,

J = 6.3 Hz, CH

N(CH

CH CH

), 139.33 (CArCH

)

), 2.19 (s, 6H, N(CH

)

), 2.09 (qt, 2H,

OD) δ 146.72

N(CH ), 54.58

CH CH );

J = 6.5 Hz, CH

(CArNO

(CH

); C NMR (126 MHz, CD

), 127.92 (CHAr), 57.19 (CH

3 2

)

(

3 × 100 mL). The organic layers was combined, dried over magnesium

OH), 46.55 (NArCH

), 45.18 (2C, N(CH

)

), 28.57 (CH

sulfate, ﬁltrated and evaporated under reduced pressure. The crude

product was puriﬁed by silica gel column chromatography (eluent:

EtOAc/EtOH, 80/20, v/v with 2% TEA) to give the imidazole 8 (4.52 g,

HRMS (ESI) m/z 229.1293 [M+H] (calculated for [C

O ]

229.1295).

3-[(5-hydroxymethyl)-2-nitro-1H-imidazo-1-N′-yl]-N,N-dimethyl-

8.8 mmol) as a brown solid. Yield 21%; Rf 0.36 (SiO

, EtOAc/EtOH,

propyl-1-amine borane complex (10-BH

12.3 mmol) was dissolved in a mixture of THF/ MeOH (30 mL, 80/20,

v/v) cooled at 0 °C. A suspension of NaBH (1.36 g, 36.9 mmol) and

). The ester 9 (3.35 g,

−

0/20, v/v + 2% TEA); mp 82–83 °C; IR (ATR) ν cm

3317, 3194,

667, 1539, 1182; H NMR (500 MHz, CD

OD) δ 7.35 (s, 1H, HAr),

.24 (q, 2H, J = 7.1 Hz, OCH

), 4.11 (t, 2H, J = 6.9 Hz, NArCH

LiBr (3.18 g, 36.8 mmol) in anhydrous THF (70 mL) was cooled at 0 °C,

water (20 mL) was added before dropwise addition to the ni-

troimidazole solution, at such a rate that the internal temperature did

not exceed 10 °C. The reaction mixture was stirred at rt for 15 h. After

cooling down to 0 °C, a saturated aqueous solution of ammonium

chloride (15 mL) was added to the reaction mixture. The pH of the

resulting solution was adjusted to 9–10 by the addition of a saturated

aqueous solution of sodium carbonate and saturated with sodium

chloride. The solution was then extracted with ethyl acetate

(6 × 150 mL) maintaining the pH of the solution at 10. The combined

organic layers were dried over magnesium sulfate, ﬁltered and evapo-

rated under reduced pressure. The residue was puriﬁed by silica gel

column chromatography (eluent: DCM/methanol, 97/3, v/v) to yield

the alcohol 10 (209 mg, 0.916 mmol, 7%) and its borane complex 10-

.31 (t, 2H, J = 6.9 Hz, CH

N(CH

)

), 2.26 (s, 6H, N(CH

)

), 1.93 (qt,

H, J = 6.9 Hz, CH

), 1.31 (t, 3H, J = 7.1 Hz, CH

);

NMR (126 MHz, CD

OD) δ 161.71 (CO

), 155.38 (CArNH

), 136.40

(

CHAr), 118.84 (CArCO), 60.85 (OCH

), 56.37 (CH

N(CH

)

), 45.08 (2C,

N(CH

)

), 42.11 (NArCH

), 28.22 (CH

), 14.69 (CH

CH );

O ]

HRMS (ESI) m/z 241.1658 [M+H] (calculated for [C11

41.1659)

Ethyl 1-N-[3-(N′,N′-dimethylamino)prop-1-enyl]-2-nitro-1H-imidazole-

-carboxylate (9). To a solution of sodium nitrite (13.0 g, 188 mmol) in

water (17 mL) cooled around 0 °C, was added dropwise a solution of the

amino ester 8 (4.52 g, 18.8 mmol) in glacial acetic acid (33 mL). The

reaction mixture was allowed to stir at rt for 2 h. The solution was

cooled at 0 °C and a saturated aqueous solution of sodium carbonate

(

90 mL) was added dropwise until pH 9–10. The reaction mixture was

(1.35 g, 5.58 mmol, 45%) as main product. Rf 0.75 (SiO , DCM/

−1

extracted with ethyl acetate (3 × 200 mL), saturated with sodium

chloride and extracted with ethyl acetate (2 × 200 mL). The combined

organic layers were dried over magnesium sulfate, ﬁltered and evapo-

rated under reduced pressure. The residue was puriﬁed by silica gel

column chromatography (eluent: EtOAc/EtOH, 90/10, v/v + 1% TEA).

The product 9 was obtained as a yellow oil (3.63 g, 13.4 mmol). Yield

MeOH, 90/10, v/v + 1% TEA); mp 104–105 °C; IR (ATR) ν cm 3216,

2386, 2363, 2314, 2271, 1489, 1333, 1166, 1036; H NMR (500 MHz,

CDCl

) δ 7.11 (s, 1H, HAr), 4.76 (m, 2H, CH

OH), 4.57–4.39 (m, 2H,

ArCH

), 2.94–2.80 (m, 2H, CH

(CH

)

), 2.61 (s, 6H, N (CH

3 2

) ),

2.44–2.24 (m, 2H, CH

); C NMR (126 MHz, CDCl

) δ 145.70

(CArNO

54.65 (CH

), 136.53 (CArCH

), 128.17 (CHAr), 61.58 (CH

(CH

) ),

1%; Rf 0.55 (SiO

, EtOAc/EtOH, 90/10, v/v + 1% TEA); IR (ATR) ν

OH), 52.11 (2C,

(CH

)

), 45.69 (NArCH ), 25.78

−

cm 1722, 1473, 1334, 1283, 1129; H NMR (500 MHz, DMSO‑d

) δ

(CH

O ]

); HRMS (ESI) m/z 243.1616 [M+H] (calculated for

243.1623), 265.1434 [M+Na]⁺(calculated for

.78 (s, 1H, HAr), 4.72–4.66 (m, 2H, NArCH

), 4.35 (q, J = 7.1 Hz, 2H,

20BN

OCH

), 2.24 (t, J = 6.5 Hz, 2H, CH

N(CH

)

), 2.06 (s, 6H, N(CH

)

19BN

NaO

]

265.1442).

.96–1.89 (m, 2H, CH

), 1.32 (t, J = 7.1 Hz, 3H, CH

), 147.37 (CArNO

N(CH

), 13.91 (CH

);

4-{5-[({amino[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-2-

NMR (126 MHz, DMSO‑d

) δ 158.48 (CO

), 133.91

nitro-1H-imidazol-1-N′-yl}-N,N-dimethylpropan-1-amine borane complex

(

CHAr), 125.91 (CArCO), 61.37 (OCH

), 56.18 (CH

)

), 46.42

CH );

(14a-BH

) was prepared from alcohol 10-BH (165 mg, 0.682 mmol) as

ArCH

), 44.69 (2C, N(CH

)

), 27.16 (CH

described for the preparation of 2a with some diﬀerences: the coupling

reaction with bis(2-chloroethyl)phosphoramidic acid dichloride (1) was

performed for 1 h before ammonia gas was bubbled through the reac-

tion for 2 min. The reaction was directly stopped by addition of water

(10 mL). The crude product was puriﬁed by silica gel column chro-

matography (eluent: DCM/ethanol, 97/3, v/v) to yield compound 14a-

HRMS (ESI) m/z 271.1400 [M+H] (calculated for [C11

O ]

71.1401).

1-{1-N-[3-(N′,N′-dimethylamino)propyl]-2-nitro-1H-imidazo-5-yl}me-

thanol (10). The ester 9 (3.63 g, 13.4 mmol) was dissolved in a mixture

of anhydrous THF/ MeOH (32 mL, 80/20, v/v) cooled at 0 °C. A sus-

pension of NaBH

(1.52 g, 40.2 mmol) in THF (75 mL) and a solution of

(282 mg, 0.634 mmol) as a beige thick oil. Yield 93%; Rf 0.20

−1

LiBr (3.51 g, 40.6 mmol) in water (20 mL) were cooled at 0 °C, in-

troduced in the same dropping funnel and added dropwise to the ni-

troimidazole solution, at such a rate that the internal temperature did

(SiO

, DCM/EtOH, 97/3, v/v); IR (ATR) ν cm

3241, 2380, 2318,

2273, 1484, 1338, 1224, 1168, 1012, 971; H NMR (500 MHz, CDCl

)

δ 7.25 (s, 1H, HAr), 5.15 (dd, 1H, ²

H-H = 13.2 Hz, ³

JH-P = 7.0 Hz,

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

OCH′), 5.02 (dd, 1H,

H-H = 13.2 Hz,

H-P = 5.9 Hz, OCH”), 4.51

(m, 4H, NHCH

Cl), 2.88–2.81 (m, 2H, CH

(CH

) ), 2.62 (s, 6H,

(

ddd, 1H, J = 13.6 Hz, J = 10.8 Hz, J = 5.4 Hz, NArCH′), 4.41 (ddd,

(CH

)

), 2.42–2.32 (m, 2H, CH

), 1.93–1.32 (m, 3H, BH

);

H, J = 13.6 Hz, J = 10.7 Hz, J = 5.5 Hz, NArCH″), 3.70–3.65 (m,

C NMR (126 MHz, CDCl

) δ 145.90 (CArNO

), 132.79 (d,

C-

H, N(CH

Cl)

), 3.58–3.44 (m, 4H, N(CH

Cl)

), 3.11 (br.s, 2H,

= 8.6 Hz, CArCH

), 129.76 (CHAr), 61.59 (CH

(CH

)

), 56.05 (d,

), 2.91 (ddd, 1H, J = J = 11.8 Hz, J = 5.0 Hz, CH′N (CH

)

C-P = 3.8 Hz, OCH

= 5.4 Hz, NHCH CH

CH CH

), 52.56 (2C, N (CH

)

), 45.78 (d, 2C,

C-

.81 (ddd, 1H, J = J = 11.8 Hz, J = 5.1 Hz, CH″N (CH

)

), 2.65

Cl), 45.55 (NArCH

), 43.17 (2C, NHCH

CH Cl),

and 2.61 (s, 3H, N (CH

)

), 2.48–2.38 (m, 1H, CH

CH′CH

), 2.38–2.27

26.14 (CH

); P NMR (202 MHz, CDCl

) δ 15,04; HRMS (ESI)

(

m, 1H, CH

CH″CH

), 1.97–1.30 (m, 3H, BH

); C NMR (126 MHz,

m/z 443.1295 [M−H]

(calculated for [C13

27BCl

O P]

[M+Na]⁺

CDCl

) δ 145.86 (CArNO

), 132.74 (d, JC-P = 9.1 Hz, CArCH

), 129.78

), 53.18,

443.1296),

467.1270

(calculated

for

(

CHAr), 61.50 (CH

(CH

)

), 55.97 (d, JC-P = 3.6 Hz, OCH

[C13

28BCl

2 6

NaO

P] 467.1272).

1.86 (2C, N (CH

5.46 (NArCH

)

), 48.94 (d, 2C, JC-P = 4.9 Hz, N(CH

Cl)

3-{5-[({amino[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-2-

), 42.70 (2C, N(CH

Cl)

), 26.12 (CH

);

nitro-1H-imidazol-1-N′-yl}-N,N,N-trimethylpropan-1-aminium

iodide

NMR (202 MHz, CDCl

) δ 16.47; HRMS (ESI) m/z 443.1296 [M−H]

(15a). To a stirred solution of amine borane complex 14a-BH

(

calculated for [C13

27BCl

P] 443.1296), 467.1272 [M+Na]

(39.9 mg, 89.6 µmol) in anhydrous THF (3 mL) was added DABCO

(50.1 mg, 447 µmol). The reaction mixture was heated at 30–40 °C for

1 h. After cooling at 0 °C, methyl iodide (90 µL, 1.45 mmol, 15 eq.) was

added and the reaction mixture was stirred at rt for 2.5 h. Methyl iodide

(30 µL, 480 µmol) was added and the reaction was stirred for an ad-

ditional 1 h. After evaporation under reduced pressure, the residue was

puriﬁed by semi-preparative ﬂash column chromatography and lyo-

philized to give the QA compound 15a (38.4 mg, 67.0 µmol) as a highly

hygroscopic yellow powder. Puriﬁcation conditions: detection at

28BCl

NaO

P] 467.1272).

-{5-[({bis[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-2-nitro-

H-imidazol-1-N′-yl}-N,N-dimethylpropan-1-amine borane complex (14b-

). To a stirred solution of the alcohol 10-BH (150 mg, 0.620 mmol)

in anhydrous THF (4.5 mL) cooled at −78 °C was added dropwise li-

thium bis(trimethylsilyl)amide (1 M in THF, 680 µL, 0.680 mmol)

under an inert atmosphere. After 5 min, a solution of the compound 12

(

306 mg, 0.840 mmol) in anhydrous THF (4 mL) previously cooled at

−

78 °C was added dropwise to the reaction mixture. The kinetic of the

254 nm and 323 nm, eluent: H O/ACN (v/v), 95/5 for 2 min, 95/5 →

reaction was monitored by P NMR. After stirring 1 h at −78 °C, the

reaction mixture was warmed to rt and allowed to stir for 20 h. The

reaction was stopped by addition of water (10 mL) and the reaction

mixture was extracted with ethyl acetate (3 × 10 mL). The organic

layers were combined, dried over magnesium sulfate, ﬁltrated and

evaporated under reduced pressure. The crude product was puriﬁed by

silica gel column chromatography (eluent: DCM/methanol, 98/2, v/v)

70/30 for 6 min, 70/30 for 2 min, 70/30 → 60/40 for 6 min, 60/40 for

3 min, 60/40 → 10/90 for 7 min, 10/90 for 4 min, retention time of

−1

14.2 min; Yield 75%; IR (ATR) ν cm 3216, 1483, 1338, 1220, 1015,

973, 923; H NMR (200 MHz, CD

OD) δ 7.32 (s, 1H, HAr), 5.19 (dd,

1H,

H-H = 13.4 Hz,

H-P = 7.7 Hz, OCH′), 5.11 (dd, 1H,

H-

= 13.4 Hz,

H-P = 7.0 Hz, OCH″), 4.61–4.46 (m, 2H, NArCH ),

3.75–3.67 (m, 4H, N(CH

Cl)

), 3.65–3.39 (m, 6H, N(CH

Cl) ,

to yield phosphorodiamidate 14b-BH

(264 mg; 0.463 mmol) as a

(d,

(CH

)

), 3.20 (s, 9H, CH

(CH

) ), 2.55–2.34 (m, 2H,

colorless thick oil. Yield 70%; Rf 0.28 (SiO

, DCM/MeOH, 98/2, v/v);

C-P

); C NMR (50 MHz, CD

OD) δ 147.08 (CArNO

), 134.88

−

IR (ATR) ν cm

2380, 2319, 2273, 1483, 1339, 1219, 1169, 1004,

8.1 Hz,

ArCH

), 129.97 (CHAr), 64.41, 64.34

64; H NMR (500 MHz, CDCl

) δ 7.28 (s, 1H, HAr), 5.17 (d, 2H, JH-

(CH

2C,

(CH

)

), 57.32 (d, JC-P = 4.2 Hz, OCH

), 54.02, 53.94, 53.87

= 8.1 Hz, OCH

), 4.54–4.47 (m, 2H, NArCH

), 3.71–3.56 (m, 8H, N

(CH

)

), 50.39 (2C, N(CH

Cl)

), 45.26 (NArCH

), 43.18 (d,

(

Cl)

), 3.41 (td, 8H,

H-P = 12.9 Hz,

H-H = 6.5 Hz, N

C-P = 1.2 Hz, N(CH

), 25.24 (CH

); P NMR

(

), 2.87–2.81 (m, 2H, CH

(CH

)

), 2.61 (s, 6H,

(202 MHz, CD

OD) δ 19.45; HRMS (ESI) m/z 445.1282 [M] (calcu-

(CH

)

), 2.34–2.25 (m, 2H, CH

CH CH

), 2.00–1.30 (m, 3H, BH

);

lated for [C14

28Cl

P] 445.1281).

C NMR (126 MHz, CDCl

) δ 145.87 (CArNO

), 132.32 (d,

C-

3-{5-[({bis[bis(2-chloroéthyl)amino]phosphoryl}oxy)méthyl]-2-nitro-

= 7.0 Hz, CArCH

), 130.09 (CHAr), 61.47 (CH

(CH )

), 56.38 (d,

1H-imidazol-1-N′-yl}-N,N,N-triméthylpropan-1-aminium iodide (15b) was

C-P = 3.8 Hz, OCH

), 52.47 (2C, N (CH

)

), 49.31 (d, 4C,

C-

prepared from amine borane complex 14b-BH (128 mg, 0.225 mmol)

4.6 Hz, N(CH

Cl)

), 45.49 (NArCH

), 42.34 (4C,

as described for the preparation of 15a with some diﬀerences: 15 eq. of

methyl iodide initially followed by two times 3.5 eq after 1 h and 1.5 h

and stirring for additional 30 min. QA compound 15b (33.4 mg,

47.8 µmol) was obtained as a highly hygroscopic yellow powder.

Puriﬁcation conditions: detection at 254 nm and 323 nm, eluent:

(

Cl)

), 26.18 (CH

); P NMR (202 MHz, CDCl

) δ

7,14; HRMS (ESI) m/z 593,1088 [M+Na]

(calculated for

[

34BCl

NaO

P] 593,1089).

-{5-[({bis[(2-chloroethyl)amino]phosphoryl}oxy)methyl]-2-nitro-1H-

imidazol-1-N′-yl}-N,N-dimethylpropan-1-amine borane complex (14c-

BH ). To a solution cooled at −78 °C of phosphorous oxychloride

200 µL, 2.15 mmol) in anhydrous DCM (5 mL), was added dropwise a

(500 mg, 2.07 mmol) and TEA

H O/ACN (v/v), 95/5 for 2 min, 95/5 → 70/30 for 6 min, 70/30 for

2 min, 70/30 → 60/40 for 6 min, 60/40 for 3 min, 60/40 → 36/64 for

1.5 min, 36/64 for 1.5 min, 36/64 → 10/90 for 2 min, 10/90 for 6 min,

(

−

solution of the compound 10-BH

retention time of 21.1 min; Yield 21%; IR (ATR) ν cm

1483, 1339,

(

320 µL, 2.30 mmol) in anhydrous DCM (4 mL). After 1 h of stirring at

1230, 1065, 1057, 1027, 971; H NMR (500 MHz, CD

OD) δ 7.38 (s,

−

78 °C, 2-chloroethylamine hydrochloride (501 mg, 4.35 mmol) was

1H, HAr), 5.31 (d, 2H, JH-P = 8.7 Hz, OCH

), 4.59 (t, 2H, J = 7.8 Hz,

added followed by the dropwise addition of TEA (1.3 mL, 9.33 mmol).

The temperature of the reaction mixture was risen gradually from

ArCH

), 3.76–3.67 (m, 8H, N(CH

), 3.52–3.43 (m, 8H, N(CH

), 2.49–2.39 (m, 2H, CH CH CH

Cl)

), 3.65–3.60 (m, 2H,

Cl) ), 3.21 (s, 9H,

); C NMR (126 MHz,

(CH

)

−

78 °C to 5 °C for 18 h. The reaction was stopped by the addition of

(CH

water (30 mL). After layers separation, the organic layer was washed

with a saturated aqueous solution of sodium hydrogenocarbonate

OD) δ 147.09 (CArNO

), 134.31 (d, JC-P = 6.8 Hz, CArCH ), 130.48

(CHAr), 64.30, 64.28, 64.24 (CH

(CH

)

), 57.86 (d, JC-P = 4.0 Hz,

(

30 mL), dried over magnesium sulfate, ﬁltrated and evaporated under

OCH

), 53.92, 53.89, 53.86 (CH

(CH

)

), 50.11 (d, 4C,

C-

reduced pressure to give a pale yellow oil. The residue was puriﬁed by

silica gel column chromatography (eluent: EtOAc/EtOH, 95/5 then 90/

4.6 Hz, N(CH

Cl)

), 45.18 (NArCH

), 43.11 (4C,

(CH

Cl)

), 25.28 (CH

); P NMR (202 MHz, CD

OD) δ

0, v/v) to yield the isophosphorodiamidate 14c-BH

(445 mg,

18.04; HRMS (ESI) m/z 569.1137 [M]

(calculated for

.00 mmol) as a beige thick oil. Yield 48%; Rf 0.15 (SiO

, AcOEt/EtOH,

[C18H

34Cl

P] 569.1128).

−

5/5, v/v); IR (ATR) ν cm

3237, 2381, 2318, 2273, 1484, 1338,

3-{5-[({bis[(2-chloroethyl)amino]phosphoryl}oxy)methyl]-2-nitro-1H-

209, 1168, 1008, 972; H NMR (200 MHz, CDCl

) δ 7.24 (s, 1H, HAr),

imidazol-1-N′-yl}-N,N,N-trimethylpropan-1-aminium iodide (15c) was

.07 (d, 2H,

H-P = 7.0 Hz, OCH

), 4.48–4.42 (m, 2H, NArCH

prepared from amine borane complex 14c-BH (90.0 mg, 0.202 mmol)

.61–3.55 (m, 2H, NHCH

Cl), 3.45–3.34 (m, 2H, NH), 3.34–3.22

as described for the preparation of 15a with some diﬀerences: 15 eq. of

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

methyl iodide was added and the reaction completed after 1.5 h at rt.

QA compound 15c (66.6 mg, 0.116 mmol) was obtained as a highly

hygroscopic yellow powder. Puriﬁcation conditions: detection at

yellowish liquid. Pure (Z) isomer (1.36 g, 3.63 mmol) and (E) isomer

(5.20 g, 13.9 mmol) were obtained as well as a mixture fraction of both

isomers (4.62 g, 12.3 mmol, (Z)/(E) = 17/83). Yield 85%; Rf (Z)

54 nm and 323 nm, eluent: H

O/ACN (v/v), 95/5 for 2 min, 95/5 →

isomer 0.50, (E) isomer 0.33 (SiO

, cyclohexane/AcOEt/MeOH, 90/6/

−

0/30 for 6 min, 70/30 for 2 min, 70/30 → 64/36 for 4 min, 64/36 for

min, 64/36 → 60/40 for 2 min, 60/40 for 2 min, 60/40 → 10/90 for

min, 10/90 for 3 min, retention time of 14.5 min; Yield 57%; IR

4, v/v/v); IR (ATR) ν cm

2954, 2922, 2871, 2581, 1455, 856;

NMR (200 MHz, CDCl

) δ (Z) isomer: 6.55 (td, 1H, Jcis = 12.6 Hz,

J = 6.2 Hz, CH]CHCH

), 6.11–5.90 (td, 1H,

cis = 12.7 Hz,

−

(

ATR) ν cm

3215, 1534, 1483, 1338, 1202, 1186, 1009, 976;

J = 1.2 Hz, CH]CHCH

), 2.90 (dd, 2H, J = 6.2 Hz, J = 1.1 Hz,

NMR (200 MHz, CD

OD) δ 7.31 (s, 1H, HAr), 5.16 (d, 2H,

H-

CH]CHCH

), 2.22 (s, 6H, N(CH

)

), 1.60–1.40 (m, 6H, Sn

CH CH CH ),

); H NMR (200 MHz, CDCl

), 5.97 (td, 1H,

= 7.9 Hz, CH

O), 4.77–4.43 (m, 2H, NArCH

), 3.70–3.53 (m, 6H,

(CH CH CH

)

3, 1.30 (h, 6H, JH-H = 7.3 Hz, Sn(CH

3 3

)

NHCH

Cl, CH

(CH

)

), 3.28–3.14 (m, 13H, NHCH

Cl,

1.01–0.81 (m, 15H, Sn(CH CH

)

(CH

)

), 2.57–2.34 (m, 2H, CH

); C NMR (126 MHz,

δ (E) isomer: 6.11 (d, 1H, Jtrans = 19.0 Hz, CH]CHCH

OD) δ 147.11 (CArNO

), 134.92 (d,

C-P = 7.8 Hz, CArCH

O),

trans = 18.9 Hz, J = 4.9 Hz, CH]CHCH

), 2.97 (d, 2H, J = 4.8 Hz,

29.89 (CHAr), 64.43, 64.40, 64.38 (CH

(CH

)

), 57.73 (d,

C-

CH]CHCH

), 2.23 (s, 6H, N(CH

)

), 1.55–1.15 (m, 12H, Sn

= 4.6 Hz, CArCH

O), 54.00, 53.97, 53.94 (CH

(CH

)

), 46.01 (d,

(CH

)

), 0.98–0.80 (m, 15H, Sn(CH

)

);

C-P = 5.1 Hz, NHCH

Cl), 45.37 (NArCH

), 44.18 (2C,

NMR (126 MHz, CDCl

) δ (Z) isomer: 146.31 (CH]CHCH ), 131.39

NHCH

Cl), 25.28 (CH

); P NMR (202 MHz, CD

OD) δ

(CH]CHCH

), 65.13 (CH]CHCH

), 45.47 (2C, N(CH

) ), 29.34 (3C,

7.12; HRMS (ESI) m/z 455.1281 [M]

(calculated for

C-Sn

20.4 Hz, Sn(CH

)

), 27.47 (3C, Sn

[

(

28Cl

P] 455.1281).

(CH

)

), 13.82 (3C, Sn(CH

)

), 10.61 (3C, JC-

4-bromo-1-N-methyl-2-nitro-1H-imidazo-5-yl)methanol (16). To

119Sn = 341.8 Hz,

C-117Sn = 326.5 Hz, Sn(CH

)

);

stirred solution of the imidazole 5 (6.01 g, 38.2 mmol) in anhydrous

DMF (45 mL) was added N-bromosuccinimide (7.56 g, 42.5 mmol). The

reaction mixture was stirred at 60 °C for 2 h 30. The resulting mixture

was cooled to rt and the reaction was stopped by addition of water

NMR (126 MHz, CDCl

) δ (E) isomer: 146.03 (CH]CHCH

), 131.76

( JC-119Sn = 378.8 Hz, JC-117Sn = 361.9 Hz, CH]CHCH

), 66.58 ( JC-

119Sn = 66.2 Hz,

C-117Sn = 64.2 Hz, CH]CHCH

), 45.23 (2C, N

(CH

)

), 29.24 (3C, JC-Sn = 20.6 Hz, Sn(CH

) ), 27.39 (3C,

(

250 mL). The yellow/green solid was ﬁltrated and washed with diethyl

C-119Sn = 54.3 Hz, JC-117Sn = 52.7 Hz, Sn(CH

)

), 13.83

ether (3 × 30 mL). The solid was dried at 30 °C under vacuum to yield

the brominated compound 16 analytically pure (5.84 g, 24.7 mmol).

The organic layer of the ﬁltrate was separated and the aqueous layer

was saturated using sodium chloride and extracted with ethyl acetate

(3C, Sn(CH

)

), 9.57 (3C,

C-119Sn = 343.3 Hz,

C-

117Sn = 328.1 Hz, Sn(CH

) ); MS (ESI) m/z 376.21 [M

+H] (calculated for [C17

H38NSn] 376.20).

(E)-3-(1-N′-methyl-2-nitro-5-{[(triisopropylsilyl)oxy]methyl}-1H-imi-

dazol-4-yl)-N,N-dimethylprop-2-en-1-amine (19). To a stirred solution of

bromo protected alcohol 17 (2.27 g, 5.79 mmol) in anhydrous DMF

(22 mL) were added tetrakistriphenylphosphine palladium (0) (1.05 g,

0.909 mmol) and N,N-dimethylamino-3-(tributylstannyl)-prop-2-en-1-

amine (18) (4.34 g, 11.6 mmol) as mixture of isomers (Z)/(E) (40/60

(

2 × 200 mL). The organic layers were combined, washed with brine

500 mL) and water (500 mL) dried over magnesium sulfate, ﬁltrated

and evaporated under reduced pressure. The resulting yellow/green

solid was dried at 30 °C under vacuum to yield a second fraction of

product 16 (1.54 g, 6.52 mmol) analytically pure by H and C NMR

analysis. Yield 82%; mp 163–164 °C; Rf 0.82 (SiO

, AcOEt/cyclo-

estimated by H NMR). The reaction mixture was stirred at 105 °C for

−

hexane, 80/20, v/v); IR (ATR) ν cm

NMR (500 MHz, DMSO‑d ) δ 4.51 (s, 2H, OCH

C NMR (126 MHz, DMSO‑d ) δ 144.31 (CArNO

13.92 (CArBr); 52.02 (OCH ); 35.06 (NArCH ).

3321, 1488, 1364, 1015;

90 min. After cooling to rt, the solution was diluted with ethyl acetate

(20 mL), ﬁltrated through a pad of Celite 545® and washed with ethyl

acetate (4 × 25 mL). Water (400 mL) was added to the ﬁltrate and the

aqueous layer obtained after decantation was extracted with ethyl

acetate (3 × 300 mL). The organic layers were combined, washed with

water (400 mL) and brine (400 mL), dried over magnesium sulfate,

ﬁltrated and evaporated under reduced pressure to yield a dark orange

oil. The crude product was puriﬁed by silica gel column chromato-

graphy (eluent: DCM/methanol, 95/5, 93/7 then 90/9, v/v) to yield

product 19 (1.51 g, 3.81 mmol) as an orange oil. Yield 66%; Rf 0.51

); 3.98 (s, 3H, NArCH

);

); 135.77 (CArCH

-bromo-1-N-methyl-2-nitro-5-{[(triisopropylsilyl)oxy]methyl}-1H-imi-

dazole (17). To a stirred solution of the alcohol 16 (9.14 g, 38.7 mmol)

in anhydrous DMF (50 mL) were added imidazole (7.92 g, 116 mmol)

and triisopropylsilyl chloride (9.1 mL, 42.5 mmol) dropwise. The re-

action mixture was stirred for 18 h at rt. Then it was cooled at 0 °C and

water (20 mL) was added dropwise until precipitation. Water (380 mL)

was added at once and the heterogeneous mixture was stirred for

−

(SiO

, DCM/MeOH, 90/10, v/v); IR (ATR) ν cm

1495, 1329, 1081,

5 min at 0 °C. After ﬁltration, the solid was washed with water

1060; H NMR (200 MHz, CDCl

) δ 6.59 (td, 1H, Jtrans = 15.5 Hz,

(

3 × 100 mL) and dried at 35 °C under vacuum to yield the protected

6.3 Hz, CH]CHCH

), 6.39 (d, 1H,

trans

= 15.6 Hz,

alcohol 17 (14.7 g, 37.5 mmol) as a yellow solid. Yield 97%; mp

CH]CHCH

), 4.80 (s, 2H, OCH

), 4.04 (s, 3H, NArCH

), 2.25 (s, 6H, N(CH ), 1.14–0.90 (m, 21H,

) δ 145.10 (CArNO ), 135.30,

), 123.98 (CH]CHCH ); 60.74

); 34.67 (NArCH );

); HRMS (ESI) m/z

), 3.08 (d, 2H,

6–67 °C; Rf 0.69 (SiO

, cyclohexane/AcOEt, 80/20, v/v); IR (ATR) ν

J = 6.2 Hz, CH]CHCH

3 2

)

−

1483, 1357, 1081, 1061; H NMR (500 MHz, DMSO‑d

) δ 4.81

Si(CH(CH

)

); C NMR (50 MHz, CDCl

(

s, 2H, OCH

), 4.00 (s, 3H, NArCH

), 1.22–1.11 (m, 3H, Si(CH(CH

)

133.09 (2C, CArC), 127.74 (CH]CHCH

.09–1.01 (m, 18H, Si(CH(CH

44.55 (CArNO ), 134.46 (CArCH

5.12 (NArCH ), 17.61 (6C, Si(CH(CH

m/z 392.1014

Si] 392.1005).

)

); C NMR (126 MHz, DMSO‑d

) δ

(CH]CHCH

), 54.01 (OCH

), 44.31 (2C, N(CH

)

), 116.61 (CArBr), 54.41 (OCH

);

17.84 (6C, Si(CH(CH

)

), 11.74 (3C, Si(CH(CH

)

2 3

)

), 11.29 (3C, Si(CH(CH

)

397.2625 [M+H] (calculated for [C19

N O

Si] 397.2629).

HRMS

(ESI)

[M+H]

(calculated

for

(E)-(1-N-methyl-2-nitro-4-[3-(N,N-dimethylamino)prop-1-enyl]-1H-

imidazo-5-yl)methanol (20). To a solution of silylether 19 (303 mg,

0.764 mmol) in anhydrous THF (6 mL) cooled at 0 °C was added tet-

rabutylammonium ﬂuoride (1 M in THF, 1.15 mL, 1.15 mmol). The

reaction mixture was stirred for 90 min at 0 °C. After concentration

under reduced pressure, the orange oil was puriﬁed by silica gel column

chromatography (eluent: DCM/methanol, 95/5, v/v then 95/5, v/

v + 2% TEA) to yield the alcohol 20 as a yellow oil (164 mg,

[

(

27BrN

3 3

Z) and (E) N,N-dimethyl-3-(tributylstannyl)-prop-2-en-1-amine (18).

a stirred solution of N.N-dimethylpropargylamine (3.80 mL,

5.3 mmol) in anhydrous toluene (150 mL), was added tributyltin hy-

dride (16.4 mL, 61.0 mmol) and azobisisobutyronitrile (751 mg,

.57 mmol). The reaction mixture was warmed to 85 °C for 3 h 30 and

then under reﬂux for 1 h. The solvent was removed by evaporation

under reduced pressure and the crude was puriﬁed by silica gel column

chromatography (eluent: cyclohexane/EtOAc/MeOH, 95/4/1, 90/8/2

then 90/6/4, v/v/v), to yield the organotin compound 18 as a

0.683 mmol). Yield 89%; Rf 0.43 (SiO

, DCM/MeOH, 90/10, v/v + 5%

−1

TEA); IR (ATR) ν cm 3133, 1499, 1332, 1035; H NMR (200 MHz,

OD) δ 6.66 (d, 1H, Jtrans = 15.7 Hz, CH]CHCH

), 6.53 (td, 1H,

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

trans = 15.6 Hz, JH-H = 6.2 Hz, CH]CHCH

), 4.71 (s, 2H, CH

OH),

53.13 (CH

(CArCH CH

OH), 51.78 (2C,

(CH

)

), 34.67 (NArCH

), 24.73

.03 (s, 3H, NArCH

), 3.24 (d, 2H, J = 6.1 Hz, CH]CHCH

), 2.36 (s,

), 24.09 (CH

); MS (ESI) m/z 255.15 [M−H]

H, N(CH ); C NMR (50 MHz, CD

)

OD) δ 146.43 (CArNO

), 136.67,

(calculated for [C10

20BN

]

255.16), 279.13 [M+Na] (calcu-

35.45 (CArC), 128.64 (CH]CHCH

), 123.98 (CH]CHCH

), 61.98

lated for [C10

21BN

NaO

279.16). Cristallographic data are avail-

(

CH]CHCH

), 52.69 (CH

OH), 44.84 (2C, N(CH

)

), 34.94 (NArCH

]

);

able in supporting information.

HRMS (ESI) m/z 241.1295 [M+H] (calculated for [C10

N,N-bis(2-chloroethyl)-{1-N′-methyl-4-[3-(N″,N″-dimethylamino)

prop-1-enyl]-2-nitro-1H-imidazol-5-yl}methyl phosphorodiamidate (23)

was prepared from alcohol 20 (93.0 mg, 0.387 mmol) as described for

the preparation of 2a with some diﬀerences: the coupling reaction with

bis(2-chloroethyl)phosphoramidic dichloride (1) was performed for

45 min before ammonia gas was bubbled through the reaction for

2 min. The reaction was directly stopped by addition of water (10 mL).

The crude product was puriﬁed by silica gel chromatography (eluent:

41.1295).

(

E)-3-(1-N′-methyl-2-nitro-5-{[(triisopropylsilyl)oxy]methyl}-1H-imi-

dazol-4-yl)-N,N-dimethylpropan-1-amine (22). To a solution of com-

pound 19 (108 mg, 0.271 mmol) in pyridine (2 mL) were added to-

sylhydrazine (255 mg, 1.37 mmol) and potassium carbonate (188 mg,

.36 mmol). The reaction mixture was heated under reﬂux for 19 h.

After cooling to rt, the reaction mixture was ﬁltrated and the solid was

washed with DCM (3 × 10 mL). The ﬁltrate was successively washed

with brine (2 × 50 mL) and saturated aqueous solution of sodium

carbonate (2 × 50 mL). The organic layer was dried over magnesium

sulfate, ﬁltered and evaporated under reduced pressure. The crude

product was puriﬁed by silica gel chromatography (eluent: gradient of

EtOAc/EtOH, 80/20, v/v, + 5% NH OH) to yield compound 23

(52.4 mg; 0.134 mmol) as an orange oil. Yield 31%; Rf 0.68 (SiO ,

−

AcOEt/EtOH, 80/20, v/v, + 5% NH

OH); IR (ATR) ν cm

3417,

3222, 1495, 1331, 1220, 1187, 1002, 977; H NMR (200 MHz,

OD) δ 6.70 (d, 1H, Jtrans = 15.6 Hz, CH]CHCH

), 6.57 (td, 1H,

MeOH in DCM 5 to 8%) to yield compound 22 (25.4 mg, 63.7 mmol) as

trans = 15.6 Hz, J = 6.0 Hz, CH]CHCH

), 5.15 (d, 2H,

H-

an orange oil. Yield 24%; H NMR (400 MHz, CDCl

) δ 4.76 (s, 2H,

= 8.0 Hz, CH

O), 4.05 (s, 3H, NArCH

), 3.53–3.33 (m, 4H, N(CH

), 2.32 (s, 6H, N(CH

), 3.72–3.58 (m, 4H, N

OCH

), 4.04 (s, 3H, NArCH

), 2.59 (t, 2H, J = 7.5 Hz, CArCH

), 1.87 (qt,

), 1.20–0.93 (m, 21H, Si(CH(CH ).

(CH

Cl)

), 3.20 (d, 2H,

.38 (t, 2H, J = 7.4 Hz, CH

N(CH

)

), 2.28 (s, 6H, N(CH

)

J = 5.9 Hz, CH]CHCH

)

); C NMR (126 MHz,

H, J = 7.5 Hz, CH

)

OD) δ 146.92 (CArNO

), 138.20 (CArCH]CH), 131.04 (d,

C-

{

4-[3-(N′,N′-dimethylamino)propyl]-1-N-methyl-2-nitro-1H-imidazo-

= 7.4 Hz, CArCH

O), 130.34 (CH]CHCH

), 123.29 (CH]CHCH

-yl}methanol (21). Conditions A: To a solution of compound 20

62.17 (CH]CHCH

), 55.91 (d,

C-P = 4.5 Hz, CH

O), 50.49 (d, 2C,

(

688 mg, 2.86 mmol) in pyridine (25 mL) were added tosylhydrazine

2.67 g, 14.3 mmol) and potassium carbonate (1.99 g, 14.4 mmol). The

C-P = 4.8 Hz, N(CH

Cl)

), 45.07 (2C, N(CH

) ), 43.12 (2C, N

(CH

Cl)

), 35.09 (NArCH

); P NMR (202 MHz, CD

OD) δ 19.37;

reaction mixture was heated under reﬂux for 3 h. After cooling to rt, the

reaction mixture was diluted with ethyl acetate (15 mL). The solid was

ﬁltrated and washed with ethyl acetate (3 × 15 mL). After evaporation

of the ﬁltrate under reduced pressure, the crude product was puriﬁed

by column chromatography on alumina (eluent: gradient from 2 to 20%

of EtOH in EtOAc, v/v) to yield compound 21 (464 mg, 1.92 mmol) as a

yellow oil. Yield 67%.

MS (ESI) m/z 443.06 [M+H] (calculated for [C14

26Cl

O P]

443.11).

N,N-bis(2-chloroethyl)-{1-N′-methyl-4-[3-(N″,N″-dimethylamino)

propyl]-2-nitro-1H-imidazol-5-yl}methyl-phosphorodiamidate (24a) was

prepared from alcohol 21 (102 mg, 0.421 mmol) as described for the

preparation of 2a with some diﬀerences: the coupling reaction with bis

(2-chloroethyl)phosphoramidic dichloride (1) was performed for 1 h 15

before ammonia gas was bubbled through the reaction for 2 min. The

reaction was directly stopped by addition of water (10 mL). The crude

product was puriﬁed by silica gel chromatography (eluent: EtOAc/

EtOH, 80/20, v/v + 5% TEA) to yield compound 24a (56.0 mg,

Conditions B: To a solution of silylether 22 (30.0 mg, 75.2 µmol) in

anhydrous THF (1 mL) cooled at 0 °C was added tetrabutylammonium

ﬂuoride (1 M in THF, 140 µL, 140 µmol). The reaction mixture was

stirred for 1 h at 0 °C. After concentration under reduced pressure, the

orange oil was puriﬁed by column chromatography on alumina (eluent:

0.126 mmol) as a yellow oil. Yield 30%; Rf 0.10 (SiO , AcOEt/EtOH,

−

EtOAc/EtOH/NH

OH, 95/4/1, v/v/v) to yield the alcohol 21 as a

80/20, v/v, + 5% TEA); IR (ATR) ν cm

3345, 3258, 1493, 1331,

yellow oil (8.5 mg, 35.4 µmol). Yield 47%; Rf 0.41 (Al

, AcOEt/

1208, 1044, 981; H NMR (500 MHz, CD

OD) δ 5.11 (dd, 1H,

H-

−

EtOH, 90/10, v/v); IR (ATR) ν cm 3290, 1487, 1327, 1020; H NMR

(CH

= 13.5 Hz, JH-P = 7.7 Hz, OCH′), 5.08 (dd, 1H, JH-H = 13.5 Hz, JH-

(

200 MHz, CD

OD) δ 4.65 (s, 2H, CH

CH ), 2.43–2.30 (m, 2H, CH

), 1.93–1.73 (m, 2H, CH

OD) δ 145.76 (CArNO ), 140.60 (CArCH

OH), 45.32 (2C, N(CH

), 25.30 (CH CH CH ); MS (ESI) m/z

243.15).

OH), 4.03 (s, 3H, NArCH

N(CH

); C NMR (50 MHz,

), 135.00 (CArCH OH),

), 34.88

), 2.63

= 7.7 Hz, OCH″), 4.04 (s, 3H, NArCH

), 3.67 (t, 4H, J = 7.1 Hz, N

), 3.52–3.35 (m, 4H, N(CH CH Cl) ), 2.68 (t, 2H,

CH ), 2.44–2.37 (m, 2H, CH

), 1.93–1.81 (m, 2H, CH

t, 2H, J = 7.5 Hz, CArCH

)

), 2.24

Cl)

s, 6H, N(CH

)

J = 7.6 Hz, CArCH

N(CH

) ), 2.27 (s, 6H,

N(CH

= 7.7 Hz, CArCH

(CH

(CArCH

)

);

C NMR (126 MHz,

9.60 (CH

N(CH

)

), 52.99 (CH

)

OD) δ 146.28 (CArNO

), 142.14 (CArCH

), 130.92 (d,

C-

(

ArCH

), 28.12 (CArCH

O), 59.72 (CH

N(CH

)

), 56.18 (d, JC-P = 4.5 Hz,

43.14 [M+H] (calculated for [C10

N O

]

O), 50.45 (d, 2C,

C-P = 4.8 Hz, N(CH

), 35.11 (NArCH

Cl)

), 45.34 (2C, N

N,N-dimethyl-3-(5-hydroxymethyl-1-N′-methyl-2-nitro-1H-imidazo-4-

). To a stirred solution of

)

), 43.13 (2C, N(CH

Cl)

), 27.96

yl)propyl-1-amine borane complex (21-BH

), 25.41 (CH

); P NMR (202 MHz, CD OD) δ

amine 21 (224 mg, 0.925 mmol) in anhydrous chloroform (4.5 mL)

cooled at 0 °C was added dropwise a 1 M solution of borane-THF

complex (4.6 mL, 4.60 mmol). The reaction mixture was stirred at 0 °C

for 1 h. Anhydrous methanol (4.5 mL) was added and the reaction

mixture was stirred 15 min at 0 °C. The reaction mixture was evapo-

rated under reduced pressure and puriﬁed by silica gel column chro-

matography (eluent: DCM/MeOH, 98/2, v/v). The amine-borane com-

19.22; MS (ESI) m/z 445.13 [M+H]⁺(calculated for

[C14H

28Cl

P] 445.13).

3-{5-[({amino[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-1-N′-

methyl-2-nitro-1H-imidazol-4-yl}-N,N,N-trimethylprop-2-en-1-aminium io-

dide (25). To a stirred solution of the phosphoramide 23 (69.2 mg,

0.156 mmol) in anhydrous ACN (5 mL) were added potassium carbo-

nate (129 mg, 0.933 mmol) and methyl iodide (59 µL, 0.948 mmol).

The reaction mixture was stirred at rt for 1 h 45 and ﬁltrated through a

PTFE (0.45 µm) ﬁlter. The solid was washed with ACN (2 mL). After

evaporation of the ﬁltrate and puriﬁcation by semi-preparative reverse

phase column chromatography the quaternary ammonium 25 (59.6 mg,

0.102 mmol) was obtained as a highly hygroscopic lyophilisate.

Puriﬁcation conditions: detection: 254 nm and 338 nm; separation

plex 21-BH

(209 mg; 0.816 mmol) was obtained as beige crystals.

Yield 70%; mp 99–100 °C; Rf 0.37 (SiO

, DCM/MeOH, 98/2, v/v); IR

−

(

ATR) ν cm

3268, 2379, 2321, 2275, 1490, 1331, 1170, 1027;

NMR (200 MHz, CDCl

) δ 4.69 (s, 2H, CH

OH), 4.06 (s, 3H, NArCH

.88 (br.s, 1H, OH), 2.79–2.68 (m, 2H, CH

(CH

) ), 2.62 (t, 2H,

J = 7.5 Hz, CArCH

), 2.54 (s, 6H, N (CH

)

), 2.20–1.98 (m, 2H,

), 1.96–0.74 (m, 3H, BH

); C NMR (50 MHz, CDCl

) δ

time: 30 min; eluent: H

O/ACN (v/v): 95/5 for 2 min; 95/5 → 70/30

44.81 (CArNO

), 139.18, 132.94 (2C, CArCH

), 63.84 (CH

(CH

)

for 6 min; 70/30 for 2 min; 70/30 → 60/40 for 6 min; 60/40 for 2 min;

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

0/40 → 10/90 for 8 min; 10/90 for 4 min; retention time: 15.4 min;

3-{5-[({bis[(2-chloroethyl)amino]phosphoryl}oxy)methyl]-1-N′-me-

−

Yield 65%; IR (ATR) ν cm

3403, 3229, 1542, 1497, 1331, 1214,

thyl-2-nitro-1H-imidazol-4-yl}-N,N-dimethylpropan-1-amine borane com-

187, 1004, 978; H NMR (500 MHz, CD

OD) δ 7.12 (d, 1H,

plex (24c-BH ). To a solution cooled at −78 °C of phosphorous oxy-

trans = 15.4 Hz, CH]CHCH

), 6.67 (td, 1H,

trans = 15.4 Hz,

chloride (38 µL, 0,408 mmol) in anhydrous DCM (5 mL), was added

J = 7.7 Hz, CH]CHCH

), 5.20 (d, 2H, JH-P = 9.6 Hz, CH

O), 4.17 (d,

), 3.69–3.58 (m, 4H,

Cl) ), 3.17 (s, 9H,

OD) δ 147.18 (CArNO ),

O), 132.31

), 68.74, 68.72, 68.70

dropwise a solution of the alcohol 21-BH (105 mg, 0.410 mmol) and

H, J = 7.7 Hz, CH]CHCH

), 4.07 (s, 3H, NArCH

TEA (63 µL, 0.452 mmol) in anhydrous DCM (2 mL). After 1 h of

stirring at −78 °C, 2-chloroethylamine hydrochloride (102 mg,

0.885 mmol) was added followed by the dropwise addition of TEA

(260 µL, 1.87 mmol). The temperature of the reaction mixture was risen

gradually from −78 °C to 5 °C for 15 h. The reaction was stopped by the

addition of water (10 mL). After layers separation, the organic layer

was washed with a saturated aqueous solution of sodium hydro-

genocarbonate (10 mL), dried over magnesium sulfate, ﬁltrated and

evaporated under reduced pressure to give a yellow oil. The residue was

puriﬁed by silica gel column chromatography (eluent: gradient of

N(CH

Cl)

), 3.49–3.33 (m, 4H, N(CH

(CH

)

); C NMR (126 MHz, CD

36.36 (CArCH]CH), 133.17 (d,

C-P = 6.5 Hz, CArCH

(

CH]CHCH

), 119.01 (CH]CHCH

CH]CHCH

), 56.04 (d, JC-P = 4.7 Hz, CH

O), 53.49, 53.46, 53.42

(CH

)

), 50.46 (d, 2C, JC-P = 4.8 Hz, N(CH

Cl)

), 43.25

OD) δ

2C, N(CH

Cl)

), 35.45 (NArCH

); P NMR (202 MHz, CD

9.38; HRMS (ESI) m/z 457.1288 [M]

(calculated for

[

28Cl

P] 457.1281).

-{5-[({amino[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-1-N′-

MeOH in DCM 0–2%) to yield the isophosphorodiamidate 24c-BH

methyl-2-nitro-1H-imidazol-4-yl}-N,N-dimethylpropan-1-amine

complex (24a-BH ) was prepared from alcohol 21-BH

.519 mmol) as described for the preparation of 14a-BH

borane

(78.0 mg, 0.170 mmol) as an oil. Yield 41%; Rf 0.26 (SiO , DCM/

−

(133 mg,

MeOH, 98/2, v/v); IR (ATR) ν cm

3338, 3235, 2381, 2318, 2271,

. The crude

1493, 1331, 1193, 1167, 1110, 999; H NMR (500 MHz, CDCl

) δ 5.03

product was puriﬁed by silica gel chromatography (eluent: DCM/EtOH,

(d, 2H,

H-P = 7.4 Hz, CH

O), 4.05 (s, 3H, NArCH

), 3.58 (t,

6/4, v/v) to yield compound 24a-BH

(150 mg, 0.629 mmol) as an oil.

J = 5.5 Hz, 4H, NHCH

Cl), 3.35–3.19 (m, 6H, NHCH

7.4 Hz,

CH CH ),

Cl),

−1

Yield 63%; Rf 0.20 (SiO

, DCM/MeOH, 97/3, v/v); IR (ATR) ν cm

2.81–2.72 (m, 2H, CH

(CH

)

), 2.67 (t, 2H,

J =

231, 2380, 2318, 2272, 1492, 1330, 1227, 1193, 1168, 1003, 980; H

ArCH

), 2.54 (s, 6H, N (CH

)

), 2.12–2.01 (m, 2H, CH

NMR (500 MHz, CDCl

) δ 5.14 (dd, 1H,

H-H = 13.4 Hz,

H-

1.77–1.32 (m, 3H, BH

);

C NMR (126 MHz, CDCl

) δ 145.11

= 7.7 Hz, OCH′), 4.99 (dd, 1H,

H-H = 13.4 Hz,

H-P = 6.4 Hz,

(CArNO

), 141.04 (CArCH

); 129.24 (d, JC-P = 7.8 Hz, CArCH

O),

OCH″), 4.06 (s, 3H, NArCH

Cl)

), 3.69–3.61 (m, 4H, N(CH

Cl)

63.82 (CH

(CH

)

), 55.03 (d,

C-P = 4.1 Hz, CH O), 51.47 (2C,

.54–3.43 (m, 4H, N(CH

), 3.08 (br.s, 2H, NH

), 2.83–2.72 (m,

(CH

)

), 45.87 (d, 2C,

C-P = 5.2 Hz, NHCH

CH Cl), 43.10 (2C,

H, CH

(CH

)

), 2.68 (t, 2H, J = 7.4 Hz, CArCH

), 2.56 and

NHCH

Cl), 34.65 (NArCH

), 24.73 (CArCH

CH ), 23.97

.54 (s, 3H, CH

(CH

)

), 2.13–2.06 (qt, 2H,

7.7 Hz,

(CH CH

); P NMR (202 MHz, CDCl

) δ 14.82; MS (ESI) m/z

); C NMR (126 MHz, CDCl

) δ 145.24 (CArNO

), 141.16

457.13 [M−H] (calculated for [C14

29BCl

P] 457.15).

(

ArCH

), 129.17 (d,

C-P

8.2 Hz,

ArCH

), 63.89

3-{5-[({amino[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-1-N′-

methyl-2-nitro-1H-imidazol-4-yl}-N,N,N-trimethylpropan-1-aminium

(26a). Conditions A: Alkylation of amine 24a (42.8 mg, 96.1 µmol)

following conditions described for the preparation of 3a (reaction time:

7 h) followed by puriﬁcation by semi-preparative reverse phase column

chromatography aﬀorded QA-compound 26a (29.2 mg, 49.7 µmol) as a

highly hygroscopic yellow powder. Yield 52%.

(CH

)

), 55.11 (d,

C-P = 4.0 Hz, CH

O), 51.55, 51.48

(CH

)

), 48.93 (d, 2C, JC-P = 5.0 Hz, N(CH

Cl)

), 42.70

2C, N(CH

Cl)

), 34.63 (NArCH

), 24.79, 24.00 (CArCH

);

NMR (202 MHz, CDCl

) δ 16.17; MS (ESI) m/z 457.08 [M−H]

(

calculated for [C14

29BCl

P] 457.15).

-{5-[({bis[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-1-N′-me-

thyl-2-nitro-1H-imidazol-4-yl}-N,N-dimethylpropan-1-amine borane com-

plex (24b-BH ). To a stirred solution of the alcohol 21-BH (120 mg,

.469 mmol) in anhydrous THF (4 mL) cooled at −78 °C, was added

dropwise lithium bis(trimethylsilyl)amide (1 M in THF, 520 µL,

Conditions B: Dissociation of the amine borane complex 24a-BH

(50.3 mg, 0.110 mmol) and alkylation according to the conditions de-

scribed for the preparation of 15a (methyl iodide 15 eq. at rt for 2 h)

followed by puriﬁcation by semi-preparative reverse phase column

chromatography aﬀorded QA-compound 26a (29.5 mg, 50.2 µmol) as a

highly hygroscopic yellow powder. Yield 46%; Puriﬁcation condi-

tions: detection: 254 nm and 338 nm; separation time: 30 min; eluent:

20 µmol) under an inert atmosphere. After 5 min, a solution of com-

pound 12 (189 mg, 519 mmol) in anhydrous THF (2 mL) previously

cooled at −78 °C was added dropwise to the reaction mixture. The

kinetic of the reaction was monitored by P NMR. The temperature

was allowed to rise gradually to 5 °C and the stirring was maintained for

H O/ACN (v/v): 95/5 for 2 min; 95/5 → 70/30 for 6 min; 70/30 for

2 min; 70/30 → 60/40 for 6 min; 60/40 for 3 min; 60/40 → 10/90 for

−

4 h. The reaction was stopped by addition of water (10 mL) and the

7 min; 10/90 for 4 min; retention time : 15.4 min; IR (ATR) ν cm

reaction mixture was extracted with ethyl acetate (3 × 10 mL). The

organic layers were combined, dried over magnesium sulfate, ﬁltrated

and evaporated under reduced pressure. The crude product was pur-

iﬁed by silica gel column chromatography (eluent: DCM/EtOH, 98/2,

3232, 1493, 1332, 1228, 979; H NMR (200 MHz, CD

OD) δ 5.16 (dd,

1H,

H-H = 13.7 Hz,

H-P = 9.3 Hz, OCH′), 5.06 (dd, 1H,

H-

= 13.7 Hz, JH-P = 9.1 Hz, OCH″), 4.05 (s, 3H, NArCH

), 3.74–3.62

CH Cl)

(m, 4H, N(CH

Cl)

), 3.54–3.35 (m, 6H, N(CH

v/v) to yield phosphorodiamidate 24b-BH

(90.7 mg, 0.155 mmol) as a

(CH

)

), 3.15 (s, 9H, CH

(CH

)

), 2.78 (t, 2H, J = 7.3 Hz,

thick oil. Yield 33%; Rf 0.31 (SiO

, DCM/EtOH, 98/2, v/v); IR (ATR) ν

ArCH

), 2.32–2.05 (m, 2H, CH

); C NMR (126 MHz,

−

cm 2381, 2318, 2272, 1493, 1331, 1219, 1167, 1088, 973; H NMR

OD) δ 146.53 (CArNO

), 140.01 (CArCH

), 131.52 (d,

C-

(

500 MHz, CDCl

) δ 5.13 (d, 2H, JH-P = 8.2 Hz, CH

O), 4.06 (s, 3H,

= 6.4 Hz, CArCH

O), 66.97, 66.89, 66.85 (CH

O), 53.82, 53.74, 53.66 (CH

CH Cl)

(CH

) ), 56.42 (d,

ArCH

), 3.68–3.54 (m, 8H, N(CH

Cl)

), 3.40 (td, 8H,

H-

C-P = 4.8 Hz, CH

)

), 50.41 (d,

= 12.6 Hz,

H-H = 6.4 Hz, N(CH

Cl)

), 2.85–2.74 (m, 2H,

2C, JC-P = 4.8 Hz, N(CH

), 43.18 (2C, N(CH

Cl)

), 35.36

(CH

)

), 2.67 (t, 2H, J = 7.6 Hz, CArCH

), 2.55 (s, 6H,

(NArCH

(202 MHz, CD

lated for [C15

), 24.07 (CArCH

), 23.33 (CH

);

NMR

(CH

)

), 2.18–1.97 (m, 2H, CH

), 1.87–1.25 (m, 3H, BH

);

OD) δ 19.45; HRMS (ESI) m/z 459.1444 [M] (calcu-

C NMR (126 MHz, CDCl

) δ 145.31 (CArNO

), 141.47 (CArCH

H30Cl

2 6

P] 459.1438).

28.53 (d, JC-P = 6.5 Hz, CArCH

O), 63.89 (CH

(CH

)

), 55.36 (d,

3-{5-[({bis[bis(2-chloroethyl)amino]phosphoryl}oxy)methyl]-1-N′-me-

C-P = 3.9 Hz, CH

O), 51.55 (2C, N (CH

CH Cl) ), 42.36 (4C, N(CH

); 24.65 (CArCH CH

)

), 49.22 (d, 4C,

C-

thyl-2-nitro-1H-imidazol-4-yl}-N,N,N-trimethylpropan-1-aminium iodide

4.6 Hz, N(CH

);

Cl)

), 34.66

(26b) was prepared from amine borane complex 24b-BH (90.7 mg,

(

ArCH

), 23.91 (CH

NMR

0.155 mmol) as described for the preparation of 15a (methyl iodide

15 eq. at rt for 1 h). QA compound 26b (38.8 mg, 54.5 µmol) was

(

202 MHz, CDCl

) δ 17.04; MS (ESI) m/z 583.14 [M−H] (calculated

for [C18

35BCl

P] 583.13).

obtained as a highly hygroscopic yellow powder. Puriﬁcation

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

conditions: detection: 254 nm and 338 nm; separation time: 30 min;

initiation of the reaction. Chemical shifts were reported relative to the

eluent: H

O/ACN (v/v): 95/5 for 2 min; 95/5 → 70/30 for 6 min; 70/

signal of a Ph

PO solution (5% in DMSO‑d ) as a coaxial reference

0 for 2 min; 70/30 → 60/40 for 6 min; 60/40 for 3 min; 60/40 → 10/

(26 ppm). The temperature of the probe was maintained at 37 °C using

the Bruker variable temperature unit. The disappearance of the starting

material as well as the appearance of the phosphoramidate anion and

their relative concentrations were determined by measuring peak areas.

0 for 7 min; 10/90 for 4 min; retention time : 21.7 min; Yield 35%; IR

−

(

ATR) ν cm

1491, 1331, 1210, 973, 959; H NMR (500 MHz,

OD) δ 5.26 (d, 1H, JH-P = 10.0 Hz, CH

O), 4.08 (s, 3H, NArCH

.76–3.64 (m, 8H, N(CH

), 3.17 (s, 9H, CH

), 2.27–2.18 (m, 2H, CH

), 140.48 (CArCH

O), 67.05, 67.03, 67.91 (CH

O), 53.84, 53.81, 53.78 (CH

CH Cl)

Cl)

), 3.51–3.38 (m, 10H, CH

(CH

) ,

N(CH

Cl)

(CH

) ), 2.81 (t, 2H, J = 7.3 Hz,

3.4. Nitroreductase assay

ArCH

); C NMR (126 MHz,

OD) δ 146.72 (CArNO

), 130.86 (d,

C-

Recombinant E. coli nitroreductase and dihydronicotinamide ade-

= 5.0 Hz, CArCH

(CH

)

), 56.98 (d,

), 50.11 (d,

nine dinucleotide (NADPH, reduced form, tetrasodium salt) were pur-

chased from Sigma–Aldrich. 10 µL of a prodrug stock solution (1.7 mM)

in ultra-pure water with 13% DMSO were added to 1000 µL of PBS

C-P = 4.5 Hz, CH

)

C, JC-P = 4.7 Hz, N(CH

), 43.06 (4C, N(CH

Cl)

), 35.43

(

ArCH

), 24.16 (CArCH

), 23.19 (CH

);

NMR

(

10 mM, pH 7.4, 37 °C) containing NADPH. The reaction was initiated

(

202 MHz, CD

OD) δ 18.24; HRMS (ESI) m/z 583.1292 [M] (calcu-

by addition of E. coli nitroreductase (25 µL of an initial solution,

lated for [C19

36Cl

P] 583.1284).

.0 mg/mL in PBS; ﬁnal concentrations: prodrug: 17 µM; NADPH:

.9 mM; nitroreductase: 25 µg/mL). Aliquots were withdrawn at var-

-{5-[({bis[(2-chloroethyl)amino]phosphoryl}oxy)methyl]-1-N′-me-

thyl-2-nitro-1H-imidazol-4-yl}-N,N,N-trimethylpropan-1-aminium iodide

ious time points over a 2 h period and the reactions were followed by

RP-HPLC as a function of time. The separation was carried out in the

same conditions as previously described for phosphate buﬀer stability.

The HPLC chromatogram peak area at 322 nm was used to calculate the

concentration of the remaining prodrug. Between the time points, all

solutions were incubated at 37 °C. Reference solutions containing 10 µL

of compound stock solution and 100 µL NADPH solution (9 mM)

completed with PBS (1000 µL ﬁnal volume), were prepared and ana-

lyzed under the same conditions in order to distinguish between ni-

troreductase-based activation and enzyme-free reaction.

(

26c) was prepared from amine borane complex 24c-BH

(67.0 mg,

.146 mmol) as described for the preparation of 15a (methyl iodide

5 eq. at rt for 2 h). QA compound 26c (47.9 mg, 81.6 µmol) was

obtained as a highly hygroscopic yellow powder. Puriﬁcation condi-

tions: detection: 254 nm and 338 nm; separation time: 30 min; eluent:

O/ACN (v/v): 95/5 for 2 min; 95/5 → 70/30 for 6 min; 70/30 for

min; 70/30 → 60/40 for 6 min; 60/40 for 3 min; 60/40 → 10/90 for

min; 10/90 for 4 min; retention time : 14.1 min; Yield 56%; IR (ATR)

−1

ν cm 3246, 1496, 1329, 1211, 1002, 990, 979; H NMR (500 MHz,

OD) δ 5.11 (d, 1H, JH-P = 9.4 Hz, CH

O), 4.06 (s, 3H, NArCH ),

.57 (t, 4H,

6.3 Hz, NHCH

Cl), 3.47–3.38 (m, 2H,

3.5. In vitro anti-proliferation assay

(CH

)

), 3.20 (td, 4H,

H-P = 12.5 Hz,

H-H = 6.3 Hz,

NHCH

Cl), 3.15 (s, 9H, CH

), 2.24–2.16 (m, 2H, CH

), 140.13 (CArCH

O), 66.92, 66.94, 66.90 (CH

O), 53.91, 53.88, 53.84 (CH

(CH

)

), 2.79 (t, 2H, J = 7.3 Hz,

Human HEMC-SS chondrosarcoma cell line was obtained from the

ArCH

); C NMR (126 MHz,

European Collection of Authenticated Cell Cultures, maintained in

DMEM/F12 medium (Life Technologies) supplemented with 10% of

fetal calf serum (Dutscher) and 4 µg/mL gentamicin and maintained in

OD) δ 146.48 (CArNO

), 131.52 (d,

C-

= 6.5 Hz, CArCH

(CH

)

), 56.57 (d,

), 45.88 (d,

Cl), 35.44

C-P = 4.8 Hz, CH

)

a 5% CO humidiﬁed environment at 37 °C. After trypsinization, HEMC-

C-P = 5.0 Hz, NHCH

Cl), 44.18 (2C, NHCH

SS cells were seeded in 96-well plates at a density of 2.10 cells in

(

ArCH

), 24.12 (CArCH

), 23.34 (CH

);

P NMR

50 µL of culture medium and allowed to adhere overnight. Increasing

(

202 MHz, CD

OD) δ 17.20; HRMS (ESI) m/z 459.1446 [M] (calcu-

concentrations of drugs diluted in DMSO (maintaining ﬁnal DMSO

concentration at 0.5% (v/v)) were added. After plate incubation for

lated for [C15

H30Cl

P] 459.1438).

4 h in normoxic (21% O

, 5% CO

, 37 °C) or hypoxic (N

, O < 0.3%,

3.2. Stability measurements in aqueous buﬀer

7 °C) conditions, the culture media was removed and cell layers wa-

shed with PBS. Cells were then left to grow for 48 h in normoxic con-

ditions. Viability of cells cultured was quantiﬁed by AlamarBlue assay.

Cytotoxic activity was expressed as the drug concentration that in-

hibited cell growth by 50% (IC50). Experiments were performed at least

Prodrugs were dissolved in Phosphate Buﬀer Saline (PBS) (10 mM,

pH 7.4, 37 °C) containing 0.13% DMSO at a concentration of 17 µM and

incubated at 37 °C. Aliquots were withdrawn at various time points

over a 24 h period and directly subjected to RP-HPLC analysis. The

HPLC chromatogram peak area at 322 nm was used to calculate the

concentration of the remaining prodrug. Analytical RP-HPLC mea-

surements were performed on a HP1100 (Agilent, Palo Alto, CA, USA).

The separation was carried out on a C18 column (Phenomenex, Luna

C18, 3.0 × 150 mm, 3 µm) using the following conditions: C18 guard

column from Phenomenex (Le Pecq, France) total experiment time:

in triplicates. Data are presented as means

SD. Statistical sig-

niﬁcance was determined using Student’s a t-Test. Results were con-

sidered signiﬁcant at p < 0.05.

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂu-

ence the work reported in this paper.

0 min, ﬂow rate = 0.4 mL/min, eluent mixture: H

containing triﬂuoroacetic acid (15 mM), gradient: 80/20 at 0 min →

5/25 at 3 min, then 75/25 → 60/40 for 2 min, then 60/40 → 50/50

for 5 min, then 50/50 → 80/20 for 1 min, then 80/20 for 9 min.

O/ACN (v:v)

Acknowledgements

.3. ³¹P NMR kinetics of chemical activation

The authors would like to thank Fernand Leal for his technical

support on IR measurement.

Prodrug (≈10 mg) were dissolved in ACN (90 µL) and cacodylate

buﬀer (300 µL, 0.1 M, pH 7.4) while sodium dithionite was dissolved in

cacodylate buﬀer. 30 µL of the solution containing 3 eq. of sodium

dithionite were added to the prodrug solution. The reaction mixture

was transferred to a 5 mm NMR tube and the data acquisition was

started. Spectra were taken at diﬀerent time intervals over a 24 h

period, and time points for each spectrum were assigned from the

Funding

This work was supported by the Ligue contre le cancer, France

(2017-2018, projet reference R16146CC/RAB16025CCA) and PRT-K,

Translational Cancer Research: INCa-DGOS, France, D-TECT (project

Y. Gerard, et al.

Bioorganic Chemistry 98 (2020) 103747

reference R15066CC / RPT15003CCA).

M. Banica, D. Jung, J. Wang, H. Ma, X. Li, Z. Yang, R.M. Hoﬀman, W.S. Ammons,

C.P. Hart, M. Matteucci, Potent and highly selective hypoxia-activated achiral

phosphoramidate mustards as anticancer drugs, J. Med. Chem. 51 (2008)

Appendix A. Supplementary material

412–2420, https://doi.org/10.1021/jm701028q .

[

21] A.V. Patterson, D.M. Ferry, S.J. Edmunds, Y. Gu, R.S. Singleton, K. Patel,

S.M. Pullen, K.O. Hicks, S.P. Syddall, G.J. Atwell, S. Yang, W.A. Denny,

W.R. Wilson, Mechanism of action and preclinical antitumor activity of the novel

hypoxia-activated DNA cross-linking agent PR-104, Clin. Cancer Res. 13 (2007)

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.bioorg.2020.103747 .

922–3932, https://doi.org/10.1158/1078-0432.CCR-07-0478 .

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