Synthesis of acyclic nucleoside phosphonates targeting flavin-dependent thymidylate synthase in Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2021.116351

Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural's cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 μM.

Full text of DOI:10.1016/j.bmc.2021.116351

Bioorg. Med. Chem. 46 (2021) 116351
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of acyclic nucleoside phosphonates targeting flavin-dependent
thymidylate synthase in Mycobacterium tuberculosis
a
a
,
*
b
b,c
Nicolas G. Biteau , Vincent Roy , Jean-Christophe Lambry , Hubert F. Becker
,
b
a,*
Hannu Myllykallio , Luigi A. Agrofoglio
a
ICOA, Univ. Orl ´e ans, CNRS UMR 7311, F-45067 Orl ´e ans, France
b
LOB, INSERM U696-CNRS UMR 7645, Ecole Polytechnique, 91128 Palaiseau, France
Sorbonne Universit ´e , Facult ´e des Sciences et Ing ´e nierie, 75005 Paris, France
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate
synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target
was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed
a new class of ANPs based on the mimic of two natural’s cofactors (dUMP and FAD) as inhibitors against
Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-
alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8%
Flavin-dependent thymidylate synthase
Acyclic nucleoside phosphonate
Ruthenium-catalyzed cross-metathesis
Sonogashira cross-coupling
inhibitory effect on ThyX at 200 M.
μ
1
. Introduction
Since the late 19th century, antibiotics have been used in the treat-
nicotinamide adenine dinucleotide phosphate (NADPH) and CH
2 4
H fo-
late to convert dUMP in dTMP. The methylene group was transferred
-
2 4
from CH H folate to the reduced form of FAD (FADH ) and then trans-
ment of life-threatening infections, however, as a result of antimicrobial
resistance (AMR), they are less effective and AMR is becoming a major
ferred to C5 of the dUMP, through a bridged methylene intermediate, to
afford the dTMP through a last flavin oxidation step. The first specific
concern of global public health, especially for Mycobacterium tuberculosis
inhibitor of FDTS, the thiazolidine analog 1 (IC50 of 0.057 M), was
μ
1
reported by Myllykalio et al.8 The same group9 found that the naph-
toquinone derivative 2 exhibited for FTDS a Ki of 28 nM. Herdewijn
et al.10 described the monophosphate nucleotide inhibitor 3 (IC50 of
(
MT). There is an urgent need for new molecules targeting new po-
tential bacterial targets for which the interest arises for their differences
between the biochemical pathways in bacteria and eukaryotic cells. The
′
′
de novo synthesis of 2 -deoxythymidine-5 -monophosphate (dTMP) is
crucial to the DNA synthesis. This synthesis can be done in bacteria by
0.91
μ
M) and its acyclic analog 4 (43% inhibition at 50 M), (Fig. 1).
μ
(SEE Fig 2.)
2
Interestingly, Johar et al.11 and Lescrinier et al.12 have shown that the
introduction of a long alkynyl side chains at the C5 position of uridine,
such as dodecynyl and tridecynyl, led to potent antimycobacterial
activity.
either the folate-dependent thymidylate synthase (TS), such as found in
Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, or
by a flavin-dependent thymidylate synthase (FDTS or ThyX; EC
2
.1.1.148),3 such as found in Helicobacter pylori, Borrelia burgdorferi,
Chlamydia species. Other pathogenic bacteria such as Bacillus anthracis,
Clostridium botulinum, and Mycobacterium tuberculosis possess both en-
A docking of dUMP and cofactors into the active site of
2
6
M. tuberculosis FDTS (PDB: 3GWC) reveals that cofactors and dUMP
were stacked together by -stacking. They were also maintained into this
4
zymes. FDTS, which is not found in humans (contrary to TS), has no
π
sequence, mechanism or structural similarities with TS.3 Thus, among
the new antibacterial targets, FDTS appears to be an excellent target for
new antimicrobial drug discovery for deadly microbes.6
,5
large and flexible pocket by hydrogen bond interactions at the pyrimi-
′
dine moiety (Arg 199 and Arg 107) and ribose (3 -OH, Gln 103 and Arg
95). The phosphate group acted as an anchor by creation of six hydrogen
bonds (Arg 107, Arg 172, Arg 87 and Gln 106). FAD shows only in-
teractions at its pyrimidine moiety (Arg 103 and Arg 95) and its
Based on the commonly approved multistep mechanism reported by
7
Kohen et al in 2016, FDTS requires flavin adenine dinucleotide (FAD),
*
Corresponding authors.
E-mail addresses: vincent.roy@univ-orleans.fr (V. Roy), luigi.agrofoglio@univ-orleans.fr (L.A. Agrofoglio).
https://doi.org/10.1016/j.bmc.2021.116351
Received 17 June 2021; Received in revised form 16 July 2021; Accepted 30 July 2021
Available online 6 August 2021
0
968-0896/© 2021 Elsevier Ltd. All rights reserved.

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
BSA. TMSCl, activated NaI and crotyl bromide were then successively
added to the reaction mixture which was then subjected to microwave
◦
irradiation at 80 C for 30 min. The corresponding N1-crotylated qui-
nazoline 9a-c were obtained, respectively, with excellent yields. A N3-
Boc protection was realized in the presence of Boc
2
O and DMAP in
◦
THF under microwave irradiation at 70 C for 10 min affording 10a-c.
Analogs 10a-c were directly engaged in a ruthenium-based olefin cross-
metathesis with dimethyl allylphosphonate by using Grubbs-II catalyst
◦
14
and DCM under ultrasonication at 55 C, 80 kHz for 24 h. The crude
reaction was not purified and directly engaged in the Boc deprotection
with TFA in DCM at rt for 6 h. Compounds 11a-c were isolated in
moderate yields with an inseparable mixture of isomers E/Z (85/15),
respectively. Phosphonate derivatives 11a-c were finally deprotected
under McKenna conditions15 in the presence of TMSBr in ACN under
◦
microwave irradiation at 70 C for 10 min to afford the corresponding
phosphonic acid derivatives 12a-c, in good yield, respectively.
Fig. 1. Structures of FDTS inhibitors.
2
.1.2. Synthesis of the C7- and C6-substituted quinazoline ANPs
In order to fill the pocket at enzyme active site, we introduced a long
alkynyl side chain (13a-c) either at the C7-position or at the C6 position
of the quinazoline moiety, (Scheme 2 & 3). Firstly, the precursor of the
chain, N-prop-2-ynyloctanamide 13a and phenylacetamidoprop-1-ynyl
1
3b were isolated by precipitation from propargyl amine 13 in the
◦
presence of the corresponding acyl chloride, DIPEA in DCM at 0 C to rt
for 2 h. Meanwhile the 1-hexyl-3-prop-2-ynyl urea 13c was obtained
from 13 and hexylisocyanate in 94% yield.16
Thus, for C7 substitution, commercially available 7-bromoquinazo-
line 14 was regioselectively alkylated to 15, which was then subjected
to the N3-Boc protection to 16. A ruthenium-based cross-metathesis of
Fig. 2. Structures of synthetized compounds.
1
6 with dimethyl allyl phosphonate, (Scheme 2), followed by the N3-
Boc removal with TFA in DCM, afforded compound 17 as an insepa-
rable mixture of isomers E/Z (85/15). Aryl bromide 17 was then reacted
diphosphate moiety (Arg 97, Arg 95, Arg 190, His 96 and His 194). No
interaction was observed between ribityl and adenine moiety. It appears
under Pd(0) cross-coupling conditions17 (CuI, Pd(PPh ) in DMF, reflux,
′
that the pyrimidine nucleobase and the 5 -phosphate moiety are
3 4
3
h) with alkynes 13a-c and Et
excellent yields. Subsequent deprotection of C7-dimethylphosphonates
8a-c with TMSBr in anhydrous acetonitrile afforded the desired
3
N to afford 18a-c, respectively, in
important in keeping the dUMP into the active site.
Based on FDTS exclusive catalytic mechanism and on the successful
development by our group of a new family of unsaturated acyclic
nucleoside phosphonates (ANPs), we describe herein new ANP analogs
bearing a quinazoline nucleobase (5) in order to interact with FAD
1
phosphonic acids 19a-c in quantitative yields, (Scheme 2).
For the synthesis of C6-quinazoline, we had to slightly modify the
chemical pathway. Side change degradation and non-separable complex
mixtures were obtained when the acidic deprotection was carried out
through -stacking and hydrophobic pairing interactions. New acyclic
π
dUMP analogs (6) substituted at C5 in order to block the methyl transfer
1
8
due to the hyperconjugation between the N1 and the alkynyl. Thus the
-bromoquinazoline (20) was converted through 21 to the N3-protected
crotyl analogue 22. Ruthenium-catalyzed cross-coupling of 22 with bis
POC)allylphosphonate 23 afforded the ANP 24 (in 58%) which it’s
were also successfully synthesized (Figure 1).
6
2
. Results and discussion
(
directly engaged into the N3-Boc deprotection to 25 in 44% yields (two
steps), which was then submitted to a Sonogashira coupling with al-
kynes 13a-c, to afford desired compounds 26a-c, respectively, in mod-
erate yields. The cleavage of POC moiety of 26a-c was realized using
NaOH 0.1 N in deionized water followed by an acidification with Dowex
2
2
.1. Chemistry
.1.1. Synthesis of quinazoline ANPs (12)
Quinazoline derivatives 12a-c were obtained in five steps starting
from anthranilic derivatives 7a-c, (Scheme 1). Reaction of 7a-c with
+
◦
13
50WX8 (H ) to led the phosphonic acid compounds 24a-c, in moderate
urea at 150 C afforded the corresponding bicycles 8a-c, respectively.
Quinazoline derivatives 8a-c were regioselectively alkylated at N1 po-
sition following a method previously developed by our group. Com-
pounds 8a-c were first subjected to a silylation step, under microwave
to good yields, respectively.
2
.1.3. Synthesis of C5-substitued uracil analogues (32a,b and 39)
◦
We then turned our attention to the introduction of the uracil acyclic
irradiation at 80 C for 5 min in the presence of molecular sieves and
◦
◦
Scheme 1. Reagents and conditions: (a) urea, 150 C, 12 h.; (b) (i) bis(trimethylsilyl)acetamide, CH
3
CN, MW, 80 C, 5 min, (ii) TMSCl, crotyl bromide, NaIact, MW,
◦
◦
◦
8
0
C, 30 min; (c) Boc
2
O, DMAP, THF, MW, 70 C, 10 min; (d) (i) dimethyl allylphosphonate, Grubbs-II catalyst, DCM anhyd.,))) 80 kHz, 55 C, 24 h, (ii) TFA, DCM,
◦
rt, 6 h; (e) TMSBr, ACN anhyd., MW, 70 C, 10 min.
2

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
◦
◦
◦
Scheme 2. Reagents and conditions: (a) RCOCl, DIPEA, DCM anhyd., 0 C-rt, 2 h; (b) isocyanate DCM anhyd., 0 C-rt, 2 h; (c) (i) BSA, CH
3
CN, MW, 80 C, 5 min, (ii)
◦
◦
TMSCl, crotyl bromide, NaIact, MW, 80 C, 30 min; (d) Boc
2
O, DMAP, THF, MW 70 C, 10 min; (e) (i) dimethyl-allylphosphonate, Grubbs-II catalyst, DCM anhyd.,)))
◦
◦
8
0 KHz, 55 C, 24 h, (ii) TFA, DCM, rt, 6 h; (f) Pd(PPh
3
)
4
, CuI, Et
3
N, alkynes 13a, 13b and 13c, DMF, 70 C, 3 h; (g) TMSBr, ACN anhyd., rt, 6 h.
◦
◦
◦
Scheme 3. Reagents and conditions: (a) (i) BSA, CH
3
CN, MW, 80 C, 5 min, (ii) TMSCl, crotyl bromide, NaIact, MW, 80 C, 30 min; (b) Boc
2
O, DMAP, THF, MW 70 C,
◦
1
7
0 min; (c) bis(POC)-allylphosphonate, G-II, DCM anhyd.,))) 80 KHz, 55 C, 24 h; (d) TFA, DCM, rt, 6 h; (e) Pd(PPh
3
)
4
, CuI, Et
3
N, alkynes 13a, 13b and 13c, DMF,
◦
+
0
C, 3 h; (f) NaOH (0.1 N), rt, 4 h, DOWEX 50WX8 (H ).
+
nucleoside phosphonate derivatives at the C5 position of uracil acyclo-
nucleoside phosphonate derivatives (Scheme 4) of various alkynyl
chains as reported in literature.11,12 Thus, the 5-iodo-N1-alkylated uracil
50WX8 (H )), in excellent yields (Scheme 4).
Finally, we developed a third series of ANPs bearing at C5 of uracil
either a benzyl or an aniline substituent. Compound 34a was obtained
1
9
21
2
8 obtained as previously described
was submitted to a ruthenium-
through a turbo Grignard reaction of 5-iodouracil (32) with MeMgCl.
◦
◦
catalyzed cross-metathesis reaction with bis(POC) allylphosphonate 23
LiCl at ꢀ 20 C for 30 min in THF, followed by the addition of iPrMgCl.
◦
in the presence of Grubbs-II catalyst in DCM at 55 C for 24 h, to give 29
LiCl at ꢀ 20 C in 30 min and stirred 2 h at room temperature, (Scheme
◦
in 57% yields, as an inseparable mixture of isomers E/Z (85/15). Two
5). Then CuI.
2
LiCl in THF was added at ꢀ 30 C and stirred at room
alkynes were introduced under Sonogashira cross-coupling conditions
temperature for 1 h. Finally, addition of benzylbromide at room tem-
(
Et
3
N, Pd(PPh
3
)
4
, rt, 16 h) to lead to 30a and 30c, in 97% and 21%
perature for 12 h, afforded the desired 5-benzyl-uracil 34a in 84%
yields, respectively. During this reaction, side products corresponding to
the known formation of fluorescent bicyclic furopyrimidine nucleosides
were observed (structures not shown).20
yields. Compound 34b22 was obtained, through a SNAr from 5-bromo-
◦
uracil 33b in presence of aniline under thermic condition at 175
C
for 20 min, in good yields. Both 34a and 34b were engaged, respec-
tively, in a regioselective N1-alkylation (35a,b) followed by a N3-Boc
protection to 36a,b in excellent yields. Ruthenium-catalyzed cross-
The desired phosphonic acid analogs 31a,c were obtained from 30a,
c, respectively, by alkaline hydrolysis followed by acidification (Dowex
◦
Scheme 4. Reagents and conditions: (a) 25, Grubbs-II catalyst, DCM anhyd., 55 C, 24 h; (b) Pd(PPh
3
)
4
, CuI, Et
3
N, alkynyl chains 13a,b, DMF, rt, 16 h; (c) NaOH
+
(
0.1 N), rt, 4 h, DOWEX 50WX8 (H ).
3

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
◦
◦
◦
Scheme 5. Reagents and conditions: (a) for (34a): (i) MeMgCl.LiCl, ꢀ 20 C, 30 min, THF, (ii) iPrMgCl.LiCl, ꢀ 20 C, 30 min, 2 h, rt. (iii) CuI.2LiCl, ꢀ 30 C to rt, 1 h,
◦
◦
◦
(
iv) BnBr, rt, 12 h.; (b) for (34b): aniline, 175 C, 20 min; (c) (i) BSA, CH
3
CN, MW, 80 C, 5 min, (ii) TMSCl, crotyl bromide, NaIact, MW, 80 C, 30 min; (d) Boc
2
O,
◦
◦
DMAP, THF, MW, 10 min, 70 C; (e) i) allylphosphonate, Grubbs-II catalyst, DCM anhyd.,))) 80 kHz, 55 C, 24 h, ii) TFA, DCM, rt, 6 h; (f) TMSBr, ACN anhyd., MW,
◦
7
0
C, 10 min.
metathesis reaction of 36a,b with dimethylallylphosphonate afforded
7a,b as an inseparable mixture of E/Z (85/15), respectively. A final
the stabilization of natural substrates in the pocket, such as Arg 199 and
Arg 95.
3
two steps deprotection gave the desired phosphonic acid compound 38a
meanwhile derivative 38b was not characterized due to its instability.
Inhibition assays were also performed without addition of FAD or at
a lower concentration of dUMP (10 M) to promote binding of molecules
μ
in the active site of Mtb ThyX, but no significant inhibitory effect was
observed. Since previous work on active ANPs against FDTS, we can
hypothesize that the four-carbon-chain linker which connects the
nucleobase and the phosphonate moiety is too short. We have thus to
optimize the length and flexibility of the linker of 19c as well as to
2
.2. Biological evaluation
A NADPH oxidase spectrophotometric assay, adapted from Basta
et al.,23 was used to test the in vitro inhibitory activities of synthesized
compounds on Mtb ThyX. As NADPH has a maximum of absorption at
′
functionalize it by to adding functional groups in order to mimic the 3 -
OH of dUMP.
3
40 nm, ThyX activity was determined by following the decrease of
absorbance at λ340. All assays used a kinetic mode of a multilabel
microplate reader with an injector, to start the reaction by injecting
3
. Conclusion
NADPH (750
are 10 M and 100
with molecules dissolved in DMSO, including DMSO alone as low-
activity control. B1-PP146, with 1,4-benzoxazine moiety and
μ
M). In the assay, final concentrations of ThyX and dUMP
FDTS of human pathogenic bacteria represent an important target for
μ
μM, respectively. The primary screen was performed
the development of new antibiotic drugs, even if its mechanism of action
is not yet fully elucidated. Twelve new compounds, analogs of acyclic
nucleoside phosphoantes, were synthesized and tested for their activity
against M. tuberculosis ThyX. The synthesized compounds are 5- and 7-
substituted quinazoline and 5-substituted uracil linked at N1 to a (E)-
but-2-enyl phosphonic acid moiety. Only the quinazoline analog 19b
a
described as tight-binding inhibitors, was used as reference com-
pound.30 The concentration of screened compounds was 200 µM
(
Table 1).
Most of the compounds exhibited low inhibition of Mtb ThyX at 200
M; C6-substituted quinazoline AMPs lack of activity compared to their
C7-substituted counterparts. Only, quinazoline analog 19c showed a
1.8% inhibitory effect on ThyX at 200 M. Docking of 19c in the active
site of Mtb ThyX shows that the quinazoline moiety parallels the FAD
through -stacking interactions, moreover the aliphatic chain seems to
showed a poor 31.8% inhibitory effect on ThyX at 200 M. Nevertheless,
μ
μ
based on 19b, future development is needed to develop an optimal
acyclic chain and identify more potent compounds.
3
μ
4
. Experimental section
π
pass over the FAD (Fig. 3 and SI). This conformation could hinder the
approach of different substrates at the N10 position of FAD which seems
4
.1. Chemistry
3
1
to be most crucial for flavin substrate binding to enzyme.
General information. Commercially available chemicals were of re-
Interactions are also formed with residual amino acids involved in
agent grade and used as received. All reactions requiring anhydrous
conditions were carried out using oven-dried glassware and under an
Table 1
2
atmosphere of dry Ar or N . Microwave reactions were carried out in a
Mtb ThyX inhibition at 200
μ
M by the NADPH oxi-
% inhibition
Biotage Initiator apparatus. The reactions under ultrasound were carried
out with Elmasonic P30H apparatus with a frequency of 80 kHz and
effective power of 100 W. The reactions were monitored by thin layer
chromatography (TLC) analysis using silica gel precoated plates (Kie-
selgel 60F254, E. Merck). Compounds were visualized by UV irradiation
and/or spraying with phosphomolybdic acid (PMA) stain, potassium
permanganate solution or ninhydrin stain, followed by charring at
dase assay.
Cmpd
1
1
1
1
1
1
2
2
2
3
3
3
2a
2b
2c
9a
9b
9c
7a
7b
7c
1a
1c
8a
15.8
26.2
26.4
14.9
14.7
31.8
0
◦
around 150 C. Flash column chromatography was performed on Silica
Gel 60 M (0.040–0.063 mm, E. Merck). Melting points were determined
with a Kofler Heizbank Reichert Type 7841 apparatus and are uncor-
rected. The infrared spectra were measured with Perkin–Elmer Spec-
0
14.9
0
1
13
trometer. The H and C NMR spectra were recorded on Bruker Avance
DPX 250 or Bruker Avance 400 Spectrometers. Chemical shifts are given
in ppm and are referenced to the deuterated solvent signal or to TMS as
internal standard and multiplicities are reported as s (singlet), d
(doublet), t (triplet), q (quartet) and m (multiplet). Carbon multiplicities
were assigned by distortion less enhancement by polarization transfer
0
23.7
a
b
B1-PP146
95.1
a
B1-PP146, a compound with a 1,4 benzoxazine
b
ring, used as positive control; value at 50 M. The
μ
standard deviations from two independent experi-
ments is between 3 and 9, as a function of the
molecules.
1
13
(DEPT) experiments. H and C signals were attributed on the basis of
H and H C correlations. High Resolution Mass spectra were
H
–
–
4

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
Fig. 3. (A) Modeling of FAD and 19c in the active site of Mtb ThyX. (B) Interactions of 19c with residual amino acids.
performed on a Bruker Q-TOF MaXis spectrometer by the “F ´e d ´e ration de
Recherche” ICOA/CBM (FR2708) platform.
20 ml). The combined organic layers were washed with Na
2 2 3
S O solu-
tion and brine, dried over MgSO filtered and concentrated under
4
reduced pressure. Pure compounds were obtained after purification on
silica gel column chromatography with DCM/MeOH (98:2) as eluent.
4
.1.1. General synthetic procedure 1 for urea cyclisation for compounds
8
a, 8b and 8c
Amino acid compound (10 g, 1 eq.) and urea (87.6 g, 20 eq.) were
4.1.2.1. 1-(But-2-en-1-yl)quinazoline-2,4(1H,3H)-dione (9a). The title
compound was prepared from 8a to afford after purification the desired
◦
grinded with a mortar pillar and then heated at 150 C for 12 h under
inert atmosphere in a dry flask. After the mixture was cooled down to
product 9a as a white solid (0.48 g, E/Z ratio = 85/15, 72%). CAS #
◦
24 1
1
00 C, 15 ml of water was added and the resultant mixture was stirred
57397–87-2.
6
H NMR (250 MHz, Acetone‑d ) δ 10.23 (bs, 1H, NH),
for another 15 min. After the reaction mixture was cooled down to room
temperature, it was filtered if necessary and aqueous solution of NaOH
8.09 (dd, 1H, J = 7.8, 1.7 Hz, H ), 7.79–7.67 (m, 1H, H ), 7.40 (d, 1H, J
5
7
= 8.5 Hz, H ), 7.29 (dt, 1H, J = 10.0, 7.8 Hz, H ), 5.84–5.37 (m, 2H,
8
6
(
1 M, 150 ml) was then added. The mixture was mixed at room tem-
H
′
′
), 4.83 (d, 1H, J = 6.6 Hz, H
′
), 4.70 (d, 2H, J = 5.2 Hz, H
′
2
,3
1 -minor
1 -
perature another 1 h. After acetic acid was added dropwise until pH 5.
Pure compounds were obtained by filtration of precipitates.
), 1.88–1.82 (m, 1H, H
). Rf : 0.58 (DCM/MeOH 95:5).
′
), 1.66 (dq, 3H, J = 6.3, 1.4 Hz, H
′
major
major
4 -minor
4 -
4
.1.1.1. Quinazoline-2,4(1H,3H)-dione (8a). Following the general
4.1.2.2. 1-(But-2-en-1-yl)-6-methylquinazoline-2,4(1H,3H)-dione (9b).
The title compound was prepared from 8b to afford after purification the
procedure 1, 8a was prepared from commercially available anthranilic
acid 7a (10 g, 1 eq., 72 mmol) and urea (87.6 g, 20 eq., 1.46 mol). The
title compound was obtained after filtration as a white solid (10.4 g,
1
desired product 9b as a white solid (E/Z ratio = 85/15, 84%). H NMR
(400 MHz, Acetone‑d ) δ 10.15 (bs, 1H, NH), 7.88 (s, 1H, H ), 7.54 (dd,
6
5
1
3 1
8
2
1
9%). CAS # 86–96-4.
xNH), 7.88 (dd, 1H, J = 8.4, 1.5 Hz, H
.5 Hz, H
), 7.33–7.04 (m, 2H, H8,7).
H NMR (250 MHz, DMSO‑d
6
) δ 11.20 (bs, 2H,
1H, J = 8.6, 2.2 Hz, H ), 7.29 (d, 1H, J = 8.6 Hz, H8-major), 7.21 (d, 1H, J
7
5
), 7.63 (ddd, 1H, J = 8.4, 7.3,
= 8.6 Hz, H
), 5.82–5.47 (m, 2H, H
′
′
), 4.83–4.77 (m, 2H, H
′
1 -
8
-minor
2 ,3
6
minor
), 4.67 (dt, 2H, J = 5.1, 1.4 Hz, H
1 -major 3
), 2.39 (s, 3H, C-CH ), 1.84
′
(
ddt, 3H, J = 7.0, 1.8, 1.1 Hz, H
4
′
-minor), 1.65 (dq, 3H, J = 6.3, 1.5 Hz,
1
3
–
–
O), 150.8
4
.1.1.2. 6-Methylquinazoline-2,4(1H,3H)-dione (8b). The title com-
H
4
′
-major). C NMR (101 MHz, Acetone‑d
6
) δ 162.4 (C
–
–
O), 140.1 (Cquat), 136.8 (Carom), 133.0 (Cquat), 129.4 (CH = CH),
pound was prepared from commercially available 7b to afford after
filtration the desired product as a white solid (95%). CAS # 62484–16-
(C
129.1 (Cquat), 128.4 (Carom), 125.8 (CH = CH), 115.8 (Carom), 44.4 (N-
1
3 1
6
.
H NMR (250 MHz, DMSO‑d
), 7.45 (dd, 1H, J = 8.3, 2.1 Hz, H
).
6
) δ 11.12 (bs, 2H, 2 × NH), 7.64 (s, 1H,
CH2-major), 40.5 (N-CH2-minor), 20.3 (C-CH
3
), 17.7 (CH-CH3-major), 13.3
+
H
5
8
), 7.06 (d, 1H, J = 8.3 Hz, H ), 2.32
7
(CH-CH3-minor). HRMS-ESI (m/z) [M + H] calcd for C13
15 2 2
H N O :
(
s, 3H, C-CH
3
231.1128, found: 231.1127. Rf : 0.63 (DCM/MeOH 95:5).
4
.1.1.3. 7-Methoxyquinazoline-2,4(1H,3H)-dione (8c). The title com-
4.1.2.3. 1-(But-2-en-1-yl)-7-methoxyquinazoline-2,4(1H,3H)-dione
pound was prepared from commercially available 7c to afford after
(9c). The title compound was prepared from 8c to afford after purifi-
filtration the desired product as a brown/orange solid (35%). CAS #
cation the desired product 9c as a white/brown solid (E/Z ratio = 85/15,
1
3 1
1
6
7
1
2484–12-2.
.75 (d, 1H, J = 8.8 Hz, H
H, J = 2.3 Hz, H ), 3.78 (s, 3H, O-CH
H NMR (250 MHz, DMSO‑d
), 6.72 (dd, 1H, J = 8.8, 2.3 Hz, H
).
6
) δ 11.02 (bs, 2H, 2 × NH),
70%). H NMR (250 MHz, Acetone‑d
6
) δ 10.03 (bs, 1H, NH), 8.00 (d, 1H,
), 6.58 (d,
J = 8.5 Hz, H
4.83–4.81 (m, 2H, H
H, O-CH
), 1.87 (d, 3H, J = 5.6 Hz, H
major). C NMR (63 MHz, Acetone‑d ) δ 166.0 (C
130.7 (Cquat), 130.6 (CH = CH), 130.1 (Cquat), 129.4 (Carom), 125.8 (CH
), 44.68
5
), 6.89–6.77 (m, 2H, H6,8), 5.88–5.50 (m, 2H, H
-minor), 4.76 (d, 2H, J = 5.6 Hz, H -major), 3.93 (s,
-minor), 1.71–1.63 (m, 3H, H
2 ,3
′ ′
),
5
6
′
′
1
8
3
1
3
3
4
′
′
4 -
1
3
–
–
–
–
O), 144.1 (C O),
4
.1.2. General synthetic procedure 2 for crotylation of N1 position for
6
compounds 9a, 9b, 9c, 15, 21, 35a, 35b
In a microwave tube, a suspension of bicyclic nucleobase (1 eq.) in
anhydrous acetonitrile (0.5 M), BSA (2.5 eq.) and 4 Å molecular sieves
were sequentially added under inert atmosphere. The tube was then
= CH), 122.2 (Cquat), 110.3 (Carom), 100.3 (Carom), 56.2 (O-CH
3
(N-CH2-major), 40.6 (N-CH2-minor), 17.8 (CH-CH3-major), 13.3 (CH3-minor).
+
HRMS-ESI (m/z) [M + H] calcd for C13
15 2 2
H N O : 247.1077, found:
◦
sealed and stirred under microwave irradiation at 80 C for 5 min.
247.1077. Rf : 0.67 (DCM/MeOH 95:5).
TMSCl (1 eq.), NaIact (1,12 eq.) and crotyl bromide (2 eq.) were suc-
cessively added. The vial was sealed and heated under microwave
4.1.2.4. 7-Bromo-1-(but-2-en-1-yl)quinazoline-2,4(1H,3H)-dione (15).
The title compound was prepared from commercially available 14 to
afford after purification the desired product as a beige solid, in 37 %
yields, as a mixture of E/Z isomers (85/15). 1H NMR (400 MHz,
◦
irradiation for 30 min at 80 C. The volatiles were removed under
reduced pressure, and the residue diluted in a solution of saturated
NaHCO
3
and EtOAc. The aqueous layer was extracted with EtOAc (3 ×
5

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
Acetone‑d
1.5 Hz 1H, H
), 5.67–5.57 (m, 1H, H
H. J = 5.8, 1.3 Hz, H -major), 1.88 (d, 3H, J = 7.0 Hz, H
-major). C NMR (101 MHz, Acetone‑d
6
) δ 10.22 (bs, 1H, NH), 8.02 (d, 1H, J = 8.4 Hz, H
), 7.54 (dd, 1H, J = 8.4, 1.5 Hz, H ), 5.89–5.75 (m, 1H,
), 4.89 (d, 2H, J = 6.0 Hz, H -minor), 4.76 (dt,
-minor), 1.70
) δ
O), 142.4 (Cquat), 138.6 (Carom),
5
), 7.69 (d, J
4.1.3.1. Tert-butyl 1-(but-2-en-1-yl)-2,4-dioxo-1,4-dihydroquinazoline-3
=
8
6
(2H)-carboxylate (10a). The title compound was prepared from 9a to
H
2
′
3
′
1
′
afford after purification the desired product 10a (E/Z ratio = 85/15,
1
2
1
′
4
′
90%) as a white solid . H NMR (400 MHz, Acetone‑d
6
) δ 8.11 (dd, 1H, J
1
3
(
dd, 3H, J = 6.5, 1.5 Hz, H
4
′
6
= 7.8, 1.6 Hz, H
= 8.8 Hz, H ), 7.34 (t, 1H, J = 7.8 Hz, H
66–5.41 (m, 1H, H
5
), 7.80 (ddd, 1H, J = 8.8, 7.8, 1.6 Hz, H
7
), 7.48 (d, 1H, J
–
–
′
1
59.6 (C
–
O), 148.8 (Cquat), 148.6 (C
–
8
6
), 5.86–5.71 (m, 1H, H
2
), 5
1
30.1 (Carom), 127.2 (CH = CH), 124.9 (CH = CH), 119.1 (Carom), 115.3
3
′
), 4.86 (d, 2H, J = 5.9 Hz, H
1
′
-minor), 4.73 (d, 2H, J =
4.7 Hz, H -minor), 1.67 (d, 2H, J = 6.4
′
Hz, H -major), 1.60 (s, 9H. HBoc). C NMR (101 MHz, Acetone‑d ) δ
4 6
(
C
quat), 45.4 (N-CH2-major), 41.4 (N-CH2-minor), 17.8 (CH-CH3-major), 14.5
1
′
-major), 1.85 (d, 3H, J = 7.0 Hz, H
4
′
+
13
(
CH-CH3-minor). HRMS-ESI (m/z) [M] calcd for
12
C H
12
2
N O
2
Br:
–
–
–
2
95.0075, found: 295.0077. Rf : 0.5 (DCM/MeOH 95:5).
160.1 (C
–
O), 148.9 (C
O), 141.3 (Cquat), 136.7 (Carom), 130.0 (CH =
CH), 129.1 (Cquat), 128.9 (Carom), 125.2 (CH = CH), 124.0 (Carom), 116.1
4
.1.2.5. 6-Bromo-1-(but-2-en-1-yl)quinazoline-2,4(1H,3H)-dione (21).
(Carom), 116.4 (Cquat), 86.5 (O-Cquat), 45.3 (N-CH2-major), 41.2 (N-CH2-
The title compound was prepared from commercially available 20 to
minor), 27.6 (3 × C-CH3Boc), 17.8 (CH-CH3-major), 13.4 (CH-CH3-minor).
+
afford, after purification, the desired product 21 as a beige solid (E/Z
HRMS-ESI (m/z) [M + Na] calcd for C17
20 2 4
H N O Na: 339.1314, found:
1
ratio = 85/15, 43%). H NMR (400 MHz, Acetone‑d
6
) δ 10.38 (bs, 1H,
), 7.85 (dd, 1H, J = 9.0, 2.5 Hz, H ), 7.39
), 5.82–5.72 (m, 1H, H ), 5.61–5.54 (m, 1H, H ),
.83 (d, 2H, J = 6.4 Hz, H -minor), 4.70 (dt, 2H, J = 5.2, 1.4 Hz, H
-minor), 1.67 (dq, 3H, J = 6.4, 1.4 Hz,
339.1314. Rf : 0.81 (PE/ EtOAc 1:1).
NH), 8.16 (d, 1H, J = 2.5 Hz, H
5
7
(
d, 1H, J = 9.0 Hz, H
8
2
′
3
′
4.1.3.2. Tert-butyl 1-(but-2-en-1-yl)-6-methyl-2,4-dioxo-1,4-dihydroquina
4
1
′
1
′
-major),
zoline-3(2H)-carboxylate (10b). The title compound was prepared from
1
.85 (dd, 3H, J = 7.0, 1.7 Hz, H
4
′
9b to afford after purification the desired product 10b as a white solid
1
3
–
–
1
H
4
′
-major). C NMR (101 MHz, Acetone‑d
6
) δ 161.2 (C
O), 150.5
(E/Z ratio = 85/15, 94%). H NMR (400 MHz, CDCl
3
) δ 7.91 (s, 1H, H
), 7.38 (d, 1H, J = 8.7 Hz, H
), 5.65–5 40 (m, 1H, H
), 4.71 (d, 2H, J = 4.7 Hz, H ), 2.41 (s, 3H, C-CH ), 1.85 (d,
5
),
–
–
(
Cquat), 150.2 (C
O), 141.5 (Cquat), 138.3 (Carom), 130.8 (Carom), 129.4
7.62 (dd, 1H, J = 8.7, 2.1 Hz, H
7
8
),
(
CH = CH), 125.3 (CH = CH), 118.3 (Carom), 115.4 (Cquat), 44.7 (N-CH2-
5.84–5.70 (m, 1H, H
1 -minor
2
′
3
′
), 4.84 (d, 2H, J = 6.0 Hz,
major), 40.7 (N-CH2-minor), 17.8 (CH-CH3-major), 13.3 (CH-CH3-minor).
H
′
1 -major
′
3
+
HRMS-ESI (m/z) [M] calcd for C12
H
12
N
2
O
2
Br: 295.0075, found:
3H, J = 7.0 Hz, H
4
′
minor), 1.67 (d, 3H, J = 6.4 Hz, H
4
′
-major), 1.60 (s, 9H,
1
3
–
–
–
O), 149.0 (C O),
2
95.0076. Rf : 0.5 (DCM/MeOH 95:5).
H
Boc). C NMR (101 MHz, Acetone‑d
6
) δ 160.2 (C
–
1
37.7 (Cquat), 137.6 (Carom), 133.8 (Cquat), 129.9 (CH = CH), 128.5
4
.1.2.6. 5-Benzyl-1-(but-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (35a).
(Carom), 125.5 (Cquat), 125.3 (CH = CH), 116.2 (Carom), 115.8 (Cquat),
86.4 (O-Cquat), 45.2 (N-CH2-major), 41.1 (N-CH2-minor), 27.6 (3 × C-
The title compound was prepared from 34a to afford after purification
1
the desired product as an oil (E/Z ratio = 85/15, 97%). H NMR (250
CH3Boc), 20.3 (C-CH
3
), 17.8 (CH-CH3-major), 13.4 (CH-CH3-minor). HRMS-
+
MHz, Acetone‑d
6
) δ 10.06 (bs, 1H, NH), 7.35 (s, 1H, H
6
), 7.31–7.11 (m,
ESI (m/z) [M + Na]
22 2 4
calcd for C18H N O Na: 353.1470, found:
5
H, 5 × Harom), 5.82–5.41 (m, 2H, H
minor), 4.26 (dt, 2H, J = 6.0, 1.1 Hz, H1 -major
.72–1.71 (m, 3H, H -minor), 1.69–1.64 (m, 3H, H
MHz, Acetone‑d ) δ 164.2 (C
quat), 130.8 (CH = CH), 129.5 (2 × Carom), 129.1 (2 × Carom), 126.9
2
′
,3
′
), 4.39 (d, 2H, J = 7.0 Hz, H
1
′
-
353.1469. Rf : 0.81 (PE / EtOAc 1:1).
′
), 3.59 (s, 2H, CH ),
2
1
3
1
4
′
4
′
-major). C NMR (63
O), 142.3 (C = CH), 140.7
4.1.3.3. Tert-butyl 1-(but-2-en-1-yl)-7-methoxy-2,4-dioxo-1,4-dihydroqui
–
–
–
–
6
O), 151.5 (C
nazoline-3(2H)-carboxylate (10c). The title compound was prepared
(
(
C
from 9c to afford after purification the desired product 10c as a white
1
CH = CH), 125.5 (Carom), 114.3 (Cquat), 49.6 (N-CH2-major), 44.5 (N-
solid (E/Z ratio = 85/15, 64%). H NMR (400 MHz, Acetone‑d
6
) δ 8.03
), 6.89 (d, 1H, J
), 5.67–5.42 (m, 1H, H ), 4.86 (d,
-minor), 4.73 (dt, 2H, J = 5.6, 1.3 Hz, H -major), 3.96 (s,
), 1.88 (d, 3H, J = 9.7 Hz, H -minor), 1.73–1.67 (m, 3H, H
major), 1.59 (s, 9H, HBoc). C NMR (101 MHz, Acetone‑d ) δ 159.6
O), 149.1 (Cquat), 143.3 (Cquat), 130.9 (Carom), 130.1
(CH = CH), 129.0 (Cquat), 125.3 (CH = CH), 111.2 (Carom), 109.2 (Cquat),
100.6 (Carom), 86.3 (O-Cquat), 56.4 (O-CH ), 45.3 (N-CH2-major), 41.2 (N-
CH2-minor), 33.0 (C-CH
2
), 17.7 (CH-CH3-major), 13.0 (CH-CH3-minor).
(d, 1H, J = 8.7 Hz, H
= 2.3 Hz, H ), 5.92–5.73 (m, 1H, H
2H, J = 5.8 Hz, H
H, O-CH
5
), 6.92 (dd, 1H, J = 8.7, 2.3 Hz, H
6
+
HRMS-ESI (m/z) [M + H] calcd for C15
H
17
N
2
O
2
: 257.1284, found:
8
2
′
′
3
2
57.1285. Rf : 0.48 (DCM/MeOH 95:5).
1
′
′
1
3
3
4
′
′
4 -
1
3
4
.1.2.7. 1-(But-2-en-1-yl)-5-(phenylamino)pyrimidine-2,4 (1H,3H)-dione
6
–
–
–
(
35b). The title compound was prepared from 34b to afford after pu-
(C
–
O), 149.2 (C
rification the desired product 35b as a white solid (E/Z ratio = 85/15,
1
9
1
7
1%). CAS # 4870–31-9. H NMR (250 MHz, Acetone‑d
H, NH), 7.44 (s, 1H, H
), 7.18 (dd, 2H, J = 8.6, 7.4 Hz, 2 × Harom),
.01–6.94 (m, 2H, 2 × Harom), 6.82–6.73 (m, 1H, Harom), 6.32 (bs, 1H,
), 4.39–4.45 (m, 2H, H -minor), 4.32 (ddt,
-major), 1.83–1.81 (m, 3H, H -minor), 1.73 –
-major). C NMR (63 MHz, Acetone‑d ) δ 162.2 (C
O), 130.9 (Cquat), 130.5 (C = CH), 129.9 (2 × Carom), 126.5
6
) δ 10.21 (bs,
3
CH2-minor), 27.6 (3 × C-CH3Boc), 17.8 (CH-CH3-major), 13.4 (CH-CH3-
6
+
minor). HRMS-ESI (m/z) [M + H] calcd for C18
23 2 5
H N O : 347.1600,
NH), 5.89–5.55 (m, 2H, H
2
′
,3
′
1
′
found: 347.1602. Rf : 0.81 (PE / EtOAc 1:1).
2
1
1
H, J = 6.0, 2.2, 1.1 Hz, H
1
′
′
4
1
3
–
–
O),
.67 (m, 2H, H
4
′
6
4.1.3.4. Tert-butyl 7-bromo-1-(but-2-en-1-yl)-2,4-dioxo-1,4-dihydroqui-
–
–
45.6 (C
nazoline-3(2H)-carboxylate (16). The title compound was prepared
(
CH = CH), 125.7 (CH = CH), 120.3 (Carom), 118.7 (Cquat), 116.5 (2 ×
from 15 to afford after purification the desired product 16 as an oil (E/Z
1
C
arom), 49.75 (N-CH2-major), 44.7 (N-CH2-minor), 17.8 (CH-CH3-major),
ratio = 85/15, 85%). H NMR (400 MHz, Acetone‑d
6
) δ 8.04 (dd, 1H, J
), 7.59–7.50 (m, 1H, H ),
), 4.90 (d, 2H, J = 6.0 Hz,
-major), 1.90 (d, 3H, J = 7.0
-minor), 1.71 (dd, 3H, J = 6.5, 1.5 Hz, H -major), 1.62 (s, 9H, HBoc).
+
1
3.1 (CH-CH3-minor). HRMS-ESI (m/z) [M + H] calcd for C14
H
16
3
N O
2
:
= 8.4, 2.7 Hz, H
5.91–5.78 (m, 1H, H
-minor), 4.78 (dd, 2H, J = 5.4, 1.4 Hz, H
Hz, H
5
), 7.68 (d, 1H, J = 2.7 Hz, H
6
8
2
58.1234, found: 258.1234. Rf : 0.4 (DCM/MeOH 95:5).
2
′
), 5.69–5.45 (m, 1H, H
′
3
H
1
′
′
1
4
.1.3. General synthetic procedure 3 for N-Boc protection for compounds
0a, 10b, 10c, 16, 22, 36a, 36b
Under inert atmosphere to a solution of N1 alkylated compound,
4
′
′
4
1
3
–
–
–
–
O), 148.8 (C O), 148.6
1
C NMR (101 MHz, Acetone‑d
6
) δ 159.6 (C
(Cquat), 142.4 (Cquat), 130.8 (Cquat), 130.6 (Carom), 129.8 (CH = CH),
respectively, (1 eq.) in dry THF (0.25 M) were successively added
127.2 (Carom), 125.0 (CH = CH), 119.0 (Carom), 115.3 (Cquat), 86.8 (O-
dimethylaminopyridine (0.5 eq.) and dicarbonate de di-tert-butyle (2
C
quat), 45.4 (N-CH2-major), 41.4 (N-CH2-minor), 27.6 (3 × C-CH3Boc), 17.8
◦
+
eq.). The sealed tube was heated under microwave irradiation at 70 C
(CH-CH3-major), 14.51 (CH-CH3-minor). HRMS-ESI (m/z) [M + Na] calcd
for 10 min. Sealed tube was degas before opened. Volatiles were evap-
orated under reduced pressure. Pure compounds were obtained after
purification on silica gel column chromatography with PE/EtOAc (8:2)
as eluent.
for C17
1:1).
19 2 4
H N O Na: 418.0422, found: 418.0422. Rf : 0.80 (PE / EtOAc
6

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
4
.1.3.5. Tert-butyl 6-bromo-1-(but-2-en-1-yl)-2,4-dioxo-1,4-dihydroqui-
6 h. After completion of the reaction, the mixture was diluted in EtOAc
nazoline-3(2H)-carboxylate (22). The title compound was prepared
3 4
and washed twice with saturated NaHCO solution, dried over MgSO
from 21 to afford after purification the desired product 22 as an oil (E/Z
filtered and concentrated under reduced pressure. Pure compounds
were purified by column chromatography on silica gel with DCM/MeOH
(97:3) as eluent.
1
ratio = 85/15) in 80% yields. H NMR (400 MHz, Acetone‑d
6
) δ 8.18 (d,
), 7.46 (d, 1H, J =
), 5.65–5.40 (m, 1H, H ), 4.86 (d, 2H,
-minor), 4.73 (dt, 2H, J = 5.6, 1.3 Hz, H -major), 1.84 (d, 3H,
-minor), 1.67 (dd, 3H, J = 6.5, 1.5 Hz, H -major), 1.60 (s,
1
H, J = 2.5 Hz, H
5
), 7.92 (dd, 1H, J = 9.0, 2.5 Hz, H
7
9
.0 Hz, H
8
), 5.88–5.71 (m, 1H, H
1
2
′
′
3
J = 6.1 Hz, H
′
1
′
4.1.4.1. Dimethyl (4-(2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)but-2-
en-1-yl)phosphonate (11a). The title compound was prepared from 10a
to afford after purification the desired product as a white/green solid (E/
J = 8.6 Hz, H
4
′
′
4
1
3
–
–
O), 148.6
9
H, HBoc). C NMR (101 MHz, Acetone‑d
6
) δ 159.1 (C
O), 148.5 (Cquat), 140.6 (Cquat), 139.1 (Carom), 130.9 (Carom), 130.1
CH = CH), 124.8 (CH = CH), 118.7 (Carom), 118.3 (Cquat), 116.1 (Cquat),
–
–
1
(
C
Z ratio = 85/15, 51% over two steps). H NMR (250 MHz, DMSO‑d ) δ
6
(
11.60 (bs, 1H, NH), 8.00 (dd, 1H, J = 7.8, 1.5 Hz, H ), 7.72 (ddd, 1H, J
5
8
6.9 (O-Cquat), 44.5 (N-CH2-major), 41.2 (N-CH2-minor), 27.6 (3 × C-
= 8.7, 7.8, 1.5 Hz, H ), 7.37 (d, 1H, J = 8.7 Hz, H ), 7.26 (t, 1H, J = 7.8
7
8
CH3Boc), 178 (CH-CH3-major), 13.4 (CH-CH3-minor). HRMS-ESI (m/z) [M
Hz, H ), 5.78–5.51 (m, 2H, H
′
′
), 4.80–4.77 (m, 2H, H
′
1 -minor
), 4.68 (t,
6
2 ,3
+
+
Na] calcd for C17
H
19
2
N O
4
Na: 418.0420, found: 418.0420. Rf : 0.80
2H, J = 4.7 Hz, H
′
-major), 3.70 (s, 3H, HPOMe-minor), 3.66 (s, 3H, HPOMe-
1
(
PE / EtOAc 1:1).
minor
), 3.58 (s, 3H, HPOMe-major), 3.53 (s, 3H, HPOMe-major), 2.93 (dd, 2H, J
1
3
=
21.7, 6.7 Hz, H
4
′
-minor), 2.66 (dd, 2H, J = 21.7, 6.7 Hz, H
4
′
-major).
–
–
O), 140.7 (Cquat),
C
–
4
.1.3.6. Tert-butyl 5-benzyl-3-(but-2-en-1-yl)-2,6-dioxo-3,6-dihydropyri-
NMR (63 MHz, DMSO‑d
6
) δ 161.7 (C
–
O), 150.0 (C
midine-1(2H)-carboxylate (36a). The title compound was prepared
135.1 (Carom), 129.2 (d, J3C-P = 14.4 Hz, CH = CH), 127.4 (Carom),
from 35a to afford after purification the desired product 36a as an oil
122.55 (Carom), 122.5 (d, J2C-P = 11.3 Hz, CH = CH), 115.7 (Cquat), 115.1
1
), 27.9 (d, J1C-P
(
E/Z ratio = 85/15) in 95% yields. H NMR (400 MHz, Acetone‑d
6
) δ
(Carom), 52.2 (O-CH
3
), 52.2 (O-CH
3
), 43.2 (N-CH
2
=
7
.47 (s, 1H, H
arom), 5.83–5.74 (m, 1H, H
.1 Hz, H
-minor), 4.31 (d, 2H, J = 6.4 Hz, H
-major), 1.55 (s, 9H, HBoc). C NMR (101 MHz,
6
), 7.30–7.26 (m, 4H, 4 × Harom), 7.23–7.16 (m, 1H,
), 5.62–5.48 (m, 1H, H ), 4.44 (d, 2H, J =
-major), 3.62 (s, 2H, C-CH ),
136.7 Hz, CH-CH
(P
2
). 31P NMR (101 MHz, DMSO‑d
6
) δ 29.58 (P
Z
), 29.25
+
H
2
′
3
′
E
). HRMS-ESI (m/z) [M + H] calcd for C14
17 2 5
H N O P: 325.2695,
7
1
′
1
′
2
found: 325.2696. Rf : 0.35 (DCM/MeOH 95:5).
1
3
1
.69 (d, 3H, J = 6.5 Hz, H
4
′
–
–
–
–
Acetone‑d
6
) δ 161.6 (C
O), 149.2 (C
O), 142.4 (C = CH), 140.1
4.1.4.2. Dimethyl (4-(6-methyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
yl)but-2-en-1-yl)phosphonate (11b). The title compound was prepared
from 10b to afford after purification the desired product as a white/
(
C
quat), 131.7 (CH = CH), 130.5 (Cquat), 129.6 (2 × Carom), 129.2 (2 ×
C
arom), 127.1 (Carom), 125.8 (CH = CH), 114.0 (Cquat), 86.3 (O-Cquat),
1
5
0.2 (N-CH2-major), 45.2 (N-CH2-minor), 33.1 (C-CH
2
), 27.6 (3 × C-
green foam (E/Z ratio = 85/15, 56% over two steps). H NMR (400 MHz,
CH3Boc), 17.8 (CH-CH3-major), 13.1 (CH-CH3-minor). HRMS-ESI (m/z) [M
Acetone‑d ) δ 10.25 (bs, 1H, NH), 7.88 (s, 1H, H ), 7.55 (d, 1H, J = 6.7
6
5
+
+
Na] calcd for C20
H
24
2
N NaO
4
: 379.1628, found: 379.1626. Rf : 0.71
Hz, H ), 7.32 (d, 1H, J = 8.5 Hz, H ), 5.82–5.62 (m, 2H, H
′
′
), 4.88 (t,
7
8
2 ,3
(
EDP/AcOEt 1:1).
2H, J = 3.6 Hz, H
′
), 4.74 (t, 1H, J = 4.6 Hz, H
1 -major
), 3.76 (s, 3H,
′
1
-minor
H
POMe-minor), 3.74 (s, 3H, HPOMe-minor), 3.63 (s, 3H, HPOMe-major), 3.61 (s,
4
.1.3.7. Tert-butyl 3-(but-2-en-1-yl)-2,6-dioxo-5-(phenylamino)-3,6-dihy-
3H, HPOMe-major), 2.92 (dd, 2H, J = 22.5, 6.1 Hz, H
J = 21.8, 6.9 Hz, H -major), 2.38 (s, 3H, C-CH
). 13C NMR (101 MHz,
O), 140.0 (Cquat), 136.8 (Carom),
4
′
-minor), 2.61 (dd, 2H,
dropyrimidine-1(2H)-carboxylate (36b). The title compound was pre-
4
′
3
–
–
–
pared from 35b to afford after purification the desired product as an oil
Acetone‑d
6
) δ 162.4 (C
–
O), 150.8 (C
1
3
(
E/Z ratio = 85/15, 96%). H NMR (400 MHz, Acetone‑d
6
) δ 7.93 (s, 1H,
133.1 (Cquat), 129.2 (d, J C-P = 15.1 Hz, CH = CH), 128.4 (Carom), 123.8
(d, J2C-P = 10.8 Hz, CH = CH), 117.0 (Cquat), 115.8 (Carom), 52.9 (O-CH3-
minor), 52.8 (O-CH3-minor), 52.7 (O-CH3-major), 52.7 (O-CH3-major), 44.4
H
6-minor), 7.91 (s, 1H, H6-major), 7.39–7.28 (m, 4H, 4 × Harom), 7.18 (t,
H, J = 7.1 Hz, Harom), 5.86–5.76 (m, 1H, H ), 5.67–5.49 (m, 1H, H ),
.50 (d, 2H, J = 6.8 Hz, H -minor), 4.37 (d, 2H, J = 6.1 Hz, H -major), 1.75
-minor), 1.70 (d, 3H, J = 6.5 Hz, H -major), 1.57 (s,
H, HBoc), 1.42 (s, 9H, HBoc). C NMR (101 MHz, Acetone‑d ) δ 159.5
1
4
2
′
′
3
(N-CH2-major), 40.7 (N-CH2-minor), 29.5 (d, J1C-P = 138.58 Hz, CH-CH2-
′
′
1
1
(
d, 3H, J = 8.0 Hz, H
4
′
4
′
major), 26.5 (d, J1C-P = 139.3 Hz, CH-CH2-minor), 20.3 (C-CH3-major).
31
P
1
3
9
6
NMR (162 MHz, Acetone‑d
6
Z E
) δ 28.76 (P ), 28.28 (P ). HRMS-ESI (m/z)
–
–
+
(
C
–
O), 154.2 (Cquat), 149.0 (C
–
O), 148.6 (Cquat), 143.5 (C = CH),
[M + H] calcd for C15
19 2 5
H N O P: 339.1104, found: 339.1104. Rf : 0.35
1
31.8 (CH = CH), 129.3 (2 × Carom), 126.7 (Carom), 126.6 (2 × Carom),
(DCM/MeOH 95:5).
1
25.6 (CH = CH), 124.72 (Cquat), 118.7 (Cquat), 86.8 (O-Cquat), 81.6 (O-
C
quat), 50.7 (N-CH2-major), 45.9 (N-CH2-minor), 28.22 (3 × C-CH3Boc), 27.6
4.1.4.3. Dimethyl (4-(7-methoxy-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
yl)but-2-en-1-yl)phosphonate (11c). The title compound was prepared
from 10c to afford after purification the desired product as an oil (E/Z
(
(
:
3 × C-CH3Boc) , 17.8 (CH-CH3-major), 14.5 (CH-CH3-minor). HRMS-ESI
+
m/z) [M + H] calcd for C24
32 3 6
H N O : 458.2285, found: 458.2285. Rf
1
0.70 (EDP/AcOEt 1:1).
ratio = 85/15, 42% over two steps). H NMR (400 MHz, DMSO‑d ) δ
6
1
1.42 (bs, 1H, NH), 7.90 (d, 1H, J = 8.7 Hz, H
5
), 6.83 (dd, 1H, J = 8.7,
4
.1.4. General synthetic procedure 4 for ruthenium-catalyzed cross-
metathesis and subsequent Boc removal for compounds 11a, 11b, 11c, 17,
7a, 37b
A mixture of protected alkylated nucleobase (1 eq.) and dimethyl
2.2 Hz, H ), 6.77 (d, 1H, J = 2.2 Hz, H ), 5.64–5.60 (m, 2H, H
′
′
),
6
8
2 ,3
4.79–4.73 (m, 2H, H
1
′
-minor
), 4.69 (t, 2H, J = 4.7 Hz, H
′
1 -major
), 3.89 (s,
3
3H, O-CH ), 3.69 (s, 3H, H
POMe-minor
), 3.66 (s, 3H, HPOMe-minor), 3.55 (s,
3
3H, HPOMe-major), 3.53 (s, 3H, HPOMe-major), 2.96 (dd, 2H, J = 22.6, 6.4
1
3
allylphosphonate (4 eq.), were stirred at room temperature in freshly
distilled DCM (0.1 M), 5 mol% Grubbs 2nd generation catalyst was
Hz, H4
′
), 2.68 (dd, 2H, J = 21.6, 6.8 Hz, H
4 -major
). C NMR (101
′
-minor
MHz, DMSO‑d ) δ 164.6 (C
(Cquat), 129.3 (Carom), 128.7 (d, J
6
–
O), 161.2 (C ), 150.3 (C O), 142.6
quat
–
◦
3
C-P
2
added. The mixture was sonicated at 55 C (80 kHz, 100 W) under inert
= 14.5 Hz, CH = CH), 122.6 (d, J
C-
atmosphere. After 2 h, 5 mol% of the Grubbs-II catalyst was added to the
= 10.9 Hz, CH = CH), 110.0 (C
arom
), 108.8 (C ), 99.3 (C ), 55.7
P
arom
quat
◦
solution. The reaction was sonicated for 2 h at 55 C before the addition
(C
–
O
–
CH ), 52.3 (O-CH
), 52.3 (O-CH3-minor), 52.2 (O-CH
3-major
),
3
3-minor
52.1 (O-CH3-major), 43.3 (N-CH2-major), 36.85 (N-CH2-minor), 27.8 (d, J1
of a third portion of 5 mol% of the ruthenium catalyst. The reaction was
sonicated 18 h under those conditions. Volatiles were eliminated under
reduced pressure. Crude product was applied on short flash chroma-
tography with EtOAc as eluent to afford desired compound which was
engaged directly on the Boc removal; thus, a solution of obtained pro-
tected phosphonate compound (1 eq.) in freshly distilled DCM (0.05 M)
was stirred with trifluoroacetic acid (20 eq.) under inert atmosphere for
C-
= 136.4 Hz, CH-CH
2-major
), 24.3 (d, J1 = 136.7 Hz, CH-CH
C-P
2-minor
+
).
P
3
1
P NMR (162 MHz, DMSO‑d ) δ 29.35. HRMS-ESI (m/z) [M + H] calcd
6
for C
1
5
20 2 6
H N O P: 355.1053, found: 355.1053. Rf : 0.35 (DCM/MeOH
95:5).
7

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
4
.1.4.4. Dimethyl (4-(7-bromo-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
lyophilized to yield the pure compound.
yl)but-2-en-1-yl)phosphonate (17). The title compound was prepared
from 16 according to the general procedure 4, affording after purifica-
4.1.5.1. (4-(2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)but-2-en-1-yl)
phosphonic acid (12a). The title compound was prepared according to
the general procedure 5 from 11a to afford after lyophilisation the
tion the desired product 17 as an oil (E/Z ratio = 85/15, 55% over two
1
steps). H NMR (400 MHz, DMSO‑d
6
) δ 10.44 (s, 1H, NH), 7.90 (d, 1H, J
1
=
8.4 Hz, H
), 5.76–5.66 (m, 1H, H
Hz, H
-minor), 4.68 (t, 2H, J = 4.5 Hz, H
.67 (s, 3H, HPOMe-minor), 3.58 (s, 3H, HPOMe-major), 3.56 (s, 3H, HPOMe-
), 2.67 (dd, 2H, J = 21.7,
5
), 7.56 (d, 1H, J = 1.6 Hz, H
8
), 7.44 (dd, 1H, J = 8.4, 1.6 Hz,
desired product as a white solid (E/Z ratio = 85/15, 95%). H NMR (250
H
6
2
′
), 5.63–5.51 (m, 1H, H
3
′
), 4.80 (t, 2H, J = 3.8
MHz, DMSO‑d ) δ 11.58 (bs, 1H, NH), 9.33 (bs, 2H, 2 × H
POH
), 7.99 (dd,
6
′
′
-major), 3.70 (s, 3H, HPOMe-minor),
1
1
1H, J = 7.8, 1.3 Hz, H ), 7.77–7.63 (m, 1H, H ), 7.38 (d, 1H, J = 8.4 Hz,
5
7
3
H ), 7.25 (t, 1H, J = 7.8 Hz, H ), 5.77–5.56 (m, 2H, H
′ ′
), 4.78–4.73 (m,
8
6
2 ,3
major), 2.94 (dd, 2H, J = 22.5, 6.5 Hz, H4 -minor
′
2H, H1 -minor), 4.69–4.59 (m, 2H, H1 -major), 2.67 (dd, 2H, J = 21.2, 5.7
′
′
1
3
–
–
13
7
.1 Hz, H -major). C NMR (101 MHz, DMSO‑d
4
′
6
) δ 161.2 (C
O), 149.8
Hz, H4
′
), 2.38 (dd, 2H, J = 21.3, 5.6 Hz, H
4 -major
). C NMR (63
′
-minor
–
–
3
–
–
–
O), 150.0 (C
–
(
C
O), 141.8 (Cquat), 129.2 (Carom), 129.0 (Cquat), 128.3 (d, J C-P
=
MHz, DMSO‑d ) δ 161.8 (C
(Carom), 127.4 (Carom), 127.1 (d, J
C-P
O), 140.8 (Cquat), 135.2
= 14.0 Hz, CH = CH), 125.0 (d,
6
4.5 Hz, CH = CH), 125.6 (Carom), 122.3 (d, J2C-P = 10.8 Hz, CH = CH),
3
1
1
2
2
4
2
17.7 (Carom), 114.9 (Cquat), 52.2 (O-CH
3
), 52.2 (O-CH
3
), 43.2 (N-CH
2
),
) δ
P:
J
= 10.2 Hz, CH = CH), 122.5 (C
), 115.7 (C ), 115.1 (C
arom
),
C-P
arom
quat
31
1
31
1
8.0 (d, J C-P = 136.3 Hz, CH-CH
2
). P NMR (162 MHz, DMSO‑d
6
43.4 (N-CH ), 32.0 (d, J
= 134.4 Hz, CH-CH ). P NMR (101 MHz,
2
C-P
2
+
+
9.16 (P
E
), 28.93 (P
Z
). HRMS-ESI (m/z) [M] calcd for C14
H17BrN
2
O
5
DMSO‑d ) δ 22.04 (P ), 21.72 (P ). HRMS-ESI (m/z) [M + H] calcd for
6
Z
E
03.0052, found: 403.0051. Rf : 0.32 (DCM/MeOH 95:5).
12 13 2 5
C H N O P: 296.0635, found: 296.0634.
4
.1.4.5. Dimethyl (4-(5-benzyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)
4.1.5.2. (4-(6-Methyl-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)but-2-
but-2-en-1-yl)phosphonate (37a). The title compound was prepared ac-
cording to the general procedure 4 from 36a to afford after purification
en-1-yl)phosphonic acid (12b). The title compound was prepared ac-
cording to the general procedure 5 from 11b to afford after lyophilisa-
1
1
the desired product 37a as an oil (E/Z ratio = 85/15, 51%). H NMR
tion the desired product as a white solid (E/Z ratio = 85/15, 97%).
H
(
(
250 MHz, Acetone‑d
6
) δ 10.43 (bs, 1H, NH), 7.65 (s, 1H, H6-minor), 7.41
NMR (250 MHz, DMSO‑d ) δ 11.48 (s, 1H, NH), 7.74 (s, 1H, H ), 7.48 (d,
6
5
s, 1H, H6-major), 7.31–7.22 (m, 5H, 5 × Harom), 5.79–5.59 (m, 2H, H
.47 (t, 2H, J = 4.4 Hz, H -minor), 4.32 (t, 2H, J = 4.4 Hz, H -major), 3.73
s, 3H, HPOMe-minor), 3.69 (s, 3H, HPOMe-major), 3.65 (s, 3H, HPOMe-major),
2
′
,3
′
),
1H, J = 8.6 Hz, H ), 7.23 (d, 1H, J = 8.6 Hz, H ), 6.00–5.59 (bs, 2H, 2 ×
7
8
4
1
′
1
′
H
), 5.61–5.48 (m, 2H, H
′
′
), 4.71–4.66 (m, 2H, H
′
), 4.65–4.52
POH
2 ,3
1 -minor
(
(m, 2H, H1
′
), 2.61 (dd, 2H J = 22.5, 7.5 Hz, H
4 -minor
), 2.32 (dd, 2H,
′
-major
J = 22.5, 7.5 Hz, H H
4
′
-major 3
), 2.27 (s, 3H, C-CH ). 13C NMR (63 MHz,
3
2
.59 (s, 2H, C-CH
2
), 2.86 (dd, 2H, J = 22.6, 6.6 Hz, H
4
′
-minor), 2.66 (dd,
1
3
H, J = 21.7, 5.8 Hz, H
4
′
-major). C NMR (63 MHz, Acetone‑d
6
) δ 164.4
DMSO‑d ) δ 162.2 (C
–
O), 150.4 (C
–
O), 139.1 (Cquat), 136.5 (Carom),
6
–
–
–
–
O), 142.5 (C = CH), 140.7 (Cquat), 130.0 (d, J3C-P
=
), 125.2 (d, J2 = 10.2 Hz,
(
C
O), 151.6 (C
132.2 (Cquat), 127.7 (CH = CH), 127.5 (C
arom
C-P
1
1
1
4.4 Hz, CH = CH), 129.4 (2 × Carom), 129.0 (2 × Carom), 126.8 (Carom),
CH = CH), 115.9 (C ), 115.6 (C
), 43.8 (N-CH ), 32.4 (d, J
C-P
=
quat
arom
31
2
2
25.2 (d, J C-P = 10.9 Hz, CH = CH), 114.3 (Cquat), 52.9 (O-CH
3
), 52.8
134.2 Hz, CH-CH ), 20.4 (C-CH ). P NMR (101 MHz, DMSO‑d ) δ
2
3
6
-
(
O-CH
3
), 49.3 (N-CH2-major), 44.8 (N-CH2-minor), 33.0 (C-CH
2
), 29.3 (d,
21.81 (P ), 21.62 (P ). HRMS-ESI (m/z) [Mꢀ H] calcd for C13H14N O P:
Z
E
2 5
1
C-P = 138.22 Hz, CH-CH2-major), 25.1 (d, J1C-P = 137.8 Hz, CH-CH2-
J
309.0645, found: 309.0645.
3
1
minor). P NMR (162 MHz, Acetone‑d
6
Z E
) δ 28.81 (P ), 28.58 (P ). HRMS-
+
ESI (m/z) [M + H] calcd for C17
22 2 5
H N O P: 365.1258, found: 365.1260.
4
.1.5.3. (4-(7-Methoxy-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)but-2-
Rf : 0.35 (DCM/MeOH 95:5).
en-1-yl)phosphonic acid (12c). The title compound was prepared ac-
cording to the general procedure 5 from 11c to afford after lyophilisa-
1
4
.1.4.6. Dimethyl (4-(2,4-dioxo-5-(phenylamino)-3,4-dihydropyrimidin-1
tion the desired product as a white solid (E/Z ratio = 85/15, 94%).
H
(
2H)-yl)but-2-en-1-yl)phosphonate (37b). The title compound was pre-
NMR (400 MHz, DMSO‑d ) δ 11.40 (s, 1H, NH), 7.88 (d, 1H, J = 8.8 Hz,
6
pared according to the general procedure 4 from 36b to afford after
H ), 6.81 (d, 1H, J = 8.8 Hz, H ), 6.76 (s, 1H, H ), 6.48 (bs, 2H, 2 ×
5
6
8
purification the desired product 37b as a white/blue foam (E/Z ratio =
H
), 5.77–5.53 (m, 2H, H
′
′
), 4.81–4.72 (m, 2H, H
′
), 4.70–4.58
POH
2 ,3
1 -minor
1
8
5/15, 53%). H NMR (250 MHz, Acetone‑d
6
) δ 10.71 (bs, 1H, NH), 7.59
(m, 2H, H1
′
), 3.87 (s, 3H, H ), 2.66 (dd, 2H, J = 20.8, 6.9 H
13
-major
13
12-
(
s, 1H, H6-minor), 7.44 (s, 1H, H6-major), 7.17 (t, 2H, J = 7.4 Hz, 2 ×
), 2.40 (dd, 2H, J = 21.4, 6.7 Hz, H
12-major
). C NMR (101 MHz,
minor
H
H
=
arom), 6.98 (d, 2H, J = 8.0 Hz, 2 × Harom), 6.75 (t, 1H, J = 7.4 Hz,
arom), 6.47 (bs, 1H, NH), 5.77 (d, 2H, J = 5.1 Hz, H ), 4.50 (d, 2H, J
5.6 Hz, H -major), 3.70 (s, 3H, HPOMe-
-minor), 4.41–4.35 (m, 2H, H
DMSO‑d ) δ 164.7 (C
–
O), 161.3 (C ), 150.4 (C
–
O), 142.8 (Cquat),
6
quat
3
C-P
), 125.1 (d, J2
′
′
2
,3
129.3 (Carom), 127.9 (d, J
= 10.3 Hz, CH = CH-
= 13.1 Hz, CH = CH
-minor
C-P
′
′
1
1
major
), 124.4 (d, J = 10.8 Hz, CH = CH ), 110.2
Z
minor), 3.67 (s, 3H, HPOMe-major), 3.63 (s, 3H, HPOMe-major), 2.87 (dd, 2H, J
3 2
(Carom), 108.8 (Cquat), 99.2 (Carom), 55.8 (O-CH ), 43.6 (N-CH ), 32.0 (d,
=
22.6, 7.6 Hz, H
4
′
-minor), 2.67 (dd, 2H, J = 21.7, 5.6 Hz, H
4
′
-major). 13
O), 145.3 (Cquat),
C
J = 134.3 Hz, CH-CH
31
), 28.3 (d, J1 = 133.1 Hz, CH-CH2-minor).
2
-major
C-P
–
–
–
–
NMR (63 MHz, Acetone‑d
6
) δ 162.5 (C
O), 150.3 (C
P NMR (162 MHz, DMSO‑d ) δ 22.19 (P ), 21.80 (P ). HRMS-ESI (m/z)
6
Z
E
3
-
1
1
30.2 (d, J C-P = 14.4 Hz, CH = CH), 130.0 (C = CH), 129.9 (2 × Carom),
[Mꢀ H] calcd for C13H14N O P: 325.0594, found: 325.0594.
2
6
2
25.1 (d, J C-P = 10.9 Hz, CH = CH)., 120.2 (Carom), 118.9 (Cquat), 116.5
(
2 × Carom), 52.9 (O-CH
3 3
), 52.8 (O-CH ) , 49.5 (N-CH2-major), 45.2 (N-
4
.1.5.4. (4-(7-(3-Octanamidoprop-1-ynyl)-2,4-dioxo-3,4-dihy-
CH2-minor), 29.4 (d, J C-P = 138.38 Hz, CH-CH2-major), 25.1 (d, J1C-P
1
=
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (19a). The title
3
1
1
37.9 Hz, CH-CH2-minor). P NMR (162 MHz, Acetone‑d
6
) δ 28.73.
compound was prepared from 18a to afford after lyophilisation the
+
1
HRMS-ESI (m/z) [M + H] calcd for C17
21 3 5
H N O P: 366.1213, found:
desired product 19a as a beige solid (qtf). H NMR (400 MHz, DMSO‑d
6
)
3
66.1213. Rf : 0.30 (DCM/MeOH 95:5).
δ 11.65 (bs, 1H, NH), 8.38 (t, 1H, J = 5.4 Hz, NH), 7.95 (d, 1H, J = 8.0
Hz, H
5
), 7.35 (s, 1H, H
8
), 7.23 (d, 1H, J = 8.0 Hz, H
6
), 5.61 (t, 2H, J =
4
.1.5. General synthetic procedure 5 for dimethyl phosphonate deprotection
′
′
′
3
.6 Hz, H2 ,3 ), 4.65 (d, 2H, J = 4.0 Hz, H
1
), 4.15 (d, 2H, J = 5.4 Hz, NH-
for compounds 12a, 12b, 12c, 19a, 19b, 19c, 38a
To dry ACN (15 ml) solution of acyclo nucleosides dia-
′
CH
2
), 2.37 (dd, 2H, J = 21.9, 4.8 Hz, H
4
), 2.11 (t, 2H, J = 7.4 Hz, CH
), 0.83 (t,
-CH ) δ 171.9 (NH-
–
–
O), 149.8 (C O), 140.7 (Cquat), 128.7 (Cquat), 127.7
2
),
a
1
.49 (p, 2H, J = 7.4 Hz, CH
2
), 1.22 (d, 8H, J = 6.3 Hz, 4 × CH
2
1
3
lkylphosphonate (1 eq.), TMSBr (15 eq.) was added under inert atmo-
3
H, J = 6.6 Hz, CH
2
3 6
). C NMR (101 MHz, DMSO‑d
◦
sphere and heated under microwave irradiation at 80 C for 10 min.
–
–
CO), 161.1 (C
3
MeOH was added and evaporated with heating. MeOH was added again,
and this procedure was repeated four times. The residue dissolved in
(
C
arom), 126.7 (d, J C-P = 14.1 Hz, CH = CH), 125.2 (Carom), 124.6 (d,
2
J
8
C-P = 10.1 Hz, CH = CH), 117.3 (Carom), 115.2 (Cquat), 91.0 (CH
2
-C),
), 31.9 (d, J1C-P = 135.3 Hz,
ultrapure H
2
O and extracted with DCM. The aqueous layer was
–
0.6 (C C-C), 43.2 (N-CH
–
2 2
), 34.9 (CO-CH
8

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
CH
CH
2
-P), 31.0 (CH
2
-CH
), 21.9 (CH
21.37. HRMS-ESI (m/z) [Mꢀ H] calcd for
P: 474.1797, found: 474.1799.
2
), 28.4 (CH
2
-CH
2
), 28.4 (NH-CH
2
), 28.3 (CH
2
-
2H, J = 5.2, 2.6 Hz, H
3
), 2.25–2.15 (m, 3H), 1.69–1.56 (m, 2H, H
7
),
3
1
2
), 25.0 (CH -CH
2
2
2
-CH
2
), 13.8 (CH
2
-CH
3
). P NMR (162
1.38–1.20 (m, 8H), 0.92–0.81 (m, 3H, H12).
-
6
MHz, DMSO‑d ) δ
C
23
H
29
3
N O
6
4.1.7. 2-Phenyl-N-prop-2-ynyl-acetamide (13b)
Propargylamine (13) (1 eq.) was dissolved in anhydrous DCM (0.41
◦
4
.1.5.5. (4-(2,4-Dioxo-7-(3-(2-phenylacetamido)prop-1-ynyl)-3,4-dihy-
M), DIPEA (1.3 eq.) was added and the solution was cooled to 0 C.
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (19b). The title
Phenylacetyl chloride (1.1 eq.) was added dropwise and the reaction
mixture was stirred at rt for 2 h. The reaction was quenched with water,
compound was prepared from 18b to afford after lyophilisation the
1
desired product 19b as a beige solid (qtf). H NMR (250 MHz, DMSO‑d
6
)
3
then diluted with DCM and washed with NaHCO solution and brine.
δ 11.64 (bs, 1H, NH), 8.66 (t, 1H, J = 5.4 Hz, NH), 7.96 (d, 1H, J = 8.1
Hz, H ), 7.36 (s, 1H, H
m, 2H, H
Organic layer was dried over MgSO4 and evaporated. The title com-
5
8
), 7.33–7.16 (m, 6H, H
), 4.18 (d, 2H, J = 5.3 Hz, NH-
7
, 5 × Harom), 5.78–5.52
pound 13b was obtained without further purification as a solid (93%).
1
6 1
(
2
′
,3
′
), 4.66 (d, 1H, J = 5.0 Hz, H
1
′
CAS # 174271–37-5.
arom), 5.54 (bs, 1H, H
H ), 2.18 (t, 1H, J = 2.6 Hz, H ).
H NMR (250 MHz, CDCl
3
) δ 7.42–7.22 (m, 5H,
), 3.60 (s, 2H,
1
3
CH
2
), 3.48 (s, 2H, CH
2
), 2.44–2.31 (m, 2H, H
4
′
). C NMR (63 MHz,
H
4
), 4.01 (dd, 2H, J = 5.3, 2.6 Hz, H
3
–
–
–
DMSO‑d
6
) δ 170.0 (NH-CO), 161.1 (C
O), 149.8 (C
–
O), 140.7 (Cquat),
6
1
1
35.9 (Carom), 130.5 (Cquat), 128.8 (2xCarom), 128.6 (Cquat), 128.1
(
2xCarom), 127.7 (Carom), 126.3 (CH = CH), 126.3 (Carom), 124.9 (CH =
4.1.8. 1-Hexyl-3-prop-2-ynyl-urea (13c)
–
CH), 117.2 (Carom), 115.4 (Cquat), 90.6 (CH
2
-C), 81.0 (C
–
C-C), 43.3 (N-
Propargylamine (13) (1 eq.) was dissolved in anhydrous DCM (0.4
CH
3
2
), 41.9 (NH-CH
2
), 31.9 (d, J1C-P = 135.3 Hz, CH
2
-P), 28.7 (CO-CH ).
2
M) under an Ar atmosphere, and the resulting yellow solution was
1
-
◦
P NMR (162 MHz, DMSO‑d
6
) δ 21.33. HRMS-ESI (m/z) [Mꢀ H] calcd
cooled to 0 C. Hexyl isocyanate (1 eq.) dissolved in anhydrous DCM
for C23
H
21
N
3
O
6
P: 466.1174, found: 466.1178.
(0.78 M) was added drop-wise, the ice bath was removed, and the re-
action mixture was stirred at rt for 45 min. The solvent was evaporated
to obtained title compound 13c as a solid (94%) without further puri-
4
.1.5.6. (4-(7-(3-(3-Hexylureido)prop-1-ynyl)-2,4-dioxo-3,4-dihy-
1
8 1
fication. CAS # 1311414–23-9.
H NMR (250 MHz, CDCl
H, H4,6), 3.99 (dd, 2H, J = 5.5, 2.5 Hz, H ), 3.22–3.12 (m, 2H, H
), 1.56–1.43 (m, 2H, H ), 1.38–1.22 (m, 6H,
9,10,11), 0.94–0.81 (m, 3H, H12).
3
) δ 4.40 (bs,
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (19c). The title
2
3
7
), 2.23
compound was prepared from 18c to afford after lyophilisation the
1
(
t, 1H, J = 2.5 Hz, H
1
8
desired product 19c as a beige solid (qtf). H NMR (400 MHz, DMSO‑d
6
)
H
δ 11.65 (bs, 1H, NH), 7.95 (d, 1H, J = 8.0 Hz, H
5
), 7.35 (s, 1H, H
), 5.61 (t, 2H, J = 3.7 Hz, H ), 4.66 (d, , 2H, J =
), 4.10 (s, 2H, NH-CH ),
), 2.97 (dt, 2H, J = 12.0, 6.6 Hz, CH
8
), 7.23
(
d, 1H, J = 8.0 Hz, H
7
′ ′
2 ,3
′
4
.1.9. General synthetic procedure 6 for Pd(0) cross-coupling under
5
.0 Hz, H
1
2
2
′
), 1.35 (q, 2H, J = 6.7, 6.3 Hz, NH-
Sonogashira conditions for compounds 18a, 18b, 18c,
2
.38 (dd, 2H, J = 21.7, 4.8 Hz, H
4
1
3
Under inert atmosphere, to a solution of bromoaryl acyclo nucleoside
phosphonate (1 eq.) in dry DMF (0.087 M) were successively added
CH
2
), 1.24 (bs, 6H, 3 × CH
2
), 0.85 (t, 3H, J = 6.6 Hz, CH
3
). C NMR
–
–
–
(
101 MHz, DMSO‑d
6
) δ 161.2 (NH-CO-NH), 157.5 (C
O), 149.9 (C
–
O),
3
copper iodide (0.2 eq.), triethylamine (3 eq.), alkyne (3 eq.), and Pd
1
40.9 (Cquat), 129.0 (Cquat), 127.8 (Carom), 126.9 (d, J C-P = 14.1 Hz, CH
2
◦
(
PPh
3
)
4
(10 mol%). The reaction mixture was heated at 70 C for 3 h.
=
CH), 125.3 (Carom), 124.7 (d, J C-P = 10.1 Hz, CH = CH), 117.3
–
The reaction was quenched with EtOAc and co-evaporated with hep-
tane. Pure compounds were obtained after purification on silica gel
column chromatography using an elution gradient of DCM/MeOH (from
(
C
arom), 115.2 (Cquat), 92.4 (CH
2
-C), 80.5 (C
-P), 31.0 (NH-CH ), 29.8 (CH
), 26.0 (CH -CH ), 22.0 (CH -CH
). P NMR (162 MHz, DMSO‑d
) δ 21.41. HRMS-ESI (m/z) [Mꢀ H]
P: 475.1751, found: 475.1751.
–
C-C), 43.2 (N-CH
-CH ), 29.7 (NH-
2
), 13.9 (CH -
2
), 31.9
1
(
d, J C-P = 134.3 Hz, CH
2
2
2
2
CH
CH
2
), 29.6 (CH
2
-CH
2
2
2
2
2
3
1
-
9
7:3 to 95:5) to give pure product.
3
6
28 4 6
calcd for C22H N O
4
.1.9.1. Dimethyl (4-(7-(3-nonanamidoprop-1-ynyl)-2,4-dioxo-3,4-dihy-
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonate (18a). The title com-
pound was prepared from 17 to afford after purification the desired
4
1
.1.5.7. (4-(5-Benzyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)but-2-en-
-yl)phosphonic acid (38a). The title compound was prepared according
product 18a and alkyne 13a as a light orange solid (E/Z ratio = 85/15,
to the general procedure 5 from 37a to afford after lyophilisation the
1
9
6%). H NMR (250 MHz, Methanol‑d
4
) δ 8.07 (d, 1H, J = 8.1 Hz, H
5
),
desired product 39a as a white foam (E/Z ratio = 85/15) in 90% yields.
1
7
.40 (d, 1H, J = 1.2 Hz, H
14.9, 4.8 Hz, H
), 5.78–5.55 (m, 1H, H
), 4.29 (s, 2H, C-CH ), 3.90 (s, 3H, HPOMe-minor), 3.85 (s, 3H, HPOMe-
8
), 7.31 (d, 1H, J = 8.1 Hz, H
6
), 5.86 (dt, 1H, J
H NMR (400 MHz, DMSO‑d
6
) δ 11.25 (bs 1H, NH), 8.89 (s, 2H, 2 ×
=
2
′
′
3
), 4.80 (d, 2H, J = 5.3 Hz,
H
POH), 7.60 (s, 1H, H6-minor), 7.57 (s, 1H, H6-major), 7.27–7.17 (m, 4H, 4
arom), 7.17–7.09 (m, 1H, Harom), 5.71–5.41 (m, 2H, H ), 4.32 (dd,
-minor), 4.24 (d, 2H, J = 4.7 Hz, H -major), 2.62
-minor), 2.48–2.38 (m, 2H, H
-major). 13
′
×
H
′ ′
2 ,3
H
1
2
2
H, J = 6.5, 3.2 Hz, H
1
′
′
1
minor), 3.77 (s, 3H, HPOMe-major), 3.73 (s, 3H, HPOMe-major), 2.77 (dd, 2H, J
′
), 2.30 (t, 2H, J = 7.5 Hz, C-CH
), 1.70 (p, 2H, J = 7.3
(
dd, 2H, J = 22.1, 7.6 Hz, H
4
′
4
′
C
=
21.9, 7.1 Hz, H
4
2
1
3
–
–
–
–
Hz, CH ), 1.46–1.28 (m, 8H, 4 × CH ), 1.01–0.88 (m, 3H, CH ).
C
NMR (101 MHz, DMSO‑d
6
) δ 163.7 (C
O), 150.7 (C
O), 142.2 (C =
2
2
3
CH), 139.9 (Cquat), 128.4 (2 × Carom), 128.3 (2 × Carom), 128.0 (d, J3C-P
–
–
NMR (63 MHz, Methanol‑d
4
) δ 175.9 (CH
2
-CO-NH), 163.5 (C
O),
1
51.9 (C
O), 142.3 (Cquat), 131.2 (Cquat), 130.2 (d, J3C-P = 14.5 Hz, CH
2
–
–
=
14.1 Hz, CH = CH), 126.0 (Carom), 125.9 (d, J C-P = 10.4 Hz, CH =
CH), 129.2 (Carom), 127.0 (Carom), 123.0 (d, J2C-P = 11.3 Hz, CH =
CH), 112.5 (Cquat), 48.4 (N-CH
2
), 32.8 (d, J1C-P = 134.3 Hz, CH-CH
2
),
).
=
–
–
–
). 3 P NMR (162 MHz, DMSO‑d
1
CH), 119.1 (Carom), 116.8 (Cquat), 90.8 (CH
2
-C), 82.2 (C
C-C), 53.6 (O-
-CH ), 30.2
3
1.9 (C-CH
2
6
Z
) δ 22.85 (P ), 22.25 (P
E
-
CH
NH-CH
CH-CH -P), 26.8 (CH
101 MHz, Methanol‑d
3
), 53.5 (O-CH
3
), 44.9 (N-CH
-CH ), 30.1 (CH
-CH ), 23.6 (CH
) δ 30.31 (P ), 22.90 (P
P: 504.2256, found: 504.2257. Rf : 0.21
2
), 36.8 (CO-CH
2
), 32.8 (CH
2
2
HRMS-ESI (m/z) [Mꢀ H] calcd for C15
16 2 5
H N O P: 305.0799, found:
(
2
), 30.1 (CH
2
2
2
-CH
-CH
2
), 29.3 (d, J1C-P = 139.8 Hz,
3
05.0800.
3
1
2
2
2
2
2 2 3
), 14.3 (CH -CH ). P NMR
(
4
E
Z
). HRMS-ESI (m/z) [M +
4
.1.6. N-Prop-2-ynyloctanamide (13a)
+
H] calcd for C25
35 3 6
H N O
Propargylamine (13) (1 eq.) was dissolved in anhydrous DCM (0.4
◦
(DCM/MeOH 95:5).
M), DIPEA (1.3 eq.) was added and the solution was cooled to 0 C.
Octanoyl chloride (1.1 eq.) was added dropwise and the reaction
mixture was stirred at rt for 2 h. The reaction was quenched with water,
4.1.9.2. Dimethyl (4-(2,4-dioxo-7-(3-(2-phenylacetamido)prop-1-ynyl)-
3,4-dihydroquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonate (18b). The
title compound was prepared from 17 and alkyne 13b to afford after
then diluted with DCM and washed with NaHCO
Organic layer was dried over MgSO and evaporated. The title com-
pound was obtained without further purification as a solid (qtf). CAS #
3
solution and brine.
4
1
purification the desired product 18b as a light orange solid (81%).
H
1
6 1
4
22284–34-2. H NMR (250 MHz, CDCl
3
) δ 5.56 (bs, 1H, H
4
), 4.06 (dd,
6
NMR (400 MHz, Acetone‑d ) δ 10.36 (bs, 1H, NH), 8.02 (d, 1H, J = 8.0
9

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
Hz, H
5
), 7.40 (s, 1H, H
H, 2 × Harom), 7.27–7.19 (m, 2H, H
.5 Hz, H
), 4.80 (t, 2H, J = 4.1 Hz, H
), 3.79 (s, 3H, HPOMe-minor), 3.76 (s, 3H, HPOMe-minor), 3.64 (s, 3H,
POMe-major), 3.62 (s, 3H, HPOMe-major), 3.58 (s, 2H, C-CH
), 2.73–2.63
) δ 170.8 (CH -CO-NH),
O), 142.2 (Cquat), 137.1 (Cquat), 130.2 (Cquat),
8
), 7.38–7.34 (m, 2H, 2 × Harom), 7.33–7.28 (m,
, Harom), 5.77 (td, 2H, J = 5.2, 4.8,
), 4.24 (d, 2H, J = 5.2 Hz, C-
2.1 Hz, H
), 7.37–7.26 (m, 4H, 4 × Harom), 7.26–7.18 (m, 1H, Harom), 5.80 (dt,
1H, J = 15.8, 5.2 Hz, H ), 5.75–5.62 (m, 1H, H ), 5.60 (d, 2H, J = 1.9
Hz, O-CH2POC), 5.59 (d, 2H, J = 1.3 Hz, O-CH2POC), 4.88 (hept, 2H, J =
6.3 Hz, 2 × OCHPOC), 4.78 (t, 2H, J = 4.8 Hz, H ), 4.23 (d, 2H, J = 5.4
Hz, C-CH ), 3.55 (s, 2H, C-H ), 1.29 (d, 12H, J =
), 2.81–2.69 (m, 2H, H
6.3 Hz, 4 × CH3POC). C NMR (101 MHz, Acetone‑d ) δ 170.7 (NH-CO),
O), 138.3 (Carom), 137.0
5
), 7.70 (dd, 1H, J = 8.7, 2.1 Hz, H
7
), 7.39 (d, 1H, J = 8.7 Hz,
2
3
7
H
8
′
′
′
′
′
3
2
,3
1
2
CH
2
H
2
′
1
1
3
(
m, 2H, H
4
–
–
′
). C NMR (101 MHz, Acetone‑d
6
2
2
2
′
4
–
–
13
1
61.9 (C
30.0 (2 × Carom), 129.7 (d, J C-P = 14.4 Hz, CH = CH), 129.1 (2 ×
arom), 128.9 (Carom), 127.3 (Carom), 125.8 (Carom), 124.1 (d, J2C-P
0.8 Hz, CH = CH), 119.2 (Carom), 116.7 (Cquat), 90.9 (CH -C), 82.3
), 43.3 (CO-CH ),
) δ
P:
O), 150.8 (C
6
3
–
–
–
–
–
O), 150.7 (C
–
1
163.2 (C
O), 154.0 (2 × C
3
C
=
(Cquat), 135.9 (Cquat), 131.6 (Carom), 130.6 (d, J C-P = 14.9 Hz, CH = CH),
1
2
130.0 (2 × Carom), 129.1 (2 × Carom), 127.4 (Carom), 122.4 (d, J2C-P
=
–
(
C
–
C-C), 52.9 (O-CH
3
), 52.9 (O-CH
3
), 44.7 (N-CH
2
2
11.8 Hz, CH = CH), 118.0 (Cquat), 117.4 (Cquat), 116.5 (Carom), 87.6
1
31
-C), 84.9 (d, J2C-P = 6.3 Hz, 2 × O-CH
–
-O), 81.3 (C
29.4 (d, J C-P = 139.3 Hz, CH-CH
2
4
2
-P). P NMR (162 MHz, Acetone‑d
6
(CH
2
2
+
1
8.32 (P
E
). HRMS-ESI (m/z) [M + H] calcd for C25
H
27
N
3
O
6
O-CHPOC), 44.8 (N-CH
2
), 43.5 (CO-CH
2
), 33.1 (d, J C-P = 139.9 Hz, CH
2
-
3
1
96.16.31, found: 496.1629. Rf : 0.23 (DCM/MeOH 95:5).
P), 30.4 (NH-CH
tone‑d
2
-C), 21.8 (4 × CCH3POC). P NMR (162 MHz, Ace-
+
6
) δ 26.49. HRMS-ESI (m/z) [M + H] calcd for C33
39 3
H N O12P:
4
.1.9.3. Dimethyl (4-(7-(3-(3-hexylureido)prop-1-ynyl)-2,4-dioxo-3,4-
700.2253, found: 700.2254. Rf : 0.31 (DCM/MeOH 95:5).
dihydroquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonate (18c). The title
compound was prepared from 17 and alkyne 13c to afford after puri-
4.1.9.6. (((4-(6-(3-(3-Hexylureido)prop-1-ynyl)-2,4-dioxo-3,4-dihy-
fication the desired product 18c as a light orange solid (E/Z ratio = 85/
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphoryl)bis(oxy))bis(methylene)
diisopropyl bis(carbonate) (26c).. The title compound was prepared
from 25 and alkyne 13c to afford after purification the desired product
1
1
5, 80%). H NMR (400 MHz, Methanol‑d
4
) δ 8.07 (d, 1H, J = 8.1 Hz,
H
5
), 7.40 (s, 1H, H
.8 Hz, H
), 5.63 (dq, 1H, J = 15.1, 7.3 Hz, H
), 4.20 (s, 2H, C-CH ), 3.86 (s, 3H, HPOMe-minor), 3.83 (s, 3H, HPOMe-
minor), 3.74 (s, 3H, HPOMe-major), 3.71 (s, 3H, HPOMe-major), 3.15 (dt, 2H, J
15.0, 7.1 Hz, NH-CH ), 1.52 (q,
H, J = 7.3, 6.8 Hz, CH ), 0.94 (q, 3H, J =
8
), 7.30 (d, 1H, J = 8.1 Hz, H
7
), 5.84 (dt, 1H, J = 15.1,
5
2
′
3
′
), 4.78 (d, 2H, J = 5.8 Hz,
26c as a light orange solid (65%). 1H NMR (400 MHz, Acetone‑d
10.37 (bs, 1H, NH), 8.05–7.98 (m, 1H, H ), 7.70–7.61 (m, 1H, H ), 7.37
(d, 1H, J = 8.7 Hz, H ), 5.90–5.65 (m, 2H, H
6
) δ
H
1
′
2
5
7
′
, H ), 5.61 (d, 2H, J = 2.0
′
8
2
3
=
2
), 2.74 (dd, 2H, J = 21.9, 7.3 Hz, CH
), 1.44–1.30 (m, 6H, 3 × CH
). C NMR (101 MHz, Methanol‑d
4
′
Hz, O-CH2POC), 5.58 (d, 2H, J = 1.3 Hz, O-CH2POC), 4.88 (hept, 2H, J =
2
5
1
2
2
6.3 Hz, 2 × O-CHPOC), 4.76 (t, 2H, J = 5.6 Hz, H
1
′
), 4.18 (d, 2H, J = 5.5
), 2.76 (dd, 2H, J = 22.6, 7.2 Hz,
), 1.29 (bd, 18H, J = 6.2 Hz, 3 × CH , 4
) δ
1
3
–
–
O),
.0, 4.5 Hz, CH
60.6 (NH-CO-NH), 151.9 (C
3
4
) δ 163.5 (C
O), 142.4 (Cquat), 131.5 (Cquat), 130.3 (d,
Hz, C-CH
2
), 3.20–3.07 (m, 2H, NH-CH
2
–
–
′
H
4
), 1.47 (t, 2H, J = 7.3 Hz, CH
2
2
3
C-P = 14.5 Hz, CH = CH), 129.1 (Carom), 127.0 (Carom), 122.9 (d, J2C-P
13
J
× CH3POC), 0.96–0.75 (m, 3H, CH
3 6
). C NMR (101 MHz, Acetone‑d
–
–
–
–
=
11.4 Hz, CH = CH), 119.1 (Carom), 116.7 (Cquat), 92.2 (CH
2
-C), 81.9
161.7 (C
–
O), 158.5 (NH-CO-NH), 154.0 (2 × C
O), 150.7 (C O),
–
–
3
(
C
–
C-C), 53.6 (O-CH
2.7 (CH -CH ), 31.2 (NH-CH
39.3 Hz, CH-CH
3
), 53.6 (O-CH
-C), 31.0 (CH
-CH
3
), 44.8 (N-CH
-CH ), 29.3 (d, J C-P
), 27.6 (CH -CH ), 23.6 (CH -CH
) δ 30.32 (P ). HRMS-
2
), 41.1 (NH-CH ),
2
141.6 (Cquat), 138.3 (Carom), 131.5(Carom), 130.7 (d, J C-P = 15.2 Hz, CH
1
2
3
1
1
2
2
2
2
2
=
= CH), 122.3 (d, J C-P = 11.8 Hz, CH = CH), 118.3 (Cquat), 117.2 (Cquat),
2
-P), 27.6 (CH
2
2
2
2
2
2
),
116.4 (Carom), 89.1 (CH
2
-C), 85.0 (d, J = 6.2 Hz, 2 × O-CH
2
-O), 80.9
). 3 P NMR (162 MHz, Methanol‑d
1
(C
–
C-C), 73.7 (2 × O-CH), 44.8 (N-CH
), 40.7 (NH-CH
), 32.3 (CH -
4.3 (CH
2
-CH
3
4
E
–
2
2
2
+
), 31.0 (d, J1C-P = 139.3 Hz, CH-
ESI (m/z) [M + H] calcd for C24
H
34
N
4
O
6
P: 505.2210, found: 505.2209.
CH
CH
2
), 31.9 (NH-CH
-P), 27.3 (CH -CH
). P NMR (162 MHz, Acetone‑d
2
-C), 31.8 (CH
2
-CH
2
Rf : 0.20 (DCM/MeOH 95:5).
2
2
2
), 23.3 (CH
2
-CH
2
), 21.8 (4 × C-CH3POC), 14.3
3
1
(
CH
2
-CH
3
6
) δ 26.61. HRMS-ESI (m/z)
46 4
H N O12P: 709.2844, found: 709.2834. Rf : 0.28
+
4
.1.9.4. Diisopropyl ((((4-(6-(3-octanamidoprop-1-ynyl)-2,4-dioxo-3,4-
[M + H] calcd for C32
dihydroquinazolin-1(2H)-yl)but-2-en-1-yl)phosphoryl)bis(oxy))bis(methy-
(DCM/MeOH 95:5).
lene)) bis(carbonate) (26a). The title compound was prepared from 25
and alkyne 13a to affording after purification the desired product 26a as
4.1.9.7. Diisopropyl ((((4-(5-(3-octanamidoprop-1-ynyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)but-2-en-1-yl)phosphoryl)bis(oxy))bis(methy-
lene)) bis(carbonate) (30a). The title compound was prepared according
1
a light orange solid (61%). H NMR (250 MHz, Acetone‑d
6
) δ 10.29 (bs,
), 7.68 (dd, 1H, J = 8.7, 2.1 Hz, H ),
), 5.81–5.78 (m, 2H, H ), 5.76–5.60 (m, 4H,
× O-CH2POC), 4.89 (h, 2H, J = 6.2 Hz, 2 × O-CHPOC), 4.80–4.65 (m,
H, H ), 2.80–2.60 (m, 2H, H ), 2.22
), 4.23 (d, 2H, J = 5.3 Hz, NH-CH
), 1.59 (q, 2H, J = 7.2 Hz, CH ), 1.30–1.04 (m,
1
7
4
2
H, NH), 7.80 (d, 1H, J = 2.1 Hz, H
5
7
◦
.37 (d, 1H, J = 8.7 Hz, H
8
2
′
,3
′
to the general procedure 6 but at 70 C, from 29 and alkyne 13a, to
afford after purification on silica gel column chromatography using an
′
′
elution gradient of DCM/MeOH (from 97:3 to 95:5) the desired product
1
2
4
1
(
t, 2H, J = 7.4 Hz, CH
0H, 4 × CH , 4 × CH3POC), 0.82–0.69 (m, 3H, CH
Acetone‑d ) δ 172.9 (NH-CO), 161.7 (C
O), 141.7 (Cquat), 138.3 (Carom), 131.5 (Carom), 130.6 (d, J C-P
2
2
30a as a light orange solid (97%). H NMR (400 MHz, Acetone‑d
6
) δ 9.99
1
3
2
2
3
). C NMR (63 MHz,
(bs, 1H, NH), 7.80 (s, 1H, H
6
), 7.43 (bs, 1H, NH), 5.90–5.71 (m, 2H,
–
–
–
–
′
′
6
O), 153.9 (2 × C
O), 150.7
H
2 ,3 ), 5.66 (dq, 4H, J = 10.7, 5.5 Hz, 2 × O-CH2POC), 4.90 (hept, 2H, J
–
–
3
′
(
C
=
= 6.2 Hz, 2 × O-CHPOC), 4.41 (t, 2H, J = 4.9 Hz, H
1
), 4.14 (d, 2H, J =
2
1
1
5.1 Hz, CH = CH), 122.3 (d, J C-P = 11.8 Hz, CH = CH), 118.0 (Cquat),
5.3 Hz, C-CH
Hz, C-CH
), 1.59 (dd, 2H, J = 9.2, 5.2 Hz, CH
Hz, 4 × CH , 4 × CH3POC), 0.92–0.83 (m, 3H, CH
Acetone‑d
2
), 2.81 (dd, 2H, J = 22.6, 6.3 Hz, H
4
′
), 2.19 (t, 2H, J = 7.5
17.2 (Carom), 116.4 (Cquat), 87.8 (CH -C), 85.0 (d, J = 6.1 Hz, 2 × O-
C-C), 73.6 (2 × O-CHPOC), 44.8 (N-CH ), 36.4 (CO-
), 30.1 (CH
), 23.2 (CH -CH
). 31P NMR (101 MHz, Acetone‑d
) δ
12P: 708.2889,
2
2
2
), 1.30 (bd, 20H, J = 6.3
–
13
CH2POC), 81.1 (C
–
2
2
3
). C NMR (101 MHz,
1
–
–
–
–
CH
CH
2
), 32.4 (CH
2
-CH
), 29.6 (CH
-CH
2
), 31.1 (d, J C-P = 139.2 Hz, CH
2
-CH
2
2
-
6
) δ 172.8 (NH-CO), 162.5 (C
O), 154.0 (2 × C
–
–
O), 148.5 (C = CH), 130.7 (d, J C-P = 15.1 Hz, CH = CH), 124.7 (d,
2
O), 150.7
3
2
), 29.9 (NH-CH
2
2
-CH ), 26.3 (CH -CH
2
2
2
2
2
),
(C
–
2
1.7 (4 × CH3POC), 14.3 (CH
6.67. HRMS-ESI (m/z) [M + H] calcd for C33
2
3
+
6
J
C-P = 11.5 Hz, CH = CH), 99.4 (CH
Hz, 2 × O-CH -O), 74.8 (Cquat), 73.7 (2 × O-CH), 49.9 (N-CH
(CO-CH ), 32.4 (CH
CH -CH ), 29.9 (NH-CH
CH
), 21.8 (4 × C-CH3POC), 14.3 (CH
tone‑d
2
-C), 90.2 (C
–
C-C), 85.1 (d, J = 6.2
), 36.4
-P), 30.4
2
), 23.2 (CH -
2
H
47
3
N O
2
2
1
found: 708.2891. Rf : 0.3 (DCM/MeOH 95:5).
2
2
-CH
2
), 31.1 (d, J C-P = 139.3 Hz, CH-CH
2
(
2
2
2
), 29.7 (CH
2
-CH
2
), 26.3 (CH
2
-CH
2
3
1
4
.1.9.5. (((4-(2,4-dioxo-6-(3-(2-phenylacetamido)prop-1-ynyl)-3,4-dihy-
2
2
-CH
3
). P NMR (162 MHz, Ace-
+
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphoryl)bis(oxy))bis(methylene)
diisopropyl bis(carbonate) (26b). The title compound was prepared ac-
cording to the general procedure 6 from 25 and alkyne 13b, affording
6
) δ 26.64. HRMS-ESI (m/z) [M + H] calcd for C29
H
45
3
N O
12P:
658.2735, found: 658.2723. Rf : 0.29 (DCM/MeOH 95:5).
after purification the desired product 26b as a light orange solid (66%).
4.1.9.8. (((4-(5-(3-(3-Hexylureido)prop-1-ynyl)-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)but-2-en-1-yl)phosphoryl)bis(oxy))bis(methylene)
1
6
H NMR (400 MHz, Acetone‑d ) δ 10.30 (bs, 1H, NH), 8.04 (d, 1H, J =
1
0

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
diisopropyl bis(carbonate) (30c). The title compound was prepared ac-
4.1.11.1. (4-(6-(3-Octanamidoprop-1-ynyl)-2,4-dioxo-3,4-dihy-
◦
cording to the general procedure 6 but at 70 C, from 29 and alkyne 13b,
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (27a). The title
compound was prepared according to the general procedure 8 from 26a
to afford after purification on silica gel column chromatography using
an elution gradient of DCM/MeOH (from 97:3 to 95:5) the desired
product 30b as a light orange solid (21%). 1H NMR (400 MHz, Meth-
to afford after lyophilisation the desired product 27a as a beige foam
1
(86%). H NMR (250 MHz, DMSO‑d
6
) δ 11.72 (bs, 1H, NH), 8.43–8.25
), 7.70 (dd, 1H, J = 12.1, 6.0 Hz, H ), 7.39
), 5.89-0.65 (m, 1H, H ), 5.53 (s, 1H, H ), 4.62 (s,
), 4.10 (d, 2H, J = 5.4 Hz, H11), 2.38–2.24 (m, 2H, H ), 2.09 (d,
2H, J = 9.5 Hz, CH ), 1.51 (q, 2H, J = 7.7, 6.5 Hz, CH ), 1.23 (bs, 8H, 4
× CH21), 0.84 (q, 3H, J = 6.2 Hz, CH ). C NMR (63 MHz,
anol‑d
4
) δ 7.86 (s, 1H, H
× O-CH2POC), 4.95 (p, 2H, J = 6.3 Hz, 2 × O-CHPOC), 4.40 (t, 2H, J =
.3 Hz, H ), 4.12 (s, 2H, C-CH ),
), 3.14 (td, 2H, J = 7.3, 3.1 Hz, NH-CH
.88 (dd, 2H, J = 22.7, 6.9 Hz, H ), 1.51 (t, 2H, J = 7.0 Hz, CH ), 1.36 (t,
8H, J = 7.1 Hz, 3 × CH , 4 × CH3POC), 0.98–0.91 (m, 3H, CH
6
), 5.90–5.74 (m, 2H, H
2
′
,3
′
), 5.72–5.63 (m, 4H,
(m, 1H, NH), 7.91 (s, 1H, H
(t, 1H, J = 8.6 Hz, H
2H, H
5
8
2
5
2
1
7
2
′
′
3
′
′
′
4
1
2
2
1
′
4
2
2
2
1
3
13
2
3
).
C
2
-CH
O), 149.7 (C
137.3 (Carrom), 130.1 (Carrom), 128.6 (Cquat), 127.4 (CH = CH), 126.0
3
–
–
–
–
–
O), 140.5, (Cquat
NMR (101 MHz, Methanol‑d
4
) δ 164.9 (C
O), 160.5 (NH-CO-NH),
DMSO‑d
6
) δ 171.9 (NH-CO), 160.9 (C
–
)
–
–
–
–
3
1
54.6 (2 × C
O), 151.5 (C
O), 149.3 (C = CH), 131.4 (d, J C-P = 15.2
2
–
Hz, CH = CH), 124.2 (d, J C-P = 12.0 Hz, CH = CH), 100.1 (CH
2
-C), 91.7
(CH = CH), 116.2 (Carrom), 115.8 (Cquat), 87.6 (CH
2
-C), 80.0 (C
–
C-C),
–
C
), 33.2 (d, J1C-P = 139.8 Hz, CH
(
C
–
C-C), 85.7 (d, J = 6.6 Hz, 2 × O-CH
quat), 50.5 (N-CH ), 41.1 (NH-CH ), 32.7 (CH
-CH
), 30.4 (d, J C-P = 139.8 Hz, CH-CH
), 23.6 (CH -CH ), 21.8 (4xCCH3POC), 14.3 (CH -CH
162 MHz, Methanol‑d
2
-O), 74.5 (2 × O-CH), 74.4
43.7 (N-CH
(CH -CH ), 28.5 (CH
CH ), 21.9 (CH -CH
2
), 33.9 (CO-CH
-CH
2 3 6
), 13.9 (CH -CH ). P NMR (162 MHz, DMSO‑d
2
2
-P), 31.6
(
2
2
2
-CH
2
), 31.82 (NH-CH
2
-
-
2
2
2
2
), 28.4 (NH-CH
2
), 28.5 (CH
2
-CH
2
), 25.2 (CH
) δ
29 3 6
H N O P: 474.1799,
2
-
1
31
C), 31.2 (CH
CH
2
2
2
-P), 27.6 (CH
2
2
2
2
). 31P NMR
18.91. HRMS-ESI (m/z) [Mꢀ H] calcd for C23
-
2
2
2
2
3
+
(
4
) δ 27.51. HRMS-ESI (m/z) [M + H] calcd for
found: 474.1804.
28 44 4
C H N O12P: 659.2676, found: 659.2678. Rf: 0.27 (DCM/MeOH
9
5:5).
4.1.11.2. (4-(2,4-Dioxo-6-(3-(2-phenylacetamido)prop-1-ynyl)-3,4-dihy-
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (27b). The title
compound was prepared according to the general procedure 8 from 26b
to afford after lyophilisation the desired product 27b as a beige solid
4
.1.10. (((4-(6-Bromo-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)but-2-
en-1-yl)phosphoryl)bis(oxy))bis(methylene) diisopropyl bis(carbonate)
25)
A mixture of protected alkylated 6-bromo quinazoline 22 (1 eq.) and
1
(
(92%). H NMR (250 MHz, DMSO‑d ) δ 11.75 (bs, 1H, NH), 8.62 (d, 1H,
6
J = 5.8 Hz, NH), 7.94 (s, 1H, H ), 7.77–7.64 (m, 1H, H ), 7.49–7.06 (m,
5
9
bisPOC allylphosphonate 23 (3 eq.), were stirred at room temperature in
6H, H , 4 × H
), 5.63 (dt, 2H, J = 9.9, 6.3 Hz, H
′ ′
2 ,3
), 4.65 (d, 2H, J =
8
arom
freshly distilled DCM (0.1 M). Grubbs 2nd generation catalyst (15 mol%)
5.0 Hz, H1
′
), 4.13 (d, 2H, J = 5.3 Hz, CH ), 3.47 (s, 2H, CH ), 2.39–2.29
13
2
2
◦
was added. The mixture was refluxed at 55 C under inert atmosphere
(m, 2H, H4 ). C NMR (63 MHz, DMSO‑d ) δ 169.8 (NH-CO), 161.0
′
6
for 24 h. Volatiles were eliminated under reduced pressure. Crude
product was applied on short silica gel flash chromatography using an
elution gradient of PE/ EtOAc (from 8:2 to 1:1) to afford the desired
(C
–
O), 149.8 (C
–
O), 140.5 (Cquat), 137.4 (Carom), 136.0 (Cquat), 130.1
(C
arom
), 128.9 (2 × C
arom
), 128.2 (2 × C
arom
), 126.6 (d, J3 = 15.3 Hz,
C-P
CH = CH), 126.4 (C
(C
arom
), 125.4 (d, J2
C-P
= 9.3 Hz, CH = CH), 116.2
+
–
–
C-C), 43.5 (N-CH ), 42.0
compound 24 as an oil (E/Z ratio = 85/15). HRMS-ESI (m/z) [M] calcd
), 115.96 (C ), 87.4 (CH -C), 80.2 (C
arom
quat
2
–
2
31
1
C-P
for C27
H37BrN
2
O
13P: 707.1200, found: 707.1211. Rf : 0.3 (EDP/AcOEt
(NH-CH ), 32.1 (d, J
= 131.6 Hz, CH -P), 28.9 (CO-CH ). P NMR
2
2
2
-
1
:1). This product is directly engaged in the deprotection step of Boc
(162 MHz, DMSO‑d ) δ 21.08. HRMS-ESI (m/z) [Mꢀ H] calcd for
6
group. Then a solution of POC phosphonate mixture (1 eq.) in freshly
distilled DCM (0.05 M), was stirred with trifluoroacetic acid (20 eq.)
under inert atmosphere for 6 h. After completion of the reaction, the
23 21 3 6
C H N O P: 466.4178, found: 466.1172.
4
.1.11.3. (4-(6-(3-(3-Hexylureido)prop-1-ynyl)-2,4-dioxo-3,4-dihy-
mixture was diluted in EtOAc and washed twice with saturated NaHCO
solution, dried over MgSO filtered and concentrated under reduced
3
droquinazolin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (27c). The title
compound was prepared according to the general procedure 8 from 26c
4
pressure. Pure compound 25 was obtained after purification on silica gel
to afford after lyophilisation the desired product 27c as a beige foam
1
column chromatography with DCM/MeOH (97:3) as an oil (44% over
(
50%). H NMR (250 MHz, DMSO‑d
6
) δ 11.71 (bs, 1H, NH), 7.90 (s, 1H,
), 5.86–5.64
), 4.04 (d, 2H,
1
two steps). H NMR (400 MHz, Acetone‑d
6
) δ 10.45 (bs, 1H, NH), 8.14
), 7.38 (d, 1H, J = 9.0 Hz, H ),
), 5.72–5.62 (m, 1H, H ), 5.60 (s, 4H, 2 ×
CH2POC), 5.57 (s, 2H, CH2POC), 4.88 (p, 2H, J = 6.2 Hz, 2 × O-CHPOC),
), 2.76 (dd, 2H, J = 22.6, 7.2 Hz, H ), 1.29 (d,
) δ 161.3
O), 141.4 (Cquat), 138.4
H
5
), 7.68 (t, 1H, J = 8.1 Hz, H
8
), 7.37 (d, 1H, J = 9.0 Hz, H
7
(
s, 1H, H
5
), 7.85 (d, 1H, J = 9.0 Hz, H
7
8
′
′
), 4.62 (s, 2H, H
1
′
(
m, 1H, H
2
), 5.51 (d, 1H, J = 15.1 Hz, H
3
5
.85–5.77 (m, 1H, H
2
′
′
3
J = 4.6 Hz, H11), 2.99 (q, 2H, J = 6.1, 5.6 Hz, H15), 2.39–2.19 (m, 1H,
H
′
H
4
), 1.35 (t, 2H, J = 7.0 Hz, CH
2
), 1.23 (bs, 6H, 3 × CH
). C NMR (63 MHz, DMSO‑d ) δ 160.9 (NH-CO-NH),
O), 140.4 (Cquat), 137.3 (Carom), 129.9 (Carom),
2
), 0.90–0.73 (m,
′
′
4
1
3
4
.77 (t, 2H, J = 5.2 Hz, H
1
3
1
1
1
H, CH
2
-CH
3
6
1
3
1
2H, J = 6.3 Hz, 4 × CH3POC). C NMR (101 MHz, Acetone‑d
6
–
–
–
–
57.5 (C
O), 149.7 (C
– –
– –
O), 154.0 (2 × C = OPOC), 150.6 (C
3
3
2
(
(
C
28.8 (Cquat), 126.1 (d, J C-P = 12.6 Hz, CH = CH), 125.8 (d, J C-P
=
C
2
arom), 130.8 (Carom), 130.5 (d, J C-P = 15.1 Hz, CH = CH), 122.4 (d,
0.3H, CH = CH), 116.4 (Carom), 115.9 (Cquat), 88.8 (CH
2
-C), 79.8
(
C
C-C), 59.3 (N-CH
P), 31.0 (CH -CH ), 29.9 (CH
-CH ), 13.0 (CH
2
), 39.8 (NH-CH
2
), 33.2 (d, J1C-P = 139.8 Hz, CH
2
-
J
C-P = 12.2 Hz, CH = CH), 119.03 (Cquat), 118.4 (Carom), 115.6 (Cquat),
–
–
8
3
5.0 (O-CH2POC), 85.0 (O-CH2POC), 73.6 (2 × O-CHPOC), 44.8 (N-CH
2
),
2
2
2
-CH
2
), 29.6 (NH-CH
2
), 26.0 (CH
2
-CH
) δ
28 4 6
H N O P: 475.11738,
2
),
1
), 21.7 (4 × CH-CH3POC). 31P NMR
2
2.0 (CH
2
2
2
-CH
3
-
). 31P NMR (162 MHz, DMSO‑d
6
1.8 (d, J C-P = 139.3 Hz, CH-CH
2
+
(
6
162 MHz, Acetone‑d ) δ 26.55. HRMS-ESI (m/z) [M] calcd for
2
0.95. HRMS-ESI (m/z) [Mꢀ H] calcd for C22
C
22 2
H29BrN O11P: 607.0687, found: 607.0688. Rf : 0.49 (DCM/MeOH
found 475.1744.
9
5:5).
4
.1.11.4. 4-(5-(3-Octanamidoprop-1-ynyl)-2,4-dioxo-3,4-dihydropyr-
4
.1.11. General synthetic procedure 7 for POC removal for compounds
imidin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (31a). The title com-
pound was prepared according to the general procedure 8 from 30a to
2
7a, 27b, 27c, 31a, 31c
BisPOC compound were dissolved in an ultrapure water solution of
1
afford after lyophilisation the desired product as a beige foam (92%). H
sodium hydroxide (0.1 M). After 4 h stirring at room temperature, the
NMR (400 MHz, DMSO‑d
s, 1H, H
), 5.80–5.65 (m, 1H, H
), 4.04 (d, 2H, J = 5.2 Hz, H
6
) δ 11.42 (bs, 1H, NH), 8.32 (s, 1H, NH), 7.96
), 5.54–5.43 (m, 1H, H ), 4.23 (s, 2H,
), 2.27 (dd, 2H, J = 20.7, 8.0 Hz, H ),
), 1.23 (bs, 8H, 4 ×
). C NMR (63 MHz, DMSO‑d ) δ
O), 148.3 (C = CH), 129.2 (CH
+
solution was passed acidified to pH 5 with DOWEX 50WX8 (H ). The
(
6
2
′
′
3
solution was filtered and the filtrate was washed two times with DCM.
Inorganic layer was lyophilized to give pure compound.
H
1
′
9
′
4
2
.08 (t, 2H, J = 7.5 Hz, H12), 1.55–1.43 (m, 2H, CH
2
1
3
CH
2
), 0.85 (t, 3H, J = 6.6 Hz, CH
2
-CH
3
6
–
–
–
1
71.6 (NH-CO), 161.9 (C
–
O), 149.6 (C
1
1

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
–
=
CH), 125.7 (CH = CH), 97.3 (Cquat), 89.7 (CH
2
-C), 74.0 (C
), 33.2 (d, J C-P = 131.0 Hz, CH -P), 31.1 (CH
), 28.4 (NH-CH ), 28.4 (CH -CH ), 25.1 (CH -CH
). P NMR (162 MHz, DMSO‑d ) δ
P: 424.1173,
–
C-C), 48.9
solution S2 (0.21 ml, 0.01 eq., 0.21 mmol) was added dropwise and the
1
(
N-CH
2
), 35.0 (CO-CH
), 28.6 (CH -CH
-CH ), 13.8 (CH
9.15. HRMS-ESI (m/z) [Mꢀ H] calcd for C19
2
2
2
-
resulting mixture was allowed to warm up to room temperature for 30
◦
CH
2
2
2
2
3
2
2
2
2
),
min. Benzyl bromide (3.21 ml, 1.3 eq., 27.3 mmol) was added at ꢀ 30 C
1
2
2.1 (CH
2
2
2
-CH
3
6
dropwise and stirred at 30 min before allowed to warm up at room
temperature for 24 h. After the completion of the reaction, it was
quenched by addition of MeOH (25 ml). The precipitate was filtered off
and dried to afford the pure compound 34a as a white solid (84%).
-
1
27 3 6
H N O
found: 424.1172.
2
2 1
4
.1.11.5. 4-(5-(3-(hexylcarbamoylamino)prop-1-ynyl)-2,4-dioxo-3,4-
CAS # 18493–83-9.
H NMR (250 MHz, DMSO‑d
6
) δ 10.86 (bs, 2H, 2
).
dihydropyrimidin-1(2H)-yl)but-2-en-1-yl)phosphonic acid (31c). The title
compound was prepared according to the general procedure 8 from 30c
× NH), 7.30–7.14 (m, 6H, H
6
, 5xHArom), 3.49 (s, 2H, C-CH
2
to afford after lyophilisation the desired product 31c as a beige foam
4.1.13. 5-(Phenylamino)pyrimidine-2,4(1H,3H)-dione (34b)
1
(
92%). H NMR (250 MHz, DMSO‑d
6
) δ 11.56 (bs, 1H, NH), 7.93 (s, 1H,
), 5.61–5.43 (m, 1H, H ),
), 2.97 (t, 2H, J = 6.6 Hz, H13),
), 1.41–1.29 (m, 2H, CH ), 1.24 (bs,
). C NMR (63 MHz,
In a dry flask under inert atmosphere, 5-bromouracil (33) (5 g, 1 eq.,
H
6
), 5.72 (dq, 1H, J = 13.5, 7.8, 6.4 Hz, H
2
′
3
′
26.18 mmol) and aniline derivatives (7.16 ml, 3 eq., 75.54 mmol) were
◦
4
2
6
.24 (s, 2H, H
1
′
), 4.05–3.93 (m, 2H, H
9
heated at 195 C for 1 h. After completion of the reaction, a mixture of
.33 (dd, 2H, J = 21.0, 7.2 Hz, H
4
′
2
2 3
H O/HCCl was added to the reaction and stirred during 15 min. The
1
3
H, 3 × CH
2
), 0.93–0.72 (m, 3H, CH
2
-CH
3
mixture was filtered off and the solid washed with water and ethanol.
The solid was dissolved in 5% aqueous NaOH and the residues were
filtered off. The filtrate was neutralized with acetic acid and the ob-
tained solid was filtered off and washed with water to afford the desired
–
–
–
DMSO‑d
6
) δ 162.1 (C
O), 157.49 (NH-CO-NH), 149.8 (C
–
O), 148.2 (C
-C),
), 32.1 (d, J C-P = 134.1 Hz,
), 29.5 (CH -CH ), 26.0 (CH
). 31P NMR (162 MHz, DMSO‑d
) δ
P: 425.1780,
=
CH), 130.0 (CH = CH), 126.2 (CH = CH), 97.6 (Cquat), 91.0 (CH
2
–
1
7
3.8 (C
–
C-C), 49.0 (N-CH
-P), 31.2 (NH-CH ), 29.8 (CH
), 22.0 (CH -CH ), 14.0 (CH -CH
2
), 41.4 (NH-CH
2
2
1 1
CH
CH
2
2
2
2
-CH
2
2
2
2
-
compound 34b as a white solid (73%). CAS # 4870–31-9.
(250 MHz, DMSO‑d
H NMR
6
) δ 10.95 (bs, 2H, 2 × NH), 7.26 (s, 1H, H
6
), 7.06 (t,
2
2
2
3
6
-
1
9.20. HRMS-ESI (m/z) [Mꢀ H] calcd for C19
H
26
N
3
O
6
2H, J = 7.7 Hz, 2xHarom), 6.83 (bs, 1H, Harom), 6.74–6.54 (m, 3H,
found: 425.1783.
2xHarom, NH).
4
4
.2. Biological assays
4
.1.11.6. [[4-(5-iodo-2,4-dioxo-pyrimidin-1-yl)but-2-enyl]-(iso-
propoxycarbonyloxymethoxy)phosphoryl]oxymethyl isopropyl carbonate
29). A mixture of alkylated 5-iodouracil 28 (1 eq.) and bisPOC allyl-
.2.1. Mtb ThyX protein expression and purification25
(
The M. tuberculosis ThyX enzyme was expressed in E. coli BL21(DE3)/
phosphonate 23 (3 eq.), were stirred at room temperature in freshly
pLysS strains containing the recombinant pET24d plasmid carrying the
M. tuberculosis H37Rv thyX gene (Rv2754c) as previously described.
distilled DCM (0.1 M). Grubbs 2nd generation catalyst (15 mol%) was
◦
added. The mixture was refluxed at 55 C under inert atmosphere for 24
Before the purification step, 200
μ
M of flavin-adenine dinucleotide
h. Volatiles were eliminated under reduced pressure. Crude product was
applied on short silica gel flash chromatography using an elution with
(
FAD) cofactor was added to the supernatant after the lysis step to in-
crease the amount of FAD bound to Mtb ThyX protein. The solubilized
protein extract was loaded on a Hi-Trap Talon 5 ml column (GE
Healthcare) previously equilibrated with equilibration buffer containing
PE/EtOAc (1:1) as eluent to give pure product as an oil (E/Z ratio = 95/
1
5
, 45%). H NMR (400 MHz, Acetone‑d
6
) δ 10.54 (bs, 1H, NH), 8.17 (s,
1
H, H6-minor), 8.03 (s, 1H, H6-major), 5.58–5.72 (m, 2H, H ,3), 5.71–5.58
2
′
3
0 mM Hepes and 300 mM NaCl at pH 8.0. The His-tagged ThyX protein
(
m, 4H, 2 × O-CH2POC), 4.89 (p, 2H, J = 6.2 Hz, 2 × O-CHPOC),
4
.56–4.52 (m, 2H, H
H, J = 23.2, 7.7 Hz, H
1
′
-minor), 4.41 (t, 2H, J = 4.9 Hz, H
1
′
-major), 2.91 (dd,
-major),
was eluted with elution buffer (30 mM Hepes pH 8.0, 300 mM NaCl, 500
mM imidazol). The fractions containing Mtb ThyX enzyme were pooled,
buffer-exchanged on Econo-Pac PD-10 columns (Bio-rad) with the
2
4
′
-minor), 2.81 (dd, 2H, J = 22.6, 6.4 Hz, H
′
4
1
3
1
.29 (d, 12H, J = 6.2 Hz, 4 × CH3POC). C NMR (101 MHz, Acetone‑d
6
)
– –
– –
O), 153.1 (2 × C = OPOC), 150.6 (C O), 149.3 (C = CH),
3 2
equilibration buffer, concentrated to a final concentration of 480
μ
M and
δ 160.5 (C
1
◦
stored at ꢀ 20 C for further use. The measured absorbance of FAD bound
to Mtb ThyX at 450 nm showed a ratio FAD to ThyX of 1 to 3 for the
purified Mtb ThyX chain.
30.1 (d, J C-P = 15.1 Hz, CH = CH), 123.5 (d, J C-P = 11.6 Hz, CH =
CH), 84.3 (O-CH2POC), 84.2 (O-CH2POC), 72.9 (2 × O-CHPOC), 67.0
(
C
quat), 49.0 (N-CH2-major), 44.7 (N-CH2-minor), 30.2 (d, J = 138.6 Hz,
CH-CH2-major), 26.2 (d, J1C-P = 140.3 Hz, CH-CH2-minor), 20.9 (4 × CH-
CH3POC). 31P NMR (162 MHz, Acetone‑d
) δ 26.87 (P
2
27IN O11P: 605.0383, found:
4
.2.2. M. Tuberculosis ThyX NADPH oxidase assay
6
E Z
), 25.89 (P ).
+
The NADPH oxidation assay for M. tuberculosis ThyX activity in 96-
HRMS-ESI (m/z) [M + H] calcd for C18
H
well plates was used to screen the synthesized compounds at a final
concentration of 200 µM. All molecules were solubilized in dime-
thylsulfoxide (DMSO) and used at a 1% final concentration of DMSO
during the test. One hundred microlitres of standard reaction mixture
6
05.0383. Rf : 0.5 (DCM/MeOH 95:5).
4
.1.12. 5-Benzylpyrimidine-2,4(1H,3H)-dione (34a)
Preparation of LiCl (solution S1): A suspension of dry LiCl (2.25 g, 53
contained HEPES 50 mM pH 8, NaCl 30 mM, FAD 50
toethanol 1,43 mM, dUMP 100 M, NADPH 750 M, and 10
rified MtbThyX. Microtitre plates were prepared and transferred to the
microplate reader Chameleon II (Hidex). Molecules at 200 M were
μ
M, β-mercap-
mmol) in dry THF (125 ml, 0.5 M) is left stirring at room temperature for
4 h under inert atmosphere until complete dissolution of LiCl. The
μ
μ
μ
M of pu-
2
solution is stored under inert atmosphere upon use. Preparation of
μ
CuI.2LiCl (solution S2): Under inert atmosphere in a dry flask, dry CuI
incubated with MtbThyX in the standard reaction mixture for 10 min at
(
0.4 g, 0.1 eq., 2.1 mmol) and LiCl (1.8 g, 2 eq., 42.0 mmol) were mixed
◦
2
5
C before starting measurements. The reactions were started by
in dry THF (21 ml, 0.1 M). 5-Iodouracil (32) (5 g, 1 eq., 21.0 mmol) was
placed in a dry flask under inert atmosphere. Applying vigorous stirring,
the substrate was dried for 15 min under reduced pressure. Solution S1
automatically injecting NADPH into individual wells and ThyX activity
was determined by following a decrease in absorbance at 340 nm for up
◦
to 20 min at 25 C. The experiment was done in duplicates and samples
(
85 ml, 2 eq., 22.0 mmol) was added and stirred 30 min at room tem-
◦
with added DMSO and enzyme-free reactions were used as positive and
negative controls, respectively. % of inhibition was calculated using the
following equation: (1-Vi/Vo)*100; Vo and Vi are, respectively, the
initial rates of the reaction without or with addition of molecule in the
assay.
perature. The solution was cooled down to ꢀ 30 C and MeMgCl in dry
THF (14.3 ml, 2 eq., 22.0 mmol) was added dropwise. After completion
◦
of the addition, the resulting solution was stirred at ꢀ 30 C for further
3
0 min, i-PrMgCl⋅LiCl in dry THF (19.23 ml, 1.2 eq., 25.2 mmol) was
then dropwised and the resulting mixture was allowed to warm up to
room temperature. After 1 h, the mixture was cooled to –30 ◦C and
1
2

N.G. Biteau et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116351
4
.3. Virtual docking
org/10.1016/j.bmc.2021.116351.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
1
3