Extension of furopyrimidine nucleosides with 5-alkynyl substituent: Synthesis, high fluorescence, and antiviral effect in the absence of free ribose hydroxyl groups

Article abstract of DOI:10.1016/j.ejmech.2020.112884

A novel methodology to access alkynyl nucleoside analogues is elaborated. Highly fluorescent 5-alkynylfuropyrimidines were synthesized (97-46%) and their antiviral properties investigated in vitro. Regiochemistry of the functionalization was achieved with the aid of 5-endo-dig electrophilic halocyclization of acetyl 5-p-tolyl- or 5-p-pentylphenyl-2′-deoxyuridine. Structure of one of the resulting nucleosides, 6-p-tolyl-5-iodo-2′-deoxyribofuranosyl-furo[2,3-d]pyrimidin-2-one, was confirmed by X-ray crystallography, and its conformation was compared to related nucleosides. Diverse alkynyl substituents were introduced at the heterobicyclic base C-5 position via Sonogashira coupling of 5-iodo-2′-deoxyribofuranosyl-furo[2,3-d]pyrimidin-2-ones. The resulting compounds had fluorescence emissions of 452–481 nm. High quantum yields of 0.53–0.60 were observed for 9-ethynyl-9-fluorenol and propargyl alcohol/methyl ether-modified furopyrimidines. These modified nucleosides, designed in the form of ribose acetyl esters, are potential tools for fluorescent tagging, studying nucleoside metabolism, 2′-deoxyribonucleoside kinase activity, and antiviral activity. Antiviral assays against a broad spectrum of DNA and RNA viruses showed that in human embryonic lung (HEL) cell cultures some of the compounds showed antiviral activity (EC₅₀ 1.3–13.2 μM) against varicella-zoster virus (VZV). The alkynyl furopyrimidine with two p-pentylphenyl substituents emerged as the best compound with reasonable and selective anti-VZV activity, confirming p-pentylphenyl potency as a pharmacophore.

Full text of DOI:10.1016/j.ejmech.2020.112884

European Journal of Medicinal Chemistry 209 (2021) 112884
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Extension of furopyrimidine nucleosides with 5-alkynyl substituent:
Synthesis, high fluorescence, and antiviral effect in the absence of free
ribose hydroxyl groups
a
b
b
a
ꢀ
Renata Kaczmarek , Dylan J. Twardy , Trevor L. Olson , Dariusz Korczynski ,
Graciela Andrei d, Robert Snoeck d, Rafał Dolot a, Kraig A. Wheeler c,
, b, *
Roman Dembinski a
a
ꢀ ꢀ
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363, Łodz, Poland
b Department of Chemistry, Oakland University, 146 Library Drive, Rochester, MI, 48309-4479, USA
c Department of Chemistry, Whitworth University, 300 W. Hawthorne Rd., Spokane, WA, 99251, USA
d Rega Institute, Department of Microbiology, Immunology and Transplantation, Herestraat 49, 3000, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2020
Received in revised form
15 September 2020
Accepted 23 September 2020
Available online 28 September 2020
A novel methodology to access alkynyl nucleoside analogues is elaborated. Highly fluorescent 5-
alkynylfuropyrimidines were synthesized (97-46%) and their antiviral properties investigated in vitro.
Regiochemistry of the functionalization was achieved with the aid of 5-endo-dig electrophilic halocyc-
lization of acetyl 5-p-tolyl- or 5-p-pentylphenyl-20-deoxyuridine. Structure of one of the resulting nu-
cleosides, 6-p-tolyl-5-iodo-20-deoxyribofuranosyl-furo[2,3-d]pyrimidin-2-one, was confirmed by X-ray
crystallography, and its conformation was compared to related nucleosides. Diverse alkynyl substituents
were introduced at the heterobicyclic base C-5 position via Sonogashira coupling of 5-iodo-20-deoxy-
ribofuranosyl-furo[2,3-d]pyrimidin-2-ones. The resulting compounds had fluorescence emissions of 452
e481 nm. High quantum yields of 0.53e0.60 were observed for 9-ethynyl-9-fluorenol and propargyl
alcohol/methyl ether-modified furopyrimidines. These modified nucleosides, designed in the form of
ribose acetyl esters, are potential tools for fluorescent tagging, studying nucleoside metabolism, 20-
deoxyribonucleoside kinase activity, and antiviral activity. Antiviral assays against a broad spectrum of
DNA and RNA viruses showed that in human embryonic lung (HEL) cell cultures some of the compounds
Keywords:
5-Alkynylfuropyrimidines
Alkynes
Modified nucleosides
Antiviral chemotherapy
Varicella-zoster virus (VZV)
showed antiviral activity (EC50 1.3e13.2 mM) against varicella-zoster virus (VZV). The alkynyl furopyr-
imidine with two p-pentylphenyl substituents emerged as the best compound with reasonable and
selective anti-VZV activity, confirming p-pentylphenyl potency as a pharmacophore.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
can be imposed by appending an alkyne, an example includes 5-
alkynyluridines [15e17]. Alkynyl modifications also provide a
Nucleoside analogues are a cornerstone for the treatment of
cancer and viral diseases [1e8]. Among the most active analogues,
furopyrimidine nucleosides stand out as potent and selective
antiviral agents with a high specific activity against varicella-zoster
virus (VZV) [9e11]. p-Alkylphenyl, particularly p-pentylphenyl,
substituents attached to C-6 give rise to the most effective struc-
tures (1a,b; Fig. 1) [12e14]. In parallel, significant biological activity
synthetic handle for further functionalization and modification of
nucleosides [18e21].
Intrigued by the combination of two active units, a synthesis of
alkynyl-substituted furopyrimidines was envisioned. Despite
extensive modification efforts, synthetic chemists have rarely
described a furopyrimidine ring with directly attached alkynes. To
this point, one report has described a C-4 attached alkyne, intro-
duced to the pyrimidine ring before cycloisomerization to furo-
pyrimidine [22]. C-6 alkynyl analogues have been prepared by
direct bromination of the furopyrimidine ring and subsequent
coupling with terminal alkynes [23e26]. However, introduction of
an alkyne at C-5 lacks a general method thus far. Formerly,
* Corresponding author. Department of Chemistry, Oakland University, 146 Li-
brary Drive, Rochester, MI, 48309-4479, USA.
E-mail address: dembinsk@oakland.edu (R. Dembinski).
https://doi.org/10.1016/j.ejmech.2020.112884
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
crystallize, which often satisfies the isolation protocol. Additionally,
iodo derivatives are expected to be the most reactive toward
functionalization. 5-Endo-dig electrophilic cyclization of 5-alkynyl-
20-deoxyuridine (2 or 3) with N-iodosuccinimide in acetone at
23 ꢀC furnished corresponding 5-iodofuropyrimidines (4 or 5).
Following this procedure iodo-functionalized nucleosides 4 and 5
were obtained with a good yield (92% and 76%, respectively;
Scheme 1). Structure of the acylated nucleoside 4 and 5 were
confirmed by 1H and 13C NMR, IR spectroscopy, and HRMS. The 13
C
NMR spectra feature characteristic iodofuro resonances C(5)-I at
53.88 and 53.95 ppm (CDCl3), which differ from non-iodinated
derivatives [40].
To explore the structure-reactivity relationship for the appen-
ded alkyne fragment, novel nucleosides were prepared (Scheme 1).
Alkynyl substituents include an oxypropynyl unit (propargyl
alcohol, 6a/7a, R’ ¼ CH2OH; 76/81% yield), its one carbon extended
homologue (6b/7b, R’ ¼ CH2CH2OH; 95/81%), tertiary aryl alcohol
(6c, R’ ¼ 9-fluorenol-9-yl; 83%), methyl propargyl ether (6d,
R’ ¼ CH2OMe; 66%), propargyl acetate (6e, R’ ¼ CH2OAc; 46%), N-(3-
butynyl)phthalimide and N-(5-hexynyl)phthalimide (6f/6g, R’ ¼
(CH2)2-phthalimide/(CH2)4-phthalimide; 93/82%). The protected
amine linkers (phthalimide) offer an opportunity for further func-
tionalization, for example to N-phosphoryl amino acids [41].
Finally, four structures containing combinations of 4-methylphenyl
(p-tolyl) and 4-pentylphenyl (antiviral active substituents, cf. Fig. 1)
were also obtained (6h/7h, R’ ¼ C6H4-p-CH3 and 6i/7i, R’ ¼ C6H4-p-
n-C5H11 70/64% and 97/72% yield). The protocol involved Sonoga-
shira coupling in the presence of tetrakis(triphenyl phosphine)
palladium(0), copper(I) iodide, and triethylamine (DMF, 23 ꢀC). The
structures of alkynyl nucleosides 6 and 7 were confirmed by 1H and
13C NMR, as well as IR spectroscopy. 1H NMR spectra exhibited one
signal in the region of the H-6, as expected (8.51e8.25 ppm). Two
signals characteristic for an alkyne were observed in 13C NMR for 6
and 7 (99.19e94.50 and 95.40e94.28 ppm). In the 13C NMR spec-
trum for 6c, an excess of five signals was observed beyond the
number of expected equivalent carbons. It is likely that the
conformation with C-2 carbonyl group anti with respect to the
ribose, alike the one observed in the crystal structure of 4 (vide
infra), exists in solution. The resulting hindered rotation of fluo-
renol, enhanced by potential interactions of the quaternary and O5’
acetyloxy group, may lead to a magnetic nonequivalency. This effect
was observed for most (five out of six per ring) of the fluorenol
aromatic carbons. Similarly, in the 1H NMR propargyl protons of
CH2OH groups in compounds 6a and 7a appeared as nonequivalent
Fig. 1. Structures of selected 6-(p-alkylphenyl) furopyrimidine nucleosides 1.
compounds containing an alkyne group attached at the C-5 posi-
tion were detected. These byproducts were isolated with meager
yields (3e9%) in reactions that targeted other structures [27e29].
Recently, an interesting synthetic approach towards triazole con-
jugates of alkynyl-modified furopyrimidine base has been reported
[30]. This methodology leads to compounds with desirable bio-
logical activity but synthetically renders mixtures containing
alkyne-free furopyrimidine. Additionally, it lacks diversity, since
both, C-6 substituent and C-5 alkyne endgroup, originate from the
same terminal alkyne reaction component.
An electrophilic cyclization reaction leading to the formation of
-halofuran rings offers a significant synthetic opportunity [31,32].
b
An analogous protocol leads to the formation of 5-
halofuropyrimidine nucleosides, which introduces the halogen in
a fully regioselective manner [33]. Addressing this so far unresolved
synthetic shortcoming in nucleoside chemistry, the extension of
the heterocyclic base conjugated system by appending an alkyne at
C-5 was sought by combining iodofuropyrimidines with various
terminal acetylenes. Considering the antiviral activity of furopyr-
imidine nucleosides 1, synthesis of a series of derivatives containing
a p-alkylphenyl group positioned at C-6 scaffold was attempted.
Interestingly, acyl-substituted nucleosides are becoming effec-
tive prodrugs due to increased lipophilicity. The isobutyryl group is
installed at the 50-hydroxyl end of N-4-hydroxycytidine derivative
EIDD-2801, which is an oral experimental antiviral drug [34]. This
synthetic nucleoside is one of only two highly effective active
compounds against coronaviruses to come out of 6 years of
screening [35]. Other groups, including 50-L-valyl ester, were
investigated to increase oral bioavailability [36,37]. We decided to
investigate a series of compounds with the acetyl group installed
on the ribose unit as its 30- and 50-ester.
dAB (coupling with OH proton).
A DEPT spectrum for the
phthalimide-derived compound 6g, which was applied to the
entire series of 13C NMR assignments, is provided in the Supple-
mentary Materials. HRMS spectra for both 6 and 7 exhibited an m/z
peak with an accurate mass [M þ H]þ for the molecular ion.
Crystallography. To gain more molecular information, crys-
tallographical analysis of furopyrimidine was attempted. Despite
the persistent synthetic pursuit of furopyrimidine nucleosides, so
far only a few and only C-6 substituted have been crystallograph-
ically characterized [20,25,38,40,42e46]. Although nucleosides are
often resistant to crystallization, efforts to obtain diffraction quality
crystals were successful for compound 4, using methanol at room
temperature. X-Ray crystallography confirmed the structure of the
30,50-di-O-acetyl-5-iodo-6-(p-tolyl)furopyrimidine (4, Fig. 2) [47].
In the solid state, the C2 carbonyl group of planar furanopyr-
imidine ring 4 adopted an anti orientation toward the ribose, with a
2. Results and discussion
Synthesis. Starting compounds containing 4-methylphenyl (p-
tolyl) and 4-pentylphenyl substituents at C-6 were selected. p-
Alkylphenyl substituents are participants of the most potent anti-
viral active structures [12]. Preparative syntheses of a starting
material 20-deoxy-5-alkynyluridines 2 and 3 were carried out from
20-deoxy-5-iodouridine (Ac-IdU) and the appropriate terminal
alkyne (Pd- and Cu-catalyzed Sonogashira coupling, Scheme 1)
[38]. The next steps combine cycloisomerization and hal-
ofunctionalization reactions. Halogens such as iodine, bromine, and
chlorine can be introduced via electrophilic cyclization of 5-
alkynyluridines [33,39]. Among the halogens, iodo derivatives are
most convenient to prepare, since they are more prone to
glycosidic bond torsion angle
c
(C2eN3eC10eO40) of ꢁ167.5(2)ꢀ, a
conformation similar to previously crystallographically character-
ized furanopyrimidine nucleosides (ꢁ120.8(5) to ꢁ172.81(18),
average ꢁ155.8; Table 1). In the molecule of 4, the N-glycosidic
bond (riboseebase distance), C10eN3 1.488(3)Å, was comparable to
2

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
Scheme 1. Synthesis and structures of 5-iodo- and 5-alkynylfuropyrimidine nucleosides 4,5 and 6,7. For summary of yields see Table S1 in the Supplementary Materials (p. S2).
previously crystallographically characterized furanopyrimidine
nucleosides (1.468(7) to 1.502(6), average 1.487; Table 1). Ribose
geometrical parameters, such as the pseudorotation phase angle (P)
were determined for 4 to be C30-exo with P ¼ 209.73ꢀ (puckering
amplitude of 27.38). The same parameters calculated for other
crystallographically characterized furopyrimidines indicate the
presence of both C20-endo and C30-exo conformation (Table 1).
Results of this analysis indicates that appending an alkyne at the C-
5 position did not meaningfully affect the base conformational
positioning, which is consistent across these characterized com-
pounds; this effect was not correlated to the ribose ring puckering
since it varies in all of the above reviewed furopyrimidine struc-
tures in the solid state, and is likely determined by packing forces.
In addition, X-ray crystallographic analysis determined the
iodine atom position at C-5 in the furopyrimidine framework,
confirming the regiochemistry of the iodocyclization reaction. If a
cycloisomerization were to precede the halogenation, then other
available sp2 carbons in the bicyclic core could potentially compete
for the iodination reagent.
publication titles [48,49]. Surprisingly, we were not able to find any
quantitative measurements in the literature beyond recent de-
scriptions of empirical and theoretical optical parameters attending
only the furopyrimidine base [50,51]. In contrast, a number of
fluorescence studies of pyrrolopyrimidines have been reported
[52,53].
Considering the importance of fluorescent nucleic acid compo-
nents [54e57], quantitative optical properties of selected furopyr-
imidine nucleosides (6a,c,d,f,h and 7a,h, as well as 4) were
determined in methanol (UVevisible and emission spectra; Figs. 3
and 4, respectively). Almost identical UVevis and fluorescence
traces were observed for 6h and 7h homologues.
An examination of UVevisible spectra indicates molar absorp-
tivity ε ranging from 8700 to 19,600 Mꢁ1cmꢁ1 (Table 2). The pentyl
substituent slightly increases ε, as compared to the corresponding
methyl-substituted homologues (7a vs 6a, and 7h vs 6h). By com-
parison, furopyrimidine without an alkyne substituent (reference
compound 1b) absorbs at lmax 280 and 352 nm (ε 23,200 and
17,300 Mꢁ1cmꢁ1, respectively). Emission wavelength (lem) values
were observed for alkynyl furopyrimidines 6a,c,d,f,h and 7a,h in
the range of 453e481 nm (lex ¼ 360 nm, Table 2), and were red-
shifted compared to the alkyne-free 1b (lem ¼ 434 nm). Slightly
Fluorescence. The fact that furopyrimidine nucleosides display
a strong optical response has been known for a while now; these
bicyclic structures have been heralded as “fluorescent”, featured in
3

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
Fig. 3. UVevisible spectra of selected furopyrimidine nucleosides (methanol, 24 ꢀC).
Fig. 2. An ORTEP view of the 4 with the crystallographical atom-labeling scheme.
Thermal ellipsoids are drawn at the 50% probability level. Selected interatomic dis-
tances [Å]: C(5)eI(1) 2.072(3); C(4a)-C(5) 1.441(4); C(5)eC(6) 1.360(4); C(6)eO(7)
1.416(4); C(7a)-O(7) 1.361(3); C(4a)-C(71) 1.397(4). Key angles [deg]: N3eC10-O4’
109.3(2); C4A-C5-I1 121.67(19); C6eC5eI1 130.9(2).
lower values were determined for the iododerivative
4
(
lem ¼ 445 nm).
The nucleosides’ fluorescence quantum yields
F
showed trends
towards a slight increase with the presence of pentyl vs a methyl
substituent (7a vs 6a, and 7h vs 6h, Table 2). The highest quantum
yields were observed for the fluorenol containing, propargyl
alcohol containing, and propargyl methyl ether containing nucle-
osides 6c, 7a and 6d (
iodine attached to the furopyrimidine core quenched the emission
of fluorescence almost completely for iodonucleoside 4 (
F
¼ 0.60, 0.59, and 0.53, respectively). The
F
¼ 0.04),
in line with identified halogen effect [58]. Overall, the quantum
yields of alkynyl furopyrimidines are competitive with those of
recently reported alkynyl fluorescent nucleosides [59,60], and are
even comparable with values for nucleosides labeled with auxiliary
chromophores [61].
Biological activity. Considering the antiviral activity of furo-
pyrimidine nucleosides, the biological activity of compounds 6 and
7 was investigated. Antiviral properties were determined using
in vitro assays against several DNA and RNA viruses, including
Fig. 4. Emission
(
lex
¼
360 nm) spectra of selected furopyrimidine nucleosides
(methanol, 24 ꢀC).
herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2),
thymidine kinase deficient (TKꢁ) HSV-1, adenovirus-2, human
coronavirus (229E), human cytomegalovirus (AD-169 and Davis
strains), varicella-zoster virus (VZV, TKþ Oka strain and TKe 07-1
Table 1
Comparison of selected conformational parameters for crystallographically characterized furopyrimidine nucleosides and 4.
CCDC deposition number
C-6 substituent
c
[ꢀ] (C2eN3eC10eO40) riboseebase distance C10eN3 [Å]
P e phase angle, amplitude)a ref.
ring puckering
175815
249882
ferrocene
ꢁ148.0(2)
ꢁ155.4(2)
ꢁ161.86(19)
ꢁ162.2(2)
ꢁ153.26(13)
ꢁ153.8(3)
1.478(3)
1.479(2)
1.483(3)
1.484(3)
1.4850(19)
1.481(4)
1.498(3)
1.497(2)
1.502(6)
C20-endo
C30-exo
164.2, 36.60 184.4, 36.10
150.6, 26.36 150.5, 26.26
42
38
p-tolylb
C20-endob
C20-endo
C20-endo
C20-endo
C20-endo
C30-exobc
C30-exo
683563
683603
683604
715301
785464
propyl
p-Bu-phenyl
170.8, 34.12
168.0, 33.62
171.1, 33.62
208.8, 33.62
182.1, 35.41
40
25
25
20
43
hexyl-pyridine ꢁ154.9(2)
deoxyuridineb
thiophene
-pyrrole
ꢁ172.81(18)
ꢁ160.0(3)
943139
1435280
1864088
propanol
ꢁ120.8(5)
ꢁ159.62(17)
ꢁ167.5(2)
1.468(7)
1.485(3)
1.488(3)
C20-endo
159.0, 37.25
173.2, 37.25
209.7, 27.38
44
45
Ph(CH2)2
C20-endo c
c
,
d
p-tolylb,
this workd
C30-exob,d
a
Calculated using pucker.py script for PyMOL, written by S. M. Law (https://pymolwiki.org/index.php/Pucker).
b
c
Di-O-acetyl derivative.
Arabino derivative.
5-Iodo derivative 4.
d
4

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
Table 2
Optical properties of selected furopyrimidine nucleosides 6,7, and 4 (lex ¼ 360 nm).
nucleoside
R0
lmax [nm]
molar absorptivity [Mꢁ1cmꢁ1
]
lem [nm]
F
6a
7a
7b
6c
6d
6f
6h
7h
4
CH2OH
CH2OH
362
362
360
365
362
364
369
370
356
8700
456
452
461
454
453
457
481
481
445
0.49
0.59
0.50
0.60
0.53
0.04
0.19
0.21
0.05
19,600
17,400
22,900
16,600
17,000
19,000
20,800
14,400
CH2CH2OH
9-fluorenol
CH2OMe
(CH2)2-phthalimide
p-C6H4Me
p-C6H4Me
n/a
strain), vesicular stomatitis virus, coxsackie virus B4 respiratory
syncytial virus, parainfluenza-3 virus, reovirus-1, sindbis virus,
coxsackie virus B4, punta toro virus, yellow fever virus, influenza A
virus (H1N1 and H3N2 subtypes) and influenza B virus. Results
from these in vitro assays indicate that some of the derivatives
showed activity exclusively against VZV in human embryonic lung
(HEL) fibroblasts (Table 3).
Diverse substituents at the propargyl carbon atom, including alkyl,
aryl, hydroxy, methoxy, and acetoxy groups, were introduced. It has
been demonstrated that the presence of an alkyne significantly
increases the fluorescence quantum yield compared to the iodo
precursor. Also, X-ray crystallographic analysis of the iododer-
ivative 4 determined the nucleoside conformation (sugar puckering
and base positioning) in the solid state (anti and C30-exo). Accord-
ingly, the characterization of the formed fused furan ring and the
regiochemistry of the iodocyclization reaction were confirmed.
Synthesis of free hydroxy groups homologues is currently in
progress.
The in vitro assays against several DNA and RNA viruses indicate
that some of the compounds showed antiviral activity only against
VZV despite protected ribose hydroxyl groups. However, inhibition
for the growth of HEL cells was also observed, and overall, the
investigated nucleosides exhibited cytotoxicity, except for the
compound with a duplicate p-pentylphenyl pharmacophore (7i).
Compound 7i with two p-pentylphenyl groups exhibited reason-
able anti-VZV activity and was not inhibitory to HEL cells growth up
In particular, compounds 6c, 6g, 6h, 7a, 7b, and 7i were inhib-
itory against VZV/TK-competent (wild-type; EC50 in the range of
1.295e7.575
range of 7.61e13
morphology up to a concentration of 100
m
M) as well as VZV/TK-deficient (mutant; EC50 in the
M). Although these compounds did not alter cell
M, they affected HEL
m
m
cells growth, except for 7i. Hence, the selectivity indices (SI) [ratio
CC50 (50% cytostatic concentration for HEL cells)/EC50 (50% effective
antiviral concentration) were ꢂ1 for 6c, 6g, 6h, 7a and 7b. In
contrast, specific antiviral effects (i.e. SI’s of 67 and 11, respectively
for VZV TKþ and TKꢁ strains) were observed for 7i, the compound
with duplicate p-pentylphenyl pharmacophore. Even though 7i
proved active against the wild-type and TK-VZV viruses, the activity
was 6-fold lower against the mutant virus than the wild-type virus.
Conclusions. The methodology of introducing alkynes at C-5 of
to a concentration of 100 mM.
the
furopyrimidine
ring
was
elaborated.
Novel
5-
3. Materials and methods (experimental)
alkynylfuropyrimidine derivatives, with biologically active sub-
stituents at C-6 (p-tolyl and p-pentylphenyl), were synthesized.
General Instrumentation. NMR measurements were carried
out on
a Bruker Avance III 500 spectrometer, operating at
13
500.13 MHz for 1H and 125.75 MHz for C, at 20 ꢀC. Mass spectra
were recorded on an Agilent 6520 Q-TOF LCMS (HRMS). FT-IR
spectra were recorded on Varian 3100 Excalibur, ThermoScientific
Nicolet 6700 ATR, and Bruker Alpha-P ATR spectrometers. All
products were stored in a refrigerator (4 ꢀC).
Table 3
Antiviral activity of furopyrimidines 6 and 7 against varicella-zoster virus (VZV) in
human embryonic lung (HEL) cells.a
Compound Antiviral activity EC50
[
m
M]b
Cytotoxicity [mM]
20-Deoxy-30,50-di-O-acetyl-5-iodouridine (Ac-IdU) [38], 20-
deoxy-30,50-di-O-acetyl-5-p-tolylethynyluridine (2) [40,62], and 20-
deoxy-30,50-di-O-acetyl-5-(4-pentylphenyl)ethynyluridine (3) [19]
were prepared by literature protocols [63].
TKþ VZV strain TKꢁ VZV strain Cell morphology Cell growth
d
OKA
07e1
(MCC)c
(CC50
)
6a
66.87
>100
>100
100
>100
100
nde
7a
6b
7.56 0.74
76.47
12.99 7.0
>100
3.8 1.1
nde
2.66 0.30
2.36 0.39
nde
3.1. Iodocyclization of 20-deoxy-30,50-di-O-acetyl-5-p-
alkylphenylethynyluridines 2 and 3. General procedure
7b
6c
6d
4.02 2.8
1.3 0.5
20
12.99 7.0
7.61 0.0
44.72
100
>100
>100
100
100
60
100
20
100
>440
>300
6e
>100
100
nde
A round bottom flask was charged with 20-deoxy-3′,5′-di-O-
acetyl-5-p-alkylphenylethynyluridine 2 or 3 (1.88 mmol), N-iodo-
succinimide (0.508 g, 2.26 mmol), and acetone (15 mL). The reac-
tion mixture was stirred at room temperature for 2 h under argon.
Solvent was removed and the residue was dissolved in chloroform
(10 mL). Silica gel column chromatography (230e400 mesh, chlo-
roform) gave a colorless fraction. The product was dried by oil
pump vacuum for 2 h.
6f
31.02
38.07
nde
6g
6h
7h
7.23 1.71
6.84 0.0
7.61
13.2 2.7
10.92 1.98
>20
6.22 0.73
8.77 0.48
nde
6i
>4
>4
nde
>100
>300
>300
7i
1.5 0.7
0.49
0.026
8.94 0.0
23.22
12.01
acyclovir
brivudin
a
For detailed Tables see Supporting Materials.
Effective concentration required to reduce virus plaque formation by 50%. Virus
b
input was 20 plaque forming units (PFU).
3.2. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-iodo-6-(4-
methylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (4)
c
Minimum cytotoxic concentration that causes a microscopically detectable
alteration of cell morphology.
d
Cytotoxic concentration required to reduce cell growth by 50%.
Not determined.
e
From 20-deoxy-30,50-di-O-acetyl-5-(4-tolyl)ethynyluridine (2,
5

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
0.800 g). White solid of 4 (0.955 g, 1.73 mmol, 92%), mp 60-63 ꢀC.
H-30), 4.71 (dd, J ¼ 11.8, 1.9 Hz, 1H, H-50), 4.61 and 4.58 (dAB,
J ¼ 16.0, 5.6 Hz, 2H, CH2O), 4.47e4.44 (m, 1H, H-40), 4.42 (dd,
J ¼ 11.9, 4.6 Hz, 1H, H-50), 3.46 (t, J ¼ 6.3 Hz, 1H, OH), 3.01 (ddd,
J ¼ 14.5, 5.5, 1.5 Hz, 1H, H-200), 2.41 (s, 3H, CH3), 2.14 (s, 3H, Ac), 2.13
(s, 3H, Ac), 2.03 (ddd, J ¼ 14.5, 7.9, 6.6 Hz, 1H, H-20); 13C 172.02,
170.60, 170.25, 156.08, 154.62, 141.02, 134.39, 129.63 (2C), 125.99
(2C), 125.28, 109.47, 97.53, 94.53, 89.02, 84.06, 74.62, 74.51, 63.96,
NMR (CDCl3,
d
): 1H 8.11 (s, 1H, H-4), 7.97 (d, J ¼ 8.1 Hz, 2H, C6H4),
7.28 (d, J ¼ 8.1 Hz, 2H, C6H4), 6.36 (dd, J ¼ 7.6, 6.0 Hz, 1H, H-10), 5.26
(td, J ¼ 6.2, 2.0 Hz,1H, H-30), 4.48 (dd, J ¼ 12.0, 2.7 Hz,1H, H-50), 4.45
(dd, J ¼ 5.4, 2.6 Hz,1H, H-40), 4.41 (dd, J ¼ 11.4, 3.3 Hz,1H, H-50), 2.97
(ddd, J ¼ 14.5, 5.7, 1.3 Hz,1H, H-200), 2.40 (s, 3H, CH3), 2.13 (s, 3H, Ac),
2.12 (s, 3H, Ac), 2.11 (partially obscured ddd, J ¼ 7.7, 7.5 Hz, 1H, H-
20); 13C 170.54, 170.48, 170.24, 154.87, 153.27, 140.93, 136.33, 129.47
(2C), 127.22 (2C), 125.17, 112.67, 88.72, 83.56, 74.48, 63.82, 53.88,
51.55, 39.56, 21.68, 21.19, 20.95. NMR (DMSO‑d6, d
): 1H 8.43 (s, 1H,
H-4), 8.01 (d, J ¼ 8.2 Hz, 2H, C6H4), 7.37 (d, J ¼ 8.2 Hz, 2H, C6H4), 6.21
(t, J ¼ 7.2 Hz, 1H, H-10), 5.53e5.49 (m, 1H, H-30), 5.24e5.19 (m, 1H,
H-40), 4.45e4.39 (m, 3H, CH2, H-50), 4.38e4.29 (m, 2H, H-500, OH),
2.63e2.57 (m, 1H, H-20), 2.46e2.40 (m, 1H, H-200), 2.38 (s, 3H, CH3),
39.55, 21.61, 21.25, 20.96. NMR (DMSO‑d6, d
): 1H 8.16 (s, 1H, H-4),
7.91 (d, J ¼ 7.7 Hz, 2H, C6H4), 7.36 (d, J ¼ 7.3 Hz, 2H, C6H4), 6.19 (t,
J ¼ 6.5 Hz, 1H, H-10), 5.25e5.21 (m, 1H, H-30), 4.47e4.42 (m, 1H, H-
40), 4.37 (dd, J ¼ 12.1, 3.0 Hz, 1H, H-50), 4.28 (dd, J ¼ 12.2, 5.2 Hz, 1H,
H-50), 2.62 (dd, J ¼ 14.2, 5.4 Hz, 1H, H-20), 2.42 (ddd, J ¼ 14.5, 7.2, 7.2
Hz, 1H, H-200), 2.36 (s, 3H, CH3), 2.12 (s, 3H, CH3), 2.08 (s, 3H, CH3). IR
2.09 (s, 3H, CH3), 2.08 (s, 3H, CH3). IR (n
, cmꢁ1, ATR) 2938 vw, 2925
vw, 1739 s, 1664 vs, 1599 s, 1573 s, 1395 m, 1220 vs, 1018 vs. UVevis
(MeOH, 23.5 ꢃ 10ꢁ6 M) lmax 297 (ε 6470), 322 (ε 4940), 362 nm (ε
8700 Mꢁ1cmꢁ1). Fluorescence (MeOH, ca. 5.39 ꢃ 10ꢁ6 M) lex
(n
, cmꢁ1, ATR) 3089 w, 3028 w, 3089 w, 2951 w, 1738 s, 1665 vs,
1590 s, 1375 m, 1220 vs, 1184 s, 1014 m. UVevis (MeOH,
21.7 ꢃ 10ꢁ6 M) lmax 257 (ε 18,500), 356 nm (ε 14,400 Mꢁ1cmꢁ1).
Fluorescence (MeOH, ca. 3.65 ꢃ 10ꢁ6 M) lex 360 nm, lem 444 nm,
360 nm, lem 456 nm,
F
¼ 0.49. HRMS (ESI-TOF) [M þ H]þ calcd for
C
25H25N2O8 481.1605, found: 481.1605.
F
¼ 0.05. HRMS (ESI-TOF) [M þ H]þ calcd for C22H22IN2O7
3.6. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(4-
hydroxybut-1-yn-1-yl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (6b)
553.0466, found: 553.0466.
3.3. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-iodo-6-(4-
pentylphenyl)-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (5)).
From 3-butyn-1-ol (0.095 mL, 1.25 mmol). Light yellow solid:
0.235 g, 0.476 mmol, 95%. NMR (CDCl3, d
): 1H 8.27 (s, 1H, H-4), 8.02
From 20-deoxy-30,50-di-O-acetyl-5-(4-pentylphenyl)ethynylur-
idine (3, 0.906 g). White solid of 5 (0.869 g, 1.43 mmol, 76%). NMR
(d, J ¼ 8.3 Hz, 2H, C6H4), 7.24 (d, J ¼ 8.3 Hz, 2H, C6H4), 6.31 (dd,
J ¼ 7.9, 5.6 Hz, 1H, H-10), 5.24 (td, J ¼ 6.4, 2.1 Hz, 1H, H-30), 4.57 (dd,
J ¼ 11.9, 2.5 Hz, 1H, H-50), 4.43e4.41 (m, 1H, H-40), 4.39 (dd, J ¼ 11.5,
4.0 Hz,1H, H-50), 3.90 (br t, J ¼ 5.8 Hz, 2H, CH2O), 2.97 (ddd, J ¼ 14.4,
5.6, 1.8 Hz, 1H, H-200), 2.97 (signal partially obscured, 1H, OH), 2.81
(t, J ¼ 6.0 Hz, 2H, CH2), 2.40 (s, 3H, CH3), 2.12 (s, 3H, Ac), 2.08 (s, 3H,
Ac), 2.05 (ddd, J ¼ 14.6, 7.8, 6.7 Hz, 1H, H-20); 13C 171.08, 170.54,
170.16, 156.21, 154.62, 140.68, 134.68, 129.53 (2C), 125.88 (2C),
125.53, 109.39, 97.26, 95.36, 88.86, 83.79, 74.52, 71.18, 63.85, 60.74,
(CDCl3,
d
): 1H 8.10 (s, 1H, H-4), 7.97 (d, J ¼ 8.4 Hz, 2H, C6H4), 7.26 (d,
J ¼ 8.1 Hz, 2H, C6H4), 6.35 (dd, J ¼ 7.8, 5.8 Hz, 1H, H-10), 5.24 (td,
J ¼ 6.1, 1.9 Hz, 1H, H-30), 4.47 (dd, J ¼ 11.9, 2.9 Hz, 1H, H-50), 4.44 (dd,
J ¼ 5.0, 2.3 Hz, 1H, H-40), 4.39 (dd, J ¼ 11.5, 3.0 Hz, 1H, H-50), 2.95
(ddd, J ¼ 14.6, 5.6, 1.3 Hz, 1H, H-200), 2.61 (t, 2H, J ¼ 7.8 Hz, CH2),
2.14e2.06 (partially obscured m, 1H, H-20), 2.12 (s, 3H, Ac), 2.10 (s,
3H, Ac),1.60 (p, J ¼ 7.4 Hz, 2H, CH2), 1.33e1.25 (m, 4H, 2CH2), 0.86 (t,
J ¼ 6.7 Hz, 3H, CH3); 13C 170.57, 170.47, 170.33, 154.88, 153.15,
145.90, 136.39, 128.83 (2C), 127.16 (2C), 125.29, 112.69, 88.72, 83.54,
74.56, 63.88, 53.95, 39.51, 35.93, 31.48, 30.95, 22.59, 21.35, 21.04,
14.14. IR (cmꢁ1, ATR) 2953 w, 2923 w, 2850 w, 1734 s, 1650 vs,
1590 s, 1381 m, 1210 m, 1181 s, 1010 s. HRMS (ESI-TOF) [M þ H]þ
calcd for C26H30IN2O7 609.1093, found: 609.1117.
39.48, 24.45, 21.63, 20.96 (2C). IR (n
, cmꢁ1, ATR) 3391 br w, 2933 w,
1740 s, 1664 vs, 1641 m, 1574 m, 1396 m, 1367 m, 1216 vs, 1188 s,
1100 m, 1032 s, 1015 s. HRMS (ESI-TOF) [M þ H]þ calcd for
C
26H27N2O8 495.1762, found: 495.1762.
3.7. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-((9-
3.4. 5-Alkynyl-6-arylfuropyrimidine nucleosides 6a-h or 7a,b,h,i.
General procedure
hydroxy-9H-fluoren-9-yl)ethynyl)-6-(4-methylphenyl)-2,3-
dihydrofuro[2,3-d]pyrimidin-2-one (6c)
A round bottom flask (25 mL) was charged with iodofuropyr-
imidine nucleoside (4, 0.276 g, 0.500 mmol; or 5, 0.304 g,
0.500 mmol), Pd(PPh3)4 (0.035 g, 0.030 mmol), CuI (0.006 g,
0.03 mmol), DMF (5 mL), Et3N (0.174 mL, 1.25 mmol), and the
respective terminal alkynes. The reaction mixture was stirred at
room temperature for 22 h. Solvent was removed (by oil pump
vacuum) and the residue was dissolved in chloroform (10 mL), and
subjected to silica gel column chromatography (230e400 mesh,
chloroform). The product was dried by oil pump vacuum for 2 h to
give 6a-i or 7a,b,h,i.
From 9-ethynyl-9-fluorenol (0.259 mg,1.25 mmol). Yellow solid:
0.261 g, 0.415 mmol, 83%. NMR (CDCl3, d
): 1H 8.37 (s, 1H, H-4), 7.82
(d, J ¼ 8.3 Hz, 2H, p-C6H4), 7.81 (dd, J ¼ 11.5, 7.4 Hz, 2H, o-C6H4), 7.69
(dd, J ¼ 7.5, 3.3 Hz, 2H, o-C6H4), 7.46 (virtual quartet, J ¼ 7.4 Hz, 2H,
o-C6H4), 7.41 (virtual triplet, J ¼ 7.4 Hz, 2H, o-C6H4), 7.07 (d,
J ¼ 8.4 Hz, 2H, p-C6H4), 6.36 (dd, J ¼ 7.6, 5.5 Hz, 1H, H-10), 5.28 (td,
J ¼ 6.4, 2.0 Hz, 1H, H-30), 4.66 (dd, J ¼ 12.2, 2.5 Hz, 1H, H-50), 4.55
(dd, J ¼ 12.2, 4.4 Hz, 1H, H-50), 4.51e4.48 (m, 1H, H-40), 4.32 (s, 1H,
OH), 3.02 (ddd, J ¼ 14.6, 5.6, 1.7 Hz, 1H, H-200), 2.35 (s, 3H, CH3), 2.15
(s, 3H, Ac), 2.11 (s, 3H, Ac), 2.09 (ddd, J ¼ 14.9, 7.4, 7.4 Hz, 1H, H-20);
13C 171.85, 170.52, 170.18, 157.07, 154.54, 146.64, 146.34, 140.93,
139.27, 139.21, 134.48, 129.89, 129.83, 129.37 (2C), 128.61, 128.52,
125.88 (2C), 125.03, 124.29, 124.20, 120.48 (2C), 109.01, 99.19, 94.28,
89.06, 84.06, 75.04, 74.52, 72.13, 63.97, 39.51, 21.58, 21.11, 20.92. IR
3.5. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(3-
hydroxyprop-1-yn-1-yl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-
d]pyrimidin-2-one (6a)
(n
, cmꢁ1, CH2Cl2) 2361 w, 2225 w, 2152 w, 1745 m, 1674 vs, 1603 m,
From 2-propyn-1-ol (propargyl alcohol; 0.072 mL, 1.2 mmol).
1576 m,1235 s, 1187 m. UVevis (MeOH,18.9 ꢃ 10ꢁ6 M) lmax 365 nm
Light yellow solid: 0.182 g, 0.379 mmol, 76%. NMR (CDCl3, d
): 1H
(ε 17,200 Mꢁ1cmꢁ1). Fluorescence (MeOH, ca. 3.67 ꢃ 10ꢁ6 M) lex
8.26 (s,1H, H-4), 8.00 (d, J ¼ 8.4 Hz, 2H, C6H4), 7.27 (d, J ¼ 8.8 Hz, 2H,
360 nm, lem 453 nm,
37H31N2O8 631.2075, found: 631.2075.
F
¼ 0.60. HRMS (ESI-TOF) [M þ H]þ calcd for
C6H4), 6.34 (dd, J ¼ 8.1, 5.5 Hz, 1H, H-10), 5.27 (td, J ¼ 6.5, 2.1 Hz, 1H,
C
6

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
3.8. 3-(30,50-di-O-acetyl-20-deoxy-
b-
D-ribofuranosyl)-5-(3-
3.11. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(4-(1,3-
methoxyprop-1-yn-1-yl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-
d]pyrimidin-2-one (6d)
dioxoisoindolin-2-yl)hex-1-yn-1-yl)-6-(4-methylphenyl)-2,3-
dihydrofuro[2,3-d]pyrimidin-2-one (6g)
From methyl propargyl ether (0.105 mL, 1.25 mmol). Light yel-
From N-(5-hexynyl)phthalimide (0.284 mg, 1.25 mmol). Light
low solid: 0.141 g, 0.285 mmol, 66%. NMR (CDCl3,
d
): 1H 8.31 (s, 1H,
brown oil: 0.267 g, 0.410 mmol, 82%. NMR (CDCl3, d
): 1H 8.25 (s, 1H,
H-4), 8.03 (d, J ¼ 8.3 Hz, 2H, C6H4), 7.27 (d, J ¼ 7.9 Hz, 2H, C6H4), 6.36
(dd, J ¼ 7.7, 5.9 Hz, 1H, H-10), 5.25 (td, J ¼ 6.5, 2.2 Hz, 1H, H-30), 4.47
(dd, J ¼ 11.6, 2.5 Hz, 1H, H-50), 4.44e4.41 (m, 1H, H-40), 4.41 (s, 2H,
CH2), 4.39 (dd, J ¼ 11.5, 3.5 Hz,1H, H-50), 3.46 (s, 3H, CH3), 2.94 (ddd,
J ¼ 14.5, 5.6, 2.1 Hz,1H, H-200), 2.40 (s, 3H, CH3), 2.16e2.10 (obscured
ddd, J ¼ 3.6, 3.6, Hz, 1H, H-20), 2.11 (s, 3H, Ac), 2.10 (s, 3H, Ac); 13C
170.47, 170.21, 170.17, 157.06, 154.54, 141.09, 134.80, 129.61 (2C),
126.11 (2C), 125.32, 109.07, 94.50, 94.43, 88.62, 83.47, 75.66, 74.34,
H-4), 7.97 (d, J ¼ 8.2 Hz, 2H, p-C6H4), 7.78 (AA’XX’, J ¼ 5.4, 3.2 Hz,
2H, o-C6H4), 7.67 (AA’XX’, J ¼ 5.5, 3.2 Hz, 2H, o-C6H4), 7.21 (d,
J ¼ 8.2 Hz, 2H, p-C6H4), 6.35 (dd, J ¼ 7.7, 5.7 Hz, 1H, H-10), 5.24 (td,
J ¼ 6.6, 2.1 Hz,1H, H-30), 4.46 (dd, J ¼ 11.9, 3.1 Hz,1H, H-50), 4.40 (dd,
J ¼ 5.9, 3.0 Hz, 1H, H-40), 4.36 (dd, J ¼ 11.9, 3.7 Hz, 1H, H-50), 3.73 (t,
J ¼ 7.1 Hz, 2H, CH2), 2.91 (ddd, J ¼ 14.5, 5.7, 2.0 Hz, 1H, H-200), 2.58 (t,
J ¼ 7.1 Hz, 2H, CH2), 2.34 (s, 3H, CH3), 2.15 (ddd, J ¼ 14.5, 7.7, 6.8 Hz,
1H, H-20), 2.09 (s, 3H, Ac), 2.06 (s, 3H, Ac), 1.87 (p, J ¼ 7.4 Hz, 2H,
CH2),1.70 (p, J ¼ 7.4 Hz, 2H, CH2); 13C (includes DEPT) 170.30,170.01,
169.95,168.24 (2C),155.86,154.46,140.35,134.61(CH),133.85(2CH),
131.89 (2C), 129.31(2CH), 125.65(2CH), 125.42, 123.05(2CH), 109.25,
98.95, 95.34, 88.51(CH), 83.30(CH), 74.35(CH), 70.30, 63.60(CH2),
39.22(CH2), 37.20(CH2), 27.72(CH2), 25.52(CH2), 21.46(CH3),
63.69, 60.53, 58.01, 39.47, 21.66, 20.94, 20.82. IR (n
, cmꢁ1, CH2Cl2)
2937 w, 1749 s, 1674 vs, 1576 m, 1234 m, 1109 w. UVevis (MeOH,
24.1 ꢃ 10ꢁ6 M) lmax 362 nm (ε 16,600 Mꢁ1cmꢁ1). Fluorescence
(MeOH, ca. 3.61 ꢃ 10ꢁ6 M) lex 360 nm, lem 453 nm,
F
¼ 0.53. HRMS
(ESI-TOF) [M þ H]þ calcd for C26H27N2O8 495.1762, found: 495.1762.
20.80(CH3), 20.64(CH3), 19.35(CH2). IR (
n
, cmꢁ1, ATR) 3000 s, 2972 s,
2940 s, 1742 vs, 1670 m, 1370 s, 1230 s, 1217 s, 1100 w, 1013 w. HRMS
(ESI-TOF) [M
652.2303.
þ
H]þ calcd for C36H34N3O9 652.2290, found:
3.9. 5-(3-Acetoxyprop-1-yn-1-yl)-3-(30,50-di-O-acetyl-20-deoxy-
-ribofuranosyl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (6e)
b-
D
3.12. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-6-(4-
From propargyl acetate (0.124 mL, 1.25 mmol). Light yellow
solid: 0.206 g, 0.395 mmol, 79%. NMR (CDCl3,
): 1H 8.34 (s, 1H, H-
methylphenyl)-5-((4-methylphenyl)ethynyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (6h)
d
4), 8.04 (d, J ¼ 8.2 Hz, 2H, C6H4), 7.30 (d, J ¼ 8.1 Hz, 2H, C6H4), 6.38
(dd, J ¼ 7.7, 5.8 Hz, 1H, H-10), 5.27 (td, J ¼ 6.4, 2.1 Hz, 1H, H-30), 4.99
(s, 2H, CH2), 4.51 (dd, J ¼ 11.9, 2.7 Hz, 1H, H-50), 4.45 (dd, J ¼ 5.7,
2.7 Hz, 1H, H-40), 4.43 (dd, J ¼ 11.9, 3.6 Hz, 1H, H-50), 2.96 (ddd,
J ¼ 14.5, 5.7, 2.1 Hz, 1H, H-200), 2.42 (s, 3H, CH3), 2.17e2.10 (m, 10H,
3Ac, H-20); 13C 172.37, 172.08.172.06, 159.50, 156.42, 143.20, 136.78,
131.54 (2C), 128.09 (2C), 127.04, 110.76, 95.87, 94.41, 90.61, 85.43,
77.79, 76.28, 65.62, 54.55, 49.08, 41.37, 23.57, 22.84, 22.74, 22.66. IR
From 4-ethynyltoluene (0.158 mL, 1.25 mmol). Light brown
solid: 0.189 g, 0.350 mmol, 70%. NMR (CDCl3, d
): 1H 8.37 (s,1H, H-4),
8.07 (d, J ¼ 8.3 Hz, 2H, C6H4), 7.43 (d, J ¼ 8.1 Hz, 2H, C6H4), 7.25 (d,
J ¼ 9.0 Hz, 2H, C6H4), 7.19 (d, J ¼ 7.8 Hz, 2H, C6H4), 6.38 (dd, J ¼ 7.6,
5.7 Hz, 1H, H-10), 5.25 (td, J ¼ 6.4, 2.1 Hz, 1H, H-30), 4.49 (dd, J ¼ 14.1,
4.8 Hz, 1H, H-50), 4.43e4.37 (m, 2H, H-40,50), 2.93 (ddd, J ¼ 14.5, 5.7,
2.1 Hz,1H, H-200), 2.38 (s, 6H, CH3), 2.15 (ddd, J ¼ 14.5, 7.6, 6.8 Hz,1H,
H-20), 2.10 (s, 3H, Ac), 2.00 (s, 3H, Ac); 13C 170.31, 170.09 (2C),
156.09, 154.42, 140.63, 139.42 (2C), 134.74, 131.31 (2C), 129.31 (2C),
129.28 (2C), 125.90 (2C), 125.44, 118.99, 108.86, 98.30, 95.23, 88.42,
(
n
, cmꢁ1, CH2Cl2) 2930,1749,1675,1603,1575. HRMS (ESI-TOF) [M þ
H]þ calcd for C27H27N2O9 523.1711, found: 523.1712.
83.32, 74.16, 63.52, 39.29, 21.49 (2C), 20.78, 20.60. IR (n ,
, cmꢁ1
CH2Cl2) 3016 w, 2970 w, 2946 w, 2210, 1749, 1674. UVevis (MeOH,
24.4 ꢃ 10ꢁ6 M) lmax 264 (ε 36,900), 369 nm (ε 19,000 Mꢁ1cmꢁ1).
Fluorescence (MeOH, ca. 3.57 ꢃ 10ꢁ6 M) lex 360 nm, lem 481 nm,
3.10. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(4-(1,3-
dioxoisoindolin-2-yl)but-1-yn-1-yl)-6-(4-methylphenyl)-2,3-
dihydrofuro[2,3-d]pyrimidin-2-one (6f)
F
¼ 0.19. HRMS (ESI-TOF) [M þ H]þ calcd for C31H29N2O7 541.1969,
found: 541.1969.
From N-(3-butynyl)phthalimide (0.249 mg, 1.25 mmol). Light
yellow solid: 0.290 g, 0.465 mmol, 93%. NMR (CDCl3, d
): 1H 8.51 (s,
1H, H-4), 7.92 (d, J ¼ 8.3 Hz, 2H, p-C6H4), 7.81 (AA’XX’, J ¼ 5.5, 3.1 Hz,
2H, o-C6H4), 7.69 (AA’XX’, J ¼ 5.5, 3.1 Hz, 2H, o-C6H4), 7.15 (d,
J ¼ 8.3 Hz, 2H, p-C6H4), 6.42 (dd, J ¼ 7.7, 5.8 Hz, 1H, H-10), 5.31 (td,
J ¼ 6.6, 2.1 Hz, 1H, H-30), 4.57 (dd, J ¼ 11.3, 2.8 Hz, 1H, H-50),
4.48e4.46 (m, 1H, H-40), 4.44 (dd, J ¼ 11.5, 4.0 Hz, 1H, H-50), 4.01
(dp, J ¼ 10.3, 4.9 Hz, 2H, CH2), 3.03e2.91 (m, 1H, H-200), 2.98 and
2.96 (AB, J ¼ 5.8 Hz, 2H, CH2), 2.35 (s, 3H, CH3), 2.26 (ddd, J ¼ 14.5,
7.8.6.8 Hz, 1H, H-20), 2.12 (s, 3H, Ac), 2.09 (s, 3H, Ac); 13C 170.48,
170.34, 170.12, 168.18 (2C), 156.16, 154.68, 140.58, 135.57, 134.21
(2C), 131.89 (2C), 129.46 (2C), 125.83 (2C), 125.42, 123.48 (2C),
109.32, 95.66, 95.04, 88.70, 83.51, 74.64, 71.84, 63.96, 39.43, 36.44,
3.13. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-6-(4-
methylphenyl)-5-((4-pentylphenyl)ethynyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (6i)
From 4-n-pentylphenylacetylene (0.243 mL, 1.25 mmol). Yield:
0.191 g, 0.320 mmol (64%, oil). NMR (CDCl3, d
): 1H 8.39 (s, 1H, H-4),
8.08 (d, J ¼ 8.3 Hz, 2H, C6H4), 7.46 (d, J ¼ 8.0 Hz, 2H, C6H4), 7.26 (d,
J ¼ 8.2 Hz, 2H, C6H4), 7.21 (d, J ¼ 8.0 Hz, 2H, C6H4), 6.39 (dd, J ¼ 7.6,
5.8 Hz,1H, H-10), 5.25 (td, J ¼ 6.4, 2.0 Hz, 1H, H-30), 4.50 (dd, J ¼ 14.7,
5.1 Hz, 1H, H-50), 4.44e4.38 (m, 2H, H-40,50), 2.94 (ddd, J ¼ 14.5, 5.6,
2.1 Hz, 1H, H-200), 2.63 (t, J ¼ 7.8 Hz, 2H, CH2), 2.39 (s, 3H, CH3), 2.16
(ddd, J ¼ 14.4, 7.5, 6.9 Hz, 1H, H-20), 2.11 (s, 3H, Ac), 2.00 (s, 3H, Ac),
1.62 (p, J ¼ 7.5 Hz, 2H, CH2), 1.36e1.28 (m, 4H, 2CH2), 0.89 (t,
J ¼ 6.8 Hz, 3H, CH3); 13C 170.48, 170.26 (2C), 156.28, 154.59, 144.62,
140.79, 134.90, 131.51 (2C), 129.58 (2C), 128.80 (2C), 126.08 (2C),
125.61, 119.35, 109.04, 98.52, 95.40, 88.58, 83.48, 77.97, 74.31, 63.68,
21.62, 20.98, 20.91, 20.03. IR (
2139 vw, 1745 m, 1719 vs, 1673 vs, 1603 m, 1575 m, 1397 m, 1237 m,
n
, cmꢁ1, CH2Cl2) 2272 vw, 2168 vw,
1113 w. UVevis (MeOH, 17.3
ꢃ
10ꢁ6 M) lmax 364 nm (ε
17,000 Mꢁ1cmꢁ1). Fluorescence (MeOH, ca. 3.52 ꢃ 10ꢁ6 M) lex
360 nm, lem 457 nm,
F
¼ 0.04. HRMS (ESI-TOF) [M þ H]þ calcd for
C
34H30N3O9 624.1977, found: 624.1977.
39.46, 35.96, 31.46, 30.94, 22.54, 21.66, 20.94, 20.76, 14.06. IR (cmꢁ1
,
7

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
ATR) 2958 m, 2924 m,1740 s, 1671 vs, 1572 m, 1402 m,1226 vs, 1058
vs. HRMS (ESI-TOF) [M þ H]þ calcd for C35H37N2O7 597.2596, found:
597.2599.
129.46 (2C), 128.99 (2C), 126.16 (2C), 125.80, 119.17, 109.13, 98.46,
95.35, 88.57, 83.48, 77.99, 74.28, 63.69, 39.52, 36.00, 31.47, 30.92,
22.56, 21.67, 20.96, 20.75, 14.05. IR (n
, cmꢁ1, ATR) 2975 m, 2927 m,
1740 s, 1671 vs, 1403 s, 1226 vs, 1058 vs. UVevis (MeOH,
3.14. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(3-
hydroxyprop-1-yn-1-yl)-6-(4-pentylphenyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (7a)
19.9 ꢃ 10ꢁ6 M) lmax 370 nm (ε 20,800 Mꢁ1cmꢁ1). Fluorescence
(MeOH, ca. 3.55 ꢃ 10ꢁ6 M) lex 360 nm, lem 481,
F
¼ 0.21. HRMS
(ESI-TOF) [M
597.2595.
þ
H]þ calcd for C35H37N2O7 597.2595, found:
From 2-propyn-1-ol (propargyl alcohol; 0.072 mL, 1.2 mmol).
Yellow solid: 0.217 g, 0.405 mmol, 81%. NMR (CDCl3,
d
): 1H 8.26 (s,
3.17. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(4-
1H, H-4), 8.02 (d, J ¼ 8.3 Hz, 2H, C6H4), 7.27 (d, J ¼ 9.1 Hz, 2H, C6H4),
6.34 (dd, J ¼ 8.0, 5.5 Hz, 1H, H-10), 5.27 (td, J ¼ 6.6, 2.0 Hz, 1H, H-30),
4.70 (dd, J ¼ 11.9, 2.0 Hz, 1H, H-50), 4.61 and 4.57 (dAB, J ¼ 16.3,
5.7 Hz, 2H, CH2O), 4.46e4.43 (m, 1H, H-40), 4.41 (dd, J ¼ 11.3, 4.3 Hz,
1H, H-50), 3.50 (t, J ¼ 6.3 Hz, 1H, OH), 3.00 (ddd, J ¼ 14.6, 5.6, 1.5 Hz,
1H, H-200), 2.64 (t, J ¼ 7.8, 2H, CH2), 2.13 (s, 3H, Ac), 2.12 (s, 3H, Ac),
2.04 (ddd, J ¼ 14.6, 7.9, 6.6 Hz, 1H, H-20), 1.63 (p, J ¼ 7.5, 2H, CH2),
1.37e1.29 (m, 4H, 2CH2), 0.89 (t, J ¼ 6.9 Hz, 3H, CH3); 13C 171.98,
170.60, 170.25, 156.14, 154.63, 146.05, 134.41, 129.00 (2C), 126.02
(2C), 125.45, 109.48, 97.54, 94.52, 89.02, 84.05, 74.61, 74.51, 63.95,
pentylphenyl)-6-(4-pentylphenyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (7i)
From 4-n-pentylphenylacetylene (0.243 mL, 1.25 mmol). Light
brown oil: 0.235 g, 0.360 mmol, 72%. NMR (CDCl3, d
): 1H 8.40 (s, 1H,
H-4), 8.12 (d, J ¼ 7.8 Hz, 2H, C6H4), 7.47 (d, J ¼ 8.0 Hz, 2H, C6H4), 7.28
(d, J ¼ 8.2 Hz, 2H, C6H4), 7.21 (d, J ¼ 8.2 Hz, 2H, C6H4), 6.39 (dd,
J ¼ 7.3, 6.1 Hz, 1H, H-10), 5.27 (td, J ¼ 6.6, 2.0 Hz, 1H, H-30), 4.50 (dd,
J ¼ 13.1, 3.7 Hz, 1H, H-40), 4.44e4.38 (m, 2H, H-50), 2.94 (ddd,
J ¼ 14.6, 5.7,1.9 Hz,1H, H-200), 2.64 (t, J ¼ 7.5, 2H, CH2), 2.63 (t, J ¼ 7.6,
2H, CH2), 2.16 (ddd, J ¼ 14.4, 7.2, 7.2 Hz, 1H, H-20), 2.11 (s, 3H, Ac),
2.00 (s, 3H, Ac), 1.63 (p, J ¼ 7.3, 4H, 2CH2), 1.36e1.29 (m, 8H, 4CH2),
0.89 (t, J ¼ 6.6 Hz, 3H, CH3) 0.88 (t, J ¼ 6.6 Hz, 3H, CH3); 13C 170.48,
170.26 (2C), 156.34, 154.60, 145.84, 144.62, 134.90, 131.53 (2C),
128.96 (2C), 128.80 (2C), 126.12 (2C), 125.80, 119.36, 109.08, 98.53,
95.38, 88.59, 83.48, 77.99, 74.30, 63.68, 39.48, 35.97 (2C), 31.46 (2C),
30.94, 30.90, 22.55 (2C), 20.94, 20.75, 14.06 (2C). HRMS (ESI-TOF)
[M þ H]þ calcd for C39H45N2O7 653.3221, found: 653.3221.
51.53, 39.54, 35.98, 31.44, 30.89, 22.55, 21.18, 20.95, 14.05. IR (n,
cmꢁ1, ATR) 3350 br w, 2968 m, 2928 m, 1739 s, 1665 vs, 1599 s, 1219
vs, 1066 vs. UVevis (MeOH, 22.2 ꢃ 10ꢁ6 M) lmax 362 nm (ε
19,600 Mꢁ1cmꢁ1). Fluorescence (MeOH, ca. 3.73 ꢃ 10ꢁ6 M) lex
360 nm, lem 452 nm,
F
¼ 0.59. HRMS (ESI-TOF) [M þ H]þ calcd for
C
29H33N2O8 537.2231, found: 537.2231.
3.15. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(4-
hydroxybut-1-yn-1-yl)-6-(4-pentylphenyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (7b)
X-ray Crystallography. See Supporting Materials.
Fluorescence. Absorbance was measured using a Cary-100
UVevis spectrophotometer (Agilent, USA) in double-beam mode
(sample and solvent blank) using a 10 mm path quartz cuvette
(Starna 23-Q-10 cell with a Teflon stopper). Fluorescence quantum
yields were determined according to the relative method [64].
Corrected fluorescence spectra were collected on a Fluorolog 3
fluorometer (Horiba Jobin-Yvon, Japan) equipped with an R928
photomultiplier tube (Hamamatsu, USA). Each nucleoside’s fluo-
rescent properties were determined using five freshly prepared
samples of varying concentration in methanol (HPLC grade) whose
absorbance at the excitation wavelength was spread between 0.1
and 0.05 absorbance units. In the same manner, five freshly pre-
pared standard samples of 9,10-dimethylanthracene (Aldrich) in
ethanol (spectrophotometric or reagent grade) were used as a
reference for calculation of the quantum yield FD ¼ 0.82 [65].
Sample preparation and measurement were conducted at 24 ꢀC.
Solvent correction was carried out using refractive index values
[66].
Biological Studies. The antiviral assays were based on inhibi-
tion of virus-induced cytopathicity or plaque formation in HEL
[herpes simplex virus 1 (HSV-1) (KOS), HSV-2 (G), vaccinia virus,
vesicular stomatitis virus, human cytomegalovirus (HCMV),
varicella-zoster virus (VZV), adenovirus-2 and human corona virus
(299E)], Vero (parainfluenza-3, reovirus-1, Sindbis virus and Cox-
sackie B4), HeLa (vesicular stomatitis virus, Coxsackie virus B4, and
respiratory syncytial virus) or MDCK [influenza A (H1N1, H3N2)
and influenza B] cell cultures. Confluent cell cultures (or nearly
confluent for MDCK cells) in microtiter 96-well plates were inoc-
ulated with 100 CCID50 of virus (1 CCID50 being the virus dose to
infect 50% of the cell cultures) or with 20 plaque forming units
(PFU). After 1e2 h virus adsorption period, residual virus was
removed, and the cell cultures were incubated in the presence of
varying concentrations of the test compounds in duplicate. Viral
cytopathicity was recorded as soon as it reached completion in the
control virus-infected cell cultures that were not treated with the
test compounds. Antiviral activity was expressed as the EC50 or the
From 3-butyn-1-ol (0.095 mL, 1.25 mmol). Light yellow solid:
0.223 g, 0.405 mmol, 81%. NMR (CDCl3, d
): 1H 8.27 (s, 1H, H-4), 8.04
(d, J ¼ 8.2 Hz, 2H, C6H4), 7.25 (d, J ¼ 8.0 Hz, 2H, C6H4), 6.31 (dd,
J ¼ 7.4, 5.9 Hz, 1H, H-10), 5.24 (td, J ¼ 6.6, 1.5 Hz, 1H, H-30), 4.57 (dd,
J ¼ 11.9, 2.2 Hz, 1H, H-50), 4.44e4.41 (m, 1H, H-40), 4.38 (dd, J ¼ 11.9,
4.2 Hz, 1H, H-50), 3.89 (AB, J ¼ 6.0 Hz, 2H, CH2), 3.03e3.00 (br s, 1H,
OH), 2.97 (ddd, J ¼ 14.9, 5.8, 1.0 Hz, 1H, H-200), 2.81 (t, J ¼ 6.0, 2H,
CH2), 2.62 (t, J ¼ 7.8, 2H, CH2), 2.12 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.06
(ddd, J ¼ 14.5, 7.8, 6.5 Hz, 1H, H-20), 1.61 (p, J ¼ 7.4, 2H, CH2),
1.36e1.28 (m, 4H, 2CH2), 0.88 (t, J ¼ 7.0 Hz, 3H, CH3); 13C 171.08,
170.54, 170.16, 156.25, 154.63, 145.72, 134.69, 128.90 (2C), 125.90
(2C), 125.71, 109.41, 97.24, 95.34, 88.88, 83.79, 74.53, 71.21, 63.86,
60.75, 39.48, 35.95, 31.45, 30.89, 20.45, 22.54, 20.95 (2C), 14.04. IR
(n
, cmꢁ1, ATR) 3380 br w, 2958 m, 2927 m, 1739 s, 1665 vs, 1572 m,
1399 m, 1219 vs, 1044 vs. UVevis (MeOH, 21.6 ꢃ 10ꢁ6 M) lmax
360 nm (ε 17,400
M
ꢁ1cmꢁ1). Fluorescence (MeOH, ca.
3.63 ꢃ 10ꢁ6 M) lex 360 nm, lem 461,
F
¼ 0.50. HRMS (ESI-TOF) [M þ
H]þ calcd for C30H35N2O8 551.2388, found: 551.2388.
3.16. 3-(30,50-di-O-acetyl-20-deoxy-
b-D-ribofuranosyl)-5-(4-
methylphenyl)-6-(4-pentylphenyl)-2,3-dihydrofuro[2,3-d]
pyrimidin-2-one (7h)
From 4-ethynyltoluene (0.158 mL, 1.25 mmol). Light brown
solid: 0.289 g, 0.485 mmol, 97%. NMR (CDCl3, d
): 1H 8.41 (s, 1H, H-
4), 8.15 (d, J ¼ 8.4 Hz, 2H, C6H4), 7.47 (d, J ¼ 8.0 Hz, 2H, C6H4), 7.30 (d,
J ¼ 8.4 Hz, 2H, C6H4), 7.22 (d, J ¼ 7.9 Hz, 2H, C6H4), 6.41 (dd, J ¼ 7.6,
5.7 Hz, 1H, H-10), 5.27 (td, J ¼ 6.6, 2.0 Hz, 1H, H-30), 4.51 (dd, J ¼ 11.7,
2.4 Hz, 1H, H-50), 4.46e4.43 (m, 1H, H-40), 4.41 (dd, J ¼ 11.8, 3.6 Hz,
1H, H-50), 2.95 (ddd, J ¼ 14.5, 5.8, 2.2 Hz, 1H, H-200), 2.66 (t, J ¼
7.8 Hz, 2H, CH2), 2.41 (s, 3H, CH3), 2.16 (ddd, J ¼ 14.4, 7.6, 6.8 Hz, 1H,
H-20), 2.13 (s, 3H, Ac), 2.01 (s, 3H, Ac), 1.64 (p, J ¼ 7.5, 2H, CH2),
1.37e1.31 (m, 4H, 2CH2), 0.89 (t, J ¼ 7.0 Hz, 3H, CH3); 13C 170.51,
170.32, 170.26, 156.41, 154.63, 145.91, 139.63, 134.84, 131.52 (2C),
8

R. Kaczmarek, D.J. Twardy, T.L. Olson et al.
European Journal of Medicinal Chemistry 209 (2021) 112884
https://doi.org/10.1021/jm000210m.
concentration required for reducing virus-induced cytopathoge-
nicity or viral plaque (VZV) plaque formation by 50%. The minimal
cytotoxic concentration (MCC) of the compounds was defined as
the compound concentration that caused a microscopically visible
alteration of cell morphology. Alternatively, cytotoxicity of the test
compounds was measured based on inhibition of cell growth. HEL
cells were seeded at a rate of 5 ꢃ 103 cells/well into 96-well mi-
crotiter plates and allowed to proliferate for 24 h. Then, the me-
dium containing different concentrations of the test compounds
was added. After 3 days of incubation at 37 ꢀC, the cell number was
determined with a Coulter counter. The cytostatic concentration
was calculated as the CC50, or the compound concentration
required for reducing cell proliferation by 50% relative to the
number of cells in the untreated controls.
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ꢀ
D. Korczynski, J.R. Green, R. Dembinski Beilstein J. Org. Chem. 16 (2020) 1e8,
Declaration of competing interest
https://doi.org/10.3762/bjoc.16.1.
[19] For
a representative work from this laboratory see: R. Kaczmarek,
ꢀ
ꢀ
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D. Korczynski, K. Krolewska-Golinska, K.A. Wheeler, F.A. havez, A. Mikus,
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgments
We thank the National Institutes of Health (NIH, CA111329), the
Statutory Funds of CMMS PAS, for support of this research. We are
also thankful to Dr. Hiroyuki Hayakawa (Yamasa Corporation, Bio-
chemicals Division) and Dr. Bruno François (Simafex, France), for a
generous supply of nucleosides and N-iodosuccinimide, respec-
tively. The NSF awards (CHE-0821487, CHE-1048719, and CHE-
1827313) and Research Excellence Fund, Center for Biomedical
Research are also acknowledged. We also thank talented under-
graduate students Susan Yang, Bo Bezeau, and Owen Hoerauf for
their assistance, and Brecht Dirix and Leentje Persoons for excellent
technical support in the antiviral assays.
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Appendix A. Supplementary data
[29] E. Coutouli-Argyropoulou, M. Tsitabani, G. Petrantonakis, A. Terzis,
C. Raptopoulou, Org. Biomol. Chem. 1 (2003) 1382e1388, https://doi.org/
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Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112884.
ꢀ
ꢀ
ꢁ ꢀ
ꢀ
ꢀ
ꢀ
[30] T. Gregoric, M. Sedic, P. Grbcic, A. Tomljenovic Paravic, S. Kraljevic Pavelic,
Contains: Preparative procedures for compounds Ac-IdU, 2, and 3,
summary of yields table for compounds 6 and 7. 1H and 13C NMR
spectra for compounds 4, 5, 6, and 7. UV-vis and fluorescence
spectra for compounds 4, 6a,c,d,f,h, and 7a,b,h. Antiviral activity
data for compounds 6 and 7, and comparison with other furopyr-
imidines. Crystallographic cif file for compound 4.
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M. Cetina, R. Vianello, S. Raic-Malic, Eur. J. Med. Chem. 125 (2017) 1247e1267,
https://doi.org/10.1016/j.ejmech.2016.11.028.
[31] R. Dembinski, Y. Li, D. Gundapuneni, A. Decker, Top. Heterocycl. Chem. 27
(2012) 65e100, https://doi.org/10.1007/7081_2011_57.
[32] A. Sniady, K.A. Wheeler, R. Dembinski, Org. Lett. 7 (2005) 1769e1772, https://
doi.org/10.1021/ol050372i.
[33] M.S. Rao, N. Esho, C. Sergeant, R. Dembinski, J. Org. Chem. 68 (2003)
6788e6790, https://doi.org/10.1021/jo0345648.
[34] B. Halford, C&EN 98 (20) (2020) 22, https://doi.org/10.1021/cen-09820-
feature2.
[35] The other drug is remdesivir, see: L.M. Jarvis C&EN 98 (13) (2020) 26e27,
https://doi.org/10.1021/cen-09813-feature4.
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