Synthesis and Cytotoxic and Antiviral Activity Profiling of All-Four Isomeric Series of Pyrido-Fused 7-Deazapurine Ribonucleosides

Article abstract of DOI:10.1002/chem.202001124

All four isomeric series of novel 4-substituted pyrido-fused 7-deazapurine ribonucleosides possessing the pyridine nitrogen atom at different positions were designed and synthesized. The total synthesis of each isomeric fused heterocycle through multistep heterocyclization was followed by glycosylation and derivatization at position 4 by cross-coupling reactions or nucleophilic substitutions. All compounds were tested for cytostatic and antiviral activity. The most active were pyrido[4′,3′:4,5]pyrimidine nucleosides bearing MeO, NH₂, MeS, or CH₃ groups at position 4, which showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism involved activation by phosphorylation and incorporation to DNA where the presence of the modified ribonucleosides causes double-strand breaks and apoptosis.

Full text of DOI:10.1002/chem.202001124

Accepted Article
Accepted Article
Title: Synthesis and Cytotoxic and Antiviral Activity Profiling of All-Four
Isomeric Series of Pyrido-Fused 7-Deazapurine Ribonucleosides
Authors: Lucia Veselovská, Natálie Kudlová, Soňa Gurská, Barbora
Lišková, Martina Medvedíková, Ondřej Hodek, Eva
Tloušťová, Nemanja Milisavljevic, Michal Tichý, Pavla
Perlíková, Helena Mertlíková-Kaiserová, Jana Trylčová,
Radek Pohl, Blanka Klepetářová, Petr Džubák, Marián
Hajdúch, and Michal Hocek
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202001124
Link to VoR: https://doi.org/10.1002/chem.202001124
01/2020

10.1002/chem.202001124
Chemistry - A European Journal
Synthesis and Cytotoxic and Antiviral Activity Profiling of All-Four Isomeric Series of
Pyrido-Fused 7-Deazapurine Ribonucleosides
Lucia Veselovská,a Natálie Kudlová,b,c Dr. Soňa Gurská,b,c Dr. Barbora Lišková,b Martina
Medvedíková,b Dr. Ondřej Hodek,a Eva Tloušťová,a Nemanja Milisavljevic,a,d Dr. Michal
Tichý,a Dr. Pavla Perlíková,a Dr. Helena Mertlíková-Kaiserová,a Jana Trylčová,a Dr. Radek
Pohl,a Dr. Blanka Klepetářová,a Dr. Petr Džubák,b,c Prof. Dr. Marián Hajdúch,b,c Prof. Dr.
Michal Hocek*a,d
a Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo
nam. 2, CZ-16610 Prague 6, Czech Republic. e-mail: hocek@uochb.cas.cz; URL:
http://hocekgroup.uochb.cas.cz/
b Institute of Molecular and Translational Medicine, Palacky University and University
Hospital in Olomouc, Faculty of Medicine and Dentistry, Hněvotínská 5, CZ-775 15
Olomouc, Czech Republic.
c Cancer Research Czech Republic, Hněvotínská 5, CZ-775 15 Olomouc, Czech Republic
d Department of Organic Chemistry, Faculty of Science, Charles University in Prague,
Hlavova 8, CZ-12843 Prague 2, Czech Republic.
Abstract: All-four isomeric series of novel 4-substituted pyrido-fused 7-deazapurine
ribonucleosides possessing pyridine nitrogen at different positions were designed and
synthesized. The total synthesis of each isomeric fused heterocycle through multistep
heterocyclization was followed by glycosylation and derivatization at position 4 by cross-
coupling reactions or nucleophilic substitutions. All compounds were tested for cytostatic and
antiviral activity. The most active were pyrido[4',3':4,5]pyrimidine nucleosides bearing MeO,
NH2, MeS or CH3 groups at position 4 which showed submicromolar cytotoxic effects and good
selectivity for cancer cells. The mechanism involved activation by phosphorylation and
incorporation to DNA where the presence of the modified ribonucleosides causes double-strand
breaks and apoptosis.
1
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Graphical abstract:
2
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Introduction
Nucleoside derivatives or analogues modified at the heterocyclic base are an important
class of molecules with wide range of biological activities. Several clinical antiviral1 and
antineoplastic2 drugs are based on base-modified nucleosides, either with or without additional
modifications at the sugar part. The need for treatment of drug-resistant leukemia and cancers,3
as well as of emerging new viruses,4 justifies the continuous interest in the development of other
novel nucleoside derivatives with potential antiviral or cytostatic effects. Particularly
interesting are modified pyrrolo[2,3-d]pyrimidine (7-deazapurine) nucleosides.5 Long-known
natural products tubercidin,6 toyocamycin7 and sangivamycin8 display significant biological
activities and served as inspiration for further structural modifications at both the heterocyclic
and ribose parts. Several types of 7-substituted 7-deazapurine nucleosides bearing 2'-methyl-
or 2'-ethynyl-ribose (or 2'-fluororibose) displayed antiviral activity against HCV or other RNA
viruses.9 Some other modified 7-deazapurine nucleosides were potent inhibitors of human or
pathogen adenosine kinases10 or have exerted antiparasitic activities.11 We have previously
reported significant cytostatic activities of 7-hetaryl-7-deazaadenine (e.g. AB-61)12 or other 6-
substituted 7-deazapurine ribonucleosides,13 which act through an unprecedented mechanism
based on activation to (ribo)nucleoside triphosphates (NTPs) and incorporation to DNA, where
they cause double-strand (ds) breaks and eventually apoptosis.14
More recently, we have designed and synthesized fused 7-deazapurine ribonucleosides.
Benzo-fused (pyrimidoindole) nucleosides were found15 to be non-cytotoxic, however they
exerted moderate antiviral activities against HCV or Dengue virus, whereas the more bulky
naphtho-fused nucleosides16 were inactive. On the other hand, the less bulky heteroanalogues,
i.e. thieno-,17 furo-18 or methylpyrrolo-fused18 7-deazapurine nucleosides exerted
submicromolar in vitro cytotoxic effects and some derivatives were selective against cancer and
leukemia cell lines. Also these compounds were found18 to be phosphorylated in the cell, got
incorporated to DNA and induced DNA damage and apoptosis. As a continuation of the
systematic SAR study of this new interesting class of biologically active nucleosides, and to
prove whether the activity of the hetero-analogues is based on their lower bulkiness or on the
presence of the heteroatoms, we designed and present here the synthesis of all-four novel
isomeric series of pyrido-fused 7-deazapurine ribonucleosides and their biological profiling.
3
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Chart 1. Structures of previously reported fused-deazapurine nucleosides
4
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Results and discussion
Chemistry.
There are four possible isomers of pyrido-fused 7-deazapurines: pyrido[2',3':4,5]- (series A),
pyrido[3',4':4,5]- (series B), pyrido[4',3':4,5]- (series C) and pyrido[3',2':4,5]pyrrolo[2,3-
d]pyrimidine (series D) (Scheme 1) and we aimed at the synthesis of all of them. Each
individual heterocycle required independent total synthesis. In all four cases, the synthesis was
designed in analogy to the previous syntheses of pyrimidoindoles (Scheme 1), but every step
needed to be optimized for each isomer.
The key isomeric 4-chloropyridopyrrolopyrimidine intermediates 9A-D were synthesized in
4 steps (Scheme 1), starting from corresponding isomeric chloronitropyridines 5A-D. The
synthesis started with a nucleophilic substitution of chlorine with ethyl cyanoacetate which in
the case of electron-poor chloronitropyridines proceeded smoothly. In case of 5A and 5C,
potassium tert-butoxide in tert-butyl alcohol was used as a base according to literature
procedure.19 In case of compounds 5B and 5D, a stronger base NaH in DMF was used to reach
good conversions.20 In each of the four series, formation of a different tautomer or mixture of
tautomers of the arylcyanoacetate 6A-D was observed (see Figure S1 in SI). Compounds 6A-
D were subsequently reduced by zinc dust in acetic acid followed by spontaneous cyclization
to obtain isomeric pyrrolopyridine products 7A-7D (94, 53, 73 and 81%). The next
cyclocondensation step with formamide afforded the tricyclic heterocycles 8A-D (86, 67, 79
and 92%).20 The next step was the conversion of the pyrimidone derivatives to 8A-D to fused
chloropyrimidines 9A-D. However, the literature procedure using POCl3 with Et3N reported for
preparation of related chloropyrimidine nucleobases20 did not give the desired
chloropyrimidine, but diethylamino nucleobase 9Ax (see SI). Therefore, these tricyclic
pyrimidone derivatives were converted to chloropyrimidine using POCl3 in presence of
dimethylaniline and benzyltriethylammonium chloride (BTEACl) (in analogy to procedure
developed previously in our group13). The desired key intermediates 9A-D were isolated in
yields of 65, 13, 52 and 59%, respectively. The synthesis of pyrido[3',4':4,5]pyrimidine 9B was
more problematic in each step due to lower stability, partial decomposition of the intermediates
and due to partial hydrolysis back to 8B in the last step. The total yield of synthesis of 9B was
only 4.5% and only ca. 400 mg of the material was prepared even after repeated attempts. The
other isomers 9A,C,D were prepared in reasonable overall yields of 52, 29 and 44%,
respectively.
5
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Scheme 1: Reagents and conditions: (i) tBuOK, ethyl cyanoacetate, tBuOH, 95 °C, 6 h; (ii)
NaH, ethyl cyanoacetate, DMF, rt, 30 min or 6 h; (iii) Zn dust, AcOH, 95 °C, 75 min or 90 min;
(iv) formamide, HCOONH4, 170 °C, 12 h; (v) N,N-dimethylaniline, BTEACl, POCl3, MeCN,
90 °C, 1 h; (vi) BSA, MeCN, 60 °C, 30 min; then 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-
ribofuranose, TMSOTf, 60 °C, 12 h.
The pyridopyrrolopyrimidine nucleobases 9A-D were then subjected to Vorbrꢀggen
glycosylation with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose15. Nucleobases were
dissolved in anhydrous MeCN and stirred with N,O-bis(trimethylsilyl)acetamide (BSA) for
30 minutes at 60 °C and then trimethylsilyl triflate (TMSOTf) and 1-O-acetyl-2,3,4-tri-O-
benzoyl-β-D-ribofuranose were added and the mixture was stirred overnight at 60 °C. This
procedure produced the key intermediates, protected 4-chloropyridopyrrolopyrimidine
nucleosides 10A-D (yields 72, 12, 58 and 57%, respectively). In case of the B-series, we also
observed formation of bis-ribosylated by-products 10Bx and 10By bearing another sugar linked
to the pyridine nitrogen (see Figure S2 in SI). These intermediates were characterized by NMR
and MS and by-product 10Bx was easily converted back to desired 10B by increased
temperature (to increase the yield to 25%, See SI). Interestingly, in case of series D, a separable
mixture of α (yield 5%) and β anomers (57%) was formed after glycosylation. Because of
overlapping signals, the anomeric configuration of the α-nucleoside 10Dα side-product could
not be determined directly and, therefore, it was transformed to methoxy-derivative 12Daα
which was fully characterized and the stereochemistry was confirmed by H,H-ROESY
experiment (see SI).
6
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Scheme 2. Reagents and conditions: (a) MeONa, MeOH or MeOH:DMF, rt–90 °C, 16 h; (b)
MeSNa, MeOH, rt–60 °C, 16 h; (c) NH3(aq), 1,4-dioxane, 120 °C, 24 h; (d) Me2NH in THF,
iPrOH:DCM 1:1, rt, 16 h; (e) Me3Al, Pd(PPh3)4, THF, 70 °C, 16 h; (f) 2-tributylstannylfuran,
PdCl2(PPh3)2, DMF, 100 °C, 4 h; (g) R-boronic acid, Pd(PPh3)4, K2CO3, toluene, 100 °C, 3–24
h; (h) R-boronic acid, Pd(PPh3)2Cl2, K2CO3, Et3N, toluene, 100 °C, 24 h; (i) MeSNa, DMF, rt,
16 h; (j) MeONa, MeOH:DMF, 90 °C, 16 h; (k) MeONa, MeOH, rt–70 °C, 16 h.
Table 1. Synthesis of 4-Substituted Nucleosides 12A–Da–h
Protected
Nucleoside [%]
Yield Final
Yield
Entry Method
R
Nucleoside [%]
1
2
3
4
5
6
7
8
(a) MeONa
OMe
SMe
NH2
Me
NMe2
2-furyl
12Aa
12Ab
12Ac
12Ad
12Ae
12Af
12Ag
12Ai
12Ah
12Ba
12Bc
62
76
53
87
81
75
78
70
69
58
41
(i) MeSNa
(c) NH3(aq)
(e) Me3Al
(d) Me2NH
(f) R-SnBu3
(g) R-B(OH)2 3-furyl
(g) R-B(OH)2 3-thienyl
(h) R-B(OH)2 2-benzofuryl 11Ah
11Ab
52
11Ad
11Ae
11Af
11Ag
11Ai
69
82
90
73
70
56
9
10
11
(a) MeONa
(c) NH3(aq)
OMe
NH2
7
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
a
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
(e) Me3Al
Me
12Bd
12Ca
12Cb
12Cc
12Cd
12Ce
12Cf
12Cg
12Ci
12Ch
12Da
12Db
12Dc
12Dd
12De
12Df
12Dg
12Di
12Dh
26b
(a) MeONa
(b) MeSNa
(c) NH3(aq)
(e) Me3Al
(d) Me2NH
(f) R-SnBu3
OMe
SMe
NH2
Me
NMe2
2-furyl
42
47
51
63
91
71
90
72
79
90
80
85
82
81
93
80
91
90
11Cd
11Ce
11Cf
11Cg
11Ci
55
67
66
84
57
64
(g) R-B(OH)2 3-furyl
(g) R-B(OH)2 3-thienyl
(g) R-B(OH)2 2-benzofuryl 11Ch
(a) MeONa
(b) MeSNa
(c) NH3(aq)
(e) Me3Al
(d) Me2NH
(f) R-SnBu3
OMe
SMe
NH2
Me
NMe2
2-furyl
11Dd
11De
11Df
11Dg
11Di
82
81
83
75
81
89
(g) R-B(OH)2 3-furyl
(g) R-B(OH)2 3-thienyl
(g) R-B(OH)2 2-benzofuryl 11Dh
a Crude compound was used directly in the next step. b Overall yield after two steps.
The key intermediates, benzoylated 4-chloropyridopyrrolopyrimidine nucleosides 10A, 10B,
10C and 10D, were subsequently modified at position 4 through nucleophilic substitutions or
cross-coupling reactions to provide four sets of desired nucleosides bearing several substituents,
such as different size heterocycles, as well as methoxy, methylsulfanyl, methyl, dimethylamino
and amino groups. The selection of substituents was based on analogy and previous experience
with other fused-deazapurine ribonucleosides.13,17,18,21 In case of intermediate 10B, which was
very difficult to obtain due to instability of intermediates and only limited amount was
available, we were able to prepare only 3 most important derivatives 12Ba,c,d. Methoxy
derivatives 12Aa, 12Ba, 12Ca and 12Da and methylsulfanyl derivatives 12Cb, 12Db were
obtained through nucleophilic substitution with sodium methoxide or sodium thiomethoxide in
MeOH. During mentioned substitution with sodium thiomethoxide in MeOH, in all three series,
mixture of methoxy and methylsulfanyl nucleosides was formed. While in series C and D these
two compounds were easily separated, in series A, inseparable mixture was formed, therefore
at first methylsulfanyl protected intermediate 11Ab was prepared and subsequently deprotected
under Zemplén conditions. To obtain amino derivatives 12Ac, 12Bc, 12Cc, and 12Dc, the key
4-chloropyridopyrrolopyrimidine intermediates were subjected to nucleophilic substitution
using aqueous ammonia in 1,4-dioxane. In all previously mentioned cases (except of compound
12Ab), removal of the benzoyl protecting groups was achieved simultaneously under the
reaction conditions. 2-Furyl derivatives 11Af, 11Cf and 11Df were prepared in good yields
8
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
from 4-chloropyridopyrrolopyrimidine nucleosides 10A, C, D by the Stille cross-coupling
reaction with the corresponding tributylstannane, catalyzed by PdCl2(PPh3)2 in DMF at 100 °C
(Scheme 2). 3-Furyl, 3-thienyl and 2-benzofuryl moieties were introduced by Suzuki−Miyaura
cross-coupling reactions with the corresponding hetarylboronic acids in toluene using
Pd(PPh3)4 as a catalyst in presence of potassium carbonate. Benzoylated intermediates 11Ag,
11Cg, 11Dg, 11Ai, 11Ci, 11Di and 11Ch, 11Dh were isolated in good yields (Scheme 2, Table
1). In the case of protected 2-benzofuryl derivative 11Ah, low yield was obtained and therefore
11Ah was synthesized using PdCl2(PPh3)2 as a catalyst and potassium carbonate and
triethylamine. 4-Methyl derivatives 11Ad, 11Cd, 11Dd were synthesized by palladium-
catalyzed alkylation of 4-chloropyridopyrrolopyrimidine derivatives 10A, 10C, 10D with
trimethylaluminium in THF at 70 °C. In case of series B, intermediate 11Bd was prepared in
the same way but it was directly deprotected to 12Bd without further purification or
characterization. Dimethylamino groups of compounds 11Ae, 11Ce, and 11De were introduced
by nucleophilic substitution with dimethylamine in the mixture of DCM and propan-2-ol at the
room temperature. Zemplén deprotection of benzoylated nucleosides 11Ab and 11A-Dd–h in
presence of MeONa in MeOH furnished desired unprotected nucleosides 12Ab and 12A–Dd–
h in good yields (Scheme 2, Table 1). Because of poor solubility of several benzoylated
intermediates in MeOH, deprotection of these derivatives were performed at increased
temperatures of 40–70 °C or in the mixture of MeOH and DMF. Yields and reaction conditions
are summarized in Table 2.
For the study of the intracellular metabolism and mechanism of action of this type of
compounds, selected most active nucleosides 12Ac, 12Ad, 12Cc and 12Cd were converted into
their 5'-O-monophosphates 13Ac, 13Ad, 13Cc and 13Cd by treatment with POCl3 followed by
addition of 2 M TEAB (aqueous triethylammonium bicarbonate). The triethylammonium salts
of products were converted to sodium salts by ion exchange on Dowex (Scheme 3).22
9
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Scheme 3. Reagents and conditions: (i) P(O)(OMe)3, POCl3, 0 ºC, 3 h, then aqueous 2 M TEAB,
ion exchange on Dowex 50 (Na+ form)
In order to obtain standards of selected 2'-deoxyribonucleotides for the study of intracellular
incorporation to DNA and RNA,14 it was required to prepare nucleoside 5'-O-monophosphates
derived from 2'-deoxy analogues of amino 12Ac and 12Cc and methyl nucleosides 12Ad and
12Cd. To synthesize the key protected β-anomeric deoxyribonucleoside intermediates 14A and
14C, the corresponding pyrido-fused nucleobases 9A and 9C were deprotonated by potassium
hydroxide or sodium hydride and then subjected to glycosylation using Hoffer’s chlorosugar
(1-chloro-2-deoxy-3,5-di-O-toluoyl-α-D-ribofuranose).10 Palladium-catalyzed alkylation of
14A and 14C with trimethylaluminum provided protected methylated intermediates 15Ad and
15Cd, which were subsequently deprotected under Zemplén conditions to give the final free
deoxyribonucleosides 16Ad and 16Cd. To obtain adenine-like 2'-deoxyribonucleosides 16Ac
and 16Cc, the key intermediates were subjected to nucleophilic substitution using aqueous
ammonia in 1,4-dioxane. Corresponding 2′-deoxy-5′-O-monophosphates 17Ad, 17Cd, 17Ac
and 17Cc were synthesized in the same manner as their ribo analogues 13Ac, 13Ad, 13Cc and
13Cd (Scheme 4).
10
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Scheme 4. Reagents and conditions: (i) 1) KOH, TDA-1, MeCN, rt, 30 min; or NaH, MeCN,
rt, 30 min; 2) Hoffer’s chlorosugar, rt, 30–120 min; (ii) Me3Al, Pd(PPh3)4, THF, 70 ºC, 16 h;
(iii) MeONa, MeOH, rt–60 °C, 16 h; (iv) NH3 (aq), 1,4-dioxane, 120 °C, 16 h; (v) 1)
P(O)(OMe)3, POCl3, 0 ºC, 3 h; 2) aqueous TEAB (2 M); 3) ion exchange on Dowex 50 (Na+
form).
We were able to determine crystal structures of four final ribonucleosides 12Ae, 12Ai, 12Ag
and 12Ci (Figure 1) and one deoxyribonucleoside 16Ac by X-ray diffraction. In some cases,
the crystal packing contained several conformers and the packing involved head-to-tail π-π
stacking of the extended nucleobases. Conformers of each nucleoside and the crystal packings
are given in Figures S4-S8 in Supporting Information.
11
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Figure 1. Crystal structures for compounds 12Ae, 12Ai, 12Ag and 12Ci.
In order to investigate if the pyridopyrrolopyrimidine ribonucleoside triphosphates are
substrates for RNA polymerases, we synthesized amino and methyl triphosphates 18Ac, 18Ad,
18Cc, 18Cd, 18Dc and 18Dd by treatment with POCl3 followed by addition of freshly prepared
solution of bis(tri-n-butylammonium) pyrophosphate and tri-n-butylamine (Scheme 5). Using
DMF as a solvent to dissolve the pyrophosphate turned out to be counterproductive in this case.
It is known that polar solvent such as DMF reacts with phosphoryl oxychloride while Vilsmeier
reagent is produced and by-product 5'-triphosphate-2', 3'-cyclic phosphate is formed.23 In an
attempt to reduce or eliminate the formation of cyclic by-product, MeCN was selected as more
appropriate solvent. The cyclic by-products 18Cdx and 18Ddx were successfully separated by
ion-exchange HPLC on Poros HQ and characterized by 1HNMR and MS (see SI).
12
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Scheme 5. Reagents and conditions: (i) 1. POCl3, PO(OMe)3, 0 °C, 1.5 h; 2) (NHBu3)2H2P2O7,
Bu3N, DMF or MeCN, 0 °C, 1 h; 3) aqueous TEAB (2 M); 4) ion exchange on Dowex 50 (Na+
form).
Fluorescence properties of pyrido-fused 7-deazapurine ribonucleosides
Many of the previously reported fused 7-deazapurine nucleosides have shown interesting
fluorescence properties and some of them were used for the preparation of fluorescent DNA
probes.16,24,25 Therefore, we have studied also the photophysical properties of the title pyrido-
fused 7-deazapurine ribonucleosides. Table 2 summarizes the results showing that the most
compounds exerted at least some fluorescence in ethanol (except of dimethylamino derivative
12Ae). Moderate to good fluorescence quantum yields (0.20–0.40) showed compounds 12Dg,
12Aa, 12Ah and 12Cc. Much brighter fluorescence with an excellent quantum yields in the
range of 0.46–0.62 showed compounds 12Df, 12Da, 12Dc and 12Dh from the D series (2-furyl,
methoxy, amino, 2-benzofuryl derivatives). These results indicate that at least some of pyrido-
fused 7-deazapurine ribonucleosides have a potential in fluorescent labelling of nucleic acids
when excited with UV lasers. On contrary, favourable excitation and emission wavelengths
enables analysis of biological activity of compounds using routinely available reporters based
on green or red fluorescent proteins.
Table 2. UV absorption and Fluorescence Spectra of Pyrido-fused Nucleosides 12A–Da–h
Compound
Absorption
Emission
λmax.[nm]
λmax [nm] (ε·10–4 [L·mol−1·cm−1])
φ
12Bc
12Ba
12Da
12Db
305
275
315
325
(0.53)
(1.06)
(1.03)
(1.26)
283
(0.82)
0.03
0.03
0.50
0.02
324
464
331
345
279
298
(1.14)
(1.37)
253
(1.82)
13
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
12Dc
12Dd
12Di
12De
12Dg
12Df
12Dh
12Aa
12Ab
12Ac
12Ad
12Ai
12Ae
12Ag
12Af
12Ah
17Ac
12Ca
12Cb
12Cc
12Cd
12Ci
12Ce
12Cg
12Cf
12Ch
320
319
303
332
302
322
365
322
336
327
287
316
340
312
326
356
326
310
302
319
323
301
319
298
328
348
(1.60)
(0.45)
(0.67)
(1.46)
(1.18)
(1.81)
(2.53)
(0.92)
(0.92)
(1.65)
(1.00)
(1.30)
(1.62)
(1.08)
(2.03)
(2.70)
(1.60)
(0.40)
(1.63)
(0.91)
(0.28)
(1.36)
(1.31)
(0.96)
(2.04)
(3.10)
0.56
0.11
0.18
0.02
0.20
0.46
0.62
0.29
0.01
0.02
0.11
0.08
0.00
0.06
0.24
0.36
0.02
0.16
0.03
0.31
0.07
0.08
0.01
0.07
0.09
0.13
339
344
383
364
374
379
399
342
343
346
362
400
364
394
404
414
345
349
390
339
371
412
503
405
423
445
282
259
(1.33)
(2.67)
254
(1.93)
260
269
348
312
324
(2.19)
(2.83)
(2.82)
(0.92)
(0.91)
275
283
256
(2.95)
(0.80)
(2.53)
254
264
250
263
269
274
(2.04)
(3.29)
(2.49)
(2.66)
(3.23)
(3.51)
280
(1.30)
290
251
257
332
(1.20)
(2.19)
(2.53)
(1.39)
259
266
(2.30)
(2.64)
UV spectra were measured in ethanol at 25 °C. Fluorescence quantum yields were measured in ethanol using
quinine sulfate in 0.5 M H2SO4 (Φ f = 0.55) as a reference. Excitation wavelength was 296 nm.
Biological activity profiling
All the final ribonucleosides 12A–D were subjected to screening of their biological activities.
Antiviral activity
The antiviral screening was performed against respiratory syncytial virus (RSV), hepatitis
C virus (HCV), herpes simplex, influenza, coxsackie, human immunodeficiency virus (HIV)
and dengue viruses (for detailed viruses strains and procedures see SI). All the final nucleosides
were found to be inactive against RSV, influenza, coxsackie, herpes simplex, HIV and dengue
viruses. The only exception were 2-benzofuryl derivatives from series C and D, which showed
weak activities against dengue (12Ch EC50 = 20.1 µM and 12Dh EC50 = 27.5 µM). The antiviral
screening of anti-HCV activities (performed as described previously)26 revealed activities
against both HCV replicons 1B and 2A in sub-micromolar concentrations (Table 3) and some
of them are even more active than Mericitabine, clinically developed nucleoside inhibitor of
viral RNA polymerase NS5B.27 The NS5B enzyme (non-structural protein 5B) builds a
replicase complex with other cellular and viral proteins in the perinuclear part of infected
cells.28 It is remarkable that nucleoside or nucleotide analogues are the only direct-acting
14
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
antivirals in clinical development displaying wide activity against all HCV genotypes, with
non-nucleoside inhibitors being exclusively or mainly active against HCV genotype 1.29 In our
study, the anti-HCV activities differed in each series. The 2-furyl, 3-furyl and 3-thienyl
derivatives 12Af, 12Ag, 12Ai, 12Cf, 12Cg from A and C series and one methylsulfanyl
derivative 12Db from D series are active only against replicon 1B. Low micromolar activity
against both HCV replicons showed 2-benzofuryl derivatives 12Dh, 12Ah and methyl
derivative 12Ad. The most potent and selective in both replicons were pyridopyrrolopyrimidine
nucleosides from the C series 12Ca, 12Cb, 12Cc and 12Cd.
Table 3. Anti-HCV Activities of Pyrido-fused Nucleosides 12A–D
HCV replicon 1B
HCV replicon 2A
EC50 (µM)
>44.44
>44.44
>44.44
1.57
>44.44
>44.44
>44.44
>44.44
15.42
Compd
EC50 (µM)
CC50 (µM)
>44.44
>44.44
>44.44
32.33
CC50 (µM)
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
38.02
12Aa
>44.44
>44.44
0.63
12Ab
12Ac
12Ad
12Ae
12Af
0.64
>44.44
0.16
0.36
0.84
5.27
>44.44
13.97
>44.44
0.46
0.59
0.09
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
34.67
>44.44
>44.44
>44.44
21.66
12Ag
12Ai
12Ah
12Ba
12Bc
12Bd
12Ca
12Cb
12Cc
12Cd
12Ce
12Cf
>44.44
28.65
>44.44
0.77
0.29
0.66
>44.44
>44.44
>44.44
>44.44
>44.44
40.13
0.16
24.47
0.41
>44.44
41.5
>44.44
>44.44
6.31
>44.44
>44.44
>44.44
>44.44
>44.44
20.29
>44.44
10.0
7.97
>44.44
3.40
>44.44
>44.44
>44.44
>44.44
15.50
12Cg
12Ci
12Ch
12Da
12Db
12Dc
12Dd
12De
12Df
12Dg
12Di
12Dh
12Daα
Mericitabine
>44.44
14.26
>44.44
42.80
>44.44
40.60
>44.44
>44.44
3.04
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
22.73
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
7.47
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
>44.44
27.5
>44.44
1.20
>44.44
>44.44
>44.44
0.99
>44.44
>44.44
Cytotoxic activity
15
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
In vitro cytotoxic activity screening of nucleosides 12A–Da–h was performed on several
cancer cell lines (A549 – human lung adenocarcinoma, HCT116 and HCT116p53–/– – colon
cancer cells with/without p53 gene, U2OS – human osteosarcoma, HeLa – human HPV positive
cervical carcinoma, HepG2 – hepatocellular carcinoma) as well as leukemic cell lines (CCRF-
CEM – acute lymphoblastic leukemia, CEM-DNR – CCRF-CEM cells daunorubicin resistant
overexpressing multidrug resistance proteins and lacking topoisomerase 2- gene, K562 –
myelogenous leukemia bcr-abl positive, K562-TAX – K562 cells paclitaxel resistant
overexpressing multidrug resistance proteins, HL60 – acute promyelocytic leukemia).30 These
cell lines were used for determination of concentration reducing cell viability by 50% (IC50) by
3-days MTS (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium)23
or
XTT
(2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide) assay31. Non-malignant fibroblast cell lines MRC-5 and BJ were used in the
MTS assay for comparison.
The cytotoxic activity screening (Table 4) showed that the presence and position of nitrogen
atom is crucial for cytostatic and cytotoxic effect. Only pyridopyrrolopyrimidine
ribonucleosides possessing nitrogen at the position 7 on the pyridine ring (series C) showed
significant submicromolar in vitro cytotoxic activity, whilst some derivatives of
pyridopyrrolopyrimidine ribonucleosides bearing nitrogen at the position 5 or 8 (series A and
D) showed moderate to good activity and the three examples of the pyrido[3',4':4,5]pyrimidine
nucleosides (series B) were entirely inactive. The most active compounds 12Ca, 12Cb, 12Cc
and 12Cd, belonging to the C series, displayed submicromolar values of activities against
leukemic and solid tumour cell lines, with low toxicity to BJ and no toxicity to MRC-5
fibroblasts, showing promising therapeutic index under in vitro conditions. It is interesting to
point out that the amino derivative 12Cc was one of the most active compounds in the study,
which makes this class of compounds different from previously reported heteroaryl-fused 7-
deazapurine nucleosides, where the 6-amino derivatives (analogues of adenine) were typically
inactive23,18. Also interesting is the comparison of these cytotoxic pyridopyrrolopyrimidine
nucleosides to previously reported benzo-fused (carba-analogues) nucleosides which did not
show any cytotoxic activity. Apparently, it is not just the bulkiness of the fused (hetero)aromatic
moiety, but also the presence and position of N atom (presumably as H-bond acceptor) are
crucial factors for biological activity of this class of nucleosides. Cytotoxic activity of tested
compounds was independent of p53 status, as cytotoxic activities were comparable in p53
proficient versus deficient HCT116 cells. However, biological activity was impaired by
expression of multidrug resistance proteins across nucleoside series 12A-Da-h, with exception
16
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
of compounds 12Cd and 12Cc, where cytotoxic activities were comparable or only slightly
different on parental versus multidrug resistant cell lines, respectively (Table 4).
17
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Table 4. Cytotoxic Activity of Nucleosides 12A–Da–h
MTS, IC50 (μM)
XTT, IC50 (μM)
CCRF
-CEM
>50
CEM
-DNR
>50
HCT116
p53-/-
>50
K562
Compound
BJ
MRC-5
A549
HCT116
K562
U2OS
Hep G2
HL 60
HeLa
-TAX
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5.6
>50
>50
9.1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
17
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
15
>50
>50
27
>50
>50
31
>50
>50
1.5
>50
>50
8.4
>50
>50
0.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.38
1.12
1.30
2.9
12Aa
12Ab
12Ac
12Ad
12Ae
12Af
12Ag
12Ai
>50
0.57
0.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5.1
>50
32
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
3.0
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.51
0.38
0.47
2.7
10
2.4
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.53
1.08
0.34
2.9
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.71
0.26
0.8
>50
>50
>50
>50
10
>50
>50
>50
>50
>50
>50
>50
>50
1.0
>50
>50
>50
>50
>50
>50
>50
>50
1.6
12Ah
12Ba
12Bc
12Bd
12Ca
12Cb
12Cc
12Cd
12Ce
12Cf
12Cg
12Ci
>50
>50
>50
0.56
0.27
0.7
1.0
0.34
0.5
0.27
0.9
1.3
0.54
0.6
1.8
2.3
3.6
7.7
>50
>50
>50
>50
>50
19
0.4
0.9
2.7
1.5
1.8
1.3
0.35
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
46
>50
42
>50
>50
>50
>50
22
>50
32
>50
>50
>50
>50
13
>50
>50
>50
>50
19
>50
>50
>50
>50
19
>50
>50
>50
>50
16
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
7.4
>50
>50
7.1
12Ch
12Da
12Db
12Dc
12Dd
12De
12Df
12Dg
12Di
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
42
>50
>50
>50
>50
>50
>50
>50
>50
29
>50
>50
>50
7.7
>50
>50
>50
>50
>50
>50
>50
>50
28
>50
>50
>50
>50
>50
35
>50
>50
>50
>50
>50
>50
>50
>50
24
>50
>50
>50
18
>50
>50
>50
>50
>50
>50
>50
>50
24
>50
>50
>50
>50
>50
>50
>50
>50
23
>50
38
>50
>50
>50
>50
27
>50
>50
10
>50
>50
27
12Dh
12Daα
>50
>50
>50
>50
>50
>50
>50
>50
>50
18
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Cell cycle studies
Pyridopyrrolopyrimidine ribonucleosides which displayed the best cytotoxic activity
against CCRF-CEM T-lymphoblastic leukaemia cells, were submitted also for cell cycle
analysis. Cells were treated with 1 × IC50 and 5 × IC50 cytotoxic concentrations of nucleoside
derivatives for 24 hours (Table 5). Most of the tested nucleoside analogues lead to a S-phase
block with corresponding decrease of G0/G1 cellular population, except for two derivatives.
The compound 12Cd, induced accumulation of treated cells in G0/G1 phase with corresponding
decrease of S-phase cells, while 12Ch showed the G2/M-phase block accompanied with
decrease of S-phase cells. Perturbations in DNA and RNA synthesis were observed for most of
tested compounds and could be attributed to cell cycle alterations and/or apoptosis. Only
analogues 12Cb, 12Cc and 12Ch demonstrated significant (>90%) and dose dependent
inhibition of RNA synthesis, which may contribute to their cytotoxic effect.
Table 5. Summary of Cell Cycle Analyses, Proportion of Apoptotic and Mitotic Cells
(pH3Ser10 Positive), DNA and RNA Synthesis in CCRF-CEM
% of total cellular populations
Compound Concentration
sub-G1
(apoptosis)
2.8
G0/G1
38.7
S
G2/M
18.5
M
DNA
synthesis
52
RNA
synthesis
51.1
pH3Ser10
1.61
12Ac
12Ad
12Ca
12Cb
12Cc
12Cd
12Ch
0
42.8
1 x IC50
5 x IC50
0
4.89
9.88
2.3
27.09 54.80 18.11
18.37 65.41 16.22
2.10
1.77
1.3
64.84
67.70
33
38.00
32.88
47
40
36.4
24
1 x IC50
5 x IC50
0
3.54
6.49
2.8
36.84 41.28 21.87
27.83 54.10 18.08
1.13
0.66
1.61
0.69
0.61
1.3
36.65
29.19
52
17.93
27.91
51.1
23.61
27.56
47
38.7
42.8
18.5
1 x IC50
5 x IC50
0
4.49
7.17
2.3
19.55 67.85 12.60
14.13 69.93 15.94
55.87
49.58
33
40
36.4
24
1 x IC50
5 x IC50
0
3.16
5.00
2
20.85 64.25 14.89
16.54 67.01 16.45
0.30
0.25
1.5
22.71
15.81
37
9.72
8.70
46
42
45
13
1 x IC50
5 x IC50
0
2.93
4.37
2.95
4.20
16.52
4.1
33.58 51.21 15.21
1.33
1.06
1.50
1.62
1.28
2.0
43.43
43.35
41
15.93
4.67
44
9.66
39
78.46 11.88
44 16.4
1 x IC50
5 x IC50
0
46.01 39.33 14.66
50.11 39.94 9.95
32.12
26.10
36.3
32.72
17.67
37
45.7
36.4
18
19
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
1 x IC50
5 x IC50
14.82
19.20
49.60 11.28 39.12
37.20 26.05 36.75
1.56
0.71
25.92
31.26
16.42
3.24
Intracellular phosphorylation
We anticipated that mechanism of action of pyridopyrrolopyrimidine ribonucleosides might
be similar to the previous cases of thieno-, pyrrolo- and furo- fused deazapurine ribonucleosides
in which the compound was first phosphorylated in cells and subsequently incorporated into
DNA or RNA. To obtain experimental evidence to prove this hypothesis, we studied
intracellular phosphorylation of methyl 12Ad, 12Cd and amino 12Ac, 12Cc derivatives from
A and C series. The corresponding ribonucleoside monophosphates 13Ad, 13Cd, 13Ac, and
13Cc were used as analytical standards. Intracellular phosphorylation was tested using BJ
(normal fibroblasts) and HCT116 (malignant) cells at different concentrations of nucleosides
(1 or 10 µmol/L) after 1 or 3 hours of treatment in vitro. The phosphorylation of all four
nucleosides proceeded in all cases, however, in contrast with previously published
thienopyrrolopyrimidine nucleosides17 the levels of monophosphates were higher in malignant
HCT116 cells than in BJ, what might explain higher selectivity of these nucleosides for cancer
cells. In addition, the ratio of pyridopyrrolopyrimidine nucleoside monophosphate versus
nucleoside was increased at higher concentration of corresponding nucleoside (10 µM), which
insinuate rate limited phosphorylation in non-malignant cells. Different level of formed
monophosphate in malignant and non-malignant cells after 1 and 3 h incubation in 10 µmol/L
concentration is compared on the Figure 2. Table summarizing exact values of monophosphate
in certain amount of cells (pmol/6 × 105 cells) in 1 or 10 µmol/L concentrations is attached in
Supporting information.
20
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Figure 2. Comparison of formed monophosphates (pmol/6 × 105 cells) in HCT116 and BJ cells
after 1 h (green) and 3 h (blue), 10 µmol/L concentration of corresponding nucleoside.
Phosphorylation and inhibition by human adenosine kinase (ADK)
After we proved that the desired pyridopyrrolopyrimidine nucleosides are effectively
phosphorylated in cells, we supposed that the enzyme responsible for phosphorylation could be
a human adenosine kinase (ADK). Therefore, we studied the in vitro phosphorylation of the
title nucleosides by human ADK32 (Table 6).
Table 6. Phosphorylation of Nucleosides 12A–Da–h by ADK and Inhibition of ADK
Phosphorylation
Inhibition
Compound
(%)
86.5
0
IC50
–
>10
>10
>10
Adenosine
12Aa
12Ab
12Ac
12Ad
12Ae
12Af
12Ag
12Ah
12Ai
12Ba
12Bc
12Ca
12Cb
12Cc
12Cd
12Ce
12Cf
0
82.3
33.1
0
9.4
0
0
0
0
0
19.4
0
0
18.6
0
0
>10
>10
245 ± 41 nM
>10
184 ± 21 nM
20 ± 1.3 µM
>10
>10
>10
>10
>10
>10
>10
>10
12Cg
12Ci
0
0
>10
>10
12Ch
12Da
12Db
12Dc
12Dd
12De
12Df
0
0
0
0
0
0
0
5.5 ± 0.9 µM
>10
>10
>10
>10
>10
>10
12Dg
12Di
0
0
>10
>10
12Dh
12Daα
0
0
2.9 ± 0.39 µM
>10
Phosphorylation: 50 µM solution of compound, 243 ng of enzyme, 30 min, 37 °C
Inhibition: 50 µM solution of adenosine, 10 µM solution of compound, 97 ng of enzyme, 20 min, 37 °C.
Unlike in the previous case of thieno-, pyrrolo- and furo- fused deazapurine ribonucleosides
where the rate of phosphorylation nicely correlated with cytotoxic activities, it was not the case
21
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
for some pyridopyrrolopyrimidine ribonucleosides. The amino derivative 12Ac is effectively
phosphorylated by ADK comparably to adenosine. Methyl derivatives 12Ad and 12Cd and
methoxy derivative 12Ca are also phosphorylated but somewhat less efficiently. Surprisingly,
two most active compounds 12Cb and 12Cc were not phosphorylated by ADK at all in our
assay. However, because all the amino and methyl derivatives from series A and C were
phosphorylated in cellulo, we can assume that these nucleosides might have been
phosphorylated by other isoforms of ADK or by some other nucleoside kinase. All nucleosides
were also tested for ADK inhibition. Generally, they are not effective inhibitors of ADK, only
derivatives 12Ai, 12Af, 12Ah, 12Ch and 12Dh displayed some moderate inhibition (Table 6).
Incorporation of nucleosides into nucleic acids of treated cells
After confirming that title nucleosides are phosphorylated preferentially in cancer cells, we
studied the incorporation of selected nucleotides (derived from methyl 12Ad, 12Cd and amino
12Ac, 12Cc nucleosides from A and C series) to DNA and RNA in living cells. CCRF-CEM
cells were incubated with 5 × IC50 concentrations of nucleosides for 2.5 h and immediately
harvested. RNA and DNA were isolated and digested. The separation and detection of modified
nucleosides and nucleotides and their concentration in nucleic acids were achieved by using
HPLC chromatography with tandem mass spectrometry (LC-MS/MS). The corresponding
ribonucleoside monophosphates 13Ad, 13Cd, 13Ac, and 13Cc as well as their 2'-deoxy
analogues (Scheme 4) were used as analytical standards.
Table 7. Incorporation of Selected Nucleosides into RNA and DNA in Treated CCRF-CEM
Cells
sample
DNA
compd
NMP content
(fmol/µg of nucleic acid)
dNMP content
(fmol/µg of nucleic acid)
12Ac
12Ad
12Cc
12Cd
12Ac
12Ad
12Cc
12Cd
471±69
539±101
494±96
407±232
172±17
nda
nda
5.5±1.8
nda
nda
RNA
180±10
nda
aNot detected. Incorporation analyzed after 2.5-hour treatment at 5 × IC50 and subsequent digestion with
nuclease P1 (data from 2–4 independent experiments)
22
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
The results show that all tested compounds are mainly incorporated into DNA as
ribonucleosides, whereas only the methyl derivative 12Ad is also incorporated into DNA as
2'-deoxyribonucleoside though in very minor quantities (2'-deoxyribonucleoside
monophosphate 17Ad found in residual quantities). All tested modified nucleosides are more
efficiently incorporated into DNA (in conversion to fmol/µg of nucleic acid) than the previously
reported AB6114 or pyrrolo- and furo-fused nucleosides.18 Interestingly, only amino derivatives
12Ac and 12Cc were detected in the RNA of treated cells. These results correlate with results
of enzymatic incorporation by T7 RNA polymerase where just amino derivatives from A and
C series are successfully incorporated into RNA (Figure 3).
Enzymatic incorporation of pyridopyrrolopyrimidine nucleotides into nucleic acids
Intracellular phosphorylation and subsequent incorporation into RNA that prevents its
function is one of the parallel mechanisms of action of cytotoxic 7-hetaryl-7-deazaadenosines.14
Therefore, selected ribonucleoside triphosphates 18Ac, 18Ad, 18Cc, 18Cd, 18Dc and 18Dd,
were prepared and tested as substrates for model T7 RNA polymerase. We used DNA templates
containing the promoter and three guanosines in the +1 to +3position according to our previous
work.33 The transcription has been performed using [α-32P]-GTP and the RNA products were
analyzed by PAGE (Figure 3) and quantified using QuantityOne software. From all six
modified ribonucleoside triphosphates we tried, just amino derivatives 18Ac and 18Cc from A
and C series were successfully incorporated (similarly as in case of incorporation by human
polymerase). The RNA products were also characterized by MALDI-TOF, which confirmed
the correct full-length modified RNA (see SI). These results suggest that only amino
pyridopyrrolopyrimidine nucleosides with the specific position of N atom on pyridine ring are
suitable substrates for RNA polymerases and are able to substitute for ATP during transcription
and to be incorporated into RNA.
23
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Figure 3. (a) In vitro transcription with amino and methyl triphosphates 18Ac, 18Ad, 18Cc,
18Cd, 18Dc and 18Dd from A, C and D series. Lanes 1, +: product of PAGE with natural ATP;
lane 2, –: product of PAGE with H2O; lane 3: 18Ac incorporated; lane 4: 18Cc incorporated;
lane 5-9: 18Ad, 18Dc, 18Cd, 18Dd not incorporated; (b) Quantifications of the relative
conversions (compared to transcription with natural ATP) of T7 RNA polymerase transcription
experiments.
In vitro pharmacology of nucleoside analogues
Absorption, distribution, metabolism, and excretion (ADME) in vitro properties of a chemical
compound are critical factors in predicting its pharmacokinetic behavior and drug-drug
interaction potential in vivo. Therefore we have also studied ADME properties of the most
active derivatives under in vitro conditions. Tested compounds showed high chemical stability
in phosphate-buffered saline (PBS pH 7.4) after 120 minutes at 37 °C. Also, all nucleosides
24
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
were found to be stable in plasma (all compounds showed more than 75% presence in plasma
after 120 min). For microsomal stability, all compounds achieved the similar results (all
compounds showed more than 73% presence in microsomes after 60 min) indicating low or
medium clearance in human microsomal fraction. Correspondingly to chemical structure, more
hydrophobic substitutions resulted in higher proportion of the compound bound to human
serum proteins. For instance, it was higher for methoxy 12Ca and methylsulfanyl 12Cb
derivative 78 and 81%, while amino derivative 12Cc was bound only from 27%. All seven
nucleosides showed moderate (– log Papp 5–6 cm/s) or low ability (– log Papp > 6 cm/s) to
diffuse across an artificial cellular membrane in the Parallel artificial membrane permeability
assay (PAMPA), suggesting alternative intracellular transport mechanism, probably involving
nucleoside transporters.34 The Caco-2 and MDCK-MDR1 permeability assays are established
models of intestinal35 and blood-brain barriers, respectively.36 Studied compounds showed low
(PappAB < 5 × 10–6 cm/s) or moderate (PappAB 5–20 × 10–6 cm/s) probability of intestinal
absorption and to cross blood-brain barriers PappAB < 10 × 10–6 cm/s CNS –; PappAB > 10
× 10–6 cm/s CNS +). Tested compounds are actively exported from the cells in both barrier
models as indicated by efflux ratios > 2. Most probably, the test compounds are exported by
MDR1 efflux pump present in both cell types, which is in good agreement with diminished
cytotoxicity in cancer cells expressing MDR1 gene (Table 4). Results from all in vitro
pharmacology testing are summarized in Table 8.
Table 8. Stability and PAMPA for Selected Cytotoxic Nucleosides
Metabolism
in vitro
Time
(min)
12Ac 12Ad 12Ca
12Cb
12Cc 12Cd 12Dc
Compound remaining (%)
Chemical
stability
0
15
30
60
120
100
98
98
95
93
100
100
92
92
90
100
100
100
100
98
100
100
100
96
100
100
100
94
100
100
100
100
100
100
100
96
94
88
90
94
Plasma
stability
0
15
30
60
120
100
96
89
92
89
100
94
96
94
93
100
90
89
93
88
100
92
91
90
88
100
96
86
87
75
100
97
90
91
88
100
90
90
84
83
0
100
94
90
100
103
93
100
109
93
100
86
87
100
96
86
100
93
89
100
100
97
Microsomal
stability
15
30
60
90
91
86
73
87
84
96
25
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
low
44
low
low
medium
low
27
low
37
low
32
Clearance
Plasma
protein
Fraction bound (%)
78 81
40
binding
Permeability
in vitro
PAMPA log Pe
–7.61 –6.63 –6.07
–5.67
–6.95 –7.17 –7.60
Category
low
low
low
medium
low
low
low
Permeability
in vivo
MDR1-MDCK
CNS (–/+)
Papp
(×10e–6)
–
–
–
–
+
–
–
7.23
1.46
9.73
9.15
10.2
1.22
1.52
Efflux ratio
active efflux
% recovery
2.22
yes
80.1
14.2
yes
59.6
3.11
yes
78.3
3.35
yes
100
1.07
no
88.4
7.70
yes
88.4
9.11
yes
91.2
Caco-2
mod.
Category
Papp
(×10e–6)
Efflux ratio
active efflux
low
low
low
mod.
10.9
mod.
12.3
low
0.90
2.67
5.68
0.84
1.42
18.5
yes
52.7
16.7
yes
55.2
11.4
yes
78.8
28.5
yes
89.3
1.72
no
87.4
2.70
yes
100.9
15.4
yes
50
% recovery
Induction of DNA damage in nucleoside analogues treated cells
To investigate potential involvement of the most active compounds 12Cc and 12Cd versus non-
cytotoxic derivative 12Dc in DNA repair machinery, the U2OS human osteosarcoma cell line
was stably transfected with the 53BP1-GFP fusion gene as reported previously.14 53BP1 protein
accumulates at DNA lesions, preferably at DNA double strand breaks, thus its GFP-conjugate
enables visualization of DNA-damage sites in nucleus. Indeed, U2OS-53BP1-GFP cells treated
with control DNA-damaging agent etoposide (topoisomerase 2 inhibitor) showed time-
dependent accumulation of 53BP1 foci in the nuclei of treated cells (Figure 4). Out of three
tested nucleosides, only 12Cc induced a minor DNA damage with peak values 15 hours post
exposure. Cytotoxic compound 12Cd and also non-cytotoxic derivative 12Dc showed no
significant interference with formation of 53BP1 foci and thus indirectly with induction and/or
repair of double-strand DNA breaks. Although the number of 53BP1 foci per nucleus in 12Cc-
treated cells (Figure 3a) was lower than in etoposide-exposed cells, the foci were larger and
resembled those reported previously for 7-(2-thienyl)-7-deazaadenosine (AB-61)14. This
corresponds well with preferential incorporation of compounds into DNA in the form of
26
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
ribonucleosides. The presence of the modified ribonucleotides in the DNA causes ds-breaks
and apoptosis.
27
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
28
This article is protected by copyright. All rights reserved.

10.1002/chem.202001124
Chemistry - A European Journal
Figure 4. 53BP1 foci in U2OS-53BP1-GFP cells exposed to a) vehicle, etoposide, cytotoxic
nucleosides 12Cc, 12Cd and non-cytotoxic analogue 12Dc. b) Time-dependence of 53BP1 foci
formation during treatment with vehicle, etoposide and the compounds. All nucleosides were
tested at 1 and 5 × IC50 concentrations, etoposide at 1 × IC50.
29
This article is protected by copyright. All rights reserved.