Design and synthesis of purine analogues as highly specific ligands for FcyB, a ubiquitous fungal nucleobase transporter

Article abstract of DOI:10.1016/j.bmc.2016.09.055

In the course of our study on fungal purine transporters, a number of new 3-deazapurine analogues have been rationally designed, based on the interaction of purine substrates with the Aspergillus nidulans FcyB carrier, and synthesized following an effective synthetic procedure. Certain derivatives have been found to specifically inhibit FcyB-mediated [³H]-adenine uptake. Molecular simulations have been performed, suggesting that all active compounds interact with FcyB through the formation of hydrogen bonds with Asn163, while the insertion of hydrophobic fragments at position 9 and N6 of 3-deazaadenine enhanced the inhibition.

Full text of DOI:10.1016/j.bmc.2016.09.055

Accepted Manuscript
Design and synthesis of purine analogues as highly specific ligands for FcyB, a
ubiquitous fungal nucleobase transporter
Nikolaos Lougiakis, Efthymios-Spyridon Gavriil, Markelos Kairis, Georgia
Sioupouli, George Lambrinidis, Dimitra Benaki, Emilia Krypotou, Emmanuel
Mikros, Panagiotis Marakos, Nicole Pouli, George Diallinas
PII:
S0968-0896(16)30789-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.055
BMC 13308
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 June 2016
31 August 2016
21 September 2016
Please cite this article as: Lougiakis, N., Gavriil, E-S., Kairis, M., Sioupouli, G., Lambrinidis, G., Benaki, D.,
Krypotou, E., Mikros, E., Marakos, P., Pouli, N., Diallinas, G., Design and synthesis of purine analogues as highly
specific ligands for FcyB, a ubiquitous fungal nucleobase transporter, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.09.055
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Design and synthesis of purine analogues as highly specific ligands for FcyB, a ubiquitous
fungal nucleobase transporter
Nikolaos Lougiakis,1 Efthymios-Spyridon Gavriil,1 Markelos Kairis,1 Georgia Sioupouli,2
George Lambrinidis,1 Dimitra Benaki,1 Emilia Krypotou,2 Emmanuel Mikros,1 Panagiotis
Marakos,1 Nicole Pouli,1* George Diallinas2*
1 Division of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian
University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
2
Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis,
Athens 15784, Greece
*Correspondence to:
Nicole Pouli, e-mail: pouli@pharm.uoa.gr
George Diallinas, e-mail: diallina@biol.uoa.gr
Key words: Aspergillus nidulans; purine transporters; structure activity relationships;
combiglide; imidazo[4,5-c]pyridine; deazaadenine
Abstract: In the course of our study on fungal purine transporters, a number of new 3-
deazapurine analogues have been rationally designed, based on the interaction of purine
substrates with the Aspergillus nidulans FcyB carrier, and synthesized following an effective
synthetic procedure. Certain derivatives have been found to specifically inhibit FcyB-mediated
[3H]-adenine uptake. Molecular simulations have been performed, suggesting that all active
compounds interact with FcyB through the formation of hydrogen bonds with Asn163, while the
insertion of hydrophobic fragments at position 9 and N6 of 3-deazaadenine enhanced the
inhibition.

1. Introduction
Fungal pathogens, and especially Aspergillus fumigatus, constitute an emerging threat
due to the increasing number of immunosuppressed patients.1,2 Most present day antifungals are
rather hydrophobic compounds which enter fungal cells via non-facilitated diffusion and target
enzymes involved in the synthesis of the plasma membrane or the cell wall.3,4 The most common
of such antifungals include azoles, polyenes and echinocardins. Due to the mechanism of non-
specific cellular uptakes, these antifungals are associated with side effects and mediocre
efficiency. In addition, resistance to these antifungals arises frequently due to genetic
mechanisms activating efflux by ABC xenobiotic exporters, or the overproduction or
modification of their target.5-7 An alternative category of antifungals is exemplified by 5-
fluorocytosine (5-FC). This highly efficient antifungal pyrimidine analogue is incorporated in
fungal cells by specific transporters and metabolically converted to the highly cytotoxic 5-
fluorouracil.3 The apparent absence of 5-FC transporters in human cells makes this antifungal
little, if not at all, cytotoxic for humans. In addition, several fungi seem to use many transporters
for 5-FC uptake, so that mutation in a single gene does not confer full resistance to the drug.8,9
Rather surprisingly, emerging knowledge on fungal transporters has not been rationally
exploited to date in relationship to the identification of novel antifungals. Ideally, a drug
recognized by a fungal transporter, but not by host transporters, as is the case of 5-FC, will also
have a highly targeted antifungal potential. As a step towards the rational design of novel
antifungals, we study structure-function relationships in nucleobase/nucleoside transporters in A.
nidulans, a genetically tractable fungus, where we have identified, cloned and fully characterized
all 7 major transporters, catalyzing the uptake of purines, pyrimidines, nucleosides and purine
analogues, namely UapA, UapC, AzgA, FurD, FurA, FcyB and CntA.10-12 The characterization
of these seven transporters has allowed the construction, through standard reverse and classical
genetics, of a ‘master mutant’ strain named Δ7, where all seven transporter genes are deleted.13
The genetic re-introduction of any selected nucleobase transporter gene in Δ7 allows the direct
and rigorous functional assessment of the corresponding transporter in a ‘clean’ genetic
background.9
In this work, we investigated whether we could rationally design, based on previously
theoretical models describing purine-transporter interactions,9,14-16 and synthesize analogues
which will be recognized by a single specific nucleobase transporter of A. nidulans. More
specifically, we wanted to test whether we can design and synthesize purine analogues
recognized solely by FcyB. We have chosen FcyB as this transporter is not only ubiquitously
present in all fungi but has also very similar transport kinetics and substrate specificity with
AzgA, both recognizing at the μM range and transporting efficiently all salvageable purines
(adenine, guanine and hypoxanthine). Thus, being able to distinguish substrates/ligands of these
two functionally similar transporters would be a rigorous test for investigating the feasibility of
future efforts for rational drug design, related to specific fungal transporters. As earlier
observations showed that purine analogues substituted at position 3 of the purine ring could still
be recognized efficiently by FcyB,17 3-deazaadenine was selected as a primary compound in hit-
lead campaign against FcyB. Our results show that, indeed, it is possible to identify purine
analogues highly specific solely for FcyB. The importance of these findings is apparent for the
future design of highly-targeted antifungals recognized by specific fungal, but not by host,
transporters.

2. Results and Discussion
2.1. Rational design of new 3-deaza analogues
Starting from the FcyB homology model structure previously constructed on the on the
Sodium-Hydantoin Transporter Mhp1 template and validated by site mutation experiments,15 we
explored the possibilities to modify 3-deazaadenine considering the binding site of the substrate
occluded structure based on the Mhp1 benzyl-hydantoin permease from M. liquefaciens. The
resulted low energy docking poses of 3-deazaadenine within the substrate binding site of the
transporter is shown in Figure 1. Two different orientations within the cavity of similar
calculated interaction energy have been considered. In the first (Fig. 1A) 3-deazaadenine
interacts with FcyB in a very similar way to adenine, through a bidentate H bond that is formed
between Asn163 amide group and ligand sites C6-NH2 and N1 (original purine numbering). In
the second (Fig. 1B) 3-deazaadenine interacts with FcyB forming H bonds through C6-NH2 and
N7, with Asn163 as well.
According to these theoretical models two major directions can be explored as targeting
regions, depicted as I and II in Figure 1 with red arrows. The first lies in the upper end of
transmembrane segment 1 (TMS1) among Val84 (TMS1), Ala162, Val166 (TMS3) and Glu397
(TMS9). The second one is a hydrophobic pocket lying near Pro353 (TMS8), Trp77 (TMS1)
and Tyr262 (TMS6). In order to explore those sites, 1 and 4 substituted 3-deazaadenine analogs
were designed using combiglide algorithm as implemented on Schrodinger Suite 2014.
Figure 1. Global minima structures of FcyB in complex with 3-deaza-adenine. Major directions I and II
for substitution are shown with red arrows.

A virtual in-house library containing 100 fragments was considered for probable
modifications, using 0 to 3 methylene groups as spacers, resulting 1600 virtual molecules for in-
silico evaluation. The resulted molecules were ranked based on GlideScore following a Virtual
Screening procedure. The 40 high ranked ligands (~3Kcal/mol from global minimum) were
selected as input for Induced Fit Docking. The output structures were then carefully inspected for
their theoretical interactions inside the binding pocket to select the most potent substrates.
4-Methylpiperazine, 2-(dimethylamino)ethylamine and benzylamine groups appeared to
be suitable modifications at position 4 of imidazopyridine to target Glu397 or Glu401 as well as
more hydrophobic residues in region I. A morpholine group has been considered as alternative to
piperazine to balance the hydrophobic part of the region. Isopropyl and cyclopentyl groups were
best fitted within the binding pocket directed to the second targeted region II and appeared to be
the most adequate moieties to be buried inside the first hydrophobic pocket near Trp77 and
Ala80. Finally, the 3,5-dimethoxyphenoxy substitution was also investigated since the resulted
molecule showed a different but interesting binding mode. The volume of 3,5-
dimethoxyphenoxy group forces the molecule to be flipped. The N1-H forms hydrogen bond
with Glu397, while the deazapurine core forms π-π stacking with Trp159 and the benzyl group
with Trp259.
2.2. Chemistry
For the preparation of the target compounds, 2-chloropyridine (1, Scheme 1) was used as
starting material, which underwent successively N-oxidation,18 nitration and reduction of both
the N-oxide and the nitrogroup to afford 4-amino-2-chloropyridine (4).19 This compound was
nitrated to result into the nitroderivatives 5 and 6.20 The selected 3-nitroderivative 5 was then
reduced with the use of tin(II) chloride and the resulting diaminopyridine 7 was ring-closed upon
reaction with triethylorthoformate to give the imidazopyridine 8.21 Acidic hydrolysis of 8
provided the hypoxanthine analogue 9. On the other hand, compound 8 was treated with the
suitable alkyl or arylhalide to provide the regio isomers 10a-c and 11a-c which were
chromatographically isolated and identified using nOe spectral data (correlation peak of the
protons of the substituent at position 1 with H-7, in the case of compounds 11). Then, derivatives
11a,b were hydrolyzed to provide the corresponding imidazopyridinones 12a,b.

Cl
Cl
Cl
Cl
Cl
Cl
O
O
NO2
N
N
N
N
N
N
a
b
c
d
+
NH2
NO2
NH2
NH2
O2N
5
6
4
1
2
3
e
Cl
Cl
Cl
Cl
R
NH2
NH2
N
N
N
N
N
N
N
f
h
N
R
N
NH
7
8
11a: R= CH(CH3)2
11b: R= C5H9
10a: R= CH(CH3)2
10b: R= C5H9
g
11c: R= 4-OCH3C6H4CH2 10c: R= 4-OCH3C6H4CH2
O
N
g, from 11a,b
HN
NH
O
9
N
HN
N
R
12a: R= CH(CH3)2
12b: R= C5H9
Scheme 1. Reagents and conditions: a) m-CPBA, CH2Cl2, r.t.; b) HNO3 (fuming), H2SO4 (98%), 90 ˚C; c) Fe,
HCl (36%), EtOH, reflux; d) i) HNO3 (fuming), H2SO4 (98%), 0 ˚C, ii) H2SO4 (98%), 75 ˚C; e) SnCl2·H2O, HCl
(36%), 50 ˚C; f) triethyl orthoformate, HCl (36%), r.t.; g) HCl (36%), EtOH, H2O, reflux; h) i) K2CO3, DMF,
r.t., ii) isopropyl bromide (for 10a, 11a) or cyclopentyl bromide (for 10b, 11b) or 4-methoxybenzyl
chloride (for 10c, 11c), r.t.
Attempts to substitute the 4-chloro group of compounds 11 with suitable nucleophiles,
either by refluxing compounds 11 with excess of the amine in the presence of ethanol or 2-
ethoxyethanol as the solvent, or by refluxing compounds 11 in dioxane in the presence of a
palladium catalyst (tris(dibenzylideneacetone)dipalladium, Pd2(dba)3) and a ligand (2-
dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl, X-Phos) in basic conditions (cesium
carbonate), were not successful since they resulted in extremely low yields of the target
compounds. Consequently, we have modified the synthetic methodology in order to insert the
suitable substituents at an earlier stage. Thus, the nitropyridine 5 reacted with a number of
primary or secondary amines as well as with a substituted phenol (Scheme 2), to provide in high

yield the intermediate nitropyridines 13a-e. These derivatives were reduced and the resulting
diamines 14a-e were cyclized without further purification to give the imidazopyridines 15a-e.
R1
Cl
R1
R1
R1
NO2
NH2
NH2
NO2
NH2
N
N
N
N
N
N
N
c
a
b
d
N
NH
NH2
O
R2
N
N
O
N
N
N
N
N
O
N
5
13a: R1=
13b: R1=
13c: R1=
14a: R1=
14b: R1=
14c: R1=
15a: R1=
15b: R1=
15c: R1=
15d: R1=
15e: R1=
16a: R1= morpholin-4-yl, R2= isopropyl
16b: R1= morpholin-4-yl, R2= cyclopentyl
CH3
N(CH3)2
N
CH3
CH3
N(CH3)2
16c: R1= morpholin-4-yl, R2= 4-methoxybenzyl
16d: R1= 4-methylpiperazin-1-yl, R2= isopropyl
16e: R1= 4-methylpiperazin-1-yl, R2= cyclopentyl
16f: R1= 2-(dimethylamino)ethylamino, R2= isopropyl
16g: R1= 2-(dimethylamino)ethylamino, R2= cyclopentyl
16h: R1= benzylamino, R2=isopropyl
N(CH3)2
HN
HN
HN
13d: R1= NHCH2C6H5
OCH3
14d: R1= NHCH2C6H5
OCH3
NHCH2C6H5
OCH3
13e: R1=
14e: R1=
16i: R1= benzylamino, R2= cyclopentyl
O
O
O
16j: R1= 3,5-dimethoxyphenoxy, R2= isopropyl
16k: R1= 3,5-dimethoxyphenoxy, R2= cyclopentyl
OCH3
OCH3
OCH3
Scheme 2. Reagents and conditions: a) amine, EtOH, reflux (for 13a-13d) or Cs2CO3, 3,5-
dimethoxyphenol, THF, reflux (for 13e); b) H2, Pd/C, EtOH, 33-55psi, r.t.; c) triethyl orthoformate, HCl
(36%), r.t.; d) i) K2CO3, DMF, r.t., ii) isopropyl bromide (for 16a, 16d, 16f, 16h, 16j) or cyclopentyl
bromide (for 16b, 16e, 16g, 16i, 16k) or 4-methoxybenzyl chloride (for 16c), r.t.
From the cyclization of compound 14d, we have also isolated the 7-amino-3-benzyl
derivative 15d1 (Figure 2) in 10% yield, which has obviously resulted upon ring-closure of 2 and
3 aminogroups of 14d.
OCH3
H3CO
O
R
N
N
N
N
N
N
NH2
17a: R= CH(CH3)2
17b: R=C5H9
15d1
Figure 2. By-product from the cyclization of 14d and regio-isomers of 16j-k.

The appropriate alkyl-group was finally inserted at position 1 of the imidazopyridines 15,
resulting into the target compounds 16a-k. In the case of the 4-(3,5-dimethoxyphenoxy)
derivative 15e, the regio-isomers 17a,b (Figure 2), were isolated, together with the
corresponding 1-substituted derivatives 16j,k.
The 4-benzylaminoderivatives 15d, 16h and 16i, were converted to the corresponding 4-
aminoderivatives 18a-c, upon treatment with ammonium formate in the presence of palladium on
carbon as catalyst.
NHCH2C6H5
N
NH2
N
N
N
a
N
N
R1
R1
15d: R1= H
18a: R1=H
16h: R1= CH(CH3)2
16i: R1= C5H9
18b: R1= CH(CH3)2
18c: R1= C5H9
Scheme 3. Reagents and conditions: a) HCOONH4, Pd/C, MeOH, reflux.
In parallel, and in order to extract more accurate structure-activity relationships, we have
also included into the biological evaluation tests adenine (19), as well as a number of selected 6-
aminosubstituted purines (20-22, Figure 3). Compounds 20-22 have been previously reported
and were prepared from 6-chloropurine.22, 23 We have synthesized those derivatives in almost
quantitative yield, following a slightly modified and convenient procedure, by refluxing 6-
chloropurine in ethanol with two equivalents of the suitable amine.
N
N
O
N
NH2
HN
N
N
N
N
N
N
N
N
N
NH
NH
NH
NH
N
N
N
19
20
21
22
Figure 3. Adenine and aminosubstituted purines.

The prepared compounds were tested, by direct in vivo transport assays, for their potential
to inhibit FcyB-mediated uptake of radiolabelled hypoxanthine or adenine.
2.3. Biological studies
2.3.1. Competition at FcyB and AzgA transporter by the synthesized derivatives
3
The new derivatives were tested as competitive inhibitors of FcyB-mediated H-adenine
uptake. As a control for testing whether these compounds are, as expected, specific for FcyB, we
3
also tested the same purine analogues as competitive inhibitors of AzgA-mediated H-adenine
uptake. In both cases, assays were performed in strains expressing solely the transporter studied
in each case, that is, in the absence of all other functionally relative nucleobase/nucleoside-
related transporters. Τhese strains (i.e. Δ7::FcyB and Δ7::AzgA), were constructed by selecting
for genetic transformants of a Δ7 mutant strain, arising from single-copy integration events of
plasmids carrying either the fcyB or the azgA gene. Transformant selection was based on
standard complementation of auxotrophic markers, PCR and southern analysis (for details see
Experimental procedures). In the case of FcyB, the transporter message is transcribed via the
medium-strength promoter of the uapA gene, rather than its own native promoter, in order to
achieve relatively higher protein expression levels necessary for uptake assays. The selected
transformants could both grow on purines as sole nitrogen sources in the medium. As expected,
Δ7::FcyB transformant was also sensitive to 5-FU, whereas the Δ7::AzgA transformant was
sensitive to 8-azaguanine.
Δ7::FcyB and Δ7::AzgA strains were used to perform competitive inhibition assays of
3
either FcyB-mediated or AzgA-mediated H-adenine uptake, using the synthesized derivatives.
Competition was carried out at 1000-fold excess inhibitors in order to identify compounds that
have even low binding affinity for FcyB or AzgA. Results are shown in Figures 4 and 5
respectively. Six deazapurine analogues, 16e, 16f, 16g, 16h, 16i and 18c competed very
3
efficiently FcyB-mediated H-adenine uptake (i.e. <10% transport compared to the non-
inhibited), but had no effect on AzgA-mediated 3H-adenine uptake rate (compare results in Fig. 4
and 5). The same result was obtained when analogues were tested as inhibitors of FcyB-mediated
3
or AzgA-mediated H-hypoxanthine uptake (results not shown). These six analogues did not
inhibit at all UapA, the third major purine transporter specific for xanthine or uric acid (results
not shown).

110
100
90
80
70
60
50
40
30
20
10
0
Figure 4. Competition of FcyB-mediated [3H]-adenine uptake by 1000-fold excess (0.5 mM) of unlabeled
purine analogues. Compounds exhibiting inhibitory activity more than 90% are depicted in red.
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
Figure 5. Competition of AzgA-mediated [3H]-adenine uptake by 1000-fold excess (0.5 mM) of
unlabelled purine analogues.
2.3.2. Kinetic characterization of competing analogues
We estimated the Ki values, using IC50 measurements,13 of the six analogues, which
specifically inhibit FcyB transport. Results are shown in Table 1. All Kis were in the 5-95 μM
range, with analogue 16h showing the highest affinity binding in FcyB (5 μΜ).

compound
16e
Ki (μΜ)
38±6
72±7
95±9
5±1
16f
16g
16h
21±4
18±2
16i
18c
Table 1. Substrate binding specificity profile of FcyB. Results are averages of at least three independent
experiments with three replicates for each concentration point.
2.4. Structure-activity relationship study
From the data presented in Figure 4 it is obvious that only the 4-amino substituted
compounds possess considerable biological activity. This is confirmed by the fact that 4-
chlorosubstituted derivatives 11a-c, as well as the corresponding imidazopyridinones 9 and
12a,b are devoid of activity. A crucial structural feature of the most potent derivatives is the
simultaneous 1- and 4- substitution since compounds 16 are considerably more active than their
mono-substituted counterparts 15, with 16h and 16i being the most active analogues. It seems
that N1-cyclopentyl substitution is preferable over the corresponding N1-isopropyl substitution.
Another important finding is that among the 4-alkylamino substituted derivatives the existence of
4-NH (compounds 16f-i) is in favor of the transporter inhibitory activity. Concerning the
remaining aminosubstituted derivatives, only the piperazine analogue 16e possesses a certain
degree of activity although moderate; however, this derivative bears a N1-cyclopentyl moiety as
well. Finally, it should be noted that although the number of derivatives is limited, our data
concerning adenine and 3-deazaadenine suggest that the 3-nitrogen is not crucial for this kind of
protein ligand interaction, since the new imidazopyridines exhibited comparable activity with the
purine derivatives 20-22. However, this would request further investigation.
2.5. Molecular modeling
Molecular simulations suggest that active compounds can interact with FcyB through
residue Asn163. This residue and Pro353 were shown to be irreplaceable for FcyB-mediated
transport.15 More specifically Asn163 proved to be critical for determining the substrate binding
affinity and/or specificity of FcyB, without affecting protein stability. All active compounds
interact with FcyB through the formation of hydrogen bonds with Asn163. The insertion of a
hydrophobic fragment at position 1 of 3-deazaadenine (18b, 18c) enhanced the inhibition,
especially for the cyclopentyl moiety as already mentioned. Those analogs bind at the same
position as adenine and the hydrophobic fragment is placed near hydrophobic region TMS1α.
(data not shown). The docking structures of the most active compounds 16h and 16f form a

bidentate hydrogen bond (HB) via C4-NH and N3 of the ligand underlining the importance of
the presence of NH at position C4 (Fig. 6). In both structures the isopropyl moiety is
accommodated between Pro353, Trp77 and Tyr262 filling the space in the hydrophobic cavity.
In the case of 16f (Fig. 6B) an extra salt bridge appears possible between the tertiary amine
positive charge and Glu397 carboxylate while in 16h (Fig 6A) the phenyl group seems to exhibit
hydrophobic interactions with Val84, Ala162, Val166. On all minima structures π-π stacking
interactions with Trp159 and Trp259 are also very important.
A
B
Figure 6. Minimum energy structures of FcyB in complex with compound 16h (A) and 16f (B).
Hydrogen bonds are shown with red dashed lines.
3. Conclusion
In conclusion, we have designed and synthesized a number of 3-deazapurines substituted
in the corresponding 6- and 9-positions of the original purine scaffold. These compounds were
tested in substrate competition assays related to FcyB and AzgA transporters, both of which
recognize and transport purines with high affinities at the low μΜ range. A number of the tested
3-deazapurines was found to be specific solely for FcyB. The Ki values of FcyB-specific
substrates/ligands were determined. Given that the synthesized 3-deazapurines were designed
rationally based on the interaction of purine substrates with FcyB, our results show that our
approach can be used successfully to design substrates/ligands highly specific for a given
nucleobase transporter. The importance of these findings is apparent for the future design of
compounds recognized specifically by all fungal, but not by host, transporters.
4. Experimental section

4.1. Chemistry
4.1.1. General
1
Melting points were determined on a Büchi apparatus and are uncorrected. H NMR spectra and
2D spectra were recorded on a Bruker Avance III 600 or a Bruker Avance DRX 400 instrument,
whereas 13C NMR spectra were recorded on a Bruker Avance III 600 or a Bruker AC 200
spectrometer in deuterated solvents and were referenced to TMS (δ scale). The signals of 1H and
1
13C spectra were unambiguously assigned by using 2D NMR techniques: H1H COSY, NOESY,
HMQC, and HMBC. Mass spectra were recorded with a LTQ Orbitrap Discovery instrument,
possessing an Ionmax ionization source. Flash chromatography was performed on Merck silica
gel 60 (0.040–0.063 mm). Analytical thin layer chromatography (TLC) was carried out on
precoated (0.25 mm) Merck silica gel F-254 plates. The purity of all the synthesized compounds
was >95% as ascertained by elemental analysis. Elemental analyses were undertaken using a
PerkinElmer PE 240C elemental analyzer (Norwalk, CT, U.S.) and the measured values for C, H,
and N were within ±0.4% of the theoretical values.
4.1.2. General procedure for the synthesis of compounds 10a-c and 11a-c
Potassium carbonate (1.9 mmol) was added into a solution of compound 8 (1.3 mmol) in dry
DMF (5 mL) under argon, and this mixture was stirred at room temperature for 20 min.
Subsequently, the suitable halide (isopropyl bromide, cyclopentyl bromide, 4-methoxybenzyl
chloride, 1.9 mmol) was added and the reaction was stirred at room temperature for 72h. Then,
the solvent was removed in vacuo and water was added to the residue, followed by extraction
with chloroform (3x50 mL). The combined organic extracts were dried over sodium sulfate,
filtered and the solvent was removed under reduced pressure. The 1- and 3- substituted isomers
were chromatographically separated.
4.1.2.1. 4-Chloro-3-isopropyl-3H-imidazo[4,5-c]pyridine (10a) and 4-chloro-1-isopropyl-
1H-imidazo[4,5-c]pyridine (11a).
These derivatives were synthesized according to general procedure described above, upon
reaction of compound 8 with isopropyl bromide. Purification was effected using a mixture of
cyclohexane / ethyl acetate (7/3, v/v) as the eluent, to provide pure 10a and 11a.
1
Data for 10a: Yield 24%. White solid, mp 93 ˚C (Et2O). H NMR (600 MHz, CDCl3) δ 8.16 (s,
1H, H-2), 8.14 (d, 1H, J=5.4Hz, H-6), 7.59 (d, 1H, J=5.4Hz, H-7), 5.38 (septet, 1H, J=6.6Hz,
CH(CH3)2), 1.62 (d, 6H, J=6.6Hz, 2xCH3). 13C NMR (151 MHz, CDCl3) δ 151.44 (C-7a),
143.62 (C-2), 140.88 (C-6), 133.61 (C-4), 127.91 (C-3a), 114.97 (C-7), 49.86 (CH(CH3)2), 23.92
(2xCH3). HR-MS (ESI) m/z: Calcd for C9H11ClN3: [M1+H]+ =196.0636, found 196.0639. Anal.
Calcd for C9H10ClN3: C, 55.25; H, 5.15; N, 21.48. Found: C, 55.39; H, 5.21; N, 21.38.
Data for 11a: Yield 76%. White solid, mp 59 ˚C (n-pentane). 1H NMR (600 MHz, CDCl3) δ
8.05 (d, 1H, J=5.5Hz, H-6), 8.02 (s, 1H, H-2), 7.27 (d, 1H, J=5.5Hz, H-7), 4.59 (septet, 1H,
J=6.6Hz, CH(CH3)2), 1.54 (d, 6H, J=6.6Hz, 2xCH3). 13C NMR (151 MHz, CDCl3) δ 142.48 (C-
4), 141.91 (C-6), 140.70 (C-2), 139.03 (C-7a), 137.44 (C-3a), 105.61 (C-7), 48.75 (CH(CH3)2),

22.29 (2xCH3). HR-MS (ESI) m/z: Calcd for C9H11ClN3: [M1+H]+ =196.0636, found 196.0641.
Anal. Calcd for C9H10ClN3: C, 55.25; H, 5.15; N, 21.48. Found: C, 55.44; H, 5.23; N, 21.30.
4.1.2.2.
4-Chloro-3-cyclopentyl-3H-imidazo[4,5-c]pyridine (10b)
and 4-chloro-1-
cyclopentyl-1H-imidazo[4,5-c]pyridine (11b).
These derivatives were synthesized according to general procedure described above, upon
reaction of compound 8 with cyclopentyl bromide. Purification was effected using a mixture of
chloroform / methanol (100/1, v/v) as the eluent, to provide pure 10b and 11b.
1
Data for 10b: Yield 17%. White solid, mp 77-8 ˚C (Et2O). H NMR (600 MHz, CDCl3) δ 8.20
(d, 1H, J=5.3Hz, H-6), 8.14 (s, 1H, H-2), 7.64 (d, 1H, J=5.3Hz, H-7), 5.50 (m, 1H, H-1’), 2.40-
2.32 (m, 2H, cyclopentyl-H), 2.05-1.97 (m, 2H, cyclopentyl-H), 1.94-1.83 (m, 4H, cyclopentyl-
H). 13C NMR (151 MHz, CDCl3) δ 151.57 (C-4), 144.07 (C-6), 140.94 (C-2), 133.91 (C-3a),
128.46 (C-7a), 114.98 (C-7), 58.43 (C-1’), 34.06 (C-2’, C-5’), 23.77 (C-3’, C-4’). HR-MS (ESI)
m/z: Calcd for C11H13ClN3: [M1+H]+ =222.0793, found 222.0797. Anal. Calcd for C11H12ClN3:
C, 59.60; H, 5.46; N, 18.96. Found: C, 59.75; H, 5.53; N, 18.78.
1
Data for 11b: Yield 76%. White solid, mp 87-8 ˚C (Et2O). H NMR (600 MHz, CDCl3) δ 8.20
(d, 1H, J=5.6Hz, H-6), 8.05 (s, 1H, H-2), 7.33 (d, 1H, J=5.6Hz, H-7), 4.74 (m, 1H, H-1’), 2.36-
2.30 (m, 2H, cyclopentyl-H), 2.06-1.99 (m, 2H, cyclopentyl-H), 1.97-1.92 (m, 2H, cyclopentyl-
H), 1.89-1.83 (m, 2H, cyclopentyl-H). 13C NMR (151 MHz, CDCl3) δ 142.71 (C-7a), 142.49 (C-
2), 140.88 (C-6), 139.66 (C-4), 137.97 (C-3a), 105.87 (C-7), 57.79 (C-1’), 32.31 (C-2’, C-5’),
23.73 (C-3’, C-4’). HR-MS (ESI) m/z: Calcd for C11H13ClN3: [M1+H]+ =222.0793, found
222.0799. Anal. Calcd for C11H12ClN3: C, 59.60; H, 5.46; N, 18.96. Found: C, 59.49; H, 5.40; N,
19.03.
4.1.2.3. 4-Chloro-3-(4-methoxybenzyl)-3H-imidazo[4,5-c]pyridine (10c) and 4-chloro-1-(4-
methoxybenzyl)-1H-imidazo[4,5-c]pyridine (11c).
These derivatives were synthesized according to general procedure described above, upon
reaction of compound 8 with 4-methoxybenzyl chloride. Purification was effected using a
mixture of dichloromethane / methanol (100/1, v/v) as the eluent, to provide pure 10c (yield
18%) and 11c (yield 45%). Compound 11c has previously been reported.24
1
Data for 10c: White solid, mp 133-4 ˚C (Et2O). H NMR (600 MHz, CDCl3) δ 8.25 (d, 1H,
J=4.5Hz, H-6), 8.10 (s, 1H, H-2), 7.70 (d, 1H, J=4.5Hz, H-7), 7.17 (d, 2H, J=8.5Hz, H-2’, H-6’),
6.91 (d, 2H, J=8.5Hz, H-3’, H-5’), 5.70 (CH2), 3.81 (OCH3). 13C NMR (151 MHz, CDCl3) δ
160.17 (C-4’), 150.16 (C-4), 146.74 (C-2), 141.65 (C-6), 128.93 (C-2’, C-6’), 126.93 (C-3a),
126.74 (C-1’), 114.74 (C-3’, C-5’), 114.62 (C-7a), 114.57 (C-7), 55.38 (OCH3), 50.25 (CH2).
HR-MS (ESI) m/z: Calcd for C14H13ClN3O: [M1+H]+ =274.0742, found 274.0745. Anal. Calcd
for C14H12ClN3O: C, 61.43; H, 4.42; N, 15.35. Found: C, 61.62; H, 4.51; N, 15.17.

4.1.3. General procedure for the synthesis of compounds 12a-b.
Concentrated hydrochloric acid (1 mL) was added dropwise into a solution of the corresponding
chloroderivative 11 (0.25 mmol) in a mixture of ethanol (2.0 mL) and water (2.0 mL), and this
reaction mixture was refluxed for 72 h. Then the organic solvent was removed in vacuo, the
residue was neutralized with sodium bicarbonate and extracted with ethyl acetate. The combined
organic layers were dried over sodium sulfate and the solvent was removed under reduced
pressure. The residue was purified by column chromatography (silica gel) to provide pure
derivatives 12a and 12b.
4.1.3.1. 1-Isopropyl-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one (12a).
This compound was synthesized according to general procedure described above, starting from
11a. Purification was effected using a mixture of dichloromethane / methanol (9/1, v/v) as the
1
eluent to provide pure 12a as a beige solid, in 16% yield. Mp 122-3 ˚C (MeOH). H NMR (400
MHz, DMSO-d6) δ 11.15 (s, 1H, NH), 8.12 (s, 1H, H-2), 7.15 (d, J=6.5 Hz, 1H, H-6), 6.62 (d,
13
J=6.5 Hz, 1H, H-7), 4.60 (septet, J=6.8 Hz, 1H, CH(CH3)2), 1.47 (d, J=6.8 Hz, 6H, CH3). C
NMR (151 MHz, DMSO-d6) δ 158.15 (C-4), 138.69 (C-2), 138.21 (C-7a), 131.50 (C-3a), 129.08
(C-6), 93.00 (C-7), 47.66 (CH(CH3)2), 22.46 (CH3). HR-MS (ESI) m/z: Calcd for C9H12N3O:
[M1+H]+ =178.0975, found 178.0980. Anal. Calcd for C9H11N3O: C, 61.00; H, 6.26; N, 23.71.
Found: C, 60.83; H, 6.20; N, 23.89.
4.1.3.2. 1-Cyclopentyl-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one (12b).
This compound was synthesized according to general procedure described above, starting from
11b. Purification was effected using a mixture of dichloromethane / methanol (95/5, v/v) as the
1
eluent to provide pure 12b as a beige solid, in 27% yield. Mp ˃250 ˚C (MeOH). H NMR (400
MHz, DMSO-d6) δ 11.17 (s, 1H, NH), 8.09 (s, 1H, H-2), 7.15 (d, 1H, J=7.1 Hz, H-6), 6.60 (d,
1H, J=7.1 Hz, H-7), 4.72 (m, 1H, H-1’), 2.17 (m, 2H, cyclopentyl-H), 1.84 (m, 4H, cyclopentyl-
H), 1.69 (m, 2H, cyclopentyl-H). 13C NMR (151 MHz, DMSO-d6) δ 158.06 (C-4), 138.96 (C-2),
138.73 (C-7a), 131.58 (C-3a), 129.08 (C-6), 92.98 (C-7), 56.50 (C-1’), 32.16 (C-2’, C-5’), 23.46
(C-3’, C-4’). HR-MS (ESI) m/z: Calcd for C11H14N3O: [M1+H]+ =204.1131, found 204.1138.
Anal. Calcd for C11H13N3O: C, 65.01; H, 6.45; N, 20.68. Found: C, 64.79; H, 6.28; N, 20.91.
4.1.4. General procedure for the synthesis of compounds 13a-d.
The suitable amine (6.3 mmol) was added into a solution of compound 5 (2.9 mmol) in absolute
ethanol (10 mL) and this mixture was refluxed for 2h. Upon completion of the reaction, the
organic solvent was removed under reduced pressure, water was added to the residue and the
precipitate was filtered in vacuo, washed with water and air-dried, to provide the pure
aminosubstituted derivatives 13a-d.

4.1.4.1. 2-(Morpholin-4-yl)-3-nitropyridin-4-amine (13a).
This compound was synthesized according to general procedure described above in 80% yield,
upon treatment of the chloroderivative 5 with morpholine. Yellow solid, mp 131-2 ˚C
(CHCl3/Et2O). 1H NMR (600 MHz, DMSO-d6) δ 7.67 (d, 1H, J=4.5Hz, H-6), 7.28 (brs, 2H, D2O
exch, NH2), 6.25 (d, 1H, J=4.5Hz, H-5), 3.61 (m, 4H, H-2’, H-6’), 3.25 (m, 4H, H-3’, H-5’). 13C
NMR (151 MHz, DMSO-d6) δ 154.75 (C-2), 151.11 (C-4), 148.60 (C-6), 119.37 (C-3), 103.35
(C-5), 65.89 (C-2’, C-6’), 47.91 (C-3’, C-5’). HR-MS (ESI) m/z: Calcd for C9H13N4O3: [M1+H]+
=225.0982, found 225.0979. Anal. Calcd for C9H12N4O3: C, 48.21; H, 5.39; N, 24.99. Found: C,
48.05; H, 5.30; N, 25.14.
4.1.4.2. 2-(4-Methylpiperazin-1-yl)-3-nitropyridin-4-amine (13b).
This compound was synthesized according to general procedure described above in 73% yield,
upon treatment of the chloroderivative 5 with N-methylpiperazine. Yellow solid, mp 166-7 ˚C
1
(EtOAc). H NMR (600 MHz, DMSO-d6) δ 7.65 (d, 1H, J=5Hz, H-6), 7.24 (brs, 2H, D2O exch,
NH2), 6.20 (d, 1H, J=5Hz, H-5) 3.25 (m, 4H, H-2’, H-6’), 2.32 (m, 4H, H-3’, H-5’), 2.18 (s, 3H,
NCH3). 13C NMR (151 MHz, DMSO-d6) δ 154.75 (C-2), 151.05 (C-4), 148.61 (C-6), 119.22 (C-
3), 102.87 (C-5), 54.36 (C-3’, C-5’), 47.26 (C-2’, C-6’), 45.68 (NCH3). HR-MS (ESI) m/z: Calcd
for C10H16N5O2: [M1+H]+ =238.1299, found 238.1292. Anal. Calcd for C10H15N5O2: C, 50.62;
H, 6.37; N, 29.52. Found: C, 50.82; H, 6.48; N, 29.31.
4.1.4.3. N2-[2-(Dimethylamino)ethyl]-3-nitropyridine-2,4-diamine (13c).
This compound was synthesized according to general procedure described above in 77% yield,
upon treatment of the chloroderivative 5 with N,N-dimethylethylenediamine. Yellow solid, mp
120-1 ˚C (EtOAc/Et2O). 1H NMR (600 MHz, DMSO-d6) δ 8.90 (t, 1H, J=4.6Hz, D2O exch, NH),
8.09 (brs, 2H, D2O exch, NH2), 7.64 (d, 1H, J=5.8Hz, H-6), 6.09 (d, 1H, J=5.8Hz, H-5), 3.51 (q,
2H, J=6.1Hz, H-2’), 2.44 (t, 2H, J=6.1Hz, H-3’), 2.17 (s, 6H, 2xCH3). 13C NMR (151 MHz,
DMSO-d6) δ 154.13 (C-4), 153.41(C-2), 151.32(C-6), 116.14(C-3), 101.13(C-5), 57.50 (C-3’),
45.13 (2xCH3), 38.87 (C-2’). HR-MS (ESI) m/z: Calcd for C9H16N5O2: [M1+H]+ =226.1299,
found 226.1296. Anal. Calcd for C9H15N5O2: C, 47.99; H, 6.71; N, 31.09. Found: C, 48.26; H,
6.89; N, 30.78.
4.1.4.4. N2-Benzyl-3-nitropyridine-2,4-diamine (13d).
This compound was synthesized according to general procedure described above in 96% yield,
upon treatment of the chloroderivative 5 with benzylamine. Yellow solid, mp 111-2 ˚C
1
(CH2Cl2/n-pentane). H NMR (400 MHz, DMSO-d6) δ 9.15 (m, 1H, D2O exch, NH), 8.12 (brs,
2H, D2O exch, NH2), 7.62 (d, 1H, J=5.7Hz, H-6), 7.35-7.20 (m, 5H, phenyl-H), 6.11 (d, 1H,
13
J=5.7Hz, H-5), 4.68 (d, 2H, J=5.7Hz, CH2). C NMR (151 MHz, DMSO-d6) δ 153.81 (C-2),
153.16 (C-4), 150.90 (C-6), 139.58 (C-1’), 128.19 (C-3’, C-5’), 127.14 (C-2’, C-6’), 126.59 (C-
4’), 116.01 (C-3), 101.34 (C-5), 44.10 (CH2). HR-MS (ESI) m/z: Calcd for C12H13N4O2:

[M1+H]+ =245.1033, found 245.1037. Anal. Calcd for C12H12N4O2: C, 59.01; H, 4.95; N, 22.94.
Found: C, 58.88; H, 4.89; N, 23.13.
4.1.5. 2-(3,5-Dimethoxyphenoxy)-3-nitropyridin-4-amine (13e).
3,5-Dimethoxyphenol (450 mg, 2.9 mmol) and cesium carbonate (940 mg, 2.9 mmol) were
added into a solution of compound 5 (500 mg, 2.9 mmol) in tetrahydrofuran (20 mL), under
argon, and this mixture was heated at 70˚C for 12h. Upon completion of the reaction, the organic
solvent was removed under reduced pressure, water was added to the residue and the precipitate
was filtered in vacuo, washed with water and air-dried, to provide the pure phenoxy derivative
13e, as a pale yellow solid in 89% yield. Mp 166-7 ˚C (EtOAc). 1H NMR (600 MHz, DMSO-d6)
δ 7.64 (d, 1H, J=5.7Hz, H-6), 7.43 (brs, 2H, D2O exch, NH2), 6.59 (d, 1H, J=5.7Hz, H-5), 6.35
(s, 1H, H-4’), 6.27 (s, 2H, H-2’, H-6’), 3.71 (s, 6H, 2xOCH3). 13C NMR (151 MHz, DMSO-d6) δ
160.89 (C-3’, C-5’), 156.49 (C-2), 154.90 (C-4), 150.77 (C-1’), 147.39 (C-6), 120.61 (C-3),
108.63 (C-5), 99.98 (C-2’, C-6’), 97.07 (C-4’), 55.43 (2xOCH3). HR-MS (ESI) m/z: Calcd for
C13H14N3O5: [M1+H]+ =292.0928, found 292.0924. Anal. Calcd for C13H13N3O5: C, 53.61; H,
4.50; N, 14.43. Found: C, 53.78; H, 4.55; N, 14.37.
4.1.6. General procedure for the synthesis of aminoderivatives 14a-e.
A solution of the nitro derivatives 13a-e (2.0 mmol) in absolute ethanol (60 mL) was
hydrogenated in the presence of 10% Pd/C (90 mg) under a pressure of 33 psi for 14d and 55 psi
for the rest compounds, at room temperature for 4h. The solution was filtered through a celite
pad to remove the catalyst and the filtrate was evaporated to dryness. The diaminoderivatives
14a-e were used immediately to the next step, with no further purification.
4.1.7. General procedure for the synthesis of imidazopyridines 15a-e.
Concentrated hydrochloric acid (0.3 mL) was added dropwise into a suspension of the diamines
14a-e (2.0 mmol) in triethyl orthoformate (5 mL), under argon, and this reaction mixture was
stirred at room temperature for 14h. The excess of triethyl orthoformate was removed under
reduced pressure, the residue was dissolved in methanol, neutralized with sodium bicarbonate
and then purified by column chromatography (silica gel) to provide pure derivatives 15a-e.
4.1.7.1. 4-(Morpholin-4-yl)-1H-imidazo[4,5-c]pyridine (15a).
This compound was synthesized according to the general procedure described above, starting
from 14a. Purification was effected using a mixture of dichloromethane / methanol (9/1, v/v) as
the eluent to provide pure 15a as a white solid, in 92% yield. The spectroscopic data of this
derivative have already been referred.25

4.1.7.2. 4-(4-Methylpiperazin-1-yl)-1H-imidazo[4,5-c]pyridine (15b).
This compound was synthesized according to the general procedure described above, starting
from 14b. Purification was effected using a mixture of dichloromethane / methanol (9/1, v/v) as
1
the eluent to provide pure 15b as a white solid, in 99% yield. Mp 234-5 ˚C (EtOH/Et2O). H
NMR (400 MHz, DMSO-d6) δ 12.61 (brs, 1H, D2O exch, NH), 8.10 (s, 1H, H-2), 7.76 (d, 1H,
J=5.5Hz, H-6), 6.86 (d, 1H, J=5.5Hz, H-7), 4.05 (m, 4H, H-2’, H-6’), 2.43 (m, 4H, H-3’, H-5’),
2.22 (s, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 151.45 (C-4), 139.90 (C-6), 139.55 (C-7a),
139.23 (C-2), 128.12 (C-3a), 99.48 (C-7), 54.97 (C-3’, C-5’), 45.81 (C-2’, C-6’), 45.96 (CH3).
HR-MS (ESI) m/z: Calcd for C11H16N5: [M1+H]+ =218.1400, found 218.1397. Anal. Calcd for
C11H15N5: C, 60.81; H, 6.96; N, 32.23. Found: C, 60.93; H, 7.02; N, 32.02.
4.1.7.3. 4-[(2-Dimethylamino)ethylamino]-1H-imidazo[4,5-c]pyridine (15c).
This compound was synthesized according to the general procedure described above, starting
from 14c. Purification was effected using a mixture of dichloromethane / methanol /
triethylamine (8/2/0.5, v/v/v) as the eluent to provide pure 15c as a white solid, in 60% yield. Mp
1
286-7 ˚C (MeOH/EtOAc). H NMR (600 MHz, DMSO-d6) δ 10.08 (brs, 1H, D2O exch, NH-1),
8.99 (brs, 1H, D2O exch, NHCH2), 8.46 (s, 1H, H-2), 7.70 (d, 1H, J=6.8Hz, H-6), 7.20 (d, 1H,
J=6.8Hz, H-7), 4.08 (m, 2H, H-2’), 3.43 (m, 2H, H-3’), 2.86 (s, 6H, 2xCH3). 13C NMR (151
MHz, DMSO-d6) δ 147.18 (C-4), 143.56 (C-2), 139.76 (C-7a), 129.59 (C-6), 126.26 (C-3a),
100.95 (C-7), 55.52 (C-3’), 42.98 (2xCH3), 37.71 (C-2’). HR-MS (ESI) m/z: Calcd for C10H16N5:
[M1+H]+ =206.1400, found 206.1393. Anal. Calcd for C10H15N5: C, 58.51; H, 7.37; N, 34.12.
Found: C, 58.68; H, 7.45; N, 33.83.
4.1.7.4. N-Benzyl-1H-imidazo[4,5-c]pyridin-4-amine (15d) and 3-benzyl-3H-imidazo[4,5-
b]pyridin-7-amine (15d1).
These compounds were obtained according to the general procedure described above, starting
from 14d. Purification was effected using a mixture of dichloromethane / methanol (100/2, v/v)
as the eluent to provide pure 15d (82% yield) and 15d1 (10% yield) as white solids. The
spectroscopic data of 15d have already been referred.26
Data for 15d1: Mp 168-9 ˚C (MeOH). 1H NMR (600 MHz, DMSO-d6) δ 8.19 (s, 1H, H-2), 7.89
(d, 1H, J=5.5Hz, H-5), 7.34-7.25 (m, 5H, phenyl-H), 6.37 (d, 1H, J=5.5Hz, H-6), 6.33 (brs, D2O
exch, 2H, NH2), 5.38 (s, 2H, CH2). 13C NMR (151 MHz, DMSO-d6) δ 147.14 (C-3a), 146.77 (C-
7), 144.77 (C-5), 140.37 (C-2), 137.64 (C-1’), 128.53 (C-2’, C-6’), 127.50 (C-4’), 127.44 (C-3’,
C-5’), 122.68 (C-7a), 102.03 (C-6), 45.92 (CH2). HR-MS (ESI) m/z: Calcd for C13H13N4:
[M1+H]+ =225.1135, found 225.1131. Anal. Calcd for C13H12N4: C, 69.62; H, 5.39; N, 24.99.
Found: C, 69.51; H, 5.30; N, 25.11.

4.1.7.5. 4-(3,5-Dimethoxyphenoxy)-1H-imidazo[4,5-c]pyridine (15e).
This compound was synthesized according to the general procedure described above, starting
from 14e. Purification was effected using a mixture of dichloromethane / methanol (100/2, v/v)
as the eluent to provide pure 15e as a white solid, in 77% yield. Mp 211-2 ˚C (MeOH). 1H NMR
(400 MHz, DMSO-d6) δ 8.35 (s, 1H, H-2), 7.81 (d, 1H, J=5.5Hz, H-6), 7.35 (d, 1H, J=5.5Hz, H-
7), 6.38-6.36 (m, 1H, H-4’), 6.38-6.34 (m, 2H, H-2’, H-6’), 3.73 (s, 6H, 2xOCH3). 13C NMR
(151 MHz, DMSO-d6) δ 161.08 (C-3’, C-5’), 156.00 (C-1’), 154.19 (C-4), 142.70 (C-2), 141.08
(C-7a), 138.58 (C-6), 128.52 (C-3a), 104.68 (C-7), 99.88 (C-2’, C-6’), 96.46 (C-4’), 55.50
(2xOCH3). HR-MS (ESI) m/z: Calcd for C14H14N3O3: [M1+H]+ =272.1030, found 272.1024.
Anal. Calcd for C14H13N3O3: C, 61.99; H, 4.83; N, 15.49. Found: C, 62.22; H, 4.96; N, 15.30.
4.1.8. General procedure for the synthesis of compounds 16a-k and 17a-b.
These compounds were synthesized following an analogous synthetic procedure to the one
described for the synthesis of compounds 10a-c and 11a-c, starting from the imidazopyridines
15a-e.
4.1.8.1. 1-Isopropyl-4-(morpholin-4-yl)-1H-imidazo[4,5-c]pyridine (16a).
This compound was synthesized according to the general procedure described above, upon
reaction of 15a with isopropyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (100/1, v/v) as the eluent to provide pure 16a as a white solid, in
1
53% yield. Mp 75-6 ˚C (n-pentane). H NMR (600 MHz, CDCl3) δ 7.93 (d, 1H, J=5.7Hz,H-6),
7.79 (s, 1H, H-2), 6.75 (d, 1H, J=5.7Hz, H-7), 4.52 (septet, 1H, J=6.8Hz, CH(CH3)2), 4.11 (m,
4H, H-3’’, H-5’’), 3.86 (m, 4H, H-2’’, H-6’’), 1.56 (d, 6H, J=6.8Hz, 2xCH3). 13C NMR (50
MHz, CDCl3) δ 151.30 (C-4), 139.60 (C-7a), 138.73 (C-6), 137.26 (C-2), 128.78 (C-3a), 97.86
(C-7), 67.10 (C-2’’, C-6’’), 48.13 (CH(CH3)2), 47.22 (C-3’’, C-5’’), 22.55 (2xCH3). HR-MS
(ESI) m/z: Calcd for C13H19N4O: [M1+H]+ =247.1553, found 247.1558. Anal. Calcd for
C13H18N4O: C, 63.39; H, 7.37; N, 22.75. Found: C, 63.56; H, 7.51; N, 22.52.
4.1.8.2. 1-Cyclopentyl-4-(morpholin-4-yl)-1H-imidazo[4,5-c]pyridine (16b).
This compound was synthesized according to the general procedure described above, upon
reaction of 15a with cyclopentyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (98/2, v/v) as the eluent to provide pure 16b as a white solid, in 90%
1
yield. Mp 97-8 ˚C (EtOAc/n-pentane). H NMR (400 MHz, CDCl3) δ 7.92 (d, 1H, J=5.1Hz, H-
6), 7.76 (s, 1H, H-2), 6.76 (d, 1H, J=5.1Hz, H-7), 4.62 (m, 1H, H-1’), 4.20 (m, 4H, H-3’’, H-5’’),
3.85 (m, 4H, H-2’’, H-6’’), 2.27-2.17 (m, 2H, cyclopentyl-H), 2.00-1.92 (m, 2H, cyclopentyl-H),
1.91-1.84 (m, 2H, cyclopentyl-H), 1.82-1.73 (m, 2H, cyclopentyl-H). 13C NMR (50 MHz,
CDCl3) δ 140.66 (C-4), 140.06 (C-7a), 139.92 (C-6), 137.46 (C-2), 129.04 (C-3a), 98.01 (C-7),
67.18 (C-2’’, C-6’’), 57.13 (C-1’), 46.96 (C-3’’, C-5’’), 32.36 (C-2’, C-5’), 23.81 (C-3’, C-4’).
HR-MS (ESI) m/z: Calcd for C15H21N4O: [M1+H]+ =273.1710, found 273.1717. Anal. Calcd for
C15H20N4O: C, 66.15; H, 7.40; N, 20.57. Found: C, 66.38; H, 7.54; N, 20.31.

4.1.8.3. 1-(4-Methoxybenzyl)-4-(morpholin-4-yl)-1H-imidazo[4,5-c]pyridine (16c).
This compound was synthesized according to the general procedure described above, upon
reaction of 15a with 4-methoxybenzyl chloride. Purification was effected using a mixture of
dichloromethane / methanol (100/1, v/v) as the eluent to provide pure 16c as a pale yellow oil, in
32% yield. 1H NMR (400 MHz, CDCl3) δ 7.87 (d, 1H, J=5.7Hz, H-6), 7.71 (s, 1H, H-2), 7.03 (d,
2H, J=8.7Hz, H-2’, H-6’), 6.80 (d, 2H, J=8.7Hz, H-3’, H-5’), 6.62 (d, 1H, J=5.7Hz, H-7), 5.13
13
(s, 2H, CH2), 4.09 (m, 4H, H-3’’, H-5’’), 3.82 (m, 4H, H-2’’, H-6’’), 3.72 (s, 3H, OCH3). C
NMR (50 MHz, CDCl3) δ 159.58 (C-4’), 151.98 (C-4), 140.32 (C-6), 140.12 (C-7a), 139.66 (C-
2), 128.84 (C-3a), 128.53 (C-2’, C-6’), 126.97 (C-1’), 114.41 (C-3’, C-5’), 97.63 (C-7), 67.14
(C-2’’, C-6’’), 55.26 (OCH3), 48.37 (CH2), 46.95 (C-3’’, C-5’’). HR-MS (ESI) m/z: Calcd for
C18H21N4O2: [M1+H]+ =325.1659, found 325.1664. Anal. Calcd for C18H20N4O2: C, 66.65; H,
6.21; N, 17.27. Found: C, 66.39; H, 6.04; N, 17.50.
4.1.8.4. 1-Isopropyl-4-(4-methylpiperazin-1-yl)-1H-imidazo[4,5-c]pyridine (16d).
This compound was synthesized according to the general procedure described above, upon
reaction of 15b with isopropyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (9/1, v/v) as the eluent to provide pure 16d as a white solid, in 84%
yield. Mp 168-9 ˚C (CH2Cl2). 1H NMR (600 MHz, CDCl3) δ 7.86 (d, 1H, J=5.7Hz, H-6), 7.73 (s,
1H, H-2), 6.66 (d, 1H, J=5.7Hz, H-7), 4.44 (septet, 1H, J=6.8Hz, CH(CH3)2), 4.13 (m, 4H, H-
13
2’’, H-6’’), 2.54 (m, 4H, H-3’’, H-5’’), 2.28 (s, 3H, NCH3), 1.49 (d, 6H, J=6.8Hz, 2xCH3). C
NMR (151 MHz, CDCl3) δ 152.09 (C-4), 140.13 (C-6), 139.36 (C-7a), 136.69 (C-2), 128.91 (C-
3a), 97.44 (C-7), 55.23 (C-3’’, C-5’’), 47.90 (CH(CH3)2), 46.10 (NCH3, C-2’’, C-6’’), 22.50
(2xCH3). HR-MS (ESI) m/z: Calcd for C14H22N5: [M1+H]+ =260.1870, found 260.1876. Anal.
Calcd for C14H21N5: C, 64.84; H, 8.16; N, 27.00. Found: C, 64.64; H, 8.11; N, 27.21.
4.1.8.5. 1-Cyclopentyl-4-(4-methylpiperazin-1-yl)-1H-imidazo[4,5-c]pyridine (16e).
This compound was synthesized according to the general procedure described above, upon
reaction of 15b with cyclopentyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (9/1, v/v) as the eluent to provide pure 16e as a white solid, in 45%
1
yield. Mp 192-3 ˚C (CHCl3/Et2O). H NMR (600 MHz, CDCl3) δ 7.90 (d, 1H, J=5.7Hz, H-6),
7.75 (s, 1H, H-2), 6.73 (d, 1H, J=5.7Hz, H-7), 4.61 (m, 1H, H-1’), 4.17 (m, 4H, H-2’’, H-6’’),
2.61 (m, 4H, H-3’’, H-5’’), 2.35 (s, 3H, NCH3), 2.25-2.15 (m, 2H, cyclopentyl-H), 1.98-1.91 (m,
2H, cyclopentyl-H), 1.89-1.82 (m, 2H, cyclopentyl-H), 1.81-1.73 (m, 2H, cyclopentyl-H). 13C
NMR (151 MHz, CDCl3) δ 152.08 (C-4), 140.17 (C-6), 139.97 (C-7a), 137.43 (C-2), 129.07 (C-
3a), 97.86 (C-7), 57.23 (C-1’), 55.27 (C-3’’, C-5’’), 46.12 (NCH3, C-2’’, C-6’’), 32.37 (C-2’, C-
5’), 23.91 (C-3’, C-4’). HR-MS (ESI) m/z: Calcd for C16H24N5: [M1+H]+ =286.2026, found
286.2032. Anal. Calcd for C16H23N5: C, 67.34; H, 8.12; N, 24.54. Found: C, 67.03; H, 8.02; N,
24.88.

4.1.8.6. 4-[2-(Dimethylamino)ethylamino]-1-isopropyl-1H-imidazo[4,5-c]pyridine (16f).
This compound was synthesized according to the general procedure described above, upon
reaction of 15c with isopropyl bromide. Purification was effected using a mixture of
dichloromethane / methanol / triethylamine (85/15/5, v/v/v) as the eluent to provide pure 16f as
1
an orange colored oil, in 84% yield. H NMR (600 MHz, CDCl3) δ 7.86 (s, 1H, H-2), 7.85 (1H,
J=6.1Hz, H-6), 6.71 (d, 1H, J=6.1Hz, H-7), 6.01 (brs, 1H, D2O exch, NH), 4.54 (septet, 1H,
J=6.7Hz, CH(CH3)2), 3.89 (t, 2H, J=6.1Hz, H-2’), 2.97 (t, 2H, J=6.1Hz, H-3’), 2.55 (s, 6H,
13
N(CH3)2), 1.58 (d, 6H, J=6.7Hz, 2xCH3). C NMR (151 MHz, CDCl3) δ 151.10 (C-4), 139.23
(C-6), 138.58 (C-2), 137.69 (C-3a), 127.79 (C-7a), 97.24 (C-7), 58.01(C-3’), 48.49 (CH(CH3)2),
44.55 (N(CH3)2), 38.07 (C-2’), 22.71 (2xCH3). HR-MS (ESI) m/z: Calcd for C13H22N5: [M1+H]+
=248.1870, found 248.1874. Anal. Calcd for C13H21N5: C, 63.13; H, 8.56; N, 28.31. Found: C,
63.31; H, 8.63; N, 28.02.
4.1.8.7. 1-Cyclopentyl-4-[2-(dimethylamino)ethylamino]-1H-imidazo[4,5-c]pyridine (16g).
This compound was synthesized according to the general procedure described above, upon
reaction of 15c with cyclopentyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (85/15, v/v) as the eluent to provide pure 16g as an orange colored
oil, in 81% yield. 1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1H, H-2), 7.85 (d, 1H, J=6.3Hz, H-6),
7.10 (d, 1H, J=6.3Hz, H-7), 4.87 (m, 1H, H-1’), 3.97 (t, 2H, J=5.3Hz, H-2’’), 3.46 (t, 2H,
J=5.3Hz, H-3’’), 3.01 (s, 6H, 2xCH3), 2.34-2.26 (m, 2H, cyclopentyl-H), 2.03-1.96 (m, 2H,
13
cyclopentyl-H), 1.96-1.90 (m, 2H, cyclopentyl-H), 1.86-1.79 (m, 2H, cyclopentyl-H). C NMR
(151 MHz, CD3OD) δ 151.79 (C-4), 142.65 (C-2), 140.54 (C-7a), 137.73 (C-6), 128.62 (C-3a),
100.37 (C-7), 60.36 (C-3’’), 59.04(C-1’), 44.06 (N(CH3)2), 39.29 (C-2’’), 33.49 (C-2’, C-5’),
25.00 (C-3’, C-4’). HR-MS (ESI) m/z: Calcd for C15H24N5: [M1+H]+ =274.2026, found
274.2030. Anal. Calcd for C15H23N5: C, 65.90; H, 8.48; N, 25.62. Found: C, 66.06; H, 8.53; N,
25.39.
4.1.8.8. N-Benzyl-1-isopropyl-1H-imidazo[4,5-c]pyridin-4-amine (16h).
This compound was synthesized according to the general procedure described above, upon
reaction of 15d with isopropyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (100/2, v/v) as the eluent to provide pure 16h as a pale yellow oil, in
86% yield. 1H NMR (600 MHz, CDCl3) δ 7.91 (d, 1H, J=5.9Hz, H-6), 7.75 (s, 1H, H-2), 7.41 (d,
2H, J=7.2Hz, H-2’, H-6’), 7.31 (t, 2H, J=7.2Hz, H-3’, H-5’), 7.24 (t, 1H, J=7.2Hz, H-4’), 6.70
(d, 1H, J=5.9Hz, H-7), 5.90 (brs, 1H, D2O exch, NH), 4.83 (d, 2H, J=5.6Hz, CH2), 4.53 (septet,
13
1H, J=6.7Hz,CH(CH3)2), 1.58 (d, 6H, J=6.7Hz, 2xCH3). C NMR (151 MHz, CDCl3) δ 151.74
(C-4), 140.74 (C-6), 139.77 (C-1’), 137.98 (C-2), 137.53 (C-7a), 128.63 (C-3’, C-5’), 127.93 (C-
2’, C-6’), 127.21 (C-4’, C-3a), 97.06 (C-7), 48.40 (CH(CH3)2), 45.20 (CH2), 22.80 (2xCH3). HR-
MS (ESI) m/z: Calcd for C16H19N4: [M1+H]+ =267.1604, found 267.1610. Anal. Calcd for
C16H18N4: C, 72.15; H, 6.81; N, 21.04. Found: C, 72.29; H, 6.89; N, 20.79.

4.1.8.9. N-Benzyl-1-cyclopentyl-1H-imidazo[4,5-c]pyridin-4-amine (16i).
This compound was synthesized according to the general procedure described above, upon
reaction of 15d with cyclopentyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (100/2, v/v) as the eluent to provide pure 16i as a pale yellow oil, in
52% yield. 1H NMR (600 MHz, CDCl3) δ 7.84 (d, 1H, J=5.9Hz, H-6), 7.66 (s, 1H, H-2), 7.34 (d,
2H, J=7.5Hz, H-2’, H-6’), 7.24 (t, 2H, J=7.5Hz, H-3’, H-5’), 7.17 (t, 1H, J=7.5Hz, H-4’), 6.64
(d, 1H, J=5.9Hz, H-7), 5.77 (brs, 1H, D2O exch, NH), 4.77 (d, 2H, J=5.7Hz, CH2), 4.57 (m, 1H,
H-1’’), 2.22-2.15 (m, 2H, cyclopentyl-H), 1.96-1.89 (m, 2H, cyclopentyl-H), 1.87-1.81 (m, 2H,
cyclopentyl-H), 1.76-1.70 (m, 2H, cyclopentyl-H). 13C NMR (151 MHz, CDCl3) δ 151.71 (C-4),
140.67 (C-6), 139.78 (C-1’), 138.64 (C-2), 137.99 (C-7a), 127.92 (C-2’, C-6’), 127.20 (C-4’),
97.30 (C-7), 57.54 (C-1’’), 45.20 (CH2), 32.55 (C-2’’, C-5’’), 24.05 (C-3’’, C-4’’). HR-MS (ESI)
m/z: Calcd for C18H21N4: [M1+H]+ =293.1761, found 293.1766. Anal. Calcd for C18H20N4: C,
73.94; H, 6.89; N, 19.17. Found: C, 74.14; H, 6.98; N, 18.83.
4.1.8.10. 4-(3,5-Dimethoxyphenoxy)-1-isopropyl-1H-imidazo[4,5-c]pyridine (16j) and
4-(3,5-dimethoxyphenoxy)-3-isopropyl-3H-imidazo[4,5-c]pyridine (17a).
These compounds were synthesized according to the general procedure described above, upon
reaction of 15e with isopropyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (100/1, v/v) as the eluent to provide the pure isomers 16j and 17a.
Data for 16j: Yield 29%. White solid, mp 127-8 ˚C (Et2O). 1H NMR (600 MHz, CDCl3) δ 8.00
(s, 1H, H-2), 7.95 (d, 1H, J=5.7Hz, H-6), 7.09 (d, 1H, J=5.7Hz, H-7), 6.44 (d, 2H, J=2.2Hz, H-
2’’, H-6’’), 6.31 (t, 1H, J=2.2Hz, H-4’), 4.62 (septet, 1H, J=6.7Hz, CH(CH3)2), 3.75 (s, 6H,
13
2xOCH3), 1.63 (d, 6H, J=6.7Hz, 2xCH3). C NMR (151 MHz, CDCl3) δ 161.26 (C-3’’, C-5’’),
155.75 (C-1’’), 155.61 (C-4), 140.72 (C-7a), 140.60 (C-2), 139.54 (C-6), 129.68 (C-3a), 102.49
(C-7), 100.30 (C-2’’, C-6’’), 97.41 (C-4’’), 55.50 (2xOCH3), 48.75 (CH(CH3)2), 22.76 (2xCH3).
HR-MS (ESI) m/z: Calcd for C17H20N3O3: [M1+H]+ =314.1499, found 314.1503. Anal. Calcd for
C17H19N3O3: C, 65.16; H, 6.11; N, 13.41. Found: C, 64.98; H, 6.02; N, 13.69.
Data for 17a: Yield 36%. White solid, mp 135-6 ˚C (Et2O). 1H NMR (600 MHz, CDCl3) δ 8.10
(s, 1H, H-2), 7.92 (d, 1H, J=5.7Hz, H-6), 7.41 (d, 1H, J=5.7Hz, H-7), 6.36 (d,2H, J=2.2Hz, H-
2’’, H-6’’), 6.33 (t, 1H, J=2.2Hz, H-4’’), 5.15 (septet, 1H, J=6.7Hz, CH(CH3)2), 3.76 (s, 6H,
13
2xOCH3), 1.64 (d, 6H, J=6.7Hz, 2xCH3). C NMR (151 MHz, CDCl3) δ 161.55 (C-3’’, C-5’’),
155.38 (C-1’’), 152.26 (C-4), 150.11 (C-7a), 142.53 (C-2), 138.71 (C-6), 119.97 (C-3a), 111.65
(C-7), 99.85 (C-2’’, C-6’’), 97.36 (C-4’’), 55.55 (2xOCH3), 49.96 (CH(CH3)2), 23.71 (2xCH3).
HR-MS (ESI) m/z: Calcd for C17H20N3O3: [M1+H]+ =314.1499, found 314.1507. Anal. Calcd for
C17H19N3O3: C, 65.16; H, 6.11; N, 13.41. Found: C, 65.34; H, 6.17; N, 13.24.

4.1.8.11. 1-Cyclopentyl-4-(3,5-dimethoxyphenoxy)-1H-imidazo[4,5-c]pyridine (16k) and
3-cyclopentyl-4-(3,5-dimethoxyphenoxy)-3H-imidazo[4,5-c]pyridine (17b).
These compounds were synthesized according to the general procedure described above, upon
reaction of 15e with cyclopentyl bromide. Purification was effected using a mixture of
dichloromethane / methanol (100/1, v/v) as the eluent to provide the pure isomers 16k and 17b.
Data for 16k: Yield 44%. White solid, mp 165-6 ˚C (Et2O). 1H NMR (600 MHz, CDCl3) δ 7.96
(s, 1H, H-2), 7.94 (d, 1H, J=5.7Hz, H-6), 7.09 (d, 1H, J=5.7Hz, H-7), 6.43 (d, 2H, J=2.2Hz, H-
2”, H-6”), 6.31 (t, 1H, J=2.2Hz, H-4”), 4.72 (m, 1H, H-1’), 3.76 (s, 6H, 2xOCH3), 2.33-2.26 (m,
2H, cyclopentyl-H), 2.06-1.99 (m, 2H, cyclopentyl-H), 1.96-1.90 (m, 2H, cyclopentyl-H), 1.85-
1.81 (m, 2H, cyclopentyl-H). 13C NMR (151 MHz, CDCl3) δ 161.24 (C-3”, C-5”), 156.77 (C-
7a), 155.77 (C-1”), 155.54 (C-4), 141.18 (C-2), 139.47 (C-6), 129.73 (C-3a), 102.72 (C-7),
100.26 (C-2”, C-6”), 97.37 (C-4”), 57.77 (C-1’), 55.47(2xOCH3), 32.55 (C-2’, C-5’), 24.00 (C-
3’, C-4’). HR-MS (ESI) m/z: Calcd for C19H22N3O3: [M1+H]+ =340.1656, found 340.1662. Anal.
Calcd for C19H21N3O3: C, 67.24; H, 6.24; N, 12.38. Found: C, 67.01; H, 6.14; N, 12.55.
1
Data for 17b: Yield 44%. White solid, mp 139-140 ˚C (Et2O). H NMR (600 MHz, CDCl3) δ
8.06 (s, 1H, H-2), 7.91 (d, 1H, J=5.7Hz, H-6), 7.40 (d, 1H, J=5.7Hz, H-7), 6.36 (d, 2H, J=2.2Hz,
H-2”, H-6”), 6.33 (t, 1H, J=2.2Hz, H-4”), 5.23 (m, 1H, H-1’), 3.76 (s, 6H, 2xOCH3), 2.33-2.26
(m, 2H, cyclopentyl-H), 2.06-1.99 (m, 2H, cyclopentyl-H), 1.91-1.84 (m, 2H, cyclopentyl-H),
13
1.80-1.74 (m, 2H, cyclopentyl-H). C NMR (151 MHz, CDCl3) δ 161.51 (C-3”, C-5”), 155.47
(C-1”), 152.42 (C-4), 150.24 (C-7a), 143.01 (C-2), 138.67 (C-6), 120.43 (C-3a), 111.62 (C-7),
99.79 (C-2”, C-6”), 97.25 (C-4”), 59.23 (C-1’), 55.51 (2xOCH3), 33.64 (C-2’, C-5’), 23.86 (C-
3’, C-4’). HR-MS (ESI) m/z: Calcd for C19H22N3O3: [M1+H]+ =340.1656, found 340.1664. Anal.
Calcd for C19H21N3O3: C, 67.24; H, 6.24; N, 12.38. Found: C, 66.98; H, 6.09; N, 12.66.
4.1.9. General procedure for the synthesis of compounds 18b-c.
Ammonium formate (150 mg, 2.5 mmol) and 10% Pd/C (70 mg) were added into a solution of
the benzylamines 16h or 16i (0.25 mmol) in methanol (5 mL), under argon, and this reaction
mixture was refluxed for 48h. An additional amount (300 mg, 5.0 mmol) of ammonium formate
was added and the reflux was continued for 48h. Upon completion of the reaction, the solution
was filtered through a celite pad to remove the catalyst and the filtrate was evaporated to
dryness. The crude product was purified by column chromatography (silica gel) to provide pure
aminoderivatives 18b and 18c.
4.1.9.1. 1-Isopropyl-1H-imidazo[4,5-c]pyridin-4-amine (18b).
This compound was synthesized according to the general procedure described above, starting
from 16h. Purification was effected using a mixture of dichloromethane / methanol (9/1, v/v) as
1
the eluent to provide pure 18b as a pale yellow oil, in 43% yield. H NMR (600 MHz,
(CD3)2CO) δ 8.12 (s, 1H, H-2), 7.71 (d, 1H, J=6.0 Hz, H-6), 6.92 (d,1H, J=6.0 Hz, H-7), 6.29
(brs, 2H, D2O exch, NH2), 4.73 (septet, 1H, J=6.7 Hz, CH(CH3)2), 1.60 (d, 6H, J=6.7 Hz,
13
2xCH3). C NMR (151 MHz, (CD3)2CO) δ 153.01 (C-4), 140.53 (C-2), 139.13 (C-7a), 138.87
(C-6), 128.39 (C-3a), 98.48 (C-7), 49.18 (CH(CH3)2), 22.76 (2xCH3). HR-MS (ESI) m/z: Calcd

for C9H13N4: [M1+H]+ =177.1135, found 177.1130. Anal. Calcd for C9H12N4: C, 61.34; H, 6.86;
N, 31.79. Found: C, 61.52; H, 6.93; N, 31.49.
4.1.9.2. 1-Cyclopentyl-1H-imidazo[4,5-c]pyridin-4-amine (18c).
This compound was synthesized according to the general procedure described above, starting
from 16i. Purification was effected using a mixture of dichloromethane / methanol (100/8, v/v)
1
as the eluent to provide pure 18c as a pale yellow oil, in 82% yield. H NMR (600 MHz,
(CD3)2CO) δ 8.12 (s, 1H, H-2), 7.72 (d, 1H, J=6.0 Hz, H-6), 6.95 (d,1H, J=6.0 Hz, H-7), 6.44
(brs, 2H, D2O exch, NH2), 4.87 (m, 1H, H-1’), 2.34-2.26 (m, 2H, cyclopentyl-H), 2.08-2.00 (m,
2H, cyclopentyl-H), 1.97-1.91 (m, 2H, cyclopentyl-H), 1.84-1.77 (m, 2H, cyclopentyl-H). 13C
NMR (151 MHz, (CD3)2CO) δ 152.81 (C-4), 141.24 (C-2), 139.75 (C-7a), 138.14 (C-6), 128.44
(C-3a), 98.76 (C-7), 58.28 (C-1’), 33.04 (C-2’, C-5’), 24.64 (C-3’, C-4’). HR-MS (ESI) m/z:
Calcd for C11H15N4: [M1+H]+ =203.1291, found 203.1284. Anal. Calcd for C11H14N4: C, 65.32;
H, 6.98; N, 27.70. Found: C, 65.44; H, 7.04; N, 27.51.
4.2. Combinatorial Design-Molecular Docking Calculations
4.2.1. Ligand Enumeration - Preparation
Based on the scaffold of 3-deazaadenine, we enumerated a virtual combinatorial library using
our in-house fragment library containing 200 chemical fragments. Moreover, we defined 2
positions for substitutions (1 and 4) combining 0 to 2 methylene groups as linkers between the
fragment and the core. Thus the intensity of the new virtual chemical library enumerated 1200
compounds. All ligands were prepared using the ligprep module as implemented on Schrodinger
Suite 2014. The geometries of the generated structures are optimized using a restricted version of
the MacroModel™ computational program, bmin, or a short conformational search is performed
to relax the structure into 3 dimensions while strongly encouraging chiral centers to adopt the
proper chirality (if the structure is highly strained). For the mono 4-substitued analogues of 3-
deaza adenine, both 1NH and 3NH tautomers were generated for further docking calculations.
4.2.2. Virtual Screening
The modeled structure of FcyB transporter is already described by our group,15 and prepared for
Virtual Screening using the protein preparation workflow as implemented on Schrodinger suite
2014. Glide energy grids were generated using the Receptor Grid Generation panel on Maestro
software with default values. Glide software was utilized for virtual screening using the SP
protocol. The enumerated database created passed through virtual screening and only the first 2%
(40 structures) of ligands based on GlideScore stored for Induced Fit Docking.
4.2.3. Induced Fit Docking

All selected molecules for synthesis were passed through exhaustive molecular docking
calculations using the IFD protocol (Induced Fit Docking protocol 2015–2, Glide version 6.4,
Prime version 3.7,Schrödinger, LLC, 2015),27,28 which is intended to circumvent the inflexible
binding site and accounts for the side-chain or backbone movements, or both, upon ligand
binding. In the first stage of the IFD protocol, softened-potential docking step, 20 poses per
ligand were retained. In the second step, for each docking pose, a full cycle of protein refinement
was performed, with Prime 3.7 (Prime, version 3.7, Schrödinger, LLC) on all residues having at
least one atom within 8 Å of an atom in any of the 20 ligand poses. The Prime refinement starts
with a conformational search and minimization of the side-chains of the selected residues and
after convergence to a low-energy solution, an additional minimization of all selected residues
(side-chain and backbone) is performed with the truncated-Newton algorithm using the OPLS
parameter set and a surface Generalized Born implicit solvent model. The obtained complexes
are ranked according to Prime calculated energy (molecular mechanics and solvation), and those
within 30 kcal/mol of the minimum energy structure are used in the last step of the process,
redocking with Glide 6.4 (Glide, version 6.4, Schrödinger, LLC, 2015) using standard precision
and scoring. In the final round, the ligands used in the first docking step are redocked into each
of the transporter structures retained from the refinement step. The final ranking of the
complexes is done by a composite score which accounts for the transporter–ligand interaction
energy (GlideScore) and solvation energies (Prime energy).
4.3. Aspergillus manipulations
Standard complete and minimal media (MM) for A. nidulans were used. Media and
supplemented auxotrophies were at the concentrations given in http://www.fgsc.net. 10 mM
NaNO3 was used as a nitrogen source. Inhibitors are added in MM dissolved in DMSO at 500
μΜ. Transformations were performed as described previously.29 Transformants of the Δ7 master
mutant, arising from single-copy plasmid integration events of vectors carrying the either fcyB or
the azgA gene, in addition to a wild-type pabA1 selection marker, were obtained by
complementation of the pabA1 auxotrophy. The Δ7 master mutant contains total genetic deletion
of all major nucleobase/nucleoside transporters (ΔfcyB::argB ΔazgA, ΔuapA, ΔuapC::AfpyrG,
ΔfurD::riboB ΔfurA::riboB, ΔcntA::riboB, pantoB100, pabA1), in addition to pabA1 auxotrophy.
Confirmation of single copy integrations, introducing intact fcyB or azgA genes, was obtained by
Southern and PCR analyses. azgA- and fcyB- containing vectors,16,17 and the Δ7 mutant strain13
have been described before. In the case of fcyB, transcription is driven by the uapA promoter for
obtaining sufficient protein levels for functional assays.17 AzgA transcription is driven by its
nature promoter.
4.4. Transport assays

Transport assays for measuring the activity of purine transporters, such as FcyB, AzgA or UapA,
is carried out in germinating conidiospores, as recently described in detail.13 For transport
competition assays, 0.5 μΜ of 3H-radiolabelled substrate (adenine, hypoxanthine or xanthine) is
added in a mix with 1000-fold excess 3-deazaadenine analogues (500 μM).13 Assays are
terminated after 1 min by freezing, immediate centrifugation and washing of cells. Ki values are
estimated from IC50 measurements using the Cheng and Prussof equation [Ki = IC50/1+ [S]/Km,
where [S] is the fixed concentration of radiolabeled substrate used] and analyzed by the
GraphPad Prism software. All experiments are carried out at three times, with each assays
performed in triplicate. Standard deviation in all cases is less than 30%. Radiolabeled purines
used are: [2,8-3H]-adenine 20.0 Ci/mmol, [2,8-3H]-hypoxanthine 27.7 Ci/mmol or [8-3H]-
xanthine 22.8 Ci/mmol, all from Moravek Biochemicals.
Acknowledgements
This research was co-supported by the European Union (European Social Fund) and Greek
national funds through the Operational Program “Education and Lifelong Learning” of the
National Strategic Reference Framework-Research Funding Program: THALES, investing in
knowledge society through the European Social Fund.
References and notes
1. Kwon-Chung, K. J.; Sugui, J. A. PLoS pathogens 2013, 9, e1003743.
2. Moye-Rowley, W. S. Frontiers in microbiology 2015, 6, 70.
3. Odds, F. C.; Brown, A. J.; Gow, N. A. Trends in microbiology 2003, 11, 272.
4. Wiederhold, N. P.; Patterson, T. F. Current opinion in infectious diseases 2015, 28, 539.
5. Lamping, E.; Baret, P. V.; Holmes, A. R.; Monk, B. C.; Goffeau, A.; Cannon, R. D. Fungal
genetics and biology 2010, 47, 127.
6. Cannon, R. D.; Lamping, E.; Holmes, A. R.; Niimi, K.; Baret, P. V.; Keniya, M. V.; Tanabe,
K.; Niimi, M.; Goffeau, A.; Monk, B. C. Clinical microbiology reviews 2009, 22, 291.
7. Prasad, R.; Rawal, M. K. Frontiers in pharmacology 2014, 5, 202.
8. Paluszynski, J. P.; Klassen, R.; Rohe, M.; Meinhardt, F. Yeast 2006, 23, 707.
9. Krypotou, E.; Evangelidis, T.; Bobonis, J.; Pittis, A. A.; Gabaldon, T.; Scazzocchio, C.;
Mikros, E.; Diallinas, G. Mol. Microbiol. 2015, 96, 927.
10. Pantazopoulou, A.; Diallinas, G. FEMS Microbiol. Rev. 2007, 31, 657.
11. Diallinas, G.; Gournas, C. Channels 2008, 2, 363.
12. Diallinas, G. Frontiers in pharmacology 2014, 5, 207.
13. Krypotou, E.; Diallinas, G. Fungal genetics and biology : FG & B 2014, 63, 1.
14. Kosti, V.; Lambrinidis, G.; Myrianthopoulos, V.; Diallinas, G.; Mikros, E. PloS one 2012, 7,
e41939.
15. Krypotou, E.; Kosti, V.; Amillis, S.; Myrianthopoulos, V.; Mikros, E.; Diallinas, G. J. Biol.
Chem. 2012, 287, 36792.

16. Krypotou, E.; Lambrinidis, G.; Evangelidis, T.; Mikros, E.; Diallinas, G. Mol. Microbiol.
2014, 93, 129.
17. Vlanti, A.; Diallinas, G. Mol. Microbiol. 2008, 68, 959.
18. Connon, S. J.; Hegarty, A. F. Eur. J. Org. Chem. 2004, 2004, 3477.
19. Searls, T.; McLaughlin, L. W. Tetrahedron 1999, 55, 11985.
20. Yin, X.; Schneller, S. W. Nucleosides Nucleotides and Nucleic Acids 2004, 23, 67.
21. Dvořáková, H.; Holý, A.; Votruba, I.; Masojídková, M. Collect. Czech. Chem. Commun.
1993, 58, 629.
22. Huang, L. K.; Cherng, Y. C.; Cheng, Y. R.; Jang, J. P.; Chao, Y. L.; Cherng, Y. J.
Tetrahedron 2007, 63, 5323.
23. Sander, K.; Kottke, T.; Tanrikulu, Y.; Proschak, E.; Weizel, L.; Schneider, E. H.; Seifert, R.;
Schneider, G.; Stark, H. Bioorg. Med. Chem. 2009, 17, 7186.
24. Crey-Desbiolles, C.; Kotera, M. Bioorg. Med. Chem. 2006, 14, 1935.
25. Schwoch, S.; Kramer, W.; Neidlein, R.; Suschitzky, H. Helv. Chim. Acta 1994, 77, 2175.
26. Krenitsky, T. A.; Rideout, J. L.; Chao, E. Y.; Koszalka, G. W.; Gurney, F.; Crouch, R. C.;
Cohn, N. K.; Wolberg, G.; Vinegar, R. J. Med. Chem. 1986, 29, 138.
27. Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. J. Med. Chem. 2006, 49,
534.
28. Sherman, W.; Beard, H. S.; Farid, R. Chemical biology & drug design 2006, 67, 83.
29. Koukaki, M.; Giannoutsou, E.; Karagouni, A.; Diallinas, G. J. Microbiol. Methods 2003, 55,
687.

NH
N
N
N
16h
specific inhibition of FcyB transporter
Ki (µM): 5±1