Novel bis‐ and mono‐pyrrolo[2,3‐d]pyrimidine and purine derivatives: Synthesis, computational analysis and antiproliferative evaluation

molecules

Article

Novel Bis- and Mono-Pyrrolo[2,3-d]pyrimidine and Purine

Derivatives: Synthesis, Computational Analysis and

Antiproliferative Evaluation

4

5

Andrea Bistrovic´ Popov ^1,2, Robert Vianelo ³, Petra Grbcˇic´

, Mirela Sedic´

, Sandra Kraljevic´ Pavelic´

,

6

1,

Krešimir Pavelic´

and Silvana Raic´-Malic´ *

1

Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,

Marulic´ev trg 20, HR-10000 Zagreb, Croatia; ab2602@cam.ac.uk

2

3

4

5

Department of Chemical Engineering and Biotechnology, University of Cambridge, Philippa Fawcet Drive,

Cambridge CB3 0AS, UK

Division of Organic Chemistry and Biochemistry, Rud¯er Boškovic´ Institute, Bijenicˇka 54,

HR-10000 Zagreb, Croatia; robert.vianello@irb.hr

Department of Biotechnology, University of Rijeka, Ulica Radmile Matejcˇic´ 2, HR-51000 Rijeka, Croatia;

petra.grbcic@biotech.uniri.hr (P.G.); msedic@biotech.uniri.hr (M.S.)

Faculty of Health Studies, University of Rijeka, Ulica Viktora Cara Emina 5, HR-51000 Rijeka, Croatia;

sandrakp@uniri.hr

Faculty of Medicine, Juraj Dobrila University of Pula, HR-52100 Pula, Croatia; pavelic@unipu.hr

Correspondence: sraic@fkit.hr

6

*

Abstract: Novel symmetrical bis-pyrrolo[2,3-d]pyrimidines and bis-purines and their monomers were

synthesized and evaluated for their antiproliferative activity in human lung adenocarcinoma (A549),

cervical carcinoma (HeLa), ductal pancreatic adenocarcinoma (CFPAC-1) and metastatic colorectal

adenocarcinoma (SW620) cells. The use of ultrasound irradiation as alternative energy input in Cu(I)-

catalyzed azide-alkyne cycloaddition (CuAAC) shortened the reaction time, increased the reaction

efﬁciency and led to the formation of exclusively symmetric bis-heterocycles. DFT calculations

showed that triazole formation is exceedingly exergonic and conﬁrmed that the presence of Cu(I) ions

is required to overcome high kinetic requirements and allow the reaction to proceed. The inﬂuence

of various linkers and 6-substituted purine and regioisomeric 7-deazapurine on their cytostatic

activity was revealed. Among all the evaluated compounds, the 4-chloropyrrolo[2,3-d]pyrimidine

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Bistrovic´ Popov, A.;

Vianelo, R.; Grbcˇic´, P.; Sedic´, M.;

Pavelic´, S.K.; Pavelic´, K.; Raic´-Malic´,

S. Novel Bis- and Mono-Pyrrolo[2,3-

d]pyrimidine and Purine Derivatives:

Synthesis, Computational Analysis

and Antiproliferative Evaluation.

Molecules 2021, 26, 3334. https://

doi.org/10.3390/molecules26113334

0

monomer 5f with 4,4 -bis(oxymethylene)biphenyl had the most pronounced, although not selective,

growth-inhibitory effect on pancreatic adenocarcinoma (CFPAC-1) cells (IC₅₀= 0.79 µM). Annexin

V assay results revealed that its strong growth inhibitory activity against CFPAC-1 cells could be

associated with induction of apoptosis and primary necrosis. Further structural optimization of

bis-chloropyrrolo[2,3-d]pyrimidine with aromatic linker is required to develop novel efﬁcient and

non-toxic agent against pancreatic cancer.

Academic Editor: Brullo Chiara

Received: 7 May 2021

Accepted: 29 May 2021

Published: 1 June 2021

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

Keywords: pyrrolo[2,3-d]pyrimidines; purine; DFT calculations; ultrasound; antiproliferative activ-

ity; pancreatic adenocarcinoma; apoptosis

1. Introduction

Cancer, deﬁned as uncontrolled, rapid and pathological proliferation of cells, is the

Copyright:

second leading cause of death, with more than 18 million cases worldwide annually [

Pancreatic cancer is predicted to become the second cause of cancer-related deaths by

2030, behind lung cancer [ ]. Pancreatic ductal adenocarcinoma is the most common

pancreatic cancer type, accounting for more than 90% of cases with a ﬁve-year survival

rate of less than 9% [ ]. Current therapy suffers from major limitations due to severe

side-effects and multidrug resistance, thereby a continued search to ﬁnd new and safer

anticancer drugs and innovate the development of new cancer treatments are required [ ].

1].

Licensee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and

conditions of the Creative Commons

Attribution (CC BY) license (https://

creativecommons.org/licenses/by/

4.0/).

2

3,4

5,6

Molecules 2021, 26, 3334. https://doi.org/10.3390/molecules26113334

https://ww w .mdpi.com/journal/molecules

Molecules 2021, 26, 3334

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More than 75% of drugs approved by the FDA and currently available on the market are

nitrogen-containing heterocycles due to their ability to easily form hydrogen bonding,

dipole-dipole interactions, hydrophobic effects, van der Waals forces and

teractions with biological targets [ ]. The naturally occurring purines play vital roles in

numerous life processes [ ]. Over the past two decades, purines and their isosteres have

appeared as important pharmacophores interacting with the synthesis and functions of

nucleic acids and enzymes [ ]. To date, 22 pyrimidine-fused bicyclic heterocycles have been

π-stacking in-

7

8

9

approved for clinical use in the treatment of different cancers [10]. Among pyrimidine-

fused bicyclic heterocycles, the pyrrolo[2,3-d]pyrimidine core can be considered as an

isosteric replacement of the biologically relevant purine heterocycle and is hence an im-

portant pharmacophore widely used in the ﬁeld of medicinal chemistry and drug design

primarily due to its anticancer properties [11

exhibited cytotoxic effects in lung and colon cancer cell lines through activation of the

mitochondrial apoptotic pathway [14 16]. Aryl-substituted pyrrolopyrimidines showed

–13]. Pyrrolo[2,3-d]pyrimidine derivatives

–

potent inhibition of the membrane bound epidermal growth factor receptor tyrosine ki-

nase (EGFR) and angiogenic inhibitors against human vascular endothelial growth factor

receptor-2 (VEGFR-2) that represent important targets in cancer therapy [17–24]. Over

the past years, some pyrrolo[2,3-d]pyrimidine derivatives were identiﬁed as inhibitors

of Mer receptor and Src non-receptor tyrosine kinases, isoform of protein kinase B (Akt),

mitotic checkpoint kinase (Mps1), Janus kinase 2 (JAK2), and phosphoinositide 3 kinase

(PI3K) with promising anticancer activity [25–33]. Design strategy in development of

purine derivatives as cytostatic agents for kinase inhibition revealed that introduction

of cyclic amines improved the activity by forming an additional hydrogen bond to ki-

nase hinge [9,34,35]. In addition, prevalence of halogenated drugs showed that halogen

bonds contribute to the stability of protein-ligand complexes [36]. Pyrrolo[2,3-d]pyrimidine

sulfonamides were recently found to act as cytotoxic agents in hypoxia via inhibition of

transmembrane carbonic anhydrases [37]. A 4-(benzylamino)-pyrrolo[2,3-d]pyrimidine

derivative exerted potent antitumour effects in vivo and induced mitotic cell blockade

by impairing both mitotic microtubule organization and dynamics [38]. To overcome

multidrug resistance in cancer patients, pyrrolo[2,3-d]pyrimidines and purine derivatives

with high lipophilicity and molecular weight were developed as potent and selective

inhibitors of multidrug resistance-associated protein 1 (MRP1, ABCC1) associated with

non-response to chemotherapy in different cancers [39]. Several heterocyclic dimers such

as bis-purines [40] and bis-benzimidazoles [41] linked in a head-to-head [42] or head-to-

tail manner [43] with acylic and cyclic spacers, have been reported to exhibit anticancer

properties by noncovalent interactions with the minor groove of DNA [44].

We previously found that bis-pyrrolo[2,3-d]pyrimidine derivatives exhibited potent an-

tiproliferative effect on pancreatic carcinoma (CFPAC-1) cells [45], therefore design strategy

led to synthesis of a symmetric series of bis-purines and regioisomeric bis-pyrrolo[2,3-

d]pyrimidines in which chlorine, amino and cyclic amino groups were introduced at

6-position of the purine and pyrrolo[2,3-d]pyrimidine scaffold (Figure 1). Besides, modiﬁed

aliphatic or π-electron rich aromatic linkers between two heterocycles were introduced to

explore further potential for improvement of anti-cancer activity.

Molecules 2021, 26, 3334

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Figure 1. Design of symmetric bis-pyrrolo[2,3-d]pyrimidines (5a–5d, 6a–6d, 7a–7d) and bis-purines (9a–9d, 10a–10d,

11a–11d).

To develop a less toxic and more environmentally friendly synthetic method, opti-

mization of the syntheses of target compounds by application of ultrasonic waves and

microwaves was performed. Computational analysis was used to elucidate kinetic and

thermodynamic aspects of the investigated reactions and identify the precise mechanistic

role of the Cu(I) catalyst. Antiproliferative evaluations of bis-purines and bis-pyrrolo[2,3-

d]pyrimidines and apoptotic mechanism of the selected compound with best antiprolifera-

tive effect were also investigated.

2. Results and Discussion

2.1. Chemistry

The novel bis-pyrrolo[2,3-d]pyrimidines(5a

10d 11a 11d were synthesized as depicted in Schemes 1 and 2. N-Alkylation of the

corresponding pyrrolo[2,3 d]pyrimidine 1a, more commonly referred to as 7-deazapurine,

with 1,2 dibromoethane in the presence of K₂CO₃afforded 2 bromoethyl derivative 2a

azidoethyl analogue 3a using NaN₃as

–5d, 6a–6d, 7a–7d and bis-purines 9a–9d,

10a–

,

–

-

,

which were converted to the corresponding 2

previously described [45] (Scheme 1).

-

Aromatic and aliphatic bis-alkynes: 1,4-bis-(propynyloxy)-benzene (4a), 4,4⁰-bis-

(propynyloxy)-1,1⁰-biphenyl (4b), 1,6-heptadiyne (4c) and propargyl ether (4d) as dipo-

larophiles, were prepared by O-propargylation of the corresponding alcohols with propar-

gyl bromide in the presence of a base [46

,

47]. Thus, obtained bis-alkynes 4a−4d were sub-

sequently reacted with azide 3a to give bis-4-chloro-pyrrolo[2,3-d]pyrimidines (5a−5d) and

mono-4-chloro-pyrrolo[2,3-d]pyrimidines 5f and 5g. 4-Chloro-pyrrolo[2,3-d]pyrimidine

dimers 5a−5d were converted to their 4-piperidine and 4-pyrrolidine- substituted ana-

logues 6a–6d and 7a–7d in very good yields of 60–97% using microwave irradiation [48].

Water, as an environmentally friendly solvent, was used in this reaction which additionally

contributed to the implementation of a green and sustainable synthetic approach.

Molecules 2021, 26, 3334

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Scheme 1. Reagents and conditions: (i) BrCH₂CH₂Br, K₂CO₃, DMF, rt, 24 h; (ii) NaN₃, acetone, reﬂux,

24 h; (iii) Method A: 1M CuSO₄, C_◦u(0), t-BuOH:H₂O = 1:1, DMF, rt, 120 h; Method B: 1M CuSO4,

Cu(0), t-BuOH:H₂O = 1:1, DMF, 50 C, US, 45 min, 1000 W; Method C: CuI, DIPEA, AcOH, CH₂Cl₂,

◦

rt., 72 h; (iv) piperidine/pyrrolidine, H₂O, MW, 400 W, 100 C, 10 min.

Scheme 2. Reagents and conditions: (i) BrCH₂CH₂Br, K₂CO₃, DMF, rt, 24 h; (ii) NaN₃, acetone,

reﬂux, 24 h; (iii) Method A: 1M CuSO₄, Cu(0), t-BuOH:H₂O = 1:1, DMF, rt, 120 h; Method B: 1M

◦

CuSO4, Cu(0), t-BuOH:H₂O = 1:1, DMF, 50 C, US, 45 min, 1000 W; Method C: CuI, DIPEA, AcOH,

CH₂Cl₂, rt., 72 h.

Molecules 2021, 26, 3334

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In the purine series, reaction of 2-bromoethyl-6-chloropurine 2b with sodium azide

afforded the diazido derivative 3b that was found to exist in two tautomeric forms, as

reported in the literature [49] and that in CuAAC reaction [50] with bis-alkyne yielded only

corresponding monomer 8h (Scheme 2).

Therefore, the synthetic strategy for the synthesis of 6-substituted bis-purines con-

nected via different spacer was changed. Thus, substitution of 6-chloro to 6-piperidino

and 6-pyrrolidino moiety was performed and then CuAAC reaction of heterocyclic azides

3c−3e with bis-alkynes 4a

–4d yielded the target 6-substituted bis-purine 9a–9d, 10a–10d,

11a, 11d) and mono-purine (9e–9g, 10f, 11e, 11g, 11h) derivatives (Scheme 2).

In order to optimize the CuAAC reaction, the reactions of bis-alkynes (4a−4d) and

heterocyclic azides (3a and 3c−3e) were carried out using different catalysts and reaction

conditions to give 6-substituted bis- and mono-purines and 7-deazapurines (Table 1).

Table 1. The scope of CuAAC reactions for the synthesis of pyrrolo[2,3-d]pyrimidines and purines

using methods A–C.

Yield (%)

Compd

Base

X

Method A ¹

Method B ²

Method C ³

5a

-

49

-

34

-

45

11

51

-

67

-

66

-

24

-

5b

5f

5c

5g

5d

-

35

14

63

35

64

-

62

-

CH₂CH₂CH₂

CH₂OCH₂

4

−

4

−

9a

9e

9b

9f

9c

8g

9d

-

28

4

48

-

40

-

55

61

-

69

-

36

29

38

31

21

10

46

-

62

-

CH₂CH₂CH₂

CH₂OCH₂

4

−

4

-

−

10a

-

10b

10f

10c

-

28

-

60

-

50

-

48

61

-

80

-

60

-

49

61

58

-

82

-

CH₂CH₂CH₂

CH₂OCH₂

4

10d

-

58

-

−

4

-

−

11a

11e

-

11g

11d

11h

-

11

-

25

-

55

8

7

CH₂CH₂CH₂

CH₂OCH₂

-

4

−

4

−

Method A: Cu(0), CuSO₄, rt, 120 h; ²method B: Cu(0), CuSO₄, US, 1000 W, 80 ^◦C, 90 min; ³method C: CuI,

1

DIPEA, AcOH, CH₂Cl₂, rt, 72 h; ⁴reaction was not carried out due to risk of forming explosive by-product; – the

product was not obtained.

Based on the known protocols [51] for CuAAC reaction that include the in situ genera-

tion of Cu(I) from a Cu(II) salt or the alternative direct utilization of a Cu(I) source using the

combination of CuI/DIPEA/HOAc, which has been found to be a highly efﬁcient catalytic

Molecules 2021, 26, 3334

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system for this reaction [52], we performed an optimization of the CuAAC reaction using

methods A–C.

The most common catalytic system, CuSO₄in the presence of metallic copper as

a reducing agent (in aqueous t-BuOH), was initially chosen in method A. For better

performance, we then investigated the application of ultrasound as a green alternative for

energy efﬁcient processes in method B. Chemical transformations induced by ultrasound

have been described previously [53,54], and it was discovered that ultrasonic irradiation

generates a large number of cavitation bubbles, which cause an increase in the local

temperature within the reaction mixture and eventually enable the crossing of the activation

energy barrier [54]. Finally, copper(I) iodide in the presence of N,N-diisopropylethylamine

(DIPEA) and acetic acid (HOAc) was employed in method C. Comparing the applied

synthetic methods A–C in CuAAC reactions of 6

with all selected bis alkynes, it can be observed that bis

in all methods, while when methods A and C were used with 4,4⁰

biphenyl (4b) and 1,6 heptadiyne (4c) the corresponding mono triazole analogues 5f and

5g were also obtained (Scheme 1, Table 1).

In the 6-piperidinyl- and 6-pyrrolidinylpurine series, the CuI/DIPEA/HOAc catalytic

system using method C was the least selective, yielding both bis- (9a 9d and 10a 10d) and

mono-heterocycles 9e 9g and 10f. The use of Cu(II) salt as a catalyst in method A afforded

-

chloro

triazole dimers 5a

bis(propynyloxy)

-

7

-

deazapurine azide derivative (3a

5d were obtained

1,1⁰

-

)

-

–

-

–

only bis-heterocycles in most cases, with exceptions for 5g and 9e when small amounts of

mono-heterocycles were isolated. However, these reactions were extremely slow and were

carried out over 7 days. It can be observed that ultrasound irradiation employed in method

B signiﬁcantly reduced the reaction time to 1.5 h. We may assume that acoustic cavitation in

the heterogeneous CuSO₄/Cu(0) system facilitated mass transfer and surface activation [55

]

and ultimately accelerated the CuAAC reaction. An additional advantage of the ultrasound-

assisted reactions was the exclusive formation of dimeric heterocyclic analogues with

improved yields (in the range of 61–82%) compared to reactions without ultrasound

irradiation in method A (yields of 28–60%) and method C (yields of 24–64%). In the case of

the adenine series, only the ultrasound-assisted reaction (method B) of 2

-azidoethyladenine

3e with bis alkyne 4a afforded bis-adenine 11a with 1,4-bis(oxymethylene)phenyl linker

-

in low yield (Scheme 2), while 3e could not react with 4b in any conditions. When this

reaction was performed using CuI, as catalyst, only mono-adenine 11e was obtained.

Also, only CuAAC reactions of azide 3e with aliphatic bis-alkynes 4c and 4d in method C

afforded mono-adenine 11g with propyl chain at C-4 of 1,2,3-triazole, and both bis- 11d

and mono-adenine 11h in low yields, respectively. Reactions of adenine azide derivative

3e and bis-alkynes (4a–4d) did not afford the target products using other applied synthetic

methods, indicating the inﬂuence of 6-aminopurine on its lower reactivity compared to its

6-piperidino- (3c) and 6-pyrrolidino-substituted (3d) purine congeners.

2.2. Computational Analysis

Computational analysis was performed to identify precise molecular mechanisms

underlying the conversion of azides and alkynes into the matching triazoles, and to reveal

the role of the Cu(I) catalysts on the reaction outcomes. For that purpose, we employed a

series of DFT calculations on several model systems (Figure 2) with the aim of providing

enough structural and electronic features to help interpreting the yields and product

distributions observed experimentally.

Figure 2. Structure of model alkyne and azide studied computationally.

Molecules 2021, 26, 3334

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To demonstrate the necessity to employ the metal catalyst in the CuAAC reaction,

we have initially investigated the uncatalyzed conversion between model azide m2 and

neutral model alkyne m1⁰, which proceeds in accordance with Figure 3. Bringing reactants

into the reactive complex is unfavorable and endergonic by 6.9 kcal mol⁻¹, from which it

takes additional 21.6 kcal mol⁻¹to reach the transition state corresponding to the concerted

formation of both C–N bonds. The latter gives the ﬁnal triazole in a single step in a very

exergonic fashion, linked with the total reaction free energy of

Still, although thermodynamically favorable, the overall reaction has a rather high kinetic

barrier,

G^‡= 28.5 kcal mol⁻¹, which renders it as very much unlikely under normal

∆

G_R=

−

51.0 kcal mol⁻¹

.

∆

conditions, and emphasizes the necessity to employ a suitable catalyst.

Figure 3. The uncatalyzed direct conversion of alkyne m1⁰and azide m2 into triazole. Relative

Gibbs free energies for isolated reactants (R), reactant complex (RC), transition state (TS) and isolated

product (P) correspond to the DMF solution (in kcal mol⁻¹).

Although alkynes are very weakly acidic systems, and their C–H acidity is typically

related with pK_avalues over 20 in water [56], one could still ﬁnd a suitable base to initiate

the deprotonation [57] and convert alkyne m1⁰into its anionic form m1^–, with the idea

to increase the electrophilicity towards azide and facilitate the reaction. Interestingly, this

does not occur (Figure S40), as it takes 7.0 kcal mol⁻¹to form the reactive complex, and,

although the kinetic barrier is reduced by 2.6 kcal mol^–1to G^‡= 25.9 kcal mol⁻¹, the

∆

reaction is thermodynamically signiﬁcantly less feasible, as the reaction free energy is made

less exergonic by 11.6 kcal mol⁻¹to

∆

G_R=

−

39.4 kcal mol⁻¹. The latter will likely prevail

and the reaction with deprotonated m1^–will be less favorable. This suggests that attempts

to improve the reaction outcomes with strong bases will likely fail.

Introducing Cu(I) ions in the system can result in their complexation with reagents,

as both acetylenes and azides are well capable of forming organometallic complexes.

Therefore, we proceeded by analyzing potential 1:1 complex with both reagents, which

might turn useful in clarifying the role of the metal in the catalysis and identifying which

reagent is being activated. The calculated interaction energies (Figure S41) show that Cu(I)

ions are much more efﬁcient in binding alkynes (∆G_INT= −11.9 kcal mol⁻¹for m1⁰) than

azides (

∆

G_INT

=

−

5.6 kcal mol⁻¹for m2). This is rationalized by electronic distribution

as m1⁰contains more electronic density within its two carbons (

−

0.34 |e|) than m2

within three of its nitrogen atoms (

electrostatic in nature.

−

0.24 |e|), thus the observed trend is predominantly

With this in mind, we analyzed a situation where the cycloaddition reaction occurs

with alkyne binding the Cu(I) ion (Figure 4). There, bringing reactants into the reactive

complex is exergonic and favorable (

one step with the kinetic barrier of

−

2.8 kcal mol⁻¹), while the reaction again proceeds in

∆

G^‡= 19.7 kcal mol⁻¹, which represents a considerable

8.8 kcal mol⁻¹reduction from the uncatalyzed reaction. This offers a product complex

with Cu(I) binding around alkyne carbons, from which it takes only 1.1 kcal mol⁻¹to

detach Cu(I) and allow the ﬁnal triazole. The obtained reaction proﬁle is highly feasible,

while the obtained reduction in the kinetic activation roughly translates into 6–7 orders of

magnitude higher rate constant, which is signiﬁcant and underlines the crucial role of the

metal catalyst in facilitating the reaction.

Molecules 2021, 26, 3334

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Figure 4. Cu(I)-catalyzed conversion of alkyne m1⁰and azide m2 into triazole with Cu(I) bound to alkyne. Relative Gibbs

free energies for isolated reactants ( ), reactant complex (RC), transition state (TS), product complex (PC) and isolated

product (P) correspond to the DMF solution (in kcal mol⁻¹).

R

On the other hand, if Cu(I) ions would be able to overcome an initially more favorable

placement around alkyne and form a complex with azide prior to reaction (Figure S42), the

formation of the reactive complex in that case, would, as anticipated, be less favorable by

5.8 kcal mol⁻¹and even endergonic (+3.0 kcal mol⁻¹). In addition to this negative aspect,

the obtained product complex featuring triazole with azide-bound Cu(I) is exceedingly ex-

ergonic (–64.4 kcal mol⁻¹), which will hinder further reaction progress. Speciﬁcally, such a

stable product complex would increase the energy requirement to ﬁnalize the reaction from

1.1 kcal mol⁻¹in the previous case to 13.4 kcal mol⁻¹here. Both of the mentioned aspects

will expectedly predominate over formally slightly lower kinetic barrier, by 1.3 kcal mol⁻¹

to ∆

G^‡= 18.4 kcal mol⁻¹, and make such a conversion less likely. As it was the case with

the uncatalyzed reaction, converting alkyne into its deprotonated form m1^–again does not

promote the reaction (Figure S43). First, both processes become less exergonic here, with

the overall reaction free energy reduced by 10.7 kcal mol⁻¹to ∆G_R= −40.3 kcal mol⁻¹

.

On top of that, when Cu(I) is bound to the anionic alkyne, the kinetic barrier becomes

increased to ∆

G^‡= 19.8 kcal mol⁻¹, and even further to ∆G^‡= 22.0 kcal mol⁻¹when the

reaction occurs with the azide-bound metal. All of this again eliminates the necessity to

employ any catalytic base in the reaction, let alone that it could, on its own, undergo the

complexation reaction with the Cu(I) ions, thus further interfere with the reaction progress

in an undesired way.

In summarizing this part, we can emphasize that obtained reaction proﬁles clearly

support the role of the metal catalyst in facilitating the conversion and demonstrate that

Cu(I) ions act by binding and activating the alkyne for a successful reaction with nucle-

ophilic azides. Also, the revealed kinetic and thermodynamic aspects seem to suggest that

increasing the reaction temperature will likely improve the reaction outcomes, which is

found in excellent agreement with a general trend that method B at higher temperatures

with ultrasound irradiation offers better reaction yields than method A. Still, to under-

stand different reaction outcomes between methods A and B, where reactive Cu(I) ions

are generated in situ, and the method C, where these are directly employed, we must

not forget that ﬁrst two approaches contain both Cu(II) ions and elementary Cu(0) within

the solution, which could, at certain cases, impact the reaction and allow different yields,

whether higher or lower. To investigate this possibility, we have repeated the analysis

considering alternative copper oxidation states.

When Cu(0) is concerned, it more favorably binds to the azide ( ,

20.5 kcal mol⁻¹

−

Figure S41), which even surpasses the most exergonic interaction that charged Cu(I) makes

with alkyne by 8.6 kcal mol⁻¹, likely being the result of the solvation effect. In that sce-

nario (Figure S44), as expected, the formation of the reactive complex is highly exergonic

(

−13.7 kcal mol⁻¹

), yet leading to a very high barrier of ∆

G^‡= 33.0 kcal mol⁻¹, which

makes this process as unfeasible and renders any impact of Cu(0) on the reaction outcomes

as insigniﬁcant. We note in passing that a much less stable reactive complex involving the

alkyne-bound Cu(0) would proceed in a stepwise fashion with both C–N bonds formed sep-

arately (Figure S44) and the rate-limiting second step. On the other hand, Cu(II) ions reveal

a very interesting trend as their ability to complex reactants is precisely identical for both

alkyne and azide at

∆

G_R=

−

13.0 kcal mol⁻¹(Figure S41), which makes both options viable.

Molecules 2021, 26, 3334

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However, the reactive complex with the alkyne-bound Cu(II) is by 6.2 kcal mol⁻¹more

favorable, which directs the reactivity towards the stepwise triazole formation (Figure S45)

where both N–C bonds are created under similar kinetic requirements. Still, the process to

form the second bond is a bit more demanding and deﬁnes the rate-limiting step, yet the

,

overall activation energy is only ∆

G^‡= 8.3 kcal mol⁻¹, which hints at a possible impact

of Cu(II) ions on the overall conversion. However, one must emphasize that this reaction

is again associated with a rather high energy cost of 12.7 kcal mol⁻¹to move from the

reactive complex and detach Cu(II) to allow the ﬁnal triazole, which might hinder any

positive effect, let alone the availability of Cu(II) to undertake the reaction in the ﬁrst place.

Which of these aspects will prevail in solution and whether the impact of Cu(II) will be

signiﬁcant or insigniﬁcant at all, is difﬁcult to say, and may likely depend on a particular

reaction condition and different electronic and structural features of the employed reac-

tants. As such, using Cu(II) salts in a combination with elementary copper, in order to

generate Cu(I) in situ, may and likely will generate slightly different conditions for the

alkyne-azide cycloaddition reaction than when Cu(I) salts are directly used, which justiﬁes

why different trends are experimentally detected and no wide-ranging conclusions can be

made in this respect.

Lastly, a somewhat general trend emerging from experiments is that 6-chloro deriva-

tives are typically more reactive than their 6-amino analogues when reaction times or

yields are compared. At ﬁrst, such differences appear surprising given a large distance

of 6-substituents from the reacting triazole to exhibit any direct impact, or the fact that

triazole is separated from the aromatic fragment by two methylene units for any indirect

inﬂuence of these substituents through resonance/inductive effects. Therefore, a plausible

explanation for the observed reactivity differences could be linked with their abilities to

form complexes with metal catalysts (Figure S46). Namely, electron-withdrawing chlorine

depletes the electron density from the aromatic skeleton, which diminishes the complex for-

mation with Cu(I). In contrast, electron-donating amines consistently increase the tendency

to complex Cu(I) in solution in both pyrimidine and purine derivatives, which could reduce

the catalytic efﬁciency of the metal, thus somewhat lower reactivity of 6-amino derivatives.

2.3. Biological Evaluations

2.3.1. Antiproliferative Evaluations

The antiproliferative results obtained for the prepared compounds on human tumor

cell lines, including lung adenocarcinoma (A549), cervical carcinoma (HeLa), ductal pancre-

atic adenocarcinoma (CFPAC-1) and metastatic colorectal adenocarcinoma (SW620) cells,

are presented in Table 2. Compounds that exhibited marked cytostatic activity (BIS-PP2

5b 5f 7a 9a 9b 9f 10b and 10f) were additionally evaluated for their cytotoxic effects

on normal human foreskin ﬁbroblasts (HFF-1), as indicated in the footnote of the Table 2

,

.

For comparison, anti-proliferative effects of chloropyrrolo[2,3-d]pyrimidine structural

analogues BIS-PP1, BIS-PP2 [45] were also included in Table 2.

Molecules 2021, 26, 3334

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Table 2. Growth-inhibition effects in vitro of bis- and mono-pyrrolo[2,3-d]pyrimidine and purine derivatives on selected

tumor cell lines and normal ﬁbroblasts.

1

IC₅₀(µM)

Compd

Base

X

A549

HeLa

CFPAC-1

SW620

BIS-PP1

BIS-PP2 ²

5a

9.4 ± 1.16

4.2 ± 1.39

>100

6.3 ± 1.2

6.9 ± 1.1

99.6 ± 1.5

>100

7.0 ± 0.64

2.3 ± 0.99

77.0 ± 2.6

3.6 ± 2.02

0.95 ± 0.28

>100

40.8 ± 3.83

6.8 ± 0.69

>100

8.0 ± 1.8

>100

5b ²

5f ²

5c

5g

5d

>100

0.98 ± 0.44

99.7 ± 5.9

75.0 ± 3.5

>100

0.79 ± 0.03

74.9 ± 3.8

19.5 ± 4.0

36.5 ± 3.9

CH₂

O

80.5 ± 14.9

>100

53.5 ± 4.8

6a

>100

6b

6c

6d

53.9 ± 2.1

59.3 ± 4.9

51.9 ± 3.0

68.5 ± 3.2

33.7 ± 8.9

28.2 ± 9.0

17.4 ± 6.3

48.7 ± 1.5

37.9 ± 3.7

24.7 ± 8.7

65.2 ± 4.8

45.5 ± 3.3

CH₂

O

7a ²

49.3 ± 6.4

6.4 ± 0.35

25.8 ± 0.98

78.0 ± 2.1

7b

7c

7d

69.0 ± 1.2

54.6 ± 5.2

80.0 ± 1.5

>100

77.4 ± 3.5

65.3 ± 5.7

>100

CH2

O

>100

9a ²

9e

53.5 ± 8.0

58.7 ± 10.2

12.3 ± 3.4

9.3 ± 0.1

>100

73.1 ± 7.7

16.2 ± 0.05

17.1 ± 1.3

3.8 ± 0.81

3.9 ± 0.69

41.2 ± 0.99

72.2 ± 9.0

34.7 ± 12.1

9.1 ± 1.3

23.4 ± 4.2

8.1 ± 1.3

43.5 ± 4.4

35.7 ± 0.79

5.2 ± 2.2

23.2 ± 3.2

>100

66.1 ± 3.0

9b²

9f²

9c

2.9 ± 1.0

53.9 ± 3.4

75.2 ± 12.2

56.8 ± 2.9

CH2

O

9g

9d

10a

95.0 ± 3.2

60.6 ± 5.9

48.5 ± 1.6

98.3 ± 7.4

10b ²

10f ²

10c

8.5 ± 0.1

62.1 ± 2.3

88.6 ± 6.4

87.3 ± 4.4

7.4 ± 0.09

8.1 ± 2.2

16.4 ± 4.6

6.5 ± 0.78

49.9 ± 1.7

52.5 ± 4.6

59.1 ± 0.34

98.0 ± 1.3

>100

CH₂

O

60.4 ± 5.0

10d

66.9 ± 11.2

63.4 ± 3.6

11a

11e

11g

11d

11h

>100

CH₂

O

1

50% inhibitory concentration or compound concentration required inhibiting tumor cell proliferation by 50%.²IC₅₀= 0.11

for BIS-PP2, IC₅₀= 0.13 0.01 M for 5b, IC₅₀= 0.62 0.16 M for 5f, IC₅₀= 0.81 0.12 M for 7a, IC₅₀= 0.76 0.20

M for 9f, IC₅₀= 0.51 0.16 M for 10b and IC₅₀= 9.4 0.30 M for 10f on normal foreskin

±

0.37

M for 9a,

µM

±

µ

±

µ

±

µ

±

µ

IC₅₀= 0.79 ± 0.18 µM for 9b, IC₅₀= 4.3

±

1.7

µ

±

µ

±

µ

ﬁbroblasts (HFF-1).

From the series of 4-chloropyrrolo[2,3-d]pyrimidines, compound 5f, with the aromatic

unit, showed the most potent cytostatic activity, particularly on HeLa (IC₅₀= 0.98 M) and

CFPAC-1 (IC₅₀= 0.79 µM) cells. Antiproliferative effects of 5f were somewhat better than

those of previously published structural analogues BIS-PP1 BIS-PP2 45]. Importantly,

this compound showed to be more selective than BIS-PP1, BIS-PP2 exhibiting lower toxicity

to normal ﬁbroblasts (HFF-1). On the contrary, compounds 5c 5d and 5g with aliphatic

µ

,

[

,

propylene and oxydimethylene central unit exhibited only moderate activity on HeLa,

CFPAC-1 and SW620 cell lines.

Molecules 2021, 26, 3334

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Among cyclic amino-substituted bis-pyrrolo[2,3-d]pyrimidines, 4-pyrrolidine ana-

logue 7a with 1,4-bis(oxymethylene)phenyl linker exhibited marked growth-inhibitory

effect (IC₅₀= 6.4

ity (IC₅₀= 17.4

µ

M) on HeLa cells, while 4-piperidine analogue 6b had strong activ-

µ

M) on CFPAC-1 cells. Only 4-piperidine pyrrolo[2,3-d]pyrimidines 6c

and 6d, with aliphatic spacers, had moderate antiproliferative activities in the range of

28.2−65.2 µM compared to corresponding analogues 5c, 5d and 7c, 7d.

In the series of bis- and mono-purines, 6-piperidinyl- (9b and 9f) and 6-pyrrolidinylpurines

0

(

10b and 10f) containing 4,4 -bis(oxymethylene)biphenyl exhibited marked, although rather

non-speciﬁc inhibitory effects. Bis-purines 9b and 10b showed the highest activity on HeLa

cell line (9b: IC₅₀= 3.8 M; 10b: IC₅₀= 7.4 M). Structural analogues 9a 9e and 10a

with 1,4-bis(oxymethylene)phenyl linker displayed decreased activity, while compounds

with aliphatic unit (9c 9g 9d and 10c 10d) showed only moderate to marginal growth-

inhibition, that is in accordance with the ﬁndings for pyrrolo[2,3-d]pyrimidines. Both

µ

,

bis- and mono-adenine derivatives showed no inhibitory activity (IC₅₀> 100

µM) on all

evaluated tumor cell lines.

Comparing to BIS-PP2, it can be observed that structural modiﬁcations in novel bis-

and mono-pyrrolo[2,3-d]pyrimidine and purine derivatives reduced their cytotoxic effects

on normal HFF-1 cells to a lesser extent. Compounds with best antiproliferative activities

also exhibited inhibitory effects on normal HFF-1 cells.

Overall, it can be observed that the linker between the heterocycle scaffolds in the

symmetrical 7-deazapurines and bis-purines had a signiﬁcant impact on antitumor activity

(Figure 5).

Figure 5. Structure–activity relationship for antiproliferative activity of bis- and mono-pyrrolo[2,3-

d]pyrimidine and purine derivatives.

Compounds with aliphatic linkers (5c

erate activity or were deprived of any antitumor activities, while introducing the 4,4⁰-

bis(oxymethylene)biphenyl linker (5b 9b and 10b) caused an enhancement of tumor cell

–7c, 5d–7d, 9c, 10c and 9d–11d) exhibited mod-

,

growth inhibition. The inﬂuence of heterocyclic scaffold was also observed showing that

series of cyclic 6-amino bis-purines (9a−9d and 10a−10d) were generally more active than

the corresponding cyclic 4-amino bis-pyrrolo[2,3-d]pyrimidine (6a−6d and 7a−7d) ana-

logues. Comparison of the antiproliferative activity of all purine derivatives showed that

cyclic amines in purine derivatives improved the activity relative to adenine derivatives

that did not exhibit inhibitory activities.

2.3.2. Apoptosis Detection

Annexin V assay was performed to determine if the antiproliferative activity of

compound 5f exhibiting the most pronounced potency in ductal adenocarcinoma cancer

cell line (CFPAC-1) could be attributed to induction of apoptosis. Obtained data are

presented in Table 3 and Figure 6.

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Table 3. Annexin V assay for apoptosis detection in CFPAC-1 cells ¹.

48 h Treatment

5f (%)

72 h Treatment

5f (%)

3.95 µM

5f (%)

1.85 µM

5f (%)

3.95 µM

CFPAC-1

Control (%)

1.85 µM

viable cells

96.58 ± 0.54

79.64 ± 2.57

68.18 ± 0.94

90.48 ± 0.02

6.28 ± 1.53

56.25 ± 6.56

27.93 ± 3.23

early apoptotic cells

late apoptotic/primary

necrotic cells

0.0

11.76 ± 3.58

21.21 ± 1.35

30.36 ± 1.49

39.63 ± 8.54

0.86 ± 0.05

2.56 ± 1.40

5.88 ± 1.44

2.72 ± 0.56

6.56 ± 0.24

4.05 ± 1.22

0.0

10.71 ± 4.89

2.68 ± 0.37

26.14 ± 2.57

6.3 ± 2.45

secondary necrotic cells

3.24 ± 0.92

¹The percentages of viable cells (Ann

−

/PI

necrotic cells (PI+) after 48 h and 72 h treatment with compound 5f at 2

is untreated cells. Data are expressed as mean ± SD from two independent experiments.

−

), early apoptotic cells (Ann+), late apoptotic/primary necrotic cells (Ann+/PI+) and secondary

×

IC₅₀(1.58

µ

M) and 5

×

IC₅₀(3.95 µM) values are shown. Control

Figure 6. Detection of apoptosis induced by compound 5f in ductal adenocarcinoma cancer cell line

CFPAC-1 using Annexin V assay after 48 (A) and 72 (B) hours of treatment at concentrations of 2 ×

IC₅₀(1.58

µ

M) and 5

×

IC₅₀(3.95

µ

M). Cells were visualized by ﬂuorescence microscope using 40

×

magniﬁcations. Shown here are bright-ﬁeld images and late apoptotic/primary necrotic cells.

Table 3 shows percentages of cells in different stages of apoptosis. It can be observed

that compound 5f showed pro-apoptotic effect in CFPAC-1 cells as early as 48 h after

treatment with both 2

48-h treatment, both concentrations of compound 5f showed pro-apoptotic activity, where

IC₅₀concentration induced decrease in cell viability by 28.4% and increase in the

×

IC₅₀(1.58

µM) and 5

×

IC₅₀(3.95 µM) concentrations. Following

5

×

percentage of cells that underwent early apoptosis by 21.21%. After 72 h of treatment, a

signiﬁcant decrease in cell viability was noticed in both treatments concomitantly with a

profound increase in early apoptotic cells after 2

×

IC₅₀and 5

×

IC₅₀treatments by 24.08%

Molecules 2021, 26, 3334

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and 33.35%, respectively, followed by a dramatic rise in the late apoptotic/primary necrotic

cells by 26.14% that occurred after 5

compound 5f induces apoptosis and primary necrosis in CFPAC-1 cells in a concentration-

×

IC₅₀treatment. Collectively, these results show that

and time-dependent manner.

3. Materials and Methods

3.1. General

All the solvents and chemicals were purchased from Aldrich (St. Louis, MO, USA)

and Acros (Geel, Belgium). Thin layer chromatography was performed on pre-coated

silica gel 60F-254 plates (Merck, Kenilworth, NJ, USA ) while glass column slurry-packed

under gravity with silica gel (0.063–0.2 mm Fluka, Seelze, Germany) was employed for

column chromatography. Melting points of compounds were determined using a Koﬂer

1

micro hot stage. H- and ¹³C-NMR spectra were recorded on a Bruker 300 and 600 MHz

spectrometers (Bruker, Billerica, MA, USA). All data were recorded in dimethyl sulfoxide

(DMSO-d₆) at 298 K. Chemical shifts were referenced to the residual solvent signal of DMSO

at δ δ

2.50 ppm for ¹H and 39.50 ppm for ¹³C. Individual resonances were assigned based on

their chemical shifts, signal intensities, multiplicity of resonances, H–H coupling constants.

The ultrasound-assisted reactions were carried out in a Bandelin Bath Cleaner (Sonorex

Digital 10 P, Berlin, Germany) with a nominal power of 1000 W and frequency of 35 kHz.

The reactions were carried out in a round-bottomed ﬂask of 25 mL capacity suspended at

the center of the cleaning bath, 5 cm below the surface of the liquid. Microwave-assisted

syntheses were performed in a Milestone start S microwave oven) (Sorisole, Italy) using

glass cuvettes at 100 ^◦C and 400 W.

3.2. Experimental Procedure for the Synthesis of Compounds

The compounds 6-(piperidin-1-yl)-9H-purine (1c) [58 ], 6-(pyrrolidin-1-yl)-9H-purine

(1d) [59 ], 7-(2-bromoethyl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (2a) [45 ], 9-(2-bromoethyl)-

9H-purin-6-amine (2e) [60 ], 7-(2-azidoethyl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (3a) [45 ], 9-

0

(2-azidoethyl)-9H-purin-6-amine (3e) [60 ], 1,4-bis(prop-2-yn-1-yloxy)benzene (4a) [46 ] and 4,4 -

0

bis(prop-2-yn-1-yloxy)-1,1 -biphenyl (4b) [47 ] were prepared according to known procedures.

3.3. General Procedure for the N-Alkylation of Compounds

The corresponding heterocyclic base 1a–1e was dissolved in dry DMF (8 mL), K₂CO₃

was added (1.2 eq) and the reaction mixture was stirred for 1 h. 1,2-Dibromoethane (1.2 eq)

was added to the mixture and stirred for 24 h at room temperature. The solvent was

evaporated to dryness and the residue was puriﬁed by column chromatography.

9-(2-Bromoethyl)-6-chloro-9H-purine (2b). Compound 2b was prepared according to

the abovementioned procedure from compound 1b (1 g, 7.40 mmol). After puriﬁcation

by column chromatography (CH₂C_◦l₂:MeOH = 50:1) compound 2b was obtained as white

crystal (1.24 g, 74%, m.p. = 110–112 C). ¹H-NMR (300 MHz, DMSO-d₆) (

δ/ppm): 8.81 (1H,

s, H2), 8.76 (1H, s, H8), 4.74 (2H, t, J = 6.0 Hz, CH₂CH₂), 4.01 (2H, t, J = 6.0 Hz, CH₂CH₂).

¹³C-NMR (75 MHz, DMSO-d₆) (

δ

/ppm): 151.9 (C6), 151.6 (C2), 149.1 (C4), 147.5 (C8), 130.8

(C5), 45.3 (CH₂), 31.1 (CH₂).

9-(2-Bromoethyl)-6-(piperidine-1-yl)-9H-purine (2c).

Compound 2c was prepared accord-

ing to the abovementioned procedure from compound 1c (550 mg, 2.71 mmol). After

puriﬁcation by column chromatography (CH₂Cl₂:MeOH = 100:1) compound 2c was ob-

tained as white solid (650 mg, 76%, m.p. = 148–150 ^◦C). ¹H-NMR (300 MHz, DMSO-d₆)

(

δ

/ppm): 8.22 (1H, s, H2), 8.19 (1H, s, H8), 4.57 (2H, t, J = 6.1 Hz, CH₂CH₂), 4.19 (4H,

bs, CH₂), 3.94 (2H, t, J = 6.1 Hz, CH₂CH₂), 1.78–1.42 (6H, m, CH₂). ¹³C-NMR (75 MHz,

DMSO-d₆) ( /ppm): 153.1 (C6), 151.9 (C2), 150.5 (C4), 139.7 (C8), 118.8 (C5), 45.6 (CH₂),

δ

44.6 (CH₂), 31.4 (CH₂), 25.6 (CH₂), 24.2 (CH₂).

9-(2-Bromoethyl)-6-(pyrrolidine-1-yl)-9H-purin (2d) Compound 2d was prepared accord-

ing to the abovementioned procedure from compound 1d (650 mg, 3.44 mmol). After

puriﬁcation by column chromatography (CH₂Cl₂:MeOH = 100:1) compound 2d was ob-

Molecules 2021, 26, 3334

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tained as white solid (650 mg, 64%, m.p. = 162–164 ^◦C). ¹H-NMR (300 MHz, DMSO-d₆)

(

δ

/ppm): 8.21 (1H, s, H2), 8.16 (1H, s, H8), 4.57 (2H, t, J = 6.0 Hz, CH₂CH₂), 4.04 (2H, s,

CH₂), 3.94 (2H, t, J = 6.1 Hz, CH₂CH₂), 3.63 (2H, s, CH₂), 1.95 (6H, s, CH₂). ¹³C-NMR

(75 MHz, DMSO-d₆) ( /ppm): 150.7 (C2), 149.4 (C4), 140.9 (C8), 119.2 (C5), 59.2 (CH₂), 45.9

δ

(CH₂), 44.7 (CH₂), 31.5 (CH₂).

3.4. General Procedure for the Synthesis of Azidoethyl Derivatives

The corresponding 2-bromoethyl derivative 2a–2f was dissolved in acetone. NaN₃(4

eq) dissolved in water (~ 3 mL) was added dropwise to the reaction mixture and stirred

under reﬂux overnight. The solvent was evaporated to dryness and the residue dissolved

in ethyl-acete (60 mL) and extracted with brine. The organic layer was dried over MgSO₄,

ﬁltered and evaporated.

6-Azido-9-(2-azidoethyl)-9H-purine (3b). Compound 3b was prepared according to the

abovementioned procedure from compound 2b (652 mg, 2.49 mmol) to give compound

◦

1

3b as white solid (230.2 mg. 63%; m.p. = 108–112 C). H-NMR (600 MHz, DMSO-d₆)

(δ/ppm): 10.13 (1H, s, H2), 8.70 (1H, s, H8), 4.62 (2H, t, J = 5.6 Hz, CH₂CH₂), 3.93 (2H, t,

J = 5.6 Hz, CH₂CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm): 145.4 (C6), 144.5 (C2), 142.4

(C4), 135.9 (C8), 119.7 (C5), 49.9 (CH₂), 43.6 (CH₂).

9-(2-Azidoethyl)-6-(piperidin-1-yl)-9H-purine (3c). Compound 3c was prepared accord-

ing to the abovementioned procedure from compound _◦2c (650 mg, 2.10 mmol) to give

compound 3c as white solid (566.3 mg, 99%, m.p. = 78–80 C). ¹H-NMR (300 MHz, DMSO-

d₆) (

CH₂), 3.81 (2H, t, J = 5.7 Hz, CH₂CH₂), 1.72–1.52 (6H, m, CH₂CH₂). ¹³C-NMR (75 MHz,

DMSO-d₆) ( /ppm): 153.1 (C6), 151.9 (C2), 150.6 (C4), 139.7 (C8), 118.9 (C5), 49.6 (CH₂),

δ/ppm): 8.22 (1H, s, H2), 8.18 (1H, s, H8), 4.35 (2H, t, J = 5.7 Hz, CH₂CH₂), 4.19 (4H, s,

δ

42.4 (CH₂), 25.7 (CH₂), 24.2 (CH₂).

9-(2-Azidoethyl)-6-(pyrrolidin-1-yl)-9H-purine (3d). Compound 3d was prepared ac-

cording to the abovementioned procedure from compound 2e (400 mg, 1.35 mmol) to

give compound 3d as white solid (345.1 mg, 99%, m.p. = 96–101 ^◦C). ¹H-NMR (300 MHz,

DMSO-d₆) (δ/ppm): 8.21 (1H, s, H2), 8.15 (1H, s, H8), 4.35 (2H, t, J = 5.7 Hz, CH₂CH₂),

4.05 (2H, bs, CH₂), 3.80 (2H, t, J = 5.7 Hz, CH₂CH₂), 3.63 (2H, bs, CH₂), 1.95 (4H, bs, CH₂).

¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm): 152.5 (C6), 152.2 (C2), 150.0 (C4), 140.2 (C8),

119.3 (C5), 49.7 (CH₂), 42.3 (CH₂).

3.5. General Procedure for the Synthesis of bis- (5a–5d, 9a–9d, 10a–10d, 11a, 11d) and Mono-

(5f, 5g, 9e–9g, 10f, 11e, 11g, 11h) Pyrrolo[2,3-d]pyrimidines and Purines

Method A: The reaction mixture of the corresponding bis-alkyne 4a

–4d (1 eq), 2-

azidoethyl base 3a 3e (2.2 eq), Cu(0) (1 eq) and 1M CuSO₄(0.3 eq) in 1 mL DMF and a

–

mixture of t-BuOH:H₂O = 1: 1 (3 mL) was stirred at room temperature for 120 h. The

solvent was evaporated and the residue was puriﬁed by column chromatography using

CH₂Cl₂, as an initial eluent, and CH₂Cl₂:CH₃OH = 10:1, as ﬁnal eluent.

Method B: The reaction mixture of the corresponding bis-alkyne 4a

–4d (1 eq), 2-

azidoethyl base 3a 3e (2.2 eq), Cu(0) (1 eq) and 1M CuSO₄(0.3 eq) in 1 mL DMF and a

–

mixture of t-BuOH:H₂O = 1:1 (3 mL) was placed in an ultrasonic bath cleaner (1000 W,

35 kHz) at 80 ^◦C for 1.5 h. The solvent was evaporated and the residue was puriﬁed by

column chromatography using CH₂Cl₂, as an initial eluent, and CH₂Cl₂:CH₃OH = 10:1, as

ﬁnal eluent.

Method C: The reaction mixture of the corresponding bis-alkyne 4a

–4d (1 eq), 2-

azidoethyl base 3a 3e (2.2 eq), CuI(0) (0.2 eq), N,N-diisopropylethylamine (4 eq) and acetic

–

acid (4 eq) in CH₂Cl₂(1 mL) was stirred at room temperature for 72 h. The solvent was

evaporated and the residue was puriﬁed by column chromatography using CH₂Cl₂, as an

initial eluent, and CH₂Cl₂:CH₃OH = 10:1, as ﬁnal eluent.

1,4-Bis{[1-(2-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)-1H-1,2,3-triazol-4-yl]methoxy}

benzene (5a). Compound 5a was prepared according to the abovementioned procedure

from compound 4a (method A: 50 mg, 0.27 mmol; method B: 22 mg, 0.11 mmol; method

C: 50 mg, 0.27 mmol) and compound 3a (method A: 132.2 mg, 0.59 mol; method B: 58 mg,

Molecules 2021, 26, 3334

15 of 26

0.44 mmol; method C: 123.2 mg, 0.59 mmol) to obtain 5a as white solid (method A: 83.5 mg,

◦

1

49%; method B: 50 mg, 67%; method C: 40.4 mg, 24%; m.p. = 234–239 C). H-NMR

(300 MHz, DMSO-d₆) (

/ppm): 8.54 (2H, s, H2), 8.01 (2H, s, H5⁰), 7.49 (2H, d, J = 3.6

Hz, H6), 6.89 (4H, s, Ph), 6.57 (2H, d, J = 3.6 Hz, H5), 4.98 (4H, s, CH₂), 4.89–4.83 (4H,

δ

m, CH₂CH₂), 4.79–4.73 (4H, m, CH₂CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm):

152.2 (Ph-q), 150.7 (C4), 150.5 (C7a), 150.3 (C2), 143.0 (C4⁰), 131.1 (C6), 124.7 (C5⁰), 116.7

(C4a), 115.6 (Ph), 98.8 (C5), 61.4 (OCH₂), 49.0 (CH₂CH₂), 44.7 (CH₂CH₂). Anal. calcd. for

C₂₈H₂₄Cl₂N₁₂O₂: C, 53.26; H, 3.83; N, 26.62. Found: C, 53.56; H, 3.95; N, 26.44.

0

4,4 -Bis{[1-(2-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)-1H-1,2,3-triazol-4-yl]-methoxy}-

1,1⁰-biphenyl (5b) and 4-chloro-7-{2-[4-(((4⁰-(prop-2-yn-1-yloxy)-[1,1⁰-biphenyl]-4-yl)oxy)methyl)-

1H-1,2,3-triazol-1-yl]ethyl}-7H-pyrrolo[2,3-d]pyrimidine (5f). Compounds 5b and 5f were pre-

pared according to the abovementioned procedure from compound 4b (method A: 50 mg,

0.19 mmol; method B: 29.4 mg, 0.11 mmol; method C: 50 mg; 0.19 mmol) and compound 3a

(method A: 93.1 mg, 0.42 mol; method B: 55 mg, 0.24 mmol; method C: 93.1 mg, 0.42 mmol)

to obtain 5b as white solid (method A: 46.4 mg, 34%; method B: 94.4 mg, 66%; method C: 26.2

mg, 35%; m.p. > 260 ^◦C) and 5f as yellow solid (method C: 8 mg, 14%; m.p. = 234–238 ^◦C).

1

5b: H-NMR (600 MHz, DMSO-d₆) (

δ

/ppm): 8.54 (2H, s, H2), 8.06 (2H, s, H5⁰), 7.54

(4H, d, J = 8.7 Hz, Ph), 7.49 (2H, d, J = 3.6 Hz, H6), 7.03 (4H, d, J = 8.7 Hz, Ph), 6.56 (2H, d,

J = 3.6 Hz, H5), 5.08 (4H, s, OCH₂), 4.90–4.86 (4H, m, CH₂CH₂), 4.79–4.75 (4H, m, CH₂CH₂).

¹³C-NMR (75 MHz, DMSO-d₆) (

δ

/ppm): 157.1 (Ph-q), ₀150.7 (C4), 150.5 (C7a), 150.2 (C2),

0

142.8 (C4 ), 132.5 (Ph-q), 131.1 (C5), 127.2 (Ph), 124.8 (C5 ), 116.7 (C4a), 115.1 (Ph), 98.7 (C6),

61.0 (OCH₂), 49.0 (CH₂CH₂), 44.6 (CH₂CH₂). Anal. calcd. for C₃₄H₂₈Cl₂N₁₂O₂: C, 57.71;

H, 3.99; N, 23.75. Found: C, 57.65; H, 3.73; N, 23.74.

5f: δ

¹H-NMR (300 MHz, DMSO-d₆) ( /ppm): 8.54 (1H, s, H2), 8.05 (1H, s, H5⁰),

7.62–7.49 (4H, m, Ph), 7.49 (1H, d, J = 3.6 Hz, H6), 7.16–6.92 (4H, m, Ph), 6.57 (1H, d,

J = 3.6 Hz, H5), 5.08 (2H, s, OCH₂), 4.91–4.84 (2H, m, J = 5.4 Hz, CH₂CH₂), 4.82 (2H,

d, J = 2.3 Hz

, CH₂CCH), 4.80–4.74 (2H, m, CH₂CH₂), 3.58 (1H, t, J = 2.3 Hz, CH₂CCH).

¹³C-NMR (75 MHz, DMSO-d₆) (

δ

/ppm): 157.1 (Ph-q), 150.5 (C7a), 150.2 (C2), 142.8 (C4⁰),

132.5 (Ph-q), 131.1 (Ph), 127.2 (Ph), 124.7 (C5⁰), 116.7 (C4a), 115.1 (Ph/C5), 98.8 (C6), 79.4

(CCH), 61.0 (OCH₂), 49.0 (CH₂CH₂), 45.7 (CH₂CH), 44.6 (CH₂CH₂). Anal. calcd. for

C₂₆H₂₁ClN₆O₂: C, 64.40; H, 4.37; N, 17.33. Found: C, 64.56; H, 4.33; N, 17.42.

1,3-Bis{1-[2-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-1H-1,2,3-triazol-4-yl}propane

(5c) and 4-chloro-7-{2-[4-(pent-4-yn-1-yl)-1H-1,2,3-triazol-1-yl]ethyl}-7H-pyrrolo[2,3-d]pyrimid

ine (5g). Compounds 5c and 5g were prepared according to the abovementioned procedure

from 1,6-heptadiyne 4c (method A: 10.5 mg, 0.11 mmol; method B: 100 mg, 1.09 mmol; method

C: 50 mg, 0.54 mmol) and compound 3a (method A: 56 mg, 0.25 mmol; method B: 534.3 mg,

2.4 mol; method C: 264.5 mg, 1.19 mmol) to obtain 5c as white solid (method A: 26.6 mg, 45%;

method B: 258 mg, 62%; method C: 183 mg, 63%; m.p. = 208–210 ^◦C) and 5g as yellow oil

(method A: 4 mg, 11%; method C: 51.5 mg, 35%).

1

5c: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.54 (2H, s, H2), 7.62 (2H, s, H5⁰), 7.52

(2H, d, J = 3.6 Hz, H6), 6.59 (2H, d, J = 3.6 Hz, H5), 4.87–4.67 (8H, m, CH₂CH₂), 2.43

(4H, t, J = 7.4 Hz, CH₂CH₂CH₂), 1.75–1.57 (2H, m, CH₂CH₂CH₂). ¹³C-NMR (75 MHz,

DMSO-d₆) (δ/ppm): 150.7 (C4), 150.5 (C7a), 150.2 (C2), 146.3 (C4’), 131.1 (C6), 122.1 (C5’),

116.7 (C4a), 98.7 (C5), 48.8 (CH₂CH₂), 45.0 (CH₂CH₂), 28.7 (CH₂), 24.0 (CH₂). Anal. calcd.

for C₂₃H₂₂Cl₂N₁₂: C, 51.40; H, 4.13; N, 31.28. Found: C, 51.66; H, 4.09; N, 31.35.

1

5g: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.55 (1H, s, H2), 7.65 (1H, s, H5⁰), 7.52

(1H, d, J = 3.6 Hz, H6), 6.60 (1H, d, J = 3.6 Hz, H5), 4.97–4.64 (4H, m, CH₂CH₂), 2.78 (1H, t,

J = 2.6 Hz, CH), 2.58 (2H, t, J = 7.4 Hz, CH₂), 2.08 (2H, td, J = 7.1, 2.6 Hz, CH₂), 1.71–1.53

(2H, m, CH₂). ¹³C-NMR(151 MHz,₀DMSO-d₆) (

δ/ppm): 150.8 (C4), 150.6 (C7a), 150.2

0

(C2), 146.1 (C4 ), 131.1 (C6), 122.3 (C5 ), 116.7 (C4a), 98.8 (C5), 84.1 (CCH), 71.4 (CCH), 48.9

(CH₂CH₂), 44.7 (CH₂CH₂), 27.9 (CH₂), 23.8 (CH₂), 17.0 (CH₂). Anal. calcd. for C₁₅H₁₅ClN₆:

C, 57.24; H, 4.80; N, 26.70. Found: C, 57.31; H, 4.90; N, 26.57.

{1-[2-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-1H-1,2,3-triazol-4-yl}methyl ether

(

5d). Compound 5d was prepared according to the abovementioned procedure from

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propargyl ether 4d (100 mg, 1.06 mmol) and compound 3a (495.6 mg, 2.33 mmol) to obtain

5d as white solid (method A: 290.1 mg, 51%; method C: 363 mg, 64%; m.p. = 206–208 ^◦C)

.

¹H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.54 (2H, s, H2), 7.93 (2H, s, H5⁰), 7.52 (2H, d,

J = 3.6 Hz, H6), 6.59 (2H, d, J = 3.6 Hz, H5), 4.89–4.83 (4H, m, CH₂CH₂), 4.80–4.74 (4H,

m, CH₂CH₂), 4.34 (4H, s, CH₂OCH₂). ¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm): 150.7

(C4), 150.5 (C7a), 150.2 (C2), 143.5 (C4⁰), 131.1 (C6), 124.3 (C5⁰), 116.7 (C4a), 98.8 (C5), 62.0

(CH₂OCH₂), 48.9 (CH₂CH₂), 44.6 (CH₂CH₂). Anal. calcd. for C₂₂H₂₀Cl₂N₁₂O: C, 48.99; H,

3.74; N, 31.16. Found: C, 49.01; H, 3.62; N, 31.16.

7-{2-[4-((Prop-2-yn-1-yloxy)methyl)-1H-1,2,3-triazol-1-yl]ethyl}-7H-tetrazolo[5,1-i]purine (8h

)

Compound 8h was prepared according to the abovementioned procedure for method B

from propargyl ether 4d (0.10 mL, 1.01 mmol) and compound 3b (513.1 mg, 2.23mmol) to

obtain 8h as white crytals (189.4 mg, 59%; m.p. = 149–148 ^◦C). ¹H (300 MHz, DMSO-d₆)

(δ

/ppm): 10.07 (1H, s, H2), 8.39 (1H, s, 8H), 7.95 (1H, s, H5⁰), 4.99–4.87 (4H, m, CH₂CH₂),

4.46 (2H, s, CH₂), 4.06 (2H, d, J = 2.4 Hz, CH₂CCH), 3.44 (1H, t, J = 2.4 Hz, CH₂CCH). ¹³C

(75 MHz, DMSO-d₆) (

δ

/ppm): 145.4 (C6), 143.3 (C4), 142.3 (C4⁰), 135.7 (C8), 124.6 (C5⁰),

119.6 (C5), 79.9 (CCH), 77.4 (CCH), 61.7 (CH₂), 56.4 (CH₂), 48.9 (CH₂CH₂), 44.5 (CH₂CH₂).

Anal. calcd. for C₁₃H₁₂N₁₀O: C, 48.15; H, 3.73; N, 43.19. Found: C, 48.08; H, 3.76; N, 43.29.

1,4-Bis{[1-(2-(6-(piperidin-1-yl)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-4-yl]methoxy}be

nzene (9a) and 6-(piperidin-1-yl)-9-{2-[4-((4-(prop-2-yn-1-yloxy)phenoxy)methyl)-1H-1,2,3-

triazol-1-yl]ethyl}-9H-purine (9e) Compounds 9a and 9e were prepared according to the

above-mentioned procedure from compound 4a (50 mg, 0.27 mmol) and compound 3c

(160.7 mg, 0.59 mmol) to obtain 9a as white solid (method A: 58.1 mg, 28%; method B:

◦

121.1 mg, 61%; method C: 67.5 mg, 36%; m.p. = 233–236 C) and 9e (method A: 4.9 mg, 4%;

method C: 34.4 mg, 29%; m.p. = 158–160 ^◦C).

1

9a: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.18 (2H, s, H2), 8.12 (2H, s, H8), 7.83

(2H, s, H5⁰), 6.90 (4H, s, Ph), 5.01 (4H, s, CH₂), 4.87 (4H, t, J = 5.7 Hz, CH₂CH₂), 4.65 (4H,

t, J = 5.7 Hz, CH₂CH₂), 4.16 (8H, bs, CH₂), 1.70–1.51 (12H, m, CH₂). ¹³C-NMR (75 MHz,

DMSO-d₆) (

δ

/ppm): 153.2 (C6), 152.4 (Ph-q), 152.1 (C2), 150.6 (C4), 143.3 (C4⁰), 139.5 (C8),

0

124.8 (C5 ), 118.9 (C5), 115.9 (Ph), 61.7 (CH₂), 48.8 (CH₂), 43.2 (CH₂), 25.8 (CH₂), 24.4 (CH₂).

Anal. calcd. for C₃₆H₄₂N₁₆O₂: C, 59.16; H, 5.79; N, 30.66. Found: C, 59.36; H, 5.64; N, 30.62.

1

9e: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.19 (1H, s, H2), 8.12 (1H, s, H8), 7.83

(1H, s, H5⁰), 6.92 (4H, d, J = 1.9 Hz, Ph), 5.02 (2H, s, CH₂), 4.87 (2H, t, J = 5.7 Hz, CH₂CH₂),

4,71 (2H, d, J = 2.4 Hz, CH₂CCH), 4.66 (2H, t, J = 5.7 Hz, CH₂CH₂), 4.17 (4H, s, CH₂),

3.53 (1H, t, J = 2.4 Hz, CH₂CCH), 1.74–1.48 (6H, m, CH₂). ¹³C-NMR (75 MHz, DMSO-d₆)

0

(

δ

/ppm): 153.0 (C6), 152.6 (Ph-q), 151.9 (C2), 151.4 (Ph-q), 150.5 (C4), 143.0 (C4 ), 138.5 (C8),

0

124.6 (C5 ), 118.7 (C5), 115.8 (Ph), 115.6 (Ph), 78.0 (CCH), 61.5 (CH₂), 55.9 (CH₂), 48.5 (CH₂),

43.0 (CH₂), 25.6 (CH₂), 24.2 (CH₂). Anal. calcd. for C₂₄H₂₆N₈O₂: C, 62.87; H, 5.72; N, 24.44.

Found: ₀C, 62.91; H, 5.77; N, 24.28.

4,4 -Bis((1-(2-(6-(piperidin-1-yl)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-1,

1⁰-biphenyl (9b) and 6-(piperidin-1-yl)-9-{2-[4-(((4⁰-(prop-2-yn-1-yloxy)-[1,1⁰-biphenyl]-4-

yl)oxy)methyl)-1H-1,2,3-triazol-1-yl]ethyl}-9H-purine (9f). Compounds 9b and 9f were pre-

pared according to the abovementioned procedure from compound 4b (50 mg, 0.19 mmol)

and compound 3c (113.8 mg, 0.42 mmol) to obtain 9b as white solid (method A: 74 mg; 48%;

method B: 105.6 mg, 69%; method C: 53.4 mg, 38%; m.p. = 221–225 ^◦C) and 9f (method C:

31.2 mg, 31%; m.p. = 184–185 ^◦C).

1

9b: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.18 (4H, s, H2; H8), 7.85 (2H, s, H5⁰),

7.53 (4H, d, J = 8.7 Hz, Ph), 7.04 (4H, d, J = 8.7 Hz, Ph), 5.12 (4H, s, CH₂), 4.88 (4H, t,

J = 5.7 Hz, CH₂CH₂), 4.67 (4H, t, J = 5.7 Hz, CH₂CH₂), 4.16 (8H, bs, CH₂), 1.73–1.48 (12H,

m, CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

150.5 (C4), 142.9 (C4 ), 132.6 (Ph-q), 127.2 (Ph), 124.7 (C5 ), 118.7 (C5), 115.1 (Ph), 61.1 (CH₂),

δ

/ppm): 157.1 (Ph-q), 153.0 (C6), 151.8 (C2/C8),

0

48.6 (CH₂), 43.0 (CH₂), 25.6 (CH₂), 24.2 (CH₂). Anal. calcd. for C₄₂H₄₆N₁₆O₂: C, 62.52; H,

5.75; N, 27.77. Found: C, 62.62; H, 5.71; N, 27.76.

1

9f: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.18 (1H, s, H2), 8.16 (1H, s, H8), 7.83

(1H, s, H5⁰), 7.55 (4H, dd, J = 8.7, 6.4 Hz, Ph), 7.05 (4H, dd, J = 8.8, 1.9 Hz, Ph), 5.12 (2H,

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s, CH₂), 4.88 (2H, t, J = 5.7 Hz, CH₂CH₂), 4.82 (2H, d, J = 2.3 Hz, CH₂CCH), 4.66 (2H, t,

J = 5.8 Hz, CH₂CH₂), 4.16 (4H, bs, CH₂), 3.58 (1H, t, J = 2.3 Hz, CH₂CCH), 1.73–1.50 (6H,

m, CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm): 157.3 (Ph-q), 156.4 (Ph-q), 153.2 (C6),

152.0 (C2), 150.6 (C4), 143.0 (C4⁰), 139.4 (C8), 133.1 (Ph-q), 132.6 (Ph-q), 127.4 (Ph), 127.3

0

(Ph), 124.8 (C5 ), 118.8 (C5), 115.3 (Ph), 115.2 (Ph), 79.4 (

CCH), 78.3 (CCH), 61.2 (CH₂), 55.5

(CH₂), 48.7 (CH₂), 43.1 (CH₂), 25.7 (CH₂), 24.3 (CH₂). Anal. calcd. for C₃₀H₃₀N₈O₂: C,

67.40; H, 5.66; N, 20.96. Found: C, 67.38; H, 5.41; N, 20.99.

1,3-Bis{1-[2-(6-(piperidin-1-yl)-9H-purin-9-yl)ethyl]-1H-1,2,3-triazol-4-yl}propane (9c

)

and 9-{2-[4-(pent-4-yn-1-yl)-1H-1,2,3-triazol-1-yl]ethyl}-6-(piperidin-1-yl)-9H-purine (9g).

Compounds 9c and 9g were prepared according to the above-mentioned procedure from

1,6-heptadiyne 4c (0.06 mL, 0.54 mmol) and compound 3c (324.1 mg, 1.19 mmol) to obtain

9c as white solid (method A: 137.3 mg, 40%; method B: 214.3 mg, 62%; method C: 78.4 mg,

21%, m.p. = 218–220 ^◦C) and 9g (method C: 20.7 mg, 10%; m.p. = 171–174 ^◦C).

1

9c: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.17 (2H, s, H2), 7.81 (2H. s, H8), 7.72

(2H, s, H5⁰), 4.80 (4H, t, J = 5.6 Hz, CH₂CH₂), 4.62 (4H, t, J = 5.7 Hz, CH₂CH₂), 4.14 (4H,

s, CH₂), 1.86–1.69 (2H, m, CH₂CH₂CH₂), 1.68–1.50 (12H, m, CH₂). ¹³C-NMR (151 MHz,

DMSO-d₆) (δ

/ppm): 153.0 (C6), 151.8 (C2), 150.5 (C4), 146.5 (C4⁰), 139.3 (C8), 122.2 (C5⁰),

118.7 (C5), 48.4 (CH₂), 43.1 (CH₂), 28.7 (CH₂), 25.6 (CH₂), 24.2 (CH₂), 24.1 (CH₂). Anal.

calcd. for C₃₁H₄₀N₁₆: C, 58.47; H, 6.33; N, 35.19. Found: C, 58.47; H, 6.25; N, 35.22.

1

9g: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.18 (1H, s, H2), 7.81 (1H, s, H8), 7.72

(1H, s, H5⁰), 4.86–4.72 (2H, m, CH₂CH₂), 4.68–4.57 (2H, m, CH₂CH₂), 4.16 (4H, bs, CH₂),

2.78 (1H, t, J = 2.6 Hz, CCH), 2.62 (2H, t, J = 7.5 Hz, CH₂CH₂CH₂), 2.12 (2H, td, J = 7.0,

2.6 Hz, CH₂CH₂CH₂), 1.77–1.43 (8H, m, CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm):

153.0 (C6), 151.8 (C2), 150.4 (C4), 146.0 (C4⁰), 139.2 (C8), 122.1 (C5⁰), 118.7 (C5), 84.0 (CCH),

71.3 (CCH), 48.4 (CH₂), 43.0 (CH₂), 27.7 (CH₂), 25.5 (CH₂), 24.2 (CH₂), 23.8 (CH₂), 17.0

(CH₂). Anal. calcd. for C₁₉H₂₄N₈: C, 62.62; H, 6.64; N, 30.75. Found: C, 58.47; H, 6.25;

N, 35.22.

{1-[2-(6-(Piperidin-1-yl)-9H-purin-9-yl)ethyl]-1H-1,2,3-triazol-4-yl}methyl ether (9d) Com-

pound 9d was prepared according to the abovementioned procedure for method B from

propargyl ether 4d (0.05 mL, 0.53 mmol) and compound 3c (301.2 mg, 1.17 mmol) to obtain

◦

9d as yellow solid (method A: 187.3 mg, 55%; method C: 156.1 mg, 46%; m.p. = 107–109 C).

0

¹H-NMR (600 MHz, DMSO-d₆) (

δ

/ppm): 8.17 (2H, s, H2), 8.03 (2H, s, H8), 7.82 (2H, s, H5 ),

4.88–4.84 (4H, m, CH₂CH₂), 4.67–4.64 (4H, m, CH₂CH₂), 4.42 (4H, s, CH₂CH₂), 4.13 (8H,

bs, CH₂), 1.67–1.62 (4H, m, CH₂), 1.57–1.51 (8H, m, CH₂). ¹³C-NMR (75 MHz, DMSO-d₆)

/ppm): 153.1 (C6), 151.9 (C2), 150.5 (C4), 142.1 (C4⁰), 139.3 (C8), 126.3 (C5⁰), 118.8 (C5),

O

(

δ

62.2 (CH₂), 48.6 (CH₂), 43.1 (CH₂), 25.7 (CH₂), 24.2 (CH₂). Anal. calcd. for C₃₀H₃₈N₁₆O: C,

56.41; H, 6.00; N, 35.09. Found: C, 56.56; H, 6.03; N, 34.98.

1,4-Bis{[1-(2-(6-(pyrrolidin-1-yl)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-4-yl]methoxy}benzene

(

10a). Compound 10a was prepared according to the abovementioned procedure from

compound 4a (50 mg, 0.27 mmol) and compound 3d (152.8 mg, 0.59 mmol) to obtain 10a

as white solid (method A: 53.2 mg, 28%; method B: 100.7 mg, 61%; method C: 99 mg; 60%,

m.p. = 253–256 ^◦C). ¹H-NMR (300 MHz, DMSO-d₆) (

δ/ppm): 8.17 (2H, s, H2), 8.09 (2H,

s, H8), 7.79 (2H, s, H5⁰), 6.90 (4H, s, Ph), 5.00 (4H, s, OCH₂), 4.94–4.76 (4H, m, CH₂CH₂),

4.76–4.59 (4H, m, CH₂CH₂), 4.01 (4H, bs, CH₂), 3.62 (4H, bs, CH₂), 1.93 (8H, bs, CH₂).

¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm): 152.4 (Ph-q), 152.3 (C6), 152.2 (C2), 149.9 (C4),

143.0 (C4⁰), 124.5 (C5⁰), 119.2 (C5), 115.7 (Ph), 61.6 (CH₂), 48.6 (CH₂), 42.9 (CH₂). Anal.

calcd. for C₃₄H₃₈N₁₆O: C, 58.11; H, 5.45; N, 31.89. Found: C, 58.15; H, 5.55; N, 32.00.

0

4,4 -Bis{[1-(2-(6-(piperidin-1-yl)-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-4-yl]methoxy}-1,

1⁰-biphenyl (10b) and 6-(piperidin-1-yl)-9-{2-[4-(((4⁰-(prop-2-yn-1-yloxy)-[1,1⁰-biphenyl]-

4-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl]ethyl}-9H-purine (10f). Compounds 10b and 10f

were prepared according to the abovementioned procedure from compound 4b (50 mg,

0.19 mmol) and compound 3d (107.3 mg, 0.42 mmol) to obtain 10b as white solid (method

A: 88.4 mg, 60%; method B: 118.4 mg, 80%; method C: 71.5 mg, 49%; m.p. = 259–262 ^◦C)

and 10f as yellow solid (method C: 60.5 mg, 61%; m.p. = 134–137 ^◦C).

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1

10b: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.17 (2H, s, H2), 8.14 (2H, s, H8), 7.80

(2H, s, H5⁰), 7.53 (4H, d, J = 8.7 Hz, Ph), 7.04 (4H, d, J = 8.7 Hz, Ph), 5.12 (4H, s, OCH₂),

4.92–4.85 (4H, m, CH₂CH₂), 4.70–4.63 (4H, m, CH₂CH₂), 4.00 (4H, bs, CH₂), 3.61 (4H, bs,

CH₂), 1.92 (8H, bs, CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

152.1 (C2), 149.8 (C4), 142.8 (C4 ), 136.3 (C8), 132.6 (Ph-q), 127.1 (Ph), 124.5 (C5 ), 119.1 (C5),

δ

/ppm): 157.1 (Ph-q), 152.4 (C6),

0

115.1 (Ph), 61.2 (CH₂), 48.5 (CH₂), 42.8 (CH₂), 41.4 (CH₂). Anal. calcd. for C₄₂H₄₆N₁₆O₂: C,

62.52; H, 5.75; N, 27.77. Found: C, 62.51; H, 5.81; N, 27.96.

1

10f: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.17 (1H, s, H2), 8.14 (1H, s, H8), 7.80

(1H, s, H5⁰), 7.55 (4H, dd, J = 8.7, 7.1 Hz, Ph), 7.04 (4H, d, J = 8.7 Hz, Ph), 5.12 (2H, s, CH₂),

4.91–4.86 (2H, m, CH₂CH₂), 4.83 (2H, d, J = 2.3 Hz, CH₂CCH), 4.70–4.64 (m, CH₂CH₂),

3.69–3.53 (3H, m, CH₂, CCH), 1.93 (4H, s, CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm):

157.2 (Ph-q), 156.4 (Ph-q), 152.5 (C6), 152.3 (C2), 149.9 (C4), 146.89, 143.0 (C4⁰), 139.8 (C8),

133.1 (Ph-q), 132.6 (Ph-q), 127.4 (Ph), 127.3 (Ph), 124.7 (C5⁰), 119.3 (C5), 115.3 (Ph), 115.3

(Ph), 79.4 (CCH), 78.2 (CCH), 61.2 (CH₂), 55.5 (CH₂), 48.7 (CH₂), 43.0 (CH₂). Anal. calcd.

for C₃₀H₃₀N₈O: C, 67.40; H, 5.66; N, 20.96. Found: C, 67.27; H, 5.71; N, 23.14.

1,3-Bis{1-[2-(6-(piperidin-1-yl)-9H-purin-9-yl)ethyl]-1H-1,2,3-triazol-4-yl}propane (10c). Com-

pound 10c was prepared according to the abovementioned procedure from 1,6-heptadiyne

4c (50 mg, 0.54 mmol) and compound 3d (305.7 mg, 1.19 mmol) to obtain 10c as white

solid (method A: 163.5 mg, 50%; method B: 150.8 mg, 82%; method C: 196.2 mg, 58%;

m.p. = 217–220 ^◦C).

10c: δ/ppm): 8.15 (1H, s, H2), 7.76 (1H, s, H8),

¹H-NMR (600 MHz, DMSO-d₆) (

7.69 (1H, s, H5⁰), 4.90–4.69 (4H, m, CH₂CH₂), 4.75–44.52 (4H, m, CH₂CH₂), 3.98 (4H, bs,

CH₂), 3.58 (4H, bs, CH₂), 2.48 (m, CH₂CH₂CH₂), 1.90 (8H, bs, CH₂), 1.83–1.70 (2H, m,

CH₂CH₂CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm): 152.4 (C6), 152.2 (C2), 149.9 (C4),

0

146.4 (C4 ), 139.8 (C8), 122.2 (C5 ), 119.2 (C5), 48.4 (CH₂), 43.0 (CH₂), 28.7 (CH₂), 24.1 (CH₂).

Anal. calcd. for C₃₁H₄₀N₁₆: C, 58.47; H, 6.33; N, 35.19. Found: C, 58.22; H, 6.19; N, 34.99.

{1-[2-(6-(Piperidin-1-yl)-9H-purin-9-yl)ethyl]-1H-1,2,3-triazol-4-yl}methyl ether (10d) Com-

pound 10d was prepared according to the abovementioned procedure for method B from

4d (50 mg, 0.53 mmol) and compound 3d (301.3 mg, 1.17 mmol) to obtain 10d as yellow

solid (method A: 164.2 mg, 48%; method C: 197 mg, 58%; m.p. = 112–115 ^◦C). ¹H-NMR

0

(300 MHz, DMSO-d₆) (

δ

/ppm): 8.16 (2H, s, H2), 8.05 (2H, s, H8), 7.81 (2H, s, H5 ), 4.86 (4H,

s, CH₂), 4.66 (4H, s, CH₂), 4.40 (4H, s, CH₂), 3.97 (4H, s, CH₂), 3.59 (4H, s, CH₂), 1.91 (8H, s

CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm): 152.5 (C6), 152.2 (C2), 149.9 (C4), 143.8

(C4⁰), 139.5 (C8), 124.4 (C5⁰), 119.2 (C5), 62.2 (CH₂OCH₂), 48.7 (CH₂), 43.0 (CH₂). Anal.

calcd. for C₃₀H₃₈N₁₆O: C, 56.41; H, 6.00; N, 35.09. Found: C, 56.78; H, 5.89; N, 35.02.

1,4-Bis{[1-(2-(6-amino-9H-purin-9-yl)ethyl)-1H-1,2,3-triazol-4-yl]methoxy}benzene (11a

)

and 6-amino-9-{2-[4-((4-(prop-2-yn-1-yloxy)phenoxy)methyl)-1H-1,2,3-triazol-1-yl]ethyl}-

9H-purine (11e). Compounds 11a and 11e were prepared according to the above-mentioned

procedure from compound 4a (100 mg, 0.49 mmol) and compound 3e (200.7 mg, 1.08 mmol)

◦

to obtain 11a as white solid (method B: 28.9 mg, 11%; m.p. > 280 C) and 11e as white solid

(method C: 47.4 mg, 25%; m.p. > 250 ^◦C).

1

11a: H-NMR (600 MHz, DMSO-d₆) (

δ

/ppm): 8.11 (2H, s, H2), 8.09 (2H, s, H8), 7.80

(2H, s, H5⁰), 7.20 (4H, s, NH₂), 6.90 (4H, s, Ph), 5.01 (4H, s, CH₂), 4.87 (4H, t, J = 5.8 Hz,

CH₂CH₂), 4.65 (4H, t, J = 5.8 Hz, CH₂CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ

/ppm):

0

155.6 (Ph-q), 152.2 (C6), 152.1 (C2), 149.3 (C4), 142.9 (C4 ), 124.0 (C5 ), 118.5 (C5), 115.7 (Ph),

61.7 (CH₂), 48.3 (CH₂), 42.6 (CH₂). Anal. calcd. for C₂₆H₂₆N₁₆O₂: C, 52.52; H, 4.41; N,

37.69. Found: C, 52.38; H, 4.17; N, 37.91.

1

11e: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.11 (2H, s, Hz, H2; H8), 7.80 (1H, s,

H5⁰), 7.20 (2H, s, NH₂), 6.92 (4H, d, J = 1.6 Hz, Ph), 5.01 (2H, s, CH₂), 4.87 (2H, t, J = 5.7

Hz, CH₂CH₂), 4.72 (2H, d, J = 2.3 Hz, CH₂CCH), 4.65 (2H, t, J = 5.7 Hz, CH₂CH₂), 3.53 (t,

J = 2.3 Hz, CH₂CCH). ¹³C-NMR (75 MHz, ₀DMSO-d₆) (

δ

/ppm): 155.9 (Ph-q), 152.6 (C6),

0

152.5 (C2), 151.4 (Ph-q), 149.5 (C4), 143.0 (C4 ), 140.5 (C8), 124.6 (C5 ), 118.6 (C5), 115.8 (Ph),

115.6 (Ph), 79.5 (CCH), 78.0 (CCH), 6.5 (CH₂), 55.9 (CH₂), 48.6 (CH₂), 43.0 (CH₂). Anal.

calcd. for C₁₉H₁₈N₈O₂: C, 58.45; H, 4.65; N, 28.70. Found: C, 58.30; H, 4.75; N, 28.87.

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6-Amino-9-{2-[4-(pent-4-yn-1-yl)-1H-1,2,3-triazol-1-yl]ethyl}-9H-purine (11g) Compound

11g was prepared according to the abovementioned procedure from 1,6-heptadiyne 4c

(50 mg, 0.53 mmol) and compound 3e (243.7 mg, 1.17 mmol) to obtain 11g as white solid

(method C: 89.3 mg, 55%; m.p. = 226–228 ^◦C). ¹H-NMR (600 MHz, DMSO-d₆) (

δ

/ppm):

8.11 (1H, s, H2), 7.78 (1H, s, H8), 7.74 (1H, s, H5⁰), 7.17 (2H, s, NH₂), 4.79 (2H, t, J = 5.8 Hz

,

CH₂CH₂), 4.61 (2H, t, J = 5.8 Hz, CH₂CH₂), 2.77 (1H, t, J = 2.6 Hz, CCH), 2.62 (2H, t,

J = 7.5 Hz, CH₂), 2.14 (2H, td, J = 7.1, 2.6 Hz, CH₂), 1.72–1.65 (2H, m, CH₂). ¹³C-NMR

(

151 MHz, DMSO-d₆) (δ

/ppm): 155.9 (C6), 152.4 (C2), 149.4 (C4), 146.0 (C4⁰), 140.4 (C8),

122.1 (C5⁰), 118.5 (C5), 84.0 (CCH), 71.4 (CCH), 48.5 (CH₂), 42.9 (CH₂), 27.8 (CH₂), 23.8

(CH₂), 17.0 (CH₂). Anal. calcd. for C₁₄H₁₆N₈: C, 56.74; H, 5.44; N, 37.81. Found: C, 56.64;

H, 5.51; N, 38.00.

{1-[2-(6-Amino-9H-purin-9-yl)ethyl]-1H-1,2,3-triazol-4-yl}methyl ether (11d) and 6-

amino-9-{2-[4-((prop-2-yn-1-yloxy)methyl)-1H-1,2,3-triazol-1-yl]ethyl}-9H-purine (11h).

Compounds 11d and 11h were prepared according to the abovementioned procedure

for method B from propargyl ether 4d (100 mg, 1.06 mmol) and compound 3e (432.9 mg,

2.12 mmol) to obtain 11d as white solid (method C: 48.9 mg, 8%, m.p. = 191–193 ^◦C) and

11h as white solid (method C: 20.8 mg, 7%, m.p. = 175–180 ^◦C).

1

11d: H-NMR (600 MHz, DMSO-d₆) (

δ

/ppm): 8.17 (2H, s, H2), 8.02 (2H, s, H8), 7.89

(2H, s, H5⁰), 7.57 (4H, bs, NH₂), 4.86 (4H, t, J = 5.7 Hz, CH₂CH₂), 4.66 (4H, t, J = 5.7 Hz,

CH₂CH₂), 4.50 (4H, s, CH₂/ppm): 155.8 (C6),

152.4 (C2), 149.4 (C4), 143.6 (C4 ), 124.1 (C5 ), 118.5 (C5), 62.1 (CH₂), 48.5 (CH₂), 42.9 (CH₂).

O

CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ

0

Anal. calcd. for C₂₀H₂₂N₁₆O: C, 47.80; H, 4.41; N, 44.60. Found: C, 47.81; H, 4.46; N, 44.59.

1

11h: H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm): 8.11 (1H, s, H2), 8.02 (1H, s, H8), 7.80

(1H, s, H5⁰), 7.20 (2H, s, NH₂), 4.85 (2H, t, J = 5.7 Hz, CH₂CH₂), 4.64 (2H, t, J = 5.7 Hz,

CH₂CH₂), 4.50 (2H, s, OCH₂), 4.09 (2H, d, J = 2.4 Hz, CH₂CCH), 3.46 (1H, t, J = 2.4 Hz,

CH₂CCH). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm): 155.9 (C6), 152.4 (C2), 149.5 (C4),

143.2 (C4⁰), 140.4 (C8), 124.4 (C5⁰), 118.6 (C5), 79.9 (CCH), 77.4 (CCH), 61.8 (CH₂), 56.3

(CH₂), 48.6 (CH₂), 43.0 (CH₂). Anal. calcd. for C₁₃H₁₄N₈O: C, 52.34; H, 4.73; N, 37.56.

Found: C, 52.47; H, 4.69; N, 37.36.

3.6. General Procedure for the Synthesis of Bis-Pyrrolo[2,3-d]pyrimidine Derivatives 6a–6d and

7a–7d

The corresponding bis-pyrrolo[2,3-d]pyrimidine 5a–5d and cyclic amine (4 eq) were

dissolved in water (3 mL). The reaction mixture was stirred under microwave irradiation

(400 W) at 100 ^◦C during 10 min. The reaction mixture was triturated with acetonitrile to

obtain the crude product.

1,4-Bis{[1-(2-(4-(piperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)-1H-1,2,3-triazol-4-y

l]methoxy}benzene (6a). Compound 6a was prepared according to the abovementioned pro-

cedure from compound 5a (100 mg, 0.16 mmol) and piperidine (0.06 mL, 0.64 mmol) to

◦

obtain 6a as white solid (68.9 mg, 59%, m.p. = 185–187 C). ¹H-NMR (600 MHz, DMSO-d₆)

0

(

δ

/ppm): 8.13 (2H, s, H2), 8.01 (2H, s, H5 ), 6.97–6.78 (6H, m, Ph; H6), 6.48 (2H, d, J = 3.7 Hz,

H5), 5.01 (4H, s, OCH₂), 4.80 (4H, t, J = 5.9 Hz, CH₂CH₂), 4.62 (4H, t, J = 5.9 Hz, CH₂CH₂),

3.92–3.74 (8H, m, CH₂), 1.69–1.50 (12H, m, CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm):

156.2 (Ph-q), 152.2 (C4), 150.7 (C7a), 150.7 (C2), 142.9 (C4⁰), 124.5 (C5⁰), 123.9 (C6), 115.6

(Ph), 102.1 (C4a), 100.9 (C5), 61.8 (OCH₂), 49.0 (CH₂CH₂), 46.2 (CH₂CH₂), 43.9 (CH₂), 25.4

(CH₂), 24.2 (CH₂). Anal. calcd. for C₃₈H₄₄N₁₄O₂: C, 62.62; H, 6.09; N, 26.90. Found: C,

62.79; H, 5.92; N, 27.15.

4,4⁰-Bis{[1-(2-(4-(piperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)-1H-1,2,3-triazol-4-

yl]methoxy}-1,1⁰-biphenyl (6b). Compound 6b was prepared according to the abovemen-

tioned procedure from compound 5b (70 mg, 0.09 mmol) and piperidine (0.04 mL, 0.37

mmol) to obtain 6b as white solid (51.9 mg, 73%, m.p. = 137–139 ^◦C). ¹H-NMR (600 MHz,

0

DMSO-d₆) (

δ

/ppm): 8.13 (1H, s, H2), 8.06 (1H, s, H5 ), 7.53 (4H, d, J = 8.7 Hz, Ph), 7.04 (4H,

d, J = 8.8 Hz, Ph), 6.88 (2H, d, J = 3.6 Hz, H6) 6.45 (2H, d, J = 3.7 Hz, H5), 5.12 (4H, s, OCH₂),

4.81 (4H, t, J = 5.9 Hz, CH₂CH₂), 4.63 (4H, t, J = 5.9 Hz, CH₂CH₂), 3.82–3.79 (8H, m, CH₂),

1.78– 1.45 (12H, m, CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm): 157.1 (Ph-q), 156.2 (C4),

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20 of 26

150.7 (C7a), 150.7 (C2), 142.8 (C4⁰), 132.5 (Ph-q), 127.2 (Ph), 124.6 (C5), 123.9 (C5⁰), 115.2

(Ph), 102.1 (C4a), 100.9 (C6), 61.1 (OCH₂), 49.1 (CH₂CH₂), 46.2 (CH₂CH₂), 43.9 (CH₂), 25.4

(CH₂), 24.2 (CH₂). Anal. calcd. for C₄₄H₄₈N₁₄O₂: C, 65.65; H, 6.01; N, 24.36. Found: C,

65.89; H, 6.11; N, 24.23.

1,3-Bis{1-[2-(4-(piperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-1H-1,2,3-triazol-4-yl

}propane (6c). Compound 6c was prepared according to the abovementioned procedure

from compound 5c (50 mg, 0.09 mmol) and piperidine (0.04 mL, 0.37 mmol) to obtain 6c as

white solid (51.3 mg, 87%, m.p. = 145–147 ^◦C). ¹H-NMR (600 MHz, DMSO-d₆) (

δ/ppm):

8.11 (2H, s, H2), 7.57 (2H, s, H5⁰), 6.92 (2H, d, J = 3.7 Hz, H6), 6.50 (2H, d, J = 3.7 Hz, H5),

4.73 (4H, t, J = 5.9 Hz, CH₂CH₂), 4.60 (4H, t, J = 5.9 Hz, CH₂CH₂), 3.98–3.64 (8H, m, CH₂),

2.47 (8H, t, J = 7.4 Hz, CH₂CH₂CH₂), 1.76–1.69 (2H, m, CH₂CH₂CH₂), 1.66–1.60 (4H, m,

CH₂), 1.56–1.50 (8H, m, CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

δ/ppm): 156.3 (C4), 150.8

0

(C7a), 150.7 (C2), 146.4 (C4 ), 124.0 (C5), 122.2 (C5 ), 102.2 (C4a), 101.0 (C6), 49.0 (CH₂CH₂),

46.3 (CH₂CH₂), 44.1 (CH₂), 28.7 (CH₂), 25.5 (CH₂), 24.2 (CH₂), 24.1 (CH₂). Anal. calcd. for

C₃₃H₄₂N₁₄: C, 62.44; H, 6.67; N, 30.89. Found: C, 62.38; H, 6.91; N, 30.97.

{1-[2-(4-(Piperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-1H-1,2,3-triazol-4-yl}methyl

ether (6d). Compound 6d was prepared according to the abovementioned procedure from

compound 5d (50 mg, 0.09 mmol) and piperidine (0.04 mL, 0.37 mmol) to obtain 6d as

white solid (34.7 mg, 59%, m.p. = 141–143 ^◦C). ¹H-NMR (300 MHz, DMSO-d₆) (

δ/ppm):

8.12 (2H, s, H2), 7.90 (2H, s, H5⁰), 6.95 (2H, d, J = 3.1 Hz, H6), 6.51 (2H, d, J = 3.1 Hz, H5),

4.84–4.75 (4H, m, CH₂CH₂), 4.66–4.58 (4H, m, CH₂CH₂), 4.38 (4H, s, CH₂OCH₂), 3.92–3.66

(8H, m, CH₂), 1.74–1.43 (12H, m, CH₂). ¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm): 156.1

0

(C4), 150.7 (C7a), 150.7 (C2), 143.5 (C4 ), 124.2 (C5 ), 123.9 (C6), 102.1 (C4a), 100.9 (C5), 62.0

(OCH₂), 49.0 (CH₂CH₂), 46.2 (CH₂CH₂), 44.0 (CH₂), 25.4 (CH₂), 24.2 (CH₂). Anal. calcd.

for C₃₂H₄₀N₁₄O: C, 60.36; H, 6.33; N, 30.80. Found: C, 60.51; H, 6.57; N, 30.88.

1,4-Bis{[1-(2-(4-(pyrrolidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)-1H-1,2,3-triazol-4-

yl]methoxy}benzene (7a). Compound 7a was prepared according to the abovementioned

procedure from compound 5a (50 mg, 0.10 mmol) and pyrrolidine (0.04 mL, 0.40 mmol) to

◦

obtain 7a as white solid (46.3 mg, 65%, m.p. = 193–195 C). ¹H-NMR (300 MHz, DMSO-d₆)

(δ

/ppm): 8.09 (2H, s, H2), 8.00 (2H, s, H5⁰), 6.89 (4H, s, Ph), 6.84 (2H, d, J = 3.5 Hz, H6),

6.48 (2H, d, J = 3.6 Hz, H5), 5.00 (4H, s, OCH₂), 4.80 (4H, t, J = 5.6 Hz, CH₂CH₂), 4.62 (4H, t,

J = 5.6 Hz, CH₂CH₂), 3.67 (8H, bs, CH₂), 1.93 (8H, bs, CH₂). ¹³C-NMR (75 MHz, DMSO-d₆)

0 0

(δ/ppm): 154.7 (C4), 152.2 (C7a), 151.2 (C2), 149.8 (Ph-q), 142.9 (C4 ), 124.5 (C5 ), 123.4 (C6),

115.7 (Ph), 102.6 (C4a), 100.7 (C5), 61.5 (OCH₂), 49.0 (CH₂), 47.4 (CH₂), 43.8 (CH₂). Anal.

calcd. for C₃₆H₄₀N₁₄O₂: C, 61.70; H, 5.75; N, 27.98. Found: C, 61.52; H, 5.90; N, 28.01.

0

4,4 -Bis{[1-(2-(4-(pyrrolidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)-1H-1,2,3-triazol-4-yl]

methoxy}-1,1⁰-biphenyl (7b). Compound 7b was prepared according to the above-mentioned

procedure from compound 5b (70 mg, 0.10 mmol) and pyrrolidine (0.04 mL, 0.40 mmol) to

obtain 7b as white solid (46.3 mg, 6%, m.p. = 187–189 ^◦C). ¹H-NMR (600 MHz, DMSO-d₆)

0

(δ

/ppm): 8.09 (2H, s, H2), 8.04 (2H, s, H5 ), 7.53 (4H, d, J = 8.7 Hz, Ph), 7.04 (4H, d, J = 8.8 Hz,

Ph), 6.82 (2H, d, J = 3.5 Hz, H5), 6.45 (2H, d, J = 3.6 Hz, H6), 5.12 (4H, s, OCH₂), 4.80 (4H, t,

J = 5.9 Hz, CH₂CH₂), 4.62 (4H, t, J = 5.9 Hz, CH₂CH₂), 3.65 (8H, bs, CH₂), 1.92 (8H, bs, CH₂).

¹³C-NMR (75 MHz, DMSO-d₆) (

δ/ppm): 157.1 (Ph-q), 154.6 (C4), 151.1 (C2), 149.7 (C7a), 142.8

(C4⁰), 132.5 (Ph-q), 127.2 (Ph), 124.6 (C6), 123.4 (C5⁰), 115.2 (Ph), 102.6 (C4a), 100.8 (C5), 61.1

(OCH₂), 49.1 (CH₂), 47.4 (CH₂), 43.8 (CH₂). Anal. calcd. for C₄₂H₄₄N₁₄O₂: C, 64.93; H, 5.71;

N, 25.24. Found: C, 64.92; H, 5.78; N, 25.12.

1,3-Bis{1-[2-(4-(pyrrolidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-1H-1,2,3-triazol-4-

yl}propane (7c). Compound 7c was prepared according to the abovementioned procedure

from compound 5c (50 mg, 0.09 mmol) and pyrrolidine (0.04 mL, 0.37 mmol) to obtain 7c

◦

as white solid (53.1 mg, 97%, m.p. = 170–172 C). ¹H-NMR (300 MHz, DMSO-d₆) (

δ/ppm):

8.07 (2H, s, H2), 7.54 (2H, s, H5⁰), 6.87 (2H, d, J = 3.5 Hz, H6), 6.48 (2H, d, J = 3.6 Hz, H5),

4.73 (4H, t, J = 5.7 Hz, CH₂CH₂), 4.59 (4H, t, J = 5.6 Hz, CH₂CH₂), 3.64 (8H, bs, CH₂),

2.46 (4H, t, J = 7.4 Hz, CH₂CH₂CH₂), 1.91 (8H, bs, CH₂), 1.79–1.62 (2H, m, CH₂CH₂CH₂).

¹³C-NMR (151 MHz, DMSO-d₆) ( /ppm): 154.6 (C4), 151.1 (C2), 149.7 (C7a), 146.2 (C4⁰),

δ

Molecules 2021, 26, 3334

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0

123.3 (C6), 122.0 (C5 ), 102.6 (C4a), 100.7 (C5), 48.8 (CH₂), 47.3 (CH₂), 43.9 (CH₂), 28.6 (CH₂),

24.0 (CH₂). Anal. calcd. for C₃₁H₃₈N₁₄: C, 61.37; H, 6.31; N, 32.32. Found: C, 61.32; H, 6.71;

N, 32.10.

{1-[2-(4-(Pyrrolidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-1H-1,2,3-triazol-4-yl}methyl

ether (7d). Compound 7d was prepared according to the abovementioned procedure from

compound 5d (50 mg, 0.09 mmol) and pyrrolidine (0.04 mL, 0.37 mmol) to obtain 7d as

white solid (39.5 mg, 65%, m.p. = 131–135 ^◦C). ¹H-NMR (300 MHz, DMSO-d₆) (

δ

/ppm):

8.07 (2H, s, H2), 7.85 (2H, s, H5⁰), 6.89 (2H, d, J = 2.2 Hz, H6), 6.50 (2H, d, J = 2.6 Hz, H5),

4.84–4.73 (4H, m, CH₂CH₂), 4.69–4.56 (4H, m, CH₂CH₂), 4.36 (4H, s, CH₂CH₂), 3.64 (8H,

bs, CH₂), 1.92 (8H, bs, CH₂). ¹³C-NMR (151 MHz, DMSO-d₆) (

/ppm): 154.7 (C4), 151.2

O

δ

0

(C2), 149.8 (C7a), 143.4 (C4 ), 124.1 (C5 ), 123.3 (C6), 102.6 (C4a), 100.8 (C5), 62.0 (CH₂), 49.0

(CH₂), 47.4 (CH₂), 43.8 (CH₂). Anal. calcd. for C₃₀H₃₆N₁₄O: C, 59.20; H, 5.96; N, 32.22.

Found: C, 59.03; H, 5.90; N, 37.56.

3.7. Computational Details

All molecular geometries were optimized using the B97D functional together with the

6–31+G(d) basis set for non-metals and the Stuttgart-Dresden (SDD) effective core poten-

tials [61] for the inner electrons of copper atoms and its associated double-

outer ones, in line with our earlier work on the copper-catalyzed organic reactions [62] and

other literature recommendations [63 64]. To account for the solvent effects, during geome-

ζ basis set for the

,

try optimization, we included the implicit SMD solvation model corresponding to DMF

(ε = 37.219). Thermal corrections were extracted from the matching frequency calculations,

so that all presented results correspond to differences in the Gibbs free energies at room

temperature and normal pressure. The choice of such computational setup was prompted

by its success in reproducing various features of different organic [65–67], organometal-

lic [68,69] and biological systems [70,71], being particularly accurate for relative trends

among similar reactants, which is the focus here. All transition state structures were located

using the scan procedure, employing both 1D and 2D scans, the latter speciﬁcally utilized

to probe the possibility for concerted mechanisms. Apart from the visualization of the

obtained negative frequencies, the validity of all transition state structures was validated

by performing IRC calculations in both directions and identifying the matching reactant

and product structures connected by the inspected transition state. All calculations were

performed using the Gaussian 16 software [72].

3.8. Cell Culturing

Human carcinoma cell lines A549 (lung carcinoma), HeLa (cervical carcinoma), SW620

(colorectal adenocarcinoma, metastatic) and CFPAC-1 (pancreatic cancer, derived from

metastatic: liver), and normal human foreskin (HFF-1) ﬁbroblasts were obtained from the

American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in hu-

◦

0

midiﬁed atmosphere at 37 C with 5% CO₂. As growth medium, Dulbecco s modiﬁed Eagle

medium (DMEM) was used with the addition of fetal bovine serum (10%), L-glutamine

(2 mM) and antibiotics: streptomycin (100 mg/mL) and penicillin (100 U/mL).

3.9. Proliferation Assay

Cells were seeded onto 96-well microtiter plates at a seeding density of 3000 cells/well

for carcinoma cell lines, and 5000 cells/well for normal human ﬁbroblasts. The next day,

cells were treated with test agents in ﬁve different concentrations (0.01–100 µM) and further

incubated for 72 h. DMSO (solvent) was tested for potential cytotoxic effect but it did not

exceed 0.1%. Following 72 h incubation, the MTT assay was performed and measured

absorbances were transformed into percentage of cell growth as described previously [73].

Results were obtained from three independent experiments. IC₅₀values were calculated

using linear regression analysis and results were statistically analyzed by ANOVA, Tukey

post-hoc test (p < 0.05).

Molecules 2021, 26, 3334

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3.10. Apoptosis Detection

Cells were seeded into 8-well chambers (Lab-tek II Chamber Slides, Thermo Fisher

Scientiﬁc, Waltham, MA, USA) in a concentration of 2

10⁴cells per well and treated with

IC₅₀and 5 IC₅₀concentrations of selected compounds for 48 and 72 h. Staining of

×

2

×

the cells was performed by Annexin-V-FITC Staining kit (Santa Cruz Biotechnology, Dallas,

TX, USA) according to the manufacturer s instructions. Cells were visualised by ﬂuorescent

0

microscope (Olympus, Tokyo, Japan) at magniﬁcation of 40×.

4. Conclusions

The novel bis- (5a–5d, 6a–6d, 7a–7d, 9a–9d, 10a–10d, 11a–11d) and mono-(5f, 5g,

9e−9g 10f 10g 11e 11g and 11h) pyrrolo[2,3-d]pyrimidines and purines were synthesized.

,

Aromatic and aliphatic spacers were introduced between N-heterocycles by the CuAAC

reaction using different catalysts and reaction conditions (methods A–C). Non-conventional

ultrasonic energy implemented in method B proved to be better in terms of selectivity,

reaction time and efﬁciency. Ultrasound-assisted reactions led to the synthesis of exclusively

symmetrical bis-heterocycles with improved yield after 1.5 h. Direct utilization of a Cu(I)

source using the CuI/DIPEA/HOAc catalytic system resulted in the formation of both bis-

and mono-heterocycles in most cases.

DFT calculations conﬁrmed that the investigated copper-catalyzed cycloaddition is

a feasible process, and revealed that triazoles are favorably formed in a concerted and

highly exergonic fashion,

uncatalyzed reaction is associated with a high kinetic barrier of

∆

G_R=

−

51.0 kcal mol⁻¹for the studied model case. Still, the

∆

G^‡= 28.5 kcal mol⁻¹

,

which renders it as very unlikely. Once Cu(I) ions are present, they bind to the alkyne

and increase its electrophylicity towards the azide, which reduces kinetic requirements to

∆G^‡= 19.7 kcal mol⁻¹, thereby allowing the conversion to occur under normal conditions.

The obtained reaction proﬁles agree that higher temperatures and ultrasound irradiation

will improve the reaction outcomes, while eliminate the option to use strong bases with the

prospect to activate alkynes through terminal C–H deprotonation. Lastly, different trends

among reactions where catalytic Cu(I) ions are generated in situ or directly introduced

could be ascribed to the presence of Cu(II) ions in the former, but this depends on their

availability and other conditions that might be operative when a large variety of reagents

is employed as was the instance here. Lastly, a somewhat general tendency of 6-amino

derivatives to offer lower reaction yields over 6-chloro analogues is likely related to their

ability to more strongly bind Cu(I) in solution, therefore hindering its catalytic efﬁciency.

Antiproliferative evaluations showed that the linker between the heterocyclic scaf-

folds had a signiﬁcant impact on antitumor activity. Thus, cyclic amino-substituted bis-

pyrrolo[2,3-d]pyrimidines 7a and purines 9b and 10b with aromatic 1,4-bis(oxymethylene)p

0

henyl and 4,4 -bis(oxymethylene)biphenyl spacer exhibited potent cytostatic activity, espe-

cially on HeLa cell lines (7a: IC₅₀= 6.4

the mono-heterocyclic derivatives, compounds 5f

µ

M; 9b: IC₅₀= 3.8

µ

M; 10b: IC₅₀= 7.4

µ

M). Among

0

,

9f and 10f with 4,4 -bis(oxymethylene)bi

phenyl unit at C-4 of 1,2,3-triazole showed the most potent antitumor activity. The 4-

chloropyrrolo[2,3-d]pyrimidine 5f showed the most pronounced inhibitory effect against

HeLa (IC₅₀= 0.98 µM) and CFPAC-1 (IC₅₀= 0.79 µM) cell lines. Conversely, the major-

ity of bis-heterocycles with aliphatic central unit had marginal activity or were devoid

of any cytostatic activity. Among heterocycles, 4-chloropyrrolo[2,3-d]pyrimidine and 6-

piperidinylpurine made a major contribution to the antiproliferative effect. Compound

5f was further investigated by Annexin V assay, which showed that its growth-inhibitory

effect on CFPAC-1 could be ascribed to the induction of apoptosis and primary necrosis.

Our ﬁndings encourage further structural optimization of purine and fused heterocy-

cle scaffolds, such as chloropyrrolo[2,3-d]pyrimidine connected through aromatic unit, as a

promising chemical entity for development of novel efﬁcient and non-toxic agent against

pancreatic cancer.

Molecules 2021, 26, 3334

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1

Supplementary Materials: The following are available online: H- and ¹³C-NMR spectra of com-

pounds (Figures S1–S39), computational analysis (Figures S40–S46).

Author Contributions: Conceptualization, S.R.-M., K.P., and S.K.P.; syntheses and structure char-

acterization, A.B.P.; antiproliferative evaluation (experimental) P.G., M.S. and (analysis) S.K.P. and

K.P.; computational analysis; R.V.; writing

−

original draft preparation, S.R.-M., R.V., A.B.P. and M.S.;

writing review and editing, A.B.P., S.R.-M., R.V., M.S., S.K.P. and K.P.; supervision, S.R.-M., S.K.P.

−

and K.P.; project administration, S.R.-M.; funding acquisition, S.R.-M. and K.P. All authors have read

and agreed to the published version of the manuscript.

Funding: Financial support from the Croatian Science Foundation under the project HRZZ-IP-2018-

01-4682 is gratefully acknowledged.

Data Availability Statement: Figures S1–S39 with 1H and 13C NMR spectra and Figures S40–S46

containing data for computational analyses are available on line.

Acknowledgments: RV would like to thank the Zagreb University Computing Centre (SRCE) for

granting computational resources on the ISABELLA cluster.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Sample Availability: Samples of compounds 5a–5d, 6a–6d, 7a–7d, 9a–9d, 10a–10d, 11a–11d are

available from the authors.

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Article Doi

Novel bis‐ and mono‐pyrrolo[2,3‐d]pyrimidine and purine derivatives: Synthesis, computational analysis and antiproliferative evaluation

DOI: 10.3390/molecules26113334

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